key: cord- -unr dvp authors: yoo, hyun jung; yoon, sung soo; park, seon yang; lee, eun young; lee, eun bong; kim, ju han; song, yeong wook title: gene expression profile during chondrogenesis in human bone marrow derived mesenchymal stem cells using a cdna microarray date: - - journal: j korean med sci doi: . /jkms. . . . sha: doc_id: cord_uid: unr dvp mesenchymal stem cells (mscs) have the capacity to proliferate and differentiate into multiple connective tissue lineages, which include cartilage, bone, and fat. cartilage differentiation and chondrocyte maturation are required for normal skeletal development, but the intracellular pathways regulating this process remain largely unclear. this study was designed to identify novel genes that might help clarify the molecular mechanisms of chondrogenesis. chondrogenesis was induced by culturing human bone marrow (bm) derived mscs in micromass pellets in the presence of defined medium for , , or days. several genes regulated during chondrogenesis were then identified by reverse transcriptase-polymerase chain reaction (rt-pcr). using an abi microarray system, we determined the differential gene expression profiles of differentiated chondrocytes and bm-mscs. normalization of this data resulted in the identification of , differentially expressed genes. to verify gene expression profiles determined by microarray analysis, the expression levels of genes with high fold changes were confirmed by rt-pcr. gene expression patterns of genes (hrad b, annexina , bmp- , contactin- , peroxiredoxin- , heat shock transcription factor- , synaptotagmin iv, serotonin receptor- , axl) in rt-pcr were similar to the microarray gene expression patterns. these findings provide novel information concerning genes involved in the chondrogenesis of human bm-mscs. mesenchymal stem cells (mscs) are present in a variety of tissues during human development, and in particular, are prevalent in adult bone marrow ( ) . mscs isolated from bone marrow (bm) and expanded in vitro in their undifferentiated phenotype, retain an extensive capacity for multi-lineage differentiation into chondrocytes, adipocytes, osteoblasts, and tenocytes under appropriate environmental cues ( ) . the presence of specific, distinct antigens identified by the monoclonal antibodies sh , sh , and sh , on the surfaces of marrow-derived mscs, that are not present on osteocytes and osteoblasts, suggests that these epitopes are developmentally regulated. the antigen which bound to sh antibody was identified as endoglin (cd ), a receptor for tgf-β , which potentially plays a role in mediating the chondrogenic differentiation of mscs and in their interactions with hematopoietic cells ( ) . chondrogenesis, the differentiation of mscs into chondrocytes, is crucial required for skeletal development and maturation, since the cartilage anlage is the model for bone formation. cartilage development thus includes the differentiation of mscs into chondrocytes, followed by their maturation, and eventual their hypertrophy and death ( ) . differential gene expression profiling has been widely performed to identify and characterize candidate genes that play potentially important roles in particular biological process ( ) . although the amount of information regarding the role of growth factors and cytokines as inducers and mediators of msc differentiation continues to increase, little is known about the gene expression profiling of msc chondrogenic differentiation. in this study, we employed abi genechips (representing > , genes) to identify genes differentially expressed during bm-msc chondrogenesis. abi is introduced technology based on nylonspotted mer oligonucleotides, that uses on oligomers to detect each gene for most genes, chemiluminescence to measure gene expression levels, and fluorescence to grid to normalize and identify microarray features. the abi gene list was compiled from information in public and celera databases ( ) . this study was designed to identify differential gene expression profiles and novel genes that might be involved in bm-msc chondrogenesis. mononuclear cells from bm aspirates were isolated by density ficoll-paque gradient separation. bm was placed in a ml syringe containing , units of preservative-free heparin, diluted : with phosphate buffered saline (pbs), resuspended in pbs to a final volume of ml, and layered over an equal volume of histopaque- , (sigma chemical co., st. louis, mo, usa). after centrifugation at , rpm for min, mononuclear cells were recovered from the gradient interface, rinsed twice in pbs, adjusted to a concentration of . × cells/ ml, and seeded onto -mm culture plates in dulbecco's modified eagle's medium-low glucose (dmem-lg; g/l glucose, jbi, seoul, korea) containing % penicillin-streptomycin (p/s; , units/ml, gibco/brl, new york, ny, usa) and % (v/v) heat-inactivated fetal bovine serum (fbs; hyclone, logan, ut, usa). total numbers of nucleated and viable cells were determined using a hemocytometer and trypan blue (gibco/brl, gaithersburg, md, usa) staining. cells were incubated at °c in a humidified % co atmosphere and allowed to adhere for hr. non-adherent cells were then removed. the medium was changed twice a week. when cells were %- % confluent, adherent cells were trypsinized ( . % trypsin, gibco/brl) at °c for min and replated in -mm culture plates. after passage , a morphologically homogenous population of adherent cells was obtained. during this expansion, medium was changed every - days. mscs that adhered to spot slide bottoms were fixed with - °c methanol ( %) for min. cells were then rehydrated in pbs for min at room temperature, washed three times with pbs, blocked with % bovine serum albumin in pbs, and incubated overnight at °c with sh (american type culture collection [atcc], rockville, va, usa) as a positive control. primary antibody (sh ) was removed by washing three times with pbs, and cells were then incubated with fluorescein isothiocyanate (fitc)labeled affinity-purified antibody to mouse igg + igm (h + l) (dinona inc., seoul, korea) for hr at room temperature. secondary antibodies were removed by washing three times with pbs. coverslips were mounted onto slides with a solution containing % pbs and % glycerol. labeled cells were observed under an axiovert (zeiss, thornwood, ny, usa). flow cytometry was performed to determine mscs positive for sh . cells were permeabilized with ice cold % methanol in pbs for min at °c, and washed three times. a fitc-conjugated sh antibody (dinona), diluted : in pbs, was then added, and cells were incubated for hr at °c. cells were analyzed within hr of staining using a flow cytometer (facscali-bur, becton dickinson, bedford, ma, usa). a total of × cells were collected for each measurement. negative control samples were stained with an isotype-matched irrelevant mab. to induce chondrogenic differentiation, , mscs were placed in a -ml polypropylene tube and centrifuged at , rpm for min. pellets were then cultured at °c in % co and μl of serum-free chondrogenic medium consisting of dulbecco's modified eagle medium-high glucose (dmem-hg, jbi) supplemented with ng/ml of transforming growth factor-β (tgf-β , r&d systems, minneapolis, mn, usa), nm dexamethasone (sigma-aldrich, st. louis, mo, usa), μg/ml ascorbate- -phosphate, μg/ml pyruvate, and mg/ml its + premix (becton dickinson biosciences, bedford, ma, usa; . μg/ml insulin, . μg/ml transferrin, . ng/ml selenious acid, . mg/ml bovine serum albumin [bsa], and . mg/ ml linolenic acid); the medium was replaced every - days for , , , or days. total rna was extracted from undifferentiated mscs and from pellets after , , , or days of differentiation using rneasy kits (qiagen, valencia, ca, usa), according to the manufacturer's instructions. and , μl of rlt buffer supplemented with beta-mercaptoethanol ( μl/ml) was added to the washed cells. rna integrity was assessed by gel electrophoresis and rt-pcr and concentrations were determined by measuring absorbance at nm. total rna was processed using the genesys applied biosystem facility (genesys, munster, germany), according to manufacturer's recommendations. each rna pool ( μg) was labeled with digoxigenin-utp using the abi chemiluminescent rt-ivt labeling kit v . . double-stranded cdna was prepared from total rna. utp-digoxigenin-labeled complementary rna (crna) was synthesized by in vitro transcription. labeled crna ( μg) was hybridized to abi human genome survey microarray v . , which was then incubated with alkaline phosphatase-linked digoxigenin antibody. phosphatase activity was then initiated to produce the chemiluminescent signal. chemiluminescent (crna) and fluorescent (spot background) signals of the crna and standard control spots were then scanned. chemiluminescent detection and image acquisition was performed using an applied biosystems , (ab , ) chemiluminescent microarrays analyzer, according to the manufacturer's instructions. using the software supplied with the ab , apparatus, the spot chemiluminescent signal was normalized over the fluorescent signal of the same spot to obtain normalized signal value. for inter-array normalization, global median normalization was ap-plied across all microarrays ( ). data analysis and data normalization were performed using the method described by quackenbush ( ) . for background correction, the mean intensities of areas surrounding spots were subtracted from spot intensities (local area background). data sets were normalized by dividing the mean intensity value of every spot (in duplicate) by sum of all spot intensities within a sample to eliminate experimental or data acquisition variations. normalized data were used to calculate the gene expression level ratios of different culture stages. a two-fold expression cut-off was applied. for hierarchical gene cluster analysis, expression ratios were calculated for all genes as described by eisen et al. ( ) . first strand cdna was synthesized using reverse transcriptase (rt) and μg of total rna. reactions were conducted in μl of buffer containing; . μl oligo (dt) - primer (gibco/brl, grand island, ny, usa), mm tris-hcl (ph . ), mm kcl, mm mgcl , mm dtt, . mm deoxynucleotide triphosphate (dntp) mixture (invitrogen, carlsbad, ca, usa), unit rnase inhibitor (gibco/brl), and units of mmlv reverse transcriptase (invitrogen). after incubation at °c for min, reactions were stopped by heating at °c for min. to remove remaining rna, μl of e. coli rnase h ( mg/ml) was added to reaction mixtures and incubated at °c for min. cdnas obtained were used as a template for pcr amplification using gene-specific primers for target genes and for glyceraldehyde -phosphate dehydrogenase (gapdh). primer sequences are listed in table . the in vitro growth pattern of msc is shown in fig. . human bone marrow-derived mscs were cultured and expanded. dur- table . ing the log phase of growth, cells proliferated with a population doubling time of hr, and this growth period was followed by a confluent growth-arrested phase. colonies were examined approximately days after initial plating. a morphologically homogeneous population of % confluent fibroblast-like cells was obtained after weeks. the cells were replated into culture dishes and cultured for weeks. the replated cells were used for subsequent experiments. the cultured mscs were positive for sh by flow cytometry (fig. ) . mscs were pelleted into micromasses and differentiated in serum-free medium in the presence of tgf-β and dexamethasone. immediately after centrifugation, the cells appeared as flattened pellets at the bottom of tubes. one day later, pellets had a thickened lip, and between days and , pellet became spherical without any increase in size. pellets then grew in size and pellet diameters increased to about -fold on days and (fig. a) . using normalized microarray data, we identified , differen-tially expressed genes (fig. a) , which included , , , , , and genes exhibiting minimum to < , to < , to < , to < and > -fold changes, respectively. to verify gene expression profiles determined by microarray analysis, the expression levels of genes with high fold changes ( - fold changes, table ) were confirmed by rt-pcr. the expression levels of the genes selected (hrad b, annexin a , bmp- , contactin- , peroxiredoxin- , heat shock transcription factor- , synaptotagmin iv, serotonin receptor- , axl, and il- ) were analyzed by rt-pcr, by using total rnas obtained from samples (fig. b) . the expression levels of genes (hrad b, annexin a , bmp- , contactin- , peroxiredoxin- , heat shock transcription factor- , synaptotagmin iv, serotonin receptor- , axl) were low in undifferentiated cells and increased in differentiated cells by rt-pcr and microarray, but the expression pattern of il- was different. expression level of il- tended to be decreased in microarray, but increased in rt-pcr (fig. ). in this study, we determined gene expression profiles in differentiated chondrocytes and bm-mscs. the microarray technology used did not allow quantitative comparisons between the expressional levels of different genes, but did allow us to compare fold changes with time and quantify differences in the expressions of multiple genes. our results show the sequences for gene expressional changes during bm-msc chondrogenesis. microarray data showed that axl, synaptotagmin iv, hrad b, peroxiredoxin- , bmp- , heat shock transcription factor- , annexin a , contactin- and serotonin receptor- expressions were maintained in differentiating bm-mscs until day . axl is overexpressed in a number of tumors ( ) , and il- is known to mediate the transactivation and upregulation of axl with subsequent activation of pi k/akt and upregulations of bcl- and bcl-xl ( ) . on the other hand, synaptotagmin iv is required for the maturation of secretory granules in pc cells ( ) . human homologues of yeast rad (hrad b) encode ubiq-uitin-conjugating enzymes, and is highly expressed in lung cancer cell. it has been reported that dna repair and uv mutagenesis are defective in saccharomyces cerevisiae rad mutant ( ) . peroxiredoxin- is the most ubiquitously expressed member of the peroxiredoxin family, and is found in the cytoplasm, nucleus, mitochondria, and peroxisomes of many cell types ( ) . furthermore, recent studies have reported high levels of peroxiredoxin- expression in the bovine bladder, seminal vesicles, testes, adrenal gland ( ) , and in the rat liver, skin, lungs and nervous system ( ) . the role of peroxiredoxin- in cell differentiation and proliferation suggests that it has a possible role in growth and development. recent studies have confirmed that bmp- is a strong chemotactic component in cartilage cells produced by mesenchymal stem cells, and it can promote cartilage cells to secrete specific extracellular matrix (proteoglycans and collagen type ii). and bmp- can induce the differentiation of bm-mscs into cartilage cells, and that it offers a greater efficiency in repairing cartilage and subchondral bone defects ( ) . heat shock transcription factor- (hsf- ) has been shown to be a transcriptional regulator of heat shock protein gene expression during the differentiation and development of eukaryotic cells in a tissue dependent manner ( ) . hsf- plays an important role in fgf- stimulated osteoclast formation, and hsf- deficiency was found to modulate gene expression in stromal/ preosteoblast cells and affect osteoclastogenesis in the bone microenvironment ( ) . annexins bind to negatively charged phos- pholipids in a ca + -dependent manner and have a conserved structure. the human annexin, annexin a (alternative names: annexin ii, p , and lipocortin ii) is expressed abundantly in various human organs, including the placenta, lungs, heart, and liver ( ) . at the cellular level, annexin a is expressed on endothelial cell surfaces and acts as a co-receptor for plasminogen and tissue plasminogen activator ( ) . furthermore, annexins are commonly dysregulated in cancer ( ) and annexin a is upregulated in a variety of tumors and cancer cell lines ( , ) . contactin- is a cell surface adhesion molecule, which is normally expressed by neurons, oligodendrocytes, and human astrocytic gliomas ( , ) . previous studies have reported that mscs express il- , essential hematopoietic growth factor ( , ) and il- is also a potent apoptosis inhibitor and has many immunomodulatory activities ( ) . the serotonin receptor is the most recently identified member of the serotonin receptor family and is found in brain, mainly in the hypothalamus, thalamus, hippocampus, and cortex ( ) . in the present study, we performed microarray analysis during bm-msc chondrogenesis in vitro. we found that over , genes were expressed by bm-mscs during chondrogenesis, and we identified genes that were differentially expressed. these data may provide novel information of the genes involved in chondrogenesis of human bm-mscs. multilineage potential of adult human mesenchymal stem cells transgene expression and differentiation of baculovirus-transduced human mesenchymal stem cells cell surface antigens on human marrow-derived mesenchymal cells are detected by monoclonal antibodies gene expression profiling following bmp- induction of mesenchymal chondrogenesis in vitro. osteoarthritis suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cdna probes and libraries cross platform microarray analysis for robust identification of differentially expressed genes open software development for computational biology and bioinformatics microarray data normalization and transformation cluster analysis and display of genome-wide expression patterns gas induces growth, beta-catenin stabilization, and t-cell factor transcriptional activation in contact-inhibited c mammary cells a promiscuous liaison between il- receptor and axl receptor tyrosine kinase in cell death control synaptotagmin iv is necessary for the maturation of secretory granules in pc cells decreased hrad b expression in lung cancer differential cellular and subcellular localization of heme-binding protein /peroxiredoxin i and heme oxygenase- in rat liver cloning of bovine peroxiredoxins-gene expression in bovine tissues and amino acid sequence comparison with rat, mouse and primate peroxiredoxins bmp induces the differentiation of bone marrow-derived mesenchymal cells into chondrocytes heat shock factor is activated during mouse heart development rank ligand expression in heat shock factor- deficient mouse bone marrow stromal/preosteoblast cells differential expression of annexins i, ii and iv in human tissues: an immunohistochemical study specific interaction of tissue-type plasminogen activator (t-pa) with annexin ii on the membrane of pancreatic cancer cells activates plasminogen and promotes invasion in vitro annexin a on lung epithelial cell surface is recognized by severe acute respiratory syndrome-associated coronavirus spike domain antibodies tenascin c and annexin ii expression in the process of pancreatic carcinogenesis redox regulation of annexin and its implication for oxidative stress-induced renal carcinogenesis and metastasis faivre-sarrailh c. f /contactin, a neuronal cell adhesion molecule implicated in axogenesis and myelination contactin is expressed in human astrocytic gliomas and mediates repulsive effects phenotypic and functional comparison of cultures of marrow-derived mesenchymal stem cells (mscs) and stromal cells gene expression profile of cytokine and growth factor during differentiation of bone marrow-derived mesenchymal stem cell death deflected: il- inhibits tnfalpha-mediated apoptosis in fibroblasts by traf recruitment to the il- r alpha chain functional, molecular and pharmacological advances in -ht receptor research mesenchymal stem cells (mscs) have the capacity to proliferate and differentiate into multiple connective tissue lineages such as cartilage and bone. in this study, using an abi microarray system, the authors determined the differential gene expression profiles of differentiated chondrocytes and bone marrow (bm)-mscs. normalization of this data resulted in the identification of , differentially expressed genes. to verify gene expression profiles of microarray, rt-pcr was also performed. gene expression patterns of genes in rt-pcr were similar to the microarray results. these findings provide novel information concerning genes involved in the chondrogenesis of human bm-mscs. key: cord- - plv q b authors: zhou, xiaoqin; li, zifu; zheng, tianlong; yan, yichang; li, pengyu; odey, emmanuel alepu; mang, heinz peter; uddin, sayed mohammad nazim title: review of global sanitation development date: - - journal: environ int doi: . /j.envint. . . sha: doc_id: cord_uid: plv q b the implementation of the united nations (un) millennium development goals (mdgs) and sustainable development goals (sdgs) has resulted in an increased focus on developing innovative, sustainable sanitation techniques to address the demand for adequate and equitable sanitation in low-income areas. we examined the background, current situation, challenges, and perspectives of global sanitation. we used bibliometric analysis and word cluster analysis to evaluate sanitation research from to based on the science citation index expanded (sci-expanded) and social sciences citation index (ssci) databases. our results show that sanitation is a comprehensive field connected with multiple categories, and the increasing number of publications reflects a strong interest in this research area. most of the research took place in developed countries, especially the usa, although sanitation problems are more serious in developing countries. innovations in sanitation techniques may keep susceptible populations from contracting diseases caused by various kinds of contaminants and microorganisms. hence, the hygienization of human excreta, resource recovery, and removal of micro-pollutants from excreta can serve as effective sustainable solutions. commercialized technologies, like composting, anaerobic digestion, and storage, are reliable but still face challenges in addressing the links between the political, social, institutional, cultural, and educational aspects of sanitation. innovative technologies, such as microbial fuel cells (mfcs), microbial electrolysis cells (mecs), and struvite precipitation, are at the trl (technology readiness levels) level, meaning that they qualify as “actual systems completed and qualified through test and demonstration.” solutions that take into consideration economic feasibility and all the different aspects of sanitation are required. there is an urgent demand for holistic solutions considering government support, social acceptability, as well as technological reliability that can be effectively adapted to local conditions. the united nations (un), many local governments, and international organizations have launched programs to deal with the negative impact on human health and the environment caused by the lack of access to adequate sanitation. in the s, un member states and at least international organizations agreed to the millennium development goals (mdgs) at the world summits. mdg (target ) was intended to halve the proportion of people without sustainable access to safe drinking water and basic sanitation by . in september , the agenda for sustainable development goals (sdgs) was adopted by world leaders at the un, which calls on . sanitation activities in scientific research . . publication patterns we identified , publications related to sanitation research in the sci-expanded and ssci databases between and , and we found a continual growth trend, as shown in fig. . there were , ( . %) papers indexed in the sci-expanded and ( . %) papers indexed in the ssci on sanitation; ( . %) were indexed in both the sci-expanded and ssci. fig. shows that after , the number of publications rose significantly. more than % of articles in web of science have, since , included abstracts, compared with only % articles in (ho et al., ) , , papers from to were selected as study samples. we grouped these works into types of documents. articles comprised . % ( , ) of the total number of works examined, making them the most common document type. the remaining publications consisted of proceedings ( ), reviews ( ), editorial material ( ), meeting abstracts ( ), book reviews ( ), news items ( ), letters ( ), book chapters ( ), corrections ( ), notes ( ), reprints ( ), discussions ( ), items about an individual ( ), biographical items ( ), retracted publications ( ), and retractions ( ). we did not use any other document types in this study because articles were the most common type of work. of the , articles, , ( %) were published in english, followed by portuguese ( ), spanish ( ), french ( ), german ( ), japanese ( ), italian ( ), polish ( ), russian ( ) , chinese ( ), croatian ( ), czech ( ), turkish ( ), hungarian ( ), korean ( ), slovene ( ), dutch ( ), romanian ( ), serbian ( ), slovak ( ), malay ( ), lithuanian ( ) and serbo-croatian ( ). we found that the number of articles published per year increased over the study period. there were articles published in and in . we created an exponential model of the cumulative annual number of articles (fig. in this model, c is the cumulative number of articles. y is the number of years that have passed since . we used this model to identify the total number of articles for a given time period. from to , our model showed that , texts were published. the quantity published in was predicted to be . in , works were published, which is about half the number predicted for . the mean number of authors per article grew from . in to . in . this was an increase of . % (table ) . however, this increase was dwarfed by the . % rise in the number of institutions per article. there was a similar increase in the mean number of references per article, . %. the average article length and average number of countries per article fluctuated slightly, with an overall mean length of . pages and average of . countries. the number of times each article was cited fluctuated, with a peak average of . citations in and an average low of less than five citations in recent years. articles most frequently had three authors; articles ( . %) had three authors, then two and four authors accounted for ( . %) and ( . %) of the total, respectively. about % of the sample consisted of five ( , . %), one ( , . %), and six ( , . %) authors together. the lancet published the article with the largest number of authors ( ) . these names all appeared on the article "global, regional, and national comparative risk assessment of behavioral, environmental and occupational, and metabolic risks or clusters of risks in countries, countries, - countries, " (forouzanfar et al., . the lancet's publication entitled "a comparative risk assessment of the burden of disease and injury attributable to risk factors and risk factor clusters in regions, - : a systematic analysis for the global burden of disease study " (lim et al., ) had contributors from countries and institutes, which reviewed the leading risk factors for global disease burden. the researchers presented evidence of key risks, including poor water quality and sanitation, vitamin a and zinc deficiencies, and ambient particulate matter pollution. the second most cited article, "global mortality, disability, and the contribution of risk factors: global burden of disease study" (murray and lopez, ) with citations, was published in lancet by who, switzerland, and harvard university in . the article "global, regional, and national comparative risk assessment of behavioral, environmental and occupational, and metabolic risks or clusters of risks in countries, - : a systematic analysis for the global burden of disease study " (forouzanfar et al., ) was also published in lancet by authors from institutes in countries; it was cited times within one year. it ranked second for the most frequently cited research article per year. this research emphasized the risks from unsafe sanitation and water; it disclosed that behavioral, environmental, occupational, and metabolic risks can explain half of the global mortality rate and more than one-third of the global-disability-adjusted life-years (dalys), indicating that there were many opportunities for prevention. the three most cited publications showed that sanitation is closely linked to health conditions for humans; poor sanitation and bad hygiene result in the spread of disease. thus, improving sanitation is key to creating a better quality of life for millions of people. the , articles we utilized for our analyses came from a wide range of journals. they appeared in publications that dealt with sanitation issues. these articles addressed a broad range of topics, including subject categories in the sci-expanded and ssci databases. we found that , or almost half ( . %), of the journals investigated had only one article that appeared in our results. other journals had more articles (in some cases, many more). of the remaining journals, ( . %) had two articles and ( . %) journals had three articles. about % of the journals ( publications) had articles, while ( . %) journals published or more. table shows our data for the top most productive journals (tp > ). this table includes the isi category as well as the position of the journal within its category. table also includes a summary of the jif, h-index, and journal's country of origin. the top most productive journals were extremely influential. the number of their published articles comprised about . % of the works we investigated. the journal of water sanitation and hygiene for development, with an h-index of , published the most articles ( ; . %), followed by the journal of food protection ( ; . %), water science and technology ( ; . %), plos one ( ; . %), and american journal of tropical medicine and hygiene ( ; . %). there were some unusual results in our analysis. for example, water research had the highest jif value ( . ) and h-index ( ); this journal was the top publication for water resources (q : / ). however, it came in eighth place in our results for its output. the journal of water sanitation and hygiene for development is a journal of the international water association (iwa), which emphasizes issues of concern in developing and middle-income countries, as well as in disadvantaged communities worldwide. the journal articles address themes that x. zhou et al. environment international ( ) - explore the water supply, sanitation, and hygiene, which potentially explains why this is the most productive journal. there were various trends exposed by the relationship between the h-index, jif, and the rank order of the most productive journals. we found that water science and technology ranked third among the most productive journals. however, this journal's jif ( . ) came in th place among the top publications. in addition, its h-index ( ) only ranked th. we found that the th-ranked journal (environmental science & technology) had a jif of . , which placed it second among the other journals. its h-index was , which then moved it to third place. this interesting pattern may be partly due to the fact that the highest impact journals are not as specialized as those with less impact. in addition, with their stronger impact, they may target higher quality studies or more often consider research that offers an original approach to a problem or idea. there was a clear supremacy of publications that came from developed nations: the uk published nine influential journals, and seven publications were from the usa. brazil had a mere two journals, which was mirrored by the netherlands ( ). there was a strong dominance of five journals. these publications were responsible for ( . %) of the articles studied. fig. shows our results for the trends at these top five publications. since journals could be assigned to multiple subject categories in this research, sanitation research actually encompassed subject categories. fig. shows that, based on the continuous increase in the number of articles per category, sanitation research mirrored the types of patterns found in other fields at the start of the new millennium. the most common subject categories were "environmental sciences & ecology", "public, environmental, & occupational health", "engineering", and "water resources", followed distantly by the second echelon of categories, which occupied a percentage between and %. "agriculture" and "food science & technology" dominated the third level. the six most common subject categories were about the environment, water, health, and food, which may be due to the fact that sanitation is always associated with water and hygiene. in addition, an increased awareness of the nutrients contained in human excreta and the recovery of those nutrients for farming are gaining increasing attention. such research is related to agriculture and food security (lienert et al., ) . by using at least one author's affiliation, we were able to identify which countries and areas were the most influential in the publication of sanitation research. we analyzed , articles with the author address information published between and , which included , institutes in countries/regions. there were ( . %) independent articles and ( . %) internationally-collaborative articles. our analysis addressed the most prolific countries. we examined the total number of works from a single country as well as internationally-collaborative research. table showed our data. this table also included the percentage of single country and internationally collaborative articles and the h-index for each area. the top countries included three asian countries/regions, ten european countries, five american countries, one oceanic country, and one south african country. the most productive countries generated the vast majority of published articles ( . %). almost four-fifths ( . %) of the sanitation articles examined in this analysis were from canada, france, germany, italy, japan, the uk, and the usa. these developed countries have superior economic capabilities and higher academic levels than those of developing nations (bell and pavitt, ) . of the regions we examined, brazil came in second, india was seventh, china was ranked tenth, and south africa was a distant th (finardi, ) . russia was not included in the top . fig. shows the global geographic distribution of authors according to the total number of articles by country/region. the usa appeared at or near the top in our results, indicating that it is a global leader in sanitation studies and publications. the usa had the highest number of total articles published ( ). it was also first for its independent articles ( ) and first authored articles ( ). the usa had an h-index of and published corresponding author articles. sixteen nations partnered with researchers from the usa, and these collaborator data are shown in table . switzerland, netherlands, and australia are the top collaborative countries. the total note: ta: quantities of articles; jif: journal impact factor; r; ranking in top productive journals; *: the category here belonged to ssci not sci-expanded. x. zhou et al. environment international ( ) - number of articles from china ranked th, and the percentages for independent and collaborated articles were . % and . %, respectively. we examined countries that had at least publications in order to highlight the trends in international cooperation. first, we placed the countries into clusters. we then used bibexcel and an algorithm from persson ( ) and graphed our results with pajek. fig. shows the results of our analysis. each country (shown as a circle) is sized according to the weight of its publications. the line weight of each circle identifies the nation's research cooperation with authors from other regions. the most productive countries are the usa and uk. they both have outsized and complex groups; belong to the usa cluster and groups belong to the uk cluster. switzerland, france, and belgium come next, but they have significantly lower numbers of articles and cooperation. the usa-uk collaborations ranked first, with cooperative articles, followed by usa-switzerland ( ), usa-canada ( ), usa-india ( ), usa-brazil ( ), and china-usa ( ) collaborations. these articles covered several aspects, including social, political, environmental, public health, technical aspects and so on, which were mainly related to the context of developing countries. the descriptions of these articles expressed three phases for sanitation development, the first phase is the baseline study, to investigate the main sanitation related issues, e.g. technologies for disinfection and hygiene, which could be effective to prevent the transmission of disease; the second phase is table the performance of the top most productive countries the realization of mdg, global estimation of the indicators according to mdgs; the third phase is the development of new technology for sanitation with the concept of closing the loop in order to reach the sdgs. for the techniques developed by the usa, during the mdgs period, the target was to get improved sanitation, intended for safe separation of humans from excreta by employing flushing toilets, septic tanks, latrines, composting toilets etc., but not a goal of hygienization or source recovery. thus, the main technologies were composting and storage processes. recently, because of the implementation of sdgs and encouragement by the bill and melinda gates foundation, some nonsewer and source oriented toilet systems have been developed, as mentioned in the reinvent the toilet challenge manuscript. from the year to , grants have been awarded for innovated toilet developers, and of them are from usa, further supporting the statements. table below lists brief descriptions of the reinvented toilet technologies developed by usa. brazil was ranked first for number of articles. this trend did not continue, however, as in , the usa moved back into the top slot. of the , articles spanning , institutes in countries, ( . %) were inter-institutionally collaborative publications and ( . %) were independent publications. the collaboration between institutes was . % higher than that between countries. the top research facilities included in the usa, in brazil, in switzerland, in the uk, and each in france and spain; of the institutes in the usa were universities. these data are shown in table . the 'center for disease control and prevention, u.s. department of health & human services', 'who' and the 'university of north carolina' are the top three institutions in h-index with high values of , , and , respectively, which further emphasizes the importance of sanitation for hygiene and disease control. over half of the institutes were universities, demonstrating that the universities are the core drivers of sanitation research. the statistical analysis of the author keywords, article title, article abstract, and "keywords plus" could be used to identify directions in science; keyword analysis is efficient for comprehending the progress of new frontiers in science (li et al., ; tan et al., ) . the analysis of author keywords across different periods is a common bibliometric technique for analyzing research trends (fu et al., ; wang et al., ; zhang et al., ; zhang et al., ; zheng et al., ) . we excluded the articles without author keyword information and analyzed , articles with the author keyword information. there were , keywords listed by authors; however, , ( . %) of the keywords were used only once and ( . %) of the keywords were used twice. there were many author keywords that were only used one time. this single use points to the utilization of comprehensive subject keywords. it also indicates that there were differences in how researchers approached their work (hou et al., ; li and zhao, ) . the bulk of the sanitation research relied on a small number of keywords. for example, ( . %) keywords appeared > times. table shows the most commonly used keywords. the changes in the rankings of words over the five-year intervals show that there were variations in the research hotspots. fig. shows the co-occurrence relationships of the most commonly-used author keywords. here, we created a co-word network to better illustrate the relationships between these terms. because the terms "sanitation" and "sanitary" were the subject words in this study, the most frequently-used keyword ("sanitation") was ignored. table and fig. show the outcomes of our keyword analysis. the three most frequently-used keywords were "epidemiology" ( ; . %), "water" ( ; . %), and "public health" ( ; . %). these top three most frequently-used substantives reflect the large amount of literature dealing with water and hygiene, especially the diseases transferred with poor sanitation, such as diarrhea, lower respiratory illnesses, and other common infectious diseases (lim et al., ) . fig. shows that "water" is closely related to "sanitation". this keyword was increasingly studied and enjoyed a rise in rank from th in - to nd in - . this increase showed a growing awareness of the importance of proper and efficient water management for sanitation improvement, especially a safe water supply and sourceoriented wastewater treatment. "water supply" ( , . %), "water quality" ( , . %), "wastewater" ( , . %), and "drinking water" ( , . %) ranked th, th, th, and th, respectively, as the most frequently-used keywords. besides "hygiene", "diarrhea", and "risk factor", "brazil" was one of the top keywords during all five-year periods, which illustrates that water, sanitation, and hygiene are ongoing concerns. the pathogenic indicator bacteria "e. coli" and "salmonella" ranked th and st, respectively, in the most frequently used keywords, which indicate that these bacterial species have been extensively used as indicator pathogens for hygienization. e. coli had an table brief information of the reinvented toilet technologies developed by usa. source: https://www.gatesfoundation.org/what-we-do/global-growth-andopportunity/water-sanitation-and-hygiene/reinvent-the-toilet-challenge. to develop a self-contained, solar-powered toilet and wastewater treatment system. a solar panel will produce enough power for an electrochemical reactor that is designed to break down water and human waste. excess power can be stored to provide energy for nighttime operation or for use under low-sunlight conditions. to develop a self-contained system that pyrolyzes (decomposes at high temperatures without oxygen) human waste into biochar. energy recovered from the biochar production process will be used for heating the system. stanford university and the climate foundation, usa to develop a self-contained toilet system that disinfects liquid waste and turns solid waste into fuel or electricity through a novel biomass energy conversion unit. to develop a solar toilet that uses concentrated sunlight, directed and focused with a solar dish and concentrator, to disinfect liquid-solid waste and produce biochar that can be used as a replacement for wood charcoal or chemical fertilizers. to develop, build, and evaluate a novel technique to treat fecal sludge using supercritical water oxidation, a process in which water is heated under pressure and then oxygen is added to burn up human waste. the reaction produces clean water, heat, carbon dioxide, benign salts, and nitrogen, all of which can be used by the community or turned into business opportunities. duke university, usa to develop an electric toilet, powered by solar power stored in batteries, that will separate liquids from solids and dewater and convert fecal matter into biochar. this approach examines using resistive heating through battery-stored solar power and is designed from existing off-the-shelf components. santec llc, usa apparent upward movement in the ranks, from th in - to th in - , which could be attributed to the fact that e. coli is commonly observed in the gut of humans and warm-blooded animals. this trend also reflects the awareness of the public's health. the names of developing countries ( , . %) ranked th in the top most frequently-used substantives in author keywords; they were initially ranked th in - , then moved to nd in - , th in - , and finally to th place in - . however, they decreased to th in - . this trend could be explained by the continuing attention given to a specific area and country, especially low-income districts. hence, "india" ( , . %) and "africa" ( , . %) ranked th and th, respectively, in the top most ( ) ( ) frequently-used keywords, and both of these keywords had a significant increase over the past years. "india" and "africa" ranked th and th, respectively, in - and th and th, respectively, in - . "child" ( , . %) ranked th in the top keywords, but it was ranked th from to and then it moved to th place in - . this trend occurred because children are the most sensitive to poor sanitation. "sustainability" has become increasingly popular in recent years, moving up in the rankings from "not available" in - to th in - , th in - , th in - , and th in - . from to , improved sanitation was a primary focus of research. it was important to ensure the hygienic separation of human excreta from human contact and the suggested facilities that could reach the targets included flush/pourflush-to-piped-sewer systems or septic tanks/pit latrines, ventilated improved pit (vip) latrines, pit latrines with slabs, and composting toilets. this explains why sustainability was barely mentioned during the first years in our study. since the awareness of protecting human health and the environment has increased, the concept of sustainable sanitation has been promoted, and sustainability has garnered increasing attention. baskerville ( ) showed that patterns and ideas in research can be identified via author keywords and the title of articles. we therefore used an algorithm to generate "keywords plus". these terms were drawn from the titles of works that appeared in the articles (garfield, ) .we also utilized a relatively new method in this analysis, mao et al.'s "word cluster analysis" (mao et al., ) . the word cluster analysis uses the author keywords, article title, article abstracts, and keywords plus to identify patterns in data. many other researchers have used this approach to determine research hotspots (fu et al., ; qian et al., ; tan et al., ) . the possible research hotspots for sanitation research were divided into categories, including "susceptible population", "epidemic disease", "specific countries", "different regions", "concerned contaminates", and "characterized microorganisms"; all the categories were closely related to hygienization. fig. shows the growth trend of hotspot-related articles during the last years in the six categories. unicef noted that clean water, basic toilets, and good hygiene practices are essential for the survival and development of children. fig. a and b show that children suffer primarily from diarrhea. the who website noted that every year, , children under five years of age die from diarrhea. the transmission of diseases is linked to poor sanitation and water contamination. one report titled "a fair chance for every child" released by unicef stressed the importance of sanitation to children's health and mortality; it noted that child mortality could be reduced by improved sanitation facilities (unicef, ). hence, improving sanitation services and basic hygiene practices for children is a critical hotspot. due to the limitations of their economies, developing countries face greater challenges in improving sanitation conditions; evidence for these challenges can be found in fig. c , which shows the hotspots of research work in specific countries. brazil, africa, and india are the top three countries/regions according to the number of articles collected from the database ( , , and , respectively). the rest of the top countries/regions are bangladesh, spain, china, mexico, south africa, kenya, and sub-saharan africa. most of these nations are defined as developing countries. the rapid increase in research in brazil may be due to the rapid urbanization of latin america in the last few decades. even though there were . million people having access to "at least basic" sanitation, and % of the population used "unimproved" sanitation (pubic or shared latrine, open pit latrine, bucket latrine), % of the population still practiced open defecation as of (watch, ) . most african countries, including south africa, sub-saharan africa, and kenya, lacked clean water, basic sanitation, and good personal hygiene, which are the results of extreme poverty. in burkina faso, only % of the rural population had access to improved household sanitation (hygienically separates human excreta from human contact); % still defecated in the open in (unicef, ) . asian countries, such as india, bangladesh, and china, show increasing improvement. india (with almost million children, % of the population) has the largest number of the world's growth-stunted children in asia, where the key issue is a lack of toilets rather than lack of food (worley, ) . thus, improving the sanitation conditions in india has garnered attention from the government and academia. in , the department of biotechnology (dbt), under auspices of the ministry of science and technology of the government of india, and the bmgf, in collaboration with india's biotechnology industry research assistance council (birac), launched "rttc-india". six grantees were chosen from applicants to develop innovative, affordable, and scalable sanitation technologies, with a total grant of $ million (mehta, ) . in china, % of the urban population has access to improved toilets (as of ); the coverage of sanitary toilets in rural areas was . % in , . % in , and . % in , indicating that great headway was made in building sanitary toilets over the last years (china, ; prc, ) . similarly, the bmgf has been funding rttc activity in china since , which encouraged x. zhou et al. environment international ( ) - researchers from universities, research institutes, enterprises, and other social groups to develop a new generation of toilets and increased the number of academic publications on the subject (liu et al., ; wang et al., ) . with the implementation of the nationwide "toilet revaluation", greater achievements are expected during china's " th five-year plan". the majority of those without improved sanitation are people who live in rural areas, thus, rural areas are characterized by the great disparity in sanitation improvements. however, fig. d shows that "rural", "city", and "urban" appear frequently in research; the number of articles with "urban" even exceeded the number of articles that included "rural" in the keywords. the percentage of the urban population living in south sudan without safe toilets accounted for . % of the total, which dominates the list, while india has million urbanites living without sanitation, ranking first for the most people without access to proper toilet facilities (wateraid, ) . the concept of sustainable sanitation not only concerns toilets, but also the proper disposal and reuse of excreta. therefore, urban areas that are far away from the main sewers urgently require innovative sanitation techniques. an announcement made on the fifth world toilet day (the iso ) titled it "non-sewered sanitation systems -prefabricated integrated treatment units -general safety and performance requirements for design and testing") indicated that who would provide international standardized regulation for sanitation systems worldwide, including urban communities without access to sewer systems, and the x. zhou et al. environment international ( ) - organization would pursue sustainable sanitation solutions (lazarte, ; lewis, ) . with the transformation of the concept of sanitation from "improved sanitation" to "sustainable sanitation", excreta and wastewater are not recognized as hazardous waste, but valuable resources that can be reused and recycled. hence, a better understanding of the composition of waste is needed. fig. e reveals that "pathogens", "sludge", "nitrogen", "microorganisms", "antibiotics", "ammonia", "phosphorus", "nitrate", "heavy metals", and "antimicrobial resistance" were the top ten research hotspots in scientific research, and they are the main components of human excreta. these keywords were increasingly being used in research articles. based on the characteristics of these pollutants, the research hotspots can be classified into three categories: (i) hygienization of excreta, (ii) resource (such as nitrogen, phosphorus, and electricity) recovery from excreta, and (iii) micro-pollutant control of excreta, such as antibiotics and heavy metals. antimicrobial resistance is a new concern from a hygienization point of view due to the increasing amount of antibiotics in water systems. (i) hygienization of excreta: the hygienization of human excreta is essential for reducing the transmission of diseases by pathogenic microorganisms. human urine contains very few, if any, pathogens, but the amount increases due to cross-contamination with feces. nevertheless, the health risk can be eliminated through natural attenuation (hoglund et al., ) or existing disinfection technologies. the behaviors of bacteria, protozoan pathogens, and viruses in stored-source-separated urine were studied, and it was demonstrated that the inactivation of pathogenic microorganisms depended on either the temperature and ph or only the temperature (hoglund et al., a , b , hoglund et al., . for safe agricultural applications, who recommends a storage period of months at °c or higher. stored urine could then be used to fertilize crops without restriction (world health organization, ) . in order to increase the inactivation efficiency and reduce storage time, researchers increased the storage temperature. the inactivation of e. coli, salmonella, and ms at these higher temperatures was faster than that which occurred in the urine stored at ambient temperature (nordin et al., ; vinneras et al., ; zhou et al., ) . the rapid inactivation of bacteria could be achieved by coupling other technologies with this process, such as acidification (andreev et al., ) and nitrification (bischel et al., ) . similarly, self-sanitization by ammonia and a high temperature is also effective for fecal sludge, but the time needed to eliminate pathogens is longer than that needed for urine due to the large amount and species of pathogens, viruses, and helminths in it magri et al., ; magri et al., ) . alkaline additives, such as ash, sawdust, and urea, could speed up the hygienization process (fidjeland et al., , niwagaba et al., a , b . thermal treatments have been applied to fecal sludge treatment, and other treatment processes, like composting and anaerobic digestion, could have an effect on pathogen inactivation at some level (vinneras, ; yin et al., ) . fig. f shows that e. coli is the primary indicator microorganism in scientific research; it is also recommended as an indicator of health risk from water contact by the us environmental protection agency (us epa). it is followed by salmonella and listeria monocytogenes. (ii) resource recovery from excreta: urine accounts for only ∼ % of the total volume of domestic wastewater, but it contains > % of the nitrogen (n) and % of the phosphorus (p) load in domestic wastewater (larsen and gujer, ) . thus, the recovery of nutrients is much more strongly promoted than their removal, especially in light of the phosphorus crisis (gilbert, ). the average amounts of phosphate, nitrogen, and potassium in human urine are approximately . kg, . kg, and . kg per person per year, accounting for %, %, and % of the global consumption of fertilizer. the main form of nitrogen in urine is ammonia due to the hydrolysis of urea during storage, while the phosphorus in urine exists as inorganic phosphate ions. feces contain some nutrients and organic material, which can act as good soil conditioners after aerobic stabilization. generally, the most direct way to use urine and feces as fertilizer for agriculture occurs after proper processing, especially in low-income areas or regions dominated by agriculture. for urine, the suggested approach for its safe reuse is long-term storage, and the storage time is dependent on the temperature. as noted earlier, who recommends months of storage before urine can be safely used for crops. the most direct approach for using feces as a fertilizer or soil conditioner is to utilize it after composting. composting is also effective for sanitizing fecal sludge. composting is a self-heating microbial process, and it is effective for pathogenic bacteria inactivation at temperatures over °c for a specific time period (niwagaba et al., a (niwagaba et al., , b . much research has been conducted to investigate the composting process, including co-composting with other carbon-rich materials, like kitchen waste, wood chips, and saw dust (mahmood et al., ; mulec et al., ; sossou et al., ) . fecal sludge recycling after anaerobic digestion is another alternative that provides fertilizer as well as energy recovery (biogas). the reuse of urine and feces after stabilization and hygienization by storage or composting/digestion has been in practice for many years since it is simple and easy to manage. however, the loss of nutrients and secondary pollution that occur during the transportation of liquids is a concern. therefore, newly-developed engineering technologies have become viable alternatives for the efficient commercial recovery of nutrients. ammonia stripping and struvite precipitation are two of the most commonly employed techniques in nutrient recovery from source-separated urine. ammonia stripping is a physicochemical process that strips ammonium to gaseous nh , which is then recovered as liquid ammonia, ammonium sulfate, or ammonium carbonate (maurer et al., ; nancharaiah et al., ) . > % of the nitrogen could be recovered by stripping under optimized operating conditions (antonini et al., , basakcilardan-kabakci et al., . kinetic analysis showed that higher air flow rates and temperatures could improve the recovery efficiency so as to decrease the unit operating cost. an extremely high ph is not recommended for the operation, even though it could increase the efficiency, because it may result in increased costs (liu et al., ) . stripping only recovers nitrogen, while struvite recovers both nitrogen and phosphorus. struvite is magnesium ammonium phosphate (mgnh po · h o, map), which is the chemical precipitation that occurs at an alkaline ph with a suitable ratio of ammonium, phosphorus, and magnesium. this process converts nutrients from liquid to solid form, and its products can be used as a slowrelease fertilizer (maurer et al., ) . the addition of magnesium is necessary for struvite formation to meet the chemical equilibrium of the constituent ions in the solution. major parameters, such as the magnesium dosage, mixing rate, and ph, have been extensively investigated at lab scale (ronteltap et al., ; tilley et al., ; wilsenach et al., ) . it was reported that % of the phosphorous and approximately % of the nitrogen could be recovered through map crystallization. a combination of ammonia stripping and struvite precipitation to improve nitrogen and phosphorus recovery has been investigated. one investigation employed ca (oh) to replace naoh to make the stripping and precipitation occur at the same time; this study showed that - % of the nitrogen and % of the phosphorus (w/w) can be harvested from hydrolyzed urine in h at °c and in h at °c (pradhan et al., ) . although % of the phosphorus and approximately % of the nitrogen could be recovered through map crystallization, nearly all of the potassium would be lost, so magnesium potassium phosphate hexahydrate (mgkpo · h o, mpp) crystallization was developed to simultaneously recover phosphorus as well as potassium (xu et al., ; xu et al., ; zhang et al., ) . the ferric ion (fe + ) has also been used for phosphorus recovery (jadhav and hocheng, ) . it was estimated that the available phosphorus from urine and feces produced in urban settings is approximately . million metric tons and will increase with population growth to over . million metric tons by (mihelcic et al., ) . thus, recovering phosphorus from urine and feces will continue to be an attractive option. other technologies that transform the wastewater treatment system, such as ion exchange, membranes, and bio-electrochemical systems (bes"s), have also been investigated. bess were a critical research hotspot recently because they provide for on-site treatment, broad contamination removal, and energy production; they are also easy to manage and operate (zollig et al., ) , since they use microorganisms to catalyze oxidation and/or reduction reactions at the electrodes (e. g., the anode or cathode) collectively (nancharaiah et al., ) . microbial fuel cells (mfcs) and microbial electrolysis cells (mecs) are two key microbial electrochemical technologies (ledezma et al., ) . it was first demonstrated in that mfcs generated electricity from diluted urine, and this electricity could be used to power cell phones (ieropoulos et al., ) . subsequently, research on enhanced mfc systems aims to improve the cathode structure, provide for more efficient nutrients/energy recovery, and support pathogen inactivation (ieropoulos et al., , merino-jimenez et al., , salar-garcia et al., , zang et al., . mecs are normally associated with hydrogen production and nutrient recovery (kuntke et al., ) , and researchers are focusing on efficient nutrient recovery by controlling the load ratio, changing the electric current conditions, and developing electrodes (dbira et al., ; rodriguez arredondo et al., ; yuan and kim, ) . to date, many technologies have been developed and modified, which demonstrates the need for innovation and specialization to meet the different requirements for various countries and regions. (iii) micro-pollutant control of excreta: sustainable sanitation takes into consideration both the reuse and safe disposal of waste after treatment, and forming a cycle (from reuse to agriculture to humans) is an ideal solution. the nutrients in excreta can return to the table through agriculture and food production. hence, with the increasing attention on food security, the investigation of unwanted antibiotics and heavy metals is as important as obtaining the nutrients from human excreta. the control of heavy metal concentrations in human-excreta-related fertilizer is important since heavy metals can accumulate in crops and throughout the food chain, creating a health risk for humans. heavy metals (such as cu, ni, zn, cd, cr, pb, and hg) have been detected in solid waste from human excreta (alvarenga et al., ; remy and jekel, ) . antibiotics are a type of antimicrobial drug used in the treatment and prevention of bacterial infections; they are difficult to degrade, and they can even impart antimicrobial resistance to pathogenic bacteria (zhu et al., ) . hence, the stabilization of heavy metals and elimination of antibiotics are newly-launched hotspots in sanitation research. nano-filtration, active carbon adsorption, or other advanced treatment processes could be alternative technologies for micro-pollutant removal. currently, photochemical processes with nanomaterials have been used as new approaches to remove antibiotics and promote antibiotic-resistant microbe removal in wastewater treatment. materials such as ag, tio , and c n have been studied (moreira et al., ; nakano et al., ; qu et al., ; thurston et al., ) , but the practical applications of such technologies for micro-pollutant removal in the sanitation sector are unclear. great effort has been made to accelerate the sanitation coverage at different scales to meet the requirements of the mdgs and the upcoming sdg. nevertheless, gaps in global sanitation still exist in the form of technical, economic, social, cultural, educational, political, and institutional challenges. the previous data show that only . % of the papers on sanitation indexed in the ssci, indicating a mainstream of technical research and a lack of social study. nevertheless, it must be mentioned that there has limited publications for ssci. to realize the targets of sdgs, government support, social acceptability, as well as technological reliability are necessary. from governmental aspects, favorable polices for the sanitation value chain stakeholders (toilet manufacturers, emptying agencies, transportation, treatment system operators, end product sellers as well as users etc.) should be established, such as subsidies, iso/national standards, regulations, in order to form a healthy market for new sanitation systems, so that everyone could play a specific and positive role in it. social acceptability is closely linked to private behavior; thus, the developed toilet should adapt to religious belief, ethnic culture, and economic level as well, which would be conducive to governmental motivation. technological reliability is the driver for sanitation development and practical application. the developed technologies should meet the requirements for safe local discharge, efficient nutrient/energy recovery, limited air emission, nonnoise, operability following non-usage/short term/long term shutdown, overload protection, expected lifetime, flexibility in ambient change, etc. table lists a brief evaluation of the current techniques described above. the comments for each technology vary according to an overview of the whole "input-operation-output" system. the trl was also applied using the standards of commercialized applications (such as a centralized treatment plant or decentralized integrated toilet system), where the economic feasibility is distinguished by the development and effective operation of the economic value chain. the relevant policy refers to sustainable market-driven mechanisms to support its development, viability, and up-scaling of the technologies, as well as policies including subsidies, import duties, and taxes from the government. institutional behavior relies on regular certified laboratory testing to ensure the end-product is safe for use. social acceptance refers to the acceptance of both the treatment process and end products. lastly, cultural tradition decides whether the innovative toilet system changes the habits of the users, such as the body-cleaning style, the education/ training of individuals, including knowledge dissemination for the stakeholders and users, and the specialized training for workers, administrators, and decision makers. for example, a composting toilet or composting infrastructure incorporating fecal sludge could presently meet the standards for commercial use; the trl of compost reaches as high as . the end products could be used in agriculture. the relevant composting policies and standards have already been established to encourage the creation of organic fertilizer via composting. therefore, composting meets the criteria for economic feasibility, relevant policy, institutional behavior, and social acceptance. however, the application of a composting toilet would be limited for non-dry toilet users who lack the awareness of its safe management. additionally, the use of such a toilet requires public education, which would mainly come from the older farming stakeholders. the other techniques have been carefully evaluated through research. these technologies still do not address all the various aspects of sanitation. we found the following challenges and drew a number of conclusions from this work. sanitation issues vary from location to location, season to season, and community to community. people who lack sanitation facilities are often living in the most challenging geographies and climates, so there is no one-fits-all solution that is best for all situations. different approaches are required for each unique situation (curry, ) . hence, many innovative toilet systems have been developed, especially in regional hotspots. more than one-third of the technologies are at the trl level (shown in table ), which means that most of the new technology is still at the level of component validation in the laboratory. without three maturing application techniques (a trl of ), the rest of the technologies would require further development for practical utilization. storage techniques for excreta have a positive effect for their hygienization. however, since a large space and long retention time are required for the process, its application is limited in an urban context. the challenges of map/mpp precipitation are related to the high recovery efficiency of nutrients and the purification of the products. the purer the products obtained, the more economic value the process will produce. though mfcs could provide renewable energy during their operation, the high cost of the electrodes might be daunting. like the mecs, chemical reagents could be replaced by electricity, but the maintenance of the electrodes is crucial. it would be difficult to balance the cost and efficiency at scale. nano-filtration, active carbon adsorption, and advanced oxidation all require high investment and maintenance costs. new technologies must balance technical effectiveness with economic feasibility. . . gaps in multilateral cooperative solutions: political, institutional, social, cultural, and educational challenges a survey of the sanitation initiatives in monkey bay, a port town on the southern part of lake malawi, showed that only . % of the ngodonated sanitation interventions were in use after two years. this low sustainability was primarily influenced by the lack of consideration of the institutional, technical, educational, operational, social, and cultural factors (gutierrez, ) . a study of timor tengah utara (utt), nusa tanagra, indonesia and the muong ang district in northwest vietnam also showed that the researched districts were areas with high poverty rates, remote households, and a low-density population without access to sewers (gero et al., ; willetts et al., ) . table shows that almost all the mentioned technologies lacked a consideration of social factors. the social, cultural, economic, political, and educational factors have a great effect on the implementation of sanitation solutions (seetharam, , uddin et al., , uddin et al., . therefore, a functional and coordinated link among these factors can support high-quality sanitation and hygiene services (rana, ) . government should play a strong leadership role and take responsibility for legislation and the coordination and definition of roles, regulations, and policy. the state should lay a foundation and enforce health standards and regulations. regulations should create conditions that will be of advantage to innovation both in terms of financing mechanisms and technology support. products, such as fertilizer, electricity, reclaimed water, biochar, and biodiesel, should be harvested using the most appropriate technology where the operating models, municipal administration, industry, and business model are in demand (evans et al., ) . in addition, the institutional support (in the form of re-training, resourcing, and reform) of government and non-government organizations are also of great importance. the relevant cultural and social factors should be taken into account during the planning and execution phases to make sanitation more effective in both urban and rural communities. therefore, it is important to understand how societies work, including households and communities. much more consideration should be given to the social, political, and economic institutions that are operating at the local or national level, which include the civil service, schools and colleges, families, and government. it is also necessary to take the various patterns and roles of individuals in societies into consideration and identify who is responsible for the family's health, water supply, and education about defecation habits and environmental hygiene. there is a severe lack of skilled staff that can effectively and efficiently facilitate the sustainable sanitation process, which hampers the continuous follow-up and monitoring of these systems. in addition, an understanding of a community's educational level would help to clarify the issues and solutions for residents and stakeholders to help improve access to adequate sanitation (gutierrez, ) . novel models of institutional, financial, contractual, and legal relationships between communities and agencies should be encouraged (ademiluyi, ) . above all, the improvement of health and sanitation services calls for a good understanding of the actors at various levels, the full involvement and continuous support of the community, and institutional, legal, and contractual linkages among communities, government, and non-government organizations in all notes: . trl level: trl (technology readiness level) are a method of estimating technology maturity of critical technology elements (cte) of a program during the acquisition process, here was divided into three column, "research & development (level , , )","technology demonstration (level , , , )" and "production & deployment (level , )". trls in the european space agency: level -basic principles observed and reported; level -technology concept and/or application formulated; level -analytical and experimental critical function and/or characteristic proof-of-concept; level -component validation in laboratory environment; level -system/subsystem model or prototype demonstration in a laboratory environment; level -system/subsystem model or prototype demonstration in a relevant environment; level -system prototype demonstration in an operational environment; level -actual system completed and qualified through test and demonstration; level -actual system proven through successful product launch. . abbreviation: map refers to mgnh po • h o; mfc refers to microbial fuel cells; mec refers to microbial electrolysis cells; mpp refers to mgkpo • h o. marks: "+" refers to the identified technique is satisfied with the corresponding evaluation aspects, the more "+", the more relevant activities it has. "−" refers to the identified technique is unsatisfied with the corresponding evaluation aspects. stages of the sanitation improvement process (ekane et al., ) . to be fully or effective implemented sustainable sanitation, the technologies developed should not only meet the technical standards, but also satisfy the social acceptance, meanwhile, the regulations for the markets and stakeholders behavior, etc. are also important for promoting a sustainable development of sanitation worldwide. in this study, bibliometric and word cluster analyses were used to evaluate sanitation research using the science citation index-expanded (sci-expanded) and the social sciences citation index (ssci) from to . a systematic analysis of global sanitation using the background, current situations, challenges, and perspectives was performed on the results. we demonstrated that researchers are focusing more on sanitation in recent years, which is supported by the increased quantities of publications. developing countries are facing more serious sanitation problems, but the usa plays a leading role in researching and developing sanitation techniques, ranking first in the quantities of articles and us-innovated toilet systems have been selected from a global pool by bmgf. there are challenges for the adoption of newer technologies in the form of the actual requirements of the people who need them as well as technical development issues. currently, sanitation encompasses water, solid waste, air pollution, human health, and food security. hence, a closedloop analysis of the energy and substance of the internal circulation, transformation, and process control is essential but still lacking in published works. moreover, although innovative solutions have been developed in terms of the hygienization of human excreta, resource recovery, and removal of micro-pollutants, gaps in the technological development and commercialization of products, as well as issues with integrated solutions that address political, social, institutional, cultural, and educational factors, all still exist. there is no one-size-fits-all approach for achieving the successful implementation of adequate global sanitation. efforts should be made from view of government support, social acceptability, as well as technological reliability to realize a holistic solution. sustainability and impact of community water supply and sanitation programmes in nigeria: an overview sewage sludge, compost and other representative organic wastes as agricultural soil amendments: benefits versus limiting factors lactic acid fermentation of human urine to improve its fertilizing value and reduce odour emissions nitrogen and phosphorus recovery from human urine by struvite precipitation and air stripping in vietnam recovery of ammonia from human urine by stripping and absorption technological accumulation and industrial growth: contrasts between developed and developing countries inactivation kinetics and mechanisms of viral and bacterial pathogen surrogates during urine nitrification toilet revolution in china bibliometric analysis of severe acute respiratory syndrome-related research in the beginning stage high-impact papers presented in the subject category of water resources in the essential science indicators database of the institute for scientific information five pressing global water & sanitation challenges the electrolytic treatment of synthetic urine using dsa electrodes multi-level sanitation governance: understanding and overcoming challenges in the sanitation sector in sub-saharan africa the potential for self-sanitisation of faecal sludge by intrinsic ammonia modeling the inactivation of ascaris eggs as a function of ammonia concentration and temperature scientific collaboration between brics countries global, regional, and national comparative risk assessment of behavioural, environmental and occupational, and metabolic risks or clusters of risks in countries, - : a systematic analysis for the global burden of disease study the most frequently cited adsorption research articles in the science citation index (expanded) mapping of drinking water research: a bibliometric analysis of research output during - china's research in chemical engineering journals in science citation index expanded: a bibliometric analysis key-words-plus takes you beyond title words. . expanded journal coverage for current-contents-on-diskette includes social and behavioral-sciences relying on markets to address human rights: sanitation supply chain analysis in low-density settings environment: the disappearing nutrient delivering pro-poor water and sanitation services: the technical and political challenges in malawi and zambia an index to quantify an individual's scientific research output japanese lung cancer research trends and performance in science citation index evaluation of faecal contamination and microbial die-off in urine separating sewage systems variation of chemical and microbial parameters in collection and storage tanks for source separated human urine viral persistence in source-separated human urine microbial risk assessment of source-separated urine used in agriculture mapping the scientific research on life cycle assessment: a bibliometric analysis waste to real energy: the first mfc powered mobile phone urine disinfection and in situ pathogen killing using a microbial fuel cell cascade system recovery of phosphorus from source separated urine by acidithiobacillus ferrooxidans culture supernatant hydrogen production and ammonium recovery from urine by a microbial electrolysis cell separate management of anthropogenic nutrient solutions (human urine) mapping a new journey for poo on world toilet day source-separated urine opens golden opportunities for microbial electrochemical technologies iso to boost global health in places without sewers bibliometric analysis of global environmental assessment research in a -year period trends in research on global climate change: a science citation index expanded-based analysis feature: toilet revolution to make india open defecation-free how farmers in switzerland perceive fertilizers from recycled anthropogenic nutrients (urine) a comparative risk assessment of burden of disease and injury attributable to risk factors and risk factor clusters in regions, - : a systematic analysis for the global burden of disease study air stripping process for ammonia recovery from source-separated urine: modeling and optimization simultaneous pollutant removal and electricity generation in a combined abr-mfc-mec system treating fecal wastewater inactivation of pathogens in feces by desiccation and urea treatment for application in urine-diverting dry toilets inactivation of adenovirus, reovirus and bacteriophages in fecal sludge by ph and ammonia co-composting of fecal matter in mongolia using two different technologies a bibliometric study of the trend in articles related to risk assessment published in science citation index treatment processes for source-separated urine combating the 'toilet challenge' with science enhanced mfc power production and struvite recovery by the addition of sea salts to urine global potential of phosphorus recovery from human urine and feces photocatalytic ozonation of urban wastewater and surface water using immobilized tio with leds: micropollutants, antibiotic resistance genes and estrogenic activity composting of the solid fraction of blackwater from a separation system with vacuum toilets -effects on the process and quality global mortality, disability, and the contribution of risk factors: global burden of disease study broad spectrum microbicidal activity of photocatalysis by tio recent advances in nutrient removal and recovery in biological and bioelectrochemical systems comparing microbial die-off in separately collected faeces with ash and sawdust additives bench-scale composting of source-separated human faeces for sanitation exploratory social network analysis with pajek pathogen and indicator inactivation in source-separated human urine heated by the sun guidelines for the safe use of wastewater, excreta and greywater: wastewater and excreta use in aquaculture v. . water sanitation health . billion people lack safe drinking water at home, more than twice as many lack safe sanitation the intellectual base and research fronts of jasis - how to use bibexcel for various types of bibliometric analysis nitrogen and phosphorus harvesting from human urine using a stripping, absorption, and precipitation process china health statistical yearbook a bibliometric analysis of global research progress on pharmaceutical wastewater treatment during - applications of nanotechnology in water and wastewater treatment urbanization and sustainability: challenges and strategies for sustainable urban development in bangladesh sustainable wastewater management: life cycle assessment of conventional and source-separating urban sanitation systems load ratio determines the ammonia recovery and energy input of an electrochemical system struvite precipitation thermodynamics in source-separated urine electricity production from human urine in ceramic microbial fuel cells with alternative non-fluorinated polymer binders for cathode construction challenges and opportunities for sanitation in developing countries inactivation mechanisms of pathogenic bacteria in several matrixes during the composting process in a composting toilet a bibliometric analysis of research on proteomics in science citation index expanded preparation and characterization of photoactive antimicrobial graphitic carbon nitride (g-c n ) films effects of storage on phosphorus recovery from urine assessment of social acceptance and scope of scaling up urine diversion dehydration toilets in kenya socio-cultural acceptance of appropriate technology: identifying and prioritizing barriers for widespread use of the urine diversion toilets in rural muslim communities of bangladesh progress on sanitation and drinking water: update and mdg assessment the state of the world's children comparison of composting, storage and urea treatment for sanitising of faecal matter and manure inactivation of bacteria and viruses in human urine depending on temperature and dilution rate global trends in soil monitoring research from to : a bibliometric analysis catalytic liquefaction of municipal sewage sludge over transition metal catalysts in ethanol-water co-solvent overflowing cities: the state of the word's toilets sanitation value chains in low density settings in indonesia and vietnam: impetus for a rethink to achieve pro-poor outcomes phosphate and potassium recovery from source separated urine through struvite precipitation china to get extreme toilet makeover to boost tourism water, sanitation, hygiene, and malnutrition in india laboratory experiments on simultaneous removal of k and p from synthetic and real urine for nutrient recycle by crystallization of magnesium-potassium-phosphate-hexahydrate in a draft tube and baffle reactor the precipitation of magnesium potassium phosphate hexahydrate for p and k recovery from synthetic urine sludge disinfection using electrical thermal treatment: the role of ohmic heating increasing phosphorus recovery from dewatering centrate in microbial electrolysis cells nutrient removal and energy production in a urine treatment process using magnesium ammonium phosphate precipitation and a microbial fuel cell technique a bibliometric analysis of world volatile organic compounds research trends a bibliometric analysis of research on upflow anaerobic sludge blanket (uasb) from to recovery of phosphorus and potassium from source-separated urine using a fluidized bed reactor: optimization operation and mechanism modeling benchmarking the scientific research on wastewater-energy nexus by using bibliometric analysis state of the art on granular sludge by using bibliometric analysis investigation on microbial inactivation and urea decomposition in human urine during thermal storage microbial mass movements removal rates and energy demand of the electrochemical oxidation of ammonia and organic substances in real stored urine the authors would like to acknowledge the financial support of the bill & melinda gates foundation in the united states(reinvent the toilet challenge-china reginal program (opp ). the authors would also like to acknowledge professor ruth elaine blake from yale university for the revision of this manuscript. the authors would also like to thank the national environment and energy international cooperation base for their support. all authors of this article declare that they have no conflict of interest regarding the publication of this paper. key: cord- -ion n b authors: de silva senapathi, upasama; abdul-cader, mohamed sarjoon; amarasinghe, aruna; van marle, guido; czub, markus; gomis, susantha; abdul-careem, mohamed faizal title: the in ovo delivery of cpg oligonucleotides protects against infectious bronchitis with the recruitment of immune cells into the respiratory tract of chickens date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: ion n b the in ovo delivery of cytosine-guanosine (cpg) oligodeoxynucleotides (odns) protects chickens against many bacterial and viral infections, by activating the toll-like receptor (tlr) signaling pathway. although the delivery of cpg odns in ovo at embryo day (ed) has been shown to reduce infectious bronchitis virus (ibv) loads in embryonic chicken lungs pre-hatch, whether in ovo delivered cpg odns are capable of protecting chickens against a post-hatch challenge is unknown. thus, our objectives were to determine the protective effect of the in ovo delivery of cpg odns at ed against ibv infection encountered post-hatch and, then, to investigate the mechanisms of protection. we found significantly higher survival rates and reduced ibv infection in the chickens following the pre-treatment of the ed eggs with cpg odns. at days post infection (dpi), we found an increased recruitment of macrophages, cluster of differentiation (cd) α+ and cd + t lymphocytes, and an up-regulation of interferon (ifn)-γ mrna in the respiratory tract of the chickens. overall, it may be inferred that cpg odns, when delivered in ovo, provide protection against ibv infection induced morbidity and mortality with an enhanced immune response. infectious bronchitis (ib) is mainly an acute and severe disease of the respiratory system of chickens [ ] . the causative agent, infectious bronchitis virus (ibv), belongs to the family coronaviridae [ ] . there is increasing evidence of ibv infection being reported in birds other than chickens [ , ] . although ibv induced changes are observed primarily in the mucosal surfaces of the respiratory tract, the virus is also known to cause pathology in the female reproductive tract and kidneys, with a varying degree of severity dependent upon the type of strain that infects and replicates in the aforementioned tissues [ ] [ ] [ ] . ever since the first record of ib in the early s [ ] , periodic ib outbreaks associated with the isolation of heterogeneous strains of ibv have been reported globally [ , ] . major losses to the broiler meat industry are due to carcass condemnation at processing, a poor feed conversion ratio resulting in poor weight gain, and mortality. ibv is considered a highly infectious agent with near % morbidity, and with mortality reaching - % [ , ] . in breeder and layer flocks, the major losses are due to reduced egg production during and after infection with ibv. the egg drop during ibv infection has been estimated to be between - % [ ] . furthermore, the downgrading of eggs because of a poor internal egg quality and egg shell quality also account for considerable production losses [ ] . the standard preventive measures, such as strict quarantine and biosecurity measures [ ] , do not seem to sufficiently control the disease. thus far, the most efficient method for controlling ibv is by vaccination [ ] . the immunization of chickens against ibv is mainly by live attenuated and killed vaccines [ ] . however, the emergence of variant ibv strains/serotypes arising from vaccinated flocks, among other factors, has led to vaccine failure and ib outbreaks. thus, the development of novel approaches as an alternative or adjunct in order to control the current measures against ibv is becoming increasingly important. toll-like receptor (tlr)s are a family of germ line encoded pattern recognition receptors (prrs) expressed on the surface or within the endosomal compartments of cells [ ] . these receptors are crucial for recognizing whole or segments of microbial pathogens, and they initiate key host immune defenses against inciting agents. among the tlrs, tlr (in mammals)/tlr (in birds) are the only receptors capable of distinguishing bacterial, parasitic, and viral dna containing cytosine-guanosine (cpg) motifs [ ] . several studies have demonstrated the immunostimulatory and therapeutic success of cpg oligodeoxynucleotides (odns) application in various host-pathogen interaction models [ ] [ ] [ ] . the protection provided by cpg odns against lethal challenges of extracellular bacteria, such as escherichia coli [ ] and salmonella typhimurium [ ] , and viruses, such as low pathogenic avian influenza virus [ ] and infectious laryngotracheitis virus (iltv) [ , ] , in chickens have been well documented. cpg odns are known to induce an array of cytokines; chemokines; and effecter molecules, such as interferon (ifn) α, β and γ, interleukin (il)- β, il- , il- , il- , tumor necrosis factor (tnf)-α, and nitric oxide (no) [ ] [ ] [ ] . these effecter molecules are believed to play a pivotal role in protecting the host against intra and extra cellular pathogens. whilst activating a variety of immune cells, it plays an integral role in bridging the innate immune system with the adaptive immune system directing immune responses toward t helper (th) response [ ] . a study that pre-treated chicken embryos with class b cpg odns at embryo day (ed) in ovo, and then challenged them with ibv ark strain the day after (ed ) , showed an increased up-regulation of ifn-γ, il- β, il- , il- , and oligoadenylate synthetase (oas) a in the embryonic spleen [ ] . additionally, the authors saw a significant reduction in the ibv nuclear (n) gene mrna expression in various embryonic tissues pre-treated with cpg odns, compared with the control, highlighting the value of cpg odn treatment in ibv control. however, they did not demonstrate whether the in ovo cpg odn delivery is effective against the ibv challenge encountered post-hatch. in this study, we determined whether the cpg odns delivered in ovo could provide protection against a post-hatch ibv challenge. furthermore, we looked into several cytokines and immune cells that may be activated with such protection. we found that in ovo delivery was protective against ibv challenge post hatch, suggesting a potential lasting protective effect of cpg odns towards ibv infection, which could be exploited for developing control measures. the specific pathogen free (spf) eggs from white leghorn layer hens were obtained from the canadian food inspection agency (cfia), ottawa, and were incubated according to the manufacturer's instructions in digital egg incubators (kingsuromax and rcom maru deluxe max, autoelex co., ltd., gimhae, gyeongnam, korea), located at the health research innovation centre (hric) , university of calgary. all of the animal care protocols as well as the use of live chickens, embryos, and spf eggs in our experiments, have been reviewed and approved by the health science animal care committee (hsacc, ac - , november ). at ed , the incubated eggs were candled in order to select viable eggs for further incubation, and the hatched birds were transported and housed in high containment poultry isolators at the prion/virology animal facility, hric, university of calgary, with access to ad libitum food, water, and necessary veterinary care. the ibv massachusetts (m) strain was purchased from the american type culture collection (atcc, manassas, va, usa) and was used in all of the experiments. nine day old spf viable eggs were used to propagate m strain of ibv, and the allantoic fluid was harvested at dpi by careful aspiration. the end point dilution assay was employed to assess the viral titers using ed spf eggs, and was expressed as a % embryo infectious dose (eid ) [ ] . the synthetic cpg odns, class b cpg motifs recognized by chicken tlr . the control odns were diluted in pbs to the same concentration ( µg in µl per egg), and were delivered via the same route (n = ). the in ovo tlr ligand delivery was carried out as described previously [ ] . on day post-hatch, the birds in both groups were infected with ibv m strain intra-trachealy, at a dose rate of . × eid per bird, and were monitored for days post-infection (dpi) for disease progression and outcome. the humane end point of the birds was determined based on the clinical score of each bird (ruffled feathers and huddling together = , droopy wings = , depression = , mild increase in respiratory rate = , increased respiratory rate with constant beak opening = , severe increased respiratory rate marked by gasping = , and body weight loss = ). the clinical score of was considered the humane endpoint. the eggs were then incubated for days until hatching. on the day of hatching, a subset of cpg odn-treated chickens (n = ) were infected with ibv m strain intra-trachealy at a dose rate of . × eid per bird, while maintaining the rest of the birds in that treatment group as uninfected controls (n = ). similarly, a subset of the control odn-treated chickens was infected with ibv m strain with the same dose (n = ), with the remaining birds being the control odn treated birds (n = ), and all the pbs treated birds (n = ) were kept as controls. the birds were weighed, wing tagged and after infection, were placed in separate isolators until the subsets of the animals were euthanized at (n = - per group) and (n = - per group) dpi. the clinical signs were observed and recorded daily as described, and the oro-pharyngeal and cloacal swab samples obtained using puritan ® unitranz-rt ® media transport systems (vwr, edmonton, ab, canada) at and dpi, and the ibv genome load were quantified following the rna extraction. simultaneously, the lung tissue was collected at and dpi in rna save ® (biological industries, froggabio, toronto on, canada), in order to determine the viral genome loads in lungs. to evaluate the ibv n antigen in the tracheal mucosal epithelium, the tracheal tissues from the dpi birds were collected and preserved in an optimum cutting temperature (oct) compound (tissue-tek ® , sakura finetek usa inc, torrance, ca, usa), and were snap frozen in dry ice until use in immunofluorescent assay. to observe the histopathology, the tracheal tissues of the dpi birds were fixed in % neutral buffered formalin (vwr international, west chester, pa, usa) and sent to the histopathology diagnostic services unit at the university of calgary, faculty of veterinary medicine, for hematoxylin and eosin (h and e) staining. additionally, the trachea and lung tissues were collected in an oct compound (tissue-tek ® , sakura finetek usa inc, torrance, ca, usa), snap frozen, and subjected to immunofluorescent assay so as to quantify the key innate and adaptive immune cells. another portion of the tissues were collected iusing rna save ® (biological industries, froggabio, toronto on, canada) for the cytokine mrna expression analysis. the animal numbers represent the total number of animals in two independent experiments. the total rna from the lungs collected at and dpi was extracted using a trizol reagent (invitrogen, canada inc., burlington, on, canada), according to the manufacturer's guidelines. for the rna extraction of oro-pharyngeal and cloacal swabs, the e.z.n.a. ® viral rna kit (omega bio-tek inc., norcross, ga, usa) protocol was adopted as per manufacturer's guidelines. the concentration of extracted rna was measured using nanodrop spectrophotometer (thermoscientific, wilmington, de, usa), with the absorbance at a / nm wavelength. two µg of total rna from the tissue samples and ng of total rna from the swab samples were used to synthesize the cdna with the use of the high capacity cdna reverse transcription kit (invitrogen life technologies, carlsbad, ca, usa), as per manufacturer's guidelines. a rt-pcr assay was carried out using fast sybr ® green master mix (invitrogen, burlington, on, canada) in order to quantify the ibv n gene and cytokine mrna expressions. rt-pcr assays were conducted in a well un-skirted, low profile pcr plate (vwr, edmonton, ab, canada), where the final reaction volume of the qpcr was maintained at µl. each qpcr run consisted of samples of interest, a positive control/s (gene specific plasmid), negative reverse transcriptase (nrt) control (cdna construct without the multiscribe reverse transcriptase enzyme), and negative template (ntc) control. all of the cdna samples originating from the tissues, along with the plasmid dilution series used to generate the standard curves, were run in triplicate. the target genes were quantified in relation to the β actin housekeeping gene. the target gene and the housekeeping gene for each sample was run on the same plate. five picomolar (pm) of different gene specific primers (forward and reverse primers) were used in each reaction (supplementary table s ). the change in the mrna expression of the cytokines was assessed using the pfaffl method [ ] . the optimum parameters used in the thermal cycler (cfx -c ) (bio-rad laboratories, mississauga, on, canada) were • c for seconds (s) of pre-incubation, • c for s, and • c for s for amplification cycles. a melting curve analysis was performed between • c and • c, with a . • c raise in temperature every s. the acquisition of fluorescent signals was performed at • c for s. for the cluster of differentiation (cd) α+ cell and macrophage (kul +) of the lung and trachea, µm thick sections were cut from the oct preserved tissues and were fixed using cold acetone for minutes (min). the tissues were then blocked by adding % goat serum diluted in a trizma buffered saline (tbs) buffer (trizma base: . g; nacl: g in l of distilled water; ph . ) at room temperature for min. after tipping off the excess blocking buffer, as the primary antibodies, the mouse monoclonal antibody specific for chicken macrophages/monocytes, kul + (southern biotech, birmingham, alabama, usa), cd α (ct- , southern biotech, birmingham, alabama, usa), was used in a : dilution in a % goat serum for min. the secondary antibody, goat anti-mouse igg (h+l) conjugated with dylight ® (red fluorescence) (bethyl laboratories inc., montgomery, tx, usa) was then used in a : in % goat serum for hour (h), followed by adding vectashield ® mounting medium with , -diamidine- -phenylindole dihydrochloride (dapi, vector laboratories inc., burlingame, ca, usa) (blue fluorescence), placing cover slips and edges sealed with lacquer as the final step. for the cd + t cell staining, before blocking the tissues with % goat serum, sections were blocked with avidin followed by biotin (vector laboratories, inc., burlingame, ca, usa), each with min incubation periods, in between washing with tbs-t for min twice and with pbs for min once. after blocking with % goat serum for min, a primary antibody, cd (ct- , southern biotech, birmingham, alabama, usa) was added in a : dilution in % goat serum for min. next, biotinylated goat anti-mouse igg (h+l) (southern biotech, birmingham, alabama, usa) was used as a secondary antibody in a : dilution in a % blocking buffer, and was incubated for min. then, dylight ® (green fluorescence) streptavidin in a : dilution was added for min, followed by a final step of mounting the slides with a vectashield ® mounting medium with dapi (vector laboratories inc., burlingame, ca, usa). all of the incubations were performed in a humidifying chamber at room temperature. each incubation with an antibody was followed by washing the slides in a tbs-t buffer for min twice and in pbs for min once. for the quantification of the tissue kul +, cd + cells, and cd α+ cells, five areas with maximum positive fluorescent signals of kul +, cd + cells, and cd α+ cells per tissue section were captured at x magnification, along with the corresponding nuclear stained (dapi) areas. the images were then subjected to fluorescent intensity quantification using image j software (national institute of health, bethesda, md, usa). the fluorescent intensities for the dylight ® (kul +, cd α+ cells) and dylight ® (cd + cells) positive signals were expressed relative to the total area (as estimated by nuclear staining with dapi), and were given as a percentage. a log-rank test was used to identify the differences in survival percentage. the kruskal-wallis test followed by the mann-whitney u test were used to identify the group differences in the clinical score data for each time point. the differences among the two groups were identified using othe student's t test. one-way analysis of variance (anova) followed by the students-newman-keuls test were used to identify the group differences in all of the other experiments. the grubbs' outlier test was performed in order to identify the outliers before the data was analyzed. the data in the graphs are shown in the original scale of the measurements. however, because of the non-normality and inability to satisfy the model assumptions of data belonging to the cell counts and cytokine mrna expression, a natural log transformation was applied to these data sets prior to analysis. model statistics were performed using graphpad prism software , la jolla, ca, usa. a normality test, generation of histograms, box plots, and q-q plots were performed in r statistical software, r studio version . . , boston, ma, usa. * = significant at p ≤ . , ** = significant at p ≤ . *** = significant at p ≤ . . we observed a significant increase in the survival rate of the cpg odn-treated chickens (p < . ) when compared with the control odn-treated chickens, as seen in figure a . also, the clinical signs in the cpg odn-treated ibv-infected group were significantly milder compared with the control odn-treated and ibv-infected group at dpi (p < . , figure b) . , and the rest were kept as in ovo cpg pre-treated uninfected controls (n = ). similarly, a subset of birds in the in ovo control odn-treated birds was infected with ibv (n = ), and the remaining birds were kept as in ovo control odn-treated uninfected controls (n = ). the in ovo pbs treated birds were kept as uninfected controls (n = ). a subset of birds from each group was sacrificed at dpi (n = - per group), and the remaining birds were sacrificed at dpi (n = - ) in order to obtain lung tissue. (c) ibv genome loads in oro-pharyngeal swabs at and dpi, (d) ibv genome loads in cloacal swabs at and dpi, and (e) ibv genome loads in and dpi lung. (f-g) the quantitative data and representative figures from the immunofluorescent assay of the trachea for ibv n antigen is presented. scale bar = µm (h) representative images of histological observations of trachea are given. control odns -ibv: severe epithelial metaplasia with severe cellular infiltration, germinal center formation is seen (a), superficial epithelial layer has become squamous with complete loss of cilia (b) and mucus glands not detected. cpg odns-ibv: pseudostratified simple columnar epithelium and intact ciliated epithelia (arrow) is evident where some have become rounded, and a few mucus secreting glands have been distorted and elongated (arrow head). cpg odns-control, control odns-control, and pbs-control: no lesions, normal pseudostratified ciliated columnar epithelium (c) with mucus secreting glands. log-rank test was used to identify the differences in the survival rate, and the kruskal-wallis test followed by the mann-whitney u test were used to identify the differences in the clinical scores at selected time points. the student's t test was performed to identify group differences in the oropharyngeal and cloacal genome loads, and one-way analysis of variance (anova) followed by the students-newman-keuls post hoc test was used to identify the differences in the lung ibv genome loads and ibv n antigen amount in the trachea. the differences were considered significant at * = significant at p ≤ . , ** = significant at p ≤ . *** = significant at p ≤ . . c-h: the animal numbers and results represent the pooled data of the two independent experiments. in order to assess the ibv genome loads in the lungs, and to determine the degree of virus shedding through the feco-oral route, the ibv n gene was quantified at and dpi in the birds that were pre-treated with in ovo cpg odns, control odns, and pbs. we observed a significant reduction in the viral genome loads in the oro-pharyngeal swabs collected and dpi (p < . ; figure c ), but did not observe a difference in the ibv genome loads in the cloacal swabs ( and dpi) between the treatment groups (p > . ; figure d ). however, significantly lower levels of lung viral genome load in the in ovo cpg odn pre-treated ibv infected group compared to in ovo control odn pre-treated ibv-infected group were observed at dpi (p < . ; figure e ). at dpi, although the control odn pretreated ibv infected lung had a significantly higher ibv genome load when compared with the uninfected controls (p < . ; figure e) , the difference of the ibv genome load in the lungs between two ibv infected groups was not significant (p > . ; figure e ). we observed a significant reduction of the ibv-n antigen in the tracheal mucosal epithelium of the in ovo cpg odn pre-treated-ibv infected birds compared with the in ovo control odn pre-treated-ibv infected birds (p < . ; figure f-g) . this was also seen in the histology of the trachea for the degree of tracheal damage following ibv infection. the mucosal epithelium of the in ovo control odn treated-ibv infected group showed severe metaplasia with severe mononuclear cell infiltration, where the superficial epithelial layer had been replaced by squamous cells. a complete erosion/loss of the entire mucosae was evident in several areas of the trachea. also, mucus secreting glands were absent from the remaining mucosae. in contrast, in the in ovo cpg odn pre-treated-ibv infected birds, the epithelium was a pseudostratified simple columnar epithelium mostly with intact cilia on the surface. mucus glands were present with some distortion and elongation (figure h ). in general, the in ovo cpg odn pre-treated ibv infected group recorded higher macrophage and cd + and cd α+ t numbers in the trachea compared with the uninfected and control odn pre-treated groups, although not all of the specific comparisons reached statistical significance (figure a-c) . similarly, the in ovo cpg odn pre-treated ibv infected group recorded higher macrophage and cd + and cd α+ t numbers in lungs compared to uninfected and control odn pre-treated groups, although not all of the specific comparisons reached statistical significance (figure d-e) . in the trachea and lungs, the macrophage and cd α+ t recruitment patterns, respectively, indicated that the in ovo delivered cpg odns are capable of increasing the recruitment of these cells in both the ibv infected and uninfected chickens (figure a the quantitative data following immunofluorescent assays done for the trachea (a) macrophages, (b) cluster of differentiation (cd) + t cells, and (c) cd α+ t cells are given. the quantitative data following the immunofluorescent assays done for lung (d) macrophages, (e) cd + t cells, and (f) cd α+ t cells are given. one-way anova followed by the students-newman-keuls post hoc test were used to identify the group differences. the differences were considered significant at * = significant at p ≤ . , ** = significant at p ≤ . *** = significant at p ≤ . . the results represent the pooled data of two independent experiments. considering that we observed a significant reduction in the ibv induced morbidity and mortality of in ovo cpg odn pre-treated birds correlating with varying degrees of increased macrophages, cd +, and cd α+ t cells in the tracheal and lung tissues, we needed to further elucidate the mechanisms by which these immune cells were efficiently recruited. several cytokine mrna expression levels in the lungs were analyzed at dpi, and our data showed a significant increase in the up-regulation of only the ifn-γ mrna expression in the in ovo cpg odn pre-treated lungs compared with the in ovo control odn pre-treated lungs, in both the ibv infected (p < . ) and uninfected (p < . ) groups (figure a-c) . in ovo delivery of poultry vaccines has been performed routinely for decades by the poultry industry [ ] . in ovo delivery targets the deposition of cpg odns in the amniotic cavity. subsequently, the ingestion of cpg odns containing amniotic fluid by the developing embryo distributes cpg odns in the respiratory and gastrointestinal tracts, leading to immune cell recruitment in these two body systems [ ] . we have shown in this study that cpg odns when delivered in ovo are capable of protecting young chickens against a post-hatch ibv infection induced ib. in ovo cpg odn-treated birds displayed reduced ibv viral loads in the lungs and a decreased ibv replication and pathology in the trachea, which is associated with high survival rates and low morbidity. we found that the macrophages in the trachea and cd + and cd α+ t cells in the lungs play important roles in this process, as increases in these cells were observed in the in ovo cpg odns treated group. lastly, we saw an up-regulation of ifn-γ mrna in the in ovo cpg odn pre-treated lungs, suggesting the critical role of this cytokine in the in ovo cpg odn-induced clearance of ibv infection. the host survival after day post-hatch ibv infection was seen as significant in the presence of cpg odn administration in ovo compared with the controls, and a similar protective effect of in ovo delivered cpg odns has been recorded against the post-hatch iltv infection [ , ] , and e. coli and salmonella thypimurium septicemia [ , ] . in the current study, the protection-mediated by the in ovo administered cpg odns was associated with significantly lower ibv replication in the trachea and ibv genome loads in the lungs at and dpi. consequently, the ibv genome loads in the oro-pharyngeal swabs were significantly reduced at and dpi. however, we did not observe a significant reduction in the ibv genome loads in the cloacal swabs at and dpi, because of the high variability of the ibv genome loads within the control odn pre-treated group. it is difficult to explain why we observed a discrepancy in the ibv genome loads between the oro-pharyngeal and cloacal swabs, as the in ovo delivered cpg odns have been shown to recruite immune cells into the gastrointestinal mucosa [ ] . our data confirm that, when delivered in ovo, the cpg odns are able to recruit macrophages into the trachea at dpi (four days of age), when compared to the in ovo delivered control odns in both the ibv infected and uninfected groups. previously, we saw that the in ovo delivered cpg odns increased the macrophages in the trachea at one day of age [ , ] . this macrophage recruitment to the trachea is associated with a lower ibv replication in the tissue, and it is possible that the cpg odn-mediated increase of the macrophages seen in the trachea in this study, played a central role in limiting the viral replication by three possible mechanisms. first, these cells may have efficiently and rapidly phagocytized the virus-infected cells and aided in virus elimination. second, they may have alerted the adaptive immune system to the invasion via active antigen presentation to the t cells. third, it may have contributed to the t and b cell activation and proliferation through the release of cytokines [ , ] . our observation of the increased recruitment of cd + and cd α+ t cells in the lungs following in ovo cpg odns delivery indicated that cpg odns could act as a mitogen, as has been shown previously [ ] . this cpg odn-mediated increased cd + and cd α+ t cell recruitment also could be due to the increased survival of these t cells in the lungs [ ] . interestingly, we saw an expansion of the cd α+ t cell population, but not the cd + t cell population in the in ovo cpg odn pre-treated-ibv infected lungs. although a portion of this increase of cd α+ cell recruitment could be potentially attributable to the ibv specific cd + t cells, we did not determine whether these cd α+ t cells are in deed ibv specific. we are at a loss as to why we did not see a similar cd + and cd α+ t cell response in the trachea, but it is possible that the in ovo delivered cpg odn-mediated cd α+ t cell recruitment is tissue specific [ ] . it is also important to note that our data is limited to dpi, and we do not know whether the in ovo delivered cpg odn-mediated cd + and cd α+ t cell recruitments in the trachea are occurring in other time points. of the examined immune mediators, ifn-γ, a dominant product of the t helper (th) type cells, was upregulated in the cpg odn pre-treated ibv infected and uninfected groups, when compared with the control odn pre-treated ibv infected and uninfected groups. the source of the lung ifn-γ mrna could be the cd + t cell lymphocytes and cd α+ cytotoxic lymphocytes [ , ] , and we observed an increased recruitment of the cd + and cd α+ t cells in the lungs in our experiment. two other immune mediators that were induced by the cpg odns and originated from the innate immune cells, such as the macrophages in the lungs, are the chemoattractant, il- β, and the no production inducer, inos [ ] . in the current study, we did not observe that the cpg odns or ibv induced the mrna expression of il- β or inos. this discrepancy in the cpg odn-mediated lack of il- β and inos expression can be explained by the difference in the time points observed. thapa et al. [ ] observed an increase il- β mrna expression in the lungs pre-hatch, and we observed a lack of il- β mrna expression post-hatch following in ovo cpg odns delivery. the significance of the observations described in our study are two-fold. first, we found that the in ovo administration of cpg odns is capable of limiting ibv replication in the lungs and trachea, leading to an increased survival and reduced morbidity in the early post-hatch birds. second, in our study, the early recruitment and maintenance of key immune cells, such as cd α+ and cd + t cells and macrophages, and the up-regulated ifn-γ mrna, exhibited not only an initiation of the early innate response, but also an effective and early adaptive host response mediated by the cpg odns, which would facilitate protection against the ibv infections encountered in birds in their immediate post-hatch life. further experiments elucidating the mechanisms of the cpg odn-mediated adoptive response, such as cell-and antibody-mediated immune responses in chickens and the duration of protection provided by this ligand against ibv, would be greatly beneficial in order to better understand the protective effects of cpg odns, and may aid in the development of more effective ibv control measures. to conclude, we show that the cpg odn-mediated protective response against post-hatch encountered ibv infection is associated with the up-regulation of ifn-γ mrna expression (in the lungs) and the enhanced recruitment of macrophages (in trachea) and cd + and cd α+ t cells (in the lungs). our findings, although preliminary, may provide a basis for developing novel control strategies in the long term against ibv infection in chickens. infectious bronchitis virus types: incidence in the united states induction of innate immune response following infectious bronchitis corona virus infection in the respiratory tract of chickens pathogenicity of australian strains of avian infectious bronchitis virus isolation of avian infectious 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oligodeoxynucleotide and double-stranded rna synergize to enhance nitric oxide production and mrna expression of inducible nitric oxide synthase, pro-inflammatory cytokines and chemokines in chicken monocytes a novel toll-like receptor that recognizes bacterial dna protection of chickens against escherichia coli infections by dna containing cpg motifs protection of neonatal broiler chicks against salmonella typhimurium septicemia by dna containing cpg motifs toll-like receptor (tlr) signalling-mediated antiviral response against avian influenza virus infection correlates with macrophage recruitment and nitric oxide production in ovo delivery of cpg dna reduces avian infectious laryngotracheitis virus induced mortality and morbidity in ovo cpg dna delivery increases innate and adaptive immune cells in respiratory, gastrointestinal and immune systems post-hatch correlating with lower infectious laryngotracheitis virus infection cpg oligodeoxynucleotides activate innate immune response that suppresses infectious bronchitis virus replication in chicken embryos cpg-odns induced changes in cytokine/chemokines genes expression associated with suppression of infectious bronchitis virus replication in chicken lungs bacterial dna-induced nk cell ifn-γ production is dependent on macrophage secretion of il- interactions between bacterial cpg-dna and tlr bridge innate and adaptive immunity a simple method of estimating fifty per cent endpoints the use of real-time quantitative pcr for the analysis of cytokine mrna levels hatchery vaccination against poultry viral diseases: potential mechanisms and limitations immunostimulatory cpg-modified plasmid dna enhances il- , tnf-α, and no production by bovine macrophages interleukin- -induced promotion of t-cell differentiation in mice immunized with killed listeria monocytogenes creating space: an antigen-independent, cpg-induced peripheral expansion of naive and memory t lymphocytes in a full t-cell compartment distinct effects of t-bet in th lineage commitment and ifn-γ production in cd and cd t cells il- up-regulates il- receptor expression on t cells, th cells, and b cells: synergism with il- for ifn-γ production this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we would like to acknowledge the staff of the prion/virology animal facility at foothill campus, university of calgary, for the experimental animal management. the authors declare no conflicts of interest. key: cord- -upwe cpj authors: sullivan, kathleen e.; bassiri, hamid; bousfiha, ahmed a.; costa-carvalho, beatriz t.; freeman, alexandra f.; hagin, david; lau, yu l.; lionakis, michail s.; moreira, ileana; pinto, jorge a.; de moraes-pinto, m. isabel; rawat, amit; reda, shereen m.; reyes, saul oswaldo lugo; seppänen, mikko; tang, mimi l. k. title: emerging infections and pertinent infections related to travel for patients with primary immunodeficiencies date: - - journal: j clin immunol doi: . /s - - - sha: doc_id: cord_uid: upwe cpj in today’s global economy and affordable vacation travel, it is increasingly important that visitors to another country and their physician be familiar with emerging infections, infections unique to a specific geographic region, and risks related to the process of travel. this is never more important than for patients with primary immunodeficiency disorders (pidd). a recent review addressing common causes of fever in travelers provides important information for the general population thwaites and day (n engl j med : - , ). this review covers critical infectious and management concerns specifically related to travel for patients with pidd. this review will discuss the context of the changing landscape of infections, highlight specific infections of concern, and profile distinct infection phenotypes in patients who are immune compromised. the organization of this review will address the environment driving emerging infections and several concerns unique to patients with pidd. the first section addresses general considerations, the second section profiles specific infections organized according to mechanism of transmission, and the third section focuses on unique phenotypes and unique susceptibilities in patients with pidds. this review does not address most parasitic diseases. reference tables provide easily accessible information on a broader range of infections than is described in the text. . this review covers critical infectious and management concerns specifically related to travel for patients with pidd. this review will discuss the context of the changing landscape of infections, highlight specific infections of concern, and profile distinct infection phenotypes in patients who are immune compromised. the organization of this review will address the environment driving emerging infections and several concerns unique to patients with pidd. the first section addresses general considerations, the second section profiles specific infections organized according to mechanism of transmission, and the third section focuses on unique phenotypes and unique susceptibilities in patients with pidds. this review does not address most parasitic diseases. reference tables provide easily accessible information on a broader range of infections than is described in the text. physician be familiar with emerging infections, infections unique to a specific geographic region, and risks related to the process of travel. this is never more important than for patients with primary immunodeficiency disorders (pidd). a recent review addressing common causes of fever in travelers provides important information for the general population [ ] . this review covers critical infectious and management concerns specifically related to travel for patients with pidd. this review will discuss the context of the changing landscape of infections, highlight specific infections of concern, and profile distinct infection phenotypes in patients who are immune compromised. the organization of this review will address the environment driving emerging infections and several concerns unique to patients with pidd. the first section addresses general considerations, the second section profiles specific infections organized according to mechanism of transmission, and the third section focuses on unique phenotypes and unique susceptibilities in patients with pidds. this review does not address most parasitic diseases. reference tables provide easily accessible information on a broader range of infections than is described in the text. emerging infectious diseases are a result of a convergence of numerous factors and comprise complex interactions among multiple variables. some of those factors are human movement, land use change, encroachment and wildlife translocation, rapid transport, and climate change. several studies demonstrate that for a pathogen to persist in a population, a minimal host population size that is specific for the type of pathogen and host population. of particular relevance to emerging infections is the pattern of rapid population growth. in the tropics, before wwii, most regional ecosystems consisted of few large cities and scattered human settlements separated by large areas of cropland, pastureland, or undisturbed forest. today, the pattern is the opposite: many large cities have developed with only patches of undisturbed forest or grassland. domestic vectors have therefore expanded their population with increasing urbanization and this markedly impacts the interactions between vectors and pathogens [ ] . human activities such as deforestation, use of pesticides, pollution, etc. lead to the loss of predators that naturally regulate rodent and insect populations. this contributes to emerging zoonotic diseases and explains why they occur more frequently in areas recently settled. in today's global economy and affordable vacation travel, it is increasingly important that visitors to another country and their the southern, with a reduction in the number of cold days per year, changes in rainfall (more winter precipitation and summer droughts) [ ] , and together these changes increase the risk of several vector-borne diseases in new areas. climate change involves not only global warming but also changes in precipitation, wind, humidity, and the location and frequency of extreme weather events like floods, droughts, or heat waves. changes in climate produce changes in pathogens, vectors, hosts, and their living environment. increases in precipitation can increase mosquitoes for example, but heavy rainfalls may cause flooding that temporarily eliminates larval habitats and decreases mosquitoes. flooding may force rodents to look for new habitats in houses and increase the opportunities of vector-human interactions, as occurs for example in the case of epidemic leptospirosis. humidity is another very important factor of climate change in the development of vector-borne diseases. mosquitoes and ticks do not survive well in dry conditions. therefore, weather impacts infectious pathogen distribution in complex ways that are not predictable by the forecast. extreme weather events can precipitate outbreaks of infection. an increase in the frequency and intensity of natural disasters like hurricanes and tsunamis, in relation to the el niño/southern oscillation phenomenon, may result in more flooded grasslands, which favor the breeding of aedes and culex mosquitoes [ ] , and impact water sanitation fostering outbreaks such as cholera. flooded areas can displace rodents leading to plague. tornados and other severe weather can stir up soil leading to infections with soil fungi leading to episodic outbreaks of invasive fungal disease such as mucormycosis such as apophysomyces trapeziformis [ ] . malaria is a common disease that can vary dramatically depending on weather, and extreme weather can alter the very landscape, providing new bodies of water to support larval development. if the melting of glaciers and the polar ice caps bring coastal cities underwater, or if overpopulation and waste cause drinkable water shortages in certain regions of the world, we can expect mass migrations. these could change the patterns of infection and drive outbreaks. migrants traversing tropical forests, or feeding with meat from game or carcasses, are but two scenarios that could be envisioned for the emergence of zoonotic infectious diseases [ ] . several predictive models have been developed to evaluate the impact of climate change on the emerging infectious diseases: climex, dymex, miasma models. nevertheless, it remains difficult to predict when and where pathogen behavior will deviate from its typical pattern. climate change primarily affects vector-borne diseases by increasing rates of reproduction and biting and by shortening the incubation period of the pathogen they carry. ticks have gained spread from the mediterranean basin to northern and eastern europe, as well as appearing at higher altitudes. increased survival, density, and activity have also been reported following shorter, milder winters. climate change has also resulted in more days of activity per year for mosquitoes. as temperatures rise, more parasites are viable within regions ranging from the mediterranean and tropical zones, up to the balkans, russia, scandinavia, and the uk. for some ticks and fleas, temperatures over °c with relative humidity of over % are optimal for their proliferation and activity throughout the year [ , , ] . for example, dengue fever is usually limited to a tropical latitude and a low altitude, since mosquito eggs and larvae lose viability with sustained low temperatures. during unusually warm summers, however, dengue has been reported as high up as m above sea level. warmer temperatures also result in smaller adult mosquitoes, which need to bite more frequently to feed themselves and be able to lay eggs, thus increasing the rate of transmission [ ] . in contrast, the incidence of malaria has followed mixed patterns of increase and decrease along recent decades, and computer models have failed to predict the spread. the explanation for this is, in part, that climate change also results in diminished survival of the vectors (warming over °c affects the survival of both parasites and vectors), and in part, that the effect of climate change is non-linear and complex [ ] . the frequency of emerging vector-borne infections varies per changes in land use, human activity, intervention maneuvers to eradicate the vector or prevent transmission to humans, drug treatment, and vaccines. both ecologic and economic changes may bring together rodents and humans. hunting activities may change vector distribution and large-scale animal movements can impact disease distribution. impoverishment of cities and overcrowding in slums, but also reforestation, golf club development, and the urbanization of rural suburbs facilitate exposure to ticks and rodents [ ] . many patients with pidd require immunoglobulin replacement. immunoglobulin products have been demonstrated to improve outcomes in hepatitis a and measles [ , ] . at least some neutralizing antibody is present directed to rsv and group b streptococci [ , ] . this raises three distinct issues for patients: ( ) difficulty in the diagnosis of infections in travelers because locally produced immunoglobulin may have antibody titers to local infections that are not typical for other countries, ( ) safety concerns about locally produced immunoglobulin, if the patient resides in the location long enough to require immunoglobulin from a local provider, ( ) the decision to use locally produced immunoglobulin products to provide superior prevention of infection compared to the patient's usual product. there are limited data on each of these subjects. serologic diagnostic testing in patients on immunoglobulin therapy will be addressed below in terms of issues related to lack of specific antibody produced by the patient (potentially) after infection. the converse can also be an issue. patients arriving from countries with significant occurrences of infections unusual in their current country may have igg antibodies to those infections simply through their immunoglobulin product and not reflecting a true infectious event in the patient. this can lead to diagnostic confusion when serologic testing demonstrates the presence of antibodies due to the infusion product. patients will often ask if immunoglobulin products from other countries are held to the same rigorous standards as their home country. today, commercially produced immunoglobulin is safe and tightly regulated. all commercially produced immunoglobulin around the world has one of the time-tested viral inactivation procedures such as nanofiltration, caprylate absorption, pasteurization, solvent/detergent, vapor heating, and low ph treatment. these procedures uniformly inactivate enveloped viruses. many emerging viruses are specifically tested for their ability to withstand the purification process. much has been learned since the transmission of hepatitis c viruses through immunoglobulin products in the early s [ ] . nevertheless, vigilance is important. in , counterfeit immunoglobulin was identified. therefore, patients should ensure that they receive only brand name products while traveling. the subject of whether a patient's interests would be best served by using a local immunoglobulin product, with antibodies to pathogens that are prevalent in the community, or have their home physician ship their usual product, for which the patient has a known tolerance is hotly debated. patients with a history of intolerance to immunoglobulin products should not switch unless necessary. however, there are compelling reasons to consider a locally produced product when patients are in a foreign country for an extended period. it is known that antibodies to west nile virus have tracked with the distribution of the virus as it has emerged in several areas [ , ] . titers in products using donors from the usa have higher neutralizing titers to west nile virus than those using donors from europe, although there is a -fold difference in titers between lots from the usa [ ] . similarly, protective antibodies to concerning pathogens may be optimal in locally produced immunoglobulin. it is critical to inquire where the plasma source is derived. in most countries, the utilized immunoglobulin is produced in europe or the usa. having a different name does not ensure that the plasma pool comes from a different country. most lots of immunoglobulin are produced from to , plasma donors. the infrastructure to support such an endeavor is not easy to establish in each geographic area. serologic testing is commonly used for the diagnosis of infection. this approach relies upon detection or quantitation of antibodies made by the host against specific pathogens. the presence of igm antibodies against a specific pathogen indicates recent infection, while igg antibodies against a specific pathogen indicate past infection. importantly, serologic testing can only be applied for the diagnosis of infection if the host can mount a specific antibody response to the pathogen. conversely, serology cannot be relied upon to diagnose infection in the setting of immune deficiency where there is impairment of the specific antibody response, such as in the case of primary antibody deficiencies, cellular immune deficiencies, combined immune deficiencies, and other secondary immune deficiencies affecting t and/or b cell function. in these situations, the causative pathogen must be identified by alternate means such as culture of the organism, antigen detection, or molecular approaches (nucleic acid hybridization, nucleic acid sequencing, or oligonucleotide probe arrays). molecular approaches are particularly relevant for the diagnosis of infection in patients with pidd. signal and target amplification techniques can be coupled with nucleic acid hybridization or probe assays to allow detection of pathogen dna or rna that is present in very small amounts in clinical samples. in patients with pidd, vaccines could play an important role in preventing infections with vaccine-preventable diseases. even pidd patients may generate some protective responses [ , ] . the decision to immunize such patients or not depends on the type and severity of pidd as well as the type of vaccine to be administered (live or inactivated) ( table ). in general, inactivated vaccines are safe for pidd patients while immunization with live attenuated vaccines is a known hazard to patients with serious immunodeficiencies of t cell, b cell, and phagocytic cell origin (table ). in less severe pidd, the vaccine can induce adequate protection as in healthy individuals or the efficacy may be reduced [ ] [ ] [ ] . of note, immunoglobulin replacement therapy induces passive immune protection to some vaccine-preventable infections, such as measles, mumps, rubella (mmr), and varicella. in addition, live viral vaccines have greatly reduced efficacy while on immunoglobulin replacement. therefore, vaccine administration in patients receiving regular immunoglobulin replacement treatment should be withheld until at least to months (depending on dose) after cessation of such treatment, if cessation and vaccination are safe. in addition, pidd patients who have received hematopoietic stem cell transplantation but have incomplete immune reconstitution or are under immunosuppression should not receive live attenuated vaccines. in general, the decision of administering live viral vaccines should be made by clinical immunology experts [ ] . in developing countries where polio is still endemic and oral polio vaccine is essential for eradicating the disease, it is of utmost importance that all pidd patients and family members should not receive live oral polio (opv) because of the reported prolonged excretion of the virus for months and even years [ ] . in addition, vaccine-associated paralytic polio is a real risk for some with pidd. these patients and family members should receive inactivated polio vaccine (ipv) instead of opv. similarly, the hazards of administering bacillus calmette-guerin (bcg) vaccine to pidd patients have been documented. in a series of bcg, vaccinated severe combined immunodeficiency (scid) patients from centers in countries, % of scid patients developed disseminated bcg infection and had the worst outcome [ ] . patients with chronic granulomatous disease vaccinated with bcg also developed local and disseminated bcg infection. recently, vaccine strains of rubella virus were found to be associated with skin granulomas in pidd patients [ ] [ ] [ ] . siblings and household contacts of patients with suspected or diagnosed pidds should receive all the national immunization scheduled vaccines. ipv should be substituted for op in families where an antibody-deficient patient exists. yearly influenza vaccination of family members is recommended in order to reduce the risk of household-social transmission [ , , ] . diseases where routine vaccination has reduced incidence can occasionally experience a resurgence in times of economic hardship with reduced attention to vaccination. diphtheria is currently seen in venezuela for this reason. war and disruption of health infrastructure are other common reasons for resurgence in vaccine-preventable diseases. in other settings, antivaccination sentiment has led to outbreaks of diphtheria, measles, and mumps. an additional consideration is that not all countries provide the same level of vaccination, and therefore, vaccine-preventable illness can still be seen regionally. these outbreaks represent a significant risk to patients with pidd. a universal consideration for patients with pidd is the concern about antibiotic resistance, which varies dramatically around the world. for certain high impact infections, the emerging antibiotic resistance patterns will be discussed below. antimicrobial resistance occurs naturally, but misuse and overuse of antimicrobials are accelerating this process. in nearly every country, antibiotics are overused and misused in people and animals leading to antibiotic resistance in every country. key resistance patterns to common bacteria include emergence of carbapenem-resistant klebsiella pneumoniae around the world with high frequency of resistance (due to different mechanisms) in the mediterranean, with greece, italy, and turkey having endemic spread of this pathogen [ ] . carbapenem-resistant strains among other genera of enterobacteriaceae have also been recognized. they are particularly common in greece, but have been found widely distributed [ ] . the new delhi metallo-beta-lactamasemediated resistance, which is endemic in the indian subcontinent but becoming increasingly spread worldwide, is a growing concern [ , ] . as a common cause of urinary tract infections, colistin is the only recourse when carbapenemresistant enterobacteriaceae, and colistin resistance has recently emerged in small outbreaks throughout the world [ ] . in these cases, the infection is essentially untreatable. fluoroquinolone-resistant escherichia coli, a common cause of urinary tract infections, now accounts for over half of the isolates in some asian countries [ , ] . t he emergence of resistance to antibiotics in grampositive pathogens has become a major international problem as there are fewer, or even sometimes no effective, antimicrobial agents available for infections caused by these bacteria. methicillin-resistant staphylococcus aureus is common in many countries and in fact has spawned a nomenclature recognized by the general public: mrsa. several studies have reported resistance to the newer antimicrobial agents like linezolid, vancomycin, teicoplanin, and daptomycin [ ] . thus far, these isolates appear to be uncommon and have been found in < % of isolates in brazil, china, ireland, and italy, with nearly undetectable rates elsewhere. vancomycinresistant enterococcus (vre) is growing in frequency and can now be a cause of primary bacteremia in immunocompromised individuals [ ] . a key message is that antibiotic resistance is increasing (generally) and travelers should be alerted to resistance to commonly encountered organisms, and if antibiotic prophylaxis is required, their prophylaxis is adjusted. neisseria gonorrhoeae is a specific organism for which resistance has become especially problematic. it has progressively developed resistance to virtually all antimicrobial agents since introduction of sulfonamides in mid- s. the current treatment guidelines recommend dual antimicrobial therapy (ceftriaxone - mg × plus azithromycin - g × ) as first-line regimen [ , ] . although dual therapy is very effective, development of concomitant ceftriaxone and azithromycin resistance is likely to emerge [ ] . the risk of untreatable n. gonorrhoeae demands better global antimicrobial surveillance system, clinical trials on combined therapy of existing drugs as well as novel agents in monotherapy, and development of a gonococcal vaccine. for pidd patients, guidance on barrier methods for the prevention of sexually transmitted diseases should be a part of any pre-travel counseling. mycobacteria tuberculosis (mtb) is an age-old pathogen with emerging resistance. drug-resistant tb, fueled by the hiv epidemic, is a global threat. in , who estimated , new cases of multidrug-resistant tb (mdr-tb) and an additional , new cases of rifampin-resistant tb (rr-tb) who would also require mdr-tb treatment. treatment of mdr-tb and mycobacterium bovis disease is beyond the scope of this text and reader is referred to recent who mdr treatment guidelines [ ] . regions of the world with > % mdr tb include regions of azerbaijan, kazakhstan, russia, uzbekistan, china, georgia, and eastern europe. extensively drug-resistant tb (xdr tb) refers to mtb resistant to isoniazid, rifampin, any fluoroquinolone and at least one second-line drug. xdr tb has been reported in countries. on average, % of patients with mdr tb have xdr tb. as is true for all types of mtb, xdr tb is contagious and small outbreaks related to person-person transmission have been reported. non-tuberculous mycobacteria (ntm) cause significant systemic infections in patients with defects of the il- / ifnγ/stat axis as well as in gata deficiency and can cause significant pulmonary infections in pidd patients with bronchiectasis. compared to tb, ntm is acquired from the environment and not from person-to-person transmission; therefore, acquisition of antibiotic-resistant strains is less common. however, in these individuals with pidd, ntm disease is often chronic and can be difficult to eradicate, and resistance can then easily develop during therapy. using combination of antibiotics is essential, and consultation with those familiar with treatment of treatment refractory ntm disease is recommended. aspergillus species are ubiquitous inhaled molds with worldwide distribution that cause opportunistic infections in immunocompromised patients [ ] . aspergillosis also occurs in pidds associated with quantitative and/or qualitative phagocyte defects; it develops most frequently in chronic granulomatous disease (cgd) patients (prevalence,~ %), while it is seen less often in patients with gata deficiency, card deficiency, and congenital neutropenia syndromes [ , ] . upon inhalation, aspergillus species cause invasive pulmonary disease in susceptible hosts with the exception of card deficiency, where aspergillosis has a predilection for extrapulmonary tissues with sparing of the lungs [ ] . diagnosis is established by fungal culture and/or histopathology showing acute-angle septate hyphae and/or detection of galactomannan, an aspergillus cell wall component released during invasive infection, in serum or bronchoalveolar lavage fluid [ ] . while azole-susceptible aspergillus fumigatus is still the most common infecting species in pidd patients, the emergence of azole-resistant a. fumigatus and nonfumigatus aspergillus species underscores the importance of a high index of suspicion in patients who do not respond to front-line voriconazole treatment [ ] . the advent of fungal molecular diagnostics has demonstrated that patients with pidds are more prone to infections by uncommon low-virulence aspergillus species with intrinsic resistance to azole antifungal agents that do not infect patients with iatrogenic immunosuppression. these primarily include aspergillus viridinutans, aspergillus tanneri, and neosartorya udagawae, which pose major diagnostic and therapeutic challenges due to their impaired sporulation and propensity for contiguous and distant tissue spread, respectively. in addition, acquired azole resistance in a. fumigatus can be seen in patients on long-term exposure to azole drugs used as treatment and/or prophylaxis [ ] . azole resistance in these strains is predominantly caused by point mutations in the lanosterol α-demethylase gene that encodes the cyp a protein, the primary target of azole drugs. importantly, infection by azole-resistant a. fumigatus strains without prior exposure of patients to azole antifungals has recently emerged as an important global health concern due to the widespread use of sterol demethylase inhibitor fungicides in agriculture that results in cross-resistance with the triazole antifungals used in clinical practice [ ] [ ] [ ] . fungicide-driven azole-resistant environmental aspergillus strains were first observed in the netherlands in and have since then been documented in other parts of europe, south and north america, the middle east, australia, africa, and asia. the prevalence of these azole-resistant aspergillus strains among clinical aspergillus strains recovered from patients in european countries was reported to be . %, while alarmingly in some areas > % of recovered strains exhibited azole resistance [ ] . the emergence of such aspergillus environmental strains poses serious threats to the treatment of immunosuppressed patients. mortality rates as high as % have been seen due to delays in diagnosis and suboptimal treatment with azole antifungals [ ] . although no prospective data exist for the treatment of patients with such resistant fungi, the use of amphotericin b-and echinocandin-based regimens are preferred over azoles [ ] . in the absence of azoles, the lack of alternative oral antifungal agents is particularly challenging for pidd patients such as those with cgd who require long-term suppressive antifungal treatment. candida species are commensal yeast fungi that colonize the mucosal surfaces of~ % of healthy individuals [ ] . when perturbations in immunity and/or microbiota occur, candida causes opportunistic mucosal or systemic infections that depend on clearly segregated immune responses for host defense. specifically, t cells of the th program are critical for mucosal and phagocytes for systemic immunity [ ] . indeed, a proportion of patients with cgd and complete myeloperoxidase deficiency develop systemic, but not mucosal candidiasis [ ] , whereas patients with monogenic syndromes of chronic mucocutaneous candidiasis due to mutations in the il- signaling pathway (il ra, il rc, il f, act ) or in other genes that adversely affect th differentiation (rorc, stat , stat , aire, dock , stk , irf ) do not develop systemic candidiasis. the only known pidd to date that results in combined mucosal and systemic candida infection susceptibility is card deficiency. systemic candidiasis in card -deficient patients has a predilection for the central nervous system, associated with brain-specific impaired recruitment and effector function of neutrophils [ ] [ ] [ ] . diagnosis of candida infections is established by culture. azole-susceptible candida albicans is still the most common infecting species in pidd patients; however, emergence of azole-resistant c. albicans is not uncommon during chronic azole use, making long-term therapy challenging due to lack of alternative oral antifungal treatment options [ ] . beyond c. albicans, non-albicans candida species can rarely infect pidd patients, some of which have intrinsic resistance to azole antifungals, including candida glabrata and candida krusei [ ] . most recently, candida auris has emerged as a global public health concern due to its resistance to antifungal drugs, high virulence potential, propensity for health careassociated horizontal transmission and outbreaks in health care settings, persistence in the human skin and hospital environment, inherent resilience to antiseptics, and misidentification by routine biochemical methods as other yeasts (most often candida haemulonii, but also candida famata, rhodotorula glutinis, or saccharomyces cerevisiae). c. auris was first recovered from the ear canal of a patient in japan in and has since then been reported to cause life-threatening infections and hospital outbreaks in europe, asia, africa, the middle east, and south and north america [ ] [ ] [ ] [ ] . most of the reported strains of c. auris have intrinsic resistance to fluconazole and other triazole antifungal agents, while a significant proportion of strains has elevated minimum inhibitory concentrations to amphotericin b and echinocandins, with some strains reportedly resistant to all three classes of azoles, polyenes, and echinocandins [ ] . avoidance of azole antifungals is important in c. auris-infected patients, and echinocandinor amphotericin b-based regimens are preferred, guided by strain-specific in vitro susceptibility patterns. this section on vector-borne infections is a major focus of this review because the infections are often more severe in immunocompromised individuals and because there are mitigation strategies that should be considered even in the absence of defined medical treatments for infection. prevention of mosquito bites depends somewhat on the endemic species but there are generalizations. the use of a mosquito repellant such as deet, oil of lemon eucalyptus, ir , or picaridin is as important as using long sleeves and long pants while in an affected area. deet and picaridin are safe in pregnancy, and some data support their greater efficacy [ ] . air conditioning and fans tend to keep mosquitoes away but netting at night is essential in mosquito-prone areas. light-colored clothing is less attractive to mosquitoes than dark clothing, and scented detergents and use of dryer sheets tend to attract mosquitoes, hence should be avoided. aedes species prefer to bite indoors and thrive in urban areas with small puddles of water. they bite most frequently around dawn and dusk. anopheles species have very similar behaviors. culex mosquitoes, in contrast, bite primarily at night. tick and fly bite prevention is focused on physical and chemical prevention. for ticks, physical inspection for biting ticks should also be incorporated. arthropod-borne viruses (arboviruses) are transmitted to humans through the bites of infected insects: mosquitoes, ticks, sand flies, or midges. some arboviruses can be transmitted through blood transfusion, organ transplantation, perinatal transmission, consumption of unpasteurized dairy products, or breastfeeding. there are > arboviruses causing human disease. most arboviral infections are asymptomatic. infectious manifestations range from mild febrile illness to severe encephalitis. arboviral infections are often categorized into two primary groups: neuroinvasive disease and non-neuroinvasive disease. tables and list the encephalitigenic viruses and the febrile/hemorrhagic disease causing viruses. in this section, we will highlight west nile virus, the most common of the encephalitogenic arboviruses. west nile virus is a single-stranded mosquito-borne rna virus of the family flaviviridae. the natural transmission cycle of the virus occurs in culex mosquitoes and birds. humans and horses are dead-end hosts for the virus. the most common mode of transmission to humans is through the bite of infected mosquitoes. other less common modes of transmission include blood transfusions, organ transplantations, occupational exposure in laboratories and mother-to-child transmission during pregnancy, childbirth, and breastfeeding. west nile virus has been diagnosed in > people in the usa with slightly more than half having neuroinvasive disease. since , > , people in the usa have been infected. it is also common in africa, europe, and asia [ ] . infection with west nile virus is asymptomatic in most individuals [ , ] . the incubation period lasts for to days. however, it can be significantly longer in immunocompromised hosts. clinical manifestations following infection develop in - % of those infected and include fever, headache, myalgia, arthralgia, vomiting, and rash. severe neuroinvasive disease leading to meningitis, encephalitis, and flaccid paralysis develops in less than % of infected individuals. the overall case fatality is approximately % which is a disproportionately high mortality in patients with encephalitis and myelitis. diagnosis of west nile virus rests on demonstration of specific antibody responses especially specific igm antibodies in the serum or csf of infected individuals by enzyme immunoassays. plaque reduction neutralization tests can differentiate cross-reactive antibodies. detection of virus in csf, blood, or tissue specimens by culture or pcr is particularly useful in immunosuppressed individuals who may have impaired serological responses. west nile virus has been reported in the context of both primary and secondary immunodeficiency. severe neurological manifestations have been reported in hiv-positive individuals, individuals receiving immunosuppressive therapy including rituximab, and individuals with pidd. infection with wnv has been reported in individuals with common variable immunodeficiency, idiopathic cd lymphopenia, gata deficiency, and a case of probable good's syndrome [ ] [ ] [ ] . individuals with antibody defects, neutropenias, and impaired t cell responses are potentially at an increased risk of severe manifestations of wnv disease including severe neurological involvement. this section highlights the four important non-neuroinvasive arboviruses based on current geographical distribution: dengue, yellow fever, zika, and chikungunya (table ) . approximately countries/territories have reported local transmission for both chikungunya and dengue viruses [ ] . dengue is due to infection with one of four dengue virus serotypes, transmitted by a mosquito (typically aedes aegypti). this febrile illness affects all ages with symptoms appearing - days after the infective bite. symptoms range from mild to high fever, with severe headache, musculoskeletal pain, and rash. severe dengue (also known as dengue hemorrhagic fever) occurs in . - % of cases and is characterized by fever, abdominal pain, persistent vomiting, bleeding, and breathing difficulty and is a potentially lethal complication [ ] . paradoxically, the main risk factor for dengue hemorrhagic fever is pre-existing antibodies. early clinical diagnosis and comprehensive management by experienced clinicians increase survival. dengue is ubiquitous throughout the tropics with the highest infection rates in the americas and asia. dengue is now endemic in countries, causing up to million infections a year and , deaths, mainly among children. over half of the world's population inhabits areas at risk for dengue infection [ ] . the presence of a. aegypti in over countries potentially puts almost the whole world at risk of becoming infected with this virus. pcr is widely used as serologic methods to diagnose dengue. immunity to one serotype does not confer protection against the other three serotypes, and heterologous antibody may be a risk factor for hemorrhagic dengue [ ] . the natural history of dengue has been studied in hiv patients where hiv did not appear to increase severity. there have been no reports of patients with pidd having dengue; however, dengue infection after solid transplantation has been reported [ ] [ ] [ ] [ ] with some patients having severe complications suggesting that t cell compromise in pidd could be a risk for severe disease. there are no antiviral medications utilized for dengue virus. care of patients with hemorrhagic disease requires meticulous approach to fluids and coagulation status. one dengue vaccine has been registered in several countries (cyd-tdv) for individuals from to (or ) years old. it is a live attenuated recombinant tetravalent vaccine with backbone of the attenuated yellow fever d virus genome with the prm and e genes that encode the proteins from the four wild-type dengue viruses. the who has suggested its use in regions where seroprevalence of dengue virus of any serotype is % or greater, but has not recommended it to hiv-infected, immunocompromised individuals, nor pregnant or lactating women [ ] . most people infected with the yellow fever virus have no illness. symptoms of yellow fever include sudden onset of fever, chills, headache, musculoskeletal pain, nausea, vomiting, fatigue, and weakness. the incubation period is typically - days, and symptoms may appear after return from travel. most people improve after the initial presentation, but % of cases progress to develop a more severe form of the disease, usually after a day of presumed recovery. the severe form is characterized by high fever, jaundice, bleeding, and eventually shock and failure of multiple organs [ ] . yellow fever virus is an rna virus that belongs to the genus flavivirus. it is transmitted from mosquitoes after biting an infected primate. it is widely distributed in the equatorial tropics [ ] . aedes species of mosquitoes are primarily responsible for transmission. large epidemics of yellow fever occur when the infection enters heavily populated areas with a high mosquito density and where most people have little or no immunity. west africa has undergone a large-scale vaccination campaign with impressive results and yellow fever is now uncommon in west africa [ ] . serologic testing for yellow fever is the diagnostic standard. pcr can be performed on tissue samples. there are no published studies of yellow fever in immunocompromised people, but the elderly, very young, people with autoimmune disease, or who are post-thymectomy are at risk from the attenuated vaccine strain. thus, it seems likely that any immunodeficiency would be associated with more severe wildtype disease. currently, no specific antiviral drug for yellow fever exists. treatment of dehydration, liver and kidney failure, and fever improves outcomes. the yellow fever vaccine is highly effective; however, immunodeficient patients should not receive it. infection with zika virus is often asymptomatic. it represents a mild infection for those who have any symptoms [ ] . the zika virus has been detected in urine, semen, and saliva of infected individuals, and transmission from transfusion and sexual relations has been reported. it is also detectable in breast milk, but breastfeeding-associated transmission has not been reported so far [ ] [ ] [ ] . contact with highly infectious body fluids from patients with severe zika infection has also been suggested as a possible mode of transmission [ ] . of tremendous importance is the presence of prolonged shedding of zika virus in a congenitally infected newborn [ ] . the main public health risk of zika virus is microcephaly in newborns from infected mothers [ ] . zika virus is capable of infecting human neural progenitor cells in vitro. infection results in disruption of cell cycle, increased cell death, and attenuated neuron growth [ ] . zika is not thought to be a major risk for people with pidd (based on the experience with hiv patients, but our recognition of zika is very recent. there is no known specific treatment for zika; however, there is an important effort to develop a vaccine. chikungunya fever is an acute febrile illness caused by the alphavirus, chikungunya virus. the incubation period is usually - days after the bite of an infected aedes mosquito. there is abrupt onset of high fever, and the fevers can be biphasic [ , ] . severe polyarthraligias develop after the onset of fever. the joint pains can affect any joint, but the pattern is usually symmetric and a true acute arthritis is not uncommon. the proportion of infected people with rash has varied across studies. when seen, the rash appears after the fever as a truncal maculopapular type of rash [ ] . cervical adenopathy is another common feature of infection. death is uncommon in chikungunya, but serious complications such as myocarditis have been seen. over half of the patients report continued joint symptoms year after acute illness and % have long-term joint symptoms [ ] . chikungunya originated in central/east africa. in forests, the virus circulates in aedes mosquitoes and non-human primates. in urban centers, the virus circulates between humans and mosquitoes similar to the pattern of dengue. there have been periodic urban outbreaks in asia and africa since with an acceleration in spread since [ ] . an important consideration is the periodic outbreaks with high attack rates in naïve populations. areas at risk currently are east africa, central africa, la reunion, india, and southeast asia. diagnostic testing utilizes pcr or serology. the threat to immunodeficient patients is not entirely clear. there are a few provocative cases where the immunocompromised appears to have been associated with fewer joint symptoms, but there were two patients, medically immune compromised, who had very severe disease [ , ] . this suggests that the presentation may be atypical and the course may be severe in immunodeficient people. treatment is supportive, although chloroquine, acyclovir, ribavirin, interferon-ɑ, and steroids have limited preclinical data to support clinical trials. babesia microti (the main species in the usa) infection can be asymptomatic, but many people develop fever, chills, headache, myalgias, anorexia, nausea, or fatigue [ ] . babesiosis often causes hemolytic anemia. b. microti is spread by ixodes scapularis ticks in the usa and babesia divergens (the main species in europe) is spread by ixodes ricinus. symptoms begin - weeks after a bite from an infected tick with b. divergens having a higher mortality rate and greater symptomatology compared to b. microti. the main geographic areas involved are the coastal eastern usa and cattle breeding areas throughout europe. the diagnosis is usually by inspection of a blood smear or through serology. a pcr test has just been developed. immunodeficiency, asplenia, and older age are recognized risk factors for severe disease and even death [ ] [ ] [ ] . thus, congenital asplenia would be considered a major risk for severe disease. a combination of atovaquone and azithromycin is generally used for therapy, although clindamycin and quinine have been used with success. patients with severe illness have been treated with exchange transfusions. five different types of plasmodium (plasmodium falciparum, plasmodium vivax, plasmodium ovale, plasmodium malariae, and plasmodium knowelsi) infect humans. malaria is transmitted primarily by female anopheles mosquitoes. symptoms vary depending on the type of plasmodium involved but usually include high fever, chills, and headache. in some cases, the illness can progress to severe anemia, kidney and respiratory failure, and death. the incubation period typically ranges from to days for p. falciparum, to days for p. vivax and p. ovale, and to days for p. malariae. in p. vivax and p. ovale infections, relapses can occur months or even years without symptoms. p. vivax and p. ovale have dormant liver stage parasites that must be specifically eradicated through medical therapy. malaria has been a global health concern throughout history and is a leading cause of death and disease across many tropical and subtropical countries. over the last years, new control measures have reduced malaria by over half [ ] . the democratic republic of the congo and nigeria account for over % of the estimated total of malaria deaths globally. high rates of malaria are seen in india as well. nevertheless, malaria exists in most tropical regions of the americas, africa, and asia [ ] . the diagnosis of malaria depends on the demonstration of parasites in the blood, usually by microscopy. the threat to immunodeficient patients is not entirely clear, but patients with hiv seem to have no additional burden of disease other than an increase in placental malaria, suggesting that t cells are not central to the defense of malaria [ , ] . asplenia is a known risk factor for severe malaria [ ] . antibodies appear to be both protective and pathologic [ , ] . treatment and prophylaxis depend on the region of the world because the parasites and resistance are highly variable and highly dynamic. therefore, it is best to consult an infectious disease specialist familiar with the prophylaxis before travel and for treatment of acute cases. leishmaniasis is due to infection with an obligate macrophage intracellular protozoa of the genus leishmania. it causes a spectrum of disease ranging from a cutaneous ulcer to mucosal disease and the most severe form, visceral leishmaniasis (vl). the liver, spleen, and bone marrow are major sites of parasite growth and disease pathology in vl [ ] . purely cutaneous leishmaniasis is most often caused by leishmania major, leishmania. tropica, leishmania aethiopica, leishmania infantum, and parasites belonging to the leishmania mexicana complex, the leishmania braziliensis complex, and the leishmania guyanensis complex. mucocutaneous disease is most often due to l. braziliensis complex, leishmania panamensis, leishmania amazonensis, and rarely by leishmania guyanensis. vl is most often caused by leishmania donovani and leishmania infantum (previously l. chagasi) [ ] . cutaneous leishmaniasis can have many variations but is most often an ulcer that develops after an indolent papule. the incubation period ranges from weeks to months. the ulcer usually heals within months to years, and there can be mild adenopathy. mucocutaneous leishmaniasis follows a cutaneous ulcer and is only caused by l. braziliensis parasites. oral and respiratory mucosa are most often involved with granulomatous lesions that may be extremely destructive. vl is associated with fever, lymphadenopathy, hepatosplenomegaly, wasting, hypoalbuminemia, and pancytopenia. this picture evolves over months to years. there can be secondary immune deficiency due to the pancytopenia. the epidemiology has changed dramatically and has been impacted by climate change [ ] . the sand flies that spread the parasite are affected by temperature and rainfall. in most endemic regions, leishmania has a patchy distribution due to micro-ecologic factors. poverty has been demonstrated to be a major risk factor for leishmaniasis [ ] . it has been estimated that up to half a million new cases of vl occur every year, but the majority are in resource-poor countries such as bangladesh, nepal, india, sudan, ethiopia, and brazil. emergence of resistance to antimony-based drugs has also led to a major resurgence of disease. the primary reservoir for leishmania is forest rodents, but dogs are increasingly important. the growing spread of leishmania is due to a combination of factors, and now countries have reported cases. immunodeficient patients are more susceptible to infection, and relapse occurs more frequently [ ] . the risk of developing vl is estimated to be between and times higher in hiv-infected than in non-hiv-infected individuals [ ] , and these patients have higher rates of treatment failure with the illness often taking a prolonged chronic course and higher mortality rates [ ] . a similar picture has been seen in patients with vl-infected post-kidney transplantation [ ] . dendritic cells, t cells, and the generation of reactive oxygen species have been shown to be essential for parasite control [ ] [ ] [ ] . pidd with impaired il- production have been associated with severe disease [ ] . a patient with cd l deficiency, associated with poor il- production, had chronic leishmania and died in spite of aggressive treatment. vl has been reported in cgd patients [ ] . six cgd patients were infected by leishmania, and they developed hemophagocytic syndrome with a poor outcome for one of them [ ] . the diagnosis of leishmaniasis is usually by visual inspection for parasites. immunofluorescence microscopy, direct agglutination, skin test, and pcr have been used. treatment is long-term and difficult. emerging resistance to first-line treatment is increasingly problematic. pentavalent antimonials are the mainstay of treatment in most countries, but liposomal amphotericin is widely used where resistance occurs. newer drugs with more favorable side effect profiles have been used in certain geographic settings: miltefosine, paromycin, and sitamaquine. rickettsiae are small gram-negative bacteria. they are obligate intracellular parasites, and the primary target in humans appears to be endothelial cells with subsequent thrombosis and clinical presentation of vasculitis [ ] . the rickettsiaceae family, originally defined by non-specific phenotypic characteristics, was reclassified into different strains and subspecies based on gene sequencing and genetic phylogeny ( table ). the clinical presentation of rickettsial disease can vary, but the classic triad of fever, rash, and headache still provides major clues for the diagnosis [ ] [ ] [ ] . however, rash is not an obligatory sign, and the incidence of rash can range between % for rickettsia conorii infection,~ % for rickettsia rickettsii, % for rickettsia africae, and less than % in the case of anaplasma phagocytophilum infection. therefore, fever in patients with exposure to a potential vector should raise a concern for a rickettsial disease, especially if there is also evidence of rash, inoculation eschar, or localized lymphadenopathy. additional supporting laboratory findings can include neutropenia, thrombocytopenia, and increase in liver transaminases. the geographic distributions of rickettsioses and ehrlichioses are mostly dependent on their vector distribution [ ] . as such, louse-borne and flea-borne are worldwide, reflecting the worldwide distribution of lice and fleas, with a tendency to parasite poor people in cold places and, characteristically, during wars. ticks, on the other hand, depend on their environment and most do not have a worldwide distribution. with the exception of the dog tick, vector for r. conorii in asia and north africa, r. rickettsii in the usa, rickettsia massiiae and erhlichia canis worldwide, most other tick-borne diseases are restricted to areas of the world correlating with the distribution of their vector [ ] . for that reason, it should be anticipated that climate and environmental changes will affect vector distribution and its reservoir host and, hence, the geography and epidemiology of tick-borne diseases [ , , ] . diagnosis presents a challenge, as it is extremely difficult to grow these organisms in culture. immunohistochemistry and pcr can be helpful. the severity of rickettsial disease varies with the causative agent and the host. r. rickettsii, rickettsia prowazekii, and orienta tsutsugamushi are considered most pathogenic. as for host factors, although severe and fatal cases have been described in healthy immunocompetent hosts [ , ] , there is evidence to suggest that children under the age of [ ] and immunocompromised hosts either secondary to hematologic malignancies, immunosuppressant treatment for organ transplantation, or hiv infection are at a greater risk to develop more severe disease with higher case fatality rates [ , ] . all rickettsiaceae are intracellular pathogens, and one could expect an increased risk for severe disease in pidds with abnormal t cell function. five to days of doxycycline is the preferred treatment for non-pregnant adults and children. treatment should not be delayed while awaiting diagnostic testing [ ] and can be given to children despite a minimal risk for dental staining. alternative treatments include azithromycin for mild disease [ ] and chloramphenicol for pregnant women. anaplasma is an intracellular bacterium that infects wild and domestic mammals, including man. a. phagocytophilum was formerly known as human granulocytotropic ehrlighiosis but is now known as human granulocytotropic anaplasmosis [ ] . e. chaffeensis infects monocytes and causes human monocytic ehrlichiosis [ ] . anaplasma and ehrlichia have historically cycled within non-human enzootic hosts, and man has become infected through increasing interactions with the environment. ehrlichia and anaplasma are transmitted by ixodes species of ticks, and their ranges include the eastern usa, south central usa, and scattered regions of europe, as far north as sweden. these infections have not been seen in humans in the southern hemisphere, but there are reports of organisms being identified [ ] . a less common mode of transmission is through transfusions. the symptoms of ehrlichia and anaplasma infections are similar [ ] . abrupt onset of an influenza-like illness occurs about days after a tick bite. ehrlichia can cause a mild rash ( % of adults and % of children), but rash is uncommon in anaplasma infections. highly suggestive laboratory features are leukopenia and thrombocytopenia. mortality in the general populations appears to be < %, but icu admission is not uncommon. hemophagocytosis has been described with anaplasma [ ] and ehrlichia [ ] . both infections are more severe in any setting of immune compromised, including asplenia [ , ] . the diagnosis is typically made by pcr, and doxycycline is the recommended treatment. intracellular inclusions can be seen on cbc smears (more often in anaplasma than ehrlichia). an uncommon but well described facet of these infections is that the tick vector can also transmit borrelia burgdorferi and babesia microti, and simultaneous infection with multiple organisms can occur. c. burnetii is a highly pleomorphic gram-negative coccobacillus and the causative agent of q fever. q fever is a zoonosis, and the most common reservoirs are cattle, sheep, and goats but many other animals can be infected by c. burnetii [ , ] . when infected, these domestic animals can shed the organism in urine, feces, milk, and especially birth products. the pathogen survives within the phagolysosome of host cells, and a spore stage has been described. this spore stage explains the ability of c. burnetii to survive in unfavorable environmental conditions, and it can be an environmental risk for months to years after shedding from an infected animal. q fever is considered an occupational disease affecting people with direct contact with infected animals; however, indirect contact through exposure to contaminated animal products has also been described to cause disease outbreaks. humans are infected by inhalation of contaminated aerosols. following an average incubation period of days, infected patients can present with severe headache, fever, chills, fatigue, and myalgia. other signs and symptoms depend on the organs involved. in contrast to rickettsial diseases described above, rash rarely occurs in the early stages of the disease. c. burnetii can cause a range of clinical symptoms. a self-limited febrile illness is probably the most common form of q fever. pneumonia, either atypical or severe, is also common and can be a part of acute q fever syndrome. in contrast, a variety of manifestations can be recognized in chronic q fever, including endocarditis, endovascular infection, osteomyelitis, hepatitis, interstitial pulmonary fibrosis, prolonged fever, and purpuric vasculitic rash. q fever diagnosis is based on serologic testing with indirect immunofluorescence being the best for differentiating between acute and chronic q fever (high antiphase i antigen titer). the treatment of choice for acute q fever is doxycycline, with co-trimoxazole, chloramphenicol, or rifampin being an accepted alternative. there is no agreement on the treatment for q fever endocarditis, and a combination of doxycycline with either fluoroquinolone or hydroxychloroquine is recommended. there is also controversy regarding the duration of treatment, ranging from years to indefinite treatment. old evidence suggests that q fever is more common in immunocompromised patients. a french study showed higher incidence of antibodies to c. burnetii in hiv positive compared to hiv-negative patients ( . vs . %). in addition, out of hospitalized patients were hiv positive ( . %), suggesting a more frequent symptomatic disease [ ] . a smaller similar study performed in central africa failed to show increased incidence of seropositivity in hivpositive patients [ ] . two case reports describe severe disease in immunocompromised patients. the first was a case of fatal q fever disease in an -year-old male with cgd [ ] . the patient was treated with broad spectrum antibiotics, but without coverage for q fever. the second case was a -yearold asplenic male who presented with fever, jaundice, and encephalopathy and was diagnosed with acute q fever [ ] . the patient was successfully treated, but the two case reports could suggest susceptibility in cases of phagocytic disorders. the bartonellaceae are fastidious, facultative intracellular gram-negative bacilli (table ). most species infect primarily non-human animals, and in most cases, human are considered incidental hosts. the documented common human pathogens include bartonella bacilliformis, bartonella henselae, and bartonella quintana, and it is believed that humans are the primary mammalian reservoir of b. quintana. infection occurs through inoculation of bartonella-infected arthropod feces into breaks in the skin. the epidemiology of bartonella infection in humans follows the distribution of the vector. as such, infection with b. bacilliformis follows the distribution of the sand fly vector (lutzomya) and is confined to the andes mountain in peru, ecuador, and colombia at heights of between and m. the human body louse pediculus humanus is the vector of b. quintana, which explains the global distribution of this pathogen and worldwide outbreaks of trench fever, mostly in conditions of poor sanitation and upon exposure to body lice. trench fever was responsible for over a million infections during world war i. fever, either abrupt or indolent in onset, with a maculopapular rash, conjunctivitis, headache, myalgias (most often affecting legs), and splenomegaly was described. urban b. quintana infections occur most often among homeless and have a distinct clinical picture with fever as the most common manifestation. endocarditis occurs in many [ ] . bartonella henselae is globally endemic, and domestic cats are a major reservoir. the major arthropod vector of b. henselae is the cat flea, which is responsible for cat-to-cat transmission. human infection, called cat scratch disease, is assumed to involve inoculation of bartonella-infected flea feces into the skin during a cat scratch. b. henselae causes primarily adenopathy and neurologic symptoms [ ] . b. bacilliformis causes a condition with two phases: the acute phase with fever, anemia, and transient immunosuppression followed by nodular dermal eruption [ ] . recently appreciated are the ongoing systemic features during the eruptive phase such as arthralgias, adenopathy, and anorexia [ ] . diagnosis of bartonella-associated diseases can be achieved by direct examination of clinical material, bacteriologic culture methods, serologic and immunocytochemical studies, pcr-based assay, or combination of these methods. bartonella infection can present differently in immunocompromised hosts [ ] . in addition to higher prevalence of bartonella infection in hiv patients [ ] , both b. quintana and b. henselae can induce neovascular proliferation which might involve the skin, lymph nodes, and a variety of internal organs including the liver, spleen, bone, brain, lung, bowels, etc. these neovascular lesions, known as bacillary angiomatosis/peliosis (ba/bp), were initially described in hiv-infected patients with advanced disease and was later described in other immunocompromised hosts secondary to immunosuppressant treatment for solid organ transplantation or hematologic malignancy [ ] [ ] [ ] [ ] . cutaneous ba lesions can vary in presentation and can be subcutaneous, dermal nodules, single or multiple papule that can be flesh colored, red, or purple. lesions may ulcerate and bleed. they can change in number and size (millimeters to centimeters; few to hundreds) and can involve mucosal surfaces or deeper soft tissues. similar variation can be seen with visceral involvement. histologically, lesions consist of lobular proliferation of small blood vessels and neutrophilic predominant cell infiltration. the term bacilliary peliosis is used to describe bloodfilled cystic spaces mostly involving the liver, spleen, and lymph node. pathogenic bacteria can be isolated from vascular lesions. while both pathogens were associated with cutaneous lesions, only b. henselae was associated with visceral bp [ ] . based on hiv literature, it is reasonable to expect an unusual presentation of bartonella infection especially in pidds involving t cell dysfunction. bartonella infection was described as a cause for hepatitis in a single cd l deficiency patient [ ] . in addition, since cases of granulomatous disease due to bartonella infection [ ] [ ] [ ] have been described, it should be considered in the differential diagnosis of pidd with granulomatous inflammation. borrelia spp. the genus borrelia belongs to the spirochaetaceae family. it includes b. burgdorferi which causes lyme disease and species that cause relapsing fever. the latter are further divided into tick-borne species and louse-borne species. louse-borne relapsing fever (lbrf) is caused only by borrelia recurrentis and is spread by human body louse. the disease was epidemic in the early twentieth century, and it is estimated that more than , died of lbrf during world war ii. with sanitation improvement lbrf is now found only in the horn of africa and among homeless people in europe. more recently, cases of lbrf in refugees and migrants were described [ , ] . tick-borne relapsing fever (tbrf) is caused by a group of pathogens which are maintained by and survive in softticks (orinthodoros genus). each tbrf borrelia species depends on one specific species of soft-body tick. except for australia and antarctica, tbrf species can be found in all continents. the animal reservoir includes small animals and rodents. since the spirochetes persist in the tick salivary gland, disease transmission occurs when humans intrude the tick's environment. tick bites are painless, and history of a tick bite is often missing. therefore, a history of potential exposure can be valuable. the major clinical symptom is relapsing fever. after a median incubation period of days, patients present with febrile episode that can last - days, followed by afebrile period of - days. patients with tbrf can have up to febrile relapses, while lbrf is usually associated with only one relapse. other symptoms include myalgia, arthralgia headaches, and vomiting, and physical findings include lymphadenopathy and splenomegaly with rash occurring only in third of the patients. a range of neurologic complications as well as systemic inflammatory response syndrome also have been described [ ] . diagnosis is based on identifying the spirochetes on blood smear. sensitivity of blood smear is higher during febrile period (about %), and a negative blood smear does not exclude rf. in lbrf, the spirochete load can be low and specific stains can be helpful. other diagnostic methods include serologic testing, pcr, and mouse inoculation. doxycycline, tetracycline, and penicillin are the preferred treatment, with most patients treated with - days of doxycycline. jarisch-herxheimer reactions with high fever and leukopenia occur in half of the patients following the first antibiotic dose and can develop into a severe reaction with hypotension, respiratory distress, and death [ ] . without treatment, tbrf carries a mortality rate of up to % with even higher % mortality rate for untreated lbrf. two cases of meningoencephalitis with borrelia miyamotoi in heavily treated immunocompromised patients have been described [ , ] . lyme borreliosis is the most common vector-borne disease in the northern hemisphere caused by a group of at least genospecies. lyme disease is a multisystem illness affecting the skin, joints, nervous system, and heart. human infection is caused mainly by three species: b. burgdorferi is the most common cause in north america but also found in europe, and borrelia afzelii and borrelia garinii which cause the disease in europe and asia. emerging infections in the mid-western usa with borrelia mayonii cause a condition similar to lyme disease. most tick species do not carry borrelia species. the vectors of all borrelia species are the ixodid tick species; this includes the deer tick, i. scapularis, in the northeast and midwest of the usa, ixodes pacificus in the west, the sheep tick, ixodes ricinus, in europe and the taiga tick, ixodes persulcatus, in asia. the ixodid tick demonstrates a complex vector ecology with preferences for different hosts in different geographical regions and at different stages of its development. more than different species, including deer, rodents, birds, and reptiles, have been described. infection rates also show seasonal variation with highest rates during lyme disease follows several stages starting with localized disease at the site of inoculation, followed by dissemination stage and, later, persistent infection [ ] . however, an individual patient can show highly variable disease progression with different patterns of organ involvement and disease severity. erythema migrans (em) is often seen at the site of the tick bite after - days of incubation. regional lymphadenopathy can be seen. secondary skin lesions represent hematogenous dissemination. at this stage, constitutional symptoms of general fatigue, fever and headaches, migratory musculoskeletal pain, conjunctivitis, and cardiac involvement occur. in total, % of untreated patients can develop frank neurologic manifestations of meningitis, encephalitis, and variable forms of neuritis with fluctuating symptoms. persistence can occur in untreated (on inadequately) patients. antibiotic refractory arthritis is well described. however, even without treatment, intermittent or persistent attacks usually resolve completely within several years. co-infection with a. phagocytophilum and b. microti can cause diagnostic confusion [ , ] . the diagnosis of lyme disease is established based on clinical symptoms, history of potential exposure, and serologic studies. although positive culture can confirm the diagnosis, it can usually be obtained only from early em lesions. pcr testing is superior to cultures and can be performed on joint fluid samples [ ] . cdc recommendations for the diagnosis of lyme disease are based on serology which might be impossible in pidd patients with abnormal humoral response. cdc guidelines require both an elisa (or comparable test) to be positive and a western blot ( out of bands ( , , or kd) on the igm or out of bands on the igg ( , , , , , , , , , kd) . most lyme manifestations can be treated with oral antibiotics, while patients with neurologic abnormalities and some patient with lyme arthritis require intravenous therapy [ ] . doxycycline is the treatment of choice for early and disseminated disease, with amoxicillin as the second-line choice. jarisch-herxheimer-like reactions can appear in the first h in about % of the patients. for patients with clear neurologic symptoms, - weeks of iv ceftriaxone is the most commonly used therapy. few cases of neuroborreliosis and hiv have been described with a good response to treatment. descriptions of lyme disease in pidd patients are lacking. zoonoses are infectious diseases that pass between animals and humans and span the spectrum of pathogens including viruses, bacteria, fungi, and parasites. zoonoses are very common and range from mild such as certain forms of tinea to lifethreatening infections such as rabies. some of the zoonoses that are vector-borne will be covered in other sections. risk mitigation strategies for zoonoses include patient education, proactive advice about risk scenarios, and avoidance of infected animals. several zoonoses are associated with contact with mammals such as rodents or domestic farm animals through direct contact or through contact with their feces. for instance, hantavirus infections are often associated with exposures to mouse droppings when staying in cabins in the western usa. occupational exposures can occur with buffalopox or parapoxvirus (causing orf infection) through direct contact with buffalo and goats/sheep, respectively [ ] [ ] [ ] [ ] . in general, there are very few cases of pidd with zoonotic infections acquired from mammals. however, there are a few special considerations. for instance, lymphocytic choriomeningitis virus is acquired through exposure to house mice primarily, with hamsters being a less common source of infection. both domestic and wild mice can carry the infection without exhibiting symptoms. although infection is rare, there have been severe cases in patients with t/ nk cell dysfunction, such as a case in xlp and cases in solid organ transplant recipients [ ] . therefore, in patients with severe t/nk defects, consideration should be given to whether small rodents are appropriate household pets. tularemia is a disease of animals and humans caused by the bacterium francisella tularensis. rabbits, hares, and rodents are the main reservoirs. humans become infected through direct contact, ingestion of contaminated water, or inhalation of organisms. ticks and deer flies can also transmit the disease through bites. fever is universal, but other features depend on the mode of transmission. a patient with cgd had a complex course suggesting myeloid defects are a risk for more severe disease [ ] . rabies is an almost universally fatal infection caused by contact with oral secretions from infected mammals, typically raccoons, bats, or foxes, and there is no suggestion that pidd or immune compromised modifies the prognosis. for individuals with high-risk exposures, such as those working with wildlife or traveling in endemic areas, pre-exposure prophylaxis is given with vaccination, and if an exposure occurs, rabiesspecific immunoglobulin is provided as well as vaccination. however, for those with humoral immunodeficiencies who cannot respond to the typical pre-exposure vaccination, there needs to be counsel on the additional risk without vaccination. in table , several of the bacterial and viral zoonoses are summarized with their typical endemic areas, which is somewhat limited by diagnostic abilities and reporting, as well as the typical clinical scenarios, known cases in immunodeficiency and an approach to diagnosis and therapy. nipah virus causes a range of infectious phenotypes ranging from asymptomatic infection to acute respiratory distress and encephalitis. nipah virus was identified in on pig farms in malaysia, leading to identification of human cases, including human deaths and the culling of one million pigs [ ] . the natural host is the fruit bat: pteropodidae pteropus. symptoms of infection from the malaysian outbreak were primarily encephalitic in humans, but later, outbreaks have caused respiratory illness, increasing the likelihood of human-to-human transmission. fever, headache, cough, abdominal pain, nausea, vomiting, weakness, problems with swallowing, and blurred vision were common. seizures were seen in % and coma in %. relapses of encephalitis have been described [ ] . increasing infections due to nipah virus is thought to be due to an increasing overlap between bat habitats and pig sties in malaysia. all outbreaks thus far have been in india, bangladesh, or malaysia. the diagnosis of nipah virus relies on pcr of fluid samples, serology in convalescent samples, and immunofluorescence of tissue. there have been no infections of immune compromised patients reported. therapy is largely supportive, although preliminary reports of ribavirin use have been encouraging. a vaccine is under development. severe acute respiratory syndrome coronavirus (sars-cov) and the middle east respiratory syndrome coronavirus (mers-cov) are two zoonotic coronaviruses. the sars pandemic in - resulted in reported cases in countries. no further sars cases were reported after the pandemic except isolated cases linked to laboratory accidents. patients usually presented with fever and respiratory symptoms, but occasionally had diarrhea and vomiting. about - % of sars patients required mechanical ventilation, with a case fatality rate of about % [ ] [ ] [ ] . mers was first noted in saudi arabia in , and countries around the arabian peninsula are now endemic for mers-cov. patients usually present with fever, cough, chills, sore throat, myalgia, and arthralgia rapidly progressing to pneumonia with over % of patients requiring intensive care. about one-third of patients present with diarrhea and vomiting, and acute renal impairment is a striking feature of mers. risk factors for poor outcome include diabetes, hypertension, and renal and lung disease. cases have been exported to at least countries with travel occasionally causing cluster of secondary outbreaks. one such example is the mers-cov outbreak involving patients in south korea, and the median incubation period was estimated to be days with a range of to days [ ] . at the end of , there were confirmed mers with a % mortality rate [ ] [ ] [ ] . bats are the natural reservoirs of both sars-cov and mers-cov. sars-cov crossed the species barrier into palm civets and other animals in live animal markets in china, which then infected human, while a mers-cov ancestral virus crossed species barrier into dromedary camels. abundant circulation of mers-cov in camels results in continuous zoonotic transmission of this virus to human, while sars-cov was not found to circulate in the intermediate reservoirs, explaining sars being a one-off outbreak and mers a continuing zoonotic disease [ ] . aerosolgenerating procedures such as intubation were associated with increased viral transmission of both covs resulting in nosocomial outbreaks [ ] . super-spreaders are responsible for large and prolonged outbreaks [ ] . the diagnosis for sars and mers include both serological tests and pcr assays that can quantify viral loads [ ] . functional genetic polymorphisms leading to low serum mannose binding lectin (mbl) are associated with susceptibility to but not severity of sars in both southern and northern chinese [ ] [ ] [ ] . mbl was shown to bind to sars-cov and inhibit the infectivity [ ] , suggesting its role as first-line defense against sars-cov. although no patients with primary immunodeficiency infected with sars-cov or mers-cov were identified, likely due to the limited number of such infections, patients with t cell defect and type interferon pathway defects could suffer a more severe disease course [ , ] . virus-based and host-based treatment strategies are largely experimental with uncertain benefits. ribavirin, type interferons, small molecules, and monoclonal antibodies that block covs entry have been explored [ ] . passive immunotherapy and multiple candidate vaccines have been tested in various animal models. convalescent plasma immunotherapy has been considered, but clinical trials are lacking in mers [ ] , while for sars a systematic review concluded convalescent serum may reduce mortality and appear safe [ ] . the filoviridae family contains three known genera, the ebolaviruses, marburgviruses, and cuevavirus. ebolavirus and marburgvirus cause hemorrhagic fever syndromes in primates and humans, with high fatality rates. cuevavirus infects only bats. the ebolavirus genus contains five species, with two of the species (zaire ebolavirus and sudan ebolavirus) being responsible for the majority of cases of human disease, while marburgviruses contain two species (marburg virus and ravn virus). filoviruses are capable of replicating in a number of cell types (with the exception of neurons and lymphocytes). upon entry into the body of the host (via breaks in the skin, parenterally, or through mucosal surfaces), filoviruses employ a variety of mechanisms to evade the activity of the immune system [ ] . the incubation period (interval from infection to onset of symptoms) varies from to days. symptoms begin abruptly, with high fever, severe headache, malaise, myalgia, diarrhea, nausea, and vomiting. a rash can occur between and days after onset of symptoms. hemorrhagic manifestations occur between and days, and fatal cases usually have some form of active bleeding. in an outbreak setting, the symptoms are unmistakable but confusion with malaria can occur early in the disease or in sporadic cases. since their original descriptions in and , respectively, for marburg and ebola, there have been a number of sporadic cases and several major outbreaks. the largest marburg virus outbreak occurred in angola in (with a fatality rate of > %), while the largest ebola epidemic happened between and in west africa (sierra leone, guinea, and liberia) and claiming over , lives (fatality rate > %). although not definitively proven in the case of ebola, bats are believed to be the natural animal reservoir for these viruses [ , ] . these viruses are transmitted via contact with blood or body fluids from an infected host; notably, certain body fluids can harbor virus for weeks to months after resolution of disease. given the recent outbreak in west africa, there has been renewed interest in understanding the pathogenesis of filovirus infections and possible therapies. literature regarding how the pathogenesis of disease may be altered in patients with pidd is lacking. however, the assumption is that in the absence of an intact cellular and/or humoral immune response, the patient with a pidd may be at increased risk of mortality in the setting where mortality is already high. these viruses induce apoptosis of lymphocytes and macrophages, and there is therefore a profound secondary immune compromised [ , ] . filoviruses can be detected in multiple body fluids via pcr. although practiced for decades, a study in guinea in failed to show a decrease in mortality among patients receiving convalescent plasma from previously infected donors [ ] . a number of additional compounds (e.g., tkm-ebola, bcx , and gs- ) and biologics (zmapp) have been shown to offer protection in animal models of ebola, but to date, no controlled and appropriately powered clinical trials have addressed their efficacy in humans. finally, a number of vaccines for ebola are undergoing clinical studies (including four in phase iii trials). importantly, in late , the rvsv-zebov vaccine was shown to have displayed high efficacy in protecting immunized adults during the guinea ebola outbreak, and the data also suggested that the vaccine may even offer bherd immunity^to unimmunized persons in proximity to recipients of the vaccine [ , ] . hepatitis e virus is a single-strand rna virus of the hepeviridae family. it is an important zoonotic disease in asia and africa, and fecal-oral spread is the usual route of transmission [ ] . handling of pig or boar meat is a risk factor, and - % of pig livers sold in grocery stores in japan and the usa are infected [ , ] . swine represent the major reservoir, although antibodies to the virus have been found in many species [ ] . the incubation period is weeks to months, and viremia disappears with the onset of symptoms. the mortality rate is - % and can reach % in pregnancy [ ] . acute hepatitis usually resolves but can lead to liver failure in severe cases. patients with hepatitis e posttransplant have had severe courses in some cases [ ] . in immune compromised patients, the course can become chronic [ ] [ ] [ ] . in these cases, cirrhosis develops. the diagnosis is by serology or pcr, and the treatment is supportive. prevention modalities for infections transmitted by humans are conceptually different than infection prevention for vector-borne infections. hand hygiene is extremely important, and avoidance of clearly infected people can be helpful. recognition of infections with fecal-oral transmission and the importance of water purity are critical for patients with pidd. in contrast, infections transmitted by aerosols require prevention strategies related to droplet precautions. in outbreak scenarios, if the risk to the patient is high, specific chemoprophylaxis may be considered. influenza viruses type a and b cause annual epidemic influenza, while type c causes sporadic mild influenza-like illness. patients present with sudden onset of fever, chills, and myalgia, followed by sore throat and cough. other less common features include diarrhea, acute myositis, and encephalopathy [ , ] . co-infection with bacteria such as pneumococci results in more severe disease [ , ] . influenza pandemics occur yearly around the world. influenza viruses infect to % of the global population, resulting in~ , deaths annually [ ] . the viruses circulate in asia continuously and seed the temperate zones, beginning with oceania, north america, and europe, then later seeding into south america [ ] . diagnosis of influenza includes direct/ indirect immunofluorescent antibody staining for antigens in nasopharyngeal aspirates and pcr. a patient with compound heterozygous null mutations of the gene encoding irf , a transcription factor for amplifying ifn-α/β, was reported to have life-threatening influenza during primary infection [ ] . fatal influenza-associated encephalopathy in both chinese and japanese children has been reported to be associated with genetic variants of thermolabile carnitine palmitoyltransferase ii [ ] . patients with scid will have prolonged viral shedding [ ] . severe pandemic influenza a virus (h n ) infection has been associated with igg and igg subclass deficiency [ , ] . in addition, influenza infection can be more severe in pidd patients with underlying lung disease, such as bronchiectasis, and antibiotic coverage of chronic colonizing bacteria (such as pseudomonas) in this setting may be helpful. inactivated seasonal influenza vaccine should be given to pidd patients even those with humoral deficiencies as their t cell response to influenza could be normal and offer protective immunity against severe influenza [ , ] . antiviral drugs include neuraminidase inhibitors (oseltamivir and zanamir) and adamantanes (amantadine and rimantadine), but resistance to adamantanes is widespread. measles is a single-stranded, negative-sense, enveloped (nonsegmented) rna virus of the genus morbillivirus. measles is highly communicable, transmitted by droplets, and less commonly by airborne spread. patients present with fever, cough, coryza conjunctivitis rash, and koplik spots. complications include pneumonia, acute encephalitis, and subacute sclerosing panencephalitis (sspe) [ ] . diagnosis of measles includes serological tests, virus isolation, and pcr. in an outbreak, the clinical features may be sufficient for diagnosis. measles vaccine has caused severe measles in children with stat and ifn-α/β receptor deficiency [ , ] , demonstrating the importance of type interferon pathway in controlling measles. immune compromised of nearly any type is associated with severe disease and higher mortality [ ] . t cell deficiency states are the most strongly associated with the development of giant cell pneumonia and inclusion body encephalitis, the most feared complications of measles. sspe is a slow encephalitis due to persistence of replication defective measles virus in the cns. it is most frequently seen when young infants are infected with measles and - years later, sspe becomes evident. there are case reports supporting the immune compromised as increasing the risk of sspe [ ] . treatment of sspe with ribavirin has shown some improvement, but the prognosis in general with sspe is very grave. patients with cgd have defective memory b cell compartment, resulting in lower measle-specific antibody levels and antibody-secreting cell numbers, but severe disease has not been reported [ ] . pidd patients may harbor the virus latently for longer than usual, leading to complications at the time of transplant [ ] . specific antiviral therapy is lacking, but ribavirin has been given to severely ill and immunocompromised children. for measles post-exposure prophylaxis, intravenous immunoglobulin (ivig) is recommended for severely immunocompromised patients without evidence of measles immunity [ ] . this would likely include patients with scid and hypogammaglobulinemia who are not yet on regular ivig. measles vaccine, given in a two-dose regimen, has brought down incidence enormously worldwide and the who is planning for eradication globally. enteroviruses (evs) are among the most common viruses infecting humans worldwide. evs are small non-enveloped, single-stranded rna viruses of the picornaviridae family. human evs are categorized into seven species that include hundreds of serotypes, such as polioviruses (pv), coxsackie viruses a, and b (cv-a and b), echoviruses, and human rhinoviruses (hrvs). of these species, many important serotypes are known to infect human such as pv - , cv-a , cv-b , ev-a , ev-d , and hrv (table ) . non-polio enteroviruses (npevs) have a worldwide distribution. infants and young children have higher incidence of infection and a more severe course of illness than adults. the mode of transmission is mainly through fecal-oral and respiratory routes. infection occurs all around the year in tropical and subtropical regions, while in temperate climates the peak incidence of infection is during summer and fall months [ ] . npevs are associated with diverse clinical manifestations ranging from mild febrile illness to severe, potentially fatal conditions. most cases are asymptomatic or have mild symptoms including fever with or without rash; symptoms of hand, foot, and mouth disease; herpangina; acute hemorrhagic conjunctivitis; upper respiratory infection; and gastroenteritis. more severe symptoms occur in infants and young children [ , ] . acute flaccid paralysis [ ] , neonatal enteroviral sepsis [ ] , myocarditis/pericarditis [ , ] , hepatitis, pancreatitis, pneumonia, and atypical hemolytic uremic syndrome [ ] are severe manifestations seen in immunocompetent people. chronic infections have been seen in immunocompromised patients [ ] . each virus may produce one or more of the aforementioned manifestations; however, some serotypes are often associated with particular features ( table ) . the definitive diagnosis of npev infection relies on pcr or virus isolation from the cerebrospinal fluid, blood, stools, urine, or throat swab [ , ] . treatment of npevs is mainly supportive since most infections are self-limited. high doses of intravenous immunoglobulin (ivig) are recommended in patients with severe symptoms. the efficacy of some new antiviral drugs (pleconaril, vapendavir, and pocapavir) is still under investigation [ ] . no vaccine has been licensed yet for npevs. however, phase clinical trials of inactivated monovalent ev-a vaccines manufactured in china showed high efficacy against ev-a in infants and young children [ ] . patients with a variety of pidds are unusually susceptible to ev [ ] . the most susceptible groups are patients with primary antibody deficiency such as xla, cvid, and hyper-igm syndrome (higms) as well as those having scid and major histocompatibility class ii deficiency [ , ] . the most severe form of infection has been described in patients with xla due to the profound deficiency of immunoglobulins essential for viral neutralization during infection. affected patients usually present with indolent but relentlessly progressive non-necrotizing meningoencephalitis. regression of cognitive skills, flaccid quadriplegia, and deafness has been described. the reported non-neurologic presentations in xla include septicemia, dermatomyositis-like disease, hepatitis, and/or arthritis [ , ] . the incidence of npev meningoencephalitis in large registries of xla cases is - % [ ] . unpublished data from the kuwait national pidd registry, which includes pidd patients, showed that nine patients had documented npev infections and two died from these infections. the two deaths were seen in scid patients (personal communication with prof. waleed al-herz, md). in addition, npev meningoencephalitis and/or septicemia were reported in few cases with either primary b cell deficiency such as b cell linker (blink) protein deficiency [ ] or acquired b cell deficiency following the administration of anti-cd (rituximab) [ , ] . in all reports, better outcome was attributed to the early administration of high doses of ivig during npev viremia [ ] . npev infection in pidd diseases remains a major threat to patients. also, the possible prolonged viral excretion and the emergence of resistant strains runs the risk of spreading infection to the surrounding community. oral polio vaccine (opv) consists of a mixture of three live attenuated poliovirus serotypes. opv induces production of neutralizing antibodies against all three serotypes, in addition to a local intestinal immune response. opv can result in vaccine-associated paralysis (vap) secondary to reversion of the vaccine strain to the neurovirulent wild-type strain. an example for such an event was demonstrated by the - outbreak in the dominican republic and haiti [ ] , believed to be driven at least in part by undervaccination of the population, which allowed the spread of the reverted vaccine strain [ ] . although rare, patients with pidd have a higher risk to develop vap. reports have shown that pidd patients with antibody deficiency can have prolonged viral replication which can persist for years and therefore theoretically increase the risk for a spontaneous reversion within the immunodeficient host [ ] [ ] [ ] [ ] . cases of vap were shown in patients with antibody deficiency and combined immunodeficiency syndromes [ , , ] . therefore, opv is contraindicated in patients with pidd, and this contraindication extends to their household contacts [ ] . beyond the obvious risk for the pidd patient, prolonged virus shedding also increase the risk for spreading vaccine-derived paralytic strain in the general population. bacterial infections have molded human behavior and altered societies over human history. today, largely ignored due to antibiotic susceptibility, they continue to cause misery and disease around the world. three infections are highlighted, and additional commonly encountered infections are listed in table . pertussis is a respiratory infection caused by bordetella pertussis that begins after a -to -day incubation period as a minor upper respiratory infection that progresses with cough. initially intermittent, it evolves into paroxysmal coughing spells usually followed by vomiting in infants and young children. it lasts to weeks and can have many complications such as syncope, weight loss, rib fracture, and pneumonia. infants under months are more severely affected, developing pneumonia, pulmonary hypertension, hypoxia, subdural bleeding, and seizures. death can occur, especially in young infants [ , ] . adults typically have a prolonged cough with fewer complications [ ] . it is transmitted via aerosolized droplets during close contact. people are most contagious during the catarrhal stage and the first half of the paroxysmal phase, totaling to weeks [ ] . the introduction of whole-cell pertussis vaccine (dpt) in the s in the usa reduced the incidence of the disease from , cases to around cases per year in the s. a resurgence in was associated with the substitution of the whole-cell vaccine by the acellular pertussis vaccine (dtap) [ ] . new strategies such as boosters with acellular pertussis for adolescents and adults with tdap and use of tdap during pregnancy seem to be effective in partially reducing the incidence of the disease [ ] ; however, pertussis cases in the usa remain higher than the s. the lack of persistence of antibody in the adult population means adults not only represent a reservoir for the disease but also do not provide sufficient titers to immunoglobulin products prepared from adult plasma pools. a relatively recent requirement in some countries is vaccination of adults every years to maintain immunity. this should, over time, improve titers in immunoglobulin products. culture of specimens obtained by nasopharyngeal swabs is the gold standard of laboratory diagnosis due to the % specificity, but polymerase chain reaction (pcr) is gaining prominence due to its higher sensitivity and speed of results; serodiagnosis can be used in the late stages of the disease [ ] . filamentous hemagglutinin (fha) and pertussis toxin (pt) antibodies were detected at peak measurements in pidd patients on regular ivig, although some of them had pt antibodies below the protective level as trough measurements [ ] . severe pertussis cases have not been reported in pidd patients, but severe disease has been seen in malignancies [ ] . antimicrobials such as azithromycin, erythromycin, and clarithromycin, if given during the catarrhal stage, may ameliorate the disease and shorten the contagious period. to avoid cases of pertussis, it is also worth emphasizing the importance of good vaccine coverage rate among the whole population, but especially among healthcare workers and family members of patients with pidd. neisseria meningitidis the onset of neisserial meningitis is associated with sore throat, headache, drowsiness, fever, irritability, and neck stiffness [ , ] . purpuric lesions are very characteristic. this pathogen can also present with sepsis which has a % mortality rate as opposed to % mortality with a meningitic presentation. this bacterium can also cause a chronic condition referred to as chronic meningococcemia. this condition is characterized by intermittent fevers lasting week to - months [ ] . a non-purpuric rash is common which may evolve into purpura. arthritis, similar to that seen with gonococcus, is common. meningococcal disease primarily affects children under years of age. n. meningitidis is a global pathogen [ ] . there are serogroups, but the majority of invasive meningococcal infections are caused by organisms from the a, b, c, x, y, or w serogroups. the annual number of invasive disease cases worldwide is estimated to be at least . million, with , deaths related to invasive meningococcal disease. serogroups b and c are responsible for most infections in europe. serogroup a has historically been the major organism in africa; mass vaccination has led to some improvement, but the emergence of group x disease is worrisome. the hajj in the middle east has seen epidemics of w- , and vaccination is now required for hajj travelers. b and c serogroups are the most common through the americas. n. meningitidis cases occur at a rate of about case per , people throughout the world [ ] , but across the sahel of africa and in china, epidemics can lead to case rates of per , [ ] . the bacterium is a natural human commensal, with carriage rates of about %. diagnosis can be by clinical examination in epidemics or by gram stain and culture. complement-deficient individuals have an increased risk of neisserial disease, but not necessarily increased mortality. hiv is associated with increased disease, suggesting that t cells are also important for defense. thirdgeneration cephalosporins are typically used for treatment. penicillin, ampicillin, aztreonam, and chloramphenicol are alternatives. there is great inter-individual variability in the development of tb disease. roughly, % of infected individuals develop clinical disease within years of infection (mostly during childhood). about % became latently infected without clinical disease, and the remaining to % develop pulmonary tb later in life, either from reactivation of latent infection or reinfection. molecular epidemiology studies from high burden areas suggest more disease results from recent transmission than from reactivation of latent tb, particularly in people living with hiv [ ] . acquired or inherited host factors may at least partially account for the variable disease course, resulting in increased susceptibility to mycobacteria infections [ ] . pidd associated with tb and ntm infections include t cell deficiencies, gata deficiency, cgd, anhidrotic ectodermal dysplasia with immunodeficiency, x-linked (xl) recessive cd ligand deficiency, autosomal recessive (ar) stat deficiency, ar irf deficiency, and ar tyk deficiency. in addition, a group of disorders with a strong susceptibility to ntm, named mendelian susceptibility to mycobacterial diseases (msmd), have been recognized since the s. these are rare inborn errors of ifn-γ signaling pathway that present with isolated predisposition to infections caused by weakly virulent mycobacteria such as bcg vaccine and environmental ntm, in otherwise healthy patients. the genetic defects involve impairment in the production of interferons (ar il rβ , ar il p , autosomal dominant (ad) irf , ar isg , xl recessive nemo) or response to interferons (ifn-γr, ad stat , ad irf , cgd) [ ] . an acquired form exits: adults with ntm infection in thailand and taiwan were found to have high-titer anti-interferongamma antibody [ ] . these individuals from southeast asia were found to have hla-drb * / : and dqb * : / : . patients suspected of having pulmonary tb should have acid-fast bacilli (afb) smear microscopy and culture performed in three sputum samples. pcr for mtb can be performed [ ] . the use of rapid tests facilitates early diagnosis, and the who has recently recommended their use. the only recommended rapid test for detection of tb with and without rifampicin resistance is the xpert mtb/rif assay (cepheid, sunnyvale, ca). the who recommends the xpert test for those suspected of having drug-resistant tb or in hiv; however, culture is still the mainstay and is not replaced by the xpert test. tb skin testing (mantoux testing) uses a purified protein derivative injected under the skin. its advantages are that it can be used for large-scale screening and it is cost effective. skin testing does have several disadvantages when used as a diagnostic test. reading the induration requires training and immunodeficiencies can alter the magnitude of the induration. immunosuppressed patients (hiv, organ transplant) are considered positive if the induration is ≥ mm. some immunodeficiencies may completely ablate the response. other causes of false-negative tests are malnutrition, concurrent infection, recent live viral vaccine administration, renal failure, malignancy, medical stress, very elderly, young infants, or with a very recent infection with mtb. conversely, the results may be falsely positive if bcg has been administered. interferon gamma release assays can be used in any setting where skin testing would be done but are considered superior in settings where the patient has had bcg vaccination and, in some cases, where skin testing has been sown to have high false-negative rates. in general, tb treatment for patients with impaired immune response, including pidd, hiv infection, and immunosuppressive therapy, is based on the standard -month regimen consisting of a -month intensive phase of isoniazid, rifampin, pyrazinamide, and ethambutol, followed by months of isoniazid and rifampin. decisions regarding treatment duration can be individualized, taking into account disease severity, organs involved, and response to treatment. significant pharmacological interaction can occur between rifampin-based mtb regimens and immunosuppressive drugs, such as calcineurin inhibitors or rapamycin, requiring strict monitoring of drug plasma concentrations [ ] . therapy for ntm is complex with highly variable drug resistance patterns and a need for biological augmentation to effectively clear the organism. aspergillus fumigatus (see above), cryptococcus gattii, histoplasma capsulatum, coccidioides immitis (or c. posadasii), blastomyces dermatitidis, paracoccidioides brasiliensis, emmonsia pasteuriana, and penicillium (talaromyces) marneffei are environmental fungi that are endemic in certain parts of the world (table ). with the exception of penicillium marneffei and emmonsia pasteuriana that only cause disease in profoundly immune compromised individuals, these fungi can cause infection in healthy individuals, ranging from mild, self-limited pulmonary disease to infection that requires antifungal therapy for eradication. on the other hand, patients with acquired defects in cell-mediated immunity such as those infected with hiv, and patients with specific monogenic disorders, particularly those involving the il- / ifn-γ/stat signaling pathways, scid, and gata depends on underlying state of immunosuppression and magnitude of environmental exposure icl idiopathic cd lymphocytopenia, aids acquired immune deficiency syndrome, stat signal transducer and activator of transcription , gata gata binding protein , scid severe combined immunodeficiency, cvid common variable immune deficiency, pidd primary immunodeficiency, il rb , interleukin- receptor subunit beta , ifngr gamma interferon receptor deficiency, are at high risk of developing life-threatening disseminated infections by these endemic fungi following environmental exposure [ , [ ] [ ] [ ] [ ] [ ] . diagnosis relies on culture of the corresponding fungus, histopathological demonstration of the fungus-specific characteristic morphologies, and/or surrogate serological and fungal antigen tests. treatment of clinical disease (as opposed to colonization) typically involves an initial induction phase with amphotericin b, followed by long-term azole maintenance therapy and secondary prophylaxis, and prognosis varies significantly depending on the fungal pathogen and underlying pidd. melioidosis is caused by b. pseudomallei, a gram-negative bacteria found in soil and water, in tropical climates of southeast asia and northern australia [ , ] . melioidosis is an emerging, potentially fatal disease ( % mortality). b. pseudomallei can be transmitted by inhalation, ingestion, or direct contact (through open skin) with contaminated soil or water. animals (sheep, goats, swine, horses, cats, dogs, and cattle) are also susceptible to infection and cases of zoonotic transmission through direct contact of skin lesions with infected animal meat or milk have been described [ ] . b. pseudomallei infections are endemic in northern australia and southeast asia. approximately % of reported infections occur during the rainy seasons. cases have also been reported in south pacific, africa, india, and the middle east. in temperate areas, infection is extremely rare and is predominantly imported by travellers or immigrants [ ] . the incubation period of melioidosis is variable from day to years, although common symptoms develop between and weeks after exposure. melioidosis presents most frequently in adults - years of age, but can occur in all ages, with one study reporting % of cases occurred in children [ ] . in australia, the average annual incidence in - was reported as . cases per , people [ ] . the incidence in indigenous australians was higher at . cases per , . a case-cluster in an australian community was associated with post-cyclonic flooding. a recent review suggests that b. pseudomallei is increasingly prevalent in the americas, with a mortality rate of % [ ] . infection in healthy individuals is uncommon, and more than % of cases occur in the setting of underlying conditions such as chronic renal disease, diabetes, chronic lung disease, and alcoholism. a recent review of melioidosis in travelers found that % of cases were acquired in thailand. symptoms usually started at days (range - days) after leaving the endemic area. traveller infections were less often associated with predisposing risk factors ( . %), diabetes mellitus being the most common ( %). melioidosis in travelers had lower mortality ( %) than infection in autochthonous cases in southeast asia [ ] . pneumonia (~ - %) is the most common presentation in adults. there is usually high fever, headache, anorexia, and myalgia. b. pseudomallei infection may also present as localized skin infection, septicemia, or disseminated infection. localized infection results in an ulcer, nodule, or skin abscess. this usually occurs from the bacterium breaching through a break in the skin. patients with renal disease and diabetes are more susceptible to sepsis. in disseminated infection, abscesses may develop in the liver, spleen, lung, and prostate. in children, primary cutaneous melioidosis is the commonest presentation ( %). bacteremia is less common in children than in adults, but brainstem encephalitis has been reported [ ] . difficulties in laboratory diagnosis of melioidosis may delay treatment and affect disease outcomes [ ] . diagnosis of melioidosis is primarily by isolation of the organism. identification of b. pseudomallei can be difficult in clinical microbiology laboratories, especially in non-endemic areas where clinical suspicion is low. although various serological tests have been developed, they are generally unstandardized bin house^assays with low sensitivities and specificities. pcr assays have been applied to clinical and environmental specimens but are not widely available and sensitivity remains to be evaluated. cases of melioidosis have been reported in patients with acquired or inherited immune deficiency. melioidosis was the presenting complaint in several patients with cgd. bacteremic melioidosis was recently reported in two patients with prolonged neutropenia, who succumbed despite appropriate antibiotics [ ] . it is likely that there is increased susceptibility in situations where innate or adaptive immunity is compromised. treatment is with intravenous antimicrobial therapy for - days, followed by - months of oral antimicrobial therapy. intravenous therapy with ceftazidime or meropenem is usually effective. oral therapy may continue with trimethoprim-sulfamethoxazole or doxycycline. free-living amoebas (fla) are protozoa found worldwide that do not require hosts to survive. fla do not employ vectors for transmission and are not well adapted to parasitism in humans. however, there are four genera/species that can cause human disease: naegleria (n. fowleri), acanthamoeba (multiple species), balamuthia (b. mandrillaris), and sappinia (s. pedata). all of these amoebae are capable of inducing cns disease in humans, but acanthamoeba species also cause various extra-cns infections, especially in immunocompromised hosts. the fla that are pathogenic in humans are reviewed below. naegleria are a diverse group of fla flagellate protozoans with a large number of distinct species. only one species, n. fowleri, has been shown to cause infection in humans. n. fowleri has a multi-stage life cycle with amoeboid and trophozoite-infective forms as well as a cyst form [ ] . n. fowleri is found commonly in warm freshwater around the world including lakes, rivers, and hot springs. humans become can become infected when swimming or diving in contaminated water. in rare circumstances, infections have also been attributed to exposure from contaminated tap water sources when utilized for religious cleansing of the nose or irrigation of the sinuses. thus, tap water should not be used for nasal and sinus irrigation. it is not possible to become infected from drinking contaminated water or from contact with an infected person, and the amoeba is not found in salt water. after entry to the nasal cavity, the amoeba travels through the cribiform plate to the olfactory bulbs and migrates to the cerebellum, resulting in primary amoebic meningoencephalitis (pam), a rapidly fatal brain infection characterized by the destruction of brain tissue. in its initial presentation, pam can mimic bacterial meningitis, further delaying accurate diagnosis and initiation of therapies that may save the patient. overall, n. fowleri infections are rare. worldwide, most cases are reported in the usa, australia, and europe; however, in developing countries, it is suspected that a large number of cases go unreported. between and , there were only infections reported in the usa with of the cases attributed to contaminated recreational water, infections following nasal irrigation with contaminated tap water, and case where a person was infected following use of a backyard slipn-slide utilizing contaminated tap water [ ] . the fatality rate associated with n. fowleri infection is over %, and between and , only of the infected persons in the usa have survived infection. initial symptoms of pam start to days after infection and can include headache, fever, nausea, or vomiting [ ] . progressive symptoms can include stiff neck, confusion, lack of attention, loss of balance, seizures, and hallucinations. cardiac arrhythmias have also been observed. the infection progresses rapidly after initial onset and causes death within to days after exposure (mean of . days). since infection often progresses rapidly to death, there is often insufficient time to mount a robust immune response. however, both the innate (neutrophils, macrophages, and complement system) and the adaptive (both t and b cells) arms of the immune system have been shown to participate in the immune response to n. fowleri [ ] . patients with pam have csf with elevated pressure that is often cloudy or hemorrhagic, with neutrophil-predominant pleiocytosis, elevated protein levels, and very low glucose. wet mounts from centrifuged csf will show motile mono-nucleated trophozoites measuring~ - μm in size. additionally, trophozoites can be identified with giemsa and wright stains of csf smears combined with an enflagellation test [ ] . confirmation can be achieved via a variety of timeconsuming methods including an immunofluorescence assay [ ] , culture of csf [ ] , or pcr-based methods [ ] . the optimal therapy for n. fowleri pam is still debated. the use of intravenous amphotericin b and fluconazole followed by oral administration of rifampin resulted in survival of a year-old child with pam [ ] . another child was shown to survive following intravenous and intrathecal amphotericin b and miconazole as well as oral rifampin [ ] . most recently, an adolescent girl was successfully treated with the combination of azithromycin, rifampin, fluconazole, and miltefosine [ ] . prevention is critical for this highly fatal infection and warning pidd patients not to use tap water for nasal irrigation is important. since its original description in , over cases of b. mandrillaris infections have been described worldwidewith most cases occurring in south america and the usa. balamuthia are found in soil, and acquisition of disease has been associated with agricultural activities, dirt-biking, gardening, and swimming in contaminated water sources. b. mandrillaris is thought to enter the body of the host through breaks in the skin and or via inhalation. the organism is believed to access the cns through hematogenous spread, resulting in a chronic, insidious, but often fatal granulomatous amoebic encephalitis (gae), which has been documented in both immunocompetent and immunocompromised hosts [ , ] . the incubation period from exposure to development of clinical symptoms is not well established and experts believe that this may occur between months and years. finally, an alternative mode of transmission via solid organ transplantation has also gained attention [ ] [ ] [ ] . in many cases, gae is diagnosed post-mortem, due to delayed diagnosis or unawareness of the clinical entity. following infection by b. mandrillaris, two clinical patterns of presentation have been described. in the first pattern, patients initially develop a skin lesion that may resemble a painless plaque that may evolve into subcutaneous nodules and rarely ulcerations [ ] . these patients may develop neurologic manifestations weeks to months later. histopathologic examination of these lesions typically reveals granulomatous reactions in the reticular dermis, associated with lymphocytic and plasma cell infiltrates as well as multinucleated giant cells, without distinct epidermal changes. skin lesions will harbor trophozoites, but these are scarce and often easily overlooked as they resemble histiocytes. it is believed that early diagnosis of b. mandrillaris infections in those presenting with skin lesions may prevent subsequent development of cns disease, but there have also been cases in which patients presenting with skin lesions have progressed to developing gae despite treatment. in the second pattern, patients present with cns involvement without a previously recognized skin lesion. patients presenting with gae may initially display fever, malaise, headache, nausea and vomiting, and frank lethargy. later, these symptoms evolve into visual abnormalities, cranial nerve palsies, seizures, focal paresis; as intracranial pressure builds, coma, and eventually death with tonsilar or uncal herniation ensue within - weeks [ ] . upon infection with b. mandrillaris, brain endothelial cells produce the proinflammatory cytokine il- , thereby initiating an inflammatory response [ ] . moreover, the amoebic trophozoites infiltrate blood vessel walls. degradative enzymes, vessel wall infiltration, and the host inflammatory responses result in tissue necrosis and infarctions in the cerebral hemispheres, cerebellum, and the brainstem. in a mouse model of b. mandrillaris infection, cd + t cells were shown to be protective [ ] , suggesting that patients with lowered number or dysfunction in cd + t cells may be more susceptible to disease by this amoeba. however, b. mandrillaris infections have been described in a variety of human hosts [ ] , ranging from the young, healthy, and presumably immunocompetent to the elderly, and those with hiv, chronic corticosteroid exposure, on post-transplant immunosuppression and even patients with cvid. as such, further research is necessary to fully delineate the susceptibility of pidd patients. in patients who develop the characteristic skin lesions, recognition, testing, and treatment for b. mandrillaris may prevent subsequent gae. as such, obtaining tissue and looking for granulomas and trophozoites is quite helpful. skin biopsies can be stained via immunofluorescence or immunoperoxidase techniques to identify b. mandrillaris [ ] . additionally, a pcr technique that identifies mitochondrial s ribosomal rna from b. mandrillaris is also available through the cdc [ ] . in patients in whom the diagnosis is confirmed via skin biopsy, wide resection and medical treatment appears to prevent development of cns disease in at least a proportion of patients. in patients presenting with cns involvement, lumbar punctures reveal csf with lymphocytic pleiocytosis, low-to-normal glucose, and mildly to significantly elevated protein levels. trophozoites are not typically found in the csf, but pcr analysis may be performed. ct or mr imaging may show multiple nodules with ring enhancement; some of these nodules may also contain focal areas of hemorrhage. biopsies of brain tissues typically reveal granulomas and foamy macrophages and multinucleated giant cells surrounded by lymphocytic infiltrates. additionally, there will be areas of necrosis filled with neutrophils, multinucleated giant cells, and lymphocytes, with balamuthia trophozoites and cysts interspersed with macrophages [ ] . as with the skin biopsies, immunofluorescent and immunoperoxidase stains may aid diagnosis and should be performed. unfortunately, the optimal medical management of cns disease is unknown. in the usa, a few patients have been successfully treated with a combination of fluconazole, flucytosine, pentamidine, a macrolide antibiotic (either clarithromycin or azithromycin), and one of the following agents: liposomal amphotericin b, miltefosine, sulfadiazine, or thoridazine [ ] [ ] [ ] ; others in peru have been treated successfully with fluconazole (or itraconazole), albendazole, and miltefosine [ ] . based on these case reports, most experts recommend treatment with a combination of medications (along with partial or complete resection of nodules) for a prolonged period of time to prevent further deterioration and death [ ] [ ] [ ] [ ] . acanthamoeba spp. the genus acanthamoeba contains at least morphologically distinct species that live in a diverse array of habitats, including soil, salt, brackish, and fresh water. acanthamoeba spp. have also been found in humidifiers, heating and cooling unit components, jacuzzis, hot water tanks, bathrooms and drains, eye wash stations and dentistry irrigation systems, and more. acanthamoeba spp. have been isolated from reptiles, birds, and other non-human mammals, suggesting a broad distribution in the environment. acanthamoeba trophozoites feed on bacteria, but have also recently been shown to harbor a number of bacteria (including legionella and burkholderia spp., e. coli, listeria monocytogenes, vibrio cholerae, mycobacteria spp., chlamydophila, and others) and at least one virus (mimivirus) as endosymbionts. acanthamoeba infections in humans can present in a variety of ways. of primary importance are cns infections. like b. mandrillaris, acanthamoeba spp. can induce gae (described above). there is a high predilection for gae in those with hiv/aids, patients on chemotherapy, and those receiving broad spectrum antibiotics [ ] . acanthamoeba are rarely found in csf, but some case reports indicate isolation of amoebae by culturing csf on bacterized agar plates. similar to gae seen with b. mandrillaris, cns histopathology may reveal edema, multiple areas of necrosis and hemorrhage, and occasional findings of angitis and blood vessel cuffing by inflammatory cells, as well as occasional trophozoites or cysts. cns disease treatment is not standardized, but several patients have been successfully treated with pentamidine, fluconazole, flucytosine, sulfadiazine, as well as miltefosine. acanthamoeba can rarely cause cutaneous infections; these lesions, like gae, are also predominantly seen in immunocompromised hosts. these lesions can start as nodules or papules on the lower extremities and develop into necrotic ulcers. histopathologic examination may reveal granulomatous dermal lesions in immunocompetent hosts, with histiocytes, as well as neutrophils and plasmacytes; trophozoites are typically visible [ , ] . the optimal management of cutaneous disease is not known, but typically involves combinational therapy with topical (e.g., chlorhexidine, gluconate, or ketoconazole) and systemic (miltefosine, sulfadiazine, flucytosine, liposomal amphotericin b, azole antifungals, etc.) drugs. additionally, nasopharyngeal and sinus infections have been seen in people with severe compromise in immunity [ , ] . patients typically present with purulent nasal discharge, and examination may reveal erosion of the nasal septum. nasopharyngeal disease can present concomitantly with cutaneous disease. treatment of nasopharyngeal or sinus disease is difficult and involves surgical debridement and combinations of systemic drugs. disseminated disease is also seen in immunocompromised hosts and typically involves concomitant pulmonary and cutaneous disease in the presence or absence of cns infection. keratitis readily occurs in immunocompetent hosts-with the major risk factor being contact lens wearing without proper adherence to recommended cleansing protocols. this infection less commonly presents as a result of direct inoculation with trauma. one of the most common reasons for contact lens wearers to acquire disease is due to the use of non-sterile tap water in preparing contact lens saline solutions [ ] , although contaminated solutions from manufacturers have also been identified. patients will have pain and photophobia. physical exam reveals conjunctival injection and epithelial abnormalities (including pseudodendritic lesions) and stromal infiltrates [ ] . the proper diagnosis can be made by staining corneal scrapings with calcofluor or wright-giemsa stains and examined by confocal microscopy, culture, or pcr analysis. prompt therapy with a combination of polyhexamethylene biguanide (or biguanide-chlorhexidine) and propamidine or hexamidine [ , ] is indicated, but misdiagnosis and delayed therapy are common. more severe cases may also require debridement. the use of topical steroids before administration of combinational therapy may result in worse outcomes and should be avoided; however, if scleritis ensues, it may be necessary to use immunosuppressants to reduce the need for enucleation. severe and/or refractory cases may result in the need for cornea transplantation. phenotypes seen in pidd like hsv- and candida [ ] . patients with ifnar deficiency seem highly susceptible to cns disease caused by mmr vaccine, an otherwise extremely rare phenomenon [ ] . recently, a case of noroviral cns disease was described associated with a novel, yet unpublished pidd, suggesting that some pidds may lead to susceptibility of the cns to viruses that normally do not exhibit neurotropism (casanova jl, personal communication) . this again favors metagenomic approaches in the study of cns sequelae in pidd patients. in pidds, the cns is also more vulnerable to virally induced immunodysregulation [ ] . conditions like primary hemophagocytic lymphohistiocytosis (hlh) may present as isolated cns disease or relapse only in the cns [ ] [ ] [ ] . almost % all human malignancies are associated with chronic infections by hbv, hcv, hpv, ebv, hhv /kshv, htlv-i, hiv- , hiv- , jcv, merkel cell polyomavirus (mcpv), helicobacter pylori, schistosomes, or liver flukes [ ] . accordingly, pidd patients' malignancies are often associated with chronic infections. mcpv-associated merkel cell carcinoma has now been described in gata and tmc (ever ) deficiencies as well as other forms of pidd [ , [ ] [ ] [ ] [ ] [ ] . large follow-up cohorts are needed to refute or confirm associations between novel pidds and malignancies, such as hyperactivating pik cd and ovarian dysgerminoma or gata deficiency and leiomyosarcoma [ , ] . recently, hymenolepis nana was found to have driven malignant transformation in an hiv patient. likely, other novel pidd-and pathogen-associated malignancies will be found in the future by those with an open and inquisitive mind [ ] . understanding the specific infection susceptibility for each pidd allows not only a better understanding of host defense, but also allows the clinician to collaborate with the microbiology laboratory to make definitive diagnoses and provide the best therapy. reviewing all of the infections for each pidd is not within the scope of this article, but there are several infections that are unique for specific pidds and require special attention from the microbiology laboratory. three examples are provided below. g. bethesdensis is a gram-negative bacterium that was identified to cause disease in patients with cgd in [ ] . g. bethesdensis is a member of the methylotroph group of bacteria, which are able to use single-carbon organic compounds as their only source of energy. they are widespread in the environment, but are rare human pathogens, and infections with g. bethesdensis have been limited thus far to patients with cgd. the organism was first detected in an adult patient with indolent and recurrent necrotizing lymphadenitis [ ] . subsequently, g. bethesdensis was isolated from nine patients with cgd, primarily causing lymphadenitis, but there have been two fatalities [ ] . treatment has been most effective with intravenous ceftriaxone. the microbiology laboratory should be alerted when there is concern for g. bethesdensis infection to allow for proper culture media. charcoal yeast extract (cye) agar and lowenstein jensen (lj) media are appropriate culture media. mycoplasma and ureaplasma spp. as molecular techniques are becoming more widely used to detect pathogens, the spectrum of infections that were previously only detected through serologic assays and research laboratories will increase. this is important especially for patients with pidd who have unique susceptibility to infection and may not have the ability to mount a serologic response. examples of infections in this group are those caused by mycoplasma and ureaplasma [ , ] . these pathogens have been known to cause osteoarticular infections for those with antibody deficiency, such as xla and cvid. recently, mycoplasma orale, typically an oral commensal, has been isolated from two patients with defects in the activated pi k delta syndrome, as chronic lymphadenitis in one and chronic splenic abscess in the other (sm holland personal communication). defects in pi kcd and pi kr are frequently associated with hypogammaglobulinemia and therefore would fit in the pattern of mycoplasma infections in those with humoral immunodeficiency. mycoplasma orale has also previously been reported as causing bone disease in a patient with cvid [ ] . in patients with xla, helicobacter, camplyobacter, and the related flexispira bacteria that are typically isolated to the gi tract can disseminate and often lead to chronic bacteremia, ulcers, and bone infections [ ] [ ] [ ] . xla patients have higher susceptibility than other humoral pidd and are thought to be due to the role that igm is playing in controlling the dissemination of these pathogens and potentially iga in providing mucosal immunity. these bacteria can be fastidious to grow, and therefore, when there is suspicion, identification needs collaboration with the microbiology laboratory. for instance, the blood culture media may allow growth (although with a longer incubation period), but then the organisms may need molecular techniques for identification, such as s sequencing, as they will not grow on the agar plates. treatment is often difficult, requiring combination antimicrobials for prolonged periods (such as year), and relapse is common. this review provides an important perspective for practicing immunologists, namely that we are a part of a global community as are our patients. this overview of emerging infections and infectious concerns for travelers serves as a foundation for practical considerations for clinicians and patients. using prevalence data, an estimation of the number of infected patients with pidd (table ) can be developed [ , , [ ] [ ] [ ] [ ] . thus, the concerns addressed in this review are not theoretical but 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x-linked agammaglobulinemia prevalence and morbidity of primary immunodeficiency diseases, united states epidemiological characteristics of spotted fever in israel over years complications of bacille calmette-guerin (bcg) vaccination and immunotherapy and their management acknowledgements the authors would like to thank thomas krell for information regarding ivig safety and david peden for recognizing global warming as central to medical knowledge. conflict of interest the authors declared that they have no conflict of interest. pidds display wide genetic and phenotypic heterogeneity [ ] . similar disease phenotypes may be caused by multiple genes, while patients' phenotypes caused by the same gene and even by the same mutations vary between individuals. importantly, after a novel pidd has been described, subsequent reports often reveal a wider variation in associated infections and cellular findings, often without clear genotype-phenotype correlations [ ] [ ] [ ] [ ] [ ] . variation may be caused by mechanisms such as other contributing genes or geographical variation in infectious exposures. geographic differences seem most pronounced in intracellular and often chronic infections. while the numbers of described pidd patients increase, at first seemingly rarely pidd-associated infections turn out to be found in a significant subset of pidd patients [ ] [ ] [ ] . for example, patients with cd l deficiency living in endemic areas display susceptibility to bartonellosis and paracoccidioidomycosis, infections not described in european and us cohorts [ , , ] .often, an infectious phenotype previously only described in secondary immunodeficiencies may reveal the possibility of an underlying primary immunodeficiency [ , , ] . increasing numbers of genetic defects causing early-onset, severe, and recurrent susceptibility to commonly circulating pathogens like pneumococci, tuberculosis, herpes simplex, and influenza viruses as well as endemic protozoans like trypanosomes and fungi are being recognized, and thus, infections with unusual pathogens require a high index of suspicion for pidd [ ] . in contrast, pidds may also manifest as suspected infection but sterile inflammation. for example, in inflammatory lesions like granulomas and necrotizing fasciitis where no clear pathogens are found, one needs to rule out aberrant host responses due to pidd [ ] .chronic viral and fungal infections may also display novel phenotypes never or rarely seen in secondary immunodefic i e n c i e s . i n f e c t i o n s l i k e d e r m a t o p h y t o s i s a n d phaeohyphomycosis deeply infiltrating the skin and lymph nodes, occasionally extending to bones and central nervous system (cns) as well as predisposition to primary cns candidiasis and extrapulmonary aspergillus slowly revealed the full phenotypic spectrum of card deficiency [ , , ] . chronic skin ulcers caused by hsv- and severe molluscum contagiosum suggest dock deficiency or gain-of-function mutations of stat [ , ] . chronic mucocutaneous candidiasis has revealed a large group of monogenic diseases (il ra, il rc, il f, stat (gof), rorx, act ), which may also be associated with recurrent bacterial infections or syndromic features [ ] . while novel diseases by newly described viruses are being discovered, one needs awareness to suspect these in pidd patients [ ] .interestingly, most novel forms of infectious disease in pidd patients have been described either in easily accessible sites like the skin or in immunologically privileged, normally sterile sites like the cns. this suggests that with the increasing use of invasive sampling and sensitive metagenomic approaches, we might find more novel infectious phenotypes. pathogens highly suggestive of certain pidds, like chronic enteroviral cns infections in xla patients are reviewed above. in hypomorphic mutations, cns seems to be especially vulnerable to chronically active and/or recurrent novel infectious bsmoldering^focal encephalitis lesions by pathogens key: cord- -hww watl authors: li, wenchao; jin, hongyan; sui, xiukun; zhao, zhanzhong; yang, chenghuai; wang, wenquan; li, junping; li, gang title: self-assembly and release of peste des petits ruminants virus-like particles in an insect cell-baculovirus system and their immunogenicity in mice and goats date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: hww watl peste des petits ruminants (ppr) is an acute, febrile, viral disease of small ruminants that has a significant economic impact. for many viral diseases, vaccination with virus-like particles (vlps) has shown considerable promise as a prophylactic approach; however, the processes of assembly and release of peste des petits ruminants virus (pprv) vlps are not well characterized, and their immunogenicity in the host is unknown. in this study, vlps of pprv were generated in a baculovirus system through simultaneous expression of pprv matrix (m) protein and hemaglutin in (h) or fusion (f) protein. the released vlps showed morphology similar to that of the native virus particles. subcutaneous injection of these vlps (pprv-h, pprv-f) into mice and goats elicited pprv-specific igg production, increased the levels of virus neutralizing antibodies, and promoted lymphocyte proliferation. without adjuvants, the immune response induced by the pprv-h vlps was comparable to that obtained using equivalent amounts of pprv vaccine. thus, our results demonstrated that vlps containing pprv m protein and h or f protein are potential “differentiating infected from vaccinated animals” (diva) vaccine candidates for the surveillance and eradication of ppr. peste des petits ruminants (ppr) is a highly contagious and economically important viral disease of domestic and some wild small ruminants, and in particular, of goats and sheep. it is notifiable to the office international des epizooties (oie). clinically, the disease is characterized by severe pyrexia, oculonasal discharges, necrotizing and erosive stomatitis, enteritis, and pneumonia. it was first described in the ivory coast, west africa, but has now become wide-spread in sub-saharan africa, arabia, the middle east, southwest asia, india, and other countries [ ] . in china, ppr was first reported in tibetin [ ] , and in december , a ppr outbreak was reported in xinjiang yili; in this outbreak, goats were infected, of which died, and goats in the susceptible population were killed [ ] . thus, ppr outbreaks can cause severe economic losses, because they often result in high morbidity and mortality; therefore, development of an effective vaccine for the prevention and control of ppr is particularly important. the causative agent, ppr virus (pprv), is a member of the family paramyxoviridae and the genus morbillivirus [ ] .the nonsegmented, single stranded, negative-sense rna genome of pprv encodes six structural proteins (nucleocapsid [n] , phosphoprotein [p] , matrix [m] , fusion [f], hemagglutinin [h] , and polymerase [l] proteins), and two non-structural proteins(c and v). proteins n, p, and l are required for reconstituting viral rna polymerase activity; m protein is required for particle formation and budding, and the two surface glycoproteins, h and f, are required for attachment and entry into the host cell. structurally, the virions are morphologically pleomorphic particles that are surrounded by a host-derived membrane, modified by the h and f transmembrane glycoproteins, as well as the m protein, which is associated with the inner surface of the viral membrane [ , ] .as in other paramyxoviruses, the c protein of pprv is expressed from an alternative open reading frame (orf) within the p gene, whereas the v protein is expressed by rna editing [ ] . these two proteins are thought to be involved in viral evasion of the host's immune responses, specifically by blocking interferon production during infection [ ] . for the control of ppr, four attenuated vaccines are available commercially. in endemic areas, the virus is currently controlled using the nigeria / strain (nig / ), which has been attenuated by serial passage in vero cells. in india, three other live attenuated vaccines are currently licensed for use: sungri , arasur , and coimbatore [ ] . however, a major disadvantage of these attenuated vaccines is that they cannot be used for differentiating infected from vaccinated animals(diva).recently, subunit vaccines have been developed based on the incorporation of pprv immunogens into various vector systems, such as sheep or goat pox, vaccinia virus, adenovirus, or baculovirus vectors [ , , , ] . virus-like particles (vlps) recently emerged as alternatives to subunit vaccines; these have the advantage that they mimic the organization and conformation of the native virus capsid and envelope, but lack the viral genome [ , ] .given their intrinsic immunogenic properties and high safety profile, vlps have been generated in different heterologous expression systems and have been tested as vaccine candidates for a variety of viral diseases [ ] . in the human vaccines market, six vlp-based vaccines, including three for hepatitis b (recombivax hb, engerix b, and genhevac b), two for human papillomavirus (gardasil and cervarix), and one for influenza a (inflexalv), have been developed [ , ] .in the veterinary field, a vlp-based vaccine against porcine circovirus type (pcv ), viz., porcilis pcv, intervet, has recently been licensed [ ] . to date, the assembly and release of pprv vlps have not been well characterized, and their immunogenicity remains unknown. it is known that the pprv glycoproteins h and f contain neutralizing antibody epitopes and several t cell epitopes [ , ] , and that pprv-specific lympho proliferative responses can be elicited in goats immunized with a recombinant adenovirus expressing the h or f protein [ ] . therefore, we hypothesized that immunization with vlps containing h or f proteins could induce a strong pprv-specific immune response. in this study, two types of vlps for pprv (pprv-h and pprv-f)were developed, using viral m protein co-expressed with h or f protein. mice and goats immunized subcutaneously with these pprv vlps developed both humoral and cellular immune responses. cell culture and viruses pprv vaccine strain (nig / ) was obtained from the china institute of veterinary drug control, beijing, china. vero cells (the american type culture collection, atcc: ccl- , manassas, va, usa) were cultured in dulbecco's modified eagle medium (dmem; invitrogen, grand island, ny, usa) containing % fetal bovine serum (fbs; invitrogen, grand island, ny, usa) at uc with % co , and were used for pprv propagation and titration. spodoptera frugiperda sf insect cells were maintained in sf- ii insect serum-free medium (invitrogen, grand island, ny, usa) as monolayer cultures or in suspension cultures maintained on temperate orbital shakers ( rpm) at uc. cloning of m, h, and f genes and construction of bacmid transfer plasmids pprv rna was extracted using trizol ls (invitrogen, carlsbad, ca, usa). reverse transcription(rt) and polymerase chain reaction (pcr) were performed on extracted viral rna with gene-specific oligonucleotide primers( table ) that had been designed according to the sequence of the pprv nig / strain (genbank accession no. x . ).following rt-pcr, the cdna fragments containing the pprv m, h, and f genes were cloned into the pmd -t vector (takara, dalian, china). the integrity of the nucleotide sequences of the m, h, and f genes was verified by dna sequencing. the transfer plasmids were generated using the pfastbac dual vector (invitrogen, grand island, ny, usa), which contains two promoters. the m gene was cloned, as a -kbxhoi-kpnidna fragment, downstream of the p promoter within the pfastbac-dual bacmid transfer vector that had been digested with xhoi and kpni. the resulting baculovirus transfer plasmid containing the pprv m gene was designated p-m. a bacmid transfer vector expressing both the m and h genes was prepared by cloning the h gene, as a . -kb bamhi-hindiii dna fragment, into the bamhi/hindiii sites downstream of the autographacalifornica multiple nuclear polyhedrosis virus (acmnpv) polyhedron(p h ) promoter in p-m. the resulting plasmid, p-mh, encoded the m and h genes downstream from the p and p h promoters, respectively. similarly, a bacmid transfer vector,p-mf, which permitted expression of both the m and f genes, was prepared by cloning the f gene, as a . -kb bamhi-xbai dna fragment, downstream of the p h promoter, into p-m (fig. ). recombinant bacmids were produced by site-specific transpositioning after transformation of bacmid transfer plasmids containing the pprv genes into e.coli dh bac competent cells(invitrogen, carlsbad, ca, usa), which contained the acmnpv baculovirus genome. the recombinant bacmids were identified by pcr using m primers together with gene-specific primers ( table ). the recombinant bacmid dna was transfected into sf insect cells using cell fectin ii (invitrogen, grand island, ny, usa), according to the bac-to-bac expression systems manual (invitrogen, grand island, ny, usa). briefly, a transfection mixture that contained mg of recombinant bacmid dna, ml of cell fectinii, and ml of unsupplemented grace's medium (invitrogen, grand island, ny, usa), was prepared. following incubation at room temperature for min, the mixture was added to sf insect cells, which had been seeded at cells per well, in -well plates. the transfection mixture and cells were incubated for h at uc; the transfection mixture was then removed and ml serum-free sf- ii medium was added. the cultures were incubated at uc for days and the supernatants were harvested as the first passage (p ) viral stock. cells were continuously passaged in order to amplify baculovirus stock with the highest viral titer. the recombinant baculovirus stock was titrated by agarose plaque assay on sf insect cell cultures in -well plates. to produce pprv-h vlps, sf insect cells were infected in a -ml volume, at a cell density of cells per ml, with rbvs expressing pprv h and m proteins, at a multiplicity of infection (moi)of . similarly, pprv-f vlps were produced by infecting sf insect cells with rbvs expressing the pprvf and m proteins. cell culture supernatants were harvested days post-infection, cleared by low-speed centrifugation ( g, min at uc)to remove cells, and vlps in the supernatants were then pelleted by ultracentrifugation using a sorvallt rotor ( , g, for h, at uc). the resulting pellets were dissolved in ml of phosphate-buffered saline solution (pbs, ph . ), and were then further purified through a discontinuous sucrose gradient ( - - % w/v, in pbs) and sedimented by ultracentrifugation in a sorvallth swinging bucket rotor ( , g, for h, at uc). as a control, we used the pprv nig / virus. the visible opalescent bands in the sucrose gradient were collected, diluted in pbs, and pelleted by centrifugation in a sorvall t rotor ( , g, for h, at uc) to remove the sucrose. the sedimented particles were resuspended in pbs and stored at uc. the concentration of the vlps was determined using a bicinchoninic acid protein assay kit (bca protein assay kit; beyotime, shanghai, china). to detect the protein reactivity of vlps, purified vlps were boiled for min in reducing laemmli sample buffer and resolved by % sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then transferred to a polyvinylidene difluoride membrane by electroblotting (bio-rad, hercules, ca, usa). the membrane was blocked with % dried non-fat milk in pbst ( . % tween- in pbs), overnight at uc, and then incubated with goat serum against pprv (diluted : in blocking buffer), for h at uc. the membrane was washed three times with pbst, for min each time, and then incubated with horseradish peroxidase (hrp)conjugated rabbit anti-goat igg antibody (diluted : in blocking buffer; zhongshanjinqiao, beijing, china). following several washes with pbst, the specific bands were detected using the substrate , -diaminobenzidine tetrahydrochloride (tiangen, beijing, china) for min at room temperature. the reaction was stopped with water and membranes were photographed. to examine budding of vlps, sf insect cells that had been infected with rbvs expressing the m and h/f proteins were fixed with . % glutaraldehyde and % osmium tetraoxide, dehydrated with ethanol, and then embedded in spi-pon resin (structure probe, west chester, pa, usa). thin sections were stained with lead citrate and uranyl acetate and then observed by electron microscopy [ ] . the presence and morphology of the purified vlps and pprv were evaluated by tem of negatively stained preparations. a carbon-coated grid was set on a -ml sample drop for min, and washed with deionized water. washed samples were negatively stained with % uranyl acetate at ph . . excess stain was removed using filter paper, and the grids were observed at magnifications ranging from , to , on a transmission electron microscope (h- , hitachi, tokyo, japan) operating at kv. the designs of the animal experiments are shown in table . the animal experiments were conducted with prior approval from the animal care and use committee of chinese academy of agricultural sciences, china. all animal experiments were carried out in accordance with the requirements of the regulations of experimental animal administration of the pr china. all efforts were made to minimize suffering. the proteins that were used to vaccinate animals were purified by sucrose gradient ultracentrigugation and the concentration was determined by using a bca protein assay kit (bca protein assay kit; beyotime, shanghai, china). for mouse experiments, female inbred balb/c mice (vital river, beijing, china), aged to weeks, were randomly divided into seven groups (n = for each group). mice were immunized subcutaneously (s.c.) with different amounts ( or mg total protein per mouse) of pprv vlps or pprv nig / diluted in ml of pbs. all groups received a booster injection of the original dosage at weeks after the primary immunization. no adjuvant was used for any of the mice. blood samples were collected by retro-orbital plexus puncture, at various time-points. after the blood samples were allowed to clot and then centrifuged, serum samples were collected and stored at ucuntil required for antibody titration. for goat experiments, outbred goats ( - months of age) were randomly divided into four groups of five each, and housed separately. none of the goats had detectable levels (titers less than : ) of pprv neutralizing antibodies. groups and were vaccinated subcutaneously with mg pprv-h or pprv-f vlps diluted in ml of pbs, respectively. group goats were inoculated with ml pprv nig / ( . tcid ), as positive control. group goats were inoculated subcutaneously with ml pbs as negative control. booster vaccination was performed using the same dose, at weeks after primary immunization. serum samples were collected at various time-points post-immunization, and stored at uc until analyzed. sera from immunized and control mice were analyzed for pprv-specific antibodies (igg, igg , and igg a) by indirect elisa. briefly, -well flat-bottomed plates were coated with mlof purified pprv whole-virus antigen at a concentration of mg/ml per well in . m carbonate/bicarbonate coating buffer (ph . ),overnight at uc, and then the plates were blocked with % skim milk in pbst, for h at uc. serum samples, serially diluted in pbst, were added to the wells, in duplicate, and incubated at uc for h. after washing with pbst three times, each well received ml of a / -diluted hrp-labeled goat anti-mouse igg, igg , and igg a (southern biotech, birmingham, al, usa) and incubated at uc for h. finally, after washing with pbst three times, ml of freshly prepared tetramethylbenzidine (tiangen, beijing, china) solution was added to each well and color was allowed to develop for min, before stopping the reaction with ml of m h so . optical density (od) values were measured at a wavelength of nm using an elisa microplate reader (multiskan ascent; thermo, vantaa, finland). the mean titers were calculated from log of the highest serum dilution that yielded a two-fold higher od value compared to that of sera from pbsvaccinated mice. all serum samples from immunized mice and goats were analyzed for the detection of pprv-specific virus neutralization antibody (vna) titers using a micro-neutralization assay, according to the oie terrestrial manual [ ] . briefly, serum samples were complement-inactivated at uc for min and were then serially diluted two-fold (mice sera, from : ; goat sera, from : ) table . experimental designs used for the animal studies. in cell culture medium. the diluted sera ( ml) were mixed with an equal volume of pprv ( tcid /ml) in a -well cell culture plate and incubated at uc for h; then, ml of vero cell suspension was added to each well and the cells were cultured at uc in the presence of % co .a series of control wells for virus and uninfected cells were also included. cells were observed for a cytopathic effect (cpe) by microscope at day after incubation, and the vna titer was calculated. vna titer was defined as the highest dilution of the test serum at which the cpe was inhibited by at least %. three mice from different groups were sacrificed at days after booster immunization and splenocyte suspensions were prepared as previously described [ ] . splenocytes were seeded in -well flat-bottom plates at a density of cells per well in rpmi medium (invitrogen, grand island, ny, usa) containing % fbs. splenocytes were stimulated in vitro with ml of uvinactivated pprv nig / ( tcid ). concanavalin a (sigma-aldrich, st. louis, mo, usa) was used as positive control, and medium was used as negative control. after days' incubation at uc in a % co atmosphere, the proliferative response was determined using a -( , -dimethylthiazol- -yl)- -( -carboxymethoxyphenyl)- -( -sulfophenyl)- h-tetrazolium, inner salt (mts) assay, using the cell titer aqueous one solution cell proliferation kit (promega, madison, wi, usa), following the manufacturer's instructions. the lymphocyte proliferation rate was quantified using the stimulation index (si), which was calculated as the ratio of the od of the stimulated cells to the od of the negative controls. goats were bled via jugular vein puncture at week after the booster injection. peripheral blood mononuclear cells were prepared as previously described [ ] . cells( cells per well) were plated in -well plates and stimulated with inactivated pprv nig / for h at uc under % co . unstimulated cells, cultured in medium alone, were used as negative control. the proliferative response was determined by mts assay, as described above. interferon (ifn)-celispot assays splenocytes from inoculated mice were obtained and plated at a density of cells per well in a -well elispot plate, which had been coated with ifn-c. one hundred microliters of inactivated pprv nig / was added as stimulus. the cultures were then incubated in a uc humidified incubator, with % co , for h, and then spots were detected as per the manufacturer's instructions (mabtech, nacka, sweden). the data were analyzed with one-way anova in graphpad prism version . (graphpad software, san diego, ca, usa); pvalues less than . were considered statistically significant. the open reading frames of pprv m, h, and f genes from pprv strain nig / were amplified by rt-pcr and sub-cloned into pfastbacdual vectors (fig. ).the insertion of the genes into the expression vector was confirmed by enzyme digestion analysis and dna sequencing. the rbvs were generated after transfection of sf insect cells with recombinant bacmids. the titers of rbvs-pprv mh and rbvs-pprv mf were titrated in a plaque-forming assay, and exhibited titers of . and . pfu/ml, respectively, at the third passage. pprv-h vlps and pprv-f vlps were produced and purified as described in the materials and methods section. the vlps bands were mainly distributed between % and %sucrose in the gradient after ultracentrifugation ( fig. a) . electron microscopic examination of negatively stained samples revealed that both pprv-h and pprv-f vlps showed spherical shapes, with spikes on their surfaces, and with a diameter of approximately - nm, which is similar to the morphology and size of native pprv particles propagated in vero cells (fig. e-g ).an ultrathin section of sf insect cells infected with rbvs at -h post-infection was investigated, revealing vlps with a diameter of approximately nm budding from the plasma membrane, and some spikeless vlps distributed throughout the cytoplasm (fig. b-c) . incorporation of the h or f proteins, as well as m protein, into vlps was confirmed by western blotting using anti-pprv antibodies, by the presence of kda, kda, and kda bands, corresponding to the m, h, and f proteins, respectively, on the blots (fig. ) . the sizes of the m and h proteins corresponded to those predicted from the deduced protein sequences, but the f protein band derived from vlps differed from the form of pprv particles propagated in mammalian cells. in the mammalian cell expression system, the f protein is synthesized as a precursor (f ) of about kda; after translation, f is glycosylated and cleaved to yield a -kda (f ) and -kda (f ) disulfide-link-competent f protein. in the baculovirus-insect cell expression systems, the pprv f protein was generated as a -kda band in the western blot membrane, indicating that the f protein was not cleaved and was generated as the f precursor, which was also glycosylated. in total, the amount of the expressed vlps was about - . mg/l in the cell culture medium. the specific antibody responses of mice immunized with pprv-h vlps or pprv-f vlps were determined by indirect elisa. the levels of total pprv-specific igg in the serum after primary and booster vaccinations were determined; the results are shown in fig. . total igg titers in all groups of mice immunized with vlps or pprv were higher after the booster than after the primary vaccination, indicating that booster immunization could stimulate the immune memory cells and induce an anamnestic response, promoting antibody production and exaltation. when mice were immunized with a lower antigen dose of mg, total igg titers of mice immunized with pprv-h vlps were significantly higher than those of mice immunized with pprv-f vlps or pprv after the primary vaccination, indicating that the titers of antibodies against pprv-h vlp proteins increased faster than the titers of antibodies against pprv-f vlps or pprv. the maximal titers detected in both vlp-and virus-immunized mice were similar after the booster vaccination. similar patterns of total igg responses were found in mice immunized with mg vlps or pprv. pprv-specific serum antibodies of igg and igg a were also evaluated by elisa. these results demonstrated that mice immunized with pprv-h vlps and pprv using a lower dose of antigen ( mg) induced high igg serum antibody titers( : to : ), and lower specific igg a serum antibody titers( : to : ) after the first immunization. in contrast, the levels of igg antibody responses were lower ( : ) ,and the igg a responses were higher ( : ),in mice immunized with pprv-f vlps relative to those observed in mice immunized with pprv-h vlps or pprv. the levels of igg and igg a from all three immunized groups of mice increased after the booster immunization (fig. a) . similar patterns of igg and igg a responses, as well as igg :igg a ratios, were found in mice immunized with mg vlps or pprv (fig. b) . taken together, the data indicated that immunization with pprv-h vlps induced predominantly specific igg serum antibodies, favoring the stimulation of a t-helper class (th- ) immune response against pprv, while immunization with pprv-f vlps induced a stronger th- immune response, despite the use of a lower immunization dose. since the neutralizing activity against pprv is an indicator of functional antibodies that confer protective immunity, we investigated whether mice and goats immunized with pprv vlps could produce neutralizing antibodies, using a micro-neutralization assay. serially diluted sera from various time-points postimmunization were complement-inactivated, and incubated with live pprv nig / ; the cpe assays were conducted in vero cells. as shown in table , pprv-h or pprv-f vlps-vaccinated mice developed vna, and the titers ranged from : to : and from : to : , after booster immunization. as shown in table , at weeks after primary immunization, the titers of vna in the pprv-h vlps-and pprv-vaccinated goat groups exceeded , a number that represents a margin considered to be protective in goats vaccinated with live attenuated pprv vaccines [ ] . moreover, this value was significantly enhanced after the booster immunization and reached values of - ; these high vna titers were maintained for at least weeks, and decreased gradually from weeks after primary immunization. the titer of the pprv-f vlps group was lower compared to that of the pprv-h vlps-and pprv-vaccinated goat groups. however, weeks after the booster immunization, all the goats that had been immunized with pprv-f vlps showed vna titers above . taken together, the data of the mouse and goat vaccination experiments showed that serum vna levels were significantly higher in the pprv-h vlps-immunized group than in the pprv-f vlps-immunized group, and were comparable to that of the whole virus-immunized group. no neutralization activity against pprv was detected when using pre-immune serum or in the sera of experimental animals injected with pbs. to investigate the cell-mediated immune responses induced by vlps, the lymphocyte proliferation response of mice was analyzed after the booster immunization. as shown in table , compared with the pbs control group, mice immunized with vlps or pprv exhibited significantly stronger lymphocyte proliferation responses (p, . ). as shown in table , the data from the lymphocyte proliferation assay of goats from the three groups immunized with pprv-h vlps, pprv-f vlps, or attenuated pprv indicated similarly strong cellular immune responses. there was no significant difference among mice immunized with different concentrations of vlps or pprv (p. . ). we also evaluated the mounting of pprv-specific t-cell responses in vaccinated mice. splenocytes, obtained at days after the booster immunization, were cultured in the presence of inactivated pprv, and ifn-c production was measured by elispot assays (fig. ). both pprv-h vlps-and pprv-f vlps-vaccination induced specific ifn-c production in response to pprv exposure; the frequency of cells secreting ifn-c positively correlated with the immunization dose. no specific production of ifn-c in response to pprv exposure was detected in control mice inoculated with pbs. altogether, these data demonstrated that inoculation with pprv-h vlps and pprv-f vlps can generate specific t-cells that recognize the pprv h and f proteins in vivo. the minimum molecular requirement for efficient assembly and release of pprv virions is still unknown. in this study, we found that the major structural genes of pprv can be efficiently expressed in recombinant baculovirus-infected sf insect cells and that these genes can then support the production of pprv vlps. the expression of m protein was necessary and sufficient for the formation of vlps (data not shown); the additional expression of the h or f glycoproteins allowed formation of budding particles with the typical morphology and size of pprv and related paramyxoviruses. a recent study demonstrated that pprv spikeless vlps could be formed by co-expression of the m and n proteins in insect cells; however, expression of m protein alone in insect cells did not result in vlp formation, as assessed by tem [ ] , which was contrary to our results. the finding that m protein alone could be released from cells and generate vlps has also been observed for some other paramyxoviruses, including human parainfluenza virus type [ ] , sendai virus [ ] , newcastle disease virus [ ] , and nipha virus [ ] , as well as for viruses in other related virus families, including ebola [ ] and vesicular stomatitis virus [ ] .in addition, some paramyxovirus m proteins, such as parainfluenza virus type [ ] and mumps virus [ ] , lack the ability to direct efficient vlp production when expressed alone in cells, but vlp production becomes highly efficient when m proteins are expressed together with other viral proteins. hence, although the requirements for efficient vlp production differ among paramyxoviruses, it appears as if, in general, m protein is the major requirement for paramyxovirus vlp production, as vlps cannot be formed in its absence [ , ] . the baculovirus expression vector-insect cell system has been utilized extensively for vlp production, and particularly for production of enveloped vlp vaccines [ , , ] . baculovirus expression of heterologous genes permits multiple post-translational modifications, like folding, oligomerization, phosphorylation, glycosylation, acylation, disulfide bond formation, proteolytic cleavage, and so on, which are similar or identical to those that occur in mammalian cells. however, in this study, the molecular size of the expressed f protein was kda, but the f protein was cleaved into f and f protein upon expression in mammalian cells; the results indicated that there are differences in the posttranslational modification in the two expression systems. furthermore, insect cells are able to grow in serum-free medium cultures; this is highly desirable, because serum may be a cause of adventitious viruses. therefore, its use requires more intensive purification processes to remove serum proteins. additionally, these cells are perfectly adapted to suspension conditions, allowing for large-scale culture [ , ] . these advantages make the baculovirus expression vector-insect cell system a promising platform for production of vlps [ ] . in this study, we chose the pfastbacdual (invitrogen) baculovirus transfer vector, which contains two promoters, so that insect cells infected with a single recombinant bicistronic baculovirus could express two proteins concurrently. the coexpression strategy is particularly important for the efficient assembly of multi-protein complexes, where simultaneous expression of multiple viral proteins is essential [ ] .co-expression and co-infection strategies have previously been compared in the production of triple-layered rotavirus vlps(vp ,vp , and vp ), and sars vlps. for production of rotavirus vlps, co-expression was more efficient than co-infection [ ] . sars vlps could not be generated in sf cells co-infected with three recombinant viruses expressing the m, e, and s proteins, but spontaneous assembly of highly stable vlps occurred when using a single recombinant baculovirus expressing all three sars proteins simultaneously [ ] . the main limitation of the baculovirus expression vector-insect cell system is the co-production of recombinant baculoviruses (enveloped viruses) during the infection process; thus, when developing pprv vlp vaccines, the challenging task is the table . peste des petitsruminants virus (pprv) neutralizing antibody titers and lymphocyte proliferation responses in goats immunized with pprv virus-like particles (vlps) or native pprv particles. purification of vlps from the recombinant baculoviruses, which share a similar density and enveloped structure [ , ] . in our study, although purification was possible by centrifugation in sucrose density gradients, residual baculoviruses were consistently detected in vlp preparations (fig. d) .given the safety concerns, vlps should be purified via chemical (binary ethylenimine) or physical(uv irradiation) inactivation treatments, to eliminate any possible baculovirus infectivity when considering clinical trials. for many viruses, recombinant vlps have been shown to yield promising vaccines, because of their robust immunogenicity, elicitation of protective neutralizing antibodies (due to the presence of conformational epitopes on vlps), and their safety profiles (as they do not contain any viral genetic material). in this study, the effectiveness of pprv vlps as an immunogen was demonstrated in mice and goats. the results indicated that high specific antibody titers against pprv could be induced by both pprv-h vlps and pprv-f vlps, which were as high as those resulting from immunization with pprvnig / . the vna titers elicited by pprv-h vlps were much higher than those induced by pprv-f vlps, and the vna titers of pprv-h vlps were as high as the responses to the attenuated virus vaccine. previous reports have shown that the h protein of pprv is a more potent inducer of high vna titers than the f protein [ ] , while the f protein seems to induce mainly a cellular immune response [ ] .our results demonstrated that mice immunized with pprv-h and pprv-f vlps exhibited a similarly high level of cell-mediated immune responses. we further showed that subcutaneous immunization of mice with pprv-h vlps at a dose of mg, in the absence of adjuvant, induced strong humoral immune responses. this immunogenicity is related to the structural features of the vlps, which are particulate structures that contain a dense and repetitive pattern of epitopes on their surfaces. in contrast, monovalent, non-particulate antigens do not always reflect the native structure of the protein, and the related antibody responses are not always specific for physiologically relevant epitopes. consequently, administration of these vaccines often requires use of strong adjuvants, as well as large and frequent doses of the antigen. in contrast, vlp-based vaccines are strongly immunogenic, often reducing or eliminating the need for exogenous adjuvants [ , ] . an additional benefit of this vlp-vaccination approach is that it may allow improved surveillance through diva, as seroconversion to n protein will occur only in animals that have been infected with pprv, but not in vlp-vaccinated animals that have not been infected. a commercially available n protein-based competitive elisa kit might strengthen the usefulness of vlp vaccines [ ] . we also detected pprv-specific antibodies in the vaccinated goats, using an n protein-based competitive elisa established in our laboratory. no n protein-specific antibodies were found in goats vaccinated with pprv h or pprv f vlps; in contrast, seroconversion to n protein was detected in goats vaccinated with the pprv nig / attenuated virus (data not shown). to our knowledge, this is the first report that demonstrated the construction of pprv vlps consisting of immunogenic proteins and that assessed their immunogenicity in two animal models, viz., mice and goats. taken together, the results of our study suggested that pprv vlps can be generated in insect cells simultaneously expressing the pprv m protein with h or f protein. the vlps had a morphology and size similar to those of native virus particles. humoral and cellular immunity against pprv were efficiently induced in mice and goats vaccinated with these pprv vlps, without the need for adjuvants. thus, pprv-h vlps or pprv-f vlps may be promising vaccine candidates for ppr, and diva tests may prove of great benefit in future control programs for this disease. . specific interferon (ifn)-cproduction in response to peste des petits ruminants virus (pprv) proteins in splenocytes from mice, as detected by elispot assay. splenocytes from mice inoculated with pprv virus-like particles (vlps), pprv per se, or phosphate-buffered saline (pbs) were isolated and cultured for h in the presence of inactivated pprv. the production of ifn-cwas measured using an elispot assay. samples were tested in triplicate and mean ifn-cvalues for each mouse are shown. doi: . /journal.pone. .g global distribution of peste des petits ruminants virus and prospects for improved diagnosis and control peste des petits ruminants virus in tibet / / : peste des petits ruminants, (people's rep. of china) classification of peste des petits ruminants virus as the fourth member of the genus 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kinetics of vector replication, mrna stability and recombinant protein production influenza viruslike particles comprised of the ha, na, and m proteins of h n influenza virus induce protective immune responses in balb/c mice development of a dual recombinant vaccine to protect small ruminants against peste-des-petits-ruminants virus and capripoxvirus infections assembly and biological and immunological properties of newcastle disease virus-like particles pandemic h n influenza virus-like particles are immunogenic and provide protective immunity to pigs development of a competitive elisa for detecting antibodies to the peste des petits ruminants virus using a recombinant nucleoprotein we thank the electron microscopy core facility of the institute of agroproducts processing, science, and technology, caas, for tem analysis. we also thank dr. huailiang ma for kindly editing this manuscript. conceived and designed the experiments: wl gl. performed the experiments: wl xs. analyzed the data: wl zz hj. contributed reagents/materials/analysis tools: jl cy ww. wrote the paper: wl zz. requested financial support: gl. key: cord- - mz w authors: xu, zhen-dong; wang, yong; liang, ge; liu, zhi-qiang; ma, wu-hua; chu, charleen t; wei, hua-feng title: propofol affects mouse embryonic fibroblast survival and proliferation in vitro via atg - and calcium-dependent regulation of autophagy date: - - journal: acta pharmacol sin doi: . /s - - -z sha: doc_id: cord_uid: mz w propofol is a commonly used intravenous anesthetic agent, which has been found to affect cell survival and proliferation especially in early life. our previous studies show that propofol-induced neurodegeneration and neurogenesis are closely associated with cell autophagy. in the present study we explored the roles of autophagy-related gene (atg ) in propofol-induced autophagy in mouse embryonic fibroblasts (mef) in vitro. we showed that atg was functionally related to propofol-induced cell survival and damage: propofol significantly enhanced cell survival and proliferation at a clinically relevant dose ( µm), but caused cell death at an extremely high concentration ( µm) in atg (−/−) mef, but not in wt cells. the dual effects found in atg (−/−) mef could be blocked by intracellular ca( +) channel antagonists. we also found that propofol evoked a moderate (promote cell growth) and extremely high (cause apoptosis) cytosolic ca( +) elevation at the concentrations of µm and µm, respectively, only in atg (−/−) mef. in addition, atg (−/−) mef themselves released more ca( +) in cytosolic space and endoplasmic reticulum compared with wt cells, suggesting that autophagy deficiency made intracellular calcium signaling more vulnerable to external stimuli (propofol). altogether, our results reveal that atg plays a crucial role in propofol regulation of cell survival and proliferation by affecting intracellular ca( +) homeostasis. autophagy-related gene (atg ) is one of the key proteins regulating autophagy activity [ ] [ ] [ ] , which is also associated with intracellular ca + homeostasis [ ] [ ] [ ] . atg has been demonstrated to affect cell survival and proliferation via its effects on autophagy [ ] [ ] [ ] . similar to other important autophagy regulators, such as mtor and beclin- , atg can be targeted to change autophagy activity and therefore affect the pathology of some diseases, such as parkinson's disease [ ] , myeloid leukemia [ ] , and some tumors [ , ] . atg knockout cells are generated [ , ] and available so that the effects of drugs on cell physiology/pathology via the function of atg and associated autophagy activity can be investigated conveniently. our previous studies suggested that propofol can regulate cell autophagy via activation of insp (insp r) or ryanodine receptors (ryr) [ , ] , with unclear mechanisms. calcium may also be involved in autophagy by affecting atg . elevation of intracellular ca + levels can activate atg , which initiates autophagy [ ] . interestingly, reducing intracellular ca + prevents the cleavage of atg , which in turn increases the levels of full-length atg and atg -atg conjugate [ ] . considering the essential role of atg in autophagy and calcium regulation [ ] , it is plausible that propofol may affect cell survival or proliferation by varying the function of atg . although propofol at high concentrations has been demonstrated to cause neurotoxicity and impairment of neurogenesis [ ] [ ] [ ] , the effects of propofol at clinically relevant low concentrations on autophagy and cell function need to be investigated. here, we studied the effects of propofol on cell survival, growth and autophagy activity in both wild-type (wt) and atg knockout cells (atg −/− ). we demonstrated that atg plays an important role in propofol effects on autophagy, cell survival, and growth due to its effects on the regulation of intracellular ca + homeostasis. cell culture wt and atg −/− mouse embryonic fibroblast (mef) cell lines purchased from riken bio resource center (tsukuba, ibaraki - , japan) were maintained in dulbecco's modified eagle's medium, supplemented with % fetal bovine serum and % penicillin/streptomycin (both from life technologies, carlsbad, ca, usa) and were cultured in a % co humidified atmosphere at °c. the culture medium was changed every two days. cells with %- % confluence were obtained from t-flask cultures using trypsinization and incubated onto the disk samples. the cell viability/proliferation was determined using the mtt ( -( , dimethylthiazol- -yl)- , -diphenyltetrazolium bromide, sigma-aldrich, st. louis, mo, usa) assay at , , and h as previously described [ ] . after washing with pbs, the cells were incubated with fresh culture medium containing mtt ( . mg/ml in the medium) at °c for h in the dark. the medium was then removed, and formazan was solubilized with dimethyl sulfoxide. the absorbance of this solution in each well was quantified by spectrophotometry at nm using a synergy™ h microplate reader (biotek, winooski, vt, usa). cell viability was further confirmed by the trypan blue assay. then, × cells per well were seeded onto -well plates. following propofol treatment, cell viability was determined by trypan blue exclusion assay. cells were mixed with . % trypan blue solution for min at room temperature. the cell number was countered with a hemocytometer in a bright field under a microscope as described previously [ ] . cells without trypan blue entry were regarded as viable. cytotoxicity was determined by lactate-dehydrogenase (ldh) detection with an ldh-release assay kit (thermo scientific, rockford, il, usa) according to the manufacturer's instructions, as described previously [ ] . briefly, μl of supernatant was collected and transferred to each well of a new -well plate. then, μl of reaction mixture was added to each well. after min of incubation at room temperature, μl of stop solution was added to terminate the reaction. the nm absorbance of the samples was measured along with background absorbance at nm using a synergy™ h microplate reader (biotek, winooski, vt, usa). cell proliferation assays wt and atg −/− mefs were plated onto cover glasses in culture medium. -bromodeoxyuridine (brdu, invitrogen, eugene, or, usa) was added to the culture medium for h before the end of treatment with a final concentration of μm. the cells were then fixed in % paraformaldehyde and permeabilized with . % triton x- . for brdu detection, acid treatment ( n hcl min on ice followed by n hcl min at room temperature) separated dna into single strands so that the primary antibody could access the incorporated brdu. after incubation with blocking solution ( % normal goat serum in pbs containing . % triton x- ), the cells were incubated with rat monoclonal anti-brdu primary antibody ( : ; santa cruz biotechnology, dallas, tx, usa) overnight at °c. after a subsequent wash with pbs containing . % triton x- , the cells were incubated with fluorescently labeled secondary antibody conjugated with anti-rat igg ( : ; invitrogen, eugene, or) for h at room temperature. cell nuclei were counterstained with ′, diamidino- -phenylindole (dapi, invitrogen, eugene, or, usa) for min at room temperature. the immunostained cells were covered and then mounted on an olympus bx tf fluorescence microscope ( ×; olympus usa, center valley, pa, usa). images were acquired using ivision . . software (biovision technologies, exton, pa, usa). five sets of images were acquired at random locations on the cover glass and were subsequently merged using imagej . v software (national institutes of health, bethesda, md, usa). the percentage of brdu-positive cells over the total number of cells was calculated and compared across different groups from at least three different cultures. the level of cytosolic ca + concentration ([ca + ] c ) of mefs with or without propofol treatment was measured by fura- /am fluorescence (molecular probe, eugene, or, usa) using a previously described method [ , ] . the assays were carried out on an olympus ix inverted microscope (olympus america inc., center valley, pa, usa) and iplab v . software (scanalytics, milwaukee wi, usa). briefly, mefs were plated onto a mm culture dish. after the cells were washed three times in hank's buffered salt solution (hbss) containing mg + and ca + and loaded with . μm fura- /am in the same buffer for min at °c, the cells were then washed twice and incubated with hbss for another min at °c. fura- am was measured by recording alternate at and nm excitation, and emission at nm was detected for up to min for each treatment. the evoked changes were recorded in response to treatment with μm versus μm propofol with or without dantrolene (dan), xestospongin c (xc) or bapta-am. the results are presented as a ratio of f /f nm and averaged from at least three separate experiments. basal calcium in the cytoplasm and er in atg −/− and wt mefs mef cells were plated on -mm round coverslips in -well plates and then cultured overnight. the next day, plasmid pcmvr-cepia er ( ; addgene, cambridge, ma, usa) was transfected with lipofectamine (l ; invitrogen, carlsbad, ca, usa) according to the manufacturer's instructions. at h post transfection, the transfection reagent was replaced by the normal culture medium and the treatment was applied. images were taken at the end of the treatment. for labeling intracellular ca + , cells were incubated with fluo- am reagent (f ; molecular probes, eugene, or, usa) at μm for min at °c. western blotting was performed according to the standard procedure and as we described before [ ] . total protein extracts from mefs cells were obtained by lysing the cells in ice-cold lysis buffer ( mm tris-hcl, mm nacl and % triton x- ) in the presence of a cocktail of protease inhibitors [ ] . after centrifugation, the supernatant was collected, and the total protein was quantified using a bicinchoninic acid protein assay kit (thermo scientific, rockford, il, usa). equal amounts of protein for each lane were loaded and separated on % sodium dodecyl sulfatepolyacrylamide gel electrophoresis (sds-page). after electrophoresis, the proteins were transferred onto a polyvinylidene fluoride (pvdf) membrane. the membranes were blocked with % fat-free milk dissolved in pbs-t for h at room temperature and then stained with primary antibody at °c overnight. after washing with pbs-t, the membranes were incubated with secondary antibodies (hrp conjugated anti-rabbit and anti-mouse igg) at : dilutions, and β-actin served as a loading control. signals were detected with an enhanced chemiluminescence detection system (millipore, billerica, ma, usa) and quantified by scanning densitometry. data analysis and statistics parametric variables were expressed as the mean ± sd and analyzed using the student's unpaired two-tailed t test, one-way or two-way anova, followed by tukey's post hoc analysis. graphpad prism software (graphpad software, inc., san diego, ca, usa) was used for statistical analyses and graph creation. p values less than . were considered statistically significant. propofol affects cell survival in an atg -dependent manner and is associated with intracellular ca + channels we first determined whether propofol affects cell viability in atg −/− and wt cells by mtt and ldh detection. after -h exposure to propofol, the mtt signal of atg −/− cells was significantly enhanced at clinically relevant concentrations ( µm); however, this effect was inverted at high pharmacological general anesthetics and autophagy zd xu et al. concentrations ( µm, fig. c ). toxic effects of atg deficiency at high doses were further confirmed by ldh detection (fig. e) . in contrast, propofol did not significantly affect cell survival at any tested concentration in wt cells. according to our prior study [ , ] , we hypothesized that intracellular calcium channels, such as inositol- , , -trisphosphate receptor (insp r) and ryr located in the sarcoplasmic/endoplasmic reticulum membrane, may be involved in propofol-induced cell survival. indeed, the ability of µm propofol to increase viability in atg −/− cells was blocked by the antagonists of insp r and ryr (fig. d) , which also blocked the toxic effects at the high µm dose (fig. d, f) . in contrast, treatment with µm propofol became harmful to wt cells after the inhibition of insp r and ryr (fig. d, f) . these results indicate that autophagy deficiency may facilitate intracellular ca + channel opening, which triggers the dual functional effects of propofol on survival in atg −/− cells. atg plays a key role in propofol effects on cell growth we next determined whether the elevation of mtt by propofol at clinically relevant concentrations in atg −/− cells (fig. c) was caused by changes in mitochondrial reductase activity (early cell damage) or an increase in cell numbers (proliferation). propofol at a low concentration ( µm) significantly increased both total viable cells (fig. c) as determined by the trypan blue assay and newly derived cells (fig. a, b) as determined by the brdu incorporation assay. in contrast, propofol at a high concentration . data in a-c are given as the mean ± sem, and d-f are given as the mean ± sd. all data are from at least three separate experiments and analyzed by two-way anova followed by the tukey's multiple comparison tests. *p < . , **p < . , or ***p < . , respectively general anesthetics and autophagy zd xu et al. ( µm) decreased the total cell numbers more significantly in atg −/− cells than in wt cells (fig. a-c) . atg is necessary for propofol-induced functional effects on autophagy activity atg plays an essential role in the induction and regulation of autophagy activity, but it is still unclear how propofol mediates functional effects by autophagy. we first confirmed that propofol up to µm did not affect atg expression and that no atg existed in atg −/− cells using western blotting (fig. a ). in addition, propofol did not produce a significant change in atg in wt cells (fig. c) . since autophagy flux, which is a dynamic process, consists of several sequential steps-sequestration, transport to lysosomes, degradation, and utilization of degradation products, it is possible that autophagy activity is altered despite unchanged atg protein expression. to further study this possibility, we applied bafilomycin a (baf a ), which can prevent fusion of the autophagosome and lysosomes. we found that µm but not µm propofol significantly increased the expression of lc ii, a biomarker to detect autophagy, by pretreatment with baf a in wt cells (fig. b, d) , indicating that high concentrations of propofol can induce autophagy. as expected, there was no lc ii protein bands formed in atg −/− cells (fig. b) . these findings suggest that the enhanced cell death in atg −/− cells treated with µm could be related to inability to activate autophagy. propofol facilitates cytosolic ca + release in atg -deficient cells our previous studies have shown that propofol increases intracellular ca + release in different types of cells [ , ] . to further confirm the change in basal ca + release in both the cytoplasm and er, we measured these effects by fluo- am dye on an inverted microscope and er ca + transfected with cepia er (fig. a) . we found that basal cytosolic and er ca + were significantly increased in atg −/− cells compared with wt cells (fig. b ). in addition, propofol-mediated ca + release could be inhibited by xc or dantrolene (fig. e , f, xc, nm, dantrolene µm), confirming the involvement of intracellular calcium channels. it is possible that the dual functional effects of propofol on atg −/− cells (fig. ) may be due to various degrees of intracellular ca + release at different concentrations. figure shows that propofol at extremely high concentrations ( µm) increased [ca + ] c more robustly than at clinically relevant concentrations ( µm) in atg −/− cells, compared with wt cells. these results suggest that autophagy-deficient cells are primed for greater lability in response to ca + mobilizing stimuli. moderate intracellular ca + release enhances cell survival/ proliferation, but over release causes cell death, which could be supported by the results shown in fig. . we have demonstrated that atg is essential for propofol effects on cell survival and growth. the above effects may be related to propofol effects on insp r/ryr activity and changes in intracellular ca + homeostasis. this study provides a detailed mechanism of propofol regulation of autophagy and associated cell survival or growth. although commonly used inhalational anesthetics (sevoflurane, isoflurane, or desflurane) [ , ] and intravenous anesthetics (propofol) [ ] all have been shown to cause ca + release from the er, their potency to affect intracellular ca + homeostasis is quite different. inhalational anesthetics at clinically relevant . all data are presented as the mean ± sd from three separate experiments and analyzed by one-way anova followed by the tukey's multiple comparison tests. *p < . , ***p < . , or ****p < . , respectively concentrations ( - minimal alveolar concentration, mac) can induce ca + release from the er via activation of insp r [ , ] , with maximum activation at mac [ ] . inhalational anesthetics at clinically relevant concentrations can also induce cell damage in different types of cells [ , , ] . however, propofol at clinically relevant concentrations ( - µm) rarely induces elevation of [ca + ] c or induced cell damage, and very high pharmacological concentrations (> µm) are usually needed for propofol to induce the elevation of [ca + ] c and cell damage [ ] . similar to previous research, the results of this study suggest that a high concentration of propofol (> µm) is needed to cause cell death in atg −/− cells or cells with dysfunctional autophagy. in addition, propofol at a clinically relevant concentration ( µm) increased cell proliferation only in atg −/− but not wt cells, suggesting that the moderately enhanced ca + release in autophagy-deficient cells crosses a threshold necessary to trigger proliferation. in combination with previous studies [ , ] , the results from this study suggest that propofol at clinically relevant concentrations (< µm) typically does not cause cell damage in the presence of normal autophagy function. however, on the basis of impaired autophagy function, such as in alzheimer's disease [ , ] , cells are more vulnerable to propofol-mediated cell cycle re-entry at low clinically relevant concentrations ( µm) and cell damage at high pharmacological concentrations (> µm). overall, these results suggest that propofol regulates cell survival and growth signaling by activating insp r/ryr, but autophagy deficiency predisposes to calcium overload at high doses of propofol. previous studies have suggested that anesthetics elevated [ca + ] c by the activation of insp r/ryr [ , , ] , but the effects of the autophagy regulatory factor atg on the propofol-mediated ca + response are not clear. in this study, we demonstrated that propofol induced a significantly higher [ca + ] c in atg −/− cells than in wt cells, and this effect can be inhibited by antagonists for insp r (xc) or ryr (dantrolene). interestingly, propofol, even at clinically relevant concentrations ( µm), increased [ca + ] c , especially in atg −/− cells. these results suggested that atg might typically buffer the activation of insp r/ryr and affect intracellular ca + homeostasis either directly or indirectly via its effects on autophagy activity [ ] . autophagy generally regulates the intracellular homeostasis of organelles such as the er by sequestrating organelles and membrane compartments. damaged or redundant er can be removed by autophagy. er is expanded when the autophagy pathway is impaired, and the ca + stores in er are also increased, consistent with the expansion of er contents in autophagy-deficient cells [ , , ] , which is in accordance with our results in the current study. however, it is not clear why cytosolic ca + is also significantly increased in autophagy-deficient cells. nevertheless, . all data are given as the mean ± sd from four separate experiments and analyzed by one-way anova followed by the dunnett's multiple comparisons test. *p < . fig. basal level ca + concentriation in both cytosolic and er significantly increased in atg −/− compared with wild type cells. the cytoplasmic ca + concentration was measured using fluo- am dye on an inverted microscope and er ca + was measured in cells transfected with cepia er (a). the ca + concentrations in both the cytoplasm and er were significantly increased in atg −/− mefs compared with wt mefs (b). all data are presented as the mean ± sd from three separate experiments and analyzed by the t test. *p < . , **p < . . all data are given as the mean ± sd from three separate experiments and analyzed by the t test. *p < . , **p < . . pfl-mediated [ca + ] c elevation was significantly inhibited by xc, dantrolene and bapta-am in both wt (g) and atg −/− cells (h), more significantly in the latter. all data are given as the mean ± sd from at least three separate experiments and analyzed by one-way anova followed by the tukey's multiple comparison tests. *p < . , **p < . , ***p < . , or ****p < . , respectively autophagy deficiency has been demonstrated to lead to an imbalance between ca + release/influx or reuptake/extrusion, resulting in higher basal [ca + ] c [ ] . in vascular smooth muscle cells (vsmcs) with defective autophagy, cytosolic ca + levels increased under basal conditions, which may be related to decreased plasma membrane ca + -atpase expression in atg −/− vsmcs, thus hampering ca + extrusion to the extracellular environment [ ] . although anesthetics have been proposed to affect autophagy and cell survival in a dose-dependent manner, with low doses stimulating autophagy and cell survival [ , ] , and high concentrations impairing autophagy flux and causing cell damage [ , ] , this may be oversimplified. in the current study, we demonstrated that high doses of propofol induces autophagy in wild type cells, but not in atg −/− cells. we propose an alternative model in which the er is expanded and ca + stores are increased in autophagy-deficient mef cells. while this enhances vulnerability to cell death triggered by calcium overload at high propofol doses, propofol at clinically relevant concentrations ( μm) induces a moderate increase in ca + release from the er into the cytosol, favoring cell survival and proliferation of autophagy-deficient cells (fig. ) . in this study, we demonstrated that the effects of propofol on calcium release are modulated by atg . furthermore, propofol at clinically relevant concentrations ( µm) promoted the proliferation of atg −/− cells through moderately elevated [ca + ] c , while at high concentrations ( µm), propofol increased the large outflow of ca + from the er, inducing the death of atg −/− cells. it is not clear whether atg has direct effects on the activation of insp r/ryr or indirect effects via the regulation of autophagy. in addition, it is not clear whether the effect of propofol on intracellular ca + homeostasis is associated with ca + influx from the extracellular space, which requires further study. our study has the following limitations: ( ) . although these two types of cells provide good tools to investigate the role of atg on the propofol effect on autophagy and its association with cell survival and growth, these cells are fibroblasts and not closely related to neurons. ( ) . whether or not atg regulates activation of insp r/ryr by either directly interacting with receptor activation or indirectly through changes in lysosome or autophagy function requires further study. ( ) . this study relied upon the conversion of lc -i (cytosolic form) to lc -ii (membrane-bound lipidated form) by immunoblotting with and without lysosomal inhibitor treatment, which is a conventional method to monitor cellular autophagic activity [ ] . now with a novel tandem construct (mrfp-gfp-lc ), autophagic flux can also be morphologically traced, although we did not perform this experiment. in summary, atg plays an essential role in modulating dosedependent propofol effects on autophagy, cell proliferation and cell death related to changes in intracellular ca + homeostasis. fig. propofol at clinically relevant concentrations promotes cell survival and proliferation in autophagy-deficient cells. the er is expanded, and the basal ca + stores are increased in autophagy-deficient mef cells. propofol at clinical concentrations ( µm) significantly induces the release of more ca + from the er into the cytosol in atg −/− cells than in wt cells via the activation of inositol- , , -trisphosphate receptor (insp r) and/or ryanodine receptor (ryr) calcium channels, favoring cell survival and proliferation the role of autophagy during the early neonatal starvation period atg is essential for the development and survival of innate lymphocytes involvement of autophagy in nk cell development and function atg and bak play important roles in the crosstalk between apoptosis and autophagy induced by influx of extracellular calcium calcium homeostasis and er stress in control of autophagy in cancer cells lysosomal calcium signalling regulates autophagy through calcineurin and tfeb the crucial role of atg in cortical neurogenesis during early brain development a critical role for the autophagy gene atg in t cell survival and proliferation autophagy limits proliferation and glycolytic metabolism in acute myeloid leukemia a novel and functional variant within the atg gene promoter in sporadic parkinson's disease atg -dependent autophagy contributes to the development of acute myeloid leukemia in an mll-af -driven mouse model protective role of autophagy and autophagy-related protein in early tumorigenesis upregulation of autophagyrelated gene- (atg- ) is associated with chemoresistance in human gastric cancer discovery of atg /atg -independent alternative macroautophagy propofol affects neurodegeneration and neurogenesis by regulation of autophagy via effects on intracellular calcium homeostasis general anesthetics regulate autophagy via modulating the inositol , , -trisphosphate receptor: implications for dual effects of cytoprotection and cytotoxicity control of basal autophagy by calpain mediated cleavage of atg autophagy, a novel pathway to regulate calcium mobilization in t lymphocytes protective effect of acetyl-l-carnitine on propofol-induced toxicity in embryonic neural stem cells down-regulation of microrna- is involved in the propofol-induced neurotoxicity observed in human stem cell-derived neurons propofol induces apoptosis and inhibits the proliferation of rat embryonic neural stem cells via gamma-aminobutyric acid type a receptor impaired autophagy in mouse embryonic fibroblasts null for kruppel-like factor promotes dna damage and increases apoptosis upon serum starvation knockdown of autophagy-related protein , atg , decreases oxidative stress and has an opposing effect on camptothecin-induced cytotoxicity in osteosarcoma cells inhalational anesthetics induce cell damage by disruption of intracellular calcium homeostasis with different potencies the common inhaled anesthetic isoflurane increases aggregation of huntingtin and alters calcium homeostasis in a cell model of huntington's disease general anesthetic isoflurane modulates inositol , , -trisphosphate receptor calcium channel opening the common inhalation anesthetic isoflurane induces apoptosis and increases amyloid beta protein levels the effect of endoplasmic reticulum stress on neurotoxicity caused by inhaled anesthetics prolonged treatment with propofol transiently impairs proliferation but not survival of rat neural progenitor cells in vitro the effects of intravenous anesthetics on mouse embryonic fibroblast viability and proliferation autophagy and alzheimer's disease endo-lysosomal and autophagic dysfunction: a driving factor in alzheimer's disease? autophagy regulates endoplasmic reticulum homeostasis and calcium mobilization in t lymphocytes defective autophagy in vascular smooth muscle cells alters contractility and ca + homeostasis in mice propofol-enhanced autophagy increases motility and angiogenic capacity of cultured human umbilical vascular endothelial cells mitigation of h o -induced autophagic cell death by propofol in h c cardiomyocytes methods in mammalian autophagy research we appreciate the valuable discussion from maryellen eckenhoff, roderic eckenhoff and editing assistance from divakara gouda at the department of anesthesiology, university of pennsylvania, philadelphia, usa. supported by grants to hw from the nih (r gm , r gm - s , and r gm - a ) and r ag - s to ctc. supported by grants to zx from shanghai municipal health bureau ( ). supported by grants to zx from the national natural science foundation of china ( ). supported by grants to yw from the national natural science foundation of china ( ). hfw designed research; zdx, yw, and gl performed research; zdx, hfw, and ctc analyze and interpret data; zdx, hfw, ctc, zql, and whm wrote the paper. competing interests: hua-feng wei is a member of advisory board on march , at eagle pharmaceutical company, new jersey, usa, which produce and sale ryanodex, a new formula of dantrolene. the dantrolene used in this study was purchased from a different company. no other authors state conflict of interest. key: cord- -n w x m authors: liu, chien-ying; liu, yun-hen; lin, shu-min; yu, chih-ten; wang, chun-hua; lin, horng-chyuan; lin, chien-huang; kuo, han-pin title: apoptotic neutrophils undergoing secondary necrosis induce human lung epithelial cell detachment date: - - journal: j biomed sci doi: . /bf sha: doc_id: cord_uid: n w x m clearance of apoptotic neutrophils by alveolar macrophages plays an important role in the resolution phase of lung inflammation. if not cleared, apoptotic neutrophils are postulated to release histotoxic granular contents. since numerous cellular proteins are degraded during apoptosis, we sought to determine whether functional serine proteinases are indeed released by apoptosing neutrophils in vitro. in a coculture system, cytokine-activated neutrophils induced detachment in the human epithelial cell line, a . this process was cd - and serine proteinase-dependent. early apoptotic neutrophils induced significant detachment, but live, senescent, resting neutrophils and terminal, secondary necrotic neutrophils had a different effect. this detachment process was cd -independent but serine proteinase-dependent. similarly, detachment occurred with primary human small airway epithelial cells. notably, epithelial cell detachment correlated with the transition of early apoptotic neutrophils to secondary necrosis and with the accumulation of elastase in the supernatant. the membrane integrity of lung epithelial cells was damaged in advance of significant cell detachment. these observations suggest that not only live activated neutrophils but also apoptosing neutrophils can reveal functional elastase activities. furthermore, the rapidity of the transition emphasizes the importance of the prompt clearance of apoptotic neutrophils before they progress to secondary necrosis at the site of lung inflammation. the utilization of neutrophilic proteinase is an important host defense mechanism against pathogenic microorganisms [ , , ] . however, abundant literature documents the potential hazards of these proteolytic enzymes in the pathogenesis of inflammatory organ injury. circulating mature human neutrophils are committed to die, even when their life spans are prolonged by various endogenous and exogenous factors. apoptotic neutrophils are recognized by professional phagocytes and semiprofessional phagocytic parenchymal cells. although definitive in vivo studies are lacking, apoptosing neutrophils are postulated to be removed without further augmentation of the existing inflammation [ , ] . this process is believed to be rapid, efficient and important during the resolution phase of inflammation [ , ] . coincident with the highly controlled process of neutrophil apoptosis is the shutdown of the degranulation response [ ] . if not phagocytosed, apoptotic neutrophils will undergo secondary necrosis, an end-stage cellular disintegration characterized by breached membrane integrity. consequently, proteinases residing in apoptotic neutrophils may be passively released and, in turn, contribute to tissue injury. granulocyte-macrophage colony-stimulating factor (gm-csf) and tumor necrosis factor (tnf)-a, both common at inflammation sites, accelerate macrophage clearance of neutrophils undergoing apoptosis in vitro [ , , , ] . however, several factors associated with chronic inflammatory diseases can markedly inhibit the ability of macrophages to recognize and engulf apoptotic neutrophils. for example, a number of granulocyte enzymes with an extracellular presence in inflammatory diseases are extremely cationic [ ] . these cationic molecules, along with a low ph, are common features of chronic inflammation and have been demonstrated to significantly inhibit recognition of apoptotic neutrophils by macrophages [ ] . in patients with cystic fibrosis, pseudornonas aeruginosa products have been reported to inhibit macrophage phagocytosis [ , ] . moreover, the ability of alveolar macrophages to phagocytose apoptotic neutrophils was found to be significantly impaired in smoke inhalation injury, a condition associated with a high incidence of both bacterial pneumonia and adult respiratory distress syndrome (ards) [ ] , and in children suffering recurrent respiratory tract infections [ ] . other investigations have suggested that macrophages which had previously ingested apoptotic cells subsequently have impaired ingestion capacity [ ] . in this situation, apoptotic neutrophils can undergo secondary necrosis, presumably resulting in the release of histotoxic granular contents [ ] . if the proteinases released remain functional, they can then escalate the inflammatory response and exacerbate lung injury. surprisingly, few studies have examined the release of functional proteinases from apoptotic neutrophils. neutrophils express several serine proteinases, including neutrophil elastase, cathepsin g and proteinase . among these, elastase is the major proteinase, and has been demonstrated to be critical in neutrophil-mediated tissue injury and extracellular proteolysis [ , , , ] . previous studies showed that serine proteinase inhibitors blocked a cell detachment induced by activated neutrophils, and that exogenous elastase restored this detachment [ ] . because the disruption and desquamation of airway and lung epithelial cells have been widely observed in a variety of immunological, infectious and inflammatory pulmonary diseases [ , , , ] , we used lung epithelial cell detachment as a bioassay in this study [ ] to investigate the effect of early apoptotic neutrophils on airway epithelial cells and the role of elastase activity in causing the effect. because cd is a f~ integrin subunit critical to the firm adhesion and the subsequent degranulation response of activated neutrophils [ , ] , and because apoptotic neutrophils have been demonstrated to accumulate cd on the cell surface [ ] , we also investigated the role of the integrin cd in the cytotoxic effect of apoptosing neutrophils. secretory leukocyte proteinase inhibitor (slpi), tnf-a and gm-csf were purchased from r&d systems (minneapolis, minn., usa). the caspase inhibitor zvad-fmk and the calpain inhibitors zlly-fmk and pf were purchased from calbiochem (san diego, calif., usa). human cd blocking monoclonal antibody (mab) . is a mnrine igg a which was obtained from serotec (raleigh, n.c., usa) [ ] . all culture medium and additives were purchased from biowhittaker (walkersville, md., usa) unless otherwise specified. human neutrophils were isolated from freshly drawn heparinized blood by dextran sedimentation and centrifugation on a ficoll-paque gradient (amersham pharmacia biotech, uppsala, sweden), as previously described [ ] . additionally, contaminating erythrocytes were lysed using . % buffered ammonium chloride. purified neutrophils of varying concentrations were then suspended in rpmi with or without % heat-inactivated fetal bovine serum (fbs; hyclone sterile, logan, utah, usa), depending on assay conditions. isolated neutrophils were consistently > % pure according to lukostat staining (fisher scientific, pittsburgh, pa., usa), and > % viable according to the trypan blue exclusion test. nologies, carlsbad, calif., usa), . rm~ sodium pyruvate and . mm nonessential amino acids at °c in a % co incubator. the cells were passaged at - °/o confluence using . % trypsin. human primary small airway epithelial cells (saecs) were purchased from clonetics (walkersville, md., usa). the saecs were expanded and maintained in saec basal medium supplemented with bovine pituitary extract, hydrocortisone, human recombinant epidermal growth factor, epinephrine, insulin, retinoic acid, triiodothyronine, gentamicin, amphotericin b and bovine serum albumin (termed saec growth medium) at °c in a % c incubator. to minimize the phenotypic changes in epithelial cells, saecs were used in a maximum of passages. screening tests for mycoplasma contamination at the beginning and at the end of this study showed no positive reaction. all cells used in this study grew well for the determined times. a cells were plated in -well plates at x cells/well in gl of minimum essential eagle medium supplemented with % fbs. following overnight culturing, confluent cells were labeled with ttm celltracker green cmfda (molecular probes, eugene, oreg., usa) for rain at °c and in darkness. cells were washed twice and incubated in fresh medium for another rain at °c. at this point, the culture medium was replaced with gl of superuatant from neutrophils cultured in phenol red-free, serum-free rpmi- medium. alternatively, fresh or cultured neutrophils resuspended at varying concentrations in fresh medium were added to the a cell monolayer. following h of coincubation, the fluorescence intensity (fi) in the wells was measured using cytofluor (perseptive biosystems, framingham, mass., usa) (with excitation at nm and emission at rim). detached and loosely adherent cells were then removed by gently washing the plate times with phenol redfree medium. next, the remaining fluorescence in the wells was determined. after subtracting background fluorescence (phenol redfree medium alone without cells), detachment was computed using the following formula: fi in test well after washing/fi in test well before washing -fi in ~~ingj ! x %, where the control well contained the a monolayer with rpmi- supplemented with % fbs for a cells and saec growth medium for saecs. detachment of the saec monolayer was determined similarly, except that saec growth medium was used. for spontaneous apoptosis, freshly isolated neutrophils were suspended in rpmi- supplemented with % fbs and incubated at a concentration of x cells/ml in -well tissue culture plates for h. apoptotic neutrophils were isolated using a macs apoptotie cell isolation kit (miltenyi biotech, auburn, calif., usa) according to the instructions of the manufacturer. briefly, neutrophits in culture were resuspended in gl of binding buffer and gl of annexin v-conjugated paramagnetic microbeads/ cells. cells were then incubated for rain at °c. after a single washing, cells resuspended in ml of binding buffer were loaded onto a midi-macs column, and the column was then irrigated with binding buffer. both the annexin v-selected (with a magnet) and flow-through fractions were collected. to ensure quality control, the purity of each fraction was determined in parallel with experiments by double labeling with annexin v-fitc (pharmingen, san diego, calif., usa) and propidium iodide (molecular probes). briefly, neutrophils in each fraction were washed once with phosphate-buffered saline (pbs). cells were then incubated with annexin v-fitc and propidium iodide in binding buffer containing mmhepes, . mmmgc and . mmcac in normal saline for min at room temperature, and analyzed by flow cytometry (epics xl, coulter, hialeah, fla., usa). elastase activity in cell-free superuatant was assessed by a modified method described previously [ ] using methoxysuccinyt-ala-ala-val- -amid- -methyl-coumarin (calbiochem). sample activity was then compared with a standard curve determined using known concentrations of purified human neutrophil elastase (calbiochem). to determine the cell membrane integrity of the adherent lung epithelial cells, propidium iodide staining was used [ ] . at each evaluation point, floating cells were removed. the remaining adherent cells were washed twice with pbs and stained with propidium iodide ( gg/ml in rpmi- ) for rain at °c. after gentle washes, cells were examined with fluorescence microscopy using a -nm filter and then trypsinized for flow cytometric analysis (with excitation at nm and emission at nm). the percentage ofpropidium iodide-stained cells in the gated lung epithelial cell region on flow cytometric plots was determined in order to quantify the damage to cell membrane integrity. data are expressed as the mean + sem. for normally distributed data, a t test was used to evaluate differences between sets. for nonnormally distributed data, the mann-whitney u test was used. graphpad prism (version . i, graphpad software, san diego, calif., usa) was used for all statistical analyses. statistical significance was defined as p < . . when cultured in serum-free media, approximately % of the fresh neutrophils lost their membrane integrity with positive trypan blue staining within h, and % of cells within h ( fig. ). fresh neutrophils, when cocultured on a monolayer of the human lung epithelial cell line a , were found to induce epithelial detachment within h ( fig. a, b) . moreover, the time required for comparable detachment to occur was significantly reduced to h if the neutrophils had previously been cultured for h ( fig. c ), suggesting a process related to neutrophil senescence and possibly cell death. next, the underlying mechanism was examined to determine whether it was soluble factor-dependent. as shown in figure a, supernatant from neutrophils cultured for h induced a cell detachment after h of coculturing. supernatant from neutrophils cultured for h also caused significant detachment. in parallel experiments, freshly isolated neutrophils or neutrophils cultured for h and resuspended in fresh serum-free medium induced minimal detachment. in contrast, neutrophils cultured for h induced significant a cell detachment in a cell number-dependent manner without exogenous neutrophil-activating stimuli ( fig. b ). there was prominent a cell detachment within h when freshly isolated neutrophils were activated with tnf-a ( fig. ). in this coculture system, detachment was noted to be almost completely abrogated by mab . , a cd -blocking antibody. to determine whether detachment induced by resting cultured neutrophils requires cd engagement, the effect of mab . on a celt annexin-fitc fig. . detachment of a cells by cultured neutrophils is cd independent. neutrophils ( x /ml) were cultured in serum-free medium for h and then collected and centrifuged. cells were resuspended in the same volume of serum-free fresh medium. two hundred microliters of cell suspension was added to each well ( x cell equivalents/well) and cocultured with a cells in the presence or absence ofmab . ( gg/ml). fresh neutrophils ( x / well) stimulated with ng/ml tnf-a were also cocultured with a cells in both the presence and absence ofmab . ( gg/ml). detachment was measured after h of incubation. results are expressed as the mean + sem of experiments performed in triplicate. *p < . compared with medium alone. ** p < . compared with neutrophils treated with tnf-a alone. detachment was tested. figure confirms the suspicion that a cell detachment induced by resting neutrophils cultured for h was not affected by mab . . neutrophils incubated in vitro undergo apoptosis constitutively. flow cytometric analysis of the approximately % trypan blue-positive neutrophils cultured for h revealed that over % of cells were annexin v positive. thus, we hypothesized that apoptotic neutrophils, if allowed to undergo secondary necrosis, would contribute to a cell detachment. to test this hypothesis, neutrophils cultured for h were separated into an annexin v-selected fraction and a flow-through fraction, each of which was then cocultured with a cells for h. neutrophils in the annexin v-selected fraction mainly occurred early in apoptosis (> % annexin v positive, propidium iodide negative, fig. a ) and induced significant detachment within h of coculturing in a cell number-dependent manner. meanwhile, > % of neutrophils in the flowthrough fraction were alive (annexin v negative, propid-ium iodide negative, fig. b ) and had a minimal effect at best ( fig. a ). utilizing human primary-lung epithelial cells, saecs, also revealed significant detachment ( fig. b ). this detachment was not affected by mab . ( fig. ) . however, the apoptosing neutrophil-induced detachment of airway epithelial cells was completely abrogated by exogenous slpi, a specific inhibitor of serine proteinases ( fig. a) . additionally, the supernatant from the apoptosing neutrophils was able to induce a cell detachment, which could be attenuated by slpi (fig. b ). to determine whether early apoptotic neutrophils release functional elastase, we examined elastase activity in supernatant from the annexin v-selected fraction cultured in vitro, and the data revealed that elastase activity continually increased with time ( fig. a ). parallel to the determination of elastase activity, the membrane integrity of cells was evaluated using trypan blue. the release of functional elastase activity from apoptosing neutrophils was temporally correlated with the progression to second- ary necrosis (fig. b ). in separate dose-response experiments using purified human neutrophil elastase, it was observed that a threshold concentration of . pg/ml was required to induce significant a cell detachment within h. in our experiments, elastase activity in the supernatant of the annexin v-selected fraction reached this level after only h. although the annexin v-negatively selected flow-through fraction also released elastase, the level of its activity was below the threshold concentration ( fig. a, b) . to further explore the effect of early apoptotic neutrophils on adherent lung epithelial cells, the annexin vselected fraction ofneutrophils cultured for h was coincubated with a cells for h. as shown in figure , the percentage of propidium iodide-stained adherent a cells increased with time, in advance of significant cell detachment, suggesting that early apoptotic neutrophils apoptotic neutrophils induce epithelial cell detachment are able to breach the ceil membrane integrity and possibly, in turn, cause cell detachment. our study demonstrates that early apoptotic neutrophils induce lung epithelial cell detachment. this detachment is strictly serine proteinase-dependent and cd independent. it also correlates with an increase in elastase activities in the extracellular milieu and the toss of membrane integrity in adherent lung epithelial cells. a healthy human adult turns over almost × neutrophils/kg of body mass daily [ ] . once recruited to an inflammatory site, neutrophit life spans were demonstrated to be extended ex vivo [ , ] . notwithstanding, mature neutrophils are committed to undergoing apoptosis. apoptosis is an intracellular remodeling process with initially limited autoproteolysis and oligonucleosomal cleavage which is accompanied by downrcgulation ofneutrophil inflammatory responses [ , , ] . this process eventually leads to cellular disintegration, with the breach in cellular membrane integrity (secondary necrosis). although histotoxic neutrophilic granular contents have been widely stated to be released under this scenario, an exhaustive literature search yielded surprisingly few peerreviewed original studies that characterized the release of functional proteinase activities from these shrinking, autoproteolysing neutrophits. utilizing annexin v-conjugated paramagnetic beads that selectively bind to exposed cell surface phosphatidylserine residues present on apoptotic but not live neutrophils [ , , ] , this study revealed more accelerated release of functional elastase activity from predominantly early apoptotic neutrophils, compared to that from predominantly live, senescent, resting neutrophils. furthermore, the timing of this release correlates with the progression of early apoptotic neutrophils to secondary necrosis and lung epithelial cell detachment in vitro. thus, this study clearly confirms that resting apoptosing neutrophils release functional elastase activity in vitro. early apoptotic neutrophils differ from live, selectively activated neutrophils in that despite increased cell surface cd , they do not require the participation of the integrin, cd , for the adhesion and release of granule contents [ ] . on the other hand, slpi, one of the endogenous serine proteinase-specific antiproteinase defenses, abrogates both live, activated neutrophil-and apoptosing neutrophil-induced lung epithelial cell detachment. importantly, exogenous slpi does not protect the membrane integrity of apoptosing neutrophils. there are relatively few investigations which have quantified the occurrence of apoptotic neutrophils in the course of acute inflammatory lung diseases in vivo. thus, the role of dying neutrophils in lung injury is unclear. in patients with ards, apoptotic neutrophils in bronchoalveolar lavage fluid are not abundantly identifiable morphologically but are present during the first few days of the initial onset of disease [ ] . this phenomenon may be consistent with the prevailing concept that apoptotic neu- trophils are rapidly recognized and ingested by professional and semiprofessional phagocytic cells [ , ] . alternatively, the rapid transition from early apoptosis to apoptotic body-tbrmation, secondary necrosis and eventual total cellular disintegration may lead to a certain degree of underestimation. furthermore, current histochemical techniques and in vitro phagocytosis assays do not distinguish whether the clearance of dying neutrophils occurs before or after the breach of membrane integrity. one recent study observed that numerous, persistent apoptotic neutrophils are present in lung abscesses formed in experimental pneumonia in vivo [ ] . this important observation implies that the balance between neutrophil apoptosis and their phagocytic clearance can be disturbed. indeed, this possibility is supported by two further ex vivo studies that specifically verified the functional impairment of alveolar macrophages, both in clearing apoptotic neutrophils in children suffering from recurrent respiratory tract infections, and in an animal model of experimental smoke inhalation injury [ , ] . although this study used a lung epithelial cell detachment assay as an in vitro bioassay to characterize the serine proteinases released from dying neutrophils, the biological effects of neutrophil proteinases are wide-ranging. for example, besides a direct role of neutrophil proteinases in experimental lung injury [ , , ] , elastase can activate neutrophil matrix metalloproteinase- [ ] , which is capable of solubilizing the human alveolar extracellular matrix [ , ] and is increased in epithelial lining fluid from patients with ards [ ] . cathepsin g can also augment inflammation through platelet activation via pmteinase-activated receptor- activation [ ] . furthermore, neutrophil elastase and a-defensins can stimulate lung epithelial cell production of il- and/or gm-csf in vitro [ , ] . conceivably, during the resolution phase of an acute inflammatory insult, functional proteinases released from apoptosing neutrophils that have escaped timely phagocytic clearance may perpetuate inflammation and contribute to tissue injury. numerous in vitro investigations have suggested that neutrophils contribute not only to host defenses against pathogenic organisms but also to defense against tissue injury. neutrophii proteinases are among the essential causes of these dual effects. moreover, the realization that robust, endogenous antiproteinase systems are generally in place further leads to the conclusion that maintaining a certain proteinase/antiproteinase balance in vivo is a biological necessity. the current paradigm of a self-limiting, neutrophil-mediated inflammatory" process entails not only the timely recruitment of neutrophils capable of active phagocytosis and degranulation, but also their subsequent efficient removal from inflammatory sites [ ] . the appreciation that multiple recognition mechanisms exist, whereby effete granulocytes are eliminated by both professional and semiprofessional phagocytes, further indicates the critical importance of this process [ , , , ] . however, confirmatory in vivo experiments that directly test the importance of neutrophil apoptosis in the resolution phase of this paradigm are not yet available. our data showed that the cell membrane integrity of a cells was significantly disrupted in advance of detachment. the molecular mechanisms of detachment of a cells or airway epithelial cells are complex and not completely known. the disruption of cell-matrix adhesion and cell-cell tight junctions, and the induction of apoptosis-necrosis have been observed on or in a cells [ , , , , , ] . the mechanisms are inducer-specific in part. the disruption and desquamation of airway and lung epithelial cells resulting from cellular damage can increase the permeability of the lung parenchyma to fluid and cells [ , , ] . many biological cascades follow, resulting in acute airway narrowing and pulmonary inflammatory edema or chronic airway remodeling and lung fibrosis [ , , ] . related symptoms, including dyspnea, cough and wheezing, can occur. the pulmonary function test will reveal airway obstruction, limited lung expansion and decreased gas diffusion [ , , ] . in conclusion, this study demonstrates that apoptosing neutrophils release, at least, functional elastase activity during secondary necrosis and induce a cytotoxic effect on airway epithelial cells. as this process is rapid, our results indicate that neutrophil apoptosis may not necessarily be benign, and emphasize the importance of removal of these disintegrating cells promptly by phagocytic cells. clearly, further study is needed to explore the molecular mechanisms of epithelial celt detachment induced by apoptotic neutrophils and to document the occurrence of neutrophil apoptosis and the kinetics of apoptotic neutrophil removal in various disease states. finally, our results suggest that a therapeutic strategy for inducing granulocyte apoptosis en masse so as to attenuate active lung inflammation should consider the simultaneous enhancement of clearance mechanisms for apoptosing granulocytes. effects of human neutrophil elastase and pseudomonas aen~ginosa proteinases on human respiratory epithelium cotton dust-mediated lung epithelial injury the injurious effect of neutrophils on pneumocytes in vitro amiodarone induces apoptosis of human and rat alveolar epithelial cells in vitro icam- -cd interaction mediates neutrophil cytotoxicity through protease release definition of a common leukocyte cell-surface antigen (lp - ) associated with diverse ceil-mediated immune functions mice lacking neutrophil elastase reveal impaired host defense against gram negative bacterial sepsis neutrophils from subjects with chronic obstructive lung disease show enhanced chemotaxis and extracellular proteolysis a novel cell culture model for studying ischemiareperfusion injury in lung transplantation macrophage engulfment of apoptotic neutrophils contributes to the resolution of acute pulmonary inflammation in vivo neutrophil kinetics in man human cd mediates recognition and phagocytosis of apoptotic cells evaluation of paf antagonists using human neutrophils in a microtiter plate assay cytokinemediated bax deficiency and consequent delayed neutrophil apoptosis: a general mechanism to accumulate effector cells in inflammation asbestos fibre length-dependent detachment injury to alveolar epithelial cells in vitro: role of a fibronectin-binding receptor regulation of cell adhesion molecule expression and function associated with neutrophil apoptosis single-step separation of red blood cells. granulocytes and mononuclear leukocytes on discontinuous density gradients of ficoll-hypaque previous uptake of apoptotic neutrophils or ligation of integrin receptors downmodulates the ability of macrophages to ingest apoptotic neutrophils macrophages that have ingested apoptotic cells in vitro inhibit proinflammatory cytokine production through autocrine/paracrine mechanisms involving tgf-beta, pge , and paf a receptor for phosphatidylserine-specific clearance of apoptotic cells activation of mmp- by neutrophil elastase in an in vivo model of acute lung injury apoptotic hepatocytes become insoluble in detergents and chaotropic agents as a result of transglutaminase action a comparison of alpha -proteinase inhibitor me-thoxysuccinyi-ala-ala-pro-val-chloromethylketone and specific beta-lactam inhibitors in an acute model of human polymorphonuclear leukocyte elastase-induced tung hemorrhage in the hamster dransfield i. molecular characterization of the surface of apoptotic neutrophils: implications for functional downregulation and recognition by phagocytes granulocyte apoptosis and its role in the resolution and control of lung inflammation impaired alveolar macrophage function in smoke inhalation injury neutrophil adherence to lung epithelial cells induce interleukin- release differential patterns of apoptosis in resolving and nonresolving bacterial pneumonia caspase-mediated proteolysis and activation of protein kinase cdelta plays a central role in neutrophil apoptosis constitutive apoptosis in human neutrophils requires synergy between calpains and the proteasome downstream of caspases annexin v for flow cytometric detection of phosphatidylserine expression on b cells undergoing apoptosis lipopolysaccharide enhances substance pmediated neutrophil adherence to epithelial cells and cytokine release endogenous nitric oxide inhibits neutrophil adherence to lung epithelial cells to modulate interleukin- release degradation of type iv (basement membrane) collagen by a proteinase isolated from human polymorphonuclear leukocyte granules neutrophil apoptosis in the acute respiratory distress syndrome effects of pseudomonas aeruginosa rhamnolipids on human monocyte-derived macrophages inhibition of macrophage phagocytosis by pseudomonas aeruginosa rhamnolipids in vitro and in vivo in vivo suppression of immune complex-induced alveolitis by secretory leukopmteinase inhibitor and tissue inhibitor of metalloproteinases neutrophil elastase in respiratory epithelial lining fluid of individuals with cystic fibrosis induces interleukin- gene expression in a human bronchial epithelial ceil line direct cytotoxicity of polymorphonuclear leukocyte granule proteins to human lung-derived cells and endothelial cells the cell biology ofleukocyte-mediated proteolysis mechanisms of neutrophil damage to human alveolar extracellular matrix: the role of serine and metalloproteases alveolar macrophages of children suffering from recurrent infections of respiratory tract are less efficient in eliminating apoptotic neutrophils cd adhesion blockade decreases bacterial clearance and neutrophil recruitment after intrapulmonary e. coli, but not after s. aureu~ effects of cystic fibrosis airway secretions on rat lung: role of neutrophil elastase proinflammatory cytokines potentiate thrombospondin-mediated phagocytosis of neutrophils undergoing apoptosis cathepsin g activates protease-activated receptor- in human platelets vitronectin receptor-mediated phagocytosis of ceils undergoing apoptosis glomerular mesangial cells and inflammatory macrophages ingest neutrophils undergoing apoptosis phagocytosis of aged human neutrophils by macrophages is mediated by a novel 'charge-sensitive' recognition mechanism macrophage phagocytosis of aging neutrophils in inflammation. programmed cell death in the neutrophil leads to its recognition by macrophages calpain and calpastatin regulate neutrophil apoptosis impaired immunity and enhanced resistance to endotoxin in the absence of neutrophil elastase and cathepsin g effect ofneutrophil serine proteinases and defensins on lung epithelial cells: modulation of cytotoxicity and il- production a novel assay for apoptosis. flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled annexin v convery cm. resting and cytokine-stimulated human small airway epithelial cells recognize and engulf apoptotic eosinophils cross-linking ofcd in human neutrophils induces an increase ofintracellular free ca +, exocytosis of azurophilic granules, quantitative up-regulation of cd , shedding of lselectin, and actin polymerization neutrophils induce damage to respiratory epithelial cells infected with respiratory syncytiai virus haslett c. impairment of function in aging neutrophils is associated with apoptosis the authors would like to thank the national science key: cord- -el wyu authors: zhang, qingxiu; zhu, wen; xu, fei; dai, xuejiao; shi, ligen; cai, wei; mu, hongfeng; hitchens, t. kevin; foley, lesley m.; liu, xiangrong; yu, fang; chen, jie; shi, yejie; leak, rehana k.; gao, yanqin; chen, jun; hu, xiaoming title: the interleukin- /pparγ signaling axis promotes oligodendrocyte differentiation and remyelination after brain injury date: - - journal: plos biol doi: . /journal.pbio. sha: doc_id: cord_uid: el wyu the repair of white matter damage is of paramount importance for functional recovery after brain injuries. here, we report that interleukin- (il- ) promotes oligodendrocyte regeneration and remyelination. il- receptor expression was detected in a variety of glial cells after ischemic brain injury, including oligodendrocyte lineage cells. il- deficiency in knockout mice resulted in greater deterioration of white matter over d after stroke. consistent with these findings, intranasal delivery of il- nanoparticles after stroke improved white matter integrity and attenuated long-term sensorimotor and cognitive deficits in wild-type mice, as revealed by histological immunostaining, electron microscopy, diffusion tensor imaging, and electrophysiology. the selective effect of il- on remyelination was verified in an ex vivo organotypic model of demyelination. by leveraging primary oligodendrocyte progenitor cells (opcs), microglia-depleted mice, and conditional opc-specific peroxisome proliferator-activated receptor gamma (pparγ) knockout mice, we discovered a direct salutary effect of il- on oligodendrocyte differentiation that was mediated by the pparγ axis. our findings reveal a new regenerative role of il- in the central nervous system (cns), which lies beyond its known immunoregulatory functions on microglia/macrophages or peripheral lymphocytes. therefore, intranasal il- delivery may represent a novel therapeutic strategy to improve white matter integrity in stroke and other brain injuries. introduction matter components. however, the potential effects of il- on white matter integrity after ischemic stroke remain elusive. in the current study, we discovered that il- is essential for white matter repair after stroke. intranasal delivery of il- nanoparticles after stroke robustly promoted oligodendrogenesis, enhanced white matter integrity, and improved long-term functional recovery. complementary studies in cell cultures and animals revealed that il- -mediated oligodendrogenesis was achievable even in the absence of microglia/macrophages in vitro or after microglia/macrophage significant depletion in vivo. furthermore, il- directly promoted opc differentiation into mature oligodendrocytes in a peroxisome proliferator-activated receptor gamma (pparγ)-dependent manner. these collective findings suggest that activation of the il- / pparγ signaling cascade may represent a novel recovery-enhancing strategy to promote longterm outcomes after ischemic stroke. to explore the potential cellular targets for il- , we used flow cytometry to assess expression of il- rα on various types of cells in the ischemic brain. consistent with previous reports [ , ] , il- rα was expressed on cd − cd b − gfap + astrocytes, cd − cd b − neun + neurons, cd intermediate cd b + microglia, cd high cd b + macrophages, and other infiltrated immune cells (cd + cd b − ) d after -min mcao (fig a and b) . notably, il- rα was also expressed in oligodendrocyte lineage cells (fig a- d ). different markers, including a b , o , and o /galactocerebroside (galc), were used to identify oligodendrocytes at different differentiation stages. il- rα was detected in cd − cd b − o + cells (fig a- d ), which were identified as preoligodendrocytes and premyelinating oligodendrocytes, and cd − cd b − o + cells (fig a and b) , which were identified as premyelinating oligodendrocytes or mature oligodendrocytes [ ] . the percentages of il- rα + o + cells were higher in the ipsilateral ischemic hemisphere (fig c and d) , suggesting a potential direct effect of il- on white matter cells. imagestream analyses confirmed that il- rα was expressed on both a b + opcs and o + preoligodendrocytes/premyelinating oligodendrocytes, as well as cd b + microglia/macrophages in the ischemic hemisphere ( fig e and s a-s c fig) . in light of these results, we evaluated whether il- influenced white matter integrity after ischemic stroke. wt and il- ko mice were subjected to transient focal ischemia induced by -min mcao. dual staining for myelin basic protein (mbp, a major myelin protein) and smi (a marker of demyelinated axons) was performed to assess the lesion in white matter (fig f- h ). the overall immunofluorescence intensity of mbp staining decreased in the infarct border along three white matter-rich brain regions (the external capsule [ec] , the cortex, and the striatum) (see "total" values in panel of the right of fig f) at d after mcao, indicating a significant loss of myelin protein after stroke. additionally, the overall immunofluorescence of smi increased in these three regions, resulting in a significant increase in the smi /mbp ratio in wt mice after stroke (fig g) . il- -deficient mice displayed exacerbation of the loss in mbp ( fig f) and a further increase in the smi /mbp ratio compared to wt mice (fig g) , suggesting that il- is important in maintaining axonal myelination or promoting axonal remyelination after stroke. stroke impacts both gray and white matter. in order to confirm the effect of il- in white matter, we explored the remyelination capacity of il- in a model of lysophosphatidylcholine/lysolecithin (lpc)-mediated demyelination using ex vivo organotypic cerebellar slice cultures. this toxicant-based experimental model of demyelination, although not attempting to fully mimic a disease with complex etiology and pathogenesis, has proven extremely useful for understanding the biology of remyelination [ ] . slices collected from wt and il- ko mice exhibited comparable extents of demyelination, as revealed by the loss of mbp staining along neurofilamentheavy (nfh + ) axonal fibers, at d after lpc (fig a) . partial remyelination was achieved by d following lpc in wt brain slices, but il- deficiency significantly delayed remyelination (fig a and b) , as shown by lower mbp staining intensity ( fig c) and less mbp + axonal coverage ( fig d) . il- supplementation significantly enhanced remyelination in wt brain slices and rescued remyelination capacities in il- ko brain slices (fig a, c and d ). il- liposome nanoparticles were prepared to facilitate the penetration of il- into the ischemic brain. the mean particle size of il- protein-loaded liposome was . ± . nm with a and the ratio of smi to mbp immunofluorescence intensity (g) in ipsilesional ec, cortex, striatum, and the total for all three areas quantified. n = - /group. data are normalized to the intensities of contralateral hemispheres. � p < . , �� p < . , ��� p < . . one-way anova and bonferroni post hoc. (h) representative images for mbp (green) and smi (red) double immunostaining in the peri-infarct areas in brains from sham, wt mcao mice, and il- ko mcao mice d after mcao. scale bar: μm. data associated with this figure can be found in the supplemental data file (s data). cns, central nervous system; ec, external capsule; fsc-w, forward scatter width; gfap, glial fibrillary acidic protein; il- r, interleukin- receptor α; ko, knockout; mbp, myelin basic protein; mcao, middle cerebral artery occlusion; mfi, mean fluorescence intensity; opc, oligodendrocyte progenitor cell; ssc-a, side scatter area; wt, wild-type. lpc ( μg/ml) was added in wt or il- ko organotypic cultures for h to induce demyelination. il- ( ng/ml) or vehicle was added after lpc removal. culture slices were fixed d and d after lpc treatment. control cultures were maintained in normal culture media for d. (a) mbp (green) and nfh (red) staining was performed to label myelin and axonal fibers, respectively. scale bar: μm. (b) mbp and nfh double staining and imaris d reconstruction. arrows indicate myelination of individual axonal fibers that are magnified in the images to the right. scale bar: μm. (c) myelin integrity was measured as the intensity of mbp immunostaining. (d) the myelination index was calculated as the % mbp + myelin coverage along the entire nfh + fibers. n = - slices/group. � p < . , �� p < . , ��� p < . . one-way anova and bonferroni post hoc. data associated with this figure can be found in the supplemental data file (s data). il- , interleukin- ; ko, knockout; lpc, lysophosphatidylcholine/lysolecithin; mbp, myelin basic protein; nfh, neurofilament heavy; wt, wild-type. polydispersity index of approximately . ± . and a zeta potential of . ± . mv, confirming high stability of the nanodispersion. the il- nanoparticles were applied through the nasal route ( μg/kg body weight), which has been increasingly utilized for cns drug delivery because of its capacity to bypass the blood-brain barrier and avoid first-pass metabolism [ ] . intranasal administration of il- nanoparticles effectively increased il- protein levels in the corpus callosum (cc) and ec, cerebral cortex, and striatum h after il- application (fig a- c ). il- remained elevated in cortex and striatum until h after delivery (fig b and c ). c bl/ j mice were subjected to -min mcao and randomly assigned to receive il- nanoparticle ( μg/kg body weight) or control nanoparticle (vehicle) treatment. nanomaterials were intranasally delivered at h after mcao and repeated at - d, d, d, and d after mcao (fig d) . magnetic resonance imaging (mri) on these animals was performed serially at . t on days , , , and after mcao. t -weighted images showed no significant difference in the brain lesion volume of il- -versus vehicle-treated mice (fig e and f) , indicating comparable gross tissue damage across treatment groups. histological staining of the neuronal marker neun further demonstrated comparable tissue loss d after stroke in il- -and vehicle-treated mice (fig e and g) . serial whole-brain in vivo diffusion tensor imaging (dti) scans were collected d, d, and d after mcao. the same animals were then killed at d after stroke, and the fixed brains were subjected to ex vivo dti scanning (fig a and b , s a and s b fig) . longitudinal observation of fractional anisotropy (fa) maps and calculation of the ratio of fa(ipsilateral)/ fa(contralateral) demonstrated progressive white matter injury (ratio < ) with a slow recovery in the ec of vehicle-treated stroke mice at - d after stroke, which was significantly improved by day following stroke in il- -treated mice (fig c) . this trend was observed until day in the ex vivo fa values (fig c) . further analysis of the d dti images revealed that il- treatment enhanced white matter integrity, as revealed by elevated fa values in the white matter-enriched ec and internal capsule (ic) (fig d, e and g ). in addition, il- treatment significantly reduced radial diffusivity (rd, λ ? ) in the ec ( fig f) and ic (fig h) , which likely indicates improved myelin integrity [ ] . no significant differences between il- and vehicle treatment groups were observed in axial diffusivity (ad, λ || ), a measurement for axonal integrity (s c and s d fig) . to further explore the biological basis of these dti results, we compared the dti results with immunohistochemical staining of mbp performed in the same animals. there were remarkable similarities between the fa maps and mbp images (fig d, i and j). il- treatment significantly increased the mbp + area in the ec (fig d and i ). there was a significant positive correlation between the mbp + areas and the fa values in the ec (fig j) . the quantification of mbp staining confirmed a significant increase in mbp intensity in the ischemic brains of il- -treated mice compared to vehicle-treated mice (fig k) . to determine whether il- can improve axonal myelination after stroke, myelin thickness was measured in the ec using transmission electron microscopy (tem) (fig a) . frequency histograms revealed much greater reduction of axon diameters in the mcao + vehicle group compared to the mcao + il- and sham groups, suggesting higher axonal degeneration in stroke mice without il- treatment (fig b) . scatter plots of the g-ratio (the ratio of the inner axonal diameter to the total outer diameter of myelinated fiber) as a function of axon diameter in sham mice or stroke mice treated with vehicle or il- revealed significant differences in myelin thickness (average g-ratio: sham = . [n = axons from animals]; mcao + vehicle = . [n = axons from animals]; mcao + il- = . [n = axons from animals]) ( fig c) . significant increases in the g-ratio were observed in both low-and high-caliber axons at d after mcao, indicating a reduction in myelin thickness ( fig d) . il- post-treatment significantly reduced the g-ratio of the middle-sized (diameter = - nm) and large (diameter > nm) axonal fibers, suggesting that il- may maintain axonal myelination or promote axonal remyelination after stroke. the g-ratio in small axonal fibers (diameter < nm) displayed a trend toward reduction with il- treatment ( fig d) . saltatory conduction of action potentials along myelinated axons relies on normal organization of the nodes of ranvier (nor), which are rich in sodium and potassium ion channels. double immunofluorescent analyses of nav . (a nodal sodium channel protein) and contactin-associated protein (caspr, a paranodal cell adhesion protein) revealed the nor in the ec (fig e) . typical nor are characterized by punctate nav . staining at nodes between the paranodal staining of caspr. the total numbers of normal nor decreased significantly after stroke in the ipsilateral ec compared to the contralateral ec (fig f) , and this was accompanied by a decrease in the caspr + paranode length (fig g) . il- treatment significantly increased the number of nor and the paranode length in the ischemic ec (fig f and g) , suggesting an improvement in nor structure. the paranodal gap between pairs of caspr staining remained unchanged between groups ( fig h) . as il- enhanced the structural integrity of white matter after mcao, we further evaluated white matter function by measuring the transmission of action potentials in the cc and ec (fig i) . evoked compound action potentials (caps) display an early peak representing fast-conducting myelinated axons (n , fig j) [ ] . the amplitude of the n segment was significantly reduced d after mcao (fig k) , indicating impaired conduction through myelinated axons. il- -treated mice exhibited less reduction in the n amplitude after mcao, suggesting myelin preservation or improved remyelination of axons compared to the vehicle-treated subjects. sensorimotor and cognitive functions were assessed by investigators blinded to group assignments. as shown in fig a and b , il- -treated and vehicle-treated stroke mice showed comparable reductions in latencies to contact and remove adhesive tape from the paw at d after stroke, suggesting similar initial deficits in dexterity across groups. however, at later time points starting from - d after stroke, il- treatment reduced the time spent touching and removing tapes in stroke mice, culminating in improved performance in the adhesive removal test (fig a and b ). long-term sensorimotor performance in the rotarod test after mcao was also improved in il- -treated mice ( fig c) . furthermore, il- -treated mice exhibited long-term improvements in cognitive function in the morris water maze test, as manifested by a reduction in the latency to find the hidden platform (improved spatial learning, fig d and e ) and increased time spent in the target quadrant when the platform was removed (improved memory, fig d and f ). there was no difference in swimming speed among different treatment groups, indicating similar gross motor skills across groups ( fig g) . next, we measured the potential correlation between white mater integrity and behavioral performance after stroke in vehicle or il- -treated mice. the results revealed significant negative correlations between mbp intensity and the latency to touch ( fig h) and remove ( fig i) the adhesive tape at d after stroke, confirming an association between histological observations of white matter integrity and long-term functional recovery of behavioral endpoints. successful differentiation of opcs into myelinating oligodendrocytes is important for remyelination [ ] . thus, we compared the regeneration of myelin-producing oligodendrocytes in mcao mice with or without il- treatment. -bromo- -deoxyuridine (brdu) was injected at - d after mcao to label newly generated cells. mature oligodendrocytes were recognized by immunostaining with anti-adenomatous polyposis coli (apc) (or cc ) antibodies [ ] . compared to vehicle-treated mice, a significantly higher ratio of brdu + apc + newly generated oligodendrocytes was observed in il- -treated mice d after mcao (fig a and b ). the total numbers of brdu + cells and the total numbers of apc + cells were also significantly increased in il- -treated mice compared to vehicle-treated mice (fig c and d ). it has been reported that higher concentrations of the apc antibody may detect some populations of astrocytes in certain pathological conditions [ ] . however, no apc signal was detected in glial fibrillary acidic protein (gfap) + astrocytes in the ischemic brain in our preclinical model of stroke (s fig) . il- is known to promote microglia m polarization, which in turn enhances opc differentiation and remyelination [ ] . we observed in our model that il- treatment robustly promoted microglia/macrophage polarization toward an anti-inflammatory phenotype (cd + iba + ) at and d after stroke (s fig). microglia depletion through genetic targeting or pharmacological treatment is a widely used approach to evaluate the contribution of this population to various biological processes [ ] . therefore, we chose plx , an inhibitor of colony-stimulating factor receptor, to deplete microglia/macrophages and identify their role in il- -afforded oligodendrogenesis ( fig e) [ ] . consistent with previous reports [ ] , plx achieved approximately % depletion efficacy within a short period. dietary intake of plx for d dramatically reduced the number of microglia in the normal brain significant depletion of microglia/macrophages did not change the number of brdu + apc + cells or total brdu + cells compared to microglia/macrophage-competent stroke mice (fig f- h ). il- treatment increased the numbers of brdu + apc + cells or total brdu + cells after stroke in mice (h) quantification of the number of brdu + cells in the peri-infarct areas. n = - /group. � p < . , ��� p < . . one-way anova followed by bonferroni post hoc. data associated with this figure can be found in the supplemental data file (s data). apc, adenomatous polyposis coli; brdu, -bromo- -deoxyuridine; ctx, cortex; ec, external capsule; il- , interleukin- ; mcao, middle cerebral artery occlusion; str, striatum. fed with the control diet. il- treatment exhibited a trend (p = . ) toward lower capacities to increase the number of brdu + apc + newly generated oligodendrocytes in microgliadepleted mice versus microglia-competent mice, supporting a potential role of microglia in il- -afforded oligodendrogenesis. importantly, il- treatment still resulted in a significant enhancement of brdu + apc + oligodendrocyte regeneration in microglia/macrophagedepleted stroke mice (fig f and g ). considering the expression of il- r on oligodendrocyte linage cells, these results indicate that il- may exert microglia/macrophage-independent effects on oligodendrocyte regeneration after demyelination. consistent with a previous study in a model of multiple sclerosis [ ] , we found that repeated intranasal il- administration did not change t-lymphocyte subpopulations or b lymphocytes in the blood (s a-s c fig consistent with a direct effect of il- on oligodendrocytes, in vitro studies showed that il- at a concentration range of - ng/ml directly stimulated the differentiation of neuron-glia antigen (ng ) + opcs into mbp + mature oligodendrocytes without the aid of microglia ( fig a and b ). il- at concentrations of or ng/ml induced opc differentiation that was comparable to the effects of a t cocktail typically used to induce opc differentiation in vitro. the effect of il- on opc differentiation was indeed il- r-dependent, because specific il- rα neutralizing antibodies (il- rabs) blocked the il- -induced increase in mrna expression of the mature oligodendrocyte marker mbp (fig c) . il- at a concentration range of - ng/ml had no effect on opc survival (s a and s b microarray analyses were performed to further elucidate the mechanisms underlying the promotion of oligodendrogenesis by il- . opcs were treated with pbs or il- ( ng/ml) for d. total rna extraction from cell pellets was used for the affymetrix clariom s rat array. principal component analysis (pca) showed clustering of samples in a treatment-dependent manner ( fig d) . in total, differentially expressed genes (degs) were identified (false discovery rate [fdr] < . ; and fold change > ), out of which were up-regulated and were down-regulated in il- -treated opcs relative to pbs-treated opcs (fig e, s and s tables). the gene ontology (go) enrichment analysis was then performed using the metascape analysis [ ] (http://metascape.org). the top enriched go terms in three categories (biological processes, cellular components, and molecular functions) were identified in the upregulated degs by il- treatment (s a fig). a network plot further captured the relationships between the main regulated biological processes, which were related to cell adhesion and motility, cytokine production, cell responses to external stimuli, cellular component organization, and cell death (fig f) . the enriched go terms among the down-regulated degs in il- -treated opcs represent biological processes such as developmental growth, small molecule biosynthetic process, vessel development, and responses to nutrients and peptides (s b fig). these results indicated changes in cell mobilization, growth, differentiation, and defense in il- -treated opcs. a kyoto encyclopedia of genes and genomes (kegg) pathway analysis g) . the ppar transcription factor assay confirmed that pparγ activity, but not pparα or pparδ activities, was up-regulated in opcs upon il- treatment ( fig h) ; these effects were abolished when il- rα was blocked with a neutralizing antibody ( fig i) . furthermore, immunostaining (fig j and k ) and western blotting (fig l- n, and s fig) demonstrated that the pparγ inhibitor gw blocked il- -induced expression of mature oligodendrocyte markers (mbp or myelin oligodendrocyte glycoprotein [mog]), suggesting that pparγ activation is essential for the opc differentiation induced by il- . finally, opc-specific pparγ ko (pparγ-opc ko) mice were used to confirm the importance of pparγ signaling in il- -enhanced oligodendrogenesis in an in vivo stroke model ( fig a) . to confirm the ko of pparγ specifically in opcs, pdgfrα + cd − opcs, glast + cd − astrocytes, cd + immune cells, and other cd − glast − pdgfrα − cns cells were sorted from the brains collected from pparγ-opc ko mice at d after -hydroxytamoxifen injections. the dna extracts were subjected to pcr using primers flanking the loxp site and primers detecting the cre cdna. the loxp band was abolished in opcs because of the targeted deletion after cre-induced recombination, while remaining intact in astrocytes, immune cells, and other cns cells sorted from the same brain ( fig b) . the expression of cre was detected in the opcs of the pparγ-opc ko mice ( fig b) . deletion of pparγ specifically in opcs exerted no impact on il- r expression on o + oligodendrocytes (s fig). vehicle or il- treatment in pparγ-opc ko mice resulted in similar neuronal tissue loss (fig c and d ) and white matter lesion (fig c, e and f) d after stroke. the lack of pparγ expression in opcs did not significantly impair oligodendrocyte replacement after stroke, as shown by comparable numbers of brdu + apc + cells in the ischemic brain from pparγ-opc ko mice and control mice at d after mcao (fig g and h) . however, the absence of pparγ in opcs reduced the capacity of il- to increase the number of newly generated brdu + apc + oligodendrocytes (fig g and h ). consistent with a reduced capacity to promote oligodendrogenesis, il- failed to improve long-term sensorimotor (fig i and j ) or cognitive (fig k- m ) deficits after stroke in the absence of pparγ. il- /pparγ signaling improves white matter repair after stroke the ischemic brain, which is attributed, at least partly, to its release from injured neurons or from infiltrated immune cells [ ] . cerebral il- levels then return to baseline within wk after stroke [ , ] . in patients, serum levels of il- are known to change following an acute stroke [ ] . furthermore, il- gene polymorphisms are related to susceptibility to ischemic stroke and subsequent functional outcomes [ , ] . finally, a recent study reported the expression of il- r surrounding axonal filaments in postmortem brain tissue from patients with multiple sclerosis [ ] . although these clinical and experimental data are correlative, the present series of in vivo and in vitro studies firmly establish a causal link between il- repletion and white matter integrity in preclinical stroke models. deficiency of endogenous il- an improvement in white matter integrity might be achieved in two ways: preservation of preexisting white matter structures or enhancement of white matter repair. our data favor the latter as the predominant mechanism of action for il- . first, longitudinal dti revealed similar reductions in fa values shortly after stroke in vehicle or il- -treated mice, indicating comparable degrees of initial white matter injury during the early/acute phase. a significant improvement in white matter microstructure was only observed in il- -treated animals in later stages of recovery ( and d after stroke). these results strongly suggest a delayed effect of il- on white matter repair, rather than preservation shortly after injury. second, electron microscopy (em) and histological studies revealed structural signs of remyelination in the ischemic brain, including thinner myelin segments than before stroke, and reduced paranode length with caspr/nav . staining [ ] . each of these structural alterations were significantly improved by il- treatment, indicating superior remyelination. third, il- treatment increased the number of new, mature oligodendrocytes (apc + brdu + ) in the ischemic brain d after stroke. such improvements in oligodendrogenesis are critical for successful remyelination. finally, the lpc model of demyelination-remyelination unequivocally confirmed the white matter-specific effects of il- in remyelination. as expected, the il- -mediated reconstitution of myelin structure culminated in functional improvements in neural conduction and behavioral performance, despite no changes in the degree of gray matter loss. although il- is important for white matter repair after brain injury, the il- ko mice do not display deficits in myelin coverage in organotypic brain slice cultures under physiological conditions. this observation is consistent with the view that different mechanisms control myelin sheath formation during normal development and remyelination [ ] . although our t scanning did not show significant reductions in brain lesion size under conditions of il- repletion, there was a trend toward lesion volume reduction at - d after stroke. as the il- treatment was initiated h after stroke, it remains possible that il- may provide some degree of early protection [ ] . it is also possible that some neun + neurons remaining within the gray matter of mice without il- might not be physiologically functional, although they are still histologically present, perhaps because their communication via white matter tracts is significantly impeded. a previous study using mixed glial cultures demonstrated that the addition of il- promotes the differentiation of opcs into mbp-expressing oligodendrocytes in vitro, an effect mainly attributed to il- -mediated anti-inflammatory effects [ ] . an additional conclusion emerging from our studies is that il- enhances white matter repair at least partially independent of its well-established anti-inflammatory effects on microglia/macrophages. in il- -treated stroke mice, we observed an anti-inflammatory phenotypic change in microglia/ macrophages, which is known to promote remyelination after cns injury [ ] . however, dramatic depletion of microglia/macrophages in the ischemic brain did not abolish the capacity of il- to enhance oligodendrocyte replacement, supporting the involvement of microglia/ macrophage-independent mechanisms. in vitro studies then confirmed that il- can also directly target white matter components and promotes opc differentiation into mature oligodendrocytes in the absence of microglia/macrophages. previous fate mapping studies revealed that opcs are responsible for generating new oligodendrocytes during remyelination [ , ] . however, the differentiation of opcs is frequently impaired in lesioned brains, mainly because of nonpermissive environmental cues [ ] [ ] [ ] . we found that il- treatment may overcome these obstacles for opc differentiation in the ischemic brain, both directly by promoting opc differentiation and indirectly through immunomodulation. notably, endogenous oligodendrocyte differentiation has been observed until as late as mo after cns injuries [ ] , and this prolonged effort may provide a sufficiently wide therapeutic window that allows delayed il- treatment to restore white matter and encourage long-term stroke recovery. in the present study, we report a previously undefined mechanism of il- , namely that it directly induces opc differentiation by activating pparγ signaling. pparγ is a master transcription factor known to promote opc differentiation and maturation though manifold mechanisms involving enhanced myelin lipid synthesis, reduced oxidative stress, and promotion of mitochondrial function [ , ] . our previous work has shown that rosiglitazone, a pparγ agonist, enhanced opc proliferation and increased the number of newly generated mature oligodendrocytes after mcao [ ] . in addition, pparγ may interact with histone deacetylases to modulate cell differentiation [ ] . we demonstrated here that pparγ inhibition abolished the effect of il- on opc differentiation in vitro and that opc-specific ko of pparγ in vivo reduced il- -enhanced oligodendrocyte replacement in the ischemic brain. these collective results indicate that il- -induced oligodendrocyte differentiation/maturation relies at least partially on the activation of pparγ activity in opcs. precisely how il- /il- r engages pparγ activity is not yet known, but several studies suggest that il- may activate pparγ in macrophages and other cell types through signal transducer and activator of transcription (stat ) signaling [ , ] . whether this pathway is also operational in opcs lies beyond the scope of the present study but warrants further investigation. aside from the cell-specific effects of il- on opcs, we note that il- r is widely distributed in many types of cns cells as well as infiltrated immune cells. several lines of evidence from our study support the capacity of il- to target multiple types of cells in addition to oligodendrocytes. first, we did not observe significant differences in the density of brdu + apc + cells between wt and pparγ-opc ko mice following mcao, suggesting that endogenous il- is able to promote white matter integrity by targeting other types of cells. second, although pparγ inhibition eliminated the effect of il- on opc differentiation in cultures, the absence of pparγ in opcs reduced, but did not abolish, the capacity of il- to increase the number of newly generated oligodendrocytes in vivo. these results suggest that pparγ expression in opcs is an important but not exclusive mechanism for il- -induced oligodendrogenesis in preclinical stroke. indeed, we observed that il- treatment robustly promoted microglia/macrophage polarization toward an anti-inflammatory m phenotype, as mentioned above. il- -stimulated microglia have been shown to release il- induced (il i ), which in turn augments remyelination [ ] . thus, direct and indirect effects on microglia/macrophages may contribute to the white matter repair afforded by il- . it has also been reported that the effects of il- on adaptive immune responses contribute partially to recovery from neurotrauma [ ] . in our hands, however, repeated intranasal il- administration did not change t-lymphocyte subpopulations or b lymphocytes in blood and did not alter the composition of infiltrated lymphocytes. these data dispute the involvement of adaptive immune responses in il- -afforded neuroprotection in our stroke model. aside from its roles in immunoregulation, il- can also directly target other types of cns cells. for example, a recent study reported that the activation of il- r signaling in neurons may increase axonal outgrowth and ameliorate axonal pathology in a model of multiple sclerosis [ ] . in this context, it is important to note that we did not observe improvements in the dti measurement for axonal integrity (ad, λ || ). in other disease models, multiple factors including inflammation, cellular infiltration, and gliosis may contribute to ad at chronic stages of disease. therefore, the measurement of λ || appears to lack sufficient resolution to distinguish axonal damage/repair versus confounding cellular responses over the course of progressive demyelination [ ] . on the other hand, our em data demonstrated an increase in axon diameter in il- -treated mice compared to vehicle-treated controls, supporting a beneficial role of il- in maintaining axonal structure. these structural data were supported by our electrophysiological studies of axon conductance. preservation of axonal structure and electrical function might provide an important feedback signal for opc differentiation and guide remyelination [ , ] , preventing wallerian degeneration of neurons after ischemia and enhancing white matter repair. in addition to these mechanisms, astrocytes are known to produce trophic factors in response to il- stimulation, including brain-derived neurotrophic factor (bdnf) and nerve growth factor (ngf), both of which are also critical for brain function and brain repair [ ] . a series of in vitro studies assessed the effective concentrations of il- in various types of cns cells. for example, il- regulates microglia activity at concentrations of - ng/ml [ , ] . il- inhibits inflammatory responses and boosts trophic factor release in astrocytes at - ng/ml [ ] . il- protects neurons against excitotoxicity and stimulates axonal outgrowth in vitro at ng/ml [ ] . in the present study, il- directly stimulated opc differentiation within a concentration range of - ng/ml. these collective studies reveal similar effective il- concentrations (in nanoscales) in glial and neuronal cells of the cns. indeed, it is precisely the multitargeting capacities of low-dose il- that may render it an excellent therapy for cns injuries characterized by a wide array of pathological mechanisms. the ultimate implementation of preclinical research into clinical treatments requires extensive information on safety, optimal dose, route, and schedule of administration, among other variables. in the present study, il- stimulated opc differentiation in cultures at a concentration range of - ng/ml, and the optimal concentration was - ng/ml. although there is neither a mathematical nor empirical method to accurately convert in vitro concentrations to in vivo intranasal dosage, these in vitro data support the capacity of il- to directly target opcs at low concentrations. previous preclinical studies have similarly documented therapeutic effects of low doses of il- in ischemic stroke and other neurological diseases. for example, subcutaneous il- application at μg/kg/d [ ] or intracerebroventricular application of il- at ng/d [ ] significantly improved long-term outcomes after stroke. vogelaar and colleagues reported that intrathecal or intranasal ( μg/d) il- treatment ameliorated clinical signs and reversed disease progression in a model of multiple sclerosis [ ] . those studies, however, did not measure il- concentrations in the brain after treatment. here, we delivered il- intranasally at a dose of μg/kg/d (about . μg/d based on . kg average body weight), which significantly elevated il- levels in key parts of the brain. this dose is similar to the effective intranasal dose for vogelaar's study and improved long-term functional recovery in our stroke model. repeated dosing regimens or controlled continuous release was employed for all in vivo il- treatment because of its rapid clearance and inactivation. the use of liposome-entrapped peptides has been reported to protect the cytokine and enhance its tissue delivery [ , ] . consistent with those findings, we demonstrated that il- liposome nanoparticle delivery elevated il- levels in the brain for at least h. further characterization of the optimal regimen of il- treatment for stroke would accelerate its clinical translation. despite growing awareness of the importance of white matter restoration in poststroke rehabilitation, there is no effective therapy to preserve white matter function or to improve its repair in humans. our study has uncovered a novel role of il- in poststroke white matter recovery-a role that lies beyond its well-known immunoregulatory functions. furthermore, we have shown that il- promotes oligodendrogenesis and white matter repair in a pparγdependent manner. therefore, it seems reasonable to continue to test il- as a therapeutic strategy for functional recovery after ischemic stroke. in particular, the intranasal route enables cns il- delivery, which is expected to significantly reduce the risk of peripheral side effects and expedite the eventual translation of il- treatment for stroke victims. all animal procedures were approved by the university of pittsburgh institutional animal care and use committee (approval number: ), performed in accordance with the national institutes of health guide for the care and use of laboratory animals, and reported in accordance with the animal research: reporting in vivo experiments (arrive) guidelines. all efforts were made to minimize animal suffering and the number of animals used. all animals were housed in a temperature-and humidity-controlled facility with a -h light/dark cycle. food and water were available ad libitum. transient cerebral ischemia was induced by intraluminal occlusion of the left middle cerebral artery (mca) for min, as described previously [ ] . sham-operated animals underwent the same anesthesia and exposure of arteries without mcao. briefly, mice were anesthetized with % isoflurane in a % o / . % n o mixture until they were unresponsive in the tail-pinch test. mice were then fitted with a nose cone blowing . % isoflurane in a % o / . % n o mixture under spontaneous breathing for anesthesia maintenance. an - monofilament with a silicon-coated tip was introduced into the common carotid artery, advanced to the origin of the mca, and left in place to limit mca blood flow for min. rectal temperature was controlled at . ± . ˚c using a temperature-regulated heating pad during surgery. regional cerebral blood flow was measured in all stroke animals using laser doppler flowmetry. animals that did not display at least % reduction in regional cerebral blood flow during mcao compared to pre-ischemia levels were excluded from further experimentation. surgeries and quantification were performed by investigators blinded to animal genotypes and experimental grouping. animals were randomly assigned to sham or cerebral ischemia groups and received randomized treatments using a lottery drawing box. a grand total of mice ( sham-operated and ischemic mice) were used in this study, including mice that were excluded from further assessments, either because of death after ischemia or failure of ischemia induction. the mortality during mcao surgery was . % in wt male mice ( / ), . % in il- ko male mice ( / ), . % in microglia/macrophage-depleted mice ( / ), and . % in pparγ-opc ko mice ( / ). there was no significant difference in mortality rate across groups. lecithin ( mg) and cholesterol ( mg) were dissolved in ml of a chloroform-methanol mixed solution ( : , volume:volume). a lipid layer was formed after evaporating the mixed solvent under vacuum distillation. recombinant il- protein ( mg, peprotech, rocky hill, nj, usa) was dissolved in ml of water and then added to the lipid layer for hydration for about min. the mixture was homogenized by ultrasonic probes for min in an ice bath and then filtered through a . -μm membrane. the free il- protein was removed by ultrafiltration with centrifugal filter units ( -kda cutoff). the particle size and zeta potential of il- protein-loaded liposomes were determined by dynamic light scattering (dls). the concentration of il- protein was determined by the bicinchoninic protein determination kit (thermo fisher scientific, pittsburgh, pa, usa). animals were randomly assigned to receive vehicle or il- ( μg/kg body weight, intranasal) nanoparticle treatment starting h after mcao and repeated at - d, d, d, and d postsurgery. briefly, under isoflurane anesthesia, five -μl drops (total μl) of il- nanoparticles were applied alternately into each nostril with a -min interval between drops. control animals received an equal volume of vehicle control using the same anesthesia and delivery regimen. in order to label proliferating cells, animals were intraperitoneally injected with the thymidine analogue brdu ( mg/kg, b , sigma, st. louis, mo, usa) twice a day (with an interval of at least h) for consecutive days, beginning at d after mcao. behavioral tests were performed by an individual blinded to experimental groups. sensorimotor functions were measured - d after mcao. the rotarod test. briefly, mice were forced to run on a rotating drum (iitc life science, woodland hills, ca, usa) with speeds starting at rpm and accelerating to rpm within s. three consecutive trials were conducted for each mouse with an interval of min. the time at which a mouse fell off the drum was recorded as the latency to fall. data were expressed as mean values from three trials. the adhesive removal test. the adhesive removal test was performed to assess tactile responses and sensorimotor asymmetries. two × -mm adhesive tapes were applied to lesioned forepaws. tactile responses were measured by recording the time to initially contact the ipsilateral paws, as well as the time to remove the adhesive tape, with a maximum observation period of s. morris water maze test. cognitive function was analyzed with the morris water maze test - d after mcao, as described previously [ ] . in the learning test, a square platform ( × cm ) was submerged cm beneath the water surface in a circular pool (diameter = cm) of opaque water. mice were placed into the pool from one of the four locations and allowed to locate the hidden platform for s. each mouse was trained on trials (with randomly assigned starting positions) per day to locate the platform for three consecutive days before mcao. at the end of each trial, the mouse was placed on the platform or allowed to stay on the platform for s with prominent spatial cues displayed around the room. the time spent to reach the platform (learning phase) was recorded. three trials were performed on each day. the memory test was performed on day . the platform was removed, and a single -s probe trial was conducted. time spent in the goal quadrant where the platform was previously located was recorded. animals were euthanized and perfused with saline followed by % pfa (sigma-aldrich; st. louis, mo, usa) in pbs. brains were cryoprotected in % sucrose in pbs. coronal brain sections ( μm) were sliced on a freezing microtome, and six equally spaced coronal brain sections encompassing the mca territory were stained with a rabbit anti-neun antibody (abn , : ; millipore, burlington, ma, usa) or rabbit anti-mbp antibody (mbp ab , : , abcam, cambridge, ma, usa). loss of neun + or mbp + tissue was analyzed by a blinded observer using nih image j software. the infarct volume with edema correction was calculated as the volume of the contralateral hemisphere minus the noninfarcted volume of the ipsilateral hemisphere. coronal brain sections were subjected to immunofluorescence staining. briefly, floating brain sections were blocked with % donkey serum in . % triton x- in pbs (pbst) for h at room temperature, followed by overnight incubation with primary antibodies at ˚c. after three washes in . % pbst, sections were incubated with the appropriate secondary antibodies for h at room temperature. the process was repeated once for double staining. image analyses were performed on one or two randomly selected microscopic fields in the peri-infarct areas of the ec, two in the cortex, and two in the striatum of each section; two sections covering the infarct area were assessed for each mouse brain. the recorded images were loaded onto image j (nih) and were manually quantified by observers blinded to grouping. positively stained cells were electronically labeled with the software to avoid duplicated counting. the infarct area was identified as the region in which the majority of dapi-stained nuclei were shrunken. the border of the infarct was determined by the loss of neun staining and the accumulation of iba + microglia/macrophage or brdu + apc + oligodendrocytes on adjacent sections. we defined the peri-infarct area as the tissue that covers a radial distance of - μm from the border of the infarct. animals were euthanized and perfused with cold saline. brains were dissected, and the ipsilateral (left) and contralateral (right) hemispheres were collected. brains were dissociated into a single-cell suspension using a gentlemacs dissociator (miltenyl biotec) following the manufacturer's instructions. the suspension was passed through a -μm cell strainer (fisher scientific, pittsburgh, pa, usa) and resuspended in % percoll (ge health). single-cell suspensions were separated from myelin and debris by centrifugation ( g, min, ˚c) on a %- % percoll gradient. cells at the interface were collected and washed with hank's balanced salt solution (hbss; sigma-aldrich, st. louis, mo, usa) containing % fetal bovine serum (sigma-aldrich, st. louis, mo, usa) and mm edta (sigma-aldrich, st. louis, mo, usa) before staining. blood cells were prepared as described previously [ ] . single-cell samples were first incubated with antibodies to surface antigens for min on ice at ˚c in the dark. after two washes, cells were fixed and permeabilized with an intracellular staining kit (thermo fisher ebioscience, pittsburgh, pa, usa) according to the manufacturer's protocol. caps were measured in the ec. mice were decapitated after co euthanasia, and brains were removed. coronal slices ( μm thick) were prepared − . mm from bregma and transferred to an incubation chamber containing pregassed ( % o / % co ) artificial cerebrospinal fluid (acsf; mmol/l nacl, . mmol/l kcl, mmol/l na h po , . mmol/l cacl , mmol/l nahco , . mmol/l mgcl , mol/l glucose [ph . ]) for min at ˚c and then for h at room temperature ( ˚c). brain slices were then perfused with acsf at a constant rate ( - ml/min) at ˚c. for recording, a bipolar stimulating electrode (intertip distance, μm) was placed across the cc at about . mm lateral to the midline. a glass extracellular recording pipette ( - mo tip resistance when filled with acsf) was placed in the ec, . , . , . , and mm lateral from the stimulating electrode. the recording at . mm from the stimulating electrode was reported. the cap signal was digitized (digidata b plus humsilencer; molecular devices, san jose, ca, usa), amplified (× k), and recorded by the axoclamp b amplifier (molecular devices, san jose, ca, usa). the signal was then analyzed by pclamp software (molecular devices, san jose, ca, usa). the input-output curves were generated by increasing the stimulating intensity by . -ma intervals from baseline, up to ma. the amplitude of the n component of the cap (representing myelinated fibers) was calculated as the variance from the first peak to the first trough. tem was used to measure myelin thickness in the cc/ec area. mice were perfused with icecold saline, followed by % pfa and . % glutaraldehyde in . mol/l pbs buffer. the cc/ec tissue near the site of ischemia was microdissected into -mm blocks. these specimens were immersion-fixed for h in % glutaraldehyde. tissue was then rinsed in pbs and fixed in % osmium tetroxide in . m pbs for min. samples were dehydrated in increasing concentrations of acetone and embedded in araldite resin. ultrathin -nm sections were cut on a leica uct ultramicrotome with a diamond knife (diatome, wetzlar, germany), stained with uranyl acetate and lead citrate, and viewed using a jem tem (jeol, akishima, japan). two sections from each animal were analyzed. three to images ( μm each) were acquired in randomly selected areas within the cc/ec from each section at a magnification of , × and analyzed with image j by an investigator blinded to experimental groups. at least random axons per animal were analyzed by tracing the axonal circumference and the whole fiber circumference in a blinded fashion. g-ratios were calculated as the ratio of the inner axonal diameter to the total outer diameter (axonal diameter + total myelin sheath thickness). for in vivo mri, mice were initially anesthetized with % isoflurane and then positioned on an animal cradle with a stereotaxic head holder. anesthesia was maintained between % and . % isoflurane throughout the experiment. respiration and temperature were monitored continuously, and temperature was maintained by a warm animal heating system (sa instruments, stony brook, ny, usa). mri was performed on a . t/ -cm aviii hd spectrometer (bruker biospin, billerica, ma, usa) equipped with a -cm high-performance gradient set and using an -mm quadrature rf transmit volume coil and a -channel receive surface rf coil and paravision . . . a t -weighted rare sequence was used to visualize the infarct, with the following setup: repetition time (tr)/echo time (te) = , / ms, field of view (fov) = × mm, acquisition matrix = × , slices with a slice thickness of . mm, averages, and a rare factor = . the dti data were collected using an echo planar imaging (epi)-dti imaging sequence with the same geometry as the t -weighted scan, using the following setup: tr/te = , / ms, fov = × mm, acquisition matrix = × , slices with a slice thickness of . mm, averages, segments, a images and noncolinear diffusion images, Δ/δ = / ms, and a b-value = , s/mm . perfusion-fixed brains were left in the skull and imaged ex vivo using a bruker av hd . t/ -mm vertical-bore microimaging system equipped with a micro . gradient set capable of , mt/m, a -mm quadrature rf resonator, and paravision . . (bruker biospin, billerica, ma, usa). dti data were collected using a multislice spin-echo sequence with a images and noncolinear diffusion images with the following setup: te/tr / , ms, averages, × matrix, × -mm fov, slices, . mm slice thickness, bvalue = , s/mm , and Δ/δ = . / . ms. this setup has been established for the acquisition of high-resolution dti scanning in small animals [ , , ] . dti data were analyzed with dsi studio software (http://dsi-studio.labsolver.org/). rois were drawn manually in a blinded manner encompassing the ec and ic in the ipsilesional and contralesional hemispheres to determine fa, ad, and rd. dec, fa, and rd maps were generated by dsi studio software. in order to verify cre-induced deletion of flanked pparγ exons, a pair of primers (forward: -tgtaatggaagggcaaaagg; reverse: -tggcttccagtgcataagtt) that flank the downstream loxp site were used to perform pcr with genomic dna. program for pcr was ˚c for min; ( ˚c for s; ˚c for s; ˚c for s) × , and then ˚c for min. a pair of primers that detect the cre cdna (forward: -gcggtctggca gtaaaaactatc; reverse: -gtgaaacagcattgctgtcactt) were used as a positive control to confirm the absence of pcr band in opc was not due to failed pcr protocol itself. microarray experiment was performed in the genomics research core in the university of pittsburgh. total rna was prepared from il- -or pbs-treated opcs using trizol (qiagen) according to manufacturer's instruction. preparation of cdna was performed using the wt plus reagent kit (thermo fisher) according to the manufacturer's instructions. reverse transcription was performed using ng total rna and dntp-t random primers. second-strand synthesis and in vitro transcription created amplified crna. cdna from a second reverse transcription reaction was fragmented and end-labeled with biotin for hybridization to the affymetrix clariom s rat array. following overnight ( h) hybridization at ˚c with rotational mixing at rpm, arrays were processed on a genechip fluidics station using manufacturer-specified protocols. to remove unbound sample, arrays were first washed with nonstringent wash buffer a. the genechips were then stained for min in stain cocktail . buffer a was again used to wash off excess stain. signal amplification was achieved by -min incubation with stain cocktail followed by a second -min incubation with stain cocktail . the chip was washed with high-stringency buffer b and filled with array holding buffer before being removed from the fluidics station and scanned using the genearray scanner. first-level image analysis was performed using affymetrix expression console. four biologic replicates were performed for each experimental condition. data resulted in , detected probes that were used for subsequent analysis using transcriptome analysis console (affymetrix) and metascape analysis [ ] (http://metascape.org). the go network plot is visualized using cytoscape, in which terms with a similarity > . are connected by edges. each node represents an enriched term and is colored by its cluster id. primary opc cultures were isolated with the differential attachment method. briefly, brains of mixed-sex sprague dawley rat pups (postnatal day - ) were triturated after removing the meninges and blood vessels. the brain tissues were dissociated with trypsin ( . %) at c for min. after washing with ice-cold dmem, the cells were passed through a -μm filter and plated onto poly-d-lysine-coated t-flasks filled with culture media (dmem/f containing % heat-inactivated fetal bovine serum, mm l-glutamine, mm sodium pyruvate, μm nonessential amino acids, u/ml penicillin, and μg/ml streptomycin) [ ] . cells were grown to confluence ( ) ( ) ( ) ) in a humidified incubator at ˚c and with % co . microglia were removed by shaking the mixed glia-containing flasks for h at rpm. after removing microglia, flasks were subjected to shaking at rpm overnight to separate opcs from the astrocyte layer. opcs were maintained for - d in a serum-free basal defined medium (bdm: dmem, . % bovine serum albumin, μg/ ml human apo-transferrin, μg/ml insulin, nm sodium selenite, nm d-biotin, nm hydrocortisone) containing ng/ml pdgf and ng/ml bfgf. for oligodendrocyte induction, opcs were stimulated with t and cntf ( ng/ml). the medium was exchanged every d. protein was isolated from in vitro cultures. western blots were performed using the standard sds-polyacrylamide gel electrophoresis (page) method. the primary antibodies used in this study include rabbit anti-mbp (cell signaling technology, , : , danvers, ma, usa), mouse mog (santa cruz, sc- , : , santa cruz, ca, usa), and mouse anti-β-actin (a , : , ; sigma-aldrich, st. louis, mo, usa). in brief, polyvinylidene difluoride (pvdf) membranes were blocked in % nonfat milk for h at room temperature and then incubated at ˚c overnight with primary antibodies. next, the membrane was incubated with secondary antibodies for h at room temperature ( : , , li-cor biosciences, lincoln, ne, usa), and scanned with li-cor odyssey infrared imaging system - u (li-cor biotechnology, lincoln, ne, usa). imagej software was used for gel analyses. nuclear extracts were prepared from opcs using ne-pe nuclear and cytoplasmic extraction reagent kit (thermo fisher, , pittsburgh, pa, usa). ppar activity was assessed using the ppar (α, δ, γ) transcription factor assay kit (abcam, ab , toronto, on, canada) or the pparγ transcription factor assay kit (cayman chemical, , ann arbor, mi, usa) following the manufacturers' instructions. the cerebellum and attached hindbrain were isolated from p c bl/ j mouse pups, sectioned sagittally at μm on a mcilwain tissue chopper, and plated onto millipore-millicel-cm mesh inserts (thermo fisher scientific, pittsburgh, pa, usa) in -well culture plates with media containing % basal medium eagle (sigma, st. louis, mo, usa, b ), % heatinactivated horse serum (gibco - ), % earle's balanced salt solution (gibco - ), . mg/ml glucose (sigma g ), % penicillin-streptomycin, and % glutamax. media were changed every - d. to induce demyelination, cultures were treated with lpc (sigma, . mg/ml) for h. slices were washed in media for min and treated for or d with pbs or il- ( ng/ml, il- was added to fresh media every other day). slices were fixed in % pfa for h. rabbit anti-mbp antibodies (santa cruz, santa cruz, ca, usa, sc- ; : ) and mouse anti-nf-h antibodies (abcam, cambridge, ma, usa, ab , : , ) were applied for h at ˚c. fluorescence-conjugated secondary antibodies were applied overnight at ˚c. slices were mounted onto glass slides and coverslipped with fluoromount-g. confocal microscopy (fluoview fv ; olympus, tokyo, japan) was used to capture images. mbp staining intensity was analyzed with imaris software (bitplane ag). morphology reconstructions were carried out with the imaris filamenttracer module. quantitative assessment of myelination was performed in a blinded manner using the imaris colocalization module. thresholds for colocalization of red (nfh + axon) and green (mbp + myelin) channels were calculated automatically and maintained across different images from the same batch of staining. a colocalization channel was created and channel statistics were calculated automatically. the myelination index was calculated as the percent of colocalized areas in the total nfh + red pixels above threshold. for each condition, at least slices collected from three independent experiments were analyzed. two to three fields were imaged from each slice. sample sizes for animal studies were determined by power analyses based on pilot studies or the literature. results are presented as mean ± standard error of the mean (sem). graphpad prism software (version . . , la jolla, ca, usa) was used for statistical analyses. the student's t test was used for comparison of two groups for continuous variables with normal distributions. the mann-whitney u rank sum test was used for continuous variables with nonnormal distributions. the differences in means among multiple groups were analyzed using one-way anova. differences in means across groups with repeated measurements over time were analyzed using the repeated-measures anova. when the anova showed significant differences, pairwise comparisons between means were tested by post hoc bonferroni tests. the correlation analyses between continuous data with normal distribution were performed using pearson correlation analysis. spearman rank correlation analyses were used to test correlations between data sets with nonnormal distributions. in all analyses, p < . was considered statistically significant. clinical prediction of functional outcome after ischemic stroke: the surprising importance of periventricular white matter disease and race the axon-glia unit in white matter stroke: mechanisms of damage and recovery demyelination as a rational therapeutic target for ischemic or traumatic brain injury white matter injury in ischemic stroke 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of learning and memory by meningeal immunity: a key role for il- . the journal of experimental medicine interleukin- is essential for microglia/macrophage m polarization and long-term recovery after cerebral ischemia. stroke; a journal of cerebral circulation il- knock out mice display anxiety-like behavior. behavior genetics /pparγ signaling improves white matter repair after stroke role of interleukin- in regulation of age-related inflammatory changes in the hippocampus neuronal interleukin- as a modulator of microglial pathways and ischemic brain damage increased brain injury and worsened neurological outcome in interleukin- knockout mice after transient focal cerebral ischemia il- in the brain: a cytokine to remember temporal oligodendrocyte lineage progression: in vitro models of proliferation, differentiation and myelination remyelination in experimental models of toxin-induced demyelination quantitative analysis of drug delivery to the brain via nasal route dysmyelination revealed through mri as increased radial (but unchanged axial) diffusion of water myelinated and unmyelinated axons of the corpus callosum differ in vulnerability and functional recovery following traumatic brain injury the oligodendrocyte-specific antibody 'cc ' binds quaking m microglia and macrophages drive oligodendrocyte differentiation during cns remyelination an updated assessment of microglia depletion: current concepts and future directions microglia are required for protection against lethal coronavirus encephalitis in mice fast direct neuronal signaling via the il- receptor as therapeutic target in neuroinflammation meta-and orthogonal integration of influenza "omics" data defines a role for ubr in virus budding reduced il- but elevated il- , il- , and ige serum levels in patients with cerebral infarction during the acute stage functional polymorphisms of interleukin and interleukin may predict evolution and functional outcome of an ischaemic stroke association of variable number of tandem repeat polymorphism in the il- gene with ischemic stroke in the chinese uyghur population regenerating cns myelin-from mechanisms to experimental medicines il- -induced peroxisome proliferator-activated receptor gamma activation inhibits nf-kappab trans activation in central nervous system (cns) glial cells and il- /pparγ signaling improves white matter repair after stroke ng glia generate new oligodendrocytes but few astrocytes in a murine experimental autoimmune encephalomyelitis model of demyelinating disease chronic oligodendrogenesis and remyelination after spinal cord injury in mice and rats myelin impairs cns remyelination by inhibiting oligodendrocyte precursor cell differentiation hyaluronan accumulates in demyelinated lesions and inhibits oligodendrocyte progenitor maturation chondroitin sulfate proteoglycans in demyelinated lesions impair remyelination peroxisome proliferator-activated receptor gamma (ppargamma): a master gatekeeper in cns injury and repair peroxisome proliferator-activated receptor-gamma agonists promote differentiation and antioxidant defenses of oligodendrocyte progenitor cells rosiglitazone promotes white matter integrity and long-term functional recovery after focal cerebral ischemia cyclin d inhibits peroxisome proliferator-activated receptor gamma-mediated adipogenesis through histone deacetylase recruitment stat transcription factor is a facilitator of the nuclear receptor ppargamma-regulated gene expression in macrophages and dendritic cells il i augments cns remyelination and axonal protection by modulating t cell driven inflammation mhcii-independent cd + t cells protect injured cns neurons via il- diffusion tensor mri as a biomarker in axonal and myelin damage neuronal activity promotes oligodendrogenesis and adaptive myelination in the mammalian brain neuronal activity regulates remyelination via glutamate signalling to oligodendrocyte progenitors functional il- receptors on mouse astrocytes: il- inhibits astrocyte activation and induces ngf secretion microglia/macrophage polarization dynamics reveal novel mechanism of injury expansion after focal cerebral ischemia cytokines in liposomes: preliminary studies with il- , il- , il- , gm-csf and interferon-gamma effect of il- and il- liposomal formulations on the induction of immune response to bovine herpesvirus type- glycoprotein d adoptive regulatory t-cell therapy protects against cerebral ischemia selective role of na(+) /h(+) exchanger in cx cr (+) microglial activation, white matter demyelination, and post-stroke function recovery tissue plasminogen activator promotes white matter integrity and functional recovery in a murine model of traumatic brain injury key: cord- -o t srim authors: chaudhari, jayeshbhai; liew, chia-sin; workman, aspen m.; riethoven, jean-jack m.; steffen, david; sillman, sarah; vu, hiep l. x. title: host transcriptional response to persistent infection with a live-attenuated porcine reproductive and respiratory syndrome virus strain date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: o t srim both virulent and live-attenuated porcine reproductive and respiratory syndrome virus (prrsv) strains can establish persistent infection in lymphoid tissues of pigs. to investigate the mechanisms of prrsv persistence, we performed a transcriptional analysis of inguinal lymphoid tissue collected from pigs experimentally infected with an attenuated prrsv strain at days post infection. a total of differentially expressed genes (degs) were detected of which degs were upregulated and degs were downregulated. specifically, genes involved in innate immune responses and chemokines and receptors associated with t-cell homing to lymphoid tissues were down regulated. as a result, homing of virus-specific t-cells to lymphoid tissues seems to be ineffective, evidenced by the lower frequencies of virus-specific t-cell in lymphoid tissue than in peripheral blood. genes associated with t-cell exhaustion were upregulated. likewise, genes involved in the anti-apoptotic pathway were upregulated. collectively, the data suggested that the live-attenuated prrsv strain establishes a pro-survival microenvironment in lymphoid tissue by suppressing innate immune responses, t-cell homing, and preventing cell apoptosis. porcine reproductive and respiratory syndrome virus (prrsv) is a positive sense, single stranded rna virus that belongs to the family arteriviridae, under the order nidovirales [ ] . based on phylogenetic analysis, prrsv was originally classified into two types: type or prrsv- , which originated in europe, and type or prrsv- , which originated in north america. the international committee on taxonomy of viruses (ictv) recently updated the arterivirus taxonomic structure in which prrsv- and prrsv- are now respectively classified as two species: betaarterivirus suid and betaarterivirus suid [ ] . prrsv infects pigs of all ages; however, clinical manifestations are more severe when the virus infects pregnant sows and young pigs, causing reproductive failure and respiratory distress, respectively (reviewed in [ ] ). prrsv is endemic in most swine producing countries worldwide, causing significant economic losses to swine producers [ ] . genes involved in innate immune response and genes encoding chemokines and receptors important for t-cell homing and trafficking were downregulated. on the other hand, genes involved in the anti-apoptotic pathway and t-cell exhaustion were upregulated. functional studies revealed that the frequencies of virus-specific ifn-γ-secreting cells are lower in lymphoid tissue than in peripheral blood mononucleated cells (pbmcs). collectively, the results shed important insight into the mechanisms of prrsv persistence in the host. the attenuated prrsv strain designated con used in this study was recovered from an infectious cdna clone constructed using viral rna extracted from the attenuated con-p [ ] . marc- cells, a monkey kidney cell line [ ] , were cultured in dulbecco's modified eagle's medium (dmem) supplemented with % fetal bovine serum (fbs) and × penicillin-streptomycin ( units/ml of penicillin, and µg/ml of streptomycin (life technologies, grand island, ny, usa). pbmcs and lymph node-derived cells were cultured in complete rpmi- medium (crpmi) supplemented with % fbs, × of glutamax- (life technologies, grand island, ny, usa), units/ml of penicillin, and µg/ml of streptomycin (sigma-aldrich, st. louis, mo, usa). mouse monoclonal antibody specific to prrsv n protein sdow was purchased from the national veterinary services laboratories (ames, ia, usa). anti-porcine cd ε (clone bb - e - c ; fitc-conjugated), anti-porcine cd (clone - - ; pecy -conjugated), anti-porcine cd (clone - - ; alexa flour -conjugated), and anti-porcine ifn-γ (clone p g ; pe-conjugated), were purchased from bd biosciences (san diego, ca, usa). alexa flour -conjugated goat anti-mouse igg was purchased from invitrogen (eugen, or, usa). the pig experiment conducted in this study was approved by the university of nebraska-lincoln (unl) institutional animal care and use committee under the protocol number . ten -week-old prrsv, porcine circovirus type (pcv ), and siv negative pigs were purchased from the midwest research swine (glencoe, mn, usa). the pigs were randomly assigned to two groups of five pigs, which were accommodated in two separate rooms in the animal biosecurity level (absl- ) research facilities at unl. after week of acclimation, pigs in group were injected with dmem to serve as negative controls, whereas pigs in groups were inoculated intramuscularly with . tcid of live-attenuated prrsv strain con . whole blood samples with anticoagulant edta were collected to obtain plasma and pbmcs. plasma samples were stored at − • c for measurement of viremia and antibody response. a portion of freshly isolated pbmcs were used for measurement of t cell responses while the rest of pbmcs were cryopreserved for future analysis. at days post-infection (dpi), sample of inguinal lymph node (iln) was aseptically collected from the pigs under anesthesia. one half of the inl was stored in crpmi for lymphocyte isolation while the other half of the inl was minced and stored in trizol reagent (life technologies, carlsbad, ca, usa) for rna extraction. viral loads in plasma and tissues were measured by a commercial rt-qpcr kit (tetracore inc., rockville, md, usa). viral loads in plasma was reported as log copies per ml, whereas viral loads in tissues were reported as log copy per µg of total rna used in the rt-qpcr reaction. for statistical purposes, samples that had no detectable levels of viral rna were assigned a value of log copies. pbmcs were isolated from edta-whole blood as previously described [ , ] . single cell suspension was isolated from iln immediately after collection as follows. the tissue was processed to viruses , , of remove connecting tissue and cut into small pieces which were placed in a -µm nylon cell strainer (corning, durham, nc, usa) in the presence of crpmi. the tissue pieces were pressed against the nylon mesh by using the plunger of a ml syringe. the resulting cell suspension was collected into a ml conical tube which was passed through a -µm cell strainer one more time to remove large tissue debris. cells were pelleted by centrifugation at × g for min at room temperature and treated with a ml red blood cell (rbc) lysis buffer (life technologies, carlsbad, ca, usa). rbc lysis reaction was ceased by adding ice cold pbs containing % fbs, followed by centrifugation at × g for min at room temperature. cell pellet was resuspended in crpmi. to determine cell concentration and viability, samples of pbmc and iln-derived cells were stained with acridine orange and propidium iodide (viastaintm aopi staining solution, nexcelom, lawrence, ma, usa) and counted using an automatic cell counter (cellometer auto , nexcelom, lawrence, ma, usa). freshly isolated cells were used for elispot and flow cytometric analysis. the remaining cells were cryopreserved in % dmso, % fbs, and % rpmi- and stored in liquid nitrogen. prrsv antibody levels in plasma were measured at the veterinary diagnostic center of the university of nebraska by using the commercial elisa idexx prrs x ab test (idexx laboratories, westbrook, me, usa) following the manufacturer's instructions. the serum-virus neutralization (svn) assay was performed as previously described [ ] using plasma rather than serum. results were expressed as the log of the reciprocal of the highest dilution that showed a ≥ % reduction in the number of fluorescent foci presenting in the control wells. the frequencies of ifn-γ-secreting cells (ifn-γ scs) in pbmcs and iln-derived cells were measured by using an ifn-γ elispot assay as previously described [ , ] . briefly two replicates of , pbmcs or iln cells freshly collected from each pig were plated into two wells of a -well plate with pvdf membrane that were coated with anti-porcine ifn-γ antibody. the cells were stimulated with µl of crpmi containing . × tcid of con . for positive control, cells were plated at cells per well, followed by stimulation with a µl crpmi containing ng/ml of phorbol myristate -acetate (pma) and µg/ml of ionomycin. for negative control, cells were simply cultured in crpmi. at h post stimulation, the plate was washed with pbs-containing . % tween (pbs-t) followed by incubation with biotin-labeled antibody against porcine ifn-γ (clone p c , bd biosciences pharmingen, san diego, ca, usa). spots were developed by using alkaline phosphatase-conjugated streptavidin (southern biotech, birmingham, al, usa) in conjunction with alkaline phosphatase substrate (vector laboratories, burlingame, ca, usa). spots were counted and analyzed using an aid elispot reader version . (aid gmbh, strassberg, germany). freshly isolated pbmcs and iln cells were ex vivo stimulated with × tcid of con as described above. a cocktail solution consisting of ng/ml of pma and µg/ml of ionomycin was included as a positive control while crpmi served as a negative control. at hrs post-stimulation, µl of crpmi containing µg/ml of golgi-plug brefeldin a (bd bioscience, san jose, ca, usa) was added and further incubated for hrs. samples were centrifuged at × g for min at room temperature. cells were resuspended in flow cytometry staining buffer (facs buffer) (pbs with % fbs and . % sodium azide), and stained with anti-porcine cd ε, cd , and cd monoclonal antibodies and incubated on ice for min in the dark. cells were washed thrice using facs buffer. the cells were fixed and permeabilized with % paraformaldehyde and . % triton x- , respectively, followed by intracellular staining with an ifn-γ antibody. cells were analyzed by using a cytek dxp cytometer (cytek biosciences, fremont, ca, usa) and acquired data were analyzed using the flowjo software (bd biosciences, san jose, ca, usa) with gating based upon fluorescence minus one (fmo) control. for each sample, , events were acquired. relevant cell population was gated on cd + prior to analyzing the cd + and cd + cells ( figure s a ). ifn-γ positive cells were counted from individual cd + , cd + , and cd + cd + double positive (dp) cells ( figure s b ). total rna was isolated from iln collected from the infected and non-infected using the trizol reagent according to the manufacturer's protocol. rna was re-purified using rneasy mini kit (qiagen, hilden, nrw, germany) according to the manufacturer instructions, followed by a dnase treatment using turbo dna-free tm kit (life technologies, v.a. graiciuno , vilnius, lithuania) to remove dna contamination. purified rna was submitted to novogene bioinformatics technology co.ltd for rna sequencing. sequencing libraries were generated using nebnext ® ultratm rna library prep kit for illumina ® (neb, ipswich, ma, usa) following manufacturer's recommendations and index codes were added to attribute sequences to each sample. briefly, mrna was purified from mg of total rna using poly-t oligo-attached magnetic beads. fragmentation was carried out using divalent cations under elevated temperature in nebnext first strand synthesis reaction buffer ( x). first strand cdna was synthesized using random hexamer primer and m-mulv reverse transcriptase (rnase h). second strand cdna synthesis was subsequently performed using dna polymerase i and rnase h. remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. after adenylation of ends of dna fragments, nebnext adaptor with hairpin loop structure were ligated to prepare for hybridization. in order to select cdna fragments of preferentially ~ bp in length, the library fragments were purified with ampure xp system (beckman coulter, beverly, ma, usa). then µl user enzyme (neb, ipswich, ma, usa) was used with size-selected, adaptor-ligated cdna at • c for min followed by min at • c before pcr. then pcr was performed with phusion high-fidelity dna polymerase, universal pcr primers and index (x) primer. finally, pcr products were purified using ampure xp system and library quality was assessed on the agilent bioanalyzer system. the libraries were sequenced on an illumina platform hiseq and approximately million raw bp/ bp paired-end) reads were generated. rna-seq reads were first trimmed with trim galore version . . [ , ] , which include filtering reads shorter than bp and low-quality ends from reads (phred score: < ) and the option of removing reads with ns. the quality of reads before and after trimming was checked with fastqc version . . [ ] . the filtered reads were aligned to the sus scrofa genome (sscrofa . ; gcf_ . ) using tophat version . . [ ] [ ] [ ] with a read mismatch of and a maximum multi hits of and further default parameters. alignment files generated by tophat were then used to generate gene counts using htseq version . . (htseq-count), with a minimum alignment quality of [ ] . a data matrix of gene counts for all samples was created using a custom python script and the data matrix was used to run the differential gene expression analysis in deseq version . . [ ] . genes with an adjusted p value smaller than . and a log fold change larger than were considered as differentially expressed. the differential expression result table generated by deseq was annotated with gene information obtained from ensembl biomart for sus scrofa, supplemented by annotation from the gtf file. to visualize the level of prrsv rna genome, reads mapped to the prrsv con genome were counted base by base, normalized to counts per million and subsequently used to generate a coverage track using deeptools version . . [ ] . gene ontology (go) in the database (http://www.geneontology.org/) is an international standardized classification system for gene function, and it supplies a set of controlled vocabulary to comprehensively describe the properties of genes and gene products. go enrichment analysis of degs was implemented by the goseq r package [ ] , in which gene length bias was corrected. go terms with viruses , , of corrected p-value less than . were considered significantly enriched by differentially expressed genes. kyoto encyclopedia of genes and genomes (kegg) is a database resource for understanding high-level functions and utilities of the biological system, such as the cell, the organism, and the ecosystem, from molecular-level information, especially large-scale molecular datasets generated by genome sequencing and other high-throughput experimental technologies (http://www.genome.jp/kegg/). kobas version . [ ] was used to test the statistical enrichment of differential expression genes in kegg pathway. virus-specific t-cell data were analyzed by unpaired t-test analysis using graphpad prism software version . . (graphpad software, llc, san diego, ca, usa). a p < . was considered significant. sequencing data from rna-seq were deposited in the ncbi geo and are available under accession number geo: gse all pigs inoculated with con became viremic starting from dpi. the viremia level increased and peaked at dpi ( figure a ). pigs inoculated with con did not displayed any clinical signs throughout the course of this study. at dpi, iln was collected and rna was extracted. rt-pcr analysis revealed that viral rna genome was detected in all five con -infected pigs but none of the sham-inoculated pigs ( figure b) . subsequently, the rna samples were subjected to rna-seq. an average of million paired-end reads were obtained for each rna sample (table s ). to confirm the presence of viral rna during persistent infection in pigs, the reads were mapped to the con genome. viral rna reads were only detected from iln of con -infected pigs which mapped throughout the viral genome ( figure c ). the results demonstrate that all five pigs in this study were persistently infected with the attenuated prrsv strain con . to examine the host responses to con persistent infection, rna reads were mapped to the reference pig genome (sscrofa . ; gcf_ . ). principal component analysis (pca) indicated that control and con -infected pigs formed separated clusters indicating that they had distinct transcriptional profiles ( figure a ). the infected pigs showed more variability than the control pigs. in addition, one pig in the con -infected group (con_ ) showed a unique transcriptional profile, as it associated closer to the control pigs. this association is likely due to the different genetic makeup of this pig than the other four animals within this group. direct comparison of rna transcripts between control and con -infected animals revealed a profound change in the transcriptional profiles in the iln tissue of con -infected pigs. out of genes in the annotated porcine genome, there were degs (fdr < %, |log | fold change ≥ ), of which degs were upregulated and degs were downregulated ( figure b and table s ). to examine the host responses to con persistent infection, rna reads were mapped to the reference pig genome (sscrofa . ; gcf_ . ). principal component analysis (pca) indicated that control and con -infected pigs formed separated clusters indicating that they had distinct transcriptional profiles (figure a ). the infected pigs showed more variability than the control pigs. in addition, one pig in the con -infected group (con_ ) showed a unique transcriptional profile, as it associated closer to the control pigs. this association is likely due to the different genetic makeup of this pig than the other four animals within this group. direct comparison of rna transcripts between control and con -infected animals revealed a profound change in the transcriptional profiles in the iln tissue of con -infected pigs. out of genes in the annotated porcine genome, there were degs (fdr < %, |log | fold change ≥ ), of which degs were upregulated and degs were downregulated ( figure b and table s ). the control animals are indicated by red dots labeled "dmem_ animal number," while the con -infected animals are indicated by cyan dots labeled "con_animal number"; (b) the volcano plot of differentially expressed genes between con -infected and control pigs. red dots denote significantly down-regulated genes (p < . , log fold change ≤− ), green dots indicated significantly up-regulated genes (p < . , log fold change ≥ ), and grey dots represent genes that were not differentially expressed. to understand their biological functions, degs were subjected to go enrichment analysis. go terms having a corrected value of p < . were considered statistically significant (table s ) . significantly enriched go terms were grouped under three categories: biological processes, molecular function, and cellular components. thirty highly enriched go terms in con -infected pigs are shown (figure a) . a large number of degs are enriched into the category of biological processes (cytoskeleton organization, multicellular organism development, regulation of intracellular signal transduction, cell communication, cell surface receptor signaling, regulation of programmed cell death etc.), followed by molecular function (cellular macromolecular catabolic processes, kinase activity, phosphotransferase activity, dna binding, metal ion binding) and cellular components (ribonucleoprotein complexes, bounding membrane of organelles). kegg pathway analysis revealed multiple enriched pathways including apoptosis, chemokine signaling, cellular, senescence, mitophagy, lysosome, endocytosis, and mapk ( figure b and table s ). detailed analysis of selected enriched pathways is presented in the respective sections below. to understand their biological functions, degs were subjected to go enrichment analysis. go terms having a corrected value of p < . were considered statistically significant (table s ) . significantly enriched go terms were grouped under three categories: biological processes, molecular function, and cellular components. thirty highly enriched go terms in con -infected pigs are shown (figure a) . a large number of degs are enriched into the category of biological processes (cytoskeleton organization, multicellular organism development, regulation of intracellular signal transduction, cell communication, cell surface receptor signaling, regulation of programmed cell death etc.), followed by molecular function (cellular macromolecular catabolic processes, kinase activity, phosphotransferase activity, dna binding, metal ion binding) and cellular components (ribonucleoprotein complexes, bounding membrane of organelles). kegg pathway analysis revealed multiple enriched pathways including apoptosis, chemokine signaling, cellular, senescence, mitophagy, lysosome, endocytosis, and mapk ( figure b and table s ). detailed analysis of selected enriched pathways is presented in the respective sections below. the innate immune system is the first line of defense against invading pathogens, with the major influence on the development of strong adaptive immune responses. con is capable of inducing type i ifns both in vitro and in vivo [ ] . in the current study, we did not detect differential expression of type i ifn or interferon stimulated genes (isg) rna transcripts, suggesting that the type-i ifn was not induced in iln of con-infected pigs at dpi. we observed an upregulation of rna-sensing molecules including tlr (dsrna), tlr , and tlr (ssrna) (figure a ). on the other hand, no differential expression of signaling molecules downstream of tlr pathways including interferon regulatory factors (irf) irf and irf , was observed (table s ). in addition, we observed an upregulation of tlr mrna in con -infected pigs. while the ligands and signaling pathway involving tlr remain poorly understood, it has been demonstrated that tlr can act as an anti-inflammatory receptor which can also suppress tlr -induced ifn-β production [ ] . interestingly, we observed an increased expression of cd (ox ) and its receptor cd r (figure a ). cd r is an inhibitory immune receptor that is expressed on myeloid cells and b-and t-lymphocytes [ ] , while cd is widely expressed on multiple cell types including endothelial cells, neurons, and lymphocytes [ ] . cd /cd r interaction suppresses cytokine production, and inflammatory responses [ ] . the innate immune system is the first line of defense against invading pathogens, with the major influence on the development of strong adaptive immune responses. con is capable of inducing type i ifns both in vitro and in vivo [ ] . in the current study, we did not detect differential expression of type i ifn or interferon stimulated genes (isg) rna transcripts, suggesting that the type-i ifn was not induced in iln of con-infected pigs at dpi. we observed an upregulation of rna-sensing molecules including tlr (dsrna), tlr , and tlr (ssrna) (figure a ). on the other hand, no differential expression of signaling molecules downstream of tlr pathways including interferon regulatory factors (irf) irf and irf , was observed (table s ). in addition, we observed an upregulation of tlr mrna in con -infected pigs. while the ligands and signaling pathway involving tlr remain poorly understood, it has been demonstrated that tlr can act as an antiinflammatory receptor which can also suppress tlr -induced ifn-β production [ ] . interestingly, we observed an increased expression of cd (ox ) and its receptor cd r (figure a ). cd r is an inhibitory immune receptor that is expressed on myeloid cells and b-and t-lymphocytes [ ] , while cd is widely expressed on multiple cell types including endothelial cells, neurons, and lymphocytes [ ] . cd /cd r interaction suppresses cytokine production, and inflammatory responses [ ] . the complement system is another crucial component of the innate immunity. it acts as an important link between innate and adaptive immune system. thus, viruses have developed a mechanism to modulate complement responses by down-regulating activation proteins and upregulation of regulatory proteins [ ] . in the current study, increased expression of regulatory proteins (cfh, cfi, and cd ) was observed from con -infected animals (figure b) . conversely, c q, a classical complement component involved in increasing virus neutralizing ability of antibodies [ ] was down-regulated ( figure b) . collectively, the data suggest that the con virus suppresses both complement activation and inflammatory responses at the site of persistent infection. apoptosis is a powerful innate immunity mechanism to curtail viral spread through eliminating virally infected cells. apoptosis can be triggered by both extrinsic and intrinsic stimuli [ ] . it is well documented that prrsv induces apoptosis in tissues of infected pigs, especially during an acute phase of the infection [ , , ] . in this study, expression of tnfrsf a (tnfα receptor ) gene that the complement system is another crucial component of the innate immunity. it acts as an important link between innate and adaptive immune system. thus, viruses have developed a mechanism to modulate complement responses by down-regulating activation proteins and up-regulation of regulatory proteins [ ] . in the current study, increased expression of regulatory proteins (cfh, cfi, and cd ) was observed from con -infected animals (figure b) . conversely, c q, a classical complement component involved in increasing virus neutralizing ability of antibodies [ ] was down-regulated ( figure b) . collectively, the data suggest that the con virus suppresses both complement activation and inflammatory responses at the site of persistent infection. apoptosis is a powerful innate immunity mechanism to curtail viral spread through eliminating virally infected cells. apoptosis can be triggered by both extrinsic and intrinsic stimuli [ ] . it is well documented that prrsv induces apoptosis in tissues of infected pigs, especially during an acute phase of the infection [ , , ] . in this study, expression of tnfrsf a (tnfα receptor ) gene that contains a death domain [ ] , was downregulated in the iln of infected pigs ( figure ) . similarly, expression of pro-apoptotic genes including aifm , chac , and osr , was downregulated ( figure ). on the other hand, expression of birc and bfl- /a (bcl a ), two apoptosis inhibitors that interfere with caspase activation [ ] , was upregulated. furthermore, expression of several negative regulators of apoptotic genes including bcl- -associated killer (bak ), damage induced apoptosis suppressor (ddias), x-linked inhibitor of apoptosis protein (xiap), mcl , api, bnip , and faim was upregulated. collectively, these results suggest that the pro-apoptotic signaling pathway was suppressed in iln tissue of pigs persistently infected with con . contains a death domain [ ] , was downregulated in the iln of infected pigs ( figure ) . similarly, expression of pro-apoptotic genes including aifm , chac , and osr , was downregulated ( figure ). on the other hand, expression of birc and bfl- /a (bcl a ), two apoptosis inhibitors that interfere with caspase activation [ ] , was upregulated. furthermore, expression of several negative regulators of apoptotic genes including bcl- -associated killer (bak ), damage induced apoptosis suppressor (ddias), x-linked inhibitor of apoptosis protein (xiap), mcl , api, bnip , and faim was upregulated. collectively, these results suggest that the pro-apoptotic signaling pathway was suppressed in iln tissue of pigs persistently infected with con . lymphocyte activation occurs in the secondary lymphoid organs. activated t-cells then migrate to the sites of infection where they exert their effector functions to eliminate infected cells [ ] . celladhesion molecules, chemokines, and receptors play a central role in regulating t-cell migration [ ] . prrsv mainly persists in lymphoid tissue of infected pigs [ , ] . in this study, expression of several important chemokine ligands (ccl , ccl , ccl , ccl , cx cl , and ccl ) and chemokine receptors (ccr and ccr ) which play an essential role in migration and localization of lymphocytes and antigen-presenting cells (apcs) to the lymphoid tissues was down regulated in iln of con -infected pigs (figure a ). on the other hand, expression of cd (pd- ), a marker of tcell exhaustion, was upregulated ( figure b) . likewise, expression of inhibitory receptors havcr (also known as tim ) and tgit, which transmit the inhibitory signals for t-cell differentiation and effector activities [ ] , was upregulated. we also found increased expression of other co-inhibitory molecules (btla, faslg, fas, and ido ) that are associated with the regulation of t-cell exhaustion during chronic viral infection (figure b) . together, the results suggest that t-cell migration to iln, one of the sites of prrsv persistence, might be affected because of the down regulation of important chemokines and receptors, and that the t-cells in iln might be exhausted. interestingly, markers of lymphocyte activation occurs in the secondary lymphoid organs. activated t-cells then migrate to the sites of infection where they exert their effector functions to eliminate infected cells [ ] . cell-adhesion molecules, chemokines, and receptors play a central role in regulating t-cell migration [ ] . prrsv mainly persists in lymphoid tissue of infected pigs [ , ] . in this study, expression of several important chemokine ligands (ccl , ccl , ccl , ccl , cx cl , and ccl ) and chemokine receptors (ccr and ccr ) which play an essential role in migration and localization of lymphocytes and antigen-presenting cells (apcs) to the lymphoid tissues was down regulated in iln of con -infected pigs (figure a ). on the other hand, expression of cd (pd- ), a marker of t-cell exhaustion, was upregulated ( figure b) . likewise, expression of inhibitory receptors havcr (also known as tim ) and tgit, which transmit the inhibitory signals for t-cell differentiation and effector activities [ ] , was upregulated. we also found increased expression of other co-inhibitory molecules (btla, faslg, fas, and ido ) that are associated with the regulation of t-cell exhaustion during chronic viral infection (figure b) . together, the results suggest that t-cell migration to iln, one of the sites of prrsv persistence, might be affected because of the down regulation of important chemokines and receptors, and that the t-cells in iln might be exhausted. interestingly, markers of regulatory t-cells (t reg ) were downregulated in iln of infected pigs (figure c) , suggesting that t reg might not be present in iln at dpi. regulatory t-cells (treg) were downregulated in iln of infected pigs (figure c ), suggesting that treg might not be present in iln at dpi. since the expression of chemokines and receptors important for t-cell migration was downregulated in iln, we sought to compare the frequencies of virus-specific t-cells in pbmcs and in iln using the ifn-γ sc elispot assay. the number of spots was similar when pbmcs and iln cells were stimulated with non-specific t-cell activator pha/ionomycin. however, the number of spots was significantly lower in iln than in pbmcs when the cells were stimulated with whole prrsv antigen (figure a ). the results clearly indicate that the frequencies of prrsv-specific ifn-γ scs were significantly lower in iln than in pbmcs. to further elucidate the phenotypes of prrsvspecific t-cells, we used a multi-color flow cytometric assay to identify subsets of t-cells that secrete ifn-γ in response to prrsv activation ex vivo. cd + cd + dp cells were the major t-cell population secreting ifn-γ, both in pbmcs and iln. swine have a significant population of extrathymic cd + cd + dp t cells that represent memory t-cells [ ] . similar to the elispot assay, the flow cytometric assay also indicated that the percentage of t-cell secreting ifn-γ was comparatively lower in iln than in pbmcs (figure b ). cells collected from dmem-inoculated pigs did not show any significant t-cell reactivities after ex vivo stimulation with con , both in elispot and in flow cytometry assays (data not shown). since the expression of chemokines and receptors important for t-cell migration was downregulated in iln, we sought to compare the frequencies of virus-specific t-cells in pbmcs and in iln using the ifn-γ sc elispot assay. the number of spots was similar when pbmcs and iln cells were stimulated with non-specific t-cell activator pha/ionomycin. however, the number of spots was significantly lower in iln than in pbmcs when the cells were stimulated with whole prrsv antigen (figure a ). the results clearly indicate that the frequencies of prrsv-specific ifn-γ scs were significantly lower in iln than in pbmcs. to further elucidate the phenotypes of prrsv-specific t-cells, we used a multi-color flow cytometric assay to identify subsets of t-cells that secrete ifn-γ in response to prrsv activation ex vivo. cd + cd + dp cells were the major t-cell population secreting ifn-γ, both in pbmcs and iln. swine have a significant population of extrathymic cd + cd + dp t cells that represent memory t-cells [ ] . similar to the elispot assay, the flow cytometric assay also indicated that the percentage of t-cell secreting ifn-γ was comparatively lower in iln than in pbmcs (figure b ). cells collected from dmem-inoculated pigs did not show any significant t-cell reactivities after ex vivo stimulation with con , both in elispot and in flow cytometry assays (data not shown). viruses , , x for peer review of a number of genes associated with th , or humoral response, were upregulated in the iln of con -infected pigs (figure a ). il- , rgs , and nuggc are involved in the development of germinal center (gc) and activation of b-cell follicles [ ] . tnfsf b and tnfsf are potent activators of b-cell lineage and ig class switching, respectively [ ] . b-cell surface antigens ms a (cd ), activation-induced cytidine deaminase (aicda) [ ] , and rafting family member (rftn ) are associated with b-cell receptor signaling. the upregulated expression of these genes in iln of infected animals suggested that the humoral immune response to con -infection was not affected. to corroborate transcriptome data, we measured both virus-specific antibody levels at various time points post-infection. high levels of non-neutralizing antibodies specific to viral n protein (measured by a commercial elisa) were detected in all pigs (figure b ). however, only minimal levels of virusneutralizing antibodies (titer : ) were detected in the serum of infected pigs at dpi (figure c ). together, both transcriptomic and serological data indicate that b-cell development and antibody production are not affected by con -infection. however, low levels of virus-neutralizing antibodies are likely due to the virus ability to escape antibody neutralization (see below). a number of genes associated with th , or humoral response, were upregulated in the iln of con -infected pigs (figure a ). il- , rgs , and nuggc are involved in the development of germinal center (gc) and activation of b-cell follicles [ ] . tnfsf b and tnfsf are potent activators of b-cell lineage and ig class switching, respectively [ ] . b-cell surface antigens ms a (cd ), activation-induced cytidine deaminase (aicda) [ ] , and rafting family member (rftn ) are associated with b-cell receptor signaling. the upregulated expression of these genes in iln of infected animals suggested that the humoral immune response to con -infection was not affected. to corroborate transcriptome data, we measured both virus-specific antibody levels at various time points post-infection. high levels of non-neutralizing antibodies specific to viral n protein (measured by a commercial elisa) were detected in all pigs (figure b ). however, only minimal levels of virus-neutralizing antibodies (titer : ) were detected in the serum of infected pigs at dpi (figure c ). together, both transcriptomic and serological data indicate that b-cell development and antibody production are not affected by con -infection. however, low levels of virus-neutralizing antibodies are likely due to the virus ability to escape antibody neutralization (see below). viruses , , x for peer review of prrsv persists in lymphoid tissue of infected pigs for several months [ , ] . the mechanism of prrsv persistence is not fully understood. we performed genome-wide transcriptome analysis of lymphoid tissue collected from pigs persistently infected with an attenuated prrsv strain using rna-seq technology that detects both host and viral rna. viral rna reads were detected in iln of all five infected pigs. it was reported previously that prrsv genome mainly exists in dsrna forms in lymphoid tissues during persistent infection [ ] . since only mrna (purified by using poly-t oligo-attached magnetic beads) was used for library construction and rna sequencing, the viral rna reads detected in this study must be derived from either viral genomic rna or sub-genomic mrna, not from dsrna. the viral rna reads map throughout the viral genome. however, we are not able to discern whether these reads are derived from genomic or sub-genomic mrna because we used a short-read rna sequencing platform. it would be interesting to use long-read rna sequencing to study the viral transcriptome at different states of infection in pigs [ ] . it was reported previously that no significant degs were observed in lymphoid tissue of persistently infected animals [ ] . in the current study, we identified a large number of degs in persistently infected animals (figure b ). this might be due to the difference in the experimental setup. in the previous study, pigs were infected with a wild-type prrsv- whereas in this current prrsv persists in lymphoid tissue of infected pigs for several months [ , ] . the mechanism of prrsv persistence is not fully understood. we performed genome-wide transcriptome analysis of lymphoid tissue collected from pigs persistently infected with an attenuated prrsv strain using rna-seq technology that detects both host and viral rna. viral rna reads were detected in iln of all five infected pigs. it was reported previously that prrsv genome mainly exists in dsrna forms in lymphoid tissues during persistent infection [ ] . since only mrna (purified by using poly-t oligo-attached magnetic beads) was used for library construction and rna sequencing, the viral rna reads detected in this study must be derived from either viral genomic rna or sub-genomic mrna, not from dsrna. the viral rna reads map throughout the viral genome. however, we are not able to discern whether these reads are derived from genomic or sub-genomic mrna because we used a short-read rna sequencing platform. it would be interesting to use long-read rna sequencing to study the viral transcriptome at different states of infection in pigs [ ] . it was reported previously that no significant degs were observed in lymphoid tissue of persistently infected animals [ ] . in the current study, we identified a large number of degs in persistently infected animals (figure b ). this might be due to the difference in the experimental setup. in the previous study, pigs were infected with a wild-type prrsv- whereas in this current study, pigs were infected with an attenuated synthetic prrsv strain [ , ] . besides, the previous study looked at a small set of selected genes while in this study we look at the genome-wide rna transcriptome. go terms and kegg analysis revealed that genes involved in the innate immune (complement and tlr) pathways, apoptosis, cytokine-chemokine signaling, t-cell exhaustion, and humoral responses are highly differentially expressed in prrsv-infected animals compared to control. the complement system is a constituent of innate immunity that serves by neutralizing cell-free viruses, lysing virus-infected cells, and boosting virus specific responses [ ] . it also links the innate and adaptive immune responses, enhances humoral immunity, regulates antibody effector mechanisms, and modulates t-cell function [ ] . many viruses have developed a strategy to evade the complement pathway by recruiting or enhancing the production of host transcription regulatory components. during acute infection ( dpi), prrsv significantly represses the expression of complement regulatory components (cd and c bpb) in lung tissue [ ] . early complement activation during acute infection facilitates the release of newly formed virions from infected cells, yet, chronic viral infection is reported to suppress the activity of complement activation proteins and increases the activity of regulatory proteins (reviewed [ ] ). in the current study, we found overexpression of cfh and cfi along with another regulatory factor cd (decay-accelerating factor). overexpression of cfh, a major soluble regulator of the alternative pathway, results in inhibition of c and c convertase enzymes. cfi suppresses the complement active proteins c b (opsonin) and c b via mediating cleavage to their inactive form [ , ] . regulatory factor cd is an inhibitor of c convertase which prevents c b deposition on the cell surface [ ] . apart from that, we also see the downregulation of c q, which increases neutralizing and hemagglutination inhibition activity of anti-influenza antibodies [ ] . available data from previous studies and the current study suggest that activation of complement pathway during acute infection helps to disseminate virus, while suppression of complement components like c q, c r, and c , and upregulation in regulatory components during persistent infection facilitates the virus to escape from the complement system to maintain persistence in lymphoid tissues. most naturally occurring prrsv strains suppress type i ifn production by inhibiting the activation and nuclear translocation of irf /irf and nf-kb [ , ] . deficiency of irf / results in severe mortality to infection with west nile virus (wnv), chikungunya virus (chikv), and ross river virus infection, and promotes viral persistence in the infected hosts [ ] [ ] [ ] . it has been reported that prrsv nsp β inhibits irf phosphorylation and nuclear translocation; thus, inhibiting ifn production [ , , ] . interestingly, the synthetic prrsv-con and its attenuated form con were able to induce type i ifns [ , ] . contrary to its nature to induce type i ifn, in the current study no upregulation of canonical type i ifn signaling pathway-associated genes was observed. however, expression of rna sensing molecules including tlr , tlr , and tlr was upregulated in con -infected animals. on the basis of available data, we hypothesize that reduced levels of viral replication and sequestration of viral rna in infected cells during persistent infection might limit further activation of type i ifn signaling pathway by preventing interaction with cytoplasmic pattern recognition receptors (prrs). apoptosis or programmed cell death is a potential host immune mechanism against virally infected cells to curtail the spread of newly formed viral progenies. apoptosis is induced by two distinct, yet inter-connected signaling pathways, the extrinsic and intrinsic pathways [ ] . in order to successfully establish persistent infection, viruses have developed mechanisms to inhibit apoptosis. for instance, adenovirus (e b- k), human cytomegalovirus (ul ), poxviruses (f l), and myxoma virus (m l) have an inhibitory effect on proapoptotic proteins bak/bax [ ] . it appears that prrsv can also modulate apoptosis. studies of pulmonary alveolar macrophages (pam) infected with prrsv in vitro reveal that the virus stimulates anti-apoptotic pathways early in infection while it induces apoptosis late in infection [ ] . on the other hand, the virus induces apoptosis in the tissues of infected animals during acute infection, but the frequencies of apoptotic cells reduced to normal levels observed in control, non-infected pigs from day post-infection [ ] . in this study, expression of multiple anti-apoptotic genes including xiap, bfl- /a (bcl a ), and birc was upregulated while expression of pro-apoptotic genes (aifm , chac , and osr ) was downregulated. xiap is an endogenous caspase and inhibitor [ ] . bfl- /a (bcl a ) is a transcriptional target of nuclear factor-kb (nf-kb) that suppresses caspase activation and apoptosis in response to death-inducing stimuli like tnfα [ ] . birc is an interaction partner to tnfrsf b and inhibits apoptosis by interfering with the caspase activation [ ] . the pro-apoptotic bcl- family member like bcl- -associated killer (bak ), which allows the release of cytochrome c via formation of homo-oligomers and stable insertion into outer mitochondrial membrane was significantly suppressed. thus, the data suggest that apoptosis was suppressed in the iln of con -infected animals during persistent infection. chemokine molecules ccl and ccl play a crucial role in migration, activation, expansion, and survival of antiviral t-cells. suppression of the ccr and ccl /ccl axis results in dysfunction of t-cells during viral infection [ , ] . ccl -ccr axis plays role in protection against multiple viruses, such as hiv [ ] , herpes simplex virus (hsv- ) [ ] , hsv- , hepatitis c virus [ ] , and pseudorabies [ ] . acute prrsv infection increases the expression of chemokines, leading to the infiltration of immune cells toward the sites of infection [ , ] . in this study, expression of both ccl and ccl was significantly downregulated in con -infected lymph node (figure a ). additionally, expression of different chemoattractant molecules (fractalkine/cx cl , ccl , ccl , ccl , ccl , and ccl ) and receptors (ccr and ccr ) was also downregulated. it is possible that downregulation of these chemokines and receptors might impair t-cells trafficking to inguinal lymph node, the site of prrsv persistence. our transcriptional data are supported by the functional data which demonstrate that the frequencies of ifn-γ sc in iln were significantly lower than in pbmcs (figure ) . chronic or persistent viral stimulation results in hierarchical loss of effector functions of t-lymphocytes, including proliferation, cytokine production (e.g., ifn-γ and il- ), and cytolytic responses [ , , ] . although the molecular signatures involved in t-cell exhaustion are not completely understood, the overexpression of cell surface inhibitory receptors (e.g., pd- , ctla- , and others) primarily mediates cd + t-cell dysfunction [ ] [ ] [ ] . t-cell exhaustion seems to be a common phenomenon caused by arteriviruses, as t-cell exhaustion was also reported in horses persistently infected with eav [ ] . recently, it was shown that prrsv infection alone or co-infection with porcine circovirus type (pcv ) can significantly upregulate surface expression of pd-l (cd ) on porcine monocytes-derived dendritic cells (modcs) [ ] . increased expression of pd-l on the surface of antigen presenting cells (apcs) possibly contributes to the ineffective t-cell responses during the prrsv infection. in the present study, expression of several markers for t-cells exhaustion such as pd- (cd ) and its ligand, cd (pd-l ) was upregulated in con -infected animals, suggesting that the t-cells in lymph nodes of persistently infected animals might be exhausted (figure b) . however, additional studies (e.g., t-cell cytotoxicity and cytokine production) are required to fully assess the functionality of t-cells during prrsv persistence. germinal centers (gcs) are the specialized locations in the secondary lymphoid tissues where b-cell maturation, differentiation, somatic hypermutation, and class switching of isotypes take place [ ] . in the current study, the gc development-associated genes, which includes il- produced by follicular helper t-cells [ ] , the regulator of g protein signaling (rgs ) [ ] , and gc-associated nuclear gtpase (nuggc) [ ] , were upregulated in the lymph node of con -infected animals. thus, prrsv infection does not seem to impair gc formation and b-cell activation. this is supported by the fact that all five con -infected pigs developed a robust antibody response, as measured by the idexx elisa ( figure b) . however, only minimal titers of virus-neutralizing antibodies were detected at dpi. perhaps, prrsv does not suppress the humoral immune response. instead, the virus evades from antibody-mediated neutralization through different mechanisms such as glycan shielding and decoy epitopes [ ] [ ] [ ] . in summary, we identified a robust host transcriptional response in the inguinal lymphoid tissue of pigs persistently infected with the attenuated prrsv strain con . genes involved in the innate immune responses are downregulated. similarly, chemokines and receptors associated with t-cell homing to the lymphoid tissue are downregulated. this might lead to the lower frequencies of virus-specific t-cells in lymphoid tissue than in peripheral blood. additionally, the genes involved in the anti-apoptotic pathways are upregulated. collectively, the data suggest that prrsv can create a pro-survival microenvironment at the lymphoid tissue which allows the virus to persist for an extended period. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s . figure s . representative flow cytometry gating strategy used to identify ifn-γ secreting cd α + , cd α + , and cd α + cd α + (dp) t cells from pbmcs and iln. table s . summary of sequencing quality control and mapping data of samples. arterivirus molecular biology and pathogenesis ictv-international committee on taxonomy of viruses. virus taxonomy: release. ec porcine reproductive and respiratory syndrome assessment of the economic impact of porcine reproductive and respiratory syndrome virus on united states pork producers effects of origin and state of differentiation and activation of monocytes/macrophages on their susceptibility to porcine reproductive and respiratory syndrome virus (prrsv) cd expression confers susceptibility to porcine reproductive and respiratory syndrome viruses in vivo detection of porcine reproductive and respiratory syndrome virus rna by in situ hybridization at different times postinfection apoptosis induced in vivo during acute infection by porcine reproductive and respiratory syndrome virus type porcine reproductive and respiratory syndrome virus infection increases apoptosis at the maternal-fetal interface in late gestation pregnant gilts open reading frame of porcine reproductive and respiratory syndrome virus as a cause of virus-induced apoptosis characterization of the porcine reproductive and respiratory syndrome virus glycoprotein (gp ) in stably expressing cells induction of apoptosis by the nonstructural protein and of porcine reproductive and respiratory syndrome virus pig immune response to general stimulus and to porcine reproductive and respiratory syndrome virus infection: a meta-analysis approach expression of toll-like receptor mrna and cytokines in pigs infected with porcine reproductive and respiratory syndrome virus analysis of the swine tracheobronchial lymph node transcriptomic response to infection with a chinese highly pathogenic strain of porcine reproductive and respiratory syndrome virus understanding prrsv infection in porcine lung based on genome-wide transcriptome response identified by deep sequencing porcine reproductive and respiratory syndrome virus: description of persistence in individual pigs upon experimental infection duration of infection and proportion of pigs persistently infected with porcine reproductive and respiratory syndrome virus humoral immune responses and viral shedding following vaccination with modified live porcine reproductive and respiratory syndrome virus vaccines simian hemorrhagic fever virus: recent advances lactate dehydrogenase-elevating virus equine viral arteritis genome-wide association study among four horse breeds identifies a common haplotype associated with in vitro cd + t cell susceptibility/resistance to equine arteritis virus infection equine arteritis virus long-term persistence is orchestrated by cd + t lymphocyte transcription factors, inhibitory receptors, and the cxcl /cxcr axis identification of factors associated with virus level in tonsils of pigs experimentally infected with porcine reproductive and respiratory syndrome virus double-stranded viral rna persists in vitro and in vivo during prolonged infection of porcine reproductive and respiratory syndrome virus development of a broadly protective modified-live virus vaccine candidate against porcine reproductive and respiratory syndrome virus enhanced replication of porcine reproductive and respiratory syndrome (prrs) virus in a homogeneous subpopulation of ma- cell line gradual development of the interferon-gamma response of swine to porcine reproductive and respiratory syndrome virus infection or vaccination location of t-cell epitopes in nonstructural proteins and of type-ii porcine reproductive and respiratory syndrome virus a -kda structural protein of porcine reproductive and respiratory syndrome virus encoded by orf b use of interleukin to enhance the cellular immune response of swine to an inactivated herpesvirus vaccine cross reactivity of immune responses to porcine reproductive and respiratory syndrome virus infection a wrapper script to automate quality and adapter trimming cutadapt removes adapter sequences from high-throughput sequencing reads a quality control tool for high throughput sequence data ultrafast and memory-efficient alignment of short dna sequences to the human genome differential gene and transcript expression analysis of rna-seq experiments with tophat and cufflinks accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions htseq-a python framework to work with high-throughput sequencing data moderated estimation of fold change and dispersion for rna-seq data with deseq manke, t. deeptools : a next generation web server for deep-sequencing data analysis gene ontology analysis for rna-seq: accounting for selection bias kobas . : a web server for annotation and identification of enriched pathways and diseases tlr is a negative regulator of both myd -dependent and -independent tlr signaling the inhibitory cd r is differentially expressed on human and mouse t and b lymphocytes cd and membrane protein interactions in the control of myeloid cells cd /cd r paired potent inhibitory molecules regulating immune and inflammatory responses; part ii: cd /cd r potential clinical applications complement evasion strategies of viruses: an overview. front influenza a virus m blocks the classical complement pathway through interacting with c qa apoptosis: a review of programmed cell death apoptosis in the lungs of pigs infected with porcine reproductive and respiratory syndrome virus and associations with the production of apoptogenic cytokines nf-kappab antiapoptosis: induction of traf and traf and c-iap and c-iap to suppress caspase- activation bfl- /a functions, similar to mcl- , as a selective tbid and bak antagonist activation, differentiation, and migration of naive virus-specific cd + t cells during pulmonary influenza virus infection regulation of t cell migration during viral infection: role of adhesion molecules and chemokines molecular and cellular insights into t cell exhaustion functional and phenotypic analysis of porcine peripheral blood cd /cd double-positive t cells speckled-like pattern in the germinal center (slip-gc), a nuclear gtpase expressed in activation-induced deaminase-expressing lymphomas and germinal center b cells member of the tumor necrosis factor family and b lymphocyte stimulator il- and il- collaborate to shape t-dependent antibody responses the architecture of sars-cov- transcriptome a synthetic porcine reproductive and respiratory syndrome virus strain confers unprecedented levels of heterologous protection viral mimicry of the complement system the complement system in regulation of adaptive immunity control of the amplification convertase of complement by the plasma protein beta h human complement c b inactivator: isolation, characterization, and demonstration of an absolute requirement for the serum protein beta h for cleavage of c b and c b in solution decay accelerating factor (cd ): a target for cancer vaccines? porcine reproductive and respiratory syndrome virus nonstructural protein beta modulates host innate immune response by antagonizing irf activation the cysteine protease domain of porcine reproductive and respiratory syndrome virus nonstructural protein possesses deubiquitinating and interferon antagonism functions regulation of interferon regulatory factor- by the hepatitis c virus serine protease infectious chikungunya virus in the saliva of mice, monkeys and humans induction of ifn-beta and the innate antiviral response in myeloid cells occurs through an ips- -dependent signal that does not require irf- and irf- interplay between interferon-mediated innate immunity and porcine reproductive and respiratory syndrome virus the cysteine protease domain of porcine reproductive and respiratory syndrome virus non-structural protein antagonizes interferon regulatory factor activation identification of viral genes associated with the interferon-inducing phenotype of a synthetic porcine reproductive and respiratory syndrome virus strain bax insertion, oligomerization, and outer membrane permeabilization in brain mitochondria: role of permeability transition and sh-redox regulation porcine reproductive and respiratory syndrome virus modulates apoptosis during replication in alveolar macrophages structural biology. controlling the caspases ccr and its ligands: balancing immunity and tolerance ccr coordinates the primary immune response by establishing functional microenvironments in secondary lymphoid organs ccl induces mucosal homing of hiv- -specific iga-secreting plasma cells in mice immunized with hiv- virus-like particles influence of ccr ligand dna preexposure on the magnitude and duration of immunity enhancement of immune responses by co-delivery of ccl /mip- beta chemokine plasmid with hcv core dna/protein immunization genetic co-transfer of ccr ligands enhances immunity and prolongs survival against virulent challenge of pseudorabies virus molecular characterization of transcriptome-wide interactions between highly pathogenic porcine reproductive and respiratory syndrome virus and porcine alveolar macrophages in vivo molecular signature of cd + t cell exhaustion during chronic viral infection t cell exhaustion t cells and viral persistence: lessons from diverse infections pd-l expression is increased in monocyte derived dendritic cells in response to porcine circovirus type and porcine reproductive and respiratory syndrome virus infections role of nf-kappa b in cell survival and transcription of latent membrane protein -expressing or epstein-barr virus latency iii-infected cells influence of n-linked glycosylation of porcine reproductive and respiratory syndrome virus gp on virus infectivity, antigenicity, and ability to induce neutralizing antibodies immune evasion of porcine reproductive and respiratory syndrome virus through glycan shielding involves both glycoprotein as well as glycoprotein identification of neutralizing and nonneutralizing epitopes in the porcine reproductive and respiratory syndrome virus gp ectodomain this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we would like to thank dirk anderson at flow cytometry core facility, nebraska center for biotechnology for assistance on flow cytometric analysis and the staff members of unl life sciences annex and veterinary diagnostic center for the care of animals. the use of product and company names is necessary to accurately report the methods and results; however, the united states department of agriculture (usda) neither guarantees nor warrants the standard of the products. the use of names by the usda implies no approval of the product to the exclusion of others that may also be suitable. the usda is an equal opportunity provider and employer. key: cord- -j qmw zy authors: nan title: chapter the need for chemotherapy and prophylaxis against viral diseases date: - - journal: perspect med virol doi: . /s - ( ) - sha: doc_id: cord_uid: j qmw zy the chapter discusses the need for chemotherapy and prophylaxis against viral diseases. it briefly mentions clinical diseases and syndromes such as influenza, respiratory tract infections, hepatitis, and arbovirus infections resulting from virus infections. viruses causing respiratory diseases, as well as many other diseases in humans are also discussed in the chapter. it describes the vaccines that are used to check the attack of different viruses as well as their cost-effectiveness. there is a list of some viruses that have been ranked according to different variables in an attempt to select a good candidate for an antiviral drug. the incidence of the viral disease is naturally an important factor, as is the severity of the disease. the incidence can be obtained for diseases being reported in accordance with local regulations, but in many cases viral diseases are not reported and the incidence has to be calculated from different surveys. also, a grading of the severity is not easy and an example is when herpesvirus infections are handled as a group, which includes both herpes encephalitis and cold sores. the greatest challenges and probably the most difficult and medically important areas for prophylaxis and therapy of viral diseases are the viruses that are rapidly changing in antigenic composition and/or viruses with animal reservoirs (influenza and arboviruses) and those forming latent infections (herpesviruses). the need for chemotherapy and prophylaxis against viral diseases marks the centenary of the first use of one of the most effective viral vaccines (rabies, pasteur, ) as well as being the famous milestone of fictional political forecasting (orwell, ) . this year period has witnessed the industrialization of countries throughout the world and, along with this social and economic development, marked changes in patterns of infectious disease both accompanying and in some cases caused by the population and social changes. excellent examples of these medical changes are shown in figs. . - . , where mortality in different age groups is compared for the two years, and in germany for infectious diseases, heart and circulation disease and neoplastic disease, respectively. a dramatic drop in mortality at all ages, but particularly in children, from infectious disease is noted between and , whereas, conversely, an increase in mortality in older persons (but not children) is noted which is caused by heart and circulation diseases and neoplasms. disease patterns are still changing today and new viral diseases are discovered regularly (witness the fevers of marburg and lassa in the last decade, and, more recently, the different viruses associated with aids). indeed there is every reason to assume that continual change in infectious diseases is to be expected in the future. even the arrival of potent antiviral compounds may not eradicate diseases such as herpes, but rather may alter the pathogenic process of the virus itself as strong selective pressures are brought to bear. a similar phenomenon has been happening with bacteria such as staphylococci or streptococci as, over the years, strong selective pressures exerted by antibiotics have led to the emergence of organisms with reduced or altered pathogenicity. in the case of malaria, wide- spread use of antimalarials on the one hand has led to a gradual and threatening build up of drug resistant strains of parasite and also the use of ddt to destroy the mosquito has led to development of resistance in the vector. therefore both eukaryotes and prokaryotes possess a dramatic ability to adapt to a changed environment, and viruses are no exception. as bacteriologists before us have done, we should ask ourselves which viral dis- eases are important causes of mortality and morbidity, and, amongst these which would be the most suitable for ultimate control or even eradication. in fact, the question is more complex than this because in different parts of the world different viral diseases are important and even the same virus (such as measles) may cause very different disease syndromes. whilst in most major european countries respiratory viruses are an important cause of morbidity and mortality, in third world countries measles, polio and diarrhoea diseases predominate in this respect. a potential for distorted programmes of control of viral disease may easily occur, with most research centred on developed countries and therefore oriented towards viral disease in those countries. an excellent example is in the area of antiviral chemotherapy. at the present time most research effort is undoubtedly in the area of herpes infections, particularly hsv- causing genital infections, and this is closely followed by the search for new compounds against influenza a virus and rhinoviruses. also, although influenza a is a pandemic virus causing mortality and morbidity throughout the world the same cannot be said for herpes viruses. although infections with herpes viruses are universal, the impact of these nevertheless pale into insignificance beside medical problems with measles, arboviruses and hepatitis a and b in third world countries. so the direction of research effort needs continuous reassessment but, more importantly, some international perspective and direction. the world health organisation has provided this perspective for the eradication of smallpox from the whole world and continues to provide direction with its integrated 'health for all by the year ' programme ( fig. . ) which calls on member states to pursue a programme aimed at attainment by all people of the world, of a level of health that will permit them to live a socially and economically produc- . piped water reaches a small community in the soloman islands: satisfying needs for water calls for a complex interlocking planning and action at many levels. c. inequality in the great cities of the world. (from 'world health', who publication.) tive life. the programme includes sections on the control of certain viral diseases causing respiratory infection and diarrhoea diseases. the who conference at alma-ata in the ussr in stressed the importance of primary health care: "essential health care must be based on practical scientifically sound and socially acceptable methods and technology made universally accessible to individuals and families in the community through their full participation and at a cost that the community and the country can afford to maintain at every stage of their develop-ment in the spirit of self reliance and self determination". this programme does not, at present, envisage eradication of any particular viral disease, as smallpox was eradicated, because this may not gain the necessary full support of all countries. individual countries might, and are, considering 'national' eradication of certain virus infections such as measles in the usa, cuba, bahamas, canada and barbados where mortality rates of less than measles death per population have now been achieved. polio is another example of a viral disease which can be eradicated nationally, but of course a 'national' eradication always means that re-introduction from outside can constantly occur. table . illustrates illness and death caused by infectious diseases (viruses, bacteria and parasites) in africa, asia and latin america. viruses are the single chief cause of mortality, amounting to many millions of persons per year. among these virus infections the worst culprits are respiratory viruses such as influenza, and measles and rotaviruses. although an estimated - million persons die of enteric infections in these three continents each year, viruses will cause only a proportion of these cases and a similar situation will be true for respiratory illness. we shall examine the methods developed for the prevention of measles, influenza, polio and rotaviruses later on (chapters , , and , respectively) but it may be mentioned here that live polio vaccines used so successfully in industrialized countries are much more difficult to apply successfully in third world countries where problems of vaccine administration, heat lability control and viral interference become very important. measles programmes are being initiated now in certain third world countries (reviewed by mckenzie, ) , but vaccines against diarrhoea diseases are in their infancy. research has been orientated towards the development of successful inactivated or live vaccines or chemoprophylactic agents against influenza for the last years with only limited success and even less success against other respiratory viruses such as respiratory syncytial virus and parainfluenza viruses. little emphasis is given in table . to mortality from arboviruses other than dengue, and from hepatitis a and b viruses. a successful yellow fever vaccine was developed in the s but is not used for mass immunization but rather for containment, whereas the development of vaccines or chemoprophylactic agents against hepatitis is only beginning and so far has only resulted in vaccines and drugs with a very limited use on a large scale because of the expense involved. rabies vaccine, on the other hand, and the topic of our introductory sentence has been a success story in developed and underdeveloped countries. cheap and effective rabies vaccines have been produced locally, with the help of the network of pasteur institutes throughout the world, to contain this terrifying disease (chapter ). in general therefore we can conclude that certain viral diseases are 'shared' between developed and underdeveloped countries (such as influenza, measles, polio and rabies) and research and technical developments in industrialized nations will hopefully be applied worldwide. an important qualification here is that new vaccines and antivirals should not be too expensive and, unfortunately, recent rabies vaccines (chapter ) and hepatitis vaccines (chapter ) are extremely costly. live attenuated polio and measles vaccines are produced very cheaply and with more attention to their delivery (chapters and ) can and should result in a dramatic decrease of polio and measles in these countries in the next decade. as we have emphasized above, deaths from infectious viral disease in industrialized nations and also in those third world countries with well organized social infrastructure (such as china in the last years) have been steadily dropping over the last years, although there is still a considerable scope for further reductions in many countries. table . shows death rates from infectious diseases in countries with 'fully developed health services' compared to those without these facilities. table . further emphasizes marked differences in mortality caused by acute respiratory infection (bacterial and viral) in children between a third world continent (africa), the usa and europe. mortality is overall times higher in africa than in the usa but nearly times higher in the - year olds. also interesting is the two fold higher mortality in children in europe compared to the usa, at least at the beginning of the s. to put mortality from infectious diseases into some perspective table . shows mortality in the usa in june from all causes. deaths from pneumonia and influenza, for example, list seventh in such an analysis. the incidence of viral dis- in summary, therefore, in the industrialized and more wealthy nations of the world infectious diseases including viral diseases play a much reduced role in causing mortality but amongst the important viruses in this respect are particularly respiratory viruses such as influenza a and b, and respiratory syncytial virus. pandemic influenza a virus, in addition, causes considerable morbidity and economic disruption. we have briefly mentioned above clinical diseases and syndromes such as influenza, respiratory tract infections, hepatitis, and arbovirus infections resulting from virus infections. but many of these disease syndromes may be caused by a multitude of viruses, making specific diagnosis without the aid of a laboratory impossible. most antiviral agents discovered to date have a very restricted range of antiviral activity and so to use these in the field it will be necessary to identify particular viruses causing a clinical syndrome rapidly and precisely. partly for this reason, methods of rapid virus diagnosis have been investigated rather intensively during the last few years and the advent of monoclonal antibodies, for example, should hasten this process. meanwhile it will be useful here to briefly summarize the range of viruses responsible for these differing clinical syndromes, and finally to list the major viruses of man in a more logical 'scientific' manner, so that the reader will not end up completely confused! a very wide range of viruses ranging from the rna-containing pleomorphic influenza viruses to the dna icosahedral adenoviruses cause respiratory infection which may be completely indistinguishable by a clinician. nevertheless some general observations are a useful guide, including the facts that rhinoviruses, echo viruses, reoviruses and coronaviruses rarely, if ever, cause lower respiratory tract infection but confine their attention to the upper respiratory tract and hence may often produce only mild respiratory illness (table . ). cytomegalovirus and varicella zoster, on the other hand, tend to produce only lower respiratory tract infections whereas influenza, measles, parainfluenza, rsv and adenovirus produce both. finally, most upper respiratory tract diseaseall ages (but not all) clinical cases of influenza are caused by influenza a and b viruses, whilst most cases of croup in infants are caused by rsv ( fig. . in chapter ). similarly, a wide range of viruses have a predilection for nervous tissue (table . ) . some viruses such as rabies, polio and certain arboviruses target upon nerve cells, but in the case of most viruses central nervous system (cns) effects result as an aftermath and complication of viral replication (e.g. mumps, herpes, rubella). certain of these disease syndromes in the cns such as rabies can be identified by clinical examination of the patient, but even polio-like paralysis can be caused by other enteroviruses (chapter ). certainly encephalitis following rash diseases such as rubella, mumps and measles presents few problems of diagnosis unless the person has been infected without a rash. " types - , and . "jc and sv -pmlviruses. types a , , -- , , , , and b - . certain rashes in their typical form can be easily diagnosed as caused by the viruses of chickenpox (varicella zoster), measles or rubella (table . ). however, diagnosis of rashes caused by certain of the enteroviruses such as echo or coxsackie a may be easily misdiagnosed as rubella. this would not be particularly important were it not for the propensity of rubella to cause foetal infections and abnormalities (congenital rubella syndrome). table . lists viruses which have been implicated in causing foetal abnormalities, abortion or postnatal infections. undoubtedly the most significant and dramatic effects on the foetus are caused by rubella virus and the many and varied deleterious effects on the developing embryo have been encompassed as the 'expanded rubella syndrome' (table . ). infection of the mother in the first week of pregnancy may result in infection and sequelae in - % of the foetuses (chapter ). herpes viruses (via infection of the cervix of the mother) may infect the foetus during delivery, whereas cytomegalovirus and vaccinia can infect the foetus in utero in the final trimester of pregnancy. increased foetal deaths or abnormalities have been noted following infection of the mother with other viruses including polio, arboviruses, measles and influenza but these effects on the foetus are more likely to be caused via constitutional upset in the mother rather than by actual infection of the foetus or foetal tissue by the virus itself. most of the above mentioned viruses cause acute infection in humans following transmission from another person or from a vector such as a mosquito or other insects. but certain viruses can afterwards establish a latent or persistent infection occurs in the presence of high levels of neutralizing antibody such vaccines may have little or no effect, unless they stimulate cell mediated immunity (chapter ). similarly, antivirals are unlikely to eliminate latent herpes viruses from the ganglia and would merely hasten the healing of a particular reactivation episode. herpes simplex venereal infections are of considerable interest to the general public, virologists and antiviral chemotherapists and data from the usa appear to suggest infections of epidemic proportions (see chapter ). data from the uk show a steady increase in hsv- genital infections but certainly not in epidemic proportions. indeed it is most useful to place this virus in a context of other venereal infections. recently published data from the uk would suggest that non-specific genital infections are increasing in incidence at least as rapidly as hsv infections. although the aetiology of non-specific genital infection is still not fully elucidated, chlamydia traclzomatis is recognized to be the commonest cause in britain, and isolation rates from the cervix of unselected women attending sexually transmitted disease clinics in britain may reach up to %. total new attendances at special (venereal disease) clinics in the united kingdom rose by . x in compared with , continuing the increase noted each year since the early s. this is less than the previous annual increase of % (which, however, was unusually large). the overall picture of sexually transmitted disease in british clinics in the past years is one of the increasing importance of new cases requiring treatment in categories other than syphilis or gonorrhoea, which now account for only % of total cases requiring treatment. the largest absolute increase in new attendances by diagnostic category in , apart from 'other conditions requiring treatment', was in non-specific genital infection: there were new attendances, an increase of ( . %) over those in , but this was about half the previous annual rise of %. there were rises in most other diagnostic categories. the number of new attendances due to herpes simplex infection increased by ( . %), those due to warts by ( . %) and those due to candidiasis by ( %). other viruses including cytomegalovirus and hepatitis b virus are transmitted in semen from infected persons, but the general significance of this is not clear. htlv-i has been implicated as a possible cause of acquired immunodeficiency syndrome (aids) in promiscuous homosexuals (see chapter ) and presumably transmission via person to person in semen might be a major factor. table . and fig. . list, rather exhaustively (but not completely), a classification of viruses causing human disease, and table . briefly summarizes information on currently used vaccines and chemoprophylactic agents against viral diseases. the epidemiology, strategy of replication and physical and antigenic structure of these viruses will be discussed as fully as possible in the ensuing chapters. however, a word of warning should be introduced here. dna technology ('genetic engineering') techniques are being introduced very rapidly indeed and are expected to revolutionize the previously used biological approaches to development of new viral vaccines. the reader can safely assume that for most viruses discussed in the following chapters, even if it is not indicated in the text, that someone is cloning the particular gene into a eukaryote or prokaryote cell. it cannot be overemphasized that with both cew vaccines and antivirals the initial discovery is often made by individuals and single groups. only during later developments are the large teams of scientists required. also, with antivirals, a new era has arrived which is seeing the first extended use in the clinic of inhibitory molecules against viral diseases, particularly herpes. so we shall undoubtedly see a plethora of new molecules each with certain biological and pharmacological advantages compared to the parent. indeed, we are witnessing this trend already with molecular derivatives of acyclovir such as dhpg and dhbg (chapter i). we have tried, in the ensuing chapters to present the reader with a review of the basic scientific knowledge and principles underlying development of vaccines and antivirals. new data should simply enhance interest in the topic and perhaps even encourage a reader to develop a vaccine or antiviral him or herself! the impact of viral diseases on society can, to some extent, be expressed in economic terms and this again illustrates the magnitude of the infectious viral disease problem. in the usa, during the period - , the annual mortality from influenza was deaths, the annual cost of treatment $ million and the annual loss of productivity $ million. the cost of less severe respiratory viral diseases, such as common cold occurring on the average more than twice yearly, is probably of the same magnitude. the distress and pain caused by recurrent labial and genital herpes infections is very large, and possibly increasing. the number of patients is difficult to express in economic terms, but an estimated number of americans are contracting genital herpes each year and approximately million episodes of labial herpes will affect the usa population each year. the impact of gastrointestinal syndromes caused by viral infection can be illustrated by an annual mortality of - million due to rotavirus infections in children in asia, africa and south america many chronic conditions are initiated in infectious diseases. hepatitis b may result in chronic infection in % of the cases, leading to chronic cirrhosis. an esti- for some communicable diseases such as rabies, although the rate of morbidity is small or non-existent, nevertheless substantial costs are incurred in surveillance, prevention and health education. also the costs for rabies extend into the agricultural sector of the community. with all communicable diseases allowance must be made for indirect costs such as production losses in the economy and these may sometimes amount to twice as much as the health service costs. indirect costs again may spread over a number of sectors of the economy, including infected animal stock or food products etc. to date very few detailed studies have been undertaken to establish the cost and none mononucleosis effectiveness of programmes for the prevention and treatment of viral diseases. moreover, these have been concerned with vaccines and no work has been published as regards specific antivirals. however, the estimated cost to develop an antiviral agent is $ - million which, in relation to common diseases such as influenza and herpes, is economically acceptable but with less frequent viral diseases can be a problem for a private company. such data as there are suggest that improved strategies of prevention of viral disease could make substantial savings and result in better health outcomes. some viral diseases such as measles and polio maintain high incidence levels unless immunization levels are constantly maintained, whereas others can be self eliminated when a threshold level of infection is reached. where the incidence of a disease changes over time, established forms of treatment may lose their justification and should be phased out or altered. the patterns of some viral diseases have changed due to medical intervention in another sphere and an example is the improvement in treatment of cancers and immunosuppression of transplant patients. this has resulted in an increasingly common situation where a successful, and often very expensive, treatment of a disease is threatened by opportunistic viral infections, mainly by latent herpes viruses. the cost of developing antiviral agents against these types of infections should be considered in the context of the total cost to manage these patients and the risk of an infection. an informal weighing of risks and benefits of immunization (e.g. for smallpox) has been carried out in some societies, like the uk, for hundreds of years. however, it is now possible to apply more precise scientific analysis to the problem. such a scientific analysis of immunization benefits in the early s led to a major alteration in national health policy in the usanamely the decision that routine smallpox vaccination should be discontinued. the last case of variola minor in the usa was in and by the risk of death from all smallpox vaccinations was per million for primary vaccinees, rising to per million for children under year of age. in addition, among primary vaccinees the combined rate of post vaccinia encephalitis and vaccinia necrosum was . per million for infants. on the other hand, the probability of a smallpox importation into the usa in was importation every years. it would probably have required smallpox importations per year to produce the same mortality which was then associated with smallpox vaccination. this is an excellent example of the direct usefulness of statistics. in the united states alone more than types of vaccines, both bacterial and viral are used and one may question how worthwhile some of these vaccines are in economic terms. cost effectiveness analysis and cost benefit analysis aggregate the net medical care costs and net health benefits from a vaccination programme and thus help to give an economic analysis. net medical care costs often include the cost of vaccine and its administration, cost of treating vaccine complications and the medical care savings due to prevention of disease. net health benefits include reduction in morbidity and mortality. also one may include the gains in productivity resulting from a reduction of absence at work. other qualitative and more difficult to cost-estimate considerations must be included to place a vaccine programme in an accurate social and medical perspective e.g. pain and anguish of illness, compensation of victims of severe vaccine reactions etc. these days, quite necessarily so, political, social, economic and medical factors are all taken into account (or at least should be by national health authorities). it is an interesting and useful exercise to see how these analyses apply to popular and seemingly useful viral vaccines (willems and sanders, ) . the salient features of this analysis which mainly refers to experience in the usa (and therefore may differ in details in european countries, for example) are presented in table . . the crucial issue for a vaccine strategy against rubella is the appropriate age of immunization. prevention of congenital rubella syndrome is the aim of this programme, because rubella itself is a mild disease scarcely worth considering if it were not for the teratogenic properties of the virus. the teratogenic effect of the virus is unique in its selectivity of action and most embryos of mothers infected during the first trimester would be affected. as an example of the community effects in the - epidemic before vaccine was introduced, therapeutic abortions were carried out and children were born with rubella syndrome in the usa alone. also excess prenatal deaths were associated with the epidemic. of the children born with congenital rubella, were deaf, deaf and blind and several thousand suffered moderate to severe mental retardation. estimated direct costs of the epidemic in the usa were $i billion, mainly ( %) as regards long term care associated with rubella syndrome. in the usa after , when vaccine became widely available, the strategy was to immunize a// children as a routine, whereas adolescents and women of childbearing age were immunized selectively. in the uk, on the other hand, mainly school girls (not boys) were immunized. the basic strategy in the usa was to displace wild virulent rubella by attenuated non-teratogenic vaccine virus. a reduced objective was aimed for in the uk and the programme to date has not been so successful. in the usa, rubella epidemics have been prevented and cases of rubella have decreased continuously in the under year age group. most cases of rubella now occur among adolescents. a study of costs and benefits found positive net benefits. in the direct cost of acute rubella for million persons was estimated at $ . million and the costs of congenital rubella syndrome in the offspring of million unprotected females was $ . million. the benefit cost ratio for vaccine to million females at years of age was : , assuming % immunization of the target population. since mumps vaccine was licensed in in the usa more than million doses h, p have been used and the incidence of mumps has dropped from rates of - per population to - per i in the late s. it may be calculated that the use of mumps vaccine for a group of a million persons would prevent cases of mumps and deaths. over a year period mumps vaccine would reduce costs by over % and the benefit cost ratio approximates to : : i . mumps vaccine in austria has a benefit cost ratio of . : and in switzerland . : (data not shown in the table) . comparable analyses appear to show that influenza vaccine is less effective in economic terms than rubella or measles vaccines but very different considerations pertain. the vaccine is recommended for special risk groups and not particularly for children. medical care costs of vaccination during - totalled $ million and l so million persons were immunized in the usa, giving a cost of $ per year of healthy life gained. moreover, the cost effectiveness of vaccination improves with increasing age of the person vaccinated (because the highest mortality occcurs in the over year age groups) so that it costs $ per vaccination for each year of healthy life gained in the under age group, to $ per vaccination in the - age group, to positive cost savings in the + age group. assuming influenza vaccine is % effective, vaccinating a million elderly persons would result in additional years of life at a net cost of $ per year of life gained. most people would agree that this is a reasonable use of medical resources. four million cases of measles occurred in the usa each year before vaccine was introduced in , with cases of encephalitis and - deaths each year. net benefits of measles immunization between and include savings of . million hospital days, million school days and cases of mental retardation. the benefit cost ratio was approximately o:i. in finland, as an example of a european country, the benefit cost ratio was . :l. however, as measles declines, marginal reductions in incidence will become increasingly costly. measles will probably be eliminated as an endemic disease in the usa during . an excellent recent example of the application of cost effectiveness of viral vaccines is being carried out in several countries at the present moment with the advent of an effective vaccine against hepatitis b (hbv) virus (szmuness et al., ) . the vaccine is rather costly because of the technical problems of purifying and inactivating antigen from human sera ($ per course of doses of vaccine) and, more important, its availability may be limited in the near future. therefore 'decision analysis' can be used to estimate likely costs and benefits of different immunization approaches in different populations at risk. a decision analysis model was constructed to compare alternatives for prevention, which were: a. immunizing all persons with no prior screening for indication of previous infec t io n b. screening all persons for indications of previous infection and then immunizing only those sero or antigen negative persons c. passive immunization of persons exposed to hbv. the estimated cost per person of hepatitis vaccination in a year period in a homosexual population with a % prevalence of hbv markers and % annual attack rate without screening and vaccinations is $ . . vaccination of all persons in this group would result in a lowering of hbv incidence from to %, costing $ . per person. however, screening followed by immunization would cost only $ . (because fewer people would be immunized). in contrast, for a group of hospital employees with relatively high exposure ( . %) and annual attack rate of %, vaccination without screening is the lowest cost strategy ($ . ). for a low risk population ( . % annual attack rate) neither vaccination nor screening followed by vaccination would result in a saving in medical care costs. indeed the net medical costs per case of hepatitis prevenfed by vaccinating the latter population would be $ . in contrast, net medical care costs per case prevented are negative when the attack rate is greater than . %. a very important caveat of the above discussion, however, is the fact that indirect costs saved by immunization, such as loss of productivity etc, are not included. if one is only interested in medical care savings then hbv vaccination should be carried out before or early during a period of unavoidable high risk as with surgical registrars, new dialysis unit patients and staff members, new prisoners, newly institutionalized mentally retarded patients, and promiscuous homosexuals. we should note that in the example given above, which is rather usa-orientated, extrapolation to conditions in europe may be questionable. in fact an excellent example of these differences has been highlighted with the current discussion about hepatitis b vaccine. for example, in greece, the prevalence of anti-hbv among health workers is - % and in medical and nursing students it was and %, respectively. screening costs around $ , whereas vaccine costs approximately $ and so it seems reasonable to vaccinate health care workers after screening, and medical and nursing students without screening. in the uk the problem of finance is very relevant because additional funds may not be made available to health authorities and so again, screening may be resorted to as a cost saving excercise. the development of an antiviral drug is a major undertaking and will require, at least for a private company, that the market for a drug is large enough to correspond to the cost and risk of development. table .i lists some viruses which have been ranked according to different variables in an attempt to select a good candidate for an antiviral drug. the incidence of the virus disease is naturally an important factor, as is the severity of the disease. the incidence can be obtained for diseases being reported in accordance with local regulations, but in many cases viral diseases are not reported and the incidence has to be calculated from different surveys. also a grading of the severity is not easy and an example is when herpesvirus infections are handled as a group, which would include both herpes encephalitis and cold sores. therefore, a rather subjective average has been used in table . . an additional important factor in deciding the targets for antiviral chemotherapy is the absence or availability of good viral vaccines and the probability of developing vaccines in the future. for viruses like rhino and influenza with many serotypes or antigenic variants, vaccine production may always be a problem especially with influenza when new antigenic types appear rapidly. in the case of hsv-i and hsv- infections the development of a successful vaccine seems unlikely when one considers that patients with frequent episodes of labial or genital herpes have high titres of neutralizing antibodies and that reinfection can occur in spite of circulating antibodies. aspects of vaccines are discussed in more detail in chapter and in connection with the different viruses. rapid diagnosis (preferably by the patient!) is of importance since an antiviral drug is likely to have a narrow spectrum of activity and the virus to have a short time period of replication. it will be necessary to know exactly which virus is causing the infection and thus which drug should be used. the self-diagnosis of recurrent diseases such as labial and genital herpes is rather easy for the patient and, from that point of view, herpesvirus infections are good targets for antiviral drugs. major points of attack when developing new antiviral drugs are viral enzymes. herpesvirus enzymes are easily accessible and well characterized, as are also influenza virus enzymes, and this feature also makes these two viruses attractive as targets for antiviral inhibitors. the use of viral enzymes as targets for antiviral drugs is discussed in chapter . in essence, we conclude from the considerations in table . that herpes and influenza should be major goals for the development of new antiviral drugs. this is also reflected in the literature where anti-herpes compounds are the most flourishing area at present. rank list of candidate viruses for the development of antiviral drugs. the listing has been made to place the virus most favourable in the development of antiviral drugs on the top of each column. the added rank numbers will then give a crude estimation of the incentive to develop an antiviral drug against each virus. the lower the number the better the target. problems easy accessible incidence with vaccine diagnosis virus enzyme aalthough not included in the six target diseases, there is a relationship between this programme and the prevention and control of diseases such as yellow fever, hepatitis, rubella and mumps. epi, expanded immunization programme; cdd, campaign against diarrhoea disease; ari, acute respiratory infections. the great need for therapy and prophylaxis of viral diseases is obvious. in recent years we have seen a swift expansion in our knowledge of the biological and chemical processes involved in viral diseases. this has made possible rational efforts to manage and prevent viral diseases, and we are now seeing the results in new vaccines and antiviral agents. the greatest challenges and probably the most difficult and medically important areas for prophylaxis and therapy of viral diseases are those viruses which are rapidly changing in antigenic composition and/or viruses with animal reservoirs (influenza and arboviruses) and also those forming latent infections (herpesviruses). the three major international co-ordinating programmes of who (table . ) include both these 'challenge' viruses and also more common viruses such as measles and polio where particular help is required in developing countries. the relative role of vaccines and antiviral drugs is difficult to predict. in cases where cheap and effective vaccines exist or can be developed as exemplified for polio, measles and hepatitis b, this is likely to be the optimum control method. in cases such as influenza, the great variability of the virus probably poses insurmountable difficulties for vaccines, and makes the antiviral drug approach more promising. finally, for viruses such as the herpesviruses, causing recurrent infections (in spite of both preexisting humoral and cell mediated immunity) vaccines seem an unlikely approach and antiherpes drugs now appear to be more promising. approaches to immunization of infants and young children against gastroenteritis due to rotaviruses target diseases for future antiviral drugs pp viral diseases in south east asia and the western pacific classification and nomenclature of viruses nouvelle communication sur la rage passive active immunization new trends and developments in vaccines cost effectiveness and cost benefit analysis of vaccines key: cord- -zf bx i authors: nan title: president’s plenary session date: - - journal: reprod sci doi: . / sha: doc_id: cord_uid: zf bx i nan hector barrera-villa zevallos, donna lai, jonathan arthur, cecilia ng, robert markham, and ian s fraser. stephen brown, elizabeth bonney, brian nielsen, renju raj, and lucia brown. burlington, vt, usa. ismail mert, and david kmak. tinnakorn chaiworapongsa, roberto romero, nandor g than, edgar hernandez-andrade, zeynep alpay savasan, adi l tarca, gaurav bhatti, zhong dong, and sonia s hassan. detroit, mi and detroit, mi, usa. delrae m eckman, charles r rosenfeld, ridhima gupta, shelton m charles, heather mertz, and lorna g moore. ; winston-salem, nc and dallas, tx. julia v gefter, carl a hubel, and robert w powers. pittsburgh, pa, usa. styliani goulopoulou, johanna l hannan, and r clinton webb. augusta, ga, usa and baltimore, md, usa. daniel kerage, randi gombos, and denise g hemmings. jennifer krupp, derek boeldt, fuxian yi, dinesh shah, and ian bird. muna noori, ann e donald, aroon d hingorani, and david j williams. london, united kingdom. concurrent session iv parturition: inflammation, infection surfactant protein (sp)-a attenuates lps-induced nf-κb activation in a mouse macrophage reporter cell probiotic attenuation of infl ammatory responses in amnion: a possible role in the prevention of preterm labor? poster session iii -douglas pavilion s- infl ammation provoking a cytokine response and disrupting the epithelial barrier is mitigated with dexamethasone: a possible mechanism of premature cervical remodeling and preterm birth immune adaptations of pregnancy as risk factors of gestational uti appropriate for gestational age-preterm neonates born to mothers with moderate, but not mild or severe, intensity of histologic chorioamnionitis, funisitis, and intraamniotic infl ammation without microbial invasion of amniotic cavity have a lower rate of rds. chan-wook park human β defensin - in human pregnancy: the effect of gestational age, parturition, and intra-amniotic infection/infl ammation. manasi patwardhan, tinnakorn chaiworapongsa, zeynep alpay savasan chronic pyelonephritis in pregnancy: intracellular survival & persistance of the gestational pathogen differential cytokine and tissue remodeling responses in chorion and decidua exposed to ureaplasma parvum disabled cervical cytokine expression in post-term women with failed labor induction infl ammation of the fetal skin following low-grade microbial stimulation s- u and u snrna downregulation in syncytiotrophoblast tal imbar, ilana ariel, and simcha yagel key: cord- -i v lyd authors: madraki, golshan; grasso, isabella; otala, jacqueline; liu, yu; matthews, jeanna title: characterizing and comparing covid- misinformation across languages, countries and platforms date: - - journal: nan doi: nan sha: doc_id: cord_uid: i v lyd misinformation/disinformation about covid- has been rampant on social media around the world. in this study, we investigate covid- misinformation/ disinformation on social media in multiple languages - farsi (persian), chinese, and english, about multiple countries - iran, china, and the united states (us), and on multiple platforms such as twitter, facebook, instagram, weibo, and whatsapp. misinformation, especially about a global pandemic, is a global problem yet it is common for studies of covid- misinformation on social media to focus on a single language, like english, a single country, like the us, or a single platform, like twitter. we utilized opportunistic sampling to compile specific items of viral and yet debunked misinformation across these languages, countries and platforms emerged between january and august . we then categorized this collection based both on the topics of the misinformation and the underlying roots of that misinformation. our multi-cultural and multilingual team observed that the nature of covid- misinformation on social media varied in substantial ways across different languages/countries depending on the cultures, beliefs/religions, popularity of social media, types of platforms, freedom of speech and the power of people versus governments. we observe that politics is at the root of most of the collected misinformation across all three languages in this dataset. we further observe the different impact of government restrictions on platforms and platform restrictions on content in iran, china, and the us and their impact on a key question of our age: how do we control misinformation without silencing the voices we need to hold governments accountable? along with the covid- pandemic crisis, an infodemic (world health organization, ) crisis has affected all aspects of human lives from elections to public health response around the world. social media has played a critical role in this infodemic crisis. first, social media offers a free and easy-access platform for users to share content (both true and false) in the form of posts, videos, pictures, and memes, all with a wide range of audiences (weinberger, ) . second, the covid- outbreak has forced people around the world to be quarantined and consequently interactions have shifted away from face-toface interactions and even more towards online/social media interactions. this means that more people are exposed to unreliable content circulating on social media and many users struggle with distinguishing between facts and lies/fictions about covid- . since the beginning of the pandemic, some official efforts have been implemented to debunk these lies and inaccurate information circulating on social media, despite substantial disagreement about which corrective measures for fact checking are practical and appropriate for massive social media platforms. still, these efforts seem not enough and there is a widespread consensus that an integrated sustainable global effort is warranted across different languages and through different platforms as are targeted in our study (dizikes, ; pennycook et al., a) . in the literature, the problematic information has been categorized into misinformation, disinformation, and malinformation, with some debate about definitions (karduni, ; wardle, ) . in this paper, we consider the following definitions: misinformation is incorrect information created without the intention of causing harm (e.g. encouraging people to wear a face shield without realizing how ineffective it could be). disinformation is incorrect information and intentionally created to hurt an individual, a group, or a country (e.g. a drug company spreading out a false rumor that its new medicine can cure covid- with an intent to increase profit.) mal-information is correct information (based on reality), but used to cause harm to an individual, a group, or a country (e.g. justifying the high rate of confirmed cases by claiming that it is because of increasing the rate of testing.) (infodemic toolkit, ; kujawski, ; wardle, ) . distinguishing between these three categories, particularly misinformation and disinformation, can be difficult or even impossible in some cases as can require assessing the intent of the creator. thus, more broadly, here in this paper, we will use the general term 'misinformation' to refer to all three categories. according to a study from oxford university, misinformation is also often true information reconfigured or recontextualize and less commonly fabricated (brennen et al., ) . this reconfigured information can circulate even faster than fully fabricated stories, taking in % of the interactions in one study (brennen et al., ) . in this paper, covid- misinformation, broadly defined to include disinformation and mal-information as well, will be investigated within multiple languages (chinese, english, and farsi) about multiple countries (china, iran, and the usa) on different social media platforms. the covid- pandemic broke out first in wuhan, china in december . then, iran became a hotspot in february . the usa has been a clear hotspot as well with % of confirmed world-wide covid cases despite only . % of the world population (dong et al., ) . we chose these three countries as a lens through which to consider major differences between covid- misinformation around the world. we show how examining social media misinformation from the perspective of one country, one language, or one platform, misses important and more holistic aspects of the pandemic. through this research, we build a more comprehensive picture of how misinformation has exacerbated the covid- crisis around the world. our research team includes native speakers of chinese, english, and farsi who were born and raised in china, iran, and the usa. to the best of our knowledge, we are the first to examine the multilingual social media landscape by using the opportunistic sampling method to collect a dataset of verified and viral covid- misinformation across languages: chinese, english, and farsi. our multicultural and multilingual team observed that the nature of covid- misinformation on social media varies in substantial ways across different languages/countries depending on the cultures, beliefs, religions, popularity of social media, types of platforms, freedom of speech, the power of people versus governments, etc. based on these observations, we proposed a novel and comprehensive categorization of the covid- misinformation based on their topics and roots such that these categories are all relevant and extendable in all three languages. it is worth noting the difference in government policies for controlling misinformation in iran, china, and the usa. china has strict government control over which platforms can be used and enforces these controls. approved platforms aggressively remove misinformation of some kinds, but not all. iran also has laws restricting which platforms can be used, but does much less to actually enforce these laws. it makes some platforms more inconvenient to use, but does less to actually prevent it. the usa has some new laws restricting the use of social media platforms, specifically chinese social media platforms. whether these laws will stand remains to be seen. there are some attempts to control the flow of some types of misinformation on platforms like twitter and facebook based on self-regulation by the platform and not government regulation. throughout our study, we examine the impact of these different approaches on the types of misinformation spreading. misinformation is not a new phenomenon, it has been around for centuries in the forms of rumors, gossip conspiracy theories, etc. (burston et al., ) . however, the emergence of the publishing industry in the th century provided an official modern platform for misinformation. the st century has been characterized by the explosion of information through the internet. the technology, particularly social media, has amplified the spread of misinformation and its adverse impacts by providing a fast and free channel to share any information whether true or false (lazer et al., ) . since the beginning of the pandemic, catastrophic and lifethreatening impacts of tremendous amounts of misinformation circulating on social media have appeared (donovan, ) . for instance, due to fake news, some individuals used toxic home remedies resulting in injury and death (vigdor, a ). misinformation has provoked many people to hoard some vital necessary products (e.g., n mask, sanitizers, toilet papers, etc.) causing a shortage of supplies for essential workers (vigdor, b the closest related work to this research is the misinfodemic report developed by meedan and written by alimardini et al (alimardani & elswah, ) . this qualitative report considers a more global response to misinformation, showcasing seven countries compared to several united states-only studies. meedan divides their report into the crumbling of public trust, informal leaders of information, and impact of the infodemic on governance. their coverage of countries is extensive, but our research adds a balance of qualitative and quantitative analysis comparing countries, languages and platforms within our sample. purely quantitative research that involves misinformation globally has been done by pew research. they surveyed social media users within several countries about how often they encounter obviously fake content (silver, ) . this study tries to point researchers toward countries that seem to be encountering misinformation more frequently. this type of quantitative research does not dive as deeply into the content, topics and root of misinformation present within each country surveyed. twitter, facebook, instagram, whatsapp, weibo, wechat and tiktok are the most popular social media platforms in china, iran, and the usa. however, due to the censorship and political reasons, some strict restrictions have been imposed on these platforms in some cases (rachman, ) . whatsapp is a cross-platform encrypted messaging application acquired by facebook, and its monthly active users have reached billion as of march (clement, b) . it has . million users just in the usa as of (andjelic, ) . whatsapp is completely banned in china. whatsapp is legally allowed to be used in iran and has become even more popular in iran after the iranian government blocked telegram in which used to be the most popular messaging application in iran. the government claimed that telegram had endangered national security (erdbrink, ) . whatsapp has become a makeshift social media platform in iran as group chats have begun including thousands of members forwarding and sharing information. although the government has blocked many social media platforms officially, many iranians still use vpn and proxy anti-filter apps/tools to access blocked social media. however, this access is more limited and requires some technical tools and skills. this has helped to drive usage towards whatsapp which is a private end-to-end encrypted messaging platform without tracing capabilities. partly as a result, whatsapp has become a major source of misinformation in iran and many other countries (alimardani & elswah, ) . whatsapp recently limited the number of times users can forward a message to only five times, in an attempt to fight against misinformation (kastrenakes, ). tiktok was developed by bytedance (字节跳动), a chinese company in beijing. tiktok has a version used only in china called douyin (抖音) to separate the domestic users from international users. due to the concern of cyber security, tiktok operation in the usa will be transferred to a new company named tiktok global and will cooperate with oracle and walmart to ensure the data safety (lin, l., ). tiktok is allowed to be used in iran. keywords and hashtags are the major methods to search on social media and we use both to collect our data set. all platforms named in this study supports both keywords and hashtag search. hashtags on whatsapp might not be as popular as other platforms however this function makes the search process very easy even through a private message chain (patkar, ) . unlike hashtags on american platforms, a hashtag on weibo is owned by a host. also, each hashtag has its own unique webpage, which is called 超级话题 (i.e., topic or super topic). since january , many super topics have appeared around covid- on weibo. figure shows a sample of super topic webpage on weibo. the topic host and the largest contributor to this topic is a state media. although english hashtags are allowed on weibo, almost all hashtags are in simplified chinese. weibo needs the symbol "#" before and after a term to function as hashtags, unlike twitter which only needs "#" before. wechat channels is a popular feature of wechat and contain public feeds of content. hashtag and keywords search can be used in wechat channels. selecting an appropriate sampling methodology is a major challenge when it comes to social media studies. nonbiased sampling from social media is often difficult because the dataset is so highly dynamic, massive in size, and difficult to filter. the sheer volume of data--facebook has over million users and twitter has a rate of , tweets per minute--makes the gold standard of data acquisition, true random sampling, challenging. this can be even more complicated when sampling for misinformation since our underlying population is not all posts, but those posts that contain misinformation that has been refuted or debunked by recognized fact-checking organizations. hashtag and keyword filters do not necessarily guarantee to find misinformation, and even if a suspected post is found, we are reliant on the efficiency of journalists in fact-checking posts and content which is also biased by the particular needs and intentions of journalists and respective media outlets (pingree et al., ) . in this study, we utilized an opportunistic sampling strategy, meaning that our sampling was determined by the population, or presence of misinformation on social media, that was available and officially debunked at the time, and our abilities to find them. we used chinese, english, and farsi hashtag and keyword filters to collect specific items of debunked misinformation that spread virally between january and august . when we encountered a particular item in one of our languages, first we used fact checking organizations to see if that claim had been debunked using fact-checking sources such as international fact checking network (ifcn), platform based factchecking tools in twitter, facebook and google, and organizations using claim review (poynter, n.d.; schema, ) . the item is logged only if we could find a verified debunking source; otherwise, it is discarded. we estimate that number of logs could be doubled if we did not have the debunking constraint. then we watched for posts with the same claim in our other languages and on other platforms. we also sought out independent media reports beyond our own direct experience that the referenced claim was spreading virally. thus, each of these items of misinformation in our study represents thousands of posts repeating the same debunked claim, often across multiple languages and platforms. out of these collected pieces of misinformation, are in chinese, in english, in farsi. we admit that our data set may be unintentionally biased considering the biased nature of social media and the fact that all authors are currently in the usa, so we might be more exposed to english misinformation. furthermore, due to the lack of copyright laws enforcements in iran, a single piece of misinformation might have been repeatedly debunked by too many sources which prolongs and complicates the search process for new pieces. figure represents a clearer breakdown of our collected data and the overlaps in misinformation across languages. strict filtering and censorship policies for chinese social media is a major factor in the notably lower totals. this can be considered as a benefit of the aggressive censoring of some types of misinformation in china. however, this could come at the expense of silencing voices that are needed to counter other kinds of misinformation. this is a key question being explored in this paper. there are , , pieces of misinformation found exclusively in chinese, english, and farsi only, respectively. out of overall pieces of misinformation, occurred in only farsi and english ( %) which is the largest overlap among possible pairs of languages while farsi and chinese have the least overlap (less than %). of the collected pieces of misinformation, % have been found in all three languages. given the complicated nature of tracking misinformation across multiple languages' social media landscapes, a comprehensive categorization over the topics of covid- misinformation was critical to analyze the catastrophic infodemic occurring during this global pandemic. we identified top level categories for the topics of covid- misinformation. we found that these proposed categories were inclusive enough to cover the chinese, english, and farsi misinformation in our collection. these categories are also extendable to also cover the possible future misinformation. the description of these categories are as follows: includes rumors, actions, and false claims to manipulate the official statistics of death and confirmed cases including exaggerating or downplaying the numbers. includes conspiracy theories and rumors spread out about other countries' roles related to the new virus. . virus transmission: includes topics related to misleading, superstitious, or fake, methods by which the virus can transmit, asymptomatic period, basic reproduction number (r ). . others: includes topics such as contact tracing (pros and cons of contact tracing, and rumors about the amount of personal information needed to be collected for the contact tracing purposes); recovery (the length of recovery periods, antibody level after the recovery, immunity of recovered patients, etc.); prediction of the pandemic; compensations, and more topics; etc. the topics for each of our collected samples were inductively examined to be specific to covid- misinformation and accordingly classified within the proposed categories. figure represents the proportion of each topic within our overall collection across languages. about a quarter of the total collected misinformation in our sample fell under the topic of prevention-individual. also, the top three topics of the collected misinformation mostly concern the individual behavior. in a few cases, the same claim has been reported in more than one category, e.g., "drinking bleach" has been circulating on social media as both a cure and prevention method. in fact, this high proportion of misinformation within the context of individual behavior verifies the vulnerability of users to misinformation to cope with uncertainty and uncontrollability of pandemic circumstances. an interesting observation on the topics of misinformation in these three languages is that the percentage of categories in english and farsi are comparable. furthermore, the top two categories for both farsi and english are "prevention-individual" and "virus transmission". this suggests that the iranian laws coupled with weak enforcement still allows misinformation of a similar kind to flow in iran as in the usa with fewer laws and restrictions. the greatest difference in english and farsi belongs to the category of misinformation related to "other countries". meanwhile, chinese and farsi misinformation within this category have similar records. since most of the chinese and farsi rumors about other countries have political roots and considering the controlling governments in iran and china, such similarity is not surprising. it is interesting that despite tight control of other kinds of misinformation in china, this category is still specifically allowed to flow. the top category of the chinese sample belongs to the topic of "origin" of the virus which is actually still a question mark for the world. we also observed a larger variety of topics in english which explains the relatively higher percentage of english misinformation categorized as "others". for instance, misinformation related to contact tracing could only be found in the usa. one of the reasons could be the fact that some laws and rules vary from one state to another in the usa (e.g., misinformation about federal and state compensation and financial support from businesses and individuals) and this confusing landscape of varying laws opens up space for misinformation to flow. in this section, we delve beyond a classification of misinformation by topic to the roots of misinformation. for example, jang et al. reported that most misinformation stems from a false statement quoted by a public figure; or is deliberate misinformation used for a particular purpose (jang et al, ) . we found that covid- misinformation also followed this same pattern. we identified the following six categories for the roots of the misinformation. it should be noted that we use the term "root", but we could have instead used terms such as "reason" for or "source" of misinformation. . political-related roots: a false statement quoted by a political figure; or related to governments and the relationship between countries; or used for political purposes, e.g., elections. . medical/science-related roots: a false statement quoted by someone claiming to be a medical expert, e.g., doctors, nurses, etc.; or a false perception related to medical research outcomes. statement quoted by a celebrity, influencer, or popular/public figure in the media field; or, a misleading/false content such as a video, photo or an article gets viral through media, e.g., tv, press, etc. . religious-related roots: a false statement quoted by a religious figure; related to religious and/or traditional and superstitious beliefs. . criminal-related roots: a false statement which has been claimed by a scammer or hackers for criminal purposes such as fraud, access to personal information. . others: any other false statements that cannot be substantiated to be related to the mentioned categories including hoaxes, jokes and other undesignated misinformation. for each of our pieces of misinformation, we attempted to track the source that led to this content going viral. the roots of misinformation collected in our sample are verified by reliable references and categorized in one of these six categories by human annotators. if a false statement has been quoted by multiple sources, then the source which made the statement goes viral will be considered as the root of the statement. as figure represents, more than one third ( . %) of the misinformation has been related to political roots which is alarming and shows the critical role of governments and political figures in the infomedic. for example, researchers analyzing -million english-language articles about the pandemic found that usa president trump was the largest driver of the infodemic over that sample (evanega et al., ) . we could not verify the roots of % of our sample, indicating the breadth and diversity of covid- misinformation; these have been categorized as "others". the three countries selected in this paper have their own unique characteristics and cultural structures and here we further discuss the roots of misinformation, particularly the religious and political roots, separately in iran, china, and the usa in the following subsections as well as a discussion of commonalities across these countries. the covid- misinformation circulating on chinese social media landscape reflects significant differences in political systems between china and the western world. for such reasons, misinformation from the western world is translated, filtered, and reflected in chinese social media. a primary root of misinformation circulation in china is the presence of fake science-based claims. when considering chinese social media, it is important to consider the systems of aggressive censorship in china that results in a strict politically filtered internet. global misinformation which has been translated and reflected in chinese social media environment can be considered politically biased since the vast majority of social media in china is tightly controlled by the state. an interesting trend observed in chinese social media is that some already debunked misinformation from the rest of the world, especially the usa, has been translated and widely shared in chinese platforms to criticize the western world's attempts to fight covid- . this pattern of disclosing the debunked misinformation is a politically manipulated misinformation per se. thus, we can only call this type of message pseudo "misinformation" (e.g. reports of president trump suggested injecting bleach to treat . in this case, it appears that the reason this misinformation is allowed to flow is more of a criticism of the usa and the susceptibility of its citizens to misinformation that appears clearly ridiculous to the average chinese citizen. through translating and sharing this message, people in china have been shocked to see such clear misinformation from the usa president and will further believe in more similar information regarding the unsuccessful control of covid- in the usa. much of the true misinformation in china are fake-science misinformation based on some assumption without sufficient evidence and scientific work to support it. though a post may point to scientific sources to back up the claim, it is difficult for most people to verify the source due to the language barrier. specifically, most people do not have the capacity of reading research publications in english or even of translating the name of the journal. this style of post can be used to give the impression of a scientific evidence that may not exist. in our chinese samples, the largest category of roots of misinformation belongs to politics ( %) which is aligned with our analysis for english and farsi samples (figure -a) . due to tight controls and censoring in china, collecting samples of covid- misinformation in chinese has been relatively harder than english and farsi. also, identifying the roots of the collected covid- misinformation is complicated. the roots of % of the chinese misinformation remain undesignated and categorized as "others". surprisingly, a large proportion of covid- misinformation in chinese has been started by celebrities and/or pop culture ( %) which is larger than the english and farsi corresponding category. unlike the iran and usa, most chinese are irreligious and atheist (huff post & briggs, ; noack, ) . however traditional chinese beliefs have been identified among covid- misinformation in chinese (only %) and categorized as having religious roots. we could not identify any misinformation in chinese associated with the criminal roots ( %). this is a strong result and could be considered one benefit of tight government control over social media in china. confronted with the impact of misinformation on western democracies (e.g. advice to drink bleach), chinese citizens could be convinced that the aggressive censorship policies help keep their society safer for criminal elements and viral misinformation. misinformation in the usa is commonly in the form of counter-expertise, the rejection of mainstream academic expertise, which dates back to the th century (douglas, ) . this form of misinformation began with christian fundamentalists rejecting evolution as it contradicted the bible (douglas, ) . since then, counter-expertise looks to distrust mainstream scientific media by promoting alternative thoughts through alternative media. this alternative media happens to be far more susceptible to misinformation (douglas, ) . such alternative media has released misinformation throughout the pandemic, with fox news ("fox's dr. marc siegel", ) stating "the virus should be compared to the flu because at worst case scenario it could be the flu". across the usa, churches have successfully resisted complying with government-led preventive measures and health orders. a recent study shows that % of protestant/evangelical ministers held in-person worship as of july , (vondracek, ) . the persistence to stay open and hold in-person worship has led to churches becoming hot spots for positive cases (conger et al., ) . father joseph illo, leader of the star of the sea church, sent out misinformation to churchgoers stating that "the news reports about covid are largely unreal" (bisacky, ) . however, not all churches within the usa are rejecting mainstream media or health orders. the current politically polarized atmosphere in the usa is also a major driver of misinformation. misinformation related to politics is often fabricated to create confirmation bias among readers, subsequently leading to heightened ingroup/party identification and polarization (douglas, ) (e.g. "the political party i identify with fought for the right thing, as opposed to the other party"). covid- misinformation spread by political figures in the usa may be a symptom of deflection and scapegoating due to inadequate administrative response. usa government sources have suggested a conspiracy theory that covid- was created in a chinese laboratory (aljazeera news, ). deflection is apparent as the usa president donald trump suggested "quick fixes" and "cures", such as hydroxychloroquine, azithromycin, and convalescent plasma (caplan, ) , that have limited testing and results as potential cures or "game changers in the history of medicine". it is necessary to acknowledge the severity and seriousness of the spread of misinformation from the usa public officials. in our collection of misinformation samples from the usa about one third ( %) have political roots ( figure -b) . while the spread of information from public officials to win elections is not new, the role of the mass media as a corrective measure on this behavior has changed. the media is now competing with social media for advertisements, which has resulted in pressure to cater content to users, further perpetuating political polarization (scheufele & krause, ) . from drastically downplaying the seriousness of the virus to stating that "one day it's like a miracle, it will disappear" (the white house, ), the usa government has contributed heavily to covid- misinformation on all media platforms. the role of celebrities in driving american culture (clemmons, ) and the power of american public figures' words should not be underestimated. in our sample, % of the collected misinformation in the usa has been directly traced back to a celebrity, influencers or popular figure. for example, on july , instagram removed madonna's post "for making false claims about cures and prevention methods for covid- " (solis, ) . false statements by self-claimed medical related crew or wrong and manipulated interpretation from medical facts are another major root of misinformation in the usa ( % of our sample represented in figure -b). covid- misinformation has hit iran harshly. two major reasons have been identified for creating and spreading misinformation: first, discourse about covid- is politically manipulated by the government (alimardani & elswah, ); second, official religious figures have interfered with covid- related issues and religion has been a barrier for ordinary hardliners to be unbiased. in general, social media reflects that people do not trust the covid information released by either government or religious officials. when the official channels of communication of information fail, people start clinging to their own unofficial channels of information gathering without monitoring the validity of the information which eventually intensifies the spread of misinformation. iran has a controlling and conservative government which micro-manages all aspects of people' lives. this characteristic of the government politicizes every subject including the pandemic. thus, it is not surprising that much of the covid- misinformation found in farsi has political roots. an ironic piece of misinformation with political roots in iran is that the government believes the covid- misinformation present in iran has been started mainly by "enemies", referring to the usa government ("bbc news persian", ). another example of political misinformation is that the iran government promoted a fake testing technology called "coronavirus remote detectors" which can detect infected individuals from a distance of yards. the unveiling ceremony on april th went viral all-over the iranian press and social media. religion has also played a critical role in spreading covid- misinformation in iran. for instance, some official religious hardliners falsely believe that sacred protection from religious shrines would prevent infection (malekian, ) . in iran, some shiite muslim religious figures often use people's faith to oppose "westernized" facts, sometimes including scientific facts. for example, on february , , a religious figure, ayatollah abbas tabrizian, advised people to rub their anuses with violet oil to prevent and cure covid- . this post on his official telegram channel (with more than , followers) has been viral on all farsi social media. users reshared this post with mixed reactions that included both adherence and ridicule (the new arab, ). as it was expected, in our collected farsi sample, the top category of roots of misinformation belongs to politics ( %) (figure -c). the next largest proportion of the misinformation has medical/science roots including both western misinformation and local and traditional persian remedies. about % of our sampled misinformation has religious roots. considering the bold role of religion in iran, this rate seems relatively low. however, the virality of this misinformation has been substantial, such that some of the misinformation is still circulating on social media, even after it officially got debunked. some however have been transformed into sarcasm to be used as a form of protest against hard-core religious figures. a b c figure : proportion of the chinese (n= ), english (n= ), and farsi (n= ) collected data across the proposed categories for roots of covid- misinformation. a recent study showed that there is an intersection between fake news and religion in societies with religious background (douglas, ) . our observation on this matter in iran is aligned with this study given the islamic background of iran ( %). however, our sample could not confirm the same result in the usa and china. given the impact of the christian community in the usa (bailey, ), % religious roots for the english misinformation was surprisingly lower than our initial expectation. when politics and government play a significant role in the destiny of a society (which is the case in iran, china, and the usa), a political polarization phenomenon will emerge (facing history and ourselves, ). political polarization has been known as an important factor to spread misinformation in a society and the meaningful relationship between misinformation and political polarization has been profoundly investigated in the literature ( the absence of misinformation with criminal roots in chinese social media is notable. this is an example of a key difference in how government strategy directly influences the types of misinformation to which the public is exposed. as a result, some societies are more vulnerable to criminal misinformation. given that politics was the largest root of misinformation across all three languages in this dataset, all societies are extremely vulnerable to government misinformation. however, some have more potential to counter government misinformation with information from private sources. in many ways, this is perhaps the key question for countries and societies around the world going forward in deciding how they want to control misinformation and infodemic. for liberal democracies, a key challenge is determining how to control misinformation without silencing the voices needed to hold government misinformation accountable. our major goal is to analyze the covid- misinformation on different social media platforms across different languages to gain a more holistic, global understanding of the misinformation's landscape. this effort is an initial step to diminish the current infodemic happening along with the pandemic. by increasing public knowledge of the adverse impacts of misinformation on public health during the pandemic, many lives could be saved. to achieve our goal, the opportunistic sampling approach was utilized to compile pieces of verified misinformation posted virally in chinese, english, and farsi across twitter, facebook, weibo, wechat, whatsapp, instagram, and tiktok between january and august . each of these pieces represented thousands of posts across platforms and often across languages. then, a classification approach was proposed to categorize the collected misinformation based on both their topics and roots. we identified high level topics being inclusive and relevant in all three languages. we also identified major categories for the roots of misinformation. our study yielded the following important results: • politics was the largest root of misinformation across all three languages in this dataset. • overall, the english and farsi samples have more in common in terms of the topic of misinformation than chinese specifically regarding individual prevention methods. • the absence of misinformation with criminal roots and fewer categories of misinformation overall in chinese social media is notable and points out a critically important tradeoff in the control of misinformation. we note important differences in how government controls on social media platforms drive usage onto some platforms and away from others, with different infrastructure for tracking and controlling misinformation. understanding how different countries utilize social media and their restrictions gives better insight as to how to regulate disruptive behavior. a key challenge going forward for all societies and countries will be in determining how to control misinformation without silencing the voices needed to hold governments accountable. overall, it is clear how focusing beyond english, beyond the usa, and beyond the usa-based social media platforms are essential to providing a clear understanding of the effects of misinformation and the effectiveness of misinformation control strategies around the world. muslims 'immune to coronavirus' some imams in somalia say, putting public at risk trust, religion, and politics: coronavirus misinformation in iran whatsapp statistics: revenue, usage, and history. fortunly christianity is declining at a rapid pace, but americans still hold positive views about religion's role in society rouhani: the conspiracy of our enemies to shut down the country for fear of the corona concerns raised after san francisco church shares misinformation on covid- types, sources, and claims of reuters institute for the study of journalism a brief history of fake news trump opened the floodgates for convalescent plasma too soon stop the spread of rumors coping with stress coronavirus: video of an undertrial in mumbai falsely viral as nizamuddin markaz attendee spitting at cop statistics: weibo monthly active users (mau) & dau. china internet watch wechat statistical highlights ; miniprogram dau> m number of monthly active facebook users worldwide as of nd quarter (in millions) number of monthly active whatsapp users worldwide from the psychology of secular saints: americans worship celebrities for better or worse churches were eager to reopen. now they are confronting coronavirus cases most popular social media sites in china the catch to putting warning labels on fake news social-media companies must flatten the curve of misinformation an interactive web-based dashboard to track covid- in real time religion and fake news: faith-based alternative information ecosystems in the the psychology of conspiracy theories coronavirus misinformation: quantifying sources and themes in the covid- 'infodemic'. the cornell alliance for science iran, like russia before it, tries to block telegram app explainer: political polarization in the united states the coronavirus doesn't discriminate, but u.s. health care showing familiar biases iran ranked world's th instagram user. financial tribune marc siegel says "worse case scenario study: rising religious tide in china overwhelms atheist doctrine an empirical investigation of network polarization a computational approach for examining the roots and spreading patterns of fake news: evolution tree analysis which countries block twitter & which no longer ban twitter? hidemyass human-misinformation interaction: understanding the interdisciplinary approach needed to computationally combat false information whatsapp limits message forwarding in fight against misinformation. the verge melanin doesn't protect against coronavirus coronavirus goes viral: quantifying the covid- misinformation epidemic on twitter combating fake news: an agenda for research and action mass polarization: manifestations and measurements tiktok owner puts deal with oracle twitter statistics every marketer should know in could covid- inspire the faithful? scholars predict spirituality surge in our future iranian hardliners accused of breaking into shrines closed to prevent coronavirus spread twitter is banned in china, so how does it have million users there? tech times taking stock with teens® − piper sandler completes th semi-annual generation z survey of map: these are the world's least religious countries the u.s. national pandemic emotional impact report secret ways to use hashtags you've never tried before. make use of the implied truth effect: attaching warnings to a subset of fake news headlines increases perceived accuracy of headlines without warnings fighting misinformation on social media using crowdsourced judgments of news source quality it was already dangerous to be muslim in india. then came the coronavirus checking facts and fighting back: why journalists should defend their profession verified signatories of the ifcn code of principles. ifcn code of principles chinese censorship is spreading beyond its borders american teens use tiktok. statista many black, asian americans say they have experienced discrimination amid coronavirus claimreview -schema.org type science audiences, misinformation, and fake news misinformation and fears about its impact are pervasive in emerging economies these celebrities have been flagged on social media for spreading coronavirus risk for asians, africans, caucasians− revealed japanese and chinese at highest risk for coronavirus applying essential oil to anus 'cures coronavirus is the new crown virus a laboratory biochemical weapon? only infect asians? why are conspiracy theories soaring? scholar analysis: the government deliberately nurturing and condoning remarks by president trump in meeting with african american leaders. the white house man fatally poisons himself while self-medicating for coronavirus, doctor says a hoarder's huge stockpile of masks and gloves will now go to doctors and nurses % of churches meeting for in-person worship, study finds. washington times information disorder: toward an interdisciplinary framework for research and policy making understanding information disorder too big to know: rethinking knowledge now that the facts aren't the facts, experts are everywhere, and the smartest person in the room is the room st who infodemiology conference. who the authors would like to kindly acknowledge assistance from gillian kurtic and yan chen for contributions to the paper including help reviewing, organizing, and formatting. we also thank dr. ricardo baeza-yates for his detailed feedback, wise advice and thought-provoking questions. we gratefully acknowledge funding from clarkson university epidemic and virus-related research innovation fund. key: cord- -v k vpi authors: st. leger, judy; wu, guang; anderson, mark; dalton, les; nilson, erika; wang, david title: west nile virus infection in killer whale, texas, usa, date: - - journal: emerg infect dis doi: . /eid . sha: doc_id: cord_uid: v k vpi in , nonsuppurative encephalitis was identified in a killer whale at a texas, usa, marine park. panviral dna microarray of brain tissue suggested west nile virus (wnv); wnv was confirmed by reverse transcription pcr and sequencing. immunohistochemistry demonstrated wnv antigen within neurons. wnv should be considered in cases of encephalitis in cetaceans. virus of the genus flavivirus that is transmitted by mosquitoes. in humans and animals, wnv has been associated with a spectrum of clinical conditions from asymptomatic infections to sudden death. these have been identifi ed in a variety of animal species. among marine mammals, wnv infection has been reported in a harbor seal (phoca vitulina) ( ) . we describe wnv infection in a killer whale (orcinus orca) and seroprevalence in conspecifi c cohort and noncohort groups. in , a -year-old male killer whale at a marine park in san antonio, texas, usa, died suddenly without notable premonitory signs. on gross examination, mild multifocal meningeal hyperemia and petechial parenchymal hemorrhage were noted in the right cerebrum and cerebellum. the left hemisphere of the brain appeared normal. focally extensive tan discoloration and fi brosis were present in the right accessory lung lobe with associated hemorrhage and congestion. both lung lobes were mildly and diffusely heavy and wet. all thoracic and abdominal lymph nodes were moderately enlarged and edematous. the second gastric chamber displayed numerous chronic and active ulcerations of . - cm. fresh and buffered % formalin-fi xed specimens were collected. fresh tissues were stored at - °c. tissues fi xed in % buffered formalin were processed routinely and stained with hematoxylin and eosin for histologic examination. histologic review demonstrated moderate multifocal subacute vasculitis and nonsuppurative encephalitis. infl ammatory lesions of the central nervous system were focused in gray matter of the medulla oblongata, pons, mesencephalan, and cerebellum. lesions were bilateral but more severe on the right side. meninges demonstrated moderate focally extensive and multifocal areas of acute meningeal congestion and hemorrhage. mild multifocal lymphocytic infi ltrates expanded the leptomeninges. blood vessels demonstrated mild to moderate acute necrosis and lymphocytic and contained plasmacytic and neutrophilic infi ltrates within vascular walls. encephalitis was characterized by perivascular lymphocytes and fewer plasma cells expanding the virchow-robbins spaces. small, scattered, perivascular ring hemorrhages were noted. a few multifocal loosely arranged glial nodules were within cerebral white matter. predominant lesions in the lungs were areas of chronic and active abscessation amid a focally extensive area of mixed infl ammation and fi brosis. there was moderate diffuse acute pulmonary edema and congestion. gastric ulcerations were present in the fi rst gastric chamber and were chronic and active. they were characterized by central ulcerations with necrosis and a mixed infl ammatory infi ltrate surrounded by variable fi brosis and a rim of epithelial hyperplasia. changes in spleen, lymph node, and kidney included acute edema, congestion, and vascular dilation. conventional diagnostic assays were performed for aerobic, anaerobic, and fungal microbes in liver, lung, kidney, cerebrospinal fl uid, and brain. all yielded minimal growth of escherichia coli. the fi nal diagnosis was fulminant peracute bacteremia and septicemia secondary to a primary viral infection associated with nonsuppurative encephalitis. published etiologic considerations for cetacean nonsuppurative encephalitis include morbillivirus and protozoal infections ( ) . a dna microarray with highly conserved sequences from > , viruses was selected to screen for known and novel viruses ( ). total rna was extracted from brain tissue and hybridized to a microarray as described ( ) . analysis of the resulting hybridization pattern demonstrated a strong hybridization signal to many oligonucleotide probes on the microarray from the family flaviviridae, in particular to wnv. consensus reverse transcription pcr primers ( ) targeting wnv were used to confi rm the microarray results. sequencing of the -bp amplicon (genbank accession no. hq ) yielded a sequence with % nt identity and % aa acid identity to wnv strain ok (genbank accession no. eu . ), a strain originally identifi ed in oklahoma, usa. to further support a wnv diagnosis, we performed immunohistochemical staining on brain tissue. the immunoperoxidase stain used was a commercial rabbit polyclonal antibody (biorelience corp., rockville, md, usa) with peroxidase-tagged goat antirabbit immunoglobulin g (dakocytomation, carpinteria, ca, usa) bridge and -amino- -ethylcarbazole (dakocytomation) as the chromogen. this staining demonstrated abundant wnv antigen within the cytoplasm of a small number of neurons and glial cells and in fewer macrophages in the brain tissue (figure) . we evaluated wnv exposure within the same cohort, as well as a geographically distant cohort of whales by using serologic testing. all testing was performed at the same laboratory by using a standard plaque-reduction neutralization test. in this assay, a % neutralization cutoff was used ( ) . a % plaque-reduction titer > was considered positive. serum from the affected whale and cohort killer whales from the same marine park in san antonio as well as whales housed at another facility in orlando, florida, usa, were evaluated. in each facility, the animals have regular contact with each other. the facilities are geographically separated so the animals do not have exposure to those in the other park. all animals from texas had % plaque-reduction titers > , ranging from to . the whales housed together in orlando had no measurable titer. we demonstrate that wnv can infect and cause disease in killer whales. these fi ndings broaden the known host tropism of wnv to include cetaceans in addition to previously known pinnipeds. although we cannot defi nitively attribute the cause of death of this whale to wnv, the observed lesions are consistent with those caused by wnv in other animals. the serologic results demonstrate that subclinical infections can occur and that exposure can be variable. we did not determine specifi c dates of exposure for these populations. both bexar county, texas, and orange county, florida, have had wnv in wildlife since . we continue annual serology on previously negative animals to document seroconversion. mosquito management practices are similar in both facilities and have been expanded since this diagnosis. differences in wnv prevalence or mosquito numbers may have played a role in the different serologic results. health evaluations of free-ranging and captive cetaceans should include wnv serology to assess exposure rates. this report focuses on killer whales, but the "loafi ng" behavior (stationary positioning at the water's surface) is commonly seen in many coastal dolphins, thereby increasing the likelihood of mosquito bites and exposure to wnv. serologic screening of bottlenose dolphins (tursiops truncatus) from the indian river lagoon demonstrated wnv titers ( ) . wnv-associated disease in these animals has not been reported. active screening for wnv may enhance diagnostic investigations. as with many species of birds and mammals, wnv infection carries a risk for zoonotic transmission. until the implications of this infection in marine mammals are better understood, biologists and veterinarians working with cetaceans should consider this possibility. potential viral shedding can occur through the oropharygeal cavity and feces as well as through blood and organs during necropsies. finally, our study demonstrates the broad applicability of using panviral microarray-based diagnostics. even though pcr diagnostics are well developed for wnv, the agent was not initially considered as a potential pathogen in this species. panviral microarray can be used not only to identify novel viruses but also to detect unsuspected agents. emerging infectious diseases • www.cdc.gov/eid • vol. , no. , august west nile fl avivirus polioencephalomyelitis in a harbor seal (phoca vitulina) crc handbook of marine mammal medicine viral discovery and sequence recovery using dna microarrays identifi cation of a novel coronavirus from a beluga whale using a pan-viral microarray universal rt-pcr diagnostic for arboviruses diagnostic procedures for viral, rickettsial and chlamydial infections serological evidence of exposure to selected viral, bacterial, and protozoal pathogens in free-ranging atlantic bottlenose dolphins (tursiops truncatus) from the indian river lagoon this work was funded by national institutes of health grant u ai .dr st. leger is the director of pathology and research for seaworld parks and entertainment, serves on the editorial board of the journal veterinary pathology, and is a board member of the cl davis foundation. her primary research interests are comparative pathology and infectious disease investigations of marine and aquatic animals. key: cord- -y zpvw authors: tan, kai sen; andiappan, anand kumar; lee, bernett; yan, yan; liu, jing; tang, see aik; lum, josephine; he, ting ting; ong, yew kwang; thong, mark; lim, hui fang; choi, hyung won; rotzschke, olaf; chow, vincent t; wang, de yun title: rna sequencing of h n influenza virus-infected human nasal epithelial cells from multiple subjects reveals molecular pathways associated with tissue injury and complications date: - - journal: cells doi: . /cells sha: doc_id: cord_uid: y zpvw the human nasal epithelium is the primary site of exposure to influenza virus, the initiator of host responses to influenza and the resultant pathologies. influenza virus may cause serious respiratory infection resulting in major complications, as well as severe impairment of the airways. here, we elucidated the global transcriptomic changes during h n infection of human nasal epithelial cells from multiple individuals. using rna sequencing, we characterized the differentially-expressed genes and pathways associated with changes occurring at the nasal epithelium following infection. we used in vitro differentiated human nasal epithelial cell culture model derived from seven different donors who had no concurrent history of viral infections. statistical analysis highlighted strong transcriptomic signatures significantly associated with and h after infection, but not at the earlier -h time point. in particular, we found that the influenza infection induced in the nasal epithelium early and altered responses in interferon gamma signaling, b-cell signaling, apoptosis, necrosis, smooth muscle proliferation, and metabolic alterations. these molecular events initiated at the infected nasal epithelium may potentially adversely impact the airway, and thus the genes we identified could serve as potential diagnostic biomarkers or therapeutic targets for influenza infection and associated disease management. the global burden of inter-pandemic influenza is high. it is estimated to affect billion people annually, with - million severe cases requiring hospitalization or intensive care treatment, resulting in approximately . million deaths [ ] . worryingly, drug-resistant influenza strains are emerging at a rapid rate that will severely hamper the ability of our healthcare systems to contain influenza outbreaks [ ] . therefore, alternative strategies are needed against severe influenza infections during both seasonal and pandemic influenza outbreaks. the normal human airway epithelium is a pseudo-stratified layer of ciliated and non-ciliated columnar cells, goblet cells, club cells, and basal cells [ ] . the airway epithelium protects against airway infection via efficient mucociliary clearance (mcc), the production of inflammatory mediators and chemokines against viruses, and the recruitment of immune cells [ ] . when the influenza virus breaches the defense of the human airway epithelium, it causes a myriad of innate responses by the infected host in response to viral invasion [ , ] . among these changes are critical factors that can determine disease severity, and which may lead to the development of diagnostic, prognostic prediction markers, or anti-influenza therapies [ ] [ ] [ ] [ ] . however, few studies have hitherto been performed in relevant models [ ] and human models of influenza are not feasible due to potential severity of the infection. therefore, the mechanistic study of viral-induced airway changes using relevant models can lead to better understanding of the development of severe complications. additionally, we need greater clarity on the different immune responses in view of the rising prevalence of chronic diseases such as diabetes mellitus and asthma. patients with these disorders are especially susceptible to severe influenza complications compared to healthy subjects [ ] . thus, the establishment of a baseline response against influenza infection of "healthy" tissue is beneficial to facilitate future comparative studies to better manage influenza in patients with co-morbidities. although the study of host responses in influenza infection is not new, current in vitro cell lines cannot accurately represent human airway infection due to the lack of key mucociliary features [ ] . hence, we have previously developed an air-liquid interface (ali) human airway epithelial cell culture that is able to sustain influenza infection [ , ] . we have also further compared the transcriptomic responses of our infected human nasal epithelial cells (hnecs) with other in vitro and in vivo influenza infection transcriptomic studies [ ] . the comparison revealed that at their peak responses against influenza, the differential transcriptome signature in hnecs was highly similar to the signatures from other influenza infection models [ ] . interestingly, compared to the homogenous cell lines tested, our heterogenous hnec model exhibited a more comparable response to the clinical influenza studies, indicating that most responses were initiated at the nasal epithelium [ ] . therefore, in this study, we aim to further utilize the hnec model as a physiologically relevant in vitro model to clarify the nasal epithelial responses against influenza h n infection, which would then facilitate the identification of the key host factors that are significant for future studies. to establish host factors that are significantly altered in the nasal epithelium as a reference of early innate responses against influenza, the dynamic expression of the genes needs to be clearly elucidated. while there are many studies that utilize microarray analysis to identify the host responses against influenza, the limitation of the microarray is its inability to determine the full extent of gene changes due to its hybridization-based protocol [ ] . the aim of this study was to utilize rna sequencing (rnaseq) technology to not only reveal the hnec responses (from multiple individuals) against influenza infection, but also to identify those genes with high magnitude changes to serve as potential reference markers of the innate responses of influenza infection. given that rnaseq functions by reading virtually all the rnas present in the samples tested, we can also discern the magnitude of each rna change and mark them as the canonical responses. in addition, as rnaseq is not constrained by probe usage as in microarrays, they are therefore more reliable in detecting novel interactions during influenza infections of hnecs. hence, rnaseq analysis will further augment the transcriptomic data established previously by microarray analysis. the augmented baseline can then be applied to future clinical studies and practice against influenza infection, especially for comparison against patients with other underlying co-morbidities that may be affected by more severe disease. approval to conduct this study was obtained from the national healthcare group domain-specific board of singapore (dsrb ref: d/ / ) and the institutional review board of the national university of singapore (irb ref: . written consent was obtained from donors prior to the collection of the tissue biopsies. at the time of collection, all subjects were free of symptoms of urti. the medical backgrounds of the subjects are summarized intable s . the hnespcs were isolated and enriched from the tissue biopsies according to a previously standardized protocol [ , ] , which normalized the hnespcs to a baseline state that differentiates into hnecs resembling healthy tissues if they pass the quality control checks for their differentiation [ ] . following enrichment, the hnespcs were expanded further and subjected to ali culture in transwells for in vitro differentiation according to previous protocol as well [ , ] . briefly, primary cells were subjected to isolation for selection of hnespcs, which were enriched and expanded with dulbecco's modified eagle medium: nutrient mixture f- (dmem/f ) (gibco-invitrogen, carlsbad, ca, usa) containing ng/ml of human epithelial growth factor (egf, gibco-invitrogen, carlsbad, ca, usa), µg/ml of insulin (sigma, st. louis, mo, usa), . nm of cholera toxin (sigma, st. louis, mo, usa), . µg/ml of hydrocortisone (sigma, st. louis, mo, usa), nm/ml of , , -triiodo-l-thyronine (t ) (sigma, st. louis, mo, usa), µl/ml of an n- supplement (gibco-invitrogen) and iu/ml of antibiotic-antimycotic (gibco-invitrogen, carlsbad, ca, usa). the expanded hnespcs were then transferred onto -well . µm transwell inserts (corning, corning, ny, usa). once confluent, growth medium was discarded and µl of pneumacult™-ali medium with inducer supplements (stemcell technologies inc., vancouver, canada) was added to the basal chamber to establish ali conditions. the cells were cultured in ali culture for weeks, with media change every - days. after - weeks of differentiation, hnecs from a total of seven donors were then subjected to influenza h n virus infection. the influenza a strain used in this study is of the h n subtype (a/aichi/ / ) (atcc, manassas, va, usa). the virus was propagated using embryonated egg culture and used for all the infection in the hnecs. prior to infection, fully differentiated hnecs were washed with × dpbs and infected with the h n influenza virus at a multiplicity of infection (moi) of . and incubated for h at • c. after the h incubation, the viral inoculum was removed and the hnecs were incubated back in • c. the control hnecs were harvested for apical wash and rna prior to the infection at h post-infection (hpi). the infected hnecs were then harvested for the apical wash and rna following , , and hpi incubation at • c. at each infection time point, µl of x dpbs was added and incubated in the apical chamber for min at • c to recover progeny viruses as the apical wash. the plaque assay for viral quantification was performed using overnight mdck cultures (atcc, manassas, va, usa) at - % confluence in -well plates. the mdck cells were incubated with µl of serial dilutions (from − to − ) of virus from apical washes at • c for h, where plates were rocked every min to ensure equal viral distribution. after incubation, the inocula were removed and replaced with ml of avicel (fmc biopolymer, philadelphia, pa, usa) overlay, and incubated at • c for - h. after incubation, avicel overlay were removed, and cells were fixed with % formaldehyde in × pbs for h. formaldehyde was then removed, and cells were washed with × pbs prior to staining with % crystal violet for min before washing the stain away. the plaque-forming units (pfu) were calculated as follows: number of plaques × dilution factor = number of pfu per µl. at each time point after the collection of apical wash, the hnecs were lysed using rna lysis buffer. total rna was then extracted from the lysate using mirvana mirna isolation kit (life technologies, grand island, ny, usa). the extracted total rna was first subjected to nanodrop analysis to first ensure the rna quality, before being submitted for rnaseq analysis. then, ng from the remaining rna was subjected to cdna synthesis using maxima first-strand cdna synthesis kit (thermoscientific, pittsburgh pa, usa). after this, qpcr analysis was performed to evaluate the transcriptional levels of host response genes selected based on previous microarray analysis using pre-designed primers (sigma aldrich). each qpcr reaction was performed in duplicate using gotaq-qpcr master mix kit (promega, san luis obispo, ca, usa), and relative gene expression was calculated using the comparative method of -∆∆ct normalized to the housekeeping gene pgk . relative gene expression levels were presented as median values and interquartile ranges, while statistical significance was determined using the wilcoxon signed-rank test. all human rnas were analyzed on the agilent bioanalyzer (agilent, santa clara, ca, usa) or the perkin elmer labchip gx system (perkin elmer, waltham, ma, usa) for quality assessment with rna integrity number (rin) or rna quality score range from . - . and median of . . cdna libraries were prepared using ng of total rna and µl of a : , dilution of ercc rna spike in controls (ambion ® thermo fisher scientific, waltham, ma, usa) using smartseq v protocol [ ] , except for the following modifications: fastq files were mapped to the human genome build hg using star. gene counts were computed using featurecounts (part of the subread package) using annotations from gencode version . differential gene expression analysis was performed using edger in a paired fashion under r version . . . multiple testing correction was done using the method of benjamini and hochberg and p-values (false discovery rate; fdr) less than . was deemed to be significant. geneset enrichment analysis using data from gene ontology (go) was performed using the bioconductor package topgo, while the analysis using reactome pathway was performed using the vioconductor package reactomepa. both analyses were run in r version . . using multiple testing-corrected significant differentially-expressed genes. tnf-α, tnf-β, vegf, eotaxin/ccl , and pdgf-aa. samples and standards were incubated with fluorescent-coded magnetic beads which had been pre-coated with the respective capture antibodies. after an overnight incubation at • c, plates were washed twice. biotinylated detection antibodies were incubated with the complex for h, and streptavidin-pe was then added and incubated for another min. plates were washed twice again, then beads were re-suspended with sheath fluid before acquiring on the flexmap ® d (luminex) using xponent ® . (luminex) acquisition software. data analysis was done on bio-plex manager™ . . (bio-rad). standard curves were generated with a -pl ( -parameter logistic) algorithm, reporting values for both mfi and concentration data. results were then expressed as mean fold change compared with uninfected control, and p-values (fdr) of less than . were considered significant. prior to analysis, the responses of all seven hnecs donors following influenza infection were plotted on a principal component analysis (pca) plot. the pca plot indicated a degree of variability in the responses between donors and time points (figure ). nonetheless, the responses were clustered tightly enough following infection to signify their consistency of infection for further transcriptomic analysis-similar to those observed in our previous study [ ] . the respective capture antibodies. after an overnight incubation at °c, plates were washed twice. biotinylated detection antibodies were incubated with the complex for h, and streptavidin-pe was then added and incubated for another min. plates were washed twice again, then beads were resuspended with sheath fluid before acquiring on the flexmap ® d (luminex) using xponent ® . (luminex) acquisition software. data analysis was done on bio-plex manager™ . . (bio-rad). standard curves were generated with a -pl ( -parameter logistic) algorithm, reporting values for both mfi and concentration data. results were then expressed as mean fold change compared with uninfected control, and p-values (fdr) of less than . were considered significant. prior to analysis, the responses of all seven hnecs donors following influenza infection were plotted on a principal component analysis (pca) plot. the pca plot indicated a degree of variability in the responses between donors and time points ( figure ). nonetheless, the responses were clustered tightly enough following infection to signify their consistency of infection for further transcriptomic analysis-similar to those observed in our previous study [ ] . significant gene expression changes (fdr < . ) of infected hnecs were detected as early as hpi, and further increased at and hpi (table ; figure a ). also, the number of genes decreased in a linear fashion as the fold change in expression increased, as seen in the x fold change genes significant gene expression changes (fdr < . ) of infected hnecs were detected as early as hpi, and further increased at and hpi (table ; figure a ). also, the number of genes decreased in a linear fashion as the fold change in expression increased, as seen in the x fold change genes indicated in figure b , where about % of the significantly altered genes remained. at hpi, there were upregulated genes and downregulated genes. the major upregulated genes were the antiviral sensors and early response genes such as ifns, ifits, and ifis. interestingly enough, interferon lambda (ifnλ) gene ifnls was the earliest response interferon of infected hnecs, as opposed to interferons alpha or beta, at hpi. at later time points, the number of gene expression changes increased substantially, with upregulation of and genes, and downregulation of and genes at and hpi, respectively. there was augmented expression of antiviral effectors and inflammatory genes at both time points. ifnl remained the interferon gene with highest expression at both time points, while a marked elevation of cytokines such as cxcl and cxcl was also observed. considering downregulated genes, proliferative and transcriptomic functions appeared to be suppressed, with diminished expression of genes such as fmo , klk , and fosb. genes associated with metabolism, cell cycle, and dna repair were further suppressed following infection at and hpi. tables s -s list the complete set of significant gene expression changes, arranged according to their fold change (log fc). in addition, we have also verified that the genes showing major expression changes by rnaseq generally concurred with rt-qpcr analyses. of the genes tested by qpcr at hpi, all of them showed the same directional changes in expression as observed by rnaseq. hence, seven of these genes showed a p-value of < . (il i , ifnl (il ), cxcl , tnfsf , ifi , ccl , and cyp a ), one gene had a p-value of < . (ctgf), while only two genes were not statistically significant (tgfa and ano ) ( figure s ). indicated in figure b , where about % of the significantly altered genes remained. at hpi, there were upregulated genes and downregulated genes. the major upregulated genes were the antiviral sensors and early response genes such as ifns, ifits, and ifis. interestingly enough, interferon lambda (ifnλ) gene ifnls was the earliest response interferon of infected hnecs, as opposed to interferons alpha or beta, at hpi. at later time points, the number of gene expression changes increased substantially, with upregulation of and genes, and downregulation of and genes at and hpi, respectively. there was augmented expression of antiviral effectors and inflammatory genes at both time points. ifnl remained the interferon gene with highest expression at both time points, while a marked elevation of cytokines such as cxcl and cxcl was also observed. considering downregulated genes, proliferative and transcriptomic functions appeared to be suppressed, with diminished expression of genes such as fmo , klk , and fosb. genes associated with metabolism, cell cycle, and dna repair were further suppressed following infection at and hpi. tables s - list the complete set of significant gene expression changes, arranged according to their fold change (log fc). in addition, we have also verified that the genes showing major expression changes by rnaseq generally concurred with rt-qpcr analyses. of the genes tested by qpcr at hpi, all of them showed the same directional changes in expression as observed by rnaseq. hence, seven of these genes showed a p-value of < . (il i , ifnl (il ), cxcl , tnfsf , ifi , ccl , and cyp a ), one gene had a p-value of < . (ctgf), while only two genes were not statistically significant (tgfa and ano ) ( figure s ). we then further compared the transcriptomic alterations in the hnecs over time, following influenza h n infection. the number of gene expression changes mirrored the viral titer changes, which peaked at hpi, and were consistent between donors ( figure a ). approximately two thirds of genes at and hpi overlapped with other time points, while about one third of genes at hpi overlapped ( figure b ). the overlapping genes displayed similar directional consistency at the significant time points. in addition, congruent with the consistent viral titer with most gene expression changes at hpi, we also noted the most consistent alterations in expression of genes across donors. this is highlighted in figure c , which portrays the heatmaps of the top genes with the smallest p-value, together with their direction and magnitude of change. based on these analyses, we proposed that hpi represents the optimal time point for the subsequent pathway analysis to ascertain influenza-specific pathway changes. we then further compared the transcriptomic alterations in the hnecs over time, following influenza h n infection. the number of gene expression changes mirrored the viral titer changes, which peaked at hpi, and were consistent between donors ( figure a ). approximately two thirds of genes at and hpi overlapped with other time points, while about one third of genes at hpi overlapped ( figure b ). the overlapping genes displayed similar directional consistency at the significant time points. in addition, congruent with the consistent viral titer with most gene expression changes at hpi, we also noted the most consistent alterations in expression of genes across donors. this is highlighted in figure c , which portrays the heatmaps of the top genes with the smallest p-value, together with their direction and magnitude of change. based on these analyses, we proposed that hpi represents the optimal time point for the subsequent pathway analysis to ascertain influenza-specific pathway changes. we then further subjected the significant gene changes to gene set enrichment using both go and reactome databases. at time points , , and hpi, there were , , and significant (adjusted p-value < . ) go biological processes (table s ) and , , and significant (adjusted pvalue < . ) reactome pathways (table s ) , respectively. at the early time of hpi, interferon- we then further subjected the significant gene changes to gene set enrichment using both go and reactome databases. at time points , , and hpi, there were , , and significant (adjusted p-value < . ) go biological processes (table s ) and , , and significant (adjusted p-value < . ) reactome pathways (table s ) , respectively. at the early time of hpi, interferon-mediated antiviral responses were elevated as expected. at hpi, the pathways appeared to be more stabilized and consistent for both go and reactome analyses, despite displaying more gene expression changes at this time point. responses to influenza virus skewing towards type i immunity were predominant in the go analysis. the expected interferon-mediated functions by the epithelium validated the authenticity of our model, where we found enriched type i interferon (go) and rig-i (reactome) pathways with upregulation of nearly all significant gene members (data not shown). besides the interferon and antiviral pathways, we identified several functions of interest initiated by the nasal epithelium that may contribute to the pathology and pathogenesis of influenza. at hpi, go pathway enrichment analysis revealed that the nasal epithelium was actively involved in initial ifnγ signaling (go: ), despite not directly producing ifnγ. we also observed enriched function in apoptosis and necroptosis (go: and go: ), immune evasion (go: ), and other pathways that may lead to complication events such as smooth muscle proliferation (go: ) and response to fatty acid (go: ) ( table ). for the reactome pathway analysis, we selected pathways that were enriched with more than significant genes present in the enriched pathway, and these were generally in agreement with the go analysis (table ). in addition to ifnγ signaling ( ) and apoptosis ( ), it also revealed changes in epithelial-initiated b cell receptor signaling ( and ) and amino acid metabolism ( ) following influenza infection. it is noteworthy that these pathways were initiated at the epithelial level without the participation of immune cells, thus highlighting the relevant genes of interest for future studies. given that rnaseq analysis facilitates more accurate expression changes following infection compared to hybridization technology such as microarray, we conducted further analysis on the levels of gene expression changes to enable more stringent and accurate transcriptomic analyses for future studies. by comparing these results to a previous study that identified influenza-specific signatures, we verified that these genes were all expressed in infected nasal epithelium later at hpi, but not at hpi. furthermore, at both and hpi, all but one of the gene signatures exhibited elevated expression of > . -fold change (> . log fc) compared to uninfected control hnecs (table ). when we applied the higher fold change cutoff, the number of significant genes decreased by approximately % (figure a) , which was also congruent with the linear association observed earlier. therefore, future studies on early transcriptional alterations could consider adopting the . -fold change in expression as a more stringent threshold, which may be more feasible, especially for large transcriptomic studies that yield large numbers of data points. in addition, when compared to the previous microarray study on a similar hnec model [ ] , both rnaseq and microarray shared a high degree of overlap, with about one third and half of total genes from rnaseq and microarray overlapping, respectively ( figure b ). the overlap was generally observed in genes with highly altered expression, such as cxcl , cxcl , and rsad , which were changed to a similar magnitude in both rnaseq and microarray (table s ). when we compared the influenza signature genes, rnaseq revealed a more consistent increase in magnitude, i.e., at hpi, the magnitude of the gene change was generally higher than that of the microarray (table ). in addition, rnaseq was also able to detect novel genes with expression changes of high magnitude that were generally higher than those found by microarray only ( genes versus genes with elevated expression greater than . -fold). genes such as heatr , pdcd , il i , art , and kcnh were altered to a higher magnitude than the . -fold threshold. hence, rnaseq-based transcriptomic analysis may augment transcriptomic findings to identify novel gene responses against influenza in the future. from rnaseq and microarray overlapping, respectively ( figure b ). the overlap was generally observed in genes with highly altered expression, such as cxcl , cxcl , and rsad , which were changed to a similar magnitude in both rnaseq and microarray (table s ). when we compared the influenza signature genes, rnaseq revealed a more consistent increase in magnitude, i.e., at hpi, the magnitude of the gene change was generally higher than that of the microarray (table ). in addition, rnaseq was also able to detect novel genes with expression changes of high magnitude that were generally higher than those found by microarray only ( genes versus genes with elevated expression greater than . -fold). genes such as heatr , pdcd , il i , art , and kcnh were altered to a higher magnitude than the . -fold threshold. hence, rnaseq-based transcriptomic analysis may augment transcriptomic findings to identify novel gene responses against influenza in the future. after deriving the transcriptomes by rnaseq, we then further investigated whether the changes in expression of genes resulted in alterations in secretory cytokines and chemokines early in the infection of hnecs. initially, we detected significant reductions in multiple cytokines at hpi, with the exception of il- which was increased ( figure s ). this may reflect the initial immune suppression during influenza infection. however, at and hpi, less significant changes were observed, i.e., only increase in tnf-a and decrease in mdc and pdgf-aa were noted at hpi. this was followed by increase in ip- (cxcl ) and tgf-a and decrease in pdgf-aa seen at hpi. this analysis highlights changes in ip- , tgf-a, and pdgf-aa to be significant early responses in secretory cytokines/chemokines following influenza infection. our study has identified epithelium-initiated host responses which are found to be involved in both innate and adaptive responses. the finding is significant as we can now focus on the primary point of contact of influenza-the nasal epithelium in the study of early host responses for identifying host factors that can be utilized for diagnostic and therapeutic purposes [ ] . in addition, our study also showed that it is important for reference databases to use relevant human models like the hnecs model, which contains the mucociliary component of the airways, in order to provide closely representative host responses. while there exists a high number of microarray studies that showed the host responses using similar hnec models, there are only a small number of equivalent rnaseq studies. compared to microarrays, rnaseq analysis can provide a more comprehensive picture of the transcriptomic landscape, and is not limited by the hybrid library variant and concentrations [ ] . hence, in order to derive accurate magnitude of gene expression changes, we performed an rnaseq analysis of h n infection using the hnecs model. h n influenza virus was selected, given that it is a major circulating subtype over long periods of time. in addition, relatively lower efficacy of vaccines against this subtype prompted us to study its interactions with the primary host target to elucidate the immune responses and association with adaptive immunity [ , ] . this model has been previously evaluated to be a highly clinically-relevant model that can facilitate controlled infection of nasal cells from multiple individuals. in addition, we have also previously shown-by microarray analysis-that the nasal epithelium is responsible for the initiation of host responses following influenza infection [ ] . this renders the hnecs to serve as a valuable tool to analyze transcriptomics from different individuals infected under the same conditions to ensure consistent and relevant responses in humans. once the magnitude of gene expression changes was considered, several interesting findings emerged. firstly, the infected hnecs were observed with strong activation of antiviral genes and early inflammatory genes leading to type i immune responses. a large number of gene expression changes were of magnitude of over -fold difference (log to log fold change). most of the genes with high-magnitude expression changes were verified by qpcr, with statistical significance congruent with the rnaseq analysis. secondly, despite the absence of immune cells, the infected hnecs were able to generate strong type i responses that may likely aid the recruitment of cytotoxic cells to clear the infected cells. thirdly, in early responses of the hnecs, ifnλ genes, which represent type iii interferons, were more strongly induced than the more frequently observed type i interferons (ifnα and ifnβ), while type ii interferons were not produced by hnecs, in agreement with previous studies [ , ] . the induction of type iii interferons may reflect an important event within the hnecs where ifnλ, the initial responders against the infection, may be more beneficial in the antiviral response [ , ] . moreover, we also observed notable suppression of expression of certain genes following influenza infection, including suppression of proliferation and dna repair genes, which may contribute to the pathology and pathogenesis of influenza [ ] . finally, rnaseq also unraveled expression changes of certain newly-discovered genes in response to influenza infection of the upper airway cells. genes such as heatr [ ] , il i [ ] , tnfsf b (baff) [ ] , and pdcd (pd- ) [ ] are recently implicated in influenza pathogenesis and mucosal defense, thereby signifying the role of the nasal epithelium against influenza infection. furthermore, rnaseq identified altered expression of art and kcnh genes that were not previously detected in influenza transcriptomes. these findings hence further reiterate the value of rnaseq in enhancing data on influenza transcriptomes for reference in future studies. via pathway enrichment analysis, we have identified known antiviral pathways to validate the hnecs responses against influenza. in addition, we have also documented the potential pathways initiated by the nasal epithelium that may contribute to influenza pathogenesis as represented by the gene expression changes listed in tables and . by analyses using literature-inferred go and reactome databases, we have demonstrated that the nasal epithelium can play a role in the main antiviral signaling, i.e., ifnγ responses despite not being a direct producer of ifnγ. the pathway enrichment indicated that hnecs may serve as important regulators of type ii interferons. even though the effects of ifnγ are vital to the robust clearance of influenza viruses [ ] , there are reports of unregulated ifnγ being a contributor to inflammatory damage [ , ] . therefore, the over-production of ifnγ response factors such as icam and cd may contribute to inflammatory damage of the epithelium. hence, production of factors such as stat [ ] by the hnecs is also crucial in ensuring appropriate regulation of ifnγ-mediated expression of influenza response genes to modulate inflammation and to minimize damage. the primary contact of influenza virus with the nasal epithelium may subsequently lead to damage to the airway epithelium as well. this is apparent with the clear enrichment of the pathways of apoptosis, mitochondrial apoptotic processes, and necroptosis that contribute to cell death and mechanical barrier loss during infection [ , ] . genes such as ifi , bak , caps , tnfsf , and fas suggest active apoptotic cell death that not only destroys cells in the epithelial barrier, but may also serve to propagate the virus and to perpetuate the damage [ ] [ ] [ ] . furthermore, during virus infection, aberrant regulation of apoptosis may also lead to further injury to the epithelium and surrounding tissues [ ] . on the other hand, necroptosis pathways have also been observed to be enriched in influenza-infected hnecs. compared to apoptosis, the study of necroptosis in influenza infection is relatively new with contradicting findings [ ] . ripk /necroptosis studies appear to generate contradictory results as to whether necroptosis protects against or is detrimental during influenza infection [ , ] . hence, its increased expression during infection of hnecs warrants further investigation on its role in influenza-induced damage. in addition, we also noted enrichment of b-cell signaling pathways by the infected hnecs which may be vital for b-cell responses during the adaptive immune response [ ] . we noted that most genes enriched in the b-cell pathways were related to antigen recognition such as proteasome subunits (psme , psmb , psma , etc.) and b-cell receptor-associated genes such as dapp and card [ ] . however, changes in expression of certain growth factors (including ereg and fgfs) following influenza infection may lead to complications involving airway remodeling and recruitment [ , ] . further, the effects of the growth factors were further confirmed by the enrichment of pathways related to the proliferation of smooth muscle cells also induced by the infection. changes to airway smooth muscle cells are usually implicated in airway remodeling [ ] [ ] [ ] , and may also contribute to post-influenza complications. hence, the genes found in this study may be crucial for elucidating the nasal-initiated responses that may contribute to the pathology and pathogenesis of influenza infection of the airways. finally, another interesting pathway that may contribute to epithelial damage is the negative regulation of innate immune responses. these genes may serve as proviral factors and aid in immune evasion. for example, adar is a proviral factor that works in synergy with influenza ns to enhance viral replication [ ] . trafd is a negative regulator of toll-like receptor signaling which is upregulated in influenza-infected hnecs [ ] . dhx is a negative regulator of rig-i/mda signaling pathway [ ] . ceacam is involved in regulation of liver inflammation [ ] and its expression appears to exert antiviral effects on influenza virus [ ] . nmi binds to influenza virus ns and inhibits irf -mediated interferon signaling [ , ] . therefore, aberrant expression of genes in this signaling pathway may directly contribute to immune evasion of influenza, culminating in viral propagation and increased epithelial damage. we summarized the identified pathways (listed in tables and ) that alluded to immune evasion (negative regulation of innate immune responses), antigen processing (metabolism of amino acids and derivatives), and immunomodulation (interferon gamma signaling, b cell receptor signaling, and response to fatty acid) that may contribute to severity of influenza. there was evidence of direct pathway enrichment of potential influenza evasion strategies and/or immunomodulation with accompanying transcriptomic changes. the genes in the pathway may be analyzed for their immunomodulatory activity and whether their expression is beneficial to the virus (immune evasion) or the host (preventing cytokine storm). in addition, the infected hnecs also revealed modified responses associated with fatty acid, with many lipid signaling molecules such as leukotrienes that mediate antiviral responses and subsequent inflammation of the airway [ , ] . such modified responses may also determine the afforded in the airway and the severity of airway inflammation and damage. in addition, the modification may also affect the lower airway responses to inflammatory mediators; hence, the changes in these pathways may also suggest a potential mechanistic link to the pathogenesis of viral-induced exacerbation of chronic inflammatory diseases. lastly, we also noted enrichment of pathways related to amino acid metabolism, which is important in antigen processing and proteasomal degradation of foreign protein. the changes in these genes at the hnecs, the target site of influenza infection, may determine the effectiveness of antiviral responses mounted and may therefore influenza disease severity. in addition, we also compared our rnaseq analysis against previously reported influenza-specific signatures in order to improve future transcriptomic analysis [ ] . in vitro transcriptomic analysis yields a large number of differentially-expressed genes that would require additional criteria to identify functionally significant genes. by means of this comparison, we discovered that almost all influenza-specific signatures exhibited differences in expression of above . -fold. hence, we propose applying fold change of > . as a threshold for future in vitro transcriptomic systems analyses, in order to increase the stringency in detecting functionally significant gene changes. finally, we observed that, unlike the transcriptome, there were notably fewer cytokines that were readily secreted during the acute phase of infection. expression of cytokines was reduced at hpi, except for il- , which interestingly is implicated in influenza-induced acute lung injury [ ] . even fewer cytokines showed altered expression at later time points. among them, only tgf-a, ip- (cxcl ), and pdgf-aa were significantly altered at and hpi. these may be significant markers that can be detected in the secretion of influenza-infected mucosal surface that may influence the severity of influenza. ip- is a well-established ifnγ response gene, and serves as a useful marker for response against influenza [ , ] . tgf-a represents an important factor involved in the secretion of il- in response to influenza, and may determine the early appropriate innate responses to prevent severe disease [ ] . on the other hand, it is also involved in pulmonary fibrosis as a ligand of epidermal growth factor receptor (egfr) and may contribute to complications in the lower airway [ ] . pdgf-aa was found to be elevated in the cerebrospinal fluid of influenza-associated encephalopathy [ ] , but was consistently reduced in hnec secretory fluid, thus warranting further investigation into its role in the infected nasal mucosa. the establishment of a reference transcriptome based on early responses of the human nasal epithelium model serves a key role in research on critical host factors involved in influenza. as the primary host contact with the virus, not only are immune responses against influenza important, but also the alterations in non-immune functions such as metabolism, cell content, and cell cycle, which may contribute to disease severity. in terms of translational potential, the model system identified gene expression changes of significant magnitude and pathways that impact responses against influenza and its severity. these genes may represent novel targets for future diagnostic and therapeutic development. under controlled conditions, the hnecs clinically establish the baseline for "normal" innate immune responses of the nasal epithelium against influenza viral infection. such a baseline can be particularly crucial when studying the changes in innate immune responses against influenza, especially in patients with underlying chronic diseases who may have aberrant airway responses against influenza. their antiviral responses may differ from "normal" subjects, and this study thus provides the basis for comparing the differential responses that culminate in more severe infections in patients with co-morbidities such as diabetes and chronic airway inflammatory diseases. such comparative clinical studies can potentially enhance the management of influenza viral infection in patients with chronic diseases. in conclusion, rnaseq technology allowed us to accurately quantify the magnitude of gene expression changes, as well as the relevant enriched pathways during h n influenza virus infection of hnecs, which can serve as a baseline for future clinical studies. the establishment of this baseline under controlled condition elucidated the antiviral innate response by the infected nasal epithelium, and highlighted the molecular factors and abnormalities in the upper airway that may contribute to influenza severity. furthermore, this study also culminated in the identification of novel gene signatures and host factors that may be harnessed for future research to develop influenza diagnostic markers and therapeutic targets. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s , figure s . real-time quantitative pcr validation of genes selected from rnaseq analyses. pcr data are expressed as log fold change using median and interquartile range. statistical significance was determined using wilcoxon signed-rank test. * p < . , # p < . ; figure s . luminex assay of secreted cytokines/chemokines in the apical supernatant. luminex data are expressed as mean fold change from uninfected control. statistical significance was determined using fdr. * p < . ; table s . information of seven donors of hnecs; table s . significant enriched pathways based on reactome pathway database analysis; table s . list of significant genes with altered expression at hpi of influenza h n infection of hnecs analyzed by rnaseq and microarray [ ] . oseltamivir resistance-disabling our influenza defenses role of il- ralpha in modulating il- -induced muc ac and ciliary changes in healthy and crswnp mucosa epithelial damage and response human nasal epithelial cells derived from multiple individuals exhibit differential responses to h n influenza virus infection in vitro comparative transcriptomic and metagenomic analyses of influenza virus-infected nasal epithelial cells from multiple individuals reveal specific nasal-initiated signatures systems-biology approaches to discover anti-viral effectors of the human innate immune response uncovering the global host cell requirements for influenza virus replication via rnai screening. microbes infect cellular networks involved in the influenza virus life cycle cd , a novel host factor of nuclear export signaling in influenza virus infection propagation of respiratory viruses in human airway epithelia reveals persistent virus-specific signatures distinction between rhinovirus-induced acute asthma and asthma-augmented influenza infection a novel three-dimensional cell culture method enhances antiviral drug screening in primary human cells comparing bioinformatic gene expression profiling methods: microarray and rna-seq the use of nasal epithelial stem/progenitor cells to produce functioning ciliated cells in vitro full-length rna-seq from single cells using smart-seq predicting clinical severity based on substitutions near epitope a of influenza a/h n effectiveness of seasonal influenza vaccinations against laboratory-confirmed influenza-associated infections among singapore military personnel in - . influenza other respir in vitro model of fully differentiated human nasal epithelial cells infected with rhinovirus reveals epithelium-initiated immune responses ifnlambda is a potent anti-influenza therapeutic without the inflammatory side effects of ifnalpha treatment interferon-lambda mediates non-redundant front-line antiviral protection against influenza virus infection without compromising host fitness influenza infection induces host dna damage and dynamic dna damage responses during tissue regeneration heatr is upregulated during influenza virus infection in lung alveolar epithelial cells the il i enzyme: a new player in the immunosuppressive tumor microenvironment. cells cigarette smoke inhibits baff expression and mucosal immunoglobulin a responses in the lung during influenza virus infection highly pathological influenza a virus infection is associated with augmented expression of pd- by functionally compromised virus-specific cd + t cells new fronts emerge in the influenza cytokine storm inflammatory impact of ifn-gamma in cd + t cell-mediated lung injury is mediated by both stat -dependent and -independent pathways production of interferon-gamma by influenza hemagglutinin-specific cd effector t cells influences the development of pulmonary immunopathology h n influenza virus infection enhances oncostatin m expression in human nasal epithelium nf-kappab-dependent induction of tumor necrosis factor fas/fasl is crucial for efficient influenza virus propagation nucleoprotein of influenza a virus negatively impacts antiapoptotic protein api to enhance e f -dependent apoptosis and virus replication influenza a virus enhances its propagation through the modulation of annexin-a dependent endosomal trafficking and apoptosis programmed cell death in the pathogenesis of influenza dai senses influenza a virus genomic rna and activates ripk -dependent cell death zbp /dai is an innate sensor of influenza virus triggering the nlrp inflammasome and programmed cell death pathways the multifaceted b cell response to influenza virus influenza virus-induced type i interferon leads to polyclonal b-cell activation but does not break down b-cell tolerance neutrophils induce smooth muscle hyperplasia via neutrophil elastase-induced fgf- in a mouse model of asthma with mixed inflammation respiratory syncytial virus infection provokes airway remodelling in allergen-exposed mice in absence of prior allergen sensitization cd , a laminin receptor showing increased expression in asthma, contributes to airway hyperresponsiveness through calcium signaling regulation of human airway smooth muscle cell migration and relevance to asthma airway smooth muscle in asthma: phenotype plasticity and function. pulm pharm the interactomes of influenza virus ns and ns proteins identify new host factors and provide insights for adar playing a supportive role in virus replication yoshimura, a. fln , a novel interferon-and lps-inducible gene acting as a negative regulator of toll-like receptor signaling rna-and virus-independent inhibition of antiviral signaling by rna helicase lgp ceacam in liver injury, metabolic and immune regulation ceacam -mediated inhibition of virus production subcellular proteomic analysis of human host cells infected with h n swine influenza virus negative regulation of nmi on virus-triggered type i ifn production by targeting irf mast cells and influenza a virus: association with allergic responses and beyond leukotriene b enhances nod -dependent innate response against influenza virus infection multi-cohort analysis identifies conserved transcriptional signatures across multiple respiratory viruses interleukin- is critical in the pathogenesis of influenza a virus-induced acute lung injury influenza induces il- and gm-csf secretion by human alveolar epithelial cells through hgf/c-met and tgf-alpha/egfr signaling overactive epidermal growth factor receptor signaling leads to increased fibrosis after severe acute respiratory syndrome coronavirus infection vascular endothelial growth factor (vegf) and platelet-derived growth factor (pdgf) levels in the cerebrospinal fluid of children with influenza-associated encephalopathy this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we thank the surgeons and staff in the department of otolaryngology, national university hospital, singapore. we thank h.h. ong and t.t. he for the subject selection and recording. we thank m.c. phoon and s.h. lau for technical assistance in viral experiments. the authors would like to acknowledge the staff of the immunomonitoring platform at sign. the authors declare no conflicts of interest. key: cord- - rzi x authors: boyd, rhea w; krieger, nancy; jones, camara phyllis title: in the us election, we can choose a just future date: - - journal: lancet doi: . /s - ( ) - sha: doc_id: cord_uid: rzi x nan when us voters go to the polls (what few are left) or attempt to mail our ballots (with what remains of the us postal service) in the election, we are not simply choosing between two parties or posturing about partisan politics. we are making a choice about the future. on the one hand, are the dangers of white supremacy, authoritarianism, and nationalism-lethal threats to our democracy, our lives, and the viability of the planet. on the other hand, is a rebuke of racist, autocratic politics, and the mandate to create a more equal, just, healthy, and habitable nation and world. the choice is stark and the stakes are high. in terms of health, the current us administration has intentionally lied about the grave risks of covid- , failed to implement a coherent national pandemic strategy, hamstrung and underfunded public health agencies, initiated the process to withdraw the usa from who, reversed and weakened health regulations, attacked abortion and contraception access, eroded transgender health protections, and aired racist, anti-asian, antiscience views. - furthermore, the us administration under president donald trump has cut food stamps, denied climate change, jeopardised the decennial census, undermined indigenous sovereignty, imposed discriminatory anti-muslim travel bans, unleashed federal force on peaceful protesters, openly appealed to white supremacists, and separated immigrant families and caged their children. - while black and indigenous communities, and people of colour more generally, are made vulnerable under the current us administration (panel), no one is fully immune to its harms. ultimately, unfettered racism, truncated rights, anaemic protections, and the resource inequities these exposures create shorten lives. anyone who doubts this needs to only consider the past months. home to only % of the world's population, the usa accounts for about a fifth of global covid- deaths. since february, , we have buried more than of our beloved neighbours, co-workers, family members, and friends. the age-adjusted covid- mortality rate among black and indigenous communities and people of colour in the usa is up to three times higher than among non-hispanic white populations. latinx and black children account for an astounding % of covid- deaths among people aged years and younger in the usa. as of oct , , more than non-hispanic white people have also died from covid- in the country. and every untimely death has occurred within the nation that spends more money on health care than any other country in the world. more exposure and less protection-eg, from racism, resource inequities, inadequate housing, and occupational and environmental hazards-not genetic differences, drive premature mortality from covid- and chronic illnesses such as heart disease and cancer. [ ] [ ] [ ] [ ] to make matters worse, in the face of the nation's cavernous inequalities, widespread economic immiseration, and the highest unemployment rate since the great depression, the current administration has proffered a one-time payment of us$ to limited households while giving tax breaks to the nation's wealthiest elites. , now, up to one in eight us households are food insecure. , an estimated - million people could risk eviction in the coming months. and in july, , the us gross domestic product had the largest drop on record. yet a month later, the stock market peaked. all of this is not a coincidence. it is a consequence of racial capitalism, the profitability of inequality, and the deadly policies of the current administration. , when it comes to this administration, "it is what it is". but seeing it clearly is essential. when torrential rain flooded parts of louisiana, extreme winds decimated parts of utah, and the us western seaboard spent weeks aflame, it is important to see this administration's enshrinement of fossil fuels as accelerants for climate catastrophe. when vice president mike pence asserted that "we will have law and order" as a condition of a second term of a trump presidency, it is crucial to see the rise in hate crimes, white vigilante terror, and human rights abuses that have been a condition of the first term of this administration. , seeing this clearly prevents tacit acceptance of a dangerous diversion. this type of diversion is how indigenous dispossession becomes "discovery", "settlement", and "columbus day". it is how vigilante mob violence against black communities become "race riots" with "fine people on both sides". in , us voters can choose a just future by first confronting our past. the usa began as a slave-ocracy, built on expropriated land and indigenous genocide. its first ruling class were white colonisers. its first enterprises were powered by slavery and premised on the ideology of white supremacy, environmental exploitation, and systems of governance, voter suppression, and policing that protected the right of the white, male, property-owning class to unilateral social, economic, and political control. this is the bedrock on which the "founding fathers" built their attempt at democracy. it is plutocracy perched atop precarity. the current administration only makes that more evident. but while the "founding fathers" anchored their fledgling democracy in inequality, the expansion of us democracy owes its fragile progress to many forebearers, chief among them are the indigenous, the formerly enslaved, and women. starting from this fuller, more inclusive understanding of who we are as a nation and how we got here, the possibilities for who we can become and where we can go are profound. racism saps the strength of the whole of society. and we each live within the limitations of the worlds we build for each other. but another world is possible. building on that conviction, since may, , black lives matter, a black-led, multiracial uprising for racial justice, has become one of the largest movements in us history. together, demonstrators across the country are asserting that our collective losses are not inevitable and our nation's deep grief need not be immutable. so whether shrouded in a booth or the privacy of our own homes this november, us citizens who can vote must affirm these self-evident truths: white nationalism and authoritarianism imperil democracy; democracy and equality are co-constitutive; equality is essential to health; the social, economic, and political conditions necessary to advance equality safeguard the planet; and thriving societies require sustainable environments and equitable economies. all these issues are on the ballot. we hope us voters choose a just future. we declare no competing interests. the views expressed in this comment are our own personal views and do not necessarily represent those of the institutions to which we are affiliated. civil and human rights rollbacks read the full transcript from the first presidential debate between joe biden and donald trump covid world map tracking the global outbreak the color of coronavirus: covid- deaths by race and ethnicity in the us sars-cov- -associated deaths among persons aged < years-united states deaths involving coronavirus disease (covid- ) by race and hispanic origin and age, by state the american health care paradox: why spending more is getting us less occupational safety and health administration (osha) and worker safety during the covid- pandemic coronavirus disease discriminates. our health care doesn't have to levels of racism: a theoretic framework and a gardener's tale trends in premature deaths among adults in the united states and latin america the employment situation the cares act sent you a $ , check but gave millionaires and billionaires far more. propublica the new york times magazine. america at hunger's edge. the new york times magazine measuring household experiences during the coronavirus pandemic national low income housing coalition. the covid- eviction crisis: an estimated - million people in america are at risk racial capitalism: a fundamental cause of novel coronavirus (covid- ) pandemic inequities in the united states dying in a leadership vacuum coronavirus is "under control". axios full transcript: mike pence's r.n.c. speech. the new york times hate-crime violence hits -year high, f.b.i. reports. the new york times trump defends white-nationalist protesters: "some very fine people on both sides one person, no vote: how voter suppression is destroying our democracy black lives matter may be the largest movement in us history. the new york times key: cord- -qjktnnn authors: wille, michelle; wensman, jonas johansson; larsson, simon; van damme, renaud; theelke, anna-karin; hayer, juliette; malmberg, maja title: evolutionary genetics of canine respiratory coronavirus and recent introduction into swedish dogs date: - - journal: infect genet evol doi: . /j.meegid. . sha: doc_id: cord_uid: qjktnnn canine respiratory coronavirus (crcov) has been identified as a causative agent of canine infectious respiratory disease, an upper respiratory infection affecting dogs. the epidemiology is currently opaque, with an unclear understanding of global prevalence, pathology, and genetic characteristics. in this study, swedish privately-owned dogs with characteristic signs of canine infectious respiratory disease (n = ) were screened for crcov and positive samples ( . %, . – . % [ % confidence interval (ci)]) were further sequenced. sequenced swedish crcov isolates were highly similar despite being isolated from dogs living in geographically distant locations and sampled across years ( – ). this is due to a single introduction into swedish dogs in approximately , as inferred by time structured phylogeny. unlike other crcovs, there was no evidence of recombination in swedish crcov isolates, further supporting a single introduction. finally, there were low levels of polymorphisms, in the spike genes. overall, we demonstrate that there is little diversity of crcov which is endemic in swedish dogs. canine infectious respiratory disease (cird) complex, colloquially referred to as kennel cough, is a contagious disease in dogs, particularly prolific in rehoming centers and kennels. dogs suffer from a dry hacking cough, which is usually cleared in - weeks, however, severe bronchopneumonia can develop (appel, ) . cird is a multifactorial disease with identified disease agents including canine parainfluenza virus (cpiv) (appel and percy, ) , canine adenovirus type (cav- ) (ditchfield et al., ) , canine pneumovirus (mitchell et al., ) , and the bacteria bordetella bronchiseptica (bemis, ) and mycobacterium cynos (mitchell et al., ) . more recently, canine respiratory coronavirus (crcov) was also identified as a causative agent of cird. this virus was first identified in the uk in a rehoming center with a high incidence of cird (erles et al., ) , however, a retrospective study has suggested that it may have circulated as early as in canada (ellis et al., ) . additional surveys have identified antibodies in dogs in the uk, ireland, italy, japan, and the us, with antibody prevalence as high as . % in the state of kentucky, usa (an et al., b; decaro et al., ; erles and brownlie, ; knesl et al., ; priestnall et al., priestnall et al., , schulz et al., ) . a small number of isolates have also been sequenced and characterized (an et al., a; erles et al., erles et al., , jeoung et al., ; yachi and mochizuki, infections in a number of avian and mammalian species. in humans, coronavirus infections range from the common cold (e.g. hcov-oc ) to more severe zoonotic diseases such as severe acute respiratory syndrome (sars) (peiris et al., ; van der hoek et al., ) and middle east respiratory syndrome (mers) (cunha and opal, ; de groot et al., ) . coronaviruses have large zoonotic potential, and the ability to cross species boundaries lies in not only mutation, but also the propensity for these viruses to recombine, with important breakpoints identified around the spike (s) gene in both birds and mammals (vijaykrishna et al., ; woo et al., ) . extensive homologous and heterologous recombination events have been documented in both human and animal group coronaviruses leading to the generation of various genotypes and strains (woo et al., ) . crcov is a group coronavirus, or betacoronavirus, which are comprised of mammalian coronaviruses. the most closely related species to crcov is bovine coronavirus (bcov), with many closely related species or strains such as sambar deer coronavirus, waterbuck coronavirus and human enteric coronavirus (hec ), illustrating the proliferation of these bcovlike viruses. in this study, we screened nasopharyngeal swabs from privatelyowned dogs in sweden with and without cird for crcov. we identified the first crcov positive dogs from sweden, and we used these to assess the evolutionary genetics of crcov, both locally and globally. specifically, we wanted to determine genetic variation and quasispecies of crcov in swedish dogs to clarify diversity within sweden and the relationship of swedish viruses to other crcov isolates in europe and elsewhere. furthermore, we aimed to understand the introduction of these viruses into sweden by using time-structured phylogeny and recombination analysis. finally, given the large number of crcov isolates sequenced in this study, we were able to better elucidate the emergence of crcov through analysis of recombination dynamics of crcov and bcov. this research was reviewed, approved and conducted in accordance with the regulations provided by the swedish board of agriculture and approved by the swedish animal research ethics board (uppsala djurförsöksetiska nämnd, reference numbers c / and c / ). between april and december , privately owned dogs with characteristic upper respiratory signs of cird (dry cough) for up to days were enrolled in a study investigating the cause of cird in sweden. maximum two dogs per households were sampled, and sampled dogs were not treated by antibiotics at the sampling. samples were taken from seven veterinary clinics across sweden, with dogs residing in swedish counties (fig. ) . nasopharyngeal swabs (e-swabs with amies medium and regular nylon flocked applicator, copan italia, brescia, italy) were collected and stored at − °c within - h of collection. a total of dogs with cird were swabbed. as controls, we also swabbed healthy dogs that had not suffered from respiratory signs for the last six months. we used generalized linear models (glm, family = binomial) to evaluate the effect of age, breed, location, and sex on crcov prevalence. explanatory variables were entered in isolation or in combination, where the resulting model improvements were χ tested for significance. statistics were done in r . . (r development core team, ) integrated into r studio . . . viral rna was extracted from nasopharyngeal swabs with the magnatrix + extraction robot (magnetic biosolutions, sweden) and vet viral na kit (nordiag asa, oslo, norway), as described in (jinnerot et al., ) . cdna was subsequently synthesized using su-perscript™ iii reverse transcriptase (invitrogen, life technologies, carlsbad, ca, usa), and the second strand was synthesized using klenow fragment™ ′-> ′ exo-(new england biolabs, m s, ipswich, ma, usa). four approaches were utilized to sequence viruses. first, using traditional pcr and sanger sequencing of the pcr products, and second, using illumina miseq to sequence pcr products of partial s gene. third, a viral metagenomics approach was done on two samples (crcov and crcov ) with the aim to get close to complete genomes. lastly, a probe-based capture method was used on one sample (crcov ) ( table a ). for the first approach, pcr reactions targeting the s, membrane (m) and hemagglutinin-esterase (he) genes were carried out using previously published primers (an et al., a; erles et al., ) (table a ) using kapa g robust hotstart readymix pcr kit (kapa biosystems, roche sequencing, pleasanton, ca, usa). thermocycling conditions were °c for min and then cycles of °c for s, a reaction specific annealing temperature for s, a reaction specific elongation time at °c, and finally °c for min (table a ). the pcr products were run on a . % agarose gel stained with gelred, visualized under uv transillumination (geldoc, bio-rad laboratories, inc., richmond, ca, usa), purified using genejet gel extraction kit (life technologies, carlsbad, ca, usa) and sequenced at macrogen europe (amsterdam, nl). a bp fragment was amplified using longamp taq dna polymeranse ( . u), dntp mix ( . mm), × longamp taq reaction buffer (new england biolabs, m s, ipswich, ma, usa), . μm of respective primer sp f and sp r (table a ) , and μl of template. thermocycling conditions were °c for s and then cycles of °c for s, °c for s, elongation time at °c for s, and finally °c for min. the pcr products were purified using the genjet pcr purification kit (thermo fisher scientific, waltham, ma, usa). sequencing libraries were made using nextera xt library preparation kit (illumina, san diego, ca, usa), normalization and pooling of nm sequencing libraries was done based on concentration measurements from agilent high sensitivity dna kit ( bioanalyzer, agilent technologies, palo alto, ca, usa). the pool of sequencing libraries was denaturated with naoh and further diluted with hybridization buffer to a final concentration of pm and spiked with % phix for diversity. paired-end sequencing was performed with miseq reagent kit v cycles on the miseq instrument (illumina, san diego, ca, usa) at the national veterinary institute, uppsala, sweden. for the viral metagenomics approach, μl of the swab media were freeze thawed twice, centrifuged at g at °c for min, the supernatant transferred to a filtrate . μm (millipore, burlington, ma, usa), and centrifuged for min at g. the filtrated sample was treated with u turbodnase in turbodnase buffer (invitrogen, thermo fisher scientific, waltham, ma, usa) and dnasei ( μl) (invitrogen, life technologies, carlsbad, ca, usa) for min at °c, followed by rnase cocktail™ enzyme mix (invitrogen, thermo fisher scientific, waltham, ma, usa) treatment for min at room temperature. thereafter, rna was extracted using trizol, chloroform and rneasy kit (qiagen, hilden, germany). the kit oviation® rna-seq system v (nugen, redwood city, ca, usa) was used to amplify the rna in accordance with the provided protocol, and the genjet pcr purification kit (thermo fisher scientific, waltham, ma, usa) was used for purification. sequencing libraries were constructed at the national sequencing infrastructure in uppsala, sweden, using the ab library builder system (ion xpress™ plus and ion plus library preparation for the ab library builder™ system protocol, thermo fisher scientific, waltham, ma, usa) and size selected on the blue pippintm (sage science, beverly, ma, usa). library size and concentration were assessed by a bioanalyzer high sensitivity chip (agilent technologies, santa clara, ca, usa) and by the fragment analyzer system (advanced analytical; agilent technologies, santa clara, ca, usa). template preparation was performed on the ion chef™ system using the ion & ion kit-chef (thermo fisher scientific, waltham, ma, usa). samples were sequenced on chips using the ion s ™ xl system (thermo fisher, waltham, ma, usa). probes were designed for feline coronavirus kb -nc_ . , canine respiratory coronavirus kb -jx . , bovine coronavirus kb -u . , kobuvirus kb -km . , porcine rubulavirus kb -nc_ . , african swine fever virus kb -benin / am . , canine parainfluenza virus kb -kc . , by agilent technologies sureselect dna advanced design wizard. this resulted in a . kbp capture based on probes. one sample that was positive for crcov with a cq-value of . as determined by qpcr was selected (crcov ). rna extracted from nasopharyngeal swabs as described above was used. in total μl of rna was converted into cdna using superscript™ iii reverse transcriptase (invitrogen, life technologies, carlsbad, ca, usa). thereafter, treated with rnase h for min at °c, and made double-stranded using, klenow fragment™ ′-> ′ exo-(thermo fisher scientific, waltham, ma, usa). kapa hyperplus library prep kit (kapa biosystems, roche sequencing, pleasanton, ca, usa) was used in combination with an unofficial protocol for using agilent's sureselectxt target enrichment system for illumina paired-end multiplexed sequencing libraries version b (june ), ng dna samples. a × bead clean-up was used on μl of double-stranded cdna prior to fragmentation. the samples were fragmented at °c for min. at the adapter ligation step the sureselect adapter oligo mix was used and the sample was incubated at °c for min. the pre-capture libraries were vacuum centrifuged to a final concentration of ng/μl. hybridization was done according to the sureselect protocol and the samples were incubated at °c for h in a proflex pcr machine (applied biosystems, foster city, ca, usa). thereafter, dynabeads myone streptavidin t (thermo fisher scientific, waltham, ma, usa) were used to capture the sureselectxt enriched libraries. an on-bead pcr with kapa hifi hotstart amplified the libraries. for this, pcr cycles were used. after a final clean up with agencourt ampure beads (beckman coulter indianapolis, in, usa), the libraries were quality assured using bioanalyzer hs dna assay (agilent technologies, santa clara, ca, usa). prior to sequencing a nm pool was made, denaturated with naoh and further diluted with hybridization buffer to a final concentration of pm and spiked with . % phix for diversity. pairedend, cycles sequencing was performed with miseq reagent kit v cycles on the miseq instrument (illumina, san diego, ca, usa) at the national veterinary institute in uppsala, sweden. reads were cleaned, forward and reverse reads aligned and a consensus per sample was made using dnastar (madison, wi, usa). the sequenced reads were demultiplexed using bcl fastq (https:// github.com/brwnj/bcl fastq.) and the adapters were removed using fastp version . . (chen et al., ) (https://github.com/ opengene/fastp). the reads were then assembled using megahit version . . (li et al., ) , with default settings. for each sample, the only contig produced corresponding to the size of the amplicon was extracted (longest contig). the reads were trimmed for quality using fastp with the a phred score threshold of and a minimum length of bp, and aligned to the longest contig obtained by megahit using bwa-mem version . . (li and durbin, ) with default parameters. the result of the alignment was converted and sorted in a bam file using samtools version . . (li et al., ) . from these alignments, single nucleotide variants were then analyzed using both shorah version . (zagordi et al., ) and quasirecomb version . (topfer et al., ) . the reads were assembled using megahit version . . (li et al., ) , with default settings. a taxonomic classification of the contigs using diamond version . . (buchfink et al., ) against the nonredundant protein database from ncbi (nr, release february ) was then performed. the produced output files (daa format) were uploaded into megan (version . . ) (huson et al., ) and all the contigs classified as betacoronavirus were extracted and inspected. the longest contigs were selected for further structural and functional annotation. reads were assembled using spades (v . . ) (bankevich et al., ) but as the coverage was too high to get a correct assembly, the dataset was randomly reduced down to , reads. those reads were then assembled using spades. the obtained contigs were then taxonomically assigned using diamond. the outputs from diamond were visualized in megan and contigs classified as betacoronavirus were retrieved. the crcov , crcov and crcov genomes were annotated using an annotation pipeline for prokaryotic and viral genomes, prokka (seemann, ) . we had previously extracted all the protein sequences belonging to the coronaviridae family from uniprotkb (release april ). this dataset was provided to prokka for annotating the newly assembled coronavirus genomes. all generated sequences from this study have been deposited to the european nucleotide archive at ebi, under the bioproject prjeb . high throughput sequencing (hts) reads have been deposited in the ebi short read archive (accession numbers: spike amplicons datasets: err -err , err -err , err -err . crcov probe-based capture dataset: err , metagenomics datasets: crcov : err and crcov : err ). final full and partial annotated genomes generated from hts have been deposited in european nucleotide archive under the accession numbers: erz (crcov ), erz (crcov ), erz (crcov ). full length genes generated through sanger sequencing have been deposited in european nucleotide archive, accession numbers erz to erz . resulting sequences were aligned using the mafft algorithm (katoh et al., ) within geneious r (biomatters, new zealand). maximum likelihood phylogenetic trees were constructed for each gene (orf ab, hemagglutinin-esterase he, s, and matrix m) using phyml . (guindon et al., ) implementing the best substitution model for each gene. for the full length s gene, we utilized beast . (drummond and rambaut, ) to better infer the evolutionary relationship within crcovs. shortly, we used maximum likelihood trees constructed in mega to test for clock-like behavior in each data set by performing linear regressions of root-to-tip distances across years of sampling in tempest (rambaut et al., ) . using beast, time-stamped data were analyzed using both the uncorrelated lognormal relaxed and strict molecular clock, the srd codon position model -a hky substitution model and a different rate of nucleotide substitution for the + codon position and the rd codon position (bahl et al., ; shapiro et al., ) . we implemented the bayesian skyline coalescent tree prior. three independent analyses of million generations were performed and convergence assessed using tracer . . independent runs were combined in logcombiner v . following a burnin of %. maximum credibility clade trees were generated using treeannotator v . . the maximum credibility clade trees were visualized in figtree v . . . to assess recombination, a concatenated sequence was generated including the viruses from which there were sequences from the partial non-structural protein a (ns), and full length he, s, envelope (e), and m, resulting in bp for analysis and the genes were placed in genomic order. to assess evolutionary patterns of all segments a splitstree network was constructed with crcov as well as bcov outgroups using splitstree (huson and bryant, ) . splitstree builds a network which takes recombination into account. to better understand the recombination process, the concatenated alignment was used in rpd to estimate break points (martin et al., ) . specifically, we used the algorithms rdp (martin and rybicki, ) and bootscan (martin et al., ) to detect the recombination window, and used additional algorithms within rpd to cross reference support for the detected window. to understand genetic variation within each sample, we utilized sequences of the partial spike gene generated using illumina miseq of nine samples. the amplicon sequenced ranged from position to the ′ end ( ) of the spike gene, so variants were only studied in this ′ region. the genetic variant population was estimated using a combination of both shorah version . (zagordi et al., ) and qua-sirecomb version . (topfer et al., ) with default parameters. we used a combination of both to ensure the accuracy of the snps found. a total of dogs were sampled as part of this study, comprising dogs with signs of disease and healthy dogs. thirteen samples collected from dogs with signs of disease were positive for crcov ( . %, . - . % [ % confidence interval (ci)]), and were collected from dogs living in the counties of stockholm ( / ), skåne ( / ), västmanland ( / ), västernorrland ( / ) and dalarna ( / ) (fig. ) . none of the healthy dogs tested positive for crcov. overall prevalence of crcov did not vary significantly by dog clinic (x = . , df = , p = . ) or county from which dogs originated (x = . , df = , p = . ). there was further no statistical difference across dog breed (x = . , df = , p = . ), sex (x = . , df = , p = . ), or age category (x = . , df = , p = . ) (fig. a ) . the largest number of positive samples were detected in winter months (n = ) compared to the autumn (n = ) and spring (n = ); no positive samples were detected in the summer (table ) . despite this detection difference, prevalence of crcov was not significantly different across season (x = . , df = , p = . ) (fig. a ) . using probe-based capture one complete genome was generated (crcov ). the complete genome of crcov was , bp and following annotation coding regions were predicted: orf a, orf ab, non-structural protein a, he, s, . kda non-structural protein, e, m and nucleoprotein (np) (fig. a ) . this full genome now brings the number of complete crcov genomes to [crcov-k (an et al., a) and crcov-bj (lu et al., ) ]. viral metagenomics resulted in partial genomes of crcov and crcov covering bp and , bp, respectively. following annotation of the crcov genome, coding regions were predicted: a partial gene coding for the he, and all full genes coding for: s, . kda non-structural protein, e, m and the np. annotation of the crcov genome predicted a partial orf ab gene and all the remaining genes located between orf ab and the ′ end of the genome. of the remaining viruses, - genes were sequenced through a combination of sanger sequencing and illumina high-throughput sequencing (table a ) . assessment of the s, e, m, ns and he genes generated using sanger and high-throughput sequencing (table a ) demonstrated high genetic similarity of viruses detected in swedish dogs, despite sampling from numerous clinics across sweden and an over a period of years. indeed, all samples for all genes were within % identity (fig. a ) . phylogenetic reconstruction of the larger genes (s, he and m) indicates that swedish viruses are most closely related to a crcov isolated in italy in (eu ), and in some trees crcov isolated in the uk in (dq ) (fig. a ) . given discordance between trees, we constructed a consensus tree from a concatenated alignment using splitstree, suggesting that all swedish viruses are likely the result of a single introduction and despite recombination being a common feature in crcov's, are most similar to the two aforementioned viruses (figs. , a ) . time-structured phylogenetic analysis of the s gene suggests that the most recent common ancestor of all swedish crcovs was introduced to sweden at the beginning of [ . with a confidence interval ranging from . to . ], with subsequence proliferation within sweden (fig. ) . as suggested with previous analysis, the viruses isolated in sweden are most closely related to an isolate from italy, but not necessarily the uk, and that crcovs found in europe may have emerged and been introduced from asia. however, due to the sparseness of sequences, we have not undertaken phylogeographic analysis, and these observations should be interpreted with caution. largely, crcov and bcov form independent lineages in phylogenetic analysis, and large number of swedish sequences in each tree help to better define the crcov clade. this lineage separation is most evident in the s tree (fig. a c) , where all crcovs form a clade that is different from bcov. however, crcov does not always fall into a distinctive clade; some genes like the he and m of some isolates are more closely related to bcov sequences than crcov. this was not the case with the viruses from sweden, which are very similar to each other, and more distantly related to bcov than other crcov sequences (fig. s a, fig. ) . when assessing pairwise identity, it is clear that the percentage identity of all global crcovs only (fig. a b) and a combination of all crcov and bcov sequences (fig. a c) for the partial ns, he and the complete m gene is rather similar. this is in contrast to the s gene, where percentage identity of all crcovs ranges from to % whereas when including bcov outgroups the range is from . to %. this is because for many genes, viruses from korea (k , k , k ) and a recent sequence from china which were mined from genbank are more similar to bcov than to crcovs detected in europe (fig. a) . recombination detection algorithms detect that there is indeed a recombination event in these previously described korean and chinese viruses, where the ns, he, e and m sequence is more similar to bcov than to other crcov sequences (region probability with mc correction: . ) ( table a ). the breakpoint, represented by the end of the he gene and start of the s gene, has a high probability ( . ); however, the certainty of the breakpoint at the end of the s gene is undetermined due to a number of potential breakpoints (fig. a ) , which is revealed in the bootscan analysis. this is because all crcovs, but especially the asian crcovs, have higher signal for bcov at the end of the s gene (fig. b) . whether this is due to recombination within the s gene, or due to evolutionary processes of conservation in this section of the s gene, is unclear given some similarity between swedish crcov and bcov in this region as well. regardless, these recombinant isolates have been maintained in asia, demonstrated by the isolation of a virus in china in sharing the same mosaic crcov-bcov pattern as viruses isolated in korea in . overall, viruses sequenced in sweden as part of this study have a different recombination profile as compared to previously described viruses from china and korea. to better understand the variation within each crcov virus detected in sweden, partial spike amplicon high-throughput sequencing of all crcov isolates except crcov , and were analyzed for variations with different methods: shorah and quasirecomb. overall, shorah identified more single nucleotide variants (snvs) compared to quasirecomb (fig. ) , however approximately half of the snvs predicted by shorah were also predicted by quasirecomb (except for the insertions/deletions that are only predicted by shorah). furthermore, regardless of method, most of the snvs observed were at a frequency lower than . . there was variation in snv's across samples. there were no variants identified in crcov with either method. in contrast, crcov contained different snv's. crcov and crcov had marked different pattern depending on method used, whereby snv's were identified with shorah but not with quasirecomb due to differences in detection of indels. there are no positions of snvs that are common to all samples. only one snv was common to crcov and crcov (position ). this variation analysis suggests that the studied region of the s gene is not variable among the crcov isolates collected from swedish dogs. in this study, we aimed to reveal the epidemiology and evolutionary genetics of crcov, one of the causative agents of canine infectious respiratory disease (cird), in sweden. cird is a major cause of morbidity in dogs and an important animal welfare condition. this disease is multi-factorial, and a recent european survey reported that crcov, canine pneumovirus and the bacteria mycoplasma cynos, besides the previously established main causes (mainly cpiv and b. bronchiseptica), all played a role in the disease (mitchell et al., ) . to date, the highest prevalence of crcov remains in rehoming centers (erles and brownlie, ; erles et al., ; mitchell et al., ) , or other instances where dogs may be cohoused, such as in racing greyhounds (sowman et al., ) . epidemiology is unclear in pet dogs, such as those sampled in our study, but we predict that occasions where many dogs meet (e.g. dog day-care centers, dog shows and other activities), m. wille, et al. infection, genetics and evolution ( ) are likely contributing to spread, similarly to other cird-causing pathogens. overall, we found that % of pet dogs with signs of cird in sweden, ranging from mild (cough only) to severe (affected general condition and deep coughing) disease signs, were positive for crcov, demonstrating the role that this virus plays in the complex aetiology of cird. importantly, we found crcov in dogs of all breeds, ages, and sexes demonstrating that this virus affects all dogs, equally. crcov was isolated between december and march; no viruses were detected in the summer months. this lack of significance of season was likely due to few tests being undertaken in the summer, resulting in a large uncertainty. the evolutionary genetics of coronaviruses is complex, largely driven by high rates of substitution, and more importantly recombination. this has allowed for interspecies variability, interspecies host jumps and novel coronaviruses to emerge under the "right" conditions (decaro et al., ; pyrc et al., ; ren et al., ; su et al., ; woo et al., ; zhang et al., ) . lineage a of betacoronaviruses is comprised of a number of closely related viruses including . splits tree of concatenated ns, he, s, e and m genes. each band of parallel edges indicates a split, which is a collection of sequence differences that separates one group of sequences from another. non-tree like evolution is expected to be from recombination. the distance between taxa represents the sum of weights of all splits that separate taxa. m. wille, et al. infection, genetics and evolution ( ) crcov, bcov, bcov-like, and hcov-oc . a study by kin et al., demonstrated that bcov and hcov-oc have > % global nucleotide identity and they reported in an earlier study that hcov-oc may be the result of a zoonotic transmission between bovines and humans (kin et al., ; kin et al., ) . furthermore, there is a . % global nucleotide similarity between crcov and bcov (erles et al., ) , but more importantly, cross-reactivity between bcov antigen with crcov antibodies (erles et al., ) , and infectivity of puppies with bcov (kaneshima et al., ; priestnall et al., ) . finally, bcov and crcov (and hcov-oc ) use sialic acids or heparan sulphate to attach to the cell surface, and use the same molecule as entry receptors (hla-i, szczepanski et al., ) . overall, this provides a lot of evidence for a very putative host switch event. our splitstree and recombination analyses of global viruses support the results in lu et al., which demonstrate high levels of recombination between bcov and previously described crcov strains, including those from uk, italy, korea and china (lu et al., ) . the viruses sequenced in this study, at a more local level, add an interesting piece to the puzzle. viruses from sweden were most closely related to a sequenced strain from italy in , and therefore the most parsimonious conclusion is that these viruses were introduced to sweden from elsewhere in europe. despite being detected in a large number of dog species, large geographic area and across years, there was very little genetic variation in crcov from swedenvery little genetic drift, few snvs and no evidence of recombination, suggesting a single, recent introduction, and subsequent spread. potentially the most interesting finding, is that the crcov circulating in sweden is the most genetically distant virus to bcov as compared to crcov previously described (particularly those from asia), with the majority of genes clustering into a "crcov clade" rather than bcov, and most genes being < % similar to bcov. whether this suggests better adaptation to dogs with time, or whether it represents geographical differences, is unclear, and will be revealed with further sequencing of these viruses, globally. recent descriptions of novel aetiological causes of cird, such as crcov, urge for further studies on epidemiology and pathogen genetics to facilitate future vaccine development and diagnostics. to date, commercial vaccines are only available for the well-established cird pathogens cpiv, cav- and b. bronchiseptica. despite frequent vaccinations in dogs, cird remains a burden for dogs and dog-owners, and as such, it is imperative to better understand the epidemiology and evolution to prevent virus introductions and spread, and to provide informed advice on quarantine decisions related to dog housing facilities, ranging from dog day-care centers to show dogs and racing kennels. fig. . recombination between crcov and bcov. (a) percentage identity between crcov and bcov strain kakegawa. white indicates no sequence from the gene was available, and grey/blue shading corresponds to the percentage identity to bcov. asterisk indicates only a partial gene sequence was available. (b) bootscan analysis of concatenated sequence illustrating a recombination where crcov from korea and china are more similar to bcov in the ns, he and m genes. genes are identified above the plot, and the statistically supported recombination window is further illustrated above the plot. lines plotted are percentage bootstrap support include only potentially recombined viruses, in addition to one of the swedish crcov viruses for reference (black). statistical support for recombination window is presented in table a and chi-squared breakpoint in fig. a . (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) the authors declare the following financial interests/personal relationships which may be considered as potential competing interests: the study was in part financed by msd animal health. this sponsor did not have any influence on the design of the study or the decision to publish the results. genetic analysis of canine group coronavirus in korean dogs a serological survey of canine respiratory coronavirus and canine influenza virus in korean dogs canine infectious tracheobronchitis short review: kennel cough sv- -like parainfluenza virus in dogs influenza a virus migration and persistence in north american wild birds spades: a new genome assembly algorithm and its applications to single-cell sequencing bordetella and mycoplasma respiratory infections in dogs and cats fast and sensitive protein alignment using diamond fastp: an ultra-fast all-in-one fastq 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diversity, phylogeny and interspecies jumping survey of dogs for group canine coronavirus infection shorah: estimating the genetic diversity of a mixed sample from next-generation sequencing data genotype shift in human coronavirus oc and emergence of a novel genotype by natural recombination we acknowledge the effort of all veterinarians involved in collecting samples for this study, as well as the animal owners and dogs enrolling.the authors would like to acknowledge support of the national genomics infrastructure (ngi)/uppsala genome center and uppmax for providing assistance in massive parallel sequencing and computational infrastructure. work performed at ngi/uppsala genome center has been funded by rfi/vr and science for life laboratory, sweden. we would also like to acknowledge slu global bioinformatics centre for providing resources for all computing.the authors would like to acknowledge the support of johanna hasmats at agilent technologies with the probe-based-capture method development and karin ullman at the national veterinary institute for support with the miseq sequencing. this study was funded by agria animal insurances and the swedish kennel club research foundation, jan skogborgs foundation, sveland foundation for animal health and welfare, msd animal health (grant number n - ) and the swedish research council for environment, agricultural sciences and spatial planning, formas (grant number - - ). supplementary data to this article can be found online at https:// doi.org/ . /j.meegid. . . key: cord- -qxu hta authors: rogers, m. a. m.; rohde, j. m.; blumberg, n. title: haemovigilance of reactions associated with red blood cell transfusion: comparison across countries date: - - journal: vox sang doi: . /vox. sha: doc_id: cord_uid: qxu hta background and objectives: the recent establishment of the national healthcare safety network hemovigilance module in the united states affords an opportunity to compare results with those of other developed nations. materials and methods: using data from national haemovigilance systems, reactions associated with red blood cell (rbc) transfusion and residual risks of transfusion‐transmitted infectious diseases were assembled from nations. country‐specific rates of adverse events were pooled using random‐effects poisson regression. results: febrile non‐haemolytic and delayed serologic transfusion reactions were the most frequent adverse events reported after rbc transfusion, occurring in patients per rbc units and patients per rbc units administered, respectively. rates of allergic, febrile non‐haemolytic and delayed haemolytic transfusion reactions in the united states were significantly greater than the pooled rates from other countries. frequencies of adverse events generated from the national haemovigilance programme in the united states were considerably lower than when obtained through active surveillance. conclusion: haemovigilance reports of adverse events in the united states are comparable to, or greater than, reports from other developed countries. rates generated from haemovigilance programmes are lower than those obtained through active surveillance. the lack of universal leucoreduction of rbc units may be a contributing factor to the higher rate of some adverse events in the united states. first established in france and japan in as a response to the vulnerability of the blood supply after the emergence of hiv [ , ] , haemovigilance systems aim to improve the safety of the blood supply through systematic surveillance of transfusion-related adverse events. such systems generate recommendations so as to encourage safety throughout the entire process, from blood donation through monitoring of recipients. several directives from the european union relate to the regulation, monitoring and safety of blood products, with the haemovigilance component helping to direct member nations' reporting of serious transfusion-related adverse reactions and events [ ] . therefore, haemovigilance systems are widespread in european nations, with prime examples being the united kingdom with its voluntary serious hazards of transfusion reporting system ( Á % participation in ) [ ] , and the netherlands with its transfusion and transplantation reactions in patients haemovigilance programme ( % participation in ) [ ] . the usa opened its first nationwide haemovigilance system in as a voluntary module of the national healthcare safety network [ ] . results from this new system are now available and can be compared with existing haemovigilance programmes. as more nations begin to implement haemovigilance programmes, an assessment of the abilities of national systems to report reactions and a comparative review of event rates can be informative. haemovigilance data on transfusion-related adverse reactions and transfusion-transmitted infections were available from the national haemovigilance systems in each of the following countries: australia [ ] , canada [ ] , denmark [ ] , finland [ ] , france [ ] , germany [ ] , ireland [ ] , japan [ ] [ ] [ ] , the netherlands [ ] , new zealand [ ] , norway [ ] , portugal [ , ] , spain [ ] , sweden [ ] , switzerland [ ] , united kingdom [ ] and the usa [ ] . data regarding adverse events after rbc transfusion were collected: allergic reactions, anaphylactic reactions, febrile non-haemolytic transfusion reactions (fnhtr), acute and delayed haemolytic transfusion reactions, hypotensive reactions, transfusion-related acute lung injury (trali), transfusionassociated dyspnoea, transfusion-associated circulatory overload (taco), delayed serologic transfusion reactions, post-transfusion purpura and transfusion-transmitted bacterial sepsis. some countries reported additional categories and these were included as well (acute transfusion-related pain, haemosiderosis). for each country, data from the most recently available report were utilized. risks of transfusion-transmitted viruses were available for agents with mandated testing. reporting methods differ by country, although in the usa, data regarding residual risk of viral transmission were derived from donor testing since long-term follow-up of recipients is not feasible [ ] . because it is theoretically possible that a donor could have newly acquired a virus but is seronegative at the time of the blood donation, seroconversion rates during this infectious window period are used to predict the residual risk of viral infection [ ] . rates of non-infectious adverse events were calculated for each country using the number of events (numerator) and the number of rbc units administered (denominator). rates were reported for each country as events per rbc units for non-infectious adverse events and events per million rbc units for infectious adverse events. canada and spain were exceptions; adverse events were recorded for components combined (rbcs, platelets, plasma). in the usa, taco was the only event in which reporting was performed for components combined (rbcs, platelets, plasma). in secondary analyses, adverse event rates from active surveillance in the usa were compared with the rates generated from the passive haemovigilance programme; in these instances, the rates were given using several denominators (per patient, per unit and per transfusion-related hospital stay). following current recommendations for summarizing rates [ ] , random-effects poisson regression models were generated to pool rates of transfusion-related adverse events with % confidence intervals. that is, the numbers of events were fit to a poisson distribution with the number of rbc units included as an offset. countries that reported zero events were included and, in such instances, a one-sided exact poisson Á % confidence interval was used for the country-specific rate. heterogeneity was assessed using a gamma density function (shape parameter = , scale = Á ) for the variance of the random intercept (between-country variance) in the poisson model. this rate index of heterogeneity (rih) is unit-independent, encompasses a range from % to % heterogeneity and is directly derived from the underlying random-effects poisson model for assessment of rates. analyses were conducted using stata/mp Á (college station, texas, usa). allergic reactions after rbc transfusion occurred in patients per rbc units administered ( % ci: Á / to Á / ) with a rih of Á % (fig. ) . the rate of allergic reactions in the usa was significantly greater ( Á / ; % ci: Á / - Á / ) than the pooled rate of the other developed countries combined ( Á / ; % ci: Á / - Á / ). some countries reported anaphylactic reactions separately and these occurred at a lower rate of Á per rbc units (rih = Á %). in the united kingdom, acute transfusion reactions were defined as instances of anaphylaxis or severe allergic reactions, severe febrile reactions, severe hypotensive reactions and severe mixed reactions; these occurred at a rate of nine patients per rbc units. there was variability in the rates of fnhtr across countries (rih = Á %). overall, the rate was patients per rbc units, although the netherlands, new zealand and the usa recorded rates in excess of patients per rbc units, or approximately one in one-thousand units (fig. ) . the rate of fnhtr in the usa was significantly greater ( Á / ; % ci: Á / - Á / ) than the pooled rate of the other developed countries combined ( Á / ; % ci: Á / - Á / ). hypotensive reactions after rbc transfusion occurred less frequently, in patients per rbc units (rih = Á %). in spain, hypotensive reactions were combined with fnhtr, yielding a rate of patients in rbc units. acute transfusion-related pain was reported separately in the swedish haemovigilance system, occurring in one patient per rbc units (fig. ) . rates of acute and delayed haemolytic transfusion reactions are shown in fig. . acute haemolytic transfusion reactions occurred in patient per rbc units administered (rih = Á %), while a delayed haemolytic transfusion reaction occurred in approximately two patients per rbc units (rih = Á %). in germany, spain and the united kingdom, both acute and delayed haemolytic transfusion reactions were combined, yielding a pooled rate of Á / units (rih = Á %). both ireland and the usa tended to have delayed haemolytic transfusion reactions at a greater frequency than other countries, although the confidence intervals for ireland were wide indicating fewer rbc units due to a smaller population. the rate of delayed haemolytic transfusion reactions in the usa was significantly greater ( Á / ; % ci: Á / - Á / ) than the pooled rate of the other developed countries combined ( Á / ; % ci: Á / - Á / ). respiratory reactions to rbc transfusion are shown in fig. . transfusion-associated dyspnoea was reported in two patients per rbc units (rih = Á %). new zealand was an outlier in this category. trali associated with rbc units was quite low (fig. ) , at a pooled rate of Á per rbc units (rih = Á %). taco occurred in approximately three patients for each rbc units transfused, although there was some variation in rates across countries ( fig. ; rih = Á %). haemosiderosis was reported separately in france and spain and occurred at a rate of Á for every units transfused (rih = Á %). the results for delayed serologic transfusion reactions are given in fig. , showing heterogeneity across countries (rih = Á %; rate = Á / units), with elevated rates in the netherlands and switzerland. posttransfusion purpura occurred rarely, at a rate of Á per rbc units administered ( fig. ; rih = %). other transfusion-related reactions, as shown in fig. , varied by country and were not described in great detail in the country-specific annual reports. documented cases of transfusion-transmitted bacterial sepsis occurred rarely, approximately once in every million rbc units administered ( fig. ; rih = Á %). anaphylaxis, hypotension and purpura, the rates were quite consistent across countries. rates of some reactions, however, were notably elevated in particular countries. rates of allergic reactions were higher in the usa and new zealand; rates of fnhtr were elevated in the usa, the netherlands and new zealand; and taco was more commonly reported in canada, france, ireland, the netherlands and the usa. new zealand reported a greater rate of transfusion-associated dyspnoea in . however, in a recent report, more than half of the dyspnoea cases were reclassified when additional information was retrieved (many being reclassified as taco) [ ] . overall, taco occurred at a rate of three cases per units when pooled from haemovigilance reports, with the incidence of taco in the usa alone being one in units. however, based on active surveillance studies from the usa, Á % to % of patients receiving rbc transfusions develop taco. this disparity between active and passive surveillance rates may stem from insufficient reporting of reactions to haemovigilance systems. for example, researchers from the mayo clinic noted that there were cases of taco in their investigation but only three were in the transfusion database that housed adverse reactionsand even then, these three cases were not labelled as taco [ ] . given the more comprehensive monitoring of fever after rbc transfusion, the variability of rates of fnhtr across countries may be a reflection of dissimilarities in patient populations, differential use of pretransfusion antipyretic medications or the preparation of rbc units. of note, the usa has not adopted universal leucoreduction ( Á % rbc units leucoreduced in )[ ] and fnhtr has been reported more frequently in patients receiving non-leucoreduced products [ , , , , ] . fnhtr rates in the usa are higher than the pooled fnhtr rates in other developed countries and the use of non-leucoreduced products could partially account for these findings. the variability in rates of allergic reactions across nations may stem from differences in case definitions. in australia, for example, a severe allergic reaction would be recorded if rash, allergic dyspnoea, angioedema, generalized pruritis or uticaria occurred within h of the transfusion [ ] . in the usa, a severe allergic reaction would be recorded if two or more symptoms (conjunctival oedema; oedema of lips, tongue and uvula; erythema and oedema of the periorbital area; generalized flushing; hypotension; localized angioedema; maculopapular rash; pruritus; respiratory distress or bronchospasm; urticaria) occurred during or within h of the cessation of transfusion [ ] . moreover, in some countries such as new zealand, both non-severe and severe allergic reactions are reported [ ] (perhaps reflecting their higher rates as shown in fig. ) while in the usa, reporting of nonsevere allergic reactions is not required [ ] . other reasons for differences in rates may be variations in hospital-specific reporting practices and the mandatory/ voluntary nature of the reporting. for events such as delayed serologic transfusion reaction and delayed haemolytic transfusion reaction, the definition hinges on laboratory testing and not symptoms. rate differences may be a reflection of the resources afforded for testing of alloantibodies and the feasibility of longitudinal observation. information regarding follow-up procedures or standardization of procedures would enhance the interpretability of rates. alternatively, haemovigilance programmes may reconsider the usefulness of expending resources to capture such data when the likelihood of consistent, meaningful information is low. perhaps periodic systematic testing in high users of transfusion would be more appropriate such as in patients with sickle-cell disease or cancer, or in participants of cardiovascular disease registries. results from this investigation indicate that the residual risks from currently tested infectious agents are very low and, in many instances, could be considered negligible. however, it is the potential risks from other infectious agents (not yet tested) which are more pertinent at this time. these include vcjd, hepatitis e virus, dengue viruses, chikungunya virus, babesia spp., west nile virus and middle east respiratory syndrome coronavirusalthough the list of potential threats includes approximately such agents [ ] . for example, in the usa, transfusion-transmitted babesiosis has been documented in more than instances and is associated with the greatest case fatality rate of transfusion-related infections [ ] . haemovigilance for transfusion-transmitted infection is meant to capture pathogen transmission from donor to recipient. the requirements include the demonstration of the pathogen in the transfused component, or in the donor at the time of donation, or in an additional component from the same donation, or in an additional recipient of a component from the same donation [ ] . also required is documentation that there are no other potential exposures to the pathogen that could be identified in the recipient and either evidence that the recipient was not infected with the pathogen prior to transfusion or evidence that the pathogen strains are related by molecular or extended phenotypic comparison testing [ ] . this is a difficult target to reach when such in-depth testing is not usually conducted with each transfusion. therefore, the pooled rate of transfusion-transmitted bacterial sepsis was very low ( Á / million rbc units). there are few published reports of prospective surveillance for comparison purposes. barrett and colleagues [ ] found a bacterial contamination rate of Á / rbc units, while dzieczkowski and colleagues [ ] reported this rate to be Á / rbc units. in a more recent report from denmark, % of rbc units contained viable bacteria which were obtained from donors years of age or older [ ] . there are avenues for improvement in existing haemovigilance systems. the addition of active surveillance components may enhance reporting. expansion of electronic medical records to incorporate more information regarding blood administration may augment comprehensiveness. adjunct programming routines could be enacted to capture pre-and post-transfusion temperatures so that detection of fnhtr could be improved. likewise, electronic capture of pre-and post-transfusion blood pressures could assist the detection of hypotensive reactions. some have already merged records from transfusion and apheresis medicine with the main electronic medical record [ ] [ ] [ ] . the scandat is a successful example of linked donor and recipient health information in sweden and denmark [ ] . in addition, international networks (e.g. international haemovigilance network [ ], global vigilance and surveillance database for medical products of human origin [ ] , world health organization global database on blood safety [ ] ) provide opportunities for surveillance and the improvement of blood safety. national haemovigilance programmes afford an important aspect of patient safety. such systems generally capture serious transfusion reactions but, with improvement, could generate more comprehensive information. crosscountry comparisons can provide the basis for discovery and continuous process improvement. decreases the incidence of febrile non- · ( · , · ) herv e p: l'organisation de l'h emovigilance en france the new japanese blood law: its impact on blood safety and usage european blood alliance: the revision of the european blood directives: a major challenge for transfusion medicine trip annual report: hemovigilance extended version transfusion-related adverse reactions reported to the national healthcare safety network hemovigilance module, united states national blood authority: australian haemovigilance report: data for - and - public health agency of canada: transfusion transmitted injuries surveillance system (ttiss): summary results for danish registration of transfusion risk (dart) hemovigilance report for adverse transfusion events in finland french agency for the safety of health products: annual haemovigilance report haeomovigilance report of the paul-ehrlich institute: assessment of the reports of serious adverse transfusion reactions japanese red cross society: nonhemolytic adverse transfusion reactions reported to jrc blood centers japanese red cross society: transfusion-related acute lung injury japanese red cross society: infectious cases of probably related to transfused blood components national haemovigilance group. national haemovigilance programme norwegian knowledge centre for the health services: monitoring of blood: haemovigilance report report of the portuguese haemovigilance system activity report of the services of blood and transfusion medicine quality and innovation: hemovigilance report from the bis -blood monitoring in sweden swissmedic haemovigilance annual report the risk of transfusion-transmitted viral infections. the retrovirus epidemiology donor study simulation-based comparison of methods for 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( )-ehc -ef transfusion-associated circulatory overload in orthopedic surgery patients: a multi-institutional study an analysis of blood management in patients having a total hip or knee arthroplasty transfusion-related acute lung injury and pulmonary edema in critically ill patients: a retrospective study incidence and transfusion risk factors for transfusion-associated circulatory overload among medical intensive care unit patients hemotherapy bedside biovigilance involving vital sign values and characteristics of patients with suspected transfusion reactions associated with fluid challenges: can some cases of transfusion-associated circulatory overload have proinflammatory aspects? characterizing the epidemiology of perioperative transfusion-associated circulatory overload transfusion-associated circulatory overload (taco) and potential risk factors among the inpatient us elderly as recorded in medicare administrative databases during reduction of febrile but not allergic reactions to rbcs and platelets after conversion to universal prestorage leukoreduction premedication with acetaminophen or diphenhydramine for transfusion with leucoreduced blood products in children a prospective, randomized, doubleblind controlled trial of acetaminophen and diphenhydramine pretransfusion medication versus placebo for the prevention of transfusion reactions febrile transfusion reaction: what blood component should be given next? characterization of reactions after exclusive transfusion of white cell-reduced cellular blood components characterization of reactions after transfusion of cellular blood components that are white cell reduced before storage prestorage universal wbc reduction of rbc units does not affect the incidence of transfusion reactions allergic transfusion reactions: an evaluation of consecutive reactions universal leukoreduction stramer sl: current perspectives in transfusion-transmitted infectious diseases: emerging and re-emerging infections blood products advisory committee, food and drug administration: strategies for implementation of antibody and nucleic acid-based testing for babesia microti in blood donors strategies for the avoidance of bacterial contamination of blood components viable bacteria associated with red blood cells and plasma in freshly drawn blood donations development of electronic medical record charting for hospital-based transfusion and apheresis medicine services: early adoption perspectives electronic health record surveillance algorithms facilitate the detection of transfusion-related pulmonary complications the new scandinavian donations and transfusions database (scandat ): a blood safety resource with added versatility the global vigilance and surveillance database for all authors contributed to the design, analysis or data interpretation; made critical revisions of the manuscript; and approved the submitted final version. key: cord- -xmq gm authors: cherednik, i. title: a surprising formula for the spread of covid- under aggressive management date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: xmq gm we propose an algebraic-type formula that describes with high accuracy the spread of covid- pandemic under aggressive management for the periods of the intensive growth of the total number of infections. the formula can be used as a powerful forecasting tool. the parameters of the theory are the transmission rate, reflecting the viral fitness and "normal" frequency of contacts in the infected areas, and the intensity of prevention measures. the duration of the period of intensive growth is essentially inversely proportional to the square root of the intensity of hard measures. a more precise formula is based on bessel functions. the data for the usa, uk, sweden, israel are provided. power law of epidemics. the simplest equation for the spread of communicable diseases results in exponential growth of the number of infections, which is mostly applicable to the initial stages of epidemics. see e.g. [ , , , ] here and below and [ ] about some perspectives with covid- . we focus on the middle stages, where the growth is no greater than some power functions in time, which requires a different approach and different equations. the equally classical logistic models of the spread, as well as the sir, sid models an generalizations, assume that the number of infections is comparable with the whole population, which we do not impose. major new epidemics were not really of this kind during the last years, which is obviously due to better disease control worldwide. the reality now is the power-type growth of total number of infections u (t) after a possible short period of exponential growth, covid- included. our approach is based on this assumption. generally, the rate of change of the total number of infections du (t)/dt is mostly related to u (t) − u (t − p), where p is the period when the infected people spread the virus in the most intensive way. assuming that u (t) = t α : du (t)/dt = αt α− and the leading term of u (t) − u (t−p) is pαt α− = pλu (t)/t, i.e. it is essentially proportional to du (t)/dt. however, if α > , there will be other terms in the expansion of u (t)−u (t−p) and the proportionality with u (t)/t can be only if either the virus transmission strength diminishes over time or we reduce our contacts over time when the total number of cases growths faster than linearly. with covid- , we attribute it mostly to the latter, to the reduction of the contacts over time of infected individuals with the rest of population, i.e. to behavioral and sociological factors. this is different from other power laws for infectious diseases; compare e.g. with [ ] . generally, if someone wants to "see" the trend of the epidemic using only the total number of infections to date, then u (t)/t is the best way of course. this is qualitative, but the corresponding differential equation du (t)/dt = c u (t)/t immediately gives that u (t) = ct c for some constant c, i.e. results in a power growth. this can be really seen for covid- and other epidemics before the active management begins. the coefficient c is approximately c ≈ for covid- initially; when the management begins, c drops and then the bessel functions describe the increase of u (t) till the "saturation", which is a technical end of the epidemic, though not its real end of course. some details. the full theory is presented in [ ] . if the number of new infections is proportional to the current number of those infected, then the exponential growth of the spread is granted. by analogy with news impact over time from [ ] , and similar sociological-type processes, it is quite likely that the number of such contacts is proportional to the current total number of the infected individuals to date divided by the time to date . the coefficient of proportionality is essentially the intensity of the spread. sociologically, it is related to the intensity of the discussion of the epidemic by the authorities in charge and everywhere, which directly influences our understanding the gravity of the situation and results in the reduction of our contacts. this can happen even before any active prevention measures begin. starting with the equation du (t)/dt = c u (t)/t for the total number of infections u (t), the coefficient of proportionality, the basic transmission rate c, is therefore a combination of the transmission strength of the virus and the "normal" frequency of the contacts in the infected place. there can be some other mechanisms for the "power law", including the biological ones. self-isolation of infected species is common not only for humans ... unless for rabies; it can grow over time and when the intensity of the spread increases. another possible mechanism can be due to the replication processes for viruses, but this is well beyond this article. anyway a sociological approach to the spread, which "explains" under some assumptions the power growth of the number of total cases, is quite natural in our work, because the active managements of epidemics is clearly of sociological nature, applicable only to humans. the coefficient c is one of the two main mathematical parameters of our theory. it can be practically seen as follows. before the prevention measures are implemented, approximately it reveals itself in the growth ∼ t c of the total number of detected infections, where t is time. for covid- , c is around , which results in the quadratic growth of total number of infections after a short period of its exponential growth. upon the active management of covid- , the growth quickly becomes t c/ , i.e. essentially linear, which is part of our theory in [ ] . this can be observed in many countries. ending epidemics. the "power law" is only a starting point of our analysis. the main problem is of course to "add" here some mechanisms ending epidemics and then prevent their possible recurrence. these are major challenges, biologically, psychologically, sociologically and mathematically. one can expect this megaproblem to be well beyond the power law itself, but we demonstrate that mathematically there is a path based on bessel functions. the power growth alone obviously cannot lead to any saturation. we are of course fully aware of the statistical nature of the problem, but the formula for the growth of the total number of infections we propose works almost with an accuracy of fundamental physics laws. this is very surprising for such stochastic processes as epidemics. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint an important outcome of our modeling is that the measures of "hard type", like detecting and isolating infected people and closing the places where the spread is almost inevitable, are the key for ending an epidemic. moreover, such measures must be employed strictly proportionally to the current numbers of infections, not to its derivative of any kind, which is the most aggressive "momentum" way to react to them, the "hardest" possible as we will explain now. for instance, if the number of infections doubled during some period, then the operation formula from [ ] requires that testing must be increased -fold. assuming that we approach the saturation, where the number of new infections becomes almost zero, the "hard way" is to keep testing at the same level as before, i.e. performing the same number of tests every day. this is in spite of almost zero number of new infections. practically, this is not always the case. if we react to the average number instead of the absolute number of infections, then we are supposed to stop testing and other measures when there are no new "cases". this is the "soft way". if this is coupled with "soft" measures (see below), then mathematically the epidemic will never reach the saturation point. the u-formula. the intensity of "hard" measures will be denoted by a in this article. upon composing the corresponding differential equations and integrating them we obtain the following. the function for the bessel function j α (x) of the first kind models the growth total number of infections, where c is the scaling parameter necessary to adjust this formula to the actual numbers. here and below time t is normalized days/ for the number of days from the beginning of the period of the intense growth of the epidemic. this functions matches the total number of infections with high accuracy. the parameter a is . for the usa and uk; it can be about . for countries with somewhat more proactive approach, like austria and israel, and it becomes . and even . for "the world" and sweden. the basic transmission rates are: c = . for the usa, c = . for uk, sweden, and it is currently . for "the world". recall that c approximately corresponds to the growth ∼ t c of total infections in the beginning of the epidemic, when no protection measures are in place. limitations. importantly, the function u(t) is proportional to t c/ + for t near t = , which is very close to ∼ t c , assuming that c ≈ . so the phase of "parabolic growth" is generally covered well by the u-formula too, though formally this does not follow from the theory in [ ] . of course there can be other reasons for u(t) to "serve epidemics"; it is not impossible that there are connections to the replication process of viruses, but this we do not touch upon. as always, there are limitations, which we will address now. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint first and foremost, the available infection numbers are for the detected cases, which are mostly symptomatic. however this is not of much concern for us. we understand managing epidemics sociologically. those who are detected mostly have symptoms, but the management is mostly focused on them too. so all our arguments fully applicable within this group; we just restrict ourselves with symptomatic cases. no assumptions on asymptomatic cases are necessary for the u-formula. we of course understand that when the number of new reported infections drops to zero, there can be many non-detected asymptomatic cases, which can potentially lead to the recurrence of the epidemic. such "saturation" is only a technical end of the epidemic. the second reservation concerns newly emerged clusters of infections and the countries where the spread is on the rise. the u-formula can be used in spite of such fluctuations, but it is now a statistical tool; see figure . the predictions must be regularly updated. the third reservation is related to the management of the epidemic. not all countries employ the protection measures in similar ways, but this is not a problem for us. the problem is if the intensity of the measures and the criteria are changed in some unpredictable ways. diminishing the "hard" measures too early or even dropping them completely at later stages is quite possible. last but not the least is the data quality . changing the ways they are collected and the criteria frequently makes such data useless for us. though, if the number of detected infections is underreported in some regular ways, whatever the reasons, such data can be generally used. thus this is sufficiently relaxed, but the data for several countries, not too many, are not suitable for the usage of our "forecasting tool". forecasting the spread. with these reservations, the first point of maximum t top of u(t) is a good estimate for the duration of the epidemic and the corresponding estimate for the top value of the total number of infections. this gives an important "forecasting tool", assuming that "hard measures" are the key in practical management of the epidemic. we note that the approximate reflection symmetry of du(t)/dt for the u(t) in the range from t = to t top can be interpreted as farr's law of epidemics under aggressive management. generally, the portions of the corresponding graph before and after the turning point are supposed to be essentially symmetric to each other. this is not exactly true for du(t)/dt, but close enough. see figure and the other ones; the turning point is at max{du(t)/dt}. as any model, our one is based on various simplifications. we assume that the number of people perceptive to the virus is unlimited, i.e. we do not consider epidemics with the number infections comparable with the whole population, as well as herd immunity . also, we disregard . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint the average duration of the disease and that for the quarantine periods imposed. the total number of infections regardless of the output is what we are going to model, which is commonly used. in spite of all these assumptions, the u-formula works unexpectedly well. we mention here a strong connections with behavioral finance ; see [ ] . for instance, practically the same u(t) in terms of bessel function as above models "profit taking" in stock markets. the polynomial growth of u(t) is parallel to the "power law" for share prices. there is a long history and many aspects of mathematical modeling epidemic spread; see e.g. [ ] for a review. we restrict ourselves only with the dynamic of momentum managing epidemics , naturally mostly focusing on the middle stages, when our actions must be as precise as possible. the two basic modes we consider are essentially as follows: (a) aggressive enforcement of the measures of immediate impact reacting to the current absolute numbers of infections and equally aggressive reduction of these measures when these numbers decrease; (b) a more balanced and more defensive approach when mathematically we react to the average numbers of infections to date and the employed measures are of more indirect and palliative nature. hard and soft measures. the main examples of (a)-type measures are: prompt detection and isolation of infected people and those of high risk to be infected, and closing places where the spread is the most likely. actually the primary measure here is testing ; the number of tests is what we can really implement and control. the detection of infected people is its main purpose, but the number of tests is obviously not directly related to the number of detections, i.e. to the number of positive tests . the efficiency of testing requires solid priorities, focus on the groups with main risks, and solving quite a few problems. even simple mentioning problems with testing, detection and isolation is well beyond our article. however, numerically we can use the following. during the epidemics, essentially during the stages of linear growth, which are the key for us, the number of positive tests can be mostly assumed a stable fraction of the total number of tests. this is demonstrated in figure . such proportionality can be seen approximately from march . however, at the end of this chart, testing was reduced below the levels required for mode (a); it must remain at least constant until the saturation of the number of total cases. the measures like wearing protective masks, social distancing, recommended self-isolation, restricting the size of events are typical for (b). this distinction heavily impact the differential equations we obtained in [ ] . however the main difference between the modes, (a) vs. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . hard measures are the key. generally, the (a)-type approach provides the fastest possible and "hard" response to the changes with the number of infections, whereas we somewhat postpone with our actions until the averages reach proper levels under (b), and the measures we implement are "softer". mathematically, the latter way is better protected against stochastic fluctuations, but (b) is slower and cannot alone lead to the termination of the epidemic, which we justify mathematically within our approach. the main objective of any managing epidemics is to quickly end them. however the excessive usage of hard measures can lead to the recurrence of the epidemic, some kind of "cost" of our aggressive interference in a natural process. this can be avoided only if we continue to stick to the prevention measures as much as possible even when the number of new infections goes down significantly. reducing them too much on the first signs of improvement is a way to the recurrence of the epidemic, which we see mathematically within our approach. some biological aspects. the viral fitness is an obvious component of the transmission rate c. its diminishing over time can be expected, but this is involved. this can happen because of the virus replication errors. the rna viruses, covid- included, replicate with fidelity that is close to error catastrophe. see e.g. [ ] for some review and predictions. such matters are well beyond this paper, but one biological aspect must be mentioned, concerning the asymptomatic cases. the viruses mutate at very high rates. they can "soften" over time to better coexist with the hosts, though fast and efficient spread is of course the "prime objective" of any virus. such softening can result in an increase of asymptomatic cases, difficult to detect. so this can . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint contribute to diminishing c we observe, though this is not because of the decrease of the spread of the disease. we model the available (posted) numbers of total infections, which mostly reflect the symptomatic cases. to summarize, it is not impossible that the replication errors and "softening the virus" may result in diminishing c at later stages of the epidemic, but we think that the reduction of the contacts of infected people with the others dominates here, which is directly linked to behavioral science, sociology and psychology. . the formula for the growth.. we will need the definition of the bessel functions of the first kind: . the key point is that measures of type (a) have "ramified" consequences, in some contrast to mode (b). namely, an isolated infected individual will not transmit the virus to many people and the number of those protected due to this isolation grows over time. combining this with our understanding of the power law of epidemics, we arrive at the differential equation for the total number of infections u(t): there is a surprisingly perfect match of the total number of infections for covid- in the usa and uk till april with our solutions u(t) above. this is from the moment when these numbers begin to grow "significantly", approximately around march for the usa and uk. epidemics are very stochastic processes, so such a precision is surprising. the site https://ourworldindata.org/coronavirus is mostly used for data, updated at : london time. we take x = days/ . the usa data. the scaling coefficient . in figure is adjusted to match the real numbers. for the usa, we set y = infections/ k, and take march the beginning of the period of "significant growth". the parameters are c = . , a = . . the red dots show the corresponding actual total numbers of infections. they perfectly match u(t) = . t . j . (t √ . ) in figure , which results in the following: the number of cases in the usa can be expected to reach its preliminary saturation at t top = . ( . days . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint from / to may ) with u top = . , i.e. with infections (it was at / ). this is of course a projection: about m of total cases at the saturation point t top near may . the black dots show the test period, till april . the predictions are of course based on the assumption that the intensity of hard measures continues to be proportional to the total number of detected infections to date, as it was clearly the case for the red dots. the jumps like the one at about x = . can be because of various reasons impossible to forecast, as well as some period of linear growth after it. however the general trend for black dots matches our u(t) well enough. obviously t = t top cannot be the end of the epidemic. the data from south korea and those from other countries that went through the "saturation", demonstrate that a linear growth of the total number of cases can be expected around and after t top , with periodic fluctuations. the obvious reasons are (a) reducing the "hard" and "soft" measures, (b) no country is isolated from infections from other places, (c) continued testing can result in finding more asymptomatic cases, (d) new clusters of disease are always possible. anyway, fluctuations closer to the "saturation" and after it are quite likely. resuming the corresponding measures is possible, but this is not always the case. our formula is just a forecasting tool, which is mostly applicable to the period when the protection measures, especially "hard" ones, are applied in a regular manner. then the match with the real data is good. covid- in uk. one of the reasons of the strong match of red dots and our u(t) can be that the usa consists of states, and therefore the total number of infections is quite an average. however this is . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint no worse for uk. the data will be from / till / ; add to our "red dots", the initial number at / , to match the actual total numbers. the black dots constitute the control period: / - / . now c = . , a = . work fine, and the scaling coefficient is . . the total number of cases is divided by k, not by k as for the usa. the expectation is now as follows: the "saturation moment" can be . , i.e about days after march , somewhere around may . the estimate for corresponding number of infections is about , with all ifs. this is assuming that the "hard" measures will be employed at the same pace as before april . the black dots confirm the trend. sweden / - / . this is an example of the country that remains essentially "open". actually, they actively do testing followed by the isolation of infected people, the key "hard" measure from our perspective. also, the strength of the health-care in this country must be taken into consideration, and that it is surrounded by the countries that fight covid- aggressively. the growth of the total number of cases was essentially quadratic for a relatively long period, which is what "power law" states for the epidemics with minimal "intervention". by now, the growth is linear, which means that what the measures they use appeared working. interestingly, our u-formula is applicable, but for record law a = . ; it is a = . for "the world". here y is the total number of cases divided by and add , the initial value, to our "dots". the projected saturation is around may . as always, it is with a reservation about their future policies, which seem sufficiently stable by now. in spite of their "soft" approach, our u-formula obviously can be used; see figure . . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . israel: "saturation". the last example we provide is what can be expected when the country went through the "saturation". israeli population is diverse, which has a potential of significant fluctuations of the number of cases and various clusters of infection. however its solid response to covid- and good overall health-system, made the growth of the spread sufficiently predictable. generally, for small countries the fluctuations can be expected higher than for countries like the usa, uk, though it depends on how "homogeneous" they are. we divide the total number of cases by , as for sweden; see figure . the red dots began march , when the total number of detected infections was , and stopped april ; the remaining period till april , shown by the black dots, was the "control one". the saturation forecast went through almost perfectly, but there were significant fluctuations in process. after april , the predicted moment of the saturation, the growth of the total number of (known) infections is supposed to be mild linear. the parameters are: a = . , i.e. the intensity of hard measures is better than with the usa, uk, and c = . . the latter means that the initial transmission coefficient was somewhat worse than c = . in the usa, uk, possibly due to the greater number of "normal" contacts. recall that a, c are parameters of our theory, related to but not immediately connected with the real factors. some discussion. recall that we consider only "total cases", the numbers of all detected infections, and begin at the moment when the "significant" growth begins, which is also essentially when the active measures start. according to our theory, the match with u(t) above can be expected in some interval around the turning point. however . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . practically it holds better than this: an almost perfect match is for about % of the whole periods of intensive growth. our restriction to the period till april , with fixing the parameters a, c, except for sweden, is not accidental. it was sufficient for the practical confirmations of the theory presented in figures , , and with other countries. the latest data, the black dots, provide the "realtime checks"; they were obtained after the parameters were fixed. the pandemic is far from over, but within the scope of this paper, the data we needed for the "forecasting tool" were essentially present by / . when the epidemic approaches the saturation, which is the first maximum of u(t) in our model, its management can be expected to evolve toward reducing and abandoning "hard" measure. this can result in a growth like ct c/ with some "mild" c and c/ ≈ and significant fluctuations, which really happens. such growth at the end is generally covered by our theory; it is not connected with bessel functions. the superb match of our u(t) during the significant part of the period of intensive growth of the spread can be of real importance for practical managing epidemics. on the basis of what we see, the best ways to use the u-curves seem as follows: ( ): determine a, c using about the first - % after the intensive growth begin, ( ): update them constantly till the turning point and somewhat beyond, ( ): try to the adjust the measures at later stages "to stay close to the curve". with ( ), the constant response is needed to new clusters of the disease, some jumps with the new cases due to the reductions of the measures and so on. generally, our forecasting tool can serve the best if the data and the measures are as uniform and "stable" as possible. then underreporting, focusing on symptomatic cases, and inevitable fluctuations with . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint the data may not influence too much the match with the u-curves; this is of course statistically and with usual reservations. also, ( ): testing the population and some "soft" measure must be continued well after the "saturation" to prevent the recurrence of the epidemic. the mathematics of infectious diseases periodicity in epidemiological models power-law models for infectious disease spread epidemic psychology: a model modeling infectious disease dynamics momentum managing epidemic spread and bessel functions artificial intelligence approach to momentum risk-taking are rna viruses candidate agents for the next global pandemic? a review a treatise on the theory of bessel functions acknowledgements. i'd like to thank eth-its for outstanding hospitality. my special thanks are to giovanni felder, rahul pandharipande. i also thank very much david kazhdan for his valuable comments and suggestions. funding: partially supported by nsf grant dms- and the simons foundation. key: cord- - g uo rd authors: tomani, jean claude didelot; kagisha, vedaste; tchinda, alembert tiabou; jansen, olivia; ledoux, allison; vanhamme, luc; frederich, michel; muganga, raymond; souopgui, jacob title: the inhibition of nlrp inflammasome and il- production by hibiscus noldeae baker f. derived constituents provides a link to its anti-inflammatory therapeutic potentials date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: g uo rd the activation of nod-, lrr-, and pyrin domain-containing protein (nlrp ) inflammasome and/or its components is associated with the physio-pathogenesis of many respiratory diseases including asthma, copd (chronic obstructive pulmonary disease), sars cov- (severe acute respiratory syndrome coronavirus ), and in several autoimmune diseases. hibiscus noldeae baker f. has been widely reported to be traditionally used in the treatment of different ailments, some of which are of inflammatory background such as asthma, wounds, headache, etc. however, the claims have not been supported by evidence at the molecular and functional levels. here, we report on the bio-guided fractionation of h. noldeae and assessment of the inhibitory properties of some fractions and purified compounds on nlrp inflammasome and interleukin (il- ). the activation of the nlrp inflammasome was determined by detecting the activity of caspase- and the production of interleukin β (il- β) in lipopolysaccharide (lps) and atp-stimulated tamm-horsfall protein (thp- ) macrophages, while the production of il- was studied in lps-stimulated raw . mouse macrophages. it was observed that hexane and ethyl acetate fractions of the crude extract of the aerial parts of h. noldeae, as well as caffeic acid, isoquercetin, and er . and er . fractions revealed significant inhibitory effects on caspase- activities, and on il- β and il- production. the er . and er . fractions downregulated the production of il- β and il- , in a similar range as the caspase- inhibitor ac-yvad-cho and the drug dexamethasone, both used as controls, respectively. overall, our work does provide the very first scientific based evidence for hibiscus noldeae anti-inflammatory effects and widespread use by traditional healers in rwanda for a variety of ailments. respiratory diseases, with asthma being the most important, are classified among the major categories of non-communicable diseases and their prevalence accounted for more than billion patients worldwide in [ , ] . asthma is a chronic inflammatory disease resulting from the malfunction of the interaction between the airways and the immune cells [ , ] . inflammation is considered to be a physiological defense mechanism which helps the body to protect itself from microbial infections, burn, toxic chemicals, allergens, or other harmful stimuli. depending on the number or the degree of events and mediators linked to that reaction, inflammatory reaction can trigger, maintain, or aggravate many diseases [ ] . sometimes the delicate balance between immune-mediated clearance and noxious stimuli is troubled and this correlates with inadvertent self-harm from chronic inflammation. as a result, the immune system inappropriately leads to inflammation against its own cells, resulting in different chronic inflammatory diseases including respiratory diseases such as asthma and chronic obstructive pulmonary disease (copd) [ ] . recently, the complicated form of sars cov- (severe acute respiratory syndrome coronavirus ) viral pneumonia outbreak was reported to be associated with a plethora of cytokines wreaking havoc throughout the body often vividly referred to as "cytokine storm", which triggers a violent inflammatory immune response that contributes to respiratory problems, multiple organ failure, and finally death in severe cases of sars-cov- infection [ ] [ ] [ ] [ ] . several studies have reported the role of different inflammatory cells and mediators in the pathogenesis of asthma where the volume and nature of involved inflammatory mediators determine the severity and exacerbations of the disease. depending on those inflammatory patterns, there are two main subtypes of asthma, the eosinophilic (diagnosed by the expression of th -derived cytokines) and the neutrophilic (diagnosed by the expression of th -derived cytokines and chemokines) [ ] . the latter asthma subset is characterized by a high proportion of neutrophils recruited by inflammatory cytokines like interleukin β (il- β), interleukin (il- ), and tumor necrosis factor alpha (tnf-α). it is well known that inflammasomes, multiprotein cytoplasmic complexes activating inflammatory responses, are involved in the upregulation of these cytokines, particularly the il- β through the activation of caspase- [ , ] . among these inflammasomes, nod-, lrr-, and pyrin domain-containing protein (nlrp ) is the most widely studied and its properties and functions are the best characterized [ ] . in fact, in response to a microbial infection or injury, the nlrp inflammasome is activated and involved in the host protection by inducing immune responses to clear injury or microbial invasion [ , ] . the activation of nlrp inflammasome requires two distinct signals. the first is a pro-inflammatory priming step, mediated by toll-like receptors (tlr), that induces the upregulation of the nf-κb transcription factor and promotes the expression of pro-il- β and the nlrp inflammasome component. the second signal is an activation step that induces the assembly of the nlrp / apoptosis-associated speck-like protein containing a caspase recruitment domain (asc)/pro-caspase- complex, ending up in the proximity auto-cleavage and maturation of caspase- which, in turn, induces a caspase- -mediated maturation of pro-il- β and release of active il- β [ ] . several pieces of evidence from research suggest that inflammasomes and/or their components and effectors are associated with the physio-pathogenesis of asthma and other respiratory diseases including copd, sars cov- , etc. [ ] [ ] [ ] [ ] . in fact, it has been reported that the excessive release of active il- β and asc (apoptosis-associated speck-like protein containing a caspase recruitment domain) specks leads to the induction of neutrophil-dominated inflammatory responses in the airways that contributes to the pathogenesis of copd and neutrophilic asthma [ , ] . the neutrophilic asthma is the most severe and has been reported to be refractory to the corticoid-based treatment [ ] . despite this resistance, the use of corticosteroids remains the basic treatment for several respiratory diseases involving cytokine storms. a recent clinical trial report revealed that the treatment with dexamethasone significantly alleviated covid- disease severity of hospitalized patients who were under invasive mechanical ventilation or oxygen [ ] . regarding the neutrophilic asthma, drugs that alleviate disease severity include specific monoclonal antibodies against key mediators of the inflammatory pathway [ ] but the associated treatment cost is not affordable for low and middle income countries (lmics). hence, urgent efforts are needed to identify new, effective, affordable, and safe anti-inflammatory drugs. a reliable therapeutic source which remains poorly explored is the traditional pharmacopeia. previously, our investigations on the anti-asthmatic activities of herbal recipes from rwandese traditional healers revealed a candidate recipe with significant inhibitory properties on the activation of caspase- using caspase-glo inflammasome assays [ ] . hibiscus noldeae, from the hibiscus genus in the malvaceae family, was one of the plants composing the recipe. plants from the same hibiscus genus, including h. acetosella, h. sabdariffa, and h. vitifolius have been reported to have anti-inflammatory potentials and some of the responsible molecules, such as gossypin, caffeoylhydroxycitric acid, delphinidin- -sambubioside, and neochlorogenic acid have been isolated [ ] [ ] [ ] . in contrast, no such studies has been reported on h. noldeae, although used by traditional healers in the treatment of asthma, urogenital infections, postpartum abdominal pain, swelling of the legs, cleaning of babies, facilitation of delivery, sprains, headache, wounds, women infertility, abortion prevention, etc. [ ] . in this study, we report on the bio-guided fractionation of h. noldeae and assessment of the inhibitory properties of some fractions and purified compounds on nlrp inflammasome and il- . the activation of the nlrp inflammasome was determined by detecting the activity of caspase- and the production of il- β in lipopolysaccharide (lps)-and atp-stimulated thp- macrophages, while the production of il- was studied in lps-stimulated raw . mouse macrophages, a well-known standard method. it was observed that hexane and ethyl acetate fractions of the aerial parts of h. noldeae, as well as caffeic acid, isoquercetin, and er . and er . fractions revealed the highest inhibitory effects on caspase- activities, and on il- β and il- production. the er . and er . , our fractions of most interest, downregulated the production of il- β and il- , in a similar range as the caspase- inhibitor ac-yvad-cho and the drug dexamethasone all used as controls, respectively. overall, our findings pave the way for the first time on the scientific based evidence on hibiscus noldeae activities underlying its potential role in the treatment of inflammatory diseases and asthma in rwanda. hibiscus noldeae (h. noldeae) has been studied by virtue of its attribute as one of the medicinal plants used by the traditional healers in rwanda to manufacture their remedies. in the context of asthma treatment, the recipe, containing different plants, that revealed the best anti-inflammatory activity [ ] comprised h. noldeae in major proportion ( %). we therefore hypothesized that this medicinal plant may harbor the therapeutic constituents. a bio-guided fractionation approach was employed and as shown in figure , a complex chemical profile for hexane and ethylacetate fractions was observed in the high performance liquid chromatography with diode-array detection (hplc-dad) analysis, indicating the presence of different major substances of different polarities. many peaks eluted between and min, a fact that demonstrates the presence of medium polarity molecules, most of which are major compounds. the transposition of the analytical method to the preparative hplc allowed for the isolation of five of the seven major compounds from ethylacetate and hexane fractions of h. noldeae. three of the five isolated compounds were pure and were identified by chromatographic and/or spectroscopic comparison with data from literature. this led to the elucidation of one flavone glycoside, namely quercetin -o-glucopyranoside, along with two polyphenols, caffeic acid and caffeoyl-hydrocitric acid (supplementary table s and figures s -s ). noldeae. an in-house hplc method used to screen the major components of crude extract was used. analytical separation was carried out on a hypersil ods ® rp column ( × . µm; particle size µm; thermofisher scientific, waltham, ma, usa). all samples were dissolved in methanol hplc-grade, filtered through a . µm pore size filter membrane and analyzed on an agilent hplc machine. samples were eluted with a nonlinear gradient method with acetonitrile and . % trifluoroacetic acid in ultra-pure water. the column temperature was maintained at °c. then µl of each sample were injected into the hplc-uv/dad (diode-array detection) system and the analysis, performed at a flow rate of . ml/min, was monitored at , , , and nm. prior to the investigation of anti-inflammatory properties of the compounds of interest, their effect on the cell viability on both thp- monocytes and derived macrophages was first determined. cells were treated with different concentrations ( , , , and µg/ml) for h, and the cell viability was determined using celltiter-glo luminescent cell viability assay kit (promega co., madison, wi, usa). up to µg/ml, all tested compounds did not exhibit cytotoxicity to both thp- and raw . cell lines ( figures s and s ). our anti-inflammatory activities were therefore determined within this range. to investigate whether the isolated compounds and fractions of interest possessed potential nlrp inflammasome inhibitory effects, their influence on the nlrp inflammasome activation, in lps and atp-stimulated thp- -derived macrophages was assessed. the levels of caspase- activities in the culture media were determined using the caspase-glo ® inflammasome assay (promega co., madison, wi, usa). as shown in figure below, it was observed as expected that lps and atp treatment significantly (*** p < . ) increased caspase- activities in the cell culture supernatant when compared with the untreated cells. however, when cells were pretreated with the isolated compounds and fractions of interest in graded concentrations ranging from to µg/ml, the caspase- activities were reduced by at least %. the tested compounds revealed their maximal inhibitory activities at µg/ml, except for the er . fraction that showed a dose-dependent inhibition of the caspase- activation (figure ) . noldeae. an in-house hplc method used to screen the major components of crude extract was used. analytical separation was carried out on a hypersil ods ® rp column ( × . µm; particle size µm; thermofisher scientific, waltham, ma, usa). all samples were dissolved in methanol hplc-grade, filtered through a . µm pore size filter membrane and analyzed on an agilent hplc machine. samples were eluted with a nonlinear gradient method with acetonitrile and . % trifluoroacetic acid in ultra-pure water. the column temperature was maintained at • c. then µl of each sample were injected into the hplc-uv/dad (diode-array detection) system and the analysis, performed at a flow rate of . ml/min, was monitored at , , , and nm. prior to the investigation of anti-inflammatory properties of the compounds of interest, their effect on the cell viability on both thp- monocytes and derived macrophages was first determined. cells were treated with different concentrations ( , , , and µg/ml) for h, and the cell viability was determined using celltiter-glo luminescent cell viability assay kit (promega co., madison, wi, usa). up to µg/ml, all tested compounds did not exhibit cytotoxicity to both thp- and raw . cell lines ( figures s and s ). our anti-inflammatory activities were therefore determined within this range. to investigate whether the isolated compounds and fractions of interest possessed potential nlrp inflammasome inhibitory effects, their influence on the nlrp inflammasome activation, in lps and atp-stimulated thp- -derived macrophages was assessed. the levels of caspase- activities in the culture media were determined using the caspase-glo ® inflammasome assay (promega co., madison, wi, usa). as shown in figure below, it was observed as expected that lps and atp treatment significantly (*** p < . ) increased caspase- activities in the cell culture supernatant when compared with the untreated cells. however, when cells were pretreated with the isolated compounds and fractions of interest in graded concentrations ranging from to µg/ml, the caspase- activities were reduced by at least %. the tested compounds revealed their maximal inhibitory activities at µg/ml, except for the er . fraction that showed a dose-dependent inhibition of the caspase- activation (figure ). then, µl of the culture supernatant were used to determine the caspase- activity using caspase-glo inflammasome assay (promega co., madison, wi, usa). the results are presented as the mean ± sd from independent experiments (n = ), each done in triplicate. a one-way anova, followed by benforroni's multiple comparison test, was used to compare the sample groups. *** means caspase- fold activation changes with a p < . . next, the production of il- β was investigated using the human il- β (enzyme linked immunosorbent assay) elisa kit (invitrogen/thermofisher, waltham, ma, usa). as controls, two distinct reference inhibitors, namely ac-yvad-cho (yvad) which specifically acts on caspase and dexamethasone (dex) which downregulates the nf-κb pathway were used. as results, it was observed as expected that lps and atp treatment significantly (*** p < . ) increased the production of il- β in the cell culture supernatant when compared with the untreated cells ( figure a ). when cells were first treated with h. noldeae constituents or the controls, the production il- β was significantly inhibited. reductions of more than % could be revealed in some cases ( figure a ). moreover, it was observed that, at equivalent dose of µg/ml, er . fraction and dexamethasone showed similar inhibitory effects, while at the same concentration other tested constituents of h. noldeae revealed inhibitory activities comparable to the caspase reference inhibitor yvad. isoquercetin was observed to have the lowest inhibitory effect on il- β production ( figure a ). cells were then stimulated by atp ( mm) for additional h. the supernatants were harvested and kept at − • c before analysis. then, µl of the culture supernatant were used to determine the caspase- activity using caspase-glo inflammasome assay (promega co., madison, wi, usa). the results are presented as the mean ± sd from independent experiments (n = ), each done in triplicate. a one-way anova, followed by benforroni's multiple comparison test, was used to compare the sample groups. *** means caspase- fold activation changes with a p < . . next, the production of il- β was investigated using the human il- β (enzyme linked immunosorbent assay) elisa kit (invitrogen/thermofisher, waltham, ma, usa). as controls, two distinct reference inhibitors, namely ac-yvad-cho (yvad) which specifically acts on caspase and dexamethasone (dex) which downregulates the nf-κb pathway were used. as results, it was observed as expected that lps and atp treatment significantly (*** p < . ) increased the production of il- β in the cell culture supernatant when compared with the untreated cells ( figure a ). when cells were first treated with h. noldeae constituents or the controls, the production il- β was significantly inhibited. reductions of more than % could be revealed in some cases ( figure a ). moreover, it was observed that, at equivalent dose of µg/ml, er . fraction and dexamethasone showed similar inhibitory effects, while at the same concentration other tested constituents of h. noldeae revealed inhibitory activities comparable to the caspase reference inhibitor yvad. isoquercetin was observed to have the lowest inhibitory effect on il- β production ( figure a) . concentrations of µm yvad and µg/ml dex were used as controls. cells were then stimulated by atp ( mm) for additional h. supernatants were harvested and kept at − °c before analysis. for the elisa analysis, samples were diluted times in the assay diluent. the concentration of il- β in the culture supernatant was measured using a human il- β enzyme linked immunosorbent assay (elisa) kit (invitrogen/thermofisher, waltham, ma, usa) according to the manufacturer's instructions. for each experiment, the amount of il- β produced without any pretreatment was considered as % and the remaining treatments were calculated as (treatment/lps) × . the results are presented as the mean ± sd of a triplicate from a representative of three independent experiments (n = ). a one-way anova, followed by benforroni's multiple comparison test, was used to compare the sample groups. *** stands for p < . . (b) immunoblotting of cell lysates by anti-proil- β (cell signaling technology, danvers, ma, usa) or anti-pro caspase- (abcam, cambridge, uk) was done twice according to the manufacturer's instructions. membranes were then stripped, reprobed with anti-β-actin antibodies and exposed again to detect the internal β-actin standard. it is well-known that the activation of the nlrp inflammasome induces the activation of caspase- that promotes the production of gasdermin d. the n-terminal domain of gasdermin d oligomerizes to form cytolytic pores and leads to the programmed cell death called "pyroptosis" [ ] . therefore, we investigated the effect of our compounds and fractions of interest on inflammasome activation-induced cell death by measuring the release of lactate dehydrogenase (ldh) in the culture concentrations of µm yvad and µg/ml dex were used as controls. cells were then stimulated by atp ( mm) for additional h. supernatants were harvested and kept at − • c before analysis. for the elisa analysis, samples were diluted times in the assay diluent. the concentration of il- β in the culture supernatant was measured using a human il- β enzyme linked immunosorbent assay (elisa) kit (invitrogen/thermofisher, waltham, ma, usa) according to the manufacturer's instructions. for each experiment, the amount of il- β produced without any pretreatment was considered as % and the remaining treatments were calculated as (treatment/lps) × . the results are presented as the mean ± sd of a triplicate from a representative of three independent experiments (n = ). a one-way anova, followed by benforroni's multiple comparison test, was used to compare the sample groups. *** stands for p < . . (b) immunoblotting of cell lysates by anti-proil- β (cell signaling technology, danvers, ma, usa) or anti-pro caspase- (abcam, cambridge, uk) was done twice according to the manufacturer's instructions. membranes were then stripped, reprobed with anti-β-actin antibodies and exposed again to detect the internal β-actin standard. it is well-known that the activation of the nlrp inflammasome induces the activation of caspase- that promotes the production of gasdermin d. the n-terminal domain of gasdermin d oligomerizes to form cytolytic pores and leads to the programmed cell death called "pyroptosis" [ ] . therefore, we investigated the effect of our compounds and fractions of interest on inflammasome activation-induced cell death by measuring the release of lactate dehydrogenase (ldh) in the culture supernatants. as expected, the pretreatment of cells by our test compounds significantly inhibited atp-induced pyroptosis ( figure s ). nlrp inflammasome-associated il- β production is a two-step process: a priming signal that promotes pro-il- β and nlrp synthesis and a secondary signal that induces the inflammasome assembly and the subsequent caspase- activation [ ] . we investigated the effect of the crude extract and fractions of interest on the expression of pro-il- β and pro-caspase- by western blot (wb). we observed as shown in figure b that cells pretreatment with er . and er . fractions led to the reduced pro-caspase- amount marked by the faint wb-signals. this suggest that our fractions of interest inhibited the expression of this protein by impacting the first priming step of the nlrp inflammasome activation or induced its degradation by a mechanism to be clarified. moreover, the pro-il- β production appeared induced as marked by the width of the wb-signals ( figure b ). knowing the critical role of caspase- in the inflammasome activation, these observations suggest that the reduced expression of the pro-caspase by both er . and er . fractions may in fact be acting to inhibit the upregulation of caspase- , hence block the pro-il- β cleavage. in addition, treatment with dexamethasone, the crude extract and the etoac fraction revealed reduced pro-il- β wb-signals. while the effect of dexamethasone is associated with the inhibition of the nf-κb activities leading to the repression of pro-il- b production, the effects of the crude extract and etoac fraction could results from the dilution of the er . and er . fractions or likely their effects on nf-kb activities by their constituents to be identified. interleukin (il- ) like il- β is a biomarker of inflammasome activation. hence, the effect of the fractions and purified compounds from h. noldeae on the production of il- in raw . macrophages supernatants by elisa was investigated. dexamethasone at concentration of µg/ml was used as control. treatment of raw . cells with lps alone resulted in a significant increase in this cytokine production as compared to the control group. as expected, a pre-treatment of cells with dexamethasone reduced about % of the production of il- . also, it was observed that the levels of il- production were significantly (*** p < . ) decreased upon pre-treatment of cells with h. noldeae constitutuents. moreover, while a saturation of inhibitory effects was already observed at either or µg/ml with the er . fraction or with the crude extract and the etoac fraction, a dose-dependent inhibition of il- production was revealed by the caffeic acid and er . fraction pre-treatment. at µg/ml, the inhibitory effect of er . fraction was similar to that of dexamethasone ( figure ). inhibition of il- production by h. noldeae constituents. cells were pre-treated with crude extract (a), etoac fraction (b) and partially purified er . (d) and er . (e) fractions, and caffeic acid (c) for h and stimulated by lps ( µg/ml) for h. dexamethasone (dex) at µg/ml was used as a control. cell-free supernatants were harvested and stored at − °c until analysis. the concentration of il- in the culture supernatant was measured using il- elisa kit (bd biosciences) according to the manufacturer's instructions. for each experiment, the amount of il- produced was considered as % and the remaining treatments was calculated as (treatment/lps) × . the results are presented as the mean ± sd from a triplicate of the representative experiment. a one-way anova, followed by benforroni's multiple comparison test, was used to compare the sample groups. *** stands for a p < . . an important body of studies have proven the associations between neutrophilic asthma and increased il- β responses induced by the excessive nlrp inflammasome activation. this raises the possibility of targeting different inflammasome components as substantial therapeutic targets in neutrophil-dominated forms of severe, steroid-refractory asthma [ ] . the analysis of inflammasome formation is mostly investigated through the monitoring of downstream processes and outcomes, the presence of cleaved caspase- enzyme being the most widely used method, as well as the caspase- activity on substrates such as pro-il- β, pro-il- , and pro-gasdermin-d [ ] . in view of determining the inhibitory properties of the h. noldeae constituents of interest on nlrp inflammasome, their effect on either caspase- activation using the caspase glo assays (promega, madison, wi, usa) and/or il- β production as hallmarks of the nlrp inflammasome was evaluated. in fact, it is well known that the inflammasome activation induces a cascade of events including the recruitment of pro-caspase- and its cleavage into a corresponding active caspase- enzyme that also cleaves pro-il- β into its mature form, il- β [ ] . in our previous study, among the plant recipes evaluated, r sz was the most potent in inhibiting caspase- activity [ ] . hibiscus noldeae, one of the plant's components of the recipe, has been cited by three different traditional healers during our investigation, indicating its possible importance in the treatment of asthma. in addition, few studies reported on the traditional use of the . inhibition of il- production by h. noldeae constituents. cells were pre-treated with crude extract (a), etoac fraction (b) and partially purified er . (d) and er . (e) fractions, and caffeic acid (c) for h and stimulated by lps ( µg/ml) for h. dexamethasone (dex) at µg/ml was used as a control. cell-free supernatants were harvested and stored at − • c until analysis. the concentration of il- in the culture supernatant was measured using il- elisa kit (bd biosciences) according to the manufacturer's instructions. for each experiment, the amount of il- produced was considered as % and the remaining treatments was calculated as (treatment/lps) × . the results are presented as the mean ± sd from a triplicate of the representative experiment. a one-way anova, followed by benforroni's multiple comparison test, was used to compare the sample groups. *** stands for a p < . . an important body of studies have proven the associations between neutrophilic asthma and increased il- β responses induced by the excessive nlrp inflammasome activation. this raises the possibility of targeting different inflammasome components as substantial therapeutic targets in neutrophil-dominated forms of severe, steroid-refractory asthma [ ] . the analysis of inflammasome formation is mostly investigated through the monitoring of downstream processes and outcomes, the presence of cleaved caspase- enzyme being the most widely used method, as well as the caspase- activity on substrates such as pro-il- β, pro-il- , and pro-gasdermin-d [ ] . in view of determining the inhibitory properties of the h. noldeae constituents of interest on nlrp inflammasome, their effect on either caspase- activation using the caspase glo assays (promega, madison, wi, usa) and/or il- β production as hallmarks of the nlrp inflammasome was evaluated. in fact, it is well known that the inflammasome activation induces a cascade of events including the recruitment of pro-caspase- and its cleavage into a corresponding active caspase- enzyme that also cleaves pro-il- β into its mature form, il- β [ ] . in our previous study, among the plant recipes evaluated, r sz was the most potent in inhibiting caspase- activity [ ] . hibiscus noldeae, one of the plant's components of the recipe, has been cited by three different traditional healers during our investigation, indicating its possible importance in the treatment of asthma. in addition, few studies reported on the traditional use of the plant in the treatment of different ailments, some of which are of inflammatory background such as asthma, wounds, headache, etc. [ , [ ] [ ] [ ] . an important question was then to determine whether h. noldeae, would have inhibitory properties on inflammasome or on il- production, a well-known downstream target of il- β. a bioguided fractionation approach to address this question was employed. many of the isolated compounds ( figure and table s ) were polyphenols, secondary plant metabolites proven to exert various biological properties including anti-bacterial, anti-inflammatory and anti-allergic activities [ ] [ ] [ ] . as far as inflammation is concerned, cell death could also lead to the activation of the inflammatory pathway in a manner similar to the inflammasome activation [ , ] . therefore, plant crude extracts, fractions and purified compouds were first tested for eventual cytotoxicity allowing to select the range of working sample concentrations in such a way that their pharmacological effects could not be biased. within a plant extract, there can be hundreds of molecules, some of which are active individually or act synergistically against a given disease. in most of the cases, major compounds are responsible of the activity of the extract though it is not always the case [ , ] . in this study, an important question was to determine whether the inhibition of nlrp inflammasome resulted from a single compound or from a combined effect of different compounds. we opted first to investigate the effect of cell pre-treatment with our various samples on the caspase- activities and observed an average reduction of about one-third which formed a plateau at the tested concentration ranges as shown in (figure ). such reduction suggested the presence of an inhibitor of caspase- in the plant extract. indeed, caffeic acid, purified from h. noldeae in this work, was reported to interact with and inhibits caspase- and asc activation [ ] . the plateau could be explained by the fact that a saturated quantity of caffeic acid is already in the lowest sample concentration used ( µg/ml). the findings that our plant products of interest had inhibitory effects on the caspase- activities prompted us to assess the production of il- β in the supernatant from the different samples. consistent with the inhibition of the caspase- activities all the tested crude extract, fractions and purified constituents of h. noldeae revealed significant inhibition of il- β production ( figure a ). to ensure that h. noldeae constituents acted on the inflammasome activation pathway, we further investigated their effects on pyroptosis. in fact, the activation of the nlrp inflammasome induces the activation of caspase- that promotes the production of gasdermin d and the latter is associated to the programmed cell death called "pyroptosis" [ ] . in contrast, compared to the production of il- β, the effect of dexamethasone was less than that of er . and er . fractions ( figure s ). this may be explained by the fact that dexamethasone is able to act at the transcriptional level or to selectively induce the degradation of il- β transcripts after their production [ ] . thus, the additional effect of these both mechanisms justifies its enhanced inhibition of il- β production in the supernatant. however, at the highest concentration tested ( µg/ml) an unexplained ease of the inhibitory effect was observed with both fractions. such unexpected reactions deserve further investigation during the characterization of er . and er . fractions. nlrp inflammasome-associated il- β production is a two-step process. our findings on both pyroptosis assays ( figure s ) and il- β production ( figure a) suggest that h. noldeae derived products interfere with the nlrp inflammasome signaling, to block cell death (pyroptosis) and il- β production. however, the western blot results showed that er . and er . fractions repressed the expression of pro-caspase- ( figure b ). capsase- is a critical component of the inflammasome and the reduced expression of this enzyme by fractions from h. noldeae indicates that er . and er . may in fact be acting to inhibit signal (priming step) of inflammasome activation by specifically blocking upregulation of caspase- . in our future research, the mode of action of the molecules contained in these fractions will be elucidated. several studies have reported in vivo anti-inflammatory properties of caffeic acid and isoquercetin as well as several corresponding derivatives [ , , ] . in this study, as suggested by our results, several polyphenolic compounds detected in the plant extracts of h. noldeae likely contribute to the inhibitory property of the plant on the inflammasome expression. however, the activity of the plant remains inferior to the activity of purified caffeic acid as well as the concentrated fractions er . and er . . this suggests that the fractionation of the plant extracts led to the enhancement of their inflammasome inhibition potency. this is in accordance with previous studies that reported the beneficial effect of fractionation in concentrating polyphenolic compounds, leading to enhanced biological activities [ , ] . we thus argue that the bioactive compounds in fraction er . , er . together with the well-known caffeic acid and isoquercetin are the main compounds involved in the regulation of the nlrp inflammasome. in the present study, the effect of our plant samples on the production of il- in raw . , a murine cell line widely used for the in vitro production of il- , was also investigated. this cytokine is a known downstream target of il- β and is consistently increased in serum from patients with nlrp inflammasome-mediated conditions. in fact, il- β has been reported to be one of the most potent activators of il- production, both in vitro and in vivo, suggesting that il- could be a therapeutic target in the treatment of il- β-mediated inflammation [ ] . in addition, abnormal expression of il- has been associated with the pathogenesis of a variety of human diseases, including asthma, cancers and autoimmune diseases such as rheumatoid arthrosis, and recently with covid- [ , ] . in asthma, it has been reported that il- is linked with mixed eosinophilic-neutrophilic bronchitis during exacerbations of the disease and worse pulmonary function in humans [ ] . moreover, cytokine-targeting therapies are gaining an increasing popularity in the treatment of inflammatory diseases including asthma and other respiratory diseases. with the covid- outbreak pandemic, it has been reported that dexamethasone and monoclonal antibody drugs targeting il- (tocilizumab) reduced the mortality rate [ , , ] . in the light of the above-mentioned role of il- in different pathologies, we investigated the effect of the h. noldeae constituents of interest on its production in raw . cell line. as shown in figure , results obtained indicate that fractions and partially purified er . and er . compounds from the plant exhibited similar trends as on il- β production ( figure a ). these findings allowed to argue that this medicinal plant is an important drug target for 'cytokine storm'-related diseases such as the neutrophilic asthma and the covid- pandemic. aerial parts of hibiscus noldeae were collected from the arboretum of ruhande, in huye district, southern province in rwanda. the plant species was identified with reference to herbarium specimens and various literature resources by nshutiyayesu samuel (from the university of rwanda, huye, rwanda) and the authentication and id verification by dr. olivier lachenaud (from the botanic garden meise/meise, belgium) as previously reported [ ] . the aerial parts of h. noldeae were cleaned of dust and debris and washed gently with tap water, and air-dried under shade. the plant material was then pulverized with a grinder. a total amount of g of the powder was extracted with the mixture of methanol: dichloromethane ( : ) as previously described [ ] . the dried crude extract ( g) was triturated in distilled water and then subjected to a liquid-liquid partitioning process using n-hexane and ethyl acetate successively. in each case, the mixture to be partitioned was vigorously shaken for min, rested for min, and then separated in a separatory funnel. each partition was conducted three times and the same fractions were combined and dried-up using rotary evaporator. at the end, the non-soluble part was dissolved in methanol to make the methanol fraction. major compounds from two fractions showing the highest inhibitory activity on nlrp inflammasome components, namely hexane and ethylacetate fractions, were further isolated on preparative hplc. to this end, an in-house hplc method used to screen the major components of crude extracts was used. in this regard, analytical separation was carried out on a hypersil ods ® rp column ( × . µm; particle size µm; thermofisher scientific, waltham, ma, usa). all samples were dissolved in methanol hplc-grade, filtered through a . µm pore size filter membrane and analyzed on an agilent (santa clara, ca, usa) hplc machine. samples were eluted with a nonlinear gradient method with acetonitrile (solvent a) and . % trifluoroacetic acid in ultra-pure water (solvent b) ( table ). the column temperature was maintained at • c. then, µl of each sample was injected into the hplc-uv/dad system and the analysis, performed at a flow rate of . ml/min, was monitored at , , , and nm. preparative hplc analysis was carried out on a variant prepstar machine. all extracts ( mg) were dissolved in methanol and then diluted with water. the amount of methanol was not allowed to exceed %. samples were filtered through a . µm filter membrane before injection. the mobile phase consisted of trifluoroacetic acid (tfa) . % in ultrapure water (a) and acetonitrile (b). the table gives the gradient used to separate the major compounds identified with hplc analytical method described above. this gradient was obtained by transposing the analytical hplc methods to preparative hplc using hplc calculator. the flow rate was ml/min and the separation was monitored at and nm. structural identification of isolated compounds was performed by d and d nuclear magnetic resonance (nmr) and infrared spectroscopy (ir). the detection was followed at and nm. human leukemia thp- monocyte and raw . murine cell lines were kindly received from the institute for medical immunology/biopark/ulb and were cultured in rpmi (life technologies, waltham, ma, usa) and dmem, respectively. all media were supplemented with % heat-inactivated fetal bovine serum (fbs; life technologies), % glutamine, and % of penicillin-streptomycin (life technologies) and % sodium pyruvate (for raw . only) and maintained at • c under % co in a humidified atmosphere. cells were passaged at least times before any experiment. for the determination of cell densities, the cell counting was done using a neubauer counting chamber with trypan blue ( . % in pbs; ph = . ; life technologies) staining. before investigating the anti-inflammatory activity of different fractions, their effect on the cell viability (thp- , monocytes and derived-macrophages, and raw . ) was screened as previously reported [ ] . the evaluation of caspase- activity was done using the caspase glo inflammasome assay (promega co. madison, wi, usa). briefly, thp- cells were grown for days in -well plates to a density of × cells/well in the presence of nm pma at • c in a humidified % co incubator. pma-supplemented medium was then discarded and macrophages were pretreated with fresh medium containing fractions at different concentrations for h. dexamethasone at a concentration of µg/ml or µm ac-yvad-cho treatments were used as controls. cells were further incubated with lps (sigma, saint-louis, mo, usa) (at a final concentration of µg/ml) for h and then with µm atp (invitrogen/thermofisher, waltham, ma, usa) for additional h. the caspase- activity was measured using the caspase-glo inflammasome assay (promega co., madison, wi, usa) according to the manufacturer's instructions. the -well plate containing treated cells was removed from the incubator and µl of the supernatant in each well were transferred to the corresponding well of a new white -well plate. an aliquot of µl of the caspase-glo reagent was then added to each well and gently mixed on a plate shaker at rpm for s. the mixture was then incubated for h at room temperature before measuring the luminescence on a berthold technologies luminometer. the culture supernatant of stimulated the thp- macrophage was used for the measurement of the cleaved form of il- β. the concentration of il- β in the culture supernatant was measured using a human il- β elisa kit (invivogen/thermofisher, waltham, ma, usa) according to the manufacturer's instructions. in all experiments, raw . cells were sub-cultured in a -well plate and incubated for h at • c and % co to acclimate before treatment. they were then pretreated with test compounds, at indicated concentrations, for h prior to h stimulation with µg/ml lps. cell-free supernatants were harvested and stored at − • c until analysis. the concentration of il- in the culture supernatant was measured using an elisa kit (bd biosciences, franklin lakes, nj, usa) according to the manufacturer's instructions. the evaluation of pyroptosis inhibition was done using the ldh-glo tm cytotoxicity assay (promega co. madison, wi, usa). briefly, thp- cells were cultured for days in -well plates to a density of × cells/well in the presence of nm pma at • c in a humidified % co incubator. pma-supplemented medium was then discarded and macrophages were pretreated with fresh medium containing fractions at different concentrations for h. dexamethasone at a concentration of µg/ml or µm ac-yvad-cho treatments were used as controls. cells were further incubated with lps (sigma, saint-louis, mo, usa) (at a final concentration of µg/ml) for h and then with µm atp (invitrogen/thermofisher, waltham, ma, usa) for additional h. then, µl of % triton x- was added to µl of vehicle-treated cells control for min before collecting the samples for ldh detection. supernatants were harvested and -fold diluted in the ldh storage buffer and kept at - • c until analysis. the ldh activity was measured using the ldh-glo tm cytotoxicity assay (promega co., madison, wi, usa) according to the manufacturer's instructions. the % cytotoxicity = × (experimental ldh release − medium background)/(triton x- ldh release control − medium background). for western blot analysis, thp- cells ( × cells/well) were differentiated in a -well plate by nm pma for days and then treated as stated above. cells were washed with µl of an ice-cold pbs, and directly lysed with a reporter lysis buffer (promega) and then centrifuged at , rpm (round per minute) for min at • c. the supernatants were kept at − • c until analysis. the protein quantitation in the supernatant was quantified by the bradford assay. an equivalent amount ( µg of protein) of each sample was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page) and electro-transferred onto nitrocellulose membranes. the membranes were immunoblotted by anti-proil- b (cell signaling technology, danvers, ma, usa) or anti-pro caspase- (abcam) according to the manufacturer's instructions. membranes were then stripped, reprobed with anti-β-actin antibodies (cell signaling technology) and exposed again to detect the internal β-actin standard. immunoblots were visualized using a chemiluminescence odyssey imaging system (odyssey ® fc, bad homburg, germany). to scan and quantify the immunoblots, we used the image studio lite ver . software (li-cor biotechnology-gmbh, bad homburg, germany). each test was done in triplicates and all the experiments were reproduced at least two times independently. for each elisa experiment, the amount of il- β or il- produced was considered as % and the remaining treatments was calculated as (treatment/lps) × . the results are presented as the mean ± sd from two or three independent experiments (n = ), each done in triplicate. data were analyzed using graphpad prism and statistical analysis data expressed as the mean ± standard deviation. comparisons of data between groups were performed by one-way anova followed by benforroni's multiple comparison test. the p-values less than . were considered to be statistically significant. considering the importance of inflammasome components in the severity of respiratory diseases in general and in asthma in particular, our preliminary results at the molecular and functional levels suggest that h. noldeae and/or its constituents may play a role in the management of asthma and other respiratory diseases if the ex vivo results obtained can be reproduced in vivo. additional purification and characterization of er . and er . fractions are of prime interest for our future study. supplementary materials: the following are available online. table s : the h-nmr chemical shifts for the purified compounds from h. noldeae. figure s : h-nmr spectrum of quercetin- -o-glucoside (isoquercetin). figure s : c spectrum of quercetin- -o-glucoside (isoquercetin) (dmso-d , mhz). figure s : h, c-hmbc of quercetin- -o-glucoside (isoquercetin) (dmso-d , mhz). figure s : effect of different h. noldeae's constituents on the thp- cell viability. figure s : effect of different h. noldeae constituents on the raw . cell viability. figure s : crude extract and the ethylacetate fraction as well as the caffeic acid and semi-purified fractions (er . and er . ), thereof exhibited significant inhibition of pyroptosis in thp- derived macrophages. the authors gratefully acknowledge the technical support of allison marotte, olivier bonnet, delphine etienne, dansala welba, and alain nyirimigabo. we are especially grateful to suzanne, a traditional healer at mamba/huye, for her cooperation and to have availed the plant material. the study could not have been completed without the cooperation of the university of rwanda's authorities, particularly the college of science and technology. the authors declare no conflict of interest. respiratory diseases in the world. realities of today-opportunities for tomorrow inflammasome-il- -th response in allergic lung inflammation the immunology of asthma and allergic rhinitis. rhinosinusitis characterization of the anti-inflammation mechanism for the ao herbal extract interleukin- β targeted therapy in severe persistent asthma (spa) and chronic obstructive pulmonary disease (copd): proposed similarities between biphasic 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(rnaemia) is closely correlated with drastically elevated interleukin level in critically ill patients with coronavirus disease therapeutic potential of anti-il- therapies for granulocytic airway inflammation in asthma anti-inflammatory agents: present and future tocilizumab in patients with severe covid- : a retrospective cohort study key: cord- -jhulvfev authors: blanchflower, david g. title: is happiness u-shaped everywhere? age and subjective well-being in countries date: - - journal: j popul econ doi: . /s - - -z sha: doc_id: cord_uid: jhulvfev a large empirical literature has debated the existence of a u-shaped happiness-age curve. this paper re-examines the relationship between various measures of well-being and age in countries, including developing countries, controlling for education and marital and labor force status, among others, on samples of individuals under the age of . the u-shape of the curve is forcefully confirmed, with an age minimum, or nadir, in midlife around age in separate analyses for developing and advanced countries as well as for the continent of africa. the happiness curve seems to be everywhere. while panel data are largely unavailable for this issue, and the findings using such data largely confirm the cross-section results, the paper discusses insights on why cohort effects do not drive the findings. i find the age of the minima has risen over time in europe and the usa. in this paper, i report on the existence of a midlife nadir in well-being. the analysis is conducted mostly at the country level with happiness and life satisfaction variables, although a number of other measures are used that relate to a household's financial situation and their living standards, satisfaction with local services, and the macro economy. all produce u-shapes in age. using country-level data, i identify u-shapes in age in advanced and developing countries. this includes of the member countries of the united nations. i find this happiness curve (rauch ) for developing and thirty-six advanced countries based on an analysis where i control for gender, education, marital and labor force status, and time. i use data from fourteen different survey series. i use these data to estimate separate country-level estimates that reach a minimum, on average, at age . . there are estimates from developed countries with an average minimum at age . and estimates from developing countries with an average minimum at . . i examine cross-section time series data at the country level rather than examining panel data. longitudinal data files that have a long run of years are restricted to the uk (bhps and ncds), germany (gsoep), and australia (hilda). in part, the concern with these surveys is non-random attrition bias and hence missing values over time with the least happy dropping out or even dying, which may well introduce measurement error. there is a small literature looking at age effects using panel data that i interpret as largely supportive of u-shapes, although there are some technical issues that must be considered. my interest is to see whether there is evidence of a midlife zenith in other countries besides the uk, germany, and australia. i examine the importance of cohort effects to determine if younger and older age cohorts are different from those in the middle and find out that they are not. i examine the data over time and adjust for cohort effects and find remarkable consistency in the findings. i find that introducing cohort effects in the samples where i have a long time series, namely, the eu commission's eurobarometer series pre and post the great recession ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) , has little impact on the results. i find the minima in europe have risen over time, from around age in to over in the most recent data. i also address the issue of possible differential response rates among older people, along with the concern that happy people live longer. to minimize that concern, i focus my analysis on people from early adulthood, which is usually age but in some samples is as low as , to under the age of . i exclude older people. it makes sense to look at as many countries as possible given the evidence that in the raw data the usa looks different. in the raw us data, essentially however measured, happiness rises initially to a peak around age and then declines into midlife and then rises again after age . this apparent m-shape disappears once controls are included and a well-defined u-shape appears. it also disappears when the sample is split into separate married and unmarried samples. these patterns are not found elsewhere in the world. this has led to a debate in the usa especially about the importance of including control variables, although less so outside the usa where it matters little. in other countries, the u-shape generally appears whether controls are included or not, although the point at which the function reaches a minimum may differ. it is also worth pursuing the possibility that the u-shape doesn't apply to poorer countries, where residents have shorter life expectancies. blanchflower and oswald ( a) find a u-shape for developing countries in world values survey sweeps - that averages out at a minimum around age when including control variables. in this paper, i find there are u-shapes in age in developing countries with minima similar to those in advanced countries regardless of how well-being is measured. i examine the presence or not of u-shapes with and without controls in the usa and find the evidence is much stronger with controls. i then turn to examining data for the uk and european countries and find there is evidence of a u-shape whether controls are included or not, with very little difference in the age minima. i then proceed to examine a series of multi-country data files. it is striking that the same finding holds across so many countries. the u-shape can be found in multiple data files and does not depend on what question is asked or how the responses are coded. i document clear patterns in the data. this paper is the mirror image of blanchflower ( b) that examined unhappiness data and finds comparable evidence using twenty different measures for an unhappiness curve that maximized with controls at age compared with a zenith of happiness estimated in this paper at age . yes, there is, despite what psychologists say. the background literature is large and there is some disagreement over whether u-shapes exist at all (see, for example, baird et al. ( ) , blanchflower ( ) , blanchflower and oswald ( ) , carstensen et al. ( ) , charles et al. ( ) , easterlin ( easterlin ( , , frey and stutzer ( ) , frijters and beaton ( ) , glenn ( ) , graham and pozuelo ( ) , hellevik ( ) , hudson et al. ( ) , lachman ( ) , leland ( ) , mroczek and kolanz ( ) , mroczek and spiro ( ) , rauch ( ) shields and price ( ) , stone et al. ( ) , steptoe et al. ( ) , wunder et al. ( ) , and schwandt ( )). a recent review by ulloa et al. ( ) goes so far as to draw the conclusion that existing studies show either a u-shaped, inverted u-shaped, or linear relation between aging and subjective well-being. other studies, such as lachman ( ) , come close to arguing that there may be a midlife dip but that it is too small to be significant. many of the studies such claims were based on had very small samples sizes and in fact did show u-shapes despite claims they didn't. an early psychology literature suggested there was no age-happiness relationship (cantril, , and palmore and luikart, ) . myers ( , p. ) argued that no time in life is notably happiest and most satisfying. in contrast, michael argyle concluded that studies of life satisfaction found that it increased with age (argyle, (argyle, , . a survey by diener et al. ( , p. ) concluded that life satisfaction often increases, or at least does not drop, with age. easterlin ( ) examined data from the general social surveys from to and claimed that "happiness is greatest at midlife but not by a great deal. on average it rises somewhat as people progress from age to and declines thereafter" ( , p. ) . a survey by diener et al. ( , p. ) concluded that recent studies converge to show that life satisfaction often increases, or at least does not drop, with age. diener and suh ( ) examined world values survey data for and argued that the raw data on life satisfaction trended up slightly through age. deaton ( ) concluded that the u-shaped relation is present solely in rich, english-speaking countries in which the elderly is relatively satisfied with their lives. (ibid., p. ). more recently, whitbourne ( ) has gone so far as to argue that the u-shape curve is a "myth." blanchflower and graham ( a) examine the evidence that psychologists have cited claiming no u-shape exists over the life span and found that many of the studies cited had very small sample sizes. examples are helson and lohnen ( ) (n = ), freund and baltes ( ) (n = ) , and hamarat et al. (n = ) to name but a few. it is hard to say much of anything about statistical differences in well-being by age with sample sizes that small. psychologists have also cited work by ingelhardt ( ) as not finding any u-shapes in age. for example, diener et al. ( ) citing ingelhardt ( ) argue that "international studies based on representative samples from multiple countries also show that life satisfaction does not decline with age." myers ( ) argued that ingelhardt showed that "age differences in well-being were trivial. does happiness then align itself more with any particular age? do young adults have more fun? surprisingly, and definitely, not" (p. ). ingelhardt ( ) examined well-being across sixteen nations using data from eurobarometers # -# (april -november and the world values survey on the usa, canada, hungary, and japan for - and argued that there was "little variation by age" in well-being (p. ). it turns out the data he used in fact show otherwise. blanchflower and graham ( a) went back to the ingelhardt book and observed he in fact reported u-shapes in the raw data in nine of the sixteen countries studied. blanchflower and graham examined the same data ingelhardt used and estimated a series of happiness equations and found there were u-shapes in age with controls in all the countries and variables ingelhardt examined. in addition, diener and suh ( ) cite work by okma and veenhoven ( ), also used eurobarometers, between and , and argued that the paper showed an almost flat line with age. from around age to , they argued it showed there was almost no change in life satisfaction. it didn't. blanchflower and graham ( a) went back to analyze these same eurobarometer files for the same years which are part of the publicly available mannheim trend file. across these nations, the average score for those under was . , reaching a low point of . at age and then rising to . at age . so, it is true that life satisfaction scores at age are not that different from age but that ignores the midlife drop. without controls in a life satisfaction equation, there is a well-defined nadir in well-being in age controlling for year and nation that minimizes at age and also one with controls-for gender, education, and marital and labor force status-that minimizes at age . i update and extend results in an earlier paper (blanchflower and oswald, a) , where it was shown that a u-shape in age existed in well-being data across a number of countries. using data on , randomly sampled americans and west europeans, the paper found that holding other factors constant, a typical individual's happiness reaches its minimum on both sides of the atlantic for both males and females in middle age. the minimum in age was broadly similar between advanced, east european, and developing nations. the function minimized on average in midlife. for example, in europe, for both men and women, it minimized at around with controls including education and marital and labor force status. for developing countries from the wvs, sweeps - , minima were for men and for women. a maximum in age in unhappiness data for europe was found at around age . some apparent exceptions, particularly in twenty developing nations along with a few western countries, mostly where there are small numbers of observations, to the u-shape were noted. subsequently, glenn ( ) argued that it was inappropriate to include controls and what mattered was the raw data; blanchflower and oswald ( ) disagreed. glenn claimed that the appearance of this u-shaped curve of well-being is the result of the use of inappropriate and questionable control variables and especially marital status. it is worth rehearsing the arguments we used there again. in many countries around the world, and especially in europe, as i illustrate in detail below, the u-shape can be found evidence for a u-shape was found in twenty-two advanced countries (australia, belgium, canada, denmark, finland, france, germany, greece, iceland, ireland, italy, japan, luxembourg, malta, netherlands, norway, portugal, spain, sweden, switzerland, uk, and usa) . second, evidence was provided for the existence of a similar u-shape through the life course in east european, latin american, and asian nations. evidence was found in fourteen ex-soviet republics (albania, bosnia, bulgaria, croatia, czech republic, estonia, hungary, latvia, lithuania, macedonia, poland, romania, serbia, slovakia) and thirty-eight developing countries (argentina, azerbaijan, belarus, brazil, brunei, brazil, brunei, cambodia, chile, china, colombia, costa rica, dominican republic, ecuador, el salvador, iraq, israel, honduras, kyrgyzstan, laos, mexico, myanmar, nicaragua, nigeria, paraguay, peru, puerto rico, philippines, russia, singapore, south africa, south korea, tanzania, turkey, ukraine, uruguay, uzbekistan, and zimbabwe. i find evidence of a u-shape in all of these countries also. that included algeria, armenia, austria, bangladesh, chile, colombia, egypt, greece, india, indonesia, iran, jordan, luxembourg, moldova, morocco, new zealand, pakistan, saudi arabia, singapore, slovenia, taiwan, uganda, venezuela, and vietnam. in this paper, i report u-shapes for all but three of them-bangladesh, pakistan, and saudi arabia. without any control variables, and a major problem with glenn's analysis was that he focused too heavily on the usa. second, we disagreed with glenn's methodological position, which seems to be that social scientists should not hold constant other factors when they study the relationship between well-being and age. ultimately, in social science, the control variables that are included in multiple regression equations we noted have to be chosen with an eye on the intellectual or policy question being answered. the summary of our argument went as follows. if the aim is to describe the data, it is reasonable to leave out most or all control variables. "smokers die at rate z" is an acceptable statement to make. but that is not the same as "smoking changes your risk by z," which requires other confounding variables to be controlled for such as diet, education, income, and exercise. we argued that would be an error to use an equation without controls to tell the public what impact aging has on happiness without separating out the effects of other variables such as, say, education, marriage, or unemployment. if the aim is to understand relationships, we concluded, "it seems, it will rarely be desirable to stop at bivariate patterns." that seems right and i don't stop at bivariate patterns in this paper either. blanchflower and oswald ( ) examined the issue of differences between the well-being and age relationship with and without controls using seven pooled crosscountry data sets, covering countries and . million randomly sampled people; the paper examines the cross-sectional pattern of psychological well-being from approximately age to age . the paper described the two conceptual approaches. one studies raw numbers on well-being and age which we termed the descriptive approach. the second studies the patterns in regression equations for well-being (that is, adjusting for other influences). this we termed the ceteris-paribus analytical approach. the paper applied each and compared the patterns of life satisfaction and happiness. using the first method, evidence of a midlife low was found in five of the seven data sets; the two that didn't were both for the usa. using the second method, all seven data sets produced evidence consistent with a midlife low. deaton ( ) reported only unadjusted estimates in part he argued because of the difficulty in applying consistent controls to the gallup data, not because the questions do not exist, but because their meaning varies so much across the globe, with different patterns of education, work, retirement, and health systems. deaton also suggested that a weightier argument is that many possible and potentially important controls are age dependent, including income and the presence of children but especially health, disability, and marital status. deaton notes that "different authors use different countries and different data sets with different swb questions, so it is possible that the age patterns in the gallup data are different from those that come from other questions and different survey protocols; it would be an important (if daunting) task to make systematic comparisons." this is what i try to do here. some psychologist have even gone as far as to argue that even if there is a u-shape it is broadly irrelevant as any change is "trivial." jebb et al. usa, usa, - and (g) latino barometer, and . in private communications, sir angus deaton suggested that he didn't have quite this in mind. he suggested, more just a look at the questions they ask, their response rates, and whether they are even grossly consistent. that "it is possible that the u-shaped (or other) curve exists but that it is so small that it is not practically meaningful. in other words, just because differences across age are statistically significant, that does not mean that these differences have practical significance. researchers in past studies have generally not taken effect size into account,… at some point, an effect size becomes so small that it is truly trivial and lacks practical significance. for our cantril ladder scale, respondents reported (and probably thought) in terms of the nearest whole scale point from to . therefore, it seemed that differences below . should be considered quite small." as blanchflower and oswald ( ) note the claim that the size of the dip is tiny does not appear to be correct. in the seven data sets, they studied the size of the drop, in well-being to the low point in the late s is equivalent in magnitude to the influence of a major life event like unemployment or marital separation. the size of the fall in wellbeing from youth to midlife is large and likely highly consequential. i should also note that i know of no evidence in any well-being data involving a change anywhere approaching . for any life event. some have argued that no u-shape exists in longitudinal data (frijters and beatton ; kassenboehmer and haisken-denew ) . in contrast, cheng et al. ( ) drawing on four data sets, and only within-person changes in well-being, build on the work of van landeghem ( ) and document powerful support for a u-shape in longitudinal data. three of the data sets are nationally representative household surveys, namely the british household panel survey (bhps, (bhps, - , the household income and labour dynamics in australia (hilda, - ) , and the german socio-economic panel (soep, (soep, - . the fourth data set comprises a relatively more homogenous sample of medical doctors from the medicine in australia balancing employment and life (mabel) longitudinal study. they measure the change in well-being of randomly selected individuals each year and then plot that against individuals' ages. on average, they find people's wellbeing gradually drops until individuals reach midlife. from then on, it picks up smoothly as people go on, in each of three countries and four data sets, to approach the age of . wunder et al. ( ) and ranjbar and sperlich ( ) both use semi-parametric methods on german soep panel data to examine the relation between age and well-being. they both get the same results. ranjbar and sperlich conclude "we find a clear, deep valley between the ages of and , typically interpreted as a midlife crisis." bleischmann ( ) also uses the gsoep and finds "mean life satisfaction is steadily declining between and . after this low, happiness increases strongly until the age of ." de ree and alessi ( ) have examined that the gsoep - found that that "the data is indeed consistent with a u-shape in age over most of the life cycle" (p. ) but have noted that age profiles are not identified without forcing arbitrary restrictions on the cohort/time profiles. there are clear issues though with the data they examine given they have to drop a quarter of households due to missing values. kroh ( ) notes that less than % of the original sample remains after . ferrer-i-carbonelli and frijters ( ) also examine gsoep data find a u-shape with controls for west german workers. the authors find the result is the same whether estimated by ols, ordered logit, or ordered probit and include controls for time, household income, children, a steady partner, and health. when they re-estimate with fixed effect, the u-shape disappears. piper ( ) uses gmm dynamic panel estimation with waves of the british household panel study on youngsters age - and found that happiness declined over that age range, a result found by comparing the coefficients of the age dummies: a result in line with the overall u-shape. furthermore, tests of the individual age group coefficients demonstrate that they are, in many cases, significantly different from each other. additionally, because the preferred model controls for the individual waves in the sample, this decline of life satisfaction with age is a life cycle effect. the life satisfaction of young people between and falls, and this seems to be something that everyone, on average, experiences. overall, his findings, piper argues, "are in line with the common u-shape finding." clark ( ) also finds, using the same data source and panel data methods controlling for fixed effects, that the data "continues to produce a u-shaped relationship between well-being and age." other commentators have expressed skepticism that the curve's trajectory holds true mainly in countries where the median wage is high and people tend to live longer or, alternatively, where the poor feel resentment more keenly during middle age and don't mind saying so. john briley in a recent op-ed argued that "the curve is not universaldata from economically struggling countries, for example, don't show the happiness rebound." arthur krystal , for example, has suggested that there may be a simpler explanation: "perhaps the people who participate in such surveys are those whose lives tend to follow the curve, while people who feel miserable at seventy or eighty, whose ennui is offset only by brooding over unrealized expectations, don't even bother to open such questionnaires." this critique of course could apply to any research based on surveys with a bias having nothing to do with age. there is zero evidence that the u-shape has anything to do with differential response bias by age especially under the age of . i have the u-shape in many data sets with various happiness measures including happiness itself and life satisfaction and cantril's ladder. it makes no difference if the dependent variable is scored, from to say or from to ; the results are essentially the same. the smaller numbers of observations for older age groups are an issue but that simply reflects the overall demographics in the country-there are fewer people age than age and especially so in countries with shorter life expectancy. helliwell ( ) recently argued that "to use a single life satisfaction question in large population-based samples might represent the best use of survey resources." following helliwell's advice, where feasible, i use life satisfaction as my well-being measure, where i can. data i examine the happiness curve using individual micro data from thirteen distinct micro survey series. these were chosen because well-being measures of various types were john briley, "does happiness in your s signal the end of ambition?," the washington post, december , . arthur krystal, "why we can't tell the truth about aging? a long life is a gift. but will we really be grateful for it?," the new yorker, october , . according to the census bureau's international population database in , there were , , individuals age in the us versus , , age . in ldcs, the ratio is smaller-in venezuela, for example, the numbers are , and , respectively, so it is times there versus times in the us. https://www.census.gov/data-tools/demo/idb/region.php?t= &rt= &a=both&y= &c=us&r= available. mostly, the questions examined are on happiness or life satisfaction. the questions used vary a little as do the number of possible responses varying from three to eleven that i call steps. other sweeps (e.g., the brfss from ), for example, did not contain happiness measures although they do contain unhappiness measures (blanchflower, a) . i also examine a broader set of questions on family life, health, trust, financial situations, living standards, and more. in most cases, i have to recode the variables such that a higher number means greater happiness. source: gallup usdt, brfss, brfss, - and gss, - . t-statistics in parentheses. sample size changes when controls are added because of missing values to the control variables *labor force status not available in , hence the smaller sample size ) ten-step happiness using sweeps - of the european social surveys (ess) - (table ) ) ten-step life satisfaction (table ) and -step step happiness from the european quality of life survey: - (table ) ) seven-step happiness from the (table ) and -step life satisfaction from the sweeps of the international social survey program (table ) ) ten-step life satisfaction from waves - of the world values survey (wvs); - (table ) ) five-step happiness from the asia barometers of (table ) ) four-step life satisfaction from the latino barometers of and (table ) ) eleven-step cantril's life satisfaction ladder from the gallup world poll ( - ) (table ) ) three-step financial satisfaction from wave of the wvs (table ) (table ) ) five-step satisfaction with living standards in the afro barometers and (table ) the issp and wvs both contain data from four large non-european englishspeaking advanced nations-australia, canada, new zealand, and the usa-plus japan. they all give u-shapes in happiness with and without controls. i use three methods to identify the u-shape. first, i run an ols regression with the dependent variable a measure of well-being, on a pooled sample of countries across all second, i then re-estimate for individual countries including the gender, education, and marital and labor force status control variables with the age of respondents limited to those under the age of . i do this for simplicity given very different life expectancies across countries and hence much smaller sample sizes for older age groups and likely variability at older ages. sample sizes are often quite small for these individual country regressions, and on average many are only around observations. i find for several advanced countries that there are insignificant results using, for example, issp data, but when using eb or ess when the samples are much larger, the significance of both age terms appears. i assume that there is a significant u-shape if there is a negative sign on the age coefficient and a positive sign on the square with the t-statistic of both above . . finally, i re-estimate the well-being equation and replace the age and age squared term with a complete set of single year of age variables which i then plot in a series of figures. this is to ensure that the quadratic i fitted is not an inappropriate functional form. this way the form is freely estimated and then plotted, with the individual coefficients added to the constant. these figures show u-shapes. the well-being variables are always coded from low to high, so a positive coefficient means happier. sometimes i use happiness data and sometimes life satisfaction and the number of options available varies by survey and year. mostly there are four options that i call -step, or eleven options from to that i call -step, plus i also use -step, -step, -step, and -step. it doesn't seem that this makes much of a difference. sample size does seem to matter although it is surprising how many ushapes are identified even with sample sizes of less than a thousand. i am also able to identify u-shapes in age in both european and african nations using a broader set of attitudinal questions on living standards as well as on an individual's financial conditions as well as the state of the national economy. i focus in particular on questions about financial situations individuals find themselves in as well as on the general state of the economy. these questions are widely used in consumer confidence surveys. respondents are asked such questions in the eurobarometers, as well as in the monthly consumer surveys run by the european commission in every eu country since the s. these surveys have started to move down sharply from march as the covid- shock hit (bell and blanchflower, ) . i also compare results of asking similar questions in europe and africa in relation to satisfaction with living standards. it seems the u-shape in age is more general than just in happiness and life satisfaction equations and applies to other attitudinal economic variables. this suggests the happiness curve has broader applicability to other attitudinal variables about the person and the economy. in this section, i report the results of estimating a series of ols well-being regressions. in each case, i report coefficients and t-statistics for the age and the age squared variables with and without controls for education, gender, marital and labor force status, country, and where appropriate where there are multiple survey years used a set of year dummies. the without controls equations include year and country dummies and in the case of the us and the uk state or region dummies when available. i calculate the minimum of the quadratic in age by differentiating with respect to age and solving which means dividing the age coefficient by the age coefficient multiplied by . hence, on row of table , the age coefficient is − . and the age coefficient is + . so the minimum is - × . /( × . ) = . both are highly statistically significant with t-statistics of and respectively. i turn first to the two countries that have micro data files with many hundreds of thousands of observations-the usa and the uk. in this paper, i report separate estimates each for the two countries, with controls and in both well-being is u-shaped and on average it minimizes in both at age (footnote ). q . "please imagine a ladder, with steps numbered from at the bottom to at the top. the top represents the best possible life for you and the bottom of the ladder represents the worst possible life for you. on which step of the ladder would you say you personally feel you stand at this time?" in the gss, the happiness q is used. q . "taken all together, how would you say things are these days? would you say that you are very happy = , pretty happy = , or not too happy = ?" (my codes). in the brfss, respondents are asked the following -step question: q . "in general, how satisfied are you with your life? very satisfied = ; satisfied = ; dissatisfied = and very dissatisfied = ." (all my codes). in table , i report the results of estimating ols regressions which include an age and an age squared term plus year dummies and state dummies for usdtp and the brfss and region dummies with the gss as that is all that is available. i then repeat including controls for gender, labor force and marital status, and education. in the case of the gusdt, the age term is negative, and the age squared term is positive without and with controls implying a minimum at and respectively. in the case of the brfss, without controls, the age term is negative, and the square term is positive, but the minimum is over . for the gss, the signs are reversed but are both significant suggesting an inverted u-shape. in both cases, when i add controls, there is a significant u-shape with a minimum of and respectively. life satisfaction was included in a subset of the brfss for louisiana, minnesota, mississippi, rhode island, and tennessee in - . i re-estimated the equation in table using these data (n = , ), with controls for age and its square, state, year, education, gender, race, and marital and labor force status and found the quadratic minimized at age . the second part of the table restricts the sample to under years of age. the major change is that the brfss data now gives a u-shape that minimizes at age versus one that minimizes at age with controls. in the case of the gss, with many fewer observations, the age squared term is insignificant and hence i don't report a minimum. it is important in the usa to look at the raw data to determine the appropriateness of fitting a quadratic to the data. fig for the brfss, - , plots the two quadratics with controls from table for all ages and for ages under . it also plots the results of replacing the two age terms with single year of age dummy variables from equations with and without controls. in each case, the individual coefficients are added to the constant. it is clear that without controls, in the raw data, there are two hills: an early dip to the early twenties and a rise to the mid-thirties and then a fall through the mid-fifties and a rise again to the early seventies before the function dips again. adding controls produces a clean and highly significant u-shape which turns over after the age of seventy and remains broadly flat thereafter. the upward slope flattens after around age and then starts turning down around age . it is clear that the quadratic for those age under , with controls, seems to fit the data better, than the one on the full sample. fig does the same with the usgdtp. the quadratic based on data under the age of seems a close approximation. of note though is that there are marked differences in the raw data in the usa between the married and the non-married that is not true elsewhere. below i report step happiness equations for the gss and -step life satisfaction equations in the brfss with only year and region controls included as below with t-statistics in parentheses. in the case of the gss, the positive age and negative age term suggest it also should be noted that there is some evidence that the minimum of the u-shape has risen over time as life expectancy has climbed. in the usa, using data for those age under , it was in versus in . the midpoint using the gss for the years - was and for the years - was . as we show below, there is also evidence of a slightly bigger rise in europe, where life expectancy in many countries grew more. i now turn to examine the data, for people under age , from the other major large cross-section survey of well-being from the most recent sweeps available for - , from the annual population surveys for the uk. earlier sweeps were used in bell and blanchflower ( ) to examine the well-being of the underemployed and the unemployed. these surveys contain data three happiness measures and overall there are about , observations on each variable. the three questions i examine are as follows. q . life satisfaction-"overall, how satisfied are you with your life nowadays, where nought is 'not at all satisfied' and is 'completely satisfied'." q . happiness-"overall, how happy did you feel yesterday, where nought is 'not at all happy' and is "completely happy'?" q . worthwhile-"overall, to what extent do you feel that the things you do in your life are worthwhile, where nought is 'not at all worthwhile' and is 'completely worthwhile?'." table shows that for all three variables, the age coefficient in all six specifications is significant and negative and the age squared term is significant and positive and all minimize in the forties. fig plots the single year of age coefficients for each of the three variables with the full set of controls included in each case. the minima are a little higher at around age . i now move to looking at data files that cover multiple countries. for simplicity, going forward, i use a quadratic in age as a reasonable approximation to the age profiles in well-being and firstly restrict the sample to those age under so that the estimated minima are not impacted by what happens in the older age groups especially as sample sizes can be small at higher ages. to report a minimum, i impose the second rule that both the coefficients on the age and age squared variables must have the right signs and t-statistics of at least . . for each of the data files, i report a pooled regression with year dummies and the full set of controls are for gender; education, and marital and labor force status which are available in broadly the same form in all of the data sets. i also fit age quadratics to each sample pooled across countries with age unrestricted and then replace the quadratic with a more flexible form of single year of age dummies. i then plot the age coefficients, added to the constant, as a check on the quadratic. i start out using data from the eurobarometer surveys (eb). concern has recently been expressed over response rates to these surveys especially in relation to the questions on respondent's views on the eu, with the concern that eurosceptics do not respond to the surveys which then suggest higher levels of support than they should. the eurobarometer surveys differ from other surveys that use the mail or the telephone; the eu commission only conducts interviews with members of the public face-to-face at home. this makes it even more difficult to achieve high response rates. the eu commission on december defended the methods of its public opinion surveys in response to criticism that the low rate of responses could lead to bias towards the eu. in the most recent eurobarometer survey for which response rates have been calculated, and obtained by the danish newspaper, the rate was % in finland, % in germany, % in luxembourg, % in italy, % in the uk, % in denmark, % in greece and france, % in ireland, % in spain, % in latvia, and % in portugal. erik gahner larsen from the university of kent in a blog noted rightly that the response rate is informative but not sufficient or even necessary in order to obtain representative samples. he finds no evidence that countries with lower response rates are much more positive towards the eu in eurobarometer compared to the european social survey. of note is that there seems very little evidence that responses to questions on life satisfaction in the eb have been impacted over time by a rise in non-response rates. table uses data on -step life satisfaction for over . million europeans from forty-two sweeps of the eb for the years - for those age under with only year dummies. the question asked is: q . "on the whole, are you very satisfied, fairly satisfied, not very satisfied or not at all satisfied with the life you lead? not at all satisfied (= ); not very satisfied (= ); fairly satisfied (= ) and very satisfied (= )". it establishes the facts in european countries, by which i mean the eu plus eight other countries (albania, iceland, norway, macedonia, montenegro, serbia, turkey, and turkish cyprus). there are six developing countries including four ex-soviet (albania, macedonia, montenegro, and serbia) that are not eu members plus turkey and turkish cyprus in that group, all of which are so-called candidate countries. "eurobarometer and euroscepticism" https://erikgahner.dk/ /eurobarometer-and-euroscepticism/ information, "new data reveals serious problems with the eu's official public opinion polls", december . https://www.information.dk/udland/ / /new-data-reveals-serious-problems-with-the-eus-officialpublic-opinion-polls and eszter zalan, "eu commission defends eurobarometer methodology," eu observer, december , . first, estimates are provided for pooled samples across all countries without controls. there is a minimum in midlife at age . separate estimates are provided by country and in all thirty-seven cases the age term is significant and negative and the squared term significantly positive. there is some variation with a low of in luxembourg and a high of for bulgaria. the average across the estimates is. table repeats the exercise adding controls and the overall equation now has a minimum of fifty-four, and there are u-shapes for every country. a set of cohort q . the situation in our country? q . the situation of the national economy? q . the employment situation in the country? q . the presence of public services in our country? q . at the present time, would you say that, in general, things are going in the right direction or in the wrong direction, in our country = things are going in the wrong direction = neither the one or the other = things are going in the right direction? i am now going to read out different aspects of everyday life. for each, could you tell me if this aspect of your life is very satisfactory (= ), fairly satisfactory (= ), not very satisfactory (= ) or not at all satisfactory (= )? q . q . in general, how would you describe your own present living conditions? possible responses include: = very bad, = fairly bad, = neither good nor bad, = fairly good, = very good? dummies are added in the second row and the minimum is largely unchanged. there are u-shapes in every country with the minima ranging from for luxembourg to in montenegro. fig uses single year of age plots with and without controls using these eb files from to , and both show u-shapes. it shows an important point that in the eurobarometer files there is always a u-shape whether controls are included or not. there is an issue raised by morgan and o'connor ( ), henceforth mo, over whether there is an m-shape rather than a u-shape in eb data. however, in blanchflower ( b) , i showed that this early bump arose because mo omitted students, who are young, and happy. once students are included, the m-shape disappears and the u-shape returns. table significant u-shapes in every year, but over time the minimum has risen as we noted it did for the usa. the minimum rises from an average of in - to over since . life expectancy for most of these eu countries rises even more rapidly over these years than it does in the usa. for example, based on oecd data between and in both france and italy, life expectancy at birth rose from to and in both germany and the uk it increased from to (see footnote above). it is perhaps surprising that the estimates from developing countries that we examine below that have lower life expectancies have broadly similar minima to advanced countries. table reports a series of happiness equations by country with controls and again restricted to age under , using eight sweeps of the european social surveys. there are over a third of a million observations overall and the question is an -step happiness variable. q . "taking all things together, how happy would you say you are, from to with zero 'extremely unhappy' and 'extremely happy?'" the ess contains our first data on four developing countries-israel, russia, turkey, and ukraine-plus twenty-five eu countries, minus malta, latvia, and romania plus iceland, norway, and switzerland. there is a minimum again in every country equation that are also in the forties and fifties and average . all four of the developing countries have a u-shape and there are eight advanced countries with no ushape (denmark, estonia, finland, iceland, italy and lithuania, poland, and slovenia is happiness u-shaped everywhere? age and subjective well-being in... all six of these countries had significant u-shapes with larger samples with the eb data. the european quality of life surveys (eqls) includes the q happiness question above but also a -step life satisfaction equation. q . all things considered, how satisfied would you say you are with your life these days? please tell me on a scale of to , where means very dissatisfied and means very satisfied. table makes use of -step life satisfaction data from four sweeps ( , , , and ) of the eqls pooled together, with controls. table now turns to look at -step happiness data in five sweeps of the asia barometers of - . the question asked is blanchflower and oswald ( a) q . "all things considered would you say that you are happy these days? -very happy = ; pretty happy = neither happy nor unhappy = ; not too happy = and very unhappy = ?" once again, the numbers refer to my codes. in each case, there is a well-defined ushape with controls and only without controls in two of the five sweeps. significant ushapes are found in fourteen asian developing countries-china, india, laos, maldives, mongolia, myanmar, philippines, singapore, south korea, sri lanka, taiwan, tajikistan, thailand, and uzbekistan. , , , and - and found a u-shape at age for men and age for women with a full set of controls, so this updates that analysis. for both and , there are well-defined u-shapes that minimize in the forties and fifties with controls. there are u-shapes for those under the age of in twelve, for bolivia, brazil, columbia, costa rica, ecuador, honduras, mexico, panama, paraguay, peru, uruguay, and venezuela. multi-country data-issp, wvs, and the gallup world poll . international social survey programme and table now moves to using -step life satisfaction data from the issp which is not limited to europe; the sample size is only , . the question asked is: q . "if you were to consider your life in general, how happy or unhappy would you say you are, on the whole? completely happy = ; very happy = ; fairly happy = ; neither happy nor unhappy = ; fairly unhappy = ; very unhappy = ; completely unhappy = ?" numbers are my coding to ensure a larger coefficient means more happiness. controls are included. all countries have significant u-shapes, mostly in the forties and fifties again. table does the same but with the issp with a -step life satisfaction question and a sample size of n = , . q . "all things considered, how satisfied are you with your life as a whole nowadays? completely satisfied = ; very satisfied = ; fairly satisfied = ; neither satisfied nor dissatisfied = ; fairly dissatisfied = ; very dissatisfied = ; completely dissatisfied = ?" there are u-shapes everywhere once again. table looks in turn at each of the five sweeps - of the world values survey in turn that all use the q -step life satisfaction equation defined above. in each of the five sweeps, there is always a minimum between forty and fifty overall with controls, and only in wave is there no u-shape without controls. in every one of the reported country estimates, for advanced and developing countries, remarkably, given the small sample sizes, there are significant happiness curves. blanchflower and graham ( b) examined data from the gallup world poll from to for fourteen countries. fourteen of those countries have significant and well-defined u-shapes in age and they are not available in any of the other data files, so in table we report results for these developing countries using the q question above for cantril's life satisfaction ladder measure. ) there are well-being u-shapes in advanced and developing countries. ) these answers seem to be similar using happiness or life satisfaction data. ) it doesn't seem to matter how many steps there are in the dependent variable; essentially, the same answer is found with a -step, -step, -step, or an -step measure. ) the answers are broadly the same whichever data file is used. ) adding cohort dummies does not remove the u-shape. ) there is a minimum around age with controls of the happiness curve in both advanced and developing countries, and a little higher than that without controls. satisfaction with financial situation: macro happiness and living standards i now move away from looking at happiness and life satisfaction directly and extend my horizons by looking at other broader measures of well-being. it was already wellknown that there were similarities between happiness data and assessment of someone's financial situation and their living standards, but i find the similarities do not stop there. remarkably, this u-shape pattern emerges when i look at assessments of the national economy as well as the quality of local services. it emerges when respondents are asked about job opportunities and time to do things and the u-shape appears to have broad applicability to a wide class of qualitative measures. , - . financial situation of the household i now turn to other ways of measuring satisfaction, which it turns out also show ushapes. all of the questions used are reported in the appendix. easterlin ( ) found evidence of a u-shape in age in the us general social survey for the years - in answers to q which relates to how an individual is doing financially. he finds that satisfaction with one's financial situation, "declines very slightly through age , but thereafter rises considerably, with the biggest increase late in life." this contrasts with his findings on happiness overall as well as happiness with the family that he found followed an inverted u-shape. i took the data easterlin ( ) used and re-estimated, with and without controls, for a longer time period, from to . t-statistics are in parentheses and i restricted the sample to those under age for simplicity. without controls, year dummies are included, with controls adds controls for gender, marital status, years of education, race, and labor force status. sample size is with controls. i confirm easterlin's findings; both happiness and family situation without controls generate inverted u-shapes in age, whereas financial situation has a u-shape in age even without controls. all three though have u-shapes once controls for education, marital status, and work status are included. the minima are for happiness, for family situation, and for financial situation with controls. it is apparent that a u-shape in these gss data seems more robust using the financial situation data than the other two measures of well-being. i explored the characteristics of this rather intriguing financial circumstance variable further as comparable data is available in wvs sweeps and for both developing and developed countries. in table , i model responses in turn from waves and of the wvs that contains a -step question on how satisfied the respondent is with the financial situation of the household q . we are interested in how people are getting along financially these days. so far as you and your family are concerned, would you say that you are pretty well satisfied (= ) with your present financial situation, more or less satisfied (= ), or not satisfied at all (= )? there are statistically significant u-shapes with controls in both developed and developing countries in both wave and wave . with controls in the country data for satisfaction with family life are only available for the years - hence the sample restriction but in what follows i used data for both happiness and financial situation for the years - . the family situation question was satfam: "for each area of life i am going to name, tell me the number that shows how much satisfaction you get from that area. your family life (my codes) - . a very great deal; . a great deal; . quite a bit; . a fair amount; . some; . a little; . none." equations with the sample restricted to those under years of age, there are u-shapes in thirty-four developing countries from around the world. table uses data from four different european data files. the first part uses eurobarometer # . for june-july . the first question relates to the financial situation examined above and finds a u-shape also that minimizes at age . i then estimate six different attitudinal questions on the individual's views on the situation in the country (q ); the national economy (q ); the respondent's own job if working (q ); the respondent's own financial situation (q ); employment situation in the country (q ); and the presence of public services in their country (q ). in every case, the age term is significant and negative, and the square term is positive. each of the variables have well-defined and statistically significant u-shapes in age and the t-statistics on age and its square are everywhere above five. a -step question is also used on the direction of the country, which is often used in polling. the age minima vary from ages - . a great deal of use is made in economics of survey responses from individuals on the general state of the economy, including in consumer confidence measures such as the michigan and conference board measures in the usa and conducted by the european commission monthly for every eu member state. for example, respondents in the eu commission survey are asked for their views on the "general situation of the economy over the next twelve months" that i have through march . these variables are then collapsed into a score. an equivalent survey from firms is available from his markit in the form of a much-watched composite pmi available monthly from through april . in fig , i plot both series for the eurozone that seem to track each other well. their decline in onwards gave early warnings as did other similar attitudinal variables that few spotted of the oncoming global recession in (blanchflower ). of note is their dramatic collapse in both in march and in april to new lows. for example, the composite pmi hit a record low of . , down from . in march and . in february. the low point in the great recession was . in february . the general economic situation measure had the biggest collapse in the history of the series that runs back to , beating the previous record collapse that occurred in august when iraq invaded kuwait. these macro happiness indicators provide a clear picture of the impact of the covid- shock in march and april that the official statistics do not (bell and blanchflower the final -step question in part of the table relates to living standards which are also ushaped with a minimum at age . the third and fourth sections of table age -step life satisfaction latinobarometers, with controls age -step life satisfaction issp with controls very little analysis has been done on how well-being and age are treated in africa. the afro barometers are a natural place to turn, but unfortunately, they don't contain any questions on happiness or life satisfaction. both the and surveys do though contain a question on living standards. this living standard, measure of wellbeing, has been widely used in the development literature for measuring well-being in africa. it was used by sulemana et al. ( ) for a study of well-being in sub-saharan africa. they justified its use arguing that "the question taps into the individual's evaluations of their life we used this construct as a suitable measure of subjective wellbeing." the authors argued that "many other studies have constructed well-being measures in the same way," which turns out to be correct. deutsch et al. ( ) used this variable from the afro barometer as did pokimica et al. ( ) and sulemana ( b) in their studies of well-being in ghana. sulemana ( a) in a study of the impact of crime on well-being in africa used data from the th sweep of the afro barometer for . sulemana et al. ( ) used this measure with the afro barometer data in their study of the relationship between corruption and well-being in africa. others have been creative in their use of measures of well-being for africa. bookwalter et al. ( ) in a study of south africa use a household level life satisfaction variable. life satisfaction in both surveys was reported at the household level. the head of the household was asked a -step question q on living standards. q . in general, how would you describe your own present living conditions? possible responses include: = very bad, = fairly bad, = neither good nor bad, = fairly good, = very good? table reports the results from estimating an ols equation with this living conditions variable as the dependent variable with and without controls by country. limiting age to less than , there are countries with significant u-shapes in and seventeen in . fig plots the single year of age coefficients added to the constant for with controls and there are obvious u-shapes again, with minima mostly in the mid-fifties. there are u-shapes for thirty african countries using the afro barometer data for those under age . the u-shape appears to have broad applicability to a range of attitudinal questions on the economy and an individual's personal economic situation as well as to their happiness and life satisfaction. there is a happiness curve. no ifs, no buts, well-being is u-shaped in age. the average age at which the u-shaped minimized across the country-level estimates reported here is . . it is in rich and poor countries. or indeed of happiness in africa, for an exception, see helliwell et al. ( ) who found evidence over the years - that happiness in the middle east and north africa had dropped steadily while sub-saharan africa had no overall trend. the authors identify how much happiness has changed over the last decade and how low it is in africa. they note big declines in happiness in rwanda, malawi, tanzania, central african republic, and botswana (their figure . i found evidence of the nadir in happiness in one hundred and forty-five countries, including one hundred and nine developing and thirty-six developed. i found it in europe, asia, north and south america, australasia, and africa. i identified it in all but six of the fifty-one european countries. i have a well-being u-shape for every one of the thirty-five member countries of the oecd. i have it for / member countries of the united nations. i found the well-being u-shape in english-speaking countries and non-englishspeaking countries. a u-shape is revealed in countries ranked highly in the cia world factbook for countries with both high and low life expectancy at birth. i found it in twelve countries ranked in the top twenty for life expectancy of or more. i also found a u-shape in ten countries in the bottom twenty for life expectancy of countries in the world according to the cia. the curve's trajectory holds true in countries where the median wage is high and where it is not and where people tend to live longer and where they don't. i found additional evidence from an array of attitudinal questions that were worded slightly differently. evidence of a u-shape was found across european countries in questions relating to an individual's finances as well as to the state of the economy and democracy and how public services work. in africa, i used a question that development scholars had used relating to living standards and found a u-shape for thirty african countries. this suggests the u-curve in age may have much broader applicability than just in well-being data. given the robustness of these findings, it remains a puzzle why so many psychologists continue to suggest that well-being is unrelated to age. people are struggling. in the usa, deaths of despair are most likely to occur in the middle-aged years, and the patterns are robustly associated with unhappiness and stress. across countries, chronic depression and suicide rates peak in midlife. those in middle age in the years since were most vulnerable to a once-in-a-generation financial shock especially if they were poor and with low levels of education. in the usa, the employment rate in was below that in . in the uk, real wages were below pre-recession levels at the onset of the covid- crash in march . the financial crisis did not suddenly create frailty in downtrodden communities but simply exposed underlying problems with deep roots in the long decades before. it seems it is normal to have a midlife dip in well-being, but for many, especially those with the least skills, with little social support and few if any savings, that was too much to bear when a giant downturn came along in . the finding of a zenith in well-being in midlife likely adds important support to the notion that being in one's forties and fifties exacerbates vulnerability to disadvantages and shocks. that is people with disabilities, less education, broken families, lost jobs, and so on are likely also to get hit hardest by the effects of aging. some might face downward spirals as age and life circumstances interact. many will not be getting the social/emotional support they need, because midlife is the worst time to present vulnerability. they will be dealing with shame and isolation, in addition to the firstorder effects of whatever they are coping with in normal times at a midlife low is tough. it is made much harder when combined with a deep downturn especially when the speed of recovery and the length of lockdown is uncertain. interdisciplinary research is clearly needed into how to stem the worst manifestations of the midlife nadir in well-being, such as depression, lack of sleep, suicide, and higher tendency to drug and alcohol abuse. the fact that the happiness zenith occurs in developed and developing countries and it has even been found in great apes (weiss et al. ) suggests there may be something deeply engrained perhaps in the genes. the pandemic is global. vulnerable individuals and communities around the world will be devastated by the shock, because of both job and income loss but also from bereavement. the prime aged with low levels of happiness already are especially at risk. the happiness curve is found in countries. no myth. i am grateful to jonathan rauch for these suggestions that he says create a "toxic brew." age minimum u-shape -step life satisfaction minima with controls by year, eurobarometers u-shape -step life satisfaction minima with controls by year, eurobarometers v -step happiness with controls -step happiness with controls, ess - is happiness u-shaped everywhere? age and subjective well-being in kazakhstan was the only country i had data for and did not find a u-shape. the remaining five i had no data for were all tiny-andorra malta ( ) eswatini ( ) causes and correlates of happiness life satisfaction across the life span: findings from two nationally representative panel studies us and uk labour markets before and during the covid- crash the well-being of the overemployed and the underemployed and the rise in depression in the uk is happiness u-shaped everywhere? age and subjective well-being in countries blanchflower dg ( b) unhappiness and age international evidence on well-being in measuring the subjective well-being of nations: national accounts of time use and well-being the mid-life dip in well-being: economists (who find it) versus psychologists (who dont)! nber working paper #w fig. living standards with controls is happiness u-shaped everywhere? age and subjective well-being in subjective well-being around the world: trends and predictors 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friends, and faith of happy people is a longer life a better life? happiness of the very old in eu countries health and social factors related to life satisfaction sliding down the u-shape? a dynamic panel investigation of the age-well-being relationship, focusing on young adults religion and subjective well-being in ghana a note on empirical studies of life-satisfaction: unhappy with semiparametrics? unmet aspirations as an explanation for the age u-shape in well-being the relationship between subjective well-being and work-life balance among labourers in pakistan exploring the economic and social determinants of psychological well-being and perceived social support in england subjective wellbeing, health, and ageing a snapshot of the age distribution of psychological wellbeing in the united states the effect of fear of crime and crime victimization on subjective well-being in africa an empirical investigation of the relationship between social capital and subjective wellbeing in ghana international remittances and subjective wellbeing in sub-saharan africa: a micro-level study a micro-level study of the relationship between experienced corruption and subjective wellbeing in africa how does subjective well-being evolve with age? a literature review is happiness u-shaped everywhere? age and subjective well-being in a test for the convexity of human well-being over the life cycle: longitudinal evidence from a -year panel evidence for a midlife crisis in great apes consistent with the u-shape in human well-being well-being over the life span: semi-parametric evidence from british and german longitudinal data publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations acknowledgments i thank three referees and the editor, angus deaton, dick easterlin, carol graham, robson morgan, kelsey o'connor, andrew oswald, and jonathan rauch for the helpful comments. conflict of interest the author declares that he has no conflicts of interest. key: cord- - s sy authors: banerjee, arinjay; rapin, noreen; miller, megan; griebel, philip; zhou, yan; munster, vincent; misra, vikram title: generation and characterization of eptesicus fuscus (big brown bat) kidney cell lines immortalized using the myotis polyomavirus large t-antigen date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: s sy it is speculated that bats are important reservoir hosts for numerous viruses, with viral families reportedly detected in bats. majority of these viruses have not been isolated and there is little information regarding their biology in bats. establishing a well-characterized bat cell line supporting the replication of bat-borne viruses would facilitate the analysis of virus-host interactions in an in vitro model. currently, few bat cell lines have been developed and only tb -lu, derived from tadarida brasiliensis is commercially available. here we describe a method to establish and immortalize big brown bat (eptesicus fuscus) kidney (efk ) cells using the myotis polyomavirus t-antigen. subclones of this cell line expressed both epithelial and fibroblast markers to varying extents. cell clones expressed interferon beta in response to poly(i:c) stimulation and supported the replication of four different viruses, namely, vesicular stomatitis virus (vsv), porcine epidemic diarrhea coronavirus (ped-cov), middle-east respiratory syndrome coronavirus (mers-cov) and herpes simplex virus (hsv). to our knowledge, this is the first bat cell line from a northern latitude insectivorous bat developed using a novel technology. the cell line has the potential to be used for isolation of bat viruses and for studying virus-bat interactions in culture. it is speculated that bats are an important reservoir host for several viruses, such as ebola virus (family filoviridae, genus ebolavirus), marburg virus (family filoviridae, genus marburgvirus), severe acute respiratory syndrome coronavirus (sars-cov; family coronaviridae, subfamily coronavirinae, genus betacoronavirus), middle-east respiratory syndrome coronavirus (mers-cov; family coronaviridae, subfamily coronavirinae, genus betacoronavirus), porcine epidemic diarrhea coronavirus (ped-cov; family coronaviridae, subfamily coronavirinae, genus alphacoronavirus) and hendra and nipah viruses (family paramyxoviridae, genus henipavirus). there is evidence that many of these viruses have been transmitted from bats to other hosts where they caused serious dis- * corresponding author. e-mail address: vikram.misra@usask.ca (v. misra). ease (drexler et al., ; changula et al., ; wacharapluesadee et al., ) . over different viruses from families have been detected in bats [reviewed in (moratelli and calisher, ) ] but most of these viruses have yet to be isolated and there is scant information regarding the biology of these viruses in bats. bats are genetically diverse and are found dispersed across much of the planet. with over species, bats display major differences in their behavior, feeding habits and the viruses they harbor [reviewed in (moratelli and calisher, ) ]. very little is known, however, about bat immune responses and if these differ across genera and species. currently a single bat cell line (tb -lu, atcc number ccl- , derived from the lung of tadarida brasiliensis) is available through the american type culture collection. research groups have established other bat cell lines, from fruit and insectivorous bats using established techniques such as using the sv t-antigen and expressing human telomerase reverse transcriptase (htert), but these are not commercially available yet (crameri et al., ; jordan et al., ; maruyama et al., ) . bats are the only mammals capable of true flight and as such they may have unique physiological adaptations. for example, they display unique strategies for neutralizing the dna-damaging byproducts of oxidative metabolism produced as a result of increased metabolic activity (shen et al., ) . zhang et al. hypothesize that bats have evolved and accumulated genetic changes as a result of their adaptation to flight. this is to limit collateral damage caused by by-products of an elevated metabolic rate (zhang et al., ) . these genetic changes may be important in the expansion and contraction of important gene families, including genes involved in the innate response pathway (zhang et al., ) . north american bat species are at risk of drastic population depletion due to white-nose syndrome (knudsen et al., ; alves et al., ) and conducting terminal in vivo experiments might not be entirely possible in future. establishing stable bat cell lines would provide an alternative for conducting in vitro host-pathogen studies. experiments using cultured bat cells could provide useful preliminary information on bat innate immune defense responses, virus-cell interactions and cellular physiology. there are several established methods for immortalizing primary cells. the first involves the introduction and stable expression of genes coding for the simian virus (sv ) large t antigen (sv tag). the large t antigen binds to and attenuates the tumor suppressor protein p and proteins of the retinoblastoma tumor suppressor family (prb, p and p ). this promotes dna replication and cell division. this method has been used to immortalize cells from a number of species including human (mayne et al., ) , rabbit (scott et al., ) and rat (lechardeur et al., ) . the second method involves the introduction and stable expression of the catalytic subunit of the human telomerase reverse transcriptase (htert). ectopic expression of htert has been successfully used to immortalize primary cells in a range of mammalian species such as goat mammary epithelial cells ) and canine schwann cells (techangamsuwan et al., ). this enzyme subunit prevents the shortening of telomeres with repeated cell divisions and thus prevents cellular senescence. here we describe a method for establishing and characterizing a kidney cell line (efk ) from eptesicus fuscus (the n. american big brown bat) using the myotis polyomavirus t antigen (mypvtag). we characterized the capacity of mypvtag to enhance dna replication in vero cells and found that it significantly increased their dna content. we then transfected mypvtag into primary bat kidney cells and sub-cloned several cell lines. we characterized the lineage of these clones and tested their expression of the interferon beta (ifn beta) gene in response to polyinosinic-polycytidylic acid (poly(i:c)) stimulation. we further tested three cloned kidney cell lines for their ability to support the replication of viruses from the families coronaviridae, herpesviridae and rhabdoviridae. the parental cell line and clones were capable of expressing ifn beta and supported the replication of viruses such as vesicular stomatitis virus (vsv; family rhabdoviridae, genus vesiculovirus), herpes simplex virus (hsv; family herpesviridae, subfamily alphaherpesvirinae, genus herpesvirus), ped-cov and mers-cov. ped-cov and mers-cov are viruses for which transmission from bats, either directly or via an intermediate reservoir, has resulted in high mortality in pigs (lee, ) and humans (coleman and frieman, ) , respectively. vsv and hsv are members of viral families that have previously been detected in bat species [reviewed in (moratelli and calisher, ) ]. although e. fuscus primary embryonic cells have been described before (qian et al., ) , to our knowledge, this is the first cell line established from a northern latitude insectivorous bat that was transformed by using a viral element (mypvtag) selected from a known bat virus. furthermore, the established kidney cell lines were able to support the replication of selected viruses from three different virus families. all procedures related to the handling and euthanasia of bats were submitted to and approved by the committee on animal care and supply of the university of saskatchewan animal research ethics board (protocol # ) and were in accordance with regulations approved by the canadian council on animal care. a moribund male e. fuscus bat submitted to the laboratory was humanely euthanized. brain, liver, lungs, spleen and kidney were harvested. each organ was finely minced, and incubated at room temperature in . % trypsin-edta (gibco, usa) with agitation. periodically cells were recovered after neutralizing trypsin with fetal bovine serum (fbs; seradigm, usa) added to %. cells were resuspended in dulbecco's minimal essential medium (dmem; corning, usa) containing penicillin (gibco, usa), streptomycin (gibco, usa) and amphotericin b (sigma, usa), placed in cm flasks (cellstar, germany) and incubated at • c in an atmosphere of % co . only kidney cells grew to form a monolayer. these cells were recovered by trypsinization, diluted / and re-plated. cell samples at various passages were cryopreserved in dmem containing % fetal bovine serum (fbs) and % dimethyl sulfoxide (emd chemicals, usa). bat kidney cells were immortalized by using viafect (promega, usa) to transfect cells with either . g of pcdna (invitrogen, usa) empty vector or plasmids expressing either sv large t-antigen (sv tag) or myotis polyomavirus large t-antigen (mypvtag). transfected cells were cultivated in dmem containing % fbs and geneticin reagent (invivogen, usa). only cells transfected with mypvtag continued to replicate. cells were confirmed to be e. fuscus cells by amplifying and sequencing a segment of mitochondrial cytochrome b transcripts (parson et al., ) . mrc cells (atcc ccl- ) were cultured in mem medium (corning, usa) supplemented with % fbs (seradigm usa), / non-essential amino acids (neaa; gibco), / ( -( -hydroxyethyl)- -piperazineethanesulfonic acid (hepes; gibco) and / gentamycin (gibco, usa). vero cells (elaine van moorlehem, vaccine and infectious disease organization -international vaccine center (vido-intervac)) were cultured in dmem with glutagro (corning, usa) supplemented with % fbs (seradigm, usa) and penicillin/streptomycin. all cell lines were checked and controlled for mycoplasma by a semi-nested pcr (described below). efk- b cells were seeded at a concentration of × in a t- flask. the cells were grown up to % confluency and treated with . g/ml colcemid (roche, usa) as mentioned previously (howe et al., ) . the cells were processed, spread on slides and chromosomes were stained with giemsa staining solution as mentioned previously (howe et al., ) . the mypvtag was amplified from the myotis polyomavirus whole genome (national centre for biotechnology information (ncbi), accession number nc . ) cloned in a topo vector (invitrogen, usa) and sub-cloned into a pcdna (invitrogen, usa) backbone. sv tag (a generous gift from ivan sadowski, university of british columbia) was also sub-cloned in pcdna . the pcdna plasmids encoding the t-antigens were used for transfection studies. to quantify dna in t-antigen (t-ag) transfected vero cells, the cells were seeded at a concentration of × cells/well in -well plates. the cells were transfected with . g of either sv tag (sv tag in pcdna ), mypvtag (mypvtag in pcdna ) or pcdna using lipofectamine (thermofisher scientific, usa). the cells were harvested and prepared for flow cytometry twenty-four hours after transfection. briefly, cells were harvested and re-suspended in dulbecco's phosphate-buffered saline (dpbs) (thermofisher scientific, usa). cells were fixed in % ice-cold ethanol for mins and stained for the respective t-antigens using . g/ml mouse anti-sv tag (cross-reactive for sv tag and mypvtag) (molecular probes, usa). the secondary antibody cocktail contained . mg/ml propidium iodide (molecular probes), . mg/ml rnase a (sigma, usa), . g/ml goat anti-mouse immunoglobulin-alexa conjugate (molecular probes) and . % triton x- (sigma, usa) in dpbs. the cells were filtered through m nylon mesh prior to analyses. for analyzing the cell lineage of the clones, intracellular staining using the commercial bd fixative (bd biosciences, usa) was carried out following the manufacturer's recommendation. murine monoclonal antibodies against vimentin ( / dilution of monoclonal anti-vimentin, clone vim . , mouse ascites fluid igm) (sigma-aldrich, usa) and cytokeratin were used ( / dilution of monoclonal anti-cytokeratin . , clone k . , mouse ascites fluid igg a isotype) (sigma, usa). secondary antibodies used were . g/ml goat anti-mouse igm ()-fitc conjugate (caltag/invitrogen, usa) and . g/ml goat anti-mouse igg a-fitc conjugate (caltag/invitrogen, usa). cells were analysed using facscalibur (bd biosciences, usa) with forward scatter detection using a photodiode with / nm bandpass filter and side scatter detection pmt with brewster-angle beam splitter. fitc was detected with a nm laser and / nm band pass filter. for each sample , events were accumulated and analyzed with cellquest pro (bd biosciences, usa). all rna extractions were performed using the rneasy plus mini kit (qiagen, germany) as per manufacturer's instructions. cdna was prepared using the quantitect reverse transcription kit (qiagen) as per manufacturer's instructions. one g of rna was used for cdna preparation. cdna was used as a template for the quantification of target genes. conventional pcr (polymerase chain reaction) to determine the cell lineage of the clones was performed using primers specific for e. fuscus vimentin (bbb-vimentin-f-tcaagaatacccgcaccaacg and bbb-vimentin-r-actgctgacggacgtcgcgc) and cytokeratin (bbb-cytoker-f-gaagacctacaaggtgtccac and bbb-cytoker-r-ccatctcgggtctcaatcttc). primers were designed using the annotated e. fuscus genome on ncbi (accession no. vimentin -xm . ; cytokeratin -xm . ). after initial denaturation for min at • c, the remaining pcr cycles were at • c/ s, • c/ s and • c/ min. the final extension was at • c for min. conventional pcr for the detection and identification of e. fuscus cytochrome b was performed using primers cytb us -cccchcchcayatyaarccmgartgata and cytb ds -tcracdg-gntgycctccdattcatgtta. after initial denaturation for min at • c, the remaining pcr cycles were at • c/ s, • c/ s and • c/ min. the final extension was at • c for min. semi-nested pcr using primers specific to the s rrna gene of mollicutes was performed for the detection of mycoplasma in cell lines. primers were designed as mentioned previously (kong et al., ; yoshida et al., ) . briefly, primers my- -acggcccadactyctacggraggcagcagta and my- -ccrtgcaccayttgtcwhhhbgwwaacctc were used for the first pcr. after initial denaturation for min at • c, the remaining pcr cycles were at • c/ s, • c/ s and • c/ min. the final extension was at • c for min. primers my- and my- -gtaatacatagctcgcaagcgttatc were used for the second pcr. after initial denaturation for min at • c, the remaining pcr cycles were at • c/ s, • c/ s and • c/ min. the final extension was at • c for min. for ifn beta quantification, qpcr assays targeting the ifn beta transcripts and the normalizer (gapdh, glyceraldehyde- phosphate) were performed for the clones. stratagene's mx p pcr (stratagene, usa) cycler was used in conjunction with quantifast sybr green pcr kit (qiagen). primers used were interferon beta (bbb ifnbeta-f-gctccgattccgacagagaagca and bbb ifnbeta-r-atgcatgaccaccatggcttc) and gapdh (bbb gapdh-f-ggagcgagatcccgccaacat and bbb gapdh-r-gggagttgtcatacttgtcatgg). primers were designed using the annotated e. fuscus genome (ncbi, accession no. interferon beta: xm . and gapdh: xm . ). samples were prepared as previously mentioned (rapin et al., ) . the products were quantified based on the amount of relative ifn beta expression. briefly, cells were either transfected with ng/ml poly(i:c) (invivogen, usa) using lipofectamine (thermofisher scientific, usa) or mock transfected. for quantifying ped-cov transcripts, primers were designed to amplify the ped-cov nucleocapsid (n) gene (genbank accession number kf ), (pedv-s gcaacaacaggtccagatctc) and (pedv-r ctccacgaccctggttatttc). for qpcr, after the initial denaturation step of • c for min, the remaining cycles were at • c/ s, • c/ min and • c/ min. the absorbance reading was taken after the • c step. relative fold change in gene expression between the two groups of cells was calculated and plotted after normalizing the ct values for ifn beta using gapdh. three housekeeping genes were tested (gapdh, beta-actin and beta- microglobulin) and none showed variation between treated and mock treated samples. thus gapdh was used for normalizing the data. difference of one ct indicates a two-fold difference in gene expression. pcr and qrt-pcr products were confirmed on a gel and sequenced (macrogen, south korea). reaction efficiencies for qrt-pcr primers were calculated to be between and %. total number of viable cells was determined by using a hemocytometer to count viable cells by trypan blue exclusion method. cells were cultivated in -well plates, trypsinized and re-suspended in media at every time point. efk parental cell line and three subclones were inoculated with vsv-indiana strain (dr. ellis' lab at the university of saskatchewan), hsv, ped-cov (dr. zhou's lab at vaccine and infectious disease organization -international vaccine center (vido-intervac)), and mers-cov (strain emc/ , dr. fouchier at erasmus medical center in the netherlands). for vsv inoculations, wst concentration of the virus was used to infect the cells, which were seeded in well plates at a concentration of × cells/well. wst was determined as that dilution of virus that produced % cell death as measured using the wst- assay. wst- assay is similar to the mtt assay (heldt et al., ) . briefly, cells were seeded in well plates at a density of × /well and analyzed with the wst assay at the indicated time points. l wst- reagent (roche, usa) was added to each well and incubated at • c for h. colour developed was measured at nm with a reference wavelength of nm using molecular devices vmax spectrophotometer. intensity of the colour developed is directly proportional to the number of viable cells in the wells. cells cultured in -well plates were inoculated with l virus (vsv) for hr, rinsed with sterile pbs and medium replaced with dmem containing % fbs. cells and the supernatant were harvested at and h post infection and frozen at − • c. after freeze-thawing the supernatant and cells three times, virus from efk parental cell line and the subclones was titrated in vero cells using the wst assay. virus was quantified using the wst assay and formula as described by heldt et al. (heldt et al., ) . for hsv titration, mrc , efk b, efk f and efk b subclones were seeded in triplicates at a concentration of . × cells/well in well plates. the cells were inoculated with hsv at a multiplicity of infection (moi) of . viral inoculum ( . ml) was replaced with complete medium and plates frozen at and h post inoculation. plates were freeze-thawed x, transferred to ml tubes and centrifuged at rpm for min. supernatant was collected and serially diluted : down to − and titrated in vero cells ( × cells/well in -well plates). for quantifying the virus, ul of diluted virus was added to the wells and incubated for an hour. the inoculum was replaced with . ml of dmem + % pooled human serum (mp biomedical cellect). plaques were counted under the microscope days later. multistep replication kinetics were determined by inoculating wells of cells in triplicate with mers-cov (strain emc/ ) with a moi of . , % tissue culture infectious dose (tcid ) per cell. one hour after inoculation, cells were washed once with dmem and culture medium replaced. culture supernatants were sampled at , , , , and h after inoculation. mers-cov was titrated by end-point titration performed in quadruplicate using vero e cells cultured in dmem supplemented with % fetal calf serum, mm l-glutamine (lonza, usa), u/ml penicillin and g/ml streptomycin. cells were inoculated with ten-fold serial dilutions of virus, and scored for cytopathic effect days later. the tcid was calculated by the method of spearman-karber (hamilton et al., ) . for ped-cov infection, cells were seeded in -well plate at a density of . × /well and cultured overnight. cells were inoculated with ped-cov at an moi of . cells were harvested and total rna extracted at , , , and h. virus replication was quantified by qrt-pcr. significance between t-ag data was calculated by mann whitney u test for two independent samples using ibm spss (version ). the sv large t-antigen is well characterized and is known to enhance dna replication in cells and immortalize primary cells (crameri et al., ) . our laboratory has previously detected a novel polyomavirus in m. lucifugus (misra et al., ) . to determine if the myotis polyomavirus t-ag shared the ability of its sv homologue to induce dna replication, we transfected vero cells with plasmids encoding genes for the two t-antigens. we then confirmed that cells expressed t antigen by immunostaining and compared the dna content of t antigen expressing cells with cells transfected with the pcdna null plasmid. fig. shows cells expressing myotis polyomavirus and sv t antigen contained more dna vero cells were transfected with plasmids expressing either sv tag, mypvtag or empty vector (pcdna). twenty-four hr after transfection, cells were immunestained for cytoplasmic t-antigen and with propidium iodide to quantify dna. the dna content of t-antigen and pcdna transfected cells was determined by flow cytometry and expressed as the fold increase in dna content relative to pcdna transfected cells. the ratio for cells transfected with pcdna was taken as ' . experiments were done in triplicate and mean values plotted. error bars represent standard deviation. statistical difference was calculated using mann-whitney u test for two independent samples. * < . . than cells transfected with pcdna plasmid. sv tag expressing vero cells showed the highest increase in dna content. we attempted to immortalize primary cells derived from the kidney of e. fuscus by transfection with plasmids expressing sv tag, mypvtag or empty vector (pcdna). we observed clusters of cells in cultures transfected with either t-ag but after several passages, only the mypvtag transfected primary cells (efk) continued to replicate. the immortalized efk cell line was cloned by limiting dilution to generate three clones (efk , and ). clones were further isolated by end point dilution of efk , efk and efk cells. we established the clones as separate cell lines and characterized representative clones from each of the three clones i.e. efk , efk and efk , along with the parental efk cells for their cell type markers, interferon beta response, virus susceptibility and cell division rates. we determined the number of chromosomes in efk- b to rule out the possibility of chromosome number abnormality in immortalized cells (supplementary fig. ). efk- b had n = chromosomes, which is normal for genus eptesicus (fedyk and ruprecht, ) . we screened the clones for cytokeratin, a lineage-specific marker for epithelial cells (xie et al., ) and vimentin, a lineage marker for fibroblasts (zschemisch et al., ) . since specific antibodies are not available for bat cytokeratin and vimentin, we used antibodies specific to the human proteins. no positive staining of the bat clones was observed with either anti-vimentin or anti-cytokeratin antibodies when cells were analyzed with flow cytometry (data not shown). we then screened the cell lines for expression of vimentin and cytokeratin transcript by conventional pcr. five of the eight clones analyzed contained detectable transcripts for both cytokeratin and vimentin. in contrast, only vimentin transcripts were detected in primary efk cells at passage (table ) . we characterized the efk clones for their capacity to respond to polyinosinic-polycytidylic acid (poly(i:c)), a synthetic analogue of double-stranded rna (mian et al., ) , through analysis of table transcripts for cell lineage markers vimentin (fibroblast) and cytokeratin (epithelial) are expressed by the efk clones. the ability of the cells to respond to poly(i:c) treatment with increased ifn beta gene expression was detected by qrt-pcr. along with the clones, a parental cell line (efk ) and primary kidney cells (efk; not transfected with either t-ag) were compared. + = pcr product detected, − = no pcr product detected. interferon beta transcription. poly(i:c)-treated primary efk cells displayed an average of , -fold increase in interferon beta transcripts when compared to mock transfected cells. all clones displayed increased interferon beta transcription following poly(i:c) treatment, with the level of increase ranging from six thousand to over sixty thousand. (table ) . all clones displayed similar multiplication rates when assayed with the wst- reagent (data not shown). we determined the cell division rates of three subclones ( b, f and b) and the parental efk cell line by counting viable cells using a hemocytometer at various time points after seeding (fig. ) . the three clones and parental cell line (efk ) did not differ from one another in their multiplication rates. we evaluated the efk parental cell line and three clones ( b, f and b) for their competence in supporting vsv, hsv, ped-cov and mers-cov replication. as a positive control, vero cells were infected with mers-cov and ped-cov and mrc cells were infected with hsv and vsv. vsv caused rounding, sloughing and detachment of mrc cells in culture, ped-cov caused syncytia in vero cells and mers-cov caused rounding and sloughing off of vero cells. cytopathic effects (cpe) such as plaques or rounding and sloughing off of cells following infection with vsv and mers-cov was observed with all efk clones. two of the three clones responded to ped-cov infection with cytopathology. in contrast, efk- f did not exhibit noticeable cpe h.p.i. with ped-cov (fig. ) . the positive control cell lines exhibited cpe h.p.i. following infection with the respective viruses. the evolutionary pressures of flight are thought to have conferred upon bats unique physiological adaptations. most viruses that have been transmitted from bats to other species have been studied in animal models of human disease or in cell lines of non-bat origin. in addition, most in vitro studies of mammalian innate immune and anti-viral responses have been performed in human and rodent cell lines. the results from these studies may not accurately represent pathogen-host interactions that occur in bats. establishing bat cell lines enable researchers to study relevant virus-host interactions in a system that more closely resembles the reservoir host. cell lines have been established from fruit bats (crameri et al., ; virtue et al., ) , myotis myotis (he et al., ) , tadarida brasiliensis and other insectivorous bats (maruyama et al., ) . primary embryonic cells have been developed from e. fuscus (qian et al., ) but an immortalized e. fuscus cell line capable of supporting the replication of viruses from three diverse viral families is not commercially available yet. historically, cells have been immortalized by either using the large t-antigen from sv , which is a monkey virus or by the ectopic expression of htert. we characterized the mypvtag and sv tag's ability to enhance dna replication in cells. both mypvtag and the sv tag significantly increased dna content in vero cells. sv t-ag is known to enhance dna content in cells (ohkubo et al., ; ahuja et al., ) and interestingly the bat polyomavirus tantigen shared similar properties. myotis polyomavirus belongs to the same family as sv . large t-ags from both these viruses were transfected to immortalize the e. fuscus kidney cells, but only cells expressing mypvtag gave rise to stable cell lines. we do not know why sv tag failed to immortalize bat cells. however, it might be possible that the expression of mypvtag, derived from a virus found in this bat, could have been favored by the cellular machinery over the sv tag derived from sv , which has not been detected in big brown bats yet. clones b, a, e, f, b expressed transcripts for lineagespecific proteins of both epithelial and fibroblast cells and clones h, a and b expressed mrna for vimentin. since primary bats cells at passage had detectable transcript for vimentin alone, it is possible that the immortalization procedure may have altered transcription in some of the clones. there is evidence, however, that tumor cells can co-express both epithelial and fibroblast markers (viale et al., ) . it is also possible that mypvtag immortalized a mixture of both epithelial and fibroblast cells and the epithelial cells had a replication advantage during the process of limiting dilution cloning. when cells were being passaged, some cells were more strongly adhered to the plastic and were not removed by trypsiniza- tion. this process could have selected for particular cell types based on their adherent properties. relatively little is known about the innate immune response of insectivorous bats to viral infection. we were able to generate an insectivorous bat cell line capable of upregulating ifn beta gene expression in response to a known innate immune stimulus. an early interferon response is known to inhibit replication of some viruses (katze et al., ) . interferon response in terms of interferon beta transcript upregulation by bat cell lines, mostly cell lines from fruit bats, has been demonstrated before (hagmaier et al., ; crameri et al., ; biesold et al., ; virtue et al., ) . we analyzed the capacity of the efk clones to respond to poly(i:c) stimulation by monitoring interferon beta gene expression. this synthetic analogue of dsrna is usually used as a pathogen-associated molecular pattern (pamp) to stimulate interferon responses (crameri et al., ) . the clones responded to poly(i:c), as we observed a several thousand-fold increase in interferon beta transcript when compared to mock treated cells. immortalization of cells is sometimes known to compromise the ability of the cells to transcribe the interferon beta gene (biesold et al., ) . the efk clones, however, retained a remarkable but variable capacity to respond to poly(i:c). west nile virus, eptesipox virus, novel group i coronavirus and american bat vesiculovirus are examples of viruses that have been detected in eptesicus fuscus (donaldson et al., ; moratelli and calisher ) . the ability of this cell line to support the replication of viruses from three different viral families demonstrates the potential application of this cell line to isolate and study viruses in bats that have previously only been detected using pcr and sequencing. further work can be done to identify receptors specific to these viruses. vsv, hsv, ped-cov and mers-cov grew to varying levels in the efk clones. clone f did not exhibit any visual cytopathology on infection with ped-cov although virus replication was detected by qrt-pcr. this could be due to a lower level of virus replication in f (fig. d ) as the images (fig. ) were taken h post-infection. at this point we do not know if f mounts a more robust interferon response or if it lacks other factors required for ped-cov replication during the initial h post-infection. in conclusion, we established a stable kidney cell line from a northern latitude bat, which has the capacity to respond to a known innate immune stimulus with transcription of ifn beta. furthermore, this cell line supported the replication of viruses from three virus families known to be harbored by bats. not much is known about innate immune responses in bats and how they are activated during viral infections. establishing well-characterized cell lines from relevant bat reservoir species is the first step in addressing the many questions that researchers have about innate immunity in bats moratelli and calisher ) . this cell line will help us better understand the bat innate responses and how they may contribute to the absence of overt disease symptoms when bats are infected with these viruses. the e. fuscus kidney cell line developed here is now available for research through kerafast, usa. the goal of this study was to generate a cell line that would allow researchers to potentially study viruses derived from bats in a reservoir in vitro model. sv large t antigen targets multiple cellular pathways to elicit cellular transformation the potential impact of white-nose syndrome on the conservation status of north american bats large t antigens of polyomaviruses: amazing molecular machines antiviral immune responses of bats: a review type i interferon reaction to viral infection in interferon-competent, immortalized cell lines from the 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in a natural host for emerging viruses a colorimetric assay for viral agents that produce cytopathic effects chromosome preparation from cultured cells authentication of the r e fruit bat cell line viruses and interferon: a fight for supremacy potential spread of white-nose syndrome of bats to the northwest: epidemiological considerations species-specific pcr for identification of common contaminant mollicutes in cell culture induction of blood-brain barrier differentiation in a rat brain-derived endothelial cell line porcine epidemic diarrhea virus: an emerging and re-emerging epizootic swine virus characterization of the envelope glycoprotein of a novel filovirus, lloviu virus efficient immortalization and morphological transformation of human fibroblasts by transfection with sv dna linked to a dominant marker length of dsrna (poly i:c) drives distinct innate immune responses, depending on the cell type detection of polyoma and corona viruses in bats of canada bats and zoonotic viruses: can we confidently link bats with emerging deadly viruses? sv large t antigen reinduces the cell cycle in terminally differentiated myotubes through inducing cdk , cdc , and their partner cyclins species identification by means of the cytochrome b gene role of the spike glycoprotein of human middle east respiratory syndrome coronavirus (mers-cov) in virus entry and syncytia formation activation of innate immune-response genes in little brown bats (myotis lucifugus) infected with the fungus pseudogymnoascus destructans maintenance of expression of differentiated function of kidney cells following transformation by sv early region dna adaptive evolution of energy metabolism genes and the origin of flight in bats transfection of adult canine schwann cells and olfactory ensheathing cells at early and late passage with human tert differentially affects growth factor responsiveness and in vitro growth coexpression of cytokeratins and vimentin in common epithelial tumours of the ovary: an immunocytochemical study of eighty-three cases interferon production and signaling pathways are antagonized during henipavirus infection of fruit bat cell lines diversity of coronavirus in bats from eastern thailand establishment and characterization of a telomerase-immortalized canine bronchiolar epithelial cell line rapid detection of mycoplasma genitalium, mycoplasma hominis, ureaplasma parvum, and ureaplasma urealyticum organisms in genitourinary samples by pcr-microtiter plate hybridization assay comparative analysis of bat genomes provides insight into the evolution of flight and immunity immortalized tumor derived rat fibroblasts as feeder cells facilitate the cultivation of male embryonic stem cells from the rat strain wky/ztm we thank nathalie berube supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/ . /j.jviromet. . . . key: cord- - k eyqx authors: diekmann, berend title: globale handelsordnung — mit den oder ohne die usa? date: - - journal: wirtschaftsdienst doi: . /s - - - sha: doc_id: cord_uid: k eyqx nan einer ausgebildeten industriearbeiterschaft. das überragende interesse der usa an einer stabilen weltwirtschaftsordnung mit offenen märkten war also durchaus auch eigennützig: nur stabile märkte mit klaren handelsregeln waren aufnahmebereit für us-waren. mit wechselnden motiven unterstützten die usa in den folgenden dekaden die weiterentwicklung der internationalen handelsordnung bis hin zum abschluss der uruguay-runde , in der die welthandelsorganisation (wto) aus der taufe gehoben wurde. wesentliche beweggründe waren die idee, die ehemaligen staatshandelsländer und etwas später auch das sich öffnende china in eine welthandelsordnung zu integrieren, die von der marktöffnung als leitmotiv bestimmt war. mit sicherheit schwang dabei die absicht mit, diese märkte für anbieter aus den usa besser zu öffnen und umgekehrt die usa selbst mit günstigen zwischen-und konsumprodukten zu versorgen. diese strategie verfolgten die usa ungeachtet des umstands, dass das verarbeitende gewerbe der usa zu diesem zeitpunkt im außenhandel nur noch zeitgespräch hänge: sie denken in ad-hoc-deals, sehen ein handelsbilanzdefi zit als monetären verlust und handelsbilanzüberschüsse als gewinn im betriebswirtschaftlichen sinn. sie beschränken sich auf die alleinige betrachtung der warenhandelsbilanz und blenden andere wichtige zusammenhänge (wie z. b. den handel mit dienstleistungen, primäreinkommen, intrafi rmenhandel) aus. die schiere größe des us-markts bewegt sie dazu, zölle bzw. generell politisches wohlverhalten wie ein "eintrittsgeld" in den us-markt zu nutzen und die gestiegene geopolitische unsicherheit bei kleineren partnern merkantilistisch auszubeuten. in ihrem betriebswirtschaftlichen, kurzfristigen kalkül gibt es sogar einen anreiz, unsicherheit zu schüren. das resultatorientierte verhalten führt zusammen mit einer betriebswirtschaftlichen auffassung von außenwirtschaft zu einem unsteten, um nicht zu sagen unzuverlässigen handelspolitischen verhalten der usa. welchen einfl uss diese entwicklung jedoch zugleich auf die haltung der usa zum freihandel ausüben konnte, war an der harten us-reaktion auf die japanischen exporterfolge in den er jahren abzulesen. japanische anbieter sahen sich einem enormen druck ausgesetzt, der zu "freiwilligen" exportbeschränkungen und mehr oder weniger erzwungenen japanischen direktinvestitionen in den usa führte. die schwankungen in der us-haltung zu einer multilateralen handelsordnung haben sich nach sowohl durch interne wie durch externe faktoren noch verstärkt. zu den internen faktoren zählten z. b. das misstrauen gegenüber der wirksamkeit von regelsystemen im welthandel und einer beschränkung der eigenen souveränität. dieses misstrauen war zwar schon immer vorhanden. hinzu trat aber nun die verstärkte wahrnehmung eines gegensatzes zwischen regeln und resultaten. die regeln waren und sind aus us-sicht entweder zu vage formuliert, die sanktionsmechanismen zu langsam und unzureichend, sie sind für neue herausforderungen (china) ungeeignet oder sie greifen zu sehr in die souveränitätsrechte ein. traditionell reagieren die usa zudem besonders empfi ndlich auf konstruktionen, die dazu führen, dass internationale entscheidungen us-recht aushebeln können (felbermayr, ) . als externe faktoren kamen die auslagerung von amerikanischer produktion ins ausland (offshoring), das phänomen eines wachstums ohne mehr arbeitsplätze (jobless growth) und ein ausuferndes handelsbilanzdefi zit hinzu. Über allem rangierten der mit immer mehr unbehagen wahrgenommene wirtschaftliche aufstieg chinas und das scheitern der integration russlands in die gruppe der marktwirtschaftlich und demokratisch verfassten länder. diese faktoren führten zu der hinwendung der usa zu einer an schnelllebigen, vorteilhaften resultaten orientierten handelspolitik. dies manifestiert sich nicht nur in klar defi nierten und inzwischen auch quantifi zierten marktzugangszielen der usa, wie schon beim von der obama-regierung formulierten ziel der verdopplung der us-exporte zu beobachten. vielmehr wuchs der merkantilismus mit dem amtsantritt der trump-administration in eine neue dimension hinein. einfl ussreiche teile der neuen us-regierung einschließlich des präsidenten selber missverstehen die wirtschaftspolitische aufgabe als betriebswirtschaftliches thema und verkürzen zusammen- insbesondere die betonte rolle der nationalen sicherheit für die handelspolitik und die explizit angekündigte aggressive durchsetzung von us-handelsrecht akzentuieren den veränderten ansatz. aber auch hinter dem ziel verbesserter handelsabkommen verbirgt sich handelspolitischer sprengstoff, denn spätestens seit dem abschluss der neuverhandlungen zum nordamerikanischen freihandelsabkommen (nafta), nunmehr us-mexico-canada agreement (usmca) genannt, ist klar, dass damit vor allem verschärfte ursprungsregeln und mehr "local content" gemeint ist, also bedingungen, die den globalen handel nicht fördern, sondern bremsen. von den nur drei jahre zuvor vom cea aufgelisteten argumenten ist kaum noch etwas übrig. mit diesem handelspolitischen ansatz können die usa die rolle des kompensierenden hegemons, die sie über dekaden im gatt und zunächst auch in der wto einnahmen, nicht mehr spielen. hegemon sind die usa, weil sie über jahrzehnte den mitgliedern des gatt/der wto nicht nur den zugang zum lukrativen us-markt zu stabilen bedingungen boten, sondern auch ihr politisches gewicht in die waagschale warfen, damit die regeln glaubwürdigkeit und gültigkeit behielten. allerdings hat die welthandelsordnung mit dem protektionistischen ansatz der usa nicht nur ihren hegemon verloren. man kann sie und die zugehörige welthandelsorganisation wto volkswirtschaftlich als klubgut charakterisieren. als klubgut werden güter bezeichnet, bei denen ausschließbarkeit von der nutzung möglich ist und keine oder eine nur geringe rivalität im konsum vorliegt. die mitgliedschaft in einem klub ist freiwillig. nicht-zahler können von der mitgliedschaft im klub/der nutzung des klubguts ausgeschlossen werden. diese defi nition trifft grundsätzlich auf die wto und ihre mitgliedschaft zu. allerdings funktioniert ein solches klubgut umso besser, je homogener die interessen der klubmitglieder sind, je homogener die leistungen ausfallen und je ausgewogener die lastenverteilung ist (langhammer, ) . die wto-mitglieder sind infolge der steigenden mitgliederzahl inzwischen in ihren interessen sehr heterogen. auch die homogenität der themen ist angesichts der ausweitung des themenspektrums in der wto nicht mehr gegeben. um ein extrembeispiel zu nehmen: aus zollverhandlungen im gatt sind zuletzt sogar diskussionen um handel und geschlechtergerechtigkeit in der wto geworden. gerade für große wto-mitglieder ist es deshalb sehr attraktiv geworden, eine homogenität des klubgutes "handelsordnung" wieder herzustellen, indem sie ihre interessen mit gleichgesinnten partnern außerhalb der wto verfolgenz. b. durch bilaterale und plurilaterale handelsabkommen. beschwerdeführer und in fällen beschwerdegegner (wto, ; eigene berechnungen). damit entfi elen auf die usa % aller fälle. zusätzlich beteiligten sich die usa in fällen als drittpartei. folglich wird nur etwa ein viertel aller streitverfahren ohne us-beteiligung geführt. in fast der hälfte aller verfahren traten die usa aktiv entweder als beschwerdeführerin oder als drittpartei auf. besser lässt sich das rege interesse der usa an der wto-streitbeilegung kaum belegen. geradezu exemplarisch zeigt sich die -je nach politischer ausrichtung der regierung -stärker denn je schwankende us-haltung zur rolle des freihandels insgesamt bei einem vergleich der argumentation in der studie des council of economic advisers (cea) zu den wirtschaftlichen vorteilen des außenhandels von mit den aussagen in der vom us-handelsbeauftragten (ustr) damit gerät allerdings auch der originäre anspruch der welthandelsordnung ins wanken: die nicht-diskriminierung auf basis der gegenseitigkeit. sie besteht aus den beiden komponenten meistbegünstigung und inländerbehandlung und ist damit so angelegt, dass auch mitgliedsländer mit geringerer marktgröße gleichberechtigt partizipieren können. zusammen mit dem gesamtverpfl ichtungsansatz (single undertaking), der es erlaubt, marktöffnungen in unterschiedlichen sektoren gegeneinander aufzurechnen, war die handelsliberalisierung ursprünglich so strukturiert, dass alle mitgliedstaaten ausreichend spielräume für erfolge und zugeständnisse hatten. da zugleich ein hegemon vorhanden war, hatte das wto-regelwerk eine vergleichsweise hohe bindungskraft und erschien nicht-kooperatives verhalten lange zeit unattraktiv. in ihrer distanzierung von der bisherigen welthandelsordnung gehen die usa aber zuletzt konzeptionell auch noch deutlich über die eigenen wege anderer großer wto-mitglieder wie der eu, die in ihren regionalen abkommen durchaus eigene akzente gesetzt hat, hinaus. dies lässt sich gut am beispiel des sogenannten phase-i-abkommens zwischen den usa und china belegen, das im januar abgeschlossen wurde: zwar wurde mit dem abschluss dieses teilabkommens eine weitere eskalation des handelskonfl ikts zwischen den zwei größten volkswirtschaften der welt erst einmal abgewendet. gleichzeitig ist das abkommen mit seinen umfassenden kaufverpfl ichtungen für china aber ausdruck eines kleinteiligen "managed trade", der konzeptionell im scharfen widerspruch zu den grundsätzen des freien und regelbasierten handels im rahmen der wto steht und im zweifel zulasten von drittländern geht. anders als durch (teil-) substitution anderer importe dürfte china insbesondere die verpfl ichtungen im agrarsektor, aber auch im industriesektor, nicht erfüllen können. erschwerend kommt hinzu: je höher der anteil von staatsunternehmen bzw. das ausmaß staatlicher lenkung in einem sektor ist, desto leichter dürfte es china fallen, bestimmte quantitativ formulierte einkaufszusagen zu erfüllen. ein hoher staatseinfl uss ist in china beispielsweise im energie-, gesundheits-oder luftfahrtsektor anzunehmen. de facto stärkt der us-ansatz eines "managed trade" mit china damit -paradoxerweise -den staatshandelscharakter und die rolle staatsnaher bzw. staatseigener unternehmen in china. zu diesem geradezu planwirtschaftlichen ansatz in der us-handelspolitik tritt ein neues, kleinteiliges buchsta- länder -auch china -derzeit dazu neigen, den export von medizinischen schutzprodukten restriktiv zu handhaben und da ein halbes jahr mit zusatzzöllen ausgereicht haben dürfte, handelsumlenkungen auszulösen, die nicht so schnell umkehrbar sind. das generell gewachsene misstrauen gegenüber der sprunghaftigkeit der us-handelspolitik könnte ein weiterer retardierender faktor sein. dass diese erfahrungen ausreichen, damit die usa ihren eigenen handelspolitischen ansatz fundamental überdenken, erscheint im lichte des us-typischen pragmatismus möglich. anstöße der wichtigsten us-handelspartner zum richtigen zeitpunkt, also unmittelbar nachdem das schlimmste überwunden ist, könnten an der stelle hilfreich wirken. trump's trade policy is hampering the us fi ght against covid- the economic benefi ts of zur rückkehr der machtpolitik in handelsfragen: theoretische Überlegungen und politische empfehlungen, perspektiven der wirtschaftspolitik unordnung in der internationalen handelsordnung: befunde, gründe, auswirkungen und therapien president's trade agenda disputes by member kranke zu versorgen -unter anderem weil medizinische schutzausrüstung in den usa knapp wird. vor dem handelskonfl ikt mit china importierten die usa medizinprodukte im wert von ca china war unter anderem hauptlieferant für schutzausrüstung, die eu lieferte in erster linie medizinisches gerät wie z. b. computertomografen oder patientenmonitore. durch die im transpazifi schen handelskonfl ikt verhängten us-zölle auf chinesische produkte erhöhten sich bisher niedrige oder nicht vorhandene zölle auf medizinprodukte massiv auf % bzw. %. hierdurch verlagerte sich die us-nachfrage nach medizinprodukten: die importe aus china sanken zwischen importe aus dem rest der welt stiegen um % eingeführt worden waren, bedeutete die entscheidung des ustr eine vollständige, der not gehorchende kehrtwende. sie belegte zugleich eindrucksvoll die fehlkalkulation der us-handelspolitik, die die eigene position (unverwundbarkeit) überschätzte, was jetzt möglicherweise auch einer breiteren Öffentlichkeit bewusst wird und zu einer gefahr für den gesamtansatz der derzeitigen us-handelspolitik werden könnte key: cord- - yb dxse authors: kang, jun-gu; jeon, kyeongseok; choi, hooncheol; kim, yuri; kim, hong-il; ro, hyo-jin; seo, yong bok; shin, jua; chung, junho; jeon, yoon kyung; kim, yang soo; lee, keun hwa; cho, nam-hyuk title: vaccination with single plasmid dna encoding il- and antigens of severe fever with thrombocytopenia syndrome virus elicits complete protection in ifnar knockout mice date: - - journal: plos negl trop dis doi: . /journal.pntd. sha: doc_id: cord_uid: yb dxse severe fever with thrombocytopenia syndrome (sfts) is an emerging tick-borne disease caused by sfts virus (sftsv) infection. despite a gradual increase of sfts cases and high mortality in endemic regions, no specific viral therapy nor vaccine is available. here, we developed a single recombinant plasmid dna encoding sftsv genes, gn and gc together with np-ns fusion antigen, as a vaccine candidate. the viral antigens were fused with fms-like tyrosine kinase- ligand (flt l) and il- gene was incorporated into the plasmid to enhance cell-mediated immunity. vaccination with the dna provides complete protection of ifnar ko mice upon lethal sftsv challenge, whereas immunization with a plasmid without il- gene resulted in partial protection. since we failed to detect antibodies against surface glycoproteins, gn and gc, in the immunized mice, antigen-specific cellular immunity, as confirmed by enhanced antigen-specific t cell responses, might play major role in protection. finally, we evaluated the degree of protective immunity provided by protein immunization of the individual glycoprotein, gn or gc. although both protein antigens induced a significant level of neutralizing activity against sftsv, gn vaccination resulted in relatively higher neutralizing activity and better protection than gc vaccination. however, both antigens failed to provide complete protection. given that dna vaccines have failed to induce sufficient immunogenicity in human trials when compared to protein vaccines, optimal combinations of dna and protein elements, proper selection of target antigens, and incorporation of efficient adjuvant, need to be further investigated for sftsv vaccine development. a a a a a severe fever with thrombocytopenia syndrome (sfts) is an emerging tick-borne infectious disease caused by sfts virus (sftsv), belonging to the phenuiviridae family of bunyavirales [ , ] . the genome of sftsv is composed of three segmented rnas: large (l) segment encoding rna-dependent rna polymerase (rdrp), medium (m) encoding the envelope glycoproteins, gn/gc, and small (s) encoding the nucleocapsid and nonstructural proteins (np and ns) [ ] . clinical manifestations include fever, gastrointestinal symptoms, leukocytopenia, and thrombocytopenia [ , ] . disease mortality of sfts patients have been estimated to be % [ ] . even though the majority of sfts cases has been reported from china [ ] , korea [ ] , and japan [ ] , sftsv infections in southern asia, including vietnam, have been recently reported in a retrospective survey [ ] . currently, no specific viral therapy nor vaccine is available. an effective vaccine is needed to combat its relatively high mortality, especially in elderly patients, and spread of sftsv between humans [ , ] . vaccine development for sfts is at an early discovery phase and there have only been a few studies on vaccine candidates using animal infection models [ ] [ ] [ ] [ ] . immunization of ns antigen with freund's adjuvant in c bl/ mice, which are naturally resistant to sftsv but partially mimic human infections [ ] , failed to enhance viral clearance, although it induced high titer of anti-ns antibodies and significantly elevated ifn-γ levels in sera upon viral challenge [ ] . vaccination of plasmid dnas encoding np and ns peptides also enhanced antigen-specific cellular immunity of t cells, such as ifn-γ and tnf-α secreting cd + and cd + t cells, in balb/c mice when applied by nano-patterned microneedles [ ] . however, the protective effect of cellular immunity induced by dna vaccination was not confirmed by in vivo infection with sftsv. recently, dong f. et al. reported that a single dose of live attenuated recombinant vesicular stomatitis virus (rvsv) vaccine expressing the sftsv gn/gc glycoproteins elicited high titers of protective neutralizing antibodies in both wild type and interferon α/β receptor knockout (ifnar ko) mice [ ] . they clearly showed that a single dose rvsv carrying sftsv gn/gc could provide complete protection against lethal challenge with sftsv in young and old ifnar ko mice, a promising infection model for severe human infection of sftsv [ , , ] . nevertheless, the potential role of cellular immunity against the viral antigens in complete protection was not examined in this study, although adoptive transfer of immune sera from mice immunized with rvsv-gn/gc to naïve ifnar ko mice provide partial (~ %) protection against lethal sftsv challenge [ ] . in this study, we developed a recombinant plasmid dna (psftsv) encoding extracellular domains of gn and gc, and np-ns fusion antigen as a dna vaccine candidate. we examined whether it could provide protective immunity against lethal sftsv infection in ifnar ko mice. in order to facilitate the processing and presentation of the sftsv antigens by dendritic cells (dcs) and enhance antigen-specific t cell responses, these recombinant antigens were fused with fms-like tyrosine kinase- ligand (flt l) [ , ] . moreover, we generated a recombinant dna encoding il- α and β in addition to the recombinant viral antigens (psftsv-il ) to further enhance cell-mediated immunity [ ] . vaccination of psftsv-il provided complete protection of ifnar ko mice upon lethal sftsv challenge, whereas immunization with psftsv elicits only partial protection, indicating that antigen-specific cellular immune responses enhanced by co-expression of il- could play a significant role in protection against lethal sftsv infection. we confirmed significantly higher levels of gn and np-specific cd + and cd + t cell responses in mice vaccinated with psftsv-il when compared to those in mock vector-immunized mice. therefore, our results indicate that enhanced antigenspecific t cell immunity against multiple sftvs antigens by dna vaccination could be a promising direction for developing an effective vaccine against sftsv infection. animal experiments were conducted in an animal biosafety level facility at seoul national university hospital and international vaccine institute. this study was approved by the seoul national university hospital and international vaccine institute institutional animal care and use committee (snuh iacuc no. - -c a and ivi iacuc no. - ) and conducted in strict accordance with the recommendations in the national guideline for the care and use of laboratory animals. in order to generate plasmids for dna vaccination, genes encoding ectodomains of gn, gc (from genebank accession no. ajo . ), and np/ns fusion protein (from genebank accession no. ajo . and aki . , respectively) were synthesized (genscript, piscataway, nj, usa) and cloned into pgx vector (genexine, seongnam, republic of korea). all these genes were fused with signal peptide of tissue plasminogen activator (tpa, uniport no. p ) and flt l (uniport no. p ) in their n-terminus [ ] (psftsv, fig ) . in addition, murine il- α and β genes (uniport no. p ) [ ] were also synthesized (genscript) and cloned into psftsv (psftsv-il- , fig ) . to confirm the expression of cloned genes in psftsv and psftsv-il , hek t cells (atcc crl- , manassas, va, usa) were transfected with pgx , psftsv, or psftsv-il plasmid using the polyethylenimine (pei) transfection method [ ] . briefly, plasmids and pei were diluted with opti-mem media (gibco, gaithersburg, md, usa) and incubated with hek t cells. after h of incubation, media were changed with dulbecco's modified eagle medium (dmem) (gibco) containing % fetal bovine serum (fbs) and incubated in humidified co atmosphere at ˚c. after h of culture, cells and culture supernatants were harvested. cells were washed with phosphate-buffered saline (pbs) and lysed with np- lysis buffer ( % np- in mm tris-hcl, ph . , mm nacl) containing protease inhibitor cocktail (sigma-aldrich, st. louis, mo, usa). lysates and supernatants were stored at - ˚c until use. for quantification of flt l-fused proteins and mouse il- in the supernatants of transfected hek t cells, human flt l and mouse il- p quantikine elisa kits (r&d systems, minneapolis, mn, usa) were used according to manufacturer's instructions. expression of gc, gn, and np/ns fusion protein in the culture supernatants and cell lysates were examined by immunoblotting using rabbit anti-gn, gc (nbp - and nbp - , novus biologicals, centennial, colorado, usa), and np (produced by custom polyclonal antibody production service via abclon, seoul, republic of korea) antibodies, respectively. each sample was separated on % polyacrylamide gels and transferred onto . μm pvdf membranes (merck, darmstadt, germany). pvdf membranes were blocked with pbs containing . % tween (pbst) and % skim milk at room temperature for h. the membranes were sequentially incubated with primary and secondary antibodies conjugated with horse radish peroxidase (hrp, invitrogen, waltham, ma, usa). the membranes were then visualized using a las- system (fujifilm, tokyo, japan). the gene encoding the np of the kasjh strain (genbank accession no. kp ) was cloned into the pet a (+) vector (novagen, gibbstown, nj, usa). np protein was then purified from e. coli strain bl (de ) harboring the recombinant plasmid. following induction with . mm isopropyl β-d-thiogalactoside (iptg) for h at ˚c, the protein was purified using histrap hp histidine-tagged protein columns (ge healthcare, chicago, il, usa) according to the manufacturer's instruction. recombinant gn and gc glycoproteins fused to fc region of human immunoglobulin heavy chain were purified as previously described [ ] . briefly, vectors cloned with gene encoding gn or gc were transfected into hek f cells (thermo fisher scientific, waltham, ma, usa) using polyethylenimine. the transfected cells were cultured in freestyle expression medium (gibco) for days. overexpressed recombinant proteins in supernatants were purified by akta start affinity chromatography system hitrap mabselect (ge healthcare) according to the manufacturer's instructions (s fig). to determine the antibody titers specific to gc, gn, and np in sera of immunized mice, immunoassay plates ( -well plates; nunc, rochester, ny, usa) were coated with ng/well of purified antigens at ˚c overnight. his-tagged gn and gc recombinant proteins were purchased from immune technology co. (new york, ny, usa). his-tagged np recombinant protein was purified as mentioned above. after antigen coating, the immunoassay plates were blocked for h at room temperature with pbst containing % skim milk. μl of seriallydiluted serum samples were incubated for h at room temperature and subsequently detected using hrp-conjugated goat anti-mouse igg (santa cruz biotechnology, santa cruz, ca, usa). , ', , '-tetramethylbenzidine peroxidase substrate solution (kpl, gaithersburg, md, usa) was then added to develop color for min, and the reaction was stopped by the addition of m h po solution. absorbance was measured at nm using a microplate reader (tecan, mannedorf, switzerland). spleen cells were released into rpmi media (gibco) by mincing the spleen through a μm cell strainer (bd biosciences, san jose, ca, usa). after lysis of red blood cell with a red blood cell lysing buffer hybri-max (sigma, st. louis, mo, usa), splenocytes were cultured for h in rpmi media containing % fbs (gibco) and % penicillin/streptomycin (gibco) in the presence of μg of purified gn or np antigens in well v-bottomed culture plates (nunc, roskilde, denmark). for intracellular detection of ifn-γ, splenocytes ( × cells/well) were treated with μg of golgiplug (bd bioscience) for the final h. cells were then blocked with ultra-block solution ( % rat sera, % hamster sera, % mouse sera (sigma), and μg/ml of anti-cd / ( . g ) (bd pharmingen, franklin lakes, nj, usa), followed by staining with anti-cd ( - c ) (bd biosciences), cd (rm - ) (bd biosciences) and cd ( - . ) (biolegend, san diego, ca, usa) antibodies conjugated to different fluorescent dyes. after surface staining, splenocytes were fixed and permeabilized with cytofix/cytoperm kit (bd bioscience) and incubated with anti-ifn-γ antibody (xmg . ) (bd pharmingen). the stained cells were analyzed on a cytoflex s flow cytometer (berkman coulter inc, brea, ca, usa). flow cytometry data were analyzed using flowjo software version . (tree star, ashland, or, usa). gating strategies for the flow cytometric analyses are summarized in s fig. sftsv (genbank accession no. mn -mn ) isolated from a korean sfts patient was propagated in vero e cells (atcc crl- ). the supernatant of infected cells was harvested at d after infection and stored at- ˚c after filtering with . μm syringe. focusforming unit (ffu) of sftsv was determined by plaque assay using methylcellulose media [ ] . briefly, the filtered supernatants were serially diluted and added to a monolayer of vero e cells and incubated for h at ˚c. viral supernatants were removed and cells were incubated under an overlay media (dmem supplemented with % fbs and % methylcellulose) at ˚c for days. cells were fixed with % paraformaldehyde (intron, seongnam, republic of korea) and % methanol (merck, darmstadt, germany). the sftsv foci were detected using rabbit anti-sfts np antibody (abclon) and goat anti-rabbit igg secondary antibody conjugated with alkaline phosphatase (thermo fisher scientific). viral plaques were visualized by incubation with nbt/bcip solution (roche, mannheim, germany). to evaluate the neutralizing activity of immune sera, a focus reduction neutralization titer (frnt) assay was performed using immunized mice sera [ ] . sftsv ( . multiplicity of infection) was pre-incubated with serially diluted sera from mice at ˚c for h. the mixture of virus and sera was added onto a monolayer of vero e cells in -well plate. after incubation for h, supernatant of cells were removed and cells were cultured under an overlay media at ˚c for days. viral foci were visualized as described above. the percentage of focus reduction was calculated as [(no. of plaques without antibody)-(no. of plaques with antibody)] / (no. of plaques without antibody) x . % focus reduction neutralization titers (frnt ) were calculated by a nonlinear regression analysis (log[inhibitor] versus normalized response method) embedded in graphpad prism software v . (graphpad software; https:// www.graphpad.com). six to eight-week-old female interferon α/β receptor knockout (ifnar ko, b bl/ ) mice [ ] were used for immunization and challenge tests. they were housed and maintained in the specific pathogen-free facility at seoul national university college of medicine. mice were intramuscularly immunized by electroporation using orbijector ep-i model (sl vaxigen inc., seongnam, republic korea) in the hind leg three times at two-week intervals. μg of purified pgx , psftsv, or psftsv-il in μl of pbs was used for each immunization. mice were also subcutaneously immunized with μg of gn-fc or gc-fc protein absorbed in aluminum hydroxychloride (alhydrogel adjuvant %, invivogen, hong kong). mice sera were collected from immunized mice at one week after the third immunization to determine the levels of specific antibody titers. two weeks after the final immunization, mice were subcutaneously challenged with x ffu of sftsv. body weight and mice survival were monitored until surviving mice fully recovered. blood and tissues of mice were collected at the indicated time after viral challenge and applied for viral quantitation using qrt-pcr, or hematological analysis. total rna was extracted from the plasma of sftsv-infected mice using trizol ls reagent (life technologies, carlsbad, ca, usa) according to the manufacturer's instruction. total rna was reverse transcribed into cdna using hisenscript rh (-) rt premix kit (intron, seongnam, republic of korea). cdna was quantified using taqman universal master mix (applied biosystems, waltham, ma, usa). qrt-pcr was performed on biorad cfx connect real-time system (bio-rad, hercules, ca, usa) under following conditions: uracil-n-glycosylase incubation at ˚c for min, polymerase activation at ˚c for min, denaturation at ˚c for s, annealing and extension at ˚c for min. amplification was performed for cycles and the fluorogenic signal was measured during annealing/extension step. primer set and detecting probe for qrt-pcr was derived from the np gene of sftsv: np forward ( '-ccttcaggtcatgacagctgg- '), np reverse ( '-accaggctctcaatcactcctg t- ') and detecting probe ( '- fam-agcacatgtccaagtgggaaggctctg-bhq - '). copy numbers were calculated as a ratio with respect to the standard control. to collect hematological data, animals were euthanized and bled by cardiac puncture. blood was prepared in tubes coated with . m edta (enzynomics, daejeon, republic of korea). prior to evaluation of platelet counts, % paraformaldehyde was added to edta-anticoagulated whole blood samples at a : ratio to inactivate virus [ ] . the platelet counts were analyzed using advia i hematology system (siemens healthimeers, erlangen, germany). data was analyzed using the graph pad prism . software (graphpad software, la jolla, ca, usa). statistical analysis was performed using two-tailed student's t-test with % confidence interval or one-way analysis of variance (anova) followed by newman-keuls t-test for comparisons of values among different groups. data are expressed as the mean ± standard deviation (s.d.). statistical analysis on survival rates were performed using the mantel-cox log rank test. a p-value of < . was considered statistically significant. four viral genes of sftsv were cloned into pgx vector to be expressed in mammalian cells (fig ) . extracellular domains of viral glycoproteins, gn and gc, were separately cloned into the plasmid vector under control of cmv promoter and ires sequence. full lengths of viral np and ns genes fused with a linker peptide (gsgsgsgsgsgra) were also cloned into the vector under control of rsv promoter. all the proteins were fused with the extracellular domain of flt l and the signal sequence of tissue plasminogen activator (tpa) in their n-terminus to promote antigen presentation and trafficking of the fusion proteins as previously described [ ] . in addition, psftsv-il plasmid also encodes murine il- α and β to enhance antigen-specific t cell responses [ ] . expression and secretion of the viral antigens and il- in hek cells transfected with the plasmids were confirmed by elisa and immunoblot analysis (fig ) . all the viral antigens were detected in cell culture supernatants, as well as in cellular lysates (fig b) . next, we assessed antigen-specific adaptive immunity against the viral antigens after vaccination in ifnar ko mice. antibody responses to np antigen was significantly elevated (mean titer ± s.d.: ± , n = ) only in mice immunized with psftsv-il , and not in mice vaccinated with mock vector or psftsv at two weeks after third immunization (fig a) . specific antibodies against gn and gc were barely detectable in all the vaccinated groups, suggesting that these dna vaccines may not efficiently induce specific antibodies against gn or gc, but produce np antibody responses in the presence of il- expression. in order to characterize the potential difference in quality of cell-mediated immunity in ifnar ko mice immunized with the dna vaccines, we analyzed antigen-specific t cell responses by assessing ifn-γ secreting t cells in an antigen-dependent manner (fig b and c ). splenocytes collected from immunized mice were stimulated with gn or np antigens and cytokine-positive t cell subsets were analyzed by flow cytometry. the frequencies of cytokinepositive cd and cd t cells induced by splenocytes of psftsv-il -immunized mice were significantly higher than those of vector-immunized mice. even though the cellular responses of immune splenocytes from psftsv-vaccinated mice were generally increased, the levels were not statistically significant, suggesting a potent role of il- co-expression in enhancing cell-mediated immunity against the viral antigens. since we observed significant elevation of t cell responses specific to the viral antigens in ifnar ko mice immunized with psftsv-il dna vaccine, we tested whether it could provide protective immunity against lethal sftsv infection. the susceptibility of ifnar ko mice to a korean sftsv isolate was examined by inoculating the mice with x to x ffu (s a fig). upon infection, all the mice gradually lost body weight and became moribund from the third to fifth day after infection, depending on the infection dose. all the infected mice died at ~ d after infection (s a fig). similar survival kinetics were previously reported in ifnar ko mice infected with other sftsv strains [ , ] , indicating that our korean sftsv isolate possesses an equivalent degree of virulence to prior chinese isolates. the platelet counts in mice infected with x ffu of sftsv significantly declined up to d after infection, and the mean platelet volumes of the infected mice were significantly higher than those of control animals (s b fig), suggesting that platelet destruction and the activation of platelet production simultaneously occur during lethal infection [ ] . to assess the protective efficacy of the dna vaccine, groups of mice were immunized with mock vector, psftsv, or psftsv-il three times and then subcutaneously challenged with a lethal dose ( ffu/mouse) of sftsv (fig ) . all the mock-immunized mice expired by d after sftsv infection. in contrast, all the mice immunized with psftsv-il were protected from lethal viral challenge, and % ( of ) of mice vaccinated with psftsv survived ( fig a) . consistently, psftsv-il -vaccinated animals lost weight until d after the viral challenge and recovered thereafter, whereas decrease in body weight of psftsv-immunized mice was observed up to d after the challenge and the surviving mice gradually recovered. when we examined the progression of thrombocytopenia during the acute phase of infection in the mice, platelet counts in vector-immunized mice rapidly declined until expiration (fig b, left panel) . however, platelet counts only marginally decreased in mice vaccinated with psftsv or psftsv-il and counts rebounded in psftsv-il -immunized mice at d after viral infection. in addition, viral loads were significantly reduced in the plasma of psftsv or psftsv-il -vaccinated mice when compared to those of vector-immunized control at d after infection. notably, despite similar initial viral loads (~ copies/ml of plasma) at d after viral challenge among the mice groups, psftsv-il -vaccinated mice (mean ± s.d. = . × ± . × ) were approximately five and fifty times lower than those of psftsvvaccinated mice (mean ± s.d. = . × ± . × ) and vector-immunized mice (mean ± s.d. = . × ± . × ) at d, respectively. since antibody responses against the viral glycoproteins, gn and gc, were poorly induced in ifnar ko mice immunized with the dna vaccines, we further investigate the potential protective role of neutralizing antibodies against gn or gc protein by immunization with the protein antigens fused with human fc fragment (fig ) . vaccination with gn-fc or gc-fc protein with alum adjuvant efficiently induced specific antibodies against the immunized antigens in ifnar ko mice; levels of antibody titers against both antigens were fairly similar (mean titer ± s.d. = , ± , , n = ). in addition, levels of neutralizing antibodies against sftsv in immune sera from mice immunized with either protein antigen were significantly higher than those from fc-immunized controls, when assessed by focus reduction neutralization test (frnt, fig b) . frnt titers of immune sera from mice immunized with gc-fc (mean titer ± s.d. = ± , n = ) and gn-fc (mean titer ± s.d. = ± , , n = ) were approximately five and twenty folds higher than those of fc-immunized mice (mean titer ± s.d. = ± , n = ). it is also notable that frnt titers of gn-fc immune sera were generally higher than those of gc-fc sera, although the difference was not statistically significant. finally, we examined the protective efficacy of the protein vaccines against lethal infection with sftsv (fig c) . all the control mice immunized with fc antigen died at five days after infection, whereas % of gn-fc immunized mice were protected from lethal challenge and regained their lost body weight. interestingly, weight loss of gc-fc vaccinated mice was delayed and observed from d after infection, whereas the control mice lost weight from d after infection. even though all the mice immunized with gc-fc succumbed to death, they survived three days more than control mice and the extension of median survival time was statistically significant (p = . ). dna vaccination has been widely investigated for various infectious diseases, especially targeting viral infections, during the last two decades [ , ] . although, human clinical trials of dna vaccines have yielded poor immunogenicity and less than optimal results, the approval of a few veterinary vaccines is a testimony of proof-of-concept and the hope that licensed dna vaccines for human use may not be too far away [ ] . while we were preparing this manuscript, kwak j.e. et al. reported a promising demonstration of dna vaccination against lethal sftsv infection in old-aged (> -years-old) ferret model [ ] . they used five plasmid dnas encoding individual viral gene, gn, gc, np, ns, or rdrp, and found that vaccination of old ferrets with a mixture of the five plasmids completely protected ferrets from lethal sftsv challenge without developing any clinical symptoms and systemic viremia [ ] . in addition, adoptive transfer of immune sera from ferrets immunized with two plasmids encoding gn and gc could not perfectly prevent the systemic viremia after sftsv infection, but provided complete protection against lethal challenge, suggesting that gn/gc may be the most effective antigens for inducing protective immunity. they also found that non-envelop (np, ns, and rdrp)-specific t cell responses also contribute to protection against sftsv infection although it failed to induce neutralizing activity [ ] . here we also observed that vaccination with a plasmid encoding gn, gc, np, and ns, together with murine il- , could provide protective immunity in ifnar ko mice against lethal sftsv challenge. however, our current dna vaccine failed to suppress initial systemic viremia and weight loss (fig ) . this might be due to the lack of type i interferon signaling and/or poor antibody responses against gn and gc glycoproteins, which are required for viral neutralization during the early stage of viral infection. inefficient generation of antibodies against gn and gc might be due to the absence of type i interferon signaling in ifnar ko mice or fusion of flt l with the glycoproteins, resulting in conformation defect of the antigens and/or dysregulation of b cell responses. given that immunization with gn-fc and gc-fc proteins can induce specific antibodies in ifnar ko mice (fig ) , the absence of type i ifn signaling might not be the cause of inefficient antibody generation. instead, flt l may suppress specific antibody responses as observed in other studies [ , [ ] [ ] [ ] . flt l may exert tolerogenic effect on cd t cells via dendritic cells, thereby promoting b cell hypo-responsiveness in vivo; however, the underlying mechanisms of cd t cell dysregulation are still unclear [ ] . nevertheless, flt l can significantly enhance cd t cell responses when fused with a target antigen and promote persistent maintenance of antigen-specific cd t cells in vivo [ , ] . in this study, we also observed significant enhancement of gn and np-specific cd t cell responses, as well as cd t cells, in the presence of il- expression (fig b and c) , which correlated well with vaccine protective efficacy in mice infected with lethal doses of sftsv. the potent effect of antigen-specific t cell responses in protection against sftsv infection is consistent with the results of the aforementioned ferret infection model [ ] . in this study, we did not include a negative control using a plasmid encoding il- alone, since it has been shown that il- alone failed to boost antigen-specific cellular immunity [ ] . finally, we evaluated the degree of protective immunity provided by the individual glycoprotein, gn or gc, after protein antigen immunization for the first time (fig ) , since previous studies showed that vaccination with both antigens retained in a live viral vector [ ] or dna vaccine [ ] can provide complete protection against lethal sftsv challenge in ifnar ko mice or old ferrets, respectively. both protein antigens are immunogenic and induce significant levels of neutralizing activity against sftsv, although anti-gn antibodies retain relatively higher neutralizing capacity than anti-gc antibodies. consistently, the protective efficacy of gn protein vaccination is relatively higher than gc protein immunization. while vaccination with either protein antigen failed to provide complete protection against lethal sftsv challenge, gc vaccine prolonged the survival time of infected mice and gn antigen can provide partial protection. considering that gn and gc glycoproteins function as viral ligands for cellular receptor binding and viral membrane fusion in host endosome, respectively [ ] [ ] [ ] , neutralization of both antigens might be required to completely block viral attachment and entry into host cells. taken together, we confirmed that antigen-specific t cell responses against sftsv induced by dna vaccination can provide complete protection against lethal viral challenge and immunization with each individual glycoprotein of sftsv can also confer partial protective immunity. given that the achilles heel of dna vaccines remains their poor immunogenicity in human trials when compared to protein vaccines [ ] , optimal combinations of dna and/or protein vaccines, proper selection of target antigens, and incorporation of efficient vaccine adjuvant, need to be further investigated for development of the best sftsv vaccine formulation for human application. the emergence of severe fever with thrombocytopenia syndrome virus taxonomy of the order bunyavirales: update epidemiological and clinical features of 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the extracellular domain of fms-like tyrosine kinase -ligand. cancer research enhanced protection against viral infection by co-administration of plasmid dna coding for viral antigen and cytokines in mice the role of phlebovirus glycoproteins in viral entry, assembly and release structure of a phleboviral envelope glycoprotein reveals a consolidated model of membrane fusion structures of phlebovirus glycoprotein gn and identification of a neutralizing antibody epitope key: cord- - u kikov authors: feehan, amy k.; fort, daniel; garcia-diaz, julia; price-haywood, eboni g.; velasco, cruz; sapp, eric; pevey, dawn; seoane, leonardo title: seroprevalence of sars-cov- and infection fatality ratio, orleans and jefferson parishes, louisiana, usa, may date: - - journal: emerg infect dis doi: . /eid . sha: doc_id: cord_uid: u kikov using a novel recruitment method and paired molecular and antibody testing for severe acute respiratory syndrome coronavirus infection, we determined seroprevalence in a racially diverse municipality in louisiana, usa. infections were highly variable by zip code and differed by race/ethnicity. overall census-weighted seroprevalence was . %, and the calculated infection fatality ratio was . %. s eroprevalence studies around the world have estimated the spread of severe acute respiratory syndrome coronavirus (sars-cov- ) to range from . % ( ) in boise, idaho, usa, to % in breves, brazil (p. hallal, unpub. data, https:// doi.org/ . / . . . ). coronavirus disease (covid- ) has also been reported to disproportionately affect black patients, but we do not know the infection fatality ratio (ifr), which requires knowing how many persons are at risk (i.e., infected). we estimated sars-cov- infections in orleans and jefferson parishes, louisiana, usa, and determined the covid- -related ifr by race. the protocol was approved by the ochsner clinic foundation institutional review board (new orleans, la, usa) and designed to enroll and test up to , persons at sites during may - , . to recruit a representative sample for this high-throughput method, a novel -step system developed by public democracy (https://www.publicdemocracy. io) considered > characteristics, including social determinants of health and us census population using a novel recruitment method and paired molecular and antibody testing for severe acute respiratory syndrome coronavirus infection, we determined seroprevalence in a racially diverse municipality in louisiana, usa. infections were highly variable by zip code and differed by race/ethnicity. overall census-weighted seroprevalence was . %, and the calculated infection fatality ratio was . %. data, to establish a pool of potential participants reflective of the demographics of the parishes, from which a randomized subset of , was selected. of these, > , volunteers were recruited through dynamic, cross-device digital advertisements, supplemented by television advertisements and a call-in number to register (appendix, https://wwwnc.cdc. gov/eid/article/ / / - -app .pdf). this volunteer pool was stratified by the same attributes and then randomly issued a text message inviting them to private testing locations. invitations were adjusted daily on the basis of response rates to achieve a representative sample. volunteers checked in with a digital code to discourage unsolicited walk-ins. we did not turn uninvited persons away but excluded them from analysis if they did not fit criteria. housemates of participants (n = ) or persons from ineligible zip codes (n = ) were excluded. six people withdrew consent. all study materials were created in english, spanish, and vietnamese. participants were offered free transportation if needed. verbal consent was electronically documented, and participants were asked a short list of questions followed by a blood draw and nasopharyngeal swab. tests approved by the us food and drug administration's emergency use authorization were used. real-time reverse transcription pcr tests of nasopharyngeal swabs were performed on the abbott m realtime system (abbott, https://www. abbott.com) and qualitative igg blood tests on the architect i sr (abbott). the igg test meets criteria described by the centers for disease control and prevention as yielding high positive predictive value, which was validated by a laboratory at ochsner health and others ( , ) . study participants for whom either or both tests were positive were considered to be infected with sars-cov- . us census values, weighted by race and parish of residence, were divided by the total sample for exposure (a pcr-positive test, an igg-positive test, or both), point prevalence (pcr-positive only), and seroprevalence (igg-positive tests regardless of pcr test result). the positive-testing population included persons with early-stage infections (pcrpositive only) and persons recovering (pcr-positive and igg-positive) and recovered (igg-positive only). early-stage infections were excluded from ifr estimation because their outcomes would not yet be registered as deaths. therefore, weighted seroprevalence was used to calculate persons presumed to be recovered ( ) . ifr was calculated by dividing cumulative deaths by race ( ) by the number of persons presumed to be recovered. methodology and symptoms observed have been described elsewhere (a. feehan, unpub. data, https://ssrn.com/ abstract= ). among the , persons in the sample, . % were female and . % were white; average age was . years, and average household size was . per-sons. among the participants who tested positive, % were black. the unadjusted exposure rate of sars-cov- in the sample population was . % ( . %, census-weighted); . % were positive for active viral shedding but had no detectable antibody. by race, seroprevalence was highest ( . %) in black participants, followed by multiracial ( . %), asian ( . %), and white ( . %) participants. hispanic participants had . % seroprevalence. we multiplied population estimates by weighted seroprevalence to generate the number of persons presumed to be recovered (table) . reported deaths ( ) were divided by number of persons presumed to be recovered plus deaths to calculate the ifr, which was . % overall. the ifr was statistically similar for white ( . %), black ( . %), and multiracial ( . %) persons but was significantly lower for asian persons ( . %). no covid- -related data on hispanic persons were collected by the louisiana department of public health during the study period. the prevalence of viral shedding (pcr-positive) and overall sars-cov- exposure (pcr-positive, igg-positive, or both) were listed and mapped by zip code across the parishes (figure) . prevalence was highly variable across the map and in some areas exceeded %. prevalence studies help to understand infection spread, especially when testing resources are limited. our study found the overall sars-cov- exposure rate in this area to be . % and confirmed a recent report of overrepresentation of black persons with covid- in the new orleans area ( ) . multiracial, hispanic, and asian persons also had higher seroprevalence than white persons. the overall ifr was . %, which is higher than ifrs found in other seroprevalence studies ( . %- . %) ( ; m. emmenegger, unpub. data, https://doi.org/ . / . . . ; p. hallal, unpub. data, https://doi.org/ . / . . . ). the similar ifr among most racial groups indicates that viral spread at least partially explains the increased number of deaths among minority populations. ministry of health singapore. communicable diseases surveillance in singapore radiological findings from patients with covid- pneumonia in wuhan, china: a descriptive study risk factors for tuberculosis infectious diseases and migrant worker health in singapore: a receiving country's perspective singapore zika study group. outbreak of zika virus infection in singapore: an epidemiological, entomological, virological, and clinical analysis performance characteristics of the abbott architect sars-cov- igg assay and seroprevalence in interim guidelines for covid- antibody testing case-fatality risk estimates for covid- calculated by using a lag time for fatality louisiana department of public health. covid- hospitalization and mortality among black patients and white patients with covid- ene-covid study group. prevalence of sars-cov- in spain (ene-covid): a nationwide, population-based seroepidemiological study the authors would like to especially thank the laboratories at the ochsner medical center jefferson highway campus for testing and keeping track of research samples; dan nichols; sarah roberts and gina mmahat for clinical site management; samantha bright, lyndsey buckner-baiamonte, and ansley hammons for research site management; emily arata for liaising with public leaders; and countless research coordinators, clinical staff, marketing personnel, medical students, and epic and it staff for making site testing possible. the ochsner health market planning and analysis team designed the maps in figure. the authors thank kathleen mcfadden for her thorough editing and mark roberts for his review and the ochsner language services department for helping to increase inclusivity. we would also like to acknowledge the new orleans mayor's office, city of new orleans office of public health, and the new orleans city council, especially council members helena moreno and jason williams for filming a public service announcement to help recruit participants. we also thank jefferson parish president cynthia lee sheng and parish council for their support.we thank george hutter and renola for their financial support. dr. feehan is a research scientist at the ochsner clinic foundation's infectious disease clinical research department. her research focuses on the gut microbiome as a treatment modality for neurologic disease, but more immediately on the sars-cov- pandemic that has greatly impacted the new orleans area. key: cord- -f p yaw authors: wang, hao; sikora, per; rutgersson, carolin; lindh, magnus; brodin, tomas; björlenius, berndt; larsson, d.g. joakim; norder, heléne title: differential removal of human pathogenic viruses from sewage by conventional and ozone treatments date: - - journal: int j hyg environ health doi: . /j.ijheh. . . sha: doc_id: cord_uid: f p yaw sewage contains a mixed ecosystem of diverse sets of microorganisms, including human pathogenic viruses. little is known about how conventional as well as advanced treatments of sewage, such as ozonation, reduce the environmental spread of viruses. analyses for viruses were therefore conducted for three weeks in influent, after conventional treatment, after additional ozonation, and after passing an open dam system at a full-scale treatment plant in knivsta, sweden. viruses were concentrated by adsorption to a positively charged filter, from which they were eluted and pelleted by ultracentrifugation, with a recovery of about %. ion torrent sequencing was used to analyze influent, leading to the identification of at least viral species, most of which belonged to families with some having unclear classification. real-time pcr was used to test for human-related viruses in inlet, conventionally treated, and ozone-treated sewage and outlet waters. the viruses identified in influent and further analyzed were adenovirus, norovirus, sapovirus, parechovirus, hepatitis e virus, astrovirus, pecovirus, picobirnavirus, parvovirus, and gokushovirus. conventional treatment reduced viral concentrations by one to four log , with the exception of adenovirus and parvovirus, for which the removal was less efficient. ozone treatment led to a further reduction by one to two log , but less for adenovirus. this study showed that the amount of all viruses was reduced by conventional sewage treatment. further ozonation reduced the amounts of several viruses to undetectable levels, indicating that this is a promising technique for reducing the transmission of many pathogenic human viruses. pathogenic human and animal viruses found in aquatic environments are usually shed from feces (enteric viruses), urine, and respiratory secretions from the infected host and enter into sewage water. the human viruses belong to different viral families. the most common viruses that are widely dispersed in sewage around the world include hepatitis a virus, hepatitis e virus, rotavirus, adenovirus, norovirus, astrovirus, parvovirus, coronavirus, poliovirus, and other enteroviruses (hellmer et al., ; laverick et al., ; lodder and de roda husman, ) . not only human enteric viruses and animal pathogens, but also other viruses can be found in waters contaminated with sewage (bosch, ; cantalupo et al., ) . if the pathogens are not removed in the treatment plants, they will be released into natural watersheds where many of them can persist for long periods (fong and lipp, ; kotwal and cannon, ) . new hosts might be infected with these viruses through direct contact with contaminated water or by drinking it or by eating animals such as mollusks that have filtered and concentrated viruses from sewage-contaminated water (nenonen et al., ) . in most western wastewater treatment plants, raw sewage is treated with combined mechanical, biological, and chemical processes such as screening, flocculation, sedimentation, and filtration. gross pollutants and most organic and inorganic solids are removed during these steps. the effluent is thereafter either discharged into a receiving water system or reused for other purposes. little is known, however, about the efficiency of removal of human viral pathogens from sewage by conventional treatment. several studies have shown that such treatments t are efficient for the reduction of the parasites giardia and cryptosporium and for bacteria but have little effect on adenoviruses and enteroviruses (li et al., ; ottoson et al., a; rodriguez-manzano et al., ) . additional disinfection after conventional treatment is applied in some treatment plants to further remove pathogens, such as treatment with peracetic acid, chlorination, and ultraviolet irradiation (das, ; kitis, ) . these treatments are efficient for inactivating and removing bacteria and protozoa, but not for most enteric viruses (freese and nozaic, ; shannon et al., ) . ozone treatment is an alternative for removing microcontaminants in sewage because ozone is an extremely reactive oxidant and thereby a powerful disinfectant. it has been used for disinfection of drinking water in europe since (rice et al., ) and has also been installed in some sewage treatment plants (oh et al., ; rakness et al., ) . the disinfecting ability of ozone treatment has been shown to be efficient for bacteria and parasites in clean water (kim et al., ; peeters et al., ) , and this treatment also has been shown to reduce the concentrations of enteric viruses and bacteriophages (burleson et al., ; kim et al., ) . however, the ability of ozonation to inactivate pathogens in wastewater might be hampered due to the high contents of organic materials in sewage (burleson et al., ) . one mechanism for reducing viable viruses in water by ozone treatment is assumed to be due to a conformation change of the viral capsid proteins by oxidation that either destroys the capsid or suppresses the virus/host cell receptor binding by changing the viral capsid proteins (shannon et al., ) . previous studies conducted in wastewater treatment plants have shown that ozone disinfection might be highly efficient in inactivating bacteria and bacteriophages after conventional sewage treatments (kim et al., ; tyrrell et al., ) , but knowledge regarding its effect for reducing human enteric viruses is relatively scarce. we used next-generation sequencing (ngs) technology and realtime pcr to investigate the efficiency of virus removal in sewage by conventional treatment and to evaluate the effect of additional ozone treatment at a full-scale pilot plant in sweden. the investigated sewage treatment plant in knivsta, which is situated km north of stockholm, sweden, uses traditional activated sludge treatment and receives primarily household waste from up to , population equivalents with a hydraulic design flow of m / h. the initial treatment is mechanical with two parallel screens and an aerated grit chamber. the subsequent biological treatment includes activated sludge and reactors with carriers of active biofilms, and this is followed by a chemical treatment step where ferric chloride is added prior to the sewage entering the two final parallel sedimentation basins. before release into the recipient river (knivstaån), the effluent passes through a pond for the removal of phosphorus-containing fine particles. in an additional ozonation step treating the entire wastewater flow was added at the end of this process line. the full-scale ozonation step is divided into two parallel lines with a total maximum capacity of m effluent wastewater per hour. the ozonation step includes lifting pumps, the production of ozone in generator units, the injection of ozone by static mixers, contact tanks, and final contact filters. each line contains two lifting centrifugal pumps (apex isf c, bristol, uk), one static mixer (nr mixer, statiflo international ltd, uk), one m stain-less steel contact tank with m water depth and two compartments, one ozone destructor for off-gas (primozone, sweden), and two contact filters with a total area of m filled with m light-expanded clay aggregates (leca, saint-gobain linköping, sweden) for potential stripping or quenching of ozone residues in ozonated wastewater. the ozone is produced from evaporated liquid oxygen with > . % o (yarapraxair, sweden) diluted to % o by addition of air in an ozone generator with a maximum production capacity of . kg o /h (gm , primozone, sweden). an ozone dose of around mg/l is added to the effluent wastewater through static mixers that transfer more than % of the added ozone to the wastewater. most of the ozone reacts or degrades rapidly after the addition to the wastewater. analysis of the water samples in the inlets and outlets of the contact tanks show ozone concentrations of - mg o /l and . - . mg o /l respectively. to verify that adequate amounts of ozone are transferred, the removal of pharmaceutical residues was calculated based on frequent inlet and outlet samples, and the results showed a typical removal efficiency for an ozone dose of - mg o /l, which is also reported in other studies and is related to total organic carbon (toc) concentrations (beijer et al., ) . the hydraulic retention time in the contact tanks was on average min and the minimum and maximum retention time was and min, respectively, during the period of ozonation that began in august and ended in february . the wastewater chemistry as well as effects on the recipient river were studied in parallel research projects before, during, and after the ozonation trial. for the present study, flow-proportional h composite samples of influent ( l per sample), effluent ( l), effluent after ozonation ( l), and effluent after the dam (outlet; l) were collected on three occasions in (november until december (week ); december until december (week ); and december until december (week / )), with time adjustment for the flow rate to represent the "same" water. all samples were cooled during sampling and then frozen at − °c until further processing. the water samples were first centrifuged at × g for min before filtration twice through nano-ceram cartridge filters (argonide, sanford, florida, usa) at an average flow rate of . l/min. the viruses were electrostatically attached to the filter from which they were eluted by ml of . m phosphate buffer containing . m glycine (ph . ). the eluate was collected, and the ph was adjusted to . by the addition of m hcl. the eluate was thereafter filtered through a . / . μm sartobran capsule filter (sartorius, göttingen, germany) to remove remaining debris and most bacteria. the filtrate was then ultracentrifugated in eight tubes at , rpm for h at °c. the pellet in each tube was resuspended in μl mm tris-hcl (ph . ) overnight, pooled, and stored at − °c until analysis. a fixed amount of human mastadenovirus (hadv- ) was added to . l raw sewage, and the sewage was concentrated by the method above. one milliliter of unconcentrated water and one ml from each concentration step was collected and analyzed for adenovirus by quantitative real-time pcr (qpcr) and isolation on cell culture. nucleic acids in the water samples were extracted from μl concentrated sample using the qiagen dna blood and tissue kit (qiagen, hilden, germany) according to the manufacturer's instructions. qpcr for adenovirus was performed in a μl reaction mix containing μl extracted nucleic acids, × universal dna master mix (thermofisher, waltham, ma, usa), . μm of forward and reverse primer, and . μm of probe (table s ). the cycling conditions were °c for min and °c for min followed by two-step cycling times at °c for s and °c for min on an abi fast real-time pcr system (applied biosystems, foster city, ca, usa). for isolation of hadv- on cell cultures, μl of -fold serial dilutions ( / to / , ) in eaglés minimal essential medium (mem, gibco, waltham, ma, usa) from each concentration step were inoculated in duplicate into wells in -well plates (thermofisher) containing confluent monolayers of a cells. the plates were incubated at °c in an atmosphere of % co for h, after which the medium containing virus was removed from each well, followed by addition of μl mem containing % fetal calf serum and % l-glutamine (gibco). the plates were incubated at °c in % co and were examined for cytopathogenic effects daily for days. . . . nuclease treatment of the dissolved pellets from the three concentrated incoming sewage samples before nucleic acid extraction, μl of the dissolved pellet was treated with u benzonase nuclease (sigma-aldrich, st. louis, mo, usa) and . mm mgcl (applied biosystems, foster city, ca, usa) and incubated at °c for h. thereafter edta was added to a final concentration of mm to inhibit the nuclease activity. dna was extracted from μl of each treated sample using the qiaamp dna mini kit (qiagen, hilden, germany), and rna was extracted from the remaining μl of each sample using the rneasy plus mini kit (qiagen, hilden, germany). the rna was reverse transcribed into cdna in a μl reaction mix containing . μl extracted rna, . μg random hexamer primers (roche diagnostics, indianapolis, in, usa), . mm dntp (sigma-aldrich), × first strand buffer (invitrogen, waltham, ma usa), mm dithiothreitol (invitrogen), u rnaseout (invitrogen), and u superscript iii reverse transcriptase (invitrogen). the synthesis was performed at °c for min followed by °c for min, and the cdna was stored at − °c until further amplification. extracted dna and cdna samples were amplified by nested pcr in triplicate. briefly, touch-up gradient pcr using primers sisp and sis (table s ) was used as the first-round amplification in a μl reaction mix containing . μl template, × taq buffer (applied biosystems), mm mgcl (applied biosystems), . mm dntp (sigma-aldrich), u taq dna polymerase (roche diagnostics), and . μm of each primer. the pcr reaction was performed for one cycle at °c for min, followed by cycles touch-up pcr with °c for s and °c for s ( °c increase per cycle), followed by cycles of °c for s, °c for s, and °c for min, and with a min final extension at °c. five microliters of the first-round pcr product were further amplified with the primers sisp and sis (table s ) in a μl reaction mix containing × taq buffer (applied biosystems), mm mgcl (applied biosystems), . mm dntp (sigma-aldrich), u taq dna polymerase (roche diagnostics), and . μm of each primer. the pcr was performed with an initial denaturation at °c for min followed by cycles of °c for s, °c for s, and °c for min, and with a min final extension at °c. the triplicates were pooled, and the pcr products were visualized by . % agarose gel electrophoresis. before library building for next-generation sequencing (ngs), the majority of the amplified products were sheared into - bp fragments by sonication using a bioruptor sonication device (diagenode, seraing, belgium). the pcr products from each sample were pooled and thereafter aliquoted into five tubes each with μl product. one aliquot was not sonicated, while the four other aliquots were sonicated for , , , and cycles, respectively (each cycle was one minute with s sonication and s without sonication). after sonication, the five aliquots were pooled, and the products were visualized by . % agarose gel electrophoresis. libraries were built from the fragmented pcr products by using the ion plus fragment library kit on an ab library builder system (thermofisher, waltham, ma usa) according to manufacturer's protocol. the newly built libraries were amplified for eight cycles and purified with ampure xp beads (beckman coulter, brea, ca, usa). samples were then further size selected to about bp by using a pippin prep (sage science, beverly, ma, usa). the recovered materials were analyzed using the agilent high sensitivity d screentape system on a tapestation (agilent, santa clara, ca, usa). the dna concentration was thereafter estimated using an ion quantitation kit (thermofisher). the libraries were diluted and pooled to reach a final concentration of pm. template preparation and chip loading was performed on the ion chef instrument using the ion pgm™ hi-q™ view chef kit according to the manual (thermofisher). an ion chip (thermofisher) was used, and sequencing was conducted on an ion torrent pgm with an ion pgm hi-q view sequencing kit (thermofisher). the sequence data obtained was automatically trimmed by the ion torrent suite software (thermofisher), and the resulting bam files were imported into clc genomic workbench . . (qiagen, hilden, germany) for analysis. the pipeline for viral identification is illustrated in supplemental figure a . low-quality reads, reads below bp, and the primer sequences were removed. the sequences were mapped to a human hg reference genome using stringent criteria, with length fraction set at . and similarity fraction set at . . the mapped reads were discarded, while unmapped reads were assembled de novo using the built-in clc de novo assembler with a word size of and a minimal contig size of bp. the contigs obtained from the de novo assembly and singleton reads longer than bp were blasted against the ncbi genbank non-redundant nucleotide database (nr/nt) using blastn and a cut-off with an e-value of − for significant hits. after blast, contigs and singletons that satisfied an e-value < − and an hsp length > bp were selected, and viral hits among them were used for further analysis. primers and probes for selected viruses related to human diseases were designed and used to verify the results obtained by ngs (table a ) and to determine the prevalence of these viruses after conventional treatment and ozone treatment. the most common viruses identified were human feces pecovirus, picobirnavirus, parvovirus, parvoviruslike virus, adenovirus genotype , astrovirus , and gokushovirus. three microliters of extracted nucleic acids from the samples were analyzed by qpcr in a μl reaction mixture containing × reaction mix (invitrogen), u rnaseout™ (invitrogen), . μl superscript ® iii/platinum ® taq mix (invitrogen), and . μm of each primer, and . μm of probe. the qpcrs for astrovirus and picobirnavirus were performed with initial reverse transcription at °c for min and °c for min. the qpcr was performed for cycles of °c for s and °c for min for most viruses, but with extension at °c for adenovirus and °c for gokushovirus and astrovirus . the qpcrs were performed in a fast real-time pcr system (applied biosystems), and all samples were analyzed in duplicate. the four concentrated water samples (incoming sewage, conventionally treated, ozone treated, and outlet water) from each of the three weeks were also analyzed by qpcr for common enteric viruses (adenovirus, astrovirus, hepatitis a virus, hepatitis e virus, norovirus gi, norovirus gii, norovirus giv, parechovirus, sapovirus, aichivirus, mengovirus, torovirus, enterovirus, and rotavirus). all primers and probes used for the detection are listed in table a . the method and conditions for the qpcr were as previously described (hellmer et al., ) . the qpcrs were performed on a fast real-time pcr system (applied biosystems), and all samples were tested in duplicate. a puc plasmid containing all viral target regions was synthesized by genescript (genescript usa inc. nj, usa), and ten-fold serial dilutions were used as the positive control in all qpcr analyses. the ct values of the samples were relative to the ct values from the dilutions of the plasmid containing the virus sequences being analyzed, and from this the numbers of viral genomes per milliliter were estimated. these values were also compared to estimated amounts of viral genomes by assuming a perfect qpcr detecting one genome by using the formula: (ct value of the sample) = − . × log genomes/ml + . the number of estimated viral genomes was not adjusted to the estimated recovery in any of the analyzed waters, but was used directly to compare the reduction of viruses by the different treatments. nested pcr was used to amplify the hexon protein-coding region of adenovirus. the μl reaction mix contained μl extracted dna, × taq buffer (applied biosystems), . mm mgcl (applied biosystems), . mm dntp (sigma-aldrich), . μm of forward and reverse primers (advf st and advr st; table s ), and u taq dna polymerase (roche diagnostics). the pcr was initiated at °c for min, followed by cycles at °c for s, °c for s, and °c for min, and a min final extension at °c. five microliters of the pcr product from the first-round amplification were used as the template for nested pcr as described above but with different forward and reverse primers (advf nd, advr nd; table s ). the fragments were purified using a qiaquick pcr purification kit (qiagen) according to the manufacturer's protocol, and the purified amplicons were sent for sequencing to gatc biotech, constance, germany. the sequences obtained were analyzed in the seqman pro program in the dnastar program package version . . (dnastar inc., madison, wi, usa). the sequences were aligned with the corresponding region of the hexon gene of adenovirus sequences representing all human mastadenovirus types obtained from genbank. evolutionary distances were calculated using the hasegawa-kishino-yano (hky) algorithm in the dnadist program in the phylip package version . (felsenstein, a; felsenstein, b) with transition/transversion ratio of . and gamma correction with alpha . . phylogenetic trees were constructed using the unweighted pair-group method using arithmetic averages (upgma) and the neighbor-joining method in the neighbor program of the phylip package. the trees were visualized with the program treeview, version . . (page, ) . the added adenovirus could be detected by qpcr and isolation on cell cultures in all concentrations steps (table ). the raw sewage water was concentrated about times (from . l to . ml) with this technique, and the adenovirus was concentrated about times, indicating a recovery of % (table ). the treatment of the pellet with nucleases before nucleic acid extraction did not significantly change the efficiency of the concentration procedure. by isolation on cell culture, it could be shown that most adenoviruses were viable, and the viruses from the final ultracentrifugation pellet could be detected up to a dilution of − , indicating a % efficiency for the technique. however, when the pelleted samples treated with nuclease were isolated on cell culture, viruses could only be detected in a − dilution, which would indicate % recovery similar to the qpcr (table ) . by ngs sequencing, , to , reads were obtained after quality control for each of the three concentrated incoming sewages (table ) . after de novo assembly, to , contigs and singletons were obtained. between and sequences were homologous to different viruses and could be classified into viral families, corresponding to about . % to . % of all contigs obtained (table ). in all, viruses belonging to different families were identified (table a ) . bacteriophages from the families of inoviridae, microviridae, myoviridae, podoviridae, and siphoviridae accounted for the largest proportion of the identified viruses. sequences similar to gokushovirus, a member of microviridae, were abundant and accounted for more than % of all viral sequences in one sample. apart from bacteriophages, viruses infecting plants, vertebrates, invertebrates, and protists were also identified. although there were fewer sequences similar to viruses that could infect vertebrates than bacteriophages, they had a high diversity and belonged to nine of the viral families. plant-related viruses, including tombusviridae, virgaviridae, alphaflexiviridae, betaflexiviridae, partitiviridae, and tymoviridae, were also widely distributed in the samples. sequences similar to a number of viruses related to human diseases were also detected. as shown in table , sequences representing some common viral families, such as adenoviridae, astroviridae, papillomaviridae, parvoviridae, and picobirnaviridae were found. contigs longer than bp identified human adenovirus f, serotype in samples qpcr was used for identification of common human enteric viruses in influent, treated sewage, ozone-treated effluents, and after passing an open dam system. three of these viruses (adenovirus, norovirus gii, and astrovirus) were detected in samples from the influent and after treatment at % to % reduced concentrations, with adenovirus being the least affected (table , fig. ). an additional four viruses (norovirus gi, sapovirus, parechovirus, and hepatitis e virus) were only detected in the inlet samples with concentrations reduced to undetectable levels after conventional treatment. based on the obtained ngs sequences, new qpcr primers and probes were designed to detect seven selected viruses related to humans in the incoming sewage, including human feces pecovirus, picobirnavirus, parvovirus, parvovirus-like virus, adenovirus type , astrovirus type , and gokushovirus (table ). for these viruses the reduction of viral genomes after the conventional treatment ranged from . % for gokushovirus to - % for parvovirus and parvovirus-like virus during weeks and / (table ). the reduction was higher, %, for parvovirus during week . ozone treatment of conventionally treated effluent lowered the amount of viruses further to undetectable levels for pecovirus, picobirnavirus, parvovirus-like virus, and gokushovirus for all three weeks. this treatment reduced the adenovirus concentration by % to %, norovirus gii by % to %, and astrovirus and parvovirus by % to % (tables and ). despite not being detected after ozone treatment, several of these viruses were found in the outlet water after the dams, as were parvovirus during weeks and and parvoviruslike virus, astrovirus , pecovirus, and adenovirus during week . for these viruses, the amount of virus identified in the outlet water was between % and % of that identified in the conventionally treated sewage samples (tables and ) . it was possible to amplify and sequence a partial region of the adenovirus hexon gene in all water samples except for the outlet samples from weeks and / . the sequence obtained for the ozone-treated sample in week could not be analyzed because it contained multiple sequences. phylogenetic analysis was performed for the remaining nine sequences and revealed that six belonged to species f and two to species a, and for the ozone-treated sample in week the species could not be determined, but it was found on the same branch as species f strains in the phylogenetic tree (fig. ) . the strains were divergent from each other, and the most similar strains were from conventionally treated sewage from weeks and within species f and were found on the same branch as the strains in the incoming and ozone-treated samples from week / (fig. ) . in this study we demonstrated a high diversity of viruses in incoming sewage. human-related viruses were strongly reduced by conventional treatment of the sewage, and the elimination of several of these was achieved by additional ozone treatment. other studies have previously shown reduction of parasitic protozoa, bacteria, and phages by conventional treatment of sewage (ottoson et al., a; tyrrell et al., ) , but little was known regarding the reduction of human enteric viruses (montazeri et al., ; ottoson et al., b,c) or their sensitivity to ozonation of sewage. while ozonation is already recognized as a promising technology for removing microcontaminants, such as pharmaceuticals, in sewage (beijer et al., ) , the additional benefit of reducing risks for the spread of human pathogenic viruses via table real-time pcr for seven of the viruses identified by ngs and the estimated numbers of genomes of these viruses per milliliter of water in untreated and treated sewage samples from three weeks in , where k numbers indicate internal sample numbers. virus week week week contaminated waters should be considered as well. the conventional treatment of sewage eliminated or reduced the amount of most viruses detected in inlet sewage in this study, but it was less efficient for parvovirus and adenovirus. additional ozone treatment lowered the amount further, for some viruses to undetectable levels. this result is in line with other studies that demonstrated a reduction of enteroviruses and f + coliphages by more than . and . log with ozone disinfection of wastewater effluents (xu et al., ) . because there is a relatively high quantity of suspended solids and organic materials in secondary effluent after conventional treatment that could affect the ozone treatment efficiency, we do not know if an increased amount of ozone or longer ozone treatment time would have reduced the amounts of viruses even further. however, in this study some viruses that were undetectable in the ozone-treated samples reoccurred in the outlet water, including parvovirus, norovirus gii, human feces pecovirus, parvovirus-like virus, gokushovirus, and hadv-f , although the amounts were significantly lower compared with raw sewage. the reason for the identification of viruses in the outlet after the dam system despite the fact that they were not detected by qpcr after ozonation is unclear, but two circumstances identified during the evaluation of the operation of the treatment plant might explain these findings. during week , the protective sieves for the lifting pumps in the ozonation step were partly clogged with large particles released by high hydraulic flow in the treatment plant leading to by-pass of % of the conventionally treated wastewater directly to the dam. in week / , the concentration of suspended solids in the conventionally treated wastewater was mg ss/l, which was % higher than in samples from week and week that contained mg ss/l. this might have allowed some of these viruses to escape the ozone treatment and to reappear in the dams. additional explanations could be that our attempts to follow the "same" water based on the flow through the treatment plant had been disrupted and that the waters analyzed from the outlet of the dams were not the same as the ozonized waters. another reason might be that the viruses were present in amounts too low to be detected in the ozonized water by qpcr. larger amounts of water might be needed to determine if these viruses were also present in the waters after ozone treatment, and further studies are also needed to determine the impact of the ozone treatment on these viruses. adenoviruses are resistant to uv disinfection (baxter carole et al., ; and were also rather insensitive to the ozone treatment used in this study. in clean water, human mastadenovirus f (hadv-f ) has been shown to be more resistant to ozone treatment than feline calicivirus, which is often used as a surrogate for noroviruses (thurston-enriquez et al., ) . in the present study, the dominant strains of adenovirus varied, both between the differently treated waters and within the sample groups. there were different hadv-f and hadv-f strains in all inlet sewage samples, while in one ozone-treated sample a divergent type f strain dominated and in some of the treated waters there were type a strains. a somewhat similar finding was also obtained by fernandez-cassi and co-workers (fernandez-cassi et al., ) . the pattern observed in this study suggests that there was a complex composition comprising multiple adenovirus types in the raw sewage, and these might have varying degrees of sensitivity to the conventional processing and ozone treatment. however, we do not know if these viruses are viable after the ozone treatment, and the oxidization might have changed the conformation of the viral head protein and thereby inhibited viral-host binding (shannon et al., ) . because the amount of viral genomes decreased significantly after the ozonation, the treatment might not only change the conformation of the viral capsids, but might also destroy or open the capsid of some viruses and thereby release the viral genomes into the water where nucleases are present. further studies are needed on ozonation of water containing adenovirus types that are easy to isolate on cell cultures in order to identify the viability of the viruses after treatment. the high diversity of viruses identified in the inlet sewage in this study is in agreement with a previous study on sewage where known viruses representing taxonomic families, as well as a large number of novel viruses, were identified (cantalupo et al., ) , confirming that raw sewage contains a vast and diverse viral pool. the identified viruses could infect bacteria, plants, vertebrates, invertebrates, and protists, with bacteriophages contributing the largest proportion. the bacteriophage gokushovirus, which primarily targets chlamydia, bdellovibrio, and spiroplasma (labonte et al., ) , accounted for most viral reads in all samples. this virus has been shown to be widespread in global environmental samples, especially in aquatic ecosystems (hopkins et al., ; labonte et al., ) , and can be used as a potential viral indicator for treatment efficacy in different waters. human disease-related viruses, which can cause diarrhea, respiratory diseases, conjunctivitis, meningoencephalitis, and infantile gastroenteritis (fong et al., ; nguyen et al., ) were also identified. some of these that might cause gastroenteritis, such as human picobirnavirus and human feces pecovirus (giordano et al., ; phan et al., ; phan et al., ) , were widely detected in the sewage samples but are usually not tested for at hospital laboratories. to understand their importance for public health, more enteric viruses identified in environmental samples should be incorporated into the routine monitoring of patients with gastroenteritis. there were also other human pathogenic viruses detected in the sewage during the whole study period, including hpv, which might cause warts, papilloma, and malignant tumors. other recent studies have also identified multiple types of hpv, including the oncogenic high-risk hpv , in raw sewage (bibby and peccia, ; cantalupo et al., ; la rosa et al., ) . the presence of this virus in large amounts in aquatic environments has caused concern about its potential for waterborne transmissions, which might cause anogenic or oral hpv infections even among people who have not been sexually active (fratini et al., ) ; however, further research into its transmission by the waterborne route is still needed. when comparing the results obtained with real-time pcr and ngs, only adenovirus and astrovirus could be identified by ngs data, and sequences corresponding to sapovirus, norovirus, and parechovirus were not found even though these viruses could be identified with realtime pcr. this might be due to their low quantity in sewage. most of the viruses detected by ngs had about one to two log higher viral genome concentrations than the overlooked viruses, indicating that ngs has some limitations when dealing with complex environmental samples and might underestimate the number of different viruses if the number reads are not high enough to exhaustively probe the sample. the concentration efficiency of about % used in this study might also have influenced the amount of viruses identified. however, this efficiency is comparable to that obtained by other concentration methods (cashdollar et al., ; fout et al., ; hellmer et al., ; ikner et al., ) . by using adsorption to charged filters, hadv- , which was used for the evaluation of this technique, has previously been used in another study with a recovery of about %, while the recovery was % to % for enteroviruses (ikner et al., ) . the low recovery of adenovirus might be due to its surface capsid structure and electrostatic charge, which might facilitate the entrapment of these viruses in the charged filters and hamper their elution from the filters (gibbons et al., ; shi et al., ) . hadv- was used in this study as an indicator of the effectiveness of the method because it could easily be detected by both qpcr and isolation on cell cultures and was shown to be viable in all concentration steps used. this method might therefore have a higher concentration efficiency for other viruses than adenovirus and thus should also be analyzed for other viruses that can be isolated on cell culture. this work expands our knowledge of the possible use of ozone treatment to eliminate viruses from water. the ability of ozonation to reduce the transmission risks of human pathogens might therefore be considered when decisions are taken as to whether it is worthwhile to install advanced sewage treatments to remove microcontaminants and bacterial pathogens. it should be acknowledged that in addition to the apparent advantages, ozonation also come with a cost, and high ozone doses in particular can sometimes lead to the formation of compounds that can negatively affect aquatic life (samuelsson et al., ) . further studies are needed to determine the most efficient methods, including different ozonation procedures, for viral elimination in sewage and whether these pathogens in treated waters are viable and can spread. the elimination of human pathogens is imperative in order to prevent waterborne viruses from re-entering into the environment and thus reduce the potential risk they pose to public health. inactivation of adenovirus types , , and in drinking water by uv light, free chlorine, and monochloramine inactivation of adenovirus types , , and in drinking water by uv light free chlorine, and monochloramine removal of pharmaceuticals and unspecified 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data associated with this article can be found, in the online version, at https://doi.org/ . /j.ijheh. . . . key: cord- -l f gp authors: nan title: oral and poster manuscripts date: - - journal: influenza other respir viruses doi: . /j. - . . .x sha: doc_id: cord_uid: l f gp nan pandemic influenza h n (h n pdm) virus of swine-origin causes mild disease, but occasionally is associated with acute respiratory distress syndrome and death. , it is important to understand the pathogenesis of this new disease. previously we showed a comparable virus tropism and host innate immune responses between h n pdm and seasonal h n influenza virus in the human respiratory tract, however h n pdm virus differed from seasonal h n influenza virus in its ability to replicate in human conjunctiva, suggesting subtle differences in receptor-binding profile and highlighting the potential role of the conjunctiva as an additional route of infection. we now compare the tropism and host responses elicited by pandemic h n with that of related swine influenza viruses and a pandemic-swine reassortant virus in ex vivo and in vitro cultures of the human respiratory tract and conjunctiva. we have used recombinant virus to investigate the role of the hemagglutinin (ha) and neuraminidase (na) of h n pdm virus in its conjunctival tropism. these findings are relevant for understanding transmission and therapy. fragments of human conjunctiva, bronchi, and lung tissues were cut into - mm fragments within h of collection and infected with influenza a viruses at a titer of tcid ⁄ ml. viruses investigated included h n pdm (a ⁄ hk ⁄ ⁄ ), swine h n virus (a ⁄ swine ⁄ hk ⁄ ⁄ ), which shares a common derivation for seven genes with h n pdm, a natural swine reassortant h n (a ⁄ swine ⁄ hk ⁄ ⁄ ), which has acquired the na gene from h n pdm and other swine influenza h n viruses. reverse genetics derived recombinant viruses with ha and na gene segments of seasonal h n and pandemic h n swapped were also studied. lung fragments were cultured at °c in culture plates; conjunctival and bronchial biopsies were cultured in air-liquid interface at and °c respectively. tissue fragments were infected for h and incubated for , , and h post infection. infectious viral yield was assessed by titration in mdck cells. the infected tissues were fixed with formalin and analyzed by immunohistochemistry for influenza antigen. cytokines profiles induced by influenza virus infected respiratory epithelial cells in vitro were measured by quantitative rt-pcr and elisa. we found comparable replication in seasonal and pandemic h n viruses in human respiratory tract, while the swine influenza a ⁄ swine ⁄ hk ⁄ ⁄ (h n ) virus and a ⁄ swine ⁄ hk ⁄ ⁄ (h n ) virus failed to infect and replicate in human lung ex vivo culture, but it replicated productively in human bronchus ex vivo. interestingly, the swine reassortant influenza h n (a ⁄ swine ⁄ hk ⁄ ⁄ ) virus (with the na from h n pdm) infected and productivity replicated in lung ex vivo and in vitro. pandemic h n pdm virus, but not seasonal h n virus, was able to infect ex vivo cultures of human conjunctiva, suggesting subtle differences in receptor binding profile in h n pdm, seasonal viruses, and the swine related h n viruses. using reverse genetics derived recombinant viruses, we were able to demonstrate that the ha and na segments of h n pdm, but not the polymerase genes, were required for the conjunctival tropism of h n pdm ( figure ). in contrast with highly pathogenic influenza h n virus, which induced high cytokine and chemokine decretion, the related swine viruses, a ⁄ swine ⁄ hk ⁄ ⁄ (h n ), as well as the swine pandemic reassortant virus, a ⁄ swine ⁄ hk ⁄ ⁄ (h n ) we studied were similar to h n pdm and seasonal influenza viruses in their intrinsic capacity for cytokine dysregulation. collectively, our results suggest that pandemic h n pdm virus differs in modest but subtle ways from seasonal h n virus in its intrinsic virulence for humans, findings that are in accord with the epidemiology of the pandemic to date. the ha and na gene segments are key to the conjunctival tropism manifested by the h n pdm virus. the pandemic reassortant influenza h n (a ⁄ swine ⁄ hk ⁄ ⁄ ) virus isolated from swine with the na from h n pdm shares with h n pdm the capacity for productive replication in lung ex vivo and in vitro. these findings are relevant for understanding transmission and therapy. isolation of influenza viruses from specimens is traditionally performed in two classical systems: embryonated chicken eggs and mdck cell culture. nevertheless, several publications are dedicated to the theme of alternative cell culture systems, which may be used for influenza virus isolation and cultivation. [ ] [ ] [ ] this is in part because mdck cells are of animal origin, which means that they cannot be used as a proper model for estimating interactions between a human virus and a human cell culture as a host. a variety of human monolayer and suspension cell cultures have been tested on their capability to support influenza virus replication. among them, some support influenza a virus growth as well as mdck cells do, others support replication of a virus, but do not enable the formation of mature viral particles, whereas others show only a weak level of replication or are not permissive at all. caco- cells, for example, represent a good substitute for mdck cells, because it has been shown that the rate of viral isolation in caco- cells is as effective as in mdck, and sometimes is even better. the success of viral replication is determined not only by the cell culture type, but also by the virus itself. despite the accepted view that it is the type of receptor that defines the interaction between the virus and the host cell, there is evidence that it is not the only factor that predetermines the fate of the cell. the fate of the infected cell can also differ. a series of articles show that apoptosis is the most probable mechanism of cell killing by influenza viruses. , influenza a viruses of different subtypes induce apoptosis to a different extent (e.g. h viruses provoke more strong apoptotic response than h viruses do ). nevertheless, it has been demonstrated that caco- cells do not follow the apoptotic pathway and die through necrosis. the sjpl cell line also dies through necrotic pathway and not apoptosis. the aim of our work was to compare growth characteristics of different flu viruses (e.g. avian, swine, and human) in various human and animal cell cultures and to evaluate their influence on cell culture growth. the parameters measured in the study were as follows: cytopathic changes of cell cultures following virus infection, hemagglutinin production, np synthesis, the dose-dependent effect of infection on cell proliferation, and the ability of viruses to induce apoptosis. influenza viruses used included: highly pathogenic avian h n a ⁄ kurgan ⁄ ⁄ , low pathogenic avian h n a ⁄ gull ⁄ kostanai ⁄ ⁄ , swine h n a ⁄ swine ⁄ ⁄ , human h n v a ⁄ california ⁄ ⁄ , human h n v a ⁄ saint-petersburg ⁄ ⁄ , human h n a ⁄ brisbane ⁄ ⁄ , and human h n a ⁄ brisbane ⁄ ⁄ . the viruses were propagated in -days embryonated chicken eggs, the allantoic fluid was collected, the aliquots were made and stored at ) °c for further use. to evaluate tcid for each virus on all cell cultures, -well plates were used. the cells were seeded ae ml per well (concentration of - ae · cells ⁄ ml). the confluent -h old monolayer was used for viral inoculation. the cells were washed twice with serum-free medium, then ae ml of tenfold viral dilutions from viral aliquots were added and left for min for contact at °c. the cells were then washed to remove the non-attached particles, and the wells were filled with tpck-trypsin ( lg ⁄ ml)-containing medium without bovine fetal serum. the plates were observed daily for cytopathic effect, and the results were evaluated at h after infection for cytopathic effect and by reaction of hemagglutination with suspension of chicken erythrocytes ( ae %). infection of suspension cell cultures was done in centrifuge tubes. cells (concentration - · ) were inoculated with viral dilutions (moi = - ). after min of contact, cells were washed, resuspended in rpmi with trypsin and fetal serum, and seeded in -well plates ( ml in each well). the results were fixed after h, calculating the number of cells grown and estimating the rate of apoptosis by hoechst- staining. cells were grown in -well plates with seeding concentration · cells ⁄ ml. one millilitre of cell suspension was placed in each well, inoculated with viral dilution (moi = - ) and left for h. after, the cells were detached from plastic with versene and calculated in fuks-rosental camera to evaluate the number of cells. the monoclonal antibodies obtained in research institute of influenza towards viral nucleoprotein np were used following the standard protocol described in. for all viruses tested, mdck turned out to be more permissive than sp cell culture. avian viruses, independently of their pathogenicity, replicated efficiently on both animal cultures tested. human h n and h n viruses demonstrated weaker replication in sp cells. the most significant differences were seen for swine influenza and pandemic h n v viruses which replicated in mdck cells at the rates comparable with other viruses, but showed poorer growth in sp cell line (see table ). human cell lines displayed clear differences in their susceptibility to viruses of various origins. avian influenza viruses replicated in all cell lines except girardi heart, and the most intense replication rate was observed for ecv- , l- , and rd lines. a- and a- were poorly infected, as well as all suspension cell lines tested. seasonal human h n , as well as h n viruses, replicated in all cell cultures tested, but the rate of infectivity was rather low in practically all cultures tested with the exception of rd and t- g cell lines. strikingly, swine influenza virus and human pandemic h n v viruses didn't replicate well in any of human lines tested. a weak replication rate was observed in ecv- , rd, and t- g, but in general, human cell lines were the titers produced by swine and pandemic influenza viruses are shaded in grey. *low-pathogenic avian influenza virus; **highly-pathogenic avian influenza virus poorly susceptible to pandemic h n v. swine influenza virus differed because it infected weakly a- and girardi heart cell cultures, which was not the case for h n v viruses. our study has shown that all influenza viruses were able to induce apoptosis in the cell cultures tested. the degradation of chromatin found in the nucleus with hoechst- staining was seen before the first symptoms of cytopathic effect (cpe) in monolayer of cells. in cell cultures where the cpe was not visible, high doses of virus still induced apoptotic response. the process of apoptosis is rather well studied in mdck cells and some other cell types, so we've focused on three human monolayer cell cultures that are relatively poorly studied: a- , ecv- , and flech. these cell cultures are less susceptible to viral infection, and besides, it was interesting to find out whether the viruses that do not cause any cpe do infect these cultures. a- turned out to be most sensitive to apoptotic response, while flech turned out to demonstrate weak reaction. time needed for apoptosis induction by different flu viruses also varied. the earliest apoptosis was noted for h n and h n viruses and h n viruses induced apoptosis at about h postinfection. it is well-known that apoptosis can be induced only by a reproducing virus, and that uv-kills viruses that are not capable of it. we tested whether swine and pandemic h n v viruses (that do not show cpe in these cultures) do replicate in them and induce apoptosis with the help of monoclonal antibodies against viral np. the obtained data show that they indeed do replicate in these cell cultures, as we observed np fluorescence, and that they also induce apoptosis (see table ). we've shown earlier thus, we've tested the ability of swine and pandemic h n v viruses in this aspect. it was shown that these viruses were comparable with the effect seen for seasonal h n virus. moreover, swine influenza virus induced stronger apoptotic response in hemablastoid cell lines in comparison with pandemic h n v viruses, which also have a swine origin. we also checked the ability of flu viruses to influence monolayer cell cultures growth. the data clearly indicated that only ecv- endothelial line and t- g glioblastoma line displayed cell proliferation in response to low moi. apoptosis wasn't registered in these stimulated cultures, apparently because the moi was very low. all the other monolayer cultures didn't respond to low moi by stimulation of their proliferation. interaction between an influenza virus particle and a host cell can follow several scenarios. cpe seen in infected cells is accompanied with high rates of viral particles production and leads to cell death. the death itself may be through apoptotic or necrotic pathways. , also, infection process in low doses can stimulate cell proliferation -the effect seen for hemablastoid lines, histiocytes, peripheral blood cell lines, , and in glioblastoma and endothelial cell lines as it was described here. considering the origin of ecv line, these cells bear all the antigenic, biochemical, and physiological traits of umbilical cord and are actively used in pharmacological tests as well as glioblastoma cells; they also are of special interest for oncogenesis studies. table . replication, apoptosis induction, and np synthesis of influenza viruses in a- , ecv- and flech cell cultures. the numbers represent the log tcid ⁄ ae ml calculated by reed-muench method as described in. the ()) symbol means that no cpe could be observed in any dilution and no hemagglutination could be registered. the (+) symbol means that apoptosis was observed with hoechst- staining though the productive replication and production of progeny viruses in human cell lines was generally low, it is evident that viral infection does occur in these cells, even for swine and h n v viruses. it can be demonstrated by the presence of np de novo synthesis and by stimulation of virus-induced apoptosis. in fact, we observe a contradiction: avian influenza viruses actively reproduce in human cell lines, but we do not see their vast spreading in human population, while h n v viruses that hardly replicate in all human cultures tested have caused the latest pandemic. influenza viruses continue to cause problems globally in humans and their livestock, particularly poultry and pigs, as a consequence of antigenic drift and shift, resulting frequently and unpredictably in novel mutant and reassortant strains, some of which acquire the ability to cross species barriers and become pathogenic in their new hosts. long-term surveillance of influenza in migratory waterfowl in north america and europe have established the importance of anseriformes (waterfowl) and charadriiformes (gull and shorebird) in the perpetuation of all known subtypes of influenza a viruses. the available evidence suggests that each of the hemagglutinin (ha) and nine neuraminidase (na) subtype combinations exist in harmony with their natural hosts, cause no overt disease, and are shed predominantly in the feces. , in this study we determined the subtypes and prevalence of low-pathogenic influenza a viruses present on the territory of kazakhstan in - and further analysed the ha and na genes of these isolates in order to obtain a more detailed knowledge about the genetic variation of influenza a virus in their natural hosts. (institute for biological safety problems, gvardeiskiy, zhambyl oblast, kazakhstan)). samples that were identified as influenza a virus positive by matrix rrt-pcr were thawed, mixed with an equal volume of phosphate buffered saline containing antibiotics (penicillin u ⁄ ml, streptomycin mg ⁄ ml, and gentamicin lg ⁄ ml), incubated for minutes at room temperature, and centrifuged at g for minutes. the supernatant ( ae ml ⁄ egg) was inoculated into the allantoic cavity of four -day old embryonated hens' eggs as described in european union council directive ⁄ ⁄ eec. embryonic death within the first hour of incubation was considered as non-specific, and these eggs were discarded. after incubation at °c for days the allantoic fluid was harvested and tested by haemagglutination (ha) assay as describe in european union council directive ⁄ ⁄ eec. in the cases where no influenza a virus was detected on the initial virus isolation attempt, the allantoic fluid was passaged twice in embryonated hens eggs. the number of virus passages in embryonated eggs was limited to the maximum two to limit laboratory manipulation. a sample was considered negative when the second passage ha test was negative. the subtypes of the virus isolates were determined by conventional haemagglutination inhibition (hi) test and neuraminidase inhibition (ni) test, as describe in european union council directive ⁄ ⁄ eec. rna extraction and pcr with specific primers rna was extracted from infective allantoic fluid using rneasy mini kit (qiagene, gmbh, germany) according to the manufacturer's instructions. the rna was converted to full-length cdna using reverse transcriptase. the rt mix comprised ae ll of dmpc water, ll of · first strand buffer (invitrogen), ae ll of mm dntp mix (amersham biosciences), ll of mm uni primer, u of rnaguard (amersham biosciences), u of mmlv reverse transcriptase (invitrogen) and ll rna solution in total volume of ll. the reactions were incubated at °c for minutes followed by inactivation of the enzyme at °c for min. pcr amplification with ha and na gene specific primers was performed to amplify the product containing the full length ns gene. twenty-five microliter pcr-mix contained · platinum taq buffer (invitrogen), lm dntp, ae mm mgcl , nm each of fw primer and rw primer, u platinum taq dna polymerase (invitrogen) and ll cdna. reactions were placed in a thermal cycler at °c for min, then cycled times between °c seconds, annealing at °c for seconds, and elongation at °c for seconds and were finally kept at °c until later use. sequences of the purified pcr products were determined using gene specific primers and bigdye terminator version ae chemistry (applied biosystems, foster city, ca), according to the manufacturer's instructions. reactions were run on a abi tm dna analyzer (applied biosystems). sequencing was performed at least twice in each direction. after sequencing, assembly of sequences, removal of low quality sequence data, nucleotide sequence translation into protein sequence, additional multiple sequence alignments, and processing were performed with the bioedit software version ae ae ae with an engine based on the custal w algorithm. the phylogenetic analysis, based on complete gene nucleotide sequences were conducted using molecular evolutionary genetics analysis (mega, version ae ) software using neighbor joining tree inference analysis with the tamura-nei c-model, with bootstrap replications to assign confidence levels to branches. [ ] [ ] [ ] [ ] ha and na sequences obtained from genbank the ha and na gene was analyzed both with selected number of influenza isolates and in comparison with virus genes obtained from genbank were used in phylogenetic studies [ ] . the nucleotide sequence data obtained in this study has been submitted to the genbank database and is available under accession numbers fj , fj ae , fj , fj , gu -gu for ha and fj , fj ae , fj , fj , gu -gu for na. avian influenza prevalence in our study h , h , and h influenza a virus subtypes were found to circulate at the same time, in the same geographic region in the kazakhstan. this finding most likely indicates the existence of a large reservoir of different influenza a viruses in kazakhstan. we analyzed the ha and na gene sequences of the eight influenza a viruses isolated in kazakhstan together with selected number of isolates, reported between year to , and previously published in the genbank. phylogenetic analysis of the h ha gene showed that all viruses separated into the american and eurasian lineages ( figure ). an evolutionary tree suggests that north american isolates have diverged extensively from those circulating in other parts of the world. geographic barriers which determine flyway outlay may prevent the gene pools from extensive mixing. the lack of correlation between date of isolation and evolutionary distance suggests that different h ha genes co circulate in a fashion similar to avian h ha genes and influenza c genes, implying the absence of selective pressure by antibody that would give a significant advantage to antigenic variants. analysis of phylogenetic relationships among the ha ha genes reported in this study clearly shows that viruses belong to the western pacific flyway, one of the major migratory flyways in this region that have subsequently spread throughout eurasia. these findings provide further evidence of the dynamic influenza virus gene pool in this region. along the western pacific migratory flyway, the influenza virus gene pool in the domestic waterfowl of southern china has 'mixed' longitudinally with viruses isolated from japan, mongolia, and siberia. however, it appears that there has also been 'mixing' latitudinally through overlapping migratory flyways, thereby facilitating interaction between the influenza virus gene pool in domestic waterfowl in the eastern and western extremities of the eurasian continent. this helps to explain the latitudinal spread of the qinghai-like (clade ae ) h n virus in the last years, while h n outbreaks in korea and japan may represent the longitudinally transmitting pathway. ha of subtype h so far has been found exclusively in shorebirds, such as gulls, and in a pilot whale (potentially a spillover from shorebirds), but not in other avian species that are natural hosts of influenza a virus, such as ducks and geese; therefore the study of the evolution of these viruses is very interesting. phylogenetic analysis h ha gene revealed three significantly different evolutionary lines: an american line, a european line, and a line comprising the isolates from america and eurasia. further we analyzed na genes of influenza viruses (figure ) . the na gene is important both because of its functional role in promoting the dissemination of the virus during infection, and because, like ha, it is a principal target of the immune system. it was shown that phylogeny of na genes of influenza have the same properties as hemagglutinin. na genes of kazakhstanian viruses belong to eurasian lineage of virus evolution. obtained data are important for surveillance and diagnostics because some of the lpai viruses examined in this study can infect and be shed by chickens and turkeys and may have epidemiology potential during further recombination with other influenza viruses. influenza virus is divided into different subtypes based on hemagglutinin (ha) and neuraminidase (na) on the virus surface. within each subtype, ha continues to mutate and produce immunologically distinct strains, as antigenic drift. the continuous mutation of influenza virus (iv) is important for annual epidemics and occasional pandemics of disease in humans. antigenic drift requires vaccines to be updated to correspond with the dominant epidemic strains. in humans, ivs show both antigenic drift frequently. in contrast, ivs from birds are in evolutionary stasis, and they show little amino acid changes. , the reason is that ivs in bird intestine are not subjected to strong immune selection. hemagglutinin (ha) gene of influenza a virus encodes the major surface antigen, which is the target for the protective neutralizing antibody response that is generated by infection or vaccination. in humans, influenza a viruses show antigenic drift with amino acid changes in the globular head of the ha so as to evade herd immunity of the population. on the contrary, avian influenza a viruses show evolutionary stasis in wild birds. h aivs have occurred frequently in chicken farms in the world. although vaccination is not permitted, h n aivs have circulated in taiwan for a time. the seroprevalence in chicken flocks reaches about % in the field. h n aivs invades internal organs, such as kidney and lung. thus, viruses in chicken flocks are pressured into antibody selection. here, we report that h n aivs in the field have showed evolutional changes instead of evolutional stasis. in response to requests from poultry farmers for diagnostic investigations of illness in poultry flocks, the authors did necropsy at the pen-site. after careful examination, tracheae were taken and kept in cold for virus isolation in the laboratory. for avian influenza virus isolation, trachea was homogenized : in tpb with antibiotics. the homogenate was frozen and thawed three times and then centrifuged at g for minutes. the supernatant was passed through a ae lm filter. the homogenate was examined for the presence of virus by inoculation into five -to -day-old specific-pathogen-free (spf) chicken eggs for two passages. thirteen h n aivs were isolated in this laboratory during and from different parts of taiwan. besides the viruses isolated in this laboratory, the ha sequences of chicken h n aivs were from the genbank. the accession numbers of hemagglutinin of aiv reference strains included in this study were as the following: g ⁄ , dq ; g ⁄ , dq ; ⁄ , dq ; na ⁄ , dq ; ⁄ , dq ; ⁄ , dq ; ns ⁄ , dq ; sp ⁄ , dq ; ⁄ , dq ; ⁄ , dq ; ⁄ , dq ; ⁄ , dq ; ⁄ , dq ; ⁄ , dq ; pf ⁄ , dq ; pf ⁄ , dq ; pf ⁄ , dq ; a ⁄ , dq ; ⁄ , dq ; ⁄ , dq ; ⁄ , dq ; ⁄ , dq ; ⁄ , dq ; ch ⁄ , dq ; ⁄ , dq ; a ⁄ , dq and ⁄ , dq . the viruses isolated were propagated in the allantoic cavities of -day-old embryonated spf eggs for hour. the virus rna was extracted using qiaamp viral rna miniprep kit (qiagen) . six-week-old balb ⁄ c mice were injected emulsion intraperitoneally with lg of purified and concentrated a ⁄ chicken ⁄ taiwan ⁄ v ⁄ (h n ) virion with complete freund's adjuvant. every two weeks, the mice were boosted supplementary five times with lg of virion in incomplete freund's adjuvant. when the mice were boosted, blood was collected from tail vein and tested by the western blot assay to check the antibody titers. the mice were then injected intraperitoneally with lg of virion at week . five days after the last injection, the splenocytes in the mice were fused with myeloma cells (sp ⁄ -ag ). one week before fusion, the myeloma cell line was expended in dmem medium (hyclone laboratories, logan, ut) with % fetal bovine serum at °c to ensure they were in the exponential growth phase. the spleen cells from immunized mice were washed, harvested, and mixed with the previously prepared myeloma cells and fused by gradually adding % polyethylene glycol- . the resulting pellet was plated into well tissue culture plates. only the fused cells grew in medium with hypoxanthine-aminopterin-thymidine (hat). with fresh medium replacement over weeks, the hybridomas were ready for screening. hightiter monoclonal antibody (mab) preparations were obtained from the ascetic fluid of mice injected with the selected hybridoma clones. the antibody from mouse ascetic fluids was purified by precipitation with ammonium sulfate, then aliquoted and frozen at ) °c, avoiding repeated freezing and thawing. eventually, six mabs were obtained and named ch -d , eb -b , eb -e , eb -f , ff -f , and ff -f , respectively. the hi test was performed following a standard method. all the viruses were diluted twofold and reacted with % chicken erythrocytes in the v-bottomed microtiter plate by the hemagglutination test. after agglutination, four hemagglutinating units of a ⁄ chicken ⁄ taiwan ⁄ v ⁄ (h n ) and ascetic fluids from the immunized mice of the six mabs were prepared for hi test. hi titers of or more were regarded as positive. the cases submitted for diagnosis from chicken farms had respiratory signs, increase in mortality, or drop in egg production (e.g. egg production dropped from % to %). the extent of drop in egg production depended on the chicken ages. for example, the age of case was weeks, a stage of increasing egg production. however, after h n aiv infection, the egg production decreased % instead of increasing and then stayed at % for a week. the infected chickens showed signs of decreasing activity, anorexia from g per bird to g per bird, and respiratory signs. case showed infection in the second floor first and then transmitted to third and fourth floor, indicating that the virus transmitted by air or human movement. however, most cases showed air borne transmission from one flock to another in spite of enforcing restrictions of persons entering the poultry pens and changing clothes and booths. in most cases, males' mortality was higher than that of female pen mates. by comparing the sequences of ha of those h n viruses, we found that amino acid changes in ha were higher than those in ha , showing that antigenic changes on the globular head of ha molecule rather than randomly on the whole ha protein, indicating that h n viruses in taiwan had been selected in the presence of antibody pressure. the aa residues and changes that showed yearly trends were the followings: a- s, i s, v i, n s, e k, l m, e d, q k, a v, or t, s n, s r, k n, y d, n t, s i, g d, l v, i v, g e, t n, g s, a v, k e, d n, i m, and m i. however, their significance on antigenic variation was previously unknown. by hemagglutinination inhibition (hi) assays, except mab ch -d , all other monoclonal antibodies elicited from v ⁄ showed different hi titers with the different h n viruses (table ). however, those mabs showed negative hi to and , the early h n strains. this indicated that the epitopes recognized by those mabs were undergoing antigenic drift. introduction aquatic birds are recognized as the natural reservoirs of the influenza a virus as all known subtypes (h -h , n -n ) have been found in them. phylogenetic analyses of influenza viruses found in other animals revealed that all were directly or indirectly derived from viruses resident in aquatic birds. however, the prevalence, movement, and evolutionary dynamics of influenza viruses in these avian hosts have not been well defined. southern china was hypothesized to be an 'epicenter' for the generation of human pandemic influenza viruses as all major influenza pandemic viruses in the th century emerged from this region. the ecological background that facilitates the occurrence of these pandemic influenza strains has not been fully explored. in the past two decades, four lineages, belonging to h n , h n , and h n viruses, have become established and long-term endemic in different types of poultry in this region. [ ] [ ] [ ] some of these viruses were disseminated to many countries in eurasia and africa and have continued to cause sporadic human infection, posing a persistent pandemic threat to the world. in the mean time, the endemic influenza lineages have undergone extensive genetic reassortment events giving rise to many variants, dramatically increasing the genetic diversity of the influenza virus in this region. questions remain as to how and where these viruses emerged, and what were the sources of the gene segments incorporated within the novel reassortant variants of the h n , h n , and h n virus lineages. to address these questions, surveillance of influenza in migratory and domestic (sentinel) ducks has been conducted since at poyang lake, the biggest fresh-water lake and the major migratory bird aggregation site in southern china. the aim of this study is to identify the prevalence, seasonality, and movement of virus between migratory and domestic ducks. migratory ducks were captured during over-wintering, from november to march. cloacal swabs and blood samples were collected from each individual bird. all birds were released after sampling. to observe the interaction between migratory ducks and domestic birds, we also sampled domestic ducks from two duck farms (designated as sentinel ducks) surrounded by rice fields and inaccessible to other types of poultry, but accessible to migratory birds. that is, the sentinel ducks share the same water body with migratory ducks and have the chance to spread viruses to each other. for sentinel ducks, sampling was conducted fortnightly, all year-round, on the two farms from august onwards. cloacal swabs and fresh fecal droppings were taken. about birds were randomly sampled fortnightly from these farmed ducks. all swabs were soaked in vials containing ae ml transport medium with antibiotics and kept on ice-packs during sampling and immediately stored in ) °c freezers for further use. blood samples from migratory ducks were treated according to methods previously described. serological survey and virus subtyping in migratory and sentinel ducks used hemagglutination inhibition (hi) and neuraminidase inhibition (ni) tests as previously described. for isolates that were not identified by reference antisera, subtypes were determined by rt-pcr using subtype specific ha and na diagnostic primers. prevalence and seasonal patterns of influenza virus in migratory and sentinel ducks during during - a total of cloacal swabs from migratory ducks and cloacal or fecal swabs from sentinel ducks were collected at poyang lake. from these specimens, influenza isolates were obtained from migra- tory ducks and from sentinel ducks; isolation rates of ae % and ae %, respectively (table ) . it was noted in sentinel ducks that virus occurrence formed a seasonal peak from november to february, which completely overlapped the over-wintering months of migratory ducks. this suggests that virus movement or transmission between migratory and sentinel ducks occurred during this period at poyang lake. thirty positive samples (hi titer ‡ ) were identified from blood samples collected during november and december in . among these, samples were positive to h , were positive to h , were positive to h , and were positive to h . one serum sample was positive to both h and h , which suggested co-infection of influenza virus in migratory ducks might occur in natural conditions. poyang lake, which is located in the northeastern part of jiangxi province, is the largest freshwater lake in china and is part of the eastern asia-australia migration route. every year, hundreds of thousands of migratory ducks congregate at poyang lake during the migration season. recent farming practice involves raising domestic waterfowl in dense populations in the poyang lake region. farmraised domestic waterfowl are allowed to feed in and share the same water body with migratory birds, thereby facilitating direct interactions between domestic waterfowl and freeranging migratory birds. this makes poyang lake an ideal site to observe the dynamics of influenza virus interactions between migratory and sentinel ducks in southern china. in our longitudinal surveillance during [ ] [ ] [ ] [ ] [ ] [ ] , the overall virus isolation rate from migratory ducks was less than %, which suggests a low prevalence of viral infection during the birds' southern migration. similar results have been observed in taiwan, which is also an important stopover site for migratory birds along the eastern asia-australia migration route during years of surveillance. the overlap in seasonal patterns of virus infection between migratory and sentinel ducks found in our study suggests that virus movement or transmission between migratory and sentinel ducks occurred during the period of time migratory birds were at poyang lake. the ha subtypes harbored in migratory and sentinel ducks were similar in our study. for migratory ducks, h , h , h were the predominant subtypes, while h , h , and h were the major subtypes in sentinel ducks. hpai h n was only detected from migratory ducks in early on two sampling occasions. from phylogenetic analyses the h n viruses isolated from migratory ducks were closely related to the viruses endemic in domestic poultry in southern china. therefore, it appears that h n viruses endemic in domestic poultry could be transmitted to migratory ducks via close contact in southern china. only lp h viruses were detected from sentinel ducks at poyang lake during this period. whether h n virus infection was absent from sentinel ducks at poyang lake needs further investigation. serological surveys provided further evidence for the prevalence of aiv in migratory ducks at poyang lake. the serological results in did not match well with the epidemiological results during [ ] [ ] [ ] [ ] [ ] [ ] , which suggests that influenza virus infection in migratory birds could be influenced by multiple factors, such as host immune status, population size, spatial and temporal variations, and migration routes. southern china has the biggest domestic duck population in the world. our study demonstrates that dynamic interactions between migratory ducks and sentinel ducks occurred frequently throughout the surveillance period. thus, sentinel ducks could be treated as intermediate hosts between the ''real gene pool'' from migratory ducks and domestic poultry in the whole influenza virus ecosystem. a sentinel duck sampling system may be a feasible method to represent the viruses in the natural gene pool and a baseline for virus or gene interactions between migratory and domestic ducks. further investigations and surveillance are required to better understand the role of the domestic duck population in facilitating virus interactions and the generation of genetic diversity. two distinct lineages of h n influenza viruses represented by a ⁄ chicken ⁄ beijing ⁄ ⁄ (ck ⁄ bei-like) and a ⁄ quail ⁄ hong kong ⁄ g ⁄ (g -like) have become established and endemic in poultry in southern china. these established h n lineages continue evolving to generate many different reassortant variants (or genotypes) , and are causing sporadic cases of human infection. , studies of h n viruses isolated from pigs in hong kong and shandong province have also raised the possibility of reassortment with human-like viruses from pigs. , in addition, h n viruses isolated beyond the late s had preferential binding with a- , -neuacgal human-like receptors. these observations suggest that the h n influenza viruses still have pandemic potential. unlike highly pathogenic h n influenza viruses that have been rarely detected in the live-poultry markets in hong kong since , h n viruses are still frequently isolated in our surveillance program. therefore, we try to understand the continuing evolution of h n viruses through genetic characterization and phylogenetic analyses of the viruses isolated in hong kong live-poultry markets from to . a total of terrestrial poultry were sampled at different live-poultry markets in the hong kong sar between january and december . of those samples, were from chickens and the others were from minor poultry species including chukar, pheasant, guinea fowl, silky chicken, and pigeon. fecal droppings, cloacal and tracheal swabs, drinking water, and environmental samples from cages were collected into transport medium. viruses were isolated in -to -day old embryonated eggs as described previously. virus isolates from positive sampling occasions were selected for sequence analysis. rna extraction, cdna synthesis, and pcr were carried out as described previously. dna sequencing was performed using bigdye terminator v ae cycle sequencing kit on an abi dna analyzer (applied biosystems) following manufacturer's instructions. all sequences were assembled and edited with lasergene ae (dnastar, madison, wi) software. sequence alignment and residue analysis were performed with the bioedit sequence alignment editor, version ae . all eight gene segments of sequenced viruses were characterized and analyzed phylogenetically together with virus sequence data available in public databases. maximum-likelihood trees were constructed using garli ae . estimates of the phylogenies were calculated by performing neighbor-joining bootstrap replicates using paup* ae . systematic surveillance of live-poultry in hong kong from to resulted in h n isolates from samples (overall isolation rate, ae %) ( table ). there were strains isolated from chicken samples (isolation rate, ae %). of these viruses, four were isolated from tracheal swabs (isolation rate, ae %), while isolates were isolated from cloacal or fecal swabs (isolation rate, ae %). an additional isolates were collected from drinking water samples (isolation rate, ae %). there were strains of h n viruses isolated from minor poultry samples (isolation rate, ae %) ( table ) . of these viruses, only one was isolated from tracheal swabs (isolation rate, ae %), whereas strains of viruses were isolated from cloacal or fecal swabs (isolation rate, ae %). the isolation rate in drinking water in minor poultry was again higher when compared with other sampling methods with strains isolated from drinking water samples (isolation rate, %). taken together, these findings suggest that the h n viruses mainly replicated in the intestinal tract of chickens and minor poultry species. also, the high isolation rate in drinking water samples could be a sensitive indicator for monitoring the prevalence of h n viruses in the field. to better understand the evolutionary pathway of h n viruses in southern china, representative viruses, isolated from hong kong live-poultry markets from to , were sequenced and genetically characterized. phylogenetic analysis of the h ha gene revealed that ck ⁄ bei-like viruses were predominant and one chicken isolate had a g -like ha gene ( figure ). this is the first time the g like h ha gene has been detected in chickens from livepoultry markets in hong kong. the ck ⁄ bei-like lineage is further divided into two subgroups as previously described. subgroup is represented by qa ⁄ st ⁄ ⁄ and subgroup is represented by dk ⁄ hk ⁄ y ⁄ . all h n viruses in this study belonged to subgroup of the ck ⁄ bei-like lineage except for the virus with the g -like ha gene. phylogenetic analysis of the na gene also showed a similar evolutionary pattern to the ha gene with all viruses clustered within the ck ⁄ bei-like lineage. these results revealed that ck ⁄ bei-like viruses are predominant in both chickens and minor poultry. all of the pb , pa, np, ns and m genes clustered with those of h n lineage viruses previously prevailing in ter- restrial poultry in southern china. phylogenetic analysis of the pb gene revealed three different lineages; g -like (n = ), ck ⁄ sh ⁄ f ⁄ -like (n = ), and unknown avian (n = ). the sh ⁄ f ⁄ -like lineage (or f ⁄ -like) was previously reported in eastern china and was used previously for vaccine production in an intensive vaccination program. this pb gene lineage was also distinguishable from the ck ⁄ bei-like lineage and its presence in the viral genome may be due to reassortment between the vaccine strain and field isolates, followed by selective establishment in terrestrial poultry. gene constellation analyses of the viruses revealed six genotypes. thirty-four of the viruses analyzed belonged to two genotypes, b and b , which were also the prevailing reassortants found in other provinces in southern china since . the remaining sixteen viruses belonged to four novel genotypes that have not been identified before in this region. characterization of h n influenza viruses isolated from live poultry in hong kong markets from a year surveillance program revealed that ck ⁄ bei-like viruses were predominant in southern china and were continuing to evolve. two recognized and four novel genotypes were identified in this study. one characterized virus, ck ⁄ hk ⁄ nt ⁄ , had a g like ha gene (the first time this has been detected in hong kong poultry markets) that showed a close relationship with two human h n strains isolated in . g -like viruses were usually detected and caused outbreaks in chickens of middle eastern and european countries, [ ] [ ] [ ] and minor poultry, mainly quail, in southern china. whether the g -like virus was transmitted from china to middle eastern and european countries, as the highly pathogenic h n virus did in the last five years, or vice versa, is still unknown. since the ck ⁄ hk ⁄ nt ⁄ strain clustered with other g -like strains isolated previously in minor poultry in southern china, the g -like viruses in chicken may be due to interspecies transmission from minor poultry species. genetic studies demonstrated that reassortants with genotypes b and b persistently occurred in either chickens or other minor poultry species from to . other genotypes that were prevalent in southern china might be being gradually replaced and four novel genotypes were identified in this study. these novel genotypes were generated through reassortment of viruses with different lineages. a newly emerged f ⁄ -like lineage originating from eastern china is responsible for generation of some of the novel genotypes found in this study. the ck ⁄ bei-like lineage is gradually being replaced by f ⁄ -like lineages which are becoming dominant in northern and eastern china. , animal experiments have also demonstrated that f ⁄ -like viruses are more effective in replication and transmission in chickens compared with ck ⁄ bei-like viruses. since the f ⁄ -like lineage of the pb gene has been introduced into southern china, this newly emerged lineage may have a higher tendency to replace the rnp genes in the circulating ck ⁄ bei-like viruses and subsequently become the endemic virus in terrestrial poultry. in vietnam, the modelling of the pandemic h n progression estimates that ( - ) pigs might be exposed to the virus on the basis of cases among swine owners ( - ). a poor level of biosecurity, high animal densities, and a mix of species could increase the risk of influenza virus flow, persistence, and emergence on swine and poultry farms. this study was set up in the red river delta, where a third of the national pig husbandry is produced. the aims are to give preliminary information of the epidemiological state of swine influenza and in order to further assess the risk of infection of swiv, through cross-species transmissions from poultry to pigs. this paper will present the preliminary results on swiv and the risk factors of pig seropositivity in vietnam. a cross-sectional study was conducted in two provinces of the red river delta in april . pig farms were randomly selected from nine communes representative of at risk area of avian h n . in each farm, pig and poultry were sampled and collected to virological and serological analyses. interviews were conducted in all farms by trained interviewees. questionnaires included closed and open questions on ª blackwell publishing ltd, influenza and other respiratory viruses, (suppl. ), - livestock husbandry ⁄ management and household characteristics, such as herd size and structure, health history and vaccination, pig housing, watering and feeding system, reproduction, purchasing of animals, biosecurity measures, pig contact with poultry, and environmental factors. the virological detection assay was performed on pools of nasal swab specimens from pigs. we investigated whether real-time rt-pcr assay could detect gene m on pools of nasal swab specimens before attempting virus isolation from individual nasal swab specimens. the poultry and pig sera were tested against influenza type a with an enzyme-like immunosorbant assay (elisa) competition test idvetª. this commercial kit is designed to specifically detect antibodies directed against the np protein antigen of influenza type a viruses. the positive serum samples were examined in hemagglutination inhibition (hi) to determine antibody titers and subtypes. the hi test was tailored for h , h , and h subtypes in pigs and h and h subtypes in poultry. seroneutralization tests by pseudo particles were used to test the presence of antibodies directed against h subtype. we analysed the data for relationships between influenza a serological status (the outcome variable) and possible risk factors using r version ae ae (r development core team). the statistical unit was the individual. initially, the quantitative variables were encoded into categorical variables according to the quartiles or median. descriptive statistics (e.g., means or medians, proportions, standard deviations) were calculated for all herd-level and commune level predictors to assist in the subsequent modeling process. we also performed the independence test among all variables to determine if variables were dependant. then, univariate analysis of potential risk factors for the pigs being positive for swiv and estimation of odds ratios were performed using generalised linear mixed models with binary outcome and logit link function for each herd-level and commune-level variable to determine which variables were individually associated with influenza a seropositivity at a significance level of p < ae . herd and commune of residence were included as a random effect to account for the correlation of observations at the herd level. the third stage of the analyses included the four herdlevel variables found to be significantly (p < ae ) associated with influenza a seropositivity. an automatic process using all possible associations between the selected variables was computed into a mixed logistic regression models, with random effects. when two variables were collinear, as determined before, only one variable was likely to enter the multivariable model, and therefore, the selection of which collinear variable to enter the model was guided by biological plausibility and statistical significance. all of the pools of nasal swabs were rt-pcr negative. the maximal possible prevalence considering perfect diagnostic tests would be of ae % at a confidence level of %, in an infinite population within these regions (win-episcope ae ). six hundred-and-nine pig sera were tested in nonvaccinating farms. the herd seroprevalence of swine influenza in the commune previously infected by the avian h n in the red river delta raised by ae % [ ae ; ae ] in april . but among seropositive farms, only four had at least two seropositive pigs. the within-herd seroprevalence is very low, and no seropositivity was detected in the majority of farms. estimates had large confidence intervals due to small sample sizes. the individual seroprevalence raised ae % [ ae ; ae ]. the subtyping of seropositive sera is still in process. descriptive statistical analyses on five major risk factors of swiv: farm size, breeding vs. fattening, purchasing, percentage of family income, and poultry production, were conducted. based on this analysis, three types of farming systems were identified and included in mixed models ( table ) . percentage of family income by pig production and poultry production were not differentiating factors for this typology. whereas types and seem to be specialized in fattening, the type produces and might sell piglets on the farm site. the exploration of the different variance components indicated that the random effect variances were mainly associated with the herd, while the commune did not seem to have any effect. therefore we included in all models only the herd as a random effect. the random effect term for herd was modelled, assuming a normal distribution with a table . typology of farming system type : large fattening farms largest scale production, with more than pigs per year specialized in fattening, and purchase more than pigs per year type : small fattening farms small scale of production, with less than pigs per year specialized in fattening, and purchase less than pigs per year type : medium breeding-fattening farms medium scale of production, with less than pigs per year breeding and fattening piglets, with rare purchase common variance [$n( ,r herd)]. the univariate analyses were conducted on variables and typology variables, with herd as random effect. some coefficient or confidence intervals were inconsistent because of small effectives, especially for the percentage of self-product culture or the pig freegrazing because of the lack of positive results in the dataset. the only one significant (p value < ae ) parameter was the percentage of pig sales in the familial annual income. surprisingly, common risk factors of swine influenza infection, such as farm size, animal movements, and sanitary parameters got low odds ratio individually (without being significant); the typology provides the hypothesis of complex interactions effects that increase the risk of infection. as shown in table , the farming system type got a higher seroprevalence of ae % [ ae - ae ] and a higher risk indicator, with or = ae (p-value = ae ) in comparison with type . this finding was not significant. in the multivariate mixed model, the percentage of familial income provided by pig production was the only one significant variable, with or = ae [ ae - ae ]. the focus on diseased animals in the winter-time is usually required in order to increase the likelihood to isolate the virus, although the isolation rate on healthy or clinical samples never exceed %. the season and the lack of disease reports might explain the difficulties to detect influenza viruses. additionally, the pooling method tends to decrease the isolation rate because of a dilution effect, potential presence of pcr assay inhibitors, or uneven distribution of virus in the sample. our seroprevalence results must be confirmed and the subtypes identified, especially because we found only one positive animal in a few farms that could be attributed to false positive results of the elisa test (performances are not known). these preliminary results are in favor of a virus circulation at low level in the spring, but must be completed by further surveys in the winter and before the new year (têt celebration) when pig production, trade, and movement increase at their maximum. no clear prior information on the expected prevalence of swine influenza in vietnam, tests sensitivity, and speci-ficity could be obtained from literature or reliable sources. bayesian methods will be carried out in the future in order to compute prevalence and ⁄ or to estimate the probabilities of freedom. the risk factors analysis was limited by the lack of positive results. further studies are necessary to identify the at-risk season and type of farming systems at risk of swine influenza infection. however, this investigation of risk factors leads to the hypothesis that medium size breeding-fattening farms had a higher risk than large or small size fattening farms. further investigation are needed to precise this typology. the risk of swiv infection increases with a combination of three major factors. poultry production does not seem to play any role on swine infection. the generalized linear mixed model afforded to take into account all the non investigated parameters at the herd level. although we investigated the most common risk factors of swine influenza infection covering different kind of fields, the herd random effect might explain risk variations. mixed models have become a frequently used tool in epidemiology. due to software limitations, random effects are often assumed to be normally distributed. since random effects are not observed, the accuracy of this assumption is difficult to check. further studies, such as case-control or cohort studies could help to identify more precisely risk factors of swine influenza seropositivity, as these study designs are more adapted than cross-sectional studies. the concept that swine are a mixing-vessel for the reassortment of influenza viruses and for the emergence of pandemic influenza viruses has been re-enforced by the emergence of the recent pandemic. the pandemic h n virus of (h n pdm) is believed to have emerged through the reassortment of north american triple reassortant and eurasian avian-like swine influenza viruses. since the immediate precursor of this pandemic virus has not yet been identified, it is not possible to be definite whether the reassortment leading to the pandemic occurred in swine, but swine influenza viruses are the nearest known ancestors of each gene segment of h n pdm. , the mechanisms of pandemic emergence are not clear. it is believed that the pandemics of and arose through reassortment of the pre-existing human seasonal influenza virus with avian influenza viruses, and swine have been proposed to be a possible intermediate host where such reassortment between human and avian viruses may take place. the pandemic was the first to arise for over years and the first to occur after the understanding that pandemics arise from animal influenza viruses. systematic studies of influenza virus ecology and evolution in swine are, therefore, important in order to understand the dynamics of pandemic emergence. furthermore, since swine are the likely host within which h n pdm virus originated, it was predicted that this virus would readily infect swine and may reassort with endemic swine influenza viruses. these predictions have now been confirmed with reports of h n pdm being detected in pigs in many countries and reassortment with endemic swine influenza virus being confirmed. while h n pdm has been genetically and antigenically stable in humans, reassortment between h n pdm, which is well adapted to transmission in humans, and other avian or swine viruses may lead to the origin of novel viruses posing a threat to public health. in addition to endemic swine virus lineages, avian influenza viruses such as h n and highly pathogenic avian influenza (hpai) h n have also been occasionally identified in pigs in parts of asia. , it has been shown that h n pdm readily reassorts with h n to generate viable progeny in vitro. it is therefore essential to monitor the ecology, evolution, and biological characteristics of swine influenza viruses so that their continued evolution and zoonotic and pandemic potential can be monitored. there is however, a paucity of surveillance data on swine influenza viruses worldwide. this is in part related to the negative commercial consequences that may arise from detection of influenza in a swine herd leading to a major economic loss to the producer. here we outline a surveillance system that has been in place in hong kong for the last decade, based on sampling animals arriving at an abattoir in hong kong. we demonstrate the feasibility of such surveillance in an abattoir setting and compare methods used for detection influenza viruses in swine. virus isolation was carried out by inoculation into mdck cells and by allantoic inoculation in embryonated eggs as previously described. virus isolates were subtyped by haemagglutination inhibition tests using specific antisera and genetically characterized by sequencing and phylogenetic analysis of the haemagglutin gene. , virus detection by rt-pcr a subset of recent specimens was tested in parallel by real time pcr using the biorobot universal system (qiagen) that enables fully-automated viral nucleic acid extraction and downstream reaction setup in a -well plate format. total viral nucleic acids were extracted in a -well plate format with the qiaamp virus biorobot mdx kit (qiagen) on the biorobot universal system (qiagen) according to the manufacturer's instructions. briefly, ll of sample was lysed in ll buffer al, supplemented with ae lg carrier rna in a s block (qiagen), which placed the samples into a well plate format. after protease digestion, samples were transferred to silica based membrane in well plate format for binding. following two washing steps, rna was eluted in ll of elution buffer (buffer ave) into a well elution microplate cl (qiagen) . for the synthesis of cdna, ll of purified rna was used in a ll reaction containing ll of · buffer, ae nm of each deoxynucleotide triphosphate (dntp), mm dithiothreitol, lg random primer, u of rnaseout recombinant ribonuclease inhibitor, and u of superscript iii reverse transcriptase (all from invitrogen). reactions were performed in the geneamp thermocycler (applied biosystems) with the following parameters: minutes at °c, minutes at °c, and soak at °c. subsequent to the reactions, ll of cdna was diluted ⁄ by adding ll of ae buffer (qiagen) . real-time pcr was performed using the power sybrÒ green pcr master mix (applied biosystems) according to the manufacturer's instructions. briefly, ll of ⁄ diluted cdna was amplified in a ll reaction containing ae ll of · power sybr green pcr master mix, nm of forward primer m c ( ¢-ctt cta acc gag gtc gaa acg- ¢) and nm of reverse primer m r ( ¢-agg gca ttt tgg aca aag ⁄ t cgt cta- ¢). the primers have been designed to amplify the sequences in the conserved region of influenza a virus matrix gene, thereby detecting viruses from different species including swine influenza viruses. real-time pcr was performed in the abi fast system (applied biosystems) with the following cycling conditions: minutes at °c once, seconds at °c, and minutes at °c for cycles, followed by melting curve analysis with seconds at °c, minutes at °c, and seconds at °c. in each assay, serially diluted plasmids containing the full length m gene cloned from a ⁄ vietnam ⁄ ⁄ (h n ) were included as standards to perform absolute quantification. a manual baseline was set from cycles - and a manual cycle threshold (ct) was set at ae . samples that were positive or unequivocal results from the real-time pcr were confirmed by performing gel electrophoresis on the pcr products. positive visual identification was made in the presence of the target pcr product at bp in length. a total of tracheal and nasal swabs were processed during the years january -april and yielded influenza virus isolates, an overall virus isolation rate of ae %. of these, were subtype h (classical swine, eurasian avian-like swine, and triple-reassortant), were human-like h viruses, and were eurasian avianlike swine h n viruses. culture in mdck cells yielded % of h subtype viruses, % of the human seasonal-like h n viruses, and ae % of the avian-like eurasian swine h n viruses. culture in embryonated eggs yielded ae % of the h subtype viruses, % of the human seasonal-like h n viruses, and ae % of eurasian avian-like swine h n viruses ( figure ). tracheal and nasal swabs each gave comparable overall virus isolation rates ( ae %). however, isolation rates for human-like h n viruses were ae fold higher in nasal swabs ( ae % versus ae % respectively; p = ae ) ( figure ) . a parallel evaluation of rt-pcr and culture was carried out in specimens. rt-pcr detected ⁄ ( %) of the culture positive specimens. rt-pcr was also positive in ⁄ ( ae %) culture negative specimens, but all these specimens had very low virus load in the rt pcr tests. virus could not be cultured from these culture negative specimens even by attempts at virus re-isolation from the frozen specimen. surveillance in an abattoir setting provides an acceptable yield of influenza viruses and is a feasible method of swine influenza surveillance. sampling in a large abattoir setting allows surveillance to be carried out anonymously with no negative consequences to the supplier. the supply-chain of pigs to the hong kong abattoir involves pigs being trucked in over long distances and may provide opportunity for virus amplification during transport. thus, virus isolation rates may be lower in more vertically integrated and homogenous production and slaughter systems where less mixing of pigs occurs. our results indicate that mdck cell culture is essential for optimizing virus isolation during swine influenza surveillance. allantoic inoculation of embryonated eggs by itself is sub-optimal for isolation of swine influenza viruses. it is however possible that inoculation of embryonated eggs by the amniotic route may lead to better isolation rates than allantoic inoculation. rt-pcr detection is an alternative method for virus detection. but the additional specimens detected by rt-pcr did not yield culturable virus, even following attempts at re-isolation and sequential passage. the rt-pcr positive ⁄ virus isolation negative specimens had very low virus load, and this may be the explanation for the inability to isolate such viruses. in addition, rt-pcr did not detect all viruses isolated by culture. tracheal and nasal swabs gave comparable isolation rates with the exception of human-like h n viruses which were more frequently isolated from nasal swabs. this may suggest that, in contrast to endemic swine influenza virus lineages, these human-like h n viruses are less adapted to replication in the lower respiratory tract. in summary, collection of nasal or tracheal swabs in an abattoir setting together with virus isolation in mdck cells provides a feasible approach to surveillance of swine influenza viruses. kong, kong, - introduction wild waterfowl are the natural reservoir of influenza a viruses (aiv), and they play an important role in the genesis of pandemic influenza. it is suggested that the pandemic virus was purely derived from avian virus, which adapted to humans and caused efficient human-to-human transmission, while the pandemics of and had acquired the viral haemagglutinin, pb polymerase, and in , the neuraminidase gene segments from the avian gene pool. the major regional outbreaks of highly pathogenic avian influenza (hpai) h n in asia, europe, and africa highlight the potential role played by migratory waterfowl in disseminating highly pathogenic influenza viruses. therefore defining the influenza virus gene-pool in wild birds is of vital importance. surveillance was carried out - times weekly from to during the winter months of october to april in the hong kong mai po nature reserve and lok ma chau, hong kong. the hong kong mai po nature reserve and lok ma chau are along the east asia-australian flyway where a peak of more than ducks and grebes congregate every winter. fecal droppings were collected and transported in vials containing ae ml of vtm, which was prepared from m ( ae g ⁄ l), penicillin g ( · u ⁄ l), polymyxin b ( · u ⁄ l), gentamicin ( mg ⁄ l), nystatin ( ae · u ⁄ l), ofloxacin hcl ( mg ⁄ l), and sulfamethoxazole ( g ⁄ l). an aliquot of ll from each swab sample was inoculated into the allantoic cavity of a -to -day-old chicken embryonated egg, and incubated for days at °c. positive ha isolates were subtyped using standard antisera , and rt-pcr was performed with the used of one-step rt-pcr assay (invitrogen) described earlier, followed by sequencing on abi prism xl dna analyzer. the determination of species of origin was performed by dna barcoding of the mitochondrial cyto-chrome oxidase i gene from dna extracted from the fecal droppings. during the -year surveillance period, a total of influenza viruses were isolated from samples collected, an overall isolation rate of ae %. a total of isolates were obtained from specimens collected during the winter period coinciding with the southern migration of waterfowl along the east asian flyway and one isolate obtained from samples collected in spring during the period when northern migration of waterfowl took place along the east asian-australasian flyway. the isolation in hong kong was slightly lower than a similar study conducted in south korea in which the isolation rate of migratory birds was ae % in - . this suggested a slightly lower prevalence of influenza virus present in hong kong as the birds migrated southwards. the viruses isolated in hong kong, representing hemagglutinin (ha) subtypes of h -h and neuramidinase (na) subtypes of n -n , were all from wild waterfowl ( table ) . out of the twelve ha subtypes isolated, h and h were the two subtypes that were isolated frequently every year for h and in six out of seven years for h , respectively. h and h viruses accounted for ae % and ae % of all virus isolated, respectively. on the other hand, h , h , and h were the least prevalence ( ae %) and were only isolated once in years. of the na subtypes, n and n were isolated most often ( ae % and ae % of all isolates, respectively) and n was the least ( ae %). november was the month that had the highest prevalence of influenza virus ( ae % of samples being positive) compared to only ae % in march. the subtype's variation was the most diverse in december during our years of surveillance. this suggested that more of these wild migratory birds may be carrying influenza virus when they arrive in hong kong. however the continued isolation of viruses suggests continued circulation of these viruses in the vicinity of mai po. the study of dna barcoding for the mitochondrial cytochrome oxidase i gene retrieved from fecal droppings revealed that the isolates originated mainly ( ae %) from birds of the order anseriform, family anatidae including eurasian wigeon, northern shoveler, northern pintail, common teal, and garganey. non-anseriformes which were found to have shed aiv viruses were cormorant, grey heron, and stint. none of the water samples collected from the ponds where these birds congregate were found to be positive for the virus. phylogenetic analyses of the ha gene of the lpai h viruses isolated in this study clustered with that of the other lpai h viruses isolated from hokkaido, mongolia, and siberia and were not closely related to the hpai h n . satellite tracking of eurasian wigeons and northern pintails in dec and revealed their flyway from hong kong to as far north as eastern russia, eastern mongolia, and northern china. no hpai h n viruses were isolated in this study from apparently healthy birds. however, as part of the surveillance of dead wild birds carried out by the department of agricultural, fisheries and conservation of the government of hong kong during this same period, over dead wild birds were tested positive for hpai h n and has been reported elsewhere. our influenza surveillance in hong kong has revealed a diversity of influenza virus subtypes the migratory waterfowl infected within the region. the result of the phylogenetic analysis correlated with the findings from satellite tracking that viruses isolated in hong kong were closely related to those isolated in areas along the migratory route. no healthy bird was isolated with hpai h n, although dead wild birds have been regularly found to have hpai h n virus, suggesting that infected birds might not live for a long period. introduction a novel swine-origin h n influenza virus emerged in mexico in april and rapidly spread worldwide, causing the first influenza pandemic of the st century. most confirmed human cases of h n ⁄ influenza have been uncomplicated and mild, but the increasing number of cases and affected persons worldwide warrant optimal prevention and treatment measures. today, almost all of the pandemic h n ⁄ viruses tested are resistant to m blockers. therefore, only the neuraminidase (na) inhibitors are currently recommended for treatment of this pandemic influenza. for the control of influenza infection, the clinical use of oseltamivir has increased substantially during the pandemic. to date, the majority of tested clinical isolates have remained susceptible to na inhibitors, oseltamivir and zanamivir, but oseltamivir-resistant variants with h y na mutation (n numbering) have been isolated from individuals taking prophylaxis, from immunocompromised patients, and from a few community clusters. , in view of the high prevalence of oseltamivirresistant seasonal h n influenza viruses in - , the isolation of resistant h n ⁄ viruses without known oseltamivir exposure raised great concern about the transmissibility and fitness of these resistant viruses. here we studied the transmissibility of a closely matched pair of pandemic h n ⁄ clinical isolates, one oseltamivir-sensitive and one resistant, in both direct contact and respiratory droplets routes among ferrets. viral fitness was evaluated by co-infecting a ferret with both the oseltamivir-sensitive and -resistant viruses. the viruses were also characterized by full genome sequencing, susceptibility to na inhibitors, and growth in mdck and mdck-siat cells. oseltamivir-resistant influenza a ⁄ denmark ⁄ ⁄ (h n ) virus (a ⁄ dm ⁄ ⁄ ) was isolated from the throat swab of a patient who had influenza-like symptoms and received post-exposure oseltamivir prophylaxis ( mg once daily). wild-type influenza a ⁄ denmark ⁄ ⁄ (h n ) virus (a ⁄ dm ⁄ ⁄ ) was isolated from a patient in the same cluster of infection as the a ⁄ dm ⁄ ⁄ virus. to assess growth kinetics of viruses, confluent mdck or mdck siat cell monolayers were infected with viruses at a multiplicity of infection (moi) of approximately ae pfu ⁄ cell (single-step) or ae pfu ⁄ cell (multi-step). supernatants were collected every h or h p.i. for time points. a modified fluorometric assay using the fluorogenic substrate ¢-( -methylumbelliferyl)a-d-n-acetylneuraminic acid (munana) was used to determine viral na activity. the drug concentration required to inhibit % of the na enzymatic activity (ic ) was determined by plotting the percent inhibition of na activity as a function of compound concentration calculated in the graphpad prism (la jolla, ca) software from the inhibitor-response curve. na enzyme kinetics were determined by measuring na activity every seconds for minutes under the same conditions as above, when all viruses were standardized to an equivalent dose of ae pfu ⁄ ml. the k m and v max were calculated by fitting the data to the appropriate michaelis-menten equations using nonlinear regression in the graphpad prism software. young adult ferrets ( - months of age) were obtained from the ferret breeding program at st. jude children's research hospital. all ferrets were seronegative for influenza a h n and h n viruses and for influenza b viruses. for transmission studies, the donor ferrets were lightly anesthetized with isoflurane and inoculated intranasally with tcid virus in ae ml sterile pbs . after the donor ferrets were confirmed to shed virus on day p.i., each donor was then housed in the same cage with two naïve direct-contact ferrets. two additional recipient ferrets were placed in an adjacent cage isolated from the donor's cage by a two layers of wire mesh (approximately cm apart) that prevented physical contact but allowed the passage of respiratory droplets. ferret weight and temperature were recorded daily for days. nasal washes were collected from donors and recipients on day , , , , , , , and p.i. by flushing both nostrils with ae ml pbs, and tcid titers were determined in mdck cells. serum samples were collected weeks after virus inoculation, and were tested for seroconvention by hi assay. full genome sequencing revealed that the pair of h n ⁄ viruses differed only at na amino acid position , where the pandemic a ⁄ dm ⁄ ⁄ virus had an h y amino acid mutation caused by a single t-to-c nucleotide substitution at codon . the wild-type a ⁄ dm ⁄ ⁄ was susceptible to oseltamivir carboxylate (mean ic : ae nm), but the a ⁄ dm ⁄ ⁄ carrying the h y na mutation had ic values approximately - times of the wild-type viruses (mean ic : nm). the ic of zanamivir was comparable for both viruses and were uniformly low (mean ic £ ae nm). the h y na mutation confers resistance to oseltamivir carboxylate but did not alter susceptibility to zanamivir. to understand the impact of the h y mutation on the na enzymatic properties, na enzyme kinetics was determined. the na of the oseltamivir-resistant virus had a slightly higher k m (mean = lm) and lower v max (mean = u ⁄ sec) than na of the sensitive virus (k m , mean = lm; vmax, mean = u ⁄ sec). the results suggested that the h y na mutation reduced na affinity for substrate and na catalytic activity, although the function of na was not severely impaired. to further evaluate the impact of the h y na mutation on virus growth in vitro, single-and multi-cycle growth studies of both viruses were performed in mdck and mdck-siat cells. in the both single-and multiple-cycle growth curves, the two viruses reached comparable levels eventually, but the initial growth of the resistant virus was significantly delayed by at least - logs in comparison to that of wild-type virus (p < ae ). the donor ferrets inoculated with wild-type a ⁄ dm ⁄ ⁄ or oseltamivir-resistant virus shed virus productively until day or day p.i., with a peak virus titer comparable to that of a ⁄ dm ⁄ ⁄ virus (table ). in a ⁄ dm ⁄ ⁄ virus group, two of direct-contact ferrets the weight loss in ferrets is the maximum percentage loss compared with the initial weight. virus shedding is indicated as number of virus-shedding animals ⁄ total number; mean peak virus titer (log tcid ⁄ ml) in nasal wash samples is indicated in parentheses. serum hemagglutination inhibition (hi) titer to homologous virus in ferret serum was determined on day p.i. duan et al. and of respiratory droplet-contact ferrets were infected through virus transmission, as indicated by the virus titers and inflammatory cell counts in their nasal washes and also by sero-conversion. under identical conditions, in a ⁄ dm ⁄ ⁄ group, only of direct-contact ferrets were infected through virus transmission, but neither respiratory droplet-contact ferrets was infected, as confirmed by the absence of sero-conversion (table ) . virus shedding in the direct-contact ferrets was lower and peaked after a longer interval in this group than in the oseltamivir-sensitive a ⁄ dm ⁄ ⁄ group (table ) , but the resistant viruses appeared to cause a similar disease course in ferrets without apparent attenuation of clinical signs. these results showed that an oseltamivir-resistant h y mutant of pandemic h n virus, a ⁄ dm ⁄ ⁄ virus could be only transmitted efficiently by direct contact. to compare the relative fitness, growth capability, and transmissibility of the sensitive and resistant h n ⁄ viruses within host, a donor ferret was co-inoculated with a : ratio of the sensitive and resistant viruses, and another two naive ferrets were housed with the donor to test direct contact. during co-infection, the pattern of virus shedding and the clinical signs were similar to those in ferrets inoculated with either a ⁄ dm ⁄ ⁄ or a ⁄ dm ⁄ ⁄ virus (table ). in the inoculated donor ferret, the virus population in the nasal washes remained mixed but wild-type viruses outgrew the resistant virus progressively ( figure ). two of direct-contact ferrets were infected through virus transmission, but only wild-type virus was detected in both direct-contact ferrets ( figure ). in summary, oseltamivir-sensitive a ⁄ dm ⁄ ⁄ virus possessed better growth capability in the upper respiratory tract than did resistant a ⁄ dm ⁄ ⁄ virus, and thus had an advantage in directcontact transmission. our study determined the comparative transmissibility of two naturally circulating oseltamivir-sensitive and -resistant pandemic h n ⁄ viruses; we demonstrated inefficient respiratory-droplet transmission of an oseltamivir-resistant h y mutant of pandemic h n virus among ferrets, although it retained efficient direct-contact transmission. we suggest that the lower fitness of resistant virus within the host along with its reduced na function and delayed growth in vitro may in part explain its less efficient transmission. notably, the h y mutant of h n ⁄ used in this study was the first oseltamivir-resistant h n ⁄ isolate from a patient on oseltamivir prophylaxis to be characterized for transmissibility. our observation in the animal model is consistent with the epidemiological data collected from humans, which showed no evidence of predominant or continued circulation of oseltamivir-resistant viruses. as this study was undertaken, additional h y mutants of h n ⁄ viruses have emerged in the absence of oseltamivir use. , the emergence of these viruses should raise concerns as to whether resistant h n ⁄ viruses will acquire greater fitness and spread worldwide as the naturally resistant h n viruses did during the - season. two independent studies have evaluated the pathogenecity and transmission of other oseltamivir-resistant pandemic h n ⁄ clinical isolates in the animal models. , one of the studies, which also used an oseltamivir-resistant virus isolated from a patient under oseltamivir prophylaxis, observed similar results as ours: although the respiratory-droplet route of transmission was not investigated, it was shown that the resistant isolate was transmitted though direct-contact route and was as virulent as wild-type virus in ferrets. in another study, two oseltamivir-resistant isolates were transmitted through the respiratory-droplet route in ferrets, and the dynamics of transmission were different between the two isolates. apparently, these two oseltamivir-resistant isolates were still unequal in their transmissibility and were disparate from the resistant isolate in our study. the isolation history of the two resistant isolates was unclear in this study, and this would be an important factor to understand the fitness of drug-resistant viruses. further studies with more clinical isolates of diverse isolation background are warranted to identify how these novel h y mutants of pandemic h n ⁄ virus have changed to retain their full transmissibility. taken together, all these related studies underline the necessity of continuous monitoring of drug resistance and characterization of potential evolving viral proteins. this study was supported by contract hhsn c from the national institute of allergy and infectious diseases, national institutes of pigs have been considered as hypothetical ''mixing vessels'' facilitating the genesis of pandemic influenza viruses. , the pandemic h n ⁄ virus (ph n ⁄ ) contained a very unique genetic combination and was thought to be of swine origin, as each of its eight gene segments had been found to be circulating in pig populations for more than a decade. however, such a gene constellation had not been found previously in pig herds all around the world. only after its initial emergence in humans has this virus been repeatedly detected in pigs, and found to further reassort with other swine influenza virus. [ ] [ ] [ ] a primary question remaining to be answered is whether the ph n ⁄ -like and their genetically related viruses could become established in pig populations, thereby posing novel threats to public health. despite the fact that ph n ⁄ first appeared in mexico and the united states, and six of its eight gene segments were derived from the established north american triple reassortant swine influenza virus (trig), its neuraminidase (na) and matrix protein (m) genes belonged to the eurasian avian-like swine lineage (ea), which had never been detected in north america previously. , likewise, the trig-like viruses were never reported in europe. in contrast, both lineages of virus were frequently detected in asia, and reassortants between them have also been documented in recent years. , this has given rise to a complicated ecological situation, i.e. the simultaneous prevalence of multiple genotypes of h n and h n viruses in pigs. , among them, two representative reassortants showed the most similar genotypic characterization to the ph n ⁄ virus, the sw ⁄ hk ⁄ ⁄ (h n ) and sw ⁄ hk ⁄ ⁄ (h n ), which respectively harbor seven and six gene segments closely related to the pandemic strains. , to understand their in vivo characteristics and zoonotic potential, these two viruses, together with a human prototype strain and a swine ph n ⁄ -like isolate, were chosen for a study of their pathogenicity and transmissibility in domestic pigs, ferrets, and mice. the prototype ph n ⁄ virus, a ⁄ california ⁄ ⁄ (ca ), was provided by the world health organization collaborating centers for reference and research on influenza (atlanta, ga, usa). three ph n ⁄ -related swine influenza viruses were isolated through our surveillance program in south china as previously described. , the a ⁄ swine ⁄ guangdong ⁄ ⁄ (h n , gd ) virus was a ph n ⁄ -like swine isolate. a ⁄ swine ⁄ hong kong ⁄ ⁄ (h n , hk ), the closest pandemic ancestor known to date, possesses an m gene derived from the ea lineage, with the other gene segments from trig viruses. a ⁄ swine ⁄ hong kong ⁄ ⁄ (h n , hk ), a recent pandemic reassortant progeny, had a ph n ⁄ like na gene (also belonging to the ea lineage), an ea-like hemagglutinin (ha) gene, and six trig-like internal genes. all viruses were propagated in madin-darby canine kidney (mdck) cells for three passages, and their titers were determined by plaque assays. all experiments with live viruses were conducted in biosafety level (bsl- ) containment laboratories. pigs ( - week old, n = - ) and ferrets ( month old, male, n = ) were intranasally infected with pfu of each virus, and mice ( ) ( ) week old, female balb ⁄ c, n = ) with a dose of pfu. naïve uninfected pigs (n = ) were co-housed in the same cage with the inoculated ones from each group. body weights and clinical signs were recorded daily. virus replication was determined by titration of the virus in nasal and rectal swabs (pigs), nasal washes (ferrets), as well as from lungs and other organs (pigs and mice). seroconversion was tested by hemagglutination inhibition (hi) assays. histopathological and immunohistochemical analysis were performed as previously described. statistical analysis was performed by mean analysis with pasw statistics (spss inc., chicago, il, usa). the probability of a significant difference was computed using anova (analysis of variance). results were considered significant at p < ae . the pathogenicity of the four viruses tested differed significantly in inoculated mice. animals infected with pfu of hk experienced the most severe body weight loss ( ae ± ae %) but started to recover after days post-infection (dpi). hk caused similar peak body weight loss ( ae ± ae % on dpi) in mice as did ca ( ae ± ae %, on dpi), but the onset of clinical signs and weight loss (on dpi) was day later than those caused by the other three viruses. the gd -infected group suffered the least body weight loss ( ae ± ae %, dpi) and was the earliest to recover. although all four viruses were detected in the lungs with comparable virus titers on dpi (p > ae ), mice inoculated with gd consistently showed the lowest lung index (lung weight ⁄ body weight, %) on , , and dpi (p < ae ), suggesting the slightest injury and consolidation of the lungs. in concordance with the body weight change, the lung index from the hk group was higher than that from any other groups on and dpi, indicating the marked virulence of hk in mice. notably, virus titer of hk in the nasal turbinate was lower than the other groups both on and dpi (p < ae ), but virus replication in the lower respiratory tract was either higher (in the trachea) or similar (in the lungs). observations of the body weight changes caused by infection of ph n ⁄ or its genetically related swine viruses in ferrets have come to a similar conclusion as that for the mouse experiment. after nasal inoculation with pfu of each virus, all groups of ferrets experienced transient body weight loss for - days, except for those infected with gd , which showed no significant weight loss (p > ae ). although ferrets from the ca -infected group reached their peak weight loss ( ae ± ae %, dpi) one day earlier than those from the hk and hk groups, they began to regain body weight quickly thereafter. hk -infected ferrets also recovered rapidly and their body weights reached the same level as those of the gd -infected group at dpi. comparatively, ferrets inoculated with hk had the most retarded body weight recovery, which did not get back to the baseline level until dpi. hk was only detectable in the nasal wash on dpi, whereas the duration of virus shedding for gd , hk , and ca was - days. by combining the data obtained from the virus titration in the mouse turbinate and ferret nasal washes, a possible conclusion can be made that hk may have lower transmissibility than the other three viruses. after inoculation or exposure by direct contact (physical contact) with the ph n ⁄ virus and its close relatives, most pigs experience no or mild symptoms, such as slight loss of appetite and inactivity. body weight loss was only recorded in pigs inoculated with hk during the second week post-inoculation, but not in their contact pigs or in the other groups. diarrhea was observed intermittently in each of the inoculated or contact groups throughout the experiment, and viruses could be recovered in the rectal swabs, saliva, drinking water, and environmental swabs (inner cage walls accessible to the pigs) at various time points. however, virus titers in the positive rectal swabs were just slightly above the detection limit, while those from the environment sometimes could be higher. whether these viruses can replicate in the digestive tract or were just carried-over by contaminated foods and water requires further investigation. although virus could be detected in the nasal swabs of all infected or contact animals, the lowest peak titer was from pigs inoculated or in contact with hk ( ae - ae log tcid ⁄ ml lower than the other groups), suggesting unfavorable replication in the nasal cavity for this virus. postmortem examination on and dpi revealed that pigs infected with hk had the most extensive gross lesions in the lungs, and histochemical staining of viral nucleoprotein (np) in lung tissues on dpi also suggested the best replication for hk in the lower respiratory tract. on days post-contact (dpc), all pigs exposed to the inoculated animals developed sero-conversions (hi = - ) except for one from the gd contact group. however, on dpc, its hi titer reached , indicating slower seroconversion. this study revealed that both the pandemic h n and its genetically related swine viruses could readily infect mice, ferrets, and pigs causing mild to moderate clinical symptoms. they could also transmit efficiently between pigs. when compared with the pandemic stains and its reassortant progeny (hk ), the hk (h n ) virus containing the ea-like m gene in the genetic context of the trig virus showed consistently higher virulence in all three mammalian models tested, but it is still unknown what might happen if such a virus further reassorts to obtain the pandemic-like or ea-like na gene. however, our findings suggest that pigs could likely maintain the prevalence of different genotypes of pandemic-related influenza viruses, and highlight the zoonotic potential of multiple strains of swine influenza virus. pandemic influenza viruses emerge from the animal reservoirs. among the three pandemics that occurred in the last century, we learned that the h n and the h n pandemic viruses emerged by reassortment between circulating human virus and avian-origin influenza virus(es). studies on the emergence of the catastrophic spanish h n virus suggest that the virus may have obtained all of its eight gene segments from the avian reservoir, , or alternatively is a reassortant between mammalian and a previously circulating human influenza virus. over years since the last pandemic, the first pandemic in the st century arose in and was caused by a swine-origin influenza virus containing a unique gene combination, with gene segments derived from the circulating north america ''triple reassortant'' (pb , pb , pa, ha, np, and ns) and the ''eurasian'' (na and m) swine influenza viruses. , analysis of the pandemic h n viruses failed to identify known molecular markers predictive of adaptation to humans. the ''triple reassortant'' swine influenza viruses emerged in late s in north america is a reassortant between classical swine (descendent of the virus after adaptation in swine population), avian, and human influenza viruses. the eurasian influenza virus was originally an avian influenza virus that was introduced into the european swine population in the late s. , while incidents of zoonotic infection with triple reassortant or eurasian influenza in humans have been reported, , sustained human-to-human transmission has never been established. these results suggest that the unique gene combination seen with the pandemic h n viruses may confer its transmissibility among humans. we have carried out systematic prospective surveillance of swine influenza in southern china over that last years through samples routinely collected at an abattoir in hong kong. during this time, the surveillance results suggest co-circulation of classical swine h n , triple reassortant h n , eurasian swine h n , and a range of reassortants between these three virus lineages. , ferrets have been reported as a suitable model for the study of influenza transmission as they are naturally susceptible to influenza infection, exhibit similar clinical signs (including sneezing), and possess receptor distribution in the airway similar to that of humans. [ ] [ ] [ ] to identify molecular determinants that enable sustained human-to-human transmission, we compared the pandemic virus with genetically related swine influenza viruses obtained from this surveillance program for their ability to transmit from ferret to ferret by direct contact or aerosol transmission. viruses human h n influenza virus [a ⁄ wuhan ⁄ ⁄ (wuhan )] and pandemic h n influenza viruses [a ⁄ california ⁄ ⁄ (ca )] were included for the study. swine influenza viruses that are genetically related with the pandemic h n virus were selected from our surveillance system, including classical swine-like influenza virus a ⁄ sw ⁄ hk ⁄ ⁄ (h n ) (swhk ), triple reassortant-like a ⁄ sw ⁄ arkansas ⁄ ⁄ (h n ) (swar ), and one reassortant between triple reassortant and eurasia swine influenza viruses [a ⁄ sw ⁄ hk ⁄ ⁄ (h n ) (swhk )]. swhk contains seven gene segments (pb ,pb ,pa,ha,np,m,ns) closely related to the pandemic h n viruses. transmissibility was tested in -to -month-old male ferrets obtained from triple f farm (sayre, pa); all ferrets were tested to have hi titer £ against human seasonal influenza h n (a ⁄ tennessee ⁄ ⁄ ), h n (a ⁄ brisbane ⁄ ⁄ ), and influenza b (b ⁄ florida ⁄ ⁄ ) prior the experiments. in each virus group, three ferrets were inoculated with tcid of the virus. at day postinoculation (dpi), we introduced one naïve direct contact ferret to share the cage with inoculated ferret, and one naïve aerosol contact ferret into the adjacent compartment of the cage separated by a double-layered perforated divider. nasal washes were collected every other day and tested for influenza virus antigen and to determine viral titers (tcid ). weight changes, temperature, and clinical signs were monitored daily. transmission is defined by detection of virus from nasal washes and ⁄ or by seroconversion (> fold rise in the post-sera collected after - days post contact). experiments were performed in the p + laboratory at st. jude children's research hospital. all studies were conducted under applicable laws and guidelines and after approval from the st. jude children's research hospital animal care and use committee. at tcid inoculation dose, all viruses replicated efficiently in the ferret upper respiratory tract with peak titers detected from inoculated ferrets at dpi. lower peak titers were detected from swhk and swhk inoculated ferrets, however, the differences were not statistically significant (table ) . tissues collected from inoculated ferrets at dpi showed that pandemic h n and swine influenza viruses replicated both in the upper and lower respiratory tract of the ferrets, while the replication of human seasonal influenza wuhan was restricted in the upper respiratory tract. direct contact transmission from inoculated donor ferrets to their cage-mates was observed for all viruses studied, albeit at different efficiency. human seasonal influenza (wuhan ) and pandemic h n viruses (ca ) transmitted most efficiently via direct contact route as the virus can be detected on dpi from direct contact ferrets, and the peak titers were detected on dpi from direct contacts. moderate direct contact transmission efficiency was detected from swar and swhk viruses as the virus can be detected from direct contact ferrets at dpi, with peak titers detected at dpi or dpi. classical swine-like swhk showed least efficient contact transmission as virus could be detected from all direct contacts only at dpi, and the peak titer detected on dpi. aerosol transmission was detected in groups of human seasonal influenza virus wuhan ( ⁄ ), pandemic h n influenza virus ca ( ⁄ ), as well as swine precursor virus swhk ( ⁄ ). transmission of wuhan and ca to aerosol contacts was detected at dpi or dpi, while transmission of swhk was detected later at dpi, suggesting that the swhk virus possessed aerosol transmission potential, but may require further adaptation to acquire efficient aerosol transmissibility. in addition to viral detection from nasal washes, we also detected viruses from the rectal swabs of ferrets inoculated or infected with pandemic h n viruses (ca ) or classical swine-like virus (swhk ), which share the common origin for the ha, np, and ns gene segments. while many of the swine influenza viruses studied were able to transmit via the direct contact route, swhk , which shares a common genetic derivation for seven genes with h n pdm, possessed capacity for aerosol transmission, albeit of moderate efficiency. swhk differed from swine triple reassortant viruses in the origins of its m gene. it is possible that the m gene derived from eurasian avian- like swine viruses also contributes to the transmissibility of h n pdm influenza viruses. outbreaks of highly pathogenic avian influenza (hpai) of the h n subtype are of extreme concern to global health organisations as human infection can result in severe acute respiratory distress syndrome, multi-organ failure, and coma. hpai viruses of either h or h subtypes contain a characteristic multi-basic cleavage site in the hemagglutinin glycoprotein as well as other virulence factors that expand the viral tropism beyond the respiratory tract of poultry. there is also emerging evidence of viral rna or antigen in multiple organs and the cns of humans infected with h n that is consistent with systemic infection , and raises the question of the role of the cleavage site in dissemination of the virus in this species. the majority of human cases with h n have involved contact with sick or contaminated poultry and exposure to respiratory secretions of birds that can be inhaled and ingested. particular risk factors for h n infection include bathing with sick birds, improper hand washing after handling sick birds, or slaughtering poultry. viral inoculum may also be consumed directly during a variety of religious and cultural practices, such as drinking contaminated duck blood and kissing of merit release birds. h n infection is lethal in % of human cases, and the pathogenetic mechanisms leading to this level of mortality are unclear. to date cases have been reported to the who, although many more people have potentially been exposed to h n through contact with infected bird populations. some studies have suggested that genetic factors may predispose an individual to severe h n disease, but little is known about the influence of route of virus exposure on morbidity and mortality. in ferrets, an animal model frequently used to study influenza because of its similar disease profile to humans, swayne et al. observed that exposure to a virulent h n strain a ⁄ vietnam ⁄ ⁄ by intra-gastric gavage did not lead to disease and did not generate an antibody response, whereas ferrets that experienced a more natural exposure by being fed contaminated meat developed severe signs of infection. in this study we further assessed the disease profile of h n following a natural oral exposure in the ferret model. to achieve this inoculation condition, conscious ferrets voluntarily consumed a liquid inoculum of h n hpai strain a ⁄ vietnam ⁄ ⁄ . as a comparison anesthetised ferrets were exposed by intranasal administration of inoculum and the ensuing disease profiles of the different routes of infection were compared. eight ferrets per group were inoculated with egg infectious dose of a ⁄ vietnam ⁄ ⁄ in a volume of ll that was given to the nares of anaesthetized ferrets to establish a total respiratory tract (trt) infection or voluntarily consumed by conscious ferrets to establish an oral infection. ferrets were culled at a predetermined humane endpoint that was defined as either a > % weight loss and ⁄ or evidence of neurological signs, discussed in ; animals that did not reach the humane endpoint were euthanased on day after challenge. nasal washes and oral swabs collected during the course of infection and organ homogenates were assessed for the presence of replicating virus by growth in embryonated-chicken eggs; viral loads were determined by titration on vero cells and expressed as tcid . tissue samples were fixed with formalin and embedded in paraffin for sectioning. viral lesions were identified by hematoxylin and eosin staining of the sections and the presence of viral antigen in the sections was determined by staining with antibody to influenza a nucleoprotein. pre-and post-exposure antibody responses were assessed by hemagglutination-inhibition assays using irradiated a ⁄ vietnam ⁄ ⁄ virus. the majority ( %) of ferrets infected by the trt route rapidly became inactive, developed severe disease, and were euthanased at the humane endpoint following infection ( figure ). ferrets infected orally had an improved chance of survival, as only % of animals developed severe disease (figure ), and the surviving ferrets were more active than ferrets infected by the trt throughout the stage of acute infection (data not shown). the improved survival rate and wellbeing of ferrets infected orally was not a result of poor infection rates by this route, as of surviving ferrets developed h specific antibodies by day post-infection, and they did not have pre-existing antibodies to h n (data not shown). the two ferrets that developed severe disease after oral infection had similar disease profiles to ferrets infected by the trt route; they both progressed to a > % weight loss and exhibited neurological signs (data not shown). viral loads in organs of these two ferrets confirmed dissemination to extra-pulmonary sites (table ) : replicating virus was detected at high titres in the spleen, pancreas, liver, and brain. similar findings were recorded in ferrets with trt infections in this study (not shown) and elsewhere. viral load in nasal washes and oral swabs taken at days , , and post-infection by the oral route did not correlate with the development of severe disease, and virus was isolated only sporadically and at low titre from the nasopharynx of these animals (data not shown). interestingly, the two ferrets with severe disease after being infected orally had no detectable viral antigen or lesions in the olfactory epithelium and bulb (table ) , whereas of ferrets culled after infection by the trt route had lesions and viral antigen in both the olfactory epithelium and bulb (data not shown). trt oral figure . percentage of ferrets that survived infection after oral or trt infection. ferrets were exposed to a ⁄ vietnam ⁄ ⁄ by the total respiratory tract (trt) route (circles) or the oral route (triangles). the percentages of ferrets that survived infection are indicated at each day following challenge. ferrets exposed orally were more likely to survive h n infection than ferrets exposed to the same dose of virus by the trt. the improved survival rates that were observed after an oral infection could be a consequence of low-level viral replication in the upper respiratory tract in combination with delivery of a substantial portion of the inoculum directly to the stomach where it may have been inactivated by the harsh environment of the gastro-intestinal tract. most ferrets infected orally developed an h -specific antibody response which differs from the studies of swayne et al. in which ferrets gavaged with a liquid inoculum neither developed signs of disease nor an antibody response. however swayne et al. administered virus to anaesthetized ferrets by gastric gavage that would have bypassed the oropharynx. in our study virus was administered to the oral cavity directly and would have had access to the oropharynx. low level of replication at this site may have been sufficient to trigger an antibody response. the two ferrets that developed severe disease following oral infection had a similar profile of viral dissemination as ferrets infected by the trt route. differences were seen in the olfactory epithelium and bulb as lesions, and viral antigen did not occur in these sites following oral infection, although cerebral involvement was identified. one route of dissemination of h n into the cns may be by transport within nerves through the olfactory bulb into the cerebrum. due to the absence of lesions and antigen in these sites following oral infection the spread of virus into the brain in these two animals may be occurring through involvement of other cranial nerves or the hematagenous routes. nasal turbinates ) ae ) ) ) ) pharyngeal lymph node interactions of oseltamivir-sensitive and -resistant highly pathogenic h n influenza viruses in a ferret model < ae b ) + + + + olfactory epithelium nd a nd ) ) ) ) olfactory bulb nd nd ) ) ) ) trachea < ae ) ) nd ) nd lung ) < ae + + ) + spleen ae ) + + ) + small intestine ) ) ) ) ) + pancreas ) ae + ) + + the pandemic potential of highly pathogenic h n influenza viruses remains a serious public health concern. while the neuraminidase (na) inhibitors are currently our first treatment option, the possibility of the emergence of virulent and transmissible drug-resistant h n variants has important implications. clinically derived drug-resistant viruses have carried mutations that are na subtype-specific and differ with the na inhibitor used. the most commonly observed mutations are h y and n s in the influenza a n na subtype (n numbering here and throughout the text); e a ⁄ g ⁄ d ⁄ v and r k in the n na subtype; and r k and d n in influenza b viruses. h n influenza viruses isolated from untreated patients are susceptible to the na inhibitors oseltamivir and zanamivir, although oseltamivir-resistant variants with the h y na mutation have been reported in five patients after , or before drug treatment; and the isolation of two oseltamivir-resistant h n viruses with n s na mutation from an egyptian girl and her uncle after oseltamivir treatment were described. the impact of drug resistance would depend on the fitness (i.e., infectivity in vitro, virulence, and transmissibility in vivo) of the drug-resistant virus. if the resistance mutation only modestly reduces the virus' biological fitness and does not impair its replication efficiency and transmissibility, the effectiveness of antiviral treatment can be significantly impaired. the recombinant wild-type h n influenza a ⁄ vietnam ⁄ ⁄ (vn-wt), a ⁄ turkey ⁄ ⁄ (tk-wt) viruses, and oseltamivir-resistant viruses with h y na mutation (vn-h y and tk-h y) were generated by using the -plasmid reverse genetics system. susceptibility to na inhibitors was tested by using a fluorescence-based na enzyme inhibition assay with munana substrate at a final concentration of lm. viral fitness was studied in vivo in a ferret model: groups of three ferrets were lightly anesthetized with isoflurane and inoculated intranasally with vn-wt, vn-h y, or mixtures of the two at a different ratios at a dose of pfu in ae ml pbs; they were inoculated with tk-wt, tk-h y, or mixtures of the two at a different ratios at a dose of pfu in ae ml pbs. respiratory signs (labored breezing, sneezing, wheezing, and nasal discharge), neurologic signs (hind-limb paresis, ataxia, torticollis, and tremor), relative inactivity index, weight, and body temperature were recorded daily. virus replication in the upper respiratory tract (urt) was determined on days , , and p.i. the competitive fitness (i.e., co-inoculation of ferrets with different ratios of oseltamivir-resistant and -sensitive h n viruses) was evaluated by the proportion of clones in day- nasal washes that contained the h y na mutation. na mutations were analyzed by sequence analysis of individual clones ($ clones ⁄ sample) created by ligation of purified pcr products extracted from nasal wash samples into a topo vector. introduction of the h y na mutation conferred high resistance to oseltamivir carboxylate in vitro; the mean ic of the vn-h y and tk-h y viruses was and times, respectively, that of the corresponding wildtype viruses. the oseltamivir ic of the tk-wt virus was $ times that of the vn-wt virus. all four recombinant h n viruses were susceptible to zanamivir. introduction of the h y na mutation reduced $ % and % of the na activity of vn-h y and tk-h y viruses, respectively, as compared to the wild-type virus activity (p < ae ; two-tailed t-test). all ferrets inoculated with either vn-wt or vn-h y virus exhibited acute disease signs (high fever, marked weight loss, anorexia, extreme lethargy), rapid progression, and death by day - p.i., and no differences in clinical signs and replication in the urt of ferrets were observed between wild-type and oseltamivir-resistant viruses ( table ) . both of the tk viruses caused milder illness than did the vn viruses, despite a much higher dose ( pfu ⁄ ferret), and the tk-h y virus caused less weight loss and fever than the tk-wt virus (table ) . however, competitive fitness experiments revealed a disparity in the growth capacity of vn-h y and tk-h y viruses as compared to their wild-type counterparts: clonal analysis established the uncompromised fitness of vn-h y virus and the impaired fitness of tk-h y virus (table ) . although, the trend towards an increase ⁄decrease in the frequency of the h y na mutation relative to the wild-type was statistically significant (p > ae ) for two studied groups only. mutations within the na catalytic (r k) and framework (e a ⁄ k, i l, h l, n s) sites or near the na active enzyme site (v i, i t ⁄ v, q h, k n, a t) emerged spontaneously (without drug pressure) in both pairs of viruses (results not shown). the na substitutions i v and e a could exert compensatory effect on the fitness of vn-h y and tk-h y viruses. the lethality and continuing circulation of h n influenza viruses warrants an urgent search for an optimal therapy. our results showed that the h y na mutation affects the fitness of two h n influenza viruses differently: the oseltamivir-resistant a ⁄ vietnam ⁄ ⁄ -like virus outgrew its wild-type counterpart, while the oseltamivir-resistant a ⁄ turkey ⁄ ⁄ -like virus showed less fitness than its wild-type counterpart. we used a novel approach to compare the fitness of oseltamivir-sensitive and -resistant influenza viruses that included analysis of virus-virus interactions within the host (competitive fitness) during co-infection with these viruses. although mixed populations were present in the urt of ferrets on day p.i., the fitness of vn-h y virus was uncompromised as compared to that of its drug-sensitive counterpart, while that of tk-h y virus was impaired. a minor population of na inhibitor-resistant variants may gain a replication advantage under suboptimal therapy in two ways: (i) preexisting variants less sensitive to the drug are selected from the quasispecies population, leading to an increase of the number of resistant clones, and (ii) outgrowing variants may acquire additional compensatory mutations that enhance their fitness. it is possible that use of antiviral drugs (particularly at suboptimal concentration) against mixtures of oseltamivir-resistant and sensitive viruses will promote the spread of drug-resistant variants * ferrets in all groups inoculated with a ⁄ vietnam ⁄ ⁄ virus died by day - p.i. and were observed once daily for days. ** results obtained from one ferret. *** by inhibiting drug-sensitive variants that are competing with them for the dominance in the infected host. the influence of multiple genes on the fitness of viruses carrying h na mutation cannot be excluded. in our study we focused on additional na mutations, and sequence analysis of individual na clones was done to identify potential host-dependent and compensatory na mutations. we found that the na mutations e a and n s, which confer cross-resistance to oseltamivir and zanamivir, , can emerge spontaneously in clade . h n influenza virus in ferrets. further, we observed that mutations at na catalytic (r k) and framework (i l and n s) sites and in close proximity to the na enzyme active site (v i, i t ⁄ v, q h, k n, a t) emerged without drug pressure in both pairs of h n viruses. compensatory mutations in na or other genes may mitigate any fitness cost imposed by resistance mutations. our study identified six potential compensatory na changes (d v, f s, i v, e a, h l, and f s) that may affect the fitness of viruses with the h y na mutation. we suggest that na mutations at residues i v and e a are of importance. interestingly, we observed differences in predominance of i v and e a na mutations in different genetic backgrounds: i v mutation was identified in a ⁄ vietnam ⁄ ⁄ (h n )-like and e a in a ⁄ turkey ⁄ ⁄ (h n )-like genetic background. moreover, i v na mutation was identified only when ferrets were inoculated with the mixtures of vn-wt and vn-h y viruses, but not in ferrets inoculated with vn-h y virus. none of the potential compensatory na mutations was identified in the original inoculum used to infect ferrets. the h y na mutation causes a large shift in the position of the side chain of the neighboring e residue, which must form a salt bridge with r to accommodate the large hydrophobic pentyl ether group of oseltamivir. residue i is located near the na active site, and although it does not alter polarity, it results in a shorter side-chain and, thus, may indirectly affect the residues in the na active site. we suggest that antigenic and genetic diversity, virulence, the degree of na functional loss, and differences in host immune response and genetic background can contribute to the observed differences in the fitness of h n influenza viruses. therefore, the risk of emergence of drugresistant influenza viruses with uncompromised fitness should be monitored closely and considered in pandemic planning. this study was supported by contract hhsn c from the national institute of allergy and infectious diseases, national institutes of health, and by the american lebanese syrian associated charities (alsac). the data presented in the manuscript have been published at: govorkova ea, ilyushina na, marathe bm, mcclaren laninamivir (r- ) is a strong na inhibitor against various influenza viruses, including oseltamivir-resistant viruses. [ ] [ ] [ ] [ ] [ ] [ ] we discovered a single intranasal administration of laninamivir octanoate (cs- ), a prodrug of laninamivir, showed a superior anti-virus efficacy in mouse and ferret infection models compared to repeated administra-tion of oseltamivir and zanamivir. [ ] [ ] [ ] this suggested that cs- works as a novel long-acting na inhibitor of influenza virus in vivo. a single inhalation of cs- proved noninferiority in adult patients and significantly superior in child patients, compared to an approved dosage regimen of oseltamivir for treatment. cs- has been commercially available as an inhaled drug, inavir Ò , for the treatment of influenza in japan since october . the long-acting characteristics of cs- are explained by several reasons. first, cs- was quickly hydrolyzed to an active metabolite, laninamivir, after an intranasal administration to mice, and was retained for a long time as laninamivir in target organs, such as lung and trachea. however, with an intranasal administration of laninamivir, it disappeared quickly and did not demonstrate its longlasting characteristics. another reason is a strong binding of laninamivir to nas of seasonal influenza viruses compared to other three na inhibitors, oseltamivir carboxylate, zanamivir, and peramivir. in the following, the tight-binding ability of laninamivir to pandemic (h n ) na, as well as to the seasonal influenza virus nas, was demonstrated. in addition, we present a hypothesis of the mechanism of the long-lasting property of cs- in mouse based on a localization of an enzyme that hydrolyzes cs- to laninamivir. the influenza viruses, pandemic(h n ) (inf ), a ⁄ new caledonia ⁄ ⁄ (h n ), a ⁄ panama ⁄ ⁄ (h n ), and b ⁄ mie ⁄ ⁄ were treated with excess na inhibitors, such as oseltamivir carboxylate, zanamivir, peramivir, and laninamivir, and then unbound na inhibitors were removed from the mixtures with a bio-spin column bio-gel p- (bio-rad laboratories, hercules, ca, usa). the na substrate, -methylumbelliferyl-n-acetyl-a-d-neuraminic acid (nacalai tesque, japan) was added to the virus-na inhibitor complex, and the na activities were followed for hours at room temperature by measuring the fluorescence at an excitation wavelength of nm and an emission wavelength of nm. the enzyme which hydrolyzes cs- to laninamivir was partially purified from rat lungs using ion exchange column chromatography, and almost all bands separated by an sdspolyacrylamide gel electrophoresis were identified by mass spectrometry. the gene expression profiles of the enzyme were investigated by the bioexpress database (genelogic inc., gaithersburg, md, usa). the enzyme gene cloned from mouse lung mrna was transiently expressed in cos cells. antiserum to the esterase was prepared by immunizing rabbits, and immunostaining was done using histomouse-tm-max kit (invitorgen corp., carlsbad, ca, usa) according to the manufacturer's manual. binding stability of na inhibitors to the four viruses are shown in figure the enzyme that hydrolyzes cs- to laninamivir in rat lungs was identified as carboxyesterase. this esterase was shown to be expressed in epithelial cells of rat lung by in situ hybridization. the mouse homolog of the rat esterase was carboxylesterase (ces ). the mrna of the mouse ces was shown to be highly expressed in lung and liver by the gene expression profile, and ces was also found to contain signal sequences for retention in endoplasmic reticulum (er) and golgi at the c-terminus. the cloned ces gene and the ces gene lacking the signal sequence were exogenously expressed in the cos cells. the cs- -hydrolyzing activity associated with the cos cells expressing ces was recovered from the culture sup of the cos cells expressing ces lacking the retention signal sequence. localization of ces was immunohistologically confirmed inside the airway epithelium cells of mice, which are the target cells for influenza virus infection. the long acting property of intranasal administration of cs- in mice can be explained both by the long retention of laninamivir in the respiratory tract and by the stable binding of laninamivir to influenza virus na. again, stable binding of laninamivir to na of pandemic (h n ) virus was also observed similar to that of seasonal h n virus. the following are speculated as the mechanisms for the long-lasting characteristics of cs- in mice. we explain the mechanism by clarifying a cs- hydrolyzing enzyme and its localization inside cells. the hypothesis of the mechanism is presented in figure . briefly, hydrophilic laninamivir may not enter easily inside cells, whereas hydrophobic cs- may enter inside cells. ces with er ⁄ golgi retention signal hydrolyzes octanoate of cs- figure . difference of binding stabilities of various na inhibitors to influenza virus neuraminidases. the na substrate was added to the influenza virus-na inhibitor complex (oseltamivir carboxylate, n; zanamivir, h; peramivir, s; laninamivir, •; distilled water, ¤), and the na reaction was followed for minutes. the background (only the na substrate [d] ) is also shown. a part of data from. to generate the hydrophilic drug, laninamivir, and then it is trapped inside er ⁄ golgi because of its high hydrophilicity. the glycoprotein, na, which matures in er ⁄ golgi, meets laninamivir there and efficiently makes a stable complex with it. there are some questions that remain. how does cs- move from the cell membrane to er ⁄ golgi? is laninamivir indeed trapped inside er ⁄ golgi, and does it make a complex with na in mice? we are now making an attempt to clarify these concerns. in our study, we have explored the antiviral potential of two newly synthesized compounds to provide protection against the novel pandemic influenza virus h n ( ) strain. the compounds were reconstituted in dimethylsulphoxide (dmso), and so the initial studies began with cytotoxicity determination of solvent on uninfected and untreated madin-darby canine kidney (mdck) cells. on obtaining an upper limit for dmso, the compounds were tested for estimation of their maximum non-toxic dose to the mdck cells. thereafter, the effective dose of the compounds was evaluated and validated by a number of assays and gene expression profiling at both nucleic acid and protein level. we found that these newly synthesized compounds possess potent inhibitory activity towards the novel pandemic influenza h n ( ) virus. these findings are being evaluated in vivo for a better understanding of their inhibitory capabilities and also their effect on the host metabolism. this will be required in the course of development of new drugs for use in the prophylaxis and treatment against the influenza virus. the mdck cell line (from nccs, pune) was maintained in · dmem media (sigma, st. louis, mo, usa) supplemented with % fetal calf serum and antibiotics viz. unit ⁄ ml penicillin and lg ⁄ ml streptomycin at °c ⁄ % co . the synthesized compounds used in this study were kindly provided by the department of chemistry, university of delhi, delhi, india. the pandemic influenza h n ( ) virus was isolated and propagated in the allantoic cavities of embryonated chicken eggs during the pandemic period. the virus stocks were prepared and stored at ) °c. plaque assay was performed as previously described by hui et al., . briefly, ae · mdck cells ⁄ ml were seeded in six-well plates and maintained in dmem for hours at °c ⁄ %co . the monolayer of the cells was inoculated with serially diluted virus samples for minutes at °c ⁄ %co . subsequently, a mixture of agar overlay was added, and the plates were incubated at °c for days or until formation of plaques. the plaques were visualized after removal of the agar plug and staining with ae % crystal violet or neutral red solution. the virus titre was expressed as plaque forming unit (pfu) per milliliter. the in vitro cytotoxicity analysis was performed to determine the % cytotoxic concentration (cc ) of the compounds on mdck cells. the compounds were dissolved in dimethylsulfoxide (dmso), and so a prior cytotoxicity analysis was performed to determine the toxic concentration of dmso on the cells. various concentrations of compounds were mixed with dmem containing % fcs before addition to the preformed monolayer of mdck cells in -well plates. a series of suitable controls for in vitro cc determination was included in every plate, and the plates were incubated in the optimum environment for mdck cell culture. the cc of test compounds was analyzed by estimation of percentage cell viability of the compound-and mocktreated mdck cells by performing a colorimetric assay using tetrazolium salt -( , -dimethylthiazol- -yl)- , diphenyl tetrazolium bromide (mtt) at end-point of hours post-incubation. the assay was performed as described by mosman . briefly, mtt stock at a concentration of ae mg ⁄ ml was prepared in · pbs. the media was aspirated from the wells and ll of mtt dye from the stock was added to each well. following incubation at °c ⁄ % co for - hours, the dye was very carefully removed from the wells, and the cells were incubated with ll of stop solution (dmso) per well at °c ⁄ % co for hour. the absorbance of the supernatants from each well was measured at nm, and the percentage cell viability was calculated. madin-darby canine kidney cells were maintained overnight in a -well tissue culture plate at °c ⁄ % co . the cells were inoculated with various virus dilutions at °c ⁄ % co for minutes and observed for cytopathic effect (cpe). the media from the experimental wells were aspirated after - hours of infection and were subjected to plaque assay. the percentage cell viability was determined by performing mtt assay. the results of both these tests were used to assess tcid of the virus. the pre-formed monolayer of mdck cells was inoculated with the -fold dilution corresponding to tcid of the virus for hour at °c ⁄ co . the experimental setup included control wells for the cells, virus, and compound. meanwhile, the concentrated stocks of the synthesized compounds were diluted with dmem (with % fcs) to various concentrations within their respective cc ranges. one hour post-infection, the cells were incubated with these diluted solutions. the cells were observed at various time intervals post-inoculation for cpe, and ll media was collected from each experimental well for performing hemagglutination test. after h, the media was collected for plaque assay and the cells were subjected to mtt cell viability assay. preformed monolayers of mdck cells were infected with virus and treated with the respective inhibitory concentration of the compounds. forty-eight to hours post-incubation, total cellular rna was isolated using ribozol (amresco, solon, oh, usa) and treated with lg ⁄ ml of dnase (promega, madison, usa). the concentration and quality of the rna from each well were determined by measuring their absorbance at and nm. one microgram of the cdna synthesized from each rna sample was used for sybr green-based real-time pcr detection of the ha gene of pandemic influenza h n ( ) virus. as a control, human glyceraldehyde- -phosphate dehydrogenase (hgapdh) was also amplified using gene specific primers. , immunoblotting immunoblotting was performed to further validate the antiviral potential of the compounds. the experimental protocol was the same as for real time rt-pcr analysis. the cells were harvested hours post-treatment with the compounds to prepare whole cell lysates in mammalian cell lysis buffer [ ae m nacl, ae m tris cl (ph ae ), ae m edta (ph ae ), m m protease inhibitor cocktail, lg ⁄ ml pmsf]. the protein concentration was determined by bca protein assay. the cell lysates were fractionated on % polyacrylamide for western blotting. the blot was developed using sheep monoclonal antibody (santa cruz biotechnology, ca, usa) against ha protein of influenza virus and horseradish peroxide conjugated rabbit-anti sheep igg ( : dilutions) as secondary antibody. the median cytotoxic concentration for compound meuh came out to be lm, and that for flh was lm. compounds showing potent antiviral effect on the pandemic influenza h n ( ) virus propagation in madindarby canine kidney cells ( figure ). the viral titres remained constant in cells treated with the compounds, while they increased in the untreated virus infected cells. ed for the compounds meuh and flh were and lm, respectively. fifty-two percent (meuh) and % (flh) inhibition against the pandemic influenza h n ( ) virus was achieved using ed of the test compounds. both the compounds were able to reduce the rna levels of the ha gene by approximately - %, whereas approximately % inhibition was seen when both the compounds were used in combination. similar results were obtained by the immunoblotting analysis ( figure ). antiviral therapy has shown to be a promising tool in the management of various respiratory diseases, including those caused by influenza viruses. we have already shown inhibition of influenza virus replication in our earlier studies using catalytic nucleic acids, which can be used as an approach in the development of new therapeutic strategy. these therapies are very useful as the influenza virus vaccines need annual renewals due to frequent genetic drifts in the viral surface proteins. in pandemic situations the existing vaccines do not provide complete protection against the novel virus as the population generally remains naïve for the newly mutated surface antigens. the antiviral drugs play an important role in the control of novel viral strains for which there are no vaccines available. however, the key obstruction in the extensive use of antiviral drugs is their cost and relative therapeutic efficacy provided. two classes of drugs were being used for treatment and control of the influenza virus infection in humans, the m ionchannel blockers , (amantadine and rimantadine), which prevent viral uncoating, and the neuraminidase inhibitors , (zanamivir and oseltmivir), which prevent the release of influenza virions from the cytoplasmic membrane. but widespread resistance to these antiviral drugs , has limited their use. thus, novel drugs are required for the effective therapy against the emerging strains of influenza virus. the novel chemical compounds used in our study were tested for their antiviral efficacy against the pandemic influenza h n ( ) virus. a reduction in the cpe in compound treated virus infected mdck cells indicated presence of antiviral activity in chemical compounds. the persistence of constant viral titers in the compound treated cells provided evidence for the interference posed by the compounds in the replication of influenza virus. inhibition in the ha gene expression further validated our hypothesis for the antiviral effect of compounds. the efficacy of these compounds in animal models is currently being validated in our laboratory. further, molecular studies are required to ameliorate the awareness regarding the mode of action of these chemical compounds against the viruses. and is now licensed in japan, while another, laninamivir, is being developed as an inhaled prodrug. resistance to nais among circulating influenza viruses was previously low (< % worldwide). [ ] [ ] [ ] however, the - influenza season was marked by a worldwide emergence of oseltamivir-resistant seasonal influenza a (h n ) viruses with the h y (h y in n numbering) in the na. [ ] [ ] [ ] [ ] [ ] [ ] the prevalence of oseltamivir resistance was even higher in the subsequent - influenza season with many countries reporting up to % oseltamivir resistance, seasonal and pandemic influenza viruses collected globally between october , and september , were submitted to the who collaborating center for surveillance, epidemiology and control of influenza at the centers for disease control and prevention (cdc) in atlanta, ga, usa, and propagated in madin-darby canine kidney (mdck) cells (atcc, manassas, va, usa). reference viruses representative of oseltamivir-sensitive and -resistant seasonal and pandemic viruses were also propagated in mdck cells. susceptibilities of virus isolates to the nais oseltamivir carboxylate (hoffman-la roche, basel, switzerland) and zanamivir (glaxosmithkline, uxbridge, uk) were assessed in the chemiluminescent ni assay using the na-star tm kit (applied biosystems, foster city, ca, usa) as previously described. additionally, subsets of virus isolates were tested for susceptibility to peramivir (biocryst pharmaceuticals, birmingham, al, usa). fifty percent inhibitory concentration (ic ) values were calculated using jaspr curve fitting software, an in-house program developed at cdc. curve fitting in jaspr was done using the equation: v = vmax · ( ) ([i] ⁄ (ki + [i]))), where vmax is the maximum rate of metabolism, [i] is the inhibitor concentration, v is the response being inhibited, and ki is the ic for the inhibition curve. box-and-whisker plot analyses of log-transformed ic s were performed for each virus type ⁄ subtype and nai using sas . software (sas institute, cary, nc, usa) to identify viruses with extreme ic values (outliers). outliers were characterized based on a statistical cutoff of ic greater than three interquartile ranges from the th percentile. outliers were subjected to genetic analysis by pyrosequencing and ⁄ or conventional sequencing to detect known or novel markers of nai resistance. those harboring previously characterized mutations in the na associated with nai resistance were considered drug-resistant; their descriptive statistics were determined separately from naisusceptible viruses. descriptive statistics to compute the mean, median, and standard deviation (sd), and a one-way analysis of variance were performed on original scale ic data, using sas . software (sas institute) for each nai and virus among seasonal influenza a (h n ) viruses tested for oseltamivir susceptibility (n = ), ( ae %) were outliers for the drug (table ) and harbored the oseltamivir-resistance conferring h y mutation in the na. by contrast, only a small proportion ( ae %) of tested h n pdm viruses (n = ) were resistant to oseltamivir. all influenza a (h n ) viruses (n = ) were sensitive to oseltamivir except for one outlier, a ⁄ ontario ⁄ rv ⁄ with d v mutation in the na, whose ic of ae nm was beyond the statistical cut-value off and > -fold the mean ic for the drug ( ae nm). all influenza b viruses (n = ) were sensitive to oseltamivir with exception of an outlier b ⁄ texas ⁄ ⁄ , with d e (d e in n numbering) mutation in the na, whose ic was beyond the cut-off, but only fourfold greater than the mean ic for the drug. all virus types ⁄ subtypes tested for zanamivir were sensitive to the drug (table ) , except for some outliers among seasonal influenza a (h n ) and a (h n ) outliers. the seasonal influenza a (h n ) outliers included a ⁄ thailand ⁄ ⁄ (h n ) and a ⁄ hawaii ⁄ ⁄ (h n ), both with combined h y and d d ⁄ g mutations in their na. the presence of concurrent mutations at na residues h and d in seasonal influenza a (h n ) virus isolates substantially enhances resistance to oseltamivir and peramivir and ⁄ or zanamivir, however, the changes at d are typically cell-derived and not present in clinical specimens. influenza a (h n ) outliers for zanamivir included a ⁄ ontario ⁄ rv ⁄ with d v mutation in the na, as well as a ⁄ maryland ⁄ ⁄ and a ⁄ vladivostok ⁄ ⁄ with d g and mixed d d ⁄ g mutations, respectively. some mild outliers for zanamivir among a (h n ) viruses with ic beyond the statistical cutoff but < -fold mean ic for the drug were also identified; their genetic analysis revealed presence of wildtype and mutant sequences at residue namely, d d ⁄ g, d d ⁄ n, or d d ⁄ a. mutations at residue d of the na are associated with reduced susceptibility to zanamivir in a (h n ) viruses, but were reported to be cell-culture derived in recent h n viruses. all virus isolates tested for peramivir (n = ) were sensitive to the drug, except for h y variants among seasonal influenza a (h n ) and h n pdm viruses, which exhibited reduced susceptibility to the drug. in addition, one influenza a (h n ) isolate, a ⁄ ontario ⁄ rv ⁄ with d v mutation in the na, showed reduced susceptibility to peramivir. the ic values determined in functional ni assays provide valuable information for detection of resistant viruses, but should not be used to draw direct correlations with drug concentrations needed to inhibit virus replication in the infected human host, as clinical data to support such inferences are inadequate. nevertheless, combining elevated ic values with the presence of established molecular markers of resistance in the na of virus isolates and their matching clinical specimens provides a reliable and reasonably comprehensive approach of identifying nai-resistant isolates for surveillance purposes. in this study, outliers with elevated ic values for oseltamivir among seasonal influenza a (h n ) and h n pdm viruses were confirmed to be oseltamivir-resistant based on the presence of the h y mutation in the na. outliers for oseltamivir and ⁄ or zanamivir among influenza a (h n ) viruses in this study were shown to harbor mutations at d , which were earlier associated with reduced susceptibility to zanamivir, and were cell-culture derived. the effects of d mutations on nai susceptibility appear to be strain-specific; however, there are no conclusive supporting data and further investigations are required. outliers among the influenza a viruses in this study exhibited changes in the na, derived naturally or through cell-culture, which altered their susceptibility to nais. however, mild outliers for oseltamivir and ⁄ or zanamivir among influenza a viruses with slightly elevated ic s, but without apparent changes in the na are sometimes identified. in such instances it is imperative to exclude the potential presence of influenza b among such outliers, using conclusive genetic tests such as real time pcr, since influenza b viruses exhibit higher ic values for oseltamivir and zanamivir than influenza a viruses. viruses exhibiting such mixes are typically excluded from statistical analyses of ic s for respective drugs and virus type ⁄ subtype. establishment of a clinically relevant ic cutoff value which could be used to differentiate statistical outliers from truly resistant viruses is imperative. global surveillance for nai susceptibility of influenza viruses circulating globally should be sustained to reflect the impact of seasonal and pandemic of influenza, given the limited pharmaceutical options available for control of influenza infections. nasopharyngeal swab specimens from patients with acute respiratory infection were collected at influenza sentinel surveillance units (outpatient and hospital-based) all over mongolia. specimens were transported to the virology laboratory, nccd, ulaanbaatar, and rt-rt pcr positive samples were grown in a mdck cell culture according to the protocol developed by cdc. and influenza virus gene segment (m genes) sequencing ( strains-genbank accession numbers: cy , cy , cy , cy , cy , cy , and cy ) and influenza virus gene segment (na gene) sequencing ( strains genbank accession numbers: cy and cy ) by the standard methods with applied biosystems xl genetic analyzer using primers supplied by who collaboration centers. a chemiluminescent na inhibition assay was performed with veritas microplate luminometer using the commercially available kit, na-star (applied biosystems, foster city, ca, usa), according to the manufacturers protocol. the na inhibitor susceptibility of influenza virus isolates was expressed at the concentration of na inhibitor needed to reduce na enzyme activity by % (ic ). oseltamivir carboxylate, was provided by f. hoffman-la roche ltd (basel, switzerland). na inhibition assay data were analyzed using robosage software comparing test data with the data produced by the reference na inhibitor sensitive and resistance strains, which were provided by the who influenza collaboration center, melbourne, australia. all viruses tested were sensitive to oseltamivir with two exceptions: a seasonal influenza virus a ⁄ ulaanbaatar ⁄ ⁄ (h n ) with ae nm ic value and a pandemic influenza virus a ⁄ dundgovi ⁄ ⁄ (h n ) with ae nm ic value ( figure ). there was oseltamivir resistance detected in ae % ( ⁄ ) of seasonal a (h n ) and in ae % ( ⁄ ) of a (h n ) pdm viruses. the oseltamivirresistant viruses were collected from untreated patients. in total, influenza b viruses were analyzed by na inhibition assay and all were sensitive to oseltamivir. the na of both oseltamivir-resistant strains contained h y mutation based on the sequencing analysis. the difference in the na amino-acid sequences between the mongolian oseltamivir-resistant viruses and the respective oseltamivir-sensitive reference viruses is shown in table all a(h n ) viruses analyzed for m channel inhibitor resistance by pyrosequencing contained the s n mutation and, thus, were resistant to this class of anti-influenza drugs. the segment sequencing revealed that seasonal a(h n ) viruses possess the common s n mutation. of note, a single strain a ⁄ zavkhan ⁄ ⁄ (h n ) contained an unusual s d change in the m protein. our study shows that the same prevalence [ ae % ( ⁄ )] of seasonal a(h n ) viruses with h y mutation in ⁄ season in mongolia with the published data for ⁄ season from japan. , however the prevalence of oseltamivir resistance in japan has dramatically increased in ⁄ season to % ( ⁄ ). the observed double mutations: h y and d g in a ⁄ ulaanbaatar ⁄ ⁄ (h n ) strain, which have been also found in japan in ⁄ season. the patient from whom the oseltamivir resistant seasonal influenza h n virus has been isolated was a -year-old boy, living in ulaanbaatar, the capital city, without history of using oseltamivir. the patient from whom the oseltamivir resistant a(h n )pdm virus was isolated was a year-old man, residing in the dundgovi, the southern province, also without history of antiviral treatment. according to the who data, isolation of the pandemic viruses carrying h y change from untreated patients has been uncommon. circulation of amantadine-resistant seasonal a (h n ) viruses has been increasing in mongolia since ⁄ influenza season. all pandemic influenza a(h n ) strains ( ) tested were resistant to m channel inhibitors due to the presence of the s n mutation in the m protein. among seasonal a(h n ) viruses, one contained a s d change whereas the others had s n, the well established marker of resistance to both amantadine and rimantadine. this is the first report of detecting the s d change in the seasonal a(h n ) viruses. according to the cdc data (unpublished), the s d change conferred the drug resistance in the a(h n ) viruses according to the virus yield reduction assay. it is essential to continue the antiviral resistance surveillance of influenza virus strains circulating in mongolia to ensure the efficiency of a proper clinical management of influenza patients. (conferred by the s n mutation). of note, the genotype and genotype dual resistant viruses from asia appear to be genetically similar to those previously reported dual resistant viruses from hong kong, sar. , the genotype virus was the only dual resistant virus with a nearly complete c genome. oseltamivir-resistance for this virus appears to be the result of a reassortment as demonstrated by the presence of the oseltamivir-resistant clade b na gene. although the detection of dual resistant seasonal influenza a (h n ) viruses is still rare, there has been an increased prevalence of dual resistance viruses during the last three seasons: . % ( of tested in - ), . % ( of in - ) , and % ( of in - ) (v p < . ). while the continued circulation or co-circulation of seasonal a (h n ) viruses is uncertain, the emergence of dual resistant influenza viruses in five countries does present a public health concern, especially since dual resistant viruses would limit the options for antiviral treatment to a single licensed antiviral drug: zanamivir. moreover, the markers of resistance seen in seasonal a (h n ) viruses also confer resistance in the more widely circulating pandemic a (h n ) virus. and, since the acquisition of mutations in influenza a viruses typically occur through drug selection, spontaneous mutation, or genetic reassortment with another drug resistant influenza a viruses, the detection of influenza a (h n ) viruses that are resistant to both adamantanes and oseltamivir warrants close monitoring, even if only detected at low frequency. new antiviral agents and strategies for antiviral therapy are likely to be necessary in the future. heightening concern that drug resistance will likewise become prominent in pandemic viral strains and highlighting the need for antiviral drug resistance surveillance. the h y mutation in h n neuraminidase is the most common mutation conferring resistance. however, due to the high mutation rates of viruses, new mutations can be expected that will also render viral neuraminidase less sensitive to antiviral drugs. pcr methods can be used to detect previously identified mutations; however, functional neuraminidase enzyme activity inhibition testing is necessary for detecting drug resistance that results from novel mutations. the two neuraminidase enzyme inhibition assays using either the fluorescent munana or chemiluminescent na-star Ò substrate are robust tools for ni susceptibility testing. the munan-a-based assay is broadly used by many groups, including many regional health organizations for ni susceptibility testing, yet no standardized protocol or dedicated kit has been in place for this assay, making comparison of data generated between different laboratories difficult. borrowing from multiple neuraminidase inhibitor susceptibility network (nisn)-published munana-based neuraminidase assay protocols, we have developed a kit-based fluorescent neuraminidase assay that offers both standardization and off-the-shelf quality-controlled reagents for ni susceptibility testing and other neuraminidase assay applications. the na-fluor tm influenza neuraminidase assay reagents and protocols were optimized in comparison to published nisn protocols according to the criteria of assay performance, ease-of-use, consideration of historically used assay conditions, reagent storage stability, and environmental impact. our optimized assay conditions consists of lm munana, ae mm mes, mm cacl , and ph ae in a ll assay volume, and performing the assay for minutes at °c following a minutes preincubation of drug with the virus. these conditions are consistent with the majority of published influenza ni screening data in publication. the standard na-fluor tm assay workflow for screening viral isolates for sensitivity to nis includes first titering the viral sample by neuraminidase activity to determine optimal virus concentration to be used in subsequent ic determination assays. the na-fluor tm assay is an ideal tool for titering virus based on neuraminidase activity in the viral coat. titering of viral samples prior to running the ic determination assays insured that assays would be performed within the fluorescence detection dynamic range of both the assay and the fluorometric instrument being used. viral titers giving rfus in the range of - were used for subsequent assays. comparison to traditional munana assays a primary goal of developing a standardized munana assay was to provide a standardized protocol and set of reagents that would allow for comparison of ni surveillance data between laboratories and over time. in addition, the assay should provide data comparable to historical data sets based on traditional munana-based protocols. to insure that our newly developed na-fluor tm assay met these criteria we performed side-by-side comparisons of the na-fluor tm assay to munana-based nisn protocols, as well as our na-xtd tm and na-star Ò chemiluminescent neuraminidase assays to compare assay sensitivity and dynamic range and for ni ic determination with multiple viral isolates. for all assay comparisons, assays were performed according to respective published protocols. for direct comparison of results, an equivalent amount of virus (and concomitant neuraminidase activity) was used for each assay. the na-fluor tm assay provides low-end sensitivity (by signal to noise ratio) and dynamic range similar to nisnpublished, munana-based protocols (data not shown). these assays all show a low-end detection of approximately ae u ⁄ well and dynamic range of - orders of magnitude when performed simultaneously side-by-side using serial dilutions of bacterial (clostridium perfringes) neuraminidase. these assays show approximately onefold less dynamic range and approximately fivefold less low-end sensitivity than chemiluminescent assays under these conditions. given the large amount of archived ni inhibition data for viral isolates over the past decade, it is very important for a standardized assay to generate data similar to established protocols so that data can be compared in relative terms. when run side-by-side, na-fluor tm assay provided oseltamivir carboxylate and zanamivir ic values similar to nisn-published, munana-based protocols. ic values vary somewhat for munana assays versus chemiluminescent assays depending on the viral isolate, as previously described. the na-fluor tm assay also exhibited similar sensitivity for detecting ni sensitive virus compared to nisn-published fluorescent assays as shown in figure . the large shift in ic values between oseltamivir-sensitive and resistant virus using the na-fluor tm assay enables detection of mutant virus in mixed viral samples ( figure ). this capability is critical for identifying resistant virus in clinical isolates presenting mixed populations of resistant and sensitive virus during ni susceptibility surveillance. several characteristics of the na-fluor tm assay make it an ideal assay for processing large numbers of viral isolates for ni sensitivity surveillance or for using the assay for high throughput screening for lead discovery of new antiviral reagents. the na-fluor tm assay signal was found to remain stable for up to hours after stop solution addition when stored at room temperature and for several days when stored at °c (data not shown). ic values did not change over these times, indicating that the assay is compatible with processing many samples in a short time frame. the na-fluor tm assay was also found to be highly reproducible giving a z' of ae or above indicating that the assay can be used confidently to identify nis in high throughput screening mode. the assay can tolerate up to % dmso, a common compound delivery reagent used in high throughput screens (data not shown). we have developed a standardized na-fluor tm assay suggested protocol that gives data similar to established mun-ana protocols. however, we have also found that several protocol adaptations can be made that generate comparable data while allowing the user more flexibility in assay mode, use of additional reagents, and to meet user-specified assay time requirements. the na-fluor tm assay can be run in either the standard minutes ⁄ °c endpoint mode described above or as real-time kinetic assay with repeated reads taken over time without the addition of stop solution, which both serves to terminate neuraminidase activity and to enhance the fluorescence of the product. for typical ni-sensitive viral strains, the rate of munana substrate turnover at °c is linear for at least hours (data not shown). as would be expected, rates of substrate turnover decrease in the presence of nis reflected in a decreased slope exhibited by real-time kinetic reads. real-time acquired rfus are typically - fold lower than rfus acquired after addition of stop solution at the same time point. ic values obtained using slope analysis for real-time assays are similar to values obtained by endpoint analysis. whether run in real-time or end-point mode, the linear rate of substrate turnover allows the user to run the assay for shorter or longer assay times than the standard protocol without compromise to assay performance. the na-fluor tm assay is also compatible with standard methods used in many laboratories to inactivate virus. we have shown that ni ic values for multiple viral strains remain unchanged when the assay is performed in the presence of ae % np- or % triton x- (data not shown). similar results are also obtained by adjusting the na-fluor tm stop solution to % ethanol prior to addition for assay termination. the assay is unaffected by phenol red concentrations present in cell culture media. we have developed a standardized munana-based fluorescent neuraminidase assay, the na-fluor tm influenza neuraminidase assay kit, which has been optimized for ni susceptibility screening. the assay provides data that can be compared to data generated using traditional munanabased protocols. the assay is economical, highly reproducible, easy to use, and environmentally friendly. the assay is flexible and amendable to user-specific adaptations including assay mode, assay timing, and reagent compatibility. trademarks ⁄ licensing ª life technologies corporation. all rights reserved. relenza is a registered trademark of glaxo- to test the prophylactic potency of h -vhhb, mice were treated intranasally with pbs, lg of h -vhhb, or negative control rsv-vhhb at , , or hours before infection with one ld of nibrg- ma virus. body weight loss was monitored daily, and on day mice were sacrificed to determine the viral load in the lungs. all mice that received h -vhhb retained their original body weight, whereas those receiving pbs or rsv-vhhb gradually lost weight (data not shown). intranasal administration of h -vhhb at or hours before challenge resulted in undetectable lung virus titers. when animals were treated with h -vhhb hours before challenge, virus titers were fold lower compared to pbs and rsv-vhhb treated mice, and three out of seven animals still had undetectable virus titers ( figure ). we next determined if h -vhhb nanobody Ò could be also be used therapeutically. we administered lg of this nanobody Ò intranasally to mice up to hours after chal-lenge with ld of nibrg- ma virus. four days after challenge, animals that received h -vhhb , , or hours after challenge had significantly higher body weight (data not shown) and lower lung virus loads than control mice. although mice treated with h -vhhb nanobody Ò hours after challenge were not clinically protected compared to control mice, they had significantly lower lung virus titers (figure ). to identify the ha amino acid residues that are potentially involved in h -vhh binding, escape viruses were selected by growth and plaque purification of nibrg- ma virus in the presence of h -vhhm or h -vhhb nanobodies Ò . the ha sequences of six independently isolated h -vhhm escape viruses revealed substitution of a lysine by a glutamic acid residue at position in ha (h numbering). in addition, two h -vhhm escape mutants carried an n d and four carried an n s substitution. the three-dimensional structure of nibrg- ha shows that n d ⁄ s and k e are close to each other as part of the corresponding antigenic site b in h ha. , interestingly, the n d ⁄ s mutations remove an n-glycosylation site, which is surmised to have evolved in h n ha as a strategy to mask an antigenic site. escape viruses selected in the presence of h -vhhb carried k n (n = ) or k e (n = ) substitutions. these results indicate that residues in antigenic site b, at the top of ha and very close to the receptor binding domain (rbd), are essential for neutralization of the virus by h -vhhm ⁄ b nanobodies Ò (figure ). the virus titer was measured in lung homogenates prepared on day after challenge. the x axis refers to the time points in hours relative to the challenge (time = hours) when ha-specific nanobodies (h -vhhb), control nanobodies (rsv-vhhb) or pbs was administered to the mice. # below detection limit, n not determined [n = - mice per condition: p values < ae (*)]. here we demonstrated that prophylactic and therapeutic treatment with llama-derived immunoglobulin single variable domain fragments is effective to control infection with h n influenza virus in a mouse model. we demonstrate that pulmonary delivery is a highly effective route of administration to treat or prevent influenza virus infection. in addition, we demonstrate that a homobivalent h -vhhb has powerful h n -neutralizing activity in vivo. it is important to note that we used a mouse-adapted derivative of the non-highly pathogenic nibrg- virus in our challenge model. nevertheless, this virus induces severe morbidity and lethality in mice. compared to conventional neutralizing monoclonal antibodies, vhhs offer the advantage that they are easy to produce in escherichia coli, typically with high yield. in addition, their small size ( kda for a monovalent vhh) and high folding capacity allow the generation of oligovalent vhh derivatives. in vitro escape selection revealed that a k e substitution in ha abolished the neutralizing effect of h -vhhm ⁄ b. a lys or arg residue at this position is conserved in all human h n virus isolates. of note, all selected escape mutants contained a glutamic acid or serine residue at position , which suggests that the conserved positively charged amino acid is important for neutralization by h -vhh nanobodies Ò . interestingly, escape mutants selected with h -vhhm also carried an n d ⁄ s co-mutation that removes an n-glycosylation site in this antigenic site of ha. the predicted n-glycosylation site at n in a ⁄ hong kong ⁄ ⁄ ha was shown to be glycosylated and may have evolved to mask an antigenic site near the rbd. , the selected amino acid changes are located near the receptor binding site of ha. therefore, it is possible that enhanced receptor binding properties of these escape viruses contribute to or are responsible for the loss of neutralizing activity of h -vhh nanobodies Ò . , we conclude that influenza virus neutralizing nanobodies Ò have considerable potential for the treatment of h n virus infections. although we focused on vhhs that presumably recognizes an epitope near the rbd, it is possible to select vhh molecules that bind to other epitopes in ha, including more conserved domains. more, a novel na (i m) substitution was discovered in a series of specimens from a patient. for the amantadine resistance, samples were tested, and all of them were confirmed to be resistant. we collected respiratory specimens from patients who had been clinically refractory to antiviral treatment since october upon ethical approval from the relevant institutions. to investigate the resistant pattern, sequence analysis to the na and matrix (m ) genes were conducted by reverse transcription (rt)-pcr and sequencing reaction. the obtained sequences were analyzed by the influenza sequences and epitopes database, which was developed in korea. eleven patients were found to be having oseltamivir-resistant pandemic (h n ) viruses with the h y substitution in the viral na genes (tables and ). some cases were associated with oseltamivir treatment on the basis of h y change from the oseltamivir-sensitive genotypes to oseltamivir-resistant genotypes in consecutive samples from the same patient. furthermore, a novel na (i m) substitution that may be associated with oseltamivir resistance was detected in specimens from one patient (patient g) who had myelodysplasia and received oseltamivir and peramivir (tables and ). in addition, we obtained viruses from clinical specimens (patients a and c) and evaluated antiviral susceptibility by measuring the dose of oseltamivir and zanamivir required for % inhibition (ic ) of na activity. these viruses (from patients a and c) were resistant only to oseltamivir (ic ae and ae nmol ⁄ l, respectively). susceptibility to zanamivir was not altered whether na contained y or h (ic ae and ae nmol ⁄ l, respectively). one isolate of pandemic (h n ) virus with an oseltamivir-sensitive genotype (h in its na) was susceptible to oseltamivir (ic ae nmol ⁄ l) and zanamivir (ic ae nmol ⁄ l). patients with oseltamivir-resistant pandemic (h n ) were treated during hospitalization with oseltamivir alone or with a combination of other antiviral drugs ( we found patients of oseltamivir resistance with h y mutation in the na gene of pandemic (h n ) virus through the surveillance of patient refractory to antiviral treatment. in addition, novel amino acid change (i to m) at position in the na gene, which might influence oseltamivir susceptibility, was detected in sequential specimens of a patient. these data showed that generation of oseltamivir resistance could be associated with oseltamivir treatment. therefore, it needs to strengthen the antiviral monitoring by supplementation of the clinical data including antiviral treatment. during the pandemic, oral oseltamivir was the primary antiviral medication used for treatment of hospitalized patients with ph n infection. many physicians worried that clinical deterioration or failure to respond to treatment with oseltamivir was due to either oseltamivir resistance or oseltamivir failure. in the united states, two investigational intravenous (iv) nais were available during - : peramivir through emergency use authorization and zanamivir by investigational new drug application. peramivir would be an option for patients with oseltamivir failure, but would not be appropriate for patients infected with h y oseltamivir resistant mutants. iv zanamivir was available in limited supply, but would be appropriate for severely ill patients infected with an oseltamivir-resistant ph n virus. during the pandemic, clinicians had few options for antiviral resistance testing in the united states. to respond to this need, the us centers for disease control and prevention (cdc) offered antiviral resistance testing for patients suspected to have clinical failure due to oseltamivir resistance. we describe the methods that cdc used to prioritize patients for testing during the pandemic and to detect markers for oseltamivir resistance, as well as the results from this testing. to facilitate decisions on which patients to test, we developed testing algorithms that were shared with state labora-tories, epidemiologists, and the emergency operation center at cdc. we prioritized patients who might benefit the most from antiviral testing given the inherent delay in providing antiviral results, e.g. patients who might have prolonged ph n shedding. patients that were critically ill [intensive care unit (icu) admission] or patients with severe immunocompromising conditions with clinical evidence for oseltamivir treatment failure (persistent detection of virus and clinical unresponsiveness to the drug) were prioritized. in addition, we tested specimens from patients that failed oseltamivir chemoprophylaxis. standard forms with information regarding specimen and minimal clinical information were collected on all patients. all protocols were validated and approved by clinical laboratory improvement amendments, e.g. quality standards to ensure accuracy, reliability, and timeliness of patient test results. information collected on patients was deemed public health response, not research, at cdc. clinical specimens, confirmed as pandemic influenza a (h n ), were tested for the h y mutation in the na using pyrosequencing. results were returned to sender within - hours of specimen receipt. from october until july , a total of specimens from patients were submitted for testing. viruses from ( %) of patients had h y mutation in the na in at least one submitted specimen. clinical information was available for patients (table ) . most patients had received oseltamivir for treatment prior to obtaining the specimen sent for antiviral testing. four patients received oseltamivir for chemoprophylaxis, all were immunosuppressed, and all had the h y mutant; duration of chemoprophylaxis until ph n infection was detected varied ( - days). among the patients with an h y mutant who were treated with oseltamivir, the median time on oseltamivir prior to collection of specimen with h y mutation was days (range - days). three patients were part of a hospital cluster of oseltamivir-resistant virus infections and were infected with h y mutants prior to oseltamivir treatment. patients with immunocompromising conditions accounted for almost half of all patient specimens tested, but they accounted for the majority of oseltamivir-resistant ph n virus infections (table ) ; among individuals with severe immunocompromising conditions and clinical failure while on oseltamivir therapy, ( %) had the h y mutant detected. among the immunosuppressed patients with an oseltamivir-resistant virus, ( %) had hematologic malignancies reported. in contrast, among the subset of icu patients without immunocompromising conditions and clinical failure while on oseltamivir therapy, we found little resistance: ( ae %) of icu patients had oseltamivir resistance detected. during the pandemic, we were able to provide timely and useful information to clinicians regarding suspected cases of oseltamivir resistance. our testing algorithm limited the number of specimens to specimens from the highest risk patients that would benefit the most from antiviral treatment. such an approach allowed us to offer this service without compromising our public health duties. in addition, the information we collected on patients from this service complimented our data on the national surveillance for antiviral resistance. we also performed national antiviral resistance surveillance from april to july . overall, resistant ph n viruses were identified from april to july in the united states among tested samples, including specimens described above, surveillance specimens, and resistant viruses reported in the literature. further studies to understand risk factors for oseltamivir-resistant ph n infection in patients with severe immunocompromising conditions are needed. while efforts to provide antiviral testing technology and materials to state laboratories are ongoing, clinicians still have limited options for such testing. rapid and inexpensive assays that could be performed by clinical laboratories, especially those caring for immunosuppressed patients, would be useful to inform patient care. the applied biosystems Ò na-xtd tm influenza neuraminidase assay kit provides the next-generation na-xtd tm , -dioxetane chemiluminescent neuraminidase (na) substrate, together with all necessary assay reagents and microplates, to quantitate sensitivity of influenza virus isolates to neuraminidase inhibitors. like the na-star Ò influenza neuraminidase inhibitor resistance detection kit, the na-xtd tm influenza neuraminidase assay provides highly sensitive detection of influenza neuraminidase activity. in addition, the na-xtd tm assay provides extended-glow light emission that eliminates the need for reagent injection and enables signal measurement either immediately or up to several hours after assay completion. the na-xtd tm assay is also used to quantitate influenza na activity directly in cellbased virus cultures to monitor viral growth or inhibition. global monitoring of influenza strains for resistance to neuraminidase inhibitors (nis) is essential for understanding their efficacy for seasonal, pandemic, or avian influenza, and studying the epidemiology of viral strains and resistance mutations. functional neuraminidase inhibition assays enable detection of any resistance mutation, making them extremely important for global monitoring of virus sensitivity to nis. the first-generation chemiluminescent na-star Ò influenza neuraminidase inhibitor resistance detection kit has been widely used for virus ni sensitivity assays, - including identification of a ⁄ h n pandemic virus resistant to oseltamivir. , in addition, this assay has been used for identification of new ni compounds, ni characterization, studies of virus transmission, drug delivery, na quantitation of virus-like particles, and cell-based virus quantitation. neuraminidase assays performed with chemiluminescent , -dioxetane substrates, including na-star Ò and na-xtd tm substrates, typically provide -to- -fold higher sensitivity by signal-to-noise ratio than assays performed with the fluorescent munana substrate. in addition, chemiluminescent assays provide linear results over - order of magnitude of neuraminidase concentration compared to - orders of magnitude with the fluorescent assay. the high assay sensitivity achieved with chemiluminescent assays enables use of lower concentrations of viral stocks, and the wide assay range minimizes the need to pre-titer virus stocks prior to ic determination. chemiluminescent reactions result in conversion of chemical energy to light energy, as light emission. the na-xtd tm substrate is a , -dioxetane structure bearing a sialic acid cleavable group. to perform the na-xtd assay, virus dilutions (from cell culture supernatant) are pre-incubated in the presence of neuraminidase inhibitor. then na-xtd substrate is added and incubated for minutes for substrate cleavage to proceed. finally, light emission is triggered upon addition of na-xtd accelerator, which provides a ph shift and a proprietary polymeric enhancer, both required for efficient light emission. chemiluminescent assays are performed in solid white microplates, and light emission is measured in a luminometer. the na-xtd tm substrate has a single structural difference from the na-star Ò substrate that provides a much longer-lasting chemiluminescent signal, with a signal half-life of approximately hours (not shown), compared to $ minutes with the na-star assay, eliminating the need for luminometer instruments equipped with reagent injectors and enabling more convenient batch-mode processing of assay plates. the na-xtd tm assay kit also provides a new accelerator solution, containing a next-generation polymer enhancer, and a triton Ò x- -containing sample prep buffer providing enhanced na activity. read-time flexibility is demonstrated by determination of oseltamivir ic values using data collected over hours after addition of na-xtd tm accelerator. although signal intensity slowly decreases over time, the ic curves and values are identical at each time point, shown using influenza b ⁄ lee ⁄ ( figure ) . triton x- detergent at % has been shown to inactivate flu virus while increasing neuraminidase activity. the addition of na sample prep buffer (containing % triton x- ) to virus stocks (at ⁄ volume, achieving a final concentration of %) provides increase in na activity up to fourfold, but is not consistently observed, and seems to be most effective with more concentrated virus stocks. ic values are unaffected by the addition of triton x- to the virus stock prior to virus dilution (not shown), so the assay is compatible with known virus inactivation reagents. assay sensitivity and ic values determined with the na-xtd assay have been compared to those obtained with both the chemiluminescent na-star assay and the fluorescent na-fluor assay (not shown). the chemiluminescent assays provide -to -fold higher sensitivity by signal-to-noise ratio, depending on the virus strain, wider assay dynamic range, and better low-end detection limit than the fluorescent assay. the wide assay range with the chemiluminescent assays enables determination of ic values over a range of virus concentrations, eliminating the need to titer virus prior to performing ic determination assays. ic values obtained with the na-xtd assay are nearly identical to those obtained with the na-star assay, with both oseltamivir and zanamivir neuraminidase inhibitors, and tend to be slightly lower than ic values obtained with the fluorescent assay. viral na quantitation provides a convenient read-out to measure viral growth or inhibition, including inhibition in the presence of inhibitory compounds or antibodies, described as accelerated viral inhibition with na as readout assay (avina). bation in the presence of varying concentrations of oseltamivir carboxylate. samples of culture media were assayed hours later. quantitation of na activity with the na-xtd tm assay demonstrates inhibition of viral growth by oseltamivir carboxylate in cell culture ( figure ). different volumes of culture media were assayed with the na-xtd assay, either in the culture plate or in a separate assay plate (not shown). performing the assay using the entire well contents ( ll) reduces assay sensitivity due to the high concentration of phenol red. assaying a smaller volume of culture medium (either in culture plate or a separate assay plate) provides higher sensitivity, and enables temporal monitoring or use of remaining culture medium for other assays. the applied biosystems Ò na-xtd tm influenza neuraminidase assay kit is a next-generation chemiluminescent neuraminidase assay providing high assay sensitivity and ''glow'' light emission kinetics for improved ease-ofuse. the applied biosystems Ò na-fluor tm influenza neuraminidase assay kit, based on the fluorescent mun-ana substrate, has also been developed to complement the na-xtd tm and na-star Ò chemiluminescence assays, for users lacking luminometer instrumentation or choosing to use fluorescence assay detection. together these kits offer: • standardized reagents and protocols • choice of detection technology • simple instrumentation requirements • high sensitivity for use with low virus concentrations • compatibility with batch-mode processing and largescale assay throughput • broad specificity of influenza detection • flexibility in assay format • additional na assay applications -cell-based viral assays, screening for new nis, detection of na from other organisms functional neuraminidase inhibition assays enable detection of any resistance mutation and are extremely important in conjunction with sequence-based screening assays for global monitoring of virus isolates for ni resistance mutations, including known and new mutations. together, these assays provide highly sensitive, convenient and versatile assay systems with standardized assay reagents, and simple assay protocols for influenza researchers. over hospitalizations and deaths in the us annually are attributable to seasonal influenza, primarily in chronically ill persons and the elderly. - following the emergence of pandemic h n influenza, severe illnesses have also been observed in children and young healthy adults. the occurrences of staphylococcal and pneumococcal pneumonia complicating influenza pandemics are well described. [ ] [ ] [ ] although temporal associations of bacterial pneumonia and influenza circulation have been reported, there is little precise data on rates of bacterial complications of seasonal or pandemic influenza. the study of bacterial lung infection has been hampered by insensitive tests for invasive disease and the difficulty of interpreting routinely obtained sputum culture results. , procalcitonin (proct), the prohormone of calcitonin, can discriminate viral and bacterial infections. this -aminoacid precursor protein normally produced by neuroendocrine cells of the lungs and thyroid gland was first shown to be elevated in bacterial infections in patients with pulmonary injury and pneumonitis. stimuli of proct include tnf-a, endotoxin, and other bacterial products. several studies indicate that bacterial infections commonly induce hyperprocalcitonemia, but that viral infections, including h n , are associated with only minimal increases. , , of note, proct induction is attenuated by viral-induced interferon-c. a meta-analysis of studies comparing proct and crp as markers for bacterial infection found that proct was more sensitive and specific than crp for differentiating bacterial from other causes of inflammation. , therefore, we measured proct levels in patients with seasonal and pandemic influenza and compared results with conventional methods for bacterial diagnosis. adults ‡ years of age admitted to rochester general hospital (rgh) from november st to june th for two winter seasons ( - ) with an admitting diagnosis compatible with acute respiratory tract infection were recruited for the study. patients were screened within hours of admission, and those with prior antibiotic use, immunosupression, or pregnancy were excluded. subjects or their legal guardian provided written informed consent. the study was approved by the university of rochester and rgh research subjects review board. at enrollment demographic, clinical and laboratory information was collected. influenza testing included nosethroat swabs (nts) for rapid antigen, viral culture, and reverse transcription-polymerase chain reaction (rt-pcr) and serology. testing for bacterial pathogens included blood cultures, sputum for culture and gram stain, nts for mycoplasma pneumoniae and chlamydophila pneumoniae pcr, s. pneumoniae antigen testing, and pneumococcal serology. if patients were unable to expectorate, sputum was induced with normal saline and bronchodilators. specimens were considered adequate by the standard criteria of > neutrophils (pmns) and < epithelial cells per high power field. serum was collected at admission and hospital day for proct measurements. influenza infection was defined a positive result for any of the following tests: . cloned proteins were coated on eia plates at ug ⁄ ml in bicarbonate buffer. after overnight incubation, plates were washed and two-fold dilutions of serum were incubated overnight at room temperature. plates were washed and incubated with alkaline phosphatase conjugate for hours, followed by substrate. a greater than or equal to fourfold rise in titer was considered evidence of infection with s. pneumoniae. urinary antigen for s. pneumoniae samples were assayed for antigen using the binax now kit. (binax inc, scarborough, me, usa). the proct was measured using time resolved amplified cryptate emission technology (kryptor pct; brahms, henningsdorf, germany). functional sensitivity is ae ng ⁄ ml (normal levels are ae ± ae ng ⁄ ml). mycoplasma and chlamydia pcr real-time pcr targeting the p adhesion gene for m. pneumoniae and the ompa gene for c. pneumoniae was used to detect atypical bacteria. results fifty-one of ( ae %) illnesses evaluated tested positive for influenza virus. of these, were due to ''seasonal influenza'' ( influenza a ⁄ h n and influenza b), and were identified as ''pandemic influenza'' ( h n ). demographics of both groups were similar: mean ages ± and ± years, respectively, and equivalent sex and racial characteristics. other than a higher incidence of underlying lung disease in the seasonal group ( % versus %, p = ae ), pre-existing medical conditions including obesity were similar. symptoms, physical findings, and discharge diagnoses did not differ, and chest radiographs (cxr) showed infiltrates in % and % of seasonal and pandemic subjects, respectively. two pandemic and one seasonal influenza patient developed respiratory failure, and none died. overall, bacterial infections were diagnosed in ( %) subjects ( -seasonal and -pandemic), and none were bacteremic. bacterial infections included: -s. pneumoniae, -m. pneumoniae, -s. aureus, and -h. influenzae. all seasonal patients were diagnosed with asthma or bronchitis, whereas three pandemic patients had pneumonia. mean serum proct (ng ⁄ ml) levels in seasonal versus pandemic patients on admission and day were: ae ± ae versus ae ± ae and ae ± ae versus ae ± ae , respectively, and were not significantly different (table ) . several patients in the pandemic group had high proct levels, and there was a trend toward more pandemic patients having admission proct values ‡ ae ng ⁄ ml than seasonal subjects [ ( %) versus ( %), p = ae ] ( figure a , b). of the four patients with proc-t > ae ng ⁄ ml, two had dense infiltrates on cxr, one had a peripheral wbc of ⁄ ml with a threefold increase in s. pneumoniae antibody, and one developed respiratory failure associated with copd exacerbation. reliable sputum samples (within hours of antibiotics) were collected in only ( %) subjects. of these, proct was ‡ ae ng ⁄ ml in two with influenza alone and three associated with bacterial infection, and < ae ng ⁄ ml in with influenza alone and five associated with bacterial infection. in the with reliable sputa and accepting the conventional bacterial diagnosis, sensitivity of a proc-t ‡ ae ng ⁄ ml for bacterial infection was %, specificity %, positive predictive value %, and negative predictive value %. notably, one patient considered to have influenza alone (proct - ae ng ⁄ ml) had group a streptococcus and s. aureus in a contaminated sputum and bilateral infiltrates on cxr. three of five patients with bacterial infections and proct < ae ng ⁄ ml had a clinical diagnosis of bronchitis. mean proct values were significantly higher in patients with infiltrates versus those with atelectasis or no acute disease on cxr ( ae ± ae ng ⁄ ml versus ae ± ae ng ⁄ ml, p = ae ). combining patients with proct values ‡ ae ng ⁄ ml with those having positive bacterial tests, rates of bacterial infection associated with seasonal and pandemic influenza were % and %, respectively. notably, antibiotics were administered to % of subjects despite % having no acute disease on cxr. in our study, bacterial infections were diagnosed in approximately % of adults hospitalized with influenza with no significant difference in rates noted between seasonal and pandemic influenza infected subjects. previous reports of bacterial infection rates of - % with seasonal influenza are difficult to compare with recent studies of pandemic influenza, because the latter tended to focus on more severely ill patients. [ ] [ ] [ ] bacterial pneumonia has been suspected or diagnosed in - % of patients in intensive care associated with h n infection and up to % of patients who died. , despite aggressive pursuit of specimens for bacterial testing, diagnoses could be confirmed in only ( ae %) of patients using conventional methodology. given the difficulty in establishing a diagnosis of bacterial infection, elevated proct values may be helpful to identify patients at high risk for invasive disease. in a study of patients with severe h n or bacterial infection necessitating intensive care, a threshold proct level of ae ng ⁄ ml, demonstrated % sensitivity and % specificity for bacterial infection. among patients with h n associated pneumonia, many of whom had respiratory failure, a threshold proct value of ae ng ⁄ ml provided a sensitivity of % and specificity of % for bacterial infection. access to samples from lower airways in ventilated patients in these studies may have improved recovery of bacteria and account for the different results we observed. it should be noted that none of our patients were bacteremic, which is a very strong stimulus for proct release. proct levels have been used successfully to guide therapy in community acquired pneumonia, and our data showing high proct levels in patients with infiltrates on cxr suggests proct may be most useful for excluding invasive disease. , elevated proct levels were not observed in patients with purulent sputum and clear cxr. it is notable that a proct level of < ae ng ⁄ ml did not exclude patients with bacterial bronchitis since proct has been used to guide antibiotic therapy in copd exacerbations. while it could be argued that healthy patients with bacterial bronchitis do not require antibiotic treatment, physician behavior in our study indicates antibiotics are frequently prescribed. combining patients with proct values ‡ ae ng ⁄ ml and those with a positive bacterial test, approximately % in patients in our study had bacterial complications associated with influenza infection. efforts should be made to curtail antibiotic use in hemodynamically stable patients with clear cxrs. given physician discomfort regarding discontinuing antibiotics, proct measurements in combination with routine bacterial cultures should be useful tools to guide therapy. influenza, mrsa, cytokines: diagnosis, treatment, prevention -a possible strategy for outpatient care we started the antiviral treatment of influenza in humans using neuraminidase inhibitors on january , in a successful attempt to cure a -year-old patient. since then, we have used the inhalant antiviral drug zanamivir, and later (october , ) changed to the use of oseltamivir with systemic bioavailability for treating patients with influenza. after years of experience with antiviral treatment of outpatients, we highlight the importance of early diagnosis and early treatment. the necessity of an earliest possible diagnosis was confirmed in the pandemic of . large hospitals reported that patients with an h n ⁄ infection had to be treated with extracorporeal membrane oxygenation. we are convinced this is due to delayed recognition of infection in most cases. valuable time is lost when the patient with a sudden onset has to be brought to a hospital for emergency treatment. the point at which the patient goes to the doctor is decisive, and this problem of timing and the delivery of early treatment is not specific to germany. in our medical office, we assessed patients with suspected influenza (to date seasonal infections, and in , h n ⁄ ) through clinical diagnosis, and then proven by point of care rapid test (quickvue; quidel, san diego, ca, usa) followed by pcr. all of the patients undergo concomitant lab tests: leukopenia, serum iron level, and the humoral inflammation status [sum of the c-reactive protein (crp) and fibrinogen levels]. because of the constant threat of a bacterial superinfection, a bacterial swab and antibiogram is carried out on every patient. in all cases positive for influenza, oseltamivir was given immediately. nowadays it is important that a double infection with influenza and mrsa must be recognized immediately and treatment started at once with antivirals and, when appropriate, with a suitable antibiotic. we pay particular attention to an extremely low iron level (signum mali ominis). in addition we monitor oxygen saturation and the course of the humoral inflammation status every - hours for every of our outpatients. among our patients with seasonal influenza, we saw within hours, within hours, and within hours after disease onset. for pandemic influenza, it was patients within hours, within hours, and two within hours. for all patients, we measured crp < ae mg and fibrinogen < mg ⁄ dl ( hours), crp < mg and fibrinogen < mg ⁄ dl ( hours), and crp > mg and fibrinogen < mg ⁄ dl ( hours, only seasonal cases). antibiotics were necessary in cases, heparin and oxygen administration in cases. one hundred forty-eight patients had a superinfection following influenza. the most common strains were haemophilus parainfluenzae and staphylococcus aureus. the subsequent use of a suitable antibiotic was only necessary in % of the patients. in all cases diagnosed, treatment (including heparin and oxygen administration) and monitoring were conducted in our medical office. none of our patients (seasonal and pandemic) had to be admitted to hospital. the early decision of whether or not antiviral and antibacterial treatment is taking effect is the only way the threat of a cytokine storm can be averted. not only does the primary care physician have to be aware of the pathophysiology involved, but also the necessary diagnostic and therapeutic options have to be made available to him. the result will lead to a saving of both lives and healthcare costs. this applies both in epidemic as well as in pandemic times. today we know that influenza leaves behind a defenceless immune system, and that the proteases of s. aureus contribute to influenza associated pneumonia. mark von itzstein, who discovered neuraminidase inhibitors, emphasized the synergistic cooperation of viruses and bacteria (personal communication, ). mrsa and influenza viruses are posing problems worldwide. the case of a -year-old boy with h n ⁄ infection demonstrates how fatal developments can be prevented. due to his constantly recurring colds, we had already detected the mrsa colonization years earlier and had always worked on boosting his general health and resistance. both the patient and his family were included in dealing with the problem. the patient was, and is, always vaccinated early with a virosomal vaccine (baxter). during the oktoberfest in munich in september , when h n infections were increasingly occurring, we learned that our patient had come down with an extremely acute feverish illness. with the help of the rapid test, we diagnosed an h n ⁄ virus infection and started treatment with oseltamivir immediately. the humoral inflammation status, which had increased very rapidly to more than ae mg ⁄ dl within hours, was treated with the effective cotrimoxazol from the antibiogram. at the same time, the patient was heparinized. the following day the patient had no fever and was symptom-free. it was only through our early knowledge of what could develop pathophysiologically that we were in a position to make the right decision at the right time. every doctor treating outpatients can follow this procedure if he is familiar with the pathophysiology of the disease and has the available tests on hand: virus rapid test, additional laboratory parameters (leukopenia, iron), and the humoral inflammation status. the decisive factor, however, is the constant clinical alertness towards the course of every acute feverish cold with acute onset. the patient has to remain in the care of the attending physician, and the chosen treatment has to be administered and monitored. this means constant spo measurements and checking the humoral inflammation status every hours. if a clinical worsening occurs during monitoring, the treatment regime has to be changed immediately, which means the administration of an appropriate antibiotic. this outpatient care on the part of the doctor has to be available days a week so that no time will be lost. reports from the netherlands and denmark show that, with the help of this preventive strategy under the motto 'search and destroy,' the dangerous, fatal course of infections reported in germany with at least four deaths a day, can be avoided. however, the doctor has to be adequately remunerated for the elaborate amount of time this intensive outpatient care requires. with our strategy, we have moved from divergence to convergence in the care of our patients. we reported on our years of clinical experience with this approach at the antivirals congress in peking. our main message was early diagnosis and early treatment. we were able to demonstrate this in outpatients with seasonal influenza and h n ⁄ outpatients. our creed is: as much outpatient care as possible and as little hospitalization as possible. virological and autopsy findings in suspected and confirmed fatal cases of h n pandemic influenza in the czech republic -preliminary results influenza viruses cause substantial morbidity and mortality. pandemic influenza may have a serious impact on certain (mainly younger) age groups in comparison with seasonal flu. influenza is one of few viral infections capable of causing a pneumonia that is difficult to cure and ⁄ or leads to sudden death. the aim of this study was to analyze and compare virological and autopsy findings in patients who died with suspected or confirmed h n pandemic influenza virus infection. there were virologically confirmed cases of pandemic influenza and deaths in the czech republic during pandemic wave. more than influenza strains belonging to the new pandemic variant were isolated in the national influenza reference laboratory. postmortem biological samples were collected from any patient who died with suspected influenza infection to test for respiratory viruses. the samples were screened for h n pandemic influenza virus by real-time pcr (rt pcr), and when rt pcr positive, by virus isolation assay. no immunohistochemical staining for influenza antigen was done on the rna pcr positive cases. other important respiratory viruses such as respiratory syncytial virus, parainfluenza viruses, and adenoviruses were detected by virus isolation assay in a suitable cell culture. epidemiological analysis of postmortem histopathologic findings in the airway tissue was carried out in of fatal cases. virological findings were subsequently correlated with histological changes and available demographic and clinical data. statistical analysis was performed by t-test using spss software. sixty-one deaths ( males, females) were analyzed. the rna of the h n pandemic influenza virus was detected by pcr in cases, while cases remained negative. five respiratory syncytial viruses and two adenoviruses were detected in the influenza negative group. the mean age of confirmed h n pandemic influenza victims was ae years, age range - years and median ae years. the mean age of influenza negative victims was ae years, age range - years and median ae years. the % ci for the difference in the age between the two groups is ) ae ; ae . the test is statistically significant at the % level. the obtained significance (p = ae ) can be explained by the relatively small size of the study group. the most common postmortem histopathologic finding in the lung tissue of the h n pandemic influenza virus-positive victims was diffuse alveolar damage (often bilateral) and ⁄ or hyaline membrane formation, possibly with signs of respiratory distress syndrome (in , i.e., ae %, of autopsied patients). in the h n pandemic influenza virus negatives, the most common finding was pneumonia or bronchopneumonia with the detection of various bacterial species (in , i.e., ae % of autopsied patients). the cause might be either primary bacterial infection or superinfection following primary infection with influenza virus that remained undetected. the h n pandemic influenza victims were younger than the patients who died with suspected but undetected h n pandemic influenza. the majority of deaths were primarily linked to rapidly developing respiratory failure. this result supports the previous reports of severe respiratory outcomes in younger age groups that are typically linked to the spread of a pandemic strain of influenza. due to limited amount of pandemic vaccine, especially at the beginning of pandemic, it is advisable to assess experiences with antiviral treatment, mainly dosing, and way of antiviral administration. primers specific for each of the eight genes of pandemic h n ⁄ were adopted from assays as described previously to discriminate against seasonal human h n and h n viral segments (table ) . the primers were allowed to cross-react specifically with the sister clade viral segments of pandemic h n ⁄ . the method we employed in this study was a -step singleplex sybr green-based real-time rt-pcr. this approach helped lower the running cost of the assays and facilitated downstream molecular analyses (e.g., sequencing) by using screened cdna samples. viral rna was extracted from viral cultures or clinical samples as described , and was converted to cdna in a universal rt-pcr. each ll rt reaction containing ae ll of purified rna, ll of · firststrand buffer (invitrogen), u of superscript ii reverse transcriptase (invitrogen), ae lg of uni ( ¢-ag-caaaagcagg- ¢), ae mm of deoxynucleoside triphosphates and mm of dithiothreitol was incubated at °c for minutes, followed by °c for minutes for heat inactivation. for each segment-specific real-time pcr, the ll reaction contained ll of a -fold diluted cdna samples, ll of fast sybr green master mix (applied biosystems), and ae lm of the corresponding primer pair. the thermocycling conditions of all eight segment-specific pcrs were optimized as °c for seconds, followed by cycles of °c for seconds and °c for seconds, and all eight assays were performed simultaneously in a sequence detection system (applied biosystems). at the end of the amplification step, pcr products went through a melting curve analysis to determine the specificity of the assay ( - °c; temperature increment: ae °c ⁄ seconds). cdna of a ⁄ california ⁄ ⁄ virus was used as a positive control. robust and specific amplification was achieved in all eight segment-specific real-time rt-pcr reactions. pcr product for each segment of pandemic h n ⁄ yielded unique melting curve pattern with distinctive melting temperature (tm), which was not observed in negative and water controls ( figure ). reactions with tm value within sds of the mean tm were determined as positive. we evaluated the assays with a number of serologically confirmed human clinical samples. all pandemic h n ⁄ samples (n = ) were positive in all eight assays, while all seasonal samples (h n = ; h n = ) were negative in all assays, as expected ( figure and data not shown). these results showed that no reassortant of pandemic h n and seasonal viruses was present in the tested human isolates. we applied these assays to our on-going influenza virus surveillance program in swine. nasal and tracheal swab samples were collected at an abattoir in hong kong and cultured in madin darby canine kidney cells or embryonated eggs as described. positive viral cultures in hemagglutination assays were tested with the established segmentspecific real-time rt-pcr assays. among swine viral isolates collected from to september , of them were recognized as pandemic h n ⁄ in all eight segments. they were confirmed to be of pandemic h n ⁄ origin by subsequent full genome sequencing analyses, showing that there were interspecies transmissions of the virus from humans to pigs. , the remaining viruses had one to seven gene segments positive in the segment-specific real-time rt-pcrs. thirty of them were selected as representative samples for full genome sequencing analyses based on the genotyping data generated in our assays. they were swine h n or h n viruses with their gene segments derived from tr or eurasian avian-like swine lineages. it should be highlighted that all of their positive gene segments in our assays belonged to the sister groups of pandemic h n ⁄ . their melting curve patterns were very similar to those derived from segments of pandemic h n ⁄ , except for ha of tr lineage. our results successfully demonstrated the use of these segment-specific real-time rt-pcrs to recognize gene segments of contemporary tr (pb , pb , pa, ha, np, and ns) and ea (na and m) swine viruses. the ha-specific assay was able to discriminate pandemic h n ⁄ from other contemporary swine viruses in the same lineage. nevertheless, to confirm the identity and to examine all the genetic variations in the viruses of interest, full genome sequencing analyses were necessary. in this study, the biggest obstacles in primer design were sequence similarity and diversity of influenza viruses. we attempted to use degenerated primers, but they were highly non-specific. the finalized non-degenerated primers crossreacted with genes from pandemic h n ⁄ and its sister clade tr (pb , pb , pa, ha, np, and ns) and ea (na and m) swine viruses with some minor sequence mismatches. three avian (h n , h n , and h n ) and classical swine (h n ) were also tested with our assays. all of these animal viruses were negative, except for ns gene of the classical swine virus. our segment-specific real-time rt-pcr assays might be used in high throughput genotyping. they detected pandemic h n ⁄ viruses and acted as a preliminary screen-ing tool to select virus reassortants of interesting genotypes for further sequencing analyses. in fact, we identified a novel reassortant in january during the course of this study. this sw ⁄ hk ⁄ ⁄ has a previously unidentified viral gene combination as shown in figure . it was confirmed to be a reassortant between pandemic h n ⁄ and other swine viruses in full genome sequencing characterization. it has a pandemic h n -like n gene, an ea-like h , and the other six internal genes derived from tr swine viruses. , the eight established real-time rt-pcrs can rapidly reveal the gene-origins of influenza viruses. we are currently using these assays in influenza surveillance in humans and other animals. it is believed that similar strategy might be applied to detect and genotype other influenza viruses and possible reassortants in the future. pandemic influenza a ⁄ h n ⁄ infects millions of people around the world. a significant fraction of the world's population may also already have been exposed to the virus and, although asymptomatic, may be at least partially immune to the disease. a precise assessment of the number of people exposed to the influenza a ⁄ h n ⁄ virus is epidemiologically relevant. however, assays typically used to estimate antibody titers against a particular influenza strain, namely hi and neutralization, require use of the actual virus. this seriously limits broad implementation, particularly in regions where high biosafety facilities are unavailable. we developed an elisa method for the evaluation of presence of specific h n influenza virus-antibodies in serum samples. mouse anti-histidine tagged antibodies ( ll; lg ⁄ ml; abd serotec Ò , uk) in pbs (ph ae ) were dispensed into standard -well plates and incubated for - hour at room temperature. excess antibody was removed by at least two successive alternate washings with pbs-tween ae % and pbs. commercial blocking solution ( ll, superblock Ò t ; pierce Ò , usa) was added and incubated for at least hour at room temperature. after successive washing steps with pbs-tween ae %, non-glycosylated histidine-tagged recombinant protein ( ll; lg ⁄ ml) was added to each well. this protein consisted of the receptor-binding domain of the hemagglutinin of the influenza a ⁄ h n virus. , after hour incubation, wells were washed for at least two alternating minutes cycles with pbs-tween and pbs. a : dilution of the serum or plasma sample to be assayed ( ll) was added to each well and incubated at room temperature for hour. after repeated alternating minutes pbs-tween ae % and pbs washes, anti-human igg antibody solution ( ll ⁄ well; : dilution in pbs-tween ae %) marked with horse radish peroxidase (pierce Ò , usa) was added and incubated for hour at room temperature. after repeated alternate washes with pbs-tween ae % and pbs), substrate solution ( ll; -step ultra tmb-elisa; pierce Ò ) was added to each well. after incubation for minutes at room temperature in darkness, the enzymatic reaction was stopped by addition of m h so ( ll ⁄ well). yellow color produced by the enzymatic reaction was evaluated by absorbance at nm in a biotek Ò microplate reader (usa). blank assays using albumin in place of human sera established the elisa background signal, which was subtracted from sample absorbance signals: abs serum sample ¼ abs serum sample before correction À abs albumin sample : absorbance values were normalized based on the average signal of non-exposed subjects (uninfected subjects), and expressed as normalized absorbance (abs norm ): where abs serum ample is the sample absorbance signal, abs albumin sample is the albumin control absorbance signal, abs non exposed subjects is the average absorbance signal of non-exposed subject samples. for ferret serum samples, the same basic protocol was followed, with minor modifications. an anti-igg anti-ferret polyclonal antibody preparation was used at a dilution of : in pbs-tween ae %. a recombinant receptor-binding domain of the ha of the influenza a ⁄ h n ⁄ virus, expressed in escherichia coli strains, was used as the elisa antigen. this kda protein, designated here as ha - -rbd, contained amino acids - of the influenza a ⁄ mexico ⁄ indre ⁄ (h n ) hemagglutinin. a sequence coding for a series of six histidines at the n-terminus of the protein was included in the genetic construct to allow purification using immobilized metal affinity chromatography (imac) and attachment to assay surfaces treated with anti-histidine antibodies (or alternatively co + or ni + ). a panel of four samples (kindly provided by st. jude from ferrets exposed to different influenza strains, namely h n , h n swine, and h n , was also tested by the elisa method using : dilutions. protein ha - -rbd specifically and selectively recognizes antibodies from serum samples from convalescent h n ⁄ influenza subjects. dubois et al. demonstrated that this protein, produced in e. coli, folds properly into a -d structure practically indistinguishable from the analogous region in the ha of the influenza a ⁄ h n ⁄ virus. ha - -rbd preserves three of the conformational immunogenic epitopes (sa, sb, and cb) described for influenza a ⁄ h n hemagglutinins. the recombinant protein was used as the antigen, attached through histidine tags to microplate surfaces treated with anti-histidine antibodies to discriminate between serum samples from subjects exposed and non-exposed to influenza a ⁄ h n ⁄ . samples collected before the pandemic onset, and therefore presumed to exhibit low specific antibody titers against influenza a ⁄ h n ⁄ , were analyzed by elisa using the antigen ha - -rbd. the histogram of normalized absorbance values from this sample set displayed a normal behavior with a standard deviation of ae units. only ae , ae , and ae % of these samples exhibited normalized absorbance values higher than ae , ae , and ae , respectively. no sample from non-exposed individuals presented an absorbance value higher than ae . variability among samples from non-exposed subjects was much lower than in samples with high specific serum antibody titers from convalescent h n ⁄ patients. exposure to the h n ⁄ influenza virus with this elisa method can be predicted by absorbance values normalized to those of abs norm ¼ ðabs serum ample À abs albumin sample Þ=ðabs non exposed subjects À abs albumin sample Þ ð Þ serum from uninfected subjects. consequently, for reliable results, inclusion of samples from non-exposed subjects on every assay microplate is necessary. figure shows the analysis of human serum samples, including samples from convalescent patients with positive diagnosis by rt-pcr. three positive (dark gray bars) and two negative controls (light gray bars) were included in the same microplate. all serum samples corresponding to convalescent subjects exhibited absorbance values ae - ae times higher than negative samples ( figure ). normalized absorbance values above ae suggested exposure to the virus, although, a more conservative threshold value of ae units is proposed for discrimination between exposed and non-exposed subjects. the elisa method described here yields adequate reproducibility and a high signal ⁄ noise ratio within determinations in the same microplate and among different microplates. using a normalized absorbance value of ae , the method was able to discriminate samples from convalescent patients, preferably after the third week of infection, and at least up to the twentyfourth week of exposure. assay sensibility was further validated against results from hi assays. a previously reported study showed that all members in a pool of fourteen samples diagnosed as positive by hi exhibited normalized absorbance values higher than ae , and % of them exhibited normalized absorbance values higher than ae . in general, high hi titers (> ) were correlated with normalized absorbance values higher than ae . figure a shows results using the ha-rbd elisa method and the hi assay on a pool of seventeen known positive serum samples corresponding to convalescent h n ⁄ patients. all samples determined as positive by hi ( samples) were also positive by elisa. while sensitivity of the hi assay was ⁄ = ae %, the elisa method recognized all samples correctly as positive ( % sensitivity) when a threshold of ae or ae was used. figure b shows that sera from ferrets infected with other influenza strains (h n , h n swine, and h n ) showed no cross-reactivity when analyzed by elisa. in summary, the ha-rbd elisa method presented here consistently distinguished influenza a ⁄ h n ⁄ infected and non-infected individuals, particularly after the third week of infection ⁄ exposure. since no actual viral particles are required, this assay can be readily implemented in any basic laboratory. in addition, should sufficient vaccine be unavailable, this elisa could determine the level of specific antibodies against the virus and presumably the extent of partial protection in a subject. therefore, the elisa protocol might allow better administration of vaccination programs during pandemic or seasonal influenza outbreaks. in april , a novel h n influenza virus emerged in north america and caused the first influenza pandemic of the st century. [ ] [ ] [ ] [ ] the pandemic h n (pdmh n ) has a unique gene constellation that was not previously identified in any species or elsewhere. it is genetically related to the triple reassortant swine h n influenza viruses currently circulating in north america, with the exception of the neuraminidase (na) and matrix (m) genes, which are derived from a eurasian swine influenza virus. swine h n influenza viruses were first isolated in and continued to circulate in north america with very little antigenic changes (classical swine h n ) until . since , however, the antigenic make up of swine h viruses has shown increased diversity due to multiple reassortment events and the introduction of h n genes from human influenza viruses. currently, four swine h clusters (a, b, c, d) are found endemic in the north american swine population. , these swine h viruses show substantial antigenic drift compared to the classical swine h viruses. cluster d swine h is derived from current human h viruses, and there is a substantial antigenic divergence between classical swine h and human seasonal h viruses. epidemiological evidence shows a two-way transmission of influenza viruses between swine and humans, and such events lead to the emergence of the pdmh n virus. , , phylogenetic analysis have suggested that possible ancestors of the eight genes of pdmh n were circulating in the swine population for at least years prior to the emergence of the pdmh n virus in humans, although the pdmh n virus itself was not isolated from pigs until after the pandemic. interestingly, pdmh n infections have been reported not only in humans and pigs, but also in other animal species such as turkeys, cats, ferrets, cheetahs, and dogs. [ ] [ ] [ ] after the first report of pdmh n infection in swine in canada, other countries, including argentina, australia, singapore, northern ireland, finland, iceland, england, united states, japan, and china reported outbreaks of pdmh n in swine as well. , [ ] [ ] [ ] the ample geographic range of pdmh n outbreaks in swine, its apparent broad host range, and the possibility of two-way transmission between swine and humans poses a tremendous challenge for controlling the virus. therefore, to differentiate pdmh n from other h strains, particularly in swine and human populations, is an important issue to ascertain the magnitude of the disease caused by the pdmh n . in this study, we developed an elisa assay to discriminate pdmh n strains from other swine and human h viruses. madin-darby canine kidney (mdck) cells (atcc, manassas, va, usa) were maintained in modified eagle's medium (mem) containing % fbs. a ⁄ california ⁄ ⁄ ⁄ h n virus (ca ⁄ ) was kindly provided by the centers for disease control and prevention (cdc), atlanta, georgia. other viruses are listed in table . viruses were propagated in mdck cells and stored at ) °c until use. viruses were titrated by the reed and muench method to determine the median tissue culture infectious dose (tcid ). three monoclonal antibodies ( b , h , and f ) against ha of pandemic h n were prepared in our laboratory following previously described methods (shao and perez et al., unpublished). purification and labeling of mabs mab b , h and f were purified on a protein g-sepharose affinity column (upstate biotechnology, lake placid, ny, usa). biotinylation of the detection antibody in the elisa was performed using sulfo-nhs-lc-biotin (sulfosuccinimidyl- -(biotinamido)hexanoate; pierce, rockford, il, usa) according to the manufacturer's instructions. purified h and f were selected as the capture antibody, and biotin-conjugated b was selected as the detection antibody, and hrp-conjugated streptavidin (abcom, cambridge, ma, usa) was developed using the tmb substrate system (kpl, gaithersburg, md, usa). in brief, the mixture of the purified h and f ( ae and ae lg ⁄ ml respectively, in carbonate ⁄ bicarbonate buffer, ph ae ) was coated to -well plates (test well, t) for h at °c. at the same time, a control antibody was coated to -well plates (control well, c). after blocking the plates with % (w ⁄ v) non-fat milk in pbs for hour at °c, the samples were diluted in extract buffer ( %tween- , ae %bsa in pbs) and added to the wells ( ll ⁄ well, each sample was table . specificity assay of the sandwich elisa result (t ⁄ c) added to four wells-two for t wells and two for c wellsand the mixture was incubated at °c for hour. after four washes, ll biotin-conjugated b ( ae lg ⁄ ml) in dilution buffer ( ae % bsa in pbs) was added to the wells and the mixture was incubated for h at °c. following three washes, ll diluted hrp-conjugated streptavidin ( ae ng ⁄ ml) in dilution buffer was added to the plates. after incubation for h at °c, the plates were washed five times, and the binding developed using the tmb substrate system for minutes. the ratio of the average od value of the t wells to that of the c wells (t ⁄ c) of individual samples was calculated. t ⁄ c values > ae were considered positive in the sandwich elisa. we developed three monoclonal antibodies, b , h , and f , against a prototypical pdmh n strain, a ⁄ california ⁄ ⁄ (h n ) (ca ⁄ ). these monoclonals were used to develop a rapid sandwich elisa for specific diagnosis of pdmh n strains. purified h and f were used as capture antibodies, whereas the biotin-conjugated b was used as detection antibody. the sandwich elisa showed strong reaction with different pdmh n strains as described in in order to evaluate if the sandwich elisa could distinguish the pdmh n from other swine h clusters (a, b, c, d), swine influenza strains spanning these clusters were tested. these viruses were first diluted : in extract buffer, and then added to the coated plates. as shown in table , the t ⁄ c ratios of these viruses were < ae , and therefore showed negative elisa result. likewise, testing of human seasonal virus strains a ⁄ brisbane ⁄ ⁄ (h n ), a ⁄ malaya ⁄ ⁄ (h n ), a ⁄ wsn ⁄ (h n ), and a ⁄ brisbane ⁄ ⁄ (h n ) also showed negative elisa results. furthermore, the sandwich elisa showed no cross reaction with avian influenza viruses, including strains of the h , h , h , h , h , h , h , h , h , h , and h subtypes. more recently, the mutation d g in the ha of some pdmh n strains has been associated with exacerbated disease and altered receptor binding. [ ] [ ] [ ] [ ] [ ] to evaluate if such mutant could be detected in our sandwich elisa, we tested a mutant of a ⁄ netherland ⁄ ⁄ (h n ) carrying the d g mutation (engineered by reverse genetics). as described in table , our elisa could still capture the d g mutant virus and showed a positive reaction, which highlights the specificity of our assay for pdmh n strains, even those with mutations. to evaluate the sensitivity of the elisa, we used the serially diluted pdmh n viruses to determine the limit of detection (lod). as shown in table , in our elisa the highest positive dilutions of nl ⁄ and ca ⁄ were : and : , respectively. the lod of the sandwich elisa by tcid was ae · and ae · tcid ⁄ ml, for nl ⁄ and ca ⁄ , respectively. it is important to note that the t ⁄ c ratio from nl ⁄ and ca ⁄ viruses showed clearly a dose dependent effect, while the t ⁄ c ratio of a ⁄ swine ⁄ iowa ⁄ (h n ) did not show the same dependence and was always < ae , corroborating the high specificity of the sandwich elisa for pdmh n strains. although we did not compare our elisa with other current commercial rapid influenza detection kits, the lod of our elisa assay is similar to other commercial kits that detect human seasonal influenza virus. comparison of the sandwich elisa with the ''gold standard'' -virus isolation in order to further evaluate the feasibility of the application of the elisa to clinical samples, nasal wash samples ae · ^ )( ae ) )( ae ) )( ae ) )( ae ) )( ae ) )( ae ) )( ae ) )( ae ) -from ferrets, of those previously infected with ca ⁄ and shown positive by virus isolation, were tested. the samples were diluted : in extract buffer and then tested using the sandwich elisa. result showed out of positive samples by virus isolation were positive also by the sandwich elisa (sensitivity ae %). the samples tested that were negative by virus isolation were also negative in the elisa, indicating % specificity for our assay. these results show not only that our elisa has high compatibility with the virus culture method, but also indicates this application can be used for clinical samples. although real time rt-pcr targeting the ha gene has been used for specific diagnosis of pdmh n with high sensitivity, [ ] [ ] [ ] [ ] [ ] [ ] it is a method that requires manipulation of the sample to extract viral rna, and it is prone to crosscontamination during the pcr steps. in this study, we described a convenient sandwich elisa based on three mabs developed against the pdmh n strain. the elisa not only shows high specificity for pdmh n strain, but also shows great sensitivity. the elisa could distinguish pdmh n strains from human seasonal h and h viruses and, more importantly, from other swine h viruses. we must note that current rapid diagnostic tests cannot be used to differentiate pdmh n from swine or human h viruses. it is also worth noting that the sensitivity of commercial rapid antigen-based diagnostic tests for detecting pdmh n is lower than that for human seasonal influenza viruses. , a study by kok et al. showed that sensitivity of the current rapid antigenic tests for pdmh n is only ae %, whereas that for seasonal influenza a is ae %. chen et al. developed a dot-elisa and increased the sensitivity for influenza rapid antigen detection. however, the dot-elisa developed by chen cannot distinguish among subtypes. the lod of our elisa is between ae · to ae · tcid ⁄ ml, comparable to the lod of rapid diagnostic tests for human seasonal influenza viruses. compared to the ''gold standard''-virus isolation-our sandwich elisa showed ae % sensitivity using ferret nasal washes. our results highlight the potential application of our sandwich elisa for the specific diagnosis of pdmh n viruses. the timely and reliable laboratory evidences are vital factors for field epidemiologists trying to control outbreaks of infectious diseases and for the practicing clinicians to properly manage disease cases. therefore, analysis of new detection methods in comparison to the routine ''classical'' methods is essential to select new methods to be introduced into health service practices, especially in developing countries. in this study we have compared rt-rt-pcr detection of influenza viruses and direct fluorescent-antibody assay using r-mix hybrid cells (a &mv lu) with the ''classical'' cell culture methods in developing country settings. in this study, we analyzed nasopharyngeal swabs col- the detection of influenza h , h , b, and pandemic influenza (h )pdm virus-specific nucleic acids was performed by rt-rt-pcr in abi fast real time pcr system using primers recommended by cdc, usa, and super-scriptÔ iii one-step rt-pcr and platinum Ò taq dna polymerase kits (invitrogen). the cycling protocol was: minutes at °c, minutes at °c, and cycles of seconds at °c, seconds at °c. rapid detection of influenza infected cells has been performed by dfa using the infected hybrid cells of r-mix within hours after inoculation, according to the manufacturers instruction (diagnostic hybrids, inc., usa). the isolation of influenza viruses was performed on mdck cell culture by the protocol recommended by cdc, usa. we detected ( ae %) influenza virus-specific nucleic acid fragments from all tested samples by rt-rt-pcr. among the positive samples, there were ae % a(h n ), ae % a(h n ), ae % influenza b, and ae % a(h n )pdm with different distributions by time series in different age-groups. inoculation of the cell lines by rt-rt-pcr positive samples selected randomly has detected influenza virus in ae % ( ⁄ ) on mdck cell culture and % ( ⁄ ) on r-mix hybrid cell culture with varying distribution for different strains. in other words, mdck cell culture technique was better for isolation for pandemic influenza viruses and dfa using r-mix hybrid cell culture technique for detection of seasonal influenza viruses (table ) . average times needed for the final results for different methods were: hours for rt-rt-pcr, hours for dfa on r-mix and days for mdck cell culture with two passages at least. the peak of the seasonal influenza a virus detection occurred in the - th weeks of , however the pandemic influenza detection peak was observed in the - th weeks of ( figure ). the outbreaks by seasonal influenza viruses was observed mostly among the children of - years of age, and pandemic influenza virus outbreak was observed mostly in the adults of - years of age. the results of this study indicate that rt-rt-pcr is the most suitable method for decision makers in epidemiological and clinical settings by sensitivity and timeliness. the final results show that r-mix dfa requires times longer, and by mdck cell culturing, times longer periods, than by rtrt-pcr. mdck cell culture technique has a higher isolation of pandemic influenza viruses, and r-mix dfa has a greater detection rate of seasonal influenza viruses by our results. according to our study, with rtrt-pcr, the isolation of positive samples by tissue culture of influenza a viruses was % and influenza b viruses was ae %, which is lower than in similar spanish study. however our study illustrates similar results with a canadian study where the sensitivity of dfa method and tissue culture technique was shown to be lower than rtrt-pcr sensitivity. as recorded by a study of american researchers, r-mix hybrid and conventional cell culture techniques have had similar sensitivity, which does not match the results of our study. however, the results of our study match with the results of italian and american scientists , where the r-mix hybrid method for seasonal influenza viruses is higher than mdck cell culture technique. background: viral kinetics is increasingly used to study influenza infectiousness. the choice of the study design, i.e. when and how many times nasal samples are to be collected in individuals depending on the sample size, is crucial to efficiently estimate the viral kinetics (vk) parameters. material and methods: we performed a model based optimal design analysis in order to determine the minimal number of nasal samples needed to be collected per subject and when to collect them in order to correctly estimate the vk parameters. the model used was a non linear mixed effect model developed with data collected from patients sampled nine times in days (initial design - samples collected), and we used d-optimization for design identification. we also computed the minimal number of participants necessary. results: considering that % of the influenza-like illness cases are not due to volunteer challenge studies have been used since the 's to provide data on virus shedding from the respiratory tract during influenza infection. recently, vk was studied in naturally acquired influenza infection. , these data are invaluable to describe the natural history of influenza-infection and to compute natural history parameters such as the latent period, generation time, or the duration of infectiousness. [ ] [ ] [ ] [ ] however, among the studies used in a meta-analysis about viral shedding kinetics, the designs varied greatly from one to another. these differences led to variable amount of available information concerning the vk. the lack of adequate sampling leads to imprecise estimates. on the other hand, intensive sampling or over-sampling, while associated with highly informative data, may lead to unnecessary discomfort for the patient and cost to the investigator. optimal design is increasingly used to conceive studies and provides cost-efficient designs. here we propose an optimised design to model vk in the case of influenza infection. we defined the number of participants, the number of samples to collect and their allocations. this design allows, at a minimum cost and discomfort, accurate vk curves and allows the natural history parameters to be well described. model a vk population model was proposed for influenza infection. this model describes with eight parameters the relations between free virus, uninfected target epithelial cells, infected epithelial cells, and early immune response. this model was built on a dataset of volunteers from which nasal samples were collected once a day over days. we call this dataset the ''original dataset''. three parameters, the induction of the early immune response, the virus production rate, and the virus clearance, did not show inter-individual variability and were precisely estimated (relative standard error below %). we considered them as fixed in this research work. five parameters were hence considered here: b the infection rate, d the infected cell mortality rate, w the effect of early immune response on virus production rate and v init the initial value of virus titre. in order to correctly estimate these parameters it is crucial to determine a design to collect informative data. optimal designs maximise the amount of information provided by the study. it involves the determination of the number and allocation of sample times per subject as well as the number of participants. d-optimization is based on the maximization of the determinant of the fisher information matrix and thus minimizes the variance of the parameters. we used the fedorov-wynn algorithm implemented in pfim . to maximize this determinant, which implies to pre-define a set of possible sample times. with the hypothesis that the inoculation occurred at : am, we chose three possible hours ( : , : , and : ) for each day with respect of the sleep-time. to validate the design, we simulated datasets of volunteers with the optimised design obtained. we then estimated the population parameters using monolix . for each of the datasets. we compared the estimated parameters obtained with the simulated datasets to the parameters used to build the optimal design. we computed the relative bias as: with n: number of successful estimations among the simulated datasets. h i : parameter value obtained with the ith dataset. h: parameter value obtained with the original dataset. we also compared the observed rse from these simulations with the rse predicted by pfim and the rse obtained with the original dataset. the rse is proportional to ffiffiffi n p , where n is the population size. we can hence deduce the smallest number of participants necessary to obtain rse below %. where rse predicted is the highest predicted rse (here rse for w) with participants and n predicted = and rse min is equal to ae . considering that % of the influenza-like illness cases are not due to influenza virus, the total number of participants should be multiplied by ae . we found that the best design was when all the participants are sampled five times: three times during the second day post-inoculation at : , : , and : hours and twice on the third day post-inoculation at : and : ( figure ). the comparison of the relative bias and rse predicted by pfim and those obtained after simulation and re-estimation of the parameters are shown in figure . v init and d in a lesser extent present bias. fixed effect parameters are precisely estimated and accordingly to pfim except for v init . we found that participants shedding virus or participants with ili symptoms are necessary if % of them are not infected with influenza virus. we propose an optimised design to accurately study the vk of influenza virus with the minimal number of samples. this design is well balanced between the amount of necessary information and the precision of estimation. we found that samples are necessary to precisely fit the vk curves, which is five times less than the number of samples collected in the original study. ??? the samples should be collected during the second and third days after inoculation. yet we showed in a previous work that the incubation period lasted ae days. ??? hence, the optimised sample times correspond to the two-first days of symptoms and this design could be applied to naturally acquired infections studies in which the inoculation time is unknown. an advantage of this design is its practicality and convenience. all samples are collected during the daytime and after the onset of symptoms. it can thus be used for studies with naturally acquired infections. the design was validated with several criteria concerning the accuracy of the estimation with the optimised design. the parameters estimates were generally satisfactory. the parameter describing the effect of the early immune response on the virus production rate was, however, less precisely estimated (predicted rse = %), and the initial value of the viral titre was very different of the one obtained with the original dataset (bias v init on figure ). this is probably due to the fact that it was measured at day post inoculation, and that the inter-individual variability is much higher than at day . furthermore, d (the infected cell mortality rate) seems also to be biased. this may be due to the fact that three parameters were fixed. the model used was developed from experimentally inoculated healthy volunteers with low serum haemagglutinin antibody titre and with virus inoculation time at : am. the applicability of the design to naturally acquired infection would depend on the pathogenicity of the virus as well as pre-existing immunity and the relevance of challenge method to natural influenza acquisition. our design could be directly used to accurately study vk during influenza infections and would reduce the discomfort of patients and the cost of the experimentation. usefulness of a self-blown nasal discharge specimen for use with immunochromatography based influenza rapid antigen test introduction influenza rapid antigen tests (irat) have become very popular and are widely used for confirming suspected clinical diagnosis of influenza in japan. most of the currently used irat that are based on immunochromatography (ic), nasopharyngeal swab, nasopharyngeal aspiration, and throat swab have been approved as specimens for japanese national health insurance purposes. but the specimen collection by these methods gives patients considerable discomfort, and sometimes appropriate specimens cannot be obtained due to patient resistance, especially by children. in the present studies, self-blown nasal discharge was used as the specimen for an irat, and the results were compared with the results of viral isolation and an identical kit primed with nasopharyngeal swab specimens for seasonal influenza viruses and pandemic (h n ) virus. patients who visited any of the clinics that belong to the influenza study group of the japan physicians association in the - and the - influenza seasons with influenza-like illnesses exhibiting findings were registered after providing informed consent. a square plastic sheet of · cm was handed to the patient. nasal discharge was collected by blowing the nose into the plastic sheet as a specimen for irat, i.e. self-blown specimen. two nasopharyngeal swab specimens were also obtained at the same time for irat and virus isolation. self-blown specimens were obtained successfully by ( ae %) of consecutive outpatients in the - season, as seen in table the sensitivity and specificity of various influenza rapid antigen tests have been reported in various settings. [ ] [ ] [ ] [ ] direct comparison of the results is difficult because of differences in patient or influenza virus, characteristics such as age, study designs, and other features. in this study of the - influenza season, the sensitivity, specificity, and accuracy of the ic kit primed with nasopharyngeal swab specimens were ae %, ae %, and ae %, respectively. these results were quite comparable to our results of the - season, in which the overall results of other ic kits were ae %, ae %, and ae %, respectively, indicating that the ic kit used is quite reliable. the sensitivity, specificity, and accuracy of an ic kit will vary by the method of specimen collection. in general, virus titer is considered to be highest with nasopharyngeal aspiration, lower with nasopharyngeal swabs, and lowest with throat swabs. practically, nasopharyngeal swab is the most popular. the sensitivity, specificity, and accuracy of the ic kit with self-blown discharge specimens compared well with those of an identical ic kit primed with nasopharyngeal swab specimens. for self-blown specimens, sensitivity and specificity were ae % and ae % for influenza a, ae % and ae % for influenza b, % and ae % for pandemic (h n ) . self-blown specimens display sensitivity, specificity, and accuracy comparable to that of conventional nasopharyngeal swab specimens. there was no significant difference in sensitivity, specificity, or accuracy between self-blown specimens and nasopharyngeal swab for influenza a, influenza b, and pandemic (h n ) . these results suggest that selfblown specimens are as useful as nasal cavity swab specimens for the diagnosis of influenza in the clinical settings. nasal discharge, obviously, cannot be collected from infants incapable of blowing their own nose or patients who do not develop a nasal discharge. in this study, self-blown specimens were obtained from ae % of the patients. the rate of successful collection was over % in the age groups of - and - years. these rates would seem to be sufficient for clinical use. the procedure of self-blown specimen collection using a plastic sheet is easy and causes no pain or discomfort. it seems to be more acceptable and safe than the other methods, especially for children. furthermore, this procedure reduces the risk of influenza transmission from patients to the medical staff members involved in sample collection. self-blown sample collection may be superior to other sample collection methods in these respects. we previously reported an inverse correlation between the amount of virus in a specimen and the time to a positive reaction. in this study, there was no significant difference in the mean time to a positive between self-blown self-blown specimens enough to be examined were obtained from consecutive outpatients, and specimens showed a tendency to be obtained large amount from children rather than the aged. there were no statistically significant differences between the ic kit results primed with self-blown discharge and nasopharyngeal swab specimens for influenza a, influenza b and pandemic (h n ) . and nasal swab specimens, suggesting that the self-blown specimens contained sufficient viral antigen for the ic kits. the influence of the presence or absence of nasal congestion on the results of the kit was assessed. the sensitivity of selfblown specimens from patients with nasal congestion was significantly lower than that from patients without nasal congestion. it is possible that insufficient capability to blow the nose due to nasal congestion might tend to lead to false negatives. the observation that the time to positive is longer for patients with nasal congestion than for patients without nasal congestion is concordant. application of self-blown specimen collection only to appropriate patients would increase the sensitivity, which would be important in a clinical setting. we tested only two commercial antigen detection kit, the quick vue rapid sp influ kit and quicknaviÔ-flu (denka-seiken co., ltd). the resulting sensitivity, specificity, and accuracy of the ic kit primed with self-blown specimens were considered adequate for clinical use. to confirm the usefulness of self-blown nasal discharge specimens, further investigation is necessary using other kits and in different settings. the usefulness of a self-blown nasal discharge specimen for an influenza rapid antigen test based on immunochroma-tography was evaluated in the - and - influenza season. results suggest that self-blown nasal discharge specimens are useful as specimens for influenza rapid antigen tests based on immunochromatography for not only seasonal influenza viruses, but also pandemic (h n ) virus. the specimen collection by the patients themselves will reduce the burden of other collection methods and the risk of infection to the medical staff. in april , a mixed-origin h n influenza virus was recognized as a new causative agent of influenza-like illnesses (ili) in humans. since its emergence, the virus has spread rapidly throughout the world and caused a pandemic. most commercial rapid antigen tests (rat) can detect influenza a or b viruses, but cannot specifically distinguish pandemic (h n ) virus with seasonal influenza. recent studies have indicated that the poor performance of the rat approach and nonspecific detec-tion of the pandemic (h n ) virus was the main obstacle to their widespread use in private clinics. , with the need for a new rapid kit with reasonable sensitivity and specificity for pandemic (h n ) virus, we developed a new rat kit in collaboration with company, standard diagnostics, inc., (yongin-si, gyonggi, korea). monoclonal antibody (mab) against haemagglutinin (ha) of the pandemic (h n ) virus was developed using korean isolate and applied to the new kit with the mab to seasonal influenza virus. we examined the detection limit of the kit using the serial dilution of korean pandemic virus isolate (a ⁄ korea ⁄ ⁄ ). during december , clinical specimens from patients with ili were collected at sentinel clinics of six provinces in korea. the specimens were tested by the new rat, and the results were compared with those of real-time reverse transcription polymerase chain reaction (rrt-pcr) by us cdc and virus isolation in mdck cell culture to determine the sensitivity and specificity for the diagnosis of pandemic (h n ) . the detection limit of the new kit against ha of a ⁄ korea ⁄ ⁄ virus was confirmed to be pfu ⁄ ml. by contrast, the detection limit against the np protein was pfu. however, when the kit was applied to clinical specimens, no difference between the two targets was found. using rrt-pcr and viral culture as the references, the performance of the ridt is shown in table . among specimens, were tested positive by rrt-pcr and were tested positive by viral culture. among the rrt-pcr confirmed cases, were positive, and among the viral culture confirmed cases, were positive with the new rat. using rrt-pcr as the reference standard, the overall sensitivity of rat was ae % ( % confidence interval (ci): ae - ae %) and specificity was ae % (ci: ae - ae %). with viral culture as the reference, the rat sensitivity and specificity was ae % (ci: ae - ae %) and ae % (ci: ae - ae %), respectively. when analyzed by the regions tested, the sensitivity ranged between ae % and ae % for rrt-pcr and between ae % and ae % for viral culture as a reference. among patients who had a record of their symptom onset and sample collection date, ( ae %) visited the clinic on the day of symptom onset, and ( ae %) visited day later. when the rat performance was evaluated by day of onset, the sensitivity was lower at three or more days after the onset of symptoms; however, the sensitivity was highest at days after onset and reasonable on the day of onset or at day after ( table ). we found that this new rat had reasonable sensitivity and high specificity compared with rrt-pcr and viral culture for detecting the pandemic (h n ) virus. in one recent study, the sensitivity and specificity of the new rat kit was % and %, respectively, and the ha protein for pandemic (h n ) was detected more sensitively than the np protein for influenza a virus. the sensitivity and specificity of our new rat were lower than those of that study. we found that the test performance varied depending on the clinics in which the tests were performed, and this might be attributable to the persons who collected the specimens. although the clinicians were trained well for *ci, confidence interval. **ppv, positive predictive value. ***npv, negative predictive value. collecting specimens, there might be some differences in performance. the new rat kit could detect pandemic (h n ) virus specifically. although the sensitivity was lower than those of rrt-pcr and virus culture, and negative rat results should be confirmed with more sensitive methods, this kit could be useful in sentinel clinics if used with caution. determination of infectious virus titres is central to many experiments designed to study the biology of influenza virus. assays based on the measurements of viral components, whether viral protein or nucleic acid, does not differentiate infectious virus from non-infectious or defective viral particles, which may have no infectivity or biological *three hundred and forty samples with a known date of onset and sample collection were analyzed. ª blackwell publishing ltd, influenza and other respiratory viruses, (suppl. ), - activity. therefore the ''gold standard'' of virus measurement requires bioassays that examine the ability of viral particles to replicate and further infect other cells. titration on madin-darby canine kidney (mdck) cells in a well plate format is commonly used to measure influenza virus titre. this method is labour intensive, subjective in their read out of cytopathic effect, and takes several days to obtain a result. microneutralization tests that quantitate neutralizing antibody titres and assays of drugs for antiviral activity also require well based assays of residual virus infectivity. therefore, technologies that improve on the titration of infectious virus will be of great benefit. this study utilized the xcelligence system (roche applied science), which adopts microelectronic biosensor technology to monitor dynamic, real-time label free and non-invasive analysis of cellular events. the system measures electronic impedance using an array of microelectrodes located at the bottom of each culture well (e-plate ). adherent cells are attached to the sensor surface of electrode arrays, and changes in impedance can be detected and recorded. the xcelligence system can monitor cell events induced by viral infection, such as changes in cell number, adhesion, viability, morphology, and motility. measured electrode impedance is expressed as dimensionless cell index and is graphically represented using software to show the phenotypic changes of a cell population over time. the aim of this study is to demonstrate that using this platform to measure real-time cell index has potential to circumvent many of the limitations of the currently established procedures of end point titration of virus infectivity and for microneutralization assays. madin-darby canine kidney cells were propagated in growth medium consisting of minimum eagle's medium (invitrogen) supplemented with % fetal bovine serum (invitrogen), ae mg ⁄ l penicillin (invitrogen), and mg ⁄ l streptomycin (invitrogen), with incubation at °c in a % co humidified atmosphere. influenza a ⁄ hong kong ⁄ ⁄ (h n ), a seasonal influenza virus from a patient who suffered from a mild febrile illness, was propagated in mdck cells maintained in virus medium consisting of minimum eagle's medium (invitrogen) supplemented with ae mg ⁄ l penicillin (invitrogen), mg ⁄ l streptomycin (invitrogen), and mg ⁄ l np-tosyl-l-phenylalaninechloromethyl ketone-treated trypsin (sigma, st louis, mo, usa), with incubation at °c in a % co humidified atmosphere. virus stocks were aliquoted and stored at °c until use, and the % tissue culture infectious dose (tcid ) of the virus stock was determined by titration in mdck cells according to standard procedures, and the tcid of the stock virus was calculated by the method of reed and muench. to perform a microneutralization assay, mdck cells seeded at a density of cells ⁄ well in an e-plate was removed from the xcelligence system after approximately hour; growth medium was then removed, cells washed, and replaced with ll virus-medium. a human serum, which is known to contain high titre antibody against the h n virus was heat inactivated for min at °c, and twofold serial dilutions were performed in virus medium. the diluted serum was mixed with an equal volume of virus medium containing influenza virus at tcid ⁄ ll. after incubation for h at °c in a % co humidified atmosphere, ll of virus-antibody mixture was added to the mdck cells to give each well an equivalent virus dose of tcid . a back titration of the virus challenge dose was performed, and a cell control (free of virus) was performed in quadruplicates. after incubation at room temperature for minutes, the e-plate was then placed back onto the xcelligence system in the incubator and maintain at °c with % co , and the cell index values were measured every minutes for at least a further hour. the same procedures were performed with cells seeded in conventional well cell culture plates for parallel comparison with the currently used standard method. in this case, cells were examined for cytopathic effect under an inverted microscope after days of infection and the lowest virus dilution, which protected the cells from viral induced cytopathic effect taken as the neutralizing end point. after hour of seeding mdck cells at cells ⁄ well, standard microneutralization assay for influenza virus was performed. integral to this assay, a serial titration of the input virus at ae log increments was carried out. wells infected with the undiluted virus ( tcid ⁄ well), the cell index commenced dropping at a steeper gradient than the no-virus cell control after approximately hour of infection ( figure ). this drop in cell index continues at a consistent slope until it flattened out when approaching zero cell index. this steep decrease in cell index with constant gradient was also observed for virus dilutions up to and including log ( -folds), and the profile shifted with increased time in proportion to the dilution made to the virus. virus dilutions beyond log have cell index profiles similar to the no virus input control, and this corresponds to the absence of cytopathic effect as determined by microscopic observation at hour after infection. hence, there was a correlation between the amount of virus used for infection, the onset of the influenza virus-mediated cytopathic effect, and the steep decline in cell index. a human serum with known microneutralization antibody titre to h n virus was used in this study to investigate the real time cell index changes that occur during the assay ( figure ). using influenza virus treated with serum dilutions up to and including a dilution of : , the cell index profile remained essentially the same as the no virus cell control, which correlates with the lack of cytopathic effects under microscopic observation at hour of infection. at a serum dilution of : , the steep decrease in cell index, which is characteristic of cellular cytopathic effect induced by the virus, became evident at around hour post infection, and this was reduced to hour when serum dilution of : was used. in contrast, for the virus -no antibody control, the onset time for this steep decrease in cell index occurs at approximately hour. for both serum dilutions of : and : , full cytopathic effect was observed microscopically at hour of infection. from microscopic observation of cytopathic effect, according to the current standard procedures, the neutralizing titre of the human serum used in this study is at : as it is the last dilution of the serum that prevented cytopathic effect from being detected. an essential part of the microneutralization assay is to confirm the titre of the input virus (normally tcid ⁄well) by performing a titration assay with decreasing serial dilutions of the virus. under normal procedures, cells are examined microscopically after hour of infection for sign of cytopathic effects. in the case of mdck cells, the cytopathic effect is cell death, which is indicative of the presence of live influenza virus infecting and replicating in the cells. therefore, the titre of the virus is taken as the last dilution in which cytopathic effect is present. parallel realtime cell index measurements demonstrated that for wells with cytopathic effects, the profile exhibits a steep gradient linear decrease in cell index after infection with the virus, which can be termed the ''cpe plunge.'' the time in which the cpe plunge became evident appears to be inversely proportional to the amount of virus, therefore the opportunity exists to utilize this aspect to calculate or compare quantitatively different virus concentrations. for unequivocal assignment of cytopathic effect, it normally requires - days after infecting the cells, with days after infection being the standard time to read virus titration and microneutralization assays. using the real-time cell index monitoring, it is found that apparent cytopathic effect can only be observed microscopically when the cell index has dropped to near zero. as the time of onset of the ''cpe plunge'' becomes evident many hours prior to observable cytopathic effect, it is possible that the time to results can be drastically reduced after some formulation of the method. we compared the current standard method in perfoming a microneutralization assay with one utilizing the real-time cell index measurement to investigate whether this approach is able to offer better performance over the existing one. the current standard neutralization assay is the microscopic observation of antibody mediated protection from virus cytopathic effect in mdck cells. this study showed that this may also be achieved by examining the profile generated from the real-time measurements of the cell index. using real-time cell index monitoring, it is possible to detect inhibitory activity at higher dilutions of the anti-serum than can be detected by the standard microscopic observation of cytopathic effect. therefore, the realtime cell index monitoring could potentially be developed to be a more sensitive method for measuring anti-viral activity. as drug resistant strains of influenza a viruses including the pandemic h n are being reported, the real-time cell based monitoring system may also have the potential to be developed for use as a diagnostic platform for drug resistance assays. this study suggests that real-time cell index monitoring has the potential to substantially reduce human resources in reading results, as well as reducing time-to-result of these assays from days to two. the saving could be substantial for work involving bio-hazard level ⁄ pathogens such as h n viruses as personnel working with these organisms are require to be highly trained and experienced. in addition, the reduction in transferring plates to and from the microscope in reading cytopathic effect will substantially reduce the possibility of accidents from occurring. furthermore, the system provides objective digital data to an otherwise subjective assay method, which can improve standardization, data exchange, and hence collaboration between different laboratories. with more detailed validation and development, real-time cell index monitoring could transform the way we study and diagnose infection with pathogens such as influenza viruses. the emergence of a novel h n influenza a virus of swine origin, the pandemic a(h n ) , with transmissibility from human to human in april posed pandemic con-cern and required modifications to laboratory testing protocols. a new protocol for universal detection of influenza a and b viruses and simultaneous subtyping of influenza a (h n ) virus, composed of two-one-step rt-pcrs, fast set infa ⁄ infb and fast set h n v (relab, italy), was evaluated and compared to the reference protocol recommended by who. fast set infa ⁄ infb was able to detect influenza a and b viruses circulating between and belonging to different subtypes and lineages, and no cross reactions were observed by either fast set infa ⁄ infb or fast set h n v. the who assay was found to have a slightly lower end-point detection limit ( ) dilution) in comparison to the new protocol ( ) ). specificity of the assays was % as assessed on a panel of stored clinical samples including adenovirus, respiratory syncytial virus, metapneumovirus, parainfluenza virus, s. pneumoniae, n. meningitidis, h. influenza, and human influenza viruses. the new assay panel allows the detection, typing, and subtyping of influenza viruses as requested for diagnostic and surveillance purposes. the high sensitivity of the protocol is coupled with capacity to detect viruses presenting significant heterogeneity by fast set infa ⁄ infb and with high discriminatory ability by fast set h n v. a rapid and sensitive assay for the detection of influenza virus in clinical samples from subjects with ili or low respiratory tract infections is a fundamental tool for epidemiological and virological surveillance, management of hospitalized patients, and control of virus nosocomial transmission. the emergence, in april , of a novel h n influenza a virus of swine origin, the pandemic (a(h n ) ), with transmissibility from human to human poses pandemic concern and required modifications to the laboratory testing protocols. molecular diagnosis of influenza is generally achieved through a twophase process: a screening phase for the detection of virus, and the subsequent strain characterization performed by either sub-type-specific rt-pcr or entire ⁄ partial genome sequencing. during a pandemic, simultaneous implementation of both the detection of influenza a and b influenza viruses and identification of the new subtype is useful for clinical and epidemiological reasons. here, we describe a new protocol including two-one-step rt-pcrs, fast set infa ⁄ infb and fast set h n v (relab, italy) that allows universal detection of all influenza a viruses and, simultaneously, all subtypes that are influenza a(h n ) . specificity and clinical sensitivity of the two-one-step rt-pcrs (fast set infa ⁄ infb and fast set h n v; relab, italy) were evaluated by testing selected specimens, including: • fifty samples collected from nasopharyngeal swabs representative of influenza viruses, belonging to differ-ent subtypes and lineages, and other respiratory viruses and bacteria circulating in italy between and . • six purified a(h n ), a(h n ), and a(h n ) strains, kindly supplied by alan hay, who influenza centre, london, uk. • two hundred-fifty influenza positive samples selected according to type, subtype, clade and viral concentration from > specimens received by the liguria influenza reference laboratory between january st and december st, . since , nasopharyngeal swabs sampled from patients suspected of having contracted the influenza virus have been collected in viral transport medium, and upon arrival into the laboratory, the samples were divided in ‡ aliquots. those not immediately processed were stored frozen at ) °c. stored samples were used for this evaluation, and all specimens were re-extracted for the study. samples collected between and included specimens positive for: no seasonal a(h n ) have been detected since january st, . furthermore, weak positive sample using fast set infa ⁄ infb, but negative at block pcr and typing ⁄ subtyping assays was tested. the analytical sensitivity of the test under investigation was determined testing ten-fold serial dilutions of seasonal influenza a(h n ), seasonal influenza a(h n ), new pandemic influenza a(h n ) , and b cell culture-grown viruses. the intra-assay reproducibility was measured by testing the same a(h n ) positive sample times in the same experiment, while the inter-assay reproducibility was confirmed by testing the same samples in independent experiments. to evaluate the performance of the protocol, all samples were tested using a block pcr confirmation test (seeplex Ò rv ace detection), and all specimens collected between january st and december st, and dilutions were also assayed using the recommended who ⁄ cdc protocol of real-time rtpcr for influenza a(h n ). typing and sub typing were performed using the who protocol and ⁄ or sequencing. viral rna was extracted from swabs using the qiaamp viral rna mini kit (qiagen) according to the manufacturer's protocol. fast set infa ⁄ infb and fast set h n v are two multiplex one-step real time pcr assays developed and evaluated by the liguria regional reference centre for diagnosis and surveillance of influenza in collaboration with relab diagnostics. both assays contain primers and a dual-labelled hydrolysis probe that targets two regions of the matrix gene (table ) . amplification conditions were as follows: reverse-transcription °c for minutes, denaturation °c for minutes, then cycles of °c for seconds, °c for seconds. the entire amplification process extended for minutes. an internal control real-time assay was also incorporated in order to detect pcr inhibition, failed extraction ⁄ pcr and technical error. the cdc realtime rtpcr (rrtpcr) protocol for detection and characterization of swine influenza includes a panel of oligonucleotide primers and dual-labelled hydrolysis (taqman Ò ) probes to be used in real-time rtpcr assays for the in vitro qualitative detection and characterization of swine influenza viruses in respiratory specimens and viral cultures. this protocol recommends three primer-and-probe sets: infa, amplifying a conserved region of the matrix gene from all influenza a viruses; sw infa, designed to specifically detect the nucleoprotein (np) gene segment from all swine influenza viruses and sw h , designed to specifically detect the hemagglutinin gene segment from a(h n ) . the seeplex Ò rv ace detection for auto-capil-lary electrophoresis is a multiplex block rt-pcr that applies dpoÔ (dual priming oligonucleotide) technology and is designed to detect major respiratory viruses, respiratory rna (influenza a and b virus, parainfluenza virus type , and , respiratory syncytial virus a and b, rhinovirus a ⁄ b, coronavirus oc and e ⁄ nl ) viruses and dna (adenovirus) virus, from patients' samples including nasopharyngeal aspirates, nasopharyngeal swabs and bronchoalveolar lavage. conventional viral culture was performed inoculating ae ml of each specimen into mdck-siat seeded into -well plates for influenza isolation. virus detection was performed by the hemagglutination test using ae % guinea pig red blood cells (rbc). specificity and clinical sensitivity results of the new protocol are reported in table . fast set infa ⁄ infb was able to detect influenza a and b virus circulating between and belonging to different subtypes and lineages, and no cross-reactions were observed by either fast set infa ⁄ infb or fast set h n v. among specimens collected between january st and december st, , all fast set infa ⁄ infb and fast set h n v high titre positive samples resulted positive using the who ⁄ cdc assay and showing reactivity using infa and sw infa primer-andprobe sets. among low titre a(h n ) positive samples at fast set infa ⁄ infb, ( ae %) were not detected by the who ⁄ cdc assay, but were positive using seeplex Ò rv . the who ⁄ cdc sw h primer-and-probe set works in ae % ( ⁄ ) and ae % ( ⁄ ) of high and low titre a(h n ) positive samples, respectively. all a(h n ) strains collected during and initially detected by fast set infa ⁄ infb were confirmed after rna re-extraction by seeplex Ò rv and who ⁄ cdc assay showing reactivity using the infa primer-and-probe set. all infa ⁄ infb were confirmed after rna re-extraction by seeplex Ò rv . one influenza a case identified by the who ⁄ cdc kit (infa primer-and-probe set, ct values: ae , sw infa primerand-probe set: negative) and new protocol (a primer-andprobe set, ct values: ae , a(h n ) primer-and-probe set, ct values: ae ) was not detected by either seeplex Ò rv or by who subtyping protocol and ⁄ or sequencing, suggesting a very low viral load or unspecific results by real time assays. the analysis of serial dilutions of cell culturegrown a(h n ) showed that the detection limit of fast set infa ⁄ infb, fast set h n v, and seeplex Ò rv was identical ( ) ) and log lower than that using the who ⁄ cdc protocol ( ) ). a similar analysis with respect to a(h n ) and a(h n ) strains indicated that fast set infa ⁄ infb sensitivity ( ) and ) , respectively) was log lower than that showed by seeplex Ò rv ( ) and ) , respectively). in comparison with the new protocol, the who ⁄ cdc assays, considering infa primer-and-probe set, was found to have a slightly lower end-point detection, detecting the ) a(h n ) and a(h n ) dilution. also in detecting influenza b virus, fast set infa ⁄ infb sensitivity ( ) and ) , respectively) was log lower than that showed by seeplex Ò rv and the who ⁄ cdc protocol. data on intra-assay and inter-assay precision, measured as cv% of ct showed that the dispersion indices observed had values of less than %. since samples were detected using the new protocol that resulted negative using the who ⁄ cdc assays. the unfortunately low quantity of low titre a(h n ) samples collected during did not allow us to highlight differences between assays fast set infa ⁄ infb, and fast set h n v positivity was always confirmed by seeplex Ò rv , which demonstrated high sensitivity, showing a detection limit comparable or lower when compared with those observed using the who ⁄ cdc assays. the high analytical sensitivity of seeplex Ò rv is reported by kim who observed a detection limit of copies per reaction for each type ⁄ subtype of influenza viruses. the high sensitivity of the new protocol is coupled with its capacity to detect viruses presenting a significant heterogeneity by fast set infa ⁄ infb and high discriminatory ability by fast set h n v. fast set infa ⁄ infb was able to identify representative influenza viruses of circulating strains during the last decade belonging to different subtypes, lineages, and clusters, and fast set h n v primerand-probe set reacted selectively with a(h n ) target. a recent report demonstrated that the sw infa assay is not specific to a(h n ) and is able to detect both human and avian (h n ) influenza a viruses and so there is the potential for misidentification. high titre (ct ae and ae at fast set infa ⁄ infb) a(h n ) viruses did not react with fast set h n v primer-and-probe set (data not shown). available human a(h n ) sequences are similar within the h n v primer-and-probe regions, but having - mismatches in the forward primer and, more notably, two of the mismatches occurred within nucleotides of the end, an important determinant for primer specificity. in conclusion, this protocol can be a powerful tool in the diagnostic laboratory setting for specific simultaneous analysis of several samples in minimal time, showing enhanced sensitivity in detecting influenza viruses, and high discriminatory ability in identifying the new pandemic a(h n ) . a university-corporate partnership to enhance vaccination rates among the elderly: an example of a corporate public health care delivery public health campaigns usually rely on governmental infrastructure and finance for vaccine implementation programs. however, there are many financial and physical barriers which preclude widespread and effective vaccine administration, especially among the elderly. on an international scale, both government agencies and citizen groups have a vested interest in searching for more resourceful methods of attaining significant immunization levels (> % of the population). in fact, it seems to have become both a grassroots civic and governmental goal, especially among developing countries. we implemented the unique strategy of enlisting the assistance of a privately-owned food market chain to address the public health issue of mass vaccination for the elderly. in this context, publix pharmacy and the university of south florida (usf) recently developed both a handbook and a training program to facilitate the administration of vaccinations. between and , the publix-usf partnership resulted in administration of over thirty thousand influenza a (h n ) vaccinations, % of which were given to adults over years of age. consequently, vaccine administration costs were decreased by using corporate resources and bypassing overly strained municipal resources. this unique university-corporate partnership successfully delivered h n vaccine to a vulnerable cross-section of society at a lower cost and with minimal side effects and morbidity. it may be safely projected that university-corporate partnerships could result in an effective method for rendering a vital service to an aging and especially vulnerable segment of the population. government policy and funding are the foundation of immunization programs on an international scale. for example, in the united states, governmental programs account for over % of the monetary outlay used for immunization. until , the global alliance for vaccines and immunizations (gavi) acted as a catalyst for implementing vaccine and immunization programs in each targeted country. under the auspices of gavi-collaborations between governments, charitable organizations, and multinational health agencies (such as uncief and the who)-many countries have increased their spending for vaccination programs. however, development of financially sustainable immunization programs geared toward reaching the majority of the population are still at a nascent level of evolution. the development of more innovative and costeffective approaches has become imperative in order to reach a greater number of vaccination candidates. administering the influenza vaccine only to the subpopulation of over year olds would save an estimated quality-adjusted life years in a cohort of approximately half the world's population. widespread public vaccination programs are made more complex by the continuing development of newer vaccines, concomitant specialized administration costs, and the logistical challenge of conveying recipients to vaccination points of service. , in spite of the increasing complexity of mass vaccination, cost-benefit analyses clearly favor annual influenza vaccination in the elderly population on an international scale. , recently, in , influenza vaccine administration was reported to reach between % and % of the elderly population, which denotes varying degrees of success within each particular country. , however, there was also a report of a uniform plateau effect at around % of the population, beyond which additional vaccination coverage was difficult to achieve. physical limitations to vaccination seem to be more insurmountable for the elderly. unfortunately, this is the population segment which could experience the most significant vaccination-associated mortality reduction. we employed the unique strategy of involving the resources of publix supermarkets, a corporate food market chain, to address the public health issue of widespread vaccination for the elderly. we took advantage of recent changes in the florida statutes, which expanded the scope of pharmacists' practice to include administration of vaccines. subsequently, publix pharmacy and the university of south florida (usf) developed a handbook and training program to facilitate and enhance vaccine administration by publix pharmacists. by using proprietary pharmacists and more practical supply storage, we were able to decrease the costs of vaccine administration. the consumer was charged $ for administration costs plus the cost of the injection itself, regardless of insurance or eligibility for governmental subsidy. although patients were initially self-selected, they were ultimately excluded if they had demonstrated prior adverse effects to influenza vaccinations or to any of the components of such vaccinations. between and , the publix-usf partnership vaccinated people against influenza a (h n ), of which were florida residents. the age range was - years old with a median age of years old. seventysix percent of the participants were over years old (see figure ). within the population surveyed, the reported side effects of the vaccine in this study were not serious, but included: vertigo, cold sweats, chills, vomiting, syncope, rash, nausea, stomach pain, elevated blood pressure, injection site reaction, inflamed bursa, and bilateral thigh discomfort. participants from all socioeconomic classes were vaccinated. an income-by-zip code analysis revealed % of those vaccinated resided in zip code areas where the average household income was <$ per year. of those remaining, % had an average income of $ -$ per year, and % had an income of >$ per year. each person vaccinated was charged ten dollars for administration costs. this represents a decrease in the administration costs ranging from one dollar to ten dollars saved per vaccine. , conclusion this unique university-corporate partnership successfully delivered h n vaccine to a high-risk population with decreased vaccine administration costs. the influenza vaccine is well-tolerated, with minimal side effects when patients who have a history of adverse reactions are excluded. we can postulate that university-corporate partnerships may indeed be effective at reaching the aging population which is a challenge in most communities. this delivery model may prove to be another tool for improving the efficiency of mass immunization by facilitating accessibility, which results in wider coverage. this model also enhances delivery of healthcare by decreasing costs of immunization regardless of whether the payer is a government, insurance company, or self-pay consumer. the gavi initiative stressed three goals for accomplishing sustainability and independence in immunization programs. the goals were to: (i) mobilize additional resources from governmental and non-governmental sources; (ii) improve program efficiency to minimize additional administration resources needed; and (iii) increase the reliability of funding. empowering privately owned corporations within the community, such as food markets or pharmacies, to administer vaccines mobilizes additional resources to readily achieve the first goal of gavi. mobilizing resources of non-healthcare, corporate vaccination locations enhances accessibility due to travel convenience. in our study, participants came from all socioeconomic classes, suggesting that ease of access is independently hindering mass vaccination, and that people of all incomes are more likely engaged when access issues are eliminated. the second and third goals were also accomplished by recruiting a corporation's resources for vaccine administration (refrigeration, storage, and employees). this minimizes the money spent from vaccine program funds to support the infrastructure of immunizations, thus improving financial efficiency and sustainability. financial efficiency implies that money is spent to safely reach as large a portion of the population as possible. by using corporate storage facilities instead of paying for independent facilities, money can be spent elsewhere. more vaccines can be purchased and more money can be spent on media communications to encourage vaccination. sustainability requires the ability to fund annual vaccination programs which reach % of the population or greater. key to the control of pandemic influenza are surveillance systems that raise alarms rapidly and sensitively. in addition, they must minimise false alarms during a normal influenza season. we develop a method that uses historical syndromic influenza data from the existing surveillance system 'servis' monitoring seasonal ili activities in scotland. we develop an algorithm based on wcr of reported ili cases to generate an alarm for pandemic influenza. wcr is defined as the ratio of the number of reported cases in a week to the number of cases reported in the previous week. from the seasonal influenza data from scottish health boards, we estimate the joint probability distribution ( figure ) we compare our method, based on our simulation study, to the mov-avg cusum and ili rate threshold methods and find it to be more sensitive and rapid. the wcr method detects pandemics in larger fraction of total runs within the same early weeks of pandemic starting than does any of the other two methods ( figure ). as shown in the table, for % pandemic case reporting rate and detection specificity of %, our method is % sensitive and has mdt of weeks, while the mov-avg cusum and ili rate threshold methods are, respectively, % and % sensitive with mdt of weeks. at % specificity, our method remains % sensitive with mdt of weeks. although the threshold method maintains its sensitivity of % with mdt of weeks, sensitivity of mov-avg cusum declines to % with increased mdt of weeks. for a two-fold decrease in the case reporting rate ( ae %) and % specificity, the wcr and threshold methods, respectively, have mdt of and weeks with both having sensitivity close to %, while the mov-avg cusum method can only manage sensitivity of % with mdt of weeks. the first cases of the pandemic were reported in scotland in the th week of the season. the wcr algorithm as well as the mov-avg cusum method detects the pandemic weeks later in week . the ili threshold method detects it week later in week . both the wcr and mov-avg cusum methods therefore outperform the ili threshold method by week in the retrospective detection of the pandemic in scotland. while computationally and statistically very simple to implement, the wcr method is capable of raising alarms rapidly and sensitively for influenza pandemics against a background of seasonal influenza. although the algorithm has been developed using the servis data, it has the capacity to be used at large scale and for different disease systems where buying some early extra time is critical. more generally, we suggest that a combination of different statistical methods should be employed in generating alarms for infectious disease outbreaks. different detection methods would provide cross-checks on one another, boosting confidence in the outputs of the surveillance system as a whole. real-time evidence being created worldwide will greatly contribute to the full understanding of influenza pandemics. here we report the real-time epidemiology and virology findings of the influenza a(h n ) pandemics in mongolia. the epidemiological and virological data collected through isss of nic, nccd, mongolia (real-time information on registered ili cases and virological laboratory results are available from the weekly updates in the nic, mongolia website: http://www.flu.mn/eng/index.php?option=com_ content&task=category&sectionid= &id= &itemid= ) were used for analysis in relation to the previous seasonal influenza activities in the country. influenza viruses were detected in naso-pharyngeal samples from ili patients by rt-rt-pcr with applied biosystems fast real time pcr system , using primers and instructions supplied by cdc, usa. influenza viruses were isolated by inoculation of rt-rt-pcr-positive samples of mdck cell culture according to the standard protocol. ten representative strains of a(h n )pdm viruses were selected for sequencing of different gene segments, namely: a ⁄ ula- , and a ⁄ dundgovi ⁄ ⁄ . sequencing of influenza virus gene segments was performed in applied biosystems xl genetic analyzer using primers and instructions supplied by cdc, usa, and bioinformatic analysis was performed with abi ⁄ seqscape v. . and mega programs. the pandemic alert in mongolia was announced by the government on april , , just after the who announcement of the pandemic alert phase, and planned containment measures were intensified. despite intensive surveillance, no a(h n )pdm virus was detected in mongolia until the beginning of october . around suspected cases, mostly arriving from the a(h n )pdm epidemic countries, tested zero by rt-rt-pcr for a(h n )pdm virus. the first a(h n )pdm case detected by the routine surveillance system in ulaanbaatar city, the capital of mongolia, was confirmed by rt-rt-pcr on october , ( st week of ). the reported ili cases escalated rapidly, reached the peak in the - th week of , and gradually decreased thereafter ( figure ). week of . however, the registered ili cases increased again from the th week of , and peaked at the - th weeks of . the viruses isolated during this nd peak were influenza b strains ( figure and table ). for the genetic characterization of the mongolian pandemic isolates, gene segments i (pb ), gene segments ii (pb ), gene segments iii (pa), gene segments iv (ha), gene segments v (np), gene segments vi (na), gene segments vii (m), and gene segments viii (ns) of the representative a(h n )pdm mongolian strains were sequenced, and all sequences have been deposited in the genbank (accession numbers: cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy ). all genes of mongolian strains were possessing ae - ae % similarity with the genbank deposited gene sequences of the original pandemic strain a ⁄ california ⁄ (h n ). the who declared the pandemic alert phase (phase iv) on april , , and was prompted to announce the pandemic phase (phase v) two days later. after days, the who declared the beginning of the pandemic peak period (phase vi) on june , . however, in mongolia, the pandemic alert period continued for days. mongolia was free of the pandemic virus during the whole first wave of the pandemics in the northern hemisphere. with the confirmation of the st influenza a(h n )pdm case on october , in ulaanbaatar, mongolia entered into the pandemic phase (phase v), and after just weeks, the registered ili cases peaked, confirming mongolia shifted into the pandemic peak period (phase vi), which i i i v i i v i v v v i i i i iii iv v vi vii viii worldwide by who in mongolia coincided with the nd wave of pandemics in many countries of the northern hemisphere (see, picture and table ). despite the relatively milder clinical manifestations, the disease burden for the health service was enormous, while the morbidity per population at the peak period was - times higher above the upper tolerant limit, and - times higher above the seasonal influenza outbreaks. in contrast to the seasonal influenza outbreaks where over % of the registered ili cases have been in the age group under , it has been observed that over % of the registered cases in this pandemic peak period were in the age group of - . on january , , we regarded the pandemic had entered into the post-peak period (phase vii) when the registered ili cases became lower than the upper tolerant limit, during which time mongolia experienced an influenza b outbreak. on may , we determined that mongolia entered the post-pandemic period (phase viii) as the influenza virus isolations were almost stopped, and after no pandemic virus detected for months. who announced pandemic vii and viii phases much later. , this first ever real-time laboratory confirmed influenza pandemics in mongolia and confirmed some variations of pandemic spread in different parts of the world. the comparison of deduced amino-acid sequence changes have shown that the mongolian strains belong to the clade , according to the classification of a(h n )pdm influenza strains suggested by m. nelson, which has circulated worldwide since july . this is also evidence that the st wave of the pandemics did not hit mongolia. the who public health research agenda for influenza is aimed to support the development of evidence needed to strengthen public health guidance and actions essential for limiting the impact of influenza on individuals and populations. each stream-specific group reviewed and discussed the proposed organization, content, rationale, and global health importance of their designated research stream. specific research recommendations were made for topics within each stream: background: a syndromic surveillance system using nonclassical data sources for detection and monitoring evolution of flu and flu-like illness (ili) in djibouti is reported here as part of the preliminary report of djibouti who-copanflu international study (wcis)**. methodology: clinical reports, over-the-counter drug sales, lab diagnosis report, and health communication trends were obtained for an integrated statistical analysis. results: transition to winter is concomitant with upsurge of ili cases and ili drug sales. in addition, more rural folks manage ili infections on self medicament than through clinical consultancy. inefficient and vague data collections were observed. a successful implementation of wcis will create a platform upon which challenges faced in djibouti health department in routine surveillance will be addressed to achieve a near-real time surveillance of flu pandemic. conclusion: innovations, prompt reporting, and instituting open source syndromic surveillance system software's in resource limited environment like djibouti will enhance early detection and evolution monitoring of pandemic flu. the spanish flu in ⁄ infected and killed millions of people, and threatened to wipe humanity off the face of planet. however, the recent scenarios of influenza h n ( ) pandemics' worldwide occurrence fell short of most scientific prediction on its magnitude and intensity. this dampened their confidence; they cannot state precisely as to when, how, where, and which of the spanish flu-like pandemic will occur in the future. in support of scientific community and governments, the who hasn't gone to slumber, but is reminding its member states to up their post pandemic surveillance and monitoring of influenza virus in circulation for advance preparedness in case of an outbreak. despite all uncertainty around the pandemic flu h n , there remains a common knowledge and understanding that this flu has shown a great potential to evolve and cause huge morbidity and mortality. although its future magnitude may be unpredictable, its recurring events have severe consequences on human health and the economic well being of everyone. and therefore, advance planning and preparedness is critical in protecting any population in the future, especially those located in resource limited environment without universal health cover and generous disaster emergency funds. . two collapsed sets of a weekly and monthly mean data (of four years period) were clustered in five categories of ili cases, drug sales, lab results, vaccine consumption, and health promotion. this was followed by a descriptive statistics analysis of cumulative weekly and monthly data to establish presence or absence of trend. time series analysis was not done due to data limitation. copanflu program: as at the time of going to press, the cohort study is at the household recruitment and inclusion phase and the study covers the djibouti city. it is in our intention to use the cohort study findings to validate or improve the niph ministry of health djibouti ili surveillance effort for better preparedness. clinical service: % of all health facilities are in djibouti city. of the ae % ( ) of the population that seeks medical care on influenza and influenza like illness each year, ae % ( ) and ae % ( ) of them are attended to at the city's public and private clinics, respectively ( figure ). the rest are attended from the regional health centers. the majority of ili incidence sharply rise with the onset of the winter season (october to april), affecting mostly the middle age group ( - years). pharmaco-surveillance: % ( ) of total prescriptions were antipyretic and antiflu drugs, ae % ( ) of which were consumed by peripheral regions, the non dji- lab diagnostics: the annual ili lab diagnosis was negligible ae % ( ), which can be attributed to less equipped virology laboratories to warrant routine service utility. documented cases were from previous bouts of avian influenza that had a human incidence from and . with support of egypt-based naval army medical research unit three (namru ), clinicians were motivated to sample all ili patients and submit to collaborating international reference influenza lab in cairo, egypt. vaccination: influenza vaccinations were undocumented, but at least ae % ( ) of population sought the service (for yellow fever and meningitis) as mandatory travel advisory or as childhood immunization need. at the time of going to the press, there were at least vaccine doses of h n ( ) virus donations yet to be administered. health promotion and hygiene: print and audiovisual risk communication remained favorite means of reaching out to urban dwellers ( ae %). while to the rural and nomadic population, person to person communications was the preferable means. to increasing public awareness that will encourage reporting of ili cases and entrench risk aversion health behavior that limits flu spread, who-copanflu international study djibouti has incorporated basic training on ili infection and personal hygiene by interviewers during household inclusion. improving national epidemic surveillance capacity and response under new international health regulation is important for any nation, including djibouti. our finding indicates the winter season predisposes one to ili infections; they therefore opt for medical services or self medication depending on their capability and ⁄ or understanding. in djibouti, almost no city dwellers favors self medication over clinical consultation, suggesting the presence of inhibitory factors like distance from the health centers and the cost of accessing consultancy. common in the absence of universal primary health care setting, it therefore calls for active innovativeness in outbreak detection, disease reporting, and preventive medicine on the part of health authority so as to achieve good population health. in respond to these, niph has turned resource limitation to a motivation instead and is working towards institutionalizing a near-real-time syndromic surveillance system as a core functional unit. it capitalizes on three major aspects within its reach: prompt accurate data generation for analysis, ehesp wcic-study input, and information technology use. prompt accurate data generation for analysis: data used in our analysis suffered from un-timeliness (weekly instead of daily basis), incompleteness (vague over-counter drug sales records), entry errors (incidence case reports), and poor collection format (most of data collection forms). use of satellite handset phones for regional health centers and mobile phones for city sentinel clinics will reduce unnecessary data delivery delays. in addition, creating awareness to data entry personnel on the importance of careful and completeness of entries is important, as is the need to reformat data collection forms to capture exact aspects of surveillance needs for relevant executable analysis. besides alerting for immediate impending epidemics, these data can also be adopted for projective predictive modeling of annual epidemics, including that for influenza. ehesp wcic-study input: djibouti wcic-study is complementary to the existing syndromic surveillance system, but with emphasis on flu and flu-like illness. various innovations as suggested above are used in seeking to overcome the prevailing challenges. while every attempt is made to realize its (wcic-study) objective and for global comparison, lessons learned from successful implementation will form a platform for future refined syndromic surveillance protocol as equally reported elsewhere in asian countries. , information technology: national institute of public health djibouti has an informatics department with sufficient working pcs and personnel to execute efficient data collection and management for epidemiological analysis. however, licensing cost of near-real time syndromic surveillance software is prohibitive, but the open access software with capacity to generate custom graphs, maps, plots, and temporal-spatial analysis output for specific syndromes should make implementation a lot easier. such output for conditions like flu (or gastroenteritis) will be essential to cause prompt response of the local public health office and international partners in saving lives and suffering of djibouti people. pandemic flu surveillance and preparedness requires multifaceted, interdisciplinary, and international approach whose efficiency and efficacy can only be refined over time. building on the health care system's swot for preparedness, the ehesp wcic-study promises to refine surveillance system operation and knowledge on individual's risk determinants to swine flu (h n ) virus infection at the household level in djibouti. these efforts are ultimately creating available control options at the time of need (pandemic occurrence), and at the same time exploring investment in quality data profiling and information technology, which will include syndrome surveillance software systems like essence, ewors, or other open sourced ones. the antibody efficacy -which compares the illness frequency between those with and those without a protective level of pre-epidemic hi antibodies ( ‡ : ) -has been proposed ; however, this index has rarely been used due to practical difficulties in confirming the strain-spe-cific disease corresponding to each of the vaccine-induced antibodies. we followed elderly individuals residing in a nursing home, whose serum specimens were obtained before and after undergoing trivalent influenza vaccination, in ⁄ influenza season (medium-scale mixed [a ⁄ h n and b] epidemic in study area, and a ⁄ h n was circulating at the nursing home). the serum antibody titre to each strain of influenza virus was measured by the hi method, using the same antigens as those in the vaccine. all participants' body temperatures, respiratory symptoms, other general symptoms, hospitalization, discharge, and death were recorded daily from november to april in a prospective manner. when the participants suffered any influenzalike symptoms, such as sudden fever ‡ ae °c, throat swabs were collected and tested using a rapid diagnosis kit for influenza, which utilizes an immunochromatographic method. the adjusted odds ratios (or adj ) for febrile illness and kit diagnosed influenza were evaluated using multiple logistic regression models adjusting for possible confounders (i.e., age, sex, coexisting conditions, and vaccine strains). after vaccination, the proportion of subjects achieving an hi antibody titre ‡ : (seroprotection level) were ae % ( ae - ae %) for a ⁄ h n , ae % ( ae - ae %) for a ⁄ h n , and ae % ( ae - ae %) for b. during the follow-up period, the a ⁄ h n strain was isolated therein, and subjects experienced sudden-onset fever ( ‡ ae °c), and eight subjects were positive for rapid diagnosis kit. patients with a seroprotection level of the hi antibody titre ( ‡ : ) had lower incidences of febrile illness (or adj , ae ; % ci, ae - ae ) and rapid kit diagnosed influenza (or adj , ae ; % ci, ae - ae ) than those with a lower titre. thus antibody efficacy ( ) or adj ) against fever related to a ⁄ h n and kit diagnosed influenza were both estimated to be %. although statistical significance was not detected due to limited sample size, these results lend support for the usefulness of antibody efficacy. some data presented within this manuscript was also published in hara et al. asia via a regional network from which epidemics in the temperate regions were seeded. the virus isolates obtained from nasopharyngeal swab specimens from outpatients were typed and subtyped by the hemagglutination (ha) inhibition assay. the emergence of a ⁄ fujian ⁄ ⁄ coincided with higher levels of influenza-like illness in korea than what is typically seen at the peak of a normal season. most of the intermediates and fujian-like strains were isolated from asian countries, and the mutational events associated with the fujian strains took place in asia. closely dated phylogeny from december , to august , showed that the antigenic evolution of the h n fujian strains had periods of rapid antigenic changes, equivalent to amino acid changes per year ( figure ). the fujian-like influenza strains were disseminated with rapid sequence variation across the antigenic sites of the ha domain. the antigenic evolution of the fujian strains was initiated by exceptionally rapid antigenic change that occurred in asia, which was then followed by relatively modest changes. some of the data presented in this manuscript was previously published in kang et al. we compared reactivity to the novel virus strain using haemagglutination inhibition (hi) assays performed on discarded plasma specimens left over from routine testing. samples were taken from healthy adult blood donors (> years) before and after the ph n influenza epidemic that occurred during the southern hemisphere winter of , and again prior to onset of the southern hemisphere influenza season. reactivity to the novel h n strain of influenza was relatively uncommon among the healthy adult population during the first australian winter wave, rising from a baseline of % to %. a further increase in the seropositive proportion from % to % was observed over the summer months, most likely attributable to immunisation. this level of immunity appears to have been sufficient to constrain the winter epidemic. together with a final serum collection, planned for late , these data will aid evaluation of the extent and severity of disease in this 'second wave' of ph n . assessment of the extent of disease due to novel influenza a(h n ) virus (ph n ) during the winter outbreaks in australia was made difficult by the generally mild nature of disease. the epidemic was experienced in a staggered fashion around the country, reflecting the considerable geographical distances between state and territory capital cities ( figure ). differences in the intensity of case-finding during the evolving pandemic response and between jurisdictions hindered comparisons of disease burden in distinct geographical regions. rates of reported hospitalisations and deaths appeared fairly similar across states but, without a consistent exposure denominator, assessment of relative severity was difficult. we conducted a national serosurvey of antibody to ph n using residual plasma from healthy blood donors collected before and after the epidemic to estimate ph n exposure. here we report the findings of that first collection, together with new data on seroprevalence of ph n antibody in specimens gathered in march-april . these latter samples were collected prior to onset of seasonal influenza activity to assess the impact of a national ph n vaccine program conducted in spring ⁄ summer ⁄ on the proportion of individuals with antibody titres deemed protective. findings informed estimates of population susceptibility to ph n prior to the influenza season and provided a baseline for a subsequent serosurvey that will be collected at the end of to assess the extent of exposure during the 'second wave.' tralian red cross blood service (the blood service) for dengue fever surveillance studies. these samples were used to provide a baseline estimate of prevalence of cross-reactive antibody to ph n in the australian population. discarded plasma specimens, taken for virologic testing from healthy adult blood service donors, were prospectively collected at two additional timepoints for measurement of antibody to ph n . collection periods were as follows: approximately plasma samples were randomly selected from donors in each of brisbane, hobart, melbourne, newcastle, perth, sydney, and townsville on each occasion. up to specimens were identified in each of the following age strata: - , - , - , - , - , and > years. at the last collection timepoint, there was deliberate over-sampling of the oldest and youngest age strata in which approximately specimens were collected (i.e., up to specimens per site). in accordance with the provisions of the national health and medical research council's national statement on ethical conduct in human research, individual consent was not required for use of these specimens, given the granting of institutional approval by the blood service human research ethics committee. reactivity of plasma against ph n was measured in haemagglutination inhibition (hi) assays using turkey red blood cells (rbc). egg-grown a ⁄ california ⁄ ⁄ virus was purified by sucrose gradient, concentrated and inactivated with b-propiolactone, to create an influenza zonal pool preparation (a gift from csl limited). plasma samples were pretreated with receptor destroying enzyme ii (denka seiken co. ltd), : (volume ⁄ volume) and tested as previously described. following hour incubation, ll % (volume ⁄ volume) of rbc was added to each well. hi was read after minutes. any samples that bound to the rbc in the absence of virus were adsorbed with rbc for hour and reassayed. samples in which background activity could not be eliminated by these means were excluded from the analysis. titres were expressed as the reciprocal of the highest dilution of plasma where haemagglutination was prevented. a panel of control sera and plasma samples was included in all assays. it comprised paired ferret sera pre-and postinfection with the pandemic virus or seasonal influenza a(h n ), a(h n ), or influenza b viruses and paired human plasma and sera collected from donors before april or after known infection with the pandemic virus or after immunisation with the australian monovalent pandemic vaccine. all assays were performed by the who collaborating centre for reference and research on influenza. for each of the three study timepoints and within each age group, the proportion of seropositive individuals (hi titres ‡ ) was calculated, with exact (clopper-pearson) confidence intervals. the contribution of individual variables (age, gender) and location to seropositive status was assessed in separate multivariate logistic regression models developed to assess the post-pandemic and pre-influenza season collections. all statistical analyses were conducted in stata . locations of specimen collection are shown in figure , together with the number of samples tested from each centre. samples with high background hi titres or discrepancies between assays were excluded at each timepoint as follows: at baseline, from the post-pandemic collection, and in early . pared with baseline was % overall, rising from % to % (table ). the only jurisdictions in which seropositive proportions were higher in october ⁄ november than in the baseline collection were hobart [ % ( % ci ae , ae )], perth [ % ( ae , ae )], and sydney [ % ( ae , ae )]. in the multivariate regression model, the only jurisdiction in which exposure appeared somewhat higher than the reference population of brisbane was hobart [or ae ( % ci ae , ae ), p = ae ]. a marked age effect on antibody status was observed at this timepoint, with an increase in the proportion of seropositive individuals in relation to the baseline collection only noted for those aged between and years (table ) . according to the multivariate model, the youngest and oldest cohorts had similar titres, with all other groups showing significantly lower seropositive proportions than the reference population of - years [e.g. - years or ae ( % ci ae , ae , p < ae )]. an overall increase in the seropositive proportion from % to % was observed between october and april , distributed throughout all jurisdictions ( ( , ) ]. antibody titres prior to the influenza season rose in all age groups, but remained significantly lower among [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] year olds than in the youngest age cohort (table ) . adjusted ors for the seropositive proportion in the multivariate model in these age groups were: - years [or ae ( % ci ae , ae )]; - years [or ae ( ae , ae )]. the relatively low titres observed in these groups reflected small incremental increases in the seropositive proportion across each of the time points studied, suggestive of both low rates of infection and vaccination. the rise in immunity observed across the population was most likely attributable to immunisation in the majority, given the absence of observed outbreaks and very few notified cases of ph n during the period between the two plasma collections. this study suggests that, while adult exposure to ph n during the southern hemisphere winter was uncommon at around %, vaccine uptake in the australian population over the period november -may was in the order of %. this latter estimate is in keeping with recently published figures for adult ph n vaccine coverage from a national immunisation survey conducted by the australian institute of health and welfare. in that survey, vaccine coverage was significantly higher in tasmania than in other states, but mostly in those over years of age, possibly in a subgroup whose health status may have differed from that of the donor population. no allowance has been made in this analysis for likely waning of natural or vaccine induced immunity, possibly resulting in lower estimates of natural and ⁄ or vaccine exposure than may have occurred over the period. regardless of such intervening processes, the seropositive proportion among australian adults at the start of the winter season appeared likely to be sufficient to constrain transmission of infection in the age groups tested. this assertion has been borne out in practice, with only modest levels of influenza reported during the late and protracted season. a final serum collection is planned for the end of the influenza season in australia from which to assess the level of exposure in relation to the baseline observed here. the need for epidemiologic studies such as this has been highlighted by groups such as the european centre for disease control to aid evaluation of the extent and severity of the 'second wave,' known to be variable from historical reports of past pandemics in disparate populations. in - , the first wave of the swine-origin novel h n flu (h n ) pandemic swept across the world, including japan. to examine the epidemiological nature of this novel infectious disease among school children within and among small regional communities, we have carried out a complete survey on the incidence of h n among school children using absentee reports provided by school health teachers in two small administrative districts (population: about in total) in japan. we then examined the epidemiological diversity on the inci-dence of h n within and among small regional communities. we investigated seventeen elementary and ten junior high schools in moroyama-town and sakado-city located in the central part of saitama prefecture. populations are: all ages, and ; elementary schools, and ; junior high schools, and , respectively. the number of school children in each school ranges from to . the surveillance system was built on an apache-and mysql-based web server using html, php, and java-script. school health teachers enter information on children absenteeism due to school infectious diseases via web browsers at each school infirmary on a daily basis. in addition to the trend graphs shown on the web browser, detailed analyses were reported to the schools and local educational boards weekly. the basic reproduction number (r ) of h n was estimated according to becker. agentbased modeling and simulations were also performed using a multi-paradigm simulator anylogic version . (xj technologies, st. petersburg, russia). by the end of march , cumulative incidence (ci) of h n among school children in moroyama and sakado reached % and %, respectively. the overall r among school children in this area was ae . vaccination rate of children in this area during the surveillance period was reported to be very low (< %). there was no considerable difference between the epidemic curves in this neighboring town and city. on the other hand, in the individual schools, the cis as of the end of march scattered from % to % ( figure ) even though the schools are closely located. to examine the cause of this diversity, we built an agent-based community model consisted of the same numbers of agents as those of children in the actual schools and people in moroyama and sakado to simulate the infection. the ratio of probability of infection in schools and the remaining places were assumed to be : or : . using a heuristic optimization scheme, we estimated the parameters for the simulations to give the overall ci of % (the ci as of the end of march ). we then performed simulations repeatedly. the cis obtained with the repetitive simulations with the assumption of higher probability of infection in schools scattered from % to %, indicating that the cis of the small population communities may vary considerably, even though all the agents were assumed to have the same susceptibility to infection at the beginning, and the other conditions were the same. the policies for surveillance ⁄ analyses ⁄ prevention of communicable diseases in local communities have generally been decided on governmental-and ⁄ or each local administrative district-basis (populations: several hundred thousands to several millions) in japan. we found the considerable variations in the cis of h n for children among much smaller areas, i.e., the school districts (populations: all ages, several thousands; school children, several hundreds). we thus conclude that the granularity of surveillance ⁄ analyses ⁄ prevention should be finer than in the past to achieve the most effective policies against influenza and similar communicable diseases in the local communities. the cause of this diversity can be explained in part by the stochastic nature of infection transmission processes in the small populations shown by the agent-based simulations. we have already conducted a complete questionnaire survey for the school children and their parents to clarify the relevance of the other issues including differences in environmental factors, preventive policies (e.g., vaccination, school closures), etc., in each school. the detailed analyses will be reported elsewhere. a www-based surveillance system for transmission of infectious diseases among school children within and among small regional communities. j epidemiol ; (s ):s . this study confirms previous findings that age, pandemic influenza vaccination, and history of ili are associated with elevated post-seasonal gmt. this study also shows that seasonal influenza vaccination may have contributed to an increase of the hai titer, especially in the elderly. further analyses in this cohort are needed to confirm and explain these first results. the follow-up of subjects involved in the copanflu-france cohort will provide data to study the risk factors for infection by the influenza virus. the first cases of the a ⁄ h n v pandemic influenza were reported in mexico and the united states in april . given the context of this new influenza virus and considering the likelihood of its pandemic spread, the cohorts for pandemic influenza (copanflu) international consortium was created in order to study individual and collective determinants of pandemic a ⁄ h n v influenza across different countries by setting up prospective cohorts of households, followed during years. this study relies on the first available data from the copanflu-france project, which is part of the copanflu international consortium. we studied factors associated with elevated haemagglutination antibody titers against a/h n v at entry in the copanflu-france cohort. we focused in this primary analysis on the association between the titers and influenza vaccination (seasonal or pandemic) across age groups. the copanflu-france cohort was set up in fall . inclusions began on december , and ended on july , . households were sampled using a random telephonic design (mitofsky-waksberg method) in a stratified geographical sampling scheme, aimed at including a sample of subjects representative of french general population. all household members were eligible to the cohort, without any age limit. the inclusion of a household required the participation of all members: the refusal of one or more member(s) prevented the inclusion of other members. the protocol was approved by a research ethics committee and written informed consent was obtained for all subjects. this study requires several visits to the households by nurses who collect written data with questionnaires and biological samples. during the inclusion visits, nurses collected from all subjects detailed data regarding medical history, including vaccination and preventive measures against influenza. blood samples were collected at entry and centralized. a standard hai technique was adapted to the detection and quantification of antibodies to the a ⁄ h n v virus. the titration endpoint was the highest dilution that exhibited complete inhibition of haemagglutination in two independent readings. the lowest read dilution was ⁄ . geometric mean titers were calculated for hai assays with the use of generalized estimating equations for interval-censored data, , taking into account a within-household correlation. multivariate models were derived from this method to identify factors associated with elevated gmts. we defined the ''gmt ratio'' (gmtr) as the multiplicative factor applied to the gmt in presence of an explanatory variable. for qualitative explanatory variables, a gmtr of n means a predicted n-fold higher gmt for subjects exposed to the considered factor compared to others. for continuous explanatory variable, the same interpretation applies to a unit difference. the following variables were included in the multivariate models: age, history of pandemic or seasonal influenza vaccination, and history of ili. age was categorized in three groups: - years (reference group), - years and over years. the definition of ili was that used by the cdc : fever ‡ ae °c and cough and ⁄ or sore throat without another known cause. history of ili was defined as an ili reported by the subject between september , (beginning of the influenza epidemic in france) and the date of inclusion. this preliminary analysis included subjects belonging to households. results reported hereafter do not account for missing data. participating households were sized - subjects, mean size = ae . in comparison, the mean size of french households is ae according to the latest national census. the median age of subjects at entry was ae years [iqr: ae ; ae ] versus ae [ ae ; ae ] for french population. the proportion of subjects reporting a history of ili since the beginning of the epidemic varied from ae % for subjects over years to ae % for subjects below years (table ) . vaccination with the pandemic strain was the highest in subjects below ( %) whereas vaccination with the seasonal strain was the highest in subjects over ( ae %). detailed data regarding vaccination is given in table . this study confirms previous findings that age, pandemic influenza vaccination, and history of ili are associated with elevated post-seasonal gmt. [ ] [ ] [ ] [ ] [ ] [ ] among non-vaccinated subjects, elevated gmt in the elderly may be the result of exposure to similar viruses in early life, whereas children and young adults with elevated gmt are likely to have been infected by the a ⁄ h n v virus. [ ] [ ] [ ] [ ] interestingly, a significant drop in the hai titer is observed during the months following vaccination with the pandemic strain. this study also shows that seasonal influenza vaccination may have contributed to an increase of the hai titer, especially in the elderly. the reason for this association is not obvious: although we cannot discard the hypothesis of a higher incidence of a ⁄ h n v infections in seasonal vaccine recipients, as described by several other studies, - the main explanation may be a cross-reaction between pandemic and seasonal strains. , , further analyses in this cohort are needed to confirm and explain these first results. the follow-up of subjects involved in the copanflu-france cohort will provide data to study the risk factors for infection by the influenza virus. in april , the cdc alerted about the appearance of a new strain of ia h n with unknown virulence. infants under years old had higher risks of hospitalization, complications, and rate of death for sari. materials and methods: a cross-sectional study was executed from may to december in . the sources were: mandatory reporting form of the province surveillance system, databases of the hospital management information system, clinical pictures reviews, and telephone daily medical reports. inclusion criteria: children under years old with diagnostic of ili or sari and confirmed cases with epidemiological nexus or laboratory confirmation (rrt-pcr, ifi). the age specific mortality rates were calculated with an estimated population for the province according to the national statistics and census institution. results: the ili rate in infants under years old was ae ⁄ people ( % ci - ) being higher in infants of years old ( ⁄ people of years ( % ci - ) ( table ) . infants had less risk of getting sick in relation to the rest of the population (rr ae [ % ci ae - ae ]) (p < ae ). the chance of sari in infants was ae ( % ci ae - ae ) compared to the rest of the population. the lethality rate was higher in infants under year old ( ⁄ people [ ⁄ ]). discussion: the evidence suggests that the infants under years old had lower risk of getting sick than the rest of the population, but had higher risk of sari if they had some past illness. the highest lethality rate was presented in infants under year old. non-medical interventions had an important role in the epidemic containment for not having a specific vaccination available. as this age group had high risks of hospitalization, it would be advisable to prioritize their vaccination. in april , the cdc alerted about the appearance of a new strain of ia h n with unknown dissemination and virulence. in june, the world health organization declared the pandemic. , the ili often presents an unspecific clinical picture in infants under years old, from mild symptoms to sari, especially in the newborn babies. infants under years old have higher risks of hospitalization, complications, and rate of death for sari. , on may th, argentina declared the first imported case of ia h n , and by the end of the month, it announced the viral circulation in the country. the epidemiological surveillance system of the province arranged that all the patients with influenza diagnosis made by a doctor must be reported. from april th to november th, suspected cases of ili in the province of tucumán were reported. the ili rate was ⁄ people, and ia h n comprised ⁄ people. the lethal rate of sari ia h n was ae ⁄ people ( ⁄ ). the objective of this research was to determine the epidemiological characteristics of the pandemic ia h n in infants under years old in the province of tucumán between may and december in . the province of tucumán is placed in the center of the northwest of the republic of argentina. it has a population of inhabitants of which are infants under years old. the crude birth rate for was ae &. the infant mortality rate was ae &. respiratory pathologies in infants under years old were the third cause of death in the province ( %). the public health system of the province is composed by three sectors: public, private, and welfare. with health facilities as a total, the average of available beds is & per inhabitants and & per neonates. a cross-sectional study was executed from may to december in in the province of tucumán, argentina. the following sources were used: mandatory reporting form of the surveillance system of the province filled by a doctor, databases of the hospital management information system, clinical pictures reviews, and telephone daily medical reports (patients with sari). inclusion criteria: • suspected case of ili: sudden appearance of fever higher than °c, cough, or sore throat. it may or may not be accompanied by asthenia, myalgia or prostration, nausea or vomiting, rhinorrhea, conjunctivitis, adenopathy, or diarrhea. ) were used for the analysis. the odds rations, risk ratio and % confidence interval were calculated to compare ambulatory with hospitalized patients, confirmed and dismissed, < years old and the rest of the population. it was considered significant a rate of p < ae . the age specific mortality rates were calculated with an estimated population for the province according to the national statistics and census institution. the epidemiological surveillance system of the province received ili reports, ae % ( ⁄ ) were infants under years old. twenty seven percent were dismissed ( ⁄ ), and % ( ⁄ ) of suspected cases were confirmed. the first ia h n case was a child of years from the province of buenos aires, in th epidemiological week, and the last suspected case was reported in october , ( figure ). the ili rate in infants under years old was ae ⁄ people ( % ci - ), being higher in infants of years old ( ⁄ people of years, [ %ci - ]). the higher ili rates in confirmed the pandemic of ia h n ( ) was detected for the first time in the province of tucumán. the evidence suggests that infants under years old had lower risk of getting sick than the rest of the population (protective factor), but had higher risk of sari if they had some past illness. the highest lethality rate was presented in infants under year old. towns with the highest demographic density had superior proportion of cases. non-medical interventions had an important role in the epidemic containment for not having a specific vaccination available. as this age group had high risks of hospitalization, it would be advisable to prioritize their vaccination. outbreak of h n influenza - : behavior of influenza h n in school children in the province of tucumá n, argentina criteria: patients treated with antiviral medication for prophylaxis, respiratory pathologies which did not justify specific medication, and incomplete forms. results: from all notifications, were cases of ili in the group aged - years old; % were males. the incidence rate in this group was ae per thousands of inhabitants. the % of laboratory samples were influenza a h n , % were confirmed as unspecific influenza, and % were dismissed. the school aged children group had a high risks of getting sick (r.r. ae [ % c.i. ae - ae ]), especially males. it appeared that school aged children had a protective factor for presenting sari (or ae [ % c.i. ae - ae ], p < ae ). the lethality rate in this group was ae ⁄ thousands. headaches, myalgia, coryza, and sore throat were very common and significantly different (p < ae ) than the rest of the population. it was reported a decrease in the ew coinciding with winter holidays (ew ). the epidemic curve was different in males compared to females during the winter holidays. discussion: school aged children got sick more than the rest of the population, although they presented less proportions of sari. however, comorbidities were decisive in order to present sari or death. the epidemic curve was different in males compared to females. through its analysis, the beneficial effect of school closure was observed, as long as children meet the recommendation to stay home. in april , different countries reported cases of influenza a h n ; mexico reported a high mortality rate associates with this disease. the world health organization (who) declared the phase influenza pandemic alert on june . several reports from different countries describe the behavior of the pandemic in school aged children. this group plays an important role in the transmission of influenza. in germany, during the summer peak, pandemic hardly spread within this group. this might be explained by the timing of the summer school holidays, which started between ew and . since mid october, after the autumn holidays, the school-aged children began to be more affected, and the proportion increased from % in the initiation period to ae % in the acceleration period. in australia, % of h n cases were school aged children ( - years), with a median age of years ( % of cases were aged - years and, and % between - years). in canada, the infection rate was highest in this group. in chile, the incidence rate was ⁄ inhabitants, although in general they had mild desease. school closure can operate as a proactive measure, aimed at reducing transmission in the school and spread into the wider community, or reactive, when the high levels of absenteeism among students and staff make it impractical to continue classes. the main health benefit of proactive school closure comes from slowing down the spread of an outbreak within a given area and, thus, flattening the peak of infections. this benefit becomes especially important when the number of people requiring medical care threatens to saturate health care capacity. it has its greatest benefits when schools are closed very early in an outbreak, before % of the population falls ill. school closure can reduce the demand for health care by an estimated - % at the peak of the pandemic under ideal conditions, but too late in the course of a community-wide outbreak, the resulting reduction in transmission is likely to be very limited. policies for school closure need to include measures that limit contact among students when they are not in school. tucumán is placed in northwest argentina and has a total area of km . the population ( census, projection ) was inhabitants; of wich were - years old. the health system of the province is composed of sectors: public, private, and welfare. it has a total of health facilities with internement available and an average of & inhabitants. influenza-like illness (ili) has seasonal and endemic behavior in this province, as evidenced by past records from the national health surveillance system and influenza sentinel surveillance unit of the province. an increase of ili was reported in , with a peak in the ew . the objectives were: general objective to describe the behavior of the influenza a h n epidemic in school aged children from the province of tucumán, argentina. specific objectives • to explore the response to preventive measures by school aged population. • to assess the effect of the suspension of classes in this group. • to estimate the magnitude and severity of the disease. • to observe the effect of co-morbidities in this group. a cross-sectional study was executed from may to december . data were gathered through mandatory reporting forms, wich were collected from all public and private health centers. inclusion criteria: patients with compatible symptoms with influenza a; school aged children - years old. exclusion criteria: patients treated with antiv- iral medication as prophylaxis, respiratory pathologies which did not justify specific antiviral medication, and incomplete forms. • suspected case of ili: cases considered by clinical criteria (fever higher than °c, cough or sore throat. it may or may not be accompanied by asthenia, myalgia or prostration, nauseas or vomiting, rhinorrhea, conjunctivitis, adenopathy, or diarrhea). • confirmed case: person with positive laboratory results for influenza a h n or unspecificed influenza a (by laboratory results through rrt-pcr or immunofluorescence techniques). • dismissed case: by negative or different laboratory results, or different clinical evolution. • comorbidities: chronic illnesses like arterial hypertension, diabetes, asthma, recurrent obstructive bronchial syndrome (robs), smoking, chronic obstructive pulmonary disease (copd), immunosuppression, hiv ⁄ aids, cancer, nephropathy, obesity; pregnancy was also considered. data were analyzed using epi software (epi infoÔ cdc, atlanta, eeuu). rates were calculated and rr was estimated with their respective confidence interval (ci). population data were taken from national census projections. an estimation based on the same census was used for the group between and years old. to observe the effects of other co-variables, the or and their ci were calculated. logistic regression was used to evaluate the influence of the comorbidities. x was used to compare proportions. respiratory samples (nasopharyngeal and faryngeal swabs) were obtained. they were analyzed at influenza sentinel surveillance unit of tucumán, and ⁄ or sent to national reference laboratory dr. c. malbrán (rt-pcr). from all notifications ( ), were cases of ili in the group aged between and years old, % ( ⁄ ) of which were males. the incidence rate was ae , and it differed according to the sexes: ae males and ae females per thousands of inhabitants (p < ae ). of all laboratory samples ( ) % were confirmed as influenza h n , % were confirmed as unspecificied influenza, and % were dismissed. the remaining percentage corresponded to the isolation of other viruses (parainfluenza, respiratory syncytial virus, and adenovirus). the school aged group had higher risk of getting sick, in relation to the rest of the population (rr ae [ % ci ae - ae ]), especially males (rr ae ) compared with females (rr ae ). the highest attack rate was observed in the capital of tucumán ( ⁄ inhabitants). according to the rest of the population, it looked like being school aged children meant a protective factor for presenting sari (severe acute respiratory infection) (or ae [ % ci ae - ae ], p < ae ). the lethality rate was ae ⁄ thousand. the risk of dying was low compared to other ages. persons with comorbidities had significantly higher risk of presenting sari (or ae [ % ci ae - ae ], p < ae ) and of dying (or ae [ % ci ae - ae ], p < ae ). respiratory comorbidities were the most fre- quent: asthma ae % ( ⁄ ) and % rors ( ⁄ ). the symptoms headaches, myalgia, coryza, and sore throat were very common and significantly different (p < ae ) than the rest of the population. if we compared the group aged - years with - years old, the epidemic curve of the first group showed a decrease in the ew , coinciding with winter holidays (ew ) (figure ). there was a slight increase in the tendency when classes began, but it showed a clear declination afterwards. the analysis of rates in school aged children by ew showed a reduction of ae % in males and ae % in females (p < ae ) at ew . however, after the first week of winter holidays, the curve in males had a significant increased to ae % compared to ew , reaching the highest weekly rate of the epidemic ( ⁄ inhabitants). the reopening of classes coincided with a significant decrease of the rate ( ae %), from to ae ⁄ inhabitants in ew (p < ae ). in females, the school closure coincided with a plateau-shaped curve, and the reopening with a significant decrease of ae % of the rate, from ae to ae in ew ( figure ). the school children got sick a lot more than the rest of the population, although they presented less proportions of sari. however, comorbidities were determined in order to present sari or death. symptoms like headache, myalgia, coryza, and sore throat were considered more conducting for the definition of cases in this population in tucumán. the epidemic curve was different in males compared to females during the winter holidays. the beneficial effect of school closure was observed as long as persons met the recommendations. the difference between males compared to females during winter holidays could mean that women would have carried out social distance recommendations much better, for example, remained at home. the significant reduction after the opening of classes is a factor to be considered as an effective intervention in the declining stage of the curve. here, we report pdmh n infection attack rate (iar) during the first wave of the pandemic. we used our iar estimates to infer the severity of the pandemic strain, including the age-specific proportion of infections that led to laboratory confirmation, hospitalization, intensive care unit (icu) admission, and death. [ ] [ ] [ ] [ ] part of these results are now available in ref. subjects of a community study, - years old between november and october , we conducted a cohort study of pediatric seasonal influenza vaccination and household transmission of influenza. one hundred fifty-one children aged - were recruited and provided baseline sera in november and december . between september and december a further children aged - were recruited and provided baseline sera for the second phase of the study. for this serologic survey, we tested the sera collected before the first wave and the sera collected after the first pandemic wave. written informed consent was obtained from all participants. parental consent was obtained for participants aged or younger, and children between the ages of and gave written assent. all study protocols were approved by the institutional review board of the university of hong kong ⁄ hospital authority hong kong west cluster. age-stratified data on virologically confirmed outpatient consultations, hospitalizations, icu admissions, and deaths associated with pdmh n from april to november were provided by the hong kong hospital authority (the e-flu database). since may , patients admitted with acute respiratory illnesses routinely underwent laboratory testing for pdmh n virus by molecular methods. sera were tested for antibody responses to a ⁄ california ⁄ ⁄ by viral microneutralization (mn). most individuals infected with influenza develop antibody titers ‡ : by viral microneutralization after recovery. we defined the pdmh n seroprevalence rate as the proportion of individuals who had antibody titers ‡ : . while mn antibody titers of ‡ are not by themselves conclusive evidence for pdmh n infection, we have assumed that the increase in cross-sectional seroprevalence between the pre-and post-first wave time periods are evidence of recent pdmhn infection. the iar was defined as the proportion of individuals infected by pdmh n during the first wave. the case-confirmation rate (ccr), case-hospitalization rate (chr), case-icu-admission rate (cir), and case-fatality rate (cfr) were defined as the proportion of pdmh n infections that led to laboratory-confirmation, hospitalization, icu admission, and death. due to containment efforts until june , all laboratory-confirmed cases were required to be hospitalized for isolation regardless of disease severity. as such, only surveillance data from june onwards were used to estimate severity measures. we estimated the iar as the difference between the prefirst-wave and post-first-wave seroprevalence rate. we used the estimated iar as the denominator for calculating the ccr, chr, cir, and cfr. we used an age-structured sir model with age classes ( - , - , - , - , and ‡ ) to describe the transmission dynamics of pdmh n in hong kong between june and november . we assumed that the mean generation time was ae days. using the age-structured transmission model, we estimated the following transmission parameters from the serial cross-sectional serologic and hospitalization data: (i) r o , the basic reproductive number; (ii) p and p , the reduction in within-age-group transmission for - and - years old during summer vacation (compared to school days during september-december ); (iii) d r , the average time for neutralization antibodies titer to reach ‡ : after recovering from infection; (iv) h a , the age-specific relative susceptibility with - years old adults as the reference group. we assumed non-informative priors for all parameters and used monte carlo markov chain methods to obtain posterior distributions of the parameters. sources of specimens: [ ] pediatric cohort study ( - april virological surveillance data suggested that the first wave of pdmh n in hong kong occurred from august to october . most of the laboratory-confirmed infections in this first wave occurred in individuals aged below years old accounting for > % of the lab-confirmed cases and hospitalizations, % of icu admissions, and % of deaths. taking into account a delay of - weeks for antibody titers to appear during convalescence, we found that these virological surveillance data were consistent with our serial cross-sectional seroprevalence data, which indicated a sharp rise in seroprevalence among the - years old from september to november and a plateau thereafter (data not shown). among individuals aged - years, the seroprevalence rates were similar across time between pediatric outpatient subjects and pediatric cohort study subjects (data not shown). similarly, for older age groups, the seroprevalence rates were largely similar between blood donor subjects and hospital outpatient subjects (except for the - years old in november-december). this provided some evidence that despite biases in our convenience sampling scheme, the resulting serologic data provided a reasonably representative description of seroprevalence in the community. the estimated pre-and post-first-wave seroprevalence rates and the corresponding iar estimates are shown in table . the severity estimates (ccr, chr, cir, and cfr) are shown in table . in summary, we estimated the iar was ae % among - years old, ae % among - years old, ae % among - years old, ae % among - years old, ae % among - years old, and ae % among - years old. overall, we estimated a population-weighted iar of ae % ( - %) among individuals aged - years through the first wave in hong kong. ccr were around ae - ae % among the - years old. chr were around ae - ae % among the - years old. cir increased from ae ( ae - ae ) per infections in - years old to ( ae - ) per infections in - years old. cfr followed a similar trend with ae ( ae - ae ) death per infections in - years old to ae ( ae - ) deaths per infections in - years old. compared to children aged - , adults aged - were ae and times more likely to be admitted to icu and die if infected. the best-fit age-structured transmission model gave the following parameter estimates: . the basic reproductive number was ae ( %ci, ae - ae ). . it took an average of ( - ) days for recovered individuals to develop neutralization antibody titer ‡ : . table . estimated age-specific proportions of individuals with pdmh n infections that were laboratory-confirmed, were hospitalized, were admitted to icu, and died. case-icu and case-fatality rates are expressed as number of episodes per infections . compared to - years old, - years old children and - teenagers were ae ( ae - ae ) and ae ( ae - ) times more susceptible to pdmh n infection, respectively. . compared to - years old, - years old older adults and - years old elderly were only ae ( ae - ae ) and ae ( ae - ae ) times as susceptible as the - years old, respectively. . compared to the school period during september-december , summer vacation reduced within-agegroup transmission by % ( - %) among - years old, but only % ( - %) among - years old. using computer simulations, we estimated that if preexisting seroprevalence is zero, real-time serologic monitoring with about specimens per week would allow accurate estimates of iar and severity as soon as the true iar has reached % (data not shown). we estimated that during the first wave in hong kong, ae % of school-age children and ae % of individuals aged - were infected by pdmh n . a serologic survey in england found similar iars in london and the west midlands. both studies highlight the importance of including serologic surveys in pandemic surveillance. the geographically compact and well-mixed population in the urban environment of hong kong permits some degree of confidence in the validity of our iar and severity estimates. the completeness of the pdmh n surveillance system, welldefined population denominator, and our large-scale serologic survey provide accurate numerators and denominators for the severity measures. we based severity estimates for pdmh n on the iar as the denominator. in most previous studies of pdmh n severity, the denominator was clinical illness attack rate, which depends on the probability of symptoms as well as medical care seeking behavior of the population. , our estimated cirs and cfrs are broadly consistent with presanis et al.'s 'approach ' severity estimates, but around - times lower than their 'approach ' estimates. our estimates of chr are - times higher than their approach estimates of symptomatic chr. however, the hospitalization-death ratio was ⁄ = as of november in hong kong, but ⁄ = as of june in new york, suggesting that the clinical threshold for admission in terms of disease severity at presentation may have been lower in hong kong. our study has a number of limitations. first, we have used antibody titers of ‡ : by viral microneutralization as an indicator of recent infection, correcting for pre-existing seroprevalence levels, but this may lead to underestima-tion of the iar if some infections led to antibody titers < : , or if some individuals with baseline titers ‡ : were infected. second, our estimates of the iar would be biased upwards if infection with other circulating influenza viruses led to cross-reactive antibody responses resulting in antibody titers ‡ : . however between august and october , % of influenza a viruses detected in hong kong were pdmh n , and only % of isolated viruses were seasonal h n viruses. third, a minority of severe illnesses associated with pdmh n infection might not be identified by molecular detection methods, for example if admission occurred after viral shedding from the primary infection has ceased, in which case we may have underestimated the disease burden of pdmh n . finally, our analyses are primarily based on seroprevalence among blood donors to the hong kong red cross, who may not be representative of the whole population. we do not have detailed data on donors to compare their risk of infection with the general population, but we did observe very similar seroprevalence rates across the three groups of subjects in our study, i.e., blood donors, hospital outpatients and participants in a community cohort (data not shown). in conclusion, around ae % of the population aged - and half of all school-age children in hong kong were infected during the first wave of pandemic h n . compared to school-children aged - , older adults aged - , though less likely to acquire infection, had ae and times higher risk of icu-admission and death if infected. thus, although the iar of pdmh n is similar to that of a seasonal epidemic, the apparently low morbidity and mortality of pandemic influenza (h n ) appears to be due to low infection rates in older adults who had a much greater risk of severe illness if infected. the reasons why older adults appear relatively resistant to pdmh n infection even though they appear to lack neutralizing antibody remains unclear. if antigenic drift or other adaptation of the pdmh n virus allows these older age groups to be infected more efficiently, the morbidity and mortality of subsequent waves of the pandemic could yet become substantial. and the national institute of allergy and infectious diseases, national institutes of health (contract no. hhsn c; adb no. n -ai- ). the funding bodies had no role in study design, data collection and analysis, preparation of the manuscript, or the decision to publish. bjc reports receiving research funding from medimmune inc., a manufacturer of influenza vaccines. the authors report no other conflicts of interest. some data presented in this manuscript were previously published in wu et al. it is well known that a primary goal of vaccination is to generate immunological memory against the targeted antigen to prevent disease in a vaccinated person. this ensures an accelerated immune response in the event of future contact with the pathogenic agent, such as a virus. therefore, it is very important to develop criteria for the assessment of vaccine immunogenicity by measuring both t and b memory cell levels from the vaccinated host. in contrast to inactivated influenza vaccines, live attenuated influenza vaccines (laivs) have been shown to provide primarily cellular and local immune responses. - to date, however, the hemagglutination-inhibition (hai) test (i.e. detection of serum antibodies) remains the method widely accepted for evaluation of an influenza vaccine's immunogenicity. improved understanding of the role of cellular and mucosal immunity and their contribution to protecting against severe illness caused by influenza infection has emphasized the need to reconsider methodologies used to evaluate the immunogenic impact of various influenza vaccines. such new assays need to include methods to measure local antibodies and virus-specific lymphocytes, especially in the case of live attenuated influenza vaccines, because of their potential to induce such broad-based immune responses. the aim of this study was to assess the ability of new russian pandemic laivs a ⁄ ⁄ duck ⁄ potsdam ⁄ ⁄ (h n ) ('ultragrivak,' registered ae ae ) and a ⁄ ⁄ california ⁄ ⁄ (h n ) ('influvir,' registered ae ae ) to induce memory t-cells in naïve human subjects and to compare results to levels of hai antibodies from each subject. a ⁄ ⁄ duck ⁄ potsdam ⁄ ⁄ (h n ) laiv was generated by : genetic reassortment of low-pathogenic avian influenza virus a ⁄ duck ⁄ potsdam ⁄ - (h n ) and master donor strain a ⁄ leningrad ⁄ ⁄ ⁄ (h n ). , the vaccine strain contains ha gene from avian virus, as well as na and internal genes from the master donor virus. a ⁄ ⁄ california ⁄ ⁄ (h n ) laiv was generated by classical ( : ) reassortment of a ⁄ california ⁄ ⁄ (h n ) with the master donor virus. the vaccine strain contains ha and na genes from a 'wild-type' h n strain and internal genes from the master donor virus. participants were aged to years and were without contra-indication of laiv vaccination. immunogenicity of a ⁄ ⁄ duck ⁄ potsdam ⁄ ⁄ (h n ) laiv was assessed in ten vaccinated persons and ten volunteers inoculated with a placebo (sterile physiological saline solution). immunogenicity of a ⁄ ⁄ california ⁄ ⁄ (h n ) laiv was estimated in vaccinated volunteers and nine volunteers inoculated with placebo. viruses or placebo were administered intranasally twice with an interval period of days at a dosage of ae ml per nostril for each vaccination. physical examination, venous blood and nasal swab samples were collected at four time points during the study: (i) before vaccination (day ); (ii) days after first vaccination (day ); (iii) days after the second vaccination (day ); and (iv) weeks after the second vaccination (day ). serum hai antibodies were measured by standard hai assay using % human red blood cells. test antigens for the assay were a ⁄ ⁄ duck ⁄ potsdam ⁄ ⁄ (h n ) or a ⁄ ⁄ california ⁄ ⁄ (h n ) to match the appropriate vaccine antigen. local iga antibodies in nasal swabs were evaluated by elisa using whole purified a ⁄ ⁄ duck ⁄ potsdam ⁄ ⁄ (h n ) or a ⁄ ⁄ california ⁄ ⁄ (h n ) viruses at hau per ae ml for absorption to elisa plates. endpoint elisa titers were expressed as the highest dilution of sera that gave an optical density (od) greater than twice the mean od of six negative controls in the same assay. percentages of virus-specific cd + cd + ifn-c + and cd + cd + ifn-c + peripheral blood memory cells were determined using a flow cytometry iccs assay performed by the published method. pbmcs were prepared with standard histopaque- gradient centrifugation from heparinized whole blood. wilcoxon matched pair test, mann-whitney u test and the students t-test were used for statistical data analysis. prior to the first vaccination (day ), gmts of hai antibodies to a ⁄ ⁄ duck ⁄ potsdam ⁄ ⁄ (h n ) and a ⁄ ⁄ california ⁄ ⁄ (h n ) laivs were ⁄ ae and ⁄ ae , respectively. in addition, gmts of siga against these specific antigens from nasal swabs were ⁄ ae and ⁄ ae , respectively. no hai antibody titers greater than : were observed prior to vaccination. background levels of virusspecific t-cells varied significantly within groups. mean levels of virus-specific cd + ifnc + cells were ae % to a ⁄ ⁄ duck ⁄ potsdam ⁄ ⁄ (h n ) and ae % to a ⁄ ⁄ california ⁄ ⁄ (h n ). for cd + ifnc + cells, initial levels were ae % and ae %, respectively. thus, background levels of virus-specific antibodies were low, but prior vaccination or virus exposure in some volunteers produced some pre-existing levels of t cells, thus they were not absolutely immunologically naïve in this sense. preexistence of h n -crossreactive antibodies and t-cells has been observed previously. [ ] [ ] [ ] effect of vaccination antibody immune responses both influenza a (h n ) and influenza a (h n ) laivs stimulated production of serum hai antibodies and local iga antibodies in nasal swabs. following the first vaccination with influenza a ⁄ ⁄ duck ⁄ potsdam ⁄ ⁄ (h n ) laiv, % percent of volunteers exhibited seroconversion of hai antibodies; after the second vaccination, % of volunteers exhibited seroconversion. after the first vaccination, a % conversion rate of siga was observed; after the second vaccination, % showed conversions in levels of siga. the first vaccination with a ⁄ ⁄ california ⁄ ⁄ (h n ) laiv showed ae % of hai antibodies seroconversions vaccination, and % seroconversion after second vaccination. for local siga, those results were ae % and ae % following the first and second inoculation, respectively. figure summarizes cellular immune responses observed in the vaccinated versus the placebo group. after the influenza a (h n ) laiv inoculation, significant differences in both cd and cd ifnc-producing t-cells were observed at day after the second vaccination (d ). these data indicate that healthy young people who never received such avian influenza vaccines and were not exposed to h n wild-type viruses were able to respond to the live attenuated h n influenza vaccine. after the first influenza a (h n ) laiv vaccination, reliable increases were observed in cd + cells only. after the second vaccination, increases in both cd + and cd + fold changes were significantly higher in vaccinated volunteers compared to the placebo group. it is noteworthy that cellular immune responses (cd + and cd + cells) were more marked in the a ⁄ ⁄ california ⁄ ⁄ (h n ). considering the long-term circulation of h -subtype viruses among humans in contrast to the novelty of h viruses, such a result would be expected. similar data were also observed following vaccination with the h n laiv. after first vaccination, the percent of people with notable increases in virus-specific cd + and cd + t-cells was % and % to h n and % and % to h n , respectively. after the second vaccination, these results were % and % to h n and % and % to h n , respectively. importantly, a significant number of vaccinated volunteers without remarkable increases ( ‡ -fold) in hai antibodies had notable increases in cd + and ⁄ or cd + memory cells. the percent of people with notable increases in virus-specific t cells after the second vaccination among hai()) volunteers was % and % to h n and h n , respectively. these results indicate that laivs were able to induce broadly responsive, key antiviral immune responses that would not have been detected by the hai assay alone. thus, it can be deduced that hai data alone fails to reveal important broad and specific immune responses to laiv. consequently, the hai test alone is not suitable for assessment of laiv immunogenicity. furthermore, vaccination with h n laiv was able to induce cross-reactive memory t-cells to a seasonal vaccine strain, a ⁄ ⁄ solomon islands ⁄ ⁄ (h n ) ( table ) . reliable increases to a (h n ) were observed in up to % of volunteers. there was an inverse dependence between levels of memory t cells before and after vaccination. authors are thankful to path for the financial support of these studies. we are also thankful to jessica d'amico and dr. rick bright for their editorial review. options for the control of influenza vii background: increased susceptibility of older populations to secondary bacterial pneumonia-like infections following influenza infection has been well documented. recent evidence in mouse models suggests that this increased risk from secondary bacterial infection occurs through a desensitization of the innate immune response. this recent finding, however, does not account for potential differences in immune responsiveness due to age. materials and methods: to address this parameter, we used three age groups (aged, adult, and young mice) to evaluate the role of age in influenza-mediated vulnerability to secondary bacterial challenge with pseudomonas aeruginosa. all mice were evaluated for multiple parameters including: (i) survival; (ii) lung bacterial load; (iii) total lung protein content; (iv) immune cell infiltration; (v) cytokine ⁄ chemokine expression; and (vi) toll-like receptor (tlr) rna expression profiles. results: prior challenge with influenza contributed to aberrant cytokine ⁄ chemokine profiles and increased lung cellular infiltrate in response to secondary bacterial infection across all age groups, supporting a critical role for influenza infection in the alteration of immune responses to other pathogens. also similar to human influenza, these changes were exacerbated by age in mice as demonstrated by increased bacterial load, mortality, and total lung protein content (an indicator of lung damage) after p. aeruginosa challenge. conclusions: these data support a potential role for virus-mediated and age-mediated alteration of innate immune effectors in the pathogenesis of influenza and the increased susceptibility of influenza virus infected mice to secondary bacterial infection. the understanding of the complex interaction of host and pathogen -and the role of age -in human influenza is critical in the development of novel therapeutics and improved vaccine approaches for influenza. our results support further examination of influenza-mediated alterations in innate immune responses in aged and non-aged animals to allow elucidation of the molecular mechanisms of influenza pathogenesis in humans. there is considerable evidence in the clinical literature to support the role of influenza infections with an enhanced risk for secondary bacterial pneumonias. [ ] [ ] [ ] given the increased pneumonia-related morbidity and mortality in both the young and elderly populations, there is rationale for gaining a deeper understanding as to the systemic changes in the pulmonary microenvironment. although there are some recent reports that account for some of the molecular mechanisms at work in this disease process, there is a paucity of experimental evidence that considers the potential effects of age. developmental changes in the immune system that occur in the aged environment have been well documented with regard to senescence of the adaptive immunity, global changes in myeloid cell function, and the establishment of a general pro-inflammatory state. , the aim of this work was to provide evidence for the contribution of the aged immune environment to the pathology of influenza mediated secondary bacterial infections. animals used in this study were housed under conditions approved by tulane university's institutional animal use and care committee. female balb ⁄ c mice used in these studies were divided into three age groups: aged ( months old), adult ( months old), and young ( months old). each age group was subdivided into two groups: influenza infected and naïve (control). mice were infected by the intranasal route with · pfu of mouse-adapted influenza a ⁄ pr ⁄ ⁄ . clinical disease was measured by body weight changes over a week period post influenza challenge, and recovery was determined as return to pre-infection weight. all mice were subsequently challenged intransally with · cfu pseudomonas aeruginosa strain pao . twenty-four hours post-pseudomonas challenge, bal with sterile pbs was performed on all mice in all groups. total rna from the cellular fraction was pooled from three experimental animals from each group. tlr mrna was detected by qrt-pcr, where expression levels were determined as relative to b-actin mrna levels. cdna was synthesized from total cellular rna from bal samples using iscript cdna synthesis kit (biorad). pcr reactions were composed of ae lg cdna forward and reverse primers according to optimized conditions and ae ll of · syber green icycler supermix (biorad), in a total vol-ume of ll and were run using a biorad icycler utilizing melting point determination. primers and concentrations used in this study included: mus_tlr f: tgctttcct-gctggagattt- nm, mus_tlr r: tgtaacgcaac agcttcagg- nm, mus_tlr f: atatgcgcttcaa tccgttc- nm, mus_tlr r: caggagcatactggt gctga- nm, mus_tlr f: ggcagcaggtggaattg tat- nm, mus_tlr r: aggccccagagttttgttc t- nm, mus_tlr f: ctggggacccagtatgctaa- nm, mus_tlr r: acagccgaagttccaagaga- nm, mus_tlr f: ggagctctgtccttgagtgg- nm, mus_tlr r: caaggcatgtcctaggtggt- nm, mus_ b-actinf: agccatgtacgtagccatcc- nm, mus_b-actinr: ctctcagctgtggtggtgaa- nm. as a measure of protein leakage into the alveolar space, total protein content in each bal was measured by bca assay of each supernatant fraction according to manufacturer's instructions (pierce). cytokine and chemokines levels were measured by multiplexed bead array (bioplex, biorad). immune cell characterization of bal was estimated by flow cytometry. lymphocyte populations were gated by forward versus side scatter and characterized as b cells (f ⁄ ) , cd + ) or t cells (cd b ) , cd + ). the myeloid population that is composed of macrophages, neutrophils, dendritic cells, and natural killer cells was enumerated by gating all but those found in the lymphocyte gate using forward versus side scatter plots. flow cytometry data was analyzed using flojo software (treestar). statistical analysis, where appropriate, was performed using a two-way analysis of variance (age versus influenza infection status) supported by bonferonni's correction for multiple comparisons. a recent finding by didierlaurent, et al., described an influenza mediated desensitization of tlr function as a primary contributor to an increase in bacterial burden when challenged after resolution of the primary influenza infection. this finding, however, was obtained using animals that were - weeks of age, where our study included two cohorts of older mice ( months and months). using whole protein content of the bal as an estimate of protein leakage into the lumen of the lung, we found elevated protein content in aged mice as compared to young and adult mice. in aged mice, a slightly lower total lung protein when comparing influenza infected to protein in the bal from influenza naïve mice challenged with p. aeruginosa (table ) . supporting previously published studies showing a generalized pro-inflammatory cytokine environment in the aged immune system, we provide evidence for significantly (p = ae ) and an increase in ifnc (p = ae ) was detected. the decrease in gm-csf correlates well with a previous report that gm-csf is less prevalent in influenza resolved animals (table ). we also report a noticeable change in the immune cell populations with respect to b-cells, cd + t-cells, and the myeloid cell populations. there is a trend of increased prevalence in cd t-cells in the post-influenza environment across all ages. b-cell numbers also trend toward increase in influenza treated animals in young and adult animals; however, there is a noticeable decrease in the bcells in aged animals. across all age groups, there is a general decrease in frequency of cells that would normally make up the myeloid cellular fraction of the bal (macrophages, neutrophils, dendritic cells, and natural killer cells) ( table ). our study also shows, as cited by others, that toll-like receptor (tlr) gene expression in the post-influenza environment is decreased in cells found in the bal after both influenza and pseudomonas infection. our data support the previous finding of a reduced expression of tlr mrna in influenza-cleared mice when we measured tlr , , , and . only tlr showed differences with respect to age with young mice showing little or no detectable change in tlr mrna expression. our results show an increase in the expression across all tlrs examined in the aged mice group (table ) irrespective of influenza infection status. these data support earlier studies performed with adult mice that showed reduced tlr mrna expression in the post-influenza environment. this study also expands the current understanding of the potential role of age in influenza mediated bacterial infection-induced mortality. the impact of these alterations in the immune microenvironment across age groups and infection status is highlighted by the ability of bacterially challenged animals to clear infection. assessment of bacterial load in the lungs of p. aeruginosa challenged mice indicated a difference in young and adult mice if previously infected with influenza virus. in aged mice, both influenza challenged and influenza-naïve mice had higher bacterial loads and less variability when comparing within the age group, supporting the risk of age alone in susceptibility to bacterial pneumonia (table , figure ). taken together, these data support the potential role for both virus-mediated and age-mediated alteration of innate immune effectors in the pathogenesis of influenza and increased the susceptibility to secondary bacterial infection that results from influenza infection in mice. these findings highlight distinct differences in the immune environment between age groups and thus reveal necessity for further examination as to the mechanisms of immunity across age with respect to current infection status. garnering a clearer understanding as to the complex interaction of host and pathogen with respect to age in influenza infections is central to the development of increased efficacy in vaccine and therapeutic strategies. prospective estimation of the effective reproduction background pandemic influenza a (h n ) virus (ph n ) emerged in early and rapidly spread to every continent. an urgent priority for international and national public health authorities was to estimate the transmissibility of the pandemic strain for situational awareness and to permit calibration of mitigation strategies. the basic reproductive number, r , is defined as the average number of secondary cases that index case generates in a completely susceptible population, and is a common measure of transmissibility. however, it is difficult to estimate r without an understanding of the degree of any pre-existing immunity in the population. the effective reproductive number, r, is defined as the average number of secondary cases that index case generates, and can be estimated over time (i.e. r t ). wallinga and teunis described a method to estimate r t based on illness onset dates of the cases while assuming that all secondary cases would have been detected, and cauchemez et al. extended the method to permit prospective estimation by adjusting for secondary cases that have not yet experienced illness onset at the time of analysis. we describe how the method can further be extended to account for reporting delays, allowing true real-time estimation of r t during an epidemic, and we illustrate the methodology on notifications of ph n and associated hospitalizations in hong kong. we obtained data on all laboratory-confirmed ph n infections ('cases') reported between may and november , to the hospital authority and center for health protection in hong kong collated in the eflu database. a subset of the cases was hospitalised. the database also included information on age, sex, illness onset date, laboratory confirmation date, and contact history (for the early cases). laboratory-confirmed ph n infection was a notifiable condition throughout our study period. we extended existing methods for estimating r t over time to allow for reporting delays between illness onset and notification, and between illness onset, notification, and hospitalisation for those cases that were hospitalised, where the reporting delay distribution were estimated empirically from the data. we further extended the methodology to allow for imported cases (infected outside hong kong) contributing to the estimation of r t as infectors but not infectees. we used multiple imputation to allow for missing data on some symptom onset dates to make best use of all available data. we used a serial interval with mean (standard deviation) of ae ( ae ) days, and in sensitivity analyses, we used serial intervals with mean ae days and ae days. statistical analyses were performed in r version . . (r development core team, vienna, austria). in late april following the who global alert, hong kong initiated containment protocols to attempt to delay local transmission of ph n for as long as possible. these measures included screening at ports, airports, and border crossings, and enhanced surveillance for people with influenza-like illness, particularly for those who had recently returned from abroad. laboratory testing capacity was substantial due to heavy investment in local infrastructure following previous experiences with avian influenza a ⁄ h n in and severe acute respiratory syndrome in . laboratory-confirmed ph n cases were isolated until recovery, and their close contacts were placed under quarantine for days. imported cases were identified sporadically through may and early june . the first case of ph n not traceable to importation (i.e. a local case) was identified on june and triggered a change to mitigation phase measures. some containment measures, including isolation of cases, were continued until the end of june to allow a soft transition between containment and mitigation phases. as an immediate measure to try to reduce community transmission of ph n , all childcare centres, kindergartens, and primary schools were proactively closed for days (subsequently extended for another - days to summer vacation in early july). any secondary schools in which one or more confirmed ph n case was identified were reactively closed for days. on june the government opened eight designated flu clinics across the territory to provide free medical consultation for outpatients with influenza-like illness and free laboratory testing for ph n . these clinics resumed regular chronic disease services in mid-august, and laboratory testing and antiviral treatment was restricted to high risk groups in september. the various interventions are highlighted in figure (a), superimposed on the epidemic curve of laboratory-confirmed ph n cases and ph n -associated hospitalizations. around % of the cases were hospitalised, and this proportion increased somewhat towards the end of the epidemic. figure (b) shows the estimates of r t based on laboratory-confirmed ph n cases. the estimated r t peaked at ae on june , and fell below between june and july (which was within the school closure period). r t fluctuated between ae and ae through the school summer vacations in july and august, it subsequently increased to around ae - ae after schools reopened in september until the epidemic peaked in late september, and then fluctuated below as the epidemic declined. the trends in r t based on h n -associated hospitalizations were similar, although with wider confidence intervals due to the smaller number of events ( figure c ). the extension of the methods to allow for reporting delays avoided substantial bias in realtime estimates of r during the epidemic for the most recent days, and closely tracked the final estimates of r t . our results suggest that ph n may have had slightly lower transmissibility in hong kong than elsewhere. for example, estimates of r t were around ae - ae in new zealand and australia. lower transmissibility in hong kong has been associated with school closures in june and july followed by summer vacations from july through august. furthermore, in hong kong the influenza virus usually does not circulate after august, and therefore seasonality could also be a cause for the lower r t . on the other hand, the interventions applied during the mitigation phase, such as the widespread use of antiviral treatment in hong kong and the pre-existing immunity in the ageing population in hong kong, may also be associated with lower transmissibility. there are some limitations to our work. first, we only used aggregated data, and we did not consider the heterogeneity among the cases in terms of sex and age or other factors. therefore our estimates can only provide a snapshot of the overall trend, but limited information for any specific subset of population. secondly, we did not consider the possibility that cases might be infected in hong kong and exported to other countries, which could lead to slight underestimation of the transmissibility. one has to be careful in translating the estimated r t to the effectiveness of any specific interventions, as interventions may not be the only factor influencing the transmissibility; for example, a depletion of the susceptible population during an epidemic can also be a factor for the decline in r t . in conclusion, real-time monitoring of the effective reproduction number is feasible and can provide useful information to public health authorities for situational awareness and planning. in affected regions, laboratory capacity was typically focused on more severe cases, and changes in laboratory testing and notification rates meant that that case counts may not necessarily reflect the underlying epidemic. a useful alternative to case-based surveillance is surveillance of the subset of severe infections, for example hospital admissions, or icu admissions, and our results show that it was feasible to monitor ph n -associated admissions in real-time to estimate transmissibility. influenza antigenic cartography projects influenza antigens into a two or three dimensional map based on immunological datasets, such as hemagglutination inhibition and microneutralization assays. a robust antigenic cartography can facilitate influenza vaccine strain selection since the antigenic map can simplify data interpretation through intuitive antigenic map. however, antigenic cartography construction is not trivial due to the challenging features embedded in the immunological data, such as data incom-pleteness, high noises, and low reactors. to overcome these challenges, we developed a computational method, temporal matrix completion-multidimensional scaling (mc-mds), by adapting the low rank mc concept from the movie recommendation system in netflix and the mds method from geographic cartography construction. the application on h n and pandemic h n influenza a viruses demonstrates that temporal mc-mds is effective and efficient in constructing influenza antigenic cartography. the web sever is available at http://sysbio.cvm. msstate.edu/antigenmap. as a segmented, negative stranded rna virus, influenza virus is notorious for rapid mutations and reassortments. the mutations on the surface glycoproteins (ha and na) of influenza viruses are called antigenic drifts, and these antigenic drift events allow the virus to evade the accumulating immunity from previous infection or vaccination and lead to seasonal influenza epidemics. a reassortment event with a novel influenza antigen may result in antigenic shift and cause influenza pandemic. for instance, the h n pandemic virus is a reassortant with a swine origin ha antigen. vaccination is the primary option for reducing the effect of influenza, and identification of the right vaccine strains is the key to development of an effective vaccination program. the antigenicity of an optimal vaccine strain should match that of the epidemic strain. in influenza surveillance program, the influenza antigenic variants are generally identified by the immunological tests, such as hemagglutination inhibition (hi) assay, microneutralization (mn) assay, or elisa. these immunological assays measure the antigenic diversity between influenza viruses by comparing the reaction titers among the test antigens and reference antisera. however, data interpretation of the data from these assays is not trivial due to the embedded challenges such as data incompleteness, high noises, and low reactors. by mimicking geographic cartography, influenza antigenic cartography projects influenza antigens into a two or three dimensional map using immunological datasets. antigenic cartography can simplify the data interpretation, and thus, facilitate influenza antigenic variant identification. recently, we developed a novel computational method, temporal matrix completion-multidimensional scaling (mc-mds), in antigenic cartography construction. in this paper, we described the details of temporal mc-mds, especially the original concepts introduced in this method, and how they can achieve the robustness in antigenic cartography construction. our method included two integrative steps: it first reconstructs the hi matrices using low rank mc method, and then generates antigenic cartography using mds with a temporal regularization. the mc concept was adapted from the movie recommendation system in netflix and the cartography concept from geographic cartography. in , netflix, an online dvd and blu-ray disc rentalby-mail and video streaming company, held a -year netflix prize contest (http://www.netflixprize.com/) on computational methods for improving its recommendation system. in its recommendation system, netflix collected the rating data from the individuals. based on his or her renting history and the ratings in the systems (e.g., from evaluators and other renters), netflix recommendation system suggests certain movies to a renter. apparently, no individuals would be feasible to provide ratings for all of the movies, as it will take hundreds of years for a single person to rate over movies available from netflix. thus, the resulting rating data is an incomplete matrix, and it can be as sparse as less as %. the challenge in netflix recommendation system is a classic mc problem. [ ] [ ] [ ] [ ] [ ] as the inspiration of netflix prize contest, many efficient low rank mc algorithms were developed, for instance, opt-space, svt, cf, bellkor, pf, and fwls. eventually, the team bellkor's pragmatic chaos won this contest. their methods combines nonlinear probe blending and linear quiz blending to come up with a predictor bigchaos. matrix completion estimates the unobserved values based on the observed values. the users can refill the missing data without repeating the experiments. furthermore, mc will help reduce the noises in the data, for instance, those biases by different individuals performing experiments. in influenza antigenic characterization, hi assay is a commonly used assay for antigenic analysis, since hi assay is relatively economic and easy to perform. however, hi is labor intensive, and it is almost impossible for any individual lab to complete the hi assays for all pairs of antigens and antisera during influenza surveillance. in addition, both testing antigens and the reference antisera are dynamic. for instance, in seasonal influenza surveillance, generally only contemporary antisera are used in experiments. thus, we will have to integrate multiple hi tables in order to evaluate the overall antigenic changes for influenza vaccine strain selection. the resulting hi tables will be incomplete, and the observed entries in the integrated hi data can be as less as %. the completion of this matrix can be formulated as a typical mc. briefly, given the combination of hi matrix with m antigens and n antisera, the hi matrix can be represented as m m·n = (m ij ) m·n , where m ij denotes the hi values from the reaction between testing antigen i and antiserum j. the low rank mc assumes that both antigen and antiserum can be embedded into a low rank space. to be specific, the low rank mc method is to seek matrix u m·r , v n·r and a diagonal matrix r r·r , where m = u m·r r r·r (v n·r ) t . in order to achieve this goal, the optimization formulation has been employed, which can be represent as following, where e denotes the observed entries in hi matrix and g(x) is a regularization function. the eqn ( ) is the standard format of a low rank mc formulation. the geographic cartography is a common technique to display the cities and their geographic distances in a map. this cartography can be generated using mds based on a geographic distance matrix. figure (a) shows the antigenic cartography generated using a distance matrix with seven cities, and figure (b) is a map for comparison. as an analog of geographic cartography, the influenza antigenic cartography maps the influenza antigens into a two or three dimensional map based on the distance matrix generated using immunological data. this incomplete matrix can be filled through mc algorithm discussed in section mc and netflix. low reactors, non-random date incompleteness, and temporal model generally, three types of data are present in a combined hi matrix: high reactor, low reactor, and missing values. among these three data types, high reactors are the most reliable data points. the low reactors are those values present in the hi matrix as ''equal to or less than a threshold h'', where h can be , , , or . low reactors have similar values in the affinity dataset but could be from different binding settings. these low reactors are present due to the detection limits of biotechnology, and they are not reliable. both these missing values and low reactors make it very difficult to analyze and interpret antigenic correlations amongst tested antigens and reference antigens. to our best knowledge, none of the existing mc method can handle the threshold values. in addition, the non-random incompleteness of influenza immunological datasets generates an additional challenge in traditional mc methods, which are based on the assumption that the observed values are randomly distributed among the matrix. in a typical combined antigenic hi data, most of the off-diagonal entries are missing values or low reactor values. in order to overcome the above issues, we incorporated a regularization function into the eqn ( ), where this indicator function is only valid for those entries with low reactor values. an alternating gradient decent method is applied to solve the optimization problem in eqn ( ) . in addition, a temporal mds method is proposed to project the antigens into a or dimensional map. x where d ij is the average distance between virus i and virus j, t i is the isolation year of virus i, d ij is the distance between virus i and virus j in cartography, d ac i is the distance between virus a and center of group i, and d c i c j is the distance between the centers of group i and group j. all the parameters are tuned by cross validation. we named this method as temporal mc-mds. by applying temporal mc-mds method in an h n dataset, low reactors. figure (a) is a three-dimensional influenza antigenic map based on this data by using mc-mds method. the reported clusters (hk , en , vi , tx , bk , si , be , be , wu , sy , and fu ) were displayed in the core of a spiral s-shape, and bk and be are located at the turning point of this s-shape. however, the antigenic distances between some viruses are incorrect. for example, the distance between hk and fu in the projection is ae units, which is close to the distance between hk and bk ( ae units). the main reason leading to those inaccurate distances is the unique distribution of hi datasets described in section . . in comparison, with the temporal model, not only the viruses in clusters have been clearly separated, but also the antigenic distances between each cluster are proportional to their isolation time interval. in this updated cartography ( figure b ), the antigenic distance between hk and fu is ae units, where the distance between hk and fu is ae units. this result suggested that the temporal information is critical for antigenic cartography construction for immunological datasets spanning a long time period. the hi data from seasonal influenza surveillance belong to this category. for seasonal influenza virus ⁄ pandemic influenza viruses within a short time span, the temporal model is probably not necessary, as there is lack of long-term immunological pressure present in the population. figure (c) is an antigenic cartography generated using a hi dataset with h n influenza viruses spanning from april of to june of . this map demonstrates that there is lack of antigenic drifts during the first wave of this pandemic influenza as all of these viruses are mixed altogether. our limited studies on h and h avian influenza viruses suggested the temporal model is not needed for avian influenza viruses. however, extensive studies are required to investigate whether there is any special data structure present in this type of data. in this study, we described in details the concepts and applications of new computational method, temporal mc-mds for influenza antigenic cartography construction. we formulate the influenza cartography as two integrative steps: low rank mc problem from the concept of netflix movie recommendation system and mds from geographic cartography construction. in order to handle two additional challenges, including low reactor and non random distribution of antigenic data, a temporal model is incorporated into mc-mds as temporal mc-mds. our applications demonstrated that temporal mc-mds is effective in constructing influenza antigenic cartography. the three dimensional antigenic cartography for a ⁄ h n seasonal influenza virus without temporal model, and the antigenic clusters were defined in ref. [ ] ; (b) the three dimensional antigenic cartography for a ⁄ h n seasonal influenza virus with temporal model; (c) the two dimensional antigenic cartography for a ⁄ h n pandemic influenza without temporal model, and these viruses were labeled in shape by the corresponding month for them to be detected. one grid is corresponding to a twofold change in hemagglutination inhibition experiment. the mechanisms driving the three waves of infection and mortality in the uk in - are uncertain. although the circulation of three distinct viruses could have generated three waves of infection, the virological evidence required to prove or disprove this hypothesis is lacking. social distancing, an alternate mechanism for generating fluctuations in the effective susceptible pool and therefore explaining multiple waves of infection, , was not generally imposed in the uk as it was in the us and australia. we are therefore motivated to explore the possible role of continual population-level changes in the average protective response against the circulating virus in generating a multi-wave pandemic, within a biologically motivated deterministic model for influenza transmission. the nature and duration of protection against further infection following recovery from influenza is uncertain and depends on the mode and tempo of viral evolution, as well as the response of the cellular and humoral arms of the adaptive immune system. for a given seasonal ⁄ pandemic strain, memory b-cells may generate a specific antibody response in a portion of the adult ⁄ elderly population, depending on the exposure to related antigenic sub-types. however neutralising antibodies are unlikely to be a widespread immunological response to a novel (pandemic) strain. memory t-cells which recognise conserved internal viral proteins may be a more common mechanism for protection; the generation of very high levels of cytotoxic cd + t-cells potentially facilitates rapid viral clearance, , and lower levels of cd + t-cells perhaps provide partial protection. in this work we explore key drivers of multi-wave pandemics within phenomenological models that incorporate different immune response mechanisms building on existing models , incorporating the role of evolving population-level protection in multi-wave pandemics. we use weekly reports of influenza mortality rates for five administrative units in the uk (blackburn, leicester, newcastle, manchester and wigan) where records from block censuses instigated by local medical officers to record the cumulative incidence of reported symptoms in each wave in a sample of or more households are also available. the symptom reporting data allows us to estimate the case fatality rate and thus use the mortality time series to constrain our transmission model. furthermore, the incidence of individuals reporting symptoms in multiple waves provides information about the acquisition and loss of immunity. we extract the death rate and symptomatic (re)infection rates predicted by our model prevalence for a given set of parameters and estimate a likelihood-based on a comparison to all the death and cumulative reported incidence data assuming a negative binomial error distribution. we utilise monte carlo markov chain (mcmc) methods with parallel tempering algorithms to maximise this likelihood and obtain parameter estimates. parallel tempering -which concurrently searches for maximal likelihood parameter solutions on a set of scaled likelihood surfaces -allows for relatively rapid exploration of the parameter space. we use bayesian information criteria (combined with qualitative assessment of biological plausibility) to aid model selection. we have implemented a deterministic compartmental transmission model, which allows for a variety of phenomenological modes of protection against the pandemic virus. to facilitate this, we stratify the population into two groups; the 'experienced' population (stratum ) who have had been exposed to an influenza virus and the 'naive' population (stratum ) who have not. in each stratum, i hosts may be classified as either susceptible s i , exposed e i and e i , having (recovered from) a symptomatic i i (r i ), or asymptomatic a i (ra i ) infection. note that the states tq i , tq i , e i , t i , and t i are included so that the hosts move between the key epidemiological states with a peaked (rather than exponential) distribution of waiting times. hosts in the experienced stratum may exhibit reduced susceptibility, infectiousness, and symptomatic proportion compared to naive hosts, parameterised by e i , e s , and e a , respectively; however note that depending on the model parameters, there may be fully susceptible hosts within the experienced stratum. in addition, we assume homogeneous population mixing and a constant basic reproduction number r with the force of infection: modulated by a sinusoidal seasonal term with amplitude b with phase chosen to maximise transmission in the winter season. here n is the total population size, and x e is the initial fraction in the experienced strata. the proportion of symptomatic cases a and the case fatality rate l are permitted to vary from wave to wave (and given indices , or accordingly). the transmission dynamics is described by the following set of coupled ordinary differential equations. where s in, = p utq i and s in, = in order to divert recovered infectious hosts from the naive stratum into the experienced stratum. the probabilities of gaining permanent protection are q = q and q = . the latent exposed period is fixed to be c = ⁄ ae days, and the rate of recovery is parameterised by m = ⁄ t inf , where t inf is the infectious period. hosts with prior sterilising protection begin in q and move into s at rate u q = ⁄ t wq . recovered hosts (r i ) migrate back to s at a rate u = ⁄ t w . the state p contains hosts with permanent protection. the modes of protection captured in this model are: i. permanent prior protection (beginning in state p ), ii. waning prior protection (beginning in state q ), iii. permanent acquired protection with probability q (moving into state p ), iv. waning acquired protection with probability ) q, and, v. partial prior protection (beginning in state s ) resulting in reduced infectiousness (e i ), susceptibility (e s ), and symptomatic proportion (e a ). in the context of this model, 'permanent' protection refers to protection which lasts for the duration of the epidemic. here we explore the results of parameter fitting to two models which differ in the nature of the assumed pre-existing protection in the community at the beginning of the pandemic. protection hypothesis assumes that the prior protection is sterilising but temporary, whilst protection hypothesis assumes that the prior protection is partial but permanent and may act on susceptibility, infectiousness, and ⁄ or asymptomatic proportion. each model allows waning acquired protection and for a proportion q of the experienced population to gain permanent protection following infection. fitted parameters common to each model are t inf , b , q, t w , a, l and the proportion beginning in p x i . prior protection hypothesis : sterilising, waning prior protection we fix x e = and fit for q (t = ) ⁄ n and t wq so that protective modes i, ii, iii, and iv are enabled ( figure ). it is important to note that due to the slow convergence of the mcmc chains, we cannot guarantee that our parameter estimates correspond to the global minimum. furthermore, parameter estimates can only be meaningfully interpreted for good fits to the data. due to the prediction of a fourth (unobserved) wave for the model fit to blackburn, we do not report these parameter estimates here. the fits to the leicester data are generated with the parameter set r = ae , a = ae , a = ae , a = ae , t w = ae years, t wq = ae years, we fix q (t = ) ⁄ n = and fit for x e , e a , e i , and e s so that protective modes i, iii, iv, and v are enabled (figure ) . the parameters corresponding to the fit in figure for leicester are r = ae , a = ae , a = ae , a = ae , t w = ae years, p (t = ) ⁄ n = ae , s (t = ) ⁄ n = ae , b = ae , t inf = ae days, q = ae , e a = ae , e i = ae , and e s = ae . our model with protection hypothesis -which, similarly to the model discussed in ref. [ ] , assumes that a sub-population has waning sterilising prior protection -is able to generate multiple waves of infection via the continual replenishment of s from an initially large proportion (over %) of hosts with prior protection in q combined with the waning of acquired immunity in around % of cases on a time-scale of months. disease severity as measured by symptomatic proportion increases from % in the first wave to above % for the second and third waves. over a quarter of the population are initially permanently immune, and a large r value of ae drives transmission in the remaining population. protection hypothesis -which assumes that prior protection offers partial susceptibility and ⁄ or reduced infectiousness or symptomatic disease -performs slightly more poorly; the fit to the leicester data has an inferior likelihood (although the mortality data only likelihood is a little larger), despite the higher dimensionality of the model. nevertheless, the model fit still mirrors many characteristics of the data, particularly for leicester. we note that for this model, a is very near the lower limit, corresponding to ubiquitous exposure in the first wave. in this scenario, refuelling of the susceptible pool to generate secondary and tertiary waves is still possible due to a shorter waning time of acquired protection (well within months) and a lower probability of gaining permanent protection following infection, when compared with the parameter estimate for hypothesis . the parameter estimates suggest that approximately % of the population initially experiences reduced disease severity (e a $ ae ), but similar susceptibility and infectiousness. a larger value for r $ ae is required to drive transmission despite low numbers beginning in p , due to the large number of hosts who acquire temporary or permanent immunity early on in the pandemic. it is clear that, at least mathematically and perhaps biologically, there are multiple possibilities for the structure of population-level protection which are compatible with the generation of multiple pandemic waves. however, whilst the models considered here are able to explain the observed mortality and reinfection data for some patterns of infection and mortality (e.g. leicester), they are not consistently able to reproduce a pandemic which dies out after three waves across the connected populations we are studying (e.g. for blackburn). it is challenging to construct a deterministic model for the spread of disease within multiple locations in the uk in , which assumes homogeneous mixing without modulation of the transmission rate by social distancing. an improved model working with these assumptions likely requires a richer structure for the host protection response than the structures we have explored thus far. we are currently seeking improved fits to the data by implementing a number of biologically defensible exten-sions to our model, including incremental immunity whereby t w increases by a factor v after each exposure to the pandemic flu, and incremental loss of prior protection whereby a increases as hosts lose their sterilising prior protection. it is important to note that the mechanism(s) generating differences in the pandemic experience recorded in geographically connected locations is an open question; true differences in demography, varying degrees of reactive social distancing, inhomogeneities in the circulation (or circulation history, i.e. prior immunity) of viral strains, stochastic variations, and ⁄ or unique socio-cultural ⁄ behavioural conditions may all contribute to this effect. the h n experience in australia and elsewhere highlighted the difficulties faced by public health authorities in diagnosing infections and delivering antiviral agents (e.g. oseltamivir) as treatment for cases and prophylaxis for contacts in a timely manner. consequently, forecasts from mathematical models of the possible benefits of widespread antiviral interventions were largely unmet. we summarise results from a recently developed model that includes realworld constraints, such as finite diagnostic and antiviral distribution capacities. we find that use of antiviral agents might be capable of containing or substantially mitigating an epidemic in only a small proportion of epidemic scenarios given australia's existing public health capacities. we then introduce a statistical model that, based on just three characteristics of a hypothetical outbreak [(i) the basic reproduction number, (ii) the reduction in infectiousness of cases governments and public health agencies worldwide, spurred by outbreaks of sars and h n , have developed preparedness strategies to mitigate the impact of emerging infectious diseases, including pandemic influenza. pandemic response plans are presently being revised in light of the h n experience. [ ] [ ] [ ] many developed countries amassed large stockpiles of neuraminidase inhibitors (nais) with the expectation that they could be used to not only treat the most severely ill, but curb transmission in the community. without relevant field experience indicating how nais should be distributed, mathematical and computational modelling has been used to inform optimal deployment policy in a pandemic scenario. - models of population transmission were used to infer likely effects on epidemic dynamics, using data from human and animal studies of experimental infection and nai efficacy trials. in the australian (and wider) context, models indicated the potential for substantial benefit at the population level if nais were distributed in a liberal manner, targeting close contacts of indentified cases. furthermore, results indicated that use of limited nai resources in this way may improve the impact of case treatment due to the effects on epidemic dynamics. however, these models did not take into account logistic and other real-world constraints, such as finite diagnostic and antiviral distribution capacities, which were identified as limiting factors during the australian h n pandemic response. [ ] [ ] [ ] in particular, if using positive pcr diagnosis as a 'decision to treat' test, delays to confirmation of diagnosis, particularly once total laboratory capacity was exceeded, prevented timely delivery of nais to both cases and contacts of cases. in previous work, we have extended our existing models to examine how diagnostic strategies [e.g. using pcr confirmation versus syndromic influenza-like illness (ili) presentation as a decision to treat], diagnostic-capacity, and nai distribution capacity each impact on the ability to deliver an effective intervention. the model uses case severity (the proportion of infections deemed severe) to determine the overall presentation proportion, and so the ability to identify individuals eligible for nai treatment and contact prophylaxis. figure (a) shows a key result from the model. for each curve shown, we simulated thousands of epidemics, sam-pling across plausible ranges of parameters describing virus, population, and intervention characteristics using a latin hypercube sampling (lhs) approach. without intervention, the proportion of the population infected either symptomatically or subclinically by the end of the epidemic is around %. if a syndromic strategy (ili presentation) is used to determine provision of nais as treatment and prophylaxis, excessive distribution of drug to individuals who are not infected with influenza occurs early in the epidemic. early stockpile expiry accounts for a marginal impact of the antiviral intervention on the final outbreak size, in the order of a few percent. the second strategy modelled (pcr ⁄ syndromic) is one where pcr confirmation of diagnosis is required early in the epidemic to make treatment decisions until such time as laboratory capacity is exceeded. from this point, individuals are treated on the basis of symptoms alone -during an epidemic phase in which a substantial proportion of ili presentations will be attributable to influenza. under this strategy, the intervention is able to control the outbreak in approximately % of the simulated epidemics given the 'base case' constraints on diagnosis and delivery assumed in the model. the results highlight that a successful antiviral intervention requires a highly sensitive diagnostic strategy in the initial stages of the epidemic and comprehensive distribution of post-exposure prophylaxis. a pcr ⁄ syndromic strategy for decision to treat and provide contacts with prophylaxis is thus optimal. the surface in figure (b) shows the percentage of simulation runs for the pcr ⁄ syndromic strategy that have a final population attack rate of < % (a substantial reduction from the no intervention case of approximately %) as a function of pcr capacity and nai daily distribution capacity. as indicated by the arrow, the estimated australian pcr laboratory capacity appears to be sufficient, while significant benefits for the public health outcome may be achieved if logistical delivery constraints for nai distribution can be ameliorated. however, the probability that such an interventioneven with substantial increases in pcr and nai distribution capacity -would successfully mitigate an epidemic is low ( - %), and consequently it is difficult to universally recommend an antiviral intervention. in this study, we introduce a statistical model that predicts whether or not an nai distribution strategy based on a pcr ⁄ syndromic antiviral distribution policy will be successful in mitigating an epidemic. we thereby provide proof-of-principle for the design of a decision support tool that may be used by public health policy makers during an epidemic when faced with formulation of context specific nai distribution policy. synthetic data of hypothetical outbreaks and interventions were generated using the lhs simulations developed in ref. [ ] . we selected a random sample of outbreaks from a total of simulated epidemics ( % of model simulations). using these data, we identified independent model parameters that were most highly rank-correlated with the final attack rate. these parameters were included in a logistic regression model to assess their ability to predict whether an influenza epidemic would be successfully mitigated by an antiviral intervention (ar < %). model predictions were then validated against the full simulated dataset. full details of the simulation model, its structure, parameterisation and parameter distributions are available in ref. [ ] . use of the lhs simulation approach, and the method of model analysis and evaluation was similar to that previously described. matlab a (mathworks, natick, ma, usa) was used for the analysis and statistical model fitting. table shows results from our logistic regression model. key parameters sufficient to predict whether or not an outbreak may be controlled by the deployment of av agents are: . r , the basic reproductive number of the outbreak (assigned values between ae and ae for this example). as the value of r increases, the epidemic progresses more rapidly and is more difficult to control, explaining the negative correlation coefficient. . e t , the relative infectiousness of treated individuals (assigned values between ae and ae ). higher values for this parameter indicate only modest drug effects on transmission, explaining the negative correlation coefficient. . g, the proportion of infections that are severe (assigned values between ae and ae ), and which in turn determines the presenting proportion (derived values between ae and ae ). as the presenting proportion increases, the ability to identify and treat cases and deliver prophylaxis to contacts also rises, increasing the impact of the antiviral intervention. the roc curve ( -specificity versus sensitivity, not shown) for the logistic regression model specified in table has an area under the curve of ae , demonstrating that the model predicts the success of an antiviral intervention extremely well. for example, with a sensitivity of % we still have a specificity of approximately %. evaluation of the pandemic response has emphasised the need for early informed decision-making to implement proportionate disease control measures. our model identifies a low probability of successful epidemic mitigation using targeted antivirals alone (figure and ref. ), in distinction to results from models that fail to account for the diagnosis and delivery constraints inherent in any public health response. the decision support tool (table ) highlights key epidemic characteristics that are predictive of a high likelihood of effective mitigation. the reproduction number was one of the earliest parameters estimated from early outbreak data during the h n outbreak. , our findings reinforce the importance of characterising epidemic severity as early and as accurately as possible, in order to inform a proportionate pandemic response. critically, a typically mild pandemic (low g), such as that experienced in , is predictably difficult to contain using a targeted antiviral strategy due to the low proportion of infectious cases that present to health authorities. the relative infectiousness of treated individuals, e t , is strongly negatively correlated with successful mitigation, perhaps a surprising result given the model's underlying assumption (based on available epidemiological and human clinical trials data) that e t lies in the range [ ae , ]. that is, nais provided as treatment have a maximum impact of just a % reduction in infectiousness. however, our previous results show a strong synergistic effect of treatment when overlayed on a contact prophylaxis strategy, explaining the observation here that e t is critical in determining likely success of an intervention. despite the limited impact of treatment at the individual-level, the model outcomes are highly sensitive to the value of the relative infectiousness of treated cases. it follows that determination of e t is important for predicting the population-level outcome of a control effort. a 'small' reduction (of the order approximately %) may be extremely valuable in terms of success of a public health control strategy, and so should not be discounted. using a mathematical model which takes into account some of the key logistic constraints that are inherent to healthcare responses, we have derived a logistic regression model for estimating the probability that an antiviral intervention based on liberal distribution of nais as treatment and prophylaxis could successfully mitigate an influenza epidemic. the model demonstrates an excellent degree of accuracy when applied to synthetic data. the choice of parameters for the regression model was restricted to those that were both highly correlated with the success of the intervention and hopefully feasible to measure during the early stages of an emerging epidemic. the model could therefore be a useful near real-time decision support tool for public health policy in the face of an influenza epidemic, although further validation on a range of synthetic data (and real-world data where available) is required. influenza to seasonal flu status to avoid overstretching the demands on healthcare services. a great deal of information has emerged as the result of the pandemic response exercises conducted by affected countries. however, uncertainties remain regarding the effectiveness of intervention measures, as well as the feasibility and the timing of their implementation. mathematical and computational models [ ] [ ] [ ] have been used to project the outcomes of influenza outbreaks under various scenarios and epidemiological hypotheses. motivated by the events of and public health measures adopted by the taiwan cdc, we use a stochastic, individual-based simulation model to study the spatio-temporal transmission characteristics of the h n virus, so as to quantitatively assess the effects of early intervention strategies. our stochastic disease simulation model builds upon a highly connected network of individuals interacting with each other via social contact groups. to represent the daily interactions of approximately million people living in taiwan, we constructed a computer-generated mock population based on national demographic and employment statistics (to derive daily commute patterns) from the taiwan census (http://www.stat.gov.tw/). each individual is created with a set of attributes, including age, sex, residence, family structure, and social standing (employment status, etc.). based on their attributes and the time of day, each individual is assigned to miscellaneous contact groups, where the potential of interactions between any two individuals resulting in flu virus transmission occurs. such epidemiological properties are defined by empirically parameterized attributes such as basic reproduction number r , transmission probability, contact probability and associated probability distributions outlining the disease's natural history. additionally, intervention measures are implemented as scheduled events that could alter control parameters during the course of a simulation run. the targeted basic reproduction number (r ) in all our simulations is ae , following the suggested range by who of ae - ae . as the latent ⁄ incubation and infectious periods for h n have not yet been reliably ascertained, we adopt the natural history of the and pandemic influenza viruses. , here, the latent period ranged from to days, with a median value of ae days. the infectious periods begin day prior to symptom onset and can continue for - days, with a median value of ae days. twothirds of the infected individuals will develop clinical symptoms, and the asymptomatic cases will have half the infectious strength. the efficacy of antiviral drugs (oseltamivir) and vaccines are based on these studies. , for the source region of the infected cases, we use the north american continent (canada, mexico and united states) with an estimated total population of and an average hours of flight time to taiwan. the average daily passenger number is based on the annual statistical report on tourism, tourism bureau, taiwan (http://admin.taiwan.net.tw/english/statistics/year.asp? relno= ). each simulation lasts days and starts with a baseline simulation of r % ae h n pdm outbreak at the source region. the outbreak was adjusted to approximate clinical attack rate (car) in the united states, april -march , . we estimate the daily number of imported cases according to average daily passenger numbers and their probability of holding a disease status. we then apply airport exit ⁄ entry screening per corresponding success rates, by subtracting the number of identified symptomatic cases. we also consider latently infected passengers with inflight disease progression, by fitting a gamma distribution to the cumulative distribution of time to onset data with hours average flight-time, as presented by pitman et al. the daily imported cases are seeded according to the traveling patterns of foreign tourists and residents returning home. from the disease's natural history, we derive that roughly % of the infected travelers present no symptoms; the percentage increases if most symptomatic individuals elect not to travel in their condition, or are stopped by airport screening. we use the official epidemic data provided by the taiwan cdc to calibrate the simulation model and perform regression analysis on scenario parameters. this data is a close estimation of the weekly new clinical cases of h n pdm patients. it consists of weekly opd (outpatient department) icd- code (influenza) tallies collected by the bureau of national health insurance, taiwanadjusted to exclude seasonal flu patients and to account for uninsured patients. we formulate our scenario settings according to events in taiwan, and establish settings to approximate the actual events. with domestic events and intervention schedules fixed in time, the start date determines the simulation outcomes and the data range for selected indicators, such as the mean car, the epidemic peak, and several significant dates for the incoming index case events. we plot the taiwan weekly h n opd cases alongside the weekly new clinical cases from our simulation results in figure . our simulations not only capture the epidemic trend, but also pick out the most likely date, may , for identifying the first symptomatic case at airport screening based on practical assumptions. we further analyze the effectiveness of various mitigation measures with february , as the empirical start date for h n pdm in north america. the simulation result confirms that by the time we identified the first symptomatic case at the border screening, infected cases had already made their way to the public. by our calculation, roughly four such cases had passed in each of our scenario settings, with the first case happening as early as weeks before detection. figure also highlights the importance of the timing for the implementation of mitigation measures; for example, a -day-delay of the identical intervention plan results in nearly an additional % of the population being infected. therefore, the rule of thumb for healthcare officials is to implement intervention measures as early as possible. in our study, we have ignored the possibility of inflight transmission and any false positive results by airport screening procedures. to assess the effectiveness of each mitigation strategy of interest and their combinations, we take the calibrated simulation model and perform simulation realizations for groups of scenarios containing only those intended mitigation measures, and analyze the averaged results. for example, in the airport exit screening policy only scenario, the first imported symptomatic case can be delayed up to months, and the epidemic peak can be delayed up to days. as the data suggests, the exit screening policy alone has very little impact on car. combining various screening success rates for both exit and entry screening allows us to quantitatively assess their beneficial ramifications on the epidemic. for example, there is very little additional benefit between % and % suc-cess rates for entry screening policies when exit screening policies are adequate, as the enhanced border screening only delayed the epidemic peak by day, and reduced car by < ae %. base on this result, the government should not attempt to exhaust all its resources in securing the border during a pandemic event, because the return of such a policy will be disappointing. instead, a response plan with a shifting focus on health resource allocation and the capacity of adjusting intervention strategies in line with the developing epidemic will be most effective. based on the same principle, we perform experiments with assorted scenarios, including relaxing entry screening policies after identifying the first imported symptomatic case, mass vaccination based on the actual vaccination schedule of h n pdm in taiwan, and altering the start dates of the vaccination schedule. our results show that with a reasonable reduction in the airport entry screening success rate, we conserve valuable healthcare resources, but loose a few days for the strategic planning and preparation of subsequent response measures. in other simulation scenarios, a national vaccination campaign has very little impact on the outcome, due to the late start of the vaccination schedule. we then explore the effect of a national vaccination campaign with various starting dates. the simulation results are illustrated in figure , where the benefit of an early start date for mass vaccination is clearly demonstrated. considering a scenario with an % airport exit screening success rate, % airport entry screening success rate and % symptomatic case tracing success rate, the combined intervention strategy results in: a % reduction in car if the vaccination campaign starts in mid-november; % reduction if the campaign starts in mid-october; % reduction if the campaign starts in mid-september; and % reduction if the campaign starts in mid-august. in retrospect, the taiwanese government's response to h n pdm proved to be effective. first and foremost, it initiated enhanced border monitoring and on-board quarantine inspection as soon as the threat of a flu pandemic became clear. at the same time, the domestic preparations towards h n pdm were escalated, such as antiviral drug stockpiling and distribution, and vaccine acquisition. as the h n cases increased worldwide, various revised plans were adopted and implemented; such as the shift from labor-extensive on-board quarantine inspection to the notifiable infectious disease reporting system and realtime outbreak and disease surveillance system in order to effectively track down symptomatic and exposed passengers, apply prophylaxis treatment and mandatory in-home quarantine. as a result, all h n pdm related statistics are well below the international average. in modern society, countries rely heavily on the global economy for their own prosperity. shutting down the border for any length of time is not only costly, but could have disastrous economic effects that linger long after the event is over. moreover, with nearly % of the infected passengers presenting no symptoms whatsoever, they are not detectable by any port authority's screening procedures, and the importation of the novel flu virus is therefore inevitable. many studies conclude that entry screening is unlikely to be effective in preventing or delaying the importation of influenza, and has negligible impact on the course of subsequent epidemic. however, these studies are based on the assumption that effective exit screening is in place. our study shows that as the exit screening success rate decreases, the sensitivity of the entry screening policy becomes more pronounced. with the same methodology, we can also study the effects of varying the length of flight time, or the disease's incubation time. lastly, the benefit of entry screening is even more crucial for a small island country such as taiwan, since all incoming traffic must go through the port authority where entry screening can be enforced. in england and wales, three waves of the pandemic struck in summer, autumn, and winter seasons of - . although the proportion of people reporting symptoms was often greater in the first wave, - a puzzling feature was the much higher mortality in the second wave, in which . % of the population died, compared with . % in the out-of-season first wave and . % in the third wave. an obvious hypothesis to explain the changes in mortality from wave to wave would be that the virus mutated to higher virulence after the (lower mortality) first wave. although pandemic virus reconstituted from the high mortality waves has proven to have high virulence in animals, it has not been possible to recover virus from the first wave in for comparative purposes. indeed it is questionable whether virulence mutation(s) occurring between wave and wave could have spread to so many different populations in the time-frames observed. furthermore, in all three pandemic waves, there was the same agedistribution of mortality, with more deaths occurring amongst younger adults than older adults. [ ] [ ] [ ] this 'pandemic signature', arguably due to immune protection of older adults who were exposed to a similar virus in the years before , , suggests that the - viruses were at least immunologically similar in all three waves. a second hypothesis would be that the higher case fatality in the later waves was due to higher rates of complicating bacterial pneumonia, to increased transmission of influenza virus in the cooler months of the year, or to other seasonal effects. we have considered a third (immunological) hypothesis to explain the greatly increased mortality in waves and . the underlying idea is that the mortality rate in the first wave was lower than in later waves because most persons were protected by prior immunity in the first wave, and that the mortality was higher in later waves because of waning of that short-lived immunity. this hypothesis builds on our earlier modelling papers suggesting that even before the first wave in , military, school, and urban populations in england and wales apparently had (short-lived) immune protection, presumably induced by recent prior exposure to seasonal influenza. [ ] [ ] [ ] we suggest that this short-lived strain-transcending protection was in addition to the longer-lasting immunity, presumably induced by exposures to a similar virus circulating prior to , that arguably reduced pandemic mortality for older adults in - . , cumulative mortality rates attributed to pandemic influenza were available for each of the three waves in - for populations in england and wales. we have built immunological models to potentially explain the variation in mortality rates across waves and populations. to show proof of principle, we have fitted these models to mortality data from a randomly selected sub-set of twenty populations. our key assumption was that the risk of a fatal infection would be limited to persons with inadequate immunity who were being exposed to the pandemic virus for the first time. persons who were exposed and who survived an earlier wave were assumed to be protected against death in a later wave. model a and assumptions (see figure ) before the first wave, we assumed that people could be fully susceptible (s ), or partially protected (q ), or fully protected (p) by prior immunity which was not necessarily specific for the new virus. we assumed that exposure to the new pandemic virus would be fatal (m) in a proportion h of fully susceptible persons who were actually exposed (e) in the relevant wave. for those surviving that first exposure, it was assumed that they would be permanently protected against death in later waves by an immune assumed that viral exposure and multiplication would induce an immune response specific for the pandemic virus that would protect them against death in that wave and in subsequent waves. in contrast, for persons with strong prior immune protection, p, the virus would not be able to multiply to induce pandemic-specific immune protection. between waves, it is assumed that due to the waning of non-specific prior immunity, persons in the p state can move to the q state, and persons in the q state can move to an s state before the next wave. the proportion (e) of susceptible persons exposed to productive infection in each population was estimated by applying the following version of the final size equation to the proportion susceptible (s & q) in each wave, for each population: note: in both figures and , we have omitted the flows out of the q and e states that removed persons from the risk of death. parameters: s = proportion fully susceptible to infection and death before wave ; q = proportion susceptible to immunising infection, but not to death from exposure in wave ; p = proportion temporarily protected against both immunising infection and death from exposure in wave ; n = proportion even more protected against both immunising infection and death from exposure in wave (model b only); r = basic reproduction number (the average number of secondary cases for each primary case) in a fully susceptible population; f = proportion moving from q to s between waves; g = proportion moving from p to q between waves; d = proportion moving from n to p between waves (model b); h = proportion of e that actually move to m and die. model a could provide a very good fit for the summer, autumn, and winter waves of the - pandemic (results not shown). however, because of the replenishment of the pool of susceptible persons over time, model a also predicted a fourth wave of influenza in the spring season of . as no such wave was seen, and as we could not find parameters values for model a that did not predict a fourth wave, we must regard model a as inadequate. model b was similar to model a, but with an additional stage of prior immunity (n), which could wane to p. model b allowed us to not only fit the three observed waves, but also to fit the imputed data (zero cases) corresponding to the absent fourth wave. following earlier work, , we used a bayesian approach with markov chain monte carlo (mcmc) procedures to estimate model parameters, and we used hyper-parameters to allow for parameter variation between populations. the initial conditions were specified by the parameters: p , q , s and n . from these and the other parameters, it was possible to simulate the behaviour of model a over three waves, and of model b over four waves, and to estimate the expected numbers dying in each wave in each population. we calculated the log likelihood of the observed numbers of deaths given the parameter estimates, and we used mcmc simulation to generate the posterior distributions of parameters. although we obtained an excellent fit between observed and expected numbers of deaths in each of the three waves for the populations for model a, we could not find parameter values for model a that would fit the three observed waves without giving rise to a fourth wave in the spring of . accordingly, in the modified model b, we allowed for an additional stage of prior immunity (figure ) , and we fitted the model to the same data, plus imputed data corresponding to 'the absent fourth wave'. we obtained a very good fit to the three observed waves and the absent fourth wave in each population. the % credibility intervals for parameter estimates, derived from the posterior distributions of the hyper-parameters were: h = . - . , s = . - . , q = . - . ; n = . (fixed); p = ) s ) q ) n ; f = . - . ; g = . - . ; d = . - . and r = . - . . this analysis had allowed all parameters to vary from population to population under the constraints of the hyper-parameters. however, several of the biologically determined parameters might be expected to be more constant from population to population, whereas those dependent on mixing history and other social characteristics which vary more widely from population to population. to test this possibility, we fixed the mean values for the more biological parameters (f = . ; d = . ; g = . ) and estimated the % credibility intervals for the others as: h = . - . ; s = . - . , q = . - . ; and as before n = . (fixed); in a subsequent paper we will be able to provide more details of the method, the robustness of the assumptions, and the results from fitting to many more populations. this short report suggests that the observed patterns of mortality in england and wales over the three waves of the - influenza pandemic , can be explained by an immunological model. in particular, the lower mortality in wave one can be explained by the assumption of protective immunity antedating the first wave, arguably induced by prior exposure to seasonal influenza. , the much greater mortality in wave two can be explained by the waning, between wave one and wave two, of that short-lived and less-specific immune protection. the somewhat lesser mortality in wave three and the 'absent fourth wave' can be explained in terms of the progressive acquisition of immunity specific to the pandemic virus. the credibility estimates for parameters are of potential interest. for example, r estimates of . - . across different populations are consistent with our earlier findings. , if all persons had been susceptible, such r values imply that the virus would have infected most people in all populations. however, even in the first wave, the proportion susceptible, s + q , was < % in all populations, so that a considerable number of persons escaped productive infection in that wave; as their immunity waned, they became susceptible to infection in the later waves. it is likely that the variation in r between populations is due to different rates of population mixing. estimates for h indicate that between % and % of infections in the most susceptible persons were fatal; the higher values of h could reflect higher rates of secondary bacterial infection in the most socially disadvantaged and overcrowded populations. although we have shown the plausibility of an immunological explanation for wave to wave changes in pandemic mortality, we cannot assume that our particular model is even approximately correct. nor can we exclude the possibility that the higher mortality in the later pandemic waves in - was because of genetic change in the virus in later waves, or because of changing rates of secondary bacterial infection or seasonal effects. nevertheless, there is growing evidence that the population spread of pandemic influenza, whether in - , or in , , can be constrained by significant prior immunity, even for viruses that are ostensibly novel. previous reports, reviewed in ref. [ , ] , support the idea of strain-transcending immune protection, which can wane over periods of a few months. this form of protection, probably induced by recent exposure to seasonal influenza, may not be mediated by hi or neutralizing antibody. in contrast, strain-specific immunity, most often mediated by hi or neutralizing antibodies can be so long-lasting that after several decades it will still provide significant protection against any closely-related virus that re-appears in the population. it has not escaped our notice that although attack-rates in the h n pandemic were low in many countries, with generally mild symptoms, the virus did cause lifethreatening illness in a small proportion of younger affected persons. it seems likely that those who were most severely affected in were doubly unlucky: they had missed out on seasonal influenza infection or vaccination in the preceding season(s), and they were born too late to have been protected by the closely-related viruses that are thought to have circulated before . during the early phases of the influenza pandemic in italy, real-time modeling analysis were conducted in order to estimate the impact of the pandemic. in order to evaluate the results obtained by the model we compared simulated epidemics to the estimated number of influenza-like illness (ili) collected by the italian sentinel surveillance system (influnet), showing a good agreement with the timing of the observed epidemic. by assuming in the model mitigation measures implemented in italy, the peak was expected on week ( % ci: , ). results were consistent with the influnet data showing that the peak in italy was reached in week . these predictions have proved to be a valuable support for public health policy makers for planning interventions for mitigating the spread of the pandemic. mathematical models have recently become a useful tool to analyse disease dynamics of pandemic influenza virus can-didates. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] as of april , after the pandemic threat emerged worldwide, it was crucial for policy makers to have early predictions on the possible spread of the pandemic influenza virus in order to support, with quantitative insight into epidemic, policy decisions. thus, after the first pandemic alert was announced by the world health organization (who) in late april , a national crisis management committee headed by the minister of health was established in italy in order to provide weekly advice to the italian ministry of health. real-time analyses using an individual based model were undertaken. the transmission model was previously used for evaluating the effectiveness of the control measures adopted in the national pandemic preparedness plan and for assessing the age-prioritized distribution of antiviral doses during an influenza pandemic. to parameterize the transmission model, we used data derived from the national surveillance system until june and estimates of key epidemiological parameters as available at that time. in order to provide a preliminary assessment of the model predictions performed during the early stages of the epidemic, we compare model predictions with surveillance data of influenza-like illness (ili) available since august . after the first pandemic alert was announced by the who in late april , a national active surveillance system for the pandemic influenza was set up from april to july . however, over the period from april to october , surveillance systems, laboratory testing, and diagnostic strategies have varied considerably in italy. since end of july , following who recommendations, the focus of surveillance activities has changed in reporting requirements, as active case-finding became unsustainable and unnecessary. for this reason, the ministry of health (ministry of health, available in italian at the website: http://www.normativasanitaria.it) requested regional health authorities to report the weekly aggregated ili cases according to a new case definition (sudden onset of acute respiratory symptoms and fever > °c plus at least one of the following systemic symptoms: headache, malaise, chills, sweats, fatigue; plus at least one of the following respiratory symptoms: cough, sore throat, nasal obstruction). by october , following the increasing number of cases, the sentinel influenza surveillance system (influ-net available at: http://www.flu.iss.it) became the official surveillance system for ili cases in italy (ministry of health, available in italian at the website: http://www. normativasanitaria.it). since , influnet is routinely based on a nation-wide, voluntary sentinel network of sentinel community based physicians in the regions and autonomous provinces of the country. incidence rates are, therefore, not based on consultations, but on the served population of each reporting physician each week. influ-net usually consists of an average of (range - ) general practitioners (including physicians and pediatricians) per year, covering about ae - % of the general population, (representative for age, geographic distribution, and urbanization level) reporting ili cases (according with a specific case definition). italian influnet surveillance system is part of the european influenza surveillance scheme (eiss). a stochastic, spatially explicit, individual-based simulation model was used. individuals are explicitly represented and can transmit the infection to household members, to school ⁄ work colleagues, and in the general population (where the force of infection is assumed to depend explicitly on the geographic distance). the national transmission model was coupled with a global homogeneous mixing susceptible-exposed-infectious-removed (seir) model accounting for the worldwide epidemic, which is used for determining the number of cases imported over time. regarding the epidemiological assumptions (e.g., length and shape of the infectivity period, which lead to an effective generation time of ae days), this study is consistent with refs [ , , , ] , but for the proportion of symptomatic individuals, which is assumed to be ae %. the basic reproductive number of the national transmission model was set to ae , according to the early estimates as obtained during the initial phase of the epidemic in mexico in a community setting. , we initialized our simulations through the global homogeneous mixing model in such a way that imported cases were generated until june . this gives a reliable way for fixing the time in the simulations and thus determining the timing of school closure and vaccination in the simulations. the model accounts for school closure for both summer and christmas holidays: we assumed that in these periods contacts among students decrease, while contacts in the general community increase, as in ref. [ ] . we also considered scenarios accounting for partial immunity in the population. in order to investigate the effects of recommendations of the ministry of health (confirmed cases coming from affected areas were isolated for - days, either in hospital or at home) established in the early phase of the pandemic (april-july ), we assumed that a fraction of the imported symptomatic cases were isolated on the first day after the symptoms onset. this recommendation was in place until july . we also assumed, according to the italian school calendar, that schools were closed from june to september for the summer holidays, and from december to january for christmas holidays. the effects of prolonged school closure were also investigated. when considering vaccination, we assumed weeks for the logistical distribution of doses of pandemic vaccine. since at the time of simulation specific recommendations regarding the administration of a single dose of pandemic vaccine from ema were not available yet, we considered the administration of vaccine doses month apart). the pandemic vaccine was considered effective after the administration of the second dose with a vaccine efficacy of %. we assumed the vaccine to be administered by priority, vaccinating first the target population accounting for essential services workers (including health care workers and blood donors), pregnant women at the second or third trimester, and at risk patients (with chronic underlying conditions) younger than years old. the vaccination coverage was assumed %. regarding antiviral treatment and prophylaxis, recommendations of the ministry of health in the initial phase of the epidemic were to administer antivirals to all confirmed cases and to their close contacts. we assumed that the surveillance system would be able to detect % of symptomatic cases. after july , recommendations changed and antiviral treatment was considered only for cases with severe complications and in case of local clusters. since it was difficult to establish the proportion of treated cases, we considered different scenarios: antiviral treatment from % to % of the symptomatic cases. consistently with ref. [ ] , both treatment and prophylaxis were assumed to start day after the clinical onset of symptoms in the index case. treatment was assumed to reduce infectiousness by %, whereas antiviral prophylaxis was assumed to reduce susceptibility to infection by %, infectiousness by %, and the occurrence of symptomatic disease by %. as of july , approximately confirmed cases have been reported to the italian surveillance system for pandemic influenza. during july, the sudden increase of ili confirmed cases suggests for sustained autochthonous transmission in italy. by analyzing the number of ili cases reported to the surveillance during the weeks from to , we found that the exponential growth rate was ae ⁄ week and thus we estimated the national reproductive number to be r = ae . this estimate of the basic reproductive number supports the choice of the value adopted in the model simulations (r = ae ). in the absence of intervention measures, the predicted cumulative attack rate was ae % ( % ci: ae , ae ), and the peak was expected on week ( % ci: , ) with a peak day incidence of ae % ( % ci: ae %, ae %). by assuming case isolation, antiviral treatment, and prophylaxis to % of symptomatic cases until july , the peak was expected on week ( % ci: , ). when considering ae % of natural immunity in the population aged more than years, the peak was expected week later than in the previous scenario, i.e., on week ( % ci: , ). to validate the model, we compared model predictions (which are based only on the available information on the early phases of the epidemic) with ili data (figure ). based on model predictions, we estimated the underreporting factor of influnet ranging from ae to ae , considering different scenarios. by aligning the simulations with the ili data adjusted by the underreporting factor, we can observe that almost all the points in the increasing phase of the epidemic lie within the % ci of the model results (both considering or not natural immunity). the decay phase of the simulated epidemics shows a small delay with respect to the ili data. when introducing single and combined mitigation measures, such as case isolation, antiviral treatment, prophylaxis, and vaccination in the model, results showed that even a low proportion of symptomatic cases treated with antiviral drugs could have led to a relevant reduction in the epidemic size (table ) . we simulated the planned italian vaccination strategy (begun on october ), obtaining a limited but not negligible reduction in the attack rate with respect to the scenarios accounting only for antiviral treatment. moreover, the effect of vaccination would be higher if coupled with antiviral treatment; vaccination would have no effect on delaying the peak incidence. model predictions produced in italy during the early phase of the pandemic influenza are in excellent agreement with italian surveillance data on the beginning of the epidemic (when case isolation, antiviral treatment of index cases, and antiviral prophylaxis to close contacts were implemented by the italian regional public health authorities) and are basically consistent with the influ- net data during the course of the epidemic. the model has been useful for predicting the timing of the epidemic, while it has overestimated the impact of the influenza pandemic for adult and elderly individuals. however, the disalignment is probably due to the model parameterization. based on literature values, , we assumed a similar fraction of cases in the different social contexts considered in the model (namely ⁄ in households, ⁄ in schools ⁄ workplaces, and ⁄ in the general community), since analysis on the relative transmissibility of the virus was not carried out for any country yet. we were also able to estimate an underreporting factor for the influnet data in the range ae - ae . if we focus our attention on the reporting factor computed by considering the total number of cases (instead of symptomatic cases), the resulting value lies in the range - ae %, which is in excellent agreement with the range estimated in ref. [ ] on previous a ⁄ h n influenza seasons, namely ae %- ae %. moreover, based on our results showing that vaccinating % of the italian population was more than adequate to mitigate the pandemic, the ministry of health decided to stockpile a limited number of vaccines. we have also shown that starting the vaccination program in october (or later) could have had only a limited effect on reducing the impact of the epidemic, although it may have been useful to prevent a possible second wave and to protect essential workers and at-risk patients. finally, our results have shown that antiviral treatment would have been the most efficient strategy to reduce the impact of the influenza pandemic, even with a limited antiviral stockpile. a population-wide passive immunotherapy program in this paper, we assume that convalescent plasma (cp) is efficacious in treating severe cases of pandemic influenza. under this premise, we test the hypothesis that a population-wide passive immunotherapy program that collects plasma from a small percentage of convalescent individuals can harvest sufficient cp to treat a substantial percentage of severe cases during the first wave of the pandemic. the proposed program involves recruiting adults (individuals age - years) to donate blood if they have experienced influenza-like symptoms more than weeks ago (to account for the time needed for neutralizing antibodies to build up). the blood samples would be screened for infectious diseases (including hiv, hbv, hcv, htlv, and syphilis, etc., as in routine blood donation screening) and neutralizing antibodies against the pandemic virus. donors whose blood samples are free of known infectious agents and contain a sufficiently high titer of neutralizing antibodies would then be invited to donate plasma by plasmapheresis or routine whole blood donation. qualified donors with higher titers may be given higher priority for plasma donation. in this paper, we use the demographic and logistical parameters of hong kong as a case study. see figure for a schematic of the proposed passive immunotherapy program. we examine the following questions regarding the logistical feasibility and potential benefits of the proposed passive immunotherapy program: (i) what percentage of convalescent individuals (donor percentage) is needed in order for the program to significantly reduce pandemic mortality? (ii) how many severe cases can be offered passive immunotherapy? (iii) what are the ratelimiting factors in the supply of passive immunotherapy? (iv) what are the epidemiologic and logistical factors that determine the demand-supply balance of passive immunotherapy? a more detailed presentation of our results is now available in ref. [ ] . transmission and natural history model for pandemic influenza we use an age-structured disease transmission model to simulate the spread of pandemic influenza. the natural history model is similar to that used by basta et al. , the most important parameter in characterizing the growth of an epidemic is the basic reproductive number r , which is defined as the average number of secondary cases generated by a typically infectious individual in a completely susceptible population. we consider values of r between ae and , which is consistent with recent estimates. , [ ] [ ] [ ] logistical model for the passive immunotherapy program we assume that q d (%) of to year-old individuals who have recovered from symptomatic infections of pandemic influenza donate their blood for screening t r = days after cessation of symptoms. follow-ups of convalescent individuals infected with h n pdm in an ongoing clinical trial of passive immunotherapy suggested that neutralizing antibodies level reaches maximal level around - days after recovery and stays at that level for months after. we assume that q s (%) of these donors are qualified for plasma donation of which q r (%) are recurrent donors who return to donate plasma every t w = days. screening involves both detection of infectious agents and neutralizing antibodies against the pandemic virus. the latter is the rate-limiting step because neutralization tests of pandemic viruses can only be done in a bsl setting. we assume that five bsl -trained technicians are available to test the blood specimens, each running viral neutralization tests in days. therefore, the capacity and turnaround time of blood screening are u s = and t s = days, respectively. hong kong currently has nine plasmapheresis machines which allow a maximal throughput of plasma donations per day (assuming -hour daily operation with each donation taking minutes). therefore, the capacity and turnaround time of plasmapheresis are u p = and t p = ⁄ days, respectively. collected cp are ready for use in transfusion after final quality check, which takes t q = days. we assume that r t plasma donations are required to treat one severe case on average. the expert panel of the abovementioned study of passive immunotherapy for h n pdm in hong kong suggested that r t < . we assume that p h (%) of symptomatic cases will be severe cases for whom passive immunotherapy is suitable. although p h will be smaller than the case-hospitalization rate (passive immunotherapy may not be suitable for some hospitalized cases), we assume that the two have similar ranges and consider p h ranging from ae % to %. because each severe case requires r t plasma donations on average, demand for cp is simply r t p h times the number of symptomatic cases. therefore, r t p h can be regarded as a single parameter, which we refer to as the lumped demand parameter. we define the outcome as the percentage of severe cases that can be offered passive immunotherapy by the proposed program during the first wave of the local epidemic. we refer to this outcome as treatment coverage and denote it by q. we consider the base case scenarios assuming q r = % and q s = %. in general, the treatment coverage q increases sharply as the basic reproductive number r and the lumped demand parameter r t p h decrease (figure a ). in particular, when r is large and r t p h is small, q is very sensitive to r t p h , but insensitive to r . similarly, when r and r t p h are small, q is very sensitive to both. with a donor percentage of q d = %, the proposed program can supply passive immunotherapy to more than % of severe cases (q > %) if r < ae and r t p h < ae %, but < % if r > ae and r t p h > ae %. in general, the treatment coverage q increases sharply as the donor percentage q d rises from %, but with rapidly decreasing marginal increase ( figure b ). when r < ae and r t p h < ae %, q > % even if q d is as low as %, which is comparable to the current average blood donation rate of ae donations per population in developed countries. when q d is > %, q becomes largely insensitive to further increase in q d in most scenarios. the treatment coverage q for q d = % is more than % that for q d = % across all values of r and r t p h considered in the base case. therefore, increasing the donor percentage q d beyond % has a relatively small impact on cp supply. this is because increasing q d can boost supply only when plasmapheresis is not yet the supply bottleneck. for the same reason, once the donor percentage q d has reached %, the treatment coverage q is insensitive to further increase in q d even when the plasmapheresis and screening capacity are doubled ( figure b , lower panel). we conduct an extensive multivariate sensitivity analysis to test the robustness of our base case observations against uncertainties in parameter values. we generate epidemic scenarios by randomly selecting parameter values from their plausible ranges using latin-hypercube sampling. although there are numerous model parameters, the treatment coverage q is mainly determined by three lumped parameters: (i) r t p h , which indicates the magnitude of demand; (ii) q s q d , which indicates the magnitude of supply; (iii) the initial growth rate of the epidemic r (results not shown). while the dependence of q on r t p h and q s q d is readily comprehensible, it is not obvious a priori that q depends on the natural history and transmission dynamics of the disease via only the initial epidemic growth rate. when the plasmapheresis and screening capacity are very large, the supply-demand dynamics is further simplified: the treatment coverage q depends on lumped demand parameter r t p h and the lumped supply parameter q s q d only via their ratio. finally, q becomes insensitive to q s q d when the latter increases beyond - %, which is consistent with our base case observations. our results suggest that with plasmapheresis capacity similar to that in hong kong, the proposed passive immunotherapy program can supply cp transfusion to treat - % of severe cases in a moderate pandemic (basic reproductive number r < ae , lumped demand parameter r t p h < ae %) when the donor percentage is - %. increasing the donor percentage beyond % has little additional benefit because cp supply is constrained by the capacity of plasmapheresis during most stages of the epidemic. increasing plasmapheresis capacity could significantly boost cp supply, especially when there is a substantial pool of recurrent donors to alleviate the dependence of cp supply on donor percentage. in an ongoing clinical trial of passive immunotherapy for h n pdm virus infection in hong kong, % of convales- cent individuals agreed to donate their plasma for the study. therefore, the donor percentage required by the proposed passive immunotherapy program ( - %) is likely to be feasible. in view of the logistical feasibility of such program, we recommend that further clinical studies are conducted to evaluate the safety and efficacy of passive immunotherapy as a treatment for severe cases of pandemic influenza virus infection. our study is based on the premise that cp will be efficacious in reducing morbidity and mortality associated with pandemic influenza. in theory, the polyclonal nature of neutralizing antibodies in cp would lower the probability of an escape mutant emerging in treated patients. further, besides providing neutralizing antibodies against the pandemic virus, cp also might carry antibodies to other bacterial pathogens, which might decrease the severity of coexisting bacterial infections. as such, cp not only might reduce the case fatality rate but might also increase the recovery rate and shorten duration of hospitalization of severe cases. the proposed passive immunotherapy program can thus significantly reduce the burden on the healthcare system, especially the intensive care unit, which will likely be stressed, if not overloaded, at the peak of an influenza pandemic wave, hence benefiting the general public and not only those receiving passive immunotherapy. although the hypothesized efficacy of cp has yet to be proven in clinical trials, our modeling results show that a public health system similar to that in hong kong has the capacity to support a population-wide passive immunotherapy program that can supply cp treatment to a substantial percentage of the severe cases in a moderately severe pandemic. we estimate that compared to other developed countries, hong kong has a relatively low plasmapheresis capacity. our conclusions regarding donor percentage needed and rate-limiting factors remain valid for plasmapheresis capacity ranging from % to % of what we have assumed in the base case (results not shown). our conclusions are robust against uncertainties in the natural history and transmission dynamics of pandemic influenza. our sensitivity analysis shows that the outcome depends on these epidemiological characteristics only via the initial growth rate of the epidemic. as such, our results are applicable not only to pandemic influenza, but also to other emerging infectious diseases for which the time-scales of disease transmission and antibody response are similar to that for influenza virus. the three determinants of treatment coverage (the initial epidemic growth rate, the lumped demand parameter r t p h , and the lumped supply parameter q d q s ) are all readily measurable in real-time during an epidemic. therefore, our methods and results can be used as a general reference for estimating the treatment coverage of the proposed passive immunotherapy program for a given plasmapheresis capacity. background highly pathogenic h n virus continues to pose a serious threat to human health and appears to have the capacity to cause severe disease in previously healthy young children and adults. at present, antiviral therapy by oseltamivir remains the mainstay for managing h n patients. while early treatment improves survival, approximately % of patients treated within days of illness still succumb to the disease. in addition to the role of viral replication, there is good evidence that the host proinflammatory responses contributes to h n pathogenesis. this suggests that both antiviral and immune-modulatory drugs may have a role in therapy. we previously demonstrated that cyclooxygenase (cox- ) plays a regulatory role in h n hyperinduced pro-inflammatory responses, and its inhibitor has potent effects at modulating this host response. now we demonstrate that, in addition to its immune-modulatory effect, a selective cox- inhibitor, ns- has a direct antiviral effect against h n infection. materials and methods human primary monocytederived macrophages or alveolar epithelial cells (a ) were pre-treated with ns- or drug-vehicle for hour before h n virus infection. h n viruses at multipicity of infection (moi) of was used to infect the cells. following virus adsorption for mins, the virus inoculum was removed, and the cells were washed and incubated in corresponding medium with ns- or drug-vehicle as controls for , , , , and hours post-infection. cells were harvested for rna isolation at hours post-infection to study viral matrix (m) gene expression. supernatants were collected for % tissue culture infection dose (tcid ) assay to determine the virus titers at , , , and hours after h n infection. results ns- was found to suppress virus gene transcription and infectious virus yield in h n -infected human cells. conclusion we demonstrate that a selective cox- inhibitor, ns- , shows an inhibitory effect on h n viral replication in addition to its immune-modulatory effect that could counter the detrimental effects of excessive proinflammatory cytokine production. the findings suggest that selective cox- inhibitors may be a therapeutic target for treating h n disease in combination with appropriate antiviral therapy. the emergence and spread of the highly pathogenic avain influenza viruses (h n ) in poultry and wild birds with repeated zoonotic transmission to humans has raised pandemic concern. at the time of writing, human cases have been reported with fatalities, an overall case fatality rate of around % (cumulative number of confirmed human cases of avian influenza a ⁄ (h n ) reported to world health organization updated to october ). our previous data demonstrated that cox- was markedly up-regulated in h n -infected primary human macrophages, and that it played a regulatory role in the h n hyperinduced host pro-inflammatory responses. such cytokine dysregulation is proposed to be a major contributor to the pathogenesis of h n disease in humans. with the use of selective cox- inhibitors, we found that the h n -hyperinduced cytokine response was significantly suppressed by the drug in a dose-dependent manner. selective cox- inhibitor is a form of a non-steroidal anti-inflammatory drug that selectively targets cox- , and it is an inducible enzyme responsible for inflammatory process and immune response. here, we report a novel finding of a direct antiviral effect of a selective cox- inhibitor, ns- , against h n infection in human primary macrophages and alveolar epithelial cells. taken together with our previous findings that suggest an immuno-modulatory effect that can modulate virus driven cytokine dysregula-tion, these findings highlight a role for cox- and its downstream signaling as potential novel targets for adjunctive therapy of severe viral pneumonia, such as that caused by h n . such therapy may be combined with conventional antiviral drugs. the h n virus used was a ⁄ vietnam ⁄ ⁄ ( ⁄ ) (h n ), a virus from a patient with h n disease in vietnam during . the viruses were grown and titrated in madin-darby canine kidney cells cells as described elsewhere. virus infectivity was expressed as tcid . all experiments were performed in a biosafety level facility. monocyte-derived macrophages: peripheral-blood leucocytes were separated from buffy coats of healthy blood donors (provided by the hong kong red cross blood transfusion service) by centrifugation on a ficoll-paque density gradient (pharmacia biotech) and purified by adherence as reported previously. the research protocol was approved by the ethics committee of the university of hong kong. macrophages were seeded onto tissue culture plates in rpmi medium supplemented with % heat-inactivated autologous plasma. the cells were allowed to differentiate for days in vitro before use in the infectious experiments. alveolar epithelial cells: a cells were obtained from atcc and maintained in culture using dulbecco's modified eagle medium supplemented with % fetal calf serum, . mg ⁄ l penicillin, and mg ⁄ l streptomycin. differentiated macrophages or a cells were pre-treated with a selective cox- inhibitor, ns- (cayman), at concentrations as indicated or drug-vehicle for hour before infection. cells were infected with h n viruses at moi of . following virus adsorption for min, the virus inoculum was removed, the cells were washed and incubated in corresponding medium with ns- or drug-vehicle as controls throughout the experiments. cells were harvested for rna isolation at hours post-infection to study viral m gene expression. supernatants were collected for tcid assay to determine the virus titers at , , , and hours after h n infection. total rna was isolated using the rneasy mini kit (qiagen) according to the manufacturer's instructions. the cdna was synthesized from mrna with poly(dt) primers and superscript iii reverse transcriptase (invitrogen). transcript expression was monitored by real-time pcr using power sybr Ò green pcr master mix kit (applied biosystems) with specific primers. the fluorescence signals were measured using the real-time pcr system (applied biosystems). the specificity of the sybr Ò green pcr signal was confirmed by melting curve analysis. the threshold cycle (ct) was defined as the fractional cycle number at which the fluorescence reached times the standard deviation of the base-line (from cycle to ). the ratio change in target gene relative to the b-actin control gene was determined by the )ddct method as described elsewhere. ns- reduced the viral m gene expression in h n infected human macrophages in a dose-dependent manner ( figure ) . similarly, production of infectious virus yield in h n infected macrophages was found to be suppressed in the presence of ns- at lm compared to vehicletreated cells (figure a) . a comparable effect of ns- was observed in h n -infected human alveolar epithelial cells ( figure b ). we have previously demonstrated that cox- expression was dramatically upregulated following h n infection in human macrophages in vitro and in epithelial cells of lung tissue samples obtained from autopsy of patients who died of h n disease. this suggests that cox- may be an important host factor involved in h n pathogenesis and also provide a possible explanation on why h n virus replication is susceptible to a selective cox- inhibitor. cox- was previously reported to play an important role in the pathogenesis of other influenza a viruses. an in vivo study has highlighted the importance of cox- in h n - infected mice. findings showed that infection induced less severe illness and reduced mortality in cox- knock-out mice than in wild-type mice. on the other hand, cox- knock-out mice had enhanced inflammation and earlier appearance of proinflammatory cytokines in the bal fluid, whereas the inflammatory and cytokine responses were dampened in cox- knock-out mice. these data suggests that cox- and cox- may lead to opposite totally contrasting effects on influenza h n infected mice. cox- deficiency is detrimental, whereas cox- deficiency is beneficial to the host during influenza viral infection. therefore in the present study, instead of blocking cox enzymes in general as reported by others, we have chosen ns- that selectively block cox- but preserve cox- activity and showed that this drug significantly reduced h n virus replication in a dose-dependent manner. taken together with our previous report suggesting its immuno-modulatory effects, we believe that selective cox- inhibitors and cox- signaling pathways deserve investigation as a promising approach for targeting therapy in h n diseases. however, a few reports have suggested the importance of cox- in the late stage of inflammation for the resulution of inflammation, [ ] [ ] [ ] and this raises concern whether inhibition of cox- may be harmful in treating diseases related to dysregulation of host inflammatory response such as acute lung injury, which is a leading cause of death in h n patients. we previously looked at the autopsy samples of lung tissues from h n patients and found that cox- expression was markedly up-regulated compared with that from persons who died of non-respiratory causes. moreover, data also demonstrated that pro-inflammatory cytokines, such as tnf-a, was markedly elevated in the h n infected lung autopsies. taken together, with the histo-pathological findings, which showed predominant features of exudative inflammatory phase in autopsy lung samples from h n patients, , we may therefore speculate that people who had fatal h n infection died during acute inflammation phase, and before the resolution could occur, especially for the cases with a short disease duration (< - days). in conclusion, the roles of cox- in both pro-inflammation and pro-resolution phases deserves detailed investi-gation. the timing of selective cox- inhibitor therapy in h n infected patients may be extremely critical. therefore a time-dependent study using selective cox- inhibitors on h n -infected animal models will be particularly important in order to address the effectiveness of this drug in treating h n disease. avian antibodies to combat potential h n pandemic and seasonal influenza highly pathogenic avian influenza a virus (hpaiv) strain a ⁄ h n with unprecedented spread through much of asia and parts of europe in poultry remains a serious threat to human health. passive immunization (transfer of protective immunoglobulins) offers an alternative and ⁄ or additional strategy to prevent and cure influenza. here, we report that virus-specific immunoglobulin y (igy) isolated from eggs of immunized hens provide protection in mice against lethal h n virus infection by neutralization of the viruses in the lungs upon intranasal administration. importantly, chicken eggs obtained from randomly selected supermarkets and farms in vietnam, where mass poultry vaccination against a ⁄ h n is mandatory, contain high levels of igy specific for a ⁄ h n virus. when administered before or after the infection, igy prevented and significantly reduced replication and spread of hpaiv h n and related h n strains. thus, the consumable eggs readily available in markets of countries that impose poultry vaccination against a ⁄ h n could offer an enormous source of valuable biological material that provides protection against a ⁄ h n virus with pandemic potential. the approach could be used to control seasonal influenza. since , hpaiv of the h n subtype has resulted in more than cases of laboratory-confirmed human infection in countries with a death rate of more than % (http://www.who.int/csr/disease/avian_influenza/). h n influenza virus remains a global threat because of its continued transmission among domestic poultry and wild birds. passive immunization (the transfer of antigen-specific antibodies (abs) to a previously non-immune recipient host) offers an alternative and ⁄ or additional countermeasure against influenza. development of human monoclonal antibodies (mabs) against h n influenza haemagglutinin (ha) using epstein-barr virus (ebv) immortalization of b cells isolated from patients infected with h n , phage display, humanized mabs, and human recombinant abs has been attempted. chickens produce a unique immunoglobulin molecule called igy that is functionally equivalent to mammalian igg. igy is found in the sera of chickens and is passed from hens to the embryo via the egg yolk. egg igy has been used to prevent bacterial and viral infections (see review ) of the gastrointestinal tract and recently for protection against pseudomonas aeruginosa infection of the respiratory tract of patients with cystic fibrosis (cf). the epidemic of hpaiv h n virus has resulted in serious economic losses to the poultry industry, mostly in southeast asia. therefore, many countries including china, indonesia, thailand, and vietnam have introduced mass vaccination of poultry with h n virus vaccines that controls the h n epidemic to some extent. chickens immunized with recombinant h and ⁄ or inactivated h n reassortant vaccines produced a high level of virus-specific serum antibodies (abs) and were protected from h n virus challenge. theoretically, these abs could be found in egg yolk and separated for use in humans to prevent and cure h n hpaiv infection and disease, respectively. here, we examined the possibility that igy isolated from consumable eggs available in supermarkets in vietnam, where mandatory h n vaccination has been implemented, provide prophylaxis and therapy of hpaiv h n infection in mice. six-to -week-old female balb ⁄ canncrl (h- d) mice (charles river and jackson laboratory) and hy-line . igy abs were extracted from egg yolks as previously described. the % egg infectious dose (eid ) was determined by serial titration of virus stock in eggs, and eid ⁄ ml values were calculated according to the method of reed and muench. human virus stocks were grown in mdck cells as described previously , with viral titers determined by standard plaque assay. the % tissue culture infectious dose (tcid ) of virus was determined by titration in mdck cells. the standard elisa was performed for detection of anti-igy in the sera of igy-immunized mice. fifty percent lethal dose (ld ) titers were determined by inoculating groups of eight mice i.n. with serial -fold dilutions of virus as previously described. for infection, ketamine-anesthetized mice were inoculated intranasally with a lethal dose with pfu ( · ld ) of a ⁄ pr ⁄ ⁄ (h n ) virus as previously described, · ld of vn ⁄ (h n ) or · ld a ⁄ aquatic bird ⁄ korea ⁄ w ⁄ (h n ) resuspended in ll pbs per animal. ketamine-anesthetized mice were treated intranasally with ll of igy before or after infection. mice were observed for weight loss and mortality. subsets of animals were scarified for virus titre. we found comparable hai titers in the sera and egg yolks obtained from a farm in vietnam that was participating in a national mass vaccination program. furthermore we found % of eggs purchased in randomly selected supermarkets in hanoi, vietnam containing h -specific igy. the hai and vn titers of pooled egg yolk igy are comparable with those of sera obtained from hens selected randomly from the farm that underwent supervised h n vaccination. in contrast, igy separated from eggs purchased in korean markets where poultry are not vaccinated against avian influenza h n has no detectable h -specific hai or vn activity. we first treated naïve mice intranasally with h n -specific igy before infection with hpaiv h n strain, a ⁄ vietnam ⁄ ⁄ , isolated from a fatal case. such treated mice displayed mild weight loss and recovered completely by the end of the first week after inoculation ( figure a ). when animals were treated once with h n specific igy after h n inoculation they exhibited minimal weight loss during the first week after inoculation, and virus titers in the lungs were substantial reduced at day after infection; however, % of treated mice succumbed to infection during the second week after inoculation ( figure b) . it is possible that not all the hpaiv a ⁄ h n viruses were neutralized upon the single treatment with igy, and escaping viruses can spread systemically to organs outside of the lungs. these viruses may reappear in lung tissue later when specific igy is absent. indeed, vn ⁄ virus injected intravenously or into the brain can spread to the lungs. to circumvent the virus escape, we administered multiple treatments with h n specific igy after the infection. as a result, all infected mice recovered completely by the second week post-infection ( figure c) , and virus titers in the lungs were substantially reduced to the level that seen in protected mice that received single prior-infection treatment ( figure d) . similarly, the protective efficacy of h n -specific igy was observed in mice infected with lethal dose of mouseadapted avian influenza virus strain a ⁄ aquatic bird ⁄ korea ⁄ w ⁄ (h n ). this virus shares . % nucleotide sequence homology with ha (h ) but has different na (n ) from the one used for mass immunization in vietnam (reassortant avian h n influenza virus a ⁄ goose ⁄ gd ⁄ -derived, strain re- ). the results indicate that h n -specific igy isolated from eggs purchased in markets have preventive and therapeutic effects against infection with hpaiv h n and the related strain h n . the findings suggest that while a single treatment with igy prior to lethal infection was sufficient to protect the animals from the infection, multiple treatment is required for complete therapeutic effect after infection with hpaiv such as vn ⁄ strain. we further examined the protective efficacy of igy isolated from eggs laid by hens immunized in the laboratory with heat-inactivated human influenza a ⁄ h n virus, a ⁄ pr ⁄ ⁄ . we found substantial levels of hai and vn abs in the sera and yolks derived from immunized hens. when naïve mice were administered intranasally with such anti-pr ⁄ igy at - hours before or after infection with lethal dose of pr ⁄ virus, they were protected from the infection or lethal disease, respectively. the virus titers in the lungs of a ⁄ pr specific igy-treated mice at day after infection were also significantly lower than those seen in untreated mice or mice receiving normal igy. intranasal administration is the most effective route as compared to oral or peritoneal or intravenous administration for protection against lethal challenge, and the presence of virus-specific igy in bronchoalveolar lavage (bal) is required for the protection. the results provide a proof-of-concept that intranasal administration of virus-specific igy prevents influenza virus infection and cures the disease. the concept could be applied to control influenza outbreaks including seasonal and pandemic influenza. the protection was correlated with hai and vn activities of the igy and reduced virus titers in the lungs after treatments, suggesting that the protection is mediated by vn. we asked if administration of igy in the respiratory tract induces anti-igy ab response in mice. if this is the case, the next question is whether pre-existing anti-igy abs block igy-mediated protection. indeed, significant levels of anti-igy were observed in animals that received single or multiple administration of igy. when igy-immune mice were treated with virus-specific igy before or after lethal challenge, the results were identical to those obtained from treated naive mice, indicating that pre-existing anti-igy abs do not interfere with the protection mediated by virus-specific igy. consistently, incubation with anti-igy serum did not interfere with hai and vn activity of the virus-specific igy, indicating that anti-igy abs do not block virus binding by virus-specific igy (figure ). the finding suggests that the igy treatment could be applied to persons who have developed anti-igy during the individuals' life, and such treatment strategy could be repeated if multiple treatment is required and ⁄ or necessary later on to protect infections with other pathogens. the approach using specific igy for prevention and therapy of hpaiv h n infection offers a practical alternative to immunotherapy using convalescent plasma and an additional therapeutic option to antiviral drugs since widespread drug resistance has been recently reported among influenza virus strains. igy is relatively stable. we found no change in protective activity after at least months storage at °c, and lyophilization does not affect the activity, making production of igy practical. the use of igy immunotherapy has many advantages, since igy does not activate the human complement system or human fc-receptors, which all are well-known cell activators and mediators of inflammation. we chose the water dilution method for preparation of igy. the method is simple, efficient and does not require any toxic compounds or any additives. such igy preparations by this method have been used in other human study. , eggs are normal dietary components, so there is minimal risk of toxic side effects, except for those with egg allergy. thus, our study demonstrated that influenza virus-specific igy can be used in passive immunization that provides great help for immunocompromised patients and elderly who have weaken immune response to influenza vaccines. importantly, the consumable eggs readily available in the markets of countries that impose mandatory h n vaccination offer an enormous source of valuable, affordable, and safe biological material for prevention and protection against potential h n pandemic influenza. parts of the information and data presented in this manuscript were previously published in http://www.plosone.org/ article/info:doi% f . % fjournal.pone. . the polyphenol rich plant extract cystus is highly introduction the ⁄ h n influenza a virus pandemic clearly demonstrates that influenza is still a major risk for the public health. although the pandemic swine origin influenza a virus (soiv) caused only mild symptoms, the control of the outbreak still remains difficult. even as vaccine is available against this virus, the possibility of reassortment between the pandemic and a seasonal or avian a ⁄ h n influenza virus strain is indeed a frightening, but a likely event. this reassortant strain might be able to transmit easily between humans causing fatal infections, and the current soiv vaccine might no longer be sufficient to protect against the reassorted virus. in such a case, we can only rely on effective antiviral drugs. today, neuraminidaseinhibitors, such as oseltamivir, represent the most common clinically approved medication against influenza a viruses. unfortunately, the frequency of reports describing the appearance of drug-resistant seasonal h n and also h n influenza a viruses dramatically increased in the recent past. [ ] [ ] [ ] [ ] drug resistance to the known antivirals highlights the urgent need for alternative antiviral compounds with novel defense mechanisms. recently, we have reported that a polyphenol rich plant extract, cystus , which showed antiviral activity against influenza a viruses in cell culture and in mice. , moreover, the antiviral activity of cy-stus against seasonal influenza virus and common colds was also demonstrated in humans. however, the efficiency of cystus against soiv and a ⁄ h n isolates was unknown so far. therefore, we investigated cy-stus effectiveness against the pandemic strain and seven natural influenza a ⁄ h n isolates detected in several avian species during ⁄ avian influenza outbreak. additionally, the potency of the most common neuraminidase inhibitor oseltamivir was also investigated against these isolates. here, we show that cystus treatment was effective in in vitro studies against soiv and a ⁄ h n influenza virus. viruses avian h n isolates were originally obtained from the bavarian health and food safety authority, oberschleissheim, germany. the soiv a ⁄ hamburg ⁄ ⁄ was obtained from the robert-koch-institut, berlin, germany. all h n viruses were further propagated in embryonated chicken eggs or mdck ii (h n v) cells at the friedrich-loeffler-institut, tübingen, germany. for the cytopathological effect (cpe) inhibition screening, in accordance with sidwell, mdck ii cells were infected with different viruses at moi of ae . virus-infected cells were then treated with antiviral compounds cystus from ae to lg ⁄ ml or oseltamivir from ae nm to mm. after incubation for hours at °c and % co , cells were fixed, and viable cells were stained with crystal violet. after extraction of crystal violet from viable cells with % methanol, the extinction was measured with an elisa reader. immediately before infection, mdck ii cells ( · cells ⁄ well) were washed with pbs and subsequently incubated with virus diluted in pbs ⁄ ba ( ae % ba) mm mgcl , ae mm cacl , penicillin and streptomycin to a multiplicity of infection (moi) of ae for minutes at °c. cystus was added in a concentration of lg ⁄ ml directly to the virus-stock and on the cell monolayer simultaneously with the infection. after minutes incubation period, the inoculums were aspirated and cells were incubated with either mem or mem containing lm oseltamivir. at indicated time points, supernatants were collected. infectious particles (plaque titers) in the supernatants were assessed by a plaque assay under avicel as described previously. in order to investigate the antiviral potential of cy-stus , ec values based on the inhibition of the cpe on mdck ii cells were determined for cystus and in addition for oseltamivir. the ec values for cystus ranged from ae to ae lg ⁄ ml. cystus demonstrated the highest sensitivity against the soiv, sn and mb isolates with ec values below lg ⁄ ml. compared to these virus strains, cystus showed a slightly increased ec value for gsb ( ae lg ⁄ ml). in contrast the ec values for bb and bb were notably elevated ( ae and ae lg ⁄ ml). thus, the weakest antiviral effect of cystus was observed against these two isolates. the ec values evaluated for oseltamivir ranged from ae to ae lm ( table ), indicating that bb ( ae ) and gsb ( ae lm) can be considered resistant against oseltamivir. to confirm these results we investigated the ability of cystus to block virus replication as published before. as a control, virus infected cells were treated with oseltamivir as described earlier. in the absence of the drugs all influenza strains showed similar growth properties (figure , black squares) . first progeny viruses were detectable between and hours post infection (figure , black squares) . treatment with cystus resulted in reduction of virus titers of all influenza virus strains (fig. a-h, open triangles) . surprisingly, oseltamivir failed to inhibit the replication of two h n influenza virus strains (gsb and bb ), supporting the data of ec values ( figure d+h , grey rhombes). we assessed the antiviral activity of cystus against the newly emerged soiv and seven avian h n influenza viruses. cystus showed efficient antiviral activity against the pandemic h n v strain and was effective to a wide range of h n viruses. furthermore, cystus demonstrated a broader and more efficient antiviral potential than oseltamivir. cystus treatment leads to a stronger reduction of progeny virus titers, and more importantly, cystus was effective against all tested viruses, while oseltamivir was unresponsive against two of seven a ⁄ h n viruses. even though the pandemic strain in general is still sensitive to oseltamivir treatment, there are increasing numbers of reports of emerging resistant variants. the treatment with cystus does not result in the emergence of viral drug resistance since the mode of action is an unspecific physical binding of the virus particle that is also beneficial to reduce opportunistic bacterial infections. , , cystus is an extract from a special variety of the plant cistus incanus, and it is very rich in polymeric polyphenols. it is well known that polyphenols exhibit protein-binding capacity. however, cystus exhibited no neuraminidase inhibiting activity. therefore, ingredients of cystus may act in a rather unspecific physical manner by interfering with the viral hemagglutinin at the surface of the virus particle as demonstrated before. while this prevents binding of the virion to cellular receptors, it does not block accessibility and action of the viral neuraminidase. since, infections with influenza a viruses are still a major health burden and the options for control and treatment of the disease are limited, plant extracts such as cystus should be considered as a new candidate drug for a save prophylactic and therapeutic use against influenza viruses. attenuation of respiratory immune responses by antiviral neuraminidase inhibitor treatment and boost of mucosal immunoglobulin a response by co-administration of immuno-modulator clarithromycin in paediatric influenza the antiviral neuraminidase inhibitor osv and zanamivir are widely used treatment options for influenza infection and are being stockpiled in many countries. although mucosal immunity is the frontline of defense against pathogens, the effects of neuraminidase inhibitor treatment on airway mucosal immunity have not been reported. the suppression of viral rna replication and viral antigenic production by these drugs may result in a limited immune response against influenza virus. macrolides, such as cam and azithromycin, have anti-inflammatory and immunomodulatory properties that are separate from their antibacterial effects. [ ] [ ] [ ] this study examined the impact of osv treatment on immune responses in the airway mucosa and plasma in mice infected with iav and pediatric influenza patients. we also assessed the immuno-modulatory effects of cam in influenza patients who were treated with or without osv. female ae -week-old weanling balb ⁄ c mice were nasally inoculated with pfu of iav ⁄ pr ⁄ h n at day . immediately after infection, mice were given lg of osv orally or vehicle at -hours intervals for days. the levels of virus-specific siga in nws and bronchoalveolar fluids (balf) and igg in plasma were measured by elisa as reported previously. a retrospective clinical study was conducted. for the study, children with acute influenza were recruited and grouped according to the treatment received: days treatment with osv (n = ), cam (n = ), osv + cam (n = ), and untreated (n = ). since parents in japan are well aware of the adverse effects of osv especially the neuropsychiatric complications, the decision on whether to administer osv or not and to prescribe cam was made by the parents and the attending paediatricians, based on their anti-viral and immuno-modulatory activity. , comparisons were made of the levels of siga against iav ⁄ h n and iav ⁄ h n , total siga, in nws and disease symptoms before and after treatment. anti-ha siga and total siga in nws of patients were determined from the standard regression curves with human iga of known concentration in a human iga quantitation kit (bethy laboratories). because an affinity purified human anti-ha-specific siga standard of each influenza a subtype is not available, the relative value of anti-haspecific siga amount was expressed as unit (u). one unit was defined as the amount of one lg of human iga detected in the assay system as reported previously. the concentrations of siga in individual nws were normalized by the levels of total siga (lg ⁄ ml). oseltamivir suppresses viral rna replication and viral antigenic protein production. to investigate the influence of daily treatment with osv on ha-specific mucosal and systemic immune responses, we analyzed ha-specific siga levels in nws and balf as well as igg levels in plasma at days and post-infection in mice treated orally with osv or methylcellulose (mc) as vehicle. the osv treated mice showed lower antibody responses in nws and balf than control mice treated with mc solution (table ) . significantly reduced ha-specific siga responses were particularly noted in the osv group at day , the period of maximal mucosal siga induction. the airway secretions and plasma from mice at day did not contain detectable levels of ha-specific antibodies. these findings were supported by other data whereby mice treated with osv displayed significantly lower numbers of ha-specific iga antibody-forming cells (afcs) in the nasal lamina propria, mediastinal lymph nodes, and lungs compared with mc-treated mice. these results clearly indicate that oral administration of osv downregulates ha-specific siga responses in mucosa. on the other hand, there were no significant differences in the elevated levels of ha-specific plasma iga and igg antibodies or the increased numbers of ha-specific iga and igg afcs in the spleen between osv-and mc-treated mice. taken together, these results implicated the oral administration of osv in a suppressed induction of haspecific siga responses in respiratory lymphoid tissues, although systemic ha-specific antibody responses were not significantly affected by osv. since cam up-regulates il- , a mucosal adjuvant cytokine in the airways, and promotes the induction of siga and igg in the airway fluids of mice infected with iav, , we assessed the impact of treatment with osv and ⁄ or cam on the levels of anti-influenza siga in nws and clinical status of influenza patients. the concentration ratio of table . anti-ha-specific siga to total siga in nws was expressed as titer: anti-ha-specific siga (u ⁄ mg) ⁄ total siga (lg ⁄ mg) · . figure shows changes in the anti-ha(h n ) siga ratio (titer) and fold of increase in siga titer in each patient during the -days' treatment for the four different treatment groups. it is noteworthy that, upon admission to the hospital, the siga titers were < in % of patients. during the days of treatment, rapid increases in the titers were observed in almost all patients in cam, osv + cam, and no treatment groups. in contrast, in the osv group, the anti-ha-specific siga titers remained unchanged or decreased in the majority of patients. the finding of significant low induction of anti-viral siga in the osv group was supported by the results of animal experiments. however, the addition of cam to osv augmented siga production and restored mucosal siga levels; % of patients treated with osv + cam showed > -fold increase in the titers during treatment. these observations suggest that cam stimulated the local mucosal immunoresponse in the nasopharyngeal region of patients treated with osv. the prevalence of disease manifestations was also analyzed. among the symptoms listed, a significant decrease in the prevalence of cough was recorded between the no treatment group and the osv + cam group and between the osv group and the osv + cam group (**p < ae ), despite the limited number of patients in each group. the duration of the febrile period was significantly shorter in the osv and osv + cam groups than the no treatment group. however, no significant difference was observed between the osv group and osv + cam group. it has been reported that osv does not affect the cellular immune responses, such as cytotoxic t lymphocytes and natural killer cells. however, the effects of osv on mucosal immunity have not been studied so far. the present study showed that osv treatment of mice infected with iav induced insufficient protective mucosal siga responses in the respiratory tract, although treated mice showed the similar levels of systemic igg and iga antibody responses in plasma to those in mice treated with vehicle (table ) . the observed effect of osv on mucosal immunity was probably due to a suppression of viral replication and viral antigen production in the mucosal layer. these observations in mice are further supported by our clinical reports of siga in nws and balf of osv treated influenza patients. the membered-and membered-ring macrolides have been found to possess a wide range of anti-inflammatory and immuno-modulatory properties, , and to be effective in the treatment of respiratory syncytia and iav infection. , the efficacy of low doses administered on the long term against pathogens that are insensitive to macrolides indicates a mode of action that is separate from their antibacterial activity. , , , in the present study, we evaluated the immunomodulatory effects of cam on mucosal immune responses in pediatric influenza. a decrease in the proportion of total siga that was anti-ha-specific siga during treatment was observed in . % of patients in the osv group (those represented by the dotted lines and closed diamonds in figure ), whereas an increase in the proportion was observed in most patients of the other groups (except for one patient of the untreated group). despite the low or unchanged induction of anti-ha-specific siga in the majority of osv-treated patients, the additional use of cam with osv boosted the mucosal immune response and restored local mucosal siga levels. we are currently engaged in detailed immunological studies of the effects of cam and osv on the levels of mediators controlling iga class switching in nws of influenza patients and airway secretion of mice infected with iav. further studies should clarify the boost mechanisms of cam and the suppression mechanisms of osv in iga class switching. our findings suggest the risk of re-infection in patients showing a low mucosal response following osv treatment and cam effectively boosts the siga production for protection of re-infection. to date there is an urgent need to develop new antivirals against influenza. most of the molecules reported target influenza proteins that acquire rapid mutations of resistance. the development of new molecules that have a broad antiviral activity and are not subjected to influenza mutation is of particular interest. our laboratory and others recently showed that proteases can participate to the innate immune response in the airways through the activation of a family of receptors called par. in particular, through the release of interferon, par agonists curbed viral replication significantly in infected cells. in this study, since erk activation is crucial for virus replication, we investigated whether par could inhibit virus replication through inhibition of the erk pathway. results showed that while influenza a infection alone or par stimulation alone induced erk activation, par stimulation does not inhibit erk activation in influenza infected cells. thus, par agonists may be a potential new drug against influenza viruses that could be used in combination with other anti flu therapy such as the inhibition of the erk pathway. respiratory tract-resident proteases are key players during influenza virus type a infection. , in addition to their direct activating effect on surface viral proteins, lung mucosal proteases can regulate cellular processes by their ability to signal through protease-activated receptors (pars). after cleavage of the receptor by proteases, the new aminoterminal sequence of par binds and activates the receptor internally. these receptors are highly expressed at epithelial surfaces, in particular in the lung, where human influenza virus replicate in vivo. pars are thus directly exposed to proteases present in the airways. among the four different pars, par acts as an antiviral through an interferondependent pathway. , thus, agonists of par are potential new drugs against a broad range of influenza viruses, which is in accordance with the broad antiviral action of interferon. however, the signalling pathway induced by par agonists in influenza a infected cells has still to be investigated. in this manuscript, we showed that influenza infection or activation of par induced erk activation, a crucial step for efficient virus replication. , however, par agonists do not impaired erk activation in influenza a virus infected cells. since the pathway of par protection is likely to be erk-independent, the use of anti erk molecules in combination with par agonists maybe of potential interest in future anti-influenza therapy. influenza viruses a ⁄ wsn ⁄ (h n ) (a kind gift from nadia naffakh) was used in the present study. mdck (madin-darby canine kidney) and the human alveolar type ii a cell were obtained from atcc and grown as previously described. for western blot analysis, the following antibodies were used: monoclonal antibody for phospho-erk ⁄ (t ⁄ y ) and for erk ⁄ antibodies from cell signaling technology (beverly, ma), horseradish peroxydase (hrp)-coupled rabbit polyclonal antibodies against mouse or rabbit igg from paris (compiègne, france). a cells were infected with iav at an moi of in emem medium, as previously described. , at various time points post infection, cells were collected and proteins were analysed as previously described. , par stimulation was performed at °c in emem medium as previously described. after infection and ⁄ or stimulation, cells were lysed in ice-cold lysis buffer. lysates were centrifuged at g for min, and total proteins of the supernatants were analyzed by western blot analysis as previously described. , results since activation of the erk pathway is essential for efficient influenza replication, we first investigated the kinetics of erk activation after influenza infection in human a alveolar epithelial cells. for this purpose, a cells were infected with influenza viruses at a moi of at different time point post-infection, and activation of erk ⁄ pathway was assessed by western blot analysis using an anti-erk antibody. results showed that erk was phosphorylated after influenza infection in a time course depen-dent manner when compared to uninfected cells. in contrast, erk phosphorylation was not observed with heatinactivated viruses, suggesting that productive infection is needed for erk activation ( figure a ). antibodies against erk ⁄ were used as controls. since erk is activated after influenza infection, we then tested whether activation of par in uninfected cells also leads to activation of this pathway. for this purpose, a cells were stimulated with the selective human (h) or mouse (m) par agonist or a control peptide for the indicated time ( figure b ). when exposed to the par agonists and compared to controltreated cells, erk phosphorylation increased over the time course of stimulation. thus, influenza infection or stimulation of par without infection in a cells induced activation of the erk pathway at different time point post-infection. since influenza infection and par stimulation induced erk activation, we then investigated whether par could inhibit erk activation in influenza infected a cells. results in figure showed that in influenza infected cells, par activation for ten minutes does not inhibit erk activation after influenza infection. thus, erk activation is not inhibited by par activation in influenza stimulated cells. in this manuscript, we studied the activation of the erk pathway after par stimulation and or influenza infection. particularly interesting is the fact that either influenza infection or par stimulation alone induce erk phosphorylation in a epithelial cells, while erk activation is not inhibited in a infected cells compared to uninfected ones after par stimulation. proteases are key factor in the pathogenicity of influenza viruses. in addition to the cleavage of ha, necessary for iav replication, extracellular proteases also play a role in the modulation of the immune system against influenza viruses through the activation of pars. particularly par , activated by extracellular trypsin-like proteases, could inhibit virus replication through the release of interferon, , thus, strengthening the immune system via agonist peptides and providing new therapeutic potential against a broad range of influenza strains. in addition, targeting the host instead of the virus could provide a way to escape from virus resistance. thus, a better understanding of how virus escapes from immune surveillance may provide new therapeutic strategies to block iav. in addition, combinations of drugs that block virus replication via different pathways are of interest. the non classical molecules hla-g maybe an interesting new target as we recently showed that it is upregulated after influenza infection, and it is a well known immunotolerant molecule. indeed, it inhibits the innate immune response as well as the adaptive immune response. , also, as previously suggested, the erk signal transduction cascade is also of potential interest since it is crucial for virus replication and particularly influenza replication. , as shown here, it is unlikely that par protection occurs through an erkdependent pathway. thus strengthening the immune response with par agonists and blocking nuclear retention of the viral ribonucleoprotein complexes with inhibitors of the mek ⁄ erk pathway may be alternative combinatory approaches for influenza therapy. in addition, since those potential drugs target the host instead of the virus, this could help in the design of new antivirals molecules more resilient to iav mutations and thus to virus resistance. the initial waves of the first influenza pandemic of the st century have passed. in june , vaccine companies estimated they could produce in months almost . billion doses of pandemic vaccine. instead, they actually produced only million doses, of which % were non adjuvanted preparations. had these doses been produced with adjuvants (i.e., . lg instead of lg ha per dose), an additional billion doses could have been made available. yet there was public opposition to adjuvants in many countries, especially by regulatory officials in the united states. misperceptions about the safety of both adjuvanted and nonadjuvanted vaccines were widespread. added to this, shortfalls in vaccine production, delays in vaccine delivery, and the ''mildness'' of the pandemic itself meant that only a few countries achieved reasonable levels of vaccine coverage. millions of doses went unused and had to be destroyed. supplies of antiviral agents were even more limited. thus, despite the best efforts of influenza scientists, health officials, and companies, more than % of the world's people did not have timely access to affordable supplies of vaccines and antiviral agents. instead, they had to rely on th century public health ''technologies.'' given current understanding of biology in the early st century, they should have had -and probably could have had -something better. this report reviews evidence for an alternative approach to serious and pandemic influenza that could be used in all countries with basic health care systems. instead of confronting the influenza virus with vaccines and antiviral agents, it suggests that we might be able to modify the host response to influenza virus infection by using anti-inflammatory and immunomodulatory agents. this idea was introduced several years ago and has been reviewed in several publications. [ ] [ ] [ ] [ ] [ ] [ ] the central importance of the host response in the pandemic, young adults had high mortality rates. ever since, influenza virologists have sought to answer the question ''why did young adults die?'' by defining the molecular characteristics of the virus that were responsible for its virulence. in doing so, they have overlooked a crucial piece of clinical evidence from the pandemic: compared with young adults, children were infected more frequently with the same virus, yet they seldom died. consequently, the more important question is ''why did children live?'' this can only be explained by recognizing that children must have had a different host response to the influenza virus than adults. physicians have long recognized that for several other medical conditions, both infectious (e.g., pneumococcal bacteremia) and non-infectious (e.g., multiple trauma), children have a more benign clinical course than adults. , a corollary of this observation is that secondary bacterial pneumonia, although commonly found in young adults in , could not have been the primary cause of death. children must have had the same or higher rates of nasopharyngeal colonization with the same bacteria that were associated with pneumonia deaths in adults, yet children seldom died of secondary bacterial pneumonia. if young adults died with secondary bacterial pneumonia, underlying host factors must have made them more susceptible. few people who die of influenza do so during the first few days of illness when pro-inflammatory cytokine levels are high. instead, like patients with sepsis, they usually die in the second week, when anti-inflammatory cytokines and immunosuppression dominate. , , influenza deaths occur more frequently in older persons with cardiopulmonary conditions, diabetes, and renal disease, but as seen in the h n pandemic, they also occur in younger adults with obesity, asthma, and in women who are pregnant. regardless of age, people with all of these conditions share one characteristic in common: they have chronic low-grade inflammation. in effect, their ''innate immune rheostats'' have been set at different, and perhaps more precarious, levels that make them more vulnerable to influenza-related complications. laboratory studies of influenza virus infection confirm the importance of the host response. in several studies in mice in which the host response has been modified (e.g., cytokine knockout), survival has been improved without increasing virus replication in the lung. in fact, severe disease can be induced without any influenza virus replication. for example, fatal acute lung injury has been induced in mice by inactivated (not live) h n virus. in this model, antiviral agents would be useless; only the host response could be responsible for disease. these observations raise the following question: could the host response be modified so patients with severe seasonal and pandemic influenza might have a better chance of surviving? influenza is associated with acute coronary syndromes, and influenza vaccination and statins reduce their occurrence. these associations led to the suggestion in that statins might be used to treat pandemic influenza. other agents that might also be effective include ppara and pparc agonists (fibrates and glitazones, respectively) and ampk agonists (e.g., metformin). , these agents have been studied in laboratory models of inflammation, sepsis, acute lung injury, ischemia ⁄ reperfusion injury, energy metabolism, mitochondrial function, and programmed cell death. the results of these studies cannot be reviewed in detail here, but the major findings for cell signaling are summarized in the table . unfortunately, the results of experimental studies are not always clear cut. for example, in one study of influenza virus infected mice, il- was necessary for containing infection, but in another study il- appeared to be harmful. nonetheless, overall understand-ing of cell signaling pathways in influenza virus infections and the actions of statins, glitazones, fibrates, and ampk agonists strongly suggest that these agents could benefit patients with severe influenza. laboratory studies in mice infected with pr (h n ) h n and pandemic h n viruses show that resveratrol, fibrates, glitazones, and ampk agonists reduce mortality by - %, often when treatment is started - days following infection. - (resveratrol is a polyphenol found in red wine. it shares with these other agents many of the same cell signaling effects.) in h n -infected mice, treatment with celecoxib and mesalazine, together with zanamivir, showed better protection than zanamivir alone. remarkably, these immunomodulatory agents have not increased virus replication. even more remarkable, in another model of a highly inflammatory and frequently fatal conditionhepatic ischemia ⁄ reperfusion injury -glitazone treatment ''rolled back'' the host response of ''young adult'' mice ( - weeks old) to that of ''children'' ( - weeks old). this unique study suggests that immunomodulatory treatment might roll back the damaging and sometimes fatal host response of young adults with influenza to the more benign and rarely fatal response of children. several, but not all, observational studies have shown that outpatient statins decrease hospital admissions and mortality due to community-acquired pneumonia. for influenza itself, preliminary evidence presented in october suggests that immunomodulatory treatment of influ- table . cell signaling targets that might be affected by immunomodulatory treatment of severe seasonal and pandemic influenza* down regulate pro-inflammatory cytokines (e.g., nf-kappab, tnfa, il- , il- ) up regulate anti-inflammatory cytokines (il- , tgfb) up regulate pro-resolution factors (lipoxin a , resolvin e ) up regulate ho- and decrease tlr signaling by pamps and damps up regulate enos, downregulate inos, restore inos ⁄ enos balance and stabilize cardiovascular function decrease formation of reactive oxygen species and decrease oxidative stress improve mitochondrial function and restore mitochondrial biogenesis decrease tissue factor and its associated pro-thrombotic state stabilize the actin cytoskeleton in endothelial cells and intracellular adherins junctions, and thereby increase pulmonary barrier integrity and decrease vascular leak differentially modify caspase activation and apoptosis in epithelial and endothelial cells, macrophages, neutrophils and lymphocytes in the lung and other organs increase the bcl- ⁄ bax ratio in influenza virus-infected cells and prevent the apoptosis necessary for virus replication. *see references , , , for details. nf-kappab, nuclear factor kappab; tnfa, tumor necrosis factor alpha; tgfb, transforming growth factor beta; ho- , heme oxygenase - ; tlr, toll-like receptor; pamp, pathogen-associated molecular pattern; damp, damage associated molecular pattern; enos, endothelial nitric oxide synthase; inos, inducible nitric oxide synthase. enza patients with severe illness could be beneficial. in a study of almost patients hospitalized with laboratoryconfirmed seasonal influenza, inpatient statin treatment reduced hospital mortality by %. in these patients, the cell signaling effects of statin treatment, summarized in the table , probably acted to reduce pulmonary infiltrates, maintain oxygenation, stabilize myocardial contractility and the peripheral circulation, reverse immunosuppression, restore mitochondrial biogenesis, and prevent multi-organ failure. achieving these clinical effects led to a decrease in mortality. because of the molecular cross-talk between statins, fibrates, glitazones, and ampk agonists, , similar clinical benefits might be expected from other members of this ''family'' of immunomodulatory agents. simvastatin, pioglitazone, and metformin are produced as inexpensive generics in developing countries. they are used throughout the world in the daily treatment of millions of patients with cardiovascular diseases and diabetes. global supplies are huge. because most people with influenza recover without specific treatment (this was true in ), not all patients would require immunomodulatory agents. instead, only those at risk of ards, multi-organ failure, and death would need to be treated. importantly, the cost of treatment for an individual patient would be less than $ . (d.s. fedson, unpublished observations). moreover, unlike vaccines they could be used on the first pandemic day. thus far, influenza scientists and the institutions that support their work (e.g., nih and cdc, national health agencies in many countries, the bill and melinda gates foundation, the welcome trust, and the world health organization) have shown little interest in immunomodulatory treatment. nonetheless, when more than % of the world's people have no access to influenza vaccines and antiviral agents, their physicians must have access to an effective ''option,'' especially one that might be lifesaving. research on immunomodulatory agents for influenza must involve investigators in many fields outside influenza science -those with expertise in the molecular and cell biology of inflammation, immunity, sepsis, cardiopulmonary diseases, endocrinology and metabolism, ischemia ⁄ reperfusion injury, mitochondrial function, and cell death. laboratory studies needed to identify promising treatment agents would probably cost $ - million (d.s. the results of these studies would inform clinical trials that critical care physicians are already eager to undertake. , this work will be especially important for people in developing countries where critical care capacity is extremely limited and not likely to improve. like critical care physicians, influenza scientists too must recognize that they cannot afford not to undertake research to determine whether generic immunomodulatory agents might be useful in managing severe seasonal and pandemic influenza. the nf-kappab-inhibitor sc efficiently blocks h n influenza virus propagation in vitro and in vivo without the tendency to induce resistant virus variants introduction influenza is still one of the major plagues worldwide. the appearance of highly pathogenic avian influenza (hpai) h n viruses in humans and the emergence of resistant h n variants against neuraminidase inhibitors highlight the need for new and amply available antiviral drugs. we and others have demonstrated that influenza virus misuses the cellular ikk ⁄ nf-kappab signalling pathway for efficient replication, suggesting that this module may be a suitable target for antiviral intervention. here, we show that the novel nf-kappab inhibitor sc efficiently blocks replication of influenza a viruses, including avian and human a ⁄ h n isolates in vitro in concentrations that do not affect cell viability or metabolism. in a mouse infection model with hpai a ⁄ h n and a ⁄ h n viruses, we were able to demonstrate reduced clinical symptoms and survival of sc treated mice. moreover, influenza virus was reduced in the lung of drug-treated animals. besides this direct antiviral effect, the drug also suppresses h n -induced overproduction of cytokines and chemokines in the lung, suggesting that it might prevent hypercytokinemia we hypothesise to be associated with pathogenesis after infections with highly pathogenic influenza viruses, such as the a ⁄ h n strains. thus, a sc -based drug may serve as a broadly active nontoxic anti-influenza agent. to assess the number of infectious particles (plaque titers) in organs a plaque assay using avicel Ò was performed in -well plates as described by mastrosovich and colleagues. virus-infected cells were immunostained by incubating for hour with a monoclonal antibody specific for the influenza a virus nucleoprotein (serotec) followed by minutes incubation with peroxidase-labeled anti-mouse antibody (dianova) and minutes incubation with true blueÔ peroxidase substrate (kpl). stained plates were scanned on a flat bed scanner and the data were acquired using microsoft Ò paint software. the virus titer is given as the logarithm to the basis of the mean value. the detection limit for this test was < ae log pfu ⁄ ml. organs of infected and control mice were homogenized and incubated over night in ml trizol Ò reagent (invitrogen) at °c. total rna isolation was performed as specified by the manufacturer (invitrogen). rna was solubilised in ll rnase free water and diluted to a working concentration of ng rna ⁄ ll. reverse transcription real-time pcr was performed using quantifastÔ sybr Ò green rt-pcr kit and quantitect primer assays (qiagen) . all samples were normalized to gapdh and fold expression analyzed relative to uninfected controls. ct values were obtained with the smartcycler Ò (cepheid). to answer the question whether the nf-kappab inhibitor sc shows antiviral properties against influenza virus, h n infected mdck cells were treated with different concentrations of the inhibitor (figure ). already treatment with nm of sc led to a reduction of viral cpe of more than %. almost % protection of cells was achieved when cells were treated with lm sc . the results indicated that sc has antiviral properties at concentrations ranging from to nm. we next tested whether sc would also be effective in the mouse model of influenza virus infection. when h n mice were treated i.v. once daily for days with mg ⁄ kg sc , survival rate of the animals increased significantly (p < ae ). the same results were found when h n influenza virus infected mice were treated i.p. with mg ⁄ kg sc (data not shown). moreover, sc treatment was not only effective when the inhibitor was given prior to h n influenza virus infection, but also in a therapeutic setup when sc was applied to the animals days after infection (data not shown). since influenza virus infected mice showed increased survival after lethal infection, we next questioned whether the amount of influenza virus was reduced in the lung. therefore, we performed quantitative real-time (qrt) pcr to detect viral mrna. mice were treated with either sc or the solvent, and hour later the lungs were prepared to perform qrt-pcr. as shown in figure a the amount of viral mrna was reduced by % in sc treated mice compared to solvent treated controls, indicating that sc leads to a reduced expression of h n specific mrna in the lung of infected mice. since infection of mice with h n leads to hypercytekinemia, we also investigated the expression of cytokines in sc treated mice. as shown in figure b the amount of il- specific mrna was drastically reduced in sc treated mice compared to solvent treated controls. moreover, also the expression of ip- was altered in sc treated h n influenza virus infected mice. here, roughly % reduction of specific mrna was detectable ( figure c ). thus, sc leads to a reduced transcription of il- and ip- in h n infected mice. there is an urgent need for new concepts to develop antiviral drugs against influenza virus. targeting cellular factors is a promising but challenging approach, and the concerns about side effects are obvious. however, it should be considered that drugs targeting viral factors, such as amantadine or oseltamivir, also exhibit a wide range of side effects in patients. thus, drug safety has to be rigorously tested in clinical trials regardless whether a drug targets a cellular or a viral factor. moreover, resistance against human h n influenza viruses and highly pathogenic avian h n virus strains to oseltamivir and amantadine have been reported. in that respect, the strategy to target cellular factors , might be one way to ensure that new drugs against influenza virus will be useful and effective for a long time without causing the development of resistant virus variants. we were able to demonstrate that the nfkappab inhibitor sc is able to reduce influenza virus activity in cell culture. moreover, the compound was also effective against highly pathogenic avian influenza viruses of the h n and h n subtypes in the mouse model. next to the reduction of virus sc was also able to reduce h n -induced overproduction of cytokines and chemokines in the lung in the lung of mice after infection with h n . most importantly, the drug did not show any tendency to induce resistant virus variants (data not shown). thus, a sc based drug may serve as a broadly active non-toxic antiinfluenza agent. [ ] [ ] [ ] [ ] [ ] in hong kong, the first confirmed case was a tourist from mexico reported on may , . the local government made its first attempt to contain the spread of h n in the local community by closing the metropark hotel where that tourist was staying, and quarantining guests and staff for days. following identification of the first local case around weeks later on june , , the government closed all kindergartens and primary schools from june until early july. fever clinics were also opened, the alarm levels in hospitals were raised to the highest, and a public education campaign was implemented. previous studies of the community responses to severe acute respiratory syndrome (sars) and human-to-human h n avian flu identified the importance of understanding the background perceptions of risk and psychological impact on the community. [ ] [ ] [ ] [ ] [ ] in this study we investigated the psychological and behavioral responses of the general local community throughout the first wave of ph n , and we also examined the factors associated with greater use of preventive measures. a total of surveys were conducted between april and november , covering the entire first wave of the ph n pandemic. computer generated random-household telephone numbers from all land-based local telephone numbers covering over % of hong kong households were used to recruit a total of local adults. one cantonese-speaking adult (age ‡ ) was invited for interview in each selected household on the basis of a kish grid. the survey instrument was based on previous experience in sars and avian influenza projects. information, including knowledge on modes of transmission, psychological responses to pandemic influenza, preventive behaviors, attitudes towards the new vaccines and socio-demographics, was collected. informed consent was obtained prior to the interview. ethics approval was obtained from the institutional review board of the university of hong kong. descriptive statistics were weighted by sex and age based on the reference population data provided by the hong kong government census and statistics department. multivariable logistic regression analyses were used to examine the association between the use of preventive measures and knowledge, perceptions and behaviors, sociodemographic characteristics, and psychological responses to pandemic influenza. multiple imputation was used to cope with a small proportion of missing data and make the best use of all available data. statistical analyses were conducted in r version . . (r development core team, vienna, austria). twelve thousand and nine hundred and sixty-five local adults were recruited throughout the study period, with a total of telephone calls being made; the response rate among eligible participants was . %. hong kong entered the containment phase after the world health organization (who) announced a global alert, and policies including border screening, tracing, and quarantine of doi: . /j. - . . .x www.influenzajournal.com suspected cases were implemented. hong kong transitioned to the mitigation phase on june , when the first local case was reported. the chronology of these and other events plus the epidemic curve of laboratory-confirmed ph n cases are shown in figure (a) . the anxiety scores and risk perception of the respondents are shown in figure (b,c) . anxiety, measured by the state trait anxiety inventory, remained steady throughout the study period. in response to the announcement made by who and the unknown nature of the new virus, a higher proportion of the respondents expressed worry (more, much more, or extremely more worried than normal) if developed ili and perceived ph n severity (same, more, or much more serious than sars) initially in early may . fewer respondents reported worry if they developed ili as the pandemic proceeded, with a slight perturbation around the first deaths in july and a steady decline to . %, while perceived severity of ph n declined more dramatically after an early high. perceived risks of infection of respondents (absolute susceptibility) and risk relative to others (relative susceptibility) were also investigated and found to remain relatively stable throughout the first wave, with no indication of an increase during the period of peak ph n activity in september (figure c) . as the first wave of ph n progressed, knowledge on modes of transmission did not improve. on the contrary, later in the epidemic increasing proportions of respondents reported oral-fecal and cold weather as modes of transmission of ph n . around - % of the respondents did not recognize direct and indirect contact or touching infected persons and contaminated objects as transmission routes for ph n throughout the first wave ( figure d ). higher proportions of respondents avoided crowded places and rescheduled travel plans in the second half of june when local kindergartens and primary schools were closed and the first ph n -associated deaths were announced. social distancing measures such as avoiding crowded places and rescheduling travel plans remained stable with slightly decreasing trends thereafter. the use of hygiene measures and other social distancing strategies was relatively stable with slightly decreasing trends during the study period ( figure ). female sex and older age were generally associated with greater reported use of hand hygiene measures, home disinfection, avoidance of crowded places, and rescheduling of travel plans. female sex was also positively correlated with use of face masks and cough etiquette. we found a negative correlation between anxiety and use of all hand hygiene measures and cough etiquette, but a positive correlation between anxiety and use of home disinfection and (c) proportion of the respondents reporting higher worry if developed flu-like symptoms (more, much more, or extremely worried), higher perceived seriousness of h n compared to sars (much more or more severe), higher probability to contract h n over the next month (certain, much more, or more likely), higher probability to contract h n over the next month compared to others outside family (certain, much more, or more likely). (d) proportion of the respondents identifying possible modes of transmission as the actual modes of transmission of h n . social distancing measures. other significant factors contributing to greater use of preventive measures were worry and knowledge. greater worry was associated with higher probability of home disinfection, social distancing measures, and use of face masks. knowledge that h n could be spread by indirect contact was associated all the investigated preventive measures, and knowledge that h n could be spread by droplets was associated with cough etiquette, but not face masks. there were no consistent trends between all the investigated preventive measures and absolute and relative susceptibility. community transmission emerged in hong kong in mid-june , and prior to emergence of community transmission, perceived risk and perceived severity were high. as ph n spread in hong kong, risk perception declined, even at the same time as incidence was increasing. anxiety was low throughout, at around . on the -point scale, compared to a maximum of . during sars on the same scale. anxiety has been showed to be positively correlated to personal hygiene measures and social distancing in previous studies; , however, we found a negative correlation between anxiety and use of all hand hygiene measures, cough etiquette, and face masks, and a positive correlation between anxiety and home disinfection. the differences in findings may be due to the fact that our anxiety measure was not specific to h n , and the score could be affected by other factors including economics. unlike hygiene measures, higher anxiety level, greater worry, and higher risk of perception were all associated with more social distancing. , , , social distancing is the most direct strategy in avoiding infection from other people, and it is commonly observed in an outbreak that the general public avoids crowded places, travelling to other countries, and social gatherings, , but the economic impact could be substantial. as community incidence of h n peaked, we did not observe any increase in use of preventive measures (figure ) . we found that face mask use peaked at the early stage of the pandemic, while hand hygiene remained fairly constant, and the knowledge on the modes of transmission of ph n did not improve over time. the lack of substantial change in preventive measures or knowledge about the modes of ph n transmission in the general population suggests that community mitigation measures played little role in mitigating the impact of ph n in hong kong. on the other hand, knowledge that ph n could be spread by indirect contact was associated with all of the preventive measures studied. consistent with reports during the sars period, , this study also showed that females and those of older age were more likely than others to use hygiene measures, avoid crowded places, and reschedule travel plans. this study has some limitations. first, this was a crosssectional study that was carried out at different time points, rather than a longitudinal study following the same individuals over time, and so the inferences on changes in behavior may need to be interpreted more cautiously. second, we recruited samples from all land-based local telephone numbers that cover % of hong kong households, but the response rate was not high enough to guarantee a representative sample, and this could be a source of selection bias. third, the responses were self-reported, and this may lead to social desirability bias in estimating knowledge, attitudes, and preventive behaviors. fourth, since the hong kong population has previously gone through unique experiences from sars in and avian flu in , our results may not be comparable to other countries or settings. in conclusion, this study revealed that the ph n pandemic failed to generate an increase use of preventive measures in the local community. there was no association between anxiety level and the events of the pandemic. with a relatively low mortality and morbidity rates compared to sars, ph n was not a matter of concern in the hong kong community. the lack of substantial change in the use of preventive measures and improvement in knowledge on the modes of transmission of ph n suggested that public health campaigns during the pandemic may not have had substantial effects on the general public. london is a major tourist destination, the seat of government and finance in the uk, and in will host much of the olympic and paralympic games. along with the rest of the global community, in and early london faced the challenges of responding to the first pandemic of the st century. at the time, nhs in london was composed of organisations, including the london ambulance service, acute hospitals, mental health and primary care trusts, and the strategic health authority. while london's nhs is well practiced at responding to large, big bang incidents, the influenza a ⁄ h n v pandemic was a rising tide event that lasted many months. significant preparatory work had been undertaken prior to april , which meant that the nhs in london was ready to respond. nhs london (the strategic health authority for london) led the response in partnership with local managers in all nhs organisations. the first uk cases of influenza a ⁄ h n v were reported in scotland on april, with the first in london on april. cases continued to increase, and the first wave peaked in london in july. cases reduced over the school summer holidays, but increased again when children returned to school at the start of september, and a second, smaller wave occurred. it is essential that the nhs learns from the ⁄ influenza a ⁄ h n v pandemic to ensure it is prepared for future challenges. nhs london provided a standardised debriefing pack to all nhs organisations in the region to identify, capture, and learn lessons. each debrief event involved health and inter-agency partners to ensure all viewpoints were considered and brought together in a single local report. all local reports were compiled in an over-arching document, which brings together common themes to inform ongoing preparedness in the region. the debrief process identified a number of common themes, such as the need for clear and appropriate communication, the importance of working with partners, and the benefits of strong and early leadership. however, differences between and within organisations were also highlighted; for example, some wanted more freedom for local decision making, whereas others would have preferred more stringently applied central direction. the following paragraphs considers individual areas assessed in the debrief process. command and control was in the main effective, with clear direction delivered from the national centre through nhs london to local nhs organisations. effective leadership is essential; the identification of senior local individuals to lead the response with teams of people to support them was critical. appropriate use of technology to communicate messages and coordinate command and control processes greatly aided the response. this included the development of the nhs london noon brief, a daily digest and associated web portal, and regular teleconferencing. key points are: • operational management at all levels must be considered in pandemic planning. • appointing an executive lead in each organisation was invaluable in the response. • pandemic flu planning for london must continue to be regionally led. communication is an essential component of the response to any incident. it must be clear, timely, and accurate. in the main, communication was excellent and met these criteria. one of the most challenging aspects was when messages from partner organisations differed, which occasionally led to confusion, unnecessary work, or frustration. the use of technology greatly aided communication across the region and supported the response; this included secure web sites, bluetooth, and text messaging etc. key points are: • regular internal communications and staff briefings are critical in the response to emergencies. • regular teleconferencing should be incorporated into future plans. • organisations should consider proactive and innovative methods for communicating during emergencies. robust partnership working was an essential component of pandemic preparedness work; however in the event, the a ⁄ h n v pandemic had little impact on sectors in london other than health. resilient communication networks between organisations, a common understanding, and the ability to make decisions were essential to the response at local level. ipcs proved an excellent mechanism to maintain local working relationships and resolve problems. clarity on the seniority of those attending these meetings and whether multi-site organisations such as mental health trusts should attend every ipc should be considered on a local and regional basis. key points are: • pandemic planning must remain part of inter-agency working. • social care resilience and planning must be embedded and integrated in health planning. 'vulnerable groups' is a universal term that covers a large and fluid group of individuals with different needs. ensuring access to healthcare during the pandemic for those who became vulnerable due to the situation, or those identified as such prior to the event, was the role of the pct in partnership with the local authorities. work continues to ensure that communication with vulnerable people is appropriate and timely in all incidents, and that organisations work together to achieve this. key points are: • planning to support the breadth of vulnerable people must continue. • pandemic preparedness for the prison sector should be further developed. • red ⁄ amber ⁄ green ratings for assessing vulnerabilities of mental health service users in an emergency should be further developed across the region. correct and appropriate usage of ppe is an essential component of reducing influenza spread, particularly in healthcare settings. london's nhs had been working towards developing local stockpiles of ppe when the pandemic commenced; however, there was little in place. the unanticipated national stockpile, while providing ppe to all organisations, was accompanied with some challenges in that it was often unfamiliar stock. key points are: • work around local stockpiling of non-standard consumables should continue. • regular training and fit testing of respirators should be embedded in all organisations. antiviral treatment was a core component of the response to influenza a ⁄ h n v, and was provided free of charge from a national stockpile. npfs reduced pressure on frontline nhs services once it was activated; however, there were concerns that patients could 'cheat' the system and obtain the drugs prior their clinical need. information about storage requirements of countermeasures must be clearly explained when they are delivered to frontline services, and the potential for recall into national stockpiles should be planned for. key points are: • regular exercising of local mass countermeasures centres and antiviral collection points (acps) should continue. • the use of community pharmacies as acps should be further considered in the capital. pandemic influenza vaccine uptake by healthcare workers was better than usual seasonal influenza uptake in the majority of nhs organisations, but could have been even better. this was largely due to the second pandemic wave not being as significant as expected, lack of clarity around when the vaccine would be delivered, and limited amounts being available initially. • gp-led and mass vaccination models for pandemic vaccination should be considered in local plans. • local lessons from the pandemic vaccination campaign should be applied to seasonal flu vaccination. the ability to maintain or increase capacity in response to a surge in demand, no matter what the cause, must be planned for. any of a number of situations could result in reduced staff or more patients, such as industrial action, transport disruption, disease outbreak, major incident, or poor weather. the work undertaken during planning for and responding to the pandemic will stand organisations in good stead for future disruptions. the importance of robust business continuity planning locally cannot be overlooked, as this is a key component of maintaining and increasing capacity. key points are: • local gp 'buddy schemes' should be encouraged for response to extreme pressure events. • organisations should regularly run staff skills audits so as to be aware of their overall capability for managing emergencies. • less emphasis should be placed on the use of retired staff when planning service continuity. reporting is a necessary but onerous task, and is often one of the most time-demanding parts of any incident response. it is also the aspect least likely to be tested through exercising. nhs london worked with organisations to endeavour to reduce reporting pressures, but much of this was dictated by central government. it is essential that future reporting requirements are proportional, informative, and realistic. while recognising it is not possible to predict the detail of information that may be requested, some broad assumptions can be made. key points are: • organisations should consider how they would collect and collate data from disparate parts of their organisation, rather than focussing on the detail of what that might be. • national and regional planning should consider the need for information and how this is balanced with the demand this places on organisations. • the introduction of the concept of a daily dashboard to identify areas of pressure should be incorporated into pandemic flu planning. the winter and pandemic influenza resilience assurance process undertaken in autumn was a useful process to inform planning for the first winter when the pandemic virus would be circulating in the uk. this consisted of a regional inter-agency exercise and a comprehensive review of the winter and pandemic plans of all nhs organisations in london. • regular assurance of pandemic flu preparedness should be maintained. • future resilience assurance processes should be undertaken in a timely and measured manner. • local organisations should continue to undertake regular pandemic flu exercises. the recovery period is as important as the response, but often receives minimal attention and has the potential to suffer as staff return to their normal jobs. one of the aspects that was not anticipated during the pandemic was the amount of stock (ppe, antivirals, and vaccine consumables) that would be recalled into national stockpiles. this proved particularly challenging for pcts who had to coordinate the process across their local areas. key points are: • the recovery period of an emergency must be given the same status and importance as the response. • future pandemic flu planning must include the recovery of national stockpiles of equipment and medicines. it is essential the lessons from the ⁄ influenza a ⁄ h n v pandemic are learnt and embedded into business-as-usual and emergency response processes in preparation for the next pandemic and other incidents. even though the a ⁄ h n v pandemic was generally milder than previous pandemics, it still presented challenges to the nhs in london. the biggest challenge that remains is to ensure that the public and nhs staff are aware that a more virulent virus could cause significantly more illness, death, and disruption, and that we must maintain our preparedness should this happen. the influenza a ⁄ h n v pandemic has been a major stimulus to business continuity planning and emergency preparedness across health in london, and many of the experiences during the pandemic proved invaluable in the unusually severe weather in early . it is important that this impetus and focus is maintained. changes to the nhs landscape in london will be considered in ongoing pandemic and emergency preparedness to ensure we remain as well prepared as possible for future events, particularly as london approaches the olympic and paralympic games. one of the major lessons learnt from all global pandemic events is that better preparedness of national health systems to deal with influenza viruses could make a significant difference. the way national health systems operate during inter-pandemic and the pandemic alert periods and the methods they use to address potential threats posed by zoonotic viruses with pandemic potential, as well as sea-sonal influenza epidemics, can clearly indicate whether the countries have enough capacities to respond adequately to unexpected influenza outbreaks. these public health decisions to ensure the maximum of efficiency require a robust scientific knowledge base. the who public health research agenda for influenza developed by the global influenza programme (gip) in cooperation with international influenza experts identified specific research topics and their importance in meeting stream-specific breakout discussion groups during the global consultation meeting included representatives of researchers and public health professionals. funding organizations were invited to observe the process with no direct participation in the deliberations. the methods used to design the research roadmap for an influenza pandemic scenario are closely related to the process of development of the final document of who public health research agenda for influenza. during a pandemic scenario, the group prioritized topics and questions relating to rapid action and response. five to key public health needs associated with a pandemic scenario have been identified for each of the research agenda streams: five priority public health topics were identified for a pandemic scenario as follows: • examination of host range and transmission dynamics of animal influenza viruses to guide surveillance, control strategies, and risk communication. • enhanced surveillance in animals and humans to monitor virus evolution: o early detection of novel reassortants or changes in genotype and ⁄ or phenotype related to virulence. o development of epidemiological and laboratory diagnostic tools and capacity building to optimize case finding. o develop a framework for surveillance in animals that address ethical, legal, and social barriers to intra-pandemic surveillance and reporting. • deconstruct the origins of the pandemic virus to identify factors that permitted efficient human transmission. • develop strategies to limit economic, social, and cultural disincentives of animal-based interventions to reduce intra-and inter-species transmission. • operational research to optimize risk communication in the early phases of the pandemic linked to animal husbandry and food safety. stream : limiting the spread of pandemic, zoonotic and seasonal epidemic influenza ten priority research topics were identified for both pandemic and inter-pandemic scenario as follows: transmissibility of influenza across the progression of infection and spectrum of disease: • relative contributions of the different modes of transmission for influenza. five priority public health topics were identified for a pandemic scenario as follows: • identification of groups at higher risk of infection and severe disease outcome through enhanced surveillance. • understanding disease severity and identification of predictors of severe outcomes. • investigation of vaccine effectiveness, especially in high risk groups in diverse geographic areas. • establishment ⁄ enhancement of pharmacovigilance, particularly for adverse events among at-risk groups. • optimization of strategies for rapid and targeted vaccine deployment. • rapid assessment to optimize acceptance of pandemic vaccine. six priority public health topics were identified for a pandemic scenario as follows: • collaboration and coordinated sharing of data, protocols, regulatory, and other implementation strategies and databases from different countries on all aspects of patient management and outcome to accelerate improvements in patient care. • development of best practices in patient management in different settings, including checklists and algorithms for clinical care and treatment, prognostic parameters, and tests to predict potential for the development of severe disease. • rapid, reliable, simple, low-cost point-of-care diagnostic tools for influenza. • best use of current antiviral drugs and optimal formulations in different target populations, such as parenteral and other routes of administration for severe infections. • use of combination therapies, including use of adjunctive therapies (e.g., use of convalescent serum and immunomodulators). • role of ongoing viral replication, host responses, and the effect of co-infections in the pathogenesis of severe disease. modern tools for early detection and monitoring of disease the group on surveillance tools concluded that the agreed topics of interest were equally applicable during a pandemic or inter-pandemic period: • studies to appraise and adapt modern technologies for early detection of influenza outbreaks in surveillance at the human-animal interface. • develop, integrate, and evaluate innovative approaches for influenza surveillance and monitoring with other existing disease monitoring systems. • study efficient mechanisms on sharing data, clinical specimens, and viruses with consideration for local, ethical, legal, and research perspectives. • examine the timeliness and quality of data required for early detection from local to national and global levels for the respective stakeholders. five priority public health topics were identified for a pandemic scenario as follows: • identify environmental determinants of seasonal variation in influenza transmissibility in tropical and temperate regions. • estimate the transmission risk associated with types of contacts by comparing measured contact patterns with outbreak data. • incorporation of validated models of behavioral responses to risk and control measures in virus transmission. • development and implementation of novel technology for real-time sero-surveillance during a pandemic. • develop experimental and theoretical framework to assess host adaptation to study host receptor, antigenicity, and virulence. modern tools for strategic communication three priority public health topics were identified for a pandemic scenario as follows: • evaluate tools to more rapidly and accurately assess and monitor knowledge, attitudes, beliefs, and practices in different population groups to guide future communication efforts; develop tools and methods to more rapidly and accurately assess and monitor knowledge, attitudes, beliefs, and practices in different population groups, and thereby, guide future communication efforts. for communicating in different cultural settings, which engage and empower individuals and communities to practice and promote appropriate risk reduction measures. implementation of the identified research priorities is expected to underpin public health decision making at all levels with proven knowledge that will help to save large numbers of lives, reduce health costs and economic loss, and mitigate potential social disruption. complemented by an analogous research roadmap for a pandemic influenza scenario, the research recommendations for an interpandemic period represent a framework to provide evidence to guide public health policies on influenza control. one of the major lessons learnt from all global pandemic events is that better preparedness of national health systems to deal with influenza viruses could make a significant difference. these public health decisions to ensure the maximum of efficiency require a robust scientific knowledge base. the who public health research agenda for influenza developed by the global influenza programme (gip) in cooperation with international influenza experts identified specific research topics and their importance in meeting public health needs for inter-pandemic periods according to its five key research streams: • stream . reducing the risk of emergence of pandemic influenza. • stream . limiting the spread of pandemic, zoonotic, and seasonal epidemic influenza. • stream . minimizing the impact of pandemic, zoonotic, and seasonal epidemic influenza. • stream . optimizing the treatment of patients. • stream . promoting the development and application of modern public health tools. stream-specific breakout discussion groups during the global consultation meeting included representatives of researchers and public health professionals. funding organizations were invited to observe the process with no direct participation in the deliberations. the methods used to design the research roadmap for an influenza inter-pandemic scenario are closely related to the process of development of the final document of who public health research agenda for influenza. during an inter-pandemic phase, a more comprehensive approach was applied to establish research topics and prioritizing a range of questions that will build a solid foundation to guide research activities to support public health decision making. five to ten key public health needs associated with an inter-pandemic scenario have been identified for each of the research agenda streams: stream : limiting the spread of pandemic, zoonotic, and seasonal epidemic influenza ten priority research topics were identified for both pandemic and inter-pandemic scenario as follows: . transmissibility of influenza across the progression of infection and spectrum of disease . relative contributions of the different modes of transmission for influenza . biological, behavioral, and social host factors that influence the risk of transmission and infection . patterns, drivers, and mechanisms affecting the seasonality of transmission . viral and population factors that influence transmission and spread of different influenza types, subtypes, and strains . strategies to reduce the transmission of influenza in community, household, and health care settings, especially in less-resourced areas . impact and cost effectiveness of social measures, such as school closures, and the role of surveillance in assessing timing of these interventions . impact, effectiveness, and cost effectiveness of individual measures, such as isolation and quarantine . role of vaccination in limiting the spread of influenza and strategies for its use . impact of antiviral treatment and prophylaxis in reducing transmission of influenza stream : minimizing the impact of pandemic, zoonotic, and seasonal epidemic influenza . identify higher risk groups and severe disease through surveillance; disease severity and identification of predictors of severe outcomes . evaluate vaccination preventable disease burden and the potential impact of immunization programs through vaccine demonstration projects . enhancement of the properties of existing vaccines, including duration and breadth of protection, safety, immunogenicity, and dosesparing . development of new vaccines and vaccine platforms, especially suitable for under-resourced country settings . study the effectiveness of vaccine strategies to reduce disease burden in children and other high risk groups in a wide range of settings . improved uptake and acceptability of vaccines for both seasonal and pandemic influenza seven priority public health topics were identified for an inter-pandemic seasonal influenza scenario as follows: inter-pandemic seasonal influenza scenario . research on the burden of severe disease with a focus on regionalspecific factors, such as the burden of tb and hiv and optimization of pandemic and management . development of new antiviral strategies and validation of surrogate endpoints which may aid in advancing understanding of disease progression . further clinical evaluation of current antiviral drugs, particularly in populations at risk . integration of seasonal influenza with pandemic preparedness; strengthen surveillance, health care systems, capacity, and preparedness planning . improving diagnostics (e.g., multiplex assays for viruses and bacteria), including antiviral resistance testing at point-of-care . dissemination of best practices, situation analysis, preparation for next epidemic (e.g., establish protocols for rotating stockpiles of antiviral drugs) . increased attention to basic science research such as studying immunomodulatory drugs five priority public health topics were identified for an inter-pandemic zoonotic influenza scenario as follows: inter-pandemic zoonotic influenza . antiviral susceptibility of circulating zoonotic viruses (e.g., h , h , h influenza viruses) . reassortment between zoonotic and human influenza viruses and the potential for inter sub-type spread of antiviral resistance and virulence modern tools for early detection and monitoring of disease the group focusing on surveillance tools concluded that the agreed topics of interest were equally applicable during both pandemic and inter-pandemic period: . identify modern technologies for early detection of influenza outbreaks as well as their application in surveillance at the human-animal interface . develop and evaluate innovative approaches for influenza surveillance and monitoring with other existing disease monitoring systems . studies to address challenges on data, clinical specimens, and viruses sharing with consideration for local, ethical, legal, and research perspectives . examine the timeliness and quality of data required for early detection from local to regional, national, and global levels role of modeling in public health decision making five priority public health topics were identified for an inter-pandemic seasonal influenza scenario as follows: . integration of genetic and epidemiological data to understand spatiotemporal spread to forecasts evolution for vaccine strain selection and to anticipate likely burden of disease . quantifying the relative contributions of different modes of transmission of human influenza and developing mechanistic modeling of transmission processes . research using data-capture technologies to characterize human contact and mobility patterns at local, regional, and global scales, and their correlation with transmission risk . integration of genetic, antigenic, and epidemiological analyses to optimize surveillance for newly emerging pathogens at the animal ⁄ human interface . identifying and quantifying human and environmental ecological, behavioral, and demographic determinants of the risk of cross-species transmission and pandemic emergence modern tools for strategic communication four priority public health topics were identified for an inter-pandemic seasonal influenza scenario as follows: . review of evidence and experience related to health crisis communication from fields to organize knowledge and support evidencebased practice in strategic communication . identify and develop tools to rapidly and accurately monitor knowledge, attitudes, and practices in different population groups and guide future communication efforts . identify and develop communication tools and approaches for cultural settings and communities to practice and promote appropriate risk reduction measures . understand the potential ethical, social, economic, and political communication in crisis and develop strategies to work within constraints while maximizing opportunities complemented by an analogous research roadmap for a pandemic influenza scenario, the research topic recommendations for an inter-pandemic period represent an important outcome of joint international efforts by who, academicians, and public health experts. implementation of the identified research priorities is expected to underpin public health decision-making at all levels with proven knowledge that will help to save large numbers of lives, reduce health costs, and economic loss and mitigate potential social disruption over a medium-tolong term period. the impacts of school resumption on the incidence of pandemic (h n ) in school students introduction school closure is one non-pharmaceutical intervention that is often suggested in pandemic preparedness plans, and it was widely implemented in pandemic (h n ) to reduce transmission amongst school students. however, from past epidemiological studies, the effect of school closure in reducing respiratory disease transmission was inconclusive. given this public health intervention causes major disruption to the education system and potentially raises childcare issues to working parents, evaluating its effect in the recent pandemic is necessary to improve future pandemic planning. in hong kong, since school closure was implemented early in the pandemic and closure was effectively continued with the commencement of summer holiday, the lack of incidence data in the absence of school closure makes it difficult to analyse its effect directly. this has prompted us to analyse the situation indirectly from the angle of school resumption after summer holiday. in hong kong, public health surveillance on pandemic (h n ) was effective from th april- th september : healthcare professionals were advised to report suspected cases of infection to centre for health protection, department of health, hksar, for further laboratorial confirmation. demographics of reported cases were subsequently recorded into a computerised system (the ''e-flu'' database). following institutional approval, a dataset of all confirmed cases diagnosed from may to september was obtained, which included the age, gender, confirmation date, and notification date of each report. all cases were classified into four defined socio-economic classes by age: pre-schoolers ( - ), school students ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) , adults ( - ), and retirees ( ‡ ). assuming cases had contracted infection on the earlier date between confirmation and notification, daily incidence in each age class was counted for epidemic curve construction. upon observing an unusual rise in the epidemic curve of school students when school season resumed in september, interrupted time series analysis (also known as intervention analysis) was applied to obtain the statistical significance of this observation. the analysis was applied to the incidence in school students from th july to th september , which covered the period from the start of summer holiday to the end of the th week of new school season. incidence in school students before summer holiday was deliberately dropped since not all schools were closed when the school closure policy was effective: all primary schools were closed proactively, whereas secondary schools were individually closed on a reactive basis if students were identified to have contracted the infection. school activity was formulated as a step function, which takes value from st september onwards (st = : t < st september, st = otherwise). a range of times series models were fitted by the maximum likelihood method and aic (akakine information criterion) was used to select the one with best fit. all computations were performed in sas version . . a total of ( ae %) pre-schoolers, ( ae %) school students, ( ae %) adults, and ( ae %) retirees were diagnosed with the infection in the surveillance period. the epidemic curves of preschoolers, school students, and adults showed a steady rise from th june onwards when local transmission of pandemic influenza was identified. an upsurge in the epidemic curve of school students can be observed in early september, coinciding with the commencement of the new school year (figure ) . interrupted time series analysis on the epidemic curve of school students returned an arima( , , ) model with equations: where st, yt, yt denote school activity, predicted and actual incidence in school students on day t, respectively. standard error and significance for model constants were: ae (se = ae , p = ae ), ae (se = ae , p = ae ). in short, the model can be interpreted as: the number of infected school students rose by ae per day on average during the entire study period, with a sharp increase by ae coming into effect when the new school year began. time series analysis showed, at the marginally significance level, that daily incidence in school students had a major increase when school season resumed. on the assumption that the increase was not caused by any change in health seeking behaviour, this result suggests that school resumption had facilitated transmission amongst school students. on the basis that school activity significantly increases incidence of pandemic influenza in school students, this study suggests closures of schools in the early phase of pandemic (h n ) and subsequently in the summer holiday probably had a major effect in mitigating transmission amongst school students. youngsters were postulated to be major vector for transmission in pandemic (h n ) . if this were true, it would be reasonable to expect the epidemic curves of the other age classes to show a similar upsurge when one is observed in school students. the absence of such observation in the epidemic curve of hong kong suggests school students were mostly disseminating the virus amongst themselves, but not to the other age groups. in november , gip convened the first global consultation on a public health research agenda for influenza to identify key research topics in each of the five main streams of public health research. during this meeting, the scientific working group (swg) of the sub-stream in ''modern tools for risk communication'' identified the requirements in research during influenza pandemics and inter-pandemic periods to provide clear, credible, and appropriate messages which meet the needs of diverse communities. the swg suggested that who hold a follow-up workshop to assess the use of modern tools related to strategic and risk communication and to further promote research in these areas. communication'' in may . one of the main objectives of the meeting was to generate a roadmap of public health research priorities related to strategic and risk communication. the research roadmap was developed by the group of invited experts on the basis of an analysis of available evidence and experience on public health and health crisis communication from relevant disciplines across global regions, as well as critical assessment of existing communication methods related to influenza control in different cultural, social, and ethnic settings. the workshop consisted of a series of presentations by experts in relation to experiences and lessons learned about communication during the sars, h n epidemic, and h n pandemic. there were also a series of group discussions on identifying research needs for pandemic and interpandemic periods in order to strengthen the research agenda. the expert group identified important public health needs in relation to communication during pandemics as well as in the inter-pandemic times. the main topics of discussion centered on communicating issues of influenza virus transmission, the use of influenza vaccines safety and efficacy, and use of antivirals as well as definition of the severity of the pandemic and the phase changes. in this context a number of research areas were identified, which can be broadly classified into four areas: understanding of communication principles and mechanisms is associated with an array of research topics covering different subject areas. one of the key questions here relates to the link between communication and ''behaviour change'' models and their application and appropriateness for different settings. the expert group defined the term ''behaviour change'' in this context as the modification of behaviour towards better health practices that are supported by clinical and scientific evidence for personal protection against infectious diseases and other adverse health risks. research topics related to these models require understanding and differentiating information and ''behaviour change'' needs of different audience segments, such as stakeholder mapping, target audience analysis, research into behaviour motivation, social norms, and the cultural, religious, social, legal, and political barriers and enablers of particular behaviors that are beneficial in influenza control. this research area also includes the analysis of media consumption among different audiences, role models, including ways to analyse how rumours and misinformation are spread, and ways to provide evidence-based information correctly. other important areas of investigation embrace methods to communicate uncertainty, learning how to build trust while communicating about a pandemic, and understanding what needs to be done before, during, and after a pandemic in order to create the best environment for influenza pandemic communication. critical key audiences identified for more intensive analysis were health workers, religious, public health, and societal (political and community) leaders. • investigation of the role of different communication channels and communication formats for different target audiences in a pandemic, particularly for groups that are ''hard-to-reach.'' • determining effects of perceptions related to pandemic influenza (severity, susceptibility, response efficacy, self efficacy, perceived social norms) on protective behaviours in different groups. • understanding audience in terms of their knowledge, preventive activities, and reasons why engaged ⁄ not engaged. • developing mechanisms to synergies between risk communication and behavior oriented approaches in the pandemic and inter-pandemic phases. • determining social, economic, cultural, and religious factors which support behaviours to limit spread and minimize impact in different settings. • identification of the key predictors ⁄ factors that influence people's behavior among different groups and populations vis-à -vis pandemic flu behaviors. • identification of elements that contribute to trust among populations and in different settings (country, public, professional, community), particularly where trust was previously compromised. • understanding psychology of different groups regarding their response to uncertainty, and finding the best way to communicate uncertainty. the research questions in this section relate to the planning, development, and evaluation of tools that can be quickly accessed and used in a pandemic situation. these may include communication materials and channels; the setting up of key stakeholder and champion communication networks; research protocols that are ready for rapid assessment during a pandemic or new communication tools. the use and understanding of terminology and language by both lay and professional groups and communities in planning for and ⁄ or reacting to a pandemic are important areas of research. acute examples, such as the naming of the viruses or the use of the word ''pandemic,'' illustrate this need well. the research focus of this area is to look at lessons learned from the a(h n ) pandemic and to document and evaluate case studies, both looking at best practices, challenges, and barriers that were experienced. different communication strategies need to be evaluated and models to be built not only in terms of reach, but also in terms of impact on thinking, emotional response, and behavioural modification. a key question was how to prepare communication for a pandemic and how can the pandemic communication contribute to longer term ''behavioural change.'' mathematical modelling on gauging outcomes of such ''behaviour change'' would provide strategic approaches in risk communication. this section aims to answer the question whether the modeling, mapping, and scenario planning are actually useful in the pandemic situation. the expert group agreed that the research on the above issues should use a variety of methods and engage a number of disciplines. this would include literature reviews, case studies, trials, ethnographic studies, modelling, surveys, network analysis, as well as any other useful methodology. in an inter-pandemic situation for actual behaviour under pandemic conditions. • study the synergies and develop priority research topics on strategic ⁄ risk communication for influenza under inter-pandemic situations that includes zoonotic and seasonal infections. the who public health research agenda for influenza initiated and facilitated a multi-disciplinary discussion for communication during pandemic and inter-pandemic situations. it focused on both theoretical and practical issues to improve practice and ensure the health of the public for influenza. critical areas for research were identified to build evidence in this field. it was recognized that there are extensive bodies of knowledge in a number of disciplines, , such as health promotion, behavioural psychology, social sciences, social and behaviour change communication, social marketing, and communication for development relating to these questions, and that these should be explored. outcomes of these research activities are expected to widen the evidence base which will support developing communication strategies for influenza by countries, institutions, and individuals and will, consequently, help to improve public health world-wide. abstract background: cytokine dysregulation contributes to the unusual severity of h n (reviewed in ). previously, we demonstrated that interferon regulatory factor (irf ) and p map kinase (p ) signaling pathways separately contribute to the induction of pro-inflammatory cytokines and chemokines in h n -infected cells. here we investigate the role of innate sensing receptors in the induction of these cytokines and chemokines in response to h n and seasonal h n infection. materials and methods: human macrophages derived from peripheral blood monocytes were infected with h n ( ⁄ ) or seasonal h n ( ⁄ ) viruses. the role of innate sensing receptors in cytokine and chemokine induction by h n virus was investigated using transient knock-down of these receptors with sirnas. the expression of innate sensing receptors in infected cells, and as a result of paracrine activation (by virus free supernatants of infected cells) of adjacent uninfected cells were also monitored by real-time pcr and ⁄ or western blotting. the involvement of janus kinase (jak) signaling pathways in these autocrine ⁄ paracrine cascades was investigated using a jak inhibitor. results: we previously showed that tnf-alpha, ifn-beta, and ifn-lambda are the key mediators directly induced by the h n virus in primary human macrophages with other cytokines and chemokines being induced as part of a secondary autocrine and paracrine cascade. here we demonstrated that retinoicacid-inducible gene i (rig-i) rather than toll-like receptor (tlr ) plays the predominant role in h n -induced cytokines and chemokines in human macrophages via the regulation of irf and nf-kb nuclear translocation. in addition to the effects on virus infected cells, paracrine interactions between macrophages and alveolar epithelial cells contributed to cytokine cascades via modulation of jak signaling and by the upregulation of sensing receptors. conclusions: h n directly induced tnf-alpha and ifnbeta mainly via rig-i signaling, and the subsequent activa-tion and nuclear translocation of irf and nf-kb in human macrophages. in addition to the effects on cytokine signaling, the innate immune sensing regulators themselves were also up-regulated by h n infection, much more so than by seasonal influenza infection, via jak signaling. the up-regulation of innate sensing receptors was not limited to the infected cells, but was also found in adjacent uninfected cells through paracrine feedback mechanisms. this may lead to broadened and amplified cytokine signals within the microenvironment of the infected lung. a more precise understanding of the signaling pathways triggered by h n virus leading to cytokine induction may provide novel options for the design of therapeutic strategies for severe human h n influenza and also for treating other causes of acute respiratory disease syndrome. human h n infection is associated with a mortality rate of more than %. the basis for the unusual severity of h n disease has not been fully explained. cytokine dysregulation has been suggested to contribute to the disease severity of h n (reviewed in ). however, signaling pathways involved in the cytokine induction by h n virus are not fully understood. previously, we demonstrated that irf and p map kinase (p ) are separate signaling pathways which contribute to the induction of pro-inflammatory cytokines and chemokines in h n -infected cells. rig-i and melanoma differentiation-associated gene (mda ) are important cytosolic sensors of nucleic acid of pathogens, while tlr and tlr also recognize nucleic acid species of pathogens, but they are localized at the endosomal membrane. rig-i was found to be responsible for the recognition of influenza a virus infection, and the transfection of vrnps induces ifn-beta expression. while many studies have shown the role of rig-i in the induction of ifn-beta by influenza virus infection, the majority of these studies used either immortalized cell lines or mouse embryonic fibroblasts. there is a lack of data on the role of these innate sensing receptors in highly pathogenic avian influenza h n infection in primary human cells in vitro, which are more physiologically relevant. furthermore, there is little data on the autocrine and paracrine up-regulation of these innate immune sensors following virus infection. human macrophages were obtained from peripheral blood monocytes by adhesion and differentiation in vitro for days in rpmi medium supplemented with % autologous plasma. the cells were infected with h n ( ⁄ ) or seasonal h n ( ⁄ ) viruses at a moi of ae . a cells were obtained from atcc and cultured in mem medium supplemented with % fcs and % penicillin and streptomycin. the role of innate sensing receptors in cytokine induction by h n and h n viruses was investigated using transient knock down of these receptors with sirnas in human macrophages as previously described using specific sirnas purchased from qiagen. immunofluorescence staining assay of irf and nf-jb was employed to detect the nuclear translocation of these transcription factors after h n infection. rabbit polyclonal antibodies against human irf and and nf-kb were obtained from santa cruz biotechnology. goat anti-rabbit igg antibody conjugated with alexa fluor was a product of molecular probes. for investigation of paracrine effects on rig-i and tlr expression, culture supernatants collected from mock, ⁄ or ⁄ infected human macrophages were used to treat uninfected cells. the supernatants were first passed through a filter with -kda cut-off. virus particles as well as molecules with a molecular weight higher than kda were retained and removed, while the filtrate was collected for treatment of uninfected cells. the expression of innate sensing receptors in infected cells and in adjacent uninfected cells following paracrine activation by virus free supernatants of infected cells was monitored by real-time pcr. the involvement of jak signaling pathways in these paracrine cascades was investigated using a jak inhibitor (calbiochem). we previously showed that tnf-alpha, ifn-beta, and ifnlambda are the key mediators directly induced by the h n virus in primary human macrophages with others being induced as part of a secondary autocrine and paracrine cascade. in this study, we demonstrate that knockdown of rig-i or tlr led to the reduction of ifn-beta and tnf-alpha in human macrophages by both ⁄ (h n ) and ⁄ (h n ) infection. as shown in figure a , ⁄ virus induced higher level of ifn-beta mrna expression than ⁄ infection. cells transfected with rig-i or tlr sirna significantly reduced the expression of ifn-beta after ⁄ infection, by % and %, respectively. rig-i silencing also significantly reduced the ifn-beta expression in ⁄ infected cells by %. in contrast, silencing of mda or tlr did not suppress the induction of ifn-beta by either ⁄ or ⁄ infection; in fact, there was a slight ( %) increase of ifn-beta in cells transfected with mda sirna. based on these results we conclude that while both rig-i and tlr contribute to h n -induced interferon-beta induction in human macrophages, rig-i plays the dominant role. in order to investigate the relationship between these innate sensing receptors and the activation of transcription factors irf and nf-jb, we next measured the nuclear translocation of irf and nf-jb in cells with rig-i or tlr silencing after h n infection. immunofluorescence staining assay on irf and nf-jb was performed and the number of cells with nuclear translocation was quantitated. the percentages of cells with nuclear translocation were plotted in figure b . we demonstrated that rig-i knockdown led to a significant reduction of irf nuclear translocation after ⁄ infection, whereas the nuclear translocation of nf-jb after ⁄ infection was significantly suppressed by rig-i or tlr silencing. these results suggest that the involvement of rig-i and tlr in the cytokine induction by ⁄ was via the regulation of irf and nf-jb nuclear translocation. since rig-i and tlr are important in influenza a virus-induced cytokine expression, we next explored the expression of these innate receptors in neighboring uninfected human macrophages by treating the uninfected macrophages with the filtered culture supernatants collected from mock, ⁄ , or ⁄ infected macrophages. as shown in figure a , ⁄ supernatant differentially induced the mrna expression of rig-i, mda , and tlr compared to ⁄ supernatant treated human macrophages. the induction of rig-i was higher than the induction of mda and tlr . in the presence of lm of jak inhibitor, the up-regulation of all three innate sensing receptors was significantly reduced showing their induction was dependent on jak activity. human lung epithelial a cells were also treated with the supernatants collected from macrophages infected with mock, ⁄ , or ⁄ virus. differential induction of rig-i, mda and tlr by ⁄ supernatant compared to ⁄ supernatant treated cells was observed (figure b) . ⁄ supernatant dramatically induced all three innate sensing receptors, while ⁄ supernatant only marginally induced rig-i and mda , but not tlr . as in human macrophages, treatment with lm of jak inhibitor caused a significant suppression of ⁄ supernatantinduced rig-i, mda , and tlr expression in a cells. these results, taken together with the direct effects on virus infected cells, suggest that paracrine interactions between macrophages and alveolar epithelial cells contributed to cytokine cascades via modulation of jak signaling and by the up-regulation of innate sensing receptors. h n directly induced ifn-beta ( figure ) and tnf-alpha (data not shown) mainly via rig-i signaling and the consequent activation and nuclear translocation of irf and nf-kb in human macrophages. these results were consistent with a previous study using beas- b cells showing the essential role of rig-i in ifn-beta reporter activity by h n influenza virus infection. while tlr also played a role in induction of ifn-beta and the activation of irf and nf-kb, it plays a less important role compared to rig-i. the reduction of irf and nf-kb activation was also confirmed with the study by le goffic showing differential regulation of irf and nf-kb by rig-i and nf-kb can also be regulated by tlr . in addition to the direct role of rig-i and tlr in sensing and signaling the presence of influenza virus, the innate immune sensing regulators were themselves also highly upregulated in both infected (data not shown) and adjacent uninfected cells by influenza virus infection. compared with seasonal h n virus, the h n viruses had a much more dramatic effect on inducing innate sensing receptors via jak signaling pathways activated by autocrine and paracrine mediators. the up-regulation of rig-i, mda , and tlr was markedly induced by virus free culture supernatants from h n -infected macrophages, while supernatant from ⁄ -infected cells induced the expression of these receptors only to a lesser degree. the soluble mediators in the virus infected cell supernatant caused paracrine upregulation of rig-i, mda , and tlr in uninfected macrophages as well as human lung epithelial cells. these effects may lead to broadened and amplified cytokine signals within the microenvironment of the infected lung. taken together these results provide, at least, part of the explanation on the hyper-induction of cytokines in h n infection. a more precise identification of the signaling pathways triggered by h n virus leading to cytokine induction may provide novel options for the design of therapeutic strategies for severe human h n influenza and also for treating other causes of acute respiratory disease syndrome. we generated mutants of y (h n ) and a ⁄ duck ⁄ hokkaido ⁄ vac generation and characterization of mutant viruses rgy sub (h n ), rgvac sub (h n ), and rgvac ins (h n ), which have a serial basic amino acid residues at their ha cleavage sites were generated by site-directedmutagenesis and reverse genetics. rgy sub (h n ) and rgvac ins (h n ) required trypsin to replicate in mdck cells, and showed similar levels of growth to their parental viruses (table ) . chickens intravenously inoculated with rgy sub (h n ) or rgvac ins (h n ) did not show any signs of disease. rgvac sub (h n ) replicated in mdck cells without exogenous trypsin, and one of the eight chickens inoculated with the virus showed slight depression at day post-infection. the h and h mutant viruses were serially passaged in the air sacs of chicks to assess their ability to acquire pathogenicity. plaque formation in mdck cells and pathogenicity in -day-old chicks and -week-old chickens are shown in table . rgy sub (h n ) replicated in mdck cells in the absence of trypsin and killed all of the chicks after six consecutive passages. two of the eight-four-weekold chickens inoculated intravenously with rgy sub-p (h n ) died within days. eventually, over % of the chickens intravenously infected with rgy sub-p (h n ) died by days post inoculation, and its pathogenicity was comparable to that of hpaivs. rgvac sub-p (h n ) was pathogenic to both chicks and -week-old chickens, and mortality increased after one more passage. rgvac ins-p (h n ) replicated in mdck cells in the absence of trypsin, killed all of the chicks, and caused % mortality among -week-old chickens. the lethal effect of rgvac ins-p (h n ) on chickens increased with one additional passage in the air sacs of chicks, as in the case of rgvac sub (h n ). to examine whether the pathogenicity of each virus via the natural route of infection correlated with that by intravenous infection or not, three -week-old chickens were challenged intranasally with the viruses at an eid of ae and observed for clinical signs until day post-infection (data not shown). all chickens inoculated with rgy sub-p (h n ) or its parental viruses survived without showing any clinical signs, and serum antibody responses were detected in the hi test. on the other hand, rgvac sub-p (h n ) and rgvac ins-p (h n ) were pathogenic as in the intravenous experiment, killing two of three chickens by day post-inoculation. one of three chickens were not infected with rgvac sub-p (h n ) or rgvac ins-p (h n ) via intranasal route (data not shown), indicating these p viruses had not been completely adapted to the host. to investigate the possibility of these p viruses to acquire further pathogenicity for chicken, rgvac sub-p (h n ) and rgvac ins-p (h n ) were obtained from the brain homogenates of the chickens that died on days post intranasal inoculation with the p viruses. although mortality rate of chickens inoculated with the p viruses was equal to that with p viruses, enhancement of pathogenicity was observed in intranasal inoculation study; all of the chickens inoculated with rgvac sub-p (h n ) were infected, and time to death was shortened to - days post inoculation in chickens with rgvac ins-p (h n ) (data not shown). to investigate whether tissue tropism of the viruses was involved in their pathogenicity, we determined viral titers in the tissue and blood samples from -week-old chickens intranasally inoculated with each virus on days post infection ( table ) . rgy (h n ) and rgvac (h n ) were scarcely recovered from the samples, and the mutant strains before passage showed broader tissue tropism than the parental viruses. none of the chickens inoculated with rgy sub-p (h n ) showed any signs of disease, and viruses were recovered from each of the samples except the brain and the blood. one chicken inoculated with rgvac sub-p (h n ) showed clinical signs such as depression, and viruses were recovered from virtually all of its organs and blood samples. two of three chickens inoculated with rgvac ins-p (h n ) showed disease signs, and one died days post inoculation. the viruses were recovered from almost all samples of the two chickens showing signs of disease. p viruses were efficiently replicated in systemic organs of the chickens as compared with p viruses. throughout the study, the viruses were recovered from the brains of all of the chickens showing clinical signs. here, we demonstrated that the h influenza virus acquired intravenous pathogenicity after a pair of di-basic amino acid residues was introduced into the cleavage site of the ha and passaged in chicks. rgy sub-p (h n ) killed % of chickens infected intravenously, and its pathogenicity was comparable to that of hpaivs (table ) . however, chickens intranasally inoculated with rgy sub-p (h n ) did not show any clinical signs of disease (data not shown). these results are consistent with a previous study in chickens that found some h influenza viruses did not show intranasal pathogenicity although their intravenous pathogenicity index was over ae , classified as hpaiv according to the definition by european union. ohuchi et al. reported that the insertion of additional basic amino acids into the h ha cleavage site resulted in intracellular proteolytic cleavage. other groups reported that h and h has tolerated amino acid mutations into their cleavage sites, and the viruses with the mutated has replicated in mdck and ⁄ or qt cells in the absence of trypsin. , the results in the present study is in agreement with these, namely, cleavage-based activation by a ubiquitous protease is not restricted to the h and h has. the intranasal pathogenicity of the h and h mutants were different (data not shown), although these viruses similarly replicated in mdck cells in the absence of trypsin and killed chickens by intravenous inoculation ( table ) . the viruses were recovered from the brain and the blood of some chickens infected with rgvac mutants (h n ), and morbidity was closely associated with viral titers in the brain (table ) . on the other hand, no viruses were recovered from the brain of chickens infected with rgy mutants (h n ), explaining why rgy sub-p (h n ) did not show intranasal pathogenicity. all the viruses passaged in the air sacs of chicks killed chicken embryos by hours post allantoic inoculation (data not shown). rgvac sub-p (h n ) and rgvac ins-p (h n ) were more pathogenic to chicken embryos than rgy sub-p (h n ); the allantoic fluid obtained from the embryonated eggs inoculated with the h viruses passaged in air sacs was turbid. it has been reported that infection of a highly pathogenic h virus were strictly confined to endotherial cells in chicken embryos or chickens. , therefore, it is suggested that endotheliotropism differed between the h and h viruses passaged in air sacs and affected their intranasal pathogenicity. taken together, it is assumed that rgvac sub-p (h n ) and rgvac ins-p (h n ) showed marked intranasal pathogenicity with high levels of viremia caused by replication in vascular endothelial cells, leading to invasion of the brain. in the intravenous experiment, rgy sub-p (h n ) easily reached systemic organs, including the brain hematogenously, replicated through the cleavage of ha by a ubiquitous protease, and then exerted its pathogenicity. further study including a pathological analysis is currently underway to test this hypothesis. for all hpai viruses of subtypes h and h known to date, the cleavage of ha occurs at the c-terminal r residue in the consensus multibasic motifs, such as r-x-k ⁄ r-r with r at position p and k-k ⁄ r-k ⁄ t-r with k at p , and leads to a systemic infection. early studies demonstrated that the ubiquitously expressed furin and pcs are activating proteases of hpai viruses. furin and pcs cleave the consensus multi-basic motif r-x-k ⁄ r ⁄ x-r with r at position p . however, replacement of p r by k and a nonbasic amino acid significantly suppresses the processing activities of furin and pcs. most of the type ii transmembrane serine protease identified so far recognize a single r at position p , but the newly isolated mspl and its transcript variant tmprss preferentially recognize paired basic residue, particularly r and k at position p , at the cleavage site. [ ] [ ] [ ] thus, mspl and tmprss can activate various bioactive polypeptides with multibasic residue motifs, including fusogenic viral envelope glycoproteins. the present study was designed to characterize the proteolytic processing of the hpai virus ha by mspl and tmprss in comparison with furin. hpai virus a ⁄ crow ⁄ kyoto ⁄ ⁄ (h n ) was isolated from embryonated eggs inoculated with tracheal homogenates from dead crows. then, the mutant ha sequence was constructed by changing r residue to k residue (n'-rkkr-c' to n'-kkkr-c') at the ha cleavage site by sitedirected mutagenic pcr as described. we used human cell line ecv , which expresses mspl and tmprss at levels below detection, and established the cells stably expressing mspl and tmprss , such as ecv -mspl and ecv -tmprss . to determine the cleavage specificities of mspl ⁄ tmprss and furin, peptides ( lg each) were incubated with ae mu mspl ⁄ tmprss for hour and furin for hours at °c, respectively. after incubation, the samples were separated by reverse-phasehigh-performance liquid chromatography (rp-hplc) with the use of a c column. the elution samples were then identified by amino acid sequence analysis and by maldi-tof-ms. we analyzed the cleavability of -residue synthetic peptides derived from ha cleavage sites of hpai strains, such as a ⁄ chick ⁄ penn ⁄ ⁄ (h n ) and a ⁄ fpv ⁄ rostock ⁄ (h n ), and low pathogenic strain a ⁄ aich ⁄ ⁄ (h n ). after incubation with human mspl or human furin, the digested samples were separated by rp-hplc, and peptide fragments were characterized by mass-spectrometry and protein sequencing. in contrast to the low cleavage efficiencies of the h ha peptide with a single r at the cleavage site ( figure a) , both the h ha peptide with the k-k-k-r motif ( figure b ) and the h ha peptide with the r-k-k-r motif ( figure c) were fully processed at the correct positions by mspl within hour. in the case of h ha peptide with multiple basic residues, mspl cleaved two carboxyl-terminal sides of r in the cleavage site sequence of n'-k-k-rfl-k-k-rfl-g-c', while furin cleaved only at a single site of r with r at position p , n'-k-k-r-k-k-rfl-g-c' in the presence of mm cacl . these cleavage site specificities of furin were consistent with that reported for the h ha peptide of hpai virus a ⁄ hong kong ⁄ ⁄ (h n ) with r-k-k-r motif. however, the h ha peptide with k at position p ( figure b) was hardly cleaved by furin under the same experimental conditions. tmprss showed similar results (data not shown). these findings suggest that mspl and tmprss cover diverse cleavage specificities, including non-susceptible specificity to furin. full length recombinant ha of hpai virus with kkkr cleavage motif was converted to mature ha subunits with membrane-fused giant cell formation in mspl or tmprss transfectant cells. in addition, this conversion was suppressed by bowman-birk trypsin inhibitor, a membrane non-permeable highmolecular mass inhibitor against mspl ⁄ tmprss . to test for the generation of infective virus, the conditioned media of -day culture of ecv -wt and ecv -mspl cells infected with wt and mutant hpai h n viruses were inoculated into newly prepared cells and cultured for hours. although spreading of wt virus infection with ha cleavage motif of r-k-k-r was detected from the conditioned medium of both ecv -wt and ecv -mspl cells, that of mutant virus with ha cleavage motif of k-k-k-r was only detected from the condition medium of ecv -mspl cells. these results strongly suggest that the expression of mspl, but not furin, potentiates multicycles of hpai virus with k-k-k-r ha cleavage motif. seasonal human influenza a virus has have consensus monobasic cleavage site sequence, n'-q ⁄ e-x-rfl-g-c', and all hpai virus has have two types of cleavage site sequences with multiple basic amino acids, n'-r-k ⁄ r-k ⁄ r ⁄ x-rfl-g-c' with r at position p in a large number of hpai viruses and n'-k-k ⁄ r-k ⁄ t-rfl-g-c' with k at position p in a small number of hpai viruses. figure shows furin efficiently cleaved synthetic hpai a ⁄ hong kong ⁄ ⁄ (h n ) ha cleavage site peptide with the r-k-k-r motif, but hardly cleaved the hpai virus a ⁄ chick ⁄ penn ⁄ ⁄ ha cleavage site peptide with the k-k-k-r motif. furthermore, cleavage of the full-length ha of hpai virus with r-k-k-r motif was detected, but cleavage of hpai virus ha with k-k-k-r motif was hardly detected in ecv -wt cells containing furin ( figure ). these substrate specificities of furin suggest that proteases other than furin and pc ⁄ play a role in the processing of has of hpai virus with k-k ⁄ r-k ⁄ t-r cleavage motif. mspl and tmprss show unique cleavage site specificities of the double basic residues at the cleavage site, and r or k at position p greatly enhanced the efficiency, which none of the other ttsps have shown similar substrate specificities so far. furthermore, infectious and multicycle viral replication along with ha processing was also noted in genetically modified mutant recombinant live hpai virus a ⁄ crow ⁄ kyoto ⁄ ⁄ (h n ) with k-k-k-r cleavage motif in ecv -mspl cells (figure ) . these results were supported by the data of two cleaved peptides by mspl in figure c . these findings suggest that mspl has diverse cleavage specificities and may cleave ha at least two sites, although multiplicity of the mutant hpai virus was observed under the conditions. these results also suggest that mspl and tmprss in the membrane might potently activate the ha membrane fusion activity of hpai viruses and promote their spread. highly pathogenic avian influenza viruses replicate in various organs in birds, and the ha processing proteases might be widely distributed in these organs. indeed, tmprss and mspl are ubiquitously expressed in almost all human organs tested and are highly expressed in lungs, leukocytes, pancreas, spleen, and placenta. , in addition, mspl and tmprss are strictly localized in the plasma membranes, suggesting that proteolytic activation of hpai virus ha occurs not only through the trans-golgi network by furin and pc ⁄ , but also on the cell surface by mspl and tmprss . the pb -f protein, which is translated from the + reading frame of the pb gene segment, has been linked to the pathogenesis of both primary viral and secondary bacterial infections in a mouse model. - a mitochondrial targeting sequence is located in the c-terminal portion of the pb -f open reading frame, and expression of full length pb -f has been associated with mitochondrial targeting and apoptosis in a monocyte dependent manner. , it has been theorized that enhanced virulence could result from mitochondrial disruption with subsequent cell death mediated by pb -f . , a suggested second function of the pb -f protein is that it enhances immunopathology by triggering the inflammatory response. , in earlier studies from our group, the pro-inflammatory phenotype was markedly upregulated when the pb -f from the pandemic strain was expressed, arguing that this protein may be an important virulence factor for highly pathogenic pandemic viruses. , in this report we analyze the pb -f protein's contribution to pathogenesis in a mouse model, examining both inflammation and cell death. pb -f proteins from a variety of epidemiologically important iav strains including all pandemic strains from the th century, a highly pathogenic avian influenza virus of the h n subtype, and representative seasonal strains were utilized to determine the relevance to pandemic disease. we demonstrate that macrophage mediated immunopathology, but not apoptosis, are relevant functions of pb -f proteins from past or potential pandemic influenza viruses. using the predicted amino acid sequences of the pb -f proteins from pr , a ⁄ brevig mission ⁄ ⁄ , ⁄ singapore ⁄ ⁄ , a ⁄ hong kong ⁄ ⁄ , a ⁄ wuhan ⁄ ⁄ , and a ⁄ vietnam ⁄ ⁄ , peptides from the c-terminal end were synthesized as described. an additional n-terminal peptide was synthesized from the pr sequence as a positive control (mgqeqdtpwilstghistqk) as described. a panel of viruses were reverse engineered as described , and included laboratory strain pr , a virus unable to express pb -f (dpb -f ⁄ pr ), or expressing the pb -f of the pandemic strain ( pb -f ⁄ pr ) or the truncated h n strain (beij pb -f ⁄ pr ). , in addition, : reassortants encoding pb gene segments from a *current address: department of immunology and microbiology, university of melbourne, melbourne, vic., australia. highly pathogenic avian influenza of the h n subtype (h n pb ⁄ pr ), or from a human h n strain (h n pb ⁄ pr ) were utilized along with their isogenic deletion mutants for pb -f (h n dpb -f ⁄ pr and h n dpb -f ⁄ pr ). cell lines and cell death assays raw . cells were grown under conditions as described. cells were infected with one multiplicity of infection (moi) of virus for - hours, or exposed to lm (final concentration) of peptides derived from the c-terminal portion of pb -f for hour. cells from the supernatant and monolayers were harvested, washed, and stained with annexin (apc) and propidium iodide (pi) (becton dickinson, san jose, ca, usa), then analysed for cell death as described. six-to eight week old female balb ⁄ cj mice (jackson laboratory, bar harbor, me, usa) were maintained in a biosafety level facility in the animal resource center and procedures approved by the animal care and use committee at sjcrh. infectious agents and peptides were diluted in sterile pbs and administered intranasally to anesthetized mice (n = - ) in a volume of ll ( ll per nare) and monitored for overt signs of illness and weight loss daily. following euthanasia by co inhalation, the trachea was exposed and cannulated with a gauge plastic catheter (bd insyte; becton dickinson, sandy, ut, usa). bronchoalveolar lavage fluid (balf) was collected, red blood cell depleted, and cellular content analyzed via flow cytometry as described. one way analysis of variance (anova) was used for multiple comparisons of cell death and cellularity of balf. a p-value of < ae was considered significant for these comparisons. graphpad prism version . for windows (graphpad software, san diego, ca, usa) was utilized for all statistical analyses. to assess the contribution of pb -f to inflammation, we utilized a panel of previously described reverse engineered viruses in the mouse infection model. , the effect of pb -f expression was observed clearly in the inflammatory infiltrate in response to infection in the lungs. deleting pb -f from pr or expression of the c-terminally truncated beij pb -f had a significantly reduced influx of macrophages ( figure a) . expression of the pb -f caused similar inflammatory effects as the pr virus. disruption of pb -f expression the virus containing the h n pb gene segment in a pr background also significantly decreased the inflammatory response compared to the virus maintaining the ability to express full length pb -f ( figure a) . however, no differences were seen that could be attributed to the h n derived pb -f . the lungs of mice infected with the panel of pb -f variant viruses were examined at hours. pathologic changes typical of pr viral infection were observed in all lungs. these typical findings included perivascular inflammation, airway necrosis, hemorrhage, and deposition of cellular debris (figure ). in the lungs of mice infected with pr or pb -f ⁄ pr , however, significantly more perivascular cuffing was noted, with a prominent increase in numbers of macrophages (figure a, c) . the overall number of inflammatory cells throughout the lungs, including both airways and alveoli, was quantitatively greater in these mice than in mice infected with dpb -f ⁄ pr or beij pb -f ⁄ pr ( figure b, d) . as the function and influence of pb -f protein on normal viral function is not currently understood, and given the abrogation of enhanced inflammation induced by the truncated pb -f beij ⁄ pr virus, we sought to elucidate whether the c-terminal domain of pb -f could alone induce this inflammatory response. mice were exposed to a panel of peptides and were euthanized hours later for collection of balf. significant influxes of macrophages into the balf were seen following exposure to c-terminal pb -f peptides derived from pr , the pandemic strains from (h n ), (h n ), and (h n ), and the h n virus compared to controls ( figure b) . similar effects were not seen with the peptide derived from a more recent h n strain, a ⁄ wuhan ⁄ ⁄ . when peptide exposed mice were followed for morbidity for days, peptides proven to induce a heightened inflammatory response correlated strongly with overt clinical signs of illness (data not shown). thus, the ability to cause lung inflammation appears to be a property of pb -f proteins of viruses containing pb gene segments reassorted directly from the avian reservoir. the pb -f protein may contribute to virulence by rendering the host cellular immune response ineffective through inducing apoptosis. we sought to determine whether this was an epidemiologically important function for combating the host immune response to infection by testing the ability of pb -f proteins from several different iav strains to cause cell death. we therefore infected raw . cells with the panel of recombinant viruses at an moi of for - hours. as has been demonstrated previously, , , pr virus induces significant cell death compared to uninfected controls ( figure c ). when raw . cells were infected with pr virus, necrotic death peaked hours after infection. viruses lacking the c-terminal portion of pb -f , including the dpb -f ⁄ pr and the beij pb -f ⁄ pr were unable to cause cell death ( figure c ). in addition, expression of the pb -f also did not cause significant increases in cell death over controls. expression of pb -f or deletion of pb -f in either an h n or h n pb gene segment background similarly did not alter the cell death phenotype. to examine additional strains for which we did not have isogenic virus pairs, we next exposed the balbcj mouse derived macrophage cell line raw . to the panel of pb -f peptides derived from pr , the pandemic strains from (h n ), (h n ) and (h n ), and the h n for hours. cell death in raw . cells was caused only by the peptides derived from the laboratory strain pr and the peptide derived from the pandemic strain ( figure d ). viability was not affected by exposure of raw . to peptides derived from other virus strains. we conclude from these data that the mechanism by which pb -f contributes to the pathogenicity of pandemic influenza is unlikely to be through its reported ability to cause cell death. these data presented here demonstrate that the lung inflammatory response is enhanced by the influenza a virus pb -f protein in a mouse model. this inflammatory response was characterized by increased cellular infiltration of macrophages into the interstitial and alveolar spaces of the lungs, as well as enhanced perivascular inflammation, airway necrosis, hemorrhage, and deposition of cellular debris. this augmentation was shown to be induced by pb -f proteins only from those strains contributing to the formation of all pandemic strains of the th century and from the currently circulating, highly virulent h n strains that constitute an imminent pandemic threat. the iav h n strains circulating in humans since around code for a truncated pb -f . these viruses may lack the cterminal residues responsible for the inflammatory effects demonstrated in this publication. additionally, recently circulating h n strains, in contrast to their pandemic forbear from , have lost the capacity to cause pb -f mediated inflammation through mutation of the c-terminus of this protein. in a novel h n iav emerged from an animal reservoir and caused a human pandemic. disease burden from this strain has been considered mild in contrast to the three pandemics of the th century. the reasons for this disparity in pathogenesis are unclear. an examination of the origins of the three th century pandemics shows that only the hemagglutinin (ha) and pb gene segments were reassorted directly from the avian reservoir in every case, suggesting gene products of one or both of these may be important. the ha surface glycoprotein provided the antigenic novelty required for the each virus to achieve pandemic status. however, the significance of inclusion of a novel pb gene segment in each of the th century pandemics is not yet understood. we show here that the pb -f of these pandemic strains contributes to virulence through induction of inflammatory responses. thus pb -f may serve as a marker of the pathogenicity of pandemic strains. since the h n strain codes a truncated pb -f of only predicted amino acids, the lack of pb -f mediated inflammation may account in part for its relatively lower virulence. , of the panel of pb -f proteins studied, only that from the laboratory strain pr was capable of rendering responding host-immune cells ineffective by induction of cell death. we therefore hypothesize that molecular signatures specific to induction of apoptosis may have been lost through genetic mutation of the pb -f gene throughout the evolution of the iavs. our findings suggest that this apoptotic function is unlikely to be important for the virulence of any of the known pandemics. rather, the inflammatory phenotype appears to be the dominant contribution of pb -f to pandemic disease. influenza virus-cytokine-protease cycles are principal mechanisms of multi-organ failure in severe influenza and therapeutic approaches introduction influenza a virus is the most common infectious pathogen in humans, causing significant morbidity and mortality, particularly in infants and the elderly. mof with severe edema is observed in the advanced stage of influenza pneumonia. however, the relationships amongst factors that induce vascular hyper-permeability in severe influenza remain unclear. it is reported that significant increases in levels of pro-inflammatory cytokine levels, such as tnf-a, il- , and il- b, affect host survival both positively and negatively. the inflammatory response affects cell adhesion, permeability, apoptosis, and mitochondrial reactive oxygen species, potentially resulting in vascular dysfunction and mof. in addition, iav infection up-regulates several cellular proteases including ectopic trypsin and mmp- . up-regulated ectopic trypsin mediates the post-translational proteolytic cleavage of viral envelope hemagglutinin (ha), which is crucial for viral entry and replication and the subsequent tissue damage in various organs. the aim of the this study was to define the pathogenic impact of cytokine storm in iav infection and the molecular mechanisms by which pro-inflammatory cytokines and proteases cause vascular dysfunction in animal model. weanling female mice aged weeks (c bl ⁄ crslc) were infected with iav ⁄ wsn ⁄ ( pfu) with and without treatment of pdtc ( . mg ⁄ kg), nac ( mg ⁄ kg), and ndga ( mg ⁄ kg). these inhibitors were administrated once daily for days after infection. the levels of cytokines in tissue homogenates were measured by elisa kits. the effect of inhibitors on viral replications was determined by real-time pcr. gelatin zymography and western blotting were conducted as reported previously. host cellular responses in the airway after iav infection figure shows schematic view of typical biological responses in the airway of mice after iav infection. an initial response before viral proliferation is significant increases in pro-inflammatory cytokine levels. immediately after cytokine inductions, there is a marked up-regulation of ectopic trypsin along with an increase in virus titer in the airway, lung, and brain. ectopic trypsin mediates the post-translational proteolytic cleavage of iav ha, which is crucial for viral entry and replication and the subsequent tissue damage in various organs. we also found that iav infection markedly induces mmp- and matrix degradation. just after the peak of viral proliferation, the innate and adaptive immune responses of protective immunity are induced for defense and recovery, or oppositely on rare occasions, mof with vascular hyper-permeability is started into the advanced stage of influenza. the levels of tnf-a and il- in the lungs were increased persistently for days after iav wsn infection, and that of il- b peaked at days - post-infection (figure a ). since these cytokine responses are associated with activation of nf-jb and ap- , we treated mice once daily for days with anti-oxidant inhibitors: pdtc and nac against nf-jb activation, and ndga against ap- activation. pdtc and ndga significantly suppressed the up-regulation of tnf-a and il- b (p < . ), and nac suppressed tnf-a (p < . ), and il- (p < . ) at day post-infection. gelatin zymography showed up-regulation of ectopic trypsin and mmp- in mice lung, brain, and heart during infection for days ( figure b ). trypsin and mmp- induction was inhibited by treatment with pdtc, nac, and ndga, probably via blockade of nf-jb and ap- binding in the promoter region of the genes. viral rna replication in various organs at day post-infection was suppressed by more than one order of magnitude by pdtc, nac, and ndga ( figure c ). suppression of viral multiplication and induction of cellular factors by pdtc, nac, and ndga, significantly improved the survival of mice at day post-infection, the late stage of infection ( figure d ). to elucidate the mechanisms underlying brain vascular dysfunction of influenza-associated encephalopathy, changes in the levels of tight-junction proteins, intracellular zonula occludens- (zo- ) and transmembrane occludin, and the matrix protein laminin, were analyzed by western blotting. marked reductions in the expression levels of tight-junction constituents were detected at day post-infection, which were partly rescued by pdtc, nac, or ndga (figure e ). no other tight-junction protein, claudin- or matrix fibronectin and type iv collagen, were affected. the present study reports several new observations: (i) proinflammatory cytokines, tnf-a, il- b, and il- , when up-regulated by iav infection, induce trypsin and mmp- expression in various organs in mice; (ii) inhibitors of nf-jb and ap- effectively suppress the up-regulation of proinflammatory cytokines, trypsin, and mmp- and improve survival rates of infected mice. based on these results, we propose the 'influenza virus-cytokine-protease cycle' hypothesis as one of the mechanisms of vascular dysfunction in mof with cytokine storm in severe influenza and influenza-associated encephalopathy. the significance of pro-inflammatory hyper-cytokinemia, or 'cytokine storm,' in the pathogenesis of iav infection remains unclear. on the positive effects, cytokines promote lymphocyte activation and infiltration at the sites of infection and exert direct antiviral effects. however, on the negative effects of excess cytokines, the hyper inflammatory process evoked by viral infection may become harmful through intracellular activation of nf-jb, ap- , and the janus kinase-signal transducers and activators of transcription signaling pathways. , [ ] [ ] [ ] the in vivo experiments presented here showed that nf-jb and ap- inhibitors markedly suppress the expression of cytokines, trypsin, mmp- , and viral replication, resulting in a significant increase in the survival of infected mice. furthermore, cytokines interact with mitochondria to increase the production of reactive oxygen species, resulting in the production ⁄ activation of vasodilatory mediators such as nitric oxide and bradykinin, and subsequent endothelial dysfunction and edema in various organs. the molecular mechanisms underlying tight-junction disruption in endothelial cells and vascular hyper-permeability following the 'cytokine storm' remain unclear. tnfa up-regulation alters the cellular redox state, reduces the expression of four complex i subunits by increasing mitochondrial o ) production and depleting atp synthesis, decreases oxygen consumption thereby resulting in mitochondrial damage, , and increases [ca + ] i atp depletion dissociates zo- from the actin cytoskeleton and thereby increases junctional permeability. endothelial dysfunction induced by 'influenza virus-cytokine-protease cycle' in the early stage of severe influenza may further affect various circulating factors, coagulation factors and complement systems, and vascular interacting cells, such as neutrophils, macrophages and lymphocytes. mof is the final outcome of metabolic and mitochondrial fuel disorder, immunosuppression, endocrine disorder, and tissue injury followed by endothelial dysfunction in many organs. another key pathway of acute lung injury in the highly pathogenic avian influenza virus h n and acute respiratory syndrome-corona virus infection reported recently involves oxidative stress and formation of oxidized phospholipids, which induce lung injury via toll-like receptor signaling pathway. in addition to these data, up-regulated trypsin and pro-inflammatory cytokines may also affect tissue destruction and immunosuppression in the late stage of iav infection. further studies are required on the role of the 'influenza virus-cytokine-protease cycle' in the pathogenesis of mof, particularly in the late stage of viral infection. though influenza a virus replication kinetics and host responses have been previously studied in umbilical vein endothelial cell or transformed endothelial cell lines, the tropism of influenza a virus including h n and pandemic h n pdm for primary human lung microvascular endothelial cell has not been well defined. in this study we employed primary human lung microvascular endothelial cells, which are more physiologically relevant for understanding pathogenesis of influenza in the lung as to obtain a better understanding of the links of endothelial cell infection to systematic virus dissemination and multiple organ involvement in severe human influenza. supernatants of cells infected at moi of two were collected for cytokine protein assays, and total rna was extracted for gene expression analysis using qpcr. we found that seasonal influenza h n and h n viruses initiated viral gene transcription and viral protein expres- sion, but did not produce infectious progeny, while the highly pathogenic avian influenza h n and the pandemic influenza h n pdm virus could replicate even with the absence of exogenous protease (figure ) . furthermore, when compared to seasonal h n and h n , the h n virus was a more potent inducer of cytokine and chemokine including ifn-b, mcp- , rantes, ip- (figure ) , and il- , in virus infected endothelial cells, whereas h n pdm induced intermediate levels of cytokine and chemokine. avian influenza h n and pandemic h n pdm virus (but not the seasonal h n and h n virus) can productively replicate in human lung microvascular endothelial cells. this is likely to be of relevant to pathogenesis and provides a possible explanation for the extra-pulmonary infection seen in animal infection models. this extra-pulmonary spread may support the previous speculation and anecdotal evidence that h n and h n pdm virus can infect the gastrointestinal tract through the virus dissemination from the infected respiratory tract as the first target cells for influenza infection. [ ] [ ] [ ] in addition, the release of proinflammatory cytokine and chemokine induced by influenza h n and h n pdm virus infection in lung microvascular endothelial cells may be important contributors to the pathogenesis of severe human influenza disease leading to endothelial cell dysfunction that contributes to severe pulmonary disease symptoms. during its replication, influenza virus utilizes the host cellular machinery for many aspects of its life cycle. characterization of such virus-host protein-protein interactions is a must to identify determinants of pathogenesis. the m ion channel protein plays a crucial role during the entry and late stages of the viral life cycle where its c-terminal domain, well conserved among influenza a viruses, is accessible to cellular machinery after fusion with endosomal membrane and during its trafficking along the secretory pathway prior to assembly and budding. the aim of the study is to identify cellular interactants of m that play important regulatory roles during influenza infection. to identify cellular partners of m we performed a genome-wide yeast-two-hybrid (y h) screening approach using the cytosolic domain of m as bait and a human placenta random primed cdna library as prey and tested more than million interactions. from the y h screening, an interesting interaction with the human annexin a (anxa ) protein, a member of annexin family proteins that binds to phospholipds in a ca + -dependent manner, was identified. co-immunopre-cipitation of myc-tagged anxa and viral m proteins coexpressed in hek t cells after transfection and infection confirmed the direct interaction between anxa and m . we further investigated whether this interaction had any functional significance with regards to influenza life cycle. using a rna interference strategy to silence the anxa gene in human lung epithelial a cells, we observed increased progeny virus titers either in a single or multiple viral growth kinetics study, suggesting a negative regulatory role for anax during viral infection (figure ). a novel interaction between m and anxa was identified. more functional studies are in progress to define precisely the potential negative regulatory role of this interaction during viral infection. a systematic dissection of the viral life cycle will be performed to identify the step(s) affected by the anxa cellular factor using specific assays such as real-time quantitative rt-pcr in a single or multiple viral growth kinetics study, cell transduction with ha-and m -pseudotyped lentiviral particles, virion attachment and internalization assay, immunofluorescence staining of np protein as a marker of viral ribonucleoproteins localization, viral polymerase activity measurement, and viral budding observation by electron microscopy. rna extraction was achieved by qiagen biorobot ez prior to respiratory multiplex pcr analysis. what remained of the extracted material of each specimen was stored by refrigeration at °c. electronic patient records were searched for parameters, such as c-reactive protein (crp), white cell count (wcc), length of admission in days, and patient co-morbidities. patients were divided into three groups according to clinical severity: mild, moderate, and severe. the 'mild' group comprised of those admitted for three days or fewer, or not admitted at all. the 'moderate' group comprised those who required admission to hospital for more than days as a result of swine flu, but who did not require admission to an intensive care unit (itu). the 'severe' group comprised those who had required itu admission. invitrogen ' · reaction mix': ae mm of each dntp + mm magnesium sulphate. primer ⁄ probe mix recipe applied biosystems fast real-time pcr system, 'respiratory multiplex' program. well content ll; thermocycler initial stage ae °c for minutes, then °c for minutes. subsequent cycles of ae °c for seconds followed by °c for seconds for cycles. sequence detection software version . (applied biosystems). of clinical isolates analyzed, all samples produced amplification of pdh material; produced amplification of both swine flu and pdh material. human male dna (lot no. at ng ⁄ l, applied biosystems) at concentration calculated at ae cells ⁄ ll was diluted from ) to ) , yielding mean average ct values of respectively ae , ae , ae , and ae . plotting log of cell number versus ct gave a y = mx + c line from which ct could be interpolated into cell numbers. for swine flu quantification, a sample of swine flu ct ae was diluted through ) to ) . it must be noted that due to variability in resultant swine flu ct values, repetitions at these dilutions were done using an rna carrier ( lg ⁄ l, qiagen; cat no. ) in place of rnasefree water. the ) concentration was positive in nine out of assays; this fraction was used in the calculation described by simmonds to obtain a copy number of targets per reaction by the equation copy value = )ln(f), where f is decimal fraction of failure rate. here, f = ⁄ = ae ; )ln ae = ae copies. a control curve was generated with ct values of ae , ae , ae , and ae giving copy values of , ae , ae , and ae , respectively. using excel (microsoft office, ), these control series were adapted into formulae to convert swine flu and pdh ct values into copy numbers of these elements per reaction. simple division derived a value for swine flu copy per pdh copy, but this was chosen to be expressed as swine flu copy number per human cells. this will be referred to as the 'c' value. forty-two patients had known clinical details; average age was ae , female to male ratio : , and average admission length of days. of the mild group (n = ), nine cases were not admitted to hospital. of the remainder, the mean average admission length was ae days. mean average c value for all samples was ae · , with a standard deviation of ae · ; geometric mean was ae , and median average was ae . log(mean average c value) is shown for each severity group and for identified risk factors in the 'mild' severity group (figures a, b respectively) . in each case variation was too great to yield statistical significance. figure shows the range of c values observed in the 'moderate' severity group. > - · ; < - · > - · ; < - · > - · ; < - · > - · ; < - · > - · ; < - · > - · ; < - · > - · ; < - · > - · ; < - · > - · ; < - · > · ; < · > · ; < · > · ; < · > · ; < · > · ; < · > · ; < · in a study by duchamp et al., no significant correlation was observed between viral ct value and presence or absence of cardiaorespiratory disease, myalgia, digestive symptoms, or upper or lower respiratory tract infection (although a trend was observed towards patients presenting with signs of upper respiratory tract infection). to our knowledge, no other study has used a dual pcr for analysis of respiratory virus concentrations, and no study has attempted to correlate biochemical markers with respiratory virus concentration. the data exhibited a spectrum of c values, from values < · ) to over · . the three severity group standard deviations all overlapped with each other, preventing statistical significance. analysis of co-morbidities showed a high mean average c value when asthma was present ( ae · ), but again this was associated with an excessive standard deviation. whereas the median average c value in the presence of asthma was higher than the overall average c value ( ae versus ae ), it was significantly lower than the median c value when no co-morbidity was documented ( ae ). there are multiple caveats that may be the cause of such variety of c values obtained. the duration between initial rna extraction and study pcr had a range of to days, with mean average delay of days. the degradation of viral rna is an important contributor to assay variance and failure; rna degradation in clinical samples has been studied. [ ] [ ] [ ] degradation of human dna in clinical samples may have occurred. several studies have chartered degradation of stored human dna. , with regards to sampling, the clinical collection of throat swabs is naturally variable according to the method of the collector. a small number of bronchoalveolar lavage samples were analyzed, yet did not amplify, presumably due to rna degradation. the upper respiratory tract may be only a physical stepping stone for the virus, and take no further role in pathogenesis of severe disease (although undoubtedly is crucial for transmission). interestingly, a ferret study of pathogenesis observed that swine flu yields from the upper respiratory tract were greater than those given by ordinary seasonal h n , with consequently increased shedding. the review by mansfield cites significant findings regarding influenza pathogenesis, including the predilection of h n strains for type ii pneumocyte cells and alveolar macrophages. it also highlights the limitation of knowledge through dearth of human autopsy studies; an exception is the recognition of haematophagocytic syndrome in severe cases. it is known that specific immunoglobulin is effective against establishment of infection in the upper respiratory tract, whereas specific cytotoxic t lymphocytes (ctls) are necessary for clearance of the virus from the lower respiratory tract. it is also suggestive that a gap of two whole days transpires between initial infection and instigation of a specific immune response. it is plausible that in the healthy individual, virus progression is confounded by efficient natural mucosal immunity, in part through good secretory immunoglobulin levels. airway inflammation associated with asthma exacerbation is known to increase both risk of respiratory viral infection and poorer outcome. it is unproven but likely that the local inflammatory processes give rise to increased virion burdens in the upper airways; however, the same effect is conceivable for epithelial cell turnover. there will likely be variance within each clinical category due to patient circumstances and clinicians' judgment of required admission. unfortunately, the duration of symptoms prior to swab collection was often omitted in the clinical notes. finally, stratification of patient group by receipt of antiviral treatment was not studied. no correlations were observed with c values and crp, wcc or admission length. trends were observed towards higher c values in 'mild' cases, but without statistical significance. the relative small study size, coupled with the intrinsic variability of the parameters studied, warrants larger, better controlled, prospective studies to elucidate clinical use of the c value for influenza illness prediction and management. in mid-april a novel variant of a(h n ) influenza virus began to spread rapidly throughout the world, causing the first pandemic of the st century. the majority of the cases associated with this new virus show to be mild, but severe and fatal cases have been reported. molecular markers associated with severity have already been identified, as is the case of the mutation d g. resistant viruses to antiviral drugs have also been identified, highlighting the importance of rapid determination of the antiviral drug profile. global a(h n ) genetic characterization, molecular evolution dynamics, antiviral susceptibility profiles, and inference of public health implications require nation and region wide systematic analysis of circulating virus. the objective of this ongoing research study was, primarily, to thoroughly characterize the genetic profile and evolution of the emergent influenza a(h n ) virus circulating in portugal and its phenotypic expression on antiviral drugs susceptibility. the cases considered in this study were obtained from the community and from two collaborating hospitals in lisbon -a reference hospital for adults (hospital de curry cabral) and a reference hospital for children (hospital dona estefânia). the cdc real-time pcr protocol, recommended by world health organization (who), was the method used to confirmed all influenza a(h n ) cases. from a total of a(h n ) positive cases diagnosed and confirmed, were selected for this study, taking in consideration that they should cover the period of epidemic activity in portugal and include cases from persons belonging to risk groups and cases associated with more severe clinical features. ninety-six a(h n ) strains were isolated in mdck-siat cells, from combined naso-oropharyngeal swabs. for the evaluation of the genetic profile of a(h n ) virus circulating in portugal, of the isolates were characterized by genetic analysis of the ha, na, and mp genes. the remaining five gene segments (pb , pb , pa, ns, and np) were also sequenced for six of this isolates. briefly, sequencing was performed according to the protocol developed by cdc and recommended by who, using bigdye terminator v. . technology. nucleotide sequences were determined in a dna automatic sequencer abi prism xl genetic analyzer. for each genomic segment, genetic analysis was performed with lasergene v. . software (dnastar inc, usa) using an average of - overlapping readings, including sense and antisense, for precise nucleotide and amino acid sequence determination. genetic mutation and phylogenetic analysis were performed by neighbor-joining method, using mega . software, against published sequences from the vaccine strain (a ⁄ california ⁄ ⁄ ) and from selected a(h n ) strains available on gisaid epiflu database. all mutations were identified with reference to the vaccine strain genome sequence. antiviral drug susceptibility profile of a(h n ) influenza virus circulating in portugal was evaluated both phenotypically and genotypically for nais and genotypically for amantadine. phenotypic evaluation to nais, oseltamivir and zanamivir, was performed for all isolates by ic determination through munana fluorescence assays. genotypic evaluation was performed by searching for mutations associated with resistance to nais in all na gene sequences. amantadine susceptibility profile was performed for all isolates by searching on m sequence for the molecular markers associated with resistance to this antiviral drug (l f ⁄ i; v a ⁄ d; a t; s n; g e). genetic characterisation of the ha subunit of ha reveals point mutations in different strains. all analysed strains present p s and i v mutations, which distinguish them from the vaccine strain ( figure a ). thirty-three of the sequenced strains group in the s t branch. this mutation is referred in the literature as being associated with the putative antigenic site ca. most of these strains ( ) further subgroup in the d e branch, this mutation being associated with one loop of the receptor-binding site. from the early to the late epidemic period, an increased circulation of virus carrying the mutation s t was observed. this is in agreement with the association between this mutation and an enhanced viral fitness that is described in the literature. additional mutations were also observed in a small number of virus, of which we highlight: regarding the genetic characterisation of na, the majority of strains analysed ( of ) presents the mutations n d and v i ( figure b) . as mutation s t in ha gene, these two na mutations are described in the literature as associated with enhanced viral fitness. the few strains not carrying these mutations have circulated in the beginning of the epidemic period. fifteen of the analysed strains further subgroup in y h branch. additionally, mutation i v was identified in two strains. for the remaining gene segments available for the six analysed strains, the observations include: (i) no previously described virulence markers in pb , pb -f , and ns were detected; (ii) pb -f protein is present in the truncated form of amino acids; (iii) the presence of mutations i v and l q in ns and v i in np; (iv) the described association of mutation i v in ns and v i in np genes with viral fitness. phenotypic evaluation of nais susceptibility revealed the existence of three minor and two major outliers to oseltamivir ( figure ). the two minor outliers exhibited a reduction of approximately twofold in the susceptibility to this antiviral drug, comparing to the baseline level, while the reduction exhibited by the two major outliers was of approximately three-and fourfold. regarding zanamivir, two minor outliers were identified with a reduction of approximately twofold in the susceptibility, compared to the baseline level. these two minor outliers (a ⁄ portugal ⁄ ⁄ and a ⁄ portugal ⁄ ⁄ ) correspond to the two major outliers identified for oseltamivir. genetic analysis revealed the presence of the mutation i v in the na sequence of these two strains. the contribution of this mutation for the profile of reduced susceptibility identified for both nais is not known, but a mutation in the same na position (i r) has been referred to as being associated with a reduction in nais susceptibility. full genome sequence analysis of these strains shows that both strains also present the v i mutation in pb gene. however, no association of this mutation with antiviral drug susceptibility is referred in the literature. concerning genetic evaluation of susceptibility to amantadine, all analysed strains present a serine in position , which is a molecular marker of resistance to m inhibitors. these preliminary results allow us to discuss several points. however, the additional data that is being obtained through this ongoing study will be essential for a more complete analysis. for example, more information is needed to determine if the mutations found alter the biology and the fitness of the virus or if there are associated with an increased prevalence of the virus. the majority of the mutations identified in ha subunit have been detected in a(h n ) strains distributed throughout the epidemic curve, not evidencing a specific evolutionary trend. this is in agreement with the genetic and antigenic homogeneity that has being described for a(h n ) virus. the occurrence of mutations in the position of the ha subunit of a(h n ) virus have been described. however, more studies are needed to clarify the outcome of these mutations, as for example in patients with severe complications. it could also be relevant to investigate the presence of single and mixed variants in viruses and in clinical specimens and the possibility of these mutations affecting the binding specificity. regarding the susceptibility of a(h n ) pandemic viruses to antiviral drugs, all analysed strains were found to be resistant to amantadine. this resistant profile was not unexpected since the mp gene from this new variant had originated in the eurasian swine lineage, which is characterised by being resistant to this antiviral drug. the majority of the a(h n ) strains analysed revealed to be susceptible to both nais, with only five strains exhibiting a profile of reduced susceptibility, three to oseltamivir and two to both nais. for these last two, the presence of the i v mutation in the na sequence could explain the reduction observed, but a more complete analysis is needed to confirm this. the french national pandemic plan includes an early containment phase followed by a limitation phase. the efficacy of such a plan depends on pre-existing surveillance and laboratory networks. the grog community surveillance network and the hospital lab networks organized by the two french nics carried out the virological monitor- the efficacy of such plan depends on pre-existing influenza surveillance and laboratory networks. in france, the community surveillance is carried through the grog surveillance network. in addition, surveillance is also carried out in hospitals by the renal network. this renal network is divided in two sub-networks: the so-called h -labs network, activated during the containment phase and the extended renal lab network activated in the limitation phase. the h -labs have bsl- facilities that can be used for diagnosis purposes. as part of the national influenza surveillance system led by the french institute for public health surveillance (invs), the grog community surveillance network and the lab networks linked to the two french nics carried out the virological monitoring of the a(h n ) pandemic from the early containment phase up until the end of the pandemic phase. during the containment phase, all suspected cases were hospitalized and declared to invs. each patient was tested on the same day by specific virological diagnosis. hospital admission was not mandatory during the limitation phase, (i) the clustered cases were monitored to study transmission chains, and (ii) the circulation of the virus in the community was monitored through grog swabs collected by practitioners. the nics organized the influenza surveillance to fulfill several objectives according to the epidemiological situation. first, rt-pcr tools (influenza a m gene rt-pcr and a(h n ) specific h and n genes rt-pcrs) were developped and distributed to the lab networks on the th of may . from the early phase, the nics and the h -lab network analyzed all the samples collected from hospitalized and community patients. during the early phase of the limitation phase, an increasing number of labs were performing the specific assays. when the pandemic wave started, all hospital labs could do the testing. results were centralised by nic and reported on a weekly basis. in addition, nics carried out the monitoring of antiviral resistance emergence (na pyrosequencing, specific h y rt-pcr, and phenotypic assays), and real-time surveillance of genetic changes involved in virus adaptation (pb ) virulence factors or antigenic variations (ha). this sequencing was carried out by the pf sequencing platform of the institut pasteur. the first imported a(h n ) influenza cases were observed from the th of april . a limited number of cases have been reported in may. local transmission could be detected end of may. clusters were observed in schools in june and in summer camps during summer. as opposed to the epidemiology of the a(h n ) virus in other european countries, no summer wave was observed in france. only a limited number of sporadic cases were reported up until october. early september, a significant number of cases presenting with influenza-like illness was reported (figure ). the virological investigation of these cases showed high prevalence of rhinovirus infection. this circulation of rhinovirus was a counfounding factor of the pandemic. the pandemic wave lasted weeks between mid-october and the end of december (week to week , figure ). the pandemic wave started week - in the ile-de-france area, and only week - in the rest of france. the peak was recorded week ( figure ). the impact of the pandemic was mainly observed in the - years group of age. overall, severe cases have been admitted to the hospital, and deaths have been recorded by the end of the pandemic wave. the major impact was observed in the - years group of age ( % of deaths recorded). amongst the severe cases and the deceased cases, % and % of cases had no risk factor, respectively. these specimens, were positives for h n , representing ae % of total influenza virus detections. only nine brisbane-like h n , brisbane-like h n , and eight b viruses have been detected in the same period of time. the weekly positive rate ranged from % to %. phylogenetic and antigenic analyses of the viruses collected during the pandemic wave did not show any emerging genetic or antigenic variants (figure a,b) . eight patients, all among cases presenting with severe illness, were infected by a virus harbouring the d g mutation in the ha. amongst the virus tested for antiviral susceptibility or screened for the h y mutation by or specific rt-pcr, only oseltamivir-resistant viruses related to the na h y mutation have been detected. one of these cases also had an i r mutation associated to a reduced sensitivity to zanamivir. all but one resistant virus were detected in treated immunocompromised patients. overall, eight patients presented a virus with the d g mutation in the ha. all these patients had a severe infection; one of these had also a h y mutation in the na asociated to oseltamivir resistance. the pandemic started by the end of april . although the first cases recorded were as early as the th of april, the epidemic wave associated with a widespread spread of the virus was only recorded in october. the french population did not have to face a summer wave, as observed in north america and in numerous european countries. , it is difficult to speculate the reasons for the lack of summer wave; the specimens collected were negative for influenza. moreover, during september, it was anticipated that school openings would be the trigger for the beginning of the pandemic wave. as a matter of fact, a significant increase of influenza-like syndromes were observed at that time, but the virological investigation carried out by the laboratories showed thta is was related to a very large epidemic of rhinovirus. the epidemic circulation of other respiratory viruses can be counfounding factors for the surveillance of the influenza epidemic clinical when the survellance is only based on collection of clinical information. the starting of the pandemic wave was heterogeneous in france. the ilede-france region (paris and its suburbean area), where the population is dense, experienced an early start as compared to the rest of france. however, once the pandemic started in the rest of the county, the epidemic curves were quite similar. the peak was reached at identical times, although it may have been delayed in some remote places in france. overall, we estimate that % of the french population consulted for an ili presentation. the impact was mainly observed in the - years groupe of. however, this age groupe represented only a limited number of severe cases and deaths. on the other hand, the - years groupe of age, where the prevalence was not high, was the age group where the majority of severe cases and deaths was recorded ( % and %, respectively). this data is consistent with the observational data reported by numerous other countries. according to the profile of hospitalized cases, a(h n ) was more aggressive than seasonal viruses. the number of admission to the hospital was ten-fold that observed during a normal influenza epidemic. even if the mortality was limited ( cases), the age distribution of the deceased patients was different as compared to seasonal influenza ( % mortality in < years of age). the lack of recordeable excess mortality has been interpreted to be the consequence of a very mild pandemic, milder than some seasonal epidemics. however, the median age of the fatal cases was much younger than those observed during the seasonal flu, leading to a mis-interpretation of the real impact of the pandemic. when the impact is measurered in loss of years of life, the impact of this pandemic is larger than seen with seasonal influenza, and is quite comparable to these of the two last pandemics. the pandemic preparadness of numerous countries, the develoment of new intensive care techniques and equipment, and the large use of antivirals have reduced the overall impact of this pandemic. these are new factors that should be taken into account when evaluating the real impact of the h n virus. the virological monitoring of the pandemic was achieved by the community-based and hospital-based sea- sonal influenza networks, reminding the importance of maintining such networks. the diagnosis of influenza in most of the patients was carried out by molecular techniques. it has been clearly stated from the beginning of the pandemic that near-patient tests were lacking of susceptibility and could not be used for patient management. the distribution of a set of validated and comprehensive techniques by the two nic was very helpfull for the monitoring of the pandemic and the patients. however, this diagnostic procedure change should not preclude maintaining virus isolation that is necessary for whole genome analysis, monitoring of antigenic changes, and phenotypic testing for antiviral testing. some of the mutants that have been recorded, including viruses with antiviral resistance phenotype or genotype, could be analysed from grown virus strains. it is striking that despite a large antiviral usage, only a limited number of isolates had mutations associated to resistance. however, the frequent isolation of such resistant virus was observed in immunocompromised patients that presented severe infections and long virus shedding. the impact of the pandemic is still under evaluation. sero-epidemiological analysis will be performed to asses for the real attack rate of the pandemic virus. as in other countries, it has been recorded that asymptomatic infections could be observed frequently. it is quite unlikely that the impact of the pandemic was reduced by the vaccination campaign, although this vaccination started on the th of november, just when the pandemic started in france. it is estimated that millions received the vaccination. pandemic strains of the influenza virus sporadically emerge, deviating from the regular endemic strains of seasonal influenza. in april , a novel pandemic influenza virus a ⁄ h n emerged, swiftly spreading across the world. immediately, domestic and international public health agencies were forced to develop containment and mitiga-tion strategies in response to the pandemic. however, the dynamics and transmission patterns of this novel virus are yet to be fully understood. simultaneously, seasonal strains of influenza (a ⁄ h n , a ⁄ h n , and b) continued to circulate in many nations. both pandemic and seasonal variants of influenza are responsible for significant morbidity and mortality. to characterize the dynamics of this disease and the variation within strains, a more detailed understanding of the patterns in viral shedding during natural infection is required. the majority of data on the patterns of viral shedding during influenza infection are a result of volunteer challenge studies. in these studies, volunteers are commonly screened for pre-existing immunity against the challenge strain and are of a certain demographic and age. information on the patterns of viral shedding in natural influenza infections, pandemic or seasonal, is limited but should provide greater generalizability. we describe the trends of viral shedding and clinical illness in community acquired cases of pandemic and seasonal strains of influenza. in , a community-based study was conducted to analyse the effectiveness of non-pharmaceutical interventions to prevent the spread of influenza in households. in , a similar community-based study was initiated to collect comparative data from individuals infected with seasonal and pandemic influenza. both studies were conducted with very similar protocols, involving households in total. the specimens and symptom data required for this study all arise from secondary infections ascertained in these two community-based studies. the recruitment process in both studies was essentially identical. index cases were first recruited from their healthcare provider if they presented with influenza-like illness (ili). this individual would be included in the follow-up if he ⁄ she tested positive for influenza virus infection by rapid antigen test (quickvue) and was the first person in his ⁄ her household that showed signs of ili in the previous weeks. follow-up consisted of three home visits that spanned approximately - days. at each home visit, nasal and throat swab (nts) specimens were collected from all household members, regardless of the presence or absence of symptoms. symptoms were recorded in daily symptom diaries provided for every household member, and digital thermometers were provided to record daily tympanic temperature. the symptoms recorded were fever ‡ ae °c, headache, myalgia, cough, sore throat, runny nose, and phlegm. influenza virus infection and subtype was identified by reverse transcription polymerase chain reaction (rt-pcr) on the nts specimens. viral shedding was quantified from the same specimens by rt-pcr to determine viral loads, as well as by quantitative viral dilutions to determine median tissue culture infectious dose (tcid ). the details concerning laboratory methods have been described in a previous study. all analyses in this study focus exclusively on secondary cases; these are household contacts of recruited index cases who acquire influenza virus infection following the initial home visit. index cases generally presented with a certain threshold of illness severity requiring medical attention, whereas infections among household contacts can vary from asymptomatic to severe representing naturally acquired influenza infections. these secondary cases must be negative for influenza for their first nts specimen, and subsequently tested positive. we analysed mean viral loads measured by rt-pcr and quantitative culture by plotting by day since acute respiratory illness (ari) onset according to strain of influenza (pandemic a ⁄ h n , seasonal a ⁄ h n , seasonal a ⁄ h n , and seasonal b). ari is the reference time point, because the day of infection is unknown and is defined as the presence of ‡ of the symptoms mentioned above. average symptom scores were also plotted according to ari onset and grouped into upper respiratory symptoms (sore throat and runny nose), lower respiratory symptoms (cough and phlegm), and systemic signs and symptoms (fever ‡ ae °c, headache, and myalgia). mean daily tympanic temperatures were also plotted since date of ari onset and according to strain of influenza virus. all analyses were conducted using r software (version . . ; r development core team). a total of households and individuals were followed-up in the two studies. of household con-tacts tested by rt-pcr, were found to be influenza positive. among these influenza infections, ( ae %) were asymptomatic (rt-pcr positive plus symptoms recorded), were subclinical (rt-pcr positive plus symptom recorded), and presented with an onset of ari during the follow-up period. from the cases with ari onset, seven pandemic a ⁄ h n , seasonal a ⁄ h n , seasonal a ⁄ h n , and seasonal b influenza virus infections were identified. the age distribution among secondary cases was observed to be largely comparable across the four strains of interest (table ). there were a lower proportion of males who acquired pandemic a ⁄ h n compared to the seasonal strains of the virus. cough was the most commonly reported symptoms during follow-up in cases of pandemic a ⁄ h n and seasonal b, whereas runny nose was most common in seasonal a ⁄ h n and a ⁄ h n cases. cumulatively, fever ( ‡ ae °c) was reported in approximately half ( %) of the secondary cases. patterns of viral shedding were analysed in a subset of influenza positive individuals who recorded an onset of ari in their symptoms diaries (figure ). household contacts that were asymptomatic, subclinical, or did not have an ari onset were excluded from the analysis. viral shedding in all three influenza a strains were recorded to occur on the day of ari onset or day post-ari onset. follow- ing the peak, measured levels of viral shedding declined steadily to undetectable levels over - days. the trend of viral shedding in influenza b infected individuals rose days before ari onset, fluctuated for around days before eventually resolving. the patterns of viral shedding over time measured by quantitative viral culture were generally similar to the patterns measured by rt-pcr. the patterns of symptoms and signs were comparable in the four strains of influenza included in this study, peaking on the day or day post-ari onset, and gradually declining over a period of - days. in all strains, systemic symptoms and signs were observed to resolved faster than upper and lower respiratory symptoms. the trend of tympanic temperature in each influenza strain was comparable to the respective symptom pattern. patterns of viral shedding observed in influenza a strain infections (pandemic a ⁄ h n , seasonal a ⁄ h n , and seasonal a ⁄ h n ) were broadly similar. the pattern differed from the observed pattern of viral shedding in seasonal influenza b infections. the majority of viral shedding in influenza a strains occurred at and near ari onset, whereas there were variable amounts of viral shedding preand post-ari onset for those with influenza b. the biological reason for this difference is yet to be clarified. these differences are consistently observed regardless of laboratory method used to quantify the viral loads. it was observed that viral shedding measured by tcid resolved more quickly than when measured by rt-pcr, suggesting that rt-pcr is more sensitive, but it could be detecting inactivated fragments of rna instead of active virus. the trends observed for the seasonal strains of influenza in this study were similar to those reported in literature. the patterns of symptoms and signs as well as tympanic temperature in the four different strains of interest in this study were found to be comparable. these patterns closely resemble the patterns of viral shedding observed in the influenza a virus strains, but not in the influenza b virus strain. the trends of viral shedding, symptom scores, and tympanic temperature for pandemic a ⁄ h n were similar to trends observed for seasonal a ⁄ h n and seasonal a ⁄ h n infections, suggesting that the dynamics of these viruses are largely the same. the clinical course of infection with pandemic a ⁄ h n influenza virus appeared to be similar to the seasonal b influenza virus, but the patterns of viral shedding over time diverges. in general, our results suggest that the dynamics of the pandemic a ⁄ h n virus were similar to the seasonal a ⁄ h n and a ⁄ h n viruses, and clinically similar to the seasonal b virus. this study faced sample size limitations; very few cases of pandemic a ⁄ h n were detected and the secondary attack rate in general was low, though a total of households were followed up. this lack of power led to the inability to analyse the differences between adult and children and other characteristics that could be correlated with amount of viral shedding. there are also biases that must be factored in during recruitment. the eligibility criteria of only healthy households could select for households with higher innate immunity. on the other hand, recruitment at health care providers can be biased towards index cases that had more severe illness that required medical attention. the strength of the study is the broad generalizability of the results due to the strict classification of secondary cases. the infections reported in this study were all community-based and should represent true natural infections. pandemic potency of the influenza virus is largely determined by its transmissibility. the first objective of this study was to model the transmission of influenza h n and h n viruses. at present, vaccination with laiv has been used as a widespread, effective public health measure for influenza prophylaxis. some unsubstantiated concerns have been raised about a potential possibility of reassortment of circulating influenza viruses with laiv viruses following vaccination with laiv. thus, another objective of this study was to assess the probability of pig-to-pig transmission of cold-adapted viruses and their potential reassortment with wt influenza strains. female albino guinea pigs weighing - g were inoculated intranasally with eid of virus without anaesthesia. transmission studies were then performed hours after inoculation. inoculated animals were housed at % relative humidity and °c in the same cage with noninfected guinea pigs or in cages placed m away from non-infected pigs. virus replication was determined by virus isolation in hen eggs and by pcr. sera were collected at and days post inoculation. seroconversions were assessed by routine hai test. genome composition of reassortants was monitored by rflp analysis. capacity of the viruses to grow at optimum, low, and elevated temperatures (ca ⁄ ts phenotype) was evaluated, and virus growth properties were observed following virus titration in hen eggs. when infected pigs were co-caged with non-infected (naïve) individuals, vn , indo ⁄ , a ⁄ california ⁄ ⁄ , and nibrg- were isolated in %, % ae %, and % of contact animals, respectively. serological confirmation of virus transmission was higher than virological data ( %, %, %, and %, respectively). in addition, it was shown that when pigs inoculated with a ⁄ california ⁄ ⁄ were co-caged with animals inoculated with nibrg- , they got infected with both viruses ( table ) . the ability of direct transmission of cold-adapted viruses was also investigated. data show that the a ⁄ ⁄ california ⁄ ⁄ laiv candidate was detected in the upper respiratory tract of ae % vaccinated pigs. the mdv was identified in % of infected animals. however, neither group of contact pigs, co-housed with the vaccinate pigs, had evidence of infection with cold-adapted viruses. in addition, none of the contact pigs had any evidence of seroconversion to the coldadapted viruses as determined by hai assay. it was also most interesting to note that pig-to-pig transmission of the highly transmittable nibrg- reassortant virus was not seen when pigs, vaccinated with mdv, were co-caged with animals infected with nibrg- virus (table ) . this strongly implies a form of interference or protection from transmissibility that was provided by the cold-adapted virus. the results show that nibrg- and indo ⁄ viruses were able to spread between cages over the m distance ( % and % naïve animals were successfully infected, respectively). a ⁄ california ⁄ ⁄ influenza and vn viruses did not transmit between infected and non-infected guinea pigs housed in separated cages (table ) . pigs with confirmed a ⁄ california ⁄ ⁄ virus replication were also infected with nibrg- virus if h n -and h n -infected animals were separated by a space. thus, influenza virus transmission from h n -to h n -infected pigs has been shown, but the reverse pattern did not occur. transmission of nibrg- or a ⁄ california ⁄ ⁄ viruses was not observed when contact pigs were first vaccinated with the mdv and housed at a m distance ( table ) . it was also shown that efficiency of transmission of nibrg- was much higher than of other studied h n viruses; it can be transmitted between naïve guinea pigs separated from infected animals at a distance of - m (data not shown). five reassortants were isolated from animals which were infected with a ⁄ california ⁄ ⁄ virus and co-caged with pigs inoculated with nibrg- . two reassortants possessed different combinations of pr , nibrg- , and a ⁄ california ⁄ ⁄ genes and demonstrated the non-ca ⁄ non-ts phenotype typical of wt viruses. unexpectedly, two other reassortants inherited ha gene from nibrg- , na gene from a ⁄ california ⁄ ⁄ , and other genes from pr became ca and ts. : non-ts reassortant inherited pa gene from pr and seven other genes from a ⁄ california ⁄ ⁄ , gained ca properties. in spite of aforesaid experimental data, we cannot exclude the theoretical possibility of simultaneous infection of human host with cold-adapted and wt influenza viruses. to better understand possible consequences of such a reassortment event, we co-infected guinea pigs with a mixture of mdv and nibrg- viruses. nasal washes were collected and cloned by limited dilutions in hen eggs in the presence or absence of immune serum to the mdv. cloning of nasal washes without antiserum led to isolation of over clones, which were all identical to the mdv (data not shown). when nasal washes were cloned in the presence of antiserum, only nine clones were isolated. genome composition analysis showed that all isolates were triple reassortants, which had inherited pb and na genes from mdv, pa gene from pr , and ha gene from nibrg- . the origin of the other gene segments (pb , np, m, ns) in the genome of guinea pig-derived reassortants varied. reassuringly, all reassortants generated in vivo had the phenotype typical of the mdv. the severity of influenza outbreaks is partly determined by efficient spreading of the causative virus strain between human hosts. however, little is known about mechanisms underlying influenza virus transmission in humans. guinea pigs have been shown to be a suitable model for influenza transmission studies. our in vivo study showed that influenza a viruses vary in their transmissibility. nib-rg- and indo ⁄ viruses were able to transmit to naïve animals caged distantly from infected animals. in contrast, cold-adapted viruses, the same as those used for licensed laivs, showed no signs of transmission from one guinea pig to another. our study also provided evidence of a lower level of transmissibility of the novel pandemic h n virus compared to the nibrg- and indo ⁄ h n strains evaluated. benefits of vaccination with laiv to aid in the control of influenza outbreaks are acknowledged by the who. in our study, the mdv inoculated into guinea pigs appeared to interfere with and even offer protection from transmission of the highly transmissible nibrg- virus. the ability to immunize with the laiv and subsequently block the spread of a homologous h n subtype and a heterologous h n subtype influenza virus between guinea pigs has been shown. interference between cold-adapted and wildtype influenza virus infection was the most likely explanation for the data observed in our study. the mdv inoculated into guinea pigs might in some way interfere with transmission of highly transmissible influenza viruses. it is believed by some that widespread use of laiv could increase the potential risk of reassortment of the vaccine strain with circulating influenza viruses immediately following vaccination. however, it was shown that any such potential reassortments would most likely lead to yet attenuated viruses. our in vivo studies have shown that introduction of mdv genes into the genome of nib-rg- virus led to the generation of triple reassortants inherited pb and na genes of mdv and ha gene of h n virus. all isolates possessed phenotypical markers associated with attenuation of mdv. our data suggest that even if a reassortment event of such rare occurrence between a laiv strain and a circulating virus were to occur, it would most likely lead to a reassortant that would retain highly attenuated phenotypic properties of the vaccine strain. our data strongly support the safety of laivs, especially those developed against highly transmissible h n and h n pandemic influenza viruses. this information builds upon databases that have clearly shown the low likelihood of transmitting an laiv, as well as the high likelihood of any field reassortment of laiv with a circulating influenza virus to retain important properties of the cold-adapted, temperature-sensitive vaccine master composition. very interestingly, we also present data that show the potential of a laiv to prevent the transmission of highly infectious influenza viruses, perhaps identifying a broader role for laiv in the overall scheme of influenza virus prophylactic use. background: schlieren imaging is a non-invasive, real-time airflow visualization technique that relies on differences in air temperatures (and the resulting changes in the refractive index) to allow exhaled human airflows to be seen clearly against the background of more-stationary, ambient air. recently, this technique, well-known to engineers, has been applied to better understand and characterize airflow behaviors associated with everyday, as well as healthcarerelated, human respiratory activities. materials and methods: as a surrogate marker for the behavior of airborne infectious agents, schlieren imaging was used to visualize the airflow patterns produced by adult human volunteers of different ages while coughing with and without the wearing of standard surgical and n masks. results: the cough plumes were generally similar in shape and range for all the adult volunteers used in this study. although both the surgical and n masks decelerated and blocked some of the forward momentum of the coughed airflows, much of the cough plume was redirected and escaped around the top, bottom, and side edges of the masks to merge with the volunteer's natural, verticallymoving thermal plume. conclusions: schlieren imaging is a safe technique for visualizing exhaled airflows from human volunteers without the need for potentially-irritant or toxic particle tracers. findings from these schlieren imaging experiments will assist the development of more effective aerosol infection control guidelines in healthcare premises where patients infected with potentially airborne infectious agents (e.g., influenza and tuberculosis) are present. these infectious agents may be transmitted to healthcare workers, other patients, and their visitors by way of exhaled airflows. with the recent influenza pandemic , and the ongoing concerns about human cases of avian influenza h n infections, there is now a very real concern about the potential for the aerosol transmission of respiratory pathogens. such concerns amongst staff and patients in healthcare environments have led to a greater emphasis on the understanding and control of infectious airflows. , previous visualization techniques have used potentially-toxic or irritant gas or particulate tracers with hazardous laser light sources that have precluded the use of human volunteers as subjects. instead, various forms of lung models that simulate human respiratory patterns with such particulate tracers have been used. , schlieren imaging is a technique familiar to engineers and offers a non-invasive (i.e., no tracer required) airflow visualization method that depends only on differences in the refractive index of the warmer, human-exhaled air and the cooler ambient air. the use of a simple incandescent or light-emitting diode (i.e., non-laser) light source is safe and allows human volunteers to be used as experimental subjects, where their exhaled airflows are then observed using a large, precise spherical or parabolic telescopic mirror and a camera, and are recorded for later analysis and presentation. [ ] [ ] [ ] the analysis of these patterns of 'real-life' human airflows will be useful in optimizing aerosol infection control guidelines, which aim to reduce the transmission of airborne infectious agents to other healthcare personnel, patients, or their visitors. the images and analysis presented here have all been obtained from the large m diameter parabolic mirror (figure ) situated at the gas dynamics laboratory of penn state (directed by gary s. settles). this large schlie-ren imaging system has been in use for over years to obtain high quality schlieren images for various engineering applications. it has only recently been applied to clinically-relevant imaging. the objective of this paper is to augment and expand upon the details of the methods and results presented in an earlier study using this same schlieren imaging system. the aim of this series of studies is to visualize and capture a series of airflow images produced by coughing from adult human volunteers of different ages ( - years old). these included males (three of years, one of years of age) and females (one of years, one of - years, and one of - years of age). each volunteer was tested with and without wearing either a standard surgical mask or n mask. more specifically, the aim was to visualize the extent and direction of leakage around the mask whilst each subject was coughing. penn state institutional approval for experiments involving human subjects was also obtained. each volunteer was asked to stand approximately m in front of the schlieren mirror, facing across the surface of the mirror on one side, and to cough several times as the real-time, color image and video footage was recorded by the operator (using a nikon d camera; nikon inc. melville, ny, usa). this process was repeated whilst each volunteer was wearing a standard surgical mask then an n mask (supplied by mÔ, st paul, mn, usa). some of the schlieren images obtained from some of these volunteers have been published previously: for a -year old male, the year-old female and a -year old male, and the - year-old female. this article completes this series of schlieren images obtained from these experiments by including the images recorded for the older, year-old man. generally, it was found that the shape of the cough plumes (shown in the figure as darker shadows emanating from the subject's mouth) produced by adult humans of different ages was relatively similar. cough plumes are roughly conical in shape and very turbulent, usually passing beyond the extent of the m mirror (figure a) . a previous detailed study of one of these images measured a maximum airflow velocity of m ⁄ second for an adult cough. similarly, the effects of wearing surgical and n masks can be generalized across different ages. wearing a surgical mask allows leakage of the coughed air from the sides, top, and bottom of the mask ( figure b ). there is also some leakage through the mask, as indicated by the darker patches of air directly in front of the mask ( figure b, c) . the useful effect of the mask appears to be a deceleration and redirection of this coughed (and potentially infectious) air into the natural, upward-rising human thermal plume, which captures it and carries it upwards where it is diluted and less likely to transmit infection to others. the effects of the n mask are similar (i.e., deceleration and redirection), yet due to its tighter (mask-fitted) face seal, more of the coughed air appears to penetrate the front of the mask ( figure c ). this penetrating air is, however, also decelerated sufficiently to allow the wearer's natural thermal plume to carry it upwards. , discussion from these series of schlieren images presented in this and other related studies, [ ] [ ] [ ] it is clear that schlieren imaging offers a safe, non-invasive, real-time technique to visualize human exhaled airflows for all age groups. it is apparent that, at least where airflow patterns are an acceptable surrogate marker for airborne transmission risks, there are beneficial effects of wearing either type of mask, even when the mask fit is relatively poor. this is often the case when n -style masks are purchased and used by the general public -in contrast to the situation with healthcare workers, who are often accurately fit-tested for this type of mask. the immediate significance of this can be seen when masks are bought by parents for their children. often, these will not be of pediatric size and the mask-fit will be loose. children are well-known to be major sources of infection in the community because of their relatively poor immunity to many types of infectious agents due to their young age and, therefore, limited past-exposure history. these images allow infection control teams to literally see how far and how fast potentially-infectious human exhaled airflows can travel from an individual. this may have significant implications for guidance on the wearing of masks for infected staff and patients, on ward bed-spacing, as well as for the types of masks to be used in different situations. the important practical potential lies in the non-intrusive visualization of airflows associated with human volunteers, to assist in heightening the awareness amongst healthcare workers of the risks and potential for the airborne transmission of infectious agents, as well as the development of more effective aerosol infection control policies. schlieren images can be analysed more quantitatively, e.g., with the 'schlieren-piv' technique, , though this additional quantitative data is probably more of research interest than being of immediate practical use to everyday hospital infection control teams. these are the subtypes that we have studied. clearly, the question arises as to whether the changes in antigenicity are coupled with changes in germicide susceptibility. we have employed a modified log-reduction method in a cell culture system employing mdck cells in serum-free ex-cellÔ medium supplemented with trypsin. microscopic examination of cpe was the marker for infectivity together with plaque assay. we confirmed antiviral potency by using specific subtype influenza identification subtype technology, quidel quickvue Ò influenza a + b test. the log inactivation and percent inactivation by bac after a second contact time for the h , h , and h pandemic strains are as follows: a ⁄ swine ⁄ iowa ⁄ ⁄ h n , ae log ⁄ ae %; a ⁄ swine ⁄ cal ⁄ h n , ae logs ⁄ ae %; a ⁄ j ⁄ ⁄ h n , logs ⁄ ae %; and a ⁄ hong kong ⁄ h n , ae logs ⁄ ae % (table ). comparable results of antiviral efficacy are obtained with the tcid and plaque assays against all subtypes studied. when performing the plaque assay the sensitivity of virus recovery was better in the vessel with a larger surface area and overall recovery was in agreement with the potency determined by tcid assay. in our plaque assay, we inoculated a ⁄ hong kong ⁄ ⁄ virus dilutions into two different vessels with hours adsorption time: -well plate and t- flask, ml inoculum per replicate. virus titers obtained were: ae · pfu ⁄ ml from -well plate and ae · pfu ⁄ ml from t- flask ( table ). the discrepancy on virus potency can possibly be explained as: the binding of virus to host cell occurs only when virus gets a chance to interact with the cell on the monolayer during adsorption time. the percentage of virus population in the inoculum that has the opportunity to bind to the cell mainly depends on the surface area where this interaction takes place. therefore, in our experiment the plaque assay in the t- flask gave higher virus recovery ae versus ae · pfu ⁄ ml. the increased virus recovery can translate into better sensitivity of the test system for disinfectant and antiviral agents. the potency of the virus used in this study was determined by tcid was · tcid ⁄ ml. rapid diagnostic testing for influenza (quickvue Ò influenza a + b test, quidel) for aj versus bac was studied. the presence of influenza viral nucleoprotein a determined by quickvue kit correlated % with the viral infection based on by cpe in viral culture. interestingly, the inactivation of viral nucleoprotein was able to be revealed with diagnostic kit in the dilutions of virus ⁄ bac reaction mixture, which possessed prominent cytotoxic effect for the host cells in viral culture system. this type of molecular testing method is useful for interpreting antiviral efficacy against a background of cytotoxicity. these experiments are intended for the sponsor to substantiate to us fda that their antiviral substances are safe and effective. the data shows that the three hemagglutinin subtypes were highly susceptible to the quaternary ammonium compound in the short term in vitro experiment. the appearance of novel subtypes in the future can be met with the assurance that disinfectant and ⁄ or antiseptic resistance will be unlikely. certainly, from the above data, although genetic reassortment of human and swine viruses may modulate influenza pathogenesis and limit existing vaccine benefit, it is not likely be a factor in control of viruses on environmental surfaces by benzalkonium-type disinfectant ⁄ cleaning agents in community or health care environments. table . comparison of viral titer obtained in different vessels using quantal tcid and plaque assay methods plaque assay tcid assay t- ( cm ) -well plate ( cm ) tcid ⁄ ml tcid ⁄ ml ae · pfu ⁄ ml ae · pfu ⁄ ml · ae · options for the control of influenza vii outbreak influenza in aged care facilities (acfs) is associated with an increased risk of poor health outcomes among residents, including death. in this paper we share our experience of managing an outbreak of viral respiratory infection in an acf very early in the influenza pandemic and also describe some of the emerging issues relating to crossreacting antibodies to the pandemic (h n ) influenza virus in the very elderly. the outbreak investigation was conducted as part of an urgent public health intervention initiated by the new south wales (nsw) department of health during the early stages of the first southern hemisphere wave of the pandemic. nose and throat swabs for nucleic acid testing (nat) plus acute and convalescent serum samples ( weeks apart) were collected from all the residents of an acf where an influenza-like illness (ili) outbreak occurred. the investigation revealed dual outbreaks of pandemic (h n ) influenza and rhinovirus infection. out of residents, three had laboratory confirmed influenza [two with pandemic (h n ) ], and had rhinovirus infection on nat. testing of acute sera collected from every subject found elevated ( ‡ : ) pandemic (h n ) hai antibody in % ( ⁄ ) subjects aged years or more (born before and median age years; geometric mean titre-gmt ae ) compared with none of the residents aged under years (born after and median age years; gmt ae , p = ae ). the acf was closed to visi-tors for days. the symptomatic residents received treatment-dose oseltamivir, and all other residents were given oseltamivir prophylaxis. more than one virus may be circulating in an acf with an ili outbreak at any one time in winter. a significant proportion of elderly residents had pre-existing cross reacting antibody to the pandemic (h n ) , which may explain the minimal clinical impact of pandemic (h n ) in this elderly population. influenza is one of the leading causes of infectious death in elderly people, principally due to co-morbidities and declining immune competence with age. it is the most important agent in outbreaks of respiratory illness. influenza in aged care facilities (acfs) is associated with an increased risk of poor health outcomes among residents, including death. the clinical presentation of influenza in residents of acfs can be subtle, with a blunted febrile response and a non-specific decline in mental and functional status. residents commonly have underlying diseases that can be exacerbated by influenza infection, and in addition, they are at higher risk of serious influenza-related complications than community dwelling elderly people. people aged over years are also at higher risk of influenza-related death, and more than % of annual influenza-related mortality is usually confined to this high risk group. in australia, influenza and pneumonia have sub-stantial health impacts; recorded as being the underlying causes of death for persons in . since the world health organization declared an influenza pandemic in june , australia has suffered one of the highest rates of confirmed infection during the first southern hemisphere wave. by late october there were reported deaths due to pandemic influenza in australia, and to date there have been about deaths reported worldwide. although disproportionately far fewer elderly people developed clinical influenza during the current pandemic than occurs with seasonal influenza, their case-fatality rate remained substantial. early in the pandemic (june ), we investigated a suspected pandemic influenza outbreak in a rural acf in the state of nsw, australia. the epidemiology (including virulence and clinical outcome in the elderly) of the pandemic (h n ) virus was mostly unknown at the time of investigation, and as time passed, this investigation provided clarity on some important issues of the influenza epidemiology in the elderly population. in this paper we share our experience of managing a dual outbreak of viral respiratory infections early in the pandemic, and also describe some of the emerging issues relating to the cross-reacting antibodies to pandemic influenza in the very elderly. the outbreak investigation was conducted as part of urgent public health intervention initiated by the nsw department of heath in conjunction with the local public health unit, the national centre for immunisation research and surveillance (ncirs), and the institute of clinical pathology and medical research (a who national influenza centre). to determine the extent and cause of the outbreak, a public health research doctor (gk) was dispatched from sydney over a weekend to assist with outbreak investigation and control. on june th , the greater southern public health unit surveillance officer (bd) received a report of a possible pandemic (h n ) outbreak in a local acf. on investigation, it was discovered that days earlier a year old female resident had become generally unwell, but without specific symptoms of influenza like illness (ili). soon after, nine of the co-residents (but no staff) had developed symptoms suggestive of influenza. one other resident had returned from a melbourne (victoria) hospital (where pandemic (h n ) was known to be circulating) the previous week after surgery, but did not have ili symptoms. on june th, the symptomatic residents had nasal swabs taken by the local doctor for influenza [including pandemic (h n ) ] nucleic acid testing (nat). there was rising concern due to reports of widespread pandemic (h n ) influenza in a local army camp just over the border in nearby victoria, where pandemic (h n ) influenza was known to be circulating widely. on june th, the year old lady proved nat positive for pandemic (h n ) , but none of the other samples were pandemic (h n ) nat positive. concern arose that there might be an outbreak of pandemic (h n ) in the facility, and that some of the swabs from other residents might be false negatives. between and june, after consent was obtained, directly or through next of kin in demented residents, all submitted to venipuncture for serology, successfully, and the other as yet un-swabbed residents were swabbed. basic demographic data were collected from every resident with clinical information on co-morbidities and current medication use. convalescent blood samples were collected after weeks on th july from of the residents. swabs were sent to icpmr where nat for influenza a [including pandemic (h n ) ] and b was performed. the acute and convalescent serum samples were tested later (in december ), using haemagglutination inhibition assay (hai) to detect pandemic (h n ) antibody. , interventions the acf was closed to visitors from th until th june. treatment of the positive case and the nine symptomatic residents, with twice daily oseltamivir, was begun on saturday june th, and all other residents were started on once daily oseltamivir prophylaxis. the facility manager and local general practitioner (gp) monitored patient health on a daily basis, and none had to stop oseltamivir due to adverse events. one resident with ili who was known to have moderately impaired renal function was given once daily rather than twice daily oseltamivir treatment. the age range of the residents was - years with a median of years. all residents had underlying medical conditions, e.g., chronic cardiac and respiratory diseases ( table ) testing of acute sera collected from every subject found elevated ( ‡ : ) cross-reacting hai antibody to the pandemic (h n ) in % ( ⁄ ) of subjects aged years or more (born before and median age years; geometric mean titre-gmt ae ). however, the hai titre was consistently < : and significantly lower (gmt ae , p = ae ) in the residents aged under years (range - years, median years) (figure ). the index case (nat positive) did not show a significant raise in hai level in convalescence (going from to ). the pandemic (h n ) case that was determined by serology was pandemic (h n ) nat negative. to our surprise, seven of the other asymptomatic residents had rhinovirus detected on extended nat (reported on june th), despite being asymptomatic at time of swabbing and remaining so. the original nine influenza nat negative samples were then tested and three of these were also nat positive for rhinovirus; in total, ten proved nat positive for rhinovirus ( ae %). the serologically confirmed pandemic (h n ) case was also positive for rhinovirus infection. of interest was that only one resident had a documented fever. this investigation illustrates some of the difficulties in managing and investigating possible influenza outbreaks in real time in the context of an influenza pandemic. finding a nat positive case of pandemic (h n ) influenza among many other symptomatic cases raised the possibility (although not the probability) that pandemic (h n ) was the cause of the outbreak. rhinovirus infection, however, was confirmed by nat in ten residents. this outbreak illustrates that more than one virus (in this case and perhaps ) may be circulating in an acf at any one time in winter. in ili outbreaks in acfs, broad laboratory testing is recommended; nat is the most sensitive method of detecting influenza or other viruses in respiratory tract samples. studies have found that the pandemic (h n ) haemagglutinin (ha) gene is more closely related phylogenetically to the h n virus and classical swine influenza a ⁄ h n viruses than more recent seasonal human influenza a ⁄ h n viruses. it is antigenically similar to the h n pandemic virus in terms of the immunodominant antibody response to haemagglutinin. [ ] [ ] [ ] it is likely that individuals alive during the emergence and initial persistence of the pandemic virus would have higher levels of cross-reacting hai antibodies to the pandemic (h n ) , which would contribute towards better clinical protection. in our investigation, % of the residents born before (aged years or above in ) had pre-existing cross-reacting hai antibody to the pandemic (h n ) . in elderly populations, severe illness may be associated with organisms typically considered to be mild, such as rhinovirus. however, studies have shown that nursing home residents may be susceptible to outbreaks of rhinovirus that may cause mild to severe respiratory illness, particularly in those with a history of lung disease. one rhinovirus outbreak in a nursing home in the usa caused fatalities. another outbreak showed residents with underlying lung disease are more likely to have longer infection, require antibiotics, develop bronchospasm, and have difficulty breathing; two residents with underlying lung disease required emergency treatment and one died. a previous influenza outbreak in a nsw aged care facility in caused significant mortality and morbidity. that outbreak resulted in hospital admissions and six deaths. in our investigation we have found that % of the residents had chronic lung disease and % had chronic cardiac conditions both considered as high risk for severe complications of both rhinovirus and influenza infection. however, there were no hospitalisations or deaths in our outbreak investigation. indeed only one resident developed fever, indicating that non-specific signs of illness (such as in our index case) may be the only, or early, indication of an ili. our own experience with managing other ili outbreaks has also taught us that staff of acfs may not be vigilant enough to detect fevers. in this outbreak, the nursing home staff, local gp, public health unit and the outbreak investigation team and supporting laboratory staff acted quickly and in a coordinated way. pre-existing cross-reacting antibody in the very elderly (aged ‡ years) probably helped to limit the spread of the pandemic virus (compared to the circulation of rhinovirus) within the acf. exposure to the pandemic (or a close variant occurring before ) appears to be responsible for a high hai titre in the very elderly, which contributed towards better clinical protection. however, wider testing early on would have alerted us more quickly to the main cause of the outbreak. treatment and prophylactic use of oseltamivir may also have contributed to halting the spread of pandemic (h n ) and also to symptom relief. pandemic (h n ) influenza virus (ah pdm) has spread worldwide since march . in a paper of ah pdm, % of infected individuals have experienced gastrointestinal symptoms such as diarrhea and vomiting, which is higher than that of seasonal influenza. however, little is known whether viable virus shed from stool and replication of viruses are ongoing in the gastrointestinal tract. , viral load and isolation of ah pdm in cell culture in stool samples has been reported. stool specimens were collected from patients suspected to have pandemic (h n ) infection from november through may . virus isolation was conducted in cell culture by using madin-darby canine kidney (mdck) cells and taqman based rt-pcr from % (w ⁄ v) stool suspension in phosphate-buffered saline. taqman based rt-pcr was conducted by using primers, probes, and positive controls provided by niid (national institute of infectious diseases of japan). to confirm presence of ah pdm viral rna, lamp (loop-mediated isothermal amplification) was used as supplemental testing. of patients, one child (case ) submitted one nasal swab and four stool samples, another one nasal swab and two stool samples, and the other one stool sample. informed consent was obtained. strand specific rt-nested pcr was performed for only case by using only one primer at the rt reaction and also assayed neu aca - gal and neu aca - gal binding specificity about isolated strain derived from nasal swab and stool. receptor binding specificity was performed using a solid-phase binding assay with the sialylglycopolymers (poly a-l-glutamic acid backbones containing neu aca - galb - glcnacb-pap or neu aca - galb - glcnacb-pap bond as described. ) nucleotide sequences of the ha gene of ah pdm viruses isolated from stool sample and nasal swab were analysed. in order to exclude the possibility of contamination, the stool samples and nasal swabs were subjected to virus isolation separately. after getting the results on the nucleotide sequence, we also confirmed no strain harboring identical sequence was isolated in our laboratory before and after the day of sample collection. ah pdm viral rna was detected in nine ( %) of the subjects from stool samples. among nine subjects, one case (case no. ) was positive for viral isolation. case , a healthy -year-old girl, experienced fever and abdominal pain, and the others had gastrointestinal symptoms without upper respiratory symptoms. in case , influenza a virus was diagnosed by rapid antigen test on the day of symptom onset. viable ah pdm virus was isolated from the stool sample and nasal swab on the second day from onset using mdck cells (table ). viral load decreased gradually after symptom onset. however, viral shedding was still present days after symptom onset. positive stranded rna was detected days after symptom onset from the stool specimen ( figure ). above two ah pdm strains (isolated from nasal swab and stool specimen) bound exclusively to human type receptor, neu aca - gal. sequence analysis demonstrated that isolated virus from stool samples was identical with that from nasal swabs in comparison of ha gene ( bp). ah pdm influenza virus was isolated from the stool and nasal swab samples in the same patient simultaneously by using mdck cells. our results suggests the detection of viral rna and viable ah pdm influenza virus from stool samples may serve as a potential mode of transmission and has important implications in understanding the context of ah pdm influenza virus. strategies to prevent transmission of influenza include use of respirators. ffp and n respirators are certified to fil-ter at least % of particles ( ae lm in diameter), and many guidelines have recommended that healthcare workers wear respirators in certain healthcare settings to protect against infection from patients with pandemic influenza. [ ] [ ] [ ] we have developed a proprietary acid-polymer formulation to coat a standard ffp respirator with an antiviral layer. we aimed to test this coated respirator for antiviral efficacy against a range of influenza viruses. a series of tests compared the antiviral efficacy of coated and uncoated respirators in conditions designed to simulate real-life exposure to influenza by varying the route of inoculation, contact time, temperature, humidity, moisture, and contaminating substances. we also investigated whether infectious viruses could be transferred from contaminated respirator surfaces to gloves. we tested human, swine, and avian influenza viruses, including influenza a and b viruses. influenza a subtypes were the a ⁄ h n pandemic strain, seasonal h n , h n , h n , h n , and h n . in each test, suspensions of influenza viruses were prepared to - log tcid ⁄ ml in mem. in some tests, organic contaminants (yeast, bsa, and mucin) were added. one set of respirators was maintained at °c and % relative humidity for hours before the viral challenge, and repeatedly sprayed with he-pes buffer to simulate respiratory secretions. for each test, three coated (glaxosmithkline actiprotect) and three uncoated (sperian willson easy fit) ffp respirator samples were inoculated with ae ml of a viral suspension, which was applied with a pipette, sprayed, or aerosolised to create airborne droplets. after minute at room temperature (on a shaker), the respirator samples were assayed for the presence of infectious viruses using standard methods. in one test, after a minute contact time of the respirator with the virus, nitrile gloves were applied with light pressure to the outer surface of inoculated respirator samples and then assayed after minute. samples were put into test medium (mem, supplemented with antibiotics [penicillin, gentamycin, or streptomycin] and amphotericin b or l-glutamine). the supernatants were vortexed, extracted, and used to prepare serial -fold dilutions in mem. each dilution was used to inoculate four wells of rmk cells in a multi-well plate, and these cultures were incubated and scored over days for cytopathic effects, cytotoxicity, and viability. (some tests substituted mdck cells; others used inoculated embryonated chick eggs.) all tests included negative cell controls, cytotoxicity controls, and neutralisation controls. the spearman-karber formula was used to calculate viral loads as tcid or eid . antiviral efficacy was calculated from the difference between the geometric mean loads of influenza virus on the coated and uncoated respirators after minute of exposure. the viral loads applied to respirators in these experiments ranged from ae to ae log tcid , and were therefore high in comparison with respiratory secretions from infected patients at the peak of influenza symptoms (range - log tcid ). tables - show that the average viral loads detected on uncoated ffp respirator samples remained high in all conditions tested, ranging from ae to ae log tcid (or ae - ae log eid ). in contrast, the average viral load on coated respirators after minute of exposure ranged from below the limits of detection to £ ae log tcid ( ae log eid ). therefore, the relative antiviral efficacy of the coating ranged from ‡ ae to ae log . table shows that the relative antiviral efficacy of the coated mask remained high in simulated-use conditions such as organic contaminants and repeated saturation at high temperature and humidity. in the experiment to test transfer of viruses from respirators, the gloves applied to regular uncoated inoculated respirators had a viral load of ae log eid (table ) . by contrast, no viruses were detected on either the coated respirators or the gloves applied to them. the relative reduction in contamination was therefore ‡ ae log . ‡ ae log viral load with organic contaminants* ae ae ae log viral load after heat, moisture, and simulated secretions** ae ae ae log viral load transferred to glove** ae £ ae ‡ ae log eid *influenza subtype was a ⁄ h n , and strain was vnh n -pr ⁄ cdc-rg. **influenza subtype was a ⁄ h n , and the strain was hong kong ⁄ ⁄ . results are mean log tcid , unless specified otherwise. results are mean log tcid , unless specified otherwise, based on an infectivity assay in triplicate. limits of detection varied. * pandemic strains. **results are mean log eid , based on a haemagglutinin assay in duplicate. options for the control of influenza vii ª blackwell publishing ltd, influenza and other respiratory viruses, (suppl. ), - strategies to prevent transmission of influenza include use of respirators, and many guidelines have recommended that healthcare workers wear respirators in certain healthcare settings for protection against pandemic influenza. - ffp respirators are certified in europe to filter at least % of nacl particles ( ae lm in diameter), and ffp and ffp respirators must filter at least % and % of these particles, respectively. influenza a viruses are typically ae lm, and can be carried in aerosolised droplets smaller than lm in diameter, which can disperse widely, remain airborne for hours, and be inhaled deeply into the respiratory tract. we have developed an acid-polymer formulation to coat the outer layer of a standard ffp respirator, in order to provide antiviral activity on the outer surface. we compared this coated respirator against standard ffp , ffp , and ffp respirators for filtration of aerosolised influenza viruses. the aim was to simulate protection against infectious viruses in droplets released when infected people cough and sneeze, and during aerosol-generating procedures in healthcare settings. the first assay compared three samples of coated ffp respirators (glaxosmithkline actiprotect) with three ffp controls (sperian willson easy fit). for each test, suspensions of influenza a (h n ) at ae log tcid ⁄ ml in ae · minimum essential medium (mem) were aerosolised with a nebulizer. the airborne droplets were introduced into a sterile chamber upstream of a respirator sample for minutes, at a flow rate of ae l ⁄ minute. constant airflow was maintained for another minutes after exposure to the virus. then the collection dish in the downstream sieve sampler (anderson) was assayed for infectious viruses using standard techniques. briefly, serial dilutions of the collection medium (mem with % fbs, % gelatine, and % hepes, supplemented with antibiotics and amphotericin b) in mem + trypsin were used to inoculate madin-darby canine kidney epithelial (mdck) cells in quadruplicate in a multi-well plate. these cultures were then incubated and scored over - days for cytopathic effects, cytotoxicity, and viability. negative cell controls and cytotoxicity and neutralisation controls were also performed. the spearman-karber formula was used to calculate tcid . the second assay compared five samples of coated respirators with five ffp controls ( m ) and five ffp controls ( m ). a suspension of influenza a (h n ), at ae tcid ⁄ ml, was nebulized for minute and seconds into the aerosol chamber, at a flow rate of ae l ⁄ minute, followed by constant airflow for minutes after exposure to the virus. then the collection medium in the downstream chamber (as before, with % nahco ) was assayed as described above. initial viral loads in the first and second assays were ae and ae log tcid , respectively, and were therefore high in comparison with respiratory secretions from infected patients at the peak of their influenza symptoms (range - log t-cid ). table shows that the average viral load that passed through the uncoated ffp respirators in the first assay was ae log tcid . the average viral load that passed through the coated respirators was ae log tcid . therefore, for active filtration of viruses, the relative efficacy of the respirator with antiviral coating was ae log greater than the uncoated respirator. for surface inactivation, the relative antiviral efficacy of the coated respirator was ae log . in the second study, table shows that the average viral load that passed through the uncoated ffp respirators was ae log tcid . in contrast, ae log tcid passed through the coated ffp respirators. by comparison with the viral load when no respirator was present ( ae log tcid ), the ffp respirators reduced the viral load by ae log , and the coated ffp by ae log . therefore, for active filtration of viruses, the respirators with antiviral coating reduced the viral load by ae log more than the ffp respirators. in this second study, the average viral load that passed through the uncoated ffp respirators was also ae log tcid . by comparison with the viral load when no respirator was present ( ae log tcid ), the ffp respirators reduced the viral load by ae log . therefore, for active filtration of viruses, the respirators with antiviral coating reduced the viral load passing through the mask by ae log more than the ffp respirators. table also shows that the coated respirators reduced the infectious viruses remaining on the mask surfaces by ae log more than the ffp respirators, and ae log more than the ffp respirators. even with a very high viral challenge, the coated respirators prevented passage of at least an additional ae log infectious viruses, compared with uncoated respirators. large numbers of infectious virions passed through all uncoated respirators tested. ffp respirators were no more effective than ffp respirators at blocking airborne influenza viruses. based on these in-vitro results, respirators with the antiviral coating could be expected to provide more protection than standard respirators from the risk of inhaling influenza viruses. strategies to prevent transmission of influenza include use of respiratory protection. ffp and n respirators are certified to filter at least % of nacl particles ( ae lm in diameter), and many guidelines have recommended that healthcare workers wear these respirators in certain healthcare settings to protect against infection from patients with pandemic influenza. , we have developed a proprietary acid-polymer formulation, designed to coat a standard respirator and inactivate influenza viruses on contact. we tested this coated respirator for cytotoxicity, skin irritation, and sensitisation potential. the antiviral coating was also tested for stability and leaching under extreme environmental conditions, such as physical abrasion and simulated breathing at different temperatures, levels of humidity and co , and saturation with contaminants. eight coated respirators were tested at standard relative humidity ( % rh) for hours, and one at elevated humidity ( % rh) for hours. four coated masks were treated with synthetic blood or oral secretions, and then tested at % rh for hour. the sample respirators were sealed onto a mannequin head inside an airtight chamber, and air at °c and ppm co was pumped through the masks by a cyclic breathing machine at l ⁄ minute. a mm glass-fibre filter was placed behind the respirator, over the mannequin's mouth opening. at the end of all tests, these filters were eluted and analysed using high-performance liquid chromatography (hplc). standard in vitro methods were used to assess the cytotoxicity of the coated polyester and uncoated polypropylene layers of the respirator (glaxosmithkline actiprotect). samples were extracted in minimum essential medium (mem), supplemented with serum, penicillin, streptomycin, amphotericin b, and l-glutamine, at °c for hours. triplicate monolayers of mouse fibroblast cells (l- ) were dosed with each extract (including a reagent control and negative and positive controls), and incubated at °c in % co for hours. after hours of incubation with samples or controls, the monolayers of mouse fibroblast cells were examined microscopically for abnormal cell morphology or cellular degeneration. samples of the coated respirator (comprising four polypropylene layers bonded to the coated polyester outer layer) were applied under occlusive patch conditions to the skin of adults. controls, including individual layers, were applied in the same way. in a separate patch test, samples of the coated polyester outer layer and controls were applied under the same conditions to adults. after hours, test patches and controls were removed. sites were then scored for itching, erythema, oedema, epidermal damage, and papular response after and hours. the patches were applied three times a week for weeks. to evaluate sensitisation, test patches were applied - days later for hours at different sites to the original samples. after this challenge, skin was assessed and graded for sensitisation potential after and hours. table shows that no residues of the antiviral coating or degradation products were detected in the air that had passed through any of the eight respirators. cytotoxicity tests showed that the coated respirator material caused % cell lysis or toxicity, classified as slight reactivity (grade ), and that uncoated material caused no cell lysis or toxicity (grade ) ( table ) . results for positive and negative controls were severe reactivity and no reaction, respectively. from the results of the two human repeat-insult patch tests, neither the coated or uncoated layers nor the fullthickness respirator fabric caused irritation (including itching, erythema, edema, vesiculation, epidermal damage, papules, or reactions beyond the patch site) or sensitisation in any of the adult volunteers at any of the time points. based on these results, in conjunction with published data on acute and repeat-dose toxicity, mutagenicity, local irritation, dermal sensitisation, and inhalation safety for all components of the antiviral coating, the potential topical or inhalation exposure to the coated antiviral respirator does not pose a safety risk. the antiviral coating is durable and stable, and stays on the outer surface of the respirator, even in extreme environmental conditions. the coated respirator is non-irritating and non-sensitising. therefore, this respirator is considered to be well-tolerated and safe for its intended use. ies were funded by gsk consumer healthcare, and gsk investigators were involved in all stages of the study conduct and analysis. knowing how influenza virus is transmitted at home and in school is the key to preventing its spread. at the previous two meetings of this conference, , we introduced our study of household transmission of seasonal influenza and reported our conclusion that protracted survival of the virus even after treatment increases household transmission, and is a major factor in the transmission of the virus to infants. on the other hand, during the recent pandemic, many schoolchildren developed serious respiratory tract disorders, which again highlights the significance of schoolbased transmission of the disease. in this study, we compared transmission of a new influenza strain at home and in school with that of seasonal influenza and proposed countermeasures. the for the analysis of school-based transmission, the epidemic status of seasonal influenza in children at six elementary schools over the past two seasons ( - and - seasons) was compared with that of pdmh in children at two primary schools. using observational data of school-based transmission, we also constructed a model for influenza transmission , and evaluated the effects of factors that could affect influenza transmission (e.g., antibody prevalence, transmission rate, non-infectious latent period, infectious latent period, school closure) through the use of simulations. in this study, a diagnosis of influenza was confirmed by rapid influenza antigen detection kit. we previously reported the high sensitivity of the kits, - not only for seasonal influenza, but also for h n pandemic compared to virus isolation and pcr. serum antibody was not investigated. most of the index patients were treated with oseltamivir or zanamivir, and patients were treated with amatadine. no treatment was done for patients. no nai therapy was done as prophylaxis within the family. the incidence of households with an initial case patient who subsequently infected another member of the household was ae % ( of households) for seasonal influenza or ae % ( of households) for pdmh . thus, the household incidence of pdmh was lower than that of seasonal influenza. in addition, the percentage of family members in households who were infected by initial case patients (household transmission rate) was ae % ( of individuals) for seasonal influenza or ae % ( of individuals) for pdmh . thus, the household transmission rate was also lower for pdmh than that for seasonal influenza. effect of family size on household incidence and household transmission rate an analysis of the effect of family size on household incidence showed that, in families consisting of - individuals, the incidence of seasonal influenza in order of increasing family size was ae %, ae %, ae %, ae %, ae %, and ae %, respectively, and the incidence of pdmh was ae %, ae %, ae %, ae %, ae %, and ae %, respectively, indicating that household incidence tends to increase with increasing family size. in contrast, no definite relationship was noted between household transmission rate and family size. transmission rates for seasonal influenza in order of increasing family size were ae %, ae %, ae %, ae %, ae %, and ae %, respectively, or ae %, ae %, ae %, ae %, ae %, and ae %, respectively, for pdmh (shown in table ). effect of age cohort of initial case patient in household on household incidence and household transmission rate an analysis of the effect of the age cohort of the initial case patient in the household on household incidence and transmission rate showed that the household incidence of seasonal influenza in c , c , c , and c was ae % ( of households), ae % ( of households), ae % ( of households), ae % ( of households), and for m and f was ae % ( of households) and ae % ( of households), respectively. therefore, household incidence was the highest in c , followed by the parents. when the initial case patient was a child, the household incidence increased with decreasing patient age. in contrast, the household incidence of pdmh in c , c , c , and c was ae % ( of households), ae % ( of households), ae % ( of households), ae % ( of households), and for m and f was ae % ( of households) and ae % ( of households), respectively. therefore, household incidence was higher when the initial case patient was a parent, rather than a child. the household transmission rates for seasonal influenza from c to f were ae %, ae %, ae %, ae %, ae %, and ae %, respectively. therefore, as for household incidence, the highest rate ( ae %) was observed in c . the corresponding household transmission rates for pdmh were ae %, ae %, ae %, ae %, ae %, and ae %, respectively, with the highest transmission rates observed for infections from parents (shown in table ). if the rate of individuals with a secondary infection transmitted from the initial case patient in a household is presented as a percentage of the total number of affected individuals, the rates for seasonal influenza and pdmh were ae % ( of individuals) and ae % ( of individuals), respectively. therefore, the rate of individuals with a secondary infection was lower for pdmh than that for seasonal influenza. by age cohort, the corresponding rates of individuals for seasonal influenza in c , c , c , and c were ae % ( of individuals), ae % ( of individuals), ae % ( of individuals), ae % ( of individuals), and for m and f was ae % ( of individuals) and ae % ( of individuals), respectively. for pdmh , the corresponding rates in c , c , c , and c were ae % ( of individuals), ae % ( of individuals), ae % ( of individuals), ae % ( of individuals), and for m and f was ae % ( of individuals) and ae % ( of individuals), respectively. these findings indicate that, especially in the case of pdmh , most secondary infections in parents tend to be transmitted from another household member. the mean annual prevalence of seasonal influenza and the new influenza strain at the elementary schools for the two seasons was ae % and ae %, respectively, whereas the prevalence determined days after appearance of the first case in school was ae % and ae %, respectively. in the recent season at the same elementary schools, however, the prevalence was a high ae %. since the prevalence at days after the appearance of the first case in school was already ae %, these data show that the influenza virus spread quickly throughout the schools. at the schools with high transmission rates in the early period of the pandemic, new infections were confirmed even days after the school closure action was taken. these findings indicate that pdmh , the current influenza virus, has a long latent period during which it becomes infectious and spreads from infected individuals to numerous others in their vicinity. we constructed a model for influenza transmission in schools and estimated the time course of changes in the number of expected cases and the expected prevalence during the season. in this model, school children were divided into six groups depending on the stage of infection: uninfected period with no immunity, non-infectious latent period, infectious latent period, onset, post-onset infectious period, and immune period. it was assumed that schoolbased transmission occurred during the infectious latent period prior to onset and that no infections occurred during the post-onset infectious period because children were absent from school. due to the long latent period of pdmh , the distribution of the non-infectious latent period of pdmh was established as (day , day , day , day ) = ( %, %, %, %) and the distribution of the infectious period as (day , day , day ) = ( %, %, %). when simulations were performed under these conditions using the model for school-based transmission of influenza in which children from classes with an outbreak were kept at home for days, the time course of changes in the number of affected individuals actually observed and the time course of changes in the number of expected cases were determined. the expected prevalence under these conditions was %. to evaluate the effect of school closure, simulations were performed based on the assumption that children from affected classes were not kept at home for days. it was shown that there was an increase in the expected number of cases during the days corresponding to the period of actual school closure and that the expected prevalence increased to %. based on these findings, it was concluded that keeping children home from classes with an outbreak is an effective means of controlling the transmission of influenza in schools (shown in figure ). if the transmissibility of pdmh virus at home is estimated based on the speed of transmission and the degree to which pdmh is prevalent in schools, it would be expected that the household transmission of pdmh is also higher than that of seasonal influenza. in fact, the opposite is the case. this paradox can be explained in two ways. . the number of children aged or more and parents with pdmh influenza as a percentage of the total number of affected individuals is lower than those with seasonal influenza ( ae % versus ae %). further, although the number of parents with a secondary infection was high at home, the percentage of the total number of individuals with pdmh was a low ae % ( of individuals), compared to that for seasonal influenza ( ae % [ of individuals]). in other words, adults are less susceptible to pdmh infections and there was a correspondingly small number of affected individuals. therefore, it was considered that the transmission rate at home was lower than that at school for this reason. . the percentage of households with more than one affected individual within the same family was higher for pdmh at ae % ( of households) than for seasonal influenza at ae % ( of households). in the patients secondarily infected with pdmh , ae % of them showed symptoms of infection days or more after the onset in the first patient, suggesting that they were not infected at home, and the actual household transmission was ae % ( of households). therefore, although the prevalence was higher for pdmh , it seems that household transmission was lower because households with an affected individual implemented satisfactory control measures against infection. seasonal influenza differs greatly from pdmh influenza in its transmissibility at home and in school. in the household transmission of pdmh influenza, both the household incidence and household transmission rate of pdmh were low compared to those for seasonal influenza. although transmission of seasonal influenza from infants to parents was marked, in the case of pdmh , the reverse was true with transmission from parents to children being predomi-nant. it should be noted that household transmission in mothers was common in all eight seasons, suggesting the need to reconsider control measures against infection when nursing unwell family members. in the case of school-based transmission, pdmh was more prevalent than seasonal influenza, indicating that the virus spread quickly throughout the schools. this difference was attributed to the long infectious latent period when pdmh rapidly became rampant in the schools. an analysis of school-based transmission using a model for influenza transmission showed that, when % of the student population is infected, schools should be closed for five consecutive days in order to minimize the spread of the disease. the effectiveness of seasonal influenza vaccine in preventing pandemic and seasonal influenza infection: a randomized controlled trial introduction household transmission has been estimated to account for one-third of all influenza transmission, , and children are at high risk of spreading the disease. with reference to previous evidence, - some vaccine deployment strategies target children to prevent them from infection and transmitting influenza. nevertheless, few studies evaluated the effectiveness of vaccinating children in reducing household transmission. , during - , a pilot randomized controlled trial was conducted to investigate such effect by studying households with school age children randomized to receive trivalent inactivated seasonal influenza vaccine (tiv). the monovalent vaccine against pandemic influenza a (h n ) (ph n ) had yet been available until the end of the first wave. various conclusions have been made as to whether seasonal influenza vaccine might possibly protect against ph n . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] we report findings on the effectiveness of tiv against ph n observed in our cohort. households were screened if they expressed interest after receiving invitation letters distributed via their children's school or an existing pediatric cohort study. to be eligible, the household had to include at least one child aged - years who was not allergic or hypersensitive to any of the tiv components. children known to have immunosuppressive conditions or other contraindications against tiv were also excluded. written consent and assent were obtained from participants aged above years and those aged - years, respectively. proxy written consent was obtained from legal guardians or parents for participants younger than years. ethical approval was obtained from the institutional review board of the university of hong kong. consented households were allocated to the tiv and placebo group (in ratio : ) according a code generated by block randomization with random block sizes of , , and . an independent nurse prepared . one child (study subjects) from each household in the tiv group received a single dose of tiv with one child from each household in the placebo group receiving a single dose of saline placebo. parents and legal guardians were asked to report any adverse reactions days following vaccination. all participants, study nurses, and other research staff were blinded to the allocation and administration of vaccine or placebo. the vaccine allocation sequence was only disclosed to the investigators at completion of the study. serum specimens were collected from subjects shortly before (november-december ), one month after vaccination (december -january ), and after the winter (april ) and summer influenza seasons (august-october ). serum specimens were obtained from household contacts at baseline and after the winter and summer influenza seasons. all household members recorded any fever ‡ . °c, chills, headache, sore throat, cough, presence of phlegm, coryza, or myalgia daily on a symptom diary. they were also invited to report to the study hotline immediately if they experienced at least of the above signs or symptoms. as a response, the study nurse would visit the households with any sick members and collect nose and throat swab from all household members. the households were also telephoned monthly or increased to fortnightly during influenza seasons to monitor for signs and symptoms and remind them to report to the hotline. supermarket or book vouchers (for children) were given to the households including us$ for each serum specimen collected, us$ . for each home visit, and us$ for completion of the study. serologically-indicated influenza infection was the primary outcome of this study. it was define as a ‡ fold rise in antibody titer within each influenza season. other study outcomes included rt-pcr confirmed influenza virus infection, acute respiratory illness (ari) (two of any of the above listed signs or symptoms), and influenza-like illness (ili) (fever ‡ . °c with cough or sore throat). antibody titers against the vaccine strains were obtained by testing each serum specimens by haemagluttination inhibition (hai). viral microneutralization (vn) using standard methods was found to be more sensitive than hai in detecting antibody response against a ⁄ california ⁄ ⁄ (h n ) in another study conducted by our group and was, therefore, used in this study. the sera was initially diluted at ⁄ and further tested in serial doubling dilutions. nose and throat swabs were tested by reverse transcription polymerase chain reaction (rt-pcr) for influenza a and b viruses. technical details of the laboratory methods have been reported elsewhere. , fisher's exact test and chi-squared tests were used to compare count data including occurrence of side effects, laboratory confirmed, and clinically defined influenza infections. wilcoxon signed-rank test were used to compare the serum antibody titers between groups. exact binomial method or the wald approximation was used to estimate % confidence intervals where appropriate. all analyses were carried out in r version . . (r development core team, vienna, austria). twenty-five primary and secondary schools in the district of the study clinic were invited to participate. to parents of three schools that agreed to take part and another study cohort, invitation letters were sent and households were enrolled. personal referrals were made from these parents to enroll additional households. among enrolled households, subject with history of epileptic seizure was assessed to be contra-indicated against receiving the vaccine. blood taking failed in another subject, and both of them withdrew from the study. eleven households did not complete the study. table shows subject and household contacts of the tiv and placebo group were similar in demographics and prior influenza vaccination history. antibody titers before vaccination were comparable between groups (data not shown). most study subjects who received tiv showed antibody titer ‡ against the vaccine strains month after receiving tiv, and the proportion was significantly higher than those who received placebo (a ⁄ h n % in tiv versus % in placebo group, p < . ; a ⁄ h n % versus %, p < . ; b % versus %, p = . ). none of the study subjects had antibody titer ‡ against ph n following receipt of seasonal tiv. no serious adverse reactions were reported, and only pain at injection sites was slightly higher in tiv group (data not shown). subjects who received tiv had lower rates of serologically confirmed seasonal influenza a(h n ) ( % versus %, p = . ), a(h n ) ( % versus %, p = . ) and b infection ( % versus %, p = . , although the differences were not statistically significant (table ) . study subjects had higher rate of serologically confirmed ph n infection ( % versus %, p = . ), yet it was not statistically significant. after adjusting for potential cross reactive antibody response, % of subjects in tiv versus % in placebo groups showed ph n infection confirmed by either serology or rt-pcr (p = . ). little differences were observed for rt-pcr confirmed infection, ari, and ili in results combining the winter and summer influenza seasons. during winter season when seasonal influenza predominated, study subjects who had received tiv showed a lower tendency to develop ili ( % versus %, p = . ) or ari ( % versus %, p = . ). an opposite tendency was seen (ili % versus %, p = . ; ari % versus %, p = . ) during summer when ph n predominated. however, these differences were not statistically significant. rates of ili in subjects infected with ph n did not differ statistical significantly between subject who received tiv and placebo ( % versus %, p = . ). the study was not powered to detect indirect benefits to household contacts of vaccines resulting from reduced household transmission. attack rates were found to be similar between household contacts of subjects received tiv and placebo (data not shown). to examine potential factors that might affect risk of laboratory confirmed ph n infection, a multivariable logistic regression model was fitted to study all subjects and their household contacts. younger participants aged below years were found to have a higher risk (< years or = . , % ci . , . ; - years or = . , % ci . , . , > or = . ). after adjusting for age, sex, and date of study completion, receipt of tiv for the - influenza season was not found to affect risk of ph n infection. however, participants who had laboratory confirmed seasonal influenza infection during the study period had % lower risk of ph n infection (infected with seasonal influenza or = . , % ci . , . ; not infected with seasonal influenza or = . ). as (see table s for winter and summer results separately). influenza-like illness (ili) defined as temperature ‡ . °c plus cough or sore throat; acute respiratory illness (ari) defined at least any two of fever ‡ . °c, chills, headache, sore throat, cough, presence of phlegm, nasal congestion, runny nose, muscle or joint pain. limited by the sample size, we were not able to differentiate between the protective effect of seasonal a(h n ) and a(h n ) infection against ph n . other details of the results from the study were published elsewhere. discussion a non-significantly higher rate of ph n infection was observed in study subjects who received tiv compared to placebo. results from a multivariable logistic regression suggested that such a pattern might be explained by more common seasonal influenza infection in placebo group prior to the pandemic, protecting the placebo group against ph n . seasonal influenza infection within - months observed in our study might have conferred better cross protection than tiv against ph n . this resembles similar previous findings on cross protection between influenza infections in human and animal studies. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] however, the same phenomenon has not been observed in some studies on seasonal influenza vaccine against ph n . , , [ ] [ ] [ ] apart from differences in study design and vaccine used, we speculate that a short time interval between ph n and most recent seasonal influenza peak activities might be crucial for the phenomenon. hong kong is a subtropical area where the pandemic was preceded immediately by summer seasonal influenza circulation and a few months apart from the winter - influenza peak. if cross protection from seasonal influenza lasts for only a short period, it might have waned below partial cross protection from tiv over time from last seasonal influenza infection. the current study is limited by a small sample size, and further studies are required to confirm our hypothesis. while tiv is only effective against matching strains, a universal influenza vaccine could provide better protection against the ever evolving influenza viruses. introduction immunisation of healthy, as well as high risk, children has been the focus of much recent attention both in prevention of seasonal influenza and during the h n pandemic. detailed information on reactogenicity, particularly for newer vaccine formulations that include adjuvants, is limited. we recently reported results of a head-to-head comparison of two h n pandemic influenza vaccines in children in the uk. here we present new, detailed analyses of reactogenicity data from that study, which has important potential implications for future paediatric influenza vaccine development and use. we compared the safety, reactogenicity, and immunogenicity of two h n influenza vaccines, one as b (tocopherol based oil in water emulsion) adjuvanted egg culture derived split virion, the other non-adjuvanted cell culture derived whole virion, given as two dose schedules days apart, in a randomised, open label trial as previously reported. the study was age stratified ( months to under years & - years) to ensure adequate data in young children. age appropriate safety data (simplified for under year olds) were collected for days after each vaccine dose and serum was collected at enrolment & days after the second dose. nine hundred-thirty seven children received vaccines as per-protocol. when comparing the two vaccines, grade ( ‡ mm) local reactions were seen more frequently following the adjuvanted than the non-adjuvanted vaccine in both age groups, after both vaccine doses. in children over years old, ae % versus ae %, p < ae , after dose one; ae % versus ae %, p = ae , after dose two, in children under years old, ae % versus ae %, p = ae , after dose one (non significant, ns); ae % versus ae %, p < ae after dose two. fever ‡ °c (axillary measurement) was seen more frequently following the second dose of the adjuvanted vaccine compared to the non-adjuvanted vaccine in < year olds ( ae % versus ae %; p < ae ). looking specifically at the adjuvanted vaccine in under year olds, comparing the second dose with the first, there were significantly higher rates of fever ‡ °c (axillary measurement) ( ae % versus ae %, p < ae ), local grade ( ‡ mm) reactions ( ae % versus ae %, p = ae ), pain ( ae % versus ae , p = ae ), use of analgesia or antipyretic medication ( ae % versus ae %, p < ae ), and decreased activity ( ae % versus ae %, p < ae ). the adjuvanted vaccine was significantly more immunogenic, most notably in the younger children. in < year olds, haemagglutination inhibition (hi) seroconversion rates were ae % versus ae %, p < ae . among all general and local reactions measured, only the maximum temperature measured during the days after the second dose of the adjuvanted vaccine showed a significant (positive) association with post vaccination hi titres. for each °c rise in temperature there was a % increase in titre (p < ae ). these reactogenicity data demonstrate a step towards the future possibility of one-dose influenza immunisation programmes for young children associated with low rates of fever and other reactions. the occurrence of fever following adjuvanted vaccine, seen particularly after a second dose in younger children, was quantitatively associated with enhanced antibody titres. this association was not seen with unadjuvanted vaccine. this apparent difference between the relatedness of the pyrogenic and immunogenic effects of the two vaccines merits further investigation. novel adjuvants appear to have the potential to overcome the relatively poor immunogenicity previously experienced with inactivated influenza vaccines in infants and young children. however, careful adjustment may be needed to optimise the balance between high protection and acceptable reaction rates. tries causing sporadic human infections. vaccination has been used as an effective public health tool for influenza prophylaxis. the goal of this study was to evaluate live attenuated influenza vaccine (laiv) vaccine candidates for subtypes h and h . the attenuated phenotype of h and h laiv candidates has been proven in experiments in ovo and in vivo. in randomized clinical trials among adult volunteers, no significant adverse reactions attributable to the live vaccine occurred. our results indicate that pandemic laiv candidates were well tolerated and elicited serum, local, and cellular immune responses. the emergence and spread of highly pathogenic avian influenza h n viruses in avian populations and concurrent infections in humans since has prompted efforts to develop vaccines for use in the event of an influenza pandemic. in , the world faced a new h n pandemic. immunization with inactivated or live vaccines is the primary measure for preventing influenza. laivs appear to be safe and efficacious, and might possibly provide broader immune responses than inactivated vaccines. our study evaluated laiv pandemic candidates as part of the global influenza pandemic preparation project outlined by the who. capacity of the viruses to grow at optimum, low, and elevated temperatures (ca ⁄ ts phenotype) was evaluated by routine technique in embryonated hen eggs. laiv and placebo were supplied by microgen (irkutsk, russia). the monovalent laiv was produced from the pandemic vaccine candidates and formulated to contain and ae eid per dose ( ae ml) of a ⁄ ⁄ california ⁄ ⁄ and a ⁄ ⁄ duck ⁄ potsdam ⁄ ⁄ , respectively. the vaccine or placebo was administered intranasally with a single-use dosing nasal sprayer. two doses were given at an interval of days. one hundred-ninety healthy adults aged - years were randomly divided into groups to receive either pandemic vaccine candidates ( ) or placebo ( ) . subjects were informed about purposes and methods of the study and potential risks associated with participation. all participants had an hai antibody titer of £ : to a ⁄ california ⁄ ⁄ (h n ) pandemic virus. in all there were and vaccines and and participants who received placebo, and were further tested for immune responses to h n or h n pandemic vaccine, respectively. another participants vaccinated with h n laiv were children between to years old. before the children were vaccinated, their parents were advised about study and their consent was required before any child was enrolled. on the advice of the national ethics committee, we did not include a placebo group in this study. individuals were not enrolled if they had an acute illness or fever at the beginning of the study or a history of egg allergy. immune responses of subjects were assessed by routine hai test (evaluation of serum igg antibodies), elisa (evaluation of iga antibodies eluted from the nasal swabs into steril pbs), and cytokine flow cytometry assay (evaluation of virus-specific cd + cd + ifnc + and cd + cd + ifnc + peripheral blood mononuclear cells). the results of phenotypic analysis in ovo showed that pandemic vaccine candidates retained the cold adapted-temperature sensitive (ca ⁄ ts) phenotype, typical of the coldadapted parental mdv. in contrast and as expected, a ⁄ california ⁄ ⁄ and a ⁄ duck ⁄ potsdam ⁄ - parental strains had the non-ts ⁄ non-ca phenotype typical of wt viruses. the h n pandemic vaccine candidate demonstrated an attenuated phenotype in mice and in java macaques and did not infect chickens. the vaccine attenuation study confirmed the attenuated phenotype of a a ⁄ ⁄ california ⁄ ⁄ pandemic laiv candidate in mouse, ferret, and guinea pig models. the phase i ⁄ ii randomized, controlled, double-blind clinical study safety evaluation of pandemic vaccine candidates in adults clinical examination of subjects who received two doses of pandemic vaccine candidates indicated that both vaccines were well tolerated. no fever reactions were observed after the first or second vaccination. after the first vaccination, ae % and ae % of reactogenicity events consisting of catarrhal symptoms, such as pharyngeal irritation or hyperemia, were observed for h n and h n vaccine candidates, respectively. after revaccination, subjects did not report local or systemic reactions. to determine whether a serological response occurred in the cohort of immunologically naïve subjects vaccinated with pandemic vaccine candidates, hai and elisa tests were used (table ) . post-vaccination geometrical mean titers (gmt) among subjects who received two doses of h n vaccine were significantly higher than pre-vaccination titers. the frequency of ‡ fold antibody rises was significantly higher ( ae %) after revaccination than after one dose ( ae %). the percentage of subjects with post-vaccination serum hai titers to h n ‡ : was ae % and for titers ‡ : , it was ae %. no seroconversions in the placebo group were detected. the virus-specific nasal iga antibody response to vaccination after two doses of the h n vaccine candidate demonstrated significant increases of ‡ fold rise iga antibodies ( %) compared to one dose. cumulative data of h n vaccination (all applied tests) showed % and % of conversions after the first and the second vaccination, respectively. increasing h n vaccine virus infectivity from ae to ae eid ⁄ dose lead to an enhancement of post-vaccination hai titers in vaccinees after the first vaccination to homologous h n antigen from ae % to ae % of ‡ fold antibody rises. values of post-vaccination serum hai antibody titers in subjects vaccinated with another pandemic vaccine candidate, a ⁄ ⁄ california ⁄ ⁄ , also proved to be rather low. after the primary vaccination, the percentage of subjects with hai protective antibody titers ‡ : were ae %. after revaccination, this parameter increased to ae %. four-fold increases in serum hai antibody titres were four-fold conversions after the first and the second vaccination was ae % and ae %, respectively. elisa antibodies in nasal swabs showed had an advantage in detecting induction of local iga as compared to serum hai antibodies. after revaccination four-fold serum hai antibody conversions were ae % vs. ae % of iga conversions in nasal swabs, respectively. taking into account cumulative data of h n vaccination (hai and elisa data), the obtained results were here and in the ae % and ae % of conversions after the first and the second vaccination, respectively. fourty-seven subjects were vaccinated with h n laiv, and who received a placebo were chosen for evaluation of cellular immune response by cytokine assays. after revaccination, the mean increases of both cd + and cd + memory cells were significantly higher in vaccinated subjects compared to the placebo group. interestingly, the same effect of vaccination was observed in vaccinees without detectable conversions of hai antibody titers. even after a single vaccination, the rate of subjects with significant increases of these cells in the blood was ae % (cd + ) and % (cd + ). after the revaccination, the percentage of subjects with significant increases in cd + and in cd + cells was ae %. immunogenicity of h n pandemic vaccine candidate in children hai antibody results among children aged to years proved to be significantly higher when compared to adult subjects: after the first vaccination, ae % of the children seroconverted; after revaccination, seroconversions reached ae % ( table ). the gmt rise to h n vaccine with primary vaccination was : ; after revaccination it increased to : . benefits of vaccination with laiv to aid in the control of influenza outbreaks are acknowledged by the who. many years of laiv seasonal trials have shown excellent tolerability and low reactogenicity. [ ] [ ] [ ] indeed, data showed that live influenza vaccines cause minimal systemic, local, and thermal reactions, generally from to %. a different situation was observed in the cohort of immunologically naïve volunteers vaccinated with pandemic vaccines. the rate of local reactions to a ⁄ ⁄ california ⁄ ⁄ and a ⁄ ⁄ duck ⁄ potsdam ⁄ ⁄ vaccine candidates increased to ae % and ae %, respectively. after revaccination no significant local and systemic reactions were observed. this confirms, indirectly, the development of a sufficiently high level of protection after the first vaccination with pandemic laiv. the most important criterion for assessing the quality of vaccines is their estimated safety, epidemic effectiveness, and immunogenicity. however, current regulatory documentation mandates that induction of serum antibodies, measured by hai, as the only criterion for a laiv immunogenicity evaluation. in addition to the standard hai assay, we determined serum (igg) and local (iga) antibodies in adult subjects vaccinated with an h n pandemic vaccine candidate. evaluation of overall results obtained in these additional serological tests, as well as those from the hai assay, showed an immune response to the vaccine in the majority of subjects ( ae % of ab seroconversions after the single vaccination and ae % after revaccination, respectively). these data show that methods used to routinely measure laiv immunogenicity should be revised to include a number of additional immunological methods such as igg and iga elisa, and cytokine assays consistent with the recently updated who recommendations on laiv monitoring. these clinical studies clearly demonstrated that pandemic laiv candidates are effective at generating pandemic specific influenza immunity. a key finding from this study is that it may be practical to give the vaccine as a single dose to both children and adults. evaluation of our laiv pandemic vaccine candidates was performed as part of the global influenza pandemic preparation project outlined by the who. it was considered that laiv could be produced in greater quantities and more rapidly than inactivated vaccines. together with the generation of herd immunity by laiv, this suggests that laiv implementation during the first wave of a pandemic may provide significant social, economic, and health benefits to the community. authors are thankful to path for the financial support of h n pandemic vaccine study. we are grateful for the the main evolutionary mechanism of influenza viruses during inter-pandemic period is the antigenic drift, but the epidemiological picture of circulating viruses is complicated by a high level of heterogeneity of strains, even though drift does not occur, due to co-circulation of drifted and old strains or to co-circulation of viruses belonging to the same type ⁄ subtype but with different antigenic patterns. [ ] [ ] [ ] [ ] [ ] [ ] lack of data exists on the impact of the wide heterogeneity of circulating strains on the seroprotection and on-field effectiveness of influenza vaccine: in particular, little is known about the ability of influenza vaccine to elicit an effective immune response against isolates with few amino acid mutations with respect to vaccine strains that represent the majority of circulating viruses. mf -adjuvanted vaccines, which are currently used for the prevention of seasonal influenza epidemics in elderly, are showed to confer higher seroprotection against homologous and drifted a(h n ) strains than non-adjuvanted vaccines. [ ] [ ] [ ] the broader immune response showed by mf -adjuvanted vaccine was measured using hi and nt assays against egg-grown drifted strains representing vaccine composition changes during the following seasons, but its ability to elicit a broader immune response against circulating viruses belonging to vaccine cluster and presenting amino acid mutations onto antigenic sites or against on-field isolates not-antigenically distant from vaccine strains has not yet been investigated. showing amino acid changes onto antigenic sites in position (n k), (n k), and (p s) with respect to a ⁄ california ⁄ ⁄ . in particular, a ⁄ genoa ⁄ ⁄ and a ⁄ genoa ⁄ ⁄ presents n d amino acid mutation detected in clade a ⁄ wyoming ⁄ ⁄ -like viruses. the ha sequences of a ⁄ genoa ⁄ ⁄ , a ⁄ genoa ⁄ ⁄ , genoa ⁄ ⁄ , a ⁄ genoa ⁄ ⁄ , and a ⁄ genoa ⁄ ⁄ fell within the clade represented by the ha of a ⁄ califor-nia ⁄ ⁄ ; among these isolates, a ⁄ genoa ⁄ ⁄ and a ⁄ genoa ⁄ ⁄ showed antigenic site sequences very close to that of the ⁄ vaccine strain, whereas ha sequences of a ⁄ genoa ⁄ ⁄ , a ⁄ genoa ⁄ ⁄ ⁄ and a ⁄ genoa ⁄ ⁄ posses amino changes onto antigenic site a(r k), c(g e) and d(r k), respectively. the ha sequences of more recent isolates fell within the clade represented by the ha of a ⁄ brisbane ⁄ ⁄ and characterized by the amino acid changes, relative to the ha of a a ⁄ california ⁄ ⁄ , g e and k i, with the exception of a ⁄ genoa ⁄ ⁄ , showing r g and l s amino acid changes present in viruses belonging to a ⁄ nepal ⁄ ⁄ clade. measure of genetic distance between vaccine and circulating strains was calculated as previously described by gupta. two blood samples were collected from each subject, just before and ± day post-vaccination. all sera were stored at ) °c. all samples were tested at the laboratory of health sciences department, university of genoa, by haemagglutination-inhibition (hi) and neutralization (nt) assays, performed following the who criteria and standardised method in our laboratory, respectively. [ ] [ ] [ ] guinea pig red blood cells were used for hi assay. all samples were assayed twice for hi and for nt. the obtained antibody titre was expressed as the reciprocal of the last sera haemagglutinating or inhibiting virus dilution. immunogenicity was determined by: geometric mean titre (gmt); mean-fold increase (mfi; ratio of post-to pre-vaccination titre); seroprotection rate (the percentage of subjects achieving an hi and nt titre ‡ iu); and seroconversion rate (percentage of subjects with a fourfold increase in hi or nt antibody titers, providing a minimal post vaccination titer of : ). post-vaccination gmt was reported as ratio, with the corresponding % confidence interval, of gmts after vaccination with mf -adjuvanted vaccine and with non-adjuvanted subunit vaccine. seroprotection and seroconversion rate % confidence interval was calculated using modified wald method. comparisons of seroconversion and seroprotection rates between subunit and mf -adjuvanted vaccine groups have been analyzed by fischer's exact test. the results were evaluated against the committee for medicinal products for human use (chmp) criteria for approval of influenza vaccines in the elderly, which require that at least one of the following criteria be met: mfi > ; seroprotection rate > %, or seroconversion rate > %. furthermore, hi titres were also transformed into binary logarithms, corrected for pre-vaccination status, as described by beyer et al. and were expressed as median titres, with the corresponding °- °i nter-quantile range. comparisons of corrected post-vaccination titers between subunit and mf -adjuvanted vaccine groups were analyzed by wilcoxon test. difference in immunogenicity profile between vaccine groups, expressed by ratio of different parameters, was correlated with genetic and antigenic distance between vaccine and viruses used in the study using spearman test. pre-vaccination titres were not significantly different between vaccine groups, for all strains (data not shown). post-vaccination gmt ratios between mf -adjuvanted and non-adjuvanted vaccine groups determined using hi and nt assays, with the corresponding % confidence interval, according to viral strain are shown in figure . both vaccines met chmp requirements for mfi (> ), seroconversion (> %), and seroprotection rate (> %) against a ⁄ wyoming ⁄ ⁄ -like, with the exception of a ⁄ genoa ⁄ ⁄ and a ⁄ california ⁄ ⁄ -like circulating viruses and against egg-grown a ⁄ wyoming ⁄ ⁄ , a ⁄ california ⁄ ⁄ , and a ⁄ wisconsin ⁄ ⁄ strains; the immune response against a ⁄ genoa ⁄ ⁄ met the requirements for mfi and seroprotection rate only in mf -adjuvanted vaccine group. requirements for mfi, seroconversion, and seroprotection rate against the a ⁄ brisbane ⁄ ⁄ -like virus a ⁄ genoa ⁄ ⁄ and the a ⁄ nepal ⁄ ⁄ -like genoa ⁄ ⁄ viruses and against egg-grown a ⁄ brisbane ⁄ ⁄ strain were reached only in subjects vaccinated with the mf adjuvanted vaccine. a similar pattern emerged from the analysis of mfi, seroconversion and seroprotection rates using nt assays. subjects vaccinated with the mf -adjuvanted vaccine showed significantly higher post-vaccination hi gmts against a ⁄ wyoming ⁄ ⁄ -like, a ⁄ california ⁄ ⁄ -like, a ⁄ nepal ⁄ ⁄ -like and a ⁄ brisbane ⁄ ⁄ like viruses, with the exception of a ⁄ genoa ⁄ ⁄ , and against egg-grown a ⁄ california ⁄ ⁄ , a ⁄ wisconsin ⁄ ⁄ , and a ⁄ brisbane ⁄ ⁄ strains, compared with individuals immunized with the non-adjuvanted vaccine ( figure ). the mf -adjuvanted vaccine also induced significantly higher seroconversion and seroprotection rates against following correction for pre-vaccination status, hi titres were significantly higher for the mf -adjuvanted vaccine group when evaluated against a ⁄ wyoming ⁄ ⁄ -like viruses, a ⁄ brisbane ⁄ ⁄ -like a ⁄ genoa ⁄ ⁄ , and a ⁄ nepal ⁄ ⁄ -like a ⁄ genoa ⁄ ⁄ strain ( figure ). pre-vaccination titre corrected response was higher in subjects vaccinated with mf adjuvanted vaccine also against egg-grown a ⁄ wyoming ⁄ ⁄ , a ⁄ california ⁄ ⁄ ⁄ , a ⁄ wisconsin ⁄ ⁄ , and a ⁄ brisbane ⁄ ⁄ . among viruses more closely related to a ⁄ california ⁄ ⁄ , subjects immunized with mf -adjuvanted vaccine showed a significantly higher corrected titres against a ⁄ genoa ⁄ ⁄ , a ⁄ genoa ⁄ ⁄ , and a ⁄ genoa ⁄ ⁄ strains compared with the non-adjuvanted vaccine ( figure ) . spearman test showed a clear correlation between the distances and the advantage offered by mf expressed by ratio between mfi, post-vaccination gmts, corrected post-vaccination median, seroconversion, and seroprotection rates calculated using hi test in the two vaccine groups. similarly, ratio between mfi, seroconversion, and seroprotection rates calculated with nt test correlated with the genetic and antigenic distance between vaccine and viruses used for the study. the ability of mf to enhance the immunogenicity and to elicit a broader immune response against drifted strains than non-adjuvanted vaccine is consistent with other findings reported during the last decade. [ ] [ ] [ ] in subjects vaccinated with the mf -adjuvanted vaccine containing a ⁄ california ⁄ ⁄ , the immune response, expressed by a number of parameters, such as crude and corrected postvaccination titers, seroconversion, and seroprotection rates calculated using hi and nt assays, is higher than that observed in individuals immunized with subunit vaccine when it is evaluated against a drifted strains, such as a ⁄ brisbane ⁄ ⁄ -like and a ⁄ nepal ⁄ ⁄ -like strains, and against egg-grown a ⁄ brisbane ⁄ ⁄ virus. for the first time in this study, the impact of heterogeneity of circulating strains antigenically close to the vaccine on the antibody response elicited by mf -and non-adiuvanted vaccines is evaluated. immune response against viruses isolated during the ⁄ season, that appear more phylogenetically close to ⁄ vaccine strain a ⁄ wyoming ⁄ ⁄ , was higher in subjects vaccinated with mf -adiuvanted vaccine as demonstrated by higher crude and corrected post-vaccination hi titres and higher postvaccination nt titres, with the exception of a ⁄ genoa ⁄ ⁄ , against whom the nt post-vaccination gmt is identical in mf and subunit vaccine groups. furthermore, hi seroconversion and seroprotection rates were higher in mf vaccine group when evaluated against a ⁄ genoa ⁄ ⁄ and a ⁄ genoa ⁄ ⁄ . as far as the immune response against a ⁄ california ⁄ ⁄ -like viruses, the small number of enrolled subjects did not allow appreciating differences using qualitative response indicators, but crude post-vaccination hi titres were higher in mf vaccine group for all the strains. interestingly, a ⁄ california ⁄ ⁄ -like viruses with at least one amino acid change onto antigenic sites, i.e. a ⁄ genoa ⁄ ⁄ , a ⁄ genoa ⁄ ⁄ , and a ⁄ genoa ⁄ ⁄ , showed a more marked difference in terms of response between the two vaccine groups. individuals immunized with mf -adiuvanted vaccine showed higher corrected post-vaccination hi titres and post-vaccination nt titres in comparison with subjects vaccinated with plain vaccine. these response indicators were similar in the two vaccine groups when the response was evaluated against a ⁄ genoa ⁄ ⁄ and a ⁄ genoa ⁄ ⁄ , which present no amino acid changes onto antigenic sites and identical hi titers respect with a ⁄ california ⁄ ⁄ at molecular and antigenic characterization, respectively. thus, the advantage offered by mf in terms of higher immunogenicity expressed by higher post-vaccination hi titres is observable also against viruses showing antigenic and molecular pattern undistinguishable from vaccine strain, but it became even more evident as the antigenic and molecular distance between vaccine and circulating strains grew. as emerged for a ⁄ genoa ⁄ ⁄ , a ⁄ genoa ⁄ ⁄ , and a ⁄ genoa ⁄ ⁄ , one amino acid was a sufficient change in antigenic sites for -fold decrease of hi titre against homologous vaccine strain to observe -fold higher post-vaccination nt titers (mf ⁄ subunit postvaccination gmt ratio range between ae and ae , figure ) and one-dilution higher corrected post-vaccination hi titers in mf vaccine group ( figure ) . finally, the correlation between the distance and the improvement offered by mf in terms of higher immunogenicity clearly emerged by spearman correlation analysis: it remains wellfounded both using a number of different response parameters obtained from hi and nt assays and calculating the distance by serological and genetic methods. outbreaks of h n pdm in pigs in commercial swine operations have been reported in several countries. in all incidents, epidemiological investigations have linked humans as the possible source of the infection to pigs. experimentally, it was established that the virus is pathogenic and transmits readily in pigs. the natural outbreaks of h n pdm and laboratory studies underscore the threat that the virus poses to the swine industry and highlight the need for developing effective control strategies. in the united states, a trivalent live attenuated influenza vaccine (flumistÒ) has been licensed for use in humans since . in swine medicine, however, temperature-sensitive laivs are not available. currently, only inactivated vaccines are available for pigs, but they provide limited protection against antigenically diverse influenza viruses. additionally, the use of inactivated vaccines has been associated with enhanced pneumonia when immunized pigs were challenged with divergent viruses. thus, the development of laivs has the potential to circumvent the drawbacks associated with commercial vaccines. with the aim of developing laiv temperature-sensitive influenza vaccines against the h n pdm virus, we have used reverse genetics to introduce attenuation markers in the polymerase genes of a swine-like tr h n influenza virus, a ⁄ turkey ⁄ ohio ⁄ ⁄ (h n ) (ty ⁄ ). we chose this isolate because it grows well in both eggs and cell culturebased substrates, displays a broad host range, and has internal genes similar to the h n pdm virus. safety and efficacy studies of the ty ⁄ att vaccine candidates in pigs demonstrated that this vaccine backbone is attenuated in swine and conferred sterilizing immunity upon an aggressive intratracheal challenge of pigs with the h n pandemic virus. thus, introduction of genetic signatures for att in the backbone of a swine-like tr influenza virus resulted in highly attenuated and efficacious live influenza vaccines with promising applications veterinary medicine. -t cells and mdck cells were maintained as previously described. a ⁄ turkey ⁄ ohio ⁄ ⁄ (h n ) (ty ⁄ ) has options for the control of influenza vii ª blackwell publishing ltd, influenza and other respiratory viruses, (suppl. ), - been previously described and it was kindly provided by yehia saif, ohio state university. a ⁄ california ⁄ ⁄ (h n ) (ca ⁄ ) was kindly provided by the centers for disease control and prevention (cdc). generation of recombinant viruses by reverse genetics (rg) was done using a previously described method. the genetic signatures for attenuation were introduced into the pb and pb genes of ty ⁄ . ny : ty ⁄ att is a : reassortant with the surface genes from the a ⁄ new york ⁄ ⁄ (h n ) virus and the ty ⁄ att internal genes. all viruses were amplified in mdck cells to produce viral stocks. twenty-five pigs were divided into five groups (n = ) and intranasally inoculated with tcid ⁄ animal of either h n : ty ⁄ att or with ny(h n ) : ty ⁄ att vaccines diluted in ml of mem. two other groups were similarly inoculated with h n : ty ⁄ wt and h n : ty ⁄ rg and served as controls, whereas a fifth group was mockvaccinated with pbs alone. clinical observations were performed as previously described. , efficacy of h n ty ⁄ att vaccine in pigs fourty pigs were divided in four groups (n = )( table ) . group was vaccinated with tcid ⁄ animal of ny(h n ) : ty ⁄ att through intranasal route, whereas group was vaccinated intramuscularly with ml of an adjuvanted uv-inactivated ca ⁄ vaccine (uvadj-ca ⁄ ). group , non-vaccinated and challenged (nv+ca ⁄ ), and group , non-vaccinated, mock-challenged (nv+mock), were also included. pigs were boosted two weeks later. fourteen days post boost (dpb), pigs from groups - were challenged intratracheally with ml of · tcid of ca ⁄ . following challenge, pigs were monitored using methods as previously described. all statistical analyses were performed using graphpad prism software version ae (graphpad software inc., san diego, ca). the differences were considered statistically significant at p < ae . the ty ⁄ att-based vaccines are attenuated in swine pigs inoculated with wt ty ⁄ viruses developed fever (> °c) that peaked hpi ( figure a) and shed large amounts of in nasal secretions ( figure b) . similarly, viral titers in bronchoalveolar lavage fluid (balf) collected at dpi ranged from to tcid ⁄ ml ( figure c ). at necropsy, the lungs from animals inoculated with these viruses had severe pneumonia ( figure d ). in contrast, none of the animals inoculated with h n or h n ty ⁄ att viruses developed clinical signs following vaccination, indicating that the ty ⁄ att viruses were safe for administration to pigs ( figure a) . correspondingly, there was - fold less virus shedding from the nose of pigs vaccinated with ty ⁄ att viruses as compared to unmodified ty ⁄ viruses. in general, ny(h n ) : ty ⁄ att -vaccinated pigs shed less virus than h n : ty ⁄ att inoculated pigs ( figure b ). in addition, viral titers in balf were significantly reduced (p < ae ) in ty ⁄ attvaccinated pigs as compared to ty ⁄ wt-infected pigs ( figure c ). although both vaccines caused mild gross and microscopic lesions in the lungs, the percentage of lung ae ± ae * ± * ± * ± * balf, bronchoalveolar lavage fluid, uvadj-ca ⁄ , uv-inactivated ca ⁄ vaccine; nv+ca ⁄ , non-vaccinated, challenged positive control group; nv+mock, non-vaccinated, non-challenged negative control group. *significantly different from nv+ca ⁄ control group at p < ae . geometric mean hi titer against ca ⁄ at the day of challenge. à percentage of macroscopic lung lesions given as mean score ± sem. § average viral titer (log ) measure as tcid per ml. -average viral titer (log ) in balf at dpc. involvement was not significantly different from mock-vaccinated pigs, corroborating the clinical findings that these vaccines are sufficiently attenuated in pigs ( figure d, e) . histopathologically, nasal turbinates and trachea obtained from pigs immunized with either vaccine were similar to control animals, as opposed to the wt-inoculated pigs ( figure e ). vaccination with h n ty ⁄ att-based vaccines provides sterilizing immunity against h n pdm in pigs the clinical performance in pigs of the h n vaccines is summarized in table . nv+ca ⁄ animals had macroscopic pneumonia, viral replication in balf and shedding in the nose. uvadj-ca ⁄ vaccine provided satisfactory protection, but this protection was not sterilizing. remarkably, animals vaccinated with ny(h n ) : ty ⁄ att had sterilizing immunity. in both vaccine groups there was a significant reduction (p < ae ) in the percentage of macroscopic lung pathology compared to the nv+ca ⁄ group. control pigs had neither significant macroscopic nor microscopic lesions in the lungs. hi antibody titers measured at the day of challenge in both vaccine groups were approximately the same (table ). in the present study, we developed for the first time, temperature-sensitive laiv for use in pigs. data from our safety studies showed that both the h n and h n ty ⁄ att vaccines were attenuated in pigs. although the ty ⁄ att vaccines were detected in balf samples, the level of viral replication was significantly reduced in comparison to unmodified virus and, more importantly, caused no overt clinical signs. a minimal amount of replication is likely beneficial for eliciting t-cell responses to internal genes that may provide heterologous cross-protection. one of the most challenging tasks in producing effective live attenuated vaccines is to achieve an adequate balance between safety and efficacy. by introducing the att modifications into the polymerase genes of a swine-like tr strain, this desirable balance was achieved. the vaccines were histopathologic scores of nasal turbinates, trachea and lungs at dpi. ny(h n ) : ty ⁄ att (a virus that carries the surface genes of a ⁄ new york ⁄ ⁄ (h n ) and ty ⁄ att internal genes). all h n viruses have their surface genes derived from ty ⁄ . values are shown as the mean ± sem. * p < ae ; **p < ae ; *** p < ae . options for the control of influenza vii ª blackwell publishing ltd, influenza and other respiratory viruses, (suppl. ), - attenuated in pigs and, more importantly, provided sterilizing immunity upon an aggressive challenge with pandemic h n as opposed to an experimental ca ⁄ inactivated vaccine, which elicited protective but not sterilizing immunity in all animals. in the face of influenza pandemics that have the ability to overcome the species barriers such as the h n , the supply of vaccines for use in agriculture could be jeopardized. our cell culture-based live att h n vaccines could be an attractive alternative for this possible pandemic vaccine shortage. because the ty ⁄ att live vaccines developed here are efficacious in swine, are easier to manufacture than inactivated vaccines, and do not require adjuvants, our study represents a major advance in vaccine development for the h n pandemic. in conclusion, our second generation of live att influenza vaccines based on modifications of the pb and pb genes of ty ⁄ retains its safety properties in vivo and can induce excellent protection against aggressive h n challenges in the swine host. influenza virus is one of the most important respiratory pathogens worldwide. , type a influenza causes an acute disease of the upper airways, and affects - million persons yearly. moreover, the threat of human influenza epidemic and pandemic has dramatically increased in recent years. vaccination is one of the crucial interventions for reducing the spread and impact of influenza. the generally used parenteral inactivated influenza vaccines induce mainly systemic antibody responses and only weak cell-mediated immunity and low levels if any mucosal immunity. on the other hand, intranasal immunization with live virus can induces a broad spectrum of both systemic and mucosal antibodies, and the immune response localized in the mucosa blocks the virus even during the first phase of infection. unfortunately, the use of live vaccines is always associated with a certain risk. the development of a crossprotective vaccine against potentially pandemic strains is an essential part of the strategy to control and prevent a pandemic outbreak. we induced intrasubtypic and intersubtypic cross-protection in balb ⁄ c mice by intratracheal (it) immunization with inactivated influenza viruses together with dead delipidated bacillus firmus (dbf) as an adjuvant. ten days after the nd immunization dose, the mice were infected with live influenza virus b ⁄ lee ⁄ lethal for mice (total infection dose corresponded to · ld ) or a⁄ pr ⁄ (total infection dose corresponded to ae - ld ). dbf adjuvant markedly increased both systemic and mucosal anti-viral antibody formation when applied together with inactivated influenza a or b viruses. protective significance was tested in vivo. mice were preimmunized with ) pbs (controls), ) dbf alone, ) virus alone, and ) vir-us+dbf. influenza b virus strains b ⁄ lee and b ⁄ yamanashi ⁄ ( years phylogenetically distant and antigenically substantially different, especially in terms of the main protective antigen -surface haemagglutinin) or two different influenza a subtypes -a ⁄ pr ⁄ (h n ) and a ⁄ california ⁄ (h n ) -were used (figures and ) . the mice were challenged with · ld of either b ⁄ lee ⁄ or a ⁄ pr ⁄ as appropriate. all controls died. the mice treated with dbf alone died with a delay or survived, which could be explained by stimulation of innate immunity. the animals immunized with virus alone were protected against homologous strains. adjuvant immunization was cross-protective: the mice immunized with a heterologous b strain (figure ) fell ill (pronounced body mass loss), but almost all survived and recovered. the mice immunized with a heterologous a subtype were excellently protected (negligible weight loss and zero mortality). intratracheal dbf ( lg per mouse) given to non-immunized mice hour before influenza infection eliminated the lethal effect in - % of infected animals depending on infection dose ( ae - ld ); in mice infected with lower than lethal doses ( ae ld ), weight loss was minimized or did not occur. the current mode of vaccination-induced immunity is mostly effective against a homologous strain of the virus used for vaccination. the attention is therefore focused on vaccines that are able to induce cross-protection and could be effective also in case of sudden appearance of a new virus variant. inactivated influenza viruses are known to be often insufficiently effective when used for mucosal immunization and for induction of cross-protection against drifted influenza viruses or novel subtypes. the drawback of vaccination with dead virus can be overcome by using a suitable adjuvant. mouse models were successfully immunized with vaccine containing inactivated virus in combination with cholera toxin or the escheria coli heat-labile toxin (lt). [ ] [ ] [ ] the use of cholera toxin in humans is precluded because of its high toxicity; a number of lt mutants that retain their adjuvant activity have been prepared; these mutants were likewise tested on the mouse model and should not cause any serious side effect in humans. for this reason, current studies aim at finding a suitable and safe mucosal and systemic immune response. dbf has been shown to be a very efficient adjuvant for mucosal immunization stimulating both innate and adaptive immunity. intratracheal immunization with inactivated influenza viruses and dbf as adjuvant induced efficient and even heterosubtypic cross-protection. dbf given hour before infection provided partial protection probably because of its strong stimulatory effect on the innate immunity. temperature-sensitive and cold-adapted candidates for live attenuated influenza vaccine with genomic composition of : based on highly pathogenic influenza a ⁄ h n viruses with pandemic potential were generated by the replacement of six internal genes from the influenza a ⁄ puerto rico ⁄ ⁄ (pr ) virus from pr -based rg-candidates for inactivated vaccine with appropriate internal genes of influenza a ⁄ leningrad ⁄ ⁄ ⁄ (h n ) master donor virus (mdv) for russian laiv by methods of classical reassortment. all attempts to capture avian n neuraminidase into the genome of the mdv laiv production were ineffective. : reassortants were not generated. step by step co-infection of triple reassortants (h n -h n -h n ) with h n mdv in some cases was the only possibility to generate influenza a ⁄ h n cold-adapted vaccine reassortants. difficulties in generating : reassortants could be explained by a substantial gene constellation in the genome of pr based h n reassortant viruses. strong coupling of pb ⁄ pr and avian n genes in a ⁄ h n -pr -rg reassortants was revealed. annually updated laiv strains are generated by classical reassortment of circulating influenza viruses with well characterized, attenuated, ts ⁄ ca mdvs. resulting attenuated reassortants inherit the relevant ha and na of wild type parental virus and six internal genes of the mdv. candidates for inactivated influenza vaccines based upon avian influenza viruses with pandemic potential are generally generated by reverse genetics methods. in these cases, like with laiv, vaccine strains are : reassortants which possess the modified ha and na from potentially pandemic virus and six internal genes from the pr virus. the pr virus is considered to be of low virulence, i.e. attenuated, for humans, yet offers properties of high seed virus growth for influenza vaccine production. the ha of avian h influenza viruses with pandemic potential is engineered to remove four basic amino acid codons from the cleavage site of ha, resulting in a virus that is considered attenuated for natural hosts and safe for people. the objective of this study was to safely generate vaccine candidates for a laiv using highly pathogenic avian influenza viruses by the replacement of six internal pr genes in the genome of candidates for inactivated vaccine subtype h n (a ⁄ h n -pr -rg) with internal genes of the laiv mdv by methods of classical reassortment. len -mdv and a ⁄ h n -pr -rg virus were co-infected in embryonated chicken eggs. five rounds of selective propagation were performed, three of which were at low temperature ( °c). the production and selection of reassortants were carried out in the presence of rabbit antiserum to len -mdv. cloning by endpoint dilution was performed in each of the last three passages. a virus sample in an open petri dish was rocked gently for sec while being irradiated with a ge watt germicidal lamp at a distance of cm from the dish. the residual infection titer was measured by titration in embryonated chicken eggs. genome composition of reassortant viruses was monitored by rflp analysis. in addition, capacity of reassortant viruses to grow at optimum, low, and elevated temperatures (ca ⁄ ts phenotype) for influenza viruses was determined by virus titration in chicken eggs. reassortment of the mdv with the vn-pr or indo-pr viruses either resulted in reassortants that contained six internal genes from len -mdv. however, all generated clones contained the na from the mdv. of ten such : reassortants based on vn-pr three reassortants had the pa gene from pr and one had ns gene from pr . : reassortants from the targeted h n composition were not generated. after repeated attempts, : temperature sensitive and cold adapted reassortants based on vn-pr and indo-pr viruses were obtained, but again, none had inherited the avian n neuraminidase (table ) . in contrast, nibrg- didn't reassort with the mdv at all. twelve unsucsessful attempts to develop : or : reassortants of nibrg- with mdv showed that the classical reassortment procedure (cloning by limited dilutions in the presence of anti-mdv serum, followed by co-infection of equal doses of two parental viruses in eggs and two selective passages at °c) did not work for this virus pair. to disharmonize the incredibly strong gene constellation of nibrg- , various modifications of the co-infection step were studied, such as: altering the nibrg- to mdv ratio (from : to : tions of anti-mdv serum alone or together with anti-pr serum. it was noted that even if the h n to mdv ratio was : , the clones obtained were presumably parental h n viruses without the transfer of any mdv-genes into genome of nibrg- . in all, clones were isolated, and of them were identical to nibrg- parental virus. in nine clones, only the pa gene from mdv was included, whereas in three clones only the 'cold' ns gene was included (data not shown). using uv inactivation of nibrg- prior co-infection was more encouraging. after the first round of co-infection of partially uv-inactivated nibrg- with mdv (at ratio : ), reassortants that inherited several internal genes of mdv were obtained in the context of the nibrg- background (b , c , c , d ) ( table ). some of them (c , c , d ) were chosen for the next round of co-infection. after the second round of co-infection, c , c , and d 'intermediate' reassortants with mdv (at ratio : or : ) : vaccine reassortants finally were obtained. live attenuated influenza vaccine is considered as one of the most promising pandemic vaccines. according to the who there is evidence that laiv might be more effective than inactivated vaccines. this study attempted the safe development of laiv for potential pandemic highly pathogenic avian a ⁄ h n viruses on the base of rg-reassortants for inactivated vaccine with modified h hemagglutinin and mdv for laiv. replacement of pr based internal genes into genome of vn-pr and indo-pr reassortants with appropriate genes of mdv was realized by the classical reassortment procedure. difficulties were encountered in obtaining : reassortants that contained both the ha and na from the wild type avian h n parental virus. in attempts to reassort the nibrg- with mdv, the classical reassortment procedure was unsuccessful. the challenge faced was to break an incredibly strong gene constellation of the nibrg- virus. partial uv-inactivation of nibrg- was encouraged in replacement of some pr internal genes with mdv genes in some cases avian-human reassortant viruses with gull h n and human influenza h n genes were difficult to generate, and reassortants with the desired genotype of six gull virus genes with human influenza a h and n genes were not isolated despite repeated attempts. the gull pb , np, and ns genes were not present in any of the gull-human h n reassortants generated. it is difficult to fully understand potential reasons for observed difficulties to reassort some avian viruses with human strains. unsuccessful attempts to develop : vaccine reassortants may be caused by an observed strong connection of pb and na genes in the genome of a ⁄ h n -pr -rg viruses. in our attempts, each reassortant that possessed avian n neuraminidase inheritied pb gene of pr as well. and vice versa, the 'cold' pb gene always appeared to be coupled with the n neuraminidase of the mdv. in some cases, step by step co-infection of triple reassortants (h n -h n -h n ) with h n mdv may be the only possibility to generate a cold-adapted vaccine reassortant. our studies demonstrate unique and significant challenges that are faced in the development of influenza vaccines for avian influenza viruses with pandemic potential. such challenges must be further studied to identify methodologies to allow for rapid development and response to emerging viruses in a crisis. it is imperative that these studies be continued and expanded to identify either mechanisms of such tight gene constellations in influenza viruses produced by rg-derived vaccine strains or inability some genes of human h n and avian h n viruses to cross. in addition, further studies to improve the efficiency of classical reassortment processes will be conducted. during the period from to , avian influenza outbreaks among humans have been registered in countries of asia, europe, and africa. morbidity and mortality of humans followed the global spread of avian influenza h n among wild and domestic birds, which caused great economic loss to the poultry industry in many regions including some highly developed countries. the global threat from avian influenza forced scientists to develop technologies for the production of a ⁄ h n human vaccine. the development of ai a ⁄ h n vaccines using strains isolated in kazakhstan and the organization of local production and creation of strategic stockpiles of effective vaccines is the an important issue for public health protection in the republic of kazakhstan. to address this, a scientific program 'influenza a ⁄ h n vaccine development for public health protection in kazakhstan' was approved and financed from to . in this article we give basic results of the development of a recombinant ai a ⁄ h n inactivated whole virion vaccine with aluminium hydroxide as adjuvant for public health protection in kazakhstan. [ ] [ ] [ ] the development of vaccine technology was conducted with the use of a ⁄ astanarg ⁄ : ⁄ (a ⁄ h n ) recombinant strain made of a ⁄ chicken ⁄ astana ⁄ ⁄ (h n ) and a ⁄ pr ⁄ ⁄ (h n ) strains by the reverse genetics. inactivation of virus containing allantoic fluid was carried out with the use of formalin in different concentrations. complete-ness of the virus inactivation was tested by -fold virus passaging in embryos. , purification and concentration of the inactivated viruscontaining allantoic fluid was conducted with the use of ultra filtration in tangential flow, which was followed by gel filtration. then we evaluated the content of total protein, hemagglutinin, and ovalbumin in purified and concentrated material. vaccine was composed of clarified and inactivated virus concentrate with the known ha dose containment, and ae % aluminum hydroxide was added in : proportions. composition components and quality control of finished vaccine was determined in the stages of semi-finished product and finished biopreparation. determination of quantitative ovalbumin content was conducted by elisa applying a strip test-system chicken egg ovalbumin elisa kit cat. n (alpha diagnostic international, usa). vaccine immunogenicity was evaluated by hai micro test in u-bottom -well plates produced by 'costar' (usa). vaccine apyrogenicity was evaluated after intravenous injection of the studied preparation to rabbits. , for confirmation of the results vaccine series were tested for bacterial endotoxins with the use of limulus amebocyte lysate produced by charles river laboratories, inc. usa. the vaccine toxicity was evaluated in white mice with body weight - gm and in rats with body weight - g both males and females according to glp principles. allergenic characteristics of the inactivated vaccine was determined in white outbred mice and guinea-pigs both males and females according to 'methodic guideline for evaluation of allergenic characteristics of pharmacological substances'. in the first series of experiments, we conducted work for obtaining influenza a(h n ) recombinant strain. bidirectional expression plasmid phw_b with full-length sequences of ha and na gene segments of the strain a ⁄ chicken ⁄ astana ⁄ ⁄ (h n ) isolated in kazakhstan were synthesized in geneart ag, (regensburg, germany). ha gene was modified by deleting the region encoding multiple basic amino acid rrrk motif in ha cleavage site. moreover, to prevent recovery of repeating basic amino acids motif due to polymerase slide, we inserted replacements g fi t and k fi t. thus the ha cleavage site consists of the following sequence ntpqgerrrkkrglfgai ntpqtetrglfgai. the basic amino acid motif of highly pathogenic strain a ⁄ chicken ⁄ astana ⁄ ⁄ (h n ) was replaced by the sequence tetr ⁄ glf, which is characteristic of low pathogenic strains of influenza h n . sequence of gene coding na in the strain a ⁄ chicken ⁄ astana ⁄ ⁄ (h n ) was cloned without modifications. the other segments pb , pb , pa, np, m and ns were obtained from influenza virus ivr- and synthesized and cloned in two-forked expression plasmid phw_b in geneart ag company, germany. the origin of genetic segments of vaccine strain a ⁄ astanarg ⁄ : ⁄ (h n ) is presented in table . vero cell culture ( passage) (who) was received from european cell culture collection (salisbury, wiltshire sp jg, great britain). the cell culture was grown in dmem ⁄ f medium with the addition of % of fetal bovine serum and mm l-glutamine. to obtain reassortant virus a ⁄ astana ⁄ ⁄ r- : , vero cells were infected with correlative plasmids by way of electroporation using nucleofector ii (amaxa) equipment. infected cells were placed in -well plates. after hour, dmem ⁄ f medium was changed into ml of opti-pro sfm (gibco) medium adding mm l-glutamine and lg ⁄ ml trypsin. two days after cytopathic effect appearance supernatant was collected and used for infection of spf-eggs. the virus a ⁄ astanarg ⁄ ⁄ - : was grown in chicken embryos, and then virus titer was determined in chicken embryos and madine-darby canine kidney (mdck) cell culture. the titer of two final a ⁄ astanarg ⁄ - : virus stocks was ae log eid ⁄ ml (chicken embryos); ae log tcid ⁄ ml (mdck cells); ha titer : . a ⁄ chicken ⁄ astana ⁄ ⁄ (h n ) virus contains motif of repeating basic amino acids in ha cleavage site. it is known that this sequence is the main determinant of ai virus pathogenicity. that is why this site was deleted in vaccine candidate strain. sequence results confirmed that influenza virus a ⁄ astanarg ⁄ ⁄ r- : strain ha gene sequence contains modified ha cleavage site and keeps mutations inserted for prevention of return to virus wild type. to confirm stability of modified ha gene sequence, five additional passages of recombinant strain a ⁄ astana rg ⁄ ⁄ - : were conducted in chicken embryos. sequencing and following phylogenetic analysis of the recombinant strain a ⁄ astana rg ⁄ ⁄ - : ha gene sequence proved the presence of modification in ha cleavage site. deletion of pathogenicity site of the obtained virus was confirmed by lethality test for chicken embryos, intravenous pathogenicity test in chicken, and in plaque-forming test with trypsin. pathogenicity test in chicken embryos showed that recombinant strain a ⁄ astanarg ⁄ - : is capable of growing up to high titers without causing embryos' death. a ⁄ astanarg ⁄ - : strain pathogenicity evaluation was conducted in - week-age white leghorns chicken, and this study proved that the strain a ⁄ astanarg ⁄ - : (h n ) is not virus pathogenicity inductor in chickens, which got intravenous injections of this virus (pathogenicity index is equal to ). h n strain ha cleavage site modification provides its cleavage capability only with tripsin-like proteases, which shows low level of pathogenicity. aiming at confirmation of ha cleavage site modification, we experimentally studied virus replication ability both with trypsin and without this enzyme. and we got the following results. in the plaque-forming test, a ⁄ astanarg ⁄ - : strain produced plaques in mdck cells only with trypsin, proving the trypsin-dependent phenotype characteristic of low pathogenic avian influenza viruses. to prove the ha subtype antigenic analyses of a ⁄ astana ⁄ ⁄ r- : strain was conducted by means of serological methods in hemagglutinin inghibition test with the use of postinfection antisera of rabbits and rats (influenza research institute swd rams), standard serum received from cdc, atlanta, usa. hai test proved that a ⁄ astana ⁄ ⁄ r- : strain belongs to h subtype. furthermore, toxicity of vaccine candidate strain was evaluated by way of subcutaneous injection of viral material to balb mice. the strain appeared to be non-toxic for white mice getting subcutaneous injection of ae ml of the preparation. the conducted research showed that according to all tested characteristics, a ⁄ astana ⁄ ⁄ r- : strain can be used for influenza a ⁄ h n inactivated vaccine production. according to its genetic characteristics, this strain belongs to the group of vaccine strains recommended by who for the development of influenza pre-pandemic inactivated vaccines. we determined basic cultivation parameters of the recombinant strain a ⁄ astanarg ⁄ - : in - day chicken embryos. the determined parameters are the following: infection dose, - eid ; cultivation period, hour; incubation temperature, °c. these cultivation parameters allow obtaining virus containing material with biological eid and hemagglutinating activity of ae - ae log eid ⁄ cm and : ha titre and even higher. in the next series of experiments, we conducted research on the determination of optimal sequence of technological stages of virus clarification, concentration, and inactivation in the order of vaccine production. samples of viral material were subjected to inactivation before and after clarification and concentration. the regimen of virus inactivation by formaldehyde with final concentration of ae %, period of inactivation of days, temperature of inactivation medium of - °c, ph of inactivation medium of - ae . on the basis of the conducted experiments we determined that the selected regimen of inactivation provides complete and irreversible inactivation of viral suspensions of the hpai strain irrespective of the kind of inactivated material. we did not observe reduction of ha activity in non-clarified viral suspensions. however, when we inactivated clarified and concentrated material, ha activity reduced by an order of magnitude. comparison of forms and sizes of virion structural elements in native (non-clarified) and formalin inactivated preparations did not reveal any significant differences. concentration of virus particles in the studied preparations was similar. the selected inactivation regimen provides obtaining completely avirulent viral suspension of the strain a ⁄ astanarg ⁄ - : , and it does not influence the structure of the virus. on the basis of the experiments results, we selected method of viral allantoic fluid inactivation without preliminary clarification. during further research, we tried to get highly clarified viral concentrate. this study resulted in the combined scheme, which includes clarification of inactivated viral allantoic fluid by low speed centrifugation at circulations per min for minutes, filtration through membrane filters with pore diameter of ae lm, ultrafilatration ⁄ diafiltration, gel filtration in b sepharose, and sterilization of viral suspension through membrane filters with pore diameter of ae lm. the experiments resulted in the development of production technology of embryonic inactivated vaccine based on recombinant strain a ⁄ astanarg ⁄ : ⁄ (h n ) contain-ing aluminium hydroxide as adjuvant. the developed influenza a ⁄ h n human vaccine has the trade name kazfluvacÒ. its composition components are presented in table . preclinical testing of the vaccine kazfluvacÒ was conducted according to the following parameters: general health condition of animals, change of body weight and temperature of immunised animals (for ferrets), presence of post vaccination antibodies response in sera, forming protective immune response against reassortant viruses of h subtype, study of acute and chronic toxicity of three experimental vaccine series in different doses and semi-finished vaccine product applying different ways of injection, study of allergic and immunotoxic characteristics of the vaccine, as well as study of pyrogenic reaction and analysis for bacterial endotoxins presence. [ ] [ ] [ ] [ ] preclinical tests of kazfluvacÒ vaccine safety showed that this vaccine does not have toxic effect on organisms of warm-blooded laboratory animals. double intramuscular injection of kazfluvacÒ vaccine in inoculative dose does not effect appearance, general health condition, behaviour of animals, their muscular strength and physical activity, does not have negative effect on biochemical parameters of blood and basic physical functions of animals organism, and does not cause pathomorphological changes. this shows the safety of the vaccine. local irritation action was not observed. the results of the vaccine allergic action study showed that the vaccine does not have allergic effect at the intravenous injection. the research also showed that the vaccine does not have negative effect on immune system of laboratory animals. research conducted on mice and ferrets showed high immunogenic activity of the vaccine at one-and two-dose regimen of injection. the research showed % of protective effect of kazfluvacÒ vaccine at two-dose injection regimen in ferrets infected by homological strain of influenza virus. the devised inactivated influenza a ⁄ h n vaccine kaz-fluvacÒ is a safe and immunogenic biopreparation that is not worse than the overseas analogues in its immunobiological characteristics. [ ] [ ] [ ] [ ] to date the whole-virion inactivated influenza a ⁄ h n vaccines of the producers such as omnivest (hungary), biken, denka seiken, kitasato institute, kaketsuken (japan), gsk biologicals (belgium), sinovac biotech (china) are registered. all of them are produced on the basis of chicken embryos and aluminum is used as an adjuvant. kazfluvacÒ differs from its analogues in the flowchart of the virus purification and concentration that makes possible to produce a safer preparation. , the results of the conducted research and preclinical testing allow starting work towards implementation of phase i preclinical tests on volunteers. it is planned to conduct a randomized blind placebo-controlled phase i study on double application of kazfluvacÒ vaccine in increasing doses. the preparation will be administered to volunteers aged - years for assessment of its safety and immunogenicity in doses of ae and ae lg of ha. when the world health organization (who) announced the sixth phase of a ⁄ h n v influenza pandemic, scientists all over the world started investigation to develop technology for production of prophylactic means against the disease. having taken into consideration the threat of a pandemic for kazakhstan, the ministry of education and science of the republic of kazakhstan launched the program ''monitoring, study, and development of diagnostic, prophylactic, and therapeutic means for influenza a ⁄ h n .'' this paper presents the experimental data obtained at the ribsp in the course of the studies towards the development of technology for production of an inactivated a ⁄ h n influenza vaccine, as well as the results of pre-clinical testing of the developed vaccine. the development of vaccine production technology was conducted with the use of who recommended vaccine strain nibrg- xp constructed by the method of reverse genetics in the national institute for biological standards and control (nibsc, great britain). the virus was inactivated with formalin at different final concentrations, and the extent of inactivation was evaluated via threefold virus passages in developing chicken embryos. the inactivated virus was purified and concentrated by the method of ultrafiltration in tangential flow followed by gel filtration. the purified and concentrated material was evaluated judging on the total protein, hemagglutinin (ha), and ovalbumin. the vaccine was prepared by pooling the purified and concentrated virus material with the certain weight content of ha and the work solution of aluminum hydroxide ( ae %) in the ratio : . the ovalbumin content was quantified in elisa with the use of the strip test system chicken egg ovalbumin elisa kit (cat. no. alpha diagnostic international, san antonio, texas, usa). weight content of the virus ha was determined according to sominina, burtseva. the content of the residual formaldehyde, aluminum (al + ) ions, and thiomersal in the vaccine was measured according to the operating instructions. the vaccine immunogenicity was assessed in the hemagglutination inhibition test, which was carried out as a microassay in -welled u-bottomed plates (''costar'', new york, usa). , apyrogenicity of the vaccine was assessed post intravenous administration of the tested preparation to rabbits. , to confirm the obtained results the vaccine batches were tested for bacterial endotoxins with use of the limulus amebocyte lysate (charles river laboratories, inc., wilmington, ma, usa). the toxicity of the vaccine was assayed in white mice weighing - g and in rats weighing - g (male and female) in compliance with the principles of good laboratory practice. allergenic properties of the inactivated vaccine were determined according to the ''operating instructions on assessment of allergenic properties of pharmaceutical substances'' in white outbred laboratory mice and guinea-pigs of both sexes. the first step in the course of developing technology for vaccine production was to determine the major conditions for influenza virus cultivation: usage of -days embryonated chicken eggs at the infectious dose within - eid , incubation temperature ( ± ae )°c, and duration of the incubation period hours. the established parameters for virus cultivation made it possible to produce virus-containing materials of infectious activity within ae - ae log eid ⁄ cm and hemagglutinating activity : and higher. in the subsequent experiments, an optimal method for virus inactivation was selected. on the basis of the experimental findings, the following conditions for inactivation of the native virus-containing material were elected: formalin of ae % final concentration as an inactivating agent; inactivation period of hours at temperature ( ± )°c. these conditions provide the complete inactivation of the virus (nibrg- xp strain) material, did not impact distinctly the structural organization of the virus, and did not reduce the antigenic activity. as it is well known, virus purification and concentration means very much in the development of technology for production of an inactivated whole-virion influenza vaccine. the investigation into optimization of the technological step of purification and concentration of the recombinant influenza virus nibrg- xp strain resulted in selection of an optimal pattern including such steps as clarification of the virus suspension by filtration through membranes with pore size ae lm, virus concentration by ultrafiltration in a tangential flow, dialysis filtration in a tangential flow, gel filtration on sepharose b, and sterilization of the viral suspension through membrane filters with pore size ae lm. the studies conducted by the ribsp specialists resulted in the development of technology for production of the first domestic whole-virion inactivated a ⁄ h n influenza vaccine with aluminum hydroxide as adjuvant and with the brand name refluvac Ò . the key processing characteristics of the whole-virion inactivated a ⁄ h n influenza vaccine vaccine refluvac Ò are shown in table . simultaneous with the performance of all process operations, the parameters such as sterility, inactivation extent, ph, vaccine specificity, total protein content, weight content of has, aluminum and formalin contents, content of thiomersal, and ovalbumin, pyrogenicity of the vaccine and its immunogenicity for mice, were optimized. the key qualitative characteristics of the designed influenza a ⁄ h n vaccine refluvac Ò are shown in table . before implementation of phase i clinical trials on volunteers, preclinical testing of three experimental batches of refluvac for immunogenic activity and safety was carried out. it was conducted in three laboratory bases of research institutions: the toxicology institute ⁄ federal medicobiological agency, russia (st petersburg), the research institute for biological safety problems (republic of kazakhstan), and the influenza research institute ⁄ north-western branch of the russian academy of medical sciences (st petersburg), with use of different animal models (mice, rats, chinchilla rabbits, guinea-pigs, ferrets). the results of the preclinical testing are as follows: • electron microscopy of the preparation has shown that the viral particles are well dispersed and do not aggregate. the portion of whole (intact) particles is over %, which is evidence of virion integrity; • assessment of polypeptide composition of the vaccine refluvac by electrophoresis in % polyacrylamide gel with sodium dodecyl sulfate has shown the vaccine to contain both surface antigens (ha, na) and highly purified inner virion proteins (np, m ) that are typespecific antigens, so the vaccine is a preparation of full immunological value; • judging on the parameters of acute and chronic toxicity for white mice and rats of both sexes, the vaccine is a non-toxic and safe preparation; • under conditions of a chronic experiment on white mice and rats, it was found that refluvac does not produce changes in behavior, somatic, or vegetative responses; • assay of hematological and biochemical blood characteristics of white mice and rats following vaccine administration did not reveal any significant differences as compared to the animals of the control group; • refluvac does not cause allergenic and immunotoxic impact; • the vaccine refluvac does not cause local irritative effect; • refluvac is apyrogenic for laboratory animals; • the pathomorphological and hystopathological analysis did not reveal any changes due to immunization in animal organs; • testing of immunogenic characteristics of the vaccine on mice and ferrets has shown formation of hemagglutinating antibodies in animals after single administration; • refluvac induces % protection in immunized ferrets at their challenge with the wild-type influenza virus a ⁄ california ⁄ ⁄ (h n v). the results of the performed preclinical testing have allowed concluding that refluvac, an inactivated whole-virion vaccine with aluminum hydroxide as adjuvant, is a safe and highly effective preparation against influenza a ⁄ h n v. the implemented study resulted in development of technology for production of the first domestic inactivated allantoic whole-virion influenza a ⁄ h n vaccine with aluminum hydroxide as an adjuvant under the brand name refluvac Ò based on the recombinant strain nibrg- xp. the devised pandemic vaccine meets who requirements as well as requirements concerning safety and immunogenicity of the national pharmacopeias of the republic of kazakhstan and russian federation. [ ] [ ] [ ] [ ] the devised technology for vaccine production differs from the previous technologies for production of allantoic whole-virion influenza a ⁄ h n vaccines in its processdependent parameters. presence of an adjuvant (aluminum hydroxide) increases significantly the vaccine immunogenicity and allows maximal reduction of the dose of the administered antigen that, in turn, results in diminished reactogenicity of the vaccine. aluminum hydroxide is an adjuvant that is most frequently used in clinical practice. to date the results of the double-centered randomized study of the europe-licensed vaccine fluval p [monovalent inactivated whole-virion influenza vaccine with aluminum phosphate based on strain a ⁄ california ⁄ ⁄ (h n ) nymc x- a (omninvest, pilisborosjeno, hungary)] that is similar to the refluvac preparation are published. the data of this research are an evidence of safety and high immunological effectiveness of the vaccine in dose lg ha at single administration both in adults and elderly persons. the results of the pre-clinical tests allow recommending carrying out phase clinical testing of the refluvac Ò vaccine for safety and immunogenicity. single immunization of volunteers with refluvac Ò in doses ae , ae , and ae lg of ha are planned. mid , respectively. the study results confirm that new h n laiv and h n laiv candidates are safe and immunogenic and confer protection from homologues influenza virus infection in mice. the recent emergence of a new pandemic h n virus and the threat of transmission of avian viruses to humans had stimulated research and development of live attenuated cold-adapted influenza vaccines against newly appeared influenza viruses. formulations of live attenuated influenza a vaccine (laiv) against pandemic influenza strains, including h n , h n , h n , and h n are currently being tested in preclinical and phase i clinical studies. the following paper describes the preclinical study of new h n and h n laiv candidates in mice. the study addressed the following three objectives: (i) to demonstrate that cold-adapted (ca) reassortant influenza a(h n ) and a(h n ) vaccine candidates are indistinguishable from the parental a ⁄ leningrad ⁄ ⁄ ⁄ (h n ) master donor strain (mds) virus with regard to replication efficiency in upper and lower respiratory tract of mice; (ii) to demonstrate the immunogenicity of different doses of cold-adapted (ca) reassortant influenza a(h n ) and a(h n ) vaccine candidates in mice; and (iii) to demonstrate the protective efficacy of cold-adapted (ca) reassortant influenza a(h n ) and a(h n ) vaccine candidates in mice against a homologous wild-type virus challenge. the a ⁄ ⁄ mallard ⁄ netherlands ⁄ ⁄ (h n ) reassortant containing the ha and na genes from a ⁄ mallard ⁄ netherlands ⁄ (h n ) and six other genes from mds, the a ⁄ ⁄ california ⁄ ⁄ (h n ) reassortant containing the ha and na genes from a ⁄ california ⁄ ⁄ (h n ) and six other genes from a ⁄ leningrad ⁄ ⁄ ⁄ (h n ) were generated by classical genetic reassortment in embryonated chicken eggs (ec). viruses were propagated in days old eggs ( °c, hours). fifty percent egg infectious dose (eid ) titers were determined by serial titration of viruses in eggs. titers were calculated by the method of reed and muench. female balb ⁄ c mice, - weeks of age were used in all experiments. mice were lightly anesthetized with ether and then inoculated intranasally (i.n.) with ll of infectious virus diluted in phosphate-buffered saline (pbs). mice were inoculated with mid ( % mouse infectious dose) of a ⁄ ⁄ california ⁄ ⁄ (h n ), a ⁄ ⁄ mallard ⁄ netherlands ⁄ ⁄ (h n ), and a ⁄ leningrad ⁄ ⁄ ⁄ (h n ) mds. viral loads were measured in respiratory and brain tissues collected at and days post-infection (dpi). tissue homogenates prepared using a disruptor and clarified supernatants were titrated on eggs at permissive temperature to determine infectious concentrations. groups of animals were inoculated with mid or mid of either h n laiv or h n laiv intranasally after collecting a pre-immunization blood sample. a second blood sample was collected at dpi. on the same day, the animals received a second intranasal inoculation with the same virus that was used for priming at dpi. to assess protection, all animals were infected dpi with either mid of a ⁄ california ⁄ ⁄ (h n ) or mid a ⁄ mallard ⁄ netherlands ⁄ (h n ) virus by the intranasal route. four animals from each group were euthanized at dpi, and the respiratory and systemic organs were harvested for virus titration. a forth blood sample was collected at dpi from the remaining animals. hi antibody titers were determined for individual serum samples collected on days , , , and . body weights were taken daily following challenge through day postchallenge. sera were tested for hi against homologous h n and h n viruses. the h n laiv, h n laiv and h n mds influenza viruses replicate in mice lungs at level ae - ae lgeid ⁄ ml at dpi (figure ). at dpi, replication of the viruses in the lungs decreased to ae - ae lgeid ⁄ ml (data not shown). in contrast, the wild-type virus a ⁄ mallard ⁄ netherlands ⁄ (h n ) demonstrated high level replication in lungs - ae lgeid ⁄ ml. the levels of replication of studied viruses in nasal turbinates were ae - ae lg eid ⁄ ml at dpi (figure ) , and ae - ae lgeid ⁄ ml at dpi (data not shown). there were no significant differences between the viruses in regard to replication in upper respiratory tract of mice. thus, it was shown that a ⁄ ⁄ mallard ⁄ netherlands ⁄ ⁄ (h n ) and a ⁄ ⁄ california ⁄ ⁄ (h n ) vaccine candidates was indistinguishable from parental a ⁄ leningrad ⁄ ⁄ ⁄ (h n ) in terms of replication in the lungs and noses of mice at and dpi. no virus was found in the brain tissue of immunized mice at and dpi (in undiluted samples tested). thus, it was shown that a ⁄ ⁄ mallard ⁄ netherlands ⁄ ⁄ (h n ), a ⁄ ⁄ cali-fornia ⁄ ⁄ (h n ) vaccine candidates are identical to a ⁄ leningrad ⁄ ⁄ ⁄ (h n ) in lacking neuroivasive capacity, and all three viruses similarly fail to replicate in the brain. it was shown that all immunized animals survived after challenge with wild-type a ⁄ mallard ⁄ netherlands ⁄ ⁄ (h n ) virus. the mice in vaccine groups showed no signs of morbidity. average weight changes were tracked from day to day in all study groups, but the changes did not exceed %. as shown in figure , the challenge virus actively replicated in respiratory tissue taken from mock immunized animals ( ae lgeid in the lung and ae lgeid in the nose), but failed to infect the brain and spleen. on the other hand, in both h n laiv vaccinated groups, all tested organs were free from presence of challenge virus. thus, immunization of mice with either mid or mid h n laiv protected the animals from the subsequent challenge infection with a homologous with wild-type h n virus. both h n and h n laiv candidates were found to be immunogenic. after one dose of mid of h n laiv, gmt of hi antibodies were ae . one dose of mid or mid h n laiv elicited hi antibody level with gmt of ae and ae , respectively. the second dose of h n laiv further stimulated serum hi antibody levels to gmt ae and ae , for mid or mid , respectively (data not shown). the mouse model is widely used to better understand the pathogenicity of avian influenza viruses for mammalian species, to be able to predict the pandemic potential of such viruses, and to develop improved methods for the prevention and control of the virus in a potential pandemic. a subset of the h viruses was evaluated for the ability to replicate and cause disease in balb ⁄ c mice following intranasal administration. h subtype viruses were able to infect mice without adaptation and manifested different levels of lethality and kinetics of replication. there is limited preclinical information available for laiv. thus, live monovalent vaccine against pandemic influenza virus h n (influvir) was tested for acute toxicity and its effect on the systems and organs of laboratory animals. according to toxicology and necroscopy results, the live monovalent influenza vaccine influvir, when applied intranasally, was safe and was well tolerated. in our current study we demonstrate that a(h n ) and a(h n ) laiv are indistinguishable from the parental mds virus with regards to replication kinetics in the upper and lower respiratory tract of mice. both h n and h n laiv candidates were immunogenic and protect mice against subsequent a challenge with the wild-type virus. live attenuated cold-adapted (ca) influenza vaccines are an effective means for the control of influenza, most likely due to their ability to induce both humoral and cellular immune responses. in our study we confirm that new h n laiv and h n laiv candidates are safe, immunogenic, and confer protection from influenza infection in mice. health organization (who) declared a pandemic by raising the worldwide pandemic alert level to phase . therefore, h n inactivated monovalent vaccine formulated with our proprietary oil-in-water emulsion based adjuvant was evaluated in ferrets for its potential to induce with low antigen dose efficient, robust, and rapid protective immunity against a wild type challenge virus (a ⁄ netherlands ⁄ ⁄ ). this adjuvant was also tested in ferrets in a h n avian influenza model for its ability to induce a cross-clade immunity and cross-protection. two independent studies (a&b) were carried out with male and female outbred ferrets (musleta putorius furo) in compliance with ''guide for the care and use of laboratory animals,'' ilar recommendations and aaalac standards. ferrets used in both studies were influenza seronegative by anti-nucleoprotein elisa and by hi assay against the pandemic and seasonal strains. in study a, four groups of seven ferrets aged approximately of months received one or two im vaccinations weeks apart of either af -adjuvanted ( ae lg of ha with af ) or unadjuvanted ( body weight loss was monitored as an indicator of disease and a mean body weight loss of % was recorded in the control group at day of necropsy. body weight loss was reduced to £ % and £ % in animals that had received and doses of either unadjuvanted or af -adjuvanted vaccine, respectively. viral lung titration showed high levels of virus replication ( ‡ ae tcid ⁄ g tissue) in the lungs of all control ferrets days after challenge. one or two administrations of unadjuvanted vaccine reduced lung viral load by and log , respectively. interestingly, ferrets that received either one or two doses of af -adjuvanted h n vaccine, showed significantly greater reduction of lung viral loads (> log ). no virus was detected in the lungs of ⁄ ( %) animals immunized with a single injection of the af -adjuvanted vaccine and in % of ferrets vaccinated twice. assessment of viral shedding from the upper respiratory tract showed that the af -adjuvanted a ⁄ h n monovalent vaccine was able to reduce the viral load in the nose and in the throat by ae and ae log , respectively, as compared to the control group. conversely, viral loads were only slightly reduced in the nose and mostly unchanged in the throat in ferrets immunized with either one or two doses of unadjuvanted a ⁄ h n monovalent vaccine. gross pathology and histology examinations revealed lung lesions consistent with influenza a ⁄ h n virus infec- however, a second dose of af -adjuvanted vaccine strongly increased hi and mn titers, which persisted for months (table ). antibody responses cross-reactive to heterologous clade . strain were elicited ferrets vaccinated with the af -adjuvanted clade . vaccine. hi antibody titers ‡ crossreactive to clade . and persistent up to d were observed in vaccinated animals. an inter-clade low crossreactive hi response to a clade strain was only detected in a few ferrets that had been vaccinated with the af -adjuvanted clade . . all af -adjuvanted clade . antigen vaccinated animals survived challenge either with the homologous or heterologous virus until euthanized day . after challenge, mean body temperature and mean body weights were monitored as indicators of disease. in the control ferrets, mean body temperature increased by - °c (depending on the challenge virus strain) h post challenge, with an accompanying mean body weight loss ranging from ae % to ae %. ferrets vaccinated with the af -adjuvanted clade . vaccine showed a lower and delayed fever compared to control ferrets that received the same viral challenge, whereas no significant differences were observed between vaccinated animals and their respective controls upon challenge with clade . or clade viruses. body weight loss was reduced in all vaccinated animals when compared to controls after challenge with either the homologous clade . strain or with one of the heterologous strains. lung virus titration showed high levels of virus replication in all control animals days after homologous challenge with the clade . virus. lung viral loads of all ferrets immunized with the af -adjuvanted clade . vaccine were reduced more than log . vaccination resulted in complete viral clearance from the lungs of % of animals assessed days after challenge. as compared to controls, a reduction of the mean viral load of about log was observed in ferrets vaccinated with the af -adjuvanted clade . vaccine after heterologous challenge with either the clade or clade virus. conversely, vaccination with af -adjuvanted clade . vaccine did not result in reduction of lung viral loads after challenge with the clade . heterologous virus strain. titration of pharyngeal swabs showed high levels of viral shedding in all control ferrets after challenge with clade . strain, whereas virus was not detected in any vaccinated animal. similarly, log reduction of viral shedding was seen in vaccinated versus control ferrets following clade heterologous challenge. lower reductions in viral shedding were observed after clade . challenge ( ae log ) and clade challenge ( ae log ). gross pathology and histology revealed lung lesions consistent with influenza a ⁄ h n virus infection all control animals challenged with the clade . , clade . or clade strains. mild to moderate lung lesions were observed in control animals following challenge with clade virus. macroscopic evaluation (percentage of affected lung parenchyma) and histopathological analysis (extent and severity of alveolitis, alveolar oedema and hemorrhage) showed that lung lesions were significantly reduced in af -adjuvanted clade . vaccinated animals after challenge with the homologous clade . virus strain as compared to controls. similarly, a reduction of the macroscopic and microscopic lung lesions was observed in vaccinated animals upon heterologous challenge with clade . and clade virus strains, whereas no differences were observed between control and vaccinated animals after challenge with clade virus. the results of these ferret challenge studies demonstrated that low doses of pandemic influenza vaccines formulated with an oil-in-water emulsion adjuvant, af , elicited strong antibody responses specific to the immunizing strain. importantly, these vaccines provided protection after homologous challenge with complete virus clearance in ferret lungs and reduced viral shedding from the upper respiratory tract suggesting an ability to reduce virus transmission. moreover, af -adjuvanted h n vaccine can provide cross-protection upon challenge with different h n clades by preventing mortality and reducing the viral burden in the lower and the upper respiratory tract. in conclusion, the results of these studies highlighted the ability of af -adjuvanted influenza vaccines to induce potent immune responses and full protection in ferrets against homologous challenge and suggested that protection may be mediated, at least in part, by antigenspecific humoral immunity. since , outbreaks of h n influenza virus infection in poultry have occurred in eurasian countries. phylogenetic and antigenic analysis of h n isolates revealed that there are three sublineages, consisting of g , g , and korean, among ha genes of the eurasian h n viruses. h n viruses do not cause severe disease in poultry, but co-infection of h n viruses with bacteria such as staphylococcus aureus, haemophilus paragallinarum, or attenuated coronavirus vaccine may exacerbate the disease. , h n viruses were isolated from domestic pigs in china and korea and from humans with febrile respiratory illness in hong kong in kong in , kong in , and it is, thus, postulated that in the present study, h virus strains were analyzed antigenically and phylogenetically to select a proper h n vaccine strain. inactivated whole virus particle vaccine was prepared, and its potency against h virus challenge was assessed in mice. viral rnas were extracted from the allantoic fluid of chicken embryos infected with viruses by using a commercial kit (trizol ls reagent; invitrogen, california, usa) and reverse-transcribed with the uni primer and m-mlv reverse transcriptase (invitrogen). the primers used for the ha gene amplification were h - f and h - r. for phylogenetic analysis, sequence data of the genes together with those from public database were analyzed by the neighbor-joining method. h influenza viruses were analyzed by hemagglutinationinhibition (hi) test. chicken hyperimmunized antisera against seven h viruses were prepared according to previous report. virus replication and pathogenicity against embryonated chicken eggs viruses were inoculated into -day-old embryonated chicken eggs and incubated for hours at °c. ha titers and % egg infectious dose (eid ) were measured every hours post-inoculation. pathogenicity of dk ⁄ hok ⁄ ⁄ against embryonated chicken eggs was evaluated by mean death time (mdt) as described previously. dk ⁄ hok ⁄ ⁄ was injected into the allantoic cavities of -day-old embryonated chicken eggs and propagated at °c for hours. the virus in the allantoic fluids ( ha) was purified by differential centrifugation and sedimentation through a sucrose gradient according to previous report. the concentration of protein was measured by od using ultrospec pro (amersham biosciences, tokyo, japan). the purified virus was inactivated with ae % formalin at °c for days. immunization of mice and challenge of immunized mice with hk ⁄ ⁄ four-week-old female balb ⁄ c mice were purchased from japan slc, inc. (shizuoka, japan). the mice were injected subcutaneously with , , ae , or ae lg proteins of inactivated dk ⁄ hok ⁄ ⁄ whole virus vaccine. two weeks later, the mice were boosted by subcutaneous injection with the same dose of the vaccine. control mice were injected with pbs. serum samples were tested by enzyme-linked immunosorbent assay (elisa) according to previous report. one week after the second vaccination, mice in each group were challenged intranasally with ll of ae eid of hk ⁄ ⁄ under anesthesia. on days postinfection, five mice in each group were sacrificed, and the lungs were separately homogenized to make a % (w ⁄ v) suspension with minimal essential medium (nissui, tokyo, japan). the virus titers of the supernatants of lung tissue homogenates were calculated in -day-old embryonated chicken eggs and expressed as the eid ⁄ gram of tissue. the other five mice in each group were monitored for body weight for days after challenge. the ha genes of h viruses were sequenced and analyzed by the neighbor-joining method. all of the h viruses were classified into the eurasian lineage ( figure ) . eleven, seven, and four strains were classified in the korean, g , and g sublineages, respectively. the h viruses of the korean and g sublineages were isolated from waterfowl, poultry, pigs, and humans in the east asian countries, and those of the g sublineage were isolated from poultry in the west asian countries. the cross-reactivity between these antisera and h n viruses were analyzed by hi test. the antisera against h viruses belonging to the korean sublineage were broadly cross-reacted to h viruses belonging to the g and g sublineages. h viruses belonging to the korean lineage were reacted to the antisera against h viruses belonging to the g and g sublineage compared with h viruses belonging to the other sublineage (data not shown). thus, it was suggested that h vaccine strain should be selected from the viruses of korean sublineage to prepare for the vaccine strain of h viruses. dk ⁄ hok ⁄ ⁄ replicated efficiently in -day-old embryonated chicken eggs (data not shown). pathogenicity of dk ⁄ hok ⁄ ⁄ against embryonated chicken eggs was determined by mdt. dk ⁄ hok ⁄ ⁄ was low pathogenic against embryonated chicken eggs (data not shown) and was selected as an h vaccine strain. to assess the potency of the vaccine against h virus infection, mice vaccinated subcutaneously with inactivated dk ⁄ hok ⁄ ⁄ were challenged intra-nasally with hk ⁄ ⁄ . immunogenicity of the inactivated vaccine was assessed by measuring the igg antibodies in mouse sera by elisa. antibody was detected in the group of mice injected lg protein after the first immunization and detected in the group of mice injected lg protein after the second immunization. thus, potency of the present inactivated whole virus vaccine was demonstrated in mice. next, to assess the protective immunity of the inactivated vaccine in mice, viral titers in the lungs was determined. the virus titers in the lungs were ae - ae eid ⁄ g in the groups of mice injected , and lg protein, and ae - ae eid ⁄ g in the other vaccinated groups. body weight reduction of mice were observed in the group of mice injected ae , ae lg protein, and control groups from dpi, and reached to % body weight loss from -to -day post-infection ( figure ). this result correlates with antibody titer in mouse sera and viral titers in the lungs. these results suggest that the test h inactivated whole vaccine confers prevent of weight loss and reduction of virus replication against h influenza virus infection in mice. recently, h n viruses of all of three sublineage have been isolated from wild birds and poultry in worldwide. h n viruses were isolated from pigs and humans in china and korea, suggesting that h n virus would be a potential for a pandemic influenza virus in human population. h n viruses were isolated from pigs in china and korea and were classified into the g and korean sublineage. in human cases, all h n virus isolated from humans in china was classified into the g sublineage. it was suggested that h n viruses isolated from pigs and humans vary in antigenicity of isolates between the korean, g , and g sublineages. therefore, it is important for the preparedness of influenza pandemic to develop h influenza virus vaccine, which could broadly cross-react to antisera of all sublineage viruses. so, we selected the vaccine candidate strain, dk ⁄ hok ⁄ ⁄ , which could broadly cross-react to antisera of all sublineage viruses, and which could replicate in this study, it was suggested that the test vaccine has potency to protect against challenge with h virus using mice for mammalian model. the challenge virus, hk ⁄ ⁄ , was isolated from human, replicates efficiently in mice, and shows pathogenicity in mice. the test vaccine inhibited viral replication and body weight loss in mice. whole inactivated vaccine produced protective immunity, supporting our approach of using whole virus particles for vaccine development. furthermore, whole particle virus vaccine could induce igg and mucosal iga levels after intranasal vaccination with whole particle vaccine. the present results may facilitate the studies of the vaccine for future pandemic caused by h influenza virus in humans. tants to attempt to improve growth. to determine whether wild type h n pdm grew better in the novartis mdck suspension cell line (mdck pf) than in eggs, isolations from h n pdm positive clinical samples were attempted in both substrates. the isolation rate of h n pdm viruses was higher in mdck pf cells ( %) ( ⁄ ) compared to allantoically inoculated eggs ( %) ( ⁄ ) . however the yields were lower than observed with seasonal viruses. little improvement in virus yield was seen with extra passaging or dilutions of h n pdm viruses isolated in mdck pf cells. with the emergence of the swine-origin pandemic h n (h n pdm) influenza in april , the need for efficient production of a suitable vaccine was a high priority. virus isolates were distributed by the who for the urgent development of suitable vaccine strains early in the pandemic. vaccine viruses can be grown in embryonated chicken eggs or in certified mammalian cells. , unfortunately wildtype h n pdm virus strains distributed by the who grew poorly in cell lines and eggs, requiring the generation of a series of conventional and reverse genetics derived reassortants to attempt to improve growth. from these reassortants, only the conventional egg derived reassortants nymc-x- a and nymc-x- (both based on one of the earliest known viruses a ⁄ california ⁄ ⁄ ) showed high enough growth and yield in eggs and cell culture to make them suitable for vaccine manufacture. these reassortants, while acceptable, still only gave haemagglutinin (ha) yields of approximately % that of seasonal h n reassortants. to determine if more recent wild type h n pdm viruses grew better in the novartis mdck suspension cell line (mdck pf), h n pdm positive clinical samples were cultured in mdck pf cells and also in embryonated hen's eggs. in addition, to improve virus yields from mdck pf isolates, extended passaging of three wild type h n pdm influenza viruses was performed using various virus dilutions at each passage level. the results were assessed using various serological and molecular biology techniques and compared to viruses isolated in eggs and conventional mdck cells. h n pdm viruses were received at the centre from who national influenza centres, who influenza collaborating centres and other regional laboratories and hospitals in australia, new zealand, and the asia ⁄ pacific region. viruses were received as original clinical specimens consisting of nasal swabs, throat swabs, nasopharyngeal aspirates, or nasal washes that had previously been shown to be h n pdm positive by real time rt-pcr. these specimens were then cultured in mdck pf cells with serum free medium containing trypzean (optaflu) and also independently inoculated into the allantoic cavity of day-old embryonated hen's eggs. virus cultures in mdck pf cells were sampled at and hour and evaluated by various means including ha titres. at hour, virus cultures were further passaged at varying dilutions ranging from ) to ) up to a total of passages. embryonated hen's eggs were incubated at °c for days and allantoic fluid was harvested and ha titres performed to determine whether a further passage was required in order to improve growth. the conventional reassortants were produced by a mixed infection of eggs or mdck pf cells with the wild type virus and a donor virus carrying the internal genes of the a ⁄ puerto rico ⁄ ⁄ virus. the reassortants were obtained by sequential passages using immuno-selective antisera against the surface antigen of the donor virus to remove virus populations carrying the ha and na protein of the donor strain. the reverse genetics viruses were rescued in vero cells using the plasmid system. both types of reassortants were generated and supplied by who collaborating centres and essential regulatory laboratories except the nvd-c- strain, which was produced by novartis. in this small study with recent h n pdm viruses, the isolation rate was higher in mdck pf cells ( %) ( ⁄ ) compared to allantoically inoculated eggs ( %) ( ⁄ ) . assessment of ha titres, however, showed higher ha titres in egg-isolated viruses compared to viruses isolated in mdck pf cells after two passages. egg generated or cell generated reassortant viruses gave higher ha titres compared to the homologous wild type viruses (table ) . no amino acid changes were observed in mdck pf isolated influenza viruses compared to original specimens or viruses isolated in conventional atcc derived mdck cells, unlike egg isolated viruses which showed a number of amino acid changes, many consistent with egg adaptation mutations (table ) . viruses isolated in mdck pf cells grouped phylogenetically with viruses isolated in conventional atcc derived mdck cells or viruses sequenced from original clinical samples, while egg isolated viruses grouped slightly differently (data not shown). as a result of the poor growth of h n pdm viruses in mdck pf cells, serial dilutions were performed over a number of passages ( figure ). based on the results obtained from the virus isolates, a ⁄ victoria ⁄ ⁄ , a ⁄ wellington ⁄ ⁄ , and a ⁄ darwin ⁄ ⁄ , a supplemental protocol was developed and used in the isolation of a ⁄ brisbane ⁄ ⁄ (figure ). only small differences in ha titer were seen between different dilutions, and copy number showed a similar trend to ha titer at each passage ( figure ). following the supplemental protocol for the isolation of a ⁄ brisbane ⁄ ⁄ results showed slightly higher ha titres with little variation between passages. the egg derived reassortants nymc-x- a and nymc-x- were also assessed for growth in mdck pf cells and were found to be superior by ha titer to other conventional reassortants (egg or cell derived), reverse genetics derived reassortants, or wild type viruses (table ) . two methods were used to determine the ratio of ha to other viral proteins: densiometric analysis using sds-page and reversed-phase hplc using a subtype specific standard. ha content in different vaccine seeds of influenza a subtypes demonstrated that the ha content per total virus protein from the nymc h n pdm reassortants was significantly different to the seasonal influenza a subtypes. for the seasonal h n the ratio of ha to p p p p p p p p p p p p p p p p n and m was ‡ %, for the h n the ratio of ha to n and m was £ %, while for the pandemic a ⁄ h n , the ratio of ha to n and m was much lower at £ % (data not shown). the results of this study has observed the growth of a series of - h n pdm viruses in vaccine suitable mdck pf cells to be generally lower than what has been seen with other seasonal influenza viruses. little improvement in virus yield was seen with extra passaging of h n pdm viruses isolated and passaged in mdck pf cells. passaging up to times in mdck pf cells using dilutions ranging from ) to ) resulted in supernatants with viral ha titres ranging from ha ⁄ ll to ha ⁄ ll. the isolation rate of h n pdm viruses was higher in mdck pf cells ( %) compared to allantoically inoculated (and passaged) eggs ( %), a trend also seen in previous work with seasonal influenza viruses. in contrast a study by hussain and colleagues found similar rates of isolation and replication of seasonal influenza viruses in mdck cells and eggs. the virus load as determined by matrix gene copy number showed a similar trend to ha titers. two of the isolates exhibited small rises and falls in ha titer during passaging, while a third, a ⁄ victoria ⁄ ⁄ gave consistently higher titers. interestingly this virus was unable to be isolated in eggs. the ha sequences of all strains were assessed at p , p , p , p , p and when available compared to the original clinical sample ha sequence. mdck pf-isolated viruses had few if any changes in their ha amino acid sequence, while the majority of egg isolates showed - amino acid changes compared to the clinical sample, with an egg adaption change (l i) evident in a number of them. the ha sequence of one of the better growing viruses, a ⁄ victoria ⁄ ⁄ , was found to have a g e change compared to the a ⁄ california ⁄ ⁄ reference virus. this change was also seen in the virus isolated in conventional, adherent mdck cells. these viruses with g e change when tested by hai have shown reduced reactivity with ferret antisera to a ⁄ california ⁄ ⁄ -like viruses, but normal reactivity with ferret antisera to h n pdm a ⁄ bayern ⁄ ⁄ -like viruses. despite this mutation all mdck pf derived viruses appeared to be a ⁄ california ⁄ ⁄ -like by hai. the h n pdm egg-derived reassortants (nymc x- a and nymc x- ) when grown in mdck pf cells were superior to wild type h n pdm viruses, reverse genetics derived reassortants, and other egg-derived reassortants. the yields of haemagglutinin from the nymc h n pdm reassortants were still below those seen with sea-sonal h n reassortants as was also seen in eggs. this trend has also been noted in other studies. in summary, attempts to improve growth and yield of the h n pdm wild types for mdck pf cells by extended passaging were not successful, and reassortants did not perform as well as seasonal h n reassortants have in the past. however, using higher dilutions for the passaging of h n pdm viruses in mdck pf cells did result in higher ha titres (a ⁄ brisbane ⁄ ⁄ ). further work is therefore required to generate pandemic h n seed viruses that grow well in a variety of cell culture and egg based vaccine production systems. the aim of this study is to evaluate antibody response to influenza virus neuraminidase (na) following immunization with live attenuated influenza vaccine (laiv). we adjusted the peroxidase-linked lectin micro-procedure previously reported by lambre, et al. ( ) to assay neuraminidase inhibition (ni) antibody in sera taken from immunized mice and from human subjects in a clinical trial. for the assay, we prepared the a(h n ) reassortant virus containing the na of a ⁄ california ⁄ ⁄ (h n ) and the hemagglutinin (ha) of a ⁄ equine ⁄ prague ⁄ ⁄ (h n ). in addition, we used an na-specific igg elisa assay to test sera from immunized mice and volunteers. in mice, one dose of laiv induced ni antibody of a geometric mean titer (gmt) of ae , compared to ae in the control group. gmt of ni from human subjects who received two doses of pandemic a(h n ) were significantly higher than pre-vaccination titers. in unvaccinated human subjects, na-specific cross-reactive antibodies to pandemic a(h n ) were detected more often than cross-reactive antibodies to ha. antibody response to influenza virus na contributes to the overall immune response to influenza and may provide partial protection against influenza infection and reduce severity of disease in the host. a number of preclinical studies using purified or recombinant na have shown that various two-dose vaccine regimens in mice may significantly reduce pulmonary virus titers following viral challenge. [ ] [ ] [ ] a plasmid dna-vaccine model demonstrated cross-reactive antibodies to human n in mice could provide partial protection against a lethal challenge against h n or recombinant pr bearing the avian n . immunogenicity of current influenza vaccines, including laivs, is measured primarily as a level of strain-specific hemagglutination inhibition (hi) antibodies. however, the who meeting on the role of na in inducing protective immunity against influenza infection ( ) specified a need to develop suitable assays for anti-na antibody detection to enhance influenza vaccine evaluation in preclinical and clinical studies. the aim of the current study was to evaluate anti-na antibodies to pandemic a(h n ) influenza virus following laiv immunization. the rn ⁄ -swine a(h n ) reassortant influenza virus containing the na of a ⁄ california ⁄ ⁄ (h n ) and the ha of a ⁄ equine ⁄ prague ⁄ ⁄ (h n ) generated by classical genetic reassortment in embryonated chicken eggs (ce). parental a ⁄ equine ⁄ prague ⁄ ⁄ (h n ) influenza virus was obtained from the center for disease control and prevention, atlanta, ga, usa. viruses were propagated in day old ce and purified by sedimentation out of the allantoic fluid, followed by ultracentrifugation on - % sucrose step gradient. for the mouse studies, week old cba mice were inoculated intranasally with one dose eid ⁄ ae ml of a ⁄ ⁄ california ⁄ ⁄ (h n ) vaccine strain or received ae ml pbs. blood samples were collected on day post inoculation. healthy young adults were immunized twice, or days apart in the fall with a ⁄ ⁄ california ⁄ ⁄ (h n ) laiv manufactured by microgen, irkutsk, russia. for the human studies, peripheral blood specimens were collected from volunteers before vaccination, days after the first vaccination, and days after the second dose of vaccine. sera from five subjects diagnosed with influenza a(h n ) were collected in december , to weeks post infection and kindly provided by e. vo ıtsekhovskaia from biotechnology laboratory, institute of influenza, rams. also, sera obtained in from unvaccinated vol-unteers were tested for presence of cross-reactive antibodies to a ⁄ california ⁄ ⁄ (h n ). sera were treated with a receptor-destroying enzyme from vibrio cholera (denka-seiken, tokyo, japan) and then were tested in duplicates for hemagglutination-inhibition (hi) h specific antibodies by standard procedures using a ⁄ ⁄ california ⁄ ⁄ (h n ) test antigen. the peroxidase-linked lectin micro-procedure previously reported by lambre, et al. was adjusted to assay ni antibody. briefly, -well plates (sarstedt, inc., nümbrecht, germany) were coated overnight with ll of lg ⁄ ml fetuin. the purified a(h n ) reassortant virus was diluted in pbs with % bsa and mm ca + to give a four times higher optical density at nm (od ) compared to control wells not containing virus. fifty-microliter volumes of serially diluted serum samples were incubated with an equal volume of prediluted virus for hour at °c. after incubation, the plates were washed and neuraminidase activity was measured by subsequently adding peroxidase-labeled lectin ( lg ⁄ ml; sigma, st. louis, mo, usa), incubating for hour at room temperature, washing the plates, and adding ll of peroxidase substrate (tmb). the reaction was stopped after minute by adding ll of n sulfuric acid. od values were measured at nm using the universal microplate reader (el x ; bio-tek instruments, inc., winooski, vt, usa). the ni titers were expressed as the reciprocal dilution that gave % od of positive control (virus, no serum control). in addition we used an igg elisa assay with ae lg ⁄ ml of purified na from a ⁄ california ⁄ ⁄ (h n ) to test sera from immunized mice and volunteers. data were analyzed with statistica software (version ae ) (statsoft, inc. tulsa, oklahoma, usa). geometric mean titers (gmt) were calculated and used to represent the antibody response. the comparisons were made within groups between pre-and postvaccinated titers (expressed as log ) after first and second vaccination using wilcoxon matched pairs test. to compare multiple independent groups we used a kruskal-wallis anova with subsequent multiple pairwise comparison based on kruskal-wallis' sums of ranks. a p-value of < ae was considered to be statistically significant. in mice, one dose of laiv induced antibody responses to both ha and na components of the a ⁄ california ⁄ ⁄ (h n ) influenza virus vaccine (table ) . geometric mean titers of ni antibody levels from vaccinated mice were ae and were significantly higher compared to those in unvaccinated control animals (p < ae ). elisa igg titers expressed as log were ae compared to ae in control group. there was good correlation between antibody rises obtained using ni or elisa tests (r = ae ). in a study during the fall of , % of examined unvaccinated subjects were negative to pandemic a(h n ) (hi titers £ : ). serum hi antibody titers to pandemic a(h n ) ‡ : were considered to be protective against *the postvaccination gmts of hi antibodies after revaccination were higher than respective prevaccination titers (p = ae ) **the postvaccination gmts of ni antibodies after revaccination were higher than respective prevaccination titers (p = ae ) serum hi and ni antibodies to a ⁄ california ⁄ ⁄ (h n ) after one or two doses of pandemic laiv were evaluated in subjects who had pre-vaccination hi titers £ : ( table ) . post-vaccination gmts of a(h n )-specific antibodies were significantly higher than pre-vaccination titers only among subjects who received two doses of laiv ( table ). the frequency of subjects with ‡ fourfold rises in hi antibody titers was higher after two doses ( ae %) compared to responses after one dose ( ae %) although the differences were not statistically significant ( table ). the highest antibody titers of hi and ni antibodies were achieved after natural infection (p < ae compared to all post-vaccination groups). all five subjects with confirmed influenza also had high levels of n -specific igg measured by elisa using purified na as the coating antigen (data not shown). influenza ha and na surface proteins are primary targets of neutralizing antibodies that provide protection against influenza infection. the correlation of strain-specific hi antibody titers ‡ : to protection of % of the subjects against influenza infection is based on a number of reports published in s. serum antibodies against viral na as result of influenza infection or vaccination also can neutralize the virus from infecting cells; however, little is known about protective levels of such antibodies. to evaluate ni antibodies directed against pandemic a(h n ) we used the reassortant a(h n ) influenza virus with mismatched ha to avoid non-specific inhibition. we demonstrated laiv immunization effectively increased levels of ni antibody, although in smaller amounts compared to influenza infection. our data suggest that an antibody to neuraminidase, resulting from an earlier infection of the circulating seasonal influenza a(h n ), evidently cross-reacted with the n of pandemic influenza virus, perhaps due to the previously reported % of conserved na epitopes in pandemic a(h n ). the peroxidase-linked lectin test using the reassortant a(h n ) influenza virus was shown to be a sensitive and time effective means of revealing homologous and cross-reactive anti-na antibodies after laiv immunization or influenza infection. this could be a useful method for influenza vaccine evaluation. significant levels of anti-na antibodies detected in peripheral serum from subjects infected with wildtype h n virus or with h n laiv. and the cross-antibody response to ph n . for calculation of geometric mean titer (gmt), a titer of < was assigned a value of . statistical significance was determined by paired t-test. cross-reactive antibody response to ph n in vaccinated populations of seasonal influenza virus table shows the antibody response to seasonal influenza viruses and ph n of participants. before vaccination, no or little antibody response to ph n had been detected in all age groups. vaccination with seasonal influenza vaccines resulted in seroresponse in over % of subjects, except children aged - years ( %) and subjects aged of - years ( %) vaccinated with - season influenza vaccine and adults aged ‡ years ( %) vaccinated with - season influenza vaccine. seroconversion was detected in over % of subjects of all ages. postvaccination to prevaccination gmt ratios for response to seasonal influenza viruses was more than ae -fold. in contrast, seroresponse to a ⁄ california ⁄ ⁄ after vaccination with - and - seasonal influenza vaccines were detected in only % and % of those aged - years, % of those aged - years, % and % of those aged - years, % and % of those aged ‡ years, respectively. seroconversion in all participants ranged from % to %, and postvaccination to prevaccination gmt ratios were < ae -fold. preexisting antibody response to ph n among subjects born before s in china according to a recent report, people who were born from to had a preexisting immunity to ph n . although only a very low level of cross-reactive antibody response to ph n had been observed among older subjects aged more than years old in china, we further analyzed these data by different age distribution of subjects, which can trace back to the previous infection that is genetically and antigenically more closely related to this new ph n influenza virus. the proportion of seroresponse to ph n with the titer of , , and (highest titer detected from participants of all ages in this study) and the value of gmt were analyzed according to the birth decade of subjects from . similarly, a peak of antibody response and the value of gmt occurred both in subjects born from to and sharply decreased afterward ( figure ). the seroresponse of subjects born in and before is significantly higher than subjects born afterward (p < ae ). similar to recent studies in some asia countries (guangxi province of china and singapore), limited antibody response to ph n had been detected in children and adults. , but, some other studies from european countries (finland, germany, the united kingdom) and the united states reported a high proportion of older individuals aged > years with pre-existing cross-reactive antibodies to ph n , which may possibly ba a result of previous exposure to antigenically related h n influenza viruses circulating in earlier decades or a lifetime of exposure to influenza a, which has resulted in broad heterosubtypic immunity among older individuals in those countries. previous infection and vaccination with a ⁄ new jersey ⁄ may also contribute to the high level of cross-reactive antibody response to ph n among adults older than years in the us. , the peak of the antibody response to ph n in subjects born between and , which is consistent with recent reports, may suggest the previous viral infections of spanish flu or closely related influenza viruses, which is before and little after the year of . recent antigenic report of new ph n viruses indicated that they are antigenically homogeneous among historical viruses, which are most similar to classical swine a(h n ) viruses. a number of reviews [ ] [ ] [ ] [ ] confirmed that the virus is the likely ancestor of all four of the human and swine h n and h n lineages, as well as the 'extinct' h n lineage. in , a(h n ) influenza viruses were first isolated from swine. they have been shown to be antigenically highly similar to the recently reconstructed human a(h n ) virus. the cellular responses may contribute to the sustaining and long term antibody response. probably, boosting by persisting antigenically related viruses in the early decades of the th century, may have contributed to the ability of these subjects to sustain memory b cells, and it is well established that a subset of plasma cells is long-lived, and these cells contribute to durable humoral immune responses, such as that observed after childhood smallpox vaccination. furthermore, t cells that recognize cross-reactive epitopes are preserved and might be enriched in the memory population; the course of each infection is influenced by the t-cell memory pool that has been laid down by a host's history of previous successive infections. our study indicated that wide transmission of this new virus or any antigenically close related influenza a(h n ) viruses may not have circulated among populations in china before the outbreak of ph n . our data also suggests the need for vaccination with ph n vaccine in all age groups. hypo-and agammaglobulinemia patients have an impaired immune system and are particularly susceptible to bacterial infections that are normally defended against by antibodies. therefore, patients routinely receive replacement therapy with immunoglobulins isolated from healthy blood donors. these patients are also prone to get viral infections, possibly due to defects in toll-like receptors and . because these patients lack an antigen specific humoral immune response, they are rarely vaccinated. the ability of hypogammaglobulinemic patients to produce a specific cell-mediated immune response upon vaccination has only been sparsely investigated. in contrast to local mucosal antibodies, vaccine-induced cell-mediated immunity is not believed to protect against pathogen entry per se, but may be sufficient to provide protection against severe disease and death following transmission of some microbes. , the aim of this pilot study was to investigate if influenza vaccination of hypogammaglobulinemic patients can induce an influenza-specific cell-mediated immune response. we therefore vaccinated hypogammaglobulinemic patients and healthy controls with pandemic h n virus vaccine and subsequently investigated the bcell and t-cell responses. the percentages of ifn-c, il- , and tnf-a cytokine producing cd + th -cells were determined, as these cytokines are important indicators of cell-mediated immunity. five a-or hypogammaglobulinemic patients were classified based on the freiburg classification : patient # is diagnosed with x-linked agammaglobulinemia, patient # and # are in group ia, patient # is in group ib and patient # is in group ii. the monovalent egg grown split virus vaccine adjuvanted with as was manufactured by glaxosmithkline (gsk), belgium. the vaccine strain was produced by reassortment between influenza a ⁄ california ⁄ ⁄ (h n ) and a ⁄ pr ⁄ ⁄ (h n ) to produce a ⁄ california ⁄ ⁄ -like virus (x a). the vaccine was mixed with adjuvant to contain ae lg haemagglutinin (ha) of a ⁄ california ⁄ ⁄ -like virus (h n ), squalene ( ae mg), dl-atocopherol ( ae mg), and polysorbate ( ae mg) per ml. healthy controls and hypogammaglobulinemia patients were vaccinated by intramuscular (im) injection. hypogammaglobulinemia patients received one or two vaccine doses days apart. the intention was to vaccinate the hypogammaglobulinemic patients with two doses of ae lg ha, but ae lg ha was inadvertently administered to the patients as the first dose. for patient # this was the second dose as he had received an initial dose of ae lg ha months prior to the study. patient # , # , and # received a second dose of ae lg ha. four healthy controls were immunised with one dose of ae lg ha according to norwegian national guidelines. peripheral blood mononuclear cells (pbmcs) were harvested and washed in pbs with % fbs. the pbmcs were resuspended in lymphocyte medium (rpmi with l-glutamine, ae mm non-essential amino acids, mm hepes ph ae , mm sodium pyruvate, iu ⁄ ml penicillin, lg ⁄ ml streptomycin, ae lg ⁄ ml fungizone and % fbs) prior to use in the enzyme-linked immunospot (elispot) and influenza-specific cd + t-cell assays. serum haemagglutination inhibition antibodies were tested by a standard method using ha units and ae % turkey erythrocytes. all samples were tested in duplicate and the test was repeated at least two times. titres < were assigned a value of for calculation purposes. for numeration of antibody-secreting cells (asc), an eli-spot assay was conducted as previously described with the following modifications. ninety-six well elispot plates were coated with lg ⁄ ml of a ⁄ california ⁄ ⁄ like (x a) h n virus diluted in pbs overnight at °c. after blocking with rpmi ( % fbs), pbmcs were added and incubated ( °c, % co ) for hour. secreted antibodies were detected with biotinylated goat anti-human igg, iga and igm specific antibody (southern biotech, birmingham, alabama, usa), incubated for hour at room temperature and developed with extravidin peroxidase and aec substrate. the numbers of spots were counted using an elispot reader (immunoscanÔ) and immunospot Ò software. the influenza-specific cd + th -cell response was measured by intracellular cytokine production of ifn-c, il- , and tnf-a. peripheral blood mononuclear cells ( per well) were incubated for hour ( °c, % co ) in ll lymphocyte medium containing lg ⁄ ml anti-cd , lg ⁄ ml anti-cd d, ae lg ⁄ ml monensin, lg ⁄ ml brefeldin a, (bd biosciences, franklin lakes, new jersey, usa), and the h n influenza split virus vaccine x a (either ae lg ⁄ ml or lg ⁄ ml ha). basal cytokine production was determined by incubating pbmcs in lymphocyte medium without influenza virus, and the percentage of cytokine positive cells without influenza stimulation were subtracted from influenza-stimulated cells. cells were stained for cd , cd , cd , ifn-c, il- , and tnf-a (bd biosciences) as previously described. finally, cells were resuspended in pbs containing % fbs and ae % sodium azide and analysed by bd facscanto flow cytometer ( - cells acquired). flowjo v ae ae (tree star, ashland, oregon, usa) was used for data analysis. five to six fold lower gmts were found in the patient group as compared to the healthy controls throughout the study ( figure a) . the lowest hi titres were obtained in patients # , # , and # , whilst patients # and # and all healthy controls fulfilled two of three european medicines agency committee for medicinal products for human use (chmp) seasonal influenza vaccine licensing criteria, by obtaining an hi titre > and a mean geometric increase of ae between pre-and post-vaccination. thus, the hi data indicate that two vaccine doses was sufficient to induce a protective hi antibody response in two out of five of the hypogammaglobulinemia patients tested in this study. the numbers of influenza-specific iga, igg, and igm asc were tested pre-vaccination and days post-vaccination with the h x a virus. few or no ascs were detected pre-vaccination (data not shown). at days post-vaccination the patient's iga, igg, and igm asc levels were significantly lower (p < ae ) compared to the healthy controls ( figure b) . but, the post-vaccination asc numbers in the patients were generally higher than at pre-vaccination stage ( - ascs). patient # had the highest iga and igg asc numbers, followed by patients # and # , whilst patient # and # had few or no asc's. these results confirm that the patients are indeed hypogammaglobulinemic and that some of the patients (# and # ) could be agammaglobulinemic in the context of producing influenza-specific antibodies. the asc levels of patients # , # , and # were lower than those of the healthy controls, but could possibly be adequate for reducing the severity of influenza disease. the influenza-specific th -cell response was evaluated by stimulating pbmcs with the influenza x a virus , , and days post-vaccination. stimulation of healthy control pbmcs with x a days after vaccination, induced ifn-c, il- , and tnf-a production by an average of ae %, ae %, and ae % cd + t-cells, respectively. patient # and # had higher responses than the healthy controls and stimulation with x a induced ae %, ae %, and ae % of t-cells from patient # to produce ifn-c, il- , and tnf-a, respectively (figure a) . the response of patient # was further boosted by a second vaccine dose, which resulted in ae %, ae %, and ae % cd t-cells producing ifn-c, il- , and tnf-a, respectively at day ( figure b ). these results show that the hypogammaglobulinemia patients studied here did not have a common impaired influenza-specific cd + th cytokine response. rather, there was a tendency towards increased responses, suggesting that the diminished antigen specific b-cell responses could induce a compensatory antigen specific th -cell response. the results from this pilot study suggest that some hypogammaglobulinemia patients may benefit from influenza vaccination. we found very different patient responses to influenza vaccination, but some of the patients (patient # and # ) did mount low influenza-specific asc responses. in addition, the vaccine-induced hi antibody titres above the protective level in patient # and # . these results are in accordance with previous publications, which described that polypeptide vaccines induce humoral responses in subgroups of common variable immunodeficiency patients. [ ] [ ] [ ] in this study, we also investigated cell-mediated immunity and found the percentages of homologous and cross-reactive influenza-specific cd + th -cells to be in the same range (for patient # , # , and # ) or higher (for patient # and # ) in the a-or hypogammaglobulinemic patients compared to the healthy controls. the higher response is probably due to the patients having received a vaccine dose of ae lg ha, whilst the controls received ae lg ha. in addition, the patients received a second booster dose, which influences the day and months responses. nonetheless, these results are the first to demonstrate that proliferation of pandemic influenza antigen specific th cells can be induced in hypogammaglobulinemic patients. in addition, vaccination induced influenza-specific asc's in some patients. the findings are promising and provide hope that hypogammaglobulineamic patients could be vaccinated against influenza and other diseases preventable by figure . peripheral blood mononuclear cells s from patients and healthy controls were isolated at day (a), (b), and day (c) and stimulated for hour with x a virus before staining and flow cytometric analysis. the figure shows the mean ± sd frequency of influenza-specific cd + cytokine producing cells (%) where the basal cytokine production from unstimulated cells has been subtracted. data for the hypogammaglobulinemia patients are additionally shown as a number for each patient. **significantly higher frequency of il- producing cd + t-cells in the patients compared to the healthy controls (students t-test p < ae ). titres are presented as the geometric mean titre ± % confidence interval. elispot data (b) are presented as the mean number of influenza-specific iga, igg, and igm ascs per peripheral blood mononuclear cells ± sem. data for the hypogammaglobulinemia patients are additionally presented by a number for each patient. *significantly higher numbers of ascs were detected in the healthy controls as compared with the hypogammaglobulinemia group (students t-test, p < ae ). vaccination. however, this hypothesis should be tested in larger clinical studies. the influenza virus undergoes antigenic evolution under intense immune selection pressure from herd immunity in humans through the process called antigenic drift and shift. , because of antigenic drift, yearly updating of vaccine strain is needed. a mismatch between the circulating strains and the vaccine strain in the subsequent season is often encountered, resulting in reduction of vaccine effectiveness and lack of protection from the circulating strain. in order to address this, a universal influenza vaccine based on a more conserved part of the influenza virus, which is not affected by antigenic change and that is conserved across all strains, remains the ultimate goal to afford cross-protection to drifted strains as well as to other subtypes of influenza which may arise from antigenic shift. , previous studies have investigated the potential of the m e. , m e has remained highly conserved since it was first isolated in . several studies have examined the use of m e as a vaccine component, using various approaches including proteins, peptides, dna vectors, and attenuated viral vectors. , [ ] [ ] [ ] [ ] [ ] [ ] although m e is a weak antigen, by linking the protein to a carrier hepatitis b virus core particle, protection against influenza has been achieved in mice particularly when administered with an adjuvant. some articles found that vaccination with m e coupled to hbc induces protective antibodies, whereas the contribution of t cells to protection was negligible. protection induced by vaccination with m e-hbc was weak overall and failed to prevent weight loss in vaccinated infected animals, and mice succumbed to high dose infection. we aimed to address the poor immunogenicity of m e-hbc by using igv as adjuvant. igv domain is common and conserved in the tim family. ligand binding sites of t cell immunoglobulin mucin (tim) located at igv domain. [ ] [ ] [ ] tim function is done by anti tim antibody which recognized the ligand binding sites of igv domain. tim family members share a common motif, including an igv domain. they are differentially expressed on th cells and th cells with the ability to regulate the immune system. , the igv domain of human b - is sufficient to co-stimulate t lymphocytes and induce cytokine secretion. soo hoo et al. vaccinated with tim- antibody and inactivated influenza and found enhanced vaccine-specific immune response. we report here for the first time the use of igv recombinant protein as adjuvant to immunize mice with influenza m e-hbc. results indicated that igv can induce the strong cellular immune response and cross reaction with different subtype influenza virus antigen. target igv may be used to develop the new method for vaccination strategies. expression and purification of recombinant igv protein rna was extracted from healthy human pbmc. one-step rt-pcr (qiagen, valencia, ca, usa) was done for the amplification igv gene. the pcr product was purified and cloned into pet a (novagen, madison, germany). the resultant construct pet a-igv has a histidine (his) tag ( his) at the n terminus. dna sequence of the insert was determined by sequencing. igv. recombinant protein was expressed in escherichia coli and was purified on a ni column (novagen). the purified protein was examined by sds-page and western blotting. six-eight weeks female balb ⁄ c mice (institute of zoology chinese academy of sciences, china) was used for the study. mice were immunized twice intradermally with ug m e-hbc (provided by cnic, china) combined with different doses of recombinant igv protein , , ug, respectively, or without igv as control. the area proximal to the tibialis anterior muscle was sterilized with % ethanol and different groups of mice were injected bilaterally with , , ug igv plus ug m e-hbc in ul phosphate buffer saline per mouse using a ml syringe with attached ⁄ ¢¢ g needle. the immunization was given at weeks intervals. four blood samples were obtained from every mouse: before immunization, after the first and second immunization, and after virus challenge by retro-orbital plexus puncture. after clotting and centrifugation, serum samples were collected and stored at ) °c prior to use for assays. mouse-adapted a ⁄ pr ⁄ ⁄ (h n ), a ⁄ brisbane ⁄ ⁄ (h n ), a ⁄ xinjiang ⁄ ⁄ (h n ), and a ⁄ guangzhou ⁄ ⁄ (h n ) were provided by chinese national influenza centre. nine to eleven days old embroynated specific pathogen free (spf) chicken eggs were inoculated with virus, and the eggs were incubated at °c for - days. the allantoic fluid was collected and purified by sucrose density gradient centrifugation, and the virus was inactivated by formaldehyde at °c overnight. to identify igg, igg , igg a against m e, elisa assays were used. in brief, -well (nunc, brunei, denmark) were coated with ul ⁄ well of m e recombinant protein (provided by gene lab of ivdc, xuanwu district, beijing, china) in carbonate buffer (ph ae ) overnight at °c. immediately before use, the coated plates were incubated with blocking solution ( % bsa in pbs) for h at °c and washed four times with pbs containing ae % tween (pbs-t). the serum samples were serially diluted and added in the plates. the detection color was developed by adding hrp-labeled goat anti-mouse igg, igg , or igg a ( figure ) . no cross-strain response was observed in the control group. the igv adjuvented groups show splenocytes stimulation with seasonal h n , h n , h n , and h n antigens. m e-hbc immunization without igv showed splenocyte stimulation, but the extent was lower than animals immunized in the presence of the igv adjuvent. these data suggested that igv had enhanced effect on priming against the conserved viral antigen matrix protein and generation cross-strain immune response. influenza is a respiratory disease causing epidemics every year. h n viruses and swine-origin h n have also infected humans in recent years. seasonal influenza vaccine cannot cope with significant antigenic drift or with the emergence of pandemic viruses of different subtypes not contained in the vaccine. the high extent of conservation of the m e makes it a promising immunogen. a vaccine based on coupling of the m e peptide to an appropriate carrier may provide a universal vaccine with effectiveness and safety. m e based vaccination induces protective antibodies not only in mice, but also in ferrets and monkeys. the carrier hepatitis b core as carrier with m e forms a virus like particle (vlp). vaccination with m e coupled to hbc induces protective antibody, whereas the contribution of t cell protection was negligible. protection induced by vaccination with m coupled to hbc was weak overall. in order to improve the vaccination effect of m e-hbc, new adjuvant igv was evaluated in combination with the m e-hbc. the tim molecules are a recently discovered class of proteins with the ability to regulate the immune system. crystal structures of the tim molecules has revealed a unique, conserved structure with ligand-binding sites in the igv domain. to determine the potential immunostimulatory molecular properties of igv, we have evaluated immune response of the igv in combination with m e-hbc vlp. previous papers reported that vlp immunized mice can induce the th and th immune response. different adjuvant combined vlp can produce biased immune response th ⁄ th mixed immune response, or th -preferred th ⁄ th profile. thus, the response following the use of igv as a new adjuvant combined with m e-hbc vlp needs to be evaluated. results indicated that igv combined groups showed th biased immune response and enhanced cross reactive t cell immune responses. this may show that igv immunized the mice and antiigv antibody can cross link the igv on t cells and enhance the cell figure . t cell proliferation assay. mice were immunized twice with , , , ug ⁄ ml igv plus m e-hbc, respectively, and naive group was immunized with pbs. three weeks after a boosting immunization, spleens were harvested from immunized and naive mice. different subtypes of inactivated virus antigen (a) h n , (b) h n , (c) h n , (d) h n were added and cocultured with different group splenocytes for h. quick cell proliferation assay kit was used to detect the cell proliferation. the - nm absorbance was read on a plate reader. data were showed were shown as mean values. the difference between naive group and different doses igv plus m e groups was determined using the student's t-test. all significance level is p < ae . response. we also evaluated the cross-protection produced by igv combined m e-hbc. we challenge with mouseadapted strain pr and prove the cross protection via reaction between the cells from the immunized animal and different subtypes of virus antigen. some subtypes of virus cannot infect the mice naturally, and therefore, virus challenge cannot be used to evaluate the effect. we co-cultured the t cells with inactivated antigen h , h , h , and h , and t cell proliferation was measured. results indicated that after immunization with igv plus m e-hbc, the t cells show cross-protection with other subtypes. this provides evidence that igv can enhance the cross protection across subtypes. the results of this study demonstrated that recombinant igv can be useful as an adjuvant and polarize the m e-hbc vlp immune response to a th profile. igv induced the m e-hbc vlp to induce t cell proliferation and cross-reactive responses to different influenza virus subtypes. this finding represents a new direction for the promotion of cell mediated immunity in m e based vaccine against influenza. a core european protocol, i-move, describing the methods to estimate influenza vaccine effectiveness (ive) was proposed by the european centre for disease prevention and control (ecdc) and epiconcept for the - season. it includes a case control method for pooled analysis based on a randomized ''systematic'' sample of swabs. , collection of swabs using a non randomized, i.e., ''ad hoc,'' sampling strategy, left at the appreciation of sentinel practitioners, provides a greater number of cases and con-trols for ive estimation more easily than using a systematic randomized sampling strategy. the french grog (groupes régionaux d'observation de la grippe) early warning network collects more than specimens yearly from cases of acute respiratory illness (ari), using both sampling methods. , during the circulation of pandemic influenza viruses in france, it gave an opportunity to compare ive estimates using systematic randomized versus non systematic ''ad hoc'' sampling. influenza vaccine effectiveness was estimated by a casecontrol methodology according to ecdc i-move protocol, using on the one hand a systematic random sampling, on the other hand ''ad hoc'' non random sampling. the study was proposed to primary care practitioners of the grog network ( general practitioners and pediatricians) trained to collect data and swabs. the study population was patients from the community of all ages consulting a grog practitioner for an influenza like illness (ili) and having a nasal or throat swab taken within an interval of < days after symptom onset. ili was defined according to the european union (eu) case definition as sudden onset of symptoms with at least one of the following four systemic symptoms: fever or feverishness, malaise, headache, myalgia; and at least one of the following three respiratory symptoms: cough, sore throat, shortness of breath. swabs were performed through usual surveillance. no ethical approval was needed, but an oral informed consent was requested. cases were excluded if they refused to participate in the study or if they were unable to give informed consent or to follow the interview in native language because of aphasia, reduced consciousness, or other reasons. an individual was considered as vaccinated against pandemic influenza if he or she reported having received a pandemic influenza vaccination during the current season, and if at least one vaccine dose occurred more than days before ili onset. the study period started with the initiation of active influenza surveillance by the grog network, i.e., days after the beginning of the influenza vaccination campaign, and finished at the end of the influenza period defined as the last week with at least one swab positive for influenza within the grog network. ''ad hoc'' sampling patients from which swabs were taken were selected by the grog practitioners during the study period. systematic random sampling during the same period, patients were selected at random as follows. an age-group - years (gps and pediatricians); - years (gps and pediatricians); - years (gps); years or more (gps) was assigned to each practitioner, who was requested to swab the first patient of the week presenting with an ili within the pre-assigned age-group. swabs were collected in appropriate transport medium (virocult Ò , viralpack Ò , utm copan Ò ) and sent by post to the laboratory in triple packaging following the international guidelines for the transport of infectious substances (category b, classification un ). laboratory confirmation of influenza was by rt-pcr to detect currently circulating influenza a (subtypes h , seasonal and pandemic h ) and b viruses. an influenza case was defined as an ili case with a respiratory sample positive for influenza during the study period. controls were cases of ili having a swab negative for influenza during the study period. the outcome of interest is laboratory confirmed influenza. confounding factors and effects modifiers identified during the i-move preliminary study were registered: risk factors, chronic diseases, severity of underlying conditions, smoking history, former vaccinations, and functional status. data on cases and controls were collected by the practitioners using a standardized questionnaire adapted from the i-move study. questionnaires were sent by the practitioners with the swab to the virology laboratory, and sent to the grog national coordination. data entry and validation were ensured by open rome through the vircases computing tool. validation steps included control of exhaustiveness of centralization of questionnaires, comparison of data entered by the labs and the national grog coordination, coherence control, and identification of missing data. analysis was done for the two sampling groups (systematic and ad hoc) on cases ⁄ controls following the european method proposed by epiconcept, using excel ª (microsoft corp. redmond, washington, usa) and stata ª . baseline characteristics of cases and controls in unmatched studies were compared using the chi-square test, fisher's exact test, or the mann-whitney test (depending on the nature of the variable and the sample size). the association between vaccination status and baseline characteristics was assessed for both case and control groups. the vaccine effectiveness was computed as ive = )or (odds ratio). an exact % confidence interval (ci) was computed around the point estimate. analysis was stratified according to age groups, time (month of onset), presence or absence of chronic disease, and previous influenza vaccination. effect modification was assessed comparing the or across the strata of the baseline characteristics. confounding factors were assessed by comparing crude and adjusted or for each baseline characteristic. a multivariable logistic regression analysis was conducted to control for negative and positive confounding factors using a complete case analysis (with records with missing data dropped) and using multiple imputation with chained equations. the complete model included age group, number of gp visits, onset week, seasonal vaccination, previous seasonal influenza vaccination, presence of chronic disease and associated hospitalizations in the previous months, gender, and smoking status. variables were tested for multi-colinearity. interactions were tested using the likelihood ratio test (or wald test) and included in the model if significant at % level. a model with fewer variables (age group, number of gp visit, onset week, and seasonal vaccination) was also tested. several models were applied to both the ''ad hoc'' and systematic sampling groups of cases and controls. as shown in table , whatever the analysis method used, the ''ad hoc'' sampling strategy led to a slightly lower estimate of ive. the ci were extended when data were missing and reduced when using multiple imputations with chained equations. however, from a statistical point of view, comparison of ''ad hoc'' versus systematic strategies is not straightforward, because ''ad hoc'' sampling is not randomized and does not allow comparisons with statistical tests using statistical distribution laws. there are more missing data with the ad hoc sampling method. this is mainly due to our validation procedure: in the case of missing data in the systematic sampling group, as required by the i-move study protocol, queries were sent to sentinel practitioners using mail and phone calls. this specific heavy workload is not usually performed during routine surveillance and has not been achieved for the ''ad hoc'' sampling group given the great number of cases and controls ( ). within the framework of the i-move study, several items were added to the grog's usual clinical form accompanying swabs (hospitalizations, number of gp visits, smoking status, help needed for bathing or walking). in - , gps explained that this added workload was not compatible with their daily additional workload due to the pandemic situation. therefore, many of them refused to fill these new items systematically and threatened to leave the network. we thus obtained that the ''i-move items'' would be filled in for the clinical forms linked to systematic sampling, but were not in a position to obtain that for ''ad hoc'' sampling. the weekly distribution of systematic swabbing is not similar to that of ad hoc swabbing. the percentage of ad hoc swabs was higher than systematic swabs during the pandemic wave (mid-november to end of december) during which time the percentage of swabs positive for influenza was also higher ( figure ). this could explain the higher rate of positive swabs within the ''ad hoc'' samples. the vaccination campaign was launched by the ministry of health on october , , and vaccination coverage increased during the surveillance period. in february, the vaccination coverage was ae % in patients swabbed in the systematic group ( ae % on imputed data) and ae % [ ae - ae ] in the ad hoc group ( ae % on imputed data). at the national level, vaccine coverage is estimated at ae %. due to the over-mediatisation of pandemic vaccination and to rumors about its poor effectiveness, overconsultation of vaccinated patients and over-swabbing of vaccinated patients in the ad hoc group are not surprising. age distribution is significantly different between our two samples (p < ae ): the rate of - years old is lower in the systematic sampling group ( ae %) than in the ad hoc sampling group ( ae %). this can be explained by the fact that for the systematic sampling procedure, each grog practitioner had to swab the first ili patient in his assigned age group, whereas for ''ad hoc'' sampling, every grog practitioner could swab any ili patient irrespective of age. given the emphasis by health authorities and media on the burden of pandemic influenza among children and teenagers, one can hypothesize that when they were able to, sentinel practitioners focused on these age groups. gps in the ad hoc sampling scheme seem to have been more likely to select cases and further, to select vaccinated cases. those patients may have consulted earlier with specific symptoms (strong headache being more prevalent among cases). over-swabbing of patients having these symptoms in the ad hoc group is likely. the - pandemic influenza season was markedly different from previous ones: vaccination rate increased during and mainly after the pandemic peak; behaviors were strongly modified by unusual media hype; clinical features and risk factors might be different. it will be necessary to see if similar results are observed during a regular influenza season during which the vaccination rate increases before the epidemic peak with usual messages about vaccination and usual clinical influenza features. influenza early warning networks can estimate ive, taking into account many covariates. from a stakeholders and patients point of view, during the - influenza pandemic wave, there were no major discrepancies between ive estimated with an ad hoc sampling strategy, based on sentinel practitioners instinct, and ive estimated with a systematic random sampling strategy whatever the multivariable analysis methodology. although from a statistical point of view, comparison of the two strategies is not readily feasible because of the non random nature of ad hoc sampling. this latter strategy seems to result in slightly lower ive estimates, which could potentially be attributed to sentinel practitioners swabbing behavior. the ability to avoid missing data is a key point to decide which sampling method must be adopted, because ci extent depends greatly on the proportion of missing data among covariates. to match ive evaluation to surveillance networks practicality, selection of only those data essential for the study endpoint and easily collected by sentinel practitioners is paramount. it will be necessary to determine if results similar to those observed during the - pandemic season are found during a regular influenza season. influenza a viruses are important pathogens which remain a major cause of morbidity and mortality worldwide, and large numbers of the human population are affected every year. the first influenza pandemic in this century broke out in humans in march , and it was declared to be pandemic by mid-june. as of august jul , the pandemic virus had caused more than deaths worldwide, according to the world health organization (http:// www.who.int/csr/don/ _ _ /en/index.html). the infection and spread of the pandemic influenza was reduced in part due to the use of vaccines. however, the lack of h n pdm vaccine early in the pandemic illustrates the need to improve vaccine production and to generate vaccines that induce stronger cross-protection. inactivated split vaccines or live attenuated influenza virus vaccines (laivs) against h n pdm viruses were approved for human use by the united states food and drug administration. both the inactivated vaccines and laivs are produced by creating reassortant viruses that generally contain six vrnas (pb , pb , pa, np, m, and ns) from a master donor strain, plus the two glycoprotein vrnas (ha and na) from a virus that antigenically matches the strain predicted to circulate in upcoming influenza season (e.g. a ⁄ ca ⁄ ⁄ ). the reference viruses containing inactivated split virus vaccines are produced in embryonated chicken eggs, and primarily result in the production of antibodies that recognize the viral glycoproteins. both of these vaccine approaches require significant lead time for vaccine production, and modern approaches to speed preparation of vaccines and improve their efficacy is a global priority. , the ns protein of influenza a virus is a multifunctional protein that plays important roles in virus replication and as potent type i ifn antagonist. , mutations and ⁄ or deletions in ns typically induce stronger ifn responses by the host; those in turn suppress the replication of influenza virus - and can enhance immune recognition. [ ] [ ] [ ] [ ] in this study, we created a panel of experimental h n pdm ns-laiv candidates that have different deletions in the ns vrna and analyzed the vaccine potential of each ns-laiv in mice and ferrets to identify the best candidate(s). wt h n pdm influenza a virus a ⁄ new york ⁄ ⁄ (ny ) was created by reverse-genetics directly from a human swab specimen collected in new york state in april . deletions were introduced into the ny ns plasmid to create three mutant ns segments: ns - , ns - , and nsd . nucleotides - (cdna of ns segment) and - were replaced by stop codons to generate ns - and ns - ; nucleotides - were deleted to generate nsd , whose open reading frames for ns and nep were maintained. recombinant viruses were generated by co-transfection of eight reverse-genetics plasmids carrying the cdna of each gene segment into t ⁄ mdck cocultured monolayer adapted from hoffmann et al. , mouse studies experiments were performed in a biosafety level laboratories approved by the u.s. centers for disease control and prevention and the u.s. department of agriculture, and were conducted under approved animal care and use protocols. groups of -week-old female balb ⁄ cj (jackson laboratory, bar harbor, me, usa) were anesthetized with isoflurane and inoculated intranasally with tcid of each recombinant virus in ll of pbs diluent, or pbs as controls. body weights and clinical symptoms of the mice were monitored daily for days. nine mice in each group were euthanized on , , and days post inoculation (dpi), and nasal washes and lungs were collected for virus titration by tcid assay in mdck cells. at dpi, mice per group were challenged intranasally with · tcid ( ld in -week-old mice) of a mouse-adapted variant of ny (a ⁄ ny ⁄ ⁄ -ma ) (accepted, journal of virology). disease symptoms and weights of the vaccinated mice were monitored for days, and four mice from each virus group were euthanized at and days post challenge. lungs were removed and homogenized for virus titration by tcid assay. the mice that became moribund or lost > % of their starting body weight were euthanized for humane reasons. male fitch ferrets (triple f farms, sayre, pa, usa), - months of age and serologically negative by hemagglutination inhibition (hi) assay for currently circulating influenza viruses were used in this study. groups of or ferrets were inoculated intranasally with ae tcid of one of the viruses: ny wt (n = ), ns - (n = ), ns - (n = ), or nsd (n = ). ferrets were monitored for clinical signs through dpi as previously described. nasal washes were collected on , , , and dpi and were titrated in mdck cells by tcid assay. serum was isolated from blood collected ae weeks after immunization and used for neutralization assays. the ferrets were challenged with pfu of a ⁄ mexico ⁄ ⁄ ae weeks postimmunization and monitored for clinical signs of disease through dpi. nasal washes were collected on , , , and dpi, and were titrated in mdck cells by plaque assay. using reverse genetics, we created three laiv candidates weight loss of wt virus inoculated mice became evident at dpi, and the mice did not recover until dpi (figure a) . in contrast, mice inoculated with any one of the vaccine candidates had no clinical signs of disease and continued to gain weight at the same rate as did the mock- inoculated mice ( figure a ). viral titers in the lungs of ns - , and ns - infected mice were $ -fold lower than titers from wt virus-infected mice at all the time points analyzed ( , , and dpi) ( figure b) . notably, the nsd laiv was cleared from the mouse lungs very rapidly, and the mean titers were $ -fold and -fold lower than the titers of the wt virus at and dpi, respectively ( figure b) . the vaccinated mice were challenged with a mouseadapted variant of ny (accepted, journal of virology) on dpi. no disease symptoms were observed in the mice immunized by any of the ns-laiv candidates or the wt control. in contrast, disease symptoms including ruffled fur, hunched posture, and weight loss were observed in the mock-immunized mice as early as days post challenge (dpc); the symptoms progressed to severe disease, and the animals showed dramatic weight loss, became moribund, and succumbed to infection by dpc (figure c ). high titers of virus ($ tcid ⁄ ml) were present in the mock-immunized mice at dpc and at dpc ( figure d ). in contrast, virus was not detected in the lungs of immunized mice ( figure d ). this challenge data demonstrates that all of the ns-laiv candidates, including the highly attenuated nsd , induced sterilizing immunity that protected mice from a lethal ny h n pdm variant. groups of ferrets were intranasally immunized with ae tcid of each vaccine candidate or the wt virus. the titer of viruses recovered from nasal washes ranged from ae to ae tcid ⁄ ml through day in the wt virusinfected group, while the ns-laivs showed various degrees of attenuation (figure a) . the viral titer of all of the ns-laivs is at least -fold lower than that of wt in the nasal wash collected at dpi. the ns - laiv was the least attenuated in ferrets, and its replication was similar to that observed in mice. relative to the wt virus, the ns - laiv showed -fold reduction in titer, and the nsd laiv was below the limit of detection (at least fold reduction) at dpi. sera from blood collected ae weeks after immunization was analyzed for the presence of neutralizing antibodies by micro-neutralization assays. the ns-laiv candidates all induced very strong neutralizing antibody responses ( - ) that were similar to the titer elicited by wt virus infection ( figure b ). the ferrets were challenged with pfu of a ⁄ mexico ⁄ ⁄ (h n pdm) ae weeks post immunization. little disease or weight loss were observed in the naïve ferrets, and the ferrets immunized by infection with wt virus or the ns-laiv candidates didn't show any disease symptoms or weight loss. in contrast to the high titer of virus detected in the naïve ferrets through dpc, the ns-laiv immunized ferrets had very low levels of a ⁄ mexico ⁄ ⁄ in their nasal washes at dpc ( figure c ). the ferrets immunized with the ny ns-laivs had $ -to -fold lower viral titers than did the naïve animals ( figure d ). in summary, the ns-laiv candidates dramatically inhibited initial replication of the h n pdm virus under stringent challenge conditions ( pfu), and that the vaccinated animals rapidly cleared the infection (to below the limit of detection, by dpc). our results demonstrate that all of the ns-laiv candidates are attenuated compared to the wt h n pdm virus, and the degree of attenuation is dependent on the specific ns mutation. ns - was the least attenuated and does not represent a good vaccine candidate; whereas, nsd and ns - were highly attenuated in both the mouse and ferret models. although they were markedly attenuated, they elicited strong neutralizing antibody responses and protected mice and ferrets from subsequent challenge. nsd has a subtle in-frame deletion ( nt) that affects both the ns (residues - ) and nep (residues - ), and is analogous to a naturally attenuated variant of a normally highly pathogenic h n virus (a ⁄ sw ⁄ fj ⁄ ). the analogous ns deletion in a ⁄ sw ⁄ fj ⁄ (residues - ) was shown to reduce binding to host cleavage and polyadenylation specificity factor (cpsf), reduce ns protein stability, and enhance the type i ifn response of this h n virus. our study indicates that deletion of these nt in the ns vrna of the h n pdm also stimulates the host ifn response, specifically, ifn-ß, ifn-k , ip , and mxa (data not shown). the role of the deletion of residues - from nep has not been elucidated, but the induction of ifn and isgs by nsd was similar to, or slightly lower than, their induction by ns- , suggesting that the nep mutation also has an attenuating effect that warrants future investigation. in summary, we have generated a panel of laivs directly from a swab specimen containing a new pandemic virus and analyzed their attenuation and immunogenicity in two animal models. our study demonstrates that nsd is a novel ns-laiv that could be used to create laivs for diverse influenza a viruses. this study also validates the use of ns-laiv candidates, which are not only highly attenuated, but they also elicit strong innate and adaptive immune responses, resulting in protection of mice from subsequent challenge with a lethal mouse-adapted variant of ny , and ferrets from challenge with a ⁄ mexico ⁄ ⁄ (h n pdm). currently, a total of approximately million doses of inactivated influenza vaccine are being produced worldwide each year. one of the limitations in vaccine production is poor growth of human isolates in embryonated chicken eggs. this is essential to develop high yield seed viruses for large scale production of influenza vaccines. influenza a vaccine production utilizes high yield reassortants carrying ha and na genes from a wild type (wt) strain with generally - internal genes from the a ⁄ pr ⁄ ⁄ (pr ) strain, an highly egg adapted high growth donor strain. influenza b vaccines, however, have been produced directly from wt strains, partly because no high yield donor analogous to pr has been identified. in recent years, reverse genetics has been used as an alternative means of developing high growth vaccine viruses. , since in this plasmid-based technology, a : reassortant (six internal genes from a donor strain and two surface antigen genes from wild type strain) can be directly rescued, reverse genetics-derived reassortant viruses were expected to grow as efficiently as those derived from classical reassortment. however, reverse genetics reassortants have not produced the expected high growth for several reasons: (i) the : configuration is not always the best for virus yield, (ii) there is no process included for positive selection of adaptive mutants from quasispecies, and (iii) cell-derived viruses are not readily adapted to grow efficiently in eggs. our laboratory at new york medical college has been preparing b reassortants for several years by classical reassortment using b ⁄ lee ⁄ as a donor. it has been possible to develop b reassortants, which produce higher virus yields than wt strains in eggs, and it was found that the np gene of b ⁄ lee ⁄ was important in producing high yield b reassortants. however, b ⁄ lee ⁄ is inconsistent in providing high yield properties to b reassortants. in this study, in an attempt to find an alternative donor, we investigated the usefulness of b ⁄ panama ⁄ ⁄ for developing high yield b reassortants. as a wt strain, b ⁄ brisbane ⁄ ⁄ was used, which is one of the recommended influenza b virus vaccine strains for the ⁄ and ⁄ seasons. we found that b ⁄ panama ⁄ ⁄ is a useful donor, and some of the resultant reassortants were considered as vaccine candidates. b reassortant viruses were prepared by the classical reassortment method described by kilbourne. the antiserum to b ⁄ panama ⁄ ⁄ hemagglutinin and neuraminidase (hana) was raised in this study by immunizing rabbits with hana isolated from b ⁄ panama ⁄ ⁄ ; purified igg was used for antibody selection. the yields of the reassortants and their corresponding parent viruses were assessed by hemagglutination assay. viral rna was extracted directly from the allantoic fluid and amplified by rt-pcr to produce cdna for analyzing the gene composition. restriction fragment length polymorphism (rflp) analyses were performed to determine the origin of each gene segment of the high yield reassortants. restriction enzyme sets for each gene segment are available upon request. in this study we investigated the usefulness of b ⁄ panama ⁄ ⁄ as a donor for transferring high yield phenotype. b ⁄ panama ⁄ is a yamagata lineage strain with high growth phenotype (ha titer: - ). b ⁄ panama ⁄ ⁄ itself was a recommended b virus vaccine strain for ⁄ - ⁄ seasons. as a wt virus, a victoria lineage strain, b ⁄ brisbane ⁄ ⁄ , was used, which is a recommended b virus vaccine strain for use in the ⁄ and ⁄ seasons. reassortants were prepared according to classical reassortment protocol. after co-infection of b ⁄ panama ⁄ ⁄ and b ⁄ brisbane ⁄ ⁄ , progeny viruses carrying surface antigens (ha and na) of the vaccine strain were negatively selected by anti-b ⁄ panama hana antibodies, followed by passages without antibodies for positive selection of eggadapted viruses and finally limited dilution cloning. nymc bx- , bx- b, bx- d, and r- a are representative of resultant reassortants, which have significantly higher ha titers than the wt strain. the complete gene compositions of these reassortants were determined by rt-pcr ⁄ rflp analyses. as shown in table , all of these reassortants contained the pb of b ⁄ panama ⁄ ⁄ . other genes of b ⁄ panama ⁄ ⁄ (np of bx- , m of bx- b, and pb of bx- d) may not be involved in the high virus yield, since no significant growth difference among these reassortants in eggs was found as assayed by hemagglutination test. accordingly, the pb of b ⁄ panama ⁄ ⁄ is considered to be the sole factor involved in the high yield phenotype donated to the vaccine strain. we previously found that the b ⁄ lee ⁄ np gene was important in producing high yield b reassortants. it was of interest to examine whether b ⁄ lee ⁄ np and b ⁄ panama pb could work together to produce even higher yields. to test this possibility, bx- b ( : reassortant: pb and m genes from b ⁄ panama and the rest of the genes from b ⁄ brisbane) was selected and further reassorted with b ⁄ lee ⁄ . despite some difficulty in removing the na gene of b ⁄ lee ⁄ (r- c, b, b in table ), by monitoring ha and na genes of resultant viruses after each antibody selection passage with anti b ⁄ lee ⁄ hana antibodies, we were able to isolate and clone a triple reassortant, nymc bx- , which contains the np gene from b ⁄ lee ⁄ and pb and m genes from b ⁄ panama; the remaining genes are from b ⁄ brisbane ⁄ ⁄ (table ). in comparison with bx- b, no significant growth enhancement (nor reduction) in eggs was found for bx- over that seen for bx- b. nevertheless, bx- stably produces high virus yield and has been utilized as a seed virus for influenza b vaccine production for the - season by one or more vaccine manufacturers. there are contradictory reports - about the usefulness of reassortment for high yield influenza b viruses. however, we have been preparing b reassortants for several years by classical reassortment using b ⁄ lee ⁄ as a donor, and have been able to generate higher virus yield than wt strains. in this study, we found that b ⁄ panama ⁄ ⁄ serves as an efficient donor in providing the high growth capacity to b ⁄ brisbane ⁄ ⁄ (a recommended vaccine virus of victoria lineage for ⁄ - ⁄ seasons), and that the pb of b ⁄ panama ⁄ ⁄ is associated with the high yield phenotype. this particular strain from yamagata lineage might be useful to prepare high yield reassortants for other victoria lineage vaccine viruses. we noticed in this study that there may be segment incompatibilities between b ⁄ panama ⁄ ⁄ and b ⁄ brisbane ⁄ ⁄ . as shown in table , the pa and ns genes of all the high yield reassortants examined are derived from wt, b ⁄ brisbane ⁄ ⁄ , not from the donor, b ⁄ panama ⁄ ⁄ . this indicates that in this reassortment, the pa and ns genes are not replaceable with that of the donor to obtain high yield viruses. this degree of incompatibility might be common in b reassortment, resulting in low donor ⁄ wt reassortants, such as : and even : reassortants that we obtained in this study. if this is the case, reverse genetics based on : configuration may not result in generating high yield b reassortants unless a variety of donor ⁄ wt combinations are designed. one can speculate that in influenza b viruses, the surface glycoproteins (ha and na) and some of the internal proteins are functionally more closely related than in influenza a virus, as was seen in that pa and ns genes of b ⁄ brisbane ⁄ ⁄ reassort together with the ha and na genes of the same parent (table ). in our recent study on a reassortment between b ⁄ lee ⁄ and b ⁄ panama ⁄ ⁄ , it appeared that ha shapes overall gene constellations of the resultant reassortants, namely the reassortants tend to have more internal genes from the same parent of ha, no matter which parent's ha is selected by antibodies against the surface antigens of the other parent (data not shown). because of success in influenza a virus reassortment with pr , it is generally believed that reassortant with : or : configuration is optimal for virus yield. this may be the case in most instances of influenza a reassortment, but is not necessarily so in b reassortment. as shown in this study, only a single donor gene is capable of improving the yield of vaccine strain by reassortment. influenza a ⁄ h n v has spread rapidly in all parts of the world in as a true pandemic. epidemic events in russia occurred during the last week of september starting from far east region (yuzhno-sakhalinsk). kaliningrad (the western most russian city) was the second starting point of the epidemic. during october the epidemic spread over the whole russian territory. in a short period the new virus started to change genetically as it began to adapt to human populations during this pandemic (http://www.who.int; http://www.euroflu.org). in the period from may to december , clinical samples (nasopharyngeal swabs and postmortem materials) of patients with influenza-like illness from different regions of russian federation were analyzed to confirm the diagnosis using real-time reverse transcription pcr (rrt-pcr). clinical nasopharyngeal swabs and bronchoalveolar lavage and post mortal fragments of trachea, lungs, bronchi, spleen from saint petersburg hospitals and basic laboratories of federal influenza center were included in this study. all specimens were taken from patients with influenza-like illness or viral pneumonia. specimens were tested by rrt-pcr according to cdc protocols, i.e. using superscript iii platinum one-step qrt-pcr system (invitrogen) with primers and probes for infa, h seasonal, and h sw (biosearch technologies). in addition, the test-systems 'amplisense influenza virus a ⁄ b-fl' and 'influenza virus a ⁄ h -swine-fl' for pcr-detection, typing and subtyping of influenza viruses were also used. these test-systems are produced by central institute of epidemiology, moscow, russia and recommended by russian ministry of health as tests for influenza diagnosis. sequencing was carried out on an abi prism -avant genetic analyzer (applied biosystems, usa) with bigdye terminator cycle sequencing kit. phylogenetic analysis was performed using programs vector nti . (invitrogen) and mega . (psu, usa) by maximum likelihood with the tim+i+g model for ha, and -hky+i+g model for na. evolutionary model was selected by akaike information criterion (aic) in model-test (posada, crandall, ). statistical reliability of tree branches was evaluated by bootstrap test ( replications). immunohistochemical study was performed using novalink antibodies to ha and np with novocastra visualization system. influenza virus a ⁄ h n v rna was detected in patients with severe form of influenza-like illness and fatal cases. out of pcr-confirmed flu recovered cases % were patients under years of age, % were aged - years, and % were older than years. mean age of recovered patients was ae years (from month to years). viral rna in postmortem materials was detected mostly in lung tissue ( % of specimens) and trachea fragments ( %), and less commonly in spleen ( %). mean age of the deceased with confirmed flu (h n v) infection was ae years with age ranging from months to years. in % of fatal cases, influenza was complicated by viral or secondary bacterial pneumonia. median time from the onset of illness until death was days. according to our data, % of patients died had diabetes, ae % were obese, and % were pregnant women in the nd or rd trimester. ha and np were detected by immunohistochemical assay in lung tissue of dead patients with confirmed influenza virus a ⁄ h n v infection. ha and np was revealed in the endothelium of different sized blood vessels (capillaries and arterioles). these influenza virus proteins were also detected in some tissue macrophages apart from epithelium and endothelium. the localization of the two proteins was different: ha is mostly localized in cell membrane and cytoplasm, and np -mostly in the nucleus. here we present data on molecular genetic characteristics of strains of pandemic virus, strains obtained from clinical specimens, and from post mortal ones isolated in the research institute of influenza. all the strains studied contain the s n substitution in m protein, which indicates resistance to the adamantane antivirals, and have no h y substitution in the neuraminidase, which indicates resistance to oseltamivir. the phylogenetic analysis showed that russian viruses were similar to influenza viruses a ⁄ texas ⁄ ⁄ and a ⁄ california ⁄ ⁄ (ha similarity ae %). all russian viruses could be divided in two clusters: the first one includes viruses similar to the reference strain a ⁄ california ⁄ ⁄ , and the second one, which is the majority of viruses analyzed includes strains with substitutions ha s t, na n d, v i, and ns i v (figure ). bootstrap support was . the isolates with ha s t substitution can be classified in one of the five minor genome variants of a ⁄ h n v viruses found in the united states and mexico in . several viruses had strain-specific substitutions in antigenic sites sb and ca and the mutation d g in ha receptor-binding site. the substitution of amino acid residue asp to gly at position of ha was found in eight of eleven isolates ( %) from postmortem lung and trachea samples and two of forty isolates ( %) from nasopharyngeal swabs of patients with severe course of the disease. appearance of amino acid substitutions in the ha receptor-binding site (d e and d g ⁄ e) could be associated with influenza virus passaging on eggs. five strains that contained g at position of ha were isolated from post mortal specimens on mdck cells in this study, thereby excluding the possibility of substitution appearance hence to virus adaptation on eggs. in order to reveal genome changes in a(h n )v, strains isolated on the territory of russian federation during the pandemic, full genome sequences from genbank, and research institute of influenza database were analyzed comparing two groups of viruses (isolated before and after sept ). nine amino acid changes observed predominantly in late pandemic strains were found. five of them (s p, s n, d g, v i, v i) reside in ha, two in na (i v, n k), two in pb (k n, t i), and one in pa (f l). towards the end of the epidemic the viral population had demonstrated statistically certain rise in number of strains containing mutations in four genes. difference between groups was statistically significant (chisquare test, p = ae ). if v > ae , than difference between early and late strains is statistically significant. additionally fisher's test determined whether 'early strains' and 'late strains' differ significantly in the proportion of 'no mutation event' and 'mutation event' attributed to them in each particular position. all calculations were performed in fisher_tk freeware by vladimir belyaev similar to calcfisher (haseeb, ) fully described here (http://www.jstatsoft.org/v /i /paper). we have selected positions with statistically significant amino acid changes in late strains (p-value ae ). according to full genome analysis of influenza virus a ⁄ h n v strains, seven clades were distinguished, but the divergence between representatives of different clades remained small. (figure ). besides the strain a ⁄ perth ⁄ ⁄ also contains substitution s f in the same ha antigenic site. according to data obtained, the epidemic in russia was caused only by influenza virus a ⁄ h n v. unlike the previous epidemic periods when most severe influenza cases were registered among the children under years and among elderly people aged over years, the first wave of pandemic due to influenza virus a (h n )v resulted in increased level of mortality mainly among the people aged - years. though all pandemic viruses showed comparative genetic homogeneity, some evolutionary trends could be outlined. for clarification of the exact pathogenic role of mutation d g in ha receptor binding site, further studies are necessary. full-genome analysis of influenza virus a ⁄ h n v strains circulating in the southern hemisphere in the new epidemic season revealed the phylogenetic subgroup distinguished by seven substitutions in inner proteins (pb , pb , np, ns ) and sa antigenic site of ha (n d). the changes revealed could be caused by adaptation of the virus to an immunized human population. nasal and throat swabs (placed in ml mem and frozen at ) °c until use for viral rna extraction and tissue culture inoculation) were collected from patients with febrile illness, i.e., > ae °c. samples were received from clinics in us embassies and us military laboratories located throughout the world since the initial who declaration of novel h n outbreaks as a global pandemic on june , . viral isolates were obtained from inoculating cultures of mdck cells with ae - ae ml viral suspensions collected in mem originated from patients after - days incubation. [ ] [ ] [ ] [ ] due to low viral titers in normal clinical samples, most of full viral genome sequences were derived from viral stocks obtained by tissue culturing passages (mdck, - times). viral rna was extracted from clarified supernatant fluid of nasal ⁄ throat swabs or mdck cultures using the 'charge-switch' rna extraction system based on the user manual protocol from the manufacturer (invitrogen inc., ca, usa). total rna was eluted into volume equal to original sample volume, i.e., ll starting viral supernatant used to yield final ll rna in molecular grade water (invotrogen inc.) and stored at ) °c until tested. generating ⁄ preparing overlapped cdnas for full genome coverage of novel h n viruses by multiple rt-pcr amplifications the first step in the high-throughput sequencing pipeline for full influenza genome sequences was to establish a robust rt-pcr amplification scheme consisting different rt-pcr primer pairs covering all rna segments to ensure % amplification coverage of full viral genomes of all the incoming targeted viruses (houng, hs. , submitted for publication). extracted viral rna ( ll), derived from mostly mdck culturing stock or clinical sample containing sufficient viral load (> infectious units per ml) was added to primer-free rt-pcr total master mixture ( ae ll) for each virus followed by adding primer pair ( ll, pmole ⁄ ll per primer). rt-pcr was then performed: rt reaction through two hold-steps ( °c, minutes and °c, minutes); cycling amplifications ( °c for seconds, °c for seconds, °c for ae minutes). specific cdna amplicons corresponding to each individual primer pair were routinely monitored and visualized by agarose gel electrophoresis. pooled cdna products ( - lg) from each viral rt-pcr amplification run were used as sequencing substrates according to the roche flx user manual and bulletins by incorporating adaptors containing individually multiplex identifier [mid]-key assigned to each individually pooled viral cdna. up to different mid-keyed viral cdna were further pooled together to be clonally amplified on capture beads in water-in-oil emulsion micro-reactors (em amplifications), and pyrosequenced using one of two regions of a · mm picotiterplate. for each individual viral genome containing multiple assemblies ( rna segments), we obtained sff file(s) containing raw sequencing reads from which nucleotide sequence data and phredlike quality scores were extracted. on average, ae - ae % of - million mid-key specific nucleotides were extracted and mapped for consensus genome sequences. roche gsmapper (v. . and . ) software was used to assemble all sequencing raw data and sff files into consensus sequences. new reference mapping projects were created to assemble each individually mid-keyed viral cdna into consensus viral sequences. one of the earliest h n genomes of california origin, a ⁄ california ⁄ ⁄ (h n ), deposited in genbank, was routinely employed as a reference genome sequence for most of gsmapper projects. the resultant consensus sequences obtained were further verified and validated through the ncbi annotation utility check and ultimately deposited to the ncbi influenza database, genbank. nucleotide sequences specific to each individual rna gene were aligned by the geneious pro . . software (http://www.geneious.com). trees were built based on the tamura-nei genetic distance model using the neighbor-joining method with no outgroup used via geneious pro . . . phylogenetic trees of the h n genomes were constructed by importing fasta files containing specific concatenated target sequences of pb , pb , pa, ha, np, na, mp, and ns from each individual virus into the geneous pro software and going through the sequence assembling and tree building steps. high-throughput pyrosequencing of pooled novel h n cdnas by roche flx system up to viral cdnas could be routinely sequenced to completion for different full viral genomes from a single roche flx picotiter plate by utilizing the combination of pico-titer plate's two distinct regions as well as different mid-keyed adaptors. the 'shotgun' sequencing approach employed in this study is a feasible method to viral isolates (n) sequence multiple pooled h n viral genomes. for each pyrosequencing experiment, approximately - passed key reads (single fragment per bead) were obtained that yielded readable nucleic acid sequences. among those close to a million passed key reads, only - passed key reads had an average sequencing read length of > bps, defined as 'long reads' ( bps · reads = total of million bases of nucleic acid sequences) that were used to assemble into influenza genome sequences. mathematically, - million bases of raw sequencing data from each single roche flx experiment would provide sufficient sequencing bases to cover full genome sequences with approximate - · of sequencing depth coverage of influenza a with average genome size of bps for the total of eight segmented rnas. so far, more than full h n genomes sampled worldwide have been successfully sequenced and deposited in the ncbi database by division of viral diseases, walter reed army institute of research (wrair). the bioinformatics derived from unique viral genome sequences generated from this study based on constant rt-pcr amplification scheme and identical roche pyrosequencing protocols provide a reliable data set in predicting the evolutionary patterns of pandemic viruses. wrair received clinical samples from us embassies and military personnel throughout the world since the initial who announcement of novel h n outbreaks. nearly equal distributions of sequenced viruses derived from three broadly categorized geographic regions, north america, central ⁄ south america, and asia ⁄ europe ⁄ africa (data not shown). besides the geographic distribution pattern of viral isolates, figure displays the viral isolation time lines of all the sequenced viruses reflecting two peaks that coincided with two waves of pandemic infections, early-mid summer and fall of . phylogenetic trees of the eight influenza a segments of all sequenced viruses were tentatively generated. it was found that the substitution frequencies per site for the ha, na, and ns genes are at much higher rate than the other five genes, pb , pb , pa, np, and mp genes (data not shown). the observed higher genetic variations for ha and na genes of h n are consistent with the historical genomic and epidemiological dynamics data of human influenza a revealing higher temporal fluctuations in ha and na genes. [ ] [ ] [ ] [ ] analysis of full influenza genomes containing concatenated eight complete rna segments revealed the existence of two distinctive genetic clades in circulation since the beginning of pandemic, as shown in fig-ure . it is noteworthy that all viruses of mexico and california origins (clade shown at the top of figure ) were isolated at the beginning of pandemic prior to the isolation of all other viruses belonging to the second genetic clade . , discussion during the past decade, the advance of dna sequencing technology, such as development of ngs, in making full viral genome sequences readily available have enabled study of far broader and more detailed aspects of evolutionary change for any new emergent infectious pathogen. the massive sequencing capacity of roche flx system allows simultaneously process and sequence millions of individual cdna molecules, in contrast to processing and sequencing individual cdna fragments by conventional sanger sequencing method. within a short period of few months since the beginning of the pandemics, wrair accomplished large number of representative h n full genomes of worldwide origins via roche flx system. sequencing data derived from this study illustrates a much higher genetic variation rate for ha and na genes of h n that is compatible to the higher temporal fluctuation rate for ha and na genes of seasonal influenza a derived from decades of intensive monitoring and comparison studies and analyses. [ ] [ ] [ ] [ ] following the mexican and us reported cases, confirmed outbreaks of swine h n rapidly proliferated and spread throughout europe, asia, africa, and south america, most probably via global airline travel. , it seemed that new cases in the us and most cases throughout the world had been clinically mild relative to the initial reported cases in mexico. [ ] [ ] [ ] [ ] here we demonstrate through the phylogenetic relationship of sequenced h n full genomes that the clinical isolates could be divided into two different clades of viruses, i.e., the clade genetic group contains only viruses isolated at the beginning (march ⁄ april , mexico and california) of pandemics and the rest of other viruses all belong to the nd genetic group, clade . thus, it's likely that the currently circulating h n of clade causing worldwide infections is genetically different from the initial h n isolates that caused the early infections in mexico and california. , introduction a pandemic influenza virus ( h n ) was recently introduced into the human population. the hemagglutinin (ha) gene of h n is derived from 'classical swine h n ' virus, which likely shares a common progenitor strain with the human h n virus that caused the pandemic in . since antigenic changes of influenza virus ha occur more slowly in swine than in humans, we hypothesized that h n might still retain an antigenic structure similar to that of h n or the early isolates of its descendants. in this study, we compared ha antigenic structures of h n and human h n viruses by a molecular modeling approach to demonstrate the existence of shared epi-topes for neutralizing antibodies. we found that has of h n and the h n virus shared a significant number of amino acid residues in known antigenic regions. from this observation, we hypothesize that the h n ha antigenic sites will be targeted by antibody-mediated selection pressure in humans in the near future. we further discuss possible directions of antigenic changes in the evolutionary process of h n . sequence data of ha genes modeller v was used for homology modeling of ha structures. after models of the ha trimer were generated, the model was chosen by a combination of the mod-eller objective function value and the discrete optimized protein energy statistical potential score. after addition of hydrogen atoms, the model was refined by energy minimization with the minimization protocols in the accelrys discovery studio . software package using a charmm force field. steepest descent followed by conjugate gradient minimizations was carried out until the root mean square gradient was less than or equal to ae kcal ⁄ mol ⁄ a. the generalized born implicit solvent model was used to model the effects of solvation. the ha model was finally evaluated by using procheck, whatcheck, and verify- d. custom-made programs were developed with the ruby language and used for investigating the numbers of potential n-glycosylation sites and candidate codons (cand ) in ha sequences. it is known that the h ha molecules have four distinct antigenic sites: sa, sb, ca, and cb. , as a result, these sites consist of the most variable amino acids in the ha molecule of the seasonal human h n viruses that have been subjected to antibody-mediated immune pressure since its emergence in , although it was absent in humans from to . to investigate the structures of these antigenic sites of h n , d structures of the ha molecules of sc , the recent seasonal human h n virus (br ), and h n (ca ) were constructed by a homology modeling approach, and compared by mapping all the amino acid residues that were distinct from those of sc ha (data not shown). we found that most of these antigenic sites of br ha predominantly contained altered amino acid residues if compared with sc . by contrast, amino acid residues at these positions were relatively conserved in ca ha when compared with sc ha. notably, the sa and sb sites, which contain many amino acids involved in neutralizing epitopes near the receptor binding pockets, remain almost intact ( table ), suggesting that antibodies raised by natural infection with sc or its antigenically related descendant viruses play a role in specific immunity against ca . these observations lead us to hypothesize that such antigenic sites involving the conserved amino acids will soon be targeted by antibody-mediated selection pressure in the human population. based on this hypothesis, we speculated that h n would undergo patterns of amino acid substitutions in ha similar to those seen in seasonal human h n viruses during its epidemic period (i.e. those that have been substituted since ) (figure ) . we then predicted possible amino acid substitutions of h n from the sequence similarity of the antigenic sites. for example, both sc and ca had an asn residue at position in the sa site. for sc , the residue at this position has altered from asn to lys since . combining these two facts, it seems reasonable to hypothesize that ca will also undergo an amino acid substitution from asn to lys at position in the future. interestingly, we found that some of the recent variants of the h n virus have indeed undergone substitutions identical to those predicted in figure . it is important to monitor whether such variants will be selected and survive in sustained circulation in humans. next, we analyzed the acquisition of potential n-glycosylation sites associated with antigenic changes. previously, we reported that cand sites, a set of three codons that require single nucleotide substitution to produce n-glycosylation sequons, were important motifs to rapidly acquire n-glycosylation sequons. therefore, we investigated the number and location of potential n-glycosylation sites and cand sites in h n ha. we found that ca also had a single n-glycosylation sequon at the same position in the globular head region of ha, and lacked the multiple n-glycosylations that have been observed in the antigenic changes of the human h n virus during the early epidemic of this virus. we also found that ca ha possessed three cand sites that were present at the same position in sc ha (positions of the first asn residue, , , and ). of these, the cand sites with positions at and had actually become potential n-glycosylation sites in human h n viruses. this result suggests the likelihood of additional n-glycosylation at these sites during future antigenic changes of h n ha. notably, some of the recent h n variants (as of march , ) have an additional n-glycosylation sequon at position , where the h n virus readily acquired an n-glycosylation site during its circulation. the present study suggests that the antigenic structure of h n ha is similar, at least in part, to that of the h n ha. the and h n has share unique three-codon motifs that are important to readily acquire n-glycosylation sequons in their globular head region. based on these similarities, we predicted possible amino acid substitutions that might be associated with future antigenic changes of h n , and confirmed that such substitutions occurred in some of the recent variants of this virus. the present study provides an insight into likely future antigenic changes in the evolutionary process of h n in the human population. influenza viruses are classified into three types, a, b, and c, based upon the antigenic properties of nucleoproteins and matrix proteins. influenza a virus infects a wide range of hosts, including human, bird, swine, equine, and marine mammal species, while influenza b and c are less pathogenic than influenza a and are mainly found in humans, although there is evidence that they can also infect other species. influenza a has evolved in association with its various hosts on different continents for extended periods of time. to survive as a successful pathogen, the influenza viruses have developed a number of mechanisms, including antigenic mutation and genome reassortment, to continuously evolve and evade the surveillance of the host immune systems. antigenic and genetic analyses have provided important insights into the molecular dynamics of influenza virus evolution. however, a comprehensive understanding of influenza viral genetic divergence and diversity remains lacking. neuraminidase (na) is a major surface glycoprotein of influenza a and b, but is absent in influenza c. it plays a key role in virus replication through removing sialic acids from the surface of the host cell and releasing newly formed virions. influenza a viral na genes are classified into nine subtypes (na -na ) based upon their antigenic properties, while na genes of influenza b are not classified into subtypes. furthermore, most na subtypes of influenza a have evolved into distinct lineages and sub-lineages, which correspond to specific hosts or geographical locations. in this study, we conducted large-scale analyses of influenza na sequences in order to infer their evolution and to identify lineages (or sub-lineages) of influenza a viruses. a total of na sequences that excluded laboratory recombinant sequences were downloaded from genbank. sequences were aligned with muscle and mafft. the alignments were adjusted manually using translatorx, based upon corresponding protein sequences. phylogenetic analyses were conducted using the maximum-likelihood (ml) method in raxml. a set of perl scripts were written by us to facilitate this computational analysis. lineages and sub-lineages were determined based on the topology of the ml trees. additional information such as hosts, geographical regions, and circulation years were also considered in the classification. we used the same lineage nomenclature as described in, but with the following modifications: a single digit is used to represent one of the nine subtypes and a letter is used to represent a lineage; a sub-lineage is also represented using a digit; a dot is used to separate a lineage and a sublineage. for example, a. means na subtype, lineage a, and sub-lineage . the time of most recent common ancestor (tmrca) was estimated using the bayesian mcmc method in beast. in all cases, we employed the gtr + u nucleotide substitution model, in which the first and second codon positions are allowed different rates relative to the third codon position. all data sets were analyzed under a relaxed molecular clock and the bayesian skyline population coalescent prior. the maximum clade credibility (mcc) tree across all plausible trees was computed from the beast trees using the treeannotator program, with the first % trees removed as burn-in. phylogenetic analysis based upon na sequences revealed two large groups corresponding to influenza a and b, respectively ( figure a ). within influenza a, two subgroups were found, one consisting of na , na , na , and na and the other consisting of the remaining five subtypes. subtype na was found to be a sister subtype of na , na being a sister subtype of na , and na a sister subtype of na . finally, each na subtype forms a distinct cluster, indicating its genetic uniqueness. influenza a and b viral na were estimated to have diverged around years ago ( figure b ). however, it had large % hpd values which ranged from years to years ago. the na subtypes of influenza a diverged from more than to several hundred years ago. the time of most recent common ancestor (tmrca) of each subtype of influenza a virus was generally recent and ranged from the calendar years to (figure b ). in addition, the tmrca for influenza b viral na was dated back to . a total of lineages were identified in influenza a (table ) . three lineages, a, b, and c, were identified for na based upon the tree topology. linage a originated from avian viruses and was further divided into sub-lineages: a. , a. , a. , a. , and a. . linage b consists of north american swine influenza viruses whereas c is a human lineage. two large lineages, a and b, were identified in na . lineage a is a human-specific lineage. interestingly, five major swine clades were observed within this lineage. lineage b is an avian-specific lineage, and consists of sub-lineages, b. , b. , and b. . three lineages were found in na . lineage a was found in north american avian, b in eurasian ⁄ oceanian avian, and c also in avian, but it does not show any geographical pattern. for na , na , and na , each was classified into lineages, one found in north american avian ( a, a, a) and the other in eurasian ⁄ oceanian avian ( b, b, b). three lineages identified respectively in na and na are north american avian ( a, a), equine ( b, b), and eurasian avian ( c, c). na was also found to have lineages: north american avian ( a), eurasian ⁄ oceanian avian i ( b), and eurassian ⁄ oceanian avian ii ( c), respectively. in this study, we conducted large-scale phylogeny and evolutionary analyses using influenza viral na sequences. the results showed that divergence between influenza a and b viruses occurred earlier than between any influenza a subtypes. this observation was consistent with previous findings based upon phylogenetic analysis of the ha gene, one of the most important genes related to host infection. within influenza a, two sub-groups were found, one consisting of na , , , and and the other consisting of the rest of five subtypes (na , , , , ) . this observation does not agree with the result described by liu et al., where na subtypes , , , , and formed one group and the remaining four subtypes (na , , , and ) formed the other group. this difference is apparently caused by the fact that an outgroup was not used in their phylogenetic analyses. in the present study, both influenza a and b viral na sequences were included in the analysis. high bootstrap values were obtained for major groups, indicating that the inferred evolutionary relationship should be highly reliable. classification and designation of the lineages and sublineages within the influenza a virus are essential for studies of viral evolution, ecology, and epidemiology. a total of lineages were identified within nine influenza a viral na subtypes and with the majority of the identified lineages found to be host or geographic specific or both. our results demonstrated a comprehensive view for the evolution of na genes and provided a framework for the inference of evolutionary history of pandemic viruses and for further exploring of viral circulations in multiple hosts. for example, the global pandemics of human h n in , h n in , the pandemic of human h n virus in , the crisis of h n hpai in hong kong in , and swine-origin h n influenza in , all can be mapped onto the lineages and sub-lineages identified in this study. such information will facilitate not only identification of known genetic origins but also early detection of novel influenza a viruses. influenza viruses constantly evolve to avoid the human immune pressure in the process of antigenic drift. through sequencing of viral genomes, the rates and direction of virus evolution can be observed. moreover, comparison of protein sequences allows us to determine amino acid substitutions that are related to immune pressure and antigenic drift. the creation of global influenza genetic databases, along with concurrent development of analytical tools, allows the comparison of multiple influenza virus strains. the main aim of this study was to perform antigenic and genetic comparison of pandemic influenza viruses (h n ) isolated during the - pandemic in ukraine and in other countries. nasopharyngeal swabs and autopsy materials collected from infected patients were received from the areas of ukraine. in addition, field isolates of influenza viruses from the ⁄ season and strain specific serum were used for identification by hemagglutinin inhibition assay. influenza viruses were identified and subtyped using real-time rt-pcr analyses using cdc primers and adopted protocols. sequencing was performed in two world health organization (who) influenza collaboration centers (centers for disease control and prevention, atlanta and national institute for medical research, london). hemagglutinin inhibition assay was conducted using chicken and guinea pig red blood cells following standard who protocols. the all ukrainian isolates of influenza viruses, which were isolated in ukraine during august-november , were identified as a ⁄ california the phylogenetic analyses confirmed the evolutionary relationship between ukrainian isolates and viruses from other countries, which were isolated during the first wave of the pandemic. high genetic and antigenic conservation of pandemic influenza viruses from ukraine and other countries also were demonstrated. considering that the emergence of the novel pandemic influenza strain occurred in countries of northern hemisphere during summer, it was very interesting and significant tracking the dynamics of genetic changes in influenza viruses, which were isolated at the beginning of epidemic and those isolated during the rise of the epidemic in ukraine. influenza a virus causes moderate to severe epidemics annually and catastrophic pandemics sporadically. due to the evasiveness of the influenza virus and the nature of its genome (eight single-stranded and negative-sense rna segments), it is essential to understand the evolution of this important pathogen. influenza virus evolves by two major mechanisms: mutation and reassortment. antigenic and genetic analyses have revealed partially the molecular dynamics of influenza virus evolution. , however, important questions, such as how many genotypes in the influenza a virus, remain unanswered. one of the major issues pertaining to this genotyping problem is how many lineages or sub-lineages can be determined for a subtype and according to what criteria. because of the unique structure of the influenza a viral genome, the computational genotyping methods developed for other viruses cannot be applied to the influenza virus. constructing phylogenetic trees is a powerful technique for the identification of evolutionary groupings (i.e., lineages ⁄ clades). however, for large trees, it is hard to determine how many lineages and the boundaries for each lineage. in this regard, multivariate analysis methods, such as multidimensional scaling (mds) and model-based hierarchical clustering, both taking advantage of dimension reduction and visualization, can complement conventional phylogenetic methods. hemagglutinin (ha), the fastest evolving segment, is recognized as the most important gene in the influenza virus that plays a key role in viral pathogenesis. however, we have only limited knowledge of lineages and sub-lineages occurring in the hemagglutinin (ha) gene of influenza a virus, although much effort has been made in assigning clades or sub-clades in highly pathogenic avian influenza (hpai) virus ha. in this study, both model-based hierarchical clustering and phylogenetic methods were used for sequence analysis. one objective for this study is to explore and develop a more accurate lineage approach for further comprehensive influenza lineage and genotype analyses. a total of hemagglutinin (ha) sequences (approximately nucleotides long), excluding laboratory recombinant sequences, were downloaded from genbank as of march, . sequences were aligned with muscle and mafft. the genetic distance matrix of all pairwise sequences was computed using the k p model under mega . . we then used the distance matrix as input to the cmdscale module in r . . for the mds analysis. the principle coordinates resulting from mds were used for the model-based hierarchical clustering analysis, again in r . . (the r foundation. available at: http://www.r-project.org/). the bayesian information criterion (bic) values were computed based upon ten different statistical data models -eii, vii, eei, evi, vei, vvi, eee, eev, vev, and vvv. the highest bic value was used to determine the number of clusters in the given sequence data. phylogenetic analysis was conducted using maximumlikelihood (ml) in raxml. raxml uses rapid algorithms for bootstrap and maximum likelihood searches and is considered one of the fastest and most accurate phylogeny programs for large-scale sequence analysis. all the analyses were conducted on the supercomputer cluster (holland computing center, http://hcc.unl.edu/main/index.php). the trees were visualized in figtree (version . . ) . lineages and sub-lineages were determined based on both the topology of the ml trees and model-based clustering results. additional information such as hosts, geographical regions, and circulation years were also considered in the classification. we used the same lineage nomenclature as described in, with the following modifications: lineage analysis was conducted for each ha subtype, which agrees with the convention of influenza virologists that ha subtypes were identified in influenza a virus; ha lineages are represented with digits and letters, where the digit(s) represent one of the subtypes and a letter represents a lineage; here, we present sub-lineages or sub-sub-lineages also in digits, with smaller numbers representing earlier lineages or sub-lineages within the same subtype (e.g., lineage occurs earlier than lineage ); the digit is used to indicate inclusion of ancestral viruses in a lineage (or sub-lineage); a dot is used to separate lineages, sub-lineages, and sub-sub-lineages. for example, a. ae means ha subtype, lineage a, and sub-lineage , and sub-sub-lineage . the sub-lineage level can be extended as necessary. the model-based clustering method corroborates commonly used phylogenetic methods in lineage and sub-lineage assignment. here we use the h subtype as an example to show the lineage and sub-lineage assignment. the bayesian information criterion (bic) reaches its maximum when the number of clusters for h equals , regardless of which mode we choose ( figure a ). therefore, based on bic, the optimal number of clusters for the h subtype is . as a result, a total of clusters based upon the vvv model were identified ( figure b) . a significant correlation was found in lineage assignments by the phylogenetic method and the model-based hierarchical clustering method ( figure b,c) . lineages a and b were identified for h , which correspond to north american avian and eurasian avian, respectively. lineage a was further divided into sub-lineages, a. , a. , where a. is the ancestral sub-lineage in a. based on both model-based hierarchical clustering and phylogenetic analyses, a total of distinct lineages were identified among subtypes, averaging out to be ae lineages per subtype ( table ). the majority of the identified lineages were found to be host or geographic specific or both. for example, three lineages, a, b, and c, were identified for ha . lineage a was further divided into two sub-lineages, a. and a. . the a. is swine-specific, whereas a. is a human pandemic h n sub-line- how to accurately identify an evolutionary lineage of influenza a viruses is challenging. one commonly used approach is molecular phylogeny, where phylogenetic trees are constructed, and the tree topology is used for lineage determination. here, we used a bayesian model-based clustering method, along with phylogenetic methods, to decide lineages and sub-lineages of influenza a viruses based upon sequence data. the results demonstrated that the modelbased clustering method corroborates phylogenetic methods and increases the accuracy of lineage assignment. one salient feature of this study is its large-scale analysis of all available influenza a hemagglutinin sequences. a total of distinct lineages and sub-lineages were classified; the majority of them were found to be host or geographic specific. this observation agrees largely with previous findings. we are conducting further analyses of other influenza a segments and expect to identify their lineages and create a comprehensive genotypes database for all influenza a viruses. such information will allow us to detect the genetic origin of newly found viruses, track their genetic changes, and identify potential genome reassortments. a hierarchical nomenclature system has been proposed and adopted for hpai ha clades and sub-clades by who influenza surveillance centers. wan et al. also proposed a hierarchical approach for influenza a viral genotypes system. the work presented here is one of the first steps towards the development of a nomenclature system for influenza a virus lineages (at the segment level) and genotypes (at the genome level). whether the naming system will be accepted and used by the influenza research community is more challenging than the lineage analysis itself. identification of the genetic origins of influenza a viruses will enhance our understanding the evolution and adaptation mechanisms of influenza viruses. the phylogenetic analysis is the traditional approach to identify the influenza progenitor. first, the nucleotide sequences are aligned using multiple sequence alignment methods, such as clustalw, muscle, and t-coffee. second, phylogenetic analysis is performed on these aligned sequences to infer their evolutionary relationship using neighbor-joining (nj), likelihood, or bayesian inference. bootstrap analyses or computation of posterior probability are usually applied to estimate the phylogenetic uncertainty. however, this phylogenetic analysis is time consuming due to intensive computations in multiple sequence alignments and phylogenetic inferences. it is difficult to perform an analysis using this method on a large dataset, for instance, with more than taxa, as is the common case for influenza studies. alternatively, blast is applied to identify the prototype genes in the database. blast determines a similarity by identifying initial short matches and starting local alignments. since influenza viral sequences have very high similarities, especially for most conserved regions, blast usually generates a large number of outputs, which will not be helpful for progenitor identification. since blast is a local sequence alignment, the results from blast may not reflect the global evolutionary information between the sequences. the blast scores cannot be used to define the evolutionary relations between viruses, especially in the context of the entire genetic pool. recently, we have developed a distance measurement method, complete composition vector (ccv), that can calculate genetic distance between influenza a viruses without performing multiple sequence alignments. , we also adapted the minimum spanning tree (mst) clustering algorithm for influenza reassortment identification. the application of this approach in the analyses of pb genes of influenza a virus showed that the integration of ccv and mst allows us to identify the potential progenitor genes rapidly and effectively. based on these results, here we develop a webserver called ipminer for influenza progenitor identification. ipminer can identify potential progenitors for a query sequence against all public influenza datasets within a few minutes. in order to improve the computing efficiency, distance matrices were pre-computed by ccv, and they include for ha (h to ), for na (n to n ), and one for each of the internal gene segments (pb , pb , pa, np, ns, and mp). these pre-computed matrices will be updated weekly. ipminer just needs to compute the query matrices for a query sequence and sequences in the database. the standalone ccv program is also available at http://sysbio.cvm.msstate.edu/ipminer. in order to identify the influenza progenitor genes, ipminer first integrates the query matrix and a corresponding pre-computed matrix into a full distance matrix, which is then clustered by mst clustering algorithm. we adapted the threshold we measured previously in mst, u + nr, where u is the average distance and r is the standard deviation of a cluster. as a result, mst will generate a hierarchical structure for the clusters. in each cluster, we will randomly select viruses or % of the cluster size if this cluster has more than viruses. ipminer will return the viruses with the smallest distances when the search reaches to the lowest level (the largest n) in this hierarchical structure. our analyses have shown that the level has generally yielded good results for influenza a viruses. to visualize the overall mst structure, ipminer applies multi-dimensional scaling (mds) method to project all the viruses in the genetic pool onto a two dimensional graph, and the precursor viruses are marked in different shapes ( figure ). the users can select other prototype viruses from the graph for further phylogenetic analyses. a single job with one query sequence takes < min. the genbank identifiers and associated genetic distances and sequence identities are displayed. the users can download the sequences for the identified precursor viruses as well as those from the prototypes viruses. in addition, for the users' convenience, ipminer generates a phylogenetic tree using nj method implemented in phylip to illustrate the phylogenetic relationship among the query sequence(s), the identified progenitors, and the selected prototypes viruses. the programs in this solution package are written in java. the shell scripts are written in korn shell script in order to achieve high performance. cascading style sheets (css) are used for a consistent look across the pages. this also enables to change the overall design just by replacing the css definition file. php has been used as server side scripting and is written in java. in order to achieve high performance for computing in a genomic scale, we apply hash function or a binary tree, which enables that the precursor identification has a time complexity of o(n). for single queries, the users can visualize the results online. for batch queries of multiple sequences, the results will be sent to the users by e-mail. ipminer has been tested on microsoft internet explorer, mozilla firefox, and safari. the users need javascript to obtain full function of ipminer server. the webserver is available at http://sysbio.cvm.msstate.edu/ipminer. in summary, ipminer webserver has three major computational features for influenza progenitor identification: (i) it calculates the genetic distances through ccv and identifies the viruses with the shortest ccv distances against the query virus to be the progenitor genes; (ii) it projects influenza viruses onto a two dimensional map, which illustrates the global relationship between the progenitor genes and other viruses in the genetic pool; and (iii) it performs phylogenetic analyses between the query virus, the identified progenitor genes, and other selected prototype viruses. ipminer provides a user friendly web service for influenza progenitor identification in real time. the gisaid initiative offers an alternative to current public-domain database models in response to growing needs of the global influenza community for the sharing of genetic sequence and associated epidemiological and clinical data of all influenza strains. gisaid's publicly accessible epifluÔ database is governed by a unique sharing mechanism that protects the rights of the submitter, while permitting ongoing research as well as the development of medical interventions, such as drugs and vaccines. for the gisaid initiative, the max planck institute for informatics (mpii) saarbrücken, germany, has developed a web portal that is accessible at http://www.gisaid.org featuring the gisaid epifluÔ database that offers a unique collection of nucleotide sequence and other relevant data on influenza viruses. the database is based on software by oracle and the dante Ò system by a systems gmbh, germany. extensive metadata are also collected for most isolates. the database provides features for searching, filtering specific datasets for download, and user friendly upload functionality. to uphold gisaid's unique sharing mechanism, all users must positively identify themselves. while access is free of charge, all users agree that they will not attach any restrictions on the data, but will acknowledge both the originator of the specimen and the submitter of the data, and seek to undertake to collaborate with the submitter. all uploaded sequence data are submitted to rigorous curation by the friedrich-loeffler-institute for animal health (fli), germany. the database has been live since september , . among its contributors are all five who collaborating centers for influenza who routinely contribute data in addition to using the epifluÔ database for their semiannual vaccine strain selection. to provide a complete picture of data, all data available in the public domain is routinely imported. as of october , , the rapidly growing gisaid dataset comprises nucleotide sequences (from isolates) with (from isolates) uniquely submitted to this database. software development is underway to continually extend the spectrum of available data analysis tools. the intergovernmental process of the nd world health assembly specifically mentions gisaid as a publicly available database for depositing virus sequence data. starting in , germany's federal ministry of food, agriculture and consumer protection will be the long-term host of the gisaid platform. the mpii will continue to develop the portal and database software and enable gisaid to act as a catalyst for the development of advanced bioinformatics software connected directly to the database. gisaid has become an indispensible resource for the international scientific community on influenza. the consortium will expand its activities and offers to catalyze research and development on a wide variety of issues pertaining to risk analysis, drug development, and therapy of influenza. options for the control of influenza vii ª blackwell publishing ltd, influenza and other respiratory viruses, (suppl. ), - the pandemic h n virus emerged in and spread rapidly throughout the world, principally affecting children and young adults. as this virus is new to the human population, it is important to determine if these influenza infections are more commonly associated with other respiratory pathogens compared to previously circulating influenza strains. co-infecting respiratory viruses may cause increased morbidity in individuals with pandemic h n , and may also be unwanted contaminants in influenza vaccines if original clinical samples containing these adventitious viruses are used to directly inoculate certified cell lines for vaccine production. to examine this issue, stored rna from original clinical samples (nasal swabs, nasal aspirates, throat swabs) from australian and new zealand subjects that were collected in that were positive for pandemic h n and samples collected in that were positive for seasonal influenza by real time pcr assay (using the cdc, usa kits), were subjected to a resplex ii -panel version . (qiagen) pathogen screen. the resplex ii assay detects common respiratory viruses, such as respiratory syncytial viruses (rsv a, b), influenza a and b viruses, parainfluenza viruses (piv - ), human metapneumo-viruses (hmpv), coxsackieviruses ⁄ echovirus (cvev), rhinoviruses (rhv), adenoviruses (adv b, e), coronaviruses (nl , hku , e, oc ), and bocaviruses. resplex ii uses a combination of multiplex rt-pcr, hybridization of pcr onto target specific beads followed by detection using luminex-xmap technology. original clinical samples were received at the center from who national influenza centers, who influenza collaborating centers, and other regional laboratories and hospitals from australia, new zealand, and the asia ⁄ pacific region. most samples were from australia and new zealand. these samples consisted of nasal swabs, nasopharyngeal swabs, nasal washes, throat washes, and throat swabs. all samples were stored at ) °c until rna was extracted. rna was extracted from ll of clinical sample using either the magnapure extraction system (roche, australia) or the qiaxtractor system (qiagen, australia) according to the manufacturer's recommendations with an elution volume of ll and stored at ) °c until used. a ll aliquot of rna was used to amplify the selected influenza virus gene using specific primers and probes as supplied by cdc (atlanta, usa) along with super-script iii platinum one-step rt-pcr reagents (invitrogen, australia). real time pcr detection was performed on a fast system with sds software (applied biosystems, ca, usa). a cut off of a cycle threshold (c t ) of or below was considered positive. resplex ii panel ver . detection the qiagen molecular differential detection (mdd) system was used, which combines qiaplex amplification (multiplex rt-pcr) with detection on the liquichip workstation (luminex's xmap microsphere based multiplexing system) and qiaplex mdd software according to the manufacturer's instructions. a low level cutoff was used ( ) to obtain maximum sensitivity. from the clinical specimens that were positive for influenza from by real time pcr, there were ( %) a(h n ) seasonal influenza viruses, ( ae %) a(h n ) viruses, ( %) b viruses, and ( ae %) viruses which were influenza a positive, but could not be typed. clinical samples from selected to study were all influenza a(h n ) pandemic positive by real time pcr. detection of influenza virus in respiratory samples was much lower with the resplex ii assay (using a low cut off of units) for pandemic influenza a virus ( ⁄ ; sensitivity ae %) and to a lesser extent for seasonal influenza a ( ⁄ ; sensitivity of ae %) and b viruses ( ⁄ ; sensitivity of ae %) when compared to real time pcr. there were relatively few co-infecting respiratory viruses with either pandemic h infections in ( ae %) or seasonal influenza infections in ( ae %) ( table ). the most common dual infection seen with pandemic h n viruses and seasonal b viruses was with cvev ( ⁄ ; and ⁄ ; , respectively) while for a(h ) viruses there were no dominant co-infecting viruses ( table ). in one case was detected with three respira- tory pathogens in the same sample, a year old female who had pandemic h n , cvev, and rhv, and in a seasonal influenza sample, one case with a triple infection was detected (bocavirus, piv and influenza b). the median age of subjects with co-infections was younger for both pandemic h n with a median age of years (range: months to years), compared to the full sample set which had a median age of years (range: months to years), while for the patients from with seasonal influenza viruses with co-infections they had a median age of ae years (range: months to years) compared to all samples which had a median age of years (range: months to years). there was good concordance in detecting influenza a and b in respiratory samples collected in between real time rt-pcr and the resplex ii system ( % versus > ae % for seasonal influenza a and b respectively). this data compares well with other studies such as li et al. who found that resplex ii had ae % sensitivity and % specificity for seasonal influenza a viruses and ae % sensitivity and % specificity for influenza b viruses. in contrast, the present study found only ae % sensitivity for the resplex ii detection of influenza a with the samples that were positive for pandemic h n by real time rt-pcr. a recent study by rebbapragada et al. also showed lower sensitivity for pandemic h n viruses in nasopharyngeal samples with the resplex ii system ( % sensitivity and % specificity) compared to other commercial platforms seeplex rvp ( % sensitivity and % specificity) and luminex rvp ( % sensitivity and % specificity). interestingly the latest version of the resplex system offered by qiagen the resplex ii plus panel ruo now has a separate target for the pandemic h n virus (mexico ). in terms of detection of other respiratory viruses such as piv- , piv- , rsv and hmpv, high sensitivities ( ae %, ae %, ae %, and %, respectively) and specificities ( ae - %) compared to taqman rt-pcr have been reported from testing of nasal wash and nasopharyngeal clinical samples. in both the seasonal influenza positive and the pandemic h n positive (by real time rt-pcr) clinical specimens, few other respiratory viruses were detected. only of the samples had another virus detectable and one had two other viruses, while in out had another virus and one had two other viruses detected from a total of influenza virus positive samples collected in each year. enteroviruses, coronaviruses, and parainfluenza viruses were most often found with both seasonal and pandemic infections. younger age appeared to be associated with co-infections with those subjects in with dual infections having a median age of only years compared to the study groups years; and similarly for , the median age for subjects with dual infections was only ae years compared to the study groups' median age of years. a study by chong et al. on nasopharyngeal swabs collected during - using resplex ii and luminex xtag rvp fast, they found dual respiratory virus infections in ⁄ ( ae %) of samples and only ( ae %) with triple respiratory viral infections; however, these were from cases with any combination of multiple respiratory viruses not necessarily influenza, although influenza positive cases were the most common respiratory virus detected ( ae % of all positive samples). given the low level and variety of viral co-infections along with both seasonal and pandemic influenza seen in this study, it is unlikely that influenza infections predispose subjects to particular respiratory viruses, but may still allow bacterial colonization, such as has been seen with severe and fatal cases with pandemic h n with various bacteria including streptococcus pneumoniae, streptococcus pyogenes, staphylococcus aureus, or haemophilus influenzae. , low levels of other respiratory viruses along with the finding that certain cell lines (like the mdck -cells used in this study) do not propagate a number of these viruses (e.g. rsv a and b, rhinoviruses, coronaviruses), but do propagate others (e.g. parainfluenza ) should make testing for unwanted viruses that might be co-isolated with influenza viruses more focused and hence easier to detect and eliminate this isolate for future vaccine production. global influenza surveillance is one of the most important approaches to combat spread of disease. current laboratory methods for characterizing influenza are time-consuming and labor-intensive, and few viral strains undergo full characterization. even fewer strains from domestic poultry and swine or from wild aquatic birds are wellcharacterized. these strains are important for global surveillance since they are thought to be the precursors to pandemic influenza strains. we have designed a highthroughput global bio laboratory to address these surveillance needs. the goal of this project was to develop highspeed and high-volume laboratory capabilities for extensive surveillance and rapid and accurate detection and analysis of influenza. the workflow consists of surveillance, sample transportation, laboratory testing, data management and analysis. five robotic systems have been designed for this laboratory: sample accessioning, biobanking, screening, viral culture, and sequencing. sample accessioning logs barcodes, centrifuges, and aliquots samples are then sent to biobanking. the robotic biobank stores samples at ) °c and reformats tubes for screening. the screening system extracts rna and confirms the presence and subtype of influenza. aliquots of positive samples are sent to the viral culturing system for scale-up. finally, cultured samples are extracted and sent to the sequencing system for full genome sequencing. the sample accessioning, sequencing, and biobanking systems have been built, delivered, and validation processes are currently being completed. robotic screening and culturing systems have been fully designed and are ready to be built. a biosafety level -enhanced containment laboratory was built to enable the flow of samples containing highly pathogenic avian influenza viruses. in full operation, this approach to surveillance is designed to enable the sequencing of up to full virus genomes per year, more than the total of all full influenza genomes sequenced to date. the design of a robotic laboratory for influenza surveillance presents unique challenges and opportunities. before a robotic system is built, each assay is worked out on the bench top, each movement of the plates and reagents is defined, and the laboratory information management system (lims) must be able to address each step of the process. alternate assays are conceived for processes that are not automation-friendly. waste streams, worker safety, and space constraints are considered. each possibility is taken to reduce processes that have the potential to aerosolize or cross-contaminate influenza samples. instruments must be found that fit the capabilities needed. detailed specifications for each of the robotic systems were written including all the parameters listed above. once the systems are built, a long validation process takes place where the processes and instruments in each system are adjusted to function together properly. finally, a validation study is performed to ensure that the system is able to produce useful data for influenza research. the entire process takes months from start to finish for each robotic system and requires complete cooperation from a diverse team of researchers. the accessioning system logs initial sample information with the lims system. samples arrive in barcoded cryotubes. the liquid handler brings all samples up to a common volume and clarifies samples by centrifugation. samples are then transferred from screw-cap sample vials into storage plates containing individually punchable storage tubes. each tube ( ae ml) is individually identifiable with a d barcode on the bottom. six archive aliquots are made, and tubes are individually weld-sealed for storage. tips for aspiration are fixed and undergo a high-pressure plasma process between each use to sterilize tips and destroy nucleic acids. samples are stored at ) °c. each module has a capacity of remp plates or $ samples. the automated freezer system can assemble requested samples as -well plates while samples remain frozen. the screening system uses magnetic bead extraction chemistry, real-time pcr, and a liquid handling system to extract samples, confirm and quantify the presence of influenza, and reformat extracted samples for input into the sequencing system. serotype of human influenza samples will be performed by real-time pcr. many samples will not have enough material for further analysis and will need to be scaled up. the culturing system combines incubators, a liquid handling platform, plate reader, and real-time pcr to culture, monitor growth, harvest, and quantify influenza. when the system is not being used for culture and scale-up, it can be used to assay previously cultured influenza samples for drug resistance. a challenge to sequencing large numbers of influenza samples is the manpower required for sample preparation. the sequencing system has the capacity to prepare up to samples for sequencing per year for sanger sequencing. sanger sequencing was chosen because it is well-established for influenza surveillance, and automation-friendly. the system is designed to work with multiple primer sets ( , , ) . robotic systems all report to the lims. each process completion, plate movement, and data point are entered and checked by an online, web-based lims. status updates, notification, reporting, and data analysis can be achieved without entering the bsl containment facility. routine data analysis such as determining whether a cultured sample is ready to be harvested will be performed by the lims. complex data analysis, while still requiring significant human input, will be made easier by the data-acquisition functions of the lims. the implementation of a high-throughput influenza surveillance laboratory will provide an influenza research and response capacity that far exceeds what is available today. with the addition of each new system, we add a new capability to the influenza community and new opportunities to foster partnerships and collaborations with government, foundations, businesses, and academic institutions. this laboratory will not only enable cutting edge research, but will also enable a more effective response of near real-time surveillance during a pandemic outbreak. pandemics of and were believed to arise from avian influenza viruses. the tropism of avian and human seasonal influenza viruses for the human lower respiratory tract deserves investigation. the target cell types that support replication of avian influenza a viruses in the human respiratory tract in the early stages of clinical infection have not well defined. in a previous autopsy studies of human h n disease, influenza a virus were found to infect alveolar epithelial cells and macrophages. in this study, viral infectivity and replication competence of human and high and low pathogenic avian influenza viruses were systematically investigated in the human conducting and lower respiratory tract using ex vivo organ cultures. we compared the replication kinetics of human seasonal influenza viruses (h n and h n ), low pathogenic avian influenza viruses (h n , h n ) with that of the highly pathogenic h n viruses isolated from human h n disease. a range of human seasonal influenza a viruses of subtypes h n and h n viruses were included in this study from to . two isolates of low pathogenic avian influenza a (lpai) (h n ) viruses from different virus lineages isolated from poultry in hong kong in , a low pathogenic influenza a (h n ) virus isolate from wild ducks in hong kong in , and two virus isolates of highly pathogenic avian influenza (hpai) a subtype h n were included. fragments of human bronchi and lung were cut into multiple - mm fragments within hours of collection and infected in parallel with influenza a viruses at a titer of tcid ⁄ ml and as control cultures were infected with ultraviolet light inactivated virus. these tissues fragments were infected for hours and washed twice with pbs and incubated for , , and h at °c. the bronchial tissue was cultured in an air-liquid interface using sponge. viral yield was assessed by titration in mdck cells. one part of the infected tissue were fixed in formalin and processed for immunohistochemistry for influenza antigen. other part of infected tissue was homogenized and underwent rna extraction, and the expression of influenza virus matrix gene was measured by quantitative rt-pcr. human bronchus ex vivo cultures supported human seasonal influenza virus to replicate efficiently. avian influenza h n virus replicated, although less efficiently than that of seasonal influenza viruses, whereas hpai h n did not productively replicate in ex vivo cultures of human bronchus. this is in agreement with our previous finding in the well-differentiated bronchial epithelial cells in vitro. on the other hand, human lung ex vivo cultures supported prominent productive replication of human seasonal influenza h n ( figure a ) and hpai h n ( figure f ) viruses. lpai, such as h n ( figure c -d) and h n ( figure e ), also replicated productively, but with a lower viral yield. surprisingly, the replication of human influenza h n viruses ( figure b ) across the last three decades was greatly inhibited. there are clear differences in viral tropism of human seasonal and avian influenza viruses for replication in the human bronchus and lung. hpai h n virus can infect and productively replicate in the lower lung, which may account for the severity of human h n disease, but not in the conducting airways. surprisingly, there are marked differences in the replication competence of seasonal influenza viruses in ex vivo lung tissues, with influenza h n viruses being able to replicate efficiently while h n viruses do not. this may be related to the more strict siaa - gal binding preference of h n viruses. on the other hand, the efficient replication of influenza h n viruses in the alveolar spaces indicates factors other than tissues tropism alone play a role in the differences in disease severity between human seasonal h n and avian h n virus infections. pre-mrnas of the influenza a virus m and ns genes are poorly spliced in virus-infected cells. by contrast, in influenza c virus-infected cells, the predominant transcript from the m gene is spliced mrna. the present study was performed to investigate the mechanism by which influenza c virus m gene-specific mrna (m mrna) is readily spliced. ribonuclease protection assays showed that the splicing of m mrna in infected cells was much higher than that in m gene-transfected cells, suggesting that viral protein(s) other than m gene-translational products facilitates the splicing of viral mrnas. the unspliced and spliced mrnas of the influenza c virus ns gene encode two nonstructural (ns) proteins, ns (c ⁄ ns ) and ns (c ⁄ ns ), respectively. the introduction of translational premature termination into the ns gene, which blocked the synthesis of c ⁄ ns and c ⁄ ns proteins, drastically reduced the splicing of ns mrna, raising the possibility that c ⁄ ns or c ⁄ ns enhances the splicing of viral mrnas. the splicing of influenza c virus m mrna was increased by co-expression of c ⁄ ns , whereas it was reduced by co-expression of influenza a virus ns protein (a ⁄ ns ). the splicing of influenza a virus m mrna was also increased by co-expression of c ⁄ ns , whereas it was inhibited by that of a ⁄ ns . these results suggest that influenza c virus ns , but not a ⁄ ns , can up-regulate the splicing of viral mrnas. pre-mrnas of the influenza a virus m and ns genes are poorly spliced in virus-infected cells. , the inefficient splicing of viral pre-mrnas can be understood partly by the fact that influenza a virus ns protein is associated with spliceosomes and inhibits pre-mrna splicing. , cis-acting sequences in the ns transcript also negatively regulate splicing. by contrast, in influenza c virus-infected cells, the predominant transcript from the m gene is spliced mrna. the present study was performed to investigate the mechanism by which influenza c virus m gene-specific mrna (m mrna) is readily spliced. the yamagata ⁄ ⁄ strain of influenza c virus was grown in the amniotic cavity of -day-old embryonated hen's eggs. cos- and t cells were cultured in dulbecco's modified eagle's medium containing % fetal calf serum. subconfluent monolayers of cos- cells were transfected with pme s containing influenza c virus m gene cdna using the lipofectamine procedure and then incubated at °c. total rna was extracted from both the transfected cells and cells infected with c ⁄ yamagata ⁄ ⁄ virus using the rneasy mini kit (qiagen). ribonuclease protection assay was performed using a ribonuclease protection assay kit rpa iii (ambion). briefly, a [ p]-labeled influenza c virus rna -specific rna probe (vrna sense) was synthesized by in vitro transcription and hybridized with the total rna at °c overnight. hybrids were digested with rnase a ( ae u) and rnase t ( u) at °c for minutes and then analyzed on a % polyacrylamide gel containing m urea. hmv-ii cells infected with c ⁄ yamagata ⁄ ⁄ and cos- cells transfected with pme s expressing influenza c virus ns were fixed with carbon tetrachloride at various times after infection and transfection, respectively. the cells were then stained by an indirect method using anti-gst ⁄ ns serum as the primary antibody and fluorescein isothiocyanate-conjugated goat anti-rabbit igg (seikagaku kogyo) as the secondary antibody. the splicing efficiency of influenza c virus m gene-specific mrna (m mrna) in infected cells was higher than that in m gene-transfected cells the ratio of m encoded by a spliced m mrna to cm encoded by an unspliced m mrna in influenza c virusinfected cells was about times larger than that in m gene-transfected cells. ribonuclease protection assays showed that the splicing of m mrna in infected cells was much higher than that in m gene-transfected cells (figure ). these data suggest that viral protein(s) other than m gene-translational products facilitates viral mrna splicing. the influenza c virus ns gene translational product may up-regulate the splicing of viral mrnas the unspliced and spliced mrnas of the influenza c virus ns gene encode two nonstructural (ns) proteins, ns (c ⁄ ns ) and ns (c ⁄ ns ), respectively. the introduction of translational premature termination into the ns gene, which blocked the synthesis of c ⁄ ns and c ⁄ ns proteins, drastically reduced the splicing of ns mrna, suggesting that c ⁄ ns or c ⁄ ns enhances viral mrna splicing. immunofluorescent staining showed that ns localized in the nucleus in the early phase of infection, and was distributed in both the nucleus and cytoplasm in the late phase of infection, raising the possibility that influenza c virus ns protein plays a role in viral mrna splicing that occurs in the nucleus. the splicing of influenza c virus m mrna was increased by co-expression of c ⁄ ns , whereas it was reduced by co-expression of influenza a virus ns protein (a ⁄ ns ) (figure a ). the splicing of influenza a virus m mrna was also increased by co-expression of c ⁄ ns , though it was inhibited by that of a ⁄ ns ( figure b ). these results suggest that influenza c virus ns , but not a ⁄ ns , can up-regulate the splicing of viral mrnas. in influenza a virus-infected cells, splicing is controlled so that the steady-state amount of spliced mrnas is only - % of that of unspliced mrnas. , the mechanisms by which influenza a virus ns pre-mrnas are poorly spliced have been investigated and the following confirmed. influenza a virus ns protein associates with spliceosomes and inhibits pre-mrna splicing. , two cis-acting sequences in the ns transcript (positions - in the intron and positions - in the ¢ exon region) inhibit splicing. by contrast, influenza c virus m gene-specific mrna (m mrna) is efficiently spliced in influenza c virus-infected cells. in this study, we examined the mechanism by which influenza c virus m mrna is efficiently spliced and the regulatory mechanism of the splicing of ns gene-specific mrna (ns mrna). the introduction of a translational pre-mature termination into the influenza c virus ns gene, thereby blocking the synthesis of influenza c virus ns (c ⁄ ns ) and ns (c ⁄ ns ) proteins, drastically reduced the splicing rate of ns mrna. we further examined whether c ⁄ ns potentially facilitates viral mrna splicing. the splicing rate of m mrna of influenza c virus was increased by co-expression with c ⁄ ns , whereas it was reduced by co-expression with influenza a virus ns protein (a ⁄ ns ) (figure a ). the splicing of influenza a virus m gene-specific mrna was also increased by co-expression with c ⁄ ns , though it was inhibited by co-expression with a ⁄ ns ( figure b ). these results suggest that influenza c virus ns can facilitate viral mrna splicing, but in no way inhibit it, which is in striking contrast to the inhibitory effect of influenza a virus ns on pre-mrna splicing. , the mechanism for splicing enhancement by c ⁄ ns also remains to be determined. we speculate that c ⁄ ns may interact with some host proteins involved in splicing, thereby leading to an up-regulation in splicing, or that c ⁄ ns may bind to pre-mrna, increasing its accessibility to the spliceosome. the spliced mrna of the influenza c virus m gene encodes the m protein, which plays an important role in virus formation and determines virion morphology. , therefore, it is speculated that the mechanism for efficient splicing of m mrna, which provides the m protein necessary for virus assembly in a redundant amount, has been maintained in the influenza c virus. by contrast, unspliced mrna from the influenza c virus m gene encodes the cm ion channel, which is permeable to chloride ions, and also has ph-modulating activity. although the role of the influenza c virus cm ion channel in virus replication remains to be determined, it is conceivable that the over-expression of the cm protein has a deleterious effect on virus replication since the fact that a high level of influenza a virus m protein expression inhibits the rate of intracellular transport of the influenza a virus ha protein and other integral membrane glycoproteins has been demonstrated. if this is the case, efficient splicing of m mrna may control the amount of cm synthesized to optimize virus replication. therefore, we speculate that efficient splicing of m mrna leads to a high level of m expression and the reduced expression of cm , thereby creating conditions that are optimal for virus replication. in this study, we provided evidence that c ⁄ ns facilitates the splicing of m mrna. furthermore, c ⁄ ns may regulate the splicing efficiency of its own ns mrna during infection, controlling the amount of c ⁄ ns and c ⁄ ns proteins in infected cells. c ⁄ ns plays an important role in the nuclear export of vrnp, and is also associated with vrnp in the later stages of infection in virus-infected cells and is incorporated into virions, suggesting that c ⁄ ns is involved, not only in the sorting of vrnp into the assembly site, but also in virus assembly. therefore, it is likely that there is a mechanism by which an appropriate amount of c ⁄ ns is provided during infection to accomplish these functions. in conclusion, c ⁄ ns , which enhances the splicing of viral mrna, may regulate both the expression level of m gene-derived m and cm proteins, and that of ns gene-derived ns and ns proteins, thereby leading to optimal virus replication. propagation of the human influenza viruses in embryonated hen's eggs always results in a selection of variants with amino acid substitutions in the hemagglutinin (ha) that affect viral receptor-binding characteristics (reviewed ). brookes et al. recently studied infection in pigs using the egg-grown virus that contained a mixture of the original a ⁄ california ⁄ ⁄ (h n pdm) and its two egg-adaptation mutants with single amino acid substitutions d g and q r ( and in h numbering system). only the original virus and the variant with g were detected in the directly inoculated animals, indicating that the variant with r failed to infect. only the original virus was detected in nasal secretions of contact infected pigs, suggesting that the d g mutant failed to transmit. in contrast, there was an apparent selection of the d g mutant in the lower respiratory tract samples from directly inoculated pigs. the d g substitution is of a special interest as it can emerge during virus replication in humans and was associated with severe and fatal cases of pandemic influenza in - - and . here we compared phenotypic properties of the original clinical isolate of h n pdm virus a ⁄ hamburg ⁄ ⁄ and its d g and d r mutants to explain observed effects of these mutations on virus replication in swine and to predict their potential effects on virus replication in humans. a ⁄ hamburg ⁄ ⁄ (ham) was isolated from clinical material by two passages in mdck cells. the virus was passaged twice in -day-old embryonated hen's eggs and plaqued in mdck cells. the plaques were amplified in mdck cells and the sequences of the viral ha were determined. the variants with single mutation d g and q r were aliquoted and designated ham-e and ham-e , respectively. the receptor-binding specificity of the viruses was assessed by assaying their binding to desialylated-resialylated peroxidase-labeled fetuin containing either a - -linked sialic acid ( - -fet) or a - -linked sialic acid ( - -fet). in brief, viruses adsorbed in the wells of -well eia micro plates were incubated with serial dilutions of - -fet or - -fet, and the amount of bound fetuin probe was quantified by peroxidase activity. the binding data were converted to scatchard plots (a ⁄ c versus a ), and the association constants of the virus-fetuin complexes were determined from the slopes of these plots. viral cell tropism and replication efficiency in human airway epithelium were studied using fully differentiated cultures of human tracheo-bronchial epithelial cells (htbe). , to determine cell tropism, cultures were infected at a moi , fixed hours after infection, and double immuno-stained for virus antigen and cilia of ciliated cells. infected cells were counted under the microscope ( · objective with oil immersion) in the epithelial segment that included - consecutive microscopic fields containing between % and % ciliated cells relative to the total number of superficial cells. percentages of infected ciliated cells and infected non-ciliated cells relative to the total number of infected cells were calculated. ten segments per culture were analyzed and the results were averaged. to compare growth kinetics of ham and ham-e, replicate htbe cultures were infected with plaque-forming units of the viruses followed by incubation at °c under airliquid interface conditions. at , , and hours postinfection, we added dmem to the apical compartments of the cultures and incubated for minutes at °c. the apical washes were harvested, stored at ) °c, and analyzed simultaneously for the presence of infectious virus by titration in mdck cells as described previously. the non-egg-adapted h n pdm virus ham, similarly to the seasonal human virus a ⁄ memphis ⁄ ⁄ (h n ), bound to - -fet ( figure a ) and did not show any significant binding to - -fet. this result contrasted with the binding of h n pdm viruses to several - -specific probes in carbohydrate microarray analysis. reduced avidity of virus interactions with soluble glycoprotein in solution as compared to its binding to the probe clustered on the microarray surface could account for these differences in the assay results. the d g mutant ham-e differed from the parent virus by its ability to bind to -fet and by its reduced binding to -fet. the q r mutant only bound to - -fet, although less strongly than did the avian virus a ⁄ duck ⁄ alberta ⁄ ⁄ (h n ). the viral cell tropism in htbe cultures ( figure b ) correlated with receptor specificity. ham and mem ⁄ showed a typical human-virus-like tropism , with preferential infection of non-ciliated cells (< % of infected cells were ciliated). the mutant with r and control duck virus displayed a typical avian-virus-like tropism (preferential infection of ciliated cells). the d g mutant displayed a cell tropism that was intermediate between those of human and avian viruses; in particular, this mutant infected significantly higher proportion of ciliated cells than ham and mem ⁄ . observed alteration of receptor specificity and cell tropism ( figure ) suggested that egg-derived mutations can affect replication of the h n pdm virus in human airway epithelium. to test this, we first compared the capacity of the viruses to initiate infection in htbe cultures. replicate cultures were infected with identical doses of the viruses, fixed hours post-infection, and immuno-stained for viral antigen. under these conditions, ham and ham-e infected comparable numbers of cells, whereas ham-e infected at least times less cells (data not shown). this result indicated that the mutation q r markedly impaired the ability of ham-e to infect human airway epithelial cultures. we next compared two other viruses ham and ham-e for their multi-cycle replication in htbe cultures and found that the original virus reached threefold higher peak titers hours post infection than did the d g mutant ( figure ). the d g mutation in h n pdm virus facilitates virus binding to - -linked receptors and alters viral cell tropism in human airway epithelium. these changes could account for increased replication of the d g mutant in the lower respiratory tract in humans - and pigs and correlation of this mutation with severe pulmonary disease. [ ] [ ] [ ] [ ] [ ] the d g mutant replicates less efficiently in human airway cultures than the original virus. this finding correlates with an apparent lack of transmission of variants with g in humans and pigs. egg-derived mutation q r abolishes virus binding to - -linked receptors and strongly decreases infection in cultures of human airway epithelium. this result agrees with poor infectivity of the q r mutant in pigs and highlights potential pitfalls of using egg-adapted viruses with this mutation for the preparation of live influenza vaccines. nin-esterase-fusion (hef), nucleoprotein (np), matrix (m ) protein, cm , and the non-structural proteins ns and ns . , cm is the second membrane protein of the virus and is encoded by rna segment (m gene). [ ] [ ] [ ] [ ] [ ] [ ] it is composed of three distinct domains: a -residue n-terminal extracellular domain, a -residue transmembrane domain, and a -residue cytoplasmic domain. , , it is abundantly expressed at the plasma membranes of infected cells and is incorporated in a small amount into virions. , cm forms disulphide-linked dimers and tetramers, and is posttranslationally modified by n-glycosylation, palmitoylation, and phosphorylation. [ ] [ ] [ ] analyses of a number of cm mutants revealed the positions of the amino acids involved in the posttranslational modifications. , evidence was obtained that the n-glycosylation was not required for either the formation of disulfide-linked multimers or transport to the cell surface, and that none of dimer-or tetramer-formation, palmitoylation or phosphorylation was essential to the transport of cm to the cell surface. in the present study, in order to investigate the effect of cm palmitoylation on influenza c virus replication, we generated a cm palmitoylation-deficient influenza c virus, in which a cysteine at residue of cm was mutated to alanine, and examined the viral growth and viral protein synthesis in infected cells. t and hmv-ii cells were maintained as described previously. , llc-mk cells were maintained at °c in minimal essential medium with % foetal bovine serum and % calf serum. monoclonal antibodies (mabs) against the hef, np, and m proteins of c ⁄ ann arbor ⁄ ⁄ (aa ⁄ ), and antisera against the aa ⁄ virion and the cm protein were prepared as described previously. , [ ] [ ] [ ] the seven pol i plasmids for the expression of viral rnas of aa ⁄ , and the nine plasmid dnas for the expression of the influenza c viral proteins were reported previously. , plasmid dna, ppoli ⁄ cm -acy(-), in which -tgt- of the m gene was replaced with -gct- , was constructed based on ppoli ⁄ m. to generate a recombinant wild-type (rwt) virus, the above-mentioned plasmids were transfected into t cells as described previously. to rescue a mutant virus, rcm -c a, a recombinant influenza c virus lacking a cm palmitoylation site, the plasmid ppoli ⁄ cm -acy(-), instead of ppoli ⁄ m, was transfected together with the other plas-mids. at hours posttransfection (p.t.), the respective culture medium of the transfected- t cells was inoculated into the amniotic cavity of -day-old embryonated chicken eggs, and a stock of the recombinant virus was prepared. the infectious titres of the stocked recombinant viruses and the supernatants of recombinant-infected hmv-ii cells were determined according to the procedure reported previously. radioimmunoprecipitation hmv-ii cells infected with recombinants were labeled with [ s]methionine or [ h]palmitic acid. cells were then disrupted and subjected to immunoprecipitation with the indicated antibodies. the immunoprecipitates obtained were then analysed by sds-page on ae % gels containing m urea, and processed for fluorography. flotation analysis was performed according to the procedure described previously. to examine whether the cm protein without palmitoylation is synthesized in rcm -c a-infected cells, hmv-ii cells infected with the recombinants were subjected to , and the lysates of the cells were immunoprecipitated with anti-cm serum and analysed by sds-page. as shown in figure , the cm protein was synthesized both in the rwt-and rcm -c a-infected cells, but no incorporation of [ h]palmitic acid into the cm proteins synthesized in the rcm -c ainfected cells was observed, indicating that cm in the rcm -c a-infected cells was not palmitoylated. the rwt or rcm -c a viruses were infected to hmv-ii cells at an m.o.i. of and incubated at °c for up to hours. the infectious titres (p.f.u. ⁄ ml) of rwt were approximately -to -fold higher than those of rcm -c a at - hours p.i. (data not shown), indicating that rwt grew more efficiently than did rcm -c a. thus palmitoylation of cm appears to have some effect on the generation of infectious virions in cultured cells. to investigate the reason(s) for the difference in growth kinetics between the two recombinants, we analysed viral proteins synthesized in the infected hmv-ii cells. pulsechase experiments of hmv-ii cells revealed no significant differences in the synthesis and maturation of the hef, np, m , and cm proteins between the rwt-and rcm -c a-infected cells (data not shown). the infected cells pulse-labeled and chased were respectively immunoprecipitated with anti-cm serum in the presence of mm iodoacetamide and analysed by sds-page in non-reducing condition. in both populations of infected cells, several bands corresponding to cm a-monomer, -dimer, and -tetramer, as well as cm b-dimer and -tetramer were detected (data not shown). these results demonstrate an absence of any significant differences between palmitoylation-deficient cm and authentic cm in terms of conformational maturation and transport in infected cells. membrane flotation analysis revealed that no significant differences in the kinetics of the hef, m , and cm proteins were observed between rwt-and rcm -c ainfected cells (data not shown). in contrast, a slight difference in np kinetics was observed. the pulse-labeled np proteins were recovered in the bottom fractions in both rwt-and rcm -c a-infected cells. in the chase experiment, the amount of membrane-associated np proteins in fractions and was % of the total np in the rwt-infected cells, which was higher than that ( %) in the rcm -c a-infected cells (data not shown). this finding may suggest that the affinity of the np protein, presumably representing the viral ribonucleoprotein (vrnp) complex, to the plasma membrane in the rcm -c ainfected cells is lower than that in rwt-infected cells, leading to the less efficient generation of infectious virions. since cm is structurally similar to m , an influenza a virus membrane protein known to be involved in infectious virus production, [ ] [ ] [ ] [ ] [ ] it is possible that the cytoplasmic tail of cm participates in the genome packaging through interaction with vrnp. in the present study, we showed that the affinity of np to the plasma membrane of rcm -c a-infected cells was slightly lower than that to the plasma membrane of rwt-infected cells. this observation may suggest that palmitoylation of cm is involved in the viral ribonucleoprotein (vrnp) incorporation, leading to efficient infectious virion generation. we hypothesize that palmitoylation contributes to proper regional structure formation in the cm cytoplasmic tail, which is competent to recruit vrnp efficiently into virions. alternatively, the cm cytoplasmic tail without palmitoylation is not likely to reach the proper conformation, resulting in reduced interaction with vrnp and less efficient generation of infectious progeny virions. the questions of if and how the m protein is involved in the interaction between the cm cytoplasmic tail and np remains to be clarified. we showed that cm synthesized in rcm -c ainfected cells was oligomerized and transported to the cell surface. this finding is consistent with the previous observation that palmitoylation is not required for the transport of cm to the cell surface in cm -expressing cos- cells. however, the use of reverse-genetics system has enabled us to conclude that the palmitoylation of cm is required for efficient infectious virus production. this suggests that the significance of the other posttranslational modifications of cm during virus replication can be clarified using recombinant viruses lacking the respective modification sites. sialic acid (sia) linked glycoproteins are the classical influenza receptors for influenza virus haemagglutinin to bind. the distribution of sia on cell surfaces is one of the determinants of host tropism, and understanding its expression on human cells and tissues is important for understanding influenza pathogenesis. previous research has shown the differences in apical versus basolateral infection and release of different influenza virus from polarized epithelial cells and correlated this with sialic acid distribution in the human respiratory tract. moreover, mass spectrometric analysis was recently employed to elucidate the glycans present in the tissue in a higher resolution in human lung. the objective of this study was to examine in detail the distribution of these sia-linked glycans at the cellular level by the use of confocal microscopy. human primary type i-like and type ii pneumocytes were isolated from human non-tumor lung tissue by tissue fragmentation, percoll density gradient centrifugation, and magnetic cell sorting. the cells were seeded on coverslips and maintained in small airway growth medium. when confluence was reached, cell monolayers were fixed with % paraformaldehyde. we used the plant lectins, sambucus nigra glutinin (sna) from roche which binds to siaa - gal, maackia amurensis agglutinin (maa)i and maaii from vector lab, which bind the siaa - gal linked glycans using vector red as fluorescent chromogen. the cells were counter-stained with dapi or with fitc-conjugated antibody against endoplasmic recticulum (protein disulfideisomerase, pdi). the cells were imaged with multi-photon excitation laser scanning microscopy using zeiss lsm. the optical cross-section pictures were reconstructed by zeiss lsm meta. we found that there was more binding of maai and ma-aii to type ii pneumocytes than type i-like pneumocytes and more overall binding of these lectins than binding of sna ( figure ). in keeping with results from other polarized cells there was more binding to the apical than basolateral aspect, thus, explaining the previously published data on apical versus basolateral infection. as sialic acid has been implicated in the targeting of proteins to the surface, the relative lack of sialic acid on the basolateral aspect can explain why there is little seasonal influenza virus dissemination to the systemic circulation in human infections. furthermore, though there was little binding of sna to the figure . primary human type i-like and type ii pneumocytes stained with lecins (red), pdi (green), and dapi (blue) and imaged captured with confocal microscope. apical or basolateral aspects of the pneumocytes, the experimental findings of infection by influenza h n virus that has a strict siaa - gal tropism suggests that there are siaa - gal glycans present, which are not readily bound by the lectin sna. the in vitro model of primary human type i-like and type ii pneumocytes system formed a polarized epithelium that has a similar lectin distribution to human alveoli in vivo which demonstrated that it is a physiologically relevant model to study the tropism and pathogenesis of influenza a virus. human disease caused by highly pathogenic avian influenza (hpai) h n virus is associated with fulminant viral pneumonia and mortality rates in excess of %. cytokine dysregulation is thought to contribute to its pathogenesis. , we previously found delayed onset of apoptosis in h n infected human macrophages and, therefore, a longer survival time of the target cells for prolonged virus replication and cytokine and chemokine secretion, which may contribute to the pathogenesis of h n disease in humans. as bronchial and alveolar epithelial cells are target cells of influenza virus because of their proximal physiological location and interaction with macrophages, we further investigated if the differential onset of apoptosis could be found in influenza h n and seasonal influenza h n infected human respiratory epithelia. we dissected the apoptotic pathways triggered by influenza virus infection. seasonal influenza h n virus (a ⁄ hk ⁄ ⁄ ), a low pathogenic avian influenza h n lineage isolated from poultry (a ⁄ quail ⁄ hk ⁄ g ⁄ ), and two virus isolates of hpai a subtype (a ⁄ hk ⁄ ⁄ and a ⁄ vn ⁄ ⁄ ) were included. primary human bronchial and alveolar epithelial cells were infected with influenza viruses at moi of and the cell monolayer was collected at , , and hours post infection for tunel assay, and supernatant were collected for ldh assay. fragments of human lung tissues were cut into multiple - mm fragments within hours of collection and infected with influenza a viruses at a titer of tcid ⁄ ml. these tissues fragments were infected for hours and incubated for hours at °c. one part of the infected tissue was fixed in formalin and processed for immunohistochemistry for influenza antigen, and the other part was homogenized and underwent rna extraction. apoptosis cdna superarray platform (sabioscience) was employed to conduct apoptosis pathway analysis. in bronchial epithelial cells, seasonal influenza h n virus induced a high percentage of apoptotic cells by tunel assay at , , and hours post infection with a peak of (figure ) . a similar observation of delayed onset of apoptosis was found in influenza h n and h n infected alveolar epithelial cells. besides, cdna array data of ex vivo infected human lung showed that both influenza h n and h n virus induced trail expression compared with mock-infected tissue (approximately folds) at hours post infection, but influenza h n virus infected lung induced significantly more trail ( folds compared to mock infected cells), albeit with a limited viral replication ( figure ). influenza h n virus infected lung also elicited more tnf-alpha and fasr transcription than either h n or h n . these observations can account for the greater apoptotic response in influenza h n virus infected lung. as little impact on the expression of intrinsic pathway components was observed, it seems that the apoptotic response to influenza virus infection in lung was mainly through the extrinsic pathways. no significant changes in the expression of anti-apoptotic protein gene was found, except for a moderate induction of birc by influenza h n virus, which may act to modulate the apoptotic response. the delayed onset of apoptosis by hpai h n and low pathogenic avian influenza h n virus infected respiratory epithelial cells may be a mechanism for the influenza viruses to have more prolonged replication within the human respiratory tract, and this may contribute to the pathogenesis of human disease. hemagglutination (ha) assay % crbc suspension was treated by mu a , -specific sialidase at °c for minutes. complete elimination of a , -receptor on sialidase-treated crbcs was confirmed by receptor staining and flow cytometry. ha assay of live viruses with % crbc or % sialidase-treated crbc were performed in bsl- facility. synthetic ¢sln-paa-biotin(pa ), ¢sln-paa-biotin(pa ), ¢sln-ln-paa-biotin(pa ) was provided by the scripps research institute (tsri). as described elsewhere with some modifications, generally, serial dilutions of sialyglycopolymers were coated in -well-flat-bottom polystryrene plates, and hau live virus ⁄ well were added. alternatively, the plates were precoated with lg ⁄ ml sialyglycopolymers, and then , , , , hau live virus ⁄ well influenza viruses were added. rabbit antisera against a ⁄ ah ⁄ ⁄ diluted in pbs containing % bsa was added into the wells. bound antibody was detected by use of hrp-conjugated anti-rabbit igg antibody and tetramethylbenzidine substrate solution. each sample was determined in duplicates and the absorbance read at nm. a total of h n virus strains were obtained from to . the name and passage history of influenza viruses used in the study are listed in table . as the same sequences of eight rna segments were detected in a ⁄ js ⁄ ⁄ and a ⁄ js ⁄ ⁄ , only a ⁄ js ⁄ ⁄ was tested here. three amantadine-resistant variants with m mutation of screening of receptor-binding preference by ha assay representative results from three sets of independent experiments are shown in table . complete ha with sialidasetreated crbcs, which were only with a , -receptors, was detected in human influenza virus (a ⁄ brisbane ⁄ ⁄ , h n ) and two human h n virus strains, a ⁄ gd ⁄ ⁄ and a ⁄ gx ⁄ ⁄ . high binding of a , oligosaccharides to h n viruses was detected ( figure a -c). and enhanced a , -binding preference was also detected in a ⁄ gd ⁄ ⁄ and a ⁄ gx ⁄ ⁄ . the a , -binding was dose dependent for sialyglycopolymers and virus titer. notably, as compared with a ⁄ gd ⁄ ⁄ of both short-and long-a , recognition, a ⁄ gx ⁄ ⁄ prefers to bind to long-a , six oligosaccharides at low viral titer ( figure b,c) . however, both of them showed strong affinity to short-and long-a , oligosaccharides at high viral loads ( figure d ). sialoside-, galactoside-, mannoside-and sulfo-os-binding are the four types of carbohydrate-binding properties of influenza virus. binding of influenza virus to the a , -or a , -linked sialylated glycans on cell surface is important for host range restriction, and the preference to a , of h n virus limited its efficient infection in human. here, dual receptor-binding preferences were detected in a ⁄ gd ⁄ ⁄ and a ⁄ gx ⁄ ⁄ , which are of clade ae ae . although there is no direct evidence supporting the occurrence of human-to-human transmission in these infection events or the association between viral virulence and receptor-binding switching, viral systemic disseminations are found in the both fatal cases (data not shown). furthermore, with the introduction of clade ae ae into the adjacent countries of china, the finding of h n virus with - binding in human should be of concern. though h n virus with human-type receptor-binding was isolated from one patient treated by oseltamivir and those viruses were with ha and ⁄ or na substitutions, whether the substitutions responsible for receptor specificity switching is pre-existed or selected in human host remains unknown. our finding that three mutant viruses bearing m mutations of a s, a t, and s n cloned from one isolate a ⁄ hb ⁄ ⁄ suggested it is likely that the resistant viruses emerged in the host environment. no variation was found in their ha and na sequence, and all of them show high affinity to a - -binding. our data suggest that the binding-specificity was not affected by the mutations on viral envelope protein m . with the adaptation from wild aquatic birds to domestic poultry or even in human host environment, influenza virus may possess broader carbohydrate-binding spectrum or topology conformation. , we demonstrated differential a , -binding property of two human h n viruses, a ⁄ gd ⁄ ⁄ and a ⁄ gx ⁄ ⁄ . though minor effect of short-a , -binding was detected in viruses a ⁄ gx ⁄ ⁄ at low virus titer, both were of high affinity to long-a , glycans, even at the low titer which are rich on apical side of human upper respiratory epithelia. notably, no evident binding preference switching was detected in the viruses isolated from the sporadic human infection cases at the early of in china (table ) . however, higher affinity to the long-a , glycans was observed in bj ⁄ ⁄ , gz ⁄ ⁄ , and xj ⁄ ⁄ (data not shown). the discrepancy from the findings obtained by sialidase-treated crbc maybe associated with a limited abundance of n-linked a - with long branches on crbc, as demonstrated in a recent study. therefore, glycan dose-dependent binding assay is valuable and should be applied in flu surveillance. the underlying cause of the tendency is unknown, and further research on receptor-binding specificity of h n viruses is required. influenza a viruses of migrating wild aquatic birds in north america towards improved influenza a virus surveillance in migrating birds european union council directive ⁄ ⁄ eec the neighbor-joining method: a new method for reconstructing phylogenetic trees confidence limits on phylogenies: an approach using the bootstrap prospects for inferring very large phylogenies by using the neighbor-joining method mega : molecular evolutionary genetics analysis (mega) software version . the influenza virus resource at the national center for biotechnology information characterization of low-pathogenic h subtype influenza viruses from eurasia: implications for the origin of highly pathogenic h n viruses h n virus outbreak in migratory waterfowl a ⁄ h and a ⁄ h influenza viruses: different lines of one precursor evolution and ecology of influenza a viruses evolutionary processes in influenza viruses: divergence, rapid evolution, and stasis antigenic and genetic conservation of h influenza virus in wild ducks biologic characterization of chicken-derived h n low pathogenic avian influenza viruses in chickens and ducks genetic and pathogenic characterization of h n avian influenza viruses isolated in taiwan between and experimental selection of virus derivatives with variations in virulence from a single low-pathogenicity h n avian influenza virus field isolate evolution and ecology of influenza a viruses is china an influenza epicenter genesis of 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antigenic and genetic characterization of h n swine influenza in china cocirculation of avian h n and contemporary ''human'' h n influenza a viruses in pigs in southeastern china: potential for genetic reassortment? h n influenza a viruses from poultry in asia have human virus-like receptor specificity characterization of h subtype influenza viruses from the ducks of southern china: a candidate for the next influenza pandemic in humans? bioedit: a user-friendly biological sequence alignment editor and analysis program for window ⁄ ⁄ nt genetic algorithm approaches for the phylogenetic analysis of large biological sequence datasets under the maximum likelihood criterion phylogenetic analysis using parsimony (and other methods) . beta a novel genotype h n influenza virus possessing human h n internal genomes has been circulating in poultry in eastern china since characterization of h n influenza viruses isolated from vaccinated flocks in an integrated broiler chicken operation in eastern china during a year period characterization of avian h n influenza viruses from united arab emirates phylogenetic analysis of influenza a viruses of h haemagglutinin subtype h n subtype influenza a viruses in poultry in pakistan are closely related to the h n viruses responsible for human infection in hong kong diversified reassortants h n avian influenza viruses in chicken flocks in northern and eastern china genotypic evolution and antigenic drift of h n influenza viruses in china from the nucleoprotein as a possible major factor in determining host specificity of influenza h n viruses pigs as the ''mixing vessel'' for the creation of new pandemic influenza a viruses origins and evolutionary genomics of the swine-origin h n influenza a epidemic pandemic (h n ) outbreak on pig farm reassortment of pandemic h n ⁄ influenza a virus in swine from where did the 'swine-origin' influenza a virus (h n ) emerge? substitution of lysine at position in pb protein does not change virulence of the 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circulating worldwide from oseltamivir-resistant influenza viruses a (h n ), norway, - influenza activity -united states and worldwide, - season emergence of resistance to oseltamivir among influenza a(h n ) viruses in europe oseltamivir-resistant influenza virus a (h n ), europe, - season widespread oseltamivir resistance in influenza a viruses (h n ), south africa and composition of the - influenza vaccine emergence of h y oseltamivir-resistant a(h n ) influenza viruses in japan during the - season pyrosequencing as a tool to detect molecular markers of resistance to neuraminidase inhibitors in seasonal influenza a viruses neuraminidase sequence analysis and susceptibilities of influenza virus clinical isolates to zanamivir and oseltamivir host cell selection of influenza neuraminidase variants: implications for drug resistance monitoring in a(h n ) viruses neuraminidase receptor binding variants of human influenza a(h n ) viruses due to substitution of aspartic acid in the catalytic site -role in virus attachment? neuraminidase inhibitor susceptibility testing in human influenza viruses: a laboratory surveillance perspective update: drug susceptibility of swine-origin influenza a (h n ) viruses comprehensive assessment of pandemic influenza a (h n ) virus drug susceptibility in vitro detection of molecular markers of drug resistance in pandemic influenza a (h n ) viruses by pyrosequencing pandemic (h n ) and oseltamivir resistance in hematology/oncology patients fluview: a weekly influenza surveillance report prepared by the influenza division development of a sensitive chemiluminescent neuraminidase assay for the determination of influenza virus susceptibility to zanamivir evaluation of neuraminidase enzyme assays using different substrates to measure susceptibility of influenza virus clinical isolates to neuraminidase inhibitors: report of the neuraminidase inhibitor susceptibility network surveillance for neuraminidase inhibitor resistance among human influenza 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for complex multipathogen interactions in acute respiratory infections performance comparison of res-plex ii and xtag rvp fast for detecting respiratory viruses clinical virology symposium communityacquired respiratory co-infection (carc) in critically ill patients infected with pandemic influenza a (h n ) virus infection bacterial co-infections in lung tissue specimens from fatal cases of pandemic influenza a (h n ) -united states a quantitative risk assessment of exposure to adventitious agents in a cell culture-derived subunit influenza vaccine john's hopkins bloomberg school of public health design of an automated laboratory for high-throughput influenza surveillance human influenza surveillance: the demand to expand influenza: an emerging disease avian-to-human transmission of the pb gene of influenza a viruses in the and pandemics proinflammatory cytokine responses induced by influenza a (h n ) viruses in primary human alveolar and bronchial epithelial cells induction of proinflammatory cytokines in human macrophages by influenza a (h n ) viruses: a mechanism for the unusual severity of human disease? influenza h n and h n virus replication and innate immune responses in bronchial epithelial cells are influenced by the state of differentiation mapping of the two overlapping genes for polypepetides ns and ns on rna segment of influenza virus genome sequences of mrnas derived from genome rna segment of influenza virus: collinear and interrupted mrnas code for overlapping proteins influenza virus ns protein inhibits pre-mrna splicing and blocks mrna nucleocytoplasmic transport the influenza virus ns protein: a novel inhibitor of pre-mrna splicing identification of cis-acting intron and exon regions in influenza virus ns mrna that inhibit splicing and cause the formation of aberrantly sedimenting presplicing complexes identification of a second protein encoded by influenza c virus rna segment influenza c virus ns protein upregulates the splicing of viral mrnas identification of an amino acid residue on influenza c virus m protein responsible for formation of the cord-like structures of the virus a mutation on influenza c virus m protein affects virion morphology by altering the membrane affinity of the protein detection of ion channel activity in xenopus laevis oocytes expressing influenza c virus cm protein evidence that the cm protein of influenza c virus can modify the ph of the exocytic pathway of transfected cells the ion channel activity of the influenza virus m protein affects transport through the golgi apparatus intracellular localization of influenza c virus ns protein (nep) in infected cells and its incorporation into virions receptor specificity, host range and pathogenicity of influenza viruses replication, pathogenesis and transmission of pandemic (h n ) virus in non-immune pigs world health organization. preliminary review of d g amino acid substitution in the haemagglutinin of pandemic influenza a (h n ) viruses 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specificity of pandemic influenza a (h n ) virus determined by carbohydrate microarray fields virology. philadelphia, pa: lippincott williams & wilkins the molecular virology and reverse genetics of influenza c virus identification of a second protein encoded by influenza c virus rna segment identification of a amino acid protein encoded by rna segment of influenza c virus influenza c virus cm protein is produced from a amino acid protein (p ) by signal peptidase cleavage a mutation on influenza c virus m protein affects virion morphology by altering the membrane affinity of the protein influenza c virus cm integral membrane glycoprotein is produced from a polypeptide precursor by cleavage of an internal signal sequence evidence that the matrix protein of influenza c virus is coded for by a spliced mrna functional properties of the virus ion channels the cm protein of influenza c virus is an oligomeric integral membrane glycoprotein structurally analogous to influenza a virus m and influenza b virus nb proteins characterization of a second protein (cm ) encoded by rna segment of influenza c virus phosphorylation of influenza c virus cm protein the sites for fatty acylation, phosphorylation and intermolecular disulphide bond formation of influenza c virus cm protein identification of an amino acid residue on influenza c virus m protein responsible for formation of the cord-like structures of the virus a human melanoma cell line highly susceptible to influenza c virus antigenic characterization of the nucleoprotein and matrix protein of influenza c virus with monoclonal antibodies construction of an antigenic map of the haemagglutinin-esterase protein of influenza c virus the synthesis of polypeptides in influenza c virus-infected cells new low-viscosity overlay medium for viral plaque assays the influenza virus m protein cytoplasmic tail interacts with the m protein and influences virus assembly at the site of virus budding the cytoplasmic tail of the influenza a virus m protein plays a role in viral assembly the influenza a virus m cytoplasmic tail is required for infectious virus production and efficient genome packaging distinct domains of the influenza a virus m protein cytoplasmic tail mediate binding to the m protein and facilitate infectious virus production influenza virus m ion channel protein is necessary for filamentous virion formation influenza h n virus infection of polarized human alveolar epithelial cells and lung microvascular endothelial cells das inhibits h n influenza virus infection of human lung tissues receptor binding specificity of recent human h n influenza viruses differential onset of apoptosis in avian influenza h n and seasonal h n virus infected human bronchial and alveolar epithelial cells: an in vitro and ex vivo study human influenza virus a ⁄ hongkong ⁄ ⁄ (h n ) infection induction of proinflammatory cytokines in human macrophages by influenza a (h n ) viruses: a mechanism for the unusual severity of human disease? proinflammatory cytokine responses induced by influenza a (h n ) viruses in primary human alveolar and bronchial epithelial cells differential onset of apoptosis in influenza a virus h n -and h n -infected human blood macrophages avian flu: influenza virus receptors in the human airway haemagglutinin mutations responsible for the binding of h n influenza a viruses to humantype receptors an avian influenza h n virus that binds to a human-type receptor evolution of highly pathogenic h n avian influenza viruses in vietnam between evolutionary dynamics and emergence of panzootic h n influenza viruses writing committee of the second world health organization consultation on clinical aspects of human infection with avian influenza a (h n ) virus recent avian h n viruses exhibit increased propensity for acquiring human receptor specificity a simple screening assay for receptor switching of avian influenza viruses glycan topology determines human adaptation of avian h n virus hemagglutinin h n chicken influenza viruses display a high binding affinity for neu acalpha - galbeta - ( -hso )glcnac-containing receptors a strain of human influenza a virus binds to extended but not short gangliosides as assayed by thin-layer chromatography overlay search for additional influenza virus to cell interactions avian flu: isolation of drug-resistant h n virus the surface glycoproteins of h influenza viruses isolated from humans, chickens, and wild aquatic birds have distinguishable properties this study was supported by the li ka shing foundation, the national institutes of health (niaid contract hhsn c), and the area of excellence scheme of the university grants committee (grant aoe ⁄ m- ⁄ ) of the hong kong sar government. this work was supported by the national institute of allergy and infectious diseases (niaid) contract hhsn c, the li ka shing foundation, and we thank all french and vietnamese field staff involved in the data collection in viet nam for their enthusiasm and support and we are grateful to the pig farmers participating in the study for their cooperation and patience. this study was a part of the gripavi project and was funded by the french ministry of foreign affairs. this research was supported in part by the national institute of allergy and infectious diseases (niaid) contract hhsn c and the area of excellence scheme of the university grants commission (grant aoe ⁄ m- ⁄ ) of the hong kong sar government. we acknowledge the food and environmental hygiene department of hong kong for facilitating the study. this work was supported by the national institute of allergy and infectious diseases (niaid) contract hhsn c, the li ka shing foundation, and the area of excellence scheme of the university grants committee (grant aoe ⁄ m- ⁄ ) of the hong kong sar government. we gratefully acknowledge our colleagues from iiii, shantou university and skleid, hku for their excellent technical assistance. the study was supported by the rfcid commissioned study (lab# ) from research fund secretariat, food and health bureau, hong kong sar; area of excellence scheme of the university grants committee (grant aoe ⁄ m- ⁄ ), hong kong sar; and by niaid contract (sjceirs, hhsn c), nih, usa.ferrets in all groups inoculated with a ⁄ turkey ⁄ ⁄ virus survived the infection and were observed once daily for days. below lower limit of detection (< ae log eid ⁄ ml).statistical cutoff of ic values for nai susceptibility, determined by x ae + iqr. outliers with ic above this cutoff and > times the mean ic for each drug were characterized as extreme outliers; those with known drug-resistance mutations such as h y were classified as resistant and analyzed separately. h wildtype, oseltamivir-susceptible isolates. h y variants, oseltamivir-resistant virus isolates. iqr, interquartile ranges; nai, neuraminidase inhibitors. we wish to thank our collaborators in the who global influenza surveillance network and united states public health laboratories for the submission of virus isolates and clinical specimens. we also thank our colleagues from the virus reference team and the influenza sequence activity, influenza division, cdc, for their valuable technical assis-the findings and conclusions of this report are those of the authors and do not necessarily represent the views of the centers for disease control and prevention (cdc). we are indebted to yonas araya, theresa wolter, and ivan gomez-osorio for their excellent laboratory techniques and animal handling assistance. we would like to thank andrea ferrero for her laboratory managerial skills. this research was possible through funding by the cdc-hhs grant ( u ci ), niaid-nih grant, (r ai ), csrees-usda grant ( - ), and niaid-nih contract (hhsn c). we thank c bazzoli for advice. this work was supported by a grant from the european union fp project flu-modcont (no. ). we thank staff at seoul, incheon, daejeon, gwangju, gangwon, and jeonbuk provincial research institute of health and environments for their laboratory testing. additionally, we would like to acknowledge the contributions of participating sentinel doctors for evaluating the new rat kit. this study was supported by a grant from the korea cdc. we thank roche applied science for providing the materials and equipment for this evaluation. this research was supported in part by the national institute of allergy and infectious diseases (niaid) contract hhsn c and the area of excellence scheme of the university grants committee (grant aoe ⁄ m- ⁄ ) of the hong kong sar government. the authors would like express their sincere thanks to cdc, usa for supporting the routine surveillance of ili in we would like to acknowledge the australian red cross blood service (the blood service) and the australian government, which fully fund the blood service for the provision of blood products and services to the australian community. we also wish to thank the donors and staff of the blood service, who have assisted in provision of specimens for testing in this protocol, as well as the staff at the who we are grateful to liping long for her assistance in map generation. this project was supported by nih niaid rc ai . cz is supported partially by canadian nserc postdoc fellowship. the authors thank the national investigation team based at the national institute of health (istituto superiore di sanita'), italy (in particular antonino bella, maria cristina rota, stefania salmaso) for providing their support in data collection, and the european union this study was supported in part by a grant-in-aid ( ) and the special coordination funds for promoting science and technology of ministry of education, science, sports and culture of japan. this study was supported in part by a grant-in-aid from the ministry of education, science, and culture of japan ( ) and the special coordination funds for promoting science and technology of mext of japan. the work described here was supported by phs grant ai- (jam) and alsac. we thank all authors for their participation in data gathering and analysis, and in writing this manuscript. the studies were funded by gsk consumer healthcare, and gsk investigators were involved in all stages of the study conduct and analysis. py, po, dw and kb are employees of glaxosmithkline. this study was funded by glaxosmithkline. we thank all authors for their participation in data gathering and analysis, and in writing this manuscript. the studies were funded by gsk consumer healthcare, and gsk investigators were involved in all stages of the study conduct and analysis. this study was funded by glaxosmithkline. we thank all authors for their participation in data gathering and analysis, and in writing this manuscript. the stud- authors are thankful to path for the financial support of this research. we would like to acknowledge jessica d'amico and dr. rick bright of path for their editorial review. this study was supported by path. the authors would like to thank rick bright, jessica d'amico, and vadim tsvetnitsky for editing assistance. the we thank dr. m. enami (kanazawa university) for generously providing plasmids containing cdnas to influenza a virus m and ns genes. we also gratefully thank dr. r. sho (department of public health, yamagata university faculty of medicine) for statistical analysis. some data shown in this study have also been presented in the reference paper. this work was supported in part by a grant-in-aid for scientific research from the ministry of education, culture, sports, science, and technology, japan, takeda science foundation, terumo life science foundation, and a grant-in-aid from the global coe program of the japan society for the promotion of science. we thank markus eickmann for his help in isolation and initial characterization of a ⁄ hamburg ⁄ ⁄ and for providing antisera against h n pdm. this study was supported by the european union fp global a(h n ) genetic characterization, molecular evolution dynamics, antiviral susceptibility profiles, and inference of public health implications require nation and region wide systematic analysis of circulating virus. in this study we analysed the genetic and antiviral drug susceptibility profiles of pandemic a(h n ) influenza virus circulating in portugal. genetic profile analysis was performed in isolates to the hemagglutinin (ha), neuraminidase (na) and mp genes, and in six of these isolates the pb , pb , pa, np and ns genes were also analysed. antiviral drug susceptibility profile was analysed for isolates, phenotypically and genotypically to neuraminidase inhibitors (nai) and genotypically to amantadine. the point mutations identified in ha, na, and mp genes of different strains do not seem to evidence an evolutionary trend. this is in agreement with the genetic and antigenic homogeneity that has being described for a(h n ) virus. all analysed strains were found to be resistant to amantadine, and five of these strains exhibited a reduced susceptibility profile to nai, three only for oseltamivir and two for both inhibitors. introduction: the dynamics of pandemic influenza a ⁄ h n compared to seasonal strains of influenza is not clearly understood. it is important to understand the patterns of viral shedding and symptoms over time in community-based infections.materials and methods: household infections were followed-up in two large community-based studies. patterns of viral shedding, symptoms and signs, and tympanic temperature were plotted over time and grouped according to strain for analysis.results: the patterns of viral shedding, symptoms and signs, and tympanic temperature in three influenza a strains (pandemic a ⁄ h n , seasonal a ⁄ h n , and seasonal a ⁄ h n ) were comparable. peak viral shedding occurred close to the onset of symptoms and resolved after - days. patterns of viral shedding in influenza b virus infections differed.discussion: the patterns of viral shedding and clinical course of pandemic influenza a ⁄ h n infections were broadly similar to seasonal influenza a ⁄ h n and a ⁄ h n . only the clinical course of seasonal influenza b infections was similar to pandemic influenza a ⁄ h n . the dynamics of pandemic influenza a ⁄ h n were observed to be largely alike to the dynamics of seasonal influenza a ⁄ h n and a ⁄ h n . the coated respirators inactivated a broad range of influenza strains within minute, including the pandemic strain and human, swine, and avian influenza viruses. antiviral effectiveness was not reduced by hot, humid conditions or repeated saturation, which might occur during prolonged use of respirators. in contrast, infectious virions were detected on the surfaces of all uncoated ffp respirators, and could be transferred to glove surfaces during handling of contaminated masks. growth of the viruses was monitored by ha titer using turkey red blood cells, by quantitative real time rt-pcr (qrt-pcr) to detect the influenza a matrix gene, and also by flow cytometry to detect virus positive cells using monoclonal antibodies (imagen influenza virus a and b). , matrix gene copy number was determined using qrt-pcr and analysed using the sequence detection software on a fast system sds (applied biosystems, california, usa). further characterisation was performed through sequence analysis and the ha inhibition (hai) assay. sequence analysis was performed using dnastar and all sequences obtained were compared with the sequence of either the original clinical specimen if available or the conventional atcc derived mdck cell isolate. the hai assay was used to characterize the viruses against a panel of known standard reference viruses and their homologous ferret antiserum.options for the control of influenza vii abstract background: we measured the cross-reactive antibody response to pandemic h n in children and adults before and after vaccination with [ ] [ ] [ ] [ ] influenza season vaccines as part of the rapid public health response to the emergence of ph n and to provide evidence for ph n vaccination policy development in mainland china. materials and methods: archived serum specimens from previous vaccine studies were detected by hemagglutination inhibition assay. results: limited crossreactive antibody response to ph n had been detected among participants of all age groups before and after they had been vaccinated with - , - influenza seasonal vaccines. vaccination with seasonal influenza viruses resulted in limited seroconversion to ph n in all age groups, compared with - % of seroconversion to seasonal influenza viruses. but similar to recent studies, a peak of cross-reactive antibody response to ph n was observed in % and % of participants born from to before and after vaccination. conclusions: in order to protect our populations in china, our study strongly suggests vaccination with ph n is required in all age groups and that older populations born before may be associated with a lower infection rate of ph n . on april and april , , cases of ph n were identified in specimens obtained from two epidemiologically unlinked patients in the united states and soon thereafter in texas and mexico. since that time, the virus has spread across the globe. assessment of cross-reactive antibody response to the ph n after vaccination with sea-sonal influenza vaccine was first reported from us centers of disease control and prevention (us cdc). according to their results, the seasonal influenza vaccines provided little or no protection against the ph n , but some degree of preexisting immunity to the virus existed, especially among adults aged ‡ years. in this study, using archived serum samples from previous vaccine studies, we measure the level of cross-reactive antibody response to ph n in children and adults vaccinated intramuscularly with trivalent inactivated vaccine developed for the northern hemi- serum specimens were collected and provided by provincial centers for disease control and prevention of china as a public health response to the emergence of ph n exempt from human-subjects review. a total of serum samples were collected from xinjiang uygur autonomous region, yunnan, and shandong provinces. all the serum specimens were grouped by the age of subjects ( - , - , - , ‡ years) and by different influenza seasons.hemagglutination inhibition assay was performed according to standard procedures in this study. [ ] [ ] [ ] as with h n components of the vaccine, the seasonal influenza viruses used in this study were a ⁄ solomon islands ⁄ ⁄ and a ⁄ brisbane ⁄ ⁄ . the ph n influenza virus used in this study was a ⁄ california ⁄ ⁄ provided by us cdc. all the viruses were propagated in specific pathogenfree embryonated chicken eggs and inactivated by & paraformaldehyde. the criteria recommended by the european agency for the evaluation of medical product was applied for the assessment of seasonal influenza vaccine gmt, geometric mean titer; hi, hemagglutination inhibition. three weeks after boosting immunization, spleens were harvested from immunized and control mice. splenocytes were prepared by lymphocyte separation media (ez-sepÔ, shen zhen, china). the cells were washed and resuspended in complete rpmi- containing fetal bovine serum (hyclone, logan, ut, usa), glutamax, um b-me. splenocytes were cultured in vitro in the presence of inactivated h n , h n , h n , and h n influenza virus antigen for h. quick cell proliferation assay kit (biovision, san francisco, ca, usa) was used to detect the cell proliferation. the - nm absorbance was read on a plate reader. all experiments have been repeated at least three times.results are presented as mean standard error of the mean (sem). comparison of the data was performed using the student's t-test. significance was defined as a p value of < ae . to evaluate the adjuvant effect of recombinant igv, the anti m e antibody subclasses was measured. igg and igg a were detected after the first and second immunization ( table ). the ratio of igg a ⁄ igg was calculated. immunization with only m e-hbc showed a lower igg a ⁄ igg ratio < ae . igv combined with m e-hbc led to a high igg a ⁄ igg ratio of up to - after first and second immunization. these igg subclass distributions indicated that igv can induce a th immune response. to determine whether the splenocytes were stimulated in vitro with different subtypes of inactivated influenza antigen after the igv plus m e-hbc antigen immunization, h n , h n , h n , h n inactivated antigen was used table . the serum igg, igg , igg a, and igg a ⁄ igg ratio were measured by elisa after first and second immunization. m e were coated on the wells plate overnight, and serial dilution sera of day , , after first and second immunization were added , ae , , , , ug ⁄ ml of igg, igg , igg a purified antibody were also added for obtaining the standard curve. hrp-labeled goat anti-mouse igg, igg , or igg a was then added, washed, and the optical density was read at nm. the results were showed at mean ± sem. day after first immunization days after second immunizationoptions for the control of influenza vii the french grog (groupes régionaux d'observation de la grippe) early warning network collects more than specimens yearly from cases of acute respiratory illness (ari), using two sampling methods: systematic randomized and non systematic ''ad hoc'' sampling. although vaccines against influenza a virus are the most effective method by which to combat infection, it is clear that their production needs to be accelerated and their efficacy improved. a panel of recombinant live attenuated human influenza a vaccines (laivs), including ns - , ns - , nsd , were generated by rationally engineering mutations directly into the genome of a pandemic-h n virus. the vaccine potential of each laiv was determined through analysis of attenuation, immunogenicity, and their ability to protect mice and ferrets. the data indicate that the novel nsd -laiv was ideally attenuated and elicited strong protective immunity. this study also shows that attenuating mutations can be rapidly engineered into the genomes of emerging ⁄ circulating influenza a viruses in order to produce laivs. the influenza virus exhibits complicated evolutionary dynamics due to multiple reasons, such as diverse hosts, high mutation rates, and rapid replications. in this study, large-scale analyses of influenza neuraminidase (na) sequences revealed influenza a and b na genes diverged first around years ago, and subsequently the na subtypes of influenza a emerged around years ago. all nine na subtypes of influenza a were genetically distinct from each other, with a total of lineages identified. in addition, five and three sub-lineages were further identified in lineage a of na and lineage b of na , respectively. the majority of lineages and sub-lineages were found to be host or geographic specific. this study provides not only a better understanding of influenza na evolution, but also a database of lineages and sub-lineages that can be used for early detection of novel genetic changes for improved influenza surveillance. although phylogenetic approaches are commonly used and often found to be powerful, how to accurately identify lineages or sub-lineages of a gene segment of the influenza a virus remains a challenging issue. in this study, we address this issue by analyzing hemagglutinin (ha) sequences using a combination of statistical and phylogenetic methods. following a hierarchical nomenclature system that uses a letter to represent a lineage and a digit for a sub-lineage, we identified distinct lineages and sub-lineages in all ha subtypes through large-scale analyses of influenza a hemagglutinin sequences. the majority of the lineages or sub-lineages were host or geographic specific or both. further analysis of other segments will allow us to construct a comprehensive database for influenza a lineages and genotypes, facilitating early detection of new viral strains and genome reassortments and hence improve influenza surveillance. identification of the genetic origin of influenza a viruses will facilitate understanding of the genomic dynamics, evolutionary pathway, and viral fitness of influenza a viruses. the exponential increases of influenza sequences have expanded the coverage of influenza genetic pool, thus potentially reducing the biases for influenza progenitor identification. however, these large amounts of data generate a great challenge in progenitor identification. clinical (nasopharyngeal swabs) and post-mortem materials (fragments of trachea, bronchi, lungs, spleen) were obtained from clinics and ⁄ or out-patients from st. petersburg and from base virological laboratories (bvls) of the research institute of influenza in different regions of the country, which cover approximately ⁄ of the territory of russia. the informed consent for the bio-materials collection and studies was obtained from research subjects or from their relatives in cases of post-mortem materials. isolation of viruses was carried out in the mdck cell culture (cdc, atlanta, ga, usa) and in -day-old chicken embryos (e). isolation was done according the standard internationally accepted methods. the reaction of hemagglutination (ha) and the inhibition of hemagglutination (hai) were performed according the who recommended standard method. for the identification of epidemic isolates, we used the hyperimmune diagnostic bovine or ovine antisera annually obtained from the who reference center (cdc). for a detailed antigenic analysis we used the hyperimmune rat antisera against epidemic and reference influenza strains during the period from july , up to april , , we have obtained swabs from clinics and out-patients in st. petersburg and swabs from the bvls. in this period, rather high incidence of lethality from pneumonia was observed, which developed on the background of the pandemic flu h n v. thus, we received from bvls postmortem materials from deceased patients which manifested pcr+ influenza h n v-specific rna. all materials were tested for a possibility of isolation of influenza virus h n v both in eggs and in mdck cells. pcr-negative materials were discarded. we isolated strains of pandemic influenza from the materials collected in st. petersburg and region, which comprised ae % of the total number of analyzed samples. at the same time, we did not isolate any other sub-types of influenza in the season - except the pandemic flu. from the swabs purchased from bvls, strains were isolated, which compose ae % of the pcr+ samples, and strains from the post-mortem materials ( ae % of the pcr+ samples).altogether in the season - , we isolated, retrieved, and analyzed in hai influenza strains. ae % of them were pandemic strains a(h n )v, and only ae % influenza b viruses. these data together with the epidemiologic data and the results of pcr-diagnostic provide evidence in favor of nearly mono-etiological character of epidemic season - in russia for pandemic influenza a(h n )v.though the isolation of pandemic viruses was fulfilled in two traditional model systems, in the case of pandemic virus, we could observe the tendency of preferential multiplication in embryos compared to mdck, especially in cases of post-mortem material for which chicken embryos are the preferential system of isolation.h n v viruses, which were isolated and passaged in mdck, even with significant ha titers, quickly lost their ha activity provided they were kept at + °c. moreover, some other tested cell lines proved to be practically nonsensitive to the pandemic viruses h n v. we used hai reaction for the typing and antigenic characterization of isolated viruses. in the course of isolation of viruses in the reported period, we produced rat polyclonal antisera to the strains a ⁄ california ⁄ ⁄ and a ⁄ st. petersburg ⁄ ⁄ (h n )v and the antisera to the strains a ⁄ new jersey ⁄ ⁄ -the virus isolated during the epidemic in the united states and also of the swine origin -and to the 'swine' strains a ⁄ sw ⁄ ⁄ and a ⁄ iowa ⁄ ⁄ . the hai results of representative strains are given in figure . table shows that the isolated strains were homogenous in their antigenic properties and interacted with the diagnostic antiserum cdc for a(h n )v and also with the antisera to the strains a ⁄ california ⁄ ⁄ and a ⁄ st. petersburg ⁄ ⁄ up to - ⁄ homologous titer. viruses that were isolated from post-mortem materials did not differ by their antigenic characteristics from those isolated from swabs of live patients. only two strains could be attributed to the drift-variants of the strain a ⁄ california ⁄ ⁄ because they reacted with the appropriate antiserum up to ⁄ homologous titer; these strains were a ⁄ pskov ⁄ ⁄ and a ⁄ belgorod ⁄ ⁄ . it is interesting that the isolated strains reacted with the antisera to the strains a ⁄ new jersey ⁄ ⁄ and a ⁄ sw ⁄ ⁄ to ⁄ - ⁄ , and some particular strains even to ⁄ homologous titer. it is even more interesting that some pandemic isolates reacted with the antiserum to the strain iowa isolated in up to ⁄ - ⁄ homologous titer. despite of the fact that since the outbreak of 'swine flu' in the usa in new jersey years had gone (and for the strain iowa this period is nearly years) the ha of these viruses and of the pandemic influenza share some common antigenic determinants as was shown in hai.one more interesting feature of a considerable part of isolated strains is their capability to react with high titers with normal equine serum heated to and to °c, while all the strains of swine origin isolated earlier were inhibitor-resistant ( figure ). russian isolates of divided, in this respect, in two clear and approximately equal in number groups: one of them is similar to the reference strain amino acid substitutions, among them more than were disclosed in antigenic sites, so the degree of similarity to this strain is %. a new site of glycosylation was also discovered in the position of ha. essential distinctions of the aminoacid sequence of ha and antigenic properties of the h n v strains as compared with actual circulating and vaccine strains is one of the factors that determine the pandemic potential of this new influenza virus.according to the literature, the mutation in the ha gene d g could cause a broadening of the spectrum of receptor specificity of influenza virus by the acquisition of the capacity to bind both the residues a( fi ) and a( fi ) of the sialic acid of cellular receptors. both types of receptors are present at the human respiratory tract, but in different parts of it, and they exist in different proportions. according to the data of the european center of disease control and prevention (ecdc), the varieties g of the h n v virus were isolated in countries from subjects deceased of influenza or who suffered a severe form of illness, as well as from those who sustained only a light course of influenza. concerning the strains isolated in rii, this mutation was discovered in nine cases: four were isolated from live patients and five from post-mortem materials. thus, there are no convincing data at present that could prove a causal relationship of the given substitution and the aggravation of a disease course. this is in accordance with previous observations. concerning the resistance of studied strains to the widely used antiviral preparations, it was shown that all tested strains possessed the substitution s n in the m protein that determine the resistance to adamantanes. there was no substitution in the position of neuraminidase (na), which determines the resistance to oseltamivir (h y). these substitutions are the characteristic indices of the eurasian lineage of swine influenza viruses. thus all studied russian h n v isolates were resistant to adamantanes (rimantadine) and sensitive to oseltamivir. respiratory clinical samples taken in and that tested positive by real time reverse transcription (rt)-pcr for seasonal influenza viruses (a and b) and pandemic h n respectively were assessed for other respiratory viruses using the resplex ii panel ver . system distributed by qiagen. results showed that co-infections with another respiratory viruses were relatively rare, with a small number of samples having another co-infecting virus present, very few samples having two other viruses detectable in their samples, and none with further viruses. this low number of co-infecting viruses and the ability of certain cell lines not to support infection with particular viruses may make primary isolation of influenza viruses in cell lines easier than might have been thought previously. cm is the second membrane protein of influenza c virus and is posttranslationally modified by phosphorylation, palmitoylation, n-glycosylation, and dimer ⁄ tetramer formation. in the present study, we generated rcm -c a, a recombinant influenza c virus lacking cm palmitoylation site, and examined viral growth and viral protein synthesis in the recombinant-infected cells. the rcm -c a virus grew less efficiently than did the wild-type virus. membrane flotation analysis of the infected cells revealed that less np was recovered in the plasma membrane fractions of the rcm -c a-infected cells than that in the wild-type virus-infected cells, suggesting that palmitoylation of cm is involved in the affinity of the ribonucleoprotein complex to the plasma membrane, leading to the efficient generation of infectious viruses. influenza c virus has seven single-stranded rna segments of negative polarity, encoding pb , pb , p , haemaggluti- both the a , linkage and its topology on target cells were critical for human adaptation of influenza a viruses. the binding preference of avian flu virus h n ha to the a , -linked sialylated glycans is considered the major factor that limited its efficient infection in human. currently, the switch in binding-specificity of human h n viruses from a , to a , -glycans did naturally occur, and limited humanto-human transmission was found. to monitor their potential adaptation in the human population, receptor-binding specificity surveillance was made in china. here, the binding specificity of human h n virus strains isolated from to was demonstrated. dual binding preference to a , and a , -glycans were found in a ⁄ guangdong ⁄ ⁄ and a ⁄ guangxi ⁄ ⁄ . furthermore, both of them showed a high affinity to the long-branched a , -glycans, which predominate on the upper respiratory epithelial in human. our data suggests that the existence of h n virus with binding specificity to humans should be of concern.introduction via envelope glycoprotein hemagglutinin (ha), influenza viruses bind to cell-surface glycosylated oligosaccharides terminated by sialic acids (sa) where their linkage is celland species-specific. differential receptor binding preference is a host barrier for influenza virus transmission. although most h n viruses have low affinity to neu aca , gal (human-type) receptor, recent findings suggested that the adaptation of h n virus to human by mutations in the receptor-binding site (rbs) do indeed happen and resulted in enhanced affinity to human-type receptor. [ ] [ ] [ ] in contrast to its putative precursor, a ⁄ gs ⁄ gd ⁄ ⁄ , diverse genotypes were presented in currently circulating h n virus, accelerating evolution and widespread occurrence. , to date, distinct phylogenetic clades ( - ) were identified based on h n ha, and the confirmed human infections were caused by clade , , ae , ae , ae , and . in china, human h n disease was mostly caused by clade ae ae , which was identified in isolates from confirmed patients from provinces since . clade and clade ae are responsible for the case in and , respectively. two current cases of and were due to clade ae ae . now information on receptor property has been documented in some h n viruses of clade , ae , and ae . [ ] [ ] [ ] little is known about h n virus of clade ae ae , particularly from human.recently, a , -specific sialidase-treated red blood cell (rbc) agglutination assay was developed and used for receptor specificity screening of h n virus. , the a , or a , -binding preference can be distinguished by the change of hemagglutination titer reacted with rbcs and enzymatic rbcs. since fine receptor specificity existed in h n viruses, , the glycan array including sulfated-, fucosylated-, linear sialosides, di-sialosides, or direct binding assay with synthetic polyacrylamide (paa)-based sialylglycopolymers was also recommended for the receptor-specificity surveillance on h n viruses. furthermore, the long-branched a , sialylated glycans were currently identified to predominate on the upper respiratory epithelial in human and the recognition of this topology, ¢sln-ln is the key determinant for the human-adaptation of influenza a virus. here, we analyzed the receptor-binding specificity of human h n viruses isolated in china from to . since , a total of h n infection cases were confirmed in china from provinces. the pharyngeal swabs and lower airway aspirations from the patients were collected within days after disease onset, maintained in viral-transport medium, and tested within hours.options for the control of influenza vii key: cord- - ughypnx authors: louis-jean, james; cenat, kenney; njoku, chidinma v.; angelo, james; sanon, debbie title: coronavirus (covid- ) and racial disparities: a perspective analysis date: - - journal: j racial ethn health disparities doi: . /s - - - sha: doc_id: cord_uid: ughypnx health disparity refers to systematic differences in health outcomes between groups and communities based on socioeconomic isolation. in the usa, health disparities among minority groups, especially african americans, limit their access to quality medical care and other beneficial resources and services. presently, the novel coronavirus (covid- ) highlights the extreme healthcare challenges that exist in the african american and other minority communities in the usa. african americans are dying at a rate nearly four times higher than the national average. with inadequate access to quality healthcare, viable resources, and information, covid- will continue to have a disastrous effect on african american communities. this communication provides a brief overview of the health inequalities resulting in african americans dying disproportionately during the covid- pandemic. the novel coronavirus (covid- ) emerged from wuhan, china, in late and quickly became a global pandemic that has already affected more than countries and territories [ ] . in symptomatic patients, this highly transmissible disease causes severe acute respiratory syndrome, which infects lower respiratory airways and results in fatal pneumonia. the effect of this pandemic is widely visible, resulting in about a . % mortality rate and causing major economic and social devastations [ ] . studies have shown that covid- is transmitted via human interactions when uninfected individuals come in contact with mucus and respiratory droplets or surfaces that contain the virus. on surfaces where it is present, the virus can remain infectious from hours and up to days depending on the surface materials. also, it can remain airborne for up to hours post aerosolization [ ] . furthermore, a small majority of carriers develop mild to no symptoms. as a result, transmission occurs rapidly and inconspicuously with both symptomatic and asymptomatic individuals unknowingly transmitting the virus, resulting in more people being infected. research data continues to show that covid- affects all age groups. older individuals and those with underlying health conditions are more prone to experiencing severe illnesses and death. data analysis of covid- from the usa highlights pre-existing health disparities among african americans as the potential cause of poor prognosis. based on reports from national health care disparities, in comparison with non-hispanic whites, african americans have a % greater chance of dying from stroke, % more likely to have asthma, % more likely to have heart disease, % more likely to have diabetes, and % more likely to be obese [ ] . this is a major concern that government and public health officials should address as it has been shown that in out of states reporting data, black people accounted for a higher share of covid- cases than their share in the population [ ] . in out of states, they accounted for a higher share of deaths than their share of the total population [ ] . in such regard, an immediate plan of action is urgently needed to address and mitigate the effects of health disparities. this communication briefly examines the health disparities among african americans in the usa during the covid- pandemic. in the early phase of the outbreak in the usa, access to testing was limited to government officials, celebrities, and a selected number of healthcare workerssymptomatic or asymptomatic. it was not until march when the federal government passed the families first coronavirus response act (ffcra), which allows free covid- testing for all individuals [ ] , that large-scale testing became available nationwide. while ffcra is a great response for all citizens, the lack of testing remains, especially in minority communities. in the usa, the racial breakdown of covid- cases and deaths is now starting to be made available. in louisiana, where % of its population is african american [ ] , % of deaths-as a result of the novel coronavirus-are among african americans. meanwhile, white americans in louisiana account for % of the population but recorded only % of covid- deaths [ ] . of the covid- deaths in illinois [ ] , the percentage of deaths among black americans ( %) is higher compared with those among whites ( %), hispanics ( . %), asians ( . %), and others ( . %). this is an area where the majority of the population is white ( %) and the black population is about %. the same phenomenon is occurring in michigan [ ] ; the black population is about % but accounted for % of covid- deaths. in new york, both hispanics and african americans are dying at a relatively higher rate in comparison with other ethnic groups. they account for % and % of new york's population, respectively. however, the reported death toll [ ] for hispanics and african americans is % and %, respectively. in connecticut, the black population is about %, but represents more than % of covid- deaths [ , ] . in milwaukee county, the black population is about % but represents % of covid- deaths [ , ] . in north carolina, % of deaths are among african americans, who represent % of the total population [ ] . the trend and commonality transcend state lines. the common denominator for the high mortality rate in all these states is race and ethnic background. of note, all data presented in this communication and fig. are subject to change as more data becomes available. for more up-to-date data, refer to the states department of public health and services. a recent study showed that in zip codes with high numbers of unemployed and uninsured residents, fewer test kits were available [ , ] . most of those zip codes have disproportionate numbers of african americans. it is no surprise that underserved communities such as the african american communities would also have less access to covid- test kits in a time that kits are scarce. assuming that african americans are seeking medical attention for covid- , they will most likely do so at minority-serving institutions which already have [ ] : (i) lower quality care due to low budgets and lack of resources (ii) shortage of critical care physicians (iii) inadequate number of medical supplies and equipment (i.e., personal protective equipment and ventilators for critically ill patients). this is in parallel to years of ongoing racial and socioeconomic discrimination in the usa [ , ] . while a series of landmark court cases such as simkins v moses h. cone memorial hospital ( ) and cypress v newport news hospital association ( ) litigated by the national association for the advancement of colored people (naacp) legal defense and education fund took legal actions against racial policies and discriminations in healthcare, the challenges for quality healthcare for african americans remain [ ] . the pursuit of legal strategies against racist policies was an essential element in a national campaign to eliminate discrimination in healthcare delivery in the usa. as covid- cases and related deaths continue to rise in the usa, data demonstrates that african american communities in various cities are the most affected ( fig. ). this is a challenge that the federal government and its covid- task force have pointed out. however, in a white house press briefing, dr. anthony fauci of the national institute of allergy and infectious diseases recently expressed, "…there is nothing we [the federal government] can do about it right now except to give them [african americans] the best possible care to avoid complications." health disparities and institutional racism [ , , [ ] [ ] [ ] [ ] [ ] ] make the covid- pandemic worse for african americans. there are studies on the effects of stress and health for african americans as stress can increase vulnerability, which in turn is a factor in determinants of health disparities. perceived discrimination can add to stress, which increases vulnerability to the health effects of environmental hazards, thus adding to health disparities. harburg et al. highlighted that darkerskinned black men having racist interactions and living in neighborhoods with high rates of social instability have an increased risk and incidence of stressful experiences in daily life, which in turn increases their likelihood of high blood pressure along with other health conditions they are predisposed to from their environment [ ] . conditions resulting from these conditions are listed as risk factors for more severe covid- cases [ ] . constant streams of statistical data (fig. ) about the novel coronavirus are showing that african americans are dying from covid- -related complications at a disproportionately higher rate than other ethnic and racial groups. racial inequities in healthcare institutions, lack of access to information, higher levels of preventable chronic diseases (i.e., diabetes, asthmas, hypertension, etc.), and covid- testing not being widely available in minority communities are among the many factors resulting in african americans dying at disproportionate numbers during this pandemic [ ] . failure to rapidly test and segregate individuals infected with covid- can result in major chain-of-transmission reactions and deaths. the socioeconomic discrimination has confined african americans to overpopulated housing estates (ghettos) and low-wage jobs. from the very genesis of this country, african americans have always been the essential or sacrificial workers used to ensure the continuity of this economy. from centuries of free slave labor to years of sharecropping to low-paying domestic jobs, african americans have always played a role in shaping the very essence of what america is [ , ] . today, a large number of african americans work in retail, home healthcare, mass transit, plant factories, and prisons/jails where social distancing is almost impossible. as a result, african americans became more vulnerable to the disease combined with inadequate access to proper healthcare in their communities [ ] . in many cities in the usa, stay-at-home orders are placed to mitigate the spread of the virus. this is a great effort, which has shown promising results. however, african americans and other minority groups account for % of the us homeless population [ ] . these individuals stand no chance against the trail of devastation that covid- will leave behind since it is almost impossible for them to follow preventative guidelines issued by the centers for disease control and prevention (cdc) and government officials. to help flatten the curve, many doctors, nurses, and other healthcare professionals from non-profit organizations are volunteering their services to the homeless community. among other preventative measures highlighted by the cdc as well as healthcare and government officials is the use of face masks in public settings. while this effort can limit and reduce the spread of covid- , this could pose a challenge to african americans and other minorities as they are more likely to be criminalized based on their appearances while wearing masks. of note, many individuals have failed to understand the zoonotic origin of covid- [ ] [ ] [ ] [ ] . as a result, significant misinformation and conspiracy theories continue to circulate on the web and social media. while the internet is an open platform to share and access information, individuals should be aware of conspiracy theories and follow credible sources for relevant information. while there is no evidence showing a correlation between covid- and the upcoming fifthgeneration ( g) technology, people continue to share misinformation linking the current pandemic to g technology. initially, there was the belief that black individuals and minorities were not susceptible to being infected with the virus. there is no available research showing that black individuals are biologically and genetically immune to the virus. without proper information, these artificial assumptions can usher individuals into disastrous directions. on a global scale, covid- is not only affecting people of african descent in the usa but across the world. minority groups everywhere are harassed and discriminated against during this pandemic. in china where the virus emerged, africans living in guangzhou are being evicted from their homes as the virus sparked fear and racial discrimination. of equal challenges, minorities and migrant workers from caribbean countries, such as haiti, living in the usa are being detained by the immigration and customs enforcement (ice) at detention centers where the virus is uncontrollable. these haitians-some of whom are infected with or are covid- carriers-are subjected to deportation to their native land of haiti, a country with a very weak public healthcare system [ ] . only a few examples are highlighted here; a comprehensive analysis is underway. altogether, african americans and individuals of african descent are more vulnerable to the impact of the virus. it is no secret that the us government has misguided and often used african americans as guinea pigs for medical research. during the time of slavery, psychologists and doctors deemed it a disease for slaves to long for freedom or want to "run away." runaway slaves were diagnosed with what was called "drapetomania," which was considered, at the time, a mental illness that amplifies the desire to become a fugitive-run away [ ] . furthermore, after slaves were freed, many american doctors continued to argue that former slaves were incapable of thriving as free members of society because their minds could not function beyond the established orders of slavery. among many other diseases, the syphilis outbreak in the south highlighted another dark chapter in african americans' relations with the healthcare system in america. many african americans infected with syphilis were promised treatment from the public health department. instead, those patients were denied treatment and turned into "guinea pigs" to monitor the progression of the disease. the patients were unaware and were lied to repeatedly about their conditions. many of those untreated "guinea pigs" of the syphilis experiment infected their wives and other women they had gotten in contact with. furthermore, many of them had unknowingly fathered children born with congenital syphilis. the syphilis experiment lasted for years, and it was a major ethical violation. the us government had broken its laws and experimented on its citizens. the us public health service did not treat the people who were experimented on even after penicillin-the effective cure for the disease-was made available. historically, this injustice has established a certain level of distrust among african americans in the us public health system; therefore, they are more reluctant in seeking routine preventative care [ ] . the time for a healthcare revolution for african americans is long overdue. this is not a surprise, as years ago, the flu pandemic highlighted the racial bias and distrust that existed in the medical community [ , ] . jim crow laws and pseudoscientific theories made the impact of the flu go unnoticed in black communities. all efforts and resources were made available to white communities as physicians believed that the virus only affected white individuals. many healthcare officials, with no reliable scientific evidence, believed that african americans were not susceptible to the flu as the linings of their "big noses" were resistant to the microorganism that affects the respiratory system. as a result, black communities were forced to battle the flu pandemic on their own, using limited resources and inadequate medical care at segregated healthcare institutions [ ] . the current covid- pandemic reflects a mirror image of the flu pandemic for african americans and other minorities in the usa [ ] . such patterns will possibly continue if the socioeconomic, health disparities, and the dividing racial lines stretch to upcoming generations. other jurisdictions have yet to publicly report their data based on race and ethnicity; regardless, the challenges remain from social and economic factors that drive health outcomes. altogether, african americans are dying at a much higher rate compared with any other ethnic group. due to various factors that affect health outcomes, a plan of action is urgently needed to respond to the challenges of this pandemic and any health threats in african american communities [ ] . access to testing and proper medical treatment is necessary to ensure the safety of african americans, the most vulnerable group. the embedded racism in the healthcare system and the socioeconomic and health disparities [ , ] continue to make the effect of the virus worst-for black americans and other minorities in the usa. overall, the lack of testing made available to the black community for covid- , the continuous poor healthcare system for african americans, and the systematic health disparity are what makes this virus more dangerous to african americans; although covid- is a new pandemic, it is deeply rooted in history in the continuous plight of african americans in this country [ ] . it is an unsettling reality for african americans to rely on the same systems that historically inflicted harm and damage on them to protect and serve them against this virus [ , , ] . more research is needed to understand the source of exposure to covid- for african americans as well as a plan of care and health outcomes of african americans in various zip codes. further research can also shed light into the type of facilities a majority of african americans receive care in, such as nursing homes, and assess their preparedness looking into their access to resources such as personal protective equipment (ppe) during the covid- pandemic, current and past documented deficiencies from state surveys, and infection control measures taken during covid- , and overall health outcome. more research is also needed to understand how the environment affects health outcome or the severity of covid- cases in terms of environmental exposures, social, and economic factors that can affect mortality, morbidity, life expectancy, healthcare expenditures, and health status amidst the covid- pandemic. this is relevant since a recent study found that living in areas with high air pollution is associated with an % increase in the covid- death rate [ ] . black leaders in all communities, historically black colleges and universities (hbcus), and minority-serving institutions (msis) should continue to engage and educate students to respond to the needs of their communities. this will require these institutions to reinvent and alter their curriculums to provide more health and medical programs (including medical history), engineering, science, and technology. currently, these institutions are operating with low resources, many are underfunded, and others are on the verge of being permanently closed. this is very unfortunate for african americans and other minority communities especially when analyzing the impact of covid- . as the pandemic forced all teaching to be done remotely, hbcus' and msis' students and institutions are at higher risks of not being able to satisfy their educational needs [ ] . many have limited to no access to technological devices and reliable internet access to fully respond to the spontaneous remote teaching caused by the effect of the virus. the gravity of the novel coronavirus covid- (sars-cov- ) pandemic is yet another event showing the racialized health inequalities that exist in the usa. african americans have systematically experienced the worst health outcomes compared with any other racial and ethnic group in the usa. the burdens of covid- are much greater among minorities living in low-income communities where access to quality healthcare and other relevant needs is scarce. the health disparities in the usa did not start with the covid- pandemic. however, the virus has significantly highlighted the pre-existing racial inequalities. altogether, the socioeconomic status and the well-being of african americans require intervention and significant improvement is needed. this will require mutual inclusions of community leaders from various organizations (i.e., churches, divine nine organizations: fraternities and sororities), local government, policymakers, and researchers to mend healthcare in low-income communities. ethics approval and consent ethical approval or consent was not applicable for this paper. the authors declare that they have no conflict of interest. world heal. organ on the novel coronavirus (covid- ): a global pandemic persistence of coronaviruses on inanimate surfaces and their inactivation with biocidal agents national surveillance of asthma: united states growing data underscore that communities of color are being harder hit by covid- louisiana state dep. heal. coronavirus disease (covid- ) in illinois test results. illinois state dep. public heal. public heal. . https:// covid tracker.health.ny.gov/views/nys-covid -tracker/ nysdohcovid- tracker-fatalities?% aembed=yes&% atoolbar=no&% atabs=n connect. state dep. public heal. the covid- pandemic: a call to action to identify and address racial and ethnic disparities covid- : county data. wisconsin state dep covid- north carolina dashboard. north carolina state div. public heal the black plague. new yorker. coronavirus philadelphia: positive tests higher in poorer neighborhoods despite six times more testing in higher-income neighborhoods, researcher says. cbs philly temporal trends in critical care outcomes in u.s. minority-serving hospitals social conditions as fundamental causes of health inequalities: theory, evidence, and policy implications racial and ethnic approaches to community health (reach ): an overview professional and hospital discrimination and the us court of appeals fourth circuit - social conditions as fundamental causes of disease the weathering hypothesis and the health of african-american women and infants: evidence and speculations the us perspective: lessons learned from the racial and ethnic approaches to community health (reach) program limitations in the use of race in the study of disease causation seeking causal explanations in social epidemiology socio-ecological stress, suppressed hostility, skin color, and black-white male blood pressure: detroit preliminary estimates of the prevalence of selected underlying health conditions among patients with coronavirus disease -united states vital signs: racial disparities in age-specific mortality among blacks or african americans -united states at the dark end of the street: black women, rape, and resistance-a new history of the civil rights movement from rosa parks to the rise of black power from slavery to freedom: a history of african americans who is homeless? natl. coalit. homeless coronaviruses: a paradigm of new emerging zoonotic diseases. pathog dis novel coronavirus takes flight from bats? bats as a continuing source of emerging infections in humans dbatvir: the database of batassociated viruses covid- ) in haiti: a call for action tuskegee and the health of black men african americans, public health, and the influenza epidemic the eighteen of - : black nurses and the great flu pandemic in the united states covid- and african americans why african-americans may be especially vulnerable to covid- exposure to air pollution and covid- mortality in the united states: a nationwide cross-sectional study beyond the face-to-face learning: a contextual analysis publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations acknowledgments thanks are due to miriame etienne, ruthonce stvil louis-jean, isaiah herbert, and magdonald aimé for their feedback.data availability all materials and data used in this communication are publicly available and are cited throughout the text. key: cord- - g zfeb authors: wanandy, troy; honda-okubo, yoshikazu; davies, noel w.; rose, hayley e.; heddle, robert j.; brown, simon g.a.; woodman, richard j.; petrovsky, nikolai; wiese, michael d. title: pharmaceutical and preclinical evaluation of advax adjuvant as a dose-sparing strategy for ant venom immunotherapy date: - - journal: journal of pharmaceutical and biomedical analysis doi: . /j.jpba. . . sha: doc_id: cord_uid: g zfeb abstract a major challenge in broader clinical application of jack jumper ant venom immunotherapy (jja vit) is the scarcity of ant venom which needs to be manually harvested from wild ants. adjuvants are commonly used for antigen sparing in other vaccines, and thereby could potentially have major benefits to extend jja supplies if they were to similarly enhance jja vit immunogenicity. the purpose of this study was to evaluate the physicochemical and microbiological stability and murine immunogenicity of low-dose jja vit formulated with a novel polysaccharide adjuvant referred to as delta inulin or advax™. jack jumper ant venom (jjav) protein stability was assessed by uplc-uv, sds-page, sds-page immunoblot, and elisa inhibition. diffraction light scattering was used to assess particle size distribution of advax; ph and benzyl alcohol quantification by uplc-uv were used to assess the physicochemical stability of jjav diluent, and endotoxin content and preservative efficacy test was used to investigate the microbiological properties of the adjuvanted vit formulation. to assess the effect of adjuvant on jja venom immunogenicity, mice were immunised four times with jjav alone or formulated with advax adjuvant. jja vit formulated with advax was found to be physicochemically and microbiologically stable for at least days when stored at and °c with a trend for an increase in allergenic potency observed beyond days of storage. low-dose jjav formulated with advax adjuvant induced significantly higher jjav-specific igg than a -fold higher dose of jjav alone, consistent with a powerful allergen-sparing effect. the pharmaceutical data provides important guidance on the formulation, storage and use of jja vit formulated with advax adjuvant, with the murine immunogenicity studies providing a strong rationale for a planned clinical trial to test the ability of advax adjuvant to achieve -fold jjav dose sparing in jja-allergic human patients. a major challenge in broader clinical application of jack jumper ant venom immunotherapy (jja vit) is the scarcity of ant venom which needs to be manually harvested from wild ants. adjuvants are commonly used for antigen sparing in other vaccines, and thereby could potentially have major benefits to extend jja supplies if they were to similarly enhance jja vit immunogenicity. the purpose of this study was to evaluate the physicochemical and microbiological stability and murine immunogenicity of low-dose jja vit formulated with a novel polysaccharide adjuvant referred to as delta inulin or advax tm . jack jumper ant venom (jjav) protein stability was assessed by uplc-uv, sds-page, sds-page immunoblot, and elisa inhibition. diffraction light scattering was used to assess particle size distribution of advax; ph and benzyl alcohol quantification by uplc-uv were used to assess the physicochemical stability of jjav diluent, and endotoxin content and preservative efficacy test was used to investigate the microbiological properties of the adjuvanted vit formulation. to assess the effect of adjuvant on jja venom immunogenicity, mice were immunised four times with jjav alone or formulated with advax adjuvant. jja vit formulated with advax was found to be physicochemically and microbiologically stable for at least days when stored at and • c with a trend for an increase in allergenic potency observed beyond days of storage. low-dose jjav formulated with advax adjuvant induced significantly higher jjav-specific igg than a -fold higher dose of jjav alone, consistent with a powerful allergen-sparing effect. the pharmaceutical data provides important guidance on the formulation, storage and use of jja vit formulated with advax adjuvant, with the murine immunogenicity studies providing a strong rationale for a planned clinical trial to test the ability of advax adjuvant to achieve -fold jjav dose sparing in jja-allergic human patients. © elsevier b.v. all rights reserved. rapid onset, systemic allergic reactions to jack jumper ant (myrmecia pilosula, jja) sting represent a significant public health problem in the south-eastern and south-western part of australia, affecting up to % of the population [ ] . immunotherapy for the prevention of stinging insect anaphylaxis involves the administration https://doi.org/ . /j.jpba. . . - /© elsevier b.v. all rights reserved. of increasing doses of purified insect venom to induce clinical tolerance, as reflected by a progressive reduction in venom-specific ige and increase in igg [ ] . jja venom immunotherapy (vit) involving a large maintenance dose of g of venom has been shown to be highly effective at preventing jja sting anaphylaxis, reducing the risk of severe systemic sting reactions by % [ ] . real-world experience shows efficacy comparable with yellow jacket and better than honeybee vit [ ] . the applicability of jja vit to the wider population, however, is limited by highly restricted jja venom (jjav) extract availability, production cost, and high incidence of adverse events [ ] [ ] [ ] . an adjuvant that could facilitate a desirable immune response to jjav and allow allergen dose reduction would have the potential to minimise adverse effects and reduce cost by reducing venom requirements [ ] . a potential candidate adjuvant for vit is the delta inulin-based adjuvant, advax tm . advax is based on the plant derived fructan inulin (␤-d-[ → ] poly(fructo-furanosyl) ␣-d-glucose) [ ] . inulin has no immunological activity when in soluble form, but once formulated into delta inulin microparticles of - m size, it has potent immune-adjuvant activity [ ] . advax has been shown in preclinical studies to enhance immunogenicity of a broad range of vaccines including influenza, hepatitis b virus (hbv), japanese encephalitis, sars coronavirus, hiv, listeria, rsv, and anthrax [ , ] . the safety and efficacy of advax have been shown in adult humans when formulated with hbv and influenza vaccines [ , , ] . when combined with honeybee vit and administered to individuals with honeybee-sting anaphylaxis, advax was shown to be safe and well tolerated [ ] , and enhanced the immunogenicity of the honeybee venom with strongly enhanced venom-specific igg responses [ ] , a potential marker of successful immunotherapy [ ] . understanding the stability of jja vit and its aggregation potential with advax microparticles in a combined formulation is essential as aggregation or decomposition of allergen components in jjav may affect their allergenic potency [ ] , which has been found to increase with storage in a previous study [ ] . the objective of the current study was therefore to explore the stability and aggregation potential of jja vit and advax in a combined formulation as a prelude to a clinical trial of low-dose jja vit with advax. although there are currently no animal models of jjav allergy, it was further necessary to demonstrate using a murine immunogenicity model that co-administration of jja vit with advax was safe and well tolerated and that the inclusion of the advax adjuvant was able to enhance the immunogenicity of low-dose jjav. chemicals, reagents and consumables used in the experiments were as previously described [ ] . chemicals used in the formulation of jja vit products were of pharmacopoeial grade; sucrose and polysorbate from avantor (centre valley, pa, usa), benzyl alcohol from pcca (houston, tx, usa), sodium chloride % solution from astrazeneca (north ryde, nsw, australia), sodium dihydrogen phosphate solution from phebra (lane cove west, nsw, australia), disodium hydrogen phosphate from safc (st. louis, mo, usa), . % sodium chloride from pfizer (west ryde, nsw, australia), water for injection from pfizer and baxter (old toongabbie, nsw, australia). microbiological media and atcc ® strains microorganisms were from oxoid (basingstoke, hampshire, uk). pooled positive sera (pps) with high jjav-specific positive ige levels was obtained from jjav allergic individuals with clinically proven history of allergy, as previously described [ ] . jjav active pharmaceutical ingredient (api) was prepared as previously described [ ] and formulated in % sucrose solution to produce clinical grade jja vit containing . mg/ml venom proteins. jjav diluent solution containing . % sodium chloride, phosphate buffer (ph . ; mm), . % polysorbate and . % benzyl alcohol was manufactured at the pharmacy department, royal hobart hospital as previously described [ ] . advax adjuvant in buffered saline (ph . - . ) was supplied by vaxine pty ltd (bedford park, adelaide, australia). the advax adjuvant was prepared according to current good manufacturing practice [ ] and supplied as a sterile suspension containing mg/ml delta inulin microparticles. for advax-free samples, jjav was diluted to g/ml using jjav diluent. where advax was included in the formulation, jja vit and advax were first diluted using jjav diluent to g/ml and mg/ml, respectively. when the two diluted components were mixed in equal quantities this gave final concentrations of g/ml and mg/ml, respectively, the planned doses to be used in the future human trial. for allergen potency studies, jjav was further diluted using jjav diluent and advax to obtain final concentrations of , and . g/ml of venom and mg/ml of advax. samples were prepared using aseptic techniques and packaged in u- insulin syringes (terumo, elkton, maryland, usa), protected from light and stored at • c and • c for up to days. baseline samples were processed within min post preparation. all stability samples were collected and processed after , , and h, and the g/ml samples were also collected at , , , , and days post storage. except for the particle size analysis, where samples were analysed without antigen extraction, jjav was extracted from the stability samples by centrifugation. samples were transferred to protein lobind microtubes (eppendorf ag, hamburg, germany) and centrifuged for min at rpm ( × g) and • c (eppendorf r microcentrifuge) to pellet the advax particles. supernatant was transferred to fresh microtubes and stored at − • c until analysis. allergenic potency in , , , and . g/ml samples was analysed using jjav ige-specific elisa inhibition assay, as previously described [ ] . briefly, -well amino immobilizer plates (nunc, roskilde, denmark) were coated overnight at • c with l of g/ml jjav in sodium carbonate ( . m, ph . ). inhibition mixtures were prepared by adding between . and ng of jjav to l pps and diluting to l with phosphate buffered saline ( . m, ph = . ) containing . % polysorbate (pbs-t) and . % bovine serum albumin (bsa). jjav in the inhibition mixtures was either baseline or stability samples as described in section . . a positive control was prepared by diluting pps : (v/v) in pbs-t with . % bsa and negative control consisted of pbs-t with . % bsa and incubated overnight at • c. subsequently, venom coating solution was discarded and the plate was washed three times with pbs-t. the venom-coated plate was incubated with l of inhibition mixtures, positive control and negative control for min at room temperature (rt). the plate was washed three times with pbs-t, and l of biotinylated mouse monoclonal anti-human ige antibody ( : (v/v) dilution in pbs-t, clone hp , life technologies, carlsbad, ca, usa) was added to each well and incubated in the dark for min at rt. the plate was washed three times with pbs-t, and then l of streptavidin-horseradish peroxidase ( : (v/v) dilution in pbs-t, bd pharmingen, san diego, ca, usa) was added to each well and the plate was incubated in the dark for min at rt. the plate was washed three times with pbs-t, and then l of -step ultra tmb-elisa (thermo fisher scientific, rockford, il, usa) was added to each well and the plate was incubated in the dark for min at rt. colour development was stopped by adding l of m sulphuric acid and absorbance was read at nm utilizing an elisa plate reader (spectramax m , molecular devices, sunnyvale, ca, usa). samples were run in triplicates, and the mean and standard deviation calculated. the percentage inhibition for each sample was calculated using the following equation: allergenic potency of jjav was measured as the amount of venom in solution that was required to inhibit % of ige binding to the jjav coated onto the plate. the stability of venom samples were calculated as a percentage relative to baseline samples. the g/ml samples were concentrated to . mg/ml using a centrifugal filter device (amicon ultra- . with kda nominal molecular weight limit, merck millipore, darmstadt, germany), after which they were mixed with sds sample buffer and analysed by sds-page or sds-page immunoblot assay as previously described [ ] . pps was used in the sds-page immunoblot assay. to assess myr p allergen peptide changes, the g/ml venom samples were analysed by uplc-uv using an acquity h-series uplc (waters corporation, milford, massachusetts, usa) coupled to an acquity photo diode array (pda) detector. an acquity uplc beh c column ( . x mm x . m particles) held at • c was used. the mobile phase flow rate was . ml/min and the solvent system was water:acetonitrile: % trifluoroacetic acid in water ( : : , v/v/v) to ( : : , v/v/v) in a linear gradient over min before re-equilibration for min to initial conditions. triplicate injections of l were made and data for quantitative measurements of peak areas corresponding to the myr p - allergen peptides were extracted at nm. under these conditions myr p eluted at . min, myr p at . min, and myr p at . min (fig s ) . data were analysed using waters masslynx and target-lynx software. the mean peak area for each allergen peptide was calculated and the analysis of remaining concentration of the allergen peptides was calculated as a percentage relative to baseline samples. . . analysis of jjav diluent stability . . . measurement of ph sample(s) ph was measured using a calibrated ph meter (hanna instrument, woonsocket, ri, usa). benzyl alcohol concentration was determined by uplc-uv using the instrument and column described above. the mobile phase flow rate was . ml/min and the solvent system was an isocratic mixture of % acetic acid in water:acetonitrile ( : , v/v). quadruple injections of l were made and data for quantitative measurements were extracted at nm to maintain on scale signals. benzyl alcohol eluted at . min and the peak area was determined using waters targetlynx software. where benzyl alcohol-free advax was added, the concentration of benzyl alcohol in the jjav diluent was reduced by %, consistent with dilutional effect. particle size of advax ( mg/ml) in the presence of jjav ( g/ml) in diluent solution was determined by dynamic light scattering (dls) using a zetasizer nano s (malvern instruments ltd., worcestershire, uk). samples were mixed by vortexing to form a uniform suspension and transferred to uvette plastic cuvettes (eppendorf ag). sample compartment temperature was adjusted to • c before measurements were taken. measurements were performed in triplicate and the average particle size (z-average size) and particle size distribution (via polydispersity index; pdi) were obtained [ ] . samples were analysed for endotoxin contents using limulus amebocyte lysate qcl- chromogenic assay (lonza, walkersville, md, usa) as previously described [ ] . the antimicrobial efficacy of benzyl alcohol in jjav diluent was evaluated using the united states pharmacopoeia antimicrobial effectiveness test (aet) [ ] . the following challenge organisms were used: candida albicans (atcc ), escherichia coli (atcc ), and staphylococcus aureus (atcc ). bacterial stock cultures were grown for - hours in tryptone soya broth and yeast stock culture was grown for - hours in sabaroud dextrose broth. microorganisms were harvested by centrifugation, washed, resuspended, and diluted in . % normal saline to obtain a microbial count of approximately × colony forming units per ml (cfu/ml). microbial suspensions were used within min of harvest. for each challenge organism, aet was conducted in five replicates. test samples consisting of l of jjav diluent containing either jjav ( g/ml) alone or with advax ( mg/ml), were inoculated with the microbial suspension at . % (v/v) to yield a final concentration between × and × cfu/ml and incubated at • c for up to days. aliquots ( l) were taken from each sample at days , and , and the number of cfus in each sample was determined using plate-count method. positive control samples ( l of . % normal saline) were inoculated with the microbial suspension at . % (v/v) to yield a final concentration between × and × cfu/ml. a l aliquot was taken from each control sample at baseline and the number of cfus was determined using plate-count method. negative control samples were growth media (from the same batches) devoid of any manipulation. to assess the potential of advax to provide jjav antigen-sparing, female balb/c mice, - week old were immunized times intramuscularly at -week intervals with jjav g (royal hobart hospital, tasmania, australia) alone or formulated with advax mg (vaxine pty ltd, adelaide, australia) versus jjav g alone in l total injection volume. blood samples were collected weeks after the last immunization for measurement of jjav-specific igg responses by elisa. . . . jjav-specific antibody elisa mouse jjav-specific igg, igg and igg a antibodies were determined by elisa, as previously described, with minor modifications [ ] . briefly, jjav ( g/ml) in coating buffer (ph . ; mm mes + mm hepes in pbs) was absorbed to well plates, blocked with . % casein, then l of : dilution of sera was incubated for h at rt, followed by washing then incubation with biotinylated anti-mouse igg, igg and igg a antibodies (abcam, cambridge, ma, usa) mixed with hrp-conjugated streptavidin (bd biosciences, franklin lakes, nj, usa) for h. washed wells were then incubated with tmb substrate (kpl, gaithersburg, md, usa) for min before the reaction was stopped with m phosphoric acid and optical density measured at nm (od nm ) using a versamax plate reader and analysed using softmax pro software (molecular devices, sunnyvale, ca, usa). average od nm values obtained from negative control wells were subtracted. statistical analysis was performed using graphpad prism . and curveexpert basic . . statistical analyses, including descriptive statistics, t-test and anova with bonferroni's post-hoc test were used as appropriate, and p < . was considered significant. image analysis was performed using imagej . r (wayne rasband, national institutes of health). for the noninferiority analysis, we performed an independent t-test on the log transformed igg data, and assessed whether the upper limit of the % confidence interval for the difference between treatments (low dose adjuvanted jjav treatment minus standard high dose jjav alone treatment) was below a predefined margin of non-inferiority of . . table i describes the acceptance criteria for the physicochemical and microbiological stability of jja vit components and advax adjuvant. the allergenic potency of jjav at baseline was not significantly affected by the presence of advax in the formulation (p = . - . ; supplementary table ). the overall allergenic potency of jjav with and without advax was acceptable for at least days when stored at and • c, with relative allergenic potencies between . - . % and . - . % for all stability samples effects of advax ( mg/ml) and storage temperature ( • c or • c) on the allergenic potency of jjav ( g/ml) stored for up to days. analysis of allergenic potency was calculated as a percentage relative to baseline samples. each sample was analysed in triplicate and presented as mean and sd. asterisks designate significant differences (* p < . , ** p < . , *** p < . ). stored at • c with and without advax respectively, and between . - . % and . - . % for all stability samples stored at • c with and without advax respectively ( figs. and ) . a trend for a progressive increase in allergenic potency was observed in the g/ml jjav stability samples, which continued to increase beyond the acceptable limits of - % after days of storage at either or • c, particularly for the stability samples with advax ( fig. ) . myr p was the least stable of the allergens that were quantified via uplc-uv, and as such, stability was limited by the stability of myr p (fig s ) . therefore, in the presence of advax, the jjav was stable for up to and days when stored at • c and • c, respectively (fig. ) . no significant differences were found between 'with' and 'without' advax for either baseline samples (supplementary table ) or for all but one of the stability pairs (fig. ) . sds-page results showed that a protein band at approximately kda disappeared slowly throughout the storage period and appeared to be replaced with new bands at approximately , and kda (fig s ) . the changes occurred regardless of sample treatments and were more obvious with prolonged storage. a similar pattern was also observed on the sds-page immunoblot results, whereby an ige-binding band at kda slowly disappeared and was replaced with a new ige-binding band at kda (fig s ) . the changes occurred regardless of advax, and were most obvious in the stability samples stored at • c for h. effects of advax ( mg/ml) and storage temperature ( • c or • c) on the allergenic potency of jjav at (a) . g/ml, (b) g/ml, and (c) g/ml stored for up to days. analysis was calculated as a percentage relative to baseline samples. each sample was analysed in triplicate and presented as mean and sd. the addition of advax did not cause a substantial change in the ph of jjav diluent (fig s ) , and storage temperature had a minimal effect on the stability of benzyl alcohol for the period studied ( fig s ) . advax delta inulin microparticles had a mean hydrodynamic diameter of . m at baseline (fig. a) , and this did not change after storage at and • c for up to and days respectively, but a two-fold increase in diameter was observed at the day study endpoint at both and • c. the polydispersity index (pdi) of advax was approximately . at baseline and did not change substantially between day and of the study period, although there were noticeable fluctuations over time, particularly in the sample stored at • c, and the pdi increased to . at day of study period at both and • c (fig. b) . the batches of jja vit, jjav diluents, and advax were virtually free of endotoxins (supplementary table ) , and sterility testing of jja vit and jjav diluents further supported the aseptic nature of the combined formulation (data not shown). all samples including jjav diluent containing only jjav and samples where advax was added met the pharmacopoeial requirements for antimicrobial activity at each sampling time point (supplementary table ). a murine immunogenicity model was used to test the hypothesis that advax adjuvant could be used for jjav dose sparing and to confirm that it had no negative effects on safety and tolerability. female balb/c mice, - week of age (n = /group), were immunized times intramuscularly at -week intervals with jjav g alone or combined with mg advax adjuvant versus jjav g fig. . particle size distribution of advax ( mg/ml) as a function of (a) z-average and (b) polydispersity index (pdi) when combined with jjav ( g/ml) and stored for up to days at either • c or • c. analysis was performed using diffraction light scattering technique. each sample was analysed in triplicate and presented as mean and sd. alone, with the aim to assess the non-inferiority of the jjav specific igg responses in the jjav g + advax low dose group when compared to the jjav g high dose group. blood samples were collected weeks after the last immunization for measurement of jjav-specific total igg and igg subtypes by elisa. while only low or undetectable levels of jjav-specific igg were seen in animals immunised with g of jjav alone, all animals in the low dose jjav ( g) + advax arm had detectable jjav-specific igg. a formal non-inferiority analysis performed using pre-specified limits, confirmed the low dose jjav + advax arm was statistically non-inferior to the high dose jjav alone group (p = . ). a subsequent test of superiority confirmed that the jjav-specific igg responses in the low dose jjav + advax arm were superior (p = . ) to those in the high dose alone arm (fig. ) . hence, advax was confirmed to provide at least -fold jjav dose-sparing. analysis of igg isotypes confirmed that the predominant effect of advax adjuvant was predominantly through significant enhancement of specific igg rather than igg a. no jjav-specific ige was detected in the sera of the immunized mice and no local (swelling, redness, hair loss) or systemic (weight loss, fever, inactivity, loss of grooming) adverse effects were observed. preclinical studies of novel adjuvanted allergen-specific immunotherapy (ait) formulations are generally required by regulatory authorities prior to initiation of clinical trials. such studies should demonstrate physicochemical and microbiological compatibility of the adjuvant with the antigenic components contained within the vaccines [ ] , together with immunogenicity data to justify the inclusion of the adjuvant in the vaccine. although many human trials have been conducted on advax adjuvant in combination with various infectious disease vaccines and one study of advax with honeybee vit [ , , ] , no information was previously available on the formulation, stability or immunogenicity of jjav antigens in the presence of advax. in the current study, we performed stability study of low-dose jja vit with and without advax adjuvant, pre-packaged in plastic syringes thereby confirming that the jja vit and advax components were compatible with each other, and minimal aggregation of delta inulin microparticles was observed in the period studied. based on the data obtained, in particular the allergenic potency studies, lowdose jja vit formulated with advax is both physicochemically and microbiologically stable for at least days when stored at • c and • c in plastic syringes. this will allow low-dose jja vit, formulated with or without advax, to be prepared at a central pharmacy location in advance of clinical trial use and then transported to the site of patient administration, removing the need to prepare the vaccine at the patient's bedside and may reduce the likelihood of errors in dosing occurring [ ] . the jjav stability results obtained in the current study are consistent with a previous report [ ] , although the conditions employed were somewhat different. in particular, the current study used u- plastic insulin syringes to store the samples and a lower concentration of jjav was used than in the previous study. measurement of total allergenic activity as determined by elisa inhibition assay is required by regulatory bodies for the standard- ization and batch control of ait products [ , ] . the slight increase in jjav allergenic activity with storage is entirely consistent with previous findings and this has been proposed to be due to conformational changes in the jjav proteins in the presence of polysorbate in the jjav diluent [ ] . despite elisa inhibition data indicating that the vast majority of allergenic activity was preserved, it does not exclude the possibility that one or more "minor" allergens might have degraded. this perhaps is better monitored using the immunoblot assay where we observed the shift down in the - kda bands, recently found to be phospholipase a enzyme [ ] , to a band at approximately kda (fig s ) . this pattern of allergen degradation is consistent with prior findings and it was not affected by the inclusion of advax in the formulation. wanandy et al. recently reported the adsorption of myr p and myr p allergens when jjav api grade products were allowed to contact rubber stoppers for more than h [ ] . even though the low-dose jjav in the current study was stored in plastic syringes with rubber plungers, negligible adsorption was identified. this difference might be due to the inclusion of polysorbate as a surface active agent in the current samples, or different adsorption capacity and surface area of the rubbers. jjav solutions above g/ml that are used for immunotherapy are intended for multi-dose use. the addition of benzyl alcohol as an antimicrobial preservative in the jjav diluent is essential to reduce the risk of bacterial contamination [ , ] . the insignificant change in the ph of the jjav diluent in the presence of advax was important as the solubility and optimal antimicrobial activity of benzyl alcohol is considerably reduced above ph [ , ] . when used as an antimicrobial preservative, benzyl alcohol is commonly added to parenteral preparations at a concentration between . - % [ , ] . a % reduction of benzyl alcohol concentration in jjav diluent due to dilutional effects from the addition of advax reduced the concentration of benzyl alcohol to below this usual range and could therefore have affected its effectiveness as a preservative. however, the aet experiments confirmed that the antimicrobial efficacy of the combined formulation against challenged organisms was maintained throughout the study period. it is possible that this was due, at least in part, to the inherent antimicrobial activities of jjav [ ] , which provided additive or synergistic effects to the preservative activity of benzyl alcohol. if this was the case, since low concentrations of venom proteins are used during the induction phase of a vit, a greater reliance on the antimicrobial efficacy of benzyl alcohol is required during this phase. a pdi of < . suggests a monodisperse preparation [ ] , and the results from these studies showed that advax had a relatively monodisperse particle size distribution at the start of the study as indicated by the pdi of . . the increase in average particle size and pdi observed at the end of study period may be caused by formation of delta inulin aggregates. however, the absence of multimodal peaks in all samples tested throughout the study period suggests that aggregation, if present, is limited under the study conditions employed and importantly the size of these particles was within the accepted range for stability (table ). advax has been reported to have minimal protein adsorptive capacity [ ] , and similarly we found no evidence that advax adsorbs jjav allergens as there was minimal difference in the sds-page and uplc-uv analysis of venom components between samples formulated with and without advax. notably, the murine immunogenicity data supported the hypothesis that advax adjuvant can provide significant antigensparing for jja vit, inducing significantly higher serum jjav-specific igg levels in the mice that received the adjuvanted venom formulation than a five times higher dose of jjav alone. whilst there is currently no animal model of jjav allergy, and hence the effects of advax on inhibition of jjav ige during vit or its ability to induce blocking igg antibodies is unable to be assessed outside of human studies, the ability of advax to enhance venom-specific igg responses when compared to immunisation with venom alone has served as a useful predictive marker of favourable human responses in previous human studies of advax-adjuvanted honeybee vit, where inclusion of advax led to an earlier and higher rise of venom-specific total igg and igg with potential blocking activity [ , ] . hence the current results support the rationale for a planned human trial of advax combined with a reduced dose of jjav ( g in maintenance phase) as compared to the currently clinically proven g maintenance jjav dose. notably, no safety or reactogenicity issues were identified with the advax-adjuvanted low dose jjav formulation in the current study. advax adjuvant provided at least -fold jjav antigen sparing in murine immunogenicity studies, with no observed issues of reactogenicity or safety. no detrimental effects of advax on jjav stability were found, supporting use of this combined formulation as a jjavsparing strategy in planned human trials. plans are in place to commence a human clinical trial to assess the ability of advax to reduce the required maintenance dose of jjav from to g per dose, which if successful would result in a -fold higher number of subjects able to be treated with jja vit with current supplies, while also significantly reducing the potential cost of therapy, which largely reflects jjav costs. towards complete identification of allergens in jack jumper (myrmecia pilosula) ant venom and their clinical relevance: an immunoproteomic approach update on mechanisms of allergen injection immunotherapy ant venom immunotherapy: a double-blind, placebo-controlled, crossover trial ant venom immunotherapy in australia: the unmet need ultrarush versus semirush initiation of insect venom immunotherapy: a randomized controlled trial von-bieren, the use of adjuvants for enhancing allergen immunotherapy efficacy carbohydrate-based immune adjuvants advax tm , a novel microcrystalline polysaccharide particle engineered from delta inulin, provides robust adjuvant potency together with tolerability and safety human phase trial of low-dose inactivated seasonal influenza vaccine formulated with advax tm delta inulin adjuvant micro-and nanotechnology in vaccine development randomized clinical trial of immunogenicity and safety of a recombinant h n / pandemic influenza vaccine containing advax tm polysaccharide adjuvant comparative safety of vaccine adjuvants: a summary of current evidence and future needs immunotherapy - . a controlled study of delta inulin-adjuvanted honey bee venom immunotherapy a randomized controlled trial to assess the safety and benefits of immunotherapy with a delta inulin adjuvanted venom extract in honey bee allergic patients encyclopedia of immunobiology factors influencing the quality of myrmecia pilosula (jack jumper) ant venom for use in in vitro and in vivo diagnosis of allergen sensitization and in allergen immunotherapy stability of myrmecia pilosula (jack jumper) ant venom for use in immunotherapy dynamic light scattering common terms defined, inform white paper development of the antimicrobial effectiveness test as usp chapter < > advax, a polysaccharide adjuvant derived from delta inulin, provides improved influenza vaccine protection through broad-based enhancement of adaptive immune responses guideline on adjuvants in vaccines for human use high-alert medications: safeguarding against errors, medication errors regulatory aspects of allergen vaccines in the us guideline on allergen products: production and quality issues monitoring bacteriostasis in allergen extract mixing: years of culture data bacteriostatic agents and sterility requirements for allergen immunotherapy antimicrobial preservative use in parenteral products: past and present excipients and their role in approved injectable products: current usage and future directions pilosulins: a review of the structure and mode of action of venom peptides from an australian ant myrmecia pilosula a novel hepatitis b vaccine containing advax, a polysaccharide adjuvant derived from delta inulin, induces robust humoral and cellular immunity with minimal reactogenicity in preclinical testing we are grateful to sandra ahokas, jenny gudden, judith hawker, and rhys gunner for their assistance in collecting the ants; judith hawker and hayley webb for their assistance in performing venom sac dissection; judith hawker, amanda bannister, roseanne ford, elizabeth jordan, and anne kaye of the pharmacy department, royal hobart hospital for their technical assistance in the manu- rjh, sgab, np and mdw conceived the study. tw, mdw and np designed the study methodologies. tw, nwd, her and yh-o performed the experiments. tw, nwd, np, rw and mdw analysed the data. tw prepared the manuscript. all authors participated in its revisions and have approved the final form. np and yh-o are associates of vaxine pty ltd which owns the advax tm adjuvant platform. tw, her, sgab, and mdw are either employee or consultants to the tasmanian health service, the organization owning the intellectual property over jja vit. all other authors declare no conflict of interest. supplementary material related to this article can be found, in the online version, at doi:https://doi.org/ . /j.jpba. . . . key: cord- -t t qqkl authors: boumans, dorine; fuest, clemens; krolage, carla; wohlrabe, klaus title: expected effects of the us tax reform on other countries: global and local survey evidence date: - - journal: int tax public financ doi: . /s - - - sha: doc_id: cord_uid: t t qqkl the tax cuts and jobs act constitutes the largest change to the us tax system since the s and thoroughly alters the way in which multinational companies are taxed. current assessments on the reform’s international impact vary widely. this article sheds light on the tax reform’s expected effects on other countries. we first use representative german business survey data to analyze the impact of the reform on german firms. many firms with substantial us revenues or capacities in the usa intend to expand us investment in response to the reform, in particular large firms and manufacturing companies. the effects on investment in germany are ambiguous: while some firms substitute between investment locations, others expand in both countries. we subsequently extend our analysis to a global level using worldwide survey data. the results suggest a negative impact on tax revenues and investment in countries with close economic ties to the usa. on december , , us president donald trump signed into law the tax cuts and jobs act. this reform constitutes the most substantial overhaul of the us tax system since president reagan's reform and changed both the corporate and the personal income tax. most notably, the reform reduced the statutory federal corporate income tax rate from to percent and thoroughly changed the taxation of multinational firms. in addition to converting from a worldwide tax system with deferral to a modified territorial tax system, the tcja introduced new international provisions (beat, fdii and gilti) affecting the taxation of multinational income. with many of the tcja's provisions targeting multinationals, the reform does not only have a far-reaching impact in the usa, but around the globe. beyond the demand stimulus expected from the tax reform, the reform may induce companies to shift investment as well as taxable profits to the usa. however, some provisions may exert countervailing effects and induce investment in other countries. so far, no clear consensus has emerged on the extent of these effects (kopp et al. ). however, when deciding whether and how to design a policy response, the international implications of the tcja are of utmost importance to policy makers. against this background, this paper gathers survey evidence to shed light on the reform's potential international effects on investment, trade and tax revenues. as the tcja's economic effects are largely contingent on firm responses to the reform, this paper mainly relies on representative survey evidence from german firm surveys. as germany is among the world's most export intensive economies (statista ) and among the largest providers of us inbound fdi (jackson ) , information on german firm responses is instructive for assessing the tax reform's international effects. our most important findings are as follows: while most german firms do not plan to alter their investment, an important share of firms with us exposure, measured by revenue generated in the usa or by having us subsidiaries, plans on increasing us investment. the effects on german domestic investment are ambiguous. while some firms intend to invest more in both countries, others intend to cut investment in germany and replace it by higher investment in the usa. companies which intend to invest more in the usa also plan to increase exports to the usa. the idea that companies will invest and produce in the usa to replace exports from germany finds little support in our data. we subsequently supplement our findings with results from a worldwide economic expert survey to gauge the reform's effects on a wide array of countries. our global survey results suggest a negative impact on tax revenues and investment in countries with close economic ties to the usa. the reminder of the paper is as follows. first, we explain some background of the tcja and review the existing literature. then, we introduce our three surveys. our survey-based results are presented along the various impacts of the tcja such as the tax burden or investment. many of the tcja's provisions exert an impact on firms around the globe, particularly in the realm of corporate taxation. first, the corporate tax rate cut directly affects after-tax profitability, leaving more cash for investment, salaries or dividends to shareholders. the reform also lowers the tax burden on pass-through entities and temporarily allows immediate expensing of short-lived capital investments. by lowering marginal effective tax rates (metr), at least for equity-financed investments (gravelle and marples ) , the tcja thereby increases incentives for domestic investment. second, the reform thoroughly alters the tax treatment of multinationals by converting to a modified territorial tax system, which exempts dividends from domestic taxation. prior to the reform, us companies faced taxation on their worldwide income. taxes paid abroad were credited against us tax. however, the taxation of foreign profits that did not qualify as subpart f income was deferred until repatriation, i.e., did not need to be paid as long as earnings were kept abroad. for this reason, many firms retained profits in their foreign subsidiaries to avoid the high tax rates upon paying dividends to the us parent. following the reform, repatriated dividends are exempt from domestic taxation, but a transition tax was levied on past foreign profits. this tax of between and . % was levied irrespective of whether repatriation takes place. exempting repatriated dividends from taxation means that cash accumulated abroad is now more easily available in the usa, for example for investment purposes, dividends to shareholders or share repurchases. however, while businesses could not use their unrepatriated earnings to engage in transactions that benefit shareholders, they could nevertheless invest these earnings in the us financial market. some analysts therefore suggest that repatriation does not make much of a difference. the varying assessments of the effects of repatriation highlight how little is known about the potential effects of the reform. third, further provisions aim at curbing tax base erosion. these measures may in some cases even increase the tax burden for us multinationals, and their incentives are not as straightforward (chalk et al. ; clausing ) . the "global intangible low taxed income" (gilti) and the "foreign-derived intangible income" (fdii) provisions were designed to remove tax incentives to shift profits derived from intangible assets to low-tax countries. gilti effectively constitutes a minimum tax on foreign earnings. while the first ten percent return on assets is tax exempt, a minimum tax of . percent is levied on earnings exceeding this threshold. this may lead to countervailing effects (clausing ; dharmapala ) : for one, the exemption provides an incentive to invest in foreign assets. also, since the tcja constitutes a global minimum tax, firms may offset earnings from tax havens with earnings from high-tax countries. for firms with substantial income from tax havens, this may actually incentivize increasing investments in high-tax countries. in contrast, the fdii is a tax deduction for export-oriented us corporations. this tax benefit applies to income that is both attributable to intangibles held in the usa and derived from foreign sales above a normal return on assets. while constituting an incentive to hold intangible assets related to exports in the usa, the fdii also encourages the offshoring of real investment (clausing ; sanchirico ) . finally, the "base erosion and anti-abuse-tax" (beat) is a minimum tax on us profits intended to limit profit shifting to other countries. this tax is charged on payments to foreign related entities above a threshold. this lowers profit-shifting incentives. fourth, the tcja contains several further provisions that impact business decisions. among others, the amortization period for r&d expenses will be extended to years from onwards, again raising the cost of investment. furthermore, the reform abolished the alternative minimum tax. it also altered loss carry over rules, eliminating loss carry backs and limiting loss carry forwards to % of subsequent years' income. in addition, the tcja also entails significant changes for the taxation of personal income. these include rate cuts, an increase in the standard deduction as well as limits on itemized deductions. as opposed to the corporate tax reform, these provisions are temporary and expire after . while this paper mainly focuses on the international implications of the corporate income tax provisions, our survey data also reflects views about the impact of the entire reform package, including the significant personal income tax cuts. this is the case as firm assessments may also account for changing us consumption patterns driven by the reform. a growing number of contributions in the literature discuss the reform's impact, partly based on macroeconomic simulation models. one strand of the literature focuses on effects on the us economy and suggests avenues for tax policy improvements (e.g., auerbach ; chalk et al. ; clausing ; gale et al. ; kopp et al. ; slemrod ) . other studies specifically target the international impact of the reform (e.g., clausing ; dharmapala ; gravelle and marples ) . some of these studies use simulation models to quantify international tax spillovers. focusing on the tax rate cut and calibrating their model with parameters found in the literature, beer et al. ( ) find declining investment and declining taxable profits of multinational firms reported in other countries. similarly, spengel et al. ( ) and heinemann et al. ( ) assess the effects of the reform on fdi flows between europe and the us based on the effective tax burden for cross-border investments. they conclude that the effective tax burden both on european fdi in the usa and on us fdi in europe falls, and additional us inbound investment from the eu rises, while outbound investment in the eu increases at a lower magnitude. low-tax countries, such as ireland, are predicted to benefit more than hightax countries, such as germany. focusing explicitly on germany, christofzik and elstner ( ) find a positive impact on german gdp and an increase in the current account using structural vector auto-regressions. however, these simulation studies abstract from many of the tcja's international tax provisions. therefore, actual effects might substantially deviate. so far, studies using firm-level responses are scarce. for one, gaertner et al. ( ) assess stock returns around the tcja's major tax reform events, finding substantial heterogeneity around the globe. while the majority of foreign firms experienced positive returns, chinese firms overwhelmingly experienced negative returns. hanlon et al. ( ) analyze company statements about actions following the tcja. while they find that % of s&p firms announced a positive investment response, responses differ by firm characteristics. notably, companies with a high ratio of cash taxes to pretax income are more likely to announce additional investment, whereas multinational companies are less likely to announce responses than companies solely based in the usa. domestic responses have also been covered by us firm surveys (see kopp et al. , for an overview). according to the nabe quarterly business conditions survey, % of firms attributed rising investment to the tcja in , while percent of small business owners surveyed by the national federation of independent business planned to expand investment with their tax savings. while these findings are broadly consistent with our survey results, the focus of our surveys differs. while other surveys capture us responses to the reform, and thereby have a domestic focus, our surveys shed light on the international implications of the tcja, focusing on german firms with considerable exposure to the us market. we assess the impact of the tcja within the scope of three surveys administered by ifo institute. while results from the two firm-level surveys help shed light on german firms' perceived impact of the reform, we subsequently complement these findings with results from a global expert survey. many studies assess tax reform implications within the scope of macroeconomic models, for example, by using vector autoregressive models (see, e.g., mertens and ravn ) . firm surveys, on the other hand, have the advantage of measuring the (perceived) impact of tax reforms at the microlevel of economic agents. while administrative tax data are typically only available with a substantial time lag, survey results provide a more readily available picture of firm responses. experts may have-potentially individually different-models under consideration and other sources of information to assess shocks to an economy. surveying experts can condense this information. both firm-level surveys are representative for german firms. as the design of the surveys differs, they jointly provide a more nuanced and in-depth picture of international firm responses. the first business survey was conducted in march as a part of the regular monthly ifo business survey. this survey is the basis for the ifo business climate index, which is considered the most important leading indicator for german economic activity. the ifo business survey is a representative monthly survey of about german firms. the four main sectors covered by the survey are industry (about answers), trade ( ), services ( ) and construction ( ). the regular questionnaire contains monthly, quarterly, biannual and annual questions and is filled in for the most parts by the owner or the ceo of the firm (sauer and wohlrabe ). the questions about the us tax reform were included as supplementary questions and were answered by firms. the largest share of responses is in the service sector with %, closely followed by industry with %. the least answers were received from the trade sector. the questions were not posed in the construction sector. as many small-and medium-sized enterprises do not operate on the us market, we show separate results for firms with us exposure. this subgroup consists of companies who derive at least % of their revenue from the usa. these firms are primarily found in the industry sector (see table ). the second business survey was conducted by ifo institute for the non-profit organization stiftung familienunternehmen (foundation family enterprises). this is a joint project where ifo executes an annual representative survey with changing main topics. the survey addressed international tax competition. based on a stratified representative sample, more than , firms were contacted either via letter or electronic mail. the survey period was between may and june . in the end, firms filled in a questionnaire, corresponding to a response rate of about %. most answers came from the service sector ( %) followed by industry ( %). the distribution of respondents across sectors and firm size is representative of the german economy as a whole. only the trade sector is somewhat underrepresented, whereas the construction sector is oversampled (table ). our firm-level analysis focuses on survey questions which specifically address the us tax reform and its potential impact on german firms. the first ifo business survey (panel a) focuses on the impact of the us tax reform on the tax burden of german firms, on their investment choices and on trade with the usa. while the second business survey (panel b) also addresses firms' tax burden and investment choices, it has a slightly different focus, notably differing with respect to the classification of firms with substantial exposure to the usa. while the first survey uses a firm's us revenue share to classify us exposure, the second survey directly identifies firms with existing us production sites. the exact wording of the questions in both surveys can be found in appendix. to broaden our analysis beyond germany, we supplement our results with a global survey of economic experts, assessed by the ifo world economic survey (wes). our global-level evidence is based on the views of around economic experts from countries who participate in the wes. in selecting economic experts for the wes panel, emphasis is placed on their professional competence in economic matters and inside knowledge of their countries. this is guaranteed by screening their education and current affiliation. each quarter, the panelists are asked to assess main macroeconomic variables in their respective country. in the strata were size, branch and legal form. the random sample was drawn from the orbis database. more details on the survey can be found in stiftung familienunternehmen ( ) . an economic expert and someone who is eligible for participating in the world economic survey is someone who works with economic data and has a good understanding of the economic developments within their own country. the wes, compiled by the ifo institute since , aims at providing a timely and accurate picture of the current economic situation and economic trends over key advanced, emerging and developing economies by polling more than experts quarterly. in selecting experts, emphasis is placed on their professional competence in economic matters and inside knowledge of their countries. see boumans and garnitz ( ) for further details. the survey has been proven to predict business cycles quite well, cf. kudymowa et al. ( ) or garnitz et al. ( ) . more studies have used the supplementary question for further research, cf. . % of the respondents have a master or phd degree and % studied economics. % of the respondents are affiliated with a university, research institution or think-thank and % work at banks or central banks. addition, the survey includes supplementary questions about political or economic issues of current interest. in the april survey, the recent us tax reform was the topic of these additional questions. in total, experts from countries participated in the april survey in . respondents answered the supplementary questions (see table for the distribution across world regions). these unique data sources offer the possibility to analyze the expected impact of the us tax reform on german businesses and to contrast these findings with other countries based on economic expert knowledge. the three surveys allow us to assess the effects along the different parameters such as tax burdens and revenues, investment choices, trade effects, as well as other relevant aspects, such as the location of intellectual property rights. to provide an overview of whether firms are affected by the tcja, we first distinguish between two aspects: whether the tcja has an impact on firms and whether firms adjust their behavior in response. that is, the first aspect encompasses changes to a firm's tax burden and, in case of the second survey, also to a firm's competitive position. these aspects do not necessarily entail any behavioral changes on the part of the firm. in contrast, the second aspect covers whether a firm actively reacts to the reform, e.g., by adjusting investment or production strategies. these different adjustment patterns will subsequently be analyzed in more detail in the following sections. responses are depicted in table . note that responses across both surveys are not readily comparable, as questions in both surveys address slightly different aspects of the reform (see questionnaire in appendix). according to the first survey, % of all firms are affected by the reform. this share increases to % among firms with at least % us revenue. across all firms, respectively, about % report to be either impacted though changes in their tax burden, or to actively react. among firms with substantial us revenue, the share of impacted firms is twice as large as the share of firms that plan to actively react. in the second survey, % of all firms report to be affected by the tcja, either through changes in the tax burden or through changes in their competitive position. this share rises to % if we only consider firms with us production sites. for all firms, but also for the firms with us production sites, it is striking that a larger fraction reports to actively react to the changes in us tax law than claims a direct impact. % of the surveyed firms plan to react to the tax reform (see table ), with details on their responses outlined in later sections. the difference between the two surveys with respect to affectedness, impact and reaction is notable. first and foremost, this is attributable to the more comprehensive set of questions in the second survey (see appendix). second, the two surveys take a different approach to measuring us exposure: raising substantial revenue is not the same as having a production plant in the usa. as many firms possibly export to the usa without any local production, this might lead to the first survey's lower perceived affectedness. to put these results in a broader light, we asked the wes panelists if they expected either benefits or losses for their own country. this initial assessment underlines the relevance of the reform for the global economy: fig. shows that experts around the world do expect their country to be affected by the tcja. colors represent the answer categories after recoding, where lose significantly was coded − , lose slightly − , no change , benefit slightly , and benefit significantly . then, an average of the answers was taken where − till − represents lose significantly, − . till − . lose slightly, − . till . no change, . till benefit slightly, and . till benefit significantly experts in the usa are envisaging a slight benefit from the tax reform, whereas negative assessments are most prevalent in countries with substantial us fdi (jackson ): in canada, germany, ireland, mexico, switzerland and the uk. in these countries, three quarters or more of respondents anticipate negative consequences. however, most respondents from the netherlands, which is one of the largest us fdi destinations, reported not to be affected by the reform ( . %), while . % expect to lose slightly. table addresses the anticipated effects of the reform on the usa and on different worldwide regions. responses clearly indicate that especially in regions with close trade ties to the usa, respondents most frequently anticipate negative outcomes (eu and other advanced economies). regions with comparably less economic integration with the usa, for example the commonwealth of independent states (cis) and eastern europe, are expected to be less affected. while respondents from the usa lean toward a positive assessment of the reform's impact, roughly a third of respondents think the usa will be negatively affected by this reform. these perceptions may be driven by several factors, including the impact the reform might have on tax planning structures, tax revenues and investment. to further assess these impacts, the next section explores these in more detail, drawing together evidence from the firm surveys as well as the expert survey. this might be explained by the bilateral investment agreement between the netherlands and the usa, the so-called dutch-american friendship treaty (daft), which provides national treatment and free entry for foreign investors. another explanation might be that although it is a large recipient of american fdi, the netherlands is also a key export platform and pan-regional distribution hub for us firms (us department of state ). the us category consists of respondents from the usa. lowering (or in some cases possibly raising) tax payments is the most immediate channel through which the tcja may affect firms. panel a of table shows the expected impact on the tax burden of firms, also distinguishing short-and longrun effects for the first survey. as expected, only a small fraction of german firms are directly affected by the reform. unsurprisingly, the share of firms envisaging a changing tax burden is larger in the subgroup of firms with us exposure. in this group, % anticipate their tax burden to decline. this number rises in the long run and is also increasing in firm size and with the share of revenues derived from the usa. more substantive long-run responses may also be due to the transition tax on past foreign profits, which could increase tax payments in the short run. in contrast, % of all firms with substantial us exposure expect a rising tax burden in the long run. this effect could be due, for instance, to the more restrictive treatment of r&d spending from onwards, or it could be related to tax avoidance measures such as beat and gilti. the share of firms expecting tax cuts is higher in the second survey ( %) and increases to % when considering only firms with us production. comparably few respondents also claim to be impacted by the changing loss carry over rules (see table panel b). responses in panel c of table show the expected impact on tax revenues across the world, assessed by the respective countries' experts. a clear majority of us respondents expects decreasing tax revenues. this is in line with the congressional budget office's ( ) and the joint committee on taxation's ( ) estimates. most respondents from other countries, however, do not expect the reform to have a substantial impact on their countries' revenues. the largest effects are anticipated in non-eu advanced economies, where % expect a decrease and % anticipate an increase in revenue. explanations are conceivable for both assessments. if profits or investments are moved toward the usa, other countries' tax revenues could possibly decrease. however, firms around the world may also benefit from increasing consumption in the usa and may even direct some of their possible revenue increases toward investment in other countries. the tcja's impact on investment constitutes one of the most important aspects of the tax reform. on the one hand, numerous provisions, such as the rate cut, make investing in the usa comparatively more attractive. this might lead to investments being shifted from other high-tax countries, such as germany, to the usa. on the other hand, some provisions may well exert a countervailing effect. notably, firms with substantial activity in tax havens may face an incentive to invest more in hightax countries, as earnings from these countries may offset earnings from tax havens under gilti. we therefore examine the tcja's effect on firms' planned investment in both the usa and germany. while a clear majority of firms in the first survey do not plan on altering their investment strategies, table shows that % of the firms with us exposure intend to invest more in the usa. this number rises to % among the firms expecting a decline in the tax burden they face, suggesting a strong firm response to tax incentives. as expected, only a few businesses plan to reduce us investment. in a similar spirit, the second firm survey asked whether firms plan to extend existing or build up new investment capacities. % of firms with us subsidies intend to expand their existing capacities, while % want to invest in new ones. this again indicates a substantial investment response to tax incentives. table also summarizes the responses regarding investment in germany. while most businesses do not plan to adjust their german investment, % of the firms with us exposure intend to invest more in germany. in addition to offsetting gilti, this may have several further reasons: expanding economic activity may require inputs produced in germany, and liquidity effects of us tax cuts may also remove constraints on investment in other countries. however, for many companies, we also find a substitution effect between investment in germany and the usa. among the firms which intend to invest more in the usa, % intend to cut back on german investment. these are twice as many as those who plan to invest more in both countries. overall, while investment effects are positive in the usa, they are more ambiguous in germany. the latter result is coherent with the expert survey. experts across different regions in the world expect a decline in investment in their own countries. especially for canada and mexico, in emerging and advanced asian economies, as well as major european economies with substantial us fdi, such as germany and ireland, experts expect a shift in investment toward the usa. in addition, negative perceptions (e.g., expecting investment to move to the us) are far more frequent in countries with moderately to high marginal effective tax rates (metr) that now exceed those of the usa (see panel d of table ). all else being equal, those countries offered lower corporate taxes than the usa before the reform, but have now lost this advantage. nevertheless, respondents from the usa are more skeptical about the effects of the tax reform (see panel c of table ). just over half of us respondents agree that investment will rise in the usa, and this while one of the main aims of the tax reform was to boost domestic investment in the usa. as a third empirical exploration, we examine survey responses regarding possible effects of the reform on exports to the usa and imports from the usa to germany. trade may be affected through changes in the location of economic activity in response to the reform and through tax deductions for exports within the scope of the fdii provision. while the effect on exports and imports is limited across the full sample (table , panel a), planned trade and investment responses are positively correlated. among the firms with growing us investment, % intend to import more from the usa and % plan on increasing their exports to the usa, compared to % who intend to export less to the usa. the idea that firms may replace exports to the usa by products produced in the usa finds little support in our survey data. along similar lines, among the firms cutting back on us investment, % intend to import less from the usa and % expect they will export less to the usa. investment and trade seem to be complements, rather than substitutes. the second firm survey (panel b) indicates only minor effects of the reform on us imports of german firms. however, a quarter of firms with us production expect to increase its us sales. yet, it is not possible to distinguish whether these originate from us production or from exports from germany to the usa. also, rising sales might likewise be due to the us personal income tax provisions stimulating us demand. the expert survey also assessed if the tax reform influenced countries' net exports. with the us president frequently criticizing the us trade deficit, trade effects also figure prominently in the political discussion. overall, assessments are more ambiguous as shown in panel c of table . while % of us experts expect besides having an impact on tax revenues, investment, and the balance of trade, the tcja's provisions affect profit shifting and encourage the location of intellectual property rights in and the repatriation of offshore profits to the usa. yet, virtually none of the firms in the first ifo business survey intend to adjust the location of their ip (not depicted here). at a first glance, this contrasts with the world expert survey: as table shows, roughly half of all us respondents expect the location of intellectual property rights to shift toward the usa. the discrepancy between both surveys may be due to the fact that ip susceptible to profit shifting would presumably be neither held in germany nor in the usa, but rather in a tax haven. negative effects on the location of ip are predominantly feared in asia and in advanced economies, including the eu- , with the most negative assessment in ireland, a country with strong incentives to hold ip, and canada. positive assessments occur more frequently in emerging economies. however, responses do not differ much between countries with and without an ip box regime. in addition to strategically locating ip rights, multinational companies have access to additional strategies, for example shifting profits to low-tax jurisdictions. % of us table survey results of the expert survey-effects of the tax reform table shows the respective answers of the ifo expert survey regarding profit shifting, location of ip rights and headquarters as well as repatriation of offshore profits. "+," "=" and "−," denote "increase," "no change" and "decrease," respectively. the numbers represent percentages respondents expect that more profits will be shifted toward the usa following the reform. the picture varies between other countries: around % of experts in advanced economies, in-and outside the eu, as well as in asian economies, expect that profits will be shifted away from their countries, while this is expected by fewer experts in other regions of the world. here, it seems to make a difference whether a country has an ip box in place. while % of experts in countries with ip regimes expect decreasing profit shifting, this only applies to % in other countries. by contrast, around % of the respondents expect that more profits will be shifted toward their country. in recent years, several large us companies raised substantial attention in the media as they relocated their legal residence to a low-tax country such as ireland (jolly ) . on average, companies reduced their effective tax burden, measured by the ratio of worldwide tax payments to profits, from % to % via corporate inversions (congressional budget office ). on the one hand, the us tax reform's shift from a global toward a territorial tax system reduces incentives to invert, as us corporations are now only liable to pay taxes on their us profits. on the other hand, some relocation incentives remain. some of the provisions of the tax cuts and jobs act specifically apply to us corporations, and corporate inversions could still be attractive to avoid gilti taxes. nevertheless, over half of us respondents believe the reform will result in an increasing number of headquarters being located in the usa. as before, countries located close to the usa like canada, mexico and some further latin american countries, as well as those with substantial us fdi, such as ireland, switzerland, the uk and germany, tend to expect the most negative impact. a similar finding applies to emerging asian countries, with chinese respondents more often expecting a relocation of headquarters than respondents in smaller asian countries. by contrast, positive evaluations are not as concentrated across countries, but tend to occur more often in emerging europe, asia and latin america. prior to the reform, the deferral of taxation until the repatriation of profits leads to a substantial accumulation of profits in foreign subsidiaries, often located in lowtax jurisdictions. moody's estimated that us non-financial corporates' offshore cash holdings amounted to $ . trillion in (moody's ) . including re-invested profits, the joint committee on taxation estimated that undistributed offshore earnings and profits even amounted to $ . trillion in (joint committee on taxation ; keightley ) . under the tcja, foreign profits are exempt from us tax. however, the transition tax charged on non-repatriated past foreign profits results in large one-time tax payments for many companies. as a result of these changes, around % of us respondents expect an increase in the repatriation of offshore profits to the usa. across the world, decreasing foreign cash holdings are expected by % of all experts, while % expect offshore profits to rise in their country. negative perceptions are particularly high in some countries. according to a congressional research service report (keightley ) , % of us corporations' overseas profits were reported in bermuda, ireland, luxemburg, the netherlands and switzerland. unsurprisingly, experts in those countries anticipate a particularly large impact, with % predicting a decrease in reported earnings in their country. among the remaining countries, experts in advanced economies and asian countries tend to expect a negative outcome. overall, negative anticipations are most frequent in countries with very low marginal effective tax rates, as well as countries with moderate tax rates that now exceed those of the usa. the tax cut and jobs act drastically altered the us tax system, also with substantial implications for multinationals with ties to the usa. we assess firmlevel survey data from germany as well as global survey data to gauge the reform's impact around the world and provide insights into policymakers on firms' responses to the reform. our results indicate that while the majority of german firms do not plan on adjusting investment, many firms with considerable us exposure respond to the reform: about a third of german firms that expect to benefit from the tax cut and/or that have capacities in the us intend to invest more in the usa. effects on german domestic investment are more ambiguous: some firms expand capacities in both locations, which may be driven by the tcja's stimulus to us demand, by firms' increase in after-tax profitability, as well as by the tcja's gilti provision incentivizing the increase of tangible assets in high-tax countries. however, other firms intend to substitute between investment in both countries. this indicates that for some firms, the tcja constitutes an impediment to germany's competitiveness in attracting multinational investment. however, while our results speak in favor of a substitution effect between us and german investment, our results do not indicate that many firms substitute us investment for german exports. overall, our results show that the reform triggers significant firm responses. when supplementing our analysis with worldwide survey data, our results also point to a negative impact on tax revenues and investment in other countries with close economic ties to the usa. since the reform, tax competition has intensified, and further countries such as france and belgium have lowered their corporate tax rates. this further worsens the competitive position of other high-tax countries, such as germany, and may warrant tax reforms that incentivize domestic investment. when designing a tax reform, policymakers should not only think about rate cuts, but consider possible interaction effects with the tcja's international provisions. measuring the effects of corporate tax cuts uncertainty and economic activity: evidence from business survey data cross-border tax effects on affiliate investment-evidence from european multinationals tax spillovers from us corporate income tax reform ifo world economic survey database-an international economic expert survey who has terror angst? perceptions of the effects of terror on the world economy us tax and trade policy-perceived impact and preferred policy responses worldwide the tax cuts and jobs act: an appraisal. imf working papers international spillover effects of u.s. tax reforms: evidence from germany. german council of econmic experts/sachverständigenrat zur begutachtung der gesamtwirtschaftlichen entwicklung profit shifting before and after the tax cuts and jobs act (ssrn scholarly paper no. id ) the budget and economic outlook the consequences of the tax cut and jobs act's international provisions: lessons from existing research monetary policy announcements and expectations: evidence from german firms der internationale steuerwettbewerb aus unternehmenssicht making only america great? non-us market reactions to us tax reform a preliminary assessment of the tax cuts and jobs act of forecasting gdp all over the world using leading indicators based on comprehensive survey data issues in international corporate taxation tax reform made me do it! tax policy and the economy implications of the us tax reform for transatlantic fdi disentangling the effects of a banking crisis: evidence from german firms and counties u.s. direct investment abroad: trends and current issues letter to the house of honorable kevin brady, chairman house ways and means committee, and the honorable richard neal jcx- - estimated revenue effects of the 'tax cuts and jobs act ireland, home to u.s. 'inversions,' sees huge growth in g.d.p. the new york times an analysis of where american companies report profits: indications of profit shifting u.s. investment since the tax cuts and jobs act of auswirkungen der us-steuerreform auf deutsche unternehmen -ergebnisse einer ifo world economic survey and the business cycle in selected countries the forecasting power of the ifo business survey the dynamic effects of personal and corporate income tax changes in the united states global implications of u moody's: us corporate cash pile to rise % to $ . trillion in ; tech sector dominates again harmful tax practices - progress report on preferential regimes: inclusive framework on beps: action , oecd/g base erosion and profit shifting project der internationale steuerwettbewerb aus unternehmenssicht. internationales steuerrecht the new us tax preference for 'foreign-derived intangible income the new ifo business climate index for germany ceo or intern-who actually answers the questionnaires in the ifo business survey? cesifo forum is this tax reform, or just confusion analysis of us corporate tax reform proposals and their effects for europe and germany top exporting countries worldwide exchange rate pass-through and credit constraints investment climate statements-netherlands publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations acknowledgements open access funding provided by projekt deal. we thank two referees for very detailed comments and constructive help. key: cord- -rzf cw x authors: wang, qidi; zhang, lianfeng; kuwahara, kazuhiko; li, li; liu, zijie; li, taisheng; zhu, hua; liu, jiangning; xu, yanfeng; xie, jing; morioka, hiroshi; sakaguchi, nobuo; qin, chuan; liu, gang title: immunodominant sars coronavirus epitopes in humans elicited both enhancing and neutralizing effects on infection in non-human primates date: - - journal: acs infectious diseases doi: . /acsinfecdis. b sha: doc_id: cord_uid: rzf cw x [image: see text] severe acute respiratory syndrome (sars) is caused by a coronavirus (sars-cov) and has the potential to threaten global public health and socioeconomic stability. evidence of antibody-dependent enhancement (ade) of sars-cov infection in vitro and in non-human primates clouds the prospects for a safe vaccine. using antibodies from sars patients, we identified and characterized sars-cov b-cell peptide epitopes with disparate functions. in rhesus macaques, the spike glycoprotein peptides s( – ), s( – ), and s( – ) elicited antibodies that efficiently prevented infection in non-human primates. in contrast, peptide s( – ) induced antibodies that enhanced infection both in vitro and in non-human primates by using an epitope sequence-dependent (esd) mechanism. this peptide exhibited a high level of serological reactivity ( %), which resulted from the additive responses of two tandem epitopes (s( – ) and s( – )) and a long-term human b-cell memory response with antisera from convalescent sars patients. thus, peptide-based vaccines against sars-cov could be engineered to avoid ade via elimination of the s( – ) epitope. we provide herein an alternative strategy to prepare a safe and effective vaccine for ade of viral infection by identifying and eliminating epitope sequence-dependent enhancement of viral infection. a novel severe acute respiratory syndrome coronavirus (sars-cov) was characterized in march after a global effort following the first epidemiological case in november . ten years later ( ), a novel middle east respiratory syndrome (mers) coronavirus emerged, which was a βcoronavirus, similar to sars-cov. sars is a deadly infectious disease and has the potential to seriously threaten public health and socioeconomic stability worldwide. serologic and genetic investigations indicate that sars-cov is of zoonotic origin, , with bats as the likely animal reservoir. since the identification of sars-cov in , there have been major advances in understanding sars-cov genetics , , and molecular epidemi-ology and in identification of host receptors , and t-cell epitopes. , models in mice, ferrets, and monkeys have been established, each with particular strengths and weaknesses. vaccine candidates have included dna, a live attenuated strain, recombinant proteins, inactivated whole virions, and vector vaccines; however, none have been approved through clinical investigation. safety concerns about a sars-cov vaccine have been raised, given the observation in vitro of antibody-dependent enhancement (ade) of sars-cov infection that could, in theory, exacerbate disease. other observations include evidence of ade reported here for the first time induced by an inactivated sars-cov vaccine in rhesus macaques ( figure ) and by antisera from sars patients (table s ) , as well as ade in other coronavirus infections. − ade in vitro has been observed for many other viruses, including yellow fever virus, west nile virus, human immunodeficiency virus (hiv), ebola virus, respiratory syncytial virus (rsv), and influenza a virus. − hiv-induced ade has been described, , but the exact mechanism is still uncertain. among other examples of ade, secondary infection with dengue virus of a heterologous serotype has been associated with an immunopathologic vascular leakage and hemorrhagic syndrome, dengue hemorrhagic fever/dengue shock syndrome (dhf/ dss). most descriptions of ade relate to fc-gamma receptor (fcγr) and/or complement components promoting viral uptake and virus replication pathways. − goncalvez et al. reported that treatment of juvenile rhesus monkeys with the cross-reactive mab a , which recognizes the fusion loop in dii of dengue virus, enhanced dv viremia by several logs within − days of infection. furthermore, a -amino acid (aa) deletion at the nterminus of the ch domain in the fc region abrogated enhancement in vitro and in vivo, confirming that ade occurred through an fcγr pathway for this class of antibodies. another study analyzed the stoichiometric relationship between antibodymediated neutralization and enhancement of west nile virus (wnv) infection in cells expressing fcγr. the results showed that there is an antibody occupancy threshold on the virion for neutralization or enhancement of virus infection. strongly neutralizing diii-lateral ridge-specific mabs inhibit at lower occupancy, whereas weakly neutralizing mabs that bind distinct epitopes require a much higher mab occupancy for neutralization. when mab occupancy falls below the threshold for neutralization, ade can occur. in subsequent studies, the same group showed that the level of antibody occupancy that promotes ade in vivo is also modulated by the binding of c q, which restricts ade in an igg subclass-specific manner. on the basis of these studies, many neutralizing antibodies may enhance infection in vitro in fcγr + cells when their concentrations fall below a key occupancy threshold, and some abs that poorly neutralize may strongly enhance over a wide dose−response range. however, for antibody isotypes that bind c q avidly, much of the enhancing effect may be minimized in vivo. other studies suggest that fcγr-dependent ade may not be the only mechanism for antibody enhancement of infection. huang et al. demonstrated ade in vitro of an anti-prm mab against denv in cell lines that lacked expression of fcγr (e.g., baby hamster kidney cell line bhk- and murine fibroblast cell linenih t ). importantly, a peptide in the m region (cpflkqnepedidcw) of prm blocked this ade in a dosedependent manner. the antibody appeared to have dual specificity and bound to dengue virus virions and/or a crossreactive hsp protein on the cell surface. thus, ade via non-fcγr-or complement-dependent mechanisms is plausible for denv, yet poorly characterized in vitro and in vivo. herein, we discovered that a peptide of the viral sequence simultaneously elicits the antibodies of disparate functions in protection and enhancement against sars-cov infection by the studies with host vero e cells in vitro and in non-human primates. vaccine in the lungs of macaques. first, we observed the ade of sars-cov infection in lungs after immunization with the inactivated virus vaccine in macaques. the monkeys with or without the immunization of inactivated sars-cov vaccine were infected by nasal cavity inoculation of live sars-cov virions ( figure ). infected macaques showed lungs with broadened interval and visible macrophage infiltration with alveolar epithelial hyperplasia. however, ade macaques after vaccination showed lungs with broadened interval, lung interval fractured with large amounts of macrophage and lymphocyte infiltration, visible fibrin, and protein-rich edema in the alveolar cavity. protected macaques showed lungs with slightly . the animals were then challenged with nasal cavity inoculation of sars-cov ( × tcid in . ml/monkey, pumc sars-cov strain) days after the boost. the animals were sacrificed days after viral challenge, and lung tissues were sampled for general and pathological observations. pathological examination procedures were as described in ref . infected macaque: lung interval broadened, visible macrophage infiltration with alveolar epithelial hyperplasia. ade macaque: lung interval broadened, lung interval fractured with large amounts of macrophage and lymphocyte infiltration, visible fibrin and protein-rich edema in alveolar cavity. protected macaques: lung interval slightly broadened, without visible abnormalities. the arrows indicate the lung lesions of infected and ade animals. broadened intervals without visible abnormalities. the results implied that the simple vaccination with inactivated whole sars-cov virions might be ineffective for protection of virus infection. identification of highly immunodominant b-cell peptides of sars-cov in humans. mapping highly immunoreactive human b-cell peptides by employing sars patient antisera was carried out by a number of research groups. consistent with previous reports, jiang's laboratory reported that the m − and m − peptides from the membrane protein and the n − and n − peptides from the n protein were highly reactive with all convalescent-phase test sera from sars patients. the laboratories of wu and che collaboratively identified three conformational (amino acids − , − , and − ) and three linear (amino acids − , − , and − ) epitopes on the full-length n protein that were immunodominant. using a "split and mix" combinatorial strategy, we synthesized an overlapping peptide library spanning all four structural proteins of the sars-cov bj strain, including the envelope (e), membrane (m), nucleocapsid (n), and spike (s). we screened this peptide library with antisera from convalescent sars patients and identified dominant peptide epitopes recognized by anti-sars igg. further experiments in this paper indicate that three peptides in the s protein (s − , s − , and s − ) were strongly bound by sars-cov-specific human igg from . , . , and . %, respectively, of the sars patients ( table ) . extension of the peptides (table s ) to further characterize the epitopes' n-and c-termini identified a new immunodominant peptide (s − ), which was recognized by antisera from % of the patients ( table ). the s − peptide specifically reduced the titer of human igg in convalescent sera from sars patients who reacted with sars-cov viral lysates ( figure a ). we studied sequentially collected sera from convalescent sars patients whose sera (obtained months after onset of infection) strongly reacted with the s − peptide. the antisera from these patients continued to recognize the s − at months [ / ( . %)], months [ / ( . %)], and months [ / ( . %)] after the onset of sars ( figure b ), implying that the s − peptide is a major immunodominant peptide in humans and elicits a long-term b-cell memory response after natural infection with sars-cov. epitope-specific antibodies of sars-cov exhibited enhancing or neutralizing functions in vitro. to study the functional significance of immunodominant peptides of the s glycoprotein, we generated monoclonal antibodies (mabs, table s ) by immunization of mice. ten of these strongly bound to the sars-cov virion ( figure a ). among them, five blocked sars-cov infection of vero e cells ( figure b ), including mab e , mab a , and mab g against s − , mab b against s − , and mab − against s − . in contrast, two mabs (mab - - and mab - - ) against s − , despite sharing some of the same isotypes and constant region configurations with neutralizing antipeptide antibodies (igg /κ, igg a/κ, and igg b/κ; table ), markedly enhanced sars-cov infection of vero e cells ( figure b ). this was particularly striking given that vero e cells lack fcγr, and the best-understood mechanism for ade involves fcmediated internalization and replication of virus. mab - - - table . peptides recognized by sars-infected patients, relevant mouse mabs, and functional classification a a *, red-, blue-, and green-colored peptides represent the epitopes that were assembled into immunogenic peptides. ζ, neu (neutralizing) or ade indicates the ability of the individual mab to block or enhance, respectively, sars-cov infection of vero e cells. #, the reactivity of individual peptides with antisera from convalescent sars patients (male, ; female, ) was determined using an elisa. commercially available elisa kits were used to confirm that % of the antisera were positive against viral lysates as antigen (cutoff value = . ). the titer of each serum was averaged from duplicate wells. and mab - - -enhanced vero e cell infection was reduced in a dose-dependent manner by s − , but not by an unrelated hbv peptide ( figure a ,b), whereas ade of human antisera from sars-cov-infected patients was also specifically blocked by the same peptide ( figure c ,d), implying that peptide-based ade occurred during the epidemic period of a sars outbreak. an antibody-peptide binding elisa indicated that mab b against s − reacts with both s − and s − , whereas mab - - against s − only bound to s − ( figure e ). the data suggest that mab b was induced by s − and is a consensus peptide of s − and s − , whereas mab - - was from the n-terminal peptide s − (lyqdvnc) immunizing antigen. synthesized peptides lyqdvn (s − ), lyqdvnc (s − ), and lyqdvnct (s − ) were all immunoreactive with the tested sera of sars-positive patients, further confirming that the s − peptide is an immunodominant one in humans ( figure s ). alanine scanning mutagenesis of the s − peptide demonstrated that l , y , q , d , and c are critical amino acid residues for interaction with mab - - ( figure e ; table s ). the s − peptide lies close to the c-terminus of the sars-cov major receptor (ace )-binding domain (rbd). we speculate that mab - - may expose specific conformations that catalyze sars-cov attachment to and/or membrane fusion with target cells. neutralization or enhancement of sars-cov infection in non-human primates by epitope-specific antibodies. low-molecular-weight peptides behave like haptens, with relatively low immunogenicity. to make them more immunogenic, we chemically ligated four copies of each peptide to a lysine core [brch co-lys -(lys)-β-ala-conh (brk ka), scheme ] to prepare a multiple antigen peptide system (map) and purified them by preparative hplc ( figure a ; table s ). molecular weight was confirmed by lc-ms/ms ( figure b ). we used these synthetic maps to vaccinate rhesus monkeys (table s ) as follows: vac , control group (received . % nacl) of four macaques randomly assigned to two groups for day or day postinfection sacrifice; vac , map-s − given to six macaques randomly assigned to two groups for day or day postinfection sacrifice; vac , a mixture of map-s − , map-s − , and map-s − given to six macaques randomly assigned to two groups for day or day postinfection sacrifice; and vac , a mixture of map-s − , map-s − , and map-s − given to six macaques randomly assigned to two groups for day or day postinfection sacrifice. the immunization protocol included one priming injection followed by three boosts at -week intervals (chart s ). antipeptide polyclonal abs (monkey igg) monitored by elisa showed high-level responses to all immunized peptides after three boosts ( figure , left). antibodies ( figure a ) against the four peptides in sera from vac -immunized animals were at background level on all blood collection days. vac gradually induced anti-s − peptide antibodies after three boosts. not surprisingly, both vac and vac immunizations elicited high levels of igg against the s − peptide because the vaccine components consisted of the s − or s − epitopes in these two animal groups. anti-s − and -s − antibodies in the vac -immunized animal group reached their highest levels before the second boost ( days after the first injection). the average titer of anti-s − igg was greater than : in vac -immunized monkeys before sars-cov challenge and was -or -fold times that of vac or vac , respectively. interestingly, when vac -vaccinated animals were challenged by sars-cov, the igg level against s − gradually decreased ( figure b ) on days and postinfection. this is different from the vac and vac immunizations, after which igg levels inexplicably increased on day postinfection and subsequently decreased on day postinfection. this observation implies that anti-s − antibodies are strongly involved in the neutralization of sars-cov in monkeys and that the existing s − epitope (in vac and vac ) may reduce the total titer of anti-s − antibodies. all immunized macaques were subsequently challenged with sars-cov (pumc strain) via nasal inhalation on the th day after the last boost, as described. at and days postinfection (dpi), the macaques were sacrificed (chart s ; table s ). the data for lung damage and viral burden in these macaques are summarized in table . gross lung pathology was evident at both and dpi in all groups. in mock-immunized macaques (vac ), the average pathology grade was grade iv ( figure a ,b; table s ), with severe diffuse alveolar damage, including alveolar shrinking and rupture of elastic fibers. massive macrophage infiltration was associated with fusion of alveolar septa. pulmonary edema, fibrin, hemorrhage, and cellular debris contributed to the severe interstitial pneumonitis. damage was similar (grade iv) for vac , somewhat reduced (grade ii−iii) for vac , and markedly diminished (grade i−ii) for vac . immunohistochemistry using mabs against the viral peptides revealed a large number of sars-cov-infected macrophages and alveolar epithelial cells in the vac group ( figure c ), averaging . ± . infected cells per high-power fields (hpf) and . ± . infected cells per hpf at and dpi, respectively. quantitative viral burden analysis by quantitative rt-pcr showed an average of , copies/mg lung tissue ( dpi) and , copies/mg lung tissue ( dpi) of sars-cov in the lungs of the vac group. compared with the vac group, the vac group showed similar histology at dpi, but the interstitial pneumonia was far worse at dpi, characterized by massive numbers of inflammatory cells in hemorrhagic septa, with extensive exudation. sars-cov-infected cells in the lungs averaged . ± . per hpf at dpi and . ± . per hpf at dpi, whereas viral copies were , /mg lung tissue and , / mg lung tissue, respectively. thus, even though s − was immunodominant in patients, antigenic in monkeys, and reactive with the neutralizing mab b , it was nonprotective and even harmful when used as a vaccine. in contrast, the immunized monkeys in the vac group had a strongly increased ability to control sars-cov infection in association with induction of high levels of anti-s − antibodies ( figure e ). at dpi, hemorrhage in septa and elastic fibers of the alveolar wall were indicated by apparent inflammation in silver staining results. alveolus septa broadening with increasing infiltration of inflammatory cells were recorded by dpi, indicating that only early, acute diffuse alveolar damage occurred. sars-cov-infected lung cells in the vac group averaged . ± . per hpf (vs vac group: p < . ) and . ± . per hpf (vs vac group: p < . ) at and dpi, respectively. the sars-cov burden averaged , copies/mg lung tissue at dpi and , copies/mg lung tissue at dpi. this represents a ratio of ∼ and ∼ between the number of sars-cov copies in the vac and vac groups at and dpi, respectively. for animals that received vac , symptoms of acute diffuse alveolar damage were visible, including fusion of thick septa and ruptured elastic fibers of the alveoli. the average number of sars-cov infected cells in the lung tissues was . ± . per hpf at dpi (vs vac group: p < . ) and . ± . per hpf at dpi (vs vac group: p < . ). the sars-cov burden the cutoff value was set to . (the average for binding of unrelated mab-hiv-p ). (b) neutralization or enhancement of the sars-cov infection in vero e cells by mabs ( . μg/ml) was specifically reduced by the corresponding immunopeptide ( . μg/ml), but not by a hepatitis b virus (hbv) peptide ( . μg/ml). hbv peptide (lldyqgmlpv) is an unrelated control peptide from an hbv surface protein. s − and hbv peptide were pre-incubated with the corresponding mabs or human antisera for min at °c before function was tested. these experiments were performed in triplicate, and the data are presented as the mean ± standard deviation. averaged , copies/mg lung tissue at dpi, that is, . times the burden of , copies/mg lung tissue at dpi. the vac group also showed a . -fold increase to vac group ( , copies/mg vs , copies/mg) of viral burden at dpi, suggesting that the presence of igg against s − facilitated sars-cov infection of immunized macaques. in a separate experiment focused on the phenomenon of ade in macaques, rhesus monkeys were divided into six groups (n = per group, table s ). three groups were separately sacrificed at or dpi; each time point included a control group and two groups that received the enhancing mab - - at doses of . mg/kg or . mg/kg day prior to challenge with the sars-cov pumc strain. gross pathologic changes were again recorded in the control group at a grade iv level ( figure a,b) . although clear pathologic changes were observed in one of the three monkeys in the . mg/kg group, we concluded that previous treatment with a dose of . mg/kg mab - - did not, on average, significantly reduce or facilitate sars-cov infection. however, macaques treated with . mg/kg mab - - showed a marked increase in lung lesions. the lung lesion area in macaque d - ( . × . cm ) at dpi was the largest in all dic experimental groups. at dpi, all lungs from the . mg/ kg group showed larger areas of necrosis, severe sheets of septa fusion, necrotic lesions at the hemorrhagic septa, and massive macrophage infiltration in the alveoli, indicating that the interstitial pneumonia was much more severe in the mab - - -treated group than in the control group ( figure c ). there were more sars-cov-infected cells in the lung tissue ( . ± . per hpf at dpi and . ± . per hpf at dpi, p < . vs control group). the average sars-cov burden in the lungs was , copies/mg lung tissue at dpi and , copies/mg lung tissue at dpi, a -fold enhancement compared with the control group at dpi and a -fold enhancement at dpi ( figure d ). this result further confirmed that enhancement of the sars-cov infection in macaques was directly related to antibodies against s − . in this study, we reported for the first time that a sars-cov inactivated vaccine could induce ade and lung pathology in experimental rhesus monkeys. four antigenic peptides (s − , s − , s − , and s − ) from the spike protein of sars-cov were identified by high cross-reactivity with a large number of antisera from convalescent sars patients. ten of mabs alanine scanning mutagenesis indicated that l , y , q , d , and/or c are critical amino acid residues and that s − is responsible for mab - - -induced enhancement, which may catalyze sars-cov attachment and/or membrane fusion with target cells by exposing specific conformations of the spike protein. s − is located at the virus rbd and can block sars-cov infection of vero e cells in vitro, probably by interfering with high-affinity attachment of sars virions to the human ace receptor at residues t and n of the rbd. , the coronavirus spike proteins have been characterized as class i fusion proteins containing highly conserved heptad repeat regions (hr and hr ). thus, interfering with hr binding to hr has become a recent target for prevention of viral fusion and entry into target cells. s − overlaps hr ( − aa) in the sars-cov spike glycoprotein. the mechanism by which anti-s − antibodies reduce the efficiency of sars-cov fusion is likely to be associated with an exposed five-turn αhelix conformation via hr binding to hr and an extended conformation formed by residues − and − of three antiparallel hr helices in an oblique orientation surrounding a parallel, trimeric coiled-coil of three hr helices. binding of mab - to the sars-cov virion was the strongest among the mabs ( figure a ), which implies that s − peptide may act as a major immunoantigen. severe lung injury occurred in one challenged rhesus monkey that had been immunized with an inactivated sars-cov vaccine ( figure ). very strong serologic reactivity of s − with antisera from . % of convalescent sars patients was observed; the response was to two tandem epitopes (s − and s − ), but each epitope generated antibodies with disparate functions regarding neutralization or enhancement of sars-cov infection. thus, in persons infected by sars-cov, enhancing antibodies and neutralizing antibodies may partly counteract each other's functions. sars-cov's ade was also reported by other research laboratories. using an hl-cz human promonocyte cell line, chen and huang found that higher concentrations of antisera collected from sars-cov-infected patients facilitated sars-cov infection and induced higher levels of virus-induced apoptosis. they further demonstrated that this phenomenon occurred via antispike protein antibodies that mediated ade, but not via anti-n protein antibodies. bruzzone and jaume's laboratory showed that antispike immune serum increased infection of human monocyte-derived macrophages by replication-competent sars-cov as well as by spike-pseudotyped lentiviral particles (sars-covpp), although they did not clarify whether this enhancement was through the fcγrii pathway. reaction a a b-cell epitopes with a free −sh sequence (containing a cys residue) were prepared directly. b-cell epitopes without a free −sh group were anchored by an additional cysteine residue at the c-terminal ( figure s ) . a four-branched, brominated, multiple-antigen core peptide (brk ka, figure s ) via lysine two α-amino and side-chain amino groups was prepared that finally conjugated with four copies of individual antigenic peptide. lentiviral particles, as well as by replication-competent sars coronavirus. jaume and collaborators proposed that ade of sars-cov utilizes a novel cell entry mechanism into immune cells. this study demonstrates for the first time that an antibody (mab - - ) targeting a specific linear epitope (s − ) of the sars-cov spike protein can mediate enhancement of virus infection both in vitro and in non-human primates via an epitope sequence-dependent mechanism. we also demonstrate that the viral peptides that induce protection from pathogenesis can be identified and distinguished from those inducing enhancement in primates. these findings reveal a new mechanism of virus evasion of host defense that potentially provides an alternative strategy to prepare safe and effective vaccines against ade of virus infections. peptide design and synthesis. new peptides from the spike glycoprotein were designed on the basis of the information gained in the first round of immunopeptide discovery. peptides were synthesized manually by standard fmoc chemistry protocols on rink-amide mbha resin (novabiochem, san diego, ca, usa; . mmol/g). all of the l-α-fmocprotected amino acids and coupling reagents (dic and hobt) were obtained from gl biochem (shanghai, china). all solvents were of analytical grade and used without further purification. each amino acid assembly was completed by using equiv of l-α-fmoc-protected amino acids and coupling reagents, and the ninhydrin colorimetric test after each coupling was carried out to ensure no detectable amino remaining. in the case that the peptide-bearing beads were colorized (positive) by ninhydrin test, the free amino group was then blocked by reacting with % acetic anhydride in dichloromethane (dcm) (v/v) for min. crude peptides without cys, met, arg, and trp residues were cleaved using a one-step tfa cleavage method under the following conditions: tfa ( . ml), deionized water ( μl), and triethyl silane ( μl). for peptides containing cys, met, arg, or trp residues, the following conditions were used: tfa ( . ml), thioanisole ( . ml), phenol ( . g), deionized water ( . ml), edt ( μl), and triethylsilane ( μl). tfa solution was treated with ice-cooled diethyl ether to precipitate the peptide. the crude peptide was washed twice by ice-cooled diethyl ether and dried in the n flow condition. in particular, peptide s − was synthesized on synphase pa ram d-series lanterns (loading μmol/lantern, mimotopes, raleigh, nc, usa). the lanterns were treated with % piperidine in dimethylformamide (dmf) for min for deprotection. the general coupling conditions for lanterns utilized a solution of % dmf and % dcm, with reagents at the following concentrations: aa, mm; hobt, mm; dic, mm. for each lantern a minimum of μl of the activated amino acid solution was required. alternatively, bromophenol blue ( μl/ml coupling solution, m) was used as an indicator to monitor the reaction. simultaneous side-chain deprotection and cleavage were carried out using . ml per lantern of a solution of . % trifluoroacetic acid (tfa)/ % thioanisole/ % anisole/ % water/ . % , -ethanedithiol for h. all crude peptides were purified by hplc to > % purity on a semipreparative rp c column (zorbax, sb-c , . × mm. agilent, colorado springs, co, usa) eluting at ml/min with a − % ch cn (buffer b) gradient in water (buffer a) containing . % tfa over min followed by a − % ch cn over min. pure peptides were identified by an shimadzu lc- at analytic hplc system with a c column ( . mm × mm, μm) and agilent lc-msd tof ( figure s ; s the branched lysine core (brch co-lys -(lys)-β-ala-conh ) scaffold was synthesized manually by standard fmoc chemistry protocols on . mmol ( mg) of rink-amide mbha resin (novabiochem, . mmol/g) as described above. n-α-fmoc-lys (fmoc) was used at the branch points. the fmoc-protected amino acids ( . equiv) were assembled by using . equiv of dic coupling reagent and hobt additive. the n-termini of the four branches of the lysine core were bromoacetylated on the resin with equiv of bromoacetic acid and dic in dmf for h. the bromoacetylated lysine core was cleaved from the resin after treatment with % h o/ % tfa for h. it was then precipitated with cold diethyl ether, lyophilized, and purified by hplc on a semipreparative rp c column (zorbax, sb-c , . × mm) eluting at ml/ (table s ). the average igg levels against peptides in each vaccine group were monitored by elisa on the day before injection or boost. igg level (b) against s − is an average of grouped macaques. the titer of anti-s − igg was greater than : in all relevant immunized monkey groups. a average gross lung pathology was determined by a standard in table s . b sars-cov-infected macrophages and alveolar epithelial cells in the vaccine groups were counted by using antipeptide antibodies. c quantitative viral burden analysis by quantitative rt-pcr (viral copies/mg lung tissue). maps were prepared for immunizations using a modified chemical ligation protocol. ligation was achieved by mixing the lysine core and peptide in μl of solvent at certain ph value. the reaction was monitored by an analytical rp c column (kromasil c , the nest group, inc., southborough, ma, usa) under uv nm wavelength. after it appeared complete, the reaction was quenched by adding . % tfa to adjust the ph to . . the final product was then purified by semipreparative hplc at a flow rate of ml/min. the ligated peptide was lyophilized and characterized by an agilent lc-msd tof. the preparative conditions of maps are listed in table s . detailed . more than antisera of sars patients and sera of healthy volunteers were involved in this study, which was conducted according to the guidelines for treatment of human subjects of the national institutes of health, usa. written informed consent was obtained from all study participants. the pepscan was carried out as described by hu et al. without major modification. the optical density was read at and nm in an optical density reader (labsystem, franklin, ma, usa). the cutoff value was determined as twice the average value of the negative controls. binding of mouse monoclonal antibodies to the sars-cov virion. the sars-cov pumc strain was diluted with coating buffer to × − tcid /ml (bicarbonate/carbonate coating buffer, . m, ph . ). the virus was then coated on well plates with μl ( × − of tcid ) of virus per well for h at °c. the coating buffer was removed from the wells, and μl of blocking buffer was added per well and incubated for h at °c ( % bsa in pbs, ph . ; gibco, carlsbad, ca, usa). the plates were washed twice with pbs (ph . ). the mouse monoclonal antibodies against sars-cov and control mab against hiv-p were diluted to . μg/μl in the blocking buffer, added to the wells separately, and incubated for h at °c. the plates were washed twice with pbs and the secondary antibody (hrp-conjugated goat anti-mouse igg, (genetex, irvine, ca, usa) diluted : in blocking buffer) was added to each well and incubated for h at °c. the secondary antibody was removed, the plate was washed three times with pbs, and then μl of tmb substrate solution (promega, madison, wi, usa) was added to each well. after incubation for min at room temperature, the absorbance of the test wells was read at nm (a ). analysis of neutralization using a neutral red staining (nrs)-based assay. mabs were serially diluted in dmem culture medium. human polyclonal antisera from convalescent sars patients were diluted to : (v/v). the sars-cov pumc strain was diluted with dmem to × − tcid . the nrs neutralization assay was performed in -well flat-bottom plates. the setup is similar to that of the cytopathic effect (cpe)-based neutralization assay. after incubation of μl ( × − tcid ) of virus with μl of antibodies or antisera in a total of μl of dmem medium per well for h at °c, vero e cells ( × cells in μl, from cell culture center of pumc) were added to each well. on day of infection, when > % of the cells showed cpe in the viral control wells (with dmem instead of antibody or antisera), the culture medium was removed from the test wells and μl of . % neutral red (sigma, st. louis, mo, usa) in dmem was added to each well. after incubation for h at °c, the neutral red medium was removed, the plates were washed twice with pbs (ph . ), and then μl of acidified alcohol ( % acetic acid in % ethanol) was added to each well. after incubation for min at room temperature, the absorbance of neutral red stained plates was read at nm (a ). percent neutralization was determined by the following formula: % neutralization = (a of antibody test wells − a of viral control wells)/(a of cell control wells − a of viral control wells) × . generation, purification, and measurement of the affinity of antipeptide monoclonal antibodies. ganptransgenic mice were originally established in a c bl/ strain as described previously. the mice were backcrossed with balb/c mice for at least eight generations and used for immunization to establish mabs. all mice were maintained in specific pathogen-free conditions by the center for animal resources and development (card), kumamoto university. as immunogens, four peptides of s − , s − , s − , and s − were synthesized and conjugated with keyhole limpet hemocyanin (klh) by operon biotechnologies (tokyo, japan). one hundred micrograms of peptide−klh emulsified in complete freund's adjuvant (cfa) was injected subcutaneously as a primary immunization and boosted after weeks with incomplete freund's adjuvant (ifa). sera of immunized mice were measured by elisa using plates coated with free peptides. the mice with highest serum ab titers were further immunized, and days later the spleen cells were obtained for polyethylene glycol-mediated cell fusion with mouse myeloma cell line x using standard procedures. the fused cells were selected with hat medium in microculture plates at a concentration of × cells/well in the presence of il- ( units/ml). the mabs binding to the immunizing peptide ags were detected with hrp-conjugated goat anti-mouse igg ab (zymed, south san francisco, ca, usa) with the substrate opd (wako chemicals, osaka, japan). the positive signals (a > . ) were selected, and hybridomas were cloned by a limiting dilution method, adapted to serum-free media, and purified by protein g-sepharose affinity chromatography (ge healthcare, buckinghamshire, uk). the purity of the antibodies was examined by sds-page and protein staining with coomassie brilliant blue. the heavy and light chain isotypes were determined using an isotyping kit (bd biosciences, franklin lakes, nj, usa). the affinity of the mabs for peptides was determined by the biacore assay. the on-and off-rate constants (k on and k off ) for binding of the mabs to s − , s − , s − , and s − peptides (table ) were determined using the biacore system (biacore international ab, uppsala, sweden). the carboxylmethylated dextran surface of the sensor chip was activated with n-ethyl-n′- ( -dietylaminopropyl) carbodiimide and n-hydroxysuccinimide (edc−ehs). each peptide was immobilized through the free thiol group of a cysteine residue that was deliberately placed at the n-terminus, by injection of μl of a μg/ml solution in mm -(n-morpholino)ethanesulfonic acid buffer (ph ) to the edc−nhs-activated surface that had been reacted with -( -pyridinyldithio)ethaneamine. the excess disulfide groups were deactivated by the addition of cysteine. the mabs were diluted in mm hepes (ph . ), mm nacl, . mm edta, and . % (v/v) biacore surfactant p and injected over the immobilized antigen at a flow rate of μl/min. the association was monitored by the increase in the refractive index of the sensor chip surface per unit time. the dissociation of the mabs was monitored after the end of the association phase with a flow rate of μl/min. kinetic rate constants were calculated from the collected data using the pharmacia kinetics evaluation software. the k on was determined by measuring the rate of binding to the antigen at different protein concentrations. macaque experiments. challenges with sars-cov. monkey challenges were performed in a bsl- facility. briefly, the animals were anesthetized with ketamine ( . ml/kg, im). the tcid − ( . ml/monkey) of the stock pumc strain virus was sprayed into the nasal cavity of the animals. pathologic examination. the lung tissues were sampled from the euthanized animals. organs were examined grossly, and photographs were taken to record the lung damage. samples of − g of lung tissues were collected from each lung and frozen at − °c. the remaining tissues were fixed in % formaldehyde for week in the bsl- laboratory. the tissues were then processed in the regular pathology laboratory by dehydration, embedding, and sectioning. for hematoxylin and eosin (h&e) staining, the sections were sequentially treated with xylene i, min; xylene ii, min; % alcohol, min; % alcohol, min; % alcohol, min; % alcohol, min; % alcohol, min; and pbs, min. the sections were then stained in hematoxylin for min; washed with tap water and double-distilled water for s, with pbs for min, with % alcohol for s, with eosin for s, with % alcohol for s, with % alcohol i for s, with % alcohol ii for s, with xylene i for min, with xylene ii for min, and with xylene iii for min; and mounted with mounting buffer (zhongshan inc., guangzhou, china). immunohistochemical analysis of the infected lung tissues. a mixture of the mouse monoclonal antibodies mab- e ( . μg/ml), mab- b ( . μg/ml), and mab- a ( . μg/ml) was used as primary antibody for the immunohistochemical analysis of the infected lung tissues. briefly, tissue sections were treated with the primary antibody at °c overnight, washed three times with pbs, incubated with hrp−goat-anti-mouse igg ( : , zhongshan inc., prc) at °c for min, and then washed three times with pbs. the dab substrate ( : ) was added and incubated at °c for min and then stained with hematoxylin for min. rt-pcr for analysis of the sars-cov burden. the tissues from cranial and caudal lobes of the left lung and cranial, middle, and caudal lobes of the right lung from each animal were sampled and pooled together, and the total rna was isolated from the lung tissues using trizol (invitrogen, usa). one hundred milligrams of lung tissue was mixed with ml of trizol extraction buffer and homogenized for s (homogenizer, ika, germany). the rna wan then extracted with chloroform and precipitated with isopropanol. rna was dissolved in μl of depc water, and the rna quality was verified by electrophoresis on agarose gel. for the reverse transcription, μg of total rna was reverse transcribed with a high-capacity cdna kit (recertaid first strand cdna synthesis kit (fermentas, glen burnie, md, usa) with random hexamer primers. the reaction was performed at °c for min and terminated at °c for min. two microliters of reverse transcription mixture was used as template. the primers for sars-cov were ′-atgaattac-caagtcaatggttac- ′ and ′-cataaccagtcggta-cagctac- ′. the pcr was normalized against monkey glyceraldehyde phosphate dehydrogenase (gapdh) (nm ). the gapdh primers were ′-tcaacagcg-acacccactc- ′ and ′-cttcctcttgtgctcttg-ctg- ′ (product size = bp). the pcr was performed with a pcr kit (taq dna polymerase, fermentas). the pcr products were detected by . % agarose gel electrophoresis. reaction conditions were as follows: × taq buffer ( μl), mm dntp ( μl), forward primer ( μm, . μl), reverse primer ( μm, . μl), taq dna polymerase ( . μl), mm mgcl ( μl), template dna (cdna, μl), h o ( . μl); step , °c for min; step , °c for s, °c for s, °c for s, for cycles, then °c for min. qrt-pcr for analysis of the sars-cov burden. the sars-cov fragment was prepared by rt-pcr. a bp fragment of sars-cov was amplified and separated by . % agarose electrophoresis. the dna fragment was recovered from the gel with a gel extraction kit (minelute gel extraction kit, qiagen stanford, valencia, ca, usa). the recovered dna was measured and adjusted to concentrations of × , × , × , × , × , and × copies/μl, which were used to generate a standard curve. this known copy number standard curve was used to calculate the copy number of sars-cov mrna in the infected lung tissues. rt-pcr for the infected samples was performed under the same conditions used for standard curves with the roche light cycler following the instructions for the sybr green realtime pcr master mix (toyobo, osaka, japan). the copy number of the virus was detected as follows. briefly, μl of the reverse transcription mixture from step was used as template. the primers for sars-cov were ′-atgaattac-caagtcaatggttac- ′ and ′-cataaccagtcggta-cagctac- ′. the reaction was continued for cycles under the conditions: × quantitect sybr green pcr ( μl), gorward primer ( μm, μl), teverse primer ( μm, μl), rnase-free water ( μl), yemplate dna (cdna, μl), total volume ( μl); step , °c for min; step , °c for s, °c for s, °c for s, for cycles and then move to the melt curve procedure. immunization of monkeys. the peptide and/or peptide mixtures were dissolved as μg/ml of each peptide in . % nacl and then formulated with an equal volume of freund's complete adjuvant (fca) (sigma, st. louis, mo, usa) according to the manufacturer's instructions. the injected dose of vaccine was . ml/ kg/monkey ( μg/ kg/monkey) given through intramuscular (im) injections at four sites on the arms and legs. rhesus monkeys were boosted at , , and weeks after the first injection with the same dose of peptides and volume of vaccine as for the first injection, but formulated in an equal volume of freund's incomplete adjuvant (ifa) (sigma). venous blood samples ( ml/each) were collected every days from each monkey. the sera were isolated by centrifugation at rpm and immediately stored at − °c. antipeptidic igg was detected by elisa. free peptides were diluted in carbonate buffer (ph . ) to a final concentration of μg/ml. one hundred microliters of the peptide solution was added to -well polystyrene high bind microplates (corning life science, santa clara, ca, usa) for coating overnight at °c . the wells of plates were then washed three times with pbs-tween buffer ( mm phosphate buffer, mm nacl, . % tween , ph . ) and subsequently blocked with % bsa in pbs-tween buffer for an additional h at °c. after three thorough washings of the wells with pbs-tween buffer, the antigen-coated microplates were incubated with diluted monkey sera at room temperature for h. after three washings with pbs-tween buffer, bound antibodies were detected with hrpconjugated goat anti-monkey igg ( : v/v, gene tex). after three washings with pbs-tween, a tmb substrate solution (promega, madison, wi, usa) was added and the color developed for min before the reaction was stopped with μl of . m h so . the absorbance at nm was read using a microtiter plate reader. macaques treated by mab - - . eighteen rhesus monkeys in six groups (table s , − years old) were examined according to the national microbiologic and parasite spf standard and were verified free of anti-sars antibody. mab - - was adjusted to concentrations of . and . mg/ml with . % nacl, respectively. a dose of . or . mg/kg of the antibody given by intravenous (iv) injection was used to treat the different groups. the same volume of . % nacl was used for the control group. the procedures were approved by the animal use and care committee of the institute of laboratory animal science, peking union medical college (animal welfare assurance no. a - , iacuc approval date september , ). statistical analysis. all data were analyzed using prism software (graphpad software). for neutralization assays and elisa, mean and standard deviation were used to determine significance. for qrt-pcr, geometric mean and standard deviation were used to determine virus burden. from sars to mers: years of research on highly pathogenic 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w.q.d., z.l.f., and k.k. equally contributed to this work. the authors declare no competing financial interest. we are grateful to professor carl f. nathan and dr. michael s. diamond for their kind discussion and manuscript preparation. we thank dr. baoxing fan for his serological evaluation of the large number of sars-cov convalescent antisera. the project described in this paper was supported by the national institute of allergy and infectious diseases (u ai ). the study was also partially supported for preparation of the monoclonal antibodies by contract research for mext for emerging and reemerging infectious diseases and promotion of fundamental studies in health sciences ( - ) of the nibio and national natural science foundation of china (no. ). the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. key: cord- -vdiaexbw authors: kshetri, nir title: china date: journal: the statesman&#x ;s yearbook doi: . / - - - - _ sha: doc_id: cord_uid: vdiaexbw nan upon soviet assistance for economic development. a soviet-style five-year plan was put into action in , but the relationship with moscow was already showing signs of strain. by the end of the s the soviet union and china were rivals, spurring the chinese arms race. chinese research into atomic weapons culminated in the testing of the first chinese atomic bomb in . mao introduced rapid collectivization of farms in . the plan was not met with universal approval in the communist party but its implementation demonstrated mao's authority over the fortunes of the nation. in he launched the doctrine of letting a 'hundred flowers bloom', encouraging intellectual debate. however, the new freedoms took a turn mao did not expect and led to the questioning of the role of the party. strict controls were reimposed and free-thinkers were sent to work in the countryside to be 're-educated'. in may mao launched another ill-fated policy, the great leap forward. to promote rapid industrialization and socialism, the collectives were reorganized into larger units. neither the resources nor trained personnel were available for this huge task. backyard blast furnaces were set up to increase production of iron and steel. the great leap forward was a disaster. it is believed that m. died from famine. soviet advice against the project was ignored and a breakdown in relations with moscow came in , when soviet assistance was withdrawn. a rapprochement with the united states was achieved in the early s. having published his 'thoughts' in the 'little red book' (as it is known in the west) in , mao set the cultural revolution in motion. militant students were organized into groups of red guards to attack the party hierarchy. anyone perceived to lack enthusiasm for mao zedong thought was denounced. thousands died as the students lost control and the army was eventually called in to restore order. after mao's death in the gang of four, led by mao's widow jiang qing, attempted to seize power. these hard-liners were denounced and arrested. china effectively came under the control of deng xiaoping. deng pursued economic reform. the country was opened to western investment. special economic zones and 'open cities' were designated and private enterprise gradually returned. improved standards of living and a thriving economy increased expectations for civil liberties. the demand for political change climaxed in demonstrations by workers and students in april , following the funeral of communist party leader hu yaobang. in beijing where demonstrators peacefully occupied tiananmen square, they were evicted by the military who opened fire, killing more than , . hard-liners took control of the government, and martial law was imposed from may to jan. . since the leadership has concentrated on economic development. hong kong was returned to china from british rule in (for the background, see page ) and macao from portuguese rule in . the late s saw a cautious extension of civil liberties but chinese citizens are still denied most basic political rights. beijing was chosen for the olympic games. china's treatment of tibet came under the international spotlight in the build-up to the games, following violent protests in tibet's capital city, lhasa. the arrest by japan of a chinese trawler in disputed waters in marked the beginning of heightened tensions between the two nations in the east and south china seas. in china became the world's second largest national economy. in nov. the communist party congress selected xi jinping to succeed hu jintao as president from march . in sept. that year, former leadership hopeful bo xilai received a life sentence for corruption in one of china's highest-profile trials in decades. in oct. the government announced the end of the country's one-child policy. a month later, the presidents of china and taiwan met for talks-the first time that leaders from the respective territories had met since . on the economic front, gdp growth in was at its lowest level for a quarter of a century. china is bounded in the north by russia and mongolia; east by north korea, the yellow sea and the east china sea, with hong kong and macao as enclaves on the southeast coast; south by vietnam, laos, myanmar, india, bhutan and nepal; west by india, pakistan, afghanistan, tajikistan, kyrgyzstan and kazakhstan. the total area (including taiwan, hong kong and macao) is estimated at , , sq. km ( , , sq. miles). a law of feb. claimed the spratly, paracel and diaoyutasi islands. an agreement of sept. at prime ministerial level settled sino-indian border disputes which had first emerged in the war of . china's sixth national census was held on nov. . the total population of the provinces, autonomous regions and municipalities and of servicemen on the mainland was , , , ( , , females, representing Á %); density, per sq. km. china's population in represented % of the world's total population. the population rose by , , (or Á %) since the census in . there were , , urban residents, accounting for Á % of the population; compared to the census, the proportion of urban residents rose by Á % (reflecting the increasing migration from the countryside to towns and cities since the economy was opened up in the late s). population estimate, dec. : , , , . china has a fast-growing ageing population. whereas in only Á % of the population was aged or over and by this had increased to Á %, by it is expected to rise to Á %. long-term projections suggest that in as much as Á % of the population will be or older. the population is expected to peak at Á m. around and then begin to decline to such an extent that by around it will be back to the level. china is set to lose its status as the world's most populous country to india in about . the un gives a projected population for of , Á m. regulations restricting married couples to a single child, a policy enforced by compulsory abortions and economic sanctions, were widely ignored, and it was admitted in that the population target of , m. by would have to be revised to , m. from peasant couples were permitted a second child after four years if the first born was a girl, a measure to combat infanticide. in china started to implement a more widespread gradual relaxation of the one-child policy. in dec. the standing committee of the national people's congress (npc) approved a resolution allowing couples to have two children if either parent was an only child. the one-child policy was formally abandoned altogether from jan. . an estimated m. persons of chinese origin lived abroad in . a number of widely divergent varieties of chinese are spoken. the official 'modern standard chinese' is based on the dialect of north china. mandarin in one form or another is spoken by m. people in china, or around % of the population of mainland china. the wu language and its dialects has some m. native speakers and cantonese m. around m. people in china cannot speak mandarin. the ideographic writing system of 'characters' is uniform throughout the country, and has undergone systematic simplification. in a phonetic alphabet (pinyin) was devised to transcribe the characters, and in this was officially adopted for use in all texts in the roman alphabet. the previous transcription scheme (wade) is still used in taiwan and hong kong. mainland china is administratively divided into provinces, five autonomous regions (originally entirely or largely inhabited by ethnic minorities, though in some regions now outnumbered by han immigrants) and four government-controlled municipalities. these are in turn divided into prefectures, cities (of which are at prefecture level and at county level), , counties and urban districts. population of largest cities in : shanghai, Á m.; beijing (peking), Á m.; shenzhen, Á m.; guangzhou (canton), Á m.; tianjin, Á m.; dongguan, Á m.; wuhan, Á m.; foshan, Á m.; chengdu, Á m.; chongqing, Á m.; nanjing, Á m.; shenyang, Á m.; xian, Á m.; hangzhou, Á m.; haerbin, Á m.; suzhou, Á m.; dalian, Á m.; zhengzhou, Á m.; shantou, Á m.; jinan, Á m.; qingdao, Á m.; changchun, Á m.; kunming, Á m.; changsha, Á m.; taiyuan, Á m.; xiamen, Á m.; hefei, Á m.; urumqi (wulumuqi), Á m.; fuzhou, Á m.; shijiazhuang, Á m.; wuxi, Á m.; zhongshan, Á m.; wenzhou, Á m.; nanning, Á m.; ningbo, Á m.; guiyang, Á m.; lanzhou, Á m.; zibo, Á m.; changzhou, Á m.; nanchang, Á m.; xuzhou, Á m.; tangshan, Á m. china has ethnic groups. according to the census , , , people ( Á %) were of han nationality and , , ( Á %) were from national minorities (including zhuang, manchu, hui, miao, uighur, yi, tujia, mongolian and tibetan) . compared with the census, the han population increased by , , ( Á %), while the ethnic minorities increased by , , ( Á %). non-han populations predominate in the autonomous regions, most notably in tibet where tibetans account for around % of the population. chang tibet relations between tibet and china's central government have fluctuated over the question of tibetan independence. the borders were opened for trade with neighbouring countries in . in a buddhist seminary opened in lhasa, the regional capital, with students, and monasteries and shrines have since been renovated and reopened. there were some , monks and nuns in . in tibetan was reinstated as a 'major official language', competence in which is required of all administrative officials. in the then chinese president, jiang zemin, said he was prepared to meet the dalai lama provided he acknowledged chinese sovereignty over tibet and taiwan. in sept. direct contact between the exiled government and china was re-established after a nine-year gap. however, in march anti-chinese protests in lhasa ended in violence, with dozens reportedly killed by the chinese authorities, and in oct. that year the dalai lama stated he had lost hope of reaching agreement with china on tibet's status. in april he announced his retirement from active politics in favour of lobsang sangay, who had been elected to lead the tibetan government-in-exile. in july , shortly after us president obama had received the dalai lama in washington, china's soon-to-be president, xi jinping, pledged to 'smash' attempts to destabilize tibet. by feb. there had been over reported cases of self-immolation by tibetans protesting against chinese rule. the estimated population of tibet at the end of had risen to Á m. from Á m. at the census. the average population density was Á persons per sq. km in , although the majority of residents live in the southern and eastern parts of the region. birth rate (per , ), , Á ; death rate, Á . population of lhasa, the capital, in was , . about % of the population is engaged in the dominant industries of farming and animal husbandry. in the total sown area was , ha. output in : total grain crops, , tonnes; vegetables, , tonnes. in there were Á m. sheep and goats and Á m. cattle and yaks. tibet has over , mineral ore fields. mining, particularly of copper and gold, has expanded rapidly since when the railway came to tibet. cement production, : Á m. tonnes. electricity consumption totalled Á bn. kwh in . in there were , km of roads ( , km in most of china has a temperate climate but, with such a large country, extending far inland and embracing a wide range of latitude as well as containing large areas at high altitude, many parts experience extremes of climate, especially in winter. most rain falls during the summer, from may to sept., though amounts decrease inland. monthly average temperatures and annual rainfall ( ): beijing (peking), jan. Á f (- Á c), july Á f ( Á c). annual rainfall Á " ( mm). chongqing, jan. Á f ( Á c), july Á f ( Á c). annual rainfall Á " ( , mm). shanghai, jan. Á f ( Á c), july Á f ( Á c). annual rainfall Á " ( , mm). tianjin, jan. Á f (- Á c), july Á f ( Á c). annual rainfall Á " ( mm). on -in , and (currently in force) . the latter was partially amended in , , , and , endorsing the principles of a socialist market economy and of private ownership. the unicameral national people's congress is the highest organ of state power. usually meeting for one session a year, it can amend the constitution and nominally elects and has power to remove from office the highest officers of state. there are a maximum of , members of the congress, who are elected to serve five-year terms by municipal, regional and provincial people's congresses. - yang shangkun - jiang zemin - hu jintao -xi jinping prime ministers since . - zhou enlai - hua guofeng - zhao ziyang - li peng - zhu rongji wen jiabao -li keqiang elections of delegates to the th national people's congress were held between oct. and feb. by municipal, regional and provincial people's congresses. at its annual session in march the congress re-elected xi jinping as president and elected wang qishan as vice-president. president xi jinping succeeded hu jintao as president in march at the th national people's congress. tipped for the role since his appointment as secretary general of the chinese communist party (ccp) and chairman of the central military commission in nov. , xi pursued a strong style of authoritarian rule at home and a proactive and muscular foreign policy in his first term. in oct. the ccp gave him the title of 'core' leader, a significant honorific bracketing him with mao zedong and deng xiaoping among previous party figures although conferring no absolute powers. then, at its five-yearly congress in oct. , the party voted to enshrine his name and ideology in the chinese state constitution. he was subsequently re-elected in march and parliament also voted to abolish presidential term limits in a major shift from precedent. xi jinping was born on june in beijing, the son of one of the first generation of communist leaders. he joined the ccp in and, after graduating from tsinghua university in with a degree in chemical engineering, he became secretary to the vice-premier and secretary-general of the central military commission. xi became the zhengding county committee deputy secretary in hebei province in and the following year was promoted to secretary. in he was made deputy mayor of xiamen city, fujian province. having undertaken various party roles in the province, he became deputy governor of fujian in and governor a year later. in he moved to zhejiang province and made his first inroads into national politics when he was named a member of the th central committee. from - he was party secretary of fujian, overseeing economic growth averaging % a year and earning a reputation as an opponent of corruption. in march xi transferred to shanghai to take the role of party secretary following the dismissal of the incumbent on corruption charges. his appointment to such an important regional post was seen as a vote of confidence from the central government and he became a member of the politburo standing committee at the th party congress in oct. . he was also made a high-ranking member of the central secretariat. on march he was elected vice-president at the th national people's congress and took on a number of high profile portfolios including the presidency of the central party school. he was also beijing's senior representative for hong kong and ahead of his presidency, xi said little about his policy ambitions. there was hope abroad and at home that he would champion political and social reform and attempt to deal with corruption and a widening wealth gap between rich and poor and between urban and rural communities. he also faced the conundrum of how to provide adequate health care to a rapidly ageing population. in jan. the prospect of greater transparency and accountability under his leadership was undermined when the authorities began criminal proceedings against anti-corruption campaigners calling for public disclosure of officials' assets. this coincided with a report by a us investigative organization claiming that relatives of some of china's top political and military figures, including xi's brother-in-law, held secret offshore financial holdings. in other social and political affairs the ccp announced plans in nov. to ease china's one-child policy (which was subsequently abandoned following an announcement in oct. , with effect from ) and to abolish the system of 're-education through labour' camps, while a party plenum called-for the first time-for markets to play a 'decisive' role in the allocation of resources. meanwhile, in xi was confronted by domestic political opposition in the form of militant attacks by ethnic uighur separatists from xinjiang region and, from sept. that year, by widespread pro-democracy and autonomy protests in hong kong. on the economic front, china's previously frenetic annual rate of growth slowed markedly in , reflecting a slump in factory production and concerns over depressed oil prices, and again in to its lowest since . it also heralded severe stock market turbulence into despite emergency government measures, which had negative reverberations throughout the world economy. nevertheless, recognizing china's rise as a global economic power, the imf in nov. voted to add the yuan as the fifth member of its special drawing rights (sdr) currency basket alongside the us dollar, japanese yen, british pound and the euro. in foreign affairs, regional concerns over china's territorial and military intentions were raised in nov. by the government's declaration of a new 'air defence identification zone' over a swathe of the east china sea including disputed islands claimed by japan and south korea. there has also been friction, regionally and with the usa, over china's sovereignty claims and land reclamation operations on islands in the south china sea, although in july an international legal tribunal ruled in favour of a challenge by the philippines to china's sovereignty assertions-a verdict beijing vowed to ignore. further afield, xi has meanwhile undertaken numerous official visits abroad, as well as attending multilateral forums, for diplomatic, trading and investment purposes. and, while in singapore in nov. , xi and president ma ying-jeou of taiwan held the first direct talks between leaders of the two estranged governments since their split in . more recently, in xi aimed to extend china's economic and military co-operation with russia and also sought to defuse tensions with india over a simmering border dispute at an informal summit with prime minister narendra modi. meanwhile, friction with the usa escalated markedly over disputed trading and commercial practices, with both countries imposing punitive trade tariffs. li keqiang took office as premier of the state council, a role equivalent to prime minister, in march , succeeding wen jiabao. he was re-elected in march . li keqiang was born on july in dingyuan county, anhui province. following graduation from high school in , he joined the ccp and in he graduated in law from peking university, serving as head of the students' federation from - . he went on to earn a master's degree and doctorate in economics and headed the university's communist youth league of china (cylc) committee. over the following two decades he rose through the cylc ranks, joining the secretariat of its central committee in the s and serving as its first secretary in the s. at this time he built up his power base and forged close ties with hu jintao, a fellow cylc committee member and future chinese president. in li became deputy party secretary for henan province and a year later was appointed henan's governor. in dec. he was named party secretary for liaoning province where he spearheaded a major coastal infrastructure project, the ' points and one line' highway development. in this template was adopted at the national level to rejuvenate industrial northeast china. he also oversaw the rehousing of Á m. shanty-town residents into new apartment blocks over a three-year period. li advanced to national level politics when he was elected to the politburo standing committee in oct. . he was appointed vice-premier of the state council in march , leading a medical reform programme aimed at creating an accessible public health care service. he also chaired an affordable housing programme and introduced tax reform plans. in nov. li was re-elected as a member of the politburo standing committee and on march became premier of the state council at the th national people's congress. regarded as the steward of the chinese economy, li has aimed to focus on securing china's long-term expansion and on the further provision of basic national health care, affordable housing, employment growth, regional development and cleaner energy. however, global confidence in china's economy has been shaken since as the country's growth momentum has slowed amid apparent policy differences and blunders, prompting rumours that li was being increasingly sidelined in the governing hierarchy. he was nevertheless re-elected to the politburo standing committee in oct. and to the premiership in march . having sought assurances that a recently revised us-mexico-canada trade agreement would not stop canada from signing deals with other countries, li met with canadian prime minister justin trudeau in nov. at an international summit in singapore with the aim of pursuing negotiations on a bilateral free trade pact. the chinese president is chairman of the state and party's military commissions. china is divided into seven military regions. the military commander also commands the air, naval and civilian militia forces assigned to each region. china's armed forces (pla: 'people's liberation army'), totalling nearly Á m. in including the paramilitary people's armed police (pap) and Á m. excluding the pap, are the largest of any country. however, active armed personnel numbers have halved since . moreover, in president xi laid out plans to reform the army structure-replacing an organization based on seven regions with one based on five 'theatre commands'-and reduce the number of military personnel by a further , . conscription is compulsory, but for organizational reasons, is selective: only some % of potential recruits are called up. service is for two years. a military academy to train senior officers in modern warfare was established in . defence expenditure in was us$ , m. (equivalent to us$ per capita). china's military spending more than trebled between and . defence spending in represented Á % of gdp, although the share has actually declined since . only the usa spent more on defence in , but china's defence expenditure totalled around a fifth of that of the usa. in march it was announced that the defence budget would rise by Á % to us$ bn. following increases of Á %, Á % and Á % in the previous three years. china is the world's third largest exporter of arms after the usa and russia, with Á % of the global major weapons total over the period - . in the period - it had only been the eighth largest exporter. as at may china had , personnel serving in un peacekeeping operations (the largest contingent of any of the five permanent members of the un security council and more than the other four combined). having carried out its first test in , there have been tests in all at lop nur, in xinjiang (the last in ). the nuclear arsenal consisted of approximately operational warheads in jan. according to the stockholm international peace research institute. china has been helping pakistan with its nuclear efforts. the army (the pla groundforce) is divided into main and local forces. main forces, administered by the seven military regions in which they are stationed, but commanded by the ministry of defence, are available for operation anywhere and are better equipped. local forces concentrate on the defence of their own regions. ground forces are divided into infantry, armour, artillery, air defence, aviation, engineering, chemical defence and communications service arms. there are also specialized units for electronic counter-measures, reconnaissance and mapping. in there were group armies covering seven military regions. they included: armoured divisions and brigades; mechanized infantry divisions, brigades and regiments; motorized infantry divisions and brigades; nine special operations units; artillery divisions and brigades; amphibious brigades and divisions; two mountain brigades; aviation brigades and regiments; and two guard divisions. total strength in was Á m. including some , conscripts. reserve forces are undergoing major reorganization on a provincial basis but are estimated to number some , . there is a paramilitary people's armed police force estimated at , under pla command. in nov. the naval arm of the pla included submarines, of which three were strategic (two jin-class and one xia-class) and tactical. by mid- two more jin-class nuclear-powered ballistic missile submarines had entered service. surface combatant forces in nov. included destroyers and frigates. sea trials of china's first aircraft carrier, liaoning (a former soviet warship purchased from ukraine), began in aug. . it entered service in sept. and was initially only used for training before being declared 'combat ready' in nov. . work on china's first domestically-built aircraft carrier began in . it was launched in april and is expected to be operational by . there is a land-based naval air force of about combat-capable aircraft, primarily for defensive and anti-submarine service. the force includes h- strategic bombers and jh- fighters. the naval arm is split into a north sea fleet, an east sea fleet and a south sea fleet. in naval personnel were estimated at , , including , in the naval air force and , conscripts. the pla air force organizes its command through seven military region air forces. the air force has an estimated , combat-capable aircraft. equipment includes j- (mig- ) interceptors (known in the west as 'fishbed'), h- chinese-built copies of tu- strategic bombers, q- fighter-bombers (evolved from the mig- and known in the west as 'fantan'), su- fighters supplied by russia (known in the west as 'flanker'), j- chinese-designed and produced fighters (known in the west as 'firebird') and j- locally-developed fighters (known in the west as 'finback'). total strength ( ) was , . in agriculture accounted for Á % of gdp, industry Á % and services Á %. industry was the largest contributor until , while services only overtook agriculture as the second largest sector in . in the late s agriculture was the largest contributor towards gdp. china's economic performance has been marked by high rates of growth for over three decades. annual gdp increases in the early s consistently exceeded % until the global financial crisis. china also holds the world's largest foreign exchange reserves, at more than us$ Á trn. in april , although they have been falling since as the central bank strives to boost the currency in the face of large capital outflows. it is among the top recipients of foreign direct investment (fdi) and is the world's largest producer and consumer of coal. in china made the transition from net receiver of foreign aid to net donor and has established itself as a key player in africa's economic development, becoming the largest export partner of sub-saharan africa in . according to the world bank, china's cumulative fdi stock in africa totalled nearly us$ Á bn. in , up from us$ Á bn. in . in china overtook japan to become the world's second largest economy after the usa. new sectors like e-commerce and online financial services are gaining momentum in an economy long dominated by export-oriented sectors. rising trade tensions between the usa and china, however, pose a threat to longer-term prospects. the first steps from a centrally-planned towards a more market-oriented economy were taken by deng xiaoping in the late s. he opened the economy to foreign trade and investment, decentralized industrial management and allowed private sector development. in china became a member of the world trade organization, establishing trade relations with many countries. private entrepreneurs and foreign investors have played an important role in developing the manufacturing sector, china's principal growth engine. even before the economy was heavily skewed towards manufacturing, but following the market-oriented transition output increased significantly. during this period there was a structural shift away from large state-owned enterprises (soes), although these still remain an important part of the economy. between and the government oversaw reform of soes, with many poorly performing businesses privatized or liquidated. stronger firms were restructured and often listed on the stock market. many more recent enterprises are labour-intensive as distinct from the capitalintensive soes. growth has been fuelled by low added value and labourintensive exports. however, chinese firms are predicted to become increasingly competitive with higher added value producers, such as south korea. although the global financial crisis reduced the rate of growth and inbound fdi, china's recovery was among the earliest. gdp growth averaged Á % in the second quarter of , up from a two-decade low of Á % in the first quarter of that year. fdi also recovered rapidly, averaging Á % of gdp annually between and . growth was rooted in a stimulus package of trn. yuan (us$ bn. or % of gdp), including fiscal spending and interest rate cuts, as well as an expansionary monetary policy. central government committed Á trn. yuan, with the rest coming from local government, banks and soes. although exports declined by around % in , other countries fared worse and china's share of world exports increased to nearly Á % in (up from % in ), making it the world's largest merchandise provider. gdp growth in stood at more than % but moderated between and , reflecting the global economic slowdown and diminishing dividends from past reforms. in aug. a devaluation of the yuan sent the shanghai stock exchange plummeting by nearly %, which was swiftly followed by a surge in capital outflows. the stock market meltdown lasted until feb. , with trading halted altogether for two days in jan. that year. nonetheless, the shanghai exchange subsequently began a recovery and had stabilized (at around , points) by feb. . despite stock market turbulence, the property market, which constitutes a quarter of china's gdp and is vital to the banking sector (as it accounts for a substantial amount of its collateral), remained buoyant. gdp growth declined to Á % in (the slowest rate in years) and fell again to Á % in as china attempted to reduce its reliance on exports, increase domestic consumption and develop its service sector. however, china remains one of the fastest growing major economies in the world, recording expansions of Á % and Á % in and , respectively, driven by exports and robust domestic household consumption. in may , for the first time since , the credit rating agency moody's downgraded china's sovereign rating. inflation was Á % in , up from Á % in , while public debt was measured by the government at Á % of gdp (although there is wide-held suspicion that the real figure is significantly higher). corporate debt reached % of gdp in , with household debt-although still low-rising by % of gdp over the preceding five years. president xi jinping has targeted spending cuts and aims to curb loans to bloated soes. rapid economic advance has brought with it a number of challenges that threaten future growth. notably, china's cost advantage has been undermined in recent years by rising wages and transportation costs, as well as weak global demand. other concerns include rising property costs, high levels of local government debt, lack of enforcement of intellectual property rights, endemic corruption at government level and credit and investment dependence, while total social financing-a broad measure of total credit-increased by % of gdp between and early . the stimulus package implemented by the government to boost growth increased total debt levels to more than double the value of gdp in . according to the imf, an increase in consumer demand and a reduced dependence on exports and investment are keys to achieving stable long-term economic expansion. china's th five-year plan (covering - ) aims to promote domestic consumption and to support innovation and entrepreneurship within a framework of balanced and sustainable development. efforts to promote domestic consumption have seen exports' share of gdp falling from % in to Á % in and a lower investment contribution to gdp. trade frictions are predicted to disproportionately affect smaller firms that are less able to squeeze profit margins to accommodate tariff hikes, as well as those geographical regions most reliant on exports. the continued decline in commodity prices coupled with china's economic slowdown has had knock-on effects for commodity-exporting nations, such as brazil, indonesia and argentina, given that china consumes about half of the world's steel, aluminium and nickel. inefficient production and outmoded equipment have meanwhile led to significant environmental problems, especially in the north of the country. air pollution, soil erosion and a declining water table are of particular concern. china has become the world's largest consumer of coal and second largest consumer of oil after the usa. the government aims to diversify its energy sources, relying less on coal and more on nuclear and alternative energy sources. there has been heavy investment in hydro-power, including the three gorges dam. since , m. people have been lifted out of poverty, yet china still has the second largest number of poor in the world after india. the world bank estimates that Á m. people lived below the national poverty line at the end of (equivalent to income less than us$ per day), located mainly in remote and resource-poor regions and particularly in the west and the interior. nonetheless, some progress has been made, with Á % of the rural population living below the poverty line in compared to Á % in . a large gap remains between living standards of the urban and rural communities, between urban zones on the chinese coast and the interior and western parts of the country, and between the urban middle classes and those who have not been able to profit from the growth of recent decades. china also faces the growing burden of an ageing population. those aged and over accounted for Á % of the total population in , up from Á % in . the currency is called renminbi (i.e. people's currency). the unit of currency is the yuan (cny) which is divided into ten jiao, the jiao being divided into ten fen. the yuan was floated to reflect market forces on jan. while remaining state-controlled. for years the people's bank of china maintained the yuan at about Á to the us dollar, allowing it to fluctuate but only by a fraction of % in closely supervised trading. in july it was revalued and pegged against a 'market basket' of currencies the central parities of which were determined every night. in july , after three years of sharp appreciation, it was repegged at around Á yuan to the dollar, leading to claims from some international observers that it was being kept unfairly low to boost exports. in june the government announced that the yuan would be allowed to move freely against the dollar as long as a rise or fall does not exceed Á % within a single day. in aug. the yuan was devalued by a total of Á % on three consecutive days. in aug. total money supply was , Á bn. yuan, gold reserves were Á m. troy oz and foreign exchange reserves us$ , Á bn. (us$ Á bn. in ). china's reserves are the highest of any country, having overtaken those of japan in . inflation rates (based on imf statistics): Á % - Á % Á % Á % Á % Á % Á % Á % Á % Á % china's economy overheated in the early s, leading to inflation rates of Á % in , Á % in and Á % in of the total revenues in central government accounted for , Á bn. yuan and local governments , Á bn. yuan. tax revenues came to , Á bn. yuan in (including domestic vat , Á bn. yuan and corporate income tax , Á bn. yuan) and non-tax revenues , Á bn. yuan. of the total expenditure in central government accounted for , Á bn. yuan and local governments , Á bn. yuan. the leading items of expenditure in were education ( , Á bn. yuan) and social safety net and employment effort ( , Á bn. yuan). the standard rate of vat is %. performance gdp totalled us$ , Á bn. in , the second highest behind the usa. china's share of world gdp has risen from % in to % in . it replaced japan as the second largest economy in . it is forecast that around china will overtake the usa to become the world's largest economy. as recently as the us economy was around eight times larger than china's. real gdp growth rates (based on imf statistics): supervising the country's banks and other deposit-taking financial institutions from the central bank. legislation in permitted the establishment of commercial banks; credit co-operatives may be transformed into banks, mainly to provide credit to small businesses. there were , rural credit co-operatives at the end of . insurance is handled by the people's insurance company. the industrial and commercial bank of china is the world's largest bank by assets (us$ , bn. as at dec. ). savings deposits in various forms in all banking institutions totalled , Á bn. yuan in ; loans amounted to , Á bn. yuan. there are stock exchanges in the shenzhen special economic zone and in shanghai. a securities trading system linking six cities (securities automated quotations system) was inaugurated in for trading in government bonds. china received a record us$ Á bn. worth of foreign direct investment in , up from us$ Á bn. in . external debt totalled us$ , m. in (up from us$ , m. in ) and represented Á % of gni. china's carbon dioxide emissions from the consumption of energy in accounted for Á % of the world total (making it the biggest emissions producer, having overtaken the usa in ) and were equivalent to Á tonnes per capita (up from Á tonnes per capita in ). carbon dioxide emissions have more than doubled since . an environmental performance index compiled in ranked china th of countries, with Á %. the index examined various factors in nine areas-agriculture, air quality, biodiversity and habitat, climate and energy, fisheries, forests, health impacts, water and sanitation, and water resources. pollution is estimated to cost china about % of gdp annually. installed generating capacity in was an estimated , m. kw, compared with m. kw in . in electricity output was , , gwh, up from , , gwh in . consumption per capita was , kwh in . rapidly increasing demand has meant that more than half of china's provinces have had to ration power. sources of electricity in as percentage of total production: thermal, Á %; hydro-electric power, Á %; wind. Á % (china is one of the world's largest producers of wind power); and nuclear, Á %. in there were nuclear reactors in use and under construction. generating electricity is not centralized; local units range between and mw of output. in dec. china formally broke up its state power monopoly, creating instead five generating and two transmission firms. the three gorges dam project on the yangtze river was launched in and is intended to produce abundant hydro-electricity (as well as helping flood control). the first three , -kw generators in service at the project's hydro-power station began commercial operation in july . the original specification was completed in oct. , although six more generators have been added in the meantime (bringing the total to ). the final two generators become operational in july , giving the dam an overall capacity of Á gw. china surpassed germany in terms of solar generating capacity in , with Á gw at the end of the year. on-shore oil reserves are found mainly in the northeast (particularly the daqing and liaohe fields) and northwest. there are off-shore fields in the continental shelves of east china. oil production was a record Á m. tonnes in and was Á m. tonnes in . china is the second largest consumer of oil after the usa. ever-growing demand has meant that increasing amounts of oil are having to be imported. a -km pipeline from skovorodino in russia to daqing in the northeast of china was inaugurated in jan. , allowing china to increase significantly its imports of oil from the world's second largest producer. the , -km turkmenistan-china gas pipeline, bringing natural gas to xinjiang in china via kazakhstan and uzbekistan, was inaugurated in dec. . this connects with china's second west-east gas pipeline. only the usa imports more oil. domestic production now accounts for only % of consumption, compared to nearly % in . proven reserves in were Á bn. bbls. the largest natural gas reserves are located in the western and northcentral regions. production was a record Á bn. cu. metres in -up from Á bn. cu. metres in -with proven reserves of Á trn. cu. metres in . china is the second largest producer of wind power after the usa, with Á bn. kwh in . in total installed capacity amounted to , mw, the highest of any country and Á % of the world total. china is one of the world's leading mineral producing and consuming countries. recoverable deposits of coal in totalled Á bn. tonnes, mainly distributed in north china (particularly shanxi province and the inner mongolia autonomous region). coal production was , m. tonnes in . annual coal production has increased every year since . growing domestic demand nonetheless meant that china became a net importer of coal in . iron ore reserves were Á bn. tonnes in . deposits are abundant in the anthracite field of shanxi, in hebei and in shandong, and are found in conjunction with coal and worked in the northeast. production in was , m. tonnes, making china the world's largest iron ore producer. it is also the largest consumer, at around % of the global total in . tin ore is plentiful in yunnan, where the tin-mining industry has long existed. tin production was , tonnes in . china is a major producer of wolfram (tungsten ore). there is mining of wolfram in hunan, guangdong and yunnan. output of other minerals (in , tonnes) in : salt, , ; bauxite, , ; aluminium, , ; zinc, , ; lead, , ; copper, , . there are also reserves of diamond, nickel, barite, bismuth, graphite, gypsum, mercury, molybdenum, silver, salt, phosphate ore and sylvite. gold production, : tonnes. china surpassed south africa as the world's leading gold producer in , since when its output has increased every year. agriculture accounted for approximately % of gdp in , compared to over % in at the time of the birth of the people's republic of china and over % in . in sown areas for major crops were (in m. ha.): corn, Á ; rice, Á ; wheat, Á ; soybeans, Á ; tubers, Á ; rapeseed, Á . intensive agriculture and horticulture have been practised for millennia. present-day policy aims to avert the traditional threats from floods and droughts by soil conservancy, afforestation, irrigation and drainage projects, and to increase the 'high stable yields' areas by introducing fertilizers, pesticides and improved crops. in aug. more than m. ha., notably in the yangtze valley, were under water as china experienced its worst flooding since the s. the flood season claimed over , lives. 'township and village enterprises' in agriculture comprise enterprises previously run by the communes of the maoist era, co-operatives run by rural labourers and individual firms of a certain size. there were , state farms in with Á m. employees. net per capita annual income of rural households, : , yuan. in there were an estimated Á m. ha. of arable land and Á m. ha. of permanent cropland; Á m. ha. were equipped for irrigation. there were Á m. large/medium-sized tractors in and Á m. small tractors. china is the world's leading producer of a number of agricultural crops. production of major products (in m. tonnes), (unless otherwise indicated): corn, Á ; rice, Á ; wheat, Á ; sugarcane, Á ; potatoes ( estimate livestock, (unless otherwise indicated): pigs, , , ; sheep, , , ; goats, , , ; cattle and buffaloes, , , ; horses, , , ; chickens ( estimate), Á bn.; ducks ( estimate), m. china has more pigs, goats, sheep, horses and chickens than any other country. it is also home to nearly two-thirds of the world's ducks. meat production in was estimated at Á m. tonnes; milk, Á m. tonnes; eggs, Á m. tonnes; honey, , tonnes. china is the world's leading producer of meat, eggs and honey. gale, fred, (ed.) china's food and agriculture: issues for the st century. powell, s. g., agricultural reform in china: from communes to commodity economy, - . forestry in the area under forests was Á m. ha., or % of the total land area. the average annual increase in forest cover of , , ha. between and was the highest of any country in the world. total roundwood production in was Á m. cu. metres, making china the world's third largest timber producer ( Á % of the world total in ). it is the highest consumer of roundwood; timber consumption in totalled Á m. cu. metres. it is also the world's leading importer of roundwood, accounting for Á % of world timber imports in . total catch, : , , tonnes, of which , , tonnes were from marine waters. china's annual catch is the largest in the world, and currently accounts for approximately % of the world total. in the annual catch had been just Á m. tonnes. china's aquaculture production is also the largest in the world, at , , tonnes in . imports of fishery commodities in were valued at us$ , m. (the third highest behind japan and the usa); exports were the most of any country, at us$ , m. china's fishery commodities exports in represented approximately % of the global total. in , the first phase of the south-to-north water diversion project opened, with a second phase opening a year later. the scheme, which by provided beijing with a third of its total supply, was estimated to have cost almost us$ bn., with a third phase still to be completed. the leading companies by market capitalization in china in march were alibaba, an e-commerce and data company (us$ Á bn.); tencent holdings, an investment holding company (us$ Á bn.); and icbc, the world's largest commercial bank (us$ Á bn.). in nov. petrochina was briefly the world's largest company after its flotation on the shanghai stock market, with a market capitalization in excess of us$ trn., although its rank has since fallen considerably. industry accounted for Á % of gdp in . cottage industries persist into the st century. industrial output grew by Á % in . modern industrial development began with the manufacture of cotton textiles and the establishment of silk filatures, steel plants, flour mills and match factories. in there were , industrial enterprises with an annual revenue of more than m. yuan. of these enterprises, , were domestically funded, , were foreign funded and , were dependent on funds from hong kong, macao and taiwan. there were , state-owned industrial enterprises in total. output of major products, unless otherwise indicated (in tonnes): cement, , Á m.; rolled steel, , Á m.; crude steel, Á m.; pig iron, Á m.; gas oil and diesel oil ( ), Á m.; gasoline, Á m.; paper and paperboard, Á m.; sulphuric acid, Á m.; chemical fertilizers, Á m.; fuel oil ( ), Á m.; yarn, Á m.; refined sugar, Á m. also produced in : cloth, , m. metres; beer, , Á m. litres; , Á m. mobile phones; Á m. notebook pcs; Á m. colour tv sets; Á m. air conditioners; Á m. home refrigerators; Á m. washing machines; Á m. bicycles; Á m. cameras; Á m. motorcycles. china is the world's leading cement, steel and pig iron manufacturer; since output of cement has trebled, and production of crude steel has increased sixfold and of pig iron fivefold (although in pig iron production fell for the first time in more than years, as did crude steel production in ). china overtook japan as the world's largest producer of motor vehicles in , and in produced Á m. cars and Á m. commercial vehicles. the employed population at the census was Á m. ( Á m. female). by it had risen to Á m. ( Á m. more than in ), of whom Á m. worked in rural areas ( Á m. fewer than in ) and Á m. in urban areas ( Á m. more than in ). in china's registered urban jobless was Á %, with Á m. registered unemployed in the country's cities. with china's fast-growing ageing population, according to the united nations the working-age population began to decline in . in china had , private industrial enterprises. it was not until the late s that the private sector even came into existence in china. the average annual wage of people working in urban units in was , yuan. china's labour law stipulates a five-day working week with no more than eight hours a day and no more than hours a week. minimum working age was fixed at in . strikes over pay have become ever more frequent in china, particularly at foreign-owned facilities. china had Á m. people living in slavery according to the walk free foundation's global slavery index, the second highest total of any country. there are five special economic zones at shenzhen, xiamen, zhuhai, shantou and hainan in which concessions are made to foreign businessmen. the pudong new area in shanghai is also designated a special development area. since joint ventures with foreign firms have been permitted. a law of april reduced taxation on joint ventures to %. there is no maximum limit on the foreign share of the holdings; the minimum limit is %. in china is the second largest trading nation in the world, accounting for Á % of global merchandise imports by value in and Á % of global merchandise exports (up from Á % when it joined the wto in ). it was the second largest importer in behind the usa and the largest exporter. as recently as the usa's total trade in goods was more than twice that of china. it overtook germany as the largest exporter of goods in . its trade surplus in goods is the highest of any country. however, it has the world's highest trade deficit in services. in imports of services totalled us$ bn. but exports only us$ bn. main imports in (in us$ bn.): machinery and transport equipment, Á ; non-edible raw materials, Á ; mineral fuels, lubricants and related materials, Á ; chemicals, Á . major exports in (in us$ bn.): machinery and transport equipment, , Á ; miscellaneous manufactured goods, Á ; light textile industrial products, rubber products, minerals and metallurgical products, Á ; chemicals, Á . the main trading partners were as follows in (in us$ m.): the total road length in was , , km, including , km of expressways (of which there had not been any as recently as the mid- s); , m. tonnes of freight and , m. persons were transported by road that year. the number of civilian motor vehicles was Á m. in , including Á m. passenger vehicles and Á m. trucks (more than double the number in , when there were Á m. civilian vehicles overall including Á m. passenger vehicles and Á m. trucks). china is the world's fastest-growing car market. there were , traffic accidents in , with , fatalities. in in jan. there were , ships of gt or over registered, totalling Á m. gt. of the , vessels registered, were bulk carriers, general cargo ships, oil tankers, container ships, passenger ships and liquid gas tankers. mainland china's busiest port in was ningbo-zhoushan (handling Á m. tonnes of cargo), followed by shanghai ( Á m. tonnes), tianjin ( Á tonnes), guangzhou (canton) ( Á m. tonnes) and qingdao ( Á m. tonnes). shanghai overtook singapore to become the world's busiest container port in and handled Á m. teus (twenty-foot equivalent units) in . shenzhen, mainland china's second busiest port for container traffic and the world's fourth busiest in , handled Á m. teus. hong kong handled Á m. teus in . in jan. the first legal direct shipping links between the chinese mainland and taiwanese islands in more than years were inaugurated. inland waterways totalled , km in ; , Á m. tonnes of freight and Á m. passengers were carried. in june the three gorges reservoir on the chang jiang river, the largest water control project in the world, reached sufficient depth to support the resumption of passenger and cargo shipping. in out of countries analysed in the fragile states index-a list published jointly by the fund for peace and foreign policy magazine-china was ranked the th most vulnerable to conflict or collapse. the index is based on indicators of state vulnerability across social, political and economic categories. six new codes of law (including criminal and electoral) came into force in , to regularize the legal unorthodoxy of previous years. there is no provision for habeas corpus. as well as treason and murder the death penalty may be used for rape, embezzlement, smuggling, fraud, theft, drug-dealing, bribery and robbery with violence. amendments to the criminal law in and reduced the number of capital crimes-which include both violent and non-violent offences-from to and further to . china does not divulge figures on its use of the death penalty, but amnesty international reports that china executes thousands of people annually and is the world's top executioner. nevertheless, western analysts believe that the number of executions now is around a fifth of the yearly total in the s. 'people's courts' are divided into some higher, intermediate and , basiclevel courts, and headed by the supreme people's court. the latter, the highest state judicial organ, tries cases, hears appeals and supervises the people's courts. it is responsible to the national people's congress and its standing committee. people's courts are composed of a president, vicepresidents, judges and 'people's assessors' who are the equivalent of jurors. 'people's conciliation committees' are charged with settling minor disputes. there are also special military courts. procuratorial powers and functions are exercised by the supreme people's procuracy and local procuracies. in march the national people's congress passed legislation developing aspects of the general principles of civil law, with effect from oct. . among its provisions was the extension of legitimate rights and interests from chinese citizens only to anyone conducting civil activities in the country. in addition, the statute of limitation was increased from two to three years. the number of sentenced prisoners in mid- was , , ( per , of national population). china was ranked th of countries for criminal justice and th for civil justice in the world justice project rule of law index, which provides data on how the rule of law is experienced by the general public across eight categories. an educational reform of brought in compulsory nine-year education consisting of six years of primary schooling and three years of secondary schooling, to replace a previous five-year system. in mainland china the population census revealed the following levels of educational attainment: Á m. people had finished university education; Á m. had received senior secondary education; Á m. had received junior secondary education; and Á m. had had primary education. Á m. people over years of age or Á % of the population were illiterate, although this compared favourably with a Á % rate of illiteracy in the census and a Á % rate in . in adult literacy was estimated at Á %; youth literacy in was Á %. in there were , kindergartens with Á m. children and Á m. full-time teachers; , regular primary schools with Á m. pupils and Á m. full-time teachers; , secondary schools (including: , senior secondary; , junior secondary; , specialized; , vocational; and , technical) with Á m. pupils and Á m. full-time teachers. there were also , pupils at , special education schools. institutes of higher education, including universities, numbered , in , with Á m. undergraduates and Á m. postgraduate level students, and Á m. full-time teaching staff. china has more than private universities, almost all of which have been established since the mid- s. a national system of student loans was established in . the number of chinese students studying abroad went up from , in to , in ; it rose above , in and , in , and by exceeded , , making china the largest source of overseas students in the world. chinese students account for a fifth of all international students in tertiary education in the oecd, but fewer than half return to china after finishing their studies. the number of chinese undergraduate students in american universities in - was times as many as in - , rising from , to , in the space of seven years. there is an academy of sciences with provincial branches. an academy of social sciences was established in . in national government expenditure on education came to , , m. yuan and accounted for Á % of national government spending. medical treatment is free only for certain groups of employees, but where costs are incurred they are partly borne by the patient's employing organization. in there were , health institutions throughout china, including , hospitals, , local health centres, , centres for disease control and prevention, and , specialized prevention and treatment centres. china's first aids case was reported in . at the end of there were , reported cases of people living with hiv/aids. the number of deaths of people who had been living with hiv/aids in was , . in the first half of china was struck by an epidemic of a pneumoniatype virus identified as sars (severe acute respiratory syndrome). the virus was first detected in southern china and was subsequently reported in over other countries. according to the ministry of health, by the time the outbreak had been contained a total of , cases had been reported on the chinese mainland; , patients were cured and discharged from hospital, and died. in water: at what cost? the state of the world's water , wateraid reported that Á % of the population does not have access to safe water. china ranked as the country with the second largest number of people living without access to safe water ( Á m. in ). in an estimated Á % of adult males and Á % of adult females smoked in china. a study from the same year estimated that chinese males smoke one-third of all the world's cigarettes. in there were , social welfare enterprises with Á m. beds. numbers (in , ) of beneficiaries of relief funds in : urban residents receiving minimum living allowance, , ; rural residents receiving minimum living allowance, , ; persons receiving traditional relief, ; persons in rural households entitled to the 'five guarantees' (food, clothing, medical care, housing and burial expenses), , . the official retirement age for men is and for women (or in the case of civil servants and professionals). the government accords legality to five religions only: buddhism, islam, protestantism, roman catholicism and taoism. confucianism, buddhism and taoism have long been practised. confucianism has no ecclesiastical organization and appears rather as a philosophy of ethics and government. taoism-of chinese origin-copied buddhist ceremonial soon after the arrival of buddhism two millennia ago. buddhism in return adopted many taoist beliefs and practices. a more tolerant attitude towards religion had emerged by , and the government's bureau of religious affairs (since renamed the state administration for religious affairs) was reactivated. ceremonies of reverence to ancestors have been observed by the whole population regardless of philosophical or religious beliefs. a new quasi-religious movement, falun gong, was founded in , but has since been banned by the authorities. the movement has claimed some m. adherents, although the chinese government has disputed this. muslims are found in every province of china, being most numerous in the ningxia-hui autonomous region, yunnan, shaanxi, gansu, hebei, henan, shandong, sichuan, xinjiang and shanxi. roman catholicism has had a footing in china for more than three centuries. two christian organizations-the chinese patriotic catholic association, which declared its independence from rome in , and the protestant three-self patriotic movement-are sanctioned by the chinese government. according to estimates (by the state-approved xinhua news agency, the chinese academy of social sciences and the state administration for religious affairs) there were m. buddhists (more than in any other country), m. christians and more than m. muslims in the country in . other official figures indicate that there are Á m. catholics, although unofficial estimates are much higher. the number of christians in china is generally thought to be far higher than official numbers indicate, with so-called 'house churches' becoming ever more popular. some analysts estimate that there are as many as m. christians overall. legislation of prohibits foreign nationals from setting up religious organizations. johnson, ian, the souls of china: the return of religion after mao. china and provides habitat for a number of rare animal species, kulangsu, a historic international settlement ( ), a tiny island located on the estuary of the chiu-lung river, qinghai hoh xil ( ), the largest and highest plateau in the world, and fanjingshan ( ), an island of metamorphic rock in guizhou province. shared with kazakhstan and kyrgyzstan, silk roads: the routes network of chang'an-tianshan corridor ( ) is a , -km section of the extensive silk roads network stretching from chang'an/ luoyang to the zhetysu region in present-day kazakhstan. china has two news agencies: xinhua (new china) news agency (the nation's official agency) and china news service. in there were , newspapers and , magazines; , m. copies of newspapers and , m. copies of magazines were published. in there were fewer than newspapers. the communist party newspaper, renmin ribao (people's daily), had an average daily circulation of Á m. in . the most widely read newspaper is cankao xiaoxi, with an average daily circulation of Á m. in . china has the second highest circulation of daily newspapers after india, with an estimated average daily total of Á m. in . as of sept. it was also home to the world's two most visited online news sites: xinhua news agency ( Á m. unique desktop users per month) and people's daily online ( Á m.). in the world press freedom index compiled by reporters without borders, china ranked rd out of countries. in , , m. volumes of books were produced. in tourist numbers totalled Á m. china was the fourth most visited destination in after france, the usa and spain. income from tourists in was us$ Á bn., ranking it third behind the usa and spain. expenditure by chinese travellers outside of mainland china for was us$ Á bn., the most of any country and more than double the next largest expenditure (that of us tourists). in both german and us travellers abroad had spent more than those from china. the lunar new year, also known as the 'spring festival', is a time of great excitement for the chinese people. the climate is sub-tropical, tending towards temperate for nearly half the year, the winter being cool and dry and the summer hot and humid, may to sept. being the wettest months. normal temperatures are jan. f ( Á c), july f ( Á c). annual rainfall " ( , Á mm). hong kong used to be administered by the hong kong government. the governor was the head of government and presided over the executive council, which advised the governor on all important matters. the last british governor was chris patten. in oct. the executive council consisted of three ex officio members and ten appointed members, of whom one was an official member. the chief functions of the legislative council were to enact laws, control public expenditure and put questions to the administration on matters of public interest. the legislative council elected in sept. was, for the first time, constituted solely by election. it comprised members, of whom were elected from geographical constituencies, from functional constituencies encompassing all eligible persons in a workforce of Á m., and ten from an election committee formed by members of district boards. a president was elected from and by the members. at the elections on sept. turnout for the geographical seats was Á %, and for the functional seats ( of which were contested), Á %. the democratic party and its allies gained seats, the liberal party and the pro-beijing democratic alliance . the remaining seats went to independents. on dec. the selection committee selected a provisional legislature which began its activities in jan. while the legislative council was still functioning. in jan. the provisional legislature started its work by enacting legislation which would be applicable to the hong kong special administrative region and compatible with the basic law. constitutionally hong kong is a special administrative region of the people's republic of china. the basic law enables hong kong to retain a high degree of autonomy. it provides that the legislative, judicial and administrative systems which were previously in operation are to remain in place. the special administrative region government is also empowered to decide on hong kong's monetary and economic policies independent of china. in july the first-past-the-post system of returning members from geographical constituencies to the legislative council was replaced by proportional representation. there were directly elected seats out of for the first elections to the legislative council following hong kong's return to chinese sovereignty, increasing in accordance with the basic law to for the election with indirectly elected. in the sept. legislative council election (and that of sept. ) of the seats were directly elected. for the election in sept. the number of seats was increased to , with directly elected and indirectly elected by functional constituencies. there were also five new functional constituency seats nominated by elected district council members. the chief executive is chosen by a beijing-backed , -member election committee ( prior to the march election), although it has been stated that universal suffrage is the ultimate aim. in a timetable was announced for hong kong to directly elect its chief executive in and its legislative council in . however, beijing insisted that only approved candidates would be allowed to stand in , prompting mass pro-democracy rallies in the territory in and formal rejection of the plan by the legislative council in june . beijing nevertheless refused to countenance amendments. in july a new accountability or 'ministerial' system was introduced, under which the chief executive nominates for appointment policy secretaries, who report directly to the chief executive. the chief executive is aided by the executive council, consisting of the three senior secretaries of department (the chief secretary, the financial secretary and the secretary for justice) and eleven other secretaries plus five non-officials. hong kong used to be administered by the hong kong government. the governor was the head of government and presided over the executive council, which advised the governor on all important matters. the last british governor was chris patten. in oct. the executive council consisted of three ex officio members and ten appointed members, of whom one was an official member. the chief functions of the legislative council were to enact laws, control public expenditure and put questions to the administration on matters of public interest. the legislative council elected in sept. was, for the first time, constituted solely by election. it comprised members, of whom were elected from geographical constituencies, from functional constituencies encompassing all eligible persons in a workforce of Á m., and ten from an election committee formed by members of district boards. a president was elected from and by the members. at the elections on sept. turnout for the geographical seats was Á %, and for the functional seats ( of which were contested), Á %. the democratic party and its allies gained seats, the liberal party and the pro-beijing democratic alliance . the remaining seats went to independents. overview hong kong has one of the world's most open economies and is an internationally important financial centre. the territory's economic rise was founded on its role as an international trade emporium, acting as a conduit for china's burgeoning exports. mainland china, the usa and japan are hong kong's major export partners, accounting for Á %, Á % and Á % of exports respectively in . the island is dependent on imports of food and other resources. in it imported % of goods from mainland china, % from taiwan and % from japan. in and the economy grew strongly on the back of a rise in chinese tourism, healthy global demand for exports and improving domestic consumer confidence. however, the global financial crisis saw the economy shrink by Á % in before rebounding with a Á % increase the following year. between and annual growth averaged Á %, supported by strong external demand. student-led pro-democracy protests in the latter months of caused major disruption in several key business districts and threatened to weaken the local economy in the short term. foreign direct investment levels have been high, averaging % of gdp between and according to world bank data, and the world economic forum ranked hong kong as the ninth most competitive economy in the world in its report. the government aims to tackle a housing shortage by providing , new housing units by the mid- s. the unit of currency is the hong kong dollar (hkd) of cents. it has been pegged since at a rate of hk$ Á to the us dollar. banknotes are issued by the hongkong and shanghai banking corporation and the standard chartered bank, and, from may , the bank of china. total money supply was hk$ , m. in july . in aug. gold reserves were , troy oz and foreign exchange reserves were us$ , m. inflation rates (based on imf statistics): environment hong kong's carbon dioxide emissions from the consumption of energy in were the equivalent of Á tonnes per capita. installed capacity was Á m. kw in . production in was Á bn. kwh. hong kong is a net importer of electricity. consumption in was Á bn. kwh. the local agricultural industry is directed towards the production of high quality fresh food through intensive land use and modern farming techniques. out of the territory's total land area of , sq. km, only sq. km is currently farmed. in local production accounted for % of live poultry consumed, % of live pigs and % of fresh vegetables. the gross value of local agricultural production totalled hk$ , m. in , with pig production valued at hk$ m., poultry production (including eggs) at hk$ m., and vegetable and flower production at hk$ m. in the total catch was , tonnes, exclusively from marine waters. the leading companies by market capitalization in hong kong in march were: china mobile, a telecommunications company (us$ Á bn.); aia group, a life insurance company (us$ Á bn.); and cnooc, an integrated oil company (us$ Á bn.). industry is mainly service-oriented. in june there were , establishments employing , , persons in service industries and , establishments employing , persons in manufacturing industries. establishment statistics by service type (and persons engaged) were mainly: import/export trade and wholesale, , ( , ); retail, , ( , ); social and personal services, , ( , ) ; professional and business services, , ( , ); financing and insurance, , ( , ); accommodation and food services, , ( , ); real estate, , ( , ) . in the size of the labour force (synonymous with the economically active population) was , , ( , , females). the persons engaged in june included , , people in wholesale, retail and import/ export trades, accommodation and food services, , in finance, insurance, real estate, professional and business services, , in the civil service, , in manufacturing and , in construction sites (manual workers only). a minimum wage of hk$ per hour was introduced for the first time on may . unemployment stood at Á % in the period sept.-dec. . in the total value of imports was hk$ , , m. and total exports hk$ , , m. the main suppliers of imports in were mainland china ( Á %), japan ( Á %), taiwan ( Á %), singapore ( Á %) and usa ( Á %). in , Á % of total exports went to mainland china, Á % to the usa, Á % to japan, Á % to germany and Á % to the united kingdom. the chief import items in were: electrical machinery, apparatus and appliances, etc. ( Á %); telecommunications, sound recording and reproducing equipment ( Á %); office machines and automatic data processing machines ( Á %); articles of apparel and clothing accessories ( Á %). the main exports in were: electrical machinery, apparatus and appliances, etc. ( Á %); telecommunications, sound recording and reproducing equipment ( Á %); office machines and automatic data processing machines ( Á %); articles of apparel and clothing accessories ( Á %). in there were , km of roads, over % of which were in the new territories. there are road tunnels, including three under victoria harbour. in there were , private cars, , goods vehicles, , buses and coaches, and , motorcycles and mopeds. there were , road accidents in , of which were fatal. a total of Á m. tonnes of cargo were transported by road in . a km bridge (the world's longest sea bridge) linking hong kong, zhuhai in guangdong province in mainland china and macao opened in oct. following a number of delays. hong kong was ranked fourth for its road infrastructure in the world economic forum's global competitiveness report - . hong kong's railways are run by the mtr corporation limited (mtrcl), a public listed company of which the government is the majority shareholder. the mtr system comprises nine railway lines serving hong kong island, kowloon and the new territories. its km network has stations and carries an average of Á m. passengers each day. mtr lines carried , m. passengers in . in addition, a light rail network ( Á km and stops) serves the local communities of tuen mun, yuen long and tin shui wai in the new territories; , passengers travel daily on the system. a high speed rail service between hong kong and guangzhou on the mainland opened in sept. . the electric tramway on the northern state of hong kong island commenced operating in and has a total track length of km. the peak tram, a funicular railway connecting the peak district with the lower levels in victoria, has a track length of Á km and two tramcars (each with a capacity of passengers per trip). it carries an average of , passengers daily. the airport express line ( Á km) opened in and is also operated by the mtrcl. it carried Á m. passengers in . in june it was estimated that Á m. passenger journeys were made daily on public transport (including local railways, buses, etc.). in the world economic forum's global competitiveness report - hong kong ranked third for quality of rail infrastructure. the new hong kong international airport (generally known as chek lap kok), built on reclaimed land off lantau island to the west of hong kong, was opened on july to replace the old hong kong international airport at kai tak, which was situated on the north shore of kowloon bay. more than airlines now operate scheduled services to and from hong kong. in cathay pacific airways, the largest hong kong-based airline, operated approximately , passenger and cargo services to destinations in countries and territories around the world. cathay pacific carried , , passengers and Á m. tonnes of cargo in . dragonair, a cathay pacific subsidiary, provided scheduled services to cities in mainland china and asia in . in air hong kong, an all-cargo operator, provided scheduled services to bangkok, beijing, ho chi minh city, manila, nagoya, osaka, penang (via bangkok), seoul, shanghai, singapore, taipei and tokyo. hong kong international airport handled more international freight in than any other airport. in , , aircraft arrived and departed and m. passengers and Á m. tonnes of freight were carried on aircraft. hong kong was second, behind only singapore, in the rankings for air transport infrastructure in the world economic forum's global competitiveness report - . the port of hong kong handled Á m. teus (twenty-foot equivalent units) in , making it the world's third busiest container port after shanghai and singapore. the kwai chung container port has berths with , metres of quay backed by ha. of cargo handling area. at the end of there were , ships ( , ocean-going) of , , gt registered in hong kong. in , , ocean-going vessels, , river cargo vessels and , river passenger vessels arrived at the port of hong kong. a total of m. tonnes of freight were handled in . hong kong was ranked third in the world economic forum's global competitiveness report - for the quality of its port facilities. in there were , , main (fixed) telephone lines (equivalent to Á per , population). the local fixed telecommunications network services (ftns) market in hong kong was liberalized in . there were , , mobile phone subscriptions in (equivalent to , Á per , population), up from , , in ( , Á per , population). the number of subscriptions doubled between and . the internet market has also seen huge growth. in there were , , wireless broadband subscriptions ( Á per , population) and , , fixed broadband subscriptions ( Á per , population). the number of fixed broadband subscriptions has been declining since as more people have wireless subscriptions instead. in march there were Á m. facebook users. the external telecommunications services market has been fully liberalized since jan. , and the external telecommunications facilities market was also liberalized starting from jan. . in the adult literacy rate was Á % ( Á % in ). universal basic education is available to all children aged from six to years. in around three-quarters of the ordinary secondary day schools teaching has been in cantonese since - , with about a quarter of ordinary secondary day schools still using english. in there were , pupils in kindergartens, , in primary schools (including international schools) and , in secondary schools (including international schools). the estimated total government expenditure on education in - was hk$ Á bn. ( Á % of total government spending and Á % of gdp). in - : Á % of total government spending and Á % of gdp. according to the oecd's pisa (programme for international student assessment) study, -year-olds in hong kong rank second among oecd and other major countries and cities in mathematics and reading, and ninth in science. the three-yearly study compares educational achievement of pupils in over countries. the department of health (dh) is the government's health adviser and regulatory authority. the hospital authority (ha) is an independent body responsible for the management of all public hospitals. in there were , registered doctors, equivalent to Á doctors per , population. in there were , dentists, , nurses and , midwives. the total number of hospital beds in was , , including , beds in public hospitals under the ha and , beds in private hospitals. the bed-population ratio was Á beds per thousand population. the chinese medicine ordinance was passed by the legislative council in july to establish a statutory framework to accord a professional status for chinese medicine practitioners and ensure safety, quality and efficacy of chinese medicine. in there were , registered chinese medicine practitioners. total expenditure on health in - amounted to hk$ , m., an increase of Á % over that in - . the hong kong act of provided for hong kong ordinances to replace english laws in specified fields. the courts of justice comprise the court of final appeal (inaugurated july ), which hears appeals on civil and criminal matters from the high court; the high court (consisting of the court of appeal and the court of first instance); the lands tribunal, which determines on statutory claims for compensation over land and certain landlord and tenant matters; the district court (which includes the family court); the magistracies (including the juvenile court); the coroner's court; the labour tribunal, which provides a quick and inexpensive method of settling disputes between employers and employees; the small claims tribunal, which deals with monetary claims involving amounts not exceeding hk$ , ; and the obscene articles tribunal. while the high court has unlimited jurisdiction in both civil and criminal matters, the district court has limited jurisdiction. the maximum term of imprisonment it may impose is seven years. magistracies exercise criminal jurisdiction over a wide range of offences, and the powers of punishment are generally restricted to a maximum of two years' imprisonment or a fine of hk$ , . after being in abeyance for years, the death penalty was abolished in . , crimes were reported in , of which , were violent crimes. , people were arrested in , of whom , were for violent crimes. the population in penal institutions was , at dec. ( per , population). social welfare programmes include social security, family services, child care, services for the elderly, medical social services, youth and community work, probation, and corrections and rehabilitation. non-governmental organizations are subsidized by public funds. the government gives non-contributory cash assistance to needy families, unemployed able-bodied adults, the severely disabled and the elderly. caseload as at aug. totalled , . victims of natural disasters, crimes of violence and traffic accidents are financially assisted. estimated recurrent government expenditure on social welfare for - was hk$ Á bn. in there were daily newspapers, of which were paid-for and four free. the newspapers with the highest circulation figures are all chinese-language papers-oriental daily news, apple daily and the sun. the english-language paper with the highest circulation is the south china morning post. circulation of dailies (including free papers) in was Á m. ( Á m. paid-for and Á m. free). there were a record , , visitor arrivals in . expenditure associated to inbound tourism totalled hk$ , Á m. in . the macao special administrative region, which lies at the mouth of the pearl river, comprises a peninsula ( Á sq. km) connected by a narrow isthmus to the people's republic of china, on which is built the city of santa nome de deus de macao, the islands of taipa ( Á sq. km), linked to macao by three bridges, colôane ( Á sq. km) linked to taipa by a -km causeway, and cotai, a strip of reclaimed land between colôane and taipa ( Á km). the total area of macao in was Á sq. km. additional land continues to be reclaimed from the sea. the population at the census was , ( , females); density, , people per sq. km. according to un estimates, the entire population lived in urban areas in . the official languages are chinese and portuguese, with the majority speaking the cantonese dialect. only about , people speak portuguese as their first language. the un gives a projected population for of , . in , , foreigners were legally registered for residency in macao. there were , legal immigrants from mainland china. social statistics : births, , ( Á per , population); deaths, , ( Á ); marriages, , ( Á ); divorces, , ( Á ). infant mortality, , Á per , live births. life expectancy at birth , Á years. sub-tropical tending towards temperate, with an average temperature of Á c. the number of rainy days is around a third of the year. average annual rainfall varies from - " ( , - , mm) . it is very humid from may to sept. macao's constitution is the 'basic law', promulgated by china's national people's congress on march and in effect since dec. . it is a special administrative region (sar) of the people's republic of china, and is directly under the central people's government while enjoying a high degree of autonomy. the legislative assembly has seats of which are directly elected, indirectly elected by functional constituencies and seven appointed by the chief executive. at the elections held on sept. the macau-guangdong union won two of elected seats with Á % of votes cast and the union for development two with Á %. ten other parties won a single seat each. turnout was Á %. fernando chui sai-on was re-elected chief executive for a second term on aug. , receiving out of votes in the election committee. chief executive: fernando chui sai-on; b. (sworn in dec. and re-elected in aug. . government website: http://www.gov.mo the gaming sector is of major importance to the economy of macao. it accounted for Á % of total gdp in and provides billions of dollars in taxes. in , Á % of the workforce was employed in gaming. in gross gaming revenue totalled us$ , m. (nearly double the figure) . however, revenues were down slightly on the total. macao overtook nevada as the world's largest gaming market in . macao's traditional manufacturing industries virtually disappeared following the transfer of much of the textile industry to the chinese mainland and, in , the termination of the multifibre arrangement, which had governed international textile trade flows for three decades. the unit of currency is the pataca (mop) of avos, which is tied to the hong kong dollar at parity. inflation was Á % in and Á % in . foreign exchange reserves were us$ , m. in . total money supply was , m. patacas in . in revenues totalled , m. patacas; expenditures, , m. patacas. revenues from gaming tax accounted for Á % of total revenue in ; current expenditure accounted for Á % of expenditure. real gdp growth was just Á % in but then rose to Á % in and Á % in . more recently the economy contracted by Á % in before growing by Á % in . total gdp in was us$ Á bn. environment macao's carbon dioxide emissions from the consumption of energy in were the equivalent of Á tonnes per capita. installed capacity was Á m. kw in ; production, Á bn. kwh. macao imported , m. kwh of electricity in . oil and gas , , litres of fuel oil were imported in . the catch in was estimated at , tonnes. although the economy is based on gaming and tourism there is a light industrial base of textiles and garments. in the number of manufacturing establishments was (food products and beverages, ; textiles and wearing apparel, ; publishing, printing and reproduction of recorded media, ). in a total of , people were in employment, including , ( Á %) in gaming and junket activities (up from , in ); , ( Á %), hotels, restaurants and similar activities; , ( Á %), construction; , ( Á %), wholesale and retail trade, repair of motor vehicles, motorcycles and personal and household goods; , ( Á %), real estate and business activities; , ( Á %), public administration and social security. employment in was Á % of the labour force; unemployment rate stood at Á %. in imports (c.i.f.) were valued at us$ , Á m., of which the main products were telecommunications, sound recording and reproducing equipment; petroleum and petroleum products; and gold, silverware, jewellery and articles of precious materials. in the chief import sources (in us$ m.) were: mainland china ( , Á ); hong kong ( Á ); japan ( Á ). exports (f.o.b.) were valued at us$ Á m., of which the leading products were articles of apparel and clothing accessories; gold, silverware, jewellery and articles of precious materials; and petroleum oils and oils obtained from bituminous minerals. in the main export markets (in us$ m.) were: hong kong ( Á ); usa ( Á ); mainland china ( Á ). in there were km of roads. in there were , passenger cars in use ( cars per , inhabitants), , buses and coaches, , trucks and , motorcycles. there were fatalities in road accidents in . a km bridge (the world's longest sea bridge) linking macao, zhuhai in guangdong province in mainland china and hong kong opened in oct. following a number of delays. an international airport opened in dec. . in macau international airport handled , , passengers and , tonnes of freight (including transit cargo). in air macau flew to bangkok, beijing, changsha, chengdu, chongqing, da nang, hangzhou, hefei, kaohsiung, nanjing, nanning, ningbo, osaka, quanzhou, seoul, shanghai, shenyang, taipei, taiyuan, tokyo, wenzhou, xiamen and zhengzhou. regular services connect macao with hong kong, km to the northeast. in there were , landline telephone subscriptions (equivalent to Á per , inhabitants) and , , mobile phone subscriptions (or , Á per , inhabitants). in , Á % of households had internet access. in march there were , facebook users. there are a judicial district court, a criminal court and an administrative court with magistrates in all. in there were , crimes, of which , were against property. there were persons in prison in dec. . there are both public and private schools. in - there were schools and colleges. number of students in the - academic year (with number of teachers): pre-primary, , ( ); primary, , ( , ); secondary, , ( , ). in - there were four special education schools with pupils and teachers. there were ten higher education institutions with student enrolment of , . in there were institutions offering vocational training courses, in which participants totalled , . expenditure on education came to Á % of gdp in and Á % of total government spending in . in there were doctors, dentists and nurses working in primary health care, and doctors, dentists and , nurses working in hospitals. in there were , hospital beds; there were Á doctors per , population. in there were an estimated , folk religionists and , buddhists according to the pew research center's forum on religion & public life. a further , people were religiously unaffiliated. there are also small numbers of catholics. the historic centre of macao was inscribed on the unesco world heritage list in . in there were daily newspapers (nine in chinese, three in portuguese and two in english) and weekly newspapers (ten in chinese and one in portuguese). tourism is one of the mainstays of the economy. in there were Á m. tourists (of which Á m. were from mainland china, Á m. from hong kong and Á m. from taiwan), up from Á m. in and Á m. in . visitor spending in totalled , m. patacas. the government-run macao international music festival featuring a wide range of chinese and western music takes place in oct.-nov. in july president lee teng-hui repudiated taiwan's -year-old 'one china' policy-the pretence of a common goal of unification-arguing that taiwan and china should maintain equal 'state to state' relations. this was a rejection of beijing's view that taiwan is no more than a renegade chinese province which must be reunited with the mainland, by force if necessary. in the presidential election of march chen shui-bian, leader of the democratic progressive party, was elected, together with annette lu hsiu-lien as his vice president. both supported independence although chen shui-bian made friendly gestures towards china and distanced himself from colleagues who wanted an immediate declaration of independence. following his wife's indictment on embezzlement charges in nov. , president chen survived three parliamentary attempts to impeach him. he was succeeded as president in by ma ying-jeou of the nationalist party. in sept. chen shui-bian received a life sentence (later reduced to a -year term) after being found guilty of multiple counts of corruption. china and taiwan signed a free trade agreement in june , which was considered a significant thawing of relations. nonetheless, tensions remained, particularly in relation to disputed sovereignty over several islands in the east china sea. in jan. ma ying-jeou was re-elected to the presidency but the election in jan. was won by tsai ing-wen, whose democratic progressive party won the most seats in legislative polls at the same timethe first occasion that the nationalist party has not been the largest party in government since . the climate is subtropical in the north and tropical in the south. the typhoon season extends from july to sept. the average monthly temperatures of jan. and july in taipei are Á f ( Á c) and Á f ( Á c) respectively, and average annual rainfall is Á " ( , Á mm). kaohsiung's average monthly temperatures of jan. and july are Á f ( Á c) and Á f ( Á c) respectively, and average annual rainfall is Á " ( , Á mm). the roc constitution is based on the principles of nationalism, democracy and social wellbeing formulated by dr sun yat-sen, the founding father of the republic of china. the roc government is divided into three main levels: central, provincial/municipal and county/city, each of which has welldefined powers. the central government consists of the office of the president, the national assembly, which is specially elected only for constitutional amendment, and five governing branches called 'yuan', namely the executive yuan, the legislative yuan, the judicial yuan, the examination yuan and the control yuan. beginning with the elections to the seventh legislative yuan held on jan. the legislative yuan has members (formerly ). of the members are elected under the first-past-the-post system in singlemember constituencies, are filled by proportional representation in accordance with a nationwide party vote and six are reserved for aboriginal candidates. since the president has been directly elected. since a resolution on the impeachment of the president or vice president is no longer to be instituted by the control yuan but rather by the legislative yuan. the legislative yuan has the power to pass a no-confidence vote against the premier of the executive yuan, while the president of the republic has the power to dissolve the legislative yuan. the premier of the executive yuan is directly appointed by the president of the republic. in dec. a law came into effect allowing for referendums to be held. professional force-a process that was originally scheduled to start in and end in but has been delayed owing to low recruitment levels. defence expenditure in totalled us$ , m. (us$ per capita), representing Á % of gdp. the republic of china army conducts ground combat missions as well as air support and airborne special operations. it was estimated to number about , personnel in , with reserves numbering Á m. its principal role is to defend against a possible amphibious assault from the chinese mainland by the people's liberation army. in addition there are paramilitary forces totalling , personnel. navy personnel in totalled , , with , reservists. the forces consist of four submarines, four cruisers and frigates. there are also missile craft for patrol and coastal defence, mine-laying vehicles and amphibious landing craft. in the air force numbered , personnel with , reservists. there were combat-capable aircraft in the same year including f- es, f- s and mirage - s. taiwan has made a successful transition from an agricultural economy to one based on high-tech electronics. economic growth averaged % per year over three decades from the s, driven primarily by high value-added manufacturing and exports, especially in electronics and computers. government-owned enterprises, including banks, have been privatized. though largely escaping the impact of the asian financial crisis, the economy went into recession in with the first year of negative growth ever recorded and unemployment reaching record highs. strong export performance stimulated a recovery, with annual gdp growth above % from - . inflation has been consistently low and unemployment, which fell below % in , has averaged between and % since the turn of the century. owing to its heavy dependence on exports, taiwan suffered a severe downturn as a result of the global financial crisis in . major export industries such as semiconductors and memory chips declined, unemployment reached its highest levels since and, in , the economy again went into recession. a us$ Á bn. stimulus package boosted recovery and in the economy recorded its highest growth rate for nearly three decades, at Á %. however, growth subsequently cooled owing to lower demand from developed nations, averaging Á % per year between and . tourism has grown in importance, with over Á m. visitors in constituting taiwan's highest annual number to date. an ageing population and high savings rates threaten to constrain domestic demand in the future. the unit of currency is the new taiwan dollar (twd) of cents. gold reserves were Á m. oz in dec. . there was inflation of Á % in and Á % in . foreign exchange reserves were us$ Á bn. in dec. . in general government revenues totalled nt$ , , m. and expenditures nt$ , , m. tax revenue accounted for Á % of revenues in ; education, science and culture accounted for Á % of expenditures, economic development Á % and general administration Á %. vat is %. taiwan sustained rapid economic growth at an annual rate of Á % from up to . the rate slipped to Á % in the s and Á % in ; taiwan suffered from the asian financial crisis, though less than its neighbours. in global economic sluggishness and the events of sept. in the usa severely affected taiwan's economy, which contracted by Á %. subsequent economic recovery led to growth of Á % in and Á % in . there was negative growth of Á % in but again the economy bounced back, and grew by Á % in and Á % in . the central bank of the republic of china (taiwan), reactivated in , regulates the money supply, manages foreign exchange and issues currency. the governor is yang chin-long. the bank of taiwan is the largest commercial bank and the fiscal agent of the government. there are seven domestic banks, commercial banks and foreign banks. there are two stock exchanges in taipei. taiwan's carbon dioxide emissions from the consumption of energy in were the equivalent of Á tonnes per capita. output of electricity in was Á m. mwh; total installed capacity was , mw. there were six units in three nuclear power stations in . crude oil production in was , bbls; natural gas, m. cu. metres. taiwan imports most of the oil and natural gas that it consumes. in the cultivated area was , ha., of which , ha. were paddy fields. rice production totalled , , tonnes. livestock production was valued at nt$ , m., accounting for % of taiwan's total agricultural production value. forest area, : , , ha. forest reserves: trees, , , cu. metres; bamboo, , m. poles. timber production, , cu. metres. the catch in was , tonnes, almost exclusively from sea fishing. the largest companies in taiwan by market capitalization in march were: taiwan semiconductor manufacturing (us$ Á bn.); hon hai precision industry, an electronics manufacturer (us$ Á bn.); and chunghwa telecom (us$ Á bn.). output (in tonnes) in : crude steel; Á m.; cement, Á m.; cotton fabrics, Á m. sq. metres; integrated circuit packages, Á trn. units; global positioning system (gps) sets, Á bn. units. in the average total labour force was Á m., of whom Á m. were employed. of the employed population, Á % worked in manufacturing; Á % in wholesale and retail trade; Á % in construction; Á % in accommodation and food services; Á % in education; Á % in agriculture, forestry and fisheries. the unemployment rate was Á %. in there were , km of roads. in , Á m. passenger cars, , buses and coaches, Á m. lorries and vans, and Á m. motorcycles and mopeds were in use. , m. passengers and m. tonnes of freight were transported in . there were , fatalities in road accidents in . in freight traffic amounted to Á m. tonnes and passenger traffic to m. total route length was , km. there are metro systems in taipei (opened in ) , kaohsiung (opened in ) and taoyuan (opened in ). there are currently two international airports: taiwan taoyuan international airport at taoyuan near taipei, maritime transportation is vital to the trade-oriented economy of taiwan. in jan. there were ships of gt or over registered, totalling Á m. gt. of the vessels registered, were general cargo ships, bulk carriers, container ships, oil tankers and nine passenger ships. there are six international ports: kaohsiung, keelung, taichung, hualien, anping and suao. the first three are container centres, kaohsiung handling Á m. -ft equivalent units in , making it the world's th busiest container port in terms of number of containers handled. suao port is an auxiliary port to keelung. in jan. the first legal direct shipping links between taiwanese islands and the chinese mainland in more than years were inaugurated. in there were , , landline telephone subscribers ( Á per , inhabitants). taiwan's biggest telecommunications firm, the stateowned chunghwa telecom, lost its fixed-line monopoly in aug. . in there were , , mobile phone subscriptions, equivalent to , Á per , persons. in there were Á mobile broadband subscriptions per inhabitants and Á fixed broadband subscriptions per inhabitants. in march there were Á m. facebook users. the judicial yuan is the supreme judicial organ of state. comprising grand justices, since these have been nominated and, with the consent of the legislative yuan, appointed by the president of the republic. the grand justices hold meetings to interpret the constitution and unify the interpretation of laws and orders. there are three levels of judiciary: district courts and their branches deal with civil and criminal cases in the first instance; high courts and their branches deal with appeals against judgments of district courts; the supreme court reviews judgments by the lower courts. there is also the supreme administrative court, high administrative courts and a commission on the disciplinary sanctions of public functionaries. criminal cases relating to rebellion, treason and offences against friendly relations with foreign states are handled by high courts as the courts of first instance. the death penalty is still in force. there were no executions in but there was one in . the population in penal institutions in april was , ( per , of national population). since there has been compulsory education for six to -year-olds with free tuition. the illiteracy rate dropped from Á % in to Á % by . there were , primary schools, , secondary schools and vocational schools in ; and universities, colleges and junior colleges. in - there were , , pupils with , teaching staff at elementary schools; , pupils and , teaching staff at junior high schools; , pupils and , teaching staff at senior high schools; and , students and , teaching staff at senior vocational schools. there were , , students in universities and colleges in - with , academic staff. in there were , physicians (one for every persons), , doctors of chinese medicine, , nurses, , dentists and assistants, and , pharmacists and assistants. in there were , medical facilities serving , persons per facility; there were , beds and Á beds per , persons. in cancers, heart diseases, cerebrovascular diseases, diabetes and accidents were the first five leading causes of death. a universal health insurance scheme came into force in as an extension to social insurance plans that cover only % of taiwan's population. premium shares among the government, employer and insured are varied according to the insured statuses. by the end of , Á m. people or % of the population were covered by the national health insurance programme. according to estimates by the pew research center's forum on religion & public life, Á % of the population in were folk religionists, Á % were buddhists and Á % christians. the remainder of the population was either religiously unaffiliated or followed other religions, including taoism. there were daily newspapers in with a circulation of Á m. and non-dailies with a circulation of Á m. the biggest circulation dailies are the liberty times and apple daily. in there were , , international visitors. receipts totalled us$ , m. the pop festival, spring scream, is held in april in kenting. xinjiang tianshan ( ), a mountainous site comprising four components covering , ha. and an important habitat for endemic and relic flora species, the grand canal ( ), a vast waterway system running from beijing to zhejiang, tusi sites ( ), the remains of tribal domains whose leaders were appointed by the central government as 'tusi', hereditary rulers of their regions from the th to the early th century, located in southwest china prime minister and president of the executive yuan: su tseng-chang vice premier: chen chi-mai. there are ministries under the executive yuan: culture; economic affairs; education; finance; foreign affairs; health and welfare; interior; justice; labour; national defence; science and technology minister of culture: cheng li-chiun examples include the mongolian and tibetan affairs commission; the mainland affairs council; the fair trade commission; the public construction commission; and the financial supervisory commission. some of these are headed by ministers without portfolio (see above). other commissions, councils and agencies are headed by: council of agriculture: chen chi-chung mainland affairs council: chen ming-tong. national communications commission: chan ting-i. ocean affairs council: lee chung-wei. overseas community affairs council: wu hsin-hsing. directorate general of personnel administration: shih ning-jye. transitional justice commission (acting): yang tsui. veterans' affairs council: chiu kuo-cheng china in world history chinese politics in the age of deng xiaoping china and the global political economy china: the rise of xi jinping the cambridge encyclopaedia of china the cambridge history of china. vols the politics of hong kong's reversion to china the european union and china the chinese economy under transition china this century the tragedy of liberation: a history of the chinese revolution china: a modern history china's deep reform: domestic politics in transition chinese capitalism deng xiaoping and the making of modern china the great chinese revolution - the politics of eu-china economic relations china's second continent: how a million migrants are building a new empire in africa china in transition: communism, capitalism and democracy sowing the seeds of democracy in china: political reform in the deng xiaoping era historical dictionary of the chinese cultural revolution how the chinese economy works the rise of modern china china: a macro history tombstone: the untold story of mao's great famine china's economy: what everyone needs to know all under heaven: a complete history of china chinese politics in the hu jintao era: new leaders, new challenges the people's republic of amnesia: tiananmen revisited chinese foreign policy in a changing world chinese economy in the s the politics of china: sixty years of the people's republic of china the party: the secret world of china's communist rulers. .-asia's reckoning: china, japan and the fate of us power in the pacific century china's war with japan, - : the struggle for survival social and political development in post-reform china age of ambition: chasing fortune, truth and faith in the new china a revolutionary life at the crossroads of post-communist modernisation: russia and china in comparative perspective a history of china wealth and power: china's long march to the twenty-first century mao's road to power: revolutionary writings - . vols china goes global: the partial power the chinese century: the rising chinese economy and its impact on the global economy, the balance of power, and your job the china-pakistan axis: asia's new geopolitics the chan's great continent: china in western minds eldest son, zhou enlai and the making of modern china china in the xi jinping era india's and china's recent experience with reform and growth critical issues in contemporary china making china strong tide players: the movers and shakers of a rising china other more specialized titles are listed under territory and popula-tion; tibet; agriculture . commissioner: ning jizhe information: the census and statistics department is responsible for the preparation and collation of government statistics. these statistics are published mainly in the hong kong monthly digest of statistics. the department also publishes monthly trade statistics, economic indicators and an annual review of overseas trade hong kong's transitions the politics of hong kong's reversion to china the end of hong kong: the secret diplomacy of imperial retreat the hong kong story the final years of british hong kong: the discourse of colonial withdrawal last post: the end of empire in the far east the politics of democratization in hong kong historical dictionary of hong kong and macau china: a political history of the british crown colony's transfer to chinese rule managing china's sovereignty in hong kong and taiwan macau, the imaginary city: culture and society, to the present taiwan statistical yearbook of the republic of china. annual. the republic of china yearbook. annual. taiwan statistical data book. annual. annual review of government administration the other taiwan: to the present day historical dictionary of taiwan national identity and status in international society taiwan's political re-alignment and diplomatic challenges managing china's sovereignty in hong kong and taiwan the shadow of china: political developments in taiwan since . national library: national central library key: cord- -iesysf l authors: eshaghi, majid; tan, wen siang; chin, wai kit; yusoff, khatijah title: purification of the extra-cellular domain of nipah virus glycoprotein produced in escherichia coli and possible application in diagnosis date: - - journal: j biotechnol doi: . /j.jbiotec. . . sha: doc_id: cord_uid: iesysf l the glycoprotein (g) of nipah virus (niv) is important for virus infectivity and induction of the protective immunity. in this study, the extra-cellular domain of niv g protein was fused with hexahistidine residues at its n-terminal end and expressed in escherichia coli. the expression under transcriptional regulation of t promoter yielded insoluble protein aggregates in the form of inclusion bodies. the inclusion bodies were solubilized with m urea and the protein was purified to homogeneity under denaturing conditions using nickel–nitrilotriacetic acid (ni–nta) affinity chromatography. the denatured protein was renatured by gradual removal of the urea. light scattering analysis of the purified protein showed primarily monodispersity. the purified protein showed significant reactivity with the antibodies present in the sera of niv-infected swine, as demonstrated in western blot analysis and enzyme-linked immunosorbent assay (elisa). taken together, the data indicate the potential usefulness of the purified g protein for structural or functional studies and the development of immunoassay for detection of the niv antibodies. nipah virus (niv) is a negative sense, nonsegmented rna virus that was first isolated from cere-brospinal fluid of the human patients (chua et al., ) . it was found to be the etiologic agent for both human and swine diseases and classified to the family paramyxoviridae under a new genus henipaviruses (chua et al., ; mayo, ) . its genome encodes six structural proteins: nucleocapsid (n), phospho-(p), matrix-(m), fusion-(f), glyco-(g) and large-(l) proteins wang et al., ) . the natural reservoir of the virus is fruit bat with no signs of infection but causes fatal encephalitis in humans and a respiratory syndrome in pigs (chua et al., ) . the infected pigs can transfer the virus to other animals such as dogs, cats, and horses (chua et al., (chua et al., , paton et al., ) . most recently, guillaume et al. ( ) found that destruction of the natural habitat of fruit bats caused the migration of these bats to live closer to humans and domesticated animals, and as a result the virus can be transferred to new species. recent outbreaks of the niv and nipahlike viruses in bangladesh (who, a,b) demonstrate the existence of a time bomb that poses a major health problem worldwide, which could destroy the economies of many countries. therefore, there is a demand for rapid detection as well as serological diagnosis of the virus for monitoring the presence of the virus and its antibodies in individuals and animals in high-risk areas. currently, production of immunological reagents for these assays require biohazard level (bl ) laboratories which are limited only to a few countries worldwide. the g protein plays a central role in the viral replication process since it is responsible for the viral attachment to sialic acid-containing host cell receptors (bossart et al., ) . thus, the g protein appears to be essential for paramyxovirus replication, and as such could represent the primary target for neutralizing antibodies as well as potential targets for antiviral agents. in this study, the extra-cellular domain of the g protein of niv was cloned and expressed in the escherichia coli system. the purified product was used as the capturing antigen in an enzyme-linked immunosorbent assay (elisa) to determine the presence of the anti-niv antibodies in serum samples collected from naturally infected swine. it was found that the purified g protein reacted only with antibodies in niv positive samples, suggesting a potential replacement for currently used whole virus antigen that requires containment facilities. swine anti-niv sera with known serum neutralizing test (snt) and inactivated niv from infected cell culture medium were generous gifts from the veterinary research institute (vri), ipoh, malaysia. a vol-ume of l of niv-infected cell culture medium was used to extract total vrna using the trizol ls reagent (life technologies, usa), as recommended by the manufacturer. extracted vrna was used as a template for cdna synthesis using the superscript ii rnaseh (−) reverse transcriptase (life technologies, usa), which was subsequently used in a pcr amplification using the platinum tm high fidelity taq dna polymerase (life technologies, usa). two synthetic oligonucleotides, tgniv forward ( -gggggatcc-atggacaatcaggccgtgatc- ) and tgniv reverse primers ( -ggggggaagcttctcaacca-atgatatgcacca- ) were used to amplify the coding sequence of the niv g gene between nucleotides and . the underlined nucleotides represent bamhi and hindiii cutting sites, respectively. these two primers were designed to eliminate the transmembrane and signal regions of the g protein of niv. the pcr product was digested with bamhi and hindiii and cloned into the prset plasmid (life technologies, usa) that had been digested with the same restriction endonucleases. the resulting construction was transformed into e. coli strain bl si (life technologies, usa). the expression was confirmed with sds-page and western blot analysis before subjecting to purification and further analysis. broad range protein markers (gibco, brl) were used in sds-page and western blot analysis. swine anti-niv polyclonal antibodies ( / dilution) were used as the primary antibody. appropriate species-specific immunoglobulin conjugated to alkaline phosphatase ( / dilution) was used as the secondary antibody. the recombinant protein was extracted from bacterial cells using the bacterial protein extraction reagent (b-per) (pierce, usa), as recommended by the manufacturer with some modifications. briefly, bacterial cells from ml culture were centrifuged at × g for min and the pellet was washed with pbs ( mm sodium phosphate, . m nacl, ph . ) and centrifuged as above. the cells were resuspended in ml of b-per reagent and the mixture was gently shaken at rt for h before centrifugation at , × g for min to separate inclusion bodies from soluble proteins. after repeating the above extraction for two times, the inclusion bodies were resuspended in ml of b-per reagent containing g/ml lysozyme (sigma, usa), and the mixture was incubated at rt for min before adding ml pbs. the inclusion bodies were collected by centrifugation at , × g for min. the pellet was resuspended in ml of b-per reagent in pbs ( : dilution) and centrifuged as above. the extracted inclusion bodies were dissolved in ml of denaturing buffer ( m guanidine hcl; mm napo , ph . ; mm nacl) and loaded onto a pre-equilibrated column packed with ml of nickel-nitrilotriacetic acid (ni-nta) agarose (life technologies, usa). the protein-bound ni-nta resin was first washed with ml of denaturing binding buffer ( m urea; mm napo , ph . ; mm nacl) followed by washing with ml of denaturing wash buffer ( m urea; mm napo , ph . ; mm nacl). the column was washed with another ml of the above washing buffer at ph . the bound recombinant protein was finally eluted with ml denaturing elution buffer ( m urea; mm napo , ph . ; mm nacl) and the sample fractions were analyzed by sds-page and western blotting. the fractions containing the purified recombinant protein were pooled and first dialyzed against m urea for h. the dialysis was continued by the addition of ml of mm tris-hcl (ph . ) every h for three times and finally the dialysis solution was replaced with mm tris-hcl (ph . ), mm nacl, . % triton x- and mm ␤mercaptoethanol, and dialyzed for another h. the purity of the sample was studied with a dynamic light scattering (dls) instrument (dynapro-ms/x, proterion inc., uk) at • c, and at least measurements were taken at each experiment. all dls measurements were performed at . nm wavelength laser at • angle. the titers of the anti-g antibodies in swine sera were determined using elisa. all washing steps were carried out three times with ttb [ . % tween- in tbs ( mm tris-hcl, mm nacl; ph . )]. all antigen and antibody dilutions were done in tbs. flat-bottom polystyrene microtiter plates were used as solid-phase adsorbents (tpp immunomax high binding flat-bottomed elisa plate, usa). the plates were first coated with anti-swine igg ( / ) (kpl, usa) for overnight at rt. after washing, the plates were blocked with l of sea block blocking buffer (pierce, usa) for h at rt. subsequently, the plates were washed and incubated for h at rt with appropriate dilution ( / ) of the swine sera from infected and noninfected animals. following another round of wash-ing, l of optimum concentration ( ng/well) of the recombinant g protein in tbs was added in the wells and the plates were incubated at rt for further h. after washing with ttb, the appropriate dilution ( / ) of mouse anti-his (amersham biosciences, uk) was added. following h of incubation at rt, the plates were washed and l of the anti-mouse igg alkaline phosphatase (kpl, usa) was added and the microtiter plates were incubated further for h at rt. subsequently, the enzyme-substrate solution, containing mg/ml pnpp (p-nitrophenyl phosphate, sigma, usa) in . m diethanolamine (sigma, usa), ph . was added. the reaction was stopped after min at rt by the addition of l of n naoh to each well and the a values were measured with a microtiter plate reader (bio rad, usa). the extra-cellular domain of the niv g gene was cloned downstream of a t promoter in the prset vector system, which allows the expression proteins via transcription by t polymerase. niv g gene was expressed as a protein attached at its amino-terminus to a polyhistidine peptide. as shown in fig. a , an extra protein band with the expected m r of approximately kda appeared in the insoluble fraction of the extract of bacteria containing the recombinant plasmid and was absent in the corresponding fraction of bacteria containing the vector without an insert. the time course study showed that the highest level of expression could be achieved after h post induction (data not shown). an immunoreactive band of approximately kda was detected by the pooled anti-niv sera on a western blot (fig. b) , confirming the expression of the recombinant g protein. the inclusion bodies, which were extracted from bacterial cells using b-per reagent, were washed several times with the same reagent to remove most of the contaminants. two reasonably pure protein bands of approximately and kda were observed in a polyacrylamide gel stained with coomassie blue (fig. a) . the lower band was detected with swine anti-nipah sera but not with anti-his (data not shown), indicating that it could be either a bacterial protein or his-tag deleted proteolytic product derived from the recombinant protein. the extracted inclusion bodies were ( ) cells containing the vector without an insert, ( ) recombinant g before purification, ( ) purified inclusion bodies, ( ) purified by affinity chromatography. (b) western blot of the above samples using swine anti-niv sera. the same number of the bacterial cells was used for the first two lanes. solubilized in m guanidine hcl containing buffer and directly purified on ni-nta matrix under denaturing conditions. subsequently, the urea was gradually eliminated from the purified protein by dialysis and the protein renatured again. the concentration of the purified recombinant g protein from the total bacterial cell proteins was estimated to be approximately . - . mg g − of the wet weight of bacterial cells. light scattering of the purified protein showed monodispersity with a hydrodynamic radius (r h ) of nm, whereas the inclusion bodies were found to be polydispersed, and polymodal regression analysis in- dicated % of one species with r h nm and % of the another species with r h nm. in order to determine the specificity and sensitivity of the recombinant g protein as antigen for detecting antibodies in niv positive swine sera a quantitive immunoassay was carried out. a total of niv positive sera and negative sera from healthy swine were tested, and the results showed that the purified g protein reacted specifically with antibodies in niv positive samples (fig. ) . this reflects the reliability of the purification procedure in eliminating bacterial contaminants from the g protein. inactivated niv is currently being used as a coating antigen in elisa for detection of the anti-niv antibodies in serum samples. the process of growing the virus in cell cultures, however, requires bl laboratories. in addition, the use of niv is restricted in certain countries due to biosecurity considerations. therefore, an elisa based on a recombinant protein, as capturing antigen, is simpler to produce and perform than elisa based on inactivated virus. the latter is hazardous and not suitable for large-scale surveys. in order to assess the suitability of the g protein as an elisa antigen for the detection of specific anti-niv antibodies, a recombinant prset vector was constructed, which could be used to express the recombinant g protein in e. coli bl si. production of such recombinant proteins in e. coli as capturing antigens for diagnosis of diseases caused by viruses have been widely used including turkey coronavirus (loa et al., ) , marburg virus (saijo et al., ) , norwalklike virus (yoda et al., ) , measles virus (warnes et al., ) , african fever swine virus (freije et al., ) , lassa virus (barber et al., ) , and hepatitis b virus (stahl et al., ) . furthermore, the advantages of using a prokaryotic host to produce recombinant g protein would be considerable due to the ease of scale-up, and the low costs involved in growing bacteria. a strong specific signal was observed in western blots confirming the specific immunoreactivity of the recombinant g protein. the results of the western blot analysis and elisa test suggest that the recombinant g protein exhibits the antigenic epitopes and conformation necessary for specific antigen-antibody recognition. the inclusion bodies before purification showed negligibly low background on western blot but when used as the coating antigen in elisa, a relatively high background was observed. this was probably either due to the presence of bacterial protein (s) and/or existence of some antibodies recognizing conformational epitopes in elisa. the results of the light scattering also confirmed the impurity of the inclusion body sample and monodispersity of the purified recombinant protein. based on the molecular mass of the recombinant g protein ( kda) and the assumption that it is spherical form, the calculated radius of the protein is about . whereas the r h of the purified protein detected by dls was about nm. this implies that the protein most probably exists in multimer forms (trimer or tetramer). in order to ensure a more consistent binding pattern of the coated antigen to the elisa plate and to avoid the high background, it was decided to use affinitypurified recombinant g protein as the coating antigen. the validation of the recombinant g protein elisa was carried out using samples of swine sera. the aim was to determine the variability of negative and positive sera using elisa based on recombinant g protein. the newly established assay was able to distinguish clearly the two groups of samples based on their reactivity. however, the result of the elisa should be interpreted cautiously, since proper standardization of the elisa requires sera from experimentally infected animals followed by testing a more significant number of field serum samples. unfortunately, it was not possible to obtain sera from experimentally infected animal sera due to lack of the bl laboratory throughout this study and also to the limited availability of the field sera. expression of lassa virus nucleocapsid protein segments in bacteria: purification of high-level expression products and their application in antibody detection functional expression and membrane fusion tropism of the envelope glycoproteins of hendra virus nipah virus: a recently emergent deadly paramyxovirus fatal encephalitis due to nipah virus among pig-farmers in malaysia isolation of nipah virus from malaysian island flying-foxes highlevel expression in escherichia coli of the gene coding for the major structural protein (p ) of african swine fever virus nipah virus: vaccination and passive protection studies in a hamster model molecular characterization of nipah virus: a newly emergent paramyxovirus expression and purification of turkey coronavirus nucleocapsid protein in escherichia coli a summary of taxonomic changes recently approved by ictv outbreak of nipah virus infection among abattoir workers in singapore enzyme-linked immunosorbent assays for detection of antibodies to ebola and marburg viruses using recombinant nucleoproteins hepatitis b virus core antigen: synthesis in escherichia coli and application in diagnosis molecular biology of hendra and nipah viruses expression of the measles virus nucleoprotein gene in escherichia coli and assembly of nucleocapsidlike structures outbreak of niv in bangladesh outbreak of nipah and hendra-like viruses in bangladesh expression of recombinant norwalk-like virus capsid proteins using a bacterial system and the development of its immunologic detection this study was supported by the grant no. - - - from the ministry of science, technology and innovation, malaysia (mosti). m. eshaghi is supported by national science fellowship (nsf) from mosti. key: cord- - guapzke authors: dodd, r. title: other emerging viral pathogens date: - - journal: isbt sci ser doi: . /j. - . . .x sha: doc_id: cord_uid: guapzke nan for many years, the greatest concerns about blood safety were focused upon viral infections, most notably hepatitides and retroviral diseases. as a result of experience with these infections, it was widely anticipated that future transfusiontransmissible infections would result from viruses causing persistent infection, most probably parenterally transmitted. however, until around the year , most effort relating to emerging infections was directed towards parasitic diseases. attention was refocused on viral infections as a result of the outbreak of west nile virus (wnv) disease in the usa along with the recognition that it was transmissible by transfusion [ ] . this paper will briefly review currently emerging viruses other than wnv, which have been shown to be, or which have the potential to be transmitted by transfusion. specifically omitted are cmv, ebv, hepatitis a virus and the b erythrovirus, which are demonstrably transmitted by transfusion, but which do not fit the current definition of emerging agents. it should be noted that most rna viruses and also hbv may mutate at a high frequency as a result of an absence of repair mechanisms during nucleic acid replication. this accounts for the large number of different strains and genotypes that are seen among these viruses, in addition to the pseudotypes that develop in individual infected patients. in the case of hbv, selection pressure can additionally lead to the appearance of so-called escape mutants that fail to express some of the antigens that are normally recognized by neutralizing antibodies [ ] . there are a number of different clades of hiv that are broadly grouped as m, o and n. the latter two groups are genetically more distant and, in the case of group o, infection is not always detected by procedures that have been developed to detect antibodies to clade b which was originally most common in the usa and other developed countries [ ] . the potential for continuing development of new clades and of recombinant viruses continues, and appropriate surveillance is necessary to ensure that sensitive tests are always available. in the usa, if a test is not validated for the detection of group o strains, donors must be deferred for a history of travel, sexual contact or residence in countries where these strains are found. similarly, there are six major genotypes of hcv which are distributed around the world in varying frequencies [ ] . although they may differ somewhat in their pathogenesis and responsiveness to therapy, there do not seem to be the same difficulties in detection as have been seen with hiv [ ] . this is also true of the eight genotypes of hbv [ ] . however, in addition to hbv genotypes, there are also a number of mutations, some of which result in the absence of effective expression of all of the epitopes, particularly on the common 'a' antigen. tests constructed with a limited repertoire of monoclonal detection antibodies may be unable to detect some of these mutant strains. again, however, appropriate surveillance and continuous improvement of test kits usually overcomes these problems. severe acute respiratory syndrome (sars) is an example of the explosive emergence of an infection apparently new to humans. the disease, in common with most emerging infections, is a zoonosis and it is thought to have been transmitted to humans in south-east china through the preparation of exotic mammals for human consumption, late in [ , ] . as its name suggests, the disease manifested primarily with respiratory symptoms, but in the more serious cases, was also systemic. it was extremely infectious by the respiratory route, and probably also by the faecal-oral route. as a result of rapid air transportation of infected individuals, it spread rapidly across the globe. between november and july , the who reported symptomatic cases, plus another for which no dates were available. fortunately, this single outbreak was contained, and apart from a small handful of cases among laboratory workers, there does not appear to have been a recurrence of the disease. the infectious agent was rapidly identified by molecular techniques as a coronavirus, novel to humans, but endemic among certain mammalian species in south-eastern china [ , ] . it is reasonable to ask why this outbreak of a new respiratory infection led to concern about blood safety. in addition, as an entirely new disease, nothing was known about it, so preventative measures were taken in accordance with the precautionary principle. the who issued recommendations to reduce the risk of transfusion transmission, as did the fda. these measures included deferral of donors with a history of the disease or contact with a patient and those with a history of travel from an affected area. even relatively casual travel exposure (such as travel through the airport in toronto) led to temporary deferral, at least in the usa. subsequent to the development of these precautions, it became apparent that there could be viraemia during symptomatic disease, but it is still unknown whether there is a presymptomatic viraemia. additionally, epidemiologic studies showed that there were asymptomatic infections, as judged by the prevalence of antibodies to the sars coronavirus [ ] . again, however, it is unknown whether such asymptomatic infection is accompanied by viraemia. there were no recorded cases of transfusiontransmission of sars during the outbreak, although it is unclear whether they would actually have been recognized if they did occur. the human herpes virus is the most recently recognized herpes virus to infect humans [ ] . it is known to be the causative agent of kaposi's sarcoma, both classical and human immunodeficiency virus-associated, and is the likely causative agent of several other rare disorders, including primary effusion lymphoma and multicentric castleman's disease [ ] . it is naturally transmitted through saliva or sexual contact and has been transmitted through organ transplantation. in some cases, recipients of infected organ transplants have subsequently developed kaposi's sarcoma [ ] [ ] [ ] . because of the pathogenic potential of this virus and because of the evident potential for blood-borne transmission, there has been a continuing undercurrent of concern about hhv- among those involved with blood safety. it is important to note, however, that hhv- is not a new agent: rather, it is newly described. presumably, blood from hhv- -infected individuals has been transfused for many years, yet there is no extant evidence of an association between transfusion and kaposi's sarcoma or other hhv- -associated disease states. evidence for transmissibility of hhv- by transfusion has been equivocal at best and there continues to be a somewhat anxious wait for the definitive answer to this key question. in , blackbourn and colleagues [ ] reported on the detection of hhv- dna in the blood of a seropositive blood donor; based on evidence of in vitro passage of the virus to allogeneic cells, the authors expressed concern about the potential for transmission by transfusion. in the same year, however, operskalski and colleagues [ ] used the tss repository to examine samples from recipients of blood from donors who were subsequently shown to be hhv- -seropositive. none of the recipients showed any evidence of infection. similarly, negative data on recipients of seropositive blood units have been reported from jamaica [ ] . in contrast, cannon and associates [ ] demonstrated an increased prevalence of hhv- antibodies among female injection drug users-a result that was interpreted as evidence favouring blood-borne transmission. other studies generated similar results. finally, there have been reports of an increased prevalence of hhv- antibodies among some, but not all, groups of blood recipients [ , ] . it is perhaps worth pointing out that there is, as yet, no clear gold standard for hhv- antibodies and that the performance characteristics of available tests differ considerably and do not seem to correlate well with the presence of viral dna [ , ] . false-positive reactions have also been noted. dollard and colleagues [ ] reported on a study of patient samples from the fact study, which was a major effort designed to determine the frequency of transfusion-associated infections in a large cohort of cardiac surgery patients from to . among the patients who were initially seronegative, had unequivocal increases in hhv- antibody titres months postoperatively. this translated to a risk of · % per unit transfused. although there were no infections among a control group of non-transfused individuals, the difference was not significant. the overall frequency of existing positive test results in the study population was quite high, at · %, although, in a different study, the donor prevalence was found to be · %. as with other studies, there is no clear donorrecipient linkage or real proof that transfusion-associated transmission had occurred. linked donor samples were not available. the study adds to the weight of circumstantial evidence, however, that hhv- may be transmissible via transfusion. more convincing evidence of transmissibility has been developed in a study in uganda, and was recently presented in preliminary form. given the variable behaviour characteristics of tests for hhv- antibodies, and the absence of clear evidence that dna testing would be effective, it is unclear how interventions for this infection will be implemented, if needed. it is, however, quite likely that leucoreduction will be able to effect a substantial, if not complete, abrogation of infectivity. dengue is caused by a mosquito-borne flavivirus which is endemic in most of the tropics. it has the potential for emergence or re-emergence in areas where the vector mosquitoes exist, including the south-eastern usa. by way of example, an outbreak of dengue has occurred in tropical queensland, australia; it was thought to have been introduced by infected travellers. although infection with dengue virus is usually acute, the example of wnv shows that this is not necessarily a barrier to transmission by transfusion. however, given the very high incidence of dengue, it is perhaps surprising that more cases of transfusion-transmitted disease have not been reported. in fact, there has been one well-documented case reported in hong kong, plus a potential case in a bone marrow transplant recipient in puerto rico. it seems likely that the infectious viraemic period must be very short, and/or that cases are not readily recognized as transfusion-transmitted in an epidemic environment. a complication of epidemic dengue for transfusionists is the fact that, because there are four circulating strains of the virus, patients may suffer a second infection. in such cases, an even more serious disease, dengue haemorrhagic fever may result [ ] . this disease state often requires platelet transfusions to correct bleeding, thus further stressing transfusion services. investigations are currently under way to determine the extent of dengue viraemia among blood donors in epidemic areas, such as brazil and puerto rico. this information will help to establish the potential risk of transmission and indicate the extent to which a testing strategy would be effective. there is considerable current concern about the possibility of an influenza pandemic. this concern is based upon the historical fact that there have been periodic pandemics associated with the circulation of new strains of the virus in humans and the current outbreak of the h n strain of avian influenza, which causes high mortality when it does infect humans [ ] . in addition, other strains of avian influenza viruses are also circulating. while there is no evidence that h n will (or will not) result in a human pandemic, its presence has triggered significant levels of pandemic preparedness. as with the sars virus, there is evidence that h n will result in viraemia during symptomatic infection, but it is unknown whether there is a presymptomatic or asymptomatic viraemia. thus, it is unclear whether there will be a risk to blood safety during an influenza pandemic. consequently, it is unclear whether donors would need to be deferred after disease, or after contact with infected individuals. there do not appear to be any documented cases of transfusion transmission of influenza during normal periodic outbreaks, but it is unclear whether such a transmission would actually be recognized. it is also unclear how an intravenously inoculated infection would be manifested. what is clear is that an influenza pandemic would have a profound effect upon the ability of the medical system to provide an adequate supply of blood in the face of widespread sickness among donors and blood centre staff. additionally, it is not entirely clear how a pandemic would impact the needs for blood. hepatitis e virus (hev) causes an epidemic form of hepatitis that is self-limited [ ] . the disease is somewhat similar to hepatitis a, although it is much more severe in pregnancy. transmission is by the faecal-oral route and is most often water-borne. the virus is related to the calicivirus group and, as such, is a non-enveloped virus that has an rna genome. although there is some evidence (based largely on seroprevalence studies) that hev is present in the usa, it is found predominantly in tropical countries. indeed, most cases identified in the usa appear to have resulted from infections that occurred in countries where hev is endemic. hev infection is self-limited and acute and it has generally been thought that there is little risk of transmission by transfusion. until recently, no cases of transfusion-associated hev infection of disease have been reported [ ] . however, a number of recent cases of such transmission have been reported from non-endemic areas, emphasizing that acute infections are also transmissible by transfusion, as long as there is an asymptomatic viraemic phase [ , ] . this has, of course, been abundantly illustrated by the example of west nile virus in the usa [ ] . there has been a continuing search for additional hepatitis viruses and, over the past few years, two different viruses or virus groups have been identified in this context. the first of such putative hepatitis viruses was identified by two separate groups in the late s. in one case, scientists at abbott laboratories looked for genomic sequences related to those of an existing isolate known as the gb virus (gbv), which had previously been associated with hepatitis in a physician. three viruses were identified, one of which (termed gbv-c) was found among a number of human sources [ ] . working in parallel, but using a different approach, scientists at gene-laboratories isolated viral rna sequences and characterized a virus they termed hepatitis g virus (hgv) [ ] . it is generally accepted that these two isolates were, in fact, representatives of essentially the same virus group, which is now known as hgv. hgv, like hcv, appears to be closely related to the flavivirus group. it is found among a relatively high proportion of the normal population, as exemplified by blood donors. its presence has been demonstrated both by seroprevalence studies, in which the frequency of antibodies is % to %, and more interestingly by detection of viral rna in the plasma of % to % of normal subjects [ ] . perhaps not surprisingly, the virus is readily transmissible by transfusion and is found at high prevalence among individuals who have undergone multiple transfusions. however, it has not proved possible to demonstrate that infection with hgv is associated with hepatitis or even with signs of mild liver disease, such as elevated alt levels. indeed, hgv appears to be a virus that is currently in search of a disease. the term 'hepatitis' in its name may be a misnomer, attributable only to the fact that the virus was found in association with hepatitis in the first place. it is also important to recognise that the worldwide distribution of hgv clearly shows that it is not a new virus but, rather, one that has coexisted with humans for many centuries. there have, however, been some intriguing observations that clearly suggest that hgv/gbv-c may have an impact on the course of hiv disease. for example, studies have shown lower mortality among co-infected individuals relative to those with hiv only [ , ] . the mechanism for this effect is unclear, but may be due to the effects of infection on the levels of a number of chemokines [ ] . curiously, another two viruses, ttv and sen-v, were separately identified among individuals with hepatitis and were also shown to be poorly, if at all, associated with hepatitis. these viruses were also found to cause prolonged viraemia and, in some cases, turned out to be present in up to % of the population. like hgv, they were readily transmitted by transfusion. both viruses were thought to be representatives of the circovirus group: small, non-enveloped viruses with a circular dna genome. this group of viruses had not previously been described among humans. workers in japan used representational difference analysis to isolate dna sequences from three patients with unexplained post-transfusion hepatitis. the sequence was established as viral, and the virus was named ttv, reflecting the initials of the patient from whom it was isolated [ ] . a considerable amount of research has revealed some of the key features of this agent [ ] . it is a small virus with a covalently closed, circular dna genome of around bases. the virus is thought to be non-enveloped and is most closely related to the circoviruses, which are responsible for a number of diseases in plants and a handful of mammalian or avian disease states. the classification of ttv is currently incomplete, and proposals have been made to place it in at least two genera. what has become apparent is that ttv is a member of an extremely diverse group of viruses, as demonstrated by considerable variation in genomic sequence. epidemiologic studies have confirmed that ttv is a widely distributed virus and have clearly established that it is transmissible by transfusion. interestingly, it also appears to be transmitted by the vertical, faecal-oral, and, perhaps, other routes. a key issue is the clinical significance of this group of viruses. although the original source of the virus and its apparent association with alt elevations implied that ttv was indeed a hepatitis virus, this identification no longer seems tenable. indeed, there are far more infections without alt elevations than with such evidence of liver disease. even in clear, transfusion-associated transmission of ttv, the recipients did not manifest alt elevations in any pattern that could be associated with the infection. thus, at this stage, there is little evidence that this virus expresses any pathogenic potential. however, it is certainly too early to conclude that this entire group of viruses is without any clinical significance. after the recognition of ttv, the search for hepatitis viruses continued, and primi and colleagues [ , ] used degenerate primers derived from ttv to probe samples from selected patients. an isolate was identified and named sen-v (after the initials of the source patient, an hiv-infected injection drug user). eventually, at least eight different strains were isolated, termed a through h. the sen group has been shown to be one branch of the ttv group, seeming to share its epidemiologic characteristics. although two of the strains (d and h) have been associated with evidence of transfusion-associated hepatitis, a causal relationship has not been established [ , ] . over the past few years, there have been a number of unexpected cases of transmission of infectious agents through organ transplantation. in some cases, such transmissions could imply that the agents may be transmissible by blood, which is certainly true of trypanosoma cruzi , a protozoan parasite that has been involved in at least three clusters of transplantassociated transmission. two viruses have been similarly involved, although it must be acknowledged that neither are currently considered to be emerging pathogens. there have been two well-documented cases of transmission of rabies by organ transplantation [ , ] , plus one cluster of transmissions of lymphocytic choriomeningitis virus (lcmv) [ ] . rabies virus infection is not thought to result in viraemia and it was considered likely that the transmission resulted from infection of, and via the sympathetic innervation of the transplanted organs. on the other hand, lcmv may generate high levels of viraemia in its normal rodent hosts and viraemia was likely in the organ donor, who was shown to have acquired the infection from a pet hamster. there have, however, been no reports of transfusion transmission of either rabies virus or lcmv. at this time, no specific interventions are recommended for blood donors who may have been exposed to either infection. concern has been expressed about the potential for transmission of simian foamy virus (sfv), a retrovirus. it has been found to infect a small number of animal handlers and it has been theorized that such individuals might be able to transmit the virus through transfusion of their blood. however, foamy viruses seem to be apathogenic, both in their natural hosts and in humans. the underlying concern is that viruses that jump species may turn out to be highly pathogenic in the new host. additionally, as discussed above, many retroviruses are prone to frequent mutation, so that pathogenic strains could emerge. the issue has been discussed by regulatory authorities in the usa, and to date, not action has been taken. however, in canada, animal handlers who have frequent contact with primates are deferred. one report details look-back to recipients of blood from an animal handler infected with sfv, but no recipient tested had any evidence of infection with the virus [ ] . at the time of writing, there has been an unexpected outbreak of mumps in the midwestern states of the usa and previously in the uk. although there have been no recorded cases of transmission of mumps virus by transfusion, it is known that there is a viraemic phase and modest precautions to reduce the theoretical risk of transmission have been recommended by the aabb in the usa. in the developed world, the major transfusion-transmitted agents (with the exception of syphilis) have traditionally been viral. interestingly, the general expectation at the end of the th century was that the next threat to blood safety would turn out to be a persistent, sexually transmitted virus. however, this was not the case. the unexpected emergence of wnv and its transmissibility by transfusion has taught us to broaden our thinking around the concept of transfusionassociated infection. nevertheless there are relatively few emerging viral infections that have shown any evidence of transmission by this route. there are certainly numerous viruses that have the potential for such transmission and continued vigilance is necessary. there are, however, no clear patterns that will define appropriate target patient groups for surveillance and we will have to continue to rely upon haemovigilance systems and astute clinicians to identify cases of emerging viral infections among blood recipients. careful and appropriate use of the precautionary principle should also help to maintain the safety of the blood supply. transmission of west nile virus through blood transfusion in the united states in molecular virology of hepatitis b virus significance of hiv- genetic subtypes hepatitis c virus genetic variability: pathogenic and clinical implications evaluation of the performance of assays used in countries with limited resources for the detection of antibodies to hepatitis c virus genetic variability in hepatitis b viruses severe acute respiratory syndrome (sars): a review of the history, epidemiology, prevention, and concerns for the future severe acute respiratory syndrome (sars): a review characterization of a novel coronavirus associated with severe acute respiratory syndrome 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graft, or sexual transmission of viruses human herpesvirus infection and transfusion history in children with sickle-cell disease in uganda multicenter comparison of serologic assays and estimation of human herpesvirus seroprevalence among us blood donors comparison of serologic assays for detection of antibodies against human herpesvirus possible transmission of human herpesvirus- by blood transfusion in a historical united states cohort dengue fever and dengue hemorrhagic fever: a review of the history, transmission, treatment, and prevention human influenza a h n virus related to a highly pathogenic avian influenza virus the discovery of the hepatitis viruses hepatitis e virus: relevance in blood donors and other risk groups transfusiontransmitted hepatitis e caused by apparently indigenous hepatitis e virus strain in hokkaido transfusion-transmitted hepatitis e in a 'nonhyperendemic' country mushahwar ik: sequence and genomic organization of gbv-c: a novel member of the flaviviridae associated with human non-a-e hepatitis molecular cloning and disease association of hepatitis g virus: a transfusion-transmissible agent emerging viral infections relevant to transfusion medicine infection with gb virus c and reduced mortality among hiv-infected patients effect of coinfection with gb virus c on survival among patients with hiv infection inhibition of hiv- replication by gb virus c infection through increases in rantes, mip- alpha, mip- beta, and sdf- a novel dna virus (ttv) associated with elevated transaminase levels in posttransfusion hepatitis of unknown etiology molecular properties, biology, and clinical implications of tt virus, a recently identified widespread infectious agent of humans sen virus infection and its relationship to transfusion-associated hepatitis genomic and molecular evolutionary analysis of a newly identified infectious agent (sen virus) and its relationship to the tt virus family investigation of rabies infections in organ donor and transplant recipients -alabama transmission of rabies virus from an organ donor to four transplant recipients update. interim guidance for minimizing risk for human lymphocytic choriomeningitis virus infection associated with pet rodents simian foamy virus infection in a blood donor key: cord- -wvlczk authors: park, hyun-jun; jang, hye rim; park, shi-young; kim, young-bum; lee, hui-young; choi, cheol soo title: the essential role of fructose- , -bisphosphatase enzyme in thermal homeostasis upon cold stress date: - - journal: exp mol med doi: . /s - - - sha: doc_id: cord_uid: wvlczk skeletal muscle is a major organ for glucose disposal and thermogenesis. while hepatic fructose- , -bisphosphatase is well known as a key enzyme for gluconeogenesis, the role of muscle fructose- , -bisphosphatase (fbp ) in glucose disposal and thermogenesis is unknown. here, using fbp knockout (ko) mice, we assessed the physiological role of fbp in energy and glucose metabolism and thermogenesis. in vivo assessments of energy metabolism, glucose metabolism, and thermogenesis were performed by indirect calorimetry, hyperinsulinemic-euglycemic clamp, and cold challenge studies, respectively. under both feeding and fasting conditions, fbp ko mice showed similar phenotypes regarding energy and glucose metabolism compared to wild-type (wt) mice. however, fbp ko mice were severely intolerant to cold challenge under fasting conditions. mechanistically, the cold-induced intramuscular conversion of lactate to glycogen (glyconeogenesis) is completely abolished in the ko muscle, which leads to a lack of glycogen source for thermogenesis in fbp ko mice. the cold-intolerant phenotype of ko mice disappeared after feeding, and the ko mice were equally as cold tolerant as the wt mice and survived during the cold challenge for three weeks. taken together, these data demonstrate that fbp is essential for muscle thermogenesis by replenishing the intramuscular glycogen pool through glyconeogenesis when the exogenous glucose source is limited. these data imply the physiological importance of fbp in thermal homeostasis and suggest a potential novel therapy targeted to increase glycogen replenishment upon cold stress. skeletal muscle is a major glucose-consuming organ that occupies~ % of body mass , and its insulin resistance is a primary defect in pandemic metabolic syndrome and type diabetes worldwide . a large body of research has been focused on muscle contraction, growth, and exercise performance due to its relevance to health and metabolic disorders ; however, these studies have placed less emphasis on the role of skeletal muscle in regulating body temperature and metabolism. generally, it has been assumed that heat production and metabolic activity in skeletal muscle are primarily caused by contractile activity because muscle shivering is recruited as the first line of defense during acute exposure to cold . shivering is a repetitive contraction-relaxation process that is activated by repeated stimulation of the neuromuscular junction that leads to an elevation of cytosolic ca + concentration, thereby activating adenosine triphosphate (atp) hydrolysis to produce heat . during shivering, heat is primarily produced by the major atp-utilizing enzymes, including na + /k + atpase, myosin atpase, and sarco/endoplasmic reticulum ca + -atpase , . lipids provide most of the heat during low-intensity shivering, whereas carbohydrates become dominant under more extreme cold conditions . the contribution from plasma glucose always remains minor; however, muscle glycogen plays an important role during intense shivering . although acute increases in exogenous glucose uptake and glycogen synthesis by insulin primarily maintain muscle glycogen size , the glycogen pool can be replenished from -carbon units, such as lactate, named glyconeogenesis , . however, whether the size of the muscle glycogen stores influences cold tolerance as well as the physiological importance of glyconeogenesis remain to be demonstrated. in mammals, there are liver and muscle isotypes of fructose- , -bisphosphatase (fbp), encoded by two separate genes, fbp and fbp . the fbp gene is mainly expressed in gluconeogenic organs, such as the liver and kidneys, and is the regulatory enzyme of gluconeogenesis . fbp plays a key role in hepatic gluconeogenesis. in animal models, the inhibition of fbp markedly inhibits gluconeogenesis and increases glucose sensitivity and utilization . in humans, fbp deficiency caused by fbp mutations decreases gluconeogenesis and leads to hypoglycemia, metabolic acidosis during fasting , , and unexpected infant death . in contrast, the fbp gene is expressed in nongluconeogenic organs and was identified in striated muscle by krebs and woodford in . in skeletal muscle, fbp catalyzes the conversion of fructose- , -bisphosphate into fructose- -phosphate and inorganic phosphate and is located in the middle of the glyconeogenesis and glycolytic pathways. several studies have attempted to clarify the physiological meaning of fbp , which is distinguished from the gluconeogenic isotype fbp . a hypothesis was proposed by newsholme in that the futile cycle between fructose- -phosphate and fructose- , -bisphosphate mediated by fbp could regulate whole-body thermal homeostasis . furthermore, the activity of fbp has been known to be increased by cold challenge as well as translocated to the mitochondria by glycogen synthase kinase inhibition , , suggesting its role in thermogenesis. however, whether fbp directly contributes to muscle thermogenesis in vivo and the underlying mechanisms have not been investigated. a study using fbp knockout (ko) animals has not been reported, and there is limited clinical evidence that fbp deficiency is associated with benign nonprogressive myopathy in humans . thus, it is a testable hypothesis as to whether fbp may affect intramuscular glycolysis, glyconeogenesis, or mitochondrial respiration, each of which could contribute to energy metabolism and thermogenesis. here, we performed comprehensive studies using fbp ko and littermate control (wild-type: wt) mice to identify the role of fbp in energy and glucose metabolism and thermal homeostasis in vivo. mice, body composition, and energy metabolism the fat and lean masses of mice were analyzed by an h-nuclear magnetic resonance system (bruker optics, billerica, ma, usa) and expressed as grams. to assess whole-body energy metabolism, a comprehensive laboratory animal monitoring system (clams, columbus instruments, columbus, oh, usa) was applied. the oxygen consumption rate (vo ), carbon dioxide production (vco ), respiratory quotient, energy expenditure, food intake and activity were monitored for h after a -h acclimation period. all experimental protocols were approved by the institutional animal care and use committee of lee gil ya cancer and diabetes institute, gachon university (permission number: - ). mice were maintained on a regular chow diet or highfat diet ( kcal%) for weeks. six to seven days before the hyperinsulinemic-euglycemic clamp, indwelling catheters beneath the back of the neck were positioned in the right internal jugular vein, which extends to the right atrium. to evaluate the basal turnover rate of glucose, [ - h]glucose (perkinelmer, waltham, ma, usa) was infused for h at a rate of . μci/min after overnight fasting. after that, a hyperinsulinemic-euglycemic clamp was performed for min with a continuous infusion of human insulin (eli lilly and company, indianapolis, in, usa). in detail, insulin was infused at a rate of mu/kg/ min for continuous infusion. in order to estimate wholebody glucose fluxes under insulin stimulation, [ - h]glucose was infused at a rate of . μci/min during the clamps. peripheral glucose uptake and metabolism under insulin-stimulated conditions were estimated by a bolus injection of -deoxy-d-[ - c]-glucose ( -dog) (perki-nelmer, waltham, ma, usa) at min during the hyperinsulinemic-euglycemic clamp. whole-body glucose fluxes and glucose uptake of tissues were calculated as previously described [ ] [ ] [ ] . individually housed mice aged - weeks were placed in an environmental chamber (dae han biolink, eumseong, korea) after h of fasting. cold stress experiments of mice at °c were performed for up to h without food but with free access to water. for the longterm cold stress experiment, mice were placed in an environmental chamber with free access to water and food ad libitum. the core temperature of the mice was measured as previously described . to obtain changes in the body temperature of mice, the rectal temperature of mice was measured by using a thermometer coupled with a probe ( , testo, lenzkirch, germany). a % (v/v) glycerol solution was used as a lubricant for the thermometer probe to prevent anal damage. snap frozen tissues from mouse hindlimb muscle were used for protein extraction. once tissues were homogenized in liquid nitrogen, tissue lysis buffer ( s, cell signaling technology, danvers, ma, usa) supplemented with protease inhibitor cocktail (p , sigma-aldrich, st. louis, mo, usa) and phosphatase inhibitor cocktail (p , sigma-aldrich, st. louis, mo, usa) was added. then, lysates were homogenized additionally and incubated for min on ice. samples were centrifuged for min at , rpm, and supernatants were used for immunoblotting. polyacrylamide gel electrophoresis and transfer of proteins to pvdf membranes (ipvh , millipore, burlington, ma, usa) were conducted for immunoblotting. a % (w/v) skim milk solution ( , difco, detroit, mi, usa) based on tris-buffered saline with . % tween- (ibs-bt , intron biotechnology, sungnam, korea) was used for membrane blocking. antibodies were purchased as below. fructose- , bisphosphatase (ab , abcam, cambridge, uk), phospho-pka substrate ( s, cell signaling technology, danvers, ma, usa), uncoupling protein and (ab and ab , respectively, abcam, cambridge, uk), and goat anti-rabbit igg antibody (ap p, millipore, burlington, ma, usa) were used. total rna isolation from cells or tissues was performed by using trizol reagent ( , life technologies, carlsbad, ca, usa) according to the manufacturer's instructions. isolated rna was subsequently reversetranscribed to complementary dna by using the topscript tm rt drymix kit (rt , enzynomics, daejeon, korea). analysis of quantitative real-time pcr was conducted with an abi (applied biosystems, foster city, ca, usa) by using topreal tm qpcr x premix (rt m, enzynomics, daejeon, korea). the relative mrna expression of each target was normalized to cyclophilin a or gapdh. mouse primer sequences are available on request (cycloa, gapdh, fbp , fbp , pygm, g pi, pfk , pgm , gys , pgc- α, tfam, nrf- , cox b, atp b, ndufv ). the snap frozen hindlimb muscle tissues from mice in cold stress experiments were used for quantitative glycogen content analysis. tissue samples were homogenized with . n perchloric acid ( , sigma-aldrich, st. louis, mo, usa) in a mixer at rpm for s. then, μl of perchloric acid homogenate was mixed thoroughly with µl of m potassium bicarbonate ( , sigma-aldrich, st. louis, mo, usa). fresh mg/ml amyloglucosidase ( , sigma-aldrich, st. louis, mo, usa) in acetate buffer was added to µl of the homogenate mixture, and the final solution was incubated at °c for h. after that, the whole mixture was centrifuged for min at , rpm, and the supernatant was used to measure the glucose concentration by using a glucose assay kit (gago- , sigma-aldrich, st. louis, mo, usa). the glycogen content was determined by differences in glucose concentrations before and after amyloglucosidase treatment to exclude free glucose from glycogen in skeletal muscle. for the cellular glycogen content assay, a glycogen colorimetric assay kit (k - , biovision, milpitas, ca, usa) was used according to the manufacturer's protocol. in detail, cell pellets were homogenized in distilled water on ice. homogenates were boiled for min to inactivate enzymes and centrifuged at , × g for min. finally, the supernatant was used to measure the cellular glycogen content according to the protocol provided by the manufacturer. the glyconeogenesis in muscle was measured in muscle tissues using a radioisotope-labeled tracer as previously described with slight modifications . individually housed mice were placed in an environmental chamber after h of fasting. basal group mice were maintained at room temperature, and cold stress group mice were subjected to °c for h with free access to water only. first, krebs-henseleit bicarbonate (khb) buffer (ph . ) containing mm sodium l-lactate (l , sigma-aldrich, st. louis, mo, usa), . μci/ml l- c(u)-lactic acid, and sodium salt (nec uc, perkinelmer, waltham, ma, usa) was gassed with oxygen for h. during oxygenation of the buffer, extensor digitorum longus (edl) muscle tissues were isolated rapidly from mice. next, muscle tissues were incubated in the buffer at °c for h and then washed three times with khb buffer. incubated tissues were added to μl of % koh solution and heated at °c for min. to isolate the glycogen pellet, μl of % w/v na so solution and μl of ethanol were added, and the total mixture was incubated in ice for precipitation. then, the total mixture was centrifuged at , × g for min, and the supernatant was discarded. the pellet was washed three times with ethanol ( %, %, and . % in sequence) and dissolved in distilled water after complete drying. the radioactivity of dissolved glycogen was measured by a liquid scintillation analyzer (tri-carb tr, perkinelmer, waltham, ma, usa). primary muscle cell and mouse embryonic fibroblast culture mouse primary myoblast and embryonic fibroblast isolation was conducted as previously described , with slight modifications. in detail, hindlimb muscle was isolated from neonatal mice ( - days old) and rinsed with cold phosphate-buffered saline (ml - , welgene, daegu, korea). isolated muscle tissue was minced with razor blades on a culture dish, and a -ml mixture of cellular and isolated mitochondrial respiration was assessed by an xf analyzer (seahorse bioscience, billerica, ma, usa) as previously described with slight modifications . briefly, the mm glucose-induced oxygen consumption rate was measured in xf assay media ( mm glucose, mm sodium pyruvate in xf basal media) for cellular respiration analysis. every reagent was diluted in xf assay media and loaded into ports of the flux plate ( μg/ml oligomycin, μm carbonyl cyanide p-trifluoromethoxyphenylhydrazone, and μm antimycin a in sequence as the final working concentrations). for isolated mitochondrial respiration analysis, mitochondria were isolated as previously described with slight modifications . in brief, hindlimb muscle was rapidly removed from overnight-fasted mice and homogenized with a dounce homogenizer in isolation buffer ( mm mannitol, mm sucrose, . % bsa, mm egta, mm hepes, ph . ). then, the homogenate was centrifuged at × g for min. the supernatant was centrifuged again at , × g for min, and the mitochondrial pellet was used for downstream analysis. mitochondria ( μg/ml) isolated from skeletal muscle were added to each well with respiration assay buffer ( mm mannitol, mm sucrose, mm kh po , mm mgcl , mm egta, mm mops, . % bsa, ph . ) supplemented with substrates ( mm pyruvate, . mm, malate, . mm glutamate, and/or mm succinate). then, every reagent was diluted in respiration assay buffer and loaded into ports of the flux plate ( mm adp, μg/ml oligomycin, μm carbonyl cyanide p-trifluoromethoxyphenylhydrazone, and μm antimycin a in sequence as the final working concentrations). all values are expressed as the mean ± s.e.m. the statistical significance of the mean value difference was analyzed by two-tailed student's t-test or anova with bonferroni post hoc analysis using graphpad prism . (graphpad software inc., san diego, ca, usa). p-values less than . were considered significant differences. fbp was abundant in white skeletal muscle, and fbp deletion changed energy metabolism first, we analyzed the expression patterns of the fbp gene in various tissues from c bl/ mice. although fbp mrna was broadly expressed in various nongluconeogenic tissues, including white and brown adipose tissues (fig. a) , the protein expression of fbp was highly specific to muscle tissues and was not detectable in the liver and adipose tissues (fig. b) . in particular, the type fiber abundant extensor digitorum longus (edl) muscle showed higher fbp gene and protein expression than the type fiber abundant soleus muscle (fig. b, c) , which is consistent with previous findings of higher fbp activity in white muscle than in red muscle . fbp gene expression was nondetectable in fbp ko mice (fig. c) . fbp ko mice showed slight but significantly lower body weight than wt mice, which was mostly accounted for by decreased lean body mass, as measured by h-nuclear magnetic resonance (nmr) (fig. d) . indeed, the weight of several skeletal muscles was significantly reduced in ko mice compared to that in wt mice (fig. e) , and the decreased muscle mass was associated with reduced locomotor activity during the comprehensive laboratory animal monitoring system study (fig. f, g) . despite the significantly decreased body weight, fbp ko mice showed reductions in oxygen consumption (vo , fig. h , i), carbon dioxide production (vco , fig. j, k) , and energy expenditure (supplementary fig. b) during the fasting period (light cycle, fig. h, j) . the respiratory exchange ratio and food intake were identical between fbp ko and wt mice ( supplementary fig. a, c) . there was no difference in energy expenditure at the feeding period between wt and fbp ko mice, inferring a specific role of fbp deletion on energy metabolism when the exogenous fuel supply was limited. fbp deletion did not alter glucose metabolism during the feeding condition because energy expenditure showed a different pattern between feeding and fasting periods, we further tested whether fbp deficiency alters glucose metabolism in the fed condition. to measure in vivo whole-body and tissuespecific glucose fluxes under feeding-mimicking conditions where insulin and exogenous glucose are supplied, we performed hyperinsulinemic-euglycemic clamp studies on wt and fbp ko mice as previously reported , . there was no difference in insulin-stimulated whole-body glucose utilization between fbp ko and wt mice, as reflected by the identical glucose infusion rates needed to maintain euglycemia during the steady state (fig. a, b) . hepatic glucose production (hgp) under basal conditions (fig. c ) and the suppression of hgp by insulin, which indicates hepatic insulin sensitivity, were not different between fbp ko and wt mice (fig. d) . insulinstimulated whole-body glucose uptake, glycolysis, and glycogen synthesis rates also showed no differences between wt and fbp ko mice (fig. e) . consistently, skeletal muscle-specific glucose uptake measured by -deoxy-d-[ - c]-glucose was identical between wt and fbp ko mice (fig. f) . we performed further hyperinsulinemic-euglycemic clamp studies under highfat-fed conditions. there was no difference in glucose fluxes between wt and ko mice during the clamp study ( supplementary fig. ). these data demonstrate that fbp deficiency did not alter glucose metabolism in vivo in either the whole-body or skeletal muscle when glucose was exogenously supplied. however, there was a tendency toward a decrease in glycogen content in various skeletal muscles after fasting (fig. b, c) . a decreased muscle glycogen content (fig. d) and energy metabolism (fig. ) in ko mice were observed only after fasting; therefore, we assumed that fbp might have more crucial roles in the severe glycogenolytic condition. thus, we further tested the role of fbp in thermogenesis under more severe glycogenolytic conditions by challenging fbp ko mice with both cold and fasting. after h of cold exposure without food, fbp ko mice exhibited more severe cold intolerance than wt mice, and most of the ko mice had to be euthanized after h of cold exposure due to the humane concern of severe hypothermia (fig. a) . fbp ko mice showed significantly decreased glycogen content in their tibialis anterior (fig. b) compared to ko mice after cold exposure. we also further tested whether glycogenolysis was accelerated by fbp deletion by measuring the muscle glycogen content at various time points after the cold challenge. consistent with the decreased body temperature patterns (fig. a) , the muscle glycogen content was significantly reduced after h of cold exposure (fig. c) in both wt and ko mice. however, during the h of cold exposure, the net amount of muscle glycogen depletion was identical between wt and ko mice (fig. c, right panel) , indicating that cold exposure did not accelerate the glycogenolysis rate by fbp deletion. however, there was a decrease in the basal glycogen content in the quadriceps muscle from overnight-fasted ko mice even at room temperature ( ± °c) (fig. d) . consistently, (see figure on previous page) fig. fbp was abundant in white skeletal muscle, and fbp deletion changed energy metabolism. a tissue-specific gene expression patterns of fbp and fbp in wt mice analyzed by quantitative reverse transcription polymerase chain reaction (rt-pcr). nd nondetected. n = per group. b tissue-specific fbp protein expression pattern in wt mice analyzed by immunoblotting. c fbp gene expression in the soleus and extensor digitorum longus muscle of wt mice analyzed by quantitative rt-pcr. n = per group. d body composition analysis by the h-nuclear magnetic resonance system including body weight, fat mass, and lean mass. n = per group. e weight of various skeletal muscles. n = per group. f locomotor activity, h whole-body o consumption (vo ), and j whole-body co production (vco ) of wt and fbp ko mice during the -h analysis. n = per group. h, day and night time averages of g locomotor activity, i vo , k vco . gas gastrocnemius, ta tibialis anterior, qd quadriceps, edl extensor digitorum longus. data are expressed as the mean ± sem. * p < . , ** p < . by student's t-test for (c-e, g, i, k). ++ p < . , +++ p < . by two-way analysis of variance (anova) with post hoc analysis for (f) and # p < . by student's t-test at each time point for h and j. ns nonsignificant. phosphorylation of pka substrates in ko muscle was sufficiently increased compared to that in cold-exposed wt muscle, even at room temperature (fig. e) . furthermore, the mrna expression of glycogenolytic genes showed an increasing trend in the ko muscle, whereas the glycogen synthase gene was decreased at room temperature (fig. f) . these data indicate that the decreased cold tolerance in fbp ko mice is accounted for by the limited fuel source due to the decreased basal content of muscle glycogen but not by an acceleration of glycogenolysis. to further differentiate the effect of exogenous fuel source on body temperature maintenance under cold stress from that of the endogenous form, we conducted a cold challenge experiment with free access to food for longer periods, up to weeks. there was no difference in body temperature (fig. g) or body weight (fig. h) during the -week experiment between wt and fbp ko mice. food intake was equally~ -fold greater and markedly increased during the cold stress experiment in both wt and ko mice (fig. i) . these results demonstrate that fbp -mediated thermogenesis did not have a substantial effect on thermal homeostasis when food was supplied but impaired whole-body thermal homeostasis when the exogenous fuel source was limited. to address the mechanism underlying the reduced glycogen content in ko mouse muscle, we next tested whether fbp plays a role in intramuscular glyconeogenesis. in the s, it was proposed that a glycogen replenishment pathway other than cori cycling may exist. a few studies have suggested that intramuscular glyconeogenesis may take part in glycogen replenishment after fig. fbp deletion did not alter glucose metabolism during feeding-mimicking conditions. a plasma glucose level, b glucose infusion rate during hyperinsulinemic-euglycemic clamp, c basal and clamped glucose level, d percentage of suppression of hepatic glucose output, and e clamped glucose uptake, glycolysis rate, and glycogen synthesis rate calculated using the hyperinsulinemic-euglycemic clamp results. n = per group. f muscle -dog uptake results. n = - per group. hgp hepatic glucose production, rd glucose disposal rate, gs glycogen synthesis rate. data are expressed as the mean ± sem and were analyzed by two-way anova with post hoc analysis for a and b and by student's t-test for c-g. ** p < . , *** p < . , ns nonsignificant. fig. fbp was important for cold tolerance during fasting conditions. a body temperature changes in wt and fbp ko mice in cold stress experiments, n = for the rt group; n = for the cold group. b glycogen content in the ta and qd muscles of wt and fbp ko mice after cold stress experiments. n = per group. c glycogen content depending on cold exposure time (left) and glycogen content decrease after cold stress experiments (right) in the ta muscle of wt and fbp ko mice. n = per group. d glycogen content in the qd muscle of wt and fbp ko mice at room temperature. n = per group. e immunoblotting results of pka substrate phosphorylation in wt and fbp ko mice in cold stress experiments. n = per group. f gene expression analysis in the gas muscle of wt and fbp ko mice after cold stress. n = - per group. g body temperature change, h body weight change, and i daily food intake of wt and fbp ko mice in cold stress experiments for weeks. n = - per group. h hour, ta tibialis anterior, qd quadriceps, pka protein kinase a, pygm glycogen phosphorylase, muscle associated, g pi glucose- -phosphate isomerase, pfk phosphofructokinase , pgm phosphoglucomutase , gys glycogen synthase . data are expressed as the mean ± sem. * p < . , ** p < . , *** p < . by student's t-test. ++ p < . , +++ p < . by two-way anova with post hoc analysis for a. ns nonsignificant. metabolic stress, such as exercise, and this pathway may be mediated by fbp in skeletal muscle , ; however, this glyconeogenesis has never been tested using fbp ko mice or by challenging with cold stress. thus, both at room temperature and after h of cold challenge, we conducted experiments using a radioisotope-labeled tracer ( c(u)-lactate) to quantitatively measure the incorporation of lactate into glycogen in the skeletal muscle of wt and fbp ko mice (fig. a) . glyconeogenesis was almost completely blocked in the edl muscle of fbp ko mice, as shown by an~ % decrease in lactate incorporation into glycogen compared to wt mice at f isolated mitochondrial respiration analysis of wt and fbp ko mice, n = per group. g cellular respiration analysis (left) and area under the curve of the oxygen consumption rate (right) of primary myoblasts isolated from wt and fbp ko mice, n = per group. edl extensor digitorum longus, ta tibialis anterior, qd quadriceps, mefs mouse embryonic fibroblasts, tg transgenic, adp adenosine diphosphate, om oligomycin, fccp carbonyl cyanide- -(trifluoromethoxy)phenylhydrazone, am antimycin. data are expressed as the mean ± sem. * p < . , ** p < . , *** p < . by one-way anova for (b), (e) and two-way anova for f, (g, left). ** p < . , *** p < . by student's t-test for c-d, (g, right). room temperature (fig. b) . these results imply that intramuscular glyconeogenesis exists even at room temperature. since the subthermoneutral temperature of mice is~ °c, room temperature (~ °c) could contribute to the decrease in muscle glycogen content in ko muscle (fig. b) . furthermore, after h of cold challenge, glyconeogenesis was markedly induced by~ % in the wt mouse muscle, whereas it remained blocked entirely in the muscle of the fbp ko mice (fig. b) , implying a more critical role of fbp under cold conditions. consistent with the edl muscle results in fbp ko mice, intramuscular glycogen replenishment was significantly decreased in the tibialis anterior (ta) and quadriceps (qd) muscles of fbp ko mice after acute cold challenge compared to wt mouse muscles (fig. c) . furthermore, using mouse embryonic fibroblasts (mefs) from fbp transgenic mice, we measured the in vitro glycogen content after cold-mimicking stimulation (isoproterenol, a nonselective β-adrenergic receptor agonist). consequently, the glycogen content was decreased by isoproterenol in wt mefs but maintained in fbp overexpressing mefs (fig. d, e) , indicating the preservation of glycogen by fbp -mediated glyconeogenesis. furthermore, we investigated whether other heat sources, such as mitochondrial uncoupling and mitochondrial respiratory function, were affected by fbp deletion in wt and ko mice. the cold challenge itself significantly increased the expression of the pgc- α gene both in brown adipose tissue and gastrocnemius (gas) muscle; however, there was no significant difference between genotypes ( supplementary fig. a) . after the cold challenge, ucp protein expression was identical between wt and ko mouse muscle, dissociating muscle mitochondrial uncoupling from the cold-intolerant phenotype of ko muscle ( supplementary fig. b, right) . the expression of pgc- α mrna ( supplementary fig. a , left) and ucp protein ( supplementary fig. b , left) in brown adipose tissues showed increasing trends in ko mice after cold challenge, which was likely a compensatory response to the decreased body temperature in the fbp ko mice. accordingly, the expression of mitochondria-related genes in the gas muscle ( supplementary fig. c ) and the mitochondrial oxygen consumption rate with isolated mitochondria from gas muscle were unchanged (fig. f) . however, with primary cultured myoblasts from the wt and fbp ko mouse muscles, cellular oxygen consumption rates were significantly decreased in ko mouse myoblasts (fig. g) , which was consistent with the decreased whole-body v o and v co rates in the indirect calorimetry study (fig. h, j) . these data suggest that fbp affects thermogenesis mainly by regulating the intracellular glycogen content rather than directly changing mitochondrial function or other thermoregulatory pathways. the physiological meaning of intramuscular glyconeogenesis has been questioned for over a decade since skeletal muscle mainly replenishes glycogen from exogenous glucose , or via liver gluconeogenesis, named cori cycling . indeed, when the oxygen supply is insufficient, typically during intensive exercise, lactate produced by anaerobic glycolysis in the muscles moves to the liver and is converted to glucose, which then returns to the muscles . we do not doubt that cori cycling is the major pathway for preventing lactic acidosis in muscle and further drives additional glycolysis reactions via nad + generation for most physiological conditions, including fasting and exercise. however, it is not clear whether intramuscular glyconeogenesis is an alternative pathway for replenishing muscle glycogen under specific conditions, such as cold exposure. furthermore, we questioned why muscle chooses the energetically inefficient method of glycogen replenishment because gluconeogenesis via the liver requires more atp consumption than glyconeogenesis in skeletal muscle, which bypasses the hexokinase step. in the present study, we discovered that acute cold challenge dramatically induced glyconeogenesis in the edl muscle of wt mice (fig. b) without increasing the muscle lactate concentration (data not shown). this suggested that muscle glyconeogenesis could have a critical contribution to muscle glycogen replenishment after cold challenge, which might mimic aerobic glycogenolytic conditions. moreover, in the present study, we found the physiological importance of fbp in the thermal homeostasis of the whole-body under acute cold stress and demonstrated intramuscular glyconeogenesis as an underlying mechanism of thermogenesis. these findings support an old hypothesis by holloszy in , which stated that the existence of intramuscular glyconeogenesis and the involvement of fbp in this pathway might contribute to glycogen replenishment from endogenous carbon sources. however, whether lactate can be resynthesized to carbohydrates in skeletal muscle under physiological conditions is controversial, and the physiological role of fbp has been unclear for two decades [ ] [ ] [ ] . this is likely due to the following three reasons. first, the majority of previous studies focused on exercise conditions , , , where most lactate was oxidized by the mitochondria rather than converted to glycogen due to the increased atp demand after physical activity. second, previous studies have focused on glycogen synthesis with exogenous glucose supply , . therefore, it might be difficult to investigate the role of intramuscular glyconeogenesis in skeletal muscle glycogen metabolism. third, experimental evidence supporting this concept , , , has never been conducted under the in vivo condition of fbp deficiency. we believe that the most important experimental design of our study that differentiated it from previous studies was the cold challenge without exogenous glucose supply. first, in the present study, we found that fbp ko mice exhibited decreased whole-body energy metabolism only during the light phase when food intake was minimized. second, we used the hyperinsulinemic-euglycemic clamp technique to mimic the feeding state, which demonstrated that there was no difference in exogenous glucose uptake between fbp wt and ko mice. third, under fasting conditions, fbp ko mice were cold intolerant and severely hypothermic after the acute cold challenge. however, when food was supplied, the fbp ko mice survived well without any decreases in body temperature even after weeks of the cold challenge. consequently, these results indicate that intramuscular glyconeogenic flux is crucial for the maintenance of the endogenous glycogen pool and muscle thermogenesis. however, our study also had certain limitations and assumptions that were not fully addressed. first, we assumed that the fructose futile cycling rate would be affected by the decreased glyconeogenesis in the fbp ko mice, but we could not provide any in vivo flux data for the futile cycling rate in the fbp ko muscle due to the technical limitations of the tracer methods. future studies with a new isotope tracer methodology may provide more direct evidence for futile thermogenesis. second, there are further questions that need to be addressed in future studies regarding whether the present findings of this study can be extended to clinical interpretation in humans. although we focused on the relationship between cold tolerance and intramuscular glyconeogenesis, the physiological role of fbp may be extended to other conditions where skeletal muscle glycogen breakdown replenishment is increased. we demonstrated in the present study that fbp is closely related to the endogenous glucose source of skeletal muscle; therefore, further experiments with aerobic exercise or prolonged fasting could provide clinical insight into fbp in various physiological conditions. in summary, the present study highlights new knowledge regarding fbp in glycogen metabolism and provides information on the importance of intramuscular glyconeogenesis on energy balance and thermal homeostasis in vivo. furthermore, the present study suggests that fbp -mediated intramuscular glyconeogenesis is a novel mechanism contributing to muscle glycogen replenishment and thermogenesis when exogenous glucose supplementation is limited. skeletal muscle mass and distribution in men and women aged - yr skeletal muscle insulin resistance is the primary defect in type diabetes sarcolipin: a key 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muscle with c nmr in vivo regulation of muscle glycogen synthase and the control of glycogen synthesis substrates for muscle glycogen synthesis in recovery from intense exercise in man lehninger principles of biochemistry Über die energieumwandlungen im muskel muscular activity and carbohydrate metabolism Über die synthes des kohlehydrats im muskel glycogen synthesis and metabolism of lactic acid after exercise synthesis of glycogen from glucose and lactate in isolated rat soleus muscle the authors declare that they have no conflict of interest.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.supplementary information accompanies this paper at https://doi.org/ . /s - - - . key: cord- - norumv authors: vere hodge, r. anthony title: meeting report: th international conference on antiviral research, in raleigh, nc, usa date: - - journal: antiviral res doi: . /j.antiviral. . . sha: doc_id: cord_uid: norumv the th international conference on antiviral research (icar) was held in raleigh, north carolina, usa from may to , . this article summarizes the principal invited lectures. john drach (elion award) described the early days of antiviral drugs and their novel modes of action. piet herdewijn (holý award) used evolutionary pressure to select dna polymerases that accept nucleoside analogs. replacing thymine by -chlorouracil led to the generation of a new form of escherichia coli. adrian ray (prusoff award) demonstrated how prodrugs can markedly improve both the efficacy and safety of potential drugs. the keynote addresses, by david margolis and myron cohen, tackled two emerging areas of hiv research, to find an hiv “cure” and to prevent hiv transmission, respectively. these topics were discussed further in other presentations – a cure seems to be a distant prospect but there are exciting developments for reducing hiv transmission. tdf-containing vaginal rings and gsk- , as a long-lasting injection, offer great hope. there were three mini-symposia. although therapy with tdf/ftc gives excellent control of hbv replication, there are only a few patients who achieve a functional cure. myrcludex, an entry inhibitor, is active against both hbv and hdv. the recent progress with hbv replication in cell cultures has transformed the search for new antiviral compounds. the hbv capsid protein has been recognized as key player in hbv dna synthesis. unexpectedly, compounds which enhance capsid formation, markedly reduce hbv dna synthesis. the development of bcx , which is active against marburg and ebola viruses, is of great current interest. this article provides an overview of the invited lectures at the th international conference on antiviral research, sponsored by the international society for antiviral research (isar), which was held in raleigh, north carolina, usa from may to , . it begins with reports of lectures by the recipients of isar's three major awards, held in memory of gertrude elion, antonín holý and william prusoff. these are followed by brief summaries of the keynote addresses and the three mini-symposia on ''hepatitis b virus'', ''research triangle park'' and ''challenges in hiv infection, treatment and prevention''. because this review article simply provides short accounts of oral presentations, it is not generally accompanied by references to the scientific literature. any descriptions of favorable treatment outcomes should not be taken as recommendations for clinical use. . gertrude elion memorial award lecture: collaborative antiviral studies for the discovery of drugs to treat cytomegalovirus infections john c. drach, ph.d., university of michigan, ann arbor, michigan, usa (fig. ) . gertrude b. (trudy) elion was born in new york city and was pleased to work for the burroughs wellcome co. when based in new york but was concerned when it transferred to research triangle park, north carolina, not many miles from this year's meeting site. however, within just a few months she declared that she was ''at home'' in north carolina. she was awarded the nobel prize in physiology or medicine in for her pioneering work in purine biosynthesis which paved the way for the discovery of drugs to treat organ rejection, cancer and viral diseases. the focus of john's presentation was on the research conducted in his own and his collaborators' laboratories that ultimately led to the invention of three compounds which were discovered to have antiviral activity against human cytomegalovirus (hcmv) and which later entered clinical trials: bdcrb pyranoside (gw x) (phase i), maribavir (phases i, ii and iii) and cyclopropavir (phase i). his major collaborators included karen biron, charles shipman, leroy townsend, and jiri zemlicka. to date, there are only five fdaapproved drugs for treatment of hcmv infections: cidofovir, fomivirsen, foscarnet, ganciclovir and valganciclovir. being inspired by the presence of a naturally-occurring , dimethylbenzimidazole nucleotide in vitamin b , research on benzimidazole nucleosides was initiated by medicinal chemists in the s and ' s. this led to the synthesis of a trichloro analog in townsend's laboratory at the university of utah and later the discovery of its activity against hcmv in john's laboratory. much work, in both their laboratories at the university of michigan, established that it and its -bromo analog (bdcrb) have excellent activity against hcmv with very low cytotoxicity. surprisingly, it was found to be inactive against other herpes viruses and it did not need conversion to a triphosphate to be active against hcmv. collaborative studies with karen biron at burroughs wellcome established that, unlike many other anti-virals that inhibit viral dna synthesis such as ganciclovir (gcv), these compounds acted by a novel mechanism, inhibition of viral dna processing. it was the viral resistance studies which revealed the viral targets, pul and pul . these two proteins, with pul , form a complex known as the terminase which cuts newly synthesised hcmv dna into unit lengths for packaging into virions. although bdcrb had many desirable properties in vitro, it had poor pharmacokinetics in mice and monkeys due to hydrolysis of its glycosidic bond; therefore it was not developed for human use. much additional work in drach's and townsend's laboratories at michigan and by biron's group at burroughs wellcome ultimately led to two potential drug candidates, bdcrb pyranoside and maribavir (fig. ) . both compounds have excellent activity against hcmv, low toxicity, and excellent pharmacokinetics. clearly, their modes of action differed markedly from that of gcv. quite unexpectedly, they have different mechanisms of action. bdcrb pyranoside has a mechanism of action very similar to its parent compound bdcrb, inhibition of dna processing. in contrast, maribavir inhibits dna synthesis, albeit indirectly. it is a isopropylamine derivative of bdcrb except that it has the unnatural l-sugar configuration. its mechanism of action involves inhibition of the viral kinase (pul ), which phosphorylates another viral protein, pul . phosphorylated pul is necessary for viral dna synthesis. thus inhibition of pul by maribavir inhibits viral dna synthesis. interestingly, pul is also the kinase that activates (phosphorylates) gcv. resistance studies confirmed that a single mutation in ul , resulting in a mutation in the kinase (leu arg), was necessary and sufficient for resistance to maribavir. in a further study of resistance, virus already resistant to bdcrb was passaged in increasing concentrations of maribavir and resistant virus was isolated. this strain grew at the same rate as the wild-type virus and was resistant to both bdcrb and maribavir. as expected, resistance to bdcrb was due to known mutations in ul and ul . however, no mutations were found in ul . further investigation showed that a single base change in ul (t c) was necessary and sufficient for resistance to maribavir. the role of the encoded protein was then unknown but the amino acid mutation (leu pro) is in the middle of the protein. similarly, biron's group detected resistance due to mutations in the ul gene. further research studies on maribavir have been summarized in previous icar scientific reports. cyclopropavir (cpv, fig. ) was synthesized in the laboratory of jiri zemlicka, karmanos cancer institute, detroit, michigan. it is a guanosine nucleoside analog which is very active against hcmv. unlike the benzimidazole nucleosides, it also inhibits epstein-barr virus (ebv) and human herpesvirus (hhv- ). like gcv, it is phosphorylated by the kinase encoded by ul . it is more potent in vitro and in vivo than ganciclovir but has a somewhat different pattern of resistance. in one resistant strain, the key mutation formed a stop codon resulting in a truncated pul kinase protein. the phosphorylation of cpv by pul is more efficient than that of gcv, with a considerably lower k m and higher v max . interestingly, the phosphorylation of cpv to its monophosphate (cpv-mp) by pul is stereoselective; only the (+) isomer of cpv-mp is formed. a single enzyme, gmp kinase, phosphorylates cpm-mp to both its di-and triphosphates. in contrast, acyclovir and gcv require additional cellular enzymes to convert their diphosphates to active triphosphates. cyclopropavir is currently in phase i clinical trials for the treatment of hcmv infections. . the antonín holý memorial award lecture: from modified nucleoside to a chemically modified genome piet herdewijn, rega institute for medical research, ku leuven, belgium (fig. ) . the icar began with a symposium, on the legacy of the late antonín (tony) holý , at which the establishment of a new isar award in medicinal chemistry was announced. the awardee is to be a senior scientist of international stature in medicinal chemistry and who has made innovative contributions impacting antiviral drug discovery or development. piet is, therefore, the first to receive this award. in the late s, the potent activities of bvdu and bvarau against herpes simplex virus type (hsv- ) and varicella zoster virus (vzv) were discovered; this work motivated piet to start antiviral research with the synthesis of carbocyclic bvdu. through to the early s, he synthesized several other nucleoside analogs with bicyclic bases having good activity against hsv- and vzv. during the s, emphasis switched to investigating the effect of modifying the sugar ring, in particular the synthesis of six-membered rings containing an oxygen or a double bond. piet showed examples of compounds with activity against hsv- , hsv- , vzv and hcmv. back in , erik de clercq showed piet a paper on aids, one of the authors being phil furman. this publication stimulated the search for anti-hiv compounds. many compounds were discovered with potent activities (and good selectivity indices) against hiv. piet worked out the first structure-activity relationships of anti-hiv dideoxy nucleosides. starting in the late s, tony holý synthesised a series of phosphonates. at the icar, erik de clercq recalled how this work led, ultimately, to tenofovir, which was to become a major success for treating hiv-infected patients. from its first introduction in , its market share has increased to well over %. in , having a single-pill regimen was agreed as a way forward to simplify, and thereby enhance, hiv therapy. this led to atripla being approved in , complera in and stribild in . tenofovir, in its various prodrug forms, is now available in over countries and is distributed widely to the known hivinfected population. in line with this research, piet synthesized phosphonate nucleosides, with a threose sugar moiety, which showed anti-hiv activity in the same range as -( -phosphonylmethoxyethyl) adenine (pmea). piet's work had taken a different pathway. it is possible to link several nucleotides together to form aptamers. for example (fig. ) , the above antiviral nucleosides, which have a -membered ring in place of the natural furanose, could be incorporated into hexitol nucleic acid (hna) aptamers. x-ray studies revealed the structures of hna-rna duplexes and hna-hna duplexes, the latter having a similar overall form to that of an rna-rna duplex with the same base sequence. hna-containing aptamers were shown to be potent and specific inhibitors of trans-activating region (tar)mediated transcription. normally, an hiv encoded protein, transactivator of transcription (tat), binds to cellular factors and to the viral tar rna regulatory element, resulting in a vastly increased rate of transcription of all hiv genes. hna-containing aptamers prevents this interaction and so inhibit hiv replication. it took four years to engineer a polymerase that would utilise hnas to assemble a strand complementary to a dna template. in line with this research, hexitol-modified sirna has shown good activity in an in vivo anti-hbv model. this success stimulated the concept that it may be possible to generate new forms of biologically active dna. in order to pursue this idea, a culture system with twin growth chambers was devised. alternative nutrient media could be fed into the chambers and the culture from one chamber could be used to seed the second chamber, the former culture being removed. in this example, the aim was to replace thymine with -chlorouracil ( fig. ) using escherichia coli. initially, the nutrient contained % -chlorouracil and % thymine. with each cycle, seeding one chamber from the previous one, the proportion of -chlorouracil was increased. after days, in which there had been about generations of e. coli, thymine had been replaced totally by -chlorouracil. an interesting outcome was that the alternative base led to a change not only in the genotype but also in the phenotype; the ''new'' e. coli cells were much longer than the original. this is the first example of a dna polymerase being adapted through evolutionary pressure to accept a nucleotide analog, resulting in the generation of a new living organism. prusoff young investigator award lecture: use of nucleotide prodrugs to enhance selectivity of anti-hiv and -hcv agents adrian s. ray, gilead sciences inc., foster city, ca, usa (fig. ) . adrian started his lecture with photos of william (bill) prusoff and reminisced of his days with bill, raymond schinazi and yung-chi (tommy) cheng. adrian presented examples to illustrate two models of how a prodrug strategy can transform a potential drug into a much improved clinical candidate. in the first, the prodrug alters the distribution of the pharmacologically active nucleotide analog to tissues where viral infection is taking place (on-target) and away from tissues resulting in adverse events (off-target). in the second, the prodrug enables one to select a drug candidate based more directly on the intrinsic properties of the active nucleotide-triphosphate analog via by-passing an inefficient activation (phosphorylation) of the corresponding nucleoside analog. sofosbuvir (sovaldi Ò ), a prodrug of -f- -c-meump, was approved in the usa on th december, for treatment of patients with hepatitis c. this is a fine example of a prodrug enhancing the activity of the parent compound. the nucleoside analogue, -f- -c-meu, is poorly active due to restricted phosphorylation to the monophosphate. sofosbuvir, a nucleotide analogue prodrug of -f- -c-meu, delivers the monophosphate into the cell and this is then further phosphorylated efficiently to give high levels of the triphosphate which inhibits hcv rna polymerase. adrian recalled being much impressed by a result reported at the meeting in of the american association for the study of liver diseases (aasld). in a phase ii monotherapy trial in patients with hcv, at day , the viral loads were reduced by log . and log . for vx- ( mg bid, n= ) and rg- ( mg bid, n= ), respectively. however, from day to , the polymerase inhibitor (rg- ) had continued to reduce the viral load, reaching a reduction of log . . on the other hand, the protease inhibitor (vx- ) did not give a sustained reduction, with the viral load starting to increase from day . at day , the viral load was only log . less than baseline. nucleotide analogues have two advantages over other classes of inhibitors. there is a high genetic barrier to resistance selection, due to the hcv rna polymerase being highly specific for its natural substrates and template. this specificity can be altered but only under extreme evolutionary pressure (see section ). also, nucleotide analogs often have pan-genotype activity because the active site of the hcv ns b polymerase is so highly conserved. as an example of how prodrugs can impact a discovery program, allowing for more targeted delivery and for the optimization of the intrinsic properties of the triphosphate, adrian presented the history of the gs- program. the c-adenine analogue ( -c-me- -aza- , -dideazaa, c-nuc ) was compared to the corresponding n-nucleoside, mk . in a genotype b replicon assay, the ec values were . lm and . lm respectively. however, their triphosphates were equally effective against hcv ns b polymerase (ic values both . lm). in the replicon system, the triphosphate of the n-nuc (mk ) was formed more efficiently than that of the c-nuc , thus explaining the lower activity of the c-nuc . however, in primary human hepatocytes, c-nuc was phosphorylated to the triphosphate more efficiently than the n-nuc (mk ). this illustrates the importance of using primary human cells. c-nuc seemed to have a benign in vitro toxicity profile, including not inhibiting the mitochondrial dna polymerase-gamma, but it had very significant toxicity in animals. in a collaboration between gilead and craig cameron at pennsylvania state university, the researchers sought to identify the toxicity target(s) for ribonucleotide analogues, including c-nuc and others that had been stopped in phase ii trials. these studies showed a correlation between c-nuc and the phase ii candidates, r , nm and bms /idx . all the latter were efficiently incorporated into rna by the mitochondrial rna polymerase (> % of the corresponding natural nucleotide). the triphosphate of c-nuc was also an efficient substrate ( % the rate of atp). in contrast, the active nucleotide analogs, formed by drugs approved for the treatment of hcv, were poor substrates. ribavirin was poorly incorporated (about %) and sofosbuvir was below the limit of detection (= . %). more extensive in vitro and cell culture evaluation of the compounds could have saved the expense of taking them into clinical trials. understanding that the mitochondrial rna polymerase is an important target for ribonucleotide toxicity, the gilead team sought analogs that were not incorporated by this polymerase. adding a cn group to the position of c-nuc did not change its activity as an hcv ns b polymerase inhibitor (ic . lm) but it did reduce incorporation in the mitochondrial rna assay (< . %). however, in the absence of a nucleotide prodrug to bypass the first phosphorylation step, the resulting di-substituted nucleoside analog would not be a drug candidate because it was not efficiently activated in cells. application of a nucleotide prodrug strategy allowed this nucleotide to be pursued further. oral absorption, delivery of the monophosphate into hepatocytes and high hepatic extraction were criteria used as part of the prodrug optimization process. a nucleotide prodrug, gs- (a mixture of diastereoisomers at phosphorous) was well absorbed in dogs (> %). comparing the pre-hepatic and post-hepatic plasma drug levels, about % of the absorbed drug was taken up by the liver. inside cells, gs- was converted to the corresponding monophosphate which was efficiently converted to the triphosphate. at h, the triphosphate levels remained about -fold above the ic value. a pure stereoisomer was selected and later named gs- . in a phase ii trial ( mg, bid days), the mean reduction in hcv load was about log . . two subjects achieved hcv rna < iu/ ml. however, the pharmacokinetics and antiviral responses were highly variable. whereas the activity results were disappointing, clinical proof of concept was observed in terms of safety. gs- did have a markedly improved safety profile relative to c-nuc , progressing through chronic toxicology studies in rats and dogs at relatively high doses. the story of gs- illustrates both how nucleotide prodrugs enable further progression of candidates and also the complexity of predicting the behavior of nucleotide prodrugs across species. one wonders what cell culture test or animal model may have predicted such variability. when selecting famciclovir as the prodrug for penciclovir, one potential prodrug was rejected because the pharmacokinetics in rats varied widely between individual animals (vere hodge et al., ) . a recent publication by adrian and his team highlights the metabolism of gs- by carboxylesterase , an enzyme highly expressed in the human small intestine but not uniformly expressed in different animal species, as a possible reason for the highly variable and suboptimal intestinal absorption of gs- in humans (murakami et al., ) . the focus of adrian's talk then switched to hiv. over the last or years in north america, the hiv-infected population has been changing, becoming older (now % over years old vs < % in ) and more likely to be obese (in every usa state, > % adults with bmip ). this has led to a shift in the focus of antiretroviral therapy (art), from solely control of hiv replication to now include tolerability in older, possibly obese, patients. the first example given for hiv was how application of a different prodrug strategy can markedly change the distribution even when delivering the same pharmacologically active nucleotide analog. the first approved prodrug of tenofovir (tfv) was tfv disoproxil fumarate (tdf). more recently, tfv alafenamide (taf) has been progressed into clinical development. a key difference in the properties of the two prodrugs is their stability in plasma, with half-lives of . and min, respectively. even with a short halflife, tdf gave better delivery of tfv into cells, as indicated by the hiv ec values in cell culture assays but there clearly was room for improvement; the ec values for tfv, tdf and taf are . , . and . lm respectively. whereas the gain in cell culture ec value may be modest, this is not the only gain. the increased stability of taf allows it to load on-target cells and tissues (e.g., lymph nodes) for a longer period of time resulting in increased lymphoid cell and tissue levels at greatly reduced circulating tfv levels, leading to less exposure to off-target tissues (e.g., kidney). in monotherapy studies after oral dosing with tdf ( mg) and taf ( mg), the plasma tfv auc is reduced from to ng.h/ml respectively whereas the reduction in hiv load from baseline is improved, from log . to log . copies/ml, respectively, reflecting the more efficient delivery of taf to target cells and tissues. clearly the lower dose of taf ( mg) relative to tdf ( mg) will give taf a marked advantage when considering combination pill therapy. understanding how marked a difference a prodrug can make from the taf example, adrian went on to describe how a prodrug approach transformed a new nucleotide project in which intrinsic properties of the pharmacologically-active nucleotide analog were optimized. their starting point was gs- (d api), which had good activity against both wild-type and resistant hiv strains but was an active inhibitor of mitochondrial polymerase-gamma. on comparing the known structures of hiv rt and mitochondrial polymerase-gamma, differences in the -binding pocket were noted. this led to gs- in which -f was added to gs- (fig. ) . compared to tfv, gs- was about -fold less active against wild-type hiv but maintained better activity against resistant strains (k r and multiple thymidine analog resistance mutations). most importantly, it was inactive (ic > lm) against mitochondrial polymerase gamma. more than prodrugs were synthesized and evaluated in metabolism studies and in dogs (intravenous and oral administration). then the enantiomers were tested separately in dogs. this led to the selection of gs- . whereas tfv is efficiently utilised by renal uptake transporters, gs- was poorly taken into the kidney. no adverse renal findings were observed with the prodrug (gs- ) in -day studies in rats, dogs and monkeys at the highest doses tested ( mg, mg and mg/kg daily, respectively). in summary, this work has given examples of the prodrug approach being used successfully both to increase selectivity (by loading on-target tissues vs off-target tissues) and to increase activity (via by-passing metabolic constraints). adrian presented cases in which a prodrug strategy was able to fulfil the full potential of a selective, active triphosphate analog and enable its further progression as a clinical candidate. the keynote speakers were david margolis and myron cohen (fig. ) . david margolis, university of north carolina, nc, usa in hiv-infected patients, there is a long-lasting reservoir of hiv in the form of integrated viral dna in resting cd + memory cells of the host immune system. therefore, even if it were possible to eliminate % of viral replication, a reservoir of hiv would remain. there may be reservoirs in other long-lived cells. to date, there is only one known hiv patient who has been cured of his infection, the ''berlin patient''. he was treated for cancer by chemotherapy followed by a bone-marrow transplant. being ccr +/À, the chemotherapy had a greater chance to remove all the ccr +ve cells. the bone marrow donor was ccr Àve. although this patient continues to have no sign of hiv infection, this is hardly a viable treatment option for most hiv-infected patients. even in subjects with hiv replication well controlled by therapy, % have detectable plasma viremia which does not appear to decay over time (at least two years). to improve the sensitivity of the assay for hiv, billion lymphocytes are mixed with antibody attached to magnetic beads. this selects for the cd + t cells, about . - billion cells. the limit of detection is copy of hiv rna/million cells, limit of quantitation is copies/million cells. to reduce the reservoir of hiv, it was suggested that activation of integrated hiv in resting cd + t cells would give renewed hiv rna synthesis and possibly result in cell death either due to viral cytopathic effects or resulting from hiv-specific immune responses. a small clinical trial was set up to test this hypothesis. vorinostat (vor), a clinically approved drug for treating certain cancers, has been shown to bind to the active site of histone deacetylases. after a single dose, there was an increase in hiv rna ( . to -fold, mean . -fold). of these subjects, elected to continue with multiple doses. from the th to nd vor dose, acetylation of histones and activation of hiv rna synthesis became refractory to therapy. also, it is not known what proportion of cells, with latent hiv, can be activated. whereas a single vor dose did increase the expression of hiv rna, this is not an effective therapy for removing the hiv reservoir. myron cohen, university of north carolina, nc, usa myron noted that there are . million new hiv infections each year. in this context, anal sex may be an important factor because just one or a few virions of hiv can be infective; within weeks, there is rapid virus replication throughout the body and latent hiv reservoirs of ''founder virus'' are already formed. although anal sex has been associated with homosexual couples, myron pointed out that it is not uncommon amongst heterosexual couples. although behavioral education should be encouraged, it can never be the whole answer. various approaches to the prevention of hiv transmission are being evaluated. monoclonal antibodies, broad neutralising antibody (bnab) and vaccines may have potential for prevention of transmission, but most progress is being made with dapivirine rings containing tdf. these are designed to stay in the vagina for a month. phase iii trials are ongoing. a long-acting hiv integrase inhibitor, gsk (generally known as gsk ), is administered i.m. once every months; a two-year safety trial will be required. phase i trial has been completed and phase ii trial is being planned. by analogy with tuberculosis therapy, in which the infectious state is disabled prior to a complete cure, one wonders if hiv transmission rates may decrease with effective art use. in , the hiv prevention trials network (hptn) initiated a study (hptn ) which enrolled , hiv sero-discordant couples (couples that have one member who is hiv-infected and the other who is hiv-uninfected), mostly ( %) heterosexual couples. the infected partner had to be well enough not to require immediate art. the couples were randomised to have either immediate or delayed art. both groups received the same care including counselling on safe sex practices, free condoms, treatment for sexually transmitted infections and regular hiv testing. in may , it had been announced that there had been hiv transmissions in the delayed art group ( couples) compared to only in the immediate art group ( couples), a % reduction. in these cases, the hiv strain was linked to the partner. this is the first randomised clinical trial to show that treating an hivinfected individual with art can reduce the risk of hiv transmission to an uninfected partner. even with ''safer-sex'' counselling, there were pregnancies in the delayed art group, despite that group having more incentive for safer-sex. following the announcement of this result, all infected participants were offered art. myron reported the th annual review of this study. in the delayed art group, there had been a total of cases of hiv transmission with the hiv strain linked to the partner and cases of unlinked transmission. in the one case of hiv transmission in the immediate art group, infection had been detected at day of the study and further investigation suggested that the infection event was on day . clearly, early art is highly beneficial. cdc guidelines now recommend that all hiv infected patients should have art. . mini-symposium: hepatitis b virus anna lok, university of michigan, mi, usa the number of people infected with hbv world-wide, as estimated by the who and cdc in , was between and million, but was declining due to vaccination. in the usa, vaccine use has led to a steady decline in the rate of new infections, decreasing from about / , residents in the s to about / , today. in contrast, the prevalence of chronic hepatitis b among immigrants remains high, with no decreasing trend. when infection is acquired early in life, chronic infection is the norm. high viral load is associated with progression to liver cancer. there are fda-approved drugs to treat chronic hbv infection, including entecavir (etv), emtricitabine (ftc) and tdf. with several years of continuous therapy, hbeag loss is achieved in about % of patients but hbsag loss (the ultimate goal, seen as a ''cure'') is still a distant prospect for most patients. however, cirrhosis can be reduced by long-term antiviral treatment. in one tdf trial at years, / patients had a liver biopsy which showed that % of patients had improved fibrosis scores (p units) and that most other patients had no worsening. tdf has now been used for years without detecting hbv resistance, making it one of the first line drugs. tdf is generally well tolerated but its rare side effects include nephrotoxicity (see above for a possible switch to taf when it is approved), reduction in bone mineral density and very rarely lactic acidosis. despite the major progress made in hbv therapy, there remain various challenges. one is cost, about $ , -$ , for -year tdf therapy. pharmacy claims show that adherence is a problem; doses used are less than doses prescribed. there is a lack of accurate prediction of how hbv disease will progress in individuals. hbv dna can be integrated into the human genome at an early stage of infection. fortunately, the integrated viral dna is usually not the complete viral genome and patients, who achieve hbsag loss, rarely relapse. stefan mehrle, university of heidelberg, germany (stephan urban, head of hepatitis b research group, university of heidelberg, was originally scheduled to give this presentation). some chronic hbv-infected subjects are co-infected with hepatitis delta virus (hdv). this is a defective virus that replicates only in the presence of hbv. current antiviral drugs do not inhibit hdv. recently, heparan sulphate proteoglycan (hspg) has been shown to be essential for binding both hbv and hdv to primary hepatocytes. in , human sodium taurocholate co-transporting polypeptide (hntcp) was identified as a functional receptor for hbv and hdv. hntcp is also designated as a solute carrier protein a (slc a ). hntcp was shown to be a binding factor for the pres domain of the hbv l envelope protein. this interaction was found to be essential for hbv and hdv infection. whereas hbv replication is poor in cell lines derived from hepatocytes (e.g. hepg and huh- ) in which hntcp is usually weakly expressed, hbv replication is possible in primary human hepatocytes. the critical discovery was that over-expression of hntcp in hepg or huh- cells conferred susceptibility to hbv and hdv infection. myrcludex-b is a lipopeptide derived from amino acid residues - of the pres region of the hbv l protein. because it quickly (within min) targets the liver, it is being developed for liver imaging and for drug targeting. it also acts as an entry inhibitor for hbv and hdv by interrupting binding between the hbv l protein and hntcp. it specifically inhibits hntcp-mediated taurocholate transport but the effect on hbv replication is much greater. myrcludex-b activity has been investigated in vivo using scid mice reconstituted with human hepatocytes. with prophylactic treatment, not one infected hepatocyte was seen. following therapeutic treatment, at week post-infection, there were a few isolated infected cells. after the end of therapy, the infection seems to spread but only to neighboring cells. myrcludex-b has been synthesised on a g scale. toxicology evaluation in chimpanzees has been completed and clinical trials have been initiated. in a phase i trial using a mg dose, myrcludex-b was well tolerated. results of a further phase i trial are due to be reported later this year ( ). a dose-ranging phase ii trial has been started. kyong-mi chang, university of pennsylvania, pa, usa anti-hbs antibodies clearly play a critical role in controlling hbv disease. their presence has been accepted as an indication of an ''effective cure''. however, these antibodies appear late in the disease course and so they must have a limited role in the early stages of the disease. what is the role of t-cell responses? in contrast to other viruses, there is a delayed onset, about - weeks rather than days. cd + t cells regulate the adaptive response, cd + t cells attack hbv-infected cells. the national institute of diabetes and digestive and kidney diseases (niddk), part of the usa national institutes of health (nih), is supporting a prospective clinical trial to investigate hbv-specific t cell responses during the course of hbv disease. there are no clear t cell differences relative to hbv genotype. the t cell responses are highest during acute hbv infection. during the chronic phase of hbv disease, t cell responses remain suppressed. in conclusion, there are a lot of players in the immune control of hbv infection but their relative contributions and how they adapt to control hbv replication are still largely unknown. . . diversifying the hepatitis b pipeline: current efforts to explore novel mechanisms andrea (andy) cuconati, institute for hepatitis & virus research, pennsylvania commonwealth institute, pa, usa current hbv therapy using nucleotide anti-virals has been highly effective in controlling the infection but a ''cure'', as defined by hbsag seroconversion, has remained elusive. at best after years, the rate is about %. other approaches are needed. myrcludex-b (see section . ) is the lead entry inhibitor. nvr- , an encapsidation inhibitor, is entering clinical trials. in addition. studies with novel nucleotide analogues are ongoing. the hbv field has been transformed recently by the introduction of cell-based antiviral assays. stefan mehrle (see section . ) has been leading the way. the assay read-out will need to be optimized for high-throughput screening (hts) but, already, the assay has shown some ''hits''. a few compounds inhibited encapsidation of viral rna. (the hbv virion contains partly double stranded (ds) dna but the reverse-transcription from rna to dna occurs within the capsid.) within the cell, hbv dna is transported into the nucleus where the viral dna forms covalently closed circles (cccdna). two specific inhibitors of cccdna formation have been found. current nucleotide anti-hbv compounds do give large reductions of hbv dna in plasma but only a minimal reduction in levels of the hbs antigen (about log . ). in contrast, one ''hit'', hbf- inhibited surface antigen production but not genomic replication. structure-activity-relationship (sar) studies have given the current lead compound, hbv- . in conclusion, the cell-based assay, with complete replication of hbv, has markedly improved the screening for anti-hbv compounds although further optimization is still needed to give hts capability. adam zlotnick, university of indiana, in, usa. over the last few years, there has been much progress towards understanding the critical role of the hbv core protein -it is much more than just a protective coat for the genome because it plays a major role in the hbv life cycle. the core protein, being amino acids long, is known as cp . the first amino acids are involved in core assembly whereas the last residues, rich in serines and arginines, bind to rna. phosphorylation of the serines, particularly s , s and s , is required for specific packaging of full length hbv rna complexed to the polymerase (reverse transcriptase -pregenomic rna; rt-pgrna). this rt-pgrna complex initiates encapsidation. the core consists mainly of cp but also includes other proteins (about . %). adam showed us a computer model of the core, using different colours to highlight the various critical components. inside the core, the area of highest density (highlighted in red) represented the polymerase which was attached to the inner surface of the core. the ''other proteins'' in the core were shown in blue. the current thinking is that the polymerase, initially acting as a reverse transcriptase, is attached to, and guided by, an ''inside railway track''. this enables the polymerase to jump to the other end of the rna to start the reverse transcription into dna and then jump again to the other end to start, but never complete, the replication of the complementary dna strand. the self-assembly of the core is an energetically ''downhill'' process. somewhat surprisingly, it is possible to get mutations in which the core is even more stable but the rt activity is reduced. the phenylpropenamide derivative, at- , fills a pocket in the core and so stabilizes it, similar to the change in amino acids in the mutants. in the presence of at- , core assembly occurs faster; hence it is known as a core assembly enhancer (as adam mentioned, not a term much loved by industry, their preference is for core assembly inhibitors). regardless, the whole capsid structure changes. the binding of only a few drug molecules is required to make the core non-functional. it seems that it is easier to find compounds to enhance core assembly than inhibitors. . . targeting cccdna to cure chronic hepatitis b massimo levrero, sapeinza universita' di roma, italy. the current hbv therapies of choice are tdf alone or with etv. these drugs have an extensive safety record with use up to years. however, as for other nucleoside/nucleotide analogs, there is only a limited (about log ) reduction in the levels of hbv cccdna. the half-life of hbv cccdna seems to be long, but is still unknown. hbv replication parallels host gene expression, in that they involve the acetylation of histones, for example h and h . both host transcription factors and viral proteins bind to the cccdna. massimo summarized various assays to study different stages of cccdna during the replication cycle. potentially, these assays would allow the study of various approaches: to reduce or clear cccdna, to silence cccdna or to prevent the formation of new cccdna so that it would eventually be removed by dilution and cell death. for proof-of-concept, known ''epigenetic'' compounds, which act as transcription inhibitors, have shown that cccdna can be silenced. by reducing histone acetylation, the cccdna becomes too compact to allow transcription. this approach mimics, partly, therapy with interferon. this research is still at an early stage. due to time constraints, the next two speakers were asked to present brief summaries. john morrey (utah state university, ut, usa) described four mouse models but all stages of the life cycle of hbv can be studied only in the chimeric mouse model, in which human hepatocytes are used. however, this model lacks the potential to study the immune system and it is very expensive. stephan menne (georgetown university, dc, usa) described the woodchuck model. woodchuck hepatitis virus (whv) resembles the human virus and the disease in animals has many similarities to that in humans. neonatal infection becomes chronic in about - % of cases. these chronic cases have virtually a % life-time risk of developing cancer, the time scale being about year of chronic infection, followed by cancer at years to . the use of microbicides is an active area of research for the prevention of transmission of hiv. david katz (duke university, nc, usa) described how mathematical models may aid drug product design. for example, if it is assumed that the microbicide gel is microns thick, the epithelium is microns and the stroma (connective tissue) is microns and if the partition coefficient between gel and epithelium in known, then it is possible to model drug transfer and suggest how various other parameters, for example the size of the subject, may modify drug delivery. it is important that different disciplines work together, for example biophysicists with behavioral scientists. biophysics can help an understanding of complex physical phenomena but human behavior can be both complex and highly variable. ralph baric (university of north carolina, nc, usa) noted that a particular infective agent, for example norovirus (nov), may cause subclinical or serious disease in different individuals. in general, animal models are designed to give consistent outcomes rather than aiming to mimic the genetic diversity found in human subjects. in a collaborative effort, mice from ''founder'' strains, including wild-derived strains, were selected. the founder laboratory strains were all derived ultimately from a single female mouse ca . the susceptibility of the founder strains to severe acute respiratory syndrome coronavirus (sars-cov) differed widely (ld p - ). the founder strains were cross-bred. although ca % of the genes was equally distributed among the new mouse lines, there were gene combinations not seen previously. after infecting mice from the different founder strains with a constant sars-cov inoculum and measuring virus load at a set time after infection, there was a correlation between virus load and disease (as measured by vascular cuffing). it was possible to relate the effect to chromosomes ( %) and ( %). hopefully, identification of the important genes may be achieved. by keeping the virus inoculum constant, this system better represents the clinical spectrum of disease. when using this system to evaluate a potential vaccine, it was found that mice, under the age of one year, could be protected. however, there was a range of effectiveness, from good protection to inactive. these variations may give a representation of human diversity. angela kashuba, university of north carolina at chapel hill, nc, usa in four clinical studies, truvada [a combination pill containing tdf and emtricitabine (ftc)] was taken once daily to prevent hiv transmission, known as pre-exposure prophylaxis (prep). the adherence rates were unexpectedly poor in all four studies, particularly low in the study including at risk women. for example in one study, ''high adherence'' was defined as subjects taking at least % of drug doses and was achieved by only % of subjects. possible reasons may have been the apparent risk of side-effects (the long consent form included pages of side-effects) and the perception that the subjects, as individuals, were not particularly at risk of infection by hiv. importantly, the trial did confirm the concept that prep could be effective. there was > % protection in those subjects generally taking doses/week and there was some protection, albeit much less, in subjects taking doses/week. adherence rates, reported by subjects, were appreciably higher than the rates evidenced by drug blood level measurements taken just before the next dose (i.e. h after previous dose). in an attempt to better understand and model these data, the drug concentrations (tdf/tfv and ftc) in various tissues were measured. the ratio between drug concentrations in blood and tissue samples differed greatly for tdf/tfv, with less variations for ftc. concentration ratios of tdf/tfv were about in rectal tissue but only . in vaginal tissue. for ftc, the ratios were . and . , respectively. when considering the possible consequences of missed doses, the time scale for hiv infection is an important factor. it is thought that hiv takes about - h to reach the epithelial cells. clearly, adherence is a critical factor for efficacy and so a real-time objective method for measuring adherence is urgently needed before further clinical studies are initiated. . . the novel nucleoside analog bcx exhibits broad-spectrum antiviral activity and confers post-exposure protection against ebola and marburg viruses travis k. warren, usamriid, fort detrick, md, usa ebola and marburg viruses are members of the filovirus family. even in recent outbreaks of these diseases, including the current ebola epidemic in west africa, care workers are becoming infected and dying. drugs, which are being investigated for treating these diseases, are progressed under the fda ''animal rule''. bcx is a c-nucleoside adenine analog (fig. ) which is being progressed by biocryst pharmaceuticals inc. (warren et al., ) . in cell culture assays, bcx is active against ebola and marburg viruses, (ec ca lm). with bcx at lm, there was no detectable incorporation into host dna or rna. in rats, bcx is efficiently activated (phosphorylated) to the triphosphate. in a primer-extension assay, there is some read-through beyond a single residue of bcx , but there is effective chain termination after the first bcx residue where the template has two consecutive uridine residues. bcx has been tested in rodent and nonhuman primate models of marburg hemorrhagic fever. in mice, there was a dose response ( , , . and . mg/dose, bid) with full protection at the two higher doses (survivors, %, %, % and % respectively). in an experiment with dosing starting at different times ( h pre-infection, , , , and h post-infection vs placebo), the placebo-treated mice died on days , and with one survivor ( %). in the treated groups, the percent survival was , , , , and , respectively. in guinea pigs, bcx (bid) with treatment starting at different times ( h pre-infection, , and h post-infection) there was full protection ( % survival) for the pre-infection and h groups, with reduced efficacy at the later start times. in cynomolgus monkeys, bcx treatment was started at , and h post-infection. in the placebo group, all animals died within days to . in all the treated groups, virus loads were reduced by more than log . there was one late death in the h group but the other monkeys survived. various markers of potential organ damage were reduced in all treated groups. encouraged by these results, -day toxicology trials have recently been completed without any serious concerns. biocryst is developing bcx under the fda animal rule and indenabling work is ongoing. when asked about viral resistance, travis explained that it is not ethically permissible to create resistant strains of marburg virus, but samples collected from the monkeys are being sequenced to look for mutations indicative of drug resistance. as yet, mitochondrial toxicity has not been examined. had similar bone marrow transplants, initially seemed to have been ''cured'' but hiv was detected after and days, respectively. latent hiv can survive in various long-lived cells for decades, especially in memory t cells. when these cells proliferate, the integrated hiv genome is duplicated as the cell divides and the cells survive so long as hiv remains silent. compounds known to activate all t-cells are too toxic to become a clinical therapy. however, latency-reversing agents (lra) have greater specificity, ideally activating only the integrated hiv, leading to the death of hiv-containing t-cells. there remains another possibility (perhaps a less popular view) that there is continued low rate of hiv replication. two clinical studies have been initiated in subjects with undetectable plasma hiv levels. raltegravir, an hiv integrase inhibitor, was added to the background therapy. latent hiv is mostly integrated into host dna but hiv may also form episomal circular dna. the proportion of the circular form increases with raltegravir treatment. in the two clinical studies, / and / subjects, respectively, had detectable hiv circles which then decayed. this implies that some de novo infection of cells is ongoing. on the other hand, art works well, with no evidence of sequence evolution in the hiv circles at weeks. is it possible that raltegravir is inducing a single round of hiv replication, to give an increase in hiv circles? derek sloan, gilead sciences, foster city, ca, usa like vorinostat, (vor), romidepsin (rmd) is a histone deacetylase inhibitor which is used clinically to treat cancer. memory cd + t cells were taken from hiv subjects on suppressive art; ex-vivo treatment with rmd ( nm) induced a -fold increase in intracellular hiv rna which persisted for h. in contrast, a much higher concentration of vor ( lm) gave a to -fold lower response which was only transient. rmd also increased levels of extracellular hiv rna and virions. encouragingly, this ex-vivo induction of latent virus was seen at rmd concentrations that are below the levels of drug achieved in humans by clinical doses of rmd. accordingly, in a phase i/ii trial in hiv-infected subjects on art, rmd gave a better and more sustained response than vor. about . % of cells containing hiv provirus were activated. although this is far too low a percentage to eliminate the latent hiv reservoir, it is hoped that combination of such lra, which give improved results in ex-vivo cell assays, may give better clinical efficacy. gilead scientists have started screening for novel lras. ''gs- '' has been identified as a hit by hts. research on this lead is at a very early stage. gilead workers are also investigating other approaches. for example, gs- is a toll-like receptor (tlr ) agonist and it acts as an immune stimulator. although it is being evaluated in phase ii studies for the treatment of chronic hbv infections, the potential effect on hiv reservoirs is being investigated. in siv-infected monkeys, oral dosing of tlr agonist induced the activation of immune effector cells such as cd + t cells and nk cells. based on these data, tlr agonists are being further investigated for their effect on latent siv reservoirs in monkeys which have good virological suppression. another approach is to use anti-envelope antibodies. broadly neutralising antibodies (bnabs) are very effective in preventing siv infection when the viral load is low but less effective against a high-load virus challenge. in addition, a prophylactic cmv-vector-based siv vaccine was effective in preventing siv infection in rhesus monkeys. this and similar vaccines are being tested in vivo for their effects on the latent siv reservoirs. in summary, lras are able to activate hiv provirus in memory cd + t cells and thereby may enhance the recruitment of immune effector cells to destroy provirus-containing cells. however, a ''cure'' for hiv infection is still a distant prospect. furthermore, latent hiv reservoirs are heterogeneous and so a combination of approaches will likely be required. gerardo garcia-lerma, centers for disease control and prevention, atlanta, ga, usa proof-of-concept studies for prep, are mostly conducted in nonhuman primates. these can be used either to model a single highdose infective challenge or repeated low inoculations, about - tissue culture infective doses (tcid ). since , rhesus macaque models have been used in a long series of investigations. in a study, in which the monkeys were treated daily with either oral tdf or tdf/ftc and given a weekly siv inoculum rectally, tdf/ftc gave a longer delay in infection than did tdf alone. when using the vaginal infection route, tdf/ ftc gave % protection. in contrast, there was far less protection in clinical trials -why? one possible reason may have been that women were having the contraceptive injection, depot medroxyprogesterone acetate (dmpa). a study, in macaque monkeys given dmpa, confirmed that dosing with tdv/ftc gave good drug levels in plasma and in vaginal secretions. therefore, this did not explain the poor protection in the clinical trial. the macaque model has been used successfully to investigate various situations that are presented in the clinic. when macaques were co-infected with siv and a bacteria and treated with tdf/ftc for weeks, there was good, but not complete, protection ( %). with ftc-resistant virus, tdf/ftc remained protective. in this case, ftc-resistant virus has increased susceptibility to tdf. with the k r mutant hiv, there was protection against a low inoculum but only partial protection (ca %) against a high inoculum. whereas daily dosing seems to be acceptable for patients living with hiv, another option for prep is desirable. gsk- (generally known as gsk- ) is an hiv integrase inhibitor. it can be formulated with nano-particles to provide an injectable drug depot. in the macaque model, gsk- , injected once monthly, gave full protection against repeated rectal and vaginal exposures. because metabolism of gsk- is much slower in humans than macaques, it was expected to remain effective in humans for up to three months. a phase i study confirmed that drug levels remained above the predicted effective level with a -week dosing interval. a phase ii trial is planned. another approach is to use vaginal rings, which have been in clinical use as contraceptive devices for years. in the macaque model, tdf-containing rings, replaced every weeks, gave full protection. a phase iii trial has just been initiated. another option, elvitegravir (evg) and taf are being evaluated in a biodegradable polymer. although daily dosing with tdf/ftc has not proved sufficiently successful as prep in clinical use, it has proved that prep is an achievable aim and this has encouraged the progression of other options. courtney fletcher, university of nebraska, omaha, ne, usa atripla was the first triple combination pill taken once daily for hiv therapy. it contained tdf, ftc and efavirenz (efv). the macaque model has been used to investigate the differing tissue distributions of these drugs and how viral replication may be continuing wherever the drug concentrations are lowest. there are two approaches: tissue homogenates and tissue cells. tissue homogenates give both the intracellular and extracellular drug amounts. from tissues, mononuclear cells (mncs) are collected and the intracellular drug concentration measured. this approach is preferred by courtney but this option may be constrained by sample size and the drug concentration may be underestimated. for exam-ple, with raltegravir, after the mncs have been washed times, the drug concentration is very low. much higher raltegravir concentrations are found when the mncs are cleaned by a rapid spin through oil. comparing an oil spin and repeated washes, the oil process gives higher drug levels, typically about % higher. following initial studies in macaques, a clinical study, in subjects, investigated distribution of the drugs from atripla in peripheral blood mononuclear cells (pbmc) and various tissues (see above). in / subjects, there are data on the time to reduce hiv load to < copies/ml. in plasma, the time was - months. in lymphoid tissues, there was a much slower rate of hiv decline. also, patient variability was noted, with the faster responders having the higher drug levels. a drug may be absorbed from the gastrointestinal tract either going via the portal vein to the liver and then into blood circulation or via the lymphoid system. blood flow is about times faster than lymphoid flow. when the water/ -octanol partition-coefficient (logp) of a drug is < , absorption tends to be via the blood route. the prodrug approach can be used to alter absorption or, as for tfv, stability of the prodrugs (tdf and taf) can influence the relative concentration in lymphoid tissues (see above). this year, the three major award lectures exemplified the strength of icar, covering very different areas of research. john drach (elion award) described his journey through the early days of antiviral research, which led to the identification of novel modes of antiviral action that had not been envisaged previously. piet herdewijn (holý award) used evolutionary pressure to select dna polymerases that accept novel nucleoside analogs. the replacement of thymine by -chlorouracil led to the generation of a new form of e. coli. i suggest that this work has important implications in conventional antiviral research. with hiv and hcv protease inhibitors, the genetic barrier is limited by the ability of the viral protease and its substrate (the viral polyprotein cleavage sites) to co-mutate so that the virus can become resistant to the antiviral drug. so far, polymerase inhibitors have not suffered the same fate but this work shows that a poor choice of nucleotide analog could result in a resistant virus with a new type of rna in which the drug replaces a natural nucleoside. adrian ray (prusoff award), describing work at gilead, demonstrated how the prodrug concept can markedly improve both the efficacy and safety of potential drugs. their progress with hiv and hcv therapies has been remarkable. the keynote addresses tackled two emerging areas of hiv research. david margolis summarized work aiming to eradicate hiv from infected subjects and myron cohen described current progress with approaches to prevent hiv transmission. i found both these presentations to be informative and stimulating. hiv ''cure'' still seems to be a distant prospect. in contrast, prior to exposure prophylaxis (prep) has been shown to be an achievable aim although the need for daily dosing is a barrier to success. gerardo garcia-lerma described recent progress which is likely to radically change the prospects for therapeutic convenience and success. tdf-containing vaginal rings, which need replacing only once a month, are being evaluated. another exciting prospect is gsk- which has been formulated as a long-lasting injection. a phase i trial confirmed that the drug may be administered at month intervals. in the absence of a proven hiv vaccine, prep with drugs has become the most promising strategy to reduce hiv infection rates among high-risk populations. this conference also included three interesting mini-symposia: ''hepatitis b virus'', ''research triangle park'' and ''challenges in hiv infection, treatment and prevention''. an innovation this year was a session devoted to the european training network, euvirna and introduced by frank van kuppeveld. all the euvirna fellows, who attended icar, gave short presentations at this session. for further information, please see the isar news ( . ) in the september issue of antiviral research for an account by frank van kuppeveld. for many years, the clinical symposium was, for me, a major highlight of icar. in my report for the icar, i expressed a hope regarding hcv therapy: ''there is the prospect that the first nucleotide analogue will be licensed by the time of our next icar meeting. the combination of a nucleotide analogue and a ns a inhibitor looks set to transform hcv therapy across all genotypes. as for hiv, single-pill, once-daily regimens are following on quickly''. on th december , sofosbuvir (sovaldi Ò ) was the first nucleotide analog to be approved in the usa by the food and drug administration (fda) for treatment of patients with hcv. approval by the european union followed soon afterwards, in january . a ns a inhibitor, ledipasvir, formulated as a single fixed-dose combination pill with sofosbuvir, is progressing quickly through clinical trials. with such remarkable progress being achieved since the icar, i was disappointed to discover that there was no presentation on this topic at this year's icar. a paper (sofia, ) , which was part of a symposium in antiviral research on ''hepatitis c: next steps toward global eradication'', emphasizes recent successes. after completing therapy, a sustained virological response for weeks (svr ) is regarded as a cure for hcv-infected patients. the combination of sofosbuvir/ledipasvir has shown remarkable results in clinical trials, with svr in the range - % across genotypes. this combination was well tolerated. a nda for the sofosbuvir/ledipasvir combination pill was submitted recently. i do not recall any previous antiviral trials in which the ''intention-to-treat'' analyses showed % success rates. perhaps similar to the hcv symposium in antiviral research, i hope that the icar, which will be held in rome, will have a mini-symposium which will include an account of this remarkable progress. it would be interesting to have an update on the clinical impact of this combination therapy for hcv and to have an assessment on the prospects for global eradication of hcv. beside this one disappointment, there were many excellent presentations and i would like to add my thanks to the isar officers and conference committee for organizing another interesting and successful icar. metabolism and pharmacokinetics of anti-hepatitis c virus nucleotide prodrug gs- beyond sofosbuvir: what opportunity exists for a better nucleoside/nucleotide to treat hepatitis c? selection of an oral prodrug (brl ; famciclovir) for the antiherpesvirus agent brl protection against filovirus diseases by a novel broad-spectrum nucleoside analogue bcx acknowledgements i wish to thank all those authors who have kindly provided me with copies of their presentations and for giving me valuable comments. also, i thank the president of isar for asking me to prepare this meeting report. key: cord- -lyrgwxit authors: winnicka, beata; o'conor, catherine; schacke, wolfgang; vernier, kaitlyn; grant, christina l.; fenteany, fiona hall; pereira, flavia e.; liang, brannen; kaur, anupinder; zhao, ran; montrose, david c.; rosenberg, daniel w.; aguila, hector l.; shapiro, linda h. title: cd is dispensable for normal hematopoiesis and myeloid cell functions in the mouse date: - - journal: journal of leukocyte biology doi: . /jlb. sha: doc_id: cord_uid: lyrgwxit the robust and consistent expression of the cd cell surface marker on very early as well as differentiated myeloid hematopoietic cells has prompted numerous investigations seeking to define roles for cd in myeloid cells. to address the function of myeloid cd directly, we created a cd null mouse and assessed the responses of purified primary macrophages or dcs from wt and cd null animals in cell assays and inflammatory disease models, where cd has been implicated previously. we find that mice lacking cd develop normally with normal hematopoietic profiles except for an increase in thymic but not peripheral t cell numbers. moreover, in in vitro assays, cd appears to be largely dispensable for the aspects of phagocytosis, proliferation, and antigen presentation that we tested, although we observed a slight decrease in actin-independent erythrocyte uptake. however, in agreement with our published studies, we show that lack of monocytic cd completely ablates anti-cd -dependent monocyte adhesion to wt endothelial cells. in vivo assessment of four inflammatory disease models showed that lack of cd has little effect on disease onset or progression. nominal alterations in gene expression levels between cd wt and null macrophages argue against compensatory mechanisms. therefore, although cd is highly expressed on myeloid cells and is a reliable marker of the myeloid lineage of normal and leukemic cells, it is not a critical regulator of hematopoietic development, hemostasis, or myeloid cell function. the development of technology to readily produce mab enabled the systematic cataloging of leukocyte cell surface molecules and prompted the first leukocyte typing workshop in [ ] . this and subsequent workshops assigned leukocytebinding mab to groups termed "clusters of differentiation", or cd antigens, by virtue of common patterns of binding to panels of primary hematopoietic cells and cell lines. in this way, the cd cell surface molecule was defined as a marker of cells of the myeloid lineage in normal and leukemic cells. subsequent molecular cloning of the cd cdna showed that it was identical to the membrane-tethered peptidase apn [ ] and was expressed on cells, in addition to those of the hematopoietic system, such as the epithelial cells of the renal proximal tubules and the intestinal brush border, cells of the brain, fibroblasts, and activated endothelial cells [ ] . cd is a large -kd type ii protein with a short cytoplasmic domain, a hydrophobic transmembrane region, and a large extracellular domain containing its enzymatic activity. as an extracellular peptidase, cd functions to cleave single neutral amino acids from the n terminus of small peptides, and therefore, its substrates and functions vary depending on the tissue where it is expressed. for example, in the brain, cd cleaves opioid peptides and enkephalins to regulate neuronal signaling, and in the intestine, it cleaves peptides to facilitate amino acid resorption [ ] . the persistent and specific myeloid expression of cd on normal cells of the hematopoi-etic lineage has prompted numerous studies that have implicated cd in a variety of myeloid cell functions, primarily in assays that correlate cd expression with a particular function, inhibit enzymatic activity with chemical inhibitors or inhibitory antibodies, or activate cd using ligand-mimicking cross-linking antibodies. to address the function of cd directly and genetically, we created a global ko of cd and tested these animals for phenotypic effects on a number of myeloid functions attributed previously to cd , such as myeloid differentiation, phagocytosis, antigen presentation, and inflammatory processes. we find that cd expression is dispensable for hematopoietic development and physiologic homeostasis and suggest that its function in myeloid cells may be dependent specifically on particular immune challenges to generate cross-linking ligands. the targeting vector (pl -cd -lox-frtneo) was constructed using bac as a source of murine genomic dna obtained from the chori bac library [ ] . we constructed a floxed cd -targeting construct with the frtneo-frt region using recombineering methods [ ] with the assistance of the gttf (uchc, farmington, ct, usa). the schematic of the targeting strategy is depicted below (see fig. ). the targeting vector was electroporated into the murine es cell line d from the ( svevtac)/cb bl/ j mouse strain, and neomycin-resistant colonies containing the targeting constructs were expanded and transferred into pseudopregnant females, resulting in chimeric mice containing the targeted cd allele. germ-line transmission of the targeted allele was confirmed, and the neo cassette was excised to produce mice carrying a floxed cd allele. finally, floxed cd animals were mated with transgenic mice expressing cre recombinase under the ubiquitous hprt gene promoter ( s -hprt tm (cre)mnn /j, jackson laboratory, bar harbor, me, usa) to produce the global cd null strain. the cd global ko animals have been backcrossed subsequently to the c bl/ strain, and experiments described in this report were performed with cd ko/ko and control mice (cd wt/wt ) backcrossed for at least six generations to the c bl/ j strain (jackson laboratory). the mice were housed in the animal facilities at the uchc in accordance with institutional and office of laboratory animal welfare guidelines. cd deletion was assessed using pcr genotyping, mrna expression analysis (rt-pcr), immunohistochemistry, and flow cytometric analysis (described below). wt and ko cd alleles were detected with the primers bwol /bwol / bwol and bwol /bwol using the pcr protocol: °c/ min ( °c/ s, °c/ s, °c/ min) ϫ , °c/ min, °c/ϰ. products were visualized after electrophoretic separation on agarose gels. primers used for genotyping: bwol (labeled p ; see bmdms and dcs were isolated from femurs and tibias in medium supplemented with csf- as described previously [ ] . thioglycollate-elicited macrophages were harvested by flushing the peritoneal cavity of untreated (resident macrophages) or % thioglycollate-treated (elicited macrophages, , , or h) mice, followed by red cell lysis. splenic dcs from single-cell spleen suspensions of -to -week-old mice were positively selected using cd c microbeads and a macs column and separator (miltenyi biotec, auburn, ca, usa), according to the manufacturer's guidelines. murine t cells were isolated from single-cell splenocyte suspensions using a negative selection kit (depletion of non-t cells), according to the manufacturer's protocol (miltenyi biotec). murine lung endothelial cells from -to -weekold animals were harvested, minced, and treated with mg/ml collagenase a (roche, indianapolis, in, usa). the digested material was serially passed through -, -, and -gauge needles, dnasei-treated (worthington biochemical corp., lakewood, nj, usa) and endothelial cells positively selected with primary rat anti-mouse pecam- antibody (mec . , bd biosciences, san jose, ca, usa), followed by sheep anti-rat igg dynabeads using the dynal-mpc™ magnetic separator (invitrogen, dynal, carlsbad, ca, usa). the final cell/bead pellet was resuspended in . ml complete endothelial cell growth medium , % fbs (lonza walkersville, walkersville, md, usa) and plated in fibronectin-coated cell-culture dishes. the medium was changed after h, and cells attached to beads became visible - h after plating. purity of the endothelial cell preparation was routinely - % vascular endothelial-cadherin-positive (santa cruz biotechnology, santa cruz, ca, usa) upon immunostaining. total rna was isolated from cells or tissues using trizol. rt-pcr reactions were performed using the superscript ii rt kit (invitrogen) and taq polymerase, according to the manufacturer's instructions. primer sets used for mcd amplification were exon-spanning mcd cdna sequences - or - as follows: mcd - f, Ј-gggttctacatttccaa-gaccc- Ј; mcd - r, Ј-ttgaactcgctcacgatgtagg- Ј; mcd - f, Ј-tcacagtgataacgggaaagccca- Ј; mcd - r, Ј-ataagctccgtctcagccaatggt- Ј; mgapdh f, Ј-accacagtccatgccatcac- Ј; mgapdh r, Ј-tccaccaccctgtt-gctgta- Ј. bm cells were flushed from femora and tibiae. single-cell suspensions were obtained by repeated pipetting in staining medium ( ϫhbss/ % fcs/ mm hepes, ph . ). single-cell suspensions from spleen and thymus were prepared after mechanical grinding of the tissues between frosted glass slides. after suspension, cells were washed with staining media; erythrocytes were lysed by hypotonic shock and filtered through a cell strainer ( mm, nytex, sefar, kansas city, mo, usa). finally, cells were counted in a hemocytometer monitoring viability by trypan blue dye exclusion. for flow cytometric analysis, with the exception of the anti-cd , which was conjugated in-house, we used commercially available, conjugated antibodies and secondary reagents (bd pharmingen, san diego, ca, usa, or ebioscience, san diego, ca, usa). for analyses of myeloid markers, we used anti-cd b (clone m / ) coupled to allophycocyanin, anti-ly- g/gr- (clone rb - c ) coupled to fluorescein, and anti-f . (clone bm ) coupled to pe. for analyses of t cell compartment, we used anti-cd (clone gk . ) coupled to pe and anti-cd (clone - . ) coupled to fluorescein. for analyses of b cells, we used anti-cd r/b (clone ra - b ) coupled to fluorescein. to analyze dcs, we used anti-cd c (clone n ) coupled to allophycocyanin. to analyze the expression of cd , we used a biotinylated anti-cd (clone r - ) followed by a second step with streptavidin coupled to pe. before analyses, cells were suspended in a solution of staining media containing g/ml pi, and dead cells that stain strongly for pi were gated out of analysis. all analyses were done in a facscalibur flow cytometer (becton dickinson, san jose, ca, usa), available through the institutional flow cytometry facility (uchc). ing a zeiss axioplan ( ϫ) attached to a zeiss axiocam high-resolution digital camera. serum from cd ko and littermate mice was incubated with the cd substrate ala-pna ( mg/ml final) at °c for h. apn/cd enzyme activity was determined by measuring the absorbance of the resulting free p-nitroaniline at nm using a microplate reader. capture antibody (r - , serotec, uk) was added ( ug/ml in bicarbonate buffer) to -well flat-bottom maxisorp plates overnight at °c. wells were washed with pbs after each incubation step. after blocking with ul % bsa/pbs for h at room temperature, serum samples ( ul) were added for . h at °c. biotinylated anti-cd mab (k -a kind gift of dr. william paul, lab of immunology, nih, bethesda, md, usa) was added for h at room temperature, followed by hrp streptavidin ( . ug/ml) for min at room temperature. trimethoxybenzoic acid substrate ( ul) was then added for the final -min incubation (room temperature) hcl ( n) was used to stop the reaction, and the plate was read on a microplate reader at nm. a cytoselect -well phagocytosis assay kit was used according to the manufacturer's instructions (cell biolabs, san diego, ca, usa). bmdms or dcs and elicited or resident peritoneal macrophages were plated at % confluency in l culture medium in -well plates. macrophages were plated day prior to the assay and incubated overnight ( °c, % co ), and dcs were plated h prior to the assay ( . ϫ cells/well). control conditions were treated with m cytochalasin d (blocks phagocytosis by interacting with actin microfilaments) for h prior to assay. sheep rbcs (cappel labs, cochranville, pa, usa) were opsonized with igg as directed. opsonized or nonopsonized erythrocytes ( l) were added to the macrophages or dcs and incubated for h at °c. nonphagocytosed erythrocytes were lysed with hypotonic buffer. the extent of phagocytosis was determined after cells were washed and lysed, followed by the addition of a proprietary erythrocyte substrate solution and absorbance measured at nm. bmdms or thioglycollate-elicited peritoneal macrophages were plated on cover slips at . ϫ cells/ml (dmem, % fbs), cultured overnight, and stimulated with g/ml human oxldl ( suspensions of responder t cells from whole spleen (wt, ko, or balb/c mice) or t cell-enriched spleen populations were negatively selected using the pan t cell isolation kit (miltenyi biotec). responder cells ( . ϫ ) were mixed with mitomycin c-treated stimulator cells ( . ϫ ; alexis biochemicals, san diego, ca, usa) and incubated for days. approximately h later, alamar blue dye (serotec) was added to mixed cultures at a : dilution ( l), and suspensions were mixed thoroughly. proliferation of responder t cells was measured days later by detecting alamar blue fluorescence at an excitation wavelength of nm and emission wavelength of nm. the proliferative stimulation index was calculated as the (respondersϩstimulators) -stimulators alone, divided by the proliferation count of responders alone [ ] . isolated murine lung endothelial cells ( . ϫ /ml) were seeded on fibronectin-coated ( g/ml) -well plates and grown to confluency for - h in endothelial cell basal medium (cambrex bio science, walkersville, md, usa). subsequently, monolayers were untreated (basal and control) or anti-cd k or control igg mab treated ( g/ml) for min and washed extensively, and calcein-labeled, elicited macrophages were added. adhesion was allowed to proceed for min. each condition was assayed in triplicate. unbound myeloid cells were washed away extensively, and the remaining cells were lysed. fluorescence was quantified in a cytofluor (perseptive biosystems, framingham, ma, usa). male mice between and weeks old were injected i.p. with ml aged % thioglycollate broth or pbs. at various time-points ( , , or h) postinjection, animals were killed and the peritoneal cavity lavaged using ml sterile pbs. cells were collected, centrifuged, and washed, the rbcs were lysed, and the total number of cells was counted. type ii collagen-specific antibodies were used to induce arthritis in female mice [ ] between and weeks of age. on day , animals were injected (i.p.) with - l/mouse of the arthrogen-cia mab cocktail, - mg total (chondrex llc, seattle, wa, usa), or pbs as control. lps ( l; g in pbs) was administered i.p. on day . starting on day , the severity of arthritis in each limb was scored every other day on a - scale: ϭ normal; ϭ swelling and/or redness in one joint; ϭ swelling and/or redness in more than one joint; ϭ swelling and/or redness in the entire paw; ϭ maximal swelling. mice recovered from lps toxicity on day , after which, the arthritis progressed rapidly. the disease peaked by days - and plateaued on days - . beginning at weeks of age, cd wt and null littermates (nϭ /genotype) were administered % dss (mp biomedical, irvine, ca, usa) dissolved in drinking water for days. a control group of mice (nϭ ) from each genotype received drinking water only during the same time period. mice were weighed and assessed for evidence of gross rectal bleeding on a daily basis during the experiment and spleens weighed at termination. colons were formalin-fixed and swiss-rolled for histological analysis and stained with h&e, and the percent of ulcerated tissue along the entire colon was calculated. analysis was performed in a blinded manner. mice were treated with ul . % croton oil (sigma chemical co., st. louis, mo, usa) in acetone applied to the outside of one ear pinnea. the alternate ear was treated with acetone alone. twenty-four hours after sensitization, mice were killed humanely, and tissue from each ear was harvested using a -mm biopsy punch to obtain an equivalent area of tissue at the edge of the pinna. ear weights were recorded, and the sample was fixed in zinc fixative (invitrogen) overnight and paraffin-embedded, and sections were stained with h&e. serum from pairs of age-matched male and female wt and cd null animals was analyzed by the research animal clinical pathology facility at the charles river laboratories (wilmington, ma, usa). total rna was isolated from peritoneal (two individual mice/genotype; four samples) or bmdms (one mouse, each genotype) from wt or cd null mice using trizol, according to the manufacturer's protocol. for quality control, rna purity and integrity were evaluated by denaturing gel electrophoresis, od / ratio, and analysis on an agilent bioanalyzer (agilent technologies, palo alto, ca, usa). total rna was amplified and purified using the ambion ilumina rna amplification kit (ambion, austin, tx, usa). briefly, ng total rna was reverse-transcribed to cdna using a t oligo(dt) primer. second-strand cdna was synthesized, in vitro-transcribed, and labeled with biotin- -utp. the labeled crna was analyzed on the agilent bioanalyzer and quantitated by nanodrop analysis. the labeled crnas were hybridized to the mouse wholegenome- beadchip (illumina inc., san diego, ca, usa) for h at °c, as per the manufacturer's instructions. the beadchips were washed, stained with streptavidin-cy , and scanned on the illumina beadarray reader. microarray data were extracted, normalized, and analyzed using illumina genomestudio software. illumina mousewg- v . expression beadchip (illumina inc.) contains -mer gene-specific oligonucleotide probes corresponding to , mouse transcript variants. there is, on average, a fold redundancy for each transcript per array. intensity data were normalized using the quantile algorithm in genomestudio. the differential expression of each gene, relative to the respective control, was evaluated using the illumina custom error model that calculates a p value as a function of intensity, differential and biological; technical; and nonspecific variation. those samples with p values Ͻ . were deleted, the relative expression of cd null versus wt calculated and expressed as fold-wt expression, and the data ranked from low to high expression relative to wt values. the genes showing the highest differential expression are listed in supplemental table . the dataset was analyzed further using ingenuity pathway analysis software (ingenuity systems, redwood, ca, usa). conditional cd null mice were produced in the uchc gttf using modifications of the recombineering method described in ref. [ ] . the mcd gene is encoded by exons spanning nearly kb on chromosome . a bac containing the cd gene identified by blast analysis was obtained from the chori bac repository and confirmed by southern blot analysis (data not shown). repeated attempts to target the Ј-most region of the cd gene resulted in no recombinants and prompted the revised strategy depicted in figure a , where loxp recombination sites were inserted in introns between exons and and and . exposure of this construct to the cre recombinase would result in a gene lacking exons - , which encode the enzymatic active site, the putative ngr-binding site [ ] , and the majority of the extracellular portion of the molecule. in addition, splicing between exons and introduces a frame-shift resulting in a stop codon early in exon , which would be predicted to produce a protein lacking exons - (as depicted in fig. b) . we have observed that relatively slight modifications to the cd protein severely impair its trafficking to the cell surface and would predict that the large alteration induced by this deletion would similarly affect cell surface expression. indeed, transfection of the c a human epithelial cell line with a mutant v -tagged cd expression construct lacking exons - showed no cell surface expression of the v tag (fig. c) . transfection of murine es cells with the targeting construct resulted in five founder lines in the c bl/ ϫ mixed background, and one h was expanded for further study. after germ-line transmission was confirmed, the mice were crossed to a transgenic strain expressing the cre recombinase under the control of the ubiquitous hprt promoter on a mixed background to create global cd null animals. homozygous kos were healthy and fertile with no overt phenotypic or serologic abnormalities (data not shown), consistent with an independently derived cd null strain [ ] . deletion of the floxed region of the cd locus in homozygous null animals was confirmed by pcr analysis (fig. d) , and evaluation of cd mrna and protein expression by rt-pcr (fig. e ) and immunohistochemistry indicated a complete lack of expression in kidney and small intestine (fig. f) , spleen, colon, and liver (not shown) tissue of the null animals as compared with abundant expression in the renal proximal tubules and brush border microvilli of wt controls. interestingly, functional assessment of the deletion with a standard colorimetric cd substrate ala-pna (fig. g , left; ref [ ] ) showed a striking retention of peptidase activity in the serum of null animals, although only background levels of the cd protein were present in these samples when assessed by elisa (fig. g , right), suggesting that this commonly used method of assessing cd levels measures other peptidase activities as well, and results using this assay should be interpreted with caution. a role for cd in hematopoietic differentiation is implied by its pattern of expression in various hematopoietic lineages. in normal and neoplastic hematopoietic populations, cd is expressed specifically on the earliest cell committed to the myeloid lineage (cfu-gm) and all of its differentiated progeny and is a reliable diagnostic marker for certain types of leukemia [ ] . more recently, cd has been shown to be highly expressed on adult pluripotent stem cells derived from multiple sites [ ] [ ] [ ] and differentiated es cells [ , ] , as well as subsets of peripheral blood progenitor cells [ ] . in addition, cd has been postulated to play a role in fetal liver hematopoiesis [ ] and myeloid cell differentiation [ ] . finally, cd expression has been reported to be controlled by various hematopoietic differentiation factors such as il- and tgf-␤ [ ] [ ] [ ] [ ] [ ] . however, its specific contribution to normal hematopoiesis is unknown. flow cytometric analysis of bm, spleen, and thymic cell populations showed an indistinguishable distribution of cells of the myeloid b and t cell populations in wt and cd null adult animals (fig. ) , with the exceptions of a significant (pϭ . ) increase in numbers of the thymic cd populations in cd null mice, which is not seen in peripheral organs, and a modest but nonsignificant trend toward an increase in cd b ϩ cells (but not cd b ϩ / f / ϩ cells) in the bm of cd Ϫ/Ϫ mice. furthermore, in vitro quantification of functional hematopoietic progenitor populations (hematopoietic stem cell, early lymphoid progenitors, and immature b cells) in spleen and bm indicated no difference in total colony-forming ability of precursors from the wt versus null animals (data not shown), suggesting that cd is not a critical regulator of developmental hematopoiesis or hemostasis. to confirm that cd is expressed in murine cells of the myeloid lineage and is deleted efficiently in these cells in the cd null animals, we isolated primary bm macrophages, thioglycollate-elicited peritoneal macrophages, and bm-derived dcs. rt-pcr analysis of mrna purified from each of these populations and control kidney tissue indicated that cd mrna is present in cells isolated from wt but not cd null animals (fig. a) , and flow cytometric analysis of cd protein on bm-derived dcs confirmed its expression on cells isolated from wt but not cd null animals (fig. b) . a similar examination of elicited peritoneal macrophages showed that cd is expressed predominantly on those populations expressing high levels of cd b protein (fig. c) . further analysis of the cd b high population with the f . macrophage marker in wt and null animals shows identical cytometric profiles (fig. d) , arguing against a developmental defect result-ing from the lack of cd in myeloid cells, and validating the use of this population in our in vitro assays. as cd has been implicated in numerous myeloid cell functions, we wished to assess the function of cd null cells in these aspects. previous reports have shown that inhibition of cd diminished proliferative capacity of t cells in vitro and in vivo [ - ] . to investigate whether the lack of cd expression alters t cell responses, we tested the ability of wt and cd null cells (h- b haplotype) to stimulate or respond to the foreign mhc molecules expressed on cells from the allogeneic balb/c strain (h- d haplotype) in the mlr, which is a measure of the ability of apcs to stimulate t cell responses and of the t cells to be activated in response to antigen. a mixture of whole splenocyte or purified t cell (fig. a , left and right, respectively) responder cells from wt or ko mice with mitomycin [ , ] and parasitic [ ] proteins, depicting the domain organization and the buried active site (red circle). structure of the truncated cd protein that would be predicted to result from the deleted allele (right). (c) anti-v antibody immunofluorescent detection of the c a human cervical carcinoma cell line transiently expressing a truncated (left, Ј, -diamidino- -phenylindole-stained, exons - ) or full-length (right) v tagged human cd cdna. cells transfected with the truncated construct showed no antibody reactivity. (d) pcr detection of the targeted genomic locus using pcr primers shown in a. (e) rt-pcr of total rna harvested from the indicated tissues of wt, heterozygous (het), and null animals (ko) with primers specific for mcd cdna or control gapdh. (f) immunohistochemical detection cd in wt and ko kidney (left) and intestine (right) using an anti-mcd polyclonal antibody shows staining of cells in the renal proximal tubules and the brush border microvilli (arrows). (g) serum from cd null animals retains the ability to cleave the colorimetric ala-pna substrate (left), although it contains nominal levels of cd protein by elisa assay (right). the graph is representative of four independent experiments with analogous results. bars represent the means and sd of four replicates. c-treated allogeneic balb/c stimulator splenocytes showed no significant difference in proliferation of either responder population to the activating stimulus. similarly, wt and ko splenocytes were capable of stimulating the proliferation of balb/c responder cells, suggesting normal levels of surfaceexpressed stimulator molecules, such as mhc i/ii, cd , and cd / , on cd null apcs, consistent with intact antigenpresenting functions in the absence of cd . cd has been reported to be present in a complex with fc␥rs on the monocyte cell surface [ ] and to act functionally as an accessory molecule to facilitate fc␥r-mediated phagocytosis [ ] . the latter study found enhanced phagocytosis of antibody-modified target cells and prolonged downstream signaling upon engagement of the fc␥r in the presence of cross-linking mab to cd in primary monocytes or myeloid cell lines, suggesting that cd may be a functional regulator of this receptor. to address the contribution of cd to this process, we isolated bm macrophages (fig. b , left) or resident (fig. b, right) or elicited (not shown) primary peritoneal macrophages from wt and cd null mice and assayed their ability to phagocytose igg-opsonized or control, nonopsonized sheep rbcs. blocking phagocytosis by perturbation of microfilament assembly with cytochalasin d illustrates that basal uptake of nonopsonized rbcs does not occur by phagocytic mechanisms. no difference was observed in the microfilament-dependent phagocytic uptake of the opsonized target cells in any of the myeloid lineages tested; thus, cd is apparently dispensable for fc␥r-mediated phagocytosis in macrophages and dcs. however, a reproducible reduction in actin-independent basal uptake by the null cells was apparent, which was possibly relevant to studies implicating cd in lipid uptake and metabolism [ ] [ ] [ ] and those proposing that cd is a target of the cholesterol uptake inhibitor ezetimibe in enterocytes and macrophages [ , ] . to address a potential role of cd in actin-independent endocytic processes, we assessed scavenger receptor-mediated uptake of oxldl, which has been reported to be unaffected by cytochalasin d treatment [ ] . although untreated cells of either genotype showed no lipid uptake (not shown), the percentage of lipidcontaining cd null bmdms (fig. c , left) or peritoneal macrophages (fig. c, right) was comparable with that seen with wt cells, again, suggesting that cd is not required for fc␥r-mediated phagocytosis or scavenger receptor-mediated endocytosis. we have demonstrated recently a new function for cd as an adhesion molecule mediating monocyte/endothelial cell interactions [ ] . cross-linking of cd with ligand-mimicking antibodies or viral ligands results in increased cell-cell adhesion and the formation of molecular complexes containing endothelial and monocytic cd , suggesting that cd may participate in some aspect of monocyte trafficking during inflammation. to address the role of cd as an adhesion molecule, we purified cd -positive primary lung microvascular endothelial cells from wt animals and assessed the ability of wt or cd null bmdms to adhere to wt endothelial monolayers, untreated or treated with anti-cd antibodies or control igg. although overall adhesion of cd null cells was slightly lower than wt to untreated or control monolayers, treatment of the endothelial monolayer with cross-linking anti-mcd antibodies significantly increased the adhesion of wt but not cd null macrophages (fig. d) , consistent with our results in human cells implicating cd as a myeloid adhesion molecule in a gain-of-function manner. the role of cd as an adhesion molecule mediating interactions between myeloid and endothelial cells suggests that it may be required for optimal inflammatory responses. consistent with this notion, inhibition of cd has been reported to lessen the severity of acute colitis in a murine model of inflammatory bowel disease, where it was reported to play a role in tgf-␤ expression in t cells [ , ] . however, induction of colitis in wt and cd null animals showed no difference in weight loss, a standard measure of disease progression (fig. a, top) . in addition, spleen weight and the percent of colonic ulceration in each genotype were comparable (fig. a , middle and bottom), arguing against a critical role for cd in this model. similarly, expression of cd has been reported to be increased in the synovial fluid of patients with rheumatoid arthritis, a process that involves the infiltration of inflammatory cells into the affected region [ ] [ ] [ ] [ ] [ ] . a second report found that synovial cd levels correlated positively with the number of infiltrating lymphocytes of arthritis patients [ ] . to assess the contribution of cd to the inflammatory phase of arthritis, we used a model in which a cocktail of four anti-collagen antibodies are injected, producing a rapid, synchronous disease independent of the mhc haplotype [ ] . assessment of the response of cd null animals in this mhc-independent caia again showed no discernable difference in the mean clinical scores between wt and null animals injected with the antibody panel at all time-points tested (fig. b) , suggesting that cd does not mediate inflammatory cell infiltration into the damaged synovium. our laboratory has shown previously that administration of mab against cd strongly diminished leukocyte transmigration in a mouse model of peritonitis [ ] . to investigate if the lack of cd resulted in fewer inflammatory cells infiltrating into the peritoneum in vivo, we treated wt or cd null mice with i.p. injections of thioglycollate broth and quantified the infiltrating cells by peritoneal lavage at , , and h posttreatment. in contrast to antibody treatment, we found no significant difference in cell numbers in the peritoneal exudates of the two genotypes at the three time-points tested (fig. a) . a similar dichotomy between antibody treatment and null animals in peritonitis has been described previously for other cell surface molecules [ , ] and is thought to be a result of an agonistic rather than blocking function of the antibody. finally, as the c bl/ strain has been shown to be able to compensate for the lack of other adhesion molecules that are critical for monocyte trafficking in other strains, we backcrossed the cd null animals onto the fvb background for generations. these mice were fully capable of supporting cell infiltration into the peritoneum (fig. b) , arguing against a strainspecific compensation in animals lacking cd . finally, we tested the cd -deficient mice in a fourth model of inflammation, the croton oil-induced atopic dermatitis model of acute inflammation, where local irritation is induced by application of croton oil to the skin of one ear with control vehicle on the other. in this model, histamine release from activated resident mast cells produces measurable edema and inflammatory cell infiltration. comparison of wt and cd null-treated ears showed equivalent levels of edema as measured by weight (fig. c) , as well as the presence of comparable numbers of phenotypically identifiable neutrophils in both genotypes, whereas little to no extravasation was observed in vehicle-treated control ears (data not shown). taken together, these data suggest that perhaps activation of cd by an as-yet-unidentified ligand is necessary to observe an effect on inflammatory trafficking or that cd participates only in response to specific inflammatory triggers. the lack of a phenotypic change in the various myeloid functional assays may be a result of compensatory effects by upregulation of the expression of other molecules that participate in these processes. to assess the mrna expression levels of relevant genes in wt versus cd null myeloid cells, we probed an illumina mouse- microarray beadchip with cdna prepared from thioglycollate-elicited peritoneal macrophages from cd wt or ko animals. subsequent analysis of the expression data of , transcript probes using genomestudio software indicated that overall, the expression of . % of the genes assayed differed by less than twofold between cd null the bars represent the mean and sem of n ϭ (whole spleen) and n ϭ (purified t cells). (b) fc␥r-mediated phagocytosis. assessment of fc␥r-mediated phagocytosis by bmdms (left) or peritoneal macrophages (right) from wt or cd ko animals. igg-opsonized sheep rbcs (opsonized) and untreated rbcs (non-opsonized) were added to phagocytes that were untreated or treated with cytochalasin d to block phagocytosis (opsonized ϩ cyt d), and phagocytosis was detected colorimetrically. bars represent the mean and sem of n ϭ . (c) scavenger receptor-mediated phagocytosis. wt and cd null bmdms (left) or peritoneal macrophages (right) were assessed for their ability to internalize human oxldl, as detected by oil red o (oro) staining. data are expressed as the percent Ϯ sd of oil red o containing cells in independent fields of n ϭ . untreated cells (no oxldl) of either genotype showed no oil red o staining (not shown). (d) cd must be present on macrophages to adhere to anti-cd antibody-activated endothelial cells. untreated, fluorescent-labeled, primary murine wt or ko peritoneal macrophages (mac) were added to monolayers of wt murine lung microvascular cells, untreated or pretreated with igg or k anti-cd -activating mab, and adhesion assessed. bars indicate mean and sd of fluorescence intensity of n ϭ independent experiments. and wt macrophages ( fig. and supplemental table ). similarly, analysis of the expression of specific genes involved in pertinent functional pathways using ingenuity pathway analysis software showed no explicatory induction of relevant myeloid genes, peptidases, adhesion molecules, etc. (data not shown), arguing against an up-regulation of compensatory genes and again, suggesting that many myeloid functions are largely independent of cd . the expression of human cd on very early myeloid-committed progenitor cells as well as their differentiated progeny strongly suggests a functional role for this cell surface molecule in myeloid development or function [ ] . although numerous reports have investigated cd functions using inhibitors or blocking or activating mab, direct genetic evidence for its role in various myeloid cell functions has not been available. in the current study, we describe the production and analysis of a mouse strain harboring a floxed cd allele crossed with the ubiquitously expressed hprt-cre transgenic to delete nearly % of the coding region of the gene, including regions specifying the enzymatic active site and the zinccoordinating region. these cd null mice are viable and fertile, suggesting few developmental or physiological defects, in agreement with studies describing an independently derived cd null strain [ ] . we find that similar to humans, cd mrna and protein are expressed on murine peritoneal and bmdms and bm-derived dcs, and this expression is abrogated completely in the null animals. nevertheless, these cells appear largely normal in many aspects of myeloid cell development and function. the lack of an overt phenotype in the cd null animals could be a result of a number of reasons unrelated to its functional role. it is formally possible that cd could be functional at a reduced level in these animals, as the deleted allele retains the promoter, transcriptional start site, and initiation codon and thus, could produce a truncated transcript/protein containing the first three exons (splicing of the remaining exons results in a frameshift to a stop codon early in exon ). however, we find that cd is undetectable at the mrna level using primers spanning the nondeleted region (nt . this agrees with our in vitro data showing that expression of a construct containing a truncated cdna with exons - does not produce a detectable, tagged protein (fig. c) , probably as a result of instability of the truncated mrna or incorrect folding of the truncated molecule. similarly, immunohistochemical, flow cytometric, and elisa assays confirm that the cd protein is not detectable in cells and tissue from these animals. the residual functional activity detected on the null cells illustrates that the widely used ala-pna assay does not detect cd activity specifically and should be used with caution. second, during development, the loss of deleted molecules often results in up-regulation of functionally similar molecules to compensate for the deficit [ ] . however, our microarray analysis of peritoneal and bmdms showed a striking similarity in expression levels among genes in the wt and null cells, arguing against compensation. with regard to enzymatic activ- (a) cd wt or null mice were administered % dss, dissolved in drinking water or water alone for days. mouse weights were recorded (top) and spleens weighed at termination (middle). the percent of ulcerated colonic tissue (bottom) was determined histologically and calculated as the percent of ulcerated tissue in the entire colon; n ϭ mice/genotype. (b) cd wt or null mice were immunized with arthrogen-cia mab blend or pbs (day ). assessment of disease severity was initiated on day , as described in materials and methods. each marker represents the mean Ϯ sem of clinical scores for five animals/genotype. ity and substrate cleavage, the argument for redundancy is more compelling, as numerous cell surface peptidases share overlapping substrate specificities. in this regard, although the preferred substrates of cd are n-terminal neutral amino acids, it can also cleave charged residues, albeit less efficiently. it is possible that another enzyme is substituting for cd to produce the necessary cleavage products. another possibility is that many of the published studies implicating cd used monocytic cell lines and human peripheral blood monocytes, and in the current study, we used primary macrophages and dcs. it is formally possible that cd plays distinct roles in monocytes and more differentiated myeloid cells, which is not addressed in the current study. finally, it has been shown that genes contributed by the c bl/ background strain can compensate for a lack of critical adhesion molecules in inflammatory responses, particularly peritonitis [ ] . this may be relevant to our studies, as the experiments described were performed on animals, where % of their background genes were contributed by the c bl/ strain. in the previous study, the effects of inflammatory challenge in pecam- (cd ) wt and null mice on the c bl/ background were indistinguishable, and animals lacking pecam- on the fvb background showed severe impairment of their inflammatory responses. however, in the current study, we found that cd is apparently not required for leukocyte infiltration in the peritonitis model in two independent inbred strains, arguing against a background influence. these caveats aside, we find that the cd null animals have normal hematopoietic development and myeloid cell function, suggesting it is not required for physiologic homeostasis. although this study is not exhaustive, we have focused on processes where cd has been implicated previously in studies using inhibitors or antibodies or where cd expression was shown to correlate with a particular function, as these would be particularly relevant to this loss-of-function analysis. for example, although treatment of animals with the cd inhibitor aprotinin showed a significant decrease in disease severity in the dss model of inflammatory bowel disease [ ] , weight loss, the percent of ulceration, and spleen weight in cd null animals are indistinguishable from that of wt animals. similarly, numerous studies have implicated a role for cd in aspects of human t cell biology, where it has been reported to be expressed on peripheral t cell subsets and activated t cells [ ] , where generally, treatment with cd inhibitors was described to diminish t cell proliferation via increases in tgf-␤ production [ - , ] . this result seems at odds with our observation of a significant increase in the numbers of cd ϩ t cells in the null thymus but may be a result of the disparate origins (peripheral vs. thymic) of the t cells studied. alternatively, it is possible that lack of cd alters the tight regulation of thymocyte selection, proliferation, or survival. identification of the specific t cell subset(s) affected, determination if the increase were t cell-intrinsic or a result of stromal effects, and a possible impact on thymocyte trafficking will be necessary to determine the exact mechanisms responsible for the increase in thymic cd ϩ cells in cd null animals. finally, although we have not focused specifically on t cell functions, the mlr measures the ability of primarily cd ϩ t cells to recognize and proliferate in response to a foreign histocompatibility antigen (mhc i or mhc ii) presented by allogeneic apcs in the culture. as such, it is a means of assessing the capacity of the cells for self-recognition as well as the levels and context of mhc and costimulatory molecules, which are important for regulating the cell interactions involved in normal immune responses. we found no significant differences in the function of cd null cells when used as responder or stimulator cells, suggesting that cd is not critical for t cell proliferation and that the levels and context of molecules required for alloantigen presentation (stimulator) cells are unaffected in the null cells. discrepancies with the published inhibitor studies may be the result of non-cd specific, off-target effects of these peptidase antagonists or again, compensatory mechanisms. we do observe a slight but significant decrease in the uptake of actin-independent, nonopsonized rbcs by cd null macrophages but not in the scavenger receptor-mediated internalization of modified ldl, which is also actin-independent [ ] . actin-independent mechanisms have been illustrated for receptor-mediated and receptor-independent endocytosis and appear to be dependent on the particular antigen and the cell type [ , ] . we [ ] and others [ , - ] have implicated cd previously in internalization of distinct surface proteins in various cell types. for example, in endothelial cells, we demonstrated that inhibition of cd impairs internalization of the bradykinin/bradykinin receptor complex by perturbing lipid raft organization, effectively inhibiting downstream signal transduction, filopodia formation, and cell invasion [ ] . it is possible that the decrease in actin-independent uptake of erythrocytes but not modified lipid suggests that cd participates in specific mechanisms of macrophage uptake, a possibility that we are pursuing currently. in contrast to consequences of cd inhibition, studies from our laboratory [ ] and others have described cd -dependent gain-of-function effects in cells treated with anti-cd cross-linking antibodies that trigger signaling cascades, presumably mimicking ligand-receptor interactions. one functional investigation into fc␥r-mediated phagocytosis in monocytes demonstrated that cd is present in phagocytic vesicles, and concomitant cross-linking of surface fc␥rs and cd molecules enhanced phagocytosis and prolonged signaling via the fcr [ ] , suggesting that cd may function as a signal regulator. however, we see no difference in fc␥r-mediated uptake of similarly opsonized targets in the presence or absence of cd . this may indicate that similar to other ligand-binding signaling receptors, cd must be cross-linked first to induce downstream signal transduction cascades. alternatively, the comparable phagocytic capacity of cells with or without cd could point to the fact that although cd cross-linking may trigger processes that complement those elicited by the fc␥r, it does not normally do so. finally, it is possible that cd may be important in the phagocytosis of certain types of opsonized antigens that are also cd cross-linking ligands, which would engage the fcr and cd and thus, be internalized more efficiently. the identification of such ligands is a current focus of our laboratory. although cd appears to be dispensable for many physiologic functions of myeloid cells, it is clearly necessary for monocytes to adhere to anti-cd -activated but not to classical, tnf-activated endothelial cells [ ] . this finding may be relevant to the defective angiogenesis reported in an independently derived, global cd ko strain [ ] . it is clear that in many pathologic settings (such as wound-healing, tumors, ischemic injury), the cytokines produced by infiltrating, inflammatory cells promote robust angiogenesis [ ] [ ] [ ] . monocyte adhesion to the endothelium at the site of injury is a prerequisite for inflammatory cell infiltration, and thus, a cd -dependent defect in adhesion would logically decrease the number of infiltrating cells, reduce proangiogenic cytokine levels, and impair angiogenesis. although the published study did not address monocytic infiltration specifically, it may well contribute to the observed phenotype. the defect in cd -dependent adhesion is also consistent with the lack of a phenotype in response to the relatively generic inflammatory challenges tested in this study and may also speak to the contradictory results from antibody-treated versus null mice in the peritonitis model. logically, specific inflammatory challenges may elicit unique molecules that bind to and cross-link cd and thus, are cd -dependent, whereas other immune challenges are not. treatment with the anti-cd antibody may mimic such a ligand and as such, would produce a phenotype distinct from that of a loss-of-function model. indeed, we have shown that cross-linking of cd with mab or with its multivalent ligand, the human coronavirus h e [ , ] , leads to a signal transduction-dependent, gain-of-function increase in monocyte adhesion to endothelium [ ] . it is possible that the virus has co-opted the normal host response to cross-linking ligands to gain access to tissues and so, may phenocopy such a ligand [ ] . thus, the role of cd in myeloid cells awaits the identification of these ligands, which will undoubtedly be facilitated by the availability of cd null animals. this work was supported by public health service grant ca- from the national cancer institute. the authors express their deepest appreciation to katie lamothe and to the members of the center for vascular biology, especially the laboratories of dr. kevin claffey and dr. timothy hla at uchc, and dr. dan wu at yale university. in addition, we thank the staff of the following uchc facilities: gttf, the flow cytometry facility, and the research histology core facility. the authors declare no conflicts. leucocyte typing human myeloid plasma membrane glycoprotein cd (gp ) is identical to aminopeptidase n hematopoietic differentiation antigens that are membrane-associated enzymes: cutting is the key! blood the ucsc genome browser database: update a highly efficient recombineering-based method for generating conditional 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cd , fc ri ␣ and chymase, and cd expression is reduced during differentiation regulation of the expression of aminopeptidase a, aminopeptidase n/cd and dipeptidylpeptidase iv/cd in renal carcinoma cells and renal tubular epithelial cells by cytokines and camp-increasing mediators potential indirect anti-inflammatory effects of il- . stimulation of human monocytes, macrophages, and endothelial cells by il- increases aminopeptidase-n activity (cd regulation of aminopeptidase-n (cd ) and fc riib (cd ) expression by il- depends on the stage of maturation of monocytes/macrophages inhibition of alanyl aminopeptidase induces map-kinase p /erk in the human t cell line karpas- role of alanyl aminopeptidase in growth and function of human t cells inhibition of alanyl-aminopeptidase suppresses the activation-dependent induction of glycogen synthase kinase- ␤ (gsk- ␤) in human t cells functional co-localization of monocytic aminopeptidase n/cd with the fc ␥ receptors cd and cd aminopeptidase n (cd ) functionally interacts with fc␥rs in human monocytes cholesterol crystallization-promoting activity of aminopeptidase-n isolated from the vesicular carrier of biliary lipids biliary lipid secretion: immunolocalization and identification of a protein associated with lamellar cholesterol carriers in supersaturated rat and human bile cholesterol crystallization-promoters in human bile: comparative potencies of immunoglobulins, ␣ -acid glycoprotein, phospholipase c, and aminopeptidase n aminopeptidase n (cd ) is a molecular target of the cholesterol absorption inhibitor ezetimibe in the enterocyte brush border membrane ezetimib influences the expression of raft-associated antigens in human monocytes mechanism of uptake of copper-oxidized low density lipoprotein in macrophages is dependent on its extent of oxidation cd is a novel mediator of monocytic/endothelial cell adhesion inhibition of alanyl-aminopeptidase on cd ϩcd ϩ regulatory t-cells enhances expression of foxp and tgf-␤ and 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transmembrane (egf-tm ) receptor cd is required for neutrophil migration and host defense new approaches for modeling cancer mechanisms in the mouse platelet endothelial cell adhesion molecule deficiency or blockade significantly reduces leukocyte emigration in a majority of mouse strains triggering endogenous immunosuppressive mechanisms by combined targeting of dipeptidyl peptidase iv (dpiv/cd ) and aminopeptidase n (apn/ cd )-a novel approach for the treatment of inflammatory bowel disease transcriptional regulation of cytosol and membrane alanyl-aminopeptidase in human t cell subsets dipeptidyl peptidase iv (dp iv, cd ) and aminopeptidase n (apn, cd ) as regulators of t cell function and targets of immunotherapy in cns inflammation multiple intracellular routes in the cross-presentation of a soluble protein by murine dendritic cells lipoprotein lipase-dependent binding and uptake of low density lipoproteins by thp- monocytes and macrophages: possible involvement of lipid rafts cd /apn regulates endothelial invasion and filopodia formation the reversion-inducing cysteine-rich protein with kazal motifs (reck) interacts with membrane type matrix metalloproteinase and cd /aminopeptidase n and modulates their endocytic pathways the coronavirus transmissible gastroenteritis virus causes infection after receptor-mediated endocytosis and acid-dependent fusion with an intracellular compartment regulation of angiogenesis by macrophages, dendritic cells, and circulating myelomonocytic cells the role of macrophage-derived il- in induction and maintenance of angiogenesis inflammation, inflammatory cells and angiogenesis: decisions and indecisions activation of human monocytes after infection by human coronavirus e human aminopeptidase n is a receptor for human coronavirus e key: cord- -kenstpja authors: nan title: china date: journal: the statesman&#x ;s yearbook doi: . / - - - - _ sha: doc_id: cord_uid: kenstpja nan yuan dynasty in , swept into southern china. the mongol yuan dynasty adopted chinese ways but was overthrown by a nationalist uprising in , led by hongwu ( - ), a former beggar who established the ming dynasty. the ming empire collapsed in a peasants' revolt in . the capital, beijing (peking), was only km from the great wall and vulnerable to attack from the north. within months the peasants' leader was swept aside by the manchus, whose qing dynasty ruled china until . preoccupied with threats from the north, china neglected its southern coastal frontier. the portuguese, who landed on the chinese coast in , were followed by the dutch in and the english in . the qing empire expanded into mongolia, tibet, vietnam and kazakhstan. but by the th century, under pressure from rural revolts ignited by crippling taxation and poverty, the qing dynasty was crumbling. two opium wars ( - ; - ) forced china to allow the import of opium from india into china, while britain, france, germany and other european states gained concessions in 'treaty ports' that virtually came under foreign rule. the taiping rebellion ( - ) set up a revolutionary egalitarian state in southern china. the european powers intervened to crush the rebellion and in british and french forces invaded beijing and burnt the imperial palace. further trading concessions were demanded. a weakened china was defeated by japan in and lost both taiwan and korea. the xenophobic boxer rebellion, led by a secret society called the fists of righteous harmony, broke out in . the guangxu emperor attempted modernization in the hundred days reform, but was taken captive by the conservative dowager empress cixi who harnessed the boxer rebellion to her own ends. the rebellion was put down by european troops in . china was then divided into zones of influence between the major european states and japan. with imperial authority weakened, much of the country was ripe for rebellion. in the kuomintang (guomintang or nationalist movement) of sun yet-sen (sun zhong shan; - ) overthrew the imperial system. the authoritarian yuan shih-kai ruled as president from to . following the overthrow of yuan, china disintegrated into warlord anarchy. in sun founded a republic in southern china but the north remained beyond his control. reorganizing the nationalist party on soviet lines, sun co-operated with the communists to re-establish national unity. but rivalry between the two parties increased, particularly after the death of sun in . after sun's death the nationalist movement was taken over by his ally chiang kai-shek (jiang jie shi; - . as commander in chief of the nationalist army from , chiang's power grew. in april his campaign to suppress the chinese communist party saw thousands of communists slaughtered. the survivors fled to the far western province of jiangxi. in chiang's army entered beijing. with the greater part of the country under chiang's rule, he made nanjing the capital. in the communists were forced to retreat from jiangxi province. led by mao zedong (mao tse-tung; - they trekked for more than a year on the , -mile long march, eventually taking refuge in shaanxi province. in the japanese invaded manchuria. by they had seized beijing and most of coastal china. the nationalists and communists finally co-operated against the invader but struggled against the superior japanese forces. during the second world war , a nationalist government ruled unoccupied china ineffectually from a temporary capital in chongqing. at the end of the war, nationalist-communist co-operation was short-lived. the soviet union sponsored the communist party, which marched into manchuria in , beginning a civil war that lasted until . although the nationalist forces of chiang kai-shek received support from some western countries, particularly the united states, the communists were victorious. on oct. mao declared the people's republic of china in beijing. chiang fled with the remains of his nationalist forces to taiwan, where he established a government that claimed to be a continuation of the republic of china. at first recognized as the government of china by most western countries, taiwan kept china's security council seat at the united nations until . chiang's authoritarian regime was periodically challenged by red china, which bombed taiwan's small offshore islands near the mainland. in the s and s, taiwan gradually lost recognition as the legitimate government and in the usa recognized the people's republic of china. in china invaded tibet, independent since . chinese rule quickly alienated the tibetans who rebelled in . the tibetan religious leader, the dalai lama, was forced to flee to india. since then, the settlement of large numbers of ethnic chinese in the main cities of tibet has threatened to swamp tibetan culture. during the s and s china was involved in a number of border disputes and wars in neighbouring states. the communists posted 'volunteers' to fight alongside communist north korea during the korean war . there were clashes on the soviet border in the s and the indian border in the s, when china occupied some indian territory. from the establishment of the people's republic of china, communist china and the soviet union were allies. communist china initially depended upon soviet assistance for economic development. a soviet-style five-year plan was put into action in , but the relationship with moscow was already showing signs of strain. by the end of the s the soviet union and china were rivals, spurring the chinese arms race. chinese research into atomic weapons culminated in the testing of the first chinese atomic bomb in . mao introduced rapid collectivization of farms in . the plan was not met with universal approval in the communist party but its implementation demonstrated mao's authority over the fortunes of the nation. in he launched the doctrine of letting a 'hundred flowers bloom', encouraging intellectual debate. however, the new freedoms took a turn mao did not expect and led to the questioning of the role of the party. strict controls were reimposed and free-thinkers were sent to work in the countryside to be 're-educated'. in may mao launched another ill-fated policy, the great leap forward. to promote rapid industrialization and socialism, the collectives were reorganized into larger units. neither the resources nor trained personnel were available for this huge task. backyard blast furnaces were set up to increase production of iron and steel. the great leap forward was a disaster. it is believed that m. died from famine. soviet advice against the project was ignored and a breakdown in relations with moscow came in , when soviet assistance was withdrawn. a rapprochement with the united states was achieved in the early s. having published his 'thoughts' in the 'little red book' in , mao set the cultural revolution in motion. militant students were organized into groups of red guards to attack the party hierarchy. anyone perceived to lack enthusiasm for mao zedong thought was denounced. thousands died as the students lost control and the army was eventually called in to restore order. after mao's death in the gang of four, led by mao's widow jiang qing, attempted to seize power. these hard-liners were denounced and arrested. china effectively came under the control of deng xiaoping. deng pursued economic reform. the country was opened to western investment. special economic zones and 'open cities' were designated and private enterprise gradually returned. improved standards of living and a thriving economy increased expectations for civil liberties. the demand for political change climaxed in demonstrations by workers and students in april , following the funeral of communist party leader hu yaobang. in beijing where demonstrators peacefully occupied tiananmen square, they were evicted by the military who opened fire, killing more than , . hard-liners took control of the government, and martial law was imposed from may to jan. . since the leadership has concentrated on economic development. hong kong was returned to china from british rule in (for the background, see page ) and macao from portuguese rule in . the late s saw a cautious extension of civil liberties but chinese citizens are still denied most basic political rights. beijing was chosen for the olympic games. china's treatment of tibet came under the international spotlight in the build-up to the games, following violent protests in tibet's capital city, lhasa. the arrest by japan of a chinese trawler in disputed waters in marked the beginning of heightened tensions between the two nations in the east and south china seas. in china became the world's second largest national economy. in nov. the communist party congress selected xi jinping to succeed hu jintao as president from march . in sept. that year, former leadership hopeful bo xilai received a life sentence for corruption in one of china's highest-profile trials in decades. in oct. the government announced the end of the country's one-child policy. a month later, the presidents of china and taiwan met for talks-the first time that leaders from the respective territories had met since . on the economic front, gdp growth in was at its lowest level for a quarter of a century. china is bounded in the north by russia and mongolia; east by north korea, the yellow sea and the east china sea, with hong kong and macao as enclaves on the southeast coast; south by vietnam, laos, myanmar, india, bhutan and nepal; west by india, pakistan, afghanistan, tajikistan, kyrgyzstan and kazakhstan. the total area (including taiwan, hong kong and macao) is estimated at , , sq. km ( , , sq. miles). a law of feb. claimed the spratly, paracel and diaoyutasi islands. an agreement of sept. at prime ministerial level settled sino-indian border disputes which had first emerged in the war of . china's sixth national census was held on nov. . the total population of the provinces, autonomous regions and municipalities and of servicemen on the mainland was , , , ( , , females, representing · %); density, per sq. km. china's population in represented % of the world's total population. the population rose by , , (or · %) since the census in . there were , , urban residents, accounting for · % of the population; compared to the census, the proportion of urban residents rose by · % (reflecting the increasing migration from the countryside to towns and cities since the economy was opened up in the late s). population estimate, dec. : , , , . china has a fast-growing ageing population. whereas in only · % of the population was aged or over and by this had increased to · %, by it is expected to rise to · %. long-term projections suggest that in as much as · % of the population will be or older. the population is expected to peak at · m. around and then begin to decline to such an extent that by around it will be back to the level. china is set to lose its status as the world's most populous country to india in about . the un gives a projected population for of , · m. regulations restricting married couples to a single child, a policy enforced by compulsory abortions and economic sanctions, were widely ignored, and it was admitted in that the population target of , m. by would have to be revised to , m. from peasant couples were permitted a second child after four years if the first born was a girl, a measure to combat infanticide. in china started to implement a more widespread gradual relaxation of the one-child policy. in dec. the standing committee of the national people's congress (npc) approved a resolution allowing couples to have two children if either parent was an only child. the one-child policy was formally abandoned altogether from jan. . an estimated m. persons of chinese origin lived abroad in . a number of widely divergent varieties of chinese are spoken. the official 'modern standard chinese' is based on the dialect of north china. mandarin in one form or another is spoken by m. people in china, or around % of the population of mainland china. the wu language and its dialects has some m. native speakers and cantonese m. around m. people in china cannot speak mandarin. the ideographic writing system of 'characters' is uniform throughout the country, and has undergone systematic simplification. in a phonetic alphabet (pinyin) was devised to transcribe the characters, and in this was officially adopted for use in all texts in the roman alphabet. the previous transcription scheme (wade) is still used in taiwan and hong kong. mainland china is administratively divided into provinces, five autonomous regions (originally entirely or largely inhabited by ethnic minorities, though in some regions now outnumbered by han immigrants) and four government-controlled municipalities. these are in turn divided into prefectures, cities (of which are at prefecture level and at county level), , counties and urban districts. tianjin, · m.; dongguan, · m.; wuhan, · m.; foshan, · m.; chengdu, · m.; chongqing, · m.; nanjing, · m.; shenyang, · m.; xian, · m.; hangzhou, · m.; haerbin, · m.; suzhou, · m.; dalian, · m.; zhengzhou, · m.; shantou, · m.; jinan, · m.; qingdao, · m.; changchun, · m.; kunming, · m.; changsha, · m.; taiyuan, · m.; xiamen, · m.; hefei, · m.; urumqi (wulumuqi), · m.; fuzhou, · m.; shijiazhuang, · m.; wuxi, · m.; zhongshan, · m.; wenzhou, · m.; nanning, · m.; ningbo, · m.; guiyang, · m.; lanzhou, · m.; zibo, · m.; changzhou, · m.; nanchang, · m.; xuzhou, · m about % of the population is engaged in the dominant industries of farming and animal husbandry. in the total sown area was , ha. output in : total grain crops, , tonnes; vegetables, , tonnes. in there were · m. sheep and goats and · m. cattle and yaks. tibet has over , mineral ore fields. mining, particularly of copper and gold, has expanded rapidly since when the railway came to tibet. cement production, : · m. tonnes. electricity consumption totalled · bn. kwh in . in there were , km of roads ( , km in ) . there are airports at lhasa, bangda and nyingchi providing external links. in , , foreign tourists visited tibet. in july a , -km railway linking lhasa with the town of golmud opened. it is the highest railway in the world. direct services have subsequently been introduced between lhasa and a number of major chinese cities, including beijing and shanghai. an extension from lhasa to shigatse, tibet's second largest city, became operational in aug. . in tibet had primary schools (with , pupils) and secondary schools of which were senior secondary schools (with , pupils), junior secondary schools (with , pupils) and eight whole secondary schools. there were also six vocational secondary schools in ( , pupils). tibet has six higher education institutes (the largest of which is tibet university), with , enrolled students in total in . the illiteracy rate of people aged and above was · % in . in there were , medical personnel (including , doctors) and , medical institutions, with a total of , beds. births, , , , ; deaths, , , . birth rate (per , population), · ; death rate, · . in the birth rate rose for the first time since . there were , , marriages and , , divorces in . in the marriage rate was · per , population and the divorce rate a record high · per , . the divorce rate has doubled since . in april parliament passed revisions to the marriage law prohibiting bigamy and cohabitation outside marriage. the suicide rate in china in was · per , population. life expectancy at birth, , was · years for men and · years for women. infant mortality, , per , live births. china has made some of the best progress in recent years in reducing child mortality. the number of deaths per , live births among children under five was reduced from in to in . fertility rate, , · births per woman (compared to over in the mid- s). annual population growth rate, - , · %. according to the world bank, the number of people living in poverty (less than us$ · a day) at purchasing power parity declined from m. in to m. in . most of china has a temperate climate but, with such a large country, extending far inland and embracing a wide range of latitude as well as containing large areas at high altitude, many parts experience extremes of climate, especially in winter. most rain falls during the summer, from may to sept., though amounts decrease inland. monthly average temperatures and annual rainfall ( ): beijing (peking), jan. · °f (- · °c), july · °f ( · °c). annual rainfall · " ( mm). chongqing, jan. · °f ( · °c), july · °f ( · °c). annual rainfall · " ( , mm). shanghai, jan. · °f ( · °c), july · °f ( · °c). annual rainfall · " ( , mm). tianjin, jan. · °f (- · °c), july · °f ( · °c). annual rainfall · " ( mm). on sept. the chinese people's political consultative conference met in beijing, convened by the chinese communist party. the conference adopted a 'common programme' of articles and the 'organic law of the central people's government' ( articles). both became the basis of the constitution adopted on sept. by the st national people's congress, the supreme legislative body. the consultative conference continued to exist after as an advisory body. three further constitutions have been promulgated under communist rule-in communist rule-in , communist rule-in and . the latter was partially amended in , , and , endorsing the principles of a socialist market economy and of private ownership. the unicameral national people's congress is the highest organ of state power. usually meeting for one session a year, it can amend the constitution and nominally elects and has power to remove from office the highest officers of state. there are a maximum of , members of the congress, who are elected to serve five-year terms by municipal, regional and provincial people's congresses. , xi pursued a strong style of authoritarian rule at home and a proactive and muscular foreign policy in his first term. in oct. the ccp gave him the title of 'core' leader, a significant honorific bracketing him with mao zedong and deng xiaoping among previous party figures although conferring no absolute powers. then, at its five-yearly congress in oct. , the party voted to enshrine his name and ideology in the chinese state constitution. he was subsequently re-elected in march and parliament also voted to abolish presidential term limits in a major shift from precedent. xi jinping was born on june in beijing, the son of one of the first generation of communist leaders. he joined the ccp in and, after graduating from tsinghua university in with a degree in chemical engineering, he became secretary to the vice-premier and secretary-general of the central military commission. xi became the zhengding county committee deputy secretary in hebei province in and the following year was promoted to secretary. in he was made deputy mayor of xiamen city, fujian province. having undertaken various party roles in the province, he became deputy governor of fujian in and governor a year later. in he moved to zhejiang province and made his first inroads into national politics when he was named a member of the th central committee. from - he was party secretary of fujian, overseeing economic growth averaging % a year and earning a reputation as an opponent of corruption. in march xi transferred to shanghai to take the role of party secretary following the dismissal of the incumbent on corruption charges. his appointment to such an important regional post was seen as a vote of confidence from the central government and he became a member of the politburo standing committee at the th party congress in oct. . he was also made a high-ranking member of the central secretariat. on march he was elected vice-president at the th national people's congress and took on a number of high profile portfolios including the presidency of the central party school. he was also beijing's senior representative for hong kong and ahead of his presidency, xi said little about his policy ambitions. there was hope abroad and at home that he would champion political and social reform and attempt to deal with corruption and a widening wealth gap between rich and poor and between urban and rural communities. he also faced the conundrum of how to provide adequate healthcare to a rapidly ageing population. in jan. the prospect of greater transparency and accountability under his leadership was undermined when the authorities began criminal proceedings against anti-corruption campaigners calling for public disclosure of officials' assets. this coincided with a report by a us investigative organization claiming that relatives of some of china's top political and military figures, including xi's brother-in-law, held secret offshore financial holdings. in other social and political affairs the ccp announced plans in nov. to ease china's one-child policy (which was subsequently abandoned following an announcement in oct. , with effect from ) and to abolish the system of 'reeducation through labour' camps, while a party plenum calledfor the first time-for markets to play a 'decisive' role in the allocation of resources. meanwhile, in xi was confronted by domestic political opposition in the form of militant attacks by ethnic uighur separatists from xinjiang region and, from sept. that year, by widespread pro-democracy and autonomy protests in hong kong. on the economic front, china's previously frenetic annual rate of growth slowed markedly in , reflecting a slump in factory production and concerns over depressed oil prices, and again in to its lowest since . it also heralded severe stock market turbulence into despite emergency government measures, which had negative reverberations throughout the world economy. nevertheless, recognizing china's rise as a global economic power, the imf in nov. voted to add the yuan as the fifth member of its special drawing rights (sdr) currency basket alongside the us dollar, japanese yen, british pound and the euro. in foreign affairs, regional concerns over china's territorial and military intentions were raised in nov. by the government's declaration of a new 'air defence identification zone' over a swathe of the east china sea including disputed islands claimed by japan and south korea. there has also been friction, regionally and with the usa, over china's sovereignty claims and land reclamation operations on islands in the south china sea, although in july an international legal tribunal ruled in favour of a challenge by the philippines to china's sovereignty assertions-a verdict beijing vowed to ignore. further afield, xi has meanwhile undertaken numerous official visits abroad, as well as attending multilateral forums, for diplomatic, trading and investment purposes. and, while in singapore in nov. , xi and president ma ying-jeou of taiwan held the first direct talks between leaders of the two estranged governments since their split in . most recently, xi urged greater trade co-operation with the usa during his first encounter with the new us president, donald trump, in april , while also increasing economic and military cooperation with russia. li keqiang took office as premier of the state council, a role equivalent to prime minister, in march , succeeding wen jiabao. he was re-elected in march . li keqiang was born on july in dingyuan county, anhui province. following graduation from high school in , he joined the ccp and in he graduated in law from peking university, serving as head of the students' federation from - . he went on to earn a master's degree and doctorate in economics and headed the university's communist youth league of china (cylc) committee. over the following two decades he rose through the cylc ranks, joining the secretariat of its central committee in the s and serving as its first secretary in the s. at this time he built up his power base and forged close ties with hu jintao, a fellow cylc committee member and future chinese president. in li became deputy party secretary for henan province and a year later was appointed henan's governor. in dec. he was named party secretary for liaoning province where he spearheaded a major coastal infrastructure project, the ' points and one line' highway development. in this template was adopted at the national level to rejuvenate industrial northeast china. he also oversaw the rehousing of · m. shanty-town residents into new apartment blocks over a three-year period. li advanced to national level politics when he was elected to the politburo standing committee in oct. . he was appointed vice-premier of the state council in march , leading a medical reform programme aimed at creating an accessible public healthcare service. he also chaired an affordable housing programme and introduced tax reform plans. in nov. li was re-elected as a member of the politburo standing committee and on march became premier of the state council at the th national people's congress. regarded as the steward of the chinese economy, li was expected to focus on securing china's long-term expansion and on the further provision of basic national healthcare, affordable housing, employment growth, regional development and cleaner energy. however, global confidence in china's economy has been shaken since as the country's growth momentum has slowed amid apparent policy differences and blunders, prompting rumours that li was being increasingly sidelined in the governing hierarchy. he was nevertheless re-elected to the politburo standing committee in oct. and to the premiership in march . the chinese president is chairman of the state and party's military commissions. china is divided into seven military regions. the military commander also commands the air, naval and civilian militia forces assigned to each region. china's armed forces (pla: 'people's liberation army'), totalling nearly · m. in including the paramilitary people's armed police (pap) and · m. excluding the pap, are the largest of any country. however, active armed personnel numbers have halved since . moreover, in president xi laid out plans to reform the army structure-replacing an organization based on seven regions with one based on five 'theatre commands'-and reduce the number of military personnel by a further , . conscription is compulsory, but for organizational reasons, is selective: only some % of potential recruits are called up. service is for two years. a military academy to train senior officers in modern warfare was established in . defence expenditure in was us$ , m. (equivalent to us$ per capita). china's military spending more than trebled during the s. defence spending in represented · % of gdp. only the usa spent more on defence in , but china's defence expenditure totalled around a fifth of that of the usa. in march it was announced that the defence budget would rise by · % to us$ bn. following increases of · %, · % and · % in the previous three years. china is the world's third largest exporter of arms after the usa and russia, with · % of the global major weapons total over the period - . in the period - it had only been the eighth largest exporter. as at may china had , personnel serving in un peacekeeping operations (the largest contingent of any of the five permanent members of the un security council and more than the other four combined). having carried out its first test in , there have been tests in all at lop nur, in xinjiang (the last in ). the nuclear arsenal consisted of approximately operational warheads in jan. according to the stockholm international peace research institute. china has been helping pakistan with its nuclear efforts. the army (pla: 'people's liberation army') is divided into main and local forces. main forces, administered by the seven military regions in which they are stationed, but commanded by the ministry of defence, are available for operation anywhere and are better equipped. local forces concentrate on the defence of their own regions. ground forces are divided into infantry, armour, artillery, air defence, aviation, engineering, chemical defence and communications service arms. there are also specialized units for electronic counter-measures, reconnaissance and mapping. in there were group armies covering seven military regions. they included: armoured divisions and brigades; mechanized infantry divisions, brigades and regiments; motorized infantry divisions and brigades; nine special operations units; artillery divisions and brigades; amphibious brigades and divisions; two mountain brigades; aviation brigades and regiments; and two guard divisions. total strength in was · m. including some , conscripts. reserve forces are undergoing major reorganization on a provincial basis but are estimated to number some , . there is a paramilitary people's armed police force estimated at , under pla command. in nov. the naval arm of the pla included submarines, of which three were strategic (two jin-class and one xia-class) and tactical. by mid- two more jin-class nuclear-powered ballistic missile submarines had entered service. surface combatant forces in nov. included destroyers and frigates. sea trials of china's first aircraft carrier, liaoning (a former soviet warship purchased from ukraine), began in aug. . it entered service in sept. and was initially only used for training before being declared 'combat ready' in nov. . work on china's first domestically-built aircraft carrier began in . it was launched in april and is expected to be operational by . there is a land-based naval air force of about combatcapable aircraft, primarily for defensive and anti-submarine service. the force includes h- strategic bombers and jh- fighters. the naval arm is split into a north sea fleet, an east sea fleet and a south sea fleet. in naval personnel were estimated at , , including , in the naval air force and , conscripts. the people's liberation army air force organizes its command through seven military region air forces. the air force has an estimated , combat-capable aircraft. equipment includes j- (mig- ) interceptors (known in the west as 'fishbed'), h- chinese-built copies of tu- strategic bombers, q- fighterbombers (evolved from the mig- and known in the west as 'fantan'), su- fighters supplied by russia (known in the west as 'flanker'), j- chinese-designed and produced fighters (known in the west as 'firebird') and j- locally-developed fighters (known in the west as 'finback'). total strength ( ) was , . in agriculture accounted for · % of gdp, industry · % and services · %. industry was the largest contributor until , while services only overtook agriculture as the second largest sector in . in the late s agriculture was the largest contributor towards gdp. china's economic performance has been marked by high rates of growth for over three decades. annual gdp increases in the early s consistently exceeded % until the global financial crisis. china also holds the world's largest foreign exchange reserves, at more than us$ · trn. in april , although they have been falling since as the central bank strives to boost the currency in the face of large capital outflows. it is among the top recipients of foreign direct investment (fdi) and is the world's largest producer and consumer of coal. in china made the transition from net receiver of foreign aid to net donor and has established itself as a key player in africa's economic development, becoming the largest export partner of sub-saharan africa in . according to the world bank, china's cumulative fdi stock in africa totalled nearly us$ · bn. in , up from us$ · bn. in . in china overtook japan to become the world's second largest economy after the usa. the first steps from a centrally-planned towards a more market-oriented economy were taken by deng xiaoping in the late s. he opened the economy to foreign trade and investment, decentralized industrial management and allowed private sector development. in china became a member of the world trade organization, establishing trade relations with many countries. private entrepreneurs and foreign investors have played an important role in developing the manufacturing sector, china's principal growth engine. even before the economy was heavily skewed towards manufacturing, but following the market-oriented transition output increased significantly. during this period there was a structural shift away from large state-owned enterprises (soes), although these still remain an important part of the economy. between and the government oversaw reform of soes, with many poorly performing businesses privatized or liquidated. stronger firms were restructured and often listed on the stock market. many more recent enterprises are labourintensive as distinct from the capital-intensive soes. growth has been fuelled by low added value and labour-intensive exports. however, chinese firms are predicted to become increasingly competitive with higher added value producers, such as south korea. although the global financial crisis reduced the rate of growth and inbound fdi, china's recovery was among the earliest. gdp growth averaged · % in the second quarter of , up from a two-decade low of · % in the first quarter of that year. fdi also recovered rapidly, averaging · % of gdp annually between and . growth was rooted in a stimulus package of trn. yuan (us$ bn. or % of gdp), including fiscal spending and interest rate cuts, as well as an expansionary monetary policy. central government committed · trn. yuan, with the rest coming from local government, banks and soes. although exports declined by around % in , other countries fared worse and china's share of world exports increased to nearly · % in (up from % in ), making it the world's largest merchandise provider. gdp growth in stood at more than % but moderated between and , reflecting the global economic slowdown and diminishing dividends from past reforms. in aug. a devaluation of the yuan sent the shanghai stock exchange plummeting by nearly %, which was swiftly followed by a surge in capital outflows. the stock market meltdown lasted until feb. , with trading halted altogether for two days in jan. that year. nonetheless, the shanghai exchange subsequently began a recovery and had stabilized (at around , points) by feb. . despite stock market turbulence, the property market, which constitutes a quarter of china's gdp and is vital to the banking sector (as it accounts for a substantial amount of its collateral), remained buoyant. gdp growth declined to · % in (the slowest rate in years) and fell again to · % in as china attempted to reduce its reliance on exports, increase domestic consumption and develop its service sector. the economy then grew by · % in . rapid economic advance has brought with it a number of challenges that threaten future growth. notably, china's cost advantage has been undermined in recent years by rising wages and transportation costs, as well as weak global demand. other concerns include rising property costs, high levels of local government debt, lack of enforcement of intellectual property rights, endemic corruption at government level and credit and investment dependence, while total social financing-a broad measure of total credit-increased by % of gdp between and early . the stimulus package implemented by the government to boost growth increased total debt levels to more than double the value of gdp in . according to the imf, an increase in consumer demand and a reduced dependence on exports and investment are keys to achieving stable long-term economic expansion. china's th five-year plan (covering - ) aims to promote domestic consumption and to support innovation and entrepreneurship within a framework of balanced and sustainable development. efforts to promote domestic consumption have seen exports' share of gdp falling from % in to · % in and a lower investment contribution to gdp. the continued decline in commodity prices coupled with china's economic slowdown has had knock-on effects for commodityexporting nations, such as brazil, indonesia and argentina, given that china consumes about half of the world's steel, aluminium and nickel. inefficient production and outmoded equipment have meanwhile led to significant environmental problems, especially in the north of the country. air pollution, soil erosion and a declining water table are of particular concern. china has become the world's largest consumer of coal and second largest consumer of oil after the usa. the government aims to diversify its energy sources, relying less on coal and more on nuclear and alternative energy sources. there has been heavy investment in hydro-power, including the three gorges dam. since , m. people have been lifted out of poverty, yet china still has the second largest number of poor in the world after india. the world bank estimates that · m. people lived below the national poverty line at the end of (equivalent to income less than us$ per day), located mainly in remote and resource-poor regions and particularly in the west and the interior. nonetheless, some progress has been made, with · % of the rural population living below the poverty line in compared to · % in . growing inequality between urban and rural regions, particularly in terms of educational opportunities, needs to be addressed, however. china also faces the growing burden of an ageing population. those aged and over accounted for · % of the total population in , up from · % in . the currency is called renminbi (i.e. people's currency). the unit of currency is the yuan (cny) which is divided into ten jiao, the jiao being divided into ten fen. the yuan was floated to reflect market forces on jan. while remaining state-controlled. for years the people's bank of china maintained the yuan at about · to the us dollar, allowing it to fluctuate but only by a fraction of % in closely supervised trading. in july it was revalued and pegged against a 'market basket' of currencies the central parities of which were determined every night. in july , after three years of sharp appreciation, it was repegged at around · yuan to the dollar, leading to claims from some international observers that it was being kept unfairly low to boost exports. in june the government announced that the yuan would be allowed to move freely against the dollar as long as a rise or fall does not exceed · % within a single day. in aug. the yuan was devalued by a total of · % on three consecutive days. in aug. total money supply was , · bn. yuan, gold reserves were · m. troy oz and foreign exchange reserves us$ , · bn. (us$ · bn. in of the total revenues in central government accounted for , · bn. yuan and local governments , · bn. yuan. tax revenues came to , · bn. yuan in (including domestic vat , · bn. yuan and corporate income tax , · bn. yuan) and non-tax revenues , · bn. yuan. of the total expenditure in central government accounted for , · bn. yuan and local governments , · bn. yuan. the leading items of expenditure in were education ( , · bn. yuan) and social safety net and employment effort ( , · bn. yuan). the standard rate of vat is %. savings deposits in various forms in all banking institutions totalled , · bn. yuan in ; loans amounted to , · bn. yuan. there are stock exchanges in the shenzhen special economic zone and in shanghai. a securities trading system linking six cities (securities automated quotations system) was inaugurated in for trading in government bonds. china received a record us$ · bn. worth of foreign direct investment in , up from us$ · bn. in . external debt totalled us$ , m. in (up from us$ , m. in ) and represented · % of gni. china's carbon dioxide emissions from the consumption and flaring of fossil fuels in accounted for · % of the world total (making it the biggest emissions producer, having overtaken the usa in ) and were equivalent to · tonnes per capita (up from · tonnes per capita in ). an environmental performance index compiled in ranked china th of countries, with · %. the index examined various factors in nine areas-agriculture, air quality, biodiversity and habitat, climate and energy, fisheries, forests, health impacts, water and sanitation, and water resources. pollution is estimated to cost china about % of gdp annually. installed generating capacity in was an estimated , m. kw, compared with m. kw in . in electricity output was , , gwh, up from , , gwh in . consumption per capita was , kwh in . rapidly increasing demand has meant that more than half of china's provinces have had to ration power. sources of electricity in as percentage of total production: thermal, · %; hydro-electric power, · %; wind. · % (china is one of the world's largest producers of wind power); and nuclear, · %. in there were nuclear reactors in use and under construction. generating electricity is not centralized; local units range between and mw of output. in dec. china formally broke up its state power monopoly, creating instead five generating and two transmission firms. the three gorges dam project on the yangtze river was launched in and is intended to produce abundant hydro-electricity (as well as helping flood control). the first three , -kw generators in service at the project's hydro-power station began commercial operation in july . the original specification was completed in oct. , although six more generators have been added in the meantime (bringing the total to ). the final two generators become operational in july , giving the dam an overall capacity of · gw. china surpassed germany in terms of solar generating capacity in , with · gw at the end of the year. on-shore oil reserves are found mainly in the northeast (particularly the daqing and liaohe fields) and northwest. there are off-shore fields in the continental shelves of east china. oil production was a record · m. tonnes in . china is the second largest consumer of oil after the usa. ever-growing demand has meant that increasing amounts of oil are having to be imported. a -km pipeline from skovorodino in russia to daqing in the northeast of china was inaugurated in jan. , allowing china to increase significantly its imports of oil from the world's second largest producer. the , -km turkmenistan-china gas pipeline, bringing natural gas to xinjiang in china via kazakhstan and uzbekistan, was inaugurated in dec. . this connects with china's second west-east gas pipeline. only the usa imports more oil. domestic production now accounts for only % of consumption, compared to nearly % in . proven reserves in were · bn. bbls. the largest natural gas reserves are located in the western and north-central regions. production was a record · bn. cu. metres in -up from · bn. cu. metres in -with proven reserves of · trn. cu. metres in . china is the second largest producer of wind power after the usa, with · bn. kwh in . in total installed capacity amounted to , mw, the highest of any country and · % of the world total. china is one of the world's leading mineral producing and consuming countries. recoverable deposits of coal in totalled · bn. tonnes, mainly distributed in north china (particularly shanxi province and the inner mongolia autonomous region). coal production was , m. tonnes in . annual coal production has increased every year since . growing domestic demand nonetheless meant that china became a net importer of coal in . iron ore reserves were · bn. tonnes in . deposits are abundant in the anthracite field of shanxi, in hebei and in shandong, and are found in conjunction with coal and worked in the northeast. production in was , m. tonnes, making china the world's largest iron ore producer. it is also the largest consumer, at around % of the global total in . tin ore is plentiful in yunnan, where the tin-mining industry has long existed. tin production was , tonnes in . china is a major producer of wolfram (tungsten ore). there is mining of wolfram in hunan, guangdong and yunnan. output of other minerals (in , tonnes) in : salt, , ; bauxite, , ; aluminium, , ; zinc, , ; lead, , ; copper, , . there are also reserves of diamond, nickel, barite, bismuth, graphite, gypsum, mercury, molybdenum, silver, salt, phosphate ore and sylvite. gold production, : tonnes. china surpassed south africa as the world's leading gold producer in , since when its output has increased every year. agriculture accounted for approximately % of gdp in , compared to over % in at the time of the birth of the people's republic of china and over % in . in sown areas for major crops were (in m. ha.): corn, · ; rice, · ; wheat, · ; soybeans, · ; tubers, · ; rapeseed, · . intensive agriculture and horticulture have been practised for millennia. present-day policy aims to avert the traditional threats from floods and droughts by soil conservancy, afforestation, irrigation and drainage projects, and to increase the 'high stable yields' areas by introducing fertilizers, pesticides and improved crops. in aug. more than m. ha., notably in the yangtze valley, were under water as china experienced its worst flooding since the s. the flood season claimed over , lives. 'township and village enterprises' in agriculture comprise enterprises previously run by the communes of the maoist era, cooperatives run by rural labourers and individual firms of a certain size. there were , state farms in with · m. employees. net per capita annual income of rural households, : , yuan. in there were an estimated · m. ha. of arable land and · m. ha. of permanent cropland; · m. ha. were equipped for irrigation. there were · m. large/medium-sized tractors in and · m. small tractors. china is the world's leading producer of a number of agricultural crops. production of major products (in m. , - . forestry in the area under forests was · m. ha., or % of the total land area. the average annual increase in forest cover of , , ha. between and was the highest of any country in the world. total roundwood production in was · m. cu. metres, making china the world's third largest timber producer ( · % of the world total in ). it is the highest consumer of roundwood; timber consumption in totalled · m. cu. metres. it is also the world's leading importer of roundwood, accounting for · % of world timber imports in . output of major products, unless otherwise indicated (in tonnes): cement, , · m.; rolled steel, , · m.; crude steel, · m.; pig iron, · m.; gas oil and diesel oil ( ), · m.; gasoline, · m.; paper and paperboard, · m.; sulphuric acid, · m.; chemical fertilizers, · m.; fuel oil ( ), · m.; yarn, · m.; refined sugar, · m. also produced in : cloth, , m. metres; beer, , · m. litres; , · m. mobile phones; · m. notebook pcs; · m. colour tv sets; · m. air conditioners; · m. home refrigerators; · m. washing machines; · m. bicycles; · m. cameras; · m. motorcycles. china is the world's leading cement, steel and pig iron manufacturer; since output of cement has doubled and production of crude steel and pig iron has quadrupled (although in both crude steel and pig iron production fell for the first time in years). china overtook japan as the world's largest producer of motor vehicles in , and in produced · m. cars and · m. commercial vehicles. the employed population at the census was · m. ( · m. female). by it had risen to · m. ( · m. more than in ), of whom · m. worked in rural areas ( · m. fewer than in ) and · m. in urban areas ( · m. more than in ). in china's registered urban jobless was · %, with · m. registered unemployed in the country's cities. with china's fast-growing ageing population, according to the united nations the working-age population began to decline in . in china had , private industrial enterprises. it was not until the late s that the private sector even came into existence in china. the average annual wage of people working in urban units in was , yuan. china's labour law stipulates a five-day working week with no more than eight hours a day and no more than hours a week. minimum working age was fixed at in . strikes over pay have become ever more frequent in china, particularly at foreign-owned facilities. china had · m. people living in slavery according to the walk free foundation's global slavery index, the second highest total of any country. there are five special economic zones at shenzhen, xiamen, zhuhai, shantou and hainan in which concessions are made to foreign businessmen. the pudong new area in shanghai is also designated a special development area. since joint ventures with foreign firms have been permitted. a law of april reduced taxation on joint ventures to %. there is no maximum limit on the foreign share of the holdings; the minimum limit is %. in china is the second largest trading nation in the world, accounting for · % of global merchandise imports by value in and · % of global merchandise exports (up from · % when it joined the wto in ). it was the second largest importer in behind the usa and the largest exporter. as recently as the usa's total trade in goods was more than twice that of china. it overtook germany as the largest exporter of goods in . its trade surplus in goods is the highest of any country. however, it has the world's highest trade deficit in services. in imports of services totalled us$ bn. but exports only us$ bn. main imports in (in us$ bn.): machinery and transport equipment, · ; non-edible raw materials, · ; mineral fuels, lubricants and related materials, · ; chemicals, · . major exports in (in us$ bn.): machinery and transport equipment, , · ; miscellaneous manufactured goods, · ; light textile industrial products, rubber products, minerals and metallurgical products, · ; chemicals, · . the main trading partners were as follows in (in us$ m.): the total road length in was , , km, including , km of expressways (of which there had not been any as recently as the mid- s); , m. tonnes of freight and , m. persons were transported by road that year. the number of civilian motor vehicles was · m. in , including · m. passenger vehicles and · m. trucks (more than double the number in , when there were · m. civilian vehicles overall including · m. passenger vehicles and · m. trucks). china is the world's fastest-growing car market. there were , traffic accidents in , with , fatalities. in in jan. there were , ships of gt or over registered, totalling · m. gt. of the , vessels registered, were bulk carriers, general cargo ships, oil tankers, container ships, passenger ships and liquid gas tankers. mainland china's busiest port in was ningbo-zhoushan (handling · m. tonnes of cargo), followed by shanghai ( · m. tonnes), tianjin ( · tonnes), guangzhou (canton) ( · m. tonnes) and qingdao ( · m. tonnes). shanghai overtook singapore to become the world's busiest container port in and handled · m. teus (twenty-foot equivalent units) in . shenzhen, mainland china's second busiest port for container traffic and the world's fourth busiest in , handled · m. teus. hong kong handled · m. teus in . in jan. the first legal direct shipping links between the chinese mainland and taiwanese islands in more than years were inaugurated. inland waterways totalled , km in ; , · m. tonnes of freight and · m. passengers were carried. in june the three gorges reservoir on the chang jiang river, the largest water control project in the world, reached sufficient depth to support the resumption of passenger and cargo shipping. out of countries analysed in the fragile states index-a list published jointly by the fund for peace and foreign policy magazine-china was ranked the th most vulnerable to conflict or collapse. the index is based on indicators of state vulnerability across social, political and economic categories. six new codes of law (including criminal and electoral) came into force in , to regularize the legal unorthodoxy of previous years. there is no provision for habeas corpus. as well as treason and murder the death penalty may be used for rape, embezzlement, smuggling, fraud, theft, drug-dealing, bribery and robbery with violence. amendments to the criminal law in and reduced the number of capital crimes-which include both violent and non-violent offences-from to and further to . china does not divulge figures on its use of the death penalty, but amnesty international reported that in china executed thousands of people and was the world's top executioner. nevertheless, western analysts believe that the number of executions now is around a fifth of the yearly total in the s. 'people's courts' are divided into some higher, intermediate and , basic-level courts, and headed by the supreme people's court. the latter, the highest state judicial organ, tries cases, hears appeals and supervises the people's courts. it is responsible to the national people's congress and its standing committee. people's courts are composed of a president, vice-presidents, judges and 'people's assessors' who are the equivalent of jurors. 'people's conciliation committees' are charged with settling minor disputes. there are also special military courts. procuratorial powers and functions are exercised by the supreme people's procuracy and local procuracies. in march the national people's congress passed legislation developing aspects of the general principles of civil law, with effect from oct. . among its provisions was the extension of legitimate rights and interests from chinese citizens only to anyone conducting civil activities in the country. in addition, the statute of limitation was increased from two to three years. the number of sentenced prisoners in mid- was , , ( per , of national population). china was ranked th of countries for criminal justice and th for civil justice in the world justice project rule of law index, which provides data on how the rule of law is experienced by the general public across eight categories. an educational reform of brought in compulsory nine-year education consisting of six years of primary schooling and three years of secondary schooling, to replace a previous five-year system. in mainland china the population census revealed the following levels of educational attainment: · m. people had finished university education; · m. had received senior secondary education; · m. had received junior secondary education; and · m. had had primary education. · m. people over years of age or · % of the population were illiterate, although this compared favourably with a · % rate of illiteracy in the census and a · % rate in . in adult literacy was estimated at · %; youth literacy in was · %. in there were , kindergartens with · m. children and · m. full-time teachers; , regular primary schools with · m. pupils and · m. full-time teachers; , secondary schools (including: , senior secondary; , junior secondary; , specialized; , vocational; and , technical) with · m. pupils and · m. full-time teachers. there were also , pupils at , special education schools. institutes of higher education, including universities, numbered , in , with · m. undergraduates and · m. postgraduate level students, and · m. full-time teaching staff. china has more than private universities, almost all of which have been established since the mid- s. a national system of student loans was established in . the number of chinese students studying abroad went up from , in to , in ; it rose above , in and , in , and by exceeded , , making china the largest source of overseas students in the world. chinese students account for a fifth of all international students in tertiary education in the oecd, but fewer than half return to china after finishing their studies. the number of chinese undergraduate students in american universities in - was times as many as in - , rising from , to , in the space of seven years. there is an academy of sciences with provincial branches. an academy of social sciences was established in . in national government expenditure on education came to , , m. yuan and accounted for · % of national government spending. medical treatment is free only for certain groups of employees, but where costs are incurred they are partly borne by the patient's employing organization. in there were , health institutions throughout china, including , hospitals, , local health centres, , centres for disease control and prevention, and , specialized prevention and treatment centres. china's first aids case was reported in . at the end of there were , reported cases of people living with hiv/ aids. the number of deaths of people who had been living with hiv/aids in was , . in the first half of china was struck by an epidemic of a pneumonia-type virus identified as sars (severe acute respiratory syndrome). the virus was first detected in southern china and was subsequently reported in over other countries. according to the ministry of health, by the time the outbreak had been contained a total of , cases had been reported on the chinese mainland; , patients were cured and discharged from hospital, and died. in water: at what cost? the state of the world's water , wateraid reported that · % of the population does not have access to safe water. china ranked as the country with the second largest number of people living without access to safe water ( · m. in ). in an estimated · % of adult males and · % of adult females smoked in china. a study from the same year estimated that chinese males smoke one-third of all the world's cigarettes. in there were , social welfare enterprises with · m. beds. numbers (in , ) of beneficiaries of relief funds in : urban residents receiving minimum living allowance, , ; rural residents receiving minimum living allowance, , ; persons receiving traditional relief, ; persons in rural households entitled to the 'five guarantees' (food, clothing, medical care, housing and burial expenses), , . the official retirement age for men is and for women (or in the case of civil servants and professionals). the government accords legality to five religions only: buddhism, islam, protestantism, roman catholicism and taoism. confucianism, buddhism and taoism have long been practised. confucianism has no ecclesiastical organization and appears rather as a philosophy of ethics and government. taoism-of chinese origin-copied buddhist ceremonial soon after the arrival of buddhism two millennia ago. buddhism in return adopted many taoist beliefs and practices. a more tolerant attitude towards religion had emerged by , and the government's bureau of religious affairs (since renamed the state administration for religious affairs) was reactivated. ceremonies of reverence to ancestors have been observed by the whole population regardless of philosophical or religious beliefs. a new quasi-religious movement, falun gong, was founded in , but has since been banned by the authorities. the movement has claimed some m. adherents, although the chinese government has disputed this. muslims are found in every province of china, being most numerous in the ningxia-hui autonomous region, yunnan, shaanxi, gansu, hebei, henan, shandong, sichuan, xinjiang and shanxi. roman catholicism has had a footing in china for more than three centuries. two christian organizations-the chinese patriotic catholic association, which declared its independence from rome in , and the protestant three-self patriotic movement-are sanctioned by the chinese government. according to estimates (by the state-approved xinhua news agency, the chinese academy of social sciences and the state administration for religious affairs) there were m. buddhists (more than in any other country), m. christians and more than m. muslims in the country in . other official figures indicate that there are · m. catholics, although unofficial estimates are much higher. the number of christians in china is generally thought to be far higher than official numbers indicate, with socalled 'house churches' becoming ever more popular. some analysts estimate that there are as many as m. christians overall. legislation of prohibits foreign nationals from setting up religious organizations. in , , m. volumes of books were produced. in tourist numbers totalled · m. the world tourism organization predicts that china will overtake france as the world's most visited destination by . it was the third most visited destination in after france and the usa. income from tourists in was us$ · bn., ranking it fourth behind the usa, spain and france. expenditure by chinese travellers outside of mainland china for was us$ · bn., the most of any country. in both german and us travellers abroad had spent more than those from china. the lunar new year, also known as the 'spring festival', is a time of great excitement for the chinese people. constituencies encompassing all eligible persons in a workforce of · m., and ten from an election committee formed by members of district boards. a president was elected from and by the members. at the elections on sept. turnout for the geographical seats was · %, and for the functional seats ( of which were contested), · %. the democratic party and its allies gained seats, the liberal party and the pro-beijing democratic alliance . the remaining seats went to independents. on dec. the selection committee selected a provisional legislature which began its activities in jan. while the legislative council was still functioning. in jan. the provisional legislature started its work by enacting legislation which would be applicable to the hong kong special administrative region and compatible with the basic law. constitutionally hong kong is a special administrative region of the people's republic of china. the basic law enables hong kong to retain a high degree of autonomy. it provides that the legislative, judicial and administrative systems which were previously in operation are to remain in place. the special administrative region government is also empowered to decide on hong kong's monetary and economic policies independent of china. in july the first-past-the-post system of returning members from geographical constituencies to the legislative council was replaced by proportional representation. there were directly elected seats out of for the first elections to the legislative council following hong kong's return to chinese sovereignty, increasing in accordance with the basic law to for the election with indirectly elected. in the sept. legislative council election (and that of sept. ) of the seats were directly elected. for the election in sept. the number of seats was increased to , with directly elected and indirectly elected by functional constituencies. there were also five new functional constituency seats nominated by elected district council members. the chief executive is chosen by a beijing-backed , -member election committee ( prior to the march election), although it has been stated that universal suffrage is the ultimate aim. in a timetable was announced for hong kong to directly elect its chief executive in and its legislative council in . however, beijing insisted that only approved candidates would be allowed to stand in , prompting mass pro-democracy rallies in the territory in and formal rejection of the plan by the legislative council in june . beijing nevertheless refused to countenance amendments. in july a new accountability or 'ministerial' system was introduced, under which the chief executive nominates for appointment policy secretaries, who report directly to the chief executive. the chief executive is aided by the executive council, consisting of the three senior secretaries of department (the chief secretary, the financial secretary and the secretary for justice) and eleven other secretaries plus five non-officials. according to the anti-corruption organization transparency international, hong kong ranked equal th in the world in a survey of the countries and regions with the least corruption in business and government. it received out of in the annual index. overview hong kong has one of the world's most open economies and is an internationally important financial centre. the territory's economic rise was founded on its role as an international trade emporium, acting as a conduit for china's burgeoning exports. mainland china, the usa and japan are hong kong's major export partners, accounting for · %, · % and · % of exports respectively in . the island is dependent on imports of food and other resources. in it imported % of goods from mainland china, % from taiwan and % from japan. in and the economy grew strongly on the back of a rise in chinese tourism, healthy global demand for exports and improving domestic consumer confidence. however, the global financial crisis saw the economy shrink by · % in before rebounding with a · % increase the following year. between and annual growth averaged · %, supported by strong external demand. student-led pro-democracy protests in the latter months of caused major disruption in several key business districts and threatened to weaken the local economy in the short term. foreign direct investment levels have been high, averaging % of gdp between and according to world bank data, and the world economic forum ranked hong kong as the ninth most competitive economy in the world in its report. the government aims to tackle a housing shortage by providing , new housing units by the mid- s. the unit of currency is the hong kong dollar (hkd) of cents. it has been pegged since at a rate of hk$ · to the us dollar. banknotes are issued by the hongkong and shanghai banking corporation and the standard chartered bank, and, from may , the bank of china. total money supply was hk$ , m. in july . in aug. gold reserves were , troy oz and foreign exchange reserves were us$ , m. inflation rates (based on imf statistics): in - revenue totalled hk$ · bn. and expenditure hk$ · bn. earnings and profits taxes accounted for · % of revenues in - and indirect taxes · %; education accounted for · % of expenditures and social welfare · %. hong kong's carbon dioxide emissions from the consumption of energy in were the equivalent of · tonnes per capita. installed capacity was · m. kw in . production in was · bn. kwh. hong kong is a net importer of electricity. consumption in was · bn. kwh. the local agricultural industry is directed towards the production of high quality fresh food through intensive land use and modern farming techniques. out of the territory's total land area of , sq. km, only sq. km is currently farmed. in local production accounted for % of live poultry consumed, % of live pigs and % of fresh vegetables. the gross value of local agricultural production totalled hk$ , m. in , with pig production valued at hk$ m., poultry production (including eggs) at hk$ m., and vegetable and flower production at hk$ m. in the total catch was , tonnes, exclusively from marine waters. the leading companies by market capitalization in hong kong in may were: china mobile, a telecommunications company (us$ · bn.); aia group, a life insurance company (us$ · bn.); and cnooc, an integrated oil company (us$ · bn.). industry is mainly service-oriented. in june there were , establishments employing , , persons in service industries and , establishments employing , persons in manufacturing industries. establishment statistics by service type (and persons engaged) were mainly: import/export trade and wholesale, , ( , ); retail, , ( , ) ; social and personal services, , ( , ) ; professional and business services, , ( , ); financing and insurance, , ( , ); accommodation and food services, , ( , ); real estate, , ( , ) . in the size of the labour force (synonymous with the economically active population) was , , ( , , females). the persons engaged in june included , , people in wholesale, retail and import/export trades, accommodation and food services, , in finance, insurance, real estate, professional and business services, , in the civil service, , in manufacturing and , in construction sites (manual workers only). a minimum wage of hk$ per hour was introduced for the first time on may . unemployment stood at · % in the period sept.-dec. . in the total value of imports was hk$ , , m. and total exports hk$ , , m. the main suppliers of imports in were mainland china ( · %), japan ( · %), taiwan ( · %), singapore ( · %) and usa ( · %). in , · % of total exports went to mainland china, · % to the usa, · % to japan, · % to germany and · % to the united kingdom. the chief import items in were: electrical machinery, apparatus and appliances, etc. ( · %); telecommunications, sound recording and reproducing equipment ( · %); office machines and automatic data processing machines ( · %); articles of apparel and clothing accessories ( · %). the main exports in were: electrical machinery, apparatus and appliances, etc. ( · %); telecommunications, sound recording and reproducing equipment ( · %); office machines and automatic data processing machines ( · %); articles of apparel and clothing accessories ( · %). in there were , km of roads, over % of which were in the new territories. there are road tunnels, including three under victoria harbour. in there were , private cars, , goods vehicles, , buses and coaches, and , motorcycles and mopeds. there were , road accidents in , of which were fatal. a total of · m. tonnes of cargo were transported by road in . a -km bridge linking hong kong, zhuhai in guangdong province in mainland china and macao has been built and was scheduled to open in july following a number of delays. hong kong was ranked fourth for its road infrastructure in the world economic forum's global competitiveness report - . hong kong's railways are run by the mtr corporation limited (mtrcl), a public listed company of which the government is the majority shareholder. the mtr system comprises nine railway lines serving hong kong island, kowloon and the new territories. its -km network has stations and carries an average of · m. passengers each day. mtr lines carried , m. passengers in . in addition, a light rail network ( · km and stops) serves the local communities of tuen mun, yuen long and tin shui wai in the new territories; , passengers travel daily on the system. a high speed rail service between hong kong and guangzhou on the mainland is scheduled to open in late . the electric tramway on the northern state of hong kong island commenced operating in and has a total track length of km. the peak tram, a funicular railway connecting the peak district with the lower levels in victoria, has a track length of · km and two tramcars (each with a capacity of passengers per trip). it carries an average of , passengers daily. the airport express line ( · km) opened in and is also operated by the mtrcl. it carried · m. passengers in . in june it was estimated that · m. passenger journeys were made daily on public transport (including local railways, buses, etc.). in the world economic forum's global competitiveness report - hong kong ranked third for quality of rail infrastructure. the new hong kong international airport (generally known as chek lap kok), built on reclaimed land off lantau island to the west of hong kong, was opened on july to replace the old hong kong international airport at kai tak, which was situated on the north shore of kowloon bay. more than airlines now operate scheduled services to and from hong kong. in cathay pacific airways, the largest hong kong-based airline, operated approximately , passenger and cargo services to destinations in countries and territories around the world. cathay pacific carried , , passengers and · m. tonnes of cargo in . dragonair, a cathay pacific subsidiary, provided scheduled services to cities in mainland china and asia in . in air hong kong, an all-cargo operator, provided scheduled services to bangkok, beijing, ho chi minh city, manila, nagoya, osaka, penang (via bangkok), seoul, shanghai, singapore, taipei and tokyo. hong kong international airport handled more international freight in than any other airport. in , , aircraft arrived and departed and m. passengers and · m. tonnes of freight were carried on aircraft. hong kong was second, behind only singapore, in the rankings for air transport infrastructure in the world economic forum's global competitiveness report - . the port of hong kong handled · m. teus (twenty-foot equivalent units) in , making it the world's third busiest container port after shanghai and singapore. the kwai chung container port has berths with , metres of quay backed by ha. of cargo handling area. at the end of there were , ships ( , ocean-going) of , , gt registered in hong kong. in , , ocean-going vessels, , river cargo vessels and , river passenger vessels arrived at the port of hong kong. a total of m. tonnes of freight were handled in . hong kong was ranked third in the world economic forum's global competitiveness report - for the quality of its port facilities. in there were , , main (fixed) telephone lines (equivalent to · per , population). the local fixed telecommunications network services (ftns) market in hong kong was liberalized in . there were , , mobile phone subscriptions in (equivalent to , · per , population), up from , , in ( , · per , population). the number of subscriptions doubled between and . the internet market has also seen huge growth. in there were , , wireless broadband subscriptions ( · per , population) and , , fixed broadband subscriptions ( · per , population). the number of fixed broadband subscriptions has been declining since as more people have wireless subscriptions instead. in march there were · m. facebook users. the external telecommunications services market has been fully liberalized since jan. , and the external telecommunications facilities market was also liberalized starting from jan. . the hong kong act of provided for hong kong ordinances to replace english laws in specified fields. the courts of justice comprise the court of final appeal (inaugurated july ), which hears appeals on civil and criminal matters from the high court; the high court (consisting of the court of appeal and the court of first instance); the lands tribunal, which determines on statutory claims for compensation over land and certain landlord and tenant matters; the district court (which includes the family court); the magistracies (including the juvenile court); the coroner's court; the labour tribunal, which provides a quick and inexpensive method of settling disputes between employers and employees; the small claims tribunal, which deals with monetary claims involving amounts not exceeding hk$ , ; and the obscene articles tribunal. while the high court has unlimited jurisdiction in both civil and criminal matters, the district court has limited jurisdiction. the maximum term of imprisonment it may impose is seven years. magistracies exercise criminal jurisdiction over a wide range of offences, and the powers of punishment are generally restricted to a maximum of two years' imprisonment or a fine of hk$ , . after being in abeyance for years, the death penalty was abolished in . , crimes were reported in , of which , were violent crimes. , people were arrested in , of whom , were for violent crimes. the population in penal institutions was , at dec. ( per , population). in the adult literacy rate was · % ( · % in ). universal basic education is available to all children aged from six to years. in around three-quarters of the ordinary secondary day schools teaching has been in cantonese since - , with about a quarter of ordinary secondary day schools still using english. in there were , pupils in kindergartens, , in primary schools (including international schools) and , in secondary schools (including international schools). the estimated total government expenditure on education in - was hk$ · bn. ( · % of total government spending and · % of gdp). in - : · % of total government spending and · % of gdp. according to the oecd's pisa (programme for international student assessment) study, -year-olds in hong kong rank second among oecd and other major countries and cities in mathematics and reading, and ninth in science. the three-yearly study compares educational achievement of pupils in over countries. the department of health (dh) is the government's health adviser and regulatory authority. the hospital authority (ha) is an independent body responsible for the management of all public hospitals. in there were , registered doctors, equivalent to · doctors per , population. in there were , dentists, , nurses and , midwives. the total number of hospital beds in was , , including , beds in public hospitals under the ha and , beds in private hospitals. the bed-population ratio was · beds per thousand population. the chinese medicine ordinance was passed by the legislative council in july to establish a statutory framework to accord a professional status for chinese medicine practitioners and ensure safety, quality and efficacy of chinese medicine. in there were , registered chinese medicine practitioners. total expenditure on health in - amounted to hk$ , m., an increase of · % over that in - . social welfare programmes include social security, family services, child care, services for the elderly, medical social services, youth and community work, probation, and corrections and rehabilitation. non-governmental organizations are subsidized by public funds. the government gives non-contributory cash assistance to needy families, unemployed able-bodied adults, the severely disabled and the elderly. caseload as at aug. totalled , . victims of natural disasters, crimes of violence and traffic accidents are financially assisted. estimated recurrent government expenditure on social welfare for - was hk$ · bn. sub-tropical tending towards temperate, with an average temperature of · °c. the number of rainy days is around a third of the year. average annual rainfall varies from - " ( , - , mm) . it is very humid from may to sept. macao's constitution is the 'basic law', promulgated by china's national people's congress on march and in effect since dec. . it is a special administrative region (sar) of the people's republic of china, and is directly under the central people's government while enjoying a high degree of autonomy. the legislative assembly has seats of which are directly elected, indirectly elected by functional constituencies and seven appointed by the chief executive. at the elections held on sept. the macau-guangdong union won two of elected seats with · % of votes cast and the union for development two with · %. ten other parties won a single seat each. turnout was · %. fernando chui sai-on was re-elected chief executive for a second term on aug. , receiving out of votes in the election committee. the economy grew by an average · % per year from - , driven mainly by the gaming sector and the ongoing construction of a number of casino resorts. however, it went into recession from following china's crackdown on government corruption. macao's traditional manufacturing industries virtually disappeared following the transfer of much of the textile industry to the chinese mainland and, in , the termination of the multifibre arrangement, which had governed international textile trade flows for three decades. the unit of currency is the pataca (mop) of avos, which is tied to the hong kong dollar at parity. inflation was · % in and · % in . foreign exchange reserves were us$ , m. in . total money supply was , m. patacas in . in revenues totalled , m. patacas; expenditures, , m. patacas. revenues from gaming tax accounted for · % of total revenue in ; current expenditure accounted for · % of expenditure. real gdp growth was just · % in but then rose to · % in and · % in . more recently the economy contracted by · % in and then by · % in -the lowest rate for any advanced economy that year-owing to a decline in tourism and gaming revenues. total gdp in was us$ · bn. there are two note-issuing banks in macao-the macao branch of the bank of china and the macao branch of the banco nacional ultramarino. the monetary authority of macao functions as a central bank (chairman, teng lin seng). commercial business is handled ( ) by banks, nine of which are local and foreign. total deposits, (including non-resident deposits), , · m. patacas. there are no foreign exchange controls within macao. macao's carbon dioxide emissions from the consumption of energy in were the equivalent of · tonnes per capita. installed capacity was · m. kw in ; production, · bn. kwh. macao imported , m. kwh of electricity in . oil and gas , , litres of fuel oil were imported in . the catch in was estimated at , tonnes. although the economy is based on gaming and tourism there is a light industrial base of textiles and garments. in the number of manufacturing establishments was (food products and beverages, ; textiles and wearing apparel, ; publishing, printing and reproduction of recorded media, ). in a total of , people were in employment, including , ( · %) in gaming and junket activities (up from , in ); , ( · %), hotels, restaurants and similar activities; , ( · %), construction; , ( · %), wholesale and retail trade, repair of motor vehicles, motorcycles and personal and household goods; , ( · %), real estate and business activities; , ( · %), public administration and social security. employment in was · % of the labour force; unemployment rate stood at · %. in imports (c.i.f.) were valued at us$ , · m., of which the main products were telecommunications, sound recording and reproducing equipment; petroleum and petroleum products; and gold, silverware, jewellery and articles of precious materials. in the chief import sources (in us$ m.) were: mainland china ( , · ); hong kong ( · ); japan ( · ). exports (f.o.b.) were valued at us$ · m., of which the leading products were articles of apparel and clothing accessories; gold, silverware, jewellery and articles of precious materials; and petroleum oils and oils obtained from bituminous minerals. in the main export markets (in us$ m.) were: hong kong ( · ); usa ( · ); mainland china ( · ). in there were km of roads. in there were , landline telephone subscriptions (equivalent to · per , inhabitants) and , , mobile phone subscriptions (or , · per , inhabitants). in , · % of households had internet access. in march there were , facebook users. there are a judicial district court, a criminal court and an administrative court with magistrates in all. in there were , crimes, of which , were against property. there were persons in prison in dec. . there are both public and private schools. in - there were schools and colleges. number of students in the - academic year (with number of teachers): pre-primary, , ( ); primary, , ( , ); secondary, , ( , ). in - there were four special education schools with pupils and teachers. there were ten higher education institutions with student enrolment of , . in there were institutions offering vocational training courses, in which participants totalled , . expenditure on education came to · % of gdp in and · % of total government spending in . in there were doctors, dentists and nurses working in primary health care, and doctors, dentists and , nurses working in hospitals. in there were , hospital beds; there were · doctors per , population. in there were an estimated , folk religionists and , buddhists according to the pew research center's forum on religion & public life. a further , people were religiously unaffiliated. there are also small numbers of catholics. the historic centre of macao was inscribed on the unesco world heritage list in . in there were daily newspapers (nine in chinese, three in portuguese and two in english) and weekly newspapers (ten in chinese and one in portuguese). tourism is one of the mainstays of the economy. in there were · m. tourists (of which · m. were from mainland china, · m. from hong kong and · m. from taiwan), up from · m. in and · m. in . visitor spending in totalled , m. patacas. the government-run macao international music festival featuring a wide range of chinese and western music takes place in oct.-nov. in sept. chen shui-bian received a life sentence (later reduced to a -year term) after being found guilty of multiple counts of corruption. china and taiwan signed a free trade agreement in june , which was considered a significant thawing of relations. nonetheless, tensions remained, particularly in relation to disputed sovereignty over several islands in the east china sea. in jan. ma ying-jeou was re-elected to the presidency but the election in jan. was won by tsai ing-wen, whose democratic progressive party won the most seats in legislative polls at the same time-the first occasion that the nationalist party has not been the largest party in government since . beginning with the elections to the seventh legislative yuan held on jan. the legislative yuan has members (formerly ). of the members are elected under the first-past-thepost system in single-member constituencies, are filled by proportional representation in accordance with a nationwide party vote and six are reserved for aboriginal candidates. since the president has been directly elected. since a resolution on the impeachment of the president or vice president is no longer to be instituted by the control yuan but rather by the legislative yuan. the legislative yuan has the power to pass a no-confidence vote against the premier of the executive yuan, while the president of the republic has the power to dissolve the legislative yuan. the premier of the executive yuan is directly appointed by the president of the republic. in dec. a law came into effect allowing for referendums to be held. national anthem 'san min chu i' ('the three principles of the people'); words by dr sun yat-sen, tune by cheng mao-yun. conscription was reduced from months to months in . the government has announced its intention to move towards a volunteer professional force-a process that was originally scheduled to start in and end in but was delayed owing to low recruitment levels. in dec. the defence minister announced that conscription would officially cease in . defence expenditure in totalled us$ , m. (us$ per capita), representing · % of gdp. the republic of china army conducts ground combat missions as well as air support and airborne special operations. it was estimated to number about , personnel in , with reserves numbering · m. its principal role is to defend against a possible amphibious assault from the chinese mainland by the people's liberation army. in addition there are paramilitary forces totalling , personnel. navy personnel in totalled , , with , reservists. the forces consist of four submarines, four cruisers and frigates. there are also missile craft for patrol and coastal defence, mine-laying vehicles and amphibious landing craft. in the air force numbered , personnel with , reservists. there were combat-capable aircraft in the same year including f- es, f- s and mirage - s. by a treaty of dec. the usa pledged to defend taiwan, but this treaty lapsed one year after the usa established diplomatic relations with the people's republic of china on jan. . in april the taiwan relations act was passed by the us congress to maintain commercial, cultural and other relations between usa and taiwan through the american institute in taiwan and its taiwan counterpart, the co-ordination council for north american affairs in the usa, which were accorded quasi-diplomatic status in . the people's republic took over the china seat in the un from taiwan on oct. . in may taiwan ended its formal state of war with the people's republic. taiwan became a member of the world trade organization on jan. . in feb. taiwan had formal diplomatic ties with countries. in aug. , of the diplomatic allies sponsored an unsuccessful proposal for taiwan to join the un. taiwan has made a successful transition from an agricultural economy to one based on high-tech electronics. economic growth averaged % per year over three decades from the s, driven primarily by high value-added manufacturing and exports, especially in electronics and computers. government-owned enterprises, including banks, have been privatized. though largely escaping the impact of the asian financial crisis, the economy went into recession in with the first year of negative growth ever recorded and unemployment reaching record highs. strong export performance stimulated a recovery, with annual gdp growth above % from - . inflation has been consistently low and unemployment, which fell below % in , has averaged between and % since the turn of the century. owing to its heavy dependence on exports, taiwan suffered a severe downturn as a result of the global financial crisis in . major export industries such as semiconductors and memory chips declined, unemployment reached its highest levels since and, in , the economy again went into recession. a us$ · bn. stimulus package boosted recovery and in the economy recorded its highest growth rate for nearly three decades, at · %. however, growth subsequently cooled owing to lower demand from developed nations, averaging · % per year between and . tourism has grown in importance, with over · m. visitors in constituting taiwan's highest annual number to date. an ageing population and high savings rates threaten to constrain domestic demand in the future. the unit of currency is the new taiwan dollar (twd) of cents. gold reserves were · m. oz in dec. . there was inflation of · % in but deflation of · % in . foreign exchange reserves were us$ · bn. in dec. . in general government revenues totalled nt$ , , m. and expenditures nt$ , , m. tax revenue accounted for · % of revenues in ; education, science and culture accounted for · % of expenditures, economic development · % and general administration · %. taiwan sustained rapid economic growth at an annual rate of · % from up to . the rate slipped to · % in the s and · % in ; taiwan suffered from the asian financial crisis, though less than its neighbours. in global economic sluggishness and the events of sept. in the usa severely affected taiwan's economy, which contracted by · %. subsequent economic recovery led to growth of · % in and · % in . there was negative growth of · % in but again the economy bounced back, and grew by · % in , · % in and · % in . the central bank of the republic of china (taiwan), reactivated in , regulates the money supply, manages foreign exchange and issues currency. the governor is yang chin-long. the bank of taiwan is the largest commercial bank and the fiscal agent of the government. there are seven domestic banks, commercial banks and foreign banks. there are two stock exchanges in taipei. taiwan's carbon dioxide emissions from the consumption of energy in were the equivalent of · tonnes per capita. output of electricity in was · m. mwh; total installed capacity was , mw. there were six units in three nuclear power stations in . crude oil production in was , bbls; natural gas, m. cu. metres. taiwan imports most of the oil and natural gas that it consumes. in the cultivated area was , ha., of which , ha. were paddy fields. rice production totalled , , tonnes. livestock production was valued at nt$ , m., accounting for % of taiwan's total agricultural production value. forest area, : , , ha. forest reserves: trees, , , cu. metres; bamboo, , m. poles. timber production, , cu. metres. the catch in was , tonnes, almost exclusively from sea fishing. the first three are container centres, kaohsiung handling · m. -ft equivalent units in , making it the world's th busiest container port in terms of number of containers handled. suao port is an auxiliary port to keelung. in jan. the first legal direct shipping links between taiwanese islands and the chinese mainland in more than years were inaugurated. in there were , , landline telephone subscribers ( · per , inhabitants). taiwan's biggest telecommunications firm, the state-owned chunghwa telecom, lost its fixed-line monopoly in aug. . in there were , , mobile phone subscriptions, equivalent to , · per , persons. in there were · mobile broadband subscriptions per inhabitants and · fixed broadband subscriptions per inhabitants. in march there were · m. facebook users. the judicial yuan is the supreme judicial organ of state. comprising grand justices, since these have been nominated and, with the consent of the legislative yuan, appointed by the president of the republic. the grand justices hold meetings to interpret the constitution and unify the interpretation of laws and orders. there are three levels of judiciary: district courts and their branches deal with civil and criminal cases in the first instance; high courts and their branches deal with appeals against judgments of district courts; the supreme court reviews judgments by the lower courts. there is also the supreme administrative court, high administrative courts and a commission on the disciplinary sanctions of public functionaries. criminal cases relating to rebellion, treason and offences against friendly relations with foreign states are handled by high courts as the courts of first instance. the death penalty is still in force. there was one execution in but none in . the population in penal institutions in april was , ( per , of national population). since there has been compulsory education for six to year-olds with free tuition. the illiteracy rate dropped from · % in to · % by . there were , primary schools, , secondary schools and vocational schools in ; and universities, colleges and junior colleges. in - there were , , pupils with , teaching staff at elementary schools; , pupils and , teaching staff at junior high schools; , pupils and , teaching staff at senior high schools; and , students and , teaching staff at senior vocational schools. there were , , students in universities and colleges in - with , academic staff. in there were , physicians (one for every persons), , doctors of chinese medicine, , nurses, , dentists and assistants, and , pharmacists and assistants. in there were , medical facilities serving , persons per facility; there were , beds and · beds per , persons. in cancers, heart diseases, cerebrovascular diseases, diabetes and accidents were the first five leading causes of death. a universal health insurance scheme came into force in as an extension to social insurance plans that cover only % of taiwan's population. premium shares among the government, employer and insured are varied according to the insured statuses. by the end of , · m. people or % of the population were covered by the national health insurance programme. according to estimates by the pew research center's forum on religion & public life, · % of the population in were folk religionists, · % were buddhists and · % christians. the remainder of the population was either religiously unaffiliated or followed other religions, including taoism. there were daily newspapers in with a circulation of · m. and non-dailies with a circulation of · m. the biggest circulation dailies are the liberty times and apple daily. in there were , , international visitors. receipts totalled us$ , m. the pop festival, spring scream, is held in april in kenting. china in world history chinese politics in the age of deng xiaoping china and the global political economy china: the rise of xi jinping the cambridge encyclopaedia of china the cambridge history of china. vols the politics of hong kong's reversion to china the chinese economy under transition china this century the tragedy of liberation: a history of the chinese revolution china: a modern history china's deep reform: domestic politics in transition chinese capitalism deng xiaoping and the making of modern china the great chinese revolution - the politics of eu-china economic relations china's second continent: how a million migrants are building a new empire in africa china in transition: communism, capitalism and democracy sowing the seeds of democracy in china: political reform in the deng xiaoping era historical dictionary of the chinese cultural revolution the rise of modern china china: a macro history tombstone: the untold story of mao's great famine china's economy: what everyone needs to know all under heaven: a complete history of china chinese politics in the hu jintao era: new leaders, new challenges the people's republic of amnesia: tiananmen revisited chinese foreign policy in a changing world chinese economy in the s the politics of china: sixty years of the people's republic of china the party: the secret world of china's communist rulers. .-asia's reckoning: china, japan and the fate of us power in the pacific century china's war with japan, - : the struggle for survival social and political development in post-reform china age of ambition: chasing fortune, truth and faith in the new china a revolutionary life at the crossroads of post-communist modernisation: russia and china in comparative perspective a history of china wealth and power: china's long march to the twenty-first century mao's road to power: revolutionary writings - . vols china goes global: the partial power the chinese century: the rising chinese economy and its impact on the global economy, the balance of power, and your job the china-pakistan axis: asia's new geopolitics the chan's great continent: china in western minds eldest son, zhou enlai and the making of modern china india's and china's recent experience with reform and growth critical issues in contemporary china making china strong other more specialized titles are listed under territory and population; tibet; agriculture further reading statistical information: the census and statistics department is responsible for the preparation and collation of government statistics. these statistics are published mainly in the hong kong monthly digest of statistics. the department also publishes monthly trade statistics, economic indicators and an annual review of overseas trade hong kong's transitions the politics of hong kong's reversion to china the end of hong kong: the secret diplomacy of imperial retreat the hong kong story the final years of british hong kong: the discourse of colonial withdrawal last post: the end of empire in the far east the politics of democratization in hong kong historical dictionary of hong kong and macau china: a political history of the british crown colony's transfer to chinese rule managing china's sovereignty in hong kong and taiwan the democratic progressive party won seats ( constituency and proportional); the nationalist party, ( constituency and proportional); the new power party, ; the people first party, ; the non-partisan solidarity union, ; ind., . current government president: tsai ing-wen; b. (nationalist party/kuomintang foreign affairs; health and welfare; interior; justice; labour; national defence; science and technology national defence: yen de-fa. science and technology: chen liang-gee. transportation and communications: ho chen-tan tang feng; lo ping-cheng coast guard administration: lee chung-wei. environmental protection administration: lee ying-yuan. fair trade commission: huang mei-ying. financial supervisory commission: koo lihsiung. hakka affairs council: lee yung-te. council of indigenous peoples: icyang parod. mainland affairs council: chen ming-tong. national communications commission: chan ting-i. overseas community affairs council: wu hsin-hsing. directorate general of personnel administration: shih ning-jye. veterans' affairs council: lee shying-jow hon hai precision industry, an electronics manufacturer (us$ · bn.); and chunghwa telecom (us$ · bn metres; integrated circuit packages, · trn. units; global positioning system (gps) sets, · bn. units. labour in the average total labour force was · m., of whom · m. were employed. of the employed population, · % worked in manufacturing · % in construction; · % in accommodation and food services buses and coaches, · m. lorries and vans, and · m. motorcycles and mopeds were in use. , m. passengers and m. tonnes of freight were transported in . there were , fatalities in road accidents in . rail in freight traffic amounted to · m. tonnes and passenger traffic to m. total route length was , km. there are metro systems in taipei civil aviation there are currently two international airports: taiwan taoyuan international airport at taoyuan near taipei, and kaohsiung international in the south there were ships of gt or over registered, totalling · m. gt. of the vessels registered, were general cargo ships, bulk carriers, container ships, oil tankers and nine passenger ships. there are six international ports statistical yearbook of the republic of china. annual. the republic of china yearbook. annual. taiwan statistical data book. annual. annual review of government administration the other taiwan: to the present day historical dictionary of taiwan national identity and status in international society managing china's sovereignty in hong kong and taiwan the shadow of china: political developments in taiwan since national statistics website the climate is subtropical in the north and tropical in the south. the typhoon season extends from july to sept. the average monthly temperatures of jan. and july in taipei are · °f ( · °c) and · °f ( · °c) respectively, and average annual rainfall is · " ( , · mm). kaohsiung's average monthly temperatures of jan. and july are · °f ( · °c) and · °f ( · °c) respectively, and average annual rainfall is · " ( , · mm). the roc constitution is based on the principles of nationalism, democracy and social wellbeing formulated by dr sun yat-sen, the founding father of the republic of china. the roc government is divided into three main levels: central, provincial/ municipal and county/city, each of which has well-defined powers.the central government consists of the office of the president, the national assembly, which is specially elected only for constitutional amendment, and five governing branches called 'yuan', namely the executive yuan, the legislative yuan, the judicial yuan, the examination yuan and the control yuan. key: cord- - n ukr authors: shapira, assaf; shapira, shiran; gal-tanamy, meital; zemel, romy; tur-kaspa, ran; benhar, itai title: removal of hepatitis c virus-infected cells by a zymogenized bacterial toxin date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: n ukr hepatitis c virus (hcv) infection is a major cause of chronic liver disease and has become a global health threat. no hcv vaccine is currently available and treatment with antiviral therapy is associated with adverse side effects. moreover, there is no preventive therapy for recurrent hepatitis c post liver transplantation. the ns serine protease is necessary for hcv replication and represents a prime target for developing anti hcv therapies. recently we described a therapeutic approach for eradication of hcv infected cells that is based on protein delivery of two ns protease-activatable recombinant toxins we named “zymoxins”. these toxins were inactivated by fusion to rationally designed inhibitory peptides via ns -cleavable linkers. once delivered to cells where ns protease is present, the inhibitory peptide is removed resulting in re-activation of cytotoxic activity. the zymoxins we described suffered from two limitations: they required high levels of protease for activation and had basal activities in the un-activated form that resulted in a narrow potential therapeutic window. here, we present a solution that overcame the major limitations of the “first generation zymoxins” by converting mazf ribonuclease, the toxic component of the e. coli chromosomal mazef toxin-antitoxin system, into an ns -activated zymoxin that is introduced to cells by means of gene delivery. we constructed an expression cassette that encodes for a single polypeptide that incorporates both the toxin and a fragment of its potent natural antidote, maze, linked via an ns -cleavable linker. while covalently paired to its inhibitor, the ribonuclease is well tolerated when expressed in naïve, healthy cells. in contrast, activating proteolysis that is induced by even low levels of ns , results in an eradication of ns expressing model cells and hcv infected cells. zymoxins may thus become a valuable tool in eradicating cells infected by intracellular pathogens that express intracellular proteases. hepatitis c virus (hcv) is a small, enveloped rna virus belonging to the hepacivirus genus of the flaviviridae family. hcv has been recognized as a major cause of chronic liver disease and affects approximately million people worldwide at the present time. persistent infection is associated with the development of chronic hepatitis, cirrhosis and hepatocellular carcinoma. a protective vaccine for hcv is not yet available and even the most recent combination of antiviral therapy is often poorly tolerated [ ] . the hcv genome encodes one large open reading frame that is translated as a polyprotein which is proteolytically processed to yield the viral structural and nonstructural (ns) proteins. the non-structural proteins include the p ion channel, the ns - protease, the ns serine protease/rna helicase and its co-factor ns a, the ns b and ns a proteins and the ns b rna-dependent rna polymerase (rdrp) [ , ] . two virally encoded proteases participate in polyprotein processing, the ns - autoprotease (which cleaves in cis at the ns - junction) and the ns - a serine protease (which cleaves at four downstream ns protein junctions). ns is an extensively studied hcv protein that possesses multiple enzymatic activities that are essential for hcv replication, making ns - a the most attractive target for anti hcv drug development [ ] . the n-terminus of ns , in complex with its endoplasmic reticulum (er) membrane-anchored cofactor ns a, primarily functions as a serine protease, which cleaves the viral polyprotein precursor downstream to ns . the remaining / of the protein has a helicase and ntpase activities, both of which are essential for hcv replication [ ] . zymogens are inactive enzyme precursors that are converted to their active form following a biochemical modification, such as proteolytic processing. among the known and important groups of enzymes that are proteolytically activated are secreted digestive enzymes like pepsin and trypsin [ , ] , the cysteine aspartic acid proteases (caspases) which play an essential role at various stages of the apoptotic process [ ] ; and blood coagulating factors [ ] . recently we described a proof of concept for potential new antiviral agents that can specifically eradicate virally infected cells (thus limiting virus production and spread). these agents, which we named ''zymoxins'' (for ''zymogenized toxins''), were composed of a fusion between the binding and translocation domains of pseudomonas exotoxin a and ns -activable modified catalytic domains of the bacterial or the plant toxins diphtheria toxin (dta) or ricin toxin (rta), respectively. upon binding and translocation into cells cytoplasm by virtue of the corresponding pseudomonas exotoxin a domains, activation of these toxins is mediated by hcv-ns protease cleavage that separates between the toxic domains and a fused, rationally designed inhibitory peptide. these zymoxins showed a higher level of cytotoxicity when applied to ns expressing cells or to hcv infected cells, demonstrating a potential therapeutic window. still, the zymoxins we described had two limitations: first, they required high levels of ns protease for efficient activation, and second, they were not totally inactive in the zymogenized state, and their basal cytotoxic activity without proteolytic activation resulted in quite narrow therapeutic windows [ ] . apparently, the zymoxins were only partially inhibited by the rationally designed inhibitory peptides. toxic proteins that are completely inhibited by a natural inhibitor can be found in bacterial ''toxin-antitoxin'' systems. natural bacterial plasmids ensure their survival within the bacterial host by replicating inside their host cell and using mechanisms that function to segregate them prior to cell division [ ] . in addition, several low copy number plasmids use a unique system called ''addiction module'' or ''toxin-antitoxin (ta) system'' which functions to prevent the proliferation of plasmidfree cells by executing the so called ''post-segregational killing effect''. the system consists of a pair of genes encoding for a stable toxin and an unstable antitoxin organized in a bicistronic operon that is transcriptionally autoregulated either by the toxin-antitoxin complex itself or by the antitoxin alone. when coexpressed in plasmid harboring cells, the antitoxin component interferes with the lethal action of the toxin. if a cell loses the plasmid, the cellular concentration of the labile antitoxin (that is degraded faster than the more stable toxin) is rapidly diminished, enabling the toxin to exert its action which eventually results in cell death (reviewed by [ , , , , ] ). toxin-antitoxin systems have also been found integrated to the chromosome of various bacteria species where their function has been the subject of considerable speculation [ , , , , , ] . one of the most studied ''genomic'' ta modules was found on the chromosome of e. coli as a negatively autoregulated bicistronic operon. the system, which is activated by several stress conditions, was denoted mazef after its two active protein components: the long lived mazf toxin and the labile maze antitoxin. mazf induced toxicity is executed by blocking denovo protein synthesis through its endoribonuclease activity that catalyze the cleavage of single-stranded mrnas at aca sequences [ ] . when coexpressed with mazf, the maze antitoxin complexed with the toxin and a catalytically inactive heterohexamer is formed in which a maze dimer is sandwiched between two mazf dimers (mazf -maze -mazf ) (reviewed by [ , ] ). the crystal structure of the maze-mazf complex indicates that the interactions between the toxin and the antitoxin are primarily mediated by the acidic c terminus of maze which wraps around the mazf homodimer crossing the edge of the dimer interface [ ] . later on, li et al had discovered that a short acidic peptide corresponding to maze c-terminal amino-acids, which they denoted ''mazep'', binds strongly to the homodimer of mazf which possesses two identical active sites. interestingly, it was found that one inhibitory peptide, occupying a single active site on the mazf homodimer, affects the conformation of both sites that consequently become catalytically inactive. this unique mechanism also explains the inhibitory activity of maze toward mazf at a : molar ratio [ ] . the discovery of the mazf mechanism of action was soon followed by demonstrations that it is also toxic to eukaryotic cells, causing bak-dependent programmed cell death in mammalian cells, suggesting it may be used as a tool for gene therapy against diseases such as cancer and aids [ , ] . here we describe the design of a different zymoxin than those described in [ ] , based on ns -activable mazf ribonuclease that is delivered as a transgene by an adenoviral vector. the delivered transgene encodes for a fusion between mazf, and a potent inhibitory peptide derived from its natural antidote maze, through an ns cleavable linker. we show that the self-inhibited, zymogenized toxin is well tolerated when expressed in naïve cells. in contrast, in ns expressing or in hcv infected cells, ns mediated cleavage separates between the toxin and its inhibitor which results in inhibition of protein synthesis followed by death of the cells. finally, we demonstrate that treatment with the mazf based zymoxin has a ''curing effect'' when applied to mixed culture of healthy and hcv infected cells, leading to specific eradication of the infected cell population. for the construction of ns -activated mazf based zymoxin, the mazf coding sequence was fused through its c terminus to the hcv p -p ' ns cleavage sequence derived from the a genotype (strain jfh ) ns a/b junction. a short inhibitory peptide corresponding to maze c-terminal amino-acids (which encompass the amino-acids inhibitory peptide (mazep) that has been described by li et al. [ ] ) was fused, preceded by a short flexible linker, to the c terminus of the mazf-ns cleavage site sequence. a flexible linker, followed by the c-terminal er membrane anchor of the tyrosine phosphatase ptp b [ ] , was than fused to the c terminus of the inhibitory peptide and the whole construct was fused through its n terminus to the monomeric red fluorescence protein mcherry [ ] (see figure ). the rationale behind the design of this construct, which was denoted ''mcherry-ns activated mazf'', was that the coupling between the ribonuclease and its antidote may enable high level of expression of the non-toxic fusion on the er membrane of uninfected mammalian cells without causing any deleterious effect. in contrast, in hcv infected cells, the fusion protein is expected to colocalize with the er membrane-bound viral ns protease (in infected cells, ns is localized to the cytosolic side of the er membrane and membranes of er-like modified compartments [ , , , , ] ). as a result, the ns cleavable linker between the toxin and its inhibitory peptide is expected to be cleaved. the toxic ribonuclease, no longer covalently tethered to its er membrane-anchored inhibitor, is now free to diffuse into the cytoplasm (which lacks the antidote) and exert its cytotoxic activity. finally, fusion to the fluorescent protein mcherry makes the whole construct trackable and facilitates the determination of its expression level and intracellular localization by fluorescence microscopy. as a control, an uncleavable construct (denoted ''mcherry-uncleavable mazf'') was constructed in which the ns cleavage sequence was replaced by a mutated amino acids cleavage sequence (p -p ') from hcv genotype a ns a/b junction in which the p valine was substituted by alanine, the p cysteine by glycine, the p cysteine by glycine and the p ' tyrosine by alanine. a schematic representation of the ns -activated mazf-based zymoxin (''mcherry-ns activated mazf'') and the hypothetical mechanism of its cleavage by ns protease on the cytoplasmic side of the er membrane are shown in figure . the aminoacid sequence of the mazf based zymoxins can be found in text s . to verify that expression of ''mcherry-ns activated mazf'' is tolerated by cells that do not express the ns protease, a colony formation assay was carried out. in this assay, the toxicity of an expressed transgene can be comparatively and qualitatively assessed by testing its effect on the competency of transfected cells to evolve into colonies under selection. hek t-rex cells where transfected with plasmids encoding either mcherry-ns activated mazf, mcherry (only the fluorescent protein) or egfp-mazf (where mazf is not fused to its inhibitory peptide). hours (h) later, expression of the encoded transgenes was confirmed by fluorescence microscopy (data not shown) and transfected cells were seeded in fold dilutions and were treated with g (to which all three plasmids confer resistance). after days of selection, surviving colonies were stained. as shown in figure , similar numbers of surviving colonies were observed when the cells were transfected with the plasmids encoding mcherry-ns activated mazf or the red fluorescent protein alone, suggesting that expression of ns -activable ribonuclease in naïve hek t-rex cells (that do not express ns ) cause minimal toxicity, if any. as expected, growth was severely inhibited when cells were transfected with the egfp-fused active (uninhibited) toxin. the er membrane-targeted zymoxin colocalizes with ns protease in vivo previously we described a hek cell line which inducibly expresses (by addition of tetracycline) a fusion between egfp and the coding sequence of the full length ns (including the helicase domain) followed by ns a from hcv a genotype [ ] . these cells, denoted ''tet-inducible full ns - a expressing cells'', were stably transfected with a plasmid encoding the ns -activated zymoxin ''mcherry-ns activated mazf'' or with the uncleavable control (''mcherry-uncleavable mazf'') encoding plasmid. following selection, stable clones that constitutively express high level of the cleavable construct (denoted ''tet-ns /activated mazf cells'') or the uncleavable control (denoted ''tet-ns /uncleavable mazf cells'') were isolated. in order to characterize the intracellular distribution of the mcherry fused zymoxins, tet-ns /activated or uncleavable mazf cells were subjected to immunofluorescence microscopy analysis. as shown in figure (a and b), both zymoxins have a similar cellular distribution, colocalizing with the er marker calnexin at the juxtanuclear region of the er. importantly, upon induced expression of ns -a in tet-ns /uncleavable mazf cells, a colocalization of the two fluorescent fusion proteins could be observed ( figure , c and d).these observations confirm that indeed both the protease and the modified ribonuclease are tethered to a common cellular compartment, presumably the cytoplasmic side of the er membrane (see scheme in figure ). since expression of active mazf was found to inhibit de-novo protein synthesis in mammalian cells [ ] , we hypothesized that such an effect may be observed also in cells in which mazf based zymoxin is proteolytically activated. in order to validate this assumption, tet-ns /activated mazf and tet-ns /uncleavable mazf cells were supplemented with tetracycline for or h, or left untreated. levels of de-novo protein synthesis were than determined by [ h]-leucine incorporation assay. as shown in figure (upper panel), a complete shutoff in protein synthesis was observed as soon as h post ns induction in cells that express the cleavable construct, indicating proteolytic activation of the zymoxin. as expected, protein synthesis was not impaired following ns induction in cells that express the uncleavable toxin. figure . schematic representation of the construct ''mcherry-ns activated mazf'' and hypothetical mechanism of activation by ns protease. the ns -activated mazf zymoxin was constructed by fusing elements in the following order (from the n terminus): monomeric red fluorescence protein mcherry, e. coli mazf ribonuclease, hcv p -p ' ns cleavage sequence derived from a genotype (strain jfh ) ns a/b junction, a short inhibitory peptide corresponding to maze c-terminal amino-acids (which encompass the amino-acids inhibitory peptide (mazep) that has been described by li et al. [ ] ) and the c-terminal er membrane anchor of the tyrosine phosphatase ptp b [ ] . after being anchored to the er membrane, the ns cleavage site that is located between the ribonuclease and the inhibitory peptide in the ''mcherry-ns activated mazf'' construct (which is active as a dimer but for convenience is illustrated here in its monomeric form) is cleaved by the hcv-ns protease which is also localized to the cytoplasmic side of the er membrane. the toxic ribonuclease, no longer covalently tethered to its er-anchored inhibitor, is now free to diffuse to the cytoplasm (which lacks the antidote) and exert its destructive activity. doi: . /journal.pone. .g in support of these findings, an immunoblot assay revealed a near complete cleavage of the zymoxin following tetracycline-induced expression of ns protease in ns -activated mazf expressing cells ( figure , lower panel). presumably, this proteolytic activation of the ribonuclease toxin resulted in a cessation of cellular protein synthesis soon after induction, what explains the detection of relatively low levels of ns protease in these cells. as expected, no zymoxin cleavage could be detected in cells expressing the uncleavable form of the toxin. a faint band, corresponding to a cleaved zymoxin, can also be detected in uninduced cells that express the ns cleavable construct. this cleavage can be attributed to the ''leakiness'' of the tet inducible system, allowing a very low figure . colony formation assay for the assessment of ''mcherry-ns activated mazf'' cytotoxicity toward naïve cells. a day before transfection, . hek t-rex cells where seeded per well in wells plate and subsequently transfected with mg of plasmids encoding either mcherry-ns activated mazf, mcherry (only the fluorescent protein) or egfp-mazf (where mazf is not fused to its inhibitory peptide). hours later, transfection efficiency was assessed by fluorescence microscopy and was determined as equal between the plasmids. transfected cells were than trypsinized, counted and seeded in fold dilutions (starting from , cells/well) in well plates and were incubated for days in the presence of mg/ml of g (to which all three plasmids confer resistance). surviving colonies were fixed and stained with giemsa (upper panel). number of surviving colonies from wells that were seeded with cells was determined by manual counting. each bar represents the mean standard deviation (sd) of a set of data from two wells (lower panel). doi: . /journal.pone. .g zymoxin-mediated removal of hcv infected cells plos one | www.plosone.org basal transcription of the protease in the absence of externally added tetracycline. such cleavage could not be detected in ''naïve'' cells following transfection with ns -cleavage substrate encoding transgene ( [ ] , data not shown). apparently, this very low, basal proteolytic activity is well tolerated by ns activated zymoxin expressing cells that shows no indication of de-novo protein synthesis inhibition in comparison to cells that express the uncleavable toxin (see cpm values in the upper panel of figure ). to evaluate the potential of the ns -cleavable mazf based zymoxin in eradication of ns expressing cells, three well plates were seeded with tet-inducible full ns - a, tet-ns /activated mazf or tet-ns /uncleavable mazf cells and were supplemented with fold serial dilutions of tetracycline starting with ng/ ml. after h, the relative fraction of viable cells was determined using an enzymatic mtt assay. as shown in figure (lower panel), the expression level of ns can be roughly tuned by modulation of the final tetracycline concentration in the growth figure . inhibition of de-novo protein synthesis by ns -activated mazf based zymoxin in ns -expressing cells. tet-ns / activated mazf or tet-ns /uncleavable mazf cells were seeded per well in -wells plate. or h later, cells were supplemented with tetracycline to a final concentration of ng/ml, or left untreated ( h tet, h tet and no tet, respectively). h after seeding, levels of de-novo protein synthesis were determined by [ h]-leucine incorporation assay, as described in ''materials and methods''. results are expressed as percent of the value obtained for cells which were not induced to express the ns protease (no tet). each bar represents the mean sd of a set of data determined in triplicates. numbers above each bar represent mean counts per minute (cpm) values for micrograms total protein samples (upper panel). micrograms of total protein from lysates of the described cells were analyzed by immunoblotting with mouse anti-mcherry (for detection of the zymoxin), mouse anti-gfp (for the detection of egfp-ns ) and mouse anti actin antibodies (loading control) followed by hrp-conjugated secondary antibodies and ecl development. solid arrow: full length zymoxin. hollow arrow: n' terminal portion of ns -cleaved zymoxin (lower panel). doi: . /journal.pone. .g media, with around ng/ml as an intermediate concentration for induction of low ns expression level. indeed, strong cytotoxicity was clearly evident when tet-ns /activated mazf cells where treated with tetracycline concentrations of down to ng/ml. no cytotoxic effect was detected when the controls tet-ns /uncleavable mazf or tet-inducible full ns - a (protease only) expressing cells were similarly treated ( figure , upper panel). these findings demonstrate the deleterious effect of the mazf based zymoxin specifically toward ns protease expressing cells, as well as its competence to be activated by very low cellular levels of protease. in order to obtain a visual insight at the cell level, tet-ns /activated mazf and tet-ns /uncleavable mazf cells were supplemented with tetracycline to a final concentration of ng/ml or ng/ml (for low and high induction levels of ns expression, respectively), or left untreated. h later, nuclei were stained and cells were examined under a fluorescence microscope. the results show that both lower and higher induction levels of ns protease caused growth inhibition and rounding of cells that constitutively expresses the cleavable mazf. furthermore, both green and red fluorescence were faint in these cells, probably as a result of the destructive ribonuclease activity of the cleaved toxin toward the ns protease and its own mrna. as expected, none of the above observations was evident when these cells were not supplemented with tetracycline or when ns expression was induced to high level in cells that constitutively express the uncleavable toxin ( figure ). adenovirus-mediated delivery of mcherry-ns activated mazf encoding cassette specifically eradicates ns expressing hepatocytes to achieve efficient dna delivery into mammalian cells, the expression cassette encoding mcherry-ns activated mazf was cloned into a ''first generation'' de /de human type adenoviral vector plasmid dna by homologous recombination in bacteria [ ] . virus particles were propagated in hek packaging cells as described in the ''materials and methods'' section. in addition, a control adenoviral vector was constructed for the delivery of a similar cassette that encodes for the . after h, cells were supplemented with fold dilutions of tetracycline starting with concentration of ng/ml, or left untreated. hours later, the fraction of viable cells (relatively to the untreated controls) was determined using an enzymatic mtt assay. each bar represents the mean sd of a set of data from six wells. lower panel: ng of total protein from lysates of tet-ns /uncleavable mazf cells that were supplemented with fold dilutions of tetracycline for h were analyzed by immunoblotting with mouse anti-gfp (for the detection of egfp-ns ) and mouse anti-actin antibodies (loading control) followed by hrp-conjugated secondary antibodies and ecl development. doi: . /journal.pone. .g uncleavable version of the construct (mcherry-uncleavable mazf). red-fluorescent comet-like adenovirus-producing foci were apparent upon infection of packaging cells with both recombinant viruses (encoding cleavable or uncleavable constructs) (see figure s ). the production yields for both viruses were , plaque forming units (pfu)/ml, after two ''cycles'' of virus amplification (see text s ). in order to evaluate the ability of adenovirus-mediated delivery of ns activated mazf encoding cassette to eradicate ns expressing hepatocytes, wild-type huh . hepatoma cells and our previously described egfp-full ns - a expressing huh . cells [ ] were infected with the ns -activated or uncleavable mazf encoding viruses. since our previous unpublished observations and studies of others have indicated that high level of transgene expression and adenoviral infection, per se, may adversely affect cell viability and growth [ , , , ] ; the cells were infected with a series of multiplicity of infection (moi) ratios in order to find an optimal moi that leads to eradication of ns -expressing cells while maintaining minimal toxicity to naïve cells. as shown in figure a (left panel), infection with the ns activated zymoxin resulted in a considerable cytotoxic effect against ns -expressing hepatocytes, leading to their almost complete eradication at moi's$ . however, infection at these ratios also adversely affected the growth of wild-type hepatocytes. in contrast, infection at moi of , decreased the viability of the ns -expressing huh . cells to about % of the untreated control without affecting the viability of the wildtype hepatocytes (see also microscopic examination in figure b ). therefore, infection at this moi was applied during the next experiments. as expected, no substantial enhancement in cytotoxicity against ns expressing huh . cells (relatively to wild-type huh . cells) was observed, at any moi, upon infection with the uncleavable mazf encoding viruses ( figure a , right panel). adenovirus-mediated delivery of ns -activated mazf encoding cassette specifically eradicates hcv infected hepatocytes to test the competence of the mazf based zymoxin to specifically eradicate hcv infected hepatocytes, we utilized the infectious chimeric virus hj - [ ] . this is one of the currently used models to study hepatitis c virus in which recombinant infectious hcv particles are produced in cell culture (hcvcc) (for review, see [ , ] ). as the main purpose of zymoxins based treatment is the specific eradication of hcv infected cells from a background of healthy cells, ''mixed culture'' experiments were carried out. in these experiments, huh . hepatoma cells were infected with the hcv a/ a chimeric virus hj - (encoding the ns protease of genotype a strain jfh [ ] ). when infection reached , % (about % of the cultured cells showed expression of the hcv-core protein, as detected by immuno-staining and fluorescence microscopy), the mixed culture and a culture of uninfected cells were treated with ns activated mazf or uncleavable-mazf encoding adenoviruses at moi of , . control cells remained untreated. h post adenoviral infection; viability assay and microscopic examination that included immunostaining for hcv-core protein were performed. as shown, treating the mixed culture with ns -activated mazf zymoxin-encoding adenovirus reduced the viable cells population to about % relatively to untreated control; while viability of the uninfected cells (''hcv negative'') was barely affected by this treatment (figure , upper panel) . microscopic examination of the treated mixed culture revealed two cell populations that differ in their appearance. while one population is characterized by a ''typical'' huh . cell morphology (hollow arrows, figure , lower panel) , the other is composed of partially detached cells with round, condensed or distorted shape (filled arrows) that are hypothesized to be zymoxin-intoxicated hcv infected cells. in order to validate this assumption, the fraction of hcv infected cells from the general population was evaluated by immunofluorescence analysis using anti-hcv core protein specific antibodies. indeed, treatment with the ns activated zymoxin showed a ''curing effect'' upon the partially infected culture, considerably reducing the fraction of the hcv infected cells from the general population ( figure ). as expected, no significant effect upon the hcv infected cell population was observed following treatment with the uncleavable zymoxin. fight against viral infections is considered as one of the most challenging areas in modern medicine. while vaccination is considered to be the most efficient method for fighting viral infections; for some viral pathogens which cause world-wide health problem like human immunodeficiency virus (hiv) and hepatitis c virus (hcv), no efficient vaccine has yet been developed. over the last years, efforts have been focused on the discovery and development of anti-viral agents that target crucial steps in the viral replication cycle which includes viral entry, rna translation and post-translational processing, reverse transcription, genome integration, viral assembly and release [ , , ] . an essential step in the replication cycle of many viruses is the processing of a polyprotein precursor by a viral-encoded protease. a partial list of human diseases associated viruses encoding protease(s) in their genome include flaviviruses such as hepatitis c virus (hcv), west nile virus (wnv), dengue fever virus (dfv) and yellow fever virus (yfv); retroviruses such as hiv- ; picornaviruses such as coxsackievirus, poliovirus and hepatitis a virus; nidoviruses such as coronaviruses (cov), including the severe acute respiratory syndrome (sars) causative sars-cov and herpesviruses such as varicella-zoster virus (vzv) and epstein-bar virus (ebv) [ , ] . therefore, a large part of antiviral drug discovery is focused on inhibiting viral proteases [ ] . our group too has published several studies where hcv replication was inhibited by intracellular expression of antibodies and peptide aptamers [ , , ] . the opposite ''side of the coin'' -taking advantage of the activity of a viral protease per-se as an antiviral approach has been rather neglected. recently we described a study that was based on the concept of ''sitoxins'' which are anti-viral agents that are designed to eradicate viral-infected cells by taking advantage of a specific viral activity instead of inhibiting it [ , ] . specifically, a sitoxin is comprised of an effector domain (e.g. a toxin); a domain bearing an intracellular signaling moiety (e.g. a degradation or an intracellular localization signal); and a domain located between the effector domain and the domain bearing the intracellular signaling moiety which specifies a cleavage site for a predetermined protease (e.g. a viral encoded protease). following the introduction of the sitoxin into the target cell (that expresses the specific protease), cleavage by the predetermined protease separates the toxic effector domain of the sitoxin from the intracellular signaling moiety, resulting either in a longer-lived (and therefore more toxic) effector domain or in an effector domain that moves from a cellular compartment where the domain is nontoxic to a cellular compartment where the domain is able to exert its effect [ ] . we developed and successfully evaluated two rationally designed viral-protease-activated chimeric toxins which we named ''zymoxins'' for ''zymogenized toxins'' [ ] . in contrast to sitoxins, in which the inhibitory activity is mediated by intracellular components which recognize a cleavable signaling polypeptide fused to a constitutively active toxin; the concept of zymoxins is based on the idea of reengineering a toxic enzyme into an inactive zymogen which is specifically activated by a predetermined protease. this general approach was demonstrated by other elegant studies in which several enzymes, including bovine rnase a, vip and the maize ribosome inactivating protein (maize-rip) were converted into protease activated forms [ , , , , ] . our previous work focused on the design of protein-delivered toxins that were converted into protease-activated forms by means of fusion to specific, rationally designed inhibitory peptides through hcv-ns protease cleavable linker. when tested in-vitro and on ns -overexpressing or hcv infected cells, a clear ns protease cleavage-dependent enhancement in activity/cytotoxicity was observed. however, these constructs had two major drawbacks, as mentioned in the introduction: the first is the incomplete inhibition of the toxic enzymatic activity that is conferred by the rationally designed fused peptide. the second relates to the necessity of relatively high level of expressed viral protease for achieving adequate zymoxin activation inside the cells [ ] . in the current study, we turned to evaluate a similar strategy for eradicating hcv-infected cells, namely, by converting a constitutively active enzyme into an ns activated zymogen. as a toxic moiety, we chose mazf, an endoribonuclease that together with its polypeptidic antidote, maze, constitute one of the most studied ''genomic'' toxin-antitoxin systems in e. coli. in order to convert mazf into a zymoxin, a fusion polypeptide was constructed in which an inhibitory peptide derived from the maze antitoxin was fused to the c terminus of the mazf toxin via an ns -cleavable linker. regarding the issue of incomplete inhibition of zymoxin's activity in uncleaved form, it should be mentioned that in contrast to our previously described constructs, in which a ''rationally designed'' peptide is appended to provide the inhibition of the toxin's activity; in the mazf based-zymoxin, a ''natural'' inhibitory polypeptide was chosen for that purpose. antidote polypeptides in toxin-antitoxin systems were ''evolutionary shaped'' to strongly inhibit the destructive activity of their toxic counterparts. thus, a very efficient inhibition may be achieved when using them as inhibitory peptides in the construction of zymoxins. indeed, our results show that the fused maze-derived inhibitory peptide diminishes the toxin's activity to such an extent that enables its non-lethal overexpression in naïve cells. in order to improve the responsiveness of the zymoxin to the presence of low levels of cellular-expressed viral protease, an er membrane ''anchoring peptide'' was fused to the c terminus of the construct, subsequently to the maze derived inhibitory peptide. by using that design, a colocalization between the er-bound ns protease and its zymoxin substrate might be achieved, resulting in an improved cleavage efficiency. indeed, such a colocalization could be observed, as shown above (figure ) . moreover, activation of the zymoxin was evident also upon expression of very low cellular levels of the viral protease ( figures and ) . it should also be mentioned that while being extremely sensitive to minute amounts of expressed ns protease, zymoxin expressing cells can still tolerate some degree of activating proteolysis (as shown above for ns -activated mazf expressing cells that show a very low, basal ns proteolytic activity without tetracycline induction (figure ) ). this property may, in fact, contribute to the specificity and general safety of zymoxin-based therapeutics, even though ''spontaneous'', unspecific proteolytic activity toward ns substrates could not be detected in naïve cells during our experiments (data not shown). as discussed, tethering the zymoxin to the er membrane through its inhibitory peptide may also enhance its cleavage-dependent toxicity. this might be achieved by enabling spatial separation between the activated toxin and its antidote following ns -cleavage and diffusion of the active mazf to the inhibitory peptide-free cytoplasm. supporting this assumption, we found that although cleaved, a cytoplasmic version of the ns -activated mazf based zymoxin is barely toxic to ns protease-expressing cells (data not shown). in contrast to our previous study, in which the toxins were delivered into mammalian cells as purified recombinant proteins; in the current research an adenoviral vector gene-delivery system was used. widely applied in gene therapy, recombinant viral vaccines and basic science studies, this system enables efficient delivery and high level of transgene expression in a wide variety of human cells. using this system, we have demonstrated specific eradication of full ns - a expressing huh . cells, sparing ''naïve'' cells which do not express the protease. furthermore, specific eradication of hcv infected cells has also been achieved, demonstrating a prominent ''curing effect'' when tested on mixed cultures of healthy and hcv infected hepatocytes. the use of viral gene delivery for treating hcv infection by eradication of infected cells has been proved successful in previous study on mice with chimeric human livers. in that study, hsu et al designed a modified proapoptotic bid molecule in which its endogenous cleavage sites were replaced by ns recognition site. as bid requires proteolytic processing in order to activate its apoptotic function, infection with adenoviral vector that delivers a transgene encoding the engineered bid molecule was demonstrated to induce activation of apoptosis in cells expressing the hcv ns protease. moreover, a significant reduction in hcv titer in the serum of the hcv infected mice was detected following injection of the engineered adenoviral vector into the jugular vein [ ] . although such an approach had proved successful; it should be taken into account that a delivered transgene which encodes for a modified form of a protein that is naturally expressed in the target cell has the potential to negatively influence normal cellular processes in which its endogenous counterpart plays a role [ , , , , ] . for example, when overexpressed in noninfected cells, an ns -cleavable apoptotic molecule, such as that described above, may act as a ''dominant negative'' by competing with the natural, endogenous protein for interactions with activators, regulators or substrates. delivery of a transgene encoding for a foreign protein which has no endogenous counterpart in target cells, such as mazf, may reduce that risk. furthermore, it should be noted that mazf possess a ribonuclease activity that is also capable of processing multiple potential cleavage sites in the hcv genome. although not evaluated in this study, a direct attack on the parasite's genetic material may represent an additional mode of anti-viral activity that works in parallel with host-cell protein synthesis shutoff. of note, the applicability of the described zymoxin may be extended by replacing the protease cleavage site that separates between the toxic moiety and the inhibitory peptide. this could facilitate eradication of cells that are infected with other protease expressing viruses other than hcv (a partial list of protease expressing viruses is given at the beginning of the discussion) or different strains of hcv that differ in their ns protease cleavage specificity. in addition, one may replace the c terminal er anchoring peptide with sequence that tethers the construct to a different intracellular location in which the viral protease resides. in that way, a colocalization between the zymoxin and the predetermined viral protease may be achieved. in conclusion, the presented anti-viral agent was designed following our previously described ''zymoxins'' concept in which a constitutively active toxin is converted into a ''zymogenized'', viral-protease activated from. the mazf-based zymoxin, that is introduced to target cells by means of adenovirus mediated gene delivery, shows very low toxicity to naïve cells and enhanced responsiveness to low viral-protease expression level, when compared to our previously presented constructs. as evident from our results, the mazf based zymoxin eradicates ns -expressing model cells and hcv infected cells with remarkable efficiency and specificity, providing further proof to the concept of zymoxins and a potential new means of fighting viral diseases. obviously, further optimizations and pharmacological assessments in animal models may be required in order to determine the safety and efficiency of zymoxins treatment in the context of the whole organism. the following escherichia coli (e. coli) strains were used: xl- blue and dh a (stratagene, usa) for plasmid propagation and bj (stratagene, usa) for the generation of recombinant adenovirus plasmid dna. recombinant dna techniques were carried out according to standard protocols or as recommended by the suppliers. nucleotide sequences were determined using the prism genetic analyzer (applied biosystems, usa) according to the supplier's recommendations. the eukaryotic cmv promoterbased gfp-fusion expression vector pegfp c , which was used for expression of mcherry, mazf and mazf-based zymoxins, was from clontech (usa). the adeasy plasmid system (pshuttle and padeasy- ) [ ] , that was used for generation of recombinant human type adenoviral vectors for gene delivery of the zymoxins expression cassettes, was a generous gift from prof. nadir arber, integrated cancer prevention center, tel aviv sourasky medical center, israel. all plasmid and dna fragment purifications were carried out with high-speed plasmid mini kit and gel/pcr dna fragments extraction kit (geneaid biotech ltd., taiwan) unless mentioned otherwise. t dna ligase and restriction enzymes were purchased from new england biolabs (usa). dna ligations were carried out at uc overnight. oligonucleotides. all the oligonucleotides that were used in this study were purchased from hylabs, israel. oligonucleotides that were used in this study are listed in table s . construction of the vector encoding for ''mcherry-ns activated mazf''. a polymerase chain reaction (pcr) was carried out using a single colony of escherichia coli strain xl- as a template, the forward primer: -clvmazf and the reverse primers: -clvmazf, -clvmazf, -clvmazf, -clvmazf, -clvmazf, clvmazf and -clvmazf. the pcr product, encoding for a fusion polypeptide composed of (from the n terminus) mazf, hcv p -p ' ns cleavage sequence derived from a genotype (strain jfh ) ns a/b junction, a short flexible linker, a short inhibitory peptide corresponding to maze c-terminal amino-acids (which encompass the amino-acids inhibitory peptide (mazep) that has been described by li et al. [ ] ), a flexible linker and the cterminal er membrane anchor of the tyrosine phosphatase ptp b [ ] was digested with xhoi and ecori and was cloned between the corresponding sites in the plasmid pegfp-c , generating plasmid ''pegfp-ns activated mazf''. next, the sequence of the red fluorescent protein mcherry [ ] was amplified by pcr from an expression cassette (kindly provided by prof. adi avni, department of molecular biology and ecology of plants, tel-aviv university, israel) using the forward primer: clvmazf and the reverse primer: -clvmazf. the pcr product was digested with nhei and xhoi and was cloned between the corresponding sites of the plasmid ''pegfp-ns activated mazf'' (replacing the egfp coding sequence), generating plasmid: ''pmcherry-ns activated mazf''. the amino-acid sequence of the encoded cleavable zymoxin can be found in text s . construction of the vector encoding for ''mcherryuncleavable mazf''. a pcr was carried out using dna of plasmid ''pmcherry-ns activated mazf'' as a template, the forward primer: -clvmazf and the reverse primers: -unclmazf and -unclmazf. the pcr product was digested with ecorv and nrui, and the digestion product of bp was cloned between the corresponding sites of the same plasmid that has been used as a template, generating the plasmid: ''pmcherry-uncleavable mazf''. this plasmid encodes for an uncleavable construct in which the ns cleavage sequence was replaced by a mutated amino acids cleavage sequence (p -p ') from hcv genotype a ns a/b junction in which p valine was substituted by alanine, p cysteine by glycine, p cysteine by glycine and p ' tyrosine by alanine. the amino-acid sequence of the encoded uncleavable zymoxin can be found in text s . construction of the vector encoding for ''egfp-mazf''. this is a mutated variant of an ''intermediate'' vector used in the construction process of the ''mcherry-ns activated mazf'' encoding vector. in this variant, a nonsense mutation was inserted instead of the tyrosine in the smsy sequence of the ns recognition site, generating the plasmid ''pegfp-mazf'' that encodes for a truncated egfp-mazf fusion protein that lacks the maze derived inhibitory peptide and the er anchor. construction of the vector encoding for mcherry. the sequence of the red fluorescent protein mcherry [ ] was amplified by pcr from an expression cassette (see construction of the vector encoding for ''mcherry-ns activated mazf'') using the forward primer: -clvmazf and the reverse primer: clvmazf. the pcr product was digested with nhei and bglii and was cloned between the corresponding sites of the plasmid pegfp c (replacing the egfp coding sequence), generating the plasmid ''pmcherry''. construction and propagation of recombinant adenoviral vectors. construction and propagation of recombinant human type adenoviral vectors for gene delivery of the mcherry-ns activated mazf and mcherry-uncleavable mazf expression cassettes was carried out using the adeasy system essentially as described in [ , ] . a more detailed description of the procedure is provided in text s . human embryonic kidney cells hek , stably expressing the tetracycline repressor protein (t-rex hek cell line, invitrogen, usa), and human hepatoma cells huh . [ ] were used throughout this study. cell lines were maintained in dulbecco's modified eagle medium (dmem) supplemented with % fetal calf serum (fcs), mm l-glutamine, u/ml penicillin and mg/ml streptomycin (biological industries, israel) in a humidified % co incubator at uc. the calcium-phosphate transfection method was applied for introducing mg of the plasmid ''pmcherry-ns activated mazf'' or the plasmid ''pmcherry-uncleavable mazf'' into t-rex hek cells inducibly expressing egfp-full ns - a [ ] seeded . cells per mm plate h before transfection. stable transfectants, inducibly expressing egfp-full ns - a and constitutively expressing mcherry-ns activated mazf (denoted ''tet-ns /activated mazf cells'') or mcherry-uncleavable mazf (denoted ''tet-ns /uncleavable mazf cells'') were selected in a medium containing mg/ml of g (a.g. scientific, usa). cell clones that express high level of the cleavable construct or the uncleavable control were identified by fluorescence microscopy and isolated. for protein extraction, h post-transfection the cells were washed with pbs, scraped and lysed in a buffer containing mm nacl, mm edta, . % np- , mm tris(hcl) ph . , and protease inhibitors cocktail (sigma, israel). following minutes of incubation on ice, lysates were cleared by centrifugation at , g for minutes, at uc. for immunoblotting, protein samples were electrophoresed on % sds/polyacrylamide gel, transferred to nitrocellulose and detected using mouse monoclonal anti-mcherry antibody (clontech, usa), mouse monoclonal anti-gfp antibody (santa-cruz, usa) or mouse monoclonal anti-actin antibody (abcam, usa), followed by horseradish peroxidase (hrp)-conjugated goat anti-mouse antibodies (jackson immunoresearch laboratories, usa) and enhanced chemiluminescence (ecl) detection using supersignal west pico chemiluminescent substrate (thermo scientific/pierce, usa). virus assays were carried out with an inter-genotypic chimeric hepatitis c virus (hcv) produced by replacing the core-ns segment of the jfh- virus genome with the comparable segment of the genotype a h virus. this chimeric virus, hj - (kindly provided by prof. stanley lemon, university of texas at galveston), contains two compensatory mutations that promote its growth in cell culture as described previously [ ] . hcv rnas were transcribed in vitro and electroporated into cells essentially as described previously [ , ] . in brief, mg of in vitro-synthesized hcv rna was mixed with huh . cells in a -mm cuvette and pulsed twice at . kv and mf. cells were seeded into well plates or -cm flasks, and passaged at -to -day intervals posttransfection by trypsinization and reseeding with a : to : split into fresh culture vessels. when infectivity reached , %, as was monitored by immunofluorescent staining with anti hcv core protein mouse monoclonal antibody (affinity bioreagents, usa) and cy -conjugated goat anti-mouse igg (jackson immunoresearch laboratories, usa) (see ''visualizing hcv infected cells (infectivity assay)'' below), the mixed culture (of uninfected and hcv infected cells in : ratio) was taken for cytotoxicity assays. confocal fluorescence microscopy analysis of t-rex hek cells inducibly expressing egfp-full ns - a and constitutively expressing mcherry-ns activated or uncleavable mazf. tet-ns /activated mazf or tet-ns /uncleavable mazf cells were seeded on poly-l-lysine coated cover-slips in a well-plate. h later, the cells were supplemented with mg/ml of tetracycline, or left untreated. h later, the cells were fixed with % formaldehyde in pbs. uninduced cells were permeabilized with triton x- ( . % in pbs) for minutes, blocked with % fetal calf serum/ % pbs at room temperature for minutes, incubated with : diluted rabbit-polyclonal anti-calnexin antibody (sigma, usa) as primary antibody for h and followed by : diluted cy conjugated anti-rabbit igg (jackson immunoresearch laboratories, usa) secondary antibody for minutes. nuclei of induced and uninduced cells were then stained by hoechst for h at room temperature. slides were washed with pbs, mounted in mowiol - solution (calbiochem, usa) (immunostained uninduced cells) or immuglo mounting medium (immco diagnostics, usa) (induced tet-ns /uncleavable mazf cells) and examined using a zeiss lsm meta laser scanning confocal microscope. visualizing t-rex hek cells inducibly expressing egfp-full ns - a (supplemented with different tetracycline concentrations) and constitutively expressing mcherry-ns activated mazf or mcherry-uncleavable mazf. tet-ns /activated mazf or tet-ns /uncleavable mazf cells were seeded on poly-l-lysine coated cover-slips in a well-plate. h later, cells were supplemented with ng/ml or ng/ml of tetracycline, or left untreated. h later, cells were fixed with % formaldehyde in pbs. following nuclear staining by hoechst for h at room temperature, slides were washed with pbs, mounted in immuglo mounting medium and examined using a fluorescence microscope. visualizing hek cells infected with recombinant adenovirus encoding for mcherry-ns activated mazf or mcherry-uncleavable mazf. hek cells were seeded per well in wells plate. when the culture reached % confluence, the growth medium was replaced by fresh medium containing fold dilutions of recombinant adenoviruses encoding for mcherry-ns activated mazf or mcherry-uncleavable mazf, starting from . pfu per well. after h, cells were fixed with % formaldehyde in pbs and examined using a fluorescence microscope. visualizing hcv infected cells (infectivity assay). huh . cells infected with hcv hj - chimeric virus were seeded into well chamber slides (nalge nunc, usa). after h, cells were fixed and permeabilized with : acetone/methanol mixture and stained with : diluted mouse monoclonal antibody c - (affinity bioreagents, usa) specific for the hcv core protein followed by staining with : diluted cy -conjugated goat anti-mouse igg (jackson immunoresearch laboratories, usa). nuclei were then stained with dapi (sigma, israel) and slides were washed with pbs, mounted (southernbiotech, usa) and examined using a fluorescence microscope. visualizing zymoxin-treated mixed culture of uninfected and hcv infected cells. see ''cytotoxicity assay of recombinant adenoviruses encoding for mcherry-ns activated mazf or mcherry-uncleavable mazf on a mixed culture of uninfected and hcv-infected huh . cells'' below. colony formation assay . hek t-rex cells were seeded per well in wells plate. h later, cells were transfected with mg of the plasmids ''pmcherry-ns activated mazf'', ''pmcherry'' or ''pegfp-mazf'' encoding for mcherry-ns activated mazf, mcherry (only the fluorescent protein) or egfp-mazf (where mazf is not fused to its inhibitory peptide), respectively. transfection was carried out using fugene reagent (roche, germany) according to the manufacturer instructions. after h, transfection efficiency was assessed by fluorescence microscopy and was determined as equal between the plasmids. transfected cells were then trypsinized, counted and seeded in fold dilutions (starting from , cells/well) in well plates and were incubated for days in the presence of mg/ml of g (to which all the three plasmids confer resistance). surviving colonies were fixed with % formaldehyde in pbs and stained with giemsa (sigma, usa). number of surviving colonies from wells that were seeded with cells was determined by manual counting. the cell-killing activities of ns -activated mazf and uncleavable mazf zymoxins were measured by a thiazolyl blue tetrazoliam bromide (mtt) assay as described below: cytotoxicity assay of intracellularly expressed mcherry-ns activated mazf or mcherry-uncleavable mazf in t-rex hek cells inducibly expressing egfp-full ns - a. tet-inducible full ns - a, tet-ns /activated mazf or tet-ns /uncleavable mazf cells were seeded in well plates ( cells per well). after h, cells were supplemented with fold dilutions of tetracycline starting with concentration of ng/ml, or left untreated. h later, the media was replaced by fresh media ( ml per well) containing mg/ml mtt (thiazolyl blue tetrazoliam bromide (sigma, israel) dissolved in pbs) reagent and the cells were incubated for further minutes. mtt-formazan crystals were dissolved by the addition of extraction solution ( % sds, % n, n-dimethyl formamide (dmf), ph . ) ( ml per well) and incubation for h at uc. absorbance at nm was recorded on an automated microtiter plate reader. the results were expressed as percentage of living cells relatively to the untreated controls. cytotoxicity assay of recombinant adenoviruses encoding for mcherry-ns activated mazf or mcherry-uncleavable mazf on full ns - a expressing huh . cells. wildtype or egfp-full ns - a expressing huh . cells [ ] were seeded per well in well plates. after h, growth media were replaced by fresh media containing recombinant adenoviruses encoding for mcherry-ns activated mazf or mcherry-uncleavable mazf at indicated multiplicity of infection (moi) ratios. control cells remained untreated. four days post infection, the media was replaced by fresh media ( ml per well) containing mg/ml mtt (except in representative wells in which cells were fixed and microscopically examined) and the cells were incubated for further minutes. the next steps were identical to theses described above. cytotoxicity assay of recombinant adenoviruses encoding for mcherry-ns activated mazf or mcherry-uncleavable mazf on a mixed culture of uninfected and hcv-infected huh . cells. uninfected huh . cells and mixed culture of hcv infected and uninfected cells at : ratio ( % infected culture) were seeded in -well plates ( cells/well). after h, cells were treated with recombinant adenoviruses (at moi of , ) encoding for mcherry-ns activated mazf or mcherry-uncleavable mazf zymoxins. control cells remained untreated. h later, the media was replaced by fresh media ( ml per well) containing mg/ml mtt (except in representative wells in which cells were fixed and microscopically examined) and the cells were incubated for further minutes. the next steps were identical to these described above. for hcv-infection immunofluorescence analysis, cells from the hcv infected and uninfected mixed culture were seeded per well into -well chamber slides (nalge nunc, usa). h later, cells were treated with recombinant adenoviruses (moi of , ) encoding for the mcherry-ns activated mazf or mcherryuncleavable mazf zymoxins. control cells were remained untreated. h post treatment, cells were fixed and permeabilized with : acetone/methanol mixture and stained with : diluted mouse monoclonal antibody c - (affinity bioreagents, usa) specific for the hcv core protein followed by staining with : diluted fitc-conjugated goat anti-mouse igg (jackson immu-noresearch laboratories, usa). nuclei were then stained with dapi and slides were mounted (southernbiotech, usa) and examined using a fluorescence microscope. for each treatment, evaluation of the fraction of the hcv-infected cells from the general cell population was performed by dividing the number of the green, hcv-core positive cells by the general number of cells (dapi stained) from five representative microscopic fields. [ h]-leucine incorporation assay tet-ns /activated mazf or tet-ns /uncleavable mazf cells were seeded per well in -wells plate. or h later, cells were supplemented with tetracycline to a final concentration of ng/ml, or left untreated. h after seeding, cells were supplemented with [ h]-leucine (perkin elmer, usa) to a final concentration of mci/ml and returned to incubation. after h, cells were scraped, washed with pbs and lysed by four freeze/thaw cycles. mg total protein form the lysate of each treatment were then add to a solution containing pbs and a final concentration of mg bovine serum albumin (bsa) in a total volume of ml. the solution was then mixed with an identical volume of ice-cold % trichloro acetic acid (tca). mixtures were then incubated on ice for minutes and centrifuged for minutes at , g, uc, after which the pellet was washed with ice-cold % tca, followed by washing with ice-cold % ethanol. the pellet was then dissolved in ml of . m naoh, transferred to a scintillation tube and neutralized with ml m hcl. ml of scintillation liquid was added and radioactivity was counted by a beta-counter device. figure s fluorescence microscopy analysis of adenovirus producing foci. hek cells were seeded per well in wells plate. when reached % confluence, cells were infected with fold dilutions of recombinant adenoviruses encoding for mcherry-ns activated mazf or mcherry-uncleavable mazf, starting from . pfu per well. after h, cells were fixed and examined under a fluorescence microscope. red fluorescent adenovirus-producing foci from wells infected with . pfu are shown. the bar represents mm. table s oligonucleotides that have been used in this study. text s amino-acid sequences of mazf based zymoxins. text s construction and propagation of recombinant adenoviral vectors. stanley lemon (university of texas at galveston, usa) for the hj - hcvcc clone. we thank prof. nadir arber (integrated cancer prevention center, tel aviv sourasky medical center, israel) for the adeasy plasmid system used for generation of recombinant human type adenoviral vectors. treatment failure and resistance with direct-acting antiviral drugs against hepatitis c virus replication of hepatitis c virus hepatitis c viral life cycle advances in the development of new therapeutic agents targeting the ns - a serine protease or the ns b rnadependent rna polymerase of the hepatitis c virus the ns / a proteinase of the hepatitis c virus: unravelling structure and function of an unusual enzyme and a prime target for antiviral therapy role of proteolytic enzymes in biological regulation (a review) mechanism of activation of the gastric aspartic proteinases: pepsinogen, progastricsin and prochymosin structure and zymogen activation of caspases a brief historical review of the waterfall/cascade of blood coagulation engineered toxins ''zymoxins'' are activated by the hcv ns protease by removal of an inhibitory protein domain bacterial mitotic machineries bacterial death by dna gyrase poisoning addiction modules and programmed cell death and antideath in bacterial cultures prokaryotic toxin-antitoxin stress response loci toxins-antitoxins: plasmid maintenance, programmed cell death, and cell cycle arrest programmed cell death in bacterial populations hypothetical functions of toxin-antitoxin systems occurrence of mazef-like antitoxin/toxin systems in bacteria toxin-antitoxin loci are highly abundant in freeliving but lost from host-associated prokaryotes bacterial toxin-antitoxin systems: more than selfish entities? mazef: a chromosomal toxinantitoxin module that triggers programmed cell death in bacteria mrna interferases, sequence-specific endoribonucleases from the toxin-antitoxin systems bacterial programmed cell death and multicellular behavior in bacteria crystal structure of the maze/mazf complex: molecular bases of antidote-toxin recognition characterization of dual substrate binding sites in the homodimeric structure of escherichia coli mrna interferase mazf the discovery of mrna interferases: implication in bacterial physiology and application to biotechnology nbk/ bik antagonizes mcl- and bcl-xl and activates bak-mediated apoptosis in response to protein synthesis inhibition characterization of the c-terminal er membrane anchor of ptp b improved monomeric red, orange and yellow fluorescent proteins derived from discosoma sp. red fluorescent protein subcellular localization, stability, and trans-cleavage competence of the hepatitis c virus ns -ns a complex expressed in tetracycline-regulated cell lines membrane association of hepatitis c virus nonstructural proteins and identification of the membrane alteration that harbors the viral replication complex structural determinants for membrane association and dynamic organization of the hepatitis c virus ns - a complex expression of hepatitis c virus proteins induces distinct membrane alterations including a candidate viral replication complex a dynamic view of hepatitis c virus replication complexes a simplified system for generating recombinant adenoviruses adenoviral vector cytotoxicity depends in part on the transgene encoded recombinant, replication-defective adenovirus gene transfer vectors induce cell cycle dysregulation and inappropriate expression of cyclin proteins effect of adenoviral vector infection on cell proliferation in cultured primary human airway epithelial cells recombinant e -deleted adenovirus vector induces apoptosis in two lung cancer cell lines compensatory mutations in e , p , ns , and ns enhance yields of cell culture-infectious intergenotypic chimeric hepatitis c virus hepatitis c virus molecular clones and their replication capacity in vivo and in cell culture studying hepatitis c virus: making the best of a bad virus review article: investigational agents for chronic hepatitis c novel targets for hiv therapy perspectives on antiviral drug development antiviral drug discovery targeting to viral proteases protease inhibitors as antiviral agents inhibition of protease-inhibitor-resistant hepatitis c virus replicons and infectious virus by intracellular intrabodies inhibition of hepatitis c virus rna replicons by peptide aptamers hcv ns serine protease-neutralizing single-chain antibodies isolated by a novel genetic screen the n-end rule toxins that are activated by hiv type- protease through removal of a signal for degradation by the nend-rule pathway a ribonuclease zymogen activated by the ns protease of the hepatitis c virus creation of a zymogen design and characterization of an hiv-specific ribonuclease zymogen from enzyme to zymogen: engineering vip , an adp-ribosyltransferase from bacillus cereus, for conditional toxicity a switch-on mechanism to activate maize ribosome-inactivating protein for targeting hivinfected cells modified apoptotic molecule (bid) reduces hepatitis c virus infection in mice with chimeric human livers mutant p exerts a dominant negative effect by preventing wild-type p from binding to the promoter of its target genes caspase- prevents sustained activation of nf-kappab in monocytes undergoing macrophagic differentiation cytochrome c and datp-dependent formation of apaf- /caspase- complex initiates an apoptotic protease cascade a c-jun dominant negative mutant protects sympathetic neurons against programmed cell death structure and activation mechanism of the drosophila initiator caspase dronc a protocol for rapid generation of recombinant adenoviruses using the adeasy system highly permissive cell lines for subgenomic and genomic hepatitis c virus rna replication adaptive mutations producing efficient replication of genotype a hepatitis c virus rna in normal huh cells production of infectious genotype a hepatitis c virus (hutchinson strain) in cultured human hepatoma cells we thank prof. matti sä llberg (karolinska institute, sweden) for the plasmid carrying the ns - a gene of the a genotype. we thank prof. key: cord- -muxrxvyo authors: markotter, wanda; geldenhuys, marike; jansen van vuren, petrus; kemp, alan; mortlock, marinda; mudakikwa, antoine; nel, louis; nziza, julius; paweska, janusz; weyer, jacqueline title: paramyxo- and coronaviruses in rwandan bats date: - - journal: trop med infect dis doi: . /tropicalmed sha: doc_id: cord_uid: muxrxvyo a high diversity of corona- and paramyxoviruses have been detected in different bat species at study sites worldwide, including africa, however no biosurveillance studies from rwanda have been reported. in this study, samples from bats collected from caves in ruhengeri, rwanda, were tested for the presence of corona- and paramyxoviral rna using reverse transcription pcr assays. positive results were further characterized by dna sequencing and phylogenetic analysis. in addition to morphological identification of bat species, we also did molecular confirmation of species identities, contributing to the known genetic database available for african bat species. we detected a novel betacoronavirus in two geoffroy’s horseshoe bats (rhinolophus clivosus) bats. we also detected several different paramyxoviral species from various insectivorous bats. one of these viral species was found to be homologous to the genomes of viruses belonging to the jeilongvirus genus. additionally, a henipavirus-related sequence was detected in an egyptian rousette fruit bat (rousettus aegyptiacus). these results expand on the known diversity of corona- and paramyxoviruses and their geographical distribution in africa. bats (order chiroptera) account for % of all mammalian species and are distributed worldwide. with the advancement in detection techniques and increased surveillance, bats are being increasingly recognized as hosts for many zoonotic viruses [ ] , including filo-, paramyxo-, corona-and lyssaviruses [ ] [ ] [ ] [ ] . regions in africa are considered a hotspot for emerging infectious diseases with more than % of recently emerging diseases originating from wildlife species on this continent [ , ] . although several surveillance studies have been implemented to detect potential zoonotic viruses in bats, including from countries in the congo basin and east africa, limited information is available for rwanda. importantly, in the bordering democratic republic of congo and uganda, marburg and ebola disease outbreaks in humans have occurred [ ] , and corona-and paramyxoviruses have been reported to circulate in bats [ , [ ] [ ] [ ] . coronaviruses are positive-sense rna viruses with the potential to cause respiratory, gastrointestinal, hepatic, and neurological diseases in their hosts [ ] and are divided into four genera namely alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus [ ] . bats host a large diversity of coronaviruses and the expanding research can be largely attributed to the emergence of novel coronaviruses of public health and veterinary importance. three such viruses emerged in the last years, including the severe acute respiratory syndrome (sars) coronavirus in , middle east respiratory syndrome (mers) coronavirus in , and the swine acute diarrhoea syndrome (sads) coronavirus in [ , ] . bat coronaviruses have been shown to be associated with particular bat genera and similar viruses have been identified throughout the geographical distribution of their hosts [ , , ] . diverse bat coronaviruses related to sars coronavirus (now termed the sarbecovirus subgenus) have been identified from the rhinolophus bat genus (horseshoe bats) in asia, europe and africa [ , , ] . continued surveillance within these bats species in china identified lineages of recombinant sars-related coronaviruses nearly identical to human sars coronaviruses, capable of using the same receptor molecules [ ] [ ] [ ] . as a result, these viruses have therefore been postulated to be capable of direct human infection [ , ] . bats from various african countries, including kenya, ghana, nigeria, tanzania, uganda and south africa, have yielded a large diversity of novel coronaviruses [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . some of the bat coronaviruses identified have been shown to be genetically related to known human coronaviruses such as hcov- e, hcov-nl , and mers coronavirus [ , , [ ] [ ] [ ] . paramyxoviruses are negative-sense single-stranded rna viruses capable of infecting a diverse host range including mammals, birds, reptiles and fishes [ ] . the taxonomic classification of viruses in the paramyxoviridae family has recently undergone several changes [ ] . in an attempt to accommodate the rapidly growing number of paramyxoviruses described, the previously known avulavirus and rubulavirus genera were elevated to the sub-family level (avula-and rubulavirinae) each with two new genera. in addition, several unclassified rodent-borne viruses were classified to newly established genera (narmovirus and jeilongvirus) in the sub-family orthoparamyxovirinae to which the henipa-, morbilliand respirovirus genera belong. several zoonotic paramyxoviruses have emerged as important public health threats in the past three decades. the emergence of hendra and nipah viruses (henipavirus genus) during the s in australia and southeast asia respectively, marked the first report of zoonotic paramyxoviruses of considerable public health importance [ , ] . these viruses are characterized by high morbidity and mortality rates and outbreaks have been reported on a near annual basis. in addition, another paramyxovirus, sosuga virus (pararubulavirus genus), emerged as the etiological agent of a single non-fatal human infection contracted in uganda, africa [ ] . the natural wildlife reservoir for these zoonotic viruses was determined to be the fruit bat species occurring in these areas, i.e., flying foxes from the pteropus genus for the henipaviruses [ , ] , and the egyptian rousette bat (rousettus aegyptiacus) for the rubulavirus [ ] . viruses related to the henipaand orthorubulaand pararubulavirus genera as well as a number of unclassified viruses have been described from countries bordering rwanda as well as other african countries [ , , , [ ] [ ] [ ] . r. aegyptiacus, hipposideros spp. and miniopterus inflatus have tested positive for paramyxoviral rna that is closely related to known human pathogens including the henipaviruses, human mumps virus, human parainfluenza virus and human parainfluenza virus [ , ] . in this study, we report the detection of a novel betacoronavirus in rhinolophus clivosus sampled in the ruhengeri cave system in rwanda. in addition, we report on the detection of jeilongvirus and related sequences in hipposideros spp. as well as a henipavirus-related sequence in the fruit bat species r. aegyptiacus. these results expand on the known diversity of these virus groups and their geographical distribution in africa. in december , a team from the university of pretoria, national institute for communicable diseases, and rwanda tourism board and national park authority visited two cave sites in ruhengeri, rwanda (gps coordinates: • . " s • . " e; figure ) where bats were caught at night using mist nets in the surrounding areas, and two bank g forest strainer harp traps (bat conservation and management, inc., usa, australia) at the cave entrances. when collecting samples in the field, personal protective equipment used included tyvek suits (dupont tm , wilmington, de, usa), disposable over gowns (stylianou medisupplies ltd, middle east), m full powered air purifying respirators ( m, maplewood, mn, usa), gumboots (bata industries ® , pinetown, south africa), double layer nitrile gloves (lasec, cape town, south africa) and leather gloves (evrigard, johannesburg, south africa). in december , a team from the university of pretoria, national institute for communicable diseases, and rwanda tourism board and national park authority visited two cave sites in ruhengeri, rwanda (gps coordinates: ° ' . "s ° ' . "e; figure ) where bats were caught at night using mist nets in the surrounding areas, and two bank g forest strainer harp traps (bat conservation and management, inc., usa, australia) at the cave entrances. when collecting samples in the field, personal protective equipment used included tyvek suits (dupont tm , wilmington, de, usa), disposable over gowns (stylianou medisupplies ltd, middle east), m full powered air purifying respirators ( m, maplewood, mn, usa), gumboots (bata industries®, pinetown, south africa), double layer nitrile gloves (lasec, cape town, south africa) and leather gloves (evrigard, johannesburg, south africa). bats were placed in individual cotton bags before processing. bats were morphologically identified [ ] and data including sex, reproductive status, forearm length and weight were also recorded. samples collected from bats included fecal and oral swabs, wing biopsies in % ethanol and blood (serum) for use in viral surveillance studies. oral swabs were collected by gently swabbing the inside of the mouth (cheeks and tongue) with a sterile swab (vwr critical swab, atlanta, ga, usa). fecal material or swabs (vwr critical swab, atlanta, ga, usa) were collected from the bat or the cotton bag, when it was available. sterile foreceps were used to collect fecal pellet(s) and place them in ml microcentrifuge tubes (sarstedt, nümbrecht, germany). urine was collected with a sterile swab (vwr critical swab, atlanta, ga, usa) from individual bats as was available. in instances where bats died during processing, necropsies were performed and various organs and tissues, including kidney, spleen, heart, pectoral muscles, liver, lung, stomach, bladder, tongue, brain and lymph nodes, were collected and placed in ml cryotubes (sarstedt, nümbrecht, germany). all samples were collected in rnalater preservative inactivation solution (qiagen, hilden, germany), stored at °c, then transported to and tested in south africa at the national institute for communicable diseases (nicd) and university of pretoria. permits were obtained from the rwanda development board/tourism & conservation and animal ethics was obtained from the animal ethics committee, university of pretoria. for viral rna detection, rna was extracted from kidney, spleen, urine, fecal, rectal and intestinal samples (table s ). rna from kidney (n = ), spleen (n = ) and urine (n = ), were extracted using the bats were placed in individual cotton bags before processing. bats were morphologically identified [ ] and data including sex, reproductive status, forearm length and weight were also recorded. samples collected from bats included fecal and oral swabs, wing biopsies in % ethanol and blood (serum) for use in viral surveillance studies. oral swabs were collected by gently swabbing the inside of the mouth (cheeks and tongue) with a sterile swab (vwr critical swab, atlanta, ga, usa). fecal material or swabs (vwr critical swab, atlanta, ga, usa) were collected from the bat or the cotton bag, when it was available. sterile foreceps were used to collect fecal pellet(s) and place them in ml microcentrifuge tubes (sarstedt, nümbrecht, germany). urine was collected with a sterile swab (vwr critical swab, atlanta, ga, usa) from individual bats as was available. in instances where bats died during processing, necropsies were performed and various organs and tissues, including kidney, spleen, heart, pectoral muscles, liver, lung, stomach, bladder, tongue, brain and lymph nodes, were collected and placed in ml cryotubes (sarstedt, nümbrecht, germany). all samples were collected in rnalater preservative inactivation solution (qiagen, hilden, germany), stored at • c, then transported to and tested in south africa at the national institute for communicable diseases (nicd) and university of pretoria. permits were obtained from the rwanda development board/tourism & conservation and animal ethics was obtained from the animal ethics committee, university of pretoria. for viral rna detection, rna was extracted from kidney, spleen, urine, fecal, rectal and intestinal samples (table s ). rna from kidney (n = ), spleen (n = ) and urine (n = ), were extracted using the trizol reagent (invitrogen, carlsbad, ca, usa), and fecal material/swabs (n = ), rectal and/or intestinal samples (n = ) (table s ) were extracted using the duet rna/dna extraction kit (zymoresearch, ca, usa) from samples homogenized in µl of phosphate buffered saline (lonza, basel, switzerland). both extraction methods were performed according to the manufacturer's instructions without deviations. dna was extracted using the dneasy blood & tissue kit (qiagen, hilden, germany) from heart tissues. confirmation of species identification of bats, in which viral rna was detected, was performed by amplifying the cytochrome b (cyt b) or cytochrome oxidase one (coi) gene region and determining the dna sequence. selection of cytochrome region for amplification was based on availability of credible comparative sequences in public databases (ncbi genbank and bold). previously reported pcr primers targeting these two regions were used or modified [ ] [ ] [ ] . following the pcr analysis, reactions were subjected to agarose gel electrophoresis on a . % agarose gel (lonza, basel, switzerland) and pcr amplicons were gel-purified using the wizard ® sv gel dna clean-up system (promega, madison, wi, usa) according to the manufacturer's instructions and without deviation. all amplicons were subjected to sanger sequencing for both the forward and reverse reactions on an abi dna sequencer (ae applied biosystems) at the sequencing facility of the university of pretoria. host gene sequences were subsequently compared to bat sequences available in the public domain (on the ncbi genbank and bold databases), results were interpreted and compared with the respective morphological field identifications. fecal, rectal and/or intestinal samples from bats (table s ) were extracted and analysed for coronavirus rna. complementary dna (cdna) was prepared using ng random hexamers (ie hplc purified, integrated dna technologies, coralville, ia, usa) with u superscript iv reverse transcriptase (thermo scientific, waltham, ma, usa). additionally, cdna was treated with u rnase h (thermo fisher scientific) incubation at • c for min and inactivated at • c for min. presence of coronavirus rna was detected with a coronavirus genus-specific hemi-nested rt-pcr assay which targets the rna dependent rna polymerase (rdrp) gene for amplification as described in geldenhuys et al. [ ] . as the hemi-nested rt-pcr assay produced only short amplicons (approximately bp), the rdrp-grouping unit (rgu) assay was used to extend the sequenced region of the identified betacoronaviruses to bp [ ] . a hemi-nested rt-pcr assay was performed using the randomly primed cdna prepared as well as forward primers from drexler et al. [ ] (sp '-ctt ctt ctt tgc tca gga tgg caa tgc tgc- ' and sp '-ata ctt tga ttg tta cga tggt ggc tg- ') in combination with a reverse primer (p beta_rev '-cat crt cas dia rda tca tcat-' ) from the geldenhuys et al. [ ] assay. assay conditions from geldenhuys et al. [ ] were used with modifications to cycling conditions including longer annealing and extension cycles ( cycles of • c for s, • c for s and • c for s). agarose gel electrophoresis and purification of all pcr products were performed as previously described for molecular host species identification. kidney from insectivorous bats, spleen from frugivorous bats and urine from both groups (n = ; table s ) were tested with the use of two broadly-reactive assays targeting the avula-rubulavirinae (ar) sub-families, and the respiro-morbilli-henipvirus (rmh) genera. for both assays, published primers targeting the conserved polymerase (l) gene [ ] were used in combination with adapted two-step hemi-nested rt-pcr protocols. samples were tested with the ar assay as previously described [ ] . for the rmh assay, the samples were tested as previously described [ ] , with minor variations in the protocol. for the first-round pcr, mm mgcl (thermofisher scientific, waltham, ma, usa) was added and the nuclease-free water (ambion, foster city, ca, usa) was adapted for a final reaction volume of µl. all cycling conditions and the protocol for the hemi-nested pcr remained the same as for ar. agarose gel electrophoresis and purification of all pcr products were performed as previously described for molecular host species identification. sequencing was performed as previously described for molecular host species identification. sequences were viewed, edited and a consensus generated using the bioedit sequence alignment editor software version . . [ ] . cipres was used for clustalx alignments, determining the best dna substitution model for nucleotide sequence analysis using the jmodeltest software and for constructing bayesian phylogenies using the beast version . software [ ] [ ] [ ] . bayesian mcmc chains were set to million iterations, sampling every steps for optimal ess scores. output files were visually inspected to check for convergence using the tracer software version . [ ] . the final phylogenies were constructed in treeannotator with a burn-in value of %. for visualization and manipulation of the phylogenetic tree, the figtree version . . software was used. pairwise similarities between sequences were analysed in mega x with complete deletion [ ] . in total, samples from bats constituting five genera were tested for coronaviruses (table and table s ). of these, two samples contained coronavirus rna, originating from two individuals of the rhinolophus genus. barcoding and molecular identification confirmed the host species to be rhinolophus clivosus (table s ). coronavirus sequences were extended to bp with rgu assay primer sets [ ] . the two sequences (rh-btcov/ /rwanda/ and rh-btcov/ /rwanda/ ) share . % nucleotide identity; pairwise similarities and phylogenetic analysis group these sequences with other lineage b betacoronaviruses (figure ). the closest relative to the rwandan betacoronavirus was reported from kenya, btcovky (tao et al. unpublished; genbank accession number ky . ), though the rhinolophus species is not specified. the sequences share very close sequence similarities ( . % nucleotide identity and % amino acid identity), suggesting that similar betacoronaviruses may be harbored by both kenyan and rwandan rhinolophus bats. full genome comparisons will be able to determine if kenyan and rwandan rhinolophus bats are infected by the same betacoronavirus species. within the analyzed conserved rdrp gene segment, this rwandan rhinolophus betacoronavirus also shares pairwise similarities of % nucleotide identity ( . % amino acid identity) to the bulgarian betacoronavirus rh-btcov/bm - /bgr/ [ ] , as well as close similarities ( . - . % nucleic acid similarity and . - . % amino acid identity) to asian rhinolophus sars-related coronaviruses such as sarsr-rh-btcov/rp and sarsr-rh-btcovwiv [ , ] . other coronavirus surveillance in rwanda, and surrounding countries such as uganda and tanzania also report sars-related coronaviruses from the rhinolophus genus [ ] . unfortunately, the sequences cannot be compared as an assay targeting a different conserved peptide of the rdrp gene was used [ ] . boldface indicates positive samples. twenty-four samples from bats were tested for paramyxovirus rna (table s ), none of which tested positive with the avula-rubulavirinae (ar) assay. an overall percentage positivity for paramyxovirus rna, detected using the respiro-morbilli-henipavirus (rmh) assay, was found to be . % (n = ). three of the viral sequences were detected in the insectivorous bat species hipposideros ruber and otomops martiensseni, while one other sequence was detected in the frugivorous bat species rousettus twenty-four samples from bats were tested for paramyxovirus rna (table s ), none of which tested positive with the avula-rubulavirinae (ar) assay. an overall percentage positivity for paramyxovirus rna, detected using the respiro-morbilli-henipavirus (rmh) assay, was found to be . % (n = ). three of the viral sequences were detected in the insectivorous bat species hipposideros ruber and otomops martiensseni, while one other sequence was detected in the frugivorous bat species rousettus aegyptiacus. host identification of positive samples was confirmed using molecular analysis (table s ). phylogenetic analysis of the sequences indicated that the insectivorous bat-borne viral sequences grouped with the jeilongvirus genus as well as in a jeilongvirus-related clade (figure ). one of the h. ruber sequences (batpv/hip_rub/up /rwa/ ) described from this study potentially groups within the jeilongvirus genus. the second h. ruber-derived viral sequence (batpv/hip_rub/up /rwa/ ) grouped with a paramyxoviral sequence detected in a bat from the same genus sampled in cameroon in , however, was not identical. the detection of two diverse viruses from bats of the same species and same population has previously also been reported in insectivorous bats sampled in other african countries [ ] . these observations can in part be explained by the generation of viral quasi-species populations due to the high mutation rate of rna viruses as a consequence of rna proofreading deficiency of the rna dependent rna polymerase [ ] . a larger pool of diverse viruses within a bat population and the co-roosting of several cave-dwelling bat species may facilitate viral sharing between different bat species [ ] . however, ongoing biosurveillance in these cave-dwelling bat species will be required before active viral sharing can be shown. the paramyxoviral sequence detected in the o. martiensseni bat (batpv/oto_mar/up /rwa/ ) was near identical to the viral sequences previously described from several individuals of the same species sampled in kenya in [ ] . these sequences shared a . % similarity on both nucleotide and amino acid level. the r. aegyptiacus-derived viral sequence (batpv/rou_aeg/up /rwa/ ) grouped within a henipavirus-related clade and was near identical to a paramyxoviral sequence detected in the same host species previously reported from kenya [ ] . sequence similarity shared between these two sequences was found to be . % and . % on nucleotide and amino acid level, respectively. to our knowledge, this study reports on the first evidence of paramyxovirus rna in bats from rwanda. two of the four viral sequences detected in h. ruber, were not closely related to the paramyxovirus sequences previously reported (sharing nucleotide and amino acid similarities of less than % and . %, respectively) and might represent novel viral species. however, a more rigorous analysis with variable genes such as the fusion and hemagglutinin gene will be required before putative species can be inferred. as observed in previous studies, viral sequences from frugivorous bats were mostly found to belong to the henipavirus genus or a related clade, while insectivorous bat-associated viral sequences have been linked to other genera including morbilliand jeilongvirus [ ] . this observation was again reflected in the current study. the detection of highly similar viral sequences from bats in rwanda and kenya, which are more than km apart, can be explained by either the phenomena of metapopulations or the hypothesis of co-evolution of paramyxoviruses with their bat hosts [ , , ] . rna viruses have an exceptionally high mutation rate commonly associated with quasi-species populations and with the potential to cross the species barrier, among others. this is evident in the high diversity of paramyxoviruses described to date and the wide host range associated with these viruses [ , , , ] . additionally, due to the emergence of sars, mers and sads, it is widely accepted that coronaviruses are capable of readily adapting to new hosts [ ] . the rwandan caves are considered an ecotourism site and guano is also mined on a small scale, providing an ideal bat-human interface. several insectivorous bat species co-roost with the egyptian fruit bats in these caves and future studies should investigate viral sharing. the egyptian fruit bat also uses these caves as a maternity roost and studies have shown that increased viral shedding is linked to reproductive cycles [ ] . longitudinal biosurveillance studies can therefore identify high risk periods in the future. as such, the detection of a sars-related bat coronavirus potentially circulating within the rhinolophus population and a henipavirus-related paramyxovirus in r. aegyptiacus in the ruhengeri region may merit further investigation to determine exposure, and the potential for spill-over events to occur. to date, emphasis of paramyxovirus surveillance has mostly been placed on fruit bats, the henipavirus genus and related viruses due to the association of other henipavirus species with zoonotic events [ , ] . however, research regarding the zoonotic potential of the insectivorous bat-associated viruses is still lacking. one major aim regarding surveillance of wildlife populations is to identify potential zoonotic agents and to evaluate any threat to the public as well as domestic animal health. though these bat-borne viruses are unlikely to pose a significant threat, it still merits continued monitoring of the chiropteran species within these caves as well as mammalian species that inhabit the surrounding area. , however, was not identical. the detection of two diverse viruses from bats of the same species and same population has previously also been reported in insectivorous bats sampled in other african countries [ ] . these observations can in part be explained by the generation of viral quasi-species populations due to the high mutation rate of rna viruses as a consequence of rna proofreading deficiency of the rna dependent rna polymerase [ ] . a larger pool of diverse viruses within a bat population and the coroosting of several cave-dwelling bat species may facilitate viral sharing between different bat species [ ] . however, ongoing biosurveillance in these cave-dwelling bat species will be required before active viral sharing can be shown. the paramyxoviral sequence detected in the o. martiensseni bat (batpv/oto_mar/up /rwa/ ) was near identical to the viral sequences previously described from several individuals of the same species sampled in kenya in [ ] . these sequences shared a . % similarity on both nucleotide and amino acid level. the r. aegyptiacus-derived viral sequence (batpv/rou_aeg/up /rwa/ ) grouped within a henipavirus-related clade and was near identical to a paramyxoviral sequence detected in the same host species previously reported from kenya [ ] . sequence similarity shared between these two sequences was found to be . % and . % on nucleotide and amino acid level, respectively. for countries where the bat-human interface is more pronounced, as a result of ecotourism, guano mining or bat hunting and consumption, surveillance is key to identify the diversity of viruses present and their potential host species. 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of the southwest indian ocean we would also like to thank the staff of the rwanda tourism board and national parks and wendy white from the kwazulu-natal bat interest group for assisting in the logistics and fieldwork. the authors declare no conflict of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results. key: cord- -ds rhbkv authors: lani, rafidah; hassandarvish, pouya; chiam, chun wei; moghaddam, ehsan; chu, justin jang hann; rausalu, kai; merits, andres; higgs, stephen; vanlandingham, dana; abu bakar, sazaly; zandi, keivan title: antiviral activity of silymarin against chikungunya virus date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: ds rhbkv the mosquito-borne chikungunya virus (chikv) causes chikungunya fever, with clinical presentations such as severe back and small joint pain, and debilitating arthritis associated with crippling pains that persist for weeks and even years. although there are several studies to evaluate the efficacy of drugs against chikv, the treatment for chikungunya fever is mainly symptom-based and no effective licensed vaccine or antiviral are available. here, we investigated the antiviral activity of three types of flavonoids against chikv in vitro replication. three compounds: silymarin, quercetin and kaempferol were evaluated for their in vitro antiviral activities against chikv using a chikv replicon cell line and clinical isolate of chikv of central/east african genotype. a cytopathic effect inhibition assay was used to determine their activities on chikv viral replication and quantitative reverse transcription pcr was used to calculate virus yield. antiviral activity of effective compound was further investigated by evaluation of chikv protein expression using western blotting for chikv nsp , nsp , and e e proteins. briefly, silymarin exhibited significant antiviral activity against chikv, reducing both chikv replication efficiency and down-regulating production of viral proteins involved in replication. this study may have important consequence for broaden the chance of getting the effective antiviral for chikv infection. phylogenetic analysis shows that chikv has three genotype variants; west african, east/central/ south african (ecsa) and asian . malaysia had its first outbreak in late at the suburb klang in the state of selangor and the chikv was identified as asian genotype. localized outbreaks and clustered cases were reported in larut matang, lama, perak. the ecsa genotype virus caused outbreaks after . in early , together with the neighboring singapore, the epidemic in johor began. this spread throughout states in peninsular malaysia and in , many cases occurred in selangor, melaka, negeri sembilan and pahang states and peaked in kelantan, kedah, terengganu, perlis and sarawak , , . from april until march , over , cases were reported in these nationwide outbreaks, although there were no fatalities . the main mosquito vectors for chikv are a. aegypti and a. albopictus , , . a. aegypti was a primary vector for the outbreak that happened in kenya, comoros, africa and asia, but the ' asian tiger mosquitoe' a. albopictus was the main vector for the reunion and subsequent indian ocean outbreak . vector switching to a. albopictus became predominant due to the substitution of alanine residue to valine residue in the chikv e protein (a v) . this convergent mutation seems to be due to the selective pressure as the impact of different and new environment or ecosystem. extensive studies continue around the world to find an effective cure, safe antiviral treatment or vaccine for chikv infection. in one study it was shown that chloroquine phosphate, an antimalarial drug, can decrease the intensity or duration of pain, and number of joints involved. however, the effectiveness of chloroquine to treat chronic symptoms could not be evaluated due to the lack of adequate control patients with these symptoms . the synergistic effect of ribavirin with interferon alpha b against chikv infection was reported only in an in vitro study . it has been shown that arbidol strongly interferes with the early stages of chikv infection by targeting the cellular membrane. however, it is only a potent inhibitor against in vitro chikv infection . flavonoids are polyphenolic compounds that are present in different plants, foods and drinks. flavonoids are well known for their different biological properties including: antimicrobial activity, anti-inflammatory activity, anti-allergic activity, and cytotoxic antitumor activity . quercetin has antiviral activity against herpes simplex virus (hsv) type- , respiratory syncytial virus, pseudorabies virus, parainfluenza virus type and sindbis virus, a type member of genus alphavirus. the mechanism of actions of quercetin includes: enhancing the antiviral activity of interferon, binding to viral proteins, and interfering with viral nucleic acid synthesis by binding to the viral polymerases. another flavonoid, kaempferol is active against hsv, human coronavirus and rotavirus replication . silymarin, extracted from milk thistle (silybum marianum), was shown to inhibit hepatitis c virus (hcv) in both in vitro and in vivo by inhibiting hcv entry, rna synthesis, viral protein expression and infectious virus production; in addition it also acts by blocking of the virus cell-to-cell spread . it becomes our concern to explain the definition for the nomenclature of 'milk thistle extract' , 'silymarin' and 'silibinin' since the findings that will be discussed further involve these three important terms. in brief, the initial extract of the crushed milk thistle seeds made up of - % silymarin and - % fatty acids, such as linoleic acid. silymarin itself, is a complex of more than flavonolignans (silybin a, silybin b, isosilybin a, isosilybin b, silychristin, isosilychristin and silydianin) and flavonoid whereas silibinin is a semi-purified, commercially available fraction of silymarin, with an approximate : mixture of diastereoisomeric compounds, silybin a and silybin b which are also referred to in the literature as silibinin a and b . here we evaluated in vitro antiviral activity of quercetin, kaempferol and silymarin against a clinical isolate of chikv. our results support the antiviral properties of silymarin as a promising compound for further investigations towards the development of an anti-chikv drug. cytotoxicity of flavonoids. the mts assay was used to determine cytotoxicity of each flavonoid for vero and bhk cells. the cc value of each compound was calculated (table ). there was no observed antiviral activity assay. primary screening. different non-cytotoxic concentrations of silymarin, kaempferol and quercetin were tested on chikv-infected vero cells to find the effective compound. a cpe inhibition assay was used at hpi. it was shown that μ g/ml of silymarin significantly inhibited the chikv-cpe presentation (p = . ) (fig. a) . this finding was not unexpected as silybin (also known as silibinin) is one of the components of silymarin and has been shown to possess anti-chikv activity . in contrast, the highest concentrations of quercetin and kaempferol only exhibited % cpe inhibitory activity (fig. a) . the results of cpe inhibition assay were further confirmed by using mts assay (fig. b) . silymarin inhibits early post-entry stages of chikv replication. from analyzed compounds only silymarin was able to suppress chikv mediated cytotoxicity. therefore it was chosen for a time-of-addition assay performed with aim to determine which stage of virus infection cycle this compound affected. as it is shown in fig. the inhibition was most efficient when silymarin was added at hpi. the time-dependence of inhibitory effect of compound is coherent with hypothesis that its anti-chikv activity may be due inhibition of some early, but probably post-entry, step of chikv replication cycle. anti-entry assay has also been performed and the result has clearly shown that there is no antiviral activity of silymarin against chikv at the anti-entry phase (fig. ). cell line contained non-cytotoxic replicon of chikv which, in addition to the virus replicase proteins, expresses also puromycin acetyl transferase, egfp and rluc markers . in this system the rluc activity is proportional to viral replicon rna replication. it was found that μ g/ml of silymarin suppressed the activity of rluc marker expressed by the chikv replicon by . % (fig. ) . the inhibition was highly significant (p = . ) confirming that silymarin affects post-entry steps of chikv infection. this data also indicates that silymarin can interfere with chikv rna replication by affecting the viral replicase system. interestingly, in previous study ic of silybin, one of the major components of silymarin, was estimated as . μ m (approximately μ g/ml). compared to this silymarin was somewhat more efficient as nearly three-fold inhibition was observed at μ g/ml indicating that other components of silymarin likely contributed to its anti-chikv activity. to confirm the post-entry antiviral activity of silymarin against chikv a virus yield assay using qrt-pcr was used. the chikv rna copy number in supernatants of cells treated with each concentration of inhibitor and that from the control cells was interpreted and depicted (fig. ). there is a significant reduction of chikv replication as treated with increasing concentrations of silymarin (p = . ), silymarin exhibits a statistically significant (p < . ) dose-dependent inhibition on the rluc activity produced by bhk-chikv-nct replicon. the rluc activity was measured at h post treatment. vehicletreated ( . % dmso) cells were used as control (" ") concentration). data from triplicate assays were plotted and analyzed from a non-parametric correlation (spearman) two-tailed test (graph pad prism version , graph pad software inc., san diego, ca). as compared to the vehicle control treated cells. based on this data it was calculated that silymarin had ic = . μ g/ml with selectivity index (si) of . . taking into account that adding silymarin hpi showed significant inhibitory effect against chikv replication confirms that silymarin acts at the post-entry stage of chikv infection. in contrast, in similar assay neither kaempferol nor quercetin showed any significant inhibitory effect against chikv replication (fig. ) . as it is shown in fig. , μ g/ml of silymarin can inhibit the chikv yield by more than %. to evaluate whether the silymarin also affects the chikv protein synthesis, a series of western blot analyses were performed using lysates of the chikv infected and silymarin treated vero cells (fig. ). as it is evident from constant levels of β actin, silymarin treatment did not result in protein degradation and, at used concentrations, the compound was not toxic for the vero cells. in contrast, dose-dependent reduction of amounts of nsp , nsp and e proteins was observed (fig. ) indicating that silymarin limited chikv replication and virus-encoded protein synthesis within the treated cells. silybin interferes with early post-entry of chikv infection in dose-dependent manner. questioned by whether the silymarin effects on the chikv replication are influenced by the fractions it is made up of, since the study done by pohjala l. et al., has proved that the silybin (one of the silymarin fraction) can suppressed the activities of rluc marker gene expressed by the chikv replicon, we have evaluated the effect of silybin against chikv intracellular replication. hence, silybin was tested for its early post-entry activity and we confirmed its anti-chikv activity in a dose-dependent manner with the ic of . µg/ml, which is higher than the ic of silymarin. this result makes us to conclude that the activity of silymarin against chikv replication might also be enhanced by other components or fractions that made it up. considering the crippling pains that persist for weeks and even years due to chikv infections and the absence of antiviral treatment for the virus, the search for effective antiviral compounds is imperative. due to their known broad spectrum anti-viral activity and low toxicity, we evaluated three flavonoids as viable candidate compounds . from these compounds only silymarin was identified as the flavonoid with significant anti-chikv activity. it was considered that it has potential for future development as its use results in ≥ % reduction of chikv infection based on cpe inhibition and mts assays (fig. ) . remarkably, the effective concentration of silymarin was much lower than mntd. in contrast, neither kaempferol nor quercetin showed significant antiviral activity against in vitro replication of chikv in both assays. to quantify the copy number of viral rna released (most probably in form of genomic rna packed into virions) from infected cells were used qrt-pcr and amplified part of nsp encoding region of chikv genome. since nsp which is involved in the negative-strand and subgenomic rna synthesis, the quantification of the negative-strand rna reflects the active viral replication, we selected the nsp as the gene target for qrt-pcr to quantify the virus load during replication [ ] [ ] [ ] [ ] . our data demonstrated that treatment with silymarin results in effective reduction of number of released viral genome indicating reduction of viral rna synthesis and/or virion formation and release. in post-adsorption assay silymarin taken at μ g/ml resulted in ≈ % inhibition of chikv replication. we therefore performed a time-of-addition assay using this concentration of silymarin to determine the stage of infection where the silymarin treatment results in biggest antiviral effect. the result was consistent with our data from post-adsorption assay; hence it can be concluded that silymarin is most effective if it is added shortly after virus infection ( hpi). the reason for lower anti-chikv activity of pretreatment with silymarin could be due to the half-life of silymarin which based on previous bioavailability study is less than hours , although, there is no data available on in vitro half-life or stability of silymarin which might be interesting for future studies. as our findings were consistent with hypothesis that silymarin effect on post-entry stages of chikv dose response analysis was also performed using a chikv replicon cell line where no virus entry or exit takes place. again, it was found that silymarin is able to suppress the activity of rluc marker expressed by chikv replicon. this finding is consistent with that from previous assay and confirms that silymarin suppresses chikv rna replication and does it in dose-dependent manner. the rluc, expressed by chikv replicon, is fused to nsp protein of the virus. accordingly, reduction of rluc activity indicates reduced amounts of nsp -rluc protein in chikv replicon cell line. to further confirmation of anti-chikv activity of silymarin, we have shown the effect of silymarin on viral protein synthesis in chikv infected cells (fig. ) . again decrease of viral protein synthesis was observed. this effect could be due to inhibition of chikv rna replication and/or transcription. however, as in virus expression and replicon cell lines the synthesis of viral rnas and proteins are coupled further study is necessary to evaluate the direct effect of silymarin on inhibition of newly synthesized chikv proteins. it is possible that some of non-structural proteins of chikv represents direct target for silymarin. the possibilities include nsp protein, which is involved in the synthesis of the negative strand of viral rna and rna capping, and nsp protein, that is another component of the viral replicase complex. down regulation of e expression may represent consequence of suppression of replication (directly or via inhibition of ns-protein(s)). however, this down regulation is clearly relevant from point of view of development of effective antiviral as e protein is one of the important virion glycoproteins and is essential for receptor binding. interestingly, a previous study showed that silymarin can inhibit the expression of ns b of hepatitis c virus (hcv) which is catalytic subunit of hcv replicase . although, the hepatoprotective property of silymarin in the context of blocking hcv infection based on in vivo studies is another important criteria for this compound to be considered as a therapeutic candidate for hepatitis c , , . this study represents an important first report on the anti-chikv properties of silymarin. previously other flavonoids such as apigenin, chrysin, naringenin and silybin, which are also one of components of silymarin, are known to be inhibitors of viral replication mainly through affecting important cellular elements for chikv replication . however, inhibitory properties of silymarin are somewhat different from that of silybin. thus our data for silymarin warrants further mechanistic studies to characterize the antiviral properties of all its components and other flavonoid compounds and head-to-head comparison of their antiviral effects at lower concentrations/ different conditions so that the best option of anti-chikv can be determined. in summary, our study showed that silymarin exhibited significant in vitro antiviral activity against chikv. it suppressed post-entry stages of viral replication, most likely chikv rna replication significantly with a dose dependent manner. coherent with this the expression of proteins, needed for rna replication, and also expression of viral structural e protein were down-regulated. in contrast to silymarin it was found that quercetin and kaempferol are unable to suppress chikv replication and accordingly are not good candidates for anti-chikv drugs. these findings warrant future mechanistic, in vivo anti-viral, toxicity and pharmacokinetic studies as part of the process for evaluation of silymarin as a potential anti-chikv therapeutic. virus and cells. the chikv isolate used in this experiment was a clinical isolate from an outbreak in johor in coded as my/ / /fn . it belongs to the ecsa genotype and has the a v mutation in e protein , . bhk- (baby hamster kidney) and vero (african green monkey kidney) cells from the american type culture collection (atcc) were used in this study. vero cells have been widely used in the in vitro antiviral research for example in the antiviral testing for the chikungunya virus, human enterovirus and coxsackievirus a , . both cell lines were cultured using eagle's minimum essential medium (emem, gibco, ny, usa) containing % inactivated fetal bovine serum (fbs) and penicillin-streptomycin, and incubated at °c with % co . the chikv my/ / /fn strain was propagated in bhk- cells and harvested after full cytopathic effect (cpe) was observed. virus stock titer was determined by the tissue culture infectious dose (tcid ) methods ; then the obtained stock was aliquoted and stored at - °c. during the time of virus propagation and antiviral assay the fbs concentration of the cell culture medium was reduced to %. vero cell line was used for further experiments such as mts assay and antiviral assays. the chikv replicon cells were grown and maintained in dulbecco's modified eagle's medium (dmem, gibco, ny, usa) supplemented with % fetal bovine serum (fbs), % tryptose-broth phosphate and penicillin-streptomycin, and incubated at °c with % co . flavonoids. silymarin, quercetin and kaempferol were purchased from sigma-aldrich corporation (sigma-aldrich, st. louis, mo, usa) and stored in − °c for future use. stock solution was diluted with emem and was filtered through a syringe filter with . μ m pore size (millipore, ma, usa) at the time of use; dmso concentration in working solutions prepared on emem was kept at . %. ( -( , -dimethylthiazol- -yl)- -( -carboxymethoxyphenyl)- -( -sulfophenyl)- h-tetrazolium) assay (promega, wi, usa) was performed to evaluate the cytotoxicity of tested compounds against vero cells and bhk cells according to the manufacturer's protocol. briefly, monolayers of vero cells were grown in -well plate and were treated with different concentrations of each compound in triplicate together with negative control (media containing . % dmso). the plate was then incubated at °c with % co for hours before the mts assay was performed. treated and control cells were kept for two days at °c, under similar conditions and duration until used for antiviral activity assay. after two days post-treatment, mts solution was added to the cells and incubated for hours at °c with % co prior to absorbance detection at nm wavelength using infinite pro multiplate reader (tecan, männedorf, switzerland). all experiments were conducted in triplicate. the half maximal cytotoxic concentration (cc ) for each compound was determined through this assay using graph pad prism (graph pad software inc., san diego, ca, usa, ). antiviral activity assays. primary assay for antiviral activity. a monolayer of vero cells were grown in -well plate in emem containing % inactivated fbs. the tested compounds were added to the wells in triplicate together with chikv at an moi = . the plate was then incubated at °c with % co for hours. the assay was conducted in duplicate for each concentration of each compound. after two days, the plate was viewed under the microscope and the degree of cytopathic effect (cpe) as measure of virus replication inhibition was expressed as the percent yield of virus control (% virus control = cpe experimental group/cpe virus control × ). the results were confirmed by performing the mts assay (promega, wi, usa) according to the manufacturer's protocols. all experiments were repeated three times independently. the statistical analysis on percentage of cpe inhibition was performed by using the gaussian populations (pearson) two-tailed assay. anti-entry assay. the procedure of the anti-entry assay was modified and performed according to lee rch et. al., . monolayers of vero cells were grown in -well plate with emem supplemented with % inactivated fbs. the vero cells were then infected with chikv and the plate was incubated for hour at °c. non-adsorbed virus was then washed with xpbs. tested compound was added in different concentrations and incubated at °c with % co for hours. the plate was again washed with xpbs and treated with citrate buffer (ph = ) to inactivate the non-internalized virus, before the plate was again washed with xpbs. the emem supplemented with % inactivated fbs was added into every wells and the plate was incubated for hours at °c with % co . chikv replicon cell line based assay. monolayers of chikv replicon cells were prepared in -well white plate (corning inc, ny, usa) and treated with different concentrations of tested compounds. after hours incubation at °c with % co the activity the renilla luciferase (rluc), expressed by chikv replicon, was detected using renilla luciferase assay (promega, wi, usa) performed according to the manufacturer's protocols. the luminescence signal was then measured using the glomax / luminometer (promega, wi, usa), plotted against the log transformation of the concentrations of compounds and a sigmoidal curve fit with variable slope was created to obtain the half maximal inhibitory concentration (ic ) value for each compound by graph pad prism (graph pad software inc., san diego, ca, usa, ). data were represent as the means ± standard error of the mean (sem) from triplicate assay from three independent experiments. the statistical analysis to determine the correlation between the concentration of silymarin and the rluc activity was performed by using non-parametric correlation (spearman) two-tailed assay. time-of-addition assay. in this experiment, a monolayer of vero cells were grown in -well plate in emem containing % inactivated fbs. the wells (in triplicate) were designated as − , − , , , , and -hour, that they are named according to the time of chikv (moi = ) infection. as for − hour, vero cells were treated with μ g/ml of silymarin. the plate was then incubated for one hour at °c with % co . one hour later, − hour wells were also treated with μ g/ml of silymarin. at the hour, all wells except the control wells were infected with chikv and again incubated for one hour at °c with % co . after -hour incubation and for every -hour after incubation at °c with % co , μ g/ml silymarin were added to − , , and hour wells respectively. the plate was then incubated at °c with % co for hours. the supernatant of the treated and control wells (treated with . % dmso was used as vehicle control) were collected two days post-treatment and analyzed using qrt-pcr. the half maximal effective concentration (ec ) of silymarin was calculated using graph pad prism (graph pad software inc., san diego, ca, usa, ). all data are from triplicate assay from three independent experiments. quantitative reverse transcription pcr(qrt-pcr). a qrt-pcr assay was used to quantify the chikv rna copy number. for this amplification of base region of nsp encoding sequence was performed as described by chiam and colleagues . the primers were nsp -f( '-gcgcgtaagtccaagggaat- ') and nsp -r ( '-agcatccaggtctgacggg- '). the cdna was first generated from the rna extracted from the supernatants (qiagen, germany) of the previous assays plate, using nsp -r primer and superscript iii reverse transcriptase (life technologies, usa) according to the manufacturer's protocol. the unincorporated primers were then digested with u of exonuclease i (new england biolabs, usa). the qrt-pcr was performed with a step-oneplus real-time pcr system (life technologies, usa) with × power sybrgreen pcr master mix (life technologies, usa), following the manufacturer's protocol, and using serially diluted standards. cycling parameters were °c for min, cycles of °c for s and °c for min. the amplified product was verified by melting curve analysis. the statistical analysis to determine the correlation between the rna copy number and the silymarin concentration was performed by using the gaussian populations (pearson) two-tailed assay. western blotting. vero cells at the density of × cells were seeded into a cm flask in emem containing % fbs and penicillin-streptomycin. the next day, each flask was infected with chikv inoculum at an moi = , placed on a rocker for minutes, and then incubated at °c with % co for h. then each flask was treated with different concentrations ( , , , . , . μ g/ml) of the trial compound, control flasks were treated with solutions containing . % dmso. all the flasks were then incubated at °c with % co until the appearance of cpe in the vehicle control. once cpe was observed, cells were scraped, washed with pbs and lysed using μ l of % triton x (sigma-aldrich, st. louis, mo, usa) containing complete protease inhibitor cocktail (sigma-aldrich, st. louis, mo, usa) at °c for min. cellular debris was pelleted out by centrifugation at , × g for min. a micro bca tm protein assay kit (thermo scientific, rockford, il) was used to quantify the protein concentration for each sample. lysate containing μ g of protein were denatured using sds-loading buffer and proteins were separated using sds-page in % gels. the gels were then equilibrated in towbin buffer ( . m tris, . m glycine % methanol) for min and proteins were transferred to a pvdf membrane using the bio-rad wet transfer system (bio rad, san francisco, ca). for detection of nsp and nsp , membranes were blocked with x pbs % casein blocker (bio rad, san francisco, ca) for h at room temperature on a shaker. the blots were rinsed three times with x pbs tween before being incubated with primary anti-chikv nsp , anti-chikv nsp or anti-chikv e rabbit polyclonal antibodies in % casein solution. the blots were then washed three times with x pbs tween for min each time. this was followed by incubation with the secondary goat anti-rabbit igg (abcam, cambridge, uk) antibodies conjugated with horseradish peroxidase (hrp) for h at room temperature on an orbital shaker. membranes were washed three times with pbs containing tween for minutes each time. for the loading control, separate blots containing the same samples were incubated with primary anti-β-actin mouse monoclonal antibody conjugated with hrp (cell signaling technology, ma, usa) dissolved in % casein for hour at room temperature on shaker. the blots were then washed three times with x pbs tween for minutes each time. membranes were developed by the colorimetric method using appropriate substrates (thermo scientific, rockford, il). a potentially emerging epidemic? biology and pathogenesis of chikungunya virus comparison of chikungunya virus infectivity in baby hamster kidney (bhk- ) cell line and c / , an aedes albopictus cell line using electron microscopy epidemiology of chikungunya in malaysia ribavirin therapy for chikungunya arthritis chikungunya virus-associated death in malaysia ph-dependent entry of chikungunya virus into aedes albopictuscells reemergence of endemic chikugunya, malaysia. emerge infect dis chikugunya infection-an emerging disease in malaysia chikungunya virus infection current situation of dengue and chikungunya in malaysia for epidemiological week arrival of chikungunya virus in the new world: prospects for spread and impact on public health a single mutation in chikungunya virus affects vector specificity and epidemic potential chloroquine phosphate treatment of chronic chikv arthritis: an open pilot study in vitro antiviral activity of arbidol against chikungunya virus and characteristics of a selected resistant mutant antimicrobial activity of flavonoids synergistic effect of flavones and flavonols against herpes simplex virus type- in cell culture. comparison with the antiviral activity of propolis multiple effects of silymarin on the hepatitis c virus life cycle silymarin for hcv infection inhibitors of alphavirus entry and replication identified with a stable chikungunya replicon cell line and virus-based assays flavonoids as nutraceuticals: a review use of a highly sensitive strand-specific quantitative pcr to identify abortive replication in the mouse model of respiratory syncytial virus disease a structural and functional perspective of alphavirus replication and assembly quantitative analysis of dengue- virus rna during extrinsic incubation period in individual aedes aegypti regulation of sindbis virus rna replication: uncleaved p and nsp function in minus-strand rna synthesis, whereas cleaved products from p are required for efficient plus-strand rna synthesis plasma concentrations of free and conjugated silybin after oral intake of a silybin-phosphatidylcholine complex (silipide) in healthy volunteers multiple effects of silymarin on the hepatitis c virus lifecycle inhibition of t-cell inflammatory cytokines, hepatocyte nf-kappab signaling, and hcv infection by standardized silymarin chikungunya virus of asian and central/east african genotypes in malaysia outbreak of chikungunya in johor bahru, malaysia: clinical and laboratory features of hospitalized patients structure-activity relationship study of arbidol derivatives as inhibitors of chikungunya virus replication antiviral effects of phyllanthus urinaria containing corilagin against human enterovirus and coxsackievirus a in vitro a simple method of estimating fifty per cent end points mosquito cellular factors and functions in mediating the infectious entry of chikungunya virus real-time polymerase chain reaction for diagnosis and quantitation of negative strand of chikungunya virus we would like to thank ministry of higher education ( key: cord- -n xp mlz authors: hall, richard j.; wang, jing; todd, angela k.; bissielo, ange b.; yen, seiha; strydom, hugo; moore, nicole e.; ren, xiaoyun; huang, q. sue; carter, philip e.; peacey, matthew title: evaluation of rapid and simple techniques for the enrichment of viruses prior to metagenomic virus discovery date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: n xp mlz the discovery of new or divergent viruses using metagenomics and high-throughput sequencing has become more commonplace. the preparation of a sample is known to have an effect on the representation of virus sequences within the metagenomic dataset yet comparatively little attention has been given to this. physical enrichment techniques are often applied to samples to increase the number of viral sequences and therefore enhance the probability of detection. with the exception of virus ecology studies, there is a paucity of information available to researchers on the type of sample preparation required for a viral metagenomic study that seeks to identify an aetiological virus in an animal or human diagnostic sample. a review of published virus discovery studies revealed the most commonly used enrichment methods, that were usually quick and simple to implement, namely low-speed centrifugation, filtration, nuclease-treatment (or combinations of these) which have been routinely used but often without justification. these were applied to a simple and well-characterised artificial sample composed of bacterial and human cells, as well as dna (adenovirus) and rna viruses (influenza a and human enterovirus), being either non-enveloped capsid or enveloped viruses. the effect of the enrichment method was assessed by both quantitative real-time pcr and metagenomic analysis that incorporated an amplification step. reductions in the absolute quantities of bacteria and human cells were observed for each method as determined by qpcr, but the relative abundance of viral sequences in the metagenomic dataset remained largely unchanged. a -step method of centrifugation, filtration and nuclease-treatment showed the greatest increase in the proportion of viral sequences. this study provides a starting point for the selection of a purification method in future virus discovery studies, and highlights the need for more data to validate the effect of enrichment methods on different sample types, amplification, bioinformatics approaches and sequencing platforms. this study also highlights the potential risks that may attend selection of a virus enrichment method without any consideration for the sample type being investigated. since the proliferation of high-throughput sequencing technologies, the search for viruses has entered a new era (lipkin, (lipkin, , . these technologies are capable of producing millions of sequence reads without a priori knowledge of the sample. sequence data generated from a sample is compared to known sequence databases in order to identify viruses. previously, virus metagenomics was accomplished by cloning and sanger-based sequencing of randomly amplified nucleic acid (breitbart et al., ; djikeng et al., ) . despite the small amount of sequence data produced, it was still possible to detect viruses due to either high concentrations, or some process of prior enrichment being applied that removes host cells and exogenous nucleic acid. the relative abundance of a virus (or viral nucleic acid) in a sample, compared to that of other organisms such as bacteria or host cells (or their genomes), is a critical factor for the discovery of viruses when using metagenomics. a higher proportion of viral sequence increases the probability that ( ) viral sequences will be represented in a metagenomic dataset and ( ) larger contigs can be assembled, increasing the likelihood of a match in the database. it is has been shown that without some type of physical enrichment method, viruses may not be present in high enough concentrations to be detected (daly et al., ) . this problem is somewhat overcome as high-throughput sequencing technologies advance in both read length and sequencing depth. nevertheless, gains in sensitivity are still possible by the application of a physical enrichment process for viruses, and may also avoid the cost of generating and analysing additional data. in the field of viral ecology there have already been significant advances in the validation of physical enrichment methods for viruses (duhaime and sullivan, ; john et al., ) , such as the methods used to concentrate and purify viruses from seawater for virus discovery by metagenomics (hurwitz et al., ) . however, only a few methodological studies have been applied to evaluate the efficiency of viral enrichment methods in metagenomics that seek to diagnose animal or human disease. a study on human liver tissue compared enrichment techniques of freeze-thaw, centrifugation and nuclease-treatment for the detection of hepatitis c virus using both roche and illumina high-throughput sequencing platforms (daly et al., ) . the abundance of viral sequences in each treatment group was compared to results obtained by quantitative real-time pcr detection of transcripts, and the effect of each treatment method on viral genome coverage was also determined. such studies show that physical enrichment methods do increase the sensitivity of detection for viruses in metagenomics. for new researchers looking to perform work on human or animal samples for the purposes of detecting or diagnosing new or unexpected viruses, it can be difficult to ascertain which virus enrichment method may be applicable to a given sample type. methods described for viral ecology studies are unlikely to be applicable. a review was undertaken of published metagenomic studies that sought to describe viruses present in human or animal samples (excluding virus ecology studies) and provide details on the enrichment methods used (table ) . most of these studies incorporate the use of low-speed centrifugation and/or filtration to remove host cells or other micro-organisms, with a final nucleasetreatment step, where dnase or rnase will destroy exogenous nucleic acid but is not thought to affect nucleic acid protected by the viral capsid or envelope. ultracentrifugation also features as a common method for the concentration of viruses from samples. the application of virus discovery methods using metagenomics has been considered for routine use in diagnostic and reference laboratories to aid in the diagnosis of human (svraka et al., ) and animal disease (belak et al., ) . the application of these techniques in a clinical setting will require that any virus enrichment methods are simple to perform, fast, robust, effective, standardised and do not require significant capital expenditure. it is noted that the vast majority of the published studies in table apply the simple enrichment techniques without any a priori justification for the selection of the technique. this study sought to examine the rapid and simple enrichment techniques for viruses that appear to be in routine use in the literature for diagnosing animal and human diseases, but for which the effects on metagenomic data have not been studied. this was achieved by examining the effect of these enrichment methods on the relative abundance of viruses in a metagenomic dataset derived from a simple and well-characterised artificial sample. human enterovirus was cultured in human rhabdomyosarcoma cell line in hanks mem (life technologies, carlsbad, ca, usa) supplemented with % foetal bovine serum (thermofisher scientific, waltham, ma, usa). human adenovirus was also cultured in the human rhabdomyosarcoma cell line. influenza a(h n )pdm was cultured in mdck-siat cells (canine) in r-mix (diagnostic hybrids, athens, oh, usa). all virus cultures were composed of cell culture supernatant and monolayer present after freeze-thaw. escherichia coli o was cultured in brain heart infusion broth (bhi) and incubated at • c overnight. human a cells were cultured in dmem (life technologies, carlsbad, ca, usa) supplemented with % foetal bovine serum (thermofisher scientific, waltham, ma, usa). an artificial sample was formulated to consist of known amounts of e. coli o , a human epithelial lung carcinoma cells (atcc ccl- ), human enterovirus , human adenovirus and influenza a(h n )pdm . aliquots of the final dilution were subjected to three freeze-thaw cycles and were frozen and stored at − • c. based upon a review of enrichment methods presented in table , five combinations of three simple methods of enrichment were selected and performed on ml aliquots of the artificial sample as follows; low-speed centrifugation in a microfuge at × g for min at • c, sterile syringe filtration at . m, nuclease treatment using . u l − turbo dnase (life technologies, carlsbad, ca, usa), . u l − rnase one (promega, fitchburg, wi, usa) and x dnase buffer (life technologies, carlsbad, ca, usa) and incubation at • c for min, or combinations of these being a -step method (centrifugation followed by filtration), or -step (centrifugation, filtration then nuclease-treatment). independent duplicates for each treatment were performed and used in all subsequent experiments. the extraction of rna was achieved using the iprep purelink virus kit (life technologies, carlsbad, ca, usa), where l of the artificial sample was extracted and eluted into l of molecularbiology grade water. all real-time quantitative pcr assays (qpcr) were performed on a stratagene mx p real-time pcr system (agilent technologies, santa clara, ca, usa). qpcr on extracted rna was used to quantify a human cells, influenza, adenovirus, e. coli o and enterovirus present in the artificial sample. the human rnase p (rnp) gene was used as a target for the detection of human a cellular rna. the nucleoprotein gene target was used for the detection of influenza a(h n )pdm rna. both assays were performed using the agpath one step rt-pcr kit reagents (life technologies, carlsbad, ca, usa) and the primers and probes for these assays have been previously described (who, ) . each l reaction contained l of nucleic acid, . l of rt-pcr buffer, l of x rt-pcr enzyme mix, . m probe and . m primers. following an initial min reverse transcription step at • c and min denaturation step at • c, a -step cycling procedure of denaturation at • c for s with annealing and extension at • c for s over cycles was used. adenovirus dna was detected using a previously published assay (brittain-long et al., ) and the agpath one step rt-pcr kit (life technologies, carlsbad, ca, usa). each l reaction contained l of dna, . l of rt-pcr buffer, l of x rt-pcr enzyme mix, . m probe and . m primers. after an initial min reverse transcription step at • c and min denaturation table virus enrichment process prior to sequencing in metagenomic studies on human and animal samples. author year journal aim of study sample step step step step step step step step step step step step step step step at • c, a -step cycling procedure of denaturation at • c for s with annealing and extension at • c for min over cycles was used. enterovirus rna was detected using a previously described assay (oberste et al., ) step system (life technologies, carlsbad, ca, usa). each l reaction contained l of rna, . l of x invitrogen reaction mix, . l of mm mgso , . l of superscript ® iii rt/platinum ® taq mix, . m probe and . m primers. following an initial min reverse transcription step at • c and min denaturation step at • c, a -step cycling procedure of denaturation at • c for s, annealing at • c for s and extension at • c for s over cycles was used. for e. coli o a one-step assay was performed using the lightcycler ® probes mastermix (roche, indianapolis, in, usa) as previously described (paton and paton, ; thomas et al., ) . each l reaction contained l of dna, l of lightcycler ® probes master, . m probe (stx ) and . m primers (o and o ). following an activation step of • c for min, a -step cycling procedure of denaturation at • c for s, annealing at • c for s and extension at • c for s over cycles was used. every assay included negative and positive controls, and rnase-free reagents and handling procedures. the real-time pcr assays were made quantitative by including a dilution series of a plasmid with known copy number, which contained concatenated primer-probe-primer target sequences for each of the five assays. dna was co-purified with rna during the nucleic acid extraction method, and thus dna was removed using ambion dna-free (life technologies, carlsbad, ca, usa), then l of this rna was reverse transcribed into cdna using a first-strand cdna synthesis kit primed by random hexamers as per the manufacturer's instructions (life technologies, carlsbad, ca, usa) including the recommended rnase h digestion. the minimum g amount of dna required for input into the illumina truseq dna library preparation protocol was not achieved. amplification of the cdna was achieved by using a whole transcriptome amplification kit (qiagen, valencia, ca, usa) as described previously (berthet et al., ; cheval et al., ) . briefly, the reverse transcription step required in the kit was not utilised, but the ligation and amplification steps were followed as per the manufacturer's instructions except that the ligation reaction was terminated by heating to • c for min, and the amplification step was performed for h followed by termination of the reaction at • c for min. for each sample more than g of dna was produced and this was sequenced on an illumina miseq instrument (new zealand genomics limited, massey genome service, massey university, palmerston north, new zealand) using an illumina truseq dna library preparation (illumina, san diego, ca, usa). water-only negative controls failed to amplify any dna. illumina miseq sequence data consisted of bp paired-end reads. quality checking and redundant-read collapsing was performed and sequence reads were then compared to the ncbi non-redundant nucleotide database (downloaded from ncbi ftp the % confidence interval. the grey line represents the copy number of the target gene when no enrichment method is applied. site in february ) using blastn (blast+ . ). an e value of . was used as the cut-off threshold value for significant hits. the blastn output files were imported and parsed in megan (huson et al., ) for taxonomic assignment. the abundance of nucleic acid from the model organisms in the artificial sample (human cells, bacteria, influenza, adenovirus and enterovirus) was determined by using quantitative real-time pcr. the effect of different enrichment methods on target gene copy number was assessed (fig. ) . in general, all enrichment methods decreased the quantity of every model organism when compared to no treatment at all. human rna was removed to a limited extent by all the methods, with the -step treatment and nuclease-only treatments being the most effective, resulting up to -fold reduction in copy number. similarly, some bacterial nucleic acid was removed by all of the enrichment methods, with the nuclease-only or the -step treatment being the most effective. the three viruses used in this experiment represent a dna virus (adenovirus), and two rna viruses one of which is enveloped (influenza) and the other non-enveloped (enterovirus). the subsequent metagenomic analysis was targeted at the detection of rna viruses, but a dna virus was included to assess the potential to detect dna viruses using this methodology. each virus also represented a different level of concentration ( ) a high concentration at × copies (enterovirus), ( ) a moderate concentration (adenovirus) at , copies and ( ) a low concentration (influenza) at , copies. all viruses showed a decrease in copy number when an enrichment method was applied. this decrease was consistent across all enrichment methods, with most showing no greater than a -fold reduction in copy number except the -step method when applied to enterovirus, where the virus copy number was reduced by -fold. the same rna extraction that was used for the qpcr was also used for a metagenomics experiment ( fig. and table ). the first replicate sample of each treatment was indexed and run on one illumina miseq run producing , , sequence reads of bp in length, the second replicate set was indexed and run on an independent illumina miseq run and produced , , sequences reads of bp in length. after submission to blastn, each sequence read was given a taxonomic assignment using megan. all five model organisms were represented in the untreated samples ( fig. and table ) and for simplicity of data representation the family level of enterobacteriaceae was chosen to represent the e.coli organism in the sample, which was the most abundant taxa identified accounting for . % of the total sequences ( , , reads; table ). the kingdom level of metazoa was chosen to represent the human cells within the untreated sample, which accounted for % of the total reads ( , reads; table ). the decision to use metazoa and enterobacteriaceae was made to facilitate simple representation of the data, but was arbitrary, as the aim of this experiment was to compare variations in the proportions of bacteria, human cells and viruses between the enrichment methods. viruses were present in the untreated sample in the following proportions; enterovirus . % ( , reads), adenovirus . % ( reads) and influenza virus . % ( reads). it is interesting that the dna virus, adenovirus, was identified in the dataset, given that the method was targeted at rna viruses. a combined total number of sequence reads for two independent physical replicates which were also run on different illumina miseq flowcells. this figure represents the collapsed sequencing data, therefore redundant reads are not represented more than once. b serial applications of treatment methods. the -step method consisted of centrifugation then filtration. the -step method consisted of centrifugation, filtration then nuclease-treatment. ( . %) that were not assigned to these aforementioned taxa were accounted for as either ( ) no hit in the blast search, ( ) no clear taxonomic assignment from the blast search due to the stringency of parameters required by megan for taxonomic assignment ( ) low complexity sequence ( ) assignment to a taxonomic level that was not captured by enterobacteriaceae, metazoa or viruses. the enrichment techniques did not greatly change the relative abundance of enterobacteriaceae, at best there was a % reduction in the number of reads assigned to this taxon when applying the -step treatment. there was an increase in the proportion of metazoa sequences when filtration was applied increasing from % to . %, with only the nuclease-treatment showing the greatest effect reducing metazoan sequence to . % (a -fold reduction). for viruses, the -step treatment was the only treatment to show a significant increase in the proportion of viral sequence, by -fold for influenza (from . % to . %) and -fold for enterovirus. the proportion of adenovirus hits appeared unaffected by all enrichment methods (fig. ) . this study compares the effect of five different viral enrichment methods on the ability to detect viruses in a metagenomic approach. enrichment techniques were deliberately chosen that have been commonly referenced in previous metagenomic studies which seek to identify new or rare viruses (table ) . these enrichment methods are often selected without prior justification. the effect of these enrichment techniques was examined by application to a highly specific artificial sample. this study does not provide a full validation of the enrichment methods, but highlights some possible risks if an enrichment method is selected based solely upon methods published by others and without consideration for the sample being examined. validation of enrichment techniques in the field of virus ecology is well developed (duhaime and sullivan, ; john et al., ) , but there is a paucity of data on the validation of enrichment methods applied to the detection of viruses in animal or human samples for the purpose of diagnosis. in the present study, an artificial sample was composed which represents the type of organisms that could possibly be observed in clinical samples i.e. a rectal swab taken from a human patient. of course, the artificial sample is unlikely to have similar characteristics to complex biological samples from humans or animals. bacteria are often a very abundant organism in de novo metagenomic datasets, and host sequence is also often present. to this end, e.coli and a human cell line were chosen, as well as two rna viruses (influenza virus, enterovirus) and a dna virus (adenovirus). the dna virus was included to assess the potential for detection when using an rna virus targeted approach, as many virus discovery projects have to create two workflows to independently target dna or rna viruses. differing amounts of each virus were placed into the artificial sample so as to represent varying concentrations. this artificial sample represents a starting point to evaluate simple and rapid viral enrichment methods for use in virus metagenomics studies that seek to detect a virus that is causing disease in humans or animals. at present, there is little guidance for researchers seeking to work in this area and published studies have often selected these simple enrichment techniques with no justification for their inclusion in the method. in general, it was observed that the choice of enrichment method such as low speed centrifugation, syringe-based filtration, nuclease treatment (dnase and rnase) or combinations of these methods, did not substantially increase the relative abundance of viruses in this metagenomics dataset, except in selected cases. despite reductions in the quantity (copy number) of bacterial and human rna gene targets as shown in the qpcr data, this did not translate into a substantially increased relative abundance for viral sequences in the metagenomic data. the qpcr method detects the absolute quantity of genome target present, whereas the metagenomic data is proportional (relative abundance). even though large gains can be made in reducing bacteria and human nucleic acid, the proportion of viral sequences in the metagenomic datasets still remained relatively low (i.e. generally less than %) except for when a -step enrichment method was applied. nevertheless, individual enrichment methods did have some effect on relative abundance of the model organisms' representation in the metagenomics data. nuclease treatment alone was successful in reducing the proportion of human (metazoa) sequences by -fold. it is hypothesised that the initial freeze-thaw processing of the artificial sample has lysed human cells and thus liberated human genome. this exogenous nucleic acid is more susceptible to digestion by nuclease than the protected bacterial and viral genomes, which are protected by membranes or capsids, which are more resistant to freeze-thaw action. the -step treatment was particularly effective at increasing the abundance of both influenza and enterovirus, both in absolute concentration as measured by real-time pcr, and also relative abundance as measured in the metagenomics data. this finding supports the use of the -step procedure for virus enrichment, and similar -step procedures have previously been employed in published virus metagenomic studies (table ) . regarding sensitivity, without an enrichment method × copies of influenza virus genome were detected using a metagenomic approach ( sequence reads; table ), and the detection of the dna genome of adenovirus present at × copies ( sequence reads; table ) was also possible. this experiment was targeted at the discovery of rna viruses but included the dna virus (adenovirus) to determine if an rna virus detection method could also co-detect dna viruses. given that the starting material used for this experiment is rna, it is possible to surmise that adenovirus mrna expressed during infection was detected. however, this may not be the correct explanation as the dnase treatment used before cdna synthesis is known to be less than % efficient, and some dna is likely to have been carried right through into the sequencing library preparation. the ability to detect dna viruses when using an rna-targeted method will also no doubt be influenced by the specific replication cycle of any given virus. it is noted that the viral metagenomic method chosen uses multiple displacement amplification, and therefore there is likely to be a bias in the amplification of larger genomes e.g. bacteria and host, which could confound results when considering the relative abundance of sequences in the metagenomic data. there are many different methods of amplification that are available and this represents only one. however, this amplification method is one that is in practical use, and has been applied in other virus discovery studies (cheval et al., ) , therefore the examination of enrichment techniques using this specific amplification method are still relevant. there is a certainly a need for future studies to expand this work into a full validation, so as to examine the effect of enrichment when using other amplification methods i.e. sispa, nextera, lamp, lasl. it would also be interesting to include a greater array of enrichment methods, sample types, sequencing platforms and bioinformatics approaches which may include the incorporation of sequence assembly methods, or the use of other search algorithms. the findings presented here should provide a starting point for those considering the use of rapid or simple enrichment methods for the purposes of diagnosing new viral diseases in human or animal samples by using metagenomics. this study also highlights the possible risks of arbitrarily selecting an enrichment method purely based upon previously published studies. rjh and mp designed the study with assistance from pec. qsh provided the design of the real-time pcr assays, and akt, sy, hs, xr, nem performed the experiments. jw, ab, and rjh analysed the data. rjh, mp, xr, nem, jw and pec interpreted the findings. rjh wrote the manuscript draft while all authors edited the manuscript. a virus discovery method incorporating dnase treatment and its application to the identification of two bovine parvovirus species new viruses in veterinary medicine, detected by metagenomic approaches phi polymerase based random amplification of viral rna as an alternative to random rt-pcr metagenomic analyses of an uncultured viral community from human feces multiplex real-time pcr for detection of respiratory tract infections evaluation of high-throughput sequencing for identifying known and unknown viruses in biological samples a viral discovery methodology for clinical biopsy samples utilising massively parallel next generation sequencing viral genome sequencing by random priming methods ocean viruses: rigorously 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comparative evaluation of taqman real-time pcr and semi-nested vp pcr for detection of enteroviruses in clinical specimens detection and characterization of shiga toxigenic escherichia coli by using multiplex pcr assays for stx , stx , eaea, enterohemorrhagic e. coli hlya, rfbo , and rfbo simultaneous identification of dna and rna viruses present in pig faeces using process-controlled deep sequencing identification and characterization of deer astroviruses metagenomic sequencing for virus identification in a public-health setting random pcr and ultracentrifugation increases sensitivity and throughput of vidisca for screening of pathogens in clinical specimens tracking verocytotoxigenic escherichia coli o , o , o , o and o in irish cattle laboratory procedures to generate viral metagenomes identification of a new human coronavirus human picobirnaviruses identified by molecular screening of diarrhea samples rapid identification of known and new rna viruses from animal tissues. plos pathog. , e . who, . who information for molecular diagnosis of influenza virus in humans -update metagenomic analysis of fever, thrombocytopenia and leukopenia syndrome (ftls) in henan province, china: discovery of a new bunyavirus this study was funded by the esr core research fund provided by the new zealand ministry of business, innovation and employment. we are thankful for the support from the esr technical staff in the clinical virology laboratory and enteric reference laboratory for providing the model organisms and protocols for real-time pcr. we also wish to acknowledge new zealand genomics limited for the provision of high-throughput sequencing services and staff at the massey genome service, in particular lorraine berry and dr patrick biggs. our kind thanks also go to the esr information technology staff ned rajanayagam and phillip mitchell for developing the computing infrastructure to support this study. key: cord- -r sjv ih authors: antas, marta; woźniakowski, grzegorz title: current status of porcine epidemic diarrhoea (ped) in european pigs date: - - journal: j vet res doi: . /jvetres- - sha: doc_id: cord_uid: r sjv ih porcine epidemic diarrhoea (ped) is a highly contagious and devastating enteric disease of pigs caused by porcine epidemic diarrhoea virus (pedv), an enveloped, single-stranded rna virus belonging to the alphacoronavirus genus of the coronaviridae family. the disease is clinically similar to other forms of porcine gastroenteritis. pigs are the only known host of the disease, and the occurrence of ped in wild boars is unknown. the virus causes acute diarrhoea, vomiting, dehydration, and high mortality in suckling piglets reaching %. heavy economic losses in the pig-farming industry were sustained in the usa between and when pedv spread very quickly and resulted in epidemics. the loss in the us pig industry has been estimated at almost seven million pigs. the purpose of this review is a description of the current status of porcine epidemic diarrhoea in european pigs and the risk presented by the introduction of pedv to poland in comparison to the epidemics in the usa. coronaviruses (covs) cause a large variety of diseases in humans and animals. in pigs, coronaviruses affect various organs, including the gastrointestinal and respiratory tracts. covs have one of the largest genomes of all rna viruses, which in combination with their high genetic diversity causes mutation and recombination, resulting in new virus variants ( ) . besides porcine epidemic diarrhoea virus (pedv), the porcine coronaviruses comprise transmissible gastroenteritis virus (tgev), porcine respiratory coronavirus (prcv), porcine haemagglutinating encephalomyelitis virus (phev), and porcine deltacoronavirus (pdcov) ( ) . the clinical symptoms of ped are diarrhoea, vomiting, and dehydration which result in very high mortality among suckling piglets and large economic losses ( , , ) . various factors influence the clinical signs of ped, mainly the age of the animals, the herd's immune status, and the virulence of the strain ( ) . multiple pedv strains are circulating on different pig farms around the world and they differ in virulence. ped was observed for the first time in in the united kingdom. the virus only infected fattening pigs and sows. subsequently, the disease spread to other european countries. emergence of a new pedv strain in china in caused serious epidemics and this strain rapidly spread worldwide. in the usa, highly virulent strains infected pigs in states between and , resulting in significant economic losses ( , , ) . the disease was characterised by rapid diarrhoea onset in pigs of all ages and mortality in suckling piglets reaching % ( ) . in europe, ped re-emerged in in germany. subsequently, highly similar strains were also found in several european countries ( , ) . ped is not a notifiable disease in the european union and is not on the list of diseases reported to the world animal health organization (oie), therefore the status of this disease is not fully recognised. in the last years, only a few countries have reported clinical cases of ped and/or seropositive animals ( ). coronaviruses belong to the coronavirinae subfamily. currently, this subfamily is classified into the four genera alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus, which fall into distinct groups on the phylogenetic tree. ( , , ) . the taxonomy of swine coronaviruses is shown in table . the coronaviruses are pleomorphic and have one of the largest rna viral genomes and a unique replication strategy ( , ) . virions are spherical with diameter of approximately nm. the most prominent and the defining feature of coronaviruses are the spike structures on the surface of the virion, which give them the appearance of a solar corona ( ) . the pedv genome is single-stranded rna ( , nucleotides in length) of positive-sense polarity containing ′ and ′ untranslated regions (utr) at both ends. two thirds of the genome from the ′ end encodes proteins necessary for rna replication, while the one third on the ′ side of the genome comprises at least seven open reading frames (orfs) that encode four structural proteins: n (capsule), s (spike), e (envelope), and m (membrane), and three nonstructural proteins (replicases a and b and orf ). in the genome, they are arranged in the order ′-replicase ( a/ b)-s-orf -e-m-n- ′. the rna genome of the virus is associated with the n protein to form a long, helical ribonucleoprotein (rnp) complex. the virus core is enclosed by a lipoprotein envelope, which contains the s, e, and m proteins ( fig. ) ( , , , ) . the m protein is the most abundant component in the viral envelope, is necessary for the assembly process, and affects production of protective antibodies with virus-neutralising activity. the small e protein plays an important role during coronavirus budding. the n protein has multiple functions in viral replication, interacts with viral genomic rna, and associates with other n proteins to protect the viral genome. it disturbs antiviral responses by antagonising interferon production ( , ) . the s protein is encoded by a gene sequence which determines the virulence of a pedv strain ( ) . this protein is composed of , amino acids (aa), significant among which are a signal peptide ( - aa), neutralizing epitopes ( - , - , - , and , - , aa), a transmembrane domain ( , - , aa), and a short cytoplasmic domain. it consists of the s ( - aa) and s ( - , aa) domains, which are responsible for binding and fusion of the virus. the s domain is involved in a specific interaction with the cellular receptor and induction of neutralising antibodies ( , ) . only one serotype of pedv has been reported. the phylogenetic analysis of the s gene indicates that pedv could be genetically divided into two groups, these being the classical genotype (gi) and the field epidemic or pandemic genotype (gii). the early european cv strains (genbank accession number af ), vaccine strains, and strains adapted to cell cultures were classified to genotype , while genotype includes strains responsible for the epidemics in the usa and asia. strains identified in the usa have been ( , , , ) . all pedv pandemic strains circulating in china after were also clustered to genotype , and they were genetically separated from other global pedv strains and from earlier chinese strains ( ) . isolation of pedv from field samples is very difficult. the virus grows in vero cells (african green monkey kidney). however, its growth there requires the presence of trypsin-supplemented cell culture medium, because this protease plays an important role in cell entry and the release of pedv virions in vero cells. trypsin does this by cleaving the s protein into s and s subunits, and it enables efficient replication and spread of pedv in vitro ( , ) . pedv adapted to the vero cell line can be successfully propagated in other cell types. ped viruses cause cell death leading to lysis of infected cells, a change observable under the microscope as a cytopathic effect (cpe) which is characterised by cell membrane vacuolisation and syncytium formation ( , , ) . the ped virus can survive for variable periods outside the host, depending on the temperature and relative humidity. the virus is stable in temperatures ranging from °c to °c but loses its infectivity above °c ( ) . survival of the ped virus in different samples is presented in table . pedv is easily inactivated by heating to °c for min. at lower temperatures, ph is a factor in pedv inactivation. the resistance of ped virus in different ph values is given in table . pedv-inactivating disinfectants are oxidising agents, bleach, % phenolic compounds, % sodium hydroxide, formaldehyde and glutaraldehyde, % sodium carbonate, ionic and nonionic detergents, iodophors in % phosphoric acid, and lipid solvents such as chloroform ( , ) . direct transmission by the faecal-oral route may also occur through ingestion of virus-contaminated vomit. indirect transmission occurs through contaminated feed trucks, service vehicles, vehicles used for the movement of pigs, and people (pig owners and farm visitors). until the introduction of pedv to the usa, feed for animals was not considered to be a vector in the spread of the virus. recent studies have shown, however, that feed can be a potential vector for pedv transmission and contaminated feed can be a source of disease ( ) . it is widely acknowledged that the most important risk factors for spreading the disease are contaminated vehicles ( , ) . oral ingestion causes viral replication in the epithelial cells of the small intestinal villi and to a lesser extent in the colonic villi, which results in degeneration of enterocytes and shortening of the villi ( , ) . pedv can infect pigs of all ages, causing watery diarrhoea and vomiting with anorexia and dehydration, which is the major cause of death in young piglets. the clinical signs depend on the age of the pigs, immune status of the herds, and virulence of the strain ( , , ) . lesions are observed in the gastrointestinal tract and are characterised by a distended stomach filled with completely undigested milk dots and thin, transparent intestine walls ( ) . pedv infection is clinically indistinguishable from other forms of porcine gastroenteritis diseases such as those caused by tgev and pdcov, therefore pedv diagnosis cannot be made only on the basis of clinical signs and has to be confirmed by laboratory tests to make diagnosis final ( , ) . a variety of pedv detection methods are applied which include immunofluorescence (if) or immunohistochemistry (ihc) tests, in situ hybridisation, virus isolation, enzyme-linked immunosorbent assays (elisa), and various reverse transcription polymerase chain reaction (rt-pcr) techniques. samples which can be used in laboratory diagnosis are fresh faeces, oral fluids, small intestine tissue, and serum (to determinate the presence of antibodies). to detect pedv rna, rt-pcr can be used for diagnosis of acute outbreaks no longer than days after the onset of the disease. for surveillance and monitoring of ped, serological diagnosis is necessary. antibodies persist for more than one year in the serum of infected pigs ( , , ) . the regenerative ability of villi is instrumental in the recovery of the pig. regeneration speed depends on age of the animals; in adult and fattening pigs, villi are restored in three to four days, while in piglets the process is longer at six to seven days ( ) . the percentage of morbidity in the course of infection can climb to %. mortality is variable and also depends on the age of animals. in suckling piglets, mortality can attain %, in piglets older than days, it is less than %, and in adult and fattening pigs, it falls below %. there is no specific treatment other than symptomatic treatment of diarrhoea, however, most growing pigs recover without treatment within - days unless secondary infections occur. reinfection may occur when the immunity wanes ( , ) . ped was first observed in the united kingdom and belgium in early s ( ) . the disease caused mortality of about % in fatteners and adult pigs. suckling piglets were not affected and remained symptom-free even when the sows had watery diarrhoea for several days. at the beginning, the disease was incorrectly diagnosed as tge because the symptoms of these two diseases are almost identical. at a later stage, tge was excluded by laboratory diagnosis. in , new cases of the disease were described in the uk. this outbreak was different in that the virus affected pigs of all ages, including neonatal and suckling piglets, inflicting around % mortality. pedv was definitively identified for the first time in in belgium and was classified to the coronaviridae family, recorded as the cv prototype strain ( , ) . in the s and s, pedv was identified as the cause of severe epidemics in japan and south korea ( ) . in the same period in europe, outbreaks of ped appeared sporadically but the virus continued to spread and persisted in an endemic form in the pig population. outbreaks of ped were observed in the netherlands in - , in hungary in , and in the uk in . typical epidemic outbreaks of ped with high mortality in neonatal piglets were also identified in italy in [ ] [ ] and china in - ( , ) . the first symptoms of the disease in the usa were observed in april and were confirmed in the laboratory in may . the ped virus had escaped from biosecurity and control systems, spread very quickly to other areas, and infected hundreds of farms. by march , the presence of pedv had been confirmed in us states. pedv epidemics resulted in significant economic loss there, and the domestic pig industry lost almost % of its population (seven million pigs) ( , ) . the ped outbreak was characterised by watery diarrhoea, dehydration, and variable vomiting. all groups of animals were affected by the epidemics, but the highest mortality was % and occurred among suckling piglets ( , ) . the strains isolated in the usa were genetically related to the chinese pedv strains reported in . pedv was probably dispersed in the usa mainly through contaminated trucks, but other factors that assist the spread of the virus cannot be excluded ( ) . two strains of pedv have been identified in the usa: highly virulent non-indel (usa/kansas / ) and the milder variant s-indel (usa/oh / ) ( ) . both variants were classified to genotype . in experimental infections, s-indel strains showed lower pathogenicity and mortality (from % to %) than the non-indel strains, where the mortality rate was up to % ( , , ) . since the ped outbreak in the usa, detection of highly virulent strains has also been reported in canada, mexico, taiwan, south korea, japan, and ukraine. detection of s-indel strains has been reported in most european countries ( , , ) . ped is not notifiable in the european union and is not on the list of diseases reported to the world animal health organization (oie), therefore, it is not possible to accurately describe the occurrence of this disease in europe. in the last years, only a few countries have reported clinical cases of ped and/or seropositive animals ( ) . the ped outbreaks in europe were significantly different from epidemics in the usa or asia. after the epidemics in in the usa, ped cases were also confirmed in austria, belgium, france, germany, italy, the netherlands, portugal, and slovenia ( fig. ) ( , , ) . viruses identified in these european countries were very similar to usa/oh / strains. in germany ped emerged in , inflicting up to % mortality and typified by acute symptoms ( ) . in slovenia ped was confirmed in the laboratory in ( ) . at almost the same time, highly virulent strains were described in ukraine. these viruses were similar to usa/kansas / . so far, ukraine is the only european country where highly virulent strains have been identified. nevertheless, taking into account the occurrence of a virulent strain in ukraine and its rapid spread, it is likely that epidemics similar to those in the usa may also occur in europe ( , ) . considering the presence of pedv in countries neighbouring poland (germany, the czech republic, and ukraine) and the rapid spread of the virus, it is highly probable that pedv will also be introduced to poland. ped is a rapidly spreading global disease and causes large economic losses in pig production around the world. frequently appearing mutations cause variability in its viral genome and can change the pathogenicity of the new pedv variants. biosecurity is a central to the prevention or spread of pedv. applying the principles of biosecurity protocols, it is possible to reduce the risk of introducing the virus into swine herds. disinfection of vehicles entering farms is highly effective because contaminated faeces or vomit can be found on the wheels. in poland, the clinical symptoms of ped have been observed, but until no studies were conducted to confirm the presence of pedv. in - , the presence of the virus and/or of specific antibodies was confirmed on several farms in poland. samples of blood, faeces, slurry, and intestines were collected in herds in which the clinical symptoms of the disease had previously been observed. so far, virus isolation in vero cells has not been carried out in poland and nor has genetic material from positive samples been sequenced. it is foreseen that pedv might be transferred in the future to polish swine herds. the severity of ped which occurred in the usa has shown that this disease should be countered with timely preventive measures and early diagnosis. therefore, the role of the national veterinary research institute in puławy, poland, in the diagnosis of ped is of paramount importance in future pig production. the authors declare that there is no conflict of interests regarding the publication of this article. porcine epidemic diarrhea virus: an emerging and re-emerging epizootic swine virus pathogenesis comparison between the united states porcine epidemic diarrhoea virus prototype and s-indel-variant strains in conventional neonatal piglets isolation and characterization of porcine epidemic diarrhea viruses associated with the disease outbreak among swine in the united states scientific opinion on porcine epidemic diarrhea and emerging porcine 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coronaviruses as the gene source of gammacoronavirus and deltacoronavirus porcine epidemic diarrhea virus variants with high pathogenicity evolutionary insights into the ecology of coronaviruses distinct characteristics and complex evolution of pedv strains molecular characteristics of the spike gene of porcine epidemic diarrhoea virus strains in eastern china in key: cord- -ocgfgx r authors: boyce, john m; cookson, barry; christiansen, keryn; hori, satoshi; vuopio-varkila, jaana; kocagöz, sesin; Öztop, a yasemin; vandenbroucke-grauls, christina mje; harbarth, stephan; pittet, didier title: meticillin-resistant staphylococcus aureus date: - - journal: the lancet infectious diseases doi: . /s - ( ) - sha: doc_id: cord_uid: ocgfgx r nan the epidemiology and the clinical management of mrsa infections have continued to evolve in recent years in the usa. the prevalence of mrsa has increased progressively since the early s, and by mrsa accounted for nearly % of nosocomial s aureus infections acquired in intensive-care units. mrsa strains have been reported to account for - % of nosocomial s aureus bloodstream infections and - % of s aureus surgical-site infections. [ ] [ ] [ ] [ ] [ ] [ ] in some facilities, the proportion of health-care-associated s aureus infections caused by mrsa is even higher. vancomycin-intermediate s aureus (visa) strains, which have vancomycin minimum inhibitory concentrations (mics) of - mg/ml, have continued to cause occasional health-care-associated infections, although transmission within health-care facilities of such strains has seldom been documented. at least three well-documented isolates of vancomycin-resistant s aureus (vrsa) that have vancomycin mics of mg/ml or greater have been recovered from patients in the usa. although it is possible that the relatively high frequency of co-colonisation by vancomycinresistant enterococci (vre) and mrsa among patients in the usa may create more opportunities for in-vivo transfer of the vana gene complex from vre into mrsa strains, it is not known if this has affected the frequency of vrsa in the usa. by contrast with the uncommon occurrence of visa and vrsa, mrsa strains that are still classified as susceptible to vancomycin (ie, mics of - mg/ml) have caused an increasing number of health-careassociated infections that respond poorly to vancomycin therapy. , , the poor response to vancomycin therapy in such cases appears to be associated with several strain characteristics. patients infected with mrsa strains with vancomycin mics of - mg/ml have responded to vancomycin therapy less frequently than patients with infections caused by strains with mics of less than · mg/ml. in some cases, poor clinical responses may have been due to strains with hetergenous resistance to vancomycin-so-called hetero-visa. fowler and colleagues found that mrsa bloodstream infections that persisted for more than days despite appropriate vancomycin therapy were significantly more likely than short-duration mrsa infections to have been caused by strains that exhibited ( ) higher rates of survival in vitro after exposure to thrombin-induced platelet microbicidal protein (p= · ), ( ) defective delta-lysin production (suggestive of loss of accessory gene regulator [agr] function; p= · ) and ( ) agr type ii (p= · ). in two prospective randomised trials of treatment of ventilator-associated pneumonia (vap), vancomycin therapy was significantly less effective than treatment with linezolid (p= · ) among a subset of patients with vap caused by mrsa. unfortunately, the microbiological characteristics of mrsa from these pneumonia studies were not described in detail. the above trends suggest that although vancomycin remains the drug of choice for most serious health-careassociated mrsa infections, clinicians in the usa may need to consider the use of other agents-eg, linezolid, daptomycin, or tigecycline-in some clinical situations based on the characteristics of the infecting strain and the body site affected. another major change in the epidemiology of staphylococcal infections in the usa is the rapid emergence of community-acquired mrsa strains since the late s. [ ] [ ] [ ] [ ] although such infections have been more common among population groups such as young children, native american and pacific islander communities, prisoners, military personnel, men who have sex with men, intravenous drug users, and individuals involved in amateur or professional competitive sports, spread within the general community is likely occurring. , [ ] [ ] [ ] a relatively small number of unique uk mrsa was first described in england in the early s, just months after meticillin had been introduced into clinical practice. mrsa has waxed, waned, and waxed again over the following decades. the international union of microbiology and who's staphylococcal centre is based in the laboratory of healthcare associated infection of the health protection agency (hpa), and was the first to identify and number epidemic mrsa strains (emrsa - ), plot their spread, and identify the existence of other epidemic mrsa strains in europe as part of the harmony network project. community mrsa are evident in england but have not yet emerged to be the problem encountered in some other countries. in the early s, about % of s aureus bacteraemias were due to mrsa; the mean figure is now about %, although there is a large range, with some hospitals encountering the organism infrequently. current english data show that the occurrence of meticillinsensitive strains has also increased, so merely focusing on the percentage of total s aureus bacteraemias can be misleading. ever since the organism was first described, hospitals have continued to vary in mrsa occurrence within and between different cities and in different wards within the same hospital. the reasons are complex and may include aspects of the reporting system; a comparison of reporting methods is underway. we know that the current prevalent uk epidemic mrsa (emrsa strains and ) differ genetically from their predecessors and also contain more or different toxins. however, we still do not understand why certain mrsa strains have this epidemic potential. when such strains spread to other countries they seem to exploit the more stressed or less "infection control compliant" hospitals or wards. mrsa are opportunistic pathogens colonising more patients than they infect. the patient case mix has also changed. mrsa has always preferably colonised and infected elderly people. improved nutrition and medical advances has meant that elderly patients are now admitted to "high-risk" units-eg, cardiovascular and orthopaedic wards with periods of care on intensive care units. such units can then "carousel" forum strains appear to be responsible for many of the community-acquired mrsa infections occurring in the usa. most of such strains contain the mobile genetic element staphylococcal cassette chromosome mec (sccmec) type iv, which is relatively uncommon among health-care-associated mrsa strains in the usa. emergence of community-acquired mrsa has also required that clinicians alter their approach to empiric treatment of community-acquired skin and soft tissue infections. physicians have been encouraged to routinely culture skin lesions to determine if community-acquired mrsa is the cause, and in geographic areas where community-acquired mrsa are relatively common, empirical therapy of such infections with clindamycin, trimethoprim-sulfamethoxazole (co-trimoxazole), doxycycline or minocycline, or linezolid is becoming common. if empiric clindamycin is administered, testing community-acquired mrsa isolates for inducible clindamycin resistance by using the "d-test" is recommended. by contrast with the netherlands and some other parts of northern europe, no standard set of measures has been adopted in all health-care facilities in the usa for controlling transmission of mrsa. measures recommended by the society for healthcare epidemiology of america for controlling health-care-associated mrsa include the use of screening cultures to detect colonised patients, placing patients in private rooms or cohorting patients, wearing gloves for room entry, gowns for substantial contact with patients or their environment, and hand hygiene before and after patient contact. however, there is considerable debate among experts in the usa about whether or not the use of screening cultures to detect patients at high risk of mrsa is necessary or practical in all health-care settings. given the considerable body of evidence that screening cultures, when combined with contact precautions, are beneficial and cost-effective, [ ] [ ] [ ] it is disconcerting that a recent survey of infectious disease consultants found that only % of respondents favoured the use of screening cultures to detect multidrug-resistant pathogens, and only % worked in facilities where screening cultures were routinely done. the costs of surveillance cultures, potential logistic problems that may result from an increased number of patients requiring isolation, and lack of controlled trials demonstrating the efficacy of screening cultures are often cited as concerns by those who do not use screening cultures. further studies are necessary to establish the relative efficacy of control measures such as screening cultures, cohort nursing, increased staffing levels, and improved hand hygiene adherence rates in controlling transmission of mrsa in health-care facilities. at present, there is no consensus regarding which measures are most appropriate for reducing transmission of community-acquired mrsa in community settings and for preventing the spread of these strains in health-care facilities. in the past year i have served as a consultant to dial corporation and woodward laboratories, who produce hand hygiene products, including alcohol-based hand rubs and soaps. i am currently a consultant to gojo industries. infectious diseases section, hospital of saint raphael, new haven, ct, usa forum mrsa-affected patients to other parts of the hospital, spreading the organisms elsewhere. there is also mrsa spread between orthopaedic wards and residential or nursing homes, which now care for more severely ill patients. another change has been the increasing involvement of paediatric populations; this is being studied specifically. it is interesting that obstetrics and paediatrics had encountered mrsa infrequently in previous decades in the uk. perhaps the development of mixed specialty wards has facilitated its introduction into this population? implementation of effective measures is being deleteriously affected by other changing environmental factors. reduced hospital bed numbers have resulted in increased interward transfers of patients, which have been shown to increase the risk of mrsa acquisition. decreased lengths of stay have resulted in patients being discharged before they present with mrsa infections. we have thus reduced the effectiveness of our alert organism laboratory surveillance systems. our data also show that inter-city transfers of patients has encouraged national mrsa spread. bed occupancy in many hospitals is over % and in some parts over %. some uk infectioncontrol teams have problems closing mrsa-affected wards because of the pressures on waiting lists. however, the relation between bed occupancy and mrsa rates is not clear-cut. insufficient numbers of skilled staff further compounds the problem. studies have shown that compliance with infection-control practices decreases as workloads increase, resulting in more mrsa crossinfection. these practices include mrsa screening, effective hand hygiene, wound and intravascular/urinary device care, and appropriate isolation measures. when we did an intra-uk comparison of hospitals in , those that were better able to control mrsa outbreaks had fewer delays in identifying mrsa patients ( · % vs %), fewer inter-hospital transferred patients ( · % vs · %), and no problems with mupirocin resistance ( % vs · %) compared with those less able to control the problem. a recent english mrsa systematic review concluded that there were many problems with the quality of the reviewed papers. absence of evidence should not be taken to mean that there is an absence of effect. the authors emphasised that the current guidelines for mrsa control should be followed until evidence emerges that other approaches are effective. prescribing of certain antibiotics probably contributes to the acquisition of mrsa, although the evidence for this is not clear-cut. the authors indicated the ways in which better studies could be designed to inform the most cost-effective means to produce sustained improvements in compliance. mrsa control guidelines were first written in and were similar to the "search and destroy" approach used in non-endemic countries. in we revised them in the light of the endemic problems encountered in many english hospitals. there has been much debate as to how to best control endemic mrsa and other writers in this forum have outlined many of these issues. our analyses of the current situation will further inform the most effective mrsa control measures. new guidelines have been written and a consultation on them is underway. several interventions will be required. the department of health and the hpa in consultation with many other bodies and health-care workers are introducing a suite of initiatives with various performance indicators and outcome measures that should enable improvements in infection control. however, these initiatives will require substantial funding. multidisciplinary involvement will be crucial, as well as ownership of infection control by all healthcare workers and sharing of good practice. there are clearly many issues that need to be discussed with policy makers; bed managers also need to work closely with infection-control teams. in some parts of england mrsa-free wards are being created, elsewhere mrsafree or fast-track orthopaedic hospitals are being assessed. i believe that effective control and real reductions in mrsa will be possible. screening strategies are critical-eg, planned screening and eradication of mrsa before admissions, monitoring of acquisitions on high-risk units and among patients who have been in hospital for prolonged periods (ie, more than a week), and rotating screening programmes on wards to provide feedback data to inform interventions such as hand hygiene improvement campaigns. a good example of this is the cleanyourhands campaign, which uses a multifaceted approach including the use of alcohol handrubs and patient empowerment. rapid mrsa detection systems are being examined for costeffectiveness. many systems have been used to isolate patients-eg, an open ward, side rooms, four-bedded cohorting, or an isolation ward. whatever strategy is used should be validated by screening strategies. modelling studies have further informed isolation strategies. the hpa and others have done much to ensure that the news media are well informed about mrsa and have provided leaflets to educate the public. however, we will also need to do more to look at infection-control standards in nursing homes and to plan the eradication of mrsa from patients better after discharge. epidemic mrsa has a long history in australia. the bacterium was first isolated in in sydney and spread rapidly through teaching hospitals over the next years, such that by the end of the s the prevalence was - % in hospitals in the cities on the eastern side of australia. annual surveillance studies by the australian group on antimicrobial resistance have allowed us to track the evolution of both epidemic and community mrsa for the past years. the current prevalence ( ) for multidrug-resistant epidemic mrsa varies for the state capital cities, and is approximately % in sydney, % in melbourne, % in brisbane, % in adelaide, and % in perth. recent epidemiological typing studies have enabled the identification of these epidemic strains as predominantly st -mrsa-iii (uk emrsa , , , vienna, portuguese/brazilian clones) with two variants, one in melbourne and adelaide and the other in sydney, canberra, and brisbane. active screening and infection-control policies initiated in the early s following a single hospital outbreak have prevented the establishment of epidemic mrsa in any western australian hospital. since , increasing numbers of st -mrsa-iv (uk emrsa ) have been detected clinically, on screening, and in the annual surveillance studies. this non-multidrug-resistant clone is the major epidemic strain in western australia, where it comprises % of all mrsa isolates and % of all epidemic strains. a similar trend is occurring on the eastern side of australia, with sydney having the highest prevalence of this strain ( % of all mrsa). nurses coming from the uk are an important source of these isolates, which are often detected on pre-employment screening. this clone has also become established in many long-term care facilities. in addition to nosocomial epidemic mrsa we have been documenting the evolution of true community-acquired mrsa strains in australia over the past years, such that now we have at least clones that have evolved independently around the country. it is somewhat ironic that western australia, the state with the lowest rate of noscomial epidemic mrsa, should now have the highest rate of communityacquired mrsa ( % of all mrsa isolates). these strains, although frequent in admitted patients, rarely seem to be transmitted to others in the hospital. when identified, patients colonised or infected with community-acquired mrsa are managed with enhanced precautions (single room if possible, attention to hand hygiene, contact precautions, and extra cleaning during admission and on discharge). to date, we have not had any major hospital outbreak with community-acquired mrsa, despite an increasing prevalence in the community. my experience with mrsa has been as a western australian, where we have successfully used a "search and destroy" policy for at least years. it is state policy that any patient who has been hospitalised outside the state in the preceding year be isolated and screened for mrsa before joining the general hospital population. in addition, staff who have worked clinically outside western australia are also screened pre-employment. this screening has led to my colleagues in sydney, melbourne, and brisbane calling us "fortress wa". we have had a number of outbreaks over the years, particularly in my own hospital, which has a lack of single rooms, but swift screening of contact patients and staff, isolation of colonised patients, attention to hand hygiene, and enhanced ward cleaning has terminated the outbreaks rapidly. we also routinely screen all patients in high-risk areas (intensive care, burns, vascular, and spinal units) on admission, discharge, and after prolonged stay. decolonisation is initiated for persistently colonised staff and patients likely to have frequent readmissions. in general, topical hexachlorophene or triclosan together with mupirocin nasal ointment is used. however, if oral carriage is detected or the topical regimen fails, systemic therapy with two agents is given. i cannot speak for my colleagues on the other side of australia who have lived with endemic nosocomial mrsa for many years, but i believe that there are a variety of practices mostly aimed at containment rather than eradication. in brisbane, where the prevalence has been very high, rates have dropped substantially with the move to new hospital accommodation and intensification of infection-control procedures. it is perhaps easiest for those of us who do not have endemic nosocomial mrsa. we have a very defined end point-no endemic mrsa-that we wish to achieve. for those with endemic mrsa, realistic end points for any containment programme must be established and measures instigated to ensure the attainment of those outcomes. containment or eradication is important-we are all aware of the emergence of vrsa carrying the vana gene complex. the coexistence of mrsa and vre provides the environment for gene transfer. policies for the control of both these organisms are therefore of paramount importance. community-acquired mrsa presents a new challenge. fortunately in western australia, most of the community strains are panton-valentine leucocidin (pvl) negative. the infections seen are therefore of similar severity to those caused by susceptible s aureus. however, there are disturbing data indicating that our most frequent community-acquired mrsa clone can, and has, acquired pvl on several occasions. we are currently contemplating what community strategies should be implemented, in particular whether we should follow the example of denmark where a search and destroy policy has been used within the community. it would not be feasible for us to do this with all our community-acquired mrsa isolates but it may be possible for those with pvl. this toxin is carried on a prophage and is therefore potentially mobile. if we can prevent wide dissemination of the pvl genes we could perhaps limit the associated disease severity. the australian public is very aware of "the golden staph" tag by which the press has labelled mrsa. there is episodic press coverage, both print and television, of personal stories and individual hospital problems. however, understanding of antimicrobial resistance of any kind is minimal within the community. providing and disseminating relevant, understandable information that will change community and prescriber attitudes is a challenge that should be met by our profession, regulatory authorities, and politicians. i declare that i have no conflicts of interest. head of the department of microbiology and infectious diseases and associate professor of the school of biomedical sciences, curtin university, perth, australia japan japan has one of the highest prevalences of mrsa in the world. among s aureus bloodstream isolates in , nearly % were meticillin resistant. to control mrsa in hospitals, it is essential to identify potential mrsa patients immediately after admission. since microbiological testing usually takes - days, the identification of those who are at high risk for mrsa carriage is required to decide whether proper precautions should be taken for each new patient. no mechanism to identify patients within hospitals currently exists in japan-there is no general database that includes previous medical and laboratory results of patients that can be shared by clinicians between different hospitals. in addition, people can attend any hospital they choose-from a large teaching hospital to a general hospital-without an appointment. without mrsa screening on admission, this situation makes it difficult to know whether the patient has already been colonised or infected with mrsa in the past. ignorance about mrsa is a huge problem among the public and media in japan. although there is some interest in the subject, the risks and safety issues surrounding mrsa are generally not that well known. mrsa carriage is seen as a "stigma" for patients and because colonisation with mrsa is not usually differentiated from an infection, the media are quick to generate a scandal and attach blame to hospitals and doctors when mrsa is reported. hence, most hospitals tend to refuse mrsa-positive patients to avoid potentially damaging repurcussions for the hospital. moreover, this situation can lead to clinicians hesitating to identify mrsa carriage of patients who are being transferred to another hospital. in many countries it is strongly recommended that clinicians at the transferring hospital should notify mrsa carriage status of the patient to the infection control team at the receiving hospital before transfer. however, this procedure has not been followed stringently in japan. an mrsa flagging system to highlight previously identified patients within the same hospital or upon readmission is expected to become available soon to most clinicians. this flagging system needs to be computerised and added on to the individual database in each hospital. however, it is still difficult to share microbiological results through a single medical computer network system. each hospital needs to develop mrsa flagging software for this purpose. although japanese hospitals are extensively computerised, the main purpose of the hospital computer system is for ordering and accounting for medical procedures. the data sorting function from previous laboratory results is usually optional. we have recently developed a small computer programme on our hospital network to indicate an "alert spot" on the individual patient record window. if a patient has a previous history of being mrsa positive, a redcoloured alert spot automatically appears on the computer display. this alert system helps us to recognise whether the patient is a potential or current mrsa carrier without checking any previous microbiological results, which is usually a tedious and time-consuming procedure for busy clinicians and hospital officers. this function will be introduced to a new hospital computer system in the future. ideally, these data will become indispensable information for inter-hospital transfers via a shared database that should be set up in the future. an alternate route of importing mrsa into hospitals is from the community. only a few studies of mrsa prevalence in the community have been published from japan. one study reported that out of ( · %) healthy children aged - years old carried mrsa in their anterior nares. this prevalence of community mrsa among children is high, and deserves further prevention efforts. mrsa surveillance is currently mandatory in large general and university hospitals but not in middle to small-sized hospitals that are the most common types of hospital in japan. for example, geriatric hospitals with less than beds are usually one of the most important reservoirs of mrsa. mrsa-positive patients are sent to us from these types of institutions without information of mrsa carriage. therefore, it is important that mrsa surveillance should be made mandatory by legislation. another less common strain of mrsa is visa, which was first reported in japan. although the hetero-visa strain (a possible precursor of visa) was identified in · % of mrsa isolates in seven university hospitals, a new visa strain has not been identified in japan so far, because therapeutic drug monitoring has become widely available from both "in-house" and commercial laboratories since . controlling the use of vancomycin means there is less chance for the emergence of vancomycin-resistant strains. educational awareness of mrsa should be provided to all health-care workers as well as the general public, and should be supported by the government or relevant associations and academic societies. mrsa carriage should be notified properly when the patient is transferred from one hospital to the next. senior lecturer, department of infection control science, juntendo university, graduate school of medicine, tokyo, japan finland is a country with low mrsa incidence. the incidence figures are based on mandatory reporting by clinical microbiological laboratories of each new mrsa isolation since and analysis of mrsa strains at the national reference laboratory at the national public health institute. in recent years, however, we have seen a worrying increase in the number of mrsa cases. the number of reported cases has increased from in to in (from · to · cases per ) in a population of · million. for many years mrsa remained below % among invasive cases, but during we saw the first signs that the figures were worsening substantially. at the same time, the coverage and efficacy of mrsa reporting has remained stable, thus we assume that we are facing a true change. strict mrsa prevention measures are taken following each newly identified mrsa case, regardless of whether it is symptomless carriage or infection. in response to three large mrsa epidemics in southern finland, national mrsa prevention consensus guidelines were adopted in . the updated version of these guidelines was published in . the new guidelines cover both acute care hospitals and long-term health-care facilities, including homes for elderly people. the main principles are promotion of hand disinfection, identification of mrsa risk patients (carriers and contacts of known mrsa cases), rapid microbiological identification of mrsa, contact isolation of mrsa cases, and treatment of mrsa infections. emphasis is placed on trying to target preventive measures at the very first steps of possible mrsa transmission. according to the guidelines, every patient who has been treated in a hospital outside of finland during the previous year and is transferred to a finnish hospital is screened for mrsa colonisation at the time of hospital admission. patient records of each mrsa case are labelled accordingly, and an "mrsa alert" is also added to electronic patient records in many institutions. these actions have proven to be effective in controlling several rather large hospital mrsa outbreaks in the s. some of the international mrsa clones have also repeatedly entered our country via patients or health-care workers who have come from abroad, but they have caused few secondary cases or outbreaks. during the past couple of years, the mrsa situation has changed in finland. in - , one-fifth of our cases showed no contact with the health-care system, and were thus considered community-acquired mrsa. usually, these mrsa strains possess only beta-lactam resistance, carry the sccmec type iv genes, and cause problems in younger age groups. however, the proportion of elderly people with mrsa has increased steadily. in , over % of cases were found in individuals aged years or older. in , over half of finnish mrsa cases were reported in long-term care facilities, and outbreaks are forum occurring in these facilities. these two settingscommunity acquisition and long-term care facilitiescreate new and demanding challenges to our mrsa prevention policy. the finnish health-care system is currently struggling with increasing output demands and decreasing funding. the nursing staff is often overwhelmed with work, and patient wards are crowded and often lack single-bed isolation rooms, creating problems for infection control. in long-term care facilities, we are witnessing the lack of trained personnel and problems in finding functional placement options for mrsa carriers. there has been a considerable amount of debate about the ethical right to isolate mrsa carriers in the nursing home setting. it is difficult to find a solid answer that would hold for every case. in our decisions about mrsa-positive people we try to respect two basic principles: the patient's right for proper medical treatment and the individual's right to live a normal life. the actions taken now and over the next few years are crucial in determining how the trend of mrsa in finland progresses. at present, we still have the chance to prevent the situation from becoming much worse. in , the finnish government launched a fund for the health districts to update their resources on emerging hospital infection issues, including mrsa prevention. sweden, denmark, norway, iceland, and finland have also started a joint initiative, provoked by the scandinavian society for antimicrobial chemotherapy, to try to find ways to keep mrsa levels below % among s aureus isolates in each country. at the very least, this cooperative effort will mean that information on the mrsa situation is exchanged, attempts are made to improve mrsa laboratory diagnostics, common reasons for why mrsa is emerging sought, and attempts made to increase awareness among the public and health-care workers. in countries where mrsa case levels have remained low, the only right and ethical decision is to try to fight hard to keep the situation from becoming worse. this battle requires new investments now, but it will save money and resources in the future. chief physician, hospital bacteria laboratory, department of bacterial and inflammatory diseases, national public health institute, helsinki, finland turkey mrsa emerged in turkey in the s as a major clinical problem in hospitals, and to date has continued to be one of the most problematic nosocomial pathogens. the extent of resistance varies nationally, regionally, and even institutionally. in addition to meticillin, these strains are also resistant to routinely used antimicrobials, and thus infections caused by such isolates cause serious treatment difficulties. unfortunately for turkey, accurate and recent population-based national surveillance for communityacquired and hospital-acquired drug-resistant microorganisms does not exist. however, data from sporadic reports of the number of cases suggest an urgent need for surveillance. available reports provide a fragmented and incomplete picture to guide our understanding of the problem. the most recent and only national resistance screening programme data supported by the turkish scientific and technological research council is from . in this study, clinical isolates both from outpatient and inpatient clinics (including surveillance isolates) from different hospitals were collected and screened. overall, mrsa resistance varied from % to % at different centres. this range of resistance may have been due to variations in patient population, hospital care practices, and infection control activities. other factors-eg, size, locality, and type of hospital-were also contributing factors to the wide disparity in resistance. among the identified isolates, nosocomial strains were randomly selected for evaluation of the mechanisms of meticillin resistance, and the presence of meca was found to be the most frequent type ( %). when various other studies from different centres were evaluated up to , it was noted that up to % of strains in some places were mrsa. in a further evaluation extending to , this figure rose to %. more recently, data from the past years from individual centres across turkey state the percentage of mrsa strains is · %. [ ] [ ] [ ] [ ] when tested, all strains were reported to be vancomycin and teicoplanin susceptible but potential emergence of resistance to these drugs should be kept in mind, since this has prompted the overuse of glycopeptides in empirical and even prophylactic therapy. , evaluation of reports showed no correlation between mrsa rates and sex, profession, hospital department, or carriage. it was difficult to evaluate the age distribution and age group at risk. ideally at each hospital multidrugresistant strains should be regularly identified. clonal typing methods should be used to identify the relations between these strains, and to see which strains predominate in the country. we evaluated our hospital's main nosocomial mrsa clone and tried to track the sources of the clonal types in different clinics by pulsed field gel electrophoresis. one major type was identified among their strains. this single clone was compared with another turkish university hospital's clone and several main international clones including the iberian and brazilian clone, and it was found to be different from them. predominance of this clone or any other clone is not known. this type of evaluation needs to be done by most hospitals. with enough funding a national netherlands yes, they do not like to see us coming: physicians, nurses, and hospital administrators may shrink away when the banner of mrsa is raised by the hospital infection-control practitioner-it always means expensive and timeconsuming isolation, may mean temporary removal of staff, or the partial or complete locking of wards that have to stop admissions. yet the incidence of mrsa in the netherlands remains one of the lowest of europe. in , mrsa was isolated from people, one-fifth of whom were health-care workers, which means that approximately patients newly acquired mrsa in the course of a year. to put this number into perspective, the netherlands has million inhabitants, hospitals, and approximately million patient-days of stay in hospital. a timely implementation of a national policy of stringent control measures, and a long-standing tradition in parsimonious antibiotic use surely contributed to keeping mrsa at bay. as in many other countries, after a short appearance in the late s, mrsa entered the netherlands in the early to mid- s. three large hospitals in rotterdam, amsterdam, and utrecht experienced outbreaks that were eventually controlled by strict infection-control measures that included isolation of patients, screening of patients and hospital staff members, and closure of wards. both in amsterdam and in utrecht, mrsa was introduced by a patient who had been transferred from a hospital abroad. these early experiences prompted the dutch working party on infection prevention (wip) to formulate national mrsa guidelines. this working group is funded by the ministry of health, and its task is to develop guidelines for infection prevention in hospitals, nursing homes and institutions for the mentally handicapped, and dental care and homecare. the guidelines issued by the wip are considered professional standards and are used as such by the dutch public health inspector; this undoubtedly contributes to the adherence to the guidelines by nearly all health institutions. the mrsa guidelines are based on three main principles. first, patients with mrsa are always isolated in single rooms, whether they have an active mrsa infection or not-carriers are also considered potential sources of transmission. isolation for being a carrier raises anxiety in the patient and their relatives. however, % of adults are staphylococcal carriers, and they develop an infection only occasionally, so, in itself, carriage of a staphylococcus that has additional resistance is not a source of worry for the individual patient. second, patients suspected of potential carriage are always placed in isolation; potential carriage is considered in all patients transferred from hospitals abroad to dutch hospitals, in patients from dutch hospitals, or from nursing homes with an actual problem of mrsa, and in patients who have been nursed in the same room as a patient in whom mrsa is detected unexpectedly. this isolation may come forum epidemiological surveillance group should be established. data emerging from various centres indicate that in turkey-like many other countries-both infections and the rate of mrsa is gradually increasing. the main factors that contribute to this organism's resistance are misuse and overuse of antimicrobials, which include constant pressure on the physician to prescribe antimicrobials when they are not indicated, the patient's failure to finish a prescribed antibiotic regimen, and also the availability of antimicrobials without a prescription. we believe that control of such factors, alongside implementing good infection-control procedures in turkey, will result in decreasing rates of mrsa. studies from other countries have shown that surveillance activities-eg, setting up a national/regional system to collect and evaluate data from all centres and updating data on antibiotic resistance patterns that will form the basis for prevention guidelines of nosocomial infections-decrease infection rates by as much as %. the negative impact of nosocomial infections on our health-care system is not well documented. data obtained from limited number of studies are not suitable for comparison with each other and neither are data from other countries. thus, a national nosocomial infection control project (nosoline), supported by the turkish hospital infections society, has recently started at the hacettepe university hospital, ankara. the project's aim is to develop a regular, standardised surveillance system, allowing electronic data transfer and online announcement of the results to the member centres. to achieve its goals, the problems of gaining financial support and recruiting qualified personnel for data evaluation and processing need to be solved, and additionally all major hospitals will need access to the system. despite the absence of extensive data, reports of recent percentages of up to % of nosocomial mrsa infections confirm the urgent need for an effective antimicrobial usage policy for our country. to prevent resistant isolates spreading within and between hospitals, proper infection control procedures should be enforced, which currently most hospitals lack. in time we hope to prevent the uncontrolled availability of antimicrobials, increase awareness of such problems to physicians and the community, and have more available funds to support programmes in hospitals. we declare that we have no conflicts of interest. as a shock to people who become ill abroad, are repatriated, and arrive with a sigh of relief in a hospital in the netherlands, only to be faced with strict isolation measures, where even the family members have to wear gowns and masks upon entering the patient's room. an often-heard reaction is "am i a leper?", and it may be taxing to the attending physician to explain the reasoning behind the precautionary measure. third, contacts of patients with mrsa-both other patients and hospital staff-are screened for mrsa carriage and treated with mupirocin nasal ointment if found positive. this screening can prove trying for staff, because they are not allowed to return to work unless negative. fortunately, mrsa is usually quickly lost in normal community life or upon treatment with mupirocin nasal ointment. on occasion, a staff member proves to be a "stubborn" carrier, and several courses of mupirocin and the use of disinfectant soap are needed to clear the carriage. this upsets personal life and the working schedules of staff colleagues, who have to fill in for their colleague during the period he or she is not allowed to work. fortunately, untreatable carriage is very unusual, but may lead to the necessity to change job. the wip guidelines for mrsa are also published in english and can be found at http:// www.wip.nl. this very active searching policy implies that many of the mrsa strains that are identified in dutch hospitals are not actually causing infections, but are merely colonisers at the moment that they are detected. indeed, of the strains isolated in the past year, one-fifth were from health-care workers, who very seldom have an active infection when they are screened; the remainder were from patients who may have had an active infection or may just have been colonised. in the netherlands, mrsa occurs mainly in isolated cases or in smaller outbreaks, and it is not yet endemic in any hospital. because of this, it is possible to institute strict control measures whenever needed, although every time they cause great upheaval in the implicated ward, which has to isolate patients and screen patients and staff. the decreased therapeutic options, the nightmare of impending vancomycin resistance, and the higher numbers of therapeutic failures that accompany infections with mrsa provide a firm ground for the dutch policy. it is therefore strongly supported by the inspectorate of health care and by all microbiologists, infectious disease specialists, and infection-control officers involved. there is the odd dissenting voice of a surgeon or intensive-care physician who points to his colleagues in the uk or the usa where mrsa is rampant-"medical life is possible over there isn't it?" part of the unease of physicians is the result of misunderstandings and some "urban myths"-more appropriately "hospital myths"about mrsa. for instance, surgeons may think that they cannot operate on a patient who carries mrsa. nothing could be further from the truth. yes, elective surgery might be postponed, but all necessary surgery should go on as planned, with extra precautions. again, it is too often forgotten that % of all adults are staphylococcus carriers, and that the carriage in itself does no harm. up to now there have been neither legal nor ethical qualms about the dutch "search, isolate, and destroy" strategy, as in the end, all sections of medical life in the netherlands-physicians, nurses, and administratorssee the value of prevention. after careful explanation, even if some measures may tax the ingenuity of administrators and staff, the experience is almost invariably one of excellent cooperation, and most people involved enjoy the process of working for the greater good. although the exact burden of disease caused by mrsa remains largely unknown, most experts would agree that mrsa infections represent an important clinical and public-health problem. we hardly need to call readers' attention to the thousands of articles published over the past three decades about epidemiological and microbiological aspects of mrsa. yet uncertainty remains about the best approach to prevent and control this worldwide plague. in this issue of the lancet infectious diseases, several authors offer insight into mrsa control approaches used in different areas of the world. countries like finland, denmark, and the netherlands have managed to keep mrsa at a low level using surveillance cultures of patients and personnel, strictly enforced contact precautions, and judicious use of broad-spectrum antibiotics. unfortu-nately, these countries are facing now the paradoxical situation that transmission of community-acquired mrsa may jeopardise their well-established strategies to control nosocomial mrsa. denmark, for instance, has seen a substantial increase in mrsa since , due to the epidemic spread of genetically distinct communityacquired mrsa strains. conversely, many middleincome countries (eg, turkey, argentina) and some highincome countries (eg, italy, greece, uk, usa) that were not able to install stringent counter-measures now have hyper-endemic mrsa and are obliged to concentrate available resources to prevent mrsa infections in highrisk populations-eg, dialysis, transplant, or critically ill patients. a few countries with endemic mrsa (eg, australia, france, belgium) have managed to stabilise or even decrease mrsa prevalence in confined geographic areas. on the other hand, several asian countries (eg, china, south korea, japan) have more or less ignored this public-health problem for a long time, resulting in some of the highest mrsa incidence rates worldwide. in these countries, financial incentives for physicians' antibiotic prescribing linked to the pharmaceutical reimbursement system have strongly influenced antibiotic overuse and increased antibiotic selection pressure on mrsa; an issue that has only recently been adequately addressed at the policy level. why do mrsa rates vary so much across countries? differences are caused largely by uneven control and isolation measures, hand hygiene practices, antibiotic prescribing behaviours, and allocation of resources. cultural and economic factors pervade all aspects of mrsa control, which can only be fully successful if strict measures and policies are installed at an early stage of mrsa dissemination, sufficiently supported by financial and staff resources. especially at the early phase of a nationwide mrsa epidemic, the full clinical impact of mrsa may not be visible, leading to misconceptions among clinicians and policy makers that mrsa may not be a threat to patient safety. do we have any hope for the future? as mrsa surveillance systems and control strategies improve in quality and become more coherent among different countries, international pressure may start to be applied to induce change in countries where infection-control policies are lax or non-existent. the situation with mrsa might become comparable to that observed for other infectious problems such as severe acute respiratory syndrome and mad cow disease-economic and political pressure may contribute to compliance and uniformity in control measures and to allocation of resources to improve patient safety. yet stringent mrsa control worldwide will remain difficult to implement and will require intensive surveillance efforts and substantial resources. to achieve this goal may be possible, as shown by several examples where successful action against mrsa has been endorsed by strong policy support. adequate hand hygiene decreases the transmission of mrsa, although the practice is difficult to enforce, because of psychological, practical, and organisational barriers. promoting hand hygiene to improve patient safety and decrease health-care-associated infections worldwide constitutes a core component of the first global patient safety challenge ("clean care is safer care") of the who world alliance for patient safety launched in . if successful, clean care is safer care will certainly have a positive impact on mrsa transmission and other antibiotic-resistant infections. low mrsa prevalence in a country is good news in that preventive measures are more likely to succeed than if endemic mrsa levels are already present. unfortunately, for key questions regarding the most cost-effective control of endemic mrsa, we have only weak or contradicting evidence. several well-conducted studies from france, germany, the uk, and the usa have recently illustrated this dilemma. [ ] [ ] [ ] [ ] [ ] whatever the final outcome of this ongoing debate, health authorities and policy makers are well-advised to put effort and money into their mrsa control efforts. mrsa is everybody's business, not only that of hospital epidemiologists and a few opinion leaders. national nosocomial infections surveillance (nnis) system report, data summary from predicting methicillin resistance and the effect of inadequate empiric therapy on survival in patients with staphylococcus aureus bacteremia occurrence and antimicrobial resistance pattern comparisons among bloodstream infection isolates from the sentry antimicrobial surveillance program outcomes analysis of delayed antibiotic treatment for hospital-acquired staphylococcus aureus bacteremia the impact of methicillin resistance in staphylococcus aureus bacteremia on patient outcomes: mortality, length of stay, and hospital charges adverse clinical and economic outcomes attributable to methicillin resistance among patients with staphylococcus aureus surgical site infection surgical site infections associated with methicillin-resistant staphylococcus aureus: do postoperative factors play a role? epidemiological and microbiological characterization of infections caused by staphylococcus aureus with reduced susceptibility to vancomycin vancomycin-resistant staphylococci and enterococci: epidemiology and control relationship of mic and bactericidal activity to efficacy of vancomycin for treatment of methicillin-resistant staphylococcus aureus bacteremia persistent bacteremia due to methicillin-resistant staphylococcus aureus infection is associated with agr dysfunction and low-level in vitro resistance to thrombin-induced platelet microbicidal protein clinical cure and survival in gram-positive ventilator-associated pneumonia: retrospective analysis of two double-blind studies comparing linezolid with vancomycin communityacquired methicillin-resistant staphylococcus aureus in children with no identified 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long-term program for controlling methicillin-resistant staphylococcus aureus in intensive care units infectious diseases society of america emerging infections network. management of inpatients colonized or infected with antimicrobialresistant bacteria in hospitals in the united states are active microbiological surveillance and subsequent isolation needed to prevent the spread of methicillin-resistant staphylococcus aureus? harmonisation of pulsedfield gel electrophoresis for epidemiological typing of methicillinresistant staphylococcus aureus by consensus in european laboratories and its application for tracing the spread of related strains methicillin-resistant staphylococcus aureus: a modern epidemic. evidence-based healthcare and public health systematic review of isolation policies in the hospital management of methicillin-resistant staphylococcus aureus: a review of the literature with epidemiological and economic modelling revised guidelines for the control of methicillin resistant staphylococcus aureus infections in hospitals automatic alerts for methicillinresistant staphylococcus aureus surveillance and control: role of a hospital information system comparison of community-and health care-associated methicillin-resistant staphylococcus aureus infection dissemination of meticillin-resistant staphylococci among healthy japanese children meticillin-resistant staphylococcus aureus clinical strain with reduced vancomycin susceptibility dissemination in japanese hospitals of strains of staphylococcus aureus heterogeneously resistant to vancomycin ohje metisilliniresistenttien staphylococcus aureusten torjunnasta communityacquired methicillin-resistant staphylococcus aureus changing epidemiology of methicillin-resistant staphylococcus aureus in finland suppression of ␤-lactam antibiotic resistance in a meticillin-resistant staphylococcus aureus through synergic action of early cell wall inhibitors and some other antibiotics resistance to meticillin of staphylococcus in turkey. th turkish microbiology and infectious diseases congress prevelance of resistant gram-positive infections protein profiles and prevalence of meticillin resistant staphylococcus aureus (mrsa) in gülhane military academy hospital in turkey the evaluation of nosocomial infection during -year-period in the burn unit of a training hospital in istanbul trends in antimicrobial resistant staphylococci in an university hospital over a -year period nosocomial infections in intensive care unit in a turkish university hospital: a -year survey new aspects of antimicrobial resistance and the resultings therapeutic dilemmas specific detection of meticillinresistant staphylococcus species by multiplex pcr molecular genotyping of meticillin-resistant staphylococcus aureus strains in a teaching hospital in turkey testing the efficacy of a molecular surveillance network: meticillin-resistant staphylococcus aureus (mrsa) and vancomycin-resistant enterococcus faecium (vref) genotypes in six hospitals in the metropolitan new york city area socioeconomic burden of nosocomial infections hospital infections: where are we? epidemiology of emerging methicillin-resistant staphylococcus aureus (mrsa) in denmark: a nationwide study in a country with low prevalence of mrsa infection control of multiply resistant cocci: do international comparisons help? antimicrobial resistance determinants and future control isolation of patients in single rooms or cohorts to reduce spread of mrsa in intensive-care units: prospective two-centre study eradication of methicillin resistant staphylococcus aureus by "ring fencing" of elective orthopaedic beds are active microbiological surveillance and subsequent isolation needed to prevent the spread of methicillin-resistant staphylococcus aureus? effectiveness of a hospitalwide selective screening programme for methicillin-resistant staphylococcus aureus (mrsa) carriers at hospital admission to prevent hospital-acquired mrsa infections successful long-term program for controlling methicillin-resistant staphylococcus aureus in intensive care units we declare that we have no conflicts of interest. infection control program, department of internal medicine, university of geneva hospitals, switzerland key: cord- -vxaqizkj authors: bouquet, jerome; tabor, david e.; silver, jonathan s.; nair, varsha; tovchigrechko, andrey; griffin, m. pamela; esser, mark t.; sellman, bret r.; jin, hong title: microbial burden and viral exacerbations in a longitudinal multicenter copd cohort date: - - journal: respir res doi: . /s - - - sha: doc_id: cord_uid: vxaqizkj background: chronic obstructive pulmonary disease (copd) is a heterogeneous disease characterized by frequent exacerbation phenotypes independent of disease stage. increasing evidence shows that the microbiota plays a role in disease progression and severity, but long-term and international multicenter assessment of the variations in viral and bacterial communities as drivers of exacerbations are lacking. methods: two-hundred severe copd patients from europe and north america were followed longitudinally for years. we performed nucleic acid detection for respiratory viruses and s ribosomal rna gene sequencing to evaluate the bacterial microbiota in sputum samples collected at stable, acute exacerbation and follow-up visits. results: similar viral and bacterial taxa were found in patients from the usa compared to bulgaria and czech republic but their microbiome diversity was significantly different (p < . ) and did not impact exacerbation rates. virus infection was strongly associated with exacerbation events (p < e- ). human rhinovirus ( . %), coronavirus ( . %) and influenza virus ( . %) constitute the top viral pathogens in triggering exacerbation. moraxella and haemophilus were -fold and . -fold more likely to be the dominating microbiota during an exacerbation event. presence of proteobacteria such as pseudomonas or staphylococcus amongst others, were associated with exacerbation events (or > . ; p < . ) but more strongly associated with exacerbation frequency (or > . ; p < e- ), as confirmed by longitudinal variations and biotyping of the bacterial microbiota, and suggesting a role of the microbiota in sensitizing the lung. conclusions: this study highlights bacterial taxa in lung sensitization and viral triggers in copd exacerbations. it provides a global overview of the diverse targets for drug development and explores new microbiome analysis methods to guide future patient management applications. chronic obstructive pulmonary disease (copd) is defined by airflow limitation but encompasses several lung diseases. this heterogeneity includes differences in clinical characteristics, source of inflammation, response to therapies and causes of exacerbation [ ] . as copd progresses, exacerbations become more frequent and more severe. exacerbation rates reflect an independent susceptibility phenotype [ ] , which could be mediated by host factors [ ] , environmental factors [ ] , viral infections and/or the bacterial microbiome [ , ] . infections are predominant causes of copd exacerbations, with approximately half reported to be caused by bacterial infections including non typeable haemophilus influenzae (nthi), moraxella catarrhalis, streptococcus pneumoniae, or pseudomonas aeruginosa, and the other half by viral infections, primarily human rhinovirus (hrv), but also influenza virus, coronavirus and respiratory syncytial virus (rsv) to name a few [ , ] . bacteria and viruses are also frequently isolated in the airways of stable copd patients [ ] [ ] [ ] . the advent of culture-independent testing has suggested viral persistence [ ] and colonization of the lower airways with a resident bacterial microbiota [ ] , implicating a role for the microbiota in disease pathogenesis, progression and treatment outcome of lung diseases [ ] . studies of the microbiome provide a new framework to understand host-pathogen interactions, which can also yield new markers for patient diagnosis and management. microbiota diversity is seen as a potential biomarker in cases where a single pathogenic organism reduces community complexity such as in bacterial vaginosis [ ] , or crohn's disease [ ] . lung microbial dysbiosis in copd is characterized by decreased diversity [ ] [ ] [ ] , which may contribute to altered immune response to environmental insults [ ] . dysbiosis at the time of copd exacerbation contributes to increased disease severity [ ] and higher -year mortality rates [ ] . geography could also be a potential covariate in copd patients microbiota. the gut microbiota has been shown to be geographically variable [ ] . previous studies in different conntries have evaluated the copd microbiota [ , ] , but the effect of geographical variations has not yet been evaluated in a single study in relation to disease severity. the present cohort stems from a study on the incidence of viral infections in copd [ ] . patients were enrolled in europe and north america and followed up for up to rsv seasons, with scheduled wellness visits and unscheduled illness visits. to further our understanding of copd exacerbation dynamics, we retrospectively evaluated the sputum bacterial microbiota from a subset of this study. the goals were to identify differences in patients with higher rates of exacerbations, to assess geographical differences in the microbiota between europe and the usa, and to determine the influence of viral infections on microbiota diversity and the frequency of exacerbations. the patient cohort is part of an observational study on the incidence of acute respiratory illness (ari) or events leading to the worsening of cardiorespiratory status in copd (clinicaltrials.gov, nct ) [ ] . the protocol was approved by independent institutional review boards, and all subjects signed written informed consent at enrollment. the study population included adults ≥ years of age with copd, recruited at sites in bulgaria, sites in czech republic, and sites across the usa from fall to spring (fig. a) . cohort from bulgaria and czech republic showed similar characteristics, and are analyzed jointly as europe for fig. sampling timeline and composition. (a) timeline from oct to may , copd patients were enrolled and sampled during scheduled wellness visits (blue arrows), and any unscheduled visits (red and purple arrows) within days of an acute exacerbation or exacerbation follow-up visit. samples were considered stable if collected days post-hospitalization or ari. dotted arrows correspond to samples collected at scheduled wellness visits that incidentally corresponded to acute exacerbation events ( . %) and exacerbation follow-up visits ( %). piecharts represent the proportion of patients from europe and usa (b), the proportion of samples collected at each disease state (c) and the proportion of samples associated with antibiotics (abx) and inhaled corticosteroids (cs) taken in the past days (d) ease of representation. subjects had scheduled wellness visits in may and october each year to obtain sputum and clinical data (fig. a) . unscheduled illness visits to collect sputum and clinical data were performed when a subject experienced an ari or acute exacerbation of copd symptoms, and during follow-up illness visits. samples were considered stable if collected at least days from the last day of hospitalization or from the last ari or acute exacerbation event if it did not require hospitalization. acute exacerbation samples were collected within h of an event, additional samples were collected - days after an acute exacerbation event during unscheduled visits (fig. a, c) . patients were characterized with a frequent exacerbator phenotype if they experienced or more exacerbation events per year. no investigational drug was administered in the study. the subject's physician prescribed and recorded all treatment deemed necessary to provide adequate supportive care. samples collected to days following treatment were considered treatment associated (fig. d) . spontaneous rather than induced sputum collection was possible in this severe copd study subset following a standardized collection visit [ ] . subjects were asked to gargle with water immediately prior to sputum collection to reduce the number of oral bacteria [ ] . subjects were asked to cough deeply and expectorate into a cup, mixed : with cold transport media and kept at - °c or below [ ] . the genmark respiratory virus panel (genmark diagnostics, inc. carlsbad, ca) was used to detect common respiratory viruses from all sputum samples. rsv detection was confirmed by rt-pcr as previously described [ ] . additionally, rt-pcr using primers against hrv vp -vp [ ] was used to detect human rhinovirus subtypes. bacterial genomic dna was extracted at a single central lab using the zymobiomics dna kit (zymo, california, usa) following manufacturer's instructions. the v hypervariable region of the s rrna gene was pcr amplified [ ] and sequenced using illumina miseq platform along with negative controls, zymobiomics microbial community and dna standards, and a phix library for quality control. s rrna gene sequences were analysed using qiime version . [ ] . dada software package [ ] , wrapped in qiime , was used for correcting sequences and obtained annotated sequence variants (asvs). taxonomy was assigned using a naïve bayes classifier [ , ] that was trained on the greengenes database version . clustered at % identity [ ] v sequences. alignment was performed with mafft [ ] , masked and used in fasttree [ ] to build the phylogenetic tree. alpha-diversity metrics [ ] , beta diversity metrics [ ] , and principal coordinate analysis (pcoa) were estimated after samples were rarefied to sequences per sample. significant features of interest were re-tested using non-rarefying alpha [ ] and beta diversity estimates [ ] . analysis of composition (ancom) was used for differential taxa abundance calculations [ ] on nonrarefied data. ancom account for the structure of the data and controls for the false discovery rate. microbiota profiles were clustered into biotypes using the biotyper package in r [ ] . longitudinal microbiota variations at stable time points were assessed by calculating the median weighted unifrac distance [ ] for patients with at least stable samples. patients were categorized into the bottom and top quartile of within-patient stable samples median unifrac distances, respectively. associations of the microbiota and viral components with demographics and clinical data were calculated in r using fisher's test for categorical variables, welsh's ttest for comparison of means in continuous variables, and chi-squared test to compare expected frequencies. odds ratio were calculated using questionr package in r [ ] . associations of microbial diversity with demographics and clinical data were calculated with qiime 's diversity plugin using grouped and pairwise kruskal-wallis test corrected for false discovery rate for analysis of alpha diversity, and permanova following permutations for analysis of beta diversity distances. the cohort was composed of patients, from europe (bulgaria and czech republic) and from the usa ( fig. and table ). all but patients presented with severe or very severe copd. patients from europe and the usa were matched by age, sex, and gold stage. at enrollment, their forced expiratory volume as a percent of predicted (fev %) and their comorbidities (congestive heart failure, diabetes, hypertension and malignancy) were similar between europe and the usa. the usa patients had significantly higher smoking history in pack-years and longer copd duration. the number of exacerbations per year and the number of frequent exacerbator phenotype, defined as or more exacerbations/year, was higher in the usa compared to europe, but not statistically significant (table ) . a total of sputum samples were collected from these patients over years, with similar proportions of samples collected at acute exacerbation in europe ( . %) and in the usa ( . %) (supplementary table ). approximately and % of samples were associated with antibiotic and corticosteroid treatment, respectively. significantly more samples collected in the usa were associated with antibiotic treatment (supplementary table ). analysis of bacterial taxa in sputum samples shows phyla commonly observed in the lung microbiota, with firmicutes, proteobacteria and bacteroidetes representing a majority (> %) of the phyla identified (fig. a) . prevotella, veillonella, streptococcus and haemophilus represented the most prevalent (> %) bacterial genera (fig. a) . samples from european patients had more bacteroidetes and less proteobacteria overall than samples from the usa patients. within the phylum firmicutes, the usa patients had more streptococcus than veillonella compared to european patients (fig. a) . microbiota predominant with prevotella, streptococcus and veillonella were found in a majority of samples ( / ; . %) (fig. b) . prevotella, streptococcus and veillonella represented the majority of the microbiota in samples collected at stable states, haemophilus and moraxella were predominant in acute exacerbation samples, and pseudomonas was prevalent in exacerbation follow-up samples (fig. b ). viral testing on sputum samples showed that . % of sputum samples were positive for at least one virus, with hrv found in the largest proportion of samples ( . %), followed by coronavirus ( . %) and influenza virus ( . %) (fig. c ). all viruses, except for adenoviruses, were detected more frequently at acute exacerbation ( . %), than at follow-up visits ( . %) or stable ( . %) (fig. c ). there were no significant differences in viral incidence between europe and the usa, with the exception of coronavirus hku , influenza b virus and rsv a (supplementary table ). alpha diversity metrics such as the number of observed asv, shannon evenness, or faith phylogenetic diversity (pd) indexes, represent the mean number of taxons in a , and their respective shannon diversity index. * p < . , **p < . , *** p < . , lower and higher statistical significant diversity compared to the average are noted in red and blue, respectively. hmpv, human metapneumovirus sample, sometimes weighted by the phylogenetic relatedness. overall, samples from the usa patients had significantly lower shannon diversity index than those from europe (fig. a) , even when corrected for antibiotic use or clinical sites (supplementary fig. and ) . years of severe copd and smoking pack-years were associated with differences in microbiota diversity but not as significantly as geography (supplementary table ). no significant differences in the number of observed asvs and in faith pd index were observed at exacerbation compared to stable samples (supplementary table ). prevotella-predominant microbiota showed the highest alpha diversity, while samples predominant with escherichia, haemophilus, and pseudomonas had the lowest alpha diversity (fig. b ). there were no significant differences in microbial diversity between samples infected or not with a virus, or between samples infected with different viruses (fig. c) . beta diversity are metrics such as weighted unifrac and robust aitchison pca used for comparing microbiota communities resulting in distance matrices. here, principal coordinate visualization of weighted unifrac distance supports the above conclusions, showing some differences in geography and predominant bacteria but not affected by the type of viral infection ( supplementary fig. ). these results were also supported by testing non-rarefied alpha and betadiversity estimates, with geography (p < . ) and dominant bacterial genus (p < . ), but not viruses (p > . ) being associated with significant changes in diversity (data not shown). the odds ratio of an acute exacerbation event and frequent exacerbations (≥ events/ year) was calculated for demographic and clinical data, viral infections and abundance of certain bacterial taxa in the lung microbiota (fig. ). viral infections were more strongly associated with an exacerbation event than with frequent exacerbations. parainfluenzaviruses (piv), influenza b virus and rsv b had the highest odds ratio of an exacerbation event (fig. a) . interestingly, influenza b virus was negatively correlated with frequent exacerbations, as it was only detected in patients that exacerbated infrequently. bacteria were more strongly associated with exacerbation frequency than with exacerbation events. presence or higher abundance of enterococcus, lactobacillus, moraxella, pseudomonas, staphylococcus and streptococcus was correlated with frequent exacerbations. neisseria, prevotella and veillonella were significantly associated with a lower exacerbation frequency (fig. b) . interestingly, top quartile abundance odds ratio of the genus haemophilus was not associated with higher exacerbation rate. this effect, however, seemed mediated by h.parainfluenzae. we noted that differential abundance of only bacterial taxa were significantly associated with exacerbation events, while taxa were associated with exacerbation frequency ( supplementary fig. ) , with high e.coli, lactobacillus, and staphylococcus in stable samples as potential predictors of frequent exacerbation ( supplementary fig. ). patients with hypertension had a significantly higher odds ratio of being frequent copd exacerbators (fig. c) . other comorbidities did not significantly influence exacerbation frequency. we quantified temporal variability of the sputum microbiota at stable state within individual subjects. for patients with more than longitudinal stable samples, we calculated their median weighted unifrac distances and categorize the top and bottom quartile patients into consistent and variable microbiota over time (fig. a) . patients with a more variable sputum microbiota (median weighted unifrac > . ) were more likely to have a higher relative abundance of bacillus, escherichia, lactobacillus, moraxella, and staphylococcus ( fig. and supplementary fig. ). microbiota variability in seemingly stable disease state were associated with higher exacerbation frequency and frequent viral infections (fig. b) . longitudinal sampling also enabled the assessment of recurrent viral infections by the same viral species, strain or subtype in consecutive samples. viruses were detected in to consecutive samples from patients (fig. ) . the time beween consecutive samples ranged from days to year. viruses that were detected within - weeks of sampling corresponded most probably to a typical single infection, and these included the detection of coronavirus oc , hrv a, piv , rsv a and b. viruses that were detected in the same patient over days apart may correspond to chronic or recurrent infections, with adenovirus c, coronavirus hku , hrv a, hrv b and hrv c detected. eighteen out of ( %) samples from these patients corresponded to acute exacerbation events, and / ( %) patients were frequent exacerbators (fig. ) . we sought to define common microbiota clusters and their association with clinical characteristics. microbiota in stable samples could be separated into biotypes, as indicated by the highest calinski-harabasz (ch) index following iterative partitioning-around-medoids clustering analysis over the jensen-shannon distance. betweenclass analysis showed major clusters, biotype represented by neisseria and veillonella, and biotype represented by streptococcus and rothia (fig. a) . samples with high relative abundance of streptococcus/rothia (biotype ) were found in greater proportion in usa patients, which was associated with longer history of copd and less frequent detection of viruses at stable visits (fig. a) . samples at acute exacerbation visits could be separated into biotypes, characterized by a high relative abundance of either prevotella (biotype ), streptococcus (biotype ), or haemophilus (biotype ) fig. risk factors of copd exacerbations. adjusted odds ratio of (a) viral infections (b) bacterial abundance (top/bottom quartile) and (c) demographics and clinical history features to be associated with acute exacerbation events or patients with frequent exacerbations (≥ events/ year). significance are presented in red (positive association) and green (negative association). orange dots represent non-significant odds ratio. horizontal bars represent the % confidence interval. genera names are in bold and species italicized (fig. b) . streptococcus and haemophilus were found in a majority of usa samples, and associated with longer copd duration, higher exacerbation frequency, antibiotics and corticosteroid use, but did not significantly correlate with higher viral infections (fig. b) . exacerbation follow-up samples were more diverse and could be optimally clustered into biotypes. biotype was characterized by a high relative abundance of pseudomonas and significantly associated with longer copd duration and antibiotic use. biotype was characterized by a high relative abundance of streptococcus or rothia and was significantly associated with higher exacerbation frequency. other biotypes were not significantly associated with clinical characteristics (fig. c) . biotyping shows that microbiota profile diversity is dynamically dependent on disease state. understanding of the presence and role of both bacterial and viral pathogens over time in the heterogeneous and dynamic copd disease [ ] is needed for patient treatment and management. the characterization of the s rrna gene microbiota and respiratory viruses from a longitudinal and international cohort of severe copd patients described in this study provides the largest survey to date on their complex associations with copd patients are particularly susceptible to respiratory infections [ ] . hrv was identified as the most prominent agent in respiratory tract infections in this cohort of copd patients. viral characterization is of particular importance as few reports exist on the diversity of respiratory viral agents in copd [ ] and viral diversity should be accounted for when designing new treatments. in particular, the newly described hrv c was detected in of , samples ( of patients) in the present cohort, which was reported only once previously [ ] . we detected viruses in % of stable samples. asymptomatic viral infections in copd patients are common [ ] . however, the role of these asymptomatic infections in disease progression is unclear. most respiratory viruses tested here showed highly significant positive odds ratio with exacerbation events, but lower significance in regards to exacerbation frequency. this may indicate that viral infections alone do not sensitize the lung to repeat exacerbations as much as the bacterial microbiota. interestingly, similar trends have been shown in asthma where respiratory viral infections in early life resulted in microbiome changes and hypersensitivity predisposition that can lead to asthma [ , ] . repeat viral detection were more frequent in patients with frequent exacerbator phenotype, but the number was small, and contradicting reports exist on the repeated detection of a single virus in copd [ ] . further complete viral genomic characterization will be needed to understand the nature of viral infection. with the advent of culture-independent techniques, it appears that all microbiomes harbor potential bacterial pathogens as characterized here and elsewhere [ ] , but that only a portion of them will develop exacerbation-prone phenotypes, a phenotype that appears independent of disease gold stage yet linked to microbiota diversity [ ] . it was previously observed that microbiota diversity in the copd lung correlated with disease severity but not disease state [ ] . here, microbiota diversity alone was not correlated with frequent exacerbations, but was highly correlated with certain bacterial taxa dominating the microbiota. microbiota predominant with escherichia, pseudomonas or streptococcus, showed significantly lower alpha diversity and significant positive odds ratio with the frequent exacerbation phenotype, suggesting a role of the microbiota in sensitizing the copd lung to acute exacerbations. this study was limited in disease severity metrics with the exception of baseline evaluation and sampling of events. longitudinal monitoring of symptoms scales would help to better understand the relation of certain bacteria to not only exacerbation frequency, but also the symptom severity and copd progression. the use of biotyping has been seldom used in respiratory microbiota research [ ] and not yet explored in copd. a complex resident bacterial community could be identified in all copd sputum samples and categorized into , and biotypes at stable, acute exacerbation and exacerbation follow-up visits, respectively. biotype at stable state was associated with higher viral infections, while biotypes and at acute exacerbation were associated with high exacerbation frequency. these findings are interesting because they mirror another study showing the partitioning of copd exacerbation samples into cytokine profile clusters [ ] with associations to specific ratios of proteobacteria, firmicutes and bacteroidetes that highlighted the heterogeneity of exacerbation profiles in copd patients. during exacerbation follow-up visits, biotype with a high relative abundance of pseudomonas was found over-represented in samples associated with antibiotics use. antibiotic treatment inadequacy is the cause for secondary infection or the emergence of multi-drug resistant p.aeruginosa [ ] . new targeted treatments, such as monoclonal antibodies, could be useful in such settings [ ] . the principal novelty of this study cohort was the long term patient follow-up. we were able to collect several sputum samples per patient at stable state over the course of years and studied the copd microbiota longitudinally. the lung microbiome is inherently variable, shaped by a process of inhalation and elimination [ ] . the lung microbiome is also personal, with large interpatient variability [ ] . previously, it was shown that microbial dysbiosis from stable to exacerbated state correlated with greater exacerbation severity [ ] . here, patients with greater microbiota variability at stable state correlated with higher exacerbation frequency. proteobacteria such as pseudomonas and moraxella were more abundant in patients with more variable microbiota at stable state. interestingly, p.aeruginosa and m.cattharalis are prominent causes of exacerbations [ ] , but their role in stable disease is less clear [ ] . it was previously shown that chronic colonization with p. aeruginosa occurs more frequently in more severe copd patients [ ] and that m.catarrhalis asymptomatic colonization was associated with a greater frequency of a sputum iga response than exacerbation [ ] . our results suggest that dysbiotic burden at stable state by pseudomonas, moraxella and others might sensitize the lung to further exacerbations and viral infections. pseudomonas and moraxella, like many opportunistic proteobacteria, are pro-inflammatory [ , ] . imbalanced inflammation can improve p.aeruginosa's fitness [ ] , allow the acquisition of new m.cattharalis strains [ ] , leading to exacerbation and possibly infections from other pathogens in a coupled cycle of inflammation and dysbiosis [ ] . microbiology clinical testing in copd patients is most often performed at exacerbation or follow-up visits. patients might benefit from clinical monitoring of these bacteria at stable state to assess their presence and/or growth which could lead to potential future exacerbations. we also noted geographical differences in copd lung microbiota. geographical differences in gut microbiota have previously been noted [ ] , but not yet in the lung. there were significant differences in alpha and beta diversity between the usa and europe, but not within countries or sites. microbiota diversity in the usa was lower and although frequent exacerbator phenotypes were more common than in europe, the difference was not significant. usa patients tended to have samples with high relative abundance of streptococcus (biotype ) and haemophilus (biotype ) associated with the frequent exacerbator phenotype. s.pneumoniae and h.influenzae are commonly associated with exacerbations [ ] , and should also be considered as potential risk factors in the frequent exacerbator phenotype. significantly more samples collected in the usa were associated with antibiotics use, but this alone did not explain differences in diversity. this observational study included a variety of standard-ofcare medications, doses and timings precluding precise treatment effect modeling on the microbiota. clinical trials exploring current and novel treatment modalities will lead to better patient management and antibiotics stewardship as reviewed elsewhere [ ] . predominance of haemophilus was over-represented in acute exacerbation samples (figs. and ), as noted in previous studies [ , , , , ] . however, interestingly, using odds ratio (fig. ) or ancom (suppplementary fig. a ) over haemophilus abundance, h.influenzae was high but not significantly associated with acute exacerbation event, whereas h.parainfluenzae was significantly higher at stable state. differences in haemophilus abundance compared to other studies might be due to the type of sputum collected, transportation media, extraction protocol or an effect of the sample size. although h.parainfluenzae can be the cause of respiratory infection in healthy subjects, it has not been associated with exacerbation in copd [ ] . h.parainfluenzae could compete for niche resources leading to overgrowth of the more pathogenic h.influenzae. previous work has shown that h.influenzae competes with s.pneumoniae [ ] and that patients colonized by nthi and acquiring hrv have more frequent and severe exacerbations [ ] . here, streptococcus species could not be resolved using s rrna v region, and although speciation of haemophilus was attempted, further validation using targeted pcr or whole genome sequencing will be necessary to ensure correct discrimination. to note, constant improvements in s databases can also affects taxonomic resolution. silva database [ ] version updated in classified reads into more genera (n = ) compared to greengenes version . updated in (n = ). however, bacterial taxa discussed in this study showed less than % variations in read classification between the database classifiers (data not shown), and conclusions were unchanged. speciations and typing of bacteria and viruses are critical to understanding their pathogenicity and complex relationships. greater taxonomic resolution will be achieved using updated databases and more comprehensive techniques like shotgun metagenomics. in summary, our study provides a broad survey of viruses and bacteria colonizing severe copd patients, providing clinicians with potential targets for clinical testing and patient treatment. it demonstrates that viral infections are strongly associated with acute exacerbation events, and that particular components of the microbiota are associated with higher exacerbation frequency. geographic and longitudinal differences in the lung copd microbiota exist and were correlated with exacerbation outcomes. stable state longitudinal microbiota monitoring and biotyping could lead to the identification of potential biomarkers indicative of future exacerbations from bacterial sensitization. comprehensive microbiota profiling and respiratory viral detection will be useful in the development of anti-microbial agents for therapeutic intervention or for better patient management. supplementary information accompanies this paper at https://doi.org/ . /s - - - . additional file : table s . sample characteristics. table s . statistical significance of alpha and beta microbiota diversity metrics against demographic and clinical variables using qiime 's diversity plugin. figure s . shannon diversity following antibiotic treatment in europe and usa patients. figure s . microbiota composition and diversity across study sites. figure s . principal coordinate analysis of weighted unifrac distances of the sputum microbiota colored by (a) geography and sample type, (b) most predominant bacterial taxa, and (c) viral infection. figure s . bacteria associated with copd exacerbation or frequency of exacerbation. (a) abundance of moraxella and h.parainfluenzae identified by analysis of composition (ancom) of microbiota between stable and exacerbated samples. (b) cladogram of bacterial taxa identified by ancom comparing frequent (> exacerbation event/ year) to infrequent exacerbator. figure s . adjusted odds ratio of bacterial abundance (top/bottom quartile) in stable samples only to be associated with frequent exacerbations. figure s . cladogram of differentially abundant bacterial taxa identified by ancom comparing consistent to variable longitudinal microbiota patient profiles at stable state. evaluation of copd longitudinally to identify predictive surrogate endpoints (eclipse) investigators. characterisation of copd heterogeneity in the eclipse cohort susceptibility to exacerbation in chronic obstructive pulmonary disease biological exacerbation clusters demonstrate asthma and chronic obstructive pulmonary disease overlap with distinct mediator and microbiome profiles factors associated with chronic obstructive pulmonary disease exacerbation, based on big data analysis respiratory syncytial virus-associated illness in adults with 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challenge for personalized medicine clinical and molecular epidemiology of human rhinovirus c in children and adults in hong kong reveals a possible distinct human rhinovirus c subgroup factors associated with symptomatic rhinovirus infection in patients with copd early-life respiratory viral infections, atopic sensitization, and risk of subsequent development of persistent asthma role of the airway microbiome in respiratory infections and asthma in children longitudinal profiling of the lung microbiome in the aeris study demonstrates repeatability of bacterial and eosinophilic copd exacerbations the nasopharyngeal microbiota of children with respiratory infections in botswana antibiotic treatment adequacy and death among patients with pseudomonas aeruginosa airway infection treatment efficacy of medi in pseudomonas aeruginosa blood stream infection and acute pneumonia rabbit models the role of the microbiome in exacerbations of chronic lung diseases analysis of the lung microbiome in 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on other inhabitants of the upper respiratory tract a prospective, observational cohort study of the seasonal dynamics of airway pathogens in the aetiology of exacerbations in copd the silva and "all-species living tree project (ltp)" taxonomic frameworks publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we would like to thank the study participants and the site investigators authors' contributions mte and mpg conceived the original study cohort. jb, det, brs, hj conceived the present study design. det and jss conducted viral testing. det and at conducted a microbiota pilot study. jb and vn conducted microbiota experiments. jb performed computational and statistical analyses. jb wrote the manuscript with critical revisions from det, jss, brs and hj. all authors read and approved the final version of the manuscript. this study was fully funded by astrazeneca.availability of data and materials data underlying the findings described in this manuscript may be obtained in accordance with astrazeneca's data sharing policy described at https:// astrazenecagrouptrials.pharmacm.com/st/submission/disclosure ethics approval and consent to participate this observational study is registered under nct at clinicaltrials.gov. the protocol was approved by independent institutional review boards, and all subjects signed written informed consent at enrollment. all authors are employees and share holders of astrazeneca. key: cord- - nyquokt authors: nemoto, manabu; schofield, warren; cullinane, ann title: the first detection of equine coronavirus in adult horses and foals in ireland date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: nyquokt the objective of this study was to investigate the presence of equine coronavirus (ecov) in clinical samples submitted to a diagnostic laboratory in ireland. a total of clinical samples were examined from equids with enteric disease in irish counties between and . a real-time reverse transcription polymerase chain reaction was used to detect ecov rna. nucleocapsid, spike and the region from the p . to p . genes of positive samples were sequenced, and sequence and phylogenetic analyses were conducted. five samples ( . %) collected in and tested positive for ecov. positive samples were collected from adult horses, thoroughbred foals and a donkey foal. sequence and/or phylogenetic analysis showed that nucleocapsid, spike and p . genes were highly conserved and were closely related to ecovs identified in other countries. in contrast, the region from p . and the non-coding region following the p . gene had deletions or insertions. the differences in the p . region between the irish ecovs and other ecovs indicated that the irish viruses were distinguishable from those circulating in other countries. this is the first report of ecov detected in both foals and adult horses in ireland. equine coronavirus (ecov) is a positive-stranded rna virus and belongs to the species betacoronavirus in the genus betacoronavirus [ , ] . the clinical signs associated with ecov infection during outbreaks in the usa [ ] and japan [ ] [ ] [ ] were fever, anorexia, lethargy and diarrhoea. the same clinical signs were also recorded in an experimental challenge study using japanese draft horses [ ] . the main transmission route is considered to be faecal-oral [ ] and ecov is usually detected in faecal samples. however, the molecular detection of ecov in faeces from horses with diarrhoea, does not prove causation. coronaviruses can cause both enteric and respiratory disease in many avian and mammalian species but ecov is less likely to be found in respiratory secretions than in faeces [ , ] . both molecular and seroepidemiology studies suggest that ecov may be more prevalent in the usa than in other countries [ ] . ecov was detected in samples collected from equids in states of the usa [ ] . in central kentucky, approximately % of both healthy and diarrheic thoroughbred foals were infected with ecov [ ] . all of the qpcr positive foals with diarrhoea were co-infected with other pathogens such as rotavirus or clostridium perfringens, suggesting that there was potential for ecov to be over-diagnosed as a causative agent in complex diseases. in contrast in japan, although an outbreak of diarrhoea occurred among ecov-infected draft horses at one racecourse [ ] [ ] [ ] , there have been no similar outbreaks subsequently, and all rectal swabs collected from diarrheic thoroughbred foals were negative. furthermore, only . % of the rectal swabs collected from healthy foals in the largest thoroughbred horse breeding region in japan were positive for ecov [ ] . in france, . % of faecal samples and . % of respiratory samples collected in counties tested positive for ecov [ ] . similar to the reports from japan and france, a low prevalence of ecov was also observed in the uk [ ] , saudi arabia and oman [ ] . the objective of this study was to investigate the presence of ecov in clinical samples submitted to a diagnostic laboratory in ireland. the samples were tested by real-time reverse transcription polymerase chain reaction (rrt-pcr) as it has been shown to be the most sensitive diagnostic method for ecov [ ] and is routinely employed as an alternative to virus isolation in diagnostic laboratories worldwide, both for timely diagnosis and in epidemiological studies [ , ] . virus isolation and biological characterisation were beyond the capacity of this study, which was similar in scope to that of the studies in horse populations in the usa, europe and asia [ , , , ] . the rrt-pcr assay was performed as previously described using a primer set targeting the nucleocapsid (n) gene (ecov- f, ecov- r and ecov- p) [ ] (table ) and agpath-id one-step rt-pcr kit (thermo fisher scientific, ma, usa) according to the manufacturer's instructions. to prove that the extraction was successful and that there was no inhibition during rrt-pcr amplification, an internal positive control primer/probe (primerdesign, southampton, uk) was added to the master mix. thermal cycling conditions were; • c for min and • c for min, followed by cycles at • c for s and • c for s. the superscript iii one-step rt-pcr system with platinum taq high fidelity (thermo fisher scientific, ma, usa) was used for sequencing analysis of two of the five ecov samples identified. there was inadequate viral nucleic acid in the other three samples for sequencing. the primer sets used to amplify the nucleocapsid (n) gene [ ] , the partial spike (s) gene [ ] , and the region from the p . to p . genes of non-structural proteins (oue, personal communication) are shown in table . the rt-pcr products were sequenced commercially by gatc biotech (cologne, germany). sequence analysis was performed using the blast and clustalw programs, and vector nti advance . software (thermo fisher scientific, ma, usa). phylogenetic analysis of nucleotide sequences was conducted with mega software version . [ ] . a phylogenetic tree was constructed based on nucleotide sequences of the k +g (n gene) and tn (s gene) using the maximum likelihood method. mega software was used to select the optimal substitution models. statistical analysis of the tree was performed with the bootstrap test ( replicates) for multiple alignments. the complete genome sequences of nc (ef ) [ ] , tokachi (lc ), obihiro - (lc ) and obihiro - (lc ) [ ] , the n (ab ) and s (ab ) genes of obihiro , the n gene of hidaka-no. / (lc ) and hidaka-no. / (lc ) [ ] , the s gene of ecov_fra_ / (kc ), ecov_fra_ / (kc ), ecov_fra_ / (kc ), ecov_fra_ / (kc ) and ecov_fra_ / (kc ) [ ] were used in sequence and/or phylogenetic analysis. the accession numbers registered in genbank/embl/ddbj are as follows: the complete sequences of the n gene; v /irl (lc ) and v /irl (lc ), the partial sequences of the s gene; v /irl (lc ) and v /irl (lc ) and the complete sequences from the p . to p . genes; v /irl (lc ) and v /irl (lc ). one six-week-old foal was the only clinical case on a public thoroughbred stud farm with approximately mares when it presented with diarrhoea. recovery took over three weeks during which it received fluid therapy, probiotics, antiulcer medication and antibiotics. the second foal was a -day-old filly, which had been hospitalised with diarrhoea two days prior to sample collection. the foal responded well to supportive treatment and at the time of sample collection, the diarrhoea had resolved. the five ecov positive samples tested negative for equine rotavirus. the nucleotide sequences of the complete n gene, the partial s gene and the region from the p . to p . genes of two positive samples ( v /irl/ and v /irl/ ) were determined. the nucleotide identities of the n and s genes of the two irish ecovs were . % ( / nucleotides) and . % ( / nucleotides), respectively. the nucleotide identities of the n gene of the two irish ecovs and the ecovs from other continents are summarised in table . phylogenetic analysis was performed for the nucleotide sequences of the complete n and partial s genes (figure ). the analysis for the n gene showed that irish ecovs were independently clustered although they were closely related to japanese viruses identified after . in the phylogenetic tree of the s gene, irish ecovs were closely related to all other ecovs analysed. the length of the region from the p . to p . genes in the two viruses was base pairs. compared with nc , irish ecovs, had a total of nucleotide deletions within p . and the non-coding region following the p . gene. compared with obihiro - and - , irish ecovs had a three-nucleotide insertion. when compared with tokachi , the irish ecovs had a -nucleotide insertion (see figure s ). the p . gene of the two irish ecovs did not have deletions or insertions, and the nucleotide identities were . - . % between these viruses and the other ecovs (nc , tokachi , obihiro - and obihiro - ). this study provides the first report of ecov circulating in ireland, the third european country with a significant horse industry where the virus has been detected in horses with enteric disease. however, detection of ecov in faeces samples from horses with enteric disease does not prove this study provides the first report of ecov circulating in ireland, the third european country with a significant horse industry where the virus has been detected in horses with enteric disease. however, detection of ecov in faeces samples from horses with enteric disease does not prove causation. in this study, samples collected between and from equids with enteric disease were tested, and only five samples ( . %) were positive for ecov. the inclusion of an internal positive control in the rrt-pcr eliminated the possibility of false negative results due to the presence of pcr inhibitors but the high content of nucleases associated with faeces samples may have caused some rna degradation. however, this low prevalence of ecov is similar to that identified in france [ ] and among thoroughbred foals in japan [ ] . although ecov has been identified on three continents, little is known about the genetic and pathogenic diversity in field viruses. in this study, sequence and phylogenetic analysis (figure ) demonstrated a high level of homology between viruses detected in a donkey and a horse in two provinces in ireland in different years. this suggests that irish ecovs may have low genetic diversity. compared with the ecovs of other countries, the n, s and p . genes of the two irish viruses were highly conserved. in contrast, the region from p . and the non-coding region following the p . gene had deletions or insertions ( figure s ). because of polymorphism in this region, this region could be useful for epidemiological investigation [ ] . the differences in the p . region between the irish ecovs and other ecovs indicated that the viruses in ireland may be distinguishable from those circulating in other countries. the positive samples were collected in november ( ), march ( ) and april ( ) in this study. higher case numbers are identified in the usa during the colder months (october to april) [ ] , and our results were consistent with the circulation period in usa. it has been reported that outbreaks mainly occurred among adult riding, racing and show horses in usa [ ] . the choice of cases to include in the current study may not have been optimal for detection of ecov as the majority of samples were from foals. however, two positive samples were collected from adult horses in a combined riding school/show jumping yard in the west of ireland. at the time of sample collection in april , the monthly mean temperatures were below long-term average and in parts of the west, were the coldest in years [ ] . cold weather may have been a predisposing factor to the ecov infection on the farm. two positive samples were collected from thoroughbred foals. a faeces sample collected from one foal with severe watery diarrhoea and inappetance was positive for ecov but a sample collected three days later tested negative. a potential difficulty in detecting ecov from naturally infected horses has been noted previously as serial samples from seven sick horses in the usa suggested that ecov only persisted for three to nine days in faeces [ ] . in both cases, the diarrhoea may have been caused by other unidentified coinfecting pathogens as has been suggested by investigators in the usa [ ] . this is the first report of ecov detection in faeces samples from both foals and adult horses in ireland. the viruses identified in ireland are genetically closely related to the japanese viruses and the results of this study give no indication of significant genetic or phenotypic diversity. in recent years, there has been an increase in awareness and testing for ecov in the usa and elsewhere [ ] . horse breeding and racing activities in ireland are the most prominent and important of any country on a per capita basis. there are over thoroughbred horses per , of population in ireland, compared to between three and five for great britain, france and the usa [ ] . thus, an investigation of ecov in ireland is pertinent not only to increase awareness nationally of the epidemiology of the virus and promote discussion on its clinical importance, but also to inform the industry globally of the health status of irish horses. ireland exports horses all over the world. by illustration, in the country was the second biggest seller of bloodstock at public auctions second only to the usa [ ] . many questions remain with regard to the clinical significance of ecov. the outbreak at a draft-horse racetrack in japan in affected of approximately horses and resulted in non-starters and the implementation of movement restrictions [ ] . however, draft horses appear to have a higher infection rate than other breeds and an outbreak of similar severity has not been reported in thoroughbred racehorses [ , ] . the much higher incidence of ecov positive thoroughbred foals identified in kentucky compared to similar populations internationally suggests an increased susceptibility to ecov infection in that population. in the past, specific environmental factors were associated with extensive reproductive loss in the kentucky area and to a lesser extent in other states [ ] , but predisposing regional factors such as differences in management, environment or husbandry have not been identified for ecov. it has been suggested that ecov is a coinfecting agent in foals with diarrhoea and clinical infections have predominantly been reported in adult horses with a mono-infection with ecov [ ] . there was no indication from the results of this study that coronavirus is a major cause of diarrhoea in irish horses but the introduction of rrt-pcr as a routine diagnostic test will assist in elucidating the significance of this virus to the irish breeding, racing and sports industries. the primary focus in future will 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attribution (cc by) license the authors are grateful to the horse owners and their veterinary surgeons for their support and discussions relating to the positive cases and also to dr marie garvey for assistance with manuscript preparation. the authors declare no conflict of interest. key: cord- -folzz nu authors: uzuner, gizem; akadiri, seyi saint; alola, andrew adewale title: tourist arrivals in four major economies: another side of economic policy uncertainty and fear date: - - journal: environ sci pollut res int doi: . /s - - - sha: doc_id: cord_uid: folzz nu in this paper, we examine the direction of causal relationships among migration-related fear, economic policy uncertainty, tourism, and economic growth in the panel economies of france, germany, the uk, and the usa. to the best of our knowledge, this study is the first to examine the interaction and interrelationship between these variables in a multivariate causality study, using a quarterly data over the period q – q via time-series causality approach as advanced by emirmahmutoglu and kose (econ model : – , ) that produces country-specific causality statistic and also captures slope heterogeneity in panel data. empirical results show that migration-related fear is linked with epu, tourism arrivals, and real income. thus, we suggest fear-induced economic policy uncertainty, fear-induced tourism, and economic policy uncertainty–induced growth hypotheses with credible policy suggestions for tourist destinations. in order to further understand the link between migration and tourism, the united nations world tourism organization (unwto ) noted certain effects arising from the migratory mobility of tourism. earlier, williams and hall ( ) noted the interconnectedness between tourism and migration and at the same time affirmed the existence of tourism-related migration patterns to include labor, return, entrepreneurial, and retirement migration. thus, the relevance of tourism to economic activities has continued to necessitate the need to further harness tourism opportunities in medical tourism, religious tourism, sport tourism, among other forms of tourism. but achieving the underlying opportunities in the tourism industry could be unattainable without decoupling the potential challenges associated the migration-related tourism. for instance, since the september terrorist attack in the usa by radical and self-acclaimed islamic group, the aftermath of the impact soon resulted to migration/immigration-related problems. in the usa and many of the european countries (such as the uk, france, and germany), the september attack has since increased backlash against the muslim community and a spike in hate-related crimes, thus compounding migration-related fear and impeding tourism development (gould and klor ; donadelli et al. ) . insight from the tourism performance of the three of europe's largest economies and the usa is an indication that the countries concerned are tourist destination countries. specifically, in , the contribution of tourism to total gdp in france, germany, the uk, and the usa is respectively given as . %, . %, . %, and . %. in the same report of the world travel and tourism council (wttc), the performance of the tourism sector (total percentage of contribution to the gdp) in france, germany, uk, and the usa is respectively expected to be . %, . %, . %, and . % by (wttc ) . similarly, the wttc indicated that travel and tourism have contributed (in total) to the employment and investment of the aforementioned countries. in france, % of total employment in was from the sector and it is expected to increase to . % in . for germany, . % of total employment in is from the sector and it is expected to increase to . % in . in the uk, total employment amounts to . % in and is expected to increase to . % in . meanwhile, . % of total employment is generated by the sector and could possibly increase to . % by . importantly, the factors associated with the tourism performance and otherwise for the aforementioned countries could either be country-specific or a spillover of events from other countries. this is an indication that a certain level of (economic) uncertainty resulting from certain factors could significantly dictate the performance of the tourism sector. in recent time, studies have widely demonstrated the link between tourism and economic growth or the investigation of the tourism-led growth hypothesis (rasekhi et al. ; akadiri et al. , fahimi et al. roudi et al. ; usman et al. ) , and the link between tourism and other factors (katircioğlu ; katircioğlu et al. ; alola a, b) . notwithstanding, the tourism outlook of france, germany, the uk, and the usa (and the global perspective) indicates the justification for the examination of the relationship between tourism and uncertainty parameters. this is necessary considering the unexpected risk associated with the ripple effects of the global financial crisis (gfc) that spread from the usa subprime mortgage market to other sectors of the economy. on this note, the current study employs the economic policy uncertainty (epu) and the migration-related index that has not been used in the extant literature and in the context of tourism. this is because of the degree of uncertainty associated with fiscal policies and the fear-induced factors in destination countries. hence, the study hypothesizes that economic policy uncertainty and migration-related fear granger cause tourism arrival in france, germany, the uk, and the usa. the use of migration-related fear is responsible for the restriction to the major economies (france, germany, the uk, and the usa). to an extent, the result of the investigation is expected to offer direction to the implementation of economicrelated policies in the examined countries. it will potentially guide on how to address potential factors that induce fear in the destination countries, in this case, major economies. the remaining sections are structured as follows. the "tourist inflows: the economic uncertainty-fear dilemma" section presents the underlying concept of fear, epu, and tourism arrivals while the "data and methodology" section highlights data description and the empirical methodologies. the empirical findings and implications for policy decisionmaking are reported in the "results and discussion" section. lastly, the concluding remarks are provided in the "conclusion" section. in addition to the effect of the us subprime mortgage market crash on the financial sector, the circumstance of the global financial crisis (gfc) of - on the international tourist arrivals globally is observed to be significant (central bureau voor de statistiek ). in reality, a high level of economic or financial risk is associated with low and effective predictability of the policy dynamics and such that characterizes the gfc. hence, the fiscal policies, especially the uncertainty in the economic policy of the destination countries, are potential determinants of tourist inflows. for instance, as the world's primary tourism destination, the european union (eu)'s tourism sector generates % of the region's gdp. subsequently, this has since triggered environmental and socio-economic policies from the member countries (european environment agency ). in addition to the impact of the economic uncertainty on the inbound tourists' decision-making, the role of fear (migrationrelated fear) on the tourists is obviously important. although fear is the feeling of a perceived threat, in this context (a migration-related fear), the threat is associated with the event within or round the destination country. as developed by baker et al. ( ) , the migration-related fear is characterized by anxiety, panic, bomb, fear, crime, terror, worry, concern, and violence. as such, these determinants of fear have been linked with tourism in previous studies (wachowiak ; bassil et al. ) . for instance, transnational terrorism and domestic terrorism are primarily associated with migrationrelated fear. nowadays, the new coronavirus (sars-cov ) has generated an unprecedented impact in most countries of the world. the virus has affected almost every country on the planet ( in total), spread to more than million people, and caused around , deaths (who ). the fear of the covid- outbreak created a huge whirlpool of uncertainty. the results of this fear created the quarantine economy which affects consumer/tourist psychology. therefore, this negative effect can be spread to travel industries. as previously indicated, evidence has shown that there exists a significant linkage between migration and tourism, thus is the migration-related fear and tourism (gould and klor ; donadelli et al. ) . for reason of "sentiment" or "mood," the decision to tour or not of a tourist (tourism consumer) is believed to change over time, thus affecting the outbound tourism and tourism demand of a destination (schwarz and clore ; dragouni et al. ). moreover, the study of dragouni et al. ( ) and many others further established the link between tourism performance and the epu. in specific, the consumer spending behavior (of a tourist) is believed to be a function of epu (gozgor and ongan ; alola et al. ) . thus, the concept of international tourism arrivals is further modified according the aforementioned studies to accommodate migration-related fear, epu, and income as determinants and illustrated accordingly as ita = f (epu, fear, rgdp). in the current study, data availability (migration-related fear data only available for the sampled countries) restricted to four ( ) major economies which include: france, germany, uk, and the usa. the series employed covers the period q - q . the epu and the migration-related fear index developed by baker et al. ( ) for the aforementioned countries and the real gdp per capita are the independent variables employed. in addition, international tourism arrivals (itas) are employed as the dependent variable. baker et al. ( ) developed the epu and the migration fear indices from the newspaper captions as illustrated in detail from http://www. policyuncertainty.com/immigration_fear.html. the world development indicators (wdi ) is the source of the itas and real gdp per capita. all the variables are used in their logarithmic forms in the estimations. it has been well-established in the literature that the standard asymptotic theory cannot be used to test for hypothesis in a level var model if the series are nonstationary (sims et al. ; toda and phillips ) . to avoid this problem, emirmahmutoglu and kose ( ) proposed an alternative method to test coefficient restrictions of a level var model. this method is based on the modified wald test in the lagaugmented var (la-var) approach with the asymptotic chi-square (χ) distribution for the estimated var (p + dmax) model. emirmahmutoglu and kose ( ) extended the la-var approach to test panel causality for heterogeneous mixed panels. where i stands for individual cross-sectional units and t is time period, k i denotes the lag structure, and dmax i is the maximal order of integration. in this study, z i, t , i = , , …n represents ita while x i, t , i = , , …n refers to epu, fear, and rgdp. three different results can be generated for the individual country from the granger causality output, such as (i) ( ) which is a standardized version of the swamy ( ) we test for slope homogeneity as shown in table . csd results as reported in table show non-rejection of the null hypothesis of the existence of csd at p < . significance level for the paneled data. similarly, results as reported in table also show non-rejection of the null hypothesis of the existence of a slope homogeneity among the variables at p < . significance level. for the stationarity test, we used the cips unit root test approach as advanced by pesaran ( ) . cips unit root test results as reported in table show that the variables under observation are all stationarity at the first difference, i.e., i ( ). finally, for long-run cointegration estimation, we make use of westerlund and edgerton ( ) bootstrap cointegration techniques. cointegration results as reported in table show support for the existence of a long-run equilibrium relationship for both individuals and across the panel. in order to achieve our research objectives, we carry out the granger causality test to examine the direction of causality relationship between the variables and whether they have predictive power over one another or not. we capture omitted variable bias in our empirical analysis by employing the real gdp per capita (a proxy for economic growth) as an additional variable for control purposes using the la-var that generates country-specific causality method as suggested by emirmahmutoglu and kose ( ) . table reveals the rejection of the null hypothesis of no causality nexus from fear to real income. results show two-way causality nexus from fear and real income in germany at p < . and p < . significance level. thus, we infer that fear has predictive power over real income and vice versa. however, we could not reject the null of no causality nexus between fear and real income in france, the uk, and the usa respectively. this indicates that migration-related fear does not necessarily influence the increase/decrease in the level of income and vice versa. thus, we suggest a fear-induced growth hypothesis in the case of germany. these findings are consistent with the findings of alola et al. ( ) . in addition, results, as reported in table , show one-way causality nexus from fear and tourism in france, germany, the uk, and the usa at p < . , p < . , and p < . significance level respectively. thus, we infer that fear predicts tourist arrivals. however, we could not reject the null of no causality nexus from tourism to fear in all countries. the findings evidently indicate that the historical values of migration-related fear do influence the present tourist decisions in choosing potential tourist destinations. it further implied that the causative circumstances of fear and especially the migration-related fear concerns in the examined economies are potentially linked with the tourism performance of the economies. thus, we suggest a fear-induced tourism hypothesis for major economies. these findings are consistent with the findings of dragouni et al. ( ) , wachowiak ( ), akadiri et al. ( ) , alola et al. ( ) , and bassil et al. ( ) . results also show two-way causality from fear to epu in the usa at p < . and p < . and one-way causality from epu to fear in the uk at p < . significance level. we thus infer that fear predicts epu. however, we could not reject the null of no causality nexus between fear and epu, as migration-related fear does not necessarily influence epu in france and germany. similarly, we found neutrality hypothesis between real income and tourism in all countries. findings indicate that tourism does not necessarily predict an increase/decrease in real income within the major countries and vice versa. these results suggest fear-induced policy uncertainty and economic policy uncertainty-induced fear hypotheses respectively. finally, results, as reported in table , show one-way causality nexus from epu to real income in germany, the uk, and the usa. these results imply that epu is a useful in this paper, we examine the direction of causality relationship among fear, epu, and tourism. we employ real income as an additional variable for control purposes. to achieve research goals, we use the la-var that generates countryspecific causality statistics as introduced by emirmahmutoglu and kose ( ) for france, germany, the uk, and the usa, using quarterly frequency data over the period q - q . empirical results show that migration-related fear is linked with epu, tourism arrivals, and real income, implying that increase in the level of perceived fear influences tourists' decision-making, enhances policy uncertainty, and, hence economic growth. in specific, the study posited a statistically significant evidence that the past history of migration-related fear is effective at explaining the trend of tourism performance vis-à-vis the international tourism arrivals of the world's four major economies. from the policy perceptive, the study posits an implication that policymakers in tourism-dependent states (both small and large) should consider the potential factors that are associated with the migration-related fear and economic policy uncertainty as among the policy concern of the state. the states could incorporate tourism-related and economic growth policies that have the potential of positively influencing the tourists' decision-making. for instance, to mitigate the increasing sentiments arising from racial, hate, xenophobic or homophobic, and other fear-related attacks, there should be more drastic and effective policy changes to address such concerns in destinations. thus, this finding provides a basis for policymakers to minimize and control fear-related attitude that potentially hampers tourist consumers' decision-making (such as investment in tourism) toward a destination. such policy is important because it is capable of re-branding the destination country from the perspective of socio-economic, political, security, and cultural situation that controls the tourists' sentiment to embark on a tour. also, considering the economic effect of emigration through the skill migration programs of some of the organization for economic cooperation and development (oecd) countries such as australia and canada, other advanced economies can reap the benefits of migration-related tourism if impediments that are fear-related are discouraged in their society. importantly, the current study can be extended in future attempt to address more economies such as the oecd. is there growth impact of tourism? evidence from selected small island states examining the causal impacts of tourism, globalization, economic growth and carbon emissions in tourism island territories: bootstrap panel granger causality analysis economic policy uncertainty and tourism: evidence from the heterogeneous panel the dynamics of tourism-refugeeism on house prices in cyprus and malta agricultural land usage and tourism impact on renewable energy consumption among coastline mediterranean countries impact of corruption and insurgency on tourism performance: a case of a developing 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consumption publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord- - aee wbu authors: kennedy, j. l.; denson, j. l.; schwalm, k. s.; stoner, a. n.; kincaid, j. c.; abramo, t. j.; thompson, t. m.; ulloa, e. m.; burchiel, s. w.; dinwiddie, d. l. title: complete genome sequence of a novel wu polyomavirus isolate from arkansas, usa, associated with acute respiratory infection date: - - journal: genome announc doi: . /genomea. - sha: doc_id: cord_uid: aee wbu we report here the complete genome sequence of a wu polyomavirus (wupyv) isolate, also known as human polyomavirus , collected in from a patient in arkansas with an acute respiratory infection. isolate hpyv /usa/ar / has a double-stranded dna genome of , bp in length. r espiratory tract infections are one of the leading causes for hospitalization in infants and young children. studies have detailed clinical diseases caused by the most important respiratory tract pathogens, including respiratory syncytial virus, influenza, parainfluenza, rhinovirus, and coronavirus. however, only recently have auxiliary infectious agents of the upper and lower respiratory tract-including human bocavirus ( ) and, most recently, wu polyomavirus (wupyv) ( ), also known as human polyomavirus (ictv species) ( )-gained clinical interest. most reports of wupyv have been coinfections or reported in immunosuppressed patients ( ) ( ) ( ) ( ) . however, studies have shown % seroconversion wupyv in the general population by months of age, and others have detected it in children with acute respiratory symptoms ( - ). wupyv has a double-stranded dna genome of~ , bp and high sequence identity ( , , ) . it is important to document novel nucleotide sequences of all viruses, including wupyv, to aide researchers and clinicians in the diagnosis and treatment of diseases caused by these potential pathogens. here, we describe a novel wupyv isolate from a nasopharyngeal swab collected from a -month-old male (hpyv /usa/ar / ) with no significant past medical history who was seen in the emergency department (ed) of arkansas children's hospital in little rock, arkansas, usa, on march . his symptoms included nonproductive cough, wheezing, and increased work of breathing. the patient was afebrile, had a heart rate of , respiratory rate of , and s a o at %. he was given vaponephrine and albuterol in the ed. he improved and was able to be discharged home with albuterol as needed. a nasopharyngeal swab was collected and stored in transport media (puritan unitranz-rt; puritan diagnostics, usa) after consent was obtained from the parents under a study approved by the university of arkansas for medical sciences institutional review board. an illumina stranded-rna sequencing library was created from isolated rna (zymo direct-zol; zymo research, usa), and hybridization-based enrichment was performed using the university of new mexico (unm) resvir (respiratory viral) panel probe set designed to be complementary to coding sequence regions of human respiratory viruses. next-generation sequencing was performed ( ϫ bp) using v sequencing chemistry on an illumina miseq platform. a total of , reads aligned to the reference sequence nc_ and resulted in . % coverage of the reference genome. generation of a consensus genome sequence of the isolate was performed using clc genomics workbench version and annotated using the vipr genome annotator ( ) . the missing base pairs of the ar genome were completed using pcr and sanger sequencing. alignment of hpyv /usa/ar / to nc_ revealed a total of variants, of which three were predicted to be nonsynonymous. phylogenetic analysis of the novel isolate was conducted using neighbor-joining with , bootstraps to known wupyv genomes and revealed isolate ar grouping closely to isolates w , np , b , and b with genbank accession numbers gu , gu , gu , and gu . alignment of our isolate to the closest related sequence, gu , showed a total of three variants, of which one was nonsynonymous. the amino acid changing variant was detected in protein add (p.glu gln). accession number(s). the whole-genome sequence of the hpyv /usa/ar / isolate has been deposited in genbank under the accession number kx . this project was supported in part by the national center for research resources and the national center for advancing translational sciences of the national institutes of health (nih) through grant numbers ul tr and kl tr to d. dinwiddie, and ul tr and kl tr to j. kennedy, as well as the centers for translational science award western consortium grant. j. kennedy also has support from the nih national institute of allergy and infectious disease (k ai ) and the arkansas biosciences institute. the content is solely the responsibility of the authors and does not necessarily represent the official views of the nih or other funding agencies. cloning of a human parvovirus by molecular screening of respiratory tract samples identification of a novel polyomavirus from patients with acute respiratory tract infections a taxonomy update for the family polyomaviridae whole-genome characterization and genotyping of global wu polyomavirus strains reactivation and mutation of newly discovered wu, ki, and merkel cell carcinoma polyomaviruses in immunosuppressed individuals presence of the newly discovered human polyomaviruses ki and wu in australian patients with acute respiratory tract infection acquisition of human polyomaviruses in the first months of life wu polyomavirus infection in children discovery and identification of wu polyomavirus in children from zhejing region complete genome sequence of a novel human wu polyomavirus isolate associated with acute respiratory infection genome analysis of the new human polyomaviruses wu polyomavirus (wupyv): a recently detected virus causing respiratory disease? viruses vipr: an open bioinformatics database and analysis resource for virology research key: cord- -ml kvl authors: kanger, laur; sillak, silver title: emergence, consolidation and dominance of meta-regimes: exploring the historical evolution of mass production ( – ) from the deep transitions perspective date: - - journal: technol soc doi: . /j.techsoc. . sha: doc_id: cord_uid: ml kvl grand environmental and societal challenges have drawn increasing attention to system innovation and socio-technical transitions. a recent deep transitions framework has provided a comprehensive theory of the co-evolutionary patterns of multiple socio-technical systems over the last years. however, so far the framework has not been subjected to systematic empirical exploration. in this paper we address this gap by exploring the co-evolutionary model linking niche-level dynamics, transitions in single systems and ‘great surges of development’, as conceptualized by schot and kanger ( ) [ ]. for this purpose, we conduct a case study on the historical evolution of mass production in the transatlantic region from to . instead of focusing on dominant technologies or common practices the development of mass production is understood as the emergence of a meta-regime, i.e. a set of mutually aligned rules guiding production activities in multiple socio-technical systems. the results broadly confirm the overall model but also enable to extend the deep transitions framework by uncovering new mechanisms and patterns in the variation, diffusion and contestation of meta-regimes. the world is confronted by a socio-ecological emergency brought on by climate change, resource depletion and loss of biodiversity. solving these crises requires rapid and deep decarbonization of a broad range of socio-technical systems including energy, housing, food, water and mobility. these challenges have drawn increasing attention to system innovation in the sustainability transitions field, including co-evolutionary dynamics extending beyond single systems. although so far the stream of studies on multi-regime interaction has remained fairly small [ ] [ ] [ ] [ ] [ ] , lately the importance of the topic has been increasingly recognized in the sustainability transitions research network agenda as well as the debates around it. the need for 'zooming out' has been advocated [ , p. ] in order to shift "emphasis away from within system dynamics that have received considerable attention to the interconnected and multi-scalar qualities of socio-technical systems that are less well understood" [ , p. ] . a recently developed deep transitions (dt) framework [ , ] attempts to go beyond the analytical limitations of studies focused on single systems. synthesizing insights from sustainability transitions studies, long wave theory and industrialization literature dt theorizes how interactions between socio-technical systems produce - year long 'great surges of development' [ ] and how successive surges, in turn, accumulate into a set of principles driving every industrial societyan industrial modernity. the authors of the dt framework argue that the co-evolution of single systems, interconnected systems and industrial modernity -the first deep transition -has created a fundamentally unsustainable trajectory of environmental degradation while not being able to solve recurring problems of social inequality. altering the situation requires a shift of comparable magnitude -the second deep transition. dt aspires to offer a comprehensive conceptual framework of the co-evolutionary patterns of multiple socio-technical systems over the last years. however, beyond the provision of selected illustrative historical examples in the original outline, the framework has yet to be subjected to systematic empirical assessment. in this paper we address this gap by exploring the co-evolutionary model outlined in schot and kanger [ ] which links niche-level dynamics, transitions in single systems and great surges of development through the emergence, consolidation and alignment of rules. for this purpose, we conduct a historical case study of the development of mass production in the transatlantic region from to . the case of mass production has been chosen because of its centrality to the th long wave beginning at the early th century [ , ] making it both a 'most-likely' case for the dt framework and an influential case with considerable environmental impact. our main research question is: how does the historical development of mass production correspond to the co-evolutionary patterns as proposed by the deep transitions framework? section defines the central concepts of the framework -rules, meta-rules, regimes, meta-regimes -and outlines a model of the co-evolutionary dynamics of rules. section describes our methodological approach. section presents a stylized narrative of the historical evolution of mass production. section assesses the extent to which the observed dynamics match the theoretical expectations and situates our findings in broader literature on socio-technical change. similarly to early case studies exemplifying the multi-level perspective on socio-technical transitions [ ] [ ] [ ] [ ] we also use our results to modify and extend the dt framework. section concludes. the dt framework [ ] was developed to address two gaps in existing knowledge on multi-system co-evolution. first, to theorize how niche-regime dynamics and transitions in single systems [ , ] relate to long waves [ , ] . second, to understand how successive long waves accumulate into major historical continuities characterizing the overall industrialization process [ ] . dt focuses on rules -"humanly devised constraints that structure human action, leading to regular patterns of practice" [ , p. ] -as a central coordination mechanism. by being embedded in the very structure of socio-technical systems rules shape the behaviour of actors and provide systems with long-lasting directionality. rules differ in terms of their scope and degree of alignment to other rules, resulting in a four-fold classification: . rule is a single prescription for action present in a single system. for example, a principle 'design for modularity' has its origins in the american housing system around s- s, where the use of modular components was seen as means to lower construction costs, reduce waste and increase efficiency [ ] . . meta-rule is a single rule present in multiple systems. for example, russell [ ] describes how metaphors such as 'architecture', 'throughput' and 'modularity' started to appear in the vocabulary of american computer engineers in the s. this indicates that the 'design for modularity' rule had crossed a boundary between housing and data processing systems. . regime is a set of aligned rules present in a single system. for example, zuboff [ ] analyses how google invented and perfected interrelated principles constituting what she calls 'surveillance capitalism'. this involved treating human experience as a free raw material, using this 'behavioural surplus' for the fabrication of prediction products and trading these predictions in behavioural futures markets [ , p. ] . from the early s this regime became paradigmatic in the communication system and was adopted by major players like microsoft or facebook. . meta-regime is a rule-set present in multiple systems. for example, by treating usergenerated data from self-driving vehicles as a commodity to be sold to advertisers [ , ] the regime of surveillance capitalism might be currently expanding from the communication system to the mobility system. drawing on these concepts, schot and kanger [ ] connected processes on niche-and system-level as described by the multi-level perspective on socio-technical transitions [ , ] , to successive 'great surges of development' as conceptualized by carlota perez. perez [ ] argues that since the late th century there have been five - year surges , each characterized by particular phases: ) gestation (pre-surge period); ) installation period (first half of the surge, lasting - years), further divided into irruption and frenzy phases; ) turning point; ) deployment period (second half of the surge, lasting - years), further divided into synergy and maturity phases. the model by schot and kanger [ ] relates the emergence, consolidation and alignment of rules to different phases of each surge, resulting in a specific pattern: . a protracted gestation period is characterized by the emergence of new rules rules in separate niches, i.e. spaces governed by specific selection criteria such as military applications prioritizing performance over cost. in some niches some of these rules may be aligned to each other. in exceptional instances, exogenous macro-level 'landscape' pressures might destabilize dominant systems, opening up a window of opportunity for niches and resulting in regime-shifts, i.e. transitions in single systems [ ] . . each surge starts with the irruption phase where emerging and incumbent rules come to compete against each other, resulting in further transitions or transition failures. early interactions between some systems might occur and some rules may turn into meta-rules as a result. overall these interactions as well as their outcomes remain ad hoc, nonstandardized and accidental in nature: at this point no lasting connections between systems are created, and thus no clear directionality in multi-system co-evolution is established. . at the beginning of the frenzy phase, many rules increasingly start to cross the boundaries of a single system, generating widespread enthusiasm about the prospects of emerging rules and associated technological opportunities but also major concerns about their anticipated societal impacts. partial alignment between different meta-rules starts to occur leading to the gradual emergence of structural and functional couplings between systems, e.g. different systems relying on the same infrastructure or forming input-output relations [ ] . this process is further amplified by the purposeful aggregation work of transnational organizations aiming to homogenize and standardize within-and between-system practices. as a result, a clearer new directionality becomes visible, yet in this phase there will still be competing options (alternative directions of evolution) available. . the competition between meta-rules and their various combinations is resolved at the turning point. major crises such as wars provide an impetus for the alignment of expectations, enabling powerful actors to tilt the playing field in favour of a particular ruleset. therefore, from this point forward one can start talking about the existence of the dominant meta-regime providing directionality across many socio-technical systems. . during the synergy phase the dominant meta-regime acts as a selection mechanism, favouring niches compatible with its logic and rejecting non-compatible ones [ ] . it continues to diffuse from one system to another, leading to the increasing take-up of its principles in various systems. because of its expansive nature the meta-regime now also starts to shape landscape structure and dynamics. . in the maturity phase new problems start to appear which cannot be fully resolved within the confines of the dominant meta-regime. the scene is set for yet another surge with new niches and systems becoming the main loci of radical innovation. as the former metaregime has now become part of the landscape, it continues to structure new niche-regimemeta-regime interactions. figure presents a visual summary of the model. j o u r n a l p r e -p r o o f as the above model focuses on developments distinctive to each phase, it implies but does not really make explicit the accumulation of phase-specific developments into long-term outcomes. an alternative way to look at the process of meta-regime building would be to separate the overall coevolutionary dynamics of rules into different sub-trends. such an analytical exercise potentially enables to observe different patterns and mechanisms related to various dimensions of meta-regime evolution that might otherwise remain unnoticed. although we recognize the difficulties with making clear-cut conceptual distinctions, we suggest that broadly speaking the co-evolutionary model of schot and kanger [ ] can be seen as a result of three interacting sub-trends: . variation: emergence of new rules or the alignment of existing rules into rule-sets (regimes/ meta-regimes) with novel qualities. from the evolutionary perspective we expect gestation, irruption and frenzy phases to be dominated by high but gradually decreasing variety, reaching its low point during the synergy phase. this would reflect a move from radical to incremental innovation and the increasing alignment of rules leaving less possibilities for the emergence of major novelty. however, towards the end of the maturity phase we expect variety to increase again as the deepening crisis of the dominant meta-regime provides an opportunity for radical niches. . diffusion: imitation and selective adoption of rules and rule-sets in different systems and various countries. although we assume the process of diffusion to occur in a piecemeal manner involving various setbacks and failures, as an aggregate trend we expect the emerging meta-regime to spread to new systems and locations throughout the surge. . contestation: debates, conflicts and struggles around emerging rules and rule-sets concerning their application, their changing relations with existing modes of production and their broader societal effects. here the implied pattern is somewhat more complex. during irruption and frenzy phases we expect that the increasing public visibility of the emerging meta-regime and the attempts to apply it in different systems would prompt increasing societal contestation up to the turning point when large-scale conflicts are resolved. during the following phases we expect the dynamics to be reversed: low contestation during the synergy phase followed by renewed contestation during the maturity phase, reflecting a gradual shift from the dominance of the meta-regime to its crisis. these phase-specific and cross-phase trends constitute a set of expectations against which empirical data on the historical development of mass production will be assessed. this will be done in sections . and . respectively. the research is based on a single longitudinal case study design [ ] , adopting a 'most-likely' case selection strategy. the literature on case selection stresses the symmetrical strengths of 'most-likely' and 'least-likely' cases for assessing the validity of an existing theory. whereas 'most-likely' cases cannot offer strong proof for a theory (as the case is expected to conform closely to theoretical propositions) they can strongly undermine it if the case does not fit the theory (because if the theory does not even explain the closest expected match it will probably perform even worse on less likely cases). conversely, 'least-likely' cases can offer strong proof for a theory (demonstrating that the theory explains even less likely cases) but they cannot strongly undermine it if the case does not fit the theory (as the case is not expected to conform strongly to theoretical propositions in the first place) [ , p. ] . where multiple theories exist for explaining a given phenomenon, the strongest supporting evidence can be obtained by choosing a case least-likely for a given theory and mostlikely for competing theories (all of which also make different predictions about the outcome of interest). the strongest weakening evidence can be obtained by choosing a most-likely case for a given theory as well as all competing theories making similar predictions, and failing to find supporting evidence for it [ , p. ] . arguments like this [ ] [ ] [ ] implicitly assume a rather mature field of research characterized by considerable knowledge base (established through numerous prior case studies) and the existence of a number of competing theories for a given phenomenon. although this might be an accurate description of the authors' own disciplines (usually political science), neither assumption holds for the current framework which establishes a new phenomenon -deep transitions -through a synthesis of existing theories (multi-level perspective and techno-economic paradigms). in this way dt goes beyond mlp and tep by design and cannot be argued to offer strictly competing explanations to either of the two frameworks. this is well evidenced by geels's seminal analysis of the transformation of factory production from the mlp perspective [ ] . not only is the study geographically and temporally restricted (usa, (usa, - , it also focuses on the breakthrough of mass production where impacts exceeding the mobility system and emerging connections with other systems are largely excluded. the most complete long-wave account of mass production [ , pp. - ] , on the other hand, largely focuses on a few leading countries (primarily usa) during the th wave only, neglecting the role niche-system dynamics in generating the surge. in contrast, the use of the dt framework necessitates the observation of interactions between niche, regime, metaregime and landscape dynamics over a much longer time-frame and broader geographical scope (see below). we suggest that in the context of scarce knowledge of the focal phenomenon (deep transitions) and the lack of competing theories offering alternative explanations, a 'least-likely' case would constitute a far too strong first empirical test of the framework. a similar reasoning, although not explicitly spelled out as such, seems to have been pursued in seminal works on socio-technical transitions which initially focused on transport, sanitation and production [ ] [ ] [ ] [ ] . it is very difficult to see what would have been gained if mlp had started out from a 'least-likely' case such as rock'n'roll instead [ ] . therefore, in our view a 'most-likely' case constitutes the best option for a first empirical test of a nascent theory. mass production was chosen as a case for three reasons. first, as the tep framework considers mass production a central element of the th surge it can be considered a 'most-likely' case for the dt framework. this means that we expect the evolution of mass production to mirror the conceptual model of schot and kanger [ ] . a failure to detect a close match with the theoretical propositions, on the other hand, would cast serious doubt on the overall validity of the dt framework. second, during the past century mass production has become increasingly central to modern lifestyles in developed and developing countries, thereby exacerbating environmental problems such as pollution, waste or resource depletion [ ] [ ] [ ] . because of its global impacts mass production constitutes an influential case the co-evolutionary dynamics of which are crucial to understand in its own right. third, practical considerations such as the availability of a broad array of secondary historical literature on the topic also played a role in the case selection. conducting indepth qualitative work with primary sources on the scale required for exploring the dt framework would have simply fallen beyond the scope of the study. the case is bounded as follows. temporally, we focus on the events between and . this covers the long gestation period of mass production ( - ), its installation during the th surge (including irruption, - , and frenzy phases, - ), turning point ( ) ( ) ( ) ( ) ( ) ( ) ( ) as well as the deployment period (including synergy, - , and maturity phases, - ) . geographically, we focus on the transatlantic region. this enables us to trace the varying origin and j o u r n a l p r e -p r o o f circulation of rules across national boundaries, including their regional adaptations. thematically, we focus primarily on the engineering-related dimension (production organization and labour control), largely excluding the dimensions of economy, policy, everyday life and culture. while the latter choice has been largely made for feasibility concerns we also recognize the resulting limitations of our study (see below). following an established tradition in transitions studies our research employs a stylized narrative explanation based on an interpretive analysis and synthesis of a broad range of secondary historical literature. as geels and schot argue, "the strength of a narrative is that it can capture complex interactions between agency and changing contexts, time, event sequences, making moves in games, and identities" [ , p. ] . although narrative-based approach is versatile, flexible and attentive to complexity, it has also been criticized for the subjectivity of researcher choices or difficulties with assessing the extent to which the results conform to expected patterns [ , ] . a similar sentiment is echoed in nuvolari's assessment of freeman and louçã ( ) as both the most articulated treatment of this [long wave] approach but also highly impressionistic and descriptive [ , pp. , ] . given our aim -to provide an early empirical assessment of a complex theoretical framework of broad scope -we argue that the strengths of a narrative-based approach outweigh its weaknesses. to address the shortcomings of the narrative approach we try to provide a clear assessment about the possible biases of our study (see below), to substantiate each statement on a general trend with empirical support (section ) and to establish clear connections between empirical data and theoretical abstractions (section ). nevertheless we readily acknowledge that ultimately our selection, presentation and interpretation of data contains a certain degree of subjectivity. data collection and analysis was conducted in four stages, involving a great degree of iteration between different steps: . beginning from authoritative works [ ] [ ] [ ] and literature search from various databases we used a snowball sampling approach to assemble a corpus of secondary literature on the history of mass production. given the analytical dimensions of interest (see section ) and the case bounding (see above) we aimed at a systematic coverage. . based on the literature we then constructed a genealogy of mass production, consisting of different rules, combinations of these rules (rule-sets) as well as several historical dead-ends (see figure ). this stage involved interpretive work, whereby various technologies, practices and principles described in the sources were transformed into rules (see box ). the emerging interpretations were discussed with two historians of technology with expertise in mass and specialty production to further improve the genealogy. . we then compiled a stylized narrative explanation of the development of the rules underlying mass production. upon detecting gaps in the narrative, additional literature search was conducted to cover the missing dimensions and to update the genealogy. . in the final stage, we used the pattern-matching technique [ , pp. - ] to compare the patterns that emerged from the stylized empirical narrative with the expected ones derived from theory. in doing so we assessed overlaps and deviations and, where necessary, developed further conceptual extensions. the pattern analysis was twofold. first, the phasespecific patterns of every period were matched to the six propositions (gestation, irruption, frenzy, turning point, synergy, maturity) described in subsection . . the results of this exercise are presented in subsection . . second, the cross-phase patterns evident throughout the periods were matched to the three overall trends (variation, diffusion, contestation) described in subsection . . these results are presented in subsection . . the limitations of our approach should be noted. firstly, as our study draws on works published in english the narrative might suffer from source bias. to address this issue we attempted to locate studies on different countries or studies with an international dimension [ ] [ ] [ ] . secondly, given our focus on the engineering dimension, the narrative might somewhat overstate the prominence of usa while downplaying the contribution of different countries to the correlates of mass production in other dimensions (economy, policy, everyday life, culture), e.g. interwar era soviet experiments with centralized planning as a precursor for large-scale state intervention in the entire transatlantic region after world war ii. it appears, therefore, that conveyors and gravity slides were adopted either immediately before the assembly line experiments or resulted from the "work in motion" principle brought to life by the assembly line [ , pp. - , on ford's experiments]. mass production of automobiles, as developed by henry ford in the half-dozen years before the first world war, depended on three basic principles: the standardisation of the product, the use of special-purpose equipment, and the elimination of skilled labour in direct production [ , p. ] . "thorough elimination of wasteful practices" is the basic concept of the toyota production system. this concept is supported by the following two fundamental principles: . "right on time"; . "automation" [ , p. ] . use hand tools for making the products the following section presents a narrative overview of the historical evolution of mass production between to . in order to establish clearer connections with the theoretical framework (see section ) the results have been divided in different phases, with each phase focusing on three trends -variation, diffusion and contestation. note, however, that because of continuous interactions (e.g. new rule-sets emerging and starting to compete with each other) the following narrative cannot always maintain as neat analytical separation of the three trends as implied by the respective titles of each sub-section. before the th century flexible specialty production was a dominant mode in the entire transatlantic region. it relied on one worker performing all tasks, the use of customizable specialpurpose machinery, the imperative to compete on novelty and/or quality (rather than price), making systematic use of shop-floor worker knowledge for production improvements, and maintaining personal and informal relations inside the enterprise and between different enterprises [ ] . from the th century experiments in various niches started to challenge these principles. the precursors of mass production can be broken down to four broad streams: standardization, continuous movement, electrification and efficiency. the first stream began with the ideal of interchangeable components in armsmaking that emerged in the french military from and was realized with hand tools in [ ] . during the first half of the th century us state armouries (harpers ferry, springfield) added two important rules: using special-purpose equipment to perform one function only and placing machines in a sequence according to the work process [ , ] . by mid- th century the alignment of these three principles constituted a rule-set of distinctive character, named american system of production by contemporary observers [ ] . closely related to the interchangeability of components was also the emerging idea of product standardization which allowed to minimize changes in design and to avoid expensive and time-consuming retooling [ , pp. - ] . another stream was related to experiments with continuous movement and flow production in various applications such as flourmilling, bakeries, breweries, cigarette-making, canning, oil and chemical industries, foundries and bicycle production. other important antecedents were the disassembly lines used in meat-processing in cincinnati (from the s, see figure ) and chicago (from the s) as well as edison's iron mining facility in ogdenburg, which was visited by henry ford [ , , p. ; ] . a third strand entailed the electrification of factories. electric motor was first introduced in the s, first as an add-on to the central steam engine but gradually moving toward unit drive, i.e. each machine having a separate electric motor. before that the central power source largely dictated the placement of the machines and also limited the precision that could be achieved. additional bonuses of electrification included improved lighting and ventilation enabling increases in precision and working time but also better health for the workers [ , , p. ] . the fourth stream aimed to increase the efficiency of labour and resource use. this included a division of production into specific tasks to be carried out by a specific worker, introduction of a clear separation between engineering, management and executive roles, and replacing workers' rules of thumb with ones devised by experts. these principles were central to the thinking of frederick winslow taylor whose work experience in steelmaking gradually led him to formulate a system of 'scientific management' [ , ] . as part of a wider efficiency movement gaining strength in industrial nations from the late th century other ideas on systems management and factory organization gradually led to an idea to optimize the work process as a whole [ , ] . similar concerns also directed attention to the need to minimize waste of resources and, where possible, reuse waste materials in the production process. the precursors of mass production originated from both europe and usa. interchangeability had a french origin but the ideas were transferred to the usa by thomas jefferson, french military experts and books. the idea to sequence machines according to the work process was developed by thomas blanchard at the us springfield armoury around the late s but the idea built on the british example of wooden blockmaking from the s [ , pp. - , ] . the benefits of subdivision of work into specific tasks had been famously described by adam smith in but the idea was taken to another level by taylor's scientific management more than a century later. in terms of sectoral origin there was even greater variety: armsmaking, mobility, food, energy and chemical industry all contained niches for experimenting with new principles of production. the initial barrier to the adoption of the american system of production was cost-related: achieving full interchangeability turned out to be more expensive than hand-fitting [ , pp. - ) . however, over the second half of the th century a number of broader factors in usa started to favour increasing mechanization of production. these included a shortage of skilled labour (thereby providing incentives for investing in machinery), road and rail network enabling nation-wide markets, fast population growth coupled with rising purchasing power, the emergence and growth of large-scale corporations able to capture various economies of scale, the rise of the engineers as a new and increasingly influential social group but also the american cultural values favouring the development of a highly accelerated society [ , , pp. - ; ] . during the second half of the th century the american system spread in a gradual and piecemeal fashion to various industries such as private armsmaking, clockmacking, sewing machines and typewriters [ ] . perhaps the most notable example of pre-fordist large-scale production was the pope manufacturing company where the automation of work on a moving assembly line, interchangeable parts across a wide variety of bicycle models and specialization of work process management in functional departments were combined for bicycle production [ ] . interchangeability and standardized parts were first introduced to the automobile industry by the cadillac company in [ , p. ]. since the precursors of mass production were dispersed in various niches interactions between these rules and specialty production remained fairly sporadic. enterprises relying on flexible production did occasionally employ new principles when it suited them, e.g. printing industry in new york being early adopters of electric drive systems [ , p. ], but overall this did not lead to a fundamental overhaul of the mode of production itself. occasionally, new rules were explicitly contested. for example, french experiments with new modes of weapon production were terminated for political reasons at the beginning of the th century [ ] . another example concerns taylor's attempts to rationalize production at bethelem steel company, leading to stiffened worker resistance and culminating in his dismissal in [ , p. ]. from henry ford and his team of engineers undertook a wave of experimentation to mechanize car production, turning simultaneous attention to gauging, fixtures, machine tool design, materials handling, factory layout and many other areas [ , p. ]. in so doing the team made use of various principles pioneered during the gestation phase (see figure and . . ) but often realized them in a novel manner. 'move work to worker' rule is a good example: whereas the th century bicycle industry had used runners to deliver parts to stationary machinists ford's engineers developed a moving assembly line for the same purpose; whereas meat-processing had used a sequential approach to disassemble the corpse, ford reversed the process. the success of ford motor company also exposed a crucial difference between 'fordism' and 'taylorism'. although both approaches offered solutions for increasing the volume of production and were seen by contemporary observers as similar and complementary, taylor focused on task optimization while ford directed his attention to the work process as a whole. the supremacy of the latter approach was clearly demonstrated early on: in ford's production exceeded the 'taylorist' packard factory by times [ , p. ]. despite taylor's search for 'one best way' turning out to be a historical dead end as a rule-set for production organization, his legacy proved to be much more enduring for labour management [ , ] . . wwi also provided a stimulus to extend the new regime to the defence system, e.g. citroën using mass production techniques to produce artillery shells (ibid.). sending men and horses to the front facilitated the mechanization of agriculture, reflected in the introduction of the fordson tractor in . however, not all attempts to apply mass production were successful, e.g. ford's failure to deliver submarine patrol boats as quickly as anticipated [ ] . high degree of task fragmentation and the accompanying routineness of work imposed severe demands on labour who were de-skilled and had very little control over the work process. as such mass production was contested from the outset. in , ford broke a strike in buffalo plant by closing it and sending the machinery to detroit; his right-hand man, charles sorensen, had to go to britain to break the metal workers union [ , p. ] . nevertheless, high labour turnover remained a pressing problem, reaching % in [ , p. ] . in order to counter this ford famously introduced a -dollar day in , prompting immediate discussions about the societal impacts of this move. whereas american socialists and radical press celebrated the rise in worker income, conservative voices in industry, media and church worried that ford's high wage policy may aggravate worker inequality and drive out other businesses thus leading to social unrest and overall decrease in local prosperity [ , pp. - ] . whereas ford's mass production was starting to push aside its competitors (e.g. taylorism) in the mobility system, specialty production continued to thrive elsewhere. in usa it contributed roughly about / of the value added in [ , pp. - ] whereas in europe the craft tradition remained largely unchallenged. in many areas such as jewellery or furniture quickly changing consumer demand made mass production techniques non-viable. however, whereas the gestation period had been characterized by loose borrowing of selected principles, some specialty producers now started to establish more symbiotic relationships with mass producers, e.g. machine shops supplying specialized single-purpose machinery for early automobile manufacturers [ , p. ]. ( - ) whereas ford had inaugurated a new regime of mass production his continued focus on productivity maximization and price-cutting of a single product made him less responsive to variations in demand. from the s general motors had begun to seek for alternatives in order to balance the economies of scale offered by mass production with diversity in demand characteristic of market economies. this led gm to introduce many business-related innovations such as the installation of multi-divisional decentralized management structure, use of consumer credit or the facilitation of used-car trade-ins. however, gm's strategy also implied changes in production leading to a new variation, 'flexible mass production' [ ] . as a guiding rule production had to be designed for annual model change, often involving minor stylistic changes. the combination of uniformity and variety was achieved by using similar components across different car models. the flexibility of production was further increased by the customization of general-purpose machinery (vs. highly specialized single-purpose machines that had characterized ford's production). the overall effectiveness of increased flexibility was reflected in the fact that in gm's chevrolet outsold ford in the american market for the first time [ , p. ]. from the s mass production consolidated its foothold in the systems of mobility (increasing diffusion of cars, especially in usa), defence (mechanization of war as an anticipated direction of evolution) and food (continued mechanization of agriculture, new techniques of fruit canning and baking, introduction of toasters). new consumer durables often acted as vehicles for introducing mass production to new systems, e.g. communication (radio sets) and housing (washing machines, vacuum cleaners, electric irons) [ , p. ] . the take-up of mass production often resulted in three types of outcomes: ) consolidating existing links within a system, e.g. oil production and car diffusion in the mobility system; ) acting as one of the building blocks for system transitions: for example, in the food system mechanization was increasingly combined with the principles of chemicalization (e.g. the use of nitrogen fertilizers and pesticides) and selection of plants and animals suitable for standardized production, gradually paving way to the intensive agriculture regime [ , pp. - ]; ) consolidating or creating new links between systems, e.g. drive-in cinemas linking mobility and communication systems or electrical appliances in the housing system intensifying production in the energy system. mass production also started to become an increasingly "powerful general concept. it quickly shed its technical and precise meaning and started to move toward an abstract ideal of standardization, mechanization and repetitive production, allied to implications of order, rationality and universality" [ , p. xvii] . this was reflected in using the term as a loose metaphor to describe various technical and social activities (energy production, education, artistic performances), the influence of machine aesthetic on industrial designers and artists (e. at the same time attempts to extend mass production through direct imitation often resulted in failures. us automotive dealers largely resisted ford's efforts to introduce routinized work procedures, division of labour, specialized machinery and progressive layout of equipment [ ] . in both europe and usa various attempts were made (and abandoned) to produce houses on the assembly line. american mechanical engineers also advocated the increasing use of mass production in woodworking and furniture industry on the grounds of increased efficiency, largely neglecting consumer preferences favouring diversity [ during the s- s the contestation of mass production gradually changed from an individual worker strategy of 'voting with one's feet' to organized resistance. added to the familiar concerns about repetitiveness and de-skilling were the various tactics of the american automobile industry for increasing the pace of work without a corresponding rise in wages, e.g. increase of line speed after installing new machinery or setting special daily production goals [ ] . until the end of the s the industry's high wages had generally neutralized the need for worker organization. however, with the great depression the power of management over labour was further increased as worker job security turned ever more fragile. this situation eventually led to the establishment of the united automobile workers union in and the introduction of sit-down strikes [ , pp. - ] . although the automotive industry started making gradual concessions to workers, the conflict was not entirely resolved by the end of the s: the link between mass production and labour organization was to be consolidated only after wwii. the relationship between mass and specialty production was characterized by important regional differences. in european countries with a strong craft tradition, mass production was claimed to be an american phenomenon that, because of smaller markets, lower incomes and diversified consumer preferences, could not be copied [ ] . in usa, however, a shift from symbiosis to competition between mass and specialty production was taking place. for example, in the early s several clothing manufacturers installed two or more assembly lines, one for standardized and the other for customized production [ ] . however, by the end of the decade the prevalent rhetoric of 'simplification', advocated by president hoover, engineering societies and managers, started to associate industrial diversity with inefficiency and waste. the federal trade commission and department of justice also countered attempts at regulating competition through price coordination and exclusive sales agreements. this effectively forced specialty producers to compete on price, making them lose one of their distinctive characteristics, and initiating the gradual waning of flexible production in usa [ ] . increasing public visibility of mass production raised wildly ranging hopes and fears about its societal effects. for example, ford's waste handling practices were initially argued to contribute to the preservation of america's timber resources [ , p. ]. an influential advocate of fordism argued that the abundance enabled by mass production would lead to the "abandonment of all class thinking and the substitution of fact-finding for tradition" [ , p. ]. mass production was also associated with the possibility of shorter working hours leading, in turn, to worries about future technological unemployment. these utopian promises initially made mass production attractive to radical political forces and totalitarian governments [ ] . however, by s widespread optimism had given way to two streams of critique. the great depression had problematized the necessity of connections between mass production, high employment, rising wages and increasing living standard. especially in europe mass production was argued to lead to the homogenization of products, consumers, personality and culture [ , pp. - ] . for example, a french satirical film "À nous la liberté" from drew parallels between industrial working conditions and life in prison (see figure ). existential threats posed by wartime conditions prompted several experiments to improve the performance of weapons and to reduce the number of defects. strategies for seeking trade-offs between product quality and quantity led to differing national strategies: whereas german and british managers preferred increasing quality and constant modifications, american and soviet industrialists stressed quantity and refrained from changes preventing the full deployment of assembly line techniques [ , p. ]. the management of this balance led to experiments with continuous improvement and just-in-time strategy in airplane production [ ] . german concentration camps were also experimenting with flexibility in uniform production combining the use of female prisoners with no special skills or training needed, state-of-the-art sewing machines and electric motors [ , p. ]. however, these experiments largely failed to spill over to the civil sector after the war when both usa and europe largely reverted to better established techniques of mass production. war requirements greatly increased the need for standardized products, including clothing, weaponry and food. government procurement, investments in factory equipment and other subsidies directly facilitated mass production [ ] as did cooperation needs between allies. especially important was the lend lease act ( ) through which usa began to provide supplies to allies, e.g. trucks, boots and food for the soviet red army or car parts to britain for the manufacturing of jeeps [ , p. ; , p. ] . whereas wwi had provided a stimulus to extend the nascent mass production regime to the defence system, wwii helped to consolidate its centrality for war in the entire region. in the american food system, where the adoption of mass produced vehicles was well underway, wwii proved to be a literal turning point (see figure ). the war also demonstrated possibilities for extending the meta-regime from one application to another. for example, the construction tycoon henry j. kaiser established the california shipbuilding corporation in for the purpose of mass producing military ships and although he had no prior experience in the field, the enterprise became the country's leading shipbuilder [ ] . another example is the hiring of henry ford's chief engineer to design the willow run production line for assembling b- heavy bombers [ ] . the established infrastructure often facilitated dual use: in the usa aircraft engines were produced in converted automobile factories [ ] whereas in germany wartime aircraft production led to the development of a strong supply network forming a basis for post-war automobile industries [ ] . wartime conditions heightened the need for 'mass loyalty' which meant putting labour conflict on hold and mobilizing the entire population in the name of cooperation and shared sacrifice [ , pp. - ; ] . in this way the combination of direct and indirect effects of wwii paved way for the post-war dominance of mass production. the lack of demand for specialty production together with state investments in mass production created a productive capacity that could be converted for civil purposes after the war. the forced savings of a substantial amount of soldiers [ ] created a possibility for post-war mass consumption. the devastation of war also raised pressing concerns for which mass production seemed to provide appropriate solution. the food system is a case in point. although organic farming had started to gain public visibility in britain shortly before wwii, severe post-war food shortages provided strong incentives for industries and newly founded international organizations such as fao to favour intensive agriculture instead [ , pp. - ] . ( - ) the post-war era can be described as the societal embedding of mass production: connecting it to horizontally integrated and multi-divisional big enterprises in the economic dimension, social contract between employers, labour and policymakers within the framework of a nationallybounded welfare state in the political dimension, individualized private mass consumption in everyday life and the ideology of material and social progress in the cultural dimension [ , ] . the combination of these features put a foundation to long-lasting economic growth in various regions of the world, enabling them to catch up with the usa. mass production continued to develop in two directions. in usa, labour unrest had prompted a managerial vision of a fully automated factory. underlying this vision were two principles: embedding the task sequences of skilled workmen in machine tools (initially through 'record and playback' systems) and using the idea of feedback to increase the flexibility of machine tools. the full realization of these principles was envisioned to result in computer-controlled assembly lines staffed with robots that would enable to replace not only semi-skilled blue-collar workers but also skilled blue-collar and white-collar employees [ , pp. - ] . whereas the rhetoric of automation was also embraced in post-war japan, experiments undertaken in toyota from focused much more on improvements in product quality, reorganization of work and increasing labour involvement. responding to constraints that had made europeans largely reject mass production in the interwar era relative lack of capital, smaller volumes of production and higher variety of models toyota started to introduce novel production principles. these included an imperative to design production for continuous product and process improvement (vs. the fairly limited flexibility of the american approach), using worker expertise as a constant input for production improvement (vs. restricting workers to the role of task execution), allowing workers to stop the production process upon spotting an error (vs. having to succumb to the dictate of the assembly line) and 'just-in-time' provision of an exact amount of supplies (vs. 'just-in-case' buffering of stocks) [ , [ ] [ ] [ ] (see figure ) . similarly to ford's early activities these rules emerged in a piecemeal fashion: by the end of the s toyota's practices had not been even formally codified yet [ ] . post-war decades were characterized by the continued deepening of mass production in systems where it had been previously employed, reflected in a move from vehicles and consumer durables to other system components. for example, in order to employ picking machines in the american food system, a standardized tomato with a thicker skin had to be bred, picked green, chemically ripened and 'enhanced' with salt and sugar to compensate for the loss of flavour [ , pp. - ) . another way of deepening involved a shift from previously attempted full-scale imitation to more selective adoption. for example, whereas the 'move work to worker' principle had been abandoned in the housing system, the use of standardized components and architecture still enabled a rapid construction of blocks of identical-looking houses [ , pp. - ] . as a result couplings between systems became consolidated in a manner that would stimulate the further deployment of mass production. this was the case for us mobility and housing systems: mass produced cars enabled to live further from the city centre facilitating urban sprawl, whereas the creation of new suburbs without efficiently functioning public transport amplified the need for vehicles. figure . 'provide supplies exactly where/when required' and 'anyone should be able to stop production upon spotting an error' rules contrasted to traditional mass production [ , p. ] . note the use of the word 'automation' for describing the latter. cold war provided another stimulus for the take-up of mass production as the competition between two superpowers also involved claims about living standard. through channels such as the marshall plan, the productivity mission, fulbright program and united states information agency mass production techniques, corporate organization structures, management styles, advertising, and consumer goods started to flood the transatlantic region. although the adoption of mass production remained selective and uneven [ ] in many ways european countries started to become more similar to usa [ ] . these influences extended beyond the iron curtain, e.g. the idea of mass produced people's car à la volkswagen beetle was present in east germany, soviet union and czechoslovakia [ ] . however, as the socialist bloc prioritized the needs of the military-industrial complex both the availability and quality of consumer goods remained notably lower [ ] . as a new direction automation was highly visible. by , usa had , 'automatic assembly machines' applied to the production of automobiles, electric motors, tv-s and radios. the role of automation in post-war industrial reconstruction was also stressed in germany, france, ussr and japan [ , pp. , - ] . in contrast, very little was known about japanese experiments in the west. toyota's engineers themselves, however, acquired information from diverse sources, including us factory visits and thorough studies of various production practices. a strive towards continuous improvements was realized through the deployment of statistical methods of quality control which had been pioneered in usa during wwii but largely abandoned thereafter. just-intime supply was derived from the supply chain practices of american supermarkets, whereas the self-stoppage mechanisms of toyota's pre-war era automatic looms inspired an idea to let the workers stop the assembly line upon spotting an error [ , pp. - ; ] . although the post-war era was initially characterized by the immediate eruption of major strike waves in the entire region [ ] , a general trend was the gradual institutionalization of labour conflict in the entire region. on one hand unions came to be acknowledged as a legitimate part of statecapital-labour nexus; on the other hand their activities were increasingly restricted by law (e.g. the introduction of taft-hartley act in usa in ). continued but decreasing level of strikes was thus accompanied by the increasingly routine and ritual nature of these activities [ ] . the increasing acceptance of mass production was also evident in its relation to possible alternatives such as specialty production. instead of asking whether to adopt mass production, different countries outside usa now focused on the question of how to make best use of these techniques in given national conditions [ , pp. - ; ] . in terms of societal effects mass production was heavily propagated as a basis for postwar reconstruction with rising productivity linked to higher living standards and social progress. the general theme of prosperity was now dominant in the entire region. however, the cultural critique of mass production also started to spread in a similar manner. increasingly influential authors of the frankfurt school argued that alienation was a consequence of the concentration of production into large-scale monopolistic corporations, the division of labour between mental and manual work and the increasing routineness and monotony of work tasks [ ] . importantly, the sources of alienation were not seen as specific to particular economic or political systems but rather the underlying features of all modern industrial societies. for example, fromm claimed that both capitalist and communist countries were developing into managerial societies that "make machines that act like men and produce men who act like machines" [ , p. ]. ( - ) in the s the excesses of mass production started to provoke radical responses from niches. counterculture activists increasingly experimented with food cooperatives and small-scale enterprises under local ownership, emphasizing home-made goods, recycling, organic food, smallscale technologies, and do-it-yourself furniture, food, energy and shelter [ , pp. - ] . in parallel, with the initiation of border industrialization program in some american corporations started to shift manufacturing jobs to northern mexico [ ] . this enabled to sustain profitability by cutting the labour costs while also decoupling production in one location from end use in another. although the broader implications were not fully understood at the time, the expansion and knock-on effects of offshoring -de-industrializing the west and putting an end to the post-war welfare state compromise -would come to exhibit significant global impacts over the following decades [ , ] . the american ideal of computer-aided design and manufacturing continued to hold sway as reflected, for example, in the development of numerically-controlled machine tools [ ] . although influential experts such as norbert wiener expected full factory automation to occur by the s, numerous technical difficulties resulted in far more modest outcomes: by mid- s the number of robots in us enterprises was still around , [ , pp. - ] . continuing large investments in r&d and various experiments in the field of information technologies nevertheless helped to establish a foundation to the th great surge of development, unfolding from the early s [ , ] . whereas automation had run into difficulties in usa, the japanese alternative was gradually maturing to take the world by surprise in the s. novel principles included a strive to implement changeover as quickly as possible (e.g. shigeo shingo's single-minute exchange dies), designing products so that they would be easy to manufacture, organizing work in teams of multi-skilled workers and maintaining close and cooperative relations throughout the entire supply chain [ , , , ] . in the mid- s the new approach was formally documented for the first time as the toyota production system [ ] . further consolidation of mass production in different systems and countries continued to be the dominant theme in the s, reflected in the diffusion of various consumer goods such as automobiles, refrigerators or tv sets (see figure ). the logic of mass production and consumption had also pervaded the socialist bloc: for example, the soviet seven-year plan for - promised to match the usa in housing and consumer durables. the resulting 'consumer socialism' [ ] attempted to emulate the west in terms of product quality and availability. as a result, one could observe the homogenization of energy and materials usage profiles in the region: whereas the consumerist west was producing increasing amounts of waste, the more limited consumption in the east was amply offset by the deployment of inefficient production technologies [ ] and general disregard of environmental impacts. the situation with american and japanese approaches to mass production continued to be similar: high international visibility of the automation discourse whereas toyota's experiments remained largely unknown outside japan. notably, the increasing reach of mass production stimulated niche responses on the same scale, e.g. the internationalization of organic farming [ , pp. - ] and appropriate technology movement [ , pp. - ] . by s two broad trends in the contestation of mass production had become visible: geographical homogenization and broadening of resistance. as european countries increasingly followed the us lead in adopting mass production, the societal critique associating mass production with cultural homogenization, loss of individuality, creativity and spontaneity was now increasingly present in the usa. by the end of the decade, the contestation of mass production had escalated into an all-out attack on industrial society and its underlying values of efficiency, standardization and businessdrivenness [ , pp. - ] . these sentiments were expressed in many influential works by herbert marcuse, jacques ellul, theodore roszak and lewis mumford [ ] [ ] [ ] [ ] . a novel strand of critique [ , ] attacked the western lifestyle for its high use of energy, materials, food and water, increasing production of waste, throw-away consumer culture, and the environmental consequences of these practices. the different tensions culminated in as mass anti-establishment protests spread over the us and europe, most notably in france and czechoslovakia, involving a wide range of people from workers to students. in the first earth day celebration in usa brought together million people, including students and housewives' organizations, further demonstrating the power of citizen activism [ , p. ]. the analysis in this section follows the structure of section where we describe our theoretical framework. we begin by comparing the empirical patterns evident in every period to the phasespecific model of dt consisting of six propositions (gestation, irruption, frenzy, turning point, synergy, maturity). we then move to comparing the patterns that we detected throughout the periods to the three overall trends prescribed by the dt framework (variation, diffusion, contestation). for all six phases we cover main trends in niches and single systems (rules/regimes), interconnected systems (meta-rules/meta-regimes) and on the landscape level. this yields cells presented in table . each cell has been colour coded to facilitate obtaining a quick visual overview of the main results. green was used when the authors agreed that the development of mass production was yellow was used when the authors either failed to find empirical support for a particular proposition or when unexpected findings were detected. these assessments reflect the authors' interpretive consensus as decisions whether the findings are 'largely in line' or 'sufficiently deviant' entail an unavoidable and irreducible amount of subjectivity. although below we try to substantiate each theoretical claim with empirical illustrations, the reader is referred back to section for more empirical detail backing up our conclusions. shortage, road and rail networks, population growth, big corporations, rise of engineering, cultural values) set the stage for the emergence of mass production - rules/regimes: alignment of various rules into mass production regime in the mobility system (ford, usa) meta-rules/meta-regimes: early experiments with mass production in food and defence systems (tractors, artillery shells), including some initial setbacks (submarine patrol boats) landscape: wwi stimulates the extension of mass production to new systems and locations (europe) - rules/regimes: 'flexible mass production' emerges in the mobility system (general motors, usa) meta-rules/meta-regimes: competition between rulesets (fordist vs. flexible mass production), selective adoption of us rules in europe resulting in regional variations (bataism, bedaux system); emergence and consolidation of links within and between systems (e.g. energy-housing, mobility-communication) landscape: great depression decreases the utopian promises of mass production, while stimulating increasingly organized worker resistance - rules/regimes: experiments with flexible and just-intime production of airplanes (usa and europe) meta-rules/meta-regimes: consolidation of mass production in some systems (food) and extension in others (ships and airplanes in the defence system); factories as structural couplings (e.g. between mobility and defence systems) landscape: direct and indirect effects of wwii (creation of productive capacity, putting the resolution of labour conflict on hold, forced savings, acute shortages) increasingly favour mass production - rules/regimes: experiments with automation (usa), and constant improvement (toyota, japan) meta-rules/meta-regimes: societal embedding of mass production, further consolidation in systems and 'americanization' of the transatlantic region landscape: facilitated by cold war, mass production starts to contribute to long-lasting post-war economic growth in the entire region rules/regimes: further development of variations of mass production (usa, japan), resurgence of radical alternatives to mass production in niches (organic farming, local production, appropriate technology) meta-rules/meta-regimes: dominant meta-regime further optimized (start of offshoring, usa) landscape: mass production increasingly associated with broad societal fears (cultural homogenization, alienation) and severe environmental impacts (high resource use, waste, pollution) in general we assess that the development of mass production largely matches the theoretical expectations in cells out of . furthermore, apart from frenzy, all phases contained minimally a / match with the expected dynamics. the frenzy phase was also the only one where yellow colour was partly used because of our inability to find supporting evidence. namely, whereas the theory stressed the role of transnational organizations in meta-regime building, our evidence pointed to the role of national organizations instead (e.g. bhpc in uk, rkw in germany). this might reflect a source bias of our study as historians of technology have fairly recently started to focus on transnational organizations [ , ] . in all other instances yellow colour denotes unexpected findings that indicate the need to extend the dt framework. first, based on our analysis of meta-regime building in the frenzy phase we propose that in addition to structural and functional couplings systems also link to each other through shared discourses or 'rhetorical couplings'. these couplings manifest themselves in three ways: ) claims about the paradigmatic system as a direct source of imitation, e.g. interwar era beliefs that assembly line techniques from car production can be applied to prefabricated housing; ) downplaying the difficulties associated with cross-system or cross-regional knowledge transfer, e.g. ford's belief that mass production can be adopted anywhere with minimal changes; ) abstracting the technical principles of the meta-regime into more general concepts, e.g. the use of mass production as a loose mobilizing metaphor or part of an overall societal philosophy. whereas the first two strategies often led to setbacks and failures when attempting to move from rhetoric to practice, the diffuseness, openness and flexibility of 'generalized fordism' enabled to connect different systems on a rhetorical level in the entire transatlantic region. somewhat paradoxically these findings suggest that, at least in early stages, rhetorical couplings facilitate meta-regime building because of the high degree of abstraction of claims involved, not because of their specificity. the rest of the deviations in each phase (from irruption to maturity) relate to the effect of landscape dynamics on the evolution of rules and rule-sets throughout the entire surge. based on our data we distinguish between three types of landscape impacts: ) stimulating new rules and rule-sets, e.g. the set of various broad factors in usa facilitating the precursors of mass production (gestation phase); ) amplifying existing rules and rule-sets, e.g. the role of wwi (irruption phase) and the cold war (synergy and maturity phases) in extending mass production to new systems and locations; ) closing off alternative directions, e.g. the role of great depression in detaching mass production from its utopian promises of societal overhaul (frenzy phase). importantly, as a turning point event wwii exerted all three types of effects, e.g. experiments with flexible production of airplanes, extension of mass production in defence and food systems, and the reduction of possibilities for alternative practices such as organic farming. our findings complement the theoretical model of dt that restricts the importance of landscape impacts largely to early stages and the turning point. they also call for more reflections on the role of the landscape in the study of single system transitions where exogenous pressures are often relegated to the role of early enablers [ , ] . moreover, the results call into question conceptualizations in long wave literature claiming surges to be driven by an internal logic, e.g. the shifting balance between production and finance capital in perez's [ ] model. this suggests two possibilities: either these literatures have so far paid insufficient attention to landscape dynamics or meta-regimes are somehow distinctive in requiring continuous landscape support. in any case, further theorization of the stimulating, amplifying and terminating effects of landscape dynamics is warranted. we now move forward to assessing the cross-phase trends related to the variation, diffusion and contestation of rules. the main results are presented on figure . note that similarly to interpretive accounts in long wave and transitions literature [ , p. ; , p. ] the curves are meant as a visual aid for the reader and do not reflect the results of a strict quantitative measurement. as with the previous section, the assessments rather reflect the authors' interpretive consensus. the results largely confirm the expected pattern in variation: gestation was characterized by high variety in different niches, irruption to synergy by the emergence, gradual improvements and societal embedding of mass production, and maturity by the resurgence of radical alternatives to mass production. however, we also found that major experiments with mass production continued in the post-war era, e.g. automation in usa and constant improvement of production in japan. this suggests that the existing dt model has somewhat overstated the incremental nature of innovation during the synergy phase. the dt framework thereby downplays the possibility that niche experiments directed at improving the existing meta-regime might eventually accumulate into a significant transformation of this meta-regime. this is precisely what happened in japan where toyota's experiments in the late s gradually grew into a distinctively novel 'lean' version of mass production that started to reshape global manufacturing from the s [ ] . more research is thus required to find out what conditions facilitate the emergence of radical internal variations of meta-regimes and what factors enable to scale up these variations. the general trend was also confirmed for diffusion: despite various setbacks, mass production spread through selective adoption to successive systems and countries throughout the entire surge. however, our analysis also revealed specific channels of early diffusion: ) mass production was more likely to move from one location to another when the underlying systems were similar, e.g. the early adoption of us techniques by french car producers; ) mass production was more likely to move from one system to another in a given location, e.g. an early extension of mass production to the food system in usa; ) in different systems mass production was first more likely to be applied to similar technologies, e.g. the use of mechanized vehicles in the american food system preceding the standardization of tomatoes. these findings are in line with evolutionary economic geography which has demonstrated the role of different forms of proximity (cognitive, organizational, social, institutional and geographical) in facilitating learning and innovation [ ] [ ] [ ] . however, explanations relying on differing forms of proximity fail to explain why radical movements and totalitarian governments were initially most receptive to mass production [ ] . in our view this observation might be better explained by the mechanism of social differentiation as used in sociological and marketing research [ , ] . works in these disciplines show that consumers use new products to express individual identity, to highlight their group affiliation and to signal their distinctiveness from other social groups. mcmeekin and southerton [ ] have applied this idea to socio-technical systems, arguing that contrasting group definitions of users can either support or hinder transitions. our findings suggest that the mechanism of social differentiation can be further extended to entire societies and to the diffusion of meta-regimes. this is in line with recent historical literature, highlighting the central role of fordism in the attempts of nazi germany and soviet union to create a societal alternative to western liberal capitalism [ , ] . in terms of contestation our findings confirmed that irruption and synergy were characterized by lower degrees of conflict than frenzy and maturity phases. however, the specific forms of contestation turned out to be quite different before and after wwii. during irruption and frenzy phases we observed a high degree of initial uncertainty about the societal impacts of mass production (e.g. celebration of and worries about the -dollar day in usa) and individualized worker resistance being reduced to fewer but regionally varying themes by the s (organized worker resistance in usa where mass production was being adopted, cultural critique in europe where mass production was resisted). during synergy and frenzy phases we first observed the homogenization of themes of contestation across the entire region (e.g. institutionalization of labour conflict in western europe, cultural critique of mass production being taken up in usa). this was followed by the generalization of contestation to broader societal and environmental themes of j o u r n a l p r e -p r o o f which mass production was only a part (e.g. revolutions of , earth day in ). we suggest that the homogenization of contestation mirrors the transatlantic diffusion of rules (i.e. equalization of both experienced issues and anticipated fears), whereas the generalization of critique reflects the impacts of the meta-regime on the socio-material landscape. in this paper we have provided the first systematic empirical assessment of the co-evolutionary rulebased model of deep transitions [ ] by studying the development of mass production in the transatlantic region between to . our main findings are as follows: . in general the historical evolution of mass production matches the expected phase-specific patterns of the model. unexpected findings include the lack of empirical evidence of the role transnational actors in the frenzy phase, the importance of landscape dynamics throughout the entire surge and the role of rhetorical couplings in connecting different systems. . expected cross-phase patterns were also largely detected in the variation, diffusion and contestation of rules and rule-sets. unexpected findings include the emergence of alternative versions of the dominant meta-regime in niches even during synergy and maturity phases, the role of various forms of proximity and social differentiation in facilitating the diffusion of meta-regimes, and contestation mirroring the increasing scale, scope and societal embeddedness of the meta-regime. . landscape dynamics were found to support the evolution of the emerging meta-regime by a) stimulating new rules and rule-sets; b) amplifying existing rules and rule-sets; and; c) closing off alternative directions. the turning point event exerts all three types of impacts. . meta-regime building is facilitated by three mechanisms: rhetorical couplings, proximity and social differentiation. interestingly, we found that the optimistic expectations maintained by rhetoric and utopian promises of societal differentiation can counterbalance the considerable difficulties of implementing the meta-regime in practice. although our results have largely confirmed the dt model, the support offered by an exploratory qualitative case study remains tentative. the next step is to move from solely qualitative assessments to mixed method approaches, combining historical narratives with text mining techniques. this enables to quantify the emergence and alignment of rules but also to interpret the findings in a historically informed manner [blinded, forthcoming]. also, mass production constituted a case most likely to follow the theoretical propositions. further work is required to find out whether the model applies to more recent meta-regimes such as digitalization or circular economy. these cases also offer opportunities to look into the operation of mechanisms uncovered by our study, e.g. the fourth industrial revolution [ ] as a rhetorical coupling connecting the digitalization of industry to the promise of sustainability, or societal differentiation as a mechanism connecting big data, ai, facial recognition software and social credit schemes to the chinese vision of 'harmonized' society [ , ] . one can also analyse the role of landscape events in shaping these meta-regimes, e.g. the role of / or the current coronavirus pandemic in facilitating the societal embedding of digitalization towards increased citizen monitoring and surveillance in usa and china respectively [ , ] . similarly to the early applications of the multi-level perspective the dt framework has so far primarily focused on the identification of broad patterns. although empirical work has begun on the role of transnational actors in shaping emerging meta-regimes [ ] , van der vleuten [ ] has argued for the need to broaden the analysis to 'system entanglers', producing cross-regional similarities and differences between systems. another theme which has not been tackled by transitions studies, long wave literature or the dt framework itself, is the continued evolution and transformation of mature meta-regimes during new surges. the interactions of mass production, it 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monitoring could be here to stay adopting and diffusing the circular economy as a policy concept: the case of the european union radical change and deep transitions: lessons from europe's infrastructure transition - this article has been discussed with various colleagues at the international sustainability transitions conference and in a series of workshops, including the deep transitions research team and invited guests. in particular we would like to thank johan schot, ed steinmueller, carlota perez, david nye and phil scranton for their informative comments. this work was supported by james anderson and baillie gifford & co.j o u r n a l p r e -p r o o f key: cord- -wfiqbu m authors: upadhyay, ranjit kumar; chatterjee, sourin; saha, satvik; azad, rajeev k. title: age-group-targeted testing for covid- as a new prevention strategy date: - - journal: nonlinear dyn doi: . /s - - -x sha: doc_id: cord_uid: wfiqbu m robust testing and tracing are key to fighting the menace of coronavirus disease (covid- ). this outbreak has progressed with tremendous impact on human life, society and economy. in this paper, we propose an age-structured siqr model to track the progression of the pandemic in india, italy and usa, taking into account the different age structures of these countries. we have made predictions about the disease dynamics, identified the most infected age groups and analysed the effectiveness of social distancing measures taken in the early stages of infection. the basic reproductive ratio [formula: see text] has been numerically calculated for each country. we propose a strategy of age-targeted testing, with increased testing in the most proportionally infected age groups. we observe a marked flattening of the infection curve upon simulating increased testing in the – year age groups in india. thus, we conclude that social distancing and widespread testing are effective methods of control, with emphasis on testing and identifying the hot spots of highly infected populations. it has also been suggested that a complete lockdown, followed by lockdowns in selected regions, is more effective than the reverse. covid- , a respiratory disease caused by a new strain of coronavirus (sars-cov- ), has spread to almost every part of the world, since first reported in december [ ] . who declared this outbreak a 'public health emergency of international concern' on january . as of april , it has caused , , deaths with , , confirmed cases of infection. till date, usa has the largest number of fatalities, followed by italy. usa reports , , cases, with cases per million people, though the statistics have still been evolving due to the large variability in testing performed by different countries as of now [ ] . in the absence of any vaccine to prevent and contain the spread of novel coronavirus disease, covid- , as well as the lack of an established treatment regimen to cure this disease (beyond mitigation of symptoms), effective non-pharmaceutical interventions are needed to contain the epidemic and minimize morbidity and mortality associated with this respiratory disease. as the covid- pandemic has swept across the globe, now affecting almost all countries, measures for mitigation have been put in place that include strict lockdown to less restrictive people movement but all aiming to achieve social distancing of different degrees to balance the socio-economic impacts of the lockdown and the disease. robust testing and tracing are key to containing the pandemic and effectively 'flattening' the infection curve, both by distributing cases over a longer period of time and by reducing the total number of cases, and thus lowering the epidemic peak. governments and health agencies have banked on mathematical models to guide towards the goal of optimizing available resources to attain maximal benefits of mitigation measures. mathematical models are often based on certain assumptions; however, these are continually improved upon through adjustments guided by emerging data, and eventually, these models become more reliable in helping navigate through such situations. here, we leverage the emerging information from covid- in different countries, mainly usa, italy and india, to develop a covid- specific model that can inform on effective interventions for coronavirus containment. as people of different age groups have responded differently to coronavirus, we utilized the age-stratified data of covid- to develop a system that can inform on more effective prevention strategies. we particularly focused on india where covid- seems to have not peaked yet despite the most restrictive lockdown imposed for over a month now. our model recommends that testing and tracing be ramped up in the - -year age-group population in india in order to flatten the infection curve in shortest time possible in the current situation. we further demonstrate this by computing the basic reproductive ratio r at different times and following an age-grouptargeted intervention. the novel coronavirus is thought to have originated in bats and eventually infected humans, due to the similarity in the genome sequence of sars-cov- to that of a bat coronavirus [ ] . human-to-human transmission has also been established. so far, we observe different transmission and fatality rates in different countries. one of the main reasons for this is differing age groups and social contact structures. in order to study this effect, we use social contact matrices, which show contact patterns of an age group with others and are used to parametrize mathematical methods to understand the transmission patterns. schenzle [ ] used an age-structured seir model with age groups to study the spread of measles, a disease which mostly affects children. this method has previously been applied to respiratory diseases like influenza [ ] , pertussis [ ] and varicella [ ] . given that covid- transmission patterns are very similar to those of respiratory diseases caused by other viruses, we can get valuable information by studying covid- disease dynamics through an age-structured model. it has been observed that social distancing, isolating infected populations and quarantine are effective ways of containing the epidemic. after receiving the best available medical intervention to date, a quarter of critically ill patients still die, signifying that the host response to the virus is an important factor [ ] . thus, the government and hospitals need to procure supportive care equipment in sufficient amounts. initiating the process of flattening the curve by these abovementioned methods provides the time to prepare for supportive care. with social distancing becoming a preventive strategy, many countries have announced partial or complete lockdowns. numerous companies are also advising their employees to 'work from home'. due to this, patterns of mixing between people change, the effects of which are hard to represent with classical compartmental disease models. however, it is essential to understand these changing contact patterns in order to more accurately model the disease dynamics. considering the differential impacts of an infectious disease on people in different age groups, perhaps due to a number of reasons including physiology, immunity, mobility, and social contact and behaviour, an infectious disease model must consider differential age-group disease dynamics. recently, some new approaches about age structure population models have been proposed in [ , ] . age-structured models offer better approximations of reality and also give health organizations better tools to develop age-group-targeted control strategies. here, we have simulated the spread of novel coronavirus using such an age-structured siqr model. we have fitted our model to the current situations in italy and usa and have estimated age-wise mortality rates. side by side, we compared this with the scenario in india. we have also analysed the success of lockdown measures adopted by these countries qualitatively and have projected the effects of further lockdowns. we examine whether starting off with a complete lock-down which is then gradually lifted in specific areas is more effective than the reverse, i.e. declaration of lockdown regionally followed by a nationwide lockdown. finally, we have proposed a novel method of age-group-targeted testing to tackle the situation and have also showed how it can help in flattening the curve effectively. this manuscript is organized as follows. in sect. , we formulate the mathematical framework of an siqr epidemic model. in sect. , we analyse the predictions made by a numerical simulation of our model. in sect. , we propose control strategies and their intended impacts. finally, in sect. , we discuss the conclusions. the basic reproductive ratio r is calculated in "appendix a", and various parameters used in our numerical simulation are tabulated in "appendix b". the mathematical framework of an age-structured siqr epidemiological model is formulated. in order to construct the model, our assumptions are stated as follows: . the entire population, n , is divided into four compartments: susceptible population, s (which are under risk of contracting the infection), infected population, i (which consists of infectious, both symptomatic and asymptomatic, or untested individuals), quarantined population, q (which are removed from all contacts within the entire population and are hence not infectious) and recovered class, r (which are recovered from infection). therefore, we have n = s + i + q + r. in order to keep track of disease-induced death, we assign an additional compartment of fatalities, f. . each population of siqr and f is further subdivided into m age classes. individuals within the same compartment interact with other individuals proportional to a coefficient of interaction c i j , which specifies the average contact between age classes i and j, with < i, j ≤ m. . there is no recruitment of the susceptible population and no natural death in any compartment. there is also no aging of individuals. . the disease is transmitted from the infected to the susceptible population with age class i, i.e. compartment s i , at a rate βλ i . here, β is the transmission probability, and λ i is the weighted coefficient fig. schematic diagram of the interacting population within a certain age class, for the model ( ) of contact of age class i with the entire infected population. individuals from compartment s i move into compartment i i and become infective immediately. . individuals from compartment i i move into compartment q i at a rate δ i and are no longer infectious. . the infected and quarantined populations, i i and q i , recover and move into the recovered population, r i , at rates and γ , respectively. . the populations i i and q i suffer disease-induced death, at a common rate of μ i . a schematic diagram of the interacting population is presented in fig. . thus, the transmission process is formulated by the following system of differential equations: the initial conditions are s i ( ) > , all the parameters in the system are positive quantities. we break down the age-structured social contact matrix, c = [c i j ] m×m , into the contributions from households, workplaces, schools (all educational institutions) and other areas (market places, restaurants, cinema halls, shopping malls, etc.), represented by c h , c w , c s and c o , respectively. each of these is weighted with coefficients α h , α w , α s and α o , which we change over time to reflect the effect of lockdown on social contact. for example, during the time period when all educational institutes are closed, we set α s = . we note that when italy and usa announced partial lockdowns, the coefficients α have been fitted to the existing data. it must also be noted that even during a complete lockdown, the contributions to the contact matrix from work and other areas are never zero, as people involved in essential services continue work and marketplaces must operate to some degree. lockdowns also induce an increase in household contact, as people are staying at home more [ ] . for numerical simulation, we have collected data on the times and nature of lockdown imposed, starting from the closure of schools and universities to complete lockdowns [ ] [ ] [ ] . a complete lockdown in india was declared on march . usa declared a national emergency on march , followed by various statewise guidelines and orders to 'stay at home'. italy had also proceeded towards lockdown step by step, with a lockdown of the northern provinces on march and a nationwide lockdown from march onwards. the parameters δ i , γ and have been assumed, using known rates of infection, recovery and the first appearance of symptoms. we assume the onset of symptoms, detection and isolation in . days on average [ ] for infected individuals. we also assume that an individual stays quarantined for days, which is the average recovery period for a symptomatic individual [ ] . in addition, we assume that unidentified infected individuals, either asymptomatic or untested, can proceed straight to recovery after days of infection on average. the parameters β and μ i have been fitted to existing case and fatality data. the values of β for india, usa and italy are . , . and . , respectively. the values of the remaining parameters are listed in "appendix b" (tables , ). it must be noted that we choose to interpret currently reported cases to belong in the quarantined compartment, q, which is a subset of the total infected population. we have also chosen our initial conditions such that i i ( ) = q i ( ), on the assumption that cases are underreported where exactly half of all infected individuals are identified (quarantined). for india and italy, we use m = age classes, equally dividing the range of ages - years. due to unavailability of data, we use m = age classes for the usa, over a range - years. for each country, we have set up m differential equations, which we have integrated using the python module 'numpy'. we have collected covid- case and mortality data up to april for each country from worldometers [ ]. we have also collected age-structure data from popu-lationpyramid [ ] and social contact data from prem et al. [ ] . the age classes and initial susceptible population data are presented in table . the infection curves predicted by our model are shown in fig. . we note that our model predicts a fairly symmetric infection curve, whose peak trails behind the peak of the quarantined population. usa and india continue an upward growth, while italy's infection curve has begun to drop. if current conditions continue without any new measures being taken, active cases are predicted to reach peak within months for usa and around months for india. we also predict that everything will be normal in months for italy, without considering the effect of herd immunity. on the other hand, it may take a year at worst for india and usa to fully recover. by 'normal', we mean that the number of infected individuals has dropped below one thousand, which represents a sufficiently small number of cases relative to the populations of the countries in consideration to be fully identified and isolated. we observe a common trend in infected age groups across all three countries, in that the young-and middleaged groups (between and years of age) have the largest numbers of infected people, relative to the initial susceptible population size of that age group. we estimate this by measuring the drop in s i from its initial value across all age groups i over time, as shown in fig. . this may be explained by larger contact coefficients α among themselves and other age groups. the population within this age group is also highly mobile, in each country. on the other hand, data suggest that infection is less common in children [ ] . dr. calum semple, professor at liverpool university, stated that 'we know that lung development doesn't finish until teenage years. ace is highly regulated in lung development. because of that the "lock" might be expressed differ-ently in kids' lungs' [ ] . hence, we have identified the - -year-old age group to be the most infected age group across all three countries (fig. ) . in our proposed control strategy, we thus place less emphasis on individuals younger than years. considering different mortality rates across different age groups has very little effect on the total infection and mortality curves, both qualitatively and quantitatively. we note that italy's mortality curve in fig. f has begun to flatten, far quicker than our model would sug- gest. we may explain this by noting that the reported mortality rates, which we used to fit our model, are likely inaccurate due to factors such as sampling bias and the changing capability of health care system. with time, as italy continues to improve medical facilities and mobilize doctors and nurses, patients receive better care and facilities are no longer swamped as they were in the early stages of the pandemic. we also note that with time, the mechanisms by which covid- causes death might be better understood by scientists and practitioners, so the observed lower mortality rate may also be explained by better, more effective treatment and the identification of drugs which improve survival chances. presently, the overall mortality rate is the highest in italy, followed by india and the usa (fig. ) . interpreting these mortality rates is complicated by the fact that pre-existing medical conditions play a major role and are somewhat correlated with age. fatality rates may also be inflated by limited testing and the resulting selection bias in which asymptomatic individuals are not accounted for. in addition, the fact that our model omits - -year age group in the usa, despite a high expected mortality rate in that group, may explain why our model predicts a lower mortality rate in usa, compared to india and italy. the basic reproductive ratio r can be interpreted as the expected number of cases directly caused by a single infected individual in a completely susceptible population. when r > , the infection spreads in the population, and it does so more rapidly with higher r . when r < , the infection eventually dies out, and the system proceeds towards the disease-free equilibrium. we have calculated r for the three countries, as presented in table . a lower r value for india initially indicates a comparatively slower spread of disease than in usa and italy. figure illustrates the decrease in r with the implementation of social distancing measures. the drop in r is significant in all cases, although only italy shows r < , which is enough for the disease to die out. concerningly, our model shows very little impact of the lockdown in india, compared to the projections without one. after fitting our model to case numbers before the implementation of social distancing measures, our model predicts that complete stop of contact between people outside their home, or even a % reduction in such contact, is not consistent with subsequent reported cases. the three different countries show different degrees of reduction in contact, in terms of different control coefficients α. this effective reduction in contact in india seems to be far less than in usa, which in turn is less than in italy. in the case of italy, data look promising, suggesting that the implementation of lockdown was more successful. the effectiveness of control strategies can be measured by the basic reproductive ratio r . the parameters which can practically reduce r are the contact coefficients, α and the rate of quarantine of infected individuals, δ i . social distancing works well at reducing interpersonal contact, but we can see that implementation issues can severely damage its effectiveness. by sufficiently increasing the number of tests carried out, we can identify and quarantine infected individuals more quickly, thus indirectly increasing δ i . this means that infected individuals would have a lower probability of infecting a susceptible individual. on the other hand, testing rates are limited by the medical resources of each country. while south korea has managed to test a large fraction of their population, in countries with very large populations such as india, randomized testing for the entire population is not feasible. hence, we suggest an age-group-targeted testing initiative, where the age groups with the largest number of infected individuals are targeted. in addition, economic activities have come to a halt during lockdown in india. barclays has estimated a loss of up to . billion usd in india [ ] . practically, it may not be possible to continue a complete lockdown indefinitely. only a multipronged approach can successfully combat an outbreak of infectious disease. though we have not provided any medical insights, they should go hand in hand with the strategies we propose here. we discuss two strategies below. . keeping in mind that individuals in the age group - years are most likely to catch infection, we emphasize testing more people from this age group rather than randomized testing. this will help in isolating infected people and restrict their disease transmission. the impact of such a strategy is evident from the curves in fig. . the corresponding change in r is illustrated in fig. b . we see that although r has not dropped below , our strategy offers a significant improvement. for this simulation, we have increased the value of δ i for the targeted age group - years, which corresponds to i ∈ { , , , , } ( table ). the initial population sizes of these age groups are shown in table . we have assumed that infected members of these groups can proceed to quarantine in . days, on average. for the remaining groups, we increase the detection period to . days. concentrating testing on groups most likely to have infected members helps bring them out of contact with the susceptible population and hence flatten the infection curves. this in turn lowers the peak number of critical cases, thus distributing the workload of medical facilities over a longer period of time. the peak is also observed far later than with normal rates of testing, although normalcy is restored not much later. . we suggest that a complete lockdown, followed by lockdowns in selected regions, is more effective than the reverse. as symptoms take time to manifest, infection can spread very rapidly to areas not under sufficient lockdown. however, if the majority of infected individuals can by identified and isolated by testing during the lockdown period, subsequent lockdowns can target those areas with larger infected populations. this would effectively reduce disease transmission across a country. we suggest that introducing a lockdown in slow phases, as was done in italy and usa, may not have been as effective as a complete lockdown introduced in the early stages of the pandemic. however, we do acknowledge differences in the socio-economic structures and dynamics of different countries, which demand differential strategies tailored individually to their underlying structures. our model is ill equipped to model lockdowns in selected regions, but we have approximated this effect with reduced contact coefficients α. our model of covid- dynamics allows us to make some useful predictions and modify pre-existing strategies to obtain better results. this model has been tuned with available data of social contact matrices and reported deaths and infected individuals available till april . methods of testing and social distancing are known to tackle this kind of situation. in the context of this novel disease, we have re-examined these methods. it must be noted that in our model, we have interpreted the number of reported cases as the number of symptomatic or tested cases, and we assume that they are transferred to quarantine as soon as they are confirmed positive. the actual number of covid- cases is much higher than reported, which is consistent with the nature of the reported infection curve. we must note that as of april , india has conducted , , tests. this is a major step up from the initial , tests before the declaration of lockdown. at this milestone of tests, india has recorded around , positive cases, as opposed to usa's , , and italy's , cases [ ] . india has also observed a comparatively lower mortality rate. this can be explained by her disproportionately young population, together with the low rate of infection and transmission in the younger age groups. the mean age of india's population is . , compared to usa's . and italy's . [ ] . we acknowledge that there are uncertainties in determining the model parameters due to unavailability of proper data and that this may lead to incorrect predictions. we note that the use of mortality data is partially motivated by the fact that such figures are more likely to be reliable, as opposed to infection numbers which suffer from under-reporting. the choice of a single transmission coefficient β across all age groups is because of the lack of age-group-specific estimates on transmission probabilities, as well as the complexity of fitting such coefficients for each of the age groups even if available. similarly, disease-induced mortality rates across the infected (both symptomatic and asymptomatic) and quarantined populations have been assumed to be the same, μ. while these rates may indeed differ among these groups in reality since severe cases are frequently quarantined, we justify this assumption with our simulation which closely mimics the trends in available data without introducing additional parameters. furthermore, we emphasize, again because of these reasons, that factors such as agedependent immunity have not been incorporated into our model, and the coefficients λ i merely reflect normalized amounts of contact between age classes. our initial conditions i i ( ) = q i ( ), which reflect the assumption that half of the cases are reported, may be adjusted with better estimates of the fraction of reported cases. the assumption that births, natural deaths and aging are absent is valid only over relatively short periods of time. we have also focused on the population below years of age, as the rest of the population is significantly small. with these assumptions, we can clearly say that our model can make short-term predictions, but cannot reliably make long-term forecasts. in this instance, we have run our simulation for a maximum time period of months. we also acknowledge that partial lockdowns of infection hot spots are not well modelled by our method, which considers a given region as a whole. with the availability of reliable data, we may be able to apply our model on smaller populations and make region-wise predictions. we also post-pone the application of targeted testing to other countries, such as usa and italy, for further study. through this study, we have offered a more efficient, country-specific covid- model for informing on strategies to contain the sars-cov- pandemic. our contribution of a new age-stratified model will aid government and health agencies and will spur further research in covd- modelling. the implications of our proposed work are timely as this is still an emerging situation in many countries and perhaps broad as well, as viral pandemics are predicted to keep re-emerging in the near future, perhaps in different shades or shapes. keeping in mind that all models are merely approximations of reality, we hope that this model can aid in developing policy, with economic and medical perspectives. here, we find the basic reproduction number using the next-generation method [ ] . we first linearize the system of equations ( ) at disease-free equilibrium, where hence, when the infected and quarantined populations are small, their dynamics are described by the following system. we collect the infected and quarantined compartments in the vector v = (i · · · i m q · · · q m ) t . we can thus rewrite the system ( ) in the form d we further break l = t − v , where the transmission matrix, t , represents the influx of newly infected individuals, and the transition matrix, v , represents the movement between the infected compartments. they are calculated numerically as follows: the basic reproductive number r = ρ(t v − ) is simply calculated as the spectral radius of the matrix t v − (table , fig. ). here, we list the parameters used in our numerical simulation (tables , and ). all population numbers are in millions covid- ) situation reports- a pneumonia outbreak associated with a new coronavirus of probable bat origin an age-structured model of pre-and postvaccination measles transmission the contribution of social behaviour to the transmission of influenza a in a human population clustering of contacts relevant to the spread of infectious disease contact network structure explains the changing epidemiology of pertussis surviving covid- nonlinear physiologicallystructured population models with two internal variables on first-order hyperbolic partial differential equations with two internal variables modeling population dynamics of two physiological structures modeling infectious diseases in humans and animals the incubation period of coronavirus disease (covid- ) from publicly reported confirmed cases: estimation and application who: report of the who-china joint mission on coronavirus disease projecting social contact matrices in countries using contact surveys and demographic data characteristics of pediatric sars-cov- infection and potential evidence for persistent fecal viral shedding scientists seek reason why coronavirus has less impact on children reproduction numbers and sub-threshold endemic equilibria for compartmental models of disease transmission key: cord- -qqb knmo authors: alayi, tchilabalo d.; tawalbeh, shefa m.; ogundele, michael; smith, holly r.; samsel, alison m.; barbieri, marissa l.; hathout, yetrib title: tandem mass tag-based serum proteome profiling for biomarker discovery in young duchenne muscular dystrophy boys date: - - journal: acs omega doi: . /acsomega. c sha: doc_id: cord_uid: qqb knmo [image: see text] blood-accessible molecular biomarkers are becoming highly attractive tools to assess disease progression and response to therapies in duchenne muscular dystrophy (dmd) especially in very young patients for whom other outcome measures remain subjective and challenging. in this study, we have standardized a highly specific and reproducible multiplexing mass spectrometry method using the tandem mass tag (tmt) strategy in combination with depletion of abundant proteins from serum and high-ph reversed-phase peptide fractionation. differential proteome profiling of year-old dmd boys (n = ) and age-matched healthy controls (n = ) identified elevated and decreased serum proteins (adjusted p < . , fdr < . ) in the dmd group relative to the healthy control group. as expected, we confirmed previously reported biomarkers but also identified novel biomarkers. these included novel muscle injury-associated biomarkers such as telethonin, smoothelin-like protein , cofilin- , and plectin, additional muscle-specific enzymes such as utp–glucose- -phosphate uridylyltransferase, aspartate aminotransferase, pyruvate kinase pkm, lactotransferrin, tissue alpha-l-fucosidase, pantetheinase, and ficolin- , and some pro-inflammatory and cell adhesion-associated biomarkers such as leukosialin, macrophage receptor marco, vitronectin, galectin- -binding protein, and prosaas. the workflow including serum depletion, sample processing, and mass spectrometry analysis was found to be reproducible and stable over time with cv < %. furthermore, the method was found to be superior in terms of specificity compared to other multiplexing affinity-based methods. these findings demonstrate the specificity and reliability of tmt-based mass spectrometry methods in detection and identification of serum biomarkers in presymptomatic young dmd patients. duchenne muscular dystrophy (dmd) remains a serious and fatal muscle disease with a worldwide incidence of : male births. dmd is due to mutations in the x-linked dystrophin gene, leading to the loss of expression of dystrophin, an essential skeletal muscle protein that maintains the integrity and function of muscle fibers. there is no cure for dmd to date except the use of corticosteroids, which are known to delay the loss of ambulation by to years without changing the disease course. , despite a number of completed and ongoing clinical trials, only two drugs received conditional approval from european medicines agency (ema) and us food and drug administration (fda) in the past years. these include ataluren, a stop codon read-through, and eteplirsen, an exon skipping antisense oligonucleotide that restores the reading frame during dystrophin mrna translation. , the slow development of new therapies in dmd has been hindered by the fact that dmd is a rare disease and that most clinical trials enroll ambulatory patients within a specific age range ( − years old), resulting in statistically unpowered studies to accurately assess the outcomes. furthermore, current clinical tests to assess disease progression and response to therapies such as min walk test, m run/walk velocity, and other physical tests are subjective and might require longer clinical trials to determine the efficacy or failure of an investigational drug. − major advances in biomarker discovery have been achieved in the past years in dmd. − owing to the multiplexing capabilities of affinity-based methods such as antibody multiplexing bead technology and somascan aptamer technology, a large number of biomarkers were discovered in dmd patients and confirmed across different laboratories and different cohorts. − although these multiplexing affinitybased assays are superior to mass spectrometry in terms of sensitivity, dynamic range, and throughput, they are inherently not quantitative and lack accuracy in determining true fold change in biomarker levels. furthermore, these multiplexing affinity methods only detect and measure targeted sets of biomarkers and are not suitable for de novo discovery of novel biomarkers or discrimination between biomarkers that exist as multiple isoforms. thus, alternative methods that are more versatile, specific, reproducible, and accurate and that can easily be implemented by other laboratories are needed. in this study, we sought to optimize and standardize a serum processing workflow in combination with tandem mass tag (tmt) multiplexing strategy to systematically survey the serum proteome of young untreated dmd boys and agematched healthy controls and identify biomarkers associated in the early stages of the disease. in this study, we focused on very young dmd boys for two reasons. first is to avoid confounding variables due to glucocorticoid use and second is to define blood-circulating biomarkers that might be associated with early stages of the disease. these biomarkers might eventually be used as tools to assess disease progression and response to therapies in this younger dmd population and facilitate their enrollment into clinical trials from which they are often excluded because of lack of outcome measures for younger patients. our method successfully confirmed some of the previously reported biomarkers but also identified novel biomarkers in young untreated dmd patients. sample preparation and quality control check. to optimize our ms-based tandem mass tag (tmt) multiplex quantitation, we evaluated each step in the sample preparation and ms analysis through the workflow depicted in (figure ). this workflow was evaluated in triplicate using commercial serum. we first checked for the depletion kit efficiency using three different aliquots of serum samples in triplicates, , , and μl containing , , and μg of total proteins, respectively. the results show a linear response between total protein used for depletion and total protein recovered after the depletion. averages of . ± . μg ( . % cv), . ± . μg ( . % cv), and . ± . μg ( . % cv) of protein were collected from the , , and μg aliquots, respectively ( figure a ). together, these findings suggest that the depletion columns have enough capacity to efficiently deplete serum-abundant proteins from serum aliquots in the to μg total protein range in a reproducible manner with an overall cv < %. moving forward, we decided to use serum aliquots containing μg of total proteins as a starting material. depletion efficiency and reproducibility were further checked using d sds-page. nondepleted serum proteins exhibited very few intense protein bands, among which human serum albumin (hsa) was the most intense band on the d sds-page ( figure b ). the technical triplicate analysis of μg of depleted serum revealed a large number of new protein bands (e.g., bands a, b, c, d, and e) on d sds-page and a considerable reduction in major protein bands such as hsa. optical density analysis of randomly selected new protein bands detected in the depleted serum sample by d sds-page (bands a, b, c, d, and e) showed reproducibility in depletion with cvs < % ( figure c ). furthermore, we checked the efficiency and reproducibility of the depletion kit using western blot analysis of two proteins ( figure d ), one of which is among the depleted proteins (haptoglobin, hp) and another one of which is not among the depleted proteins (gelsolin, gsn). the depletion process resulted in an enrichment of gsn protein by approximately -fold (cvs < %) and in a depletion of hp protein by approximately fold (cvs < %), in comparison to nondepleted serum as expected ( figure e ). we then checked the reproducibility and stability of the entire workflow using μg of total serum proteins as a starting material. aliquots of − μl of each sample were processed for top abundant protein depletion kit and μg of depleted sample was processed for tmt -plex tagging, as described in the method. the obtained peptides were labeled with tmt reagent, which covalently modifies the primary amines of the n termini and lysine side chains of peptides. equal volumes of tagged samples were taken and mixed together and fractions were collected from the high-ph reversed-phase spin column and analyzed by lc−ms/ms, as described above (figure ). figure . chart depicting the overall workflow from sample preparation to mass spectrometry analysis to data processing. serum samples ( μg per aliquot) from year-old dmd patients (n = ) and age-matched healthy controls (n = ) were processed for most abundant proteins depletion. resulting samples were in-solution-digested and randomized for tmt tagging, as shown in the figure. one of the control samples (dark blue) was used as a reference to normalize the data. each sample mixture was further fractionated using a high-ph reversed-phase column and each fraction was analyzed by lc−ms/ms. data were processed as described in the method. the stability and reproducibility of the overall workflow ( figure ) including serum depletion step, tandem mass tag (tmt) multiplexing step, and high-ph reversed-phase peptide fractionation were evaluated by lc−ms/ms in triplicate analysis using commercial serum. after removing contaminant proteins and missing data, this standardized tmt ms method enabled the identification and quantification of serum proteins across the triplicate experiment with cv < % for proteins ( % of protein quantified), cv < % for proteins ( % of protein quantified), and cv < % for proteins ( figure f ). the complete list of quantified proteins by lc−ms/ms and associated normalized protein abundance ratios, means of normalized abundance ratios, standard deviation, and cvs of three technical replicate analysis of commercial serum are shown in table s . serum protein biomarker screening in young dmd patients. serum samples from year-old glucocorticoid naive dmd patients (n = ) and age-matched healthy controls (n = ) were processed for proteome profiling using our standardized multiplexing tmt-based mass spectrometry method described above. a total of four experiment sets of tmt msbased assays were run in parallel including two -plex figure . quality control of the immunoaffinity depletion of the highly abundant proteins from serum samples. aliquots of serum samples with different amounts of total proteins ( , , and μg) were used in triplicate to examine the variability and quality of the immunoaffinity depletion process. (a) linear response between the different starting amounts of total serum proteins and recovered total proteins in depleted samples. approximately . μg (cv . %), . μg ( . %), and . μg (cv . %) of proteins were recovered from serum samples containing , , and μg of total proteins, respectively. (b) sds-page showing side-by-side nondepleted serum ( μg) and depleted serum samples in triplicate ( μg each). the nondepleted serum proteins exhibited very few intense protein bands, among which human serum albumin (hsa) was the most intense band on the d sds-page, while the depleted serum samples revealed large and reproducible number on new protein bands (ex. bands a, b, c, d, and e). (c) detailed optical density analysis of randomly selected new protein bands observed on the depleted serum d sds-page (bands a, b, c, d, and e) allowed the comparison of the technical triplicate depletion of serum and estimated the cv < . %. (d) western blot analysis of depleted proteins (haptoglobin, hp) and nondepleted proteins (gelsolin, gsn). (e) quantitative analysis of western blot shows an increase in gsn by approximately -fold (cvs < %) in comparison to nondepleted serum and a decrease in hp by approximately -fold (cvs < %), confirming the effectiveness and the reproducibility of the depletion kits. (f) quality control of in-solution digestion and tmt tagging efficiency. a total of serum proteins were identified and quantified. the coefficient of variation between these three technical replicates is depicted in the pie chart with the majority of quantified proteins ( %) showing cv < %, with % of proteins showing cv < % and % of proteins showing cv < %. experiments comparing three controls and three dmd samples each, one -plex experiment comparing two dmd samples and three controls, and one -plex experiment comparing one dmd sample and three controls. to control for intra-and inter-experiment variation, the same amount of digest of tmt -labeled control serum sample was used as an internal standard in all four experiments ( figure ). using our optimized and standardized workflow, proteins were quantified across all samples with only % of missing values in all samples (table s ). further statistical analysis identified significantly elevated proteins and significantly decreased proteins (student's t-test, adjusted for multiple testing, p < . ) in serum samples of year-old dmd boys compared to year-old healthy controls ( figure a ,b and table ). twenty-nine proteins out of the potential biomarker candidates had missing values in some samples and these are shown in figure s . fold change in protein levels in serum of young dmd patients compared to healthy controls. serum proteome profiling was performed on yearold dmd patients (n = ) and age-matched healthy controls (n = ) using the tmt method. significance was defined between the two groups by independent two-sample t-test (two-sided) corrected with adjusted p value and by permutation-based fdr of . . significantly elevated and significantly decreased proteins in dmd patients relative to controls are presented in green and red, respectively. (b) hierarchical clustering of serum proteins whose levels were significantly altered between the year-old dmd (n = ) group and age-matched healthy control (n = ) group. intensities of proteins are normalized abundance ratios, which were log -transformed. as expected, a large number of identified biomarkers ( %) that were found to be elevated in sera of these young dmd boys relative to the healthy controls were of muscle origin based on gene ontology molecular function annotations ( figure s ) and information collected using mass spectrometry data analysis tools. most of these muscle-associated proteins were previously reported by other research groups and us , , − except telethonin (tcap), plectin (plec), smoothelin-like protein (smtnl ), myosin- (myh ), pdz and lim domain protein (pdlim ), tropomyosin alpha- and beta chain (tpm , tpm ), troponin c skeletal muscle (tnnc , tnnc ), troponin i slow skeletal muscle (tnni ), myomesin- (myom ), and fructose-bisphosphate aldolase c (aldoc), which are novel to this study. the fold change difference between dmd patients and healthy controls for this class of biomarkers ranged from . to . ( figure a ,b) and likely underlie the well-known sarcolemma instability in dmd and release of muscle-specific proteins. , , the second class of elevated serum proteins found in young untreated dmd patients relative to controls included the well-known heme carrier protein myoglobin (mg) and muscle-specific enzymes such as creatine kinase band m-type (ckb, ckm), fructose-bisphosphate aldolase a (aldoa), and carbonic anhydrase (ca ). additionally, novel enzymes in this class such as fructose-bisphosphate aldolase c (aldoc), kda type iv collagenase (mmp ), utp−glucose- -phosphate uridylyltransferase (ugp ), aspartate aminotransferase, cytoplasmic got , pyruvate kinase pkm (pkm), atp-dependent -phosphofructokinase muscle type (pfkm), proteasome subunit beta type- (psmb ), and glutathione s-transferase mu (gstm ) (table , figure b , and figure s ) were also found. , , an example of a panel of these newly identified dmd biomarkers using mass spectrometry is shown in figure c ,d. serum proteins that were found to be decreased in young untreated dmd patients relative to healthy controls can be classified into three major groups. one group consisted of proteases such as beta-ala-his dipeptidase (cndp ), previously reported by others and us, − and others newly identified proteases such as lactotransferrin (ltf), tissue alpha-l-fucosidase (fuca ), pantetheinase (vnn ), plasminogen (plg), carboxypeptidase n catalytic chain (cpn ), glutamyl aminopeptidase (enpep), exostosin- (ext ), ficolin- (fcn ), and transketolase (tkt) ( figure a ,b and figure s ). the second group of decreased biomarkers consisted of actin-binding proteins such gelsolin (gsn), thymosin beta- (tmsb x), coactosin-like protein (cotl ), and cofilin- (cfl ). circulating gsn was previously reported by others and our group to be decreased in dmd patients relative to controls, − while the decrease in the blood levels of the remaining actin-binding proteins is novel to this study. finally, the last group of serum protein biomarkers that were found significantly decreased in young dmd patients relative to age-matched controls are also novel to this study, which include proteins involved in cell signaling such as leukosialin (spn), macrophage receptor marco (marco), vitronectin (vtn), galectin- -binding protein (lgals bp), and prosaas (pcsk n). the list of prominently decreased proteins in young dmd boys relative to healthy controls with a minimum . -fold change is shown in table , and a box plot example of these decreased biomarkers is shown in figure e ,f. the complete list of proteins identified and quantified in year-old dmd (n = ) versus age-matched controls (n = ) and associated statistical analysis are shown in table s . biomarker correlation map reveals biomarker groups associated with different pathobiochemical pathways of muscle pathogenesis in dmd. serum or plasma protein levels can provide an indication on irregularities in the muscle integrity and health in dmd. for example, creatine kinase, lactate dehydrogenase, aldolase, troponin, and carbonic anhydrase caiii are commonly used serum markers associated with muscle injury. , , to examine if newly identified protein biomarkers correlate with well-known muscle injury proteins or other group of proteins, we performed pairwise correlation analysis of biomarkers, which resulted in a data matrix of proteins for participants ( dmd patients compared to age-matched healthy control candidates). correlation of biomarkers to each other together with hierarchical clustering based on normalized intensity ( figure b ) of each protein led to a map of pearson correlation coefficients in total for both dmd patients and healthy controls. interestingly, one of these groups (orthogonal rectangles indicated with black arrows, figure a ) containing muscle-centric proteins such ttn, myom , tnnc , myh , aldoa, and ckb is positively correlated in dmd patients (red rectangle indicated with black arrows, figure a , pearson correlation coefficient range between . and . ) and negatively correlated in healthy controls (blue rectangle indicated with black arrows, figure a , pearson correlation coefficient range between − . and − . ). furthermore, we observed an opposite correlation of the remaining muscle-derived proteins and skeletal muscle enzymes such as tnnc , eno , ckm, and tpm with the other group of proteins. meanwhile, this group of proteins was positively correlated with the other groups of proteins in the normal control (red rectangle indicated with green arrow, figure a , pearson correlation coefficient range between . and . ). these proteins turned out to be negatively correlated or have complete loss of correlation in dmd with other proteins such as plasma membrane antigen (kctd ) known for its implication in protein−protein homo-oligomer formation, e ubiquitin-protein ligase (rnf ) involved in apoptotic processes, and lactotransferrin (lft) known for its each protein is listed with its fold change (dmd/healthy controls) and two-sided p value corrected with adjusted p value and by permutationbased fdr of . . b isoform. c adjusted. http://pubs.acs.org/journal/acsodf article antimicrobial activity (blue rectangle indicated with green arrow, figure a , pearson correlation coefficient range between − . and . ). in drosophila, knockdown of rnf (drnf ) has been reported to promote mitochondrial biogenesis, improve exercise capacity in muscle, and extends climbing time to exhaustion in moderately aged flies. the reduction of rnf in serum of dmd could be due to a compensatory mechanism in humans where the reduction of rnf levels in serum in dmd correlated to the muscle protein increase in the serum due to muscle damage. more importantly, we found a group of mostly significantly decreased extracellular matrix proteins in dmd (e.g., igfals, plg, c bpa, c bpb, cpn , gc, vtn, and c ) to positively cluster with each other in dmd ( figure b , pearson http://pubs.acs.org/journal/acsodf article correlation coefficient range between . and . ) while exhibiting variable correlation in healthy controls. these proteins are involved in cell adhesion and tissue remodeling (e.g., igfals, vtn, and plg) as well as complement cascade activation and inflammation (c bpa, c bpb, c , and cpn ). this finding suggests a regression or deficit in immunity in dmd, as reported previously. we also found a group of proteins for which levels decrease in dmd and whose positive correlation in normal controls is disturbed in dmd patients ( figure c ). these proteins are mostly located in the extracellular matrix and plasma membrane. proteins such as agt, cpn , basp , spp and cndp , fcn , lgals bp, and defa are involved in vasoconstriction, extracellular matrix assembly, complement activation, cell defense response, organ tissue development (e.g., diaphragm), biomineral cell differentiation (e.g., osteoblast), and positive regulation of bone resorption. these proteins are positively correlated to each other in the normal control (pearson correlation coefficient range between . and . ), while in dmd, their levels were significantly decreased, and the correlation was disturbed ( figure c ). this latest finding suggests that organ growth and bone development in natural defense systems are significantly impaired in dmd. this result corroborates with clinical observations from other groups. − confirmation of a subset of newly identified dmd biomarkers using elisa. to verify the reliability of our new multiplex workflow method approach, we performed elisa assays to confirm a set of biomarker candidates using the same cohort of dmd patients and healthy controls as in ms analysis. the confirmation analysis was performed using elisa assay for cfl , fcn , and plec. elisa analysis was performed in duplicate for both the standard curve and the samples with cv < % in all analysis. the elisa data agree with mass spectrometry data and show that cfl and fcn were both significantly decreased in dmd ( figure d ,e) by approximately . -fold (p = . ) and -fold (p = . ), respectively. contrary to mass spectrometry data, plec was found to be slightly decreased by . -fold (p = . ) using elisa in dmd patients compared to healthy subjects ( figure f ). plectin is a large actin-binding protein (highly expressed in muscle and heart) that links intermediate filaments (if) to dystrophin−glycoprotein complexes (dgc) and integrin complexes and anchors intermediate filaments to desmosomes. , the elisa result of plectin analysis could be explained with the lack of specificity or interference observed with the immunoassay. , indeed, the antibody could recognize a common sequence of a protein present in different proteoforms without being able to distinguish between them. human plectin protein (approximately kda) has several isoforms (nine isoforms in universal protein resource (uniprot)) with % sequence homology produced by alternative splicing. antibody specificity for a specific isoform is needed for data interpretation but is not often possible. an example of such interferences has been reported for antibodies against the glycated hemoglobin form hba c that also crossreacted with the glycosylated form of hemoglobin. mass spectrometry was able to quantify two to six unique peptides of isoform- of plectin- (plec) and aqqqaeaer and qveeaer peptide sequences across all samples used for statistical analysis. development of blood-accessible biomarkers in dmd had become attractive in the past few years owing to the growing number of clinical trials and the need of reliable and sensitive outcome measures to assess disease progression and response to therapies. current outcome measures such as timed functional tests, although important, are often subjective, can be used for ambulatory patients only, and might require longer clinical trials to observe meaningful changes. − , serum biomarkers are rather objective and can be used as monitoring tools in clinical trials if associated with the disease outcomes and drug effect. in the past few years, affinity-based serum proteome profiling methods such as somascan aptamer-based technology and antibody bead array have contributed to the discovery of a large number of serum protein biomarkers in dmd. , , although they are highly multiplexing and powerful, these techniques are inherently less quantitative and suffers from the epitope effect and specificity for certain target proteins that exist as multiple isoforms. , furthermore, potential biomarker candidates for which there are no aptamers or antibodies available can be overlooked. in this study, we optimized and standardized a tmt multiplex assay workflow featuring high specificity and reproducibility. the workflow consisted of depletion of the most abundant serum proteins, followed by in-solution digestion, tmt tagging, high-ph fractionation, and lc−ms/ms analysis. the overall workflow tested with three different starting amounts of total serum proteins and in triplicate for each experiment was found to be reproducible and stable over time. more than proteins on average were identified and quantified across all samples with cvs < % for the serum protein depletion process and cv < % for % of protein quantification using the tmt-based mass spectrometry approach. our standardized workflow was implemented to compare the serum proteome profiles of untreated year-old dmd patients (n = ) and age-matched healthy controls (n = ). we reliably identified elevated and decreased proteins in serum samples of the dmd group relative to the control group (p < . , adjusted for multiple testing). as expected, the majority of elevated proteins were of muscle origin and reflect sarcolemma instability even in young preasymptomatic patients. although % of these muscle injury-associated biomarkers were previously reported by others and our group, , , , − additional muscle-associated proteins were identified and are novel to this study. these included telethonin (tcap), plectin (plec), smoothelin-like protein (smtnl ), pdz and lim domain protein (pdlim ), and tropomyosin alpha- and beta chain (tpm , tpm ), which to the best of our knowledge, were not described before. importantly, the fold change of these muscle injury proteins in dmd patients relative to controls was somewhat lower than the fold changes reported in earlier studies using affinity-based assays such as antibody bead arrays or somascan aptamer assay. for instance, circulating ck-m and ca were found to be -fold and -fold higher in young dmd patients relative to age-matched controls using the somascan aptamer method, while these same proteins were found to be only . -fold and . -fold higher in dmd patients relative to controls using the tmt mass spectrometry method. the . fold value in ca levels in dmd patients relative to controls determined by the tmt method was closer to the -fold acs omega http://pubs.acs.org/journal/acsodf article value obtained for this same protein using an absolute quantitative assay. the discrepancy in fold change observed for ck-m between tmt mass spectrometry data and previously reported somascan data could be due to the fact that different techniques measure different things. in dmd, circulating creatine kinase exists as ck-m and ck-b and as homodimers and heterodimers. somascan might lack specificity and might measure both ck-m and ck-b and the different dimers. indeed, we have superimposed somascan data of ck-m and ck-b using the same set of samples and the r was almost equal to (data not shown), indicating that somascan was not able to distinguish between circulating ck-m and circulating ck-b. mass spectrometry, on the other hand, enabled measurement of ck-m and ck-b separately. further experiments using highly reliable absolute quantification methods are needed to resolve this discrepancy seen in the ck fold changes between dmd patients and age-matched healthy controls using these different techniques. other elevated biomarker candidates identified in this study included some glycolytic enzymes such as atp-dependent pfkm, ugp , gstm , and psmb . these enzymes are also abundant in skeletal muscle and their release into the blood circulation could reflect sarcolemma instability. in this study, we have also identified proteins whose circulating levels were significantly decreased in young dmd boys compared to controls. several of these decreased biomarker candidates in dmd patients relative to controls, namely, cnpd , agt, spp , gsn, omd, and plg, were previously reported to be decreased in young dmd patients relative to controls using an independent somascan technique. additionally, several proteins that were found to be decreased in dmd patients relative to controls using tmt technology were reported to be unchanged in their levels between dmd patients and controls using the somascan technique. these included carbonic anhydrase (ca ), tkt, fcn , and galectin- -binding protein (lgals bp). this discrepancy between the tmt mass spectrometry assay and somascan assay could be due to the epitope effect that might hinder the binding of the aptamer in the somascan assay in case the target is in complex with another protein and thus provide different quantitative results than mass spectrometry where all proteins are denatured and dissociated before analysis. the newly identified biomarkers that decreased in untreated young dmd patients relative to controls included tmsb x, cotl , cfl , tkt, blvrb, among others. the serum biomarker proteins that were found to be decreased in young dmd patients relative to controls have mostly enzymatic activity, for instance, already described dmd biomarkers such as cndp but also new biomarkers such as ltf, fuca , vnn , plg, cpn , enpep, ext , and tkt. cpn is an enzyme that protects the body from potent vasoactive and inflammatory peptides. cpn cleaves cterminal arginine and lysine residues from complement anaphylatoxins (inactivation of c a and c a with significant reduction of c a, kinins, and ckm) found in the bloodstream. , we hypothesize that the decrease in circulating cpn is to reduce the complement activation cascade, a retro control mechanism used to reduce inflammation and attack of organ following fibrosis, muscle damage, and protein leakage in the bloodstream. − a small proportion of decreased serum biomarkers are of muscle origin, for example, known dmd biomarkers such as gsn but also new biomarkers such as tmsb x, cotl , and cfl . these groups of proteins all interact with and bind actin. as reported for circulating gsn, these proteins might be involved in scavenging toxic circulating actin. their decline in the circulation could be attributed to the fact that the formed complex with actin is cleared by the liver. the last group of decreased biomarkers includes those involved in g protein-coupled receptor binding or transmembrane signaling receptor activity and scavenger receptor activity. this latest protein group includes new biomarkers such as spn, marco, lgals bp, pcsk n, and vtn. vtn is a glycoprotein largely found in serum or plasma, which is produced mainly in the liver. it has different roles in complement system regulation. reduced level of vtn in serum has been shown to be related to liver disease. indeed, in dmd and becker muscular dystrophy (bmd), several severe cases of defects in hepatocytes and nonalcoholic fatty liver disease were reported in pediatric patients. , recently, cases of a reduction of the plasma vtn were reported in patients with myasthenia gravis (mg), a neuromuscular disease with similar symptoms as dmd, mainly muscle weakness and rapid fatigue. another protein found in this group, prosaas (pcsk n), is a neuroendocrine with multiple functions including fetal neuropeptide pro. prosaas is known to inhibit proprotein convertase (pc / ), a major protein produced in neuroendocrine cells, which regulates the proteolytic cleavage of neuroendocrine peptide precursors. , pcsk n is enzymatically cleaved into different neurosignaling peptides including pen, len, and saas with roles as neurotransmitters. the alteration in pck n levels can possibly affect pc / and lead to endocrine system dysregulation reported in dmd , or in other neuromuscular diseases such as fibromyalgia (fm). as prosaas-derived peptides, two peptides were identified and quantified, one of which was specific to prosaas (aeaqeaedqqar) and not to pen, len, or saas fragment of neurotransmitters. this suggests that the change in the level of prosaas is driven by this peptide and not by the neurotransmitter peptides, which can bias prosaas protein quantitation. taken together, these findings add deeper insight into the understanding of the dmd disease mechanism and pathology in young patients. we correlated all serum biomarkers filtered based on % quantitative data completeness. this led to a data matrix of proteins for participants. the biomarkers' correlation to each other together with hierarchical clustering based on normalized intensity of each protein generated a map of pearson correlation coefficients in total for both dmd patients and healthy controls. this unbiased analysis led remarkably to the clustering of proteins into two main groups. our findings revealed impairments in the natural defense system, translated with decreases in the levels of a group of proteins in sera of dmd patients relative to healthy controls. these proteins are involved in cell adhesion and tissue remodeling (e.g., igfals, vtn, and plg) as well as proteins involved in complement cascade activation, regulation, and inflammation (c bpa, c bpb, c , and cpn ). this finding suggests a regression or deficit in immunity in dmd, as previously reported. also, we identified a group of proteins mostly located in the extracellular matrix and plasma membrane, such as agt, cpn , basp , spp and cndp , fcn , lgals bp, and defa . these proteins are involved in vasoconstriction, extracellular matrix assembly, complement activation, cell defense response, organ tissue development (e.g., diaphragm), biomineral cell differentiation (e.g., osteoblast), and positive regulation of bone acs omega http://pubs.acs.org/journal/acsodf article resorption. this finding indicates that organ growth and bone development are significantly disturbed in dmd. this result corroborates with clinical observation from other groups. − we recognize that sample size is one of the limitations of our current study. this is a recurrent question, especially when dealing with a rare disease such as dmd. we had some challenges enrolling and collecting more samples from yearold boys during the period of the study. however, in this pilot study, although it is preliminary and uses a small sample size, we have confirmed previously reported biomarkers. we have also identified novel biomarkers that were significantly different between young dmd boys and age-matched healthy controls with a good p value adjusted for multiple testing. we have not noticed any large variations in the level of candidate biomarkers between patients. nevertheless, follow-up studies using a larger sample size are needed to validate these novel biomarker candidates and define their physiological significance and relation with early-stage dmd pathogenesis. the second limitation of our study is the identification and quantification of a lower number of serum proteins after the depletion step compared to previous studies. , this is mainly due to differences in sample preparation between this study and previous studies. indeed, previous studies used intensive sample fractionation (> fractions per sample) and online chromatographic separation during the fractionation step, requiring more resources and machine time than our current workflow where only fractions were collected from the spin column and analyzed by lc−ms/ms. our simplified method will be easy to implement by others for future validation studies. the third limitation of our study is the lack of a specific and validated elisa assay to confirm newly identified candidate biomarkers. this is also a recurrent question when it comes to validating proteomics data. biomarker discovery and validation are an ongoing effort and more specific and sensitive elisa assays or alternative orthogonal methods are needed to achieve this goal. we implemented and standardized a serum proteome profiling workflow, which implements serum-abundant protein depletion and the sensitivity gained from high-ph reversed-phase peptide fractionation and ms-based tandem mass tag (tmt) multiplex quantitation approach. we evaluated our workflow on year-old untreated dmd cases and age-matched healthy controls to define candidate biomarkers associated with the early stage of the disease. a large number of these biomarkers confirmed the previous studies performed by other groups and us. however, several new biomarkers were identified in this study. these findings support the efficiency, reliability, sensitivity, and capability of our serum biomarker study workflow in biomarker discovery. also, we brought new insight into the assessment of the disease pathogenesis in young dmd patients with a new panel of biomarker signatures such as proteases and actin-binding proteins. further examination of serum protein biomarkers using our workflow in presymptomatic very young dmd patients less than years old might provide further insight into muscle pathogenesis and provide tools to assess disease severity and response to intervention in dmd infants. collection. in this study, we used serum samples from a subset of young dmd patients (average age, . ± . years old; n = ) and from age-matched healthy controls (average age, . ± . ; n = ). dmd patients were enrolled through the cooperative international neuromuscular research group−duchenne natural history study (cinrg-dnhs). the study protocol was approved by institutional review boards at all participating institutions and informed written consent was obtained from the parents of the participants or their legal guardians. serum samples from healthy control pediatric donors were obtained from a third party company (bioivt, hicksville, ny, usa). detailed patient demographics and characteristics are listed in table s . serum samples were prepared following a rigorous standardized operating protocol and stored in small workable aliquots at − °c in polypropylene cryogenic vials (thermo scientific nalgene) to avoid repetitive freeze-thaw cycles. the commercial serum (catalogue #h ) was purchased from sigma-aldrich. top serum-abundant protein depletion and quality control. protein concentration was determined using the pierce bca protein assay kit (thermo fisher scientific, ll, usa) according to the manufacturer's instructions and measurements were performed using the spectramax i x microplate reader (molecular devices, san jose, ca). serum aliquots containing , , or μg of total proteins (corresponding to − μl of serum) were processed for depletion of the top serum-abundant proteins using pierce top abundant protein depletion spin columns (thermo fisher scientific, ll, usa). briefly, the depletion spin columns were equilibrated at room temperature ( min), the column's screw caps were removed, and an adequate amount of total protein serum was directly added to the resin slurry into each column. columns were recapped and inverted several times until the resin was completely suspended in the solution and then incubated with gentle end-over-end mixing for min at room temperature using a tube revolver (thermo fisher scientific, ll, usa) set at rpm. after the incubation, the bottom closure of the columns was twisted off and the cap was loosened. the column was then placed in ml protein lobind tubes (eppendorf ag, hamburg, germany) and centrifuged at g for min to collect the remaining serum depleted sample ( − μl). sample processing quality control was checked by gel electrophoresis using precast nupage − % bis-tris protein gels (thermo fisher scientific, ll, usa). aliquots containing μg of total proteins of depleted serum samples were mixed with μl of nupage lds sample buffer and μl of mm pierce dithiothreitol (dtt) (thermo fisher scientific, ll, usa) and heated at °c for min. samples were uploaded into the wells and gel electrophoresis was run for h using mops sds running buffer (thermo fisher scientific, ll, usa) at a constant voltage of v in an xcell surelock electrophoresis cell (thermo fisher scientific, ll, usa). the gel was stained with ml of bio-safe coomassie (bio-rad laboratory, ca, usa) and then destained overnight with distilled water. gel images were taken using an azure c imager (azure biosystems, ca, usa) and analyzed using open-access software (gelquant.net). in-solution digestion of depleted serum samples and tmt derivatization. all the reagents used in this experiment were from tmtsixplex isobaric mass tagging kit (thermo fisher scientific, ll, usa) unless indicated. aliquots of − μl containing μg of total proteins from each sample were vacuum-dried to μl and denatured with μl of % sodium dodecyl sulfate (sds) heated at °c for min. disulfide bonds were reduced by adding μl of mm tris( -carboxyethyl) phosphine (tcep) followed by incubation at °c for h. free thiols were then alkylated by adding μl of mm iodoacetamide (iaa) followed by incubation at °c for h in the dark. proteins were then precipitated by adding μl of pure chilled acetone (− °c), followed by centrifugation at rpm for min using an eppendorf centrifuge (eppendorf ag, hamburg, germany). the precipitate from each sample was kept at − °c overnight ( h). the mixture was then centrifuged again the next day and the supernatant was gently removed from the protein pellet. then, μl of mm triethyl ammonium bicarbonate (teab) was added to the protein pellet and vortexed for min. for protein digestion, a total of μl of . μg/μl pierce trypsin protease, ms grade (thermo fisher scientific, ll, usa), was added to each sample in two steps for a total enzyme/protein ratio of : (w/w). for the first h of proteolysis, only μl of the trypsin was added to each sample and the remaining μl was added to each sample for overnight protein digestion. during the entire digestion process, samples were incubated at °c using a thermomixer c (eppendorf ag, hamburg, germany) set rpm. the peptide derivatization with tmt label reagents (thermo fisher scientific, ll, usa) was performed according to the manufacturer's procedure with slight modification. vials of tmt reagent (containing . mg of each tag reagent) were reconstituted in μl of anhydrous acetonitrile and μl of resulting solutions was added to the corresponding digested samples ( μg of protein digested) and incubated at °c for h with shaking at rpm. to quench each reaction, μl of % hydroxylamine was added to each sample and incubated for h at °c with shaking. to multiplex the sample before the lc−ms/ms analysis, μl of each tmt-labeled sample was combined in either -plex, -plex, or -plex in new eppendorf tubes, dried, and stored at − °c until high-ph reversedphase fractionation. for the year-old serum study, combined samples shared in common the same digested control sample labeled with tmt tag, which was selected for the normalization of data. high-ph reversed-phase peptide fractionation. vacuum-dried samples were reconstituted in μl of . % trifluoroacetic acid (tfa) and fractionation was performed using pierce high-ph reversed-phase peptide fractionation kit (thermo fisher scientific, il, usa) according to the manufacturer's procedure with slight modification. briefly, columns were conditioned with successive cleaning steps using pure acn ( μl twice) and washing steps with . % tfa ( μl twice) with centrifugation set at g for a min duration. each sample ( μl) was loaded onto the columns and centrifuged at g for min. the operation was repeated once again and the flow-through (ft) was collected. the columns were then washed with water ( μl twice) by centrifugation at g for min, and the wash (w) was then collected. peptide elution was performed with μl of different elution buffer mixtures made with acn and . % triethlyamine (tea) at g centrifugation force for min each. in total, consecutive fractions were collected with elution buffers gradually increasing by % acn increments up to %. the ft, w, and collected factions were dried using a speedvac vacuum concentrator and stored at − °c until nanolc−ms/ms analysis. liquid chromatography−tandem mass spectrometry analysis. collected peptide fractions were reconstituted in μl of water containing . % formic acid (fa) (v/v) and μl of samples was injected. peptide separation was carried out using an ultimate rslcnano system (dionex/thermo fisher scientific). for each analysis, the sample was loaded into an acclaim pepmap trap column ( cm × μm inner diameter, c , μm, a; dionex, ca, usa) at . μl/min with aqueous solution containing . % fa and % acn (v/v). after min, the trap column was set online with an easy-spray acclaim pepmap rslc analytical column ( cm × μm inner diameter, c , μm, a; dionex, ca, usa). peptides were eluted by applying a mixture of solvents a and b. solvent a consisted of hplc-grade water with . % fa (v/ v), and solvent b consisted of hplc-grade acetonitrile ( % acn) with . % fa (v/v). separations were performed using a linear gradient of to % solvent b at nl/min over min followed by a min linear increase of acn percentage up to a washing step ( min at % solvent b) and a min linear decrease of acn percentage up to an equilibration step ( min at % solvent b). the total analysis run time was min. lc−ms/ms data-dependent acquisition was performed using a q-exactive hf mass spectrometer (thermo scientific, bremen, germany) in positive mode. for ionization, an easy-spray es (thermo scientific, bremen, germany) was used with a voltage set at kv, and the capillary temperature set at °c. full ms scans were acquired in the orbitrap mass analyzer over an m/z − range with a resolution set at , for m/z . the target automatic gain control value of × was used with a maximum allowed injection time (maximum it) of ms. for ms/ms, an isolation window of . m/z was utilized. the most intense peaks with a charge state between and were selected for fragmentation using high-energy collision-induced dissociation with a stepped normalized collision energy of − . the tandem mass spectra were acquired with fixed first mass of m/z in the orbitrap mass analyzer with a resolution set at , for m/z and an automatic gain control of × . the ion intensity selection threshold was . × , and the maximum injection time was ms. the dynamic exclusion time was s for the total run time of min. mass spectrometry data processing, protein identification, and quantitation analysis. all data files were collected and processed with a specific workflow designed in proteome discoverer . (thermo fisher scientific). searches were performed against homo sapiens (taxid = ) protein sequence database downloaded from www.uniprot.org on november ( , entries) using sequest ht (thermo fisher scientific) with precursor and fragment mass tolerance respectively set at ± ppm and ± . da and the following dynamic modifications: carbamidomethyl on cysteine, acetyl on protein n terminus, oxidation on methionine, tmt -plex modification on lysine, and n terminus of peptides. the targetdecoy database search allowed us to control and estimate the false positive discovery rate at % for the peptide and protein as well. quantitation of -plex ( , , , and ), -plex ( , , , , and ) , and -plex ( , , , , acs omega http://pubs.acs.org/journal/acsodf article , and ) were set up in proteome discoverer by the activation or deactivation of unused channels from original tmt -plex quantification method. the reporter ion quantifier tolerance was set at . da with the integration method as the most confident centroid. the peptides used for the quantitation are only unique peptides and the spectra with missing channels were rejected. the reporter quantitation was corrected with isotopic impurity of reporter values for minimum signal-to-noise ratio (s/n) set at . . the protein abundance was calculated as the sum of abundance of the corresponding protein in each fraction. all data were normalized to the same control sample labeled with tmt , which was used across all multiplexing analyses. the mass spectrometry proteomics data are made available via proteomexchange under the project named dmd biomarkers in year-old boys, dmd patient vs control study, with identifier pxd . enzyme-linked immunosorbent assay (elisa). a subset of identified biomarker candidates including cofilin- (cfl ), ficolin- (fcn ), and plectin (plec) were examined using elisa assay with kits corresponding respectively to catalogue numbers okeh , okdd , and okbb (aviva systems biology, san diego, ca, usa) on the same serum samples analyzed by mass spectrometry. serum samples from dmd patients and agematched healthy controls were diluted at : for cfl , : for fcn , and : for plec and the analyses were performed according to the manufacturer's instructions using the spectramax i x microplate reader (molecular devices, san jose, ca). western blot. aliquots containing μg of total proteins from each sample including molecular weight markers, ibright prestained protein ladder ranging from to kda, were loaded in different wells of precast criterion xt % bis-tris ( mm, -well) gels. gels were run at v for h and transferred onto the nitrocellulose membrane (thermo fisher) at . a for h. membranes were then washed with pbst and cut at the kda ladder marker for separate antibody incubations for haptoglobin and gelsolin. the membranes were then washed in bio-rad blotting-grade blocker and separately incubated in anti-haptoglobin (ab ) and anti-gelsolin (ab ) at : and : dilutions, respectively. membranes were washed again and incubated in anti-rabbit igg-hrp (na v) antibodies at : dilutions. membranes were revealed using an azure c imager (azure biosystems, ca, usa) and image-analyzed using open-access software (gelquant.net) with fluorescence settings at s of absorbance. the supporting information is available free of charge at https://pubs.acs.org/doi/ . /acsomega. c . supporting table descriptions; hierarchical clustering of other biomarkers found with missing data; gene ontology molecular function analysis (pdf) dmd patient and age-matched healthy control demographics; list of proteins identified and quantified in triplicate experiment using commercial serum; list of proteins identified and quantified in serum samples of young dmd patients relative to healthy controls; go molecular function ontology (mfo) analysis of the elevated and decreased proteins in serum samples of dmd patients relative to healthy controls (xlsx) ■ author information corresponding author united states; email: yhathout@binghamton.edu authors tchilabalo d. alayi − department of pharmaceutical science, school of pharmacy and pharmaceutical sciences united states holly r. smith − department of pharmaceutical science, school of pharmacy and pharmaceutical sciences and department of biochemistry children's national health system children's hospital of richmond at vcu the cinrg-dnhs was funded by the u.s. department of education/nidrr (#h b and #h b ) evidence-based path to newborn screening for duchenne muscular dystrophy the pathogenesis and therapy of muscular dystrophies corticosteroid treatments in males with duchenne muscular dystrophy: treatment duration and time to loss of ambulation advances in the treatment of duchenne muscular dystrophy: new and emerging pharmacotherapies restoring dystrophin expression in duchenne muscular dystrophy: current status of therapeutic approaches the -minute walk test and other endpoints in duchenne muscular dystrophy: longitudinal natural history observations over weeks from a multicenter study revised north star ambulatory assessment for young boys with duchenne muscular dystrophy discovery of serum protein biomarkers in the mdx mouse model and cross-species comparison to duchenne muscular dystrophy patients proteomics profiling of urine reveals specific titin fragments as biomarkers of duchenne muscular dystrophy muscle-derived proteins as serum biomarkers for monitoring disease progression in three forms of muscular dystrophy affinity proteomics within rare diseases: a bio-nmd study for blood biomarkers of muscular dystrophies aartsma-rus, a. tracking disease progression non-invasively in duchenne and becker muscular dystrophies longitudinal serum biomarker screening identifies malate dehydrogenase as candidate prognostic biomarker for duchenne muscular dystrophy the fundamental flaws of immunoassays and potential solutions using tandem mass spectrometry cinrg investigators. the cooperative international neuromuscular research group duchenne natural history study-a longitudinal investigation in the era of glucocorticoid therapy: design of protocol and the methods used iii isobaric labeling-based relative quantification in shotgun proteomics proteomics software suite for in-depth mass spectrometry data analysis using grid computing biochemical markers of muscular damage mass spectrometry-based identification of muscle-associated and muscle-derived proteomic biomarkers of dystrophinopathies role of complements and immunoglobulins in duchenne muscular dystrophy short stature in duchenne muscular dystrophy: a study of patients patterns of growth in ambulatory males with duchenne muscular dystrophy plectin isoforms as organizers of intermediate filament cytoarchitecture plectin links intermediate filaments to costameric sarcolemma through -synemin, -dystrobrevin and actin advantageous uses of mass spectrometry for the quantification of proteins a review of variant hemoglobins interfering with hemoglobin a c measurement aptamers they trust: the caveats of the somascan biomarker discovery platform from somalogic. circulation carbonic anhydrase iii in serum in muscular dystrophy and other neurological disorders: relationship with creatine kinase a pleiotropic regulator of inflammation structure and function of human plasma carboxypeptidase n, the anaphylatoxin inactivator targeted disruption of the gene encoding the murine small subunit of carboxypeptidase n (cpn ) causes susceptibility to c a anaphylatoxin-mediated shock plasma gelsolin: indicator of inflammation and its potential as a diagnostic tool and therapeutic target role of plasma gelsolin and the vitamin d-binding protein in clearing actin from the circulation the complement factsbook quantitation of vitronectin in serum: evaluation of its usefulness in routine clinical practice the role of liver in muscular dystrophy duchenne and becker muscular dystrophy presenting as nonalcoholic fatty liver disease plasma vitronectin is reduced in patients with myasthenia gravis: diagnostic and pathophysiological potential coexpression of proprotein convertase spc and the neuroendocrine precursor prosaas a novel function for prosaas as an amyloid anti-aggregant in alzheimer's disease prohormone convertase / is essential for processing of the glucose-dependent insulinotropic polypeptide precursor endocrine aspects of duchenne muscular dystrophy muscular dystrophy tracking and research network md star. bone health and endocrine care of boys with duchenne muscular dystrophy: data from the md starnet systematic analysis of the cerebrospinal fluid proteome of fibromyalgia patients quantitative workflow for sensitive biomarker discovery in plasma yields novel candidates for early myocardial injury the pride database and related tools and resources in : improving support for quantification data key: cord- - gej co authors: marcekova, zuzana; psikal, ivan; kosinova, eva; benada, oldrich; sebo, peter; bumba, ladislav title: heterologous expression of full-length capsid protein of porcine circovirus in escherichia coli and its potential use for detection of antibodies date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: gej co a capsid protein of porcine circovirus (pcv ) serves as a diagnostic antigen for the detection of pcv -associated disease known as a postweaning multisystemic wasting syndrome (pmws). in this report, a bacterial expression system was developed for the expression and purification of the full-length pcv capsid (cap) protein from a codon-optimized cap gene. replacement of rare arginine codons located at the ′ end of the cap reading frame with codons optimal for e. coli was found to overcome the poor expression of the viral protein in the prokaryotic system. the cap protein was purified to greater than % homogeneity by using a single cation-exchange chromatography at a yield of mg per litre of bacterial culture. despite the failure of the e. coli-expressed cap protein to self-assemble into virus-like particles (vlps), the immunization of mice with recombinant cap yielded antibodies with the same specificity as those raised against native pcv virions. in addition, the antigenic properties of the purified cap protein were employed in a subunit-based indirect elisa to monitor the levels of pcv specific antibodies in piglets originating from a herd which was experiencing pcv infection. these results pave the way for a straightforward large-scale production of the recombinant pcv capsid protein and its use as a diagnostic antigen or a pcv subunit vaccine. porcine circoviruses (pcv) are small non-enveloped, singlestranded circular dna viruses of the circoviridae family . two distinct types of pcv have been described: the non-pathogenic pcv type (pcv ) (tischer et al., ) and the pathogenic pcv type (pcv ) nayar et al., ) , which is associated with a newly emerged disease called postweaning multisystemic wasting syndrome (pmws) (clark, ) . four-to twelve-weeks old piglets which are affected with pmws display clinical symptoms of wasting, respiratory distress, anaemia, diarrhoea, jaundice and enlarged lymph nodes (for review, see chae, ) . pmws is considered to be an important porcine disease worldwide which is reported to have a serious economic impact on the global pig farming industry. the . kb pcv genome contains three functional open reading frames (orfs) . orf encodes several forms of non-structural replicase proteins (mankertz and hillenbrand, ; mankertz et al., ) , orf encodes the capsid protein (nawagitgul et al., ) , and orf encodes a -amino acid protein which appears to be involved in virus-induced apoptosis of infected cells (liu et al., ) . the capsid protein is a unique structural protein of the viral coat (nawagitgul et al., ) that is formed by protein subunits in an icosahedral t = capsid structure (crowther et al., ) . the n-terminus of the capsid protein is rich in basic amino acid residues and displays a nuclear localization signal (nls) which is important for capsid assembly (liu et al., a) . the capsid protein is highly immunogenic and reacts strongly with serum from pcv -infected pigs (mahe et al., ; fenaux et al., ) , and thus, it is the preferred antigen in a variety of serological tests (nawagitgul et al., ; blanchard et al., ; liu et al., ; shang et al., ) . the expression of recombinant capsid protein was achieved successfully in the baculovirus system (nawagitgul et al., ; mahe et al., ; blanchard et al., ; fan et al., ) , where the expressed capsid protein assembled spontaneously into virus-like particles (vlps) (nawagitgul et al., ; liu et al., ) . vlps represent non-infectious structures which completely lack the dna or rna genome of the virus. therefore, vaccines based on vlps trigger an immune response to the host immune cells by mimicking native virus morphology (ludwig and wagner, ) . however, the production of vlps for vaccination in eukaryotic systems is costly for veterinary applications. on the other hand, heterologous protein expression in e. coli offers an alternative to the production of large amounts of protein. the expression of full-length capsid protein in a standard bacterial expression system, such as e. coli bl (de ), has not been reported. only certain regions of the cap protein (wu et al., ) or a fusion protein with maltose-binding protein (liu et al., b) or truncated variant of cap lacking the nls (zhou et al., ; trundova and celer, ) have been expressed in e. coli. in this study, a genetic approach which takes advantage of a codon-optimized synthetic oligonucleotide and a synthetic codonoptimized gene is described to obtain the high-level production of the full-length pcv capsid protein in e. coli bl (de ) cells. the purified capsid protein is used as antigen to develop an indirect elisa for monitoring the levels of pcv specific antibodies in piglets originating from a herd experiencing pcv infection. the czech field-strain isolate of porcine circovirus type (l- , brno, czech republic) was used in this study. the virus stock was prepared from the supernatant of organ homogenate from a pig which fulfilled the diagnostic criteria for pmws (sorden, ) . samples of enlarged lymph nodes were pooled and homogenised in a fivefold volume of phosphate-buffered saline (pbs, ph . ). two volumes of chloroform were added to volumes of the homogenate and the mixture was shaken at • c for min and centrifuged at × g for min. the supernatant was subjected to a cushion of cscl density gradient ( . g/ml) and centrifuged at , × g for h in a beckman sw ti rotor (beckman coul-ter, fullerton, usa). the purified pcv virions were resuspended in pbs and subsequently used to infect the circovirus-free pk cells which were maintained in a d-mem medium (paa laboratories, pasching, austria) supplemented with % heat-inactivated fetal calf serum (gibco, invitrogen, carlsbad, usa) at • c with % co . the viral dna was purified from pk -infected cells using a dnazol genomic dna isolation reagent (molecular research center, cincinnati, usa) according to manufacturer's instructions. the genomic dna of pcv isolate was sequenced by abi prism xl analyzer (applied biosystems, foster city, usa) using bigdye terminator . cycle sequencing kit (applied biosystems, foster city, usa). the nucleotide sequence encoding the cap protein was . % identical with that of orf of pcv strain fd (genbank accession no. ay ) (de boisséson et al., ) . a bp sequence encoding the cap protein was amplified using polymerase chain reaction (pcr) with the following primers: the upstream primer -ccccatggcgatgacgtatccaaggaggc- containing the ncoi site and the downstream primer -tgtctcgagagggttggggggtc- containing the xhoi site. the purified pcr product was cut with ncoi and xhoi (new england biolabs, ipswich, usa), cloned into the pet b expression vector (novagene, merck kgaa, darmstadt, germany), and the vector was designated as pet b-cap-his (fig. b) . to generate a truncated version of the cap protein ( cap-his) lacking the first n-terminal amino acid residues, the pet b-cap-his was used as a template for pcr with the following primers: the upstream primer -agaccatgggcagcc-atcttggccagatc- containing ncoi site and downstream primer -tgtctcgagagggttggggggtc- containing the xhoi site. the bp pcr product was cut with ncoi and xhoi, cloned into the pet b vector, and designated as pet b-cap-his (fig. b) . for the construction of a vector carrying the codon-optimized nucleotide sequence of the first amino acid residues of the cap protein, the pet b-cap-his expression vector was cut with ncoi and msci. the resulting fragment was ligated with a dsdna adaptor (sense oligonucleotide -catgacctatccgcgccgccgttacc-gccgccgccgccaccgcccgcgcagccatctggg- and anti-sense oligonucleotide -cccagatggctgcgcgggcggtggcggcggcg-gcggtaacggcggcgcggataggt- ) creating the ncoi and msci overhangs at and end of the adaptor molecule, respectively. this construct was designated as pet b-o-cap-his (fig. b) . codon-optimized gene encoding the pcv capsid protein was obtained from genscript (piscataway, usa) and the nucleotide sequence was deposited in genbank (accession no. eu ). the plasmid carrying the nucleotide sequence of the cap protein was cut with ncoi and xhoi and cloned into pet b expression vector. to enhance the expression of the synthetic cap gene in e. coli, a pet b expression vector was modified by replacement of the nucleotide sequence between the ndei and saci sites with a dna adaptor molecule annealed from a pair of synthetic oligonucleotides (sense oligonucleotide -tatgcaccaccaccacc-accacgccatgggagct- and anti-sense oligonucleotide -cccatggcgtggtggtggtggtggtgca- ) which encoded the n-terminal histidine tag of the synthetic cap gene. this construct was cut with ncoi and xhoi and ligated with the synthetic cap gene which had been cut with the same restriction enzymes to obtain the expression vector carrying s-cap with the flanking n-and cterminal histidine tags (pet b-s-cap) (fig. b) . all the constructs were confirmed by dna sequence analysis with abi prism xl analyzer (applied biosystems, foster city, usa) using a big dye terminator cycle sequencing kit. recombinant proteins were expressed in e. coli bl (de ) (novagene, merck kgaa, darmstadt, germany). cells containing the expression plasmid were grown at • c in luria-bertani (lb) medium supplemented with g/ml kanamycin to the optical density of . at nm and then isopropyl ␤-dthiogalactopyranoside (iptg) (alexis corporation, lausen, switzerland) was added to a final concentration of mm. after h of growth, cells were harvested by centrifugation ( × g for min) and resuspended in mm tris-hcl (ph . ) containing mm edta. the expression in e. coli bl -codonplus(de )-ripl strain (stratagene, agilent technologies, santa clara, usa) was done under the same conditions, except for antibiotics concentrations: kanamycin ( g/ml), chloramphenicol ( g/ml) and spectinomycin ( g/ml). this strain contains extra copies of the argu, iley, prol, and leuw genes for rare trnas, which can rescue protein production restricted by either agg/aga or ccc codons. the expression of desired proteins was documented by sds-polyacrylamide gel electrophoresis (sds-page) and western blot analysis. bacterial pellet was washed and resuspended in sonication buffer ( mm mes, ph . , mm nacl) containing mm phenylmethylsulfonyl fluoride. the cells were disrupted by sonication ( w, misonix sonicator , misonix, farmingdale, usa) on ice and the homogenate was centrifuged at , × g at • c for min. solid urea was added to a final concentration of m, and mixed gently for min. after centrifugation at , × g for min at • c, the supernatant was loaded to a unosphere-s cation-exchange column (bio-rad, hercules, usa) which was connected to an akta prime chromatography system (ge healthcare, chalfont st. giles, united kingdom). after washing with column volumes of sonication buffer, the cap protein was eluted with a continuous gradient of nacl ( - m) in a buffer containing mm mes, ph . and m urea. pooled fractions of purified cap protein were concentrated by using centricons (amicon mw cutoff, millipore, billerica, usa), and stored at − • c for further use. total protein concentration was determined by the bradford assay (bio-rad, hercules, usa) using bovine serum albumin as a standard. proteins were separated by sds-page using % polyacrylamide gels and either stained by coomassie brilliant blue r- or transferred to a nitrocellulose membrane (pall corporation, new york, usa) in a transfer buffer ( mm tris-hcl, ph . , mm glycine, . % sds, % methanol) using a te xp semi-dry transfer unit (hoefer, holliston, usa) at v/cm for h. the membranes were blocked with % non-fat milk in pbs-t (phosphate-buffered saline containing . % tween- ), incubated with anti-his monoclonal antibody ( : ) (sigma, st. louis, usa) followed with a peroxidase-conjugated anti-mouse antibody. the signal was developed using an enhanced chemiluminiscence system (ge healthcare, chalfont st. giles, united kingdom). the experimental work with mice was conducted according to the certificate of animal welfare authority commission of the ministry of agriculture of the czech republic no. mze / in accordance with current czech legislation on animal welfare. a total of balb/c mice ( weeks of age) were used in the experiment. a group of mice was bled before the immunization to obtain pre-immune serum. a group of mice received intramuscular injection with recombinant s-cap ( g protein per mouse) mixed with a complete freund's adjuvant (sigma, st. louis, usa). three weeks later, the mice were boosted intramuscularly with the same dose of recombinant cap protein mixed with incomplete freund's adjuvant (sigma, st. louis, usa). the mice were euthanised and bled days after the last injection prior to the sera were screened for the presence of the cap-specific antibodies by using an immunoperoxidase monolayer assay (ipma). for ipma, confluent monolayers of pk cells were mixed with a reference pcv isolate (stoon- , after passages) and incubated at • c for h prior to fixation with % paraformaldehyde. the fixed cells were then incubated with mice sera ( : dilution in pbs-t buffer) at • c for h, followed by incubation with a peroxidase-conjugated goat anti-mouse secondary antibody ( : dilution in pbs-t buffer) and the peroxidase activity was detected with -chloro- -naphthol (bio-rad, hercules, usa) as a substrate. e. coli cells were washed twice in mm phosphate buffer (ph . ) and fixed in a solution containing % glutaraldehyde in mm phosphate buffer (ph . ) for h. after three washes, the cells were post-fixed in buffered % oso ( mm phosphate buffer, ph . ) for h, washed three times in mm phosphate buffer (ph . ), dehydrated in ethanol series, and then transferred into absolute acetone and embedded in vestopal w resin (sigma, hercules, usa). ultrathin sections were cut with glass knife using a lkb ultratome (lkb, bromma, sweden) and mounted on formvar-coated copper grids. the grids were stained in saturated aqueous uranyl acetate followed by lead citrate. samples were examined under philips cm electron microscope (fei, eidhoven, the netherlands) at kv. images were recorded by using a megaviewii slow scan camera controlled by analysis . software (olympus soft imaging solutions, münster, germany). pcv antibodies were determined with an indirect enzymelinked immunosorbent assay (elisa) using the purified s-cap and cap-his proteins as coating antigens (cap-elisa). the -well elisa plates (nunc, thermo fisher scientific, waltham, usa) were coated with l of mm sodium carbonate buffer (ph . ) containing g/ml of the proteins and incubated overnight at • c. the plates were washed with phosphate-buffered saline (pbs) containing . % tween- (pbs-t) and blocked with a blocking buffer (pbs containing % bovine serum albumin) at • c for h. after washing, l of each serum sample (diluted : ) was added into each well and tested in quadruplicate: two wells for a negative control antigen glutathione s-transferase (sigma) and two parallel wells for the antigens. the plates were washed with pbs-t and l of blocking buffer containing a peroxidase-conjugated goat anti-swine antibody (bethyl laboratories, montgomery, usa) was added into each well and incubated for h. finally, the plates were washed with pbs-t three times and the colour reaction was developed with , , , -tetramethylbenzidine (tmb) as a substrate. the optical density at nm (od nm ) was read using a microplate reader. the final od nm value was calculated by subtracting the mean value of od nm of wells containing a negative antigen from that of the parallel wells containing the capsid proteins. the cut-off value was determined by using specific pathogen free (spf) pig's serum samples diluted at : which were negative for anti-pcv antibodies as determined by ipma. the mean od nm value was . with a standard deviation (s.d.) of . and the final cut-off value was calculated by adding the mean od nm value + s.d. to the value of . . pig serum with a high titre of pcv antibodies : , as determined by ipma, was used as a positive control (kindly provided by annette mankertz from robert-koch institut, berlin, germany). the spf pig sera were negative for pcv and used as negative controls in each plate. a limited serological and genomic load survey of specific pcv antibodies and equivalent pcv genome copy numbers, respectively, was carried out on pig sera collected at , , , and weeks of age. selected piglets were selected from a pig herd experiencing pcv infection, marked with tags and monitored during their nursery and fattening period. at the regular intervals, a minimum of ml of blood was collected from each piglet and the sera were stored at − • c for further use. total genomic dna was extracted from a l volume of each serum sample using a nucleospin blood isolation kit (macherey-nagel, düren, germany) according to manufacturer's instructions. quantification of pcv dna levels was performed using a realtime pcr by roche light cycler (roche, basel, switzerland) in a taqman format as previously described (brunborg et al., ) . in short, the taqman probe was labelled with -fam and -bhq fluorophores (generi biotech, hradec kralove, czech republic) and the primers were designed to amplify a bp dna segment within the nucleotide sequence of the pcv cap gene. the absolute quantification of pcv dna was carried out using calibration curves generated by means of external standard dna obtained by cloning the pcv cap gene into a pcr . vector (invitrogen, carlsbad, usa). standard curves for pcv dna quantification were gener- ated using tenfold dilution of the linearised plasmids in the range of log . statistical analyses were carried out using the one-way analysis of variance (anova) test running in spss software (base . , spss, chicago, usa). values of p < . were considered significant. the open reading frame encoding cap protein was amplified using pcr from genomic dna of pcv (l isolate) and the bp long dna fragment was cloned into pet b expression vector (fig. b) . the expression was documented in crude cell lysate by sds-page and western blotting. as demonstrated in fig. , no expression of cap-his was detected in coomasie-stained gels and by western blot using anti-his antibody. frequently, an insufficient production of heterologous proteins in e. coli is due to the presence of codons that are used rarely in bacterial proteosynthesis (zahn, ) . forced high-level expression of genes with rare codons may lead to the depletion of the endogenous pools of the corresponding trnas, resulting in the abortion of the translation process and the degradation of the mrna (mcnulty et al., ) . examination of the nucleotide sequence of the cap gene revealed a cluster of rare codons within the first codons of the gene (fig. c) . the presence of such rare codon tandems near the end of a coding sequence has been reported to cause ribosomal frameshifts and codon skipping with a strong inhibitory effect on protein translation (gurvich et al., ) . in order to eliminate the cluster of rare codons from the end of the cap gene, the expression vector encoding a truncated variant of cap protein ( cap-his), which was lacking the first amino acid residues, was constructed (fig. b) . expression of the truncated gene in iptg-induced e. coli bl (de ) cells resulted in the production of a protein with an apparent molecular weight of kda ( fig. a) which corresponded to the cap-his protein. this suggested that the removal of the first codons from the end of the cap gene allowed the production of cap protein in e. coli. in previous report, the expression of the truncated variant of cap protein lacking the nuclear localization signal (nls) has been reported (zhou et al., ; trundova and celer, ) . the nls consists of amino acid residues at the n-terminus of cap and the nls-defective constructs have been expressed also as fusion proteins with glutathione s-transferase (zhou et al., ) or maltose-binding protein (liu et al., b) . based on these results, the authors concluded that the expression of the capsid protein in e. coli was inhibited by the nucleotide sequence encoding the nls domain. however, the expression of the truncated cap protein, which lacks the first amino acids, was achieved which suggested that the production of the full-length capsid protein in e. coli was hindered only by the occurrence of rare arginine codons near the end of the cap gene. the heterologous expression of the entire cap gene was examined also in e. coli bl -codonplus(de )-ripl cells. this strain contains extra copies of the argu, iley, prol, and leuw genes for rare trnas, which rescue protein production restricted by either agg/aga or ccc codons. the expression of the cap gene in iptginduced e. coli bl -codonplus(de )-ripl yielded the production of a protein with an apparent molecular weight of kda which corresponded to the full-length cap protein ( fig. a) . this indicated that the expression of rare aminoacyl-trnas allowed the production of the full-length cap protein. however, the use of e. coli bl -codonplus(de )-ripl for a high-yield production of recombinant proteins is inconvenient as the expression system depends on using a combination of three different antibiotics: one for the maintenance of the expression vector, and the other for the maintenance of plasmids carrying trna genes. moreover, due to the presence of high amounts of antibiotics the growth rate of bacterial cells is reduced and expensive. a promising approach to maximise heterologous production of proteins in e. coli is codon optimization strategy which makes codon usage in the gene of interest to match the available trna pool within the cells (jana and deb, ; peti and page, ; burgess-brown et al., ) . to facilitate the cap gene expression in e. coli bl (de ), the sequence of the first codons was optimized as described in fig. c . ten codons, rare in e. coli, were replaced by the most frequent ones, including six arginine, two proline, and single leucine and threonine codon substitutions. the codon-optimized sequence was introduced into pet b-cap-his using ncoi and msci restriction sites to obtain pet b-o-cap-his (fig. b) . the expression of the o-cap-his gene in e. coli bl (de ) cells yielded the production of a protein with a molecular weight of kda (fig. a) . the corresponding band was recognised by the anti-his antibody which confirmed the presence of the o-cap-his protein (fig. b) . these data indicated that the replacement of the first codons of the cap gene with a codon-optimized sequence enabled the production of cap protein in a conventional prokaryotic expression system, such as e. coli bl (de ). different bioinformatics tools are available for codon optimization which considers many factors, such as rna secondary structure, gc content, repetitive and rare codons (grote et al., ) . these approaches were used to design a synthetic cap gene with nucleotide sequence optimized for prokaryotic expression in e. coli (genbank accession no. eu ). the synthetic gene with introduced ncoi and xhoi restriction sites was cloned into pet b vector carrying the c-terminal polyhistidine tag and the expression of this construct was analyzed by sds-page. however, no protein product was detected in coomasie-stained gel (data not shown). to force the expression of the capsid protein from the synthetic gene, additional codons encoding the polyhistidine tag were added at the end of the gene. the expression of this construct (pet b-s-cap) in e. coli bl (de ) yielded the s-cap protein with a molecular mass of kda, which was a slightly higher (+ kda) than the o-cap-his protein due to the presence of the additional polyhistidine tag at the n-terminus of the protein (fig. a) . the levels of the s-cap protein expression were comparable to those of the o-cap-his protein which indicated that codon optimization of the entire sequence of the cap gene did not enhance significantly the heterologous expression of the capsid protein in e. coli. the analysis of protein distribution in e. coli cells revealed that all the cap proteins were recovered in the soluble (cytoplasmic) fraction of the iptg-induced cell lysate. however, initial attempts to purify native cap proteins gave unsatisfactory results. using standard chromatographic procedures, no significant binding of cap proteins was detected while loading the material on either cationexchange or nickel immobilised affinity columns in native buffer conditions (data not shown). to enable purification of cap proteins, denaturating conditions in the presence of m urea were utilised. the cytosolic extract of the cell lysates was supplemented with solid urea to a final concentration of m and the mixture was loaded onto a unosphere-s cation-exchange column at ph . . a typical elution profile of protein fractions from the unosphere-s column is shown in fig. a . fractions - contained large amounts of unrelated bacterial proteins, while the later fractions, eluting after min, were enriched with cap proteins (fig. b) . the identity of the cap proteins was confirmed also by western blotting with anti-his antibody (fig. c) . however, some apparent differences between the s-cap protein and the remaining capsid proteins were observed during purification procedures. while the s-cap protein was recovered in a high purity in the distinct peak eluting at min (fig. b, fraction ) , both the o-cap-his and cap-his proteins were eluted from the column in a shorter time (about - min). moreover, these fractions were enriched partially with a protein with a molecular weight of about kda and some low molecular weight proteins below kda (data not shown). the original aim of using the polyhistidine tag was to isolate the cap proteins on a metal affinity chromatography column. however, the presence of two polyhistidine tags within the s-cap protein resulted in the increase of the basicity of the protein, which enabled simple purification of the protein by a single step cation-exchange chromatography. the next purification steps did not increase the purity of proteins (data not shown) and these steps were omitted in the purification protocol. the purity of the s-cap protein was greater than % after the cation-exchange chromatography and approximately mg of purified protein was obtained from l of bacterial cell culture. these data suggested that the yield of the s-cap protein is much higher than the yield of the o-cap-his and cap-his proteins in terms of purity, time and costs for large-scale production of the recombinant pcv capsid protein. to examine the immunogenicity of the s-cap protein, mice were immunized with the purified protein and the collected sera were analyzed for the presence of cap-specific antibodies. western blot analyses revealed that a single -kda band in the bacterial lysate enriched in the s-cap protein was recognised by the cap-positive sera, but not by the pre-immune sera (data not shown). moreover, a typical dense nuclear staining of pcv -infected pk cells was seen using the cap-positive sera compared to the pre-immune sera by in situ immunohistochemistry (data not shown). these results documented the immunogenicity of the s-cap protein isolated under denaturating conditions. the majority of vlps have been produced using insect or mammalian cell expression systems, but some vlps have been found to assemble also in e. coli cells (noad and roy, ) . in order to detect whether the pcv capsid protein is able to self-assemble into vlps in e. coli, the ultrastructure of the bacterial cells expressing the o-cap protein, which lacks any of the additional tag (fig. ) , was examined by using electron microscopy (em) of ultrathin sections. as shown in fig. , no vlps were observed in either non-induced cells or e. coli cells expressing the o-cap protein. in contrast, in vivo vlps assembly was detected unambiguously following the expression of a fusion protein containing the capsid and nucleocapsid protein obtained from the gag polyprotein of mason-pfizer monkey virus (ulbrich et al., ) (fig. a and b ). in addition, the sucrose density gradient fractionation of cytosolic content of e. coli cells expressing o-cap protein did not reveal any presence of o-cap protein in the form of vlps. the absence of vlps was corroborated also by em of negatively stained preparations (data not shown). these results demonstrated that the pcv capsid protein was unable to self-assemble into vlps in the cytosol of e. coli cells. in contrast, self-assembly of the pcv capsid protein into vlps has been observed repeatedly in baculovirus expression systems (nawagitgul et al., ; kim et al., ; liu et al., ) . the vlps were of similar morphology as the intact pcv virions and appeared to be empty capsids with a less ordered structure (nawagitgul et al., ) . however, the question why the pcv capsid protein does form vlps in insect cells and not in e. coli remains to be addressed. sequence analysis of the pcv capsid protein revealed amino acid residues with consensus patterns for potential posttranslational modifications (n-glycosylation, phosphorylation) (liu et al., b) , which are known to occur in eukaryotic but not in prokaryotic expression systems. another explanation could be a specific need for chaperones, scaffolding proteins, or ssdna, which might play important roles in a proper pcv assembly (ludwig and wagner, ) . a plausible hypothesis is that the n-terminal portion of the pcv capsid protein is enriched with basic amino acids that could bind preferentially to the bacterial genomic dna, and therefore, prevent a regular arrangement of the capsid subunits into vlps. this hypothesis would be supported by the observation that the e. coli cells expressing cap protein produced large amounts of outer membrane vesicles (fig. d ) compared to the non-induced cells or the cells expressing the capsid protein of mason-pfizer monkey virus. indeed, the release of outer membrane vesicles has been attributed to a novel envelope stress response (mcbroom and kuehn, ) , which might result from the binding of the cap protein to genomic dna. specificity and sensitivity of both the full-length capsid protein (s-cap) and its truncated variant ( cap-his) to pig pcv -positive sera was determined using an indirect elisa format (cap-elisa). using a chequer-board titration of pcv -positive serum (ipma titre of ), optimal antigen concentrations and serum dilutions were selected to be g/ml and : , respectively. when the positive/negative cut-off value was set to . (see section ), all the pcv -positive sera tested were % specific (data not shown). cap-elisa showed a low background, and significant differences between pcv -negative and pcv -positive pig sera were detected (fig. ) . cap-elisa did not exhibit any cross-reactivity with pig sera obtained from spf pigs which were infected experimentally with coronavirus causing a transmissible gastroenteritis (tge) or a porcine epidemic diarrhoea (ped), respectively. moreover, no significant differences in specificity and sensitivity of cap-elisa were observed between the s-cap and cap-his proteins when used as coating antigens, indicating that the n-terminus of the capsid protein is not specifically recognised by antibodies in the pcv positive sera (wu et al., ) . this showed that the recombinant full-length cap protein could be used as coating antigen to develop the indirect cap-elisa for specific and sensitive detection of pcv antibodies. pcv antibodies are detected currently by indirect immunofluorescence , ipma , competitive elisa (walker et al., ) and by indirect elisa based on either pcv viral particles (nawagitgul et al., ) or recombinant pcv capsid protein expressed in baculovirus (nawagitgul et al., ; blanchard et al., ; liu et al., ) . however, while both the indirect immunofluorescence and ipma assays are highly demanding and not suitable for large-scale survey of pcv infection, the cap-his proteins ( g/ml) was analyzed by a colorimetric reaction at the optical density at nm (od nm). od nm of . represents a cut-off value for seropositivity of the samples. tge-specific and ped-specific pig sera were obtained from spf pigs which were experimentally infected with the coronavirus causing transmissible gastroenteritis (tge) and porcine epidemic diarrhoea (ped), respectively. data represent the means ± s.d. for the three independent experiments. current elisa formats are less specific and display antigenic crossreactivity to non-pathogenic pcv (magar et al., ) . recently, an indirect elisa based on the recombinant nls-truncated pcv capsid protein expressed in e. coli has been established by shang et al. ( ) . although a direct comparison in the sensitivity and specificity of the full-length (this study) and the nls-truncated (shang et al., ) proteins as antigens would be interesting, it is very likely that both elisa formats are comparable in the detection of pcv antibodies. considering these data, the cap-elisa based on the recombinant full-length cap protein as coating antigen would provide a simple and reliable tool for standard serodiagnosis of pcv infection. to further analyze the capacity of cap-elisa to detect the pcv specific antibodies, a limited serological and genomic load survey of pcv infection was monitored in piglets during the first weeks of age. the piglet's antibody response along with total amounts of pcv dna per ml of serum was determined by cap-elisa and quantitative pcr, respectively. as shown in fig. a , the levels of pcv -specific antibodies showed an initial drop within the first weeks of age, from then on the levels increased gradually until th week. in contrast, the dna copy numbers of pcv increased gradually during the st weeks of age to reach the maximum level in the th week, after which they decreased slightly until the th week (fig. b) . these results are in good agreement with the time course dynamics of pcv infection in newborn piglets (mckeown et al., ) . a dramatic decrease of the pcv -specific antibodies in the th week of age corresponds to the period in which the passive intake of maternal antibodies from breast milk during the weaning period (weeks - ) is discontinued. this results in the onset of pcv infection after the th week of age prior to the active production of the intrinsic pcv -specific antibodies (weeks - ). taken the final values were expressed as a signal-to-positive (s/p) ratio. s/p value was determined according to the following formula: (od nm of sample − od nm of negative control)/(od nm of positive control − od nm of negative control). s/p value of . represents the cut-off signal for seropositivity of the samples. (b) time course analysis of the pcv dna copy numbers per ml of serum as detected by the quantitative pcr. total genomic dna was extracted from l of each serum sample and quantified by the pcr amplification of bp dna segment within the nucleotide sequence of the pcv cap gene using a taqman format. results of statistical analysis using one-way analysis of variance (anova) between serum samples obtained at given time intervals from piglets are represented by the box plots. the median value for each dataset is indicated by the black center line. the vertical height of each box indicates the - % data range. the upper and lower bars denote the largest and smallest data values. the marker ( ) denotes the extreme value of the individual serum sample that is > . times the inter-quartile range from the upper and lower quartile. the marker (᭹) denotes the extreme value of the individual serum sample that is > times the inter-quartile range from the upper quartile. data represent the means ± s.d. for the three independent experiments. together, these data confirmed that cap-elisa was developed with a high specificity and sensitivity for detection of pcv antibodies in pig sera and could be used for large-scale surveys of pcv infection at low cost. in summary, a bacterial expression system has been developed for the production of the full-length recombinant capsid protein of porcine circovirus type . here, the codon optimization strategy was used to obtain high yields of the recombinant capsid protein in an inexpensive cultivation system. purification protocol based on the single step cation-exchange chromatography provided a cost effective procedure to obtain substantial quantities of the cap protein in a high purity. although the recombinant capsid protein expressed in e. coli did not self-assemble into vlps, the antigenic properties of the cap protein resembled that of intact pcv virions. in addition, the recombinant full-length cap protein was used as antigen to develop the indirect cap-elisa for specific and sensitive detection of pcv infection. porcine circoviruses: a review isolation of porcine circovirus-like viruses from pigs with a wasting disease in the united states of america and europe an orf protein-based elisa for porcine circovirus type antibodies in post-weaning multisystemic wasting syndrome quantitation of porcine circovirus type isolated from serum/plasma and tissue samples of healthy pigs and pigs with postweaning multisystemic wasting syndrome using a taqman-based realtime pcr codon optimization can improve expression of human genes in escherichia coli: a multi-gene study postweaning multisystemic wasting syndrome: a review of aetiology, diagnosis and pathology post-weaning wasting syndrome comparison of the structures of three circoviruses: chicken anemia virus, porcine circovirus type , and beak and feather disease virus molecular characterization of porcine circovirus type isolates from post-weaning multisystemic wasting syndrome-affected and non-affected pigs isolation of circovirus from lesions of pigs with postweaning multisystemic wasting syndrome construction and immunogenicity of recombinant pseudotype baculovirus expressing the capsid protein of porcine circovirus type in mice immunogenicity and pathogenicity of chimeric infectious dna clones of pathogenic porcine circovirus type (pcv ) and nonpathogenic pcv in weanling pigs jcat: a novel tool to adapt codon usage of a target gene to its potential expression host expression levels influence ribosomal frameshifting at the tandem rare arginine codons agg-agg and aga-aga in escherichia coli strategies for efficient production of heterologous proteins in escherichia coli characterization of the recombinant proteins of porcine circovirus type field isolate expressed in the baculovirus system nuclear localization of the orf protein encoded by porcine circovirus type bacterial expression of an immunologically reactive pcv orf fusion protein development of an elisa based on the baculovirus-expressed capsid protein of porcine circovirus type as antigen characterization of a previously unidentified viral protein in porcine circovirus type -infected cells and its role in virus-induced apoptosis efficient production of type porcine circovirus-like particles by a recombinant baculovirus virus-like particles-universal molecular toolboxes retrospective serological survey of antibodies to porcine circovirus type and type differential recognition of orf protein from type and type porcine circoviruses and identification of immunorelevant epitopes replication of porcine circovirus type requires two proteins encoded by the viral rep gene identification of a protein essential for replication of porcine circovirus release of outer membrane vesicles by gramnegative bacteria is a novel envelope stress response effects of porcine circovirus type (pcv ) maternal antibodies on experimental infection of piglets with pcv mistranslational errors associated with the rare arginine codon cgg in escherichia coli characterization of novel circovirus dnas associated with wasting syndromes in pigs detection and characterization of porcine circovirus associated with postweaning multisystemic wasting syndrome in pigs modified indirect porcine circovirus (pcv) type -based and recombinant capsid protein (orf )-based enzyme-linked immunosorbent assays for detection of antibodies to pcv open reading frame of porcine circovirus type encodes a major capsid protein virus-like particles as immunogens strategies to maximize heterologous protein expression in escherichia coli with minimal cost development and validation of a recombinant capsid protein-based elisa for detection of antibody to porcine circovirus type update on porcine circovirus and post-weaning multisystemic wasting syndrome (pmws). j. swine health prod a very small porcine virus with circular single-stranded dna expression of porcine circovirus orf gene requires codon optimized e. coli cells distinct roles for nucleic acid in in vitro assembly of purified mason-pfizer monkey virus canc proteins overexpression of an mrna dependent on rare codons inhibits protein synthesis and cell growth in vitro expression, monoclonal antibody and bioactivity for capsid protein of porcine circovirus type ii without nuclear localization signal expression of the porcine circovirus type capsid protein subunits and application to an indirect elisa development and application of a competitive enzyme-linked immunosorbent assay for the detection of serum antibodies to porcine circovirus type the excellent technical help of sona charvatova, hana kubinova, zuzana vecerkova and petra klodnerova (proteix, s.r.o.) is acknowledged. we also wish to thank pavel ulbrich (institute of chemical technology, prague, czech republic) for providing mason-pfizer monkey virus canc expression vectors. this work was supported by grants gacr / /p (l.b.), npvii b (p.s.) of the ministry of education, youth and sports of the czech republic, npv b (i.p.) and (i.p.) of the ministry of agriculture of the czech republic, and institutional research concept av z and av z . key: cord- -v q spmm authors: resende, talita pilar; medida, ramya lekha; guo, yue; vannucci, fabio a.; saqui-salces, milena; gebhart, connie title: evaluation of mouse enteroids as a model for lawsonia intracellularis infection date: - - journal: vet res doi: . /s - - - sha: doc_id: cord_uid: v q spmm lawsonia intracellularis, an obligate intracellular bacterium, is an important enteric pathogen in pig herds and horse farms worldwide. the hallmark feature of l. intracellularis infection is the proliferation of epithelial cells in intestinal crypts. a major limitation to the study of l. intracellularis infection is the lack of an in vitro model that reproduces the changes observed in proliferative enteropathy. here we investigated the suitability of mouse enteroids as a model to study l. intracellularis infection. mouse enteroids were microinjected with l. intracellularis, filter-sterilized l. intracellularis culture supernatant, or sterile cell culture media (dmem). l. intracellularis antigen was detected in mouse enteroids by immunohistochemistry and was located mostly in the basal region of the epithelium. there was no differential growth of enteroids among treatment groups, and cellular proliferation was not increased in l. intracellularis-infected enteroids in relation to non-infected enteroids based on immunofluorescence staining. l. intracellularis infection did not induce changes in gene expression of ki- (proliferation marker), sox (marker for transit amplifying cells) and muc (marker for goblet cells). these results indicate that although l. intracellularis antigen is detectable in mouse enteroids, indicating susceptibility to infection, mouse enteroids fail to replicate the cellular proliferation and gene expression changes observed in proliferative enteropathy. nevertheless, we have successfully demonstrated that mouse enteroids can be used to model days-long intracellular pathogen infection, serving as potential models for the study of other pathogens of interest in veterinary medicine. pig herds and horse farms worldwide are regularly challenged by proliferative enteropathy (pe). this disease is caused by lawsonia intracellularis and is characterized by the thickening of the small intestinal mucosa due to proliferation of intestinal epithelial cells. pe causes diarrhea and compromised weight gain in pigs [ , ] . in weaned foals, pe causes diarrhea and hypoproteinemia, occasionally resulting in death [ ] . other species are also affected by pe, including ratite birds, rabbits, non-human primates, rats and mice [ ] . lawsonia intracellularis is a gram-negative bacterium that requires an intracellular culture system and a specific atmosphere to be propagated in vitro [ ] . traditional single cell cultures (cell lines) are permissive to l. intracellularis propagation, but they have failed to reproduce the increased cellular proliferation observed in pe-affected animals [ ] . the lack of an in vitro model that represents the in vivo progression of pe has limited advancement in knowledge of the pathogenesis mechanisms utilized by l. intracellularis. hence, identification of new alternatives for control and prevention of pe has been difficult, and pig producers and horse owners have been relying on available commercial vaccines (whole cell vaccines) and antimicrobials to prevent and control pe [ ] [ ] [ ] [ ] . the costs associated with producing whole cell vaccines is reflected on the commercial price of the vaccines, hampering the access to some producers to the commercial vaccines, and, therefore preventing pe. in addition, the use of antimicrobials as growth promoters in swine production has raised concerns about the selection of antibiotic-resistant organisms in treated herds [ ] . therefore, a deeper understanding open access *correspondence: resen @umn.edu department of veterinary and biomedical sciences, college of veterinary medicine, university of minnesota, st. paul, mn , usa full list of author information is available at the end of the article of pe pathogenesis is necessary for the development of novel non-antimicrobial based prevention and treatment methods such as recombinant vaccines or antimicrobial alternative strategies [ ] [ ] [ ] [ ] . the currently held hypothesis is that l. intracellularis infects intestinal epithelial cells, especially in the crypt compartment, leading to cellular proliferation and decreased differentiation in goblet cells along with increased apoptotic events [ , , ] . the mechanisms involved in epithelial changes during the course of pe are still unclear. thus, knowledge about interactions between l. intracellularis and intestinal epithelial cells, determined using a controlled environment, could generate a better understanding of how l. intracellularis induces cellular proliferation. a promising alternative to single cell cultures are tridimensional multi-cell type cultures, also known as organoids. one of the most relevant advantages of organoids in relation to the traditional in vitro single cell culture is the similarity of the organoids to their respective tissue of origin [ , ] . enteroids, small intestinal organoids, for instance, possess not only enterocytes, but also enteroendocrine cells, transit amplifying cells, goblet cells and stem cells. all these cells form structures with cell distributions that are similar to crypts and villi found in the small intestine, and possess a lumen where cell debris and secretion are shed continuously [ ] . enteroids have been recently used to study host-pathogen interactions of important enteric bacteria in human medicine [ ] [ ] [ ] [ ] [ ] , but they have not been investigated as a model for bacterial infections in the field of veterinary medicine [ , ] . the objective of this study was to evaluate mouse enteroids as an in vitro model for l. intracellularis infection. infected enteroids were monitored over time by size along with changes in expression of genes that have been reported to change during pe. we found evidence of infection with l. intracellularis in enteroids followed for up to days post-infection (dpi). the genetic profile of infected mouse enteroids, however, diverged from the gene expression changes observed in pe. mouse enteroid preparation and maintenance were performed as described elsewhere [ ] (iacuc approval number - a). the formulation of the enteroid culture medium was as described previously [ ] . a l. intracellularis isolate (phe/mn - , atcc pta- , manassas, va, usa) at low (≤ ) passage [ ] was propagated in mccoy mouse fibroblast-derived monolayers (atcc ® crl- ™ ). mccoy cells were cultured as described elsewhere [ ] . cells were cultured in dulbeco's modified eagle medium (dmem, gibco thermofisher, waltahm, ma, usa) with % fetal bovine serum (fbs; heat inactivated, corning ™ cv, corning, ny, usa). mccoy cells at % confluency were infected with about l. intracellularis and incubated at °c under a controlled microaerophilic atmosphere created with a mixture of gas containing % hydrogen, % carbon dioxide, and % nitrogen [ ] . seven days after infection, cell monolayers were mechanically lysed by passage through a -gauge needle coupled to a syringe after immersion in sterile . % potassium chloride and then centrifuged at × g to separate cell debris from l. intracellularis organisms. the pellets were discarded and the remaining supernatant was then filtered through . µm sterile filters (millipore sigma, burlington, ma, usa) to remove any remaining mccoy cells and nuclei. after a final centrifugation at × g at °c the bacterial pellet was either used to re-infect mccoy cells, or to prepare the inoculum suspended in approximately µl of its own supernatant and used as inoculum. as l. intracellularis growth requires special atmospheric conditions [ ] , we first tested whether the atmosphere conditions required for l. intracellularis growth would impact growth and development of mouse enteroids. one plate with enteroids was maintained under atmospheric conditions specific for l. intracellularis i.e., % hydrogen, % carbon dioxide, and % nitrogen [ ] while another plate was maintained at % co as a control. both plates were incubated at °c. since l. intracellularis manipulation in vitro requires an antibiotic-free sterile system, antibiotics (penicillin/streptomycin) were excluded from both plates to assess sterility of the cultures. the plates were observed every h for weeks, with media replacement and enteroid passage as described previously. to standardize conditions for infection of mouse enteroids with l. intracellularis, we adopted the "incubation and seed" method [ ] . briefly, immediately after enteroid passage and before plating, enteroids were directly mixed with l. intracellularis suspended in µl of enteroid culture medium (resulting in an inoculum with ~ l. intracellularis organisms/ml), with occasional gentle agitation, and incubated at °c under special atmospheric conditions generated with a gas tank of % hydrogen, % carbon dioxide, and % nitrogen for min. the infected enteroids were briefly centrifuged at × g for min, mixed with - µl ice-chilled matrigel (corning, usa), and then seeded into culture plates. enteroids were harvested days post-infection (dpi) by removing culture media and suspending the matrigel in phosphate buffered saline (pbs). after centrifugation for min at × g, the pellet was suspended in % formaldehyde and incubated for h at room temperature. enteroids were then washed in pbs, centrifuged at × g for min, suspended in histogel (thermofischer, usa) and placed into a biopsy mold. histogel blocks were inserted into a cassette and maintained in % alcohol until they were processed for histological evaluation. four-micrometer sections were placed onto glass slides and used for hematoxylin and eosin (h&e) staining and immunohistochemistry [ ] . although l. intracellularis antigen was detected by ihc in the infected enteroids by this method (figure ), we opted to employ the microinjection approach described below to facilitate contact of l. intracellularis with the cellular apical membrane in an effort to improve the level of infection. mouse enteroids were cultured and passaged until a sufficient number of enteroids (average of enteroids per treatment group, per time point) with µm diameter were obtained for the trial. wnt a protein, which regulates the signaling pathways related to the cellular proliferation in the intestinal crypt compartment [ ] , was removed from the enteroid culture media at least days before infection to enable cells to better differentiate. twenty-four hours prior to infection, enteroids were divided into three treatment groups: dmem: sterile antigen is associated mostly with cell debris (arrow heads), × . b l. intracellularis antigen is associated with cell debris (arrow heads) and in close proximity to enteroid cells (black arrows), × . c l. intracellularis antigen is observed in close association with mouse enteroid cells (black arrows), × . d a few l. intracellularis antigens are observed in this image, some close to mouse enteroid cells (black arrow), and some associated with cell debris (arrow head), × . enteroid lumen is indicated by an asterisk (*). culture media; sn: sterile-filtered l. intracellularis-culture supernatant; l. intracellularis: suspension with about l. intracellularis organisms/ml. each enteroid received approximately nl of each respective treatment. treatments were placed directly into the lumen of the enteroids by microinjection using a microinjector (nanoject ii, drumond scientific, broomall, pa, usa) operated by a trained technician. experiments were performed in four independent replicates. microinjected enteroids were monitored for week, from the day prior to infection until dpi. images were captured at × magnification using an inverted microscope coupled with a digital camera immediately after injection ( dpi), and at , and dpi. the enteroid area was measured using an image software (nih imagej software . r, national institute of health, bthesda, md, usa). the relative enteroid area (enteroid growth) was measured by subtracting the enteroid area from the area recorded at its previous measurement. enteroids from each treatment group were harvested at , and dpi and prepared for immunofluorescence. matrigel was disrupted with a sterile pipet tip and the contents in each well were transferred to a . ml tube and centrifuged at × g for min. the supernatant was discarded, and the pellet suspended in ml of pbs. this step was repeated two times to remove matrigel. the resulting enteroid pellet was suspended and fixed in % paraformaldehyde for h at room temperature. after fixation, enteroids were washed three times in pbs and centrifuged at × g for min. fixed enteroids were embedded in histogel (richard-allan scientific histogel, thermofischer, usa) and then either placed in optimum cutting temperature (oct, tissue-tek; sakura finetek, beaver creek, co, usa) for preparing cryosections or dehydrated in % ethanol and embedded in paraffin. paraffin-embedded sections ( μm) on charged slides were used for ihc for detection of l. intracellularis using specific rabbit polyclonal antibodies and a previously described method [ ] . stained sections were evaluated by bright field microscopy. oct-embedded blocks were sectioned ( µm), placed on charged glass slides and stored at − °c until immunofluorescence staining. anti-ki- immunofluorescence was performed as described elsewhere [ ] . briefly, slides were hydrated with tris buffered saline (tbs) containing . % triton-x (tbs-t) for min and then blocked with % normal goat serum (zc , vector laboratories, burlingame, ca, usa) for min to prevent non-specific binding. sections were then incubated with anti-ki- antibody ( : in tbs-t, crm b, biocare medical, pacheco, ca, usa) for h, followed by a min wash in tbs-t and incubation with cy -labeled goat antirabbit antibody ( : , ab , abcam, burlingame, ca, usa) for min. all incubations were carried out at room temperature. slides were washed and mounted with prolongold (thermofisher, usa) and covered with glass coverslips. the number of ki- -positive cells and the total number of cells were counted in at least random fields at × using an olympus bx fluorescence microscope. enteroids were harvested at , , and dpi, released from matrigel as described above, lysed with . ml of trizol (thermofisher, usa) and stored at − °c until rna extraction. rna extraction was performed using chloroform and isopropanol precipitation following trizol protocols and then cleaning with the rneasy mini kit (qiagen, germantown, md, usa) according to the manufacturer's instructions. total rna was quantified using a nanodrop (thermo scientific, usa) and ng of rna were transcribed to cdna using the high capacity cdna reverse transcription kit (applied biosystems, foster city, ca, usa) with random hexamer primers. quantitative pcr reactions were performed using sybr green pcr master mix (applied biosystems) according to the manufacturer's instructions. amplification was performed with the following conditions: initial activation at °c for min, followed by cycles of denaturation at °c for s and annealing at °c for s. expression of the housekeeping gene gapdh was monitored as reference. relative gene expressions were normalized to gapdh using the primer efficiencies. the expression levels of ki- (marker for cellular proliferation), sox (marker for transit amplifying cells) and muc (marker for goblet cells) in enteroids of all treatment groups were measured at , and dpi. fold change in each case was calculated in relation to expression in enteroids from day (control). all primers used are listed in table . results are presented as a mean ± standard deviation of the mean (sem) for each group. two-way anova followed by geisser-greenhouse correction was used to verify statistical differences with p < . considered statistically significant. graphpad prism . software for windows (graphpad software, la jolla, ca, usa) was used to perform the analysis. there was no difference in growth or morphology between enteroids maintained for week in the microaerophilic atmosphere relative to those maintained under regular conditions, i.e., % co incubator (data not shown), indicating that growth conditions required for l. intracellularis do not impact enteroid viability or growth. mouse enteroids were infected with l. intracellularis by microinjection. enteroids were harvested and analyzed by ihc for l. intracellularis antigen at , , and dpi. l. intracellularis antigen presence was observed at all time points, indicating successful infection with l. intracellularis. unexpectedly, although l. intracellularis antigen was observed in the cytoplasm of enteroid cells, most of the antigen, especially at dpi, was found in the basal region of epithelial cells (figure ). this is in contrast to the location of l. intracellularis in vivo, which is in the apical region of the epithelial cells [ , ] . to determine whether l. intracellularis infection would lead to increased size or secretory activity of infected enteroids, the area of enteroids in each treatment group was measured at , , and dpi. l. intracellularisinfected enteroids did not have higher area compared with sn or dmem treatment groups ( figure a ). representative images of enteroids from each treatment group are shown in figure b . to determine whether l. intracellularis infection induces changes in the proliferation and differentiation of enteroid epithelial cells, as observed in the swine intestine, we evaluated expression of ki- , sox and muc in enteroids harvested at , and dpi relative to expression in enteroids at dpi. there were no significant differences in expressions of any of the genes analyzed in the treatment groups independent of the time point (figure ) . immunofluorescence staining confirmed that none of the treatment groups showed increased ki- expression overtime, although in the l. intracellularis infected group considerable variability in the in ki- expression throughout was noted ( figure ). classical single cell culture systems have been extensively used in studies to understand host-pathogen interactions. although they provide valuable information, these models are limited by their inability to represent the tissue organization observed in vivo since most of the single cell systems are either cancer-derived or transformed immortalized cell lines [ , ] . these limitations make it difficult to assess the proliferative effects of l. intracellularis infection [ ] . the mechanism by which l. intracellularis causes proliferation of intestinal epithelial cells remains unclear. recently, the effects of l. intracellularis infection on the proliferation of monocultures of non-intestinal, non-epithelial cells, as well as intestinal epithelial cells were investigated under various culture conditions [ ] and the findings supported previous observations that cell monocultures infected with l. intracellularis do not accurately represent proliferation observed in lesions in the intestine of affected animals. in vivo, l. intracellularis accesses the cell cytoplasm via the apical membrane and the organisms are observed in the cytoplasm throughout the course of pe. l. intracellularis is more frequently observed in crypt cells than cells of the villus [ , , ] . therefore, we hypothesized that enteroids would be a suitable model to further investigate l. intracellularis pathogenesis in vitro by providing polarized epithelial cell culture with all the crypt and villus epithelial cell types found in the mammalian intestine and similar cell exchange ratio to the in vivo intestine [ ] . enteroids were firstly developed in [ ] and have been used to study intestinal morphophysiology as well as pathogenesis of some enteric diseases [ , , , ] . human enteroids can be obtained by culture of intestinal crypts isolated from biopsies. mouse enteroids are more widely used in research as the ethical and biosafety regulations for human enteroids are more restrictive. additionally, mouse intestinal physiology is well-characterized and molecular tools to study intestinal cell proliferation and differentiation are available. the presence of l. intracellularis dna has been detected in feces of mice trapped in pig farms and in the surroundings of horse farms [ , ] , suggesting that mice are susceptible to infection. furthermore, mice experimentally infected with l. intracellularis develop intestinal lesions that resemble lesions in affected pigs and horses, although the lesions in mice are less extensive, less severe and mostly localized in the large intestine when compared to the lesions in pigs and horses [ ] [ ] [ ] . in addition, l. intracellularis has been successfully propagated in vitro using mccoy mouse fibroblast cells [ , , ] . based on this knowledge, we hypothesized that mouse enteroids may serve as a feasible model to investigate the progression of l. intracellularis infection and the mechanisms involved during cell proliferation. mouse enteroids in the tridimensional culture conditions, as spheres composed of polarized epithelium, have their cellular apical side oriented to the center of the enteroid. hence, l. intracellularis infection in this study was performed by microinjecting the bacterial suspension directly into the lumen of the enteroid. microinjection is a technique that has inherent limitations in controlling the bacterial inoculum, i.e., the exact number of bacteria each enteroid was exposed to. although we plated enteroids to obtain an average of enteroids per well, the actual number of enteroids per well and their size at the time of injection was found to be variable. these technical limitations may have contributed to the high variability observed in our results and constitute one of the limitations of using tridimensional enteroids as models for the study of luminal pathogens. others have used mouse enteroids [ , ] and human enteroids [ , ] to study bacterial pathogenesis using microinjection. however, none of these studies evaluated infection times longer than h [ , ] . because of the nature of l. intracellularis infection, longer durations, i.e., several days, are needed to observe the expected changes. to our knowledge, this is the first enteroid infection experiment reporting successful bacterial infection of enteroids for up to dpi. whether the length of the infection times had a significant effect on the number of bacteria present and variability on the evaluated parameters remains to be defined. one of the most intriguing findings in the present experiment was the unexpected location of l. intracellularis in the infected enteroids. while l. intracellularis antigen was observed at all time points tested ( , and dpi), the antigen was mostly observed localized in the extracellular space proximal to the basal region of the epithelium of the mouse enteroids. in vivo, l. intracellularis organisms are consistently observed on the apical side of the cytoplasm at similar infection time points to those used in this study [ , ] . a possible explanation for this difference in localization is that, in contrast to conditions in vivo, nutrients necessary for enteroid growth and maintenance are placed over the matrigel, in a region that corresponds to the basal lamina in vivo and not directly in the enteroid lumen. we speculate that the gradient of nutrients on the exterior of the enteroid could be causing the l. intracellularis to move towards the nutrients to support their growth. it is also possible that conditions of the enteroid culture environment could result in less permissible conditions for l. intracellularis closer to the lumen. another possibility is that the fibroblasts and immune cells in the basal region of the intestinal epithelium in vivo may have a regulatory effect on l. intracellularis traffic in the intestine, and the lack of those cells in the mouse enteroid culture would modify l. intracellularis traffic in vitro. immunohistochemistry for l. intracellularis is considered the gold standard method for diagnosis of proliferative enteropathy [ , ] . in the present study, we used immunohistochemistry to verify whether the mouse enteroids, microinjected with l. intracellularis suspension (~ l. intracellularis organisms/ml), were infected and to visually demonstrate the level of infection in these microinjected enteroids. our results were not only surprising in regards to the localization of l. intracellularis antigen, as discussed before, but also in regards to the amount and morphology of the stained antigen. generally, l. intracellularis antigen stained by immunohistochemistry is observed as small bacilli in the cytoplasm of cell cultures [ , , ] and in the apical cytoplasm of intestinal epithelial cells of naturally and experimentally-infected pigs. in the present study, instead of observing similar morphology of l. intracellularis antigen, we observed mostly the antigen as a "bundle" accumulated on the basal region of the enteroid cells. nevertheless, occasional well-shaped l. intracellularis antigens were observed in the cytoplasm of some enteroid cells ( figure d ), which is an indication that after the microinjection in the lumen of the enteroid, l. intracellularis organisms gained entrance into the cell cytoplasm. the events involving the changes in the morphology of the antigen and its final location in the basal side of enteroid cells remains unclear and may deserve more investigation. the homeostasis of the intestinal epithelium involves various signaling pathways of which canonical wnt signaling is responsible for maintaining a balance between the pool of undifferentiated cells with proliferation capacity, named transit amplifying cells, and differentiated cells [ , ] . in vitro, when enteroids are cultured in the presence of wnt a in the media, the transit amplifying cells stay undifferentiated for a longer period of time in relation to in vivo conditions [ ] , resulting in enteroids in a pro-proliferative state. in the present study, one of the main objectives was to verify the effects of l. intracellularis on cell proliferation, therefore we removed wnt a from the culture media days before microinjection and during the experiment to allow the epithelial cells to differentiate, and better reproduce the proportion of proliferative cells of the normal intestinal epithelium. however, we failed to detect increased proliferation in l. intracellularisinfected enteroids by rt-qpcr or immunofluorescence. it is possible that the removal of wnt a days before microinjection was not sufficient to reduce proliferation to a level where we could detect changes induced by l. intracellularis. another possible explanation for this lack of difference in proliferation between treatment groups was the small amount of l. intracellularis delivered to the enteroid lumen, thus requiring a longer time for the bacteria to replicate in numbers to produce a detectable change in proliferation markers. the low amount of antigen detected in the microinjected enteroids indicate that l. intracellularis did not encounter an ideal environment for propagation as it does in mccoy cells and in the intestines of mice and other rodents [ , , , , ] . the infection efficacy of enteroids derived from the intestines of more l. intracellularis susceptible species, such as pigs and horses, would help to confirm this hypothesis. changes in the area of enteroids can indicate an expansion on the number of cells or an increase in secretory activity. secretory activity of enteroids has been demonstrated by swollen enteroids as a result of accumulation of luminal secretions after stimulation with forskolin [ ] . in the present study, we expected to observe increased enteroid areas of infected enteroids that could be due to increased numbers of cells (proliferation) or secretory activity. in contrary to our expectations, we observed an increased volume from dpi to dpi on the sn group without changes in proliferation, suggesting the possibility of a secreted product with capacity to induce intestinal secretion. further confirmation of this phenomenon is required. l. intracellularis is a unique bacterium, requiring intracellular conditions and special atmospheric conditions for propagation in vitro. little is known about l. intracelllaris survival in the cellular cytosol, or the host-cellular pathways that are disrupted during l. intracellularis infection. by using mouse enteroids as an in vitro model for l. intracellularis infectioin, our aim was to develop a tool to further define several aspects of l. intracellularis pathogenesis. the localization of l. 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peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold open access which fosters wider collaboration and increased citations maximum visibility for your research: over m website views per year • at bmc salmonella-infected crypt-derived intestinal organoid culture system for host-bacterial interactions interaction of salmonella enterica serovar typhimurium with intestinal organoids derived from human induced pluripotent stem cells intestinal organoids model human responses to infection by commensal and shiga toxin producing escherichia coli characterization of porcine intestinal enteroid cultures under a lipopolysaccharide challenge porcine intestinal enteroids: a new model forstudying enteric coronavirus porcine epidemic diarrhea virus infection and the host innate response establishment of human epithelial enteroids and colonoids from whole tissue and biopsy dietary fiber sources and non-starch polysaccharidedegrading enzymes modify mucin expression and the immune profile of the swine ileum attenuation of virulence of lawsonia intracellularis after in vitro passages and its effects on the experimental reproduction of porcine proliferative enteropathy comparison of intestinal mucosa homogenate and pure culture of the homologous lawsonia intracellularis isolate in reproducing proliferative enteropathy in swine an alternative method for cultivation of lawsonia intracellularis adenovirus-mediated efficient gene transfer into cultured three-dimensional organoids preparation and characterization of polyclonal and monoclonal antibodies against lawsonia intracellularis extrinsic control of wnt signaling in the intestine the third dimension bridges the gap between cell culture and live tissue evaluation of porcine ileum models of enterocyte infection by lawsonia intracellularis transmissible lleal hyperplasia of hamsters. ii. ultrastructure adult intestinal stem cells: critical drivers of epithelial homeostasis and regeneration single lgr stem cells build crypt-villus structures in vitro without a mesenchymal niche human enteroids as a model of upper small intestinal ion transport physiology and pathophysiology adenovirus infection of human enteroids reveals interferon sensitivity and preferential infection of goblet cells lawsonia intracellularis in the feces of wild rodents and stray cats captured around equine farms colonisation and shedding of lawsonia intracellularis in experimentally inoculated rodents and in wild rodents on pig farms infection of sparrows (passer domesticus) and different mice strains with lawsonia intracellularis evaluation of the involvement of mice (mus musculus) in the epidemiology of porcine proliferative enteropathy validation of an immunoperoxidase monolayer assay as a serologic test for porcine proliferative enteropathy comparison of different methods for diagnosis of porcine proliferative enteropathy entry of the bacterium ileal symbiont intracellularis into cultured enterocytes and its subsequent release characterization of lawsonia intracellularis gen. nov., sp. nov., the obligately intracellular bacterium of porcine proliferative enteropathy the intestinal crypt, a prototype stem cell compartment infection of different strains of mice with lawsonia intracellularis derived from rabbit or porcine proliferative enteropathy a functional cftr assay using primary cystic fibrosis intestinal organoids publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors acknowledge the technical support provided by marta ferrandis-vila, caitlin kupiec, ashley manicor, amanda g. de souza daniel, erika vasquez, and lacey marshall-lund. of the basal side of the enteroid epithelial cells may indicate nutrients or a microenvironment preferred by l. intracellularis and merits further investigation. likewise, little is known about mouse enteroids maintained with a pathogenic enteric bacterium for a period longer than h [ , ] . more information is needed to determine if long-term infection of enteroids mirrors the dynamics of epithelium in vivo in terms of secretion, differentiation and apoptosis observed in chronic enteric diseases. finally, yet importantly, although we were able to demonstrate infection of mouse enteroids, we did not observe the changes associated with l. intracellularis infection that have been noted in vivo in more susceptible species. testing l. intracellularis infection in enteroids from other more susceptible species will provide further insight to species susceptibility to pathogenesis. authors' contributions tpr contributed to the acquisition, analysis and interpretation of data and preparation of the manuscript. rlm contributed to execution of the experiments, data acquisition and interpretation. yg contributed in the execution of the experiments. cg, fav and mss contributed to the conception, design, data analysis and interpretation of the study. all authors read and approved the final manuscript. the authors disclose receipt of the following financial support for the research, authorship, and/or publication of this article: tpr was supported by the brazilian government sponsoring agency coordenadoria de aperfeiçoamento de pessoal de nível superior (capes). the project was partially supported by nifa min- - (mss). the data collected in this project is shown in our results section. further information can be available upon request. mouse manipulation was approved by the university of minnesota institutional animal care and use committee (iacuc approval number - a). the authors declare that they have no competing interests. key: cord- -ug ler e authors: ramos-rincón, josé m.; pinargote-celorio, héctor; belinchón-romero, isabel; gonzález-alcaide, gregorio title: a snapshot of pneumonia research activity and collaboration patterns ( – ): a global bibliometric analysis date: - - journal: bmc med res methodol doi: . /s - - - sha: doc_id: cord_uid: ug ler e background: this article describes a bibliometric review of the scientific production, geographical distribution, collaboration, impact, and subject area focus of pneumonia research indexed on the web of science over a -year period. methods: we searched the web of science database using the medical subject heading (mesh) of “pneumonia” from january , to december , . the only document types we studied were original articles and reviews, analyzing descriptive indicators by five-year periods and the scientific production by country, adjusting for population, economic, and research-related parameters. results: a total of , references were retrieved. the number of publications increased steadily over time, from publications in to in (r( ) = . ). the most productive country was the usa ( . %), followed by the uk ( . %) and japan ( . %). research production from china increased by more than %. by geographical area, north america ( . %) and europe ( . %) were most dominant. scientific production in low- and middle-income countries more than tripled, although their overall contribution to the field remained limited (< %). overall, . % of papers were the result of an international collaboration, although this proportion was much higher in sub-saharan africa ( . %) and south asia ( . %). according to the specific mesh terms used, articles focused mainly on “pneumonia, bacterial” ( . %), followed by “pneumonia, pneumococcal” ( . %) and “pneumonia, ventilator-associated” ( . %). conclusions: pneumonia research increased steadily over the -year study period, with europe and north america leading scientific production. about a fifth of all papers reflected international collaborations, and these were most evident in papers from sub-saharan africa and south asia. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. acquired pneumonia (cap) remains the primary cause of death from infectious disease globally, and its high impact on morbidity and mortality is especially concentrated in children under five and the elderly [ , [ ] [ ] [ ] . the world health organization (who) predicted that deaths from lower respiratory tract infections would remain among the top four causes of deaths up to at least [ ] . antibiotic-resistant strains have also been on the rise, although resistance does not appear to be related to mortality. however, pneumonia is associated with high rates of hospitalization and length of hospital stay. moreover, it has considerable long-term effects on quality of life, and long-term prognosis is worse in patients with pneumococcal pneumonia [ ] . despite the public health importance of the disease, few studies have evaluated research in the area using bibliometric methods. indeed, only head et al. ( ) have analyzed publications on pneumonia, and their work was limited in geographical scope to the uk [ , ] . in this study, by analyzing scientific papers on pneumonia published in the main international scientific journals, we aimed to identify the scientific contribution of different countries to the worldwide research effort, the most cited landmark articles, the degree and nature of scientific collaboration, and the topics addressed. this bibliometric description can provide relevant information for researchers in the field, particularly new scientists, giving a snapshot of strong research areas in pneumonia and global health as well as possible gaps requiring additional investments [ ] [ ] [ ] . the paper also provides clues for addressing the weaknesses observed, such as the need to promote north-south collaborations and other research initiatives with countries that have relatively little scientific development on the topic [ , ] . the aim of the present study is to assess the scientific literature on pneumonia that is indexed in the web of science (wos). specifically, we will analyze: ( ) the evolution of scientific production; ( ) its distribution by countries and regions; ( ) the impact of the research papers; and ( ) the degree of international collaboration. finally, we will present details on the subject area focus of different publications according to the medical subject headings (mesh). for the performance of the study, we opted to identify documents about pneumonia by means of the mesh thesaurus in the medline database because this is a detailed instrument for controlled terminology. the thesaurus employs both a human team of specialist indexers to analyze each article and assign medical subject headings to it, plus automated processes to improve indexing; the result is a highly consistent system of classification for research topics [ ] [ ] [ ] . the pneumonia descriptor was introduced in as a disease of the respiratory tract and the lung, and it was defined as "infection of the lung often accompanied by inflammation" [ ] . synonyms of this descriptor (and therefore also included in search results) are "lung inflammation" and "pulmonary inflammation". additional file : table s shows the mesh tree structure for "pneumonia". the next step was to identify the documents assigned with the medline descriptor of "pneumonia" indexed in the wos. this body of research constitutes the population of documents for the present study. conceived by eugene garfield but now maintained by clarivate analytics, wos is the top scientific citation search and analytical information platform worldwide, serving both as a multidisciplinary research tool supporting a variety of scientific tasks and as a dataset for large, dataintensive studies [ ] . the use of the wos databases enables the analysis of all institutional affiliations reported in the documents and the calculation of citation indicators. the wos brings together the most visible literature at a global level. these qualities justify its choice as the database platform used in this study despite some limitations related to covering non-english biomedical journals [ ] . although initially no limitations were imposed on our search, to calculate the bibliometric indicators we considered only two types of documents, articles and reviews, as these are the primary references for researchers. the study period was limited to - , as delays associated with assigning mesh descriptors to documents mean that information on the most recent articles on pneumonia is not updated. the searches took place on the clarivate analytics wos platform, which includes medline database, on march , . for each of the retrieved documents, data on the following bibliographic characteristics were extracted: year of publication, journal of publication and wos subject category, document type, authorship, citations, institutional affiliation(s), and mesh descriptors. data were then standardized: institutional affiliations corresponding to england, northern ireland, scotland and wales were grouped together under "united kingdom," while affiliations in overseas france, british overseas territories, and island dependencies were also assigned to their ruling countries (for example, the documents signed by authors from french polynesia, guadeloupe, martinique, new caledonia, and reunion were assigned to france), although regional designations correspond to geographical rather than political criteria. scientific production from taiwan, which in wos is considered independently from the democratic republic of china (china) but whose status is disputed at an international level, was analyzed separately. countries responsible for publications were categorized according to their world bank classification by income level: low-income (< usd ), lower-middleincome (usd to usd ), upper-middle-income (usd to usd , ) , and high-income (≥ usd , ) countries. each of the countries identified was assigned to a macro geographical (continental) region according to the groups established by the world bank based on geopolitical and economic criteria and reflected in the world bank country and lending groups (see additional file : tables s and s ) [ ] . two kinds of indicators were obtained: descriptive indicators for the evolution of scientific production production by country, adjusted for demographic and economic parameters as well as for human resources dedicated to research activities we determined standardized indicators for each country's productivity with respect to: -population: number of publications per million inhabitants (population index). data were obtained from world development indicators in the world bank online databases [ ] . we calculated a mean value for each indicator based on available data from the study period. the analysis was limited to countries participating in the top articles in the field of pneumonia in order to facilitate comparison between countries' scientific production, demographic indicators, and economic development. results for the top articles are shown in the main text, while those for the top are provided in additional file . we calculated the following citation indicators by journal, country, and geographic region: -citation of the publications. absolute number of citations received. -citation rate (cr). number of citations divided by number of publications. -hirsch index (h-index). the h-index is a semiqualitative proxy measure to assess the impact of an author's or country's research output on the scientific community [ ] . an h-index of indicates that out of published papers have been cited at least times. in order to assess the differences in the distributions of the publications according to the prestige of the journals, we performed a specific analysis of a sub-sample of publications in journals occupying the top % in the impact factor ranking in their respective subject categories in the journal citation reports ( edition). we analyzed participation in these "prestigious journals" according to geographical location (regions and countries), collaboration level and number of citations. we calculated the percentage of documents produced in international collaboration and the evolution by quinquennium in order to estimate the scope of cooperative practices at a global level, considering the whole population of documents analyzed (research field) by country and geographic region. to specifically analyze collaboration between countries, collaboration networks were generated for each of the three quinquenniums using pajek software. to specifically analyze collaboration between countries, collaboration networks were generated for each of the three quinquenniums using pajek software. the collaboration network is a graphic representation (graph), wherein the nodes represent authors' countries (as determined from their institutional affiliations) and links between the nodes represent coauthorships between countries, that is, an international collaboration in published research. the more intense the collaboration, the thicker the links between the nodes. the spatial distribution of the nodes responds to the execution of the kamada-kawai algorithm in pajek, which places the most prominent nodes (those with a greater number of documents and collaboration links) in the center of the map, and the nodes with a smaller number of publications and degree of collaboration towards the periphery. based on an analysis of mesh terms, we identified the main research focus of the studies in the area, generating density maps using the vosviewer program with a spatial description of the main mesh terms for each type of pneumonia [ ] : (a) "pneumonia, aspiration" (b) "pneumonia, bacterial," (c) "pneumonia, ventilator-associated," (d) "pneumonia, viral," and (e) "pneumonia, pneumocystis"). the process of generating and interpreting the maps proceeded as follows: -determination of the co-occurrence of the descriptors assigned to the documents and generation of a matrix of absolute values. the joint assignment of two descriptors in a single document implies a thematic affinity, as both aspects are addressed simultaneously in the same paper. this affinity will be more intense as it is repeated a greater number of times in the collection of documents analyzed. -elimination of generic descriptors. in order to facilitate the analysis, we eliminated some excessively generic descriptors (like "humans" or "animals"), along with geographical descriptors and those related to age groups. these descriptors showed very high-density relationships, complicating the analysis and the interpretation of the results, so we analyzed their frequency more specifically. -visual representation of the network. to establish the main topics that exist for each type of pneumonia and to represent them visually, we used a clustering algorithm in the vosviewer program, which helps to detect the communities (clusters) within a network, made up of groups of homogeneous items that are strongly related to each other. the different groupings, in the form of "islands" in red tones, represent the main clusters of the thematic networks, while the chromatic gradation illustrates the areas with a lower density of relations between the mesh in yellow and green tones. the spatial distribution of the mesh and their proximity to each other responds to the intensity of co-occurrence between them. all data used to perform the study, including the information downloaded from the database as well as that derived from the treatment of the bibliographic entries, are available in the dataverse project, an open access public repository [ ] (https://dataverse.harvard.edu/, doi: https://doi.org/ . /dvn/ bune). due to the nature of the study and dataset, it was not necessary to obtain informed consent or approval from an institutional ethics committee. the search yielded a total of , documents published between and and assigned with the descriptor "pneumonia" in the medline database. of these, , ( . %) were indexed in the wos core collection databases; , ( . %) of them were classified as articles and ( . %) as reviews. thus, the population of study documents was a dataset of , articles and reviews, which we used to calculate the indicators presented below. letters (n = ; . %), editorials (n = , ; . %), news (n = ; . %), proceedings (n = ; . %) and other document types (n = , . %) were excluded from the analysis. the number of publications rose from in to in .the evolution of scientific production by year was fitted to a linear growth model, showing an r value of . . overall, the study period saw a two-fold increase in scientific production (additional file : figure s ). the country with the greatest number of documents was the usa ( . %), followed at some distance by the uk ( . %), japan ( . %), germany ( . %) and france ( . %). table shows the number of documents and the evolution of scientific production in the most productive countries by quinquennium (see additional file : table s for results on the top countries). although the usa ranks first in all periods, its relative contributions have declined, from . % of all documents in - to . % in - . on the other hand, china's emergence is highly notable, with a . % share of total scientific production in the first period (rank = ), compared to a . % share in the third (rank = ). south korea has also seen considerable growth, contributing just . % to total research production in - (rank = ) but . % in - (rank = ). likewise, taiwan and brazil have increased their production from . and . %, respectively, to . and . %. scientific production in different countries and geographic regions, and its evolution by quinquennium, is concentrated in north america and europe & central asia; together these regions are responsible for . table ) (see additional file : figure s for a visual representation of density equalizing mapping projections). table ranks the production of the top countries, adjusted for demographic and economic indicators (see additional file : table s for results on the top countries). when normalized by population, the most productive countries were switzerland, the netherlands, iceland, and denmark. adjusted for the gdp index, the most productive lmics were the gambia, malawi, uganda, and guinea bissau. if we calculate the ratio of pneumonia publications to gni per capita index, the usa, china, india, malawi y brazil were the most productive. adjusting by r&d expenditure index, the usa ranked first, followed by spain, the uk, china, and italy. in relation to the researchers in r&d index, the usa also leads the ranking, followed by india, uganda, and china. (see additional file figure s and figure s for a visual representation of density equalizing mapping projections of the number of documents and world development indicators, by gni per capita index, gdp index and population index plus r&d expenditure index). figure shows the collaboration networks between different countries by quinquennium. the most prominent countries in all time periods, occupying central positions in the networks with multiple cooperative links, are the usa, canada, the uk, germany, france, and the netherlands. the presence of south american and african countries is scarce in all periods. only south africa has a notable presence in the third quinquennium (fig. a) . a few other countries also "emerge" with a high degree of collaborative links in the second period, like spain, greece, italy, australia, china, and japan, although the latter two countries are not fully integrated in global networks, showing collaborative ties only with the usa (fig. b) . finally, other european countries, while present throughout all three periods, stand out to a greater degree in the third period. this is the case of sweden, switzerland, belgium, and austria. at the same time, china and japan seem more implicated in the network in this third period, while india and south korea also gain relevance (fig. c) . the documents we analyzed were published in scientific journals. twelve journals accounted for . % of the pneumonia literature table table s for results on the top journals with highest absolute and relative citations). the comparative analysis of the scientific production and crs of different journals is noteworthy in that some journals (such as the american journal of respiratory and critical care, critical care medicine, and intensive care medicine) present a very high cr in relation to their total scientific production (additional file : figure s for the top journals producing the most research on pneumonia, plus citation rates). with regard to the subject categories to which the journals are assigned, the most prominent are "infectious diseases" ( . % of the documents), "respiratory system" ( . %), "immunology" ( . %), "microbiology" ( . %), and "critical care medicine" ( . %) table . many of the most productive journals in pneumonia also fall into these subject categories. moreover, over the course of the three study periods, nearly all of the subject categories saw a moderate decrease in their relative contribution, as research articles became more dispersed and made headway into different disciplines producing less research on pneumonia table . the analysis of the documents published in the top % of prestigious journals shows a higher participation from the usa ( . %, compared to . % in the overall body of documents) and from some other european countries like the uk or spain. in contrast, the weight of asian countries, particularly japan and china, is much lower (table ) . overall, international collaboration in these journals (n = , . %) was sensibly higher than in the overall body of documents ( . %), and the greater degree of collaboration was much more pronounced for countries like brazil, japan, china, and even european countries like italy and germany ( table ). the high degree of collaboration was also confirmed between regions in the publications appearing in these journals (table ). with regard to the degree of citation, we observed notable increases in the citation rate of the usa and the european countries; these were even more significant for countries in the middle east & north africa, and for sub-saharan africa when they participated in these journals ( table ). with regard to types of pneumonia studied, the mesh terms to appear most frequently were "pneumonia, bacterial" ( . %), followed by "pneumonia, pneumococcal" ( . %), and "pneumonia, ventilator-associated" ( . %). table shows the number of documents assigned to each term describing the different types of pneumonia (additional file : table s for the top general mesh). table ranks the top countries in crude numbers of retrieved articles, stratified by types of pneumonia (additional file : table s for information on the most productive countries). for "pneumonia, aspiration", the main countries were the usa, japan, and germany; for "pneumonia, bacterial", the usa, france, and spain; for "pneumonia, pneumocystis", the usa, france, and the uk; for "pneumonia, ventilator-associated", the usa, france, and spain; and for "pneumonia, viral", the usa, china, and japan. table shows the relationship between mesh terms referring to age groups with those corresponding to different types of pneumonia. the closest associations for "aged, and over" and "aged" were with "pneumonia, aspiration" ( . and . %, respectively), while "pneumonia, viral" was the most frequent topic for studies in pre-adults ("infant", "child", "child, preschool" and "adolescent"). the one exception to this was "infant, newborn", where the highest proportion of articles was about "pneumonia, pneumocystis." in "adult" and "middle aged" people, studies most frequently focused on "pneumonia, bacterial" and "pneumonia, ventilator-associated." figure shows the subject area maps with the main mesh terms in the documents on (a) "pneumonia, aspiration"; (b) "pneumonia, bacterial"; (c) "pneumonia, ventilator-associated"; (d) "pneumonia, viral"; and (e) "pneumonia, pneumocystis." the principal mesh term related to "pneumonia, aspiration" is "deglutition disorder", but research is linked to a broad array of topics, including epidemiological aspects ("incidence", "risk factor", "retrospective studies"), treatment approaches in intensive care, and surgical techniques procedures facilitating breathing, swallowing, and feeding (fig. a) . the two main mesh terms that appear most frequently with "pneumonia, bacterial" are "community-acquired infections" and "anti-bacterial agents", reflecting the central focus that research has taken to identify risk factors and test different therapeutic approaches. mesh terms related to specific bacteria and infections, such as streptococcus, chlamydia, acinetobacter, and haemophilus influenzae, are also prominent (fig. b) . for its part, research on "pneumonia, ventilatorassociated" seems more disperse, although three areas of interest can clearly be differentiated: (a) epidemiological studies, clinical protocols, and treatment in intensive care units (the term "intensive care unit" is the most prominent in this area); (b) treatment outcomes ("treatment outcome" and "anti-bacterial agents"); and (c) cross infections ("cross infection") (fig. c) . research on "pneumonia, viral" also shows a disperse nature, with different areas of interest. epidemiological aspects are covered under terms such as "communityacquired infections" and "hospitalization", while at a researcher level, interests reside in the virus "influenza, human" and "orthomyxoviridae infections" (fig. d) . with regard to "pneumonia, pneumocystis", one prominent subject focus is on "aids-related opportunistic infections" and another is on "pneumocystis jirovecii" (fig. e ). our analysis shows that the number of publications on pneumonia increased notably over the study period, with annual research outputs doubling from to . different factors may have contributed to this. the first of these is the growing research relevance of pneumonia as a clinical entity, as this disease is one of the community-acquired infections with the highest incidence and is an important cause of hospital admissions. it is also associated with a high global burden of morbidity and mortality in both children and adults [ ] [ ] [ ] ] . the second potential factor relates to advances in basic immunological and microbiological research along with deepening knowledge on the pathogenesis of the disease with regard to aspects like microbiological resistance and preventive interventions (e.g. vaccines) [ ] . thirdly, increased funding has been directed toward research and particularly "proactive investments for emerging infectious threats" [ , ] , and finally, the increase in scientific production could be related to scientific development and international dissemination of scientific research in the wos databases. this is particularly the case of china and other emerging economies like brazil, where the rates of growth were highest relative to their respective regions [ ] [ ] [ ] . we observed a substantial increase in research worldwide, but particularly in some geographical regions and countries of south asia, east asia & the pacific, latin america & and the caribbean, and sub-saharan africa. to a great extent, this increase is simply a reflection of the limited contribution to global research that these countries made in the first period analyzed ( ) ( ) ( ) ( ) ( ) . the bulk of scientific production continues to come from countries with more economic and scientific development in europe and north america (together, these countries participated in % of all publications). despite the striking increase in scientific production across lmics, the relative contribution to pneumonia research remains very modest, and the fact that some countries rank highest in demographic and economic indicators may not be a positive feature, but rather a reflection of the scant development in their scientific systems. furthermore, the increase in international collaboration could have played a role in these indicators, multiplying the assignment of articles to different countries and possibly inflating some values, masking the real contribution of countries with less scientific development in research activities [ ] . the usa is undoubtedly the main reference for pneumonia researchers in quantitative terms, as it produces by far the largest volume of publications-four times that of the next most productive country in the last period. other european countries with important scientific systems (e.g. the uk, germany, france, and spain), along with other countries like japan, canada, china, india, and brazil, also stand out in relation to some of the indicators of scientific production and economic development (gni per capita index, and r&d expenditure index). the other significant aspect in the analysis of how scientific production evolved over the study period is the emergence of china, which in the last period of study ( ) ( ) ( ) ( ) ( ) trailed only the usa in research output. this growth has come about in large part from the investments and scientific policies to foster openness that have been implemented over the past several decades to promote internationalization [ , ] . the level of international scientific collaboration that we have observed in the field of pneumonia ( %) is below that seen in other areas of knowledge [ , , , [ ] [ ] [ ] [ ] . thus, even though the trend is toward increased international cooperation, rising from to % over the study period, implementing new strategies that favor collaboration is still necessary [ ] . initiatives promoting research could include those launched by international organizations, such as the world health organization (who) and the bill & melinda gates foundation, which have both invested considerable resources to investigate the etiology of childhood pneumonia in low-income countries [ ] [ ] [ ] . however, these initiatives [ ] are also collaborating in different projects related to hiv, tuberculosis, and malaria, and these organizations are largely responsible for the important degree of collaboration between european and sub-saharan african countries [ ] . research for operational health services is necessary to improve the distribution and accessibility of pneumonia treatments, including antibiotics in primary healthcare centers and oxygen in hospitals. likewise, new vaccines still need to be developed for strains of pneumococcus that current multivalent conjugate vaccines do not protect against [ ] . in addition to programs focused on financing and implementing collaborative north-south and south-south projects, other efforts could be directed toward reducing obstacles associated with publication processes fig. subject area maps with the main mesh terms associated with different types of pneumonia-(a) "pneumonia, aspiration" (b) "pneumonia, bacterial, " (c) "pneumonia, ventilator-associated, " (d) "pneumonia, viral, " and (e) "pneumonia, pneumocystis" groupings in the form of "islands" in red tones represent the main clusters of the thematic networks, while the chromatic gradation in yellow and green tones illustrates the areas with a lower density of relations between the mesh. the spatial distribution of the mesh and their proximity to each other responds to the intensity of co-occurrence between them that limit the dissemination of lmics through the main international scientific journals. the literature has described obstacles related to linguistic skills and methodological deficiencies, which highlights the need to improve these areas in particular [ , ] . other authors have pointed to the costs associated with publishing in open access journals, so it is worth assessing whether the programs to support open access publishing implemented at an institutional level and by publishers such as plos, biomed central, or the lancet journals, are sufficient [ ] [ ] [ ] . with regard to the impact of research, although europe and north america are balanced in terms of the absolute number of citations, north america holds an advantage in terms of the citation rate. research from sub-saharan africa also has a very high citation rate, which almost reaches that achieved in europe. the fact that these african countries present a high degree of collaboration with researchers in the usa and europe, who represent the "mainstream" international research interests, could help explain the high citation rates seen in this region. on the other hand, latin america & caribbean, south asia, and east asia & pacific are all regions with generally lower citation rates, although this difference is not so pronounced in the case of papers produced in collaboration, as reported elsewhere [ ] . by country, the hegemony of the usa and several european countries in terms of the number of citations received was evident, as was the lower ranking of some asian countries, such as japan and china, in relation to their scientific production. the positioning of china as a reference for scientific production and participation in international research networks does not correspond to its ranking with regard to citation indicators, despite their improved standing over the past several years [ ] . on these indicators, china still lags behind the usa as well as the leading european countries, canada, australia and even nearby countries such as japan. for now at least, the countries that have traditionally occupied the "mainstream" of scientific research still maintain their hegemony [ ] . as with the relative indicators of scientific production adjusted for economic and demographic parameters, some countries surpass the major scientific systems with regard to the citation rate, which links the degree of citation with the volume of scientific production [ ] . these countries may have participated in certain highly relevant contributions, or they may be small countries with highly developed scientific systems, such as vietnam, switzerland, south africa, new zealand, and saudi arabia. these countries also stand out for their high levels of international collaboration, which is a factor associated with more citations. the high mean citations received by publications produced in sub-saharan africa, and the participation of different emerging countries like vietnam and south africa in some of the highest cited papers we identified, underlines the capacity of these countries to contribute to high-impact and excellent-quality scientific studies. this result is consistent with previous studies that have also demonstrated these countries' capacity to participate in emerging research topics [ ] . these specialists therefore represent an excellent asset, strengthening the human capital from high-income countries and enabling the advancement of research [ , ] . in general, the most prestigious journals show a greater concentration of research from the usa and europe, with greater collaboration and impact when countries from other geographical regions also participate [ ] . bacterial pneumonia is the main branch for the multidisciplinary and multipathological mesh of "pneumonia", with the main areas of interest ("community-acquired infections", "anti-bacterial agents" and "treatment outcome") reflecting the focus of research on identifying risk factors and assessing different treatments and their outcomes. in publications pertaining to the mesh "pneumonia, ventilator-associated," the main axes of the subject content according to the mesh terms were the group of epidemiological studies and clinical and treatment protocols in intensive care. "pneumonia pneumocystis," is closely related to infection due to hiv and immunodepression. the main areas of research interest for "pneumonia, viral," were the epidemiological aspects related to the setting for the infection ("community-acquired infections" and "hospitalization") along with the viruses responsible ("influenza, human" and "orthomyxoviridae infections"). finally, for the mesh "pneumonia, aspiration" the main research focus is "deglutition disorder". the main limitation of this present study is its analysis of only the documents included in the wos databases and medline ( % of the documents). thus, a number of papers were excluded from the study, particularly those written in languages other than english, as well as the proceedings included in wos, as our searches were based on the journals included in medline. on the other hand, our approach also allowed us to precisely characterize collaboration in the area, as only recently has medline begun to include all the institutional affiliations of the authors. we were also able to analyze the citations of the publications, with a focus on the journals with the highest impact and dissemination at an international level [ ] . in conclusion, pneumonia research increased steadily over the -year study period, with europe and north america leading scientific production. about a fifth of all papers reflected international collaborations, and these were most evident in papers from sub-saharan africa and south asia. additional file : table s . descriptors included under the mesh "pneumonia" in pubmed. table s . top countries in crude numbers of retrieved articles in "pneumonia, aspiration", "pneumonia, bacterial", "pneumonia pneumocystis", "pneumonia, ventilator-associated", and "pneumonia, viral" mesh. figure s . evolution of scientific production on pneumonia ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . clinical and economic burden of communityacquired pneumonia among adults in europe burden of community-acquired pneumonia in north american adults global, regional, and national life expectancy, all-cause mortality, and cause-specific mortality for causes of death, - : a systematic analysis for the global burden of disease study impact of age and comorbidity on cause and outcome in communityacquired pneumonia the burden of community-acquired pneumonia in the elderly: the spanish evan- study burden of pneumococcal community-acquired pneumonia in adults across europe: a literature review all cause mortality estimates for - . who mapping pneumonia research: a systematic analysis of uk investments and published outputs - investments in respiratory infectious disease research - : a systematic analysis of uk funding scientometrics analysis of research activity and collaboration patterns in chagas cardiomyopathy evolution of cooperation patterns in psoriasis research: co-authorship network analysis of papers in medline bibliometric analysis of leishmaniasis research in medline ( - ) investment in pneumonia and pneumococcal research medical subject headings used to search the biomedical literature mesh now: automatic mesh indexing at pubmed scale via learning to rank years on -is the nlm medical text indexer still useful and relevant? 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publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we gratefully acknowledge the assistance of meggan harris in translating our manuscript from spanish.authors' contributions jmrr: study conception, study, design, data analysis, manuscript writing and final manuscript approval; hpc: data collection, data analysis, manuscript writing and final manuscript approval; ibr: study conception, manuscript writing and final manuscript approval; gga: study conception, study design, data collection, data analysis, manuscript writing and final manuscript approval no funding was received for this work. all data used to perform the study, including the information downloaded from the database as well as that derived from the treatment of the bibliographic entries, are available in the dataverse project, an open access public repository [ ] (https://dataverse.harvard.edu/, doi: https://doi.org/ . /dvn/ bune). due to the nature of the study and dataset, it was not necessary to obtain informed consent or approval from an institutional ethics committee. the authors give consent to publish the manuscript. the authors declare that they have no competing interests. key: cord- -wdc p bc authors: gonzález-scarano, francisco; rima, bert title: infectious etiology in multiple sclerosis: the debate continues date: - - journal: trends microbiol doi: . /s - x( ) - sha: doc_id: cord_uid: wdc p bc ‘demonstrating infectious cause: viral and bacterial infections in ms and related disorders’ was held in brighton, uk, – august . m ultiple sclerosis (ms) affects over americans, britons and perhaps in excess of two million people worldwide, and has long been suspected to have an infectious etiology. however, despite intensive investigation, no single infectious agent or group of agents has been identified. 'demonstrating infectious cause: viral and bacterial infections in ms and related disorders' brought together immunologists, virologists, epidemiologists and clinicians to review the current knowledge about the role of infectious agents in ms, and to identify key concepts and questions for research. nineteenth-century descriptions of the neuropathology of ms emphasized the prominent infiltration of leukocytes and other inflammatory cells into its characteristic central nervous system (cns) lesion, which is now called a plaque. although it is now well accepted that inflammatory responses often have non-infectious causes, there are a number of clues that point towards an exogenous, possibly infectious, factor or factors as an important component of ms. for example, studies of twins with ms indicate that monozygosity is associated with a disease concordance of ~ % which, although suggesting a strong genetic component (the concordance for dizygotic twins is in the order of %), also indicates that the disease is influenced by additional, possibly environmental, factors. furthermore, there is a strong geographical influence on ms prevalence: the rates range from - per in northern europe and in the northernmost usa, whereas in southern europe the prevalence drops to ~ per , similar to the rates in the southern usa. remarkably, a large number of epidemiological studies have shown that individuals maintain the 'signature' incidence associated with the geographical area in which they spent their youth, even after migrating from an area of high incidence to one of low incidence, or vice versa. this has been interpreted as evidence that exposure to a microorganism prior to adulthood predisposes people to the development of ms (ref. ). the most persuasive evidence that foreign antigens are associated with ms, however, is the indication of a humoral immune response within the cns. detectable as 'oligoclonal' bands in electrophoresed samples of concentrated cerebrospinal fluid (csf) proteins, antibodies produced by plasma cells within the cns are usually seen in infectious diseases such as syphilis or aids, where they are directed against antigenic components of the invading microorganism. their presence and persistence in ms (ref. ) suggests that a chronic infection underlies the clinical syndrome, although it is also possible that these antibodies are directed against as-yet-unidentified self-antigens. additionally, analysis of tissue samples from around the plaques themselves also indicates antibody production and deposition. most investigators believe that identification of the antigens against which these antibodies are directed will provide important information about ms, perhaps even identifying its cause. although there were calls from conference 'break-out' sessions for better stratification of ms patients in epidemiological studies, the consensus of studies pointing in the direction of an environmental cause, as well as the evidence from the oligoclonal immune responses, convinced the majority of delegates that ms is likely to be caused or triggered by an infectious agent. the infectious agent: known or unknown? the majority of studies concerning specific agents in ms are based on the assumption that the microorganisms persist in the patient, probably in the cns but perhaps in other organs. a number of new technologies such as representational difference analysis (rda), differential display, consensus pcr and phage display are now available for identification of unknown or difficult-to-culture organisms. with that in mind, donald gilden (university of colorado, denver, co, usa) has adapted phage-display technology to analyse the antigen specificity of oligoclonal bands by extracting the rna surrounding an ms plaque and amplifying the immunoglobulin heavy and light chains using consensus primers. after cloning into an expression vector, the immunoglobulins are displayed on the coat of a bacteriophage. these immunoglobulins can then be specifically amplified, and the antigen against which they are directed can subsequently be identified. gilden has already achieved success using this approach for at least one chronic infection caused by measles virus (subacute sclerosing panencephalitis; sspe). his laboratory is now directing its attention to the ms lesions and the cns areas where the csf is produced. other presentations concerning molecular technologies gave the overall impression that the new technologies have not yet been applied in a systematic fashion to pathologically well defined ms tissue, and that there was room for more work using these approaches. some investigators felt that the agent that induces ms has already been identified. subramamiam sriram (vanderbilt stallworth rehabilitation hospital, memphis, tn, usa) presented data indicating that the csf of % of ms patients (compared with % of patients with other neurological diseases) contains genomic material from the bacterium chlamydia pneumoniae, as shown by pcr amplification. sriram was also able to culture c. pneumoniae from the csf of ms patients. furthermore, oligoclonal bands from ms csf were adsorbed by incubation of the fluid with a c. pneumoniae antigen, but not with antigens from herpes simplex or measles viruses. these data support his view that the immune response in those patients was directed against c. pneumoniae. sriram also suggested that a therapeutic trial with antibiotics directed against c. pneumoniae might be required to demonstrate a causal association between this microorganism and ms. konstance knox (institute for viral pathogenesis, milwaukee, wi, usa) presented equally compelling data supporting earlier suggestions that human herpesvirus six (hhv- ) is associated with ms. immunohistochemical analysis of ms plaques has revealed a concentration of an hhv- antigen in oligodendrocytes, the cns cells responsible for the formation and maintenance of myelin (myelin destruction is a key component of the ms lesion). knox and collaborators found hhv- expression in out of patients with 'active' disease, compared with only three out of patients with 'inactive' ms. there were also dramatic differences in viremia. twenty seven out of patients with ms had active hhv- viremia, compared with none of the controls. other investigators presented data supporting potential roles for human endogenous retroviruses (herve perron, biomerieux sa, lyon, france), and delayed exposure to epstein-barr virus (tove christensen and sven haar, aarhus university, aarhus, denmark) as potential triggers , . however, as with many such reports, in most individuals there is evidence of prior or current infection with these agents whether or not they have ms, and there are no moredefinitive tests such as viremia or analysis of oligoclonal bands to differentiate patients with ms from those with other diseases. further research will be needed in order to clarify and reconcile these potentially conflicting findings, as several presentations outlined an association with a specific agent in over half of the cases. it is entirely possible that multiple microorganisms are each responsible for a small proportion of this disease, perhaps indirectly, by inducing immune responses that result in an immune attack on the cns, without persisting in the affected patient. several animal models of virally induced demyelination, particularly those associated with murine coronaviruses or with theiler's murine encephalomyelitis virus (tmev), support such an idea. in these models, demyelination is related to the presence of an identified virus within the cns; in tmev-mediated disease, the extent of demyelination depends not only on viral load but also on a locus on mouse chromosome (michel brahic, institut pasteur, paris, france). although an intact immune system is necessary for the disease to manifest in its full form, which arm of the immune response is most critical varies from model to model, and even within models, apparently depending on the viral strain. demyelination after tmev infection of mice transgenic for a human leukocyte antigen (hla) was shown to be modulated by the hla transgene (moses rodriguez, mayo clinic, rochester, mn, usa), a finding that might relate to the preponderance of certain hla subtypes in ms. interestingly, by careful injection, foreign antigens can be introduced into the brain of mice and not be 'seen' by the immune system until after a subsequent peripheral challenge, which leads to detection and commencement of an immunopathological process (hugh perry, university of southampton, southampton, uk). whether or not a single agent is indeed responsible for this disease, there are multiple ways in which an infectious agent could be associated with, or trigger, ms. in some of these, the microorganism might no longer be present by the time the disease manifests but, for example, it affects the cns by changing the intrathecal cytokine environment and promoting pro-inflammatory reactions or by stimulating autoreactive t cells. matthias von herrath (scripps clinic, san diego, ca, usa) indicated that, in an autoimmune diabetes model, the growth kinetics of different strains of lymphocytic choriomeningitis virus determined whether the disease was exacerbated by a second peripheral challenge or whether this was, in fact, protective. balances between immune reactions also produce different responses in borna virus infection in rats (luther stitz, institute of animal health, tüebingen, germany). one of the more popular arguments has been that a microorganism could stimulate an autoimmune response by mimicking an endogenous antigen (antigenic mimicry) (uwe liebert, university of leipzig, leipzig, germany; henry mcfarland, national institutes of health, bethesda, md, usa); circumstantial evidence indicates that such a mechanism could lead to the development of autoimmune, reactive t-cell clones , . alternatively, an exogenous agent could act as an mycobacterium tuberculosis infection is complex and multifaceted. as indicated in the review by murray , both cd ϩ and cd ϩ t cells are essential to control tuberculosis (tb) in mice [ ] [ ] [ ] [ ] , and recent data support a role for both types of t cells in human tb resistance [ ] [ ] [ ] . macrophage activation, which is likely to occur within a granuloma, is the key to controlling tb. granulomas consist of macrophages and lymphocytes, but the role(s) of each cell type, the types and kinetics of the cytokines produced by different cell populations, and the evolution of the granuloma remain poorly understood. murine studies have revealed essential roles for certain cytokines, including interferon ␥ (ifn-␥), interleukin (il- ) and tumour necrosis factor ␣ (tnf-␣). however, there is little data regarding the importance of these cytokines in human tb. humans with mutations in the genes encoding ifn-␥ or il- , or their receptors, often succumb to mycobacterial infections other than tb (ref. ). although it is probable that these cytokines are significant in human tb, their relative importance, specific roles and cellular targets remain less clear. most patients infected with m. tuberculosis have cd ϩ t-cell responses that are expected to be effective in activating macrophages to kill intracellular mycobacteria (e.g. ifn-␥ production). however, because most infected individuals harbor the organism for many years, perhaps for a lifetime, this response is apparently not completely effective in eliminating virulent m. tuberculosis. in fact, in vitro, it has been difficult to demonstrate ifn-␥-induced killing of m. tuberculosis within human macrophages, although similarly activated macrophages eliminate other intracellular pathogens. a recent study has shed some light on this phenomenon however, the infection dramatically inhibits the association of the transcription factor stat (signal transducers and activators of transcription ) with the transcriptional coactivators cbp [camp-response-element-binding protein (creb)-binding protein] and p , an association that is essential for ifn-␥-induced transcription. live bacteria are not necessary for this effect, as it can be reproduced using cell walls and it does not appear to be mediated by lipoarabinomannan. neutralization of transforming growth factor ␤ (tgf-␤) or il- did not reverse the effects of m. tuberculosis infection, suggesting that these cytokines are not the only contributors to macrophage deactivation. this phenomenon might also occur in mice, because mice do not eliminate m. tuberculosis, even though a strong immune response involving both cd ϩ and cd ϩ t cells and production of ifn-␥, tnf-␣ and other cytokines is present. m. tuberculosis is an enormously successful pathogen that has evolved numerous mechanisms for evading elimination by the host immune response. we are still in the process of learning which immune responses are important in controlling m. tuberculosis during the acute and latent phases of the infection. in most cases, the host maintains control of the infection, but occasionally the bacterium gains the upper hand. dissecting these interactions at an immunological level in both animal models and human systems remains the challenge for tb research in the future. the development of better vaccines ultimately depends on our increased understanding of the host-pathogen interaction. dept of molecular genetics and biochemistry, university of pittsburgh school of medicine, pittsburgh, pa , usa adjuvant, perhaps by incorporating a self-antigen into its structure, much like retroviruses can incorporate cellular proteins into their envelopes . steve miller (northwestern university medical school, chicago, il, usa) presented data to indicate that only antigenpresenting cells from tmevinfected mice with pre-existing myelin damage and not from acutely infected or control mice were able to present proteolipid apoprotein (plp) epitopes to specific t-helper type cell lines. taken together, the presentations and discussion at the brighton conference summarized an impressive amount of information, leading to the conclusion that there are indeed exogenous agents involved in the development of ms. however, it is also clear that at this point there are insufficient data to identify a single culprit, and future research will be directed at replicating some of the most promising findings to date, and at understanding the possible pathogenic mechanisms by which foreign antigens could trigger an immune attack on the cns. why is ifn-␥ insufficient to control tuberculosis? multiple sclerosis key: cord- -li x m authors: hung, ling-chu title: the monoclonal antibody recognized the open reading frame protein in porcine circovirus type -infected peripheral blood mononuclear cells date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: li x m the purpose of this study in the context of the open reading frame (orf ) protein of porcine circovirus type (pcv ) was especially its location and its relation to the capsid protein and the apoptosis protein in pcv -infected porcine peripheral blood mononuclear cells (pbmcs). to detect the orf protein, monoclonal antibodies (mabs) were generated in this study. the mab d binds to the orf peptide (residues – ) and the native orf protein in pcv -infected pbmcs, as shown by immunofluorescence assay (ifa). the data show that – % of pbmcs were positive for orf protein or p protein. further, – % of pbmcs were positive for the capsid. this study confirmed the orf protein not only colocalized with the capsid protein but also colocalized with the p protein in pbmcs. immunoassays were conducted in this study to detect the capsid protein, the orf protein, anti-capsid igg, and anti-orf igg. the data show the correlation (r = . ) of the orf protein and the capsid protein in the blood samples from the pcv -infected herd. however, each anti-viral protein igg had a different curve of the profile in the same herd after vaccination. overall, this study provides a blueprint to explore the orf protein in pcv -infected pbmcs. the porcine circovirus (pcv) is a small virus and contains closed circular single-stranded dna [ ] . previous studies indicated that porcine circovirus type (pcv ) is non-pathogenic to porcine herd [ , ] . however, porcine circovirus type (pcv )-infected pigs showed dullness, thinness, lymphadenopathy, jaundice, hepatomegaly, and other manifestations [ ] [ ] [ ] [ ] [ ] . lymphocyte depletion and apoptosis of lymphocytes were the significant histopathological lesions in lymphoid tissues of pcv -infected pigs [ , ] . pcv infection causes a reduction of t-and b-lymphocytes in the pbmc [ ] . likewise, pcv -associated disease (pcvad) manifested as severe swine herd problems [ , [ ] [ ] [ ] . recently, pcvad has become one of the significant swine diseases worldwide, and it impacts pork production [ ] . at least four commercial pcv a vaccines have been widely used to reduce viremia and pcv infectious pressure in swine herds [ ] [ ] [ ] [ ] . that might have caused the global trend shift from pcv a to pcv b first [ ] [ ] [ ] [ ] , and then pcv d (pcv b mutant strain) has become a predominant genotype globally [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the open reading frame (orf ), open reading frame (orf ), and open reading frame (orf ) genes are the three principal orfs among orfs in pcvs [ ] . the orf encodes for the replicase peptide c and peptide n were appended with an n-terminal cysteine during synthesis, which was required for conjugation with maleimide-activated carriers. animal experiments in this study were performed following the current legislation on ethical and welfare recommendations. the experiments followed the standards of the guide of the care and use of laboratory animals. all animal work was approved by the institutional animal care and use committee (iacuc) of the livestock research institute, and the iacuc of the animal health research institute. the iacuc approval numbers lriiacuc - (for swine and murine experiments), a (for murine and rabbit experiments), and a (for murine and rabbit experiments) were given in this study. antisera against pcv were generated by immunizing new zealand white rabbits (from livestock research institute, council of agriculture, taiwan). rabbits were maintained in isolation rooms, and the room temperature was at - • c. they were fed with a commercially pelleted diet (fwusow in., taiwan), and pure water was available ad libitum. then, animals were immunized with peptide immunogen (the peptide n conjugated with klh) or commercial vaccine five times at two-week intervals. for peptide immunization, an initial dose of µg of the conjugated peptide was mixed with complete freund's adjuvant (sigma-aldrich, st. louis, mo, usa) at the first injection. each rabbit was re-immunized with the same amount of immunogenic mixture with incomplete freund s adjuvant (sigma-aldrich, st. louis, mo, usa). for virus-like particles (vlp) of pcv immunization, the rabbit was injected intramuscularly in the legs with . ml of the pcv vaccine (circoflex ® , boehringer ingelheim). the blood was harvested two weeks after the last injection. the antibody titers were measured by indirect enzyme-linked immunosorbent assay (ielisa) [ ] (performed as described previously for mouse anti-pcv elisa, with the exception that peroxidase-conjugated donkey anti-rabbit igg (h + l, jackson immunoresearch, west grove, pa, usa) secondary antibody was used). hybridomas were generated following established methods, as previously described [ ] . briefly, four five-week-old, female, balb/cbyjnarl mice were purchased from a specific pathogen-free (spf) colony (the national applied research laboratories, taiwan). the mice were maintained in isolation rooms in filtertop cages, and the room temperature was at - • c. mice were fed with a commercially pelleted diet (labdiet, , st. louis, mo, usa), and pure water was available ad libitum. mice were inoculated with µg of immunogen (the peptide n conjugated with klh) emulsified in freund's adjuvant. subsequently, mice were boosted fortnightly with the same amount of immunogen. three days after the final booster, the spleens of the mice were harvested for hybridoma generation. hybridomas were screened for the secretion of anti-orf -specific mabs by ielisa [ ] using the peptide n as a coating antigen. that secondary antibody solution (hrp-conjugated goat anti-mouse iga/igg/igm, h/l chain (novus, saint charles, mo, usa) was used at : dilution for hybridoma screening. the hybridomas that secreted anti-n mabs were subcloned by limited dilution of the cells. then, ascites containing mabs were collected as previously described [ ] . subsequently, the mabs were purified from the ascites by using the nab protein g spin column (thermo scientific, rockford, il, usa), then collecting solutions were exchanged for the buffer and concentrated using the amicon ultra k (millipore, burlington, ma, usa). the concentration of mabs was determined using the nano photometer ® (implen, munich, germany). the heavy chain and the light chain of the two mabs secreted by each hybridoma were determined by using an sba clonotyping system/hrp (southern biotechnology, birmingham, al, usa). it was performed according to the manufacturer's instructions and a previously published method [ ] . briefly, the nunc maxisorp flat-bottom -well plate (thermo fisher scientific, inc., waltham, ma, usa) was coated with the capture antibody (goat anti-mouse ig, µg/ml) in . -m bicarbonate buffer (sigma-aldrich, st. louis, mo, usa) at • c overnight. after three washes with pbs containing . % tween (pbst), the plate was blocked with the blocking buffer (pbst containing % casein hydrolysate) at • c for min. after washing, µl of each culture supernatant of hybridoma were added sequentially, and the plate was incubated at • c for h. after washing, µl of hrp labeled goat anti-mouse igg , -igg a, -igg b, -igg , -iga, -igm, -κ light chain, and -λ light chain were added to appropriate wells of the plate. pbst served as background. the plate was then incubated at • c for h and after that washed with pbst. finally, µl of , -azino-bis ( -ethylbenzothiazoline- -sulfonic acid) diammonium salt (abts, sigma-aldrich, st. louis, mo, usa) buffer were added to each well of the plate. after min, the optical density (od) of each well was measured at nm using a spectramax m microplate reader (molecular devices, san jose, ca, usa). the epitope mapping of these mabs was performed by ielisa [ ] using truncated peptides (as shown in table ) as coating antigens. briefly, peptides contained the sequence of orf protein of pcv b between residues and , associated -mer peptides, truncated derivatives, and the control (peptide c ). ninety-six-well maxisorp plates were coated with each peptide ( µg/ml) in bicarbonate buffer and incubated at • c overnight. after three washes in pbst, the plates were blocked with the blocking buffer at • c for min. after washing, the culture supernatant of hybridoma was added, and plates were again incubated at • c for h. the anti-peptide n mouse serum (at a : dilution) was used as the positive control, and the anti-orf (peptide c ) mab ( h ) was served as the negative control. after rinsing three times with pbst, the secondary antibody solution was applied to the appropriate wells. peroxidase-conjugated goat anti-mouse igg (subclasses + a + b + , fcγ, jackson immunoresearch, west grove, pa, usa) was used at a : dilution for binding to the mab d . hrp-conjugated goat anti-mouse lambda light chain (novus, saint charles, mo, usa) was used at a : dilution for binding to the mab d . after h, the plates were washed three times. the colorimetric reaction was developed by using the abts solution. following a -min incubation, the od was measured at nm. both of the isotype and molecular weight of the mab d were determined by western blotting. the culture supernatant of the hybridoma (clone d ) was separated by bolttm bis-tris plus gel (invitrogen, carlsbad, ca, usa). one gel was stained with fastain protein staining solution (yeastern biotech, taiwan). the other gel was transferred to the polyvinylidene difluoride (pvdf) membranes (millipore, burlington, ma, usa) with fast semi-dry transfer buffer (thermo, waltham, ma, usa) by using a yrdimes semi-dry transfer system (wealtec, new taipei city, taiwan). the membrane was blocked with the blocking buffer for min at • c. after three washes in pbst, each strip of the membrane was incubated with one appropriate horseradish peroxidase (hrp)-conjugated antibody to detect mab at • c overnight and then at room temperature for h. peroxidase-conjugated goat anti-mouse iga/igg/igm, h/l chain (novus, saint charles, mo, usa) at : , hrp-conjugated goat anti-mouse iga (southern biotechnology, birmingham, al, usa) at : , hrp-conjugated goat anti-mouse lambda light chain (novus, saint charles, mo, usa) at : , hrp-conjugated goat anti-mouse igm, µ chain (jackson immunoresearch, west grove, pa, usa) at : , and hrp-conjugated goat anti-mouse igg (subclasses + a + b + , fcγ, jackson immunoresearch, west grove, pa, usa) at : were used in this assay. then, they were washed three times with pbst, min each wash. each strip was incubated with , -diaminobenzidine (dab, horseradish peroxidase substrate, millipore, burlington, ma, usa) buffer for min. protein molecular weight markers were included in each western blot analysis. after revelation with the dab substrate, the target protein was visualized and calculated the molecular weight by using molecular weight markers. homemade immunofluorescence assay (ifa) slides were performed as previously described [ ] . pcv -infected peripheral blood mononuclear cells (pbmcs) were collected from the pcv -infected conventional piglet. the pig serum was detected as seropositive for pcv by anti-capsid peptide (c ) specific immunoassay [ ] . the whole blood sample was confirmed as positive for pcv antigen by using the ingezim pcv das kit (ingenasa, madrid, spain). briefly, pbmcs were isolated from ethylenediaminetetraacetic acid (edta)-treated whole blood samples by ficoll-paque plus (ge healthcare bio-sciences, uppsala, sweden) and according to the manufacturer's instructions. then, mononuclear cells were drawn from the interface of the ficoll-paque plus-containing tube and resuspended in ml pbs for washing and centrifugation ( × g for min at • c). after washing three times with pbs, the cell pellet was resuspended in . ml of fetal bovine serum. an aliquot ( µl) of cell suspension was loaded onto a glass slide for the cell smear. after the cell smear, the slides were air-dried at room temperature. thereby, the pbmcs were stuck on slides. these slides were fixed with pbs containing % paraformaldehyde at • c for min and then washed three times with pbs. following that, the slides were soaked in pbs containing . % triton x- at • c for min. subsequently, slides were washed with pbs and prepared for ifa. two anti-capsid (the peptide c ) mabs ( h and b ) [ ] , one anti-pcv mab a (ingenasa, madrid, spain), one anti-p protein (wt-p ) rabbit polyclonal antibody (bioss, woburn, ma, usa), the homemade anti-orf peptide (n ) mab ( d ), and rabbit antisera (as the method mentioned above) were used in this study. the anti-capsid peptide mabs ( h and b ) recognized native capsid proteins and reacted with core peptides (p , kdpplnp ); however, the mab h bound the three minimal linear epitopes (dpplnp, dpplnpk, and lkdpplkp) and the mab b bound two minimal linear epitopes (kdpplnp and kdpplnpk). these mabs produced a variable definite staining pattern in pbmcs by ifa [ ] . the pcv -infected pbmcs slides were incubated with a : dilution of rabbit antiserum and a : dilution of mab (ascites). after incubation at • c for h, the slides were gently rinsed briefly in pbs and then soaked for min in pbs at • c. the slides were then incubated at • c with tritc-conjugated goat anti-mouse igg (subclasses + a + b + , fcγ) at a : dilution and fitc-conjugated goat anti-rabbit igg (h + l) (all from jackson immunoresearch, west grove, pa, usa, and each minimal antibody cross-reaction to other animal's serum protein) at a : dilution. after min of incubation, the slides were washed with pbs and then incubated with , -diamidino- -phenylindole, dihydrochloride (dapi, aat bioquest, sunnyvale, ca, usa) at a : dilution in pbs at room temperature for min. the slides were mounted under % glycerol and observed with an olympus bx fluorescence microscope and spot flex camera (diagnostic instrument, model . mp, usa). terminal deoxynucleotidyl transferase dutp nick end labeling (tunel) assay was performed according to the manufacturer's instructions (the cell meter™ tunel apoptosis assay kit, aat bioquest ® , inc., sunnyvale, ca, usa) to detect excessive dna breakage in the pbmc. briefly, the pcv -infected pbmcs slides were incubated with the tf -dutp/reaction buffer. after min of incubation, the slides were washed with pbs. they were then incubated with hoechst solution at room temperature for min. the slides were mounted under % glycerol and observed with an olympus bx fluorescence microscope and spot flex camera. blood samples were collected from the pcv -infected herd with the following vaccination at two weeks of age with a pcv vaccine ( ml, porcilis ® pcv, msd animal health) in the conventional pig farm with the farrow-to-finish operation. pigs were selected from animals of different ages ( , , , , , , , , , and ≥ weeks of age) at one time. then, four samples were randomly chosen per age group. blood was collected in the vacutainer ® edta tubes (becton and dickinson bv) and sored at − • c. all whole-blood samples had been treated by the freeze-thaw process twice before the test. the capsid protein was detected from µl of whole-blood sample by using the ingezim pcv das kit (ingenasa, madrid, spain) according to the manufacturer's instructions, and the absorbance of each well was measured at nm. meanwhile, the homemade antigen-elisa was conducted in this study for detecting the orf protein in whole-blood specimens. it was based on the double-antibody-sandwich principle. ninety-six-well maxisorp plates were coated with µl/ml ( µl per well) of the trapping sera (rabbit anti-orf peptide) in bicarbonate buffer. the plates were incubated at • c overnight and washed three times with pbst. then, the plates were blocked with viruses , , of the blocking buffer at • c for min. after the plates were washed, µl of pbst and µl of whole-blood samples were mixed and added to the well. the plates were incubated at • c for h and then washed in pbst. then, µl/well of mab d at µg/ml were added to the plates, and the plates were incubated at • c for h. after the plates were washed, µl/well of peroxidase-conjugated goat anti-mouse igg fcγ at a : dilution were added, and the plates were incubated at • c for h. after washing, abts buffer was added to each well. following a -min incubation, the od was measured at nm. . . detection of the specific antibodies against the capsid protein or the orf protein in plasm samples from the pcv -infected herd with a pcv vaccine plasm samples were separated from the aforementioned fresh whole-blood samples and sored at− • c. the antibody titers were measured by ielisa [ ] (performed as described previously for pig anti-peptide elisa, with the exception that the capsid protein (circoflex ® , boehringer ingelheim) or peptide p was used as the coating antigen, and pig plasm at a : dilution was used). the subsequent procedures of blocking, pig antibody, washing, secondary antibody, substrate, and the od reading, were the same as described previously. the data were analyzed by using one-way analysis of variance (anova) and tukey's studentized range (hsd, honestly significant difference) multiple comparisons test using the sas enterprise guide . ® software (sas institute inc., cary, nc, usa). a p-value < . was considered significant. the orf peptide n or vlp of pcv was capable of inducing each specific antibody. the antibody titer of each post-immune serum (two weeks after the fourth booster) was detected at the dilution : ( figure s ). the binding of the rabbit antiserum to the virus was confirmed by using an immunofluorescence assay (ifa) ( figure s ). although pre-immune serum had a low background (od value less than . ) at a dilution of : , the pre-immune serum did not bind the virus as detected by ifa (data not shown). the author checked for the cross-reactivity of post-immune antiserum to other antigens. the result shows they have a minor cross-reaction at a dilution of : ( figure s ), but the od value of cross-reaction has not significantly higher than that of the negative control serum (p ≥ . ) ( figure s ). following the fusions, hybridoma supernatants were screened for the presence of anti-orf (peptide n ) specific antibodies by ielisa. among them, hybridomas supernatants reacted with the peptide n at the first screening (data not shown). after repeatedly subcloning by the limiting dilution and selection, two stable hybridomas secreting anti-orf mabs ( d and d ) were obtained. the hybridoma produced igg mab ( d ) with kappa-light chains. the other produced mab ( d ) with a lambda-light chain ( figure a) ; however, no heavy chain was detected in the supernatant of clone d by sba clonotyping system/hrp assay. the supernatant of a hybridoma (clone d ) was separated directly by bolttm bis-tris plus gel to confirm the heavy chain of this mab. the gel showed a broad staining region within - kda. the author speculated that the supernatant of a hybridoma contains abundant serum proteins from the fetal bovine serum-containing medium. that is to say, the supernatant of a hybridoma (clone d ) contains a little mab d (light chain). further, the western blotting was used to confirm the heavy chain of mab d and its molecular weight. the hrp-conjugated antibodies (goat anti-mouse iga/igg/igm, h/l chain, and goat anti-mouse lambda light chain) gave specific reactions with the supernatant. two bands were observed at about (between and ) and kda, respectively ( figure ). the lower band ( kda) implied that some degradation of this mab. however, other anti-ig antibodies (goat anti-mouse ig a (α chain), goat anti-mouse igm (µ chain), and goat anti-mouse igg (fcγ)) gave negative reactions with the supernatant. this study confirmed mab d contains a lambda light chain, but it does not include any intact heavy chain. since the molecular weight of the light chain was kda, it may be assumed that the mab d might contain a short fragment of a protein. a peptide scan analysis was performed to determine the binding site for each mab ( figure ). the mab d bound to two linear peptides (peptide n and p ). the peptide n was appended with a cysteine residue at the n-terminal of the orf peptide (residues - , p ). both of them contained the sequence of the orf protein of pcv b between residues and . however, the non-igg mab ( d ) not only reacted with peptide n but also showed minor reactions against other peptides, which compared with the mab d in the elisa test ( figure ) . interestingly, the anti-peptide n mouse serum reacted all test peptides and bound firmly to six linear peptides (peptide n , p , p , p , and p ). notably, anti-n poly antibodies reacted with these peptides (expect p ) which contained the common core peptide (p and tllhfpahfq ). nevertheless, this study did not get any mab which bound firmly to the peptide p . viruses , , x for peer review of a peptide scan analysis was performed to determine the binding site for each mab ( figure ). the mab d bound to two linear peptides (peptide n and p ). the peptide n was appended with a cysteine residue at the n-terminal of the orf peptide (residues - , p ). both of them contained the sequence of the orf protein of pcv b between residues and . however, the non-igg mab ( d ) not only reacted with peptide n but also showed minor reactions against other peptides, which compared with the mab d in the elisa test ( figure ) . interestingly, the antipeptide n mouse serum reacted all test peptides and bound firmly to six linear peptides (peptide n , p , p , p , and p ). notably, anti-n poly antibodies reacted with these peptides (expect p ) which contained the common core peptide (p and tllhfpahfq ). nevertheless, this study did not get any mab which bound firmly to the peptide p . anti-orf mabs ( d and d ) bound the linear peptide spanning from residues to . the antipeptide n mouse serum was used as the positive control, and the anti-orf (peptide c ) mab ( h ) served as the negative control. the mab binding was tested by using an ielisa. peptides contained the sequence of the orf protein between residues and , associated -mer peptides, truncated derivatives, and the control (peptide c ) (as shown in table ). the experiments were performed three times. data represent the mean ± standard error (se). for these homemade pbmcs slides, all slides were made from the same batch and contained the same proportion of pcv -infected cells. all wells contained both negative and positive cells (pcv infected cells) confirmed by ifa, as previously noted in a similar study [ ] . in this study, pcv viral protein (orf protein and capsid protein) locations were initially assessed by ifa. the previous study indicated that the mab h bound the three minimal linear epitopes (dpplnp, dpplnpk, and lkdpplkp), which were located at carboxyl-terminus (cterminus) of the capsid proteins of pcv b, pcv d, and pcv a, respectively [ ] . likewise, the mab b bound two minimal linear epitopes (kdpplnp and kdpplnpk), which were located at cterminus of the capsid proteins of pcv b, and pcv d, respectively [ ] . first, the percentage was determined by counting the number of positive cells per nuclei in an ifa slide. the data show that - % of pbmcs were positive for anti-capsid peptide mab ( h ) staining (pcv a-, pcv b-, the anti-peptide n mouse serum was used as the positive control, and the anti-orf (peptide c ) mab ( h ) served as the negative control. the mab binding was tested by using an ielisa. peptides contained the sequence of the orf protein between residues and , associated -mer peptides, truncated derivatives, and the control (peptide c ) (as shown in table ). the experiments were performed three times. data represent the mean ± standard error (se). for these homemade pbmcs slides, all slides were made from the same batch and contained the same proportion of pcv -infected cells. all wells contained both negative and positive cells (pcv -infected cells) confirmed by ifa, as previously noted in a similar study [ ] . in this study, pcv viral protein (orf protein and capsid protein) locations were initially assessed by ifa. the previous study indicated that the mab h bound the three minimal linear epitopes (dpplnp, dpplnpk, and lkdpplkp), which were located at carboxyl-terminus (cterminus) of the capsid proteins of pcv b, pcv d, and pcv a, respectively [ ] . likewise, the mab b bound two minimal linear epitopes (kdpplnp and kdpplnpk), which were located at cterminus of the capsid proteins of pcv b, and pcv d, respectively [ ] . of pbmcs were positive for anti-orf mab ( d ) staining ( figure s ). it might be due to different abundance of the protein, the degradation of the protein, different pcv strains, conformational epitopes, or linear epitopes, which interacted with the antibody. interestingly, the anti-vlp of pcv rabbit serum produced cytoplasmic staining in pcv -infected pbmcs ( figure b,f) . when pcv -infected pbmcs were co-stained with anti-vlp of pcv rabbit serum and anti-capsid peptide mabs ( b ), some vlp/capsid peptide dual-positive cells were shown ( figure d ). the overlap between the anti-vlp rabbit serum staining and the anti-orf mab ( d ) staining is presented in figure h , and the anti-orf polyclonal antibody and anti-capsid mab ( a ) overlapped the same in figure p ; however, the polyclonal antibody staining was brighter than the mab staining. this result shows orf protein overlaps the capsid protein (or vlp) of pcv ( figure h,p) . both the capsid proteins and the orf protein were detected mainly in the cytoplasm. according to the previous study, the n-terminal half of orf protein (residues - ) was discovered in the cytoplasm, and the c-terminal half of orf protein (residues - ) was majorly located in the nucleus [ ] . the medial region of orf protein (residues - ) might be primarily located in the cytoplasm in this study. of pcv ( figure h,p) . both the capsid proteins and the orf protein were detected mainly in the cytoplasm. according to the previous study, the n-terminal half of orf protein (residues - ) was discovered in the cytoplasm, and the c-terminal half of orf protein (residues - ) was majorly located in the nucleus [ ] . the medial region of orf protein (residues - ) might be primarily located in the cytoplasm in this study. a previous study suggested that orf protein led to increased p protein levels and apoptosis of the infected cells [ ] . to identify the subcellular location of the orf protein in pcv -infected pbmcs, anti-capsid, anti-orf , and anti-p antibodies were used. the p protein also colocalized with capsid peptide (figure l ), and the orf peptide ( figure x ). the p protein was mainly distributed in the cytoplasm and around the nucleus ( figure j,v) . the orf protein, as detected with mab d , seemed to colocalize quite nicely with the p protein ( figure x ). the p /orf dual-positive cell presented a segmented nucleus ( figure w,y) . the nuclei of negative cells were round or oval, and nuclear segmentation was very rare in negative cells ( figure y ). the apoptosis in pcv -infected pbmcs was observed by using a terminal deoxynucleotidyl transferase dutp nick end labeling (tunel) assay (figures and s ) . the data show that - % pbmcs were positive for tunel staining. it is very similar to the percentage of anti-orf mab ( d ) staining. however, these similar percentages are not sufficient proof to show that the orf protein of pcv causes dna damage. a more careful analysis would be needed to reveal whether pcv infection causes dna damage. unfortunately, it was not easy to label both the orf protein and the signals of dna breakage simultaneously by ifa and tunel assay (data not shown). further research would be needed to determine whether the orf protein colocalizes with the signals of tunel. a previous study suggested that orf protein led to increased p protein levels and apoptosis of the infected cells [ ] . to identify the subcellular location of the orf protein in pcv -infected pbmcs, anti-capsid, anti-orf , and anti-p antibodies were used. the p protein also colocalized with capsid peptide (figure l ), and the orf peptide ( figure x ). the p protein was mainly distributed in the cytoplasm and around the nucleus ( figure j ,v). the orf protein, as detected with mab d , seemed to colocalize quite nicely with the p protein ( figure x ). the p /orf dual-positive cell presented a segmented nucleus ( figure w,y) . the nuclei of negative cells were round or oval, and nuclear segmentation was very rare in negative cells ( figure y ). the apoptosis in pcv -infected pbmcs was observed by using a terminal deoxynucleotidyl transferase dutp nick end labeling (tunel) assay ( figure and figure s ). the data show that - % pbmcs were positive for tunel staining. it is very similar to the percentage of anti-orf mab ( d ) staining. however, these similar percentages are not sufficient proof to show that the orf protein of pcv causes dna damage. a more careful analysis would be needed to reveal whether pcv infection causes dna damage. unfortunately, it was not easy to label both the orf protein and the signals of dna breakage simultaneously by ifa and tunel assay (data not shown). further research would be needed to determine whether the orf protein colocalizes with the signals of tunel. b (a); mab h (i); and mab a (m)), and the orf peptide of pcv (mab d (e,u)). staining with spf mouse serum was used as the negative control (q). (b,f,j,n,v) staining with rabbit antiserum (green) was used to identify the vlp of pcv (b,f), the p (j,v), and the orf peptide of pcv (n). (c,g,k,o,s,w,y) nuclei were stained with dapi (blue). the arrow points to the positive staining cell (z) and its irregular shaped nucleus (y), and the enlarged image in the sixth row (x,w). (d,h,l,p,t,x,z) the merged images are also shown. the scale bar is μm. the viral proteins were detected in the whole-blood samples using antigen-elisa techniques. the cut-off value in the commercial pcv capsid elisa and the orf protein elisa were . and . , respectively. the capsid protein of pcv was mainly detected in pigs at , , , and ≥ weeks of age in this study farm ( figure a ). noticeably, these groups (at , , , and ≥ weeks of age) showed more elevated standard error of od value than other groups in this statistical analysis. this result implies that pigs at that age would be susceptible to pcv infection. interestingly, non-vaccinated piglets showed the highest od value of capsid protein at one week of age. after piglets were inoculated with the pcv vaccine at two weeks of age, the od value of capsid protein gradually decreased until weeks of age. however, the od value of capsid protein was raised at ≥ weeks of age (two gilts and two sows). there is evidence to suggest that the vaccine could not protect pigs against pcv infection at weeks postimmunization. the orf protein of pcv showed the same result except for od values of the orf protein were lower than the capsid protein. the correlation coefficient (r) was . . the mean od values of the orf protein were significantly higher for suckers at one week of age than postweaning ones at - weeks of age (p < . ; figure b ). there is an apparent probability that the amount of orf protein was fewer than the amount of capsid protein in pcv -infected blood from suckers at one week of age. protect pigs against pcv infection at weeks postimmunization. the orf protein of pcv showed the same result except for od values of the orf protein were lower than the capsid protein. the correlation coefficient (r) was . . the mean od values of the orf protein were significantly higher for suckers at one week of age than postweaning ones at - weeks of age (p < . ; figure b ). there is an apparent probability that the amount of orf protein was fewer than the amount of capsid protein in pcv -infected blood from suckers at one week of age. figure . assessment of the capsid protein and the orf protein in the whole-blood samples from pigs of different age groups. this study used the commercial capsid kit (a) and the orf protein elisa (b) to measure each specimen, and the absorbance was measured at or nm, respectively. the peptide n and psbt were used as a positive (pos) control and a negative (neg) control, respectively, in the orf protein elisa. the data represent the mean ± se. treatments with different letters have statistically significant differences (p < . ). figure . assessment of the capsid protein and the orf protein in the whole-blood samples from pigs of different age groups. this study used the commercial capsid kit (a) and the orf protein elisa (b) to measure each specimen, and the absorbance was measured at or nm, respectively. the peptide n and psbt were used as a positive (pos) control and a negative (neg) control, respectively, in the orf protein elisa. the data represent the mean ± se. treatments with different letters have statistically significant differences (p < . ). the anti-viral protein-specific antibodies were detected in the plasm samples using ielisa techniques. the cut-off value of in the anti-capsid igg elisa and the anti-orf peptide (p ) igg elisa were . and . , respectively. non-vaccinated suckers showed the highest mean od value of anti-capsid igg and anti-orf igg at one week of age. it may mean that non-vaccinated suckers absorbed large amounts of maternally derived antibodies after birth. notably, the non-vaccinated group showed the most elevated standard error of od value (anti-capsid igg) in this statistical analysis. a gradual decrease of the mean od value of anti-capsid igg for piglets from the suckling period (one week of age) to the weaning period (six weeks of age) was observed. from to weeks old, the mean od value of anti-capsid igg increased significantly (p < . ; figure ). the mean od value of anti-capsid igg also increased significantly for pigs from to weeks old (p < . ) and reached a plateau at weeks old. the same trend was observed in the result of the anti-orf igg elisa test, except for od values of anti-orf igg were lower than anti-capsid igg. notwithstanding, it is worth mentioning that there was a sharp decrease of the mean od value of anti-orf igg for suckers from one to three weeks old. the mean od value of anti-orf igg increased significantly for pigs from six to nine weeks old (p < . ; figure ) and reached a plateau at nine weeks old. there was a weak correlation (r = . ) between the od value of anti-capsid igg and the od value of anti-orf igg in the plasm samples. it points to the probability that the amount of anti-capsid igg reflects humoral immunity in pcv -infected pigs before and after the injection of a pcv vaccine. besides, the amount of anti-orf igg reflects humoral immunity in pigs, which were caused by pcv infecting only. the mean od value of anti-capsid igg also increased significantly for pigs from to weeks old (p < . ) and reached a plateau at weeks old. the same trend was observed in the result of the anti-orf igg elisa test, except for od values of anti-orf igg were lower than anti-capsid igg. notwithstanding, it is worth mentioning that there was a sharp decrease of the mean od value of anti-orf igg for suckers from one to three weeks old. the mean od value of anti-orf igg increased significantly for pigs from six to nine weeks old (p < . ; figure ) and reached a plateau at nine weeks old. there was a weak correlation (r = . ) between the od value of anti-capsid igg and the od value of anti-orf igg in the plasm samples. it points to the probability that the amount of anti-capsid igg reflects humoral immunity in pcv -infected pigs before and after the injection of a pcv vaccine. besides, the amount of anti-orf igg reflects humoral immunity in pigs, which were caused by pcv infecting only. figure . assessment of the anti-capsid igg and the anti-orf peptide (p ) in the plasm samples from pigs of different age groups. the experiments were repeated three times, and data represent the mean ± se. treatments with different letters at the same kind of antibody assay have statistically significant differences (p < . ). according to a previous study, the orf peptide (residues - ) of pcv interacts with the p -binding domain of ppirh [ ] . the peptide n (residues - ) of the orf protein was synthesized and used in this study. subsequently, anti-orf mabs were generated and characterized in this study. this work created one hybridoma producing the mab d with igg , and the other producing the defective-ig mab ( d ) with a lambda-light chain. the mab d (igg ) bound to the linear peptide n (c hndvyislpi tllhfpahfq kfsqpaeisdkr ) and p ( hndvyislpi tllhfpahfq kfsqpaeisdkr ). however, the defective-ig mab d minorly reacted with all truncated peptides. this finding is similar to the previous study [ ] . the defective-ig mabs likely have broad binding, moderate specificity, and low affinity with the associated peptide. there were concerns about this defective-ig mab since this seems to be very rare. further, the molecular weight of the mab d was determined by western blotting. two bands were observed figure . assessment of the anti-capsid igg and the anti-orf peptide (p ) in the plasm samples from pigs of different age groups. the experiments were repeated three times, and data represent the mean ± se. treatments with different letters at the same kind of antibody assay have statistically significant differences (p < . ). according to a previous study, the orf peptide (residues - ) of pcv interacts with the p -binding domain of ppirh [ ] . the peptide n (residues - ) of the orf protein was synthesized and used in this study. subsequently, anti-orf mabs were generated and characterized in this study. this work created one hybridoma producing the mab d with igg , and the other producing the defective-ig mab ( d ) with a lambda-light chain. the mab d (igg ) bound to the linear peptide n (c hndvyislpi tllhfpahfq kfsqpaeisdkr ) and p ( hndvyislpi tllhfpahfq kfsqpaeisdkr ). however, the defective-ig mab d minorly reacted with all truncated peptides. this finding is similar to the previous study [ ] . the defective-ig mabs likely have broad binding, moderate specificity, and low affinity with the associated peptide. there were concerns about this defective-ig mab since this seems to be very rare. further, the molecular weight of the mab d was determined by western blotting. two bands were observed at about and kda, respectively. the small one ( kda) implied some degradation of this mab. since the molecular weight of the light chain was kda, the author suggested that the mab d might contain a short fragment of a protein. interestingly, the author also tested the molecular weight of another defective-ig mab c [ ] (which against peptide c ), and two molecules at about and kda were again shown (data not shown). according to previous documents, human patients with abnormal serum immunoglobulin-free light chain production should only be due to monoclonal plasmaproliferative disorders (included multiple myeloma, light chain myelomas, and light chain amyloidosis) [ ] [ ] [ ] . arguably, the defective-ig mab ( d ) might be related to the phenotype of splenocytes, which fused with the myeloma cell. further research should be carried out using advanced techniques (such as liquid chromatography with mass spectrometry and spectroscopic techniques) to investigate the constitution of the mab d . based on previous reports, lymphocyte depletion and apoptosis in lymphoid tissues are histological hallmarks in pcv -infected pigs [ ] [ ] [ ] . interestingly, these histopathological lesions are similar to marek's disease and african swine fever [ , ] . more recent evidence shows that these viruses caused an early cytolytic infection in lymphocytes by apoptosis [ ] [ ] [ ] . therefore, this study hypothesized that lymphocyte and pbmcs lineage cells should be the primary target cells for pcv infection. to the best of the author's knowledge, the study of the orf protein by indirect immunofluorescence assay in pcv -infected pbmcs has never been performed, and only a single article mentions the transient expression of the orf in porcine pbmcs and detecting apoptosis with a tunel assay [ ] . therefore, the author explored the relation of the capsid, p protein, and the orf protein by ifa and shed light on p protein (the marker of apoptosis) accompanied the peptide (residues - , p ) of the orf protein in pcv -infected pbmcs. it is worth highlighting that - % of pbmcs were positive for some antibodies staining, including anti-orf mab ( d ) staining, anti-p protein rabbit polyclonal antibody staining, and tunel assay ( figure s ). there is a strong probability that - % of pbmcs were undergoing the process of apoptosis. however, the data revealed that the variable percentage ( - %) of pbmcs was positive for anti-capsid mabs ( h , b , and a ) staining and anti-vlp rabbit serum staining. it was because these mabs or antiserum recognized different epitopes of proteins presented in pcv -infected cells. that means that the variety of interaction between antigen-binding sites of antibodies and epitopes, which includes linear form epitopes, conformational epitopes, degradation, and different pcv strains. curiously, this study ( figure s ) showed that the percentage of pcv -infected pbmcs (by mab h staining) was about -fold the rate of orf -positive pbmcs (by mab d staining) or the percentage of p -positive pbmcs. it seems likely that not all pcv -infected cells contained orf proteins or p protein. this finding concurs with the previous study, which indicated that the apoptosis statistically decreases in the initial pcv -infected pigs [ ] . on the other hand, this study showed the orf protein colocalized with the capsid protein marker of pcv . moreover, the p protein was also colocalized with the orf protein. remarkably, the p /orf dual-positive cell presented a segmented nucleus ( figure w ). however, these p /orf dual-positive cells were very few in these samples. this finding will be confirmed by flow cytometry in the future. although a previous study indicated that pcv -infected pigs had a reduction of lymphocytes in the peripheral blood [ ] , the mechanism of lymphocyte lysis was still unclear. this study confirmed that the orf protein was related to the p protein (apoptosis marker) in pcv -infected pbmcs, while other factors (proliferative activity [ ] , caspases and [ , ] , granzymes [ ] , or corticosteroids [ , ] ) causing lymphocyte lysis or depletion need to be considered. although the experiment on apoptosis in pcv -infected pbmcs was carried out by the tunel assay (figure ) , it did not clarify the relationship between the tunel result and the orf protein in pcv -infected cells. only the ifa data confirm previous reports that exogenous orf protein was related to the accumulation of p protein [ , ] , but more proof are needed to make sure the orf protein is a major factor leading to apoptosis in pcv -infected cells [ , , ] and depletion of lymphocytes. pcv is one of the most critical pathogens in modern swine production and causes endemic disease in pig farms [ ] . the commercial vaccines were confirmed protective in the field against pcv and mainly administered to suckers in herds with pcv infection [ , , [ ] [ ] [ ] . most researchers utilized the quantitative real-time pcr to detect pcv nucleic acid, and then they evaluated the efficacy of vaccination in regard to pcv viremia in pigs [ , , ] . however, little is known about the capsid protein or the orf protein in blood from the pcv -infected herd with vaccination. although the capsid antigen-elisa could detect capsid protein, this assay could not differentiate from native viral proteins or vaccine ones in blood. the orf protein-elisa, by contrast, only identified native orf proteins in pig blood since no commercial vaccine contains the orf protein of pcv . for these purposes, this study used the commercial capsid antigen-elisa and homemade orf protein-elisa (anti-n polyclonal antibodies and mab d based) to detect viral proteins in pig blood. this study found non-vaccinated suckers had the highest of pcv proteins in blood at one week of age. the author suggests that in these suckers it was caused by pcv infection, and these viruses majorly stemmed from the sow-to-newborn transmission [ ] . after inoculated with the pcv subunit vaccine, the capsid protein, and the orf protein were gradually decreased. however, these proteins were detected again in gilts and sows (≥ weeks of age). that means pcv viral proteins (the capsid protein or the orf protein) in gilts and sows were higher than that of pigs at - weeks of age. to put it another way, vaccinated-pigs reduced viremia compared to non-vaccinated suckers, and the immunity could not continue to protect vaccinated pigs after weeks post-vaccination. since pcv is highly resistant to environmental conditions, being able to remain in the farm environment and thus represent a risk for infection maintenance [ ] , even mass pcv vaccination (without implementing further farm management practices or biosafety measures) was not able to clear out pcv infection, and the virus became detectable again when vaccination was stopped [ , ] . another key point to mention is that pcv -specific antibodies response play roles in the pcv -infected herd. it is worth noting that there were two different antibody profiles in this pcv -infected herd with the pcv vaccine. the data show that pcv -infected herd had a higher od value of anti-capsid igg at age , , and ≥ weeks, compared with - weeks. in addition, pcv -infected herd had a higher od value of anti-orf igg at age and ≥ weeks, compared with - weeks. according to previous studies, newborn suckers received colostral antibodies from seropositive sows and showed various levels of maternally derived antibodies [ , ] . these maternally derived antibodies might interfere with the viral protein-specific antibodies response while suckers immunized with the pcv subunit vaccine. it may be assumed that the artificial capsid protein (pcv vaccine) elicited anti-capsid igg producing while maternally derived antibodies progressed to degrade. the total level of anti-capsid igg decreased slowly but still maintained a high level of anti-capsid igg during the age of - weeks. this phenomenon could neutralize the pcv virus and prevent piglets from suffering pcv infection. in contrast with dual-source capsid proteins (pcv virus and subunit vaccine), the piglet's immunity response to the orf protein was only caused by pcv virus infection. the total level of anti-orf igg decreased quickly at - weeks of age. then, it increased significantly at - weeks of age. the weak correlation (r = . ) between the anti-capsid igg and the anti-orf igg is worth mentioning. it might reflect two kinds of antibody responses in virus infection and post-immunization. these data interpret the interaction of the viral protein with the host immune system. according to previous reports, orf encodes a -kda protein that is involved in viral capsid formation and contributed to self-assembled virus-like particles (vlp) [ ] . the monomer structures were assembled into a vlp model consisting of capsid subunits to form an icosahedron [ ] . vaccination with this vlp (subunit vaccine) induced both humoral and cell-mediated responses against pcv [ , ] . however, orf encodes an . -kda protein [ ] that is the non-structural protein and involved in pcv -induced apoptosis [ , , ] . until now, no report mentions that the orf protein was solely used as the vaccine against pcv , reducing viremia or pathological lesions in pcv -infected herds. these differences contributed to their application in different ways [ , ] . in this study, the percentage of orf -positive pbmcs was significantly lower than capsid-positive pbmcs. similarly, the antigen-elisa result shows that the amount of orf protein was less than the amount of capsid protein in pcv -infected blood from suckers at one week of age. that might be because mab d only recognizes the orf protein (peptide p ) for pcv b strain (genbank: aac . ), and it causes low detection. however, pcv d (pcv b mutant strain) and pcv b are still predominant genotypes in the field farms. besides the similar percentages of pbmcs were positive for these antibodies (mab d and anti-p protein rabbit polyclonal antibody) staining or tunel assay. in contrast to the orf protein, the detection of the capsid protein could be the variable result in pcv -infected pbmcs via different antibodies, due to different pcv strains [ , ] , conformational epitopes [ ] , or linear epitopes [ , ] . previous studies indicated that antibody binding residues were often on the exterior of the capsid of pcv [ , , , , , ] . although an epitope might be on the surface of the single capsid unit, it could bury in the vlp and be inaccessible to the antibody [ , ] . in general, these results confirm that mab d recognized the native orf protein in pcv -infected pbmcs. this study used mab d or its minimal linear epitope to design various immunoassays. these assays evaluate the viral load and the immune response of viral proteins stimuli. these may serve as surveillance tools for monitoring natural pcv infection in the herd. overall, the author generated anti-orf mabs against the orf peptide (residues - ) of pcv . this study confirmed the defective-ig mab ( d ) with a lambda-light chain, and its molecular weight was about kda. the mab d contained heavy chains (γ ) and kappa-light chains, and it bound to one minimal linear epitope (hndvyislpitllhfpahfq kfsqpaeisdkr). the data show that - % of pbmcs were positive for orf protein or p protein. otherwise, - % of pbmcs were positive for anti-capsid peptide mab ( h ) staining. this study confirmed the orf protein colocalized with the p protein in pcv -infected pbmcs. the author devised the orf protein-elisa (anti-orf antisera and mab d -based) to detect the orf protein in blood samples. the results show that the amount of orf protein was less than the amount of capsid protein in pcv -infected blood from suckers at one week of age. the correlation between the orf protein and the capsid protein is worth noting (r = . ). furthermore, the antibody level of anti-capsid igg and anti-orf igg could imply the immunity response of pig herd, but the sample size needs to be considered. the author declares no conflict of interest. a very small porcine virus with circular single-stranded dna characterization of novel circovirus dnas associated with wasting syndromes in pigs studies on epidemiology and pathogenicity of porcine circovirus experimental reproduction of severe wasting disease by co-infection of pigs 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med doi: . /j.jepm. . . sha: doc_id: cord_uid: wg hew b the practice of veterinary medicine is facilitated by appropriate equipment, and exotic pet medicine is no exception. exotic practitioners use standard or modified veterinary and human equipment, and now even enjoy the benefit of specialized products manufactured specifically for exotic pet practice. i n years past, specialized equipment for the practice of exotic animal medicine was nonexistent. today, equipment is modified from what is available for human or traditional pet medicine, or even manufactured specifically for exotic patients. this has provided the practitioner with tools to greatly improve quality of care and offer services at the same high standard of medicine that were previously only available for dogs and cats. blood sample collection techniques used for exotic mammals and reptiles are modified from traditional pet medicine and utilize a variety of needles, syringes, and other collection devices and containers. techniques for blood collection are described in detail elsewhere. most reference laboratories are willing to provide the practitioner with specimen containers designed for smaller volume samples, such as micro-gel separator tubes, both plain and coated with lithium heparin, and smaller calcium or sodium ethylenediamine tetraacetic acid tubes for submission of whole blood. submitting small volume samples in standard-sized tubes containing anticoagulant may result in dilutional artifact because of improper ratio of anticoagulant to sample (fig ) . human neonatal-or pediatric-sized culturettes are available from reference laboratories for the collection of culture specimens from locations such as the nasal cavity of rabbits or the ear canal of rodents. radiography in very small exotic patients is greatly facilitated by high-speed cassettes and film or mammography film. techniques for ultrasonography of small exotic mammals and various reptile species have also been described. ultrasound machines should be equipped with a transducer suitable for the size of the animal, with a small footprint or contact area. transducers with frequencies from . to mhz will be the most useful. many standard ultrasound machines can be fitted with probes of this frequency. color doppler is advantageous for assessing blood flow, but it is harder to interpret. most new ultrasound machines are capable of digital recording to facilitate further review after contact with the patient is completed. ultrasound-guided biopsy of organs can be performed as in traditional pet species. another useful diagnostic modality that has recently received increased attention is diagnostic endoscopy of exotic animals. rigid endoscopy for determination of sex is well recognized in birds maintained in pet and zoo collections. recently, more attention has been given to diagnostic endoscopy and the collection of endoscopic-guided samples. , diagnostic endoscopy uses rigid endoscopes, most commonly . and . mm in diameter. endoscopes use a high-intensity light source for illumination. the endoscope and light source are the minimum equipment required for visualization of the oral cavity, ear canal, and nasal cavity of small exotic mammals (fig ) . however, more advanced techniques such as endoscopic evaluation of the urethra, bladder, and vagina, and abdominal or thoracic endoscopy require a diagnostic sheath to allow insufflation of sterile fluid or air to enhance visualization. the diagnostic sheath also allows introduction of a variety of instruments for sample collection for cytology, culture and sensitivity, and/or biopsy. all endoscopic procedures benefit from the addition of a camera to allow viewing of images on a monitor, and an image capture system to save images for education, documentation, or teaching purposes. in reptiles, diagnostic endoscopy is most often used to examine the oral cavity, coelomic cavity, vent, urinary tract, and even portions of the reproductive tract of female animals. , collection of diagnostic samples is only half the challenge of obtaining meaningful data. samples must be submitted to laboratories that are familiar with the process and have some degree of expertise in the evaluation of exotic animal specimens. many veterinary diagnostic laboratories are willing to accept these samples, and a number of facilities actively develop unique tests specifically for exotic animals (for example, tests for viral or bacterial pathogens). a few facilities are even dedicated solely to exotic animal testing. see table for a list of some laboratories offering specialized tests specifically for exotic animals. injectable anesthetic protocols require no specialized equipment other than small syringes and needles, and the ability to perform dilutions of drugs for use in very small patients. induction and maintenance of gas anesthesia are facilitated by modified or specialized equipment. several manufacturers make smaller, inhalant anesthetic cones and endotracheal tubes with narrow interior diameters (for example, . mm). in exotic mammal medicine, intubation of rabbits, ferrets, and larger exotic pets is common. laryngoscopes with straight miller blades sizes or facilitate intubation and visualization, particularly in small carnivores. intubation of smaller mammals is technically much more challenging than that for the larger exotic species previously mentioned. endoscopically guided intubation may be useful in these difficult cases. johnson recently described this technique in rabbits, ferrets, prairie dogs, chinchillas, and guinea pigs using a semirigid . -mm endoscope (focuscope; mds inc., brandon fl usa). this may be exceedingly more difficult to impossible to perform in chinchillas and guinea pigs using a true rigid endoscope that is incapable of making the slight bend required to direct the endoscope and tube into the trachea. other equipment that may aid intubation includes a variety of mouth gags, one designed specifically for ferrets (nazzy ferret mouth gag; usi/bsi, universal surgical instruments, glen cove, ny usa). maintenance of core body heat during surgery is critical, especially in smaller exotic patients. warm water-circulating heating pads, radiant heat bulbs, and forced-air heating systems have all been used successfully in exotic pet practice. some small animal ventilators can be used to control depth and frequency of respirations in small exotic mammals. use of the ventilator necessitates proper intubation (fig ) . careful and attentive monitoring of anesthetized exotic patients is critical, and no monitoring device or equipment is more important than a dedicated veterinary anesthetist. clear, transparent drapes allow one to view smaller patients to monitor respiratory rate, depth, and other important parameters. monitoring devices used regularly in exotic pet medicine include vascular doppler, a flexible, constant readout temperature probe, electrocardiogram, pulse oximetry, and indirect measurement of blood pressure in larger patients (figs and ) . recently, techniques have been developed for measurement of blood pressure with a cardiac doppler and small blood pressure cuffs in patients weighing less than kg. standard aseptic technique is critical in exotic mammal and reptile surgery, despite the fact that surgical site preparation is often more difficult. less-than-optimal preparation carries the same risk of infection and wound dehiscence found in traditional pet surgery. clipping of fur from delicate skin must be done carefully to avoid iatrogenic skin lacerations. small clippers with fine blades are appropriate for these cases. skin is prepared with warmed dilute ( . %) chlorhexidine solution followed by warm saline solution rinse, because cold scrub solutions and alcohol can produce hypothermia in small patients. a number of materials can be used as sterile drapes in these patients. however, most exotic animal surgeons and anesthetists prefer transparent drapes for reasons described above. some transparent drapes are self-adhering or can be secured to the patient with smaller, less traumatic towel clamps. many of the surgical instruments used in small mammal and reptile surgery are actually human pediatric, vascular, or ophthalmic instruments (fig ) . a number of manufacturers and distributors market these directly to exotic animal veterinarians. standard instruments are adequate when working with larger exotic pets, but fine, delicate instruments are essential when considering procedures such as laparotomy in rodents or small reptiles. an example of a typical small exotic mammal/reptile surgical pack is described in table . radiosurgery has been described for use in exotic mammals and reptiles, and is an effective method for initial incision and for intraoperative hemostasis (surgitron; ellman international, oceanside, ny usa). wire-tip electrodes are ideal for making incisions, and ball electrodes are useful for coagulation. bipolar forceps are especially useful for the coagulation of individual vessels. access to the surgical field makes these surgeries extremely challenging. a number of specialized retractors allow adequate access. the most versatile retractor is a plastic ring device with a number of completely adjustable stay hooks that can be arranged in any number of useful configurations (lonestar retractor; lonestar medical products, stafford, tx usa) (fig ) . magnification is often of critical importance in smaller patients. a variety of surgical loops, telescopes, and operating microscopes are available. in most cases, magnification of . to . ϫ is adequate. magnification allows greater visibility of tiny vessels and other structures, but surgeons must practice regularly and become accustomed to this ocular aid to maximize the benefits. care must be taken to use a magnification system that allows the surgeon to maintain a normal, comfortable posture, and not be forced to lean over or significantly bend the neck. improper posture leads to fatigue, thereby contributing to back and neck pain. , hemostasis during surgery is of extreme importance in smaller patients because of overall decreased blood volume. blood losses acceptable in larger species are rapidly fatal in small exotic patients. sterile, cottontipped applicators are useful for application of direct pressure. radiosurgical hemostasis is mentioned above. metal hemostatic clips are manufactured in a variety of sizes and are extremely useful in these patients, especially in situations where manipulation of suture material is extremely difficult to impossible (hemoclip; weck, triangle park, nc usa) (figs and ). other useful hemostatic aids include sterile, absorbable gelatin sponge or cellulose products (gelfoam; pharmacia and upjohn co, kalamazoo mi usa; surgicel; ethicon, inc., johnson and johnson, somerville, nj usa). these hemostatic aids are especially useful for situations in which the exact source of mild hemorrhage cannot be identified and directly ligated. recently, a vessel heat-sealing device has been described for use in small exotic patients. the ligasure vessel-sealing system has been used for vessel ligation and procedures such as castration and hysterectomy in a variety of species, including lizards and chinchillas (ligasure; tyco health care valleylab, boulder, co usa). the hand-held hemostat comes in a variety of sizes useful for smaller patients. the device can be used via a diagnostic port for endoscopic surgeries, but is currently too large for traditional small exotic endoscopy equipment. smaller instruments of this type are currently under development. closure of surgical sites follows principles similar to those described for traditional pets. an important exception is the closure of skin in reptile patients. skin is the holding layer in reptile coelomic surgeries and must be closed with an everting pattern. most closures are made with -to- -sized to -to- sized suture material. catgut suture material has been found to produce marked inflammatory responses in most species, including reptiles. although surgeon preference often drives selection of suture material, choices typically include monofilament absorbable materials such as polyglyconate. suture material must be swaged on to small, less traumatic needles to reduce trauma and excessive movement of small exotic patients. postsurgical suture disruption is more common in some rodent species, whereas rabbits, ferrets, and guinea pigs tend to be more tolerant of external sutures. for this reason, many surgeons prefer subcuticular closure of skin, followed by tissue adhesive. application of rigid endoscopy in small exotic mammals hernandez-divers sj: modern endoscopy equipment and advanced endoscopy techniques in birds and reptiles endoscopy of the distal urogenital tract as an aid in differentiating causes of urogenital bleeding in small mammals endoscopic evaluation of turtles, tortoises and terrapins over the endoscope endotracheal intubation of small exotic mammals lichtenberger m: shock, fluid therapy, anesthesia and analgesia in the ferret. exotic dvm preparation and equipment useful for surgery in small exotic pets ergonomics in avian and exotic practice use of a blood vessel sealing instrument in exotic animal surgeries equipment for diagnostics and surgery key: cord- -napw pxe authors: paul, swarna kamal; jana, saikat; bhaumik, parama title: a multivariate spatiotemporal model of covid- epidemic using ensemble of convlstm networks date: - - journal: j doi: . /s - - -x sha: doc_id: cord_uid: napw pxe the high r-naught factor of sars-cov- has created a race against time for mankind, and it necessitates rapid containment actions to control the spread. in such scenario short-term accurate spatiotemporal predictions can help understanding the dynamics of the spread in a geographic region and identify hotspots. however, due to the novelty of the disease there is very little disease-specific data generated yet. this poses a difficult problem for machine learning methods to learn a model of the epidemic spread from data. a proposed ensemble of convolutional lstm-based spatiotemporal model can forecast the spread of the epidemic with high resolution and accuracy in a large geographic region. the feature construction method creates geospatial frames of features with or without temporal component based on latitudes and longitudes thus avoiding the need of location specific adjacency matrix. the model has been trained with available data for usa and italy. it achieved . % and . % mean absolute percent error for total number of predicted infection cases in a -day prediction period for usa and italy, respectively. wuhan city in china initially observed an outbreak of covid- disease caused by sars-cov- . eventually it became a pandemic and more than countries are fighting hard to contain the infection. one of the best ways to contain the infection is rapid identification of positive cases and isolation. forecasting regional spread can help identify future hotspots and distribution of infection which would eventually help to take containment measures. a spatiotemporal epidemic spread model can accommodate both spatial and temporal correlations in data. however, most of the models either require diseasespecific domain knowledge [ ] or are too spatially coarse [ ] . deep learning models can learn the dynamics of epidemic spread with high spatial resolution and high degree of accuracy with minimal initial bias due to its capability of high nonlinear representation. deep neural network-based spatiotemporal models [ ] have already been applied to predict epidemic spread. however, this model is experimented on a small localized region and influence of external factors are ignored. deep learning models also tend to overfit due to its high representational capability. due to availability of very limited dataset, the problem of overfitting looms large in this case. thus, modelling of covid- spread in a wide region with high spatial and temporal resolution is challenging. to address the problem of spatiotemporal prediction of covid- spread in a large geographical region with high resolution, an ensemble of convolutional lstm [ ]based model is proposed which needs to be trained with multilayer temporal geospatial data, transformed as sequence of frames. each layer of the geospatial data corresponds to a causal factor that might influence the spread of the epidemic. an ensemble of models helps reduce variance in errors and overfitting on small training dataset. experimentation is carried out with data of usa and italy and achieved country-level mean absolute percent error (mape) of . % and . %, respectively, on forecasting of total infection cases in days period. the paper is organized as following. a brief discussion on modelling the epidemic spread and feature engineering is presented in sect. . in sect. , the ensemble of convolutional lstm model and performance measurement metrics are presented. section is about experimental results. the following section concludes the paper. disease spread is a complex dynamical system, and numerous factors contribute to the dynamics of spread making it non-stationary. covid- is no different. geographical location, weather conditions [ ] , human mobility [ ] , and population statistics might be some of the impacting factors changing the dynamics of the spread. epidemic spread is correlated in time as well as spatial dimensions. however, it may be spatially autocorrelated in a small localized region but not across wide regions. thus, a large geographic region is divided into relatively smaller grids and model is trained with samples drawn from local distribution of infection cases. the objective of the model is to forecast new cases of infection on daily basis in different regions across a country which can be added up to calculate total cases of infection. all the observations in the dataset are mapped to a spatial region bounded by defined latitude and longitude. the region is geospatially divided in m n grids of equal sizes bounded by calculated latitudes and longitudes. figure a illustrates a grid bounded by latitudes and longitudes. the box represented by the dotted line is called as frame. the frames have overlapping areas in all directions. the overlap allows flow of spatial influence from neighbouring grids to another. each frame is in turn divided into l l pixels which includes the overlapping area. each pixel represents a bounded area in geospatial region. the values in each pixel are mapped to certain feature in the bounded geospatial region. separate frame matrices are constructed for each feature and concatenated through channels. for example, new infection count and population are two features and they represent two separate l l matrices in a frame concatenated across a third axis. each pixel in the infection count matrix contains the count of new infections (di) in the pixel area in a day. infection count is distributed both in spatial and temporal dimensions. to reduce the skewness, the infection count in a pixel is log transformed and normalized in - scale. considering r factor of covid- between and , it is calculated that % of the population (p) in an area needs to get infected to attain herd immunity and reduce further spreading [ ] . similar to the sir model [ ] , the total population p is compartmentalized into susceptible (s) and infected/recovered/deceased (i) group. susceptible population at any day is calculated as : p À i: a pixel value is calculated as ln di þ ð Þ=ln s þ ð Þ. total population is distributed spatially in similar fashion, and it is assumed time invariant within a short interval. pixel value of population matrix is calculated as Þ . each frame is represented as tensor of dimension t l l c, where t is the total time span and c is number of channels or features. as shown in fig. b , each training sample in a frame is fig. a illustration of overlapping frames obtained by spatially dividing a geographical region. the bold lines represent latitudes and longitudes which separate the grids. the box with dotted line represents the overlapping frame and it is represented as an image for training the model. each grid is divided in certain number of pixels. the margin refers to number of pixels in the overlapping region. b illustration of sequence of images in a frame. t- is the most recent image. x train , y train are the training samples and x test , y test are testing samples generated by sliding a time window size of w ? by , leaving behind a test case sample of time window size of w in the most recent period. number of training samples in a frame can be calculated as t À w À w À . thus, total number of training samples s train for all frames can be calculated as s train ¼ t À w À w À ð Þ Ã m * n. the forecasting problem is framed as supervised learning problem. given a sequence of observed matrices of spatial data as images x ; x . . .x t , the final objective of the model is to predict the next image x tþ . the training samples are divided into input sequences and output sequences each of length w. the input sequence is time lagged images of output sequence. the model forecasts the normalized log transformed new infection count in each pixel in a frame for each timestep. thus, the output frame consists of only channel. the input training dataset (x train Þ can be represented as a tensor of size s train  w  l  l  c and the output dataset (y train ) as s train  w  l  l  . for training, the input sequences are selected from all frames having nonzero total infection count. figure b illustrates the sequence of images in a frame. the image t- to t- represents an input training sequence (x train Þ of length w. the output image sequence (y train ) for this training sample is t- to t- . other training samples are generated by sliding the window w backwards in time by . the most recent images t- and t- represent the test output images (y test ), and immediate sequence of images t- to t- is the test input sample (x test ). the test set x test is represented by a tensor of size m * n ð ÞÂw  l  l  c and y test by m*n ð ÞÂw  l  l  . the model comprises of a convolutional lstm network [ ] configured to take multichannel input such that distinct features can be passed through different channels. multiple convolutional lstm layers are stacked sequentially to form a network with high representational capability. the network terminates with a d convolutional layer having one filter which constructs a single channel output as the next frame prediction. a single model may be prone to overfitting on training dataset and loose stability in terms of prediction made. creating an ensemble of diverse models intended to solve the same task, and combining the predictions made by them typically improves test accuracy and stability [ ] . we used bootstrap aggregation [ ] to create an ensemble of models. % random samples are drawn with replacement from the original training dataset, and an ensemble of five models is trained individually. during prediction the output of each of the models is weighted as per following equation. o is output of the ensemble, o n is output of the model n, e is set of all models in the ensemble, i n train is number of infected patients in the training samples of model n, mse n is mean squared error of model n on validation dataset, and r is softmax function. the weights are proportional to the amount of positive cases used for training the model and inversely proportional to the validation error. during testing the model is given a sequence of most recent frames as input, and the next frame is predicted. the predicted frame is temporally appended to the input sequence of frames and fed to the model again to obtain the next predicted frame. this continues until required number of future frames are predicted. the accuracy of a model is tested with the metric ''mean absolute percent error'' (mape) and kullback-liebler (kl) divergence [ ] . the pixel values are transformed to di and summed up cumulatively to calculate total infection cases i, up till a specific day. mape is calculated at pixel level for total infection cases at the end of prediction period and averaged. the pixels with susceptible population count are ignored while calculating mape. pixel mape is calculated as per eq. , where g p is set of all unique pixels in all grids such that the frame for each corresponding grid has nonzero total infection count, w is prediction time period, w ¼ t À w is total time period in training set, p is a pixel from a set of unique pixels in the total region having nonzero actual susceptible population, p i is predicted pixel value on i th day, s i p is susceptible population at pixel p on i th day calculated from p i and s iÀ p , di i p is actual new infection count at pixel p on i th day, and n p is total number of pixels p in the region.Î p and i p are total predicted and actual infection cases, respectively. kl divergence at pixel level is calculated for total infection cases at the end of prediction period to measure the dissimilarity of distribution of predicted infection cases with respect to actual. r is softmax function applied after scaling a series in to scale, and p x ð Þ is probability distribution of x. softmax is applied to convert total infection cases as probability distribution across pixels. mape is also calculated at country level with respect to total predicted infection cases across the region during the prediction period. experiments have been carried out to predict the future new infection cases in italy for a period of days and days and in usa for a period of days and days. data have been collected from harvard dataverse [ ] [ ] [ ] . for usa the data collection period is ' - - ' to ' - - ' and for italy it is ' - - ' to ' - - '. test data period for italy data is ' - - ' to ' - - ' and for usa it is ' - - ' to ' - - '. figure a , b shows the region of usa and italy which has been divided into grids. the length w of each training input sequence is taken as days. for both the countries, the frames containing at least a single covid- infection case are considered for training and testing the model. each frame in turn is divided in pixels with an overlap margin of pixel. the model consists of ensemble of convolutional lstm networks. for italy each network contains hidden layers with sigmoid activation. the output layer is a convolutional d layer with exponential linear unit as activation. the models are trained for epochs with mean squared error as loss function. the input and hidden layers have filters. the input layer is configured to take images of size . the second channel is fed with normalized population data. the region in usa is approximately times than italy, and the distribution of covid- cases in usa is geospatially highly skewed. thus, grids are divided in four equal sections by latitude and longitude with each section containing grids. a set of heterogenous ensembles are trained for each of the sections. the configuration of networks is same as that of italy except they contain hidden layers and each network is trained for epochs. table shows the performance of the models in terms of kl divergence and mape. for both usa and italy, low kl divergences state that the predicted geospatial probability distribution of total infection cases nearly matches with the actual probability distribution. the pixel level mape for italy stays below %. for usa in -day forecasting period mape is % as there are many pixels in usa with low total patient count. a slight deviation in the prediction for these pixels shoots up the mape. country level mape is low for both italy and usa. figures a and a shows predicted versus actual total covid- cases for a period of and days in usa and an ensemble of convolutional lstm-based spatiotemporal epidemic spread model has been proposed for shortterm forecasting of covid- spread. experiments done on data obtained for usa and italy reveal acceptable prediction accuracy with high resolution. since the model has option to fed in any number of external features, we are experimenting with multiple external features that might influence the spread. this might help to find important optimal vaccine distribution in a spatiotemporal epidemic model with an application to rabies and raccoons demographic variability, vaccination, and the spatiotemporal dynamics of rotavirus epidemics a deep residual network integrating spatial-temporal properties to predict influenza trends at an intraurban scale convolutional lstm network: a machine learning approach for precipitation nowcasting temperature and latitude analysis to predict potential spread and seasonality for covid- modelling and prediction of the coronavirus disease spreading in china incorporating human migration data herd immunity: a rough guide qualitative analyses of communicable disease models why m heads are better than one: training a diverse ensemble of deep networks bagging predictors on information and sufficiency us covid- daily cases with basemap publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations log transformed predicted versus actual total covid- cases after a period of days in each pixel in italy. b log transformed predicted versus actual total covid- cases after a period of days in each pixel in usa key: cord- -hkq wti authors: haukkala, hiski title: nonpolar europe? examining the causes and drivers behind the decline of ordering agents in europe date: - - journal: int polit doi: . /s - - - sha: doc_id: cord_uid: hkq wti is europe headed towards nonpolarity? what would that entail for the future stability and security of the continent? taking its cue from ir debates about the effects of polarity on international order, the article develops conceptual tools and an analytical narrative concerning europe during the post-cold war era. it refines the concept of a great power by suggesting the notion of ‘ordering agents’ and ponders whether europe is in danger of drifting towards a period of nonpolarity with no power being able to shoulder the responsibility of ordering and providing good stewardship over europe. for the past five centuries, europe has been the veritable centre of the world. at first the europeans developed the modern conceptions of statehood and international politics and then actively projected these innovations through the application of a 'standard of civilisation' well beyond the continent's boundaries (bull and watson ) . in the process, an increasingly global international system was formed, and it has been one where the europeans have continued to enjoy a pride of place. the europeans have also been one of the main sources as well as beneficiaries of western international order. since the end of the cold war, it has rested on us primacy, but it is a wider western phenomenon that has resulted in a liberal and rulebased world order (ikenberry ) . in europe, it has been manifest in a concentric hegemonic order where the us global primacy and regionally preponderant security role through nato have been complemented by the european union (eu) that has assumed the position of a regional normative hegemon in its own right ( ) with a view of ordering the continent along essentially unipolar lines by envisaging a europe of concentric circles revolving around 'brussels' (aalto ; diez and whitman ) . in europe, the usa and the eu have formed a 'matryoshka hegemony' (deyermond ) of sorts that has sought to, and largely succeeded in ordering the continent in a largely unified, or at least complementary manner. for many, these developments have been welcome and benign. unarguably europe has never been as stable and secure as it has been for the past three decades. yet there are growing signs that this era is coming to an end. on the global level, the usa has entered a period of retrenchment with tensions increasing with the rapidly rising china (allison ) . under president donald trump, it has also largely abdicated its global leadership role and the liberal rule-based international order is under an immense and growing strain (sørensen ) . china together with russia and other smaller regional powers is challenging the current international order on the global level while staking their claims for regional spheres of influence. this is visible also in europe where russia has for some time been making the case for a more equitable bipolar setting. this process of contestation culminated in the ukraine conflict in that has resulted in a low-simmering conflict between the west and russia. taking its cue from international relations (ir) debates about the effects of polarity (the number of great powers) on the evolution of international order (for a chronological lineage, see waltz ; kegley and raymond ; mearsheimer ; brooks and wohlforth ; monteiro ) , this article poses the question whether europe is experiencing not only a period of 'normal' geopolitical contestation, but could it in fact be in a transition towards a state of affairs where it will become void of great powers capable of effectively ordering the continent, let alone the wider world. in a word, is europe headed towards a period of nonpolarity? and if so, what would it entail for the future of cooperation, stability and even security on the continent? the article makes three main contributions. firstly, it links european studies with ir debates concerning polarity and the ongoing change in the structure of international politics. it situates itself in the so-called third wave of hegemony studies (ikenberry and nexon ) , but concentrates on examining the unravelling of western hegemony in europe. secondly, it seeks to conceptually expand our understanding of great powerhood by introducing the notion of 'ordering agents'. the article seeks to add nuance to the extant literature that has largely simply assumed that great powers are always also capable of generating order. instead, the article draws attention to a whole gamut of prerequisites for successful ordering role in contemporary international politics. moreover, it is argued that these requirements are particularly salient in europe that is undoubtedly the most highly institutionalised region in the world (diez and whitman ) . thirdly, the article presents an analytical narrative concerning the developments in europe during the post-cold war era. it does so in two stages: firstly recounting the story of western attempts at hegemonic ordering of europe and russia's growing contestation of it, and then moving on to analysing the main causes and drivers behind potential nonpolarity in europe. the argument will be developed in four stages. firstly, the concepts of (non) polarity and great powerhood are introduced and refined with a view of introducing the concept of 'ordering agents'. secondly, the roles that the usa, the european union and russia have played in ordering europe during the post-cold war era are analysed. thirdly, the causes, drivers and consequences of nonpolarity in europe are discussed. finally, the article concludes by pondering the question whether europe indeed is headed towards a period of nonpolarity and what that could entail for the future of the continent. it ends with a policy relevant point by warning about the possible unintended consequences of current contestation and institutional atrophy in europe. the article suggests that the current trajectory is not in the best interest of any party and engages in some normative argumentation by contending that in the twenty-first century a great power worthy of the name should be held to a higher standard in terms of providing building blocks for stability and order on both the regional and global levels than is currently the case. often order is the norm in everyday life. this is the case in our domestic societies where the monopoly on the legitimate use of violence enjoyed by the states ensures a hierarchical and, at least most of the time, also orderly conduct of our national politics. the anarchical international system does not lend to a clear-cut hierarchy along the domestic lines, but it is not entirely without order, either, that for the purposes of this article is defined as (sub-)systemwide structures and practices-institutions, norms, values and principles-that govern and regulate the conduct of international politics. in international politics, order stems mainly from two sources (see young : - from which the following is taken). it may emerge spontaneously generated by the 'hidden hand' of international anarchy. oran young has called these self-generated orders, the classic example of which is the principle of balance of power that has at times been rather successful in moderating interstate conflict (little ). yet for the purposes of this article, and indeed the increasingly interdependent and global international system in which we find ourselves, this is too rudimentary and in effect shallow conception of order. therefore, the other, and arguably more important, avenue for generating order is that of premeditated design. in young's terminology, these imposed or negotiated orders require intentional agency on the part of key actor(s) that take the lead, or act in concert, in generating, upholding and, at times, modifying and even undoing orders. there is nothing automatic in this latter type of order generation, as it takes effort and the use of power to achieve. in the final analysis, it requires not only great power but great power(s) to generate and uphold an international order. but being a great power is not in itself enough, as one must also be both able to conduct the ordering role and willing to carry the associated burden. therefore, it is suggested in this article that order-generating great powers are a particular subset of the category that should be called ordering agents that are defined here as a great power that has the capacity, ability and willingness to establish, uphold and project (sub-)systemwide ordering structures and practices-institutions, norms, values and principles-and, as a consequence, to have intended/desired ordering effects. the essential building blocks of a successful ordering agent are summarised in table . let us begin with capacity. this refers to power that is perhaps the most essential concept in the study of international politics (baldwin : ) . it is also one of the most contested ones. this is not the occasion to try to settle these debates; suffice it to say, that a multifaceted conception of power is called for. the key question here pertains to the quantity and quality of power as well as its varying effects when employed in ordering the international. according to waltz ( : ) , great powers require a whole spectrum of attributes: size of population and territory, resource endowment, economic capability, military strength, political stability and competence. traditionally the master variable has been military power, which in an anarchic setting is the ultimate arbiter of conflicts between states. yet it is possible to overstate its relevance, especially in our global and interdependent world (nye ) . other forms of power, ranging from economic to 'soft' and normative, play an increasingly important role (baldwin ; nye ; manners ) . it is also noteworthy that military power does not ensure reliable control over outcomes across the board of relevant issues in contemporary international politics: states can employ strategies that offset military power differentials and they can have meaningful geopolitical effects despite not being fully credible peer competitors. indeed, an actor may have the power to affect the behaviour of others through disruptive forms of power-power to avoid nonpreferred outcomes or simply acting as a spoilerwithout necessarily being fully able to coerce others into adopting a stance preferred by the actor. also, if military power is the hammer, not every issue of consequence in contemporary international politics is a nail. on the contrary, there are plenty and perhaps even increasingly issues in the world today that cannot be solved, nor necessarily even contained with the use of military power. the question of climate change as well as other systemic risks, such as pandemics, is a case in point (goldin and mariathasan ) . the same applies to the disruptive forms of power, as they do not generate collective capacity for problem-solving and governance. therefore, identifying the existence of great power capacity in the abstract is only the first step in the analysis. without the ability to channel, or translate, that capacity into actual influence they remain only resources, void of actual ordering effects (see also sørensen : ). analysing power differentials is imperative, yet it does not tell us anything about the quality of interaction and the nature of consequent contestation and ordering between the great powers. the exercise of power is always an empirical issue: it is an outcome of interaction and it cannot be deducted from power differentials alone, regardless of how significant they may be (arreguin-toft ) . to gain a more nuanced view of the processes at play, one can turn to the english school and the seminal work of hedley bull. he was one of the first theorists who argued that an order is not merely a pattern that grows out of mechanical interaction between like units, but it is an intentional and in the final analysis also inherently social process where the key actors take the lead in generating and upholding a particular conception of order that rests on a set of shared values, norms and institutions (bull ) . for bull, it was indeed the existence of shared institutions, such as balance of power and international law, that formed the basis of what he called table key characteristics of a successful ordering agent in international politics material capability competence, ability to act political willingness to act in an ordering role ideational/ideological foundation for organising principles external appeal/legitimacy of ideas, norms and values promoted 'international society'. but for him the common institutions represented something shared and universal. halliday ( : ) has pointed out how bull's international society is 'communitarian', implying a group with shared values. he also did not address the question of change, but adopted a rather conservative stance, advocating relative perpetuity in the constitution of the international society (see friedner parrat ). in bull's reading, norms, values and institutions were static and, by and large, seen at the same time as both prerequisites for and in their operation beneficial to the existence of an international society. the point is not that bull was entirely off the mark, however. international order always serves the interests of the wider international society, but does so unevenly. it is important to bear in mind anna leander's ( : ) insightful words that an order is never neutral: it always works to the advantage of some and to the (at least potential) disadvantage of others, inviting us to ask whose international order we are talking about (see also williams : ) . in this interpretation, an international order does not only serve certain useful functions for the society at large, but it also reflects the interests and ideas of certain key players of that society. therefore, an international order is never neutral, nor is it necessarily an entirely voluntary exercise on the part of all its members: a certain amount of co-optation and coercion, at times perhaps even domination is to be expected. the stance taken here is that when it comes to the question of order in international politics, norms, practices and institutions should be viewed as sites of contestation and struggle: in the final analysis, it is their content that reflects both the power and the ideas of those who are able to affect change in them (wiener ; see also ikenberry and nexon ; wivel and paul ). in this reading, a choice between power and ideas is a false dichotomy: it is not either ideas or power but both that will be required to make sense of any given international order (sørensen ) . although at times a single actor-or a group of actors acting in concert-may be powerful enough to dictate the rules of the game on others, brute strength alone is usually not enough to recalibrate international order. all these point towards the role of intentional agency. order does not change by itself, but it is changed by wilful actors who are able to affect that change. nor does an order sustain itself on its own, but it requires agents that seek to uphold it. this refers to the third variable in our definition, namely the willingness to act as an ordering agent and to carry the associated burden. here, ikenberry's ( ) treatment of international order as a process where the leading state(s) has sought to lock other states into a certain set of institutionalised practices is a useful starting point. in addition, order has always entailed a viable domestic model and the projection of its essential elements beyond borders to generate that very order. this is the case in two different respects. firstly, a viable domestic model is a prerequisite for effective agency: you can only function as an ordering agent internationally if you first function to a satisfactory degree internally. secondly, the reconstitution of an international order must also rely on legitimacy: of the actors involved and the normative foundation promoted (cf. hoffmann : - ; philpott : - ) . in the words of georg sørensen ( : ), 'a stable and legitimate order is founded on a fit among a power base…, a common collective image of order expressed in values and norms, and an appropriate set of institutions'. the last conceptual issue that needs to be discussed in this context deals with the relationship between great power politics and polarity, i.e. the number of great powers in any given international system (waltz : ) . power is obviously the key consideration in deciphering the number of great power poles: to count as one, their relative capabilities must be roughly commensurate across all domains with the most powerful state in the world. according to kegley and raymond ( : ) in a bi-/multipolar system, great powers must be near, but not necessarily absolute equals. the number of poles has varied historically, but in general multipolarity-the existence of more than two poles in the system-has been the norm (see kegley and raymond ) . indeed, the post-world war ii eras stand out in a sense that they witnessed, first, a period of intense rivalry between two superpowers locked into a bipolar setting during the cold war, followed by the period of unipolarity with the usa enjoying unrivalled primacy and hegemony during the post-cold war era (monteiro ; brooks and wohlforth ) . see table for a summary of great powers in europe during the modern era. table shows how although polarity has varied historically, the issue of nonpolarity has not arisen during the modern era. at first sight, the whole notion of nonpolarity might seem odd: surely we are not anticipating the wholesale collapse of the state-centric international system and the disappearance of great powers with it. to better understand how nonpolarity in the european context might be possible and perhaps even likely, we must examine the question of polarity in light of our definition of ordering agents. in its established usage, the relationship between great powers and polarity is almost tautological-great powers are poles and vice versa. but for the present purposes this is not analytically very useful. instead, the article contends that polarity in its traditional reading is a bird's-eye view of international politics based on resources and potential, whereas a more useful analytical lens would take into account the other aspects of agency, namely the ability and willingness to act as an ordering agent in the sense defined above. this entails that to count as a pole, the agent in question must be in possession of a host of attributes: a material power base, ideational foundation and perceived external legitimacy to begin with, but also a political variable concerning the essential competence to act as an ordering agent in international politics and willingness to carry the associated burdens. in light of this discussion, we may define nonpolarity as the lack of great powers that are capable, able and willing to act as ordering agents in a given international system or its sub-system. the term nonpolarity was introduced by richard n. haass who in his article written for foreign affairs over a decade ago defined it loosely as an 'international system… characterised by [too] numerous centres with meaningful (haass ; see also bremmer who spoke about a 'g- world'). yet the use of the term in this article differs from haass's conception in two respects. first, it is regional in scope, drawing attention to europe instead of overall global politics. second, it does not assume that power has drifted away to or been diffused by a host of nonstate actors to a degree that would account for nonpolarity. on the contrary, the article argues that the potential period of nonpolarity stems from changes that are taking place within the key actors at play: their indigenous capacity and willingness to act as ordering agents are atrophying. in addition, nonpolarity is also an outcome generated by the current interaction and growing contestation between the main contenders for an ordering role. in short, nonpolarity is a situation where great powers are either incapable or unwilling, either alone or in concert, to apply power to successfully create or uphold an international order in europe. the dissolution of the soviet union and the end of the cold war division opened (geo)political space in europe. the rigid bipolar confrontation gave way to a much more fluid setting where fresh opportunities and challenges rapidly mushroomed. this called for new policies on the part of both the usa and the emerging eu. for the usa, the four main objectives were: ( ) managing the transition to a new post-cold war order in a peaceful and orderly fashion; ( ) facilitating the emergence of russia as a successor state of the soviet union as a responsible and constructive player, including the development of cooperative threat reduction with russia to deal with the toxic assets left behind by the soviet union; ( ) ensuring the primacy of nato-and by extension also the usa-in european security, and downplaying the eu's potential to emerge as a fully independent security actor; while ( ) using that very eu as a proxy to organise political and economic integration and consequent transition in the emerging wider europe. (for a discussion concerning the key tenets of us policy, see goldgeier and mcfaul ; talbott ; stent .) the usa took the lead in ordering the post-cold war setting along liberal lines, first in europe and then increasingly also globally (mearsheimer ) . for quarter of a century, the usa was able to secure an unrivalled position at the top of international hierarchy. it was also successful in stabilising the conflicts in europe during the s while locking most of the continent into its preferred security structure through the expansion of nato (mastanduno ) . even if russia made some dissatisfied noises at the time, there was an expectation that it, too, could be successfully placated by offering moscow some privileged forms of partnership with the west and the usa in particular (asmus ) . the eu's rise to prominence dovetailed these developments. in the early s, it started to express ambitions and develop capacities for indigenous views about international affairs and to develop them into policies and actions on the world stage. the adoption of the common foreign and security policy (cfsp) in the maastricht treaty of was of particular significance, as it created fresh instruments for external action and institutionalised a cooperative culture that over time have resulted in impressive, although perhaps needlessly cumbersome, finesse and complexity in today's eu (see keukeleire and delreux ) . in the process, the eu was effectively thrust into assuming a leading role in responding to the economic effects of the dissolving soviet empire. the objectives of the nascent 'european foreign policy' and those of the usa were largely compatible and even complementary to each other. one way to characterise the role the two played in ordering europe is to think of 'matryoshka hegemony' where the us global primacy and regionally preponderant security role through nato set the broad liberal framework within which the eu took the lead in ordering the european continent through the expansion and outward projection of its policies, norms and values (haukkala ) . consequently, the usa and the eu, working in tandem, met practically all the requirements for a successful ordering agent in europe: they enjoyed unrivalled material preponderance while portraying both competence and political willingness to act as ordering agents. they also promoted a coherent set of idea(l)s and did so during a period when their leading role enjoyed a great deal of external legitimacy, reflected in the long queues of accession candidates in front of both nato's and the eu's doors. having established this, one should exercise some caution in assigning too much strategic intentionality on the part of the west. both nato's and the eu's eventual sprung nach osten were more reactions and responses to events and demands beyond the west's control or initial appetite rather than preconceived programmes to order or subjugate the whole continent to their will (see asmus ; smith ; hill ) . the hegemonic underpinnings of their ordering role were arrived at in a piecemeal, almost haphazard manner. this gives the western unipolar hegemony over europe a paradoxical quality. to a degree, this also explains why it fizzled out so quickly in the face of growing russian resistance. for quite some time, russia remained an outlier to the western attempts at ordering europe. it was not fully in nor fully out, while it was trying to figure out its own national interests and international identity. in the process, russia has developed an increasingly strained relationship with both the usa and the eu and adopted a highly belligerent approach towards the concentric order they have been propagating. for the russians, the crux of the issue was that it lacked a voice and a role that would have been commensurate with its own self-image as a great power. one way to interpret the events during the last quarter century in europe is to view it as a process of increasing frictions and tensions between the western nato/eu-centric attempts at ordering the continent and russia's growing frustration and even hostility towards its inability to secure a place for itself that would have allowed it a voice and a veto (marten ) . in the words of hill ( ) , in the eyes of moscow the european architecture has left 'no place for russia'. related to this is the wider russian complaint concerning the role the usa has played globally. the us post-cold war primacy in general and the way washington responded to the / terrorist strikes in accentuated the russian impression of a rampant usa bent on dominating the world unilaterally. in vladimir putin's resentful words, uttered already in , the usa had 'overstepped its national borders in every way' (putin ) . positioning itself as a counterforce to a reckless and domineering washington became the leitmotif of putin's rhetoric and russian foreign policy well before the dramatic events in ukraine. although not felt as keenly at the time, the eu enlargement also created its own frictions between russia and the west. in particular, the question of a 'common/ shared neighbourhood' created in the aftermath of the 'big bang' eastern enlargement of proved to be a source of tensions. ukraine's orange revolution-which took both the west and russia equally by surprise-changed moscow's tack concerning the eu's role in the region. moscow's previous benign neglect subsided, as it began to view the eu's growing role and the western orientation of the cis countries with increasing suspicion (gretskiy et al. ) . although it was not appreciated at the time, the orange revolution was the starting gun for the preparation of operations and practices witnessed first in georgia in and then in crimea and eastern ukraine since (franke ) . over time russia's grudging acquiescence to western liberal hegemony morphed into outright opposition. yet the current conflict between russia and the west was far from inevitable. on the contrary, in early s both parties were still looking for ways to make the relationship work. (for accounts that testify to this effect, see forsberg and haukkala ; stent .) these repeated attempts at 'resets' and other fresh beginnings were marred by the underlying and largely irreconcilable tension where the western unipolarity, however, well intended, ran counter to the growing russian calls for a more equitable and essentially bipolar setting in europe. by the end of s, russia started to take much more assertive steps to promote its preferred vision of order beyond its borders. in the first instance, this took the form of the eurasian economic community (eurasec) that soon became the eurasian economic union (eaeu) through which russia started to invest in a more institutionalised bipolar setting in europe, with moscow as the leading power in the other half of the continent with the expectation of attracting the majority, if not all, of the post-soviet states under russia's leadership (see dragneva and wolczuk ) . although the eaeu did gain some initial momentum, as exemplified by the expansion of its members from the original three to current five, the fact that russia felt compelled to resort to open blackmail and coercion to attract new members to the eaeu speaks volumes about the power of attraction of the new regional bloc. the conflict in ukraine was the culmination of these unhappy trends (haukkala ) . with its actions, russia made abundantly clear that it views eastern europe as its primary sphere of interests (trenin ) that both the eu and nato must respect and that it is willing to use all the means at its disposal to enforce this policy. russia also signalled its readiness to pay a high price in terms of economic hardship and international, although mainly western, opprobrium for doing so (menon and rumer ; wilson ) . the reasons for this are myriad, but the main point worth stressing is that the near-existential nature of russian interests in and over eastern europe creates an asymmetry that is unfavourable to the west: no matter how hard it pushes its version of order on the east, moscow is always willing to push back a little harder-and accept the eventual price for doing so. in other words, russia has been stable, able and ruthless enough to act as a spoiler in europe, but, apart from the eaeu that contains severe internal tensions and contradictions, as exemplified by the catastrophic outcome of trying to co-opt ukraine to join the organisation in - testified, it is not competent, capable or even willing to act as an ordering agent in europe. on the contrary, the dramatic escalation in ukraine in has been followed by a pattern where russia has proved ruthless in the application of power to challenge and to a degree overturn the current order on the regional level. admittedly, compared with the west russia is lacking in many of the key attributes of power. its population is stagnating and its economy is weak and almost entirely reliant on the sale of hydrocarbons on increasingly turbulent world markets. yet what it may suffer in the aggregate, it amply makes up in the skill and will to employ the wherewithal at its disposal (see baldwin : ) . when this is factored in, russia's record of accomplishment, and indeed short-to midterm potential, looks more promising. russia has also shown great acumen in projecting military means to achieve political ends beyond its borders both in its immediate vicinity in ukraine and even beyond as the events in syria have shown. this continued contestation is also a factor that feeds into the potential emergence of nonpolarity in europe, discussed next. to a degree, the above narrative is compatible with traditional forms of ordering in international politics. it is possible that the process we are currently witnessing is merely 'normal' turbulence associated with a period of intense contestation that will be followed by the creation of a new or at least amended order in europe (gilpin ). yet in recent years a myriad of factors and developments have warranted asking the question whether the notion of nonpolarity is in fact more applicable. by examining the factors outlined in the definition of an above ordering agent, we can see how the main contenders in europe portray varying drivers for the rapid decline in the current order and augur the possible emergence of nonpolarity on the continent. to begin with, one must note how the unified, even if concentric, western hegemony in europe is rapidly unravelling. both the usa and the european union portray much less cohesion and ability to act in tandem as successful ordering agents than previously. under trump, the transatlantic partnership has experienced growing strains, even signs of serious erosion. (the process is documented in brookings .) the trump administration views the eu and its institutions with hostility and its european allies with growing suspicion. over time, the europeans, too, have started to lose faith in their partnership with the americans. the relations between the eu and the usa have atrophied with no meaningful dialogue or interaction currently taking place between brussels and washington. consequently, the ability of the two to coordinate their activities and channel their respective energies in the same direction has been lost. as a result, it is difficult to talk about western hegemony in europe anymore. what is more, the whole idea of a unified 'west' is increasingly being questioned on both shores of the atlantic (kimmage ; tcherneva ) . this is a development that affects the european order negatively and has a major impact on the evolution of relations on the global level as well. if the two former hegemons have found it hard to work together, the same applies to their roles individually in europe. in the us case, the issue is not the wholesale atrophy in the capacity to act as an ordering agent. no one is suggesting that in terms of overall capacity or competence the usa is completely lacking or that it lacks economic or military power. but what it increasingly is lacking, is the ability, through the erosion of perceived legitimacy and the lack of essential willingness to uphold the current order globally and in europe. this is due to the political variable that stems from the current period of soul-searching revolving around the perceived need to recalibrate the us global role and posture in light of the new era of 'great power competition' that is seen as being in the offing. the trump presidency has accentuated and aggravated these trends, but he is not the root cause behind them. kagan ( ) might be right in arguing that trump has struck a stake through the heart of the liberal world order, but the problem is not just him. the us role would be changing in any case. in the short term, a lot will depend on the presidential elections in november . but even if trump is unseated, it will most probably affect mainly the mood and atmospherics in transatlantic relations. that can be significant, but will not undo nor necessarily even repair the damage that stems from deep-seated structural factors and tensions between the usa and the eu (polyakova and haddad ) . turning to the eu, we find it increasingly isolated, alone and adrift, essentially clinging to the vestiges of its preferred order globally and regionally without the necessary means to project or protect that very order. the fact that the eu does not seriously register as a military power would seem to exclude from the list of possible poles, yet it would be erroneous to discount it as an ordering agent. economically the eu is still in a very strong position, even if the problems of governing the european common currency cast a shadow on its prospects. in addition, waltz's ( : ) verdict concerning the europeans two decades ago seems to hold: the eu still lacks the organisational ability and the collective political will to translate its sizable power potential into credible international actorness. moreover, there are mounting signs that the eu is no longer acknowledged as a key player in global affairs, if indeed it ever was. on the contrary, european perspectives are increasingly sidelined in global politics. increasingly, the eu is not the actor, but the stage upon which others act. the eu's inability to project order, or at least stymie growing instability, is most visible around its own borders. the european neighbourhood policy (enp) that was meant to result in a 'ring of friends' around the eu effectively collapsed in the ukraine conflict. the aftermath of the so-called arab spring resulted in the tragic civil war in syria and completed the current outcome that the economist dubbed a 'ring of fire' around the union (haukkala ) . none of this to argue that these developments are primarily the eu's fault-but to act as a successful ordering agent, one needs to be able to project stability and not just protest instability around you. internally the eu's situation is equally precarious. during the last decade, it has repeatedly succumbed into crises that have sapped its energies and legitimacy even in the eyes of its own citizens (wivel and waever ) . at times, it seems as if the europeans are facing growing problems in keeping even their own eu house in order, as exemplified by brexit and the growing internal fissures concerning economic governance and the rule of law in europe (krastev ; zielonka ) . it should, however, be pointed out that we are not talking about the potential collapse of the eu itself. yet there exists serious doubt whether and to what extent the eu can keep up its role as an ordering agent even among its own ranks, let alone aspire to a successful ordering role externally. the final contender, russia, demonstrates very few or perhaps not at all signs of playing a constructive ordering role in europe for the moment. on the contrary, it does not even aim to generate order elsewhere than at home and seems busy wielding a geopolitical wrecking ball to hasten the demise of the current order. considering our definition for an ordering agent, russia has fallen short during post-cold war era, and even before, as exemplified by the collapse of the soviet union and its empire. currently it does not have the economic power base to act as an ordering agent nor does it offer any coherent alternative to the order it is criticising. currently moscow's actions do not add up to a positive agenda nor does it seem to aspire to one. consequently, the external appeal and legitimacy of russia's agenda in europe is very limited. taken together, none of these attributes translate into a capacity to act as an ordering agent in the european setting. this discussion is tentatively summarised in table : tentatively, because to assess these findings with full confidence would obviously require more rigorous analysis. that said, it is nevertheless hoped that the thesis has enough prima facie plausibility so that it merits to be taken seriously and is subjected to more rigorous research and analysis in the future. europe has entered a period of relative decline and its role and heft in global politics is rapidly shrinking. there are deep structural forces at play, and the diminution in europe's importance is inevitable. at the same time, these developments are compounded by the fact that the very order undergirding europe's own security and stability is deteriorating. this article argues that this not only is due to normal geopolitical contestation, but is a sign that europe is in danger of losing ordering agents, i.e. great powers capable, able and willing to create or sustain order on the continent. the main driver behind these developments is the changing us power and role in europe. under trump, washington has lost its appetite to act as the bulwark of stability in europe. this has resulted in the ongoing collapse of the 'matryoshka hegemony', the previously complementary concentric us and eu ordering roles in europe. to make matters worse, the eu is not up to the task on its own and, it seems, the final contender for the ordering role in europe, russia, is not even interested in it. in addition, the internal trajectories within both the eu and russia point towards decreasing capacity for successful ordering agency. therefore, it is time to put forward the question whether the conflict between the west and russia is contributing to the relative decline of europe in world politics at large, and whether the unintended consequence of this contestation could be hastening the arrival of a period of nonpolarity in europe, with no power being able nor necessarily even willing to shoulder the responsibility of ordering and providing good stewardship in and over europe. we have no historical track record of nonpolarity in the modern era, yet we can surmise about its possible effects based on conceptual and empirical understandings weak of international politics. it is argued that a period of nonpolarity would pose serious problems, for two reasons. firstly, europe is living in an era of high and constantly rising interaction capacity that creates both demands and challenges in terms of coordination and cooperation (buzan , ) . trade and other flows, both legal and illicit, as well as the increasingly important cyber space all require governance and joint management. secondly, we are living in an era of high complex interdependence (keohane and nye ) that also includes mounting risks and challenges, some of them potentially catastrophic, even on a global scale (ord ) . handling these issues requires order and ordering agents. order is the prerequisite for governance without which the risks inherent in our current world become very difficult, practically impossible to control or contain. the haphazard and uneven response to the covid- coronavirus pandemic is a case in point. these are global trends, but it is argued that in europe these issues are even more pertinent because for the last three decades, and more, europe has generated its own web of densely institutionalised forms of cooperation, rules and regulations that require capable ordering agents for their continued existence. russia might find some of these entanglements too close for comfort, but, it is argued, they, along with the us military overlay, have been instrumental in keeping the peace on the continent. a way to read the current situation is to envisage it as a dialectical process where the western thesis has now been fully countered by the russian antithesis. the synthesis remains still to be seen-but it is not an easy one to locate or arrive at due to the essential incommensurability of current positions. indeed, the crux of the potential for nonpolarity in europe is the fact that currently no one can impose an order anymore, while the sides are deeply divided and unable to agree on a new one. one avenue forward might be a return to some form of a balance of power in europe. yet the discrepancies between the various forms of power are probably too large to allow for that. moreover, the internal dynamics within the main potential ordering agents are such that the situation is unlikely to remain stable enough to allow for a sustainable recalibration of the european setting. on the contrary, a safer prognosis seems to be the continued and perhaps even expanding turbulence in europe. over time, the lack of good stewardship over europe will result in further erosion of norms and institutions, generating growing friction and potential for conflict. this means missed economic opportunities and increasing inability to work successfully together to tackle wider problems facing europe and the world. more worryingly, the hard-won gains of the past years are at risk of being undone. an open conflict between russia and the west cannot be excluded. this should be a cause for concern and rallying call for more responsible policies and politics. the emergence of a nonpolar europe cannot or should not be in anyone's interest. what is more, in the twenty-first century a great power worthy of the name should be held to a higher standard in terms of providing building blocks for stability and order on both the regional and global levels than is currently the case. this is not a new idea: as brown ( ) has argued, great powers, simply due to the virtue of their size and importance, have always been held to a higher standard than other powers. in the final analysis, the advent of possible nonpolarity in europe depends on political choices by the main protagonists. indeed, this article is not a prognosis that nonpolarity in europe is inevitable. three questions in particular stand out that will affect the future developments: ( ) the evolution of the us role globally and in europe; ( ) the eu's ability to overcome its current crises and to develop more robust forms of international actorness; and ( ) the future of russia's conflict with the west as well as the evolution of its appetite to seek more constructive roles and openings in europe. nonpolar europe can still be avoided by political choices and responsible policies by the main actors that foster the return of capable, credible and committed ordering agents in europe. european union and the making of a wider northern europe destined for war: can america and china escape thucydides's trap? how the weak wins wars? a theory of asymmetric conflict opening nato's door. how the alliance remade itself for a new era power analysis and world politics: new trends versus old tendencies paradoxes of power power and international relations every nation for itself: what happens when no one leads the world world out of balance: international relations and the challenge of 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k.; kabra, rajesh; natale, andrea; poe, stacy; saha, sandeep a.; russo, andrea m. title: covid- and cardiac arrhythmias: a global perspective on arrhythmia characteristics and management strategies date: - - journal: j interv card electrophysiol doi: . /s - - - sha: doc_id: cord_uid: vi fb t background: cardiovascular and arrhythmic events have been reported in hospitalized covid- patients. however, arrhythmia manifestations and treatment strategies used in these patients have not been well-described. we sought to better understand the cardiac arrhythmic manifestations and treatment strategies in hospitalized covid- patients through a worldwide cross-sectional survey. methods: the heart rhythm society (hrs) sent an online survey (via surveymonkey) to electrophysiology (ep) professionals (physicians, scientists, and allied professionals) across the globe. the survey was active from march to april , . results: a total of respondents completed the survey with % of respondents from outside the usa, representing countries and continents. of respondents, ( %) reported having covid- -positive patients in their hospital. atrial fibrillation was the most commonly reported tachyarrhythmia whereas severe sinus bradycardia and complete heart block were the most common bradyarrhythmias. ventricular tachycardia/ventricular fibrillation arrest and pulseless electrical activity were reported by . % and . % of respondents, respectively. there were of ( . %) respondents who reported using anticoagulation therapy in all covid- -positive patients who did not otherwise have an indication. one hundred fifty-five of ( %) reported regular use of hydroxychloroquine/chloroquine (hcq) + azithromycin (azm); concomitant use of azm was more common in the usa. sixty of respondents ( . %) reported having to discontinue therapy with hcq + azm due to significant qtc prolongation and ( . %) reported cases of torsade de pointes in patients on hcq/chloroquine and azm. amiodarone was the most common antiarrhythmic drug used for ventricular arrhythmia management. conclusions: in this global survey of > ep professionals regarding hospitalized covid- patients, a variety of arrhythmic manifestations were observed, ranging from benign to potentially life-threatening. observed adverse events related to use of hcq + azm included prolonged qtc requiring drug discontinuation as well as torsade de pointes. large prospective studies to better define arrhythmic manifestations as well as the safety of treatment strategies in covid- patients are warranted. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. the novel coronavirus (sars-cov- ) and the resulting respiratory tract infection (coronavirus disease or covid- ) is a pandemic with over , , cases globally, resulting in , deaths at the time of this writing [ , ] . following initial reports in wuhan, china, viral progression culminated in over , cases in china during january/february [ , ] . the subsequent global spread has involved more than countries [ ] . the usa has reported > . million confirmed cases and over , deaths, the highest in the world [ ] . as this global pandemic continues to rage, cardiovascular, especially arrhythmic, manifestations associated with covid- have become evident [ ] [ ] [ ] . a recent report from wuhan, china, noted that . % of hospitalized and . % of icu patients with covid- had arrhythmias [ ] . although arrhythmias appear to be common in hospitalized covid- patients, arrhythmia mechanisms and characteristics as well as antiarrhythmic therapies and their outcomes have not been welldefined. to better understand the cardiac arrhythmic manifestations and treatment strategies employed in hospitalized covid- patients through a worldwide cross-sectional survey of arrhythmia professionals. the heart rhythm society (hrs) conducted a global survey that was developed by the hrs communications committee with input from the hrs covid- rapid response task force. the survey was active from march , , to april , . the audience for the survey was electrophysiology (ep) professionals (physicians, nurse practitioners, physician assistants, nurses, ep lab technicians, scientists, and other allied professionals) across the globe. the survey consisted of questions (supplemental appendix). demographic questions included primary occupation, practice setting, and practice location (state/province and country). the goal of the survey was to understand the cumulative experience as well as the variability in incidence and management strategies of arrhythmias associated with covid- . the survey was administered using surveymonkey (surveymonkey, palo alto, ca, usa), and the survey link was disseminated to the hrs membership through a dedicated email, keeping pace weekly email, and also through the covid- webpage on the hrs website and hrs member open forum. additionally, it was disseminated to other arrhythmia societies across the world through email to their leadership and to the larger arrhythmia community through social media (twitter, facebook, and linkedin). the first respondents were all hrs members who received the survey link through a dedicated email. the subsequent respondents represented a combination of hrs members as well as self-identified respondents who received the survey link either from their respective arrhythmia societies or through social media channels. continuous variables are reported as mean ± standard deviation or median (interquartile range). categorical variables are reported as frequency and percentages. student's t test, or mann-whitney u test, was used to compare continuous variables, and categorical variables were analyzed using χ tests. all tests are two-tailed and a p value < . indicates statistical significance. statistical analysis of the responses was performed using statistica . (tibco software, palo alto, ca). a total of respondents completed the survey. seventyfour percent of the respondents were physicians and % were allied professionals. twenty-six percent were in academic practice, whereas % and % were in private and hospital-based practices, respectively. fifty percent of respondents were from outside the usa and represented countries and six continents. practice locations of us respondents represented states. demographic characteristics are detailed in table . of the respondents, ( %) reported having covid- -positive patients in their hospital. for those who reported hospitalized covid- , the reported total number of hospitalized covid patients at the time of the survey was , , with a mean and median number of patients of . ± . and (interquartile range, - ), respectively. of the respondents who reported at least one covid- patient in their hospital, . % had < patients, % had - patients, . % had - patients, . % had - patients, and . % had ≥ patients. a variety of supraventricular and ventricular arrhythmias were reported in covid- patients. of the respondents, ( %) reported cases of atrial fibrillation, ( . %) reported atrial flutter, ( . %) reported sustained atrial tachycardia, and ( . %) reported paroxysmal supraventricular tachycardia. among ventricular arrhythmias, frequent monomorphic premature ventricular contractions were reported by ( . %) respondents, multimorphic premature ventricular contractions by ( . %), and non-sustained ventricular tachycardia (vt) by ( . %). sustained monomorphic vt was reported by ( . %), polymorphic vt/torsade de pointes by ( . %), vt/ventricular fibrillation (vf) arrest by ( . %), and pulseless electrical activity by ( . %) respondents respectively ( fig. and table ). of respondents, ( %) reported significant sinus bradycardia, ( %) reported complete heart block, ( . %) reported first-or second-degree av block, and ( . %) reported bundle branch block or intraventricular conduction delay in covid- patients ( fig. and table ). one hundred and forty of ( . %) respondents reported using empiric anticoagulation therapy in all covid- positive patients who did not have an indication otherwise. of those who used empiric anticoagulation, of ( %) reported using intravenous heparin or subcutaneous low molecular weight heparin, ( %) reported using novel oral anticoagulants, and ( %) reported using warfarin. reported use of empiric intravenous heparin or subcutaneous low molecular weight heparin was more common outside the usa ( . % vs . %, p = . ). of respondents, ( . %) reported acute pericarditis; small pericardial effusion was reported by ( . %), whereas moderate and large pericardial effusions were reported by ( . %) and ( . %) of respondents, respectively. of respondents, ( . %) reported having patients on hydroxychloroquine (hcq)/chloroquine. twenty-seven ( . %) reported using it only in - % of patients, whereas ( . %) reported using it in - % of covid- patients. of respondents, ( %) respondents reported using hcq/chloroquine in combination with azithromycin (azm). thirty-six ( . %) reported using the combination only in - % of patients, whereas ( . %) reported using it in - % of covid- patients. for hcq monotherapy, in the usa, % responded that they had not used hcq in any patients, % had used it in some, but not all, patients, and % reported using it in essentially all patients. outside the usa, % had not used it at all, % had used in some patients, and % reported using hcq in essentially all patients (p = . ). for hcq/azm combination therapy, in the usa, % reported not using at all, % in some, and % in essentially all patients. outside the usa, % of respondents had not used combination therapy at all, % had used in some patients, and only % responded using in essentially all patients (p = . ). based on these results, use of either hcq or hcq in combination with azm appears to be more common in the usa (fig. ) . of respondents, ( %) reported using a qtc monitoring protocol for patients on hcq/chloroquine, with no significant difference between those from the usa versus outside the usa ( % vs %, p = . ). among individuals using a qtc monitoring protocol in the usa, % were in academic practice whereas outside the usa, only % of those using a qtc monitoring protocol were in academic practice (p = . ). twenty percent of respondents reported using magnesium supplementation in all patients on hcq/chloroquine. qtc prolongation ≥ ms (≥ ms with qrs duration > ms) was reported by of respondents ( %) ( table ). sixty ( . %) of respondents reported having to discontinue combination therapy with hcq/chloroquine and azm due to significant qtc prolongation. twenty ( . %) respondents reported cases of torsade de pointes in patients on hcq/chloroquine and azm. prophylactic amiodarone use was rare (reported by only ( . %) of respondents). in covid- patients with ventricular arrhythmias, ( %) of the respondents reported not using any class i, ii, or iii antiarrhythmic agents whereas ( %) used amiodarone and ( . %) used lidocaine/mexiletine. sotalol and dofetilide use was infrequent, reported by ( %) and ( . %) of survey respondents, respectively. the major findings of this global survey include the following: (a) in hospitalized covid- patients, ep professionals across the globe reported a wide variety of arrhythmic manifestations, with several reporting potentially life-threatening ventricular arrhythmias (sustained monomorphic vt, polymorphic vt/torsade de pointes, vt/vf arrest) as well as fig. difference between us and non-us respondents regarding the percentage of hospitalized covid- patients being treated with hcq/chloroquine + azithromycin fig. characteristics of bradyarrhythmias observed in hospitalized covid- patients pulseless electrical activity. (b) atrial fibrillation was the most common cardiac arrhythmia noted in these patients. severe sinus bradycardia and complete heart block were the most common bradyarrhythmias. (c) twenty-two percent of respondents used therapeutic anticoagulation in covid- patients without established indications, with use of intravenous heparin/low molecular weight heparin more prevalent outside the usa. (d) there was wide variation in use of hcq/ chloroquine and azm, with concomitant use of azm more common in the usa. discontinuation of hcq/chloroquine + azm due to qtc prolongation and torsade de pointes was reported by . % and . % of respondents, respectively. (e) amiodarone was the most common antiarrhythmic drug used for managing ventricular arrhythmias. currently, limited information is available regarding arrhythmic manifestations associated with covid- . in one study of patients, . % reported palpitations at presentation [ ] . wang et al., in a single-center retrospective analysis of consecutive patients admitted with covid- in wuhan, china, reported arrhythmias in . % of hospitalized patients, with a much higher incidence ( . %) in those needing intensive care. however, no definition as to what constituted an arrhythmia was provided [ ] . guo et al., in another single-center retrospective study of patients from wuhan, china, evaluated the association of underlying cardiovascular disease and myocardial injury on fatal outcomes in patients with covid- . they noted that % of patients had myocardial injury as evidenced by elevated troponin t (tnt) levels. incidence of vt/vf was . % and increased to . % in patients with elevated tnt [ ] . the reported percentage of vt/vf in our survey is comparable with the data from guo et al. moreover, our survey also provides additional information on pulseless electrical activity (reported by . % respondents), underscoring the potential influence of covid- on life-threatening cardiac arrhythmias and likely pump failure. atrial fibrillation was the most common reported arrhythmia in covid- patients. although we do not have demographic details of patients and do not know how many had de novo versus pre-existing atrial fibrillation, this is not surprising as the majority of sicker covid- patients are older and have underlying comorbidities, predisposing to atrial fibrillation [ ] . multiple mechanisms could lead to the increased incidence of brady-and tachyarrhythmias associated with covd- infection. arrhythmic manifestations could be secondary to direct myocardial inflammation and injury. severe hypoxic lung disease from covid- can trigger atrial arrhythmias. viral infection and associated increased metabolic demand and cytokine activation can trigger atrial and ventricular arrhythmias in patients who develop acute myocarditis or inflammatory response and in those with underlying coronary or other structural heart diseases. our data show that % of respondents are using anticoagulation, either oral, subcutaneous, or intravenous, in patients who did not otherwise have an indication for anticoagulation. this highlights the concern regarding the prothrombotic potential of covid- . the relatively common presence of atrial fibrillation further raises the need to address anticoagulation. these aspects as well as duration of anticoagulation following recovery from covid- require further study. at the time of this writing, except for the emergency use authorization of remdesivir, no other fda-approved treatments are available for covid- . there has been great interest in hcq/chloroquine ± azm, for inpatient treatment of covid- ; however, available data have been conflicting and randomized studies are lacking [ ] [ ] [ ] . the combination, however, poses a significant risk of qtc prolongation and torsade de pointes [ ] . a randomized, double-blind, currently non-peer-reviewed study from brazil assigned patients to a low-and high-dose chloroquine regimen; all patients received ceftriaxone and azm. at -day follow-up, . % had a qtc > ms ( . % in low-dose and . % in high-dose arm (p = . )). two out of patients ( . %) had vt from qtc prolongation [ ] . in a retrospective study of patients given hcq + azm, % had a qtc > ms [ ] . information from this survey closely mirrors data from these recent studies [ , ] and shows that the risk for arrhythmic adverse events is not inconsequential, and suggests a cautious approach and close monitoring of qtc when using these, yet to be proven, therapies. this study has several limitations. the findings represent cross-sectional data from a survey completed by arrhythmia professionals. given that the survey was disseminated to the global arrhythmia community, it is difficult to assess a response rate. the opinions of the survey respondents may not fully represent the entire ep community and may not represent all practitioners who care for covid- patients. the survey findings are subject to recall bias as respondents may tend to remember the sicker patients. reports of arrhythmias are from ep professionals, likely representing selection bias for sicker covid- patients at a higher risk for developing arrhythmias. since the survey was disseminated to ep professionals and not to institutions, it is possible that respondents working in the same institution may be referencing the same patient or group of patients. although a thorough review of the data did not reveal any duplicate responses, the fact that the survey was also disseminated through social media, it is not possible for us to be absolutely sure that only one response was obtained per respondent. the information on various arrhythmias presented here may not represent the accurate incidence of arrhythmias in the covid- population, but simply serve to provide a broad overview of arrhythmic manifestations and therapeutic strategies employed by ep professionals across the globe. the way the survey questions were worded, it was not possible to determine whether a reported brady-or tachyarrhythmia was de novo or pre-existing. this survey did not assess for comorbid conditions or the level of medical care the covid- patients were receiving (regular ward vs intensive care, ventilator use); hence, no association could be made between underlying conditions or severity of illness and arrhythmic manifestations. with regard to empiric subcutaneous or intravenous anticoagulation, although the intent was to assess the use of therapeutic doses, the wording of the survey question may not distinguish between prophylactic and therapeutic use. these results should be confirmed by future prospective studies or registries. in this global survey of data from > ep professionals regarding hospitalized covid- patients, a variety of arrhythmic manifestations were observed, ranging from benign to life-threatening. observed adverse events related to use of hcq + azm included prolonged qtc requiring drug discontinuation as well as torsade de pointes. these findings underscore the need for large, prospective studies to better define arrhythmic manifestations as well as the safety and efficacy of treatment strategies in covid- patients. covid- interactive map a pneumonia outbreak associated with a new coronavirus of probable bat origin interim clinical guidance for management of patients with confirmed novel coronavirus ( -ncov) infection association of cardiac injury with mortality in hospitalized patients with covid- in wuhan, china clinical characteristics of hospitalized patients with novel coronavirus-infected pneumonia in wuhan, china cardiac and arrhythmic complications in patients with covid- clinical characteristics of novel coronavirus cases in tertiary hospitals in hubei province cardiovascular implications of fatal outcomes of patients with coronavirus disease (covid- ) case-fatality rate and characteristics of patients dying in relation to covid- in italy breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid- associated pneumonia in clinical studies in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus (sars-cov- ) no evidence of rapid antiviral clearance or clinical benefit with the combination of hydroxychloroquine and azithromycin in patients with severe covid- infection chloroquine diphosphate in two different dosages as adjunctive therapy of hospitalized patients with severe respiratory syndrome in the context of coronavirus (sars-cov- ) infection: preliminary safety results of a randomized, double-blinded, phase iib clinical trial (clorocovid- study) the qt interval in patients with covid- treated with hydroxychloroquine and azithromycin publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations acknowledgments the authors would like to convey their sincere appreciation to all the arrhythmia professionals who shared their experience by completing this survey as well to the heart rhythm society for supporting this endeavor. key: cord- -l heb or authors: nagarajan, kiran koushik; bali, atul; malayala, srikrishna varun; adhikari, ramesh title: prevalence of us-trained international medical graduates (img) physicians awaiting permanent residency: a quantitative analysis date: - - journal: nan doi: . / . . sha: doc_id: cord_uid: l heb or background: international medical graduates (imgs) contribute to about % of the physician workforce in the usa. certain us-trained imgs face long wait times for transitioning to a permanent resident status, which limits their ability to work to fullest capacity, especially during a public health emergency. objectives: to estimate the number of us-trained imgs awaiting permanent residency. study design: data were obtained from national residency matching program (nrmp) to quantify the number of imgs who secured residency training in the us from to . estimates of physician demographics were based on nrmp/ecfmg match data and federation of state medical boards (fsmb) physician census data. results: between and , a total of , non-us imgs who were not us citizens successfully matched to residency training programs. applicants from india and china were noted to be impacted by delays in adjustment to permanent resident status. per our estimate, there are between , and , us-trained physicians from china currently awaiting permanent residency, with applicants waiting since october , and between , and , us-trained physicians from india currently awaiting permanent residency, with applicants waiting since june . conclusions: the total number of us-trained immigrant physicians in active practice awaiting permanent residency to the usa is estimated to be ranging between , and , . each year, multiple physicians from all over the world attempt to secure residency training in the usa [ ] . physicians who graduate from international medical schools are called international medical graduates (imgs) [ ] . many of these physicians require visa sponsorship for legal entry into the usa (us) to initiate their residency training [ ] . after completion of their training, they become eligible to apply for a permanent resident status in the usa, often colloquially referred to as a 'green card' [ ] . however, due to the limited annual quota of green cards, which are allocated based on the country of birth of the applicant, the wait times can vary widely among applicants. due to the sheer number of applicants from countries like india and china, expectedly higher from the two most populous countries in the world, these applicants have the longest wait times before they become eligible to adjust their status to that of a permanent resident [ ] . while every applicant seeking an employment-based immigrant visa must demonstrate that they possess specific skills, advanced degrees or extraordinary abilities for their eligibility, physicians constitute a small but particularly highly trained subset of these applicants. notably, these physicians are perhaps the few immigrants where federal tax dollars have already been utilized for their training, as they navigate through years of grueling residency training, often followed by even more specialized fellowship training. given the resource-intensive process of training a physician, there are several legislative programs in places such as the appalachian regional commission (arc) program and the conrad program, with the primary purpose of retaining these assets to serve areas of the usa with inadequate physician access [ ] . furthermore, if these highly trained physicians are unencumbered by restrictions imposed by their visa status, they could be mobilized to areas of higher need. this flexibility could prove crucial amidst a public health crisis, such as the covid- pandemic [ ] . therefore, it is imperative to quantify the number of immigrant physicians who are awaiting permanent residency status due to the reasons mentioned above. to the best of our knowledge, the usa citizenship and immigration services (uscis) does not publish or track profession-specific data pertaining to applicants with an approved i- (immigrant petition for alien workers), who have not yet become eligible for applying for an adjustment of status [ ] . in the absence of this information, one can only estimate the number of immigrant physicians currently awaiting permanent residency status. for this reason, we have attempted to quantify the prevalence of ustrained immigrant physicians awaiting permanent residency. we formulated a stepwise approach to the problem, by first quantifying the number of non-us imgs (international medical graduates who were not us citizens), who had sought residency training in the usa, from until . residency training in the usa has a minimum duration of years, with some physicians pursuing subsequent fellowship training of minimum years duration, only after completion of which would the physician start working in the capacity of an attending physician and become eligible to apply for permanent residency [ ] . most physicians qualify for the 'eb- ' immigrant visa category, which specifies that the applicants must be 'professionals holding advanced degrees and persons of exceptional ability' [ ] . as of may , based on data from the us department of state, the subset of eb- applicants most impacted by delays in transition to permanent residency had applied in july and onwards [ ] . to account for years of training as elucidated above, we chose as the earliest data point for the sake of our analysis. the national residency matching program (nrmp) is a private, non-profit organization that utilizes a sophisticated algorithm to allow medical students to be matched with their preferred residency training programs[ ]. with rare exceptions, the overwhelming majority of applicants utilize the nrmp match to secure a residency training position. we collected data from the nrmp residency match data and reports to determine the cumulative number of non-us imgs who had successfully matched for each year since [ ] [ ] [ ] [ ] [ ] . determining the country of birth of practicing physicians posed a unique challenge, as this demographic information is not evaluated by agencies tracking data pertaining to the us physician workforce. while there are exceptions, most individuals complete their medical education prior to residency in their countries of birth. therefore, we used the country where the physician completed their medical school graduation as a surrogate for their country of birth. based on data from the biennial census of physicians with active licenses conducted by the federation of state medical boards (fsmb), we determined the percentage of practicing physicians who completed their medical school graduation from various countries [ ] . these numbers were used to get an estimate of physicians who had likely applied for immigrant petitions currently practicing in the usa but were awaiting permanent residency. the cumulative number of non-us imgs who had successfully matched for residency training from to was , [ ] [ ] [ ] [ ] [ ] . based on the may [ ] visa bulletin released by the us department of state, all eb- applicants with an approved i- were eligible to adjust their status, other than those who were born in india and china [ ] . thus it was important to determine the percentage of non-us imgs from these countries who had secured residency training since . the fsmb census data suggested that while the proportion of physicians from china was relatively small and was not quantified, the majority of licensed imgs that were currently in practice in the us had graduated from medical schools in india, with their numbers being estimated at , ( %) (figure ) [ ] . upon review of prior biennial census data from the fsmb, this percentage of imgs from india has remained remarkably stable over several years. furthermore, match data obtained by a joint effort from the nrmp and the educational council for foreign medical graduates (ecfmg) in , also obtained every applicant's citizenship at birth. review of these demographic data suggested that . % ( / ) of matched applicants were indian citizens at birth. therefore, given the stability of this percentage despite the influx of medical graduates each year, we operated under the assumption that the composition of imgs matching to us residency programs was similar to fsmb census data. we thereby estimate approximately between , and , us-trained physicians from india currently awaiting permanent residency, with some applicants waiting since june (figure ). since the fsmb census data did not assign a definitive number of us-trained chinese physicians, we revisited the nrmp-ecfmg match data and noted that . % ( / ) were chinese citizens at birth (figure ) [ ] . additionally, we utilized data from a study published by duvivier et al. in april [ ] . the authors procured data from the american medical association physician masterfile, noting that the total number of chinese-educated physicians had risen modestly from , in , to , in . additionally, they noted that as compared to data where physicians were in residency training, only physicians were in residency training positions in . they inferred that there had been a relative decline in the influx of physicians who completed their medical school graduation in china. according to data from the may visa bulletin, eb- applicants from china with an approved i- from october are eligible for adjustment of status to permanent residency [ ] . assuming the modest rate of influx of chinese physicians as noted in the duvivier study, with a relatively higher rate noted in the nrmp-ecfmg data, there would be approximately between , and , us-trained physicians from china currently awaiting permanent residency, with some applicants waiting since october . combining the estimates listed above, the total number of us-trained foreign physicians in active practice awaiting permanent residency to the usa is estimated to be ranging between , and , . the robust clinical and research experience offered during residency and fellowship training attracts thousands of foreign physicians to the usa each year. this has resulted in nearly a quarter of practicing physicians in the us to be imgs. american medical association (ama) suggests that about . % of the licensed us physicians are imgs. owing to the ever-expanding population and the retirement of the existing physician workforce, the [ ] number of imgs in practice has grown by nearly , since , a . % rise and that figure is bigger than the % rise in us. medical graduates over that same time period [ ] . in a report published by the ama in october , it was noted that % of imgs speak two or more languages fluently, helping patients overcome linguistic and cultural barriers that can impede care. imgs tend to work mostly in the primary care specialties (about %) and that is about double the % of all us. physicians who work in a primary care specialty, helping to meet a critical workforce need. this influx of highly trained immigrants has positively impacted the physician shortages across the country. due to programs redirecting these us-trained foreign physicians such as the arc program and conrad program, these physicians serve in higher proportions in rural and underserved areas, where there are greater african american, hispanic and non-white populations [ ] . this suggests that these actively licensed physicians are caring for the most medically vulnerable americans. in this manner, physician immigration into the usa is mutually beneficial. however, the inability of us-trained immigrant physicians to secure permanent resident status for prolonged periods, in comparison to their peers who trained in other countries, increases physician dissatisfaction and may serve as a disincentive to continue to practice in the usa. furthermore, their visa restrictions prevent them from pursuing additional opportunities such as offering new medical services or hiring additional staff members, which would benefit the communities they serve from both healthcare and economic standpoint. for this reason, it is important to be able to quantify this untapped resource and introduce means to expedite the permanent residency status of these highly skilled immigrants working towards the nation's best interests. there are nearly , us-trained foreign physicians in the usa who are awaiting permanent resident status and are consequently limited in their scope due to visa restrictions. this cohort represents a large pool of us-trained, licensed physicians who are not being utilized to their fullest potential. devising means to expedite their legal immigration would bolster the healthcare workforce and prevent growing physician shortages in the usa, during and even after the ongoing global public health emergency. there are several limitations and potential sources of error that we would like to acknowledge, which limit the precision of our estimates. while the eb- immigrant visa is the pathway that most physicians are eligible for, a few others may have obtained permanent residency via alternative means which greatly reduced wait times, such as the eb- immigrant visa (alien of extraordinary ability) or the eb- immigrant visa (immigrant investor) category. there are personal circumstances which may alter our estimate, such as dual foreign physician households (only one family immigrant petition would be required), foreign physicians married to us citizens, foreign physicians who accepted a residency position outside of the nrmp match, and us-trained foreign physicians who may no longer be in practice (left the us, became disabled, or deceased). these factors are difficult to quantify based on publicly available databases. however, our effort appears to be the only publication attempting a quantitative analysis of this unique problem afflicting the us physician workforce. no potential conflict of interest was reported by the authors. http://orcid.org/ - - - x srikrishna varun malayala http://orcid.org/ - - - ramesh adhikari http://orcid.org/ - - - x about . % of the nrmp match applicants are from india, followed by china, . %. rest of the world constituted about . %. international medical graduates in the us physician workforce international medical graduate (imgs) immigration information for international medical graduates -year wait for indian immigrants with advanced degrees foreign medical graduates: a brief overview of the j- visa waiver program events as they happen, world health organization requirements for foreign medical graduates green card eligibility criteria ] national residency matching program. results and data: main residency match® results and data: main residency match® national resident matching program and educational commission for foreign medical graduates. charting outcomes in the match for international medical graduates main residency match® advance data tables for the main residency match® fsmb census of licensed physicians in the usa the contribution of chinese-educated physicians to health care in the usa how imgs have changed the face of american medicine american immigration council. foreign-trained doctors are critical to serving many u.s. communities key: cord- -q x xb authors: nan title: th icar abstracts: date: - - journal: antiviral res doi: . /j.antiviral. . . sha: doc_id: cord_uid: q x xb nan the society was organized in as a non-profit scientific organization for the purpose of advancing and disseminating knowledge in all areas of antiviral research. to achieve this objective, the society organizes an annual meeting. the society is now in its th year of existence, and has about members representing countries. for membership application forms or further information, please contact dr. amy patick, secretary, isar; pfizer global r&d, department of virology, science center drive, san diego, ca ; phone + ; fax + ; e-mail amy.patick@pfizer.com. membership application forms will also be available at the conference registration desk, or from our website www.isar-icar.com. enzymes of the pol gene of hiv have been identified as important viral targets for the discovery anti-hiv therapeutic agents. while the viral targets, hiv reverse transcriptase and hiv protease, have been successfully investigated for the development of clinically useful therapeutic agents, research efforts on drug discovery on the third enzyme of the pol gene, hiv integrase, have not resulted in a single fda-approved drug. nevertheless, as integrase is essential for hiv replication, it remains an attractive target for the discovery of new anti-hiv agents. in this presentation, we report the discovery of a conceptually new beta-diketo acid, constructed on a nucleobase scaffold, that is a potent inhibitor of both the -processing and strand transfer steps of recombinant hiv integrase. this inhibitor and the positive control compound, azt, were tested in a pbmc cellbased, microtiter anti-hiv assay against the clinical isolate, hiv- teki (nsi phenotype) and hiv- nl - (si phenotype), and in a magi-x assay against hiv- nl - with hela-cd -ltr-beta-gal cells. our integrase inhibitor was found to have highly potent in vitro anti-hiv activity and efficacy. the discovery of this remarkably active molecule, representative of a unique set of diketo acids bearing nucleobase scaffolds, has uncovered a new chapter in the chemistry and biology of integrase inhibitors and their potential therapeutic applications. berta bosch , imma clotet-codina , julia blanco , eduard pauls , gemma coma , samandhy cedeño , francesc mitjans , anuska llano , margarita bofill , bonaventura clotet , jaume piulats , jose este retrovirology laboratory, fundacio irsicaixa, badalona, spain; laboratorio de bioinvestigación, merck farma y química, barcelona, spain macrophages are key cells for hiv infection and spreading inside the organism. macrophages cultured in vitro can be successfully infected after differentiation with cytokines such as macrophage colony stimulating factor (m-csf). in the monocyte to macrophage differentiation process with m-csf, av-integrins are upregulated concomitantly to the capacity of hiv to generate a productive virus infection. in the present study we show that an anti-av antibody, e , inhibited hiv- infection of primary macrophages. the effect of e on r or x hiv- replication in acutely infected macrophages was dose-dependent, with a % effective concentration (ec ) of ± g/ml in the absence of cytotoxicity. similarly, a monoclonal antibody targeting the avb integrin ( d .f ) also inhibited hiv- infection in this cell type. e reduced the detection of hiv- bal proviral dna in acutely infected macrophages but was completely ineffective against hiv- bal production in chronically infected macrophages, suggesting that e inhibited hiv infection at an early stage of the virus cycle. finally, a small molecular weight antagonist of the avb integrin reduced hiv replication at subtoxic concentrations. therefore, our results suggest that av-containing integrins could play a role in hiv replication in macrophages and indicate that small molecular weight compounds may be developed to interfere with hiv replication in macrophages through the interaction with av integrins. andrew vaillant , hong lu , shuwen liu , carol lackman-smith , roger ptak , jean-marc juteau , shibo jiang replicor inc., laval, que., canada; f. lindsay kimball research institute, new york blood center, new york, ny, usa; southern research institute, frederick, md, usa the sequence independent antiviral activity of phosphorothioate oligonucleotides in inhibiting hiv- by blocking interactions between the v loop and cd has been previously described. this activity was attributed to their polyanionic activity. here we show that ps-ons (and their fully -o-methylated derivatives) are also potent inhibitors of hiv- -mediated membrane fusion and hiv- replication in a sequence-independent, sizedependent (optimal size ∼ - bases) and phosphorothioation dependent manner (independent of stabilization). ps-ons interact with the heptad repeat regions of gp and the hiv- fusion inhibitory activity of ps-ons is closely correlated with their ability to bind to these heptad repeats and block gp six-helix bundle formation, a critical step during the process of hiv- fusion with the target cell. the requirement for ps-on interaction was also found to be dependent on phosphorothioation, suggesting that the v loop/ps-on interaction may also have a hydrophobic component. the increased hydrophobicity of longer (≥ base) ps-ons may contribute to their inhibitory activity against hiv- fusion and entry because these longer ps-ons have a greater hydrophobicity and are more potent in blocking the hydrophobic interactions involved in the gp sixhelix bundle formation than shorter ps-ons (< bases). this novel antiviral mechanism of action of long ps-ons has important implications for therapy against infection by hiv- and other enveloped viruses with type i fusion proteins. chris meier , soenke jessel , bastian reichardt , olaf ludek , jan balzarini university of hamburg, institute of organic chemistry, hamburg, germany; rega institute for medical research, katholieke universiteit leuven, leuven, belgium carbocyclic nucleoside analogues like abacavir showed very interesting antiviral properties. therefore, we are interested in a convenient stereoselective access to this class of compounds as potential antiviral agents. by using a new convergent synthetic strategy, starting from a chiral cyclopentenol, enantiomerically pure carbocyclic thymidine (carba-dt) was obtained as a key intermediate for further variations at the -position. this pathway allows an entry to d-and l-configurated nucleoside analogues. however, using this approach a mixture of side products avoids the formation of the product in very high yields. however, we will present that the side products can be recovered by a stereoselective hydroboration leading to one intermediate only that can be use as well for the synthesis of carbocyclic nucleosides. the condensation of the carbocyclic moiety and different pyrimidine and purine nucleobase was achieved by a mitsunobu reaction. various analogues have been prepared via this strategy, e.g. d-and l-carba-bvdu, nucleoside analogues known to be antivirally active against hsv- . additionally, carbocyclic ␣nucleosides and carbocyclic iso-nucleosides are accessible by this reaction sequence. all new nucleoside analogues were tested for their antiviral activity. particularly carba-dt was found to be a potent anti-hiv active derivative showing no toxicity. however, it can not be excluded that a non-activity of a compound is related to a missing phosphorylation to the monophosphate. in order to prove that, all nucleosides were converted into their cyclosal-phosphate trimesters and transferred into the nucleotides. detailed chemistry, enzymatic and antiviral activity data will be presented. in some cases the nucleotide releasing system showed improved antiviral activity as compared to the parent nucleoside. michela pollicita , candace pert , maria-teresa polianova , alessandro ranazzi , michael ruff , carlo-federico perno , stefano aquaro university of rome tor vergata, italy; georgetown university, washington, dc, usa monocytes/macrophages (m/m) are a strategic reservoir of hiv- commonly infected by ccr -using (r ) strains of hiv- . ccr is an attractive target for inhibition of ccr mediated hiv- entry. thus, ccr antagonists are expected to be a power-ful new class of receptor-based therapeutic agents against hiv- infection. d-ala-peptide t-amide (dapta) is an octapeptide derived from the gp v region of hiv- , able to bind ccr . dapta acts as selective viral entry inhibitor, displacing the binding of gp with ccr . dapta anti-hiv- activity was evaluated in m/m infected with two different hiv- r strains, bal and a, in presence of several doses of the compound. dapta inhibited hiv- replication in m/m (> % compared to control), measured by the p gag ag released in the cell culture supernatants, at concentration of - nm. pcr analysis of integrated hiv- proviral dna on cultured m/m proved that dapta is able to block hiv entry and so, to prevent hiv infection in m/m. moreover, the capability of different hiv- r strains produced and released by infected m/m in affecting neuronal homeostasis was assessed in a neuroblastoma cell line, sk-n-sh, expressing ccr . in sk-n-sh were evaluated cell morphology, propidium iodide binding and fluorescenceactivated cell sorting (facs) analysis. dapta, at concentration of - and - nm, strongly inhibited apoptosis in sk-n-sh of and %, respectively, compared to control. unexpectedly, tak- (a nonpeptidic ccr antagonist with potent anti-hiv- activity) inhibited apoptosis only of % compared to control. our results suggest that the development of new anti-hiv- compounds, such as dapta, could be important in synergistic combination with other antiretroviral treatments in prevent both central nervous system hiv-infection and the consequent neural damage. the mechanisms of dapta inhibition may include both suppression of hiv- r strains in the brain as direct inhibition of hiv- replication in m/m and gp related damage by ccr binding. pradimicin a (prm-a) is an antifungal non-peptidic benzonaphtacenequinone antibiotic that specifically inhibits human immunodeficiency virus (hiv) in cell culture. it markedly suppresses a variety of different hiv- clades in pbmcs, in primary macrophages and several hiv- and siv strains in laboratory cell lines (range of % effective concentrations: . - g/ml; % cytostatic concentration: > g/ml). prm-a also inhibits syncytium formation between persistently hiv- -infected hut- cells and uninfected sup t cells. prm-a behaves as an artificial lectin that selectively binds mannose-containing glycans. consequently, biacore experiments revealed that it binds to gp of hiv- /mn in the presence of ca + . prm-a is endowed with a high genetic barrier with regard to drug resistance development against hiv- . a variety of multiple mutations at n-glycosylation sites in hiv- gp are required before the virus looses marked sensitivity to the drug. there is no clustered pattern of hiv- gp glycan deletions that occur under prm-a drug pressure. the resistance spectrum and mode of action is unique among any of the existing anti-hiv drugs and warrant further (pre)clinical investigations. acknowledgement: this research was supported by the flemish "fonds voor wetenschappelijk onderzoek," the centers of excellence of the k.u. leuven (no. ef/ / ), and the european commission (empro). jan muench , ludger ständker , knut adermann , axel schulz , michael schindler , raghavan chinnadurai , wolf-georg forssmann , frank kirchhoff department of virology university of ulm, albert-einstein allee , ulm, germany; ipf pharmaceuticals gmbh, feodor-lynen-strasse , hannover, germany a variety of components in human blood might influence hiv- replication in infected individuals. peptide libraries derived from hemofiltrate (hf), an aqueous blood solution, contain essentially all circulating blood peptides with a molecular mass below kda, including chemokines, defensins, and cytokines. to identify the most potent natural occurring factors inhibiting hiv- replication, we screened a hf-derived peptide library for antiviral activities. the most active fraction contained a -residue peptide corresponding to a c-terminal fragment of ␣ -antitrypsin (␣ -at), a highly abundant serine proteinase inhibitor. further analysis of the corresponding chemically synthesized peptide, termed virus inhibitory peptide (virip), demonstrated that it inhibits infection by all hiv- variants tested, independently of their subtype and coreceptor usage. notably, virip also blocked multi-resistant hiv- variants and primary isolates. virip specifically inhibited hiv- env function, and did not affect infection by virions containing hiv- , siv, mlv, hcv, ebola or vsv env proteins. the antiviral activity proved to be highly specific for the -residue virip sequence since structurally closely related peptides were inactive. we found that virip inhibits hemolysis of erythrocytes induced by the hiv- gp fusion-peptide (fp). nmr spectroscopy confirmed that virip interacts directly with synthetic gp fp. our observations are evidence that a naturally occurring human substance inhibits hiv- infection by a new mode of action, i.e. binding of the highly conserved fp. furthermore, we performed a structure-activity-relation study with more than virip analogs and found that specific amino acid changes enhanced the antiviral potency of virip by up to two orders of magnitude. experiments in cell culture and animal models further demonstrated that virip exerted no cytotoxic effects. thus, virip derivatives might become a new class of hiv- entry inhibitors. stefano aquaro , valentina svicher , roberta d'arrigo , ubaldo visco-comandini , andrea antinori , mario santoro , giovanni di perri , sergio lo caputo , pasquale narciso , carlo-federico perno , university of rome tor vergata, italy; inmi l. spallanzani, italy; university of turin, italy; sm annunziata hospital, florence, italy to investigate gp -variability and correlation with viroimmunological parameters in hiv-infected patients (pts) receiving t added as a single active drug to a failing regimen. two hundred and ten hiv-gp sequences and clinical follow-up from t -treated patients were analyzed from baseline up to weeks (weeks) of treatment. the association of mutations with viremia (vl)/cd count (c/ul) was assessed by mann-whitney test. the addition of t to the failing antiretroviral regimen induced at weeks a significant vl decrease from . log (stable in the last weeks prior t ) to . log (p = . ) and a significant cd increase from c/ul (decreasing in the last weeks prior t ) to c/ul (p = . ). while vl rebounded to . - log at - weeks, respectively, cd increased to c/ul at weeks. t resistance mutations, absent at bl, occurred shortly after treatment and usually alone. v a was the most common sign of t failure ( . % of pts). the viroimmunological outcome of t -treated pts varied according to gp -mutations. v a/e ( . % of pts) was associated with a cd increase from bl ( c/ul) of . -fold ( c/ul) at weeks and . -fold ( c/ul) at weeks (p = . and . compared without v a/e, respectively). no significant correlation with vl was observed (from . log at bl to . - . at - weeks). by contrast, q h + l m ( . % of pts) was associated with cd loss from c/ul at bl to c/ul at weeks (p = . ), without significant changes in vl (from log at bl to log at weeks). mutation n k (observed in pts, but never found at bl) abrogates the th gp -glycosylation site and correlated with . -fold cd increase at weeks. conformational changes induced by v a/e in the highly conserved giv motif of gp -hr , are tightly related with a loss of hiv-induced damage of immune system. this facilitates cd -recovery through mechanisms that can be virus-(loss of fusion efficiency) and immune-mediated (exposure of new epitopes) not applicable to protease/rt-inhibitors, and thus important for innovative therapeutic strategies. the spread of highly pathogenic h n influenza viruses in humans in asia, with high mortality rates among infected individuals is a major public health concern. in the absence of a vaccine antigenically matching the pandemic virus, antiviral drugs can play an important role. in the present study we reported the antiviral activity of neuraminidase inhibitor oseltamivir against lethal h n influenza virus infection in ferrets, an appropriate animal model that closely resembles clinical signs of human influenza. inoculation of young adult ferrets with a viral dose as low as eid of a/vietnam/ / (h n ) influenza virus caused high fever ( . - . • c), weight loss ( . % of initial), anorexia, extreme lethargy and death of animals on days - post-virus inoculation (p.i.). oral administration of oseltamivir at a dose of mg/kg/day for days twice daily initiated h p.i. inhibited the febrile response, reduced weight changes ( . % of initial) and, most importantly, completely protected ferrets from lethal h n infection. in the treatment groups, virus replication in the upper respiratory tract of ferrets was prevented, whereas untreated animals shed virus at titers of . - . log eid /ml on days , and p.i. systemic spread of the h n virus was observed in untreated ferrets: virus was detected in multiple internal organs, including the brain. treatment with oseltamivir resulted in complete inhibition of virus replication in the lungs and small intestine on day p.i. in the brains of treated animals virus was detected in one of the two animals tested with > -fold reduction of titer. sequence analysis showed no amino acid substitutions at conserved residues in na or ha subunit in viruses isolated from ferret's internal organs after treatment. these results suggest that oseltamivir earlier treatment can prevent h n mortality in ferrets, however, further studies investigating optimal doses and treatment durations required to achieve protection against infection with highly pathogenic influenza viruses are much needed. natalia ilyushina, erich hoffmann, rachelle salomon, robert webster, elena govorkova st. jude children's research hospital, memphis, tn , usa in the present study we tested in the mouse model the hypothesis that combination chemotherapy with drugs targeting dif-ferent virus proteins may lead to more potent and beneficial effects. we applied plasmid-based reverse genetics technique to generate two recombinant a/vietnam/ / -like (h n ) viruses. one virus possessed asparagine at position of the m protein that was found in the naturally circulating virus (rgvn- ) and confers resistance to amantadine. the other recombinant virus possessed serine at that position and was sensitive to amantadine (rgvn- sens) . balb/c mice were administered oseltamivir ( or mg/kg/day) and amantadine ( . or mg/kg/day) twice daily for days by oral gavage; the first doses were given h before inoculation with mld of h n virus. combination treatment with mg/kg/day oseltamivir and mg/kg/day amantadine was given on the same schedule. single-drug oseltamivir produced a dose-dependent antiviral effect against both recombinant h n viruses (p < . ). treatment with oseltamivir at dosage of mg/kg/day significantly inhibited virus replication in the lungs, brain, spleen, and blood of mice at days , , and after inoculation (p < . ), but resulted in low survival rate ( %). single-drug amantadine showed dose-dependent effect only against rgvn- sens strain. notably, risk of death for mice that received mg/kg/day of amantadine or mg/kg/day of oseltamivir was similar (p < . ). in contrast, prophylactic treatment of mice with combinations of oseltamivir and amantadine completely inhibited virus replication in the animals infected with rgvn- sens (p < . ) compared to singledrug usage and protected % of animals. importantly combination chemotherapy completely protected h n virus spread to the brain of the mice: virus was not detected in brain of treated animals on days , , and after inoculation and neurological symptoms were not observed. our results suggest that combination chemotherapy provides an advantage over single-agent treatment. this strategy could be an option for the control of influenza virus infection, and combinations with other novel drugs should be explored. françois jean , reid asbury , meera raj , david lawrence , martin petric the university of british columbia, vancouver, bc, canada v t z ; ge healthcare bio-sciences, piscataway, nj , usa; british columbia center for disease control, vancouver, bc, canada v z r in late , severe acute respiratory syndrome (sars) became the first new severe and easily transmissible human disease to emerge in the st century. although it abated after six months, sars serves as a modern paradigm for human emerging infections with deaths reported from countries. the causative agent was found to be a new sars-associated coronavirus (sars-cov) . while the sequence of sars-cov genome was first reported by the bc genome sciences center, the full set of viral and cellular proteins that compose the sars-cov virion remains unknown. to approach this problem, we have utilized two-dimensional gel electrophoresis and liquid chromatography-tandem ms (lc-ms/ms) to identify the viral and cellular proteins in purified sars-cov virions obtained from human infected cells [huh : human liver] and primate (veroe : monkey kidney) infected cells. interestingly, analysis of the proteins from purified sars-cov preparations has revealed that the enveloped virions contain not only the predicted viral structural proteins (e.g. spike glycoprotein, nucleocapsid protein, and membrane glycoprotein) but also an important number of differentially incorporated host cellular proteins into or onto the newly formed viruses. we have unambiguously identified over host cellular proteins in sars-cov virions by lc-ms/ms. these proteins include members of the annexin superfamily, cytoskeletal proteins, chaperones, vesicular transport proteins, uracyl-dna glycosylases, and aldehyde oxidoreductases. this study provides the first comprehensive and comparative analysis of the viral and cellular proteins that compose infectious particles of sars-cov obtained from human and primate infected cells. the functions of these newly identified hostspecific proteins are currently being investigated using rna interfering systems; their contributions to structure, viral productive replication, and pathogenicity will be discussed. acknowledgement: supported by an early career ubc operating grant (f. jean) and cihr (m. petric) . dale barnard , craig day , robert montgomery , kevin bailey , matt heiner , larry lauridsen , robert sidwell , kurt berg institute for antiviral research, utah state university, logan, ut, usa; panum inst., immi, the ifn-lab, copenhagen, denmark severe acute respiratory syndrome (sars) is a life-threatening respiratory illness caused by sars-cov. there are no approved therapies for sars. some drugs inhibit sars-cov replication in vitro including human interferons and selected antiinflammatory agents (chihrin and loufty, . , - ) . interferons are very promising because of their potent in vitro inhibition of sars-cov. although anti-inflammatory agents are not very active in vitro, it is thought that they might be efficacious in reducing any deleterious inflammatory response associated with virus infections such as sars infections in humans. for example, troxerutin, a flavenoid with anti-inflammatory properties, is in clinical trials for treating rhinovirus (rv) infections, ameliorating rv-induced inflammation (turner et al., . apmis , - ) . therefore, troxerutin was tested for inhibition of sars-cov replication in the lungs of infected mice using a mix of four hydroxyethylrutosides that included troxuretin. in addition, mouse interferon-alpha, used as a model compound for human interferon-alpha, was evaluated for inhi-bition of virus lung titers. both mouse interferon-alpha administered i.p. daily beginning h pre virus exposure at doses of , and , iu and the hydroxyethylrutoside mix ( and mg/kg) administered i.p using the same schedule reduced virus replication in the lungs of mice to below detectable limits. when a hydroxyethylrutoside mix was given to mice in the drinking water at . mg/ml (likely equivalent to an i.p. dose of mg/kg, assuming that the mice drank freely), virus lung replication was also completely inhibited. all treatments appeared to be well tolerated, since all groups of mice gained weight. we also report on the efficacy of various combinations of two doses of these drugs administered i.p., using the same dosing regimen as described. these data support the supposition that interferon might be a useful therapy for treating human sars infections and that hydroxyethylrutosides should be investigated further as a potential therapy. acknowledgement: supported by contract no. n -ai- from the virology branch, niaid, nih. treatment options for human respiratory syncytial virus (rsv) are limited. an effective vaccine is not yet available. neutralizing polyclonal antibody (respigam tm , medimmune) and a humanized monoclonal antibody (synagis tm , medimmune), are licensed for prophylactic use. ribavirin is the only approved antiviral against rsv, but its efficacy is controversial and its use is limited to treatment of high-risk patients. there is a clear need for new anti-rsv therapeutics with improved efficacy and ease-of-use. many early efforts to identify anti-rsv compounds focused on blocking the process of fusion. we have developed a cell-based screening platform to identify antivirals that inhibit rsv transcription and replication. the assay does not require infection with wild-type virus. it is based on an rsv subgenomic replication system in baby hamster kidney (bhk- ) cells that express the essential viral replication proteins (n, p, l and m - ). the readout is expression of the reporter gene lacz from a subgenomic rna. screening of the apath small molecule library yielded compounds (hit rate = . %) with ec values ≤ m and with selectivity index (si) values ≥ . seventy-two compounds demonstrated antiviral activity against wild-type rsv (strain a ) in a cytopathic effects inhibition assay (ec < m; si > ). these anti-rsv compounds represent nine different chemical classes. two compounds, a -aminoquinoline (ec = . m) and a thienopyrimidine (ec = . m), were shown to have desirable pharmacokinetic profiles and were chosen for efficacy testing in the cotton-tail rat model of infection. sar studies to identify the pharmacophore of the compounds have been initiated. preliminary studies to characterize the mechanism-ofaction in virological assays will be discussed. acknowledgement: supported by nih r ai - . we have previously reported bicyclic furano pyrimidine nucleoside analogues (bcnas) as exquisitely potent and selective inhibitors of varicella zoster virus (vzv) , with subnanomolar activity for p-alkylphenyl substituted analogues such as lead compound cf (cf- ) ( ) . these compounds have entered preclinical development with fermavir pharmaceuticals. we now report the first chromatography-free synthesis of these agents, their scale up to multi-gramme amounts, and their pre-clinical characterisation. in addition, we were keen to address potential solubility and bioavailability issues of these highly lipophilic agents by the synthesis of more polar analogues in two categories; side-chain ethers ( ) as new analogues in their own right, and -phosphates ( ) as potential more soluble prodrugs. we report data on both of these new families at this meeting. finally, we note the application of our phosphoramidate pro-tide approach to this family, with a series of bcna protides ( ) designed as intracellular phosphate delivery forms to bypass the essential vzv thymidine kinase-mediated first phosphorylation step. graciela andrei , joos van den oord , pierre fiten , ghislain opdenakker , erik de clercq , robert snoeck rega institute for medical research, katholieke universiteit leuven, leuven, belgium; pathology department, u.z. leuven, leuven, belgium varicella (chickenpox) , the primary infection caused by vzv, is characterized by viremia and skin lesions. reactivation of the latent virus results in skin lesions characteristic of herpes zoster (shingles). as keratinocytes are one of the main target cells for productive infection in vivo for vzv, human epithelial cells represent a relevant model for the study of vzv pathogenesis and evaluation of antiviral compounds. organotypic epithelial raft cultures permit full differentiation of keratinocytes via culturing of the cells on collagen matrix at the air-liquid interface. we have previously shown that the susceptibility of cultures to infection with vzv depends on the stage of differentiation of the rafts. we have now quantified the activity of reference anti-vzv compounds by measuring viral dna load by realtime pcr. quantitative pcr for vzv dna was performed by using specific primers and a mgb-probe for the orf gene (single-stranded dna binding protein) by the taqman method. two series of raft cultures were infected with the wild-type oka strain after days of differentiation and treated with serial dilutions of the test compounds. at days post-differentiation one series of the cultures was processed for histology and the other one for viral dna quantification. acyclovir (acv), penciclovir (pcv) and brivudin (bvdu) at and . g/ml, foscarnet (pfa) at g/ml and cidofovir (cdv) at , . and . g/ml inhibited viral dna content by more than %. these results were in agreement with histological examination of the rafts, no cytopathic effect being observed at these concentrations. as expected, only cdv and pfa inhibited the replication of the thymidine-kinase deficient (tk-) - strain. a correlation between the degree of protection as determined by histological examination and viral quantification could also be demonstrated for cdv and pfa against the tk- - strain. since no animal model is available for the in vivo evaluation of antiviral agents against vzv, the organotypic cultures may be considered as a valuable ex vivo model to evaluate the efficacy of new anti-vzv antivirals. jae-seon hwang , oliver kregler , john c. drach , , leroy b. townsend , elke bogner institut für klinische und molekulare virologie, erlangen, germany; department of biologic and materials sciences, school of dentistry; interdepartmental graduate program in medicinal chemistry, college of pharmacy, university of michigan, ann arbor, mi, usa dna packaging is the key step in viral maturation and involves binding and cleavage of viral dna containing specific dnapackaging motifs. this process is mediated by a group of specific enzymes called terminases. we have previously demonstrated that the hcmv terminase is composed of two subunits, the large one encoding pul and the small pul , where each protein has a different function. while the large subunit mediates sequence specific dna binding and atp hydrolysis, pul is only required for duplex nicking. inhibitors targeting pul and/or pul are attractive alternatives as hcmv antivirals since mammalian cell dna replication does not involve cleavage of concatameric dna. we now have screened several members of the benzimidazole ribonucleoside class of replication inhibitors in order to determine if a compound has the capacity to block the atpase activity of the large terminase subunit pul . analysis by bioluminometric atpase activity assays identified bdcrb and one more compound [ -bromo- , , -trichloro- -( , , -tri-o-acetyl-␤-dribofuranosyl)benzimidazole (btcrb)] with inhibitory effects. although only btcrb and bdcrb were inhibitors of the atpase activity, two other compounds, dbdcrb and cl rb, inhibited virus replication in a plaque-reduction assay, thus indicating that those have a different mode of action. in addition, by electron microscopy of thin sections we observed that in the presence of btcrb only b-capsids and dense bodies were formed. furthermore, spherical capsids accumulated in the perinuclear cisternae indicating a block in nuclear egress thereby providing additional evidence that closely-related benzimidazole d-ribonucleosides may have differences in their antiviral modes of action. human cytomegalovirus (hcmv) is the cause of significant morbidity and mortality in a variety of immunocompromised patients. currently available anti-hcmv drugs interfere with dna replication; however, these drugs are highly toxic, pre-cluding their long-term use in humans. interrupting hcmv viral entry is largely unexplored as an antiviral drug development strategy and is potentially an ideal and tractable goal. hcmv is believed to rely upon formation of ␣-helical coiled coils in the viral glycoproteins gb and gh to promote virus-host membrane fusion; peptides encompassing heptad repeat sequences in these two proteins inhibit viral infection. we have explored nonnatural oligomeric molecules ("foldamers") that are designed to mimic elements of the putative ␣-helical segment of gb. this effort has led to the discovery of oligomers of ␤-amino acids ("␤-peptides") that block hcmv infection. the ␤-peptide scaffold offers several advantages for the design of protein-protein interaction inhibitors, as ␤-peptides are amenable to modular synthesis, resist proteolytic degradation, and can display large and tailored molecular surfaces. the most potent ␤-peptide inhibitor blocks hcmv infection with a micromolar ic in a cell-based assay. these compounds show specificity for hcmv relative to closely related viruses. mechanistic studies suggest that these inhibitors interfere with membrane fusion between hcmv particles and host cells. current efforts are focused on understanding in greater detail the origin of the observed biological activity, exploring other foldamer scaffolds as bases for inhibitor design, and developing specific fusion inhibitors for other herpesviruses. previous reports have indicated that herpes simplex virus (hsv) activates nuclear factor-kappab (nf-kb) during productive infections. nonsteroidal anti-inflammatory drugs (nsaids) have significant inhibitory effects on nf-kb. therefore, two nsaids, indomethacin and aspirin, were assayed for antiherpetic effects and utilized as tools to further study the role of nf-kb in hsv- infection. we report that indomethacin and aspirin inhibited hsv- replication at non-cytotoxic doses. in vero cells, um indomethacin and mm aspirin reduced hsv- titers . and . %, respectively. electromobility shift assays revealed that hsv- activation of nf-kb is inhibited by the nsaids at doses that coincide with reduction of hsv- titers. to investigate a pathway for nf-kb inactivation, protein levels of ikb-alpha, a cytoplasmic nf-kb inhibitor, were examined. ikb-alpha protein was present in uninfected samples, but decreased over time in all hsv samples, regardless of chemical treatment, suggesting localization of nf-kb to the nucleus. immunohistochemistry studies verified that p , a component of the dimeric nf-kb complex, translocated to the nucleus of hsv- infected cells in the presence or absence of the nsaids. finally, direct effects on viral gene activity were assayed by real-time rt-pcr analysis. indomethacin and aspirin reduced mrna for icp , an essential hsv immediate-early gene, . and . -fold, respectively, resulting in significant decreases of icp protein. but transcriptional analysis revealed that synthesis of mrna for thymidine kinase, an hsv early gene, was unaffected by chemical treatment. however, mrna for glycoprotein c, an hsv late gene was undetectable in indomethacin and aspirin treated samples. cumulatively, these data indicate that: (i) indomethacin and aspirin block hsv- replication and (ii) the in vitro anti-herpetic effects of nsaids may reside in their ability to block nf-kb activity within the nucleus, impairing activation of essentials hsv genes. increasing species-specificity constraints preclude study of human cytomegalovirus (hcmv) in animals, necessitating the use of rodent cmvs to model human disease. however, the susceptibility of animal cmvs to clinically useful antivirals is unpredictable. for example, the guinea pig cmv (gpcmv), a uniquely valuable virus for modeling congenital cmv infection, is highly resistant to ganciclovir (gcv) at medically relevant doses. we used a molecular virological approach to test the hypothesis that gcv susceptibility could be conferred on gpcmv by insertion of the human ul phosphotransferase gene into the gpcmv genome. the gpcmv genome, cloned as a bacterial artificial chromosome in e. coli, was modified by site-specific recombination, using a shuttle plasmid targeting the gp locus, and carrying the ul gene from hcmv strain towne. the resultant chimeric virus was replication competent, and was found to contain the hcmv ul by southern-blot and sequence analyses. northern-blot revealed that a hcmv ul -specific transcript was expressed with late gene kinetics. western-blot, using a hcmv ul -specific polyclonal antibody, detected protein in virus-infected cells. the chimeric virus was gcv-susceptible, compared to wild-type gpcmv, with an ic of m. chimeric virus also exhibited increased sensitivity to maribavir (mbv), exhibiting a -log reduction (compared to wild-type virus) in the presence of m mbv, and an ic of m. to study the in vivo pathogenesis of chimeric virus, cyclophosphamide-immunocompromised strain two guinea pigs were challenged intraperitoneally, resulting in evidence of disseminated infection, and mortality. ganciclovir treatment ( mg/kg/day) resulted in reduced weight loss, and mortality, compared to placebo. these studies confirm the key role of ul in cmv antiviral therapy, and demonstrate that a 'humanized' gpcmv can be generated with altered antiviral susceptibilities. genital herpes infections are a global health problem and impact hiv/aids epidemic. strategies to prevent transmission include treatment of infected subjects to suppress shedding and prophylaxis with vaginally-applied microbicides. we examined the in vitro and vivo activity of rep , a fully degenerate mer phosphorothioated oligonucleotide against hsv- infection of human cervical cells and in a vaginal murine model. rep has broad-spectrum anti-herpetic activity with potent in vitro activity against hsv- , hsv- , hcmv, vzv, ebv, and hsv- (vaillant et al., submitted for publication). at a concentration of m, rep inhibited -logs of hsv- infection if present during the entire experiment. synchronized infectivity assays demonstrate that, unlike sulfonated polyanions in clinical trials, which primarily block hsv attachment, rep acts at multiple steps and inhibits binding, entry and post-entry gene expression. in our in vivo studies, mice were treated once intravaginally with rep or pbs control at various times prior to vaginal challenge with a lethal dose of hsv- strain ( log pfu). rep prophylaxis provided protection to mice from hsv- infection and disease. protection was significant when challenged min after treatment (p < . ). additionally, treatment with an analog of rep , which cannot activate tlr- mediated immune stimulation, was at least as active as rep , suggesting that direct antiviral activity and not stimulation of innate immunity is the mechanism of action in vivo. utilizing this analog, protection was significant when challenged min after treatment (p < . ) with a trend toward protection when administered min prior to challenge (p = . ). in summary, treatment with the rep analog which has superior resistance to low ph and nuclease degradation was more effective than rep , in some experiments protecting % of mice from viral infection and disease. the testing of this ph resistant rep analog in a gel formulation is currently underway. acknowledgement: supported by contract no -ai- from the virology branch, niaid, nih. a phosphorodiamidate morpholino oligomer (pmo) designed to hybridize to a highly conserved region including the aug translation start site of hcv, called avi- , has been evaluated for efficacy, toxicity, and pharmacokinetic properties. avi- inhibits translation initiated at the aug start site with ec of nm ( . ug/ml) and shows positive cooperativity. this pmo retains most of the activity in the presence of point mutations in the hcv genome. huh- cells were incubated with normal human serum (nhs) or hcv infected human serum (is) and hcv replication observed by rt-pcr. avi- produced robust inhibition of hcv in a dose and sequence-specific manner. studies conducted in vivo with avi- in the hcv infected trimera mouse (xtl) show reduction in viral titer which is dose dependent with approximately % of mice with undetectable viral titer and the remaining mice show log reduction in viral titer with . mg/mouse/day for consecutive days. the fractional bioavailability of avi- from a sq dose is approximately . the apparent elimination half life in rat, nonhuman primate and humans was . , . and . h, respectively. the volume of distribution ranged from . to . l/kg and the cmax is linearly related to the dose in mg/m . a phase i study in healthy volunteers in which daily sq doses of and mg has been completed. no serious adverse events have been observed. treatment of infected patients is currently planned. inhibition of hcv polyprotein synthesis is anticipated to contain therapeutic benefis of both protease inhibitors and polymerase inhibition. hcv infection can progress to fibrosis, reduced liver function, hepatocellular carcinoma, and death. currently, the standard treatment for hcv infection involves treatment with pegylated interferon in combination with the nucleoside analogue ribavirin. this treatment regimen effects a cure in approximately - % of the genotype- (gt- ) population; therefore a significant unmet clinical need exists in hcv therapy. virus-encoded polymerases have proven to be excellent molecular targets for chemotherapeutic intervention in numerous viral mediated diseases. in the case of hiv, hbv and herpes virus infections, deoxy-nucleoside analogues, which act as chain terminating agents, have been shown to have invaluable clinical utility. by analogy, appropriate ribonucleoside analogues might be expected to inhibit the essential rna polymerase (ns b) encoded by hcv. here we describe the preparation of nucleoside analogues as inhibitors of the hcv polymerase. in our design of nucleoside analogs as potential anti-hcv agents, we chose to investigate the effect of -substituted ribonucleoside derivatives. we reasoned that after incorporation of a ribonucleoside containing a -substituent, a disruption in elongation of the growing rna could be effected through either steric hindrance or via a conformational change of the carbohydrate moiety. our investigations on several such analogues will be presented. of particular interest is -azido-cytidine, which shows good activity in the genotype b sub-genomic replicon (ic = . m) with no measurable cytotoxic or cytostatic behavior. in addition, we have shown that the triphosphate of -azido-cytidine is a potent and highly selective inhibitor of ns b (ic = . m). joanna e. boerner, sue ma, choilai tiongyip, michael p. cooreman, teresa compton, kai lin novartis institutes for biomedical research, technology square, cambridge, ma , usa current drug discovery efforts for hepatitis c virus (hcv) focus on developing specific inhibitors of two viral enzymes, ns b polymerase and ns - a protease. however, resistant viral mutants are likely to emerge during therapy, compromising the effectiveness of these inhibitors. an alternative and complementary strategy is to target host factors that are also essential for viral replication. cyclophilins, a family of peptidyl-prolyl isomerases and the cellular targets of cyclosporin a (csa), present such an opportunity. it was reported recently that cyclophilin b bound to hcv ns b polymerase and stimulated its rnabinding activity, and that these functions were blocked in the presence of csa (watashi k. et al., molecular cell ) . nim , a csa derivative, is a more suitable candidate for hcv therapy because it binds to cyclophilins with higher affinity than csa while lacking the immunosuppressive activity associated with csa. using the hcv replicon system we demonstrated that nim exhibited potent anti-hcv activities in vitro. moreover, the combination of nim with a specific non-nucleoside inhibitor of hcv polymerase led to synergistic antiviral effects with no significant increase of cytotoxicity. resistant clones against both inhibitors were obtained in vitro, however, it was much more difficult to generate resistance against nim than the polymerase inhibitor. also, there was no cross-resistance between the two inhibitors. finally, addition of nim to the hcv polymerase inhibitor drastically reduced the emergence of resistance compared to polymerase inhibitor alone. taken together, nim , with a novel mechanism of action and a favorable pharmacokinetics and safety profile, represents a promising clinical candidate for treating hepatitis c and provides a rationale for specific combination therapy. the nucleoside analog r was identified as a specific inhibitor of hcv replication in subgenomic hcv replicon cells. r -tp is a competitive inhibitor of cmp incorporation by hcv polymerase ns b. in a transient replicon system r inhibited hcv rna replication driven by genotype b polymerase with similar potency as compared to that driven by genotype a polymerase. r -tp inhibited native hcv replicase and recombinant ns b from genotype a and b with similar potency. in contrast, r -tp did not inhibit human dna polymerases alpha, beta or gamma, including reverse transcriptase activities of dna polymerases beta and gamma, which were highly sensitive to inhibition by azt-tp and tc-tp. no significant inhibition was observed with human rna polymerases i, ii and iii derived from hela cells. in addition, the functionally related native influenza virus rna dependent rna polymerase (rdrp) activity in vitro was not inhibited by r -tp at concentrations up to mm, suggesting high selectivity for the hcv rdrp. thus, r was identified as a potent and highly selective inhibitor of hcv polymerase mediated rna synthesis. guangxiang luo, zhaohui cai, chen zhang, kyung-soo chang, jieyun jiang microbiology, immunology, and molecular genetics, university of kentucky college of medicine, lexington, ky, usa the study of hepatitis c virus (hcv) replication and the search for specific antiviral agents against hcv infection have been hampered by the lack of an efficient stable cell culture system of hcv infection and propagation. we have successfully constructed stable human hepatoma cell lines that contain a chromosomally integrated-genotype a hcv cdna and constitutively produce and secrete high titers of infectious virus into the culture media. transcriptional expression of the full-length hcv rna genome is under the control of a cellular pol ii polymerase promoter at the end and a hepatitis delta virus ribozyme at the end. the resulting hcv rna was expressed and replicated efficiently, as shown by the presence of high levels of hcv proteins as well as hcv rna in the stable huh cell lines. hcv secreted from the stable cell lines was infectious, as determined by antibody neutralization, blockage of putative hcv receptors, and inhibition of hcv replication by interferon. our findings demonstrate the establishment of a stable cell culture system of infectious hcv production and propagation, which allows the study of the entire hcv infectious cycle. the stable hcv-secreting cell lines are now being pursued to develop high throughput screens for effective hcv inhibitors. additionally, we established a novel and powerful hcv replication system in the mouse hepatocyte and mouse embryo fibroblasts (mef). hcv rna was found to replicate efficiently in both pkr +/+ and pkr −/− mef cells, demonstrating that hcv rna replication in mef cells is a powerful system to study host-virus interaction by using diverse gene-knockout animals. interestingly, hcv rna replicates more efficiently in the pkr −/− cell than in the pkr +/+ cell, suggesting a role of pkr in the control of hcv rna replication. however, ifn inhibited hcv rna replication in the pkr −/− cell with an efficacy similar to that in the pkr +/+ cell, suggesting a pkr-independent antiviral mechanism. clearly both pkr-dependent and pkrindependent antiviral mechanisms are important for the control of hcv replication and the mediation of the ifn-induced anti-hcv response. our studies set a stage for the development of transgenic mouse models of hcv replication and open up new avenues to study hcv and host interactions in mefs derived from diverse gene-knockout animals. andrea cuconati , haitao guo , gael westby , anand mehta , timothy block , institute for hepatitis and virus research; drexel institute for biotechnology and virology research, doylestown, pa, usa the high levels of hepatitis b surface antigen (hbsag)-bearing non-infectious particles in the serum of infected individuals is thought to play a role in suppressing hepatitis b virus (hbv)specific immune response by titering out hbv-specific antibodies and lymphocytes. current hbv therapeutics do not directly reduce this viral antigenemia. our group has focused on the enhancement of the immune response through the inhibition of viral antigen secretion in the infected hepatocytes, with the therapeutic goal being the use of hbv vaccination for the treatment of acute and chronic infection. the high-throughput screening of a small molecule library of , drug-like compounds was undertaken to discover novel inhibitors of hbsag secretion. using the stably hbv-transfected, human hepatoma cell line hepg . . , we developed an hts-compatible elisa protocol for the detection of hbsag secreted in the culture media. the screen resulted in initially positive hits, a hit rate of . %. subsequent retesting for activity and toxicity by mtt assay has narrowed the number of confirmed, non-toxic hits to , currently categorized in twelve chemical series. we have previously reported on a trio of related pyrazolo-pyridines with ec measurements below . m and cc measurements > . m. nascent structure-activity relationship (sar) suggests that a central moiety of the molecules is essential to activity, with an aromatic side group contributing to potency. among recently confirmed inhibitors, two currently under investigation include: ( ) an isobutyl-acetamide with an ec of . nanomolar, and a cc of > m, and ( ) a carbothiamide with an ec of . micromolar and a cc of > m. measurement of secreted hbv l and m antigens and cellular markers indicated that the pyrazolo-pyridines are not specific inhibitors of viral antigens, while the isobutyl-acetamide and the carbothiamide are indeed specific. measurement of intracellular viral dna indicated that none of these molecules are inhibitors of replication. we will be reporting on our studies of the potency, specificity, and potential mechanisms of action of these novel anti-hbv compounds. background: entecavir (etv) is a potent competitive inhibitor of hepatitis b virus (hbv) polymerase with activity versus all three enzymatic functions including priming, minus, and plus strand dna synthesis. virologic rebound due to etv resistance (etvr) has only been observed in lamivudine resistant (lvdr) hbv (m v/i ± l m), and requires at least one additional change in the reverse transcriptase domain (rt) at residues t , s , or m . these substitutions surround the dntp binding site or primer grip of rt. the objectives of this work were to further characterize etvr and its mechanism(s) using cell culture, in vitro enzyme, and molecular modeling studies. methods: hbv cell culture assays used transfected hepg cells and quantitation of released, immunocaptured hbv nucleocapsids. gradient-purified intracellular nucleocapsids were used for in vitro rt assays. a d homology model based on the hiv- rt structure was used to model resistance changes in hbv. results: reduced etv susceptibility of etvr hbv was observed both in culture and enzymatically in vitro. kinetic studies showed various etvr substitutions in lvdr hbv selectively reduced etv-triphosphate (etv-tp) binding (k i ) to rt without markedly changing the affinity for dgtp (k m ) or inhibition by ddgtp. etvr rts also displayed reduced enzymatic activity (k cat ) relative to wildtype and etvr hbv appeared growth impaired. modeling studies suggested a novel etv-tp binding pocket in hbv rt that became constrained with etvr changes. m changes in the primer grip region of rt were unique in that resistance was primarily seen during synthesis of minus strand dna. etvr changes in the absence of lvdr substitutions had greatly reduced impacts on etv susceptibility, confirming models suggesting etvr is imparted through lvdr changes. summary: etv provides a high genetic barrier to resistance, requiring additional changes at residues t , s or m along with pre-existing lvdr substitutions m v/i ± l m. kinetic parameters and molecular modeling indicated that etvr substitutions selectively affected etv-tp binding and reduced the replication capacity of hbv. a nonhuman primate (nhp) model of classical, lesional smallpox has been used to test the efficacy of intravenous (iv) cidofovir treatment. cynomolgus macaques were infected with a high dose ( pfu iv) of variola to produce an artificial primary viremia, and then treated with cidofovir at , , or h postinfection (pi). later treatment times were not evaluated. treatment at or h pi halted increases in peak blood viral genome titers measured by quantitative taqman-mgb real-time pcr, which were more than -fold less in cdv-treated animals compared to placebo. historically, the number of pox lesions provided the best correlation with human smallpox clinical severity, and cdv treatment in our model significantly reduced maximum pox lesion counts by > %; the number and size of skin lesions, and in untreated animals contributed significantly to the total viral burden with lesions containing - genomes/g. to better understand the role of viral burden and disease progression in major organ systems, a serial sample study was undertaken. in untreated animals at h pi, viral replication in spleen exceeded genomes/g while liver and bone marrow yielded genomes/ml. in comparison, titers in other tissues ranged between and genomes/g and blood yielded genomes/ml at h, suggesting that the liver, spleen, and marrow may be initial sites of replication. levels of virus in the bone marrow reached a peak of approximately genomes/g at day , then decreased to quantities consistent with those in blood. viral load in the blood increased with time, peaking around days - at genomes/ml. virus was also detected in intestine, skeletal muscle, and late in infection, testes. the ability to successfully treat with cdv h pi despite early extensive organ infection in the accelerated nhp variola model suggests that this treatment could be effective in reducing viremia and mortality after onset of symptoms in human smallpox, which demonstrates a more protracted disease course. work involving variola virus conducted in who-sanctioned cdc, atlanta bsl- laboratory. earl kern , kathy keith , robert jordan , dennis hruby , debra quenelle department of pediatrics, university of alabama school of medicine, birmingham, al, usa; siga technologies, inc., corvallis, or, usa although cidofovir (cdv) has been approved as an investigational new drug for emergency treatment of smallpox, its lack of oral activity and dose limiting toxicity dictates a need for continued development of better therapeutic agents for this potential bioterror disease. it has been reported previously that st- , a low-molecular weight compound, inhibits replication of all the orthopoxviruses in vitro and protects mice infected with vaccinia or ectomelia virus. in the present study, we have utilized cowpox virus (cv) and vaccinia virus (vv) infections in vitro and in vivo to evaluate the efficacy of st- for treatment of orthopoxvirus infections. in plaque reduction assays in human foreskin fibroblast cells, both cv and vv were inhibited by about . - . um of st- . for in vivo studies, st- was administered once daily by oral gavage to mice using mg/kg for , , , or days beginning or h after intranasal inoculation with vv or cv. st- was highly effective (p < . ) in preventing mortality due to vv or cv even when treatment was delayed up to h post-infection. a dosing duration of days was adequate for vv infected mice, but duration of days or longer was required for efficacy in cv infected mice. when st- was given once daily for days at , , or mg/kg daily at , , or h post-cv inoculation, mortality was significantly altered at all dosage levels and time points. to determine the effect of treatment on virus replication in target tissues, mice were inoculated with cv or vv and treated once daily with mg/kg of st- . on various days post-infection tissues were harvested and assayed for virus. in cv or vv-infected mice, st- treatment successfully reduced virus titers from to logs in liver, spleen, and kidney. little effect was noted in lung tissue. these results indicate that st- has significant activity against vv and cv infections in vitro and in vivo and may be a potential chemotherapeutic agent for treatment of human orthopoxvirus infections. cidofovir (hpmpc) is a broad-spectrum anti-viral agent that is used (vistide ® ) to treat aids-related cmv retinitis. currently, cidofovir is of particular interest as a potential therapy for orthopox virus infections, including smallpox. an important limitation of cidofovir and analogous nucleotide drugs in a therapeutic role is their low oral bioavailability and poor transport into cells. in principle, bioavailability of a drug can be improved by structural modification targeting transporters expressed in human intestine. to be effective, the transported prodrug must be cleaved by endogenous enzymes to its parent compound. we will present synthetic studies of novel cidofovir and cyclic cidofovir (chpmpc) prodrugs incorporating amino acids or small peptides, comparing different drug-amino acid linkage strategies. the compounds were evaluated for transporter-mediated uptake and cellular and plasma hydrolysis. the results will be compared with similar studies carried out on a series of peptidomimetic conjugates of foscarnet, the trisodium salt of phosphonoformic acid (pfa), an anti-viral agent that also has very low oral bioavailability and poor cell penetration. the question addressed in this study is if wnv-reactive antibody can improve disease signs in a hamster model after the virus is demonstrated to be in the brain. the hypothesis is based on the high activity of a humanized monoclonal antibody, he , in a mouse model when administered later in infection (oliphant et al., . nat. med. , ) . in this study, virus was demonstrated to be in the brains of hamsters at days post-viral injection (dpi) by cell culture assay, quantitative rt-pcr, and immunohistochemical staining of wnv in neurons. eighty percent of hamsters treated i.p. dpi with mg/kg of humanized monoclonal antibody, he , survived wnv disease, whereas, % of placebo-treated hamsters survived ( *** p < . ). if administered at dpi, % survived. we tested the hypothesis that he is effective if delivered directly into the brain instead of by peripheral administration. the antibody was delivered into the brain dpi using convectionenhanced delivery through a cannula implanted into the brain. the he was detected in the cns, but none was detected in the kidney. the survival of he -treated hamsters was % as compared to % of placebo-treated animals ( *** p < . ). for additional proof, the majority of hamsters having wnv in their cerebrospinal fluid, a marker for cns infection, were protected with he administered i.p. at dpi. this humanized monoclonal antibody, therefore, is a possible treatment for the post-exposure, wnv-infected humans that develop signs of neuroinvasive disease. acknowledgement: supported by contract no -ai- from the virology branch, niaid, nih, and grant -u ai - from the rocky mountain regional centers of excellence, nih. hemorrhagic fever viruses are of serious worldwide health concern as well as potential biological weapons. lassa fever virus in particular annually infects several hundred thousand individuals in west africa, and the export of this pathogen outside of this region, either intentionally or unintentionally, presents a serious risk to the developed countries of the world. the cdc and niaid have identified lassa fever virus as a category a priority pathogen, indicating the highest degree of threat to public health. no arenavirus-specific antiviral drugs are currently approved for use in humans. the purpose of siga's biodefense program is to develop safe and effective drugs for preventing and treating diseases caused by category a viruses. to that end, a large and diverse library of small molecule compounds was screened using a viral pseudotype assay to identify inhibitors that target the essential lassa surface glycoprotein (gp) and thus block viral entry into the host cell. twenty-six compounds were identified as quality hits, as defined by potency, selectivity, and chemical tractability. antiviral activity against authentic lassa fever virus was assessed in cell culture through a collaboration with colleagues at usamriid. a number of these potent antiviral compounds and their related analogs have exhibited informative chemical structure-biological activity relationships (sar). two potential lead compound series have emerged from these studies, each with % effective concentrations (ec s) of less than nm against lassa fever virus and with ec s of less than nm against lassa gp-pseudotyped virus. characterization of the in vivo properties of these compounds is underway. the in vitro antiviral potency and selectivity, animal pharmacokinetics, and the development process will be presented. these inhibitors represent an important step toward the development of a small molecule antiviral drug for lassa fever virus. sven enterlein , pramila walpita , allison groseth , heinz feldmann , ramon flick university of texas medical branch, department of pathology, galveston, tx, usa; national microbiology laboratory, public health agency of canada, winnipeg, man., canada nipah (niv) virus (family paramyxoviridae) is a recently emerged human and animal pathogen that can cause severe encephalitis with fatality rates of up to %. since no treatment or vaccination is available, and cross-species spread was observed, the virus has been classified as biosafety level (bsl- ) agent. to avoid bsl- containment for the study of cis-acting signals as target for antiviral strategies, we used an optimized plasmid-driven t minigenome rescue system (without the need for recombinant vaccinia virus mva-t ) as well as an newly established rna polymerase i-based approach. minigenome rescue is based on transfection of the minigenome niv-cat and the plasmids encoding for the three nucleocapsid proteins n (nucleoprotein), l (polymerase), and p (phosphoprotein) and measured by enzymatic cat assays. we used the established plasmid-based minigenome rescue systems to screen for potential antiviral compounds. in a first step we tried to determine the optimal strategy for the delivery of small hairpin (sh) interfering rna molecules. for this we compared three shrna delivery systems against another bsl agent-reston ebolavirus (family filoviridae); (i) plasmid-mediated pol i and (ii) pol iii-driven shrnas, and (iii) exogenously (t ) produced shrna, for their ability to induce gene silencing. interestingly, beside the in vitro-generated or pol iii-driven shrnas, pol i transcripts showed very efficient inhibition of minigenome rescue. however, the most efficient delivery method was transfection of in vitro transcribed shrnas. we will present the results of this comparison and, based on the most efficient approach, also first results of shrnas targeted either to niv n, p, and l genes or to the leader/trailer noncoding regions to interfere with minigenome replication. conformative data with live virus experiments under bsl conditions will be included. filoviruses, which include ebola virus and marburg virus, are among the most notorious human pathogens because they cause sporadic outbreaks of severe hemorrhagic fever. unfortunately, very few therapeutic agents are available to treat infections with these viruses. antiviral screening methods that determine the effect of compounds on viral replication involve working with infectious virus, which is obviously not practical for these biosafety level (bsl- ) agents. we developed an antiviral screening method based on a cell-based, infection-independent, ebola subgenomic replication system in which the expression of an easily measurable enzyme is dependent on the rna replication and transcription factors of ebola virus. using this system we screened a synthetic compound library for antiviral activity against ebola virus and have identified a number of inhibitors. we also used it to identify a peptide inhibitor directed against vp . anti-ebola virus activity for many of the inhibitors was confirmed in a viral replication assay using a gfp-expressing zaire ' strain of ebola virus. fifty-two small molecule inhibitors from at least six classes of compounds had ec values in the low micromolar range and good selectivity. several of these compounds have promising chemical, biological, and pharmacological profiles to pursue as potential anti-filovirus drugs. we are currently preparing to test these compounds in a mouse model of ebola virus. we have also begun a lead optimization program to improve antiviral potency and selectivity of aryl sulfonamide and -aminoquinoline compounds. acknowledgement: supported by nih r ai - and r ai - . human papillomavirus (hpv) has been a difficult virus to target by traditional antiviral methods due to its small size, its small number of obvious therapeutic targets, and its resistance to propagation in vitro. nevertheless, antiviral compounds that reduce hpv dna load have the potential to prevent carcino-genic progression in infected patients. to that end, we developed an approach that dramatically reduces the hpv episomal dna load of keratinocytes in vitro by targeting viral dna sequences. pyrrole-imidazole polyamides, with some containing fluorescent probes to aid in cell localization studies, were designed to recognize the hpv ori. all fluorescent compounds rapidly localized to the nucleus of cultured keratinocytes following addition to the culture media. the compounds were then tested for their ability to alter keratinocyte hpv episomal dna content. two of the compounds caused a dose-dependent reduction in hpv episomes as measured by taqman tm realtime pcr. while control and vehicle-treated cells maintained ∼ copies of hpv per cell, compounds -ta and -ta both reduced hpv dna levels to below copies per cell after h incubation with m compound. an alternative taqman tm amplicon within the hpv e gene produced identical results. a multiplexed taqman tm real-time pcr reaction that followed the ratio of hpv dna to the human apoe gene also demonstrated dramatic loss of hpv dna copies, further confirming our initial observations. finally, cells were treated with polyamides for h, polyamide-containing media was removed, and episome levels were followed for days. at day , days after removal of polyamide and days after sub-culturing of the cells, viral episome levels remained approximately % lower than control samples. by day , days after removal of polyamide, viral dna levels were beginning to recover but still remained significantly lower than control samples. together our results demonstrate that targeting the hpv origin of replication with dna-binding compounds dramatically reduces episomal dna levels. small interfering rnas (sirnas) are potent tools for gene down-regulation but are minimally stable in cells. to improve the efficacy of sirna, we replaced non-bridging oxygens in the phosphodiester linkages of natural rnas with bh groups. the resulting boranophosphates have unique properties, including enhanced nuclease resistance, altered hydrogen bonding of the phosphate, different interactions with metal ions, and increased thermal stability of rna:rna and rna:dna duplexes. anti-egfp sirnas containing boranophosphate modifications were prepared by in vitro transcription with t rna polymerase from ribonucleoside -(alpha-p-borano)triphosphates, as well as normal and phosphorothioate sirnas. after confirming the presence of the borane modifications with maldi-ms, several properties of borano-modified sirnas were investigated: ( ) the double stranded rna with borane modifications maintained the a-form conformation characteristics according to the circular dichroism (cd) spectra; ( ) the borane groups in the sirnas increased the thermal stability, with an enhancement of t m by . - . • c per modification; and ( ) sirnas with borano-modifications were shown to be at least -fold more resistant to rnase a digestion than normal ones. when these modified sirnas were used to down-regulate egfp expression in hela cell cultures, it was found that: ( ) borano-modified sirnas were consistently more effective than sirnas containing the corresponding phosphorothioate modifications; ( ) borano-sirnas were more effective than normal sirnas provided that the center of the antisense strand was not heavily modified; ( ) borano-sirnas were more potent than normal or phosphorothioate sirnas at lower concentrations; and ( ) finally, the silencing activity of boranophosphate singlestranded sirna (ss-rna) was comparable to that of unmodified ds-sirna. the borano ss-rna had excellent maximum silencing activity and was highly effective at low concentrations, and silencing activity was durable up to one week after transfection. results with anti-hpv sirnas will be discussed. boranophosphate modification is a potential new class of anti-viral therapeutic agents. this report describes the antiviral structure activity relationships that led to the discovery of phosphonomethoxy- -fluoro- , dideoxydidehydroadenosine (fd ap, gs ), a novel ntrti, with an excellent resistance profile toward hiv- variants containing major n(t)rti resistance mutations. methods: phosphonomethoxy analogs on purine and pyrimidine dideoxydidehydro (d ) and dideoxy (dd) ribose scaffolds were prepared. antiviral activity was measured against wildtype and n(t)rti-resistant recombinant viruses using cytopathic assay in mt- cells. mitochondrial toxicity was assessed in hepg cells by measuring mitochondrial dna content. results: the d scaffolds displayed superior antiviral activity compared to the dd scaffold and adenine was superior to other nucleobases. phosphonomethoxy- , dideoxydidehydroadenosine (d ap) inhibited hiv- replication with a mean ec of . m and an . -, . -, and . -fold change in potency against viruses containing m v, k r, and thymidine analog mutations (tams), respectively. further exploration of d ap was limited by its mitochondrial toxicity, which was then addressed in ways: (i) preparation of l-d ap or (ii) fluorine substitution. l-d ap exhibited an ec of . m but had substantially reduced potency ( -fold) toward m v mutant viruses. fd ap exhibited an ec of . m, with . -, . -, and . -fold change in potency against viruses containing m v, k r, and tams, respectively. no cytotoxic effects were measured up to mm in mt- cells and no effects on mitochondrial dna were detected up to m in hepg cells for both fd ap and l-d ap. conclusion: fd ap is a novel phosphonate ntrti with antiretroviral activity toward wild-type and resistant mutant hiv- strains. compared to d ap, the -fluorine atom significantly improved the in vitro toxicity profile while retaining the favorable resistance profile. in subsequent studies, the monoamidate prodrug strategy was applied to fd ap to achieve optimal in vivo pharmacokinetic properties. entry inhibitors, and ccr- antagonists in particular, have become one of the most actively pursued treatments for hiv within the pharmaceutical industry. recently, multiple groups have disclosed piperidine-based ccr- antagonists that -to the medicinal chemist's eye -might appear to share a common three-point pharmacophore comprised of a tertiary amine, a phenyl ring, and a carboxamide or sulfonamide group. in several of these cases, these pharmacophoric elements are tethered together by a flexible, aliphatic chain. we sought to improve the potency of and introduce structural novelty into this class of compounds by rigidifying this tether. herein, we describe stereoselective syntheses and sar of a series of ccr- antagonists wherein the tether has been replaced with four stereochemical isomers of a rigidified cyclopropyl scaffold. the regulation of hiv transcription is a complex, multistage process that requires the concerted action of viral and cellular proteins. we discovered the n-aminoimidazoles (naims) as a unique class of hiv inhibitors targeted at the viral transcription level. a prototype naim, nr- , prevents the reactivation of dormant virus by inhibiting both the hiv- p and viral mrna production from latently hiv- -infected cell lines upon stimulation with tnf-␣, pma, or tsa. extensive research revealed that nr- was unable to inhibit the nf-b activation pathway or chromatin remodeling at the viral promoter, both known to be crucial for viral transcriptional activation. focusing on the viral transcription process, chromatin immunoprecipitation (chip) experiments revealed that nr- was able to inhibit the ser phosphorylation of the c-terminal domain (ctd) of rna polymerase ii. this step is mediated by the cdk subunit of p-tefb, which is recruited to the viral promoter by the hiv- tat protein. since we did not find an inhibition at the level of cdk activity or tat-mediated transcription in tat-expressing cell lines transiently transfected with a ltr-gfp construct, we infer that nr- must interfere with the transcription process by a unique mode of action. evidence points towards a kinase, not belonging to the cdk family, to be the target of the naims, resulting in an antiviral action at the level of retroviral transcription. clara e. cases-gonzález , sandra franco , miguel a. martínez , luis menéndez-arias centro de biología molecular "severo ochoa", csic-uam, madrid, spain; fundació irsicaixa, hosp. university germans trias i pujol, badalona, spain a ser-ser insertion at codons - together with substitutions t s and t y in the reverse-transcriptase (rt)-coding region of hiv- are known to confer resistance to zidovudine (azt) and stavudine (d t). phenotypic resistance correlates with increased atp-dependent phosphorolytic activity on inhibitor-terminated primers. we have previously shown that an rt derived from a clinical isolate (ss rt) that contained the insertion and additional mutations related to drug resistance (including t y) showed > -fold increased unblocking activity on azt-and d t-terminated primers, when compared with an rt containing the insertion together with mutations t s and t y, in an otherwise wild-type bh sequence. these results suggested that other mutations associated with the complex t sss/t y in clinically relevant rts contributed to increase atp-mediated excision activity and conferred high-level resistance to azt and d t in phenotypic assays. to identify residues increasing the excision activity, we obtained recombinant enzymes bearing ss rt residues - and wild-type bh rt residues - (l rt), or residues - of the bh rt and - of the ss rt (l rt), as well as an l rt variant with the substitution t y (l rt) and an l rt derivative with t sss (l rt). additional rts containing mutations m l, a v, or k r together with the combination t sss/t y in the bh background were also obtained. atp-mediated excision activities on azt-and d tterminated primers were determined and the effects of mutations were tested in phenotypic assays using recombinant hiv- . the l rt containing mutations t sss/t y and additional changes in the n-terminal region showed the highest atp-dependent phosphorolytic activity on blocked primers, giving values similar to those reported for the ss rt. results were consistent with phenotypic data. in contrast, l , l , and l rts displayed low-level activity. further experiments revealed that three amino acid changes at the n-terminal region of the polymerase (m l, a v and k r) were responsible for the increased excision activity shown by rts bearing mutations t sss and t y. from a series of phenyl-substituted thiazolobenzimidazoles, several compounds were identified as selective inhibitors of coxsackie b virus replication in vero cells. a structure-activity relationship was established, from which the -trifluoromethyl substituted analogs emerged as the most potent congeners. the compounds were active against all six coxsackie b strains tested. the in vitro antiviral activity of one of the most selective compounds, i.e. chi- , was assessed by (i) mts-based cytopathic effect assays, (ii) virus yield reduction assays, (iii) real-time quantitative pcr (rt-qpcr) and (iv) by monitoring viral antigen expression. in all assays a clear concentration-response effect was obtained. the % effective concentration (ec ) was . ± . g/ml, while the cc ( % cytotoxic concentration) of chi- for vero cells was more than g/ml, thus resulting in a selectivity index of > . detailed single cycle time-of-drug-addition studies (in which viral replication was monitored by means of rt-qpcr) revealed that the compound interacts with viral replication at a time that coincides with the onset of intracellular viral rna synthesis. chi- resistant virus is being generated by culturing the virus in the presence of increasing drug concentrations. drug-resistant virus will be genotyped, which should allow us to identify the (putatively viral) molecular target of this class of compounds. retroviruses hiromichi tanaka , kazuhiro haraguchi , hiroki kumamoto , takao nitanda , masanori baba , ginger e. dutschman , yung-chi cheng school of pharmaceutical sciences, showa university, tokyo, japan; center for chronic viral diseases, kagoshima university, kagoshima, japan; school of medicine, yale university, new haven, ct, usa our recent research program on the development of synthetic methods for -carbon-substituted nucleosides has led to a new strategy, ring opening of , -epoxy-nucleosides with organoaluminum and organosilicon reagents. this enabled us to introduce alkyl, alkenyl, and alkynyl groups to the -position. as a result of this study, -ethynylstavudine ( -ed t) was found to be more anti-hiv active than the parent compound stavudine (d t). this compound ( -ed t) has several additional appeals as a promising anti-hiv agent: much less toxic to various cells and also to mitochondrial dna synthesis, better substrate for human thymidine kinase than d t, very much resistant to catabolism by thymidine phosphorylase, its activity enhances in the presence of a major mutation k n known for nnrti-resistant hiv. in this conference, we present the synthesis and sar studies of -ed t analogues modified mainly in the sugar portion. negatively charged polymers (np) possess a broad immunoadjuvant and antiviral activity topically useful for vaccine, drug, and microbicide development. but their efficiency is limited over a reversibility of electrostatic kind of interference with virusspecific nano-objects. to overcome this limitation the purposemade intra-molecular modifications of np were studied among non-toxic maleic acid co-polymers (npsa), dextran and chitin derivatives (npps) within varied alicyclic modifiers application. the configurationally flexible alkyls (i), as non-alicyclic control, are ineffective synergist for np antiviral potency. monocycles (ii) are moderate active too. on the contrary the hardconformation frame-structured spheroids (iii-vi) exhibit ability (at optimal macromolecular parameters) to be super-effective synergists for strength and diapason of np antiviral action. unlike small molecular iii/iv-containing prototypes (amantadin, rimantadin, deitiforin, etc.), narrowly-effective inhibitors mainly of influenza a viruses, the np-coupled modifications become effective also against many other viruses, including the drugs resistant strains [antivir. res. ( ), ]. in focus of the anti-hiv potency the ivs provide a - -fold elevation of np activity. the more available and less toxic iii species are similarly active, but iii* (with spatial-optimally contactable double bond due to the exo-configuration) turns out the best synergist - -fold amplifying the anti-hiv- selectivity up to is∼ . augmentation of the frame cycles from iii-iv toward v-vi results in no essential enhancement of antiviral activity, but stimulates toxicity. the recently involved in the investigation vii, cholesterol-like systems, as tools for novel raft-targeted strategy, demonstrate capacity for at least -fold amplification of anti-hiv- potency our earlier studies showed that esterification of cidofovir (hpmpc) with alkoxyalkanols increased antiviral activity by more that two logs and promoted oral bioavailability. to evaluate this approach with purine based nucleoside phosphonates, we synthesized several alkoxyalkyl esters of acyclic purine phosphonates such as , ,-diamino-( -[ -phosphonomethoxyethyl]purine (pme-dap) and -amino- -cyclopropylamino-( -[ phosphonomethoxyethyl]-purine (pme-cpr-dap) these purine phosphonates have been reported to be active against a wide range of viruses such as human immunodeficiency virus (hiv- ), other retroviruses, herpesviruses, poxviruses and hepatitis b virus. for this study several alkoxyalkyl analogs of acyclic , diaminopurine nucleoside phosphonates were synthesized and evaluated against hiv- . the alkoxyalkyl esters were more inhibitory than the unmodified compounds in p reduction assays in mt- cells infected with hiv- . for example, hexadecyloxypropyl (hdp) and oleyloxyethyl (ole) esters of pme-cpr-dap were > logs more active than unmodified pme-cpr-dap. in spite of increased cytotoxicity in mt- cells, the selectivity indexes are more than -fold higher then for unmodified compound. in conclusion, esterification of pme-dap and pme-cpr-dap with hexadecyloxypropyl-or oleyloxyethyl-residues greatly increased their antiviral activity and selectivity against hiv- in vitro. victor kuz'min , eugene muratov , anatoly artemenko , ludmila koroleva , vladimir silnikov , v. lozitsky , a. fedchuk a.v. bogatsky physical-chemical institute, odessa, ukraine; institute of chemical biology and fundamental medicine, novosibirsk, russian federation; ukrainian mechnikov research anti-plague institute, odessa, ukraine "chemical" ribonucleases hold promise as tools for studying the structures of rnas and rna-protein complexes, as reactive groups in conjugates intended for cleavage of particular rnas, as therapeuticals inactivating virus genome rnas or certain mrnas, and as a promising antiviral agents. drug design and development of new medicines directed against hiv are permanently actual tasks. the usage of modern quantitative structure-activity relationship (qsar) methods could allow us to solve these problems more effectively. the objective of the present work is qsar analysis of antiviral activity of various tetrapeptides-artifical ribonucleases and consequent molecular design of new antiviral agents. qsar approach based on simplex representation of molecular structure (sirms) has been used for the solution of the formulated problem. usage of sirms allows us to develop the molecular design of the new effective antiviral agents. thorough researches of relationship between antiviral activity (hiv- , % of rna p-o bond cleavage) and a structure of artifical ribonucleases have been carried out. statistic characteristics for pls (partial least squares model) are quite satisfactory (r = . , q = . ). on the base of these models the molecular fragments with positive or negative influence on the explored property have been determined. thus, for example, guanidine and triethylenediamine fragments promote antiviral action. it gives a possibility to realize based on elucidated rules molecular design of compounds with the high level of antiviral activity. the results of prognosis are verifying by the experimental investigations. thus, quite adequate simplex qsar model "anti-hiv activity-artifical ribonucleases structure" was obtained and used for drug design. the cyclotriazadisulfonamide (cada) compound specifically down-modulates the cd receptor expression on the surface of lymphocytes and monocytes/macrophages, the primary receptors utilized by hiv for infection of its target cells. cada thus inhibits the entry of hiv and hhv- (vermeire et al., . virology , - ) . cada chemotherapy may not be susceptible to the production of drug resistant strains of viruses, as its mechanism of action is completely different from those of any other anti-hiv drugs currently in clinical use. the cd down-modulating and antiviral potencies of more than cada analogs have been described (vermeire et al., . mol. pharmacol. , - ) . structural modifications of cada were made to increase potency, reduce cytotoxicity, and improve physical properties. several head group analogs were synthesized with polar groups and good leaving groups ( fig. ) . the anti-hiv and cd down modulation activities of these compounds are being studied. some of these head groups may regenerate the double bond of cada by elimination reactions, potentially producing water-soluble pro-drugs. isocada (sa ), an isomer of cada, was synthesized by cyclization of , , -triazabicyclo-[ . . ]dec- -ene (tbd) (fig. ). this structural modification may reveal a relationship between the symmetry of the molecule and its biological activity. two new fluorine-containing analogs were also synthesized by modifying the toluenesulfonamide side arms (fig. ) . the anti-hiv and cd down modulation activities of these new cada analogs are summarized. the center for drug discovery, university of georgia, athens, ga , usa drug discovery targeted at the elusive viral enzyme, hiv integrase, has not resulted in a single fda-approved drug. in this presentation we describe our molecular modeling studies with conceptually novel inhibitors of hiv integrase that also possess potent in vitro anti-hiv activity. docking was performed on the catalytic core of integrase represented by chain c of pdb structure code bl . building of molecules and primary modeling was done with sybyl . on a silicon graphics onyx (r ) workstation. the program gold . (genetic optimization for ligand docking) was used extensively in evaluating the docking poses of these compounds with the active site of hiv integrase and to give information on key residues involved in the recognition and binding of these ligands. the gold function consists of three basic components: protein-ligand h-bonding energy, protein-ligand van der waals energy, and ligand internal energy. post-processing gold output was done with the program silver . , a utility program supplied with gold for evaluating hydrogen-bonding interactions, metal coordination and van der waals factors. for comparison purposes, additional docking was performed using other docking protocols, notably the sybyl module flexx. data obtained from these and related studies including binding poses, binding affinities, functional and conformational considerations, and gold function scores will be presented and explained. the center for drug discovery, university of georgia, athens, ga , usa hiv integrase is essential for hiv replication and is an attractive target for drug discovery against aids. however, research efforts on drug discovery pertaining to hiv integrase have not resulted in a single fda-approved drug for which mechanism of action is inhibition of hiv integrase. recently, we have been exploring a novel class of diketo acids that are constructed on nucleobase scaffolds and that have a specific arrangement of the functional and hydrophobic group on the scaffold. these compounds are inhibitors both key steps of hiv integrase. one lead compound from this group has also been found to have remarkable in vitro anti-hiv activity. however, the syntheses of the inhibitors are quite challenging. this presentation will describe the synthetic methodologies specifically developed in our laboratory for the preparation of some representative examples of these integrase inhibitors. purification approaches to produce highly purified compounds for biological studies will be explained. structural, functional and conformational data obtained from extensive spectroscopic studies will be discussed. representative anti-hiv integrase data and in vitro anti-hiv screening results will be presented. we have recently reported the synthesis and antiviral activities of a ring-expanded ("fat") nucleoside analogue, called nz- , that inhibits both hcv and hiv in vitro with ec values ranging in micromolar concentrations or less, with little or low toxicity to the host cells. in this part i of the presentation on this subject, we report our preliminary findings on the mechanism of anti-hiv activity of this compound, along with the synthesis and antiviral activity of a few additional analogues. in view of the fact that a number of hiv patients also suffer from hcv as a major coinfection, and that a number of them ultimately die of end-stage hcv-related complications including liver cirrhosis and hepatocellular carcinoma, a drug with dual inhibitory characteristics against both viruses is highly desirable and timely. marina burshtein , alexander serbin , alissa bukrinskaya d.i. ivanovsky institute of virology, moscow, russia; health research and development found, moscow, russia introduction: amantadine is a well-known effective antiinfluenza drug. it was modified to enhance its antiviral activity by chemically linkage with the water-soluble polyanionic matrix via different spacer groups. the other group of used compounds was norbornene derivatives, as norbornene is an adamantane analogue on anti-influenza activity. methods: the absence of cytotoxic effect was shown by mtt test for estimating cytotoxic dose (ctd ). the antiviral effect of the compounds was analyzed in lymphoblastoid mt- cells and in hela cd +/b-galactosidase cells ("magi" cells). the effect of the compounds was registered by immunoblotting of cell lysates and by measuring of b-galactosidase activity. results: the strong inhibition of hiv- replication was observed when the compounds were added with the virus and was expressed even when the compounds added with the virus were removed h after infection. the anti hiv- effect of the compounds was gradually decreased if they were added and h after infection, no inhibition was observed when the compounds were added h after infection. the compounds did not impair the virion structure. adamantane and norbornene derivatives were shown also to inhibit azt resistant viral strains. conclusion: adamantane and norbornene were shown to be active hiv inhibitors with the high selectivity index. the compounds are promising candidates for further investigation including preclinical studies. less is known about the effect of their intracellular half-lives on the maintenance of antiviral activity. to investigate this question, we developed a novel in vitro antiviral persistence assay. measurement of the antiviral persistence of tenofovir (tfv) and abacavir (cbv) was coupled to measurement of the half-lives of their tfv-dp and cbv-tp anabolites. methods: mt- cells or stimulated primary cd + t-cells were incubated with graded concentrations of tfv or cbv for h (h); then extracellular drug was removed by washing. cells were further incubated without drug for - h and then infected with hiv- (iiib or bal). p was quantified on day ; inhibition of hiv- replication due to intracellular drug persistence (pc ) was determined relative to a standard ec . decay of intracellular dp/tps in cd + t-cells was measured using lc/ms/ms. results: in mt- cells, the pc value for tfv h after drug removal remained unchanged relative to the ec (< -fold shift) whereas the pc for cbv shifted > -fold, indicating less persistence of cbv. in cd + t-cells, the pc value for tfv also showed a minimal shift relative to the ec ( . -fold) h after drug removal. cbv showed a much larger relative shift (> -fold). quantification by lc/ms/ms of intracellular tfv-dp and cbv-tp in cd + t-cells in vitro demonstrated that tfv-dp had the longest intracellular half-life of the two drugs (tfv-dp, h versus cbv-tp, h). conclusions: a novel antiviral persistence assay was developed to study the relationship between intracellular nrti halflives and antiviral activity. in both mt- cells and primary activated cd + t-cells, tfv had the longest persistence of antiviral activity. in cd + t-cells, tfv-dp also had the longest half-life of the two nrtis. cbv-tp had a much shorter half-life than tfv-dp and showed less antiviral persistence. although both drugs are approved for qd dosing, the half-life of intracellular tfv-dp maintains antiviral suppression in vitro over a timeframe most consistent with qd dosing. karen m. watson, tracy l. hartman, lu yang, robert w. buckheit jr. imquest biosciences, inc., frederick, md, usa isis is a phosphorothioate oligonucleotide with a molecular structure of t g t . the g-quartet possessing molecule has been shown to be a potent inhibitor of hiv attachment and cell-cell fusion and acts by specifically interacting at the v loop of gp . mapping studies with monoclonal antibodies targeting epitopes in and around the v loop have been used to define the binding site of isis . in vitro, isis inhibits all laboratory and clinical strains of hiv- and hiv- tested, including representative subtype viruses, drug resistant viruses (including mdr viruses) and viruses that utilize the cxcr and ccr chemokine receptors. serial passage of virus in the presence of increasing concentrations of the oligonucleotide did not result in the selection of drug resistant virus strains and combination assays resulted in additive to synergistic interactions with other approved hiv inhibitors. the antiviral and toxicity profiles of isis resulted in the performance of human clinical trials for the therapeutic use of the oligonucleotide to treat hiv infection. the antiviral properties and mechanism of action of isis suggest that it may be an excellent anti-hiv topical microbicide. isis was found to be highly active in a cervical explant model of hiv infection with highly significant inhibition of ccr -tropic strains of virus. activity was also observed in cell-free and cell-associated virus transmission assays, as well as in cd -dependent and cd -independent acute infection inhibition assays. in microbicidal specific combination assays, significant efficacy has been observed with isis used in combination with other microbicidal compounds. the results of these studies suggest that isis may represent a new and novel anti-hiv topical microbicide. karen m. watson, tracy l. hartman, lu yang, robert w. buckheit jr. imquest biosciences, inc., frederick, md, usa though a variety of compounds are being developed as anti-hiv topical microbicides, such as polyanionic molecules, surfactants, natural products, peptides, proteins, heterocycles, and virucidal agents, clinical efficacy studies that demonstrate the ability of these agents to impact virus transmission are still in progress. it has been estimated that a microbicide that is only % effective would have the capacity to prevent millions of new infections each year. thus, one of the challenges in hiv drug development is the discovery of compounds that will inhibit the sexual transmission of infectious organisms between sexual partners. the rapid mutability of hiv and the known presence of drug resistant viruses in wild type virus populations suggests that microbicide development will suffer from the same problems that exist for all hiv therapies, namely the selection of resistant virus strains that will bypass the microbicide barrier and infect target cells in the vaginal or rectal environment even in the presence of the microbicide. thus, it is likely that haart-like combination drug therapies will become the most effective means of inhibiting the sexual transmission of hiv. we have evaluated a wide variety of anti-hiv and anti-sti compounds in vitro alone and in combination with one another and have demonstrated that certain patterns of inhibition (additivity, synergy, antagonism) occur between the various classes of compounds. recently, we have compared the combination anti-hiv activity of microbicide compounds in fresh human pbmcs infected with clinical isolates of hiv to the combination activity of the same test agents in cem-ss-based cultures. in general, these two assay systems yield similar combination assay results. to provide a rationale for the combination use of the compounds in a microbicide setting, the same combination of compounds was evaluated in a microbicide-like virus transmission assay. these combination results suggest that higher levels of synergy between virus attachment and reverse transcriptase inhibitors might be expected in the microbicide environment compared to levels predicted for the systemic therapeutic environment. the results of the combination assays with various microbicides will be presented. during the onset of the hiv disease, hiv rna is continually produced in the face of treatment with haart in circulating reservoirs and rt inhibitors are almost ineffective in the postintegration events. among the classes of anti hiv- drugs, protease inhibitors (pi) are the unique to inhibit the hiv- production in chronically infected macrophages. in the progression of hiv infection, the role of the monocytes-derived macrophages (m/m) is further confirmed as they represent chronologically the first cytotype where the viral replication restarts as a consequence of failure or interruption of antiviral therapy. aim of the work was to evaluate the rebound of hiv- production when pi have been removed in hiv- chronically infected m/m and, moreover, to verify the effect of this removal on virus maturation, infectivity and ability to trigger apoptosis in uninfected peripheral blood lymphocytes (pbl). a rebound of p gag protein was measured starting from h after drug removal yet virus infectivity remained log lower than control up to week. inhibition of hiv- replication was still % and % upon amprenavir and m, respectively. these data were confirmed by western blotting and electronic microscopy showing production and release of immature viral particles. moreover, pi (amprenavir and indinavir) treatment dramatically reduced apoptosis of pbl co-cultured with chronically infected m/m and kept cd /cd ratio above the levels of untreated controls until the th day of co-culture. taken altogether, these findings suggest a wide clinical importance for amprenavir and indinavir for their relevant long-lasting antiviral effect in persistently-infected reservoirs of hiv even in case of drug interruption and/or when hiv infection can restart in districts where drugs find not sufficient concentration. moreover, these results strengthen the evidence for an unique positive utilize of pi against ongoing and productive hiv infection. weili jin, salvatore santino, michael wang gilead sciences, foster city, ca, usa background: effective inhibition of hiv reverse transcriptase (rt) currently represents a crucial objective of antiretroviral therapy. capravirine is a second-generation non-nucleoside rt inhibitor (nnrti) that is capable of blocking the replication of certain nnrti-resistant strains of hiv and was recently in clinical development. in this study, we report on the in vitro selection and characterization of viral resistance to capravirine. methods: viral resistance selection experiments were performed in mt- cells with the hiv iiib isolate and increasing concentrations of capravirine. viruses were analyzed genotypically by population sequencing and by single genome sequencing (sgs). recombinant viruses with nnrti mutations were generated from proviral dna clones. phenotypic analyses were performed in mt- cells. results: capravirine resistance selections were initiated at nm (ec of . nm for capravirine). following nine passages in the presence of increasing concentrations of capravirine, the l i mutation emerged in rt and additional passaging led to v d and f c mutations at higher concentrations ( - nm). further increases in capravirine concentrations led to the emergence of a l i + v d + f c triple mutant, which confers > -fold resistance to capravirine. sgs of mixed viral populations from different passages showed that l i, v d and f c were present on the same genome, with l i as the primary mutation, and f c and v d were acquired sequentially at later passages. through sgs analysis, a l i + k r + v d + f c quadruple mutation on the same genome was also observed at higher capravirine concentrations (> nm). recombinant viruses carrying these mutations were produced to assess their susceptibilities to capravirine. conclusions: after extensive in vitro passaging of hivinfected cells in the presence of capravirine, neither k n nor y c mutations in rt were observed. instead, the l i mutation was initially acquired, followed by mutations f c and v d. addition of the k r mutation to the triple mutant genome, l i + v d + f c, appears to further enhance hiv resistance to capravirine. oluwafemi olawuyi , adeyemi falegan medical microbiology, university college hospital, ibadan, nigeria; dentistry, university college hospital, ibadan, nigeria issue: the percentage of aids/hiv is increasing every year in the third worlds, and this is reinforced by the factor that majority of youth in third worlds do not know his/her hiv status. description: a self developed validated and reliable questionnaire [r = . ] was used to collect the data and percentage was used to analyze the data. the population of the study was made up of youth [female and male] in higher institutions, working places, market places and community streets in nigeria, , -sample size, selected through simple random sampling technique. the mean age is . years old. relative risk [rr] calculated is . , i.e. rr > , indicating that the factor is the risk factor, and the confidential interval [ci] for rr at % significant level is . < . < . from the formula, ci lower limit < rr < ci upper limit. lessons learned: seventy percent of the sample population did know his/her hiv status and had had sexual intercourse in the past before, out which % had the unprotected intercourse once or more, % had protected sex while % were not sure of using protection means. while, % have knowledge about own hiv status and had had sexual intercourse before. ten percent have no knowledge about own hiv status and had no sexual intercourse before. conclusion: aids/hiv still remains a killer disease in the third world. however, the lack of knowledge of individual's hiv status remains the only highest risk factor for the spread of the disease in the third worlds. yuichiro habu , , jacob barnor , , norio yamamoto , kahoko hashimoto , , naoko miyano-kurosaki , , koichi ishikawa , naoki yamamoto , david ofori-adjei , hiroshi takaku , , department of life and environmental sciences, chiba institute of technology, chiba, japan; high technology research center, chiba institute of technology, chiba, japan; japan foundation of aids prevention; department of molecular virology, bio-response, tokyo medical and dental university, tokyo, japan; aids research center, national institute of infectious disease, tokyo, japan; department of virology, noguchi memorial institute for medical research accra-ghana, accra, ghana; bach tech corp. rna interference (rnai) is a potentially strong gene interference tool, which had been successfully used to silence many pathogenic viruses including hiv. however, many recent reports have shown that, in long-term assay cultures involving rna viruses such as hiv, escape mutants breakthrough the silencing effect. in the light of this conundrum, it had been proposed that, vector designed to target multiple genes in a synergistic manner, may address the problem. hence, we designed a chimeric rna expression vector which express vif shrna and decoy tar rna by combining vif shrna and decoy tar rna with linker to which dicer was able to recognize for cleavage, as a second generation rnai expression vector system. the synergistic effect of these molecules enhanced the inhibition of hiv- replication in a long-term transduced pbmcs, h , and jurkat cell culture assays ( weeks) and prevented virus breakthrough associated with sirna-mediated escape variants. notably, hiv- replication was similarly suppressed in the control cells expressing only vif shrna for about weeks, but an increase in virus replication was observed afterwards. hiv viral rna extracted and sequenced at this point indicated escape mutants in the cells expressing the vif target in hiv. we confirmed substitution of bases in the vif shrna target sequence. on the other hand, the incidence of mutation was not observed in a sequence of viral rna from the culture expressing the vif shrna-decoy tar rna at the fourth week. interestingly, virus production was inhibited for a long-term by an effect of decoy tar rna, through the rna-protein interaction. combining shrna with decoy tar rna as second-generation anti-hiv shrna may provide practical basis for applying sirna-based gene therapy to the treatment of hiv/aids. introduction: this is a designed efficient gene therapy against aids/hiv. the novelty of this aids vaccine design/concept is seen in the fact that the 'pol' gene encoding for nonstructural proteins (polyproteins that generate three enzymes: reverse transcriptase, integrase and protease) is cloned in a suitable retroviral vector and adult stem cells are transfected by this and reinfused into the circulation to effectively counter hiv replication and antigenic variation. method: the mrna are isolated from adult stem cells and transcribed into cdna with reverse transcriptase. the cdna are then cloned in a suitable retroviral vector (vacinia) carrying 'pol' gene that confers resistance to a strong reverse transcriptase inhibitor drug. the adult stem cells are transfected by the recombinant mixture, and reinfused into the circulation of hiv infected person. result: the transfected stem cells are reinfused to provide renewable source of more and better empowered normal blood cell types that would disrupt and half hiv replication in the circulation. there would be efficient induction of both humeral and cellular mediated immunity with prolonged expression of antigens and protective immunologic memory generation against hiv antigenic variation. conclusions: this aids vaccine design would lead to both efficient prophylactic and therapeutic therapy against aids in that it would effectively take care of the problematic factor of hiv antigenic variation which has long been the main obstacle to potent aids vaccine development. because of the real risk of interspecies transmission and/or reassortment between avian, swine and human influenza a strains, drug susceptibility monitoring of circulating avian and porzine virus strains appears to be warranted for effective application of antiviral drugs like amantadine. this study was designed to gain insight into amantadine susceptibility of avian and porcine influenza a viruses isolated in germany between and . virus strains were isolated in embryonated chicken eggs and passaged one time in mdck cells. plaque reduction assays were applied to examine virus susceptibility to amantadine. genotyping was used to confirm drug resistance. in the result of these antiviral studies, only of the porzine isolates but all avian isolates were shown to be amantadine-susceptible. interestingly, the three amantadinesensitive porzine strains were isolated between and . all porzine influenza a viruses isolated later on were drugresistant and contained the aa substitutions g e, s n, and r q in the matrix protein (m ). additionally, l a was detected in two h n strains. s n and/or l a are well known amino acid substitutions in m that confer amantadine resistance. the role of the pig as an intermediate host of avian and human influenza a viruses, the possible involvement of genetic reassortment, and the high incidence of naturally amantadineresistant porcine influenza a viruses suggest a real risk of emergence of amantadine resistant human viruses. therefore, further studies are ongoing now to evaluate the circulation of the resistant phenotype in pigs, birds and human. recently much attention has been devoted to searching for effective chemotherapeutic agents and vaccines for eradication of this notorious disease. at present only chemotherapy is available to combat avian flu, for instance, tamiflu, approved for the treatment by the us-fda. development of a simple, novel molecule with potential antiviral activity against is essential to treat avian flu viral infection. isatin ( , -dioxoindole), is a versatile lead molecule for designing of potential antiviral agents and its derivatives were reported to possess broad spectrum antiviral activity. methisazone (nmethylisatin- -thiosemicarbazone) was first clinically approved for treatment of pox viral infections, and its derivatives were documented to have anti-influenza activity. based upon this evidence, the present work was initiated to determine the antiviral activity of novel isatin derivatives against avian flu (h n ) in mdck cells. antiviral activity was studied by virus yield assay (ec ), and cytotoxicity by neutral red uptake assay by uninfected mdck cells. all five compounds of a series inhibited the replication of avian flu (h n ) virus replication in mdck cells and compounds spiii- h and spiii- cl were most active (ec . g/ml, cc > g/ml and si > ). details of these studies and results of treatment of influenza-infected mice are discussed. acknowledgement: supported in part by contract noi-ai- and noi-ai- from virology branch, niaid, nih]. arginine-rich peptide conjugated phophorodiamidate morpholino oligomers (arp-pmo) are nuclease resistant antisense compounds that hybridize to target rna in a sequence-specific manner resulting in disrupted rna function. eight arp-pmo were designed to base-pair with various regions of a/pr/ / (h n ) rna and were then evaluated by hemagglutination and plaque assays for their ability to inhibit fluav production in vero cell culture. arp-pmo targeting the aug translation start site of the np or pb segment mrnas, or the -terminus of their respective vrnas, were highly effective, reducing influenza virus titer by - orders of magnitude in a dose-dependent and sequence-specific manner over a period of days. two of the p-pmo, targeting the pb translation start site region (pb -aug) and the terminus of np vrna (np v ), were evaluated by endpoint dilution (tcid ) or elisa assays against another h n strain (a/wsn/ ), as well as a/memphis/ / (h n ) and a/thailand/ (kan- )/ (h n ). the pb -aug arp-pmo generated over % specific reduction of virus level, regardless of viral subtype or methodology, at concentrations in the range of - m. the np v p-pmo yielded similar results, with the exception of considerably lower efficacy against the h n strain, with which it has two base mispairings. studies are planned to further evaluate of at least two arp-pmos in animal models for h n and h n fluva subtypes. macroheterocyclic compounds containing crown fragments and nitrogen atoms show large-scale biological activity. we synthesized series of aza-crown ethers and their derivatives. we also studied anti-influenza and antiherpetic action of some of them. anti-hsv action of studied compounds was tested using cyto-morphological method. hep- cells were infected with hsv- strain us in dose ifu/cell. the cells were incubated in eagle's medium that contained compounds in a dose of − m in experimental samples, or without them in control samples. then cells were fixed with % ethanol and stained with . % acridine orange solution. the amount of infected cells with dna-containing virus inclusion bodies was counted by fluorescent microscopy. anti-hsv activity of compounds was calculated as the difference between of the percentage of infected cells in treated cell cultures to the percentage of infected cells in untreated cell cultures. anti-influenza activity was studied on the model of replication of a/hong kong/ / (h n ) strain in tissue culture of chorio-allantoic membranes of chicken embryos. compounds were used in a dose of − m during the study of their anti-influenza action. diaza- crown- and two of its derivatives have showmen neither anti-hsv nor anti-influenza activity. diaza- crown- derivatives that contain -oxyethyl-or ethoxycarbonyl-fragments decreased amount of cells infected by hsv- by and %, respectively. both of these compounds inhibited replication of influenza virus on . log tid aza- crown- did not show antiviral activity, but both its derivatives proved to be active inhibitors of hsv and influenza virus reproduction. aza- crown- derivatives that contain -amino- -phenyl-propanoyl-or -benzyloxy- -oxapentyl-fragments decreased amount of cells infected by hsv- with virus-specific intranuclear inclusions by and %, respectively. first compound inhibited replication of influenza virus on . log tid and the second one decreased virus amount on . log tid . the results of this study show that aza-crown ethers are the perspective class of compounds for search of new antiviral agents. acknowledgement: this work was partially supported by stcu (grant # ) . robert w. sidwell , kevin w. bailey , min-hui wong , donald f. smee , dale l. barnard , shanta bantia institute for antiviral research, utah state university, logan, ut, usa; biocryst pharmaceuticals, inc., birmingham, al, usa the cyclopentane neuraminidase inhibitor, peramivir (bcx- , rwj- ) has striking inhibitory effects on a spectrum of influenza viruses in vitro, and has also demonstrated significant effects against influenza a (h n , h n ) and b virus infections when administered orally to mice and ferrets. unfortunately, clinical trials with the drug administered orally were not successful, probably due to low blood levels obtained after oral administration. significant plasma drug levels of peramivir persist up to h after intramuscular (i.m.) injection; more importantly, however, is the observation that peramivir remains tightly bound to influenza virus n neuraminidase for over h, suggesting single i.m. or intravenous (i.v.) therapy with the drug may be highly effective against an influenza infection. experiments now in press have indicated that single i.m. peramivir therapy administered up to h after virus exposure was protective to mice infected with influenza a (h n ) virus. in the present study, peramivir was administered i.m. or i.v. in a single injection h pre-virus exposure in separate experiments to mice infected with an influenza a (h n ) virus; efficacy was compared to similar dosages of oseltamivir and oseltamivir carboxylate run in parallel. dosages of and mg/kg of peramivir administered by either route significantly prevented deaths, lessened arterial oxygen (sao ) decline, inhibited development of lung consolidation, and inhibited lung virus titers. the lung assays were performed at varying times after virus exposure. oseltamivir and oseltamivir carboxylate, which do not have the same neuraminidase binding abilities seen with peramivir, were less efficacious in these experiments. delaying the single i.v. therapy up to h after virus exposure also significantly inhibited the virus infection. peramivir appeared to be well tolerated in toxicity control animals run concomitantly with these studies. these data indicate parenterally administered peramivir may hold promise as a therapy for clinical influenza a (h n ) virus infections. acknowledgement: supported by contract no -ai- from the virology branch, niaid, nih. hiroshi saitoh , naoko miyano-kurosaki , , hiroshi takaku , department of life and environmental sciences, faculty of engineering, chiba institute of technology, chiba, japan; department of life and environmental sciences, faculty of engineering and high technology research center, chiba institute of technology, chiba, japan background: influenza virus causes widespread infection in the human respiratory tract, but existing vaccines and drug therapy are of limited value. recently, small interfering rnas (sirnas) are a powerful tool for sequence-specific, post-transcriptional gene silencing and have a potential therapeutic and prophylactic application against cancer, as well as infectious diseases. here we show that short interfering rnas (sirnas) specific for conserved regions of the viral genome can potently inhibit influenza virus production in cell lines. the influenza virus np gene is a potential target for rnai technology. on the other hand, the baculovirus (acmnpv) can infect a variety of mammalian cells, facilitating its use as a virus vector for gene delivery in viral entry into cells. in this study, we describe the inhibition of influenza virus production by baculovirus-mediated shrna expression vectors. methods: the psv neo-u plasmid vectors and pvl based baculovirus vectors were used in this study. the influenza virus a and b np genes were made into the target and the shrna expression plasmid vectors were constructed under the control of the human u pol iii promoter. the shrna expression plasmids or shrna expression baculovirus vectors introduced into mdck cells, and h later the cells were infected with either a/pr or b/ibaraki virus at a moi of . . at h postinfection, culture supernatants were harvested and assayed to determine the virus titer by plaque assay. conclusion: the findings reveal that newly synthesized np proteins are required for influenza virus replication and provide a basis for the development of shrnas expression plasmids as prophylaxis and therapy for influenza infection in humans. julia serkedjieva, ekaterina krumova, tsvetanka stefanova, nadja nikolova, maria angelova institute of microbiology, bulgarian academy of sciences, sofia, bulgaria a semi-standardized polyphenol-rich extract (pre), obtained from geranium sanguineum l., inhibited the reproduction of influenza viruses types a and b in vitro and in ovo and protected mice from mortality in the experimental influenza virus infection (serkedjieva and manolova, ) . the selective in vitro virus-inhibitory activity of pre was fairly modest and this was in contrast with the significant protection in vivo. thus, the therapeutic effect of pre needed explanation. it was presumed that it might be attributed to a combination of more than one biological activities known for natural polyphenols. we have demonstrated previously that pre manifested strong antioxidant and radical-scavenging activities in model systems (sokmen et al., ) . the current study was undertaken to investigate the effect of the plant extract on the levels of the antioxidant enzymes superoxide dismutase (sod), catalase (kt) and peroxidase (po) in mice lungs during influenza virus infection as well as the effect of pre on the production of reactive oxygen species (ros) and reactive nitrogen intermediates (rni) by alveolar macrophages in influenza virus infected mice. mice were challenged intranasally (i.n.) with - ld of a/aichi/ / (h n ) influenza virus. pre was administered by i.n. instillation h before infection in the dose of mg/kg. it was established that influenza infection induced an increase in sod, kt and po production and on days and after infection their levels reached - % of placebo control. the application of pre brought enzymes values to control levels. influenza infection caused also a significant increase of h o , o •− and no production by alveolar macrophages; the generation of ros and rni peaked on day . pre-treatment before viral challenge reduced this excessive production. in conclusion, the obtained results outlined the antioxidant and radical scavenging properties of the plant extract; pre beneficially modulated the oxidative stress response in influenza virus-induced pneumonia. this alternative mechanism of action might contribute to the overall protective effect in the lethal murine experimental influenza infection. the antiviral activity of s , a natural herb extract, ji-sun kwon , hyun-jeong lee , chi-ung moon , jong-hwan kwak , youn-jeong lee , chang-seon song avian disease laboratory, college of veterinary medicine, konkuk university, seoul, korea; hanyang university, seoul, korea; sungkyunkwan university, seoul, korea; national veterinary research and quarantine services, seoul, korea the antiviral activity of s , one of the traditional korean medical herb extract, against influenza virus was investigated. the % effective concentration (ec ) using plaque reduction assay was . ug/ml and the mean % cytotoxic concentration (cc ) using wst- assay in the mdck cells was ug/ml. oral gavage treatment of the s to balb/c mice infected with a/pr/ / (h n ) influenza virus showed the therapeutic effects as delaying clinical signs, significant inhibition of death and reduction of lung virus titers. to identify the lead molecules, the s was subjected to further fractionation, purification, and isolation of active compounds. the antiviral activity of these natural herb compounds will be discussed. these results suggest that the s is a possible candidate for the development of new antiviral medicine for influenza therapy. hiroshi takaku , , , takayuki abe , hitoshi takahashi , naoko miyano-kurosaki , department life environ. sci., chiba inst. tech., chiba, japan; high tech. res. center, chiba inst. tech., chiba, japan; res. inst. microbial dis., osaka university, osaka, japan; bach tech corp background: the baculovirus autographa californica nuclear polyhedrosis virus (acnpv) has long been used as a biopesticide and as a tool for an efficient recombinant protein production in insect cells. in this study, we examined the immunization of a recombinant baculovirus expressing the influenza virus hemagglutinin (ha) against lethal influenza infection in mice. protection was observed in mice immunized intranasally with not only the recombinant baculovirus but also a wild-type baculovirus. baculovirus was also shown to induce secretion of inflammatory cytokines, such as tnf-␣ and il- , in murine raw . macrophage cell line. results: a varied route of immunization with a recombinant baculovirus expressing the influenza virus hemagglutinin protein of a/pr/ / (h n ) virus against lethal influenza infection was examined in mice. the recombinant baculovirus encoding the hemagglutinin gene under the control of chicken ␤ actin promoter was inoculated twice, weeks apart, at a dose of . × pfu per mouse by intramuscular, intradermal, intraperitoneal, and intranasal routes. mice intramuscularly and intraperitoneally immunized with the recombinant exhibited higher level of production of serum anti ha antibody than those immunized via the other routes, but protection was only achieved by the intranasal immunization. surprisingly, mice immunized with a wild-type baculovirus with intranasal route were also protected from the lethal influenza virus challenge. sufficient protection in mice was achieved by the intranasal immunizations with pfu of either the recombinant or wild-type baculovirus, as evaluated by the reduction of virus titer, production of inflammatory cytokines, and pulmonary consolidations in the lung. these results indicate that infection with a baculovirus induces a strong innate immune response and protection of mice from lethal influenza virus infection. conclusion: baculovirus (cpg motifs) induces a strong innate immune response and protection of mice from lethal influenza virus a and b infection. andrew vaillant , annie lebel , nathalie goyette , guy boivin , jean-marc juteau , phil wyde replicor inc., laval, que., canada; chuq-chul and laval university, st. foy, que., canada; baylor college of medicine, university of texas, houston, tx, usa potent antiviral activity of phosphorothioate oligonucleotides (ps-ons) was observed against influenza viral infections. antiviral activity was sequence-independent, size dependent (optimally active ps-ons were ≥ bases in length) and dependent on the presence of the phosphorothioate modification (hydrophobicity). binding studies showed that rep (a mer degenerate ps-on) interacts with both neuraminidase and hemagglutinin although the sialidase activity of neuraminidase was not affected, suggesting that the structural interactions of these proteins required for influenza activity are the target for this compound. the requirement for hydrophobicity further suggests that the alpha helical regions of hemagglutinin are one of the regions of interaction. the antiviral activity of rep was conserved in many influenza a and b strains suggesting potential therapeutic activity against avian flu and other newly emerging influenza strains. rep aerosol has excellent characteristics for lung deposition and aerosol treatment with rep was well tolerated and highly effective against infections with influenza a both in prophylaxis and h after infection. these results demonstrate the therapeutic potential of aerosolized ps-ons against influenza infection. acknowledgement: supported by nih contract no -ai- . irina v. alymova , y. sudhakara babu , allen portner virology division, department of infectious diseases, st. jude children's research hospital, memphis, tn , usa; biocryst pharmaceutical, inc., birmingham, al , usa bcx is a novel selective inhibitor of human parainfluenza virus infections, which design was based on the threedimensional structure of the hemagglutinin-neuraminidase (hn) protein of newcastle disease virus. compound exhibited striking activity against parainfluenza viruses in vitro and in vivo, and was efficacious in prophylaxis of lethal synergism between parainfluenza virus and streptococcus pneumoniae in a mouse model. present study was conducted to determine if bcx 's resistant variants of the recombinant sendai virus whose hn gene was replaced with that of human parainfluenza virus type (rsev(hpiv- hn) could be selected in tissue culture and animals. for this purpose virus was serially passaged in llc-mk cells at moi . in the presence of increasing (from to m) concentrations of compound; infected × /svj mice were treated with mg/kg/day of bcx twice for five days. treatment started h before infection. individual clones of viruses were analyzed for the presence of mutations. one mutation, e k, on the globular head region of the hn protein was selected in tissue culture after the fifth and eleventh passages of rsev (hpiv- hn) . several mutations in hn gene of rsev (hpiv- hn) were selected in an animal model after the second passage of virus from mice treated with bcx . two mutations, n s and p q, were located in the cytoplasmic domain of hn protein; mutations n s and t a were found on the globular head region of the glycoprotein. only nonconserved amino-acid residues of hn protein were involved in substitutions. all isolated mutant viruses were stable after the five passages in llc-mk cells without drug; did not develop other substitutions in the presence of drug and displayed no resistance to bcx both in vitro and in vivo. infectivity of all mutants was not altered to compare with the wild type of rsev (hpiv- hn) virus. taking together our results indicate that prophylaxis/treatment of human parainfluenza virus infections with bcx may not lead to appearance of clinically significant variant of viruses. kie-hoon jung , michelle mendenhall , lawrence m. blatt , robert w. sidwell , brian b. gowen institute for antiviral research, utah state university, logan, ut, usa; intermune, brisbane, ca, usa hantavirus pulmonary syndrome (hps) is an acute human respiratory disease with remarkably high case fatality rates ( - %) for which the etiological agents are members of the bunyaviridae family, genus hantavirus. maporal (map) virus is a recently identified hantavirus isolated in western venezuela, which is most similar phylogenetically to hantaviruses known to cause hps in southern regions of south america. despite the lack of evidence that map can productively infect humans and cause hps, infection of hamsters closely resembles disease manifestations associated with human hps. hantaviruses, in general, are known to produce little to no cytopathic effect (cpe) in cultured cell lines. unexpectedly, we found that map produces remarkable cpe in several vero cell lines facilitating the evaluation of known antiviral agents, ribavirin and interferon alfacon- . both drugs were highly effective at reducing cpe, as determined by visual examination and neutral red dye uptake, associated with map infection. since much of the observed cpe may be due to apoptosis of uninfected bystander cells, we also developed a quantitative (q)rt-pcr assay to detect copies of map genomic sequence to more directly assess the inhibition of viral replication. data obtained using the qrt-pcr-based assay were consistent with the visual cpe reduction and neutral red-uptake cytotoxicity findings. the development of in vitro antiviral testing methods for map are essential to the evolution of the in vivo hamster disease model of hps. the latter is of utmost importance considering the current need for effective antivirals for the treatment of hps and the lack of a suitable model that does not require biosafety level containment facilities. acknowledgement: supported by contract no -ai- from the virology branch, national institute of allergy and infectious diseases, national institutes of health. nucleoside analogues are widely used in antiviral and anticancer chemotherapy. for this class of drugs, intracellular conversion of the nucleoside analogue into the corresponding mono-, -di-, and -triphosphate after target cell penetration is a prerequisite for biological activity. because of the structural differences from natural nucleosides, this conversion is often inefficient and, as a consequence, therapeutic efficacy is sometimes limited. the free phosphates, or nucleotides, have limited utility in therapy on account of their poor membrane permeability and chemical stability. one approach to improve the therapeutic potential of nucleoside analogues is the delivery of the corresponding nucleotide entities via neutral, lipophilic prodrugs, or protides. the nucleoside aryl phosphoramidate approach, developed by mcguigan and co-workers ( ) has been successfully applied to a number of different nucleosides (azt, d t, dda, d a). the general structure of aryl phosphoramidates encompasses two masking groups, an amino acid ester and an aryl moiety bonded to the phosphate group. in order to apply this protide technology to nucleosides with the potential for anti-hepatitis c virus (hcv) activity, we have undertaken studies designed to probe the effect of varying the natural and unnatural amino acid esters and the aryl groups used as masking groups in the target phosphoramidates. these compounds have been synthesised and evaluated using genotipe b sub-genomic hcv replicon. we have prepared a variety of arylphosphoramidate derivatives from a range of -substituted nucleosides, including azido-cytidine, -azido-uridine, and , -protected variants. with certain nucleoside phophoramidates, we have observed dramatic enhancement (> -fold) of replicon activity relative to the parent nucleoside. the synthesis, biological activity and sar of these compounds will be presented. reference mcguigan, d. cahard, balzarini, j., . mini-review. med. chem. , - . we have identified a series of novel anthranilic acid derivatives that are potent, reversible inhibitors of hepatitis c virus (hcv) ns b polymerase, an essential enzyme for viral replication. the micromolar ns b polymerase inhibitors belong to the n-phenoxyacetylanthranilic acid chemotype. x-ray crystallography determined that the inhibitors bound to ns b between the thumb and palm regions adjacent to the active site. guided by crystallography, subsequent modifications to the hydrogen bonding and lipophilic regions of the inhibitors resulted in greatly improved activity against ns b. further sar studies revealed a second, more potent sub-series where the phenoxy group was replaced by an anilino group. analogs in both subseries showed antiviral activity in a cell-based replicon model of hcv. andrea brancale , dimitrios vlachakis , maria chiara barbera , romano silvestri , colin berry , johan neyts cardiff university, the welsh school of pharmacy, cardiff cf xf, uk; universita' degli studi "la sapienza", dipartimento di studi farmaceutici, roma, italy; cardiff university, cardiff school of biosciences, cardiff cf us, uk; rega institute for medical research, k.u. leuven, b leuven, belgium hepatitis c is a viral infection that affects million people worldwide, including million in the united states and million in europe. the virus establishes a chronic infection in - % of cases and % of affected individuals develop cirrhosis. at the moment there is neither a vaccine nor an effective antiviral therapy available and efforts to identify a specific anti-hcv inhibitor have dramatically intensified in the last few years. many research groups have focused their interest on the enzymes involved in the viral replication and, among these enzyme, the viral helicase/ntpase has proven to be a suitable target for developing novel anti-hcv compounds. compound is a potent inhibitor of the hcv helicase and, although its mode of action is still uncertain, it has been proposed that it acts as competitive inhibitor of rna binding. starting from this hypothesis, we have prepared a series of novel compounds based on the structure of where the benzimidazole moiety has been replaced by different chemical groups, including the negatively charged carboxylate moiety, which should mimic the phosphate backbone of the nucleic acid. the synthesis, the enzyme inhibition and the biological evaluation in replicon of these novel compounds will be presented and analyzed. dale r. cameron migenix inc., wesbrook mall, vancouver, bc, canada v s l hepatitis c virus (hcv), a leading cause of liver disease, continues to be an attractive target for new drug development. among the more favourable approaches to developing new hcv drugs is to target the rna-dependant rna (rdr) polymerase (ns b), which has been shown to be an essential enzyme for replication. there are several published non-nucleoside inhibitors of this polymerase (some in clinical development) and several published allosteric binding pockets on the protein they target. to be successful, traditional lead identification can be timeintensive, costly and have large infrastructure requirements. increasingly, a push towards effective computational-based screening has led to the development of virtual screening tools. such tools allow investigation of large quantities of compounds in silico for particular properties without the need for compound synthesis or high throughput screening. moreover, these techniques require only a modest infrastructure investment and are very efficient. we employed the openeye set of screening tools (omega, rocs and eon) in concert with publicly available hcv inhibitor information, and commercial databases to identify novel leads. the inhibitor coordinates from a protein-inhibitor complex crystal structure were utilized as the target. available compound databases (asinex and chembridge) were utilized as the testset of compounds. filtered compound conformers were generated using omega and compared with the template using rocs with post-analysis by eon. visual analysis to maximize particular desirable binding features while minimizing protein-inhibitor steric clash allowed the list of potential hits to be further narrowed. multiple classes of compound were identified from the above procedure and after sourcing a subset of the actual compounds or close analogs, they were tested for enzymatic inhibition activity and further characterized. iteration of the process resulted in the identification of a lead compound class containing multiple active compounds, one with reasonable replicon activity. in conclusion, readily available structural and database information and virtual screening tools can be successfully utilized to identify novel inhibitors of hcv rdr polymerase which, in turn, can serve as novel leads for developing new therapies for treating hcv. synthesis, antiviral activity, and cytotoxicity of some novel quinazolin- ( h)-one derivatives a series of novel -bromo/ , -dibromo- -( -oxo- -phenyl- h-quinazolin- y-l)-benzenesulphonamides were synthesized by condensation of -substituted benzo[ , ]oxazine- -ones and sulphonamide. their chemical structures were assigned by means of spectral analysis (ft-ir, pmr, ms). synthesized compounds were screened for in vitro antiviral activity against human pathogenic viruses (hiv, hcv, hsv, vv). -bromo- -( -oxo- -phenyl- h-quinazolin- y-l)benzenesulphonamide (sps-ii) and -( -oxo- -phenyl- hquinazolin- y-l)-benzenesulphonamide (sps-i) inhibits the replication of hiv- in acutely infected mt- cells at a concentration of approximately g/ml, while not being toxic to the host cell at a concentration of or > g/ml (selectivity index: and > ), respectively. in huh - cells sps-i inhibited hcv rna synthesis at ec of g/ml, while at cc for cell growth g/ml. sps-ii inhibited the virus-induced cytopathicity in human embryonic lung (hel) cell infection with hsv- , hsv- or vaccinia (vv) at a concentration of g/ml, while not being toxic to the cells up to a concentration of g/ml. further molecular modification in this series of compounds may help in optimising their antiviral activity. wengang yang, yongnian sun, avinash phadke, milind deshpande, mingjun huang achillion pharmaceuticals, new haven, ct , usa hcv nonstructural protein ns b is the catalytic subunit of the replication complexes, possessing a motif characteristic of rna-dependent rna polymerases. biochemical assays using recombinant ns b have been used to investigate ns b nonnucleoside inhibitors. however, the inhibitory effect of compounds often varies with the forms of recombinant ns b and the concentrations of the template and/or primer used in the assays. in addition, it does not always correlate to that obtained with replicon-containing cells. these observations have cast concerns about the validity of these cell-free assays. in the report, we explored replication complexes, isolated as crude membrane fractions from replicon-containing cells, for their competency to synthesize viral rna in vitro as well as their responsiveness to ns b inhibitors. after optimizing the experimental conditions, two species of nascent viral rna, one double-stranded and the other single-stranded, were readily detected. the addition of ns b nucleotide inhibitor blocked synthesis of both species. the presence of nonnucleoside inhibitors, however, inhibited mostly single-stranded rna (ssrna) synthesis. in addition, the replication complexes isolated from the cells containing a replicon that carried a resistant mutation in ns b to the nonnucleoside inhibitor were able to synthesize the same amount of ssrna in vitro regardless of the presence or absence of the inhibitor, demonstrating that the phenomenon is due to the specific inhibitory effect of the compound on ns b. combining with kinetic studies that ssrna synthesis was inhibited only when the nonnucleoside inhibitor was present during the pulse period, we conclude that ssrna synthesis catalyzed by the replication complexes in vitro is likely derived from the de novo initiation. we have recently reported the synthesis and antiviral activities of a ring-expanded ("fat") nucleoside analogue, called nz- , that inhibits both hcv and hiv in vitro with ec values ranging in micromolar concentrations or less, and little or low toxicity to the host cells. in this part ii of the presentation on this subject, we report our preliminary results on mechanistic studies of anti-hcv activity of this compound, along with the synthesis and antiviral activity of a few additional analogues in the series. in light of the fact that hcv is a major co-infection in patients infected with hiv, and that a number of them ultimately die of end-stage hcv-related complications including liver cirrhosis and hepatocellular carcinoma, a drug with dual inhibitory characteristics against both viruses is highly desirable and timely. nigel bourne, ronald veselenak, richard pyles, minkyung yi, stanley lemon the university of texas medical branch, galveston, tx, usa more than million people worldwide are estimated to be infected with hepatitis c virus (hcv). in the majority of these people a chronic infection is established which can result in serious long-term liver damage including progressive fibrosis, cirrhosis and hepatocellular carcinoma. in fact, hcv is believed to cause more than , cases of liver cancer annually worldwide and accounts for at least % of liver transplants in the us. current treatment options are limited and there is a high treatment failure rate. thus, there is a real need for new treatment options. amantadine has been evaluated as a treatment for chronic hcv infection in a number of clinical studies both as a monotherapy and in combination with other therapeutics. however, the results of these trials have been contradictory and at this time the clinical potential of amantadine as a therapy for chronic hcv infection remains unclear. recent studies have shown that the small hydrophobic hcv p protein forms an amantadine sensitive ion channel providing a possible basis for antiviral activity. we examined the ability of amantadine to reduce hcv replication in both subgenomic and full-length hcv replicons of genotypes a strain h c and genotype b strain n. in these studies amantadine failed to reduce viral rna replication in any of the replicons tested. further, in infectious virus assays using hcv genotype a strain jhf- um amantadine failed to reduce viral rna levels under any of the conditions tested. however, in these infectious virus studies, when the viral inoculum was treated with amantadine prior to infection of cell monolayers, or when the amantadine was added to cells h after virus adsorption there was a significant reduction in the number of infectious viral foci observed after h incubation (p < . and < . , respectively). these results suggest that even in the absence of a direct impact on rna replication amantadine has antiviral activity. we are currently evaluating amantadine for activity in infectious hcv genotype a assays to further define its antiviral spectrum of activity. studies to more fully define its mechanism of action in the virus life cycle are also underway. dominique dugourd, raymond siu, jeremy fenn migenix inc., vancouver, bc, canada celgosivir is an alpha glucosidase inhibitor that is being developed for the treatment of hepatitis c virus (hcv) infections in humans. the purpose of this study was to evaluate the in vitro antiviral activity of celgosivir and its primary active metabolite, castanospermine, when combined with current approved therapies (ribavirin, interferon ␣- b, or both) in a surrogate model of hcv (bovine viral diarrhea virus (bvdv)). compounds alone or in combination were tested against bvdv in infected madin-darby bovine kidney (mdbk) cells. synergies were analyzed using isobolograms and volume of synergy measurements (macsynergy ii tm software). the celgosivirinterferon ␣ b combination was significantly more synergistic than the celgosivir-ribavirin combination (∼ -fold), or the ribavirin-interferon ␣ b combination (∼ -fold). similarly, the castanospermine-interferon ␣ b double combination was more synergistic than the castanospermine-ribavirin combination (∼ -fold), or the ribavirin-interferon ␣ b combination (∼ . -fold). the combinations of celgosivir-interferon ␣ b or castanospermine-interferon ␣ b led to significant decreases in the ec s of celgosivir (up to > -fold) and castanospermine (up to > -fold). the effective ec s of celgosivir or castanospermine were further reduced by the addition of ribavirin. the cytotoxicity of the double and triple combinations was additive or less than additive, indicating that combinations of celgosivir or castanospermine with ribavirin and/or interferon ␣ b were generally less toxic than expected. these results indicate that the combination of celgosivir with interferon ␣ b or with interferon ␣ b and ribavirin may be effective in the treatment of hcv. pegylated interferon ␣ plus ribavirin is the current standard of care for the treatment of chronic hepatitis c virus (hcv) infections. this regimen results in sustained virologic response in only about % of patients and is associated with significant treatment-associated toxicities. a number of approaches are being used to identify novel therapeutic combinations with better tolerability and/or efficacy. inhibitors of endoplasmic reticulum (er) ␣-glucosidase have been shown to inhibit viral replication and secretion and may have utility as part of new multi-drug treatment cocktails. the ␣-glucosidase inhibitor celgosivir is currently being evaluated in combination with pegylated interferon ␣ and ribavirin in humans. the purpose of this study was to evaluate the antiviral effects of combinations of celgosivir and castanospermine, the primary active metabolite of celgosivir, with other antiviral agents having diverse mechanisms of action. the effect of the combination of celgosivir or castanospermine with the nucleoside analogue nm- , amantadine, and another iminosugar, n-butyl-deoxynojirimycin (nb-dnj) was determined in a cytopathic assay using the hcv surrogate virus bovine viral diarrhea virus in madin darby bovine kidney cells. synergies were analyzed using isobolograms and volume of synergy measurements (macsynergy ii tm software). volumes of synergy indicated that the castanospermine and nb-dnj combination was additive, while the celgosivir and nb-dnj combination was synergistic at high nb-dnj concentrations (> m). celgosivir and castanospermine were synergistic with both amantadine and nm- , with volumes of synergy between and m%. isobologram analysis confirmed these synergistic interactions. these results indicate that celgosivir could be considered in combination regimens containing drugs that directly target viral replication like nm- . mechanism(s) of synergy are under investigation. department of biotechnology, yonsei university, seoul - , korea hepatitis c virus (hcv) is an enveloped virus with positivestranded rna genome of approximately . kilobases and a major cause of non-a and non-b hepatitis, leading to liver cirrhosis and hepatocellular carcinoma. combination of interferon-␣ (ifn-␣) and ribavirin is the current standard therapy for the treatment of hcv infection, but there is no specific antiviral therapy available. the hcv viral genome encodes a single polyprotein of approximately amino acids, which is proteolytically processed by a combination of host and viral proteases into at least distinct structural and nonstructural proteins. the structural proteins include c, e , e , and p and the nonstructural (ns) proteins include ns , ns , ns a, ns b, ns a, and ns b. as new hcv specific therapies, small-molecule inhibitors against hcv enzymes including ns b protein, the viral rna-dependent rna polymerase (rdrp), and ns protease are in clinical tests. however, rapid emerging of drugresistant mutants has been hampering their practical clinical applications. recently, we have shown that phosphorylation of hcv rna polymerase by protein kinase c-like (prk ) regulates virus rna replication. hcv rna replication was inhibited when prk expression level was down-regulated by using a prk -specific sirna. in this study, we investigated the anti-hcv effect of prk inhibitors in an hcv subgenomic replicon system. treatment of the replicon cells with prk inhibitors suppressing the endogenous prk activity inhibited the phosphorylation of hcv rna polymerase and resulted in suppression of hcv rna replication in a dose-dependent manner. furthermore, the prk inhibitor in combination with ifn-␣ more effectively inhibited hcv rna replication than ifn-␣ alone. because the prk inhibitor did not show cytotoxicity in the cell-based drug inhibition studies and cellular proteins rarely get mutated, prk can serve as a cellular target for therapeutic intervention of hcv replication. specific inactivation of prk activity will provide an opportunity to interfere with hcv rna replication. haitao guo , tianlun zhou , ju-tao guo , andrea cuconati , anand mehta , timothy block drexel university college of medicine, doylestown, pa, usa; nucleonic inc., irvine, ca, usa; hepatitis b foundation, doylestown, pa, usa more than million people worldwide are chronically infected with hepatitis b virus (hbv). the major complication of chronic hepatitis b is the development of primary hepatocellular carcinoma (hcc), which causes an estimated , deaths annually. currently clinical treatments (␣-interferon and nucleoside analogs) of chronic hepatitis b rarely cure the virus infection. this is due, at least in part, to their failure to eliminate viral covalently closed circular (ccc) dna from the nuclei of infected hepatocytes. hbv cccdna is essential to the virus life cycle by serving as the template for the transcription of the pregenomic rna and of the subviral rna species. its elimination during chronic infection is considered critical to long-term therapy. however, cccdna has not previously been targeted in high throughput screens of small molecule libraries. to screen compound libraries for antiviral drugs targeting cccdna, we set out to develop a cell-based assay suitable for high throughput screening. since cccdna is time-consuming to assay, it was desirable to use a viral gene product that could serve as a reporter for intracellular cccdna level. we predicted that the secretion of hbv e antigen (hbeag) by hepad cells, a hepg -derived tetracycline inducible hbv expression cell line, would be cccdna-dependent. this is because a large portion of pre-core mrna leader sequence in the terminus of integrated viral genome was deleted, preventing hbeag expression from transgene, but could be restored from the terminal redundancy of pre-genomic rna during viral dna replication and subsequent cccdna formation. our experimental results showed that following induction, hepad produced and accumulated cccdna, which became detectable between and days. hbeag synthesis and secretion into culture fluid were dependent upon and proportional to the level of cccdna detected. therefore, the secretion of hbeag by hepad cells could potentially serve as a convenient reporter for the high throughput screening of novel antiviral drugs targeting hbv cccdna. kathy aldern, james beadle, karl hostetler university of california, san diego and the veterans medical research foundation, san diego, ca, usa (s)-hpmpa is a broad spectrum antiviral active against orthopoxviruses, hbv, cmv, hsv, and other herpes group viruses. we have shown that hdp-(s)-hpmpa has greatly enhanced antiviral activity against these viruses. in addition, while hpmpa itself is nearly inactive against hiv, we showed that hdp-(s)-hpmpa exhibited an ec > logs less than unmodified hpmpa in mt- cells by p reduction assay. to evaluate the metabolic basis for the increased antiviral activity, we studied and compared the cellular uptake of radiolabeled cdv, (s)-hpmpa and their hdp-esters and conversion to hpmpa-diphosphate (hpmpapp) and cdv-diphosphate (cdvpp) in mrc- human lung fibroblasts using hplc partisil sax ion exchange chromatography. cellular uptake of hdp-cdv and hdp-(s)-hpmpa was similar. however, when cells were exposed to the respective drugs for , and h, hpmpapp appeared much earlier than cdvpp and reached levels several fold greater than observed with hdp-cdv. drug wash out experiments were carried out in mrc- cells exposed to radiolabeled hdp-cdv and hdp-(s)-hpmpa. after h, the culture medium was removed and replaced with complete medium without drug and the levels of hpmpapp and cdvpp were measured by hplc every days for - days. levels of the diphosphates declined slowly with a t / of - days. in conclusion, hdp-(s)-hpmpa is converted to its diphosphate more rapidly than hdp-cdv and reaches higher intracellular levels. this may explain, in part, its greater antiviral activity. the antiviral activity and oral bioavailability of cidofovir (cdv) is enhanced when the phosphonate is esterified with various straight chain alkoxyalkyl groups. the length of this chain is an important determinant of antiviral activity and selectivity. however, in some cases, rapid metabolism to an inactive short chain metabolite was observed. to enhance the metabolic stability of these esters, we synthesized cidofovir alkoxyalkyl esters bearing methyl groups on the penultimate carbon of the alkyl chain. enzymatic stability of -me-hdp-cdv ( ) was tested in liver s fractions from various species. in mouse and human liver s fractions, compound was completely stable for min while - % of the straight chain hdp-cdv was metabolized. the branched alkoxyalkyl esters were then evaluated in cells infected with vaccinia, cowpox and ectromelia viruses. the branched methyl analogs were substantially more active than cdv and equal to or slightly more active than the straight chain analogs. compound retained full activity compared to hdp-cdv and compound showed greater activity against orthopoxviruses compared to its unbranched analog. we believe that the structural modification of the alkyl chain slows the formation of inactive metabolites, possibly by interfering with oxidation and may result in better pharmacokinetics and more potent antiviral activity against orthopoxvirus infection in vivo. cidofovir ([ -(s)- -hydroxy- -(phosphonomethoxy)propyl]cytosine, hpmpc) is a broad spectrum antiviral agent clinically used for treatment of aids-related cmv retinitis. cidofovir has limited oral bioavailability (< %), attributed to ionization of its phosphonic diacid moiety under physiological conditions. we have shown that masking of this group by conjugation of the cyclic form of the drug (chpmpc) via a ser side chain p-o ester linkage with x-ser dipeptides, where x = a hydrophobic amino acid, can result in prodrugs that afford significantly improved biological availability of parent drug in an animal model. here we describe the total synthesis of novel cyclic cidofovir prodrugs ab of l-val and l-phe using an alternative conjugation strategy, viz. via an ethylene glycol link utilizing p-o and c-o ester bonds. the preparation of the hpmpc synthon from r-glycidol used our modification of the literature procedure (brodfuehrer et al., ) , involving reaction of tritylated (r)-glycidol directly with unprotected cytosine to achieve regiospecific opening of the epoxide ring, followed by reaction with benzoic acid anhydride to obtain the desired n-benzoyl intermediate needed to continue the synthesis. pybop was used as condensing agent in a convenient, one-pot conversion of hpmpc to chpmpc and subsequent esterfication of the latter by the ethylene glycol-modified amino acids. the prodrugs were converted to drug by cellular (caco- , hff) and tissue (liver and intestinal) homogenates, but did not show enhanced oral bioavailability when evaluated in a rat model, suggesting that such compounds may be useful for understanding the effectiveness of in drug delivery. cidofovir (cdv) is a broad-spectrum anti-viral agent that is used to treat aids-related cytomegalovirus (cmv) retinitis and other cmv infections. cdv has good in vitro activity against orthopox viruses, including smallpox; however, its use is limited because of the drug's low oral bioavailability and poor transport into cells. in order to improve its oral bioavailability, our group has synthesized a series of dipeptide and amino acid prodrugs of the cyclic analog of cidofovir (ccdv). in the current project, we examined the cytotoxicity and antiviral activity of the prodrugs, showing that the compounds are not cytotoxic and have diverse activity against hcmv and orthopox viruses (vaccinia and cow pox) with % inhibitory concentrations ranging from . to . and m and greater, respectively for the two virus types. in vitro and in situ perfusion studies established that the permeability of the prodrugs is enhanced more than -fold and that the transport is mediated, at least in part, by the intestinal dipeptide transporter. we also have found that the bioavailability of the prodrugs is dependent upon the prodrug structure and that we can achieve up to an eight-fold increase in bioavailability over the parent compound in vivo. drug stability experiments showed that in gastrointestinal and liver homogenates, the ccdv prodrugs are enzymatically hydrolyzed to the parent compound. it is clear from this work that the biologically benign dipeptide moiety, strategically linked to the drug to mask its anionic properties, significantly enhances intestinal transport of ccdv, creating the possibility of an orally bioavailable form of ccdv with low toxicity. acknowledgement: supported by funds from tsrl inc, the university of michigan, and nih grants r ai and u ai . lawrence trost , bernhard lampert , lloyd frick , merrick almond , george painter chimerix, inc., durham, nc, usa; dmpk advisor, chimerix, inc., durham, nc, usa foscarnet, a pyrophosphate analog approved for the treatment of cmv retinitis and acyclovir-resistant herpes infections in immunocompromised patients, is active against highly drug resistant strains of hiv- . however, the clinical utility of foscarnet is limited because it requires controlled intravenous infusion and is associated with high risks of renal impairment and seizure caused by alterations in plasma minerals and electrolytes. lipid conjugation has been shown to increase the in vitro activity, improve oral bioavailability, and reduce the toxicity of several antiviral drugs requiring intravenous administration because of poor bioavailability. in the case of foscarnet, conjugation to methylbatyl alcohol (cmx ) decreases the apparent ec value against hiv- by up to -fold. cmx was esterified to produce cmx in order to increase solubility and to protect against decarboxylation of the foscarnet moiety during passage through the stomach. here we present the results of a preliminary toxicology and toxicokinetic study of the methylbatyl alcohol conjugate of foscarnet methyl ester (cmx ). rats were given oral doses of , and mg/kg cmx daily for days. there were no clinical signs of toxicity. body weight and food consumption were comparable to controls and serum biochemistry, hematology, coagulation parameters and urinalysis were normal. there were no gross findings at necropsy, no effects on organ weights and no findings by histopathological examination of a wide range of tissues. importantly, there were no changes in serum biochemistry parameters or histopathological examination that were indicative of the renal impairment or serum electrolyte changes that are associated with foscarnet. oral dosing resulted in significant plasma exposure to cmx (c max > g/ml), the biologically active deesterified form of cmx . in conclusion, cmx is absorbed after oral administration, converted to cmx , and has a good preliminary toxicity profile. these results support the development of cmx for the treatment of drug resistant hiv infection. zhiqian wu , julie breitenbach , ulrika erickson , john hilfinger , john drach , gordon amidon department of pharmaceutical sciences, college of pharmacy, the university of michigan, ann arbor, mi, usa; school of dentistry, the university of michigan, ann arbor, mi, usa; tsrl, inc., ann arbor, mi, usa vaccinia virus is a surrogate model system for study of pox virology and development of antiviral therapeutics. the potent anti vaccinia virus activity and various shortcomings of vidarabine make it a good candidate for improvement by utilizing prodrug strategy. vidarabine is a polar nucleoside drug with low membrane permeability and rapid degradation by adonesine deaminase. -monoester prodrugs of vidarabine with various amino acids promoieties (l-valine, l-isoleucine, l-phenylalanine. laspartic acid, l-proline) are synthesized and evaluated for their stability, permeability and activity against vaccinia virus. prodrugs exhibit different hydrolysis rate in caco- cell homogenate (t / : - min). -l-isoleucyl and -l-valyl monoester prodrugs exhibit comparable bio-conversion rate and hpept mediated uptake as well as caco- permeability with valacyclovir, a commercially marketed oral amino acid ester prodrug. both prodrugs have potent activity against vaccinia virus and are resistant to ada . preliminary animal study shows -lisoleucyl vidarabine results in > -fold increase in circulating vidarabine level. the results suggest that it may be possible to use amino acids prodrug strategy to improve vidarabine as anti vaccinia virus agent. vidarabine [ -␤-d-arabinofuranosyl)adenine or ara-a) was originally investigated as an anti-tumor agent and was later found to be active against herpes simplex virus (hsv) types and . it was the first fda-approved drug for treatment of systemic hsv infections. although replaced by acyclovir and analogs for most applications, vidarabine remains an alternative therapy for acyclovir-resistant hsv and varicella-zoster virus infections. despite its proven efficacy, vidarabine suffers some limitations including: (i) metabolism by adenosine deaminase (ada) to its inactive hypoxanthine homolog (ara-h); (ii) low lipophilicity and membrane permeability and (iii) poor aqueous solubility, thus limiting options for parenteral and peroral delivery. our recent interest in vidarabine was triggered by our discovery that it was ∼ -fold more active against vaccinia (vv) and cow pox (cpv) viruses than was cidofovir in plaque reduction assays. its activity was enhanced about -fold by combination with m -deoxycoformycin (pentostatin, a potent inhibitor of ada) thereby providing significant superiority to cidofovir. from these results and our earlier studies on -substituted vidarabine analogs (lipper et al., . mol. pharmacol. , - ), we determined that minimizing metabolism of vidarabine by synthesizing -amino acid substituted prodrugs gave a significantly more potent anti-pox virus agent. we found that amino acid ester prodrugs of vidarabine are active against vv at non-cytotoxic concentrations. further, using cell homogenates, purified enzyme and intact cell systems, we showed that the prodrugs are resistant to inactivation by ada. the prodrugs also had enhanced transport potential, most likely targeting the intestinal dipeptide transporter. finally, oral delivery of the prodrug to the small intestine resulted in a -fold increase in vidarabine plasma levels when compared to unsubstituted vidarabine. these properties make the prodrugs of vidarabine good candidates as orally bioavailable anti-pox virus agents that are stable in the presence of ada. acknowledgement: supported by funds from tsrl inc. and the university of michigan. ulf goerbig , anne baum , jan balzarini , chris meier university of hamburg, institute of organic chemistry, hamburg, germany; rega institute for medical research, katolieke universiteit leuven, leuven, belgium the cyclosal pronucleotide system has been designed for an intracellular delivery of therapeutically active nucleoside monophosphates. as part of recent work on the cyclosal approach, the interaction of cyclosal nucleotides with cholinesterases has been investigated. it is known that organo-phosphates may act as irreversible inhibitors of cholinesterases (suicide mechanism). in the case of cyclosal nucleotides, cholinesterase inhibition could lead to unwanted side effects in a possible therapeutic application. there are two types of cholinesterase found in humans, the highly specific, physiologically important acetylcholinesterase (ache) and the much more unspecific butyrylcholinesterase (bche) of unknown physiological importance. fortunately, no inhibition of ache was observed for a variety of different cyclosal nucleotides. in contrast, bche inhibition was found in some cases. the anti-hiv-active , -bis-tertbutyl- -fluoro-cyclosal-d t monophosphate is the first cyclosal derivative combining three desired properties: successful intracellular delivery of nucleotides, sufficient hydrolytic stability and strongly reduced inhibitor activity towards bche. because of the promising properties of this compound, we combined this mask developed for d t with the antiviral active nucleoside analogues like d a, dda, azt and acyclovir. in this contribution we present the synthesis, hydrolysis stability, inhibition behaviour towards bche and anti-hiv data of these new compounds. henning jessen , wolfgang fendrich , tilmann schulz , jan balzarini , chris meier university of hamburg, institute of organic chemistry, hamburg, germany, rega institute for medicinal research, katholieke universiteit leuven, leuven, belgium cyclosal-pronucleotides are used for the delivery of antivirally active nucleotides into cells via a ph triggered selective hydrolysis. to distinguish between intra-and extra-cellular environment enzyme-cleavable side chains were introduced in the aromatic moiety of the pronucleotide to enrich the compound inside cells. this behavior will further be described as "lock-in"effect. many other different cyclosal pronucleotides have been designed, all showing different hydrolysis properties and antiviral data, both originating from the nature of the cyclosal-moiety as well as of the nucleoside. to examine these differences, analytical tools of high accuracy and sensitivity were needed, being structurally as close as possible to the lead compounds. these requirements are met by intrinsically fluorescent nucleosides coupled to different cyclosal masking groups. for analysis of the purine-type nucleosides iso-da with high intrinsic fluorescence properties was chosen and converted into iso-a, iso-dda and iso-d a. for the pyrimidine-type series the fluorescent nucleoside m k was synthesized as well as the dideoxy-compound dm k. these nucleosides were transformed into different cyclosalpronucleotides and tested for their suitability for fluorescence analysis. in fact, an improvement of sensitivity by a factor of compared to uv-detection was found for some of the compounds (pmol detection). for all compounds fluorescence and absorbance spectra were recorded to determine the absorption and emission maxima. the new compounds lacked activity against hiv- and hiv- strains. however, the compounds showed low cytotoxicity, which is important for their usability as fluorescent probes in cells. due to the analytical sensitivity, a simple model uptake study could be carried out, employing an u-tube with two aqueous phases, which were separated by an unpolar organic solvent simulating a diffusion barrier. the properties of the aqueous phases were varied and an enzyme-driven enrichment of a "lock-in"-modified intrinsically fluorescent cyclosal-pronucleotide passing the diffusion barrier could be simulated. nicolas gisch , jan balzarini , chris meier university of hamburg, institute of organic chemistry, hamburg, germany; rega institute for medical research, katholieke universiteit leuven, leuven, belgium cyclosal-pronucleotides efficiently deliver therapeutically active nucleoside monophosphates in human cells. "lock-in"-cyclosal-pronucleotides -the so-called second generation of cyclosal-compounds -have been designed to trap the compound by intracellular cleavage of esterase-cleavable moiety. one disadvantage of the "lock-in"-compounds is their high chemical stability, which leads to a delayed drug delivery. therefore, conceptually different, enzymatically activated cyclosalpronucleotides have been developed. in this concept lipophilic donor substituents attached to the aromatic ring are converted into a polar acceptor substituent by intracellular enzymatic cleavage. as a consequence the liberated acceptor group leads to a strong decrease in hydrolysis stability and a rapid formation of a charged intermediate is the result. from the phosphodiester intermediate the nucleotide is released subsequently. the concept, synthesis, characterization and in vitro antiviral evaluation of the third generation of cyclosal-pronucleotides will be presented. tomas cihlar , richard mackman , adrian ray , dean boojamra , lijun zhang , deborah grant , hon hui , jennifer vela , neil parkin , yolanda lie , kirsten white , michael miller , gerry rhodes , manoj desai gilead sciences, foster city, ca, usa; monogram biosciences, so. san francisco, ca, usa background: n(t)rtis are currently used as a backbone of antiretroviral combination therapy. however, their long-term benefit can be limited by adverse effects, resistance development, drug-drug interactions, and sub-optimal efficacy in treatment-experienced patients. therefore, we searched for novel nucleotide analogs with improved pharmacological profiles. methods: phosphonomethoxy- -fluoro- , -dideoxydidehydroadenosine (gs ) was selected from a broad range of nucleoside phosphonate analogs. phosphoramidate prodrug technology previously explored with tenofovir was applied to gs , resulting in the identification of gs (ethylalaninyl phenyl ester of gs ). results: gs exhibits potent anti-hiv- activity in primary lymphocytes and t-cell lines (ec < nm). low cytotoxicity (cc > m) was observed in multiple cell types including renal cells. diphosphate metabolite of gs was shown to act as an obligatory dna chain terminator and a competitive inhibitor of hiv- reverse transcriptase (rt) (k i = . m). unlike ddi, d t, or d fc, neither gs nor its prodrugs inhibited mitochondrial dna replication in hepg cells. in a phenosense assay, gs retained its full activity against hiv- variants with k r, m v or l v mutations in rt. viruses with ≥ thymidine analog mutations showed ≤ fold reduced susceptibility to gs , a shift that was smaller than that of any other tested nrti. following an oral dose of mg/kg gs in dogs, the bioavailability of prodrug exceeded %, resulting in high intracellular levels ( . ± . m) and prolonged retention (t / > h) of gs diphosphate in blood lymphocytes. conclusions: both gs and its prodrug gs exhibit favorable in vitro pharmacological profiles including less resistance due to rt mutations than approved nrtis. gs possesses good in vivo pharmacokinetic properties and thus represents an attractive development candidate with potential for clinical efficacy in both treatment-naive and nrti-experienced patients. martin mcdermott, gabriel birkus, ruth wang, holly macarthur, xiaohong liu, nilima kutty, tomas cihlar, craig gibbs, swami swaminathan, arnold fridland, william lee gilead sciences, inc., foster city, ca, usa gs- and gs- are alkylalaninyl phenyl ester prodrugs of tenofovir (tfv; -[( -phosphonomethoxy)propyl]adenine) and a novel nucleotide analog fd ap (phosphonomethoxy- -fluoro- , -dideoxydidehydroadenosine), respectively. both gs- and gs- exhibit potent in vitro anti-hiv- activity, favorable resistance profile, and low cytotoxicity. compared to tenofovir disoproxil (viread), both prodrugs are significantly more stable in plasma and deliver > -fold greater levels of active diphosphate metabolites into pbmcs in vitro and in vivo. the initial step in the intracellular activation of gs- and gs- is the hydrolysis of the alanine carboxyester by an unknown hydrolytic enzyme. the isolation and identification of this enzyme from human pbmcs is reported here. results: a major enzyme capable of cleaving gs- and gs- was purified from human pbmcs and was separable from esterases able to cleave alpha napthyl acetate (ana). the increase in specific activity of prodrug hydrolase achieved was -fold. sds-page analysis showed the presence of a prominent protein band at kda, which was identified by ingel tryptic digestion and ms/ms sequencing of the resultant peptides as lysosomal carboxypeptidase a (cathepsin a, ec . . . , cata). the biochemical properties of purified prodrug hydrolase matched those of cata. recombinant cata and the isolated prodrug hydrolase displayed nearly identical susceptibility to hydrolase inhibitors and substrate preference against a panel of tenofovir phosphoramidate prodrugs. incubation of both enzymes with [ c]gs- and [ h]difluorophosphonate resulted in the labeling of an identical kda protein (catalytic subunit). both labeled bands reacted with polyclonal antibodies specific for human cathepsin a. finally, following incubation with gs- , approximately - -fold lower intracellular concentrations of tfv metabolites were detected in fibroblasts from patients expressing non-functional cat a (cat a-cells) compared to normal control fibroblasts (cat a+ cells). center for drug delivery and nanomedicine, university of nebraska medical center, omaha, ne, usa nucleoside -triphosphate (ntp) is the biologically active form of many antiviral nucleoside analogs capable of efficiently blocking the production of viral nucleic acids in infected cells. we describe novel microparticulate formulations for encapsulation of ntp, drug delivery and antiviral therapy of respiratory infections. polymer networks (poloxagels) consisted of crosslinked poloxamers and cationic polymer molecules were designed, synthesized and characterized by loading with ntp and interaction with cells. poloxagel-ntp formulations could be obtained by simple mixing of the aqueous solution of ntp with the aqueous dispersion of poloxagel and subsequent lyophilization. drug loading was equal up to % by weight. in this form, phosphates groups of ntp are complexed with amino groups of polycationic backbone of poloxagels, and the formulations could be stored at room temperature for many months without degradation of ntp. the particle size of aqueous poloxagel-ntp dispersions was low, with a hydrodynamic diameter of . - . m. the rate of passive drug release in physiological solution was from to % of loaded drug during the -h period. these formulations were effectively consumed by many types of cells. significant amounts of drug and poloxagels were detected in the cellular interior after only - h of incubation. in the presence of cellular membranes drug release from poloxagel-ntp formulations was dramatically increased. we attribute this effect to the triggered release of the bound ntp as a result of competitive interaction of polycationic backbone of poloxagels with phospholipids of cellular membranes. mucoadhesive properties of poloxamers may additionally enhance binding of poloxagels with airways/lung epithelium. -triphosphate of -␤-d-ribofuranosyl- h- , , -triazole -carboxamide (ribavirin) was synthesized using phosphorylation with a tris(imidazolyl) phosphate in a convenient one-pot approach. formulations of different poloxagels with the ribavirin -triphosphate were prepared and characterized as prospective antiviral formulations for prophylactic and therapeutic treatments of respiratory infections including influenza a virus. aerosolic route of application of these antiviral formulations and associated problems are discussed. edwin gong , ebrima gibbs , joel oger department of pharmacology and therapeutics, the university of british columbia, vancouver, bc, canada; neuroimmunology lab, ubc hospital, department of medicine, the university of british columbia, vancouver, bc, canada ifn-alpha and ifn-beta are currently employed in the treatment of many viral diseases, especially chronic hepatitis. ifn-beta is also employed for the treatment of multiple sclerosis (ms), a chronic and often debilitating disease of the central nervous system. however, as with other protein therapeutics, long-term ifn therapy can lead to the development of binding and neutralizing antibodies to ifns and thus lead to deceased clinical effect of ifns. in order to measure the bioavailability of ifns and the level of neutralizing antibody, we have developed a realtime rt-pcr (taqman) assay by quantitating the expression of mxa (an ifn-induced protein) mrna. the nucleotide sequences of mxa deposited in the genebank were aligned, and a pair of primers and the hybridization probe were designed based on the conserved regions. a house keeping gene, gapdh, was used as a calibrator for relative quantitation. the rna standards were generated by in vitro transcription from cloned mxa gene in a plasmid vector. the reaction parameters were optimized. the assay was validated using pbmcs of ms patients that were treated with ifn-beta. for evaluation of the ifn bioavailability, the total rna was extracted from pbmcs and quantitatively detected by one-step rt-pcr for both mxa and gapdh. the results calculated by the (− ct) method showed that the difference (signal-to-noise ratio) between samples with neutralizing antibodies and samples from untreated ms patients or healthy donors were approximately - -folds. this indicates that our assay is a reliable method for determination of ifn bioavailability. v. lozitsky , i. kravchenko , v. larionov research anti-plague institute, odessa, ukraine; national university, odessa, ukraine transdermal delivery (td) of drugs is a novel method for treatment of diseases. td is carried out by the help of transdermal therapeutic systems (tts), which are multilayer plasters that contain active ingredients. td have a number of advantages, such as: ( ) prolongation of the drug's action; ( ) drug's concentration is maintained in therapeutic range; ( ) there is no trauma to patient's skin while using td; ( ) removing tts from the skin immediately stops drug's entering to the organism; ( ) first-pass effect in the liver is reduced; and ( ) many highly active drugs are irritating the gastro-intestinal tract if administered orally, others have a short half-life time-these drugs do not have downsides mentioned above if used as tts. in our previous research we elaborated tts containing rimantadine (ttscr) and studied its efficacy during experimental influenza in mice. we had established that transdermal delivery of rimantadine is more effective than oral administration. the aim of this work was to increase the efficacy of ttscr. to solve this task we studied the influence of some permeability enhancers on anti-influenza efficacy of ttscr during experimental infection. applied tts had adhesion hydrogel matrix (polyvinyl alcohol and . -propylenglycol). they consisted of a base and a plastificator, which improves the administration of active substances through the skin and does not induce irritation. ttscr ( mg/mouse) were applied on shaven backs of experimental animals. tts for other groups additionally contained one of such permeability enhancers as: mg/mouse of dmso or mg/mouse of octanol or mg/mouse of papain. tts were applied on shaven backs of mice and stayed there from day before infection to th day after challenge. mice of all groups were infected intranasally with influenza virus a/pr/ / (h n ), which is highly pathogenic for them. challenge was carried out using four animals for each virus dilution within the range of − to − . deaths of animals were recorded for days. the results showed that proteolytic enzyme papain increased the anti-influenza efficacy of ttscr on log tid . dmso and octanol did not demonstrate such activity. mikhail dobrikov , serguei vinogradov , barbara ramsay shaw department of chemistry, duke university, durham, nc, usa; center of drug delivery and nanomedicine, and college of pharmacy, university of nebraska, omaha, ne, usa nucleoside reverse transcriptase inhibitors (nrti) are widely used in the antiviral chemotherapy. most nrtis require stepwise phosphorylation to the respective nucleoside triphosphates, which inhibit the viral dna synthesis. however, the emergence of hiv- reverse transcriptase-dependent drug resistance limits the effectiveness of treatment by nrtis. the ␣-p-borano-nucleotide analogues show several unique physico-chemical and biological properties: (i) enzymatic studies indicate that the rp-isomer of ␣-p-borano- , -ddndps is a better substrate for cellular ndp kinase than the parent ddndp; (ii) neither isomer of the ␣-p-borano-ddndps is a substrate for mammalian pyruvate kinase and shows very poor inhibitory properties to this enzyme; (iii) the rp-(␣-p-borano)-ddntp isomers are better inhibitors of drug-and multidrug-resistant viral reverse transcriptases and are poor substrates for dnadependent dna polymerises; and (iv) after incorporation into viral dna the borano-ddnmp residues are more resistant to atp-dependent removal from viral dna than parent ddntps. to by-pass inefficient phosphorylation of the nrtis, several prodrugs of ␣-p-borano-nucleotide analogues have been previously synthesized. a more efficient delivery system for ␣-p-boranonucleotide analogues based on nanosized cationic polymeric gel (nanogel) is proposed. selective inhibition of drug-and multi-drug resistant viral rts, poorer inhibition of intracellular kinases and dna polymerases by the ␣-p-borano-nucleotide analogues, and their specific delivery into infected cells in the complex with nanogel particles suggest a new approach to the design of more powerful antiviral drugs. acknowledgement: this work was supported by the nih grant r al to b.r.s. we have developed a high-throughput, cell-based assay to address the critical need for antiviral drugs for the treatment of influenza. in consideration of the demand to screen high volumes of compounds, we targeted the development of a microtiter plate format for the assay. in this assay, the inhibition of the influenza-induced cytopathic effect (cpe) in mdck cells was assessed using the celltiter-glo luminescent cell viability assay by promega. this reagent measures the amount of atp present in cells, which is directly proportional to the number of metabolically active cells. validation studies were executed to establish optimal cell density, viral concentration, dmso tolerance for compound dilution, incubation time for virus-induced cpe and effective control drug concentration. additional parameters, such as assay variability, reagent and read stability, edge effects, and ic stability were also investigated during validation. we are currently initiating use of the assay to screen chemical libraries and will report our findings from library screens in addition to the aforementioned validation. we believe the approach will also provide a mechanism for discovery of new antiviral leads for influenza as well as avian flu. fundacio irsicaixa, hospital universitari germans trias i pujol, badalona, spain we have developed bacteriophage lambda based genetic screen that can be used to isolate and characterize site-specific proteases. this genetic screen system is based on the bacteriophage lambda ci-cro regulatory circuit, in which the encoded repressor ci is specifically cleaved to initiate the lysogenic-to-lytic switch. we have adapted this simple, safe and rapid genetic screening system to predict the activities and phenotypes of human immunodeficiency virus type (hiv- ) proteases in the course of viral infection and antiretroviral therapy. a specific target for the hiv- protease, p -p , was inserted into the lambda phage ci repressor. the target specificity of the ci-hiv repressor was evaluated by coexpression of this repressor with an hiv- protease construct. upon infection of escherichia coli cells expressing the two constructs encoding the ci-hiv- repressor and hiv- protease, lambda phage replicated up to -fold more efficiently than in cells that did not express the hiv- protease. this assay responds appropriately to well-known hiv- proteases inhibitors and can be used to search for new proteases inhibitors. the high level of specificity of this system, in which modest differences in catalytic efficiency can be quantified, should be also useful for the characterization of different mutant viral proteases. we further demonstrated the broad applicability of this protease assay using other viral proteases and their cognate cleavage sites, including hepatitis c virus (hcv) ns protease and severe acute respiratory syndrome (sars) coronavirus (cov) (scov) c-like protease. compared with other protease assay methods, this assay has the following advantages: safe, highly sensitive, highly specific, easy quantification, and rapid generation of different protease cleavage substrates using molecular cloning and expression. this system may be useful for the development of a screening method to identify viral protease inhibitors and should be also useful to characterize cellular, viral, or other infectious agent proteases with different activities and specificities. karen m. watson, todd b. parsley, robert w. buckheit jr. imquest biosciences, inc., frederick, md, usa a virus transmission and rapid resistance selection assay has been developed in order to quickly evaluate the biological properties of anti-infective test compounds and rationally prioritize them for further development. the transmission assay specifically evaluates the ability of test agents to suppress and clear virus replication from cultures during the serial passage of virus in the presence of fixed concentrations of the test compounds alone or in combination. the growth and expansion of virus in the infected cultures has been shown to occur through the replication of originally infected cells in the absence of virus spread, through direct virus to cell infection, and through cell to cell transmission. in order to sterilize a culture a test compound must possess the ability to specifically and potently interfere with virus replication by each of these three methods and must be able to inhibit the replication of resistant viruses which pre-exist in the viral inoculum and which rapidly grow in the presence of the fixed concentrations of the test agent. twelve pyrimidinediones being evaluated for potential use as both anti-hiv therapeutic agents and topical microbicides were evaluated for their ability to inhibit virus transmission and to define their ability to rapidly select for resistant virus strains. these compounds were evaluated in parallel with known anti-hiv agents that inhibit virus entry (t ) and reverse transcription (sustiva, uc and azt). the results of the transmission assays suggest that significant biological differences exist between antiviral compounds and even between highly related congeners of the same class of pyrimidinediones, suggesting that the transmission and rapid resistant selection assay measures important antiviral properties of anti-hiv agents. biological studies that evaluate the mechanisms of virus growth in the presence of high concentrations of test compounds will be described. laure deflubé , kerstin angner , anna overby , david stein , patrick iversen , ramon flick utmb, department of pathology, galveston, tx, usa; avi biopharma, inc., corvallis, or, usa in the family bunyaviridae, several members on the genus phlebovirus have been reported to cause disease in humans or livestock. among these, rift valley fever (rvf) virus is an important human/animal pathogen. its widespread geographic distribution and its ability to produce severe human disease makes this virus a worldwide public health concern. the phosphorodiamidate morpholino-oligomers (pmo) are a class of dna-like antisense agents typically synthesized to a length of about subunits and contain purine or pyrimidine bases attached to a backbone composed of six member morpholine rings joined by phosphorodiamidate intersubunit linkages. they have been shown to be effective antiviral compounds for different virus families, e.g. coronaviridae and flaviviridae. pmo bind to rna preventing translation of the viral rnas. we used our recently developed plasmid-based minigenome rescue systems for uukuniemi (phlebovirus model virus) and rvf viruses to screen antiviral compounds based on the morpholino antisense oligonucleotide approach. for this the antiviral compounds were appraised on the basis of reporter gene activity (fig. b) . the inhibitory effects of the same compounds were also tested by measuring reduction in virus titer (fig. a) , by monitoring changes in viral antigen production using an indirect immunofluorescence procedure and facs analysis, and analysis of genome transcription/replication by rt-pcr. indeed several pmos could be identified with interfering effect at a low ic on bunyaviral minigenome rescue as well as virus proliferation. based on these results, we plan to confirm antiviral activity of the most promising compounds in suitable animal models. we have shown that the fractal approach to the problem of viruscell interaction gives the unique possibility to process the data through the sequence of the direct and inverse fourier transforms. the studies were carried on the herpes simplex virus us- interacting with the hep- sensitive cell culture. the object was imaged as system of bright peaks formed as a result of laser diffraction on the structural elements of the virus-cell system. the whole virus-cell interaction information is inserted into computer in a fastest parallel way. the laser intensity peaks, which form the speckle image of the system under consideration, could be transformed into the hierarchical system of the circles (or squares) according to the choice of the researcher, but conserving the same d value, which depends only on the true intermolecular interaction potential. this potential, being characteristic for every stage of virus-cell interaction, is responsible for the given structure of the dynamic virus-cell system and the unique, but the typical form of the fractal cluster corresponding both to the system itself and its image as well, was processed by computer techniques. the hierarchical fractal design of the virus-cell system, proposed here for the first time, gives the universality, needed for the quantitative description of any possible combination of the virus and corresponding sensitive cell. it should be noted, as well, that the fractal microscope use for viruscell dynamic system imaging have all the properties, required from all other experimental tools of monitoring, including the reliability, reproducibility and preciseness. this device could be used in drug design biological test stages with the scope of time and efforts economy during the compounds libraries screening. the fractal microscope combined with the qsar drug design technique makes the antiherpetic drug design more competitive as compared to the regular approaches. acknowledgement: the authors are indebted to the partial support of the stcu grant # . we have investigated experimentally the fractal properties of diffraction images obtained by laser irradiation of virus-cell system. it was shown that the diffraction process is mathematically equivalent to the direct fourier transform of the said system's components modeled with simple geometrical figures (e.g. circles). each viral family could be coded and described quantitatively with the average size of the free viral particle and the type of its symmetry. we propose here to use the inverse fourier transform of the virus-cell system in order to get the real enlarged image of the viruses attacking the sensitive cell as well as the cell's structural transformation caused by the sequent stages of virus reproduction process. the set of bright and dark spots, which forms the virus-cell system's diffraction image, could be coded into set of numbers (matrix form of correlation vector-function) using the quantification procedure. the correlation function was used as presented in polar coordinates because the system has the axial symmetry (laser beam taken as main physical axis). the full information included into the image peaks' diameters and color index is transformed using inverse fourier technique into set of intersecting bright and dark circles. the full in vitro dynamics of the structural changes of the virus-cell system are described by the changes of circles' diameters and the area of their intersection. it was shown, also, that the magnification of the fractal microscope could achieve , × to , ×, depending on the laser power used. proposed fractal microscope could be applied as well in vivo experiments until the required magnification will not make us to use projection laser with the output exceeding mw. we have shown that the fractal microscope based on the inverse fourier transform could be applied successfully in pharmaceutical antiviral drug design, laboratory and clinical trials of new antiviral preparations, especially effectively in hierarchic qsar research. acknowledgement: authors are grateful to the support under the stcu grant # . we have previously reported bicyclic furano pyrimidines as potent and selective inhibitors of varicella zoster virus (vzv) , with subnanomolar activity for palkylphenyl substituted analogues . these compounds however are highly lipophilic and poorly soluble in water. we then reported a series of p-alkyloxyphenyl compounds containing a phenolic ether aiming to enhance water solubility whilst retaining antiviral activity (mcguigan et al., ) . we will now report the synthesis, characterisation and antiviral evaluation of a novel series of p-alkyloxyphenyls where there is at least one methylene spacer between the phenyl and ether group to potentially boost the pharmacokinetic profile. the alkyl chain length was fixed to retain a high clogp value, a parameter that has previously been shown to correlate with high antiviral potency . the target structures were prepared by the pd-catalysed coupling of a series of para-substituted alkoxyphenyl acetylenes with -iodo- -deoxyuridine, to give intermediate -alkynyl nucleosides, which were subsequently cyclised in the presence of cui to give the desired bicyclic systems. the antiviral activity, cytotoxicity, and solubility of these compounds are to be reported. we have previously reported on some novel nucleoside analogues containing and unusual furano bicyclic pyrimidine base and long side chain , which were discovered to be both potent exquisitely and selective towards the varicella zoster virus. following this discovery, three main sites for modification were identified and explored: ( ) the side chain; ( ) the bicyclic base; and ( ) the sugar moiety. modification to the side chain by insertion of a phenyl ring, led to the most potent anti-vzv nucleoside to date (ec nm) . the investigation into modifications at the three sites stated above has continued and we herein report further adjustments to these analogues. replacement of the furo oxygen with sulfur on the parent nucleosides bearing an alkyl side chain has been reported to retain antiviral activity. however, those bearing a phenyl alkyl side chain are here shown to give a slight reduction in anti-vzv activity. modifications to the phenyl ring of the side chain have included halogen substitutions, and the fluorine in particular has produced some intriguing results in that, while the ortho and meta substitutions show some anti-vzv activity, the para analogue is completely devoid of antiviral activity. we now report further studies which include the di and tri substituted phenyl analogues. finally, we have also investigated sugar modification that has included substitutions of the hydroxyl group. previous modifications which have replacements of the hydroxyl groups, resulted in loss of activity against vzv . we now present some new substituted analogues which have provided interesting biological results. we have previously reported bicyclic furano pyrimidines as potent and selective inhibitors of varicella zoster virus (vzv) with subnanomolar activity for palkylphenyl substituted analogues srinivasan et al., ) . the sar is now further explored via the substitution of phenyl derivatives with electron withdrawing and electron donating groups. we now report the synthesis, characterisation, and biological evaluation of a novel series of mono substituted phenyl derivatives in order to probe the structure activity relationships in this region. the target compounds were synthesised under sonogashira conditions where a series of substituted phenyl acetylenes were coupled with -iodo- -deoxyuridine, to give intermediate alkynyl nucleosides that were subsequently cyclised in the presence of cui to give the desired bicyclic systems. diseases caused by herpes simplex virus (hsv) are widely distributed. prophylaxis and treatment of these infections are important health care tasks that require also the search, design and development of new antiherpetic drugs overcome drug resistance and toxic side effects of existing drugs. drug selection simply based on results of empirical screening is not very effective. computer-based technologies may help to optimize the structure of antiviral compounds as well as to design and develop new drugs. the objective of the present work is the quantitative structureactivity relationship (qsar) analysis of antiviral activity of various n,n -(bis- -nitropyrimidyl)dispirotripiperazines in connection with consequent drug design. the well-established simplex representation of molecular structure (sirms) qsar approach has been used to fulfill this objective. it allows the molecular design of new effective antiviral drugs. thorough investigation of the relationship between: (a) cytotoxic (hela cells and gmk cells, cc , g/ml); (b) antiherpetic activity (hsv- strain kupka, ic , g/ml); and (c) selectivity index (ratio of cc to ic ) and the structure of n,n -bis- -nitropyrimidyl derivatives of dispirotripiperazine have been conducted. statistic characteristics for pls (partial least squares) models are quite satisfactory (r = . - . , q = . - . ). the results are confirmed by experimental data. based on the obtained models, molecular fragments that promote and interfere with antiviral activity were defined. additionally, these models provide the possibility to predict molecular fragments that will enhance antiherpetic activity and to design new well tolerated highly virus-specific drugs. in summary, the developed simplex approach is an effective instrument for prediction and design of novel effective antiherpetic agents. several representatives of a series of -arylethynyl- deoxyuridines ( a) bearing bulky aryl groups were recently shown to possess unexpected activity towards hsv- . unlike common anti-hsv drugs, these compounds retain activity towards kinase-deficient acyclovir-resistant strains. therefore, an unusual mechanism of antiviral action is assumed. in order to investigate the mechanism and to discover more potent analogues we synthesized several novel -deoxy ( a) and -arabino ( b) uridine derivatives possessing different -arylethynyl substituents. dinucleosides containing two uridine moieties coupled to a single polycyclic aromatic hydrocarbon (e.g. pyrene) represent another type of structural variation of nucleosides a. these compounds as well as some of a and b possess bright fluorescence that can be used in biological evaluations. cytomegalovirus (cmv) is a wide spread opportunistic pathogen which belongs to the beta subfamily of the herpesviridae. primary infection is generally asymptomatic resulting in life long latency. however, morbidity and mortality rates post-transplantation are greatly increased following reactivation or recrudescence of cmv. ganciclovir (gcv) and cidofovir (cdv) have both been successful in suppressing cmv viral replication in immunocompromised patients. although sustained use of these drugs has resulted in the emergence of multi-drug resistant strains of virus. in this study we used plaque reduction assays to determine the antiviral efficacy of two ribonucleotide reductase inhibitors, didox (dx; , dihydroxybenzohydroxamic acid) and trimidox (tx; , , trihydroxybenzamidoxime) in inhibiting both wild type and drug-resistant strains of murine cmv (smith strain). the results presented here demonstrate that both dx and tx inhibit viral plaque formation in a dose dependent manner in both wild type and the resistant strain. a -and -fold increase in drug dose was required for cdv and gcv respectfully to inhibit plaque formation by % in the resistant strain (cdv wt: . m, r: . m/gcv wt: . m, r: . m). this compared to only a moderate increase in drug dose required for dx and tx to achieve % inhibition in the resistant strain (dx wt: . m, r: . m/tx wt: . m, r: . m), corresponding to a . -and . -fold increase respectfully. further work is currently underway to determine the possible mechanism of antiviral actions and toxicity profiles of these novel virostatics. in patients with human immunodeficiency virus (hiv) infection, coinfection with herpesviruses continues to be a problem for patients receiving antiviral hiv therapy. since treatment can be affected by the large number of drugs required for multiple infections, it would be useful to have antivirals that are active against both hiv and the herpesviruses. we reported previously that alkoxyalkyl ester prodrugs of cidofovir (cdv) are several logs more active against herpesvirus replication than unmodified cdv. to determine if this strategy would be effective for other acyclic nucleoside phosphonates which are active against hiv infections, hexadecyloxypropyl (hdp) esters were synthesized from -(phosphonomethoxyethyl)-cytosine (pme-c), -(phosphonomethoxyethyl)- -bromo-cytosine (pme- brc), -(phosphonomethoxyethyl)- -fluoro-cytosine (pme- fc), -(phosphonomethoxyethyl)- , -diaminopurine (pme-dap) and -(phosphonomethoxyethyl)- -amino- cyclopropylaminopurine (pme-cprdap) and assayed for activity against herpesvirus replication. overall, the hdp esters were more active than the unmodified acyclic nucleoside phosphonates, indicating that this is a useful strategy for increasing the antiviral activity of acyclic nucleoside phosphonates. one of the most active compounds was hdp-pme-cprdap which had ec values of . , . , and . m in hff cells infected with hsv- , hsv- or hcmv, representing a - -fold increase in efficacy over the parent pme-cprdap. another promising compound was hdp-pme-dap, which had ec values of . , . , and . um in hff cells infected with hsv- , hsv- , and hcmv, representing a - -fold increase over the parent pme-dap. the results presented here indicate that modified acyclic nucleotides with antiviral activity against hiv also inhibit the replication of some of the herpesviruses. further evaluation of their activity against other herpesviruses that are a problem in hiv-infected patients, such as human herpesviruses type and , is warranted and may provide new therapeutic options for patients with coinfections. julie m. breitenbach , katherine z. borysko , jiri zemlicka , john c. drach biologic & materials sciences, school of dentistry, university of michigan, ann arbor, mi, usa; karmanos cancer institute, wayne state university school of medicine, detroit, mi, usa we previously described first (qiu et al., . j. med. chem.) and second generation (zhou et al., . j. med. chem.) methylenecyclopropane purines that have potent and selective activity against hcmv. strains selected separately for resistance to first-generation analogs (synadenol, synguanol) were - -fold resistant to several first-generation purine analogs. similar resistance was observed to the second-generation guanine analog cyclopropavir [ic 's in plaque assays = . and m, respectively for wild-type (wt) and synguanol-resistant ( r) virus]. likewise a ul deletion mutant (prichard et al., . j. virol.) was resistant to both first and second-generation compounds (ic 's = . and . m in wt; and m in ul del , respectively for synguanol and cyclopropavir). ul from the hcmv strain selected for resistance to the synadenol was sequenced and two mutations were identified: m i and c y. because hcmv with either m i or the related c y mutation alone was sensitive to synadenol and synguanol (baldanti et al., . antiviral res.), we hypothesize that two mutations are required for resistance to first-and second-generation analogs. this hypothesis was tested by construction of three strains of hcmv from hcmv ad bac with one, the other, or both mutations in ul . as expected, the two strains with the single mutations were -to -fold resistant to ganciclovir but had little resistance to the first generation compounds synadenol and synguanol ( . -to -fold). both strains were somewhat more resistant to the second-generation compound cyclopropavir ( to -fold) but less so than observed in the r virus with two mutations. study of the resistance of the constructed virus with two mutations is underway. we conclude that a functional ul is required for activity against hcmv and that is likely that two mutations in ul are required for significant resistance. acknowledgement: supported by grants p -ai and r -ai from nih and funds from the university of michigan. svitlana zagorodnya , nadiya nesterova , inna alexeeva , larisa palchikovskaya , galina baranova , alexander kobko , anna golovan zabolotny institute of microbiology and virology of nas of ukraine, kiev, ukraine; institute of molecular biology and genetics of nas of ukraine, kiev, ukraine search of new effective preparations capable to inhibit herpesviruses reproduction is stipulated by their certain resistance to different groups of chemical preparations. new triazine bearing tricyclic bases and their n-glycosidic derivatives structures are widely used as potential antiviral agents. the objective of the present investigation was to study the activity triazine bearing tricyclic bases nos. and , as well as n-glycosidic derivatives no. against epstein-barr virus-lymphotropic and oncogenic virus from herpesviridae family. as a model of ebv-infection in vitro we used the line of lymphoblastoid b-cells raji, which infected by ebv. an inhibition of reproduction of ebv in a cell culture by no , no , and no was determined by reduction of a number of genome-equivalents of ebv dna on a cell, which were revealed by quantitative pcr with use of primers and reagents "amply-senc- r" (russia). the first stage of investigation of substances was the analysis of their cytotoxicity for cell line raji. they have been studied in concentrations of , , , , , , , , , . and . g/ml. the concentrations that inhibited the quantity of live cells on % (id ) were equal to substances no. - g/ml, no. - g/ml and no. - . the minimal inhibiting concentration (mic) of nos. , , and was equal to g/ml, because the amount of genome-equivalents of dna ebv on a cell was reduced with . up to . hence, the index of selectivity (is) was equal to and for triazine bearing tricyclic bases nos. and , for n-glycosidic derivatives- . in addition these compounds were also tested in transcription and replication model systems in vitro. our results indicate that bases and their n-glycoside derivatives effect rna and dna synthesis in different manner. r. sgarbanti , l. nencioni , g. macrì , c. nucci , u. benatti , m. magnani , e. garaci , a.t. palamara department public health sciences, university rome "la sapienza," rome, italy; department biopathology, physiopathological optics, university rome "tor vergata," rome, italy; department exp. medicine biochemistry section, university genova, genoa, italy; inst. biochemistry, university urbino, urbino, italy; department exp. med. biochem. sciences, university rome "tor vergata," rome, italy several studies have demonstrated that different viruses induce an imbalance in the intracellular redox state through a depletion of glutathione (gsh), the main intracellular antioxidant. the imbalance in the intracellular redox state represents a key event in the development of viral infection. indeed, our previous data showed that treatment with gsh prevents a decrease in intracellular gsh and inhibits replication of different rna and dna viruses in vitro and in vivo. our recent data demonstrated that a butanoyl derivative of gsh (gsh-c ), with increased hydrophobic properties, inhibited in vitro parainfluenza- and hsv- replication more efficiently than gsh. for this reason we evaluated the effectiveness of topical gsh-c administration in hsv- -induced keratitis in rabbits. for infection, the corneal epithelium, previously scratched, was inoculated with × pfu/ml of hsv- . gsh-c , dissolved in a saline solution ( mm, ul/eye), was administered as eyedrops four times daily for ten days. a saline solution was used for the control group. the clinical evaluation of conjunctival and corneal involvement, performed by using . % fluorescein sodium eyedrops and a slit lamp fitted with a cobalt blue filter, demonstrated that gsh-c treatment was effective in reducing the severity and progression of keratitis and conjunctivitis. moreover, in gsh-c treated animals, conjunctival hsv- titre, assayed by tcid on day post-infection, was significantly reduced as compared to that of control animals (mean = . × units/ml versus . × units/ml, n = for group). accordingly, similar results were obtained by measuring virus titre from the corneas of gsh-c -treated animals versus placebo animals (mean = . × units/ml versus . × units/ml, n = per group). such results highlight the antiviral activity of gsh-c in vivo and suggest that topical gsh-c treatment could be considered as complementary therapy of hsv- -induced keratitis. debra quenelle , deborah collins , latisha pettway , caroll hartline , james beadle , w. wan , karl hostetler , earl kern university of alabama school of medicine, birmingham, al, usa; department of medicine, university of california, san diego and veterans medical research foundation, san diego, ca, usa cytomegalovirus (cmv) can cause a wide variety of clinical manifestations in immunocompromised hosts or transplant recipients. we have utilized severe combined immunodeficient (scid) mice implanted with human fetal tissue and subsequently infected with hcmv or balb/c mice infected with mcmv to evaluate new antiviral therapies against cmv infection. in the current studies we used these two models to determine the efficacy of (s)- -[ -hydroxy- -(phosphonomethoxy)propyl]adenine ((s)-hpmpa), hexadecyloxypropyl-(s)-hpmpa (hdp-(s)-hpmpa), or octadecyloxyethyl-(s)-hpmpa (ode-(s)-hpmpa). in the hcmv model, human fetal thymus and liver (thy/liv) tissues were implanted under the kidney capsule of mice and inoculated - weeks later with pfu of hcmv. tissue samples were obtained at various time points for quantitation of hcmv titers by plaque assay. in general, replication of the toledo strain of hcmv in the implant tissue increased through - days and then gradually decreased to undetectable levels by weeks post-infection. to determine efficacy of these compounds, oral treatment with vehicle or mg/kg of (s)-hpmpa, hdp-(s)-hpmpa or ode-(s)-hpmpa was initiated h after infection and continued for days. cidofovir (cdv) at mg/kg was administered i.p. daily as a positive control. results indicated that (s)-hpmpa, hdp-(s)-hpmpa and ode-(s)-hpmpa were highly effective in significantly reducing replication when compared to the vehicle control. in mcmv infected mice, hdp-(s)-hpmpa was highly effective in preventing mortality when administered orally at or mg/kg beginning h post-viral inoculation and mg/kg when treatment was delayed until h postviral inoculation. these data indicate that these compounds were highly efficacious in two animal models of cmv infection and should be evaluated for use in hcmv infections in humans. cytomegalovirus (cmv) is a ubiquitous ␤-herpesvirus that asymptomatically infects immunocompetent individuals but leads to serious illness in immunocompromised individuals, such as transplant recipients, neonates and aids patients. thus, the need for well-tolerated and potent antiviral compounds with activity against cmv is well recognized. in our current studies, we have evaluated the in vivo activity of rep , a fully degenerate mer phosphorothioated oligonucleotide against murine cytomegalovirus infection (mcmv) in mice. rep has potent in vitro activity against hsv- , hsv- , hcmv, vzv, ebv, and hsv- (vaillant et al., submitted for publication). in our initial studies, infected mice were treated with rep and compared to saline-treated infected control mice. compound was administered intraperitoneally for consecutive days at mg/kg, starting at days prior to infection. mice were infected with × pfu mcmv on day , at h post-treatment. sera were collected at − h, at hpi, and at dpi for elisa analysis of ifn␥ production. spleens and livers were collected at dpi for determination of virus titers. at dpi, virus titers in the spleens and livers were significantly reduced by rep treatment as compared to control mice. splenomegaly was observed in infected mice treated with rep but not in saline treated, infected mice or in rep treated, uninfected mice. ifn␥ levels in mice treated with rep peaked at hpi compared to hpi for saline-treated control mice. these data suggests that immune stimulation might contribute to the antiviral activity of ps-ons, perhaps through ifn␥ levels. a second study comparing the in vivo activity of rep with two oligonucleotide analogs that do not activate tlr- mediated immune stimulation suggests that direct antiviral activity of rep and the analogs was the predominant therapeutic mechanism in vivo. moreover, one rep analog exhibited even greater antiviral activity than rep while causing no splenomegaly. additional experiments are underway to provide insights into the mechanism of action against mcmv infection. acknowledgement: supported by contract no -ai- from the virology branch, niaid, nih. kathy keith , joseph maddry , namita bansal , kochurani jacob , secrist john , earl kern department of pediatrics, university of alabama school of medicine, birmingham, al, usa; southern research institute, birmingham, al, usa a series of novel antiviral agents was prepared based on lead compounds related to acyclic nucleoside phosphonates. these agents consist of a purine nucleus bearing a pendant phosphonic acid group. the design strategy was two-fold: ( ) following the approach of the hostetler group, to mask or partially mask the anionic phosphonate as a lipophilic ester and/or as an amino acid phosphonamidate prodrug that could enhance cellular uptake and be cleaved intracellularly; and ( ) to investigate new substituents at the purine -and -positions. for proof of concept, a phosphonomethoxyethyl adenine (pmea) scaffold was employed. over analogs of pmea substituted at the purine -or at the adenine n- site have been synthesized and evaluated for activity against orthopoxvirus infections. many n- substituents other than the previously recognized n-cyclopropyl have shown antiviral activity, and these structure-activity relationships are being investigated. an exciting finding has been that introduction of several novel moieties at the purine -position particularly the hydrazino, hydroxylamino, or the cyclopropylamino groups resulted in several compounds with excellent in vitro activity. for example, octadecyloxyethyl (ode) -amino-n( )-cyclopropyl pmea had ec values of . - . m and ode -hydroxylamino-n( )-cyclopropyl pmea had ec values of . - . m against cowpox and vaccinia viruses, respectively, using a plaque reduction assay in hff cells. under these conditions the parent molecule, pmea, was completely inactive. these two compounds had cc values of - m giving selective indices of - . these studies indicate that several modifications in the pmea scaffold can result in good antiviral activity against orthopoxvirus infections in vitro and the most active compounds are currently being scaled up for evaluation in animal models. isatin ( , -dioxoindole), a versatile lead molecule for designing of potential bioactive agents, and its derivatives have been reported to possess inhibitory activity against a variety of pathogenic viruses. methisazone(n-methylisatin- thiosemicarbazone) was one of the first synthetic antiviral agents used clinically for the treatment of orthopox virus infections. the presence of the thiosemicarbazone, however, can result in immunosuppresion and we have attempted to replace the thiosemicarbazone with a sulphonamide in order to modify the antiviral activity. the present work was performed to evaluate the antiviral activity and cytotoxicity of some novel -[( , dihydro- -oxo- h-indol- -ylidene)amino]-n-( , -dimethyl- pyrimidiny)-benzene sulphonamides against pox viruses such as vaccinia and cowpox virus in human fibroblast cells and the activity was compared with cidofovir(cdv). among the compounds tested, -[( -methyl- , -dihydro- -oxo- h-indol- -ylidene)amino]-n-( , -dimethyl- -pyrimidiny)-benzene sulphonamide(spiii- me), was the most active compound with an ec value of mol, compared with cdv, which had an ec of mol against vaccinia virus. all the compounds were non-toxic (> lm)using a neutral red uptake assay. substitution of a halogen atom in th position of isatin was found to abolish the antiviral activity. this compound should be evaluated in orthopox infections in animal to determine its potential for development as an effective agents for treatment of these infections. acknowledgement: supported in part by contract no -ai- from virology branch, niaid, nih, usa. evgeny belanov , svetlana kotovskaya , nikolay bormotov , sergey balakhnin , olga serova , nataliya perova , zina baskakova , galina dzhumbaeva , valerii charushin , oleg chupakhin state research center of virology and biotechnology "vector", koltsovo, novosibirsk reg., russia; ural state technical university, yekaterinburg, russia; institute of organic syntheses, yekaterinburg, russia during this study, we synthesized a series of , , -benzotriazine ( fig. ) derivatives in order to evaluate the structural features required for anti-orthopoxviruses activity. these derivatives were tested for cytotoxicity and activity against the vaccinia, cowpox, mousepox, monkeypox, and in some experiments with variola viruses in vero and mk- cells. the results from studies of structure-activity relationship revealed that only compounds containing phenyl group at c- and the alkoxy and fluoro substitutes in the benzene ring of benzotriazines showed anti-orthopoxviruses activity. the antiviral activity was reduced or lost after substitution with other substitutes. thus, we find a new class of heterocyclic compounds with antiviral activity. acknowledgment: this research was funded by istc project # . yali chen, guang yang, kady honeychurch, dennis hruby, robert jordan siga technologies, inc., sw research way-suite , corvallis, or , usa we have recently discovered a highly specific and potent antiorthopoxvirus compound (st- ) via high throughput screening (yang et al., . j. virol. , - ) . marker rescue of st- resistant variants localized compound resistance to the f l gene that encodes a major orthopoxvirus envelope protein (p ), which is required for extracellular viral particle formation. p participates in wrapping of intracellular mature virus (imv) in membranes derived from the trans golgi or late endosomal compartment to produce intracellular enveloped virus (iev) that are transported to the cell surface to form extracellular virus particles. to gain insight into the mechanism of action of st- , we examined the effects of st- on the production of the extracellular viral particles in bsc cells infected with recombinant vaccinia virus containing a gfp-tagged p protein. in the presence of st- , iev particle formation was dramatically reduced, plaque formation was almost completely inhibited, and imv particles appeared to be retained in intracellular vesicles as revealed by electron microscopy. furthermore, st- prevented the intracellular localization of p to the late endosome compartment as measured by confocal microscopy. in contrast, st- did not affect localization of p expressed from a st- resistant virus variant. more intriguingly, the compound did not affect the intracellular localization of p in transfected cells. these results suggest that st- inhibits an unknown virusspecific activity that requires f l. this work underscores the exquisite specificity of st- and supports continued development of st- as a potential anti-orthopoxvirus drug. guang yang, chris harver, dennis hruby, robert jordan siga technologies, inc. sw research way, suite corvallis, or , usa st- is a potent, orally bioavailable anti-orthopoxvirus compound that is active in vitro and in vivo. the frequency of naturally occurring st- resistant variants was measured by fluctuation analysis and found to be . × − . marker rescue of drug resistant variants localized changes associated with reduced compound susceptibility to the vaccinia virus f l gene. the spectrum of mutations that confer st- resistance was determined using an error-prone pcr procedure to increase the frequency of compound resistance by -fold relative to the frequency of naturally occurring resistance. using this procedure, random point mutations were introduced into the f l coding sequence by error-prone pcr and the mutated f l alleles were transferred into wild-type virus genome by marker rescue. sequence analysis of the input error-prone pcr products prior to marker rescue identified numerous nucleotide changes in the f l coding sequence, some of which created nonsense mutations. virus recombinants were selected that formed plaques in the presence of drug selection. this powerful selection procedure enriched for viruses that produced functional, st- resistant, f l proteins. sequence analysis of the compound resistant f l alleles identified numerous silent mutations scattered throughout the f l coding sequence and point mutations leading to amino acid changes that clustered around amino acid positions - within the f l gene. seven of these mutations resulted in single amino acid changes and could be correlated with reduced compound susceptibility. taken together, these results suggest that: ( ) mutations in at least positions within f l can confer resistance to st- and ( ) st- resistant mutations cluster to a amino acid domain in a region of the protein of unknown function. several -substituted pyrimidine analogs were identified as having antiviral activity against cowpox virus (cv) and vaccinia virus (vv) in primary human foreskin fibroblast cells. molecules containing benzopyran, cyanovinyl, and pyrazolone moieties at this position exhibited significant antiviral activity against both these viruses. three compounds in this series had ec values below m in a plaque reduction assay against both cv and vv. the antiviral activity of these compounds was also determined against herpes simplex virus (hsv) in a plaque assay. two compounds with cyanovinyl derivatives at the position had ec values below m against both hsv- and hsv- , whereas other substituents at this position exhibited weaker activity against one or both of these viruses. analogs containing the benzopyran substituents were the most effective against varicella zoster virus (vzv) and yielded ec values below m in a plaque reduction assay. none of the compounds were active against human cytomegalovirus. interestingly, all of the compounds were much less effective in a thymidine kinase (tk) negative strain of cv suggesting that the activation by this enzyme was important in their mechanism of action. tk deficient strains of hsv were also comparatively resistant to some of the compounds. the tk dependence of these compounds in cv and hsv taken together with the lack of activity against cytomegalovirus replication suggests that activation by a viral tk is important in their mechanism of action. these results indicate that pyrimidine analogs with large substituents at the position are substrates for the distinct tk homologs encoded by the herpesviruses and orthopoxviruses and suggest that they may be effective against infections with these viruses. synthesis and testing of additional analogs is warranted and should help identify the most potent analogs for in vivo testing. department of pediatrics, university of alabama school of medicine, birmingham, al , usa n-methanocarbathymidine ((n)-mct) is a conformationally locked nucleoside analog that is active against some herpesviruses and orthopoxviruses in vitro. this compound inhibits the replication of herpes simplex virus (hsv) with ec values below g/ml, and vaccinia virus (vv) and cowpox virus (cv) with ec values of . and . g/ml, respectively. assays using a thymidine kinase (tk) negative strain of cv yielded ec values -fold greater than a tk positive isolate. similarly, a tk negative strain of hsv- was -fold less sensitive to the drug than wild-type strains. thus, the antiviral activity of this molecule is dependent on the type i tk in hsv and the type ii tk expressed by vv and cv viruses, suggesting that it is a substrate for these divergent forms of the enzyme. the drug is also a good inhibitor of viral dna synthesis in both viruses and is consistent with inhibition of the viral dna polymerase once it is activated by the viral tk homologs. it is also possible that the phosphorylated forms of the drug may inhibit other enzymes such as thymidylate synthetase and inhibit viral dna synthesis indirectly. the interesting tk dependence of this molecule explains the rather unusual spectrum of activity that includes orthopoxviruses, alphaherpesviruses, epstein-barr virus (ebv), and human herpesvirus (hhv- ), since these viruses all express molecules with tk activity that could phosphorylate and thus activate the drug. conversely, n-mct is ineffective against the betaherpesviruses because they do not encode tk homologs. the compound is also highly effective in reducing the mortality of mice infected with cv, vv, and hsv when treatment is initiated h after infection and at doses as low as mg/kg. these results indicate that (n)-mct is active in vitro and in vivo and its mechanism of action suggests that the molecule may be an effective and selective therapeutic for orthopoxvirus and certain herpesvirus infections and that it warrants further development. we have previously reported the isolation and characterization of drug-resistant mutants obtained following repeated passages of the vaccinia virus (vv, lederle strain) in the presence of increasing concentrations of cidofovir (cdv). cdvr mutants encoded two mutations (a t and a v) not related to genetic polymorphism. we have now introduced these mutations in the pathogenic strain w western reserve and characterized the drug-susceptibility profile of the recombinant viruses and their pathogenicity in mice. both the a t and the a v recombinant viruses proved to be resistant to cdv and related compounds, such as cyclic cdv and -propoxy]pyrimidine}. the virus bearing both substitutions proved to be more resistant to cdv than the single mutants. interestingly, the a t and the a v mutants differed in their sensitivity to phosphonoacetic acid (paa); the a t and the a v mutants being, respectively, hypersensitive and resistant to paa. in contrast, the double mutant showed no change in sensitivity to paa as compared to the wild-type strain. unlike the a v mutant that showed only a two to three-fold decrease in susceptibility towards the -hydroxy- -phosphonomethoxypropyl (hpmp) purine derivatives, the a t mutant showed cross-resistance to the hpmp purine derivatives. it should be noted that in the process of selection of cdv-resistant mutants in the presence of increasing concentrations of the compound, the a t mutation appeared before the a v substitution, and the latter mutation only occurred in conjunction with a t. when tested for virulence in a lethal intranasal infection model in mice, all cdvr recombinant viruses proved to be attenuated, suggesting that cdvr mutations are associated with reduced pathogenicity. furthermore, we found that cdv at a dose of mg/kg/day for days was still able to protect mice (in terms of body weight loss) against an intranasal challenge with the a t + a v recombinant virus. evaluating the use of cpg dna as an antiviral therapy amanda phelps, linda eastaugh, amanda gates, david ulaeto, arthur krieg defence science and technology laboratories (dstl), salisbury, wiltshire, uk; coley pharmaceuticals ltd., ottawa, ont., canada at present there are no licensed antivirals against orthopoxvirus infections such as variola or vaccinia virus (vacv). although a stockpile of smallpox vaccine exists and has utility as a post-exposure treatment to infection, it is a live viral vaccine and as such cannot be administered to those with contraindications. bacterial dna contains unmethylated motifs that, together with their flanking regions, can stimulate an innate immune response. synthetic cpg dna mimics the immunostimulatory activity of bacterial dna and is recognised by intracellular toll-like receptor . there are four classes of cpg dna all of which have different properties, eliciting distinct initial immune responses. previous studies using an established lethal respiratory model of poxvirus infection demonstrated that a class b cpg dna ( ) could provide protection from lethality against vacv in balb/c mice when administered up to days prior to challenge. in order to evaluate efficacy balb/c mice were challenged intra-nasally with vacv and treated with doses of ranging between and ug/mouse. treatment was administered intra-nasally under light anaesthesia either on the day of challenge, , , , or days post-challenge. efficacy was determined by percentage body weight loss post-challenge. the optimum survival rate observed was % when treated with ug day post-challenge ( mld challenge). a survival rate of % was observed when treated with ug days post-challenge ( mld challenge). the delay of treatment to either or days post-challenge was ineffective, indicating that the window of opportunity for delivery of is within days. multiple doses of were used to attempt to extend this window of opportunity, delivering twice within a -day period. interestingly, this had a considerable detrimental effect, actually accelerating the onset of disease and ultimately death. further work is required to optimise the use of cpg dna as a potential antiviral therapy, and there is evidence to suggest that they may have immense utility as part of a co-administration therapy with other antiviral compounds, an area of work currently under investigation. © crown copyright dstl . department of virology, hebrew university, hadassah medical school, jerusalem, israel the pathogenicity and immunogenicity of the lister (elstree) strain of vaccinia virus, used for vaccination against smallpox, was studied in the mouse model. the virus did not reach the brain when inoculated intranasally, but when injected intracranially at a dose of × plaque forming units (pfu), was lethal for % of the mice. lower doses of virus caused the mice to initially loose some weight but they completely recovered thereafter. a significant level of protection against a lethal dose of the wr strain was achieved in mice following immunization with the lister strain, while higher doses and repeated vaccination procedure, were required with modified vaccinia virus ankara (mva). we found that the lister vaccine strain applied in israel is comprised of heterogeneous virus population. we isolated and plaque-purified three virus variants differing in their plaque size in bs-c- cell cultures. they were named: l-large plaque, m-medium plaque and s-small plaque variants. these isolates could be neutralized by rabbit antibodies prepared against the western reserve strain of vaccinia virus and their one-step growth curves in bs-c- cells were quite similar. however, they differ in their pathogenicity to mice following intranasal inoculation of pfu, or an intracranial injection of × pfu; the s variant being more virulent than the other two variants and resembles the pathogenicity of the lister strain. activity was also determined against a thymidine kinase (tk) deficient vaccinia virus in mouse and monkey cells. the potency of (n)-mct was similar to that seen with wild-type virus, suggesting that a cellular enzyme may be more important than viral tk to phosphorylate the compound. mice were intranasally infected with cowpox and vaccinia viruses followed h later by intraperitoneal treatment with (n)-mct ( x/day for days) or cidofovir ( x/day for days). (n)-mct treatment at and mg/kg/day resulted in and % survival from cowpox virus infection, respectively, compared to % survival (placebo). statistically significant reductions in lung virus titers on day occurred in , , and mg/kg/day treated mice. these doses did not spare mice from lethal vaccinia virus challenge, however, but the and mg/kg/day treatments significantly reduced day virus titers and lung weights, and the mg/kg/day treatment reduced lung consolidation. cidofovir ( mg/kg/day) protected all animals from death in both models. the evaluation of (n)-mct may be limited to mice based upon its greatly reduced efficacy in the cells of higher animals. acknowledgement: supported in part by contract no- -ai- from the virology branch, niaid, nih. chelsea byrd , elena sbrana , shu-yuan xiao , marina siirin , robert tesh , dennis hruby , robert jordan siga technologies, inc., corvallis, or, usa; university of texas medical branch, galveston, tx, usa st- is a potent small molecule inhibitor of orthopoxvirus replication that has been shown to protect mice from lethal challenge with vaccinia and ectromelia viruses. here we report the results of preliminary trials that show efficacy of st- against severe monkeypox virus infection in the ground squirrel model. ground squirrels infected with less than pfu of monkeypox virus develop a fulminant disease resembling human hemorrhagic smallpox: the most severe and lethal form of the disease. oral administration of st- at mg/kg once per day for days protected ground squirrels from a lethal challenge with and pfu of monkeypox virus. compound treated animals showed no weight loss or evidence of disease, and blood chemistry values were similar to uninfected animals. in contrast, placebo-treated animals showed elevated liver enzyme (alt and ast) levels and all animals died by day post-infection. when treatment with , , and h, % protection was observed in the , , and h groups, and % protection in the h group. severe pathologic changes were observed in the organs of the animals receiving placebo, especially in the lungs, liver, and spleen. in contrast, the organs of the animals receiving st- at , , , and h postinfection appeared grossly and microscopically normal. thus, st- appears to be a promising candidate for continued development as a therapeutic agent for severe orthopoxvirus infection. inge vliegen , guang yang , dennis hruby , erik de clercq , robert jordan , johan neyts rega institute for medical research, k.u. leuven, belgium; siga technologies, inc. corvallis, or, usa st- is a potent inhibitor of the replication of various orthopoxviruses. resistance of cowpoxvirus to st- maps to a mutation in v , which is homologous to vaccinia virus f l (yang et al., . j. virol.) . the latter encodes the envelope protein p required for production of extracellular virus. deleting f l resulted in a virus ( f l-vac) that is replicationcompetent in cell culture but that produces smaller plaques than the wild-type wr-vac. whereas intravenous (i.v.) inoculation of nmri mice with × pfu of wr-vac resulted in ± pox tail lesions per mouse, the same inoculum of f l-vac caused no lesions (p < . ). athymic nude (nu/nu) or scid mice inoculated iv with × pfu f l-vac did not develop tail lesions. the mean day of death in nu/nu mice inoculated with f l-vac was ± days as compared to ± days for wr-vac-infected mice (p < . ); scid mice survived the infection. we next studied whether f l-vac is able to protect mice against a subsequent infection with wr-vac. to mimic the human vaccination protocol, nmri mice were infected intracutaneously (i.c.) by means of scarification at the lumbosacral area with × pfu f l-vac or placebo. none of the infected mice developed lesions at the inoculation site. one week later, animals were infected ic with × pfu of wr-vac. all placebo animals, but none of the f l-vac animals developed poxvirus lesions. in a second set of experiments, mice were again inoculated ic with placebo or f l-vac and were infected one week later with × pfu of wr-vac by the iv route. placebo animals developed an average of ± pox tail lesions; no lesions developed in the f l-vac animals (p < . ). in a third set of experiments, nmri mice were inoculated iv with either × pfu of f l-vac or placebo, and none of the mice developed lesions. one week later, animals were inoculated iv with × pfu wr-vac. the placebo group developed an average of ± lesions as compared to . ± . lesions in the f l-vac mice (p < . ). f l-vac may thus be considered as a severely attenuated virus that may have potential for use as a smallpox vaccine. ji yuan , travis lim , shuan coughlin , dexin qiu , zhen liu , dave stein , decheng yang the james hogg icapture centre for cardiovascular and pulmonary research, university of british columbia, vancouver, bc, canada; avi biopharma, inc., corvallis, or, usa background: coxsackievirus b (cvb ) is the most common cause of viral myocarditis, but existing drug therapy is of limited value. antisense oligonucleotides (asons) designed to pair with viral rna could inhibit viral replication. however, the effectiveness of traditional asons is limited due to poor cellular uptake and degradation by nucleases. phosphorodiamidate morpholino oligomers (pmos) contain backbone modifications, which make pmos more resistant to nucleases. in addition, an arginine rich peptide (p ) is conjugated to the end of the oligomer to improve its delivery into cells. these features make p -conjugated pmos (p-pmos) promising candidates for the inhibition of cvb infection. methods: total p-pmos targeting distinct regions of viral genome and one scrambled sequence were designed and chemically synthesized. fitc labeled p-pmos were used to observe their distribution of by confocal microscopy. viral protein vp , viral titre, and cell viability were measured by western-blot, plaque assay, and mts assay, respectively. results: p-pmos showed increased cellular uptake compared to non-conjugated pmos. among the p-pmos, p-pmo- , targeting the internal ribosomal entry site in the utr, showed the most potent anti-cvb ability in a dose-dependent manner. both infected hela and cardiomyocytes hl- cells treated with p-pmo- showed drastically reduced vp production and . log decreases in viral titres as compared to the controls. cell viability assay revealed that and % of treated hela and hl- cells were still alive as compared to and % of control-treated cells and % antiviral activity still existed after days treatment. in addition, cells treated post-infection showed similar inhibition of viral replication. furthermore, the specificity of the p-pmos was demonstrated by their inability to inhibit rsv infection in hela cells. we have showed that p-pmos can effectively inhibit viral replication in vitro, providing a new possibility for antiviral intervention. picornaviruses are responsible for various human viral diseases including common cold, encephalitis, meningitis, myocarditis, etc. up to now, there is no specific antiviral therapy to treat or prevent such viral disease. the usage of modern computer technologies may help to solve this problem more effectively. the objective of the present study was the quantitative structure-activity relationship (qsar) analysis of antiviral activity of a set of [(biphenyloxy)propyl]isoxazole derivatives that inhibit cvb replication in hela cells. based on results from qsar, the structure of new potential antiviral agents should be predicted by using consequent molecular design. the qsar approach applied is based on simplex representation of molecular structure (sirms). the relationship between: (a) antiviral activity against the pleconaril-sensitive clinical cvb isolate - (ic , g/ml); (b) cytotoxicity in hela cells (cc , g/ml); and (c) selectivity index (si = ratio of cc to ic ), and structure of [(biphenyloxy)propyl]isoxazole derivatives has been studied systematically. quite adequate qsar models (r = . - . , q = . - . ) have been obtained using pls (partial least squares) method for all parameters studied. the models are in close correlation with experimental data. structural fragments with positive or negative influence on cytotoxicity as well as antiviral activity have been determined on the base of these models. for example, qsar analysis of antiviral activity of [(biphenyloxy)propyl]isoxazole derivatives revealed that the presence of m-nitrophenyl or p-trifluorophenyl fragment has distinctly negative influence on antiviral action. compounds with strong antiviral activity have to contain an oxadiazole fragment. moreover, our data allow the virtual screening and molecular design of new well-tolerated compounds with strong anti-cvb activity. ivanka nikolova , roumena petkova , boris atanassov , stoyan chakarov , angel s. galabov institute of microbiology, bulgarian academy of sciences, sofia, bulgaria; scientific technological service, ltd., sofia, bulgaria; institute of organic chemistry, bulgarian academy of sciences, sofia, bulgaria analysis of the rna sequence of the disoxaril-resistant mutants of the coxsackievirus b was carried out. the wild-type disoxaril-sensitive strain (connecticut ) and two disoxarilresistant mutants (one of them produced in fl cells and the other one isolated from brains of newborn mice) infected with coxsackievirus b and treated with disoxaril and a disoxarildependent mutant strain obtained from the resistant strain by passages in cell culture were included in the present study. a rt-pcr assay with primer sets selected from a region of the coxsackievirus b genome coding for the capsid protein vp was carried out. a parallel comparative analysis of the sequences of resulting fragments from the disoxaril mutants studied and the genbank sequence of origin of the vp gene of coxsackievirus b was performed with the blast alignment tool. distinct alterations in the vp locus of the disoxaril-resistant and the disoxaril-dependent mutants compared to the sequence of origin from the genbank (namely, a deletion of uug at ntt. - and an insertion of uuu at nt. ) were observed. high-degree similarity ( %) between the resistant mutant produced in cell cultures and the dependent strain was observed, while the similarity to the wild strain was only %. the resistant mutant obtained in mice was found to be very similar to the strain, developed in cell cultures. a putative -d model of the spatial folding of the target protein in disoxaril mutants is proposed. ralitsa vassileva-pencheva, angel s. galabov in previous study of ours we presented a new approach to combined application of antivirals-consecutive administration of the partners. this schedule could be considered especially suitable for treatment of enteroviral infections, in which the development of resistance is very rapid due to the extremely high viral mutation rate. this approach aims to restrict the resistance development in experiments in vivo, using antivirals with proved high efficiency in experiments in cell cultures. the screening of various double, triple, and quadruple combinations that we carried out showed that two of the triple combinations, namely disoxaril (win compound)/oxoglaucin (a new antiviral drug, developed in our laboratory)/ptu- (a classic enteroviral inhibitor) and disoxaril/oxoglaucin/guanidinehydrochloride (a classic enteroviral inhibitor) manifest significant effect of protection in newborn mice with neurotropic coxsackievirus b infection. in the current study the role of the chronology of arrangement of the antivirals included in the combinations was investigated. in the experiments carried out with the triple combination disoxaril/oxoglaucin/guanidine-hydrochloride, it was found that the optimal treatment course should start with disoxaril. the treatment course is quite successful when disoxaril is followed by guanidine-hydrochloride. the effect of the triple combination starting with oxoglaucine, followed by guanidine-hydrochloride was moderate. the course starting with guanidine-hydrochloride proved to be ineffective. furthermore, we studied the virus sensitivity to the inhibitorspartners (ic values) and some other phenotypic characteristics of the brain isolates, e.g. the size of the plaques and the pathogenicity for mice. recently our contribution to the development of new antipicornavirus agents has led to the discovery of methylthio- -aryl-isoxazole- -carbonitrile derivatives whose in vitro anti-coxsackievirus b activity were dependent on the nature of the substituents on the para position of the phenyl ring. particularly, compounds -methylthio- -[ -( -phenyl- -propoxy)phenyl]isoxazol- -carbonitrile (on- ) and -methylthio- -[ -( -phenoxy- -buthoxy)phenyl]isoxazol- -carbonitrile (on- ) exhibited an interesting antiviral activity with high selectivity indexes. in the present study, we investigated on the mechanism of action of these compounds. studies on time of addition experiments suggested that these compounds exert a different interference with an early step of the viral replicative cycle. in fact, compound on- was effective when added within h after the end of the adsorption period and no reduction was observed if it was added during the adsorption period. whereas a reduction of virus titer was observed for on- when was added during the adsorption period, while no reduction was observed if the compound was added after this period (time ). the influence of the compounds on virus adsorption step, studied by the infective center assays, indicated that on- primarily interferes with coxsackie b cellular attachment. at a concentration times the id , inhibition of adsorption of coxsackievirus by on- was complete, while similar concentration of on- had no effect. our experiments on neutralization of viral infectivity and on thermal stabilization demonstrated that the compounds were able to directly inactivate coxsackievirus, and the infectious titer was restored to the original value after extraction of the compound with chloroform. however, the compounds did not protect the viral infectivity against heat inactivation at the different concentrations used. the blood-brain barrier (bbb) fulfills a vital protective function by limiting entry of potential pathogens, toxins, and inflammatory cells into the central nervous system (cns). disruption of the bbb is a common component of many cns diseases, including viral diseases such as that caused by west nile virus (wnv). transforming growth factor-␤ (tgf-␤ ) has previously been shown to improve the function of an in vitro model of the bbb. we evaluated the role of the bbb in wnv infection in mice by determining the ability of intraperitoneally (i.p.) administered sodium fluorescein to move from the circulating blood to the central nervous system. to demonstrate bbb permeability a mean and normal range of permeability values was determined in non-infected c /bl mice. in subsequent experiments, any animal expressing a permeability value greater than sd above the mean was considered abnormally high. we determined that elevations in bbb permeability can be detected in mice days after subcutaneous inoculation with wnv. wnv inoculated animals were treated with doses of , , or ng/kg/day of tgf-␤ or with drug vehicle once daily via the i.p. route on and days post-virus inoculation (dpi), and then assayed for bbb permeability on dpi. sixty-two percent ( / ) of placebo-treated animals had abnormally high permeability values, while animals treated with and ng/kg/day of tgf-␤ had % ( / ) and % ( / ) of animals with abnormally high permeability values, respectively. in contrast, none of the animals treated with ng/kg of tgf-␤ ( / ) expressed abnormally high permeability values, which was significantly lower (p < . ) than placebo-treated animals. these results suggest that tgf-␤ may improve the function of the blood-brain barrier in wnv infected mice. acknowledgement: supported by grant -u ai - from the rocky mountain regional centers of excellence, nih. and contract no -ai- from the virology branch, niaid, nih. people infected with west nile virus (wnv) usually see their physicians after showing symptoms suggestive of neurological infection. wnv infects the central nervous system (cns) of rodents - days after s.c. viral challenge. yet, most published animal studies begin therapeutic treatments before or soon after viral challenge. the question addressed in this study is if neuroprotective agents can be efficacious when administered early before brain infection or later after the virus is demonstrated to be in the brain. the drugs evaluated in wnvinfected rodents were nmda and ampa receptor antagonists, modulators of nitric oxide synthase (nos) and nitric oxide production, and riluzole for reducing glutamate excitotoxicity. serial doses of diethyldithiocarbamate (ddtc) and n(g)monomethyl-l-arginine (l-nmma), an inducer or inhibitor of nos, respectively, administered i.p. daily for days beginning h before viral challenge slightly improved survival of mice, but the difference was not statistically significant. tolerated doses of two nmda-receptor antagonists, flupertine ( mg/kg) and mk- ( mg/kg), and one ampa-receptor antagonist, gyki ( mg/kg), were administered twice daily (b.i.d.) on though days post-virus inoculation (dpi). gyki slightly improved mouse survival and weight gain, but the difference was not statistically significant. talampanel, an ampa-receptor antagonist and a derivative of gyki , slightly improved hamster survival (p ≤ . ) when treatment began on dpi, but repeated experiments using different doses and slightly different protocols gave mixed results. riluzole, the only drug shown to improved survival of amyotrophic lateral sclerosis (als), presumably by reducing glutamate excitotoxicity, was not effective against wnv disease when administered b.i.d. beginning dpi. overall, neuroprotective agents did not consistently improve wnv disease, although slight improvements in animal survival might be relevant to improvement of neurological sequelae in wnv-patients. acknowledgement: supported by contract no -ai- from the virology branch, niaid, nih. hamster and mouse models for west nile virus (wnv) disease were used in this study to identify infected cells of the central nervous system (cns) early in the course of infection. this information may be relevant to therapeutic strategies since most wnv-infected people visit their physicians after showing symptoms suggestive of neurological infection. we subcutaneously infected adult mice and hamsters using . tissue culture infectious doses of wnv. tissues of infected and control animals from to days post-viral injection (dpi) were fixed by cardiac perfusion using % paraformaldehyde. we localized wnv, neuronal and astroglial markers in the paraffin embedded tissue sections by immunofluorescence. the images were captured using the confocal microscope (bio-rad, mrc ). we observed the presence of wnv antigen in cns tissues of mice and hamsters as early as and dpi, respectively. a strong wnv-specific immunofluorescence staining was observed in the cytoplasm of neurons from the spinal cord, cerebellum, cerebral cortex, and midbrain of these rodents. the wnv-specific staining co-localized with neuron-specific markers; however, astroglial markers were not co-localized with wnv antigen in brain sections. the lack of tropism by wnv for astrocytes was also confirmed in primary murine astrocyte cultures. interestingly, infected neurons in the midbrain of -day infected hamsters co-localized with calbindin, which is a calcium-binding protein and mostly expressed in the interneurons of the cns. therapies were evaluated in hamsters or mice at a time-point when wnv-stained neurons were identified in the cns. acknowledgement: supported by grant -u ai - from the rocky mountain regional centers of excellence, nih. laboratory of virology, department of biological chemistry, school of sciences, university of buenos aires, argentina dengue virus (denv) is an arthropod-borne flavivirus that has re-emerged in recent years as an increasingly important public health threat with nearly million infections occurring each year. at present neither specific antiviral therapy nor vaccine exists for the treatment and prevention of denv infections. carrageenans are sulfated galactans that can be extracted from red seaweeds and comprise diverse structures with a wide range of biological properties useful in biomedicine. in a previous study we have demonstrated the antiviral activity of commercialand -carrageenans against denv type and in vero (monkey kidney cells) and hepg (human hepatoma cells), showing inhibitory concentration % (ic ) values in the range . - . g/ml and selectivity indexes (cc /ic ) in the range - . in the present work we studied the mode of action of these polysulfates against denv- in vero and hepg cells, first analyzing the influence of time of addition of compounds on anti-denv activity by an infectious centre assay. the highest inhibitory effect was observed when the compounds were added during adsorption or at h p.i., being ineffective at later times. then, the effect of compounds on virus adsorption and internalization was studied separately by a virus yield inhibition assay. significant antiviral efficacy was attained if compounds were present either only during denv- adsorption or internalization. the possible effect of carrageenans on viral protein synthesis, the subsequent stage of the virus cycle occurring during the first hour of infection, was analyzed by pulse-labeling with ( s)-methionine. no alterations in denv protein synthesis in carrageenan-treated cells were observed. when cells were transfected with purified denv- rna in the presence of -carrageenan no inhibition in fluorescent cell focus formation and virus production was detected. besides, no significant direct virucidal effect on denv- was shown by the compounds. these results indicate that both denv adsorption and internalization seem to be the main target for these compounds, lacking effect on the steps that occur once the viral genome is inside the cell during in vitro infection of human and monkey cells. multiple members of the flavivirus genus of the family flaviviridae cause lethal hemorrhagic fever or encephalitis. the public health significance of the hemorrhagic fever and encephalitis caused by such flaviviruses is enormous and global and there is a tremendous need for antivirals. imino sugar glucosidase inhibitors have been shown to have selective antiviral activity against viruses such as bovine viral diarrhea virus (bvdv) and west nile virus (wnv) that have common requirements for their glycoprotein processing during virus production. we are developing imino sugar deoxynojirycin (dnj) derivatives through chemical synthesis of compounds with various alkyl side chains and antiviral testing against bvdv and wnv as well as in wnv subgenomic replicon assays. briefly, using a single step growth (virus yield reduction) assay for bvdv and wnv, a series of dnj derivatives containing various conformational locking side chains were shown to have antiviral activity. pre-liminary structure-activity relationships (sar) were obtained for further modification of the alkyl side chain and improvement of these dnj derivatives. the activity and mechanisms of action of these compounds will be presented. several flaviviruses cause life-threatening diseases in man. currently, there is no therapy available for these infections. in recent years, several highly selective inhibitors of the replication of hepatitis c virus (hcv) were designed. most small molecule inhibitors of hcv that are in preclinical or clinical development are either protease or polymerase inhibitors. most of these compounds are highly selective for hcv and are unlikely to exhibit activity against flaviviruses. nucleoside polymerase inhibitors, however, may have the potential to inhibit the replication of flaviviruses as well. we evaluated in vitro whether the active component of the anti-hcv compound valopicitabine, i.e. -c-methylcytidine inhibits the replication of flaviviruses in cell culture. the compound was found to exhibit specific antiviral activity against yellow fever virus d (ec = . g/ml in cpe reduction assays and > % reduction at g/ml as assessed by qpcr) and dengue fever virus type (ec = . g/ml in cpe reduction assays). the compound also efficiently inhibited west nile virus replication (> % at g/ml as assessed by qpcr and > % by plaque reduction neutralization test at g/ml). in the absence of any drugs for the treatment of flavivirus infections, it may be envisaged that nucleoside polymerase inhibitors, when marketed for the treatment of hcv infections, could be used off-label for the treatment of lifethreatening flavivirus infections. even if such drug would not be able to completely inhibit flavivirus replication, a partial reduction of the viremia during the acute phase of the infection may be sufficient to prevent the development of a fulminate disease and thus protect against virus-induced mortality. yuri klimochkin , andrew shiryaev , igor moiseev , v sabynin , larisa rustamova , alexandr petkevich state technical university, samara, russia; institute of epidemiology and microbiology, minsk, belaruss arenaviruses are one of the most dangerous tools of bioterrorism in the view of pathogenicity and epidemiological threat. for the purpose of searching new remedies for treatment are-naviruses infections the synthesis of new derivatives of cage compounds has been carried out. the prepared compounds are bridgehead derivatives of cage compounds bearing different functional groups such as hydroxy, acylamino, alkoxycarbonylamino, alkylthiocarbonylamino groups as well as iminoalkyl adamantane derivatives, some adamantylated heterocycles and compounds containing two adamantane moieties in a molecule. the antiviral activity of the cage compounds has been studied in respect to arenaviruses lassa (sierra-leone strain) and pichinde (an- strain) on the vero cells culture. different level of antiviral activity was shown by compounds. the most active compounds are monosubstituted adamantane derivatives having sufur and nitrogen-containing substituent in the bridgehead position. the data on the activity confirm the availability of searching inhibitors of arenaviruses reproduction in the cage compounds series. brian gowen , donald smee , min-hui wong , anne pace , kie-hoon jung , kevin bailey , lawrence blatt , robert sidwell institute for antiviral research, utah state university, logan, ut, usa; intermune, brisbane, ca, usa several arenaviruses endemic to the south american (junin, machupo, and guanarito) and african (lassa) continents are known to cause frequently fatal hemorrhagic fever. with the exception of ribavirin, which has demonstrated efficacy in cases of lassa fever, there are no other effective therapeutics for the treatment of arenaviral hemorrhagic fever. the outcome of treatment is ultimately dependent upon early diagnosis and the tolerability of ribavirin by patients at the high doses required for effective antiviral activity. we have recently demonstrated that consensus interferon-alpha (ifn-alfacon ) can protect hamsters from lethal pichinde virus (pcv) infection (gowen et al., . antimicrob. agents chemother.), which serves as a model for acute arenaviral disease in humans. here we demonstrate highly effective therapy through the combined use of ribavirin and ifn alfacon- for the treatment of pcv infection in hamsters. ribavirin was given orally, twice per day for days, and ifn alfacon- was administered intraperitoneally, once per day for days. treatments were initiated - days post-infection with various dose combinations, many which were less than optimal when the drugs were given independently. combining suboptimal doses of ribavirin ( - mg/kg/day) with ifn alfacon- ( - mg/kg/day), we were able to show increased protection from mortality, reduced viral burden and liver disease, and greatly extended survival times as compared to treatments where drugs were administered alone. our data indicate that synergistic activity resulted from combination therapy and that this activity may slow down the progression of disease and decrease fatality rates seen with severe arenaviral infections. further, combination therapy reduces the effective dosage of ribavirin, which would serve to limit its toxicity. acknowledgement: supported by contract no -ai- from the virology branch, national institute of allergy and infectious diseases, national institutes of health. slobodan paessler , laure deflubé , andrew vaillant , jean-marc juteau , ramon flick department of pathology, university of texas medical branch, galveston, texas, usa; replicor inc., laval, que., canada rift valley fever virus (rvfv; genus phlebovirus, family bunyaviridae) is an arbovirus transmitted by many species of mosquitoes. this virus is a major public health concern in sub-saharan africa and egypt, which spread to yemen and saudi arabia. in this area, rvfv is responsible for dramatic epidemics/epizootics underlining the need for efficient antiviral/prophylactic measures. rep is a mer phosphorothioate oligonucleotide, which has previously been shown to have broad-spectrum activity in several viruses (vaillant et al., submitted for publication) . we used a vaccine strain (mp ) as well as the wild-type rvfv (zh ), to test the ability of rep to inhibit bunyavirus proliferation. in vitro data showed reduction of virus titer for both strains using rep at nanomolar concentrations. moreover, the absence of the phosphorothioate modification in a stabilized rep analog resulted in a loss of antiviral activity, suggesting that as in other viruses, the increased hydrophobicity of rep is essential for its antiviral activity. based on the inhibitory activity observed in vitro, we started with in vivo efficacy studies by utilizing a validated mouse model used in our laboratory. more animal experiments are ongoing to confirm the in vitro results and to evaluate the antiviral effect of the rep . adriana garozzo , rossella timpanaro , aldo stivala , gianna tempera , christian c.c. cutrì , angelo castro department of microbiological sciences, university of catania, via androne , catania, italy; department of pharmaceutical sciences, university of catania, viale a. doria , catania, italy our previous studies described the synthesis and the antiviral activity of , , -trisubstituted isothiazole derivatives that were found to be particularly effective against picornaviruses. compound -methylthio- -phenyl- -isothiazolecarbonitrile (is- ) exhibited an interesting anti-poliovirus activity with high selectivity index. in the present study, we investigated on the mechanism of action of this compound. studies on the time of is- addition to poliovirus type infected cells suggested that the compound may inhibit some early processes of viral replication. in order to determine its mechanism of action, we evaluated the rate of attachment and internalization of purified [ h]uridine-labeled poliovirus to hep- cells in the presence or absence of is- . no effect on poliovirus adsorption and internalization to host cells was detected. we also investigated the influence of the compound on virus uncoating using labeled poliovirus and measuring the radioactivity of oligoribonucleotides formed from viral rna susceptible to ribonuclease. these experiments demonstrated that poliovirus uncoating is influenced by is- action. justin julander , aaron olsen , john morrey , lawrence blatt , kristiina shafer , robert sidwell institute for antiviral research, utah state university, logan, ut, usa; intermune, brisbane, ca, usa alpha togaviruses are medically important arboviruses, with clinical cases occurring each year in north, south, and central america. the recent increase in the threat of the use of these viruses as bio-terrorism agents has led to increased efforts to develop therapeutic agents for treatment of these viruses. venezuelan (vee) and western equine encephalitis (wee) viruses have been listed as category b priority pathogens by the national institute of allergy and infectious disease (niaid). the goal of these studies was to characterize animal models for vee and wee for use in evaluation of antiviral therapies. c h/hen mice were infected through the intranasal (i.n.) route with a vaccine strain of vee, tc- . virus was detected in the brain days post-viral injection (dpi). brain titers increased to a peak titer of . % cell culture infectious doses per gram tissue (ccid /g) on dpi, maintained a titer of ccid /g through dpi, and dropped slightly to . ccid /g by dpi. virus was also detected in spleen, liver, and kidney. treatment of vee-infected mice with interferon alpha b/d, a human consensus interferon, resulted in % survival, whereas all placebotreated animals died by dpi. syrian golden hamsters were infected with ccid wee through intraperitoneal (i.p.) injection. morbidity, including hind limb paralysis, tremors, nasal bleeding, and hunching, and some mortality were seen as soon as dpi. the majority of deaths occurred on dpi. virus was detected in all organs assayed (brain, liver, and spleen) with peak titers occurring dpi. interferon alfacon (ifn alfacon), a human consensus interferon, active in hamsters, was effective in significantly reducing mortality (p < . as compared to placebo). there was a trend for reduction of brain titers in ifn alfacon-treated animals (mean titer . ccid /g) as compared with placebo (mean titer . ccid /g), although this difference was not statistically significant. these models will be useful in screening potential antiviral agents for efficacy against vee and wee. acknowledgement: supported by contract no -ai- from the virology branch, niaid, nih. justin julander , kristiina shafer , john morrey , lawrence blatt , robert sidwell institute for antiviral research, utah state university, logan, ut, usa; intermune, brisbane, ca, usa yellow fever virus (yfv) has caused significant morbidity and mortality for centuries. primates were the only animal models for visceral yfv. recently, hamsters were found to have morbidity and mortality when injected with a hamster-adapted jimenez strain of yfv (tesh et al., j. infect. dis. , - . the objective of this study was to characterize this model of yfv viscerotropic disease for the study of effects of antiviral compounds and to test compounds with known efficacy for use as a positive control. animals were challenged with a − dilution (a dilution previously shown to cause high mortality) of a liver homogenate made from livers taken days post-viral injection (dpi) from hamsters challenged with the jimenez strain. there was % mortality in animals challenged with the virus up to dpi. virus titers in the liver peaked dpi as determined by qrt-pcr. a significant increase in serum levels of alt ( dpi), alkaline phosphotase ( dpi) and bilirubin ( dpi), and a significant decrease in amylase ( dpi), albumin ( dpi), and glucose ( dpi) were observed. hepatic icterus was observed in hamsters that exhibited disease signs at the time of necropsy. hamsters were treated with ribavirin or interferon (ifn) alfacon , a consensus interferon. ribavirin and ifn alfacon both significantly (p < . ) reduced mortality as compared with placebo-treatment. there was also significant reduction in weight loss with ribavirin (p < . ) and ifn alfacon (p < . ) treatment as compared with placebo. disease signs, such as lethargy and lying prostrate, were also reduced with treatment of ribavirin and ifn alfacon . viral liver titers from treated animals were not significantly different from titers in placebotreated animals. the hamster model of yfv disease will serve as a suitable model for the evaluation of antiviral compounds for efficacy against the virus. acknowledgement: supported by contract no -ai- from the virology branch, niaid, nih. polyomaviruses are small dna tumor viruses that depend on the host cellular dna polymerase for their replication. three polyomaviruses have been associated with tumor formation in humans: jc virus (jcv), bk virus (bkv) and simian vacuolating virus (sv ). in addition, some of them have been associated with viral diseases. jcv can cause progressive multifocal leukoencephalopathy in immunosuppressed patients, while bkv is considered to be the causative agent of polyomavirusassociated nephropathy, which leads to kidney transplant failure. sv has not been associated with a well-defined disease, but viral dna sequences and protein expression have been detected mostly in central nervous system (cns) tumors which strengthens the evidence for the association of this virus with human cancer. the activity of various acyclic nucleoside phosphonates (anps) such as cidofovir and adefovir against murine polyomavirus and primate sv in vitro has already been demonstrated (andrei et al., . antimicrob. agents chemother. , - ) . here, the activity of a new class of anp's, namely -[ -(phosphonomethoxy)alkoxy]- , diaminopyrimidines, against polyomaviruses was assessed. confluent uc -b cells were infected with either of the four murine polyomavirus strains mn/rde toronto, pta, pta or lid- , while bsc- cells were infected with either the primate sv strain a , the sv pml- strain ek or the sv pml- strain dar. after removal of the residual virus, serial dilutions of the test compounds were added. the viral cytopathic effect was recorded microscopically after - days (murine polyoma virus) or - days (sv ). hpmpo-dapy ( , -diamino- -(r)-[ -hydroxy- -(phosphonomethoxy)propoxy]pyrimidine) and pmeo-dapy ( , -diamino- -[ -(phosphonomethoxy)ethoxy]pyrimidine) were less active/selective than cidofovir and adefovir against the three sv strains tested. hpmpo-dapy and pmeo-dapy proved to be equally active as cidofovir and adefovir against the murine polyomaviruses. naresh sunkara , sylvester mosley , brian bakke , joshua sadler , katherine seley(radtke) , sunny zhou university of maryland-baltimore county, baltimore, md, usa; washington state university, pullman, wa, usa inhibition of biologically significant enzymes critical to nucleotide metabolism and viral replication is a well-established chemotherapeutic approach to the treatment of many diseases. transcriptional -capping of viral mrna has been implicated as an "elongation checkpoint" critical to the replication cycle of many viruses. this capping process is accomplished by various methyltransferases, therefore disruption of methylation becomes an attractive target for therapy. this can be accomplished in several ways; in particular, by direct inhibition of methyltransferases (metase) and/or indirect inhibition of s-adenosyl-l-homocysteine hydrolase (sahase), both established cellular targets for antiviral, antiparasitic and anticancer agents. modified nucleosides, in particular carbocyclic nucleosides, have exhibited potent inhibitory activity against both sahase and metase. inspection of the recent literature has revealed a close correlation between sahase inhibition and potent biological activity against negative stranded (−)-rna viruses (i.e. arenaviridae, paramyxoviridae, rhabdoviridae), double stranded (ą)-rna viruses (reoviridae), poxviridae, as well as hiv- , thus supporting the importance of sahase as a viable chemotherapeutic target. herein we report the design, synthesis, and preliminary biological activity of a new class of structurally novel carbocyclic nucleosides. phosphorylation of ␣-p-borano substituted nucleoside diphosphates charlotta wennefors, mikhail dobrikov, barbara ramsay shaw chemistry department, duke university, durham, nc - , usa most nucleoside antiviral agents require stepwise phosphorylation to their respective triphosphates in order to be activated in the cell. ␣-p-borano substituted nucleoside triphosphates are of interest because they have proven to be good substrates for hiv- reverse transcriptase (rt) and may therefore be useful antiviral agents. studies in our laboratory have indicated that the ␣-p-borano substitution of -dideoxycytidine triphosphate (ddctp) resulted in a -fold increase in efficiency of incorporation by mmlv rt compared to non-substituted ddctp. however, the potency of these ␣-p-borano substituted nucleoside analogs as anti-viral drugs highly depends on their ability to be activated to nucleoside triphosphate (ntp). the phosphorylation of nucleoside analog diphosphates to their respective triphosphates has remained largely unexplored. here, the roles of several phosphorylating enzymes are examined. in our laboratory, nucleoside diphosphate kinase, creatine kinase, and pyruvate kinase are being evaluated for their specificity towards nucleoside analog diphosphates. the effects of nucleobase, ribose, ␣-phosphate substitution and stereochemistry of the boranophosphate group are of interest. the binding affinities of the substrates for creatine kinase (ck) and pyruvate kinase (pk) were determined using a fluorescence-quenching assay, which allowed us to investigate the substrate affinity in the pre-steady state. rabbit muscle ck and pk were titrated with a wide range of ndps and ntps by monitoring a decrease in enzyme intrinsic fluorescence. the affinities of these substrates were determined to establish a structure-activity relationship for ck and pk and to evaluate the effect of a substrate ␣-p-borano modification. ck showed stereospecificity towards the sp isomer of adp␣b whereas pk showed stereospecificity towards the rp isomer of adp␣b. negative cooperativity was observed for all studied substrates. steady-state experiments are also being performed directly following the product formation using uv-visible spectroscopy and high performance liquid chromatography (hplc). these kinases were investigated because they may serve as a means for activation of antiviral ␣-p-borano substituted ndps. traditional antiviral targets encoded by the small human papillomavirus (hpv) genome are lacking. for this reason, we chose to target dna sequences within the hpv genome in an effort to identify compounds that would block viral dna replication in cells. we chose compounds known as polyamides, which are related to distamycin and other natural products, as our dna binding agents. unlike many literature studies where polyamides were designed to block formation of the transcription complex for a particular gene, we chose to target sequences within the origin of replication (ori). thus, pyrrole-imidazole polyamides, with some containing fluorescent probes to aid in cell localization studies, were designed to recognize the hpv ori. the principles used to design these compounds will be described. we used "traditional" hairpin polyamides and some more unusual structures related to very recent literature reports. from the focused library that we prepared, two highly active molecules were identified. the rest of the molecules had minimal or zero activity. no cellular toxicity was observed, either in this project or in a related program where polyamides were used to affect cox- transcription (and subsequent expression) in rheumatoid synovial fibroblasts. of particular interest is the difference between the active molecules and two closely related compounds that were inactive: the active species bind and recognize two more hpv dna base pairs than do the related but inactive structures. this presentation will provide detailed chemistry background and structural information to complement our cell work that is also being presented at the meeting. discovery of the chemokine receptor ccr as a co-receptor for hiv- infections revealed a novel approach to hiv- treatments and preventions. ccr , a member from the family of tm g-protein coupled receptors, thus became an attractive target pursued in the pharmaceutical industry. with the recent successful developments of several small molecules in clinic, these ccr antagonists hold great promise to be the next generation of anti-hiv medicines. this poster will describe our efforts at the n-terminal piperidine ring of template a to improve pharmacological properties of derived molecules. according to current models, proteolytic processing of hiv- gag precursor occurs within the virions which detach from infected cells. meanwhile, the viral protease is activated much earlier, and gag p cleavage initiates in infected cells. we followed the fate of matrix protein cleaved in infected cells (cma) in comparison with ma cleaved in the virions (vma) and showed that both forms differ in their localization in the infected cells and in the virions, both forms are involved into virus pathogenesis and represent the targets for antiviral compounds. mt- cells were labeled with [ h]-leucine or myristic acid, and h after labeling protease inhibitor was added to separate the cleavage of cma from vma. cma was found in the nuclear and membrane fractions of infected cells while cca resided in cytoplasm. - h after labeling cma was found in the virions localizing in the cores. vma was located under lipoprotein envelope of the virions. new membranotropic antiviral compounds based on adamantane-and norbornene-related derivatives not toxic for the host cells were added to mt- cells before infection or - h later and at concentration - ug/ml blocked reverse transcription, the transport of cma into the nuclei, and the production of infectious virus. the compounds inhibiting very early step of virus life cycle are optimal candidates for microbicides. to enhance their antiviral activity, we plan to associate polyanionic matrix with ma imitating peptides and cholesterol-like fragments. kurt vermeire , thomas bell , sreenivasa anugu , noah duffy , roger le grand , erik de clercq , dominique schols rega institute for medical research, katholieke universiteit leuven, leuven, belgium; department of chemistry, university of nevada, reno, usa; service de neurovirologie, fontenay-aux-roses, france the cyclotriazadisulfonamide (cada) compounds were shown to be potent inhibitors of hiv replication in human t-cell lines, pha-stimulated pbmcs, and monocytes/macrophages (ec : . - . m). the prototype compound, cada, had consistent activity against laboratory adapted and primary clinical isolates of hiv- , irrespective of chemokine receptor preference (r , x , r /x ). cada acted synergistically when evaluated in combination with various other hiv drugs, such as reverse transcriptase (rt), protease, and virus entry inhibitors. flow cytometric analysis revealed a significant decrease in the cell surface and intracellular expression of the cd receptor in human cells after cada-treatment. moreover, the anti-hiv activity of cada correlated with its ability to down-modulate the cd receptor in human t-cells. here, we report the consistent antiviral activity of cada against: (i) drug-resistant viruses (i.e. viruses resistant to rt inhibitors, protease inhibitors, and enfuvirtide); (ii) different hiv- subtypes (a, b, c, d, a/e, f, h, o); and (iii) various hiv- strains examined. in addition, cada potently inhibited sivmac infection of pbmcs isolated from macaques (ec : . m). comparable results were obtained in human cells infected with sivmac . flow cytometric analysis also demonstrated a significant and dose-dependent down-regulation of the cd receptor expression at the cell surface of simian pbmcs after treatment with cada. the combination of cada with cellulose acetate , benzenedicarboxylate (cap), an enteric coating polymer for capsules and tablets, resulted in a synergistic antiviral activity. in summary, our data indicate that cada may qualify as a potential anti-hiv microbicide drug candidate for the prevention of the sexual transmission of hiv. the preparation of gel formulations of cada (as single drug and in combination with cap) for vaginal administration in non-human primates is currently under investigation. department of micro & immuno, suny upstate medical university, syracuse, ny, usa varicella zoster virus (vzv, human herpesvirus ) infection causes chicken pox, latency is established in neurons, and reactivation leads to shingles. acyclovir and its derivatives are the treatment of choice for both manifestations of vzv. new therapeutics are needed because acyclovir-resistant strains exist, and treatment must begin within h. we have studied the anti-vzv properties of roscovitine, a cyclin dependent kinase (cdk) inhibitor. here, we tested more compounds that block the cell cycle and determined that vzv is acutely sensitive to them. their effects on vzv replication were tested in human foreskin fibroblasts (hffs) because these primary cultures should have a normal cell cycle (unlike tumor cell lines). the cytotoxicity of the drugs was determined by neutral red dye uptake assays. hffs were inoculated with a low moi ( . ) of vzvinfected cells, which remains entirely cell-associated, and then treated with drugs or diluent for h. vzv spread and replication were measured by infectious focus assay and quantitative real time pcr. all of the drugs tested (acyclovir [acv], phosphonoacetic acid [paa], aphidicolin, aloisine a, purvalanol a, roscovitine, r-roscovitine, s-roscovitine, indole- -carbanol [i c], l-mimosine, dichloro-␤-d-ribofurano-sylbenzimidazole [drb]) had some anti-vzv activity. the selective indices of aphidicolin ( ), purvalanol a ( ), and i c ( ) were greater than the positive controls acv ( ) and paa ( ). aphidicolin inhibits mammalian dna polymerase and is in clinical use for cancer; purvalanol a, a , , -trisubstituted purine, primarily inhibits cdk ; and i c is derived from cruciferous vegetables and inhibits cell proliferation. the concentrations of these compounds that inhibited vzv replication were much less than those needed to cause cell cycle arrest, suggesting that vzv depends on the enzyme activities targeted by these compounds and not on cell proliferation per se. these three drugs will be studied next in skin organ culture and in the scid-hu mouse model of vzv pathogenesis. the results presented here demonstrate that targeting cell functions can inhibit vzv replication and help us better understand virus-host interactions. the viruses could be identified as supra-biopolymeric nanoscale complexes, parasitic intervention in cells of which occurs on an inter-polymeric reactions level. so the antiviral safety can not be fully provided without adequate nano-responsible antivirals (nav). here we discuss a strategy and methodology for the multi-functional nav development by rational macromolecular sar-cooperation of: ( ) polyelectrolyte-specific interferon induction and immunomodulation; ( ) electrostatic-selective prevention of viruses absorption on plasma membranes; ( ) membrane-targeted blocking of post-absorption steps (fusion); ( ) macromolecular prevention of structure-specific interactions of viral and cellular receptors; as well as ( ) polymericassociated disruption of the latest stage of viral replication (virions assembly and maturation). a cooperation of the ( ) and ( ) functions was explored by synthesis and sar-optimization of succinate and carbohydrate polymeric derivatives modified with controllable combinations of anionic (a /a ) groups. the immune-mediated protectors against tick-born, rabies, and other viral infections in vivo, and hiv- absorption inhibitors in vitro, were developed. this pre-nav generation was used as a macromolecular platform to step-by-step targeted design and synthesis toward high effective multi-functional nav where virusresponsible nano-selectivity was achieved by rational intra-or inter-molecular cooperation of virus-specific membranotropic vectors (bi), raft-targeted anchors (ci), and peptide-kind mimickers of virus usable receptors of human cells (pi), particularly ccr /cxcr . as a result, the novel nano-sensitive systems possessed unique wide multi-synergistic antiviral potency on a high level selectivity up to si = , (against hiv- strains) were created and purposed for advancement of antiviral vaccines, drugs, and microbicides. marina kukhanova , alexander ivanov , , georgii galegov , valeria andronova , maxim jasko engelhardt institute of molecular biology, russian academy of sciences, moscow, russia; centre for medical studies, university of oslo, moscow, russia; ivanovsky institute of virology, russian academy of medical sciences, moscow, russia novel acyclic purine phosphonate derivatives bearing a double bond conjugated with the nucleic base, namely, (z)-and (e)- -[ -(phosphonomethoxy)prop- -en- -yl]purines, were synthesized, and their efficacies against hiv- and hsv- were evaluated in cell cultures. the activity of (z)isomer was higher against hiv than that of the reference -[ -(phosphonomethoxy)ethyl]adenine (adefovir) and comparable in respect to the activity of adefovir against hsv. the (e)-isomer showed low antiviral activity against both viruses. the compounds were less toxic towards cell cultures if compared to adefovir. the diphosphates (z)-and (e)- -[ -(phosphonomethoxy)prop- -en- -yl]purines were evaluated as substrates towards hiv- reverse transcriptase and hsv dna polymerase. (z)-isomer was shown to be a more efficient substrate for both enzymes than the (e)-isomer. human dna polymerase alpha could incorporate neither of the diphosphates into the -end of the growing dna chain. available to this virus. jev genome is an approximately -kb single-stranded positive-sense rna that has a cap structure at its terminus but lacks a poly(a) tail at its -terminus. the coding region of the genome is flanked by -and -untranslated region (utr). the -utrs on both plus-and minus-strand jev genome contain important cis-acting elements required for the replication of the viral rna genome. peptide nucleic acid (pna) is a synthetic oligonucleotide, in which the phosphodiester backbone of dna/rna is replaced with a polyamine-( -aminoethyl) glycine skeleton. pna offers a potentially powerful approach for recognition of rna and silencing of gene expression. in this study, we investigated the antiviral effect of the pnas targeted to the -utr region of jev genome. to evaluate the pnamediated inhibitory effect on rna synthesis in vitro, the rnadependent rna polymerase (rdrp) of jev, ns protein, which plays a major role in replication of the viral genomic rna, was expressed in escherichia coli and purified to near homogeneity by sequential column chromatographies. the recombinant jev ns protein exhibited a primer-dependent rdrp activity in vitro on a homopolymeric template, poly(a). in addition, it was able to accept both plus-and minus-strand -utrs as templates for rna synthesis in the absence of an exogenous primer. it could utilize the -end -nt of jev genome as a minimal template. in vitro rdrp assays using this functional recombinant jev rdrp in the presence of the pnas targeted to the jev -utr -nt showed a dose-dependent rna synthesis inhibition. delivery of the inhibitory pnas to the jev-infected cells suppressed jev replication, as determined by western-blot analyses and plaque assays. our results showed a sequence specific inhibition of jev replication by antisense pnas, suggesting the possible application of pna as a novel anti-jev agent. julia serkedjieva , reneta toshkova , milena nikolova , reneta tsvetkova , stefka antonova , ivana roeva , munnever sokmen , bektas tepe , medine gulluce , fikrettin sahin , atalay sokmen institute of microbiology; institute of experimental pathology and parasitology; institute of botany, bulgarian academy of sciences; faculty of biology, department of microbiology, sofia university, sofia, bulgaria; faculty of art and science, department of biology, cumhuriyet university, sivas, turkey; faculty of art and science, department of biology, atatürk university, erzurum, turkey natural products can be an important source of new pharmaceuticals. research on antivirals of natural origin is mainly focused on plants, since, among other reasons, they can be selected on the basis of their ethnobotanical use. plant extracts and natural plant products exhibit also a variety of biological activities with pharmacophoric utility. the population of the balkan peninsula, like people from all continents, has long applied poultices and imbibed infusions of hundreds of indigenous plants. the present report summarizes the antiviral screening study of plant products, obtained from bulgarian and turkish medicinal plants. they were tested for inhibitory effect on the reproduction of selected influenza virus (flu) strains in mdck cells and herpes simplex virus (hsv) strains in mdbk cells. the reduction of virus-induced cpe and infectious virus yields were used as measures of viral inhibition. fifteen samples ( . %) inhibited flu reproduction, and twelve samples ( . %) were active against hsv. the anti-flu activity was confirmed in vivo for all tested samples. the most effective products were tested further for their antiproteolytic, antioxidant and immunogenic capacities and for potential antibacterial and antifungal effects. the following correlations among the variety of biological and pharmacological activities of the plant products were observed: the anti-flu effect was associated with anti-hsv effect and vice-versa in . %; the antiviral effect was connected with antioxidant activity in %; the anti-flu effect was associated with immunogenic properties in %; there was found no correlation between the antiviral effect and the antiproteolytic capacity, the anti-viral properties and bacterial or fungal inhibition, the anti-viral activity and the polyphenol contents. our previous investigations have revealed antiviral activity of some proteolysis inhibitors such as e-aminocaproic acid (e-aca) and para-aminomethylbenzoic acid (pamba) in vitro, in vivo and in clinic. construction of qsar computer-assisted hierarchical system for the effective anti-herpetic (anti-hsv) and anti-influenza (anti-flu) agents' selection as well as the elaboration of new methods of their synthesis are permanently the object of keen interest of our team. the objective of this study was to investigate the efficacy , -di-substituted pyridines and their analogs combined with the fragments of proteolysis inhibitors in the framework of the qsar approach. molecules of new compounds consisted of "nucleus" (py or ar) and two symmetrical fragments: e-aca-carbonyl or pambacarbonyl taken from the inhibitors' molecules. anti-flu activity in dose - m was studied in vitro on the model of a/hong kong/ / (h n ) reproduction in tissue cultures of chorioallantoic membranes of days old chick embryos. anti-hsv activity in doses - m was studied on models of reproduction of hsv- on cell culture hep- . compounds with py-nucleus, contained pamba-carbonyl or e-aca-carbonyl fragments, demonstrated sufficient anti-hsv activity ( . and % of reduction of intra-nucleus virus-specific inclusions on infected cells account accordingly). , -dihydrazine-carbonyl- , -dimethylpyridine showed high anti-hsv ( %) activity. the efficacy of the designed antiherpetic compounds obtained with the combined efforts of qsar computer-assisted design, properties prediction, synthesis, and biological testing as well as the correction introduced after the iteration circle passsage have proven to be the efficient modern way of drug design. acknowledgement: this research was supported in part by stcu grant # and all the authors are indebted to stcu foundation courtesy. lubomira nikolaeva-glomb , angelina trifonova , stephan filipov , angel s. galabov * institute of microbiology, bulgarian academy of sciences, sofia, bulgaria; institute of organic chemistry, bulgarian academy of sciences, sofia, bulgaria a series of aporphinoid alkaloids isolated from glaucinum flavum l. or obtained synthetically, were tested in vitro for antiviral activity against viruses belonging to picorna-, orthomyxo-, paramyxo-and herpesviruses. one of them, oxoglaucine, manifested a well-pronounced inhibitory effect on poliovirus replication in fl cells measured by the semi-quantitative agardiffusion plaque-inhibition test. in virucidal activity testing the compound did not show direct virucidal effect on the extracellular virus. oxoglaucine's % inhibitory concentration for poliovirus (mahoney) was found to be . g/ml in the cpeinhibition test and . g/ml in the classical plaque-inhibition test. similar values were obtained for the vaccinal poliovirus type strain, lsc- ab. the antiviral effect of oxoglaucine on the replication of viruses belonging to another enterovirus species was tested, i.e. coxsackie and echoviruses (hev-b). cva- , the six coxsackie b viruses and echoviruses were tested for their sensitivity against the antiviral effect of oxoglaucine by the endpoint dilution method in the multi-cycle cpe inhibition set-up in fl cells. oxoglaucine revealed a marked inhibitory effect on all tested enteroviruses. the concentrations that reduced virus titer by lg ranged from . to . g/ml. selectivity index was greater than and even greater than for some of the viruses tested. time-of-addition study by the one-step virus growth cycle set-up showed strong virus inhibition during the early periods of virus replication. milka mileva , angel s. galabov department of medical physics and biophysics, medical university, sofia, bulgaria; institute of microbiology, bulgarian academy of sciences, sofia, bulgaria in this study an investigation and comparison of the effects of plant flavonoid polyphenols quercetin and its sugar-containing homologue (rutinoside) rutin on the "oxidative stress" in liver, isolated from influenza virus a/aichi/ / (h n ) ( . of ld ) inoculated mice, is carried out. it was found that experimental influenza virus infection is accompanied with graduated oxidative disturbances in the liver of mice, despite the absence of virus and inflammation in this tissue. it was found that experimental influenza virus infection is accompanied with a significant increase of lipid peroxidation products, a decrease of natural antioxidants (vitamin e, glutathione) and cyp, an inhibition of cytochrome c-reductase and liver monooxygenases (analgin-ndemethylase and amidopyrine-n-demethylase) as compared to control (non-infected) animals. the preliminary ( days) supplementation of mice with rutin, quercetin or its combination, and their subsequent virus inoculation influence significantly all analyzed parameters as compared to controls. the protective effect of rutin against influenza virus-induced lipid peroxidation and activities of cyp and liver monooxygenases in liver was better expressed than the effect of quercetin may be due to containing of rutinoside part or difference of its metabolism. hyun-jeong lee , ji-sun kwon , chi-ung moon , jong-hwan kwak , youn-jeong lee , chang-seon song avian disease laboratory, college of veterinary medicine, konkuk university, seoul, korea; hanyang university, seoul, korea; sungkyunkwan university, seoul, korea; national veterinary research and quarantine services, seoul, korea one of the traditional korean medical herb extract named s was investigated to determine the anti-influenza virus activity in vitro and in vivo. the s showed potent antiviral activities against a/pr/ / (h n ) influenza virus with the % effective concentration (ec ) values of . g/ml and the % cytotoxic concentration (cc ) values of . g/ml in mdck cells. treatment with the s appeared capable of significantly ameliorating the influenza virus infection in mice by oral gavage treatment. the s treated mice showed significantly higher survival rate and lower pathogenic index as well as lung virus titers than untreated control mice. further, the s was extracted with chcl , etoac and n-buoh for isolation of active compounds. the anti-influenza effects of these active compounds will be discussed. the antiviral effect of aqeous and ethanol extracts of ocimum gratissimum (og), terminalia catappa (tc), gynostemma pentaphyllum (gp), newbouldia laevis (nl), aspilia africana (aa) and phyllantus amarus (pa) leaves was examined by cultivation of virus and extracts in embryonated chicken eggs. extracts were inoculated immediately after virus (zero time) or h after virus inoculation. virus replication was compared with those of controls by haemagglutination assay. at zero time, aqeous extracts of og, tc, pa, and gp inhibited virus growth by , , , and %, respectively whereas those of nl and aa did not. ethanol extracts of og, tc, pa and gp at same time inhibited by , , , and %, respectively whereas nl and aa did not. at two h after virus inoculation aqeous extracts of og, tc, pa and gp inhibited virus growth by , , , and %, respectively whereas nl and aa had no effect. ethanol extracts of tc, pa and gp inhibited the virus by , , and %, respectively whereas those of og, nl, and aa did not. the herbs were studied because they were being used by some herbalists in the treatment of human infectious diseases. the th international conference on antiviral research will be held in the palm springs, california area. the conference will begin on sunday, april , and will end on thursday afternoon, may , . all scientific sessions will be held at the westin mission hills resort, rancho mirage, ca. the purpose of the international conference on antiviral research is to provide an interdisciplinary forum at which investigators involved in basic, applied, and clinical research worldwide can meet to review recent developments in all areas of antiviral research. specific topics to be covered in the program include synthesis and chemistry, biochemistry and mechanism of action, molecular biology and drug targeting, in vitro evaluation, animal models, pharmacokinetics, toxicology, and clinical trials. within these areas of interest, there will be invited overview speakers, oral presentations, and poster presentations. the famous el paseo shopping district of palm desert and downtown palm springs offer not only a large variety of galleries, boutiques and shops too numerous to mention, but there are restaurants for virtually every palate. whether your tastes run to burgers, sushi, pizza, escargot, steak, mexican or continental you will find it here with a california flourish in every price range. we hope you will take advantage of this opportunity to combine an important learning experience with a magnificent travel experience and join us in palm springs, california for the th international conference on antiviral research. isar conference committee future conferences acknowledgement: the study was supported by rfbs - - and russian ministry of sciences (lot ). key: cord- -ikepr p authors: tulchinsky, theodore h.; varavikova, elena a. title: expanding the concept of public health date: - - journal: the new public health doi: . /b - - - - . - sha: doc_id: cord_uid: ikepr p ancient societies recognized the needs of sanitation, food safety, workers’ health, and medical care to protect against disease and to promote well-being and civic prosperity. new energies and knowledge since the eighteenth century produced landmark discoveries such as prevention of scurvy and vaccination against smallpox. the biological germ theory and competing miasma theory each proved effective in sanitation, and immunization in control of infectious diseases. non-communicable diseases as the leading causes of mortality have responded to innovative preventive care of health risk factors, smoking, hypertension, obesity, physical inactivity, unhealthful diets, and diabetes mellitus. health promotion proved effective to modern public health in tackling disease origins, individual behavior, and social and economic conditions. the global burden of infectious and non-communicable diseases, aging and chronic illness faces rising costs and still inadequate prevention. the evolution of concepts of public health will have to address these new challenges of population health. the development of public health from its ancient and recent roots, especially in the past several centuries, is a continuing process, with evolutionary and sometimes dramatic leaps forward, and important continuing and new challenges for personal and population health and well-being. everything in the new public health is about preventing avoidable disease, injuries, disabilities, and death while promoting and maximizing a healthy environment and optimal conditions for current and future generations. thus, the new public health addresses overall health policy, resource allocation, as well as the organization, management, and provision of medical care and of health systems in general within a framework of overall social policy and in a community, state, national, transnational, and global context. the study of history (see chapter ) helps us to understand the process of change, to define where we came from and where we are going. it is vital to recognize and understand change in order to deal with radical transformations in direction that occur as a result of changing demography and epidemiology, new science, evolving best practices in public health and clinical medicine, and above all inequalities in health resulting from societal system failures and social and economic factors. health needs will continue to develop in the context of environmental, demographic and societal adjustments, with knowledge gained from social and physical sciences, practice, and economics. for the coming generations, this is about not only the quality of life, but the survival of society itself. over the past century there have been many definitions of public health and health for all. mostly they represent visions and ideals of societal and global aspirations. this chapter examines the very base of the new public health, which encompasses the classic issues of public health with recognition of the advances made in health promotion and the management of health care systems as integral components of societal efforts to improve the health of populations and of individuals. what follows in succeeding chapters will address the major concepts leading to modern and comprehensive elements of public health. inevitably, concepts of public health continue to evolve and to develop both as a philosophy and as a structured discipline. as a professional field, public health requires specialists trained with knowledge and appreciation of its evolution, scientific advances, concepts, and best practices, old and modern. it demands sophisticated professional and managerial skills, the ability to address a problem, reasoning to define the issues, and to advocate, initiate, develop, and implement new and revised programs. it calls for profoundly humanistic values and a sense of responsibility towards protecting and improving the health of communities and every individual. in the twenty-first century, this set of values was well expressed in the human development index agreed to by nations (box . ). public health is a multidimensional field and therefore multidisciplinary in its workforce and organizational needs. it is based on scientific advances and application of best practices as they evolve, and includes many concepts, including holistic health, first established in ancient times. the discussion will return to the diversity of public health throughout this chapter and book many times. in previous centuries, public health was seen primarily as a discipline which studies and implements measures for control of communicable diseases, primarily by sanitation and vaccination. the sanitary revolution, which preceded the development of modern bacteriology, made an enormous contribution to improved health, but many other societal factors including improved nutrition, education, and housing were no less important for population health. maternal and child health, occupational health, and many other aspects of a growing public health network of activities played important roles, as have the physical and social environment and personal habits of living in determining health status. in recent decades recognition of the importance of women's health and health inequalities associated with many high-risk groups in the population have seen both successes and failures in addressing their challenges. male health issues have received less attention, apart from issues associated with specific diseases, or those of healthy military personnel. the scope of public health has changed along with growth of the medical, social, and public health sciences, public expectations, and practical experience. taken together, these have all contributed to changes in the concepts and causes of disease. health systems that fail to adjust to changes in fundamental concepts of public health suffer from immense inequity and burdens of preventable disease, disability, and death. this chapter examines expanding concepts of public health, leading to the development of a new public health. public health has evolved as a multidisciplinary field that includes the use of basic and applied science, education, social sciences, economics, management, and communication skills to promote the welfare of the individual and the community. it is greater than the sum of its component elements and includes the art and politics of the funding and coordination of the wide diversity of community and individual health services. the concept of the interdependence of health in body and in mind has ancient origins. they continue to be fundamental to individuals and societies, and part of the fundamental rights of all humans to have knowledge of healthful lifestyles and to have access to those measures of good health that society alone is able to provide, such as immunization programs, food and drug safety and quality standards, environmental and occupational health, and universal access to high-quality primary and specialty medical and other vital health services. this holistic view of balance and equilibrium may be a renaissance of classical greek and biblical traditions, applied with the broad new knowledge and experience of public health and medical care of the nineteenth, twentieth, and the early years of the twenty-first centuries as change continues to challenge our capacity to adapt. the competing nineteenth-century germ and miasma theories of biological and environmental causation of illness each contributed to the development of sanitation, hygiene, immunization, and understanding of the biological and social determinants of disease and health. they come together in the twenty-first century encompassed in a holistic new public health addressing individual and population health needs. medicine and public health professionals both engage in organization and in direct care services. these all necessitate an understanding of the issues that are included in the new public health, how they evolved, interact, are put together in organizations, and are financed and operated in various parts of the world in order to understand changes going on before our eyes. great success has been achieved in reducing the burden of disease with tools and concepts currently at our disposal. the idea that this is an entitlement for everyone was articulated in the health for all concept of alma-ata in . the health promotion movement emerged in the s and showed dramatically effective results in managing the new human immunodeficiency virus (hiv) pandemic and in tackling smoking and other risk factors for non-communicable diseases (ncds). a health in all policy concept emerged in promoting the concept that health should be a basic component of all public and private policies to achieve the full potential of public health and eliminate inequalities associated with social and economic conditions. profound changes are taking place in the world population, and public health is crucial to respond accordingly: mass migration to the cities, fewer children, extended life expectancy, and the increase in the population of older people who are subject to more chronic diseases and disabilities in a changing physical, social, and economic climate. health systems are challenged with continuing development of new medical technologies and related reforms in clinical practice, while experiencing strong influences of pharmaceuticals and the medicalization of health, with prevention and health promotion less central in priorities and resource allocation. globalization of health has many meanings: international trade, improving global communications, and economic changes with increasing flows of goods, services, and people. ecological and climate change bring droughts, hurricanes, arctic meltdown, and rising sea levels. globalization also has political effects, with water and food shortages, terrorism, and economic distress affecting billions of people. in terms of health, disease can spread from one part of the world to others, as in pandemics or in a quiet spread such as that of west nile fever moving from its original middle eastern natural habitat to the americas and europe, or severe acute respiratory syndrome (sars), which spread with lightning speed from chinese villages to metropolitan cities such as toronto, canada. it can also mean that the ncds characteristic of the industrialized countries are now recognized as the leading causes of death in low-and middle-income countries, associated with diet, activity levels, and smoking, which are themselves pandemic risk factors. the potential for global action in health can also be dramatic. the eradication of smallpox was a stunning victory for public health. the campaign to eradicate poliomyelitis is succeeding even though the end-stage is fraught with setbacks, and measles elimination has turned out to be more of a challenge than was anticipated a decade ago, with resurgence in countries thought to have it under control. global health policies have also made the achievements of public-private partnerships of great importance, particularly in vaccination and acquired immunodeficiency syndrome (aids) control programs. there have been failures as well, with very limited progress in human resources development of the public health workforce in low-income countries. the new public health is necessarily comprehensive in scope and it will continue to evolve as new technologies and scientific discoveries -biological, genetic, and sociological -reveal more methods of disease control and health promotion. it relates to or encompasses all community and individual activities directed towards improving the environment for health, reducing factors that contribute to the burden of disease, and fostering those factors that relate directly to improved health. its programs range broadly from immunization, health promotion, and child care, to food labeling and fortification, as well as to the assurance of well-managed, accessible health care services. a strong public health system should have adequate preparedness for natural and human-made disasters, as seen in the recent tsunamis, hurricanes, biological or other attacks by terrorists, wars, conflicts, and genocidal terrorism (box . ) . the concepts of health promotion and disease prevention are essential and fundamental elements of the new public health. parallel scientific advances in molecular biology, genetics and pharmacogenomics, imaging, information technology, computerization, biotechnology, and nanotechnology hold great promise for improving the productivity of the health care system. advances in technology with more effective and less expensive drug and vaccine development, with improved safety and effectiveness, and fewer adverse reactions, will over time greatly increase efficiency in prevention and treatment modalities. the new public health is important as a conceptual base for training and practice of public health. it links classical topics of public health with adaptation in the organization and financing of personal health services. it involves a changed paradigm of public health to incorporate new advances in political, economic, and social sciences. failure at the political level to appreciate the role of public health in disease control holds back many societies in economic and social development. at the same time, organized public health systems need to work to reduce inequities between and inside countries to ensure equal access to care. it also demands special attention through health promotion activities of all kinds at national and local societal levels to provide access for groups with special risks and needs to medical and community health care with the currently available and newly developing knowledge and technologies. the great gap between available capabilities to prevent and treat disease and actually reaching all in need is still the the mission of the nph is to maximize human health and well-being for individuals and communities, nationally and globally. the methods with which the nph works to achieve this are in keeping with recognized international best practices and scientific advances: . societal commitment and sustained efforts to maximize quality of life and health, economic growth with equity for all (health for all and health in all). collaboration between international, national, state, and local health authorities working with public and private sectors to promote health awareness and activities essential for population health. . health promotion of knowledge, attitudes, and practices, including legislation and regulation to protect, maintain, and advance individual and community health. . universal access to services for prevention and treatment of illness and disability, and promotion of maximum rehabilitation. . environmental, biological, occupational, social, and economic factors that endanger health and human life, addressing: (a) physical and mental illness, diseases and infirmity, trauma and injuries (b) local and global sanitation and environmental ecology (c) healthful nutrition and food security including availability, quality, safety, access, and affordability of food products (d) disasters, natural and human-made, including war, terrorism, and genocide (e) population groups at special risk and with specific health needs. . promoting links between health protection and personal health services through health policies and health systems management, recognizing economic and quality standards of medical, hospital, and other professional care in health of individuals and populations. . training of professional public health workforces and education of all health workers in the principles of ethical best practices of public health and health systems. . research and promotion of current best practices: wide application of current international best practices and standards. . mobilizing the best available evidence from local and international scientific and epidemiological studies and best practices recognized as contributing to the overall goal. . maintaining and promoting equity for individual and community rights to health with high professional and ethical standards. source of great international and internal national inequities. these inequities exist not only between developed and developing countries, but also within transition countries, mid-level developing countries, and those newly emerging with rapid economic development. the historical experience of public health will help to develop the applications of existing and new knowledge and societal commitment to social solidarity in implementation of the new discoveries for every member of the society, despite socioeconomic, ethnic, or other differences. political will and leadership in health, adequate financing, and organization systems in the health setting are crucial to furthering health as an objective with defined targets, supported by well-trained staff for planning, management, and monitoring the population health and functioning of health systems. political leadership and professional support are both indispensable in a world of limited resources, with high public expectations and the growing possibilities of effectiveness of public health programs. well-developed information and knowledge management systems are required to provide the feedback and information needed for good management. it includes responsibilities and coordination at all levels of government. non-governmental organizations (ngos) and participation of a well-informed media and strong professional and consumer organizations also have significant roles in furthering population health. no less important are clear designations of responsibilities of the individual for his or her own health, and of the provider of care for humane, high-quality professional care. the complexities and interacting factors are suggested in figure . , with the classic host-agent-environment triad. many changes have signaled a need for transformation towards the new public health. religion, although still a major political and policy-making force in many countries, is no longer the central organizing power in most societies. organized societies have evolved from large extended families and tribes to rural societies, cities, regions, and national governments. with the growth of industrialized urban communities, rapid transport, and extensive trade and commerce in multinational economic systems, the health of individuals and communities has become more than just a personal, family, and/or local problem. an individual is not only a citizen of the village, city, or country in which he or she lives, but a citizen of a "global village". the agricultural revolutions and international explorations of the fifteenth to seventeenth centuries that increased food supply and diversity were followed only much later by knowledge of nutrition as a public health issue. the scientific revolution of the seventeenth to nineteenth centuries provided the basics to describe and analyze the spread of disease and the poisonous effects of the industrial revolution, including crowded living conditions and pollution of the environment with serious ecological damage. in the latter part of the twentieth century, a new agricultural "green revolution" had a great impact in reducing human deprivation internationally, yet the full benefits of healthier societies are yet to be realized in the large populations living in abject poverty in sub-saharan africa, south-east asia, and other parts of the world. global water shortages can be addressed with new methods of irrigation, water conservation and the application of genetic sciences to food production, and issues of economics and food security are of great importance to a still growing world population with limited supplies. further, food production capacity can and must be enlarged to meet current food insecurity, rising expectations of developing nations, and population growth. the sciences of agriculture-related fields, including genetic sciences and practical technology, will be vital to human progress in the coming decades. these and other societal changes discussed in chapter have enabled public health to expand its potential and horizons, while developing its pragmatic and scientific base. organized public health in the twentieth century proved effective in reducing the burden of infectious diseases and has contributed to improved quality of life and longevity by many years. in the last half-century, chronic diseases have become the primary causes of morbidity and mortality in the developed countries and increasingly in developing countries. growing scientific and epidemiological knowledge increases the capacity to deal with these diseases. many aspects of public health can only be influenced by the behavior of and risks to the health of individuals. these require interventions that are more complex and relate to societal, environmental, and community standards and expectations as much as to personal lifestyle. the dividing line between communicable and non-communicable diseases changes over time. scientific advances have shown the causation of chronic conditions by infectious agents and their prevention by curing the infection, as in helicobacter pylori and peptic ulcers, and in prevention of cancer of the liver and cervix by immunization for hepatitis b and human papillomavirus (hpv), respectively. chronic diseases have come to the center stage in the "epidemiological transition", as infectious diseases came under increasing control. this, in part, has created a need for reform in the funding and management of health systems due to rapidly rising costs, aging of the population, the rise of obesity and diabetes and other chronic conditions, mushrooming therapeutic technology, and expanding capacity to deal with public health emergencies. reform is also needed in international assistance to help less developed nations build the essential infrastructure to sustain public health in the struggle to combat aids, malaria, tuberculosis (tb), and the major causes of preventable infant, childhood, and motherhood-related deaths. the nearly universal recognition of the rights of people to have access to health care of acceptable quality by international standards is a challenge of political will and leadership backed up by adequate staffing with public health-trained staff and organizations. the challenges of the current global economic crisis are impacting social and health systems around the world. the interconnectedness of managing health systems is part of the new public health. setting the priorities and allocating resources to address these challenges requires public health training and orientation of the professionals and institutions participating in the policy, management, and economics of health systems. conversely, those who manage such institutions are recognizing the need for a wide background in public health training in order to fulfill their responsibilities effectively. concepts such as objectives, targets, priorities, cost-effectiveness, and evaluation have become part of the new public health agenda. an understanding of how these concepts evolved will help the future health provider or manager to cope with the complexities of mixing science, humanity, and effective management of resources to achieve higher standards of health, and to cope with new issues as they develop in the broad scope of the new public health for the twenty-first century, in what breslow called the "third public health era" of long and healthy quality of life (box . ). health can be defined from many perspectives, ranging from statistics on mortality, life expectancy, and morbidity rates to idealized versions of human and societal perfection, as in the world health organization's (who's) founding charter. the first public health era -the control of communicable diseases. second public health era -the rise and fall of chronic diseases. third public health era -the development of long and high-quality life. preamble to the constitution of the who, as adopted by the international health conference in new york in and signed by the representatives of states, entered into force on april , with the widely cited definition: "health is a state of complete physical, mental and social well-being and not merely the absence of disease or infirmity". this definition is still important conceptually as an ideal accepted as fundamental to public policy over the years. a more operational definition of health is a state of equilibrium of the person with the biological, physical, and social environment, with the object of maximum functional capability. health is thus seen as a state characterized by anatomical, physiological, and psychological integrity, and an optimal functional capability in the family, work, and societal roles (including coping with associated stresses), a feeling of well-being, and freedom from risk of disease and premature death. deviances in health are referred to as unhealthy and constitute a disease nomenclature. there are many interrelated factors in disease and in their management through what is now called risk reduction. in , claude bernard described the phenomenon of adaptation and adjustment of the internal milieu of the living organism to physiological processes. this concept is fundamental to medicine. it is also central to public health because understanding the spectrum of events and factors between health and disease is basic to the identification of contributory factors affecting the balance towards health, and to seeking the points of potential intervention to reverse the imbalance. as described in chapter , from the time of hippocrates and galen, diseases were thought to be due to humors and miasma or emanations from the environment. this was termed the miasma theory, and while without a direct scientific explanation, it was acted upon in the early to mid-nineteenth century and promoted by leading public health theorists including florence nightingale, with practical and successful measures to improve sanitation, housing, and social conditions, and having important results in improving health conditions. the competing germ theory developed by pioneering nineteenthcentury epidemiologists (panum, snow, and budd), scientists (pasteur, cohn, and koch), and practitioners (lister and semmelweiss) led to the science of bacteriology and a revolution in practical public health measures. the combined application of the germ (agent-host-environment) and miasma theories (social and sanitary environment) has been the basis of classic public health, with enormous benefits in the control of infectious and other diseases or harmful conditions. the revolutionary changes occurring since the s have brought about a decline in cardiovascular and cancer mortality, and conceptual changes such as health for all and health in all to bring health issues to all policies at both governmental and individual levels. the concepts of public health advanced with the marc lalonde health field concept (new perspectives on the health of canadians, ) , stating that health was the result of the physical and social environment, lifestyle and personal habits, genetics, as well as organization and provision of medical care. the lalonde report was a key concept leading to ideas advanced at the alma-ata conference on primary care held in and more explicitly in the development of the basis for health promotion as articulated in the ottawa charter of on health promotion. this marked the beginning of a whole new aspect of public health, which proved itself in addressing with considerable success the epidemic of hiv and cardiovascular diseases. in the usa, the surgeon general's reports of on smoking and health, and of defining health targets as national policy promoted the incorporation of "management by objectives" from the business world applied to the health sector (see chapter ). this led to healthy people usa and later versions, and the united nations (un) millennium development goals (mdgs), aimed primarily at the middle-and low-income countries (box . ). the identification of infectious causes of cancers of the liver and cervix established a new paradigm in epidemiology, and genetic epidemiology has important potential for public health and clinical medicine. in the basic host-agent-environment paradigm, a harmful agent comes through a sympathetic environment into contact with a susceptible host, causing a specific disease. this idea dominated public health thinking until the midtwentieth century. the host is the person who has or is at risk for a specific disease. the agent is the organism or direct cause of the disease. the environment includes the external factors which influence the host, his or her susceptibility to the agent, and the vector which transmits or carries the agent to the host from the environment. this explains the causation and transmission of many diseases. this paradigm (figure . ), in effect, joins together the contagion and miasma theories of disease causation. a specific agent, a method of transmission, and a susceptible host are involved in an interaction, which are central to the infectivity or severity of the disease. the environment can provide the carrier or vector of an infective (or toxic) agent, and it also contributes factors to host susceptibility; for example, unemployment, poverty, or low education level. the expanded host-agent-environment paradigm widens the definition of each of the three components ( figure . ), in relation to both acute infectious and chronic noninfectious disease epidemiology. in the latter half of the twentieth century, this expanded host-agent-environment paradigm took on added importance in dealing with the complex of factors related to chronic diseases, now the leading causes of disease and premature mortality in the developed world, and increasingly in developing countries. interventions to change host, environmental, or agent factors are the essence of public health. in infectious disease control, the biological agent may be removed by pasteurization of food products or filtration and disinfection (chlorination) of water supplies to prevent transmission of waterborne disease. the host may be altered by immunization to provide immunity to a specific infective organism. the environment may be changed to prevent transmission by destroying the vector or its reservoir of the disease. a combination of these interventions can be used against a specific risk factor, toxic or nutritional deficiency, infectious organism, or disease process. vaccine-preventable diseases may require both routine and special activities to boost herd immunity to protect the individual and the community. for other infectious diseases for which there is no vaccine (e.g., malaria), control involves a broad range of activities including case finding and treatment to improve the individual's health and to reduce the reservoir of the disease in the population, and other measures such as bed nets to reduce exposure of the host to vector mosquitoes, as well as vector control to reduce the mosquito population. tb control requires not only case finding and treatment, but understanding the contributory factors of social conditions, diseases with tb as a secondary condition (substance abuse and aids), agent resistance to treatment, and the inability of patients or carriers to complete treatment without supervision. sexually transmitted infections (stis) which are not controllable by vaccines require a combination of personal behavior change, health education, medical care, and skilled epidemiology. with non-infectious diseases, intervention is even more complex, involving human behavior factors and a wide range of legal, administrative, and educational issues. there may be multiple risk factors, which have a compounding effect in disease causation, and they may be harder to alter than infectious diseases factors. for example, smoking in and of itself is a risk factor for lung cancer, but exposure to asbestos fibers has a compounding effect. preventing exposure to the compounding variables may be easier than smoking cessation. reducing trauma morbidity and mortality is equally problematic. the identification of a single specific cause of a disease is of great scientific and practical value in modern public health, enabling such direct interventions as the use of vaccines or antibiotics to protect or treat individuals from infection by a causative organism, toxin, deficiency condition, or social factor. the cumulative effects of several contributing or risk factors in disease causation are also of great significance in many disease processes, in relation to infectious diseases such as nutritional status as for chronic diseases such as the cardiovascular group. the health of an individual is affected by risk factors intrinsic to that person as well as by external factors. intrinsic factors include the biological ones that the individual inherits and those life habits he or she acquires, such as smoking, overeating, or engaging in other high-risk behaviors. external factors affecting individual health include the environment, the socioeconomic and psychological state of the person, the family, and the society in which he or she lives. education, culture, and religion are also contributory factors to individual and community health. there are factors that relate to health of the individual in which the society or the community can play a direct role. one of these is provision of medical care. another is to ensure that the environment and community services include safety factors that reduce the chance of injury and disease, or include protective measures; for example, fluoridation of a community water supply to improve dental health, and seat-belt or helmet laws to reduce motor vehicle injury and death. these modifying factors may affect the response of the individual or the spread of an epidemic (see chapter ). an epidemic may also include chronic disease, because common risk factors may cause an excess of cases in a susceptible population group, in comparison to the situation before the risk factor appeared, or in comparison to a group not exposed to the risk factor. these include rapid changes or "epidemics" in such conditions as type diabetes, asthma, cardiovascular diseases, trauma, and other non-infectious disorders. disease is a dynamic process, not only of causation, but also of incubation or gradual development, severity, and the effects of interventions intended to modify outcome. knowledge of the natural history of disease is fundamental to understanding where and with what means intervention can have the greatest chance for successful interruption or change in the disease process for the patient, family, or community. the natural history of a disease is the course of that disease from beginning to end. this includes the factors that relate to its initiation; its clinical course leading up to resolution, cure, continuation, or long-term sequelae (further stages or complications of a disease); and environmental or intrinsic (genetic or lifestyle) factors and their effects at all stages of the disease. the effects of intervention at any stage of the disease are part of the disease process (figure . ). as discussed above, disease occurs in an individual when agent, host, and environment interact to create adverse conditions of health. the agent may be an infectious organism, a chemical exposure, a genetic defect, or a deficiency condition. a form of individual or social behavior, such as reckless driving or risky sexual behavior, may lead to injury or disease. the host may be immune or susceptible as a result of many contributing social and environmental factors. the environment includes the vector, which may be a malaria-bearing mosquito, a contaminated needle shared by drug users, lead-contaminated paint, or an abusive family situation. assuming a natural state of "wellness" -i.e., optimal health or a sense of well-being, function, and absence of disease -a disease process may begin with the onset of a disease, infectious or non-infectious, following a somewhat characteristic pattern of "incubation" described by clinicians and epidemiologists. preclinical or predisposing events may be detected by a clinical history, with determination of risk including possible exposure or presence of other risk factors. interventions, before and during the process, are intended to affect the later course of the disease. the clinical course of a disease, or its laboratory or radiological findings, may be altered by medical or public health intervention, leading to the resolution or continuation of the disease with fewer or less severe secondary sequelae. thus, the intervention becomes part of the natural history of the disease. the natural history of an infectious disease in a population will be affected by the extent of prior vaccination or previous exposure in the community. diseases particular to children are often so because the adult population is immune from previous exposure or vaccinations. measles and diphtheria, primarily childhood diseases, now affect adults to a large extent because they are less protected by naturally acquired immunity or are vulnerable when their immunity wanes naturally or as a result of inadequate vaccination in childhood. in chronic disease management, high costs to the patient and the health system accrue where preventive services or management are inadequate, not yet available, or inaccessible or where there is a failure to apply the necessary interventions. the progress of diabetes to severe complications such as cardiovascular, renal, and ocular disease is delayed or reduced by good management of the condition, with a combination of smoking cessation, diet, exercise, and medications with good medical supervision. the patient with advanced chronic obstructive pulmonary disease or congestive heart failure may be managed well and remain stable with smoking avoidance, careful management of medications, immunizations against influenza and pneumonia, and other prevention-oriented care needs. where these are not applied or if they fail, the patient may require long and expensive medical and hospital care. failure to provide adequate supportive care will show up in ways that are more costly to the health system and will prove more life-threatening to the patient. the goal is to avoid where possible the necessity for tertiary care, substituting tertiary prevention, i.e., supportive rehabilitation to maximum personal function and maintaining a stable functional status. as in an individual, the phenomenon of a disease in a population may follow a course in which many factors interplay, and where interventions affect the natural course of the disease. the epidemiological patterns of an infectious disease can be assessed in their occurrence in the population or their mortality rates, just as they can for individual cases. the classic mid-nineteenth-century description of measles in the faroe islands by panum showed the transmission and the epidemic nature of the disease as well as the protective effect of acquired immunity (see chapter ). similar, more recent breakthroughs in medical, epidemiological, biological, and social sciences have produced enormous benefit for humankind as discussed throughout this text, with some examples. these include the eradication of smallpox and in the coming years, poliomyelitis, measles, leprosy, and other dreaded diseases known for millennia; the near-elimination of rheumatic heart disease and peptic ulcers in the industrialized countries; vast reduction in mortality from stroke and coronary heart disease (chd); and vaccines (against hepatitis b and hpv) for the prevention of cancers. these and other great achievements of the twentieth and early part of the twenty-first centuries hold great promise for humankind in the coming decades, but great challenges lie ahead as well. the biggest challenge is to bring the benefits of known public health capacity to the poorest population of each country and the poorest populations globally. in developed countries a major challenge is to renew efforts of public health capacity to bear on prevention of chronic conditions such as diabetes and obesity, considered to be at pandemic proportions; and the individual and societal effects of mental diseases. in public health today, fears of a pandemic of avian influenza are based on transmission of avian or other animal-borne (zoonotic) prions or viruses to humans and then their adaptation permitting human-to-human spread. with large numbers of people living in close contact with many animals (wild and domestic fowl), such as in china and south-east asia, and rapid transportation around the world, the potential for global spread of disease is almost without historical precedent. indeed, many human infectious diseases are zoonotic in origin and transferred from natural wildlife reservoirs to humans either directly or via domestic or other wild animals, such as from birds to chickens to humans in avian influenza. monitoring or immunization of domestic animals requires a combination of multidisciplinary zoonotic disease management strategies, public education and awareness, and veterinary public health monitoring and control. rift valley fever, equine encephalitis, and more recently sars and avian influenza associated with bird-borne viral disease which can affect humans, each show the terrible dangers of pandemic diseases. ebola virus is probably sustained between outbreaks among fruit bats, or as recently suggested wild or domestic pigs, and may become a major threat to public health as human case fatality rates decline, meaning that patients and carriers, or genetic drift of the virus with possible airborne transmission, may spread this deadly disease more widely than in the past (see chapter ). the health of populations, like the health of individuals, depends on societal factors no less than on genetics, personal risk factors, and medical services. social inequalities in health have been understood and documented in public health over the centuries. the chadwick and shattuck reports of - documented the relationship of poverty and bad sanitation, housing, and working conditions with high mortality, and ushered in the idea of social epidemiology. political and social ideologies thought that the welfare state, including universal health care systems of one type or another, would eliminate social and geographic differences in health status and this is in large part true. from the introduction of compulsory health insurance in germany in the s to the failed attempt in the usa at national health insurance in (see chapters , and ) and the more recent achievements of us president obama in - , social reforms to deal with inequalities in health have focused on improving access to medical and hospital care. almost all industrialized countries have developed such systems, and the contribution of these programs to improve health status has been an important part of social progress, especially since world war ii. but even in societies with universal access to health care, people of lower socioeconomic status (ses) suffer higher rates of morbidity and mortality from a wide variety of diseases. the black report (douglas black) in the uk in the early s pointed out that the class v population (unskilled laborers) had twice the total and specific mortality rates of the class i population (professional and business) for virtually all disease categories, ranging from infant mortality to death from cancer. the report was shocking because all britons have had access to the comprehensive national health service (nhs) since its inception in , with access to a complete range of services at no cost at time of service, close relations to their general practitioners, and good access to specialty services. these findings initiated reappraisals of the social factors that had previously been regarded as the academic interests of medical sociologists and anthropologists and marginal to medical care. more recent studies and reviews of regional, ethnic, and socioeconomic differentials in patterns of health care access, morbidity, and mortality indicate that health inequities are present in all societies including the uk, the usa, and others, even with universal health insurance or services. the ottawa charter on health promotion in placed a new paradigm before the world health community that recognized social and political factors as no less important ion health that traditional medical and sanitary public health measures. these concepts helped the world health community to cope with new problems such as hiv/aidsfor which there was neither a medical cure nor a vaccine to prevent the disease. its control came to depend in the initial decades almost entirely on education and change in lifestyles, until the advent of the antiretroviral drugs in the s. there is still no viable vaccine. although the epidemiology of cardiovascular disease shows the direct relationship of the now classic risk factors of stress, smoking, poor diet, and physical inactivity, differences in mortality from cardiovascular disease between different classes among british civil servants are not entirely explainable by these factors. the differences are also affected by social and economic issues that may relate to the psychological needs of the individual, such as the degree of control people have over their own lives. blue-collar workers have less control over their lives, their working life in particular, than their white-collar counterparts, and have higher rates of chd mortality than higher social classes. other work shows the effects of migration, unemployment, drastic social and political change, and binge drinking, along with protective effects of healthy lifestyle, religiosity, and family support systems in cardiovascular diseases. social conditions affect disease distribution in all societies. in the usa and western europe, tb has re-emerged as a significant public health problem in urban areas partly because of high-risk population groups, owing to poverty and alienation from society, as in the cases of homelessness, drug abuse, and hiv infection. in countries of eastern europe and the former soviet union, the recent rise in tb incidence has resulted from various social and economic factors in the early s, including the large-scale release of prisoners. in both cases, diagnosis and prescription of medication are inadequate, and the community at large becomes at risk because of the development of antibioticresistant strains of tubercle bacillus readily spread by inadequately treated carriers, acting as human vectors. studies of ses and health are applicable and valuable in many settings. in alameda county, california, differences in mortality between black and white population groups in terms of survival from cancer became insignificant when controlled for social class. a -year follow-up study of the county population reported that low-income families in california are more likely than those on a higher income to have physical and mental problems that interfere with daily life, contributing to further impoverishment. studies of the association between indicators of ses and recent screening in the usa, australia, finland, and elsewhere showed that lower ses women use less preventive care such as papanicolaou (pap) smears for cervical cancer than women of higher ses, despite having greater risk for cervical cancer. many factors in ses inequalities are involved, including transportation and access to primary care, differences in health insurance coverage, educational levels, poverty, high-risk behaviors, social and emotional distress, feeling a lack of control over one's own life, employment, occupation, and inadequate family or community social support systems. many barriers exist owing to difficulties in access and the lack of availability of free or low-cost medical care, and the absence or limitations of health insurance is a further factor in the socioeconomic gradient. the recognition that health and disease are influenced by many factors, including social inequalities, plays a fundamental role in the new public health paradigm. health care systems need to take into account economic, social, physical, and psychological factors that otherwise will limit the effectiveness of even the best medical care. the health system includes access to competent and responsible primary care as well as by the wider health system, including health promotion, specific prevention and population-based health protection. the paradigm of the host-agent-environment triad (figures . and . ) is profoundly affected by the wider context. the sociopolitical environment and organized efforts at intervention affect the epidemiological and clinical course of disease of the individual. medical care is essential, as is public health, but the persistent health inequities seen in most regions and countries require societal attention. success or failure in improving the conditions of life for the poor, and other vulnerable "risk groups", affect national or regional health status and health system performance. the health system is meant to reduce the occurrence or bad outcome of disease, either directly by primary prevention or treatment as secondary prevention or by maximum rehabilitation as tertiary prevention, or equally important indirectly by reducing community or individual risk factors. the the effects of social conditions on health can be partly offset by interventions intended to promote healthful conditions; for example, improved sanitation, or through good-quality primary and secondary health services, used efficiently and effectively made available to all. the approaches to preventing disease or its complications may require physical changes in the environment, such as removal of the broad street pump handle to stop the cholera epidemic in london, or altering diets as in goldberger's work on pellagra. some of the great successes of public health have been and continue to be low technology. examples, among many others, include insecticide-impregnated bednets and other vector control measures, oral rehydration solutions, treatment and cure of peptic ulcers, exercise and diet to reduce obesity, hand washing in hospitals (and other health facilities), community health workers, and condoms and circumcision for the prevention of stis, including hiv and cancer of the cervix. the societal context in terms of employment, social security, female education, recreation, family income, cost of living, housing, and homelessness is relevant to the health status of a population. income distribution in a wealthy country may leave a wide gap between the upper and lower socioeconomic groups, which affects health status. the media have great power to sway public perception of health issues by choosing what to publish and the context in which to present information to society. modern media may influence an individual's tendency to overestimate the risk of some health issues while underestimating the risk of others, ultimately influencing health choices, such as occurred with public concern regarding false claims of an association between the measles-mumps-rubella (mmr) vaccine and autism in the uk (see the wakefield effect, chapter ). the new public health has an intrinsic responsibility for advocacy of improved societal conditions in its mission to promote optimal community health. an ultimate goal of public health is to improve health and to prevent widespread disease occurrence in the population and in an individual. the methods of achieving this are wide and varied. when an objective has been defined in "social justice is a matter of life and death. it affects the way people live, their consequent chance of illness, and their risk of premature death. we watch in wonder as life expectancy and good health continue to increase in parts of the world and in alarm as they fail to improve in others. a girl born today can expect to live for more than years if she is born in some countries -but less than years if she is born in others. within countries there are dramatic differences in health that are closely linked with degrees of social disadvantage. differences of this magnitude, within and between countries, simply should never happen. these inequities in health, avoidable health inequalities arise because of the circumstances in which people grow, live, work, and age, and the systems put in place to deal with illness. the conditions in which people live and die are, in turn, shaped by political, social, and economic forces. social and economic policies have a determining impact on whether a child can grow and develop to its full potential and live a flourishing life, or whether its life will be blighted. increasingly the nature of the health problems rich and poor countries have to solve are converging. the development of a society, rich or poor, can be judged by the quality of its population's health, how fairly health is distributed across the social spectrum, and the degree of protection provided from disadvantage as a result of ill-health." preventing disease, the next step is to identify suitable and feasible methods of achieving it, or a strategy with tactical objectives. this determines the method of operation, course of action, and resources needed to carry it out. the methods of public health are categorized as health promotion, and primary, secondary, and tertiary prevention (box . ). health promotion is the process of enabling people and communities to increase control over factors that influence their health, and thereby to improve their health (adapted from the ottawa charter of health promotion, ; box . ). health promotion is a guiding concept involving activities intended to enhance individual and community health and well-being (box . ). it seeks to increase involvement and control by the individual and the community in their own health. it acts to improve health and social welfare, and to reduce specific determinants of diseases and risk factors that adversely affect the health, well-being, and productive capacities of an individual or society, setting targets based on the size of the problem but also the feasibility of successful intervention, in a cost-effective way. this can be through direct contact with the patient or risk group, or act indirectly through changes in the environment, legislation, or public policy. control of aids relies on an array of interventions that promote change in sexual behavior and other contributory risks such as sharing of needles among drug users, screening of blood supply, safe hygienic practices in health care settings, and education of groups at risk such as teenagers, sex workers, migrant workers, and many others. control of aids is also a clinical problem in that patients need antiretroviral therapy (art), but this becomes a management and policy issue for making these drugs available and at an affordable price for the poor countries most affected. this is an example of the challenge and effectiveness of health promotion and the new public health. health promotion is a key element of the new public health and is applicable in the community, the clinic or hospital, and in all other service settings. some health promotion activities are government legislative and box . modes of prevention l health promotion -fostering national, community, and individual knowledge, attitudes, practices, policies, and standards conducive to good health; promoting legislative, social, or environmental conditions; promoting knowledge and practices for self-care that reduce individual and community risk; and creating a healthful environment. it is directed toward action on the determinants of health. l health protection -activities of official health departments or other agencies empowered to supervise and regulate food hygiene, community and recreational water safety, environmental sanitation, occupational health, drug safety, road safety, emergency preparedness, and many other activities to eliminate or reduce as much as possible risks of adverse consequences to health. l primary prevention -preventing a disease from occurring, e.g., vaccination to prevent infectious diseases, advice to stop smoking to prevent lung cancer. l secondary prevention -making an early diagnosis and giving prompt and effective treatment to stop progress or shorten the duration and prevent complications from an already existing disease process, e.g., screening for hypertension or cancer of cervix and colorectal cancer for early case finding, early care and better outcomes. l tertiary prevention -stopping progress of an already occurring disease, and preventing complications, e.g., in managing diabetes and hypertension to prevent complications; restoring and maintaining optimal function once the disease process has stabilized, e.g., promoting functional rehabilitation after stroke and myocardial infarction with long-term follow-up care. health promotion (hp) is the process of enabling people to increase control over, and to improve their health. hp represents a comprehensive social and political process, and not only embraces actions directed at strengthening the skills and capabilities of individuals. hp also undertakes action directed towards changing social, environmental, and economic conditions so as to alleviate their impact on public and individual health. health promotion is the process of enabling people to increase control over the determinants of health and thereby improve their health. participation is essential to sustain health promotion action. the ottawa charter identifies three basic strategies for health promotion. these are advocacy for health to create the essential conditions for health indicated above; enabling all people to achieve their full health potential; and mediating between the different interests in society in the pursuit of health. these strategies are supported by five priority action areas as outlined in the ottawa charter for health promotion: regulatory interventions such as mandating the use of seat belts in cars, requiring that children be immunized to attend school, declaring that certain basic foods must have essential minerals and vitamins added to prevent nutritional deficiency disorders in vulnerable population groups, and mandating that all newborns should be given prophylactic vitamin k to prevent hemorrhagic disease of the newborn. setting food and drug standards and raising taxes on cigarettes and alcohol to reduce their consumption are also part of health promotion. promoting a healthy lifestyle is a major known obesity-preventive activity. health promotion is provided by organizations and people with varied professional backgrounds working towards common goals of improvement in the health and quality of individual and community life. initiatives may come from government with dedicated allocation of funds to address specific health issues, from donors, or from advocacy or community groups or individuals to promote a specific or general cause in health. raising awareness to inform and motivate people about their own health and lifestyle factors that might put them at risk requires teaching young people about the dangers of sexually transmitted diseases, smoking, and alcohol abuse to reduce risks associated with their social behavior. it might include disseminating information on healthy nutrition; for example, the need for folic acid supplements for women of childbearing age and multiple vitamins for elderly, as well as the elements of a healthy diet, compliance with immunization recommendations, compliance with screening programs, and many others. community and peer group attitudes and standards affect individual behavior. health promotion endeavors to create a climate of knowledge, attitudes, beliefs, and practices that are associated with better health outcomes. international conferences following on from the ottawa charter were held in adelaide in , sundsvall in , jakarta in , mexico in , bangkok in , and nairobi in . the principles of health promotion have been reiterated and have influenced public policy regarding public health as well as the private sector. health promotion has a track record of proven success in numerous public health issues where a biomedical solution was not available. the hiv/aids pandemic from the s until the late s had no medical treatment and control measures relied on screening, education, lifestyle changes, and supportive care. health promotion brought forward multiple interventions, from condom use and distribution, to needle exchanges for intravenous drug users, to male circumcision in high-prevalence african countries. medical treatment was severely limited until art was developed. the success of art also depends on a strong element of health promotion in widening the access to treatment and the success of medications to reduce transmission, most remarkably in reducing maternal-fetal transmission (see chapter ). similarly, in the battle against cardiovascular diseases, health promotion was an instrumental factor in raising public awareness of the importance of management of hypertension and smoking reduction, dietary restraint, and physical exercise. the success of massive reductions in stroke and chd mortality is as much the result of health promotion as of improved medical care (see chapter ). the character of public health carries with it a "good cop, bad cop" dichotomy. the "good cop" is persuasive and educational trying to convince people to do the right thing in looking after their own health: diet, exercise, smoking cessation, and others. on the other side, the "bad cop" role is regulatory and punitive. public health has a serious responsibility and role in the enforcement of laws and regulation to protect the public health. some of these are restrictive box . elements of health promotion . address the population as a whole in health-related issues, in everyday life as well as people at risk for specific diseases. . direct action to risk factors or causes of illness or death. . undertake activist approach to seek out and remedy risk factors in the community that adversely affect health. . promote factors that contribute to a better condition of health of the population. . initiate actions against health hazards, including communication, education, legislation, fiscal measures, organizational change, community development, and spontaneous local activities. . involve public participation in defining problems and deciding on action. . advocate relevant environmental, health, and social policy. . encourage health professional participation in health education and health advocacy. . advocate for health based on human rights and solidarity. . invest in sustainable policies, actions, and infrastructure to address the determinants of health. . build capacity for policy development, leadership, health promotion practice, knowledge transfer and research, and health literacy. . regulate and legislate to ensure a high level of protection from harm and enable equal opportunity for health and well-being for all people. . partner and build alliances with public, private, nongovernmental, and international organizations and civil society to create sustainable actions. . make the promotion of health central to the global development agenda. of individual rights that may damage other people or are requirements based on strong evidence of benefits to population health. readily accepted are food and drug standards, such as pasteurization of milk, and iodization of salt; requirements to drive on the right-hand side of the road (except in some countries such as the uk), to wear seat belts and for motorcyclists to wear safety helmets; and not smoking in public places. enforcement of these and similar statutory or regulatory requirements is vital in a civil society to protect the public from health hazards and to protect people from harm and exploitation by unscrupulous manufacturers and marketing. cigarette advertising and sponsorship of sports events by tobacco companies are banned in most upper income countries. the use of transfats in food manufacturing and baking is now banned and salt reduction is being promoted and even mandated in many us local authorities to reduce cardiovascular disease. advertising of unhealthy snack foods on children's television programs and during child-watching hours is commonly restricted. banning high-sugar soda drink distribution in schools is a successful intervention to reduce the current child obesity epidemic. melamine use in milk powders and baby formulas, which caused widespread illness and death of infants in china, is now banned and a punishable offence for manufacture or distribution in china and worldwide. examples of this aspect of public health are mentioned throughout this text, especially in chapters and on nutrition, and environmental and occupational health, respectively. the regulatory enforcement function of public health is sometimes controversial and portrayed as interference with individual liberty. fluoridation of community water supplies is an example where aggressive lobby groups opposing this safe and effective public health measure are still common. this is discussed in chapter . equally important is the public health policy issue of resource allocation and taxation for health purposes. taxation is an unpopular measure that governments must employ and enforce in order to do the public's business. the debate over the patient protection and affordable care act (ppaca or "obamacare"), discussed elsewhere in this and other chapters, shows how bitter the arguments can become, yet the goal of equality of access to health care cannot be denied as a public good, demonstrably contributing to the health of the nation. primary prevention refers to those activities that are undertaken to prevent disease or injury from occurring at all. primary prevention works with both the individual and the community. it may be directed at the host to increase resistance to the agent (such as in immunization or cessation of smoking), or at environmental activities to reduce conditions favorable to the vector for a biological agent, such as mosquito vectors of malaria or dengue fever. landmark examples include the treatment and prevention of scurvy among sailors based on james lind's findings in a classic clinical epidemiological study in , and john snow's removal of the handle from the broad street pump to stop a cholera epidemic in london in (see chapter ). primary prevention includes elements of health protection such as ensuring water, food and drug, and workplace safety; chlorination of drinking water to prevent transmission of waterborne enteric diseases; pasteurization of milk to prevent gastrointestinal diseases; mandating wearing seat belts in motor vehicles to prevent serious injury and death in road crashes; and reducing the availability of firearms to reduce injury and death from intentional, accidental, or random violence. it also includes direct measures to prevent diseases, such as immunization to prevent polio, tetanus, pertussis, and diphtheria. health promotion and health protection blend together as a group of activities that reduce risk factors and diseases through many forms of intervention such as changing smoking legislation or preventing birth defects by fortification of flour with folic acid. prevention of hiv transmission by needle exchange for intravenous drug users, promoting condom usage, and promoting male circumcision in africa, and the distribution of condoms and clean needles for hivpositive drug users are recent examples of primary prevention associated with health promotion programs. primary prevention also includes activities within the health system that can lead to better health. this may mean, for example, setting standards and to reduce hospital infections, and ensuring that doctors not only are informed of appropriate immunization practices and modern prenatal care or screening programs for cancer of the cervix, colon, and breast, but also are aware of their vital role in preventing cardiovascular and other non-communicable diseases. in this role, the health care provider serves as a teacher and guide, as well as a diagnostician and therapist. like health promotion, primary prevention does not depend on health care providers alone; health promotion works to increase individual and community consciousness of self-care, mainly by raising awareness and information levels and empowering the individual and the community to improve self-care, to reduce risk factors, and to live healthier lifestyles. secondary prevention is early diagnosis and management to prevent complications from a disease. public health interventions to prevent the spread of disease include the identification of sources of the disease and the implementation of steps to stop it, as shown in snow's closure of the broad street pump. secondary prevention includes steps to isolate cases and treat or immunize contacts so as to prevent further cases of meningitis or measles, for example, in outbreaks. for current epidemics such as hiv/aids, primary prevention is largely based on education, abstinence from any and certainly risky sexual behavior, circumcision, and treatment of patients in order to improve their health and to reduce the risk of spread of hiv. for high-risk groups such as intravenous drug users, needleexchange programs reduce the risk of spread of hiv, and hepatitis b and c. distribution of condoms to teenagers, military personnel, truck drivers, and commercial sex workers helps to prevent the spread of stis and aids in schools and colleges, as well as among the military. the promotion of circumcision is shown to be effective in reducing the transmission of hiv and of hpv (the causative organism for cancer of the cervix). all health care providers have a role in secondary prevention; for example, in preventing strokes by early identification and adequate care of hypertension. the child who has an untreated streptococcal infection of the throat may develop complications which are serious and potentially life-threatening, including rheumatic fever, rheumatic valvular heart disease, and glomerulonephritis. a patient found to have elevated blood pressure should be advised about continuing management by appropriate diet and weight loss if obese, regular physical exercise, and long-term medication with regular follow-up by a health provider in order to reduce the risk of stroke and other complications. in the case of injury, competent emergency care, safe transportation, and good trauma care may reduce the chance of death and/or permanent handicap. screening and high-quality care in the community prevent complications of diabetes, including heart, kidney, eye, and peripheral vascular disease. they can also prevent hospitalizations, amputations, and strokes, thus lengthening and improving the quality of life. health care systems need to be actively engaged in secondary prevention, not only as individual doctors' services, but also as organized systems of care. public health also has a strong interest in promoting highquality care in secondary and tertiary care hospital centers in such areas of treatment as acute myocardial infarction, stroke, and injury in order to prevent irreversible damage. measures include quality of care reviews to promote adequate longterm postmyocardial infarction care with aspirin and betablockers or other medication to prevent or delay recurrence and second or third myocardial infarctions. the role of highquality transportation and care in emergency facilities of hospitals in public health is vital to prevent long-term damage and disability; thus, cardiac care systems including publicly available defibrillators, catheterization, the use of stents, and bypass procedures are important elements of health care policy and resource allocation, which should be accessible not only in capital cities but also to regional populations. tertiary prevention involves activities directed at the host or patient, but also at the social and physical environment in order to promote rehabilitation, restoration, and maintenance of maximum function after the disease and its complications have stabilized. the person who has undergone a cerebrovascular accident or trauma will reach a stage where active rehabilitation can help to restore lost functions and prevent recurrence or further complications. the public health system has a direct role in the promotion of disability-friendly legislation and standards of building, housing, and support services for chronically ill, handicapped, and elderly people. this role also involves working with many governmental social and educational departments, but also with advocacy groups, ngos, and families. it may also include the promotion of disability-friendly workplaces and social service centers. treatment for conditions such as myocardial infarction or a fractured hip now includes early rehabilitation in order to promote early and maximum recovery with restoration to optimal function. the provision of a wheelchair, walkers, modifications to the home such as special toilet facilities, doors, and ramps, along with transportation services for paraplegics are often the most vital factors in rehabilitation. public health agencies work with groups in the community concerned with promoting help for specific categories of risk group, disease, or disability to reduce discrimination. community action is often needed to eliminate financial, physical, or social barriers, promote community awareness, and finance special equipment or other needs of these groups. close follow-up and management of chronic disease, physical and mental, require home care and ensuring an appropriate medical regimen including drugs, diet, exercise, and support services. the follow-up of chronically ill people to supervise the taking of medications, monitor changes, and support them in maximizing their independent capacity in activities of daily living is an essential element of the new public health. public health uses a population approach to achieve many of its objectives. this requires defining the population, including trends of change in the age and gender distribution of the population, fertility and birth rates, spread of disease and disability, mortality, marriage and migration, and socioeconomic factors. the reduction of infectious disease as the major cause of mortality, increased longevity coupled with declining fertility rates, resulted in changes in the age composition, or a demographic transition. demographic changes, such as fertility and mortality patterns, are important factors in changing the age distribution of the population, resulting in a greater proportion of people surviving to older ages. declining infant mortality, increasing educational levels of women, the availability of birth control, and other social and economic factors lead to changes in fertility patterns and the demographic transition -an aging of the population -with important effects on health service needs. the age and gender distribution of a population affects and is affected by patterns of disease. change in epidemiological patterns, or an epidemiological shift, is a change in predominant patterns of morbidity and mortality. the transition of infectious diseases becoming less prominent as causes of morbidity and mortality and being replaced by chronic and non-infectious diseases has occurred in both developed and developing countries. the decline in mortality from chronic diseases, such as cardiovascular disease, represents a new stage of epidemiological transition, creating an aging population with higher standards of health but also long-term community support and care needs. monitoring and responding to these changes are fundamental responsibilities of public health, and a readiness to react to new, local, or generalized changes in epidemiological patterns is vital to the new public health. societies are not totally homogeneous in ethnic composition, levels of affluence, or other social markers. on one hand, a society classified as developing may have substantial numbers of people with incomes that promote overnutrition and obesity, so that disease patterns may include increasing prevalence of diseases of excesses, such as diabetes. on the other hand, affluent societies include population groups with disease patterns of poverty, including poor nutrition and low birth-weight babies. a further stage of epidemiological transition has been occurring in the industrialized countries since the s, with dramatic reductions in mortality from chd, stroke and, to a lesser extent, trauma. the interpretation of this epidemiological transition is still not perfectly clear. how it occurred in the industrialized western countries but not in those of the former soviet union is a question whose answer is vital to the future of health in russia and some countries of eastern europe. developing countries must also prepare to cope with increasing epidemics of non-infectious diseases, and all countries face renewed challenges from infectious diseases with antibiotic resistance or newly appearing infectious agents posing major public health threats. demographic change in a country may reflect social and political decisions and health system priorities from decades before. russia's rapid population decline since the s, china's gender imbalance with a shortage of millions of young women, egypt's rapid population growth outstripping economic capacity, and many other examples indicate the severity and societal importance of capacity to analyze and formulate public health and social policies to address such fundamental sociopolitical issues. aging of the population is now the norm in most developed countries as a result of low birth and declining mortality rates. this change in the age distribution of a population has many associated social and economic issues as to the future of social welfare with a declining age cohort to provide the workforce. the aging population requires pension and health care support which make demands of social security systems that will depend on economic growth with a declining workforce. in times of economic stress, as in europe, this situation is made more difficult by longstanding short working weeks, early pension ages, and high social benefits. however, this results in unemployment among young people in particular and social conflict. the interaction of increasing life expectancy and a declining workforce is a fundamental problem in the high-income countries. this imbalance may be resolved in part through productivity gains and switching of primary production to countries with large still underutilized workforces, while employment in the developed countries will depend on service industries including health and the economic growth generated by higher technology and intellectual property and service industries. the challenge of keeping populations and individuals healthy is reflected in modern health services. each component of a health service may have developed with different historical emphases, operating independently as a separate service under different administrative auspices and funding systems, competing for limited health care resources. in this situation, preventive community care receives less attention and resources than more costly treatment services. figure . suggests a set of health services in an interactive relationship to serve a community or defined population, but the emphasis should be on the interdependence of these services with one other and with the comprehensive network in order to achieve effective use of resources and a balanced set of services for the patient, the client or patient population, and the community. clinical medicine and public health each play major roles in primary, secondary, and tertiary prevention. each may function separately in their roles in the community, but optimal success lies in their integrated efforts. allocation of resources should promote management and planning practices to assist this integration. there is a functional interdependence of all elements of health care serving a definable population. the patient should be the central figure in the continuum or complex of services available. effectiveness in use of resources means that providing the service most appropriate for meeting the individual's or group's needs at a point in time are those that should be applied. this is the central concept in currently developing innovations in health care delivery in the usa with organizations using terms such as patient centered medical home, accountable care organizations (acos), and population health management systems, which are being promoted in the obamacare health reforms now in process (see chapter ) (shortell et al., ) . separate organization and financing of services place barriers to appropriate provision of services for both the community and the individual patient. the interdependence of services is a challenge in health care organizations for the future. where there is competition for limited resources, pressures for tertiary services often receive priority over programs to prevent children from dying of preventable diseases. public health must be seen in the context of all health care and must play an influential role in promoting prevention at all levels. clinical services need public health in order to provide prevention and community health services that reduce the burden of disease, disability, and dependence on the institutional setting. health was traditionally thought of as a state of absence of disease, pain, or disability, but has gradually been expanded to include physical, mental, and societal well-being. in , c. e. a. winslow, professor of public health at yale university, defined public health as follows: "public health is the science and art of ( ) preventing disease, ( ) prolonging life, and ( ) winslow's far-reaching definition remains a valid framework but is unfulfilled when clinical medicine and public health have financing and management barriers between them. in many countries, isolation from the financing and provision of medical and nursing care services left public health with the task of meeting the health needs of the indigent and underserved population groups with inadequate resources and recognition. health insurance organizations for medical and hospital care have in recent years been more open to incorporating evidence-based preventive care, but the organization of public health has lacked the same level of attention. in some countries, the limitations have been conceptual in that public health was defined primarily in terms of control of infectious, environmental, and occupational diseases. a more recent and widely used definition is: "public health is the science and art of preventing disease, prolonging life, and promoting health through the organized efforts of society." this definition, coined in in the public health in england report by sir donald acheson, reflects the broad focus of modern public health. terms such as social hygiene, preventive medicine, community medicine, and social medicine have been used to denote public health practice over the past century. preventive medicine is the application of preventive measures by clinical practitioners combining some elements of public health with clinical practice relating to individual patients. preventive medicine defines medical or clinical personal preventive care, with stress on risk groups in the community and national efforts for health promotion. the focus is on the health of defined populations to promote health and well-being using evidence-based guidelines for cost-effective preventive measures. measures emphasized include screening and follow-up of chronic illnesses, and immunization programs; for example, influenza and pneumococcal pneumonia vaccines are used by people who are vulnerable because of their age, chronic diseases, or risk of exposure, such as medical and nursing personnel and those providing other personal clinical services. clinical medicine also deals in the area of prevention in the management of patients with hypertension or diabetes, and in doing so prevents the serious complications of these diseases. social medicine is also primarily a medical specialty which looks at illness in an individual in the family and social context, but lacks the environmental and regulatory and organized health promotion functions of public health. community health implies a local form of health intervention, whereas public health more clearly implies a global approach, which includes action at the international, national, state, and local levels. some issues in health can be dealt with at the individual, family, or community level; others require global strategies and intervention programs with regional, national, or international collaboration and leadership. the social medicine movement originated to address the harsh conditions of the working population during the industrial revolution in mid-nineteenth-century europe. an eminent pioneer in cellular pathology, rudolph virchow provided leadership in social medicine powered by the revolutionary movements of , and subsequent social democrat political movements. their concern focused on harsh living and health conditions among the urban poor working class and neglectful political norms of the time. social medicine also developed as an academic discipline and advocacy orientation by providing statistical evidence showing, as in various governmental reports in the mid-nineteenth century, that poverty among the working class was associated with short life expectancy and that social conditions were key factors in the health of populations and individuals. this movement provided the basis for departments in medical faculties and public health education throughout the world stressing the close relationship between political priorities and health status. this continued in the twentieth century and in the usa found expression in pioneering work since the s at montefiore hospital in new york and with victor sidel, founding leader of the community health center movement the usa from the s. in the twenty-first century this movement continues to emphasize relationships between politics, society, disease, and medicine, and forms of medical practice derived from it, as enunciated by prominent advocates such as harvardbased paul farmer in haiti, russia and rwanda, and in the uk by martin mckee and others (nolte and mckee, ) . similar concepts are current in the usa under headings such as family medicine, preventive medicine, and social medicine. this movement has also influenced sir michael marmot and others in the world health commission of health inequalities of , with a strong influence on the un initiative to promote mdgs, whose first objective is poverty reduction (commission on inequalities report ). application of the idea of poverty reduction as a method of reducing health inequalities has been successful recently in a large field trial in brazil showing greater reduction in child mortality where cash bonuses were awarded by municipalities for the poor families than that observed in other similar communities (rasella, ). in the usa, this movement is supported by increased health insurance coverage for the working poor, with funding for preventive care and incentives for community health centers in the obamacare plan of for implementation in the coming years to provide care for uninsured and underserved populations, particularly in urban and rural poverty areas. the political aspect of social medicine is the formulation of and support for national initiatives to widen health care coverage to the percent of the us population who are still uninsured, and to protect those who are arbitrarily excluded owing to previous illnesses, caps on coverage allowed, and other exploitative measures taken by private insurance that frequently deny americans access to the high levels of health care available in the country. the ethical base of public health in europe evolved in the context of its successes in the nineteenth and early twentieth centuries along with ideas of social progress. but the twentieth century was also replete with extremism and wide-scale abuse of human rights, with mass executions, deportations, and starvation as official policy in fascist and stalinist regimes. eugenics, a pseudoscience popularized in the early decades of the twentieth century, promoted social policies meant to improve the hereditary qualities of a race by methods such as sterilization of mentally handicapped people. the "social and racial hygiene" of the eugenics movements led to the medicalization of sterilization in the usa and other countries. this was adopted and extended in nazi germany to a policy of murder, first of the mentally and physically handicapped and then of "racial inferiors". these eugenics theories were widely accepted in the medical community in germany, then used by the nazi regime to justify medically supervised killing of hundreds of thousands of helpless, incapacitated individuals. this practice was linked to wider genocide and the holocaust, with the brutalization and industrialized murder of over million jews and million other people, and corrupt medical experimentation on prisoners. following world war ii, the ethics of medical experimentation (and public health) were codified in the nuremberg code and universal declaration of human rights based on lessons learned from these and other atrocities inflicted on civilian populations (see chapter ). threats of genocide, ethnic cleansing, and terrorism are still present on the world stage, often justified by current warped versions of racial hygienic theories. genocidal incitement and actual genocide and terrorism have recurred in the last decades of the twentieth century and into the twenty-first century in the former yugoslav republics, africa (rwanda and darfur), south asia, and elsewhere. terrorism against civilians has become a worldwide phenomenon with threats of biological and chemical agents, and potentially with nuclear capacity. asymmetrical warfare of insurgencies which use innocent civilians for cover, as with other forms of warfare, carries with it grave dangers to public health, human rights, and international stability, as seen in the twenty-first century in south sudan, darfur, dr congo, chechnya, iraq, afghanistan, and pakistan. in , kerr white and colleagues defined medical ecology as population-based research providing the foundation for management of health care quality. this concept stresses a population approach, including those not attending and those using health services. this concept was based on previous work on quality of care, randomized clinical trials, medical audit, and structure-process-outcome research. it also addressed health care quality and management. these themes influenced medical research by stressing the population from which clinical cases emerge as well as public health research with clinical outcome measures, themes that recur in the development of health services research and, later, evidence-based medicine. this led to the development of the agency for health care policy and research and development in the us department of health and human services and evidence-based practice centers to synthesize fundamental knowledge for the development of information for decision-making tools such as clinical guidelines, algorithms, or pathways. clinical guidelines and recommended best practices have become part of the new public health to promote quality of patient care and public health programming. these can include recommended standards; for example, follow-up care of the postmyocardial infarction patient, an internationally recommended immunization schedule, recommended dietary intake or food fortification standards, and mandatory vitamin k and eye care for all newborns and many others (see chapter ). community-oriented primary care (copc) is an approach to primary health care that links community epidemiology and appropriate primary care, using proactive responses to the priority needs identified. copc, originally pioneered in south africa and israel by sidney and emily kark and colleagues in the s and s, stresses medical services in the community which need to be adapted to the needs of the population as defined by epidemiological analysis. copc involves community outreach and education, as well as clinical preventive and treatment services. copc focuses on community epidemiology and an active problem-solving approach. this differs from national or larger scale planning that sometimes loses sight of the local nature of health problems or risk factors. copc combines clinical and epidemiological skills, defines needed interventions, and promotes community involvement and access to health care. it is based on linkages between the different elements of a comprehensive basket of services along with attention to the social and physical environment. a multidisciplinary team and outreach services are important for the program, and community development is part of the process. in the usa, the copc concept has influenced health care planning for poor areas, especially provision of federally funded community health centers in attempts to provide health care for the underserved since the s. in more recent years, copc has gained wider acceptance in the usa, where it is associated with family physician training and community health planning based on the risk approach and "managed care" systems. indeed, the three approaches are mutually complementary (box . ). as the emphasis on health care reform in the late s moved towards managed care, the principles of copc were and will continue to be important in promoting health and primary prevention in all its modalities, as well as tertiary prevention with followup and maintenance of the health of the chronically ill. copc stresses that all aspects of health care have moved towards prevention based on measurable health issues in the community. through either formal or informal linkages between health services, the elements of copc are part of the daily work of health care providers and community services systems. the us institute of medicine issued the report on primary care in , defining primary care as "the provision of integrated, accessible health care services by clinicians who are accountable for addressing the majority of personal health care needs, developing a sustained partnership with patients and practicing in the context of the family and the community". this formulation was criticized by the american public health association (apha) as lacking a public health perspective and failing to take into account both the individual and the community health approaches. copc tries to bridge this gap between the perspectives of primary care and public health. the community, whether local, regional, or national, is the site of action for many public health interventions. moreover, understanding the characteristics of the community is vital to a successful community-oriented approach. by the s, new patterns of public health began to emerge, including all measures used to improve the health of the community, and at the same time working to protect and promote the health of the individual. the range of activities to achieve these general goals is very wide, including individual patient care systems and the community-wide activities that affect the health and well-being of the individual. these include the financing and management of health systems, evaluation of the health status of the population, and measures to improve the quality of health care. they place reliance on health promotion activities to change environmental risk factors for disease and death. they promote integrative and multisectoral approaches and the international health teamwork required for global progress in health. the definition of health in the charter of the who as a complete state of physical, mental, and social well-being had a ring of utopianism and irrelevance to states struggling to provide even minimal care in severely adverse political, economic, social, and environmental conditions (box . ). in , a more modest goal was set for attainment of a level of health compatible with maximum feasible social and economic productivity. one needs to recognize that health and disease are on a dynamic continuum that affects everyone. the mission for public health is to use a wide range of methods to prevent disease and premature death, and improve quality of life for the benefit of individuals and the community. the world health organization defines health as "a state of complete physical, mental and social well-being, not merely the absence of disease or infirmity" (who constitution, ) . in at the alma-ata conference on primary health care, the who related health to "social and economic productivity in setting as a target the attainment by all the people of the world of a level of health that will permit them to lead a socially and economically productive life". three general programs of work for the periods - , - , and - were formulated as the basis of national and international activity to promote health. in , the who, recognizing changing world conditions of demography, epidemiology, environment, and political and economic status, addressed the unmet needs of developing countries and health management needs in the industrialized countries, calling for international commitment to "attain targets that will make significant progress towards improving equity and ensuring sustainable health development". the object of the who is restated as "the attainment by all peoples of the highest possible level of health" as defined in the who constitution, by a wide range of functions in promoting technical cooperation, assisting governments, and providing technical assistance, international cooperation, and standards. in the s, most industrialized countries were concentrating energies and financing in health care on providing access to medical and hospital services through national insurance schemes. developing countries were often spending scarce resources trying to emulate this trend. the who was concentrating on categorical programs, such as eradication of smallpox and malaria, as well as the expanded program of immunization and similar specific efforts. at the same time, there was a growing concern that developing countries were placing too much emphasis and expenditure on curative services and not enough on prevention and primary care. the world health assembly (wha) in endorsed the primary care approach under the banner of "health for all by the year " (hfa ) . this was a landmark decision and has had important practical results. the who and the united nations children's fund (unicef) sponsored a seminal conference held in alma-ata, in the ussr ( kazakhstan), in , which was convened to refocus health policy on primary care. the alma-ata declaration stated that health is a basic human right, and that governments are responsible to assure that right for their citizens and to develop appropriate strategies to fulfill this promise. this proposition has come to be increasingly accepted in the international community. the conference stressed the right and duty of people to participate in the planning and implementation of their health care. it advocated the use of scientifically, socially, and economically sound technology. joint action through intersectoral cooperation was also emphasized. the alma-ata declaration focused on primary health care as the appropriate method of assuming adequate access to health care for all (box . ). many countries have gradually come to accept the notion of placing priority on primary care, resisting the temptation to spend high percentages of health care resources on high-tech and costly medicine. spreading these same resources into highly costeffective primary care, such as immunization and nutrition programs, provides greater benefit to individuals and to society as a whole. alma-ata provided a new sense of direction for health policy, applicable to developing countries and in a different way than the approaches of the developed countries. during the s, the health for all concept influenced national health policies in the developing countries with signs of progress in immunization coverage, for example, but the initiative was diluted as an unintended consequence by more categorical programs such as eradication of poliomyelitis. for example, developing countries have accepted immunization and diarrheal disease control as high-priority issues and achieved remarkable success in raising immunization coverage from some percent to over percent in just a decade. developed countries addressed these principles in different ways. in these countries, the concept of primary health care led directly to important conceptual developments in health. national health targets and guidelines are now common in many countries and are integral parts of box . declaration of alma-ata, : a summary of primary health care (phc) . reaffirms that health is a state of complete physical, mental, and social well-being, and not merely the absence of disease or infirmity, and is a fundamental human right. existing gross inequalities in the health status of the people, particularly between developed and developing countries as well as within countries, are of common concern to all countries. . governments have a responsibility for the health of their people. the people have the right and duty to participate in planning and implementation of their health care. . a main social target is the attainment, by all peoples of the world by the year , of a level of health that will permit them to lead a socially and economically productive life. . phc is essential health care based on practical, scientifically sound, and socially acceptable methods and technology. . it is the first level of contact of individuals, the family, and the national health system bringing health care as close as possible to where people live and work, as the first element of a continuing health care process. . phc evolves from the conditions and characteristics of the country and its communities, based on the application of social, biomedical, and health services research and public health experience. . phc addresses the main health problems in the community, providing promotive, preventive, curative, and rehabilitative services accordingly. . phc includes the following: (a) education concerning prevailing health problems and methods of preventing and controlling them (b) promotion of food supply and proper nutrition (c) adequate supply of safe water and basic sanitation (d) maternal and child health care, including family planning (e) immunization against the major infectious diseases (f) prevention of locally endemic diseases (g) appropriate treatment of common diseases and injuries (h) the provision of essential drugs (i) relies on all health workers … to work as a health team. . all governments should formulate national health policies, strategies and plans, mobilize political will and resources, used rationally, to ensure phc for all people. national health planning. reforms of the nhs -for example, as discussed in chapter , remuneration increases for family physicians and encouraging group practice with public health nursing support -have become widespread in the uk. leading health maintenance organizations, such as kaiser permanente in the usa and district health systems in canada, have emphasized integrated approaches to health care for registered or geographically defined populations (see chapters - ). this approach is becoming common in the usa in acos, which will be fostered by the obamacare legislation (ppaca). this systematic approach to individual and community health is an integral part of the new public health. the interactions among community public health, personal health services, and health-related behavior, including their management, are the essence of the new public health. how the health system is organized and managed affects the health of the individual and the population, as does the quality of providers. health information systems with epidemiological, economic, and sociodemographic analysis are vital to monitor health status and allow for changing priorities and management. well-qualified personnel are essential to provide services, manage the system, and carry out relevant research and health policy analysis. diffusion of data, health information, and responsibility helps to provide a responsive and comprehensive approach to meet the health needs of the individual and community. the physical, social, economic, and political environments are all important determinants of the health status of the population and the individual. joint action (intersectoral cooperation) between public and non-governmental or community-based organizations is needed to achieve the well-being of the individual in a healthy society. in the s and s, these ideas contributed to an evolving new public health, spurred on by epidemiological changes, health economics, the development of managed care linking health systems, and prepayment. knowledge and self-care skills, as well as community action to reduce health risks, are no less important in this than the roles of medical practitioners and institutional care. all are parts of a coherent holistic approach to health. the concept of selective primary care, articulated in by walsh and warren, addresses the needs of developing countries to select those interventions on a broad scale that would have the greatest positive impact on health, taking into account limited resources such as money, facilities, and human resources. the term selective primary care is meant to define national priorities that are based not on the greatest causes of morbidity or mortality, but on common conditions of epidemiological importance for which there are effective and simple preventive measures. throughout health planning, there is an implicit or explicit selection of priorities for allocation of resources. even in primary care, selection of targets is a part of the process of resource allocation. in modern public health, this process is more explicit. a country with limited resources and a high birth rate will emphasize maternal and child health before investing in geriatric care. this concept has become part of the microeconomics of health care and technology assessment, discussed in chapters and , respectively, and is used widely in setting priorities and resource allocation. in developing countries, cost-effective primary care interventions have been articulated by many international organizations, including iodization of salt, use of oral rehydration therapy (ort) for diarrheal diseases, vitamin a supplementation for all children, expanded programs of immunization, and others that have the potential for saving hundreds of thousands of lives yearly at low cost. in developed countries, health promotions targeted to reduce accidents and risk factors such as smoking, high-fat diets, and lack of exercise for cardiovascular diseases are low-cost public health interventions that save lives and reduce the use of hospital care. targeting specific diseases is essential for efforts to control tb or eradicate polio, but at the same time, development of a comprehensive primary care infrastructure is equally or even more important than the single-disease approach. some disease entities such as hiv/aids attract donor funding more readily than basic infrastructure services such as immunization, and this can sometimes be detrimental to addressing the overall health needs of the population and other neglected but also important diseases. the risk approach selects population groups on the basis of risk and helps to determine interventional priorities to reduce morbidity and mortality. the measure of health risk is taken as a proxy for need, so that the risk approach provides something for all, but more for those in need, in proportion to that need. in epidemiological terms, these are people with higher relative risk or attributed risk. some groups in the general population are at higher risk than others for specific conditions. the expanded programme on immunization (epi), control of diarrhoeal diseases (cdd), and acute respiratory disease (ard) programs of the who are risk approaches to tackling fundamental public health problems of children in developing countries. public health places considerable emphasis on maternal and child health because these are vulnerable periods in life for specific health problems. pregnancy care is based on a basic level of care for all, with continuous assessment of risk factors that require a higher intensity of follow-up. prenatal care helps to identify factors that increase the risk for the pregnant woman or her fetus/newborn. efforts directed towards these special risk groups have the potential to reduce morbidity and mortality. high-risk case identification, assessment, and management are vital to a successful maternal care program. similarly, routine infant care is designed not only to promote the health of infants, but also to find the earliest possible indications of deviation and the need for further assessment and intervention to prevent a worsening of the condition. low birth-weight babies are at greater risk for many short-and long-term hazards and should be given special treatment. all babies are routinely screened for birth defects or congenital conditions such as hypothyroidism, phenylketonuria, and other metabolic and hematological diseases. screening must be followed by investigating and treating those found to have a clinical deficiency. this is an important element of infant care because infancy itself is a risk factor. as will be discussed in chapters and and others, epidemiology has come to focus on the risk approach with screening based on known genetic, social, nutritional, environmental, occupational, behavioral, or other factors contributing to the risk for disease. the risk approach has the advantage of specificity and is often used to initiate new programs directed at special categories of need. this approach can lead to narrow and somewhat rigid programs that may be difficult to integrate into a more general or comprehensive approach, but until universal programs can be achieved, selective targeted approaches are justifiable. indeed, even with universal health coverage, it is still important to address the health needs or issues of groups at special risk. working to achieve defined targets means making difficult choices. the supply and utilization of some services will limit availability for other services. there is an interaction, sometimes positive, sometimes negative, between competing needs and the health status of a population. public health identifies needs by measuring and comparing the incidence or prevalence of the condition in a defined population with that in other comparable population groups and defines targets to reduce or eliminate the risk of disease. it determines ways of intervening in the natural epidemiology of the disease, and develops a program to reduce or even eliminate the disease. it also assesses the outcomes in terms of reduced morbidity and mortality, as well as the economic justification in cost-effectiveness analysis to establish its value in health priorities. because of the interdependence of health services, as well as the total financial burden of health care, it is essential to look at the costs of providing health care, and how resources should be allocated to achieve the best results possible. health economics has become a fundamental methodology in policy determination. the costs of health care, the supply of services, the needs for health care or other health-promoting interventions, and effective means of using resources to meet goals are fundamental in the new public health. it is possible to err widely in health planning if one set of factors is overemphasized or underemphasized. excessive supply of one service diminishes the availability of resources for other needed investments in health. if diseases are not prevented or their sequelae not well managed, patients must use costly health care services and are unable to perform their normal social functions such as learning at school or performing at work. lack of investment in health promotion and primary prevention creates a larger reliance on institutional care, driving health costs upwards, and restricting flexibility in meeting patients' needs. the interaction of supply and demand for health services is an important determinant of the political economy of health care. health and its place in national priorities are determined by the social-political philosophy and resource allocation of a government. the case for action, or the justification for a public health intervention, is a complex of epidemiological, economic, and public policy factors (table . ). each disease or group of diseases requires its own case for action. the justification for public health intervention requires sufficient evidence of the incidence and prevalence of the disease (see chapter ). evidence-based public health takes into account the effectiveness and safety of an intervention; risk factors; safe means at hand to intervene; the human, social, and economic cost of the disease; political factors; and a policy decision as to the priority of the problem. this often depends on subjective factors, such as the guiding philosophy of the health system and the way it allocates resources. some interventions are so well established that no new justification is required to make the case, and the only question is how to do it most effectively. for example, infant vaccination is a cost-effective and cost-beneficial program for the protection of the individual child and the population as a whole. whether provided as a public service or as a clinical preventive measure by a private medical practitioner, it is in the interest of public health that all children be immunized. an outbreak of diarrheal disease in a kindergarten presents an obvious case for action, and a public health system must respond on an emergency basis, with selection of the most suitable mode of intervention. the considerations in developing a case for action are outlined above. need is based on clinical and epidemiological evidence, but also on the importance of an intervention in the eyes of the public. the technology available, its effectiveness and safety, and accumulated experience are important in the equation, as are the acceptability and affordability of appropriate interventions. the precedents for use of an intervention are also important. on epidemiological evidence, if the preventive practice has been seen to provide reduction in risk for the individual and for the population, then there is good reason to implement it. the costs, risks and benefits must be examined as part of the justification to help in the selection of health priorities. health systems research examines the efficiency of health care and promotes improved efficiency and effective use of resources. this is a vital function in determining how best to use resources and meet current health needs. past emphasis on hospital care at the expense of less development of primary care and prevention is still a common issue, particularly in former soviet and developing countries, where a high percentage of total health expenditure goes to acute hospital care with long length of stay, with smaller allocation to preventive and community health care. the result of this imbalance is high mortality from preventable diseases. new drugs, vaccines, and medical equipment are continually becoming available, and each new addition needs to be examined among the national health priorities. sometimes, owing to cost, a country cannot afford to add a new vaccine to the routine. however, when there is good evidence for efficacy and safety of new vaccines, drugs, diagnostic methods or other innovations, it could be applied for those at greatest risk. although there are ethical issues involved, it may be necessary to advise parents or family members to purchase the vaccine independently. clearly, recommending individual purchase of a vaccine is counter to the principle of equity and solidarity, benefiting middleclass families, and providing a poor basis of data for evaluation of the vaccine and its target disease. on the other hand, failure to advise parents of potential benefits to their children creates other ethical problems, but may increase public pressure and insurance system acceptance of new methods, e.g., varicella and hpv vaccines. mass screening programs involving complete physical examinations have not been found to be cost-effective or to significantly reduce disease. in the s and s, routine general health examinations were promoted as an effective method of finding disease early. since the late s, a selective and specific approach to screening has become widely accepted. this involves defining risk categories for specific diseases and bearing in mind the potential for remedial action. early case finding of colon cancer by routine fecal blood testing and colonoscopy has been found to be effective, and pap smear testing to discover cancer of the cervix is timed according to risk category. screening for colorectal cancer is essential for modern health programs and has been adopted by most industrialized countries. outreach programs by visits, telephones, emails or other modern methods of communication are important to contact non-attenders to promote utilization, and have been shown to increase compliance with proven effective measures. these programs are important for screening, follow-up, and maintenance of treatment for hypertension, diabetes, and other conditions requiring long-term management. screening technology is changing and often the subject of intense debate as such programs are costly and their cost-effectiveness is an important matter for policy making: screening for lung cancer is becoming a feasible and effective matter for high-risk groups, whereas breast cancer screening frequency is now in dispute; while nanotechnology and bioengineering promises new methods for cancer screening. the factor of contribution to quality of life should be considered. a vaccine for varicella is justified partly for the prevention of deaths or illness from chickenpox. a stronger the right to health public expectation and social norms argument is often based on the fact that this is a disease that causes moderate illness in children for up to weeks and may require parents to stay home with the child, resulting in economic loss to the parent and society. the fact that this vaccination prevents the occurrence of herpes zoster or shingles later in life may also be a justification. widespread adoption of hepatitis b vaccine is justified on the grounds that it prevents cancer of the liver, liver cirrhosis, and hepatic failure in a high percentage of the population affected. how many cases of a disease are enough to justify an intervention? one or several cases of some diseases, such as poliomyelitis, may be considered an epidemic in that each case constitutes or is an indicator of a wider threat. a single case of polio suggests that another persons are infected but have not developed a recognized clinical condition. such a case constitutes a public health emergency, and forceful organization to meet a crisis is needed. current standards are such that even one case of measles imported into a population free of the disease may cause a large outbreak, as occurred in the uk, france, and israel during through , by contacts on an aircraft, at family gatherings, or even in medical settings. a measles epidemic indicates a failure of public health policy and practice. screening for some cancers, such as cervix and colon, is cost effective. screening of all newborns for congenital disorders is important because each case discovered early and treated effectively saves a lifetime of care for serious disability. assessing a public health intervention to prevent the disease or reduce its impact requires measurement of the disease in the population and its economic impact. there is no simple formula to justify a particular intervention, but the cost-benefit approach is now commonly required to make such a case for action. sometimes public opinion and political leadership may oppose the views of the professional community, or may impose limitations of policy or funds that prevent its implementation. conversely, professional groups may press for additional resources that compete for limited resources available to provide other needed health activities. both the professionals of the health system and the general public need full access to health-related information to take part in such debates in a constructive way. to maintain progress, a system must examine new technologies and justify their adoption or rejection (see chapter ). the association between health and political issues was emphasized by european innovators such as rudolf virchow (and in great britain by edwin chadwick; see chapter ) in the mid-nineteenth century, when the conditions of the working population were such that epidemic diseases were rife and mortality was high, especially in the crowded slums of the industrial revolution. the same observations led bismarck in germany to introduce early forms of social insurance for the health of workers and their families in the s, and to britain's national health insurance, also for workers and families. the role of government in providing universal access to health care was a struggle in individual countries during the twentieth century and lasting into the second decade of the twenty-first century (e.g. president obama's affordable health care act of ). as the concept of public health has evolved, and the cost effectiveness of medical care has improved through scientific and technological advances, societies have identified health as a legitimate area of activity for collective bargaining and government. with this process, the need to manage health care resources has become more clearly defined as a public responsibility. in industrialized countries, each with very different political make-up, national responsibility for universal access to health has become part of the social ethos. with that, the financing and managing of health services have developed into part of a broad concept of public health, and economics, planning, and management have come to be part of the new public health (discussed in chapters - ). social, ethical, and political philosophies have profound effects on policy decisions including allocation of public monies and resources. investment in public health is now recognized as an integral part of socioeconomic development. governments are major suppliers of funds and leadership in health infrastructure development, provision of health services, and health payment systems. they also play a central role in the development of health promotion and regulation of the environment, food, and drugs essential for community health. in liberal social democracies, the individual is deemed to have a right to health care. the state accepts responsibility to ensure availability, accessibility, and quality of care. in many developed countries, government has also taken responsibility to arrange funding and services that are equitably accessible and of high quality. health care financing may involve taxation, allocation, or special mandatory requirements on employers to pay for health insurance. services may be provided by a state-financed and -regulated service or through ngos and/or private service mechanisms. these systems allocate between percent and percent of gross national product (gnp) to health services, with some governments funding over percent of health expenditure; for example, canada and the uk. in communist states, the state organizes all aspects of health care with the philosophy that every citizen is entitled to equity in access to health services. the state health system manages research, staff training, and service delivery, even if operational aspects are decentralized to local health authorities. this model applied primarily to the soviet model of health services. these systems, except for cuba, placed financing of health low on the national priority, with funding less than percent of gnp. in the shift to market economies in the s, some former socialist countries, such as russia, are struggling with poor health status and a difficult shift from a strongly centralized health system to a decentralized system with diffusion of powers and responsibilities. promotion of market concepts in former soviet countries has reduced access to care and created a serious dilemma for their governments. former colonial countries, independent since the s and s, largely carried on the governmental health structures established in the colonial times. most developing countries have given health a relatively low place in budgetary allotment, with expenditures under percent of gnp. since the s, there has been a trend in developing countries towards decentralization of health services and greater roles for ngos, and the development of health insurance. some countries, influenced by medical concepts of their former colonial master countries, fostered the development of specialty medicine in the major centers with little emphasis on the rural majority population. soviet influence in many ex-colonial countries promoted state-operated systems. the who promoted primary care, but the allocations favored city-based specialty care. israel, as an ex-colony, adapted british ideas of public health together with central european sick funds and maternal and child health as major streams of development until the mid- s. a growing new conservatism in the s and s in the industrialized countries is a restatement of old values in which market economics and individualistic social values are placed above concepts of the "common good" of liberalism and socialism in its various forms. in the more extreme forms of this concept, the individual is responsible for his or her own health, including payment, and has a choice of health care providers that will respond with high-quality personalized care. market forces, meaning competition in financing and provision of health services with rationing of services, based on fees or private insurance and willingness and ability to pay, have become part of the ideology of the new conservatism. it is assumed that the patient (i.e., the consumer) will select the best service for his or her need, while the provider best able to meet consumer expectations will thrive. in its purest form, the state has no role in providing or financing of health services except those directly related to community protection and promotion of a healthful environment without interfering with individual choices. the state ensures that there are sufficient health care providers and allows market forces to determine the prices and distribution of services with minimal regulation. the usa retains this orientation in a highly modified form, with percent of the population covered by some form of private or public insurance systems (see chapters and ). modified market forces in health care are part of health reforms in many countries as they seek not only to ensure quality health care for all but also to constrain costs. a free market in health care is costly and ultimately inefficient because it encourages inflation of provider incomes or budgets and increasing utilization of highly technical services. further, even in the most free market societies, the economy of health care is highly influenced by many factors outside the control of the consumer and provider. the total national health expenditure in the usa rose rapidly until reaching over . percent of gross domestic product (gdp) in , the highest of any country, despite serious deficiencies for those without any or with very inadequate health insurance (in total more than percent of the population). this figure compares to some . percent of gdp in canada, which has universal health insurance under public administration. following the defeat of president clinton's national health program, the conservative congress and the business community took steps to expand managed care in order to control costs, resulting in a revolution in health care in the usa (see chapters and ). in the - decade health expenditure in the usa is expected to rise to . percent of gdp, partly owing to increased population coverage with implementation of the ppaca (obamacare). reforms are being implemented in many "socialized" health systems. these may be through incentives to promote achievement of performance indicators, such as full immunization coverage. others are using control of supply, such as hospital beds or licensed physicians, as methods of reducing overutilization of services that generates increasing costs. market mechanisms in health are aimed not only at the individual but also at the provider. incentive payment systems must work to protect the patient's legitimate needs, and conversely incentives that might reduce quality of care should be avoided. fee-for-service promotes high rates of services such as surgery. increasing private practice and user fees can adversely affect middle-and low-income groups, as well as employers, by raising the costs of health insurance. managed care systems, with restraints on fee-for-service medical practice, have emerged as a positive response to the market approach. incentive systems in payments for services may be altered by government or insurance agencies in order to promote rational use of services, such as reduction of hospital stays. the free market approach is affecting planning of health insurance systems in previously highly centralized health systems in developing countries as well as the redevelopment of health systems in former soviet countries. despite political differences, reform of health systems has become a common factor in virtually all health systems since the s, as each government searches for costeffectiveness, quality of care, and universality of coverage. the new paradigm of health care reform sees the convergence of different systems to common principles. national responsibility for health goals and health promotion leads to national financing of health care with regional and managed care systems. most developed countries have long since adopted national health insurance or service systems. some governments may, as in the usa, insure only the highest risk groups such as the elderly and the poor, leaving the working and middle classes to seek private insurers. the nature and direction of health care reform affecting coverage of the population are of central importance in the new public health because of its effects on allocation of resources and on the health of the population. the effects of the economic crisis in the usa are being felt worldwide. while the downturn has largely occurred in wealthier nations, the poor in low-income countries will be among those affected. past economic downturns have been followed by substantial drops in foreign aid to developing countries. as public health gained from sanitary and other control measures for infectious diseases, along with mother and child care, nutrition, and environmental and occupational health, it also gained strength and applicability from advances in the social and behavioral sciences. social darwinism, a political philosophy that assumed "survival of the fittest" and no intervention of the sate to alleviate this assumption, was popular in the early nineteenth century but became unacceptable in industrialized countries, which adopted social policies to alleviate the worst conditions of poverty, unemployment, poor education, and other societal ills. the political approach to focusing on health and poverty is associated with jeremy bentham in britain in the late eighteenth century, who promoted social and political reform and "the greatest good for the greatest number", or utilitarianism. rudolf virchow, an eminent pathologist and a leader in recognizing ill-health and poverty as cause and effect, called for political action to create better conditions for the poor and working-class population. the struggle for a social contract was promoted by pioneer reformists such as edwin chadwick (general report on the sanitary condition of the labouring population of great britain, ), who later became the first head of the board of health in britain, and lemuel shattuck (report of a general plan for the promotion of public and personal health, ) . shattuck was the organizer and first president of the american statistical association. the social sciences have become fundamental to public health, with a range of disciplines including vital statistics and demography (seventeenth century), economics and politics (nineteenth century), sociology (twentieth century), history, anthropology, and others, which provide collectively important elements of epidemiology of crucial significance for survey methods and qualitative research (see chapter ). these advances contributed greatly to the development of methods of studying diseases and risk factors in a population and are still highly relevant to addressing inequalities in health. individuals in good health are better able to study and learn, and be more productive in their work. improvements in the standard of living have long been known to contribute to improved public health; however, the converse has not always been recognized. investment in health care was not considered a high priority in many countries where economic considerations directed investment to the "productive" sectors such as manufacturing and large-scale infrastructure projects, such as hydroelectric dams. whether health is a contributor to economic development or a drain on societies' resources has been a fundamental debate between socially and market-oriented advocates. classic economic theory, both free enterprise and communist, has tended to regard health as a drain on economies, distracting investment needed for economic growth. as a result, in many countries health has been given low priority in budgetary allocation, even when the major source of financing is governmental. this belief among economists and banking institutions prevented loans for health development on the grounds that such funds should focus on creating jobs and better incomes, before investing in health infrastructure. consequently, the development of health care has been hampered. a socially oriented approach sees investment in health as necessary for the protection and development of "human capital", just as investment in education is needed for the long-term benefit of the economy of a country. in , the world bank's world development report: investing in health articulated a new approach to economics in which health, along with education and social development, is seen as an essential precondition for and contributor to economic development. while many in the health field have long recognized the importance of health for social and economic improvement, its adoption by leading international development banking may mark a turning point for investment in developing nations, so that health may be a contender for increased development loans. the concept of an essential package of services discussed in that report establishes priorities in low-and middle-income countries for efficient use of resources based on the burden of disease and cost-effectiveness analysis of services. it includes both preventive and curative services targeted to specific health problems. it also recommends support for comprehensive primary care, such as for children, and infrastructure development including maternity and hospital care, medical and nursing outreach services, and community action to improve sanitation and safe water supplies. reorientation of government spending on health is increasingly being adopted, as in the uk, to improve equity in access for the poor and other neglected sectors or regions of society with added funding for relatively deprived areas to improve primary care services. differential capitation funding as a form of affirmative action to provide for highneeds populations is a useful concept in public health terms to address the inequities still prevalent in many countries. as medical care has gradually become more involved in prevention, and as it has moved into the era of managed care, the gap between public health and clinical medicine has narrowed. as noted above, many countries are engaged in reforms in their health care systems. the motivation is largely derived from the need for cost containment, but also to extend health care coverage to underserved parts of the population. countries without universal health care still have serious inequities in distribution of or access to services, and may seek reform to reduce those inequities, perhaps under political pressures to improve the provision of services. incentives for reform are needed to address regional inequities, and preserving or developing universal access and quality of care, but also on inequities in health between the rich and the poor countries and within even the wealthy countries. in some settings, a health system may fail to keep pace with developments in prevention and in clinical medicine. some countries have overdeveloped medical and hospital care, neglecting important initiatives to reduce the risk of disease. the process of reform requires setting standards to measure health status and the balance of services to optimize health. a health service can set a target of immunizing percent of infants with a national immunization schedule, but requires a system to monitor performance and incentives for changes. a health system may also have failed to adapt to changing needs of the population through lack, or misuse, of health information and monitoring systems. as a result, the system may err seriously in its allocation of resources, with excessive emphasis on hospital care and insufficient attention to primary and preventive care. all health services should have mechanisms for correctly gathering and analyzing needed data for monitoring the incidence of disease and other health indicators, such as hospital utilization, ambulatory care, and preventive care patterns. for example, the uk's nhs periodically undertakes a restructuring process of parts of the system to improve the efficiency of service. this involves organizational changes and decentralization with regional allocation of resources (see chapter ). health systems are under pressures of changing demographic and epidemiological patterns as well as public expectations, rising costs of new technology, financing, and organizational change. new problems must be continually addressed with selection of priority issues and the most effective methods chosen. reforms may create unanticipated problems, such as professional or public dissatisfaction, which must be evaluated, monitored, and addressed as part of the evolution of public health. literacy, freedom of the press, and increasing public concern for social and health issues have contributed to the development of public health. the british medical community lobbied for restrictions on the sale of gin in the s in order to reduce the damage that it caused to the working class. in the late eighteenth and the nineteenth centuries, reforms in society and sanitation were largely the result of strongly organized advocacy groups influencing public opinion through the press. such pressure stimulated governments to act in regulating the working conditions of mines and factories. abolition of the slave trade and its suppression by the british navy in the early nineteenth century resulted from successful advocacy groups and their effects on public opinion through the press. vaccination against smallpox was promoted by privately organized citizen groups, until later taken up by local and national government authorities. advocacy consists of activities of individuals or groups publicly pleading for, supporting, espousing, or recommending a cause or course of action. the advocacy role of reform movements in the nineteenth century was the basis of the development of modern organized public health. campaigns ranged from the reform of mental hospitals, nutrition for sailors to prevent scurvy and beriberi, and labor laws to improve working conditions for women and children in particular, to the promotion of universal education and improved living conditions for the working population. reforms on these and other issues resulted from the stirring of the public consciousness by advocacy groups and the public media, all of which generated political decisions in parliaments (box . ). such reforms were in large part motivated by fear of revolution throughout europe in the mid-nineteenth century and the early part of the twentieth century. trade unions, and before them medieval guilds, fought to improve hours, safety, and conditions of work, as well as social and health benefits for their members. in the usa, collective bargaining through trade unions achieved wage increases and widespread coverage of the working population under voluntary health insurance. unions and some industries pioneered prepaid group practice, the predecessor of health maintenance organizations and managed care or the more recent acos (see chapters and ). through raising public consciousness on many issues, advocacy groups pressure governments to enact legislation to restrict smoking in public places, prohibit tobacco advertising, and mandate the use of bicycle helmets. advocacy groups play an important role in advancing health based on disease groups, such as cancer, multiple sclerosis, and thalassemia, or advancing health issues, such as the organizations promoting breastfeeding, environmental improvement, or smoking reduction. some organizations finance services or facilities not usually provided within insured health programs. such organizations, which can number in the hundreds in a country, advocate the importance of their special concern and play an important role in innovation and meeting community health needs. advocacy groups, including trade unions, professional groups, women's groups, self-help groups, and many others, focus on specific issues and have made major contributions to advancing the new public health. the history of public health is replete with pioneers whose discoveries led to strong opposition and sometimes violent rejection by conservative elements and vested interests in medical, public, or political circles. opposition to jennerian vaccination, the rejection of semmelweiss by colleagues in vienna, and the contemporary opposition to the work of great pioneers in public health such as pasteur, florence nightingale, and many others may deter or delay implementation of other innovators and new breakthroughs in preventing disease. although opposition to jenner's vaccination lasted well into the late nineteenth century in some areas, its supporters gradually gained ascendancy, ultimately leading to the global eradication of smallpox. these and other pioneers led the way to improved health, often after bitter controversy on topics later accepted and which, in retrospect, seem to be obvious. advocacy has sometimes had the support of the medical profession but elicited a slow response from public authorities. david marine of the cleveland clinic and david cowie, professor of pediatrics at the university of michigan, proposed the prevention of goiter by iodization of salt. marine carried out a series of studies in fish, and then in a controlled clinical trial among schoolgirls in - , with startlingly positive results in reducing the prevalence of goiter. cowie campaigned for the iodization of salt, with support from the medical profession. in , he convinced a private manufacturer to produce morton's iodized salt, which rapidly became popular throughout north america. similarly, iodized salt came to be used in many parts of europe, mostly without governmental support or legislation. iodine-deficiency disorders (idds) remain a widespread condition, estimated to have affected billion people worldwide in . the target of international eradication of idds by was set at the world summit for children in , and the who called for universal iodization of salt in . by , nearly percent of households in developing countries consumed adequately iodized salt. china and nigeria, have had great success in recent years with mandatory salt fortification in increasing iodization rates, in china from percent to percent in years. but the problem is not yet gone and even in europe there is inadequate standardization of iodine levels and population follow-up despite decades of work on the problem. professional organizations have contributed to promoting causes such as children's and women's health, and environmental and occupational health. the american academy of pediatrics has contributed to establishing and promoting high standards of care for infants and children in the usa, and to child health internationally. hospital accreditation has been used for decades in the usa, canada, and more recently in australia and the uk. it has helped to raise standards of health facilities and care by carrying out systematic peer review of hospitals, nursing homes, primary care facilities, and mental hospitals, as well as ambulatory care centers and public health agencies (see chapter ). public health needs to be aware of negative advocacy, sometimes based on professional conservatism or economic self-interest. professional organizations can also serve as advocates of the status quo in the face of change. opposition by the american medical association (ama) and the health insurance industry to national health insurance in the usa has been strong and successful for many decades. the passage of the ppaca has been achieved despite widespread political and public opposition, yet was sustained in the us supreme court and is gaining widening popular support as the added value to millions of formerly uninsured americans becomes clear. in some cases, the vested interest of one profession may block the legitimate development of others, such as when ophthalmologists lobbied successfully against the development of optometry, now widely accepted as a legitimate profession. political activism for reform in nineteenth-century britain led to banning and suppressing the slave trade, improvements in working conditions for miners and factory workers, and other major political reforms. in keeping with this tradition, samuel plimsoll ( - ), british member of parliament elected for derby in , conducted a solo campaign for the safety of seamen. his book, our seamen, described ships sent to sea so heavily laden with coal and iron that their decks were awash. seriously overloaded ships, deliberately sent to sea by unscrupulous owners, frequently capsized, drowning many crew members, with the owners collecting inflated insurance fees. overloading was the major cause of wrecks and thousands of deaths in the british shipping industry. plimsoll pleaded for mandatory load-line certificate markers to be issued to each ship to prevent any ships putting to sea when the marker was not clearly visible. powerful shipping interests fought him every inch of the way, but he succeeded in having a royal commission established, leading to an act of parliament mandating the "plimsoll line", the safe carrying capacity of cargo ships. this regulation was adopted by the us bureau of shipping as the load line act in and is now standard practice worldwide. jenner's discovery of vaccination with cowpox to prevent smallpox was adopted rapidly and widely. however, intense opposition by organized groups of antivaccinationists, often led by those opposed to government intervention in health issues and supported by doctors with lucrative variolation practices, delayed the implementation of smallpox vaccination for many decades. ultimately, smallpox was eradicated in , owing to a global campaign initiated by the who. opposition to legislated restrictions on private ownership of assault weapons and handguns is intense in the usa, led by well-organized, well-funded, and politically powerful lobby groups, despite the amount of morbidity and mortality due to gun-associated violent acts (see chapter ). fluoridation of drinking water is the most effective public health measure for preventing dental caries, but it is still widely opposed, and in some places the legislation has been rescinded even after implementation, by wellorganized antifluoridation campaigns. opposition to fluoridation of community water supplies is widespread, and effective lobbying internationally has slowed but has not stopped progress (see chapter ). despite the life-saving value of immunization, opposition still exists in and harms public health protection. opposition has slowed progress in poliomyelitis eradication; for example, radical islamists killed polio workers in northern nigeria in , one of the last three countries with endemic poliomyelitis. resistance to immunization in the s has resulted in the recurrence of pertussis and diphtheria and a very large epidemic of measles across western europe, including the uk, with further spread to the western hemisphere in - (see chapter ). progress may be blocked where all decisions are made in closed discussions, not subject to open scrutiny and debate. public health personnel working in the civil service of organized systems of government may not be at liberty to promote public health causes. however, professional organizations may then serve as forums for the essential professional and public debate needed for progress in the field. professional organizations such as the apha provide effective lobbying for the interests of public health programs and can have an important impact on public policy. in mid- , efforts by the secretary of health and human services in the usa brought together leaders of public health with representatives of the ama and academic medical centers to try to find areas of common interest and willingness to promote the health of the population. in europe too, increasing cooperation between public health organizations is stimulating debate on issues of transnational importance across the region, which, for example, has a wide diversity of standards on immunization practices and food policies. public advocacy has played an especially important role in focusing attention on ecological issues (box . ). in , greenpeace, an international environmental activist group, fought to prevent the dumping of an oil rig in the north sea and forced a major oil company to find another solution that would be less damaging to the environment. an explosion on an oil rig in the gulf of mexico in led to enormous ecological and economic damage as well as loss of life. damages levied on the responsible company (british petroleum) amount to some $ . billion dollars and several criminal negligence charges are pending. greenpeace also continued its efforts to stop the renewal of testing of atomic bombs by france in the south pacific. international protests led to the cessation of almost all testing of nuclear weapons. international concern over global warming has led to growing efforts to stem the tide of air pollution from fossil fuels, coal-burning electrical production, and other manifestations of carbon dioxide and toxic contamination of the environment. progress is far from certain as newly enriched countries such as china and india follow the rising consumption patterns of western countries. public advocacy and rejection of wanton destruction of the global ecology may be the only way to prod consumers, governments, and corporate entities such as the energy and transportation industries to change direction. the pace of change from fossil fuels is slow but has captured public attention, and private companies are seeking more fuel efficiency in vehicles and electrical power production, mainly though the use of natural gas instead of fuel oil and coal for electricity production or better still by wind and solar energy. the search for "green solutions" to the global warming crisis has become increasingly dynamic, with governments, the private sector, and the general public keenly aware of the importance of the effort and the dangers of failure. in the latter part of the twentieth century and the early twenty-first century, prominent international personalities and entertainers have taken up causes such as the removal of land mines in war-torn countries, illiteracy in disadvantaged advocacy is a function in public health that has been important in promoting advances in the field, and one that sometimes places the advocate in conflict with established patterns and organizations. one of the classic descriptions of this function is in henrik ibsen's play an enemy of the people, in which the hero, a young doctor, thomas stockmann, discovers that the water in his community is contaminated. this knowledge is suppressed by the town's leadership, led by his brother the mayor, because it would adversely affect plans to develop a tourist industry of baths in their small norwegian town in the late nineteenth century. the young doctor is taunted and abused by the townspeople and driven from the town, having been declared an "enemy of the people" and a potential risk. the allegory is a tribute to the man of principle who stands against the hysteria of the crowd. the term also took on a far more sinister and dangerous meaning in george orwell's novel and in totalitarian regimes of the s to the present time. populations, and funding for antiretroviral drugs for african countries to reduce maternal-fetal transmission of hiv and to provide care for the large numbers of cases of aids devastating many countries of sub-saharan africa. rotary international has played a key role in polio eradication efforts globally. the public-private consortium global alliance for vaccines and immunization (gavi) has been instrumental in promoting immunization in recent years, with participation by the who, unicef, the world bank, the gates foundation, vaccine manufacturers, and others. this has had an important impact on extending immunization to protect and save the lives of millions of children in deprived countries not yet able to provide fundamental prevention programs such as immunization at adequate levels. gavi has brought vaccines to low-income countries around the world, such as rotavirus vaccine, pentavalent vaccine in myanmar, and pneumococcal vaccine for children in countries in sub-saharan africa, including dr congo. the bill & melinda gates foundation pledged us $ million in to establish gavi, with us $ million per year and us $ billion in to promote the decade of vaccines. international conferences help to create a worldwide climate of advocacy for health issues. international sanitary conferences in the nineteenth century were convened in response to the cholera epidemics. international conferences continue in the twenty-first century to serve as venues for advocacy on a global scale, bringing forward issues in public health that are beyond the scope of individual nations. the who, unicef, and other international organizations perform this role on a continuing basis (see chapter ). criticisms of this approach have focused on the lack of similar effort or donors to address ncds, weak public health infrastructure, and that this frees national governments from responsibility to care for their own children. no one can question, however, that this kind of endeavor has saved countless lives and needs the backing of other aid donors and national government participation. consumerism is a movement that promotes the interests of the purchaser of goods or services. in the s, a new form of consumer advocacy emerged from the civil rights and antiwar movement in the usa. concern was focused on the environment, occupational health, and the rights of the consumer. rachel carson stimulated concern by dramatizing the effects of ddt on wildlife and the environment but inadvertently jeopardized anti-malarial efforts in many countries. this period gave rise to environmental advocacy efforts worldwide, and a political movement, the greens, in western europe. ralph nader showed the power of the advocate or "whistle-blower" who publicizes health hazards to stimulate active public debate on a host of issues related to the public well-being. nader, a consumer advocate lawyer, developed a strategy for fighting against business and government activities and products which endangered public health and safety. his book unsafe at any speed took issue with the us automobile industry for emphasizing profit and style over safety, and led to the enactment of the national traffic and motor safety act of , establishing safety standards for new cars. this was followed by a series of enactments including design and emission standards and seat-belt regulations. nader's work continues to promote consumer interests in a wide variety of fields, including the meat and poultry industries, and coal mining, and promotes greater government regulatory powers regarding pesticide usage, food additives, consumer protection laws, rights to knowledge of contents, and safety standards. consumerism has become an integral part of free market economies, and the educated consumer does influence the quality, content, and price of products. greater awareness of nutrition in health has influenced food manufacturers to improve packaging, content labeling, enrichment with vitamins and minerals, and advertisement to promote those values. low-fat dietary products are available because of an increasingly sophisticated public concerned over dietary factors in cardiovascular diseases. the same process occurred in safe toys and clothing for children, automobile safety features such as mandatory use of car seats for infants, and other innovations that quickly became industry standards in the industrialized world. dangerous practices such as the use of lead paint in toys and melamine contamination of milk products from china capture the public attention quickly and remind public health authorities of the importance of continuous alertness to potential hazards. consumerism can also be exploited by pharmaceutical companies with negative impacts on the health system, especially in the advertising of health products which leads to unnecessary visits to health providers and pressure for approval to obtain the product. the internet has provided people with access to a vast array of information and opinion, and to current literature otherwise unavailable because of the often inadequate library resources of medical and other health professionals. the very freedom of information the internet allows, however, also provides a vehicle for extremist and fringe groups to promote disinformation such as "vaccination causes autism, fluoridation causes cancer", which can cause considerable difficulties for basic public health programs or lead to self-diagnosis of conditions, with often disastrous consequences. advocacy and voluntarism go hand in hand. voluntarism takes many forms, including raising funds for the development of services or operating services needed in the community. it may take the form of fund-raising to build clinics or hospitals in the community, or to provide medical equipment for elderly or handicapped people; or retirees and teenagers working as hospital volunteers to provide services that are not available through paid staff, and to provide a sense of community caring for the sick in the best traditions of religious or municipal concerns. this can also be extended to prevention, as in support for immunization programs, assistance for the handicapped and elderly in transportation, meals-on-wheels, and many other services that may not be included in the "basket of services" provided by the state, health insurance, or public health services. community involvement can take many forms, and so can voluntarism. the pioneering role of women's organizations in promoting literacy, health services, and nutrition in north america during the latter part of the nineteenth and the early twentieth centuries profoundly affected the health of the population. the advocacy function is enhanced when an organization mobilizes voluntary activity and funds to promote changes or needed services, sometimes forcing official health agencies or insurance systems to revise their attitudes and programs to meet these needs. by the early s, canada's system of federally supported provincial health insurance plans covered all of the country. the federal minister of health, marc lalonde, initiated a review of the national health situation, in view of concern over the rapidly increasing costs of health care. this led to articulation of the "health field concept" in , which defined health as a result of four major factors: human biology, environment, behavior, and health care organization (box . ). lifestyle and environmental factors were seen as important contributors to the morbidity and mortality in modern societies. this concept gained wide acceptance, promoting new initiatives that emphasized health promotion in response to environmental and lifestyle factors. conversely, reliance primarily on medical care to solve all health problems could be counterproductive. this concept was a fundamental contributor to the idea of health promotion later articulated in the ottawa declaration, discussed below. the health field concept came at a time when many epidemiological studies were identifying risk factors for cardiovascular diseases and cancers that related to personal habits, such as diet, exercise, and smoking. the concept advocated that public policy needed to address individual lifestyle as part of the overall effort to improve health status. as a result, the canadian federal government established health promotion as a new activity. this quickly spread to many other jurisdictions and gained wide acceptance in many industrialized countries. concern was expressed that this concept could become a justification for a "blame the victim" approach, in which those ill with a disease related to personal lifestyles, such as smokers or aids patients, are seen as having chosen to contract the disease. such a patient might then be considered not to be entitled to all benefits of insurance or care that others may receive. the result may be a restrictive approach to care and treatment that would be unethical in the public health tradition and probably illegal in western jurisprudence. this concept was also used to justify withdrawal from federal commitments in cost sharing and escape from facing controversial health reform in the national health insurance program. during the s and s, outspoken critics of health care systems, such as ivan illytch, questioned the value of medical care for the health of the public. this became a widely discussed, somewhat nihilistic, view towards medical care, and was influential in promoting skepticism regarding the value of the biomedical mode of health care, and antagonism towards the medical profession. in , thomas mckeown presented a historicalepidemiological analysis showing that up to the s, medical care had only a limited impact on mortality rates, although improvements in surgery and obstetrics were notable. he showed that crude death rates in england averaged about per population from to , declining steeply to per in , per in , and per in , when medical care became truly effective. mckeown concluded that much of the improvement in health status over the past several centuries was due to reduced mortality from infectious diseases. this he related to limitation of family size, increased food supplies, improved nutrition and sanitation, specific preventive and therapeutic measures, and overall gains in quality of life for growing elements of the population. he cautioned against placing excessive reliance for health on medical care, much of which was of unproved effectiveness. this skepticism of the biomedical model of health care was part of wider antiestablishment feelings of the s and s in north america. in , milton roemer pointed out that the advent of vaccines, antibiotics, antihypertensives, and other medications contributed to great improvements in infant and child care, and in the management of infectious diseases, hypertension, diabetes, and other conditions. therapeutic gains continue to arrive from teaching centers around the world. vaccine, pharmaceutical, and diagnostic equipment manufacturers continue to provide important innovations that have major benefits, but also raise the cost of health care. the latter issue is one which has stimulated the search for reforms, and search for lower cost technologies such as in treatment of hepatitis c patients, a huge international public health issue. the value of medical care to public health and vice versa has not always been clear, either to public health personnel or to clinicians. the achievements of modern public health in controlling infectious diseases, and even more so in reducing the mortality and morbidity associated with chronic diseases such as stroke and chd, were in reality a shared achievement between clinical medicine and public health (see chapter ). preventive medicine has become part of all medical practice, with disease prevention through early diagnosis and health promotion through individual and community-focused activities. risk factor evaluation determines appropriate screening and individual and community-based interventions. medical care is crucial in controlling hypertension and in reducing the complications and mortality from chd. new modalities of treatment are reducing death rates from first time acute myocardial infarctions. better management of diabetes prevents the early onset of complications. at the same time, the contribution of public health to improving outcomes of medical care is equally important. control of the vaccine-preventable diseases, improved nutrition, and preparation for motherhood contribute to improved maternal and infant outcomes. promotions of reduced exposure to risk factors for chronic disease are a task shared by public health and clinical medical services. both clinical medicine and public health contribute to improved health status. they are interdependent and rely on funding systems for recognition as part of the new public health. during the s, many new management concepts emerged in the business community, such as "management by objective", a concept developed by peter drucker at general motors, with variants such as "zero-based budgeting" developed in the us department of defense (see chapter ). they focused the activities of an organization and its budget on targets, rather than on previous allocation of resources. these concepts were applied in other spheres, but they influenced thinking in health, whose professionals were seeking new ways to approach health planning. the logical application was to define health targets and to promote the efficient use of resources to achieve those targets. this occurred in the usa and soon afterwards in the who european region. in both cases, a wide-scale process of discussion and consensus building was used before reaching definitive targets. this process contributed to the adoption of the targets by many countries in europe as well as by states and many professional and consumer organizations. the usa developed national health objectives in for the year and subsequently for the year , with monitoring of progress in their achievement and development of further targets for and now for . beginning in , state health profiles are prepared by the epidemiology program office of the centers for disease control and prevention based on health indicators recommended by a consensus panel representing public health associations and organizations. the eight mdgs adopted by the un in include halving extreme poverty, reducing child mortality by twothirds, improving maternal health, halting the spread of hiv/aids, malaria, and other diseases, and providing universal primary education, all by the target date of . the mdgs form a common blueprint agreed to by all countries and the world's leading development institutions. the process has galvanized unprecedented efforts to meet the needs of the world's poorest, yet reviews of progress indicate that most developing nations will not meet the targets at current rates of progress. the united nations development programme (undp) global partnership for development report on the mdgs states that if the national development strategies and initiatives are supported by international development partners, the goals can be achieved by . the mdgs were adopted by over nations and provided guidance for national policies and for international aid agencies. the focus was on middle-and low-income countries and their achievements have been considerable but variable (see box . and chapter ) . as of july , extreme poverty was falling in every region, the poverty reduction target had been met, the world had met the target of halving the proportion of people without access to improved sources of water, and the world had achieved parity in primary education between girls and boys. further progress will require sustained political commitment to develop the primary care infrastructure: improved reporting and epidemiological monitoring, consultative mechanisms, and consensus by international agencies, national governments, and non-governmental agencies. the achievement of the targets will also require sustained international support and national commitment with all the difficulties of a time of economic recession. nevertheless, defining a target is crucial to the process. there are encouraging signs that national governments are influenced by the general movement to place greater emphasis on resource allocation and planning on primary care to achieve internationally recognized goals and targets. the successful elimination of smallpox, rising immunization coverage in the developing countries, and increasing implementation of salt iodization have shown that such goals are achievable. while the usa has not succeeded in developing universal health care access, it has a strong tradition of public health and health advocacy. federal, state, and local health authorities have worked out cooperative arrangements for financing and supervising public health and other services. with growing recognition in the s that medical services alone would not achieve better health results, health policy leadership in the federal government formulated a new approach, in the form of developing specific health targets for the nation. in , the surgeon general of the usa published the report on health promotion and disease prevention (healthy people). this document set five overall health goals for each of the major age groups for the year , accompanied by specific health objectives. new targets for the year were developed in three broad areas: to increase healthy lifespans, to reduce health disparities, and to achieve access to preventive health care for all americans. these broad goals are supported by specific targets in health priority areas, each one divided into four major categories: health promotion, health protection, preventive services, and surveillance systems. this set the public health agenda on the basis of measurable indicators that can be assessed year by year. reduce child mortality -progress on child mortality is gaining momentum. the target is to reduce by two-thirds, between and , the under- -year-old mortality rate, from children of every dying to of every . child deaths are falling, but much more needs to be done in order to reach the development goal. revitalizing efforts against pneumonia and diarrhea, while bolstering nutrition, could save millions of children. l mdg . improve maternal health -maternal mortality has nearly halved since , but levels are far removed from the target. the targets for improving maternal health include reducing by three-quarters the maternal mortality ratio and achieve universal access to reproductive health. poverty and lack of education perpetuate high adolescent birth rates. inadequate funding for family planning is a major failure in fulfilling commitments to improving women's reproductive health. l mdg . combat hiv/aids, malaria, tuberculosis, and other diseases -more people than ever are living with hiv owing to fewer aids-related deaths and the continued large number of new infections. in , an estimated . million were living with hiv, up percent from . this persistent increase reflects the continued large number of new infections along with a significant expansion of access to lifesaving antiretroviral therapy, especially in more recent years. l mdg . ensure environmental sustainability -the unparalleled success of the montreal protocol shows that action on climate change is within grasp. the th anniversary of the montreal protocol on substances that deplete the ozone layer, in , had many achievements to celebrate. most notably, there has been a reduction of over percent in the consumption of ozone-depleting substances. further, because most of these substances are also potent greenhouse gases, the montreal protocol has contributed significantly to the protection of the global climate system. the reductions achieved to date leave hydrochlorofluorocarbons (hcfcs) as the largest group of substances remaining to be phased out. l mdg . a global partnership for development -core development aid fell in real terms for the first time in more than a decade, as donor countries faced fiscal constraints. in , net aid disbursements amounted to $ . billion, representing . percent of developed countries' combined national income. while constituting an increase in absolute dollars, this was a . percent drop in real terms over . if debt relief and humanitarian aid are excluded, bilateral aid for development programmes and projects fell by . percent in real terms. equitable and sustainable funding of health services. . developing human resources (educational programs for providers and managers based on the principles of the health for all policy). . research and knowledge: health programs based on scientific evidence. . mobilizing partners for health (engaging the media/ television/internet). . policies and strategies for health for all -national, targeted policies based on health for all. a - review has been commissioned by the european office of the who to assess inequalities in the social determinants of health. while health has improved there are still significant inequalities. factors include variance in local, regional, national, and global economic forces. the european union and the european region of who are both working on health targets for the year . there are competing demands in society for expenditure by the government, and therefore making the best use of resources -money and people -is an important objective. the uk has devolved many of the responsibilities to the constituent countries (england, wales, scotland, and northern ireland) within an overall national framework (box . ). of the health consequences of their decisions and to accept responsibility for health. health promotion policy combines diverse but complementary approaches, including legislation, fiscal measures, taxation, and organizational change. it is a coordinated action that leads to health, income, and social policies that foster greater equity. joint action contributes to ensuring safer and healthier goods and services, healthier public services, and cleaner, more enjoyable environments. health promotion policies require the identification of obstacles to the adoption of healthy public policies in non-health sectors, and ways of removing them. built on progress made from the declaration on primary health care at alma-ata, the aim was to make the healthier choice the easier choice for policy makers as well. the logo of the ottawa charter has been maintained by the who as the symbol and logo of health promotion. health promotion represents activities to enhance and embed the concept of building healthy public policy through: l building healthy public policy in all sectors and levels of government and society l enhancing both self help and social support l developing personal skills through information and education for health l enabling, mediating, and advocating healthy public policy in all spheres l creating supportive environments of mutual help and conservation of the natural environment l reorienting health services beyond providing clinical curative services with linkage to broader social, political, economic, and physical environmental components. (adapted from ottawa charter; health and welfare canada and world health organization, ) an effective approach to health promotion was developed in australia where, in the state of victoria, revenue from a cigarette tax has been set aside for health promotion purposes. this has the effect of discouraging smoking, and at the same time finances health promotion activities and provides a focus for health advocacy in terms of promoting cessation of cigarette advertising at sports events or on television. it also allows for assistance to community groups and local authorities to develop health promotion activities at the workplace, in schools, and at places of recreation. health activity in the workplace involves reduction of work hazards as well as promotion of a healthy diet and physical fitness, and avoidance of risk factors such as smoking and alcohol abuse. in the australian model, health promotion is not only the persuasion of people to change their life habits; it also involves legislation and enforcement towards environmental changes that promote health. for example, this involves mandatory filtration, chlorination, and fluoridation for community water supplies, vitamin and mineral enrichment of basic foods. primary care alliances of service providers are organized including hospitals, community health services serving a sub-district population for more efficient and comprehensive care. these are at the level of national or state policy, and are vital to a health promotion program and local community action. community-based programs to reduce chronic disease using the concept of community-wide health promotion have developed in a wide variety of settings. such a program to reduce risk factors for cardiovascular disease was pioneered in the north karelia project in finland. this project was initiated as a result of pressures from the affected population of the province, which was aware of the high incidence of mortality from heart disease. finland had the highest rates of chd in the world and in the rural area of north karelia the rate was even higher than the national average. the project was a regional effort involving all levels of society, including official and voluntary organizations, to try to reduce risk factors for chd. after years of follow-up, there was a substantial decline in mortality with a similar decline in a neighboring province taken for comparison, although the decline began earlier in north karelia. in many areas where health promotion has been attempted as a strategy, community-wide activity has developed with participation of ngos or any valid community group as initiators or participants. healthy heart programs have developed widely with health fairs, sponsored by charitable or fraternal societies, schools, or church groups, to provide a focus for leadership in program development. a wider approach to addressing health problems in the community has developed into an international movement of "healthy cities". following deliberations of the health of towns commission chaired by edwin chadwick, the health of towns association was founded in by southwood smith, a prominent reform leader of the sanitary movement, to advocate change to reduce the terrible living conditions of much of the population of cities in the uk. the association established branches in many cities and promoted sanitary legislation and public awareness of the "sanitary idea" that overcrowding, inadequate sanitation, and absence of safe water and food created the conditions under which epidemic disease could thrive. in the s, iona kickbush, trevor hancock, and others promoted renewal of the idea that local authorities have a responsibility to build health issues into their planning and development processes. this "healthy cities" approach promotes urban community action on a broad front of health promotion issues (table . ). activities include environmental projects (such as recycling of waste products), improved recreational facilities for young people to reduce violence and drug abuse, health fairs to promote health awareness, and screening programs for hypertension, breast cancer, and other diseases. it combines health promotion with consumerism and returns to the tradition of local public health action and advocacy. the municipality, in conjunction with many ngos, develops a consultative process and program development approach to improving the physical and social life of the urban environment and the health of the population. in , the healthy cities movement involved countries with cities in europe, canada, the usa, the uk, south america, israel, and australia, an increase from cities in . the model now extends to small municipalities, often with populations of fewer than , . networks of healthy cities are the backbone of the movement, with more than member towns and cities across europe. the choice of core themes offers the opportunity to work on priority urban health issues that are relevant to all european cities. topics that are of particular concern to individual cities and/or are challenging and cutting edge for innovative public health action are especially emphasized. healthy cities encourages and supports experimentation with new ideas by developing concepts and implementing them in diverse organizational contexts. a healthy city is a city for all its citizens: inclusive, supportive, sensitive and responsive to their diverse needs and expectations. a healthy city provides conditions and opportunities that encourage, enable and support healthy lifestyles for people of all social groups and ages. a healthy city offers a physical and built environment that encourages, enables and supports health, recreation and well-being, safety, social interaction, accessibility and mobility, a sense of pride and cultural identity and is responsive to the needs of all its citizens. the apha's formulation of the public health role in , entitled the future of public health in america, was presented at the annual meeting in . the apha periodically revises standards and guidelines for organized public health services provided by federal, state, and local governments ( table . ). these reflect the profession of public health as envisioned in the usa where access to medical care is limited for large numbers of the population because of a lack of universal health insurance. public health in the usa has been very innovative in determining risk groups in need of special care and finding direct and indirect methods of meeting those needs. european countries such as finland have called for setting public health into all public policy, which reflects the vital role that local and county governments can play in developing health-oriented policies. these include policies in housing, recreation, regulation of industrial pollution, road safety, promotion of smoke-free environments, bicycle paths, health impact assessment, and many other applications of health principles in public policy. public health involves both direct and indirect approaches. direct measures in public health include immunization of children, modern birth control, and chronic disease case finding -hypertension, diabetes, and cancer. indirect methods used in public health protect the individual by community-wide means, such as raising standards of environmental safety, ensuring a safe water supply, sewage disposal, and improved nutrition (box . ). in public health practice, the direct and indirect pproaches are both relevant. to reduce morbidity and mortality from diarrheal diseases requires an adequate supply of safe water and waste disposal, and also education of the individual in hygiene and the mother in use of ort, and rotavirus vaccination of all children. the targets of public health action therefore include the individual, family, community, region, or nation, as well as a functioning and health system adopting current best practices for health care and health protection. the targets for protection in infectious disease control are both the individual and the total group at risk. for vaccine-preventable diseases, immunization protects the individual but also has an indirect effect by reducing the risk even for non-immunized persons. in control of some diseases, individual case finding and management reduce risk of the disease in others and the community. for example, tb requires case finding and adequate care among high-risk groups as a key to community control. in malaria control, case finding and treatment are essential together with environmental action to reduce the vector population, to prevent transmission of the organism by the mosquito to a new host. control of ncds, where there is no vaccine for mass application, depends on the knowledge, attitudes, beliefs, and practices of individuals at risk. in this case, the social context is of importance, as is the quality of care to which the individual has access. control and prevention of noninfectious diseases involve strategies using individual and population-based methods. individual or clinical measures include professional advice on how best to reduce the risk of the disease by early diagnosis and implementation of appropriate therapy. population-based measures involve indirect measures with government action banning cigarette advertising, or direct taxation on cigarettes. mandating food quality standards, such as limiting the fat content of meat, and requiring food labeling laws are part of the control of cardiovascular diseases. the way individuals act is central to the objective of reducing disease, because many non-infectious diseases are dependent on behavioral risk factors of the individual's choosing. changing the behavior of the individual means addressing the way a person sees his or her own needs. this can be influenced by the provision of information, but how someone sees his or her own needs is more complex than that. an individual may define needs differently from the society or the health system. reducing smoking among women may be difficult to achieve if smoking is thought to reduce appetite and food intake, given the social message that "slim is beautiful". reducing smoking among young people is similarly difficult if smoking is seen as fashionable and diseases such as lung cancer seem very remote. recognizing how individuals define needs helps the health system to design programs that influence behavior that is associated with disease. public health has become linked to wider issues as health care systems are reformed to take on both individual and population-based approaches. public health and mainstream medicine have found increasingly common ground in addressing the issues of chronic disease, growing attention to health promotion, and economics-driven health care reform. at the same time, the social ecology approaches have shown success in slowing major causes of disease, including heart disease and aids, and the biomedical sciences have provided major new technology for preventing major health problems, including cancer, heart disease, genetic disorders, and infectious diseases. technological innovations unheard of just a few years ago are now commonplace, in some cases driving up costs of care and in others replacing older and less effective care. at the same time, resistance of important pathogenic microorganisms to antibiotics and pesticides is producing new challenges from diseases once thought to be under control, and newly emerging infectious diseases challenge the entire health community. new generations of antibiotics, antidepressants, antihypertensive medications, and other treatment methods are changing the way many conditions are treated. research and development in the biomedical to improve the quality of public health practice and performance of public health systems sciences are providing means of prevention and treatment that profoundly affect disease patterns where they are effectively applied. the technological and organizational revolutions in health care are accompanied by many ethical, economic, and legal dilemmas. the choices in health care include heart transplantation, an expensive life-saving procedure, which may compete with provision of funds and labor resources for immunizations for poor children or for health promotion to reduce smoking and other risk factors for chronic disease. new means of detecting and treating acute conditions such as myocardial infarction and peptic ulcers are reducing hospital stays, and improving long-term survival and quality of life. imaging technology has been an important development in medicine since the advent of x-rays in the early twentieth century. technology has forged ahead with high-technology instruments and procedures, new medication, genetic engineering, and important low-technology gains such as impregnated bed nets, simplified tests for hiv and tb, and many other "game changers". new technologies that can enable lower cost diagnostic devices, electronic transmission, and distant reading of transmitted imaging all open up possibilities for advanced diagnostic capacities in rural and less developed countries and communities. molecular biology has provided methods of identifying and tracking movement of viruses such as polio and measles from place to place, greatly expanding the potential for appropriate intervention. the choices in resource allocation can be difficult. in part, these add political commitment to improve health, competent professionally trained public health personnel, the public's level of health information, and legal protection, whether through individuals, advocacy, or regulatory approaches for patients' rights. these are factors in a widening methodology of public health. the centers for disease control and prevention (morbidity and mortality weekly report) in summarized great achievements of public health in the usa, with an extension of the lifespan by over years and improvements in many measures of quality of life. they were updated in a similar summary report in , showing continuous progress, and a global version which was also encouraging in its scope of progress (table . ). these achievements were also seen in all developed countries over the past century and are beginning to be seen in developing countries as well. they reflect a successful application of a broad approach to prevention and health promotion along with improved medical care and growing access to its benefits. in the past several decades alone, major new innovations are leading to greater control of cardiovascular disease, cancer prevention, and many other improvements to health affecting hundreds of millions of people. a similar report by the cdc shows global progress in the first decade of the twenty-first century, while mdg reports show progress on all eight target topics, although not at uniformly satisfactory rates. these achievements are discussed throughout this text. this successful track record is very much at the center of a new public health involving a wide range of programs and activities, shown to be feasible and benefiting from continuing advances in science and understanding of social and management issues affecting health care systems worldwide. public health issues have received new recognition in recent years because of a number of factors, including a growing understanding among the populace at different levels in different countries that health behavior is a factor in health status and that public health is vital for protection against natural or human-made disasters. the challenges are also increasingly understood: preparation for bioterrorism, avian influenza, rising rates of diabetes and obesity, high mortality rates from cancer, and a wish for prevention to be effective. health systems offer general population benefits that go beyond preventing and treating illness. appropriately designed and managed, they: l provide a vehicle to improve people's lives, protecting them from the vulnerability of sickness, generating a sense of life security, and building common purpose within society l ensure that all population groups are included in the processes and benefits of socioeconomic development l generate the political support needed to sustain them over time. health systems promote health equity when their design and management specifically consider the circumstances and needs of socially disadvantaged and marginalized populations, including women, the poor, and groups who experience stigma and discrimination, enabling social action by these groups and the civil society organizations supporting them. health systems can, when appropriately designed and managed, contribute to achieving the millennium development goals. the mdgs selected by the un in have eight global targets for the year , including four directly related to public health (discussed above, box . ). these are a recognition and a challenge to the international community and public health as a profession and as organized systems. formal education in newly developing schools of public health is increasing in europe, including many countries of eastern europe, and beginning to develop in india and sub-saharan africa. but there is delay in establishing centers of postgraduate education and research in many developing countries which are concentrating their educational resources on training physicians. many physicians from developing nations are moving to the developed countries, which have become dependent on these countries for a significant part of their supply of medical doctors. progress in implementation of the mdgs is mixed in sub-saharan africa, making some progress in immunization, but falling back on other goals. proposals to renew global health targets following the end-stage of the mdg health goals will need to add a focus on ncds, which account for percent of global deaths, including . million premature deaths below the age of (undp). economic growth has been hampered by the global recession since , which will affect continued progress with many other factors of changing population dynamics, the economics of prevention versus expensive treatment costs, and the high costs of health care. environmental degradation with high levels of carbon dioxide contamination is a growing concern, with disastrous global warming and consequent effects of drought, flooding, hurricane, and elevated particulate matter-induced asthma and effects on cardiovascular disease. the potential for the development of basic and medical sciences in genetics, nanotechnology, and molecular biology shows enormous promise for health benefits as yet unimagined. at the same time, the effectiveness of health promotion has shown dramatic successes in reducing the toll of aids, reducing smoking, and increasing consciousness of nutrition and physical fitness in the population, and of the tragic effects of poverty and poor education on health status. the ethics of public health issues are complex and changing with awareness that failure to act on strong evidence-based policies is itself ethically problematic. the future of public health is not as a solo professional sector; it is at the heart of health systems, without which societies are open to chronic and infectious diseases that are preventable, affecting the society as a whole in economic and development matters. there is an expanding role of private donors in global health efforts, such as the rotary club and the polio eradication program, gavi with immunization and bed-nets in sub-saharan africa, and bilateral donor countries' help in reducing the toll of aids in sub-saharan africa. the new public health has emerged as a concept to meet a whole new set of conditions, associated with increasing longevity and aging of the population, with the post-world war ii baby-boom generation reaching the over- age group facing the growing importance of chronic diseases. inequalities in health exist in and between affluent and developing societies, as well as within countries, even those having advanced health care systems. regional inequalities are seen across the european region in an east-west gradient and globally a north-south divide of extremes of inequality. the global environmental and ecological degradation and pollution of air and water present grave challenges for developed and developing countries worldwide. yet optimism can be derived from proven track records of success in public health measures that have already been implemented. many of the underlying factors are amenable to prevention through social, environmental, or behavioral change and effective use of medical care. the new public health idea has evolved since alma-ata, which articulated the concept of health for all, followed by a trend in the late s to health in all policies and establishing health targets as a basis for health planning. during the late s and early s, the debate on the future of public health in the americas intensified as health professionals looked for new models and approaches to public health research, training, and practice. this debate helped to redefine traditional approaches of social, community, and preventive medicine. the search for the "new" in public health continued with a return to the health for all concept of alma-ata (renewed in ) and a growing realization that the health of both the individual and the society involves the management of personal care services and community prevention, with a comprehensive approach taking advantage of advancing technology and experience of best practices globally. the new public health is an extension of the traditional public health. it describes organized efforts of society to develop healthy public policies: to promote health, to prevent disease, and to foster social equity within a framework of sustainable development. a new, revitalized public health must continue to fulfill the traditional functions of sanitation, protection, and related regulatory activities, but in addition to its expanded functions. it is a widened philosophy and practical application of many different methods of addressing health, and preventing disease and avoidable death. it necessarily addresses inequities so that programs need to meet special needs of different groups in the population according to best standards, limited resources, and population needs. it is proactive and advocates interventions within legal and ethical limits to promote health as a value in and of itself and as an economic gain for society as well for its individual members. the new public health is a comprehensive approach to protecting and promoting the health status of the individual and the society, based on a balance of sanitary, environmental, health promotion, personal, and community-oriented preventive services, coordinated with a wide range of curative, rehabilitative, and long-term care services. it evolves with new science, technology, and knowledge of human and systems behavior to maximize health gains for the individual and the population. the new public health requires an organized context of national, regional, and local governmental and non-governmental programs with the object of creating healthful social, nutritional, and physical environmental conditions. the content, quality, organization, and management of component services and programs are all vital to its successful implementation. whether managed in a diffused or centralized structure, the new public health requires a systems approach acting towards achievement of defined objectives and specified targets. the new public health works through many channels to promote better health. these include all levels of government and parallel ministries; groups promoting advocacy, academic, professional, and consumer interests; private and public enterprises; insurance, pharmaceutical, and medical products industries; the farming and food industries; media, entertainment, and sports industries; legislative and law enforcement agencies; and others. the new public health is based on responsibility and accountability for defined populations in which financial systems promote achievement of these targets through effective and efficient management, and cost-effective use of financial, human, and other resources. it requires continuous monitoring of epidemiological, economic, and social aspects of health status as an integral part of the process of management, evaluation, and planning for improved health. the new public health provides a framework for industrialized and developing countries, as well as countries in political-economic transition such as those of the former soviet system. they are at different stages of economic, epidemiological, and sociopolitical development, each attempting to ensure adequate health for its population with limited resources. the challenges are many, and affect all countries with differing balances, but there is a common need to seek better survival and quality of life for their citizens (table . ). the object of public health, like that of clinical medicine, is better health for the individual and for society. public health works to achieve this through indirect methods, such as by improving the environment, or through direct means such as preventive care for mothers and infants or other atrisk groups. clinical care focuses directly on the individual patient, mostly at the time of illness. but the health of the individual depends on the health promotion and social programs of the society, just as the well-being of a society depends on the health of its citizens. the new public health consists of a wide range of programs and activities that link individual and societal health. the "old" public health was concerned largely with the consequences of unhealthy settlements and with safety of food, air, and water. it also targeted the infectious, toxic, and traumatic causes of death, which predominated among young people and were associated with poverty. a summary of the great achievements of public health in the twentieth and in the early twenty-first century in the industrialized world is included in chapter and throughout this text. these achievements are reflective of public health gains throughout the industrialized world and are encourage and leverage national, state, and local partnerships to build a stronger foundation for public health preparedness and investigate health problems and health hazards in the community . inform, educate, and empower people about health issues . mobilize community partnerships to identify and solve health problems . develop policies and plans that support individual and community health efforts . enforce laws and regulations that protect health and ensure safety evaluate effectiveness, accessibility, and quality of personal and population-based health services vision, mission and goals guidelines on food fortification with micronutrients. who, geneva. alliance for health policy and systems research essential public health services healthy communities, . model standards for community attainment of the year national health objectives determinants of adult mortality in russia: estimates from sibling data commission on social determinants and health. closing the gap in a generation: health equity through action on the social determinants of health compression of morbidity in the elderly institute of medicine. who will keep the public healthy? educating public health professionals for the st century global alliance for vaccine and immunization (gavi) chronic disease prevention and the new public health the evolution, impact and significance of healthy cities/healthy communities world health organization. ottawa charter for health promotion: an international conference on health promotion behavioral and social sciences and public health at cdc. mmwr health in all policies: seizing opportunities, implementing policies. ministry of social affairs and health new perspectives on the health of canadians: a working document new perspective on the health of canadians: years later the us healthy people initiative: its genesis and its sustainability mortality from cardiovascular and cerebrovascular diseases in europe and other areas of the world: an update strategic review of health inequalities in england post. department of health primary care (extended version): ten key actions could globally ensure a basic human right at almost unnoticeable cost public health in europe: power, politics, and where next health: a vital investment for economic development in eastern europe and central asia. european observatory on health systems and policies. who, european regional office it is not just the broad street pump addressing the epidemiologic transition in the former soviet union: strategies for health systems and public health reform in russia what is the "new public health"? millenium development goals: progress chart united nations development programme, millennium development goals. eight goals for healthy people healthy people. the surgeon general's report on health promotion and disease prevention the millennium development goals: a cross-sectoral analysis and principles for goal-setting after selective primary health care: an interim strategy for disease control in developing countries declaration of alma-ata. international conference on primary health care healthy cities networks across the who, european region preamble to the constitution of the world health organization as adopted by the international health conference regional office for europe. health -health for all in the st century. who regional office for europe, copenhagen. world health organization, . regional office for europe. who european healthy cities network. available at:. who regional office for europe leading health indicators selected for incorporate the original objectives in healthy people , which served as a basis for planning public health activities for many state and community health initiatives. for each of the leading health indicators, specific objectives and subobjectives derived from healthy people are used to monitor progress. the specific objectives set for healthy people are listed in box . . thirteen new topic areas are listed for , such as older adults, genomics, dementias, and social determinants of health. these provide guidelines for national, state, and local public health agencies as well as insurance providers, primary care services, and health promotion advocates. a key issue will be in reducing regional, ethnic, and socioeconomic health disparities.the process of working towards health targets in the usa has moved down from the federal level of government to the state and local levels. professional organizations, ngos, as well as community and fraternal organizations are also involved. the states are encouraged to prepare their own targets and implementation plans as a condition for federal grants, and many states require county health departments to prepare local profiles and targets.diffusion of this approach encourages state and local initiatives to meet measurable program targets. it also sets a different agenda for local prestige in competitive terms, with less emphasis on the size of the local hospital or other agencies than on having the lowest infant mortality or the least infectious disease among neighboring local authorities. the who european region document "health -health for all in the st century" addresses health in the twentyfirst century, with principles and objectives for improving the health of europeans, within and between countries of europe. the health targets include: . closing the health gap between countries. . closing the health gap within countries. . a healthy start in life (supportive family policies). . health of young people (policies to reduce child abuse, accidents, drug use, and unwanted pregnancies). . healthy aging (policies to improve health, self-esteem, and independence before dependence emerges). . improving mental health. . reducing communicable diseases. . reducing non-communicable diseases. . reducing injury from violence and accidents. . a healthy and safe physical environment. . healthier living (fiscal, agricultural, and retail policies that increase the availability of and access to and consumption of vegetables and fruits). . reducing harm from alcohol, drugs, and tobacco. . a settings approach to health action (homes should be designed and built in a manner conducive to sustainable health and the environment). . multisectoral responsibility for health. . an integrated health sector and much stronger emphasis on primary care. . managing for quality of care using the european health for all indicators to focus on outcomes and compare the effectiveness of different inputs. the uk national health service (nhs) has semi-autonomous units in england, scotland, wales, and northern ireland. they are funded from the central uk nhs but with autonomy within national guidelines. the nhs has defined national health outcomes for improvements grouped around five domains, each comprised of key indicators aimed at improving health with reducing inequalities. l preventing people from dying prematurely from causes amenable to health care for all ages: l the target diseases include cardiovascular, respiratory, and liver diseases, and cancer (with focus on cancer of breast, lung, and colorectal cancer) l reducing premature death in people with serious mental illnesses l reducing infant mortality, neonatal mortality, still births, and deaths in young children l increasing -year survival for children with cancer. health improvement; help people to live healthy lifestyles, healthy choices, reduce health inequalities, protection from major incidents and other threats, while reducing health inequalities. l health care, public health and preventing premature mortality; reduce the numbers of people living with preventable ill-health and people dying prematurely, while reducing the gap between communities.source: uk department of health. available at: https://www.gov.uk/government/organisations/department-of-health/about#our-priorities, https:// www.gov.uk/government/uploads/system/uploads/attachment_data/ file/ /improving-outcomes-and -supporting-transparency-part- a.pdf. pdf, and https://www.gov.uk/government/uploads/system/uploads/attach-ment_data/file/ / -nhs-outcomes-framework- - .pdf. pdf [accessed june ] . national policy in health ultimately relates to health of the individual. the various concepts outlined in the health field concept, community-oriented primary health care, health targets, and effective management of health systems, can only be effective if the individual and his or her community are knowledgeable participants in seeking solutions. involving the individual in his or her own health status requires raising levels of awareness, knowledge, and action. the methods used to achieve these goals include health counseling, health education, and health promotion (figure . ).health counseling has always been a part of health care between the doctor or nurse and the patient. it raises levels of awareness of health issues of the individual patient. health education has long been part of public health, dealing with promoting consciousness of health issues in selected target population groups. health promotion incorporates the work of health education but takes health issues to the policy level of government and involves all levels of government and ngos in a more comprehensive approach to a healthier environment and personal lifestyles.health counseling, health education, and health promotion are among the most cost-effective interventions for improving the health of the public. while costs of health care are rising rapidly, demands to control cost increases should lead to greater emphasis on prevention, and adoption of health education and promotion as an integral part of modern life. this should be carried out in schools, the workplace, the community, commercial locations (e.g., shopping centers), and recreation centers, and in the political agenda.psychologist abraham maslow described a hierarchy of needs of human beings. every human has basic requirements including physiological needs of safety, water, food, warmth, and shelter. higher levels of needs include recognition, community, and self-fulfillment. these insights supported observations of efficiency studies such as those of elton mayo in the famous hawthorne effect in the s, showing that workers increased productivity when acknowledged by management in the objectives of the organization (see chapter ). in health terms, these translate into factors that motivate people to positive health activities when all barriers to health care are reduced.modern public health faces the problem of motivating people to change behavior; sometimes this requires legislation, enforcement, and penalties for failure to comply, such as in mandating car seat-belt use. in other circumstances it requires sustained performance by the individual, such as the use of condoms to reduce the risk of sti and/or hiv transmission. over time, this has been developed into a concept known as knowledge, attitudes, beliefs, and practices (kabp), a measurable complex that cumulatively affects health behavior (see chapter ). there is often a divergence between knowledge and practice; for example, the knowledge of the importance of safe driving, yet not putting this into practice. this concept is sometimes referred to as the "kabp gap". the health belief model has been a basis for health education programs, whereby a person's readiness to take action for health stems from a perceived threat of disease, a recognition of susceptibility to disease and its potential severity, and the value of health. action by an individual may be triggered by concern and by knowledge. barriers to appropriate action may be psychological, financial, or physical, including fear, time loss, and inconvenience. spurring action to avoid risk to health is one of the fundamental goals in modern health care. the health belief model is important in defining any health intervention in that it addresses the emotional, intellectual, and other barriers to taking steps to prevent or treat disease.health awareness at the community and individual levels depends on basic education levels. mothers in developing countries with primary or secondary school education are more successful in infant and child care than less educated women. agricultural and health extension services reaching out to poor and uneducated farm families in north america in the s were able to raise consciousness of safe self-health practices and good nutrition, and when this was supplemented by basic health education in schools, generational differences could be seen in levels of awareness of the importance of balanced nutrition. secondary prevention with diabetics and patients with chd hinges on education and awareness of nutritional and physical activity patterns needed to prevent or delay a subsequent myocardial infarction. the who sponsored the first international conference on health promotion held in ottawa, canada, in ( figure . ) . the resulting ottawa charter defined health promotion and set out five key areas of action: building healthy public policy, creating supportive environments, strengthening community action, developing personal skills, and reorienting health services. the ottawa charter called on all countries to put health on the agenda of policy makers in all sectors and at all levels, directing them to be aware a typical healthy city has a population in the multiple thousands, often multilingual, with an average middleclass income. a healthy cities project builds a coalition of municipal and voluntary groups working together in a continuing effort to improve quality of service, facilities, and living environment. the city is divided into neighborhoods, engaged in a wide range of activities fostered by the project. municipalities have traditionally had a leading role in sanitation, safe water supply, building and zoning laws and regulation, and many other responsibilities in public health (see chapter ). the healthy cities or communities movement has elevated this to a higher level with policies to promote health in all actions. some examples are listed of municipal, advocacy group, and higher governmental activities for healthier city environments: working with senior levels of government, other departments in the municipalities, religious organizations, private donors, and the ngo sector to innovate and especially to improve conditions in poverty-afflicted areas of cities is a vital role for health-oriented local political leadership. human ecology, a term introduced in the s and revived in the s, attempted to apply theory from plant and animal life to human communities. it evolved as a branch of demography, sociology, and anthropology, addressing the social and cultural contexts of disease, health risks, and human behavior. human ecology addresses the interaction of humans with and adaptation to their social and physical environment.parallel subdisciplines of social, community, and environmental psychology, medical sociology, anthropology, and other social sciences contributed to the development of this academic field with wide applications in health-related issues. this led to the incorporation of qualitative research methods alongside the quantitative research methods traditionally emphasized in public health, providing crucial insights into many public health issues where human behavior is a key risk factor.health education developed as a discipline and function within public health systems in school health, rural nutrition, military medicine, occupational health, and many other aspects of preventive-oriented health care, and is discussed in later chapters of this text. directed at behavior modification through information and raising awareness of consequences of risk behavior, this has become a longstanding and major element of public health practice in recent times, being almost the only effective tool to fight the epidemic of hiv and the rising epidemic of obesity and diabetes.health promotion as an idea evolved, in part, from marc lalonde's health field concepts and from growing realization in the s that access to medical care was necessary but not sufficient to improve the health of a population. the integration of the health behavior model, social ecological approach, environmental enhancement, or social engineering formed the basis of the social ecology approach to defining and addressing health issues (table . ).individual behavior depends on many surrounding factors, while community health also relies on the individual; the two cannot be isolated from one another. the ecological perspective in health promotion works towards changing people's behavior to enhance health. it takes into account factors not related to individual behavior, which are determined by the political, social, and economic environment. it applies broad community, regional, or national approaches that are needed to address severe public health problems, such as controlling hiv infection, tb, malnutrition, stis, cardiovascular disorders, violence and trauma, and cancer. beginning to affect the health situation in countries in transition from the socialist period. countries emerging from developing status are also showing signs of mixed progress in the dual burden of infectious and maternal/child health issues, along with growing exposure to the chronic diseases of developed nations such as cardiovascular diseases, obesity, and diabetes. the new public health synthesizes traditional pub lic health with management of personal services and community action for a holistic approach. evaluation of costeffective public health and medical interventions to reduce the burden of disease also contributes to the need to seek and apply new approaches to health. the new public health will continue to evolve as a framework drawing on new ideas, science, technology, and experiences in public health throughout the world. it must address the growing recognition of social inequality in health, even in developed countries with universal health programs with improved education and social support systems. for a complete bibliography and guidance for student reviews and expected competencies please see companion web site at http://booksite.elsevier.com/ bibliography key: cord- -k w h authors: carr, paul r. title: shooting yourself first in the foot, then in the head: normative democracy is suffocating, and then the coronavirus came to light date: - - journal: postdigit sci educ doi: . /s - - - sha: doc_id: cord_uid: k w h this text starts with the premise that ‘normative democracy’ has rendered our societies vulnerable and burdened with unaddressed social inequalities. i highlight three central arguments: ( ) social media, and, consequently, citizen engagement are becoming a significant filter that can potentially re-imagine the political, economic, and social worlds, which increasingly bleed over to how we might develop and engage with ‘democracy’; to this end, i introduce a brief case study on the nefarious interpretation of the killing of jamal khashoggi in to underscore the tension points in normative democracy; ( ) capitalism, or neoliberalism, needs to be more fully exposed, interrogated, and confronted if ‘normative, representative, hegemonic, electoral democracy’ is to be re-considered, re-imagined, and re-invented; the perpetuation of social inequalities lays bare the frailty of normative democratic institutions; ( ) covid- has exposed the fault lines and fissures of normative democracy, illustrating here the ‘common sense’ ways that power imbalances are sustained, which leaves little room for social solidarity; i present herein the case of the economic/labor dynamic in quebec during the coronavirus. ultimately, i believe the quest to re-imagine a more meaningful, critically engaged democracy, especially during a context that is imbued with a political, economic, and public health crisis, cannot be delayed much longer. ; thésée, carr, duclos, and potwora ) , and contextualizes how we think (and act) about the subject. to further clarify this context of how normative democracy manifests itself, i highlight the following examples from my research projects with colleagues over the years, which involved studies in some countries with roughly teacher education and educator participants (carr and thésée ) : & we found that the vast majority did not have a robust, significant democratic experience in their own education; & that this has affected how they consider democracy and education; & that social justice is, for many, a difficult and problematic area to cultivate in and through education owing to a weakly asserted, structured, and supported institutional culture based on normative democracy; & that most considered that the space for inclusive and critical engagement in and through education is constrained, limited, and fraught with obstacles; & that racialized participants had significantly higher levels of experience, conscientization, and engagement with, for example, racism, antiracism, and efforts to address racial inequities, which further underscores how normative democracy closes down fundamental debate, dialog, teaching and learning as well as transformative education while presenting the posture and framework of democracy. when democracy and education are considered to be naturally disconnected while not leaving room for a more critically engaged democracy, it is not difficult to imagine the suffocating nature of normative democracy. normative elections, the ones that have been so effectively presented by the usa as the backbone to any meaningful democracy, have been jettisoned into a cesspit of turmoil and intractable debate that often neglects problematizing some of the most intractable and germane issues (achen and bartels ; howe ; torcal and ramon montero ) . not everyone involved in elections is corrupt or corrupted, or is afflicted with unsightly motivations, and people who go to the polls are not simply sheep being led to the proverbial slaughterhouse. there is a great deal of complexity as to why we vote and why we hope that there will be some hope in participating in mainstream democracy, but the faith in electoral democracy is waning almost everywhere (torcal and ramon montero ; carr and thésée ). yet, these normative elections, which are often ordered to measure with the threat of massive (real and rhetorical) carpet-bombing and worse, if not realized, are replete with all kinds of paradoxical anti-democratic maneuvers, starting with who can be elected, how much money plays into the process, how media can control and shape the message, manipulation, and diversion is a fundamental component, how seeking to win is more a priority than seeking to build a meaningful democracy, and how capitalism is the enormous, indelicate, meandering proverbial pound gorilla in the room (amico ; carr and thésée ) . added to this is the role, the purpose, and place of education in supporting, cultivating, and building a critically engaged democracy as well as critically engaged citizen participation. it is extremely difficult to have one without the other (democracy without education, for example, or, rather, meaningful, critically engaged democracy without meaningful, critically engaged education). (see carr , and carr and thésée , as well as the unesco chair dcmÉt website at uqo.ca/dcmet/ for an archive of publications.). and then, starting in late , the world started to feel the indelible, intractable, and (in)visible perturbations of the coronavirus, which emanated in china, and has quickly disseminated throughout all regions, making it a global pandemic. the number of people affected, contracting the virus, and ultimately succumbing to it, is increasing daily at this time but there is much analysis and data-crunching indicating that, in many areas, after several weeks of self-distancing, hygienic measures, increasing testing, closing down all but 'essential services', and enhancing medical and health care measures, the 'curve' may be flattening. however, few people believe that the virus will disappear, nor that the cost, in terms of human life, will be entirely negligible. so what is the connection to democracy, capitalism (or perhaps more correctly neoliberalism), and covid- ? the vulnerabilities, inequalities, and fault lines that existed prior to the coronavirus have been exacerbated, and the virus has disproportionately impacted racialized, marginalized, and lower income communities. the contraction and death rates are higher, and the economic, labor, living, and social conditions have worsened, notably for already vulnerable communities. this pandemic, sadly, provides a tremendous and significant impetus to re-consider and re-calibrate our thinking around democracy (diamond ; roy ) . this text starts with the premise that 'normative democracy' has put us in a pickle, and that, although there are ways out if it, this will require breaking out of the glass box that has a great many of us believing that there is no alternative. i highlight three points related to democracy in this text, formulating the following central arguments: ) social media and, consequently, citizen engagement are becoming a significant filter that can potentially re-imagine the political, economic, and social worlds (outside of and beyond normative democracy), which increasingly bleed over to how we might develop and engage with 'democracy' (garrett ) ; to this end, the advent of 'fake news' is a worthy subject to explore here because a functioning democracy, to a certain degree, is dependent on media/political literacy, critical engagement/participation, and the capacity to communicate, analyze, and disseminate nuanced perspectives, ideas, and information; i introduce a brief case study on the nefarious interpretation of the killing of jamal khashoggi in (bbc news ) to underscore the tension points in normative democracy; ) capitalism, or neoliberalism, needs to be more fully exposed, interrogated, and confronted if 'normative, representative, hegemonic, electoral democracy' is to be re-considered, re-imagined, and re-invented (lydon ) ; the perpetuation of social inequalities lays bare the frailty of normative democratic institutions; ) covid- has exposed the fault lines and fissures of normative democracy, illustrating here the 'common sense' ways that power imbalances are sustained, which leaves little room for social solidarity (human rights watch ); i present here a small case study of the economic and labor dynamic in quebec during the coronavirus. ultimately, i believe the quest to re-imagine a more meaningful, critically engaged democracy, especially during a context that is imbued with a political, economic, and public health crisis, cannot be delayed much longer. capitalism, in addition to acknowledged and unacknowledged hegemony, is central to this model or framework, and a natural order and superiority flows effortlessly through thinking and believing that this is the only way to be, exist and function. democracy . , which considers more fluidly agency, power crystallizations, social justice, and individual as well as collectivist media and citizen engagement, is much messier than democracy . , which connects more directly with normative, representative, hegemonic, and electoral machinations (carr, hoechsmann, and thésée ) . social media is an exemplary feature of this new environment and can help us draw out the fundamental question if greater media, communication, and online involvement can lead to more robust, critical democratic forms of citizen participation. elsewhere, with colleagues (carr, daros, cuervo, and thésée ) , i describe some of the overlapping components, processes, and concerns that help frame the context for social media, fake news, and citizen participation. it would appear that everyone today is somehow connected to social media, even if one does not have an account for one or many of the social networks that pervade, link and smother the socio-cultural landscape (keating and melis ) . there are networks for an untold array of information sharing and gathering. nouns have become verbs as in 'youtubbing,' 'blogging,' 'vlogging,' 'googling,' 'facebooking,' etc.. the reach is significant, and the digital imprints (and footprints) are equally commensurate (sun, wang, shen and zhang ) . one can do a search for a pair of shoes on amazon.com, and, magically, there will be ads for shoes on the personal facebook feed immediately afterward. algorithms are increasingly programming what we see, and aligning at least some of our attention on 'stuff,' for lack of a better word, where we might not otherwise be interested. this surveillance, usurpation and data-gathering was significantly exposed in , with facebook being highlighted for a particularly negative watershed year (sutton ; wong and morris ) . among the litany of events, problems and phenomena that have plagued facebook, which are clearly not limited to this one, albeit prominent social network, were the following claims, findings and evidence, amongst other issues: algorithms connected to the 'negative effects to referral traffic,' unregulated ads that underscored the mueller investigation that has, as it focus, in large part, the russian involvement in the us presidential election, the cambridge analytica scandal that 'obtained the data of tens of millions of facebook users without their knowledge or consent to help build a powerful political influencing tool,' privacy and security issues, 'special data-sharing arrangements with tech manufactures like amazon, apple and samsung,' hacking of accounts, and regulation problems (sutton ) . (carr et al. : ) fake news has leaped into mainstream consciousness over the past few years as if it is the problem hampering democracy. emphasizing that fake news is rarely neatly packaged within a singular category, the report cited above cautions that deception needs to be interrogated at various times while viewing media messages. with the avalanche of fake accounts, fake (bot) users, and fake (or tampered with) algorithms, the terrain is fertile for fake news. this is especially the case if users, consumers, and citizens are conditioned to not question or verify what comes their way, are reluctant to disbelief 'official' sources, are ignorant, are disinterested, or are enveloped in turbulent news cycles with complex, nuanced, voluminous information, for which they are unable to decipher the diverse and divergent realities emanating from a particular situation, event, or reality (carr, daros, cuervo, and thésée ) . citizen participation requires critical engagement, and constructing media/political literacy, however defined, needs to be considered in order to better underpin meaningful forms of democracy (carr, cuervo, and daros ) . the hailstorm of misinformation, misdirection, and disinformation during the early phases of the coronavirus mirrors the general online landscape, serving as both a tremendous opportunity and a mud-slide concurrently, and highlighting the potential for meaningful solidarity as well as, conversely, marginalization and xenophobia (ali and kurasawa ) . i am also drawn to the nuanced layers that mackenzie and bhatt ( ) add to this debate, suggesting that '[b]ullshit is different from lying and it need not undermine trust, particularly when it is blatant'. (the literature around the notion and proliferation of 'bullshit' is linked to, and builds on, the work of harry g. frankfurt, notably the book aptly entitled on bullshit.) this is extremely relevant in contemporary times, given populist movements, xenophobic manifestations, and the denunciation of human rights, and the quest to diminish 'news' as being 'fake' as a basic principle emanating from some powerful leaders in the western world as well as elsewhere. at the same time, i acknowledge that the traditional media is anchored in biases and hegemonic trappings but am troubled that the 'fake news' caravan seeks to whitewash anything that may bring contrary dimensions to the debate, especially in relation to revealing, exposing, and countering mainstream narratives related to war, conflict, racism, inequalities, and the like. democracy . had a relatively controlled audience, whereas democracy . has let the floodgates open, and this means that there are now opportunities for critique, solidarity, and mobilization that may not have been as readily available previously, including diverse social movements that have taken off through social media (carr, daros, cuervo, and thésée ) . these social movements can be a force of change in society at the local, national, and global levels where governments and international institutions are unwilling, unable, or unmotivated to respond to the needs of the population. for instance, black lives matter (mundt, ross, and burnett ) , #metoo (botti et al. ) , occupy, and idle no more have all had a significant social media influence, and environmental, peace, and other movements have also been influential at diverse levels in mobilizing solidarity that surpasses cultural, linguistic, geographic, and political boundaries carty ) . within the quickly evolving media/social media landscape, i can think of what appear to be several major (media and/or social) events in recent times-noting full-well that by the time this article is published, they may not even be recognizable-including the khashoggi killing, the covington school student debacle, the parkland shooting, the kavanaugh nomination, the thai cave recue, the (british) royal wedding, the manifestations in haiti, the never-ending quest to build a 'wall/barrier/fence' between the usa and mexico, and the political/humanitarian crisis in venezuela, among many others. i apologize for the usa-centric focus here. as a canadian, i am fully cognizant of the depth and reach of usa tentacles, thinking, control, power, and influence in and on my own work, as well as on many others, even though i collaborate widely with colleagues in diverse jurisdictions and contexts, notably in latin america. the usa and its interests bleed over to every region of the world, and, although united statesians (the concept of 'american' is hotly contested and does not cover all of the peoples of the 'americas') may not be talking collectively (in a central way) about the world or may not be collectively immersed in ingratiating the usa into the infinite number of political and economic issues, concerns, and cultural representations of the other countries and peoples, the world is watching, listening, and being consumed by the behemoth of usa empire. the covid- pandemic also squarely places the usa within the core of the action, with daily pronouncements about blaming china, cutting off funding to the world health organization, downplaying the spread of the virus, boasting about how the virus has been beaten back, and spreading the political and economic reach of this country far and wide, in military, diplomatic, commercial, and (potentially) humanitarian ways. it seems as though the reality of this being a (global) pandemic, a far-reaching health crisis, is only partially the story, and the present manifestations in the usa of people demanding that 'isolation' be stopped, while so many are being infected and even dying, is almost incomprehensible, and social media concurrently exposes, denounces, disseminates, and provides an echo chamber for what is taking place. so i question what becomes news, indeed viral, and how does it become more than click-bait, algorithmic entertainment, the bouncing around in limited, like-minded networks, tepid sharing, and a platform for trolls? is it about numbers, the quantity of clicks, views, shares and reads, or something more substantive? at the same time, what are the true dimensions of the issue(s)? who frames it, how, and why, and to what end? what is omitted, downplayed, obfuscated, how and why? in the list of issues in the previous paragraph, we can think of many pitfalls, foibles, and problematic concerns as to what 'issues' look like in democracy . . all issues are not simply a usa problem, but connections to elites, hegemony, power differentials, and media framing are, i believe, worth establishing and interrogating. what is clear is that power differentials are at play in how fake news is constructed, disseminated, understood, and engaged with. the more volatile social media can push up against normative media in further determining how fake news can be projected, masked, embellished, and consumed. concerning the jamal khashoggi killing in october in istanbul (bbc news ), we can follow the usual process of focusing on hegemonic interests and avoiding contextual factors and backdrops. several significant and pivotal factors were down-or under-played in reporting on this tragedy. for example, the relationship to the saudi kingdom, human rights, billions upon billions of dollars in armaments sold to the saudis, the unimaginable assault by saudi arabia against yemen, and the impending famine and genocide in yemen as a result, women's rights, journalistic freedom, and an unending series of beheadings by the quasi-untouchable saudi regime. undoubtedly, information, discussion, debate, reports, and mobilization on all of these fronts can be located and advanced through social media, in spite of the mainstream, hegemonic vision. the point here is that central, controlled, and 'manufactured' debate, at least within a condensed and constrained optic and timeframe, shined a light on the actual killing of khashoggi in turkey, who did it, how, and why. yet, significantly, it was only weakly concerned with the other, what could/should be considered to be, highly pertinent and central issues that are/were intertwined within this quagmire. why such deference was paid to the saudi leadership in this case, when this same deference is not paid in many other instances, especially when the faulty regime is not an ally, is quite pertinent. the lack of historical, political, and economic context, combined with the propensity to avoid latching onto 'research', and a plurality of visions, perspectives, and experiences seem to be a predominant feature of how these stories crystallize. the khashoggi example, like others, contains an evolving set of circumstances and frames, as well as questions, and we are cognizant of how some segments of social media can provide differing narratives that can, consequently, re-shape the 'official' story. yet, the social media dimensions can also counter the formal hegemonic narrative, and this is where alternative forms of 'democracy' can start to take hold (jenkins, shresthova, gamber-thompson, kligler-velenchik, and zimmerman ) . why the more critical dimensions within the khashoggi case (or the venezuela situation or others, for that matter) were/are not more broadly taken up by democracy . relates, i believe, to the hegemonic shaping/framing of the issues. it is also combined with a weakly focused mainstream media, whose reach is now consumed within the 'fake news' bubble, and a still questionable place, at least among many formal political leaders and their business sector supports, of uncontrolled social movements and social media within formal political spheres. however, i do believe that this last factor-social movements and social media being a mobilizing force-is, and will continue to potentially be, central to conceptualizing, developing, cultivating, building, and elaborating a more decent, meaningful, robust, and critically engaged democracy, in spite of the status quo aiming to maintain and sustain its hegemonic place. social media movements can also lead to dictatorship, genocide, and an infringement of rights (sapra ) . for instance, gayo-avello ( ) hypothesizes that social media may contribute but is not the central feature to democratization: in short, social media is not a democratizing catalyzer per se. it is just one of many factors, in addition to great tactical tools, provided the conditions in the nondemocratic country are suitable. moreover, there are many variables which can negatively affect the outcome of any uprising, even without the regime tampering with social media. in other words, social media does not make people free; freedom requires people taking risks and organizing themselves. (gayo-avello : ) social media cannot magically lead to class consciousness, anti-racism, peace, and social solidarity. however, it may be able to provide an outlet and legs to important stories, events, and realities for people who were only previously loosely connected. this could have a dual effect of further questioning and delegitimizing normative democracy, and also providing space and voice for marginalized interests, perspectives, and arguments. social media is now indelibly a part of the citizen participation landscape. what is the point of living in a 'democracy' if you are one of those living in abject poverty, are homeless, and are working tirelessly to make ends meet but never achieve economic justice (ely yamin )? of course, the notion of having the 'freedom' to pursue your dreams, as in 'the american dream', is sufficiently grounded within normative debates to ensure that questioning entrenched, systemic, institutional, deeply grounded social inequalities will be quickly snuffed out. within the usa context, amadeo ( ) highlights the increasing social inequalities as follows: structural inequality seems to be worsening. between and , after-tax income increased % for the wealthiest % of households. it rose % for the top fifth. the bottom fifth only increased by %. that's true even adding all income from social security, welfare, and other government payments. during this time, the wealthiest % increased their share of total income by %. everyone else saw their share shrink by - %. as a result, economic mobility worsened. the financial crisis saw the rich get richer. in , the top % of earners took home % of all income. (amadeo ) powers, fischman, and berliner ( ) have highlighted how research on poverty and social inequalities is poorly understood or operationalized, which further underpins weak policy responses to entrenched and systemic problems. similarly, it is helpful to problematize how wealth has been accrued historically through genocide, slavery, imperialism, war and conflict, colonialism, and a host of racialized, sexist, and other machinations in addition to piketty's ( ) welldocumented treatise capital in the twenty-first century. mclaren (see pruyn and malott ) has highlighted marx's theory on surplus value and the limited mobility between the social classes, and the crushing blow of capital against labor; ultimately, the value of what is produced encounters hyper-inflation in the hands of investors, owners, and speculators without real production, which may seem locked into the days of children being exploited in coal mines over a century ago but there are still many parallels today. giroux ( ) has coined 'casino capitalism' to label the politicoeconomic system that enraptures the vast majority of formal, and to varying degrees, informal activity that underpin mass exploitation. he further elucidates the danger of continuing on the one-way neoliberal path before us: neoliberalism has put an enormous effort into creating a commanding cultural apparatus and public pedagogy in which individuals can only view themselves as consumers, embrace freedom as the right to participate in the market, and supplant issues of social responsibility for an unchecked embrace of individualism and the belief that all social relation be judged according to how they further one's individual needs and self-interests. matters of mutual caring, respect, and compassion for the other have given way to the limiting orbits of privatization and unrestrained self-interest, just as it is has become increasingly difficult to translate private troubles into larger social, economic, and political considerations. one consequence is that it has become more difficult for people to debate and question neoliberal hegemony and the widespread misery it produces for young people, the poor, middle class, workers, and other segments of societynow considered disposable under neoliberal regimes which are governed by a survival-of-the fittest ethos, largely imposed by the ruling economic and political elite. (giroux ) mclaren (see pruyn and malott ) and giroux (see giroux, sandin, and burdick, ) have also made a compelling case to interpret today's reality as a politicoeconomic context that is launching us into hyper-sophisticated forms of fascism. within this backdrop, i believe that there is a great need, as there always has been, to be more fully engaged with (and in) education, in political circles and in public debate, in general, in relation to the philosophy and operationalization of capitalism and, in particular, to the all-encompassing mercantilization of all public and private goods, services, and experiences enveloped within neoliberalism. the covid- context has expedited and underscored the slippery slope toward authoritarianism, stripping away rights while creating socio-economic cleavages that are even more serious than before . democracy . is tethered to democracy . conceptualizations of the world, but the door is (slightly) open to develop a new world, despite the titanic hegemonic vice-grip that maintains a stranglehold on education and public debate. as alluded to in the previous section, the collective 'we' are free to surf the web, consume, create, diffuse, comment, and cajole the other, whether the 'other' knows us, sees us, or cares about us or not. we are not frontally impeded from opening our eyes and ears. on the contrary, many movements have been stimulated from doing so-including the arab springalthough the aftermath re-captured regressive hegemonic features of what preceded it. the dilemma is that the corporate/business politico-economic (hegemonic) world has grown into this concurrently in-your-face and stealth, quickly-evolving, dynamic context seamlessly stamping its imprint in every way possible. the interplay between democracy . and democracy . , thus, offers tremendous potential for citizen participation and engagement while, simultaneously, presenting the quicksand mirage that we may not be as 'free' as we think we are, or we may not be as 'engaged' as we think that we are. neoliberalism has many people around the world gasping for air. now mired in a pandemic that vacillates from signs of encouragement that the 'curve is flattening' to fears that 'community transmission' is rapidly spreading through asymptomatic contact, there is enormous stress about when there will be an effective vaccination, how the health context will play out, and, increasingly, when will the 'economy get back to normal.' at this point in time-although we are aware of massive numbers (the information is not hidden, anyway) of unnecessary deaths in 'developing' countries related to hunger, disease, poverty, and conflict-we can see the extreme concern within local, national, and international governments and institutions to get the economy working. while most of the world has emphasized 'social distancing' as a key measure to diminish the dissemination and transmission of covid- , an eerily bizarre phenomenon has taken hold in the usa (wong, vaughan, quilty-harper, and liverpool ) . disparate, semi-organized protests against 'self-isolation' are taking place in diverse locations, often replete with a range of arms and placards enunciating the right to, among other things, 'haircuts' and to 'play golf.' is it pure insanity, a case of hubris beyond all limits, an anti-science ideology that needs to play out in every sector-including the environment-or complete indifference to human suffering? while the usa situation deeply underscores the anxiety and agitation around the health/economy dichotomy, i present below a brief illustration of the neoliberalization of the political and economic convergence through an example of the coronavirus in québec (canada). québec, a predominantly french-speaking province of . million people in canada, provides an interesting illustration of how a jurisdiction within a federal framework has worked to mobilize, sensitize, and activate a range of health, economic, political, and education measures to confront covid- . there are daily press briefings, information sessions, directives, a vast media campaign, testimonies, and a host of consultations, which all serve to educate the public and to engage the citizenry concurrently. it would be disingenuous to simply criticize where there have been gaps and problems; the reality is that many people have worked diligently and courageously to create a sense of the gravity of the problem and to diminish the extent of the propagation of the virus. having a universal healthcare system has been, i believe, indispensable to understanding how to assess, allocate, distribute, and organize resources. this is not an individual problem but a vast, insidious collective one. it should be acknowledged as well that what we know is shifting and re-calibrating in real time, and decisions made on march were questioned and re-assessed by march and so on. moreover, what we know now cannot always be fully understood until later, and decisions that are taken in that light can lead to nefarious situations and the rampant spread of the virus. hindsight is / as the proverb goes so a fulsome diagnosis of what we are doing today will be more effectively critiqued once we are through to the other side of the pandemic. the situation in québec, one that is surely not unknown elsewhere, underscores the fragility of 'normative' democracy; this is, i believe, a question of normative democracy working the way that it does. one heart-wrenching issue that we are observing at this time is that the vast majority of deaths in québec, like elsewhere, is among those years of age and older, and particularly the + age-group. moreover, what many of us did not know or fully consider, the vast majority of deaths up until now within québec are among those who are in long-term care residences (in french, they are called cshlds), roughly %, which are essentially senior's residences for people with health issues. the transmission within these residences is extensive and rapid, with an increasing number of personnel, nurses, and doctors also being affected. one residence, for example, experienced an overwhelming amount of infection (herron, discussed below), and there are others that have also been deeply affected. one might say that there are two public health crises at this time: one for the general population and another for these particular residences with this specific group. on the one hand, the population is astonished, sickened, and in shock ('how could this happen?,' 'especially to "our elderly"?'). on the other hand, this was a serious politico-economic cocktail being mixed for a couple of decades, massaged through diverse political parties within the normative democracy that adjudicates such matters. (why was there such sustained neglect and under-funding? why was this not flagged as a serious catastrophe in the making?). i would like to underscore that this is not a problem of one person, one political party, one decision, one law, or one particular model: it is the consequence of systemic, institutional failure/negligence as well as the thinnest wedges of normative democracy carrying the day over the broader public interest and good. i briefly present some of the specific underlying conditions that lay the groundwork for what is playing out within this vulnerable population at this time: a lack of monitoring, under-paying workers, and diminished policy importance and planning. media accounts provide information on the tragedy unfolding before our eyes. in one case, at herron, in western montréal, the chsld there, which is privately owned, experienced serious staffing shortages, insufficient equipment, poor oversight, inadequate support from oversight bodies, and unacceptable communications with health authorities. mckenna ( ) provides a sense of the chaos and suffering there: nurses were getting sick, too: six out of the seven registered nurses on staff were experiencing covid symptoms, and of seven licensed practical nurses (lpns), only four were still healthy. (…). about a quarter of the orderlies (préposés aux bénéficiares, or patient attendants) had also stopped workingeither because they were experiencing covid symptoms or because they felt it was no longer safe to work at chsld herron. within weeks, a quarter of those patient attendants would test positive for covid- . (…). bedridden residents were lying in sheets stained brown up to their necks in excrement, so long had it been since their diapers had been changed. some were dehydrated and unfed. (…). the head of professional services at the ciusss, dr. nadine larente, is the doctor who went to help. she told the french-language newspaper la presse the place was in chaos: one lpn and two patient attendants were trying to care for residents. food trays had been placed on the floor, dishes untouched because residents with mobility issues could not reach them. (mckenna ). about double the number, proportionately, of seniors in québec opt for long-term care residences compared with the rest of the country, which could be a function of culture, policy, economics, and options available, and the rapidly aging québécois population is a further aggravating factor preparing the context (dougherty ). one expert (see lowrie ) noted that the spatial configuration 'with long corridors and residents sharing rooms, have a harder time isolating sick residents from uninfected ones, compared to residences with house-style layouts, where residents live in smaller wings' is another factor that helps explain the extreme transmission of the virus in chslds. with staff falling ill or refusing to come to work, there has been a massive campaign to recruit retired nurses and also to bring doctors and specialists into the overburdened long-term care system; the premier of the province has also asked for the military (over a thousand troops) to further provide support within these seniors' residences. social class and political power are fully intertwined in the quickly unraveling situation involving seniors' residences in québec. raising the minimum wage in québec, for example, was vigorously opposed by the present government and others along the way, fearful that employers, especially small businesses, could not afford it. while there is no maximum wage being regulated, those struggling with the minimum wage are often obligated to work two or three jobs, to seek assistance elsewhere, and face other severe challenges, including in relation to housing, childcare, education, and the cost of living. the chsld situation brought everything to a head, with it being clearly obvious that those designated as 'essential services' were often those being paid the least in society. the premier took the almost unprecedented measure of apologizing for underpaying workers when it became difficult and problematic to staff these residences: 'i know a lot of quebecers are asking themselves how we could have got ourselves in this situation,' a sombre legault said at his friday briefing, addressing the catastrophe unfolding in covid- -stricken long-term seniors' residences (chslds). 'i myself have spent several days and nights asking what i should have done differently.' 'if i was able to redo one thing, i would have increased the wages of orderlies faster, even without the accord of the unions. i assume full responsibility. we entered this crisis ill equipped, and clearly the situation deteriorated for all kinds of reasons. the virus got in.' (authier ) . the premier also took a series of steps to increase pay for healthcare workers. as part of its effort to improve working conditions in the health-care system, quebec announced that nearly , employees in both the public and private sector will be getting temporary pay increases. workers who are in direct contact with the diseasesuch emergency-room professionals and nurses in coronavirus testing centreswill receive an % boost in their salaries. those working in long-term care homes, known as chslds, will also be among the , workers to benefit from the % raise…. another , people who work in the health-care system but aren't as directly exposed to the disease, such as the nurses who staff the health line, will get a salary increase of %. and workers in private long-term care homes, many of whom make little more than minimum wage, will get an additional $ per hour. that measure appears designed to discourage these workers from quitting and staying home, to take advantage of federal financial assistance that's worth $ a month. (shingler, stevenson, and montpetit ) . one question that arises here is how these workers could have been underpaid for so long, and what the effect may have been, for them, the people receiving the care, the healthcare institutions and system, and society as a whole. did it dissuade qualified workers from pursuing careers or staying in them? what were the other priorities that negated remunerating fairly such indispensable and 'essential' workers? on the economic side of the ledger, how efficient is it to underpay some employees and over-compensate others who have not actually done the work or who are, ironically, considered to be disproportionately fundamental? is a : ratio for salaries at the top and the bottom reasonable or should it be : or : ? in canada, in general, the wage differentials are less extreme and odious than the usa, but the issue of social (in)equality is also a significant concern there. one study (mishe and wolfe ) focused on usa compensation provides some backdrop to how public services and priorities can be disproportionately affected. ceo compensation is very high relative to typical worker compensation (by a ratio of -to- or -to- ). in contrast, the ceo-to-typical-worker compensation ratio (options realized) was -to- in and -to- in . ceos are even making a lot more-about five times as much-as other earners in the top . %. from to , ceo compensation grew by . % ( . % under the options-realized measure), far outstripping s&p stock market growth ( . %) and the wage growth of very high earners ( . %). in contrast, wages for the typical worker grew by just . %. there is a lot of complexity to how covid- is analyzed, and comparing diverse sites/ jurisdictions/systems and how data are compiled and evaluated may not reveal the true breadth and scope of the reality. similarly, there are many moving parts and lots of people (remunerated and volunteer) involved and engaged, and there are also all kinds of activities aimed at supporting a solidified, vigorous response. my intention in presenting this case study is not to admonish or diminish those serious and important efforts. on the contrary, it is my hope that this pandemic will reveal a silver lining somewhere in that extreme vulnerabilities and shortcomings need to be rectified in order to ensure, as much as possible, that economics will not suffocate political considerations in the future. and i have not emphasized here the race, gender, and other pivotal underlying factors underpinning this pandemic, but they are also a significant piece of the puzzle. this text has underscored what 'democracy' we are trying to achieve, to cultivate, and to ingratiate. the focus and direction of my central arguments about the lack of bone fide democracy within a normative, mainstream political framework that preaches that we live in a developed democracy has, i believe, become accelerated and accentuated as a result of covid- . i have highlighted some of the fundamental issues and problems with 'normative, representative, hegemonic, electoral democracy,' and also emphasized the pivotal contextual shifts and cornerstones embedded in democracy . as well as democracy . . i have also made the case for more robust, critically-engaged citizen participation, which would require or, at the very least, benefit from new forms of education and media/political literacy. the social media equation was brought to light since it serves as an unruly, uncontrolled, and rapidly evolving microcosm of the world, its diversity, its problems, its challenges, and its potential. i was careful to not make a definitive declaration related to achieving democracy through the potentially transformative technologies that now shape how we live and function and relate to the world. despite everything, we are still mired in conflict(s), in inequitable power relations, and in 'democracies' that are not very 'democratic. ' we are still straddling democracy . , in which formal political declarations are fabricated with partisan political interests at the fore, the stock market is seemingly central to everything, and business elites are catered to at every level. similarly, tax cuts-regardless of political stripe-figure into everything, political parties shamelessly line up to receive 'donations' (does anyone believe that they come with no strings attached?), tax breaks for companies must be considered as much as lower tax rates for the rich (does anyone believe that rich people will create more employment based on having more cash? if so, why are there so many off-shore accounts in tax-havens intended to not pay tax?), and (military) might is (still) right for many. the further the coronavirus expands, the more there is discussion about needing an economic balance to 'get back to normal,' and indicators such as the stock market are central to supposedly gaging what is happening (karabell ) . of course, there have been lots of (incremental) changes, and lots of new laws, policies, practices, and shifts in cultural norms that have benefitted, generally speaking, women, racialized minorities, the poor, and the vulnerable. yet, social inequalities, despite massive technological and others changes, not only persist but, in many regards, are increasing. how could this be when there is so much wealth? why do so many people leave their countries in complete desperation, why is there still so much military conflict-most of which goes unreported-why do so many problems of poverty and discrimination persist in the most vulgarly palpable ways, why is there such little global outrage over the state and fate of indigenous peoples (the loss of land, language, culture and autonomy), and why is the 'environment' not the priority? this very partial list of questions is noteworthy because neoliberalism is, definitely, an accelerator to many of the problems we are facing . to be clear here, this is not a binary proposition, and avoiding confronting real problems with real people will not address real suffering, oppression and marginalization (gray and gest ) . we might ask: why are there (recurrent, entrenched) problems when there are so many people, projects, forces, and movements fighting for a more decent, robust, and (even) alternative democracy . , one that could place neoliberalism within a new, different and alternative landscape? how should hegemony be understood today when (many) people so freely believe that they have complete agency over their actions, thoughts and experiences, and when (many) people believe that voting is the (only) key? i would stress here that the binary capitalist-socialist, rich-poor binary is not the most productive lens through which to examine the complexity of such extreme power imbalances around the world. the debate around 'democracy,' i believe, needs to be more all-encompassing, involving all of the tentacles and blockages of neoliberalism into the class, race, gender, cultural, and other pivotal sociological markers of identity, and it also needs to carve out a place for how power works, is distributed and recreated. this debate needs to leave open the door for unknown questions and answers as well as (creative and alternative) processes and deliberations, accepting that the normative elections in place are most likely not very beneficial for most people, and, most definitely, the massive numbers of people who do not participate, willingly or unwillingly (van reybrouck ). it is important to connect the local with the global, as we can through covid- . ely yamin ( ) provides a sense of the need to address global issues globally and to be leery of not considering the complexity of the linkages between complex problems. but that and many other challenges requires weaving human rights praxis-human rights for social change-into broader social movements, as well as working across disciplinary silos. the problems facing the rights movement are too complex for any one set of advocacy tools or any one field's expertise. of course, there is no single monolithic 'human rights community' just as there is no unified 'health and human rights community'. those tropes are used from the 'inside' to police the boundaries of orthodoxy and from the 'outside' to caricaturize sets of actors and strategies. yet, there are dangers of circling the wagons defensively around our professional tribes. the complexity of the challenges posed by rampant inequality, the spread of authoritarianism and illiberalism, distrust in multilateralism, and climate cataclysm call for embracing justified critiques and opening up to new ideas and perspectives-and uniting with labor, environmental and many other social justice movements. (ely yamin ) inspired by paulo freire's transformative work (freire ) , i would advocate for more openness and acceptance of political realities that shape our lived experiences as well as an extremely healthy dose of humility as means to being able to understand, engage with, and be with the 'other.' i explore more fully the interconnections and inspiration of freire's work with my colleague gina thésée (carr and thésée ) . the hard-wired, testosterone-induced, keep-fundraising-at-all-times political systems that have been put in place all over the place need to be re-imagined before they suffocate themselves and everyone else. people will slowly divest themselves from the voting game, leaving it as an empty shell filled with a bunch of white guys in suites. (yes, there are some openings for other identities in this equation but the game was made by and for these guys.) freire wrote of conscientization, and i believe that to get there, we need to focus on peace, not war, social, and cultural development as opposed to economic development, solidarity, and emancipation rather than exclusively on individual rights and liberties, and the recognition that we are (all) human beings. as human beings, we are not required to be racist (no baby is racist but we learn to be so), sexist (a totally learned behavior), classist (exploiting one's neighbor is not an obligation), kill one another (who gets killed anyway? the rich or the poor, and who are they? do we care?), or live with so much misery, hatred, and oppression. ultimately, we are in the same boat (or world) together. one could see the glass half full with lots of progress all over the place, and, yet, the empty side of the glass contains real people living through unimaginable (for the full side of the glass) realities; the wage discrepancies and gaps in the québec example exemplify this reality. the quest for a meaningful democracy aimed at both sides of the glass would be a more conducive option, and re-imagining democracy will require more fully and, even disproportionately, considering the empty side of the glass. taking a stand against democracy . and 'normative, representative, hegemonic, electoral democracy' is a necessary condition to moving forward for this re-imagined democracy. donkervoort ( ) underscores that the pandemic has been exploited by 'autocrats' but that citizens can resist and coalesce around global initiatives to weaken and confront hegemonic forces. this could mean enhanced civil society engagement across all boundaries with an eye to unmasking and dismantling the concentration of wealth and power. covid- has exposed the need for a different universe, not only in terms of public health but also, importantly, in relation to democracy and citizen engagement (roy ) . so while my foot, to return to the title, may be in taters, i'm hopefulindeed, it may be the only way out if this-that my head will not be the ultimate causality as we strive to either sustain or re-imagine a democracy that can not only take us out of a pandemic but, rather, into social solidarity that will remove our bodies and minds (and souls) from imminent disaster. open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creativecommons.org/licenses/by/ . /. democracy for realists: why elections do not produce responsive government #covid : social media both a blessing and a curse during coronavirus pandemic. the conversation structural inequality in america: how structural inequality stifles the american dream do democracy and capitalism really need each other? harvard business review the struggle for democracy in education: lessons from social realities covid- analysis: legault offers a mea culpa -but did he really have a choice? montreal gazette jamal khashoggi: all you need to know about saudi journalist's death the #metoo social media effect and its potentials for social change in europe. brussels: feps -foundation for european progressive studies does your vote count? critical pedagogy and democracy citizen engagement in the contemporary era of fake news: hegemonic distraction or control of the social media context? postdigital science and education social media and the quest for democracy: faking the re-awakening? democracy . : media, political literacy and critical engagement it's not education that scares me, it's the educators…': is there still hope for democracy in education, and education for democracy? social movements and new technology america's covid- disaster is a setback for democracy. the atlantic while autocrats exploit the pandemic % of quebec's covid- deaths are in long-term care, seniors' residences. ipolitics putting human rights at the centre of struggles for health and social equality on bullshit pedagogy of the oppressed social media's contribution to political misperceptions in u.s. presidential elections social media, democracy, and democratization the mad violence of casino capitalism. counterpunch the covid- pandemic is exposing the plague of neoliberalism the new henry giroux reader: the role of the public intellectual in a time of tyranny silent citizenship: the politics of marginality in unequal democracies citizens adrift: the democratic disengagement of young canadians by any means necessary: the new youth activism as covid- spreads, listen to the stock market for now social media and youth political engagement: preaching to the converted or providing a new voice for youth? many factors behind covid- outbreaks hitting quebec's long-term care homes noam chomsky: neoliberalism is destroying our democracy: how elites on both sides of the political spectrum have undermined our social, political, and environmental commons. the nation why are american elections so long? orderlies worked without ppe, covid- patients wandered herron's halls for days after health agency took over lies, bullshit and fake news: some epistemological concerns ceo compensation has grown % since : typical worker compensation has risen only % during that time. economic policy institute scaling social movements through social media: the case of black lives matter capital in the twenty-first century making the visible invisible: willful ignorance of poverty and social inequalities in the research-policy nexus this fist called my heart: the peter mclaren reader (volume i) the pandemic is a portal the last decade showed how social media could topple governments and make social change -and it's only getting crazier from here no one 'is more deserving,' says legault, raising wages of , health-care workers as covid- cases climb location information disclosure in location-based social network services: privacy calculus, benefit structure, and gender differences facebook's terrible, horrible, no good, very bad year: and youthought you had a rough conjuguer démocratie et Éducation : perceptions et expériences de futurs-es enseignants-es du québec. citizenship education research journal/revue de recherche sur l'éducation à la citoyenneté political disaffection in contemporary democracies: social capital, institutions, and politics against elections: the case for democracy it's complicated: facebook's terrible . the guardian covid- latest: cdc director warns us second wave could be even worse key: cord- - v kuq authors: boylan, brandon m.; mcbeath, jerry; wang, bo title: us–china relations: nationalism, the trade war, and covid- date: - - journal: fudan j doi: . /s - - - sha: doc_id: cord_uid: v kuq the trade war between the usa and china has shocked many across the world. a disruption to the interdependence of the two largest economies seemed unfathomable. however, in an effort to thwart china’s economic practices and boost the us economy, president trump’s administration levied tariffs on chinese imports shortly after taking office, moving us foreign economic policy from liberalism, practiced for decades, to protectionism. china has retaliated, and the trade war continues today. with conceptual insights from the nationalism literature, we explore the nationalist roots of the trade war from both the us and chinese perspectives. in the usa, the trump administration’s plan to achieve energy autonomy, decrease reliance on foreign resources, and reinvigorate the manufacturing sector has led to protectionist policies, the othering of china, and hence the trade war. although reluctant to enter the conflict, china has rebuffed the usa, resisting and counterattacking us actions, owing to a long-felt sense of persecution in the global space and an eagerness to participate fully, and lead in some issue areas, in international affairs. the conflict continues into the covid- era, marked by us scapegoating of china and hits to economic performance. until both sides are convinced they have achieved their goals, or the usa undergoes an administration change, the conflict will likely continue. linking the two largest economies, the us-china trade relationship is the world's most important. however, in recent years, nationalism in each country, the trade war, and covid- have strained this relationship. guided by the literature on nationalism, we explore how rising nationalist frustrations in both the usa and china have contributed to the trade war, a conflict exacerbated in the covid- era. while they have common elements, the sources of nationalism in the two countries are different. in the usa, a growing segment of the domestic public, believing it lacked political representation and protection of its values, responded to an "america first" agenda, culminating in the election of donald trump. in return, president trump has continued to oppose china for domestic political support, particularly from the manufacturing and agricultural sectors and from an ethnically and religiously homogeneous base. in china, political leaders resented the lack of global political status commensurate with rapid economic growth and rising military might. they demanded more respect for their sphere of influence, and they mobilized against us threats to chinese sovereignty. indeed, china has aspirations to global leadership status on par with the usa and russia. in this article, we first provide the main features of the us-china trade war. we then highlight insights from the nationalism literature to frame the ways in which nationalist grievances in the usa and china have contributed to the ongoing trade dispute. from the us side, we examine the push for energy independence, the revival of manufacturing, and internal and external xenophobia-factors that have led to the scapegoating of china and motivated the conflict. from the china side, we assess historical victimization, economic reforms and internationalism, and leadership in the international community. feeling a sense of historical repression, and with a goal of being a full participant in global affairs, china has asserted economic and political power in the trade conflict. throughout, we discuss how the covid- pandemic has influenced the trade war. during his presidential election campaign, donald trump routinely condemned china for what he and others believed were unfair trade practices and decried its trade surplus with the usa (bbc news ). promoting an "america first" foreign policy agenda marked by us nationalism, protectionism, and unilateralism, and appealing simultaneously to large corporations (through tax cuts) and the us manufacturing and agricultural sectors (through shoring up jobs), he vowed to take a hard line on china in response to its reported currency devaluation, export subsidies, and theft of us intellectual property (long ; daniels ) . as president, trump oversaw the establishment of economic policies that launched a trade war, placing tariffs and nontariff restrictions on chinese imports. in early , his administration imposed tariffs on imported solar panels and washing machines (lynch ) and later on steel and aluminum (donnan ). although the tariffs applied to imports from many countries, chinese goods were seemingly the primary target. in retaliation, china imposed tariffs on an array of products from the usa. from july to december , the tariff war escalated in a tit-for-tat fashion. after fragile progress of negotiations in early , the trump administration raised tariffs from to % on $ billion worth of chinese goods in summer (bryan ) . china retaliated with its own tariffs. in august , china suspended the purchase of new us agricultural products, and the u.s. treasury department declared china a "currency manipulator" (u. s. department of the treasury ). the two countries made progress in repairing trade relations in fall and agreed to a trade deal in january . however, despite the deal, in which china promised to import more us agricultural goods, it has not met its purchasing targets, especially in the wake of covid- (bermingham ) . china's decreased importation of agricultural products from the usa has harmed us farmers. these products include soybeans, grain sorghum, pork, cotton, and cattle hides. although china purposefully targeted the us agricultural base in the hope that farmers would pressure the trump administration (li et al. ), farmers continued to support the president. based on surveys of corn and soybean farmers in , zhang, rodriguez, and qu argue that three factors account for their support (zhang et al. ) : first, the trump administration has given billions in assistance to offset profit losses. second, farmers believe that decline in profits in the short term will lead to better gains in the long term. third, china has been inconsistent over time in its purchases of agricultural goods, according to farmers. thus, despite chinese efforts and some economic setbacks, farmers could continue to support the president. the trump administration has also attempted to thwart chinese theft of intellectual property and espionage. in may , it banned us companies from working with huawei, a large chinese multinational telecommunications and electronics company, over concerns it was stealing intellectual property and spying on companies and the government (paletta et al. ). a month later, it targeted five supercomputing companies-chengdu haiguang integrated circuit, chengdu haiguang microelectronics technology, higon, sugon, and the wuxi jiangnan institute of computing technology-over fears they were using their technology for military purposes (leonard and donnan ) . in june , president trump proposed visa restrictions on chinese students and scholars associated with china's "militarycivil fusion strategy" in the belief that the chinese government used them to acquire intellectual property illegally from the usa (redden ). in late july , the federal bureau of investigation (fbi) arrested chinese students who had not disclosed they had connections with the people's liberation army (pla) when applying for admissions and student employment positions at various us universities. chen, chen, and dondeti contend that "the trade war is not, in fact, about trade but about technological dominance, and that both sides might fall into a 'thucydides's trap,' the pattern of large-scale conflict when a rising power challenges a dominant one" (chen et al. ) . amid the covid- outbreak, president trump referred to the disaster as the "chinese virus" (rogers et al. ) and later, at a rally in tulsa, oklahoma, as "kung flu." he said that the us government is determining if the virus originated from the wuhan institute of virology. he has also accused the world health organization (who) of acting like the "public relations agency for china" and has withdrawn us funding from the institution (al jazeera ). trump's former national security advisor john bolton accused him of asking president xi jinping for domestic support in his bid for reelection (bolton ) . in one example, bolton argued that trump pressured china to purchase large amounts of soybeans to boost farmers' support as the presidential election neared. indeed, the president has had a difficult balance to achieve: remaining politically and rhetorically tough on china for failing to keep many of its trade promises and for cracking down on hong kong, while encouraging a market rebound before the election, itself partly contingent on the progress of us-china trade relations. meanwhile, his credibility and trustworthiness seem to fluctuate in accordance with this inconsistent behavior. from the chinese perspective, the trade war has come as a surprise. in earlier administrations, and especially since china's entry into the wto in , trade relations had been amicable and permissive. chinese were thus shocked at president trump's unconventional approach to negotiations by threatening to increase tariffs on chinese goods and to do so swiftly. chinese leaders interpreted this as bullying or baling in mandarin ("ba" meaning a "tyrant," and "ling" meaning "insulting"). "face" or mianzi is an essential virtue in traditional chinese culture, and trump's coercive and bullying approach awakened memories of the " years of humiliation," when foreign powers transgressed chinese sovereignty. for many, the chief culprit became the usa. however, the chinese state media employed a moderate approach in covering the trade conflict, criticizing the trump administration for its protectionist policies instead of blaming the usa as a whole for economic aggression (zeng and sparks ) . chinese commentators on the growing trade hostilities took different stances. hu xijin and jin canrong, both with large bases of support, voiced incendiary economic nationalist memes. moderate opinion leaders, such as yao yang (a professor at beijing university), called for a rational and cooperative approach in the management of us-china trade disputes (yao ). spokespersons for the chinese ministry of foreign affairs occasionally used tit-for-tat discourse as the conflict unfolded, which foreign audiences probably thought was undiplomatic. ironically, economic nationalists welcomed this type of rhetoric that targeted a domestic audience. however, it was subject to misperception by foreign actors (wang and mcneil ) . the chinese ministry of commerce is less visible to the chinese public than the ministry of foreign affairs is, and it is largely free from the pressure of chinese nationalism. during the trade negotiations, it played a constructive role as the economic and commercial expertise while its officers built confidence across the negotiating table. the outbreak of the covid- pandemic, first in china and then in europe, the usa, and elsewhere, made bilateral trade negotiations more complicated. controversies outside the trade conflict-for example tensions in the south china sea, the chinese national government's promulgation of national security articles in hong kong, us-china disputes concerning the who, and the like-added fuel to the flames of nationalism. upon the rapid deterioration of political and security relations between the two countries, first foreign minister and chinese community party (ccp) political bureau member wang yi, and then yang jiechi (also a ccp political bureau member), delivered speeches dismissing talk of us-china disengagement. yang also visited the usa and met with secretary of state mike pompeo to ease tensions. speeches of chinese leaders expressed a willingness to cooperate and compromise with their us counterparts, and they also served to bridle disorderly domestic nationalist impulses. in this section, we review concepts and literature related to nationalism to develop a foundation to analyze the drivers of the us-china trade war. these concepts include nationalism, state nationalism, economic nationalism, and american exceptionalism. elites have long employed nationalist ideas to differentiate from others, and at times scapegoated and blamed these differentiated others, to achieve their strategic objectives. nationalism is a devotion to the nation and its people, identity, and culture. it stems from a national consciousness and prioritization that is in opposition to or comes at the expense of other nations. often, social movements spring from nationalist convictions and aim to protect and promote the nation. gellner ( ) argues that the principal goal of nationalism is to achieve the congruence of the national and political units. infrequently do the boundaries of nations and states completely align, however, and movements to build nation-states often fail. civic nationalism is a movement to advance the political community, while ethnic nationalism aims to advance the political, economic, and cultural interests of the ethnic group. smith argues that nationalism is "an ideological movement to attain and maintain autonomy, unity, and identity on behalf of a population, some of whose members believe it to constitute an actual or potential 'nation'" (smith , ) . state nationalism is a form of civic nationalism that attempts to transform the sovereign state to a nation, what can be called the "state-nation." state nationalists contribute to this agenda by othering and marginalizing both internal and external actors. to build the state-nation, leaders and their followers scapegoat ethnic and racial minorities, immigrants, and political and cultural challengers within the state, while at times paradoxically embracing some of these ethnic and political "others" if they support the nation building project. state nationalists blame global forces, international institutions, and other countries and peoples for their challenges, revisioning them as threats to the state's advancement of their interests and to state expansionism. leaders and followers in the state-nation project have a complex and mutually reinforcing alliance. leaders reward their constituents with rhetorical and material benefits in exchange for their support to exact a domestic and foreign policy agenda to move the state to a state-nation. this alliance is often tenuous: leaders' failure to deliver these rewards could weaken constituent support. state nationalism ranges from benign patriotism on the one end of the spectrum to violent fascism on the other. one form of state nationalism is state economic nationalism (or mercantilism). economic nationalism is an ideology that prioritizes the state's economy over all others. economies elevate state security and military power according to the ideology. economic wealth is seen in realist and zero-sum ways. a leading international relations realist, and scholar of economic nationalism, gilpin ( , ) writes, "its central idea is that economic activities are and should be subordinate to the goal of state-building and the interests of the state." he further argues, it "recognizes the anarchic nature of international affairs, the primacy of the state and its interests in international affairs, and the importance of power in interstate relations" (gilpin , ) . contrasting economic liberalism, it opposes globalization and free trade and what is deemed as their destructive forces. instead, economic nationalists aim to safeguard and control their own economies through protectionist measures like tariffs, quotas, and excessive regulations. economic nationalism aims for economy diversification, while economic liberalism aims for specialization. often, industrialization processes and the manufacturing and agricultural sectors are prioritized in the economic nationalist perspective. economic nationalists routinely scapegoat others for economic losses. such others include economic liberals and other countries. (a racialized version also blames immigrants and ethnic minorities.) china's version of economic nationalism has been state corporatism, in which the government intervenes heavily in the market and owns or partially owns many corporations. although nationalist movements are pervasive around the world, nationalism in the usa holds a distinctive place in world history. a young state but old democracy, the usa is often characterized by its "exceptional" status-a label first applied by de tocqueville ( ) . as old as the country itself, american exceptionalism is rooted in the american revolution and u.s. constitution. according to lipset ( ) , americans are committed to a common set of values: individualism, antistatism, populism, and egalitarianism. as the "first new nation" (lipset ) , other states are not born out of the same suite of ideals. based on a conviction that the usa is not only exceptional but superior, the usa believes it has a special mission to transform the world, which has justified international interventionism and democracy promotion since world war i. inherent in american exceptionalism is a recognition of how the usa differs from other countries-both enemies and allies. as we show in the next sections, the trump administration has both galvanized and capitalized on domestic economic frustrations to perpetuate a state nationalist project to secure support for its domestic and foreign policy goals. in the international sphere, this project has targeted primarily china. meanwhile, china's recent economic and political rise is also driven by nationalism and an antagonism toward the usa. although nationalism in each country contributes to the trade war, the roots and character of these nationalisms differ. nationalism in the usa has contributed to the othering of china and the ongoing trade war. in this section, we consider three expressions of us nationalism: energy independence, revival of manufacturing, and internal and external xenophobia and then assess how each contributes to the trade conflict. the trump administration's push to become more energy independent, decrease the country's reliance on foreign resources, and protect manufacturing jobs has led to the resurgence of economic protectionism. veiled in xenophobic rhetoric, china has been made a target in this context in an attempt to increase domestic support. the trump administration has striven for energy independence. it has aggressively pursued fossil fuels and minerals development in the country, with critical financial and political support early in the administration ( - ) and rollback of environmental laws and rules considered obstacles to energy independence and economic development before and during the covid- pandemic. shortly after his election to the presidency in , trump promised a new era of "energy dominance," a term he coined, perhaps to imply that there would be no obstacles to increasing production of conventional and tight oil and gas, making coal "king" again for electricity generation, and expanding nuclear energy and hydropower (council on foreign relations ). this was a much more aggressive agenda than that of previous republican presidents, and it responded to the interests of large oil companies, allied interests (e.g., oil field service firms), and constituent groups such as older caucasian voters, the lower middle class, and less educated workers. having promised during his presidential campaign that he would end the "war on american energy," trump, three years into his presidency, claimed that the usa had attained energy dominance. as the economic recession and covid- pandemic spread in the usa, the trump administration went to great lengths to further the objectives of the fossil fuel industry. in forming a task force called the "great american economic revival" of industry groups to "reopen" the economy, he appointed nine executives from oil and gas corporations, two from the utility sector, one from oil mining, but no one from renewable industries. then, in early when oil and gas prices decreased, trump played a broker role seeking a reduction in global oil production from saudi arabia and russia. the president tweeted, "we will never let the great oil & gas industry down … i have instructed the secretary of energy and secretary of the treasury to formulate a plan … so that these very important companies and jobs will be secured long into the future" (dillon ). he sought to reduce the global oil glut and ease pressure on us oil and gas, reserve prices for which were briefly negative. when administration plans to purchase millions of barrels of oil for the strategic petroleum reserve (spr) conflicted with congress, the u.s. department of energy leased space providing aid to the industry (lee and clark ). the u.s. federal reserve board of governors increased liquidity in the economy, and then the president and congress offered the largest stimulus program since the great depression (by mid- , amounting to nearly $ trillion). hundreds of billions in loans-much of which did not need to be repaid-went to businesses harmed by the pandemic, including energy companies (cahlink and koss ) . republicans, and industries such as tobacco, oil and gas, coal, and chemicals, have long advocated for revision of federal agency, especially epa, rulemaking. the trump administration placed regulatory reform high on its agenda and planned an unprecedented rollback of environmental regulations, but the reform process was interrupted by the transition in power as democrats gained control of the house after the midterm elections and then the impeachment proceedings. regulatory reform gained urgency for the upper echelons of the administration in , especially as energy prices fell and the stock market failed. the trump administration's targets of regulatory reform have long been icons of the us environmental movement: the clean air act, the national environmental policy act (nepa), the clean water act, as well as waters of the us (wotus) regulations. critics viewed these regulations as impeding energy and other apparently hazardous proposed developments. too, regulatory reforms limited the application of the endangered species act (esa) protections of migratory routes and curbed the length of public comment periods for responding to proposed environmental impact statements (eis) reviews. altogether these rollbacks challenged the environmental law and regulatory system established in a generation, although environmental groups challenged all reforms in court (see, for example, farah and hijazi ). the trump administration also sought to expand access to federal lands for oil and gas drilling, coal mining, and mining of strategic minerals such as rare earth elements (see hao and liu ) . most remaining us public lands are in the west, and since the sagebrush rebellion of , elected federal and state officials in western states have resisted "federal overreach." thus, the administration's efforts garnered praise, while environmentalists and a few other interests (e.g., cattle ranchers, fisheries) were in opposition. moreover, lands once reserved from development of rare, precious, and threatened species, such as the western sage grouse, were opened as well. by mid- , the usa produced percent of the fossil fuels it consumed. energy independence is a subset of economic nationalism. the trump administration aims to decrease its reliance on other states for fossil fuels (e.g., oil, gas, and coal) and use us resources to provide power to factories, businesses, and residences. energy independence increases national power in competition with global rivals (e.g., russia) that are self-sufficient in fossil fuels. it increases national economic development and wealth, as few expensive resources need to be imported. and it increases diplomatic flexibility, as preferred outcomes (e.g., support for israel) are not constrained by resource dependence. fossil fuel dominance also protects and rewards large oil and gas corporations that have bankrolled republican leaders including president trump, and it ensures continued employment for hundreds of thousands of oil/gas/coal field workers and sustenance of communities in which they live. this expression of nationalism is linked directly to trade protectionism and thus the us-china trade war. the decline of us manufacturing was an issue in the s, and the circumstances regarding manufacturing enterprises have worsened since then (pierce and schott ) . in the election, candidate donald trump called for a renaissance of manufacturing. as the economic downturn set in, and then the covid- pandemic erupted, the focus became primarily the automobile industry and the global supply chain that undergirded it. the us automobile business was once dominated by the "big "-general motors (gm), chrysler, and ford. however, the detroit auto industry's continued production of high fuel consumption vehicles, when oil shocks pushed gas prices skyward, created openings for inexpensive, fuel-efficient foreign automobiles. to cultivate political support, increasingly foreign manufacturers opened plants in the usa. notwithstanding attempts by some manufacturers to limit foreign sourcing of auto parts, the supply chain for vehicles became more global than national. the detroit auto industry lost considerable political support when gm and chrysler, having declared bankruptcy, gained bailouts of $ billion from the federal government in , as corporate executives flew by lear jets to washington, dc for hearings (lassa ) . given their global linkages, us multinationals were at odds with president trump's rhetoric. for example, in a cost-cutting action gm announced it would close five factories and lay off , workers in north america. in response, the president tweeted that he was considering ending electric vehicle (ev) subsidies: "general motors made a big china bet… when they built plants there … don't think this bet is going to pay off. i am here to protect america's workers!" (joselow ) . as president trump began his reelection campaign in , he claimed, "many, many plants are now under construction in michigan and ohio, pennsylvania, north carolina, south carolina, florida. they hadn't built one in decades and now they're all over the place." although little new plant construction occurred under the trump administration (robertson ), the industry had become compliant with the president. as auto sales fell, and the covid- crisis erupted in early , trump administration agencies allowed exemptions to warehouse and factory safety restrictions so that workers would not lose their jobs. when in march us car sales seemed likely to drop more than %, and us factories closed, the industry received loan guarantees, tax deductions for employees' paid leave, and deferred corporate tax payments. the president also used the emergency provisions of the defense production act to configure factories of gm, ford, and tesla to produce ventilators and other medical supplies (joselow ) . through action on new fuel efficiency standards, the trump administration made its strongest efforts to revive the auto industry. the trend since had been to reduce the amount of gasoline or diesel fuel consumed by cars and light trucks through raising standards of miles-per-gallon. shortly after taking office, the trump administration proposed a rollback of obama administration targets which would have the effect of increasing us greenhouse gas emissions, while reducing the prices of cars and trucks. the auto industry was divided about the proposal. the catalyst was the koch brothers and their conservative network, but most environmental nongovernmental organizations (ngos) were opposed. a new york times investigation contended that the motivation behind the campaign to lower fuel efficiency standards was the surplus of fossil fuels in the usa and the need to increase demand for their products (and fossil fuel dominance) (tabuchi ) . this change, along with historically low gas prices, gave us automakers strong incentives to produce more big "dirty" cars (ferris ) . manufacturing nationalism is another form of economic nationalism. although it increases consumer costs, as us labor and most material costs are higher than those in mexico and other developing nations, reversing the hollowing out of us corporations avoids the flight of us jobs to mexico (through the north american free trade act) and emerging economies (through the world trade organization). it increases national wealth and power by spurring innovation and technology, which lead to productivity growth. increasing exports and reducing imports improves economic health by reducing reliance on other states. too, reduction in balance of payments deficits makes the usa more competitive vis-à-vis its global rivals, especially china. new manufacturing enterprises add hundreds of thousands of jobs to the economies of depressed regions and states and electoral success to national leaders. this form of economic nationalism inspires protectionism and has contributed to the us-china trade war. xenophobia in the usa can be internal or external. the internal, or inward, dimension refers to actions and events within the country and involves the scapegoating of persons or groups along racial, ethnic, religious, or gender lines. the external, or outward, dimension refers to the racialized and hostile treatment of other countries. for example, in the george w. bush administration, an "axis of evil" (north korea, iran, and iraq) preoccupied us policymakers. in the trump administration, the fixation has been on mexico and china. political elites use both forms of xenophobia in an attempt to garner domestic support. during the presidential campaign, trump lambasted previous administrations for failing to enforce border security and stop drug smugglers and other criminal elements from entry into the usa. the most potent message of his campaign was to "build a wall," and this appealed strongly to caucasians (and even some latinos) in his base. during the first two years of his administration, construction of new sections to the already existing wall was a regular story on the public agenda. most dramatic was conflict between the executive and the house of representatives when the latter body, under democratic control after the midterm election, refused to appropriate funds to pay for wall construction. the ensuing government shutdown was the longest in us history. the administration did stop the flow of immigrants and began the return of some asylum seekers, but us law made instant resolution impossible. republican leaders focused on the "flood" of illegal immigrants, most from central america transiting through mexico to cross the us border. the numbers had escalated since the previous immigration reform legislation, totaling at least million. in addition to their lack of legal status, illegal immigrants, according to the critics, used us public services (schools, hospitals, and reduced transportation and housing fares) at taxpayers' considerable expense and took jobs from us citizens. supporters of legalizing immigrants' status offered evidence that immigrants on balance contributed toward the services they received and in most cases worked in jobs (e.g., farm labor, domestic cleaning, and health care) that most americans did not desire. economic and political conflicts with china changed the dynamics of xenophobia in the usa. as mentioned, conflicts over trade rules and policies grew during the second and third year of the trump presidency, and relations worsened greatly when the covid- pandemic reached the us homeland. president trump and several conservative pundits accused china of genetic manipulations in wuhan laboratories, causing outbreak of the virus and then failing to disclose it, a pattern repeating the chinese regime's actions in the - sars epidemic. then, us leaders accused china of infiltrating us laboratories, research institutes, and universities; stealing intellectual property; and hacking us computer systems. several chinese students who were apprehended had worked for the china's people's liberation army (pla). by mid-year , us-china relations had become the worst since . in addition to trade sanctions, risky military encounters (e.g., chinese ships in the us continental shelf), the cessation of nearly all flights into the usa from china (and most of those from the usa to china), and the closure of the houston consulate exacerbated relations. the us labeled huawei technologies co. and its telecom companion zte as a national security threat. secretary of state mike pompeo said that the relationship between the two nations should be based on the principle of "distrust and verify" and went on to say that the diplomatic opening of china by president nixon in had undermined us interests (wang and myers ) . both political and economic dimensions of nationalism stimulated xenophobic responses within the usa and in its international behavior. the political malaise was the expression of endless wars fought by the usa without clear victory or just compensation for its contributions. economic discontent was the result of several decades of globalization that left large numbers of citizens behind (e.g., less educated, laid-off manufacturing workers). political leaders and particularly donald trump exploited this dissatisfaction and its victims and found targets to blame: foreigners (e.g., central americans, mexicans, chinese, africans) and democrats (who supported "illegal" immigrants and were the elites of the "deep state" who grew rich from globalization). scapegoating of these groups relieved emotional stress of those who believed they had been forgotten. once mobilized, leaders then directed the anger of the "forgotten" to others, and the states appearing to represent them, such as china. this strategy has become a dominant feature of president trump's reelection campaign. although china's history as a civilization extends many thousands of years into the past, its modern identity as a nation dates to the nineteenth century, and it is expressed in four forms: economic, ethnic, cultural and political. nationalist sentiments in china, as in the usa, have contributed to the trade war. from a longfelt perspective of being persecuted, and with a desire to participate fully in global affairs and exert leadership over some issue-areas, china has rebuffed us demands and contributed to the trade war. an ancient source of chinese nationalism is its long history as a civilization. china prides itself on the longevity of the chinese language and culture and the ancient belief that china was the central power of the world. chinese leaders are keen to create state nationalism and unity in light of the multiethnic composition of the population. chinese nationality formally includes all of the state's official ethnic groups. although more than % of the people are han chinese, leaders state that cultural inclusiveness is an important value (gao and zhu ) , despite the reality. china celebrates an elaborate tribute system, brought up to the present in the belt and road initiative. it also has a global diaspora, which connects mainland china to hundreds of thousands of chinese communities throughout the world. most western researchers, including ethnic chinese researchers in the west, emphasize china's memory of humiliation from foreign invasion, particularly the " years of humiliation" from the opium war to the end of world war ii and past grievances at the hands of foreign enemies. from this follows the memory of identity as a victimized people, who should "never forget national humiliation" (wang ; zhao ) . western scholars' research in other contexts has suggested that nationalism is one of several factors driving public support for aggressive external policies. some see a rise in aggressive nationalist rhetoric from both official and popular sources (see, among others, whiting ) . in the s, the media provided greater opportunity and incentives for grassroots nationalist expressions (he ) . they featured angry and sometimes violent protests following international incidents involving the usa and japan (gries ) . this has led several western observers to characterize chinese nationalism as irredentist, insecure, and virulently anti-foreign (whiting ) , and this played a role in the increase of regional and bilateral tensions (cotillon ; yahuda ) . on the other hand, some chinese opinion leaders believe china has been subject to provocation analogous to that of germany and japan before world war ii. nationalist commentators, such as hu xijin (chief editor of global times), jin canrong (professor at renmin universitiy), hu an'gang (professor at tsinghua university), and kong lingdong (professor at beijing university), appear to subscribe to radical nationalist beliefs and have large internet followings. in either case, a nationalism centered on a long-felt sense of persecution has contributed to the ongoing trade war. china's history of being othered has created a "we" and "they" mentality and an insecurity that four decades of economic growth cannot easily remedy. modern chinese economic reform emerged at the fall of the qing dynasty in - and the rise of the republic of china, notwithstanding political turmoil until the establishment of the people's republic of china (prc) in . private entrepreneurship, which had long been suppressed under imperial rules, thrived, as evidenced in the slogan, "entrepreneurship saves china" (shiye jiuguo). after three decades of marxist and maoist rule, china's reforms and open door policies were adopted upon deng xiaoping's return to power in . more recently, china has strategized to invest ambitiously in locations all around the world, from africa to the arctic, and trade with a host of partners, all supported by the belt and road initiative, a strategy to develop roads, railways, and ports in several countries around the globe. economic reforms established state capitalism as a system to create wealth and power in china. state-owned enterprises remained pillars of the economy, in sectors of national security importance such as transportation, energy, natural resource extraction and production, electricity, and several types of manufacturing. china's doors were opened to multinational corporations and importation of their technology and capital. china's integration into the global economy brought great pride in the chinese nation as it became the world's premier production center. chinese competitiveness was essential for reduction of poverty and the rise of a middle class, but this came at the cost of national independence-as noted in xenophobic responses associated with both cultural and ethnic nationalism and directed at groups like the uyghers, tibetans, and taiwanese. china has been able to provide elementary school education to nearly all youth. middle school education is not yet universal, and access to high school and college is limited to those passing entrance examinations. still, young people today are much more likely to be knowledgeable about the global community than they were fifty years ago. the increase in educational opportunity facilitates independent and critical thought about the linkage of china's national interest and economic interdependence (qin and zhu ) . further, scholars generally agree that economic reforms increasing competitiveness and promoting global integration have been beneficial to china's national interest (chen ; brien and he ) . therefore, as the trump administration sought to sever economic and cultural ties with china, the majority of chinese, including elites, objected. they wanted to prevent a trend leading to disengagement from global affairs. when chinese considered the degree to which china had advanced as a consequence of ties with the outside world and particularly the usa, they harnessed nationalist instincts in the developing trade war. the chinese communist party was founded on marxism, and during the first three decades of the prc the regime followed the soviet model of a state-owned and managed economy. during that period, ideological disputes within the fabric of marxism and maoism prompted the great leap forward to surpass the usa and uk, followed by the cultural revolution which attempted to purge capitalist elements from society. less than two decades later, leaders staged a "patriotic education campaign," designed to shore up support for the ccp in the wake of the student movement of and the setback to marxism as represented in the fall of the berlin wall and ultimate dissolution of the soviet union in . western scholars' research on chinese nationalism focuses on public attitudes and foreign policy making (weiss ; sinkkonen ) . for chinese leaders, the politics of nationalism are more complicated and have both domestic and foreign targets. president xi jinping envisions china and the global community as sharing a common destiny and expects china to play a positive and active leadership role in global governance. in xi's vision of the chinese dream, china will exercise responsibility in producing more public goods, including harmonious interactions between humans and nature (bhattacharya ) . thus, xi is under pressure to end the trade war and reassert china's growing leadership role in international relations. this vision of china's role in global governance, along with china's visible benefits from participation in the global economic system, most notably with the usa, are key factors balancing against the bitter feelings chinese harbor toward the trump administration for disrespectful treatment during the course of trade negotiations. china and the usa have different political and ideological belief systems. china is an outsider to the global security network, which features a us-led alliance network. these differences will be difficult to eliminate in the short term, and for this reason trade relations increasingly appear risky. when these factors become dormant, trade issues will be easier to resolve. of course, increasing global economic benefits would ease trade hostilities, but the slowdown in global growth due to the covid- pandemic limits the ability of both china and the usa to soften in their trade positions. a number of uncertainties remain for both countries regarding the role nationalism plays in trade relationships. in the usa, domestic politics are polarized, and there are problems in economic recovery. in china, there are concerns in the process of domestic political development as to whether the open door and economic reform policies should continue. too, there is the question of china's identity as a responsible global actor. thus, china counteracts us aggression in the trade war, not only to protect its economic interests but also to assert power to international audiences. although the trump administration initiated the trade war, china has retaliated with tit-for-tat measures and exerted a great deal of power to protect its economic and political interests and its burgeoning leadership role in global affairs. both sides' participation in the conflict rests on several nationalist factors. in the usa, president trump has pushed to wean the country off foreign sources of oil and other natural resources and has promised to bring manufacturing jobs back to the rust belt and other areas. both of these agenda items have led to economic nationalist and protectionist steps and to the weakening of the us-china trade relationship. in this context, trump and other high-level administrators have used xenophobic rhetoric in an attempt to blame china for us economic ills. the rhetoric ramped up at the onset of the covid- pandemic (and in the year of a presidential election campaign). for the trump administration, the pandemic was an opportunity to further blame china for domestic economic problems long in the making. initially surprised at the high tariffs imposed on its goods, china has stood up to us pressure, in part owing to its identity of being historically marginalized and its desire to continue to grow economically and politically on the world stage. what have been the effects of the us-china trade conflict? it has led to higher consumer prices, lower corporate profits, unstable markets, and slower economic growth (norland ; ungarino ; lawder ) . one analysis shows how president trump's tweets related to the trade conflict have adversely affected the stock market (burggraf et al. ) . account deficits and manufacturing decline in the usa are the result of low savings, high labor costs, and rising service sectors rather than imports from china (sheng et al. ) . of greater alarm, the trade war has exacerbated account deficits and eroded the usa's comparative advantage in the technological sector and human capital by pressuring china to invest in high technology industries (sheng et al. ) . moreover, deficit reductions have not promoted economic growth (moosa ) , and the trade deficit reduction with china will likely lead to deficits with other countries (lai ). it appears that the usa will not reduce its trade deficit with china, and even if it could do so, the usa will not achieve strong economic progress but instead develop deficits with other countries. the trump administration, as long as it is in power, will likely not let up in its pressure toward china. its othering of china continues to be relevant as long as the usa continues to maintain unsatisfactory wages and societal interruptions owing to the pandemic. although the usa has legitimate reasons for pressuring china, the country is also a convenient target for domestic grievances. although china would prefer to return to the status quo, it will resist and counterattack us actions to both prevent economic decline and showcase power to domestic and foreign audiences. funding we did not receive funding for this research. we do not use a software program for this paper and do not have separate coded data. bbc news. trump accuses china of 'raping' us with unfair trade policy china falling further behind us trade deal energy targets, even as crude oil imports soar to record volumes chinese nationalism under xi jinping revisited the scandal of trump's china policy. the new york times trump just ramped up tariffs on $ billion worth of chinese goods. here are all the products that will get hit political news and stock prices house sends $ . t stimulus bill to trump wto and economic nationalism: china's car industry in the st century the us-china trade war: dominance of trade or technology? territorial disputes and nationalism: a comparative case study of china and vietnam council on foreign relations (cfr) us files trade complaint over china's 'excessive' ag subsidies, cnbc clean cars rollback adds to evs from national identity to national identity in a multi-national country nations and nationalism the political economy of international relations global political economy: understanding the international economic order tears of rage: chinese nationalist reactions to the belgrade embassy bombing rare earth minerals and commodity resource nationalism history, chinese nationalism, and the emerging sino-japanese conflict the us-china trade war, the american public opinions, and its effects on china lawder, david. drag from trump's trade wars continues to ripple through trump says demand is back trump blacklists more china tech companies days before xi summit lessons from previous the first new nation: the united states in historical and comparative perspective american exceptionalism: a double-edged sword trump imposes tariffs on solar panels and washing machines in first major trade action the thucydides trap as an alternative explanation for the us-china trade war trump administration cracks down on giant chinese tech firm, escalating clash with beijing: the president also signed an executive order to protect u.s. networks from foreign espionage, a move that appears to target china the surprisingly swift decline of us manufacturing employment neo-internationalism and china's diplomacy trump proclamation bars entry of certain chinese students. inside higher ed trump's repeated falsehood on auto plants trump defends using 'chinese virus' label, ignoring growing criticism. the new york times why will trump lose the trade war? nationalism, patriotism, and foreign policy attitudes among chinese university students ethno-symbolism and nationalism the oil industry's covert campaign to rewrite american car emissions rules. the new york times as trump's trade war escalates to department of the treasury. treasury designates china as a currency manipulator never forget national humiliation: historical memory in chinese politics and foreign relations officials push u.s.-china relations toward point of no return. the new york times china voices strength, pushes nationalism around trade war powerful patriots: nationalist protest in china's foreign relations chinese nationalism and foreign policy after deng china's new assertiveness in the south china sea it is dangerous to advocate china-us disengagement popular nationalism: global times and the us-china trade war reasons midwest farmers hurt by the u.s.-china trade war still support trump. the conversation a nation-state by construction: dynamics of modern chinese nationalism conflict management and peace science, nations and nationalism, and studies in conflict and terrorism, among others. his collaborative projects have been funded by the national science foundation, us department of commerce, and norwegian centre for international cooperation in education. he teaches a range of undergraduate and graduate courses in international relations, comparative politics, and research methods. he is faculty advisor for model united nations and model arctic council at uaf mcbeath is professor emeritus of political science at the university of alaska fairbanks (uaf) his publications include approximately sixty journal articles and book chapters and sixteen books, the most recent of which are big oil in the united states (mcbeath, ) and environmental education in china (mcbeath and mcbeath, with tian qing and huang yu he has published dozens of peer reviewed journal articles in both chinese and english. he has also led several research programs granted by apec, china's national social sciences foundation, and other state and provincial foundations we have no conflicts of interest or competing interests in this project. our data are open source and cited. key: cord- -baqaqsli authors: dreesen, david w. title: animal vaccines date: - - journal: rabies doi: . /b - - / - sha: doc_id: cord_uid: baqaqsli rabies in terrestrial animals, primarily carnivores, is caused by the classic genotype rabies virus. even though the widespread vaccination of domestic dogs has been the one most effective factor in the reduction of human rabies, the number of human deaths worldwide is greater than that of the combined deaths from polio, meningococcal meningitis, japanese encephalitis, yellow fever, severe acute respiratory syndrome and avian influenze (bird flu).tools are available in highly efficacious and safe animal and human vaccines. multiple factors can, however, prevent their use effectively in many areas of the world. for several decades, virtually all rabies nerve tissue origin (nto) vaccines were inactivated with phenol using the method described by semple. the nto vaccines currently in use for mass vaccination campaigns in africa, latin america, and the caribbean are primarily produced from rabies virus-infected suckling mouse brains or lamb brains. these vaccines are shown to be effective in campaigns. however, nto-killed vaccines for dogs and other animals have often, in the past, resulted in post-vaccinal nervous system reactions that could result in the death of the vaccinated animals. in his quest for a means to prevent rabies in humans, louis pasteur initiated research into animal rabies vaccine in france in the early s (bunn, ) . virus obtained from a rabid dog was first serially passed in rabbits by intracerebral inoculation at specified time intervals. dogs were then vaccinated at various time intervals and challenged with rabies virus. although this method produced acceptable results, pasteur found that by serial intracerebral inoculation of monkeys with the dog origin virus, the incubation period increased while the virulence of the virus decreased. by using this regimen, pasteur demonstrated that dogs vaccinated were resistant to subsequent challenge with virulent street (non-laboratory propagated) rabies virus. in , pasteur attenuated, or weakened, the virus by desiccation (bunn, ) to improve on the safety of these early attempts to produce a rabies vaccine. in a review by friedberger and frohner ( ) , it was reported that hogyes and protopopoff and others conducted further studies to improve on the safety and efficacy of vaccines for dogs and to reduce the number of doses needed. in , the first international rabies conference recommended that fixed virus for canine rabies vaccines be completely inactivated or attenuated so that they caused no disease in dogs vaccinated either subcutaneously (sc) or intramuscularly (im) (schoenig, ) . for the next several decades, virtually all rabies nerve tissue origin (nto) vaccines were inactivated with phenol using the method described by semple (bunn, ) . the nto vaccines currently in use for mass vaccination campaigns in africa, latin america and the caribbean are primarily produced from rabies virus-infected suckling mouse brains or lamb brains. these vaccines have been shown to be effective in campaigns (who, ) . however, nto killed vaccines for dogs and other animals have often, in the past, resulted in post-vaccinal nervous system reactions that could result in the death of the vaccinated animals (bunn, ) . better vaccines were needed. embryonated chicken eggs were used by koprowski and cox ( ) for serial passage of the flury strain (a human rabies virus isolate). the virus was initially passed times in -day-old chicks. vaccine produced from the th to the th chicken embryo passage lost its viscerotropic properties but retained some neurotropic properties. this was designated as flury low-egg passage (lep). while effective in dogs, the vaccine occasionally caused rabies in young pups, cats and cattle (bunn, ) . to increase the safety of the vaccines in these species, koprowski et al. ( ) increased the passages of the flury strain in embryonated eggs until the virus was found to be non-pathogenic for dogs when inoculated intracerebrally following the th passage. this flury high-egg passage (hep) vaccine was declared safe for im use in cats and cattle as well as puppies months of age. however, since cases of vaccine-induced rabies occurred in cats administered im with the flury-hep vaccine, it was later withdrawn from the market (cabasso etal., ; dean and guevin, ) . the flury and kelev strains of the rabies virus are used to produce chick embryo origin (ceo) modified live virus (mlv) vaccines. tissue culture (tc) vaccines, such as those derived with the street alabama dufferin (sad) strain, which was adapted to hamster kidney cells (fenji, ) and the evelyn-rokitnicki-abelseth (era) strain, are grown on porcine kidney cells (abelseth, ) and are commonly used to produce mlv vaccines (reculard, ) . several other mlv vaccines have been produced over the years. these mlv vaccines, especially those using the ceo, sad and era strains are still used extensively in asia and africa and parts of europe and have been adapted for oral immunization of carnivores, including domestic dogs and cats (blancou and meslin, ) . the tc mlv vaccines produce fewer allergic reactions than the ceo vaccines. potency tests for mlv vaccines for animal use consist of measuring the titer of infectious virus in a animal vaccines sample from each filling lot (see section . . ). if the titer is as high as that proved efficacious in the species of animal for which the vaccine is intended, the vaccine is released for use (sizaret, ) . even though mlv vaccines have been trustworthy over the years, the use of inactivated (killed) cell culture vaccines is increasing in areas of the world where mlv vaccines are still in use. the who does not recommend mlv vaccines for parenteral use in animals (who, ) and no mlv rabies vaccines are currently licensed for use in the usa. the concept of oral rabies vaccines (orv) was first proven to be successful in (baer et al., ) . using the sad berne strain of virus adapted from the era strain, several types of mlv orv vaccines have been produced for use in baits for free-ranging animals that serve as vectors for the maintenance and transmission of the disease in wildlife species . orv have been used extensively in europe since and in canada from with considerable success (isara et al., ; aubert et al., ) . unfortunately, the live-virus sad vaccines contained some degree of residual pathogenicity for wild rodents (artois et al., ) and resulted in partially impaired immune responses in fox cubs < weeks old born from sad b -vaccinated vixens, resulting in insufficient protection against rabies (muller et al., ) . since the early to mid- s, the sad strain used in vaccine has been replaced by the sag- and sag- (sad-avirulent-gif) strains in the development of vaccines. the sag- strain, the strain of choice, is a double mutant isolated from the sad berne strain after two successive selection steps utilizing anti-glycoprotein monoclonal antibodies. this strain is avirulent following intracerebral inoculation of immunocompetent mice and protects the mice against challenge with challenge virus standard (cvs) (lafay et al., ) . the sag- strain of rabies virus, packaged in chicken-head baits, has successfully protected captive african wild dogs against rabies challenge (knobel et al., ) . in studies conducted at the centers for disease control and prevention in atlanta, georgia, usa, the sag- vaccine produced no clinical illness in laboratory vaccinates (beagles) and residual sag- virus was isolated from only one of oral swabs from the dogs (fekadu, et al., ; orciari et al., ) . no orv derived from sad/sag origin vaccines are currently licensed for use in the usa (compendium, ) . a recombinant vaccinia virus expressing the rabies virus glycoprotein gene (v-rg) was developed by inserting the cdna of the glycoprotein gene of the era strain into the thymidine kinase gene of the copenhagen strain of vaccinia virus. initial studies of this new orv were conducted to determine whether the v-rg recombinant virus vaccine satisfied the various criteria that had to be met for such a vaccine to be distributed in the wild (kieny et al., ; wiktor et al., ) . criteria included: the vaccine would be effective when delivered by an oral bait; the baits would be readily accepted by target species but would be rabies virusfree; the vaccine in the baits would have reasonably long-term genetic and thermal stability; the vaccine would be biologically contained in the host; oral exposure to baits with the vaccine would produce full protection against rabies virus challenge; that no non-target species would develop rabies if they ingested the baits; and that the baits were clearly identified and safe for contact with humans. in an extensive series of trials carried out in the usa and in france, these criteria were met and the vaccine was licensed in for raccoons to prevent spread of raccoon rabies (and later to prevent the spread of mexican dog rabies to texas coyotes along the south texas border with mexico) by the us department of agriculture (usda) (wiktor et al., ; rupprecht et al., ; brochier et al., brochier et al., , brochier et al., , desmettre et al., ) . the single licensed product is produced only by merial, inc., athens, ga, usa as raboral v-rg tm for use by governmental (state public health) agencies as an orv for raccoons and coyotes. in the usa, raboral v-rg tm is currently delivered to raccoons and coyotes in an extended fishmeal polymer bait, which contains mg of tetracycline hydrochloride as a bone biomarker and a plastic sachet containing . ml of the vaccine. an extruded poultry-based bait with identical vacdne content has been shown to be more effective for targeting gray foxes (merial, inc., athens, ga, usa). the successful use of the orv to achieve containment or elimination of rabies in some terrestrial wildlife animals in the usa and canada is indicated by the effective containment to near elimination of red fox rabies in southern ontario (macinnes et al., ) , canine rabies in south texas (fearneyhough et al., ) and raccoon rabies in ohio (krebs et al., ) , southern ontario (rosatte et al., ) and eastern new brunswick (slate et al., ) . in , over million baits were distributed in states in the usa (slate et al., ) . new and potentially more effective oral vectored vaccines and more effective baits, including a fishmeal coated sachet bait, are being developed for orv (slate et al., ) . a canarypox-rabies glycoprotein recombinant vaccine was developed and found to be as effective as other poxvirus-rabies glycoprotein recombinants (taylor et al., (taylor et al., , . live canarypox virus that expresses the rabies virus glycoprotein has been licensed in the usa as a parenteral monovalent vaccine for cats and as a combination rabies vaccine for cats with feline panleukopenia virus, feline parvovirus and feline calicivirus vaccines included in the product. a combination canarypox-rabies vaccine with the whole-cell bacterin of neorickettsia risticii included is also licensed for use in the prevention of potomac fever in horses. these are the only rabies virus glycoprotein vaccines currently licensed in the usa (compendium, ) . a recombinant adenovirus-vectored vaccine expressing rabies virus glycoprotein (adrab.gp) was shown to be capable of inducing antibody immune responses in greyhound dogs immunized either subcutaneously or intramuscularly. the dogs had been previously vaccinated for rabies but had low or no rabies antibody titers (tims et al., ) . this vaccine holds promise as a rabies virus vaccine for dogs. the inactivated vaccines require that the rabies virus be produced in high concentrations. this is initially done by growing the virus strain (primarily cvs- , pittman-moore (pm)-nil and pasteur virus (pv)-bhk strains) in the brain tissue of rabbits, baby hamster kidney (bhk) cells, suckling mouse brains (smb), guinea pig brain cells, chick embryo cells (ceo), vero cells or other substrates (precausta and soulebot, ; reculard, ) . neonatal mice can be used as they lack the immunogenic (or allergenic) myelin that caused encephalomyelitis occasionally noted in animals vaccinated with earlier smb nto killed vaccines. the production methods used for the tco rabies vaccines have allowed less allergenic but more immunologic products (greene and rupprecht, ) . various methods, which are still valid, have been used to render the virus non-pathogenic or essentially inactivated (killed) as vaccines. these include, but are not limited to, beta propiolactone (bpl), uv light, and acetylethylamine as well as other amines. phenol and formaldehyde are no longer recommended for virus inactivation (reculard, ) . the most commonly used inactivating agent is bpl. once inactivated, adjuvants are added in order to increase the immune response to the antigen. the most common adjuvants are aluminum hydroxide, aluminum phosphate, saponin (in cattle vaccines) and, rarely, oil adjuvants (precausta and soulebot, ) . much of the information on cell lines, inactivating methods and adjuvants is proprietary and cannot be reported specifically for any one vaccine. the stability of these inactivated cell culture vaccines has allowed the rabies vaccine to be combined with other vaccines and bacterins such as canine distemper, canine adenovirus type , leptospira and parvovirus for canines. for cats, the combination vaccines include feline panleukopenia-virus, feline parvovirus and feline calicivirus. a combined rabies and foot-and-mouth disease vaccine is available for cattle, sheep and goats (who, ) . the potency and safety of the inactivated rabies vaccines have proven to be quite good. in , the national institutes of health (nih) of the us department of health and human services (dhhs) adopted a mouse inoculation test to measure the potency of inactivated vaccines (seligmann, ) . this was necessitated because of the poor performance of the initial manufactured tissue culture origin vaccines (bunn, ) . although a number of other tests to measure vaccine potency are used throughout the world, the nih test is considered the 'gold standard' for measuring the ability of an inactivated vaccine to protect a mouse against virus challenge. the nih test relies on challenge exposure of immunized mice to one virus strain (cvs), a strain thought to be derived from the original pasteur isolate (baer, ) . this test has some inherent bias towards vaccine from the same virus strain origin when comparing vaccine efficacy across the variety of strains (i.e. sad, flury strain vaccines) used to prepare vaccines (barth et al., ) , but this bias does not occur when non-pasteur stain vaccines are tested for protective potential against wild virus strains (baer, ; wunderli et al., a) . in addition, the nih uses two doses of vaccine administered at a one-week interval by an intraperitoneal challenge two weeks later. this vaccination route is quite different from that used for routine administration of rabies vaccine. the second dose prevents an evaluation of the vaccine's primary immunologic potential and the challenge results in a disruption of the blood-brain barrier, allowing neutralizing antibodies in the serum to prevent infection. as a result of these limitations, the who has acknowledged that the nih test needs some improvements or further suggesting that a new rabies potency test may be needed (who, , i ). two recent reports have proposed an alternative method that avoids these shortcomings (wunderli et al., a (wunderli et al., , b . due to the higher antigenic mass and the use of adjuvants, inactivated rabies vaccines have produced post-vaccinal local and systemic reactions. the most common non-neurologic reactions include soreness, lameness and regional lymphadenopathy in the injected limb. fever and anaphylaxis have also been reported (dreesen, ; greene and rupprecht, ) . focal vasculitis and granulomas have been seen - months after vacdnation (greene and rupprecht, ) . post-vaccinal sarcomas may develop as a result of sustained inflammatory reactions at the site of the vaccination that involve the underlying dermas. such postvaccinal sarcomas are often aggressive and invasive, espedally in cats, months to years following vaccination (dubielzig et al., ; kass et al., ; greene and rupprecht, ) . a review of cases of fibrosarcomas in cats following single vacdnation showed that % of the cats with vaccination-site tumors had received rabies vaccine, % were administered a non-rabies combination vaccine and % received a feline leukemia vaccine (hendrick et al., ) . it is not unusual for palpable lesions to occur in cats administered killed vaccine subcutaneously (schulze et al., ) . adverse incidence rates for reactions to rabies vaccination in a retrospective study of ferrets was % when the rabies vaccine was given alone and . % when given in combination with distemper vaccine. the most common adverse events were vomiting and diarrhea (moore et al., ) . the new generation of vectored recombinant vaccines now appearing on the market, such as the avipoxvirus vaccine recently licensed for use for cats in the usa (a rabies glycoprotein, live canarypox vectored vaccine) appears to produce few, if any, allergic or neoplastic reactions (greene and dreesen, ; greene and rupprecht, ) . the who's world survey of rabies reported that there are at least countries or territories that reported producing animal rabies vaccines during . for the production of animal rabies vaccines, countries use cell culture, seven use neural tissue and six countries use embryonated eggs (who, ) . four countries produced more than one type of vaccine. both mlv and inactivated vaccines are produced worldwide. the who world survey of rabies reported that brazil is the major producer of nto rabies vaccines for animal use followed by bangladesh, romania, tunisia and e salvador (who, ) . these five countries account for . % of the . million doses of nto vaccine, primarily smb origin (fuenzalida strain), reported produced for the year. this same survey reported that the usa produced approximately million doses of tco rabies vaccines, % of all tco animal vaccines produced. vietnam is reportedly the primary source of embryonated egg-origin animal vaccine, producing % of this vaccine produced worldwide. it should be noted here that argentina, france, germany, india and a number of other countries that presumably produce animal rabies vaccines did not contribute to the who report. during the -year period - , the availability of rabies vaccines for dogs and cats in latin america grew by . % and the total doses of vaccine administered for these species rose by . % (redipra, ) . vaccine coverage increased from . % in brazil to . % in the southern cone (argentina, chile, paraguay, uruguay). however, there was a . % decline in the andean area (bolivia, columbia, ecuador, peru, venezuela) and a . % decline in central america. this same report denotes that, in the andean area, % of the canine population was vaccinated, in the southern cone . %, brazil %, central america %, mexico % and latin caribbean %. the who recommends that % of dogs in a population should be effectively immunized to prevent an epidemic of canine rabies (coleman and dye, ) . there were laboratory confirmed canine rabies cases in all of latin america during and during . during the same periods, cattle accounted for and cases and other domestic animals accounted for and cases respectively. many types of rabies vaccines are currently marketed in the usa for use in domestic animals. there are inactivated monovalent rabies vaccines licensed for dogs and cats, two for ferrets, four for horses, four for cattle and five for sheep. two inactivated vaccines are combined with other biologics for use in horses. in , a new generation of vaccines was licensed for use in cats. these are the live canarypox-rabies virus glycoprotein recombinant vectored vaccines, either monovalent (one licensed vaccine) or in combination with feline panleukopenia virus, feline parvovirus and feline calicivirus vaccines (compendium, ) . a live vaccinia-rabies virus glycoprotein recombinant vectored vaccine is licensed for restricted use in wildlife raccoons and coyotes. as stated earlier, there are no mlv (attenuated) rabies vaccines licensed for use in the usa. all currently licensed killed rabies vaccines intended for use in carnivores must protect of or of (or a statistically equivalent number) animals from an im challenge with a rabies virus for days post challenge and % of controls must die from the challenge (code of federal regulations, ). alternative challenge requirements have been outlined when the test animals are of a species other than carnivores (code of federal regulations, ). the us department of agriculture (usda), animal and plant health inspection service (aphis), center for veterinary biologics has jurisdiction over licensure of rabies vaccines in the usa. the national association of state public health veterinarians (nasphv) publishes annually the compendium of animal rabies prevention and control (compendium, ) this compendium is a basis for animal rabies programs and the nasphv issues it as recommendations. some states (e.g. georgia) and various cities and counties adopt the recommendations in the compendium as regulations for animal rabies control and prevention. the inactivated tco vaccines should be used in animals at months of age or older and then again one year later. this minimum age precludes maternal antibody blockage and recognizes the immature immune system's often poor response (greene and dreesen, ) . depending on the vaccine, the animal species and, at times, local regulations, the animals should be vaccinated annually or triennially thereafter (compendium, ) . depending on the vaccine type and the species, the vaccine is administered either im or sc, while some vaccines can be administered either way. the minimum age for animal vaccination is weeks of age for the licensed vectored vaccines. regardless of the rabies vaccine type, only when the antibody response peaks, at approximately days after primary vaccination, is the animal considered fully immunized, if vaccination has been administered in accordance with the manufacturer's recommendations. from an epidemiologic viewpoint, the effectiveness of canine rabies prevention and control programs can be measured by comparing reports of rabies in dogs with reports of increases in cat rabies. this was apparent during the recent raccoon rabies epidemic in the middle atlantic and northeastern usa (krebs et al., ; hanlon and rupprecht, ) . the increase in rabies cases in cats, while dog rabies cases remained substantially unchanged, reflects the vaccine status of the two populations as well as the number of feral animals in the two populations (eng et al., ; petronek, ; dreesen, ) . of respondents in a survey of state and community health officials by johnson and walden ( ) , % stated that canine rabies vaccination was required by state law while only % stated that cat vaccination was state law. the need for cat vaccination and feral population control cannot be overemphasized (dreesen, ) . johnson and walden's survey ( ) also noted that over-the-counter sales of rabies vaccines was permitted in states and that, at that time, vaccination of wolfhybrids was permitted in states; however, in all but two of these states the owner must sign a liability statement. fourteen other states did not address the wolf-hybrid issue at all. in , after extensive studies at the centers for disease control and prevention (niezgoda et al., ) , a rabies vaccine for ferrets was approved by the usda, aphis. the ferret should be treated in a similar manner as a dog or cat in regard to vaccination and post-exposure management (compendium, ) . vaccination of wolf-hybrids with canine rabies vaccine is still a matter of considerable debate. in a meeting of taxonomists in , it was concluded that rabies vaccines for dogs would probably protect wolves and their hybrids as they are genetically virtually indistinct from the domestic dog (dreesen, ) . at least one well-documented case of rabies has occurred in a properly vaccinated wolf-hybrid (jay et al., ) . this animal was vaccinated with a -year vaccine at months of age and received other vaccines and bacterins and an antihelminthic on the same day. six months later the animal was found with a dead skunk in its mouth. within weeks the animal developed signs suggestive of rabies, was euthanized and rabies was confirmed in the laboratory. currently, there are no licensed rabies vaccines for wolf-hybrids and the compendium states that wild animals and hybrids (offspring of wild animals crossbred to domestic animals) should not be kept as pets. an animal can be considered to be immunized against rabies virus exposure approximately days after the primary rabies vaccination, which is consistent with a peak antibody response (compendium, ) . thus, an animal is considered immunized if the primary vaccination was administered at least days previously and the follow-up vaccinations have been administered as recommended by the package insert and/or the compendium ( ). the nasphv (compendium, ) recommends that unvaccinated dogs, cats and ferrets exposed to a known or suspected rabid animal should be euthanized immediately. if not euthanized, the animal should be placed in strict quarantine for months and vaccinated either upon entry into isolation or one month prior to release. animals with expired vaccinations should be evaluated on a case-by-case basis. currently, vaccinated dogs, cats and ferrets should be revaccinated immediately following exposure and kept under control and observation for days. it has been shown that there is some evidence that the use of vaccine alone will not reliably prevent rabies from occurring in an unvaccinated domestic animal (hanlon et al., ) . vacdnated livestock exposed to rabies should be revaccinated and observed for days (compendium, ) . if not previously vaccinated, food animals should be slaughtered within days with disposal of tissues in the exposed area. if not slaughtered within this time period, the animal should be closely observed for months. as previously mentioned, the compendium ( ) is issued as recommendations only. some states do not strictly adhere to the recommendations. for example, the texas health and safety code originally followed the previously noted recommendations for animals exposed to rabies (clark and wilson, ) . however, in , the code was amended; unvaccinated domestic animals exposed to a rabid animal were to be euthanized or vaccinated immediately after exposure, kept in isolation for days and given booster vaccinations in the third and eighth week of isolation. this regimen was based loosely on recommendations for humans exposed to rabies virus. a retrospective study conducted by clark and wilson ( ) found that . % of unvaccinated animals did not develop rabies during the - period during which the recommendations of the nasphv were followed. two pep failures did occur ( . %). for the period - , after the texas code was amended to allow pep for unvaccinated animals exposed to rabies, of animals ( . %) that received the pep booster vaccinations did not die of rabies. there was no statistical difference between the two regimens under conditions followed in texas. in a follow-up study for the years - , wilson and clark ( ) found only four of ( . %) domestic animals that received the pep protocol, as recommended, during the previous -year period developed clinical rabies. they concluded that this is an effective pep protocol and 'has been proven to be effective for the control of rabies in animals'. this alternative method of pep for unvaccinated domestic animals exposed to rabies, as practiced in texas, has not been endorsed in the compendium ( ) . propagation of rabies virus in pig kidney cell culture partial pathogenicity for rodents of vaccines intended for oral vaccination against rabies: a comparison oral wildlife rabies vaccination field trials in europe, with recent emphasis on france evaluation of an animal rabies vaccine by use of two types of potency tests oral vaccination of foxes against rabies nih test, a problematic method for testing potency of inactivated rabies vaccine modified live-virus rabies vaccination for oral immunizations of carnivores elimination of fox rabies from belgium using a recombinant vaccinia-rabies vaccine: an update large-scale eradication of rabies using recombinant vaccinia-rabies vaccine (letter) use of a vaccinia-rabies recombinant virus for the oral vaccination of foxes against rabies canine and feline rabies vaccines, past and present vaccination of cats against rabies postexposure rabies prophylaxis and preexposure vaccination failure in domestic animals title immunization coverage required to prevent outbreaks of dog rabies compendium of animal rabies prevention and control rabies vaccination of cats use of vaccinia rabies recombinant for oral vaccination of wildlife preexposure rabies immunization mycoplastic sarcoma originating at the site of rabies vaccination in a cat rabies surveillance, united states results of an oral vaccination program for coyotes efficacy and safety of an oral rabies vaccine (sag- ) in dogs propagation of rabies virus in a culture of hamster kidney cells friedberger and frohner's veterinary pathology rabies. in: infectious diseases of the dog and cat rabies and other lyssavirus infections the reemergence of rabies postexposure prophylaxis for prevention of rabies in dogs comparison of fibrosarcomas that developed at vaccination sites and at non-vaccination sites in cats: cases ( - ) sylvatic rabies in italy: epidemiology studies on chick-embryo-adapted rabies virus. vi. further changes in pathogenic properties following prolonged cultivation in the developing chick embryo rabies surveillance in the united states during rabies surveillance in the united states during vaccination against rabies: construction and characterization of sag , a double avirulent derivative of sadbern elimination of rabies from red foxes in eastern ontario incidence and risk factors for adverse events associated with distemper and rabies vaccine administration in ferrets effect of maternal immunity on the immature response to oral vaccination against rabies in young foxes lyssavirus p gene characterization provides insights into the phylogeny of the genus and identifies structural similarities and diversity within the encoded phosphoprotein pathogenesis of experimentally induced rabies in the domestic ferret rapid clearance of sag- rabies virus from dogs after oral vaccination free roaming and feral cats -their impact on wildlife and human beings vaccines for domestic animals cell-culture vaccines for veterinary use report of the viii meeting of directors of national rabies control programs in latin america emergency response to raccoon rabies introduction into ontario oral vaccination of wildlife against rabies: opportunities and challenges in prevention and control oral immunization and protection of raccoons (procyon lotor) with a vaccinia-rabies glycoprotein recombinant virus vaccine experimental studies with killed canine rabies vaccine repeated physical and cytologic characterizations of subcutaneous postvaccinal reactions in cats potency tests requirements of the united states national institutes of health (nih) general considerations in testing the safety and potency of rabies vaccines status of oral rabies vaccination in wild carnivores in the united states biological and immunogenic properties of a canarypoxrabies recombinant, alvac-rg (vcp ) in non-avian species efficacy studies on a canarypox-rabies recombinant virus adult dogs receiving a rabies booster dose with a recombinant adenovirus expressing rabies virus glycoprotein develop high titers of neutralizing antibodies world health organization expert consultation on rabies world health organization. requirements for rabies vaccine for veterinary uses (requirements for biological substances no world survey of rabies no. for the year . p. i. geneva: world health organization world survey for rabies no. for the year . whoicdsicsriephi . p. . geneva: world health organization world health organization expert consultation on rabies immunogenic properties of vaccinia recombinant virus expressing the rabies glycoprotein protection from rabies by a vaccinia recombinant containing the rabies virus glycoprotein gene rabies control in south and southeast asia postexposure rabies prophylaxis protocol for domestic animals and epidemiologic characteristics of rabies vaccination failures in texas: - effects of vaccine route and dosage on protection from rabies after intracerebral challenge in mice effect of heterogeneity of rabies virus strain and challenge route and efficacy of inactivated rabies vaccines in mice rabies virus key: cord- -y pc qfc authors: zhou, bo-ya; wang, wen-bo; wu, xiao-li; zhang, wen-jie; zhou, guang-dong; gao, zhen; liu, wei title: nintedanib inhibits keloid fibroblast functions by blocking the phosphorylation of multiple kinases and enhancing receptor internalization date: - - journal: acta pharmacol sin doi: . /s - - -y sha: doc_id: cord_uid: y pc qfc keloid is a benign skin tumor characterized by its cell hyperproliferative activity, invasion into normal skin, uncontrolled growth, overproduction and deposition of extracellular matrices and high recurrence rate after various therapies. nintedanib is a receptor tyrosine kinase inhibitor targeting vegf, pdgf, fgf, and tgf-β receptors with proved efficacy in anti-angiogenesis and in treating various types of cancers. in this study, we investigated the effects of nintedanib on keloid fibroblasts in both in vitro and ex vivo models. keloid fibroblasts were prepared from keloid scar samples in active stages collected from patients. we found that nintedanib ( − μm) dose-dependently suppressed cell proliferation, induced g( )/g( ) cell cycle arrest, and inhibited migration and invasion of keloid fibroblasts. the drug also significantly inhibited the gene and protein expression of collagen i (col- ) and iii (col- ), fibronectin (fn), and connective growth factor (ctgf), as well as the gene expression of other pathological factors, such as alpha smooth muscle actin (α-sma), plasminogen activator inhibitor- (pai- ), fk -binding protein (fkbp ), and heat shock protein (hsp ) in keloid fibroblasts. furthermore, nintedanib treatment significantly suppressed the phosphorylation of p , jnk, erk, stat , and smad, enhanced endocytosis of various growth factor receptors. using an ex vivo tissue explant model, we showed that nintedanib significantly suppressed cell proliferation, migration, and collagen production. the drug also significantly disrupted microvessel structure ex vivo. in summary, our results demonstrate that nintedanib is likely to become a potential targeted drug for keloid systemic therapy. keloids are characterized by uncontrolled growth beyond the original lesion, hyperproliferative fibroblasts and overproduction and deposition of extracellular matrices (ecm). thus, keloids are considered as benign skin tumors [ , ] , and are suitable for anticancer strategies [ , ] . in addition, chemotherapy agents targeting signaling pathways are good drug candidates for cancer therapy. previous studies showed the involvement of abnormal signaling pathways, including the transforming growth factor-β (tgf-β)/smad, phosphatidylinositol -kinase (pi k)/mammalian target of rapamycin (mtor) and mitogen-activated protein kinase (mapk)-signaling pathways, in keloid development [ , ] . additionally, enhanced cell proliferative and migratory abilities, abnormal angiogenesis, and vascular hyperpermeability have also been found to be enhanced by vascular endothelial growth factor (vegf), platelet-derived growth factor (pdgf), and fibroblast growth factor (fgf) in keloid development [ , ] . recent progress in this field also demonstrated that inhibitors of multiple signaling pathways are the preferred targeted drugs due to their synergistic mechanisms of antagonizing different signaling pathways [ ] . nintedanib (bibf- ) is a receptor tyrosine kinase inhibitor (tki) targeting vegf receptors (vegfrs), fgf receptors (fgfrs), and pdgf receptors (pdgfrs) [ ] with proven efficacy in antiangiogenesis and in treating various types of cancers, including hepatocellular carcinoma (hcc), renal cell carcinoma (rcc), colorectal cancer (crc), prostate cancer, and gynecologic malignancies [ ] . for example, nintedanib reduced vessel density, vessel permeability, and tumor growth by inhibiting the pi k-akt and mapk-signaling pathways [ ] . nintedanib was also reported to reduce fibrotic progression by inhibiting early events in tgf-β signaling, involving phosphorylation of the type ii tgf-β receptor, activation of smad , and p mapk phosphorylation [ ] . in particular, nintedanib is the first targeted chemotherapy drug for idiopathic pulmonary fibrosis (ipf) therapy in patients, indicating the therapeutic potential of tkis in the treatment of nonmalignant diseases. previous studies have demonstrated that pdgfr, vegfr, and fgfr are usually localized at low-density, detergent-resistant membrane domains called lipid rafts/caveolae [ , ] . lipid rafts/ caveolae are small cholesterol-enriched domains present on the cell membrane. when cholesterol is depleted, receptors are retained in the residual elements of the caveolae, and remain able to bind the ligand, but the downstream cascades are blocked due to steric inhibition and the inability to form a signaling complex [ ] . it has been shown that the keloid mechanism involves multiple signaling pathways, such as vegf, pdgf, fgf, and tgf-β, but there has been no report on a drug that could target more than three growth factors among the studies on drugs targeting keloid cell behavior [ , , ] . this study aimed to explore the targeted drug effect of nintedanib on antagonizing multiple signaling pathways related to vegf-r, fgf-r, pdgf-r, and tgf-β-r and examine its therapeutic effect on keloid fibroblast behaviors, including cell proliferation, cell cycle progression, overproduction of extracellular matrix, cell migration, and cell invasion, using in vitro and ex vivo models. in addition, we also investigated whether disruption of the lipid raft/caveolae structure is involved in the drug effects. patients and keloid samples fifty-four keloid scar samples in active stages were collected from patients without any previous treatment before surgery (see details listed in table ). keloid specimens derived from surgical excision were donated by the patients for research purposes only with written informed consent. the procedures for processing human tissues and cells were approved by the ethics committee of the shanghai jiao tong university school of medicine. chemical reagents nintedanib (selleck chemicals, houston, tx, usa) was dissolved in % dimethyl sulfoxide (dmso) to a stock concentration of mm and stored at − °c under light-protected conditions. the reagent was diluted directly to the desired dose upon use, with an identical final concentration of dmso in both the experimental and control groups. the final concentration of dmso was maintained below . % (v/v) in all of the following experiments to ensure no cytotoxic effect on the cultured kfs. isolation and culture of keloid fibroblasts isolation and culture of keloid fibroblasts were performed according to a previous protocol [ ] . keloid tissues were treated with collagenase nb (serva electrophoresis, heidelberg, germany) dissolved in dulbecco's modified eagle's medium (dmem, hyclone, logan, ut, usa, . % v/v) for h at °c with rotation. after digestion, cells were collected and resuspended in dmem supplemented with % fetal bovine serum (fbs, gibco-invitrogen corp., grand island, ny, usa) and penicillin/streptomycin (gibco) and seeded onto -cm culture dishes. the primary, first-passage cells were utilized in the subsequent experiments. to better represent the keloid cases, kfs derived from three patient tissue samples were mixed as a pooled cell sample. cell counting kit- (cck- ) assay the drug effect on kf proliferation was analyzed by cck- (dojindo laboratories, mashiki, japan). briefly, primary cultured kfs were seeded in -well plates per well and starved for h. then, the medium was replaced with culture medium with or without nintedanib, and the cells were tested with cck- reagent at days , , , , and . the medium optical density value of each well was measured at nm using a microplate reader (thermo fisher scientific, waltham, ma, usa). the assay was carried out in quadruplicate and repeated with three independent pooled cell samples. cell cycle analysis as previously described [ ] , kfs treated with nintedanib at different concentrations ( , . , , , μm) for days were collected. the cells were then fixed in % ethanol at °c overnight followed by staining with a cell cycle analysis kit ( sea biotech, shanghai, china). afterwards, a flow cytometer (beckman coulter, fullerton, ca, usa) equipped with modifit lt v . software was used for flow cytometric analyses, and the experiment was repeated in four independent pooled cell samples. female asian chest female asian arm male asian chest female asian shoulder female asian shoulder male asian chest female asian chest male asian chest female hoechst-positive cells (blue) were imaged under a fluorescence microscope (olympus, tokyo, japan) and counted using image-pro plus (ipp) . software (media cybernetics, silver spring, md, usa). the incorporation ratio of edu-positive cells is shown as the percentage of edu-positive cells of the total hoechst-positive cells. the results were derived from the average cell counts in five randomly selected fields in a well and repeated with three independent pooled cell samples. cell migration assay for the scratch assay, as previously described [ ] , the cell monolayer was scratched using a -μl pipette tip when the cells reached more than % confluence, and the cells were then cultured in serum-free medium with or without nintedanib for h. images were acquired at , , and h after scratching. the results were derived from measured areas in six randomly selected fields in a well and repeated with six independent pooled cell samples. a transwell system ( -μm pore size) was also utilized for the migration assay as reported [ ] . in brief, × serum-starved kfs were seeded in the upper compartment of the boyden chamber in a -well plate in serum-free medium containing nintedanib or vehicle control. after incubation for h, the migrated cells were fixed with % paraformaldehyde and stained with ′, -diamidino- -phenylindole (dapi, sigma, chemical co., st. louis, mo, usa). five high-power fields were randomly selected, and the migrated cells were counted by image-pro plus. this assay was performed in three independent pooled cell samples. furthermore, an oris tm cell migration assay kit (platypus technologies, madison, wi, usa) was used as reported [ ] . briefly, . × kfs were seeded in each well, and after h, the medium was replaced with μl serum-free medium containing different concentrations of nintedanib after removing the stoppers. after another -h incubation, cells were stained with calcein-am (maiyuer bio, shanghai, china). the cell number in the migration zone was counted by image-pro plus. this assay was performed with three independent pooled cell samples. cell invasion assay for the transwell assay, × serum-starved kfs were placed in the upper well of a boyden chamber coated with matrigel (corning). after a -h incubation, invaded cells were fixed and stained with dapi. cell numbers in five randomly selected highpower fields were counted by image-pro plus. the assay was repeated with three independent pooled cell samples. furthermore, an oris tm d embedded invasion assay (platypus technologies) was performed to confirm the inhibitory effect of the drug on kf invasive ability as previously described [ ] . briefly, . × kfs per well were seeded, and after h, the medium was replaced by μl rat-tail collagen i (col- ) solution. after incubation for min, serum-free medium containing nintedanib or vehicle control was added to each well. images of kfs stained with calcein-am were obtained at h post treatment. cells in the detection zone were counted by image-pro plus. this assay was performed with three independent pooled cell samples. ex vivo explant culture of human keloid tissues keloid tissue after epidermal removal was minced into mm × mm × mm fragments. the dermal fragments were equally treated, seeded onto three -cm culture dishes and incubated in dmem containing % fbs until attachment. then, the medium was replaced with fresh medium containing nintedanib or vehicle control. representative images were acquired at the same location on days , , , and after the kfs migrated from the edge of the tissue. on day , the tissue fragments were collected for rt-qpcr and western blotting assays, and the kfs growing in the dishes were collected and counted using a hematocytometer. rna isolation and real-time quantitative pcr after treatment with nintedanib or vehicle control for h in dmem plus % fbs, the cultured kfs were harvested for total rna extraction using trizol reagent (invitrogen life technologies inc., grand island, ny, usa) as previously reported [ ] . for the ex vivo explants, the tissues were minced with scissors and ground by a pro- tissue homogenizer (pro scientific, monroe, ct, usa) to a homogeneous lysate and prepared for rna isolation using trizol reagent. complementary dna (cdna) was synthesized from µg total rna per sample using amv reverse transcriptase (promega, madison, wi, usa). cdna was amplified using rt-qpcr in a realtime thermal cycler (stratagene, la jolla, ca, usa) with power sybr green pcr master mix ( ×) (applied biosystems, foster city, ca, usa) as previously described [ ] . the housekeeping gene β-actin was used as an internal control. each assay was performed in triplicate and repeated with three independent pooled cell samples. the human primers for real-time qpcr analysis are displayed in table . immunofluorescence assay briefly, × kfs per well (for alpha smooth muscle actin, α-sma detection) or × kfs (for col- detection) were seeded in sixwell plates and preincubated for h in regular culture medium. afterwards, the medium was replaced with dmem containing % fbs with or without nintedanib for h (for α-sma detection) or days (for col- detection) followed by fixation in % paraformaldehyde overnight. the primary and secondary antibodies (table ) were diluted according to the instructions. dapi was used for nuclear counterstaining. images of α-sma-positive or col- -positive (green) and dapi nuclear-stained (blue) cells were obtained under a fluorescence microscope (olympus). this assay was performed with three independent pooled cell samples. keloid specimens with a size of mm × mm × mm were fixed in % paraformaldehyde at °c overnight after being cultured in dmem plus % fbs with nintedanib or vehicle control for days. afterwards, the specimens were embedded in paraffin and sectioned to μm thickness. following incubation of the sections with antibodies against col- , fibronectin (fn), cd , and cd (see details in table ), , ′-diaminobenzidine (dab, g , servicebio, wuhan, china) was used as a chromogen to visualize the bound antibodies, and the slides were counterstained with hematoxylin. cd + and cd + vessels were counted in five randomly selected fields under a microscope for semiquantification. this assay was performed with four independent samples. western blotting analysis for ex vivo explants, mg/each of treated or nontreated tissues were washed and cut into small pieces in pbs. the tissues were then homogenized with a pro- tissue homogenizer (pro scientific) in lysis buffer supplemented with a protease inhibitor cocktail (thermo fisher scientific, rockford, il, usa). the lysate was centrifuged at , × g for min at °c. for protein production in cells, kfs were treated with or without nintedanib ( , , μm) for or h to examine cellular signaling molecules or other antigens. protein extraction and western blotting analysis were performed as previously described [ ] . detailed information on the antibodies is listed in table . this assay was performed with three independent samples. to determine whether lipid rafts/caveolae were related to the inhibitory effect of nintedanib on keloid fibroblasts, disruption of lipid rafts/caveolae was carried out by incubating cells in the presence of mm mβcd (sigma) dissolved in double-distilled water (ddh o). cck- and scratch assays were used to measure cell proliferation and migration, respectively. western blotting was also performed to examine the expression of receptors. cells were treated with vehicle control, mβcd ( mm) or nintedanib ( μm) alone or in combination in growth medium. for the combination treatment, the cells were pretreated with mm mβcd for min at °c before the experiment. for the cck- assay, cells were tested on days , , and , and for the scratch assay, images were obtained at and h after scratching. for western blotting assays, cells were collected days after treatment. detailed methods are described above. all the data are presented as the mean ± standard deviation (sd) and were statistically analyzed with one-way anova followed by student-newman-keuls (s-n-k) post hoc test after confirming the normal distribution of the data. for ratio data of the edu assay, nonparametric kruskal-wallis test plus dunn's post hoc test with bonferroni correction was employed. all statistical analyses were performed with the statistical software spss (version . ; spss, inc., chicago, il, usa). a p-value < . was considered statistically significant. nintedanib suppressed kf proliferation and induced g /g cell cycle arrest as shown in fig. a , the cck- assay demonstrated a significant inhibitory effect of nintedanib on kf proliferation in a dosedependent manner during the -day time period compared to the vehicle control group. anova plus the s-n-k test demonstrated significant differences among the groups ( supplementary fig. s , p < . ). next, cell cycle analysis revealed a dose-dependent increase in the cell percentage in g /g phase, whereas a dose-dependent decrease in the cell percentage was found in s phase with significant differences among the groups treated with , and μm nintedanib (p < . , fig. b, c) , indicating that nintedanib induced cell cycle arrest at g /g phase. additionally, an eduincorporation assay also showed significant inhibition of dna synthesis in kfs by nintedanib treatment because much less edu labeling was observed in the treated cells than in the control cells (fig. d) . semiquantitative analysis showed a reduction in edu labeling among the groups (p < . , fig. e ). nintedanib attenuated the migratory capacity of cultured kfs after and h of nintedanib treatment, the scratch assay demonstrated a significant inhibitory effect on kf migration compared to that in nontreated cells when observed under a microscope (fig. a) . at both time points, quantitative analysis further confirmed a significant dose-dependent reduction in cell migration in the treated groups (p < . , fig. b) . similarly, the transwell assay also revealed the inhibitory effect of nintedanib on cell migration by reducing the number of cells that migrated to the bottom surface of the upper chamber in the treated group at h post treatment (fig. c) . semiquantitative analysis showed a dose-dependent reduction in the migrated cell numbers with significant differences among the groups, as revealed by anova plus the s-n-k test (p < . , fig. d) . furthermore, the oris migration assay also confirmed the inhibitory effect of nintedanib on cell migration by both microscopic observation (fig. e ) and quantitative analysis (fig. f) , which revealed a dose-dependent reduction in migrated cell numbers with significant differences among the groups after h of nintedanib treatment (p < . ). nintedanib inhibited the invasive capacity of kfs both the transwell assay and oris invasion assay combined with matrix coating were also performed to investigate the drug effect. as shown in fig. a , b, much fewer cells were able to successfully traverse the matrigel-coated chamber after nintedanib treatment compared to the control group at h post treatment, and the effect was dose-dependent. a similar trend was also confirmed in the oris invasion assay, which used col- coating as the matrix material. as shown in fig. c (microscopic view) and fig. d (quantitative analysis) , a dose-dependent decrease in cell numbers in the detection zone was found with increasing drug concentrations (p < . ). nintedanib suppressed the migration and proliferation of cultured keloid explants to further verify the inhibitory effect of nintedanib on cell migration and proliferation, an ex vivo keloid explant culture model was also established. as shown in fig. e , f, spindle-shaped kfs of the vehicle group migrated out of the edges of the cultured keloid explants and gradually spread over the petri dish within a week with a total cell number of . × after days of culture. however, treatment with increasing concentrations of nintedanib for days significantly inhibited kfs from migrating out of the tissue explants in a dose-dependent manner with significant differences from that of the control group (p < . ). interestingly, the inhibitory effect of the drug was also supported by decreased gene expression of matrix metalloproteinases (mmps). as shown in fig. g , h, mmp and mmp gene levels were significantly reduced in the drug-treated groups, as demonstrated by rt-qpcr analysis (p < . ). nintedanib suppressed the expression of fibrotic factors related to keloid pathogenesis using an in vitro cell culture model, the drug effects on the expression of fibrotic factors at both the gene and protein levels were investigated. as presented in fig. a , at the transcriptional level, treatment with the drug at concentrations of , , and μm for h significantly suppressed the gene expression of col- , fn, connective tissue growth factor (ctgf), plasminogen activator inhibitor- (pai- ), and fk -binding protein (fkbp ) in a dose-dependent manner, with significant differences among the groups (p < . ). the suppression of α-sma and heat shock protein (hsp ) occurred at higher concentrations, and no significant effect was found on col- . consistent with the gene expression results, western blotting also showed significant suppression of the protein production of col- , fn, and ctgf, as shown in fig. b , and semiquantitative analysis revealed significant differences among the different groups (fig. c) . the suppressed protein production of α-sma and col- was also confirmed by immunofluorescence staining of kfs treated with different concentrations of nintedanib (fig. d, e) , which showed decreasing protein expression with increasing drug concentrations. in addition, the drug treatment also disrupted the intracellular collagen pattern (fig. e) . nintedanib inhibited collagen production and disrupted angiogenesis in keloid tissue explants to better mimic the clinical scenario, an ex vivo model of tissue explants was generated for the investigation. immunohistochemical staining of the keloid tissue explants exposed to nintedanib showed a marked decrease in the intensity of col- staining and a reduced fn staining area, which indicated significantly decreased production of col- and fn (fig. a) . consistent with these findings, qpcr and western blotting analyses of tissue explants also showed a reduction in col- and fn expression at both the transcriptional and translational levels (fig. b-d) . furthermore, a reduced number of cd -positive and cd positive microvessels as well as disrupted capillary structure were also observed with nintedanib treatment (fig. a , e, f). to further define the potential mechanism of the above findings, the effect of nintedanib on various signaling pathways was investigated using an in vitro cell culture model. as shown in fig. , treatment of kfs with nintedanib significantly inhibited the phosphorylation of smad and smad at higher concentrations. nintedanib as a multiple signaling blockade for keloids by zhou et al. it also significantly inhibited the phosphorylation of mapk pathway components including p , jnk, and erk, as well as signal transducer and activator of transcription (stat ), in a dosedependent manner (fig. a) . cholesterol is the major structural component of lipid rafts/caveolae; thus, cholesterol depletion was performed to determine whether lipid rafts/caveolae mediate the cellular response to nintedanib. among various approaches, mβcd is the most common method for removing cellular cholesterol [ ] . cck- and scratch assays were used to measure proliferation and migration. the results showed that mβcd treatment alone could have a minor inhibitory effect on cell proliferation and migration, which was significantly different from the control treatment. nevertheless, mβcd was able to rescue to a certain degree the inhibitory effect of nintedanib on kf proliferation and migration when compared to treatment with nintedanib alone (fig. b-d) . interestingly, western blotting assays demonstrated fig. nintedanib inhibits the proliferation of treated keloid fibroblasts. a the cck- assay was used to analyze the proliferation of kfs treated with different concentrations of nintedanib at days , , , , and . b flow cytometry analysis of the cell cycle of treated keloid fibroblasts. c representative cell cycle data were plotted for each group. d after treatment with nintedanib ( , , μm) or vehicle control for h, kfs were incubated with edu for an additional h. the samples were imaged under a fluorescence microscope at × (bar = μm). e the ratio of edu-positive cells to dapi-labeled cells in each group was determined. *p < . vs control, # p < . vs . μm, $ p < . vs μm. abbreviations: g /g , period between the end of m phase and the start of s phase; s, dna duplication phase; g , period between the end of s phase and the start of m phase; m, mitosis. increased protein expression of growth factor receptors in the mβcd and nintedanib combined treatment group relative to the expression levels of nintedanib only treatment group (fig. e) , providing supporting evidence that nintedanib may affect the functions of growth factor receptors by enhancing their endocytosis, which can be disrupted by removing cellular cholesterol in the lipid raft domain. keloids are benign skin tumors in which keloid fibroblasts display tumor cell-like biological behaviors [ , ] . keloids inevitably recur after various treatments, and thus, new therapeutic approaches are needed. targeted therapy is considered an effective strategy for cancer treatment and includes various targets for drug intervention, such as rhodopsin-like g protein-coupled receptors (gpcrs), ion channels, protein kinases, and nuclear hormone receptors, with kinase inhibitors being an important part of targeted drugs for cancer therapy [ , ] . keloid formation is driven by multiple growth factors, including tgf-β, pdgf, and vegf. tgf-β promotes keloid fibroblast proliferation, collagen production, inhibition of mmps [ , ] , and the smad-signaling pathway [ ] . pdgf and vegf also contribute to keloid pathogenesis by stimulating collagen production and angiogenesis [ ] . additionally, pdgf contributes to the chemotaxis of fibroblasts, cell proliferation, migration, and collagen synthesis during wound healing [ ] . studies also demonstrated elevated pdgf receptor expression in keloid fibroblasts, which responded to pdgf more potently than normal dermal fibroblasts [ ] . elevated vegf in keloid tissue and circulation of keloid patients [ ] could lead to imbalanced angiogenesis and contribute to keloid formation [ , ] . moreover, bfgf is also indicated to be involved in keloid angiogenesis [ ] . preclinical data also demonstrated that the inhibition of fgfr signaling could suppress the proliferation of cells derived from many tissue types [ ] [ ] [ ] . this collected evidence provides an excellent strategy for targeted drug therapy of keloids. there have been several studies on the targeted therapy of keloids. palomid (p ), which inhibits the pi k/akt/mtor pathway by targeting both mtor complex (mtorc- ) and mtorc- signaling, caused keloid tissue shrinkage, growth arrest, and apoptosis [ ] . sorafenib, a potent tki targeting vegfr and pdgfrβ, has been shown to effectively inhibit keloid cell proliferation, migration, invasion, and collagen production [ ] . antisense therapy specific to tgf-β mrna transcripts demonstrated downregulation of mmp- in keloid fibroblasts [ ] . however, these studies focused on only one or two targets. we thus hypothesized that the use of a chemical compound that targets multiple signaling pathways would be a more efficient approach to keloid-targeted therapy. nintedanib was reported as an angiogenesis inhibitor of pdgfr-α and β, vegfr- , , and and fgfr- , , and and was proven effective in the therapy of many types of cancers [ ] . moreover, the antifibrotic effects of nintedanib have also been utilized in treating several fibrosis-related disorders, such as ipf and systemic sclerosis [ , ] . recent studies also showed that nintedanib likely targets the tgf-β-signaling pathway [ ] . as tgf-β, vegf, pdgf, and bfgf are involved in keloid mechanisms [ , ] , and nintedanib is a good candidate for targeted drug therapy for keloids, it would be interesting to explore the roles of nintedanib in inhibiting various pathological activities of keloid fibroblasts as preclinical experimental evidence for future clinical studies. this study demonstrated that nintedanib could inhibit various pathological phenotypes, including inhibition of cell proliferation, cell cycle arrest at g /g phase in vitro (fig. ) and inhibition of kf migration and invasion in vitro and ex vivo (figs. and ). in addition, it also significantly inhibited the gene expression of col- , fn, α-sma, and their protein production in vitro (fig. ) . the reduced protein production was also confirmed in an ex vivo model (fig. ). in addition, matrix degradation is likely to be enhanced by drug treatment, as the gene expression of pai- , a serine protease inhibitor that is often highly expressed in keloid fibroblasts [ ] , was significantly inhibited (fig. ) . in addition, nintedanib also significantly inhibited the gene expression of fkbp and hsp , both of which are highly expressed in fibrotic tissues [ , ] . moreover, this study showed that mmp- and mmp- gene expression was inhibited by the drug, which may also partially account for the inhibited cell migration in cultured tissue explants (fig. ) , in which matrix degradation was also required for cell migration out of the tissue explants. as these pathological aspects of keloids are mediated by growth factors, including tgf-β, pdgf, vegf, and bfgf, drug treatments are likely to inhibit the functions of these factors. for example, ctgf, a growth factor downstream of tgf-β [ , ] , was found to be downregulated at both the gene and protein levels, which may account for the inhibition of cell proliferation and matrix production. as further evidence of the inhibition of multiple signaling pathways, the results of fig. demonstrated that nintedanib noticeably inhibited the phosphorylation of smad and smad at higher concentrations and the mapk pathway dose-dependently, including the pathway components p , erk, and jnk. a previous study showed that the mapk pathway is involved in tgf-β transcription, in which p participates in the phosphorylation of smad / in kfs, while erk and jnk promote smad / / complex translocation and then regulate the expression of related genes, such as pai- [ , ] . consistent with the elevated smad and smad phosphorylation levels in keloid fibroblasts which were related to cell proliferation, matrix synthesis, and mmp regulation in keloids [ , ] , downregulation of smad and smad by rna interference resulted in a significant decrease in procollagen expression in keloids [ , ] . thus, the observed inhibition of cell migration and invasion as well as reduced matrix production is likely mediated by drug inhibition of smad-related pathways. moreover, increase in stat activation was also found in kfs and may contribute to keloid formation by promoting cell proliferation, migration, and collagen production [ ] , while stat phosphorylation was inhibited by the drug (fig. a) . in addition, vegf, pdgf, and bfgf are also involved in keloid pathogenesis by enhancing angiogenesis, cell proliferation, and migration through the p , pi k/akt, and mapk pathways. the ex vivo model study demonstrated that nintedanib significantly inhibited the expression of cd and cd and disrupted microvessel formation (fig. ) , as previously reported [ ] . overall, this study demonstrated that nintedanib could exert its therapeutic effects on various aspects of keloid pathogenesis likely by attenuating various signaling pathways and thus could serve as a blocker of multiple signaling pathways. according to the literature, most protein kinase antagonists are competitive inhibitors that interact with the atp-binding pocket to interfere with receptor dimerization and block the phosphorylation of kinases and downstream signaling cascades [ , ] . however, it has also been reported recently that certain drugs indirectly block signaling pathways by promoting receptor internalization and degradation [ ] . most of the protein kinases are localized in regions of cholesterol-rich lipid rafts/caveolae on cell membranes [ ] . studies have shown that mβcd can block receptor internalization by removing cholesterol [ ] . our results also demonstrated that mβcd was able to rescue nintedanibmediated receptor internalization to a certain degree, suggesting a dual mechanism for nintedanib-mediated inhibition of signaling pathways, i.e., it blocked the kinase atp-binding sites as the main mechanism, while it also promoted kinase internalization. the drawback of this drug is that it also inhibited the proliferation of normal dermal fibroblasts (data not shown) and likely affected other physiological activities. a previous study on pulmonary fibrosis also showed that nintedanib had similar inhibitory effects on both normal pulmonary fibroblasts and fibroblasts in ipf [ ] in terms of cell proliferation, α-sma expression, and myofibroblast appearance. this evidence suggests that nintedanib is less likely to be keloid cell specific and that certain toxic effects on normal cells are possible. therefore, localized use of this drug for keloid treatment would be an ideal method. in summary, this study demonstrates that inhibition of the tgf-β/smad and mapk-signaling pathways by nintedanib can effectively inhibit the proliferation, migration, invasion, and ecm deposition of kfs by blocking kinase atp binding and by enhancing kinase internalization. although further exploration of the detailed mechanism is still needed, the results revealed by the current study support the conclusion that nintedanib is likely to become a promising drug candidate for keloid-targeted therapy, which deserves clinical study in the future. human skin keloid fibroblasts display bioenergetics of cancer cells overexpression of 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tgf-beta on keloid fibroblast proliferation and collagen synthesis critical role of transforming growth factor beta in different phases of wound healing tgf-beta-induced fibrosis and smad signaling: oligo decoys as natural therapeutics for inhibition of tissue fibrosis and scarring on the nature of hypertrophic scars and keloids: a review altered angiogenic balance in keloids: a key to therapeutic intervention upregulation of transforming growth factor-β and vascular endothelial growth factor in cultured keloid fibroblasts: relevance to angiogenic activity molecular pathways: fibroblast growth factor signaling: a new therapeutic opportunity in cancer fibroblast growth factors (fgfs) in cancer: fgf traps as a new therapeutic approach inhibition of pdgf, vegf and fgf signalling attenuates fibrosis nintedanib inhibits fibroblast activation and ameliorates fibrosis in preclinical models of systemic sclerosis pai- in tissue fibrosis fk -binding protein , a potential novel drug target for idiopathic pulmonary fibrosis effect of heat shock protein on collagen accumulation in keloid fibroblast cells pathogenesis of fibrosis: role of tgf-beta and ctgf adiponectin is involved in connective tissue growth factor-induced proliferation, migration and overproduction of the extracellular matrix in keloid fibroblasts p map kinase mediates transforming growth factor-β transcription in human keloid fibroblasts mechanisms of transforming growth factor β /smad signaling mediated by mitogen-activated protein kinase pathways in keloid fibroblasts aspidin pb, a novel natural anti-fibrotic compound, inhibited fibrogenesis in tgf-β -stimulated keloid fibroblasts via pi- k/akt and smad signaling pathways inhibition of smad expression decreases collagen synthesis in keloid disease fibroblasts modulation of collagen synthesis in keloid fibroblasts by silencing smad with sirna stat contributes to keloid pathogenesis via promoting collagen production, cell proliferation and migration mode of action of nintedanib in the treatment of idiopathic pulmonary fibrosis sorafenib suppresses tgf-β responses by inducing caveolae/lipid raft-mediated internalization/ degradation of cell-surface type ii tgf-β receptors_ implications in development of effective adjunctive therapy for hepatocellular carcinoma pirfenidone and nintedanib modulate properties of fibroblasts and myofibroblasts in idiopathic pulmonary fibrosis this study was financially supported by the national natural science foundation of china (no. ). the authors also thank prof. jung huang from the biochemistry department of st. louis university school of medicine (usa) for his valuable suggestion on part of the experimental design and valuable discussion on related scientific issues. the online version of this article (https://doi.org/ . /s - - -y) contains supplementary material, which is available to authorized users.competing interests: the authors declare no competing interests. key: cord- - lds nti authors: ecker, jeffrey w.; kirchenbaum, greg a.; pierce, spencer r.; skarlupka, amanda l.; abreu, rodrigo b.; cooper, r. ethan; taylor-mulneix, dawn; ross, ted m.; sautto, giuseppe a. title: high-yield expression and purification of recombinant influenza virus proteins from stably-transfected mammalian cell lines date: - - journal: vaccines (basel) doi: . /vaccines sha: doc_id: cord_uid: lds nti influenza viruses infect millions of people each year, resulting in significant morbidity and mortality in the human population. therefore, generation of a universal influenza virus vaccine is an urgent need and would greatly benefit public health. recombinant protein technology is an established vaccine platform and has resulted in several commercially available vaccines. herein, we describe the approach for developing stable transfected human cell lines for the expression of recombinant influenza virus hemagglutinin (ha) and recombinant influenza virus neuraminidase (na) proteins for the purpose of in vitro and in vivo vaccine development. ha and na are the main surface glycoproteins on influenza virions and the major antibody targets. the benefits for using recombinant proteins for in vitro and in vivo assays include the ease of use, high level of purity and the ability to scale-up production. this work provides guidelines on how to produce and purify recombinant proteins produced in mammalian cell lines through either transient transfection or generation of stable cell lines from plasmid creation through the isolation step via immobilized metal affinity chromatography (imac). collectively, the establishment of this pipeline has facilitated large-scale production of recombinant ha and na proteins to high purity and with consistent yields, including glycosylation patterns that are very similar to proteins produced in a human host. recombinant proteins are an accepted vaccine platform and are components in a range of commercially available vaccines including the hepatitis b (hbv) and human papilloma virus (hpv) vaccines [ ] [ ] [ ] . herein, we describe an approach for developing stable transfected human cell lines expressing recombinant influenza virus hemagglutinin (ha) and influenza virus neuraminidase (na) proteins for the purpose of in vitro and in vivo vaccine development. ha and na are the major glycoproteins expressed on the surface of influenza viral particles and the major targets of the humoral response [ , ] . the benefits of using recombinant proteins for in vitro and in vivo assays include ease of use, high level of purity and the ability to scale-up production. additionally, vaccine strategies based on administration of recombinant proteins enable focusing of the immune response toward antigens associated with protective immunity [ ] . moreover, usage of recombinant proteins as immunogens circumvents issues associated with alternative influenza vaccine production systems, such as the occurrence of adaptive mutations using embryonated hen eggs for virus growth [ ] . for this reason, current under-development next generation influenza vaccines mostly rely on a recombinant protein-based platform [ , ] . additionally, the here described protein production platform can be considered safer and more affordable as compared, for example, to transducing systems such as those relying on recombinant lentivirus [ ] . in fact, lentiviral-based systems often require a higher biosafety capacity [ ] . several vectors are commercially available for the expression of proteins in mammalian cell lines. as described in figure , commercially available mammalian expression vectors to recombinantly express ha or na ( figure ) were utilized. key features of these plasmids include a human cytomegalovirus (cmv) promoter for expression in mammalian cell lines and a polyadenylation signal and transcription termination sequence downstream of the multiple cloning site for enhanced mrna stability. furthermore, these plasmids have appropriate antibiotic resistance selection markers for: ( ) maintaining the plasmid in escherichia coli (e. coli) using ampicillin; and ( ) confirming incorporation of the plasmid into the mammalian genome using zeocin or neomycin, respectively. the n-terminus of the truncated ha protein (blue) is modified by the foldon trimerization domain (salmon) followed by the avi-tag (light blue) and × his-tag (violet) regions. separating dotted line inside ha indicates the ha head region (n-terminus) versus the ha stem region (c-terminus) also depicted in light blue and salmon, respectively, on the d drawing on the right. (b) schematic of the design of the recombinant tetrameric neuraminidase. the cd signal sequence (grey) is at the n-terminus of the protein, followed by the × his-tag (violet) and the thrombin cleavage site (light blue) sequences, which are followed by the tetrabrachion domain (salmon) linked to the stem region of the truncated neuraminidase protein (blue). dotted line indicates the head region (c-terminus) and stem region (nterminus) also depicted in light blue and salmon, respectively, on the d drawing on the right. figure generated with biorender. for adherent growth, expi f cells (thermo fisher scientific) were passaged at - % confluency. cells were dissociated using a trypsin-edta ( . %) solution (thermo fisher scientific), and then adjusted to cells/ml before seeding into falcon cm rectangular canted neck cell culture flasks (corning, corning, ny, usa) with vented cap with dulbecco's modified eagle medium (dmem, thermo fisher scientific) supplemented with % fetal bovine serum (fbs, atlanta biologicals, flowery branch, ga, usa) and % penicillin-streptomycin (thermo fisher scientific) in a °c incubator with % co and high humidity. for stable cell lines, medium containing geneticin or zeocin (at the appropriate concentration detailed in section . ) was replenished every - days. for suspension cell culture, expi f cells were maintained in suspension cultures in expi the n-terminus of the truncated ha protein (blue) is modified by the foldon trimerization domain (salmon) followed by the avi-tag (light blue) and × his-tag (violet) regions. separating dotted line inside ha indicates the ha head region (n-terminus) versus the ha stem region (c-terminus) also depicted in light blue and salmon, respectively, on the d drawing on the right. (b) schematic of the design of the recombinant tetrameric neuraminidase. the cd signal sequence (grey) is at the n-terminus of the protein, followed by the × his-tag (violet) and the thrombin cleavage site (light blue) sequences, which are followed by the tetrabrachion domain (salmon) linked to the stem region of the truncated neuraminidase protein (blue). dotted line indicates the head region (c-terminus) and stem region (n-terminus) also depicted in light blue and salmon, respectively, on the d drawing on the right. production of recombinant proteins from cell lines has become a routine procedure, and it can be achieved at either a large industrial or a smaller laboratory scale. through this process, in-house production of proteins for a multitude of uses has become far more affordable and accessible using yeast, insect, plant and mammalian cells in suspension [ , ] . additionally, protein-expression systems using baculoviruses and insect cells have been licensed in the usa since for the production of rha-based vaccines such as flublok, originally developed by protein sciences corp. and now owned by sanofi pasteur (paris, france) [ ] . as described, mammalian cells have been used for expression, specifically, the human epithelial kidney (hek) -derived cell line expi f by thermo fisher scientific (waltham, ma, usa). this system has the benefit to obtain post-translation modifications (e.g., glycosylation) that are more similar to modifications on wild-type viral particles as compared to expression of the same proteins in yeast, insect and plant-based systems [ ] . they also provide the benefit of being developed into stable cell lines that can be cryopreserved for long-term storage and subsequent expansion for future protein expression needs. equivalent recombinant protein production systems based on other cell lines are also available, such as the expi f inducible cells [ ] . however, this system would require the further addition of an inductor to the culture, e.g., tetracycline, for the initiation of the recombinant protein expression and thus affecting the overall production costs. once a protein is expressed, immobilized metal affinity chromatography (imac) can be used as a highly specific and efficient means of protein isolation from culture supernatants. proteins purified through the imac method can yield very high purities, especially since few cellular proteins have a sufficiently strong affinity to remain associated with the immobilized metal matrix following a washing step with buffer containing a low concentration of imidazole. due to the poly-histidine (his)-tag being of a relatively small size in comparison to the recombinant ha or na proteins, and its location being at the c-or n-terminus of the protein, its incorporation into the protein sequence should not disrupt their native conformation or biologic activity of the protein. in this context, generation of physiologically relevant recombinant ha and na proteins, such as in the case of other recombinant proteins, requires that the proteins remain soluble in the culture medium and that they maintain an equivalent conformation as the analogous proteins expressed by the corresponding wild-type influenza viruses. likewise, recombinant ha and na proteins will ideally maintain functional activity after purification, and even after prolonged storage. in the case of ha, which is responsible for viral attachment to cells of the upper respiratory tract [ ] , this entails binding of sialic acid and the ability to agglutinate erythrocytes. for na, which is responsible for viral egress, this includes enzymatic activity and cleavage of terminal sialic acid moieties. herein, we detail an expression pipeline at key checkpoints that facilitated large-scale production of recombinant ha and na proteins to high purity and with consistent yields across production runs. moreover, we evaluated these proteins using a variety of laboratory techniques to assess their conformational structure, temperature stability and functional activity. plasmids for expressing recombinant wild-type ha influenza proteins were human codon optimized and ordered from genewiz (south plainfield, nj, usa) or cloned from a full-length ha gene into a pcdna . /zeo (+) vector (thermo fisher scientific) using the bamhi and hindiii restriction sites. alternatively, ha possessing the y f mutation to attenuate sialic acid binding (ha∆sa) were also cloned into the pcdna . /zeo (+) vector [ ] . the end of ha genes were truncated starting from amino acid (h n a/california/ / ha numbering) and modified to include a foldon of t fibritin trimerization domain to promote its expression as a soluble trimeric protein, as previously described [ ] . an avi-tag sequence followed by a × his-tag was also included downstream of the trimerization domain to enable site-specific biotinylation [ ] and purification of the recombinant ha by imac, respectively ( figure a ). plasmids encoding human codon optimized full-length influenza na proteins were synthetically synthesized by genewiz. the truncated na genes were pcr amplified with kpni and ecori overhang primers into a pcdna . -topo vector (thermo fisher scientific). the resulting plasmids were sequence confirmed. similar to previously described na designed proteins [ ] , the gene for na was truncated at the end starting from amino acid (h n a/texas/ / na numbering) removing the transmembrane domain and a portion of the stem, which was replaced with the tetrabrachion domain from staphylothermus marinus and separated from the na gene by three linker amino acids (gsg or gtg). a thrombin cleavage site was incorporated upstream of the tetrabrachion domain and preceded by a × his-tag and lastly a cd signal sequence ( figure b) . for dna plasmid amplification, chemically competent top or dh α e. coli cells (thermo fisher scientific) were used for bacterial transformation using - ng of the original dna. e. coli chemically competent cells were transformed following the instructions provided by the manufacturer (zymo research, irvine, ca, usa) and plasmids purified from a single transformed bacterial colony. in brief, e. coli transformed colonies were grown at • c in luria-bertani (lb) broth with aeration by growing them in a shaker incubator set at rpm. ampicillin ( µg/ml) and kanamycin ( µg/ml) were used for antibiotic resistant selection depending on the plasmid. a nanodrop spectrophotometer (denovix, wilmington, de, usa) was used to quantify plasmid preparations and measure purity. a / absorbance ratio greater than . was considered acceptable for downstream steps. dna plasmids were digested using restriction enzymes (bamhi/hindiii for ha and kpni/ecori for na) according to the manufacturer's instructions (new england biolabs, ipswich, ma, usa) and then resolved by gel electrophoresis using o'generuler kb plus dna ladder (thermo fisher scientific) for reference to confirm their identity. sybr safe dna gel stain (thermo fisher scientific) was incorporated into % w/v seakem le agarose (lonza, basel, switzerland) prior to casting and gels were run at - v until loading dye reached the end of the gel before imaging under uv light using the chemi-doc imaging system (bio-rad, hercules, ca, usa). for adherent growth, expi f cells (thermo fisher scientific) were passaged at - % confluency. cells were dissociated using a trypsin-edta ( . %) solution (thermo fisher scientific), and then adjusted to cells/ml before seeding into falcon cm rectangular canted neck cell culture flasks (corning, corning, ny, usa) with vented cap with dulbecco's modified eagle medium (dmem, thermo fisher scientific) supplemented with % fetal bovine serum (fbs, atlanta biologicals, flowery branch, ga, usa) and % penicillin-streptomycin (thermo fisher scientific) in a • c incubator with % co and high humidity. for stable cell lines, medium containing geneticin or zeocin (at the appropriate concentration detailed in section . ) was replenished every - days. for suspension cell culture, expi f cells were maintained in suspension cultures in expi expression medium (thermo fisher scientific) at • c, % co and high humidity on a shake platform set to rpm. as per manufacturer recommendation, cell density was maintained at . - × cells/ml in polycarbonate vented erlenmeyer flasks (corning) containing a medium volume of / - / of the total volume of the flask. for stable cell lines, medium containing geneticin or zeocin (at the appropriate concentration detailed in section . ) was replenished every - days. sp / mouse myeloma cell line (kindly provided by dr. l. wysocki, university of colorado at denver, denver, co, usa) and previously generated b-cell hybridomas ( h , g , a and f ) were cultured as already described [ ] . in detail, mouse cell lines were maintained in b cell medium (bcm) consisting of rpmi medium (sigma-aldrich, saint louis, mo, usa) containing % fbs (atlanta biologicals), . mm sodium bicarbonate (thermo fisher scientific, waltham, ma, usa), . mm -( -hydroxyethyl)- -piperazineethanesulfonic acid (hepes; amresco, solon, oh, usa), mm penicillin g (tokyo chemical industry, tokyo, japan), mm streptomycin (sigma-aldrich), mm -mercaptoethanol (sigma-aldrich), nonessential amino acids (thermo fisher scientific) and mm sodium pyruvate (thermo fisher scientific). for monoclonal antibody (mab) production, hybridoma cell lines were grown in bcm containing % super low igg fbs (hyclone, logan, ut, usa). transient transfection typically requires - days from culture expansion to purified protein ( figure ). expi f cultures with > % viability were centrifuged and resuspended in fresh medium at × cells/ml within h prior to transfection. in this case, cells were transfected using the expifectamine transfection kit according to manufacturer's instructions. dna was diluted in % of final culture volume while in a separate conical tube, expifectamine was diluted in % of the final culture volume to achieve a final culture concentration of µg dna/ml and . µl/ml, respectively. diluted mixtures were incubated for min at room temperature (rt). dna mixture was added to expifectamine mixture and incubated at rt for min before addition to cells. twenty hours post-transfection, % and . % of final culture volumes were added of enhancer and enhancer , respectively, as per manufacturer's instructions. as an example, ml of final culture volume requires µg of dna diluted in ml of medium and µl of expifectamine diluted in ml of medium. after h, ml of enhancer and . ml of enhancer are added to transfected flasks. protein expression in culture supernatant was confirmed - h post-transfection and harvested days post-transfection or when viability reached < %. ha and na recombinant proteins were also expressed through the generation of stable transfected cells ( figure ). expi f cells were seeded in -well plates at a density of~ × cells/well. after - h, corresponding to a % cell confluency, they were transfected with the lipofectamine (thermo fisher scientific) transfection kit following the instructions provided by the manufacturer. briefly, a mixture of µg of dna, µl of opti-minimum essential medium (mem) i (thermo fisher scientific) and µl of p reagent was prepared. a second mixture of µl lipofectamine and µl of opti-mem was prepared and both were incubated at rt for min. the mixture containing dna was added to the mixture of lipofectamine, gently mixed and incubated an additional min prior to addition to wells containing . ml of opti-mem i. culture supernatants were collected - h post-transfection and assessed for protein expression. following transfection, -well ha or na transfected cells were supplemented with µg/ml of zeocin (invivogen, san diego, ca, usa) or µg/ml geneticin (thermo fisher scientific), respectively, based upon previous calculation of the minimal inhibitory concentration. transfected cells were periodically expanded under drug selection and expression of recombinant ha or na was periodically checked by western blot as described below. to select stable transfected cells expressing higher yields of rha, a limiting dilution approach was performed in -well plates. in brief, × cells were seeded in well a containing µl selective growth medium and a -fold serial dilution of cells was performed vertically and a -fold dilution horizontally. selective drug medium was replenished every - days until at least wells having at a~ % confluence and containing viable and a single subpopulation-derived cell colony were screened for expression through western blot as described in section . . wells showing the highest level of expression as per western blot screening were expanded as adherent cultures (as described in section . ) and were then re-adapted to grow in suspension by passaging and growing them in expi expression medium (as detailed in section . ) under drug selection. culture supernatants containing secreted or purified proteins or influenza viruses, propagated in embryonated chicken eggs as previously described [ ] , were mixed with × laemmli sample buffer (bio-rad) and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page). in the same gel, a spectra multicolor broad range protein ladder (thermo fisher scientific) was also included as a reference. additionally, ng of his-tagged rha or rna standards were also loaded in the same gel as positive controls for the western blot and as references to evaluate the level of rha and rna expression, respectively. in brief, precast % sds gels (thermo fisher scientific) were electrophoresed at v for min. proteins were transferred to polyvinylidene difluoride (pvdf) membranes using a trans-blot turbo device (bio-rad) using the preset high molecular weight conditions and membranes were processed using the ibind device (thermo fisher scientific) following the manufacturer's instructions. primary, mouse anti-his-tag (clone j b , biolegend, san diego, ca, usa) at : , a group -specific mouse mab (clone b ; cat. no. it- - m , immune technology corp., new york, ny, usa) or a group -specific mouse mab (clone c ; cat. no. it- - m ; immune technology corp.) at : and a secondary horseradish peroxidase (hrp)-conjugated goat-anti-mouse igg (southern biotech, birmingham, al, usa) antibody at : were used. visualization was accomplished using clarity ecl substrate (bio-rad) and membranes were imaged using the chemi-doc imaging system (bio-rad). cell debris and macrovesicles were removed from conditioned media by centrifugation at , × g for min at • c in a fixed-angle rotor, followed by filtration through . -µm pes membrane filter units (corning). recombinant his-tagged proteins were then purified by imac using histrap excel columns (ge healthcare, chicago, il, usa) and an automated purification Äkta pure system (ge healthcare). in brief, columns were first equilibrated with column volumes of -mm napo and . -m nacl buffer using a flow rate of ml/min/ml of bead resin. as per manufacturer's recommendation, a flow rate of ml/min/ml of bead resin was also used for sample application and all subsequent washing and elution steps. in greater detail, following completion of sample application, histrap excel columns were washed with column volumes of a mm imidazole, mm napo and . m nacl buffer to remove non-specifically bound proteins. his-tagged recombinant ha or na proteins were then eluted using column volumes of a mm imidazole, mm napo and . m nacl buffer and collected as column volume ( ml) fractions ( figure ). protein fractions were then concentrated and dialyzed against pbsa (phosphate buffered saline supplemented with . % w/v nan ) using amicon ml k centrifugal concentrator (corning) by spinning at × g for min. protein samples were then quantified using a spectrophotometer by measuring absorbance at / nm and a bicinchoninic acid assay (thermo fisher scientific). to assess purity, protein samples were run on sds-page for min at v and stained with coomassie blue (thermo fisher scientific). for experiments involving the use of previously described mabs, their purification was performed as already described [ ] . to assess the thermal stability of rha protein, rha from a/california/ / (ca/ ) was subjected to different temperature incubations ( - • c range) for h using a multigene optimax thermal cycler (labnet international, edison, nj, usa). the different treated rha were then immediately used to coat elisa plates as described below. to assess the antigenicity of generated rha, its trimerization and possible conformational changes due to different temperature incubations, enzyme-linked immunosorbent assays (elisa) were performed as previously described [ , , ] . in brief, immulon hbx plates (thermo fisher scientific) were coated overnight at • c in a humidified chamber with µl per well of a carbonate buffer solution (ph . ) containing µg/ml of the native, the different temperature-treated in-house expressed and purified rha from ca/ , as well as commercial trimeric (protein sciences, meriden, ct, usa) and monomeric (immune technology) rha from the same strain. for the thermal stability experiments, the murine mab clones b (immune technology corp.), f [ ] and j b (biolegend) and the human mab cr (creative biolabs, shirley, ny, usa) [ ] were diluted in blocking buffer ( % bovine serum albumin [bsa] and % gelatin in pbs/ . % tween- ; all supplements from vwr international, radnor, pa, usa) at a final concentration of µg/ml. alternatively, a ca/ -specific polyclonal mouse serum obtained from a previous study [ ] was diluted : in blocking buffer. to assess the thermal stability of rha protein, rha from a/california/ / (ca/ ) was subjected to different temperature incubations ( - °c range) for h using a multigene optimax thermal cycler (labnet international, edison, nj, usa). the different treated rha were then immediately used to coat elisa plates as described below. to assess the antigenicity of generated rha, its trimerization and possible conformational changes due to different temperature incubations, enzyme-linked immunosorbent assays (elisa) were performed as previously described [ , , ] . in brief, immulon hbx plates (thermo fisher scientific) were coated overnight at °c in a humidified chamber with µl per well of a carbonate buffer solution (ph . ) containing µg/ml of the native, the different temperature-treated in-house expressed and purified rha from ca/ , as well as commercial trimeric (protein sciences, meriden, ct, usa) and monomeric (immune technology) rha from the same strain. for the thermal stability experiments, the murine mab clones b (immune technology corp.), f [ ] and j b (biolegend) and the human mab cr (creative biolabs, shirley, ny, usa) [ ] were diluted in blocking buffer ( % bovine serum albumin [bsa] and % gelatin in pbs/ . % tween- ; all supplements from vwr international, radnor, pa, usa) at a final concentration of µg/ml. alternatively, a ca/ -specific polyclonal mouse serum obtained from a previous study [ ] was diluted : in blocking buffer. for the experiments assessing the trimeric conformation of the generated rha, the murine mab clones a [ ] and at . . (irr) were -fold diluted starting from µg/ml and used as primary antibodies. binding of all primary antibodies was performed for h at °c. plates were washed five times with pbs, µl per well of hrp-conjugated goat anti-human or anti-mouse igg (southern biotech) diluted : in blocking buffer was added, and plates were incubated at °c for h. finally, plates were washed five times with pbs and , ′-azino-bis( -ethylbenzothiazoline- - for the experiments assessing the trimeric conformation of the generated rha, the murine mab clones a [ ] and at . . (irr) were -fold diluted starting from µg/ml and used as primary antibodies. binding of all primary antibodies was performed for h at • c. plates were washed five times with pbs, µl per well of hrp-conjugated goat anti-human or anti-mouse igg (southern biotech) diluted : in blocking buffer was added, and plates were incubated at • c for h. finally, plates were washed five times with pbs and , -azino-bis( -ethylbenzothiazoline- -sulfonic acid) (abts) substrate (vwr international) was added. plates were incubated at • c for - min. colorimetric conversion was terminated by addition of % sds ( µl per well), and od was measured at nm (od ) using a spectrophotometer (powerwave xs; biotek, winooski, vt, usa). the hemagglutination assay was used to assess functionality of rha to agglutinate erythrocytes. hemagglutination assays were performed similarly to previously described protocol [ ] . in brief, µl of duplicate two-fold serial dilutions of representative wild type rha from a/singapore/ / (sing/ ) and a/new caledonia/ / (nc/ ), their corresponding y f mutant versions (ha∆sa) and influenza viruses (positive controls), and bsa (negative control) were diluted in pbs, starting from µg/ml for proteins or . × and . × plaque forming units per milliliter (pfu/ml) for sing/ and nc/ influenza viruses, respectively, and incubated for min with an equal volume of . % turkey erythrocytes diluted in pbs. erythrocytes were washed and used the day of the assay. the plates were mixed by agitation and covered, and the erythrocytes were settled for min at rt. the hemagglutination titer was determined by the reciprocal dilution corresponding to the rha concentration of the last well that contained agglutinated erythrocytes. images of hemagglutination assays were acquired using the immunospot s ultimate (cellular technology limited, shaker heights, oh, usa). neuraminidase activity assays were performed to evaluate the sialidase activity of na and similarly to a previous described protocol for the enzyme-linked lectin assay (ella) [ ] . high affinity immunoblot recombinant ha proteins possessing the y f mutation to attenuate sialic acid binding (ha∆sa) were dialyzed into mm tris-hcl (ph . ) and total protein concentration adjusted to~ mg/ml after bca estimation. ha∆sa proteins were biotinylated with biotin protein ligase (bira) (avidity biosciences, la jolla, ca, usa) following manufacturer recommendations and confirmed by direct elisa. high binding elisa plates (costar, washington, dc, usa) were coated overnight with to ng/well of bira conjugated ha∆sa protein in carbonate buffer (ph . ) at • c. elisa plates were blocked and biotinylated ha∆sa proteins detected with hrp-conjugated streptavidin (southern biotech) followed by addition of the abts substrate. biotinylated protein standard and a negative control protein were purchased from avidity biosciences (la jolla, ca, usa; cat. no. bis- ). fluorescently-conjugated ha∆sa probes were generated by stepwise addition of high concentration ( mg/ml) pe-or apc-conjugated streptavidin (sa-pe or sa-apc) (biolegend) to a final -fold molar excess, in ice-cold pbs with protease inhibitor cocktail set i (calbiochem, san diego, ca, usa; cat. no. ). ha∆sa probes were stored at • c in the dark up to two days. the previously characterized ha-specific g hybridoma cell line [ ] ( × ) was stained with ng of apc conjugated rha∆sa probes in µl of staining buffer (pbs/ % fbs) for min on ice and protected from light. cells were then washed by centrifugation, resuspended in µl of staining buffer, and µl of -aminoactinomycin ( -aad) live/dead staining was added min prior to acquisition in a lsrii flow cytometry analyzer (bd biosciences, san jose, ca, usa). cell surface bcr expression was evaluated by staining with apc-conjugated goat anti-mouse igg at : (biolegend, cat. no. ). the sp / myeloma and the ha-negative h hybridoma cell lines were similarly stained and used as negative controls. alternatively, × of banked cryopreserved peripheral blood mononuclear cells (pbmcs) from a previously identified highly reactive influenza vaccinated individual [ ] , collected days post vaccination, were first treated with fc receptor blocking solution (biolegend; cat. no. ) and then simultaneously stained with ng of phycoerythrin (pe)-conjugated ca/ ha∆sa and ng allophycocyanin (apc)-conjugated a/hong kong/ / (hk/ ) ha∆sa. excess unbound ha∆sa probes were washed by centrifugation and human pbmc were then stained with an antibody cocktail for b cell immunoprofiling as previously described [ ] . data were acquired on a facsaria fusion flow cytometer (bd biosciences) and analyzed using the flowjo software (bd, franklin lakes, nj, usa). all graphically represented results are reported as absolute mean values plus standard deviations. differences in binding of antibodies to rha treated at different temperatures as compared to the reference rha were analyzed using the ordinary one-way anova test. all statistical analysis was performed using prism v. . . software (graphpad, san diego, ca, usa), and a p value lower than . was considered statistically significant. as wild-type ha and na are trimeric and tetrameric protein complexes, we incorporated a trimerization or a tetramerization domain, respectively, to promote and stabilize their oligomerization [ ] (figure ). as shown in figure a , following purification and coomassie staining of sds-page loaded proteins, the monomer fraction of rha constitutes the major species, with a size of~ kda (red arrow). lower intensity bands are also visible at~ and~ kda, and represent rha oligomers, dimers (yellow arrow) and trimers (white arrow), respectively. another lower intensity band is visible at~ kda, possibly representing a less glycosylated rha monomer isoform (blue arrow). additionally, the migration pattern of rha is similar to ha derived from influenza viral particles, as shown in the western blot depicted in figure . comparable composition ( figure a ). furthermore, as depicted in figure b -d, purified rha were specifically recognized by group -and group -specific mabs for h n and h n rha, respectively. (a) (b) rna from h n tx/ , respectively. for rha, arrows indicate monomers (red), dimers (yellow) and trimers (white) and a less glycosylated rha monomer isoform (blue). for rna, arrows indicate dimers (green), tetramers (orange) and monomers (pink) (a). representative yields from culture media of transfected expi f cell lines stably expressing rha proteins from h n cobra p , ca/ , brisb/ and h n switz/ , hk/ and sing/ (b). to assess their functional activity and capacity to agglutinate red blood cells (rbcs), rha proteins and the corresponding h n or h n influenza virus were evaluated in parallel. as shown in figure , rha from sing/ and nc/ exhibited hemagglutination activity, with minimal effective concentrations of . and . μg/ml, respectively. in agreement with previous reports [ , ] , sing/ and nc/ rha proteins possessing a y f mutation (haΔsa), along with the bsa negative control, lacked detectable hemagglutination activity. accordingly, the corresponding sing/ and nc/ influenza viruses possessed hemagglutination activity at a minimal viral titer of and × pfu/ml, respectively. the major bands of rna are~ (green arrow) and~ kda (orange arrow), corresponding to rna dimers and tetramers, respectively. a low intensity band is also visible at a kda approximate size (pink arrow), corresponding to the rna monomer. on average, the yields from rha or rna stable transfected cell line ranged nearly -fold ( . - mg rha/l). as shown in figure b , the maximal rha yields were achieved by the previously described computationally-optimized broadly reactive antigen (cobra) p ha protein [ , ] stable transfected cells. variable, but consistent, rha yields were achieved with ca/ , a/brisbane/ / (brisb/ ), a/switzerland/ / (switz/ ) and hk/ rha stable transfected cell lines. unfortunately, despite undergoing zeocin selection and limiting dilution, the sing/ rha stably transfected line remained a poor producer. importantly, consistent recombinant protein expression levels were maintained with all the stable cell lines, even after approximately five months of passaging (the longest time period we were able to test) following their generation (data not shown). as an additional means of characterizing the purified rha, their size and migration patterns following sds-page were further characterized through western blot analysis. in comparison to native ha present on the surface of influenza virions, the corresponding rha demonstrated a comparable composition ( figure a) . furthermore, as depicted in figure b -d, purified rha were specifically recognized by group -and group -specific mabs for h n and h n rha, respectively. to assess their functional activity and capacity to agglutinate red blood cells (rbcs), rha proteins and the corresponding h n or h n influenza virus were evaluated in parallel. as shown in figure , rha from sing/ and nc/ exhibited hemagglutination activity, with minimal effective concentrations of . and . µg/ml, respectively. in agreement with previous reports [ , ] , sing/ and nc/ rha proteins possessing a y f mutation (ha∆sa), along with the bsa negative control, lacked detectable hemagglutination activity. accordingly, the corresponding sing/ and nc/ influenza viruses possessed hemagglutination activity at a minimal viral titer of and × pfu/ml, respectively. in figure , rha from sing/ and nc/ exhibited hemagglutination activity, with minimal effective concentrations of . and . µg/ml, respectively. in agreement with previous reports [ , ] , sing/ and nc/ rha proteins possessing a y f mutation (haΔsa), along with the bsa negative control, lacked detectable hemagglutination activity. accordingly, the corresponding sing/ and nc/ influenza viruses possessed hemagglutination activity at a minimal viral titer of and × pfu/ml, respectively. an important consideration and logistical barrier to mass vaccination efforts against seasonal influenza and other pathogens, especially in under-developed countries in which maintenance of cold-chain may not be feasible, is vaccine stability and immunogenicity. to this end, we sought to evaluate the thermal stability, and specifically preservation of conformational epitopes, of representative recombinant ha (ca/ ) proteins following incubation at increasing temperatures. specifically, following incubation at increasing temperatures ( - °c) , the ca/ rha protein was used to coat wells of an elisa plate and probed with either polyclonal or mab preparations to monitor the structural integrity of conformational epitopes. as shown in figure , binding of conformation-sensitive mabs (cr and f ) and polyclonal serum is higher or similar to that achieved to the reference rha, at temperatures ranging from to °c. higher temperatures ( - °c) caused a linear decrease of the binding activity of polyclonal serum and of ha stem-and headdirected conformation-sensitive mabs ( figure c,d) . interestingly, the binding of mabs directed to an ha stem linear epitope ( b ) or to the his-tag (j b ) was also affected by the different temperature incubations ( figure a,b) . however, differently from conformation-sensitive mabs and polyclonal serum, their magnitude of binding was not linearly affected by the different temperature treatments. figure . hemagglutination activity of rha expressed by stably-transfected cell lines. representative wild type rha and corresponding ha∆sa from sing/ (a) and nc/ (b) were evaluated for their ability to agglutinate turkey rbcs. all proteins were tested in duplicate starting from µg/ml and two-fold serially diluted (from left to right). bsa was used as negative control and the corresponding influenza viruses from sing/ and nc/ were used as positive controls. an important consideration and logistical barrier to mass vaccination efforts against seasonal influenza and other pathogens, especially in under-developed countries in which maintenance of cold-chain may not be feasible, is vaccine stability and immunogenicity. to this end, we sought to evaluate the thermal stability, and specifically preservation of conformational epitopes, of representative recombinant ha (ca/ ) proteins following incubation at increasing temperatures. specifically, following incubation at increasing temperatures ( - • c), the ca/ rha protein was used to coat wells of an elisa plate and probed with either polyclonal or mab preparations to monitor the structural integrity of conformational epitopes. as shown in figure , binding of conformation-sensitive mabs (cr and f ) and polyclonal serum is higher or similar to that achieved to the reference rha, at temperatures ranging from to • c. higher temperatures ( - • c) caused a linear decrease of the binding activity of polyclonal serum and of ha stem-and head-directed conformation-sensitive mabs ( figure c,d) . interestingly, the binding of mabs directed to an ha stem linear epitope ( b ) or to the his-tag (j b ) was also affected by the different temperature incubations ( figure a,b) . however, differently from conformation-sensitive mabs and polyclonal serum, their magnitude of binding was not linearly affected by the different temperature treatments. to assess the functional activity of purified na recombinant proteins, a neuraminidase activity assay was performed to evaluate their ability to cleave fetuin-borne sialic acid. as reported in figure , representative proteins from h n and h n strains are endowed with neuraminidase activity, as demonstrated by the na dose-dependent increased specific binding of peanut agglutinin to the sialic acid-cleaved fetuin protein. interestingly, the order of na activity featured by na recombinant proteins is similar to those exerted by the corresponding na on the influenza viral particles. in particular, h n recombinant na from a/brisbane/ / (brisb/ ) and a/texas/ / (tx/ ) had an ec of ng/ml. alternatively, h n a/chile/ / (chile/ ) and brisb/ showed an ec of ng/ml. the h n ca/ na had the lowest ec corresponding to ng/ml. differently, h n recombinant na from tx/ and switz/ showed an ec of ng/ml. on the other hand, the one from hk/ showed an ec of ng/ml. to assess the functional activity of purified na recombinant proteins, a neuraminidase activity assay was performed to evaluate their ability to cleave fetuin-borne sialic acid. as reported in figure , representative proteins from h n and h n strains are endowed with neuraminidase activity, as demonstrated by the na dose-dependent increased specific binding of peanut agglutinin to the sialic acid-cleaved fetuin protein. interestingly, the order of na activity featured by na recombinant proteins is similar to those exerted by the corresponding na on the influenza viral particles. in particular, h n recombinant na from a/brisbane/ / (brisb/ ) and a/texas/ / (tx/ ) had an ec of ng/ml. alternatively, h n a/chile/ / (chile/ ) and brisb/ showed an ec of ng/ml. the h n ca/ na had the lowest ec corresponding to ng/ml. differently, h n recombinant na from tx/ and switz/ showed an ec of ng/ml. on the other hand, the one from hk/ showed an ec of ng/ml. to assess the functional activity of purified na recombinant proteins, a neuraminidase activity assay was performed to evaluate their ability to cleave fetuin-borne sialic acid. as reported in figure , representative proteins from h n and h n strains are endowed with neuraminidase activity, as demonstrated by the na dose-dependent increased specific binding of peanut agglutinin to the sialic acid-cleaved fetuin protein. interestingly, the order of na activity featured by na recombinant proteins is similar to those exerted by the corresponding na on the influenza viral particles. in . recombinant na proteins from h n strains and h n strains were evaluated for neuraminidase activity measured as the ability to cleave sialic acid displayed on the fetuin glycoprotein. lod represents the limit of detection. to confirm the foldon-driven trimerization of rha, an elisa was performed. in detail, a representative in-house expressed and purified rha, a trimeric recombinant h n protein component of the commercial influenza flublok vaccine formerly by protein sciences (now sanofi-pasteur) and a commercial recombinant ha monomer were probed with rha-specific mabs. as shown in figure , the a mab, whose binding activity is specific for the trimeric form of the ha [ ] , binds in a dose-dependent manner to either the in-house rha or the commercial vaccine-derived trimeric rha. no binding activity was confirmed against the ha monomeric rha. differently, the at . . mab clone, whose binding activity is specific for the monomeric form of the ha, shows a dose-dependent binding activity against the rha monomer and is not able to bind either the in-house rha or the commercial vaccine-derived trimeric rha. to confirm the foldon-driven trimerization of rha, an elisa was performed. in detail, a representative in-house expressed and purified rha, a trimeric recombinant h n protein component of the commercial influenza flublok vaccine formerly by protein sciences (now sanofi-pasteur) and a commercial recombinant ha monomer were probed with rha-specific mabs. as shown in figure , the a mab, whose binding activity is specific for the trimeric form of the ha [ ] , binds in a dose-dependent manner to either the in-house rha or the commercial vaccine-derived trimeric rha. no binding activity was confirmed against the ha monomeric rha. differently, the at . . mab clone, whose binding activity is specific for the monomeric form of the ha, shows a dose-dependent binding activity against the rha monomer and is not able to bind either the inhouse rha or the commercial vaccine-derived trimeric rha. identification of ha-specific b cells using rha proteins possessing the y f mutation has been reported previously [ ] . to demonstrate that our in-house expressed rhaΔsa proteins could also be used for similar purposes, we utilized an ha-reactive murine b cell hybridoma that have been reported previously [ ] . the g hybridoma line expressed a comparable level of surface igg to another hybridoma line ( h ) known to be not specific for ha, and thus were utilized as a simplified model for assessing the specificity of rhaΔsa staining. in accordance with previously published data for the corresponding g mab [ ] , the g hybridoma cells stained positive with both the fluorescently-labeled cobra p and ca/ rhaΔsa reagent ( figure a ). in contrast, the h hybridoma cell line failed to yield definitive staining with the same probes ( figure a ). collectively, the observed rhaΔsa reagent staining patterns support their usage for identification of ha-specific b cells in more complex samples, such as human pbmcs. to further validate the use of rhaΔsa probes for the identification of ha-specific b cells, pbmcs from a donor that mounted a broadly-reactive response following seasonal influenza vaccination were stained with rhaΔsa probes representing the respective h n and h n vaccine strains. as expected, a well-defined plasmablast population (cd + cd ++ ) was present seven days identification of ha-specific b cells using rha proteins possessing the y f mutation has been reported previously [ ] . to demonstrate that our in-house expressed rha∆sa proteins could also be used for similar purposes, we utilized an ha-reactive murine b cell hybridoma that have been reported previously [ ] . the g hybridoma line expressed a comparable level of surface igg to another hybridoma line ( h ) known to be not specific for ha, and thus were utilized as a simplified model for assessing the specificity of rha∆sa staining. in accordance with previously published data for the corresponding g mab [ ] , the g hybridoma cells stained positive with both the fluorescently-labeled cobra p and ca/ rha∆sa reagent ( figure a ). in contrast, the h hybridoma cell line failed to yield definitive staining with the same probes ( figure a ). collectively, the observed rha∆sa reagent staining patterns support their usage for identification of ha-specific b cells in more complex samples, such as human pbmcs. to further validate the use of rha∆sa probes for the identification of ha-specific b cells, pbmcs from a donor that mounted a broadly-reactive response following seasonal influenza vaccination were stained with rha∆sa probes representing the respective h n and h n vaccine strains. as expected, a well-defined plasmablast population (cd + cd ++ ) was present seven days after vaccination, and a subset of these cells were labeled with one or more rha∆sa probes. similarly, % of the cd + igd − b cell compartment (class-switched memory b-cells) were also labeled by the fluorescently-labeled rha∆sa probes ( figure b) . importantly, the overall frequency of rha∆sa-labeled cells was higher for both b-cells compartments in high vaccine-reactive subjects as compared to low vaccine reactive subjects ( figure c ). after vaccination, and a subset of these cells were labeled with one or more rhaΔsa probes. similarly, % of the cd + igd -b cell compartment (class-switched memory b-cells) were also labeled by the fluorescently-labeled rhaΔsa probes ( figure b) . importantly, the overall frequency of rhaΔsa-labeled cells was higher for both b-cells compartments in high vaccine-reactive subjects as compared to low vaccine reactive subjects ( figure c ). recombinant protein production, through the processes of cell culture transfection and stable cell lines, remains a viable technique for increasing yields of distinct proteins that are used as tools in a wide variety of applications, but mainly as antigens for diagnostic and prophylactic purposes [ ] . production of large protein quantities can also be achieved, while also moderating the overall cost, in comparison to purchasing similar commercially available proteins on the open market. furthermore, the creation of stable cell lines reduces the budget and time required to perform multiple large-scale transfections and allows for a more consistent product over multiple runs. this protocol is a set of guidelines for production and purification of soluble recombinant proteins through transient transfection and stable cell line production in mammalian cells from the initial plasmid creation to the final isolation step via imac. through these means, we are able to create a product of high purity, as well as, high levels of consistency between runs. in addition, due to being synthesized in mammalian human-derived cells, the proteins have more similar glycosylation and functional activity to the native proteins expressed on the surface of the influenza virions as compared to non-mammalian cells [ ] . as demonstrated by hemagglutination and neuraminidase assays, the produced rha and rna maintain similar functional activity as their native counterparts displayed on the surface of influenza viral particles. additionally, crystallography study of cobra- rha protein generated through this expression pipeline was structurally preserved at the atomic level [ ] . likewise, both human or mouse mabs recognizing conformational epitopes exhibit strong binding of rha proteins isolated using similar methods [ , , ] . owing to their stability and structural integrity, the ha∆sa proteins serve as an excellent tool for specific labeling of ha-reactive b cells. this was evident through labeling an ha-specific b cell hybridoma ( g ) [ ] as well as plasmablasts and memory b cells present within a responder following influenza vaccination. in fact, the use of recombinant antigens as "probes" for capturing antigen-specific b cells has important implications not only in an antibody discovery setting [ ] , but also in the context of investigating defined antigen-specific b cell populations (e.g., plasmablasts and memory b cells) following infection or vaccination [ , ] . the native conformation of our in-house produced rha was largely maintained despite exposure to elevated temperatures. in fact, binding of mabs recognizing conformational epitopes was generally preserved across a temperature range of - • c relative to rha that was unmanipulated. however, a linear decrease in binding for these same mabs was observed following treatment of the rha at higher temperatures ( - • c), suggesting a disruption of the corresponding conformational epitopes. interestingly, the binding of mabs directed against a linear epitope or the his affinity tag were also affected by the temperature changes. however, alteration in binding signal did not follow a pattern, rather suggesting that increasing the temperature of rha may have induced molecular dynamics and consequent modifications in the display of the relevant epitopes [ ] . additionally, as previously described, rha conformational changes do occur following a low ph buffer treatment, as demonstrated by the generally decreased affinity of a panel of mabs against the treated protein as compared to the native rha [ ] . introducing a series of quality control steps into the expression pipeline, such a western blot of culture supernatants, along with sequence analysis of plasmids and stable cell lines, serve as important checkpoints to ensuring the identity of the expressed and purified product. in this regard, we utilized commercially available mab reagents to confirm the proteins expected size and reactivity with subtype-specific mab probes ( figure b-d) . of additional importance, when multiple proteins are expressed and purified at the same time, during protein isolation via imac, it is also important to keep all protein supernatants free of cross contamination due to the common his-tag found on all of our cell-line produced proteins. if cross contamination were to occur anywhere during our protocol, it would become nearly impossible to discern between proteins of the same subtype via these western blot analyses. instead, proper identification of any contaminating protein would require sequencing using mass spectrometry. for this reason, we encourage the use of protein-specific columns to avoid potential carry-over between successive purification. while at this time our protocol is solely being used for recombinant protein production, transfection of suspension expi f cells can be used for additional applications. as an example, we have confirmed that it may also be used for more affordable and larger scale virus-like particles (vlps) and mab production. this can be achieved through the transfection of the mammalian cells with multiple plasmids simultaneously, such as those encoding for the scaffold and envelope proteins or antibody heavy and light chains [ ] , respectively, yielding cells expressing multiple viral proteins and a higher yield as compared to adherent cell lines [ ] . additionally, our protocol is well-suited for scalability. in fact, through the use of multiple chromatography columns plugged to different or multi-channel peristaltic pumps, it is possible to run different conditioned media for the simultaneous purification of distinct recombinant proteins. the availability of an expression platform endowed of a high yield capacity accompanied by an accurate recombinant protein expression as compared to the native counterpart is fundamental not only for the common practice in research laboratories, but also in a company setting aimed at producing large amounts of good manufacturing practices (gmp) quality-grade recombinant proteins as vaccine formulations or as high-quality antigens to be used for diagnostic purposes [ ] . as a further example, the recombinant proteins generated through this described protocol have been extensively utilized in a pre-clinical setting to evaluate the breadth of the antibody response following immunization with universal influenza vaccine candidates [ , ] . in particular, the breadth of recognition of ha-specific b cells as well as polyclonal antibodies and mabs, has been assessed by using rha as antigens in immunological assays, such as elisa and elispot. likewise, ha∆sa probes can be used to for the isolation of ha-reactive plasmablasts and memory b cells using fluorescence-activated cell sorting (facs) [ ] . current studies are aimed at evaluating their antigenicity and immunogenicity, as sole recombinant proteins or conjugated to nanoparticles, for the pre-clinical evaluation of universal influenza vaccine candidates based on the main influenza virus surface glycoproteins [ ] . however, the here described pipeline can be easily adopted not only to produce a wide variety of other influenza virus proteins (such as m and np) [ ] , but also as an affordable, rapid and scalable methodology for recombinant protein-based vaccine or diagnostic platforms [ ] . in this regard, this protein production pipeline has been easily converted by our group for the rapid large-scale production of sars-cov- recombinant proteins [ , ] . collectively, the proteins generated through the expression pipeline detailed here are intended not only to serve as core reagents for the assessment of antibody breadth and durability elicited by universal influenza vaccine candidates, but also as prototype immunogens for next generation vaccines based on recombinant proteins. next generation vaccines for infectious diseases van der meeren, o. persistence of antibodies y after vaccination with a combined hepatitis a and b vaccine. hum. vaccines immunother elicitation of broadly protective antibodies following infection with influenza viruses expressing h n computationally optimized broadly reactive hemagglutinin antigens. immunohorizons beta -microglobulin-deficient mice can be protected against influenza a infection by vaccination with vaccinia-influenza recombinants expressing hemagglutinin and neuraminidase defining b cell immunodominance to viruses recombinant hemagglutinin influenza vaccine provides broader spectrum protection towards a universal influenza vaccine: different approaches for one goal prospects and challenges in the 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analytical approach high-throughput igg reformatting and expression characterization of influenza h n gag virus-like particles and extracellular vesicles co-produced in hek- sf recent developments in bioprocessing of recombinant proteins: expression hosts and process development h n vaccine-elicited memory b cells are genetically constrained by the ighv locus in the recognition of a neutralizing epitope in the hemagglutinin stem development of a pan-h influenza vaccine development of universal influenza vaccines based on influenza virus m and np genes novel platforms for the development of a universal influenza vaccine sars-cov- seroconversion in humans: a detailed protocol for a serological assay, antigen production, and test setup recombinant receptor-binding domain of sars-cov spike protein expressed in mammalian, insect and e. coli cells elicits potent neutralizing antibody and protective immunity we thank the international reagent resource (irr), influenza division, world health organization collaborating center for surveillance, epidemiology and control of influenza, centers for disease control and prevention (atlanta, ga, usa) for providing the mab clone at . . (fr- ). the authors declare no conflict of interest.vaccines , , key: cord- -gp u kh authors: song, xiang; hu, wei; yu, haibo; zhao, laura; zhao, yeqian; zhao, yong title: high expression of angiotensin-converting enzyme- (ace ) on tissue macrophages that may be targeted by virus sars-cov- in covid- patients date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: gp u kh angiotensin-converting enzyme- (ace ) has been recognized as the binding receptor for the severe acute respiratory syndrome coronavirus (sars-cov- ) that infects host cells, causing the development of the new coronavirus infectious disease (covid- ). to better understand the pathogenesis of covid- and build up the host anti-viral immunity, we examined the levels of ace expression on different types of immune cells including tissue macrophages. flow cytometry demonstrated that there was little to no expression of ace on most of the human peripheral blood-derived immune cells including cd + t, cd + t, activated cd + t, activated cd + t, cd +cd +cd low/− regulatory t cells (tregs), th cells, nkt cells, b cells, nk cells, monocytes, dendritic cells (dcs), and granulocytes. additionally, there was no ace expression (< %) found on platelets. compared with interleukin- -treated type macrophages (m ), the ace expression was markedly increased on the activated type macrophages (m ) after the stimulation with lipopolysaccharide (lps). immunohistochemistry demonstrated that high expressions of ace were colocalized with tissue macrophages, such as alveolar macrophages found within the lungs and kupffer cells within livers of mice. flow cytometry confirmed the very low level of ace expression on human primary pulmonary alveolar epithelial cells. these data indicate that alveolar macrophages, as the frontline immune cells, may be directly targeted by the sars-cov- infection and therefore need to be considered for the prevention and treatment of covid- . the epidemic of a new coronavirus infectious disease is wreaking havoc worldwide, caused by the severe acute respiratory syndrome coronavirus (sars-cov- ). currently, this virus has been globally spreading for over months, with over million confirmed cases and , deaths. due to the lack of effective antiviral drugs, most patients may be treated only by addressing their symptoms, including reducing fevers and coughs. preliminary results from the double-blind, randomized, placebo-controlled trial of intravenous remdesivir showed the reduced median recovery time ( days) for hospitalized covid- patients [ ] . based on this evidence, the united states food and drug administration (fda) has approved remdesivir under an emergency-use authorization for the treatment of adults and children with severe covid- . despite this approved treatment, high mortality rates among patients have persisted. as remdesivir is an antiviral drug, the treatment is not sufficient to control covid- [ ] . to date, no pharmacological treatments have been shown effective for the treatment of covid- [ , ] . consequently, understanding the pathogenesis and finding an alternative treatment to improve clinical outcomes is extremely urgent as a global top priority. sars-cov- is an rna virus that displays high similarities, in both genomic and proteomic profiling, with sars-cov that first emerged in humans in after transmitting from animals in open-air markets in china [ ] . the betacoronaviruses are divided into four lineages (i.e. a-d). both sars-cov and sars-cov- belong to lineage b, with single strand rna ( , and , bp respectively) [ , ] . most viruses enter cells through pattern recognition receptors (prr) mediated endocytosis. angiotensin-converting enzyme (ace ), with a multiplicity of physiological roles such as a negative regulator of the renin-angiotensin system [ ] , has been recognized as the prr for sars-cov- infecting host cells [ ] , which is similar to the sars-cov [ ] . the ace expression has been mainly distributed in microvilli of the intestine and renal proximal tubules, gallbladder epithelium, testicular sertoli cells and leydig cells, glandular cells of seminal vesicle and cardiomyocytes [ ] . the human respiratory system is primarily affected by the sars-cov- infection. using the polyclonal anti-serum-based immunohistochemistry, the expression of ace was reported on type ii alveolar epithelial cells [ ] . however, a single cell-rna profiling analysis showed that only . % of lung type ii alveolar epithelial cells expressed ace at rna level [ ] , with no expression of ace protein [ ] . thus, there are fundamental knowledge gaps underlying the pathogenesis of covid- that need to be clarified. to understand the immunopathology and advance the strategies for the prevention and treatment of covid- , we examined the levels of ace expression on different types of immune cells. our data demonstrated that the activated macrophages and alveolar tissue macrophages, among others, displayed high levels of ace , while most of the immune cells were negative or displayed very low expressions of ace . this data highlights the importance of macrophages in the pathogenesis and treatment of covid- . to determine the expression of ace on different types of immune cells, human buffy coat blood units (n = ; mean age of ± . ; age range from to years old; males and females) were purchased from the new york blood center (new york, ny, usa). human buffy coats were initially added to ml chemical-defined serum-free culture x-vivo tm mediums (lonza, walkersville, md, usa) and mixed thoroughly with ml pipette. next, they were used for isolation of peripheral blood-derived mononuclear cells (pbmcs). pbmcs were harvested as previously described [ ] . briefly, mononuclear cells were isolated from buffy coats blood using ficoll-paque tm plus (g= . , ge healthcare, chicago, il, usa). next, the red blood cells were removed using ack lysing buffer (lonza, walkersville, md, usa). after three washes with saline, the whole pbmc were utilized for flow cytometry. to isolate monocytes, monocytes were purified from pbmc by using cd + microbeads (miltenyi biotec, bergisch gladbach, germany) according to the manufacturer's instruction, with purity of cd + cells > %. to generate th cells, cd + t cells were initially isolated from pbmc using cd + microbeads (miltenyi biotec, bergisch gladbach, germany) according to the manufacturer's instruction, with purity of cd + t cells > %. the purified cd + t cells were seeded at ´ cells/well in the anti-cd monoclonal antibody (mab) ( µg/ml, bd pharmingen, franklin lakes, nj, usa) precoated -well tissue culture-treated plate, in the presence of soluble anti-cd mab ( µg/ml, bd pharmingen, franklin lakes, nj, usa), interleukin (il)- ( ng/ml, biolegend, san diego, ca, usa), il- β ( ng/ml, biolegend, san diego, ca, usa), transforming growth factor (tgf)-β ( ng/ml, biolegend, san diego, ca, usa), il- ( ng/ml, biolegend, san diego, ca, usa), penicillin-streptomycin ( µg/ml, sigma, saint louis, mo, usa), the neutralizing antibodies anti-il- mab ( µg/ml, bd pharmingen, franklin lakes, nj, usa) and anti-ifn-γ mab ( µg/ml, bd pharmingen, franklin lakes, nj, usa), in x-vivo serum-free medium ( µl per well), at °c, % co conditions. after culturing for days, the cells were stimulated with the cell activation cocktail of . mm monensin sodium salt, . mm phorbol -myristate -acetate and . mm ionomycin calcium salt (bio-techne corporation, minneapolis, mn, usa) at µl. stock solution was diluted with ml of the culture medium for hours at °c, % co conditions. finally, the th cells were harvested for flow cytometry analysis. adult human platelet units (n = ; mean age of . ± . ; age range from to years old; males and female) were purchased from the new york blood center (new york, ny, http://nybloodcenter.org/). the mitochondria were isolated from platelets using the mitochondria isolation kit (thermo scientific, rockford, usa) according to the manufacturer's recommended protocol [ ] . the concentration of mitochondria was determined by the measurement of protein concentration using a nanodrop spectrophotometer (thermofisher scientific, waltham, ma, usa). for mitochondrial staining with fluorescent dyes, mitochondria were labeled with mitotracker deep red fm ( nm) (thermo fisher scientific, waltham, ma) at °c for minutes according to the manufacturer's recommended protocol, followed by two washes with pbs at rpm ´ minutes [ ] . finally, the mitochondria were harvested for flow cytometry analysis. to generate m macrophages and determine ace expression, monocytes were purified from human pbmc by using cd microbeads (miltenyi biotec, bergisch gladbach, germany) according to the manufacturer's instruction, with the purity of cd + cells > %. the isolated monocytes were seeded in the six-well tissue culture-treated plate at ´ cells/ well in the attached monocytes were washed twice with pbs to remove floating cells and cellular debris, followed by treatment with ng/ml mcsf (sigma, saint louis, mo, usa) in x-vivo serum-free medium at °c, % co . after culturing for days, the m macrophages were treated with µg/ml lipopolysaccharides (lps) (sigma, saint louis, mo, usa) or ng/ml il- (biolegend, san diego, ca, usa) in duplicates respectively for hours in x-vivo serum free medium. consequently, cells from different groups were collected for evaluations. phenotypic characterization of pbmc, monocytes, macrophages and th cells were to determine an expression of ace protein on different types of pbmc, macrophages, th cells, platelets or platelet-derived mitochondria, the indirectly-labeled immunostaining with mouse anti-human ace mab (novus biologicals, catalogue# nbp - - µg, clone #ac f, littleton, co, usa) was utilized in combination with above lineage-specific fluorescence-labeled mabs. briefly, samples were first pre-incubated with human bd fc block to block non-specific binding (bd pharmingen, franklin lakes, nj, usa) for minutes at room temperature, before being directly aliquoted for different antibody stainings. cells were initially incubated with mouse anti-human ace mab at : dilution, at room temperature for minutes. next, cells were washed with pbs at × g minutes and then stained with fitc-conjugated affinipure donkey or cy -conjugated affinipure donkey anti-mouse nd abs (jackson immunoresearch laboratories, west grove, pa, usa) at : dilution for minutes at room temperature. cells only with nd ab staining served as control. after finishing the nd ab staining, cells were washed with ml pbs to remove residual nd ab. consequently, cells were immunostained with above lineage-specific fluorescence-labeled mabs, as previously described [ ] [ ] [ ] female nod/ltj mice (aged weeks) and nod-scid mice (aged - weeks) were purchased from jackson laboratories (bar harbor, me, usa) and maintained under pathogen-free conditions, according to a protocol approved by the institutional animal care committee (acc). tissue samples (e.g., small intestine, lung, liver and kidney) were fixed in % formaldehyde, processed, and embedded in paraffin. tissue sections were cut at mm thickness. tissue sections from nod/ltj mice ( weeks old) were utilized for immunohistochemistry including lung tissue sections (n = ), small intestine tissue sections (n = ), liver tissue sections (n = ), and kidney tissue sections (n = ). immunostaining was performed as previously described with minor modifications [ ] . to block non-specific staining, sections were incubated in a buffer containing . % horse serum (vector laboratories, burlingame, ca, usa) and mouse fc block (bd pharmingen, franklin lakes, nj, usa) for minutes at room temperature. tissue sections were initially immunostained with ace rabbit polyclonal antibody (abcam, cambridge, ma, usa) at : dilution for hours at room temperature. next, tissue sections were stained with cy conjugated affinipure donkey anti-rabbit nd ab (jackson immunoresearch laboratories, west grove, pa, usa) at : dilution and combined with fitc-conjugated rat anti-mouse f / mab (ebioscience, san diego, ca, usa) at : dilution at room temperature for hour. for every experiment, only tissue sections with nd ab staining served as negative control. finally, the slides were covered by using mounting medium with dapi (vector laboratories, burlingame, ca, usa) and photographed with a nikon a r confocal microscope on a nikon eclipse ti inverted base, using nis elements version . software. statistical analyses were performed with graphpad prism (version . . ) software. the normality test of samples was performed by the shapiro-wilk test. statistical analyses of data were performed by the two-tailed paired student's t-test to determine statistical significance between untreated and treated groups. the mann-whitney u test was utilized for non-parametric data. values were given as mean ± sd (standard deviation). statistical significance was defined as p < . , with two sided. to explore the direct action of sars-cov- on immune cells, we examined the ace expressions on different types of immune cells from human peripheral blood (n = ). they were characterized and gated with cell type-specific surface markers [ ] : cd for t cells, cd + cd + for cd + t cells, cd + cd + for cd + t cells, cd c + cd for myeloid dendritic cells (mdc), cd -cd + for plasmacytoid dc (pdc), cd for monocytes, cd for b cells, cd + cd + cd low/for regulatory t cells (tregs), cd + cd + for nkt cells, cd -cd + for nk cells, and cd -cd b + for granulocytes ( figure a-c) . flow cytometry demonstrated that there were no expressions of ace on most types of immune cells, or with a background level (< %) ( figures d) . the percentages of ace + cells for nk and nkt cells were only . % ± . % and . % ± . % respectively (n = ) ( figure d ). the activated cd + hla-dr + t cells displayed only . % ± . % of ace + cells (n = ). the activated cd + hla-dr + was only . % ± . % (n = ) ( figure d ). the data suggests that sars-cov- virus may not directly attack blood immune cells lacking the ace expression. t-helper type (th ) cells are important pathogenic mediators for several autoimmune diseases, potentially contributing to the pathogenesis of covid- . rorγt (retinoic acid receptorrelated orphan nuclear receptor gamma t) belong to nuclear hormone receptors (nhrs) and act as a crucial transcription factor for the differentiation and function of th cells both in humans and mice [ ] . using rorγt, interleukin- a (il- a), il- f, and ccr as specific th markers [ ] , the purity of il a + rorgt + th cells was . % ± . % (figure a ). the percentage of il a + il f + th cells was . % ± . % ( figure a ). the purity of il a + ccr + th cells was . % ± . % ( figure a ). the gated th cells failed to express ace (figures b, n = ). this data implies no direct interaction between th cells and sars-cov- . increasing clinical evidence demonstrated the coagulation abnormalities in covid- subjects including disseminated intravascular coagulopathy (dic) and low levels of platelet count [ , ] . to determine whether platelets were directly targeted by sars-cov- or trigged by viral inflammatory reactions, we examined the ace expression on the highly-purified cd b + cd a + platelets from human peripheral blood ( figure a our previous work established that platelets could release mitochondria contributing to the immune modulation and islet b-cell regeneration [ ] . to explore the mitochondrial function in viral infection, flow cytometry indicated that while the purified platelet-derived mitochondria did not express ace (figures d), they strongly display the mitochondrial antiviral-signaling protein (mavs) with the percentage of mitotracker red + hsp + mavs + mitochondria at . % ± . % ( figure e , n = ) [ ] . this data suggests that platelets may have potential to improve antiviral immunity through the releasing mitochondria. macrophages have been characterized with type macrophages (m , inflammatory) and type macrophages (m , anti-inflammatory), according to their phenotypic differences such as spindlelike morphology and high expression cd and cd on m macrophages [ ] . initially, flow cytometry established that cd + monocytes from human peripheral blood failed to express ace ( . % ± . %, n = ) ( figure a ). m macrophages were then generated in the presence of ng/ml macrophage colony-stimulating factor (m-csf) with the percentage of spindle-like cells at . % ± . % (n = ). to evaluate the ace expressions on macrophages, m macrophages were activated by the treatment with lipopolysaccharide (lps) [ ] and interleukin- (il- ) respectively [ ] . phase contrast image showed significant differences in the morphology between two groups ( figure b ). lps-treated m macrophages exhibited pseudopod-like protrusions compared to the spindle form of il- -treated m macrophages ( figure b, left) . flow cytometry demonstrated that the level of ace expression was higher on the lps-activated m macrophages than that of il- -treated m macrophages ( figure c-e) . this finding was further confirmed by the confocal microscopy and image analysis ( figure f ). therefore, the data suggests the upregulation of ace expression on the activated m macrophages. to determine the expression of ace on tissue macrophages, we initially performed doublestaining with mouse macrophage marker f / through the immunohistochemistry in the small intestine, lung, liver, and kidney tissues of -week non-obese diabetic (nod) mice. using an expression of ace in the small intestine as a positive control ( figure a) , the data revealed that most ace expressions were co-localized with the f / + tissue macrophages in the lung and liver ( figure b and c) , which are known as dust cells (alveolar macrophages, figure b ) and kupffer cells ( figure c ) respectively. unexpectedly, there was little to no expression of ace on the alveolar epithelial cells ( figure b ). additionally, we observed the strong expressions of ace on the proximal tubules, with some scattered f / + macrophages double-positive with ace staining ( figure d ). to further determine the distribution of ace expression on alveolar macrophages, tissue sections were examined by utilizing non-inflammatory lung tissue from nod-scid mice ( figure e ). immunohistochemistry confirmed the marked co-localization of ace expression on f / + alveolar macrophages ( figure f ), with a low expression of ace on alveolar epithelial cells. next, we utilized the primary human pulmonary alveolar epithelial cells (hpaepic) to define their level of ace expression. flow cytometry proved the low level ( %) of ace expression on human pulmonary alveolar epithelial cells ( figure g ). therefore, these data indicate the high expression of ace on tissue macrophages. the human pulmonary system is primarily targeted by sars-cov- . ace and proteases such as tmprss (transmembrane protease, serine ) or cathepsin b/l were utilized for host cellular entry of sars-cov- [ ] . ace may act as a limiting factor for viral infection at the initial stage [ ] . sungnak et al. reported the high expression of ace in nasal epithelial cells [ ] . our current studies demonstrated little to no expression of ace on both primary human pulmonary alveolar epithelial cells and mouse lung alveolar epithelial cells, which is consistent with previous reports [ , ] . notably, we found that high expression of ace was colocalized with tissue macrophages of the lung (alveolar macrophages) and liver (kupffer cells), and up-regulated on the activated m macrophages. however, most immune cells in human peripheral blood were negative for ace expression, including the freshly-isolated monocytes. therefore, these data highlight the importance of alveolar macrophages during the pathogenesis of lung damage in covid- subjects. based on this evidence, we propose that lung macrophages may be directly clinical autopsies from sars-cov-infected patients demonstrated that there were major pathological changes in the lungs, immune organs, and small systemic blood vessels with vasculitis. the detection of sars-cov was primarily found in the lung and trachea/bronchus, but was undetectable in the spleen, lymph nodes, bone marrow, heart and aorta [ ] . this evidence highlights the overreaction of immune responses induced by viral infection which resulted in significant harm, as evidenced by pathogenesis of the lungs, immune organs, and small systemic blood vessels. the pathological study in covid- patients revealed that the majority of infiltrated immune cells in alveoli were macrophages and monocytes, with minimal lymphocytes, eosinophils and neutrophils [ ] . there were abundant proinflammatory macrophages in the bronchoalveolar lavage fluid of severe covid- patients [ ] . thus, the virus-infected alveolar macrophages play a critical role in the pathogenesis of covid- and sars [ ] [ ] [ ] and may recruit the lung infiltration of additional immune cells through predominantly releasing cytokines and chemokines [ , ] , resulting in pulmonary edema and hypoxemia: the hallmark of acute respiratory distress syndrome (ards) ( figure ). consequently, the viral inflammation disrupts the homeostasis and causes multiple dysfunctions including lymphopenia [ ] , coagulopathy [ , ] , diarrhea [ ] , liver injury [ ] , manifestations of cardiovascular [ , ] and central nervous system [ ] , and renal failures [ ] . additionally, the percentage of inflammation-associated th cells was markedly increased in the peripheral blood of covid- patients [ ] . th cells act as important pathogenic mediators for several autoimmune diseases including type diabetes (t d), multiple sclerosis, rheumatoid arthritis, alopecia areata (aa), psoriasis, and even the insulin resistance in type diabetes (t d) [ ] [ ] [ ] . thus, the viral inflammation caused by sars-cov- is very similar to the majority of autoimmune diseases in humans caused by overactive immune systems. therefore, immune modulation strategy may be potentially beneficial to enhance antiviral immunity and efficiently reduce the viral load, improve clinical outcomes, expedite patient recovery, and decline the rate of mortality in patients after being infected with sars-cov- . macrophages are widely distributed in human tissues and organs with pleiotropic functions in maintaining homeostasis. therefore, dysfunctions of macrophages may increase vulnerability to the viral infection. for example, the phenotype of intestinal macrophages may be changed during the chronic intestinal inflammation and infection [ ] . due to high expression of ace in the epithelial cells of the intestine and the utilization of immune-suppression regimens, patients with chronic inflammatory bowel disease (ibd) might have an increased risk of the sars-cov- infection [ ] . additionally, increasing clinical evidence demonstrates that the chronic metabolic stress-induced inflammation causes multiple dysfunctions of macrophages, leading to the insulin resistance in type diabetes [ ] . clinical studies have shown that type diabetes is one of the major risk factors for covid- [ , [ ] [ ] [ ] [ ] , while an interstitial subset of cd + lung-resident macrophages, primarily located around the airways in close proximity to the sympathetic nerves of the bronchovascular bundle, exhibit the characteristics of type macrophages and antiinflammatory effects [ ] . therefore, these two types of macrophages play an essential role in the immune surveillance and maintenance of homeostasis of the pulmonary system. considering all current approaches for the prevention and treatment of covid- , there are no therapies, either being tested or at the beginning of the pipeline, that directly focus on the modulation of macrophages. to address the overreaction of immune responses caused by viral infection, immune suppression regimens (e.g., chloroquine, hydroxychloroquine, jak inhibitors, anti-cytokine therapy, and anti-il- r antibody) are being tested in clinical trials, which may be not sufficient to treat covid- as they make subjects more vulnerable to the sars-cov- infection, in addition to their associated side effects. rather, the immune modulation strategy like that of traditional chinese herbs lian-hua-qing-wen granule [ ] may potentially enhance anti-viral immunity and improve clinical outcomes in patients after being infected with sars-cov- . additionally, over the last years, dr. yong zhao at tianhe stem cell biotechnologies has developed the stem cell educator (sce) therapy by utilizing the immune modulation of human cord blood-derived multipotent stem cells (cb-sc) for the treatment of type diabetes and other inflammationassociated diseases. clinical safety and efficacy have been demonstrated for sce therapy through multicenter clinical trials [ ] [ ] [ ] [ ] . recently, mechanistic studies established that cb-sc-released exosomes could differentiate the purified cd + monocytes into m macrophages [ ] , suggesting that sce therapy may have the potential to treat covid- (clinicaltrials.gov: nct ). targeting alveolar and other tissue macrophages through immune modulations may be potentially beneficial to correct the viral inflammation, effectively ameliorate anti-viral immunity, efficiently reduce the viral load, improve clinical outcomes, expedite the patient recovery, and decline the rate of mortality in patients after being infected with sars-cov- . authors are grateful to mr. poddar and mr. ludwig for generous funding support via hackensack umc foundation. all authors have no financial interests that may be relevant to the submitted work. ace protein was primarily displayed on alveolar macrophages of lung, with no or low expression on alveolar type (at-i) and type (at-ii) epithelial cells. upon entering the pulmonary alveoli, healthy alveolar macrophages may directly kill the virus, with asymptomatic or mild clinical symptoms. at this earlier stage , the infected alveolar macrophages may alternatively recruit other immune cells to build up the antiviral immunity through releasing cytokines (e.g., il- , il- , il- , and tnfa) and chemokines (e.g. cxcl and cxcl to recruit granulocytes, cxcl to recruit t cells, nk cells, and dcs). for instance, the recruited cd + t cells may secret interferon (ifn)-g to strengthen the antiviral immunity of alveolar macrophages and minimize the viral load. however, if this first line of defense is broken, the more cytokines and chemokines are released from the dead cells or dead-cell engulfed macrophages (stages and ), the more immune cells are infiltrated into pulmonary systems, leading to patients experiencing a rapid deterioration and the development of ards with high fatality in the clinic. remdesivir for the treatment of covid- -preliminary report pharmacologic treatments for coronavirus disease (covid- ): a review should chloroquine and hydroxychloroquine be used to treat covid- ? a rapid review systematic comparison 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findings of covid- associated with acute respiratory distress syndrome the pathogenicity of th cells in autoimmune diseases rd. th cells in immunity and autoimmunity the potential pathogenic role of il- /th cells in both type and type diabetes mellitus origin, differentiation, and function of intestinal macrophages viral screening before initiation of biologics in patients with inflammatory bowel disease during the covid- outbreak macrophages, inflammation, and insulin resistance case-fatality rate and characteristics of patients dying in relation to covid- in italy diabetes is a risk factor for the progression and prognosis of covid- clinical course and risk factors for mortality of adult inpatients with covid- in wuhan, china: a retrospective cohort study identification of a nerve-associated, lung-resident interstitial macrophage subset with distinct localization and immunoregulatory properties efficacy and safety of lian-hua qing-wen granule for covid- : a protocol for systematic review and meta-analysis reversal of type diabetes via islet beta cell regeneration following immune modulation by cord blood-derived multipotent stem cells hair regrowth in alopecia areata patients following stem cell educator therapy targeting insulin resistance in type diabetes via immune modulation of cord blood-derived multipotent stem cells (cb-scs) in stem cell educator therapy: phase i/ii clinical trial differentiation capacity of peripheral naive t cells in rheumatoid and psoriatic arthritis key: cord- - qwtvm x authors: chikhlikar, priya; de arruda, luciana barros; maciel, milton; silvera, peter; lewis, mark g.; august, j. thomas; marques, ernesto t.a. title: dna encoding an hiv- gag/human lysosome-associated membrane protein- chimera elicits a broad cellular and humoral immune response in rhesus macaques date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: qwtvm x previous studies of hiv- p gag immunization of mice have demonstrated the usefulness of targeting antigens to the cellular compartment containing the major histocompatibility complex type ii (mhc ii) complex molecules by use of a dna antigen formulation encoding gag as a chimera with the mouse lysosome-associated membrane protein (mlamp/gag). in the present study, we have analyzed the magnitude and breadth of gag-specific t-lymphocyte and antibody responses elicited in rhesus macaques after immunization with dna encoding a human lamp/gag (hlamp/gag) chimera. elispot analyses indicated that the average gag-specific ifn-γ response elicited by the hlamp/gag chimera was detectable after only two or three naked dna immunizations in all five immunized macaques and reached an average of spot-forming cells (sfc)/ ( ) pbmcs. high ifn-γ elispot responses were detected in cd (+)-depleted cells, indicating that cd (+) t-cells play a major role in these responses. the t-cell responses of four of the macaques were also tested by use of elispot to overlapping -amino acids (aa) peptide pools containing ten peptides each, encompassing the complete gag protein sequence. the two mamu immunized macaques responded to eight and twelve of the pools, the mamu b to six, and the other macaque to five pools indicating that the hlamp/gag dna antigen formulation elicits a broad t-cell response against gag. additionally, there was a strong hiv- -specific igg response. the igg antibody titers increased after each dna injection, indicating a strong amnestic b-cell response, and were highly elevated in all the macaques after three immunizations. moreover, the serum of each macaque recognized of the peptides of a -aa peptide library covering the complete gag amino acid sequence. in addition, hiv- -specific iga antibodies were present in the plasma and external secretions, including nasal washes. these data support the findings of increased immunogenicity of genetic vaccines encoded as lamp chimeras, including the response to dna vaccines by non-human primates. several human immunodeficiency virus type (hiv- ) antigenic formulations have been tested in animal models and clinical trials, but the immunogenicity and protection achieved to date are still far from the desired goals for an hiv- vaccine. while the correlates of immune protection are still only vaguely defined, it is now generally recognized that an effective hiv- vaccine must elicit strong t-cell responses as well as neutralizing antibody [ ] [ ] [ ] [ ] . it has been postulated that the immunogenicity of a protein antigen can be enhanced by targeting the antigen to the major histocompatibility complex type ii (mhc ii) processing compartment of professional antigen-presenting cells (apcs). one of the approaches is the use of the lysosomal associated membrane protein- (lamp) as a lamp/antigen chimera to target endogenous antigens to the mhc ii processing compartment [ ] [ ] [ ] [ ] [ ] [ ] . many laboratories have reported that lamp targeting can greatly enhance the immune responses against a number of antigens, including hpv- -e and e proteins; human telomerase reverse transcriptase (htert); west nile virus prem-e; the thyroid hormone receptor (tshr); hiv- gag, env gp , env gp , and nef; human melanosomal antigen (mage- ); dengue prem-e; listeriolysin o; and sars coronavirus n protein [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . several approaches have been used to design dna vaccines encoding lamp/antigen chimeras. one frequently used construct contains the transmembrane and cytoplasmic (tmcy) domains of lamp added to the c-terminus of the protein antigen. more recently, we have found that some antigens, including hiv- gag, must be incorporated into the entire lamp molecule in order to effect enhanced antigen expression and trafficking to the lysosomal academic editor: douglas nixon, university of california, san francisco, united states of america compartment. several lamp/antigen chimeras, including proteins of west nile, dengue, sars cov, hpv and hiv- , have been shown to co-localize in vitro with mhc ii, lamp- , lamp- , and h- m in multiple cell types by confocal imunofluorescense microscopy, and/or by immunogold electron microscopy [ ] [ ] [ ] [ ] [ ] [ ] ] . the immunological benefits of lamp-targeted antigens have been demonstrated in several mouse strains and, most importantly, also in humans [ ] [ ] . lamp/antigen chimeras have been shown to induce increased cd + responses to the antigens in several assay systems, producing increased secretion of il- , il- , il- and ifn-c cytokines; increased proliferative responses; a greater number of spot-forming cells (sfcs) in elispot assays; intracellular cytokine staining (ics); higher precursor frequencies; increased functional avidity and a broader response repertoire [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the increased cd + -mediated responses produced by the lamp/chimeras are thought to play an important role in modulating b-cell, cd + responses and the development of immune memory [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . when compared to the non-targeted molecules, lamp/antigens elicited greatly increased antibody titers, viral neutralization, antibody affinity and numbers of b-cell epitopes recognized. cd + responses were also enhanced in several lamp/antigen chimeric systems, as assessed by tetramer staining, ifn-c elispot, chromium release, and the functional avidities and t-cell response repertoires of cd + cells. the longevity of the immunological memory of b cells and cd + cells is also increased in animals immunized with lamp/chimeras. previous studies of hiv- dna antigen formulations have described an hiv- p gag dna vaccine that elicited strong, broad and poly-functional cellular and humoral immune responses in immunized mice when the gag sequence was incorporated into the complete lamp cdna sequence and the coding sequences were bracketed by the inverted terminal repeat (itr) sequences of adeno-associated virus (aav) [ ] [ ] [ ] . these observations in mice led us to investigate the immunogenicity of a naked dna encoding the hiv- lamp/gag antigen formulation in rhesus macaques, a relevant animal model for testing hiv vaccines for potential use in humans. we found that immunization of these primates with a human lamp/gag (hlamp/gag) dna vaccine promoted a humoral and cellular immune response, which was associated with a sustained activation of b-lymphocytes as well as cd + and cd + t cells. furthermore, gag-specific t cells were detected after only two immunizations and were further expanded by booster injections of hlamp/gag chimera. these results substantiate the usefulness of the lamp vaccination strategy as a means of effectively eliciting t-and b-cell responses to naked dna immunization of rhesus macaques. the mouse lamp/gag (mlamp/gag) plasmid was constructed as described previously [ ] : nucleotides - of the hiv- hxb p gag gene (genbank tm accession number k ; hiv sequence database, los alamos national laboratory theoretical biology and biophysics, los alamos, nm, usa) was inserted into the pitr vector [ ] , which contains the aav-itr flanking the expression elements (cytomegalovirus promoter and bovine growth hormone polyadenylation signal). the p gag sequence was inserted between the luminal domain and the tmcy domain of mouse lamp (mlamp) (genbank tm accession number j ), as described previously [ ] . in the present study, the complete mlamp cdna described above was replaced by the human lamp- (hlamp) cdna (genbank tm accession number nm ). the control plasmids, consisting of mlamp and hlamp, were constructed using the respective complete lamp sequences without gag. the hlamp/gag plasmid used for vaccination of rhesus macaques was produced by quality biological inc. (gaithersburg, md, usa), with a dna purity of % and endotoxin level of . eu/mg. human cells were plated in -well plates ( cells/well) and transfected with plasmid dna ( mg) using the fugene tm (roche applied science, indianapolis, in, usa) transfection reagents according to the manufacturer's instructions. the western blot analysis was done as previously described [ ] . evaluation of hlamp/gag targeting by confocal microscopy mhc ii (i-ek)-expressing cells (dcek.icam.hi , a gift of dr. susan swain, the trudeau institute, saranac lake, ny, usa) [ ] were plated onto poly-lysine-coated coverslips in -well plates ( cells/well) and incubated overnight. the cells were transfected with the dna plasmids using lipofectamine tm transfection reagent (invitrogen life technologies, carlsbad, ca, usa), and - h later they were stained to evaluate cellular localization. coverslips were fixed in % paraformaldehyde in phosphate-buffered saline (pbs) for min and washed with pbs, then blocked and permeabilized with pbs containing % normal goat serum and . % saponin. for detection of gag expression, the cells were incubated for h with mouse anti-gag monoclonal antibody (provided by dr. james hildreth, johns hopkins school of medicine, baltimore, md, usa) at a : dilution. after three washes with . % saponin in pbs, the cells were incubated for h with texas red-labeled goat anti-mouse igg (bd pharmingen, san diego, ca, usa) at a : dilution. co-localization of the hlamp/gag chimera with mhc ii of the transfected dcek cells was performed by double immunostaining, first for the hlamp/ gag chimera proteins as described above and followed by anti-mhc ii, by incubating the cells for h with fitc-labeled goat antimouse i-ek ( - - s) (bd pharmingen) at a : dilution. the cells were then washed three times with pbs, and the coverslips were mounted onto glass slides using prolong antifade reagent (molecular probes, eugene, or, usa). confocal microscopy was performed using a wallac confocal laser scanning microscope. colocalization of the proteins with mhc ii was thus determined by merging fluorophore images individually captured and digitally colored by use of adobe photoshop . (adobe system corp., san jose, ca, usa). immunization of mice female balb/c mice, - weeks of age, were obtained from charles river (kingston, ny, usa). mice in groups of eight were each immunized twice ( days apart), intramuscularly (i.m.), with mg of the indicated plasmid and sacrificed days later. for cd + experiments, the mice were challenged days after the first immunization with a vdk- vaccinia virus plasmid containing the native gag sequence ( plaque-forming units; nih aids research and reference reagent program, rockville, md, usa). antibody responses mouse serum was obtained from the tail vein days after the second immunization. serum igg levels were measured by enzyme-linked immunosorbent assay (elisa) as described previously [ ] . preparation of splenocytes for assaying the t-cellmediated immune responses of immunized mice singlecell suspensions depleted of red blood cells were prepared from freshly isolated mouse splenocytes in culture medium (rpmi medium supplemented with % v/v fbs, u/ml penicillin/streptomycin, mm glutamine, mm mercaptoethanol, and . m hepes buffer). splenocytes were counted and resuspended at cells/ml in culture medium for t cell-mediated assays. t-cell responses of immunized mice were measured as described previously [ ] . rhesus macaques five healthy -to -kg male rhesus macaques were maintained in the non-human primate facility of southern research institute, frederick, md, usa. animal care and treatment were in accordance with standards approved by the institutional animal care and use committee, according to the principles set forth in the guide for the care and use of laboratory animals, national research council, national academy press, . immunization of macaques each animal was immunized intramuscularly (i.m.) five times at weeks , , , and with mg of hlamp/gag dna plasmid using a biojector, and blood samples were drawn over the week experiment period. mucosal samples including mouth swabs, nasal and rectal washes were collected after four dna immunizations. blood and mucosal samples of non-immunized macaques were supplied periodically as controls. isolation of peripheral blood mononuclear cells (pbmcs) pbmcs were isolated by centrifugation ( g, min) on a ficoll-hypaque gradient (amersham biosciences piscataway, nj, usa). mononuclear cells were collected from the interface and washed three times in cold phosphate-buffered saline with ca + and mg + (gibco, grand island, ny, usa). residual red blood cells were removed with ack lysing buffer (quality biological inc., gaithersburg, md, usa). pbmcs to be used for cytokine analysis or elispot assay were resuspended in supplemented culture medium (rpmi medium containing % fbs, u/ml penicillin/streptomycin, and mm lglutamine). cd + lymphocyte separation positive selection of cells expressing cd + antigen was performed with monoclonal antihuman cd + conjugated to r-phycoerythrin (pe) and microbeads conjugated to monoclonal anti-pe antibodies (cd microbead kit, miltenyi biotec, bergisch gladbach, germany) as described by the manufacturer. the cd + cells were . % pure as assessed by flow cytometry. fractionated cells were suspended in rpmi- medium and used on the same day for elispot assay. hiv gag-specific ifn-c elispot assay the frequency of ifn-c-producing cd + or cd + t cells from immunized rhesus macaques was measured by elispot assays, using the ifn-c elispot set from bd-biosciences pharmingen according to the manufacturer's protocol. initially, elispot plates were coated with anti-human ifn-c ig at mg/ml and incubated at uc overnight. after blocking with rpmi- containing % fbs for h at room temperature (rt), total pbmcs ( cells/well) were cultured with hybridoma serum-free medium (gibco) supplemented with % fbs, u/ml penicillin/streptomycin and mm l-glutamine, in the presence of mg/ml of hivsf p gag recombinant protein (rgag); or hiv gag -amino acids (aa) peptides overlapping by -aa; or -aa overlapping by -aa. a negative control included in each assay consisted of medium lacking hiv antigen. phytohemagglutinin (pha) (sigma, st. louis, mo, usa) at ng/ml with ng/ml ionomycin (sigma) was included as a positive control for each rhesus macaque. after h of culture, the plates were washed and incubated with biotinylated anti-human ifn-c for h at rt, followed by hrpconjugated avidin for h at rt. the reaction was developed with -amino- -ethylcarbozole substrate (calbiochem-novabiochem corporation, san diego, ca, usa). analysis of the ifn-c levels was performed using an immunospot image analyzer (cellular technology limited, cleveland, oh, usa). background spots obtained with medium alone were subtracted from each experimental value. all results were expressed as mean number of sfc per pbmcs. for each antigen, a responder rhesus macaque was defined as one exhibiting a significant number ifn-c sfc cells over the background value or value for non-immunized macaques, at any time point over the immunization course. analysis of il- secretion by capture elisa total pbmcs ( cells) from immunized macaques were cultured in a well plate (nunc, roskilde, denmark) containing recombinant human il- ( u/well). except for the negative and positive control wells containing culture medium alone and ng/ml pha (sigma) containing ng/ml ionomycin, respectively, pbmcs were stimulated with mg/ml hivsf p rgag. after a -h incubation at uc in % co , the supernatants were collected for detection of secreted cytokines and stored at uc until further use. il- concentrations were determined using a monkey il- opteia tm set (bd pharmingen). hiv gag-specific humoral and mucosal immune responses serum samples were tested throughout the experimental period for the presence of binding antibody to hiv gag. nunc plates were coated with mg/ml hiviiib lysate in sodium carbonate-bicarbonate buffer, ph . (pierce, rockford, il, usa). after overnight incubation at uc, the solution was removed, and the plates were washed six times with pbs containing . % tween- (pbs-t) wash buffer. the plates were then incubated for h at uc with blocking buffer (pbs-t with % fbs) and then washed three times with pbs-t. igg responses were measured with four serial : dilutions, starting at : , in blocking buffer. iga responses in serum, nasal, mouth and rectal washes were measured with ml of -fold diluted sample added to the blocked plate in duplicate. the plates were incubated overnight at uc. after six washes, ml of horseradish peroxidase-conjugated rabbit anti-monkey igg (sigma) diluted : in blocking buffer was added to each well. for the detection of iga antibodies, ml of goat antimonkey iga (nordic immunology, tilburg, netherlands) diluted : in blocking buffer was added to each well. the plates were incubated for h at uc and washed eight times with washing buffer. turbo tmb substrate solution (bd pharmingen) was then added to each well and incubated for min at rt. the reaction was stopped by adding ml of m sulfuric acid, and absorbance at nm was measured in a bio-rad model microplate reader. b-cell epitope mapping hiv- gag -aa peptides, spanning residues to with a -aa overlap (nih aids research and reference reagent program), were diluted in . m sodium carbonate-bicarbonate buffer, ph . , to give a concentration of mg/ml, and ml of the individual peptide was added to each well of a -well plate, in duplicate. after overnight incubation at uc, the solution was removed, and the plates were washed six times with pbs-t. they were then incubated with blocking buffer for h at uc and washed three times. serum samples ( ml of a : dilution in blocking buffer) were added to each well, and the plates were incubated overnight at uc. after the plates were washed six times, ml of hrp-conjugated rabbit anti-monkey igg (sigma), diluted : in blocking buffer, was added to each well. the plates were incubated for h at uc and then washed. turbo tmb substrate solution (bd pharmingen) was added to each well and incubated for min at rt. the reaction was stopped by adding ml of m sulfuric acid, and absorbance at nm was measured in a bio-rad model microplate reader. statistical analyses all the graphs were made using graphpad prism version . a for macintosh (graphpad software, san diego, ca, usa). lamp/gag expression and cellular trafficking to the cellular mhc ii compartment has been repeatedly demonstrated in studies with murine lamp chimeras [ ] [ ] [ ] [ ] [ ] [ ] . the present study confirmed the similar expression and trafficking of the human lamp/gag protein chimera. western blotting with anti-gag antibody showed the presence of , kda lamp/gag protein at comparable levels in cells transfected with either mouse or human lamp/gag ( figure a ). there appeared to be increased degradation of gag in the hlamp/gag transfected cells; however, the significance of this is not known as the results of other similar studies of mlamp/gag transfected cells have shown variable levels of gag degradation products [ ] [ ] [ ] . co-localization of the hlamp/gag chimera transgene product with the cellular mhc ii was confirmed with transfected dcek cells stained with anti-gag and anti-mhc ii monoclonal antibodies ( figure b) . verification of the in vivo immune responses to the hlamp/gag chimera and a comparison of the responses to mlamp/gag were examined with mice immunized on days and with mg dna of the pitr plasmid vectors encoding the mlamp/gag and hlamp/gag chimeras. total anti-gag igg responses were assayed with blood collected on day (figure a ). the response of mice immunized with the hlamp/gag construct was considerably greater than the response to the mlamp/gag. splenocytes of mice sacrificed after the two immunizations were assayed for t-cell ifn-c responses. the mhc ii-targeted mouse and human lamp/gag plasmids elicited comparable ifn-c + responses ( pg/ml and pg/ml, respectively) ( figure b) . assays of cd + t-cell responses were carried out with mice immunized once with mg of the plasmid dna, followed days later by in vivo expansion of gag-specific t cells through inoculation with recombinant vaccinia-gag-pol (rvvgag-pol). five days later, the mice were injected with an immunodominant h- kdrestricted gag peptide epitope and sacrificed after h for the ex vivo assay. mice immunized with both the mouse and human lamp/gag chimeras uniformly developed significantly strong cd + responses as measured by epitope-specific cd + tetramer binding, intracellular ifn-c staining, and ctl lysis of peptidepulsed target cells ( figure c ). the larger fraction of cd + t cells expressing ifn-c (. %) following inoculation with recombinant vaccinia virus encoding gag, in comparison with the proportion of tetramer positive cells, is attributed to the massive in vivo expansion of gag-activated cd + t cells reactive against several other gag mhc i epitopes, besides the immunodominant h- kd-amqmlketi peptide-tetramer epitope complex. the magnitude of the vaccine-induced t-cell responses varied from macaque to macaque; however, in every case, ifn-c secretion was detected after the second immunization at week , reached the highest level of to sfc/ cells at week following the third immunization, and declined gradually until further immunizations were given at weeks and ( figure ) . these studies included an analysis of the relative efficiency of the -or -aa peptides in stimulating t-cell responses in vitro, and we observed that pbmcs of all the five immunized macaques stimulated with the -and -aa gag peptide pools separately, showed no significant difference in the ifn-c response ( figure ). cd + -specific responses were also studied by elispot analyses with cd + -depleted pbmc stimulated with rgag. all five macaques showed a response in the range of - ifn-c + sfc/ cd + -depleted cells, indicating a potent cd + -mediated response ( figure ). the repertoire of peptide-specific responses elicited by lysosomal targeting in balb/c mice has been shown to include the same immunodominant epitopes as those elicited by the native antigen; however, the chimeric antigen commonly elicits enhanced and additional t-cell responses that were not detected with the native antigen [ ] . in an analysis of the potential effect of lamp targeting on the breadth of the t-cell repertoire in macaques, pbmcs from the immunized macaques were tested at week , after dna immunizations, for t-cell responses to different pools consisting of ten peptides each, of the -aa gag peptide set (figure ) . t-cell responses to specific peptides were detected with each of the four immunized macaques that were included in this study. three of the immunized macaques showed responses to five to eight of the peptide pools. one of the animals, macaque # , showed activation of ifn-c + cells in response to all of the peptide pools. collectively, these data suggest that hlamp/gag can prime a broad t-cell response in primates. one remarkable feature of the dna-encoded lamp-targeted antigens has been the dramatic increase in antibody-mediated responses of immunized mice. similar findings have been obtained with the five immunized macaques, each of which showed strong humoral immune responses (figure ) . the strength of these responses by the individual macaques followed the same pattern as the t-cell responses. high anti-gag igg antibody titers were present in serum after immunization with hlamp/gag, indicating effective priming. four of the five vaccinated macaques (# , , and ) developed antibodies against hiv- after the second immunization and high antibody titers were achieved by these four macaques following the third dna immunization at week . the remaining macaque (# ), which showed the weakest t-cell response, also elicited a relatively weak antibody response as compared to the other macaques ( figure a ). the third dna immunization worked as a booster for all the macaques, and antibody titers reached their highest values after the th injection (at week ). the end-dilution titers of the igg antibodies achieved after four hlamp/gag immunizations ranged from , to , ( figure b ). serum samples of the immunized macaques were also assayed for iga production, significant levels were detected in three (# , and ) of the immunized macaques ( figure a ). il- is a key cytokine for terminal differentiation of b-cells into iga-secreting plasma cells in both the mouse and human systems [ ] [ ] . interestingly, il- production was seen in the same three macaques ( figure b) , correlating with the plasma iga response. the presence of gag-specific iga in external secretions, including mouth swabs and nasal and rectal washes collected after four dna immunizations, was also assayed ( figure c ). the levels of iga antibodies detected in mouth swabs and nasal and rectal washes varied remarkably among the individual macaques; however, comparatively higher iga antibodies were found in nasal washes of all the five macaques. serum samples collected from the five macaques at week , after four dna immunizations, were analyzed individually in elisa assays against each of the -aa gag peptides, overlapping by -aa ( figure ). the repertoires of peptides recognized by each of the macaque sera were very similar; all of the animals reacted with the same of the gag -aa peptides tested. three peptides containing b-cell determinants were located at the aminoterminal, whereas, most of the peptides recognized were located in the carboxy-terminal region of the gag protein. these results from immunized macaques support the conclusion that vaccination with an hlamp/gag chimera can prime a broad b-cell response [ ] . this study demonstrates that rhesus macaques immunized with a dna plasmid vaccine-encoding gag as an hlamp/gag chimera develops strong antigen-specific humoral responses as well as cd + and cd + t-cell responses. previous studies by others with candidate dna vaccines have shown protection of small animals against pathogenic challenges [ ] ; however, their performance in primates has generally been disappointing. most naked dna vaccines have produced sub-optimal immune responses, even with repeated boosting, with only moderate t-cell responses when compared to live-attenuated virus or recombinant virus vaccines and very low or no humoral responses. in contrast, hlamp/gag dna immunization elicited potent cd + t-cell as well as gagspecific cd + t-cell responses in macaques after only a few dna immunizations. the responses included highly significant igg antibody titers after two dna immunizations in four of the five immunized macaques and iga responses in serum and nasal washes. all of the macaques showed a high igg antibody titer after three dna immunizations, with a rapid and enhanced immune memory response with the fourth and fifth immunization. the breadth of the t and b cell repertoire elicited by dna hiv vaccines is thought to be important for pathogen control, perhaps by preventing the selection of escape mutants. we have previously reported that immunization of balb/c mice with lamp-targeted gag increases the breadth of b-and t-cell responses. moreover, others have shown that human dendritic cells transfected with lamp-targeted hiv nef are able to induce the activation of an increased repertoire of t-cells isolated from infected patients [ ] . in this study, each of the four macaques tested, produced gag-specific ifn-c + responses to many gag aa peptide pools, indicating a broad range of t-cell responses. a question that remains to be investigated is whether this result reflects a true increase in the number of new epitopes being recognized as a result of hlamp/gag administration or whether the ability of hlamp/gag to increase the overall magnitude of the cellular immune response simply enhances our ability to detect epitopes that would otherwise have been below the limit of detection. we also mapped b-cell epitopes of linear, -aa gag peptides with serum samples obtained from the macaques after three dna immunizations with the hlamp/gag chimera. the results of these analyses indicated that some of the peptides behaved as dominant epitopes and induced strong b-cell activation in all five macaques. these include two dominant peptides located in the aminoterminal portion of the molecule, and peptides located in the carboxy-terminal portion of the gag protein. the amino-terminal region is known for its antigenic properties [ ] [ ] [ ] and has been reported to contain both b and t-cell epitopes and to be relatively conserved among european hiv- isolates [ , ] . the carboxyterminal region is also known to bind antibodies developed during natural hiv- infection in humans [ ] . our studies also showed that iga antibodies were present in the serum and external secretions of the hlamp/gag-immunized macaques. furthermore, il- production was detected in three of the five immunized macaques. il- is known to play a key role in the terminal differentiation of iga-committing b cells into igasecreting plasma cells in both mice and humans [ ] [ ] . in this study, the increased production of il- by gag-specific cd + t cells is consistent with the induction of hiv-specific iga b-cell responses in addition to systemic igg antibody responses. iga and igg antibodies may function as a first line of defense, preventing hiv/siv adherence to the mucosal surface or interfering with viral replication through secretory iga [ ] . although t-cell immune responses induced by dna immunization are generally moderate, previous studies have demonstrated that dna prime is very important in heterologous prime-boost immunization regimens with viral vectors. the prime-boost formulations have been shown to induce strong cellular immune responses and protection in malaria [ ] [ ] and sivmac [ ] [ ] models. moreover, the dna prime and viral boost approach has been shown to be important in increasing the breadth of the response repertoire of viral vector vaccines [ ] , thereby contributing to the overall immunological protection. there is growing consensus that plasmid dna represents a particularly good prime immunogen. therefore, considerable work is now being done to explore the use of bimodal vaccine regimens in which the plasmid dna is used to prime the immune response and a live recombinant vector is used to boost that immunity [ ] . these studies collectively suggest that more work is needed before a dna approach alone or a dna prime followed by a viral vector boost is able to completely control the pathogenic challenge in these model systems. a recent report has shown consistent and strong ctl responses to gag in macaques immunized with dna_crl -adjuvant gag plasmids and boosted with rad encoding gag [ ] . the studies have established that a significant percentage of humans respond to both the dna and adenovirus approaches, and more evaluation of further potent dna vaccines is clearly warranted. the current study was limited by the small number of animals and the lack of an hiv challenge system to assess the protective efficacy afforded by the hiv hlamp/gag dna vaccine. a further goal is to test the effect of lamp targeting on additional relevant antigens of the siv challenge model, in collaboration with the g. pavlakis and b. felber group (human retrovirus section, basic research laboratory, national cancer institute, frederick, md, usa). the rational design of an aids vaccine correlates of immune protection in hiv- infection: what we know, what we don't know, what we should know vaccination preserves cd memory t cells during acute simian immunodeficiency virus challenge preserved cd + central memory t cells and survival in vaccinated siv-challenged monkeys the motif tyr-x-x-hydrophobic residue mediates lysosomal membrane targeting of lysosome-associated membrane protein major histocompatibility complex class ii compartments in human and mouse b lymphoblasts represent conventional endocytic compartments segregation of mhc class ii molecules from mhc class i molecules in the golgi complex for transport to lysosomal compartments the role of endosomes and lysosomes in mhc class ii functioning involvement of miic-like late endosomes in b cell receptor-mediated antigen processing in murine b cells transport of peptide-mhc class ii complexes in developing dendritic cells west nile premembrane-envelope genetic vaccine encoded as a chimera containing the transmembrane and cytoplasmic domains of a lysosome-associated membrane protein: increased cellular concentration of the transgene product, targeting to the mhc ii compartment, and enhanced neutralizing antibody response /lamp chimera targeted to the mhc class ii compartment elicits longlasting neutralizing antibodies sars coronavirus nucleocapsid immunodominant t-cell epitope cluster is common to both exogenous recombinant and endogenous dna-encoded immunogens dna vaccine encoding human immunodeficiency virus- gag, targeted to the major histocompatibility complex ii compartment by lysosomal-associated membrane protein, elicits enhanced long-term memory response inverted terminal repeat sequences of adeno-associated virus enhance the antibody and cd (+) responses to a hiv- p gag/lamp dna vaccine chimera hiv- p gag encoded in the lysosome-associated membrane protein- as a dna plasmid vaccine chimera is highly expressed, traffics to the major histocompatibility class ii compartment, and elicits enhanced immune responses targeting antigen in mature dendritic cells for simultaneous stimulation of cd + and cd + t cells induction of primary carcinoembryonic antigen (cea)-specific cytotoxic t lymphocytes in vitro using human dendritic cells transfected with rna synergistic neutralizing antibody response to a dengue virus type dna vaccine by incorporation of lysosome-associated membrane protein sequences and use of plasmid expressing gm-csf lysosome-associated membrane protein- -mediated targeting of the hiv- envelope protein to an endosomal/lysosomal compartment enhances its presentation to mhc class ii-restricted t cells the enhanced immune response to the hiv gp /lamp chimeric gene product targeted to the lysosome membrane protein trafficking pathway enhanced induction of telomerase-specific cd (+) t cells using dendritic cells transfected with rna encoding a chimeric gene product engineering an intracellular pathway for major histocompatibility complex class ii presentation of antigens dendritic cell-lysosomal-associated membrane protein (lamp) and lamp- -hiv- gag chimeras have distinct cellular trafficking pathways and prime t and b cell responses to a diverse repertoire of epitopes telomerase mrna-transfected dendritic cells stimulate antigen-specific cd + and cd + t cell responses in patients with metastatic prostate cancer expansion of hiv-specific cd + and cd + t cells by dendritic cells transfected with mrna encoding cytoplasm-or lysosome-targeted nef gene delivery to skeletal muscle results in sustained expression and systemic delivery of a therapeutic protein costimulatory requirements of naive cd + t cells. icam- or b - can costimulate naive cd t cell activation but both are required for optimum response human and murine interleukin induce high rate iga secretion in iga-committed b cells human appendix b cells naturally express receptors for and respond to interleukin with selective iga and iga synthesis heterologous protection against influenza by injection of dna encoding a viral protein monoclonal antibodies to conserved regions of the major core protein (gag ) of hiv- and hiv- characterization of murine monoclonal antibodies directed against the core proteins of human immunodeficiency virus types and highly conserved epitope domain in major core protein p is structurally similar among human, simian and feline immunodeficiency viruses human immunodeficiency virus type p production and antigenic variation in tissue culture of isolates with various growth characteristics mapping of igg subclass and t-cell epitopes on hiv proteins by synthetic peptides comparison of iga versus igg monoclonal antibodies for passive immunization of the murine respiratory tract enhanced immunogenicity for cd + t cell induction and complete protective efficacy of malaria dna vaccination by boosting with modified vaccinia virus ankara boosting with recombinant vaccinia increases immunogenicity and protective efficacy of malaria dna vaccine effective induction of simian immunodeficiency virus-specific cytotoxic t lymphocytes in macaques by using a multiepitope gene and dna primemodified vaccinia virus ankara boost vaccination regimen simian immunodeficiency virus dna vaccine trial in macaques enhanced breadth of cd t-cell immunity by dna prime and adenovirus boost immunization to human immunodeficiency virus env and gag immunogens control of a mucosal challenge and prevention of aids by a multiprotein dna/mva vaccine vaccineinduced immunity in baboons by using dna and replication-incompetent adenovirus type vectors expressing a human immunodeficiency virus type gag gene we thank dr. susan swain, from the trudeau institute, saranac lake, ny, for the fibroblast cell line dcek.i-cam.hi ; dr. deborah mcclellan for editorial assistance; dr. james hildreth, for providing the mouse anti-gag monoclonal antibody; and betty hart and delores henson for their excellent technical assistance. several reagents were obtained through the aids research reagents program, division of aids, niaid, national institute of health: -and -aa gag peptides and purified p gag protein. key: cord- -llb f a authors: ji, wen-jie; ma, yong-qiang; zhou, xin; zhang, yi-dan; lu, rui-yi; guo, zhao-zeng; sun, hai-ying; hu, dao-chuan; yang, guo-hong; li, yu-ming; wei, lu-qing title: spironolactone attenuates bleomycin-induced pulmonary injury partially via modulating mononuclear phagocyte phenotype switching in circulating and alveolar compartments date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: llb f a background: recent experimental studies provide evidence indicating that manipulation of the mononuclear phagocyte phenotype could be a feasible approach to alter the severity and persistence of pulmonary injury and fibrosis. mineralocorticoid receptor (mr) has been reported as a target to regulate macrophage polarization. the present work was designed to investigate the therapeutic potential of mr antagonism in bleomycin-induced acute lung injury and fibrosis. methodology/principal findings: we first demonstrated the expression of mr in magnetic bead-purified ly g-/cd b+ circulating monocytes and in alveolar macrophages harvested in bronchoalveolar lavage fluid (balf) from c bl/ mice. then, a pharmacological intervention study using spironolactone ( mg/kg/day by oral gavage) revealed that mr antagonism led to decreased inflammatory cell infiltration, cytokine production (downregulated monocyte chemoattractant protein- , transforming growth factor β , and interleukin- β at mrna and protein levels) and collagen deposition (decreased lung total hydroxyproline content and collagen positive area by masson’ trichrome staining) in bleomycin treated ( . mg/kg, via oropharyngeal instillation) male c bl/ mice. moreover, serial flow cytometry analysis in blood, balf and enzymatically digested lung tissue, revealed that spironolactone could partially inhibit bleomycin-induced circulating ly c(hi) monocyte expansion, and reduce alternative activation (f / +cd c+cd +) of mononuclear phagocyte in alveoli, whereas the phenotype of interstitial macrophage (f / +cd c-) remained unaffected by spironolactone during investigation. conclusions/significance: the present work provides the experimental evidence that spironolactone could attenuate bleomycin-induced acute pulmonary injury and fibrosis, partially via inhibition of mr-mediated circulating monocyte and alveolar macrophage phenotype switching. idiopathic pulmonary fibrosis (ipf) is a chronic, progressive, interstitial fibrotic lung disease characterized by chronic lung inflammation, disruption of alveolar structure, interstitial fibroblast proliferation, and excessive extracellular matrix synthesis and deposition [ ] [ ] [ ] . although evidence showed that the persistent inflammatory response is associated with progressive development of ipf, therapies currently used for ipf, namely anti-inflammatory or immunosuppressive drugs, are largely ineffective [ ] . therefore, novel therapies capable of targeting inflammation without compromising body's immunity can still be a challenge in this area. macrophages in lung tissue play an important role in the clearance of pulmonary pathogens and steady-state homeostasis maintenance. emerging evidence suggests that there is a causal link between lung macrophage mediated inflammation and excessive tissue destruction elicited by variety of exogenous stimuli, i.e., silica and asbestos exposure, virus infection, etc., which will ultimately lead to a failure of inflammation resolution, a key feature that progressively promotes the development of lung fibrosis [ ] [ ] [ ] [ ] . on the other hand, macrophages are a cell population with high plasticity, and display functional diversity during different stage of inflammatory response [ , ] . the activation state of macrophage can be generally characterized as classical activation (m polarization) that is associated with a th immune response, or alternative activation (m polarization) that is associated with th immune response [ ] . in lung tissue, m -like macrophages are the first line defense in acute lung injury and are later replaced by m -like macrophages that contribute to tissue repair and fibrosis. it is generally believed during inflammation, myeloid ly c hi monocytes contribute to lung macrophage replenishment [ , ] . the results from recent basic studies indicate that manipulation of macrophage phenotype switch might be a potential target for many macrophage mediated disorders [ ] [ ] [ ] . recently, usher and colleagues demonstrated that macrophages from mice lacking myeloid mineralocorticoid receptor (mr), exhibit a transcription profile that mimic alternatively activated macrophages, and are protected against angiotensin ii (angii) induced cardiac hypertrophy and fibrosis [ ] . this work provides evidence indicating that mr in mononuclear phagocytes might be a potential target for therapeutic purpose. based on current evidence, we speculated that pharmacological inhibition of mr with clinically approved drug, may regulate lung macrophage phenotype switching, as well as their progenitors, bone marrow-derived circulating monocytes, and may confer novel therapeutic potential in a murine model of bleomycin-induced acute pulmonary injury and fibrosis. eight to ten weeks male c bl/ mice, weighing to validate the mrna expression of mr in mouse circulating monocytes, circulating monocytes from c bl/ mice were purified from peripheral blood using a magnetic bead-based kit (easysep tm mouse monocyte enrichment kit, cat no. , stemcell technologies, vancouver, bc, canada). the purity of enriched monocytes was confirmed by flow cytometry (see below). detailed methods for total rna isolation, reverse transcription, and real-time pcr analysis are shown below. to validate the protein expression of mr in circulating monocytes and alveolar macrophages, the purified monocytes and cells from bronchoalveolar lavage fluid (balf) were seeded on glass slides for immunohistological detection of mr. briefly, the cells were fixed with methanol, followed by permeabilization with . % triton x- . then, the cells were incubated with the primary anti-mouse mineralocorticoid receptor monoclonal antibody ( : , ab , abcam, cambridge, ma, usa) at °c overnight. to ensure specificity, isotype control (igg a) was prepared. for alveolar macrophages, the cells were further incubated with the primary anti-mouse f / antibody ( : , ab , abcam) at °c for h. after washing with . m pbs, the cells were incubated with tetramethylrhodamine isothiocyanate (tritc)-conjugated goat anti-mouse secondary antibody [for alveolar macrophage, fluorescein isothiocyanate (fitc)-conjugated goat anti-rat secondary antibody was also added] in dark. then, cell nuclei were stained by , -diamidino- -phenylindole (dapi, sigma-aldrich, st. louis. mo, usa) with light protection. images were visualized by a fluorescence microscope (eclipse i, nikon, tokyo, japan). the unstained samples and samples stained with the secondary antibody without incubation with primary antibodies were used as negative controls and showed no signal during analysis. to induce pulmonary fibrosis, mice were lightly anesthetized by inhalation of ether. bleomycin a ( . mg/kg body weight in μl saline) or saline was administered by oropharyngeal instillation as described previously [ ] . animals were then randomly allocated into four treatment groups: ) . % normal saline (ns) only; ) bleomycin (blm) only; ) bleomycin plus . % normal saline (blm+ns); ) bleomycin plus mg/kg of spironolactone (blm+sp). from the day of the administration (day ), vehicle ( . % saline), sp (dissolved in . % saline) were delivered by oral gavage once daily, and continued for days. at , , , or days, animals were sacrificed by exsanguinations under sodium pentobarbital anesthesia ( mice each time point). blood, balf and lung tissues were collected for the following assays. the balf was collected through an intratracheal cannula with three sequential ml of . % sterile saline and centrifuged at g for min at °c. the cell-free supernatant was stored at - °c for analysis of cytokines. the cell pellet was resuspended in sterile . % saline for total cell counts, differential cell counts, immunohistochemical staining, and flow cytometry analysis. the left lung (from which no balf was harvested) was fixed in % paraformaldehyde solution for h. after embedding in paraffin, μm sections were prepared and stained with hematoxylin-eosin or masson's trichrome, and examined on a light microscope (e pol, nikon, tokyo, japan). for detection of myofibroblasts, α smooth muscle actin (α-sma, : , a , sigma-aldrich, st. louis. mo, usa) immunofluorescent staining was carried out as previously described [ ] . for the evaluation of inflammatory response induced by bleomycin, semi-quantitative scoring criteria by szapiel and coworkers were used in a blinded fashion [ ] . fibrosis and collagen was determined from non-overlapping fields by using digital quantitative analysis (image pro plus software version . , media cybernetics, silver spring, md, usa). the lung fibrosis index was defined as the sum of the total area of collagen in the entire visual field divided by the sum of total connective tissue area in the entire visual field. the collagen content in the whole left lung was determined by analysis of hydroxyproline as previously described [ ] . in brief, lung lobes were homogenized in ml of phosphate buffered saline (pbs, ph= . ) and then hydrolyzed in ml of n hydrochloric acid for hours at °c, and neutralized to ph . with naoh. chloramines t reagent ( ml of . mol/l) was then added and the samples were left at room temperature for minutes. then % p-dimethylaminobenzaldehyde solution (dissolved in . n perchloric acid) was added to each sample, and the mixture was incubated at °c for minutes. absorbance was measured at nm on a nanodrop c spectrophotometer (thermo scientific, waltham, ma, usa). cells from blood, balf and lungs were subject flow cytometry analysis on a cytomics fc cytometer (beckman coulter, miami, fl, usa). all antibodies were obtained from biolegend (san diego, ca, usa). all data were analyzed with flowjo software (treestar, ashland, or, usa). for validation of the purity of magnetic bead-enriched circulating monocytes, anti-mouse cd b-phycoerythrin (pe) (clone m / ) and anti-mouse ly g-percp-cy . (clone a ) were used. for analysis of circulating monocyte subsets, ethylenediaminetetraacetic acid (edta) anti-coagulated whole blood was stained with anti-mouse cd b-phycoerythrin (pe) (clone m / ) and anti-mouse ly c-fitc (clone hk . ), incubated for min at room temperature in the dark. following red cell lysis, samples were analyzed. for immunophenotypic analysis of alveolar macrophages (am), cells isolated from balf were first centrifuged ( min at g at room temperature), and the supernatant was discarded to remove dead cells. for each flow cytometry analysis, the cells were first suspended in . % trypan blue in pbs, and the number of live and dead cells was measured using an automatic cell counter (counterstar tm , rui yu biotechnology co.,ltd, shanghai, china). by this method, the number of live cells in each sample is more than %. for subsequent flow cytometry analysis, the cells were incubated with anti-mouse f / -pe-cy (clone bm ), anti-mouse cd c-pe-cy (clone n ) and anti-mouse cd -pe (clone c c ). following incubation, flow cytometry analysis was carried out. for immunophenotypic analysis of interstitial macrophages (ims), lung single-cell suspensions were prepared from lavaged lung (from which the balf was harvested) to reduce the contamination of am. in brief, the lower lobe of right lung were minced and incubated with . mg/ml collagenase solution (type i, sigma-aldrich) at °c for min. after filtering through μm nylon mesh, similar procedure to remove dead cells was carried out as did during sample preparation for am analysis, then the cell suspension was stained anti-mouse f / -fitc (clone bm ), anti-mouse cd c-pe-cy (clone n ) and anti-mouse cd -pe (clone c c ). following incubation, samples were analyzed with flow cytometer. isotype antibodies (clone rtk for f / ; clone htk for cd c; clone rtk for cd ; clone rtk for cd b; clone rtk for ly c; clone rtk for ly g) were used to detect nonspecific binding. the gating strategies for analyzing am and im were according to previous report [ ] . total rna from purified blood monocytes and lung tissue was isolated using trizol reagent (invitrogen, carlsbad, ca, usa) according to the manufacturer's instructions. total rna ( μg) was reverse-transcribed into the cdna using a reverse transcription assay (promega, madison, wi, usa) in μl of reaction volume according to the manufacturer's instructions. real-time pcr was performed with sybr green pcr master mix (roche diagnostics, indianapolis, in, usa) on an abi prism sequence detection system (applied biosystems, foster city, ca, usa) in triplicate and according to a two-step pcr protocol ( min at °c, cycles for s at °c, min at °c). the primer sequences are shown in table . relative expression of real-time pcr products were normalized for expression of the β-actin and expressed as transcript fold change over ns mice using the -△△ct method [ ] . the levels of transforming growth factor β (tgf-β ), monocyte chemoattractant protein- (mcp- )/chemokine (c-c motif) ligand (ccl ), interleukin- (il- ), and interleukin- β (il- β) in the balf were measured by commercially available elisa kits (r&d systems, minneapolis, mn, usa), according to the manufacturer's instructions. all data are presented as the mean ± standard error of mean (sem). statistical analysis was performed using graphpad prism . software (graphpad, san diego, ca, usa). statistical comparison of multiple groups was performed by one-way anova with bonferroni post-hoc test or kruskal-wallis test followed by dunn's multiple comparisons (inflammation score and fibrosis index). a two-tailed p value less than . was considered statistically significant. by using magnetic bead-based monocyte enrichment method, more than % of the harvested cells were ly g-cd b+ ( figure a) . then we confirmed mr mrna expression in these cells by real-time pcr and pcr product electrophoresis ( figures b) . then, the mr protein expression of enriched monocytes was further validated by immunofluorescent staining (figure c) . using mouse balf, we also confirmed mr expression in alveolar f / + macrophages ( figure d) . these results suggest that mr is expressed in mouse mononuclear phagocytes, which provides a basis for pharmacological intervention. figure shows the detailed research protocol of in vivo pharmacological intervention study. figure (a to h) shows the representative h.e. stained lung sections on day , which represents the peak magnitude of lung inflammatory response following bleomycin instillation. spironolactone treatment could significantly reduce the inflammatory response induced by bleomycin ( figure i) . panel j in figure shows the results of differential cell counts from the balf that harvested on day . typically, the total fluid recovery was over % in all animals and the percentages of fluid recovered were not significantly different across all treatment groups. in agreement with histological findings, spironolactone treated lungs exhibited decreased total cell, macrophage, lymphocyte, neutrophil infiltration and esosinophils in alveoli. next, we measured the levels of inflammatory and profibrotic cytokines in the balf and determined related gene expression levels in lung tissue. as shown in figure , compared with blm and blm+ns groups, spironolactone treatment was associated with downregulated ccl /mcp- , tgf-β and il- β both at the mrna and the protein levels. in addition, markers for m polarization, such arginase- (arg- ) mrna level in lung tissue (figure g) , and il- protein content in balf ( figure f) were downregulated by spironolactone. figure shows the profibrotic response using lung tissue that harvested on day . the histological analysis showed that mr antagonism was associated with reduced collagen deposition and α-sma positive cells (myofibroblasts). compared with ns group, the expression of type i and type iii collagen mrna in the lungs from blm and blm+ns groups were significantly upregulated, whereas spironolactone treatment could partially regress bleomycin-induced collagen expression upregulation, which was consistent with the histological findings. we next evaluated the effect of spironolactone treatment on circulating monocyte subset change. figure a shows the gating strategies for circulating monocyte subset analysis. as shown in figure b , compared with ns group, blm treated mice exhibited a significant increase of ly c hi monocytes, starting from day , reaching the plateau level on day , then followed a gradual decrease till day . spironolactone treatment could significantly reduce bleomycin-induced the ly c hi monocyte pool expansion on day and thereafter. the reciprocal changes of ly c lo monocyte subset is shown in figure c . using enzymatically digested lung tissue, we evaluated interstitial macrophage phenotype changes during drug intervention. as shown in figure b , one day after bleomycin challenge, the majority (more than %) of interstitial macrophages presented with a m -like phenotype (f / +cd c-cd -), followed by a gradual decreasing trend of the proportion of m -like macrophages, and this trend reached statistical difference on day . moreover, compared with blm and blm+ns groups, spironolactone has no obvious influence on interstitial macrophage phenotype switching induced by bleomycin. then we investigated the impact of spironolactone on alveolar macrophage phenotype changes. as shown in figure b , alveolar macrophages in ns group were mainly (more than %) presented with a m -like phenotype (f / +cd c +cd -). after bleomycin challenge, there was a quick decrease of m -like macrophage with a concomitant increase of m -like phenotype (f / +cd c+cd +). whereas in spironolactone treated mice, this trend was partially normalized, indicating an inhibitory effect on alternative activation by mr antagonism. recent studies showed that the renin angiotensin aldosterone system (raas) plays an important role in the pathogenesis of lung injury [ ] [ ] [ ] . in addition, the therapeutic efficacy of drug intervention targeting this system has been reported in bleomycin-induced lung injury models [ ] [ ] [ ] [ ] [ ] [ ] . zhao and coworker first demonstrated the therapeutical potential of spironolactone in ameliorating bleomycin-induced lung fibrosis [ ] , which is also supported by a recent study [ ] . a growing body of evidence suggests that manipulation of the mononuclear phagocyte phenotype switching could be a feasible approach to alter the severity and persistence of pulmonary injury and fibrosis in experimental models [ ] [ ] [ ] . it has been demonstrated that mr plays an important role in regulating myeloid cell phenotype switching in different disease conditions [ , [ ] [ ] [ ] [ ] . to our knowledge, the role of mononuclear cell mr in mediating acute lung injury induced pulmonary fibrosis has not been addressed. the present work confirmed that mr antagonism by a clinically approved drug, spironolactone, could attenuate bleomycin-induced acute lung injury and fibrosis. specifically, mr inhibition partially attenuates ly c hi monocyte expansion in circulating compartment and normalizes disturbed balance of macrophage polarization in alveolar compartment, leading to reduced alveolitis and collagen deposition in lung tissue. these findings highlight mononuclear phagocyte mr as a promising target for ameliorating acute lung injury and profibrotic response in lungs. the raas is a hormone system which acts on multiple physiologic pathways by regulating blood pressure and fluid balance. as the terminal effector of the raas cascade, the role of aldosterone/mr signaling has been recently implicated the pathogenesis of cardiovascular diseases, insulin resistance and diabetes, and chronic inflammation associated fibrosis [ ] [ ] [ ] . these effects are supported by the fact that in addition to the kidney, there is a wide tissue distribution of mr, such as cardiomyocytes, endothelial cells, vascular smooth muscle cells, adipocytes and macrophages [ ] . here, we demonstrated that mr is expressed both in purified murine circulating ly g-/cd b+ monocytes and in f / + alveolar macrophages, providing a basis for mr regulation of monocyte/ macrophage phenotype switching. macrophages are professional phagocytic cells with different transcriptional profiles and functional capabilities depending on their origins from various organs [ ] . broadly speaking, the lung tissue contains two tissue-resident macrophage compartments, i.e., alveolar macrophages and interstitial macrophages. the traditional belief that tissue-resident macrophages are derived from circulating monocyte progenitors has been challenged by recent fate mapping studies by showing that the steady-state turnover of alveolar macrophages is extremely low: to months after bone marrow transplantation, %- % of alveolar macrophages are host derived [ , ] . in addition, recent studies demonstrated that lung alveolar macrophages are established prior to birth and maintains themselves subsequently during adulthood independent of replenishment from circulating monocyte input in steady state [ , ] . on the contrary, during acute lung inflammatory response, circulating monocytes have an important impact on the lung macrophage dynamics. in general, recent studies are in agreement with the notion that following injury, there is an increased accumulation of m -like mononuclear cells in alveoli [ , [ ] [ ] [ ] [ ] . moreover, in patients with chronic obstructive pulmonary disease, a skewing of alveolar macrophages from an m to m phenotype has been observed [ , ] . however, with regard to the origin of these m -like cells, some controversy existed. in an endotoxin-induced lung inflammation model, maus and coworkers showed that despite a rapid recruitment of monocytes in lung tissue, the resident alveolar macrophage pool remained static throughout the duration of inflammation and the expansion of the lung macrophage pool was mainly mediated by an influx of the circulating monocytes, followed by their differentiation into tissue macrophages [ ] . in agreement with this finding, recently osterholzer et al [ ] , using a gene-targeted alveolar injury model, demonstrated an increased exudate macrophages and their progenitors, ly c hi monocytes, both exhibiting m polarization in alveoli. in another study [ ] , gibbons and colleagues adoptively transferred ly c hi monocytes into bleomycin-treated mice during the progressive phase of lung fibrosis, which led to an exacerbation of disease progression and an increased accumulation of m -like macrophage in the lung. surprisingly, these alternatively activated macrophages were host derived and not from the donor ly c hi monocytes. as a corollary, regardless of their origins, our current knowledge points to a general scheme of their relationship: initially, acute lung injury induces a rapid expansion and infiltrating ly c hi monocytes in lung tissue, which contributes to a paralleled increase of m like macrophages (by direct differentiation or by paracrine effects) in alveolar compartment, and the severity and persistency of m polarization in alveolar macrophages would ultimately influence inflammation resolution and fibrosis. the above model highlights the circulating ly c hi monocytes as a therapeutic target. although we did not use a monocytetargeted approach to suppress ly c hi monocytosis, it is likely that spironolactone would also exert its major pharmacological effect on circulating monocyte pool since the efficacy of orally administered drug is significantly compromised by its inability to reach alveolar space at an appropriate concentration [ ] . additionally, because evidence shown that monocyte infiltration would facilitates alveolar neutrophil emigration and determines the ongoing neutrophil influx in the persistent phase of acute lung injury [ ] [ ] [ ] , suppression of ly c hi monocytosis by spironolactone would concomitantly lead to a decreased tissue accumulation of neutrophils, which is also observed in our study. the present work has the following limitations. first, because spironolactone has anti-androgen effect, the observed effects of this work cannot be totally ascribed to mr antagonism. indeed, there is a sex discrepancy in bleomycin-induced lung fibrosis, and estrogen may have protective effect on this model [ , ] . in this regard, mr knockout mice are preferred to address this issue. second, we did not observed significant changes in lung interstitial macrophages by spironolactone. previous study showed this population might have a role in limiting inflammation and fibrosis [ ] . thus it remains unclear whether cd is an appropriate m marker for this population as recent study showed that the change of cd is modest after bleomycin challenge [ ] , or this population is insensitive to mr inhibition, or due to enzymatic digestion-induced surface marker loss during sample preparation, a commonly encountered technical issue. third, due to the wide distribution of mr in the body, the mechanistical explanation of global mr antagonism is fairly complex. for example, aldosterone has been implicated in the pathogenesis of pulmonary hypertension [ ] , and spironolactone has been shown to attenuate experimental pulmonary hypertension via mr inhibition in pulmonary artery smooth muscle cells [ ] .admittedly, bleomycin is also a frequently used tool drug to induce pulmonary hypertension [ , ] . the downregulation of α-sma by spironolactone observed in this study, also support an antifibrotic effect of spironolactone on fibroblasts. moreover, the functional expression of mr has been demonstrated in neutrophils [ ] , which may also participate in spironolactone induced amelioration of lung fibrosis, as shown by reduced neutrophil count in balf. thus, in addition to its effect on mononuclear phagocytes, the mechanisms underlying therapeutic effect of systemic use of spironolactone on bleomycin-induced lung injury is multifactorial. forth, it seems obscure to interpret the effect of mr antagonism on alveolar macrophage polarization, since macrophages lacking myeloid mr exhibit alternative activation (m polarization), whereas our results showed that mr inhibition could reduce alveolar m polarization. it should be noted the long-established binary classification of macrophage in terms of classical (m ) and alternative activation (m ) is based on in vitro studies [ ] . indeed, a recent study demonstrated eplerenone, another clinically approved mr antagonist, promotes alternative activation in human monocyte-derived macrophages [ ] . however, macrophages in vivo maintain their plasticity and can alter their phenotype based on the microenvironment, including cytokine milieu among other factors [ ] . as pointed early, drug administration via oral route, cannot reach alveolar space at an appropriate concentration. therefore, the alterations in alveolar macrophage polarization state cannot be ascribed to mr antagonist's direct effect. it is conceivable that suppression of inflammatory (ly c hi subset) monocyte expansion should be the direct effect by spironolactone, which ameliorates lung injury via the "ly c hi directed pulmonary alterative activation" mechanism [ ] . thus, future monocyte-targeted approaches, as well as in vitro studies are warranted to elucidate the molecular mechanism underlying suppressed ly c hi monocytosis by mr antagonism. finally, the algorithms used in this study are relatively simple, and may inadvertently contain dendritic cells and eosinophils. more rigorous and sophisticated algorithms have been published and suggested [ , ] . in conclusion, the present work provides the experimental evidence that mr antagonism by spironolactone could attenuate bleomycin-induced acute pulmonary injury and fibrosis, partially by reducing circulating inflammatory ly c hi role of the chemokine receptor cxcr in bleomycin-induced pulmonary inflammation and 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murine lung injury male sex hormones exacerbate lung function impairment after bleomycin-induced pulmonary fibrosis age and sex dimorphisms contribute to the severity of bleomycin-induced lung injury and fibrosis what is the clinical relevance of different lung compartments? aldosterone inactivates the endothelin-b receptor via a cysteinyl thiol redox switch to decrease pulmonary endothelial nitric oxide levels and modulate pulmonary arterial hypertension mineralocorticoid receptor antagonism attenuates experimental pulmonary hypertension lung extracellular superoxide dismutase overexpression lessens bleomycin-induced pulmonary hypertension and vascular remodeling therapeutic hypercapnia prevents bleomycin-induced pulmonary hypertension in neonatal rats by limiting macrophage-derived tumor necrosis factor-alpha aldosterone abrogates nuclear factor kappab-mediated tumor necrosis factor alpha production in human neutrophils via the mineralocorticoid receptor transcriptional regulation of macrophage polarization: enabling diversity with identity eplerenone promotes alternative activation in human monocyte-derived macrophages identification of myeloid cell subsets in murine lungs using flow cytometry key: cord- -l c x tb authors: klomp, richard w.; jones, laurie; watanabe, emi; thompson, william w. title: cdc’s multiple approaches to safeguard the health, safety, and resilience of ebola responders date: - - journal: prehosp disaster med doi: . /s x sha: doc_id: cord_uid: l c x tb over , people were sickened by ebola and over , people died between march of and june of . the us centers for disease control and prevention (cdc; atlanta, georgia usa) was one of many public health organizations that sought to stop this outbreak. this agency deployed almost , individuals to west africa during that timeframe. deployment to these countries exposed these individuals to a wide variety of dangers, stressors, and risks. being concerned about the at-risk populations in africa, and also the well-being of its professionals who willingly deployed, the cdc did several things to help safeguard the health, safety, and resilience of these team members before, during, and after deployment. the accompanying special report highlights innovative pre-deployment training initiatives, customized screening processes, and post-deployment outreach efforts intended to protect and support the public health professionals fighting ebola. before deploying, the cdc team members were expected to participate in both internally-created and externally-provided trainings. these ranged from pre-deployment briefings, to preparing for work overseas (pfwo) and public health readiness certificate program (phrcp) courses, to incident command system (ics) , , and courses. a small subset of non-clinical deployers also participated in a three-day training designed in collaboration with the center for the study of traumatic stress (csts; bethesda, maryland usa) to train individuals to assess and address the well-being and resilience of themselves and their teammates in the field during a deployment. participants in this unique training were immersed in a virtual reality environment (vre) that simulated deployment to one of seven different types of emergencies. the cdc leadership also requested a pre-deployment screening process that helped professionals in the cdc’s occupational health clinic (ohc) determine whether or not individuals were at an increased risk of negative outcomes by participating in a rigorous deployment at that time. when deployers returned from the field, they received personalized invitations to participate in a voluntary, confidential, post-deployment operational debriefing one-on-one or in a group. implementing these approaches provided more information to clinical decision makers about the readiness of deployers. it provided deployers with a greater awareness of the kinds of challenges they were likely to face in the field. the post-deployment outreach efforts reminded staff that their contributions were appreciated and there were resources available if they needed help processing any of the potentially-traumatizing things they may have experienced. the ebola outbreak in guinea, sierra leone, and liberia sickened over , people between march of and june of , and resulted in over , confirmed deaths. it attracted the attention of people all around the world and required the dedicated efforts of countless caring individuals from diverse organizations and numerous countries. almost , public health professionals at the united states centers for disease control and prevention (cdc; atlanta, georgia usa) deployed to west africa. they tested patient samples, communicated health messages, educated health care workers, advised travelers, trained officials, and interviewed people who might have been in contact with ebola patents. approximately , additional cdc personnel contributed by working around the clock in cdc's emergency operations center (eoc) in atlanta, georgia. senior cdc leadership tasked their office of safety, security, and asset management's (ossam) worklife wellness office (wwo) to set up a pre-deployment screening process to reduce the likelihood of deploying someone at-risk of negative mental health outcomes in an emergency response. the cdc added this new screening to pre-deployment safety and resilience briefings, classroom resilience training, and post-deployment outreach initiatives that have been a part of cdc deployments since . the cdc leadership prioritized providing rapid, stigma-free assistance for all individuals who might desire it, who had worked in inhospitable and potentially dangerous environments. the wwo resilience program submitted a request for project determination and approval to the cdc institutional review board (irb). the irb provided a waiver for the report of this resilience-related project. in this report, resilience is defined as "the ability to withstand, recover, and grow in the face of stressors and changing demands." the cdc strongly recommended a variety of trainings focused on topics such as travel safety, personal security, and the incident command system (ics; ics , , and ) for cdc staff preparing to deploy to the field. multiple cdc offices had developed deployment preparation courses and asked wwo to conduct resilience-enhancing segments of those classes. the cdc's three-day "preparing for work overseas" (pfwo) class, which addressed learning objectives from the us department of state (washington, dc usa), included a one-hour section on "physical and mental resiliency while traveling." in that section, instructors shared resilience basics along with diaphragmatic breathing and muscle relaxation techniques designed to combat the well-known fight-or-flight response. the cdc also hosted a four-day training titled: "public health readiness certificate program" (phrcp). this training included a one-hour segment on "deployment resiliency." almost , cdc staff completed these kinds of resilience-related trainings. records show that , staff participated in other wellness offerings, such as on-site support for eoc staff, weekly physical activity sessions, and stress management classes during the ebola response timeframe. pre-deployment trainings also provided valuable information, presented a variety of common responses to unusual situations, and encouraged potential deployers to prepare mentally and emotionally for a deployment. however, for many years, the cdc lacked personnel specifically trained to provide resilienceenhancing support services in-country to deployed cdc staff. several years ago, cdc resilience experts conducted an environmental scan to see what other federal agencies did to protect workers placed in inhospitable or potentially dangerous environments. the cdc's resilience team reached out to the bureau of alcohol, tobacco, firearms, and explosives (washington, dc usa); us border patrol (washington, dc usa); us coast guard (washington, dc usa); drug enforcement agency (springfield, virginia usa); environmental protection agency (washington, dc usa); federal bureau of investigation (washington, dc usa); national aeronautics and space administration (washington, dc usa); and the department of defense (virginia usa). the scan revealed no standardized inter-agency processes. it did appear upon preliminary review that each agency based their approach on some type of psychological model combined with a peer-support component. to develop an impactful intervention, the cdc collaborated with the center for the study of traumatic stress (csts; bethesda, maryland usa) at the uniformed services university of the health sciences. , leaders at csts already had conducted key informant interviews at the cdc in . they had recommended then that the cdc develop training to enhance cdc emergency responder resilience. the csts also recommended that the cdc consider using psychological first aid (pfa) as the foundation for its resilience-supporting training initiative; pfa could be described as a pragmatic, evidence-informed, public health or population-based framework designed to help non-clinicians organize a response to trauma at the individual or community level. it was developed by the national center for post-traumatic stress disorder (washington, dc usa) to assist people immediately after a disaster to reduce initial distress and foster both short-and long-term adaptive functioning. the wwo worked closely with csts psychiatrists to develop a three-day course titled: "deployment safety resiliency team" (dsrt) training that incorporated pfa. the training included two days of pfa principles including: peer support, coping skills, stress management, triage, and proper referral processes ( figure ) . a third day highlighted the basics of disaster site safety including: blood borne pathogens, personal protective equipment, respiratory protection, radiation basics, and fatigue mitigation. the wwo excerpted relevant safety information from a well-established disaster site safety course and a collateral duty course. the focus was on the kinds of safety risks that were likely to be encountered in the field during a deployment. five experienced trainers also shared relevant insights about public health deployments they had gained during prior emergency responses. this innovative, highly-interactive, educational approach also incorporated small group analysis of three realistic, deploymentbased scenarios. the culmination of class included immersion in one, -minute virtual reality environment (vre). the vre options included a simulated deployment to: a rural african village, a city devastated by a hurricane, a town rocked by an earthquake, a community hit by a radiological dispersal device, a location dealing with a pandemic, a deliberate release of a toxic substance, or a food-borne infectious disease outbreak. during the -minute immersive experience, which introduced potentially stressful scenarios, up to trainees could see, hear, and "interact" with characters on a large screen in a darkened training room. the vre's pre-recorded characters addressed typical health, safety, and resilience challenges in a particular scenario. training participants, in three-person teams, used hand-held electronic devices to test their knowledge of relevant course content. they made decisions within their teams about how to assess available assets and threats, and then address these realistic challenges. they also had the opportunity to apply several of the principles they studied during the course in a safe, virtual environment. the wwo resilience team reviewed among other things the military's total force fitness framework to understand if or how it might be adapted to the cdc's workforce. the framework uses the connection between mind, body, spirit, environment, and relationships to holistically build and maintain health, readiness, and optimal performance of the us armed forces. it also assesses soldiers' resilience before and after deployment. the wwo's review included the popular press and media focused on resilience-related concepts and processes. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the goal was to conceptualize, create, and implement a screening process to help safeguard the health, safety, and resilience of staff being considered for deployment. the wwo resilience team convened an internal, -person expert panel to consider ways to address dozens of obstacles the cdc faced as it prepared to implement an assessment process for civilians. the panel was composed of psychometricians, an ethicist, mental health professionals, attorneys, epidemiologists, and experienced deployers. panel members reviewed numerous assessment tools designed to create a current snapshot of an individual's resilience. they eventually agreed upon a focused battery of brief and relatively unobtrusive assessment instruments. the panel report included these recommendations to cdc leadership: • adopt three specific assessment instruments; • develop standard operating procedures (sops) to ensure professional, confidential, stigma-free collection of pre-deployment assessments; and • encourage collaboration with the cdc occupational health clinic's (ohc) medical professionals to make a team-based recommendation to inform deployment decisions. the panel recommended that cdc implement a pre-deployment assessment battery comprised of: the expert panel determined that using these kinds of tools would screen for thoughts and behaviors that might indicate staff would be at elevated risk of a negative mental health outcome during deployment to potentially dangerous environments. the tools were brief enough to encourage completion while avoiding assessment fatigue. the panel also considered the battery broad enough to identify individuals who were struggling with issues that might jeopardize work productivity and personal well-being in the field. between november , and december , , there were , deployments by cdc staff in response to the ebola outbreak in west africa. records indicate that almost of the total deployments were by repeat deployers. the assessment scores of approximately different deployers were outside the norms for the externally validated assessments that were used. in accordance with the sops that the wwo resilience team developed, a licensed mental health professional within the cdc's resilience assessment and maintenance program (ramp) held a confidential conversation with those individuals about factors that might be negatively impacting their assessment scores at that time. during most of those confidential conversations, an exchange of pertinent information made it clear to both parties whether or not a deployment at that time was in the best interests of the individual and the organization. for example, if a person's pregnant spouse was only a couple of weeks from their due date, a deployment probably would be contraindicated. or if a person had just lost their father, it might not be advantageous to the cdc or in the individual's best interests to deploy right away. there were a few occasions when it was advisable for a ramp mental health professional to consult with a cdc ohc medical professional who had completed a physical assessment of the potential deployer. during those consultations, the mental health and medical professionals determined deployment eligibility jointly. the ramp clinicians ensured that all information related to assessments, concerns, and conversations remained secure and confidential in the ohc's electronic medical record system. they also referred several individuals to the cdc's employee assistance program (eap) for support with temporary challenges they faced. the cdc subject matter experts provided pre-deployment briefings for everyone who participated in a deployment managed by the eoc in atlanta. briefings ranged from minutes to minutes, depending on the nature of the response (eg, outbreak, humancaused, or natural disaster) and types of environments and challenges eoc leaders anticipated the deployers might encounter. for several years, eoc leadership requested that the resilience team provide an overview of basic resilience-related principles in those briefings. the resilience briefer highlighted physiological, cognitive, and behavioral symptoms of stress and emphasized the importance of self-care and social support. in virtually every briefing, the resilience briefer made this norm-setting statement: "emergency response is much more like a marathon than a sprint, so it's important to pace yourself while you're deployed." the briefing coordinator shared with briefing participants additional written material and references to supplemental resources, including contact information for eap professionals. during the past dozen years, cdc mental health professionals have reached out to deployers who had worked on challenging and stressful assignments to see how they were doing. for example, the resilience team contacted hundreds of cdc professionals when they returned from deployment to the marburg hemorrhagic individual level-the cdc sought to ensure that returning responders had multiple opportunities to speak with caring colleagues who could provide a non-judgmental listening ear. responders also received access to relevant information and, if desired, additional support after an especially challenging deployment. also, ramp developed a plan and implemented a process for team members to send personalized emails to returned ebola responders. the ramp team member conducting the outreach obtained from the cdc's eoc a list of staff getting ready to return from the field. they sent those individuals a standardized email inviting them by name to participate in a voluntary, confidential, non-clinical conversation about what they experienced in the field. between september , and june , , ramp team members emailed invitations to , different individuals who had deployed as part of the cdc's ebola response. of that group, individuals ( %) chose to participate in a conversation, which typically lasted - minutes (appendix ; available online only). the ramp team members informed responders that relevant information they shared would be aggregated, summarized, de-identified, and included in a report shared regularly with internal stakeholders at the cdc. recipients included the division of emergency operations (deo), incident management leadership, and the ossam. the deo and ossam had the opportunity to use insights and recommendations in these reports to identify gaps or redundancies in the deployment process, improve procedures, and fine-tune the deployment experience to increase the health, safety, and resilience of future deployers. as a result, the cdc provided additional information to potential deployers about proper clothing to wear, hotel conditions, computer and technology issues in the field, and types of personal equipment to bring. because of feedback, the cdc drafted and shared additional guidance about what to do if a deployed team member became ill. group level-many people were willing to share positive and negative feedback about processes and programs in a oneon-one format. the ramp team members believed that others might be more comfortable, and consequently more likely, to ask pertinent questions or share helpful process improvement information in a group setting. voluntary post-deployment group conversations were offered as part of the cdc's eoc activation for ebola from to . approximately individuals participated in one of these weekly or bi-weekly group meetings that typically lasted between and minutes. the ramp team members designed the group sessions to be facilitated conversations. to protect their anonymity, the group session leader asked participants not to share their names. he asked them to share their insights and observations in response to a handful of general questions about their deployment experience (appendix ; available online only). the group session leader informed responders that relevant information they shared would be aggregated, summarized, de-identified, and included in a report shared regularly with internal stakeholders at the cdc to identify gaps or redundancies in the deployment process, improve processes, and fine-tune the deployment experience for future deployers. for example, in response to this input from returned deployers, presenters updated information in security briefings. emergency response leaders tried to reduce the volume of email sent to deployed staff. the practice of having an in-country technology specialist become more standardized. after returning from the field, more deployers had access to help completing complicated travel vouchers and reimbursement paperwork. the cdc granted usb drive exceptions for computers in the field when deemed helpful. feedback collected and shared by resilience team members also supported development of improved checklists of steps in the deployment process and helpful packing lists. approximately individuals completed the three-day dsrt training during the ebola response. as part of this course conducted at the cdc since , the ramp team administered assessments to provide the opportunity for the cdc to evaluate training effectiveness. more specifically, ramp administered pre-and post-training assessments to participants in the following areas: . knowledge of resilience-enhancing principles and processes ( figure ); . knowledge of basic disaster site safety principles and processes ( figure ); . sense of self-efficacy as measured by a -item general self-efficacy scale ( figure ); . overview of course content; and . general effectiveness of the training via a standard training assessment form. the -item dsrt course content survey was intended to assess mastery of constructs related to suicidal ideation, pfa, compassion fatigue, dsrt principles, resilience, support, and referral recommendations (appendix ; available online only). the -item self-efficacy survey was an externally-developed, self-assessment of how an individual thought they could manage challenging situations (appendix ; available online only). to assess the effectiveness of the training, ramp assessed the change in the total score for the dsrt course content survey. they observed a statistically significant improvement in the total mean scores. at baseline, participants, on average, scored a . on the -item test. at post-test, they scored an average of . , an increase of . points ( % ci, . - . ). analysis of the individual items demonstrated that the responses to a large majority of the items improved between baseline and the post-test. for example, it was found for the two items "something you might do if you suspected a person was having thoughts of suicide" and "core principles of psychological first aid (pfa)," there were statistically significant increases in the percent correct (p < . ). for the items "which of the following is part of the 'five steps to getting support?'" and "which of the following can be influenced by compassion fatigue," there were not statistically significant improvements. in the future, ramp will consider revising or removing items that did not demonstrate a statistically significant improvement. the self-efficacy survey total score showed a significant improvement in overall self-efficacy. the individual items were likert scales that ranged from one (do not agree) to four (completely agree). at baseline, on average, individuals scored a . across all items. at post-test, individuals' scores improved to . , which was a statistically significant improvement (change = . points; % ci, . - . ). all individual items demonstrated a statistically significant improvement over the baseline responses. this suggests participants gained useful knowledge of resilience principles and strategies from the course content. the cdc has implemented several evidence-informed approaches to safeguard the health, safety, and resilience of its responders. these included additional pre-deployment trainings, a new pre-deployment assessment process, expansion of training to provide support in the field during a deployment, and multiple postdeployment outreach initiatives. these efforts were in addition to the three-day dsrt training, which the cdc had implemented several years earlier. since , over individuals have completed this unique resilience-focused training, which has created a cadre of individuals who can assist fellow deployers in real time. leadership at ramp trained these primarily non-clinician participants in the basics of assessing and addressing their own and their colleagues' resilience during a field deployment. in addition, ramp helped prepare participants to accomplish these tasks by: • clarifying expectations about their roles in the field; • providing information about core actions and core principles of pfa; • encouraging small groups to analyze and apply their experience and what they learned in class to address challenges presented in three different realistic scenarios; and • sharing an overview of relevant concepts and potentially helpful questions from five valid and reliable assessment tools. also, ramp made presentations highlighting this resilienceenhancing training developed in collaboration with csts at conferences in san diego, california; atlanta, georgia; little rock, arkansas; washington, dc; charleston, south carolina; san juan, puerto rico; mexico city, mexico; and tel aviv, israel. additionally, ramp shared a two-day version of this training with public health agency of canada colleagues in ottawa, canada, and a one-day version with national institute of occupational safety and health (niosh) colleagues in cincinnati, ohio and morgantown, west virginia. introducing a pre-deployment assessment process for the cdc's responders improved the quality and quantity of relevant information to which cdc's clinic staff had access. it also addressed management's directive that a screening be implemented to mitigate the risk of deploying someone who might neither contribute to, nor benefit from, a deployment. access to timely and relevant data allowed better planning and allocation of resources for staff. in addition, ramp gave deployers additional points of contact from whom they could receive support or share input and feedback about emergency planning and coordination. it expanded both the depth and breadth of information available to cdc's clinical staff to help consider the variables that affect medical clearance. it also gave potential deployers the opportunity to proactively explore and prepare for some unintended consequences in the field. this includes fatigue or distress that might negatively impact their work and family dynamics while deployed. the intent was to improve their professional and personal success and happiness in the field. the post-deployment outreach initiative provided on-going, process-improvement data to the cdc's eoc and deployment coordination team. an added benefit of the outreach was that it served as a consistent, unobtrusive vehicle through which the cdc could emphasize the organization's gratitude for personal and professional sacrifices and contributions made during the ebola response. it reminded them about the meaningfulness of their professional contributions in the field and provided an additional opportunity for deployers to connect with supportive resources, if needed. the abbreviated resilience-related training provided during pre-deployment briefings and participation in more general trainings helped increase participant awareness of challenges and opportunities in field deployments. analysis of pre-and post-training assessments of graduates of the cdc's three-day dsrt course indicated, with statistical significance, that they acquired relevant knowledge and that their self-efficacy was increased. finally, ramp determined that the assessment data supported their operating assumptions that it made sense to take multiple approaches to klomp © prehospital and disaster medicine safeguard the health, safety, and resilience of individuals deployed to inhospitable and potentially dangerous environments on an emergency response. to view supplementary material for this article, please visit https:// doi.org/ . /s x ebola outbreak in west africa control strategies, and lessons learned in the cdc response to the - ebola epidemic virtual classroom immersion training: safeguarding the health, safety, and resiliency of emergency responders chairman of the joint chiefs of staff instruction intervention and resilience after mass trauma center for the study of traumatic stress psychological first aid (pfa) field operations guide: nd edition total force fitness army master resilience training course provides valued instruction stronger: develop the resilience you need to succeed the power of resilience: achieving balance, confidence, and personal strength in your life the resilience handbook: approaches to stress and trauma the resilience factor learned optimism: how to change your mind and your life the survivor personality: why some people are stronger, smarter, and more skillful at handling life's difficulties : : : and how you can be too /a-ready-and-resilient-workforcefor-the-department-of-homeland-security-protecting-americas-front-line.aspx key: cord- - uaj hmx authors: desmonts de lamache, d.; moges, r.; siddiq, a.; allain, t.; feener, t. d.; muench, g. p.; mckenna, n.; yates, r. m.; buret, a. g. title: immuno-modulating properties of tulathromycin in porcine monocyte-derived macrophages infected with porcine reproductive and respiratory syndrome virus date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: uaj hmx porcine reproductive and respiratory syndrome virus (prrsv) is a positive-stranded rna virus that grows in macrophages and causes acute pneumonia in pigs. prrsv causes devastating losses to the porcine industry. however, due to its high antigenic variability and poorly understood immunopathogenesis, there is currently no effective vaccine or treatment to control prrsv infection. the common occurrence of prrsv infection with bacterial infections as well as its inflammatory-driven pathobiology raises the question of the value of antibiotics with immunomodulating properties for the treatment of the disease it causes. the macrolide antibiotic tulathromycin (tul) has been found to exhibit potent anti-inflammatory and immunomodulating properties in cattle and pigs. the aim of this study was to characterize the anti-viral and immunomodulating properties of tul in prrsv-infected porcine macrophages. our findings indicate that blood monocyte-derived macrophages are readily infected by prrsv and can be used as an effective cellular model to study prrsv pathogenesis. tul did not change intracellular or extracellular viral titers, not did it alter viral receptors (cd and cd ) expression on porcine macrophages. in contrast, tul exhibited potent immunomodulating properties, which therefore occurred in the absence of any direct antiviral effects against prrsv. tul had an additive effect with prrsv on the induction of macrophage apoptosis, and inhibited virus-induced necrosis. tul significantly attenuated prrsv-induced macrophage pro-inflammatory signaling (cxcl- and mitochondrial ros production) and prevented prrsv inhibition of non-opsonized and opsonized phagocytic function. together, these data demonstrate that tul inhibits prrsv-induced inflammatory responses in porcine macrophages and protects against the phagocytic impairment caused by the virus. research in live pigs is warranted to assess the potential clinical benefits of this antibiotic in the context of virally induced inflammation and tissue injury. a a a a a responsible for estimated losses exceeding us$ million/year in the usa alone, porcine reproductive and respiratory syndrome (prrs) is a devastating disease in the swine industry [ ] . first identified in europe and north america in the late s [ ] , this syndrome is currently prevalent in most swine-producing countries [ ] . its causative agent, the porcine reproductive and respiratory syndrome virus (prrsv), is a small enveloped positive-sense singlestranded rna virus, member of the arterivirus genus [ ] . sequence comparison between viral isolates demonstrated that prrsv exists in at least two distinct genotypes, the european genotype (eu type or type i) commonly referred to as prrsv- , and the north american genotype (na type or type ii) known as prrsv- [ ] . prrsv has a very narrow cell tropism, and may induce persistent asymptomatic infections [ , ] . in its natural host, the virus targets alveolar macrophages (am) [ , ] , and is able to infect most cells of the monocyte-macrophage lineage such as intravascular and lymph node macrophages [ , , ] . these cells play a crucial role in immune surveillance, pathogen killing and adaptive immune response stimulation [ ] . prrsv impairs macrophage phagocytic and bactericidal functions, induces host cell death often resulting in an inflammatory response, and perhaps most importantly predisposes the pig to secondary infections [ ] [ ] [ ] [ ] [ ] . indeed, opportunistic pathogens, whether viral-swine influenza virus, pseudorabies virus-or bacterial-streptococcus suis, bordetella brochiseptica-potentiate prrsv-induced pneumonia [ ] . these synergistic effects promote a self-sustaining inflammatory response increasing the severity and the duration of the disease [ ] [ ] [ ] [ ] [ ] . the common occurrence of prrsv infection with bacterial infections combined with the lack of efficient vaccines begs the question of the value of antibiotics for the treatment of prrs. traditionally, antibiotic efficacy is evaluated solely based on their antimicrobial properties. however, some macrolides have been found to modulate ros and pro-inflammatory cytokines such as cxcl- and il- , and to alter the production of lipid mediators that regulate inflammation [ ] [ ] [ ] [ ] [ ] [ ] . these antibiotics accumulate within leukocytes at concentrations that may reach times the systemic levels, which in turn allows them to be transported directly to the site of infection and confers them superior pharmacodynamics [ ] . there is little evidence supporting a direct anti-viral property for macrolides, but their effects on leukocytes support the hypothesis that such macrolides may be beneficial in the context of viral infections such as prrsv [ , ] . in an attempt to uncover new mechanisms whereby macrolides may protect against the detrimental effects of prrsv, the present study investigated the effects of tulathromycin in porcine monocyte-derived macrophages. tulathromycin is a triamilide in which its lactone-ring is comprised of polar amine groups. it is used for the treatment and prevention of swine respiratory diseases associated with actinobacillus pleuropneumoniae a gram-negative bacteria often found in prrsv-infected pigs [ ] . a. pleuropneumoniae exerts cytotoxic effects in macrophage and neutrophils and increases the production of pro-inflammatory il- , cxcl- -also known as interleukin- -and leukotriene b , which ultimately leads to severe pulmonary tissue damage and death [ ] [ ] [ ] [ ] [ ] . recent studies have demonstrated that in addition to its antimicrobial effects, tulathromycin inhibits cxcl- and ltb production in stimulated neutrophils and macrophages [ , , ] . in addition, tulathromycin promotes the apoptotic death of neutrophils and their phagocytic clearance by macrophages -a phenomenon known as efferocytosis-both crucial processes in the resolution of inflammation [ , , [ ] [ ] [ ] . we hypothesized that tulathromycin may generate immunomodulatory benefits in prrsv-infected monocyte-derived macrophages. the findings indicate that tulathromycin, in the absence of a direct anti-viral effect, is able to restore the phagocytic function and to attenuate the pro-inflammatory phenotype of prrsv-infected monocyte-derived porcine macrophages. the african green monkey kidney cell line marc- (crl- ) which is highly permissive to prrsv, was used for viral passage and plaque titration assay, as validated previously [ , , ] marc- cells were cultivated in dulbecco's modified eagle's medium (dmem; thermo fisher scientific, waltham, ma, usa) supplemented with % fbs (invitrogen, carlsbad, ca, usa) and iu/ml penicillin-streptomycin (thermo fisher scientific, waltham, ma, usa). the cells were maintained at ˚c, % co and passaged twice weekly. prrsv- isolate nvsl - (genbank accession no. ay . ) was used in all experiments as previously described [ ] . viral titration was performed via plaque assay. briefly, marc- cells were seeded in well plates (costar; sigma aldrich, saint-louis, mo, usa) and grown until confluency. once at confluency, cells were infected with prrsv, for hour in serum-free dmem to allow attachment of viral particles. following attachment, marc- were overlaid with a solution of x mem diluted : with . % agarose. infection was carried for h and plaques were revealed with neutral red (sigma-aldrich, saint-louis, mo, usa). dr. r. m. yates from university of calgary generously provided both marc- cell line and prrsv- isolate nvsl - . all animal experimental practices and care were conducted according to the standards of the canadian council of animal care guidelines and approved by the university of calgary life and environmental science animal care committee. blood was collected from healthy large white and landrace cross -to weeks old ( -to kg) female and castrated male piglets. the animals were housed at the veterinary science research station (university of calgary) at ˚c ± ˚c with % humidity, light cycles consisted of hours continuous light exposure followed by hours of darkness. piglets were fed twice with the antibiotic-free feed % hog grower (hi-pro feeds, okotoks, ab, canada), water was provided ad libitum. after weeks, animals were euthanized and tissues made available for secondary teaching and research use. in accordance with the standards of the canadian council on animal care, pigs were euthanized by intracardiac injection with sodium pentobarbital. monocytes were obtained and differentiated into macrophages as described previously [ ] . briefly, blood was pooled and centrifuged for minutes at x g, ˚c in a heraeus megafuge r (thermo fisher scientific, waltham, ma, usa). the plasma was removed, and the buffy coat layer was collected into and diluted : in filter-sterilized . % nacl. sterile polysucrose and sodium diatrizoate gradient solution (histopaque; sigma-aldrich, saint-louis, mo, usa) was added into each tube before centrifugation for minutes at x g, ˚c. pbmcs located at the opaque interphase were then collected, washed with sterile-filtered x hank's balanced salt solution (hbss; thermo fisher scientific, waltham, ma, usa) and centrifuged for minutes at x g, ˚c. contaminating erythrocytes were removed by three hypotonic lysis cycles with sterile ice-cold double-distilled water for seconds followed by the addition of x hbss to restore tonicity. pbmcs were then resuspended in serum-free iscove's modified dubelcco's medium (imdm; thermo ficher scientific, waltham, ma, usa) supplemented with iu/ml penicillin-streptomycin. cells were counted using a hemocytometer and viability was assessed by . % trypan blue exclusion (flow laboratories). pbmcs purity was determined by diff-quick staining on cytospin slides (cytospin cytocentrifuge, thermo fisher scientific, waltham, ma, usa). the cells were then plated in tissue-culture treated , , and well plates (costar; sigma aldrich, saint-louis, mo, usa) or in labtek chamber slides (thermo fisher scientific, waltham, ma, usa) at a concentration of . x cells/ ml for two hours to allow attachment. following adhesion, non-adherent mononuclear cells were washed with warm hbss ( ˚c). subsequent adherent monocytes were incubated for days at ˚c, % co in imdm supplemented with % heat inactivated(hi)-pig serum (ge healthcare, chicago, il, usa), iu/ml penicillin-streptomycin and % l supernatant to allow for differentiation into monocyte-derived macrophages (mdms). l -conditionned medium is commonly used to potentiate monocytes to differentiate into homogenous populations of mature macrophages [ , ] . culture media was changed every days. flow cytometry was used to quantify the number of cells expressing cd (a known cluster of differentiation of monocytes and macrophages). more than % of the isolated cells expressed cd (s fig) . on day , as described previously [ ] , macrophage differentiation was monitored by microscopic morphological changes using diff-quick, and esterase staining, a well known feature allowing to distinguish between monocytes and mature macrophages [ ] . at day , more than % of the cell preparations were differentiated macrophages (data not shown). seven days-old differentiated macrophages were incubated with tulathromycin (draxxin; zoetis, parsippany-troy hills, nj, usa) diluted in imdm + % hi-pig serum at a concentration of . mg/ml or mg/ml or with vehicle control (imdm + % pig serum), as established recently [ , ] . at these concentrations and time points, the drug exhibits immunomodulating properties in bovine macrophages without inducing apoptosis [ ] . antibiotics like tulathromycin accumulate within leukocytes at concentrations that may reach > times the systemic levels, which in turn allows them to be transported directly to the site of infection, and hence confers them with superior pharmacodynamics [ , ] . this phenomen is critical to the mode of action of tulathromycin. the drug concentrations used in these present experiments are consistent with this knowledge, and with previous studies that showed that tulathromycin has immunomodulating effects in bovine and porcine neutrophils and macrophages [ , , ] . these recent studies have reproduced the same immunomodulating effects seen in vitro at these drug concentrations than when using live infected cattle and pigs given tulathromycin at the recommended therapeutic dosage [ , , ] . hence the concentrations used here reflect the physiological conditions in which the drug accumulates at high concentrations within these leukocytes. indeed, comparison of intracellular drug concentrations in treated versus untreated animals have been published previously [ ] . using lc/ ms ms, in animals given the recommended dose of . mg/kg body weight, studies have measured the rapid and prolonged distribution of the drug into lung homogenates, pulmonary epithelial ling fluid (pelf), as well as in pelf cells. macrophages are the major constituents of pelf cells in such preparations. drug levels measured in pelf cells reached concentrations times greater than those in plasma [ ] . it is believed that this great affinity for cellular uptake may be related, in part, to the tri-basic chemical structure of the drug and the trapping of ionized drug within acidic phagolysosomes. macrophages were infected with prrsv minutes after tul or vehicle treatment, or not infected (uninfected controls) and incubated for h at ˚c, % co to allow virus attachment and entry (time ; t = ). prrsv was diluted in serum-free dmem to reach a multiplicity of infection (m.o.i) ranging from . to depending on the experiment. culture media was replaced by pre-warmed imdm supplemented with % pig serum for all experimental groups. prrsv infection was performed for another to hours depending on the experiment. all functional assays contained the following experimental groups: untreated and uninfected control (control); tulathromycin-treated (tul); untreated and prrsv-infected (virus); tulathromycin-treated and prrsv-infected (tul+virus); lps-activated; pro-apoptotic positive control (staurosporine; μm) (sts); or pro-necrotic positive control ( . % triton-x) (trit-x) where appropriate. to avoid l cytokine-induced polarization of macrophages, all macrophages activation experiments were performed on monocytes that were grown in l supernatant free. the effects of tulathromycin and prrsv on macrophage differentiation and activation was determined via microscopic observations and cytokine quantification. mdms were treated with tulathromycin ( . or mg/ml) for hour and infected with prrsv (m.o.i. of . ) for , , or h at ˚c, % co . supernantants were collected and frozen at - ˚c until processed and macrophages were stained with diffquick (electron microscopy sciences, hatfield, pa, usa) and observed with a nikon eclipse t microscope to assess morphological changes. images were taken with a retiga x camera (q imaging, surrey, bc, canada) on a leica dmr fluorescent microscope (leica, wetzlar, germany) and analyzed using imagej software. individual macrophage morphology was assessed and classified as "resting" or "fibroblast-like" morphology. at least macrophages per group in independent experiments were assessed. supernatants were processed to measure interleukin- (cxcl- ) and interleukin- (il- ) concentrations using the porcine cxcl- quantikine enzyme-linked immunosorbent assay (elisa; p , r&d systems, minneapolis, mn, usa) and the il- quantikine elisa (p , r&d systems, minneapolis, mn, usa) respectively. samples were processed as per manufacturer's instructions. ros production by mdms following tulathromycin treatment and/or prrsv infection was monitored with the oxiselect intracellular ros assay kit (cell biolabs, san diego, ca, usa). experiments assessed ros production in resting cells, as well as in a group of cells induced by lipopolysaccharide (lps) to determine the effects of the various stimuli under basal conditions in these cells, as well as when they were activated. mdms were infected for , , , or h (m. o.i of . ) or uninfected (uninfected control). the same treatments were performed on macrophage stimulated with lipopolysaccharide ( μg/ml lps from e. coli o :b (sigma-aldrich, saint-louis, mo, usa). mdms were exposed to ', '-dichlorodihydrofluorescin diacetate (dcfh-da) a cell-permeable fluorogenic probe oxidized to highly fluorescent ', '-dichlorodihydrofluorescein (dcf) by ros. fluorescence intensity, proportional to ros levels within the cytosol was measured using a spectramax m e microplate reader (molecular devices, san jose, ca, usa) reading at nm (excitation) and nm (emission). phagocytic capacity of mdms was assessed using non-opsonized zymosan particles and opsonized latex beads. non-opsonized phagocytosis was monitored using fluorescently labelled saccharomyces cerevisiae zymosan a particles (texas red; sigma-aldrich, saint-louis, mo, usa). mdms seeded on labtek chamber slides or on coverslips at x cells/ml were infected for or hours (m.o.i of . ) or not infected (uninfected control). following infection, experimental groups were incubated with zymosan a particles diluted in control media to a final ratio of : (zymosan:cells) for hour. after exposure, extracellular zymosan a particles were washed away with warm pbs and the cells were fixed in ice-cold % acetone solution. actin was stained with the alexa fluor phalloidin antibody (thermo fisher scientific, waltham, ma, usa) and the nucleus was revealed with dapi (thermo fisher scientific, waltham, ma, usa). enumeration of intracellular zymosan was performed using a leica dmr fluorescent microscope. fc-mediated phagocytic index was measured using carboxylate-modified μm diameter latex or silica beads (kisker biotech, steinfurt, germany) covalently coated with bsa and human igg (sigma-aldrich, saint-louis, mo, usa). the beads were subsequently incubated with macrophages for minutes at a : (beads:cells) ratio. following phagocytosis, extracellular beads were washed away with warm pbs and the cells were stained with diffquick (electron microscopy sciences, hatfield, pa, usa) before microscopic observations. macrophages containing one or more zymosan particles or latex beads were considered as 'positive cells', the phagocytic index was calculated as the ratio of positive macrophages versus total macrophages. a minimum of cells per experimental group were counted from randomly selected fields. all pictures were taken using leica dmr fluorescent microscope with a retiga x (q imaging, surrey, bc, canada) and analyzed using imagej software. in order to prevent any counting bias slides labellings were covered with tape prior to microscopic observations. the pro-apoptotic effects of tulathromycin and prrsv were assessed using a cell death detection elisa kit (roche) according to the manufacturer's instructions as previously described [ , ] . absorbance was measured using a spectramax m e microplate reader (molecular devices, san jose, ca, usa) set at nm. mdms were incubated with tulathromycin ( . or mg/ml) for minutes and infected with prrsv (m.o.i. of . ) for , or h at ˚c, % co . similar experimental treatments were conducted with cells stimulated with lps ( μg/ml from e. coli o :b ; sigma-aldrich, saint-louis, mo, usa) to assess apoptosis in activated macrophages. for all experiments, cells incubated with imdm containing % hi-pig serum or staurosporine ( μm) were used as negative and positive controls respectively. annexin v staining (roche) was performed on the same experimental groups to further assess apoptotic cell death. staining was performed as per manufacturer's instructions and fluorescence was observed using a leica dmr fluorescent microscope equipped with a hcx pl fluotar x objective (aperture = . ). images were taken at , and hours post infection (p. i) with a retiga x (q imaging, surrey, bc, canada). importantly, experiments measuring cytokines and ros followed those in which we measured apoptosis. this allowed to adjust concentrations of tulathromycin and virus to levels at which apoptosis was not detected in the cells, and hence cell death would not be a factor in the cellular cytokine and ros responses. necrosis was assessed through the determination of lactate dehydrogenase (ldh) levels using a cytotoxicity detection kit (roche). mdms were treated with vehicle medium alone (control) or with tulathromycin ( mg/ml) for minutes. cells were then infected with prrsv for , , or h (m.o.i. . ) or supplemented with control medium, and % triton x in media was used as positive control. supernatants were collected and processed following manufacturer's instructions. a spectramax m e microplate reader (molecular devices, san jose, ca, usa) was used to measure ldh concentrations in each sample at nm. necrosis was expressed as the absorbance ratios of the experimental cell lysates versus absorbances from controls arbitrarily set at . ( %). all experimental groups were assessed in duplicates. to assess the potential anti-viral effects of tulathromycin, extracellular and intracellular viral particles counts were monitored with plaque titration assays. for extracellular counts, supernatants were harvested at , , and hours p.i and incubated with confluent marc- cells for h as described above. for intracellular counts, macrophages were washed twice with warm ( ˚c) phosphate buffer saline (pbs; sigma-aldrich, saint-louis, mo, usa) and lysed with double distilled water exposure and thorough mixing. cellular debris were spun down at , x g for minutes and supernatants were harvested and incubated with confluent marc- cells as described previously. prrsv staining was performed to further characterize potential antiviral effects. marc- cells were seeded in labtek chamber slides, grown to % confluence and infected with prrsv (m.o.i. . ) for h at ˚c, % co . prrsv foci numbers and size were revealed using the sr- f antibody (rti, llc, brooking, sd, usa). fluorescence ratio was calculated using imagej. five fields of view per well were counted per sample. expression levels of prrsv receptors in mdms in the presence and absence of tulathromycin were assessed by immunofluorescence. seven days old mdms cultivated with or without l conditioned medium were treated with hbss (control) or tulathromycin ( mg/ml for hours). the murine l fibroblast cell line, known to secrete macrophage-colony stimulating factor (m-csf) is widely used to induce macrophage differentiation from monocytes and prevent differentiation into monocyte-derived dendritic cells [ ] . prior to staining, l -grown cells were washed times in ice-cold pbs to remove all l media and then fixed in % paraformaldehyde in pbs for minutes. fixed cells were then washed times in cold pbs and stained for hour with a r-phycoerythrin (rpe) conjugated anti-cd antibody (bio-rad, hercules, ca, usa) and a fluorescein isothiocyanate (fitc) conjugated anti-cd antibody (bio-rad, hercules, ca, usa) at a dilution of to and to respectively. following staining cells were washed times in cold pbs and observed under leica dmr fluorescent microscopy. fluorescence ratios from randomly selected fields were calculated using the software imagej. images were taken with a retiga x camera. to prevent any bias, slide labelling was covered with tape prior to microscopic observations. all statistical analyses were made using prism software and data were expressed as means + standard error from mean (sem). all data sets were tested for normality. data with parametric distribution were compared using student's t-test, or one-way anova with tukey's multiple comparision anlaysis where appropriate. non-parametric data were compared with a kruskal-wallis test. for every assay, a minimum of separate, independent experiments were conducted with all experimental groups assayed in duplicates or triplicates. statistical significance was established at p < . . in order to determine whether blood monocytes and mdms are susceptible to prrsv we isolated blood monocytes from healthy pigs and cultured them for a period of days in medium supplemented with pig serum to mimic biological conditions. after plating, adherent monocytes exhibited a round shape morphology and were approximatively μm in diameter ( fig a) . by day , the cells displayed a larger, macrophage-like, morphology with characterisitic cytoplasmic vacuoles (fig c) . monocyte differentiation was also measured using non-specific esterase (nse) staining. by day more than % cells were esterase-positive cells. oneday-old monocytes were significantly less susceptible to prrsv compared to days old differentiated mdms (fig ) . prrsv viral particle numbers increased by a . log ( -fold increase) in mdms, and by a . log ( -fold increase) in blood monocytes, between and hours p.i. in both cell types, prrsv infection reached a plateau at h p.i. (fig ) . to optimize the macrophage differentiation protocol, mdms were also cultured in a l -conditioned medium. monocytes cultivated in l -conditioned medium showed the same morphology as those cultivated in medium devoid of l -factors. consistent with previous data, microscopic observation and nse staining showed that by day , more than % cells were macrophages. viral titers in monocytes incubated with l were significantly higher at hours p.i. compared to viral titers in monocytes cultivated without l supernatant (fig ) . numbers of prrsv infectious particles were significantly elevated in mdms cultivated with l -supernatants at all time points of the infection (except from the hours p.i. time point) versus mdms cultivated in medium supplemented with hi-pig serum alone (fig ) . in l -cultivated mdms, prrsv infection peaked at h p.i. and declined afterwards, whereas it continued to increase at hours in l -cultivated monocytes (fig ) . based on these observations, we chose to use l -cultivated mdms for functional experiments, unless stated otherwise. considering that l supernatants may contain cytokines other than m-csf (such as il- and il- ) that could influence macrophage polarization and confound our studies, we decided to grow our cells in medium containing only pig serum when assessing macrophage pro-inflammatory signaling. moreover, to limit the impact of tulathromycin and prrsv-induced apoptosis on macrophage numbers and functions, we treated our cells with tulathromycin at a concentration of . mg/ml and decreased prrsv m.o.i from . to . . at these concentrations, neither the virus nor the drugs significantly induced mdm apoptosis at the experimental time points (data not shown). if the cells were treated at a concentration of mg/ml, functional analysis were performed before hours of incubation. prrsv infection induced a sharp fibroblast-like morphological alteration and pseudopod projections in mdms (fig a) . in uninfected cells (control and tul), less than % of cells exhibited this change in morphology, while nearly % of the cells exhibited this phenotype upon prrsv infection (fig b) . tulathromycin pre-treatment significantly inhibited this morphological change in mdms (fig b) . since macrophage shape and function are correlated [ ] , we hypothesized that prrsv-induced morphological changes were associated with a change in macrophage activation. infection of mdms with prrsv caused a -fold increase of cxcl- secretion after hours (fig ) . prrsv-induced cxcl- secretion was significantly inhibited when mdms were pretreated with tulathromycin (fig ) . the positive control lps, also significantly increased the production of cxcl- (fig ) . we then measured the production of mitochondrial ros, a hallmark of pathogenic oxidative damage in inflamed tissues [ , ] . prrsv infection significantly increased intracellular ros (fig ) . tulathromycin treatment abolished prrsv and lps-induced intracellular ros production, however, it did notrestore ros levels to control values in cells exposed to both lps and prrsv (fig ) . interestingly, intracellular ros levels were significantly lower in mdms incubated with lps and prrsv compared to mdms stimulated only with lps (fig ) . as our results indicated that tul inhibited macrophage pro-inflammatory signaling, another set of experiment assessed the effects of the drug on il- , a cytokine with potent anti-inflammatory properties [ ] . resting mdms produced approximately pg/ml il- throughout the course of the experiments (fig ) . prrsv infected cells secreted significantly less il- compared to control cells at and hours p.i. (fig ) . il- levels did not significantly change versus controls when uninfected cells were treated with tulathromycin alone. however, immuno-modulating properties of tulathromycin in prrsv-infected porcine monocyte-derived macrophages prrsv-induced il- inhibition was abolished when the cells were pre-treated with tulathromycin at and hours post infection (fig ) . tulathromycin ( mg/ml), as well as prrsv alone (m.o.i = . ), or the positive control staurosporine induced mdms apoptosis h post-infection (fig ) . combined pre-treatment with tulathromycin ( mg/ml; h) and prrsv (m.o.i = . ) for hours showed an additive effect to induce further mdms apoptosis versus single treatments (fig a) . to confirm these data, cells were stained with annexin v, a phospholipid-binding protein with high affinity for the early apotptic marker phosphatidylserine (ps) [ ] . at hours, both tulathromycin alone or prrsv alone induced significant levels of apoptosis compared to controls ( -fold increase vs. control) (fig b and c) . when cells were exposed to the combination of tulathromycin immuno-modulating properties of tulathromycin in prrsv-infected porcine monocyte-derived macrophages and prrsv, levels of apotosis were almost double those measured in cells exposed to single treatments (fig b and c ). prrsv infection (m.o.i = . ) significantly increased the levels of ldh produced during necrosis and hours p.i. (fig ) . treatment with tulathromycin ( mg/ml) significantly reduced prrsv cell necrosis at hours (fig ) . this effect of tulathromycin could no longer be detected at hours. tulathromycin alone did not alter levels of necrosis (fig ) . triton-x (trit-x), used as a pro-necrotic positive control, induced necrosis in mdms (fig ) . another set of experiments assessed the effects of prrsv, and of tulathromycin, on the nonopsonized and opsonized phagocytic functions of mdms. prrsv infection significantly immuno-modulating properties of tulathromycin in prrsv-infected porcine monocyte-derived macrophages inhibited both phagocytic functions of the cells (figs and ). prrsv-induced phagocytic inhibition was inhibited by tulathromycin (figs b and b ). tulathromycin treatment alone did not alter mdms phagocytosis versus controls (figs and ). during phagocytosis, macrophages can engulf multiple antigens at the same time [ , ] . additional experiments assessed mdms that engulfed less than particles (ie with basal phagocytic indices) versus cells that ingested more than particles (i.e. with elevated phagocytic indices). prrsv infection significantly reduced the number of cells with high phagocytic indeces, an effect that was abolished by tulathromycin (figs c and c) . tulathromycin inhibited the prrsv-induced reduction of basal and high phagocytic indices (figs and ). tulathromycin alone did not change either of the mdms phagocytic indices versus controls. the same results were obtained when the cells were infected for hours (fig ) . another set of experiments assessed whether the effects of tulathromycin described above were associated with direct antiviral properties of the antibiotic, in porcine mdms (fig a) or marc- cells (fig b) . upon incubation with prrsv, extracellular and intracellular viral particles were enumerated via plaque assay. tulathromycin did not change intracellular or extracellular viral titers in either of the cell models (fig a and b ). to verify these results, marc- cells were stained with fitc-conjugated anti-prrsv nucleocapsid antibody sr f antibody. size and numbers of viral foci were calculated in presence or absence of tulathromycin ( fig c) . again, tulathromycin pre-treatment did not alter viral titers compared to exposure to prrsv alone (fig c and d ). to further examine the effects of tulathromycin on prrsv infectivity, experiments measured viral receptor expression in mdms. to date, two major prrsv receptors have been extensively studied (cd and cd ) and it is not entirely clear which one of these two receptors is essential for prrsv infection [ ] [ ] [ ] . since l -conditioned medium increases viral titers, we hypothesized that it might be due to an increase in cell permissivity resulting from an increase in prrsv receptor expression. to test this hypothesis, we cultivated monocytes in medium containing pig serum alone or in l -conditionned medium for days and then treated them with tulathromycin. mdms differentiated in medium devoid of l -supernatant expressed both receptors. approximatively % of cells expressed cd and % of cells expressed cd . tulathromycin treatment did not significantly change the percentage of cd and cd positive cells (respectively % and % of positive cells) (fig a; upper panels; fig b) . mdms incubation in l -supernatant supplemented medium was sufficient to significantly increase the number of cd positive cells (more than % of mdms were cd positive versus less than % in pig serum supplemented medium alone). in addition, following l -supernantant exposure we were not able to detect any cd positive cells (fig a; lower panels; fig b) . tulathromcyin treatment following l -incubation did not have any significant effect on viral receptor expression in these experiments (fig a; lower panels; fig b) . prrs is one of the most devastating diseases of the porcine industry [ , ] . treatment options to control prrs outbreaks are limited and the efficacy of vaccines is thwarted by the antigenic variability of prrsv [ ] . disease severity is closely related to the ability of the virus to dysregulate macrophages functions and induce inflammation. therefore, we hypothesize that targeting either of these components may represent a critical element of novel therapeutic approaches. anti-inflammatory and immunomodulatory properties of macrolides have been well established [ - - ] . whether these effects may be beneficial in the context of viral diseases such as prrs remains obscure. the present study assessed the anti-viral and immunomodulating properties of tulathromycin (tul) in prrsv-infected porcine macrophages. the findings indicate that tul inhibits prrsv-induced inflammatory responses in porcine monocyte-derived macrophages and protects against the phagocytic impairment caused by the virus, in the absence of any direct anti-viral effects. the two most common cellular models are pams and marc- cells [ , ] . both have significant limitations. the isolation of pams requires bronchoalveolar lavages, and the function of these cells depends on the age and environment of the animal [ ] . moreover, shortly after the initiation of a respiratory infection, alveolar macrophages are replaced by monocytederived macrophages, which therefore represent a key cell population in host-prrsv immuno-modulating properties of tulathromycin in prrsv-infected porcine monocyte-derived macrophages interactions. monkey marc- epithelial cells do not originate from pigs. therefore, the present experiments developed and used a simple porcine monocyte-derived macrophage model system to characterize the impact of tulathromycin on prrsv infection. previous in vitro studies have shown that the virus could infect blood monocyte-derived macrophages (mdms) [ ] . consistent with previous findings, monocytes were less susceptible to prrsv than differentiated monocyte-derived macrophages [ ] . the addition of l supernatant during macrophage differentiation significantly increased their susceptibility to the virus compared to macrophages cultivated in medium supplemented with pig serum alone. it has been well established that l supernatant is a source of m-csf and is used to induce macrophage differentiation [ ] . immunostaining of prrsv receptors showed that the addition of l immuno-modulating properties of tulathromycin in prrsv-infected porcine monocyte-derived macrophages strongly upregulated cd ( % positive cells to % positive cells) but abolished cd expression. these results indicate that l supernatant modulate the expression of prrsv receptors, and that cd alone is sufficient for prrsv infection. this is consistent with recent observations showing that cd , but not cd , enabled non-permissive cells to become susceptible, and that increased cd correlates with increased susceptibility to prrsv [ , , ] . l supernatant is known to contain m-csf, but very little is known about other cytokines and chemokines present in this supernatant [ ] . considering that cd and cd expression can be induced by il- and ifn-γ respectively, and that il- treatment increases prrsv infectivity while ifn-γ decreases it [ , , ] , the role of these cytokine in the modulation of macrophage susceptibility to infection requires further investigation. the present findings demonstrate that mdms can readily be infected by prrsv, and hence represent a useful cellular model to study prrsv pathogenesis, as suggested recently [ ] . a hallmark of prrsv pathogenesis resides in its ability to alter macrophages survival and function, hence predisposing the host to secondary infections [ , ] . there is correlation between macrophage morphology and function, hence providing an easy way to monitor immuno-modulating properties of tulathromycin in prrsv-infected porcine monocyte-derived macrophages changes in macrophage polarization [ ] . in this study we found that prrsv infection dramatically altered monocyte-derived macrophage morphology, inducing an elongated phenotype with numerous cytoplasmic pseudopods. recent findings indicate that these pseudopods promote intercellular junctions allowing prrsv to evade host immunity through direct intercellular spread [ ] . tulathromycin pre-treatment was sufficient to prevent the prrsvinduced pseudopod formation and morphological alterations in macrophages. whether tul may prevent intercellular junctions and thus hinder prrsv immune evasion requires more research. to test the hypothesis that change in macrophage morphology was associated with altered function, we measured the production of pro-and anti-inflammatory cytokines (cxcl- and il- respectively) as well as the production of mitochondrial ros. cxcl- is a potent neutrophil chemoattractant secreted by macrophages and other cell types, and is a critical mediator of neutrophil infiltration in inflamed tissues [ ] . the present findings demonstrate that prrsv is a potent inducer of cxcl- in monocyte-derived macrophages. virally induced cxcl- secretion was inhibited by tul. studies in live animals are warranted to assess whether these observations suggest that tul might attenuate prrsv-induced inflammation through cxcl- inhibition. mitochondrial ros production is a hallmark of cell stress and inflammation, and contributes to prrsv-induced tissue damage [ , ] . other reports showed that mitochondrial ros production was implicated in prrsv-induced apoptotic death of marc- cells [ ] . here we demonstrate that prrsv indeed induces ros production in porcine monocyte-derived macrophages, and that this production is inhibited when the cells are pre-treated with the antibiotic. tulathromycin was also able to restore ros levels to control in lps-stimulated cells but not in lps and prrsv exposed to both lps and prrsv. interestingly, in these conditions mdms showed a decrease in ros production compared to cells exposed only to lps. this suggest that prrsv may inhibit intracellular ros production of mdms during bacterial infections.another set of studies sought to determine whether tul inhibition of the viral-induced pro-inflammatory cxcl- coincided with an increase in antiinflammatory signaling. we found that the virus alone was able to inhibit il- secretion, and that tul blocked this effect. these data are in contrast with others from the scientific literature. indeed, it is generally accepted that prrsv induce il- production to increase its infectivity [ ] . in fact, il- activated cells are more permissive to prrsv than unstimulated m -polarized macrophages [ ] . more research is necessary to explain the mechanisms whereby prrsv regulates the production of il- . tulathromycin alone did not induce il- secretion suggesting that cxcl- and mitochondrial ros inhibition by tul was not dependent on il- production. taken together the present findings strongly support the hypothesis that tulathromycin may attenuate prrsv-induced inflammation by inhibiting production of pro-inflammatory cxcl- , and by preventing the suppression of anti-inflammatory il- . consistent with previous studies, we found that prrsv and tul induced macrophage apoptosis [ , , , ] . the present findings also illustrate that tul and prrsv haver additive pro-apoptotic effects. morevoer, the data indicate that prrsv leads to cell necrosis, an effect that was inhibited by tul. necrosis is known to exacerbate local inflammation, to induce the release of cytotoxic molecules, and to lead to extensive tissue damage, while cell apoptosis contributes to the resolution of inflammation [ , ] . more research in live prrsv-infected animals will help determine whether tul is able to promote the resolution of prrsv-induced pulmonary inflammation at least in part via such a mechanism, as well by shifting local cytokine release from pro-inflammatory to anti-inflammatory mediators. it is well established that prrsv infected pigs are often infected by secondary pathogens [ , ] . at present, the mechanisms resulting in the increase of secondary infections during prrsv infections remain incompletely understood. studies have shown that prrsv is directly able to impair macrophage phagocytosis, which in turn may represent a key element of the development of secondary infection [ , , ] . macrophage phagocytosis is triggered when phagocytic receptors including opsonic receptors (fcr) or pattern recognition receptors such as the mannose receptor, are activated [ , ] . using non-opsonized zymosan particles or igg-coated latex beads, the present findings demonstrate that prrsv significantly inhibits both phagocytic pathways. these results are consistent with previous reports showing decreased phagocytosis of latex beads, or live bacteria (streptococcus suis) upon prrsv infection [ , ] . recent findings suggest that prrsv- inhibits phagocytosis through its interaction with sialoadhesin (also referred to as cd ). however, in our model system, l cultivated mdms were negative for cd suggesting either that mechanisms for inhibition of phagocytosis are strain and/or genotype dependent, or that prrsv may inhibit phagocytosis through multiple pathways [ ] . another report recently demonstrated that the same nsvl- - strain as used here may impair phagosomal maturation and nadph oxidasemediated respiratory burst, both implicated in the antimicrobial properties of macrophages [ ] . tul blocked the prrsv-induced inhibition of non-opsonized and igg-mediated macrophage phagocytosis. these observations pave the way towards studies in vivo to assess whether this antibiotic might help control secondary infections during prrsv infections through this mechanisms in addition to its direct anti-microbial properties. the mechanisms whereby tul protects against prrsv-induced inhibition of phagocytosis require further elucidation. some macrolides such as tilmicosin and tylvalosin have been recently demonstrated to possess direct anti-viral effects against prrsv [ , ] , while others like erythromycin do not [ ] . in the experiments described herein, tul did not exhibit any direct anti-viral properties, nor did it significantly alter the expression of the two receptors used by the virus for entry, cd and cd . together, the data indicate that in porcine mdms, tul is able to block prrsvinduced pseudopod formation, necrosis, pro-inflammatory cxcl- and mitochondrial ros production, and inhibition of macrophage phagocytosis. in addition tul also synergized with prrsv to induce pro-resolution cell apoptosis and the production of anti-inflammaotry il- . the results also show that the protective modulation of macrophage structure, function, and behavior by tul occurs in the absence of a direct anti-viral effect. the present observations pave the way towards further studies with a prrsv- strain to determine whether the effects we observed in this study are conserved with the other prrsv genotype. studies in vivo will help determine whether and how these effects may translate into clinical benefits. assessment of the economic impact 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induces apoptosis of porcine neutrophils and macrophages, promotes efferocytosis, and inhibits pro-inflammatory cxcl- , il α, and ltb production, while inducing the release of pro-resolving lipoxin a and resolvin d dual infections of prrsv/influenza or prrsv/actinobacillus pleuropneumoniae in the respiratory tract virulence factors of actinobacillus pleuropneumoniae involved in colonization, persistence and induction of lesions in its porcine host immunomodulatory effects of tulathromycin on apoptosis, efferocytosis, and proinflammatory leukotriene b production in leukocytes from actinobacillus pleuropneumoniae-or zymosan-challenged pigs il- as a keystone cytokine in health and disease host-pathogen interplay at primary infection sites in pigs challenged with actinobacillus pleuropneumoniae anti-inflammatory benefits of antibiotic-induced neutrophil apoptosis: tulathromycin induces caspase- -dependent neutrophil programmed cell death and inhibits nf-κb signaling and cxcl transcription. 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pathway through iκb degradation. virology porcine reproductive and respiratory syndrome virus induces apoptosis through a mitochondria-mediated pathway biology of interleukin- quantitation of apoptosis and necrosis by annexin v binding, propidium iodide uptake, and flow cytometry the cell biology of phagocytosis phagocytosis: receptors, signal integration, and the cytoskeleton sialoadhesin and cd join forces during entry of the porcine reproductive and respiratory syndrome virus modulation of cd receptor expression and replication of porcine reproductive and respiratory syndrome virus in porcine macrophages an intact sialoadhesin (sn/ siglec /cd ) is not required for attachment/internalization of the porcine reproductive and respiratory syndrome virus improved vaccine against prrsv: current progress and future perspective enhanced replication of porcine reproductive and respiratory syndrome (prrs) virus in a homogeneous subpopulation of ma- cell line variability of neutrophil and pulmonary alveolar macrophage function in swine establishing porcine monocyte-derived macrophage and dendritic cell systems for studying the interaction with prrsv- cd expression confers susceptibility to porcine reproductive and respiratory syndrome viruses human monocytes express cd , which is upregulated by il- and identical to p interferon-inducible cd / siglec attenuates anti-hiv- effects of ifn-α immune responses in pigs infected with porcine reproductive and respiratory syndrome virus (prrsv) porcine reproductive and respiratory syndrome virus utilizes nanotubes for intercellular spread adenoviral-expressed gp of porcine respiratory and reproductive syndrome virus differs in its cellular maturation from the authentic viral protein but maintains known biological functions apoptosis and porcine reproductive and respiratory syndrome virus corpse clearance defines the meaning of cell death differential effects of apoptotic versus lysed cells on macrophage production of cytokines: role of proteases effects of porcine reproductive and respiratory syndrome virus (isolate tw ) on porcine alveolar macrophages in vitro in utero infection with prrs virus modulates cellular functions of blood monocytes and alveolar lung macrophages in piglets mechanisms of phagocytosis in macrophages porcine reproductive and respiratory syndrome virus productively infects monocyte-derived dendritic cells and compromises their antigen-presenting ability transcriptional analysis of prrsv-infected porcine dendritic cell response to streptococcus suis infection reveals up-regulation of inflammatory-related genes expression antiviral activity of tilmicosin for type and type porcine reproductive and respiratory syndrome virus in cultured porcine alveolar macrophages tylvalosin exhibits anti-inflammatory property and attenuates acute lung injury in different models possibly through suppression of nf-κb activation antibiotic-mediated inhibition of porcine reproductive and respiratory syndrome virus (prrsv) infection: a novel quinolone function which potentiates the antiviral cytokine response in marc- cells and pig macrophages the authors thank troy feener, barbara smith, and the staff at the veterinary sciences research station at the university of calgary for their help with animal handling. we also thank dr. constance finney and dr. edina szabo for their help with flow cytometry. buret. key: cord- -v m l wz authors: nan title: neurocritical care society (th) annual meeting date: - - journal: neurocrit care doi: . /s - - - sha: doc_id: cord_uid: v m l wz nan in this exploratory analysis, csf levels in the progesterone treated group were variable and not as supratheraputic compared to serum levels. this study highlights an additional factor that needs to be considered in the designing of clinical trials in tbi. not only does the heterogeneity of the injury and subsequent outcome measures need to be refined, but the biomarker of pk levels also needs to be analyzed in csf as well as serum to determine if the treatment is reaching the target organ, the brain. arctic ground squirrels (ags) are extreme hibernators capable of withstanding months of freezing temperatures by suppressing metabolic rate. hibernation is characterized by hypoxia and low cerebral blood flow and interrupted by bouts of arousal in which perfusion is quickly restored. curiously, ags do not experience reperfusion injury which is hypothesized to be reflected in altered transcriptional signatures in an in vitro model of reperfusion injury. to investigate the influence of ischemia/reperfusion on ags neuronal stem and neural progenitor cells (nsc/npcs), we exposed ags and murine nsc/npcs to control conditions, hypoxia, oxygen and glucose deprivation or glucose deprivation alone or following return to normal conditions to model reperfusion. cell viability and cell cycle state were assessed by automated cytometry; metabolic phenotype by in vitro oxygen consumption and extracellular acidification rate. to determine novel genes involved in ags resilience to reperfusion injury, a cdna library was constructed in a mammalian expression vector and introduced into murine nscs that were then assayed for viability after ischemia/reperfusion. ags nsc/npcs demonstrated marked resistance to ischemia/reperfusion injury compared to murine nsc/npcs. this survival phenotype is associated with suppressed mitochondrial oxidation and altered cell cycle regulation. ags genes regulating mitochondrial function strongly modulated murine nsc/npc viability following ischemia/reperfusion injury. a dynamic ability to suppress mitochondrial oxidation may underlie resilience to reperfusion injury in ags by promoting a quiescent cell cycle phenotype. development of therapeutic agents suppressing mitochondrial oxidation may induce a protective phenotype and promote survival following reperfusion injury. high throughput imaging of motor system connectivity in the mouse brain. stroke results in profound alterations to architecture in the brain, particularly the corticospinal tract (cst). some plasticity may contribute to functional recovery, while other changes may be maladaptive. studies of cst connectivity have been limited by standard imaging methods which do not allow visualization and analysis of global axonal connectivity in the brain. we employed two novel imaging methods to visualize axonal projections to forelimb musculature. first, a pseudorabies viral (prv) vector carrying green fluorescent protein (gfp) was injected into the left forelimb flexor in naïve - week-old c mice. prv was transported retrogradely and transynaptically, labeling neurons in the motor cortex and other regions of interest. whole slide imaging was performed using an automated slide scanner (nanozoomer, hamamatsu photonics k.k., hamamatsu city, japan) producing images of serial coronal sections, allowing visualization of multiple levels of the brain in a single slide image. for serial two-photon tomography (stpt), utsw whole brain microscopy facility used a tissuecyte imaging system (tissue vision, somerville, ma) which uniquely performs automated sectioning and fluorescent imaging of the brain to produce -dimensional images with micronlevel resolution. this allows for unprecedented visualization of axonal connectivity in the whole brain. we have imaged motor systems in the brain of uninjured mice using two different methods, each with distinct benefits. whole slide imaging allows for quantification of regions of interest in the brain on a single slide, while stpt produces a highly detailed image that improves our understanding motor systems in -d space. future directions will investigate changes in connectivity following stroke injury and during recovery, allowing a greater understanding of the complexity of plasticity and how it contributes to beneficial and pathological circuit remodeling after injury. andexanet alfa (anxa) is a modified recombinant factor xa (fxa) derivative that sequesters direct fxa inhibitors and reverses their anticoagulation effects. non-specific prothrombin complex concentrates (pccs) have been proposed as potential reversal strategies. the objective of these studies was to compare -factor (bebulin) and -factor (kcentra®) pccs, approved for reversal of warfarin, with anxa in reversing anticoagulation effects of rivaroxaban in a rabbit model of bleeding. nzw rabbits were treated with rivaroxaban (iv, mg/kg) and min later, either pccs ( -factor pcc: or mg/kg; -factor pcc: , , or iu/kg) or anxa ( or mg/rabbit) was administered iv. liver injury was then induced with -cm incisions following laparotomy, and blood loss was measured for min. plasma concentrations of unbound (active) and total rivaroxaban, as well as pharmacodynamic (pd) parameters (anti-fxa activity, pt, and aptt) were determined. anxa reduced blood loss in rivaroxaban-anticoagulated rabbits to levels seen in non-anticoagulated rabbits. in rabbits treated with anxa, anti-fxa activity and unbound rivaroxaban were reduced dosedependently by > % and > %, respectively, within minutes, and both parameters correlated with reduction in blood loss. in contrast, -factor or -factor pccs had no significant effect on any of these markers in rivaroxaban-anticoagulated rabbits. anxa effectively reversed the anticoagulation activity of rivaroxaban in a rabbit model of bleeding. in contrast, pccs showed no reversal activity as assessed by blood loss or pd markers. these results suggest that use of non-specific pccs as reversal agents for direct fxa inhibitors are not likely to be as effective as specific reversal agents that target fxa inhibitors. investigation of anxa vs. pcc to reduce hematoma expansion in models of intracranial hemorrhage is warranted. outcomes after resuscitation from cardiac arrest (ca) remain poor. preventable secondary injury from ongoing brain tissue hypoxia (bth) may worsen injury burden. unfortunately, markers to allow individualized, real-time care optimization are lacking. we performed a randomized crossover trial in a swine model of opioid-induced ca to ) determine the prevalence of bth with standard care (stdc), and ) test whether neuromonitor-guided goal-directed care (ngdc) can prevent bth. female swine ( - kg) were anesthetized with propofol and fentanyl. we placed femoral arterial and venous sheaths, a continuous cardiac output pulmonary artery catheter (edwards lifescience) and a right frontal intracranial access bolt (hemedex) with probes for brain tissue oxygen (pbto ), pressure (raumedic), microdialysis (mdialysis ), cerebral blood flow (cbf) (hemedex), and an -contact electroencephalographic depth electrode (adtech). we induced apnea with mcg/kg fentanyl, extubated the animal and began acls min after apnea. after h stabilization, animals with return of spontaneous circulation (rosc) were randomized to three alternating h care blocks: stdc (mean arterial pressure> mmhg, oxygen saturation - %, cardiac output> % baseline) or ngdc (pbto > mmhg, cbf> ml/ g/min). animals were euthanized at h post-rosc. our primary outcome was the effect of care block on pbto , which we analyzed at min resolution using generalized estimating equations with robust standard errors. overall, of animals achieved rosc after ± min. pbto was higher during ngdc than stdc (p< . ) and did not differ during ngdc from pre-arrest. pbto was < mmhg more during stdc than ngdc ( % of minutes vs %, p< . ). cbf was lower during ngdc than stdc (p< . ), and lower in both arms than pre-arrest (both p< . ). brain tissue hypoxia was common in this cardiac arrest model and prevented by neuromonitor-guided goal-directed care. lower cbf and higher pbto during goal-directed care implies preserved hypoxic cerebral vasodilation and diffusion-limited oxygen delivery. future work will incorporate electroencephalographic and metabolic injury markers. sage- is a proprietary formulation of the endogenous neurosteroid allopregnanolone, being studied as a potential adjunctive therapy for the treatment of super-refractory status epilepticus (srse) . srse refers to a condition of persistent seizures that have failed treatment with first-, second-and third-line treatments. in preclinical models, prolonged seizures reduce the surface expression of synaptic gabaa receptors, exacerbating neuronal excitability and limiting target sites for gabaergic treatments (eg. benzodiazepines). here we present preclinical data describing the pharmacological properties of sage- that support its further development as a potential treatment for srse. gaba-evoked currents were examined in vitro with whole cell patch clamp recordings in cells expressing -pilocarpine model of refractory status epilepticus (pouliot ) was used to examine in vivo anticonvulsant activity. sage- or pentobarbital was administered intravenously minutes after the onset of pilocarpine-induced seizures, a time point when benzodiazepines are ineffective in animal models (pouliot ). sage- potentiated both synaptic-vitro, with ec s of nm and nm, respectively. the concentration-gabaa receptors by sage- was , -fold more potent than that observed with pentobarbital alone. when sages were also observed in the rat model of rse. when sub-active doses of sage- and pentobarbital were combined, electrographic seizure activity was significantly reduced. in vitro, sage- potently modulated both synaptic-type and extrasynaptic-type gabaa receptors, and the maximal potentiation at these receptors was further augmented by the co-application of pentobarbital. this enhanced in vitro potency and maximal effect at gabaa receptors provides further support for the development of sage- as a potential treatment for srse. vasospasm has long been considered the primary mechanism underlying delayed cerebral ischemia (dci) in subarachnoid hemorrhage (sah), but increasing evidence shows that other processes such as cortical spreading depressions and inflammation. we propose that abnormal neural activity in the form of epileptiform abnormalities, we term ictal-interictal continuum abnormalities (iicas), may contribute to dci. these abnormalities may increase metabolic demands in injured brain tissue, thereby contributing to metabolic crisis and secondary neuronal injury. here, we investigate whether the presence of iicas predict dci development. we analyzed eeg reports from icu patients with moderate-severe non-traumatic sah. continuous eeg data was recorded with daily review to identify electrographic seizures and interictal patterns. we tallied daily seizures, sporadic epileptiform discharges, lateralized or generalized periodic discharges (lpds and gpds), and lateralized or generalized rhythmic delta activity (lrda and grda). delayed cerebral ischemic events were also marked. cumulative distribution curves and iica-to-dci time plots were calculated. iicas are more prevalent in patients who develop dci, especially when they begin several days after the onset of sah. all iica types except generalized rhythmic delta activity occur more commonly in patients who develop dci. in particular, iicas that begin later in hospitalization correlate with increased risk of dci (lrda day , ed day , lpds day , gpds day ) most iicas also precede the onset of dci. we next trend features of discharges to identify those most closely associated with dci and will present our preliminary findings. iicas represent a new marker for identifying early patients at increased risk for dci. moreover, iicas might contribute mechanistically to dci and therefore represent a new potential target for intervention to prevent secondary cerebral injury following sah. up to % of patients resuscitated from cardiac arrest remain in a coma, and the ability to predict longterm neurologic recovery in these patients is limited. quantitative analysis of electroencephalography (qeeg) is objective and may facilitate outcome prediction. consecutive patients with hypoxic-ischemic coma were enrolled. continuous eeg was obtained on all patients. eeg was post-processed and analyzed by fourier transform. spectral analysis was conducted on artifact-free contiguous -minute eeg epochs from each hour. whole band ( -- hz) --suppression ratio were computed as quantitative metrics of eeg for the entire eeg recording, and then statistically compared during the last hours of eeg. sedation, level of arousal, and body temperature were also analyzed. good outcome (good neurologic outcome, gno) was defined as consciousness recovery at any point in the acute hospitalization. ten subjects were included in the study, with ceeg durations ranging from - hours of recording. the mean age was . years ( - ). there were significant differences in alpha power ( . ( . - . ) vs . ( . - . ), median (iqr), p< . , gno vs poor neurologic outcome [pno] ), delta power ( . ( . - . ) vs . ( . - . ), median (iqr), p= . , gno vs pno), burst suppression ratio ( . ( . - . ) vs . ( . - . ), median (iqr), p= . , gno vs pno), and multiple measures of variability between gno and pno patients. quantitative spectral analysis of continuous eeg may be predictive of consciousness recovery in patients with hypoxic-ischemic coma. higher alpha power, lower burst suppression ratio, and higher variability were all correlated with good outcome. because the media plays an important role in educating the public and impacting public perception on medical topics, we sought to evaluate whether mainstream media provides education or misinformation to the public about brain death through review of articles on two recent highly publicized brain death cases: ) the jahi mcmath case, in which a teenage girl was declared brain dead and her family refused to allow organ support to be discontinued; and ) the marlise muñoz case, in which a pregnant woman was declared brain dead and the hospital refused to terminate organ support until they were ordered to do so by a judge. media websites of using the search terms, "jahi mcmath" and "marlise muñoz." each article was evaluated to determine whether it contained ) teaching points, or ) misinformation, defined as misleading, incomplete, or incorrect information. we reviewed unique articles. the subject was referred to as being "alive" or on "life support" in % ( ) of the articles, % ( ) of which also described the subject as being brain dead. a definition of brain death was provided in % ( ) of the articles. only % ( ) of the articles noted that organ support should be discontinued after brain death declaration unless a family has agreed to organ donation. reference was made to wellarticles and % ( ) of these implied both patients were in the same clinical state. mainstream media provides poor education to the public on brain death. because public understanding of brain death impacts organ and tissue donation, it is important for physicians, organ procurement organizations, and transplant coordinators to improve public education on this topic. neurocrit care ( ) :s -s post-operative hemorrhage: a possible predictor of delirium in brain tumor patients post-operative delirium after brain tumor resection is frequent, difficult to manage, and may increase chart review of patients admitted to the neurologic intensive care unit (nicu) after brain tumor resection. we also evaluated the effect of agitated delirium on length of stay. medical records of nicu admissions form - were reviewed to identify cases and controls. cases were defined as patients with no pre-existing neuropsychiatric history who experienced significant agitated delirium post-operatively, defined by requirement for treatment with neuroleptics (quetiapine or dexmedetomidine) < hours after surgical resection. we compared these patients to a control group comprised of randomly selected patients admitted after brain tumor resection who did not experience agitated delirium. in a multifactors: sex, age, tumor location, pathology, postpost-operative hemorrhage, use of steroids and prophylactic anti-epileptics, particularly levetiracetam. there were cases and controls. multivariate analysis revealed male sex (o.r . ; % ci . - . ; p= . ) and a post-operative course complicated by hemorrhage within the resection cavity (o.r . ; % ci . - . ; p< . ) as significant predictors of agitated delirium. the icu length of stay was significantly longer in those with agitated delirium ( . ± . days vs. . ± . days; p< . ). neurointensivists caring for post-operative patients with brain tumor may consider resection site ctor for developing agitated delirium. future studies may investigate -operative bleeding and delirium and the long term outcome of these patients. cardiac arrest is a leading cause of death and disability, and predicting outcome in these patients is a challenge. optic nerve sheath diameter (onsd) on brain ct correlates closely with intracranial pressure. therefore in this study we studied correlation between onsd measured on the initial brain ct in patients after cardiac arrest and outcome. this is a retrospective study of patients with cardiac arrest admitted to the medical intensive care unit at our hospital between and . pati arrest were included. demographics, neurological status on arrival and day and outcomes were collected. onsd on brain ct was measured bilaterally mm behind the optic nerve head and averaged for each patient. a total of patients were included. mean age was ± . years, patients ( %) were male. patients ( %) suffered out-of-hospital cardiac arrest. mean glasgow coma scale (gcs) on admission was . ± . (range - ; median ). return of spontaneous circulation (rosc) time was . ± . intervention. patients ( . %) underwent therapeutic hypothermia. patients ( . %) had seizures. average modified ( . %) had a good outcome (mrs - ). average duration from rosc to ct was . ± . days. mean onsd in patients with gcs - at day was . ± . mm, while in those with gcs - at day , onsd was . ± . mm (p= . ). mean onsd in patients with mrs - at discharge was . ± . mm, while in those with mrs - was . ± . (p= . ). there does not appear to be a significant correlation between the onsd o cardiac arrest and outcome at day . traumatic brain injury (tbi) is a major public health problem. while the association between subarachnoid hemorrhage and systolic dysfunction (sd) has been established, the effect of tbi on the incidence of early sd in previously healthy patients following moderate-severe tbi, and ) to describe the longitudinal change in we conducted a prospective cohort study among mild and moderate-severe tbi patients admitted to a level trauma center with these inclusion criteria: ) age < years, ) no severe non-tbi injuries, ) no prior cardiac disease, and ) minimal comorbidities. transthoracic echocardiograms were performed at < hours, - days, and - days following tbi. systolic function was assessed using fractional shortening (fs), and sd was defined as fs< %. descriptive statistics were used to compare the mild and moderate-severe tbi groups. multivariable linear regression was used to compare fractional shortening between groups. patients were studied ( mild tbi and moderate-severe tbi). both groups were young ( . years mild tbi and . years moderate-severe tbi) and mostly male ( % mild tbi and % moderatesevere tbi). early sd was present in ( %) moderate-severe tbi patients and ( %) mild tbi patients (p< . ). on multivariable regression, moderate-severe tbi was associated with an absolute . % reduced fs compared to mild tbi ( % ci . % - . %, p= . ). all patients with early sd recovered to normal systolic function by - days injury ( figure ). sd is common early after moderatehospitalization. tbi severity is independently associated with worse systolic function. early echocardiography is a safe, applicable, and feasible procedure following tbi and may aid clinicians with hemodynamic management post tbi. sophie samuel. department of pharmacy. memorial hermann medical center, houston, tx, usa. paroxysmal sympathetic hyperactivity (psh) is a neurological condition that occurs most frequently after traumatic brain injury (tbi). sympathetic hyperactivity can manifest as increased heart rate, blood pressure, respiratory rate, temperature, sweating and posturing activity. in , a consensus statement and probability of diagnosis. the objective of this study is to report the incidence, frequency and severity of psh symptoms in the acute setting after tbi using the new diagnostic criteria. this was a retrospective study from july -august . included were all adult patients admitted to a tertiary care intensive care unit with a primary diagnosis of tbi and a length of stay longer than days. symptoms were recorded. the most frequent symptom was tachycardia ( %), followed by tachypnea ( %), hypertension ( %), symptoms occurring simultaneously and % had symptom - ) vs ( - ); p= . ] and at day ; [ ( - ) vs ( - ); p< . .] medications often used to control symptoms included, anti-pyretics, opioids, beta--- ) vs ( - ); p= . ], but no difference in mortality. symptoms of sympathetic hyperactivity were seen commonly after tbi in the acute setting. using a admitted with a hospital length of stay greater than days. hypertonic saline (hts) appears to be more effective than mannitol in reducing raised intracranial pressure (icp) after severe traumatic brain injury (tbi). in this study we investigated which agent had superior combined effects on icp and cerebral perfusion pressure (cpp). the brain trauma foundation tbi-patients who received only hts were identified and matched with patients who received mannitol only ( : and : match). the two groups were matched for age, pupillary reactivity, glasgow coma scale (gcs), ct abnormality, craniotomy and occurrence of hypotension on day . univariate analysis was performed to compare combined average and duration of icp> mmhg (icphigh) and cpp< mmhg a total of patients with severe tbi, who received only hts were identified and matched with ( : ) and ( : ) patients who received mannitol only. in the : group hts patient was excluded, as there was no corresponding match in mannitol group. the mean age, gcs, incidence of abnormal pupils, hypotension, abnormal ct, craniotomy and day of icp insertion were similar in the groups. there was no difference in number of days of icp monitoring (p= . , . ; : , : groups). osmolar doses were comparable; all patients in hts group received % hts except one who received . %. in : match, number of days with cpplow ( . ± . vs. . ± . , p= . ) was significantly lower in the hts group. in p= . ) were significantly lower in the hts group. these results were reproduced in the : analyses. hts is superior in its combined effect on icp and cpp after severe tbi when compared to mannitol. statins constitute a class of medications commonly used in the treatment of elevated cholesterol. however, in experimental studies statins also have other non-cholesterol mediated mechanisms of action, which may have neuroprotective effects. the purpose of this study was to determine if administration of atorvastatin for days after injury would improve neurological recovery in patients with mild traumatic brain injury (mtbi). the hypothesis was that atorvastatin administration would reduce post-concussion symptoms and also -injury would be safe. patients with mtbi were placebo for days starting within hours of injury. assessments of post-concussion syndrome, postthe rivermead post-concussion symptoms questionnaire at months was the primary outcome. enrollment in the trial was stopped early because of difficulty in recruiting sufficient numbers of subjects. patients with mtbi were enrolled; patients received atorvastatin and received placebo. the mean rivermead score was . for the atorvastatin group compared to . for the placebo group at months post-injury [f( , ) = . , p=. )]. the change in the rivermead score between baseline and months was also analyzed. the mean change in score was a decrease of . for the atorvastatin group and . for the placebo group [f( , )=. , p=. ]. no serious adverse events occurred, and there was no significant difference in the incidence of adverse events in the two treatment groups. atorvastatin administration for days post-injury was safe, but there were no significant differences in neurological recovery after mtbi with atorvastatin. association between comorbidities, nutritional status, and anticlotting drugs and neurological outcomes in geriatric patients with traumatic brain injury an essential part of the management of traumatic brain injury (tbi) is the mitigation of secondary insults to the brain such as sustained increases in intracranial pressure (icp). it would be beneficial to be able to predict increased icp so as to facilitate safe transport of patients. given the role of neuro-inflammation in increases in icp. after admission. receiver operating characteristic (roc) curves were used to compare the predictive elevation of icp above or mmhg for min or more in the following hours. serum samples from patients were matched to subsequent hour periods of monitoring.. ni-vs the predictive capacity of a combined model of ni-vs and il level over ni-vs alone in predicting icp elevation to > mmhg ( . vs . , p mmhg ( . vs . p < . ). levels when combined with physiological data. even without invasive monitoring, predictions about measurements. head injury neuroworsening (nw) after traumatic brain injury (tbi) is a major cause of added morbidity, however, there is no reliable way to predict nw. we hypothesized that autonomic nervous system dysfunction (ans) measured by analysis of plethysmograph variability (ppgv) in the first hour after presentation may predict nw in the initial hours after tbi. and head abbreviated injury score(ais)> . patients with systemic trauma were excluded. nw was defined as any of the following occurring in the first hours: new asymmetric pupillary dilatation (> mm), point gcs decline, interval worsening of ct scan as assessed by the marshall score, or requirement for neurosurgical intervention. the beat-to-beat variation of the ppg, and ppg morphologic features were calculated to quantify the ans impact on the physiological status. multivariate stepwise logistic regression was used to develop predictive models of nw. there were patients (mean age years old, gcs , iss , % women) who met criteria between december and may . nw occurred in ( %) patients. ppgv analysis at (ppg ) and (ppg ) minutes post-admission demonstrated predictive capability for nw(p< . ). ppgv was able to better discriminate nw as compared to a baseline model of age, sex, initial vs (roc . v. . , p= . ). ppgv better discriminated future nw as compared to the model of age, sex, admission vs and gcs (roc . v . ,p= . ), and marginally better than a model combining admission vs, gcs, and marshall score on ct(roc . v . ,p= . ). ans dysfunction assessed by continuous ppg waveform analysis in the first hour represents a nonclinical factors to more accurately predict nw, potentially leading to automated algorithms for earlier therapeutic interventions. tanzania severe traumatic brain injury (tbi) is the number one cause of death and disability among young adults worldwide. formulation and subsequent adherence to the brain trauma foundation (btf) guidelines has been associated with reduced mortality after severe tbi. in this study we studied epidemiology and treatment of severe tbi at a tertiary referral hospital in tanzania in reference to the btf guidelines. patients with tbi hospitalized at bugando medical centre, a tanzanian tertiary referral hospital were recorded in a prospective registry. demographics, cause of trauma, clinical characteristics, hospital care, and mortality were recorded for days and on the day of discharge. between september and october , of patient . %) were hospit pressure (icp) monitoring were not performed for any patient. thirty-eigh - pre-hospital and routine icu care, ct imaging, blood pressure and icp monitoring are underutilized or unavailable in the management of severe tbi in the tertiary referral hospital setting. tbi associated mortality is significantly higher than that in high-income countries. improving outcomes after severe tbi will require concerted investment in pre-hospital care as well as improvement in availability of neuroimaging, icu resources and expertise in multidisciplinary care. establishment of comprehensive traum elevated intracranial pressure (icp) is thought to mediate secondary brain injury by decreasing cerebral perfusion pressure (cpp) and reducing cerebral blood flow. clinical trials targeting icp thresholds have not demonstrated benefit. we hypothesized that stratifying elevations in icp based on their effect on cpp would be feasible with the use of continuous, time-resolute neuromonitoring data. we studied a convenience sample of five patients undergoing intracranial neuromonitoring after severe traumatic brain injury per our institutional protocol. patients had a parenchymal icp monitor time-synced with continuous arterial blood pressure. waveform data were recorded into moberg cns monitors, and second-bywere identified and their duration was measured along with cpp. elevations were stratified into those that exhibited a reciprocal decrease in cpp (icp[neg] ) and those with stable or elevated cpp (icp [preserved] ). the mean patient age was ; three were male. a total of individual elevations in intracranial pressure were observed over a monitoring duration of , minutes. we found icp elevations that reciprocally decreased cpp (icpneg) were identifiable using bedside neuromonitoring devices, and that these elevations resulted in a cpp drop of nearly mmhg, despite a similar maximum icp despite a shorter duration. the majority of these icp elevations occurred in patients with poor outcome, and may represent a target for aggressive icp lowering therapy. mild traumatic brain injury (mtbi) is defined as an initial glasgow coma scale (gcs) - . current recommendations include a follow-up computed tomography (ct) scan of the head prior to discharge. often, imaging and neurological exam remains stable, questioning the role of routine repeat imaging. a retrospective chart review was completed on tbi patients evaluated at a level trauma center between august and december . inclusion criteria included: initial gcs - , blunt head injury, and available repeat imaging. exclusion criteria included gcs < , penetrating trauma, those that required immediate surgery, or those without repeat imaging. a total of patients were included in the analysis. statistics were done with mann-u whitney or chi-square testing. age was . ± years. there were males and females. the most common mechanism was falls ( . %), followed by motor-vehicle collision ( . %), motor-cycle accident ( . %), assault ( . %), pedestrian--related ( . %). polytrauma occurred in . %, better, . % were stable, . % were worse. only patients ( . %) exhibited neuro-exam changes, where patients received repeat imaging which ultimately demonstrated stable findings. ultimately, only one patient required a neurosurgical procedure, an external ventricular drain, due to significant decline. age, gender, mechan neuro-exam changes. on the other hand, neuro-exam changes significantly correlated with ct changes (p = . ). repeat imaging tend to show worse findings when associated with neurological changes. on the other hand, results for repeat imaging were variable without neurological changes and generally did not alter repeat imaging is only warranted for neurological changes that may necessitate a neurosurgical procedure. create a meaningful tool, we explored family members' and physicians' perspectives on prognosis communication during goals-of-care discussions for citbi patients employing mixed-methods. we conducted semi-structured interviews with citbi surrogate decision-- trauma centers, and attending physicians representing geographic (northeast,mid-atlantic,south,west,midwest] and subspecialty diversity (neurocritical care,neurosurgery,trauma,palliative care). two independent reviewers analyzed transcribed interviews using deductive and inductive approaches (nvivo-software). the sample size was determined by theme saturation. prognosticated outcomes expressed as percentages, and % preferred prognosis in a "more direct" manner. surrogates favored percentages because they were "more clear, more concise, and less confusing". in contrast, % of physicians stated that they do not use precise percentages when discussing prognosis in citbi due to distrust in the predictive accuracy of existing data: "better have damn good data to do that with, and most often, we do not." physicians also voiced concern over families' judgment…they become simplified and [these numbers are] used against you later." the dissimilar preferences for the use of percentages and numbers during prognostication represent an important difference between surrogates and physicians. these findings have a direct impact on the design of a goals-of-care sdm tool for citbi. a future goals-of-care decision aid will require iterative decompressive craniectomy (dc) is not a new procedure, however, it has gained momentum in recent years, in the management of refractory intracranial hypertension. however, the timing of dc has not been -based guidelines for the optimal timing of dc has resulted in a wide variability in practice patterns. in most instances, dc has been performed based on neurosurgical evaluation of the patient with or without intracranial pressure (icp) monitoring and evidence of increased shown variable outcomes in emergency (within hours of injury) neurosurgical procedure in traumatic brain injury (tbi). the purpose of this study was to evaluate the impact of emergency dc on in-hospital mortality following blunt tbi. craniectomies performed on patients with a blunt mechanism of head injury within twenty-four hours of admission, were included in the study. in-hospital mortality was the main outcome of interest. patients qualified for the study. patients ( %) underwent a craniectomy within hours (emergency group)and patients ( %) had craniectomy performed between > hours hours to hours following hospital arrival (late group). propensity matched analysis identified pairs of patients in both groups. the mean standardized differences were less than % after matching. there were no significant differences in mortality [odd ratio . , ci ( . [ . , % ci (- . , . ), p= . ] and length of stay between the groups [hazard ratio, . , % ci ( . , . ], p= . ]. no difference was seen on in-hospital mortality between patients operated within hours versus patients operated between and hours of admission. spreading depolarizations (sds) are pathological waves of neuronal depolarization that occur in % of patients with traumatic brain injury (tbi) who require surgical treatment of focal lesions. the incidence of sds in non-surgical tbi i (deeg) placed at bedside via burrhole. we hypothesized that the incidence of sds recorded using deeg in non-surgical patients would be similar to that documented in surgical tbi. -penetrating tbi who did not require urgent neurosurgery on admission. all patients underwent bedside burrhole placement of intracranial pressure, tissue oxygen, regional blood flow and deeg monitors via a single quad-lumen bolt per institutional protocol. data were recorded on moberg cns with dc-coupled amplifiers. over a -- ; % male; gcs range - ) underwent monitoring for a mea - . hours, beginning . hours (median, quartiles: . -( %) monitoring devices were placed in nonpatients had focal pathology in the monitored lobe, w died, including those with sds. there were no significant hematomas or infections related to invasive neuromonitoring. the incidence of sds detected with deeg placed in non-dominant frontal lobe was lower than previously reported with injury-targeted placement of subdural strips in surgical tbi patients. this may be due to targeting to injured periinnate incidence of sd in patients with non-surgical or more diffuse injuries. targeted placement of subdural electrodes through burr holes may be warranted in non-surgical tbi patients. university of utah general surgery, salt lake city, ut, usa. traumatic brain injury (tbi) is a prevalent condition that is responsible for a significant amount of disability and healthcare expenditures. clinicians can and do use the impact prognostic calculations to inform o examine self-reported individual and institutional use of the impact prognostic calculations in an effort to identify trends and effects on treatment. we conducted an international and multidisciplinary survey examining self-reported awareness and use of the impact prognostic calculator. factors associated with awareness and use of the calculator including provider specialty, years in practice, personal and institutional volume of tbi patients treated, and institutional trauma level were als voluntary and anonymous survey in an email. study data was collected and managed using redcap. respondents ( . %) were aware of the calculator, only . % ( ) said that they often, and . % ( ) said they sometimes used it. volume of tbi cases and specialty both were positively associated with awareness and use of the calculator. providers often or sometimes used calculator, . % ( ) stated that it had some influence on their care for the patients. . % used the information to better of care and % used it to provide more aggressive care. of those aware, still only slightly more than half ( %) used it. the use of the impact calculator was mainly to better communicate with patient family, but a portion, . % and % of providers, said it influenced their care in other ways. these results provide direction to increase awareness and use of the impact prognostic calculations. the brain trauma foundation guidelines suggest individualizing cerebral perfusion pressure (cpp) goals based on tissue oxygenation (pbto ), pressure reactivity (index; prx), and metabolism (lactate-pyruvate ratio; lpr). our objectives were to investigate practices pertaining to bedside hemodynamic and neuromonitoring in tbi patients, and to analyze differences among "neurointensivists" (nis; defined as clinical electronic survey of items including a tbi case-scenario; endorsed by sccm ( , recipients) and esicm (on-line newsletter) in . chi-square test was used to compare proportions of responses between nis and ois with a significance p< . . there were responders ( % completion rate); ( %) were classified as ois and ( %) as nis. use of neuromonitoring-derived variables to optimize cpp in patients with severe tbi, for the entire cohort: pbto ( %), transcranial doppler(tcd) ( %), jugular venous bulb ( %), ct perfusion(ctp) ( %), prx ( %), and lpr ( %). nis use more pbto ( % vs. %, p= . ) and ctp ( . % vs. . %, p= . ). more nis have a hemodynamic protocol ( . % vs. . %, p= . ) for tbi, use more arterial waveform analysis ( % vs. %, p= . ), and bedside ultrasound ( % vs. . %, p= . ), while more ois monitor mixed venous oxygen saturation ( . % vs. %, p= . ). in the case scenario of raised icp, low pbto , and preserved pressure autoregula (vasopressor use . % nis vs. % ois, p= . ). "neurointensivists" employ more hemodynamic and neuromonitoring to patients with tbi. intracranial pressure and cpp remain cornerstones of management, however the use of other physiologic variables -specific pp goals. the predominant experience of penetrating traumatic brain injury (ptbi) derives from battlefield settings, but the civilian experience in western settings in patients treated after is limited to only small and single-center studies. as a result, outcome predictors of civilian ptbi in modern trauma and neurocritical care settings are poorly defined. the aim of this study was to identify predictors associated with survival in a contemporary, large, diverse two-center ptbi cohort, and to develop a parsimonious survival prediction score for civilian ptbi. our cohort comprised ptbi patients retrospectively identified from the local trauma registries at two u.s. level- trauma centers, of which one was predominantly urban and the other predominantly rural. predictors of in-hospital and -month survival identified in univariate and multivariable logistic regression were used to develop the simple surviving penetrating injury to the brain (spin) score. at hospital discharge and -months post ptbi was . %. motor glasgow coma sub-score, pupillary reactivity, self-inflicted injury, transfer from other hospital, female sex, injury severity score and inr were independently associated with survival (all p< . ; area-under-the-curve . ). important radiological factors associated with survival were also identified but their addition to the full multivariable would have resulted in model overfitting without much gain in the area-under-the-curve. we developed the spin score, a logistic regressionafter ptbi. while external validation is warranted, this clinical survival prediction tool may provide important information to guide families and physicians during intervention-and goals-of-care decision- real-time visualization of the cumulative pressure and time dose of intracranial pressure in individual traumatic brain injured patients. the 'dose' of intracranial hypertension, a summary measure of duration and intensity of elevated intracranial pressure (icp) episodes, is associated with worse outcome in traumatic brain injury ( retrospective analysis of minute-by-minute icp monitoring data from a large multicenter database of tbi population-based color-coded plots by güiza et al, where 'bad' icp episodes are red, and 'good' icp episodes are blue. the icp insult currently experienced by the patient, together with his icp episodes of the previous hours, and the cumulative icp burden since icu admission, are shown. when playing these minute-by-minute snapshots consecutively, an animation is created showing the current and cumulative burden of icp of the patient. we present the clinical course of patients, with good outcome (gos ), and who died (gos ). clinical trials aiming at aggressively treating icp below a fixed threshold of mmhg have given thresholds, could present a new way to define secondary injury by icp, and a future target for therapy. the proposed method visualizes the current and cumulative time and pressure burden of icp for individual patients, which could help a neuro-intensivist in identifying when a patient is currently in a state of potentially harmful elevated icp, or when his outcome is at a turning point. acute blood pressure variation and mortality in severe traumatic brain injury we retrospectively evaluate temperations ( hematomas) from april to march operated by one young neurosurgeon. during the operation, we made sure to put the burr hole the highest, not to drainage tube into the right position. we scaled the amount of hematoma by hand free roi using head ct before operation and also the next day to evaluate the improvement rate. the recurrence rate was . % ( cases). the average amount of air contaminated was . ml and the improvement rate was . %, both of which didn't relate to the recurrence. no relevance among the recurrence rate, the amount of air contaminated and the hematoma improvement rate could be found. though the amount of air contaminated during temperation doesn't relate to the recurrence rate, it was useful to scale the hematoma improvement rate and the amount of air in order to objectively assess the d recurrence became less, which suggest that we need to investigate the operation for chronic sundial hematoma further after we operate more. the aim of this prospective observational study was to evaluate if trans-cranial doppler (tcd) ultrasonography can be used as an inference tool of cerebral hypoxic episodes in patients with moderate to severe traumatic brain injury. recruited patients had serial tcd studies to assess blood flow velocity of the middle cerebral artery (mca). measurements were done on bilateral mcas as soon as logistically possible after the insertion of pbto monitoring, once a day for a total of days, and during dynamic challenge tests when feasible. multiple physiologic parameters were registered concomitantly with each tcd measurement, with a particular focus on determinants of pbto and potential confounding factors. we studied consecutive patients with a total of tcd studies, of which ( %) were performed - . h) after tbi. when considering all readings, we found no correlation between pbto and mca's readings > h. for value level, icp, and cpp. to correlate with brain tissue hypoxia and could be use as a screening tool to help minimise timesensitive secondary injury during that period. otherwise, vmean is not correlated to pbto . a precise assessment of brain condition after severe traumatic brain injury (tbi) is crucial to reduce secondary injuries and sequelae. multimodal neuromonitoring permits to assess multiple systemic and brain parameters, but these data are complex to interpret continuously, especially in the overwhelmed environment of intensive care unit (icu). computerized decision support systems (cdss) can assist the clinicians in optimizing care. this study aims to evaluate an algorithm for classifying the cerebral condition, as a first step in the development of a cdss. the study was approved by the local research ethics committee. patients with severe tbi (glasgow coma score < ) with a monitoring of intracranial pressure and brain tissue oxygenation pressure were eligible. data were extracted from the existing icu electronical medical records (semi solutions médicales). an incremental learning fuzzy minparameters online was implemented. the different cerebral status categories included: control condition, ntracranial hypertension. previously validated and published datasets were used to train the system. the system was then tested with the patients' data and compared to a classification made by two clinical experts. eight -hour recording periods from adults with severe tbi were analyzed. the pathophysiological status was appropriately classified by the cdss in (median) % (interquartile: - %) of time. every critical event was detected, but brief misclassifications were frequently observed during the transition periods. in this preliminary cohort of patients with severe tbi, the cdss was able to adequately classify the brain condition in a large proportion of time, but some errors occurred during brief transitional periods. further training of the cdss with a larger dataset may improve the system accuracy, which should be tested in a larger patient population. mild traumatic brain injury (tbi) is a commonly seen pathology at trauma centers. neurosurgical consultation is a routine practice; however, the vast majority do not require surgical intervention or invasive monitoring during the entirety of their hospital stay. in certain trauma centers, neurologycentered neurocritical care solely evaluate and manage mild tbi. we provide a retrospective analysis of this practice at our level trauma center. a retrospective chart review was completed on tbi patients evaluated at a level trauma center between september and december . inclusion criteria included; initial gcs - , blunt head injury, available repeat imaging, and management by neurology-centered neuro-intensivists. exclusion criteria included gcs < , penetrating trauma, those that needed immediate surgery, those with neurosurgical consultation, and those without available imaging. a total of patients were included in the final analysis. age was . ± years. there were males and females. the most common mechanism was falls ( . %), followed by motor-vehicle collision ( . %), motor--related ( . %), assault ( . %), and pedestrian- . % were stable, . % were worse. only patients ( . %) exhibited neuro-exam changes, where patients received repeat imaging which ultimately demonstrated stable findings. no patients required a neurosurgical procedure. average hospital stay was . ± . days. neurology-centered neuro-intensivists can manage mild tbi appropriately without official neurosurgical consultation. this practice can streamline tbi management and potentially reduce hospital costs. bulic, natasha n. renda, may m. kim-tenser, gene g. sung, benjamin b. emanuel. usc, los angeles, ca, usa. measurements of optic nerve sheath diameter (onsd) using bedside ultrasound (us) have been shown to correlate with clinical and radiologic signs and symptoms of increased intracranial pressure (icp). eleven patients ( males, females) with traumatic brain injury (tbi) and gcs< were evaluated. all patients had extraventricular drainage (evd) monitors, right and left. three patients had right decompressive hemicraniectomy, had left hemicraniectomy, had suboccipital craniectomy and did not have decompression. a total of examinations were obtained with invasive icp measurements, pulsatility indices (p balance were recorded. twenty-nine ocular uss were performed on individual patients. in ons assessments, bilateral onsd was . mm, while icp was mmhg, however, later developed icp mmhg within hours. another patient had bilateral ons . mm, while icp was mmhg, however, later developed icp of > mmhg within hours. two patients had bilateral ons measurement> . mm with corresponding icp > mmhg all patients with onsd mmhg had ons > . mm. although, patients with ons > . mm and icp mmhg within the next h. there was no correlation between pi on tcd, thus tcd was not useful in this dataset. there was no correlation between increased temperature or elevated blood pressure with icp> mmhg. this small sample size suggests that onsd may predict future icp elevations, however, a larger sample size is needed to confirm these results. the precise threshold differentiating normal and elevated intracranial pressure (icp) is variable among individuals. in the context of several pathophysiologic conditions, elevated icp leads to abnormalities in global cerebral functioning and impacts the function of cranial nerves (cns), either or both which may contribute to ocular dysmotility. the purpose of this study is to assess the impact of elevated icp on eye watching a -second continuously playing video moving around the perimeter of a viewing monitor. pupil position was recorded at hz and metrics associated with each eye individually and both eyes together were calculated. linear regression with generalized estimating equations was performed to test performed at icp levels ranging from - to mm hg in twenty-three patients (twelve female, eleven male, mean age . years) on fiftycorrelating with cranial nerve function linearly decreased with increasing icp (p-value mm hg was . . intracranial pressure. increasingly elevated icp was associated with increasingly abnormal eye tr physiologic impact of elevated intracranial pressure. this represents a new non-invasive automatable means for assessing the physiologic impact of elevated icp. use of shared decision--of-care decisions in critically-ill traumatic brain injury (citbi) offers the hope to decrease variation and bias in goals-of-care discussions. sdm guidelines demand the inclusion of an evidenceand acceptance of the "international-mission-for-prognosis-and-analysis-of-clinical-trials-in-tbi"(impact)-model by physicians holding such discussions with citbi families. we conducted a mixed-methods study with semi-structured interviews in attending physicians representing geographic (northeast,mid-atlantic,south,west,midwest) and subspecialty diversity (neurocritical care, neurosurgery, trauma, palliative care). we explored methods of prognosis derivation and communication, citbi outcome model use, and, specifically, awareness and perceived utility of the impact-model. we analyzed transcripts in nvivo-software with the investigator-triangulated-inductive--approach. theme saturation determined the final sample size. overall, % of physicians use the impact-model, % were not aware of it, and % don't rely on any tbi outcome models. positive impact-model views included: "helpful in getting an idea where your confidence should be"; "to ground physicians a little bit"; "reduces the variability of prognosis that a large some participants "do not thin only"; "those calculators are about populations; they're not about individuals, use of those calculators for this purpose is a perversion of the original construct". we identified substantial physician variability in the awareness of, use, and attitude toward the impactmodel, which crucially informs the development and successful implementation of future goals-of-care sdm tools in citbi. the analyses of acute subdural hematoma using acute subdural hematoma (asdh) is associated with cerebral contusion and laceration of bridging veins following a head injury, however a few cases of asdh without head injury had been reported. the purpose of this study was to detect the difference of traumatic asdh and non-traumatic one. cases of asdh hospitalized at our institute from march to march were retrospectively reviewed. traumatic groups were patients ( . %), and non-traumatic groups were patients ( . %). the results were statistically analyzed by logistic regression to use the various factors: age, gender, glasgow coma scale (gcs) score, the presence of light reflection, danti--coagulation agents, neurological outcome and so on. neurological outcome was evaluated using glasgow outcome scale, and it was classified into two groups: the good prognosis group (gr, md) and the poor prognosis group (sd, vs, d). traumatic groups were mean age . ± . years, and were comprised of males and females. non-traumatic groups were mean age . ± . years, and were comprised of male and females. there were significant difference in patients` characteristics, the presence of light reflection, d-dimer and neurological outcome (p < . ). non-we will be described the detail of them and collect further cases in the future. traumatic brain injuries (tbi) are of significant importance due to increased morbidity and mortality. we retrospectively analysed tbis to assess clinical profile and factors predicting in-hospital mortality. electronic database at a private, urban tertiary care centre was screened ( to ) to include all tbi -hospital mortality outcome were assessed. mean age of population was . ± . years with . % being males. most injuries ( %) resulted from road traffic accidents (rtas). bleeding from either ear, nose or throat (ent) was most common presentation ( . %) followed by vomiting ( . %) and convulsions ( . %). on presentation, severe brain d subdural ( . %) haematoma was most common followed by sub-arachnoid ( . %), extradural ( . %) and intracerebral ( . %). threatened airway was observed only in . % cases. bony trauma ( %) followed by face ( . %) ,chest ( . %), and spine injuries ( . %) were associated injuries. in-hospital in-hospital mo . , . ; p< . ), tachycardia (or . , % ci . , . ; p= . ) and with development of hyponatremia (or . , % ci . , . ; p< . ) or fever (or . , % ci . , . ; p= . ) during hospitalization. ventilator support was necessary in ( %) cases out of which ( %) died. hospital stay (days) did not vary significantly in survivors and non-survivors ( . ± . vs . ± . , p= . ). development of hyponatremia or fever and requirement of assisted ventilation were associated with -hospital mortality. casey we established a team that included attending physicians, fellows, advanced practice providers (app), specific guidelines. we surveyed staff regarding team communication and discussion of qsis during rounds. we designed a dgt that defined team member roles, structured communication patterns, and prompted standard discussion of qsis. following implementation, we evaluated team compliance with the dgt, as well as rates of catheter-associated urinary tract infections (cauti) and ventilatorassociated pneumonias (vap). % respiratory therapists). for most qsis, a minority of staff reported that these were always addressed during morning rounds (% staff indicating qsi "always" addressed: % pressure ulcers, % code status, % cam icu, % mobility goal, % central line catheter removal, % urinary catheter removal, % dvt prophylaxis plan, % ventilator weaning). shared understanding of daily goals between nurses and physicians was reported by % of staff, with a significant difference between nurse %), p = . . dgt audits spanning patient days demonstrated median compliance greater than % for discussion of all qsis. there was an % reduction in cautis ( % ci %, %), p = . , and a trend towards reduction in vap that was not significant. team discussion of qsis can be enhanced by dgts. by promoting adherence to evidence-based best practice, dgts may reduce hospital-associated infections. follow-up is ongoing to determine the impact of dgts on clinical outcomes and team communication. catheter-associated urinary tract infection (cauti) is the most common health-care associated infection accounting for > , nosocomial infections annually (gould, ) . according to the center for disease control, cautis are also a leading cause of secondary blood stream infection resulting in development related to cognitive, motor, and sensory deficits. neuroicu's goal was to eliminate cautis, defined as a rate of zero. with initial efforts ( ( catheter days (titsworth et al, . in february, neuroicu launched a patient-centered quality improvement effort to further reduce cautis. the neuroicu interdisciplinary comprehensive unit safety program (cusp) performed an extensive literature review of evidence based best practices specific to urinary catheter management. a preexisting, staff nurse driven urinary catheter management protocol was revised to better fit the needs of the neurocritical care population, including assessment of and interventions for acute and chronic scan assessments to every hrs, revision of urinary d-ofconducted daily rounds to evaluate the necessity and management of indwelling urinary catheters. after three months of implementation, compliance with use of the neuroicu's urinary catheter management algorithm (ucma) was greater than %, urinary catheter utilization was reduced from % to %, and cauti rates were reduced to zero. implementing a neurocritical care patient-centered, interdisciplinary approach to urinary catheter management significantly impacted urinary catheter utilization, cauti rates, and unit culture. dedicated neurointensivists have previously been shown to improve various outcome measurements in patient and family satisfaction. the purpose of this study is to evaluate the impact of newly appointed neurointensivists on quality outcome measures in a nsicu. this is an observational cohort study of adult patients (> years) in a -bed nsicu at an academic, tertiary care center evaluating quality outcome measures pre-and post-neurointensivists. outcome measurements include catheter associated urinary tract infection (cauti), central line blood stream infection (clabsi), ventilator associated pneumonia (vap), patient acuity, mortality, and length of stay (los). patient satisfaction questionnaires from discharged patients were compared to historical controls. tentiveness of doctors; ) recommend the hospital to others. statistics include -sample binomial and n- chi-squared (categorical) and t-test (continuous). for questionnaire data, considered significant. total patient days occurred pre-and days post-neurointensivist coverage. patient acuity decreased . % (p= . ). cauti ( %, p= . ), clabsi ( %, p= . ), central line days ( . %, p< . ), ventilator days ( . %, p= . ), and vap ( %, p= . ) also decreased. these saved the hospital an estimated $ , based on health services advisory group data. questionnaires were returned. patient satisf on physicians' attentiveness (p= . ). patients recommending the hospital to others increased % (p= . ). dedicated neurointensivists positively impact quality outcome metrics, particularly significantly improving patient satisfaction. future studies should evaluate the direct impact of neurointensivists on medicare reimbursement from improved patient satisfaction. bertan hallacoglu, tanmayi t. oruganti, chandran c. seshagiri. research & development, boston, ma, usa. cephalogics has developed a wearable diffuse optical tomography (dot) imaging device to help clinicians monitor perfusion and oxygenation from multiple brain regions on the bedside in disease states the system to changes in cerebral tissue oxygenation (scto ) induced by hyperventilation in a pig and human subjects. dot sensor was positioned on the pig's head along the sagittal line, and the second sensor was positioned on the hind leg muscle for monitoring systemic tissue oxygenation (ssto ). dot measurements were performed continuously during baseline ( mins, paco = mmhg), transient hypercapnia ( mins, paco = mmhg), and recovery ( min, paco = mmhg) periods controlled by the respiratory rate of the ventilator. dot data were recorded to a laptop for off-line analysis. sto -sd) were computed for comparison of results across measurements. the approach was also investigated in three human volunteers, who were instructed to hyperventilate during dot recordings. -- %), consistent with reports of reduced cerebral blood flow during hypocapnia. in contrast, ssto estimates - %), indicating elevated systemic perfusion. both parameters fully recovered to baseline values during the recovery period. scto response to hyperventilation in human volunteers were consistent with the results in pig. hypercapnia induced reduction in scto was noninvasively imaged in human subjects and a pig despite the large scalp-cortex distance in pig. the results of this study demonstrate the sensitivity of the cephalogics' dot system to scto values and its ability to separate scto from systemic perfusion. brittany doyle, michael m. rogers, daiwai d. olson, venkatesh v. aiyagari. ut southwestern, depts. of neurological surgery, neurology and neurotherapeutic, dallas, usa. multidisciplinary rounds play a pivotal role in optimizing care in the neurosciences critical care unit (nccu). care providers were frustrated with inconsistency and the need for manual data entry to conduct multidisciplinary rounds. the purpose of this project was to develop an integrated computerized form that incorporated "smart" features within epictm. the nccu nursing council collaborated with the physicians to design a standardized system-based approach to multidisciplinary rounds, and the elements that would be addressed within each system. input was collated from nursing, neurology, neurosurgery, pharmacy, and critical care into a paper version which was beta tested before the epic tool was officially rolled out. the rounds template is now a perpetually editable note with fields that include drop-down menus, copy--populate with up-to-date data (e.g., icp values, lab values). while there was a learning curve to the use and un -led rounds became much more efficient, comprehensive, and less frustrating for the entire healthcare team. within four months, nursing management saw the benefit of such a standardized tool for clear communication and the tool also became the standard for nurse-to-nurse handover at change of shift. efficient and orderly presentation of information during multidisciplinary rounds is greatly assisted by the use of a standardized electronic tool. having all of the current, relevant data available in a single location has greatly improved the quality of nurse-led neurocritical care rounds. this tool can be replicated and customized to the needs and patient populations of other units and hospitals. the degree of burst suppression on continuous critical care eeg (cceeg) monitoring is used to guide dosing of intravenous anesthetic drugs (ivads) in the treatment of refractory elevated intracranial pressure (icp) and refractory status epilepticus (se). however, medication titration is performed only as frequently as cceeg review (potentially as little as - times a day). quantitative eeg (qeeg) may provide a continuous, objective assessment of the level of burst suppression that would allow for more precise and rapid titration of ivads. compared software-generated qeeg burst suppression ratio (qeeg bsr) with three raw eeg variables as determined by two board-certified neurophysiologists (r and r ): bursts per minute (bpm), total burst duration per minute (bd) and average inter-burst interval (ibi) for one minute segments per patient. a total of eeg segments were analyzed. agreement between readers was very high for the three raw eeg variables: bpm, bd and ibi (correlation coefficient . , . , and . , respectively). the best correlation was observed between bd and qeeg bsr (- . for both r and r ). the correlation between bpm and qeeg bsr (- . r and - . r ) and ibi and qeeg bsr ( . r and . r ) was not as strong. left and right hemispheric qeeg bsr did not differ statistically from the generalized qeeg bsr (p= . and . , respectively) despite the presence of focal intracranial pathology. the depth of therapeutic burst suppression can be accurately assessed by generalized qeeg bsr. although cceeg bursts per minute is the most commonly used cceeg metric of burst suppression, it shows a lesser correlation with qeeg bsr than total burst duration per minute. there is little operational data on optimal neuro-icu physician staffing. this prospective study evaluated the introduction of night-time in-hospital neurocritical care fellows (ncfs) in an urban academic hospital. the goal was to determine if the new staffing model enhances patient care and provider and patient satisfaction. irb approval was obtained. the new staffing model was rolled out on - - . providers (ncfs; neurology residents and attendings; neurosurgery residents, physician assistants, advanced practice nurses and attendings; neuro-icu nurses) were surveyed. a pre-roll out survey of providers' perceptions of the existing model (night-time ncf coverage from home with on-call in-hospital neurology residents and neurosurgery physician assistants or residents, not dedicated to the neuro-icu) was administered prior to new model roll-out. two follow-up surveys, a separate night-shift nurses survey, and patient (or surrogate) surveys were administered between - - and - - . surveys were electronic and responses were voluntary and anonymous except for the night-shift nurses survey which was paper, required, and anonymous. response rates were % (pre-roll out), % (first follow-up), and % (second follow-up). % of providers indicated pre-roll out that night-time in-hospital ncfs would be beneficial; % (first follow-up) and % (second follow-up) indicated satisfaction with the new staffing model. major reasons for satisfaction included: physician response to emergencies, physician -nurse communication, and patient outcome. the reason for dissatisfaction was decreased educational opportunity for residents. % of night-shift nurses reported preference for the new model. % of patients (or surrogates) agreed to participate: % reported satisfaction with the care they received in the icu; dissatisfaction was not more than % of providers were satisfied with a new staffing model featuring night-time in-hospital ncfs. additional studies are necessary to determine optimal neuro-icu physician staffing with increasing patient and treatment complexity and decreasing resources. therapeutic plasma exchange (tpe) is a first-line therapy for guillain-barre syndrome, myasthenia gravis, chronic inflammatory demyelinating polyneuropathy and polyneuropathy associated with paraproteinemias. tpe is also a second-line therapy for neuromyelitis optica, chronic focal encephalitis, and acute multiple sclerosis. the therapy is also used in autoimmune encephalopathies, refractory status care service has provided a neurointensivist run tpe program using membrane-based technology with the gambro® prismaflex system. a benefit of a neurointensivist managed membrane -based service is lower direct costs compared to centrifugal-based therapies. since a consultation to another service (often available during business hours) is avoided, delays in therapy can be reduced (potentially further decreasing costs) and the therapy is also available for emerging indications such as refractory status epilepticus. this review is of the initial patients treated with tpe in our neurocritical care unit between april and may . all patients were treated with % albumin with a targeted exchange of . to . plasma volumes. overall dosing, timing, indications as well complication were reviewed. indications for therapy included guillain-barre syndrome, myasthenia gravis, autoimmune encephalitis, refractory status epilepticus, autoimmune vasculitis, and parainfectious transverse myelitis. all patients received a minimum prescription of . plasma volumes and had the therapy started sooner than historical controls. no changes in nurse staffing were required for the therapies and we report no bleeding, infectious or access related complications. mild coagulopathy was seen in most patients after previously not available. a neuro-intensivist lead tpe program is feasible and safe. the therapy can be delivered more timely and can be offered for a wider variety of indications. a cost analysis of the program is now underway. specialty neurocritical care improves hospital length of stay and mortality in patients with critical neurologic illness. however, clinical practices are often informed by studies focused on mortality or gross functional outcome. both staff and family members face uncertainty about neurological outcome, which performing detailed followup for all patients admitted to the university of cincinnati neurosciences intensive care unit (nsicu). all patients admitted during a twotrained outcome scale-extended (gose), cognitive outcome using the teleph and quality-of-life using the euro-qol. we assessed the duration of each follow-up call in order to determine feasibility. n= patients were admitted to the nsicu; mean age was ; % were male. overall follow-up was obtained in %; % of survivors were contacted. disposition from nsicu included: home ( %), acute rehab ( %), long-term care ( %), hospice ( %). in-hospital mortality was %. median gose (iqr) was ( - ) and median mrs (iqr) was ( -- . an average of : (mm:ss) was required for each assessment; comprehensive assessments required : . overall, a total time of approximately hours was required for patients. our pilot study demonstrated feasibility of following patients admitted to the nsicu. these findings have the potential to guide in-hospital care and out-of-hospital resources when used as a quality improvement metric, and to provide valuable information for retrospective research. our neurocritical care unit restructuring provided an opportunity to revise our staffing model. the prior model ratio of : resulted in at least one nurse experiencing a : ratio when high acuity patients -specific data to support a higher staffing. prospective observational study of nursing time using the -item therapeutic intervention scoring system (tiss- ) and manual timing of discrete nursing interventions including off-unit transport. baseline data was obtained to include measures of nursing experience. measures of central tendency and regression analysis was performed using sas v . . the average time to complete a neurologic assessment was . minutes; the average time to chart a neurologic assessment was . minutes. for time spent off the unit the average time spent traveling to ct was < minutes, average time spent in mri was minutes twice a day, average time in ir was . minutes. we found that nursing experience was not associated with patient acuity, duration of assessment, nor time spent doc ents. assessment time was associated with documentation time. moreover, higher acuity was a predictor of both assessment time and documentation time. the data were used to support an increased acuity model with nurses having planned : ratio. neurologic critical are patients require more hands on nursing care and time spent in diagnostic testing than an average critical care patient. neurocritical care units staffing ratios should be adjusted to provide time to ensure nursing care is complete. melissa panter, sonja s. stutzman, daiwai d. olson, venkatesh v. aiyagari. utsouthwestern/neurocritical care, dallas, usa. venous access is a basic yet critical component of care. determining which venous access devices (vad) to use depends on the type, duration, and frequency of infusion. prolonged continuous infusion of drugs or fluids, benefit from midline peripheral catheter (midline) or a peripherally inserted central catheters (picc). midlines are safe and effective but use is declining in favor of piccs, which have similar insertion costs and added benefits, such as the delivery of toxic drugs harmful to peripheral veins. however, an infection associated with a picc is classified as a central line associated blood stream infection which has important financial implications for the hospital. therefore, it is important that the type of vad be specifically tailored based on -physician collaborative study to design and test a decision support tool to assist clinicians in determining the appropriateness of the piccs vs midlines for patients. this study has three phases. first, a retrospective chart review of vad decisionexamine the current degree of appropriateness for each type of vad for each specific patient scenario. next, we will design a decision support tool to help decide which vad should be used. the tool will be developed based on expert clinician, pharmacist review and a review of the literature. finally, we will their patients. the first phase of the study is ongoing and we will present the results of the retrospective part of the study and the decision support tool at the annual neurocritical care society meeting. a decision support tool to guide clinicians choosing between different types of vad will help improve current clinical practice and patient outcomes. clinical trials in neurocritical care require a predictable set of baseline, monitoring, treatment, and clinical outcomes data. however, interoperability standards restrict automated real-time streaming of this data, resulting in inefficiencies performing clinical trials, preventing real-time clinical trial oversight and constraining collaborative research. we investigated available data systems and developed a conceptual e. examining current traumatic brain injury interventional trials, we considered technical interoperability -based clinical trial oversight and collaborative analytic research. we elaborated a vendor-neutral interoperability schema for data extraction, repositories, analysis, annotation, and visualization. the proposed conceptual solution is described. nodes of data acquisition include: ) continuously streaming devices including physiologic monitors and infusion pumps; ) discrete data from highpenetrance ehr and laboratory platforms; and ) biospecimen, radiology, and clinical outcomes repositories. an application program interface performs function calls to utilize individual episodes of data. a data management system queries and manages multiple patient records for batch processing of ondemand or prefor real-time or post-hoc assessment of raw and derived parameters (e.g., percent time in target range or on-protocol compliance). we enumerate the variety of current nodes requiring interoperability interfaces, and propose an open standard to promote a highly efficient platform for n -based clinical research, featuring automated case report form data extraction, a programmable interface for oversight and early warning detection, and a platform for annotation and crowdsourcing of novel algorithms. this conceptual architecture for a modular, vendor-neutral, data collection and management system for -based clinical trials in neurocritical care and offer new functionality for real-time oversight and collaborative analytics. improving cancer is a devastating illness; with a rise in brain and spine tumors specialized care is more important that ever. with advances in technology and treatment strategy, those too ill to previously receive care reased acuity has translated to the need for higher levels of hospital care. recognizing the unique expertise required to care for this emerging population, the decision was made to merge neurology and oncology specialties within critical care. the purpose of this poster is to describe the admission criteria, patient population, utilized technology, staffing model, and patient outcomes of a newly created neuro-oncology critical care unit (nccu). in january , a bed neurocritical care unit was opened in a midwestern academic medical center. six of the beds housed within this bed unit were designated and budgeted to the oncology medical center. separate nursing staff and management teams were created to support the care within the unit while maintaining close collaboration with university hospital nccu nursing teams. while nursing teams are distinct, nurse practitioners, neurointensivist, and other multidisciplinary team members are shared between both services. to evaluate the benefit of patient diagnosis, care needs, nccu length of stay (los), number of ventilator acquired pneumonias (vaps), urinary tract infections (utis), and central line acquired bloodstream infections (clabsis). at the time of this submission, specific results are still being tabulated. merging access to academic research trials and oncologic and neurointensive specialists, this has created an environment that promotes care reflective of the most up to date evidence based practice. through the utilization of creative staffing and focused onboarding, this unit has been able to treat this subspecialized population holistically utilizing a multidisciplinary approach to minimize hospital acquired complications and los. in , the neurocritical care society (ncs) published a consensus statement in support of multimodal neuromonitoring, emphasizing the essential need for systems to integrate data in meaningful ways to t barrier to the integration of data from multiple group on neurocritical care informatics was established in ; it includes experts from the clinical and research side of neurocritical care and representatives from medical device manufacturers. through an open meeting and continued discussion, a recommendation document "medical device connectivity" was produced. its objective is to provide guidance to medical device manufacturers desiring to design a communication protocol that allows external systems to acquire data from their devices. our research on this project uncovered a high percentage of errors in medical device protocols as well as undocumented characteristics and safety issues. the group developed recommendations for providing robust communications as well as address usability and safety concerns. the document outlines the content that should be transmitted which includes device identification, protocol version identification, patient identifier, events, alarm conditions, system status, data labels, and units. finally, it must be thoroughly documented and validated by the manufacturer. multimodal monitoring (which requires medical device connectivity) reportedly improves quality of care through reduction of errors and increased detection of adverse events. however, widely adopted connectivity are on the horizon. in the meantime, the document created in this project will provide guidance for manufacturers in their communications protocol development. in doing so, they will then further the ncs's recommendations on device connectivity for multimodal monitoring. warfarin-related intracranial hemorrhage (ich) is associated with increased mortality due to higher rates of hematoma expansion. current guidelines recommend rapid anticoagulation reversal using intravenous vitamin k and prothrombin complex concentrate (pcc). previous studies show high prevalence of incomplete anticoagulation reversal with -factor pcc. we therefore sought to assess the impact of pcc type in reversing warfarin in ich patients. this was a retrospective study of ich patients ( traumatic and non-traumatic) with warfarinassociated coagulopathy who were admitted to a level ii trauma center between january and september and received at least one dose of -factor or -factor pcc. post-pcc inr of £ . was considered successful inr reversal. multivariable model using logistic regression was performed to assess the impact of pcc type on successful inr reversal after adjusting for age, sex, bmi, and baseline inr. overall, the prevalence of successful inr reversal was . %. there was a higher proportion of patients with successful inr reversal in those who received -factor pcc than -factor pcc ( . % vs. . %, respectively, p= . ). in the multivariable model, -factor pcc (or . ; % ci: . to . ) and baseline inr (or per unit of inr . ; % ci: . - . ) were independent predictors of successful inr reversal. the change in inr post-pcc was significantly greater in those who received -factor pcc than -factor pcc ( . ± . vs. . ± . respectively, p< . ). -factor pcc more reliably reversed warfarin in ich patients compared to -factor pcc. cortical spreading depolarizations (csds) as highly active metabolic event commonly occur in patients with intracerebral hemorrhage (ich) and may contribute to secondary brain injury. fever is an independent predictor for unfavorable outcome after ich and may trigger csds. here, we investigated the dynamics of brain-temperature (tbrain) relative to csds and core-temperature (tcore). twenty comatose patients with ich and multimodal electrocorticograpy (ecog) monitoring were prospectively enrolled. a subdural ecog strip was placed adjacent to the evacuated ich. a combined intracranial pressure (icp) and tbrain probe was inserted in the white matter ipsilateral to the ich. monitoring data were averaged to -minute-means for longitudinal analysis and to one-hour-means. -burden was defined as % of temperature > . ° c per -hours. data were analyzed using gee-models and are presented as median and interquartile range (iqr). during hours ( hours [ -csds occurred in clusters. baseline tcore and tbrain were . °c ( . - . ) and . °c ( . - . ), respectively. tbrain but not tcore significantly increased minutes preceding the csds by a median of . °c ( . -- . ]; p< . ) but not tcore (p= . ) was higher during clusters compared to episodes of single csds. csds probability was highes or= . per %; tcore: p< . ; or= . per %) independent of map and icp. csds were triggered during episodes of fever. our data suggest an association between csds and cerebral heat production, especially during clusters. integration of ecog monitoring in trials investigating prophylactic normothermia after ich may help to understand the potential beneficial effect of this intervention. anticoagulation reversal is recommended for patients with intracerebral hemorrhage (ich) on vitamin k antagonists. we propose a window for reversal of vitamin k antagonists, in which ich volume remains below the average growth in a control population on follow up imaging a retrospective review of neuro icu patients at henry ford from - was conducted on patients with the icd code for diagnosis of ich. inclusion criteria: brought to ed at onset of symptoms, either not on anticoagulation or were on warfarin with therapeutic inr, and had received pcc administration. fifty eight total patients were identified. patients were approximately matched for gcs on admission, ich w - cc difference in volume estimation. forty-seven control patients were identified: mean age ( - ), gcs on admission ( - ), sbp , ich volume on admission . cc, ich volume on repeat scan . cc, change in ich volume . %, and mean time between initial and stability scans was minutes. eleven patients on vkas were identified who underwent reversal with -factor pcc. mean age ( - ), gcs on admission ( - ), sbp , ich volume on admission . cc, ich volume on repeat scan . cc, and time between scans was minutes. average inr on presentation was . . all patients on warfarin patients who had pcc administered before minutes ( ) had a mean change in ich volume of . %. patients who had reversal completed after the minutes ( ) had a mean change in ich volume of . %. (p value= . ). we propose a potential "recommended reversal time" of less than minutes for vitamin k antagonists in our institution. limitations of study include small sample size. the full outline of unresponsiveness (four score) is a validated scale that provides the essentials of a coma examination by incorporating motor response, eye opening and eye movements, brainstem reflexes, and respiratory pattern. we incorporated the four score into the existing ich score and consecutive patients admitted to our institution from - with spontaneous ich were reviewed. using patient age, hemorrhage location, hemorrhage volume, evidence of intraventricular extension and gcs, the ich score was calculated. the four score was then incorporated into the ich score as a substitution for the gcs (ich-four score). the ability of the two scores to predict mortality at month was then compared. in total, patients met inclusion criteria. the median age was years (iqr - ) and ( . %) were male. overall mortality at one month was . % (n= ). the area under the roc curve was . ( % ci . - . ) for the ich score, and . ( % ci . - . ) for the ich-four score. for ich scores of , , and , one-month mortality was . %, . %, . % and . %. in the ich-four score model, mortality was . %, . %, . % and . % for scores of , , and , respectively. the ich score and the ich-four score predict -month mortality with comparable accuracy. as the four score provides additional clinical information regarding patient status, it may be a reasonable substitute for the gcs into the ich score. depressive symptoms in patients with intracerebral hemorrhage (ich) are common and are associated with worse outcomes. it is not well described how often depressive symptoms are appropriately ascertained and treated in a multicenter cohort, and whether this is a potential target for improving outcomes. we retrieved diagnostic codes from four university health systems across chicago (multicenter cohort). separately, we prospectively screened for depressive symptoms (nih patient reported outcomes measurement information system, promis, t score ), in patients at one prospectively assessed cohort center at one, three and twelve months after ich onset. we compared detection rates of depressive symptoms between the two samples. diagnostic codes for depressive symptoms up to three months after ich onset were recorded in of ( . %) of the multicenter cohort versus of ( . %) in the prospectively screened cohort (or . , % ci . - . , p < . ). results were similar considering depressive symptoms up to months after ich, of patients in the multicenter ( . %) versus of prospective patients ( . %; or . , % ci . - . , p < . ). in the multicenter cohort less than % of patients months of ich onset. the prevalence of depressive symptoms in survivors of ich is more common than would be suggested treated and ssris may be an under-utilized therapeutic option. vitamin k antagonist (vka)are associated with higher mortality than primary ich. prompt reversal of international normalized ratio (inr) with prothrombin complex concentrate (pcc) may promote hemostasis and decrease hematoma impact of an electronic order set designed to standardize and facilitate more timely reversal of coagulopathy in vka-associated ich. we identified all adults that received pcc for vka-associated ich from june to march at ucsf medical center, which included a period before and after an electronic order set became available in . we abstracted baseline demographics and clinical data from electronic medical records. the primary outcome was time from radiographic identification of ich to administration of pcc. secondary outcomes included pcc dosing accuracy based on actual weight and baseline inr as well as time from pcc order to follow-up inr. we identified patients that received pcc for vka-associated ich, including patients before and patients after the order set became available. baseline demographics and clinical features were similar. order set use was associated with a significant decrease in the time from identification of ich on imaging to the administration of pcc (median vs. minutes; p= . ), more accurate doses delivered ( . % vs . %; p< . ), and a shorter time from the pcc order to follow-up inr (median vs minutes, -hospital mortality were similar in the two groups. an electronic order set for administering pcc for vka-associated ich was associated with significantly faster time to pcc administration and increased accuracy in dose administered. andrew naidech, alan a. long, kathryn k. muldoon, rajbeer r. sangha. northwestern medicine, chicago, il, usa. crucial to identify patients. both active contrast extravasation ("spot sign") and lower platelet activity have been associated with hematoma growth. we tested the hypothesis that patients with a spot sign had lower platelet activity. we prospectively identified patients with acute ich, measured platelet activity on admission, routinely obtained ct angiography and graded the presence or absence of a spot sign. we limited the analysis to patients who underwent ct angiography within hours of ich symptom onset. platelet activity was measured with the verifynow-asa (accumetrics, ca). non-normally distributed data were compared with the mann-whitney test, and binary variables with chi-squared or logistic regression. - . years, % were women and % had a history of hypertension. sixteen ( . %) had a positive spot sign. a spot sign was associated with lower platelet activity ( [ - ] vs. [ - ] aru, p= . , where <= aru indicates an aspirin effect). of patients with a spot sign, had platelet activity <= aru. platelet activity <= aru was associated with increased odds of a spot sign (or . , % ci . - . , p= . ). the presence of a spot sign on ct angiography was associated with lower platelet activity, suggesting a hematoma growth. introduction: clot dissolution is a biochemical process catalyzed by enzymatic proteins, requiring a specific temperature range for optimal function. clear iii, a randomized, double-blinded, placebocontrolled trial tested whether extraventricular drainage (evd) plus intraventricular alteplase improved outcome by removing ivh compared to evd plus saline. methods: retrospective assessment of prospectively collected temperature data q h over first days post randomization. blinded assessment o functional outcome (mrs - ). results: median (interquartile range) daily temperature was . ( . , . )°c and did not differ between - (vs. at days [ . ( . , . ) vs. . ( . , . ), p . °c was significantly associated with faster ivh clot lysis rate (spearmans rho . ; p= . ), but despite significantly higher temperatures, patients with mrs - (vs. mrs - ) had significantly lower average percentage ivh removal at day ( . ± . %vs. . ± . %, p< . ) and ( . ± . % vs. . ± . %, p= . ). temperature functional outcome of . ( %ci: . - . ) and . ( %ci: . - . ) at and days respectively, and was an independent predictor of poor functional outcome at day , but not after adjustment for early infection and other severity predictors. associated with faster clot lysis rate, but also with poor functional outcome. negative associations with of ich patients, patients ( %) had waich and received kcentra. the inr ranges were: . - . in patients ( %), . - . in ( %), . - . in ( %) and > . in patients ( %). within minutes of -minute repeat inr ranges were . - . and . - . , respectively. ffp was administered to ( %), ( %), ( %) and ( %) patient in each group for a persistently elevated inr. at h post-pcc, inr reversal occurred in %, % (rest had inr . ), %, and %, respectively. at h and one had dic following pcc administration. in this small case series of waich patients, inr correction with kcentra was adequate except for those with inr . - prevalence of stroke following craniotomy or craniectomy for spontaneously intracranial hemorrhage intensive care resources, particularly, neuroscience intensive care resources are limited and costly. in most institutions in the country, all intracerebral hemorrhage (ich) patients are admitted to the neuroscience intensive care unit. we sought to identify what criteria will allow us to determine which primary intracerebral hemorrhage patients will not need admission to an intensive care unit (icu). we studied retrospectively patients with primary ich from january to the end of dec . we reviewed multiple admitting characteristics: demographics, hematoma volume, location of hemorrhage, any brain compression, blood pressure, respiratory status, inr, glasgow coma score (gcs). the reasons for neuroscience intensive care unit admission requirements are the need for mechanical ventilation, hydrocephalus, increased intracranial pressure, low gcs score, hematoma expansion, or the unit, and who did not require any icu care intervention. this group had the following characteristics: supratentorial ich, ich volume . we called this criteria the "non-admission criteria for patients with primary intracerebral hemorrhage". patients were identified as primary ich. patients ( . %) fulfilled the non-admission criteria to the neuro-intensive care unit. of patients patients ( . %) discharged home, patients to acute rehab facility ( . %), patients discharged to snf ( . %), patient died ( . %); elderly patient with existing dnr comfort care orders. none of the patients had to be readmitted to neuroscience intensive care unit, and none required neurosurgical procedure. we propose that ich patients that fulfill the non-admission criteria do not have to be admitted to an icu and can safely be monitored in a step down unit this represented about % of ich patients at our tertiary academic medical center. future prospective studies are required to validate the criteria. joseph r. blunck, justin j. shewmaker. saint lukes hospital / pharmacy, kansas city, mo, usa. current guidelines recommend the use of -factor prothrombin complex concentrate (pcc) rather than fresh frozen plasma for reversal of warfarin-related intracranial hemorrhage (wrich). there is no consensus regarding an optimal pcc dosing strategy, but limited data suggest that pcc dosing should be based on weight and international normalized ratio (inr). in november of , our health system implemented a wrich reversal protocol with a -factor pcc dosing nomogram that utilized fixed-dose options of , above or below . the purpose of this study is to evaluate the effectiveness and safety of this simplified -factor pcc dosing protocol. patients given -factor pcc for wrich reversal in our health system were retrospectively identified with billing codes. chart review was completed to evaluate the primary endpoint of achieving a post treatment evidence of a venous thromboembolic event, time to goal -hospital mortality. we identified wrich patients from november through april that were reversed with our simplified -factor pcc dosing protocol. seventy-seven ( . %) patients achieved a post reversal goal -six ( . %) patients received a pcc dose equivalent to rounded pi dosing and the mean (sd) dose difference between groups was ( ) units vs. ( ) units, protocol vs. pi dosing, respectively. one patient ( . %) had a thromboembolic event that occurred days post reversal. a simplified -factor pcc dosing protocol is a safe and effective strategy for wrich reversal. -hospital, year and year mortality, and has been influences functional outcomes. we sought to evaluate the association between cci and outcomes in patients with primary intracerebral hemorrhage (ich). patients admitted to our center with primary-ich from - were included. demographic and clinical data were collected. the primary outcome measures were the proportion of patients with discharge mrs (dmrs) of - , death and poor discharge disposition (any disposition other than home or inpatient-rehabilitation). crude and adjusted logistic regression were used to evaluate the association between cci and outcomes. patients were identified. there were ( . %) patients with a cci of or , ( . %) patients with a cci of - , and ( . %) with a cci of or greater. while the continuous cci was not significantly associated with a dmrs of - (or . , % ci . - . , p= . ), it was associated with disposition. the odds of poor disposition increased % with each increase in cci (or . , % ci . - . , p= . ). the odds of death increases % with each point increase in cci (or . , % ci . - . , p= . ). after adjusting for baseline ich score, cci remains significantly associated with poor disposition (or . , % ci . - . , p= . ), however the association between cci and death was not statistically significant (or . , % ci . - . , p= . ). in contrast to previous studies, cci was not associated with poor short-term functional outcome or inhospital mortality in ich patients after adjusting for ich score. however, it was significantly associated with poor discharge disposition. this suggests that cumulative comorbidities only predict disposition in ich, because the ich score strongly impacts poor functional outcome and in-hospital mortality. accurate assessment of renal function remains a unique challenge in patients with intracerebral hemorrhage (ich). mathematical estimates of creatinine clearance (crcl) routinely used are often inaccurate in this setting. subsets of critically ill patients have been shown to exhibit a hyperdynamic response leading to an enhanced renal clearance. no studies exist evaluating the directly measured creatinine clearance of patients with ich. this was a single-center prospective observational study of adult patients with ich admitted to the nsicu between january and july . eight-hour urinary creatinine clearances were performed daily to directly measure crcl until the patient no longer had a foley catheter or the patient left the nsicu. urinary -gault equation. statistical significance was defined as p-value < . . thirty patients with ich were enrolled in the study. the study sample was % male with a mean age of ± . years. the median admission ich score was (iqr - ) with a mean ich volume of ± . ml. the median admission gcs was . (iqr - ) and median admission sofa score was . (iqr -additionally, the mean urinary crcl was significantly higher than the estimated crcl each individual study patients with ich consistently experienced urinary crcl greater than estimated crcl predicted based on -gault equation. as renally eliminated medications are routinely dosed based on mathematical estimates of renal function, further study is needed to optimize medication regimens in this patient population to prevent underexposure. the cognitive reserve hypothesis suggests that variations in patient pre-morbid status such as education, occupation and brain morphology influence outcome. this has been extensively validated in patients with alzheimer's disease. an important component of cognitive reserve is brain morphology, which can be quantified with measures such as whole brain-or gray matter volume. this study examines use of novel measures of brain morphology to measure cognitive reserve in patients with sdh. patients with ct-documented sdh were screened. we identified patients who had a clinically indicated post-morbid mri, telephone interview for cognitive status (tics) and barthel index (bi) at discharge and follow-up. mri was used to measure several volumes such as whole brain-, gray matter-, left vs right caudate-, hippocampal and intraventricular csf volume using a freesurfer pipeline. primary outcome measures were tics at and bi at months results: -up. regional - ) (sd), ---- at discharge and - . at follow-up ( -- . ) at mo. we found a positive correlation between intraventricular csf volume and bi at mo (r = . , p= . ). there was also a significant correlation between left caudate volume and bi at months. brain morphology did not correlate with tics outcome at months. quantitative imaging can be used to predict functional outcomes in patients with intracranial hemorrhages. as we continue enrollment we hope to generate meaningful no and brain tumors. christian hernandez, vivien v. lee, bichun b. ouyang, torrey t. birch. rush university medical center/department of neurological sciences, chicago, il, usa. it remains unclear which patients with intracerebral hemorrhage (ich) benefit from surgical hematoma evacuation, and the patient factors associated with the decision to pursue surgical intervention are largely early surgical intervention for ich and to investigate if an age bias exists at our institution. one hundred and twenty consecutive patients who were admitted to the neurosciences intensive care unit with primary ich between april and january were retrospectively reviewed. multivariate regression analysis was used to analyze if select patient factors were associated with the decision to pursue early surgical intervention. in total patients were analyzed; patients ( %) were female (mean age . ± . ), patients ( %) had supratentorial ich, and patients ( %) underwent hematoma evacuation. in univariate analysis age, race, and gender were not significantly a intervention. the only factors associated with hematoma evacuation were gcs score and ich volume. after controlling for significant variables, multivariate analysis showed that the only factor associated with surgical intervention was ich volume (or . , p= . ) . surgical intervention did not affect discharge disposition (p= . ), but was associated with a longer length of stay ( vs days, p= . ). in this analysis, ich volume was the only predictor of hematoma evacuation in patients with primary ich. age and sex did not influence patient selection for surgical intervention. surgical treatment did not affect patient disposition at discharge, but was associated with longer hospitalizations. further investigation is needed to determine which patients with ich benefit most from early hematoma evacuation. studied. we investigate factors associated with admission systolic blood pressure (sbp), including hemorrhagic transformation (ht) and discharge outcome. this is a retrospective study of consecutive ais patients presenting from april to march . demographic and clinical data were collected. admission sbp was divided into three tiers: . the primary outcome measure was in-hospital mortality. seven hundred seventy six patients were included (meanwere patients with sbp> ( . %), with sbp - ( . %), and with sbp were ry of hypertension ( . %; p . premorbid use of antihypertensives did not differ among the three groups. there were no differences in proportion of ht ( . % vs. . % vs. . %; p= . ). although patients treated with iv-tpa were evenly distributed among tiers ( . % vs. . % vs. . %), more patients with sbp had mrs - (p= . ). compared to sbp was associated with lower odds of in-hospital mortality (or . , %ci= . - . , p= . ). this remained significant after adjusting for age and nihss (or . , %ci . - . , p= . ). normal presenting sbp (< ) in patients with ais was associated with worse discharge functionaloutcome and higher in-hospital mortality. these differences may be related to other associated medical conditions such as pre-existing heart failure. further research is needed to define the ideal range to maintain sbp after ais. between april and february , patients with non-traumatic, non-aneurysmal and nonmalignant parenchymal cerebral icb were identified. the associations of nihss at presentation (nihssp), nihss at hours (nihssd ), size of icb, comorbidities, and infection with los were investigated retrospectively. the mean age for the patients was . ± . years and % were male while most patients were white ( %). the most prevalent comorbidity was hypertension ( % nihssd is a useful measure of los and should be collected for patients with icb. nihssd at upper teens present most challenges to discharge and should be the aggressive focus for discharge planners. incidence of infection and ventilator need is high in this population, adding to the challenges. although oral factor xa inhibitors (fxai), used for the prevention and treatment of venous racranial hemorrhage (ich) than warfarin in clinical studies, intracranial bleeding is still associated with high morbidity and mortality. moreover, there are no specific guidelines for managing these bleeds other than empirical institution-based hemorrhage protocols. there is a need to understand the real-world management, outcomes, and resource utilization of fxai-associated major bleeding in order to potentially improve morbidity and mortality in these patients. five us medical centers participated in a retrospective study of patients admitted to the hospital with lifethreatening bleeding on or after january while on apixaban, rivaroxaban, or low molecular weight heparin (lmwh). baseline characteristics, treatment patterns, outcomes, and resource utilization were assessed. ich patients were reviewed and are reported specifically. this interim report includes major bleed patients, including ich. the majority of ich were hypertensive [ ( %)], ( %) were on concomitant anti-platelet with fxai, ( %) were diabetic and e ich was spontaneous ( %) and trauma ( %). in the management of ich bleeding, % of patients received clotting factors (e.g. pcc), whereas % received interventions (e.g. radiological embolization). within days of discharge, ( %) died, of which occurred during the admission. for the patients who did not die, length of hospitalization was . ( . - . ) days, with only discharged patient restarting on an anticoagulant. this study provides a real-world picture of patients receiving fxais or lmwh, experiencing lifethreatening intracranial bleeds. despite efforts to restore hemostasis, mortality remains high and substantial healthcare resources are expended. this highlights the need to develop specific strategies for managemen prophylactic seizure medications are not recommended by guidelines for patients with intracerebral hemorrhage, yet are prescribed to nearly % of them. there are few data on disparities in their use. we tested the hypothesis that there are differences in the administration of seizure medication, specifically we electronically retrieved information from patients who were diagnosed with intracerebral hemorrhage from healthlnk, a multi-center electronic repository in chicago, il, from - ("multicenter cohort"). from through , we prospectively identified patients with intracerebral hemorrhage at one site ("prospective cohort"). there were , patients in the multicenter cohort from four sites. the use of levetiracetam varied with -americans (or . , % ci . - . , p< . ). in the prospective cohort (n= ), hematoma location, older age, depressed consciousness, larger hematoma volume and no alcohol abuse were -americans to receive levetiracetam (or . , % ci . - . , p= . ). african-americans were more location was independently associated with levetiracetam administration (p< . ) disparities in the use of levetiracetam and they are confounded by hematoma location, a datapoint not typically available in administrative datasets. deviation from guidelines for the use of seizure medications is common and rational, and any impact on outcomes is uncertain greater gains in late recovery for intracerebral hemorrhage patients with more debilitating initial injury. multiple intracerebral hemorrhage (ich) studies have examined differences between discharge and month outcome. however, few studies have examined late recovery specifically between -and month endpoints. the aim of this study was to identify potential factors predicting late recovery in ich patients. twenty-four patients diagnosed with primary ich at yale-new haven hospital were prospectively enrolled between july , and july , . outcomes were assessed using barthel index (bi) at discharge, months, and months. repeated-measures regression analysis was conducted using age, admission glasgow coma scale (gcs), ich volume, intraventricular extension, and ich location, to compare outcomes at discharge, months, and months. there was a significant improvement across time points (p = . ), with follow-up testing showing improvement between discharge and months (p < . ), discharge and months (p = . ), and months and months (p = . ). regression testing resulted in a significant relationship of time (p = . ), time x gcs (p = . ), time x ich volume (p < . ), time x ich location (p < . ). plots show increased late recovery (i.e., between and months) for patients with lower gcs scores, larger ich volume, and deep ich location. patients with more devastating initial ichs show greater gains in late recovery between and months. these results suggest initial disability at months may not represent overall recovery and support continual follow-up out to one year. while extensive studies have examined the outcomes of intracerebral hemorrhage (ich) patients under the age of , few studies have examined outcomes in an elderly cohort (> years). the aim of this case-control study was to determine the independent effects of age on outcome after ich. nineteen ich patients prospectively identified at yale-new haven hospital above the age of were matched against patients below age , based on ich location (lobar, deep, cerebellar, brainstem), ich volume (> cc), presence of intraventricular hemorrhage (ivh), and admission gcs ( - , - , - ). the matched groups were compared via univariate analysis to examine differences in morbidity while there was no difference in pre-ich disability (mrs > ; p > . ), at months elderly patients exhibited higher morbidity (mrs > , p = . ). despite these differences, there were no differences in overall mortality between groups at discharge or at months and no differences between barthel index at months. despite greater morbidity as defined by mrs, elderly patients with ich do not significantly differ from younger patients in mortality rates or ability to perform activities of daily living at discharge and months. these results may warrant further studies to provide more accurate prognostication after ich in elderly populations. hypertension after intracerebral hemorrhage (ich) is associated with hematoma expansion, morbidity and mortality. there are currently no recommendations to standardize the critical care approach to acute blood pressure (bp) management in ich. we performed a large retrospective cohort study to examine practice variability in bp management in acute ich. care center using local get with the guidelines data. we abstracted time-stamped clinical variables including all documented bp measurements, and medications administered, as well as hematoma location from the electronic medical record. all acute ich patients are admitted to the neuroicu and comanaged by neuro-intensivists and vascular neurologists. we used descriptive statistics to summarize overall population and treatment characteristics. - ) years, and % were female. and % other. electronic medical record data were available in subjects, of which ( %) received continuous infusion for bp control within hours of admission ( % nicardipine, % clevidipine). a systolic bp goal was charted in ( %) and ranged from to mmhg. these goals were modified in % during the same admission ( % increased, % decreased). overall, unique oral antihypertensives were administered ( % received a beta-an ace inhibitor, and % a diuretic). hypertension requiring continuous antihypertensive infusion is common after ich. there is high practice variability in bp targets and choice of antihypertensive medications. a prospective study of a systematic and protocolized approach to antihypertensive medication use in ich is necessary to determine if reducing practice variability improves outcomes. intracranial hemorrhage (ich) has long been thought to be a devastating consequence in the setting of end-stage liver disease. due to its association with abnormal coagulation, the prognosis is thought to be poor, and frequently leads to withdrawal of care. our aim with this study was to assess the true overall mortality rate of ich in end-stage liver disease and compare it to mortality of ich in the general population. all patients > years of age admitted to mayo clinic from to with a subsequent diagnosis of non-traumatic ich and end-stage liver disease were identified. patients presenting with primary epidural, subdural, intraventricular, or subarachnoid hemorrhage were excluded. using actuarial methods, day, day, and year mortality rates were calculated. patients with simultaneous diagnosis of ich and end-stage liver disease were identified. of the patients were female ( %) and patients were male ( %). the mean age at diagnosis was years. the mean systolic blood pressure in the mortality cohort was mmhg, compared to mmhg in the survival cohort. the day, day, and year mortality rates were %, %, and % respectively. ich associated mortality in end-stage liver disease does not significantly differ when compared to ich mortality in the general population. the mean systolic blood pressure at presentation did not specifically correlate with an increased incidence of ich associated mortality. these findings are important in the practice of neurocritical care, confirming that poor prognostication and expedited withdrawal of care should be reconsidered. future directions will include mortality adjusted by hemorrhage location, size, and presence of intraventricular extension. kcentra® (human prothrombin complex concentrate) is the first fda-approved non-activated -factor prothrombin complex concentrate for the urgent reversal of vitamin k antagonist agents in adults with acute major bleeding. the recommended dosing is based upon the units of factor ix, which can vary within each vial (range from vial size to reduce waste and some based upon the exact factor ix content. this variation in dosing may be associated with complications in care and has never been evaluated. underdosing of the medication can lead to suboptimal response and overdosing the medication can cause thromboembolic events. the purpose of this observational trial is to assess the current prescribing practices of kcentra® in neurocritical care unit patients across the united states and evaluate the impact on patient response and safety. additionally we plan to characterize current approaches to repeat dosing. this was a retrospective observational study of adult patients across centers who received kcentra for reversal of warfarin-related bleeding between january , and december , . descriptive statistics and tests for comparison will be utilized to evaluate differences in dosing, outcome, and the occurrence of adverse events. statistical significance will be defined as p-value < . . data collection is ongoing but the full results will be presented during the meeting. result of this study will document the real-world use of kcentra® in patients with severe life threatening bleeding and assess the impact of variations in prescribing practices on patient responses. there is a paucity of data regarding this topic and combining data from multiple neurocritical care units will be timely in identify optimal dosing strategies. perihematomal edema (phe) associated with intracerebral hemorrhage (ich) has been suggested to have an impact on both mortality and functional outcomes in spontaneous, supratentorial ich. there have been no studies examining the impact of phe in infratentorial hemorrhage. the aim of this study was to evaluate the impact of absolute phe volume as well as phe expansion rate in cerebellar hemorrhages at the time of discharge and at months. patients diagnosed with primary cerebellar ich at yale new haven hospital were prospectively enrolled between july , and july , . patients were evaluated using mrs and bi at discharge and months. ich and edema volumes on ct were measured using a semi-automated threshold based approach and phe expansion rate was the difference between initial and follow-up phe volumes divided scale (gcs), ich volume, intraventricular (ivh) volume, edema (phe), and phe expansion rate from baseline to first follow up ct scan before decompression ( h window). at discharge, patients with higher morbidity (mrs > ) exhibited higher ich volume (p = . ) and phe volume (p = . ) on admission ct and a trend for greater rates of phe expansion (p = . ). these differences were not significant at months and when adjusted for ich volume. plots suggest an association between higher phe rate and lower bi scores at months. phe may have a clinically significant impact in patients with cerebellar ich at time of discharge, but these results are limited by a small sample size. these results warrant further studies and suggest therapies to ameliorate edema may be a treatment option for cerebellar ich. yahia thrombolysis and adjunctive stent retriever thrombectomy (srt) is associated with better perfusion and outcomes. despite benefit, % to % of patients had poor outcomes. thrombectomy in ais with lao within hours is performed as secondary after iv thrombolysis, which may be associated with delay. the purpose of our study is to evaluate the safety, feasibility, recanalization rate and outcome of primary str within hours without intravenous thrombolytic in ais from lao. srt as an alternative to iv rtpa. consecutive patients who underwent primary srt for lao within patients with lao; mean age . ± . years and mean nihss ± ; chose primary srt after informed consent. near complete (tici b in ) complete (tici in ) was observed in all ( %) patients. recanalization from symptoms and groin puncture was . ± . and . ± . minutes respectively. immediate post-thrombectomy, hour and day nihss score was . ± . , . ± . and . ± . respectively. asymptomatic perfusion related hemorrhage developed in patients ( %). days outcomes; mrs %, mrs . %, and mrs . %. our study demonstrates that primary srt in ais from lao occlusion is not only safe and feasible, but associated with complete recanalization and good outcome. further study is required. currently, no reliable predictive tools are available to determine which patients with a large hemispheric infarction (lhi) will progress to cerebral herniation (ch). we sought to determine whether continuous measurements of blood pressure variance (bpv), heart rate variance (hrv), and entropy within hours of admission would enhance the ability to predict future ch in lhi patients. patients presenting within hours of onset from an internal carotid artery (ica) or middle cerebral artery and november . patients with ch were matched : by age and nihss with patients who did not have ch. shannon entropy and standard deviation were used to measure the instability of hr and between groups. a model predicting ch utilizing the admission factors of age, gender, nihss, intraarterial (ia) therapy, and thrombolysis was compared to an admission model enhanced with bpv, hrv and entropy hours after admission. data from patients were analyzed (median age years old, median nihss , % women). there were no differences in the proportion of patients with a left hemisphere syndrome ( %), undergoing ia therapy ( %) or thrombolysis ( %) between groups. ch was observed a median of days after the ictus. at hours after admission, median measures of bpv, hrv and entropy were significantly higher in the ch group (p< . ). a model of admission factors enhanced with physiologic data was better able to predict ch than a model with admission factors alone (roc: . v. . , p= . ) poster early recognition of which patients with large hemispheric infarction (lhi) will develop malignant cerebral however, the early time-course of edema has not been adequately studied. we applied volumetric in the first hours, prior to development of midline shift (mls). we identified lhi patients with scans within six hours of onset and subsequent scans early ( - while all recently published endovascular stent-retriever randomized clinical trials (rcts) were positive, their designs differed considerably particularly with regard to the extent of intravenous rt-pa use prior to thrombectomy. here, we assessed whether rt- we adapted a method previously published by us to develop a pooled outcome model relating percent utilization of rt-presenting > , subjects and a range of - % utilization of rt-pa. we correlated percent rt-pa and baseline - ) and mortality. this model includes ± p<. statistical interval surfaces to assess whether a trial's outcomes surpasses the variability of the pooled sample (neurology : - , ) . stent retriever rcts were compared against the model. the mrs model showed excellent fit: r-square= . , p< . . each stent retriever trial's outcomes exceeded mrs improvement varied dramatically according to %rt-pa, with the greatest improvement in those with % rt-pa use. when we included all case series and testing their outcomes at their baseline nihss and percent rt-pa use, shortest overall treatment times also related to improved outcomes (p=. ). mortality trends were similar in that lowest mortality was seen in those trials with highest rt-pa use and the trial with higher than expected mortality had the lowest rt-pa use. an outcome model including % iv rt-pa permitted analysis of stent-retriever therapy compared to a large sample. best outcomes were related to higher rt-pa utilization and shorter treatment times, suggesting a strong treatment interaction between modalities. based on these results, until studied prospectively, iv rt-pa, if administered rapidly, should not be bypassed prior to thrombectomy. intra-arterial mechanical thrombectomy (iamt) is currently considered the standard therapy for acute thrombectomies performed under general anesthesia (ga) may adversely affect functional outcomes. we report our experiences with iamt performed routinely under ga at the montreal neurological hospital (mnh). this is a retrospective analysis of adult patients admitted to the mnh from january to april with ais from proximal intracranial occlusions of the anterior cerebral circulation. all patients were assessed both clinically and radiologically. forty-two patients underwent iamt for ais. twenty-eight patients were included for analyses ( had procedures under conscious sedation, had missing months assessment). clinical outcomes were (mrs) outcomes (mrs --good and poor outcome groups respectively. sixty-eight percent of the good outcome group received iv tpa, as compared to % in the poor outcome group. patients in the good outcome group were also more % in the poor outcome group]. intraprocedural map drops below mmhg observed in patients in the good outcome group versus non in the poor outcome group. in our small retrospective single centre study about thrombectomy for ais under ga, our -month outcomes are comparable to larger studies where thrombectomies were done under conscious sedation. pending the results of ongoing prospective trials about the use of ga during iamt for ais, our results do not support the reservations derived from recently published retrospective data on the use of ga in this context. conservative initial management of young adults with severe hemispheric stroke in a comprehensive stroke center reduces decompressive craniectomy rates pooled european trial results of early decompressive craniectomy (dc) did not require radiographic mass effect at the time of dc. early surgery for supratentorial cerebral hemorrhage does not improve recovery or survival compared to initial conservative medical management. early vs delayed dc for hemispheric a prospective inpatient neurosurgical database from october to march was queried for neurocritical care admissions for hemispheric - under irb approval. a retrospective chart review was conducted using a structured questionnaire using the electronic medical record. we identified patients who met the inclusion criteria for the pool were managed with medical treatment only (mto) with average maximal septal shift of . mm and pineal shift of . mm. twelve patients ( %) underwent dc with average maximal septal shift of . mm and group, mto, and dc were respectively: mr - % vs % vs %; mr - % vs % vs %, and death % vs % vs %. four patients in the mto group declined dc; died and one survived with mr of . no patients developed brainstem herniation prior to referral for decompressive craniectomy. surgical complications death or survival with severe disabilities. time of recanalization since symptoms is a strong predictor of outcome in patients who underwent stent retriever thrombectomy from middle cerebral artery occlusion. hours since symptoms have not been clearly investigated especially, those with large artery occlusion (lao) and underwent stent retriever thrombectomy (srt) . objective: to identify the predictors of outcome in ais patients from middle cerebral artery (mca) occlusion with large clot burden (lcb > mm) and underwent srt, who recanalize less than hours versus more than hours since symptoms. software was used to analyze the data. ais patients who underwent srt in mca; age . ± . years and mean admission nihss ± . complete (tici ) and partial (tici b) recanalization was observed in . % and . % respectively onset was ± minutes. presenting nihss of . dropped to , and at immediate, hours and days post srt respectively. good univariate analysis, recanalization time, immediate and hours post srt nihss were predictors of outcome (p-value= . , . and . respectively). in multivariate analysis, time of recanalization since symptoms (p-value= . ) and baseline mrs (p-value= . ) continued to be the predictors of good outcome. our study demonstrates that patients with lao from mca who recanalize less than hours of symptoms onset have good chance of good outcome compared to those who recanilize more than hours. therefore, all ais patients with lao should offer early srt to achieve a good functional outcome. further studies are required. jennifer a. frontera. neurological institute, cleveland clinic, cleveland, oh, usa. prospectively collected data of heartmate ii (n= ) and heartware (n= ) lvad patients from a single blood stream infection [bsi]), specific pathogens mann-whitney u, chi--wise logistic regression analyses. of patients, lvad infection occurred in ( %) including: bsi in ( %), wound infection in infect p< . ). driveline and wound infection were not a - . , associated with bsi (aor . , %ci . - . , p= . ). there was no association with any specific infectious pathogen. precautions to mitigate i demonstrate a causal relationship. the frequency of dysphagia is greater than %. the early clinical evaluation of swallowing disorders can help define approaches and avoid oral feeding, which may be detrimental to the patient. the aim of this study was to identify predictive clinical factors associated with enteral tube feeding in acute ischemic our database were reviewed. clinical early ct score (asp association. of the patients, used enteral feeding tubes ( . %). the mean age ( . years -sd . ), mean gcs ( . -sd . ), mean nihss ( . -sd . ), and aspect score ( . -sd . ) were significantly higher in the tube group. logistic regression showed that only age (odds ratio [or], . ; % confidence interval [ci], . - . . p= , ), nihss score (or, . ; % ci, . - . , p= , ) and nihss (dysarthria) subscore (or, . ; % ci, . - . , p= , ) were independent predictors of enteral tube feeding. a - in conclusion, combining information about age, nihss, nihss subscore, may be a useful predictor kyushu university, fukuoka, japan. tissue plasminogen activator (te designed and developed an information and communication ipads, pcs, and bigdepartments before the patient's arriva number of calls is reduced. we compared the number of times that emergency room (er) nurses called for computed tomography (ct) or magnetic resonance imaging (mri) between before and after the system introduction. before this system, er nurses called for ct or mri an average . and . times, respectively; after system introduction, the average number of times decreased to . and . , respectively. therefore, this system -pa. also, it automatically records the transitions between could improve treatment times for iv tclinical trial to confirm the tool's efficacy. an important and controversial issue of peri-(est) is the management of sedation and airway. according to retrospective data the widely favored intubation and general anesthesia (ga) appears associated with worse functional outcome compared to "conscious sedation" (gs) in the non-intubated state. siesta is a prospective, monocentric, outcome assessor-blinded, : randomized, parallel-group interventional study comparing non-intubated vs. intubated patients receiving est for acute ischemic endpoint is the improvement of the national institute of health s enrolment of the intended patients has been completed. sixty of the recruited patients are female these patients, % received pre-procedural rtpa. seventy-eight patients were randomized to cs, ( %) of these had to be converted to ga during the procedure. we will present preliminary results of the study, including the primary endpoint improvement in nihss after hours and selected secondary endpoints. the aim of this study is the prospective randomized investigation of potential advantages of the non- in young chung. departments of neurology , seoul national university bundang hospital, seongnam, korea, republic of. cerebral edema during therapeutic hypothermia us hypothermia. the authors retrospectively reviewed patients with large hemispheric infarction who were treated with therapeutic hypothermia and hyperosmolar therapy from to . patients who were dead or underwent hemicraniectomy was defined as failure of therapeutic hypothermia. infarction size was measured as sum of restricted area in diffusion weighted imaging which were performed on admission. b -ct was carried out regularly after onset of therapeutic hypothermia. shift of septum pellucidum, pineal gland and choroid plexus calcification were measured in b-ct. seventeen patients were enrolled after exclusion of patients whose b-ct was inadequate to evaluate. ten patients were successfully treated with therapeutic hypothermia (group success, n= ). six patients were dead and patient had hemicraniectomy (group failure, n= ). initial infarction size between two groups was not significantly different. both septum pellucidum shift (sds) and pineal gland shift (pgs) were significantly different in groups on . ± . days after onset of therapeutic hypothermia (mean sds . vs. . mm ; mean pgs . vs. . mm). specificity and positive predictive values for the failure calcification shift was not significantly different in groups during therapeutic hypothermia. degree of progression of cerebral edema on . ± . days after onset of therapeutic hypothermia helps to excellent in predicting fatal outcome. the main limitation of this study include its retrospective singlecenter nature, which may limit generalizablility of the study. aminocaproic acid for reversal of tissue plasminogen activator (tpa) related hemorrhagic transformation in acute ischemic stroke. -thrombolytic ich. aminocaproic acid (aca) inhibits binding of plasminogen to fibrin, hence inhibiting fibrinolytic property of tpa. there is limited report a case series of aca use for reversal of post-tpa ht. we reviewed the and identified patients treated with iv tpa. patients with post-tpa ht who received iv aca were identified. data on demographics, clinical characteristics, nihss, ich score, new thrombotic events during hospitalization, and hospital and intensive care unit (icu) length of stay (los) were collected. a total of patients developed post-tpa ich, of which received aca. % of patients were male, mean age of . ± . years. patients received tpa within a mean time of ± minutes from symptom onset, pre-tpa mean nihss was . ± . . mean time for ht after tpa administration was . ± . hours, with a hematoma volume of . ± . mm . in addition to aca, % received cryoprecipitate, % platelets, and % fresh frozen plasma transfusions. % of patients had no hematoma expansion and % developed a new thrombotic event. mean hospital los was ± days and mean icu los was ± days. at the time of discharge % had an mrs of , % mrs and % mrs . in this retrospective case series % of patients had hematoma expansion despite receiving aca, while % had a new thrombotic event. further research is warranted to determine the utility of aca for the treatment of post-tpa ht. large vessel occlusion (lvo). while studies have analyzed difference in blood clot constructs, limited data is available understanding the effect of prior anti-platelet use on endovascular therapy (evt) for ias patients with lvo in the middle cerebral artery (mca). we aimed to determine if prior anti-platelet use had effect on evt procedure time, recanalization rate, and functional outcome measured by the modified a retrospective chart review was conducted of consecutive ais patients who underwent evt of lvo of mca at the university of kansas medical center from - . outcomes were measured using total procedural and fluoroscopy time, procedural recanalization score using the modified thrombolysis in cerebral infarction score (tici), time to recanalization, incidence of sich, and -month mrs. univariable and multivariable analysis were performed. . for all) were similar between antiplatelet use versus those without. in separate multivariable models (adjusting for all significant variables), antiplatelet use was not associated with tandem ica occlusion, total procedure time, fluoroscopy time, good recanalization, or -month mrs. no impact was found of prior antioutcomes in ais patients undergoing evt. these findings should be further confirmed in a larger database and prospective cohort study. the study evaluated the effect of a neurology-specific heparin infusion protocol with more frequent ptt monitoring and a narrower goal ptt range ( . x normal) on rate of hemorrhagic or thromboembolic events. this is a retrospective cohort study evaluating patients before (october -september ) and after (october -september ) implementation of a neurology-specific heparin infusion protocol. all patients > years old receiving intravenous heparin with a diagnosis of acute ischemic st evaluated for inclusion. primary outcomes are time to first therapeutic ptt and time to therapeutic ptt range. secondary outcomes include rate of intracranial hemorrhage, rate of thromboembolic events, protocol compliance, number of subtherapeutic and supratherapeutic ptt values, time to initiation of oral anticoagulation, duration of heparin infusion, and number of heparin titrations. time to therapeutic ptt range was . hours in the pre-protocol group (n= ) and . hours in the post-protocol group (n= ) (p= . ). number of ptt values per patient was . in the pre-protocol group and . in the post-protocol group, of which . % and . % were therapeutic, respectively. percentage of supratherapeutic ptt values was . % and %, respectively (p= . ). time to first ptt, time to first therapeutic ptt, and percentage of subtherapeutic ptt values were not significantly different. assessment of secondary clinical outcomes is ongoing. our neurology-specific heparin protocol resulted in a faster time to therapeutic ptt range with a higher percentage of therapeutic ptt values and fewer supratherapeutic ptt values. investigation regarding change in incidence of hemorrhagic and thromboembolic complications is ongoing. hospital moyses deutsch, in the southern city of são paulo is indicated for the use of rtpa intravenously ctive to demonstrate experience the use of intravenous retrospective study, in all cases of isch to february . protocol indicates the use of alteplase patients with inclusion criteria, the period between the onset of symptoms and hospital admission up to . hours and no contraindication to the use of thrombolytics, nihss calculated on admission and hours after thrombolysis. computed tomography (ct) on admission and after hours.evaluation required by neurological telemedicine hospital israelita albert einstein shortly after the conclusion of the tc cranio. trough has been triggered in cases. time between onset of symptoms and drug administration, patients less than minutes, patients between and minutes, patients - minutes .the average nihss at admission was , with patients showed a reduction of or more the points nihss score within the first hours. patient non-symptomatic intracranial hemorrhage and symptomatic intracranial hemorrhage and deaths during the period. all patients receiving alteplase in the recommended time interval and underwent ct cranio control. some cases were not triggered by the evaluation of neurology telemedicine. there was improvement in nihss score similar percentage observed in reference studies. the protocol implementation has been adequate excellent support of telemdicina neurology team. good profitability of time and therapeutic efficacy. the mortality that correlated with the severity of patients and the nihss admission. this data ratifies the intracerebral hemorrhage (ich) is approximately % and non-ich bleeding over %. hypofibrinogenemia occurs in approximately % of tpa-treated patients and i cryoprecipitate is often used to restore fibrinogen levels, despite limited published evidence. cryoprecipitate has several limitations, including the need for abo matching, thawing, and concerns regarding potential transmission of viral pathogens. riastap, a purified fibrinogen concentrate, is a promising alternative to cryoprecipitate for the reversal of hemorrhage post-tpa. the objective of this study was to evaluate the safety and efficacy of riastap for the treatment of post-tpa hemorrhage. a single-center retrospective observational analysis was conducted to evaluate patients who received riastap for the treatment of postmeasure was reversal of hypofibrinogenemia. hypofibrinogenemia was defined as a fibrinogen level < hospital mortality. eleven patients were included in our analysis. the average dose of riastap administered was , units. five patients had hypofibrinogenemia prior to riastap administration, with a mean fibrinogen level (iqr to ). the six patients who were not hypofibrinogenemic at baseline had minimal effect on fibrinogen levels post-- . to ). one patient was diagnosed with a deep vein thrombosis days post-riastap administration and no infusion reactions were reported. in-hospital mortality occurred in . % of our patient population. riastap administration successfully and safely treated hypofibrinogenemia in patients with post-tpa hemorrhage. casey catheter-associated urinary tract infections (cauti) are the most prevalent hospital-acquired infections (hai), and account for more than , cases annually and , deaths per year. cauti is the most common hai in neuroscience intensive care units ( immobility and urinary retention. we implemented a team-driven multimodal quality improvement initiative to reduce cauti and catheter-utilization rates in the nsicu. we convened a multidisciplinary cauti prevention team including nurses, advance practice providers, physicians, and infection control specialists. we developed a cauti surveillance program that involved review cauti and catheter utilization rates. we applied root cause analysis to target improvement opportunities, and implemented interventions including best-practice catheter insertion techniques, modification of bowel regimen, and guidelines for timing of catheter removal. we also implemented a daily goals tool to prompt standardized team communication surrounding catheter removal on morning interdisciplinary rounds. we performed poisson generalized linear model analyses, controlling for linear time trends and testing with sandwich errors. we analyzed data before and after implementation of interventions, spanning a time period of months. -- . ), as did mean catheter days per -- . ). we observed a % reduction in cauti rate adjusted by catheter days ( % ci %, %), p = . . there was an % reduction in catheter utilization rate adjusted by patient days ( % ci %, %), p = . . a team-driven multimodal approach to cauti reduction resulted in significant decreases in cauti and catheter utilization rates in the nsicu. team-driven interventions enhance communication and shared -up is ongoing to evaluate sustainability. non-neurological complications involving a single or multiple organ systems during intensive care in critically ill patients of traumatic brain and spine injuries is significant cause of poor prognosis but often not well managed. the aim of this study was to assess the frequency of such complications in neuro icu and assess their impact on morbidity and mortality. a prospective observational study on patients of varied demographic profile admitted in neuro icu over a period of months for injury and associated multisystem involvement was conducted. significant predefined parameters addressing the non-neurological complications occurring during their icu stay were recorded including disturbances and bleeding complications. the study period was from admission to the icu till the discharge from the icu or demise. % of patients developed respiratory complications in the form of chest infiltrate ( %) and atelectasis ( . %). . % of patients suffered from cardiovascular complications. % of patients had dyselectrolytemia, commonest being hypernatremia due to hypovolemia ( %). sepsis was observed in . %. bleeding diathesis and acute renal injury were observed in % & . % of patients respectively. % of the patients succumbed to injury out of which . % was due to non -neurological cause. further results will be discussed in detail with inferences at the meeting. intensivists in neuro icu must consistently assess and treat the non-neurological complications in traumatic brain and spine-injured patients and deliver appropriate care to bring down the mortality and morbidity and improve outcome. neurocrit care ( ) :s -s transcranial doppler (tcd) is a useful ancillary test in neurologic critical care for monitoring patients at with elevated intracranial pressure and cerebral vascular resistance. the normal values of cerebral blood flow velocity and pis are significantly distorted by nonpulsatile blood flow, as in patients on venoarterial extracorporeal membrane oxygenation (va-ecmo) circulation. this analysis evaluates changes in pi measurements in patients on va-ecmo following cerebral vasodilation, vasoconstriction, increased intracranial pressures, or cerebral circulatory arrest. data from tcds in patients on va-ecmo in the cedars sinai medical center cardiac surgical icu were reviewed. mean pis were calculated for each patient using gosling's pi formula. the values obtained were compared with ejection fractions (ef) obtained within hours of tcd. pis were globally low or absent in all tcds. the non-demonstrable pi seen in one patient is from severely diminished cardiac function, resulting in tcds were performed at the initiation and conclusion of va-ecmo cannulation. the pi values for these tcds correlated directly with changes in efs. also, an abrupt rise in pi to normal value was seen with placement of a total artificial heart and return of pulsatile circulation. we demonstrate that patients on mechanical circulatory support demonstrate low-absent pis on tcds. ion or cerebral circulatory arrest. moreover, rising pis in patients with improving cardiac function should not be confused with elevated intracranial pressures. venous thromboembolism (vte) prophylaxis in underweight patients with neurologic injury remains unaddressed by recent guidelines and primary literature. this study aimed to describe vte prophylaxis strategies employed in this population and compare the impact of underweight and non-obese patients on thrombotic and bleeding events. underweight and non--care unit from september , to july , were retrospectively identified. underweight was defined as a body -obese as a bmi . excluded if they received > vte prophylaxis regimen, had an icu length of stay < hours, or received vte prophylaxis for < hours. patients were stratified to non-obese and underweight groups and subsequently matched : , on age and diagnosis. prophylaxis regimen, prevalence and type of the most common regimen in the underweight (n= ) and non-obese (n= ) groups was unfractionated (ufh) units subcutaneously every hrs ( . % vs. . %; p= . ). only underweight patients received ufh units subcutaneously every hrs ( . % vs. . %; p< . ). non-obese and underweight patients had no difference in the proportion of overall bleeding ( . % vs. . % p= . ) and thrombotic events ( . % vs. . % p= . ) while receiving vte prophylaxis. further analyses revealed a statistically significant difference in the proportion of underweight patients that developed intracranial hematoma expansion while receiving prophylaxis versus non-obese patients arge dispositions were seen between groups. current practice does not reflect a consistent dose reduction for neurologically-injured, underweight patients. caution should be considered when using increased doses of ufh in neurologically-injured patients. continued assessment of vte prophylaxis is needed to confirm these findings. patients in the neuro intensive care unit (nicu) commonly need vasopressor infusions for various reasons. the traditional approach is to insert central venous catheters (cvc) for this purpose. cvcs carry among others. phenylephrine is a commonly used vasopressor in the nicu. the purpose of this study was to evaluate the safety of phenylephrine infusion through peripheral intravenous catheter (iv). retrospective review of consecutive patients admitted to the neuro icu and administered phenylephrine infusion through peripheral iv line. one hundred patients, mean age years (sd ± ) were included in the analysis. fifty-four ( %) were men. eightydisease. the most common indications of phenylephrine were hemodynamic augmentation ( %), multifactorial transient post-operative hypotension ( %) and hypotension due to other causes ( %). most common location of iv line was proximal upper extremity ( % antecubital, % forearm) with gauge of the iv line between ( %) and ( %). average maximum rate of phenylephrine infusion duration of hours (sd ± , range to ). central line was eventually placed in % due to physician preference and in another % due to a change of vasopressor to norepinephrine. there were any complications. infusion of phenylephrine through peripheral iv appears safe when used in moderate doses for the short term and can be considered in lieu of placing a central line solely for this purpose. this may reduce the complications associated with central lines. osmotic therapy continues to be standard care in the medical management of cytotoxic cerebral edema. the long term use of monotherapy is often limited by side effect profile. the combination of low dose mannitol and hypertonic saline may provide synergistic effect by combining mechanisms of action, while limiting dose-related toxicities of either agent. we investigated safety and efficacy endpoints for combination therapy. a single-center retrospective cohort study from august to december . identified patients were administered combination mannitol and hypertonic saline for > hour duration. the primary outcome criteria, sodium fluctuation, and central pontine myelinolysis. patients (mean age ± , % male) were identified. underlying neurological injury included % brain injury. % had neurosurgical management. the average number of mannitol doses given was , and the average duration of hypertonic saline was hours. the range of mannitol dose was . - . percentage of osmotic therapy doses were held for pred mannitol, % held hypertonic saline). aki occurred in ( %) patients ( -stage aki, -stage central pontine myelinolysis. low dose combination osmotic therapy was tolerated with no central pontine myelinolysis and rare sodium fluctuations; however transient low grade aki was common. further study is needed to evaluate the relative efficacy of single and combination osmotic therapy in the neurocritical care population. clinical characteristics of nonconvulsive status epilepticus diagnosed by simplified continuous eeg monitoring at an emergency intensive care unit. the clinical characteristics of nonconvulsive status epilepticus (ncse) presenting in icu in japan is limited. our institute provides a noninvasive monitoring system of two-channel simplified continuous eeg (seeg) for the bedside monitoring of cerebral activities. the present study aimed to elucidate the clinical characteristics of ncse in patients with altered mental status (ams). this single-center retrospective study comprised patients who were hospitalized between march , and september , at the emergency intensive care unit (icu) of the kagawa university hospital. primary outcome was the ncse incidence. the secondary outcome was the comparison of duration of icu stay, hospital stay, and a favorable neurological outcome (fo), as assessed using the tal between the groups with and without ncse. fo and poor neurological outcomes (po) were defined as mrs scores of - and - , respectively. simplified continuous electroencephalogram (seeg) was monitored in patients (median age, years; . % males) with acute ams. ncse was observed in ( . %) of the patients with ams. rates of fo, duration of icu stay, and hospital stay were not significantly different between the ncse and non-ncse groups (p = . , p = . , and p = . , respectively). approximately % of the patients with ams admitted to emergency icus developed ncse. the outcomes of ams patients with and without ncse did not differ significantly when appropriate medical attention and antiepileptic drugs were initiated. seeg monitoring may be recommended in patients with ams in emergency icu to obtain early detection of ncse followed by appropriate intervention. approximately , people per year will need mechanical ventilation secondary to neurological injury resulting in significant mortality. delaying liberation in neurologically impaired patients otherwise ready for liberation is a source for significant hospital charges. there is no clear guideline to suggest one spontaneous breathing trial (sbt) over another in predicting the liberation success. zero pressure support and zero positive end expiratory pressure (peep) or zeep is a traditional method assessing patient's readiness for mechanical ventilation liberation. however, neurologically injured patients with was to assess mechanical ventilation liberation in patients who failed zeep and subsequently passed pressure support trial. retrospective analysis of liberation in intubated patients in a neurosciences intensive care unit. all patients were initially challenged with zeep. if passed, patients were liberated from mechanical mcnemar's exact test. p value < . was considered significant. adult (> years old) patients were included. the majority of patients were successfully liberated from mechanical ventilation using minute zeep trial alone (n= , . %). eleven ( . %) patients failed . %) required reintubation. ten ( . %) this study shows that the majority of patients can be successfully liberated from mechanical ventilation successful liberation from mechanical ventilation. neurocrit care ( ) :s -s vancomycin establishey using nonmem software by the department of pharmacy of nanjing drum tower hospital in neurosurgical intensive care unit patients. according to the patient's gender, age, body weight, serum creatinine (scr), serum albumin (alb), the actual measured value. during the period from march to march , patients including male and female, whose age is ± years old ( - years old), were grouped and copies of blood concentration of vancomycin were measured. the average concentration was . m the actual measured value (r= . , p< . ), the mean absolute percentage error (mape) was . . neurosurgical intensive care unit patients for drug value prediction and drug dosage guidance. but because of coma, the body weight estimation has errors (about %). the renal function sometimes changed by contrast agent and diuretic drug has an impact on predictive results. by adjusting methods, accurate prediction rate increased to nearly %. xi liu-deryke, sindhuri s. avula, jason j. vilar. florida hospital orlando/pharmacy department, orlando, fl, usa. little data exists concerning clevidipine in this population. large variations in bp during the first hours is an independent predictor for poor outc aneurysmal subarachnoid hemorrhage (asah) admitted to neuroscience intensive care unit from january through december were identified retrospectively. patients were included if they received clevidipine or nicardipine for initial acute bp management, and bp goal was defined by the prescribers. bp variability was measured by standard deviation (sd) of mean arterial pressure (map) over the first hour of therapy. seventy three patients were included in the analysis (clevidipine n= ; nicardipine n= ). admission and % asah. baseline map between clevidipine and nicardipine group was comparable ( vs. mmhg). the number of bp recordings was similar between groups (clevidipine vs. nicardipine ; p= . ) and the average time to goal was minutes and minutes, respectively (p= . ). the average map during the first hours was similar (clevidipine vs. nicardipine mmhg; p= . ). although not statistically significant, clevidipine group had a higher percentage of bp above goal compared to nicardipine group ( . % vs . %; p= . ). there was no significant difference in bp variability between clevidipine and nicardipine group (sd . vs. . mmhg; p= . ). our study did not find a difference in bp variability between clevidipine and nicardipine following acute long corrected qt interval (qtc) has been associated with malignant ventricular arrhythmias specifically present in neuro intensive care unit (nicu) patients. in addition to medical causes, acute neurologic insult has been shown to cause multiple neuro-cardiac manifestations including qtc prolongation. prevalence ge and surgical icu patients which have different disease processes compared to nicu. retrospective review of consecutive patients admitted to the neuro icu and having abnormal qtc interval. ninety-five patients, mean age years (sd ± ) were included in the analysis. fifty four ( %) were men. average duration of hospitali ( %), subdural hemorrhage ( %), and cerebral hemorrhage ( %). fiftycardiovascular disease, % had abnormal ejection fraction. thirty-seven patients ( %) needed abnormalities were observed throughout the hospitalization and patients frequently received qtc prolonging drugs. mean qtc was ms (sd ± , range - ). there were episodes ( %) of nonsustained ventricular tachycardia which did not lead to any immediate consequences. one patient had cardiac arrest following anesthesia for hemicraniectomy. initial rhythm was asystole followed by fine ventricular fibrillation and therefore could not be clearly attributed to prolonged qtc. there were no episodes of tdp. -sustained ventricular tachycardia was observed without leading to cardiac arrest. no episodes of tdp were observed in these patients. deep venous thrombosis (dvt) of the lower extremities is a common cause of morbidity and mortality among neurologically injured patients. the data on incidence and prevalence rates of dvt among high medical or surgical intensive care unit with very limited information on patients in neuro-intensive care units (nicu). the aim of the present study is to assess the incidence and prevalence of deep vein thrombosis among patients admitted with acute neurologic injury. our institution routinely conducts ultrasound screening within hours of admission and -month period. data was abstracted and analyzed to assess the prevalence of dvt in this period. we excluded patients presenting with superficial vein thrombosis, hematoma and chronic venous scarring. over a period of one year; the prevalence of dvt was . % (n= ). of the cases that were diagnosed with dvt; more than one-half ( . %) presented with dvt at the time of admission. patients ( . %) acquired dvt during hospitalization. majority of the patients with dvt at the time of admission are caucasian males with mean age and mean saps ii score of . , ranging between and . prevalence of dvt at the time of presentation to the neuro icu is relatively high. further research is s neurocrit care ( ) :s -s practice guidelines recommend that practitioners should not prescribe prolonged prophylactic systemic antibiotics (ppsa) after neurosurgical procedures, even if drains are left in place. we sought to evaluate ) current practice patterns related to ppsa administration to neurosurgical patients with drains and devices and ) practitioner perception about the need for ppsa in this population. we surveyed members of the neurocritical care society on use of ppsa (defined as maintenance antibiotics after the time of insertion) and personal perception about the need for ppsa in patients with intraparenchymal monitors, subdural drains, subgaleal dra -pratt spinal drains, and lumbar drains. of respondents, routine institutional use of ppsa was reported by fewest respondents reported use with subgaleal drains and the most respondents reported use with -pratt spinal drains with instrumentation. respondents had varying personal opinions on the need while the lowest ( %) was for patients with subgaleal drains. it is on the use of ppsa in patients with neurosurgical drains is necessary to optimize patient care. transition to comfort measures only (cmo) in an intensive care unit (icu) is a common but delicate process that requires a well-organized multi-disciplinary and multi-professional care model. the goal of this survey was to understand potential deficiencies and inconsistencies in the transition to cmo in order to develop a process to improve the quality of care provided to patients at their end-of-life. after obtaining irb approval, a web-based questionnaire was distributed to attending physicians, residents, fellow trainees, bedside nurses, respiratory therapists and spiritual care team members, who deliver care to patients in neurological, medical, trauma-surgical, and burn intensive care units at the university of washington's harborview medical center. overall survey response was . % ( out of ). the concept that transition to cmo is a multidisciplinary process was not universal with only . % of all bedside nursing and respiratory therapists feeling invited and actively engaged in the discussion about cmo. the majority of respondents ( %) encountered at least one 'less than ideal' transition to cmo. deficiencies identified included gaps d interprofessional conflict ( . %). most participants ( %) agreed that a formalized process might reduce round the transition to cmo. we identified several barriers towards an optimal, collaborative transition to cmo in icus at a large academic medical center, highlighting the need for a formalized process. such a process would ensure communication between various disciplines and professions, and offer healthcare providers opportunities for dialogue to address all the issues resulting in a smooth transition to cmo. dexmedetomidine's propensity to cause bradycardia is well documented in non-neurocritically ill patients. e units (icu) when defined as heart rate (hr) < bpm. neurocritically ill patients have been excluded from all randomized trials. the aim of this study is to assess the development of bradycardia in patients with neurologic injury who have received dexmedetomidine for sedation in the icu. was done via nursing driven protocol with no loading doses. primary outcome was the incidence of bradycardia (hr < bpm) during first administration. secondary outcomes were percent decrease in hr from baseline and time to event analysis using cox regression. mortality in the icu was collected. a total of patients were included ( % male, mean age years, mean saps ii ). the most bradycardia occurred in patients ( . %). the average maximum dose was higher in patients who infusion duration did not vary. baseline hr was lower in bradycardic patients ( ± bpm vs. ± bpm, p= . ) and a larger mean percent decrease in baseline hr was observed ( . % ± . vs. . % ± . ). median time to first bradycardic event was hours [ . - . ] which was significantly impacted by baseline hr (hazard ratio . ; % ci, . - . ; p= . ). mortality was significantly lower in patients who developed bradycardia, . vs. . % (p= . ). these data indicate that bradycardia associated with dexmedetomidine occurs considerably among the neurocritically ill. future assessment of clinicall development would further contribute to the limited data of dexmedetomidine use within this patient population. medical complications after subarachnoid hemorrhage. medical complications occur frequently after subarachnoid hemorrhage (sah). their impact on outcome has been previously described, but was not validated in international series of sah. we evaluated consecutive patients admitted to a tertiary hospital in brazil with sah from january - ) at discharge. we calculated the frequency of medical complications according to prespecified criteria and eva of poor outcome. thirty-six% had a poor outcome; mortality was . %. the most frequent complications were hyperglycemia ( %), fever ( %), pneumonia ( . %), hypotension (< mm hg systolic) treated with vasopressors ( . %) and venous thromboembolism ( . %). hyperglycemia (odds ratio [or], . ; % confidence interval [ci], . - . ; p= . ) significantly predicted poor outcome after adjustment for age and hunt-hess grade. hyperglycemia affected more than one third of patients with sah and was significantly associated with poor functional outcome. critical care strategies directed at maintaining normoglycemia may improve outcome after sah. s neurocrit care ( ) :s -s blood pressure (bp) can be measured in critically-ill patients using non-invasive (oscillometric) blood pressure (nibp) and intra-arterial blood pressure (iabp) monitoring. the accuracy of nibp compared to the "gold standard," aibp, has been questioned. nibp monitors generally tend to over-read at low values and under-read at high values compared to iabp. previous studies exploring nibp-iabp correlations have generally been performed on patients not receiving continuous infusions of vasoactive medications. since many critically-ill patients receive vasopressors and antihypertensive agents, we wanted to study the relationship between simultaneously-measured nibp and iabp recordings in this patient population. we prospectively identified patients (n= , target n= ) admitted to a neurosciences icu, who had simultaneous iabp and nibp monitoring while receiving intravenous infusions of manually abstracted via retrospective chart audit. covariate and demographic variables were also abstracted and entered into an electronic spreadsheet. statistical analysis performed using sas v . . initial results from subjects ( % caucasian, % male, mean age . years, mean bmi . ), observations. independent-samples t-tests showed a significant difference between nibp vs iabp readings: ([sbp: m= vs mmhg respectively; p mmhg ( . %)]. bland-altman plots demonstrated good inter-method agreement between nibp-iabp measures (when visually excluding outliers) and -aibp sbp differences at higher blood pressures. preliminary analysis indicates a statistically significant difference between nibp-iabp readings for patients on vasoactive medications. yet when visually excluding outliers, there is good inter-method agreement. data from the entire cohort will be available for presentation at the ncs annual meeting and will be helpful in choosing appropriate bp monitoring methods for patients on vasoactive infusions. new-onset refractory status epilepticus (norse) is an important syndrome often associated with a poor outcome. the aim of the present study was to review norse cases in our hospital and to determine the main factor that may improve patient outcomes. we retrospectively reviewed our hospital medical records and database of electroencephalograms (eegs) over a years period (may -may ). in our facility, we performed -h continuous eeg monitoring using the international - system. of the monitored patients, we excluded those who were meningitis, herpes encephalitis, and history of epilepsy. we discussed their causes and neurological outc (go) was defined as a mrs score of - , whereas a poor neurological outcome (po) was defined as a score of - . moreover, we attempted to determine the main factor that influenced the neurological outcomes. we identified patients who had undergone eeg, and identified six norse patients among them. the on arrival was . all patients were diagnosed with limbic encephalitis and all had nonconvulsive status treatments, such as steroids, were delayed in all po patients. on the basis of our data, the cause of all norse cases was limbic encephalitis. in po patients, definitive treatments, such as pulse steroid therapy, were delayed. this is a relatively small study. further research is needed to identify the factors which could improve outcomes. multi-drug resistant organisms (mdro) are an increasing concern in health systems. pathogens such as pseudomonas aeruginosa, acinetobacter baumanii, and carbapenamase-producing enterobacteriaceae hold highest mortality rates especially when the central nervous system is involved. when mdros are cultured treatment options are becoming limited and reliance on medications such as colistin and aminoglycosides is becoming more prevalent. however, penetration of these therapies into the central nervous system is concerning therefore local administration is a potential concomitant therapy. this study was a retrospective chart review from to for all patients with documented mdros who received intraventricular colistin. seven patients from to met inclusion criteria. the average age of the patients included was years old, were males, and the median length of intensive care unit stay was days. the dose of colistin used for each patient was mg via intraventricular route. the duration of therapy ranged from - days and all cerebrospinal fluid cultures were sterile at days after administration of colistin. each patient received concomitant systemic antibiotics while receiving intraventricular colistin. six of the seven the use of intraventricular colistin was not associated with any reported adverse events. the use of intraventricular colistin was associated with positive clinical outcomes with no reported adverse effects. myasthenic crisis: epidemiology, economics and opportunities for change -a single center retrospective analysis. avinash b. kumar, vikram v. tiwari, kevin k. scharfman, justin j. calabrace. vanderbilt university medical center, nashville, tn, usa. myasthenia gravis (mg) patients are admitted to the icu for myasthenic crises characterized by immunoglobulin (ivig) or plasmapheresis and supp and care flow maps of patients admitted to our institution this is an irb-approved, retrospective cohort study of patients admitted to a tertiary neuro icu. we included adult (age > years), with a diagnosis of mg who received plasmapheresis or ivig therapy. the demographics and clinical data were summarized for patients in the ivig and plasmapheresis cohorts. we also compared the icu and hospital los and in addition the hospital cost data for patients in both cohorts. the final cohort included hospital encounters for individual patients ( female) admitted between - . the mean age on admission was . ± . y. there was no significant difference between c ventilation; the median duration of mv was . d (range - ). the median readmission rate was . ± . . ). patients had multiple crisis readmissions (> ). this cohort was socially challenged ( divorced, analysis included patients ( in ivig cohort and in plasmapheresis cohort). the mean hospital costs (variable direct-technical) in ivig cohort was approx. $ more than the plasmapheresis cohort. there was no statistically significant difference between in the limited financial analysis. . % of patients were either medicare or medicaid patients, . % wer the disease burden on patients and hospitals of this orphan condition are significant and continues beyond the icu. evidence based care pathways need to be explored for the management of this high resource utility disease. botulism is a rare potentially fatal and treatable disorder caused by a bacterial-produced toxin that affects the presynaptic synaptic membrane resulting in a characteristic neuromuscular dysfunction. it is caused by either the ingestion of the toxin or the bacteria, inhalation, or wound infection. we present our we report consecutive cases of botulism presenting to university medical center of el paso. medical records where reviewed to obtain demographic information, clinical presentation, treatment and outcome. in popping and had abscesses in the administration areas. by history the most common %, ophthalmoplegia %, ptosis %. interestingly enough, in those patients with the documentation the pupils were reactive in %. all patients required mechanical ventilation and all were treated with the trivalent antitoxin. thirteen patients were disc derivatives (mostly -monoacetylmorphine and -monoacetylmorphine) was associated with the development of botulism. its presence in the us-mexican border is not surprising since is frequently produced in latin america. its association with the development of botulism should be recognized early to allow a prompt diagnosis and treatment with the antitoxin. a clinical feature worth noting is the presence of normal pupillary light reflex in nearly half of patients thus a normal pupillary response should not be used as a finding to exclude botulism. in clinical trials limits the discovery of effects that may be particularly relevant to underrepresented populations. clear iii, a presented an opportunity to evaluate african american (aa) enrollment. investigators across u.s. hospitals screened , patients over a -year period: % aa; . % asian; . % native american; . % pacific islander; . % white; . % mixed race; and . % not reporting. the mean age for aas was younger at . (sd: . ) vs. . (sd: . ) for whites (p= . ). the randomized-to-screened ratio for aas was . vs. . % for other racial groups (p< . ). higher . %, p= . ); northeast ( . % vs. . %, p< . ); south ( . % vs. %, p= . ); and west ( . % vs. . %, p=nonsignificant). african americans were less frequently excluded due to non-hypertensive etiology ( . % vs. %, p< . ), not having ventricular drainage ( . % vs. . %, p= . ), dnr status ( . % vs. . %, p= . ) and unstable bleeding ( . % vs. . %, p= . ); and more frequently excluded for prior disability ( % vs. . %, p= . ), larger hemorrhages ( . % vs. . %, p= . ), and by investigator decision ( . % vs. . %, p< . ). of the patients who refused consent, aas accounted for . % vs. . % of whites. in an unadjusted logistic model, the odds ratio for successful enrollment of aas was . (p< . ) vs. whites, and . (p< . ) after adjustment for age and hispanic ethnicity. the age < , - and - subgroups maintained higher adjusted odds ratios than whites at . (p< . ), . (p< . ) and . (p< . ) respectively; the above subgroup was not significantly different. others have reported difficulty enrolling aas into clinical trials. clear iii suggests this may be a misperception s neurocrit care ( ) :s -s pipeline that utilizes machine-learning algorithms to integrate clinical data and quantitative eeg (qeeg) trends, providing continuous estimation of prognosis. a collaboration involving two academic centers in the u.s. assembled a retrospective clinical and eeg database of adult subjects with cardiac arrest and return of spontaneous circulation who underwent continuous eeg monitoring. four qeeg features were included in the model: regularity, tsalis entropy, alpha-to-delta ratio, and voltage < uv. only the first hours of eeg data were evaluated in this analysis. poor outcome was defined as cerebral performance category of - at discharge. ten fold cross validation resampling method was utilized, and model performance evaluation metrics were area under roc curve (auc), sensitivity, and specificity. the algorithm provided an hourly estimation of poor clinical and eeg data was available for a total subjects. mean age was . years and overall mortality was . %. one hundred and twenty subjects ( . %) had poor outcome. our multiparametric qeeg method achieved optimal performance for mortality prediction at hours (auc . ), with a sensitivity of % and specificity of %. optimal poor outcome prediction performance was achieved at hours (auc . ), with a sensitivity of % and specificity of %. at a false-positive rate of %, the sensitivity for poor outcome was %. alpha-delta ratio and voltage < uv were independently associated with mortality and poor discharge outcome at hours (p< . ). employment of machine-learning methods in qeeg analysis allows early and robust outcome prediction in cardiac arrest. this approach has potential to facilitate real-time individualized prognostication in cardiac arrest. secondary brain injury may be a significant barrier to survival following extracorporeal membrane oxygenation (ecmo) for, otherwise reversible, cardiorespiratory failure. prevalence of brain injury phenotypes on neuroimaging were described in our prior wor neurological injury on outcomes in adult patients on ecmo. a retrospective cohort of ecmo-treated adults. clinical and outcome data was obtained from electronic chart abstraction of clinical and physi of decannulation. % (n= ) of ct scans and % (n= ) of mris had abnormal findings. intracranial hemorrhage was seen in % patients with neuroimaging. in addition, % of mris revealed diffuse significant difference in survival to hospital discharge and mean modified with or without neuroimaging during ecmo ( % vs. %, p= . ; mrs, . ± . vs. . ± . , p= . ). however, in the group undergoing neuroimaging, normal scans were associated with better survival to hospital discharge ( % vs. % p< . ) and lower mrs ( . ± . vs. . ± . , p= . ). ( . %) of survivors who did not get neuroimaging and ( %) of those who got neuroimaging achieved abilty to perform independent adl at discharge (p= . ). all patients with ability to perform independent adl in neuroimaging group had normal scans. ( %) of survivors who did not get neuroimaging and ( %) of those who got neuroimaging were discharged home (p= . ). all surviving patients in the neuroimaging group who were discharged home had normal scans. secondary brain injury in ecmosurvival and functional outcomes. a prospective study has been planned to better understand mechanisms mediating this effect. neurocrit care ( ) :s -s re-verse ad is an ongoing, phase , cohort study evaluating the extent to which idarucizumab, a humanized fab fragment specifically reverses dabigatran's anticoagulation effect in patients with serious bleeding or requiring urgent interventions. dabigatran is a direct acting oral anticoagulant approved for -valvular atrial fibrillation and venous thromboembolism treatment and prevention. in re-ly, dabigatran and mg bid were associated with significantly lower annualized rates of intracranial hemorrhage (ich) than warfarin ( . %, . % and . %, respectively). nonetheless, the mortality rate with ich in the context of any anticoagulation remains high, probably reflecting the effect of hematoma expansion. whether idarucizumab improves clinical outcome in dabigatran-treated patients this analysis of the first patients enrolled in re-verse ad focuses on patients with ich. patients presenting with ich were given intravenous idarucizumab g as two . g bolus infusions administered effect, based on central laboratory determination of dilute thrombin time (dtt) or ecarin clotting time (ect). we compared the clinical outcome of this re-verse ad interim analysis with dabigatran-treated ich patients in re-ly. in this interim analysis, patients with dabigatran-associated ich were enrolled in re-verse ad. complete reversal of anticoagulation was observed by dtt and ect within minutes of idarucizumab administration. preliminary results from this interim analysis indicate that the mortality rate of ich patients re-ly. idarucizumab reversed anticoagulation in ich patients and appears to improve mortality rates in dabigatran-treated patients with ich versus historical controls from re-ly. results from additional patients in re-verse ad will provide further information on the effects of idarucizumab reversal in patients with ich. ****permission was not granted to print this abstract**** s neurocrit care ( ) :s -s callie electroencephalography (eeg) has clinical and prognostic importance for comatose survivors of cardiac arrest. recent interest in quantitative eeg (qeeg) analysis has grown. the qualitative effects of sedation ing effects of sedatives on qeeg are poorly characterized in anoxic injury. we hypothesize that sedation would decrease amplitude-integrated eeg would predict neurological recovery. we routinely monitor comatose post-arrest patients with eeg for this prospective study, we included consecutive eeg-monitored patients who had protocolized sedation interruptions, excluding those with contraindications to interruption such as seizure or hemodynamic instability. we used persyst v to quantify sr, aeeg, and adr and calculated medians for min immediately prior to sedation interruption and the last min of interruption. we used nonparametric tests to determine if the qeeg signal changed pre-to post-and whether this differed by outcome (cerebral performance category - at hospital discharge vs - ). of screened subjects, met inclusion criteria (median age years, % male). sedation regimens varied ( propofol; fentanyl; midazolam). median duration of sedation interruption was min, and did not differ by sedative type. pre-interruption, higher adr and aeeg and lower sr predicted favorable outcome. post-interruption, sr decreased (median change - . , iqr: - . to ), aeeg increased ( . , (p= . ), but aeeg and adr changes did not differ by outcome. in acute anoxic brain injury, sedation increases sr and decreases aeeg. larger decreases in sr with sedation interruption predict worse outcomes, which may reflect a susceptibility of deafferentated cortex to suppress in response to sedation. ashley r. hedges, gary g. davis, brianne b. wolfe, erin e. lingenfelter, gregory g. hawryluk, safdar s. ansari. university of utah hospital and clinics, salt lake city, ut, usa. for patients presenting with subarachnoid hemorrhage (sah) or traumatic brain injury (tbi), levetiracetam has begun to emerge as a preferred alternative to phenytoin for seizure prophylaxis following initial presentation. however, the optimal dose of levetiracetam has not been determined. a retrospective review of electronic medical records identified patients that received levetiracetam for seizure prophylaxis for sah or tbi in a level one trauma center from may , to october , . the goal of this research was to quantify the combined seizure incidence (including both clinically observed seizures and those confirmed by electroencephalogram) in patients receiving levetiracetam mg twice daily compared to levetiracetam > mg total daily dose. among patients captured, % were male, with a mean age of years old. for patients receiving seizure incidence was observed, . % vs . %, in patients receiving levetiracetam mg twice daily this was observed despite no difference in potential confounders, includ trend towards increased levetiracetam failure rates was observed in the lower dosing scheme of mg twice daily. t electroencephalograms ordered ( vs , p= . ). no difference in adverse effects were observed our project suggests that patients may benefit from a standardized levetiracetam dosing scheme of mg twice daily. treating intraventricular hemorrhage (ivh) with a fibrinolytic (ivf) therapy such alteplase via a catheter is becoming an increasingly popular intervention. with the conclusion of the clear iii trial a larger cohort of patient data is available to update past meta analyses. mortality and good functional outcome after thrombolytic treatment was reviewed in patients with hypertensive ivh. a literature search was performed from to march to collect all literature on ivh treatment with ivf. seventeen papers meeting our inclusion and exclusion criteria were collected for further analysis. ivh patients with vascular abnormalities and traumatic injuries were excluded. mortality and functional outcome was assessed to compare ivf treated and control groups in all publications. there was a significant difference in mortality favoring the treatment group at days, days and days ( %ci, . - . ; p= . ; %ci, . - . ; p= . ; %ci, . - . ; p= . ). pooling mrs and gos good functional outcomes, there was a significant difference favoring the treatment group at , , and days ( %ci, . - . ; p= . ; %ci, . - . ; p= . ; %ci, . - . ; p= . ). there was a non-significant trend in mrs scores favoring the treatment group. a significant difference in gos score favoring the treatment group was found at , , , and days ( %ci, . - . ; p= . ; %ci, . - . ; p= . ; %ci, . - . ; p= . ; %ci, . - . ; p= . ). treatment of hypertensive ivh with thrombolytic may improve functional outcome and reduce mortality compared to control groups as early as days, a trend that continues to days for mortality and days for functional outcome. different effect sizes are generated when different functional outcome tools, such as mrs and gos, are used. an aging population and increasing use of anticoagulants and antiplatelet agents to prevent ischemic hematoma (ssdh) and sdh related to trauma (tsdh). we sought to study the association of antiplatelet agent and warfarin use in sdh patients admitted to our neurosurgical icu. warfarin were studied. neurosurgical methods of sdh evacuation and re-evacuation were studied as well as hemostatic factors such as international normalized ratio (inr) within the first hrs, blood products and hemostatic agents given to reverse coagulopathy or antithrombotic effects. demographic information such as age, gender and comorbidities were noted and indication for antithrombotic agent. we excluded major trauma (level ) associated with sdh at our center. from january to may , we admitted sdh patients, of which . % required evacuation ( -> , mean = . ), and on ( %) underwent redo evacuation (range in days -(date range - days). hour inr rates in warfarin related sdh were all < . except for . of these , only one patient required re-operation. mort in our patient population, sdh was associated with a need for evacuation in . % of all patients, of which a higher rate of reagents. mortality was also higher in the warfarin associated sdh patients. consecutive patie complications were prospectively enrolled. medical critical care attendings (micu), neurocritical care attendings (nicu), residents (res), and nurses (rn) predicted the following: ) -month functional -month quality of life (qol). patients were followed up at months and their functional status and qol were compared to the predicted values. functional outcomes were dichotomized to good (mrs - ) vs. poor (mrs - ). (of ) patients had -month mrs predicted by all provider groups. fifty-four ( %) patients had good outcome and ( %) had poor outcome. the micu, nicu, res, and rn providers had similar predictive values ( % ci) for accurately predicting good outcome ( % ( - ), % ( - ), % ( - ), and % ( - ), respectively). nicu was most accurate in identifying poor patient outcome, % ( - ), followed by micu % ( - ), rn % ( - ), and res % ( - ) (p= . , . , and < . , respectively). when patients who transitioned to comfort measures only (n= ) were excluded from the analysis, the nicu team was more accurate at predicting poor outcome. fifty-three survivors had qol predicted by all provider groups. the accuracy of qol predi neurocritical care attendings are better than healthcare providers without neurological training at predicting poor -month functional outcome in neurocritical care patients. however, the overall predictive accuracy for -month mrs and qol was similar between healthcare provider teams. there are significant limitations in providers' ability to predict long-term functional outcomes. patients with severe acute brain injury (sabi) raise important palliative care considerations associated with sudden, devastating injury and uncertain prognosis. the goal of this study was to explore how family members, nurses and physicians experience the palliative and supportive care needs of patients with sabi receiving care in the neurosciences intensive care unit (neuro-icu). design: semi-structured in-person interviews were audiotaped, transcribed, and analyzed using thematic analysis. setting: thirty-bed neuro-subjects: forty-seven interviews were completed regarding patients receiving care in the neuro-icu with family members (n= ), nurses (n= ) and physicians (n= ). hope varied depending on the par away, generally in the process of conveying prognosis, while families expressed hope as an action that supported coping with their loved one's acute illness and its prognostic uncertainty. ( ) participants described the loss of personhood through brain injury, the need to recognize and treat the brain-injured patient as a person, and the importance of relatedness and connection, including personal support of families by clinicians. in their pursuit to recognize and preserve personhood, physicians used stories from patients and families to inform them about patient identities, while nurses focused on providing supportive, empathetic care to patients and families. support for hope and preservation of personhood challenge care in the neuro-icu as identified by families and clinicians of patients with severe acute brain injury. specific practical approaches can address these challenges and improve care to meet the needs of patients and families in the neuro-icu. despite increasing evidence that early mobilization strategies are effective, we showed patients were not adequately mobilized in two argentinean hospitals. we implemented a progressive-mobility protocol and examined its feasibility, safety and applicability in our neurocritical patients. prospective observational implementation study of a progressive-mobility protocol for neurocritical patients admitted to icus of two university hospitals in argentina. all patients were evaluated twice daily for level of movement and clinical stability. patients progressed as tolerated from passive movement implementation baseline to icu patients admitted in months - after implementation began (two month start-up phase not analyzed). there were pre-implementation patients with assessments ( % post-operative, % traumatic -implementation patients with measurements ( % postpopulations: median age years ( % ci - ), nearly % were men. mobilization was . times - . ) after protocol implementation. two thirds of pre-implementation patients ( . %) were not mobilized compared with only . % post-implementation (p< . ). among mechanically ventilated patients, . % of pre-implementation assessments showed no mobilization vs. . % post. post-implementation patients with an endotracheal tube had a lower rate of mobilization ( . %) than ventilated patients with a tracheostomy ( . %). passive movement, turns and full assistance to sit up and transition out of bed to chair was achieved for . % and . % achieved higher levels ( , , ) . mobility sessions with the physical therapist were < minutes in % of the cases. no mobility-related adverse events occurred. mobilized following protocol implementation. this prospective study demonstrated that early and progressive mobility among neurocritical care patients in argentina is feasible and safe. psychiatry, geriatrics, and oncology have adopted comprehensive approaches to predict outcomes accounting for important constructs such as spirituality and resilience. critical illness often occurs as a sudden catastrophic event leaving patients with significant long-term cognitive, behavioral and neurological disturbances. impact of resilience and spirituality on recovery in this setting has not been investigated. we have designed a study to validate two important scales, connor davidson resilience scale- and brief rcope spirituality scales for surrogate responders. hours with one or two surrogate responders will be included. this prospective cohort study will collect demographic, laboratory and radiographic data in a redcap database. for every patient enrolled, the cd-risc-and a behalf, themselves, and for each other. each patient will complete two resilience and two spirituality scale cd-risc and three spirituality scales . the scales will be administered to the patient ,if possible, prior to discharge; at months and at months. if patient ing followup. cars study has screened patients in days, enrolling patients. common diagnoses include subarachnoid hemorrhage ( ), cns malignancy ( ), intracranial hemorrhage ( ), unruptured aneurysm ( ), subdural hematoma ( ). based on current enrollment, this unique methodology for surrogate validation of scales is feasible. by august , an anticipated subject will be recruited. surrogate validation of quantitative measurements of resilience, spirituality can provide new insight into prognostication and patient centered critical care. active family engagement in the intensive care unit (icu) could improve patients' and families' experience with care, interactions with the healthcare team, and outcomes. this study examined the perceptions and attitudes of family members regarding increased engagement with passive mobilization of neurocritical care patients. an educational video on passive mobilization of icu patients was developed to engage family members to participate as valued members of the healthcare team. an anthropologist and a nurse or physician (study team) invited family of neurocritical care patients in an academic medical center icu in argentina their perceptions of engagement with care. a multidisciplinary team ( mds, rns, anthropologists) completed the analysis. thirty-two family members ( % female) of icu patients participated. the study team observed and the family participants reported to be positively surprised by: the format of learning by video that was different from how they usually received daily information; the information in the video was more detailed than anything previously taught; the invitation to watch the video and engage in patient's care was new and unexpected as was the opportunity to provide opinions on how to improve the video instruction. a newly authorized them to touch the patient and participate in care. they reported increased hope about prognosis and perceived an improved relationship with the healthcare team. only one family member considered the video irrelevant. family members' attitudes and perceptions toward this low cost approach to engagement were positive. this approach to teaching and engagement may help humanize the complex icu environment. - % of americans believe in the concept of miracles. we hypothesize that a belief in miracles leads to consultation rate of palliative care. addressing a patient or family's belief in miracles and understanding what a miracle signifies early in the neuro- the critical illness resilience and spirituality (cars) study is a prospective cohort study currently -icu at mount sinai hospital with an expected length of stay of at least hours and surrogate responders. as part of the study, all recruited patients and their families primary outcome is tracheostomy and peg tube placement. secondary outcomes include length of stay, full code status, and palliative care consultation. these groups will be matched with regards to the disease specific sever regression will be used to compare rates of the primary and secondary outcomes. the cars study has screened patients screened and enrolled patients so far. of those enrolled, the most common diagnoses include subarachnoid hemorrhage ( ), cns malignancy ( ), intracranial hemorrhage ( ), unruptured aneurysm ( ), subdural hematoma ( ). this is an ongoing study, we anticipate recruiting patients by the end of august, . belief in miracles could potentially influence continuation of aggressive measures in a shared decisionparadigm in the neuro-icu. mary m. barden, teddy t. youn, carolina c. maciel, sonya s. zhou, david d. greer. department of neurology, yale-new haven hospital, yale school of medicine, new haven, ct, usa. withdrawal of life-sustaining therapy (wlst) for predicted poor neurological outcome is a common cause of death among post-cardiac arrest patients. recent guidelines recommend against wlst before hours post-arrest. early wlst perpetuates a self-fulfilling prophecy that may contribute to premature death in some patients who otherwise would have survived with good neurological recovery. a retrospective cohort of resuscitated cardiac arrest patients from january to march at a single tertiary academic medical center was reviewed. patients were evaluated for outcomes at hospital discharge and (when applicable) the timing of and reason for wlst. prognostic indicators including clinical examination, electrophysiology, and neuroimaging were analyzed and findings were compared to day of wlst. of patients, ( %) had wlst due to perceived poor neurological prognosis. median day of wlst for this reason was post-arrest day . when stratified according to treatment with targeted temperature management (ttm), the median day of wlst remained day for both ttm-treated and non ttm-treated groups. of patients with wlst, the phrase "no chance for meaningful recovery" was used in documentation for ( %), mri results were cited as indicative of poor neurological prognosis for ( %), and pupillary light reflex was present day post-arrest (or day post-complete rewarming) in ( %). in a retrospective cohort of resuscitated cardiac arrest patients, wlst for predicted poor neurological outcome was the most common cause of death. the median day of wlst was post-arrest day . many patients with wlst had present pupillary reflexes on day post-arrest (or day post-complete wlst in the setting of indeterminate prognostic indicators undermines accurate neurological prognostication of post-cardiac arrest patients and perpetuates a self-fulfilling prophecy of poor outcome. brain injury global hypoxic ischemic brain injury (hibi) is a major cause of death and disability worldwide. invasive monitoring of brain function enables goal-directed treatment strategies that optimize cerebral physiology, reduce secondary brain injury (sbi), and potentially improve outcomes. we report a series of patients with hibi where intracranial monitors were placed to guide clinical management. retrospective analysis of patients with hibi cared for at a large academic center over a year period. all patients received therapeutic hypothermia (th) to °, continuous eeg monitoring, and had a bundle of invasive monitors placed through a multi-lumen cranial bolt. the full bundle consisted of an icp monitor, brain oxygen (pbto ) monitor, cerebral blood flow (cbf) probe, and cerebral microdialysis probe. patients received the full bundle, while the others received a partial bundle. patients were treated using a tiered algorithm designed to optimize cerebral physiological parameters. precipitants of hibi included cardiac arrest ( patients), airway occlusion during anesthesia induction ( patient), and hanging ( patient). mean patient age was years. average time between initial injury and probe placement was hours. average duration of monitoring was . days. no adverse events occurred after monitor placement. episodes of deranged cerebral physiology-including intracranial hypertension, brain hypoxia, cerebral glycopenia, metabolic crisis, and reduced perfusion leading to treatment changes occurred in of patients. they occurred up to days after initial injury, and in all cases would have otherwise been clinically silent. of patients died in the hospital. the surviving patients all regained consciousness and were discharged to acute rehabilitation facilities. we did not find invasive intracranial monitoring after hibi appears safe and identifies physiological states associated with sbi. goal directed treatment utilizing multi-modality monitoring in hibi merit further study. is associated with worse patient outcomes; however, it can be difficult to reliably detect. delirium prevention is therefore a potentially beneficial strategy and is most effective in patients who are at high to evaluate whether the advanced practice providers (app's) would both use the dps and also find the dps easy to use. during a --bed neurocritical care unit at a large -stratify consecutive admissions of patients with ais a descriptive statistics. the apps completed a -item questionnaire that included the system usability scale (sus) and open-ended questions to determine the usability of the dps, as well as to assess for facilitators and barriers for the use of the dps. no individual patient data was collected. patients admitted with ais and ich (n= ) were assessed by the app's using the dps. compliance with dps use was of apps (n= ). the sus score ( . ) was mid-point between "acceptable" and "excellent." facilitators and barriers for use of the dps were identified. the dps was easy to use and was consistently used by the app's. adoption of the dps with this patient population can be a first step to identify the most atthis vulnerable population. the intensive care unit is a complex learning environment with variability in a number of external factors. prior studies of neurology residency training in the neurological intensive care unit have focused on general exposure. this study aims to evaluate resident perception of neurocritical care training. an online survey was sent to program directors and neurocritical care members for distribution to neurology residents. the survey consisted of free-text or selection style questions that focus on resident perception of neurocritical training. statical analysis for group differences was completed with t or fisher exact tests a total of responses ( . % response rate) was obtained. of those responders, completed a freetext question regarding needed improvements to neurointensive care training. % responded with needs for educational changes, and these responders did not differ from other responder in average required practitioners ( % vs % p= . ), and neurocritical care attendings ( % vs % p= . ). this is the first study to examine neurology residents' concerns with neurocritical care rotations. there is little neurocritical care educational materials focused to neurology residents, but the emergency assess the neurocritical care educational training priorities during neurology residency are warranted. our institute had several cases of conflict come to light in the evaluation of patients being evaluated for death by neurological criteria. provider understanding and awareness of clinical guidelines was found to be low across all sub-specialties. it was deemed important to follow appropriate procedures based on published guidelines and a standardized process to provide appropriate care for each patient, optimize icu resource utilization and strengthen provider and public trust. due to medical, legal and ethical issues involved , an institutional standard was called for . -specialties about discrepancies, we revised the institutional policy to reflect emphasis of educational gaps and reflect the latest published guidelines and practice updates . we created an education module , a standardized template in electronic medical to allow escalation in case of conflicts . the project led to increased participation and satisfaction amongst the clinical providers in the icus when n education source to evaluate patients with a consistent approach based on published practiced parameters. we observed a trend in decrease in length of stay and variance for brain dead patients since donation referrals as well timely initiation and effectiveness of family discussions in irreversibly confirming this is in a survey model. a standard care pathway towards evaluation of patients with death by neurological criteria can be successfully implemented at an institutional level in a tertiary care academic medical center. establishing and maintaining optimal brain perfusion is a crucial endpoint for resuscitation and postcardiac arrest care. a recently fda-approved device that employs laser and pulsed doppler now provides clinicians with the cerebral flow index (cfi), a non-invasive measure of brain perfusion. we sought to determine if cfi provided by the ornim c-flow device can be used as a simple and valid measurement of brain perfusion after resuscitation in cardiac arrest patients. we performed a single-center prospective observational inception cohort study of adult patients with cardiac arrest starting in october . comatose patients with sustained return of spontaneous circulation (rosc) within minutes of maneuvers were included. the ornim cflow was connected as soon as feasible after rosc. clinicians were blinded to cfi values. primary outcome was survival at discharge and secondary outcome was neurological assessment using the cerebral performance categories (cpc) scale at discharge. a total of patients have been enrolled as of may . half ( . %) were out-of-hospital arrests and neurological outcome (cpc or ). mean interval between arrest and start of monitoring was hours with a mean duration of hours. adequate signal was available . % of the monitoring time. mean cfi in survivors was . , compared to . in non-survivors (p value . ). patients with good neurological outcome at discharge also had a higher mean cfi, although the small sample size precludes any conclusion. our results demonstrate that cerebral perfusion monitoring using the ornim cflow after cardiac arrest is feasible. it also suggests that higher cfi might be associated with survival at discharge. as enrollment progresses and more data are collected, further insight on the potential role of cfi as a neuromonitoring tool might emerge. tuberculous meningitis (tbm) is the most devastating form of tuberculosis, yet rates of neurological complications and mortality are uncertain in high-income countries. we used administrative claims data on all admissions at nonfederal hospitals to identify adult patients with tbm in california between -- , and florida between - . our outcomes of interest were mortality and the fo seizure, hydrocephalus requiring a ventriculoperitoneal shunt, vision impairment, and hearing impairment. kaplan-meier survival statistics were used to assess the cumulative rates of neurological complications and death. we identified patients with tbm, of whom . % ( % ci, . - . %) developed at least one neurological complication or died. more than two-thirds of these complications occurred during the initial hospitalization for tbm. individual neurological complications were not uncommon: the cumulative rate of - . %), the rate of seizure was . % ( % ci, . - . %), and the rate of ventriculoperitoneal shunting was . % ( % ci, . - . %). vision impairment occurred in . % ( % ci, . - . %) of patients and hearing impairment occurred in . % ( % ci, . - . %). the mortality rate was . % ( % ci, . - . %). nd death even in high-income countries such as the united states. neuropalliative care in peru: emergence from the conspiracy of silence the development of palliative care in peru remains limited, particularly for non-oncologic services such as palliative and end-of-life care in patients, families, nurses and physicians in a specialized neurological institute in lima, peru. we used a mixed methods approach consisting of surveys and qualitative, semi-structured interviews that were recorded, transcribed and analyzed using thematic analysis. surveys identified a substantial need for palliative care in the neurological institute ( % of doctors and of do emerged from qualitative interviews evolved around communication about end-of-life choices in neurologic disease. knowledge about advance directives was limited among both clinicians and families, and prognosis, and who should tell them. however, the perception that a physician should be honest, and that suffering and pain should be avoided at all times was unanimous. barriers to transparency in patientphysician communication included ( ) expectation of cure with medical treatment; ( ) families' trust in god training in communication, symptom management and end-of-life care; and ( ) a paternalistic culture. participants identified several challenges specific to palliative care in neurologic disease. in a country without a palliative care training program and no legal basis for advance directives, families and clinicians are emerging from a culture of silence about serious diagnoses and end-of-life care choices. our findings emphasize the need for palliative care education for neurology providers and the public in peru. a trained physician must perform the brain death examination in a systematic fashion in order to recognize and prevent potential sources of error. given the infrequency at which brain death presents in a hospital setting, clinicians may not always have the opportunity to observe a brain death examination during their training. in this study, we plan to evaluate the effect of medical specialty and expertise on documentation errors. we performed a retrospective chart review of brain death examinations between jan. to july st at the university of pittsburgh medical center presbyterian. physician specialty and training level, documentation errors, and confirmatory tests such as cerebral blood flow (cbf), electroencephalography (eeg), and computed tomography angiography (cta) were collected from medical records. exams. ams carried out. the most common completion of documentation by at least one examiner. attending physicians, residents and fellows were responsible for % ( neurology and neurosurgery residents have limited exposure to the brain death examination. regardless communication with patients and their families is of central concern in healthcare. however, evidence shows that it is often poorly addressed, especially at times of rapid health status changes and periods of clinical uncertainty. acute neurological emergencies pose an inherently unique challenge in communication. while emerging studies have addressed communication gaps and strategies to improve them in various critical care settings, none have assessed this issue in acute neurological emergencies ongoing irb approved prospective observational study in a bed neurocritical care unit in tertiary care academic medical center all patients admitted to the unit and all clinical providers participating in their care screened for inclusion. direct observation of discussions between clinical providers and families by a questions addressing the satisfaction, understanding of treatment options, impact on health care decisions and ways to improve communication five patients have been enrolled in the study, so far. the results are analyzed for concordance between tween answers is considered between all participants for general satisfaction with the communication (as well as family's understanding of treatment options explained to them by the physician. some disagreement on the impact of the discussion on health care decisions. qualitative domains identified by families as areas of good ical radiographic images. domains identified as needing improvement included explanation of medical circumstances and need for private room for discussions. we observed general satisfaction with communication. further enrollment will help elucidate any definitive areas of improvement and impact of communication on health care decisions. laith maali, sheema s. khan, mahmoud m. ismail, rhys r. brooks, vishnumurthy v. shushrutha hedna. the university of new mexico, albuquerque, nm, usa. cerebral venous thrombosis (cvt) usually accounts for < in their demographics, etiology, clinical features, radiological presentation, and mortality have not been previously explored. a systematic search was performed for publications in pubmed usi thrombosis", "cerebral vein thrombosis" and "cortical vein thrombosis". a total of relevant studies were abstracted with strict selection criteria and a total of patients' data were used for the final analysis. linear correlation was used for our descriptive analysis. cases reported were europe- , asia- , north america- , africa- , australia- and south america- . overall male to female ratio was : . , among clinical characteristics headache was the most common symptom and hematological factors were the most common etiology. location of the thrombosis was described mostly in the transverse sinus. intercontinental differences in relation to demographics, etiology, clinical features, radiological presentation, and mortality were identified. cvt can have significant disparity in their demographics, etiology, clinical features, radiological presentation, and mortality when compared from one continent to another. it is important for the worldwide physicians to recognize these differences and to follow the most recent guidelines, diagnostic methods and treatment to insure the best outcome and prognosis. timely communication is critical for high quality care in the intensive care unit(icu). published literature in surgical icus quotes up to % of patient caregivers receive prognostic information with mean prognostic interval . ± . days since icu admission prognostication in acute neurological injuries is challenging and uncertainty may delay communication. we assess occurrence and timeliness of goals of care communication in a neurocritical care unit prospective observational study by surveying nurses in a bed neurocritical care unit in tertiary level eriod. data was also collected during daily morning multidisciplinary huddle and verified by verbally surveying the nurses. survey results were analyzed for patients and patient encounters. in . % encounters , the nurses felt the patient's treatment plan matched patient-centered goals of care in . % encounters, a provider family discussion had occurred in the last hours. within the st hours of icu admission, . % patients were identified to need goals of care discussion in the multidisciplinary huddle, only . % had such a discussion. for patients needing goals of care addressed, a discussion occurred on an average . ± . days since icu admission. dichotomized by age, . % patients younger than years old had a discussion , if one was needed, while only % older than years had one. when dichotomized by gender, . % of males and . % of females had a provider discussion. . % females compared to % of males received a discussion on goals of care if identified as needed within st hours of icu admission. our data shows timely communication of goals of care in the neurocritical care unit with a mean time comparable to published literature. however, there appear to be demographic disparities that warrant further research. cerebral vasomotor reactivity reflect prognosis after cardiac arrest sungeun lee. ajou university school of medicine / department of neurology, suwon, korea, republic of. neurological prognostication after cardiac arrest is a difficult problem. since several studies reported good effect of target temperature management (ttm), prognostication after cardiac arrest was delayed and became complex. recently, some reports presented that impaired cerebral autoregulation was correlated with neurologically poor outcome. the aim of this study was to determine whether vasomotor reactivity (vmr) test by transcranial doppler (tcd), reflecting cerebral hemodynamic status, affected accuracy of neurological prognostication in post cardiac arrest patients. since january , patients were enrolled after cardiac arrest. patients who performed vmr test during ttm period were included and patient with unstable vital sign or malignant findings in brain ct, such as massive subarachnoid hemorrhage or severe brain edema, or poor temporal windows. primary outcome was cerebral performance category scale (cpc) at discharge. vmr test used breath-holding method during seconds. carbon d analysis. other conventional prognostication test, such as eeg, sep, et al., was performed after hours from rewarming time. we divided patients between good (cpc - ) and poor (cpc - ) outcome group and compared results from prognostic test between two groups. potential, and electroencephalography after hours from rewarming time were presented favorable results in good outcome group. (p< . ) vmr during breath-holding technique during ttm period also was more increased in good outcome group at right ( . ± . % vs. . ± . %, p< . ) and left ( . ± . % vs. . ± . %, p< . ) middle cerebral arteries. the present study shows that vasomotor reactivity is preserved in patients with neurological good outcome. to evaluating cerebral hemodynamic status by vmr test seems to be useful tool for early prognostication after cardiac arrest. michelle l. lozano, susan s. yeager. the ohio state university wexner medical center, columbus, oh, usa. as the numbers and opportunities for advanced practice providers (apps) in neurocritical care units (nccus) has increased, the integration of these providers into the health care setting has become a greater challenge. currently no data exists to support h comfort levels before and after completion orientation. this prospective, pre and post observational study was sent to newly hired nccu apps within an academic medical center. a one hundred-item survey was created to evaluate self-reported experience cus. baseline data was collected from each app. next, apps were integrated into the nccu utilizing a three month orientation program which fused a series of didactic, simulated, and precepted experiences. after e survey tool. student's t test statistics were utilized to compare before and after experience and comfort levels with items identified as necessary to perform in the nccu app role. as utilization of apps in the nccu becomes more prevalent, integration processes need developed to practice. a structured approach enables identification of high priority areas to assist with initial and and comfort levels. results indicate that further education and exposure to items such as neurologic imaging may be helpful. limitations of this study include subjective data from a small, self-reported, single institutional sample. further research of larger, more diversified sample representation is needed to validate whether these results can be generalized to other nccus. yasuhiro kuroda, kenya k. kawakita, toru t. hifumi. department of emergency medicine, kagawa university, miki, japan. brain damage after return of spontaneous circulation (rosc) varies among studies and patients despite an established modality enabling proper evaluation. evaluation of brain injury after rosc is needed for the determination of the inclusion criteria of neurocritical care, especially of targeted temperature management. literatures are reviewed and summarized. the association between admission glasgow coma score (gcs) motor score and neurologic outcome after rosc (day ) is an independent predictor of good neurologic outcome at days in patients sustaining out-of-hospital cardiac arrest who receive therapeutic hypothermia: gcs motor score , n= ( . %); score - , n= ( . %); score - , n= ( . %), p< . (hifumi ). recently no significant differences of neurologic outcome at days after hospital admission was observed between mild therapeutic hypothermia and control in the subgroup of gcs motor score or . these data show that initial gcs motor score examination immediately after rosc can at least provide baseline objective prognostic data for decisions by healthcare professionals. neurological signs such as gcs, brain stem reflex, respiratory status, and degree of shivering are potential variables that can be incorporated into a predictive model for a more precise evaluation of brain injury in cardiac arrest survivors undergoing ttm. effect of targeted temperature management should be evaluated depending on the brain injury in pcas. cydni n. williams, jennifer j. wilson. oregon health and science university, department of pediatrics, portland, or, usa. -level estimates of et utilization in pediatric ais, and explore demographic and clinical characteristics, associated interventions, and outcomes. retrospective cohort analysis of the kids' inpatient database evaluated et utilization in children with ais and age > days, identified by diagnosis and procedure codes. analyses were weighted for national estimates and compared with chi-square and t-tests. among pediatric ais patients, ( %) received et. anterior circulation occlusions were seen in % of et patients. et patient age ranged versus %, p<. ) was more common and seizure was less common ( % et versus %, p=. ) in et patients. average age was higher with et ( versus years, p<. ). other patient demographics, hospital characteristics, and critical care procedures were similar. thrombolytic agents (tpa) were common with et ( % et versus % overall). intracranial hemorrhage was similar ( % et versus %, p= . ), and varied by tpa ( % et with tpa, % tpa only, % et only, % neither). there was a nonsignificant trend toward poor outcome (death, discharge to nursing facility, tracheostomy, or gastrostomy) was seen between poor outcome and et ( % et versus %, p=. ). et in pediatric ais is uncommon, utilized mostly in older children and those with paresis. though hemorrhage was uncommon, this data suggests caution with et and tpa combination. associations between et and poor outcome may reflect disease severity bias. more research on outcomes with et in pediatric ais is needed. luis p. lee, michael m. leoncio, balagangadhar b. totapally. nicklaus children's hospital / pediatric critical care department, miami, fl, usa. and cerebral edema is the most serious complication leading to morbidity and mortality. we queried a nationally representative database to determine epidemiologic data of cerebral edema in children with dka. an analysis of the healthcare cost and utilization project's kids inpatient database for the year was performed. the database was filtered using icd- diagnosis codes for dka ( . , . , . , . ) and cerebral edema ( . ) from the age of month to years. we examined these procedures, outcome and mortality rates. sample weighing was employed to produce national estimates. chi-square test, mann whitney u test and binary regression analysis were performed using spss to analyze the data. a total of , patients with dka were discharged during . females were %. racial distribution - ) years. cerebral edema was present in ( . %) children. the overall mortality rate was . %, but the mortality rate in children who developed cerebral edema was higher at . % (or: ; % ci: - ). mortality was higher in children who had a major operative procedure ( . % vs . %; or , % ci: - ) and in those with medicaid compared to private insurance ( . % vs . % p= . ) and lower in number of chronic conditions, and hospital charges were significantly higher among non-survivors but there was no difference in the age. the overall mortality rate in children admitted with dka is . %. cerebral edema prevalence is . % and it increases mortality significantly. mullai baalaaji, sunit s. singhi, muralidharan m. jayashree, arun a. bansal. pediatric intensive care unit, department of pediatrics, pgimer, chandigarh, india. near-infrared spectroscopy (nirs), a non-invasive modality to measure regional cerebral oxygenation (rso ), is being increasingly used to monitor cerebral tissue oxygenation. we studied relationship of rso with cerebral perfusion pressure (cpp) and intracranial pressure (icp) in children with acute cns infections to determine if rso could be used as non-invasive surrogate for cpp. in a prospective observational study we enrolled children, aged < years, with raised icp due to acute cns infections after approval by institutional ethics committee. they were monitored simultaneously for rso of both frontal-- c, covidien-iic), invasive blood pressure, and icp using intraparenchymal fibre-optic catheter (codman). linear trends and correlation coefficients were used to define relation of rso with icp and cpp. a total of paired values of rso , icp and cpp were analysed. the linear trends during the first hours revealed no significant correlation between changes in rso and changes in icp and cpp from baseline (r = . , . for icp and cpp respectively). however, the trend was not uniform - % patients had no correlation between rso and cpp, % showed a positive correlation and % showed a negative correlation. subgroup analysis revealed that strength of correlation between rso and - . ,p mmhg and normal cpp were . ( . - . ,p % respectively. rso has complex interaction with icp and cpp; the changes in icp and cpp could not predict changes in rso . however, the odds for normal cpp was significantly higher when rso > % and this cut-off could be used as a non-invasive target for age appropriate cpp. refractory status epilepticus is persistent seizure activity despite treatment with one first-line and one second-line anti-epileptic medication, while seizure activity > hours is considered super-refractory. functional outcome for children with these conditions is not well defined. this study describes functional outcome for children with refractory and super-refractory status epilepticus proposing that prognosis will be variable with high mortality. survivors will be and technology dependence. this retrospective chart review evaluated children age - years who received pentobarbital infusion at texas children's hospital pediatric intensive care unit from - for status epilepticus. outcome was defined using pediatric cerebral performance category score (pcpc) at time of discharge and at the most recent clinical evaluation per the medical record. additional measures included mortality, need for medical technology (tracheostomy or gastrostomy tube), seizure burden, and number of seizure medications at discharge. children met inclusion criteria. in-hospital mortality was %, secondary to withdrawal of support ( %), brain death ( %), or cardiac arrest ( %). highest mortality occurred in acute hypoxic ischemic injury (p= . ). of survivors, % returned to baseline pcpc at discharge while % demonstrated tracheostomy and children underwent gastrostomy tube placement. seizures persisted at discharge for most patients with no prior frequency. most children required additional home seizure medications. long-term follow-up was documented for survivors up to years after discharge. % demonstrated improved pcpc and % showed decline including additional deaths. mortality in this population was high. functional outcome in survivors was variable. some children returned to neurologic baseline by time of discharge and for those who did not, continued functional improvement was possible over time. their s -injury. in addition to standard anatomic imaging, mr sequences obtained "often or always" included: diffusion--perfusionpediatric tbi subjects received an acute mri within days post-injury. fifteen adapt sites, accounting for over % of adapt enrollment, committed to recruit adapt subjects for a non-sedated mri scan at one year post -tbi. conclusion: collection of - acute mri scans from the subjects enrolled in adapt to study associations between acute mri findings and functional outcome is potentially feasible. allowing for % mortality and % recruitment rate, recruitment of - adapt subjects from adapt sites for a follow-up mri to study relationships between advanced mri measures and neurocognitive function is potentially feasible and would represent the largest such study conducted to date. antimicrobial prescribing practices and antibiotic resistance following neurosurgical drain placement: a single-center observational study andrea j. passarelli, hasan h. alhasani. christiana care health system department of pharmacy, newark, de, usa. the use of systemic antibiotics for the duration of neurosurgical drain placement has not been associated with reduced rates of drain related infection (dri) and may contribute to the development of antimicrobial resistance and clostridium difficile infection (cdi). we sought to describe antimicrobial prescribing practices, incidence of dri, and development of antimicrobial resistance and cdi after neurosurgical drain placement at our institution. this was a single center study including adult patients status post ventriculostomy or ventriculoperitoneal shunt or ommaya reservoir, and use of an antibiotic impregnated drain. bacterial cultures and c. difficile pcr during the index admission and days post-discharge were collected. antibiotic resistance was defined as an organism resistant to the prophylactic agent. prolonged prophylaxis was defined as antibiotics continued for > hours after drain placement. eighty-one patients with drains were included. the median duration of prophylaxis was . days and cefazolin was most commonly prescribed agent ( %). three of patients with evds developed dri. prolonged vs. perioperative prophylaxis. of non-dris % were resistant to the prophylactic agent used. e. coli, k. oxytoca, and s. aureus had higher rates of resistance to cefazolin compared to our institutional antibiogram, although not statistically significant. no patients developed cdi. the use of prolonged prophylaxis was not associated with a reduced reduction in dri. most bacterial isolates were resistant to the prophylactic agent used. we suggest that antibiotic prophylaxis for neurosurgical drain placement be limited to one preoperative dose within minutes of the procedure the objectives of this study are to evaluate our institution's practice for initiating seizure prophylaxis postoperatively and establish a standard of care. adult patients who underwent cerebrovascular surgery from august to july were screened for study inclusion. patients who received lev postoperatively were compared to those who did not receive lev. clinical seizures and data were obtained from retrospective review of electronic medical records. the primary outcome was seizure occurrence in the first days after surgery. secondary outcomes of the patients included in the study, there were in the no lev group and in the lev group. two seizures occurred in the no lev group while no seizures occurred in the lev group ( vs , p= . ). there were no differences between surgery type, intraoperative blood loss or proportion of asah. of the patients with asah, % were not on lev and seizure occurred. of patients with intraparenchymal or intraventricular extension, % were not on lev. average length of stay was prolonged for the lev group ( vs days, p< . ). the majority of patients did not receive lev postoperatively and there was no difference in seizure developing a standardized approach for initiating lev may decrease variability in practices and streamline postoperative care. post-operative pain control after craniotomy: a meta-narrative review craniotomy is commonly performed for the treatment of a variety of conditions including brain tumors, aneurysms, and vascular malformations. despite significant advances in the quality and efficacy of neuroanesthetic care, there are no evidence-based guidelines for the management of post-operative pain after craniotomy. uncontrolled poststay, increased hospital care costs, and poor health-dencebased clinical decision rules, clinicians often rely on institutional or expert-based opinions to guide their decisionon opioid use, there an urgent need to evaluate existing pain management protocols. hence, we conducted a meta-narrative to evaluate heterogeneity in current practices regarding management of postoperative pain after craniotomy. a meta-narrative review was performed utilizing th terms "pain" and "craniotomy". a total of articles and systematic reviews were resulted. inclusion criteria were studies from - , randomized controlled trials, retrospective studies, systematic reviews, case reports, case series published in english were included. of these, articles and systematic reviews were included in the final analysis. there is a paucity of randomized controlled trials to develop evidence based peri-operative pain management protocols in craniotomy patients. there is evidence to suggest that scalp infiltration with local anesthetic may improve post-operative pain scores immediately after surgery. the perioperative use of nonsteroidal anti-inflammatory medications may improve pain scores without a subsequent increase in management of post-operative pain after craniotomy remains a challenging problem for clinicians and patients. there is an urgent need to conduct well designed randomized controlled trials to guide perioperative pain management in craniotomy patients and to use opioid sparing techniques for improving patient outcomes. symptomatic plateau waves are characterized by paroxysmal neurological symptoms suggestive of elevated intracranial pressure such as depressed level of consciousness, pupillary dilatation, and dysautonomia in a patient with an intracranial mass lesion. cli seizures, syncope, or new brain injury. noninvasive cerebral blood flow can be measured using ultrasound-tagged infrared spectroscopy; continuous eeg is sensitive to changes in blood flow. we report two patients without invasive intracranial pressure monitoring who demonstrated changes in blood flow and eeg during symptomatic plateau waves. case series. case was a year-old man with fungal ventriculomeningitis. after a prolonged hospital course, he developed an entrapped th ventricle and began to experience periods of complete unresponsiveness with anisocoria, clonus, and tachy-or bradycardia lasting between and minutes. episodes resolved after decompression and ventricular stent placement. case was a year-old woman with intraventricular meningioma who underwent partial resection with entrapment of the right lateral ventricle. on post-operative day she developed multiple episodes of unresponsiveness, diaphoresis, clonus, tachy-or bradycardia lasting to minutes, culminating in a persistent episode requiring urgent craniotomy. in both patients, ceeg was started to assess for seizures and ornim device was used to characterize blood flow. in each, symptomatic plateau waves were accompanied by decreased blood flow, followed by attenuation of faster frequencies on the ceeg. symptomatic plateau waves may be characterized noninvasively by using surface measurements of blood flow and ceeg. this case series demonstrates that decreases in bifrontal blood flow lead to depressions in ceeg during these symptomatic plateau waves. noninvasive measurement of blood flow in conjunction with ceeg provides an adjunct to invasive icp monitoring in patients with mass lesions at an open--sseefficacy of sage- , a proprietary formulation of allopregnanolone, in patients with super-refractory status epilepti line agents (tlas) while sage-(none attributed by the safety committee to sage- ). post-hoc analyses evaluated the pharmacological effects of sage- and the effect of sage- administration in the context of multiple antiepileptic drugs (aeds), pressors, and tlas. here we examine the hemodynamic properties of sage- in the study patients, with the goal of further understanding the clinical context of sage- administration in this critically ill population. burst suppression we maintenance of the tla. key exclusion criteria were anoxic brain injury and very short life expectancy. at enrollment, patients received an average of . aeds and . tlas with an average status epilepticus duration of . days. hemodynamic measurements (heart rate, systolic and diastolic blood pressure) were collected at screening, pre-dose, during sage- treatment ( , , , minutes; , , , , , , , hours) and followwas examined. twenty-five patients received treatment with sage- . during the study, mean changes in hemodynamic parameters from baseline were limited, both for patients receiving the standard (n= patients) and high (n= patients) sage- dose. regarding hemodynamic parameters, sage- was well tolerated in the srse patients studied, suggesting for further study that sage- may not elicit immediate or sustained hemodynamic changes in srse patients. real-world studies regarding use of benzodiazepines in pre-hospital and emergency department (ed) was to analyze benzodiazepine usage patterns in se by emergency medical services (ems) and the ed of an inner-city hospital. and september to ems and hospital ed were reviewed. the associated outcomes of interest were endotracheal intubation, hospital admission, and seizure recurrence. data was analyzed via descriptive statistics. of patients analyzed, ( . %) had a history of epilepsy. benzodiazepine utilization varied; ems preferred midazolam ( . %) while the ed used lorazepam most often ( . %). benzodiazepine dosages used were lower than recommended; median dose of midazolam administered by ems was only mg and median dose of lorazepam in the ed was mg. patients received . ± . benzodiazepine doses on average. seizure activity was aborted with benzodiazepines alone in ( . %) patients and recurred in ( . %). twenty-three ( . %) patients were intubated, all post-arrival. there was no observed correlation between number of benzodiazepine doses given and baseline characteristics, decision to intubate, or incidence of seizure recurrence. all patients were admitted and ( . %) were admitted to the icu. we observed consistent underdosing of benzodiazepines used for the treatment of se by both ems and the ed. there was lower than expected achievement of seizure cessation and intubation rates were higher than reported in previous studies. further investigation is needed to identify the barriers to optimal benzodiazepine selection and dosing for se patients at our institution. super-refractory status epilepticus (srse) refers to a condition of persistent seizures that have failed treatment with first-, second-and third-line treatments. sage- , a proprietary formulation of the endogenous neuroactive steroid allopregnanolone (a potent positive allosteric modulator of synaptic and extrasynaptic gabaa receptors in animal models), is being developed for the treatment of patients with srse who have not responded to standard treatment regimens. -sse- was an open-label, phase - in patients with srse. the present analysis explores the pk properties of sage- over the course of the trial. sage- was administered as a -day continuous intravenous infusion to patients with srse and receiving third line agents (tlas) for seizure or burst suppression. patients received either a standard dosing regimen (n= ) or a high dose regimen (n= ) and were subsequently weaned off tlas and sage- . the standard dose was chosen, based on a modeling approach, to achieve a mean plasma exposure roughly equivalent to the highest endogenous concentrations measured in the third trimester of pregnancy (~ nm). since women tolerate this endogenous level without apparent adverse effects, mean (sd) steadyand for the high dose regimen wa -state concentrations were approximately dose proportional between the standard and high doses, indicating that clearance was dose-independent of infusion to allow determination of half-life or volume of distribution. in this study of patients with srse, sage- clearance was not dose-dependent and plasma concentrations were in line with target exposures. jennifer a. creed, christa c. swisher. duke university medical center / department of neurology, durham, nc, usa. ****permission was not granted to print this abstract**** seizures after resuscitation from cardiac arrest predict worse outcomes, but there is no evidence that treating seizures improves outcomes. we leveraged existing practice variation to compare the effectiveness of aggressive electroencephalography (eeg) and antiepileptic drug (aed) use to infrequent spot eeg and aed use. we performed a retrospective cohort study including comatose post-arrest patients at two academic centers from - . the same critical care group staffs both, but center uses continuous eeg (ceeg) monitoring and aggressively treats malignant eeg patterns while center uses infrequent spot eegs and rarely treats with aeds. we classified each patient's daily eegs from admission until death, malignant," or "not performed." we abstracted covariates and outcomes from our prospective registry, e rhythm, arrest location, survival to discharge and functionally favorable survival. we used multi-level mixed-effects logistic models to test for an association of center with outcomes after adjusting for eeg and clinical covariates. we included subjects (center : , center : ). center subjects were younger, arrested more often out-of-hospital and had higher illness severity (all p< . ). overall, ( %) center subjects were eeg-monitored (median days (iqr - d)), ( %) had a malignant pattern observed and median of d (iqr - d), ( %) had malignant patterns observed (less frequent myoclonic status epilepticu center comparisons). in multilevel modeling, there was no significant center effect on outcomes. after cardiac arrest, treatment at a center using aggressive ceeg monitoring and aed treatment is not associated with better outcomes at discharge. phenytoin dosing adjustment for obesity may not be necessary effective loading with weight-based dosing of phenytoin for therapeutic levels is necessary in several emergent settings. practices for dosing obese patients, those > . x ideal body weight (ibw) vary, including using total body weight (tbw), adjusted body weight (adjbw; correction factor . ), and the abernathy formula (correction factor . ). our objective was to determine whether dose adjustments were necessary for obese patients. charts were reviewed retrospectively from two tertiary medical centers from september to august . we included all admitted patients older than years of age, initiated on iv fosphenytoin for any reason, with therapeutic post-load level (total phenytoin of -in pre-mean weight-based loading doses were compared for obese and non-obese patients who achieved postload levels in the therapeutic and high therapeutic range (total level - , free . - . ), using welch's two-sample t-tests. a total of patients, non-obese and obese, met inclusion criteria, including non-obese and obese patients who achieved high therapeutic levels, desired for ongoing status epilepticus. the mean -obese and . ( % ci: . , . ) for obese patients (t = . , p = . ). dose to achieve high therapeutic levels was . ( % ci: . , . ) for non-obese and . ( % ci: . , . ) for obese patients (t = . , p = . ). our results suggest that adjustment calculations of fosphenytoin loading dose for obese patients may not be necessary, thus can all patients into high therapeutic range, especially desirable in status epilepticus, while not harmful in other patients. stephen sage- is a proprietary formulation of allopregnanolone. sage- was studied in an open-label nical study of patients with super-refractory status epilepticus (srse). the primary - . - in resolving srse in these patients. to further understand the specific patient and treatment-related parameters which may affect outcomes in this study, we performed a post-hoc analysis on the completed data set. in this open-label, single-treatment with sage- . key efficacy outcome measures were: ) successful wean off of tla(s) after hour ; and ) subsequent successful taper off sage- after hour , without recurrence in the hour period following treatment. a total of patients received open-label treatment with sage-sage- . response rate appeared consistent across varying patient demographics (gender, age, ethnicity) and baseline treatment regimens. overall, % of patients experienced at least serious adverse event (sae) and patients died during the trial. no saes and no deaths were attributed by the safety committee to sage- administration. there was little evidence of a relationship between response rate and patient demographics (gender, -trial supports further investigation of sage- in srse, and can inform inclusion criteria for future trials. the clinical efficacy and safety of sage- in the treatment of srse is being evaluated further in an ongoing phase , randomized, placebo-controlled trial. jocelyn y. cheng. drexel university college of medicine, philadelphia, pa, usa. in catastrophic neurologic injury, withdrawal of care (woc) is often considered. while woc is based on the perception of poor prognosis, the question of whether it creates a self-fulfilling prophecy has been raised. though studied in traumatic brain injury epilepticus (se) is unclear. the goal of this study was to describe the final cause of death in adults with se, and determine the impact and associated clinical characteristics of woc on mortality rates. a single-center retrospective study at an urban academic medical center was conducted between age;gender;glasgow coma scale (gcs);acute physiology and chronic health evaluation-ii (apache-ii);history of epilepsy;etiology of se;refractory se (rse);in-hospital mortality; and cause of death. -tests were used as appropriate. binary logistic regression analysis adjusted for covariates, and p < . was considered significant. of subjects, male, mean age years, there were ( . %) in-hospital deaths, ( %) due to woc. the remaining causes were cardiogenic(n= , %) and respiratory(n= , . %), with sepsis, brain death and seizures individually comprising . %(n= each). excluding woc, in-hospital mortality fell to -group without cardiac arrest (ca), inof which %(n= ) was due to woc; mortality decreased t the total cohort, there was no significant difference in baseline characteristics excepting metabolic seizure were more common in woc subjects. metabolic etiology (or: . ,p= . ) and ca (or: . ,p= . ) remained significantly associated with woc after univariate but not multivariate adjustment. withdrawal of care is a major determinant of mortality in se, and is associated with metabolic dysfunction clinical decision- an open--sse- ) evaluated the safety and efficacy of sage- , a proprietary formulation of allopregnanolone, in patients with super-refractory status epilepticus (srse). entry criteria were designed to maximize patient treatment opportunities. the objective of this post-hoc analysis was to demonstrate sage- activity despite heterogeneity of srse causes and high comorbidity burden. d-line agent (tla; with anoxic brain injury or very short life expectancy were excluded. - years) and mean status epilepticus duration was . days (range - days). probable srse causes included infection, hemorrhage, worsening cgi-s score. all patients required - tlas and - aeds at baseline. up to weans from tlas were sage- at the end of da six patients ( %) died from underlying srse cause or associated comorbid conditions. no saes were attributed by the safety committee to sage- . mean numbers of baseline wean attempts, comorbid conditions, and srse episode duration were comparable between responders and non-responders. activity despite the heterogeneity of cause of srse and comorbidity burden. delayed neurologic deterioration (dnd) from vasospasm is associated with poor outcome after subarachnoid hemorrhage. continuous eeg (ceeg) monitoring has lead to detection of eeg patterns of uncertain clinical significance -ictal-interictal continuum (iica). these have been associated with acute brain injury but variably predict outcome. we describe the types and prevalence of iica eeg patterns in patients who develop angiographic vasospasm and discharge outcomes retrospective study of adult patients with non-traumatic subarachnoid hemorrhage admitted at emory university hospital neuro-icu from january -february who underwent ceeg. ceeg were recorded using - electrode placement and interpretation of the iica eeg patterns using the american clinical neurophysiology society research terminology. in sah patients, ( %) were female and hypertensive ( %). majority had poor grade sah ( % hh grade , % grade and % grade ). moderate to severe angiographic vasospasm were detected in ( %) patients . days after admission. ceeg was initiated . days after admission. periodic discharges (pds) occurred in ( %) patients, ( . %) of which were generalized and ( . %) lateralized. rhythmic delta activity (rda) occurred in ( . %) with ( . %) generalized. stimulus induced rhythmic discharges (sirpids) were seen in ( . %) and electrographic seizures in ( . %) patients. vasospasm was common in patients with any iica patterns ( . % vs. . % p= . ), pds ( . % vs. . % p= . ) and rdas ( . % vs. . % p= . ). rdas were common in patients with discharge mrs - ( . % vs. . % p= . ) and pds were equally seen across all outcomes ( % vs. % p= . ). the observed trends were not statistically significant. iicas such as pds and rdas were common in patients who developed vasospasm but seen equally with a larger sample size is needed to support these findings. ncs and (ncse) have been reported in - % of critically ill patients. whether patients with ncse should be treated as aggressively as patients with convulsive status remains controversial. this study sing on its correlation with patients' outcome and possible predictors. in this retrospective study n= patients underwent ceeg at mount sinai neurological and neurosurgical icu (nsicu). ceeg data according to acns guidelines was collected. outcome was evaluated by mortality, glasgow outcome scale (gos), glasgow coma scale (gcs). we compared seizures were detected in % of subjects and % experienced of cg (p= . or . ci . of cg (p= . , or . , . considering clinical predictors, only gaze deviation and subtle facial movements were significant (p= . or . , p= . , . mortality and mean hospitalization length were not different. outcome was significantly different in nsicu with mean gcs being in ng and in cg (s for p= . ), but was not significantly different at discharge, as mean gos was in ng and . in cg (p= . ). our findings show that patients' history of epil rstand prognostication in these patients. raquel farias-moeller, archana a. pasupuleti, luca l. bartolini, amy a. kao, brittany b. cines, jessica j. carpenter. children's national health system, washington, dc, usa. super refractory status epilepticus (srse) ensues when there is no improvement of seizure control in response to anesthetic therapy or seizure recurrence after reduction of anesthetic agents. there is no consensus on standard of care for srse. ketogenic diet (kd) has reported success but technical challenges exist including inability to feed patients, concomitant steroid use, persistent acidotic states and ur step-by-step approach to initiation and continuation of kd in the picu. patients with srse who had kd initiation in the picu were identified from a prospective neurocritical care database with irb approval. data from the hospital course was supplemented by review of the electronic medical record. descriptive analysis was performed. neurointensivists used our step-by-step guideline to start patients on kd. nine children with srse who had kd initiated in the picu were identified. the mean age was . years (sd . ). median number of days to start kd from detection of seizures was . mean time nine children remained on the kd for months or longer. the median number of aeds trialed before kd was started was [iqr - ] and the median number of continuous infusions was [iqr - ]. after initiation of kd most patients were weaned off continuous we demonstrated the feasibility of a practical approach to initiation of kd in the picu for children with srse. these children were successfully weaned off continuous anesthetic infusions. larger studies, both in children and adults, are needed to determine the effectiveness, safety and tolerability of kd in the management of srse as well as its ease of implementation. although overall mortality of status epilepticus is high, baseline patient characteristics and co-morbidities may help to predict outcomes and shape treatment decisions. two previously published scores exist to predict outcomes: the status epilepticus severity score (stess) and the epidemiology-based mortality score in status epilepticus (emse). however, a comparison of the two scores has not previously been completed in an american population. we hypothesize that both scores will adequately predict the primary outcome of in-hospital death. we performed a retrospective analysis of all cases of status epilepticus admitted to the neuro-critical collected data on age, comorbidities, eeg findings, and seizure history. the primary outcome was inhospital death. a sensitivity and specificity analysis was completed, in addition to a student's t-test for a comparison of the two scores. forty-six patients were admitted to the nccu for management of status epilepticus during june and january , of which experienced in-hospital death. the median age of the sample was , with approximately half of the sample ( . %) having or more comorbidities. the two most common etiologies were cryptogenic (n= ) and acute cerebrovascular events (n= ). while the sensitivity of both emse and stess were very high ( % and % respectively), the specificities were very low ( . % and . % respectively). a student's t-test between those who experienced in-hospital death and those who did not was only significant for emse at the p< . level (p= . ). the specificity of emse and stess for our external validation did not correlate with previous studies; however, both tools are sensitive. the emse and stess may be useful to predict outcomes of status epilepticus in populations with few comorbid conditions, but are less helpful when patients have multiple medical problems. in , the acns published critical care eeg terminology in an effort to improve clinical research and management of patients requiring continuous eeg (ceeg) monitoring. we sought to understand the familiarity of providers in our neurocritical care (ncc) program with this terminology two years after implementation at our institution. we administered a question web-based survey to ncc fellows, advanced practice providers (apps), tanding of acns terminology, and clinical eeg application. there were attending physicians, ncc fellows, and apps. attending physicians and apps had a median of (range: , ) and years (range: . , ) experience since most recent post graduate training, respectively. all data is reported for apps and physicians respectively as percentage correct. highest rated component of the ceeg report that influenced patient management was "conversation with lected by . % of apps and % of physicians. set forth by the acns and highlight the importance of communication between ncc providers and epileptologists as well as areas of potential education for providers of all training levels. carbamazepine (cbz), an oral antiepileptic drug (aed), is a potent inducer of cytochrome p (cyp) (eg, phenytoin, fosphenytoin, phenobarbital, valproic acid, levetiracetam, or lacosamide) to reduce the -drug metabolism (reduced efficacy or toxicity). an iv cbz formulation has been developed; study ov- (nct ) evaluated bioequivalence and a (nct ) evaluated tolerability. both studies were similarly designed. eligible adult patients received a stable oral cbz regimen ( daily dosage (divided doses q h) during the confinement period (ov- : -or -min infusions q h for days, patients in the -min group were eligible to receive four -to -min infusions on day ; a: -min infusions q h for days, then one -min infusion on day ). oral cbz was resumed for days ( a: days). bioequivalence of iv to oral cbz was evaluated in ov- ; tolerability data were pooled. in ov- , -min iv cbz infusions were within the %- % bioequivalence range vs oral cbz; min infusions exceeded the upper limit for maximum plasma concentration. in both trials, patients switched to iv cbz ( -min: n= ; infusion was dizziness ( %); infusion-site reactions ( %) were the only new aes experienced by patients vs oral cbz. seizure control was maintained during the switch. to avoid potential drug toxicity reactions, it is beneficial to maintain patients on cbz. iv cbz administered as multiple -min infusions was bioequivalent to oral cbz. iv cbz was well tolerated. treatments for aneurysmal subarachnoid hemorrhage (asah) remain inadequate. eg- is a sustained release formulation of nimodipine for intraventricular delivery in order to avoid dose-limiting -label, dose escalation study of a single intraventricular dose of eg- that was designed to determine the maximum tolerated glasgow outcome scale (www.clinicaltrials.gov identifier: nct ). subjects with asah repaired by clipping or coiling were randomized within hours of asah to eg- or oral nimodipine if they were world federation of neurological surgeons grade to and had a ventricular catheter. cohorts of subjects received , , , , or mg eg- ( per cohort) or oral nimodipine ( per cohort). plasma nimodipine concentrations were sustained for days. the maximum concentration, steady state concentration and area under the curve for the first days increased with increasing dose of egbetween males and females. plasma nimodipine concentrations following eg- administration did not exceed plasma concentrations of oral nimodipine mg every hours at steady state. cerebrospinal fluid nimodipine concentrations with eg- were orders of magnitude higher than in plasma or with oral nimodipine. subjects treated with eg- (n= ) had a median intensive care stay . days less and hospital length of stay . days less than subjects treated with enteral nimodipine (n= , table) . intraventricular eg- produced sustained, dose-dependent nimodipine plasma concentrations and shortened intensive care and hospital length of stay. improved clinical outcome support conduct of a pivotal phase study of eg- . increasing exposure to ionizing radiation for medical diagnostics and treatment has raised questions about possible long term effects. this study describes the effective dose of ionizing radiation exposure in patients with aneurysmal subarachnoid hemorrhage (asah). thirty-five asah patients admitted to a university hospital between jan , and december , , greater than years old, and discharged alive were included. the primary objective was to calculate the mean total effective dose of ionizing radiation (tedir) in asah patients. secondarily, the number of asah patients with a calculated tedir greater than the annual and cumulative maximal permissible radiation dose (mprd) as described by the national council of radiation protection and measurements (ncrp) and the international commission on radiological protection (ircp) was determined. factors associated with greater than maximal exposure limits were evaluated. tedir exposure ranged from . - . millisievert (msv), with a mean (sd) of . ( . ) msv. seven , the presence of vasospasm (p< . ), external ventricular drain (evd) (p < . ), or ventriculo-peritoneal shunt (vps) (p< . ) were statistically significant factors for increased tedir in asah. demographic data, previous medical history, and location of aneurysm were not statistically significant. univariate analysis representing the degree to which tedir increases for each of these factors revealed hh class ( or ) . (p< . ), vasospasm . (p< . ), evd . (p< . ), vps . (p< . ). in multivariate analysis representing the degree in which the tedir increases, only vasospasm . (p< . ) and evd . (p< . ) were statistically significant factors. following asah, patients with severe disease requiring vasospasm treatment and shunting are at warranted. patients with aneurysmal subarachnoid hemorrhage (sah) have high morbidity and mortality related to cerebral ischemia and infarction. in this study we explored the reversibility of reduction in cerebral blood flow (cbf) after sah. we conducted a retrospective analysis using prospectively collected ct perfusion (ctp) data from sah patients. patients were grouped as good (hunt hess - ) and poor grades . ctp data were compared at baseline ( - days after aneurysm rupture) and follow-up (> days). cbf at baseline was comparable between good and poor grade patients ( . ± . vs. . ± . -up there was an improvement from baseline in both groups ( . ± . and . ± . respectively, p= . ). however, in hypoperfused areas, rcbf was significantly lower in poor grade patients compared to good grade ( . ± . vs. . ± . , p= . ) and significantly lower than global cbf in both groups (p< . ). at follow-up, only poor grade patients demonstrated an increase in rcbf ( . ± . , p= . ) while in good grade patients, rcbf remains unchanged ( . ± . , p= . ). the absolute improvement in rcbf was significantly greater in poor grade patients ow-up in both groups was not statistically different in the two groups (p= . ) but significantly lower than global cbf (p< . ). regional hypoperfusion occurs after sah. in good grade patients there is no recovery in rcbf while in poor grade patients there is partial recovery implying a biphasic response with a reversible and an irreversible reduction in rcbf. this has not been previously described in the literature and may implicate two distinct mechanisms responsible for rcbf reduction after sah. aneurysmal subarachnoid hemorrhage (asah) is an important cause of morbidity and mortality, with patients susceptible to a variety of medical complications. external ventricular drains (evds) are commonly used for intracranial pressure monitoring and csf drainage; however, this puts asah patients -associated infections (vais). many preventative strategies have been proposed and implemented over the last years. using the nationwide inpatient sample (nis) database, this study examines trends in evd usage, vai rates, and mortality over a -year period. in this retrospective analysis, data from the nis was obtained for the period of january , through december , using international classification of diseases, th revision (icd- ) codes. analysis was performed using sas . surveymeans. primary outcomes of interest were rates of evd use, vai and in-patient mortality, as well as hospital length of stay. during the study period, there were , asah admissions, with evd placement reported in . % of cases. there was no change in either evd use or rate of vai (mean vai rate of . % over the years). no change in hospital length of stay was observed. from january , to december , , despite a variety of vai-preventative strategies advocated for and implemented, the national vai rate in asah patients has not changed; however, mortality has declined by a mean arr of . % per year over the study period. this may reflect improved neurointensive care provided to this critically ill population. stable vai rates may reflect incomplete adoption of effective preventative strategies, or use of ineffective o study has previously examined these trends in asah. the purpose of this study was to determine the effect of routine use of dexamethasone on delayed cerebral ischemia and poor outcome (death and severe disability) after aneurysmal subarachnoid hemorrhage (asah). this is a single center, observational cohort study comparing patients with asah admitted to a tertiary referral center from to . a variation in practice patterns for the use of dexamethasone - mg every hours after asah exists in our institution depending on neurosurgeon preference. patients were followed prospectively for the occurrence of complications including delayed cerebral ischemia (dci), e (bad outcome defined as a mrs of - ) and months (bad outcome defined as mrs of - ). out of the patients, ( %) patients received dexamethasone during the first hours of admission. significant factors associated with steroid use were females ( % v %;p= . ) and aneurysm clipping verses coiling ( % v %;p< . ). there was no difference in hh, fisher grade, incidence of infections, or incidence of dci ( % v %;p= . ). steroid use was significantly associated with bad outcome at discharge ( % v %;p= . ), but no difference at months ( % v %;p= . ). when examined separately for coiled and clipped patients similar trends were found in both subgroups. steroid use was associated with a longer hospital length of stay (los). in multivariable regression analysis steroid use was significantly associated with worse outcome (or . ;p< . ) when controlled for age, hh grade and type of surgical intervention. the study did not detect any benefit in the use of steroids in reducing the incidence of delayed cerebral ischemia in acute aneurysmal subarachnoid hemorrhage. however, steroid use was significantly associated with longer los, and worse functional outcome at discharge. antiplatelet therapy for the prevention of peri-coiling thromboembolism in high risk patients with ruptured intracranial aneurysms thromboembolic events (tee) during or after coiling of intracranial aneurysms is the most frequent procedural complication, resulting in permanent neurologic disability in a subset of patients. in unruptured aneurysm patients, there is evidence supporting the use of periprocedural antiplatelet therapy to prevent tee. whether patients with ruptured aneurysms and subarachnoid hemorrhage should also be given peri-coiling antiplatelet therapy is less clear. we reviewed a prospective registry of endovascularly treated aneurysm patients to delineate angiographic features associated with periprocedural tee. we then performed a controlled before-andangiographic feature associated with tee) to evaluate whether selective aspirin administration would reduce the rate of periprocedural thromboembolism without increasing major hemorrhagic complications. small parent artery diameter, an incorporated branch, intraprocedural thrombus formation, and parent rate of periprocedural tee, from . % in the control group to . % in the aspirin-treated group (p = . ). tee reduction in the aspirin-treated group continued to be statistically significant even when ith tee in other large studies with an adjusted or of . ( % ci . - . ). there were no major systemic hemorrhagic -bleed, symptomatic intracranial hemorrhage, or major external ventricular drain (evd)-associated hemorrhage (p = . ). significantly reduce the rate of peri-coiling tee without increasing major systemic or intracranial hemorrhages. neurocrit care ( ) :s -s muhammad k. athar, umer u. mukhtar, umer u. shoukat, david d. boorman, fred f. rincon, matthew m. vibbert, syed s. shah, jacqueline j. urtecho, jack j. jallo. thomas jefferson university hospital, philadelphia, pa, usa. fever is frequent in patients with subarachnoid hemorrhage (sah). differentiating infectious fever from central fever can be challenging. it is important to diagnose the cause of fever in the neurological intensive care unit (nicu) because of the detrimental effects of fever on brain injured patients. we hypothesized that procalcitonin (pct) could be useful to distinguish central fever from infectious fever in patients with sah. prospective, chart review study conducted in the nicu between december and september . was clinical infection defined as positive cultures (blood, urine, sputum, mini bal, csf, and c. difficle toxin) or infiltrate on chest x-ray within days of onset of fever. sixty-twenty-- . , and had pct > . . out pct > . . using multiple logistic regression, pct between . - . had an odds ratio of . ( % ci . - . ), pct > . had an odds ratio of . , and a maximum temperature odds ratio of . (ci . - . ). using pct > . alone had an odds ratio of . ( % ci . - . ). -pv: . % with a sample prevalence of . %. roc curve area: . %. fever in sah patients. the test has high specificity and npv so it can be a valuable toll to rule out infectious fever in nicu. intraventricular hemorrhage (ivh) due to subarachnoid hemorrhage (sah) has been associated with fever, hydrocephalus, and shunt dependence. the modified graeb score (mgs) as an enhanced measure of intraventricular hemorrhage has been shown to correlate wit intracerebral hemorrhage (ich) as well as shunt dependency in sah. we evaluated the mgs's association to complications during hospital stay and impact on functional independence at discharge in sah patients. retrospective review was performed of prospectively collected data for consecutive sah patients enrolled into the university of maryland recovery after cerebral hemorrhage (reach) study. hunt and hess (hh) grade, global cerebral edema (gce), and infarct on admi team of neurointensivists. mgs was calculated from each patient's admission ct scan and dichotomized according to a cutoff value based on the median value for our sample. clinical complications during each admission were recorded, and independence of performing adl's was obtained from physical and occupational therapy notes. statistical analysis was performed using univariate and multivariate logistical regression. ninety-eight sah patients from july to november were reviewed for this study. mgs was calculated in patients and dichotomized based on a median cutoff value of . hh, gce, and admission infarcts were not found to be significantly associated with high mgs. on univariate analysis, elevated mgs was significantly associated with hospital acquired infections (uti, pneumonia, and sepsis, p< . ), fever (p= . ), hypotension (p= . ), hypernatremia (p= . ), symptomatic vasospasm (p< . ), and new i independence with adl's (p= . ). severity of ivh as measured by mgs is associated with multiple in-hospital complications. the mgs can be used as an independent predictor of loss of independence of adl's on discharge for patients with sah. ed cerebral ischemia (dci) and brain injury following subarachnoid hemorrhage (sah). while systemic corticosteroids may mitigate inflammation and promote fluid and salt retention following sah, there is limited evidence on the impact of corticosteroid administration on outcomes following sah. corticosteroids are frequently administered in clinical practice following sah for the management of post-operative cerebral edema and refractory headache. our goal was to examine the impact of corticosteroid use following sah on the occurrence of dci and poor functional outcome at discharge. retrospective analysis of data from a single center sah registry on patients admitted between - who survived > hours. a logistic regression model was created with multiple potential predictors of outcome and steroid use, and with corticosteroid use as the response variable. patients were divided into quartiles based on the propensity score. the impact of corticosteroid use on the outcome of interest (dci then poor functional outcome at discharge) was then determined while controlling for the propensity score quartile. co-variates in analysis included age, gender, ethnicity, history of diabetes or statin use, aneurysm location, aneurysmal vs non-aneurysmal bleed, treatment modality, hunt hess, modified fisher. a total of patients with aneurysmal and nonaneurysmal sah were included in this analysis. corticosteroids were administered in ( . %). dci occurred in ( . %). poor outcome (mrs> at discharge) occurred ( . %). following propensity score analysis, corticosteroid use was not associated with dci (p= . ) but was associated with a significant reduction in poor outcomes at discharge (p= . , or . , % ci . - . ). corticosteroid use following sah was not associated with a reduction in dci but was associated with an approximately % reduction in the odds of poor functional outcome at discharge. a clinical trial of corticosteroids initiated in the early period following sah may be warranted. besides the impact of the initial bleeding, cv remains the leading cause for mortality and morbidity after successful cian therapy? data of patients with cian were analyzed with regard to onset of multimodal neuromonitoring, if one or both hemispheres were monitored and for the integration of neuromonitoring values in decision months after sah using the glasgow outcome scale (gos). ct-scans were reviewed for infarctions at time of discharge. patients were in the favourable outcome group (gos - ), patients showed unfavourable outcome (gos - ). in patients of the gos - group neuromonitoring was implanted in the hemisphere with the highest transcranial doppler (tcd) values. additional monitoring was installed contralaterally if tcds increased. in of those patients, contralateral pbto values were ischemic and angiography revealed severe cv in the non cian treated hemisphere. ct scans of those patients revealed significant infarctions in the hemisphere that was not initially monitored. the patients with gos - were monitored bilaterally at early timpo small infarctions but no territorial infarct was seen at discharge. a delay in bilateral multimodal neuromonitoring might facilitate delayed cerebral ischemia (dic). this might be due to a delayed detection of a mismatch between oxygen supply and consumption. in addition severe cv is not always detectable in tcd and might thus be diagnosed too late to initiate a successful cian therapy. in contrast, new severe cv or relaspses of cv after stop of cian therapy are detected efficiently if bilateral neuromonitoring is in place and the values are acted upon accordingly. the effects of short versus longer duration prophylaxis of levetiracetam on cognitive / functional outcomes in aneurysmal subarachnoid hemorrhage and risk of development of delayed seizures tamara majic, dela d. amoussou, chrystal c. reed, asma a. moheet. cedars sinai medical center, los angeles, ca, usa. chart review performed on patients admitted from january to december with asah, who received levetiracetam seizure prophylaxis mg bid or greater for < days versus days or more. we compared the length of icu stay, delta gcs at discharge, mrs ( vs days), and incidence of delayed seizures -- . ; p < . ) lengths of icu stay for short-duration levetiracetam therapy was -- . for long-duration (p< . ). length of icu stay in low dose -- . days (p < . ). preliminary data for early and late onset seizures delayed seizures occurred with longer duration prophylaxis preliminary data suggests delayed cerebral ischemia was universally present in patients with delayed seizures. the incident rate of dci was higher in patients with early seizures ( %) vs without seizures ( %) between low dose and high dose, and between short duration and long-duration levetiracetam therapy. length of icu stay is shorter in subjects treated with low dose levetiracetam vs high dose, which may suggest that a low dose levetiracetam may have a lower adverse effect profile. the presence of delayed ischemia may warrant a longer duration prophylaxis. the longer duration prophylaxis does not seem to reduce the incidence of delayed seizures, although a longer duration of study is warranted. in spite of improvements in mortality and physical disability for aneurysmal subarachnoid hemorrhage for 'delayed brain injury' often attributable to the direct neurotoxic and neuroinflammatory influence of the initial hemorrhage burden. these processes can result in global brain atrophy and commonly manifests as new cognitive disability including deficits with memory, executive function, and language. heparin exerts a wide range of interactions postulated to antagonize multiple pathophysiological mechanisms implicated in asah. here we review low-dose iv heparin (ldivh) as a promising treatment for preventing 'delayed brain injury' in asah survivors and inform on a new multi-center randomized trial. recent studies evaluating ldivh in asah are reviewed. the astroh study is an open-label, blindedadjudication, randomized phase ii trial. the primary efficacy outcome is mean montreal cognitive assessment (moca, - , normal - ) scores at the -day follow-up and patients will be enrolled over years at academic medical centers. the primary safety outcome is any major bleeding or clinically relevant non-major bleeding. one-year outcomes are also being assessed. ldivh significantly reduced neuroinflammation, demyelination, and transsynaptic apoptosis in a rat sah model. in a retrospective study ldivh patients were compared to well-matched controls. ldivh subjects had % clinical vasospasm and % vasospasm related infarction compared to % and % respectively in controls (p= . and p= . ). in another retrospective cohort study ldivh patients (n= ) had mean moca of . compared to . in controls(n= ) (p= . ). multivariate analysis confirmed ldivh positively influenced moca scores when controlling for factors that negatively influenced cognition. the astroh study is active and enrolled its first subject in april, . ldivh is a promising treatment for asah and is currently being investigated in a multi-center randomized trial (astroh), nct . gastrointestinal bleeding (asah) patients and to determine the effect of gib on in-hospital complications and outcomes. gib in asah patients and to determine the effect of this complication on other in-hospital complications and outcomes. the incidence of gib in asah hospitalizations (n= , ) was , per , patients with . % requiring blood transfusions. multivariate independent predictors of gib included: age - gib in asah is uncommon and is influenced by patient demographics and preexisting comorbidities and significantly increases disability and mortality. twenty-six consecutive asah patients undergoing multimodal neuromonitoring including cmd were studied. interventions of full-strength enteral nutrition (en) after > hours without any feeding preceding en were identified. parameters of systemic and cerebral metabolism and insulin dose were timeand analyzed together with continuous variables to study the effect of en on brain metabolism (glucose, lactate, pyruvate and glutamate). out of interventions in total, were excluded because of simultaneous parenteral supplementation or missing values, leaving interventions in patients eligible for analysis. the mean en--glucose significantly increased from perfusion pressure (cpp), baseline serum and brain glucose levels, the baseline metabolic profile [brain metabolic di and independent of the insulin dose given during the intervention. the increase of cmd-glucose was strongly dependent on the delta increase of serum glucose (median during the intervention (p< . ). although probe location influenced absolute cmd-glucose-levels (p< . ), significant increases were even observed in perilesional brain tissue (p< . ). no change in cmd-lactate, cmd-pyruvate, cmd-lpr or cmd-glutamate levels were observed (p over . ). brain glucose levels increased during enteral feeding independent of cpp, baseline glucose levels, insulin administration, and probe location. despite this increase, no additional metabolic improvement was observed. the clinical benefit of interventions ta needs to be investigated in a prospective approach. mean platelet volume (mpv) is a common daily laboratory investigation in subarachnoid hemorrhage diseases and is postulated to signify increased systemic thrombogenicity. similarly, diabetics have elevated mpv suggestive of associated vasculopathic complications through increased thrombogenicity. with non-aneurysmal sah (nasah) as compared to aneurysmal sah (asah). hence, we investigated if vasculopathy. we reviewed charts of patients admitted with the diagnosis of sah between january and december . we compared proportions using fisher's exact tests, and constructed roc curves to find threshold values for admission mpv that had the best combination of sensitivity and specificity to predict nasah versus asah. of the patients who met the inclusion criteria were asah. diabetic patients who presented with diabetic patients, a threshold value for mpv of . fl yielded the best combination of sensitivity and specificity to predict asah vs nasah (auc= . ; % ci . , . ). using this threshold, sah is more -similar mpv association was not observed among diabetic patients presenting with sah. mean mpv at admission did not differ between diabetic patients who presented with asah ( . fl) and those who presented with nasah ( . fl, p= . ). nonng with sah. aneurysmal subarachnoid hemorrhage (asah) is associated with mortality rates up to %, and up to one half of survivors suffer from long term neurologic disability. though several clinical scores have been developed to predict in-hospital mortality and long term outcomes, there is no universally accepted score. create a new predictive model. we conducted a retrospective chart review of patients admitted with asah to a single neurocritical care unit from september to february . we excluded patients with non-aneurysmal sah (including trauma, avms, and mycotic aneurysms). demographic and clinical variables collected included age, admission gcs, admission apache ii score, hunt and hess score, presence of delayed cererbral ischemia, and hospital acquired infections. our outcome measure was glasgow outcome scale at discharge. we created a penalized logistic regression model to determine predictors of outcome. we assessed performance by estimating the area under the roc curve (auc). of patients reviewed, met inclusion criteria. the mean age of the cohort was years. . % (n= ) of patients were female. the mean apache ii score on admission was . (median . ). majority of patients (n= , . %) had a discharge gos of . a combination of predictors performed optimally: age, admission apache ii, gcs, use of mechanical ventilation and presence of hospital acquired infections. the mean auc of the model was %. at the point of maximum-accuracy on the roc curve, the sensitivity was %, and specificity was %. clinical features at admission and during hospitalization can predict outcomes in patients with asah. clinical characteristics from the first few days of the hospital admission, such as hospital acquired infections, can be added to existing models, to improve outcome prediction scores. subarachnoid hemorrhage (sah) patients may experience supply-demand mismatch of cerebral metabolism from seizures, vasospasm, cortical spreading depolarization, hydrocephalus, or cerebral edema. previous studies have focused on non-neuronal measures of cerebral autoregulation. we examine the impact of various neurocritical interventions by examining anecdotally identified intracranial eeg (ieeg) responses considered clinically impactful as well as systematic examination of repeated interventions within patients. sah patients of hunt-hess grade - underwent ) clinical multimodality neuromonitoring utilizing brain tissue oxygen cerebral oximetry, cerebral blood flow, spencer depth electrode, and fiberoptic icp through a quadconsent using time-synchronized monitoring (cns- , moberg research). we reviewed clinician anecdotes of treatment responses to vasopressors, endovascular vasodilators, anti-seizure pharmacotherapy, nimodipine, and ventriculostomy adjustments. we then assessed each patient's response to multiple grouped interventions using spectral features including alpha-to-delta ratio (adr) normalized to pre-intervention baseline (nadr). paired t-tests and scatter plots, respectively, demonstrated the impact of interventions and blood pressure on nadr. patients had available post-sah ieeg data over months. of patients with post-procedural brain responded with an increase in ieeg alpha activity power. two patients developed a decline in adr associated with asah-related vasospasm, one who had eeg improvement after endovascular spasmolysis. two patients developed scalp-negative ieeg seizures, both electroclinically improving with anti-seizure pharmacotherapy. grouped interventions showed heterogeneous responses to vasopressors and one patient with a significant, repeated response. nimodipine had no consistent discernible peri-dose impact on nadr. we display scatter plots showing the peri-intervention patient-specific correlation between mean arterial pressure and nadr. patients with aneurysmal sah may develop neuronal impairment rescuable by neurocritical care interventions. our data show these responses are patient-and statemodels of dynamic sah pathophysiology. introduction: (dci), may be an important determinant of outcome following subarachnoid hemorrhage (sah). potentially, early treatment measures such as control of intracranial pressure, blood pressure management and initiation of nimodipine may mitigate ebi. our objective was to study the impact of delayed presentation to medical care on the occurrence of dci and poor outcomes following sah. retrospective analysis from a single center sah registry. patients admitted between and for nontraumatic sah, who survived more than hours were eligible for inclusion. [vr ] the explanatory variable of interest was time from symptom onset to diagnostic ct, dichotomized at hours. covariates included age, gender, ethnicity, hunt-hess grade, modified fisher grade, hypertension, aneurysm location and treatment modality. the primary outcome of interest was poor functional outcome at discharge (defined as models were constructed with the outcomes of interest as the response variables. a total of patients were included. the median time to diagnosis was . hours (interquartile range . - . ). twenty-four patients ( . %) presented greater than hours from onset. poor functional outcome at discharge occurred in ( . %) and dci in ( . %). multivariate analysis revealed no association between delayed presentation and either dci (p = . ) or poor functional outcome at discharge (p = . ). hours from symptom onset to diagnosis as a continuous variable also did not reveal a significant association with dci or poor functional outcome. delayed presentation to medical care beyond hours is not associated with either dci or poor functional outcome at discharge following subarachnoid hemorrhage. a treatment bundle including extracorporeal cardiopulmonary resuscitation (ecpr) combined with targeted temperature management (ttm) may improve outcome of cardiac arrest (ca) patients, however, prognostication for these patients still remains challenging. we sought to examine the prognostic value of amplitude-integrated electroencephalogram (aeeg) for ca patients during ecpr and ttm. this was a single-center, retrospective analysis of adult ca patients treated with ecpr and ttm under aeeg monitoring with subhairline montage. intra-arrest cooling was immediately initiated with cold fluid infusion and extracorporeal cooling method and maintained at °c for h. patents underwent intraaortic balloon pumping (iabp) and percutaneous coronary intervention (pci) if needed. neurological outcome was assessed with the cerebral performance category (cpc) scale at hospital discharge. ecpr was conducted in patients (age . [ - ] years, % male) amongst ca or post-ca comatose patients since november . the initial cardiac rhythm was refractory ventricular fibrillation in , pulseless electrical activity in , and asystole in . the cause of ca was cardiogenic; underwent pci and needed iabp support. collapse-to-ecpr time was . min. initial aeeg patterns were; flat trace (n= ); low voltage (n= ); suppression-burst (sb) (n= ); electrographic status epilepticus (ese) recovery (cpc - ). their aeeg pattern was continuous in , low voltage in , and ese in . among rn of spontaneous circulation. patients with ese recovered after antiepileptic administration. ecpr was withdrawn in patients based on clinical and prolonged flat aeeg findings. continuous aeeg adds early prognostic information for ca patients with ecpr under ttm. the suppression ratio (sr) is a processed eeg variable estimating the percent of an eeg epoch ( - ) that is suppressed. sr has been associated with neurologic outcome after several types of brain injury and using different technologies including full montage eeg recordings and simplified processed eeg monitors. we compared sr during targeted temperature management (ttm) after cardiac arrest, using two independent blinded assessment tools. a convenience sample of adult patients treated with ttm after cardiac arrest were enrolled to compare and the full montage continuous eeg using natus equipment with persyst magicfor . seconds). machine times were recorded to synchronize, and sr results were recorded once for each subject at a time without stimulation or artifact using correlation and altman-bland analysis. adults were enrolled in this study with a median age of years, ( %) were male. during sr - ) for persyst sr . ( . - ). comparing medtronic and persyst sr, the spearman correlation was . (p< . ), and altman bland testing revealed a bias of . with % limits of agreement - . to . . bedside estimation of suppression ratio during ttm after cardiac arrest showed excellent agreement when measured with the medtronic bispectral index monitor and the full montage natus ceeg monitor though the impact of therapeutic hypothermia on neurological outcomes remains controversial, there is strong evidence that pyrexia is detrimental. posthypothermia fever experienced by cardiac arrest patients is of particular concern. this abstract examines the ability of an esophageal heat transfer device (ehtd) to maintain core temperature below °c in critical care patients, with a focus on posthypothermia fever in post cardiac arrest (pca) patients. de-identified data for subjects who received temperature management using an ehtd were collected with a condition appropriate for active temperature management. core temperature readings for each patient were recorded at least hourly; if measurements were recorded more frequently, temperature over an hour span was averaged. patient data was analyzed to determine what proportion of measurements were above °c. data from a total of patients was collected, including post-cardiac arrest patients and fever reversal cases. a total of core temperature measurement events (over an average of . h per patient) were included in the analysis. ( . %) were below °c, recorded measurements exceeded °c, and no data were recorded for time points. of the measurements recorded posthypothermia, ( . %) remained below °c. esophageal temperature modulation using an ehtd appears to be an effective method for fever prevention and reduction. visual representations of the pca subset showed an upward trend in temperature after - hours of maintaining target temperature, but before active cooling ended. this suggests that many of these patients might have become febrile in the absence of active temperature management. achieving and maintaining normothermia (nt) after subarachnoid hemorrhage (sah) or intracerebral hemorrhage (ich) often requires surface or intravascular cooling devices that are associated with a significant burden of shivering. we describe a new, closed loop esophageal cooling device (ecd: -- . c) and the shiver burden during the maintenance of nt. we enrolled mechanically-ventilated patients with sah or ich with refractory fever (> . c). temperature and bedside shivering assessment scale (bsas) were recorded every minutes for the time above c, median bsas and cumulative number of anti-shivering interventions per patient was recorded prospectively. all patients received magnesium, buspirone, and acetaminophen as baseline anti -shivering interventions. ten patients ( ich, sah) were enrolled between october and april . the median gcs at initiation was ( ---- . m , and % were women. there was a temperature reduction at minutes (mean . c to . c, p= . ) and % of patients achieved nt (median time = . hrs.; range: . - hours). nt was maintained for median -- %) time above > e time. the median number of total shiver interventions per patient was ( - ) throughout the ttm time period. no device related complications were noted. the ecd successfully achieved and maintained nt with a low shiver burden and may be a feasible option for nt in this critically-ill population. we present a case of toxic leukoencephalopathy in a young woman taking a thermogenic dietary supplement. a year old female with unremarkable pmh except being on a diet drug "remuvik" presented with a day history of severe headache, blurry vision, photophobia, phonophobia, nausea, vomiting and brief intermittent hand spasms. neurological exam was notable for mild right finger-to-nose ataxia and diffuse hyperreflexia. initial mri demonstrated extensive bilaterally symmetric t hyperintensities of the corpus callosum and periventricular white matter. csf profile was unremarkable. labs were unremarkable except for serum sodium of meq/l. approximately hours later, patient became unresponsive with bilaterally fixed-dilated pupils and decerebrate posturing. she was intubated and gms of mannitol was emergently administered with concern for cerebral edema. iv lorazepam was also given. a stat ct head showed diffuse cerebral edema. an external ventricular drain was placed emergently and % nacl was started. continuous eeg was negative for seizures. next day she started following commands and on day she was discharged home with normal neurological exam. given her presentation and mri findings, she was diagnosed with acute toxic leukoencephalopathy due to thermogenic diet pill "remuvik". patient had been taking the diet drug for months and had lost lbs. the main ingredients in remuvik are listed as resveratrol, caffeine-free green tea and l -carnitine while the remaining ingredients are unknown. similar presentation with another diet drug "thermatrim" has been previously reported. these products are not fda regulated and are easily available to the general public. the acute cerebral edema with decompensation was thought to be due to hyponatremia caused by remuvik. while the mechanism of leukoencephalopathy is not well understood and further investigation is needed, spreading awareness is the key to prevent serious adverse effects of such unregulated products. baclofen is a frequently used muscle relaxant. we report a case of, low dose baclofen causing reversible gped's (generalized periodic epileptiform discharges). on review of literature, baclofen toxicity/overdose has been associated with burst suppression patterns on eeg, with one case report of baclofen toxicity causing gped's. to the best of our knowledge there have not been reports of low dose baclofen induced significant eeg changes. case reportthe patient is an year old woman, with poor baseline functional status from advanced dementia and limb contractures, on coumadin for old dvt/pe, with sub-therapeutic inr, was admitted with new onset seizures secondary to venous infarcts over bilateral parieto-occipital areas, due to extensive venous sinus thrombosis. she was monitored on continuous video eeg and initiated on antiepileptic medications, vimpat and dilantin. eeg recording initially demonstrated occasional sharp waves, maximal in the left frontal region. however, because of excessive emg artifact caused by hypertonia, the patient was started on baclofen mg. within hours patient's mental status deteriorated and eeg recording demonstrated gped's with periods of suppression. due to concern for drug adverse reaction, baclofen was discontinued. the eeg reverted to pre-baclofen pattern, while her mental status slowly improved. she was provided supportive care and ultimately discharged to a rehabilitation facility. in this elderly dementia patient, with low seizure threshold from the acute cerebral insult, low dose of baclofen was enough to induce encephalopathy and gped's. the absence of any metabolic disturbances along with rapid resolution of clinical and electroencephalographic abnormalities after discontinuation of the drug supports the hypothesis that these findings may be the direct cerebral toxic effect of baclofen. iatrogenic encephalopathy with baclofen should be considered in the differential for elderly patients with low cognitive reserve rotational vertebrobasilar insufficiency, also called bow hunter's syndrome after the symptom-inducing head position adopted when aiming a bow, is a rare cause of posterior circulation ischemia. we present a case of an -year-old woman who presented to barnes-jewish hospital with several days of episodic vertigo and gait instability. two weeks prior to presentation she had fallen and struck her head. imaging revealed a complex c fracture as well as an odontoid fracture with posterior displacement. she began having positional spells characterized by loss of consciousness, gaze deviation, fencer posturing, and sonorous breathing. review of clinical records and literature review. the spells were initially highly concerning for seizures. the patient was monitored on continuous video eeg, however no seizures were detected during typical spells. a ct angiogram revealed an occluded right vertebral artery at the level of c with diminutive vs. absent posterior communicating arteries isolating the posterior circulation. subsequent mr angiography revealed a patent right vertebral artery with no evidence of stroke. catheter cerebral angiography demonstrated a patent left vertebral artery. turning the head degrees during the procedure, however, elicited a typical spell and completely occluded the left vertebral artery. the patient underwent occipitocervical fusion, but unfortunately suffered a multifocal posterior circulation stroke and was discharged with hospice care. we present an unusual case of rotational vertebrobasilar insufficiency that mimicked a classic sezure semiology and presented several diagnostic dilemmas in the icu. in this case, traumatic injury resulted in likely bilateral positional vertebral artery occlusion with resultantly profound brainstem ischemia. bow hunter's syndrome should be considered in all cases of positional neurological spells, particularly in the setting of neck injury. optic nerve sheath diameter (onsd) measurement using ultrasound has been proposed as a reliable method for non-invasive assessment of intracranial pressure (icp). we report a case of using onsd to monitor icp in a tbi patient with elevated icp undergoing medical treatment with acetazolamide. we hypothesize that a difference in onsd could be detected with ultrasound before and after treatment. patient is a year old man with mild tbi due to assault. his head ct reveals a long calvarial fracture extending along the superior sagittal suture line and posteriorly into the left parietal bone, as well as a large epidural hematoma overlying the frontoparietal vertex near midline, and causing inferior displacement and extrinsic compression of the superior sagittal sinus. his physical exam reveals a young man with right orbital ecchymosis who is sleepy but easily arousable with a gcs of and no motor deficits. dilated fundus exam by ophthalmology reveals grade - papilledema consistent with elevated icp. the patient complains of persistent headaches and nausea that is unremitting. acetazolamide was started to decrease icp. we measured onsd with a sonosite ultrasound device prior to start of acetazolamide and days afterwards. two measurements were taken on each eye, one in the horizontal and vertical orientation each. the average onsd was . mm on the right eye and . mm on the left eye prior to initiation of treatment. on the day after treatment onsd was . mm on the right and . mm on the left eye. the patient's headache improved and nausea resolved. the next day onsd was . mm on the right and . mm on the left eye. headache and nausea completely resolved. this case report affirms that ultrasound measurement of onsd could be used reliably to assess icp noninvasively during the course of treatment for elevated icp. manoj k. mittal. kansas university medical center/ neurology, kansas city, ks, usa. timing of brain death evaluation could be crucial in maintaining organ perfusion for donation. a new bedside cerebral blood flow monitor (cflow monitor from ornim) has not been previously studied for determining the timing of brain death examination. we present here a case illustrating the role of bedside blood flow monitoring in determining the timing of brain death evaluation. a year-old-woman presented with acute right middle cerebral artery stroke and bilateral internal carotid artery occlusions. she was not a candidate for intravenous thrombolysis or endovascular therapy due to unknown time of symptoms onset. her initial nihss was (right gaze deviation, mild aphasia, mild dysarthria, left facial droop, left hemiparesis, left sided decreased sensation and neglect). day- , she got intubated for hypoxic respiratory failure. day- , ct head showed cerebral edema with midline shift of mm. patient was not a decompressive hemicraniectomy candidate. day- , patient was comatose. day- , patient lost bilateral pupillary reflex. ct head showed worsening midline shift of mm with right uncal herniation, bilateral anterior cerebral artery and left posterior cerebral artery stroke, and brainstem compression. day- , bedside cerebral blood blood flow monitoring was started with right sided cerebral blood flow index (cfi) of and left side cfi of . patient met criteria for brain death except that she was still breathing over the ventilator. patient was extubated for comfort measures. after minutes patients stopped breathing. her cfi dropped < bilaterally. patient underwent cardiac arrest after minutes and then both cfi were < . patient was not a candidate for organ donation. bedside cerebral blood flow monitoring may assist in determining the timing of brain death evaluation in comatose patients with imminent brain death. patients with cfi < may be considered for brain death evaluation. our finding needs further confirmation. aneurysmal subarachnoid hemorrhage patient. icus are high cost in the u.s., comprising about % of the us gdp. pressure is being placed on hospitals and intensivists to reduce costs, including earlier palliative care engagement to shorten length of stay.. as the u.s. migrates to a value-based system, further pressure will be made on reducing prolonged and expensive icu interventions, similar to quality adjusted life year (qaly) cutoff values to justify costs. a young year old man presented with worst headache of his life, and was found comatose by ems and referred to our neuroicu. he had a . cm giant basilar asah with intraventricular hemorrhage. the aneurysm was coiled endovascularly with external ventricular drain placed. he required therapeutic hypothermia, osmotherapy, induced hypertension and balloon angioplasty and intraarterial verapamil for refractory basilar and bilateral middle cerebral artery vasospasm. he had refractory intracranial pressure from global cerebral edema and around post-operative day # required bifrontal craniectomy. later percutaneous tracheostomy, peg tube, and ventriculoperitoneal shunting were performed. his total costs exceeded $ , u.s. dollars. one year later, his modified rankin scale was zero, and he went to college. his qaly (quality adjusted life year) for the rest of his year was . given a utility of . his physicians felt he should live to a normal life expectancy of years of age, q is quality of life weight = (perfect health, utility = ), l is residual life expectancy = more years. his qal-expectancy , is about life-years gained which divided over his life span is about $ , /year and less than the current cms reported value of $ , per year. this case exemplifies how high cost care can be delivered to deliver cost-effective, high quality care and underscore the need for integrated high-complexity neuroicu care. early mobility in the intensive care unit setting is associated with a number of positive effects including improved quality of life. though there is a strong body of evidence supporting early mobility in medical intensive care units, the benefits of very early mobilization after acute stroke are yet unclear as early hemodynamic variability in patients with impaired cerebral auto regulation is of concern. another potential barrier to early mobilization is the presence of an external ventricular drain (evd) for cerebrospinal fluid diversion and intracranial pressure (icp) monitoring. this case demonstrates hemodynamic and icp responses to progressive, device assisted mobility interventions during the acute phase of intracerebral hemorrhage (ich) in the setting of persistent elevations of icp requiring two evds. a year-old man was admitted to the neuroscience critical care unit with an acute thalamic ich and intraventricular hemorrhage requiring placement of two evds. starting on day following ich onset, the patient underwent progressive mobilization following the johns hopkins nccu activity and mobility algorithm. range of motion exercises were performed initially, progressing to supine cycle ergometry followed by incremental verticalization using the tilt table (sara combilizer®arjo huntleigh inc.,il). blood pressure, heart rate, oxygen saturation, and icp were recorded before, during and after the mobility interventions. no adverse neurologic effects were noted during these mobility interventions. recorded hemodynamic variables and icp remained within the set goals throughout. moreover the patient was able to tolerate degrees of verticalization on the tilt table. progressive, device assisted early mobilization was feasible when titrated by skilled healthcare professionals in a critically ill hemorrhagic stroke patient with evds. studies on larger patient samples are needed to improve our understanding of the hemodynamic and neurophysiologic responses to establish safety of progressive early mobilization of critically ill patients with acute stroke. anand venkatraman, ayaz a. khawaja, angela a. shapshak. university of alabama at birmingham / department of neurology, birmingham, al, usa. we describe a case of a patient with uncontrolled hypertension (htn) and prior intracranial hemorrhage (ich) who developed an intracranial hemorrhage shortly after consuming redline, a heavily-caffeinated energy drink. a -year old caucasian male with prior history of ich and chronic untreated htn was transferred to our service for evaluation of . x . cm ich in the left thalamus. blood pressure had been elevated in the emergency room there and he had been started on a nicardipine infusion. nih stroke scale was . ich score was . admission labs were normal. urine drug screen was negative. on questioning, patient revealed that symptoms had started within hours of consumption of bottle of redline, an energy drink. he was not a regular user but did consume it whenever he needed to get a lot of work done. mri of the brain did not show any vascular malformation or other lesion. multiple remote hemorrhages were seen in the subcortical areas. we stared lisinopril and weaned off his nicardipine infusion. he was discharged with minimal deficits. the high caffeine content is the most likely component of the drink that led to the ich, given that high caffeine consumption is linked to increased risk of hemorrhagic strokes. caffeine also has effects on platelet aggregation and endothelial function that could raise stroke risk. generalizability is limited by the fact that the patient had uncontrolled htn and prior ichs. however, given that % of adults in the usa have uncontrolled htn, and given that ich account for - % of all strokes, a large population is at risk. to our knowledge this is the first report of intracranial hemorrhage following consumption of an energy drink. consumers must exercise caution, especially in the setting of uncontrolled risk factors. elena schmidt, varada v. nair, gene g. latorre. suny upstate university hospital / department of neurology, syracuse, ny, usa. often times medications given in emergency cases have unintended consequences, sometimes posing even more harm than the reason for their administration. we report a case of a young lady with history of anaphylactic reaction who received i.m. epinephrine after developing allergic reaction to antibiotic, resulting in bilateral intracerebral hemorrhage (ich). our case is of a year old female with history of anaphylactic reaction who had been recently started on cephalexin for orbital cellulitis. she was sent to ed after having a syncopal event in the doctor's office. while in ed, the patient was administered . mg of : , epinephrine i.m. because of suspected anaphylactic reaction. shortly after, she complained of nausea, vomiting and developed right sided weakness and numbness. immediate ct head revealed two areas of ich, within the left parietal and right occipital lobes. extensive work-up ensued, with cta head and neck (negative for vessel anomalies), cerebral dsa (negative for vasculitis), mri brain w/wo contrast (negative for malignancy or amyloid angiopathy), ct thorax and abdomen (negative for malignancy). serum studies for vasculitis work-up were also unrevealing. echocardiogram did not show evidence for chronic hypertension such as lv hypertrophy. although there have been reports in the literature of spontaneous intracranial hemorrhage (intraparenchymal or subarachnoid) after various ways of epinephrine administration, in our patient's case, the extensive work-up done to exclude other etiologies stands out. this strengthens the hypothesis that epinephrine, causing an acute spike in blood pressure, ultimately led to spontaneous ich. in addition, the case of our patient is unique in that she developed two areas of ich, in a location typical for posterior reversible encephalopathy syndrome (pres), a syndrome known to be caused by significant elevation in blood pressures, suggesting a common underlying pathophysiology. careful consideration of indications should occur before administering such potentially harmful treatments. "last known normal" (lkn) time remains the standard for determining the onset of acute ischemic stroke and appropriateness of providing acute therapies. as older adults become more familiar with social media platforms, these applications may become a source of recognizing when a patient was lkn. we report an year-old woman who was "found down" at home. the patient lived independently, and was able to crawl to a telephone for help. on arrival to the emergency department, she had a right middle cerebral artery syndrome with an nih stroke scale of . she had a decreased level of arousal and severe dysarthria which precluded assessment of her lkn. the patient's son reported that he had last seen her normal hours prior, placing her outside the time window for acute therapies. however, the patient's granddaughter reported that the patient had been logged into facebook < hour prior to her admission "chatting" and commenting on photos. "timestamps" of comments left on photos by the patient provided exact times of the patient's activity. the only logical means of being able to perform these relatively high-functioning tasks would have been if the she was normal at the time of posting, thereby establishing her lkn. the patient was treated with systemic t-pa followed by endovascular therapy for a proximal m occlusion. the patient had rapid improvement of her stroke symptoms. she was discharged home with an nih stroke scale of . this patient's recent use of the social media was critical in determining the patient's lkn, leading to lifesaving acute stroke therapy. providers should be aware that social media may serve as a useful source of symptom onset information. in this case, it led to good outcome and discharge home. bilateral recurrent artery of heubner (rah) infarctions have been seldomnly reported in the literature. even more so for those cases that have occurred subsequent to neurosurgical extensive resections of large invasive olfactory groove meningioma. rah, a branch of the anterio-inferior cerebral artery, supplies anterior limb of the internal capsule, anterior caudate, putamen and globus pallidus. infarction typically results in contralateral paresis of the arm and face. other symptoms can occur i.e. choreiform movements, abulia, attention disorder, impaired memory, apathy, decreased spontaneity, depression, dementia etc. we present a case of bilateral rah infarcts as a complication of a large olfactory groove meningioma resection. we did an extensive chart review of our patient during post-operative neurointensive care unit stay, rest of the hospital stay and discharge follow up at month. our patients brain mri done as a part of routine post-operative imaging showed bilateral caudate head infarcts in the territory of rah. post-operative exam was significant for a left hemianopsia and right super quadrantopsia with color desaturation. patient did not experience any new weakness or movement related problems. he did have changes in cognition (forgetfulness & irritability) along with a subjective loss of sense of smell but these were consistent with his pre-op assessment. olfactory groove meningioma's comprise % of all intracranial meningiomas, are slow growing and tend to engulf and compress neighboring structures. most common complications of olfactory groove meningioma resections are post-operative cerebral edema, csf leak, seizures, cns infections, hydrocephalus and rarely brain ischemia. bilateral rah infarction, although rare has been reported in literature in association with vascular anomalies and other stroke risk factors. cerebral infarction involving the aca territories remains a known adverse complication of large olfactory groove meningioma resections, but bilateral infarcts due to these have not been reported before. angioinvasive aspergillus associated stroke in an immunocompetent host. aspergillus vasculitis is an under-recognized cause of stroke in immunocompetent hosts, especially when other risk factors are present. we present a case of autopsy proven angioinvasive aspergillus causing strokes in an immunocompetent host, and review the characteristic imaging findings to aid diagnosis. -year-old female developed cardiogenic shock after three-vessel-coronary artery bypass grafting (cabg) using saphenous vein grafts requiring intra-aortic balloon pump placement. this was complicated by aortic dissection, and she underwent replacement of the ascending aortic arch. refractory cardiogenic shock ensued for which she underwent placement of veno-arterial extracorporeal membrane oxygenation. postoperatively, she was noted to be in coma, and a non-contrast ct of the brain showed small multiple small ischemic strokes bilaterally. with persistent multi-organ failure, she was ultimately transitioned to comfort care and passed. autopsy revealed multiple perivascular petechial hemorrhagic infarcts involving white matter, deep gray matter and cerebellum on gross specimen. histopatholgic study showed aspergillus associated acute and chronic inflammation of blood vessel, and surrounding gliosis. aspergillus was also found in coronary grafts and kidneys. aspergillus associated cerebral vasculitis was considered less likely, as cabg, extracorporeal membrane oxygenation (ecmo) device-related thrombosis and acute mi were the leading differentials for stroke here, and no obvious immunosuppression was evident. cerebral aspergillosis can occur from direct spread from sinus infections or through hematogenous mode, and seemed to have originated from coronary grafts in this case. aspergillus has a predilection for posterior circulation arteries, and lacunar-type infarcts or petechial hemorrhages within the midbrain, thalami, or corpus callosum are characteristic. these findings should raise suspicion for aspergillus, especially without objective evidence of other mechanisms of stroke. early initiation of anti-fungal therapy may improve the likelihood of survival, and confirmatory testing in the form of blood vessel imaging, csf analysis and fungal blood cultures should be performed in suspected cases. autoimmune ganglionopathy: a rare cause of cardiac arrest kelly braun. neurological institute, cleveland clinic, cleveland, oh, usa. autoimmune autonomic ganglionopathy is a rare disorder characterized by pandysautonomia that occurs as a result of autoantibodies to ganglionic nicotinic acetylcholine receptors. we describe a year old male with autoimmune ganglionopathy previously treated with ivig who suffered cardiac arrest and anoxic brain injury as a complication of this disorder. the patient had a history of multiple autoimmune diseases (dm , autoimmune hepatitis, hashimoto's thyroiditis, celiac disease, antiphospholipid syndrome and ulcerative colitis). to alleviate his pre-syncopal lightheadedness related to dysautonomia, he would typically kneel and place his head on his folded arms. the patient was found unresponsive in this position in pea arrest. he underwent cpr followed by therapeutic hypothermia ( °c x h). the initial exam off sedation showed an obtunded patient with intact pupillary and corneal reflexes, but no tracking or command following. though he moved all extremities spontaneously, the movements were not purposeful and had a choreiform quality. notable labs were an elevated achr ganglionic neuronal antibody ( . nmol/l on hospital day and . nmol/l on hospital day ; normal < . nmol/l). mri brain showed symmetric diffusion restriction and flair changes throughout the brainstem, thalami and cerebellum, however there was no cortical diffusion restriction. eeg showed generalized intermittent rhythmic slowing, which was maximal bifrontally. he was treated with methylprednisolone mg daily for days and transitioned to prednisone mg daily. at the time of discharge to an acute rehabilitation facility, the patient followed most simple commands and moved all of his extremities against resistance, though he was noted to have diffuse hypotonia. to our knowledge, this is the first reported case of cardiac arrest attributed to autoimmune autonomic ganglionopathy. while rare, this is a serious complication of this syndrome. bibhukalyani das, shantanu s. shubham. institute of neurosciences kolkata, kolkata, india. global burden of tuberculosis is still high particularly in developing world. india is the largest tb burden country accounting for / th of the global incidence.cns tuberculosis is the most severe form of infection with microbacterium tuberculosis.emergence of mdr(multi drug resistant) tuberculosis has compounded the risk and adverse outcome. fatality rate of mdr tb meningitis is % with significant functional impairment in most of the survivors. mortality > % if patient is hiv positive. we report a case of yrs old girl from eastern india case of mdr -cns tuberculosis with a protracted clinical course of years. she developed a whole range of complications including hydrocephalus, optochiasmatic arachnoiditis with secondary optic atrophy, multiple tuberculomas, cerebellar and brainstem tubercular abscesses and siadh with hyponatraemia. our case is notable for few rare complications in the form of transverse sinus thrombosis secondary to chronic meningitis necessitating oral anticoagulation . the patient also developed various side effects of long term att such as -(i) att induced hepatitis. (ii) moxifloxacin induced seizures and re-adjustment of antiepileptics due to interaction with antitubercular drugs. she was managed with antitubercular drugs ( first line drugs) along with second line drugs (amikacin, levofloxacin, cycloserine, ethionamide) her clinical course was complicated by obstructive hydrocephalus requiring evd, vp shunt and shunt revision . developed acute sdh possibly secondary to shunt and required surgical drainage . later she developed posterior fossa tubercular abscess and needed craniectomy. pus from tubercular abscess grew mtb resistant to rifampicin and isoniazid. so the patient was maintained on second line drugs, ultimately succumbed to hospital acquired pneumonia. cns tuberculosis if associated with multiple medical, surgical complications, impose real critical care challenges compounded by mdr which often encountered in a developing country like india. megan lange, rebecca r. horrell. university of maryland medical center, neurocritical care unit, baltimore, md, usa. super-refractory status epilepticus, defined as seizures persisting despite anesthetics, is associated with high morbidity and mortality. here we present two cases of super-refractory status epilepticus intractable to aggressive therapies, including but not limited to anesthetics, electroconvulsive therapy, and immunotherapy. in both cases, the patients developed sepsis and cardiac arrest following prolonged hospitalizations with subsequent termination of seizure activity and improvement in electroencephalogram findings and neurologic exams. a review of the literature revealed a variety of publications describing super-refractory status epilepticus as a result of sepsis or cardiac arrest, but there is limited data describing either complication as therapeutic for status epilepticus. we propose that the systemic effects associated with profound sepsis, or the brief electrographic silence occurring in the setting of cardiac arrest could have played a role in halting seizures in these patients. we describe two theories regarding the potential mechanism by which cardiac arrest or sepsis could play a role in termination of seizures. exploration into specific mediators involved in these conditions and their relationship to status epilepticus could uncover therapeutic targets. targeted therapies could demonstrate promise in effectively treating super-refractory status epilepticus, thereby improving morbidity and mortality rates. ticagrelor is approved for prevention of cardiovascular events in adults with acute coronary syndrome (acs) . we present a patient with sah who developed thrombus during coiling procedure that was treated with abciximab followed by ticagrelor and aspirin, with potentially devastating consequences. a -year-old male presented after sudden onset severe headache. imaging revealed sah from a ruptured basilar tip aneurysm. the patient was neurologically intact with mild confusion, but declined during transfer and required intubation. an external ventricular drain was placed for hydrocephalus. during cerebral angiogram with coil embolization, a thrombus formed on the coil. intra-arterial abciximab was used with resolution of thrombus. he was extubated post procedure. aspirin and ticagrelor ( mg twice daily) were prescribed. the following day, the patient became increasingly lethargic with an increased respiratory rate ( s). he reported no perception of increased work of breathing. portable chest radiograph demonstrated only mild pulmonary edema. he did not have an oxygen requirement. venous blood gas demonstrated a ph . with a pco of , suggesting a respiratory alkalosis. transcranial dopplers demonstrated normal velocities, but the patient was considered for cerebral angiogram given a high concern for vasospasm with his neurologic exam. ticagrelor was stopped the following day. the patient's tachypnea and mental status rapidly improved. ticagrelor reversibly inhibits the platelet p y adenosine phosphate receptor and is indicated for prevention of cardiovascular events in adults with acs. in patients with both cerebral hemorrhage and a need for antithrombotic therapy, this reversible agent may become more widely used. dyspnea is a known side effect of ticagrelor, occurring in . % of patients (p< . ). dyspnea causes respiratory alkalosis and the resulting hypocapnea results in vasoconstriction. in this case, mental status change after administration of ticagrelor suggests that patients at risk for vasospasm may be particularly vulnerable to its side effects. over the past years, the americas have experienced waves of emerging and re-emerging arboviruses that cause neuroinvasive disease, including west nile virus, chikungunya virus, zika virus, and dengue virus. these viruses pose great challenges for traditional candidate-based infectious disease diagnostics that already fail to identify a causative pathogen in approximately % of encephalitis cases. we present the case of a year-old girl with a history of renal transplant managed with mycophenolic acid, tacrolimus and prednisone who presented to an emergency department with two days of high fevers, chills, upper back, neck pain and rash followed by encephalopathy. one month prior to presentation she attended summer camp by a lake in the angeles national forest, california. her hospital course was complicated by status epilepticus. cerebrospinal fluid (csf) analysis demonstrated a mixed neutrophilic and lympocytic pleocytosis. mri of the brain demonstrated symmetric t hyperintensities and edema in the bilateral thalami and leptomeningeal enhancement in the thalamus, cerebellum, brainstem, cervical spine and caudal equine. an extensive diagnostic work-up for infectious causes of encephalitis was performed and only identified epstein-barr virus. research protocol. unbiased mds of rna extracted from her csf and processed through a custom bioinformatics pipeline identified west nile virus. subsequently, convalescent serum serologies confirmed west nile virus infection. this case provides a first proof-of-principle that mds can detect even low level arbovirus burden in the csf of a patient with acute meningoencephalitis. given the rapidly changing landscape of viral causes of encephalitis in the americas, the ability of mds to comprehensively detect a huge array of microbes with a single assay may make it an optimal method for early identification of emerging causes of viral encephalitis, including in the transplant patient population. cladophialophora bantiana is a dematiaceous mold with a predilection for causing central nervous system infection, particularly in normal hosts. there is no standard therapy and mortality rates from this disease remain extremely high approaching %. here we describe a case involving a year-old immunocompetent man who presented with new onset seizures. brain imaging revealed bifrontal ring enhancing lesions concerning for abscess.the patient underwent surgical debridement of the lesions and bilateral intracavitary treatment with amphotericin b using ommaya reservoirs for several months. after approximately months of treatment which included surgical debridement , oral voriconazole and intracavitary amphotericin b, our patient is off all antifungals and no longer receiving intracavitary treatment. he remains fully functional with a nonfocal neurologic exam, being monitored with serial brain mris. due to rare incidence of cerebral phaeohyphomycosis, there are no clinical trials to help formulate standardized treatment guidelines despite its high mortality . this case places emphasis on an early aggressive multimodal approach for treatment of cerebral phaeohyphomycosis using a combination of surgical debridement, intracavitary antifungal injection, and oral antifungal therapy. does neurocritical care need to improve outreach to non-neuro specialties? firas abdulmajeed, mb. chb, bart b. nathan, md, fcns. university of virginia/ department of neurology, charlottesville, va, usa. the number of neuro-critical care(ncc) fellowship positions has been increasing yearly. the number of applicants has plateaued over the last years, leaving many programs with unfilled fellowship positions. the demand for neurointensivists is on the rise. trainees have come from neurology traditionally, with a limited number from internal medicine (im). we hypothesize that the relative paucity of im fellows was in part due to insufficient knowledge of ncc. we surveyed im residency programs in the united states, asking program directors to forward our survey to their residents. on another survey, ncc fellowship directors were asked: ) how many trainees and how many faculty hires from im and/ or em have they had within the last five years internal medicine residencies survey results: we obtained individual responses, responses were complete. how long is the ncc fellowship? n= % year % years % years residents of what specialty can apply to a ncc fellowship? n= . % (neurology, neurosurgery, anesthesiology, im and em) % neurology only % neurology, neurosurgery and anesthesia knowledge of san francisco matching system? n= % yes. % no do you know about the application cycle for the ncc match? n= % yes. % no knowledge of emergency neurological life support? n= % yes % no ncc fellowships' directors survey results: of the programs responded fellows with im/em background that were trained within the last years: / neuro-intensivists with im/em background hired: / im residents appear to have little knowledge of ncc fellowship. a lack of awareness of enls could affect the quality of care provided for neurological emergencies. additionally, for the specialty to grow and fill unmatched fellowship positions,current training and outreach strategies to non-neurology trainees may need to be improved somatosensory evoked potentials (sseps) are a sensitive, minimally invasive technique used to identify injury from the posterior columns of the spinal cord to the somatosensory cortex. the role of sseps as a neuromonitoring tool, in the neuroicu has not been well established. we present a case using sseps as a neuromonitoring tool illustrating electrical improvement along with clinical and radiographical improvement in a symptomatic chiari i malformation. year old female who was months postpartum after vaginal delivery with epidural analgesia presented with headaches, diplopia and nausea/vomiting. after arrival to er, she acutely developed flaccid quadriparesis with ophthalmoplegia and loss of airway while awake and following commands. given the concern for intracranial hypotension, we administered mannitol, hyperventilated and placed in trendelenburg position. head ct showed cisternal effacement in the setting of a likely pre-existing chiari malformation with cerebellar tonsillar. decompressive surgery was not an option initially given her dysautonomia and neurologic instability whenever the patient was not in trendelenburg. during her prolonged course, she had two mris of her brain and spinal cord which showed chiari i malformation with syrinx at c and presyrinx down to t . there was cervical spine venous engorgement and csf block at the level of the foramen-magnum. she was evaluated with serial sseps which initially showed low amplitude n response that improved with her clinical improvement. ssep is a minimally invasive method to electrically assess the somatosensory pathway integrity from the spinal cord, brainstem and cortex. given its sensitivity to the function of the dorsal columns of the spinal cord and medial lemniscus of the brainstem, sseps may be a useful monitoring adjunct to follow the evolution of posterior fossa lesions in patients that may not tolerate other means of monitoring and/or transportation, such as mri. review of prospectively maintained patient database identified one case of status epilepticus in a patient with cns-ptld. we present a case report with literature review. a -year old hispanic woman with a history of renal transplant years prior, presented with episodic confusion and gait ataxia progressing over two weeks. she was on immunomodulation with mycophenolate. patient had witnessed periods of behavioral arrest. continuous electroencephalography (ceeg) demonstrated right temporal sharps and - second epochs of bi-frontal - hz activity, some of which were associated with non-stereotyped movements of her left shoulder and trunk, suggestive of se. she received benzodiazepines followed by levetiracetam (renal dose) and phenytoin load for seizure control. mri brain without contrast demonstrated multifocal infiltrative t -hyperintense white matter lesions, most prominent in right temporal lobe. csf analysis demonstrated rbc, (l %) wbc, protein, glucose, culture and gram stain were negative. there were unmatched csf bands with an unremarkable cytology. csf pcr was positive for ebv and viral load was detected at copies/nl. other csf microbial assays including jcv were negative. stereotactic right temporal brain biopsy demonstrated areas of necrosis, axonal disruption, loss of myelin with polytypic plasma cells, cd and cd positive b cells and cd positive t cells on immunohistochemistry consistent with a diagnosis of polymorphic ptld. despite treatment with dexamethasone and rituximab, patient continued to remain critically ill and eventually received palliative measures. among transplant recipients, pcns-ptld is rare but debilitating with varied neurological presentation. high degree of suspicion, early diagnosis and treatment are paramount for survival. cortical myoclonus caused by activation of cortical areas subjacent to multiple subdural hematomas is an unusual mechanism of epilepsy. we report the case of a patient with an extra axial bleeding and myoclonic seizures evaluated with ictal fdg-pet. case report a year old male was admitted to our hospital because of worsening symptoms of cardiac failure of chagasic etiology. due to hemodynamic instability he was treated with the placement of an intraortic balloon pump. sixteen days after hospital admission, he presented intermittent generalized myoclonic jerks. on initial examination he was alert and oriented to time and place, had preserved strength in all limbs, although presenting with very frequent clusters of myoclonus. initial investigation with a head ct showed multiple foci of extra axial bleeding, distributed over the frontal and parietal areas. the electroencephalogram (eeg) confirmed the suspected diagnosis of myoclonic seizures, exhibiting generalized polispike-slow wave complex. due to the multiplicity of bleeding sites, with no obvious reason for spontaneous bleeding other than regular anticoagulation, the patient was submitted to a whole-body fdg-pet in order to exclude the possibility of dural metastatic implants. fdg-pet showed areas of cortical hypermetabolism adjacent to the bleeding foci, probably reflecting an epileptogenic mechanism of cortical activation. there was no evidence of hypermetabolism directly over the extra-axial areas of bleeding, what ruled out the hypothesis of dural metastasis. a diagnosis of spontaneous subdural hematomas associated to anticoagulation was given after all other causes were excluded. the patient was treated with sodium valproate and had sustained improvement of the myoclonic seizures. the bleeding areas were eventually reabsorbed, but the patient died from complications of cardiac failure. our report is the first to illustrate the mechanism of cortical activation leading to epileptic status in a patient with multiple subdural hematomas detected by ictal fdg-pet. to present a case of early onset myoclonic status epilepticus (mse) after cardiopulmonary arrest with incomplete resolution of myoclonus and good cognitive outcome. a year-old man presented status post cardiopulmonary arrest and cpr in the field with return of spontaneous circulation (rosc) after arrival to the hospital and cardioversion. the patient was intubated and treated with therapeutic hypothermia, but developed clinical mse with normal eeg within hours. he was aggressively managed with propofol, levetiracetam, and fentanyl. initial mri demonstrated diffuse hypoxic ischemic injury. mri on the th day of admission demonstrated improvement but a new white matter lesion in the splenium of the corpus callosum. after multiple unsuccessful attempts to discontinue fentanyl and days of treatment, the patient was given a poor prognosis based on the aan mse practice parameters and was placed on do not escalate care orders. propofol was slowly decreased; however the patient improved significantly throughout hospitalization with improved language and cognitive examination and only mild residual reflex myoclonus at the time of discharge. mri imaging had completely resolved by the th day of hospitalization. the patient's final diagnosis is lance-adams syndrome of action myoclonus incompletely controlled with levetiracetam. aggressive and prolonged treatment including therapeutic hypothermia in young patients with early onset mse was effective despite aan practice parameters. this patient survived with good cognitive outcome and with relatively modest deficits. further research is needed to assess whether improvements in intensive care unit capabilities over the past decade may contribute to improved outcome in young patients with cardiac arrest and whether practice parameters should be revised. at the start of this protocol, the serum sodium was and one-hour urine output was liter. this protocol was continued for hours. endocrinology was consulted and recommended changing to ddavp. serum sodium was [np ] and one-hour urine output cc prior to first dose of ddavp university of washington, department of surgery, seattle, wa, usa. earlier feeding results in improved outcomes in adults with severe traumatic brain injury (tbi) and in the overall pediatric intensive care unit (picu) population. current practices of nutrition initiation in children with tbi are not well described. this multicenter study evaluated timing and factors associated with nutrition initiation in children admitted to picus with tbi. we hypothesize that severely brain injured patients would have a delay in initiation of enteral nutrition. we retrospectively analyzed the multicenter pediatric trauma assessment and management database (ptam) from . patients with severe tbi were defined as glasgow coma scale (gcs) < with n in this group was compared injury, abdominal procedures were compared between the two groups. chi square and fisher exact tests were used for dichotomous variables; non-parametric tests were used for continuous variables. multivariable regression analysis with a stepwise procedure was performed to ascertain the best set of variables associated with delayed initiation of enteral nutrition. of patients admitted to the five ptam picus with severe tbi, ( %) were fed < hours from admission. patients with gcs < were fed a median . hours from admission (iqr . - . ) compared to . hours (iqr . regimen, higher injury and illness severity scores and lower minimum gcs were significantly associated with feeding initiation > hours. on multivariable analysis, scheduled bowel regimen, higher prism score and lower minimum gcs were significantly associated with nutrition initiation > hrs. lower gcs is independently associated with delayed initiation of enteral nutrition in children with tbi, independent of severity of injury or abdominal injury. all patients that suffered ca within hours of sah onset were identified from a prospectively collected characteristics, and outcomes of those with and without ca in the setting of sah using binary logistic regression. only % (n= ) of sah patients had ca within hours of the bleed. % (n= ) of those with ca had f these patients died while in the hospital. three patients had a ventricular fibrillation (vfib) arrest, and one of these patients (n= ), and half of these patients survived. % of patients were comatose after the arrest, most of which underwent cooling (goal temperatures - ). % of deaths in our cohort were from withdrawal of life support (n= ). increased aneurysm size (or . for each mm, % ci . - . ), amount of sah (or . , , and global cerebral edema (or . , ci . - . ) were associated with noncomatose patients and those with vfib arrests may have a better prognosis. acute herniation at the time of bleeding as indicated by large volume sah and global cerebral edema may be the underlying mechanism of most early cardiac arrest in sah patients. early identification of delayed cerebral ischemia (dci) following aneurysmal subarachnoid hemorrhage (sah) could allow more effective intervention. statistical methods that predict dci using variables collected routinely during icu care such as trends in vital signs and laboratory values have shown promise in recent studies. however, these studies have not all employed methods to guard against model overfitting. in this study we use cross validation to obtain minimally-biased estimates of the value of passively collected icu variables for predicting dci. early identification of delayed cerebral ischemia (dci) following aneurysmal subarachnoid hemorrhage (sah) could allow more effective intervention. statistical methods that predict dci using variables collected routinely during icu care such as trends in vital signs and laboratory values have shown promise in recent studies. however, these studies have not all employed methods to guard against model overfitting. in this study we use cross validation to obtain minimally-biased estimates of the value of passively collected icu variables for predicting dci. dci occurred in % of patients. penalized logistic regression selected features for inclusion in the final predictive model, derived from gcs, heart rate, mean aterial blood pressure, respiratory rate, spo , ventricular drainage, and sodium data. the mean auc of the model was %. potentially clinically relevant (sensitivity, specificity) points on the roc curve included ( , )% and ( , )%. dci occurred in % of patients. penalized logistic regression selected features for inclusion in the final predictive model, derived from gcs, heart rate, mean aterial blood pressure, respiratory rate, spo , ventricular drainage, and sodium data. the mean auc of the model was %. potentially clinically relevant (sensitivity, specificity) points on the roc curve included ( , )% and ( , )%. subarachnoid hemorrhage (sah) remains a highly morbid disease leading to > -related year of life lost before age . mechanisms of sah-related early brain injury and vasospasm remain microrna (mir)- a is released in response to hypoxia and promotes angiogenesis. we hypothesize that higher levels of mir- a is associated with outcome in human sah. functiona -up every months. good functional outcome is defined as mrs % reduction in caliber of any vessel on post-sah day cerebral angiogram. in sah subjects we compared csf and plasma mir- a by quantitative pcr on post-sah days , and between outcome groups. data are normalized using log-transformation and then compared using student's t- study population has mean age of . % has hunt and hess (hh) grade > . good outcome at months is associated with higher plasma mir- a levels on post-sah day (p= . ) and day (p= . ). after adjusting for important predictors of outcome (hh grade; age), plasma mir- a on post-sah day remains strongly associated with outcome (p< . ). plasma mir- a levels were not associated with vasospasm. mir- a is present in csf and is elevated in sah compared to controls (p< . ), but csf mir- a showed no association with functional outcome or vasospasm status. higher plasma mir- a level at post-sah day is independently associated with -month sah outcome. mechanistic experiments are necessary to determine whether mir- a expression is neuro-protective in sah. validation studies in larger, independent cohorts are necessary to validate mirna- a as a accurate assessment of renal function remains a unique challenge in patients with aneurysmal subarachnoid hemorrhage (asah). mathematical estimates of creatinine clearance (crcl) routinely used are often inaccurate in this setting. patients with asah have been shown to exhibit a hyperdynamic response leading to an enhanced renal clearance. no studies exist evaluating the directly measured creatinine clearance of patients with asah over time. this was a single-center prospective observational study of adult patients with asah admitted to the nsicu between january and july . eight-hour urinary creatinine clearances were performed daily to directly measure crcl until the patient no longer had a foley catheter or the patient left the nsicu.-gault equation. statistical significance was defined as p-value < . . fifty patients with asah were enrolled in the study. the study sample was % female with a mean age of . ± . years. the median hunt and hess grade was (iqr - ) and the median modified fisher grade was (iqr - ). additionally, the median admission gcs was . (iqr - ) and median admission sofa score was (iqr - ). the mean urinary crcl over the study period was . ± . patients with asah consistently experienced urinary crcl greater than estimated crcl predicted based on -gault equation. as renally eliminated medications are routinely dosed based on mathematical estimates of renal function, further study is needed to optimize medication regimens in this patient population to prevent underexposure. agitated delirium is frequently encountered after acute brain injury, but data is limited in patients with nces of agitation in these patients. via records of antipsychotic or dexmedetomidine administration, and agitation was confirmed via chart study team. outcome was assessed at months using interview for cognitive status (tics), and lawton-iadl score. agitation developed in of patients ( . %) and was most common in the first hours after admission, and in patients with hunt and hess grades and . agitated patients were significantly more in half of these patients a complication appeared to occur within hours of the onset of agitation. patients with agitation had increased icu and hospital lengths of stay, but this was not significant after controlling for other predictors of length of stay. for patients with hunt and hess grades - , agitation was not independently associated with functional impaired at months compared to those without agitation after controlling for other predictors (lawton > ; p = . , or . , % ci . - . ). patients with sah frequently experience agitation requiring medical treatment, especially early in their clinical course, and especially in non-comatose patients with higher clinical grades. agitation is also associated with the development of multiple hospital complications, and may have an independent impact on long-term outcomes. seizures after subarachnoid hemorrhage (sah) are a frequent complication. sah patients are typically prescribed prophylactic anti-epileptic drugs (aed) for three to seven days. phenytoin has fallen out of favor as aed prophylaxis due to its association with worsened outcome as well as drug interactions. newer aeds including levetiracetam are more commonly used despite an incomplete understanding of their effect on outcome. retrospective analysis was performed of prospectively collected data for consecutive sah patients enrolled into the university of maryland recovery after cerebral hemorrhage (reach) study between -hess (hh) and modified fisher score (mfs) was adjudi a team of neurointensivists. retrospective analysis of cumulative dose of levetiracetam was divided into groups of low-dose (= , mg) using the median as a cutoff. concordance and discordance was noted. pearson chi-square was used. association of levetiracetam dose and quetiapine use as a surrogate of in-hospital delirium was also investigated. multi-variate logistic regression was used to determine predictors of ability to perform activities of daily living (adls) in survivors. asah patients from july to november were reviewed for this study. cumulative levetiracetam dose was calculated in patients and dichotomized into high-dose (>= , mg) or lowdose groups. hunt-hess was found to be significantly associated with high-dose levetiracetam. on multivariate analysis, high-there is a trend towards increased use of quetiapine in the high-dose levetiracetam group. full analysis will be provided at time of presentation. an extended course of levetiracetam is an independent predictor of loss of independence in activities of daily living after sah. there is also a trend toward increased delirium. larger, prospective studies are necessary for a more complete understanding of the impact of seizure prophylaxis on functional outcome after subarachnoid hemorrhage. myocardium: a case series and review of the literature. intra-aortic balloon pump (iabp) counterpulsation has been used to maximize cerebral blood flow in patients with subarachnoid hemorrhage (sah), refractory vasospasm and evidence of cardiac dysfunction. neurogenic stunned myocardium (nsm) pr lv dysfunction. we present cases with sah, vasospasm and iabp placement, including cases with nsm. we also reviewed the literature with the goal of examining the safety of iabp for cardiac dysfunction after sah, outcomes and selection criteria for its use. we searched for cases of sah and iabp placement at the university of kansas medical center (kumc) from to . patients met criteria and all had a secured aneurysm, refractory vasospasm and echocardiograms prior to iabp placement. we collected demographics, vitals, ekg, troponin, medications, iabp and icu complications, discharge and follow-up mrs. however, at follow-r outcome. literature review identified patients -up. our results indicate that patients that have iabp placement in the setting of sah, vasospasm and cardiac dysfunction may have a good outcome if they are younger, have evidence of reversible nsm and avoid icu complications including pe, uti and sepsis. the patients level of mobility and independence at discharge may not be indicative of overall functional improvement. a significant complication of non-traumatic sub-arachnoid hemorrhage (nt-sah) is the development of delayed cerebral ischemia associated with cerebral vasospasm. milrinone, an inotrope and a phosphodiesterase inhibitor, has been used intravenously, intra-thecally and intra-arterially as a delayed cerebral ischemia treatment and prophylaxis. the purpose of the current study is to systematically review the available evidence on its efficacy for that indication. articles from medline, embase, cochrane library, clinicaltrials.gov, reference lists of relevant articles, and gray literature were searched. study selection criteria were used and strength of evidence was graded. neurological outcomes and side effects were assessed. of articles identified, studies met the selection criteria and analyzed. the level of evidence varied and was generally low. this systematic review helped determine the current state of evidence for the efficacy and safety of milrinone in the management of delayed cerebral ischemia in the context of nt-sah. the available evidence is promising but of generally low quality suggesting the need for a randomized controlled trial. blood lactate variability: a strong independent predictor of neurological outcomes in patients with aneurysmal subarachnoid hemorrhage blood lactate levels during intensive care unit (icu) management of patients with aneurysmal subarachnoid hemorrhage (sah) can be used as an indicator of not only volume status but also aerobic glycolysis caused by excessive catecholamine levels and impaired lactate clearance. to determine whether blood lactate variability (lv) can predict neurological outcomes in patients with sah, we assessed the standard deviation (sd) of blood lactate level of each patient during icu stay. we retrospectively reviewed all patients at the age of years or older who were consecutively hospitalized in kagawa university hospital with sah and at least five arterial lactate measurements between january , and may , . patients were divided into two groups with a mean lactate to identify independent predictors of unfavorable neurological outcome. unfavorable neurological outcomes occurred in . % of a total of patients. in both groups, there were increases in unfavorable neurological outcomes with increasing sd of lactate (quartile , %; that sd of la correlated with unfavorable neurological outcomes (p < . ). multiple logistic regression analysis showed that sd of lactate (odds ratio, . ; % confidence interval, . - . , p < . ), age, and h&k grade were independent predictors. this study demonstrated that increased lv was an independent predictor of unfavorable neurological outcomes in patients with sah. the main causes of mortality and morbidity after aneurysmal subarachnoid hemorrhage (saha) are rebleeding and delayed cerebral ischemia secondary to cerebral vasospasm. the use of milrinone, an inotropic and vasodilator agent, is described in as one option to treat vasospasm in patients with refractory symptoms. our objective was to describe the experience of our neurocritical care service with the use of milrinone in accordance with the montreal protocol for patients with refractory vasospasm. a retrospective study based on data obtained from medical records of patients suffering from saha and refractory vasospasm treated with milrinone from february to february . from saha patients admitted to our hospital during the study period, were identified with refractory were female and % of patients were pre-hypertensive. a total of % of the patients had hunt-hess scores between - and % scored or in the modified fisher scale. vasosespam was identified after -- . days. in % of the patients hypertension was induced with norepinephrine as an initial treatment. the mean duration of the treatment - . days. two cases were treated with intra-arterial milrinone and angioplasty. the most common adverse event during the use of milrinone was hypotension ( %). death occurred in patients. favorable functional outcome at the discharge was observed in % of the cases. in conclusion, the use of milrinone seems to be a safe option in the treatment of delayed cerebral ischemia secondary to vasospasm, especially in services where the availability of endovascular treatment is not a routine. (sah). however, pathomechanism and etiology of this elevation leading to poor outcomes remains uncertain. this study investigated the effect of troponin elevation on multi-organ dysfunction and outcomes in patients with sah. admitted to the neuroscience intensive care unit from july to january . among patients, patients were eligible for inclusion with investigation of serum troponin level at admission. troponin elevation (> elevation were older ( . ± . vs . ± . years; p < . ) and more often had a loss of consciousness ( . % vs . %; p < . ), symptomatic hydrocephalus ( . % vs . %; p = . ), and a higher hunt-hess score ( . ± . vs . ± . ; p < . ) and modified fisher score ( . ± . vs . ± . ; p < . ) at ictal period. during hospitalization, patients with troponin elevation more often had a respiratory failure ( . % vs . dysfunction ( . % vs . %; p = . ) and more often treated with vasopressure ( . % vs . %; p = . ) and longer duration of mechanical ventilation ( . ± . vs . ± . day; p = . ) than those without troponin elevation. troponin elevation in the acute stage of sah is associated with multi-organ dysfunction. thus troponin to mitigate early brain damage in subarachnoid hemorrhage (sah), we have been treating world federation of neurological surgeons grade (wfns) grade patients with therapeutic hypothermia (th) for days immediately after onset. management after rewarming was problematic since fever in sah is associated with vasospasm and poor outcome. we studied the feasibility and safety of endovascular cooling to maintain prophylactic normothermia following initial th in patients with severe sah. th (core body temperature . °c) was initiated, using surface cooling, immediately after the diagnosis of wfns grade sah was made. the ruptured aneurysm was surgically clipped as soon as feasible. around postoperative day , after rewarming to °c, an endovascular catheter with cooling balloons jugular vein and connected to xp® temperature management system (asahi kasei zoll medical corp.) for days. prospectively collected data were analyzed. . days. nine patients developed shivering with increased temperature and were given acetaminophen and dexmedetomidine. there was no evidence of vasospasm or additional cerebral infarction during endovascular cooling, and no catheter-related sepsis or thromboembolic event. after removal of the cooling catheter, vasospasm-related cerebral infarction and fatal bacterial meningitis related to spinal drainage occurred. three-month outcomes were good recovery (n= ), moderate disability (n= ), severe disability (n= ); vegetative state (n= ), and death (n= ). elimination of fever burden in the first days after onset was safe and feasible with combined surface and endovascular cooling in patients with wfns grade sah. disease processes. this study examines: ) the relationship between admission lactate and the clinical and radiographic severity of asah, and ) whether levels predict outcomes including vasospasm, delayed cerebral ischemia (dci), and inpatient mortality. this is a retrospective analysis of consecutive asah patients with lactate drawn on admission.compared to those with normal levels. differences between groups were compared using chi-square tests for categorical variables, and independent t-tests for continuous variables. spearman correlations were calculated between lactate levels and mean values for continuous variables. elevations in lactate were associated with admission gcs, hunt & hess (hh) grade, fisher score, serum white blood cell count (wbc), troponin i (tn), glucose, and ventilator-free days (vfd). positive correlation was found between lactate and hh, fisher score, wbc, tn, and glucose. an inverse correlation existed between lactate and gcs, and vfd. compared to survivors, non-survivors had significantly higher lactate levels. all results were considered significant with a p-value < . . no association between lactate and the development of vasospasm or dci was found. higher admission serum lactate is positively correlated with hh grade, fisher score, serum wbc, tn and glucose, but negatively correlated with admission gcs and vfd. presence of an elevated lactate was also predictive of inpatient mortality. this is the first report of correlation between early lactate and asah severity, and conflicts with prior results suggesting an association between lactic acid and the development of dci. further studies are needed to determine whether lactate elevations relate to hypovolemia, acute inflammatory response, elevated sympathetic outflow, or other cause. with malignant cerebral infarction with high osmotic pressure therapy. some patients need decompressive hemicraniectomy or expire due to cerebral herniation after ttm. hence this study was performed to determine associated factors in case of failure of ttm in patients with malignant cerebral infarction. from january to december , a study was performed in patients with malignant cerebral infarction occurred within hours at neurological intensive care unit. all patients were diagnosed hyperosmotic fluid therapy and ttm. we defined failure of ttm to cases treated by decompressive hemicraniectomy or death due to brain herniation. a total of patients, ttm were failed in patients. failures of ttm were common in patients without recanalization after thro ( fever occurs in - % of critically ill neurological patients, and small temperature elevations are correlated to increased morbidity. it is therefore crucial to acutely control the temperature of such patients. systems currently available are resource intensive and not always readily available in units, resulting in delays in treatment. emcools pads and are composed of multiple cooling units filled with graphite and water, with an adhesive underside that allows for efficient heat transfer. pads are stored at - c, and are available for immediate use. the retrospective analysis of the emcool device included all subjects that had the device applied in the neuroscience intensive care unit (nsicu), with consistent temperature data recorded. preliminary subarachnoid hemorrhage ( %), intracerebral hemorrhage ( %), subdural hemorrhage ( %), and pituitary tumor ( %). all subjects were febrile ( treatment period. the bedside shivering assessment scale was recorded at each application. c, t avg = . c) drop in temperature at mins c) achieved at mins. unconscious patients displayed a much higher rate of cooling at t as c). of the total subjects, % had shivering events upon application (bsas ), device. preliminary results show the emcools pads are an effective and safe method to control temperature elevations in neurologically critically ill patients. all even numbered posters will present on friday all odd numbered posters will present on saturday all poster sessions are in prince george exhibit hall a from : pm superior sagittal sinus thrombosis (ssst) accounts for only . - % of all strokes, with a traumatic etiology representing an uncommon occurrence. current guidelines advocate treating ssst with anticoagulation regardless of etiology, though efficacy is controversial and not yet studied in the traumatic brain injury (tbi) patient population. we recognize the importance of alternate treatment modalities of post-traumatic ssst, particularly surgical alternatives, and the dilemmas faced with anticoagulation therapy in the trauma population. we report a case of a -year-old male admitted with ssst who suffered severe tbi secondary to a pedestrian versus automobile collision. imaging demonstrated bifrontal and right temporal lobe hemorrhagic contusions, scattered subarachnoid hemorrhage, diffuse cerebral edema, multiple nondepressed skull fractures, and ssst. on post trauma day two, the patient clinically deteriorated; an external ventricular device (evd) was placed, and therapeutic heparin drip was started, despite the presence of intracranial hemorrhage and risk of evd-related hemorrhage. the patient developed refractory elevated intracranial pressure (icp) mandating initiation of pentobarbital to achieve burst suppression on continuous electroencephalography (ceeg) and serial administration of . % hypertonic bolus and mannitol for two weeks. hemicraniectomy and endovascular treatment were entertained though not pursued due to anticipated complications associated with concomitant anticoagulation therapy. anticoagulation was briefly interrupted for evd removal on post trauma day . he was extubated on post trauma day and transitioned to warfarin. repeat imaging showed complete recanalization of the superior sagittal sinus. the patient was discharged to inpatient rehabilitation after a -day hospital course. management of ssst secondary to tbi remains controversial as these patients present with multiple confounding factors, further complicated by the lack of treatment guidelines. further studies are needed to determine which independent or combined medical and surgical treatment modalities will decrease morbidity and mortality in this patient population. takotsubo cardiomyopathy (tc) is known to occur in patients with subarachnoid haemorrhage (sah) but is rarely reported in patients with traumatic brain injury (tbi). here we present a tbi patient with complicated clinical course developing severe tc and compared to previously published reports. case report and literature review. a years-old-woman was admitted to our tertiary care hospital because of tbi with admission glasgow coma scale score of . computed tomography (ct) scanning of the brain revealed an acute subdural hematoma and traumatic sah over left hemisphere and a small left frontal hemorrhagic contusion. six hours later she deteriorated and head-ct showed significant progression of right frontal hemorrhage with intraventricular expansion and a midline shift. hematoma evacuation was immediately performed. postoperatively the patient developed cardiogenic shock necessitating an increasing dose of noradrenaline, neosynephrine and dobutamine to achieve a cerebral perfusion pressure of > mmhg. echocardiography demonstrated severe left ventricular myocardial dysfunction suggestive for tc, supported by raised troponin-t and nt-probnp levels and abnormalities in ecg. continuous infusion of levosimendan was added and maintained for hours which led to stabilization allowing noradrenalin to be decreased the following days. repeated echocardiography days later showed normalized cardiac function. the patient's condition gradually improved and was extubated after days fully awake with mild left facial-brachial weakness. here we present a complicated case of tc with tbi developing cardiogenic shock within hours of admission. we will compare the patient's tc characteristics and clinical course with published cases (n = ) of tc with tbi. further studies of tc in patients with tbi and the utility of levosimendan is warranted. management of post-operative central diabetes insipidus (di) is focused on replacing urine output with free water. this may not always be sufficient, and desmopressin (ddavp) is needed. the use of ddavp, however, is known to cause profound changes in sodium particularly if the triphasic response postpituitary surgery is occurring. herein, we report a case using a dilute vasopressin bolus protocol in managing hypovolemia in acute, post-operative, central di. case report comparing two protocols for di management. statistical analysis was performed on serum sodium, urine specific gravity, and urine output using student t-test. p< . was considered significant. anti-programmed cell death (pd- ) antibodies are an effective treatment option for nsclc and other cancer entities. anti pd- antibodies including nivolumab can induce immune-related adverse events (iraes) in a number of organ systems. neurological iraes can be life-threatening and necessitate appropriate investigation and management by a neurologist. myasthenic syndromes have rarely been described. here we present a case of a man who developed a lambert eaton myasthenic syndrome thought to be a nivolumab-related immune adverse event. a -year-old man on nivolumab for metastatic nsclc developed asymmetrical ptosis followed by facial diplegia, dysarthria and dysphagia and fatigable limb weakness. he had clinical features of lambert eaton myasthenic syndrome with striking truncal, shoulder and pelvic girdle fatigable weakness that demonstrated a temporary increase in strength during the first few contractions. he developed neuromuscular respiratory failure requiring noninvasive positive pressure ventilation. his muscle reflexes were initially absent and after repeated attempts could be elicited. his mri brain was normal, csf was within normal limits and his serum and csf antibodies against ach receptors, musk and voltage gated calcium channels were negative. he weakened after an initial mg/kg dose of prednisone. plasma exchange resulted in a significant improvement of his weakness and the ability to wean the patient off noninvasive positive pressure ventilation. it is important to recognize that neurologic immune-related adverse events associated with nivolumab can cause lambert eaton myasthenic syndrome. early recognition and aggressive treatment with plasma exchange can be life-saving. neuroendocrine tumor (net) of middle ear is extremely rare. many names have been ascribed to these seemingly benign neuroendocrine lesions including middle ear adenoma (mea), adenomatous tumor and carcinoid tumor (ct). 'neuroendocrine adenoma' has also been used to better describe the histologic nature of these tumors. here we present the first case of carcinoid tumor of middle ear (ctme) complicated by dural sinus thrombosis and bony metastasis. a year old man presented with right sided facial palsy for hours and progressive hearing loss for months. physical examination revealed perforated right tympanic membrane with visible purulent material. ct scan of the head showed a small right cerebellar infarct. the mri demonstrated right cerebellar hemorrhagic venous infarct and a heterogeneous middle ear mass extending into the internal auditory canal. mrv revealed thrombus in the right sigmoid and transverse sinus.cervical spine mri revealed diffuse osseous metastases .the histology and immunohistochemistry (ihc) after surgical resection was consistent with ctme. proliferation rate of> % was seen by ki staining. he suffered massive intracranial bleed on heparin therapy and passed away. the nets of head neck region are divided in categories based on histology and ihc. ) well differentiated ct, / hpfs and ki- > %. although regional metastasis is not uncommon, only cases of distant metastasis have been reported in the past. our patient was diagnosed to have atypical carcinoid but the ki- was > % which is consistent with small cell cancer. the new classification system that takes the ihc and presence of metastasis into consideration to classify these tumors is much more clinically relevant. more research is necessary to find out the biological markers for better prognostication of this rare cancer. sung m. cho.neurological institute, cleveland clinic, cleveland, oh, usa. acute transverse myelitis is an acquired inflammatory spinal cord disorder, which can be due to infection, autoimmune disorders, or malignancy, however, the cause is often unknown despite an extensive workup. we describe a rare case of acute transverse myelitis caused by coxsackie b /b virus. a -year-old male with no past medical history presented with a viral prodrome of flu-like symptoms followed by severe headache, neck stiffness, photophobia, encephalopathy and paraplegia. mri of the brain with contrast was unremarkable, but mri of the spine showed an extensive longitudinal, nonenhancing t cord signal change from c -t without hemorrhagic components. lumbar puncture revealed rbc, wbc, protein, and glucose ( mg/dl serum glucose). extensive serum and csf work-up was negative for hiv, wnv, ebv, cmv, lyme, vzv, hsv, nmo antibody, ace, paraneoplastic panel, cytology, cryptococcus, and csf bacterial and fungal cultures. ct chest and blind transbronchial needle lymph node biopsy were negative for sarcoidosis. serum coxsackie b ( : ) and b (> : ) antibody titers were significantly elevated. the patient was treated with intravenous methylprednisolone mg for days along with plasmapheresis for sessions. the patient had improvement in lower extremity strength during his hospital stay and was discharged to a rehab facility on a steroid taper. at one-month follow up, the patient had complete recovery of lower extremity strength as well as bowel and bladder function and was ambulatory. at -month follow-up, mri and csf studies were markedly improved. transverse myelitis due to coxsackie has been reported in serotypes b , b , a , a , however only three cases of b or b related transverse myelitis have been reported and this is the first case, to our knowledge, with both b and b related transverse myelitis. external ventricular drains (evds) are necessary for select patients admitted to the neurointensive care unit (nicu). evds are critical to the management of diseases such as subarachnoid hemorrhage, traumatic brain injury, and acute hydrocephalus. we report a case of a woman with poor-grade aneurysmal subarachnoid hemorrhage who experienced inadvertent intraventricular non-iodinated contrast injection during vasospasm evaluation with ct angiography. we provide a review and analyses of adverse evd related injections reported in the literature and summarize management recommendations. a pubmed search was performed for unintended evd injections from to . unique cases were selected and classified by the type of inadvertent injection and location of the event. acute management was categorized by the use of evd manipulation, lumbar drain placement, and supportive medical therapies. cases involving ventriculoperitoneal shunts, ommaya reservoirs, or other intrathecal device systems were excluded. a total of seven unique cases were identified, four involving contrast administration and three involving medication administration. the sentinel event in five cases occurred outside of the nicu. acute management with evd manipulation was employed in six cases; three of these cases also used lumbar drains. three cases necessitated intubation. our patient received prophylactic dexamethasone and levetiracetam, underwent immediate evd manipulation, and placement of a lumbar drain. continuous eeg monitoring and daily head cts were performed until intraventricular clearance was noted at hours. acute management of inadvertent intraventricular injections entails immediate evd manipulation and possible lumbar drain placement to facilitate rapid csf clearance of injected substances. intubation may be required immediately depending on exposure. inadequate distinction between compatible drain tubing and relative inexperience of providers managing the evds likely contributed to the errors in these cases. the increased incidence of these events outside of the nicu suggests additional safety measures may be warranted when patients with evds travel off-unit. an unusual presentation of isolated brain abscess in non-traumatic convexal subarachnoid hemorrhage: a case report to present a unique case of isolated brain abscess presenting as non-traumatic convexal subarachnoid hemorrhage (csah) six days before radiologic signs could be seen. to our knowledge only one other case of csah due to brain abscess has been reported thus far. a -year old man with no past medical history or prior trauma presented with acute onset of transient left hemianesthesia lasting ten minutes. computed tomography (ct) of the head revealed csah. we were presented with a diagnostic dilemma when magnetic resonance imaging (mri) of the brain, magnetic resonance angiography (mra) of the head and neck, magnetic resonance venography (mrv) and conventional angiography failed to show the cause of csah. the patient was discharged in a stable condition, but returned six days later with worsening symptoms, including left hemiparesis. repeat mri with contrast revealed a cerebral abscess in the same location as the prior csah. csah without clear evidence of trauma due to abscess is a rare occurrence. we suggest in cases of csah where imaging techniques present no abnormalities, follow-up imaging within seven days should be considered. jonathan marehbian, diane d. chan, david d. greer.yale new haven hospital, department of neurology, new haven, ct, usa. spinally-mediated reflex movements can be present in brain dead patients. however, abnormal movements have long been a challenge in the clinical determination of brain death. in this report, we describe delayed plantar extension with noxious nail bed stimulation that has not been previously described in brain death. a -year-old male suffered severe anoxic brain injury following respiratory failure due to heroin overdose. his clinical exam and apnea testing were consistent with brain death with the exception of a reproducible delayed plantar extension with noxious nail bed stimulation. ancillary testing with technetium m nuclear scan (spect) demonstrated no cerebral blood flow, confirming that the finding was spinally-mediated. novel movements are important to document in order to aid in the timely determination of brain death, and to avoid unnecessary and potentially confounding ancillary testing. the mechanism underlying delayed plantar extension is likely spinally mediated. vascular perforation during a neuroendovascular procedure is an unexpected and feared complication, which can lead to fatal outcomes. a prompt recognition and initiation of treatment are paramount. endovascular strategies to address this complication have been widely described. however, the goals of therapy in the neurointensive care unit (neuroicu) remain unclear. we report two cases in which endovascular strategies associated with aggressive intensive care resulted in a good clinical outcome at discharge. case report. vessel perforation occurred in patients during neuroendovascular interventions: a left-carotid stenting and an ica-aneurysm embolization with balloon-assisted coiling. once contrast extravasation was demonstrated, heparin was immediately reversed and endovascular strategies were performed to minimize the complications. sah was present in the initial head ct in both cases. in the neuroicu, targets of therapy were a) systolic blood pressure (sbp) < mmhg, b) mechanical ventilatory support, and c) seizures and vasospasm prophylaxis with phenytoin and nimodipine respectively. one of the patients developed mild hydrocephalus and left upper extremity weakness, with mri showing tiny right hemispheric strokes, while the other one, did not show any focal deficit. both patients were discharged home few days later with mrs of and , respectively. iatrogenic vascular perforation is an uncommon complication that occurs secondary to inadequate manipulation of the catheter, guide wire, devices, or forceful contrast injection. clinical care strategies aim to prevent fatal outcomes. we recommend reversing heparin; provide an adequate ventilatory support, maintain a strict control over the sbp, and seizure and vasospasm prophylaxis. but, larger studies are required to determine the standard of care since endovascular therapy is rapidly becoming first-line of treatment for neurovascular conditions. cerebral hyperperfusion syndrome (chs) symptoms range from severe unilateral headache to seizures, focal symptoms and intracerebral hemorrhage, usually occurring follow carotid endarterectomy (cea)/ carotid stenting (cas). we describe a case of a patient who developed chs after spontaneous recanalization of carotid intra-stent thrombosis. case report. years old african american male who had recently placed left cervical stent was transferred to baptist medical center with new evidence of intra-stent thrombosis. he was started on heparin drip and sbp was augmented with levophed for sbp> . two days later, the patient developed excruciating headache followed hours later by acute onset of right upper extremity and face twitching associated with severe hypertension. the bp was controlled, protamine was given to reverse heparin and keppra was loaded to treating seizures. cta at this time showed recanalization of carotid in-stent stenosis and ctp confirmed hyperperfusion of frontal and parietal lobe with neither evidence of new ischemic area nor bleeding. fortunately, inspite a delay in diagnosis by hours, patient did not suffer intracranial hemorrhage. most patients who develop chs will have complete recovery if it is discovered and treated early.aggressive prophylactic blood pressure control is the main treatment. for those who are diagnosed late and those progressing to ich, the prognosis can be devastating with mortality rates up to %. considering the importance of blood pressure control in the cerebral hemodynamic, studies have been done, trying to find a better tool to predict the best bp target in order to prevent chs. near infrared spectroscopy (nirs) is a non-invasive and reliable technique that monitor the cerebral hemodynamic. had nirs been deployed during anticoagulation, the diagnosis of cerebral hyperperfusion syndrome would have been made at the onset of headaches and would have avoided the development of seizures and potentially a life threatening hemorrhage. the triphasic response: water imbalance after neurosurgery : a case reportbibhukalyani das, indranil i. ghosh.institute of neurosciences kolkata, kolkata, india. water balance disorders after neurosurgery are well recognized, but detailed reports of the triphasic response are scarce. we describe a -year-old woman, who developed the triphasic response with hyper and hyponatraemia after resection of craniopharyngeoma. a -year-old female (no previous medical history, no medication, normal electrolytes and endocrine parameters) with mri showing s/o craniopharyngeoma underwent neurosurgery using a subfrontal approach to resect the craniopharyngeoma while leaving the pituitary stalk intact (according to the surgical notes). within a few hours of an uneventful postoperative recovery she started having polyuria and hypoosmolar urine with hypernatremia. being normoglycaemic and not on any diuretics a diagnosis of central diabetes insipidus was made treated with desmopressin nasal spray and drinking water ad libitum. improvement occurred over the next postoperative day when desmopressin was discontinued. on the third day she developed with hypoosmolar hyponatremia along with seizure treated with % saline and fuid restriction. two days later polyuria returned and was ultimately discharged with desmopressin tablets and advice to take fluids ad libitum. na this case illustrates the dramatic and sudden changes in water balance that may occur after neurosurgery. the pathophysiology of the triphasic response appears to be early hypothalamic dysfunction, subsequent release of vasopressin from the degenerating pituitary and, finally,depletion of vasopressin stores. it has been difficult to identify patients at risk, but predisposing factors appear to relate both to the disease (macroadenoma, microadenoma, craniopharyngioma) and to the surgery (degree of manipulation). successful prevention probably involves a psychological switch by not waiting until frank dysnatraemia has developed, but to act as soon as urine output and tonicity change. this requires an index of suspicion for treating and consulting physicians and specific instructions to nursing staff, especially in nonintensive care settings. refractory status epilepticus (rse) has high mortality and is difficult to treat. when traditional therapies fail ketamine may be considered. ketamine is associated with limited reports of adverse cardiac events during anesthesia, but not during treatment for rse. we evaluated occurrences of cardiac arrhythmias associated with ketamine. retrospective chart review of neurocritical care patients in a tertiary academic medical center who received ketamine infusion for rse between october and april . ten patients were admitted to a neurologic intensive care unit and received ketamine infusion for rse. etiology of rse included autoimmune/infectious process ( ), ischemic stroke ( ) and subarachnoid hemorrhage ( ). of the ten patients who received ketamine, three had documented cardiac events without prior cardiac history. one patient remained clinically stable and did not require intervention. another patient required escalating doses of ketamine infusion (maximum mg/kg/hr) for rse secondary to presumed leptomeningeal disease, had an asystolic event and expired. the third patient was on low dose ketamine ( . mg/kg/hr) for rse secondary to subarachnoid hemorrhage, and developed multiple arrhythmias including recurrent episodes of asystole. once ketamine was discontinued the patient stabilized. arrhythmias are not uncommon in critically ill patients, but this is the first report of cardiac arrest associated with the use of ketamine for rse. although sympathomimetic properties of ketamine may provide vasopressor sparing effects, which reduce the need for vasopressors to counteract the hypotension commonly seen with other anesthetics used in rse, it may put patients at risk for cardiac arrhythmias. in addition, ketamine has direct negative ionotropic effects and may raise pulmonary artery pressures. caution should be employed when ketamine is used in rse in patients with other independent risk factors for cardiac events. a year-old veterinary technician with headache and fever for days presented with altered mental status and myoclonic jerking. initial lp showed white cells and elevated protein ( mg/dl). recurrent clinical seizures occurred for days prior to transfer to our institution. his exam demonstrated diffuse hyperreflexia and coma; eeg demonstrated up to . hz frontally-predominant rhythmic delta but no unequivocal seizures. extensive workup revealed no evidence of infectious, toxic, or immune-mediated encephalitis. mri demonstrated bithalamic injury and a region of questionable periventricular nodular heterotopia in the right parietal region. subsequently, he developed recurrent clinical and unequivocal electrographic seizures from the right parieto-occipital region. five periods each lasting > hours of anesthesia-induced eeg burst suppression failed to stop seizures, qualifying him as super-refractory status epilepticus. after weeks of failure to wean from anesthetia, invasive monitoring for seizure localization was carried out using strips and depth electrodes. multiple seizures were recorded, localizing to the medial occipital lobe, which was subsequently resected along with the region of pathologyconfirmed heterotopia. seizures gradually improved, requiring anti-seizure drugs and a ketogenic diet. he regained consciousness with preserved higher cognitive functions (language, memory) and personality months later as his antiseizure drugs were successfully decreased. his motor recovery was limited by critical illness myoneuropathy. new-onset focal super-refractory status epilepticus may respond to surgical resection in extreme cases. close collaboration with a multidisciplinary team of epileptologists and neurosurgeons can lead to resolution of seizures and eventually recovery. in patients with leptomeningeal metastases (lm) and elevated intracranial pressure (icp), transient neurologic events secondary to plateau waves -temporary elevations in icp -may occur. there is a paucity of clinical reports correlating video-eeg with definite or presumed plateau waves in patients with lm. case report and literature review. pubmed was queried for 'leptomeningeal metastases and eeg', 'leptomeningeal metastases and plateau waves' and ' leptomeningeal metastases and intracranial pressure'. a -year-old woman was transferred to our hospital for further care of a pituitary microadenoma noted on outside imaging. she had a history of hodgkin lymphoma treated with radio-chemotherapy. during her admission, she developed episodes of confusion and video-eeg monitoring was initiated. while there were no electrographic seizures, several events, between to minutes in duration, of delayed or absent verbal responses, eye rolling, staring and alternating gaze preferences to both sides were noted. all these coincided with abrupt onset of marked background slowing, evident through generalized - hz delta, mixed with some theta frequencies. review of outside and repeat imaging with mri of the brain with contrast revealed brain and leptomeningeal metastases. cerebrospinal fluid examination showed raised opening pressures and malignant cells, leading to a subsequent diagnosis of primary signet ring cell cancer. we found one additional report describing video-eeg correlates of presumed plateau waves in patients with lm. our clinical description of transient neurologic events in this patient adds to the current literature of paroxysmal manifestations owing to raised icp in patients with lm. awareness of this clinical phenomenon may serve as a surrogate of raised icp before clinical signs of the same develop in patients with lm. it may also help delineate the cause of raised icp due to cns metastases in a patient with a corresponding history of cancer. background: terson's syndrome is the development of intraocular hemorrhage (ioh) in association with subarachnoid hemorrhage (sah). we report a case of terson's syndrome and review the literature. case report: a yr woman presented with severe neck pain and somnolence. ct imaging showed fisher grade sah with aneurysms in the right internal carotid and posterior communicating arteries. she developed low pressure hydrocephalus treated with ventriculostomy. on hospital day she developed vision loss. ophthalmologic examination demonstrated bilateral vitreous hemorrhages with near complete fundoscopic resolution by day . over the next months she underwent pars plana vitrectomy (ppv) of the left eye two times. current vision od / , os / . discussion: the incidence of terson's syndrome among patients with sah is documented to be between - %. in prospective studies, ioh was found in up to % of patients with sah compared to only % in retrospective studies suggesting under-reporting. this is likely because ioh is found more often in higher severity bleeds where patients cannot self-report visual loss due to decreased loc. patients with ioh are more likely to have worse neurologic outcomes and die more often than those with lower grade bleeds without the development of ioh. along with fundoscopy, hand held ultrasound may be used for bedside diagnosis. erm development is the most common intraocular complication from terson's syndrome and occurs in - % of patients. complete or near complete return of visual acuity is less likely without surgical intervention. visual acuity has been show to recover better and faster if ppv is performed within days. there is no literature on incidence of visual loss after acute phase of sah. conclusions: routine evaluation with imaging and fundoscopy may help in detecting ioh sooner in the clinical course and has the potential to decrease long-term morbidity. iatrogenic underfeeding in critically ill patients is often unrecognized and underestimated. an international prospective study of critical care units showed patients received only . % and . % of prescribed calorie and protein needs. the inability to initiate enteral nutrition within - hours of icu admission or frequent interruptions of the enteral regimen lead to insufficient nutrient delivery and a compounding energy deficit. an increase in infectious complications is associated with negative energy balance in patients with subarachnoid hemorrhage (sah). a quality initiative project was developed at mayo clinic florida to measure time to reach enteral nutrition target and common interruptions of enteral nutrition. the target subjects were mechanically ventilated patients in medical and transplant icu; however the quality measure has recently extended to the neurocritical care unit. data collected included clinical diagnosis, sofa and apache ii score, subjective global assessment (sga) score, nutric score, enteral tube type and regimen, and reason and duration for interruption of nutrition. a year old subject with sah and posterior fossa avm resection was deemed low nutrition risk with sga score a and nutric score . enteral nutrition was initiated via nasoenteric tube within hours of intubation. target enteral goal rate was reached within hours. the patient received % of calorie/protein needs of the days. the most common enteral interruption was for procedure; primarily head ct, for longest duration of minutes. the interruption of enteral nutrition in neurocritical care patients is likely unavoidable due to procedures. these disruptions, however, need not result in iatrogenic underfeeding. neurocritical care units may utilize volume-based enteral protocols to allow nurses to compensate for lost nutrition with increased enteral rate. neurogenic pulmonary edema is challenging to manage in the context of aneurysmal subarachnoid hemorrhage (asah) due competing priorities between organ systems. we present a case of refractory neurogenic pulmonary edema due to asah necessitating extracorporeal membrane oxygenation (ecmo). case report. a year-old female with a history of hypertension and diabetes presented neurologically intact with hh f asah due to a left posterior communicating artery aneurysm. she underwent coil embolization on sah day and remained intubated after the procedure due to development of flash pulmonary edema. transthoracic echo demonstrated normal left ventricular function. on sah day after unplanned extubation, she was temporized on noninvasive ventilation until reintubation the following day. she progressed to severe ards requiring high-dose sedatives and paralytics which obscured her neurologic exam. on sah day , a day course of intrathecal nicardipine was initiated for elevated left mca transcranial doppler velocities. on sah day , her respiratory status further declined and veno-venous ecmo was initiated as rescue therapy after head ct did not demonstrate new hemorrhage or infarct. throughout the day ecmo course, a low-dose heparin infusion was utilized along with vasopressors to optimize cerebral perfusion pressure (cpp). she was decannulated on sah day . surveillance ct head demonstrated left-sided ischemic infarcts in multiple vascular territories. on sah day , she was discharged to an outside facility for ventilator weaning. upon discharge, she was alert and followed commands with her left arm, however she was aphasic without movement of her other extremities. veno-venous ecmo was performed in an asah patient after coil embolization. although the patient did not develop intracranial hemorrhage, her course was complicated by severe vasospasm and delayed cerebral ischemia (dci). while ecmo is a rescue therapy for severe hypoxemia, its effect on cpp remains uncertain and may potentiate dci. key: cord- -k y fz k authors: belli, simone; mugnaini, rogério; baltà, joan; abadal, ernest title: coronavirus mapping in scientific publications: when science advances rapidly and collectively, is access to this knowledge open to society? date: - - journal: scientometrics doi: . /s - - - sha: doc_id: cord_uid: k y fz k the covid- pandemic is creating a global health emergency. mapping this health emergency in scientific publications demands multiple approaches to obtain a picture as complete as possible. to progress in the knowledge of this pandemic and to control its effects, international collaborations between researchers are essentials, as well as having open and immediate access to scientific publications, what we called “coopetition”. our main objectives are to identify the most productive countries in coronavirus publications, to analyse the international scientific collaboration on this topic, and to study the proportion and typology of open accessibility to these publications. we have analyzed , articles indexed in web of science. we performed the descriptive statistical analysis in order to explore the performance of the more prolific countries and organizations, as well as paying attention to the last years. registers have been analyzed separately via the vosviewer software, drawing a network of links among countries and organizations to identify the starred countries and organizations, and the strongest links of the net. we have explored the capacity of researchers to generate scientific knowledge about a health crisis emergency, and their global capacity to collaborate among them in a global emergency. we consider that science is moving rapidly to find solutions to international health problems but access to this knowledge by society is not so quick due to several limitations (open access policies, corporate interests, etc.). we have observed that papers from china in the last months (from january to march ) have a strong impact compared with papers published in years before. the united states and china are the major producers of documents of our sample, followed by all european countries, especially the united kingdom, germany, the netherlands, and france. at the same time, the leading role of saudi arabia, canada or south korea should be noted, with a significant number of documents submitted but very different dynamics of international collaboration. the proportion of international collaboration is growing in all countries in – , which contrasts with the situation of the last two decades. the organizations providing the most documents to the sample are mostly chinese. the percentage of open access articles on coronavirus for the period – is . % but if we focus in the figures increase up to . %, due to the commitment of commercial publishers with the emergency. keywords coronavirus · covid- · sars-cov- · -ncov · scientific collaborations · open access · bibliometric analysis in december , a new type of virus of the family coronaviridae, currently called sars-cov- (former -ncov), was identified as the cause of an outbreak that was later on january , declared a public health emergency of international concern (pheic) by the international health regulation emergency committee by which tens of thousands of cases with relevant associated mortality have been reported. on march , , the world health organization declared the pandemic. since then, the covid- outbreak is creating a global health emergency. effective control must be done not only by reinforcing containment measures, but also by promoting basic research on the disease, trying to anticipate it, and developing treatments and vaccines that prevent new scenarios of generalized contagion. also, it is crucial to acquire the maximum possible knowledge about the sars-cov- virus, as well as the possible clinical-healthcare and public health measures that allow its immediate incorporation to protect people's health and contain the spread of the disease covid- . bibliometric studies on coronaviruses are few. the first of them was from chiu et al. ( ) , and collected articles indexed in the science citation index (sci) in the period march-july , analyzing traditional indicators (authorship, collaboration, journals, language, document type, organization, times cited, etc.). from here other similar studies have been published such as an analysis of papers on sars in medline (yang and yang ) , articles on sars indexed in sci in the period - (kostoff and morse ) , articles on mers published between and in pubmed (wang et al. ) , articles about mers between and in scopus (zyoud ) or articles in scopus of various infectious pathologies (sweileh ) . more recently, a short letter to the editor (bonilla-aldana et al. ) has been published that provides a very succinct description of , articles on coronavirus in sci, scopus, and pubmed. on the other hand, larivière et al. ( ) have emphasized the importance of "opening" all scientific literature and also paying attention to articles that are not written in english to facilitate the rapid advancement of research in an emergency situation such as today. according to their data, , articles have been published on the topic of coronaviruses since the late s in wos but more than half are closed access. with a wider perspective, zhang et al. ( ) measure the response of academia to six international public health emergencies since (sars, influenza a(h n ), ebola, zika, and they also present a preliminary analysis of the academic response to covid- based on publications indexed in wos, pubmed and cnki until april . although the sources and time coverage are different, in results section we will compare some of their results with ours. bibliometric techniques have also been applied to the analysis of press news about coronavirus. the first one was from chan et al. ( ) and the treatment of sars was focused on six newspapers (china, hong kong, canada, and europe) to compare the presence of news in the press with the official figures of dead and infected people, provided by who. this was followed by others of a similar nature focused on the press in china (hong ) , the usa (bomlitz and brezis ) or south korea (you and ju ) . in general, the bibliometric studies on coronaviruses published so far mainly extract data from wos, scopus, and medline. they are based on few articles and carry out a traditional bibliometric analysis (authors, collaborations, countries, organizations, journals, etc.) . in our case, in addition to compiling a greater number of articles, we want to place emphasis on two focus that ensure the rapid advancement of science: international collaborations (previously studied) and the availability of content in open access (a newer indicator in this type of study). the availability of scientific production openly accessible related to it is, beyond a desired phenomenon, an indicator that the openness of science is a reality that can be framed if needed. open access is an essential instrument based on the new strategies to share knowledge and cooperative work using digital technologies showing the world that investigators are achieving attempts that otherwise would not be possible without collaborative networks and technological tools combined together . the spreading of the oa movement in several countries, exemplified by the growth of regional and national initiatives, such as the creation of oa digital journal libraries and the establishment of supportive governmental policies (abadal et al. ; minniti et al. ) , provides evidence of the significant role oa is playing in reducing the scientific gap between countries and improving their participation in the so-called "global knowledge commons" (chan et al. ) . open science is the best method because it is an approach based on collaborative work, openness, and transparency in all stages including not only publication, but also data collection, peer review, and assessment. in this way, open science aims to bring research closer to society. open science entails a radical transformation in the way scientific research is conducted and in its assessment system. although the term open science includes various components, such as open research data, open access to publications, open peer review, citizen science, and new metrics, we focus on two of them. the main benefits and opportunities of open science include transparency (all phases can be monitored), greater effectiveness (rapid knowledge transfer helps avoid duplication and increases the speed of research), reproducibility (thus preventing fraud) and greater output. the main aim of the present paper is to analyze scientific publications on coronavirus listed by web of science to analyze two main characteristics of open science, as international collaboration and open access to publications. the data obtained will be useful to determine the evaluation of coronavirus publications in the world and the principal characteristics of these scientific publications. this article explores the capacity of researchers to generate scientific knowledge about an international health crisis emergency, and their global capacity to collaborate between them. to progress in the knowledge of this pandemic and to control its effects, international collaborations among researchers are essentials, as well as having open and immediate access to scientific publications. also, the paper identify the countries with the most scientific publications, their international scientific collaboration, their impact, and the percentage of open accessibility of these publications. we offer a general search in all databases available at web of science (wos) platform and a deeper bibliometric analysis of recent coronavirus scientific publications indexed in its core collection. the general search covers all registers present in all databases, while the core collection search considers the period from to . also, we consider the major trend from to to map the number of publications, but the specific analysis will be treated both in the two last decades and two last years to compare what is happening in the present moment. the bibliometric analysis of the wos database aims at mapping the use and distribution of oa in articles and cited references, among countries and organizations. the main reason we focused our mapping on wos is the availability and structuring of the cited references offered by its core collection and especially the information provided about the accessibility of each record ("open access indicator" field). besides that, and considering the fact that a great part of its collection is represented by journals with restricted access, it seemed interesting to see how much of the articles are opening in the present. the data cover all documents related to the following search expression (topic search: "coronavirus or covid- or sars-cov- or -ncov") retrieved from web of science (from clarivate analytics). we proceeded a manual download ( registers each time), divided into two datasets retrieved respectively on march th and rd, with the following search limits: year published: no limit defined document type: (dt = article or dt = proceedings paper or dt = review) databases: kci-korean journal database, russian science citation index and scielo citation index (we filtered the regional indexes and discarded the others, like derwent innovations and current contents connect) year published: (py = or py = or … or py = document type: (dt = article or dt = proceedings paper or dt = review or dt = early access) collection citation indexes: sci-expanded, ssci, a&hci the article was considered as the unity of analysis, and its references were used to calculate percentage citations to oa journals and the price index (percentage of citations to articles until the past years). in order to make a comparison between the recent publications about the covid- and coronaviruses in general published in the last two decades, we considered two periods: - and - . the bibliometric analysis was focused on the following characteristics of the citing articles, considering the information of various bibliographic fields presented in wos: . database the first three characters of the "accession number" (ut) were used to identify the source database; and those ut begun with "wos" were used to match to the following dataset, in order to identify the intersection between both. agency and grant number" (fu) field; . international collaboration or not based in the "author address" (c ) field, the following criteria were applied: if the article has authors from more than one country it was classified as international collaboration; . affiliation country and organization using the "author address" (c ) field, each country and organization were identified. then we processed the information from the "cited references field" (cr) of core collection dataset, which consisted on separating the different cited references, discarding non-journal articles and separating each piece of information that composes the cited reference. an algorithm was applied (mugnaini et al. ) to match the string respective to the cited journal, considering all variations written by the authors in order to identify if it was present in doaj. finally, we identified the references whose year was not older than years with respect to the citing article in order to calculate its percentage (price index). we performed the mainly descriptive statistical analysis in order to explore the performance of the more prolific countries and organizations, as well as paying attention to the last years. registers have been analyzed separately via the vosviewer software (van eck and waltman ) drawing a network of links among countries and organizations to identify the starred countries and organizations, and the strongest links of the net. the reason for choosing the vosviewer software tool to build and represent large networks form "open access status is provided across the web of science platform as a result of a partnership with impactstory, a not-for-profit organization that recently launched a knowledgebase of open access (oa) content. this knowledge base makes it possible to discover and link to legal gold or bronze (free content at a publisher's website) and green (e.g., author self-archived in a repository) oa versions. this partnership improves discoverability and access to article-level oa versions not only by adding more links to oa content, but also by prioritizing the links to the best version of oa content when multiple versions of an article are available". retrieved from: https ://image s.webof knowl edge.com/image s/help/wos/hp_resul ts.html#dsy -trs_open_acces s. bibliometric data among other tools, like gephi or pajek (van eck and waltman ), is the significant display quality, the choice of depicting the density of links and the possibility of generating overlay maps adding data batches. these features are relevant in order to conduct our research. similar criteria could be observed in the study of vargas-quesada et al. ( ) and belli and baltà ( ) , who also prefer vosviewer (version . . ) over other software tools. it should be noted that the wos data has been modified in order to unify and group country names in the c and rp parameters. specifically, the denomination "peoples r china" has been replaced by "china"; the joint denomination "united kingdom" has been added to the registers of scotland, wales, north ireland, and england. also, for regional comparison, the name "european union" has been added after the name of the countries that are currently part of it, to which we have added the united kingdom despite the recent departure of the eu on january . the general search in the all databases dataset resulted in , registers, from which , were articles published in journals. a reduced part of the production ( . %) was found in regional databases (citation indexes from korea, russia and ibero-america "scielo"). the great amount of production came from wos core collection, from which we differentiate three types of records, distributed in two databases. one database is the emerging sources citation index-esci (with records or . %), that contains journals of regional importance and in emerging scientific fields, but that were not accepted yet to the core collection. it means that the impact factor is not calculated to esci journals, representing a huge negative effect by authors from countries where evaluative processes give special importance to this indicator. another type of record was created using the same search expression we used to conceive the core collection dataset, that is to say, those representing the intersection between both datasets (with , or . % of the articles). finally, we have the records that are also present in core collection, but cannot be retrieved with our search expression in that database (representing . %). figure reveals differences over time, mainly after , when both numbers of articles show growth, but more significantly to the retrieval in both wos sources, meaning that the terms in our search expression settle. that was the year just after the first sars crisis began, resulting in that the queried terms retrieved . % of the articles in core collection before that , but . % afterward. the differences above are important to be taken into account when investigating the coronavirus research in wos, but we decided to pay deeper attention just to the , articles queried in core collection (including their , cited references), from the last two decades. one reason is simply due to the fact that the authors have appointed the terms in the title and/or abstract of their articles and the other is the completeness of metadata in core collection when downloading data from its interface, compared to the data delivered by wos platform. our objective is to analyze the composition by countries and organizations of publications on coronavirus in the period - and in the most recent period january -march , covering the sars crisis of ( - ) and that of - . in this way, we will be able to capture the most prolific countries and the composition of organizations around coronavirus research, attending to their productivity, the degree of international collaboration and the number of citations accumulated. the network map generated by the vosviewer software for the , documents by countries for the period - ( fig. ) offers us a fairly clear image of three main clusters. the first one is the usa in green, involved in the . % of the documents. it is followed by china in yellow, with the . %. finally, it is the cluster formed by the different - - european countries in red, where contributions from germany ( . %), uk ( . %), the netherlands ( . %) and france ( . %) stand out above the rest. to these, we must add a fourth cluster made up of different asian countries such as japan ( . %), south korea ( . %) or taiwan ( . %), among others. paying attention to the links between countries we can identify, based on co-authorship, certain recurring relationships. it highlights the high interaction between european countries and their preferential relationship with the usa over china. in fact, the different european countries and the usa appear as preferential partners in the network, with each other and with the rest of the countries for the period - . by covering two decades of publications, we understand that its composition suggests a relatively established structure of leadership and recurring relationships in research around the coronavirus. on the other hand, trying to capture the characteristics of the publications related to the covid- , we focus on the period between january and the twelve first weeks of . since the search strategy used uses the different names of the virus, it is assumed that a large part of the documents does not refer to the recent pandemic that began in the months of november and december of that year in the chinese region of wuhan and spread through different areas of china, asia, europe and the rest of the world during the first trimester of . despite this, we consider this sample as related and representative of the first research incomes of the second wave of sars. the map generated by vosviewer (fig. ) for the documents for the period - shows fewer countries involved than those accumulated for the last two decades and a somewhat different distribution of the clusters. compared to the - map (fig. ) , china is involved in the . % of the documents and gains a more central position in the network in close relationship with the usa, which participates in the . %. again, both countries are the main producers of documents and relationship nodes in the network, together with a group of european countries led by the united kingdom ( . %), germany ( . %), france ( . %), and the netherlands ( . %). likewise, there are differentiated groups of countries whose main partner is the usa but which do not maintain relations with each other. one of them, in green color, is the one formed by canada, involved in the . % of the documents. in this group, we also have italy ( . %), india ( . %) and iran ( . %). the other is the one formed by saudi arabia, with an important contribution of documents ( . %), egypt ( . %) and lebanon ( . %). we can also identify a representation of the asian cluster, in blue in the - network map ( fig. ) , in which south korea, involved in the . % of the documents, gains prominence above japan, involved in the . %. the number of documents provided by each country, the number of citations accumulated by these documents and the total link strength (tls), that is, the total sum of all links generated by each document, are considered for both periods. a higher value of the tls in proportion to the number of documents is an indicator of greater international co-authorship in the publication of scientific documents and of a greater recurrence of international collaboration in research in a given country. on the other hand, a lower tls value suggests a greater weight of internal scientific production. for the - period (table ) , the value of the tls in proportion to the number of documents provided is especially low in countries such as japan, south korea, taiwan or brazil, with . , . , . and . links per document and . %, . %, . % and . % of documents resulted from international collaboration respectively. at the other extreme, we find especially high values in egypt, with . links per document and . % of documents fruit of international collaboration, switzerland, with . links per document and , % of international collaboration, having who's headquarters or saudi arabia, whose organizations have been characterized in recent years by the hiring of foreign research personnel in compatibility with their origin affiliations (bhattacharjee ; messerly ) , with . links per document and . % of documents fruit of international collaboration. the countries that accumulate a greater number of links, the most connected in the network previously analyzed, also tend to accumulate a greater number of citations. it stands out the highest proportion of citations collected in relation to the number of documents in countries such as the netherlands, canada or switzerland, which in turn stand out for a large number of accumulated links. in any case, it should be kept in mind that this indicator would only serve as a gateway for a detailed study of the specific publications that accumulate the most citations. it would be fallacious to assume an equal distribution of the number of citations according to the number of documents published. the usa appears as the country with the most documents contributed to the sample ( ), the most accumulated citations ( , ) and the strength of the link ( ). china appears in second place with documents that accumulate , citations and generate a total of links. both countries present figures above the rest of the countries in the sample, although their size and composition make them hardly comparable to the rest. in this sense, if we compare their figures with the group of the countries of the european union, we see how they are in the second position after the usa and above china, with documents that accumulate , citations and generate a total of links. a previous study about mers-cov research publications from to in scopus databases (zyoud ) found that the netherlands produced the greatest proportion of publications with international research collaboration ( . %) followed by the uk ( %) and germany ( . %) out of the total number of publications for each country. in our study, covering a wider timespan, these countries also present high values ( . %, . % and . % respectively). therefore, they are among the most contributing countries to the sample behind the usa and china (table ) . despite this, the data for the set of eu countries present values more similar to the usa and china. this is explained by less international collaboration in the rest of the countries of the european union that are not highlighted and the lower proportions presented by germany, the united kingdom, the netherlands or france alone. similarly, it is to be assumed that the level of international collaboration for the different united states of america or chinese regions does not present the same distribution. comparatively, china has a lower percentage of documents resulting from international collaboration ( %), the proportion of links ( . ) and citations ( . ) per document than the usa, with the % of internationally co-authored documents and . links and . citations per document, and the eu, also with the % of documents co-authored with non-eu countries and . links and . citations per document. comparing these figures for the two last decades with results of other previous studies related to the first sars crisis, the same countries are among the most productive varying their positions depending on the databases. for instance, the usa, canada, the united kingdom, and germany accumulated the % of publications about sars in the science citation index (sci) database from the beginning of the outbreak, in march, until july , . the usa had about the % of the total share, followed by hong kong with the % (chiu et al. ) . another example with a short time-span, sept- to aug- , and a sample of documents from medline database sets china as the most contributive country accounting the % of the total share followed by the usa with the % (yang et al. ) . a wider study between and with papers retrieved from sci database shows china as the most contributive country followed by the usa. china increases the number of publications over the years getting closer to the first position and leading the public health response (kostoff and morse ) . our study covers a more extensive time-span ( ( -march . we show that the most contributive countries, with the highest number of publications, around the two sars crisis are also the two most contributive countries in sars-related publications: china and the usa. kostoff and morse ( ) also suggest that the countries hit hardest by sars increase the number of publications concerning the topic. according to this, our sample time-span specifically points to the first publications related to the second sars crisis (covid- ) outbreak. if we put the number of documents provided in relation to the total number of links generated with other countries, we see proportionally higher values than in the sample for - , going from an average of . links generated by documents in the total period to . links for the countries with the largest contribution of documents to the sample for each period. so, there is a higher level of international collaboration in the - period, with . % of the documents, than in the - period with . % of internationally co-authored documents. the most notable exception is china, which falls from . to . % of the documents ( . links to . links per document). japan increases the number of documents internationally co-authored from . to % in - , despite the ratio decreases from . to . links per document, meaning that there are fewer countries involved. on the other hand, italy increases from . to . links per document but keeps around % of international documents in both periods, so more countries are linked in the most recent publications. other countries that offer a low proportion of links throughout the whole period show a slight rise in - , such as south korea that goes from . to . links per document and . % to . % of documents fruit of international collaboration, or brazil that goes from . to . links per document and . % to . % of international documents. in the same way as for the period - , for the period - (table ) , there is some correspondence between a higher total link strength and the number of citations collected by country, although the value of citations is lower due to the recent publication of the works. despite this, the exception of china stands out, which despite offering a proportionally low value of . links per document and . % of internationally coauthored documents, accumulates a greater number ( ) and proportion ( . ) of citations of its registers than the other countries. the - sample shows even sharper ratio differences when we focus on the comparison between the values of china, the usa and the countries of the european union as a whole. the number of citations in proportion to the documents provided increases in china and the usa ( . citations and . links per document with the . % of international collaboration) compared to those of the european union, which increase to a lesser extent ( . citations and . links per document with the . % of international collaboration). contrasting these three specific cases, the proportion of citations accumulated per document is not related with the number of links generated and the international collaboration percentage for documents dated in the - period. our explanatory hypothesis for this significant difference in citations is, on the one hand, the initial impact of the covid- pandemic in china and that the data treated in our study reflects the first studies carried out. on the other hand, there is the more than likely presence of works that have served as a reference for further research, such as publications of chinese origin describing the covid- person-to-person transmission (chan et al. ) . we understand these data as a conjunctural description of the first impact of the covid- pandemic on international scientific research to be compared in the future with the total number of records for , which it is supposed to show a greater number of accumulated citations and a wider number and distribution of links between countries. we found interesting to add each organization's country to see how the network changed when compared to the rest. thanks to this, we can map which countries have the most collaborative organizations about coronavirus and when they have started. in addition, thanks to the number of collaborations, we can identify if individual organizations have decided to work on this topic or if it was a country's decision and if different organizations from the same country do the same. also, we can observe if in international collaborations, organizations assume a central role due to political decisions from their countries or if it is an initiative of researchers of the organizations. some of the organizations from these networks are supranational or depend on scientific political decisions from different countries. in fig. , the network shows the main organizations that have collaborated in the scientific production from to . we can observe the total production (the size of the node) and the number of collaborations (the size of the link) with other organizations. it is divided into several clusters of different colors. the cluster in the middle of the map is represented by organizations with a central role in collaborations. many of them are based in the united states, and as we observe, collaborate with different parts of the world, mainly with the european union and china. the red cluster contains chinese organizations. they have a major number of publications as the nodes show, but they do not have a central role in scientific collaboration. in the periphery of this network, we can observe how several organizations compose small clusters of scientific collaborations. at the bottom of the map, for example, there is the pink cluster composed by korean universities with a medium number of publications and a very active collaboration between them, but with few cooperation with organizations from other countries. the same situation is for the yellow cluster, composed by brazilian and italian organizations, with the difference that they work in a more isolated dynamic. in this graph (table ) , we can observe with more detail what the previous network introduced. the university of hong kong and the chinese academy of science have the largest number of publications with a big number of link strengths. many of these publications were produced by collaboration between them. for this reason, in the network the two nodes appear very close. also, these two organizations collaborate with other centers but they do not have a main role like the organization that we meet in the second position, the center for disease control and prevention of the united states. this research organization has a high number of documents and of total link strength, many of them with different international organizations. for this reason, its position on the map is in the middle. a similar situation is for the ministry of health of saudi arabia. its position is close to the middle of the map and has the third position in total link strength. it is the first non-chinese organization with more scientific collaborations. the university of hong kong has the leadership in the number of documents ( ), total link strength ( ), and as we observe in this last graph, in the number of citations from to . its leading position, with , citations, is evident. the number is more than double than the center for disease control and prevention ( , citations), and the chinese academy of science ( , citations), respectively in the second and the third position with a similar number of citations. the first european organizations are the university of utrecht, which has a high number of documents ( ) and citations ( ). this organization is close to the middle of the previous network (fig. ) , which means it has a high number of scientific collaborations with the rest of international organizations too. thanks to this graph, it is possible to understand that the impact of these organizations follows a close relation with the number of publications and the number of collaborations. it is not surprising that the university of hong kong, the organization that has the leadership in the last years, is the world benchmark organization for coronavirus research. in this study, it was important to compare the results of the last two decades with the results of the last months, and the first weeks of , when covid- has become a global pandemic with more than a million of contagions and several thousands of dead until april . the interests of researchers and organizations have moved to study coronavirus and to find a solution. in this network (fig. ) , we can observe how the clusters of chinese organizations (blue, pink, and red) have moved in the middle of the map. this means that the number of collaborations has increased and also it is more distributed the research on covid- in different chinese centers and not only in a few like in the last two decades. moreover, the relevant role of these chinese organizations has increased in relation to the rest of the world, with different chinese clusters that are connected with different other clusters. the map has changed also if we compare the position of the us cluster with the previous map. now, us organizations move to a more peripheral zone of the map and have lost their leader place in the middle of it. also, we can observe how more us organizations have appeared in these last years with a stronger collaboration with chinese organizations, a situation that previously did not happen. the most relevant data that we can observe in this comparison is how china has increased the number of publications distributed in different scientific organizations, and not only in a few centers. in these last months, eleven chinese organizations are leading the area with several publications (table ). this affects the main role of the university of hong kong and the chinese academy of science, which however occupy the first two positions, but their number of publications is not so high compared with the other organizations. on the other hand, for the number of collaborations with foreign organizations, we discover that the trend has moved to more collaboration than the last two decades. outside china, it appears a south korean organization, the seoul national university in the fourth position, and the ministry of health of saudi arabia with a relevant role in collaborations. the first european university is the university of utrecht, confirming its leading role as in the global trend ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) , also highlighted due to cooperation with foreign organizations. as we have observed in the previous network, the us organizations have in this section, our primary objective is contributing to the mapping of oa coronavirus publications and their cited references, observing how it improves along the period. for this, it is important to analyse if the oa blooms of the research on coronavirus is funded or not. moreover, we observe if it is due to international collaboration or not. another aspect of interest is related to the obsolescence of the literature that supports the research under consideration-we hypothesise it increases due to higher productivity in times of epidemic/pandemic. finally, we explore how the most prolific countries move forward and widen access to coronavirus scientific knowledge. the cited references, in turn, indicate whether the greater availability of openly accessible literature is actually being used for its own advancement. we can observe the number and percentage of articles in oa (table ), concluding that it is growing almost every year, especially in , when it grows . % in relation to the previous year. when comparing the percentages among different oa types, we see that bronze predominates in the first years, when green takes the lead until and again. in and from to gold-doaj is the most preferred, sharing the first with bronze-the latter really taking the lead-in the present year. this huge increase is due to the announcement made for many commercial publishers to give free access to covid- and coronavirus related articles published in their subscription journals. it is also interesting to highlight the actual prominence of doaj journals to widen access to coronavirus scientific knowledge. in the end of this section, we compare the relative importance of the other types against gold-doaj, in relation to the different countries. figure presents the temporal evolution of oa in coronavirus literature as well as its obsolescence. as we can observe, the total amount of articles shows some variation, decreasing between one and another epidemics. the joint analysis of the presence of oa among cited references and articles is presented in fig. a shows that from to the percentage of oa increases practically in a constant manner, from ~ to ~ % among articles, and ~ % to ~ % among cited references. in the previous interval, its variation was somewhat erratic. in the current year improves significantly in publications, but decreases in citations. on the right side, the percentage of oa articles is considered between funded or not research, and internationally collaborated or not (fig. b) . funding is more associated to oa publication than those publications that do not mention acknowledgment to funding, especially years with highest gap between both (~ %): , and . as observed about table , through these years the green type is growing while bronze is dropping. after that the doaj-gold takes the scene until , and the gap of percentage of a articles between research funded or not narrows crescently. in due proportion, the presence of foreigners in the collaboration is also significant to oa publications (the average gap between and is around %). finally, it is important to highlight that the lines converge in , when the differences among categories become null, reaching its maximum value, and surpassing %. figure c , d focus on the cited references. the former, measures the average percentage of oa in relation to the presence (or absence) of funding, showing no significant difference between the trends. by the way, the presence of foreign co-author shows some positive difference, meaning that more cited references relate to oa journals, mainly in the - period. however, we have observed that the journals in oa are scarcer, mainly until the middle of the period, when they leave the level of % and reach about % in . if the higher, recent and crescent availability of articles influences this tendency among cited references, the age of the references must show that it is getting younger-as to say, that the price index is increasing. as pointed out by larivière et al. ( ) , the scarcity of scientific production implied a decrease in the price index (as they observed in the two world wars). so, in the opposite way, fig. d permits to observe three peaks, coincident with periods when the scientific production about the epidemics were high: one in , following sars crisis; the second between and , after the mers-cov, and now, during the sars-cov- pandemic. in this specific case, we call the attention to the increase of cited references to oa journals by not funded articles. considering the scientific production of the most prolific countries, in each of the periods, we have considered the average percentage of oa in cited references versus percentage of oa in articles. in order to analyse the changes in both oa variables, by the countries, we have ran a cluster analysis to group them according to similar behavior profiles, considering the relative growth, between the periods, in each variable (fig. ) . also, the changes between periods to all countries in each cluster are presented below (table ) , where the other variables can be analyzed: international collaboration, funding and price index. the seven oa clusters were identified with markers in the scatter plot with different geometric shapes, while the periods are differentiated by different shades of the same color (fig. ) . in a general manner we observe that in the last years the group of countries grows in both variables, with the exception of belgium (alone in the cluster ), that decreases the percentage of oa articles in the recent period. this can be due to the few - - . subtitle: in order to guarantee consistent percentages in articles cited references, we have discarded articles with less than references (corresponding to ~ %) number of articles in the recent period, denoting outlier behavior, that is also the case of singapore, taiwan and vietnam (for this reason they do not present data bars in table ). taiwan is the other cluster ( ) with just one country, due to its remarkable increase in the percentage of cited references in oa journals, but have to be analyzed carefully. subtitle: in order to guarantee consistent percentages in articles cited references, we have discarded articles with less than references (corresponding to ~ %) the fourth cluster is formed by canada and italy (shades of purple), increasing at least % their percentage in each of the variables. also, in table we observe that canada presents an increase in the percentage of international collaboration (we have observed it presents increase in its tls in tables , ) and funding, while italy increases in the price index. it is interesting to compare the previous cluster with the third one (shades of green), formed by brazil and singapore, that present similar patterns of increase in cited references, less in articles. however, in percentage of international collaboration and funding singapore improves better and brazil with the second highest increase in the price index (table ). the situation is not so different to cluster (shades of red), differing due to its lower increase in percentage of publications in oa in the recent period-being formed by india, spain switzerland, usa and vietnam. in table we observe that spain and switzerland increases their percentage of funded research and india and usa in international collaboration. cluster (shades of brown) is composed by egypt and saudi arabia, which present the biggest percent of cited references with oa journals, showing no increase in the recent period. egypt decreases its percentage of international collaboration, while both of them increase modestly the percentage of funded research. but their percentage of cited references in the price index deserves attention, due to a decrease of more than %, meaning less attention to recent articles in the recent period. they are probably more focused in the literature respective to the previous epidemics. finally, cluster (shades of blue) is the biggest one, with australia, china, the majority of european countries, japan and south korea. they present an increase in both variables of oa, tending to improve better in the cited references. about the other variables (table ) we highlight a general increase in international collaboration (excepting china, that retroceeds, as observed in table ) and funding (with exception of sweden), while the price index presents few increases (australia and south korea, while sweden have to be considered carefully due to its few publications in the recent period). as we observe, oa is playing an important role in all countries and getting more attention in recent years. but this growth comes together to the tendency to cite more recent literature just in the case of australia, brazil, india, italy and south korea. one variable whose the change between periods correlates, despite negatively with these, is the funding, that increases less in all case (except india that decreases). finally, we focus on the relative importance of the other types against gold-doaj (fig. ) . considering that part of the articles in the other types can be openly accessible after payment by the author (robinson-garcia et al. ) it is important to compare the share of this part of the production with the share in doaj journals. the same authors find that the green type is strongly represented in europe and north america, while in south america gold has a comparable level of importance. we can observe in fig. that the majority of the european countries, usa and canada are distributed along the x-axis (related to the complete period), with a ratio of at least . (spain and usa almost . ). japan is the positive outlier related to this direction, showing that the doaj-gold type is not its usual option (they prefer to publish in hybrid journals). on the opposite side we find brazil, egypt and saudi arabia, to whom the doaj-gold is the favourite choice. china is in the middle, not so far from taiwan and australia. considering the y-axis, we highlight those countries that increase the ratio in the recent period, which are brazil and italy. in the opposite situation, we find egypt, canada, japan and spain, whose ratio decreases significantly. we have explored the capacity of researchers to generate scientific knowledge to face a health crisis emergency, and their global capacity to collaborate between them in a global problem. we have also observed how different well-established clusters of countries and organizations are maintained over the two studied periods. the united states and china are the major producers of documents of our sample, followed by all european countries, especially the united kingdom, germany, the netherlands and france. at the same time, the leading role of saudi arabia, canada or south korea should be noted, with a significant number of documents submitted but very different dynamics of international collaboration. the case of the usa and china deserves attention, since their outputs are proportional to the magnitude of their built scientific infrastructure. one must therefore consider the rise of china, which in (leydesdorff and wagner ) takes second place in the ranking of articles (in wos). on the other hand, if we observe the production of these countries in the periods considered in our study, we observe in the incites platform (of clarivate analytics): that usa presents . % of the world share in - and . % in - ; while for china these percentages are, respectively, . % and . %.we note therefore the significant rise of china ( . %), and the decrease of the usa (− . %). on the other hand, when we update our search in wos on coronaviruses, usa presents . % for - and . % for - (− . %), while china presents respectively . % and . % ( . %). just over months of previous searches, usa takes the lead in research in covid- . this change of positions is to be expected in a context where science reacts so quickly to face the pandemic. by the way, the proportion of international collaboration is growing in the great majority of countries in the last period, which contrasts with the situation of the last two decades. in this sense, china is the great exception, decreasing the share of articles in international collaboration (− %, as shown in table ), even if this does not allow us to directly link this change with the impact of the health emergency generated by covid- . when we look closely at the details of organizations, we see how things change. the organizations that provide more documents to the sample are mostly chinese. the proportions of international collaboration are very different between the different organizations in the same country, especially in the case of china and, to a lesser extent, the us. however, if we look at the organizations that have the highest number of citations in their works, we can see that they have high international collaboration rates and that they increase proportionally in organizations from all countries, as well as in chinese organizations with high contributions to the sample. a comparison with previous studies (kostoff and morse ; zyoud ; zhang et al. ) shows that new countries have emerged in this network only in the last years. small and emerging countries have taken advantage of international collaborations to enhance their visibility in scientific scenarios. these new countries have started to connect with other parts of the world, like china, south korea and saudi arabia. the important role that open science plays is increasingly evident to have immediate access to scientific publications. the percentage of open access articles on coronavirus for the period - is . % but if we focus in , the figures increase up to . %, due to the commitment of commercial publishers with the emergency (they have provided free access to their contents). not by chance, our analysis has shown that fully open journals have become the main open access type until . on the other hand, it is worth noting that the pandemic phenomenon gives way to the bronze type, that is, without guaranteed perennial open access, and depending on the goodwill of commercial publishers. these are very relevant figures, never achieved before. commercial publishers are assuming implicitly that open access ensures best results and effectiveness. it is important that this practice will continue after the covid- pandemic and that can be extended to all the topics. plan s (coalition ) can be a good strategy and may be it can be followed by a greater number of funding agencies. it was important to observe that the coronavirus research is giving importance to the openly accessible scientific literature, since more of its cited references are being directed to fully open journals. this indicates that growing impact is being presented by them. therefore, the consideration of plan s on the possible absence of quality of open access journals (in certain areas of knowledge), and the consequent responsibility of funders to empower them (coalition ), must be taken seriously. in addition, we can say that this analysis sheds light on that, since the great majority of countries are improving their attention to them. however, the group of countries formed by australia, brazil, india, italy and south korea, which in the recent period of the pandemic are giving more emphasis to recent literature, caught our attention. in addition, their last publications denote that they are neither funding depending, except australia, nor they have depended too much on international collaboration, except india. such characteristics may result from a clear and almost selfsufficient reaction to the situation. several limitations affect this study. the first one is the restriction regarding the database used, wos, that do not include other journals in other languages that are indexed in scopus and scielo among others. wos data sources have some biases (packer ; mongeon and paul-hus ; lucio-arias et al. ) , as publications in english, publications about natural sciences, ecology and bio-medicine, and publications authored by researchers from the netherlands, united kingdom, and the usa are overrepresented. in contrast, all other countries and languages are underrepresented (belli and baltà ) . the second limitation is the lack of reliability of publications in oa before in wos due to a not well-defined specification distinction between not-oa and oa publications before that year. the third limitation is the impossibility to capture the most recent publications in these last weeks when the covid- outbreak was declared a pandemic by the world health organization. although the replicability possibilities of the searches carried out are taken into account, it must be underlined that the wos databases are constantly updated and expanded through works not previously contemplated too. wos databases are updated not only by the most recent works but also by older ones. as a consequence, nowadays, if any researcher reproduces the same searches, the number of registers for these samples and the timespan will increase. however, we are sure that more bibliometric analysis will be offered in this journal in the next months to follow this study. from the beginning of , when the covid- pandemic created a global health emergency, the scientific community has concentrated its efforts to promote research on the disease from different parts of the world. even if the coronavirus research has started many years earlier, only in the last period the number of scientific publications has increased significantly. thanks to our bibliometric analysis, we have mapped an unknown territory until the moment, useful to determine the evaluation of coronavirus publications in the world and the principal characteristics of these scientific publications. we consider, observing the tendency of coronavirus publications, that science is moving rapidly to find solutions to international health problems but access to this knowledge by society is not so quick due to several limitations such as open access policies and corporate interests. open science is the best way to face global emergencies as covid- pandemic. international collaboration in research and open access to publications, two of their main characteristics, can contribute decisively in obtaining more rapid and effective results. this crisis shows the importance of these open science practices. this type of study allows us to imagine and build future scenarios for scientific collaboration, creating a "common vision" and scientific policy recommendations to improve the numbers 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structure in graphene research a bibliometric analysis of pubmed literature on middle east respiratory syndrome a bibliometric study on sars in medline. paper presented at the proceedings of issi news media's surveillance and gatekeeping in representing health risk how scientific research reacts to international public health emergencies: a global analysis of response patterns global research trends of middle east respiratory syndrome coronavirus: a bibliometric analysis acknowledgements the work presented here was supported by "union iberoamericana de universidades ", by "atracción de talento (modalidad ), comunidad de madrid" and by cnpq (research productivity grant # / - ). the authors would like to thank claudia valeria alonso (uam, spain) for commenting and revising this text. the authors express their gratitude to fábio castro gouveia (fiocruz, brazil) and bob green (clarivate analytics, uk) for many discussions of the findings. key: cord- -qboon uv authors: zheljazkov, valtcho d.; sikora, vladimir; dincheva, ivayla; kačániová, miroslava; astatkie, tess; semerdjieva, ivanka b.; latkovic, dragana title: industrial, cbd, and wild hemp: how different are their essential oil profile and antimicrobial activity? date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: qboon uv hemp (cannabis sativa l.) is currently one of the most controversial and promising crops. this study compared nine wild hemp (c. sativa spp. spontanea v.) accessions with registered cultivars, eight breeding lines, and one cannabidiol (cbd) hemp strain belonging to c. sativa l. the first three groups had similar main essential oil (eo) constituents, but in different concentrations; the cbd hemp had a different eo profile. the concentration of the four major constituents in the industrial hemp lines and wild hemp accessions varied as follows: β-caryophyllene – % and . – . %; α-humulene . – . % and . – . %; caryophyllene oxide . – . % and . – . %; and humulene epoxide , . – . % and . – . %, respectively. the concentration of cbd in the eo of wild hemp varied from . to . % of the total oil while cbd in the eo of the registered cultivars varied from . to %; cbd in the eo of the breeding lines and in the cbd strain varied from . to % and . to . %, respectively. the concentrations of δ -tetrahydrocannabinol (thc) in the eo of the three groups of hemp were significantly different, with the highest concentration being . %. the eo of wild hemp had greater antimicrobial activity compared with the eo of registered cultivars. this is the first report to show that significant amounts of cbd could be accumulated in the eo of wild and registered cultivars of hemp following hydro-distillation. the amount of cbd in the eo can be greater than that in the eo of the usa strain used for commercial production of cbd. furthermore, this is among the first reports that show greater antimicrobial activity of the eo of wild hemp vs. the eo of registered cultivars. the results suggest that wild hemp may offer an excellent opportunity for future breeding and the selection of cultivars with a desirable composition of the eo and possibly cbd-rich eo production. hemp (cannabis sativa l.) is a new-old crop, one of the most controversial and promising crops due to its multiple utilizations, and contains a wide array of biologically active substances synthesized and accumulated in different plant parts [ ] . industrial hemp has been grown for grain and fiber for many decades in europe, asia, and north america. in addition, there is wild hemp (c. sativa spp. spontanea vavilov, also known as spontaneous), which is native to both central and eastern europe as well as parts of asia, and is found as a weed in agricultural fields. hemp essential oil (eo) can be extracted using various extraction methods, the simplest and most natural using either steam or hydro-distillation, as is the case with many other eo crops. hydro-distilled or steam distilled hemp eo are generally preferred by consumers and can be incorporated into a number of certified organic products; the market for organic food and non-food products reached us$ billion in in the usa alone [ ] . currently, cannabidiol (cbd) production and markets in many countries are depressed due to the overproduction and the covid- situation. for example, hemp production in the usa has increased rapidly and by mid- , there were around , licensed acres to grow hemp [ ] . that acreage in would have generated an estimated us$ . billion income, or around % of the total value of all cash crops in the usa [ ] . however, due to the depressed markets and expensive feminized seed (us$ /seed), there is now significant interest in hemp eo from industrial hemp cultivars and even from wild hemp. indeed, a number of producers and processors in the usa are developing new products based on naturally extracted hemp eo or cannabinoids. overall, research suggests that the hemp eo has medical significance and may also be utilized as an ingredient in commercial insect repellents and biopesticides [ , ] . some cultivars such as finola have been employed for commercial production of eo, as hemp eo has commanded high prices in recent years. hemp terpenes in the eo contribute to the aroma of various cannabis genotypes, and so far, around different terpenes have been reported in this plant [ , [ ] [ ] [ ] . current thinking is that terpenes have played a key role in the selection of medical/recreational and cbd type cannabis because their concentration is positively correlated to some of the cannabinoids [ ] . the hemp eo profile depends on genotype, growth conditions, and extraction method (steam, hydro, co , or solvent extraction) [ ] . of the various groups of terpenes, monoterpenes such as limonene, β-myrcene, α-pinene, β-pinene, and linalool (containing carbon units) comprise the major portion of the volatile oil fraction [ ] . these eo constituents are widely found in the eo of other plant species such as spices, eo crops, and medicinal herbs; they are not specific to cannabis. sesquiterpenes (with carbons) such as β-caryophyllene, α-humulene, caryophyllene oxide, and β-phellandrene are also present in higher concentrations in hemp extracts [ , ] . monoterpene composition can distinguish between monoecious and dioecious hemp cultivars [ ] . furthermore, certain terpenoids are highly correlated to the concentration of cbd and ∆ -tetrahydrocannabinolic acid (thca); consequently, they have been proposed as a chemotaxonomic classification tool and to distinguish drug-type cannabis in nevada [ ] . overall, hemp varieties (cultivars) with a higher concentration of monoterpenes have a more pleasant aroma compared with the varieties with higher amounts of sesquiterpenes. the monoterpenes pinene and limonene are the determinants of cannabis aroma in the immediate vicinity of the plant [ ] . hydro-distilled eo from industrial hemp cultivars may contain cbd [ ] ; the interaction of environment and genetics plays a role in the hemp eo profile. hemp eo (distilled from leaves, inflorescences, and thinner stems) has shown biological activity against several targets of pharmaceutical interests s. aureus, h. pylori, candida, and malassezia spp., enzymes, and cancer cell lines [ ] . the eos (collected from inflorescences after blooming) of cvs. carmagnola, fibranova, and futura have shown significant antimicrobial activity against g + and gbacteria and yeast, and the effect depended on the cultivar and seeding date [ ] . this study addresses a knowledge gap and current industry interest towards hemp eo with different origins and profile. the hypothesis was that wild hemp would have a different eo content, composition, and antimicrobial activity compared with the eos of registered industrial hemp cultivars, new hemp breeding lines, and a hemp strain (unregistered cultivar) that is currently used for the commercial production of cbd. the objective of this study was to compare nine wild hemp accessions (c. sativa spp. spontanea) sampled from agricultural fields in northeastern serbia with eu registered cultivars, eight breeding lines, and one cbd hemp strain (belonging to c. sativa) with respect to their eo profile and antimicrobial activity. the eo yield of the wild hemp accessions varied from . to . ml/ g for air-dried material and the yield of the breeding lines was . to . , while the eo yield of the registered cultivars was . to . ml/ g of dried material. however, the overall differences in oil yield between the three groups of hemp were not significantly different, with an overall mean of . ml/ dried material (table ) . table . analysis of variance (anova) p-values that show the significance of the effect of cultivar on constituents, square root of mean squares error (root mse) that represents the common standard deviation, and the overall mean oil content (v/w, volume oil per dry weight) and concentration (%) of the five constituents with no significant difference between the cultivars and cbd. the p-values that show significant (p < . ) and marginally significant ( . ≤ p < . ) effect and need multiple means comparison are shown in bold. overall, the eos of the wild hemps and registered cultivars in this study were similar to those reported previously: . to . % in fresh inflorescences [ ] , . to . % depending on the collection time with higher eo yield from plants sampled earlier (in september than in october) [ ] , and . % in stems and . % in the leaves of wild hemp from austria [ ] , respectively. however, the eo content of the usa hemp strain, utilized for cbd production in the usa and grown near the field trials was . to . %. for the wild hemps, two locations (kovacica and susara) were randomly selected among the nine locations (slavka, kovacica, buro, daleka zemlia, susara, saykaj, perez, titelski breg, and paluka) to be used as two replications to represent wild hemp in the statistical analyses. as indicated in the materials and methods section, one-way anova was completed to determine the significance of differences between the mean constituents obtained from nine hemp cultivars from northeast serbia. the constituents were: α-pinene, β-pinene, isocaryophyllene (γ-caryophyllene), β-caryophyllene, α-(e)-bergamotene, (z)-β-farnesene, caryophyllene oxide, humulene epoxide , selina- -en- -ol, caryophylla- ( ), ( )-dien- α-ol, caryophylla- ( ), ( )-dien- β-ol, -hydroxy-(z)-caryophyllene, β-bisabolol, α-bisabolol, cbd, and δ -tetrahydrocannabinol. overall, the eo profile of the wild hemp was different from that of one of the registered cultivars and the new breeding lines (supplementary tables s -s ). the eo constituents whose concentrations were significantly different are shown in bold in table . the mean concentrations of the five constituents and cbd from the above list that did not have significant differences between the wild and the registered cultivars are shown in table . the constituents whose means were significantly different are shown in table . the comparative concentrations of the eo constituents in this and previous reports cited here are summarized in table . table . mean concentration (%) of isocaryophyllene (γ-caryophyllene) [ ] , β-caryophyllene [ ] , α-(e)-bergamotene [ ] , caryophyllene oxide [ ] , humulene epoxide [ ] , selina- -en- -ol [ ] , caryophylla- ( ), ( )-dien- β-ol [ ] , β-bisabolol [ ] , α-bisabolol [ ] , and δ -tetrahydrocannabinol (dronabinol) [ ] obtained from the cultivars. the concentration range of α-pinene (bicyclic monoterpene) in the eo was from non-detected (n.d.) in three accessions of wild hemp to . % in wild hemp slavka; from n.d. to . % in the registered cultivars; and . to . % in the new breeding lines (supplementary tables s -s ). however, when grouped together, the concentration of α-pinene in the eo was not statistically different between the three groups of hemp. the concentration of α-pinene in the eo of the usa hemp strain (grown in the vicinity of the registered cultivars in this study and extracted the same way for the same time duration) was . to . % of the total eo. the monoterpenes pinene and limonene are the determinants of cannabis aroma in the immediate vicinity of the plant [ ] . α-pinene concentration in the eo in this study was comparable to previous reports from studies that used either steam or hydro-distillation [ ] [ ] [ ] [ ] , but was lower than that in other reports [ , , ] (table ). the eo of wild hemp from austria contained % of this compound in stems, but a much lower concentration in the leaves [ ] , while the eo of spontaneous (wild) hemp from hungary contained . , . , and . % of this compound in the leaves, male, and female flowers, respectively. these differences may be due to the environment (growing conditions including latitude and altitude), the plant parts analyzed, and/or the genetics (cultivar). the concentration range of γ-caryophyllene in the eo was . to . % in wild, . to . % in the registered cultivars, and . to . % in the eo of the breeding lines (supplementary tables s -s ) . γ-caryophyllene concentration in the eo of the usa strain was . %. the concentration of β-caryophyllene was to % in the eo of wild hemp, to % in the registered cultivars, and to % in the eo of the breeding lines (supplementary tables s -s ) . overall, statistically, the highest concentration of β-caryophyllene was found in the eo of cv. spic and the lowest in the eos of cvs. simba and dioica (table ) . β-caryophyllene in the usa strain eo was . to . %. β-caryophyllene in the eo of this study was similar to those reported in previous studies [ , , , , , ] (table ) . the data from this study and previous reports suggest that the concentration of β-caryophyllene could vary significantly depending on the cultivar. (e)-β-caryophyllene is known as the major eo constituent in hemp [ ] . this is one of the c. sativa eo caryophyllane-and humulane-type sesquiterpenes that include sesquiterpenes (e)-β-caryophyllene, (z)-β-caryophyllene, caryophyllene oxide, and the ring-opened isomer α-humulene (α-caryophyllene) [ ] . this eo compound has been shown to function in vivo as a non-psychoactive cb receptor ligand in foodstuff [ ] . the concentration of α-(e)-bergamotene (bicyclic sesquiterpene) ranged from . to . % in the wild hemp, . to . % in the registered cultivars, and . to . % in the breeding lines (supplementary tables s -s ) . overall, the concentration of α-(e)-bergamotene was statistically higher in cvs bacalmas and helena, and the lowest in cv. cs (carmagnola selezionata) ( table ). the concentration of this compound in the usa strain eo was . %. overall, the concentration of this eo compound in wild hemp from this study was similar to that in the literature reports on wild hemp from austria and hungary [ , ] . the concentration of caryophyllene oxide (bicyclic sesquiterpenoid) was . to % of the total eo in wild hemp, . to . % in registered cultivars, and . to % in the eo of the breeding lines (supplementary tables s -s ). the concentration of caryophyllene oxide was statistically higher in the eos of cvs. bacalmas, helena, and sequieni, and lowest in the eo of cv. carmagnola ( table ). the concentration of this compound in the usa strain eo was . to . %. caryophyllene oxide in the eo of wild hemp from austria was . % [ ] , while its concentration in the eo of spontaneous hemp from hungary was . , . , and . % in the leaves, male, and female flowers, respectively [ ] . the concentration of humulene epoxide (bicyclic sesquiterpenoid) was . to . % in wild hemp, . to . % in registered hemp cultivars, and . to . % in the eo of the breeding lines (supplementary tables s -s ) . overall, the concentration of humulene epoxide was statistically higher in the cvs. bacalmas and sequieni, and the lowest in cv. spic ( table ). the concentration of this compound in the usa hemp strain eo was . %. the concentration of this compound in the eo of wild hemp from austria was < . % [ ] , while in the eo of spontaneous hemp from hungary, it was . , . , and . % in the leaves, male, and female flowers, respectively [ ] . selina- -en- -ol (bicyclic sesquiterpenoid) was . to . % in the eo of wild hemp, n.d. to . % in the eo of the registered cultivars, and . to . % in the eo of the breeding lines (supplementary tables s -s ). its concentration was statistically highest in the eo of cv. dioica and lower in the eos of cvs. bacalmas and spic (table ) . hydro-distillation (hd); steam-distillation (sd); headspace solid-phase microextraction (hs-spme); solvent extraction (se); microwave-assisted extraction (mae). the concentration of caryophylla- ( ), ( )-dien- β-ol (bicyclic sesquiterpenoid) was . to . % in the eo of wild hemp; from n.d. to . % in the eo of the registered hemp cultivars; and . to . % in the eo of the breeding lines (supplementary tables s -s ). its concentration was statistically the highest in the eo of the wild hemp and the lowest in the eo of cv. spic ( table ). the concentration of this compound in the usa strain eo was . to . %, while its concentration in the eo of spontaneous hemp from hungary was . , . , and . % in the leaves, male, and female flowers, respectively [ ] . the concentration of β-bisabolol (monocyclic sesquiterpenoid) was . to . % in the eo of wild hemp; from n.d. to . % in the eo of the registered cultivars; and . to . % in the eos of the breeding lines (supplementary tables s -s ; table ). overall, β-bisabolol was higher in the eos of cvs. bacalmas, sequieni, and in the wild hemp, and the lowest in the eo of cv. spic ( table ). the concentration of this compound in the usa strain eo was . %. the concentration of α-bisabolol (monocyclic sesquiterpenoid) was . to . % in the eo of wild hemp, n.d to . % in the registered cultivars, and . to . % in the eo of the breeding lines (supplementary tables s -s ). α-bisabolol was statistically the highest in the eo of cv. cs and lower in wild hemp and cvs. bacalmas, sequieni, and spic (table ) . α-bisabolol in the usa strain eo was . to . %. epi-α-bisabolol was reported in the eo of spontaneous hemp from hungary [ ] . the major eo constituents of the usa hemp strain that was grown in close vicinity had a different chemical profile, with major constituents of myrcene ( . to %), β-caryophyllene ( . to . %), limonene ( . to . %), β-(e)-ocimene ( . to . %), and α-bisabolol ( . to . %). some previous reports identified a different number of eo constituents in hemp eo (e.g., eo constituents, with myrcene, α-pinene, and β-pinene as the main monoterpenes, and β-caryophyllene as the main sesquiterpene) [ ] ; eo constituents were identified in wild (spontaneous) hemp by nagy et al. [ ] . the latter authors named these hemp plants spontaneous forms, with the main eo constituents of the leaves, male and female flowers being e-caryophyllene ( . %), α-humulene ( . to . %), β-selinene ( . to . %), and α-selinene ( . to . %) [ ] . apparently, the spontaneous hemp plants from hungary had a different eo chemical profile compared with the eo of the wild hemp of this study that was collected in the northeastern part of serbia. the hydro-distilled leaf eo of wild hemp in austria contained mainly β-caryophyllene ( . %), α-humulene ( . %), β-selinene ( . %), caryophyllenen oxide ( . %), and α-selinene ( . %) [ ] . in the same study, stem eo contained α-pinene ( . %), β-caryophyllene ( . %), β-pinene ( . %), and myrcene ( . %) [ ] . apparently, the eo of wild hemp in austria had a similar composition to some, but not to other wild hemp eos, in this study. previous research has suggested that metabolic fingerprinting can be used for chemotaxonomic purposes in c. sativa [ ] . however, this and earlier reports on wild hemp [ , ] have demonstrated that the eo profile of wild hemp can vary significantly. therefore, genetic analyses may be needed to ascertain if the spontaneous or wild hemps in europe originated from some of the old industrial hemp cultivars that have been grown in europe over the last few centuries or from medical cannabis, or are products of spontaneous crossings between the two groups. in this study, the concentration of cbd in the eo of wild hemp varied from . to . % of the total oil, the cbd in the eo of the registered cultivars was from . to . %, while the cbd concentration in the oil of the breeding lines was from . to . % (supplementary tables s -s ) . however, when we grouped them together, because of the high variation, there were no significant differences between the wild accessions, registered cultivars, and the breeding lines with overall means of . % (table ). the concentration of cbd in the eo of the usa strain (which is commercially grown for cbd production) varied between . and . % of the total eo, which is an interesting result. the cbd concentrations in the eo of wild hemp slavka, kovacica, susara, perez and titelski breg were . , . , . , . , and . %, respectively, while the cbd concentrations in the eo of buro and saykaj were . and . %, respectively. therefore, this and previous studies [ , ] support the notion that wild hemps can be used as a source for the commercial production of cbd-enriched eo. this is the first report on such a high concentration of cbd in hydro-distilled hemp eo. overall, the concentration of thc in the eo was significantly different between the three groups of hemp ( table ). the thc concentration was significantly higher in the eo of wild hemp accessions, with an average of . % of the total oil, and a range between n.d. (in daleka zemlia) and . % (in kovacica) (supplementary tables s -s ). interestingly, it was much higher than the thc ( . %) in the eo of the usa hemp strain, which was actually developed from marijuana type hemp. the thc concentration in the eo of most of the registered cultivars varied from n.d. (e.g., in cvs. spic and bacalmas) to over . % (in the eo of cv. chameleon) ( table ). the thc concentration in the eo of the breeding lines was generally low, n.d., or below . % with the exception of line sk , where it reached . % of the total oil. previous research has shown that hydro-distilled eo from industrial hemp varieties contained cannabidiol (cbd) [ ] . overall, the content of monoterpenes fluctuated from n.d. to . % in the eo of wild hemp, . to % in the eo of the registered cultivars, and . to % of the eo of the breeding lines (supplementary tables s -s ). monoterpene concentration in the eo of the usa hemp strain was to % of the oil. sesquiterpenes were the largest group of chemical constituents. the content of sesquiterpenes was to % of the eo of wild hemp, to % of the eo of the registered cultivars, and to % of the eo of the breeding lines (supplementary tables s -s ). sesquiterpenes constituted to % of the eo in the usa hemp strain. cannabinoids also comprised the second largest group of chemical constituents in the wild hemp. the concentration of cannabinoids was to % of the eo of wild hemp, . to % of the eo of the registered cultivars, and . to % of the eo of breeding lines (supplementary tables s -s ) . cannabinoid concentration in the eo or usa hemp strain were . to . %, surprisingly low. most of the strains for thee commercial production of cbd were selected from marijuana (drug-type hemp with to % of ∆ -thc %), however, they were selected to have < . % total thc in dried biomass in order to be compliant with the current rules and regulations that are evolving [ ] . in a study of spontaneous hemp, nagy et al. [ ] reported that the eo was majorly composed of sesquiterpene hydrocarbons ( . to . %), followed by cannabinoids ( . to . %) and oxygenated sesquiterpenes ( . to . %). overall, the results from this study suggest that wild/spontaneous hemp in europe is chemotaxonomically related to the industrial hemp varieties (cultivars) grown in europe and deviate from the chemical profile of the usa hemp strain that was developed from marijuana-type cannabis for the commercial production of cbd. the usa hemp strain used in this study was started with feminized seed, which guarantees the production of female only plants, which may be one of the reasons for its much higher eo content. the pharmacological power of hemp is based on the content of ∆ -tetrahydrocannabinolic acid (thca) and cannabidiolic acid (cbda) [ ] . other major cannabinoids include cannabinolic acid (cbna), cannabigerolic acid (cbga), cannabichromenic acid (cbca), and cannabinodiolic acid (cbnda) [ , ] . current hemp eo and cannabionoid production systems in the usa have been scaled up from marijuana production. like with marijuana production, hemp 'christmas tree' production systems rely on feminized seed, because female plants with non-fertilized flowers (flower bracts) accumulate significantly higher concentrations of secondary metabolites such as cannabinoids and terpenes compared with fertilized flowers [ ] . currently, hemp chemical production is based on non-registered hemp 'strains' that were originally developed by marijuana breeders, which must meet government regulations for hemp with less than . % ∆ -tetrahydrocannabinolic acid (thca) in the dried biomass. due to the rapidly growing market for non-psychoactive cannabinoids, mainly cannabidiol (cbd), most of production has been focused on cannabidiolic acid (cbda). newer hemp strains bred for these characteristics are more likely to be compliant, and breeding companies are working on the registration of a number of hemp strains with high cbd content as commercial cultivars. the results from this study demonstrate that wild hemp may be a good source for further selection and breeding of cultivars with high concentration of cbd and low concentrations of thc. we used antibiotics as a positive control: cefoxitin for gram-negative (g -) bacteria and gentamicin for gram-positive (g + ) bacteria, and fluconazole for yeast. from gwe used se, salmonella enterica subsp. enterica; pa, pseudomonas aeruginosa; and ye, yersinia enterocolitica. from g + , we used sa, staphylococcus subsp. aureus; ef, enterococcus faecalis; and sp, streptococcus pneumoniae. from yeast, ca, candida albicans; ck, candida krusei; and ct, candida tropicalis were used. the method has been described previously [ ] . the eos of different hemp cultivars, accessions, and strains had differential antimicrobial activity that can be explained with differences in the eo profile. some of the eos had similar (although lower) activity to the antibiotics gentamycin, cetofoxin, and fluconazole. the antibiotic gentamycin was used as a positive control in the data presented in figure . the eo of wild hemp buro was the most potent against staphylococcus subsp. aureus (sa), followed by the eo of wild hemp saykaj ( figure a) . similarly, the eos of wild hemps buro and saykaj showed the highest antimicrobial activity against enterococcus faecalis (ef). the eos of wild hemp buro and cv. dioica were the most potent against streptococcus pneumoniae (sp) ( figure b) . the eos of wild hemp buro and the registered cv. dioica had higher antimicrobial activity against streptococcus pneumoniae compared with that of other eos ( figure c) . furthermore, the eos of wild hemp buro and the registered cv. dioica had higher antimicrobial activity against pseudomonas aeruginosa compared with that of the eos of the other hemps ( figure a ). cetofoxin (antibiotic) was used as a positive control for the data in figure . the eo of wild hemp susara had the highest activity against the g − yersinia enterocolitica ( figure b ). the eo of wild hemp buro had the highest activity against the g − salmonella enterica subsp. enterica, lower than the eos of wild hemps daleka zemlia and saykaj, and the lowest in the eo of other hemps ( figure c ). the antibiotic fluconazole was the positive control for the data presented in figure . the eo of wild hemp saykaj had the highest bioactivity against candida albicans (ca), the bioactivity of eo of wild hemp perez was not significantly different, while the bioactivity of the eos of the other hemp eos was lower than that of saykaj ( figure a ). the eo of wild hemp susara and the eo of registered cv. cs had the highest bioactivity against candida krusei (ck), while the bioactivity of the eo of sequieni was not different from the above ( figure b ). the eo of wild hemp paluka had the highest bioactivity against candida tropicalis (ct) ( figure c) . previously, hemp eo has shown biological activity against several targets of pharmaceutical interest: s. aureus, h. pylori, candida and malassezia spp., enzymes, and cancer cell lines [ ] . in another study, the eos (collected from inflorescences after blooming) of cvs. carmagnola, fibranova, and futura showed significant antimicrobial activity against gram (+) and gram (−) bacteria and yeast, and the effect depended on the cultivar and seeding date [ ] . the antibiotic fluconazole was the positive control for the data presented in figure . the eo of wild hemp saykaj had the highest bioactivity against candida albicans (ca), the bioactivity of eo of wild hemp perez was not significantly different, while the bioactivity of the eos of the other hemp eos was lower than that of saykaj ( figure a) . the eo of wild hemp susara and the eo of registered cv. cs had the highest bioactivity against candida krusei (ck), while the bioactivity of the eo of previously, hemp eo has shown biological activity against several targets of pharmaceutical interest: s. aureus, h. pylori, candida and malassezia spp., enzymes, and cancer cell lines [ ] . in another study, the eos (collected from inflorescences after blooming) of cvs. carmagnola, fibranova, and futura showed significant antimicrobial activity against gram (+) and gram (−) bacteria and yeast, and the effect depended on the cultivar and seeding date [ ] . overall, the findings in this study are consistent with the ones in a recent report [ ] . this study provides new information on the antimicrobial activity of the eos of the registered and wild hemps. overall, the findings in this study are consistent with the ones in a recent report [ ] . this study provides new information on the antimicrobial activity of the eos of the registered and wild hemps. good antimicrobial activity against enterococcus, listeria, and staphylococcus growth were found, which were compared to the conventional antibiotics that were used [ , ] . novak et al. [ ] found that the eo of five different cultivars of hemp had modest antibacterial activity. the gram-positive bacterial strains tested demonstrated high sensitivity toward cannabidiol, with slightly lower effects by cannabidiolic acid [ ] . due to significant antimicrobial potency of cbd against mrsa, the synergy with conventional antibiotics was tested. however, cbd was not able to revert the resistance pattern or demonstrate synergy with any of the conventional antibiotics tested [ , ] . certified industrial hemp (cannabis sativa l.) seeds were obtained from the institute for field and vegetable crops in novi sad, serbia. field experiments were set up at the alternative crops and organic production department in backi petrovac, serbia (n e ) using different cultivars of industrial hemp ( table ) , some of them included in the european list of approved hemp varieties (cultivars) [ ] . the cultivars used in this study included cs (carmagnola selezionata), spic, dioica, helena, carmagnola, squieni, bacalmas, simba, silesia, chameleon, fibrol, futura, and lovrin ( table ). the results from the first eight varieties were used in the statistical analyses and in the tables. in addition, new hemp breeding lines (named sk to sk ) were seeded adjacent to the above experiment and subjected to the same growth conditions. although the botanical classification of hemp is controversial [ ] , wild populations belong to uncultivated narrow leaf cannabis sativa ssp. spontanea vavilov [ ] , which is considered native to central and eastern europe and parts of asia. wild hemp samples in this study were collected from the edges of agricultural fields in the same region that have been used to grow other crops, but not hemp. these were agricultural fields where no hemp has been grown for the last years. however, around - years ago, at some of the collection sites, there were hemp processing factories or hemp seed storage facilities. generally, wild hemp is phenotypically different from plants of either old or new commercial hemp cultivars; wild hemp also has a shorter stature, with fewer branches than plants from registered cultivars. the locations of the wild hemp samples were named after the names of the nearby villages: buro, daleka zemlia, paluka, perez, saykaj, susara, slavka, kovacica, and titelski breg ( table ). the wild hemp samples were dried under the same conditions, and the eo was extracted via the same method and during the same time as the hydro-distillations of other hemp samples. hemp was grown as a rainfed crop without irrigation, as is traditional for the region, and the production technology, which is typical production system for commercial production of hemp in middle and southern europe, was applied. hemp was seeded with a corn planter on march, with cm spacing between rows and at a seeding rate of kg/ha. the soil type was alluvial chernozem with ph . ., previous crop was millet. the soil preparation prior to seeding included deep plowing on november, and fine seedbed preparation on march, . the experimental design was completely randomized with three replicates, with the size of the experimental plots being m × m. fertilizer (npk : : ) was applied as a broadcast treatment at kg/ha before deep plowing. an additional kg/ha of nitrogen was applied in the spring before sowing. weed control was conducted using burnout with glyphosate prior to seeding. mechanized weed control was performed twice during the first four weeks of vegetation stage though the use of sweep-type cultivators. hemp closes the canopy at - weeks after emergence and hence, suppresses weeds very well after that. the trial of the breeding lines was at the same field, approximately m from the main trial, and was subjected to the same agricultural conditions. plants in both trials were cultivated the same way including seeding time, seeding depth, fertilization, and weed control. fresh biomass samples (around kg fresh, in three reps) were obtained on june, at the beginning of flowering of the male plants. hemp tissue samples were generated by cutting the top . feet ( cm) from the top of female plants (male plants were not included in the samples). fresh weight was measured, then the samples were hung in a shady area (tobacco dryer) until constant air-dried weight and then extracted. the eo extraction was conducted via hydro-distillation of hemp air-dried material using -l hydro-distillation clevenger-type units as described previously for another plant material [ ] . the sample size was up to g dry weight (dw) biomass in . l water. all distillations were performed in two replicates, which was sufficient for statistical analyses. beginning of the distillation was noted when the first drop of eo was deposited in the collection part of the clevenger apparatus. all samples were distilled non-stop for min. at the end of the distillation, the heat source was removed, the eos (along with some water) were collected in glass vials, and placed in a freezer. after all distillations were complete, the eo was separated from water, measured on an analytical scale, and kept in a freezer until the gas chromatography (gc) analyses could be performed. hemp volatile compounds were analyzed by gc-fid and gc-ms as described previously [ ] , with the following modifications: the column temperature was initially set at • c, then increased to • c at a rate of • c/min, which was held for min. the flow rate of the carrier gas (he) was maintained at . ml/min. the injection volume was . µl at a split ratio of : . the temperatures of the ionization source, the transfer line, and the injector were • c, • c, and • c, respectively. the msd was operated in full scan mode. all mass spectra were acquired in electron impact (ei) mode with ev in the m/z range of - . the injector and detector temperature (fid) was set at • c and • c, respectively. all constituents present in the eo samples were identified by comparing their linear retention indices (lri) and ms fragmentation patterns with those from the national institute of standards and technology (nist ) and adams mass spectra library. the estimated lri were determined using a mixture of a homologous series of aliphatic hydrocarbons from c to c under the same conditions described above. for the agar disc diffusion method, a µl of cfu/ml bacterial suspension after incubation was spread on the mueller hinton agar (mha, oxoid, basingstoke, uk). filter paper discs ( mm in diameter) were infused with µl of the eo, tested, and placed on the inoculated mha. mha was kept at • c for h and then at • c for h. for yeasts, µl of the yeast suspension was spread on sabouraud agar (sa, oxoid, basingstoke, uk) and agars were cultivated at • c for h. after the incubation period, the diameter of inhibition zones was measured (mm). growth inhibition was compared with the standard drugs. the standard drugs cefoxitin for g − bacteria, gentamycin for g + bacteria and fluconazole for yeasts were used as positive controls. tests were performed in three separate experiments, and the means were calculated. one-way analysis of variance (anova) with two replications was completed to determine the significance of differences among the mean oil content and constituents obtained from nine cultivars. the constituents were: α-pinene, β-pinene, isocaryophyllene (γ-caryophyllene), β-caryophyllene, α-(e)-bergamotene, (z)-β-farnesene, caryophyllene oxide, humulene epoxide , selina- -en- -ol, caryophylla- ( ), ( )-dien- α-ol, caryophylla- ( ), ( )-dien- β-ol, -hydroxy-(z)-caryophyllene, β-bisabolol, α-bisabolol, cbd, and δ -tetrahydrocannabinol (dronabinol). this completely randomized design has two replications. the differences among the mean antimicrobial activities (sa, ef, sp, pa, ye, se, ca, ck, and ct) obtained from the seven registered cultivars (bacalmas, carmagnola, cs, dioica, helena, sequieni, and simba) and seven wild accessions (buro, daleka zemlia, paluka, perez, saykaj, susara, and titelski breg) were also compared using one-way anova with three replications. the analyses were completed using the mixed procedure of sas [ ] . since the effect of cultivar was significant (p-value < . ) or marginally significant ( . ≤ p-value < . ) on most of the constituents, further multiple means comparison was completed using the lsmeans statement of proc mixed (equivalent to fisher's least significant difference [lsd]) at % level of significance and letter groupings were generated. the overall mean was calculated for the constituents where cultivar effect was not significant. for the antimicrobial activities, cultivar effect was highly significant (p-value < . ) on all nine activities, and considering the large number ( ) of cultivars and accessions compared, multiple means comparison was done using tukey's multiple range test, which controls the type ii experiment-wise error rate. for each response variable, the validity of model assumptions was verified by examining the residuals as described in montgomery [ ] . the results from this study demonstrated that the essential oil (eo) of wild hemp from serbia is different in its chemical profile and bioactivity from the eo of registered industrial hemp cultivars, the breeding lines, and the hemp strain grown for cbd production in north america. wild hemp eos were also somewhat different from the eo profile of wild and spontaneous hemps collected in austria and hungary, as reported in the literature. however, although having been named differently, the wild hemp in this study, the wild hemp collected in austria, and the spontaneous hemp from hungary might have common origins; the collection sites were approximately in the same region (with a dimeter of approximately km) although collected in three different countries. these wild/spontaneous hemps have been exposed to significant environmental and agricultural (pesticide) pressure over the last few decades. these populations may all belong to cannabis sativa var. spontanea vavilov, (a synonym of cannabis sativa l.), considered native to central and eastern europe and parts of asia. however, the taxonomy of hemp is still debatable and there is no consensus among taxonomists as to whether it is a single species with several subspecies or multiple species. the concentration of the four major constituents in the industrial hemp lines and wild hemp varied as follows: β-caryophyllene to % and . to . %; α-humulene (α-caryophyllene) . to . % and . to . %; caryophyllene oxide, . to . % and . to . %; humulene epoxide , . to . % and . to . %, respectively. the major eo constituents in the usa hemp strain that was grown in the vicinity of the field trials had different chemical profiles, with the major constituents myrcene ( . to %), β-caryophyllene ( . to . %), limonene ( . to . %), β-(e)-ocimene ( . to . %), and α-bisabolol ( . to . %). overall, the eo of wild hemp has shown greater antimicrobial activity against staphylococcus susp. aureus, enterococcus faecalis, and g−yersinia enterocolitica, salmonella enterica subsp. enterica, and yeasts candida albicans and candida tropicalis compared with the eo of registered cultivars. this is the first report to show that a significant amount of cbd can be accumulated in the eo of wild and registered cultivars of hemp following hydro-distillation. one of the wild hemp eos showed a very high concentration of cbd. the eo of the wild hemp had a significantly higher concentration of thc relative to the one from the registered eu cultivars of industrial hemp breeding lines. in addition, it was interesting to see that wild hemp had a higher concentration of cbd in the eo relative to the eo from a strain grown for commercial production of cbd in the usa therefore, wild hemp collected in serbia could be used for the development of varieties (registered cultivars) with specific desirable chemical composition, and may also provide excellent material for the selection and breeding of hemp cultivars with high cbd for the commercial production of cbd and other high-value chemicals. supplementary materials: the following are available online, table s . essential oil constituents of wild hemp accessions, in % of total oil. table s . essential oil constituents of new hemp breeding lines in % of total oil (area under the curve). table s . essential oil constituents of registered hemp cultivars. cannabis sativa: the plant of the thousand and one molecules organic trade association the field of dreams. in an economic survey of the united states hemp cultivation industry repellent activity of essential oils from seven aromatic plants grown in colombia against sitophilus zeamais motschulsky (coleoptera) taming thc: potential cannabis synergy and phytocannabinoid-terpenoid entourage effects chemistry and analysis of phytocannabinoids and other cannabis constituents method for the analysis of cannabinoids and terpenes in cannabis evolution and classification of cannabis sativa (marijuana, hemp) in relation to human utilization metabolic fingerprinting of cannabis sativa l., cannabinoids and terpenoids for chemotaxonomic and drug standardization purposes fiber hemp inflorescences; from crop-residues to essential oil production terpenoid chemoprofiles distinguish drug-type cannabis sativa l. cultivars in nevada terpene synthases from cannabis sativa chromatographic analyses, in vitro biological activities, and cytotoxicity of cannabis sativa l. essential oil: a multidisciplinary study characterization and antimicrobial activity of essential oils of industrial hemp varieties (cannabis sativa l.) essential oil composition obtained from cannabis sativa growing wild in the urban area of vienna, austria genetics and selection of hemp the crop-residue of fiber hemp cv. futura : from a waste product to a source of botanical insecticides chemical characterization of leaves, male and female flowers from spontaneous cannabis (cannabis sativa l.) growing in hungary new methods for the comprehensive analysis of bioactive compounds in cannabis sativa l. (hemp). molcules cannabis sativa and humulus lupulus essential oils as novel control tools against the invasive mosquito aedes albopictus and fresh water snail physella acuta beta-caryophyllene is a dietary cannabinoid the essential oil from industrial hemp (cannabis sativa l.) by-products as an effective tool for insect pest management in organic crops variation in the compositions of cannabinoid and terpenoids in cannabis sativa derived from inflorescence position along the stem and extraction methods valorizing industrial hemp (cannabis sativa l.) by-products: cannabidiol enrichment in the inflorescence essential oil optimizing sample pre-treatment prior to distillation cannabidiol-enriched hemp essential oil obtained by an optimized microwave-assisted extraction using a central composite design production of ∆ -tetrahydrocannabinolic acid from cannabigerolic acid by whole cells of pichia (komagataella) pastoris expressing ∆ -tetrahydrocannabinolic acid synthase from cannabis sativa l constituents of cannabis sativa. in handbook of cannabis cannabis guide grinding and fractionation during distillation alter hemp essential oil profile and its antimicrobial activity chemical characterization and evaluation of the antibacterial activity of essential oils from fibre-type cannabis sativa l. (hemp) essential oils of different cultivars ofcannabis sativa l. and their antimicrobial activity. flavour fragr isolation, purification, and antimicrobial characterization of cannabidiolic acid and cannabidiol from cannabis sativa l synergistic effects of antimicrobial peptides and antibiotics against clostridium difficile azithromycin synergizes with cationic antimicrobial peptides to exert bactericidal and therapeutic activity against highly multidrug-resistant gram-negative bacterial pathogens agricultural species-varieties. hemp-cannabis sativa controversial taxonomy of hemp cannabis: evolution and ethnobotany chemical composition of the essential oil of the endemic species micromeria frivaldszkyana (degen) velen user's guide sample availability: samples of the essential oils are not available from the authors. © by the authors the authors are grateful to the institute for field and vegetable crops in novi sad, serbia; oregon state university, usa; the global hemp innovation center (ghic) in corvallis, or, usa.; university of novi sad, department of field and vegetable crops; the plant genetic research group, agrobioinstitute in sofia, bulgaria; slovak university of agriculture in nitra, slovakia; the university of rzeszow, poland; and the agricultural university, plovdiv, bulgaria. these institutions provided significant in-kind support such as access to laboratories and fields, and access to other infrastructure and research instrumentation. authors also thank michelle jeliazkova for critically reading and editing the final version of the manuscript. the authors declare no conflict of interest. key: cord- - v y j authors: nishiura, hiroshi; klinkenberg, don; roberts, mick; heesterbeek, johan a. p. title: early epidemiological assessment of the virulence of emerging infectious diseases: a case study of an influenza pandemic date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: v y j background: the case fatality ratio (cfr), the ratio of deaths from an infectious disease to the number of cases, provides an assessment of virulence. calculation of the ratio of the cumulative number of deaths to cases during the course of an epidemic tends to result in a biased cfr. the present study develops a simple method to obtain an unbiased estimate of confirmed cfr (ccfr), using only the confirmed cases as the denominator, at an early stage of epidemic, even when there have been only a few deaths. methodology/principal findings: our method adjusts the biased ccfr by a factor of underestimation which is informed by the time from symptom onset to death. we first examine the approach by analyzing an outbreak of severe acute respiratory syndrome in hong kong ( ) with known unbiased ccfr estimate, and then investigate published epidemiological datasets of novel swine-origin influenza a (h n ) virus infection in the usa and canada ( ). because observation of a few deaths alone does not permit estimating the distribution of the time from onset to death, the uncertainty is addressed by means of sensitivity analysis. the maximum likelihood estimate of the unbiased ccfr for influenza may lie in the range of . – . % within the assumed parameter space for a factor of underestimation. the estimates for influenza suggest that the virulence is comparable to the early estimate in mexico. even when there have been no deaths, our model permits estimating a conservative upper bound of the ccfr. conclusions: although one has to keep in mind that the ccfr for an entire population is vulnerable to its variations among sub-populations and underdiagnosis, our method is useful for assessing virulence at the early stage of an epidemic and for informing policy makers and the public. when an emerging influenza virus appears in humans, an early concern is whether the virus has the potential to cause a devastating pandemic, i.e., the global spread of an infection killing a substantial number of people. to assess the pandemic potential, two critical aspects need to be studied: the transmission potential and the clinical severity of the infection [ ] [ ] [ ] . it is widely known in epidemiology that the former aspect, the transmission potential, can be quantified by the reproduction number, i.e., the average number of secondary cases generated by a single primary case [ , ] , by characterizing the heterogeneous patterns of transmission (e.g. age-specificity) [ ] , and by measuring other epidemiological quantities such as household secondary attack rate. there are two different approaches to assessing the latter aspect of a pandemic, the virulence of infection. one is to explore specific genetic markers of the virus that are known to be associated with severe influenza (e.g. the pb gene) [ ] , although the absence of a known marker, as was for example the case in a novel swine-origin influenza a (h n ) virus (s-oiv), does not necessarily indicate that the virus is benign [ ] . another is an epidemiological approach to quantification of the case fatality ratio (cfr), the conditional probability of death given infection (or disease; see below). the cfr in general is vaguely defined as the ratio of deaths to cases, whose denominator should ideally be the total number of infections, but is frequently taken to be only the diagnosed cases due to the impossibility of counting all infected individuals. because in the early phase of an outbreak information is often limited to confirmed cases, we concentrate on confirmed cases only, and refer to the cfr as the confirmed cfr (ccfr) for clarity. as the world has experienced a global spread of s-oiv since april , methods have been sought for the real-time assessment of virulence by measuring the ccfr which is a representative of the epidemiological measurements of virulence [ , ] . nevertheless, a much-used crude estimate of the ccfr, i.e. the ratio of the cumulative number of deaths to cases at calendar time t, tends to yield a biased (and mostly underestimated) ccfr due to the time-delay from onset to death [ ] ; similar estimates of such a biased ccfr for severe acute respiratory syndrome (sars) have shown how such estimates can vary substantially as an epidemic progresses, stabilizing only in the later stages of the outbreak [ , ] . in the following we will use the terms biased and unbiased ccfr when we refer to this particular source of bias. improving an early epidemiological assessment of an unbiased ccfr is therefore crucial for the initial determination of virulence, shaping the level and choices of public health intervention, and providing advice to the general public [ ] . to obtain an estimate of the ccfr, the lesson from the sars outbreak is that a statistical technique is required that corrects the underestimation, e.g. a technique addressing censoring [ , , ] . nevertheless, in the case of novel s-oiv, an early unbiased estimation of the ccfr has appeared particularly challenging. initial reports from the government of mexico suggested a virulent infection, whereas in other countries the same virus was perceived as mild [ ] . in the usa and canada there were no deaths attributed to the virus in the first days following a declaration of a public health emergency by the world health organization. even under similar circumstances at the early stage of the global pandemic, public health officials, policy makers and the general public want to know the virulence of an emerging infectious agent. that is, a simple method for assessing ccfr is called for, even when only a few deaths have been reported, or even when there has been no report of deaths. except for another unbiased ccfr estimate in mexico ( . %, range . - . %) [ ] , this early assessment has been missing. in the usa, a technical discussion has taken place on the crude measurement of the biased ccfr using the cumulative numbers of deaths and confirmed cases so far [ ] . in line with this, an epidemiological method and its practical guide for early assessment of virulence are called for. the present study aims at developing a simple method to assess the virulence of an emerging influenza virus at the early stage of the epidemic, even when there have been only a few deaths or none at all. the method takes into account the time from the onset of symptoms to death, while differing from previously published statistical methods which employ censoring techniques [ , ] . as an example, we give an early prediction of the ccfr of s-oiv infection in the usa and canada, and show that the unbiased ccfr, as estimated by our method at the early stage of the epidemic in these countries, was in fact comparable to that estimated for mexico [ ] . our unbiased estimation of the ccfr does not address all sources of error in data (e.g. underdiagnosis of infected individuals) and we summarize the relevant issues in the discussion. we assess the virulence of s-oiv by measuring the risk of death, expressed as the ccfr. the ccfr is interpreted as the conditional probability of death given confirmed diagnosis [ ] . since the data of s-oiv infection we use in the present study are only confirmed cases, we have replaced ''infection'' in the denominator of cfr by confirmed diagnosis of infection (see discussion). accordingly, an unbiased estimator of ccfr would be the proportion of deaths among confirmed cases at the end of an epidemic. although one could instead assess the virulence by measuring the proportion of hospitalized cases among a total number of confirmed cases, criteria for hospital admission are not universal, being influenced by isolation policies and in some regions by cultural and social differences. in the following, the notation used to represent the three different statistical measurements of ccfr is: (i) b t , which is a crude, biased estimate of the ccfr calculated at time t; (ii) p, which is an unbiased ccfr to be estimated in the present study, and is the unknown parameter that governed the outbreaks; and (iii) p t , a random variable, which yields an estimator of p (see below) and is regarded as the realized value in one particular outbreak. first, b t , a crude and biased estimate of ccfr, calculated at time t, is given by the ratio of the cumulative number of deaths d t to the cumulative number of confirmed cases c t : during the outbreak of severe acute respiratory syndrome (sars) in - , it was shown that this estimator, b t , considerably underestimates the ccfr [ ] . this is easily demonstrated by relating c t and d t to the incidence function c t (i.e. the number of new confirmed cases on day t), and the conditional probability density function f s of the time from onset to death, given death. first, c t is the cumulative number of confirmed cases up to time t: second, d t is the cumulative number of deaths up to time t: as we mentioned above, p t is the realized proportion of confirmed cases to die from the infection, and is a random variable, which would be an unbiased estimator for p. therefore, b t can be rewritten as as can be observed in equation ( ), the estimator b t is smaller than the realized p t , because the time delay from onset to death, expressed in the double summation in the numerator, results in the numerator being smaller than the denominator (note that f s is a probability distribution). therefore we refer to b t as the biased estimator of the ccfr: it gives a biased estimate, calculated on day t, of the ccfr [ , ] . when we observe the entire course of an epidemic (i.e. tr'), b t tends to p t and becomes an unbiased estimator. the aim is to obtain an unbiased estimator ''well before'' observing the entire course of the outbreak. an adjustment of the estimator b t by a factor of underestimation is achieved by rearranging equation ( ): we use p t as the unbiased estimator of p, which is informed by three pieces of information: the cumulative number of deaths d t ; the incidence c t ; and the distribution of the time from onset to death f s . the former two are observed during the course of an epidemic. when there are a few deaths or none at all, an assumption has to be made for f s , e.g. from literature based on previous outbreaks (see below for detailed descriptions of f s ). we call the multiplicative factor in equation ( ) the factor of underestimation, u t , defined by the estimator p t can be written as p t = b t /u t . figure depicts the concept of the sampling scheme. the cumulative number of cases c t is regarded as the total population size. of these, only a proportion u t has been at risk for dying by time t, whereas the outcome for the remaining proportion -u t is still unobserved. among the u t c t cases that have been at risk, d t have died and u t c t -d t have survived the infection. this is a sample from a binomial distribution with sample size u t c t and probability p: an alternative way of deriving this probability is by first considering the total number, y, of people in the sample c t that will ultimately die from infection, which is binomially distributed with sample size n = c t and probability p. however, because of the time delay from onset to death, we do not observe this outcome by time t: only for a proportion u t is the outcome observed. hence our observation is a hypergeometric sample from a population of size c t , with sample size u t c t , and number of deaths y [ , ] : which is equivalent to equation ( ). we can use equation ( ) as a likelihood function to obtain the maximum likelihood estimate of p t : the % confidence interval of p t is derived from the profile likelihood. further technical details, especially where an exponential growth of incidence is observed, are given in the supporting information s . for calculation of the factor of underestimation u t , two pieces of information are needed: the incidence function c t and the distribution of time from onset to death f s . for c t , we use the published dates of onset among confirmed cases, while f s is assumed known. we analyze empirical datasets of two different infectious diseases: sars in hong kong ( ) and s-oiv infection in the usa and canada ( ). first, we examine a simplified version of our method by using only deaths and cases from an early stage of the sars epidemic, and compare our estimate against the eventual stable estimate at the end of the epidemic. for simplicity, we employ an exponential distribution for the distribution of the time from onset to death, f(s), with a mean of . days [ ] , and f s is subsequently calculated as the daily increase in f(s), i.e., f s = f(s) f(s ). second, we use the most recent published datasets of s-oiv epidemics in which the dates of illness onset for confirmed cases are known [ , ] . the latest such reports for the usa and canada were at may and june , , respectively. in the usa, there were confirmed cases by may , with known dates of onset ( figure a ). among confirmed cases, cases resulted in death by may . in canada, there were confirmed cases, with known dates of onset by june , among which cases died by june ( figure b ). the biased ccfr estimates, b t in these countries were . % ( = / ) and . % ( = / ), respectively. the six deaths are insufficient to determine the distribution of time from onset to death for these countries. we therefore employ a gamma distribution for f(s) (to calculate f s ), with reference to historical data for h n [ ] , with a mean length of days and a variance of . days (coefficient of variation %, shape parameter . ) [ ] . to address the uncertainty, we examine the sensitivity of our unbiased ccfr estimate to different means ( - days) and variances ( - days ). see supporting information s for further technical details. for the unbiased ccfr, we use and cases, respectively, as our c t in equation ( ) for the usa and canada. the population and sampling process for estimating the unbiased confirmed case fatality ratio during the course of an outbreak. at time t we know the cumulative number of confirmed cases and deaths, c t and d t , and wish to estimate the unbiased case fatality ratio p, by way of the factor of underestimation u t . if we knew u t we could specify the size of the population no longer at risk (u t c t , shaded), although we do not know which surviving individuals belong to this group. a proportion p of those in the group still at risk (size ( -u t )c t , unshaded) is expected to die. because each case no longer at risk had an independent probability of dying, p, the number of deaths, d t , is a sample from a binomial distribution with n = u t c t , and p t = p. doi: . /journal.pone. .g similarly, d t is and deaths, respectively. nevertheless, since the adjustment of underestimation requires dates of symptom onset, we use and cases for computing u t . although this has little impact on the estimate for the usa, the ccfr in canada is likely to be underestimated by our estimator, because the majority of the cases whose dates of onset have yet to be clarified, may have experienced their symptom onset close to the latest time point of observation. we subsequently compare ccfr estimates between the usa and canada by means of fisher's exact test. for the hypothesis testing, the number of deaths, d t , as well as the number of those survived, calculated as u t c t d t , is compared between two countries. the factor of underestimation u during the exponential growth phase is independent of time t and given by where m(-r) is the moment generating-function of f(s), given the exponential growth rate r which is estimated via a pure birth process (see supporting information s ). that is, when f(s) is the density of an exponential distribution with mean t, we have u = m( r) = /( +rt). figures a and b show the cumulative numbers of cases and deaths of sars, and figure c the observed (biased) ccfr estimates as a function of time, i.e. the ratio of the cumulative number of cases to deaths at time t. due to the delay from onset of symptoms to death, the biased estimate of ccfr at time t underestimates the realized ccfr at the end of an outbreak (i.e. / = . %). nevertheless, even by only using the observed data for the period march to april, equation ( ) yields an appropriate prediction (figure d ), e.g. the unbiased ccfr at mar is . % ( % ci: . , . ). an overestimation is seen in the very early stages of the epidemic, but the % confidence limits in the later stages include the realized ccfr (i.e. . %). when only a few deaths have been reported at the early stage of an epidemic, the unbiased ccfr estimate is given by minimizing the negative logarithm of the likelihood (see equation ( )). given and deaths in the usa and canada, respectively, and employing a gamma-distributed time from onset-to-death, the unbiased estimates of the ccfr are . % ( % confidence interval (ci): . , . %) and . % ( % ci: . , . %) in the usa and canada, respectively. the estimate in the usa appears significantly higher than that in canada (fisher's exact test; p, . ). the uncertainty bounds on the unbiased ccfr estimates in both countries overlap with that estimated for mexico [ ] . sensitivity analysis suggests that the expected values may lie in the range of . - . % and . - . % in the usa and canada, respectively ( figure ). even when there has been no observation of death by time t, it would be useful for policy makers to understand the implication of no deaths for interpreting virulence in a conservative way. when d t = equation ( ) simplifies to: which would result in an unbiased ccfr estimate of . because sampling a finite number of cases during the course of an outbreak cannot prove that infection never results in death, a more useful result would be the maximum ccfr with a certain level of confidence if no deaths are observed after c t cases. to obtain this result, we rearrange equation ( ) to obtain where p max is the maximum ccfr given c t cases and no deaths, at a confidence level of -a, e.g. % if a = . . equation ( ) is useful for obtaining a conservative estimate of virulence (i.e. upper bound of possible ccfr estimates) when no deaths have been reported by time t. in particular, during the early exponential growth phase the factor of underestimation, u, is independent of t. assuming that the exponential growth phase of influenza continued until april and , , respectively, in the usa and canada, r in these countries is estimated at . ( % ci: . , . ) per day and . ( % ci: . , . ) per day, it should be noted that confirmed cases include substantial numbers of imported cases from abroad. in canada, a few cases whose dates of onset were unable to be traced are also included according to their dates when a specimen was collected (the exact number of such cases is not known). assuming that their impact on our estimation procedure is negligibly small, we regard all cases in b as representing the dates of onset. doi: . /journal.pone. .g respectively (see supporting information s ). the resulting p max in the usa and canada (based on and cases and no deaths) is shown in figure . these upper bounds are examined for confidence levels at % and %. if the mean and variance of the time from onset to death are days and . days , and we employ a gamma distribution, p max is estimated at . % and . % at a = . and . in the usa. similarly, p max in canada is estimated at . % and . % at a = . and . , respectively. we propose a new epidemiological method for assessing the virulence of an emerging infectious disease at the early stage of an epidemic. the results with the hong kong sars dataset prove the usefulness of this method that corrects the biased ccfr estimator which is simply the ratio of cumulative deaths to cases. early in the epidemic, the ultimately realized ccfr is within the confidence interval obtained by our method. the proposed method is particularly useful when an epidemic curve of confirmed cases is the only data available (i.e. when individual data from onset to death are not available, especially, during the early stage of the epidemic). our estimates suggest that the virulence of s-oiv h n infection is comparable to the virulence observed in past influenza pandemics of the th century (, . % for the - pandemic and, . % for the - pandemic [ ] ). although our estimates may not be as high as . %, and even though the unbiased ccfr estimate for the usa is a likely overestimation (see below), we should emphasize that antiviral treatment and other medical interventions have been instituted from the beginning of this pandemic. our results show that the few observations of death in the usa and canada give us no reason to believe that the unbiased ccfr, and therefore the virulence of the novel pandemic strain, is smaller in the usa and canada than in mexico. nevertheless, given that the cfr of seasonal influenza is equal to or less than . % [ ] , our estimates (with the lower bound of ccfr close to the . %) do not offer conclusive results to indicate that the s-oiv is more virulent than seasonal influenza, but do point in that direction. it should be noted that our method only adjusts underestimation due to time delay from onset to death, and other epidemiological characteristics associated with unbiased estimation of the ccfr have yet to be addressed. in the present study, we estimated the ccfr as the proportion of deaths among confirmed cases. this definition was chosen, because of our aim to use the minimally available data, and so we were not able to estimate the proportion of deaths among all symptomatic cases, and not able to estimate the proportion of deaths among all those infected (symptomatic and asymptomatic). the issue of defining the correct denominator population can never be completely resolved, but it is essential to realize how the obtained estimate relates to other situations [ ] . by only using confirmed cases, it is clear that all cases will be missed that do not seek medical treatment or are not notified, as well as all cases that are asymptomatic. this means that our ccfr estimate is higher than the proportion of deaths among infecteds, and may be considered an overestimate. however, when relating our estimate to previous pandemics, it should also be realized that the current pandemic is the first where many confirmatory diagnoses of influenza have been recorded using rt-pcr techniques, allowing improved precision of ccfr estimates over those for previous influenza epidemics. whereas the use of rt-pcr in the current pandemic may yield a smaller denominator (and thus an overestimate of cfr compared to previous pandemics), other pandemics could have involved substantial numbers of falsepositive cases in the denominator. developing a method which permits comparable assessment of virulence is ongoing. the comparisons between the realized ccfr (horizontal grey line), the unbiased ccfrs based on observations by calendar time t, and the biased ccfr estimates, b t , given by the ratio of deaths to cases. each prediction was obtained by using the exponential growth rate r up to time t and the cumulative numbers of deaths and cases at time t, and the mean time from onset-to-death of . days [ ] which is assumed to follow an exponential distribution. overestimation is seen in the early stages of the epidemic, but the % confidence limits in the later stages include the realized ccfr. doi: . /journal.pone. .g figure shows the time course of biased ccfr estimates in the usa and canada based on the reporting date of confirmed cases and deaths to the world health organization. note that the estimates in figure c are different from our b t due to unavailability of the date of onset, although they give an approximate indication of the time-course of the biased ccfr. it is striking to see that the biased ccfr during the very early stage (i.e. from late april to mid-may) showed a declining trend following a single spike. the biased ccfr estimates at later time points show a slight increase as a function of time, which is consistent with our knowledge of underestimation of the ccfr [ ] . the early spike may be explained by a time-varying coverage of confirmed diagnoses which could have increased as a function of time (i.e. cases in the very beginning of the epidemic were less likely to be confirmed). other plausible explanations include ( ) demographic stochasticity, ( ) effective treatment, and ( ) heterogeneous risk of death among subpopulations. as for ( ), because the number of deaths in the usa and canada was very small during the early stage, the spike may reflect (unpredictable) probabilistic variations in the number of deaths among a small number of confirmed cases. if that is the case, our unbiased ccfr estimate for the usa (with data until may ) may be too high, not because of a systematic bias but just by chance. in relation to factor ( ), it is plausible that cases diagnosed in later stages of the epidemic receive treatment at an early stage of illness (or even before symptom onset). with respect to ( ), the risk of dying is likely to be different for different subpopulations [ , , , ] . it should be noted that the composition of sub-populations (e.g. agegroups and those with a specific underlying disease) is likely to vary as a function of time, and a ccfr estimate for the entire population, such as ours, is influenced by this variation. these points need to be addressed in future studies. to fully clarify the virulence and its epidemiological characteristics (e.g. variable risks by age and underlying diseases), two lessons for surveillance and data sharing should be noted. first, rather than updating the data based on date of reporting, it is critically important to summarize the data according to the date of onset both at local and global levels. knowing the date of symptom onset is a key to applying our proposed estimation framework to empirical observation. second, epidemiological data should be updated in a precise reporting interval at least during the early stage of an epidemic (so that the data permit estimation of the unbiased ccfr). given that mean time from onset to death is around days, weekly data do not enable us to make our explicit adjustment. optimal reporting for the early ccfr estimation may be incorporated into official pandemic response plans. moreover, in addition to using death as an outcome of virulence, the usefulness of other epidemiological measurements of severe manifestation (e.g. the number of admissions to intensive care unit) needs to be explored. despite a need to further clarify heterogeneous risks of death for the s-oiv pandemic, early assessment of virulence by means of our unbiased ccfr estimator is useful for informing policy makers and the general public about the potential severity of an infectious disease (of course, one needs to ensure an understanding of the above mentioned bias among non-experts). we have shown that underestimation can be adjusted in a very simple manner, and our approach enabled us to obtain an unbiased ccfr estimate by only minimizing a binomial deviance. these methods are particularly useful when there have been only a few deaths or even no death at all by time t during the course of an epidemic. uncertainties surrounding the unbiased estimate of ccfr based on a few deaths can partly be addressed by sensitivity analysis of the estimate to different lengths of time from onset to death. an observation of zero deaths in a given country (or a specific setting) should not be deemed a signature of a ''benign'' virus without observing a substantial number of cases. we have shown that a conservative upper bound of ccfr is a more useful interpretation of the observed number of cases without death. in this way, given that we have some prior knowledge or a few observations of death which permit us to assume f(s) is known, epidemiologists and biostatisticians in each country or locality can directly apply our method to assess the virulence of an infection at the early stage of any emerging infectious disease. during the final stages of revision, it came to our attention that an epidemiological study on ccfr of s-oiv with similar techniques and statistical philosophy has been published online [ ] , indicating that the preliminary estimate of ccfr for a combination of the usa, canada and mexico is . % and emphasizing a need to accurately capture the cases for the denominator. supporting information s given by the ratio of deaths per confirmed cases. the data were extracted from irregular situation updates of the world health organization [ ] , and the horizontal axis (time) corresponds to the date of reporting. therefore, it should be noted that the estimate suffers reporting delay, and in this sense, the calculated biased ccfr is different from our b t (based on date of onset) in the main text. the most recent report was made on june . since the interval of update has been irregular, the cumulative number of cases and deaths is kept the same as the latest report when there was no update on the corresponding date. doi: . /journal.pone. .g pandemic potential of a strain of influenza a (h n ): early findings how severe will the flu outbreak be? swine flu goes global mathematical epidemiology of infectious diseases: model building, analysis and interpretation transmission potential of the new influenza a(h n ) virus and its age-specificity in japan 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transmissibility of pandemic influenza in prussia textbook of influenza update: novel influenza a (h n ) virus infections -worldwide serum cross-reactivity antibody response to a novel influenza a (h n ) virus after vaccination with seasonal influenza vaccine assessing the severity of the novel influenza a/h n pandemic epidemic and pandemic alert and response (epr), world health organization ( ) influenza a(h n ) key: cord- - e n n authors: bünte, claudia title: china und die wirtschaft: der riese erwacht nicht, er ist längst auf der Überholspur date: - - journal: die chinesische ki-revolution doi: . / - - - - _ sha: doc_id: cord_uid: e n n die chinesische und die westlich geprägte welt sind deutlich unterschiedlich. um die innovationskraft verstehen und richtig einordnen zu können, die gerade bei ki von china ausgeht und weiter ausgehen wird, zeigt dieses kapitel, wie sich china im vergleich zur westlichen welt wirtschaftlich entwickelt und bis voraussichtlich entwickeln wird. dabei wird deutlich, dass china den westen bereits in einigen punkten rasant überholt hat und dass dieses Überholen weitergehen wird, insbesondere (aber nicht nur) durch den chinesischen -jahresplan rund um „made in china “. wie es sich u. a. anfühlt, diese entwicklung hautnah seit jahren mitzuerleben, berichtet anschließend beatrix frisch, general manager china von mackevision cg technologies im interview. das kapitel zeigt anschließend die unterschiede im datenschutz sowie dem datenschutzgefühl (also welche daten wie schützenswert sein sollten) im westen und in china. es diskutiert die damit verbundenen auswirkungen auf die implementierungsgeschwindigkeit der chinesischen gesellschaft in bezug auf neue, datenverarbeitende technologien, wie künstliche intelligenz eine ist. es legt dar, dass genau diese unterschiede im datenschutz einer der entscheidenden faktoren dafür sein wird, dass china im bereich künstliche intelligenz die weltweite führung übernehmen wird. anschließend werden die drei wichtigsten -jahrespläne vorgestellt und diskutiert, also: „made in china “, das die industrieproduktion chinas optimieren und mit hochwertigen erzeugnissen chinas position als wichtige produktionsmacht weiter ausbauen soll; der -jahresplan chinas von , der die zeit von bis umfasst und der china an die spitze der führenden wirtschaftsmächte bringen soll; der plan des zentralkommitees zur rolle der ki von , der ki als den entscheidenden faktor für china auf dem weg zur führenden globalen macht definiert. das kapitel zeigt weiter, wie stark china den bereich ki bereits heute unterstützt. es liefert zahlen zu den finanziellen mitteln im eigenen land, zur globalen unterstützung von ki-startups (hier ist china global führend) und bringt beispiele für die unbürokratische unterstützung der behörden, sobald ein thema im fokus der chinesischen planung steht. außerdem gibt einen breiten Überblick über das schul- und universitätsgrogramm chinas, das so angelegt wird, dass es die nötigen expert(∗)innen hervorbringt, um zu einer „ai first“-nation zu werden. es folgt ein interview mit matthias müller, dem general manager des german center of industry and trade in shanghai, über seine erfahrungen nach über jahren in china. das kapitel schließt ab mit einem fazit, warum es sich lohnt, nach china zu schauen. die chinesische und die westlich geprägte welt sind deutlich unterschiedlich. um die innovationskraft verstehen und richtig einordnen zu können, die gerade bei ki von china ausgeht und weiter ausgehen wird, zeigt dieses kapitel, wie sich china im vergleich zur westlichen welt wirtschaftlich entwickelt und bis voraussichtlich entwickeln wird. dabei wird deutlich, dass china den westen bereits in einigen punkten rasant überholt hat und dass dieses Überholen weitergehen wird, insbesondere (aber nicht nur) durch den chinesischen -jahresplan rund um "made in china ". wie es sich u. a. anfühlt, diese entwicklung hautnah seit jahren mitzuerleben, berichtet anschließend beatrix frisch, general manager china von mackevision cg technologies im interview. das kapitel zeigt anschließend die unterschiede im datenschutz sowie dem datenschutzgefühl (also welche daten wie schützenswert sein sollten) im westen und in china. es diskutiert die damit verbundenen auswirkungen auf die implementierungsgeschwindigkeit der chinesischen gesellschaft in bezug auf neue, datenverarbeitende technologien, wie künstliche intelligenz eine ist. es legt dar, dass genau diese unterschiede im datenschutz einer der entscheidenden faktoren dafür sein wird, dass china im bereich künstliche intelligenz die weltweite führung übernehmen wird. anschließend werden die drei wichtigsten -jahrespläne vorgestellt und diskutiert, also: "made in china ", das die industrieproduktion chinas optimieren und mit hochwertigen erzeugnissen chinas position als wichtige produktionsmacht weiter ausbauen soll; der -jahresplan chinas von , der die zeit von bis umfasst und der china an die spitze der führenden wirtschaftsmächte bringen soll; der plan des zentralkommitees zur rolle der ki von , der ki als den entscheidenden faktor für china auf dem weg zur führenden globalen macht definiert. das kapitel zeigt weiter, wie stark china den bereich ki bereits heute unterstützt. es liefert zahlen zu den finanziellen mitteln im eigenen land, zur globalen unterstützung von ki-startups (hier ist china global führend) und bringt beispiele für die unbürokratische unterstützung der behörden, sobald ein thema im fokus der chinesischen planung steht. außerdem gibt einen breiten Überblick über das schul-und universitätsgrogramm chinas, das so angelegt wird, dass es die nötigen expert * innen hervorbringt, um zu einer "ai first"-nation zu werden. es folgt ein interview mit matthias müller, dem general manager des german center of industry and trade in shanghai, über seine erfahrungen nach über jahren in china. das kapitel schließt ab mit einem fazit, warum es sich lohnt, nach china zu schauen. typischerweise schauen wir in europa bei technischen innovationen in die usa. und tatsächlich lohnt sich auch bei ki und digitaler transformation der blick "über den großen teich". allerdings sind die entwicklungen auf den gebieten der ki und der digitalen transformation in asien, allen voran china, deutlich schneller und massiver. aber warum fällt das in europa und den usa so wenig auf? der sogenannte "westen" behandelt den sogenannten "osten" noch häufig wie die zeitgenossen von queen victoria im . jahrhundert: die westliche kultur und wirtschaft dünken sich noch immer überlegen, china und indien scheinen vergleichsweise zurückgeblieben und brauchten ggf. sogar hilfe. redewendungen wie "developed world" und "developing world" sind dafür nur ein erstes, sprachliches zeichen. ab etwa bis in die er-jahre mag dieses bild von china noch richtig gewesen sein: der untergang des kaiserlichen chinas und damit das westliche bild war geprägt vom opiumkrieg und drogenabhängigen, von günstigen arbeitskräften aus china für den bau der us-eisenbahnen in der zweiten hälfte des . jahrhunderts, der sogenannten "hunnenrede" wilhelms des ii. angesichts des boxeraufstandes in china und der abschottung chinas nach dem erstarken des kommunismus und dem "großen sprung vorwärts" von mao zedong. dieser sogenannte große sprung sollte den zweiten fünfjahresplan - inhaltlich ausfüllen und die chinesische wirtschaft auf augenhöhe mit der westlichen welt bringen. tatsächlich musste der plan gestoppt werden, da die auswirkungen in eine hungersnot mündeten, bei der nach vorsichtigen schätzungen rund millionen menschen durch hunger und gewalt ums leben kamen (gottschalk ) . so sehr das westliche bild von diesen rund jahren geprägt sein mag, so falsch ist es im historischen vergleich. denn die jahre vor und die jahre seit etwa passen gar nicht in das bild einer im vergleich zum westen unterentwickelten kultur oder eines zurückgebliebenen wirtschaftsraumes. im gegenteil: china war viele jahrhunderte räumlich, kulturell und wirtschaftlich eines der führenden, wenn nicht das führende land auf der welt. unter der mongolischen herrschaft war china ab etwas das nach fläche größte land der erde, ganze vier mal so groß wie das römische reich. zeitgleich gab es hier die größten städte der welt. man kannte den buchdruck jahre vor guttenberg, und schießpulver und papiergeld wurden bereits genutzt (zdf history ohne jahresangabe). zu beginn der ming-dynastie, direkt nach den mongolen gibt es um ca. herum im reich der mitte die größte schiffsflotte der welt (dabringhaus ) etwa seit den er-jahren erholt sich china wirtschaftlich von der jahre dauernden fremdherrschaft und inneren machtkämpfen, ohne dass dies vom westen ausreichend registriert wurde. das, was man ab den er-jahren von chinas wirtschaft hauptsächlich wahrnahm, wirkte zunächst nicht wie die leistungen einer sich wirtschaftlich entwickelnden industrienation: die in den westen exportierte billigware "made in china" machte china zur "werkbank" des westens und die qualitätswahrnehmung war gering. die studentenaufstände auf dem platz des himmlischen friedens in peking und deren blutige zerschlagung mit den anschließenden, recht halbherzigen, wirtschaftlichen sanktionen des westens wirkten im westen wie ein weiteres kapitel einer politik, die keine demokratie zulassen wollte und wirtschaftlich abhängig vom westen war. dieser kurze abriss macht verständlich, warum der westen größtenteils zum gefühl einer gewissen Überlegenheit tendierte, sowohl in moralischer, wirtschaftlicher wie kultureller hinsicht. "wie sollte china also jemals in der lage sein, vorreiter in wirtschaft und kultur zu werden" hätte man sich fragen können. diese sicht ist heute mehr als überholt. china holt nicht nur auf, es hat den westen in vielen bereichen längst überholt und weite teile der weltwirtschaft bereits von sich abhängig gemacht. das gleiche gilt, mit in zeitlicher verzögerung, für indien. ich bin daher genau wie hans rosling ( ) der meinung, dass eine einfache aufteilung der welt in "die" und "wir" oder "westen" und "osten", "developing world" und "developed world" längst nicht mehr ausreicht, um die komplexität der welt zu erklären. schauen wir uns das näher für china an: zum einen zeigt china ein enormes wirtschaftliches wachstum. zwischen und wuchs das bruttoinlandsprodukt pro kopf, kaufkraftbereinigt, um den faktor , während das bip deutschlands und der usa nur um den faktor bzw. zulegten, zugegeben von einem sehr hohen niveau kommend. das bip indiens, dem bevölkerungsreichsten land nach china, wuchs im selben zeitraum um den faktor (siehe abb. . ). gleichzeitig sank die zahl der menschen in extremer armut -das sind menschen, die bis us-dollar am tag zur verfügung haben: lebten in indien und china noch % der bevölkerung in extremer armut, ist dieser wert in beiden ländern gesunken: in indien fiel er auf absolut %, das sind millionen menschen weniger menschen, die in armut leben. in china reduzierte sich der wert sogar auf , % absolut -das sind rund eine halbe milliarde menschen, die nicht mehr in absoluter armut leben müssen (rosling , s. zum diesem "chinesischen traum" gehört auch der ausbau der sogenannten "neue seidenstraße", das größte infrastrukturprojekt chinas außerhalb der eigenen grenzen. der plan sieht vor, einen seeweg und einen landweg von china bis nach europa zu schaffen. china will angeblich milliarden us-dollar in dieses vorhaben investieren (steinmetz ) und bietet den betroffenen ländern großzügige kredite an, wenn sie sich daran beteiligen. länder haben von diesem angebot bereits gebrauch gemacht. china hat parallel dazu den hafen von piräus in griechenland gekauft und hält % der anteile des flughafens von toulouse in frankreich (lepault und franklin ) . in dschibuti, einer kleinen republik an der ostküste von afrika, hat china die erste militärbasis außerhalb chinas eröffnet. dschibuti liegt strategisch günstig am eingang zum roten meer, einem teil der seeseitigen seidenstraße (steinmetz ) . china engagiert sich ebenfalls in afrika und vergibt dort kredite für infrastrukturprojekte (lepault und franklin ) , kauft global wasservorräte und land auf und war bereits nach der us-notenbank der größte gläubiger der usa (hackhausen ) . mit mehr als einer billion us-dollar in liegt die verschuldung der usa bei china in schon bei , billionen us-dollar (manager magazin ). uns mag nicht alles gefallen, was wir sehen, angefangen von fehlender mitbestimmung der bürger * innen am politischen geschehen über den aus westlicher sicht mangelnden datenschutz bis hin zu einem unguten gefühl in westlichen wirtschaftskreisen, die eigenen innovationen, einmal in china auf den markt gebracht, würden nicht mehr so geschützt, wie man es von einem europäischen patentschutz erwarten könnte. aber nur, weil es uns nicht gefallen mag, heißt dies nicht, dass es nicht stattfinde oder uns nicht betreffe. es gibt stimmen, die meinen, dass sich china, um wirtschaftlich weiter erfolgreich zu sein, in zukunft mehr um seine bürger * innen kümmern müsse. dyer, ein journalist der financial times und vormaliger büroleiter der financial times in peking, schrieb dazu im jahr : "on the economic side, the growth model that relies on funneling cheap bank loans to investment projects is running out of steam and needs to be replaced by more emphasis on consumers. most of the goals that china has set itself for the coming decades, weather it is encouraging more innovation or winning cultural recognition, are rooted in the initiative of private citizens. the next chinese era will be less about the state and much more about the individual. xi's challenge is to chart a series of reforms that start to address these demands without provoking a backlash from the party's many and powerful vested interests" (dyer , s. ) . sollte dies umgesetzt werden -und es sieht ganz danach aus -wird es dem einzelnen chinesen und der einzelnen chinesin wirtschaftlich immer besser gehen und der individuelle konsum wird weiter zunehmen. e-commerce, b c-marketing und ki als unterstützendes tool kommen in diesem spiel eine schlüsselrolle zu, die ich im folgenden noch eingehender erörtern werden. chinas wachsende bedeutung in der wirtschaft wird uns in der sogenannten "entwickelten" welt umdenken lassen müssen. in nur wenigen jahren wird ein normaler einerseits ist die annahme von innovation generell positiver und willkommener denn in der westlichen bzw. entwickelten welt (japan gehört hinzu, liegt aber im osten). jedoch ist dies auch ein natürliches phänomen, wenn man bedenkt, dass jahrhundertelanges abschotten sowie die kulturrevolution mit den folgenden jahren des chaos keine modernisierung ermöglichten. mit deng xiaopings vorstoß, den fortschritt proaktiv ins land zu lassen, wurde das innovationsklima zunächst nur langsam genährt. vorwiegend kamen veraltete technologien und produkte ins land, was sicherlich auch mit dem zunächst vergleichsweise geringerem einkommens-und entwicklungsniveaus in china zu tun hatte. schnell jedoch kam die lust am neuen, auch mit wachsender möglichkeit, ins ausland zu reisen und zu guter letzt natürlich auch mit der rasanten verbreitung des internets, das weitere impulse setzte. daraus entwickelte sich jedoch ein hunger nach dem nächsten neuen trend, der zu einer relativ schnellen müdigkeit mit blick auf das gerade erreichte führte. insofern gab es oft wenig chancen, das, was gerade neu und in war, vernünftig und nachhaltig weiter zu entwickeln. im gegensatz hierzu hat sich das marketing nicht so schnell und auch nicht in der gleichen art entwickelt, wie in der "westlichen" welt. der unterschied liegt darin, dass chinesinnen und chinesen ein vermehrtes maß an informationen zu einem produkt haben wollen. dies liegt zum einen am gewissen mangel an einer durchgängigen erfahrung mit den produkten oder serviceangeboten, zum anderen durchaus auch an der anderen sprachkultur, die eher visuell orientiert ist (chinesische schriftzeichen sind keine buchstaben, sondern stammen von piktogrammen). das marketing in china muss also sehr visuell und erläuternd sein. experience marketing hat in china daher einen weitaus höheren stellenwert als im westen. ein weiteres phänomen ist die rasante entwicklung des online-handels und die rolle des digitalen marketings. dies ist vor allem der größe des landes geschuldet, die eine durchgängige und gleichmäßige durchdringung mit gütern und services erschwert. zudem ist durchaus eine differenzierung von bildungsständen und einkommen zu bedenken, die sich wiederum in zeitlich versetzten adaptionen von innovationen zeigt (tier -städte bzw. megalopolen gegenüber tier und ) marketing wurde in der anfangszeit vor allem durch die ausländischen firmen geprägt; die chinesischen marken entwickelten ihr marketingverständnis zuerst nachahmend aus, denn was die ausländer ausführten, galt als vorbild und "state of the art". eine kopierte marketingbotschaft konnte fast schon eher als hommage oder kompliment für die originale marke gesehen werden. in der zwischenzeit und mit wachsendem nationalstolz entwickelte sich dann eine china- im prinzip ist meiner einschätzung nach china definitiv vor europa anzusiedeln, alleine basierend auf dem fast unbegrenzten zugang zu einem der größten datenpools der welt. in china kennt man keine gesetzliche restriktion à la dsgvo, es ist maximal der export personenbezogener daten ins ausland geschützt und ab einem umfang von . persönlichen datensätzen gesetzlich verboten. mit der vorgabe von echtnamen-registrierungen für telefon und internetkonten vor einigen jahren wurde dieser zugang zu echtdaten möglich und einfach. alleine aufgrund der bevölkerungsgröße und der massiven durchsetzung der nutzung von mobilen diensten und anwendungen im alltag kann man von einem guten entwicklungsstand ausgehen. zumal auch die bevölkerung hier keine aversionen zeigt, einfach gesagt, weil sie nichts anderes kennt. wieweit allerdings die tatsächliche nutzung und anwendung der daten im bereich ki vorangeschritten ist, ist für mich schwer einzuschätzen, da ich hier keine direkten erfahrungen besitze. jedoch ist davon auszugehen, dass die meisten chinesischen unternehmen einfacher auf die anwendung von ki eingehen können, da sie weniger etablierte strukturen vorweisen. auch die belegschaft ist im schnitt jünger und eher der digitalen generation zuzuordnen, was die einbindung von ki vereinfacht und somit die allgemeine entwicklung in und mit dieser ressource vorantreiben kann und wird. was wäre aus ihrer sicht die größte veränderung, die marketing-und salesmanager*innen in europa in den nächsten jahren erfahren werden? der bedarf und die anwendungsmöglichkeiten für programmatic marketing, von diensten zur individualisierung und die integration zwischen verschiedenen industrien werden sich rasant weiterentwickeln. in unserem eigenen bereich der d-visualisierung mit computer-generierten bildern (cgi) erleben wir einen der schnellsten wandel der letzten fünf jahre. bedarf und verfügbarkeit an ressourcen (ki, mensch und maschine) treiben die entwicklung fast schon in wochenschritten voran. sie erleben seit jahren die positive wirtschaftliche entwicklung in china mit. hätten sie ein paar beispiele aus ihrem alltag, die diese entwicklung zeigen? die mobilität ändert sich: vor drei bis vier jahren konnte man noch ein taxi an der straße heranwinken. mittlerweile geht dies nur noch mit einer der gängigen (und mehr werdenden) mobilitäts-apps wie didi. bezahlen mit bargeld wird als veraltet angesehen, man nutzt hier nur noch das mobiltelefon mit wechat pay oder alipay -das gilt auch für den straßenverkäufer. flugzeuge sind vorwiegend mit chinesischen passagieren gebucht, ausländer sieht man nur noch selten. und: chinesische kollegen nehmen mittlerweile tatsächlich ihren urlaub, um die welt zu erkunden. das prozedere für visa (reise-oder arbeitsvisa) ist für ausländer komplexer und schwieriger geworden -china scheint die einreise von ausländern generell beschränken zu wollen, und für arbeitsvisa gerade wegen einer wachsenden eigenen arbeitsbevölkerung. das stadtbild ändert sich: die alten gebäude weichen modernen mit wenig chinesischen merkmalen. einerseits ist es nachvollziehbar, dass alte (marode) gebäude verschwinden; doch gleichzeitig verlieren die städte auch an kultureller identität. doch bei allem fortschritt bleiben einige phänomene auch unverändert: bankgeschäfte zum beispiel bedürfen einer vorort-präsenz mit einem zeitaufwand von mindestens einer stunde (ausgenommen ist der transfer zwischen konten bis zu einer gewissen summe innerhalb chinas). woran liegt diese positive entwicklung aus ihrer sicht? was macht china richtig? das hat nichts mit richtig oder falsch zu tun, sondern hat zum einen mit dem enormen bedarf, der nach den langen jahren der abschottung erkannt wurde. zum anderen ist es natürlich die tatsache, dass china ein ein-parteien-staat ist, der in sozialistischer manier eine planungsökonomie betreibt und somit alle entscheidungen in zentraler art trifft, mit roll-out in die provinzen. sämtliche infrastruktur ist staatlich, innovationsprojekte werden staatlich geplant und gelenkt. das einzelschicksal hat sich einerseits dem unterzuordnen, auch ggf. die natur. für die hochgeschwindigkeitsstrecken werden z. b. gerade strecken geplant, so weit möglich; liegt ein dorf, feld oder wald im weg, so gibt es keine umweltschützer, die sich in den weg stellen. andererseits ist die chinesische bevölkerung stolz auf die entwicklung und unterstützt sie, auch wenn es einmal gegen den eigenen willen und das eigene wohl geht. seine nichtexistenz und die unterordnung unter das allgemeinwohl. es erscheint mir außerdem oft so, dass in china das berühmte "bigger picture" nicht gesehen wird oder in zu engen grenzen, den chinesischen grenzen. die tatsache, dass andere länder und kulturen anders funktionieren, scheint auch den chinesischen marketern fremd oder schwierig zu sein. wenn sie marketingmanager*innen und werber*innen in europa einen tipp geben sollten, was sie mehr/besser machen sollten, um wettbewerbsfähig zu sein, was wäre das? sich informiert halten, was in den diversen regionen der welt geschieht. anzunehmen, dass überall deutsche, westliche oder amerikanische verhältnisse bestehen, ist genauso falsch, wie anzunehmen, dass das deutsche marketing für amerikanische konsument * innen passt. die kulturellen und sprachlichen belange und bilder sind divers und diffizil. im ausland zu arbeiten, vor allem in einem land, das weniger bekannt oder entwickelt ist, erweitert den horizont. zudem sollte man danach streben, relevant zu bleiben, zu wissen: "was sind trends, technologische bedingungen und bedarfe, ohne die sich die welt in der nahen zukunft nicht mehr bewegen wird? erst hatte chinas staats-und parteichef xi jinping die cybersicherheit zu einer frage der nationalen sicherheit erklärt (alsabah ) . seitdem gibt es in china viel bewegung im datenschutz, eine regelrechte aufrüstung. das ist insbesondere vor dem hintergrund der rasant wachsenden nutzung von onlinediensten wichtig und nötig. millionen internetnutzer gab es in in china (alsabah ) und es musste festgelegt werden, wer wann wie diese daten verarbeiten und nutzen darf. das cybersicherheitsgesetz vom . juni regelt einen teil davon. es soll für datensicherheit, den schutz kritischer infrastrukturen sowie zu einem kleineren teil für den schutz der privatsphäre chinesischer bürger sorgen. auf diese weise zwingt es auch ausländische unternehmen zu einem hohen grad an transparenz. bei nichteinhaltung drohen empfindliche strafen bis zum entzug der gewerbeerlaubnis. nach alsabah ergeben sich daraus für westliche firmen vier herausforderungen (alsabah ) : erstens müssten laut dem cybersicherheitsgesetz von it-produkte, die zu den sogenannten "kritischen infrastrukturen" zählen, eine staatliche sicherheitsprüfung bestehen. das neue gesetz klassifiziere folgende infrastrukturbereiche als kritisch: kommunikationswesen, energie, transport, wasserversorgung, finanzwesen sowie e-government-dienstleistungen. darüber hinaus werde nicht genauer definierte bereiche genannt, die auswirkungen auf die "nationale sicherheit", "das wohlergehen der bürger" oder das "öffentliche interesse" haben. diese vagen formulierungen erlaubten es den behörden, weitere bereiche als "kritisch" einzustufen. die sicherheitsanforderungen würden für alle produkte gelten, die mit digitalen daten zu tun haben. ausländische technologien betrachtet man besonders kritisch. zweitens seien unternehmen verpflichtet, bestimmte daten in zukunft lokal zu speichern. (nutzer-)daten, die von betreibern "kritischer infrastrukturen" gesammelt werden, müssen nach dem cybersicherheitsgesetz innerhalb chinas gespeichert werden. drittens zwingt das antiterrorismusgesetz von unternehmen, staatlichen behörden zugriff auf die daten von terrorverdächtigen zu gestatten. das könne sich als äußerst problematisch erweisen, etwa für unternehmen, welche die sogenannte ende-zu-ende-verschlüsselung einsetzen. sie verfügten damit nicht über die technischen möglichkeiten, die daten ihrer konsument * innen einzusehen und könnten einer entsprechenden aufforderung daher nicht nachkommen. das gesetz lasse hier keine ausnahmen zu. viertens schreibe die "verordnung über den kommerziellen einsatz von verschlüsselung" ( ) (dorloff ) . vor einem kauf die agbs gründlich durchlesen oder mühevoll formulare ausfüllen, will offenbar kaum jemand. auch bei lee finden sich entsprechende kommentare: "but people in china are more accepting of having their faces, voices, and shopping choices captured and digitized. this is another example of the broader chinese willingness to trade some degree of privacy for convenience." (lee , s. obwohl dieses gesetz sehr weitereichend aussieht, werden auch hier wieder primär die firmen und die nutzer haftbar gemacht und nicht die regierungsbehörden (shi-kupfer und chen, massenhaft nutzer -mangelhafter datenschutz, , s. ). die chinesische regierung ist auf den reibungslosen aus-und aufbau eines nationalen datennetzwerkes angewiesen. dieses datennetzwerk bildet den grundpfeiler des von der zentralregierung vorangetriebenen gesellschaftlichen bonitätssystems (social credit system), mit dem das verhalten von firmen und einzelpersonen überwacht und gesteuert werden soll. mit strafen und belohnungen will peking unternehmen und bürger * innen zu regelkonformem verhalten erziehen. das social-credit-system befindet sich derzeit in der pilotierung, soll es flächendeckend eingeführt werden. obwohl zur zeit die teilnahme daran noch freiwillig ist, soll es angeblich schon von über % genutzt werden (ryssel "wohlverhalten" ist z. b. die pflege älterer familienangehöriger, positiv auf die nachbarschaft einzuwirken, blut zu spenden, aber auch, sich lobend über die regierung in sozialen medien zu äußern. als "fehlverhalten" wird angesehen, zum beispiel bei rot über die ampel zu gehen oder betrunken auto zu fahren, in online-spielen zu betrügen, aber auch, sich regierungskritisch in sozialen medien zu wort zu melden oder "illegal" gegen die behörden zu demonstrieren. dass diese Überwachung im verkehr bereits live geschaltet ist, zeigt abb. . . die aufnahme hat die autorin im november auf einer ganz normalen straße in peking aufgenommen. an der kreuzung filmen kameras, welche fußgänger * innen bei rot über die ampel gehen. deren gesichter werden direkt an der straße auf einer großen videowand gezeigt. die augen der verkehrsünder * innen werden dabei noch mit einem schwarzen balken belegt, so dass nicht unmittelbar erkenntlich ist, welche person sich gerade regelwidrig verhalten hat. der film, aus dem die bilder kopiert sind, findet sich hier: geschränkten zugang zu öffentlichen dienstleistungen rechnen. aus westlicher sicht besonders besorgniserregend ist die idee, menschen mit geringem punktestand mit name, id und foto im öffentlichen raum an den pranger zu stellen oder der plan eine tonwarnung vor dem freizeichen einzubauen, wenn man eine entsprechende person anrufen will (benrath und bartsch, asien in zahlen -teil ii -punktabzug für zu seltene besuche bei den eltern, ). erste nachteile wurden schon eingeführt: , millionen chinesen und chinesinnen durften im jahr nicht fliegen, , millionen nicht mit dem schnellzug fahren, weil ihre sozial-punkte zu niedrig waren (wurzel ) . chines * innen, die die sicherheits-app von qihoo auf ihrem smartphone installiert haben, sollen in zukunft gewarnt werden, wenn sie es mit jemanden am telefon zu tun haben, der auf der schwarze liste des bezirksgerichts chaoyang steht (trivium china , s. ). auch die wichtigsten tech-firmen chinas sind auf die eine oder andere art und weise am system beteiligt: "that is to say: we haven't found any evidence to suggest that behavioral shopping data (like what items people purchase online), and social media data (like what people say in chat rooms), will feed directly into the national social credit database. you won't get a black mark on your record for buying cigarettes. however, private tech platforms are participating in the social credit system, particularly in the areas of fintech and ecommerce. it's a little difficult to tease the concepts of fintech and ecommerce apart, because many of china's major ecommerce players are also china's biggest online lenders, and they operate financial services platforms" (trivium china , s. in der ersten stufe soll china in zehn jahren zu einer wichtigen produktionsmacht aufsteigen: bis will man die industrialisierung erreichen, die fertigungskraft konsolidieren und die digitalisierung der fertigung erheblich vorantreiben. bis soll sich die gesamtqualität der fertigung und die innovationskapazität deutlich verbessern, die arbeitsproduktivität insgesamt soll sich erheblich erhöhen und die integration der it in die industrie ein fortgeschrittenes niveau erreichen. die zu erreichenden wirtschaftsindikatoren und ihre wachstumsraten bis werden ebenfalls definiert. hierbei wird z. b. angestrebt, dass die breitbandabdeckung für das internet von % im jahr auf % im jahr ansteigen soll. in der zweiten stufe bis soll das chinesische verarbeitende gewerbe ein mittleres niveau unter den weltherstellern erreichen. in der dritten stufe bis zum jahr , dem hundertsten jahrestag der gründung des sogenannten "new chinas", also der volksrepublik china, soll chinas status im verarbeitenden gewerbe konsolidiert und china zum führenden land unter den fertigungsländern der welt geworden sein. die drei wichtigsten politischen instrumente sind dabei technologieaustausch mit ausländischen unternehmen für den marktzugang zu china, beschränkter zugang für ausländer und der aufkauf von ausländischen unternehmen und deren knowhow. die zehn strategischen wirtschaftsbereiche, auf die man sich fokussieren will, sind . interessant ist außerdem, dass in diesem text kein einziges mal das wort künstliche intelligenz genannt wird, sondern damit verwandte begriffe wie "iot" (internet of things) und "digitalisierung". dass nur zwei jahre später ein ganzes strategiepapier zu ki erscheint, zeigt, wie dynamisch china sich auf regierungsebene den schnell weiterentwickelnden marktbedingungen anpasst (webster et al. ) . in dieser rede stellt xi einen -stufen-plan für china vor. in der ersten stufe wolle man bis eine insgesamt mäßig prosperierende gesellschaft schaffen, in der zweiten stufe bis china grundsätzlich modernisieren und bis aus china ein auf allen gebieten hervorragendes, modernes sozialistisches land machen. ziele, die bis erreicht sein sollen, sind: • "china's economic and technological strength has increased significantly. china has become a global leader in innovation. • the rights of the people to participate and to develop as equals are adequately protected. the rule of law for the country, the government, and society is basically in place. institutions in all fields are further improved; the modernization of china's system and capacity for governance is basically achieved. • social etiquette and civility are significantly enhanced. china's cultural soft power has grown much stronger; chinese culture has greater appeal. • people are leading more comfortable lives, and the size of the middle-income group has grown considerably. disparities in urban-rural development, in development between regions, and in living standards are significantly reduced; equitable access to basic pu-blic services is basically ensured; and solid progress has been made toward prosperity for everyone. • a modern social governance system has basically taken shape, and society is full of vitality, harmonious, and orderly. • there is a fundamental improvement in the environment; the goal of building a beautiful china is basically attained" (xi , s. - ) drei dinge fallen hier auf: zum einen wird der plan wie schon das strategiepapier zu "made in china " mit klaren, messbaren zielen verbunden. zum zweiten sind die formulierten ziele erneut nicht nur gesellschaftlicher oder wirtschaftlicher natur, sondern allumfassender, da auch die umwelt, das regierungssystem, chinesische kultur und das rechtswesen mit einbezogen werden. und zum dritten will china nun schneller, nämlich bis , weltweit führend sein bei innovationen. noch , also nur zwei jahre vorher, wollte man bis führend sein. die ziele, die china bis erreichen soll, sind noch anspruchsvoller: • "new heights are reached in every dimension of material, political, cultural and ethical, social, and ecological advancement. • modernization of china's system and capacity for governance is achieved. • china has become a global leader in terms of composite national strength and international influence. • common prosperity for everyone is basically achieved. • the chinese people enjoy happier, safer, and healthier lives." (xi , s. ) auch hier fällt auf, dass die ziele breit angelegt sind, allerdings auch weniger greifbar, vermutlich, weil jahre im voraus geplant wird. dennoch ist der anspruch, führend in der welt zu sein, klar erkennbar. dass ki dabei eine wichtige rolle spielen soll, geht aus der letzten der drei quelle hervor: in diesem plan erläutert das zentralkomitee der kommunistischen partei chinas die strategische wichtigkeit von ki als grundsätzlich weltverändernd und essentiell für chinas entwicklung auf dem weg zur führenden globalen macht: "the rapid development of artificial intelligence (ai) will profoundly change human society and life and change the world. to seize the major strategic opportunity for the development of ai, to build china's first-mover advantage in the development of ai, to accelerate the construction of an innovative nation and global power in science and technology, in accordance with the requirements of the cpc central committee and the state council, this plan has been formulated." (webster et al. zwar sehen, wie beschrieben, auch die führenden westlichen industrienationen in ki eine der schlüsseltechnologien dieses jahrhunderts (faz ) und unterstützen sie mit finanziellen mitteln -die eu z. b. bis ende mit geplanten mrd. euro aus privaten und öffentlichen händen (john ) , die deutsche regierung bis mit mrd. euro aus öffentlicher hand (heide ) . gegen die finanziellen ressourcen, die china bereitstellen will, wirken diese summen aber recht klein: china, das bis führend auf dem gebiet der ki führend sein will, schätzt diese neue branche auf einen wert von milliarden euro (hua und jahn ) und plant u. a. einen , milliarden teuren ki-gewerbepark in peking (neuerer ) . hat china , % der staatlichen haushaltsausgaben daher in r&d investiert, die usa dagegen nur , % (manuel ). dies finanzielle unterstützung geht gar nicht mal nur von der zentralregierung aus, sondern von den vielen lokalen funktionären, die darum wetteifern, der partei zu zeigen, wie gut sie in der lokalen umsetzung sind. ein gutes beispiel dafür sind die investitionen in xiong'an, eine region etwa km entfernt von peking, die so umgestaltet wird, dass sie, wenn alles nach plan läuft, die weltweit erste stadt sein wird, die speziell auf autonom fahrende autos ausgerichtet ist. baidu hat einen vertrag mit der lokalen administration geschlossen, dort eine solche ki-stadt zu entwickeln, mit verkehrsmanagement, autonomen fahrzeugen und bestimmten umweltschutzmaßnahmen. schätzungen gehen von einer investition von milliarden us-dollar in die infrastruktur von xiong'an aus (lee , s. - ) . man muss nicht viel phantasie haben, um sich vorzustellen, dass ein staat, der ein bonus/ malus-system für unternehmen und bürger * innen eingeführt, ein vergleichsweise laxes persönlichkeitsdatenschutzgesetz und klare -jahrespläne inkl. budget für das thema ki entwickelt und kommuniziert hat, in der lage ist, den entsprechenden firmen und startups rund um ki bei genehmigungen und anderen bürokratischen prozessen bevorzugt weiterzuhelfen. an zwei beispielen wird dies exemplarisch deutlich: shenzhen, bis dahin eher landwirtschaftlich geprägt, wurde unter deng xiaoping im mai zur ersten sonderwirtschaftszone chinas, unter anderem wegen seiner günstigen lage direkt gegenüber von hongkong. bis dahin war die stadt rund . einwohner groß. seitdem shenzhen sondergenehmigungen und unterstützung vom staat erhält, ist die stadt bis auf millionen einwohner angewachsen. sie besitzt eine eigene börse. nicht nur einige der wichtigsten chinesischen hightec-firmen, wie etwa byd, dingoo, gionee, hasee, huawei, skyworth, tencent, xunlei oder zte siedelten sich dort an, auch viele ausländische it-unternehmen haben niederlassungen in der stadt. dazu zählen beispielsweise siemens und der weltgrößte elektronikhersteller foxconn, der mit mehr als . mitarbeiter * innen ipods für apple und bauteile für sony, nintendo und hewlett-packard herstellt (wikipedia a, b) . ganz nebenbei hat shenzhen auch die erste flächendeckende ausrüstung mit gesichtserkennungskameras weltweit; die stadt beobachtet ihre bewohner rund um die uhr (lohse ). die dabei gesammelten daten werden in einem system namens "skynet" zentral gesammelt. bereits heute ist die polizei in shenzhen damit in der lage, per foto eines gegenübers und dem abgleich mit einer app, den betreffenden zu identifizieren. selbst, wenn er oder sie den ausweis nicht dabeihat, oder nicht zeigen will, kann sie feststellen, ob er oder sie polizeilich gesucht wird (orth ) . alle gesammelten daten werden in einer schaltzentrale der stadtverwaltung zusammengeführt. hier ist dokumentiert, wie die wasser-, strom-und gesundheitsversorgung aktuell aussieht und bei welchen häusern illegale bautätigkeiten stattfinden. Über bodycams der polizist * innen und mitarbeiter * innen werden auch nebenstraßen kontinuierlich überprüft. wer heute als autofahrer * in an einem zebrastreifen in shenzhen nicht anhält, um fußgänger * innen über die straße zu lassen, bezahlt direkt, in echtzeit, die dafür vorgesehene strafe per app im smartphone -so vernetzt sind autodaten und smartphonedaten in echtzeit. abb. . zeigt die beschriebene schaltzentrale der stadtverwaltung im jahr . der wirtschaftliche erfolg bleibt dabei nicht aus. bereits heute hat shenzhen dieselbe wirtschaftsleistung eines landes wie schweden (wolff und yogeshwar ). peking hat gerade in nur jahren bauzeit und baukosten von milliarden euro (ankenbrand, china will weltgrößten flughafen eröffnen, b) einen zweiten in-ternationalen flughafen errichtet. dieser flughafen ist der nach fläche größte weltweit und etwa doppelt so groß wie der geplante neue flughafen in berlin (ber). die geplanten fluggastzahlen sollen den bisher größten flughafen der welt, atlanta, übersteigen. der zeitplan wurde bis auf wenige monate eingehalten. der bau dieses flughafens ist nicht nur ein prestigeobjekt, sondern war auch wirtschaftlich nötig geworden, da der alte flughafen bereits aus allen nähten platzt und die anzahl der passagiere weiterwachsen wird. er ist teil eines großen umstrukturierungsprojektes für peking. peking soll nur noch die für eine hauptstadt üblichen aufgaben übernehmen, wie etwa die staatsregierung, internationale kommunikation, forschung und entwicklung. alles anders soll ausgelagert werden in das umland, in dem eine neue metropolregion, "jing-jin-ji" genannt, entsteht (ingenieur.de ). es liegt nahe anzunehmen, dass hier die schnelligkeit von genehmigungen eine priorität für alle behörden ist, die mit dem bau beschäftigt sind. im vergleich zu den auflagen, verzögerungen und kosten des berliner flughafens ist die chinesische geschwindigkeit sehr hoch: der berliner flughafen wurde genehmigt, der spatenstich folgte , die fertigstellung war für geplant, sollte also auch innerhalb von jahren erfolgen (stern der ki-plan der regierung von sieht explizit vor, dass in bildung und ausbildung von ki investiert werden soll, (webster et al. ) wörtlich aus dem chinesischen übersetzt steht dort: "construct an ai academic discipline improve the disciplinary layout of the ai domain. establish ai majors. promote the construction of a discipline in the domain of ai. establish ai institutes as soon as possible in pilot institutions. increase the enrollment places for masters and phds in working in ai and related disciplines. encourage colleges and universities to broaden the content of ai professional education on an original basis. create a new model of "ai + x" compound professional training, attaching importance to cross-integration of professional education for ai and mathematics, computer science, physics, biology, psychology, sociology, law, and other disciplines. strengthen cooperation in production and research. encourage universities, research institutes, enterprises and other institutions to carry out the construction of an ai discipline." in summe sollen also ai-universitätsabschlüsse und ai-universitätsinstitute geschaffen werden sowie ausbildungen von "ki + x", die ki mit mathematik, informatik, physik, biologie, psychologie, soziologie, recht und andere disziplinen kombinieren. außerdem wird dazu angehalten, produktion und forschung bei ki stärker zu verknüpfen. die ersten sichtbaren erfolge dieser spezifischen ausbildungen in china werden sicherlich noch ein paar jahre auf sich warten lassen werden. allerdings kann man schon heute absehen, dass die investitionen der letzten jahre in das bildungssystem ein gutes fundament darstellen, um darauf aufbauend junge talente in ki auszubilden: china verbessert offenbar seit jahren die schulbildung und seine universitäten. der "bertelsmann tranformationsindex" (bti) attestiert china im bereich "bildungspolitik/ forschung und entwicklung" im jahr sechs von möglichen punkten, sieben und schon acht von "bildungspunkten". damit liegt china vor staaten wie brasilien, indien, russland und südafrika (bertelsmann-stiftung ). im neusten bti erreicht china erneut acht von punkten. vergleiche mit deutschland oder den usa können leider nicht gezogen werden, da der bti diese länder nicht mit einbezieht (bertelsmann-stiftung . die positiven ergebnisse der bildungspolitik sind auch sonst sichtbar: im aktuellsten globalen pisatest von / führt china vor deutschland und den usa. während china und deutschland sich von bis verbesserten, fallen die usa sogar weiter ab (siehe abb. . ). allerdings muss man die zahlen mit vorsicht genießen. der pisatest weist für china mehrere regionen aus: shanghai, hongkong und die sonderverwaltungszone macao. die zahlen des pisa-testes für shanghai und , hier lag shanghai auf platz , sollen nicht repräsentativ sein, weil sie lediglich an eliteschulen erhoben worden seien (erling ) . daher habe ich diese für die abbildung bewusst nicht ausgewählt. außerdem muss darauf hingewiesen werden, dass das primäre offizielle bildungsziel chinas, hier formuliert von xi, weiterhin sei, erbauer des sozialismus und nachfolger heranzuziehen, die die auferstehung chinas als großer nation beförderten. lehrer hätten als "ingenieure der seele" den patriotismus der jugend zu wecken (erling ) . der staat dämmt folglich derzeit offenbar die privaten initiativen der boomenden nachhilfeindustrie wieder ein. diese branche ist in den vergangenen jahren deutlich gewachsen. es gibt . private organisationen in china, die auf außerschulische nachhilfe und fortbildung spezialisiert sind (erling ) . aktuell liegen die privat finanzierten ausgaben für nachhilfe im schulischen bereich bei umgerechnet rund milliarden euro. darüber hinaus legt das chinesische schulwesen weiter großen wert drauf, dass die schüler * innen die lehrenden möglichst gut kopieren. eigene weiterentwicklungen der schüler * innen sind zumindest an den allgemeinbildenden schulen in china nicht sehr verbreitet. außerdem gibt es ein für sich entwickelnde länder übliches ungleichgewicht zwischen dem bildungsstand von stadt-und landbewohnern: während der bildungsstand in städten mit dem anderer länder mithalten kann, brechen offenbar zwei von drei schulkindern auf dem land die schule vor ende der schulpflicht ab (derzeit schuljahre). bisher war das geringere bildungsniveau für die chinesische wirtschaft nicht unmittelbar von nachteil, weil der größte teil der produktion und die haupteinnahmen durch arbeiten als "verlängerter werkbank" für die westliche welt generiert wurde. derselbe geringe bildungsstand wird aber aller voraussicht nach die weiterentwicklung des landes behindern, so der stanford-Ökonom rozelle (ankenbrand, bildungsproblem in china: zwei von drei chinesischen landkindern brechen schule ab, a). denn um führend bei innovationen, ki und in der wirtschaft zu werden, braucht es viele, selbständig denkende und weiterentwickelnde geschäftsleute mit einer hochwertigen ausbildung. allerdings scheint die qualität der chinesischen universitäten noch nicht auf einem ähnlich hohen niveau im vergleich zu anderen ländern zu sein. laut bertelsmann-stiftung sind im times ranking der weltweit besten hochschulen - nur zwei chinesische universitäten zu finden (bertelsmann-stiftung , s. ) . das ändert aber nichts dran, dass die anzahl der studenten an chinesischen hochschulen jedes jahr um % steigt. vielleicht erlaubt china wegen der noch nicht optimalen qualität der inländischen hochschulen seinen talenten außerdem zunehmend, im ausland zu studieren, wie abb. . zeigt. auch deren anzahl wächst jährlich um jeweils %. schätzungen gehen davon aus, dass in china zwischen und millionen chinesen englisch lernen. das ist eine größenordnung, welche der bevölkerung der usa entspricht. (dyer , s. ) . im jahr werden, laut vorhersagen, % der wissenschaftler * innen in der welt aus china kommen, nur , % aus den usa und , % aus deutschland (wolff und yogeshwar ) . selbst wenn man die bevölkerungszahl dieser länder zur gewichtung und damit zum fairen vergleich heranzieht, bleibt die tatsache, dass ein land, das % der wissenschaftler * innen vereinigt, wahrscheinlich dann mehr und auch bessere forschung und patente hervorbringen wird. das legt schon das gesetz der wahrscheinlichkeit nahe. bereits lag der chinesische anteil der weltweiten patentanmeldungen bei , %, während die usa als zweitgrößter anmelder auf , % kam (world intellecutal property organization , s. ). nehmen in den usa in der regel strukturiert zur verfügung stehen, während sie in china eher unstrukturiert vorliegen. welle : perception-ki: hier liegt aktuell china vorne in einem von lee geschätzten verhältnis von : . er rechnet mit einem wahrscheinlich noch deutlicheren vorsprung von china vor den usa in jahren, ( : ) , weil chinesische bürger * innen eher als us-amerikaner * innen bereit sind, auf die persönlichen schutzrechte ihrer daten zu verzichten, um einen eigenen convenience-vorteil zu erzielen. welle das mckinsey global institute kombiniert zahlen und experteneinschätzung und bewertet mit einer sehr ausführlich beschriebenen logik die heutige "readyness" einzelner staaten für ki (mckinsey global institute , s. - ) . dafür gebe es ki-indikatoren, zu denen gezählt werden: investitionen in ki, ki-forschungsaktivitäten und potentieller produktivitätsanstieg durch ki und automatisierung. als sogenannte "ki-enabler" werden betrachtet: digitale absorber, z. b. breitbandausbau, innovationsgrundlagen, humankapital, z. b. mathematisch-wissenschaftliche fähigkeiten von schüler * innen, austauschfähigkeit des landes mit anderen ländern, arbeitsmarktstruktur und -flexibilität). mck trennt nach der analyse die untersuchten länder in vier gruppen, abhängig von ihrer "ai-readyness" (siehe abb. . ). in der ersten gruppe -"aktiv global führende länder"-befinden sich nach dieser analyse nur die usa und china, die aktuell auch die spitzenpositionen einnehmen. es lohnt sich, die originalzusammenfassung der mck-analyse dazu zu zitieren (mckinsey global institute , s. ): "these two countries are currently leading the race to supply ai, and they have unique strengths that set them apart from all others. scale effects enable more significant investment, and network effects enable these economies to attract the talent needed to make the most of ai. together, they are responsible for the vast majority of ai-related research activities. they are a long way ahead of other countries on ai-related patents, publications, and citations. they also make substantial investment in ai. in terms of external investment abb. . grad der ki-aufnahmefähigkeit eines landes (mckinsey global institute , s. . der zweite teil der analyse (landesgruppen und ) wird hier aus platzgründen nicht gezeigt. zu den in der grafik gezeigten fußnoten führt mckinsey aus: ) for the threshold, we calculated a global average and then measured standard deviation. if countries are generally one standard deviation above the average, we categorized them as "above" and one standard deviation below average as "below"; we categorized the rest as being "within." for certain dimensions where values for leading countries are far higher than the average, we lowered the threshold to show relative differences clearly. ) vc = venture capital; pe = private equity; m&a = mergers and acquisitions. ) pisa = programme for international student assessment, oecd; stem = science, technology, engineering, and math; ghci= global human capital index; wef= world economic forum. ) gtci= global talent competitiveness index. ) the score is calculated based on a weighted average of each area that can have a different degree of impact on gdp growth per their elasticity. note: the contents of this table are indicative. countries in each group are listed in alphabetical order) (investment from one firm to another), including venture capital, private equity, and m&a, the united states accounted for percent, while china was a distant second with percent in . however, china's share is growing rapidly. these countries also have solid enablers. in , they invested about to percent of gdp in overall r&d. depending on national priorities and business opportunities, these huge r&d investment capacities could be channeled into ai. although china's capacity to innovate is increasing, the economy is digitizing quickly, and investment in ai is substantial. china and the united states are also the large contributors to global trade (in terms of both exports and imports), responsible for more than percent of all the value being traded globally." mck simuliert dann die ki-absorption der einzelnen länder in der zukunft (nicht ohne darauf hinzuweisen, dass die ergebnisse der simulation nur indikativ zu verstehen seien) und sagt voraus, das die aktuelle "ki-absorptionsfähigkeit" eines landes der treiber für den zukünftigen wirtschaftlichen erfolg sein wird. ein weiteres beispiel ist sicherlich auch das dienstleistungsangebot, welches insbesondere in den letzten jahren aufgrund der digitalisierung nicht umfassender sein könnte. alles, was das leben und den alltag einfacher und effizienter macht, gibt es i. d. r. per app. alleine mit wechat kann ich überall zahlen, flüge buchen, steuern und strafzettel bezahlen, taxis rufen, essen bestellen, stromrechnung begleichen, kinotickets buchen, und, und, und. china arbeitet mit -jahresplänen. können sie kurz erklären, was im aktuellen -jahresplan steht und was mit "made in china " gemeint ist? was ist der "chinesischen traum"? beim aktuellen -jahresplan geht es im wesentlichen um die transformation der wirtschaft, der umwelt bzw. den umweltschutz und vor allem auch gesellschaftliche themen. daran kann man sehr gut ablesen, wie sich chinas interessen und prioritäten seit ende der er-jahre von deng xiaoping eingeleiteten Öffnungspolitik verändert haben. heute geht es um ein kontrolliertes wirtschaftliches wachstums, die gezielte förderung von innovationen und hi-tech. "made in china " ist das erklärte langzeitziel chinas, die weltweite technolgieführerschaft zu erreichen -insbesondere in den bereichen digitalisierung, robotertechnik und automatisierung. vor allem aber hat china seine umweltprobleme erkannt und geht diese gezielt an, z. b. weg von der kohle hin zu alternativen energien. aus meiner sicht allerdings sind die gesellschaftlichen themen die spannendsten. wenn man sich überlegt, in welcher geschwindigkeit china sich komplett gewandelt hat … das geht an einer gesellschaft nicht spurlos vorbei. nicht umsonst heißen die aktuellen schlagworte und ziele im -jahresplan "harmonische gesellschaft", "soziale stabilität" usw. der "chinese dream" unterscheidet sich eigentlich nicht so sehr vom "american dream". prinzipiell ermöglichen die politischen rahmenbedingungen den chines * innen alle freiheiten, karriere zu machen und es zu materiellem reichtum zu bringen bzw. den lebensstandard zu verbessern. aber anders als in den usa geht es nicht so sehr um das individuum, sondern um die verwirklichung des "chinese dream" aus dem chinesischen kollektiv heraus, mit einer starken nation und der kommunistischen partei im rücken. welche generell bereitet bürokratischer aufwand nirgendwo in der welt viel freude. aber ich kann deutlich erkennen, dass china sich auch hier ständig weiterentwickelt und mittlerweile sehr, sehr viel digital bzw. online abgewickelt werden kann. die Ämter werden immer moderner und kundenfreundlicher gestaltet. und auch wenn eine behörde voller menschen ist, läuft die abwicklung doch sehr zügig und organisiert. und dass auf meinem steuerbescheid "thank you for contributing to china's prosperity" steht, ist einfach eine nette geste, über die ich immer noch schmunzeln muss. china wird in den deutschen medien sehr oft hart angegangen. das ist aus meiner sicht nicht immer fair und hängt zu allererst davon ab, aus welcher perspektive man beurteilt. viele stimmen westlicher industrienationen fordern von china z. b. demokratie. ein mitspieler aus meiner fußballmannschaft hier in china, der aus einem kleinen armen afrikanischen land kommt, sieht zuerst, dass china es geschafft hat, die grundbedürfnisse von , milliarden menschen zu befriedigen, oder anders ausgedrückt, seine bevölkerung "durchzufüttern". china hat seit anfang der er-jahre über mio. chinesen aus der armut geführt. china exportiert weder terrorismus noch wirtschaftsflüchtlinge. und die umfassende modernisierung der infrastruktur, die stete erhöhung des wohlstands, der ausbau des sozialversicherungssystems und die stärkung der arbeitnehmerrechte sind weitere errungenschaften. auch mit der gewachsenen internationalen verantwortung geht china meines erachtens gut um, ist eine gemäßigte stimme im un-sicherheitsrat und stellt u. a. ein der größten blauhelm-kontingente. wenn sie marketingmanager*innen in europa einen tipp geben sollten, was sie mehr/ besser machen sollten, um wettbewerbsfähig zu sein, was wäre das? na ja … die marketingmanager * innen können ja letztendlich auch nur vermarkten, was die europäischen firmen an produkten zu bieten haben. ich denke vielmehr, dass europa aufpassen muss, nicht den anschluss zu verlieren, insbesondere bei der digitalisierung und bei innovationen. wenn europa aus gemeinsamer stärke heraus agiert und international mit einer stimme spricht, werden die firmen auch wettbewerbsfähig bleiben. unabhängig davon sollte man immer ein auge auf die entwicklung und fortschritte im asiatischen raum und vor allem in china haben. beispielsweise macht uns china gerade bei der g-technologie vor, wie es geht. fazit: darum wird der sack reis, der in china umfällt, wichtig für uns das kollektive kulturelle erbe macht es für die menschen in china leichter als für jene in westlichen gesellschaften, mit neuen technologien kollektiv umzugehen. der geringe personendatenschutz und die tatsache, dass china das bevölkerungsreichste land der erde ist, spült deutlich mehr daten zum analysieren in die ki-systeme, als dies im westen derzeit möglich ist. der strengere chinesische landesdatenschutz bringt es außerdem mit sich, dass einerseits die eigenen daten im lande bleiben und andererseits der knowledgetransfer nach china hinein besser funktioniert als aus china heraus. die regierung unterstützt darüber hinaus mit viel geld die ausbildung von ki-expert * innen im in-und ausland, die optimierung des schul-und ausbildungsssystems auch in bezug auf ki-fähigkeiten und erleichtert bürokratische vorgänge rund um ki. es kommen in china also die drei faktoren zusammen, die es braucht, um auf dem sektor der ki führend zu werden: ki-technologie und fachpersonal, große mengen miteinander verknüpfter daten und ein genügend hohes budget, um daraus neues zu entwickeln. kein wunder, dass die "big three", also baidu, alibaba und tencent den vergleich mit den "big four" der westlichen welt, google, apple, facebook und amazon, nicht scheuen müssen. hier werden die nächsten ki-innovationen entstehen, hier werden wir hinschauen müssen, um lernen zu können. es wird für uns im westen wichtig werden, ob und wie der sprichwörtliche sack reis in china umfällt. wo wir tatsächlich heute schon im bereich marketing von china lernen können, zeigen die nächsten kapitel. peking will gläserne unternehmen bildungsproblem in china: zwei von drei chinesischen landkindern brechen schule ab oktober asien in zahlen -teil ii -punktabzug für zu seltene besuche bei den eltern asien in zahlen -teil -made in china bti transformationsindex the race for ai supremacy mitte der welt auf dem weg zur totalen Überwachung. . juli the new era of competition with china drillen bis zum umfallen macron setzt auf künstliche intelligenz spotlight on china retail income per person der wahn des tyrannen wie china den dollar knacken will flughafen berlin (ber) kosten. . juli bundesregierung will milliarden in ki investieren und den usa und china zuvorkommen wie china bei der künstlichen intelligenz zur supermacht aufsteigt world economic and financial surveys creativity is china's greatest future resource ai superpowers: china, silicon valley, and the new world order china -die welt des xi jinping merkel in china: wo die kameras alles sehen januar how to win the technology race with china notes for the frontier: modeling the impact of ai on the world's economy anzahl der studenten aus china, die im ausland studieren in den jahren deutsch-französische kooperation -china investiert massiv in künstliche intelligenz dezember private equity investment in artificial intelligence the world in -the long view: how will the global economic order change by ? factfullness: wie wir lernen, die welt so zu sehen, wie sie wirklich ist sozialpunktesystem in china: eine welt ohne datenschutz? massenhaft nutzer -mangelhafter datenschutz made in china was china in dschibuti vorhat bau von ber-flughafen: erinnern sie sich an diese nachricht? understanding china's social credit system world population prospects full translation: china's ‚new generation artificial intelligence development plan‛ märz a). digital ecosystem der große umbruch -wie künstliche intelligenz unser leben verändert world intellectual property indicators . märz secure a decisive victory in building a moderately prosperous society in all respects and strive for the great success of socialism with chinese characteristics for a new era key: cord- - iu qx n authors: ronquillo, jay g; lester, william t; zuckerman, diana m title: using informatics to guide public health policy during the covid- pandemic in the usa date: - - journal: j public health (oxf) doi: . /pubmed/fdaa sha: doc_id: cord_uid: iu qx n background: current and future pandemics will require informatics solutions to assess the risks, resources and policies to guide better public health decision-making. methods: cross-sectional study of all covid- cases and deaths in the usa on a population- and resource-adjusted basis (as of april ) by applying biomedical informatics and data visualization tools to several public and federal government datasets, including analysis of the impact of statewide stay-at-home orders. results: there were . cases and . deaths per million residents, respectively, in the usa with variable distributions throughout divisions, regions and states. forty-two states and washington, dc, ( . %) had statewide stay-at-home orders, with the remaining states having population-adjusted characteristics in the highest risk quartile. conclusions: effective national preparedness requires clearly understanding states’ ability to predict, manage and balance public health needs through all stages of a pandemic. this will require leveraging data quickly, correctly and responsibly into sound public health policies. the coronavirus disease (covid- ) pandemic caused by severe acute respiratory syndrome coronavirus (sars-cov- ) presents complex challenges to health professionals, researchers and policymakers. there has been a global effort to make relevant technologies, resources and information available that would accelerate data-driven solutions for all aspects of this pandemic. , however, the current focus on raw counts of cases and deaths in the usa, while necessary, is not sufficient to fully assess the risks, resources and policies to guide better public health decision-making, now and in the future. , our study analyzes important pandemic characteristics in the usa on a population-and resource-adjusted basis using several publicly available datasets and visualization tools in order to provide deeper insight into critical issues for the current pandemic. this was a cross-sectional study of daily report and time series data for the usa from the study date ( april ), that was downloaded from the github covid- data repository hosted by the center for systems science and engineering at johns hopkins university in comma-separated values file format. specific fields from the daily report file used for analysis include the name of the state (province_state), country (country_region), total number of covid- cases (confirmed) and total number of covid- deaths (deaths). similarly, specific fields from the time series file include the name of the state (province_state) and country (country_region), along with a single field for each date from january up to the study date april for confirmed covid- cases and deaths. population-adjusted characteristics were calculated by dividing us state-level totals for covid- (i) cases and (ii) deaths, respectively, by state population estimates from the us census bureau (https://data.census.gov/). resource-adjusted characteristics were calculated by dividing state-level cases by (i) estimated state-level physician totals from the agency for healthcare research and quality compendium of us health systems (https://www.a hrq.gov/chsp), and (ii) published state-level estimates for mechanical ventilators as described in the society of critical care medicine report on us icu resource availability for covid- . [ ] [ ] [ ] each state in the usa is responsible for setting their own policies regarding pandemic risk mitigation. using information from available publication and news sources, we identified states with and without stay-at-home or similar nonpharmaceutical intervention (npi) orders that were implemented statewide as of the study date. [ ] [ ] [ ] for each state with a statewide stay-at-home order, we calculated the number of days between the effective date of the order and (i) the date of a state's first reported case, (ii) the date of a state's first reported death and (iii) the study date. summary statistics were collected as medians with interquartile range (iqr). quartiles for covid- case and death characteristics were calculated for each state and visualized as choropleth maps generated in plotly (version . . ). all data were integrated using an informatics pipeline built via a jupyter notebook running python (release . . ), and analyses were performed using microsoft excel version . . (redmond, wa). as of april , there were reported covid- cases and deaths in the usa. broken down by us census region, there were cases and deaths in the midwest, cases and deaths in the northeast, cases and deaths in the south and cases and deaths in the west. overall, there were . cases and . deaths per million residents, respectively. population-and resource-adjusted characteristics by us census region and division are shown in table . trends in cases and deaths adjusted for state populations and resource estimates are visualized in figure . forty-two states and washington, dc, ( . %) had statewide stay-at-home orders for all residents, implemented at a median (iqr) of . ( . - . ) days after first reported case and . ( . - . ) days after first death, and have been in place for a median of . ( . - . ) days as of the study date. at the time that their respective orders became effective, states had a median of . ( . - . ) cases per million residents and . ( . - . ) deaths per million residents. at these thresholds, all states without statewide orders (arkansas, iowa, nebraska, north dakota, oklahoma, south dakota, utah, wyoming) would be ranked in the highest quartile for cases per million residents, as well as in the highest quartile for deaths per million residents. most statewide stay-at-home orders were implemented roughly - weeks after a state's outbreak was first detected, and have only been in place for a few weeks at the time this study was performed. the influenza pandemic triggered nonpharmaceutical intervention orders in many us cities lasting several months, had multiple (e.g. two or more) waves of pandemic infections, and killed more than half a million people in the usa and tens of millions worldwide. , , yet even for that pandemic, us cities with npi orders that were (i) started soon after an outbreak was detected, (ii) longer in duration, and (iii) broader in scope had better overall outcomes than cities without those characteristics. , it remains concerning that most states currently without stayat-home orders have population-adjusted case and death metrics that are just as grave as the rest of the country. we strongly recommend that all states implement or continue to implement responsible mitigation strategies focused on protecting their most vulnerable populations. the us food and drug administration (fda) is responsible for regulating medical devices and laboratory developed tests (ldts) essential to address the current pandemic, including products like mechanical ventilators and diagnostic tests for covid- . the traditional regulatory approval process requires that companies provide evidence of a product's safety, effectiveness and performance; however, the fda recently issued several emergency use authorizations which lower regulatory standards in order to quickly address urgent supply shortages. , as the pandemic continues to intensify, it will be critical to improve the quality of all medical devices (including ldts) reaching patients. covid- diagnostic tests with low specificity (and high false positive rates) could lead to unnecessary quarantines, mental stress and wasted hospital resources while low sensitivity (high false negative) tests could lead to multiple waves of uncontrolled community transmission. until there is a robust supply of accurate, validated diagnostic tests to gauge community spread, the high-risk (upper quartile) states in our figure highlight where greater healthcare resource capacity is needed to prevent overwhelmed hospital systems from increasing patient mortality and putting healthcare employees at risk of infection. , , , what this study adds effective national preparedness requires clearly understanding states' ability to predict, manage and balance public health needs through all stages of a pandemic. , , the rapid spread of sars-cov- has exposed the limited availability of key resources, from personal protective equipment to mechanical ventilators to the diverse healthcare providers at the front lines of clinical care. looking beyond raw case and death counts by adjusting for publicly accessible data on populations and resource estimates can help clarify risks and inform public health policy. our study had several limitations. first, the numbers of covid- cases and deaths are likely underestimated, given the current shortage of adequate diagnostic tests across the usa. second, provider and ventilator estimates are not updated as frequently as the pandemic counts, and thus only provide general guidance on resource availability. third, our study focuses primarily on state-level data but future research should leverage finer levels of granularity, including data about counties, population density, race, age and social determinants of health. finally, our work integrates and harmonizes pandemic reports with fragmented data from various federal agencies and published outlets. however, effective public health solutions for covid- and future pandemics will require access to interoperable public health data at all levels. the covid- pandemic is neither the first nor last major health challenge that the world will face but lasting success will depend on synthesizing data and information quickly, correctly and responsibly into sound national and international public health policies. dr. ronquillo reports working for syapse, which had no role in the study, has received cloud research grants from google and microsoft during previous work as a medical school faculty member and has received cloud research funding from the google cloud for startups program. the authors have no other competing interests to declare. priorities for the us health community responding to covid- geographical tracking and mapping of coronavirus disease covid- /sars-cov- epidemic and associated events around the world: how st century gis technologies are supporting the global fight against outbreaks and epidemics covid- and health care's digital revolution defining the epidemiology of covid- -studies needed an interactive web-based dashboard to track covid- in real time population, population change, and estimated components of population change mechanical ventilators in us acute care hospitals icu resource availability for covid- public health interventions and epidemic intensity during the influenza pandemic see which states and cities have told residents to stay at home. the new york times thinking globally, acting locally-the u.s. response to covid- nonpharmaceutical interventions implemented by us cities during the - influenza pandemic transmissibility of pandemic influenza software-related recalls of health information technology and other medical devices: implications for fda regulation of digital health policy for diagnostic tests for coronavirus disease- during the public health emergency: immediately in effect guidance for clinical laboratories, commercial manufacturers, and food and drug administration staff guidance for industry and fda staff: statistical guidance on reporting results from studies evaluating diagnostic tests covid- -navigating the uncharted potential association between covid- mortality and health-care resource availability governmental public health powers during the covid- pandemic stay-at-home orders, business closures, and travel restrictions health it, hacking, and cybersecurity: national trends in data breaches of protected health information public health and epidemiology informatics: recent research and trends in the united states none. all authors included in the manuscript provided substantial contribution to (i) conception and design, acquisition of data, or analysis and interpretation of data, (ii) drafting the article or revising it critically for important intellectual content and (iii) final approval of the completed manuscript. jgr had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. the funders had no role in the design and conduct of the study; collection, management, analysis and interpretation of the data; preparation, review or approval of the manuscript; or decision to submit the manuscript for publication. none. key: cord- -epsp bbm authors: luppa, peter b.; müller, carolin; schlichtiger, alice; schlebusch, harald title: point-of-care testing (poct): current techniques and future perspectives date: - - journal: trends analyt chem doi: . /j.trac. . . sha: doc_id: cord_uid: epsp bbm point-of-care testing (poct) is a laboratory-medicine discipline that is evolving rapidly in analytical scope and clinical application. in this review, we first describe the state of the art of medical-laboratory tests that can be performed near the patient. at present, poct ranges from basic blood-glucose measurement to complex viscoelastic coagulation assays. poct shortens the time to clinical decision-making about additional testing or therapy, as delays are no longer caused by transport and preparation of clinical samples, and biochemical-test results are rapidly available at the point of care. improved medical outcome and lower costs may ensue. recent, evolving technological advances enable the development of novel poct instruments. we review the underlying analytical techniques. if new instruments are not yet in practical use, it is often hard to decide whether the underlying analytical principle has real advantage over former methods. however, future utilization of poct also depends on health-care trends and new areas of application. but, even today, it can be assumed that, for certain applications, near-patient testing is a useful complement to conventional laboratory analyses. a large number of laboratory analyses support correct diagnosis in over % of all diseases, in addition to aiding the monitoring of drug therapy in many other cases. laboratory medicine is therefore a vital component in differential diagnosis in the clinic and in local general practice. an affordable, competent system of laboratory diagnostics, in the doctorÕs office and hospital, has been made possible through centralization of analysis in purpose-built laboratories or in facilities provided by large hospitals. in contrast to this centralization and increased efficiency in laboratory diagnostics, there has been a recent trend towards a more decentralized diagnostic analysis, so-called point-of-care testing (poct), which occurs directly at patientsÕ beds, in operating theatres or outpatient clinics, or at sites of accidents. this modern variety of laboratory medicine is characterized by minimizing instrument size and procedures and the increasing use of current information technology. there are analytical devices available that make it possible to process a whole blood sample in a simple manner, allowing untrained staff to carry out laboratory diagnostics. it is clear that the use of poct shortens the time between sample acquisition and analysis (turnaround time). however, this type of diagnosis is only useful if the results produced lead to immediate therapeutic decisions [ ] . however, there is still a paucity of evidence supporting the use of poct and improved patient outcomes [ ] . nevertheless, it is not difficult to predict that there will be growth in the development of poct diagnostics for parameters that are at present available only in central laboratories. this growth is supported by new analytical technologies amalgamating issues (e.g., miniaturization, nanoparticle techniques, multiplexing, wireless connectivity, and novel biomarkers). clinical pathology as a discipline needs to be responsible for this field, since adherence to quality-management systems ensures accurate, reliable biochemical-test results for optimal patient care and safety, regardless of whether the individual test is performed in a central laboratory or as poct at the bedside [ ] . poct is mainly characterized by proximity to the patient, quantitative or semi-quantitative single measurements, short turnaround time, no sample preparation, no pipetting, use of pre-made reagents, user-friendly dedicated analytical instruments and instant, resultdeduced therapeutic action [ , ] . all outpatient or ward personnel will be expected to be able to use these analyzers. no previous knowledge in sample analysis should be required. however, there is a continual transition between poct and laboratory-based methods. a generally accepted definition of poct is still under debate. for example, viscoelastic coagulation tests, described below in this article as ''type '' poct, are to be performed near the patient in cases of active bleeding. the results have to be available quickly to allow instant therapeutic action. however, the caregiver is mostly overstrained when operating the viscoelastic apparatus, so often these analyses are performed in a central laboratory with online transmission of the sensorgrams to the operating room; so these analyzers can be called ''pseudo-poct''. currently, the annual turnover for poct in vitro diagnostics in europe stands at approximately € bn, germany contributing € . bn of this; the majority comes from blood-sugar-test strips and devices for home testing in diabetes. in the past few years, the market for poct systems grew yearly by more than %, but this rapid growth has been slightly dampened since . nevertheless, a marked increase is expected mid-term and long-term [ ] . table gives insight into the variety of parameters available at present. . current techniques: categorization of the stateof-the-art poct devices poct devices often employ biosensors (fig. ) . according to the iupac definition, a biosensor is an analytical device for the detection of analytes that combines a biological component with a physicochemical-detector component. this generally occurs through the use of miniaturized analysis systems, where biological components are immobilized on a solid-state surface, which, in turn, interacts with the analyte [ ] . these interactions may be detected by using either electrochemical or optical methods. in the latter case, a large role is played by fluorescence or reflection spectroscopy. different parameters, being characteristic for certain diseases, can be detected by using specific biomolecules. poct analyzers currently available can be separated into various groups. we attempt to categorize the instruments according to their practical use in poct and moreover by the following attributes: sensor characteristics, complexity, measuring mode, underlying detection principle and/or sample matrix. here is the simplest form of quantitative poct device, with most of the analysis taking place on the respective test strips. the reader is used only to read the result from the strips where the reaction has already taken place. these test strips are one-use articles. examples include figure . the biosensor as the basis of analysis in many point-of-care testing (poct) instruments. note that the recognition layer may comprise attached recognition elements (antibodies, receptors, aptamers . . .) or immobilized enzymes. in the latter case, the addition of substrates is essential. not illustrated is the fact that the amplified signal is finally processed by microelectronics and displayed. glucometers for home and the hospital poct stations, as well as the i-stat abbott (abbott park, il, usa), a multi-parameter unit-use poct instrument [ ] . blood-sugar analysis is, both historically and financially, the most commonly used poct technique. this is valid for in-patient and out-patient care, and for home testing by patients. although generally either capillary or venous blood is used, the results can be presented as blood-glucose or plasma-glucose level, depending on the calibration by the manufacturers. this can make a relevant difference of over %, potentially resulting in false therapeutic measures. to minimize the risk of confusion between whole-blood and plasma-glucose results, the international federation of clinical chemistry and laboratory medicine (ifcc) suggested in that glucose values should be given as plasma levels only, independent of sample type or measuring method. the usa and other countries have already followed this recommendation, and it is also now being implemented in the german-speaking regions [ , ] . the strong correlation between the international normalized ratio (inr) and the clinical outcome of oral anticoagulation with vitamin k antagonists (warfarin, marcumar) has led to a tight therapy-monitoring regime using inr poct devices. inr testing may be performed at the doctorÕs office or by the patient (''home testing''). for the most popular devices see table . all these systems use unit-use test strips and whole blood obtained by finger prick, making them most convenient for the patient [ ] . different types and sensitivities of the thromboplastins used and the different signaling types (micromechanical versus electrochemical) cause systematic differences between results obtained with the different inr devices [ ] . however, the between-day precision of the devices seems to be satisfactory [ ] , so the monitoring should be performed with one single type of device. these instruments are generally more complex than unit-use machines and use different analytical principles [ ] : spectrophotometric substrate and enzyme-activity measurement; hematological particle counting; immunoassay; and, sensor-based blood-gas analysis tailored for special poct applications. they use antibody-based immunoassay methodologies. bgas use potentiometric/amperometric or optical sensors for ph, po and pco . additional ion-sensitive electrodes for the measurement of electrolytes and other substrates are available. a selection of systems from the seven marketdominating manufacturers is given in table . optional is the configuration of a bga with a cooximetry unit. this is a most remarkable technical development. the co-oximetry unit [ ] is a miniaturized multi-wavelength spectro-photometer, which measures the typical absorption spectra of the various hemoglobin (hb) species in order to distinguish o -hb (oxy-hb) from other hb species and to determine the o -hb saturation [i.e. the percentage of o -hb compared to the total amount of hb, including co-hb, o -hb, deoxygenated hb (hhb with fe + ), and met-hb (hhb with fe + )]. all leading companies on the market have sophisticated oximetry units on board their blood-gas analyzers, which have no counterpart in the central medical laboratory. the only technological diversification is the factor that the hb species are found only inside the erythrocytes. hence, some systems use a cell-lysis step prior to spectrophotometry, whereas others eliminate the erythrocyte-caused light scattering by applying matrixassisted algorithms. these poct compatible machines show a high degree of complexity. although they are valid for use in poct, only qualified personnel should operate them (e.g., a laboratory physician or a trained technical assistant). the combined analysis of plasma clotting, thrombocyte function and fibrinolysis is termed viscoelastic coagulation testing [ ] . examples include the rotem (tem international, munich, germany) [ ] or the sonoclot from sienco inc (arvada, co, usa). it is also possible to analyze platelet function in terms of in vitro bleeding time or via optical aggregometry [ ] . examples here are the pfa from siemens healthcare diagnostics (eschborn, germany) or the verifynow from accumetrics (san diego, ca, usa) [ ] and [ ] . as described in the introduction, these analyzers have to be called pseudo-poct. the most common example here is continuous glucose monitoring [ ] . such analyzing and application systems are already available commercially. they are likely to replace the invasive, intravenous electrode by the minimally invasive location of a microdialysis catheter in subcutaneous tissue (fig. ) . conversely, other non-invasive methods (e.g., microporation or optical techniques in direct transcutaneous measurement of metabolic parameters) are at least unlikely to prevail. this is mainly due to the broad range of human-skin characteristics concerning thickness, pigmentation and hairiness as well as physiological phenomena (e.g., humidity and salt content). currently available systems for continuous glucose monitoring are the guardian rt from medtronics (minneapolis, mn, usa), the glucoday from a. menarini . . type -molecular biology-based poct devices to detect infectious agents at present on the market, there are many qualitative test strips to detect infectious pathogens. the basic principle in most systems is immunochromatography of a specific microbial antigen (or, more rarely, antibody) in the patient sample (urine, swab, or whole blood). there have also been some attempts to use molecular biological methods [mostly the polymerase chain reaction, (qrt)-pcr] for poct, although these are technically demanding (with a dna/rna-extraction step required), so that they are no rapid tests in the strict sense (yet). due to the complexity of the test procedures and the challenging interpretation of results, future rapid nucleic-acid testing (nat) is more likely to be located in the central laboratory rather than at the bed-side [ ] . nevertheless, rapid quantification of dna/rna from various infectious agents will be beneficial for clinical diagnostics. at present, the genexpert system from cepheid (sunnyvale, ca, usa) is the cutting-edge device to automate and to integrate all real-time pcr-based nat steps: sample preparation, dna amplification and detection. the system is a - -site, random-access instrument, integrating real-time cycler, amplification and fluorescence detection. the use of multiple fluorescence dyes permits multiplex assays for detection of several targets within a single sample. the system provides pcr-test results from a raw clinical sample in about h, enabling time-critical nat at the point of need. there is a series of different unit-use microfluidic cartridges to be inserted into the analyzer {e.g., the ''xpert sa nasal complete'' cartridge is designed to detect s. aureus and mrsa colonization from nasal pharyngeal swabs flu a-panel cartridge is already under evaluation [ ] }. the currently available novel poct systems are in part forerunners of a new generation of laboratory systems, which will dominate the market in - yearsÕ time (fourth-generation laboratory systems). these methods are characterized through miniaturization, parallel analyses and networking via information technology (it) [ ] . . . . miniaturization. microfluidics, chip technology and electrochemical detection have enabled the construction of miniaturized systems, with which even dna may be directly analyzed in a few microliters of sample. immunoassays and other methods of protein determination may also be carried out by ''micro-total analysis systems'' (ltas). the advantages of miniaturization are obvious: fluid volumes in the nl and pl range reduce not only the amount of reagents required but also the time needed for basic analytical processes (e.g., mixing and equilibrating) [ ] . . . . parallelization. chip technology opens up the possibility of measuring multiple channels or multiple time points in the smallest space; photolithographic techniques allow reaction reservoirs and liquid channels to be etched on a wafer to enable or more measurements to be taken simultaneously. an even higher density of reactions has already been realized in gene analysis with the use of microarrays. appropriate microautomation systems allow multiple laboratory profiles to be established rapidly and cost effectively, thereby opening up the possibility of obtaining an overview of a patientÕs situation in just a few minutes. the use of a pattern of laboratory values -in contrast to single parameters -may, at least in principle, open up new diagnostic and prognostic avenues. however, the attendant problems in evaluating and interpreting the massive amounts of data thus obtained remain unsolved. networking. next-generation laboratory systems will generally be networked via it, and an international communication standard (poct -a) already exists for this purpose. however, current pointto-point communications (e.g., that between decentralized blood-glucose monitors and the laboratory it system) show only a modest beginning. for the future, there have been discussions on individual electronic patient files, in which laboratory values from various sources can be directly input and linked to other results. at present, a wave of novel analytical principles and instruments can be envisioned for the near future, including alternative biological detection elements (scaffolds, aptamers, or anticalins in place of antibodies), sophisticated applications of optical-signal technologies (total internal reflection fluorescence, surface plasmon resonance, reflectometric interference spectroscopy) [ ] and new dedicated microarrays for inflammation, malignancies and autoantibody diagnostics. this process will be encouraged through innovation from the communication industry, particularly via wireless networking (wifi) technology. the following details refer to new instruments that are on the market or will shortly to be analyzer. this cartridge-based instrument allows the determination of hba c, albumin/creatinine ratio (acr), and crp by reflectometry. for testing, whole blood, plasma and urine samples can be used. the advantages of this device are short assay times, all-inone reagent cartridges and its ease of application. tech. this is a hand-held immunoassay machine, which carries out immunological reactions using magnetizable nanoparticles. the particles are labeled with antibodies and exposed to a variable magnetic field. detection is performed with a ''bound-from-free'' separation of reaction components, currently using a frustrated total internal reflection method. nanoparticles can also be used in the so-called giant magnetoresistance effect in signal generation [ ] . we expect that the instrument will provide a multi-analyte detection mode, with a spectrum of parameters (e.g., cardiac markers, parathormone and sepsis parameters). this company is developing a new signal-generation and detection technology. the poct instrument uses a piezo film made from polyvinyl fluoride, which acts as a pyroelectric film, thereby enabling an ultrasensitive non-separation assay design for immunoassays (e.g., cardiac markers, thyroid hormones). when led light strikes the piezo film, it causes thermal disturbance with subsequent changes in the charge (signal). however, this is only the case when a carbon particle-labeled immuno complex was previously bound. a labeled antibody, free in the solution and not bound to the surface, does not cause a change in the charge of the piezo film. analyzers. an innovative technology based on an established detection principle is realized in the new multiplate analyzer (dynabyte, munich, germany). the device uses the impedance aggregometry principle [ ] for the assessment of platelet function in whole blood. blood thrombocytes are non-thrombogenic in their resting state, but expose receptors on their surface when being activated. this allows them to attach on vascular injuries and artificial surfaces (e.g., the sensor wires). when the platelets aggregate on the multiplate wires, they enhance the electrical resistance between them, which can be continuously recorded. in order to enhance the resistance on the sensor wires, tight attachment of the platelets is required. a number of plateletstimulating test reagents are available for specific, comprehensive platelet diagnostics. . . . type -continuous measurement with poct systems. for a continuous mode of poct measurements, minimally-invasive microdialysis systems have become accepted as best approach. nevertheless, the most desired clinical access for such probes, the intravenous line, was, for a long time, an unfulfilled clinical desire. only recently, the microeye system of probe scientific ltd. (coventry, uk) enabled continuous, automated monitoring of substances in the circulating blood stream without withdrawing blood from the patient by applying an intravenous microdialysis catheter (http:// www.probescientific.com/microeye/). many companies are currently working on different poct-suitable dna-amplification techniques (e.g., the quantitative detection of the influenza a panel or of enterovirus strains). . . . . nanosphere (northbrook, il, usa). the verigene system uses -nm gold-nanoparticle-probe technology for the detection of infectious dna or rna. the probe technology uses the change in optical properties of these gold nanoparticles that occurs when two or more nanoparticles are brought into close proximity. the wavelength of light scattered from the surface of the particles is within the visible range and can easily be detected. details of the distancedependent optical properties of gold nanoparticles are portrayed in fig. . . . . . enigma (salisbury, wiltshire, uk). this company is working on providing the next generation of integrated and automated real-time pcr-based diagnostics systems. . . . . dxna (st. george, ut, usa). a mobile poct technology (genestat) provides rt-pcr-based detection of severe acute respiratory syndrome (sars) or highly pathogenic avian influenza. . . . . idaho technology (salt lake city, ut, usa). the filmarray system is a user-friendly automated multiplex pcr. . . . . iquum (marlborough, ma, usa). the liat analyzer offers an innovative lab-in-a-tube platform for detection and genotyping of dna/rna viruses and bacteria. a number of novel parameters for poct applications are under discussion. potentially interesting À but still clinically unevaluated À new markers for poct applications are as follows. . . . ngal. neutrophil gelatinase-associated lipocalin (ngal) tests for acute renal failure [ , ] . . . . galectin- . this b-galactoside-binding protein tests for fibrosis and adverse cardiac remodeling [ , ] . copeptin. this c-terminal residue of proavp is for early diagnosis of myocardial infarction [ , ] . the combination of placental growth factor (pigf), soluble fms-like tyrosine kinase (sflt- ) [ ] [ ] [ ] , and endoglin are for early diagnosis of preeclampsia [ , ] . many of the possibilities of extending the application of poct depend on the wishes, the needs and the practicalities arising from using poct, as well as the advantages and the disadvantages for the patient. many people should be involved in this discussion: clinical and laboratory medical personnel, doctors in practice, political and regulatory authorities, state and private health insurers, industry and, not least, the patient. further-more, applications of poct must be scientifically grounded, evidence based {e.g., via outcome analysis, which has only partly been the case to date [ ] } and financially viable. financial viability is strongly associated with the respective national reimbursement system for the various tests, which is, in most countries, mainly a question of healthcare politics. the quality of poct can be assured only through a poct-coordination system, in which the clinical laboratory plays a pivotal role. all poct activities should be discussed and agreed upon between the clinics and the clinical laboratory [ ] . there are many examples of fruitful cooperation even today. however, a future necessity will be the more structured search protocol for a new poct method with agreement on the core laboratory method so that the clinical colleagues can assume clinical acceptability of the result variations caused by the poct method. new rules are needed for assessing agreement on a certain analyte result between two different methodologies [ ] . in some analytical cases (e.g., step b: the dna probes are hybridized to a dna target in solution. when they are spotted onto the glass slide (step c) these hybridized gold nanoparticles scatter with h · m . the plasmon efffect induces a red light shift with m < m . the scheme presented is in accordance with [ ] . blood-glucose or prothrombin-time monitoring), errorgrid analysis can be used to determine cut-offs where differences between central laboratory and poct results may impact clinical decisions [ ] . novel statistical approaches are yet to be defined. data management of the poct results is also fundamental to quality [ ] . tight surveillance of poct data can show error trends before they affect the results. however, a prerequisite for this is comprehensive connectivity of all poct systems used within a hospital-wide network. newer poct devices have data-storage functions that can collect key information at the time the test is performed and later transmit the result together with the internal quality-control data to a server-based poct data manager software linked to the information system of the hospital. recently developed connectivity standards (e.g., poct -a) enable different poct devices to share a common interface. poct data-management software programs can be categorized into proprietary and non-proprietary. examples of the first category are cobas it from roche, radiance from radiometer or rapidcomm from siemens healthcare. non-proprietary software solutions in the eu are pocelerator from conworx (berlin, germany), or, in the usa, quick-linc from telcor (lincoln, ne, usa) [ ] . wireless real-time communication of test results and quality data will become a reality in the near future [ ] . the number of patients requiring inpatient treatment in a hospital, together with the length of their stay, is likely to decline further in the near future -to the advantage of outpatient treatment and patient observation in their home environment. the importance of poct will increase in these areas. in outpatient care, it is clear what advantage immediate diagnostic and therapeutic decisions offers: the patient may not need to be further investigated. an enlargement of the current parameter spectrum is also important for the doctor in practice; here, in addition to the tests now available [ ] , a means of identifying infectious agents would be required. home-care will be extended -apart from glucose and inr monitoring -when new tests for chronic diseases become readily available. the most important trend in this context is termed ''personalized medicine''. in this case, chronic diseases (e.g., diabetes or atherosclerosis) would be diagnosed earlier than by traditional clinical diagnosis, which is focused on the patientÕs clinical signs and symptoms. the therapeutic strategies would then be tailored to each patient individually, thus improving the results of treatment (fig. ) . ''telemedicine'' is increasingly important in this area. instruments used to monitor the patient can, in the right circumstances, send the data obtained directly to the relevant medical service point, which can intervene (e.g., when critical values are exceeded). the effectiveness of home monitoring and thus patient safety are improved. though poct may be used in principle at ''any'' locale, its use today is mainly centered on various areas of the hospital, general practitioners and patient self-monitoring of glucose and inr. above and beyond this, other potential uses have been discussed, or have indeed been, albeit mostly in small numbers, already realized. these include: mobile emergency paramedical care (blood mobiles, mobiles for public events); transport vehicles (e.g., ambulances and helicopters); sport medicine or competitive sport; outbreaks of disease and catastrophes [ , ] ; remote areas, ''third world'' countries; military use; at the pharmacy; prisons; alternative-medicine practitioners; corporate healthcare operations; nursing homes; veterinary medicine; fitness studios; and, home healthcare in many cases, the equipment currently available, as well as the spectrum of parameters, is only partly suitable; but, future developments will raise the acceptance level and therefore the use of poct in these specific areas. the availability of medical treatment to the population as a whole is particularly important in developing countries. the current western systems (e.g., structured around a large central hospital) are largely unsuitable; rather a decentralized health system offers the most effective means. in this case, a decentralized diagnostic process with poct systems can play a very useful part. there have been a few successful movements in this direction (e.g., in reserves of the australian aborigines) [ ] . however, widespread use of the currently available systems is hindered by costs, complexity and unreliability under extreme conditions of heat or humidity. the parameters offered often do not meet those required in these situations. the american national institute of biomedical imaging and bioengineering (nibib) has therefore begun co-operation with india, the aim of which is to develop devices, which are suitable for use at the point of need [ ] . the international council for standardization in hematology (icsh) is working on a set of guidelines, which will be applicable worldwide for poct standardization in hematology and which can also be used in developing countries [ ] . specialized, novel rapid microbiological tests must be developed to recognize the common infective agents in these countries. the bill and melinda gates foundation has sponsored research in this direction in order to establish a simple poct application for aids patients. this so-called cd initiative should make it possible to count the cd positive t-lymphocytes in hiv-infected patients, with a view to better control of anti-retroviral therapy. the value of poct in epidemic disease in developing countries can be demonstrated in the following example. tuberculosis treatment is hampered by slow, insensitive diagnostic methods, particularly for the detection of drug-resistant forms. an early diagnosis is essential to reduce mortality and to interrupt transmission, but the insufficient healthcare infrastructure in poor countries limits their accessibility and effect. boehme et al. [ ] assessed the performance of the automated molecular identification of mycobacterium tuberculosis (mtb) and the testing of resistance to rifampin (rif) on the genexpert mtb/rif system (mentioned above), applying sputum samples of patients from peru, azerbaijan, south africa, and india. as a result, the authors stated that the mtb/rif test provided sensitive detection of tuberculosis and rifampin resistance directly from untreated sputum in less than h. the test was specific in of patients without tuberculosis ( . %). as compared with phenotypic drug-susceptibility testing, mtb/rif testing correctly identified of patients ( . %) with rifampinresistant bacteria and of ( . %) with rifampin-sensitive bacteria. the device is definitely applicable in rural areas without being compromised by a lack of analytical quality. all future projects should take into account that poct can in principle be brought simply to the patient, but not necessarily the expertise of the laboratory doctor. it is difficult to imagine how complex panels of analyses (e.g., tumor markers or genetic susceptibility tests), supplied via poct, could be interpreted without adequate technical expertise and knowledge. it is therefore vital, in the interest of the patient, that poct is employed by laboratory medical personnel who can suggest appropriate tests and carry them out satisfactorily. a specific problem involves the problem of quality assessment. the wide use of poct in areas outside of the standard hospital environment or by general practitioners can lead to new problems in quality assurance. it is clear today, that use of poct without relevant trained personnel leads to unsatisfactory results and that the end-user has to rely on a competent laboratory medical advisory service, at least when difficulties arise. further development of the relevant equipment is likely to result in inbuilt quality control, so that the current procedures for internal and external controls will become obsolete. however, the problems of end use by untrained or unsupervised personnel will increase, particularly with respect to pre-analysis (patient preparation, sample acquisition, recognition of invalid samples) and post-analysis (documentation, data protection) [ ] . this is notably the case where poct is used in areas far from a laboratory (e.g., developing countries). it is an important task for laboratory medicine as a whole to make it clear that poct analysis that meets the medical requirements rests on more than reliable analysis systems. principles and practice of point-of-care testing point-of-care testing key: cord- - hsn us authors: poitiers, niclas frederic title: institutionelle aspekte einer neuen handelsordnung date: - - journal: wirtschaftsdienst doi: . /s - - - sha: doc_id: cord_uid: hsn us nan der französischen unabhängigkeit in der landwirtschaft, dem gesundheitswesen, der industrie und der technologie" (macron, ) . vertreter der industrie gehen noch weiter und fordern "sanitäre unabhängigkeit", womit gemeint ist, dass vollständige wertschöpfungsketten wieder nach europa verlegt werden sollen (hudson, ) . peter navarro, der wirtschaftsberater des us-präsidenten, fordert die schaffung ganzer wertschöpfungsketten in den usa (politi, ) . es wurden in vielen ländern, inklusive der eu und den usa, exportkontrollen von medizinischen gütern eingeführt, die insbesondere kleinere und ärmere länder vor eine schwierige situation stellen. dies erschwert die diskussion um die zukunft des welthandelssystems und die suche nach lösungen gegen den verfall der internationalen wirtschaftsordnung. dabei war der welthandel schon vor der corona-pandemie im rückzug. seit der finanz-und wirtschaftskrise ist der anteil des handels am globalen bip zurückgegangen. dies hatte verschiedene gründe, technologische und politische, hatte jedoch vor allem aber mit den auswirkungen der finanzkrise zu tun (darvas, ; hoekman, ) . die eu befi ndet sich inmitten des chinesisch-us-amerikanischen konfl iktes, und ist direkt und indirekt von diesem betroffen (jean et al., ) . so wurden erste europäische güter direkt von den usa mit zöllen belegt und weitere zölle, insbesondere für den gerade für deutschland wichtigen automobilsektor, angedroht. zölle werden von der us-amerikanischen regierung inzwischen auch als drohmittel außerhalb von handelsstreitigkeiten benutzt (euobserver, ). indirekt betrifft der handelskrieg europa aber auch dadurch, dass die zölle und sanktionen, mit denen chinesische produzenten belegt werden, auch auswirkung auf ihre europäischen geschäftspartner haben. am größten ist jedoch das risiko, dass die usa das internationale handelssystem in richtung eines systems von staatlich gelenktem handel (managed trade) führen. dies würde bedeuten, dass anstatt von angebot und nachfrage staatsregierungen die gehandelten mengen bestimmen würden. anstelle von markteffi zienz würden politische abwägungen den internationalen handel leiten. große Ökonomien wie die usa könnten mit geopolitischen mitteln andere länder unter druck setzten, güter politisch wichtiger produzenten zu kaufen. der chinesisch-us-amerikanische handelsvertrag gibt einen vorgeschmack hierfür. darin wurde festgelegt, dass das "phase- -abkommen" ein art waffenstillstand erreicht. doch dieses abkommen löst keine der grundursachen des konfl ikts. selbst vor der sars-cov- -pandemie war es hoch fragwürdig, ob die zusagen, die china in diesem abkommen gemacht hat, erreichbar waren (bown, ) . diese sind jetzt endgültig nicht mehr einzuhalten, und die wichtigsten beschwerden der usa gegenüber china sind in dem abkommen noch gar nicht adressiert worden. ein kollateralschaden dieses handelskriegs erleidet die internationale wirtschaftsordnung, da die usa internationale regeln zur verhinderung von zöllen ignoriert und die schiedsgerichtsbarkeit der welthandelsorganisation (wto) lahmgelegt haben. nun verstärkt die sars-cov- -pandemie die negativen trends im weltweiten handel weiter. die prognosen des internationalen währungsfonds (iwf) sagen inzwischen für eine globale rezession mit einem weltweiten wirtschaftswachstum von - % voraus, und für das bruttoinlandsprodukt (bip) der eurozone erwartet der iwf einen rückgang von , %. die wto prognostiziert, dass der welthandel im optimistischen fall um , % einbrechen wird. all diese prognosen sind mit enormen unsicherheiten behaftet. je nachdem, wann die pandemie eingedämmt werden kann, könnte der rückgang der wirtschaftstätigkeit und des welthandels auch weitaus dramatischer ausfallen. in ihrem pessimistischen szenario sieht die wto einen rückgang des welthandels um , % voraus, vergleichbar wäre dies nur mit der großen depression der er jahre. gleichzeitig haben die auswirkungen der pandemie die fronten zwischen china und den usa weiter verschärft. china übt sich in einer "maskendiplomatie", indem es publikumswirksam schutzkleidung exportiert, und schürt gerüchte, dass das virus us-amerikanischen ursprungs sei. währenddessen deklariert us-präsident trump die krankheit als "chinesisch" und setzt zahlungen an die weltgesundheitsorganisation (who) aus, da die organisation zu viel chinesischem einfl uss ausgesetzt sei (hass und dong, ; trump, die eu ist als großer "dritter block" zu bezeichnen, da sie es als einzige Ökonomie von der größe her mit china und den usa aufnehmen kann. jedoch fehlen der eu viele der wirtschafts-und außenpolitischen kompetenzen, um außerhalb von handelsfragen mit dem gleichen nachdruck wie china und die usa zu agieren. gerade deshalb ist die institutionalisierung des internationalen handels auch jenseits von seinen grundsätzlichen vorzügen so wichtig für die eu. sie erlaubt es, handelsfragen in einem relativ engen rahmen innerhalb von eu-kompetenzen zu erörtern. wenn jedoch handelspolitik mit außen-und sicherheitspolitischen erwägungen verbunden wird, fehlen der eu die formellen kompetenzen, um ihre interessen wirksam durchzusetzen. viele kleinere länder, die nicht das wirtschaftliche gewicht der eu haben, sind noch viel abhängiger als diese von einer regelbasierten internationalen wirtschaftsordnung, in der nicht das recht des stärkeren vorherrscht. daher versucht die eu in einigen bereichen eine "koalition der willigen" zu bilden und z. b. durch multilaterale abkommen den von den usa lahmgelegten appellate body zu ersetzen. leider sind viele dieser länder aber auch direkt wirtschaftlich oder militärisch von den usa abhängig. insbesondere chinas aggressive politik im südchinesischen meer hat dazu geführt, dass länder wie japan, südkorea oder taiwan sich stärker vom militärischen schutz der usa abhängig sehen (council on foreign relations, ). daher wird die bereitschaft dieser länder, eine koalition mit der eu gegen die interessen der usa einzugehen, nur sehr begrenzt sein. außerhalb von einigen, weniger politisierten, bereichen, wie z. b. e-commerce, gibt es wenig hoffnung, dass es zu grundlegenden reformen der struktur des welthandels kommen wird. um ihre handelspolitischen interessen zu erreichen, wird die eu weiterhin stark auf bilaterale handelsabkommen setzen müssen. in den letzten jahren hat die eu bereits eine beeindruckende zahl solcher abkommen abgeschlossen. aber gerade gegen die größeren abkommen wird in europa häufi g opponiert. themen, die im grunde nicht direkt mit dem abbau von handelsbarrieren zu tun haben, sind immer stärker in den fokus gerückt: china , billionen us-$ zusätzlich an landwirtschaftlichen gütern aus den usa einführen muss (landwirte stellen eine für die republikaner wichtige wählerschaft dar). dies ist zum nachteil nicht nur chinas und der meisten us-bürger, die im gegenzug höhere preise für chinesische güter zahlen müssen, sondern auch anderer exporteure dieser güter, insbesondere brasiliens (bown, ) . weiterhin verhindert es, dass die vorteile von freihandel durch komparative vorteile und spezialisierung realisiert werden. insbesondere deutschland, als eines der vom welthandel am stärksten abhängigen länder der eu, hat ein interesse daran, dass institutionelle lösungen für diesen konfl ikt gefunden werden. die geschichte internationaler institutionen im allgemeinen und der wto im besonderen zeigt aber, dass diese sich nur extrem langsam fortentwickeln, und dass ohne die zustimmung der usa nur sehr wenig zu erreichen ist. viele der vorstellungen der eu für eine reform des handelssystems scheinen auf absehbare zeit nicht realisierbar. stattdessen muss eine agenda entwickelt werden, wie europa seine interessen in einem stärker durch großmachtkonfl ikte geprägten handelsumfeld vertreten kann. die us-administration scheint beschlossen zu haben, dass die regeln der wto ihrem vorgehen chinas gegenüber hinderlich sind, und dass sie ihren konfl ikt mit china weitgehend außerhalb des institutionellen rahmens der wto austragen. die eu hingegen ist aufgrund ihrer beschaffenheit weniger agil in ihrer außenpolitik und hat multilateralismus und regelbasiertes handeln in ihrer dna. daher ist die priorität der eu, die wto zu reformieren und den konfl ikt zwischen den usa und china zu entschärfen. leider scheint dies ein struktureller konfl ikt zu sein, denn auch unter den us-amerikanischen demokraten wird china als neuer gegenspieler wahrgenommen. selbst wenn der handelskrieg beendet werden sollte, wird eine wiederherstellung des status quo ante bellum kaum möglich sein. langjährige fundamentale vorbehalte der usa gegen zwischenstaatliche organisationen haben sich mit dem vorwurf gepaart, dass internationale institutionen von china zu seinem vorteil ausgenutzt werden. so gehen z. b. die us-amerikanischen vorbehalte gegen den appellate body, der letzten instanz des schiedsgerichtshofs für zwischenstaatliche konfl ikte in der wto, zeitgespräch schen handelspolitik bestehen. um europas wirtschaftspolitische interessen wirksam zu vertreten, ist es wichtig außen-und sicherheitspolitik stärker zu koordinieren und zu europäisieren. all dies bedeutet nicht, dem multilateralismus den rücken zu kehren. dieser stellt einen hohen wert an sich dar und sollte auch weiterhin die grundlage europäischer außen-und handelspolitik sein. jedoch müssen wir anerkennen, dass auf absehbare zeit eine reform des welthandelssystems eher unwahrscheinlich ist. dieses droht sogar weiter zu erodieren. deshalb muss die eu strukturelle schwächen abbauen, die die europäische verhandlungsposition gegenüber china aber auch den usa angreifbar machen (leonard et al., ; dadush und wolff, (philippon, ) . das gleiche gilt für die offenen märkte in europa, da sie eine einbindung anderer länder in die wirtschaft ermöglichen und effi zienzgewinne durch spezialisierung und wettbewerb schaffen. daher sollte europa protektionistischen refl exen widerstehen und auch in diesen schwierigen zeiten für einen regelbasierten offenen welthandel eintreten. umwelt-und arbeitnehmerschutz (sogenannte "level playing field"-klauseln) sowie die institutionelle struktur von investitionsschiedsverfahren (isds). die einbindung dieser themen in handelsabkommen erlaubt es der eu, ihre politische agenda in diesen themen international voranzutreiben und um rückhalt für diese abkommen unter den eu-bürgern zu werben. dies trägt aber auch das risiko in sich, dass handelsabkommen überfrachtet werden. insbesondere die "level playing field"-klauseln werden von den vertragspartnern der eu teilweise als bevormundung und protektionismus wahrgenommen. viele vorschläge auf der agenda der eu, die das institutionelle gefüge der wto verbessern könnten, werden mittelfristig nicht umsetzbar sein. der weg zu einer weitgehenden reform des appellate body scheint bis auf weiteres versperrt. auch die erfolgschancen für die bildung eines internationalen investitionsgerichtshofs für isds sehen eher schlecht aus. viele dieser vorschläge gehen in die richtung einer weiteren "institutionalisierung" des welthandels, was auf erheblichen widerstand in den usa stößt. diese sehen sich von solchen institutionen in ihrem handeln beschränkt, haben aber auch ernstzunehmende einwände hinsichtlich deren legitimation. während die mitgliedstaaten der eu es gewohnt sind, nationale souveränität mit multilateralen institutionen zu teilen, sehen die usa dies als undemokratische einschränkung der kompetenzen des us-kongresses, der us-gerichte und der us-exekutive. der welthandel steht durch den usa-china-konfl ikt an einer wegscheide, und durch die sars-cov- -pandemie ist die unsicherheit noch weiter gestiegen. ob sich der handelskrieg noch weiter verschärft, wird nicht nur davon abhängen, wer die us-präsidentschaftswahl gewinnt, sondern auch davon, wie schnell sich die verschiedenen regionen von der pandemie erholen. sollten sich pessimistische szenarien für den westen bewahrheiten, könnte dies zu weiteren sozio-ökonomischen verwerfungen führen, die die eu und die usa schwächen und zu einer stärkeren rückkehr zum protektionismus führen. auf der anderen seite kann es sein, dass es in china zu einer schweren rezession kommt, die das politische system dort infrage stellt. es wäre zu hoffen, dass die staaten die krise als das erkennen, was sie im grunde ist: ein externer schock enormen ausmaßes, der global und koordiniert bekämpft werden sollte. bisher machen jedoch die exportbeschränkungen von medizinprodukten eher den gegenteiligen eindruck. auch wenn die politik gerade "auf sicht" agieren muss, so bleiben die langfristigen herausforderungen der europäi- unappreciated hazards of the us-china phase one deal, piie trade and investment policy watch us-china strategic competition -the quest for global technological leadership the state of china-european union economic relations the european union's response to the trade crisis resisting deglobalisation: the case of europe trump threatened eu-tariffs over iran, germany confi rms companies must move supply chains further from china, nikkei asian review, . februar the us, china and asia after the pandemic: more, not less, tension the global trade slowdown: a new normal, voxeu.org ebook l'europe doit construire sa souveraineté sanitaire the dispute settlement crisis in the world trade organization: causes and cures, peterson institute for international economics international trade under attack: what strategy for europe? redefi ning europe's economic sovereignty rede an die nation the great reversal: how america gave up on free markets us trade adviser seeks to replace chinese drug supplies report to congress on china's wto compliance key: cord- -kf a hix authors: mendenhall, emily title: the covid- syndemic is not global: context matters date: - - journal: lancet doi: . /s - ( ) - sha: doc_id: cord_uid: kf a hix nan what is driving coronavirus to move through the population in the usa and interact with biological and social factors, however, differs from other contexts. us political failures have driven covid- morbidity and mortality, and this cannot be divorced from our historical legacy of systemic racism or our crisis of political leadership. this matters because in other contexts covid- is not syndemic. new zealand's political leadership in response to the crisis has been exemplary. covid- is not syndemic there. in this sense, syndemics allow us to recognise how political and social factors drive, perpetuate, or worsen the emergence and clustering of diseases. recognising contexts are different matters a great deal. for instance, contexts throughout sub-saharan africa are doing much better than the most burdened contexts, like the usa, brazil, and india. many people have questioned, why? some have argued that this reflects a racist frame thinking that african contexts should suffer more. yet, many african governments acted more swiftly and confidently than wealthier countries. the political leadership in these contexts, therefore, prevented the extensive death tolls, compared to contexts like the uk and the usa, where political leadership failed. recognising political determinants of health is central to the syndemic construct. by calling the covid- syndemic global, we miss the point of the concept entirely. i do not write this to dampen horton's use of the term, as i believe covid- is syndemic in my country (the usa). this is precisely because pre-existing conditions such as hyper tension, diabetes, respiratory disorders, systemic racism, mistrust in science and leadership, and a fragmented health-care system have driven the spread and interacted with the virus. these synergistic failures have caused more death and devastation than many other contexts. recognising failures of wealthy countries is imperative as we think about where global knowledge and power sit within fields like global health. syndemic frames provide us with an opportunity to do this. i declare no competing interests. science, technology, and international affairs program, edmund a walsh school of foreign service, georgetown university, washington, dc, usa offline: covid- is not a pandemic syndemics and the biosocial conception of health beyond comorbidity: a critical perspective of syndemic depression and diabetes in cross-cultural contexts racial capitalism: a fundamental cause of novel coronavirus (covid- ) pandemic inequities in the united states understanding the us failure on coronavirus-an essay by drew altman three reasons why jacinda ardern's coronavirus response has been a masterclass in crisis leadership how to talk about covid- in africa key: cord- -ntme w i authors: nan title: speaking up against inequity and racism date: - - journal: nat cancer doi: . /s - - -x sha: doc_id: cord_uid: ntme w i inequalities are prevalent across the spectrum of cancer research and patient care, with destructive repercussions for people and society. we cannot ignore them and must act against the social injustices that perpetuate them. that science, research and medicine strive for the benefit of humankind is a truism. that they are the products of the societies in which they operate, and thus reflect the privileges and shortcomings of those societies, is also a factone that often remains in the background of scientific discourse. the unvarnished reality is that inequalities cut deeply through biomedical science and patient care, with a devastating impact on individuals and communities. the cancer field is not immune to such disparities. racial, ethnic and socioeconomic factors weigh heavily on cancer prevention, diagnosis, access to and quality of care and, ultimately, outcomes. in the usa, members of minority racial and ethnic groups suffer disproportionately from cancer. this is well documented for many affected communities, including black americans, who experience higher cancer mortality rates than those of the white population. for colorectal, prostate and female breast cancer in particular, both incidence and mortality are higher for black people . black patients also have lower participation in clinical trials, even when these are testing treatments for cancer types that are highly prevalent in their population. as a result, they are denied access to potentially life-extending therapies, and clinical findings become skewed toward a non-representative, white majority. race is a social construct that does not bear a clear relationship to genetics and biology. however, the lack of a sampling diversity that corresponds to the real-world population remains a pervasive concern in research, as it can obscure links with disease traits and therapy response and thereby reinforce health disparities , as highlighted in the field of human genomics . the lack of appropriate representation is also reflected in the composition of the medical workforce, with only % of physicians and . % of oncologists self-identifying as black or african american, despite the fact that black americans make up . % of the us population. similarly, minority ethnic or racial groups reportedly comprise only - % of biomedical research faculty in the usa , even though they are better represented at the doctoral and postdoctoral levels. the causes of these disparities are complex and include historical and structural racism, implicit racial and social biases, entrenched economic, educational and healthcare inequities, and the cultural and behavioral trends of communities and individuals. when considering these latter behavioral factors, it is important to acknowledge the mistrust of many african americans toward the medical system in light of the exploitation and discrimination to which they have been subjected historically. the notorious tuskegee syphilis study is one such example, as is the case of henrietta lacks in the cancer fielda story that is as much about the general lack of bioethical standards at the time as it is about suffering and dying of cancer in the racially segregated us society of the s. the scientific community has been working toward understanding and addressing these issues. in the usa, the national institutes of health (nih) have a long-held policy on the inclusion of minorities in clinical research. the more recent launch of the nih-driven all of us research program also aims to engage participants from traditionally underrepresented groups so that contributed health data will be representative of the diversity in the us population . among other efforts by the american association for cancer research, the by initiative aims to address cancer disparities in the african-american population by collecting genomic and clinical data from african-american patients with cancer. the us national cancer institute's center to reduce cancer health disparities is dedicated to decreasing the disproportionate cancer burden in society through research, training, education and mentoring efforts. the american society of clinical oncology has announced a strategic plan to increase racial and ethnic diversity in the oncology workforce. on a political level, policies such as the affordable care act have enhanced healthcare equity by expanding coverage and access to medical care to disadvantaged groups, including black people . affirmative changes in policy could in fact drive rapid and meaningful change. it has been estimated that % of the cancer deaths in in the usa could have been avoided had these patients had access to and quality of health care and treatment similar to that of college-educated people . such efforts are important, but they are not yet enough. from access to diagnosis, treatment and care, to population representation in the patient cohorts that inform research findings and drive clinical discovery, racial and ethnic minorities remain disadvantaged around the world. the covid- pandemic has shone new light on these health disparities. although race and ethnographic data continue to be limited, emerging analyses show that in the usa, minority communities, including african americans and latino americans, bear an unequal burden when it comes to infection and mortality rates . the underlying reasons await detailed study, but the fact that historical and systemic inequality and discrimination have been affecting these communities in terms of financial means, access to healthcare insurance and diagnostic and treatment centers, quality and security of housing, and their ability to avoid virus exposure through work, cannot be denied. against this backdrop of health inequity, rooted in large part in racial and social exclusion and discrimination, came the recent killing of george floyd, an african american man, at the hands of police. this was not an isolated or unprecedented incident but rather one event in a long string of injustice and brutality due to racial discrimination in the usa. at the time of writing, on the day of george floyd's minneapolis memorial service, the words of the poet paul laurence dunbar come to mind: "a pain still throbs in the old, old scars / and they pulse again with a keener sting. " systematic and institutionalized racism continues to ripple through society, and its corrosive effects cannot be ignored. inequality and discrimination have many incarnations, but one of their common drivers is the neutrality of a majority, the indifference and passive acceptance that perpetuates injustice. the street protests that have swept through the usa during the past ten days have reignited a much-needed public dialog about racism. biomedical science has an important part to play in this discussion. as nations reopen their economies and the research enterprise gears up again, we should not seek to restart, picking up from where we left off and returning to our own definition of normality. rather, we should redouble efforts to alter the conditions that permit inequity to persist in academia and the industry, in healthcare systems and clinical practice, and in science communication and publishing. how do we catalyze this change? how do we transform the heartfelt expressions of solidarity into something more tangible than words and more meaningful than symbolic gestures of inclusivity? a first, essential step is to give the voices that are being drowned out the space, attention and respect they are due. we need to listen, learn and reflect. moreover, beyond denouncing the overt, poisonous hate that sustains racism, we need to talk about the latent intolerance, the implicit biases, the passive neutrality and the entrenched privilege that permit racism to persist. they are more difficult to discern and therefore are the hardest to uproot. recognizing and addressing their presence in our daily lives and the social and professional structures in which we operate is essential. it may also be uncomfortable, but that is a good thing. change does not come from a place of comfort. we must remember and act on this long after the street protests end and the news cycle moves on. nature cancer stands with the black community and minority and underrepresented groups against discrimination and intolerance. we are committed to supporting work on health disparities and diversity and highlighting these issues through our pages. we pledge to increase diversity in our reviewer pool and to amplify the voices of underrepresented minority authors. finally, we promise to continue educating ourselves, so that we may contribute to the efforts to level inequalities in a meaningful manner. to that end, we welcome the comments and ideas of our readers at cancer@nature.com. ❐ published: xx xx xxxx https://doi.org/ . /s - - -x seer cancer statistics review the all of us research program investigators key: cord- -yfaji cv authors: kim, yong-kyun; sohn, hong-gyoo title: disaster theory date: - - journal: disaster risk management in the republic of korea doi: . / - - - - _ sha: doc_id: cord_uid: yfaji cv to find a conclusive definition for contemporary purposes and uses, we look at many of the various definitions of disasters through cataclysmic events, historical records, public policies, laws, and organizational usage. our natural progression leads us to modern theories of disaster and disaster risk management (drm) that have had to tackle new types of disasters that are being brought about by the interconnectivity of societies, people, diseases, technology, etc., increasing in magnitude and complexity like what was seen in fukushima, japan, in and on-setting disasters like climate change. after looking at all the historical evidence, we come to a definition for the term disaster for modern usage and what it means for policy implications. risk as "the probability of harmful consequences, or expected loss (of lives, people injured, property, livelihoods, economic activity disrupted or environmental damaged) resulting from interactions between natural or human induced hazards and vulnerable conditions" (undp ) . in the national infrastructure protection plan (nipp ), department of homeland security (dhs) considered risk elements as "threat nature and magnitude, vulnerability to a threat, and consequence that could result." haddow et al. ( ) noted that risk is composed of ( ) the probability and frequency of a hazard occurring, ( ) the level of exposure of people and property to the hazard, and ( ) the effects or costs, both direct and indirect, of this exposure. the korean word for risk is "wiheom" or "wiheomdo." finally, the origin of the word "crisis" is the greek word "krinein (separate; critical moment);" and it has been used as the medical term of "decisive moment of life and death." quarantelli ( ) viewed that a crisis of a certain organization appears in the three following interrelated conditions: ( ) a type of threat including organizational value, ( ) sudden occurrence of an unexpected event, and ( ) need to respond collectively as the outcome may seem more negative otherwise. the term "crisis" is defined as "a difficult or dangerous situation that needs serious attention" in the merriam-webster's learner's dictionary (webster dictionary ) . the korean word for crisis is "wigi." the term indicates a dangerous crucial moment or time and the state to make the final decision on a critical matter. the term "wigi" is a combination of the two characters: "wi" meaning danger and "gi" meaning chance. in the same context, lee et al. ( ) defined crisis as an "incomplete state that desperately needs a significant change or the turning point to determine whether to proceed with, modify, or end an event or a behavioral process." disaster means a condition where the damage cannot be overcome without national or external assistance due to lack of capacity or resources of the damaged community or local/state governments uniqueness korea a presidential decree regulates the magnitude of disaster, which can be financially supported by the national government usa disaster is classified into emergency and major disaster declaration depending on the magnitude of the damage japan disaster caused by natural hazard was historically emphasized in act unisdr a comprehensive approach considering human, physical, economic, or environmental losses and impacts is emphasized fritz considers disaster as the disruption of the essential functions of the social system quarantelli considers disaster as what is caused by natural or technological hazard that is beyond the knowledge of modern society jeong ( ) saw the word "crisis" as natural disasters such as typhoons, heavy snowfalls, and floods; human and technical disasters such as explosion, traffic accidents, and collapse; comprehensive risk situations in various contexts such as terrorist attacks, status of diplomatic relations with north korea, failure of various policies, financial difficulties of firms, stores, and state, crimes, spread of diseases, and various scandals. the terms related to disaster can be summarized as table . . summarizing the discussions above, "disaster" can be conceptualized as follows: • a state that cannot be recovered to the original state with the ability of the community due to great damages in human life and property • inclusive of disasters triggered by natural, technological, or social hazard the term "disaster" will be used embracing natural, technological, and social disasters that the whole community need to cope together reflecting the characteristics of contemporary societies. therefore, in this book, we will define "disaster" as "a status of community or nation's being seriously damaged by natural, technological or social cause and difficult to recover from the damage with its own resources, requiring the whole community to cope together." this definition is similar to the definition of disaster by unisdr and consistent with the definition by laws in korea, japan, and the usa. originates from the old french word "sauf" meaning "not damaged or not harmed," or the latin word "salvus" meaning "not damaged or healthy" the condition of being safe from undergoing or causing hurt, injury anbo security originates from se (¼without, liberation) + cura (¼care, concern, or distress) the state of being protected or safe from harm wiheom (do) originates from the spanish word "risque," known to be used from the mid-seventeenth century a probability of harm that encompasses threat/hazard, vulnerability, and consequence wigi crisis originates from the greek word "krinein" (separate) dangerous crucial moment or time in this context, referring to "jaehae" as natural disaster and "jaenan" as human-caused disaster is not appropriate, which had been commonly used in korea from to ; it is still partially used. both "jaehae" and "jaenan" should be used as terms pointing out natural, technological, and social disasters. to be more precise, "jaehae" means the damage caused by hazards, and "jaenan" means the situation that creates or is likely to create damage. like the concept of disaster, its categorization is also very diverse. the typical one is a categorization by disaster cause. the other ways include categorization by the characteristics of the event, phase-related categorization, and categorization by surgical and medical disaster. in korea, the current framework act on the management of disasters and safety classifies disasters into natural and social disaster. its original version enacted in classified disasters into natural, human-caused, and social disasters, but the same act when it was amended in integrated "human-caused disaster" and "social disaster" into "social disaster." thus, disaster in the current korean law is divided into "natural disaster" and "social disaster." the categorization by disaster cause is a typical way about disaster type. most of renowned organizations dealing with disaster data, such as center for research on the epidemiology of disasters (cred) and munich re, and many scholars have used disaster cause as the principle of disaster categorization. the official categorization of disasters in korea is "natural disaster" and "social disaster" regulated by disaster-related laws, which are differentiated by causing phenomenon. the countermeasures against natural disasters act, formerly the disaster control act and the disasters and safety act, defined disaster-related terms as below: • countermeasures against natural disasters act (act no. , amended on march , ) the purpose of this act is to prescribe necessary matters concerning natural disaster prevention or recovery and other countermeasures against natural disasters, in an effort to preserve national land and to protect lives, bodies, and properties of nationals as well as key infrastructures from disasters caused by natural phenomena, such as typhoons, floods, etc. this act defined "jaehae" as any damage caused by "jaenan" and encapsulated "jayeon-jaehae," meaning natural disaster and "pungsu-hae," meaning disaster triggered by hydrological hazard as specific examples of "jaehae." article (definitions) the definitions of the terms used in this act shall be as follows: . the term "jaehae" means any damage caused by "jaenan" under subparagraph of article of the disasters and safety act (hereinafter referred to as the "framework act"); . the term "jayeon-jaehae" means any disaster caused by typhoon, flood, heavy rain, strong wind, wind wave, sea wave, tidal water, heavy snowfall, lightning, drought, earthquake (including any earthquake-caused tsunami), yellow dust or other natural phenomena corresponding thereto, from among disasters falling under subparagraph ; . the term "pungsu-hae" means any disaster caused by typhoon, flood, heavy rainfall, strong wind, wind wave, sea wave, tidal water, heavy snowfall or other natural phenomena corresponding thereto. • disaster control act (enacted on july , , act no. and abolished on june , ) this act, abolished when the disasters and safety act was established on march , , states that the purpose of the act is to establish a disaster and safety control system of the state and local governments, establish a system for prevention of and countermeasures against disasters as well as emergency rescue, declare a special disaster area, and define matters such as emergency measures necessary for disaster management in order to protect the lives and property of citizens from disasters due to artificial causes. this act was enacted to prepare for human-caused disasters, such as arson and building collapse, rather than natural disasters, such as drought and floods. thus, it targets technological disasters, such as explosions, collapse of bridges, traffic accidents, and chemical, biological, and radioactive accidents, like the leakage of hazardous material and environmental pollution incidents. • framework act on the management of disasters and safety (enacted on march , , act no. ) the framework act on the management of disasters and safety that took effect in categorized disaster into natural, human, and social disaster and defined them as follows: article (definitions) the terms used in this act shall be defined as follows: . the term "disaster" means any of the followings, which actually causes or is likely to cause any harm to the lives, physical safety and property of citizens and the state: (a) disasters caused by a typhoon, flood, downpour, strong wind, wind and waves, tidal wave, heavy snowfall, lightning, drought, earthquake, sandy dust, red tide, ebb and flow and other natural phenomena equivalent thereto; (b) damage beyond the scale prescribed by presidential decree, such as a fire, collapse, explosion, traffic accidents, chemical, biological and radioactive accidents, and environmental pollution incidents and other accidents similar thereto; (c) damage caused by the paralyzation of the state's backbone systems, such as energy, communications, transportation, finance, medical treatment and water supply, and by a spread, etc. of infectious diseases. . the term "overseas disaster" means those which actually cause or are likely to cause any harm to the lives, physical safety and property of citizens of the republic of korea outside the territory of the republic of korea, and which require the government to take measures thereon. • framework act on the management of disasters and safety (partially amended on august , , act no. ) types of disasters were categorized into natural and social disasters in the disasters and safety act partially amended on august , , defining them as follows. the korean government classifies standardized disaster types on the standard crisis management manual as shown in table . according to the statutory classification of disasters summarized above. article (definitions) the terms used in this act shall be defined as follows. . the term "disaster" means any of the followings which actually causes or is likely to cause any harm to the lives, physical safety and property of citizens and the state. (a) natural disasters: disasters caused by a typhoon, flood, downpour (the korean word "howoo"), strong wind, wind and waves, tidal wave (the korean word "hae-il"), heavy snowfall, lightning, drought, earthquake, sandy dust (the korean word "hwangsa"), red tide (the korean word "jeokjo") outbreak, ebb and flow (the korean word "josu"), and other natural phenomena equivalent thereto (b) social disasters: damage beyond the scale prescribed by presidential decree, such as a fire, collapse, explosion, traffic accidents, chemical, biological and radioactive accidents, and environmental pollution incidents and other accidents similar thereto, and damage caused by the paralyzation of the state's critical systems, such as energy, communications, transportation, finance, medical treatment and water supply, and by a spread, etc. of infectious diseases under the infectious disease control and prevention act, and contagious animal diseases under the act on the prevention of contagious animal diseases enforcement decree article (scope of disasters) "damage beyond the scale prescribed by presidential decree" in subparagraph (b) of article of the disasters and safety act (hereinafter referred to as the "act") means the following harm: . harm to human life or property for which measures at the level of state or local government are required; . other harm deemed by the administrator of the national emergency management agency to be necessary for disaster control, and which is equivalent to the harm referred to in subparagraph damage on cultured marine products and fishery production facilities due to a discoloration of seawater to red or light brown caused by an explosive growth in phytoplankton density ※ article of the act on the prevention of and countermeasures against agricultural and fishery disasters 「methods regarding the monitoring, prediction of red tide, and damage prevention」(directive of ministry of maritime affairs and fisheries) drought a prolonged shortage of water supply in a certain region due to a period of below-average precipitation or insufficient available water resources: it can be categorized as a meteorological drought, a hydrological drought, an agricultural drought, or a socioeconomic drought depending on its usage tidal wave damage by level rise due to the effects of tides, weather tidal waves, swells, and abnormal waves in costal or estuarine waters social disaster forest fire trees, weeds, fallen leaves, etc. in a forest or in an area adjoining to a forest are burned by a fire artificially or naturally ignited ※ article of the forest protection act chemical accident all situations that occur because a chemical flows out or leaks out to humans or the environment due to the fault of a worker as at the time he/she works, such as replacement of facilities, defects in facilities or deterioration of facilities, a natural disaster, a transport accident, etc. ※ article of the chemicals control act (continued) disaster type definition water pollution accident that oil, toxic substance, or sewage or wastewater is discharged to river, causing continuous large-scale suspension of water intake or perish of fishes and significant effects on the people's living and natural ecosystem large-scale marine pollution accident accident caused by the crack of oil tank due to sinking, stranding, or collision of a ship resulting in a significant and extensive damage to the people's living and natural ecosystem and requiring comprehensive measures at the government level utility-pipe conduit accident occurred on the facility installed underground for smooth maintenance, aesthetic improvement, preservation of road structure, and smooth traffic flow by accommodating underground facilities (electricity, gas, waterworks supply facilities, communication facilities, drainage system, etc.) commonly collapse of dam accident of leak or overflow occurred due to natural disaster such as large-scale flood or earthquake or structural defect of dam, concerning the collapse of dam large-scale subway accident accident occurred due to the collision, derailment, fire, explosion or flooding of electric train in operation, causing casualty or expecting the suspension of train operation for a long period of time large-scale high-speed railroad accident accident occurred due to the collision, derailment, fire, or explosion of high-speed train in operation, causing casualty or expecting the suspension of train operation for a long period of time large-scale fire at multiuse facility accident at a facility used by the many and unspecified general public concerning which has a high concern of large casualty and property damage in case of large-scale fire radiation exposure from a neighboring country situation requiring national protection measures due to a large-scale radiation exposure or radiation contamination exposure accident from a nuclear facility of neighboring country marine vessel accident accident occurred due to the collision, fire, explosion, stranding, sinking of vessel, loss of loaded cargo, or other hull damage large-scale casualty accident at workplace accident of large-scale casualty such as physical explosion related to tasks at the workplace, largescale collapse accident at construction site, choking accidents due to oxygen deficiency, acute poisoning accident due to exposure to chemicals, or other equivalent accidents multiuse facility large building collapse accident accident at a facility used by the many and unspecified general public concerning large casualty and property damage in case of collapse of building (continued) paralysis of essential national financial information function due to natural disaster, strike, terrorism, and electronic infringement on the financial information system which is the key national infrastructure safety of nuclear facility disaster caused by radiation leak or radioactive contamination from a nuclear power plant and research reactor facility electric utility serious impediment to national life and paralysis of national functions due to anxiety about the electricity demand and supply caused by increased demand, defected facilities, and social conflicts demand and supply of oil serious hazard to the national safety and the stability of national economy due to imbalance between demand and supply of oil caused by foreign oil producing countries health and medical services serious hazard to national health due to the paralysis of healthcare system drinking water national crisis situation such as suspension of drinking and industrial water supply due to water pollution, destruction of facility due to natural and artificial disasters, and strike and cyber terror cargo transport situation which may cause or lead a significant crisis in the national logistics system due to refusal of (continued) by cred and munich re the cred is a research institute founded in by lechat, a professor in belgium and has been studying international disasters and disputes for over years. it has been a world health organization (who) collaborating center since and is operating an international disaster database called emergency events database (em-dat). the disaster classification system of em-dat is in four levels: generic group, subgroup, main type, and subtype. generic group, the highest level of the system, classifies disaster into natural and technological disasters. the natural disaster category is divided into six subgroups: biological, geophysical, climatological, hydrological, meteorological, and extraterrestrial disasters. each subgroup in turn covers disaster types and more than subtypes. more detailed information can be found in the em-dat report (cred ) . natcatservice provided by munich re group in germany is another database related to disasters that wins global recognition, using a natural disaster classification system similar to em-dat. natcatservice enters all natural disasters that cause human and physical damages regardless of scale, but it does not mention artificial/technological disasters (munich re ) . in , cred and munich re developed and presented a joint standard classification system for natural disasters (cred and munich re ). this can be considered the international standard classification system for natural disasters. this system classifies disasters into five levels, dividing disaster into two generic disaster groups of natural disasters and technological disasters. natural disasters are then divided into six disaster groups, geophysical, meteorological, hydrological, climatological, biological, and extraterrestrial, each of which is then classified into main type, subtype, and sub-subtype. however, this system is limited in that it cannot present a detailed classification system for artificial/technological disasters, as munich re does not have the classification for such disasters. threat and hazard identification and risk assessment the threat and hazard identification and risk assessment (thira) is a four-step common risk assessment process that has been designed by the dhs in the usa. this process helps the whole community identify risks, and enables the community to build local resilience to the risks. according to thira, threats and hazards are divided into three disaster type definition people in cargo truck transport business to cargo transport as a group without justifiable grounds resulting in suspension or setbacks of cargo transport gps signal interference service interruption of national infrastructure due to intentional or unintentional interruption of gps signal reception for national core infrastructure and service using gps cosmic radio wave disaster related to radio wave occurred due to change in electromagnetic energy existing outside of the earth's atmosphere types as listed in table . . the natural hazards are the types that result from acts of nature, such as flood, earthquakes, drought, pandemics, epidemics, or more. the technological hazards are the types that result from the accidents or the failures of systems and structures, such as transportation accidents, collapse, hazardous materials spills, or more. the human-caused incidents are the types that result from the intentional actions of an adversary, such as terrorism, sabotage, cyber incident, chemical attack, biological attack, or more. classification of disasters by their cause has been useful for deciding governmental actions toward disaster recovery because it clearly indicates the responsibility of who will pay for what, e.g., supporting disaster victims and/or providing disaster relief. however, the categorization is not useful for disaster response because the response way is not different depending on its cause. therefore, a new prism is needed. kim et al. ( ) have analyzed the occurrence of large-scale disasters in korea since and the government's response to them and have proposed new optics for classifying disaster types for advancing disaster responses. to this end, two important elements were considered: the evolutionary or devolutionary pattern of the magnitude of disaster damage and the roles and responsibilities of responding agencies. regarding the first element of the pattern of disaster damage, three types can be derived: ( ) events with measurable prediction of large-scale damage, ( ) events with unpredicted high social shocks with quick evolution and devolution independent of its magnitude, and ( ) events slowly evolving to large scale. large-scale damage caused by typhoons, torrential rain, and heavy snowfall can be predicted according to weather forecasts and precedence. train crashes and building collapses are types of disasters that suddenly evolve and devolve, leaving not only physical damage but also a shock to the social fabric. finally, foot-andmouth disease, infectious disease, and red tide are disasters that commence on a small scale, or with little notice, but over time become considerable disasters that require large-scale disaster response tactics. the reason why this classification is important is that the response system, such as the timing of the operation of the central disaster and safety countermeasure headquarters (cdschq) and the central disaster management headquarters (cdmhq), is different depending on the progress or evolution of the damage. secondly, the scope of the overall coordination that cdschq should undertake varies depending on whether there are one or more disaster management authorities. the role of the coordinating authority becomes important for typhoons, torrential rain, and heavy snowfall, since they are types of disasters that simultaneously cause damage to various facilities, which will result in various agencies, such as the ministry of construction, the ministry of agriculture, the ministry of environment, etc., engaging the disaster together. for effective response to these types of disasters, it is important for the coordinating authority to support the human and material resources necessary for the various disaster responses by the disaster management agencies. on the other hand, for the disasters such as aircraft accidents, infectious diseases, and dust storms, expertise and experience are concentrated in specialized disaster management authorities, meaning it is efficient for the agencies responsible for those disasters to respond. the category by kim et al. ( ) is very useful in developing an optimum disaster response system fitting the disaster cause and evolutionary path. in this book, we will propose the disaster category for effective disaster response based on the category by kim et al. ( ) as outlined in table . . dombrowsky ( ) suggested event-related concept and phase-related concept as disaster categorization. the event-related concept is composed of time, space, and severity, while the phase-related concept is composed of pre-emergency phase, emergency phase, warning, and post-emergency phase. methods of classifying disasters include one in terms of emergency and medicine. in the field of emergency and medicine, disaster is classified into surgical and medical disasters. the purpose of this categorization is to treat efficiently patients during disasters by securing effectiveness of first aid on-site and hospital treatment. surgical disasters are mostly those in which victims are injured, and they refer to disasters in which damage from physical disasters appears in the form of injury. medical disasters or disease disasters refer to chemical disasters that cause hindrance to respiratory organs and metabolic systems due to the leakage of chemicals, radioactive matters, or toxic agents. this classification method has significance in that it is possible to predict the conditions of the patients in disasters and effectively manage medical resources accordingly (kim and lim ) . in case of instant disaster with single primary response agency, such as dam failures, major rail accidents, and maritime accidents, the cdschq and the cdmhq are established simultaneously; the cdmhq takes charge of response, and the cdschq coordinates the pan-governmental support primary response agency takes charge of its own responsibility in the early stage of the event. as the damage evolves, the charge is transferred to the cdschq. this type of event includes infection, animal disease, red tide, and green tide contemporary society is becoming vulnerable to new types of disasters, such as new epidemics including ebola virus and middle east respiratory syndrome coronavirus (mers-cov), new animal diseases including bovine spongiform encephalopathy (bse) and hpai, and complex disasters such as the tohoku earthquake and tsunami in along with typical types of disasters like typhoon, fire, and building collapse. in addition, the characteristics of complexity in modern times require new and innovative approaches that are different from current response ways. there has recently been much research in various fields, such as sociology and science of public administration, in order to understand what increases disaster complexity in modern times. this section will review representative disaster theories and find how these theories explain major disasters that occurred in korea and around the world. based on this understanding, we will recommend the desirable policy change for effective disaster response in korea. representative theories, such as heinrich's law, normal accident, risk society, and complexity theory, will be reviewed in the following section. herbert william heinrich, who was an assistant superintendent at an american insurance company, presented an important study in based on his analyses of a wide range of accidents. he coined the notion of heinrich's law, which states that, for every major accident, there have been preceding minor accidents and signs of anomaly (heinrich ). heinrich's law is therefore also known as the law of : : . in other words, an accident that is large enough for people to notice is only a small tip of an iceberg and is always antedated by countless other accidents and happenings that warn of the upcoming disaster. through a scientific statistical approach on industrial disasters that were once believed to take place unexpectedly, heinrich found that massive disasters occurred due to negligence on minor defections. originally, the heinrich's law was applied to industrial disasters, but it is now extended to rules related to accidents, disasters, and failures throughout all kinds of areas in contemporary society. the : : rule is meaningful in that it sets up a rule to explain quantitatively the process of an evolutionary incident, starting from a small accident and resulting in a catastrophic event. he also applied the domino theory to disaster areas and indicated that in order for disasters to take place, an inappropriate "direction of flow" occurs sequentially: many causes interact with complexity, and those causes generate interwoven influences. as a result, phenomenon called disaster or accident takes place, and ultimately it causes human and physical disasters, which is a major argument of the domino theory. the theory emphasizes fundamental elements before accidents to take place and explains there are three potential elements as follows: the first condition -human genetic component or socially undesirable phenomena the second condition -flaws caused by the first condition the third condition -unsafe actions, mechanical and physical, according to the second condition among the three conditions that cause a disaster, the first condition of inherited elements or social environment and the second condition of inherited or acquired human defects are hard to be rectified; however, the risk by the third condition can be significantly reduced through safety education and strengthened safety devices. in other words, heinrich insisted that if the third condition is effectively eliminated, disaster can be prevented before it occurs (heinrich ) . the sampoong department store collapse accident in korea is a typical example of the heinrich's law. the accident happened because no countermeasures had been taken even though there were many signals before the accident. the department store opened its business with inborn structural problems during the construction process: about tons of installation equipment was installed on the rooftop, which was four times more than its original design load, and reinforcing bars were out of place. along with problematic construction, poor management was another cause of the accident: minor symptoms such as cracks in the ceilings and damage to the floor of the rooftop (potential elements of ) were overlooked by staff and maintenance crews. in addition, although customers and employees raised several concerns about the general health of the building, such as vibrating sound from an air conditioner and many cracks in the wall, no specific actions were taken, even after receiving evaluations from experts ( small accidents). neglecting potential elements eventually led to one huge accident with casualties (one massive accident) (lee et al. ) . we found that most of the major social disasters, such as the seongsu bridge collapse, the sampoong department store collapse, the daegu subway fire, and the sewol ferry sinking accident, have common things to cause the disasters: improper alteration of use, insufficient safety culture, insufficient safety inspection, and insufficient safety infrastructure. heinrich's law indicates the direction of disaster response that korea should follow: institutional reforms for strengthening disaster and safety management, improvement of safety inspection, expansion of safety education, and enhancement of disaster and safety infrastructure. in the book normal accidents, charles perrow, a professor of sociology at yale university, insisted that complex systems, such as nuclear power plants, chemical factories, aircrafts, ships, dams, and gene manipulation, hold a risk of tragedy. thus, there are some unavoidable accidents called normal accidents that have a high probability of occurring regardless of safety measures and devices (perrow ) . charles perrow proposed the normal accident theory based on the empirical evidence of the three mile island (tmi) nuclear power plant accident that took place in pennsylvania in , the first nuclear power plant crisis to have occurred. perrow ( ) defines a normal accident, which he also refers to as a system accident, as "an event that involves the unanticipated interaction of multiple failures, reflecting the characteristics of high-risk system in which multiple and unexpected interactions of failures are inevitable." perrow's theory insists that accidents in today's cutting-edge technological societies are closely related with complex technological and mechanical structures with built-in risks and those risks are therefore a normal part of our lives. an extremely complex system, in which individual technologies interact with one another closely and inseparably through an endless loop, is prone to catastrophe caused by a series of breakdowns whenever one of the interwoven elements begins to dysfunction. it is nearly impossible for humans to predict such technical failures (park ) . perrow noted that conflicts of interests might exist between a given organization and its members and that organizations are also subject to external political and social environments. technical solutions are therefore not enough, particularly as it is impossible to ensure the total control and containment of risks in such highly complex private facilities as nuclear power plants and petrochemical factories, at which minor errors can lead to complete failures (perrow ). the three mile island accident is considered as the typical example of normal accident. this brought widespread panic to the usa as large portions of one of the reactor's cores partially melted, releasing radioactive gases and hazardous iodine into the surrounding environment. the direct cause of the accident was officially recorded as a mistake by operational staff; however, the complexity of the disaster is a hidden root cause of the catastrophic event. therefore, as perrow indicated, complex systems with built-in intensive risk, such as nuclear power plants, require that centralized and decentralized management should be considered simultaneously. there have been a lot of industrial accidents around the world. accidents, such as the india bhopal chemical factory accident, the chernobyl nuclear power plant radiation leak, and the bp gulf oil spill, indicate that industrial safety measures need constant improvement through comprehensive understanding about high-risk technology and systems. some scholars criticized that the normal accident theory overemphasizes the vulnerability of high-risk facilities; the high reliability theory is a typical example of the critics. professors from uc berkeley and weick, an organizational theorist, proposed the high reliability theory based on research of organizations fraught with disaster risk, such as national aeronautics and space administration (nasa), the nuclear industry, aviation, nuclear carriers, swat, and massive petrochemical facilities in the usa. the high reliability theorists insist that sophisticated quality controls, a settled safety culture, built-in safety by cross-check, and continuous education and training can prevent disasters at high-risk facilities. irrespective of these critics, perrow's normal accident theory provides a beneficial lens through which we may view and analyze risks in contemporary society. because the theory explains risks as a matter of organizational characteristics of the ecosystem, it overcomes the shortcomings of the science and technology-centered approach. it provides sociological explanations for organizational risks that are likely to cause massive collateral damage, such as in petrochemical factories and nuclear power plants (jeong ). after the earthquake that occurred on september , in korea, social interest in the safety of high-risk facilities such as nuclear power plants and old industrial complexes has increased. in accordance with etkin ( ) , who noted that careful thought should be given to the construction of complex tightly coupled systems, the korean government should entirely overhaul the safety of risky facilities such as nuclear power plants, industrial complexes, and chemical plants. moreover, there is a need for society-wide attention and efforts to provide a more sophisticated safeguard system. around the s, an in-depth exploration on new risks that occurred in modern society was conducted by european scholars, such as luhmann, giddens, and beck (lee ) . ulrich beck, the german sociologist, suggested the concept of risk society as a solution to structural and deep-rooted problems of industrial societies, including science and technical safety issues, which started in the mid- s (lee et al. ) . in his book world risk society ( ), ulrich beck argued that the modern society is a "risk society" replete with risks all throughout and emphasized the multiplication of cross border risks and international dangers that single nationstates cannot tackle on their own (beck ) . during his lecture at seoul national university in , beck pointed out the similarities between the asian financial crisis in the s and the chernobyl nuclear plant accident in , stressing the importance that all nations must work together to reduce the global risk on the basis of a common understanding that they are facing the same global risk. beck also considers this emerging risk as a result of the de-bounding of traditional national boundaries in the spatial, temporal, and social dimensions. beck defined risk society as a society where socially produced risk is inherently accompanied by socially produced wealth (oh ) . he also insisted that industrial society should be addressed through a comprehensive perspective, which includes social, historical, and technological views (beck ). as the world entered into a contemporary society, new types of risks, combined with secondary, supernatural, and artificial uncertainty, had arisen, and those risks were beyond a dimension of traditional response methods (lee ) . the background with such phenomena includes a complex structural change that is understood as the term of postmodernity. generally, risk, unlike danger that indicates direct and physical loss, is based on the probability of prediction or control: a new concept of "uncontrollable risk" needs urgent attention because this type of risk denies the applicability of currently available risk theories and risk control mechanisms, arising new threats (beck ). the concept of risk society by beck has a critical meaning to korea's drm. the increased risk can be also found in urbanization in korea. lee et al. ( ) emphasized the increased risk due to urbanization by showing the increased disaster risk following urban development with the change of green space to paved road. korea has also suffered from newly emerging disaster risks, such as severe acute respiratory syndrome (sars), foot-and-mouth disease, and mers-cov. therefore, an innovative and cooperative approach to disaster risk suggested by beck needs to be reflected in designing resilient future. complexity theory began from researching complex natural phenomena such as meteorology. just as lorenz proved with the butterfly effect, the contemporary society embedded with complex network needs to take different countermeasures against disaster. the fukushima nuclear power plant accident of japan, a great flood in thailand, and new types of epidemics such as mers-cov are representative examples of disasters that the contemporary society is newly facing. the complexity theory is useful for understanding the characteristics of contemporary disasters and finding appropriate countermeasures. on march , , the fourth strongest earthquake struck japan, since japan started to observe earthquakes, with a mega tsunami and aftershock that caused more than , deaths. also, the earthquake destroyed the nearby fukushima nuclear power plants and caused radiation leakage. this disaster showed signs of complex disaster. due to the fukushima accident, air, soil, ocean, and underground water were exposed to radioactivity, and damages from contamination influenced largely not only japan but also the whole world continuously. the severe flood in thailand is a typical example that a disaster that happened in one country affected the regional economy. due to a heavy rain, combined with low topographic characteristics of thailand and high tide of seawater, two thirds of the land in thailand, including parts of bangkok, was flooded, causing significant damage to the entire manufacturing industry. the flood did not affect just thailand. for example, many japanese companies' production facilities located in the central region, having developed the area for the manufacturing of automotive and electronic goods, were inundated during the flood. the intensity of the flooding caused all of the facilities to be shut down, which caused tremendous economic loss for both countries. additionally, the shutdown of the automotive plants caused a reduction of japanese auto and parts deliveries to other major markets like japan, the usa, and europe. in case of the electric and electronic industry, hard disks became in short supply due to flooding of hard disk manufacturing factories. due to this, the production of semiconductors by intel became inadequate, resulting in a short supply of semiconductors to the world's semiconductor market. moreover, it caused a price increase of computers worldwide. also, since the severe flood occurred right before the tourist season, the number of tourists in the fourth quarter decreased by approximately %, and additionally induced damages occurred in other industries, such as transportation and food services. due to the influence of the heavy flood, the estimated economic growth rate of thailand in was lowered from . % to . % (korea institute for international economic policy ). table . shows the summary of damage that occurred to the japanese economy due to the heavy flood in thailand. this table shows that the flood was not just problematic for thailand but indicates that a disaster occurring in one country does influence other countries. the contemporary society is facing new types of epidemics and animal diseases, such as sars ( ), h n virus ( ), foot-and-mouth disease ( ), and mers-cov ( ), which did not exist in the past, and such symptoms have a high possibility of becoming more frequent due to increase in trade and traveling with foreign countries. pelling ( ) argued that complexity theory possesses a very important lesson to understanding ways to cope with disasters. for example, one of the characteristics of complexity theory is "emergence," which shows a trait of disasters that take place with unexpected causes at an unexpected place. according to drabek and mcentire ( ) , "emergence" appears while people change organizations in the process of making temporary organizations and responding to disaster situations. beck's risk society and perrow's normal accident theory share the similar understanding with complexity theory in that the emerging risk in modern society is closely related with its increased complexity. therefore, the core elements of complexity theory, such as nonlinearity, self-similarity, fractal, self-organization, and emergence, are essential in understanding disaster characteristic and innovating disaster response methods in modern society. as disasters started to become complexed and intensified, scholars and stakeholders began to look for new theories and methods to improve disaster risk knowledge and organizational capabilities. starting in the s, several theories, such as petak's four-phase model, mcloughlin's comprehensive disaster management procedures, and the new public administration theory, were developed to deal with these new complex and intensified disasters and to help modernize and reform institutions that were becoming too antiquated to deal with modern disasters. organizations such as the usa's fema, created in the s, used the progressing knowledge and concepts to help it mature in the s, and the un and world stakeholders, who came together in the early s to produce the hyogo framework for action (hfa), used the foundations' insights to increase disaster resilience around the world. investigating the historical progress of such knowledge and the evolution of response organization is important in figuring out what logical steps should be made for a resilient future as well as in strengthening organizational capabilities to better deal with complex and intensified disasters. in , petak proposed a four-phase model to identify the role of governments and stakeholders in each disaster management phase. he divided pre-disaster management and post-disaster management according to the progress of disasters and countermeasures and explained disaster management procedures in a timesequential manner: . disaster mitigation . disaster preparedness . disaster response . disaster recovery petak emphasized that the clear delineation of roles and responsibilities of all levels of governments and stakeholders is essential for effective disaster management four main areas in japan reduced production by % nippon steel corporation adjusted release of the crude steel in japan due to effect of reduced automobile production honda suspended the operation of four-wheeled vehicle plant in malaysia toshiba had trouble in hard disk drive (hdd) production pioneer had trouble in the production of car navigation system, relocated from thailand to malaysia deteriorated business results expects billion yen decrease in sales automobile possibility of sales decrease of billion yen for five automobile companies suspended distribution suspended the transport based on flooded areas paid insurance tokio marine and fire insurance co., ltd. began investigation to provide insurance to japanese client companies (petak ) . petak's model influenced the basic structure of the framework act on the management of disasters and safety (hereafter "disasters and safety act") in korea, which is shown in fig. . . the new public administration theory emphasizes that a government can provide better public service to citizens by adopting business management principles to public management (rosenbloom and goldman ) . the theory aims to overcome the problems of a typical bureaucracy and to improve the efficiency of the public sector by adopting business management skills and by emphasizing output and outcome of public policy rather than the input to implement the policy (rosenbloom and goldman ) . the theory was accepted through the national performance review project during the clinton administration, and laid the foundation for fema's reformation (waugh and streib ) . the demand for the reformation of fema started from hurricane hugo, which hit north carolina and the virgin islands in , causing $ billion in damages. in the aftermath of hurricane hugo, the loma prieta earthquake struck california, a fire broke out in oakland, and hurricane andrew struck florida and louisiana, dealing a massive blow to the two states. disappointed by fema's ineffective response to these massive natural disasters, political groups and citizens in the usa raised the need to improve fema's performance, which was endorsed by the clinton administration in (anna et al. ). james lee witt, appointed as director of fema by president clinton, emphasized disaster mitigation and shifted from recovery-oriented policy to preventionoriented policy. additionally, he insisted that disaster mitigation investment should be based on the assessment of the effectiveness. consequently, government policy has continued to embrace this line of thinking. to quantify the future savings of hazard mitigation activities, the multihazard mitigation council (mmc) of the national institute of building sciences (nibs) analyzed three major hazard mitigation grant programs: the hazard mitigation grant program, project impact, and the flood mitigation assistance program. the independent study proposed the following significant findings: ( ) for every dollar spent on mitigation, four dollars was saved from future spending; and ( ) fema mitigation grants beget nonfederally funded mitigation activities (nibs ) . the new public administration theory affected the development of disaster management in korea: the increased investment to structural and nonstructural measures for disaster mitigation and the evaluation of the effectiveness of disaster mitigation and recovery projects in the s are typical examples of the effect of the theory. although the theory receives criticism in that it does not consider the characteristics that disaster management has as a public service, it can provide a useful prism to improve the effectiveness of disaster management policy by focusing on the outcome or performance of the policy, rather than the input. public governance has been acknowledged as a way to improve public-private partnership in disaster management. in the public governance theory, rules govern the behavior of actors not as the result of official authority or market equilibriums, but according to the consensual process among participants, based upon networks and cooperation. the concept of collaborative networks in disaster management entails the assumption that parties involved in disaster management have diverse cultural backgrounds and are bound to experience conflicts. effective cooperation in this setting requires cultural sensitivity and mutual understanding from all participants. collaborative networks, moreover, are becoming important because, in modern society with emerging and complex risks, it is not possible to ensure perfect preparations and resources for all possible disasters and because a specific organization or one single agency cannot perfectly control all response agencies involved in managing disasters (waugh and streib ) . the advantage of public governance theory is that it strengthens shared responsibility by enabling diverse social groups to participate in the decisionmaking process so as to tackle uncertainty with social intellect and make policy decisions based on the social consensus. the theory emphasizes cooperation, public participation, problem-solving, and openness as key principles of disaster management. it aims to form a collaborative risk governance system consisting of diverse actors, including governments, businesses, and civil society organizations, in which national government serves as facilitator in promoting the development of a decentralized collaborative network among local governments, nonprofit organizations, and various public services. however, there is an opinion that the public governance theory is hard to be applied to emergency response. in other words, in an urgent disaster response process where there is not enough time to make a consultation, the process of negotiation through mutual discussion and consultation is not appropriate (waugh and streib ) . the argument seems reasonable, but recent researches have shown that interagency cooperation is becoming more important in an emergency situation. moynihan pointed out that establishing network governance in advance are an essential element in achieving the two objectives of "interagency cooperation" and "coherent response" in a crisis situation for effective disaster response (moynihan ). public governance theory is also important in the development of the disaster response system in korea. the recent major disasters in korea, such as the mauna ocean resort gymnasium collapse accident, the sewol ferry sinking accident, and the middle east respiratory syndrome, taught korea important lessons that cooperation among all relevant organizations, such as on-site response agencies, local disaster and safety countermeasure headquarters (ldschq), various line ministries involved in the cdmhq, and the cdschq, is essential for effective disaster response and relief. therefore, the korean government should develop an interagency cooperation plan and execute a joint field training program; demanding the participation of all relevant organizations specified above. in late , more than federal organizations related to civil engineering and defense had fragmented responsibilities for hazard mitigation and disaster response, resulting in no coordinating organization taking the full responsibility for the entire phase of disaster management. president jimmy carter created fema in for this reason. the establishment of fema made it possible to unify the fragmented responsibilities of emergency preparedness and response resources (anna et al. ) . mcloughlin ( ) proposed an integrated emergency management model. considering disaster as an incident or condition that threatens the survival of organizations, he was concerned with the fact that troubles in cooperation among related agencies repeatedly occurred during disaster response since various public and private groups had been engaged in disaster response without comprehensive coordination. he argued that a comprehensive and integrated emergency management system could sustain administrative capabilities during emergencies and protect property and life through a series of circulation processes under the cooperation of the federal, state, and local governments. this model emphasizes that each local government and the federal government should cooperate to protect life, property, and government functions through a program of mitigation, preparedness, response, and recovery (cho ) . quarantelli ( ) also emphasized that comprehensive disaster management is essential in modern times in order to build a comprehensive and unified organization that manages all types of disasters. he argued that a dispersed or separated approach, by which each line ministry is responsible for its own disaster; devoid of a coordinating agency, cannot deal with complex and intensified disasters in modern times. additionally, he asserted that a comprehensive and integrated method be used when managing disasters because: first, disasters have become more complex and capable of destroying the functionality of typical community operations, meaning that dividing disasters into natural or human-caused ones is inadequate for effective response; second, there is an underlying commonality among governmental departments to respond to disasters; therefore, comprehensive measures are required for a unified effort; third, the planning process and its contents for disaster response by each department has commonality, thus, it is ineffective that each department develops its own response plan; finally, the shared response resources among the governmental departments reveals that a move to a comprehensive management format is feasible since vital resources are similar in each department (quarantelli ). global cooperation for effective disaster relief had been a pivotal issue among the international society. to improve the international cooperation for disaster relief to affected nations, the united nations established a responsible agency, labelled as "the united nations disaster relief organization (undro)," in . since then, the international community has been working for developing a systematic disaster management framework; disaster response plan, disaster prevention measures, and scientific and technical solutions to disaster risk have been discussed and adopted. in particular, technical approaches, such as vulnerability analysis for disasters and early warning measures, have been tried. additionally, the scientific approach played a key role in the evolution of disaster management through a variety of research on how to identify hazard and assess vulnerability (unisdr ). in , the international society began to prepare the international decade for natural disaster reduction framework to promote more systematic disaster management (unisdr ). the international community established the un humanitarian emergency assistance and the international search and rescue advisory group (insarag) mechanism in . also in , systematic mechanisms for disaster response were introduced by building the united nations disaster assessment and coordination (undac) mechanism. the international conference held in in japan adopted the yokohama strategy that emphasized the paradigm shift from recovery-oriented policies to prevention-oriented policies. in , the name of the international strategy for disaster reduction was adopted with the title "safer world in the st century: disaster and risk reduction." since then, a comprehensive policy framework to cope with increasing global risk has been developed in earnest (unisdr ). in december , a tsunami hit countries in the eastern-western-southern asian regions, such as indonesia, sri lanka, and india. in the wake of the catastrophic event, national governments, international organizations, un agencies, and other stakeholders agreed to adopt "the hyogo framework for action (hfa): building the resilience of nations and communities to disasters," to mitigate global disaster risk for - . with the hfa, disaster management moved toward building resilience based on social consensus and strengthening shared responsibilities among all stakeholders. in march , all national governments also agreed to the sendai framework for disaster risk reduction - (sfdrr), which is the first major agreement on disaster management in line with post- development agenda. the sfdrr aims for the significant reduction of disaster risk and losses with the following four priority actions: understanding disaster risk, strengthening disaster risk governance to manage disaster risk, increasing investment in disaster risk reduction for resilience, and enhancing disaster preparedness for effective response and to "build back better" in recovery, rehabilitation, and reconstruction. the drr-related global agenda in various fields will be further reviewed and analyzed in chap. in order to link them with disaster resilience in the future. in the s, massive disasters continued to occur throughout the world. national governments have reformed their disaster response system to deal with massive disasters. the usa reformed its drm system after / in , shifting from a typical natural and human-caused disaster management paradigm to comprehensive security (park and cho ) . within this context, since the s, civil defense and civil protection became important concepts in disaster management. civil defense was originally designed to protect citizens from calamities, such as war, but it was gradually expanded to encompass drm, reducing civilian casualties during disasters. alexander ( ) suggested that the role of the state and the civilian in disaster preparedness be shifted from civil defense to civil protection. this is because civil protection is an appropriate concept to reinforce the protection of people against external risks, meeting the need to protect people from typical disasters, such as natural and technological disasters, as well as emerging risks, such as new infectious diseases, terrorism, and cyber attacks, while civil defense was useful to cope with the invasion of an outside force which was a severe threat during the cold war era. one of the most important features of civil protection suggested by alexander is a shift from providing public safety service by the government to encouraging public participation. when a national government provides public safety services, it usually prefers to use a top-down approach: command and control, a hierarchical decision-making system excluding citizen's participation, strengthening law and order, and rules through the principle of confidentiality. however, the civil protection concept by alexander put stress on a bottom-up approach: emergency preparedness and response ensuring public participation and cooperation, problemsolving approach, and openness principles. as a result, risk governance is the most critical issue in civil protection, ensuring the cooperative interaction among the government, the private sector, and civil organizations to replace the existing hierarchical bureaucratic system. alexander ( ) addressed hurricane katrina as a typical failure case of civil defense with a top-down approach, requesting a shift from a top-down approach to a bottom-up approach, along with improved risk governance. this section will describe disaster response institutions in korea, the usa, japan, and other nations. the analysis of one nation's disaster response institution requires huge efforts and a large amount of work. due to limited time and space, this section will focus on the basic structure of disaster management organizations for normal times and emergency situations at national and local levels. the disaster response institutions in korea, the usa and japan will be analyzed first, and then those in australia, germany, switzerland, and the united kingdom (uk) will be analyzed. in korea, the ministry of public safety and security (mpss) is responsible for the overall coordination of the nationwide disaster response based on the disasters and safety act. after the presidential election on may , , the ministry of interior (moi) has been preparing for the revision of the government organization act, which includes the establishment of ministry of public administration and safety (mopas) by integrating mpss with moi and the establishment of korea fire service and korea coast guard as ind ependent agencies. the act is expected to be reviewed and decided by the national assembly in late . in addition, the crisis management center under the national security council in the blue house (presidnet's office) is expected to work as a control tower for national crisis, such as the sewol tragedy. the korean government has developed its disaster response system suitable for normal times and emergency situations at the national and the local levels. organizations for normal times are composed of disaster management agencies, disaster-management supervision agencies, emergency rescue agencies, emergency rescue, and relief support organizations. in addition, central, city/do, si/gun/gu committees are being operated to deliberate matters on disaster and safety management under their responsibilities. disaster management agencies take charge of all phases of disaster management activities that are related to their responsibilities. the agencies include national administrative agencies, local governments, local administrative agencies, public institutions and organizations, and other organizations prescribed by presidential decree. disaster-management supervision agencies are responsible for disaster response when a disaster or an accident occurs in accordance with the responsibilities prescribed by presidential decree. for example, the ministry of education is responsible for disasters in schools and school facilities, the ministry of environment is responsible for environmental pollution accidents, and the ministry of employment and labor is responsible for large-scale human accidents occurring in places of work. table . shows the disaster-management supervision agencies by disaster or accident type in korea. emergency rescue agencies are responsible for carrying out life rescue, first aid, and other necessary measures to protect the lives and property of citizens when a disaster is likely to occur or when a disaster occurs. the agencies in charge include fire hqs and coast guard hqs, under the mpss, city/do fire headquarters and si/gun/gu fire stations, and regional headquarters of the korea coast guard and coast guard stations. to support rescue activities, presidential decree prescribed emergency rescue and relief support agencies, which are equipped with human resources, installations, equipment, operation systems, etc., that are necessary for emergency rescue and relief service. to deliberate and decide crucial matters related to disaster and safety management at the national level, the central safety management committee, chaired by the prime minister, is operated. in addition, the safety policy coordination committee, chaired by the minister of public safety and security; the central disaster broadcasting consultative committee, chaired by a person appointed by the minister of the ministry of science, ict and future planning; the central private-public cooperative committee, chaired by the vice minister of public safety and security; and a civilian representative are being operated to advise, consult, deliberate, or sometimes decide disaster and safety management issues under their responsibilities. at regional and local levels, a city/do safety management committee, a si/gun/gu safety management committee, a city/do disaster broadcasting consultative committee, and a si/gun/gu disaster broadcasting consultative committee are operated. when disaster occurs or is likely to occur, emergency response organizations are established and operated to take timely and proper measures at national, regional, and local levels. the emergency response organizations at the national level include the cdschq, chaired by the mpss; the cdmhq, chaired by the head of the relevant disaster-management supervision agency; and the central emergency rescue control group (cercg), chaired by the head of the central fire headquarters for disaster occurring on land and the chief of the central rescue center for disaster at sea, respectively. the local emergency response organizations include the city/do disaster and safety countermeasure headquarters (city/do dschq), chaired by mayor or governor, and the si/gun-gu disaster and safety countermeasure headquarters (si/gun/gu dschq), chaired by the head of si/gun/gu. for rescue activities, the local emergency rescue control group (lercg), chaired by the head of the fire headquarters and a chief of a fire station, is operated; when disaster occurs at sea, the head of a si/gun/gu emergency rescue control group and the head of a city/do emergency rescue control group shall be respectively construed as the chief of a regional rescue center and the chief of a metropolitan rescue center under article of the rescue and aid at sea and in the river act. when disaster occurs or is likely to occur, the mpss will immediately hold a situational meeting to supervise the initial response, rescue, and first aid operations. in particular, in the event of a major disaster, the ministry will operate the cdschq and coordinate the intergovernmental disaster response activities. in case of an oversea disasters, the minister of foreign affairs shall exercise the authority of the head of the central countermeasure headquarters, and in cases of radioactive disasters, the chairperson of the nuclear safety and security commission shall exercise the authority of the head of the central countermeasure headquarters, respectively. in case government-wide integrated response is necessary, the prime minister may exercise the authority of the central countermeasure headquarters. in such cases, the minister of public safety and security, the minister of foreign affairs (limited to cases of overseas disasters), or the chairperson of the nuclear safety and security commission (limited to cases of radioactive disasters) shall be the vice head. when a disaster occurs in a jurisdiction, the mayor or provincial governor and the head of a si/gun/gu shall set up their own dschq and coordinate the response and recovery operations. in order to efficiently operate the cdmhq under article - ( ) of the disasters and safety act, the head of the disaster-management supervision agency shall predetermine necessary matters for organizing, operating, etc. of the cdmhq and exercise the authority of the head of the headquarters. also, local disaster management headquarters shall be established to work as an action team of cdmhq in the disaster area. the disaster response organizations during emergency situations is shown in fig. . . the disaster response plan in korea consists of three parts: the standard risk management manual, the working-level manual for risk response, and the manual for actions-at-scene. as of may , kinds of standard risk management manuals, working-level manuals for risk response, and , kinds of manuals for actions-at-scenes have been prepared and utilized. the standard risk management manual, prepared by a disaster-management supervision agency, delineates roles and responsibilities of related agencies in disasters at the national level, which shall be the guidelines for preparing the working-level manual for risk response. the working-level manual for risk response is a document stipulating the measures and procedures necessary for responding to actual disasters in accordance with the functions and roles of the disaster-management supervision agency and support agencies, which are stipulated in the standard risk management manual. the manual for actions-at-scene, prepared by implementing agencies, such as local governments, stipulates in detail the procedures for actions to be taken by an agency that directly performs its duties at a disaster scene. the standard risk management manual shall be prepared by each disaster management supervision agency that is regulated in table . . however, the standard risk management manual for disasters involving many disaster management authorities, such as typhoon and drought, can be prepared by the minister of mpss. the working-level manual for risk response shall be prepared by related support agencies that are designated by the standard risk management manual. the manual for actions-at-scene shall be prepared by an agency designated by the working-level manual for risk response. the head of the si/gun/gu may develop several disaster types of manuals for actions-at-scenes in consolidation as needed. the manuals delineate roles and responsibilities of the disaster-management supervision agency, related support agencies, and implementing agencies. on the other hand, the operational functions of disaster response of each agency shall be designated by action plans for disaster response by function under article - of the enforcement decree of the disasters and safety act. the functions for disaster response, similar to the emergency support function (esf) of national response framework (nrf) in the usa, are described below: . managing disaster situation . supporting emergency livelihood stabilization . supporting emergency communications . emergency restoration of facilities damage . restoring damaged energy supply facilities . supporting disaster management resources . traffic countermeasures . supporting medical and disinfection services . environmental arrangement at disaster scenes . supporting and managing volunteer work . maintaining social order . searching, rescuing, and emergency support at disaster areas . publicity of disaster management in summary, the two axes of the disaster response plan in korea are three levels of manual and functional action plans. the manual describes the roles and responsibilities of primary response ministries and related agencies, and the functional action plans describe how each agency performs its key response functions in line with its roles and responsibilities. the disaster response organizations during emergency situation are: cdschq, city/do dschq and si/gun/gu dschq for overall coordination; cdmhq and ldmhq for the implementation of their own responsibilities; cercg and lercg for search and rescue; and support agencies. both the establishment and evolution of fema and dhs in the usa had influenced the disaster management system in korea; in particular, the establishment of the national emergency management agency (nema) in and the establishment of the mpss in . in addition, the integrated disaster management formed by the dhs, the nrf, and the national incident management system (nims) has also influenced the evolution of the disaster response system in korea. dhs, established in , is responsible for national security and disaster management. fema under dhs is in charge of all phases of disaster management, including national preparedness, public and private capacity assessment, mobilization of resources for emergency management and disaster relief, and long-term recovery plans. at the state level, the disaster management department focuses on strengthening the linkage between the federal government, the state, and the local governments and assisting the local government in disaster prevention, preparedness, response, recovery, and relief. when a disaster occurs, the emergency operations center (eoc) commences operation and responds to disasters in accordance with a preestablished disaster response plan (eop, emergency operation plan, or cemp, comprehensive emergency management plan). local governments have a primary responsibility for the whole process of the localized disaster, prevention, preparedness, response, and recovery of disaster, and for the activation of eop followed by executing the eoc to respond to disasters. when a disaster that exceeds the capacity of a local or a state government occurs, federal government's assistance and involvement is requested and is provided through the joint field office (jfo). the usa performs disaster management through nrf and the nims. be that as it may, the catastrophic events on september , ( / ), and august , (hurricane katrina), saw the federal government's failure to provide proper support to state and local governments for effective disaster response. after that, there is an increasing demand for the federal government to respond to disasters proactively by federal emergency declaration to large-scale disasters and pre-deployment of federal resources to states. however, the basic principle of disaster management in the usa is still that local governments are primarily responsible for all disasters, with the support from state and federal governments concerning disasters that exceed the capacity of local governments. the disaster management organizations of the usa are summarized in fig. . . the dhs was established in january to integrate the prevention of terrorism and the function of disaster management under one department's coordination in the wake of the september , terrorist attacks and the mailing of anthrax spores. dhs took charge after the commencement of the national strategy for homeland security and the homeland security act. the dhs carries out tasks that include the suppression of terrorists' attack, minimization of damage, prevention, preparation, response, and recovery in the emergency plans for all domestic and international dangers that threaten the usa. the dhs consists of the office of intelligence and analysis (oia), the information analysis and infrastructure protection (iaip), the chemical and biological (cb) weapon management service, the state affair safety service, secret service (ss), and fema. fema, which is the most well-known organization among the various organizations of the dhs, is responsible for various tasks that include the disaster risk reduction at all levels, the reduction of property loss through various risk-based emergency management programs for the preparation, response, and recovery, and the protection of people's lives and main facilities. fema was established in as an organization coordinating the response and recovery of disasters during the carter administration. however, the initial phaseout of fema was not that high. it had grown into a member of the administrative cabinet and a ministerial level organization during the clinton administration through the establishment of an integrated response system and the strengthening of preventative and mitigation programs after failing to respond effectively to several large-scale disasters such as hurricane hugo. through the establishment of fema, various agencies related to disaster response were integrated into one agent under the direct control of the president and capable of comprehensively responding to various human-caused disasters and natural disasters. after the dhs was established, fema was incorporated and operated as a bureau under the dhs. in the early days, under the dhs, fema's status was weakened since antiterrorism and security were the first priority of the nation. however, after hurricane katrina, the importance of disaster management has been highlighted, and fema has also been strengthened as the independent deputy-minister level agency. fema, headquartered in washington, dc, operates local offices and provides regular disaster support personnel who can respond immediately in case of a disaster. the central organization for when an emergency occurs includes the jfo. the jfo is established by fema for coordinating between state governments and federal agencies after the president declares a state of emergency, and jfo plays a pivotal role in providing coordination between federal, state, and local governments and non-governmental organizations (ngos) and private sector accident supports. the head of a jfo is called the federal coordinating officer (fco), who is designated by the president and carries out the comprehensive coordination and the management of support activities using the resources secured by the federal government. in general, the state and local governments have an organization dedicated to disaster management: a disaster management department at the state level and an emergency office at the local level. a local emergency office (leo) normally consists of divisions handling emergency operation, information and communication, administrative task related to disasters, prior identification of risk, and the modification and supplementation of a risk management plan. in the event of a disaster, the eoc is set up to coordinate or support the disaster response at the site and responds to the disaster in accordance with the incident command system (ics). disaster response plans in the usa are developed at the federal, state, and local levels. at the federal level, the nrf and the nims constitute overall responsibilities of the dhs, fema, line ministries, and other agencies. the purpose of the nrf was to connect government agencies with ngos and the private sector and transparently assign and coordinate key roles and responsibilities nimbly. the nrf consists of the base document, the esf annexes (esf annex), the support annexes, and the incident annexes. table . shows the organization of the nrf. the local disaster response plan is carried out through the preparation of an eop by each area. the plan incorporates all aspects of disaster management in any given area, and it guides the roles and responsibilities of all related agencies for disaster response, depending on the size and complexity of a disaster. eop consists of the basic plan enclosed with the annex including the esf, the administrative and financial support plan, and the incident annex. nims provides national response doctrine for the whole community to work together based on the principle of the nrf. the nims defines standardized command and control principles to enable various response agencies to coordinate in the event of a terror and disaster. it provides a standardized response principle that allows federal, state, local governments, and nongovernmental organizations to respond consistently to a disaster regardless of the cause, size, location, or complexity of the disaster. to this end, it defines concepts, principles, organizations, rules, procedures, and terminology that provide a structured framework that is flexible, applicable, comprehensive, and geographically balanced for all types of disasters. the on-scene commander of the nims directs and controls the response at the incident site, and the head of the eoc is responsible for supporting the incident site while coordinating and managing local resources from outside the incident site. the components of the nims include preparedness, communication and information management, resource management, technical support, continuous management, and maintenance. in , fema developed "state nims integration" to facilitate states to adopt the nims. the guidelines require states to develop both an "emergency operation plan" and a "procedural document." the eop is a response plan that adapts the disaster response principles and the emergency support functions specified in the nrf tailored to the situation of the state. the procedural document consists of general principles for disaster response, standard action procedures, on-site operation guides, and job aids. each standard operating procedure (sop), the instruction for carrying out esf tasks to enable the smooth support for disaster response in the field and how to carry out those disaster response tasks, contains the guiding principles listed in the esf. it can be drawn from fema's actions that the modified us disaster management system would emphasize comprehensive, integrated, and mutual cooperation among the relevant organizations and stakeholders. to solidify this new emphasis, fema announced in seven guiding principles to cope with national emergencies: comprehensive, progressive, risk-driven, integrated, collaborative, coordinated, flexible, and professional (emergency management ). describe key roles and responsibilities around the nation and structures for implementing nationwide response policy and operational coordination for all types of domestic emergency events emergency support function annexes specify the federal resources and capabilities to provide emergency support for functional areas and identify coordinator, primary agency, and support agency for each functional area support annexes describe common and basic supports to the majority of incidents: critical infrastructure and key resources support, financial management support, international coordination support, etc. describe the response methods for seven incident categories: biological, catastrophic, cyber, food and agriculture, mass evacuation, nuclear/radiological, and terrorism. the basic act for disaster management in the usa is the stafford disaster relief and emergency assistance act. it gives the state the right to request support from the federal government and the right of the federal government to supplement resources to requesting state. additionally, it gives the president the right to declare emergency or major disaster in order to provide federal assistance. the homeland security act and the post-katrina emergency management reform act are also important disaster-related laws. japan has been exposed to various natural and human-caused disasters, such as earthquake, typhoon, and hazardous material contamination. to cope with those threats, japan has developed a comprehensive disaster management system at the national and local levels. the "basic act on disaster control measures," enacted in , functions as the backbone of the disaster management system in japan. the japanese government enacted the law in the wake of typhoon vera (isewan typhoon), which caused deaths, , injures, and property damage of approximately . trillion yen. since then, the "basic act on disaster control measures" has become the foundation for carrying out all measures related to disasters including emergency countermeasures and recovery as well as the disaster prevention, and this law handles disaster prevention measures in each field comprehensively. after the great hanshin-awaji earthquake on january , , japan revised the master plan for disaster prevention completely, and the "basic act on disaster control measures" was partially revised to reflect the lessons learned from the earthquake. the great hanshin-awaji earthquake, which led to a full modification of the master plan for disaster prevention, resulted in deaths, , houses destroyed completely, and property damage of trillion yen, which accounted for . % of gross domestic product (gdp) at that time. with the occasion of several huge disasters, the disaster management system in japan has been appropriately modified so that a more systematic disaster response can be provided through central disaster prevention meetings. a central disaster prevention meeting (chairman, prime minister) consists of the prime minister, the minister of disaster prevention, related ministers, the representatives of designated public agencies, and people with knowledge and experience designated by the prime minister. this meeting carries out the preparation and implementation of a master plan for disaster prevention and emergency measure plans and the examination of important items regarding disaster prevention according to the advice of the prime minister. the fire and disaster management agency under the ministry of internal affairs and communications, which is equivalent to the central firefighting headquarters in korea, was established based on article , paragraph of the national government organization act and article of the firefighting organization act. the fire and disaster management agency under the ministry of internal affairs and communications takes charge of planning and drawing up firefighting administration procedures and policies of various laws and standards. this agency has no direct right to command firefighting at the local level but handles tasks in an adversarial, instructional, and coordination role. the general affairs and planning department (bureau) and disaster prevention sections in the general affairs division have been established to carry out cooperation and coordination tasks since the cooperation and coordination between relevant local divisions are important for disaster management. the local disaster prevention meeting is divided into prefectures and municipalities. the local crisis meeting of prefectures consists of heads of local administrative agencies, self-defense forces, and superintendents of education with the prefecture governor as the manager. the head of the municipality takes charge of the municipality local crisis meeting. these two agencies take charge of contact and coordination between relevant agencies in case of a disaster and play a role in establishing and implementing a disaster prevention plan to handle each step effectively including disaster prevention, emergency disaster measures, and disaster recovery. the local emergency organization is the local disaster relief center. the local disaster relief center is installed according to a local disaster prevention plan in case a disaster is expected or a disaster occurs. the local disaster relief center is responsible for carrying out disaster prevention and emergency disaster measures related to the relevant prefecture or municipality according to the local disaster prevention plan of the relevant prefecture or the local disaster prevention plan of the municipality. the central/local and normal times/emergency response institutions of japan are summarized in table . . the disaster management system in japan has been established into central and local disaster management systems and regular and emergency disaster management systems to enable a smooth communication among divisions. japan, where various disasters occur frequently, has established various systematic disaster response plans just as the disaster management system. the disaster response plan consists of a "master plan for disaster prevention" for the central government and a "local disaster prevention plan" for the local level. the "master plan for disaster prevention" is a comprehensive long-term plan for disaster prevention prepared by the central crisis meeting, and it is based on the basic act on disaster control measures. it is responsible for determining the comprehensive and long-term plan for disaster prevention, the key points for the disaster prevention task plan and the local disaster prevention plan, and the preparation standard for the disaster prevention task plan and the local disaster prevention plan. based on such disaster prevention plans, japan has prepared for disaster prevention in three steps including prevention, emergency measures, and recovery and redevelopment. each step contains the basic policy for disaster prevention, mutual linkage around the country between public agencies and local governments, and measures to share disaster prevention information between disaster prevention agencies and residents. the central disaster prevention plan and the local disaster prevention plan are executed identically. disaster management in australia utilizes a comprehensive and integrated approach (ema ) . each provincial and local government, in accordance with federal guidelines, establishes a disaster management act that puts forward how local disaster entities should actively and effectively prepare against regional risks. such a decentralized legal system gives each state or local government the responsibility and the flexibility to carry out disaster countermeasures tailored to the characteristics of disasters, which have occurred or are to occur in each area of australia. one of the most influential events that moved opinion for the improvement of disaster management in australia was a large-scale fire called the tasmanian bushfire that occurred on february , . this large-scale fire resulted in deaths, displaced from families, and , ha of land damaged. this fire made the australian government recognize the importance of a disaster management system at the federal level, which resulted in the establishment of the natural disaster organization (ndo) in . this organization was strengthened in due to the needs of more systematic disaster preparedness, and its name was also changed to emergency management australia (ema). currently, ema performs its duty as the standard central organization for disaster management and, based on the commonwealth government disaster response plan (comdisplan), takes charge of planning and the coordination for disaster management at the state, district, and local governmental levels in australia. disaster management in australia is divided into four levels: federal, state, district, and local. the disaster management system is handled by ema at the federal level, whereas disaster management groups (dmg) -state/district/local -manage the disaster management system at the state, district, and local levels. at the federal level, the australian government has established separate disaster management systems suitable normal times and emergency situations, respectively. the ema division in the attorney general's department carries out normal disaster management: national disaster management, planning, coordination, -h disaster status monitoring, international support, and cooperation tasks. in case of emergency situations, the federal government carries out disaster management through the australian government crisis coordination centre (ccc), an emergency center coordinating disaster responses for all line federal departments and state, district, and local governments. the state, district, and local governments in australia also have two types of disaster management systems suitable for normal times and emergency situations, respectively. during normal times, the dmg is the primary organization to deal with disaster management, particularly focusing on prevention and preparedness, which is managed by the department of the premier and cabinet (premier of state government), consisting of the premier of each state government, all state ministers, and the army commander of each state government. the dmg develops and operates disaster management plans, strategies, and policies and supports state, district, and local management groups. in case of an emergency, the manager of the dmg should appoint a disaster coordinator in advance for the cooperation and coordination between the federal government, other state governments, and relevant organizations within state governments. on the other hand, the disaster coordination centre-state/district/local (dcc) was established to handle and operate a disaster management system during emergencies more systematically. the dcc consists of divisions representing each state government, the bureau of meteorology under the federal government, the australian defense force, the australian red cross, and the australian government ) insurance parliament at the senior officer level, and is responsible for decisionmaking and coordination regarding support of resources to local, district, and state governments according to the level of the disaster situation occurrence. the disaster response plans in australia have also been separated into a federal level and a local-level disaster response plan. the disaster response plan of the federal government is the comdisplan, which specifies competent disaster organizations in the six states and seven territories of australia, and the contents and procedures for resource support and cooperation between state governments. the disaster response plan at the state, district, and local levels is called the disaster management plan (dmp) or guideline-state/district/local and consists of four parts: prevention, preparedness, response, and recovery for strategic policies to deal with disaster by delineating roles and responsibilities for each phase in disaster management. the roles and responsibilities of each department and agency are specifically described in esfs. for more specific response activities, the response phase is subdivided into four steps: alert, lean forward, stand up, and stand down. two record-breaking disasters have occurred in australia since . the black saturday bushfires that occurred on february , , resulted in large-scale forest fire damage in the southeastern region of australia. more than forest fires occurred simultaneously resulting in a more significant damage occurrence, and these forest fires resulted in deaths and forest loss of , ha (victorian bushfire reconstruction and recovery authority ). a series of floods called the queensland floods occurred in western brisbane, queensland, on december , . these floods resulted in more than dead, more than missing, and approximately , homes and businesses flooded (queensland floods commission of inquiry, ). these floods were recorded as the largest floods within years, and the continuous rain for weeks made the damage greater. the australian government is in the process of improving its disaster management system in the wake of these two catastrophic events, which is worthwhile to be monitored. according to germany's basic law, the primary responsibility for disaster management rests on local and state governments, and the federal government provides financial, human, and physical assistance to local and state governments when a large-scale disaster occurs that exceeds the capacity of the local government or the state government. in the event of a disaster beyond the capacity of the state, the state will be supported by police departments and military forces in the neighboring states. and if necessary, the state receives the support of the police department, military forces, and technical support from the federal government. the federal ministry of the interior (bmi, bundesministerium des innern) is in charge of coping with major disaster or nonmilitary crisis that require the intervention of the federal government. in particular, the federal office of civil protection and disaster assistance (bbk, bundesamt für bev€ olkerungsschutz und katastrophenhilfe), established in may under the ministry of the interior, is responsible for responding to disasters in an integrated manner through systematic cooperation with other federal, state, and local governments in the event of largescale disaster. bbk has been expanded from the federal office of administration (bundesverwaltungsamt) as the importance of civil protection has increased in germany, in the wake of the september terrorist attacks in the united states in and the floods of the elbe river in germany in august . the main responsibilities of the bbk include the development of a comprehensive civil protection plan, dissemination of emergency information to citizens in crisis situations, critical infrastructure protection, and education and training for civil defense. the joint situation and information center (gmlz, gemeinsames melde-und lagezentrum von bund und ländern) in the bbk is responsible for monitoring disaster situations, disseminating disaster information, and international requests for help. when a large-scale disaster occurs in germany, the federal ministry of home affairs calls up the crisis task force (krisenstab). the task force works jointly with relevant departments within the federal government, agencies under the ministry of interior, and state liaison officers. in the event of a major type of catastrophic event that can be a national crisis, the ministry of interior will form the joint task force with responsible ministries. for example, if a nuclear accident or an illegal use of radioactive materials happens, the bmi and the federal ministry for the environment, nature conservation, building and nuclear safety (bmub, bundesministerium für umwelt, naturschutz, bau und reaktorsicherheit) will form the joint crisis management task force. when pandemics or biochemical terrorism occurs, the bmi and the federal ministry of health (bmg, bundesministerium für gesundheit) will form the joint task force. the german local system consists of the state (länder) government, the city or county (kreis) government, and the municipal government (gemeinde). disaster management organizations in germany's local system are different depending on the characteristics of each region; however, departments dealing with internal affairs mostly take charge of disaster management. in recent years, there have also been a growing number of states establishing disaster risk protection departments (gefahrenabwehr) by experiencing increased terrorism and massive natural disasters due to climate change. the central/local and normal times/emergency institutions of germany are summarized in table . . each state government develops and operates its own disaster response plan. for example, hessen is the state government that has the disaster response plan under the title of katastrophenschutzpläne (emergency plans). katastrophenschutzpläne (emergency plans) of hessen specifies the contents regarding necessary information in the event of disaster and means to be used. also, in case of a very severe disaster such as nuclear risk that targets a specific object, it is required to establish and operate sonderschutzpläne (special protection plans) separately. figure . shows the katastrophenschutzpläne (emergency plans) in hessen, germany, and it shows the contents regarding the sonderschutzpläne (special protection plans). the english translation of fig. . is: § disaster protection plans the disaster protection plans shall contain, in particular, the necessary information on the emergency aid, the alarm, and the means of assistance available in a disaster. they must be coordinated with the neighboring disaster control authorities. special protection plans are to be drawn up for special dangers. the uk, based on the tradition of local autonomy and accountability of citizen and local governments, utilizes a bottom-up disaster management system. in , the cabinet office, a guide to emergency response and recovery was released with eight core principles: anticipation, preparedness, subsidiarity, direction, information, integration, cooperation, and continuity (cabinet office a). the basic act for disaster management in the uk is the civil contingencies act (cca), . the uk has developed its disaster management system suitable for normal times and emergency situations at the federal and at the local levels (korean association for local government studies ) . at the federal level, the civil contingencies secretariat (ccs) was established in to take charge of the overall coordination of disaster management during normal times, whereas the cabinet office briefing room (cobr) and the civil contingencies committee (ccc) take charge of disaster response in case of emergency situations. the ccs is headed by the permanent secretary, vice-minister level official in the cabinet office, and it takes overall responsibility for disaster management in the uk. if needed, the minister of the home office (or the cabinet office) reports to the national assembly for sharing emergency information or hearings after hit by disaster. the ccs carries out the emergency response tasks including anti-terrorism and disaster restoration tasks and is responsible for identifying and making preparations for a crisis during an emergency situation. the cobr and the ccc are activated for the federal government to act when catastrophic events occur. the activation process and procedures are decided depending on the level of emergency, specifically at level and level ; and at . disaster theories and progress of disaster management in modern times level , the responsible minister takes the leading role as chairman, and at level , the prime minister serves as chairman. table . shows the three levels for disaster response, in which the roles and responsibilities of each agency at the federal and the local levels are delineated differently. the cobr and the ccc focus on cooperation between the federal and local governments and support tasks rather than command and control. also, highranking officials can participate in the cobr, receiving and processing a report of situation, for prompt disaster management. figure . shows the block diagram of cobr. the cca categorizes agencies for disaster response into two categories according to their roles and duties with different obligations. the category responders are the agencies that have priority for response. the category responders consist of police services, fire and rescue services, health bodies, maritime and coastguard agency, local authorities, and environment agency. the police normally control and coordinate the activities at and around the scene. there are, however, exceptions, for example, the fire and rescue service takes the responsibility at the scene of a major fire. category responders consist of a wide range of private sector bodies that have an important role, but not routinely involved in the core of multi-agency emergency response and recovery work. utilities, telecommunications and transport providers, highways agency, strategic health authorities, and health and safety executives are included in the category responders. a local resilience forum (lrf) is a private and public joint organization of local governments, military officials, corporations, and civic groups that analyzes risk factors and establishes the risk management plan. the main aim of the lrf is to assist multi-agency and multi-sectoral cooperation. the lrf has regular meetings at least once every months to strengthen regional resilience, and category responders should participate in the meetings. the lrf is not a legal entity, nor does the lrf have powers to direct its members. nevertheless, the cca and the regulations provide that responders, through the forum, have a collective responsibility to plan, prepare, and communicate in a multi-agency environment. the police serve as chairman for the lrf, and a manager is assigned to each of the areas classified according to the police administrative district. a regional resilience forum (rrf) is a high-level organization of the lrf to coordinate plans for large-scale emergencies that are difficult to handle locally and to coordinate with the central government. the rrf divides areas into local units, and a table . disaster step as the standard of disaster management system in the uk ((cabinet office, b)) step disaster support system by step step (significant) the competent agency provides supports and cooperation at central government level step (serious) the national crisis committee is organized with the competent minister as the chairman step (catastrophic) the national crisis committee is organized with the prime minister as the chairman local government office is located in each local unit, and the regional director of government offices serves as chairman. the strategic coordinating group (scg) and the regional civil contingencies committee (rccc) are local emergency organizations for coordinating multiagency cooperation. the scg is a type of accident response and recovery center consisting of government branches, military, police, fire, hospitals, and private local committees in the relevant area at the local level for accident response, and all government officials dispatched to the jurisdiction of the central government are under the direction and control of the local director. the rccc is established when an emergency crisis exceeds the local level, and its composition and system are similar with the rrf. the disaster management in the uk is shown in table . . the disaster response plan in the uk is also operated separately into national and local disaster response plans. the national disaster response plan in the uk is carried out through the national contingency plan (ncp). the ncp describes initial response, accident response, responsibility and compensation, role and responsibility of central government, international support, and cooperation for each type of disaster, and it becomes the foundation for the disaster plan of the central government. the uk has also prepared a disaster response plan for flood under the ncp since flood occurs frequently, which is the guidance for accessing specialist flood rescue mutual aid. the guidance for accessing specialist flood rescue mutual aid specifies the strategic approach of the government for preparing for and responding to a flood, and it is responsible for providing comprehensive emergency response guidelines to all flood rescue service providers including public and private volunteer organizations including the utilization of existing and future flood relief assets. also, the guidance for accessing specialist flood rescue mutual aid presents the role and responsibility for each agency regarding floods as well as the action procedure (sop) to enable prompt response in case of a flood. the local disaster response plan in the uk is carried out through the strategic emergency plan (sep). the purpose of the sep is to present the direction of regional response activities and methods, through which the disaster response strategy and disaster response plan of each cooperation organization is prepared. the sep is managed through the lrf organized in each area, and the lrf is responsible for developing a more detailed implementation plan on the basis of what was given by rrf. in switzerland, cantons and communes have the authority and responsibility for disaster response, and the federal government intervenes only in the event of a national crisis. particularly, switzerland has a disaster management system based on a close cooperation among the federal, state, and local governments, a proactive role of the military in disaster response, and a strong civil defense system. the basic act of disaster management in switzerland is the federal civil protection and civil defence act (bzg, bundesgesetz über den bev€ olkerungsschutz und den zivilschutz). at the federal level, the federal office for civil protection (babs, bundesamt für bev€ olkerungsschutz) under the federal department for defense, civil protection and sport (vbs, eidgen€ ossisches departement für verteidigung, bev€ olkerungsschutz und sport) is responsible for disaster management. under babs, national alarm center (naz, nationale alarmzentrale) operates h all around the year in order to monitor situations, disseminate disaster information, and respond to disasters when necessary. since its establishment in , naz has designated radioactivity, chemical accidents, and the collapse of bridges due to a natural disaster as major disasters, making preparations for such disasters, and naz has also established the linkage system between major facilities and competent authorities to judge the situation in case of a disaster. the office for civil protection, sport, and military (bsm, amt für bev€ olkerungsschutz, sport und militär) takes charge of the normal-time disaster . rccc (regional civil contingencies committee) . lrf (local resilience forum) . scg (strategic coordinating group) management system at the local level. bsm is divided into four departments, and the department related to disaster management is included in the citizen and nation protection division. the manager of bsm changes according to the conditions of the state government. the five local-level emergency services utilize an "integrated system" (verbundsystem) which allows for an autonomous or a cooperative response, depending on the severity of the situation. in an extended or severe disaster situation, any of the police, fire brigades, health and ambulance services, technical agencies, or civil defense organizations can partner up to handle the crisis; this can be done at the communal or at the cantonal level. additionally, based on federal government guidelines, which includes the basic principle of disaster management and civil defense, the physical plan for disaster response, and the technical and structural countermeasures, state and local governments develop and manage their own disaster response plan. for planning and preventative measures for natural hazards, the five civil-security agencies follow the national platform for natural hazards (planat) guidelines. the swiss civil-security agencies, basing their planning activities on "risk-based planning" and "integral risk management," have developed common prioritization procedural guidelines (riko) and an online tool (econome). the guideline and the online tool, based on "protection objectives" (schutzziele), are also designed to keep natural hazard management projects cost-effective. when it comes to implementing a project, the babs have developed a tool (kataplan) to identify and classify various risks and the planning of response measures by cantonal agencies. the babs have also developed a learning program (lernrisk) and assessment software (riskplan) (babs ; bfu and babs ) . until now, we have described the disaster management system in six countries. table . shows the national and local disaster management organizations during normal time and emergency situations. social advancements in contemporary society beget new techniques and technologies. thus, new risks due to the application of the new technologies are continually springing up, and difficulties predicting uncertainties increase in parallel with new risks. furthermore, due to the development of information technology, the rise of interdependence increases propagation or chain properties to expand continuity in type, range, and scope (oh ) . the contemporary society can be featured with a change in natural environment such as climate change, socioeconomic environmental change such as aging, urbanization, and polarization, international environmental change such as each country being networked, and an entrance of complexity which indicates characters such as emergence, self-organization, and adaptation. due to these changes in environment, the contemporary society can be represented as increase of complex general set up the strategic objective for national safety, crisis management, and disaster relief and established the comprehensive drm system. in summary, most developed countries are in the process of shifting from a top-down, fragmented, and hazard-oriented disaster risk management approach into a comprehensive, integrated, and human-centered approach. while this is happening, special attention is being paid to "low probability and high impact" focusing events that are located in the long tail of the power-law distribution. symbolic and practical interpretations of the hurricane katrina disaster hazard mitigation and preparedness babs ( ) leitfaden kataplan: kantonale gefährdungsanalyse und vorsorge bfu and babs ( ) pragmatisches risikomanagement mit riskplan online risikogesellschaft: auf dem weg in eine andere moderne. frankfurt am main, suhrkamp beck u ( ) world risk society cabinet office ( b) responding to emergencies the uk central government response concept of operations disaster data: a balanced perspective national incident management system dhs ( ) threat and hazard identification and risk assessment guide: comprehensive preparedness guide again and again: is a disaster what we call a "disaster? emergent phenomena and the sociology of disaster: lessons, trends, and opportunities from the research literature principles of emergency management supplement emergency management australia (ema) ( ) emergency management in australia: concepts and principles disaster theory: an interdisciplinary approach to concepts and causes disaster relief and emergency assistance act, as amended, and related authorities fema ( ) national response framework: third edition fritz ce ( ) disaster and community theory introduction to emergency management, th edn hessisches gesetz über den brandschutz, die allgemeine hilfe und den katastrophenschutz national comprehensive crisis management: theory and reality research on effective disaster relief planning disaster classification for optimal disaster response in korea economic loss due to thailand flood in and policy implications to korea business korean association for local government studies ( ) a study on the comparison of disaster and safety management system in major advanced countries study on the change of risk structure in korea and its social and cultural effect a terminological and etymological study about safety damage situation in thailand big flood and implications for korean companies building an integrated emergency management system in korea introduction of disaster prevention. donghwa tech, hwaseong-si mcloughlin d ( ) a framework for integrated emergency management the network governance of crisis response: case studies of incident command system standard crisis management manual munich re ( ) natcatservice, global distribution of insurance premiums per capita crisis management system in korea: focusing on nuclear power plant and oil transportation the role of u. s. military forces and its implication for the korean military during disaster management natural disaster and development in a globalizing world. routledge, london perrow c ( ) normal accidents: living with high risk technologies what is a disaster? the need for clarification in definition and conceptualization in research technological and natural disasters and ecological problems: similarities and differences in planning for and managing them queensland disaster management arrangements public administration: understanding management, politics, and law in the public sector victorian bushfire reconstruction and recovery authority ( ) victorian bushfire reconstruction and recovery authority day report living with hazards, dealing with disasters: an introduction to emergency management collaboration and leadership for effective emergency management oecd studies in risk management: innovation in country risk management most developed countries have put stress on developing an effective disaster response system to cope with emerging risk interwoven with complexity and climate change. the policy implications through the comparative analysis can be summarized as follows:first, many countries aim for an integrated organization that considers both natural disasters and social disasters. the usa has endeavored to build an integrated disaster management system through the establishment of fema, and since the / terror attack, the dhs has been integrated to take charge of disaster management and terrorism. the dhs set the national preparedness goal to reflect the insights and lessons learned from hurricane katrina and to build a comprehensive organizational capacity for the entire nation that includes federal, state, and local governments and the private sector.japan and the uk have an organization that control and coordinate disaster and emergency response directly under the prime minister. the cca of uk and the cabinet office of japan are both small in size, but they are characterized by a very high level of authority for the overall coordination of disaster policies. according to the types in the reorganization of disaster management proposed by the organization for economic cooperation and development (oecd), the case of the usa corresponds to the first case, "super-ministry," and japan and the uk correspond to the second type, "a relatively small and highly influential body under direct authority of the head of government" (wyman ). disaster response plans are also being developed within national and local governments using an integrated disaster management style for all hazards approaches (waugh ) .second, all stakeholder engagement for disaster response is highly recommended. the usa successfully established the disaster risk management system by promoting the engagement and commitment of all levels of governments, citizens, and ngos. australia and japan also developed standard operating procedures indicating citizenry roles and activities in case of emergencies. in addition, australia developed checklists to enhance citizenry participation for disaster preparedness. japan has enhanced citizenry participation and preparedness by strengthening citizenry education and training. korea should improve the participation of local governments, ngos, and citizens to drm by providing guidelines and incentives and enhancing the governance-based approach.finally, many countries have stressed the clear accountability of each government and interlink between the national government and local governments. most countries, such as australia, germany, japan, and the usa, empowered local governments for disaster response and engaged in the national crisis situation. in addition, the interlink between national and local disaster response planning was also emphasized. the usa improved interlink between nrf and nims at the federal level and eop at the state and local levels. in australia, the attorney key: cord- - vzwc d authors: nan title: proceedings of scanning /seems charleston, south carolina, usa date: - - journal: scanning doi: . /sca. sha: doc_id: cord_uid: vzwc d nan the widespread application of monte carlo electron trajectory modeling has never been realized in the electron microscopy community because of the computation-intensive nature of the monte carlo algorithm (e.g., many hours of computer time were required to run one simulation which might improve one's ability to interpret data if sufficient statistics were obtained). massively parallel supercomputers with multiple-instruction multiple-data (mimd) architecture are a good platform for monte carlo electron trajectory codes without the complications of vectorization of the computer code required on vector supercomputers (e.g., cray-ymp). the nist monte carlo electron trajectory simulation code has been adapted to run on a massively parallel computer. for the first time, the increased speed of the calculations has made monte carlo calculations a real-time tool for data interpretation. the increased speed achieved by the parallelization of the monte carlo code results in the ability to model small probability events due to the massively parallel monte carlo (mpmc) code's ability to simulate large numbers of electron trajectories quickly. applications include both thin-film and bulk x-ray microanalysis and examples of contrast in backscattered electron images obtained in the sem. this work was supported by the united states department of energy under contract #de-ac - al . in electron probe x-ray microanalysis, "secondary fluorescence" refers to the emission of characteristic x-rays following photoelectric absorption of the primary x-rays generated by the electron beam. while generally a minor contribution to the total x-ray emission, secondary x-ray fluorescence can become an important issue, when the spatial resolution of analysis is of interest, because of the significantly greater range of x-rays than of electrons. thus, for a nickel- % iron alloy excited with a kev electron beam, the range of excitation of the primary radiation will be a hemispheric volume with a diameter of about one micrometer, while the range of secondary fluorescence of iron k-shell radiation by nickel k-shell radiation will be a hemispheric volume with a diameter of approximately micrometers for % of the total secondary radiation. such long-range production of secondary radiation is important when the measurement of trace element distributions near phase boundaries is attempted in situations where an analyte appears at high concentration across the boundary. monte carlo electron trajectory simulation provides a powerful tool for the calculation of the three-dimensional distribution of the primary radiation. to calculate the spatial distribution of the secondary fluorescence due to the absorption of the primary radiation, a second calculation is needed that integrates the x-ray absorption equation in three dimensions. the resulting hybrid calculation can give the total secondary radiation induced for a given structure, such as a planar interface between two materials. the transport phenomenon of incident electrons in solid materials is an important subject in microscopy, microanalysis, and microlithography. it has been treated by either an analytical method, the transport equation, or monte carlo simulation. because of the flexibility of modeling, monte carlo simulation has been developed extensively. various models for monte carlo simulation proposed so far are classified into four groups from the aspect as to how inelastic interactions are treated. the most simple model is the one which uses the inelastic mean free path. this model is applied, for example, to spectroscopic analyses of elastically reflected electrons, auger electrons, and photoelectrons. the most popular model is the single scattering model in which the step length is taking the free path of elastic scattering of electrons, and an energy loss during their traveling is calculated by the continuous slowing-down approximation of bethe, [de/ds] bethe . the most complicated model is the direct simulation model in which all inelastic interactions are taken into consideration as the discrete process. this model requires all differential cross sections for inelastic scattering and, thus, a long computational time. the last one, a compromise model, is the hybrid model which introduces partially the discrete process of inelastic scattering. in the model the continuous energy loss [de/ds] ( ) where [de/ds] dis includes both energy losses due to core electron ionization and valence electron excitation. a variety of the cross sections has been used for inelastic scattering. the fast secondary production model uses the moller equation for the cross section, assuming that all atomic electrons are free (murata et al. ). the authors have published a hybrid model by using the differential inelastic cross sections of vriens for core electron ionization and the moller cross section for free electron excitation (murata et al. ). the mott cross section is used for elastic scattering. typical results for backscattering have shown fairly good agreement with experimental data. the present paper gives a brief survey of monte carlo modeling and discusses the validity of the hybrid model mentioned above. figure shows a typical example of the calculated depth distribution of generated x-rays in an au target in comparison with the experimental result of castaing and descamps ( ) . agreement is good. also shown is the result without the discrete processes of inelastic scattering. an appreciable deviation is seen in the vicinity of the peak. however, the effect of the energy straggling is not so significant. we have also done calculations for other x-rays and have obtained reasonable agreement with experimental data. the effect of the energy straggling will appear more significantly in electron scattering in a thin film. figure shows the energy distribution of transmitted electrons, with a comparison between two monte carlo results and the experimental data of shimizu et al. ( ) . the agreement is not as good; it seems that the disagreement comes from insufficient discrete processes because we still keep the continuous energy loss process. we also propose a new model to improve the discrepancy in figure , caused by energy straggling. the straggling is impor-tant in thin-film analysis, especially at initial energies near the ionization energy for x-ray production. discussions of ways to improve monte carlo simulations have usually concentrated on topics such as the effect of the choice of scattering cross section, or the appropriate model for electron stopping power at low energies. much less attention has been given to considering whether or not we actually have sufficient experimental data to make it possible to demonstrate by a comparison of simulation and experiment that one model, or a portion of that model, actually performs better than another alternative. the earliest experimental data on electron-solid interactions was published years ago (by starke in germany in , and by campbell-swinton in england in ), and numerous workers since then have contributed to the literature on this topic. unfortunately, there never seems to have been any attempt to collect and collate all of this material, and consequently workers seeking data such as the variation of the secondary electron yield with energy for silicon have been forced to conduct a random search of the literature to find what values are available. it is not surprising that such a search typically finishes as soon as a set of data plausibly matching the simulated values is found. in order to try and remedy this deficiency, a systematic search of published data has been carried out to generate material for a rudimentary database, with the hope that this will provide some of the necessary numbers against which monte carlo simulations can be tested. in its current form the database, derived from separate references, is divided into four segments arranged by atomic number (or compound) and comprising secondary electron yields, backscattered electron yields, x-ray ionization cross sections, and electron stopping power. only experimental values are included; thus interpolated, extrapolated, or normalized data sets, or values not specifically indicated by the author to be experimental, have been removed. this restriction unfortunately eliminates most of the voluminous secondary electron data, since such published values have invariably been normalized in order to facilitate fitting to a yield curve equation. no attempt has been made to judge the quality of any of the data since any such assessment would be premature until a sufficient number of independent values are available to permit obviously erroneous, or possibly dubious, results to be safely identified and eliminated. the database is available on request from the author both in printed form, or as a set of cricketgraph ™ files for the apple macintosh. while the quantity and quality of data available for a given element vary widely, they are mostly sparse and scattered. for about half the elements in the periodic table, no experimental values ever appear to have been published, and data are conspicuously absent for even the most common alloys and compounds. even for an element such as silicon ( fig. ) , the scatter in the data is too large to make it possible to be certain to better than a factor of what the se yield is at some energies. thus, unless a major source of quantitative results has been overlooked, it is fair to conclude that the experimental data presently available are not good enough to allow the merits of competing monte carlo models to be judged, or even to permit specific numerical comparisons (e.g., the backscattered yield of carbon, copper, and gold at kev) to be made with any satisfactory level of certainty. on the positive side, however, the database does give some indication of interesting trends (e.g., the variation of secondary yield with atomic number) that have not been easily accessible previously. school of electrical engineering and the national nanofabrication facility, cornell university, ithaca, new york, usa two simulation programs have been developed at cornell, seel [ ] [ ] [ ] (for simulation of electron energy loss) and pyra-mid. seel is a two-dimensional monte carlo program for the simulation of electron trajectories in complex, multi-material nanostructures; the program incorporates energy loss models that include quantum mechanical cross sections for energies down to < ev. the second program, pyramid, , is a very fast electron beam proximity correction program that can perform proximity correction (electron scattering correction) for complex ultra-large-scale integrated circuit patterns with nm minimum feature sizes (mfss). pyramid uses seel to generate the point spread function or radial exposure distribution (red) as the input. seel simulations have been tested against numerous published electron energy loss data and pyramid has been tested on ulsi density ( nm mfs) exposures using . µm pmma. simulation results are compared with experimental data to evaluate pyramid's performance. a more recent application of seel is the evaluation of signal-noise ratio (snr) for time-resolved microscopy of microelectromechanical structures (mems) . for this application, we are interested in estimating the snr for high-speed video recording , of moving high-aspect-ratio mems oscillating > mhz, particularly applications of nanoelectromechanical structures for metrology and time-resolved characterization. iv- scanning vol. , supplement iv ( ) simulation of image formation and detection systems in the sem is a vital link in performing image analysis to obtain precise measurements, to provide the necessary connection between image parameters and structural dimensions, and to reflect important microscope beam and detector parameters. monte carlo methods allow a wide degree of freedom in specifying simulation conditions for sample composition and geometry. published examples often limit the simulation to two-dimensional structures, with a zero-diameter electron beam and all secondary or backscattered electrons (bse) collected. a more useful and realistic approach will take into account the effect of beam diameter, and detector geometry and gain characteristics. these effects for simulated bse images for three-dimensional patterned structures of carbon on a silicon substrate are shown in figure . the monte carlo simulation used here is based on a single-scattering procedure with the rutherford scattering cross section and the bethe energy loss formula modified for low-energy primary electrons. the program is a further development of a single-scattering monte carlo program (in pascal) widely distributed by david joy and modified later by russ et al. the program is written in c language and runs on a sun workstation. the program can scan the beam position in x and y directions over an arbitrary multielement and multilayer sample with topographical features to produce images. using the monte carlo procedure, it is possible to extract information about the position, energy, and direction of the electron at each scattering point. this allows tracking bses as a function of angle. if the dependence of detector gain on electron energy is added, then the details of signal formation can be modeled. the effect of electron beam shape and diameter on the image can be added by convolution after an ideal image is generated. figure shows the matrix representing a gaussian electron beam used in this work. an example of a simulated bse-sem image is shown in figure for a structure with carbon features on a silicon substrate. fig. . contour plot of gaussian shape electron beam with standard deviation of Å was used to convolute the data obtained for a zero-width electron beam. fig. . bse image simulated using a zero-diameter electron beam (left) and after convolution using the gaussian beam width shown in figure (right). the simulation was performed for kev primary electrons with trajectories for each point. the image represents × pixels. monte carlo simulations of electron scattering in a target normally use one of two elastic cross sections, either the screened rutherford cross section or tabulated partial wave expansions of the mott cross section. the screened rutherford cross section gives acceptable results for high energies and low atomic numbers, but mott cross sections are required for low to medium incident energies ( . - kev) and high atomic number targets. however, computations tend to be slow using tabulated data due to the need to interpolate between data points. empirical equations for the total and differential electron/atom elastic scattering cross sections have been found that can be substituted for tabulated mott cross sections in predicting backscattering coefficients. the total elastic mott scattering cross section is fitted by similar form to the screened rutherford cross section but contains three terms in energy in the denominator. the empirical total elastic scattering cross section is valid for atomic numbers up to and for energies from ev to kev: ( ) the fit to the differential mott cross sections is decomposed into two parts, one part being of the same mathematical form as the screened rutherford cross section (σ r ), and the second part being an isotropic distribution (σ i ). the screened rutherford part of the differential scattering cross section is first fitted to the half angle of the mott cross sections. this fit of the differential screened rutherford is in turn reduced to a fit of the screening parameter alone over energy and atomic number. in marked contrast to the screened rutherford cross section, the tabulated mott cross sections show only a small overall downward trend in half angle with increasing atomic number(z). implying an average rutherford screening parameter for all z, with e the electron energy, of: the ratio of the total cross sections (σ r /σ i ) between the screened rutherford part of the differential scattering cross section and the isotropic part of the distribution is fitted to the backscattering coefficients calculated directly from tabulated mott cross sections. the ratio of rutherford to isotropic cross sections is: ( ) figure shows a comparison of the calculated backscattering factors using the present empirical fit (solid lines) with those calculated using mott cross sections. the fit for al, cu, and au is good over the entire energy range. the fit for ag is moderate and the fit for c is high. however, most deviations are similar to differences because of the use of different atomic models in the mott cross sections and are acceptable. there are two major reasons why the simple monotonic eqs. - work well. first, the scattering of the electrons in a solid is a multiple scattering process. thus, many of the complex quantum interference effects are averaged out. second, the elastic backscattering is monotonic with atomic number. these two factors serve to smooth out the effects of the complex multidimensional cross sectional surface that is being fitted over z, e, and θ. reference iv- scanning vol. , supplement iv ( ) σ rutherford σ isotropic = e − z / z + z × e σ t = . × − z . (e + . z . e . + . z / e . ) a scanning interference microscope may be constructed by allowing light which has probed the object and light which has not probed the object (the reference beam) to interfere on a suitable detector. if the detector is large in extent, a conventional scanning interference microscope results, whereas if a point detector is used we have a confocal scanning interference microscope. the image in both cases may be regarded as a superposition of three terms. the first represents a normal conventional or confocal image depending on the kind of interferometer used. the second we will call an interference term image, whereas the third represents a constant background. it is possible to design a system in which the interferometer term image is identical for both conventional and confocal scanning systems in all respects, including optical sectioning. a simple scheme permits the interference term image to be detected separately and then processed in a variety of ways. let us consider two specific geometries. the first is an almost common path confocal system based on a single mode optical fibre. the interferometer in this case is confocal. simple image processing then permits surface profilometry to be performed. the second implementation is a conventional scanning interference system. the interference term image from this conventional image may again be isolated and processed to give a confocal image. in biology, the description and precise representation of microstructural forms is of increasing importance for threedimensional ( -d) computer image understanding, in particular to enhance -d visualization and analysis. the intermediate level of computer vision, located between the bottom layer (signals) and the top layer (model) is best suited as a starting point to improve multidimensional image understanding. we implement the concept of -d topology embedded in the bottom-up structure of data processing to enhance subsequent rendering and specific quantitative analysis. segmented and contoured serial section images require sophisticated algorithms to generate correct surface-rendered views. we have, therefore, developed a method to evaluate connectivities between sections, based on bijective correspondance analysis of the center of gravities of contours. these connections indicate nods and branches of -d structures and can be interactively edited in exploded views of the contour stack. because of the strong data reduction, such procedure can be implemented even in computer graphic systems with entry graphics. the topological skeletons are the backbone to render correctly biological structures of free forms, in particular those featuring frequent branching patterns. examples where such an analysis is successful include microvessel networks, dendritic trees, lung anatomy, or dental root canals (baumann et al. ) . moreover, the automatic labeling of connected structures gives rise to the analysis of topological criteria, which was only available in stereological methods until now. this includes criteria such as connectivity and branching angle, or higher representations of branching schemes. in return, topological connectivities can be used to improve the segmentation of images in the top down-process of data processing, or it can serve as embedded analytical graphics to improve volume renditions. the mammalian central nervous system (cns) contains at least ten times more glial cells than neurons. the three major populations of cns glia are astrocytes, oligodendrocytes, and microglia. astrocytes and oligodendrocytes together are often referred to as macroglial cells and arise from primitive neuroectodermal precursor cells, while microglia are derived from the mesodermal germ layer. it is thought that common precursor cells, known as o- a progenitors, can differentiate into either astrocytes or oligodendrocytes during gliogenesis. in contrast, the origin of microglial cells remains enigmatic, although there is substantial evidence that microglial cells arise from primitive hematopoietic stem cells that gain access to the cns at a very early stage of development. much of our knowledge about glia and glial cell function has been derived from histochemical and electron microscopic studies. astrocytes can be visualized reliably using immunohistochemical methods with antibodies against the glial fibrillary acidic protein (gfap). gfap is an abundant constituent of intermediate filaments which can seen ultrastructurally in most astrocytes. in addition, astrocytes also contain enzymes, such as nadph diaphorase and glucose- -phosphatase, which are readily detected by enzyme histochemical methods. we have focused much of our attention on developing methods for detecting oligodendrocytes and microglial cells in sections of rat brain. lectin histochemistry has been a particularly useful tool in studying these types of cns glial cells. using lectins i and ii from griffonia simplicifolia seeds with carbohydrate specificities against α-d-galacatose and nacetyl-d-galactosamine (glcnac), we were able to demonstrate selective labeling of microglia and oligodendrocytes, respectively. the b -isolectin from griffonia simplicifolia coupled to horseradish peroxidase (gs i-b -hrp) can be used to detect a membrane-bound glycoprotein on the microglial cell surface at all stages of cns development ranging from the early embryonic age to adulthood. in contrast, oligodendrocytes were found to express glcnac-containing glycoproteins in the perinuclear cytoplasm using biotinylated gsl ii. the perinuclear staining was determined ultrastructurally to be associated with golgi complexes. biochemical analyses using tricine/sds-polyacrylamide gel electrophoresis and western blotting with gsl ii showed the glcnac-containing glycoproteins to be insoluble, with molecular masses ranging from to kd. having available specific markers for the three major glial cell groups, we were able to combine lectin histochemistry with immunohistochemistry to perform double-labeling studies demonstrating specificity of each stain for a given glial cell type. following the study of glia in the normal cns, we went on to investigate glial cell reactions that occur as a consequence of nervous system injury or disease. it became immediately ap-parent that microglial cells were the major glial cell type responding to neuron injury. microglia not only proliferate and change their morphology in response to cns damage, but they can also vary their membrane phenotype by expressing new molecules on their surface. we found that antigens of the major histocompatibility complex (mhc), which are largely absent from the normal cns, are expressed de novo on microglia and related perivascular cells responding to neuron injury. our studies, which have included various neuropathologic conditions including stroke and brain tumors, have shown that the expression of mhc antigens, as well as other related immunomolecules, is always restricted to cells of the microglial lineage. these findings strongly suggest a role for microglia as indigenous immunocompetent cells of the cns. jeremiah r. lowney national institute of standards and technology, gaithersburg, maryland, usa a scanning electron microscope (sem) can be used to measure the dimensions of the microlithographic features of integrated circuits. however, without a good model of the electron-beam/specimen interaction, accurate edge location cannot be obtained. a monte carlo code has been developed to model the interaction of an electron beam with lines lithographically produced on a multilayer substrate. the purpose of the code is to enable one to extract the edge position of a line from sem measurements. it is based on prior codes developed at nist but with a new formulation for the atomic scattering cross sections and the inclusion of a method to simulate edge roughness or rounding. the code is currently able to model transmitted and backscattered electrons, and the results from the code have been applied to the analysis of electron transmission through gold lines on a thin silicon substrate, such as used in an x-ray lithographic mask. there is provision for both transmitted and backscattered electron detectors. by comparing the predictions of the code with measured data, it is possible to obtain edge positions to the order of nm, which is needed for the advanced lithography projected for the year . the uncertainty of these measurements is limited by the sample geometry and surface roughness and not by the measurement process. much of the improved code is devoted to the treatment of boundary crossings by the electrons. the present code allows for a substrate of at most three layers and one or two identical lines with a trapezoidal cross section on top. there is also provision for a symmetrical jog (i.e., a discontinuous change in the width of the trapezoid) along the edges to simulate edge roughness and rounding. the three layers that form the substrate, which are typical of an x-ray mask membrane, are silicon, polyamide, and chromium. the lithographically produced lines are gold, and the chromium improves adherence of the lines to the substrate. the code is easily modified to model other media by simply changing the atomic parameters in the input subroutine. the x-ray lithography mask, which has been used as a test sample, is a very good model system for the development of accurate sem standards because it provides a measurement of both transmitted and backscattered electrons for comparison with the predictions of theoretical models. a plateau in the transmitted and backscattered electron signal occurs as the beam traverses the sloping edge of the line trapezoid. this effect can be blurred by edge roughness, and the effects on the plateau of various widths and heights for the jog can be shown as well as the effects of rounding at the bottom of the lines and the calculated noise level for various numbers of trajectories. direct comparisons with measured transmission through an x-ray mask of gold on a silicon membrane can be used to demonstrate the determination of the edge of the gold line. figure shows the transmission (measured downward) along the axis of a gold line indicating the plateaus in the data near the middle of the edges. figure shows a simulation of the data with the plateaus at nearly the same location along the edges as in the data. this work shows how high-resolution metrology of the features produced by advanced lithography can be obtained with an sem. extensions of this code to modeling secondary signals as well as the effects of charging are planned. at philips research, a monte carlo program for electron microscopy is developed, which simulates electron-solid state interactions in the energy range . - kev. it is based on the program published by l. reimer (scanning , , ) . in particular, to ensure proper operation in the low-voltage region, mott cross sections for elastic scattering are calculated by numerically solving the dirac equation. the model for inelastic scattering treats inner shell ionizations as discrete events, described by a scaled gryzinski formula, whereas the effect of valence and/or conduction electrons is incorporated as a continuous bethe loss. this model, which differs from similar models presented in the literature, provides good fits to experimental stopping power data. the program is extended to include multilayer specimens, each layer composed of multiple elements. one of our objectives is the modeling of cd linewidth measurements in a high-resolution low-voltage scanning electron microscope. our monte carlo program can generate top-view backscattered electron (bse) and secondary electron (se) video profiles of lines with variable slope and pitch. we use the se generation model of d.c. joy (j microsc, , , ) , which is extended to account for the regions near the corners of the line profile. furthermore, the recollection of se by the sample is accounted for. as a result, realistic pure se profiles are generated. the primary beam parameters include the probe size and the depth of focus. determination of the top and bottom linewidths via the commonly used heuristic algorithms reveals the systematic errors of these methods. a simple example is the probe size dependence of the peak-to-peak width, which can be related to the strong asymmetry of these peaks. we expect that improved algorithms can be developed, which use a modest database acquired by monte carlo simulation. a scanning electron microscope (sem) fitted with a helium-neon laser interferometer is used to measure the widths of features on photomasks. in this way the magnification of the sem can be known very precisely. algorithms yielding good measurement repeatability, which use the back-scattered electron (bse) signal, have been developed (nunn , nunn and turner ) but in order to be able to relate measurements made on the image to the physical dimensions of the artefact, it has been necessary to model the image formation process. the essential details of the "plural scattering" model used in this work have been described by d.c. joy ( ) . typical geometries and materials of photomasks have been modelled along with a range of accelerating voltages and beam diameters to observe how the image is affected by the different parameters. more important, the modelled image intensity profiles are studied to relate the position of the physical foot of the sloping edges of actual physical lines to the broadened image intensity profile of the edges. although the "single scattering" model, also described by joy ( ) , is more rigorous and more applicable to the type of samples used in this work, it was not used in the first instance because of the much greater computer time required. results from the modelled profiles suggest that the position of the % thresholds of the bse image intensity profiles are located a few tens of nanometres away from the physical position of the edge. the exact offset depends on the angle between the sloping sides and the horizontal. the modelled offset increases from approximately nm for a vertical edge to nm for an edge angled at ˚ to the horizontal. the effect of the bse detector size and geometry on the signal detected has also been modelled. the model shows that for best performance the detectors should collect the largest possible solid angle, but that as long as the detector is placed symmetrically, the only adverse effect of a smaller detector on the image is a loss of signal to noise. measurement comparisons between optical microscopy and the sem corrected by the "plural scattering" modelled offsets have been encouraging (nunn and turner ) ; nevertheless, if time permits, a more rigorous study using more appropriate models should be pursued. audio enthusiasts carefully read the test reports when looking for their new equipment. however, usually no one tries to find out the frequency response, distortion or signal-to-noise ratio of a very expensive, complex system such as a scanning electron microscope (sem). sems were once used mainly as image-gathering devices, and in spite of certain obvious and sometimes serious problems they served as acceptable and effective instruments for many applications. these "image grade" microscopes are now being replaced with much better quality "measurement grade" instruments. the guaranteed resolution is no longer enough to ensure good performance and other factors must be considered as well. even though these newer systems have field emission of lab electron guns, the main advantage is in the built-in, computer controlled, digital image and data-processing capabilities. there are several alternatives to upgrading an existing microscope with an external, usually desktop computer-based measuring system. soon the sem, similar to a scanner or camera, will just be a part of an imaging network, and automatic image enhancement and processing will be commonplace. this presentation deals with a short description of the electrical properties of different components of an sem; the beam scanning circuitry, the video signal chain, sampling, analog to digital (ad) and digital to analog (da) conversion. it will relate their possible influences to the detected and observed signal to improve the imaging. this will also make possible better comparisons of measured data to the computer-modeled data. essentially, the main parts of an sem essentially are the electron source, electromagnetic coils, dc power supplies, (scan) generators, detectors, amplifiers and, finally, the displays. the quality of an sem image depends on the characteristics of the primary electron beam, the correctness of the beam scanning, signal detection, and the fidelity of the video signal chain. the correctness of the beam scanning not only means good linearity and proper amplitude and direction of the deflection of the primary and displaying electron beam, but the displaying or data assigning has to be in correct synchrony also. the deflection coils are fundamentally nonlinear and inherently have hysteresis; hence, correcting circuitry is indispensable. the total distortion caused by improper magnification, nonlinearity, and hysteresis can be greater than % and it is difficult to keep it below % . the fidelity of the video signal chain depends on its transfer function, noise, and distortion figure. the main parts of the video chain of a modern sem are the detector, amplifier, ad converter, computer memory (disk, imaging network), da converter, amplifier, and the display or printer. all of these parts make their contribution and form the overall characteristics of the video chain. to produce good quality images at a certain scan rate, the bandwidth of the video chain must be high enough to show the finest possible details of the image. the transfer function that describes the frequency or bandwidth and phase characteristics of a detector or an amplifier is very useful to characterize the video chain of an sem. for good quality tv frequency imaging, - mhz bandwidth is required, but for slow-speed image collection this bandwidth can be surprisingly low, that is, - khz. noisy signal means loss or lack of information. at a certain noise level, details that otherwise would be in the video signal cannot be seen. the visual effects of noise are closely tied to the resolution, appearance, contrast, brightness, and other aspects of the image, as well as intensity distribution and other properties of the noise itself. a db ( - ), signal-to-noise ratio gives a reasonably clean image, but more noise degrades the ability to differentiate two areas of different brightness by eye. to turn an analog signal to digital data, ad conversion is needed. usually there is a sampling and hold circuitry that keeps the analog signal unchanged for the time of conversion. depending on the design, this results in smaller or larger signal loss because just a fraction of the analog signal is collected due to the relatively short sampling and long conversion time. well-designed circuitry, for example, a gated integrator, can improve the signal-to-noise ratio, but the quantization process in the ad converter itself introduces noise also. modeling data with monte carlo or other techniques has to include the shortcomings of the real, nonideal measuring tool, that is, the sem with all its associated components. by knowing the transfer function, noise, and distortion figure in digital form, it is relatively easy to obtain more accurate comparison of the measured and calculated signal (fig. the calculation of image contrast in the scanning electron microscope (sem) can be done using monte carlo techniques if the electron trajectories can be calculated through the composition profiles in the specimen. this has been done for the case of a discrete one-dimensional composition variation in the incident beam direction , but most of the programs that are available are designed to calculate signal intensities from samples of uniform atomic number and density (e.g., reimer and stelter ) . nevertheless, the calculation of electron trajectories in a three-dimensional heterogeneous microstructure has not been reported. the basic idea behind a monte carlo calculation is that an electron, in penetrating a solid, will undergo a series of predictable elastic collisions (electron-atom) which change the direction and inelastic collision (electron-electron) which change the energy. in figure , which is the example of a calculation in a lead tin eutectic with somewhat unrealistic geometry, the electron enters the sample and is scattered to a new coordinate location. since the directional change and distance scattered are sensitive to the variations of atomic number, atomic weight, and density, the electron might travel to location in the block of the lead-rich position of the pbsn eutectic, scatter into the position , and then go on to position of the tin-rich matrix. thus, depending upon the details of the microstructure, that is, the size and location of the second phase, the image will appear different. the anticipated backscattered intensities from this rather unrealistic microstructure are shown, for example, in figure as the block varies both in size and location below the sample surface. we clearly see that the backscattered signal in this case is much more sensitive to the position in the specimen of the sec- iv- scanning vol. , supplement iv ( ) fig . the geometry of the sample and a schematic of the electron trajectory for the calculation. intensity ond phase rather than the size distribution, but for more realistic microstructural geometries such a calculation would be useful in determining the presence of alternative phases in a complex assemblage. terference microscopy - is based on the observation that a simple single-arm interferometer can be constructed by allowing light from a laser to be back-reflected from a target and to reenter the laser resonant cavity to produce a modulation of the laser light-intensity. [ ] [ ] [ ] the resultant light modulation is dependent on the optical phase (optical path length traveled) and amplitude of the reentry light. if the target is moved over several wavelengths, the laser light intensity displays a cos(z) dependence; for a mirror target, the light modulation index (i-i o /i+i o ) can be as great as . . these observations were denoted as laser-feedback interferometry (lfi); this phenomenon has been characterized and analyzed several times in the past years. [ ] [ ] [ ] [ ] [ ] [ ] [ ] the theory of lfi , , provides an understanding of the harmonic content of lfi signals; they follow a bessel-function dependence, a property that is useful in designing electronic feedback circuits necessary to produce a practical laser-feedback microscope. lfi can employ either diode or gas lasers as the interactive laser source/detector. using a he-ne laser with a high-reflectivity output coupler mirror (≤ %), it is possible to measure surface profiles of highly-reflecting surfaces (e.g., metals or high index-of-refraction materials such as silicon) with axial detail as small as nm and surface vibrations up to - mhz can be measured down to picometer amplitudes. in addition, weakly backscattering materials (e.g., biological cell surfaces and cell components) give sufficient signals to provide a useful method of high-resolution imaging in biology. a versatile laser-feedback microscope (phoebe) has been constructed having the following properties. the he-ne ( . nm) laser incorporated is a milliwatt, linearly-polarized output unit with special mounting to minimize output mirror movement. access to the low-power rear laser beam allows the laser intensity to be measured directly by a silicon photodetector. the main beam is expanded to fill the back aperture of any type of microscope objective (air-, water-, or oil-immersion) suitable for the microscopic examination being undertaken. an x,y scanning stage moves the sample under the laser beam in a raster fashion; pixel × pixel image frames are obtained in < s. axial (z) motion, furnished by a tubular piezoelectric transducer, provides both a modulation signal input for the electronic feedback circuit ( khz) and repositions the sample at each point. the electronic feedback circuit maintains a fixed distance between the output coupler mirror of the laser and a point in the object being imaged. the correction (output) signal of the feedback circuit then gives a measure of the surface topography of the sample. concomitantly, the laser intensity modulation gives a measure of the surface reflectivity of each point on the sample. these two images are digitized and stored in the computer memory. all microscope control functions have been consolidated in a dedicated small computer allowing samples to be run without interruption by the resetting or realignment of the microscope. phoebe scans for biological use range from µm × µm to µm × µm. when an air objective is used, surface topography images display a quantitative measure of height; for fluid-immersion objectives, the measured heights are shortened by a factor dependent on the index-of-refraction of the fluid (water or oil). the confocal property of this scanning microscope is due to the requirement that only backscattered light reentering the cavity-resonator mode (tem oo ) of the laser is effective; the through-focus response of lfm verifies this property in comparison with a confocal pinhole placed at the focus of the laserbeam expanding lens. the lateral resolution of phoebe is nm as determined by imaging silicon-based resolution standards. an important result of measurements with the phoebe lfm is that high-contrast images can be obtained from biological samples placed on a plastic or glass substrate (e.g., a microscope slide or petri dish). in contrast to images of samples with sharply defined index-of-refraction boundaries, what is measured in this case is a point-by-point optical path length difference with the substrate furnishing the majority of the back-reflected, lfi-measured light. other imaging modes include the use of interferometric optical sectioning and the buildup of three-dimensional structures from a series of twodimensional sections. because of the coherence requirement of lfm, the use of fluorescent labels and the detection of fluorescence is precluded; however, other reflective labels may be employed to gain back some of the advantages of that method. - , - ( ) . sarid d, iams d, weissenberger v, bell ls: compact scanningforce microscope using a laser diode. opt lett , lett , - lett , ( university of california, irvine, california, usa nmr microscopy is one of the newly emerging tools for the high resolution three-dimensional ( -d) imaging of live animals and plants for biological as well as medical research. more recent applications and developments include such things as porous materials and microflow, essential for oil research. one of the main interests of nmr microscopy, however, lies in the fact that the method is truly a noninvasive -d high resolution imaging tool with which µm resolution can be achieved. however, nmr imaging, especially nmr microscopy, has a number of formidable difficulties, namely, small signal-tonoise ratio due to the inherently small object size, diffusion and bandwidth limitations, and other inhomogeneity effects such as chemical shifts and susceptibility. among others, diffusion problems due to the random brownian motion of (water) molecules appear to be one of the fundamental physical limitations to nmr imaging, especially in high-resolution microscopy where these molecular diffusion distances are close to the resolution limits. although the diffusion effect in nmr has been studied extensively, the effect on nmr microscopy has not as yet been observed experimentally due to the current limitations of the nmr microscope. theory, however, suggests substantial resolution broadening in nmr microscopy if molecular self-diffusion prevails, especially when we deal with molecules with large diffusion coefficients. resolution limits on nmr microscopy especially include diffusion limits, namely, effects of phase variation due to molecular self-diffusion during data acquisition, effects of signal attenuation and related line broadening, and some diffusion effects of molecules which, when confined in boundaries or walls, exhibit anomalies in resolution observation in nmr microscopy. the first two are based on free-water molecules, while the third is based on the model of water molecules confined by boundaries, which have significant physical consequences. diffusion effects limit resolution in microscopic imaging, as does the phase factor which is related to the finite bandwidth of the imaging instrumentation: that is, the available gradient strength and acquisition time, both of which are intimately related to the diffusion effect. an x-ray microtomographic system is being developed at our laboratories amil & arts. a generalized feldkamp cone-beam reconstruction algorithm was already developed for our system (wang et al. ) . the generalized algorithm is approximate, but quite accurate and computationally efficient. under some feasible conditions, the generalized algorithm produces exact volumetric reconstruction for longitudinal invariant specimens and exact transaxial reconstruction for a point source contained in that transaxial plane. in the generalized feldkamp cone-beam reconstruction, a transaxial slice is reconstructed using projection data collected from a scanning turn of ˚ angular range. in fan-beam reconstruction, there actually are two complete sets of projection data over a full-scan range. exact reconstruction can be achieved using only projection data corresponding to a half-scan. in our cone-beam system configuration, it can be appreciated that there are "approximate redundancies" in data acquired along geometric rays that would be identical after projection onto a transaxial plane. there are various weight functions for half-scan fan-beam reconstruction. with gullberg and zeng's weight function, a half-scan generalized feldkamp cone-beam algorithm is obtained for less longitudinal blurring: ( ) where ( ) ∆ is an additional angle for a smooth transition between essential and duplicated radon regions, and φ(z) is an offset specific to the longitudinal coordinate z. optionally, cone-beam projection values associated with appropriate pairs of opposite rays can be lineraly interpolated to synthesize needed fan-beam projections for transaxial reconstruction. at an additional computational cost, the interpolation-based cone-beam algorithm allows exact reconstruction if the longitudinal specimen variation is linear. numerical simulation results demonstrate the feasibility of our extended algorithms. wang g, lin th, cheng pc, shinozaki dm: a general cone-beam reconstruction algorithm. ieee trans med imag ( ), - ( ) biological specimens can be preserved by rapid freezing, a process which takes just a few milliseconds and is termed "cryofixation." it is an alternative to chemical fixatives which may take many minutes to penetrate a specimen and even longer to fully stabilise cellular components, thereby compromising their ultrastructural and chemical integrity. when cryofixation is performed properly, the water molecules in the specimen do not have time to form ice crystals and the specimen is preserved in a near life-like state. the time-scale of cryofixation means that short-lived phenomena can be captured and preserved. when this aspect is combined with electrical or chemical stimulation in a controlled experiment, then cellular processes can be studied by freezing the experiment at predetermined time intervals, so that a series of transient steps are preserved. besides electrical and chemical stimulation, other methods have been combined with cryofixation; these have involved electrophoresis, electroporation, temperature-jump, and flash photolysis. electrically stimulated muscle was slam-frozen by sjöström et al. ( ) and van harreveld et al. ( ) . their methods held specimens in defined physiologic states: true time-resolved freezing was introduced by heuser et al. ( ) , who showed that ultrastructural differences could be observed in synaptic events at neuromuscular junctions between and ms after stimulation. chemical stimulation is effectively performed by a chemical flow method, involving the rapid mixing of small specimens with a stimulant from different syringes and flowing them for a predetermined time along a reaction tube before spraying the reactants into a coolant, thus quenching the reaction (knoll et al. , rand et al. . electrophoresis involves the movement of particles in an electrical field and was used with freeze fracture to study the diffusion rate of intramembrane particles (sowers and hackenbrock ) . electroporation uses a radio frequency field to induce transient pores in cell membranes (chang and reese ) . optical stimulation has been used to cause a temperature jump to investigate temperature effects, for example, a ˚c jump after ms exposure to a xenon lamp, on the ultrastructure of lipid specimens (chestnut et al. ) . flash photolysis methods have been used to release caged photolabile chemicals in the study of the dynamics of the actomysin cycle (funatsu et al. , ménétret et al. . when the available stimulation methods are considered with the available freezing methods (namely, slam, plunge, jet, and microdroplet-spray freezing), then it becomes clear that there is promising scope for the study of dynamic cellular processes using electron microscopy. the combined methodology integrates the temporal resolution of rapid freezing with the spatial resolution of the electron microscope. chang dc, reese ts: changes in membrane structure induced by electroporation as revealed by rapid-freezing electron microscopy. biophys j , - ( - ( ) universität des saarlandes, homburg-saar, germany contrary to standard preparation at ambient temperature, there is some confusion on what is optimal in cryopreparation of biological specimens for subsequent electron microscopy (em) investigation. this results mainly from the diversity of methods as well as instruments described and the rapid development of these techniques in the preceding years. since the big advantages of cryopreparation in various fields are often hidden by the choice of unsuitable or even antiquated techniques, it seems to be justified to report about the recent developments in this field and to classify different methods for different purposes. this tutorial is based mainly on experience in transmission electron microscopy (tem). nevertheless, most of the information may also be useful for scanning electron microscopy, since a proper preparation has the same importance in both fields. without doubt, cf is the most important first step in most cryopreparations with a big variety of different techniques. the simplest procedure (plunging or immersion cryofixation = icf) is best suited for vitrification of thin suspension layers ("bare grid" or "ice embedding") for subsequent cryo-tem in the frozen hydrated state. high pressure freezing (hpf) reduces ice segregation in suitable specimens (e.g., thin plant leaves or rigid tissues such as cartilage). the main problem of hpf in soft tissues results from the indispensable dividing of the specimens into small pieces. impact freezing on a metal mirror (mmf) is well suited for suspensions such as hpf or double jet (djf), but mmf has an advantage for soft tissues, since it is suitable for larger specimens, that is, tissue slices > mm . as far as liquid cryogens are concerned (icf, djf), ln or partially frozen n -slush are not to be recommended; ethane gives the best results. propane is well suited and less expensive for routine applications. as an alternative to cryosectioning, these more time-consuming preparations gain continuously in importance for cytochemistry. even element analysis is possible in certain cases. only for simple morphology or morphometry additives (e.g., oso , uo -acetate, aldehydes) are short drying times in freeze substitution (fs) or freeze drying (fd) possible. proper drying by fs or fd without additives seems only possible for small objects (diameter < . mm) in periods between - days at − ˚ to − ˚c. otherwise, severe artifacts by thermal collapse phenomena and redistribution effects result. a suitable instrumentation is of great importance for this long drying time. the results are excellent and the advantages are striking as long as sufficiently long drying times are employed. in addition, low-temperature embedding (lte) in special acrylics improves the results. care has to be taken in handling these monomeric resins, since some of them are strong allergens. sugar protected specimens are easy to section on the dry knife at − to − (− )˚c for subsequent histochemistry of macromolecular components. overall morphologic preservation is rather poor in comparison with fs/fd/lte. larger areas are mostly not obtainable, but speedy work and results within hours instead of days or weeks are possible. sectioning can be considerably improved by the use of cryo-diamond knives together with an ionizer (useless without antistatic tool). sectioning below − ˚c down to − ˚c is possible with cryo-diamond knives on an ionizer, if the specimen is well frozen (no ice segregation) and the cutting area << . × . mm . trimming with a diamond trimming tool is advantageous. good preconditions are given after hpf or mmf. the advantage of mmf is the mirror-like, well-frozen surface, which can be easily trimmed and orientated to the knife edge. cryotransfer to the tem/stem or proper fd for fedx pose no severe problems (fd for > h at − ˚c). most of the different actual cryomethods (except fresh frozen cryosectioning < − ˚c for cryo-tem/stem) allow routine work, if the most suitable method is carefully selected and modern instrumentation is available. in all cases, the additional effort and investigation are definitely justified by better and more reliable results (literature and reprints on request). paul walther, renÉ hermann, martin mÜller laboratory of em , department of cell biology, eth zurich, zurich, switzerland the reason for using low temperatures for preparation and microscopy is the decreased mobility of atoms and molecules reducing the danger of artefact formation. for scanning electron microscopy (sem) cryotechniques have the following advantages. . cryofixation (rapid freezing) is the fastest way to immobilize a biological sample at a defined physiologic state. thereby, all processes in a cell are arrested within milliseconds. chemical fixation, in contrast, takes seconds or minutes to act, leading to unpredictable osmotic effects and redistribution of cellular compounds. . a frozen biological sample behaves like any other bulk specimen, and redistribution of substances is almost excluded. inner structures can be made amenable to the electron beam by cryofracturing or cryosectioning. . the conductive metal layers have a finer grain size when applied to cold samples, because diffusion of the metal atoms on the surface is reduced. . the frozen sample is analyzed in the sem by use of a cold stage. this prevents volume changes due to drying artifacts. in addition, hydrocarbon contamination due to irradiation by the electron beam is greatly reduced at cold temperatures. for most applications in biology, cryotechniques are only used for some but not for all preparation steps; for example, samples are cryofixed and then dehydrated by either freezedrying or freeze-substitution and afterward stored and imaged at ambient temperature. for high resolution sem, it is advantageous to coat also dry samples at cold temperatures in order to obtain a finer grain size and to observe the samples at cold temperatures in the sem to reduce hydrocarbon contamination. ( ) a major limitation of cryotechniques is distortion of the ultrastructure due to ice crystal formation during freezing. high pressure freezing allows for direct cryofixation of living samples within minimal or no ice crystal artefacts up to a thickness of several µm. ( ) the problems of water vapour contaminants that condense on the sample during preparation and microscopy were well investigated for the tem freeze-etching technique in the s and s. during the last years, this knowledge has been adapted for the construction of cryo preparation systems for the low-temperature sem. ( ) drying artefacts such as shrinkage are omitted by observing fully hydrated frozen samples. on the other hand, the ice covers many structures of interest and therefore often needs to be partially removed, either by freeze-drying or by freezesubstitution. however, removal of water bears the risk of drying artefacts. ( ) hydrated samples are extremely sensitive to the electron beam. the high surface-to-volume ratio inherent in particulate samples, and the fact that we already live in a sea of possibly contaminating particles, necessitates special care and understanding in particle preparation. selection of reagents and handling practices can be critical. for instance, in a hypothetical case, a particle analyst reports that his sample consists of approximately five major inorganic phases. the first has a rounded morphology with crystalline overgrowths, the light element content of the second is depleted, the crystalline structure of the next is damaged, the fourth occurs at a % volume level, and the fifth consists of glass shards. in this example, these results are essentially worthless. phase one was soluble in a liquid used in the preparation process and then reprecipitated upon evaporation. the light elements of the second were leached because of the use of the same solvent. the third was attacked by hf formation during ultrasonification in a fluorinated solvent. the fourth was originally present at a % volume level, but was preferentially attracted to the sides of the storage container prior to sample preparation, biasing the sampling; and the fifth is not part of the original sample, but comes from the ground glass neck of one of the reagent bottles. decisions on reagents and methods are crucial but unfortunately cannot satisfy every concern. for instance, reagents chosen for inorganic preparation should be nonpolar when easily leached elements such as boron or lithium are of interest, or if surface oxidation or ionic dissolution is a concern. but nonpolar solvents can increase particle agglomeration problems due to the lack of charge dissipation. therefore, wise choices in preparation methods are strongly tied to the objectives of the analyst, and sometimes multiple methods performed in parallel with additional analyses may be required to obtain truly representative results. contamination often is a concern, especially when performing high resolution work on a trace particle constituent. suspended urban air particulate is typically - µg/m , mostly in the . - . µm range and usually with less than particles per cubic centimeter above µm. supermicrometer-sized par-ticles can constitute µg/m . these particles may settle out gravitationally, electrostatically, or may simply happen to intercept the surface of the sample mount or sample particles. a large background can accumulate on unprotected substrates. the use of easily charged containers such as polystyrene disposable petri dishes or polyethylene centrifugation cones can further complicate dust collection or an additional problem, sample losses, due to electrostatic forces. the use of light microscopes for particle preparation makes possible a variety of preparation methods including micromanipulation. uses include ensuring even particle distributions on mounts, precise particle positioning, particle size reduction, washing unwanted films or residues off of particles, and many more. a light microscope can also be used to make some helpful observations such as specific gravity, population densities, refractive index, morphology, size, solubility, film thickness, and color. "micro world" effects often have to be provided for when performing preparation on a microscale with a microscope. for example, the polarity of solvents sometimes must be sacrificed for lower evaporation rates. reduction in evaporation rate can be aided by the use of small transparent covers, air movement shields, or by the choice of a combination of substrate and solvent that have poor affinity for one another (the resulting bead-ing-up action decreases the surface-to-volume ratio). the insertion of a "reservoir object" such as a probe tip at °"holding" a liquid droplet against the substrate, or a coverslip from the edge of which a supply of underlying liquid can be obtained, are examples of creative procedures that may be used (fig. ) .recommended polar solvents include heptane and cyclohexane. these avoid hfc environmental concerns, evaporate readily, and avoid halogen interactions. isoamyl acetate, flexible collodion, and formvar in ethylene dichloride are preferred micromanipulation and mounting media. microscale fume "hoods" protect microscope optical coatings and personnel. for containers, glass or static dissipator-coated plastics are preferred to ordinary uncoated plastics. if clean rooms are not available, static dissipators (physical or chemical), laminar flow work benches, single hepa-filter curtained areas, and modified work practices are inexpensive solutions. syringe filters help provide easy to handle point-of-use contamination control for reagents. recommended micromanipulation probes include tungsten and specific animal hairs. intermediate substrate surfaces may employ temporary teflon or paraffin coatings. other procedures include ashing, centrifugation, filtration, ultrasonification, and the exploitation of effects observed during micromanipulation. an approach for the indirect visualization of biological material in transmission electron microscopy (tem) is the freezeetch or freeze-fracture/replica method. the preparations steps ( fig. ) of this purely physical method include: (a) fast fixation and stabilization by quick freezing (in ms vs. min necessary for conventional chemical fixation); (b) creation of clean fracture faces with a fracturing cryotome (high vacuum required); (c) replication with electron beam evaporation (ebe, high vacuum required); (d) three-dimensional imaging of fracture faces and surfaces (with structural resolution of - nm); (e) high stability of the pt-c replicas in the electron beam (selection of relevant details) and possible long-term storage (weeks to months). the important drawbacks are that the replicas have to be cleaned (subsequent changing of several cleaning solutions over hours or days), picked up (disintegration of highly structured replicas happens frequently), and can be viewed only as a result of one single fracturing event. specimens providing iv- scanning vol. , supplement iv ( ) fig highly redundant structures and relatively smooth fractures, such as cell suspensions or o/w emulsions, were investigated using freeze fracture/replication and ambient temperature transmission electron microscopy (at-tem). freeze-drying is comparable to freeze-etching ( fig. ) , but with the important difference that all the water is removed from the specimen by sublimation. in tem, scanning electron microscopy (sem), or scanning tunnelling microscopy (stm), small cell components or particles, such as viruses, can be imaged together with the replication layer on top. in freeze-fracturing for low-temperature sem (lt-sem), most advantages of tem cryopreparation are kept. the preparation includes: (a) quick freezing; (b) fracturing under high vacuum conditions (c) coating with a planar magnetron sputter source (pms, ar as working gas); (d) -d imaging and observation of changed conditions (in situ etching); (e) high signal/noise ratio of pt or w coatings for secondary electron imaging and low yield from cr for backscattered electron detection of immuno-gold labelled specimens. the specimens can be imaged directly eventually after multiple fracturing (i.e., searching of distinct structures) in a different situation (i.e., fully hydrated or partly freeze-dried, coated or uncoated), in a "close to nature state." however, new problems have to be solved in order to get thin reproducible, conductive coatings, without superimposition of specimen and coating film structures and direct imaging, reducing contamination and beam damage, or long-term low temperature storage. moreover, the coating film thickness, the ice in the frozen bulk specimen, and, most important of all, the type of sem are now limiting the structural resolution. the biological relevant details of results obtained with low-temperature sem in a conventional fieldemission sem can be compared with those achieved with routine freeze fracture/replication and tem. according to honig and hook, the physics of water in high vacuum play an ultimate role in the application of these techniques (fig. ) ; therefore, only this knowledge in combination with cold stages and high vacuum technology enable the application of state of the art preparations of biological material for electron microscopy. the cryo-jet freezing technique provides a means for the in situ preservation of diffusible ions in mouse spinal cord explants after trauma. using the technique, the effects of trauma, in particular the intracellular shifts of calcium and other diffusible ions, can be analyzed and recorded by qualitative and quantitative electron probe microanalysis (epma). chemical fixation must be avoided if one intends to attempt the epma localization of diffusible substances in cells and tissues. the goal of the cryo-jet method is to confine axonal components and chemical elements to a biologically natural position. trau- matized mouse embryo cord explants were compared with the intact rat spinal cord trauma model, using epma studies of perturbations of calcium and other ions that are reported to be located in the axonal cytoskeleton. icr mouse embryo spinal cord explants were grown to - -day maturation in maximow chambers. cords that survived stringent elimination examination criteria and exhibited minimal degeneration or spontaneous necrosis were randomly divided into four groups. group i served as experimental controls; group ii was traumatized by dropping a mg weight through a . cm glass pipette positioned directly over the culture and cryojetted h after trauma. representative explants from i and ii were freeze-substituted and embedded in epoxy for comparison. by light microscopic examination of the whole mount, living culture, the site of impact could be determined within - min. the impact zone became markedly dense when compared with the surrounding, uninjured tissue. toluidine blue of the freeze-substituted sections revealed the site of impact on the surface and path of the trauma damage through the explant. control cultures with spontaneous necrosis could be distinguished from the experimental group, because the traumatic necrosis was confluent and often even wedge-shaped. within the traumatized zone, nerve fiber alterations similar to those observed in in vivo spinal cord trauma and calcium toxicity could be found, that is, granular degeneration of axoplasm, pleomorphic spheroids, tubulovesicular profiles, and myelin showing adaxonal, periaxonal, and intramyelinic vacuolization. light microscopy sections from the freeze-substituted explants were noted to be well preserved at the surface and into the culture for - µm (fig. ) ; however, some ice crystal damage was noted deep within the body of the culture. previous studies that compared metal mirror and plunge freezing results with the propane jet methodology reported here confirmed that propane jetting is the method of choice for preserving organotypic spinal cord tissue cultures. x-ray microanalysis of cryosections of the cytoskeletal tubules, cross bridge structures, mitochondria, and the myelin sheath of in vitro spinal cord explants, compared with the in vivo model of spinal cord trauma, has yielded unique data concerning the ionic changes that occur as a result of trauma or experimental manipulation. after trauma, populations of axons were found that could be distinguished by their elemental compositions. one group was similar in elemental composition to controls, although the morphology was dissimilar, while a second group had - % lower k and - -fold elevated ca (fig. ) . the response of axons and the associated myelin was paired, that is, loss of k and gain of na within the axon was always accompanied by comparable changes in the myelin and vice versa. the morphologic appearance of axons was not a predictor of the elemental composition. the cardiomyopathic (cm) hamster manifests cardiac dysfunction from an early age, with the disease ultimately progressing to congestive heart failure. on the basis of significant elevation in the ca + content of the cm hearts, as well as significantly increased ca + in the mitochondrial (mt) fractions and an increased density of voltage-sensitive ca + channels, previous studies suggested a ca + overload in the cm hearts (sen et al. ). evidence has also been presented for an increased sensitivity of cardiac muscle cells of cm hamster hearts to an external ca + "stress," that is, any manipulation which increases ca + influx into the cells (hano and lakatta , sen et al. ). on the other hand, ultrastructural studies have shown localized focal lesions from an early age, believed to be due to local cell injury. the cardiac myocytes within these areas suffer irreversible damage, becoming ca + overloaded as demonstrated by measurements of mt and a-band (ab) ca + content by electron probe microanalysis (epma) in a previous study from our laboratory (bond et al. ). this study also demonstrated that the vast majority of cardiac myocytes throughout the cm hearts not only had a normal ultrastructural appearance, but also showed no elevation of subcellular ca + content. an alternative explanation for impaired contractile function may be a decreased amount or availability of ca + stored in the sarcoplasmic reticulum (sr) for stimulated release and activation of contraction. to investigate this question, we have utilized epma to measure ca + content directly in the junctional sr (jsr), as well as in mt and ab of the cm hamster heart, either under control conditions or, alternatively, after pretreatment with the ca + channel agonist, bay k , in order to increase ca + influx into the cardiac muscle cells. isolated papillary muscles from normal and cm hearts at days of age were stimulated to contract electrically, and parameters of isometric contraction were recorded at l max . muscles were randomly assigned to one of three protocols: ( ) eleven cm and normal papillary muscles were used to construct dose/response curves to the ca + channel agonist, bay k. after stabilization at l max , cumulative doses of bay k ( - m to - m) were added to the muscle bath. all contractile parameters were recorded from the muscle at each dose. ( ) for epma, five cm muscles were frozen, after stabilization at l max , at peak +dt/dt and five muscles during relaxation. ( ) ten cm papillary muscles were incubated with single dose of - m bay k in order to elicit a maximal inotropic effect. once the contractile response to drug addition had stabilized, the muscles were frozen either at peak +dt/dt (n= ) or during relaxation (n= ). ultrathin cryosections were cut from the surface of the frozen muscles and freeze-dried overnight. subcellular ca + content (ab, mt, and jsr) of cm muscles was measured by epma in stem mode in a philips cm scanning transmission electron microscope (fig. ) . measurements of baseline contractile function revealed a significant decrease in develop tension (dt) (from . ± . g in cm muscles to . ± . g in normals), +dt/dt ( . ± . g/s to . ± . g/s) and a decrease in −dt/dt ( . ± . g/s to . ± . g/s) in cm versus normal hamster. there was no significant difference in values of resting tension (rt). the inotropic response to increasing doses of bay k was markedly blunted in the cm muscles compared with controls, suggesting that even when ca + entry into the cardiac muscle cells is increased, force development is still impaired. a comparison of elemental content (na, mg, p, s, cl, k, and ca) of ab and mt between experimental groups revealed no statistically significant differences. in addition, no differences in elemental composition of ab and mt were observed compared with our previous measurement on normal hamsters frozen during contraction and relaxation (moravec and bond ) . the amount of ca + stored in the sr of the cm muscles that were rapidly frozen during relaxation (in absence of bay k) was . ± . mmol/kg dry weight ( fig. ) (left panel, rel); however treatment with the ca + channel agonist bay k significantly increased the size of the store to . ± . mmol/kg dry weight (right panel, rel). thus we conclude that ca + uptake into the sr of cm muscles was enhanced as a result of bay k treatment. the contractile data show that the amount of ca + that can be released from the jsr of cm muscles during a cardiac twitch is very small (equivalent to the difference between the relaxed and contracted values) in "control" (untreated cm hamsters, left panel); however, treatment with bay k increases the sr ca + load in the relaxed muscle, with little demonstrable effect on the amount of ca + remaining in the sr at peak +dt/dt (right panel, cont), resulting in a significant increase in the amount of releasable ca + in the sr store. the total ca + content measured in the relaxed bay k treated muscles was, nevertheless, considerably less than previously measured in normal papillary muscles rapidly frozen during relaxation. in summary, these data suggest that ( ) a ca + deficit, as opposed to a ca + excess or ca + overload, may be an important factor contributing to the cardiac dysfunction in cm hamsters, and ( ) that, specifically, impaired ca + regulation by the sr may result in this ca + deficit. the application of automated scanning electron microscopy (sem) to the analysis of particulate populations is in many ways a unique application of microanalytical instrumentation. when applied to the analysis of large numbers of particles, the sem is used as both a microanalytical and a macroanalytical instrument. as a microanalytical instrument, the sem provides single-particle compositional and morphologic information that is not available from the conventional macroanalytical techniques used in particle analysis such as atomic absorption and instrumental neutron activation analysis. as a macroanalytical instrument, the sem, when used for automated particle analysis, provides information from which population characteristics can be inferred. for example, automated sem information often is used to determine the percent of an aerosol that originates from a given source by extrapolating the single-particle results (e.g., the number of particles containing major fe) to the entire particle population, that is, the air filter. this value is then extrapolated to the sampled air volume. while providing the analyst with a wealth of information, the dual role of the sem in automated particle analysis has several limitations that must be considered when doing an analysis. these limitations involve, among other things, the spatial dispersion of the particle sample; particle size distribution; analytical parameters such as magnification, accelerating voltage and electron dose; and the algorithms for determining particle composition and particle groupings. at nist we have been conducting a series of experiments to study the limitations associated with the quantitative elemental analysis of particles during an automated run. specifically, we have been evaluating the relationship of measured x-ray intensity to the accuracy, precision, and detection limits of automated x-ray analyses. analytical accuracy, precision, and detection will have a pronounced effect on the ability to separate particles with similar but different elemental compositions into groups. for this experiment, we developed an analytical glass series containing six glasses with varying amounts of uranium and lead ( table i) . samples of the different glasses were prepared as bulk-polished specimens and as particles. the results from the analyses of the bulk samples represented the "best case" that could be expected under a given set of analytical conditions since there were no particle effects involved. all analyses were done on an electron probe and a sem at kev and na beam current. dead times for the bulk and particle analyses were between and %. two separate counting times were selected for the experiment , s and s. these times resulted in the x-ray peak intensities for pb and u m xrays that are shown in table ii . glass k- was used as the standard for the quantitative analyses of the different runs. for the particles, both bulk and particle forms of k- were used as standards. the concentration of oxygen was determined by stiochiometry. the results of the experimental runs on the bulk and particle forms of the glasses are shown in figure a and b as plots of the u versus pb concentrations in wt.% (normalized to % total analysis) for the s data. the bulk plot shows a complete separation of the six different glasses, while the particle results show an overlap of adjacent glasses even at the s count time. the s data show a much stronger overlap between adjacent glasses for both bulk and particle morphologies. the analytical data were also processed with a clusteranalysis algorithm to determine the average silhouette width (asw) for both bulk and particle forms of the glasses at the different counting times, (fig. ) (kaufman and rousseeuw ). the asw is a robust measure of the cluster strength with a value approaching representing the maximum association among cluster members. since there are six glasses in the series, the asw for six clusters should be the highest. of all the different runs on both bulk glasses and particles, only the s data on the bulk glasses has the highest asw for six clusters. the results of this study indicate that the uncertainties associated with the shorter counting times in asem analysis may severely limit the ability to distinguish correctly between similar groups of materials. in addition, the uncertainties associated with particle analyses are considerably greater than those from bulk analyses due to absorption and mass effects. these greater uncertainties for particles underscore the need to define the strengths and limitations of asem analysis and to design automated methods that will maximize the information that can be obtained from a sample. botany department, university of georgia, athens, georgia, usa the monoblepharidales (chytridiomycetes) produce asexual motile reproductive structures known as zoospores through the cleavage of cytoplasm in the zoosporangium. although different aspects of zoosporogenesis have been studied in a number of zoosporic fungi, little is known about the events of zoospore formation in the sporangium of the monoblepharidales, or the chytridiomycetes in general. earlier studies of other zoosporic fungi proposed various spore formation events that have been challenged recently (hyde et al. ) , especially with respect to the relationship of vesicles, golgi, and cleavage furrows. hyde et al. ( ) concluded that all eukaryotic cleavage events may need reinvestigation. we are studying the various aspects of zoosporogenesis in monoblepharella using both chemical fixation and cryofixation methods to determine whether the cleavage events in this chytridiomycete are similar to those found in the study by hyde et al. ( ) . questions that remain include how the nuclei accumulate in the sporangium, how the cytoplasmic domains are established, and how each zoospore obtains its usual complement of cellular components. mycelia were induced to form sporangia by transferring the cultures to distilled water. sporangia were chemically fixed at different stages of development using a sequential aldehyde/ osmium fixation protocol. the resulting tissue was embedded in araldite/embed . freeze substitution fixation was also used for comparison of vesicle and cleavage furrow profiles. when the tissue was to be used for cytochemical localizations, osmium was omitted and lr white was used as the embedding medium for both fixation protocols. various dyes, lectins, and antibodies were used to localize organelles within the sporangium. nuclei were close to the plasma membrane as they moved from the subtending hyphal strand to the swelling tip of the developing sporangium. the centrioles were closely associated with the nuclei and oriented toward the periphery of the sporangium. nuclei then moved a short distance from the centriole toward the center of the sporangium. microtubules, originating from the centriolar region ( fig. ), were seen around the nucleus and extended into the cytoplasm, possibly delimiting the boundary of the forming zoospore. membranes forming the cleavage furrows appeared around the forming flagella near the kinetosome (fig. ) and also at specific sites along the periphery of the sporangium. the furrows continue to expand at both sites as extending sheets of membrane. dictyosomes with large numbers of vesicles were closely associated with the nuclei, suggesting a source of the membrane needed for furrow extension. er was first seen in large sheets in the central region of the sporangium. later the strands of er surrounded the nuclei prior to ribosomal aggregation. iv- scanning vol. , supplement iv ( ) fig. nucleus (n) with associated centriole (arrow) and microtubules (arrowheads) at periphery of sporangium. fig. developing cleavage furrow and flagellum (f). note coated region of furrow (arrowhead) and radiating microtubules (arrows). hyde gj, lancelle s, hepler pk, hardham ar: freeze substitution reveals a new model for sporangial cleavage in phytophthora, a result with implications for cytokinesis in other eukaryotes. j cell sci , ( ) center of ultrastructural research, barrow hall, university of georgia, athens, georgia, usa cryptocaryon irritans, a parasitic ciliate of many species of seawater fishes, has a complex life cycle consisting of feeding, resting, dividing, and infective stages. the disease, termed white spot disease, can cause extreme fish loss in marine aquaria and mariculture environments. cryptocaryon irritans attaches to the epidermis of the fish and feeds on epidermal cells. this form of the ciliate termed the "trophont" appears as large white spots on the fish. the trophonts grow in size and eventually leave the host. the free-living trophont settles down to the substrate and secretes a cyst wall. while in this resting stage known as the "tomont," the cell undergoes a series of unequal palintomic divisions to form daughter cells called "tomites." the cyst wall ruptures and free swimming ciliates known as "theronts" are released. the theronts represent the infective stage as they search for new hosts. different procedures were used to prepare tomite, theront, tomont, trophont, and cyst stages. organisms were fixed with glutaraldehyde and osmium tetroxide for transmission electron microscopy (tem). parduc's fixation (oso +saturated hgcl) was applied for the scanning electron microscopic (sem) work. the parasite's cytoskelatal framework was stained by using indirect immunofluorescence technique. for this purpose monoclonal anti-α-tubulin was used as a primary antibody, and rhodamine-labeled goat antimouse igg as a secondary antibody. fluorescently labeled cells were examined by laser scanning confocal microscopy. the trophont has an elongate body shape with a broad anterior end, a tapered posterior end, and is completely covered with somatic cilia. the cytostome is apically located and trophonts often were observed moving with their mouth part leading. kineties were arranged in a parallel fashion along the longitudinal axis of the cell and terminated in a ring around the cytostome. there is no oral membrane at the oral region. there are cirri-like structures around the oral opening as described by cheung et al. ( ) . these cilia are shorter ( - µm) and wider than somatic cilia. the cytopharynx is surrounded by ridges or oral ribs (nonciliated lining). large bundles of microtubules support the oral region. the theronts are oval to teardrop-shaped and completely covered with cilia. they have a ventral mouth with a slit-type structure in the middle. the cytostome covers almost / - / of the body. the cirri-like structures are found around the mouth and are similar to those in trophonts. the oral ribs are present but they are layered. based on sem and tem results, it was found that there were some structural differences in the cytostomes of trophonts and theronts. the mouth probably does not become functional until the theront has entered a host. the short and stiff cilia seem to be used for burrowing and the gathering of food particles. the role of the pellicle and cyto-plasm is discussed in relation to penetration into fish epidermis. we found that there are mucocysts in both trophont and theront, but not in tomont. theront mucocysts are concentrated around the slit type structure of the mouth. the secretion of mucocysts might contain enzymes that help the theront to penetrate into fish epidermis. they also might aid trophonts in feeding on fish tissue and have a function in cyst wall formation at the later stage. penetration into the fish tissue probably is started by mucocyst secretion that either enables the parasite to stick to the tissues or to help it enter the tissue by enzymatic reaction over the irritated areas. the theront may then utilize its relatively stiff oral cilia for burrowing into the these irritated areas. after burrowing into the epidermis of fish, the theront develops its oral apparatus and increases its size. trophonts eventually leave the host when they reach a certain size ( - µm). this study is supported in part by the national aquarium, baltimore, md. over the years that the fbi has practiced sem/edxa, the technology has grown in importance to be considered an essential tool for investigative forensic exams. because of the wide variety of applications, there is a corresponding vast array of preparation methods and analytical techniques. analytic techniques and sample preparation methods are inextricably linked, and although many are routinely applied, often only imaginative approaches serve to fulfill the desired analytic result. the scanning electron microscope (sem) practitioner is expected to be knowledgeable about the applications and proficient at the methods of preparation in order to utilize the sem to its greatest advantage. the main types of analysis practiced at the fbi include ( ) visualization of structure, including surface features and internal structure, ( ) inorganic elemental characterization, ( ) particle analysis, and most recently ( ) the use of a compositional data base for identification and association. sem is a powerful complement to light microscopy (lm) for low magnification morphology characterization and is unsurpassed for applications requiring greater depth of field than are available with lm. toolmark and fracture exams can be enhanced by stereo analysis. preparation can be minimal, and several electronic signals (bei, sei) are available. the preparation of cross sections permits the study and comparison of heterogeneous materials. embedment usually is necessary to support the object during cross sectioning. hard materials generally are polished by an adaption of metallurgical polishing methods, and soft materials generally are microtomed. sectioning often is possible by manual methods, without the use of a microtome. this method can reveal complex structures such as plating layers and document laminates. the most routinely applied application of edxa is the qualitative exam. it is part of the inorganic analysis scheme for material characterization. the qualitative exam frequently is combined with elemental distribution mapping to provide "compositional pictures." particle populations often are indicative of an environment and can be used to associate an item or individual with an activ-ity. too small for individual manipulation, they are most easily sampled by adhesive lift. additional methods involving separation and concentration often are effective. compositional characteristics of materials are stored in a database to permit comparison and identification of a questioned material. standard spectra are collected and a specific peak for each element is integrated above background and ratioed to the sum peaks from all elements. this value representing % x-ray counts is stored in a "periodic table" data base including standard information. in addition, the original spectrum is stored on disk and a hard copy is filed. the standard files include metal alloys, building materials, paints, tape adhesives, fingerprint powders, and cosmetics. the reference list can be queried for comparison to an unknown for alloy matching, identification of an unknown material, or manufacturer identification. the effectiveness of this method depends upon the compositional uniqueness of the material and the variation of composition within the class of materials to which it belongs. this project is in its infancy, with only several hundred entries to date. data entry currently is manual, although software currently is being developed to extract required data from spectra automatically and to export it to the database. the need for a vehicle for information exchange has been expressed within the community of sem users in crime labs. since an electronic medium such as internet was not feasible because most forensic laboratories are not electronically linked, a "newsletter" was produced to link laboratories involved with sem in forensic science and related areas, as well as individuals in industry and academia. timely and informal, it augments the professional publications and attempts to bring practical methods, reprints from obscure journals, translations from foreign publications, and questions/answers directly to the user. hamilton county coroner's laboratory, cincinnati, ohio, usa many examinations in the crime lab involve comparing questioned material from a suspect to known material from a victim. by characterizing the material it may be possible to establish a link between the suspect and victim. our trace evidence section characterizes materials by using a combination of analytical instruments. the infrared microspectrometer is used to determine the organic constituents, and the scanning electron microscope-energy dispersive x-ray spectrometer (sem/edxa) is used to analyze the inorganic composition. this approach is routinely applied to paint particles because paint formulations include both organic and inorganic constituents. analysis by sem/edxa is very valuable when more than one layer is present in the particle. the instrument can then be used in line scan mode to analyze each layer individually without separation. in addition to comparisons, the sem/edxa is used to identify materials. in bombing cases it may be difficult to identify the explosive as well as other components. large amounts of potassium and sulfur in residues from a pipe bomb indicate black powder as an explosive. if chlorine is also present then "pyrodex," a black powder substitute, may have been used. other applications, such as matching small fractures, make use of the superb imaging capabilities of the sem. the capability of maintaining excellent depth of field at high magnifications is particularly important when matching the ends of wires that have been pulled apart. this is exactly what was done in a case of tape players that were jerked out of victims' vehicles. small fractured surfaces also have been encountered in the investigation of hit-and-run accidents when pieces of chrome trim were knocked loose and left at the scene. clearly modern instrumental means of analysis, such as the sem/edxa, are critical to the work of the forensic scientist. the increased sensitivity of the instrumentation, however, may raise questions as to the relevance of the evidence found. if a single, very small paint particle is found on the jeans of a pedestrian struck in a hit-and-run accident, could the particle be from the striking vehicle, or merely from the roadway debris at the scene? such questions indicate that increases in instrument sensitivity require sensitivity on the part of the analyst to questions of contamination and weight of evidence. research division, office of laboratories and scientific services, u.s. customs service, washington, d.c., usa the u.s. customs service laboratory system provides a variety of analytic services to control the commerce and assure enforcement of numerous regulations at the border. any item which is imported into the united states may need to be analyzed to determine the answer to any number of questions. what is the item? is the item correctly described? does the item infringe upon a u.s. patent? the scanning electron microscope (sem) and eds x-ray system can be utilized to answer some of the questions which arise. several examples follow. a u.s. company holds a patent on a feature incorporated into an electronically programmable read-only memory (eprom) cell. they allege to the international trade commission (itc) that another company is incorporating this patented feature in their eproms without the patent holder's permission. they win their case and the itc issues an exclusion order. it is at this point that the u.s. customs service becomes involved. we are charged with enforcement of the exclusion order. the incorporated feature is exceedingly small on a visible scale. how will we know which shipments of eproms should be excluded from entry into the u.s.? now the ability of the sem to produce images easily at very high magnifications comes into play. with the help of the sem, the laboratories are able to determine if the infringing feature is present or not. another u.s. company holds a patent for a denim (textile) finishing process. they bring a complaint before the itc that their patent is being infringed upon. the complaint is upheld by the itc and an exclusion order is issued. the u.s. customs service laboratory system must now develop a method to differentiate among various finishing processes in use. research at the headquarters laboratory found that a combination of imaging with a stereomicroscope and an sem could make the differentiation. the sem samples were au-pd sputtercoated with a hummer vi a sputtering system (anatech ltd.). the sputtercoated denim samples clearly showed distinctive features not easily seen with an optical microscope. a sample purported to be eelskin was submitted to the headquarters laboratory for conformation of its identity. it was thought that the product might be embossed plastic or possibly leather of mammalian origin. the top surface of the sample was au-pd sputtercoated and then examined with the sem. the features displayed by this examination were convincing evidence that the sample was indeed leather. a cross section of the sample was then prepared and au-pd sputtercoated. the sem images showed cell patterns consistent with reptilian or marine origin. another sample arrived courtesy of a foreign customs service. their inspectors had seized a large statue of a roman gladiator and his horse and chariot. the original reason for suspicion was that the declared value for the statue was much higher than one would expect for an object of this type; it appeared to be a cheap plastic statue. it had been dismantled and tested for the presence of drugs. the results were negative. using our princeton gamma-tech eds x-ray system, an elemental analysis of the exterior covering of the portion of the statue we received was performed. it showed the presence of large amounts of silver. this would explain the high declared value of the statue. when the u.s. customs service ran tests on narcotics particle detection systems, it was noted that sampling for heroin was more difficult than sampling for cocaine. a brief study of samples of each narcotic using the sem showed that in general the average particle size for heroin is less than that of cocaine. this may help to account for the sampling difficulty for heroin. allan n. walters u.s. postal inspection service, forensic laboratory, dulles, virginia, usa currently, the two main applications of sem/edax are explosive residue analysis and alloy quantitation. most explosives encountered are from improvised explosive devices (ieds) and commonly are low explosives such as black powder, smokeless powder, pyrodex, and flash powder. pyrodex and smokeless powder are commercially manufactured, while black and flash powder may be either of commercial or improvised (homemade) manufacture. the following table illustrates the composition of the common low explosives: device components are placed in the sem, and edax is performed before disturbing the residue. the resulting elemental profile is then used to guide further analyses with other instrumentation such as xrd, ftir, hplc, tlc, and chemical spot tests. sputter coating of samples is not performed so as to avoid modifying the sample and interfering with subsequent analyses and examinations. quantitative analysis is performed on alloys which are of interest to the u.s. postal service engineering and development center and is required to ensure that the materials meet the re-quired specifications. samples have included lock bodies, lock springs, keys, and lock tumblers. reverse engineering using quantitative analysis of current collectors for mobile electrification systems (mail sorting machines) has been conducted. quantitations are performed using a standardless method. scanned probe microscopy has evolved significantly over the last years. beginning with the first commercial scanning tunneling microscopes (stm) and continuing through the sophisticated "multifunction" microscopes of today, the "probe" has been one of the most critical and, in some instances, the least understood component of these systems. depending on the type and geometry of the sample, the properties of a probe can be optimized to reduce imaging artifacts. examples of this will be given using two well-known techniques, scanning tunneling microscopy (stm) and atomic force microscopy (afm). also, new innovations in probes for these two techniques (and others) will be presented. scanning tunneling microscopy, as first introduced, used a probe constructed of a chemically sharpened tungsten wire. later, it was found that a suitable probe could be formed from a mechanically sharpened wire (usually pt/ir). both of these probes proved that they could produce self-consistent images under specific sample and environmental conditions. however, problems arose with the tungsten probe while imaging in air because of the formation of native oxide. in addition, the mechanically formed probe tended to produce significant imaging artifacts if used on samples with topography > nm. in many cases the distortion produced by these artifacts made the data uninterpretable. a solution to these problems was to use a chemically etched probe (to control the shape) made from an inert material with physical properties suitable for the samples involved. musselman et al. developed the techniques necessary to chemically etch pt/ir wire into tips with a controlled geometry. these probes were capable of imaging surface structures greater than µm peak-to-valley depths with significantly reduced tip-related artifacts. this controlled geometry shape also made it more advantageous for use as a coated tip for electrochemical imaging. still, the aspect ratio of this particular probe was not suitable when imaging high-aspect ratio features such as pits in optical discs, contact vias in ics, fracture surfaces, etc. to obtain images from these types of structures, it was necessary to "machine" (in a controlled way) the probe described above. by using a focused ion beam (fib) as a machining tool, it was possible to create a probe to image the above features ( fig. ) . this fib "nanomachining" technique has opened up many possibilities for specialty probes. the majority of atomic force microscopy still utilizes the basic silicon nitride (si n ) triangular cantilever and pyramidal probe combination. the base of the pyramid is on the order of µm with the sidewalls extending upward at an approximate o angle to the apex. again, for samples with features < nm, such as mica, these probes have provided very good image repeatability. in general, however, for structures much greater than nm, a convolution of the probe and the sample surface will again occur. these probes can be modified, using the fib technique, to increase their aspect ratio significantly. because of their "hollow" design, these modified probes are still limited to topographies of ≤ . µm. most of the advances in probe manufacturing (on a wafer level) have come about by utilizing silicon as the probe material. several silicon probe types are now available, which provide significantly sharper tips with aspect ratios on the order of to . for imaging structures of higher aspect ratios, such as vias or deep trenches, longer and thinner probes are needed. these are made possible by using a combination of fib and electron beam techniques to "grow" a thin probe using an existing si n pyramidal probe as the base (fig. ). these probes are available in lengths up to µm with aspect ratios of ≥ to . utilizing a combination of fib milling of existing structures and electron beam growth, probes with lengths of - µm are feasible. an obvious problem with probes of this type (and with any long, thin structure) is "flexing" during imaging. analysis of one such probes' physical characteristics has shown that the elastic modulus is relatively low (e~ . gpa), while the coefficient of friction on most surfaces is extremely low (µ< . ). thus, probes of this type should be kept as short as possible while exceeding the maximum peak-to-valley distance to be imaged. other scanned probe techniques now in the prototype phase include thermal, magnetic force, near field optical, and probes capable of imaging undercut sidewalls. probes for all of these methods have been shown to be feasible, although manufacturing techniques to provide large quantities, reliably and repeatably, have yet to be developed. when imaging with an atomic force microscope (afm), the image resolution is a complex function of the relative tip and sample geometries. when imaging or measuring high-aspect ratio features, sharp and slender tips offer the possibility of probing down into extremely small topographical features. the most commonly used contact mode afm tips are batchfabricated si n thin-film cantilevers with an integrated pyramidal structure used as the tip. it has been shown that microtips, which are fabricated by electron beam-induced growth of carbonaceous material on the apex of the pyramid, can reduce the artifacts associated with integrated pyramidal afm tips. graph of a whisker of electron beam-grown contamination, or microtip, grown on the apex of an integrated pyramid, is shown in figure . an obvious problem with the use of a long slender microtip is the lateral deflection of the microtip as it is scanned across the sample surface. the proper use and interpretation of artifacts associated with electron beam-grown microtips demands an understanding of the mechanics of microtip deflection. it has been observed that long, slender microtips scanned over flat surfaces (rms roughness of < nm) produce hysteresis in the fast-scan direction. a model has been developed to explain the observed hysteresis loop in terms of a mechanical cantilever beam deflecting because of lateral frictional forces induced by repulsive imaging forces. this model is shown schematically in figure . by applying cantilever beam mechanics to the model of microtip deflection, a method of calculating the elastic modulus of microtip material has been developed. to find the elastic modulus of microtip material, a series of experimentally determined microtip deflection distances and the respective microtip lengths are required. microtip deflection distances have been experimentally determined for different microtip lengths on two different flat surfaces; fusion deposited boro-silicate glass and polished silicon. the elastic modulus of the microtip material has been determined from the deflection data to be approximately . gpa. once the elastic modulus has been determined, the coefficient of friction between microtip material and a sample sur-face can be calculated. the coefficient of friction between a microtip and the sample surface will indicate if the sample material is suitable for afm imaging with electron beam-grown microtips. the elastic modulus of microtip material and the coefficient of friction data lead to a better understanding not only of microtips but of electron beam-induced contamination in general. the low elastic modulus rules out the possibility of the material being diamond-like and suggests a polymeric material. the use of microtips can greatly improve image resolution; however, it is important to note that since microtip deflection increases with increasing microtip length, the microtip used to image a sample surface should be as short as possible while remaining long enough to image the largest peak-valley structure on the sample surface. the magnitude of observed microtip deflection should be reduced substantially by the use of ac mode microscopes where surface friction is less of a concern. because of their ability to achieve high resolution simultaneously in all three dimensions in a wide range of ambient conditions, scanning probe microscopes are promising candidates for performing measurements of surface topography. crosssectional and perspective views can be generated, nondestructively, at any location once an image has been acquired. surface topography measurements fall into two basic classes: position (or pitch) measurement and size (or critical dimension) measurement. the ability of a microscope to perform position measurement depends more on the quality of its design and construction than on the fundamental interaction of probing beam or stylus with the sample. size measurement, on the other hand, depends strongly on the probe-sample interaction. modern manufacturing, especially semiconductor lithography, often produces high-aspect ratio, submicron structures whose size and shape must be known with tiny uncertainties. surprisingly, stylus profilometers in the guise of scanning probe microscopes can perform some of these measurements at a level unmatched by any other type of microscope. to obtain size and shape measurements in semiconductor manufacture, we have developed a scanning probe microscope with several refinements, depicted in the figure. we use capacitance-based sensors for probe force sensing iv- scanning vol. , supplement iv ( ) cantilever and microtip at rest and for probe position measurement. , many of the samples that we scan are at least partially electrically insulating. for this reason, our microscope is used primarily as a scanning force microscope, although it is capable of operating as a tunneling microscope also. our force sensor employs a small silicon beam that pivots in one dimension about a pair of magnetically constrained ball bearings. the beam forms a pair of capacitors that both sense the position of the beam and maintain its balance with a suitable servo loop. this force-balance technique allows high-force sensitivity without sacrificing the stiffness required to resist surface forces. capacitors are also used to measure the position of the probe tip. the piezoceramic tube used as a scan actuator exhibits strong hysteresis and creep, so the drive voltage is an unreliable measure of probe position. the capacitors monitor the probe position in all three dimensions during the scan, and these data are collected along with the topograph. the most important factor determining the quality of the measurements is the shape of the probe tip. geometry alone makes the probe-sample interaction strongly nonlinear. in surface roughness measurements, a blunt probe can severely limit the range of spatial frequencies that can be detected. in scans of high-aspect ratio features, the probe shape determines what parts of the features can be measured. when scanning deep trenches and holes on a patterned surface, we use either a conical probe or a cylindrical probe, depending of what part of the feature is most important. the conical probes are made using focused ion beam sputtering of iridium. a chemical etch is used to form the cylindrical probes. if the probe shape is well known, then it is possible to determine what parts of a scan were distorted by the probe tip and, in some cases, this distortion can be removed. , the probe microscope itself can be used to determine the probe tip shape if a suitable structure is available for probe characterization. since the pioneering work of binnig and rohrer in the early s on scanning tunneling microscopy (stm), the stm has evolved into a powerful tool for spectroscopy, metrology, electrochemistry, and nanolithography. many other instruments have also evolved from the stm technology under the family of scanning probe microscopes (spms) with applications in atomic force, electric potentiometry, and magnetic force imaging. in the magnetic force microscopy (mfm) mode, the technique has been applied to the imaging of magnetic bit patterns in recording media (grütter et al. , mamin et al. ) and the mapping of static and dynamic magnetic fields of recording heads (martin and wickramasinghe ) . mfm is typically performed in the noncontact atomic force microscopy (afm) mode using a silicon cantilever which is coated with a thin film magnetic material, usually co, ni-fe alloy, or co-pt-cr alloy. the force exerted on the magnetic tip by stray fields from the sample causes the deflection of the cantilever which is subsequently measured. using a novel variation of the stm technique with a flexible iron tip, rice and moreland ( ) have also performed mfm imaging on magnetic bit patterns on a hard disk in a tunneling-stabilized mfm (tsmfm) mode. standard mfm images, however, reflect both topographic and magnetic information, with the relative strengths of each signal depending on the tip-to-sample spacing. using a differential interferometric technique, schönenberger et al. ( ) have shown that the topographic and magnetic information can be reasonably separated. in this abstract, results of some applications of the spm for topographical and magnetic force imaging of magnetic materials are presented. as the critical dimensions of magnetic devices are getting smaller, the surface topography and magnetic morphology of the recording head and media are becoming increasingly important with respect to optimization for best performance. the ability of the spm to obtain submicron topographical and magnetic information makes this an invaluable metrology and failure analysis tool for the magnetic recording industry. compared with other techniques for highresolution magnetic imaging, such as lorentz microscopy, electron holography, scanning electron microscopy with polarization analysis, mfm has the advantages of ease of sample preparation and the ability to operate in air. typical resolutions of - nm are obtainable in the mfm mode, although resolutions of - nm have also been achieved with considerable effort. in our work, topographical imaging was performed in the contact mode using a v-shape silicon nitride cantilever of length l = µm, width w = µm, thickness t = µm and force constant k = . n/m. the sensing tip at the end of the cantilever is pyramidal in shape, with a × µm square base and : aspect ratio. the tip radius is typically smaller than Å. for magnetic force imaging, the instrument was operated in the noncontact amplitude resonance mode by oscillating the cantilever and measuring changes in its resonant frequency using either phase or amplitude detection. this method of detection is responsive to the force gradient. for each point of the recorded image, the cantilever was first brought to a large tipto-sample distance of nm or greater to acquire the magnetic force image and subsequently moved close to the sample surface to acquire the topography image. this allows the simultaneous acquisition of topography and magnetic force images. a - v specimen bias was also applied to provide a linearizing and stabilizing force to the servo feedback loop. the cantilever used for the mfm imaging is a diving boardshape probe made of ( ) silicon. the geometry of the cantilever are l = µm, w = µm, t = µm and k = n/m. the sensing tip is conical in shape with a height of about µm, an aspect ratio of : , and a tip radius of < Å. the cantilever, which was sputter-coated with a Å thin-film cobalt material and magnetized inside a - gauss solenoid field, has a typical resonant frequency of khz. figure shows a three-dimensional topographic profile of a defective thin-film recording head, in which the pole tips protrude slightly from the surface, using the contact afm mode. the dimensions of the two rectangular pole tips are about µm × µm and µm × µm. the gap width was measured to be about . µm. dirt particles can also be seen and these are due to the cleaning process during sample preparation whereby the head was lightly swabbed with a cotton tip soaked in methanol. figure shows the magnetic force image of the surface of a recorded computer hard disk. the bright and dark lines represent regions of different magnetization or bits on the hard disk surface. the separation between each pair of bright-dark lines was measured to be about . µm and the track width is about µm. the striations in the topographic image (not shown), representing the texture lines, are approximately perpendicular to the recorded bit patterns. the line profile of the bit patterns in figure (not shown) is very similar to the calculated force gradient contours by mamin et al. ( ) in which both the horizontal and vertical magnetization components of the tip are are being sensed. iv- scanning vol. , supplement iv ( ) fig. three-dimensional topographic profile of a defective thin-film recording head using contact afm, in which the pole tips protrude slightly from the surface. in a good recording head, the pole-tips are supposed to be slightly recessed from the surrounding region. image resolution in traditional far-field microscopy is limited by diffraction caused by the primary aperture in the optical system of the microscope being used. in practice, diffraction limits lateral resolution to approximately λ/ , or about . micron for optical microscopes. to image smaller cellular structures and biological materials which are smaller than . micron, biologists have used microscopies which "illuminate" the specimen with radiation of smaller wavelength, such as an electron beam (i.e., using an electron microscope). the nearfield scanning optical microscope (nsom) breaks the diffraction limit to lateral resolution by illuminating (or collecting light from) the specimen through a sub-wavelength aperture which is held a small fraction of a wavelength above the surface of the specimen. using this nearfield technique, resolution far better than λ/ has been demonstrated on biological specimens and resolution better than λ/ has been demonstrated on standards (betzig and trautman ) . with nsom we have recently obtained optical images of tobacco mosaic viruses ( nm diameter) and of photoreceptor rod outer segments at a sub-wavelength resolution. betzig e, trautman jk: near-field optics; microscopy spectroscopy and surface modification beyond the diffraction limit. science , in june , the initial incident involving a report of a syringe found in a canned pepsi ® product received nationwide publicity. during the following months, law enforcement authorities investigated hundreds of additional claims of tampering from nearly every state in the united states. more than cases involving foreign objects allegedly found in soft drink containers were processed by the national forensic chemistry center (nfcc). to date, the forensic and law enforcement efforts of the fda have resulted in numerous arrests and convictions for charges related to product tampering and felonious reporting of product tampering. scanning electron microscopy (sem) has been valuable, and in several cases crucial, in providing conclusive evidence of fraudulent reports of product tampering. the items reportedly found inside of suspect cans were primarily hypodermic syringes (with and without needles), but other reported objects included nails, screws, bullets, pins, sewing needles, tacks, glass and plastic shards, and rodent carcasses. since most of the cases involved syringes, primarily insulin-type syringes, the first analyses were designed to determine if the syringes were contaminated and if they contained human blood, tissue, drug, or insulin residue. syringe/needle rinses were examined by a number of techniques including sem, which was used to find any intact blood cells and/or tissue. energy dispersive x-ray analysis (edxa) was performed on residue preparations. edxa was able to detect a small k αl peak for zinc in several syringe rinses which was consistent with edxa analysis of dried residues containing some types of insulin. type identification of insulin from syringe rinsing was performed by lc/mass spectrometry in a method developed at nfcc. in another case, a broken pin was reportedly found in a soft drink can. a cross-sectional analysis of the suspect pin by edxa showed the pin was a nickel-plated, iron-core straight pin. the absence of observed iron corrosion provided evidence that the object had not been in contact with the soft drink from the time the canned product was produced until the time the pin was allegedly discovered. the most common question requested by investigators was "how long had the syringe been in the soft drink can?" to answer part of that question, a number of experiments were conducted at nfcc. one sem/edxa method investigated the corrosion of the aluminum crimp (found on some syringe needles) submerged in diet cola. new aluminum crimp needles were sealed in diet cola and removed at -h intervals. stereoscopic light microscopy was initially used to examine each needle crimp. it was discovered that the submersion produced a brown to black "corrosion flower" in less than h of soaking. of specific interest, the submersion repeatedly produced a single point of corrosion on the crimp. the single point of corrosion suggested possible electrolysis of the aluminum crimp in the soft drink. this corrosion point continued to enlarge and penetrate more deeply into the aluminum crimp over time. backscattered electron (bse) imaging and x-ray mapping were used to highlight the region of corrosion in the sem. figure shows the secondary and backscattered electron image (sei and bei) of an aluminum crimp on a syringe needle after h submersion in diet cola. the sei shows the corrosion of the aluminum crimp as pitting in the crimp surface. the bei shows the corrosion area as a darker region caused by surface oxides blocking bse generation from the aluminum below. the study also demonstrated that the corrosion of the aluminum crimp was dependent upon the amount of time the crimp was submerged in the pressurized soft drink container. figure shows a point of corrosion on an aluminum crimp after weeks of submersion. at this time interval, several points of corrosion often developed. the pitting of the aluminum mater-ial immediately beneath the corrosion was extensive. sem/edxa analyses have shown that the aluminum crimp on syringes submerged in diet cola produced a characteristic single point of corrosion after only h. the corrosion resulted in damage to the crimp surface and the production of a metal oxide. the location of the oxide was identified and plotted by x-ray mapping for oxygen (via edxa). the analysis of samples related to the pepsi "tampering" cases of are continuing to date. in many cases, the circumstances and sample condition are unique and generated specific questions which require further forensic research using additional time-related studies, multielement x-ray mapping, and/or image analysis. confocal microscopy can be used to investigate the properties of rough surfaces. based on the kirchhoff approximation (sheppard et al. a,b) , the three-dimensional ( -d) confocal image can be modelled using the -d coherent transfer function (ctf). the form of the -d ctf for confocal reflection and transmission have been derived using a high-angle scalar theory (sheppard et al. ) . in both cases the ctfs can be expressed analytically. using these allows the profile of the rough surface to be reconstructed. in many cases we need to know only the statistics of the rough surface, rather than the actual profile. ways in which the statistical properties can be extracted directly have therefore also been considered (sheppard iv- scanning vol. , supplement iv ( ) fig . a. castenholz in previous in vivo studies based on vital microscopic and fluorescence microscopic techniques it was possible to observe lymph flow in initial lymphatics and regional lymph nodes. [ ] [ ] [ ] [ ] as an expression of immunological mechanisms, the studies carried out in the rat tongue under various issues showed some remarkable phenomena such as cell traffic along lymphatic pathways and phagocytotic activity of the lymphatic endothelium. since these phenomena could not be defined with traditional fluorescence microscopy on the cytological level, confocal laser scanning microscopy (clsm) was applied to the living tissue and fixed specimens). moreover, in morphologic studies on the lymphatic and blood vascular system, clsm has proved a very suitable tool also for the representation of resin-injected tissue, which is commonly processed as corrosion casts for scanning electron microscopy. all these approaches, also including the application of special fluorescent markers, should be outlined here. in vivo studies with the clsm have been designed for the functional morphologic analysis under normal conditions and in a state of inflammation and experimental edema. after staining the lymphatic endothelium with dimethyl-carboxfluorescein, clsm revealed a distinct pattern of the so-labelled initial lymphatics consisting of bright (cytoplasm) and dark zones (nuclear portions). for labelling tissue macrophages passing the lymphatic pathways and other phagocytotic cells, fluorescent microbeads (latex standard particles and liposomes) were applied by interstitial injection. thus, clsm enabled a certain identification of moving particle-laden cells and also gave evidence of phagocytotic activity of the endothelium of initial lymphatics. in long-term experiments, phago-cytosis of cells lining the sinuses of lymph nodes could be clearly recognized. tissue (tongue, skin) and lymph nodes from different sites of the rat were examined in the clsm as unfixed or fixed (glutardialdehyde) thick sections. cells labelled with fluorescent microbeads or stained with fluorescent dyes thus could be easily detected and identified. thereby, it was possible to determine the location of single beads at the endothelial surface or within the endothelial cytoplasm (fig. ). if the tissue was conventionally stained with hematoxilin eosin or acridine orange, optimum information could be obtained from unlabelled structures in these specimens as well. by means of the two-detection system, clsm also was able to distinguish two or more different fractions of microbeads incorporated by macro-phages ( fig. ) or lying in tissue spaces as free elements. in hemal lymph nodes of rats, erythrophagocytosis related to sinus macrophages was successfully represented by the clsm after vital staining of erythrocytes with the fluorescent celllinker pkh . in this approach, mercox (acrylate) stained with rhodamin and fluorescent yellow was used to create high fluorescence in the lumen of blood or lymphatic vessels. if the resin was injected into the tissue, the interstitial spaces were filled up by resin. clsm of sections from such resin-injected specimens enabled exact distinction between casts related to the blood or lymphatic system and those of the interstitial spaces, when two differently stained resins were simultaneously injected from the arterial system and into the interstice. clsm of resin-injected specimens was also applied as a suitable method for the control of corrosion casts of sem. proceedings of scanning /seems iv- today, clsm has become an established tool in many fields of biological sciences. because of its abilities to produce images with optimum resolution and clarity, the application in experimental lymphology is striking as well. some experiments in techniques related to in vivo experiments, supra vital and fixed tissue, and resin-injected specimens have been reported here. these approaches, also comprising the application of new fluorescent markers for living cells, may demonstrate how wide the spectrum of application is spanned for clsm in experimental lymphology and immune research. this study was supported by a grant of dfg (deutsche forschungsgemeinschaft, bonn) daniel chin, ph.d agouron institute, la jolla, california, usa the regulated metabolism and distribution of nucleic acids is required for endogenous antisense or rna regulatory systems. recent interest has focused upon using exogenous agents as antisense therapeutics to treat viral infections or metastatic diseases. both endogenous and therapeutic antisense regulation of gene expression requires the formation of a hybrid between the antisense molecule and the message or gene sequence. it is not surprising that detection of such hybrids in living cells has not been reported to date. we have used fluorescence resonance energy transfer (fret) to study hybrid formation and dissociation after microinjection of oligonucleotides (odns) into living cells. two systems were examined: one system characterized the kinetics of hybrid dissociation of two synthetic odns while a second system examined the distribution of hybrid complexes formed by hybridization of injected odns with endogenous mrna. in the first system, a -mer phosphodiester odn (+pd) was synthesized and labeled with a ′ rhodamine (+pd-r). the complementary, antisense ′-fluorescein labeled phosphorothioate odn (-pt-f) was specifically quenched by addition of the +pd-r, as detected by both absorbance and fret in solution. rapid and specific hybridization between the odns occurred at µm within min and the preformed hybrid slowly dissociated (t / ≈ h) in the presence of a -fold excess of the unlabeled odn. upon microinjection into the cytoplasm of cells, preformed fluorescent hybrids dissociated with a halftime of min, which is attributed to the degradation of the phosphodiester. formation of the hybrid from sequentially injected odns was detected by fret transiently in the cytoplasm and later in the cell nucleus, where nearly all injected odns accumulate. diffuse, specific nucleoplasmic fret could be distinguished from nonspecific fret in punctate nuclear structures which may result from concentration effects similar to fret seen in late endosomes or lysosomes between endocytosed, inert fitc-dextran and +pt-r odn. a second set of experiments with two adjacent -mer pt odns complementary to mouse b-actin mrna was performed in mouse t fibroblasts or n neuroblastomas. the upstream odn was ′ labeled with fitc and the downstream odn was ′ labeled with rhodamine. when hybridized to synthetic actin mrna or a complementary -mer pd odn, quenching of fitc and fret was observed in solution. injection studies in t cells showed transient odn accumulation and fret in filopodia and extended processes. in n neuroblastomas, fitc quenching was dependent upon cytoplasmic compartmentalization. after min, both odns accumulated into the nucleus. in summary, these experiments suggest that antisense odns can hybridize to intracellular targets in both the cytoplasm and the nucleus. the goal of this work was to increase the sensitivity of the electron backscattering pattern (ebsp) technique by introducing an electron energy filter to increase contrast and improve pattern visibility. energy filtering previously has been shown to increase contrast in selected area channelling patterns (joy ) . energy filtering differs from arithmetic background subtraction in signal processing, because filtering before detection has a physical basis and eliminates the unwanted background without increasing the noise component in the signal. the electrostatic retarding filter used in this experiment was constructed as a cone with five coaxial electrodes. the primary purpose of the electrodes was to create symmetric fields that would not distort the pattern, yet allow only backscattered electrons with the least energy loss to contribute to the pattern. analysis (courtesy e. munro, micro electron beam software, ltd.) of the electron trajectories indicated that for kev electrons, filtering voltages of up to kv could be used before chromatic aberrations became unacceptable. the angular field-of-view of the filter was approximately o . another feature of the detector is the use of a microchannel plate as the initial sensing component. microchannel plates have high gain and are very sensitive to the low-energy electrons that pass through the retarding field filter. in a configuration similar to that of venables ( ) , the output of the microchannel plates was proximity-focussed to a phosphor screen that displayed the microdiffraction patterns. the gain of the assembly exceeded a simple phosphor by more than times. the light output of the assembly was focused on a kodak megaplus . ccd camera using either a macrolens alone or a hybrid fiber optic-lens combination. no vignetting was observed across the field. the digital output of the camera was displayed and analyzed on a macintosh iix using nih image. results were obtained on a variety of materials including single crystal silicon, al-sic metal matrix material, alumina, aluminum foil, and aluminum used in aluminum cans. the high voltage design limited the maximum retarding potential to − kv so that meaningful data were obtained with incident electron beam energies between and kev. filter potentials above % of the primary beam accelerating voltage increased the kikuchi band contrast relative to the background. figure contains a very low contrast pattern for alumina obtained with essentially no filtering. the effect of filtering to within kv of the beam accelerating voltage is shown in figure for the same specimen. the best patterns were obtained when the filter voltages were between and % of the accelerating potential. filtering above % degraded pattern resolution while less filtering usually produced less contrast. up to four-fold improvements in the contrast ratio were achieved on some specimens. the detector also had the ability to obtain energy-filtered backscattered electron images in sem raster mode. one advantage of this configuration is that the sem characterization of a given feature and the ebsp data obtained from it can be obtained from the same perspective. another is that filtered im-ages appeared to have improved surface detail and greater crystallographic contrast. venables ( ) showed that a detector of this kind could be used for "dark field" sem imaging. this work demonstrated the feasibility of employing an energy-filtering detector with high gain to increase the sensitivity of microdiffraction measurements in the sem. further work is planned to optimize the filter and improve pattern quality and the range of filtering voltages. the authors gratefully acknowledge the contributions of munro electron beam software, ltd. and of mark vaudin at nist, as well as the support by the national science foundation under award number iii- , and the department of commerce under contract number -dkna- - . submicron-sized elements are of great interest in physics and technology. there exists a variety of methods to produce them by using conventional microelectronics technology as well as new methods for deposition of such elements directly from the desired material onto a substrate. the current onestage maskless techniques, for example, laser-induced cvd or laser-induced etching , do not ensure resolution below nm because of their physical limitations imposed by the laser wavelength. focused ion beams, which generally allow the formation of submicron-sized elements, often are unacceptable as they can cause radiation damage in the material. moreover, the cost of the necessary equipment is high. these circumstances stimulate the development of techniques for direct formation of submicron-sized patterns with the necessary geometry from an arbitrarily chosen material directly at substrates based on electron-beam-induced cvd . the cvd experiments were performed in a temscan jem- cx electron microscope at an accelerating voltage of kev and a beam diameter ~ nm. the electron microscope was equipped with a specially designed pressure cell and an oil-free system for pumping and reagent vapor inlet. this allows to maintain a well-defined atmosphere of chemical compounds (metal carbonyls and halides, freons) around the specimen. two electronically controlled needle valves are capable of supplying two different reagents simultaneously into the specimen holder. the sample itself is placed inside a heater which provides temperatures up to ˚c required for the local electron-beam-induced etching. contacts are made to the sample, which allow in situ measurement of its electrical characteristics. the holder is supplied with two differential apertures (through which the electron beam passes) placed above and beneath the sample. these apertures keep the gas pressure around the sample up to pa without breaking the vacuum in the microscope column. w(co) and rez(co) were used as reagents. structure and composition analysis both were performed in a jem- fx electron microscope equipped with a link an- / s system for edx analysis. self-supporting rods of - nm in width, containing tungsten or rhenium, respectively, were grown up to nm long at a speed of the electron-beam movement - nm/s and at a vapor pressure near the sample of . pa. provided a constant speed of the beam is maintained, the thickness of the produced rods was found to be inversely proportional to the beam speed but decreasing towards the rod's end. this result is similar to that obtained by electron-beam-induced fabrication of self-supporting carbon-containing rods electron microscopic observation of the inner structure of these self-supporting metal-containing rods revealed many individual fibers mutually aligned in parallel. thus, the mor- iv- scanning vol. , supplement iv ( ) fig. tem image (a) and sad pattern (b) from a self-supporting tungsten containing rod section after annealing in vacuum at ˚c for min. the accelerating voltage is kv, the diffraction length on the (b) is cm. phology of these rods is similar to that of the well-known carbon rods . this observation suggests similar formation mechanisms independent from the rod material. selected area diffraction (sad) patterns show the prepared rods as being amorphous. in situ annealing of the tungsten-containing rods at ˚c for min in the heating holder inside the microscope column transforms the amorphous structure into a nanocrystalline one (fig. ) . sad patterns reveal the presence of a set of different phases: in addition to pure crystalline tungsten there are various tungsten-containing compounds. among them, in particular, two new cubic phases with lattice constants ± pm and ± pm could be identified. after annealing chemical and phase compositions of the rods grown from w(co) and rez(co) were found to differ depending on the conditions of their formation (reagent vapor pressure, electron current, beam speed). the rods of nano-sized width, grown from rez(co) are considered to be promising as tips for scanning tunneling microscopes because of their chemical stability. an important requirement for developing high-speed highpower devices is fabrication of thermally stable ohmic contacts with low resistance and smooth interfaces. metal semiconductor interface inhomogeneities, such as lateral interface phases and spiking protrusions, lead to nonuniform current flow and consumption of the gaas substrate. this type of interface morphology is not acceptable for device applications where a large electric field or shallow contact is required. in particular, devices such as a heterojunction bipolar transistor (hbt) cannot tolerate contacts with lateral and vertical interface inhomogeneities. recently, we have introduced a novel metallization scheme, pt/ti/ge/pd, which yields thermally stable and low resistance ohmic contacts to both n and p + -gaas (han et. al. ) .to insure processing reproducibility and contact reliability, one must identify and understand the mechanisms responsible for this contact's electrical per-formance. this study employed cross-sectional transmission electron microscopy (tem), auger electron spectroscopy (aes), and electrical measurements (transmission line mode) to investigate the structural, chemical, and electrical properties of this contact. the interface morphology, phase composition, and elemental diffusion were examined and correlated with the measured contact resistances at annealing temperatures which yielded the best slight degradation and the worst electrical performance. annealing at ˚c yielded the lowest specific contact resistance, ~ . × - Ω-cm . the metal-semiconductor interface was planar and structurally abrupt. the pd and ge reacted to form a pdge phase. directly beneath the pdge was a thin, discontinuous, ga-rich pd-ga-as ternary phase. the presence of this ga-rich ternary compound has important implications for contact formation on the n-gaas substrate. formation of this interface phase creates excess ga vacancies in the gaas substrate. upon heating, ge diffuses into the gaas, occupying ga vacancies at dopant levels, to form an n + surface layer, thus allowing considerable tunneling at the metal-semiconductor interface. the existence of the ga vacancies is crucial, because otherwise ge would not readily diffuse into the n-gaas to form this n + -gaas layer. for the p + -gaas, this ge indiffusion occurs also, but the ge concentration level is not such that it has a detrimental effect, that is, it does not approach that of carbon (the p-type dopant) ~ × cm - . the ti/pt layers remained stable, hence no surface degradation was present. annealing at ˚c resulted in a slightly higher specific contact resistance, ~ . × - Ω-cm . there was significant elemental diffusion within the contact metal and minor elemental diffusion into the substrate. the interface exhibited a roughness on the order of nm and possessed large areas where spiking single-phased pdgega protrusions spatially dominated the metal-semiconductor interface region. the nonplanar nature of the interface lends itself to several explanations: ( ) nonuniformity in the heating associated with the annealing process, ( ) material defects or pitting in the gaas substrate such that the contact metallization covered and filled the pitted area as it would a flat gaas surface, and/or ( ) new phases being formed as a result of the ti layer beginning to break down as a diffusion barrier. apparently, this interface nonuniformity (spiking) does not cause significant deterioration in the specific contact resistance, since the contact resistance maintains reasonable electrical integrity. it is speculated that the compositional uniformity of the protrusion phase, which is close to that of the pdge phase formed during the ˚c anneal, is responsible for this behavior. annealing at ˚c proved to have a detrimental effect on the specific contact resistance, ~ - Ω-cm . this degradation was accompanied by strong chemical intermixing between the contact and the substrate, resulting in laterally continuous and vertically deep (> nm) multiphased protrusions spiking into the gaas substrate. the surface of this contact possesses surface anomalies (~ . µm in size) with a density of ~ %. the anomalies are somewhat enriched in pt and as and depleted in ge. this nonuniform surface morphology demonstrates that pt no longer represents a smooth surface for bonding. our results demonstrate that annealing temperatures between - ˚c are of practical interest for hbt device processing. the thin base region ( Å) and narrow distance between the contact and emitter ( . - . µm) make the ˚c anneal impractical for the hbt. specifically, the composition, extent, and magnitude of the interface spiking would be totally detrimental to this device design, that is, spiking areas would penetrate through the base region, into the collector, and short the device. the present paper is a review of diagnostic availabilities as well as of physical data taken by color cathodoluminescence scanning electron microscopy (ccl-sem) used for investigation of structural, polytypic, and impurity inhomogeneities of sic crystals, epitaxial layers, and devices (saparin and obyden ) . as examined objects, the "alpha" and "beta" sic crystals, different polytypes ( h, h, c, r) sic epitaxial layers and devices have been studied. the epitaxial layers were grown by the sublimation "sandwich" method (vodakov et al. ) in the vacuum or ar-media under temperatures between and ˚c. from results of experimental studies it is possible to enumerate the following that are impossible to prove by other techniques: ( ) space distribution of impurities; ( ) static and dynamic characterization of polytype transformation. multitransformation of polytypes during the growth process was observed by way r → c → h. it was concluded that the growth conditions of epitaxial layers and surface perfection of the substrate markedly influence polytype variations. ( ) space distribution of radiation defects on dependence of annealing temperature; ( ) action of mechanical defects on the spatial distribution of luminescence centers; ( ) availability to observe the transient layers in sic devices; ( ) -d analysis of epitaxial layers with polytype transformation. saparin gv, obyden sk: colour display of video information in scanning electron microscopy: principles and applications to physics, geology, soil science, biology, and medicine. scanning , - the examination of chemical vapour deposition (cvd) diamond films by the scanning electron microscope (sem) (secondary electron mode with spatial resolution - nm) shows that the morphologies of films are strongly affected by the synthesis conditions, especially the substrate temperature, the methane concentration, total pressure in the deposition chamber, etc. studies demonstrate that the changes of ch for concentrations in the range of . - % vol. create the shape variation of crystals determined by the ratio of the apparent growth rates of the ( ) and ( ) faces r( )/r( ) in cubo-octaedrons interval from . - . . the exact cubooctahedral shape observed very frequently is determined by the value of ratio . and preferable orientation of the ( )-or ( ) faces. thus, the secondary electron mode of the sem is a very useful diagnostic technique for diamond films. less frequently researchers use the second diagnostic availability of the sem (saparin and obyden ) : cathodoluminescence (cl) mode (spatial resolution . - . mkm) with monochromatic and panchromatic (real color) images and local cl spectra. diagnostic possibilities of this mode identify the quality of diamond films in comparison with natural diamond. the cl spectra of undoped epitaxial films show the dependence on the face upon which the epitaxy was done. cl discriminates between different types of diamond. there are distinct spectral differences between natural, synthetic diamonds and cvd films. the cl emission of the a-band was associated with donor-acceptor pair recombination (blue and green regions); vacancy-rich material emits in the red spectral region ( - nm). we would like to note that the cl spectra of natural diamonds (types ia, ib, iia, and iib) lie in the range of - nm. variations in these cl spectra were attributed to differences in the defect structures formed during the growth of material. cl images and local spectra allow one to recognize, for doped and undoped films, the small amounts of impurities, such as al( ppm) and b( . ppm) and nitrogen remaining in the gas, which could be a possible source of donors. thus, the sem investigations of morphology, crystallinity, and cl emission of films led to the correlative estima-tion of diamond film quality in comparison with the natural diamond as standard. the etching process of a crystal surface during its dissolution is well-known. the process of etch pits formation, as a result of crystals dissolution, usually is explained by scientists by the presence of dislocations inside the crystal structure. however, the same process occurring near the crystal edges is studied less, although such investigations can give additional scientific knowledge for a better understanding of the dissolution mechanisms of solids. this brief communication continues the series of investigations (dorozhkin , dorozhkin et al. the results show that here can be various surface structures taking place under geometric interaction among the growing etch pits and dissolving fap crystal surfaces. as the fap has a hexagonal crystal structure, the growing etch pits mainly have a hexagonal shape also (dorozhkin , dorozhkin et al. ). on the other hand, the investigated fap crystals were obtained from natural fap-containing rock after its disintegration and concentration stages. therefore, the crystals had a very irregular shape and a lot of dislocations inside. figure shows an example of a typical hexagonal etch pit having only three faces instead of the necessary six; three lacking faces (left side) have already been dissolved by the acid. a very similar situation is presented in figure . this is another example of a former hexagonal etch pit having only three faces (center). it is easy to restore events which occurred with the pits some minutes before. the pits began to form and grow from the moment when the outputs of dislocations on the fap crystal surface began to interact with the acid solution. as the pits are always faced only by fast dissolving faces, their dissolution rate is greater than the one for the crystal surface in common. on the other hand, the growing of etch pits and the fap crystal surface dissolution are always occurring simultaneously. as a result, a layer of fap substance between the growing pit, which is situated close to any vicinal dissolving crystal face, and the vicinal crystal face itself were getting increasingly thinner. finally, this layer disappeared completely, resulting in the pits' formation having only three faces (figs. , ) . if one had dissolved the fap crystal for a few more seconds, its surface would have reached the bottom of the pits and they would have disappeared entirely. figure shows an example of interaction between etch pits with an irregular shape and a surface of the fap crystal. for this purpose a very thin and sharp spallation fragment of the fap crystal was chosen which was then etched by phosphoric acid solution. inasmuch as the spallation fragment was equal to a random and unknown crystallographic face, the irregular etch pits were obtained. it is easy to see four different states of such pits (fig. ) : they point to a pit which ( ) ( ) is only running through the fap crystal but is situated relatively far from the dissolving crystal face. finally it should be noted that all the above-mentioned cases of interaction among pits and crystal surface may only take place when directions of dislocations inside the dissolving crystals are close to lines parallel to any nearest vicinal crystal face. k. habib and p. g. caceres* materials applications department; *central analytical laboratory kisr, safat, kuwait a fundamental study of co-based metallic glasses has been conducted. the study focused on understanding the changes of the properties and structures of an fe-b-si glass as a function of co, co-ni, co-mn, and co-ni-mo additions. the separate addition of co, co-ni,co-mn, and co-ni-mo elements was successful in a way four new metallic glasses were produced. the compositions of the new glasses are fe co b si, co fe ni b si , co fe mn b si , and co fe ni mo b si . consequently, an evaluation of the physical and magnetic properties was determined. furthermore, the internal and surface structures of the glasses have been characterized by a transmission electron microscope and a scanning tunneling microscope, respectively. a comparison between the internal and surface structures of the glasses was carried out on both amorphous and crystallined forms. as a result, a correlation between the properties and structures of the glasses is established. for instance, figures a and a show surface structures of the co fe ni mo b si metallic glass in the amorphous and annealed crystalline forms, respectively. on the other hand, figures b and b show the corresponding x-ray diffraction patterns of the amorphous (fig. a) and the annealed structures (fig. a) , respectively. figures a and a are basically three dimensional line plots of the surface profile. it is clear that the amorphous structure (fig. a) represents a complete rough surface along nm × nm scanning area. in contrary, the annealed structure (fig. a) exhibits a surface profile with less surface roughness than the annealed structure. this observation is in agreement with work done by the author on other metallic glasses cited elsewhere (habib et al. ). the current status of external funding for most academic and research facilities throughout this country is meager at best. many institutions are being forced to seek financial support from sources other than the conventional governmental agencies, private funding, or contractual agreements. this facility has, for the past years, derived the bulk of its operational budget through third-party payments for services rendered as hospital charges for diagnostic pathology services. while we have been fairly successful in maintaining a relatively consistent level of service, the overall cost of this operation continues to rise. as cost containment became the buzz word and hospital admissions declined, the number of requests for diagnostic procedures also began to decline. this pattern was observed not only in the electron microscopy (em) lab but in many other specialized laboratories throughout the hospital and our affiliated clinics. as the health care debate accelerated and new concepts such as hmos, emergency care clinics, managed competition, and regional alliances all geared to assure "more health care for the dollar came into being," it became apparent that an entire new concept was needed to provide high-quality diagnostic services for these newer group practices and smaller clinics that were being created to meet these new demands. the diagnostic referral service * has been established in the department of pathology, medical college of georgia, to offer a range of state-of-the-art diagnostic services to external referral sources, including private practitioners, group practices, pathology laboratories, and hospitals. these specialized services are designed to supplement other routine analyses and assist in the diagnosis, prognosis, and clinical management of patients with a wide range of diseases. the laboratories involved are fully cap-accredited, and all diagnoses are evaluated by a pathologist certified by the american board of pathology. the following is a listing of the laboratories contributing to this endeavor and a brief summary of some of the diagnostic offerings. this laboratory provides cytogenetic analyses to identify specific chromosomal abnormalities. these are useful not only in establishing a diagnosis of malignancy, but also in classifying certain malignant disorders, monitoring remission and progression, deciding on treatment regimen, and estimating prognosis. chromosomal analysis can be conducted on bone marrow aspirates, peripheral blood, and lymph node biopsies. this laboratory offers standardized transmission electron microscopic analysis of biopsies from any organ or tumor, as well as specialized procedures for examination of cell suspensions such as bone marrow and lung aspirates and blood samples. em analysis is particularly useful in conjunction with light and/or immunohistochemical microscopy for the diagnosis of tumors which cannot be classified by conventional light microscopy. working in close collaboration with the histology laboratory, the immunohistochemical laboratory, and the renal biopsy service, the em laboratory provides standardized, reproducible ultrastructural analysis which allows a consistent comprehensive approach to the interpretation of biopsy pathology. immunophenotyping by flow cytometry details the presence or absence of surface antigen markers of cellular maturation which define cellular subsets present in specific forms of leukemia and lymphomas. this technique provides precise information to aid in the diagnosis, prognosis, and clinical management of patients thought to have varying forms of lymphoproliferative disorders. in addition, quantitative dna ploidy and cell cycle analysis in combination with standard histopathologic and cytochemical methods provides the most comprehensive assessment of clinicopathologic status, tumor aggressiveness, and the likelihood of disease progression for patients with breast, colon, and ovarian cancer. this facility performs state-of-the-art diagnostic immunohistochemical and in situ hybridization techniques for the morphologic analysis of cellular and molecular events. these techniques are adjuncts to histopathologic examination and can be applied to the study of neoplastic, infectious, and other diseases. the laboratory offers more than immunohistochemical markers as special decision-making tests to resolve differential diagnoses. special histochemical stains and molecular probes for colorimetric in situ hybridization are also provided by this laboratory. this laboratory offers a full service for examination and consultation on kidney biopsies involving a wide range of disease processes. in addition to routine histopathology, all renal biopsies are examined by immunofluorescence for the identification of immunoglobulins, albumin, and c deposits, and by transmission electron microscopy for the detection of early or submicroscopic abnormalities. this laboratory presently offers complete dau screening and gc/ms confirmation for pre-employment/employee drug testing. upon completion of certification as a forensic urine drug lab, this will be the only such certified lab in this area. department of pathology, duke university and va medical centers, durham, north carolina, usa microprobe analysis in biomedicine is usually done on an electron microscope (em) equipped with an energy-dispersive x-ray detector (edx). this type of analysis is commonly referred to as electron probe microanalysis (epma). there are also other fundamentally different new techniques for microprobe analysis which involve the use of laser or ion beams. however, these are not yet commonly employed for diagnostic studies (ingram et al. ) . whereas biomedical epma primarily used to be a research tool, that is no longer the case. epma findings now often have diagnostic, therapeutic, and/or legal significance for the patient (baker et al. , shelburne ). in addition, since much of the current work involves conditions such as the pneumoconioses, the findings frequently have public health and/or industrial medicine implications as well as implications for single patients (shelburne et al. ) . currently the most commonly studied clinical conditions include the pneumoconioses, especially asbestosis and related conditions, "hard metal" pulmonary fibrosis, and other min-eral-induced pneumoconioses (roggli and shelburne ). a second major application is the use of this technology for the analysis of stones, particularly renal stones. microprobe analysis can be more sensitive than x-ray diffraction or chemical techniques, particularly for the identification of small components of complex stones. another major application is the use of microprobe analysis to identify unexplained pigments or deposits and to study unexplained granulomas (kupke et al. , pickett et al. . as is evident from the foregoing discussion, most applications involve the study of insoluble particulates. accordingly, conventional histologic processing with chemical fixation and paraffin or plastic embedding is acceptable. an obvious limitation of this approach is that electrolytes cannot be studied. currently we are exploring the feasibility of utilizing flash freezing techniques to permit studies on electrolytes utilizing cryoultramicrotomy. one way to gauge the usefulness of this technology is to study the use of microprobe analysis in a single hospital system, that of the veterans administration medical centers. currently there are va medical centers in the united states. within these hospitals there are diagnostic electron microscopy laboratories. of these, only five currently utilize epma as a diagnostic technique. at each of these laboratories, conventional transmission electron microscopic studies are the predominant type of analysis. epma studies constitute less than % of our electron microscopy laboratory workload. nevertheless, as the chemical information available from epma is better understood by clinicians, and as cryotechniques are shown to be useful, we anticipate increased usage. in conducting these studies, it is important to be aware of several artifacts that are common problems. the major types are those caused by poor specimen fixation. not only does traditional chemical fixation remove electrolytes from the tissue, it is common in electron microscopy laboratories to add heavy metals such as osmium, uranium, and lead. these elements may produce peaks in the final spectrum that can obscure important elements of physiologic significance. for example, the m-alpha line for lead obscures the k-alpha line for sulfur. in conducting an epma study, it is important to identify all peaks obtained so that the investigator is not mislead by a contaminant. in addition, it is important to utilize several controls. the investigator should not only probe the feature of interest, but also the cytoplasm adjacent to that feature and the blank stub. only in this manner can artifacts contributed by, for example, metal in the microscope column be understood and eliminated from consideration. all living things are infected/affected by viruses. whether the subject is a tissue culture, an animal being used in research, or a human, it behaves differently when infected with a virus. in research subjects virus identification is important to prevent erroneous data due to the presence of a foreign organism. in the case of human viral illness, it is increasingly important to identify pathogens so that appropriate viral therapy can be initiated. several antiviral agents are already on the market, and many more are presently in clinical trials. advantages of using electron microscopy (em) in viral diagnosis are that it is rapid; specific standards and reagents are unnecessary; and infectious particles are not required. disadvantages include the facts that a fairly high concentration of virions must be present in liquid samples to visualize them, and that solid tissues may have focal infections that must be included in the sampling. identification of viruses by direct em is performed by two techniques: negative staining of liquid samples and thin sectioning of tissues, cells, and tissue cultures. negative stains most used in virology are phosphotungstic acid (pta) and uranyl acetate, although many others have been described. for thin sectioning, any fixation method used successfully for em of tissue will preserve viruses; this includes some form of glutaraldehyde and osmium fixation, usually followed by uranyl acetate. detailed preparatory techniques have been described. in negative stains, morphology questions used in identification are: is the virus naked (always icosahedral) or enveloped (pleomorphic); if naked, what is its size, and does it have distinguishing capsid (outer) markings; if enveloped, does it have visible spikes or fuzz around the outside; what is its size, and is the nucleocapsid (the core) visible in particles that have been penetrated by stain; what is the shape of the nucleocapsid, if visible? naked human viruses are all icosahedral; these pathogens fall into three size ranges: - nm, - nm, and - nm. the small viruses may or may not have surface substructure; those that do not are not morphologically distinguishable and have been referred to as small round viruses (srv) (fig. a) if smooth, or small round structured viruses (srsv) if rough. others may have characteristic surface markings that permit precise morphologic identification. the medium-sized (fig. b) and large (fig. c, d) viruses are identifiable. enveloped viruses (those that have a pliable covering) are harder to identify, especially if mixed together with cellular debris. if they have surface fuzz or spikes (fig. e) , they are more readily distinguished. the genetic material inside is sometimes packaged into a distinct form such as an icosahedron, similar to the naked viruses (fig. a-d) or helical filament (fig. f ). if the negative stain penetrates the membrane, this nucleocapsid may be recognizable. however, some viruses do not have a morphologically characteristic nucleocapsid. in thin sections of infected cells, dna viruses are usually seen in the nucleus ( fig. a, b) where they are constructed, and rna viruses are usually found in the cytoplasm where they are formed (fig. c, d) , but there are exceptions. enveloped viruses can be seen associated with or budding through cell membranes; the membrane type is a further clue to identity. finally, the shape of the nucleocapsid within enveloped viruses is a key. possibilities are icosahedral (round in sections, fig. a, b) , helical or filamentous (like worms in sections, fig. c ), complex (pox viruses), or morphologically nondescript. recognition of viral particles and differentiation from cellular components and debris is paramount. once the presence of a virus has been determined, one may consult an atlas to confirm identification. [ ] [ ] [ ] [ ] [ ] for specific concentration or identification of viruses, some antiviral antibodies are available. these reagents can be used to aggregate, to coat, or to gold-label viruses. use of antibodies requires an a priori hint of the identity of the potential pathogen for selection of the proper reagent. charles d. humphrey, cynthia s. goldsmith, luanne h. division of viral and rickettsial diseases, cdc, atlanta, georgia, usa an unexplained acute pulmonary illness resulting in the death of previously healthy individuals was recognized in may . the cause of the illness was quickly identified serologically, pathologically, and genetically as a close relative of prospect hill virus (a rodent-transmitted hantavirus). recently, we isolated the virus from trapped rodents near the homes of patients, cultivated it in vero e cells, and determined that it was identical genetically and morphologically to the causative infectious agent. preparations for electron microscopy (em) were made by extracting, clarifying, and concentrating the virus from unfixed and . % glutaraldehyde-fixed, supernatant fluids of infected vero e cells . uninfected supernatants were prepared similarly as controls. concentrated virus suspensions were applied to glow-discharge treated formvar-carbon grids, blotted, and stained with . % uranyl acetate (ua) or with % phosphotungstic acid (pta), ph . . infected and noninfected cells were prepared for thin section by washing with . m phosphate buffer (po ) ph . , fixing in situ with . % glutaraldehyde in po , scraping, and pelleting. cell pellets were postfixed en bloc in po buffered % osmium tetroxide, stained in . % ua, dehydrated, infiltrated, and embedded in epon-substitute araldite. thin sections were stained with ua and in lead citrate. the extracted negatively stained virus resembled a hantavirus. the nm - + nm viruses were spherical, generally nonelongated particles with typical hantavirus grid-like surface features to which surface projections were attached (fig. ) . iv- scanning vol. , supplement iv ( ) fig our previous studies have demonstrated that application of scanning electron microscopy (sem) imaging for examination of transplanted hearts gave new possibilities for evaluation and early detection of acute rejection (jakobczak et al. ) . continuation of our studies confirmed the benefits of sem for diagnostics and basic investigations in cardiac transplantations. the aim of this study was to investigate coronary vessels during acute rejection in experimentally transplanted hearts. allogeneic heterotopic heart transplants were performed in ether anaesthetized rats, using microsurgical techniques according to the ono-lindsey method. male rats of the inbred long-evans strain were used as recipients, and male inbred sprague-dawley rats served as donors. the anastomoses-the donor aorta to the recipient abdominal aorta and the donor pulmonary artery to the inferior vena cava of the recipient (end to the side)-were performed under the operating microscope (wild m ) with - microsutures (davis-geck). cardiac allograft survival was estimated daily by electrocardiogram and palpation of ventricular contractions. rejection was considered complete at the time of cessation of a palpable cardiac beat. rejection was confirmed with laparotomy and histologic examination. the animals did not receive immunosuppression. allograft survival ranged from to days. transplanted hearts were perfused with . % nacl and . % glutaraldehyde solution in . m sodium cacodylate buffer ( mosm; ph . ). hearts fixed in glutaraldehyde were cut into thin sections, mm thick slides orientated perpendicularly to interventricular and interatrial septums. slides were treated with % osmium tetroxide for h at room temperature. then the specimens were dehydrated in an ascending series of ethanol solutions, ending in rinses in % ethanol and in pure acetone. thereafter, slides were criticalpoint dried using co , mounted on aluminum stubs with conductive silver paint and coated with a thin layer of gold. for examination a jeol jsm c sem was used. microcorrosion casts of the coronary arteries and veins and myocardial microvasculature of transplanted hearts were prepared by infusion of methacrylate casting medium (mercox). following infusion of approximately ml of casting material for one heart, the preparations were left for min to allow polymerization to occur. then, the tissue was macerated, leav- empty nm particles were often seen. virus particles were seen by thin section em as shells coated with barely distinct surface projections and enclosing hair-like strands of nucleocapsid material (fig. ) . particle size ( - nm) was smaller than that seen by negative stain em. spherical particles with elongated tubules (fig. ) were often observed by both negative stain and thin-section em. we conclude that deer mice trapped near the homes of humans with unexplained acute pulmonary illness harbor hantaviruses that likely are the causative agent for the human illness. ing a microcorrosion cast. dried preparations were mounted on stubs, coated with gold, and examined with sem (murakami , potter et al. . investigations of the coronary vessels of the right and left ventricular walls, septum, and the apex region were performed. tissue fragments were taken from various heart regions each day during the first postoperative week for studies with sem, and remnant tissues were used for histologic procedure. grading of the severity of cardiac graft rejection was based upon the stanford classification. in addition, the coronary vessels of transplanted hearts (of the other experimental group) were reproduced with a casting medium and examined with sem. the observations were compared with views of nontransplanted and syngeneically transplanted control hearts. the applied vascular casting method enables one to study the three-dimensional architecture of the vascular network of the rejecting myocardium. besides the large areas with minimal damage and readily distinguishable impressions made in the cast material by endothelial nuclei, there were the regions where various signals of vascular pathology were present: changes of diameter of coronary arteries and veins; unusual size and shape of the vessels; indentations on the cast surface caused by adhesion of blood cells to the vessel wall; characteristic occlusion of the vessels; irregularity of capillary network with changes in diameter and cast surface; capillary destruction with accompanying extrusion of casting material into the interstitial tissue. the observed vasculopathies varied in mild, moderate, and severe rejection and in several areas of the hearts. studies of coronary vessels of transplanted hearts using sem imaging have made significant advances in the understanding of rejection vasculopathy possible. sem observations of sectioned transplanted hearts correlate with results obtained by application of the microcorrosion casting technique. data showed that the highest rate of acute rejection occurs in the interventricular septum, and the development of rejection in various heart regions differs significantly. observed differences in localization and dynamics of development of vasculopathies in the septum and left and right ventricular walls of transplanted hearts confirm the unequal character of rejection. sem imaging is a valuable method for investigation of vascular pathology during acute rejection in transplanted hearts. the authors are very grateful to prof. a. miodonski, director of the sem laboratory of ent dept. of the n. copernicus academy of medicine, cracow, for the enabling of sem observations. preservation of the ureteral blood supply in kidney transplant surgery and ureteral reconstruction is of paramount importance in preventing urological complications of the ureter. the incidence of complications in ureteral surgery has declined in recent years, in part due to greater understanding of the ureteral blood supply and improved surgical technique. however, the ureteral vasculature has been described primarily at the gross level and only recently received attention at the microvascular level. because of its critical role in the success of renal transplants and its potential vulnerability to surgical trauma, the ureteral vasculature merits further investigation. we studied the microvascular anatomy of the ureteral vasculature (uv) in male new zealand rabbits using transmission electron microscopy (tem) and scanning electron microscopy (sem) of vascular corrosion casts and alkalitreated tissue samples. the uv was perfuse-fixed with buffered glutaraldehyde via the abdominal aorta, washed in buffer, and treated with alkali according to the method of takahashi-iwanaga for sem. for tem, fixed tissue was embedded in epon, sectioned, and stained with uranium and lead. for vascular corrosion casts, the uv was washed free of blood with buffered saline and filled with plastic resin (mercox•/methylmethacrylate, / ) at physiologic temperature and pressure. resin-filled tissues were digested with % koh, washed in water, critical-point dried, mounted on stubs, and sputtercoated with gold palladium for sem examination. the blood supply to the male rabbit ureter originates primarily from ureteric branches of the renal artery, the testicular artery, and the vesicular artery. these intrinsic vessels run within the wall of the adventitia the full length of the ureter. perforating arterioles and venules pass through the muscle wall and divide further in the lamina propria to supply a dense plexus of continuous capillaries in the mucosa. although numerous arterioles and venules populate the lamina propria, the ureteral musculature does not possess a rich capillary bed. the capillary plexus is positioned between the transitional epithelium and the lamina propria and uniformly extends the entire circumference of the ureter. capillary orientation occasionally follows the longitudinal axis of the ureter, but is primarily plexiform and exhibits multiple "y" and "t" shaped anastomoses (fig. ) . at the points of junction with the underlying arterioles and venules of the lamina propria, the capillaries commonly exhibit "kinks" and "bends." alkali treatment revealed that the capillary bed is supported by a dense, fibrous network of collagen (fig. ) . pericytes were occasionally associated with the capillary network. intercapillary distance ranged from - µm and capillary diameters typically were - µm. venous valves were not observed in the ureter. the combination of vascular corrosion casts, alkali treat-ment, and tem provides a clear and comprehensive perspective of the microvasculature of the rabbit ureter. the limited number of intrinsic vessels supplying the ureter wall and capillary bed emphasize the importance of understanding the blood flow in this organ in related surgical procedures. rabbits were perfused with glutaraldehyde and mercox ® via the abdominal aorta and subsequently processed for preparation of vascular replicas according to a previously described method. the angioarchitecture of the tibia was studied with use of the scanning electron microscope. at low magnification the pattern of vascularisation to the tibia could be established. they are richly supplied with arteries which pass into the bone substance proper from the periosteum. the main supply is from the nutrient arteries, which divide longitudinally in a dichotomous manner after passing through a foramina in the compact bone, major branches running both proximally and distally. this is the general case in all long bones. these branches reach the epiphysis and diaphysis of the tibia. in addition, separate arterial entities supply specific regions of the tibia: i. periostal vessels, ii. epiphyseal vessels, and iii. diaphyseal vessels. arteries and veins can be readily distinguished due to their characteristic nuclear impression present on the surface of the replicas. arterial nuclear impressions are oriented longitudinally to the long axis of the vessel, whereas in veins they are situated circumferentially (miodonski et al. ) . for further details about methods, see syed ali et al. . at higher magnification it was possible to demonstrate the microangioarchitecture of the epiphyseal region, which consisted of a very fine meshwork of capillaries arising from a main epiphyseal artery. a connection between arteries of the epiphysis was observed and consisted of medium-sized arteries. the capillaries of the epiphyseal meshwork show a rich anastomosing network building open, sinus-like dilatations. capillaries are also richly present in the areas of the growth plate and stressed areas of the epiphysis of the tibia. the so-called sinuses are situated under the cartilaginous plate. the venous drainage takes place through the vessels running parallel to the corresponding arteries. the animals were premedicated with chloroform and were then anesthetized with ketanest ( . ml/kg body weight) via intraperitoneal injection. after examining the abdominal reflex the rabbits were fixed on an operating table. all preparations were carried out at room temperature. the abdomen was shaved and opened cranially at the xyphoid process down to the pubic symphysis. after gently displacing the intestines and mesenterial convolutes, the abdominal aorta and the inferior vena cava were exposed very carefully caudal to the kidney vessels. a knob cannula was inserted through a small incision in the abdominal aorta and held in place by a ligation around the cannula distal to the point of incision. these procedures should be performed very quickly, otherwise the blood pressure will fall. the hindlimb vessels were washed through the cannula with an isotonic nacl solution containing heparin ( i.e.) and sodium nitroprusside ( mg/l) in ml. the inferior vena cava was opened with an incision as exit for fixing fluid and clamped when required. soon thereafter, . % glutaraldehyde in phosphate buffer ( . m, ph . ) was passed through the same cannula. after completing the perfusion, about ml mercox (methacrylate) was applied through the cannula and observed until the mercox came out of the inferior vena cava. it was then clamped to avoid unnecessary backflow. the animals were left overnight at room temperature and then placed in a water bath for h for rapid polymerization. the bones were dissected free from other tissues and left in a % koh solution, which was changed every day. the specimens were washed with distilled water very carefully; it is very important to avoid every kind of mechanical disturbance. the solution and water can be changed with the help of a small water pump. the best results were achieved with an alternative change of % koh and % trichlor tetra acetic acid solutions. after complete maceration, the preparation was checked under a stereo microscope to eliminate all rest tissue. it was then frozen at − °c and freeze-dried. the preparation was contrasted with % os o vapour in a desiccator for about h to increase the contrast in the microscope. the specimens were fixed on scanning stubs with conducting silver and sputtered with gold ( min, - ma). they were then viewed in a psem (philips) microscope and photographed with kodak film. iv- scanning vol. , supplement iv ( ) fig. low magnification view of a rabbit tibia from the epiphysial zone with its specific end capillaries arrangement, the proliferation zone, and the connecting capillaries between the epiphysis and metaphysis ( apart from the ability to capture three-dimensional ( -d) images of microscopic structures, a confocal laser scanning microscope (clsm) equipped with multiple detectors allows one to add an extra dimension to the data acquisition process. a typical example are the ongoing studies in our laboratories. in this paper, we discuss the channel spill-over problem associated with acquiring two-color clsm images and present image processing techniques that analyze multichannel image data. for double labeling of dna replication sites, mouse t cells, exponentially grown on cover slips or synchronized at specific times in -phase, were pulsed for brief times ( - min) with cldu (chlorodeoxyuridune). the pulsed cells were then fixed and processed for fluorescence microscopy using monoclonal antibodies, appropriate extraction conditions, and fluorochrome-conjugated secondary antibodies which enabled differential recognition of sites of cldu versus idu incorporation into newly replicated dna. under the conditions used, there is no measurable cross reaction between the antibodies for cldu-versus idu-labeled replication sites. optical sections of labeled cells were collected with an olympus lsm gb- clsm with a m w ar ion laser. the microscope is operated in high resolution mode ( × pixels) with three fluorescence channels and a transmission channel. the microscope is controlled by a - mhz computer with mb ram and . gb hard drive. the digital confocal optical sections were transferred via ethernet to a dedicated sun sparc / with mb memory for further analysis. during image capture, a combination of band-pass and high-pass filters are used to mask unwanted emission at each detector. a major problem with the dyes used is their overlapping emission spectra. this seemed to contribute a significant amount of spill-over from the green channel (fitc) to the red channel (texas red or rhodamine) and only an insignificant amount in the other direction. because of the unidirectional nature of the spill-over between red and green channels, we were able to apply a correction factor to the red channel based on the green-channel intensity. to obtain the correction coefficients, a set of calibration images were obtained by using a sample which is identically labeled in both color fluorescent dyes and captured at different gain and offset parameters. for each of these images, the mean spill-over value in to the red channel (r i ) is calculated for each pixel value (i) in the green channel as follows: ( ) where i r (x,y,x) and i g (x,y,z) denotes the image intensities of the red and green channels, respectively, and n i indicates the number of pixels in the green channel having value i. a third order polynomial is fitted to the data set (<i, r i >,i= , , ... ) and the corresponding coefficients (a o a ,a ,a ) are estimated using the least square error criteria. the correction factor for each pixel of the red channel is then computed as follows and subtracted from the original intensity of the red channel to obtain the corrected image. ∆i r (x,y,z)= a o + a i g (x,y,z) + a i g (x,y,z) + a i g (x,y,z) ( ) figure shows the channel spill-over for different filter sets. the corrected images were analyzed using a set of image processing routines developed at our laboratory to obtain the boundary of replication sites in individual channels. these boundary data are then used to detect the overlapping particles between channels, and the overlap area is calculated for each overlapping particle. the results are presented in several forms including a two-dimensional histogram of particle volume and overlap percentage. the image processing routines are able to generate boundary data of a × × image with particles in approximately mins and the overlap estimation using boundary data takes approximately s. currently we are working on visualization techniques of raw and processed multichannel -d images. these fringes, these dark, granular, linear structures shed dark particles which then fused with opposite lines. a stream of particles trafficking between cells in contract, particularly at overlaps, was noticed. this pointed to a rather intense exchange of very small particles between cells of the same origin on contact. we conclude that interference reflection mode, video-rate, laser scanning confocal microscopy is a useful tool for intravital analysis of the intracellular structural dynamics in relationship to cell type, function and pathophysiological state. department of plant pathology and department of botany, university of georgia, athens, georgia, usa powdery mildew diseases of plants are caused by a group of obligately parasitic fungi belonging to the order erysiphales, class ascomycetes. the somatic hyphae of most of these fungi grow exclusively on the surfaces of infected plant organs, most typically leaves. these hyphae form specialized structures known as appressoria that attach to the host surface. each appressorium gives rise to a tiny penetration peg that penetrates the wall of the underlying epidermal cell and invaginates the host cell plasma membrane. the penetration peg then develops into a specialized structure known as a haustorium that absorbs nutrients from the host cell. in this study transmission electron microscopy was used to examine the haustoria of the powdery mildew fungus erysiphe lagerstromemiae and the relationship of these structures to epidermal cells of infected leaves of crape-myrtle (lagerstromemia indica). the haustorium of e. lagerstromemiae (fig. ) possesses a slender neck region and an expanded body that contains a single prominent nucleus. much of the neck is surrounded by a collar of host cell wall material. a single septum with a tiny central pore with which woronin bodies are associated is present in the distal portion of the neck near the haustorial body. the haustorial body is divided into numerous small, coiled branches. the entire haustorial apparatus is separated from the host cell cytoplasm by an extrahaustorial membrane (figs. , ) . though continuous with the host cell plasma membrane, the extrahaustorial membrane is much thicker than the plasma membrane and is highly convoluted in certain regions. the haustorial branches are separated from the extrahaustorial membrane by a region known as the extrahaustorial matrix. at this point it is unclear whether the finely fibrillar material comprising this matrix originates from the fungus, the host cell or both. although the haustorial apparatus of e. lagerstromemiae occupies a considerable portion of the overall volume of an invaded cell, the host cell organelles appear normal. however, numerous golgi bodies and many structures resembling microbodies are concentrated in the host cytoplasm very near the extrahaustorial membrane (fig. ) . pavel vesely and alan boyde* institute of molecular genetics, academy of sciences of the czech republic, prague, czech republic; *department of anatomy and developmental biology, university college, london, u.k. video-rate laser scanning confocal microscopy in the reflection interference mode (irm) enables the visualisation of fine intracellular structures in living cells in vitro and the observation of rapid changes of shapes, the trafficking of very small particles, and exchange of material. the dynamics of this intracellular activity can be established by optical sectioning with objectives of high magnification and numerical aperture, from within the bottom level of the cell contact to the substrate through to the top cell surface: these levels may be up to µm apart for large and/or tall cells. in the present studies, three video rate confocal microscope systems (lasertec lm , noran odyssey and biorad dvc ) were used to compare the ultramicroanatomy and dynamics of motion of intracellular structures in established cell lines (k , k , t , and a on). k cells are clonal descendents of spontaneously in vitro transformed lew/cub rat fibroblasts which give rise to low metastatic sarcomas in vivo: k cells are rous sarcoma virus transformants of the k line and both t and a on cells developed from k by neoplastic progression to a higher metastatic capacity. previously observed differences in the incidence of cells with rapidly oscillating and trafficking particles between k and the other cell lines were analysed at improved resolution. using the noran odyssey system, with zoom factor and a / . nikon lens, this phenomenon was observed in almost all the cells of all the cell lines compared. this finding provided an explanation of the previously described difference between k and the other cells which was obtained using a nikon / . wi lens (but not with an olympus / . wi using the lasertec system, zoom factor ) from the level of the cell periphery adjacent to the culture surface only. the present observations indicate that there may be a difference in the extent of this type of intracellular activity towards the periphery of the cell. more accurate mechanical targetting of the optical probe will be needed before it will be possible to measure this effect. at the free, top surface of some k cells, small pinocytic opening and closing was seen. similar images of opening and closing near the bottom sides of some cells in all cell lines were evidently produced by vertical oscillatory movements of particles in small clusters. when a . mm pinhole diameter (odyssey) was used to improve confocality, the interference fringes were observed to move, during focussing, along the steep slope of the high bodies of the k cells, optically transforming granular, linear, or randomly oriented structures inside the cell into concentric lines. above and below florida institute of technology, department of biological sciences, melbourne, florida, usa acid phosphatase (ap) is well known as one of the representative lysosomal enzymes. ap activity is recognized at the light microscopic level as intensely stained granules. although the localization and distribution of ap activity in the mammalian inner ear were researched by several investigators, we found only one report about the occurrence of ap in the avian inner ear (marmo ) . the objective of the present study is to demonstrate the occurrence of ap activity in the membranous labyrinth of the chick's inner ear, using the simultaneous coupling azo dye method (barka and anderson ) , and to discuss the significance of these findings. after newly hatched chicks were sacrificed, their ears were fixed for h in . % glutaraldehyde (ga) or . % paraformaldehyde (pfa). the membranous labyrinths were dissected out, dehydrated in either a graded series of acetone (used only for . % ga fixation specimens) or n,n-dimethylformamide (dmf), and embedded in jb- (polysciences, warrington, penna.), a methacrylate plastic. these sections were incubated for or h at °c in an incubation medium (ph . ) containing naphthol as-bi phosphate as substrate and hexazonium pararosaniline as a coupler that used the azo-coupling method. in some instances, intact, fixed whole specimens were incubated in an aliquot of the same incubation medium as that of sectioned specimens and were then dehydrated with dmf, embedded in jb- or lr white, and sectioned. for control specimens, either a substrate-deficient medium or an incubation medium containing cupric sulfate ( . m) was used. methyl green was used as a nuclear stain. intense ap activity, as represented by dense dye deposits, was detectable in the supranuclear area of almost all hair cells in the basilar papilla and vestibular sensory hair cells (fig. ). this result was in agreement with the finding of marmo ( ) . there were no differences in the distribution pattern of ap activity of the hair cells from the distal to the proximal region in the basilar papilla. in particular, hair cells of lagenar macula were most often characterized by intense ap activity in the subcuticular area (fig. ) . the functional significance of high levels of ap in the hair cell is still a matter of conjecture. for example, ishii and balogh ( ) demonstrated no morphologic evidence for phagocytic or secretory activities in hair cells, although their electron micrographs of hair cells reveal lipofuscin-like substances within lysosomes. it has long been contended that nonsensory hair cells such as stria vascularis, external spiral sulcus cells, and those around the macula and cristae ampullares help to regulate the ionic composition of endolymph (kimura et al. , kikuchi and hilding ) . in the present study, the columnar cells and the cells of the tegmentum vasculosum showed moderate to strong ap activity, and the transitional epithelia of the cristae ampullares also showed strong ap activity . the dark cells help to regulate the ionic composition of endolymph and perilymph (kimura ). in the present study, intense ap activity of dark cells was concentrated in the supranuclear area or diffusely in the cytoplasm. these results show that ap activity is highest in cells with highest levels of transport activities on the production of endolymph (marmo ) , although the metabolic linkage between ap and the transport enzymes is uncertain. the wall cells and supporting cells of the vestibular labyrinth showed no enzyme reaction. the statoacoustic and vestibular ganglion cells showed various degrees of ap activity. the sections of statoacoustic and vestibular ganglion cells of specimens that had been embedded in lr white displayed more intense ap activity than those sections from specimens embedded in jb- . ap activity was also stronger in specimens fixed in . % pfa than in . % ga. the syncytiotrophoblast layer covers the fetal villous tree which is in direct contact with maternal blood. it contains many microvilli, resembling a carpet surface, which are responsible for the absorption, excretion, and synthesis of many key hormones which are important for fetal development, secretion, and exchange of gases; of course, many earlier scanning electron microscopy (sem) studies of normal and pathologic placental villi have been described and many investigations on the histology, ultrastructure, and three-dimensional features of human placental villi have been carried out. [ ] [ ] [ ] [ ] technical handicaps causing structural deformations on the placental villi have been indicated. all of these studies depended on the normal development of placental villi but were not found to be comparable with tubal pregnancies. the purpose of this study is to describe the development of the human placental villous trees emerging from chorionic plate during the early periods of uterinal and tubal pregnancies, and to compare their three-dimensional structures using sem technique. iv- scanning vol. , supplement iv ( ) fig samples of human placentas have been studied. six early specimens obtained by curettage or by hysterectomy have been staged as described in our previous study. four additional specimens dated according to anamnestic data in comparison with the measurement of fetal weight and length at , , , and weeks p.c. were obtained from clinically normal pregnancies interrupted by legal curettage or by hysterectomy, and two samples aged and weeks p.c. from tubal ectopic pregnancies. all specimens were fixed and prepared for sem. the specimens were dehydrated in ascending concentrations of ethanol, critical-point (bio-red e ) and air dried, and coated with a layer of gold particles using a bio-red sc super coater. observations were made with a jeol-jem sem. ectopic tubal placental villi trees are not as well developed as normal uterinal villi trees. villi formation and ramification were very rare depending on the physiologic causes. some villi shoots were thinned gradually from base to its apex, and at the top these developing villi showed very interesting properties; they were curved, thinned, and crossed each other (fig. ) . the predominant villi types were immature, intermediate, and mesenchymal villi. [ ] [ ] [ ] the surface of these villi trees was completely covered by small and deformative curved microvilli and by fibrinoid particles compared with the normal uterinal villi trees. the trophoblastic shell of some villi was peeled off and degenerated. developmental retardation of placental villus trees is clearly seen. some villi were curved and crossing, and gradually thinned to the apex; on the villi surface, many wrinkles and furrows resembling very old skin were observed (fig. ) . it is well known that the placental development shows a parallel regulation due to the desidual properties. cellular and extracellular interactions take place between uterine and trophoblast during initial stages of placentation. , - khong and robertson suggested that the deficient decidua is the main reason why tubal pregnancies do not reach to term. the findings they describe may be explained as being due to absent decidua rather than being its cause. moreover, another reason for the usual failure of ectopic pregnancies to reach term is placenta accreta which becomes placenta percreta with bleeding. according to our sem observations, in tubal pregnancies the formation and development of placental villi trees were found in some patterns observed in uterinal placental samples. villi formation patterns-buds, tendrils, and shoots-are very rare on the surface of villi trees (fig. ) . placental villi shoots do not show uniform composition compared with to the uterinal villi samples. these are very thinned to the apex of villi, and are compressed in a common configuration, resembling a racket which does not appear as an alive condition. these observations are very interesting and original. the university of iowa central electron microscopy research facility (cemrf) was established in to support all faculty, staff, and students needing this technology. initially, the cemrf operated with one transmission electron microscope (tem), one scanning electron microscope (sem), and three staff members, and it supported about projects annually. during the past years, all instrumentation predating has been replaced and now includes two tems, two sems, two eds systems, a scanning probe microscope, a laser scanning confocal microscope, and all necessary supporting equipment. the facility presently consists of staff members and supports over projects yearly from departments in colleges and industrial laboratories. one of the unique strengths of the cemrf is that both biomedical and physical scientists use the facility. the development of the university of iowa cemrf was made possible due, in large part, to the central administration's support of equipment acquisition over the past decade. of the $ , , invested in equipment, more than % was provided by the graduate college and the vice president for research, with the other % obtained through equipment grants and user fees. of the operating expenses for the facility, % are recovered from a large and well-funded group of investigators who are charged on an at-need, fee-for-service, first-come, first-served basis. faculty recognize that they have a facility available to them that provides immediate access to state-of-the-art equipment and techniques, as well as one that provides training and supervision. in addition to supporting research, the cemrf offers four formal courses, as well as assisting with sections of eight other classes. the facility annually organizes at least two workshops and provides about tours for visiting scientists, faculty and student recruitment, high school students, business groups, and politicians. the facility also serves as the business office for the iowa microscopy society. concurrent with the development and success of the cemrf, several departments voluntarily divested themselves of their own electron microscopy (em) equipment ( ems on campus in compared with ems in ). this represents a savings of more than $ , annually ( dollars), as well as the release of or more rooms for other use. in addition, the interaction between the remaining six em laboratories is more positive as a result of this downsizing and centralization. the availability of user-friendly quality equipment in the cemrf assures that em is now accessible to all university faculty, staff, and students. in addition, with many investigators sharing instruments, it is easier to justify acquisition of new equipment for the cemrf. accurate dimensional metrology of the submicrometer gold absorber structures of x-ray masks can be accomplished in the scanning electron microscope (sem) with the use of electron beam modeling (postek ) . accurate metrology is possible because the x-ray masks present a unique measurement object from most other semiconductor structures viewed in the sem. this occurs because the silicon support membrane is x-ray transparent by design. this characteristic can be used as a distinct advantage in electron beam-based mask metrology since, depending upon the incident electron beam energies, substrate composition, and substrate thickness, the membrane can also be essentially electron-transparent. the areas of the mask where the absorber structures are located are essentially x-rayopaque, as well as electron-opaque. viewing the sample from a perspective below the mask by placing an electron detector beneath the mask provides excellent electron signal contrast between the absorber structure and the base membrane. the present technique utilizes a broad acceptance angle detector which is different in concept from other transmission electron (te) detectors used in the sem. in this case, the broad angle is used to detect as many of the transmitted electrons as possible (i.e., whether scattered or not) that have an energy above some predetermined threshold which is usually several kiloelectron volts. then, the electrons are physically filtered both by the signal threshold characteristics of the detector and an by an electron energy filter in front of the detector. energy filtering of the transmitted electrons excludes the highly scattered and thus lower-energy electrons from entrance into the detector. this greatly improves the contrast level over the conventional transmitted-electron detection mode for this type of application, and greatly simplifies the required electron beam/sample interaction modeling necessary for edge determination. it is, in fact, this change in electron detection philosophy that makes the present te approach so attractive for dimensional metrology and inspection of x-ray masks. monte carlo modeling of the transmitted electron signal was used extensively to support this work in order to determine the optimum electron detector position and characteristics, as well as to determine the position of the edge in the image profile. the monte carlo modeling is more accurate in this work, in contrast to the secondary electron detection mode, because in the transmitted electron detection mode the modeling of the electron beam/specimen interaction becomes far less difficult than in the modeling of typical secondary electron images of other opaque objects. the generated low-energy secondary electrons (which are complex to model) are excluded from the detector and, therefore, do not need to be included in the calculation. combining all of these factors provides a modeled signal that is extremely sensitive to wall slope. wall slope variation can result in large differences in the modeled profiles. the comparison between the data from the theoretically modeled electron beam interaction and actual fitted experimental data is shown in figure for a wall angle of ˚. the theoretical profiles were shown to agree extremely well with experiment, particularly with regard to the wall slope characteristics of the structure obtained from the sem images and video profiles. a plateau in the transmission is seen in the modeled profile as the beam traverses the edge, which can be used to identify the loca-tion of the edge of the absorber line and thus allows accurate measurements to be made. this work provides an approach to improved x-ray mask linewidth metrology and a more precise edge location algorithm for measurement of feature sizes on x-ray masks in commercial instrumentation. the transmitted electron detection mode is also useful in both mask inspection and mask repair, because the high contrast of the image allows for rapid determination of mask defects and high-density contamination particle detection because the transmitted electrons simulate transmitted x-rays. this work represents the first time that electron beam modeling has been used to determine the accurate edge location in an sem image. this also represents an initial step toward the first sem-based accurate linewidth measurement standard from nist, as well as providing a viable metrology for linewidth measurement instruments of x-ray masks for the x-ray lithography community. postek using monte carlo models that take into account the gaussian beam width of the incident electron beam, a noticeable broadening of the linewidth measurements is simulated for aluminium linewidths under glass. this simulated effect correlates closely with measurements of linewidth using energy dispersive x-ray analysis of an aluminium ka line under glassivation. detailed simulation of a full linewidth measurement has been performed using a point source and gaussian incident electron beam for the multiple scattering and single scattering models. the modeling effort was undertaken to better understand the effect that incident electron beam width would have on the accuracy of scanning electron microscope measurements. the multiple scattering (ms) model was adapted from pas-cal code for a single material to simulate layered materials, specifically, sio over aluminium. further, application-specific code was added to simulate an electron beam scanning across a linewidth on a fabricated microelectronic circuit. the linewidth selected for these simulations was one micrometer and one micrometer thick with a selectable sio thickness for the overcoat. in this case, the sio thickness was . micrometers. the single scattering (ss) model was adapted also from available pascal code for layered materials with the same application-specific code used in the multiple scattering model. the differences in the results from the two models used for the simulation are shown in figures and . the results for the ms and ss models at k trajectories are shown in table i . the gaussian beam width was selected to be . micrometers. the gaussian beam noticeably broadens linewidth measurement over point source, based on the results in table i . the linewidth measurement was taken at fwhm by extrapolation between points. the large differences between the ss and ms models is under further study. the calculation of the image contrast from samples with surface topography can be done using monte carlo techniques as long as the electron trajectories can be calculated through a surface profile. the image simulations that are described here were done using the same methodology that has been applied to the calculation of the electron backscattered signal from samples where the composition variation is taken into account (ly and howitt and these proceedings) . the design of the program is such that the scattering cross sections are reassessed as the electron passes from one region of the specimen into another, which in this case includes free space. the program takes longer to run than that for a homogeneous specimen with a flat surface because the parameters, such as the atomic number and atomic weight and density, need to be constantly updated at each point in the calculation. depending upon the image resolution that is required, we divide the three-dimensional specimen into blocks of either specimen or vacuum. the modifications to the conventional monte carlo approach to such calculations include not only the specimen geometry but also the determination of the energy of the electron that is backscattered and the direction in which it travels. in this way the signals at the backscattered and secondary detectors can be distinguished because almost all the low-energy electrons find their way to the secondary electron detector, whereas only those in the line of sight to the backscattered detector contribute to this signal. in most practical situations, where the geometry of the specimen is difficult to predict, it is useful to have specimens of standard shape, such as a sphere cone or box, to compare directly with the images. the calculation of the image at the secondary electron detector for spheres of the same size but of different atomic weight is shown as an example in figure . the spheres are assumed to be on a graphite substrate and the electron-beam energy is kev. the signals are displayed as they would appear in a micrograph, that is, in an intensity scan across the image and as a two-dimensional profile of the image intensity. we have also found that pseudo color images, where the various gray levels are replaced with different colors, are very useful when it comes to comparing calculated and experimental images. calculated secondary electron images from nm spheres of silicon, titanium, nickel, and molybdenum on a carbon substrate at an electron beam energy of kev. the signals are displayed as they would appear in a micrograph, that is, in an intensity scan across the image and as a two-dimensional profile of the image intensity. pierre hovington, dominique drouin, raynald gauvin, david c. joy * department of mechanical engineering, university de sherbrooke, sherbrooke, québec, canada; * em facility, university of tennessee, knoxville, tennessee, usa quantitative analysis of resolution < nm can be achieved at low accelerating voltages. however, to exploit all the emitted signals (x-rays, secondary, and backscattered images) fully, specialized programs have to be used. in this paper we present some results that can be achieved with such programs. it is shown that our single scattering monte carlo program can effectively predict the effect of surface oxidation on aluminum (al), model the backscattered coefficient even at very low energy (< kv) and accurately predict the backscattered profile around a spherical inclusion. at low voltages, mott elastic cross section has to be used. the tabulated mott scattering cross section of czyzewski et al. ( ) , combined with the energy loss of joy and luo ( ) , is used as the physical basis of the program. hence, the program can be used accurately for simulation even at very low energy (e < kv) (fig. ) . numerical experiments can be made with one or several regions of different composition and shapes. the regions can be defined as horizontal (i.e., layered samples) or vertical (i.e., grain boundary). in addition, based on the results of gauvin et al. ( ) , spherical inclusions can also be modeled. the program is written in c language and makes use of a fully graphic environment both for input and output of data. in figure , we present a typical output of the program of a simulation on a multilayer electronic component [a nm ga ( % at ) al ( % at) as ( % at) on a gaas substrat] covered with nm of contaminated carbon. the effect of the backscattered electrons on the resolution can be clearly determined since their paths are marked with a different color. in addition, at the end of the simulation, the interaction volume ranges at , , , and % are plotted. to increase the range of applicability of our program, backscattered, secondary, and x-ray images (k, l, and m lines) can be generated. generated and experimental images can then be used for metrology and microanalysis. we present in figure a backscattered linescan of a nm diameter beam (eo = kv) with a hypothetical nm diameter hemispherical mns inclusion in a fe matrix. it is important to note in figure that the difference between the "screen dimension" and the "real dimension" is over % . in figure , we present an experimental backscattered profile taken across an mns inclusion in steel at kv. we note the similarity of the theoretical and the simulated backscattered profile (slight decrease between the center and the end of the inclusion and the presence of peaks at the interface between the matrix and the inclusion). iv- scanning vol. , supplement iv ( ) fig . when low voltages are used, the geometry and composition of the surfaces greatly influence the resulting signals. in figure , we present an experimental spectrum from a pure aluminum (al) sample taken at kv with a windowless eds detector and a theoretical generated spectrum (hovington et al. ) . the difference between the two spectra is mainly due to the presence of oxide (al o ) at the surface of the sample. in figure , we compute the j(rz) curves for an oxide thickness of and nm and for a nonoxidized sample. the decrease of the al k intensity for the and nm compared with the nonoxidized sample is and %, respectively. the experimental to theoretical ratio found on the spectra (fig. ) is approximately %, indicating that the oxide layer is < nm thick. it is important to note that because the standard and the unknown may not have the same thickness of al o , the use of theoretical stan-dards may become critical in quantitative x-ray microanalysis at low voltages. material investigation methods based on the interaction of the electron beam with a solid requires detailed information about the electron distribution. among the various approaches which allow one to calculate the electron distribution, the monte carlo simulation seems to be most suitable. this is due to the fact that the monte carlo method generally can be applied to any target. the main problem of this approach is the accuracy of the electron distribution approximation. one important criterion of the quality of the approximation is the coincidence of the calculated and experimentally observed x-rays in depth distribution. note that the correct calculation of this function plays the essential role in epma data interpretation. as have been shown in the monte carlo process developed by murata et al., which is based on the mott cross section for elastic and on a knock-on model for inelastic interactions, it provides a rather good agreement between the calculated and experimental φ(ρz) function for cdlα line in al and au targets at the electron beam energy e = kev. but a more careful comparison of φ(ρz), obtained according to this model with the experimental data for sikα in al, ni, ag, and au at e = , , kev, respectively, and cukα at e = , kev, reveals some discrepancy, especially in the tail part. this discrepancy increases when the atomic number grows and the electron beam energy decreases. the origin of this discrepancy can be connected with the errors of the approximation of the differential cross sections for elastic and inelastic interactions, as well as with the ionization cross section. as the mott formula for elastic cross section uses the model of the atomic potential, then the approximation of this potential can influence the results of the scattering. to avoid the errors connected with the choice of the atomic potential approximation, we have used the elastic differential cross section obtained by riley et al. with the help of the static approximation theory (sat). the main features of the developed monte carlo program are as follows: . the elastic interaction is calculated according to the sat cross-section . the energy dissipation process is described by the knockon model. the bethe formula for de/ds is used in the high-energy region (e > . j i ). in the low-energy region (e < . j i ), the equation used in [ ] is exploited instead of the bethe law . the fast secondary electrons produced by electron-electron interactions in an inelastic collision are taken into account . the mean ionization potential is chosen according to the berger and seltzer formula . to calculate the ionization cross section, the formula of gryzinski is used. we have calculated the energy and angular spectra of the electrons transmitted through the films of the various compositions and thicknesses. comparison of these results with the data obtained with the help of the mott cross section shows that the use of the sat approximation for the elastic interaction does not influence essentially the electron distribution and cannot improve the accuracy of its determination. the comparison of φ(ρz) functions which are found in accordance with these two models confirms this conclusion. therefore, one can state that the details of the mott cross section together with the errors in atomic potential approximation cannot influence the electron distribution in targets. the factor which can affect the φ(ρz) in targets independently of electron distribution is the ionization cross section. to take into account this factor, we have used the experimental results obtained by long et al. during differentiation of the heart there appears to be a sequential pattern to the formation of individual muscle fibers. phenotypic changes result in the expression, synthesis, and organization of complex proteins to form the terminally differentiated myocytes. the formation of the basic pattern of myofibers ultimately determines the physiologic performance of the final form of the heart. pattern formation involves both intracellular events associated with myofibrillogenesis and extracellular events associated with cell:cell and cell:matrix interactions. this basic pattern is repeated to form complex layers which results in the final form of the heart. it is essential to understand the sequence of expression associated with these patterns of fibers in order to understand errors that may result in congenital defects in heart formation. in this study time pregnant ( . - . days of gestation, ed) sprague dawley rats were obtained from harlan sprague dawley laboratories (indianapolis, ind.). animals were anesthetized with % chloral hydrate in normal saline solution and the individual embryos, including uterine tube, were removed and placed in . m phosphate buffered saline solution containing . % azide (pbs-a) at °c. the embryos were removed from the uterine tube under a dissecting microscope and fixed for - h in % paraformaldehyde in . m hepes (n- -hydroxyethylpiperazine-n'- -ethanesulfonic acid) at ph . . the embryos were scored and gestational ages were assigned. the embryos ( . - . days of gestation) were stained using a modification of the procedure described by tokuyasu and maher ( ) . after fixation, the embryos were placed in pbs-a for h and immersed in . % triton x- in pbs-a for h. after washing for an additional hour in pbs-a, the embryos were incubated in units/ml bovine testicular hyaluronidase for min, washed in . % triton x- with . m glycine in pbs-a for h and immersed in % bovine serum albumin (bsa) in pbs-a for h. the individual embryos were incubated overnight with rhodamine-labelled phalloidin from molecular probe, eugene, ore. ( µl of stock solution/embryo), or control buffer. following incubation, the embryos were washed in pbs-a at °c for h and immersed in % bsa for h. hearts from . - . day gestation embryos were removed and fixed as above. after fixation, the embryos were encased in % polyacrylamide gel using a modification of the procedure described by hansen and dryer ( ) . after fixa-tion, the hearts were washed in pbs-a for h and immersed in . % triton x- in pbs-a for / h. the hearts were embedded individually in blocks containing % polyacrylamide with . % bis-diamine which was polymerized with % ammonium persulfate. the polyacrylamide blocks containing the hearts were sectioned at µ with an oxford vibratome model g before being stained as described above. the tissue was examined using a bio-rad mrc- confocal laser scanning microscope (biorad, cambridge, mass.) using × (na= . ) and × (na= . ) objectives. at ed . to . , the cells of the myocardium are round and tightly packed with no discernable pattern. the first indication of cell orientation into fiber patterns occurred in the outflow tract and ventricle. the outflow tract myofibers developed circumferentially and maintained this pattern throughout the study time. the ventricular myofibers also developed circumferentially; however, they gradually changed into a net-like pattern ( fig. ) followed by a thickening of the ventricular wall and protrusion of trabeculae. with continued maturation, the ventricular trabeculae appeared to coalesce especially in the re-gion of the muscular interventricular septum (fig. ) . the myofiber pattern developed later in the atria. the atria expressed the same early pattern seen in the ventricles; however, they retained the net-like appearance throughout development. the results indicate that there are region-specific differential changes in the orientation of myocytes that result in the unique myofiber patterns of the heart. these regional pattern changes may be correlated with mechanical forces and hemodynamic alterations during development. the preparation of thick, optically clear sections of fragile tissue structures greatly aids the power of confocal laser scanning microscopy in imaging three-dimensional structures. hale and matsumoto ( ) have presented confocal images of agarose-embedded, vibratome-sectioned tissues; earlier workers have embedded soft tissues in polyacrylamide gels for frozen sectioning and lectin or immunohistochemical staining of structures in - micron sections (bronson et al. , hausen and dreyer , johnson and blanks . we report here conditions for embedding, sectioning, and staining embryos in polyacrylamide gels for a variety of confocal imaging techniques. in general, infiltration of - mm specimens for - h in a mixture of - % acrylamide monomer ( part bisacrylamide cross linker to parts monomer) yields, upon polymerization (with . volume of % ammonium persulfate), blocks that cut easily by vibratome between - microns. these conditions work well for tissues previously stained (with fluorescent probes or dii tattoos) or for staining gel sections water-soluble fluorochromes with low molecular weight (e.g., propidium iodide, phalloidin). for immunostaining after sectioning, the acrylamide concentration must be reduced to - % acrylamide to facilitate access of immunoglobulins to antigenic sites, and the gel must be supplemented with % agarose to aid sectioning and handling. illustrated below are confocal images from acrylamide sections of a stage chick embryo, fluorescence-immunostained in whole mount for cadherins. this specimen was fixed in % dmso/ % methanol (dent and klymkowsky ) and incubated overnight with a : dilution of commercial antiserum against a synthetic peptide common to all known cadherins (sigma, pan cadherin, #c ). rinsing preceded overnight reaction with tritc-conjugated secondary antibody, brief formaldehyde fixation, and embedment in % acrylamide. a survey ventral view was taken before transverse vibratome sectioning across the entire embryo at micron intervals, yielding eight sections that were spanning the thoracic region and were mounted in % glycerol for closer inspection. figure illustrates a stereo pair of projections from a series of confocal images across the atrioventricular junction. this gel-embedding and vibratome-sectioning method yields abundant, optically clear, and easily handled sections for confocal examination of fluorescent structures in water-miscible media. greater detail concerning procedures and technical problems with this technique are provided elsewhere (germroth et al. in press) . department of anatomy and cell biology, suny health science center, syracuse, new york, usa endothelial cells arise in the early embryo from precurser cells called angioblasts. in the quail embryo, the emergence of these cells can be observed as an epithelial to mesenchymal conversion of cells from the mesoderm which may be observed by scanning electron microscopy (sem) after removal of the endoderm or by immunolabelling the whole embryo or sections with a monoclonal antibody (qh- ) which labels angioblasts. the process of angioblast migration and assembly into the solid cords, which are the rudiments of the earliest blood vessels, is called vasculogenesis. simple embryological manipulations have been used to distinguish the role of cell migration in the early vessel rudiments. the dorsal aortae arise ventrolateral to the forming somites, and inserting blockages revealed that little cell migration is involved in their formation. the endocardial cells which form the endothelial cell lining of the heart, in contrast, undergo an extensive migration from more lateral regions of the embryo which has been studied by blockages and the construction of quail/chick chimeras. iv- scanning vol. , supplement iv ( ) the first rudiments of veins in the embryo are the cardinal veins. these form beneath the ectoderm of the embryo in an area with few angioblasts. the early stages of cardinal vein formation have been studied by using immunogold-labelled secondary antibodies after qh- labelling and imaging in the sem backscatter mode after silver enhancement. these studies revealed that small groups of angioblasts appear over the lateral mesoderm, possibly by migration from the angioblastrich mesoderm adjacent to the endoderm, and then migrate medially to assemble into a solid cord of cells in association with the developing wolffian duct. the ability of scanning probe technology to image atomic topography of a surface, to manipulate individual atoms, and even to probe the internal structure of a molecule confirms the significance of these super-resolution microscopies used at the nanometer-scale analysis of molecular systems. so far, a number of interactions between the sample and the probe tip gives access to a variety of local properties (wickramasinghe ) . the latest applications of the local probe microscopies on organic molecules include photoemission, tunneling potentiometry, electrostatic, elastic and tribo logic properties, or near-field thermal measurements and as many other phenomena on which various contrast formation mechanisms are based. topics include imaging of individual biomolecules, highly ordered molecular assemblies, and polymeric materials under various environments. the development of additional imaging techniques,based on near-field electromagnetic interaction between the probe tip and the surface (betzig and trautman ) , or even by means of magnetic resonance (rugar et al. ) , indicates that new technologies with subnanometer spatial resolution could be achieved in principle. recent advances in near-field optical microscopy (nsom) confirmed spatial resolution in the range of - nm but still limited by the diameter and optical penetration depth of the aperture. here, we demonstrated a new concept for an aperture near-field scanning optical microscope which combines force microscopy and optical scattering for imaging the sample at subwavelength resolution without the use of an aperture. the end of a silicon tip is illuminated in transmission mode by a laser beam through a transparent substrate. both the tip and the sample undergo perpendicular motion, each at a different frequency, with amplitudes chosen comparable to the desired measurement resolution. the scattered light from the tip and the surface is detected at the difference frequency for imaging and sample at sub-wavelength resolution without the use of an aperture. we describe the novel experimental scheme and present the most recent results obtained from our system. the resolution demonstrated to date is nm using helium-neon wavelength and optical line scans are shown in figure . finally, a consideration of the basic theory demonstrates that much higher resolution can be easily anticipated. these preliminary results firmly establish the great potential of this new near-field optical microscopy for biological research. high-resolution scanning electron microscopy (sem) studies of enamel crystals from remineralized enamel have provided clues as to the changes in crystal diameters within specific zones of artificially created carious-like lesions. , these data have supported the evidence obtained through polarized light microscopy of zones of demineralized and remineralized enamel. the dark zone, viewed in polarized light, has been identified as the zone of remineralization. these initial studies were conducted using sputtered coatings of gold/palladium (au/pd). high-resolution images of the crystals within the zone of remineralization revealed large crystallites in excess of µm, often with what appeared to be remineralized "nodules." however, it was difficult to determine whether these were one crystal, or two crystals which had fused during the remineralization phase. a study was conducted using an ultrathin coating of sputtered chromium (cr) film and se-i imaging to provide topographic contrasts of crystal surfaces. since the majority of the se-i information should be produced close to the surface, the resultant image is specimen-surface specific. , artificial caries-like lesions were prepared in noncarious human teeth (second and third molars) and sectioned into µm sections which were then viewed in polarized light to identify the dark zone of remineralization. the sections were then fractured through the dark zone producing a spicule of enamel with an edge of fractured enamel free from sectioning artifacts. each specimen was then mounted onto supports with silver paste with its fractured edge facing up. measurements were then made to determine the location of the dark zone so that it would be easily located within the sem. specimens were degassed and then sputtercoated with nm au/pd and nm cr. , specimens were imaged at high magnification in the se-i mode by placement on the condenser/objective (c/o) lens stage of an isi ds- /lab sem at kv and a hitachi s- cold cathode field emission (fe) sem operated at kv. high magnification se-i images (s- fe sem) of au/pd coated enamel crystal surfaces within the dark zone, revealed particulate features decorated by metal giving it a continuous granular appearance (fig. ) . analysis of a specimen coated with a . nm cr film revealed crystal surfaces with well defined particulate features that were clearly delineated and separate from one another. this study documents the effect au/pd coating has upon the examination and evaluation of enamel crystals at high magnifications. results obtained document that an ultrathin coating of sputtered chromium film of - nm does not produce the coating artifacts found with conventional sputtered au/pd. an ultrathin coating of cr, together with the use of se-i imaging, allows surface features to be more accurately imaged and measured. iv- scanning vol. , supplement iv ( ) fig . previous results have shown that the ability of intercalative dyes to modulate the antiviral activity of poly r(a-u) was related to the groove through which the dyes intercalated into the poly r(a-u). when poly r(a-u) was combined with the minor groove intercalating dyes such as ethidium bromide (eb) or the minor/major groove intercalating dyes, optimum enhancement of antiviral activity was observed at a dye/ribonucleotide ratio of / . no enhancement of antiviral activity was observed when poly r(a-u) was combined with the major groove intercalating dyes. when eb was combined with poly r(a-u) and then added to human foreskin fibroblast (hsf), the % effective dose of the poly r(a-u) was -fold lower. the results of additional studies demonstrated that the enhanced antiviral activity was not due to superinduction of interferon, direct viral inactivation, or host cell cytotoxicity. phase contrast, fluorescence, and confocal micrographs of hsf cells following a -h exposure to µm eb alone or a µm eb/ µm poly r(a-u) combination stained the nucleolus, but not the chromatin. negatively stained transmission electron microscopy (tem) preparations (fig. a) and replicas (fig. b) (fig. a,b) which may be associated with the enhanced antiviral activity of this combination. under the conditions employed in this study, poly r(a-u) exhibits an elongated conformation ( - nm in length) that possesses a number of hairpin loops as well as single-stranded and double-stranded domains (fig. a,b) . the double-stranded domains are found predominantly at the base of nm hairpin loops. in contrast to the poly r(a-u) alone, micrographs of the eb/poly r(a-u) combination illustrate the presence of condensed structures with diameters ranging from to nm. results from scanning force microscopy corroborate the results of both tem preparations. tem of unstained and uranyl-stained eb/poly r(a-u)-treated hsf cells illustrate the endocytosis of electrondense material into acidic compartments of the hsf cells. subsequently, the electron-dense material escaped from the acidic compartment and formed electron-dense bodies with dimensions that closely approximate the dimensions of the eb/poly r(a-u) combination visualized in the negative staining preparations. these electron-dense bodies are detected near the nuclear membrane and in the nucleolus. the nucleolus of an unstained, eb/poly r(a-u)-treated hsf cell demonstrates the segregation of the nucleolar components so that the fine fibrillar component, which comprises the nucleolar organizer region, is being peripheral to a dense granular material occupying the major central area of the nucleolus. the results of the current study and our previous work suggest that the elevated antiviral activity of the eb/poly r(a-u) combination may be related to the ability of the eb to complex with poly r(a-u) and condense it into a conformation with dimensions that can be accommodated by endocytotic vesicles. exit from the acidic compartment is promoted by unbound eb that induces endosomal or lysosomal swelling. subsequently, changes in the topology and surface charge of the poly r(a-u) induced by the eb allow increased access to the nucleolus which results in the modulation of additional cellular processes, especially rrna synthesis and processing. aromatic constituents in plant cell walls are associated with other wall components and affect strength and other characteristics of plant cell walls. ultraviolet (uv) absorption microspectrophotometry has been useful in characterizing aromatics within specific cell types in plant organs. , , this technique was applied to walls of bran and endosperm cells in a series of hard and soft wheats. kernels from hard and soft cultivars were fixed in glutaraldehyde ( % in . m cacodylate buffer at ph . ), and sections were cut with a microtome at µm thickness. the sections were mounted on quartz slides in glycerin and covered with a quartz cover slip. cell types in the sections were analyzed for uv absorption using a computer-controlled zeiss umsp microspectrophotometry system. transmitted illumination was provided by a xenon lamp (xbo w) with a connecting grating monochromator using a bandwidth of nm. a -× quartz objective lens with a final aperture of . µm, which was delimited within about onethird of the area of a field-limiting diaphragm to reduce stray light, was positioned over walls of selected cell types. absorbance of transmitted uv illumination was recorded over a range from - nm at -nm increments. the system was standardized at µm. spectra were collected, displayed, and evaluated using the zeiss lambda scan software. uv absorption maxima or distinct shoulders occurred near , , and nm. while the absorption near nm is not defined, absorption near nm is believed to be due to lignin (i.e., polymerized phenolic constituents) and that near likely is due to ester-linked ferulic acid. synthetic lignin, from polymerization of coniferyl alcohol with horseradish peroxidase, has a strong absorption at nm and no absorption at higher nm. further, ferulic acid linked to arabinoxylans, which has been isolated from bermudagrass cell walls, gave a shoulder near nm and a strong max at nm. other work, using the addition of cinnamic acids to milled lignins or removal of phenolic esters by alkali treatment, supports the above spectral interpretations. the kernel of wheat consists of several distinct cell types (fig. ) . spectra were obtained of walls of these various cell types (fig. ) . epidermal and parenchymal walls gave the highest absorption and both had spectra indicative of lignin (near nm) and also of ester-linked ferulic acid (near nm). nucellar epidermis walls have spectral patterns similar to the cell types above, but absorption was much less. aleurone walls gave spectral patterns indicative of mostly ester-linked ferulic acid and less lignin than previous cell types; the side walls gave a considerably higher absorbance than upper or lower walls. endosperm cell walls, which enclose the starch/protein matrix, were totally lacking in uv absorption maxima, indicating no aromatic constituents in these walls. variations occurred in uv absorption and spectral patterns for walls of various cell types in wheat kernels. however, no consistent differences occurred among the various kernels that might relate to hard-or soft-rated wheats. iv- scanning vol. , supplement iv ( ) fig . food microscopists are continually searching for noninvasive methods of examining the internal structure of foods, especially those with delicate labile structures. x-radiation is of considerable potential because of the great penetrative power and the minimal need for sample preparation. examination can be carried out at atmospheric pressure, and there is no intrinsic reason why temperature-controlled or temperature-ramped studies are not possible. the term x-ray microscopy covers several techniques and can be defined as the use of x-rays to produce a magnified image, or to give information about specific microscopically identifiable parts of a specimen. our current equipment consist of an electron gun, two electromagnetic lenses (condenser and objective), and x-ray target. x-rays are generated from a µm spot on a tungsten target. the target usually is positioned so that the microfocused electron beam glances the side of the target. x-rays from the microfocal spot form a cone which diverges through a thin window. focusing is achieved by placing a grid with bar widths of and µm into the beam and adjusting the current in the condenser and objective lenses. the image is captured on a tv monitor. finally, test radiographs are recorded on x-ray sensitive film. the x-ray radiograph is a shadowgraph and, where the sample is thick, the overlaying shadows will be complicated to interpret. two methods can be used to help interpret the image. one method is to take stereo pairs of the sample and view these images with a suitable stereo viewer which will show a threedimensional image. the other method is tomography. in the projection x-ray microscope, the entire specimen is in focus at once with the same resolution, determined by the spot size, although different depths within the specimen are magnified by different amounts. three-dimensional images can be made by moving the specimen either by tilting or translating it between exposures in the divergent x-ray beam. these images are then viewed with a suitable stereo viewer which will show a three-dimensional image of the structure removing some of the confusing overlay of detail to be found. we have also obtained images from pieces of tissue subject to tensile elongation. in the design of our x-ray microscope there is considerable distance separating the x-ray film from the specimen, and this space allows the apparatus for tomography to be set up. tomography is a form of imaging whereby a section or "tome" can be taken through a specimen by using x-rays, and details above and below the sectional area of interest will be blurred out, giving a fairly clear image of this area. this technique is nondestructive to the sample. the method is similar to that de- scribed by lindegaard-andersen and thuesen, originally proposed by watson. in their method, the x-ray source is stationary and the subject and recording film are rotated synchronously. the x-ray beam strikes the film at near glancing incidence producing transaxial summation images. however, the associated point spread function has a /r radial dependency, which means that smearing of detail will have occurred. the results are sufficiently encouraging, nevertheless, to stimulate the search for improved contrast and resolution, with provision for three-dimensional mapping of internal structures. the images shown here are of food products containing large air cells, giving maximal contrast in the x-ray beam. we have obtained comparable success with water-filled cellular structures such as vegetable and fruit tissue. in the future, we anticipate further enhancement of contrast and resolution by the use of improved x-ray flux, image capture, and image processing. centre for food and animal research, agriculture and agri-food canada, ottawa, ontario, canada over the past years, electron microscopy (em) has proven to be a useful tool in assessing the effects of various physical and chemical treatments on the microstructure of egg components (related to the production of egg products, baking, behaviour in various food systems, nutrient bioavailability, etc.) much of this information has been acquired using transmission electron microscopy (tem) to view yolk samples which have been biochemically extracted after removal from the egg. the author studied microstructural changes in yolk during its in vivo digestion by the epidermal cells of the chick embryo yolk sac. yolk is digested within these cells, as signified by its presence in varying degrees of microstructural alteration, compartmentalized within cell organelles. a temporal sequence can be inferred with the degree of microstructural change observed. it was possible to follow the fate of yolk granules and low density lipoprotein (ldl) particles during digestion, because yolk granules retain their integrity during preparation for em. ldl particles were made visible by crosslinking action of the fixatives and by the use of a specific enhancement technique. initial work focused on the microstructure of native, undiluted yolk which had been fixed using conventional and novel fixatives. thin sections were examined by tem. these experiments allowed us to optimize fixation conditions and confirmed earlier results by other workers that yolk is composed of granules which have a subgranular structure and lack a boundary membrane. the granules sit in a suspension of closely packed ldl particles. yolk granules from incubated eggs were identical to those from unincubated eggs and showed no microstructural changes to indicate that digestion had taken place outside of the cell. after clarifying that yolk is digested intracellularly, the next phase of the study was the microstructural description of intracellular yolk and yolk sac epithelial cells during incubation, prepared using half-strength karnovsky's fixative and the imidazole-buffered osmium tetroxide protocol (ibo) of angermuller and fahimi ( ) which enhanced lipid staining. epithelial cells were examined systematically using em, from apex to base, to study the three processes associated with digestion: uptake, breakdown, and transport. using tem, granules and ldl particles were observed to enter the cells in uptake structures, the microplicae and coated pits, respectively. when samples were viewed using sem (fig. ) , these uptake structures appeared as fossae of different sizes, which have not previously been described in the literature. yolk granules and ldl particles were observed by tem, within the cells, in phagosomes, endosomes, and acid phosphatase-containing vacuoles (secondary lysosomes), components of an active intracellular digestion system as it is presently known. the secondary lysosomes contained yolk granules exhibiting various degrees of microstructural alteration (fig. ) . the microstructure of these intermediates in yolk digestion appears to be very similar to that appearing in the literature describing known biochemical manipulations of yolk ex ovo, and indicates that a re- iv- scanning vol. , supplement iv ( ) fig. sem of apices of yolk sac epithelial cells from an embryo incubated for days. yolk granules have entered the cell, and a fossa (f) is observed. mp = microplicae. lationship may exist among structural, functional, and biochemical information. the results of these individual experiments were used to propose a scheme of yolk digestion based on progressive microstructural changes of intracellular yolk. in addition, a transport system for yolk lipid and its digestion products to the embryo was microstructurally demonstrated using ibo. this transport system shared ultrastructural characteristics with that reported for lipid transport in intestinal cells, especially with respect to the formation and transport of chylomicronlike particles. laser scanning confocal microscopy and digital image processing were used to visualize the pattern of de novo blood vessel formation in the quail embryo. stage quail embryos were fixed on the yolk, excised, then immunolabeled with qh , an antibody marker for angioblasts and vascular endothelial cells . after incubation in fluorescein-conjugated secondary antibodies, the embryos were mounted in pbs/glycerol ( : ratio) under a # glass coverslip. specimens were examined on a biorad mrc confocal laser scanning device equipped with zeiss optics. a × objective lens was used to image the entire caudal half of each embryo during the laser scanning step. ten µm optical sections were acquired in a plane parallel to the embryonic axis (en face). the ten optical sections were then collapsed into one composite image using biorad's proprietary software (fig. ) . the image was imported into photoshop ™ software on a macintosh quadra . using this software, the single composite fluorescence image was processed with the "emboss" tool (fig. ) . other image processing routines such as edge tracing, sharpening, and contrast enhancement can also be applied with ease (not shown). in contrast to conventional microscopy, this procedure yields a map of the entire endothelial network of the quail embryo in sharp focus, and with a highly favorable signal-to-noise ratio (fig. ) . while some geometric distortion occurs when ten sections are collapsed into one imaginary plane, the flat- ( ) and ldl particles ( ) are found in separate organelles, and are also found together within the same organelle ( ) . myelin bodies (m) and lipid drops (l), products of intracellular digestion of yolk, are also observed. fig . this immunofluorescence image depicts a map of all the vasculature elements in the caudal half of a developing quail embryo (stage ). the wide vessels on each side of the embryonic axis are the dorsal aortae; lateral to the aortae are two fields of rapidly forming vascular networks. tened nature of the early avian embryo minimizes this problem. also, since the qh epitope (a carbohydrate) is present on multiple cell surface molecules, the image gives a reasonable approximation of vessel morphology and the protrusions of the vascular endothelial cells. the rendering of the data into a pseudo three-dimensional relief provides the observer with a more familiar visual format (fig. ). the type of image shown in figure facilitates comparison of endothelial sprouts and anastomotic foci during formation of the first vasculature in the quail embryo. based on images from embryos at progressively older developmental stages, we suggest that morphogenesis of the lateral anastomotic network occurs by mechanisms that involve angioblastic tractional structuring of the extracellular matrix (madri et al. , montesano et al. , stopack and harris . this hypothetical mechanism more recently has been elaborated upon by vernon and colleagues ( ) . according to the latter workers, cellular responses to morphogenic cues within the highly planar extracellular matrix underlie early vascular patterning. in addition, the digital imaging approach shown here offers an improved method of comparing normal vasculogenesis with experimentally produced malformations (drake et al. ) . ( . - . ). chemical composition and ph did not change during extended heating. the shmp sample was the firmest and the sc sample was initially the softest. the firmness of the sc and tsp samples increased with extended heating whereas the firmness of the dsp sample did not change. meltability decreased with extended heating in all samples, except the shmp sample which had the lowest meltability of all samples throughout the experiment. there were marked differences in the microstructure of the process cheese protein matrices after heating. osmiophilic areas gradually developed during heating and their incidence was related to the melting salt used. after h of heating, they were numerous but considerably smaller in the dsp sample ( fig. ) than in the tsp sample (fig. ) . the highest and the lowest incidence was in the shmp and the sc samples, respectively, which were the samples with the lowest and highest meltability. new york, usa mozzarella cheese contains parallel protein fibres created by stretching the curd in hot water during manufacture. the protein fibres are oriented parallel to the direction of extrusion and are separated by milkfat or whey (kaláb , taneya et al. . by varying manufacturing parameters and storage time, cheeses with different sensory/functional properties, as well as with different microstructural characteristics, can be produced (kiely et al. ). in the present study, the impact of varying the stretching temperature ( °c, °c, and °c) and storage time ( days, weeks, and weeks) at °c on the microstructure of mozzarella cheese was investigated using light microscopy (lm) and scanning electron microscopy (sem). for both lm and sem, samples were fixed in % glutaraldehyde in mm pipes containing mm cacl , before cryosectioning ( µm sections). sections were cut both parallel to the protein fibres (longitudinal) and across the fibres. for lm, sections were stained with ice-cold oil red o and methyl- ene blue. because the fat is not well fixed by aldehyde fixation, slides were held on a bed of ice during staining to prevent fat migration. for sem, the sections were stained with uranyl acetate in methanol, washed in methanol, and stained with lead citrate, dried, mounted on aluminum stubs, and gold-coated. this procedure removed most of the free fat and water/whey, allowing the association of the protein fibres to be clearly visualized. increased stretching temperature increased the size of the fat globules, which was particularly noticeable in cross sections. with °c stretching, the fat globules were relatively small and uniformly distributed across the section, while with °c and °c stretching there were almost two populations of globules: some small and some very large. in longitudinal sections, the protein strands formed during stretching at °c and °c were slender and smooth, while those formed during stretching at °c were thicker and less regular. little difference was seen with storage time in either longitudinal or cross sections for the three different stretching temperatures using lm. with sem, however, after removal of the fat, the ability of the fibres to hold together decreased with storage time. this is best seen in longitudinal sections, where the protein fibres are closely associated in cheese stored for days (fig. ) , and are able to withstand the stress of drying, while in cheese stored for weeks (fig. ) , the fibres have been weakened (presumably by proteolytic activity from coagulant and culture enzymes) and pulled apart as the section dried. indian dairy association, sector-iv, r.k. puram, new delhi, india milk is a canvas of seemingly silent molecular structure. there is a large variety of "resident molecules" with molecular harmony in milk which are of mammary gland origin. in general, the "structural matrix" of milk comprises three classes of biomolecules: molecules in suspension (casein micelles), molecules in solution (lactose, proteins, vitamins, and salts) , and molecules in emulsion (fat globules). because of its excellent nutritive composition, milk is ubiquitous and the most popular "ready-to-consume" food from time immemorial. today, in iv- scanning vol. , supplement iv ( ) fig . different parts of the world, milk from various species of animals is used for food. in the u.s., however, the cow furnishes virtually all of the available market milk, whereas in my country more than % of the total milk produced is of buffalo origin. hence, structural studies particularly on milk and milkfood products made from various animal species are more fascinating and challenging from a global angle. it is the local consumer habits that determine the final structural orientation of the product in the country of origin, especially in a country such as india where to % of milk produced is converted into a variety ( plus) of traditional milk products, using processes such as coagulation (heat and/or acid), desiccation, and formulation. hence, a presentation on the "structural style" of milkfoods which are developed on the basis of "environment-friendly green technology" in the tropical countries might generate a new dimensional need for future studies on milkfood products. a comprehensive literature scan of published papers in the areas of "food" and "food structure" during the last years reveals the following publication status: during the period to : . global: of the total number of , food articles published, , are related to "food structure." the number on "food microstructure" is . when i think of their contributions, i claim no merit for my presentation in this conference today. these data provide a "food-for-thought" why food structural studies do not receive the desired attention. we may ponder this issue. while preparing this presentation, the excellent review article by kalab on food structure and milk products was extensively consulted. keeping in view the existing and reported studies on the subject, the situation in the tropical countries, particularly regarding indian milk such as buffalo milk, deserves special attention. according to kalab the structure of milk and milkfoods determines their properties such as firmness, spreadability, elasticity, viscosity, and susceptibility to syneresis, which are globally recognized as texture. the understanding of the processes both conventional and traditional and their relationship between "structural style" and "textural mood" is important in product development. in the tropical countries, the preprocessing exposure of milk to the environment, that is, temperature, microbes, and humidity has to receive extra attention. the dynamic status and kinetics of molecular interaction in different species of milk should be a vital area of structural studies in asian countries. the structural studies on milk and milkfood are generally conducted by use of microscopy. the methods used are optical microscopy such as fluorescence microscopy and confocal scanning laser microscopy, and electron microscopy consisting of two major types: scanning electron microscopy (sem) and transmission electron microscopy (tem). these techniques meet the particular requirements of the study. the application and findings derived using sem and tem in various milk food systems have been extensively covered recently by kalab . repeating and reproducing the published data may not be necessary for the participants in this conference. however, a brief microcosm might be refreshing, and is hence documented below: . milk casein micelles ( - nm), being small, cannot be seen using a light microscope, but tem shows them as globules that are apparently composed of submicelles. . the casein micelles, if deprived of their stabilizing factor, aggregate and form a gel of a regular structural "crowd." . in the case of acid-gel, the micelles start disintegration as a result of the solubilisation of their structural partners such as calcium phosphate. . there is an interstructural and intrastructural relationship between the suspended casein micelles and soluble blactoglobulins. heat disturbs their stability equilibrium and their molecular relationship. . among the milkfoods, cultural milk products like yoghurt are highly influenced by heat in relation to their "set-style" and "stirred-style." the indian counterpart, "dahi," offers its advantage when made from buffalo milk because of its high curd tension due to high calcium level. this area deserves a close look using sem technique. . as far as cheese is concerned, because of its inherent multinatural processing steps using chymosin on one hand and starter culture on the other, it is an evergreen exploratory platform using either sem or tem. . dried milkfoods as spray-dried milk particles are globular of larger diameter fat-globules, however, they undergo some changes as revealed by sem studies. lactose plays a commanding role in a so-called instantisation process. while making a presentation before a "highly-structured" qualified scientist using sem and tem, it is tempting for me to present our own work of limited nature. in our laboratory, during the last decade, we carried out a sem study on buffalo milk and its casein. the micelles are of larger size and have higher-bound calcium unlike cow casein micelles. regarding the casein fractions, an elaborate study on the b-casein in relation to structure and function as affected by heat and enzymic cleavage were carried out. work relating to microstructure development on another indian milk product, namely "paneer," from buffalo and cow milk has been reported. more recently, while working on casein micelles in csiro dairy research laboratory in australia, the author made an original contribution when ascertaining the micellar integrity of casein micelles in milk. he used a very simple technology not dependent on sem or tem. it was on the "colour communication" property of molecules as an integrated system or in isolation. using this new technique, it is possible to declare the miscellar integrity and to identify milk from different animal species, based on the different miscellar structure-dependent light-reflecting phenomena. the time has come to develop a "global strategy" on milk structural studies with a view first to understand the local product and then to ensure marketing of "rightly-structured milkfoods" of the right type to meet the countries' marketing needs. starch is a major component of many foods and is commonly used to modify the texture of a system. starch gels are composites of swollen gelatinized granules embedded in a continuous amylose network. the rheological characteristics of the starch pastes or gels depend on the shape and swelling power of the granules, amount of amylose and amylopectin leached outside the granule, network entanglement, and interaction between the paste components. many food systems also include in their formulations a lipid phase, thus, starch-lipid interaction becomes a major concern in starch paste rheology. the interactions of triglycerides might be different from those of monoglycerides due to their amphiphilic character, dispersion states, and steric hindrances. triglycerides owe their characteristics to their fatty acid composition (chain length and insaturation) and distribution (davis et al. ) . the objectives of this work were to analyze the effect of different lipid phases on the swelling power of the starch granules, as well as to analyze the rheological behavior of the starch pastes (apparent viscosity and viscoelasticity). commercial corn starch (cs) (refinerías de maíz, argentina) was used as thickener. the lipid phases used were: ( ) shortening (sh) (molinos río de la plata s.a., argentina) containing . % of mono-and . % of polyunsaturated and . % of saturated fatty acids; ( ) sunflower oil (so) (molinos río de la plata s.a., argentina) containing . % of monoand % of polyunsaturated and % of saturated fatty acids, and ( ) commercial glycerol monostearate (gms) mivaplex (eastman kodak co., u.s.a.), which contained % of monoglycerides and % of diglycerides. seven percent starch suspensions with and without lipid phase ( % of sh or so or % of gms) were prepared using a modification of the method of eliasson ( ) . swelling power was calculated as the weight of sedimented gel divided by the original dry weight of starch. samples were placed on slides and micrographed in a leitz ortholux ii microscope with a photographic camera leitz vario orthomat (leitz, germany). the suspensions were gelatinized in a thermostatic bath at ± . °c under standardized conditions. a rotational viscometer haake rotovisko rv (germany) with a sensor mvip of concentric cylinders was used. the measurements were performed at °c and the transient shear stress curves (τ vs. time) were obtained at different constant shear rates (d) from to s − . apparent viscosities were calculated as τ/d ratio at d= s − . iv- scanning vol. , supplement iv ( ) table i shows the swelling power obtained from the starch suspensions with and without lipid phase. when either of both triglycerides, sh or so, was added, the swelling power values were higher than the value of the control (cs). the so, with the highest insaturation content, presented the higher swelling power. the addition of the monoglyceride (gms) led to the lowest value. the relative sizes of the swollen granules shown on the micrographs of figure confirm these observations. gms forms a helical inclusion complex with amylose, which might delay the transport of water into the granule and consequently decreases the swelling power (eliasson ) . the formation of inclusion complexes between the triglycerides and the amylose seems to be difficult because of the steric characteristics of these lipids. apparent viscosities correlated well with the swelling power (table i) ; the larger the swelling power the higher the viscosity obtained from the rheological curves at long shear times. bird-leider model (dickie and kokini ) was used to analyze the viscoelastic behavior and the shear time dependence of the different pastes: where n and m are the power law parameters and b and c are adjustable parameters related to the viscoelasticity and to the structure breakdown of the samples. a satisfactory goodness of fit was obtained (r min= . ) (fig. ) . when the swelling power increases, b parameter decreases (table i) . since viscoelasticity (b) depends mainly on the network entanglement (leached amylose) and the volume of the swollen granules, both factors should be considered to explain this behavior. because of the health risk and other environmental factors that airborne grain dust presents to the working population, our laboratory has initiated studies on the isolation, identification, and characterization of bacteria that possess multiple resistance to a series of antibiotics and other compounds (e.g., insecticides and pesticides) at the molecular level. samples of grain dust were collected from various grain elevators in the duluth-superior regions of the u.s. during the diverse growing seasons. each sample collected consisted of a heterogenous population of constituents that vary with encountered geographic, climatic, and handling differences. in addition, the geographical growth regions and the mechanism of storage of grains appear to be directly associated with the microbial flora and occurrence of toxic substances. scanning electron micrographs of the concentrated grain dusts were morphologically consistent with the observation of previous investigators. the dust from various plants consisted of a distinct assortment of particles; small husk fragments or pericarp (seed coat in case of flax) and "trichrome-like particles" were also present. numerous bacteria spores were seen at high magnification, particularly durum wheat and barley. light photomicrographs showed a heterogenous population of both gram negative and gram positive bacteria. the bacteria consisted of different shapes such as short rods, long rods, and cocci. transmission electron photomicrographs revealed the isolated strains to consist of one or more flagella attached to the membrane surfaces. at least three of the bacterial strains isolated were encapsulated. we exposed, selectively, twelve of the isolated bacterial strains to a variety of antibodies, pesticides, and insecticides. as a result, of the bacterial strains tested showed resistance to both ampicillin and bacitracin ( µl/mg). of the strains, showed resistance to insecticides (sevin) and the pesticides (enforcer) as high as µl/mg. three of the five strains that were resistant to both ampicillin and bacitracin were also resistant to the insecticide sevin at high concentrations ( µl/mg). these data suggest that bacteria found in grain dust may be directly or indirectly related to occupational health disorders. chlordiazepoxide (librium) is a commercial antianxiety agent, but its use has not been associated with any striking health improvements. nevertheless, millions of chlordiazepoxide (cdz) prescriptions have been written and the drug is widely employed clinically as a muscle relaxant, anticonvulsant, anxiolytic, and hypnotic. adverse effects vary from skin rash, nausea, headache, and impaired sexual functions to vertigo and lightheadedness, as well as complications from the drug's administration during pregnancy. the ciliated protozoan, tetrahymena pyriformis, has been of significant importance to research since its successful axenic culture years ago. the drug's tranquilizing effects on humans may possibly be elucidated by investigating cdz-induced impairment of the protozoan's growth and motility through modifying microtubular-directed ciliary function (bell et al. ) . here, uptake, recovery, and attempted localization of cdz, as well as its alterations of cellular ultrastructure are reported. thin layer chromatography of cdz (hoffman laroche, nutley, n.j.) in three different mobile phases revealed optimal cdz stability when dissolved and stored in isoamyl alcohol; benzene. after days of liquid culture, t. pyriformis removed % of the radioactivity from [ c] -cdz in the growth medium. liquid scintillation counting of cell washes during the -day time course suggested surface, nonspecifically-bound radioactivity. following days of culture, [ c] -cdz together with its metabolites and/or breakdown products were recovered from homogenates of tetrahymena. to detect the intracellular sites of cdz localization and possible action, both transmission electron microscopy and immunoelectron microscopy were performed. the former revealed that cdz disrupted cytoplas-buffer or cacodylate-buffered dopa revealed dopa/ppo reaction product but failed to reveal a definitive, substrate localization of the enzyme. instead, cytoplasmic morphologic distortions of buffered dopa-treated hyphae were apparent, mandating modification of the employed higher-plant, cytochemical procedure for localizing fungal ppo. thus, attempts to establish the route of ppo secretion to the growth medium utilizing liquid cultured hyphae may be of limited value as the sheath appears to be sloughed during shake culture. in this connection, c. versicolor grows upon a solid substratum when decaying timber in situ. support: doe-bctr program. the evaluation of surface topography is conventionally done with the scanning electron microscope (sem) by visually reconstructing stereo images in a stereo viewer. the monocular clues in an sem image, shown for example in the image in figure , can also be used to map the topography of a sample surface. the first attempt to identify the surface gradient from a secondary electron detector in this way was reported by suganuma ( ) who found that ( ) and described the inclination of the surface to the specimen plane, where a and b are the signal outputs from the two detectors and a n and b n are the signal outputs from the same material when it is perfectly horizontal. although it is possible to add additional detectors and to modify their signal outputs in a conventional electron microscope, the technique can also be implemented in a straightforward manner by using the differences in signal intensity from the two halves of a split backscattered detector. thus if a and b are the signal outputs from the two different halves of the detector, the backscattered signal, which is usually collected as topographic information (a − b) or composition information (a + b), can be multiplied together (a − b ) to produce the basis of the empirical relationship described by suganuma iv- scanning vol. , supplement iv ( ) fig . ( ) to fit the contrast variations. indeed, since the denominator in this equation is also a constant and there is invariably a flat spot somewhere in the image, it is easier to simply use the expression to try and describe the surface gradient. this function can then be scaled to an absolute magnitude of about to take maximum advantage of the contrast variations that exist. the simplest way to evaluate the surface topography without actually modifying the microscope is to process a digital image in a computer. such an image can be collected directly or a polaroid micrograph can simply be scanned into the machine. in either event the processing is fairly straightforward to accomplish the use of software such as the nih image program. the surface profile is obtained by integrating the surface gradient over the scan distance, and in figure we show, by way of an example, a faceted surface displayed as a conventional secondary image, as a topographic profile, and as a grey scale image indicating the faceted surfaces with the same orientation to the electron beam direction. suganuma t: measurement of surface topography using sem with two secondary electron detectors. j electron microsc ( ), - ( ) corn kernels of an unknown italian cultivar were surfacesterilized in full strength bleach ( . % sodium hypochlorite), rinsed in sterile distilled water, and germinated on difco potato dextrose agar (pda) under fluorescent light at room temperature. isolation of bacteria was made either directly from the rhizosphere of seedlings or from colonies produced on agar. bacteria were identified by their fatty acid profiles (microbial identification system, newark, del.) and diagnostic biochemical test (micro-id, durham, n.c.) . fusarium moniliforme used to test for bacterial antagonism were isolated from corn. experiments designed to infect seedling roots with the isolated bacterium were performed on corn kernels that were subjected to a double sterilization process in which the kernels were surface-sterilized with bleach and then subjected to a mild heat treatment to remove internal bacteria and fungi. this process produced sterile seedlings which remained so at least up to the to leaf stages of growth. the corn cultivars used were trucker's favorite, silver queen, reid's yellow dent, and an inbred cultivar, pr. samples were fixed in % glutaraldehyde in sodium cacodylate buffer and postfixed in % osmium tetroxide in buffer. the tissues were dehydrated in an ethanol series, critical-point dried and coated with gold-palladium or embedded in spurr's medium and sectioned. samples were examined using a philips scanning or jeol cx transmission electron microscope. the bacteria isolated from roots of the italian corn cultivar were gram-negative rods and were further characterized as being positive for voges-proskauer reaction, nitrate reductase, and ornithine decarboxylase and b-galactosidase. the isolate fermented arabinose and malonate. it was negative for phenylalanine deaminase, lysine decarboxylase, urease, h s, and indole production. it lacked the ability to utilize adonitol, inositol, and sorbitol, and was negative for esculin hydrolysis. these biochemical characteristics served to place this bacterium in the family enterobacteriaceae, the tribe klebsielleae, and it was identified as e. cloacae according to ewing ( ) . microscopic studies established that the bacterium was endophytically associated with the roots of the corn cultivar. scanning and transmission electron microscopy demonstrated that the bacterium was distributed uniformly over the corn root epidermis but was randomly distributed intercellularly in the root cortex and outer margin of the pericycle, usually adjacent to phloem cells. although there was a proliferation of bacterial cells in the intercellular spaces of roots (figs. , ) , there was no evidence of damage to host cells, decline in seed germination, nor seedling growth from e. cloacae infection during the week observation period. the presence of the bacterium on kernels of several corn cultivars enhanced the growth of corn seedlings and inhibited growth of f. moniliforme when the bacteria-infected kernels were germinated in fungal-amended soil. this indicated that e. cloacae is biologically associated with corn plants. the bacterium exhibited strong antagonism to several f. moniliforme isolates when grown on nutrient agar and pda plates. the fungus growth was inhibited and restricted to aerial growth on agar plates. the endophytic nature of e. cloacae in corn roots and its antagonism against isolates of f. moniliforme indicate that this bacterium has potential for the biocontrol of f. moniliforme, a corn pathogen. tracheal organ cultures have been widely used for the demonstration of the cell-and organ-destroying capacities of bacteria and viruses colonizing the respiratory tract. however, the assessment of the cell-damaging abilities of fish parasites, the gill organ culture appeared to be a suitable tool (stadtländer and kirchhoff ) . piscine gill epithelium represents the relevant target tissue for cytotoxicity studies of fish parasites, but this tissue is diffi- iv- scanning vol. , supplement iv ( ) fig cult to cultivate in vitro. excessive mucus production complicates the investigation with the light and electron microscope. this is even more difficult when infected specimens are investigated. here, cell debris due to released cells and cell fragments require special attention of the investigator to obtain interpretable electron micrographs. a fine balance is required between preservation of the status quo of the in vitro situation and the necessary procedures (multi-step preparation) for scanning electron microscopy (sem). in this abstract, we describe the detailed and improved preparation of piscine gill epithelium for sem. this will allow other investigators using piscine tissue for research to obtain scanning electron micrographs with high resolution at high magnification. gill filaments were obtained from rainbow trouts (salmo gairdneri, richardson) and were infected in vitro with mycoplasma mobile k, a wall-less prokaryote causing severe damage in gill organ culture (stadtländer and kirchhoff ) . each gill filament (noninfected and infected) was rinsed in several changes of . m cacodylate-trihydrate [cacodylic acid buffer (cb)] at ph . to free the tissue surface from mucilage, medium, and unattached mycoplasmas. the washing step turned out to be critical for obtaining satisfactory results. excessive washing led to complete removal of cell debris and did not reveal the same status quo of infection as seen with the light microscope during the in vitro cultivation of gill filaments. on the other hand, insufficient washing complicated the interpretation of electron micrographs, especially the identification of m. mobile on gill epithelium, due to excessive mucus and cell debris released from infected tissue. the pre-fixation was performed with . % glutaraldehyde (v/v) (in cb) for h at ˚c. the aldehyde was removed by careful rinsing in cb (three times for min). samples were not postfixed, but dehydrated in acetone covering a graded series of solutions (acetone/water) from through % (v/v). all specimens were critical-point dried (cpd) using carbon dioxide as the transitional fluid. cpd was performed in a critical point drying apparatus e (polaron equipment ltd., england) by filling the chamber with / of liquid carbon dioxide. the heating process was conducted in ˚c per min steps until the critical point was reached ( o c and bar). samples were immediately placed in an exsiccator containing anhydrous calcium chloride to avoid rehydration of samples by air contact. after being attached to aluminum mounting stubs using double-sided sticky tape, each specimen was coated with a layer of to nm of gold in a hummer v sputter coater (technics inc., alexandria, va., usa). samples were examined in an etec auto scan b (etec, calif., usa) operating at an accelerating voltage of . - kv. results were documented on a polaroid × land film (type , positive-negative). the method described above allows the detailed investigation of noninfected and infected piscine gill epithelium at high magnification with an sem (fig. , ) . good structural preservation of noninfected, apparently intact secondary lamellae (fig. ) and destroyed lamellae after infection with m. mobile (fig. ) document the usefulness of the described method for the study of the infectious process with this mycoplasma on fish tissue and will certainly also give convincing results in investigations with other fish pathogens. the arrows in figure indicate the presence of the flask-shaped mycoplasma on damaged piscine gill epithelium. bars represent figure : µm, figure : µm. for this study, the sem was superior to light microscopy (lm) and transmission electron microscopy (tem). lm does not demonstrate details in the damaged tissue and tem is much more laborious and time consuming. the mammalian bladder functions in urine storage and expulsion and is thus subject to alternating distension and contraction. blood flow in the bladder wall is compromised by distension (dunn , levin et al. , and acute and chronic overdistension result in ischemia and necrosis, respectively. the latter often leads to loss of mucosal integrity. yet, the vasculature of the bladder wall has rarely been studied (inoue and gabella ) except at the gross level. in this study we describe several unique features of the bladder microvascular anatomy using light microscopy (lm), transmission and scanning electron microscopy (tem and sem), vascular corrosion casting, and alkali digestion (takahashi-iwanaga ). twenty four male new zealand white rabbits were anticoagulated, anesthetized, and cannulated via the abdominal aorta. for routine lm, tem, and sem, the bladder vasculature was flushed with buffered saline at physiologic temperature and pressure, then perfuse-fixed with buffered glutaraldehyde; for corrosion casting, the vasculature was filled with resin (mercox/methylmethacrylate monomer, / ); and for alkali digestion, tissue was treated with n naoh at ˚c for min. thin sections were stained with lead and uranium, and casts and digested tissues were mounted on sem stubs with silver paste or colloidal carbon and viewed at - kv. the bladder is supplied by left and right vesicular arteries, branches of the internal ( %) or external ( %) iliac arteries. within the adventitia, the vesicular arteries send coiled branches dorsally and ventrally over the bladder surface. secondary arteries penetrate the muscularis to supply a rich capillary plexus (fig. ) closely apposed to and located in grooves within the base of the transitional epithelium (fig. ) . capillaries measure - µm in diameter and are often fenestrated and invested with pericytes. veins exhibit abundant valves primarily in the basal half of the bladder. the lamina propria of the bladder consists of very loose, flexible, collagenous connective tissue. unusual capillary "glomeruli," associated with accessory vessels paralleling the primary bladder vessels, are present in the adventitia on either side of the bladder wall. these glomeruli consist of one to four contiguous capillary spheres. these various methods provide a clear, three-dimensional view of the microvasculature of the rabbit bladder, reveal the very close association between the urothelium and the underlying capillary plexus, and describe the fine structure of the mucosal capillaries. several unique features of the bladder vasculature including capillary glomeruli require further char- iv- scanning vol. , supplement iv ( ) acterization. the latter may be associated with sensory ganglia related to pressure sensation, but their function has not been determined. these results form the basis for comparison of normal bladder vasculature with that of experimentally compromised vasculature. the epithelial cell layer lining the nasal turbinates of humans is functionally and histologically consistent with that of the lower airways. this anatomic site is easily sampled using an inexpensive, disposable curette, and the epithelium obtained can be evaluated for both clinical and experimental objectives. the original rationale for ultrastructural evaluation of clinical specimens of nasal epithelium in this facility was to document index ciliary lesions consistent with the diagnosis of primary ciliary dyskinesia (pcd) (figs. , ) among individuals considered at risk. patients referred for study encompassed both adult and pediatric populations and had lifelong histories of chronic sinusitis, bronchiectasis, and/or otitis media, but with normal immunoglobulin levels. several patients presenting with situs inversus, polysplenia, or infertility problems-clinical findings considered risk factors for pcd-also were evaluated. approximately nasal biopsies are submitted annually for evaluation, of which approximately % demonstrate impaired ciliary motion and ultrastructural abnormalities that could be the basis for altered ciliary motion and a diagnosis of pcd. this finding is consistent with the suspected prevalence of pcd in the general population although the rate appears higher among individuals of scandinavian descent, pacific islanders, and inbred populations. the diagnosis of pcd is confirmed by the electron microscopic documentation of any of three ultrastructural level lesions of airway cilia. these lesions are: ( ) dysmorphology of dynein arms, ( ) absent radial spokes, and ( ) microtubular transpositions. in our experience, dysmorphology of the dynein arms represents the major form of ciliary abnormality associated with pcd. missing radial spokes and microtubular transpositions have not been documented among any of our patients. these clinical studies also have provided a unique opportunity for defining a spectrum of ciliary abnormalities which distinguish the heritable primary ciliary abnormalities associated with pcd from acquired cil- iary abnormalities associated with chronic disease or acute injury due to infectious processes or exposure to irritant gases in the ambient air. in addition, other histopathologic features such as mucus cell hyperplasia have been encountered occasionally among children presenting with chronic respiratory disease and referred for evaluation. in summary, our experience shows that ultrastructural evaluation of nasal epithelium can provide a clinically significant perspective on respiratory health. this work was supported by scor grants hl , hl , and hl from the national heart, lung, and blood institute and by u. s. environmental protection agency cooperative agreement cr to philip a. bromberg. this is an abstract of a proposed presentation and does not necessarily reflect epa policy. using conventional light microscopy, silicone has been described as a translucent, clear, mucoid, refractile material which is often difficult to visualize. it is not birefringent, as is silica, by polarized light microscopy. silicone gel tends to form homogenous, rounded "globules," "vacuoles," or "droplets" unlike the angulated, sharp spicules observed with silica. in paraffin-embedded tissue sections, silicone gel often appears to be slightly out of the plane of focus. in addition, silicone gel "globules" occasionally drop out of standard µm histologic sections during tissue processing, leaving partially or totally vacant holes or "cysts." because silicone extravasation and deposition into surrounding fibrous breast capsules is difficult to visualize by standard light microscopy techniques, the electron microscopist must often "blindly" examine multiple fields/grids in a labor-intensive fashion. to decrease the time generally required for the positive identification of silicon by electron probe microanalysis (epma), alternative light microscopy techniques for preliminary screening purposes were investigated. six periprosthetic capsulectomy specimens, synovium from a previously reported silicone breast augmentation patient with arthritic pain and a silicone granuloma from another patient with a ruptured prosthesis were utilized in this study. sections were cut at , , , and µm and mounted on glass slides. in addition to standard permount mounting media, aqua-mount was stained with ink preparations: black stamp pad ink, india ink and aniline artist dyes-black, blue, brown in a : mixture for coverslip application. each series was stained with a battery of common histochemical stains. the sections were then viewed with a zeiss axioplan microscope utilizing conventional incidental light, polarized, non-koehler, phase contrast, and darkfield microscopy. a commercial silicone gel and silicone gel extracted from a previously implanted silicone breast prosthesis were smeared and examined unstained or stained with papanicolaou and "diff quik." silicone was noted to be refractile, nonpolarizable, and nonstainable. the results confirm the refractile, nonpolarizable, and nonstainable properties of silicone in histologic and cytologic preparations (fig. , scale bar = µm). decolorizing techniques (toluidine blue o, etc.) that were not completely differentiated, especially with thicker sections, occasionally demonstrated nonspecific dye "trapping" on the larger silicone globules. the relative ease of silicone localization was greatly increased in histologic specimens with non-koehler, phase contrast, and darkfield microscopy when compared with conventional light microscopy. although standard h&e staining was adequate for silicone localization in tissue sections, uniform dark staining with toluidine blue o increased the contrast between the stained tissue and the unstained refractile silicone. the contrast was also enhanced by aqua mount black stamp pad ink mounting media, with the silicone appearing milky white. in thicker sections, negative staining was slightly accentuated because of the increased concentration of stain as well as thicker, more refractile silicone globules, especially with darkfield microscopy. by utilizing these sensitive screening techniques, we were then able to sample paraffin-embedded tissue selectively, which dramatically decreased the time and effort for correlating confirmatory identification of silicon by epma (fig. ) iv- scanning vol. , supplement iv ( ) the majority of biological samples are high in water content. preparing tissues for scanning electron microscopy (sem) requires removal of this water and often produces severe structural distortions due to surface tension forces during phase transitions. this can result in bulk shrinkage artifact, surface cracking, curled cell borders, clumping or flattening of cilia, and collapse of surface vesicles. failure to postfix biological material with oso can lead to extraction of surface membrane lipids during solvent dehydration. it is likely that many shrinkage artifacts are related to incomplete or improper fixation and drying. critical-point drying (cpd) has become the standard procedure for most biological materials. although it produces a relatively intact end product, wollweber et al. have reported shrinkage of as much as % after cpd of glutaraldehyde and osmium-fixed macrophages and lymphocytes. the use of mordant techniques to complement or enhance oso and uranyl binding may aid in preserving fine structural details regardless of the drying method. numerous substitution/transition fluids have been introduced for both critical-point and direct-evaporative drying, some with more success than others. an advantage in using solvent drying techniques as alternatives to cpd is the ability to process large numbers of samples simultaneously. early attempts at direct drying of nonosmicated samples from ethanol and other solvents produced a generalized cell shrinkage and collapse. freon evaporative drying was shown to be a useful rapid drying technique by liepins and de harven. gamliel further refined the freon direct-drying technique to include the use of guanidine hcl as a bifunctional mordant, prior to osmification, to minimize shrinkage of leukocytes. direct drying from hexamethyldisilazane(hmds) was introduced by nation to dry insect tissue and has subsequently been used for drying various other tissues (adams et al. ) . peldri ii has since been shown to be an effective solvent drying medium. it is a solid at room temperature. a comparative study by bray et al. of peldri ii, hmds, and cpd, using both plant and animal tissues, has produced identical results with animal tissues but not plant tissues. the purpose of this study is to introduce acetonitrile as a potential solvent for direct evaporative drying. acetonitrile has been used as a less toxic propylene oxide replacement transition fluid for transmission electron microscopy (tem) dehydration and infiltration (edwards et al. ) . to date, no studies have been published indicating the of use of acetonitrile as both a dehydration and intermediate transition fluid for direct solvent-drying of tissue-cultured cells for sem. this study compares solvent drying of kb (hela) cells from freon , peldri ii, hmds, acetonitrile, and ethanol to critical pointdried cells grown on thermanox coverslips. the mordant (gtgo) technique of gamliel is included to illustrate its usefulness in shrinkage control. solvent-plasticware compatibility should always be tested before preparing cultured cells for sem; whenever possible, the use of glass is best. cells grown on thermanox coverslips were fixed in . % glutaraldehyde, . m cacodylate, . m sucrose, . mm cacl , ph . . the gtgo fixation protocol of gamliel was found to be the most useful one in preserving cell architecture (figs. , ) . in figure , cells were evaporative-dried under a mild aspirator vacuum after gtgo fixation and dehydration in acetonitrile ( %, %, %, %- ×, min each). as a comparison, cells in figure were identically fixed and criticalpoint dried after ethanol dehydration. the overall appearance of the cells from both treatments is similar at low magnification (figs. a, a) , with some shrinkage evident in either case. under these conditions, it appears that the cpd cells retain more fine structural details than those evaporative dried from an (figs. b, b) . the results of this study are encouraging and may lead to future studies to develop a simple fixation protocol which enables evaporative drying from solvents directly miscible with water such as acetonitrile. snow, which may occasionally cover up to % of the earth's land, supplies about one-third of the water that is used for irrigation and the growth of crops (gray ) . for this reason, estimating the amount of water in winter snow pack is an extremely important forecast activity that attempts to predict the amount of water that may be available for the following growing season. unfortunately these estimates can be easily iv- scanning vol. , supplement iv ( ) fig . confounded by the sizes and shapes of the snow crystals that comprise the snowpack. a snow crystal is a single frozen ice grain that generally results from a process known as nucleation in which atmospheric water vapor condenses on a solid particle or nucleus at temperatures below °c. when nucleation occurs, the water molecules form a hexagonal crystal lattice resulting from the specific orientation and binding that occurs between the oxygen and hydrogen atoms. depending on the temperature and moisture that prevails during formation and descent of snow crystals, nucleation may result in plates, stellar crystals, columns, needles, or dendrites-all of which are based on the hexagonal lattice structure. an individual snow crystal may range in size from µm to mm (gray ); aggregations of two or more of these crystals form a snowflake. the shapes of snow crystals have been extensively studied and photographed with the light microscope (bentley and humphreys , nakaya ) . although these studies have resulted in a classification system that currently recognizes distinct classes and over subclasses of snow crystals (hobbs ), detailed examinations have been hampered by the difficulty of working with a frozen specimen, which is susceptible to sublimation and melting, and by the limiting resolution of the light microscope. for these reasons, an attempt was made to determine whether snow crystals could be collected/stored and prepared for observation and recording in the low-temperature sem. attempts to allow snowflakes merely to settle on a precooled specimen holder were unsuccessful; the snowflakes tended to "bounce" off the holder and those that did alight did not remain attached during subsequent handling. a successful procedure consisted of placing a thin layer of methyl cellulose solution on a holder and precooling it to the prevailing outside temperature during a snow fall. next, snowflakes were allowed to settle on the surface of the methyl cellulose solution. after a few minutes, the holder was plunged into liquid nitrogen and transferred to the laboratory where it was retrieved from liquid nitrogen, mounted on the transfer rod of an oxford ct- hr cryosystem, moved into the prechamber for sputter coating with au/pd, and then inserted into a hitachi s- field emission scanning electron microscope (sem) equipped with a cold stage that was maintained at − °c. these procedures allowed us to observe several forms of the individual snow crystals as well as their nucleation centers. at low magnification, the specimens, which did not appear to be altered by the sputter coating, resembled those that had been previously photographed with the light microscope. the snow crystals were stable in the beam, did not sublime, and could be observed at magnifications of , × or more to reveal microcrystalline water deposits or rime on the surface of some the snow crystals. this procedure, which was used to collect specimens during several snow falls in beltsville, maryland during the - winter season, was also capable of preserving sleet, graupel, and hail. furthermore, storage holders were devised that allowed capture of the snowflakes and their storage in liquid nitrogen until the specimens could be processed for examination in the sem. finally, the specimen stage of the sem allowed specimen tilt so that stereo images of the snow crystals could be recorded (fig. ) . in conclusion, low-temperature sem is a viable technique for examining snow crystals at magnifications that far exceed the resolution of the light microscope. furthermore, the ability to collect and store samples enables investigators to accumulate samples from numerous locations or different time intervals so that detailed observations and comparisons can be done in a convenient and orderly manner. these were identified and returned into the corresponding reconstruction sections in an automatic tracing procedure, programmed and executed on vidas . (zeiss/kontron germany). in addition it is possible to obtain a plastic, -d-like impression by applying the rcm with oblique illumination. this is achieved by decentralization of stach's slide (central diaphragm). . two main problems in -d reconstruction of histologic specimens are the horizontal distortion during the preparation of serial thin-tissue slides and the following vertical readjustment (alignment). we have recently shown that optical sections can be obtained by rcm within thick tissue-slides. its confocal-like principle provides an alternative to mechanical slices. the preserved integrity of the examined object allows the precise movement within the optical axis in one (vertical) dimension, thus avoiding manual alignment. applying the rcm on histochemical and immunohistochemical stains, reflections can be observed within the tissue. the depth of penetration of the light beam amounts to approx. µm, equivalent to about optical tomolevels (fig. ) . using the example of neurons of the supraoptic nucleus of the rat, we demonstrate this new technique on chrome-alum haematoxylin stained neurosecretion. the reflections of the dye particles associated with neurosecretory granules allow the precise localization of these subcellular structures. the visualization of the neurosecretion and its distribution is more distinct and of sharper contrast than in bright-field microscopy. reflected light is like a binary signal and therefore generates a suitable prerequisite for automatic discrimination in greyscale image analysis. thus identified black and white negatives were reconstructed with the module rec d on vidas . . this paper introduces two extended applications of the rcm for -d reconstruction. the generated optical slices and isohypses allow qualitative and quantitative investigations of intracellular structures and surfaces. quantitative chemotaxis is of great interest in a broad field of cell research (e.g., receptor-ligand interaction, ionic channels, cytoskeletal and metabolic processes, embryogenesis) as well as in clinical studies (e.g., immune reaction, wound healing, infection, tumor invasion). the most accepted assay to measure chemotactic behaviour of cells is the boyden filter assay, in which cells move against a gradient of chemical or biological substances. the chemotactic response is analyzed by measuring either the distance travelled by the leading front of cells or by quantifying the number of cells on the lower surface of the filter. this method is laborious and limited by the observer's subjective errors. we have developed a computer-based image analysis system to estimate the three-dimensional distribution of the migrated cells inside the filter. using one-micron optical sectioning, we can determine the position, size, and shape of many individual cells. within min, the position of thousands of cells can be recorded and the migration profile in the filter can be determined. it is possible to distinguish between different cell populations by selecting particular cell parameters. the system consists of a nikon labophot- a microscope with video microscopy and programmable focus control, used in connection with a hasotec-image processor with fast data acquisition and user-friendly software (mswindows). the high throughput, the consistent accuracy, and the simple operation of the system optimize all aspects of routine chemotaxis analysis in basic research and clinical studies. the atomic force microscope (afm) is being used increasingly in the life-sciences field. with this increase in usage, a concomitant increase in the need for both better developed specimen preparation techniques and better defined operational parameters for the afm instrument has occurred. lifesciences afm methodology can be divided into three main areas: ( ) choosing the appropriate support substrate for the specimen, ( ) choosing the most appropriate immobilization techniques for attaching the specimen to the support substrate, and ( ) selection of the optimum instrument scan parameters to ensure reliable transfer of data from the specimen to the image. of central importance to life-science afm is the nature of the substrate to be used. the most reliable substrates are those with a well-documented surface, whose features are at least an order of magnitude smaller than the specimens of interest. furthermore, the material should be transparent so that other forms of microscopy can be used (i.e., light, fluorescence, near-field scanning optical, etc.) and the surface chemistry should be both well documented and susceptible to chemical derivitization. the two most common substrates to fit the above descriptions are glass and freshly cleaved mica. both of these surfaces can easily be manipulated to produce a wealth of reactive primary amines, either by coating the substrate with poly-l-lysine or by treatment with -aminopropyltriethoxy silane (aptes). an analysis of the modified and unmodified substrates show that treatment with either aptes or poly-l-lysine resulted in an increase in surface roughness (ra). based on the roughness information, we recommend modified mica as being suitable for biological material ranging from whole cells to dna, whereas modified glass is unsuitable for samples with heights < . nm. the red blood cell (rbc) cytoskeleton was examined by atomic force microscopy (afm). samples were placed on either glass or mica and imaged in air. no fixative or stain was used; this allowed modification of the samples between images so that molecular components could be identified. the meshlike structure, which is observed when the rbcs are lysed, is identified as a complex of the cytoskeletal integral proteinsspectrin, actin, and band . . the identification was accomplished by imaging the intact cytoskeleton, then treating the sample to selectively remove these proteins and re-imaging the sample. to support the identification of the cytoskeletal proteins further, images were obtained of samples which had been treated with detergent to remove the lipid membrane and leave the cytoskeleton behind. this is the first study of the intact rbc cytoskeleton which identifies specific proteins. more generally, it shows that afm is a useful tool for examining biological systems in their native state since sample preparation is simple and, once attached to the substrate, the sample can be treated in a variety of ways. we have reported some features about the structural and morphologic changes in nylon transformed from β-alanine single crystal and nylon polymerized from ε-amino-ncaproic acid one, which are members of the ω-amino acid family, through the solid-state polycondensation procedure. it was also found that p-aminohippuric acid [n-( -aminobenzoyl)glycine] single crystal could be converted into an aromatic polyamide crystal by heat treatment below its melting point. in this report, crystalline and morphologic structures of polyglycine produced from glycine (the simplest amino acid) single crystal were examined mainly by means of scanning electron microscopy (sem) and x-ray diffraction technique. the monomer solution was prepared by dissolving g of commercial glycine powder into ml of distilled water at °c. the monomer single crystal was precipitated at °c from the solution, which was transparent and prism-shaped. definitive cleavage planes were observed in the monomer crystal, which is found to be parallel to the a-c plane in the αform crystal of glycine. it has been reported that such cleavage is caused by the characteristic structure of α-form glycine crystal. the monomer single crystal was used as the original specimen for the polycondensation reaction, where the original specimen was annealed in decaline at and °c up to h. morphology of the polymerized material was observed by using a hitachi s- sem with accelerating voltage of kv after being coated with gold. x-ray photographs were taken by a flat camera mounted on an x-ray generator with ni-filtered cu-kα radiation, where the crystal took three orientational positions so that a-c (cleavage plane), a-b, and b-c planes made a right angle with the incident beam, respectively. figure shows two types of x-ray diffraction patterns from a specimen polymerized at °c for h with the a-c plane perpendicular (fig. la) and parallel (fig. lb) to the incident beam, where patterns reflected from the polymerized material were observed as well to have spots from the original monomer single crystal. in figure la , two strong arcs are observed, which are indexed as (inner reflection) and (outer reflection) from type-i modification of polyglycine crystal. a more complicated diffraction pattern is shown in figure lb , which seems to be a fiber diagram. almost all molecular chains in polyglycine-i crystal are considered to be normal to the a-c plane and parallel to the hydrogen bond in the original monomer single crystal. figure a shows a sem photograph for the surface of the original monomer crystal. lamination layers of lamellae are observed on the cleavage plane, edges of which are parallel to the b-axis of the monomer crystal. voids were observed in the grain boundary or crack region. a sem picture of the specimen polymerized at °c for h is shown in figure b , where the laminal materials are seen to overlap each other crosswise. such intersecting laminal structure seems to be responsible for the biaxial crystalline orientation observed in the x-ray diffraction pattern shown in figure la. in the case of the specimen polymerized at higher temperatures, the fibrillar structure was observed over the surface. figure c shows the results for the specimen annealed at °c for h. hiroshi toyoda, takashi itoh, hiroshi sakabe, takashi konishi department of polymer science, kyoto institute of technology, kyoto, japan it is well known that polytetrafluoroethylene (ptfe) is produced by emulsification polymerization in the form of powder or aqueous dispersion to be processed industrially. the dispersion-type material is mainly composed of fibrillar and/or lamellar crystallites, where the spherulite structure generally is unseen because of stiffness of the molecular chain. such morphologic feature is considered to be responsible for the remarkable repellent property of the polymer to liquid. the authors prepared thin films of ptfe from the aqueous dispersion to observe change in the fibrillar structure through heat treatment, considering that from such a viewpoint the fibrillar morphology in ptfe is the aspect that is most different from other polymeric materials. the polymerized ptfe used in this study was produced by dupont ltd. a glass plate was dipped into the solution and then withdrawn vertically. the thin film was prepared by drying the solution on the plate at room temperature. the heat treatment was performed at , , , , and °c for h in an air-drying oven. the samples were immediately quenched in ice water (cooling rate: , °c/min) or cooled in air at controlled rates ( , , l, . , and . °c/min). any serious oxidation and/or degradation effects were not observed even during annealing at °c. a scanning electron microscope (sem)(jeol, jsm- lv) was mainly used to observe the au-coated surface structure of the sample, when an accelerating voltage was set at kv to make the resolution of the sem high. composite materials were produced by coating ptfe on textile of glass-fiber and that of carbon fiber. after the coating and subsequent annealing at °c for min, the material was cooled to room temperature. the surface was found to be covered with balls of a diameter of about nm, composed of several hundred nodules of a diameter of about nm as shown in figure . in this sample, the degree of crystallinity of ptfe was about % and no fibrillar structure was observed. by increasing the annealing temperature to > °c, the fiber bun- iv- scanning vol. , supplement iv ( ) dles grew up. such phenomena may be explained by stiffness of the molecular chain including f instead of h, that is, the chain does not fold even if the molecular motion is stimulated at elevated temperatures. any sheaf-like or microspherulitic structure (a precursor of the spherulite), which is often observed in thermoplastic polymers such as polyethylene, did not appear in ptfe. when decreasing the cooling rate, the fibrils grow laterally and interfibrillar space tends to become larger. it is unknown how such space affects the physical properties of the material. thinner fibrils appeared, which connected the original thick ones, in the case of . °c/min cooling as shown in figure . such morphologic structure characteristic of ptfe seems to be responsible for the processing efficiency. schott glaswerke, department of instrumental analysis and mineralogy, mainz, germany fused-cast refractories of the zac-type (zirconia-alumina cast) are of great importance in the manufacturing of glass. especially glass-tanks for the melting of specialty glasses are built with these materials. zac refractories are produced by melting the raw materials at very high temperatures and subsequently casting them into suitable molds. on cooling, part of the material crystallizes, forming al o (corundum) and zro (zirconia, baddeleyite), where the zirconia crystals are also growing within or into the corundum crystals. besides the crystalline phases there also exists a glassy phase, composed mainly of sio , al o , and zro , and small amounts of alkali (na o, k o), tio , and fe o . this microstructure of the material must have an influence on the corrosion behavior when subjected to the aggressive melt in a glass tank. the aim of this work was to establish a method to characterize quantitatively the microstructure of these refractories. the inhomogeneous nature of the material can already be discerned in the light microscope; however, the resolution is not sufficient to measure the sometimes very small (< µm) crystal sizes of zro , not to mention the determination of a form factor! therefore, the electron microprobe (epma) was used to perform this work. the instrumentation used was a jeol jsm- scanning electron microscope, equipped with an optical microscope for reflected and transmitted light, motorized stage (x, y, z), and coupled with a combined eds/image analyzing system voy-ager from noran, which also controls the stage movement. the following parameters of the microstructure, for example, the geometric and chemical properties of the crystalline and glassy phases, were to be determined: maximum and minimum diameters, area of the individual phases and fractional area within the material, form factors and orientation to a reference plane, and the chemical composition of the glass phase. furthermore, it was necessary to distinguish between the zirconia within the corundum and the more isolated crystals. since the material is inhomogeneous, a rather large number of image fields had to be analyzed (at magnifications of × and higher the individual image field is very small!); as a consequence, the whole procedure had to be automated as much as possible. sample preparation: a flat and polished surface of a section of the material had to be produced. although the grinding and polishing was done with diamond wheels and paste, a certain amount of relief between the hard corundum and the "soft" glass is unavoidable. the first step in analyzing the microstructure is to recognize/discriminate the three phases in the material. the signal used for image formation and subsequent phase discrimination is the backscattered electron (bse) image; however, the abovementioned relief due to differences in hardness made the distinction between glass and corundum impossible, since (a) brightness differences between glass and corundum are not very strong in the first place, and (b) edge effects in the corundum crystals showed brightness values of the glass, and vice versa (discrimination, i.e., tranformation of a particular phase into a binary image, is based on such brightness differences). contrast enhancement or image filters were not sufficient for clear separation of these two phases. therefore, for the recognition of the glass phase, an element distribution for si (x-ray map) was used; combined with the image of zro , which is easily discriminated from other phases because of its high average atomic number/high backscatter signal, it yields the inverse of the corundum image. this sounds quite simple; however, since the resolution of the x-ray signal is considerably less than the bse signal, quite some effort had to be put into the treatment of the si-distribution to give correct areas and outlines of the glass phase. another rather complicated step was the recognition of the zirconia crystals grown within or into the corundum. crystals fully enclosed by the corundum presented no problem, but those growing from the edge into the al o were difficult to discriminate as to belonging to this particular structure. the problem here is to tell the machine what the eye and judgement of the operator consider to be part of this feature. an additional treatment of the glass phase (binary image filters) was necessary to solve this task. the binary image of the glass phase is used as a template for the determination of the chemical data of the glass; it provides the control for stepping the electron beam only over the desired areas while acquiring data with the energy dispersive spectrometer (eds). all image analysis procedures, including the storage of images and eds spectra together with the evaluation set-ups (selection of properties to be determined), were combined within a schedule. the stage control and automation software then connects the stage movement to predetermined points (or a number of points along a line) with the execution of the schedule at each of these points on the sample. after completion of the analysis run, which needs about hours for image fields and is therefore done unattended overnight, the large amount of stored data are statistically evaluated (for the zirconia particles alone, there are easily more than , data sets, consisting of seven measured or derived properties each!). this task is performed using the lotus - - calculation program. the chemical data are extracted from the stored spectra with the zaf-correction procedure to yield wt-% oxide data, and also statistically evaluated with the lotus program. although the aim of this work was only to set up an analysis procedure, the test runs on various samples have already shown differences in the microstructure as determined by the geometric and chemical properties of the individual phases. the application of this procedure to "real" samples will then establish correlations between the microstructure and the properties of the material with respect to their use in glass melting. approximately , tons of electric furnace flue dust accumulated in an industrial area in tifton, georgia. vehicles transporting the flue dust, classified as k hazardous waste, initially dumped the material in a warehouse. once the warehouse was full, the flue dust was dumped in an uncovered pile. run-off from the pile and wind-driven particles contaminated nearby industries, residential buildings, and soils over a period of many years. scanning electron microscopy-energy dispersive x-ray spectrometry (sem-eds) was used to compare the morphology and chemical composition of fly ash dust from the suspect pile ( fig. ) with samples collected from the surrounding buildings and soil. post-it notes (millette et al. ) , modified with a strip of conductive carbon tape, were used to collect dust that had accumulated in buildings surrounding the fly ash dump site. suspect dust particles were analyzed by sem-eds to compare with known dust particles from the fly ash pile. soil samples were sieved, with "fines" from the dry soil analyzed by sem-eds and compared with samples from the fly ash pile. particles similar in chemical composition and morphology were identified in most of the buildings sampled that surround the fly ash dump site. soil samples from areas surrounding the dump site were also found to contain fly ash iv- scanning vol. , supplement iv ( ) fig. backscattered electron image of fly ash spheres from the flue dust pile. scale bar = µm. particles similar in morphology and chemical composition to fly ash from the suspect pile. in conclusion, soil and dust samples taken from homes and outdoor areas surrounding the fly ash pile were found to contain particles similar in morphology and chemical composition to particles from the fly ash pile. scanning electron microscopes (sems) are widely used in detection and quantification of material microstructure and imperfections. results obtain with sems need a high expertise to be fully exploited. specialized programs such as a single-scattering monte carlo simulation can effectively predict the electron beam interaction with solids and thus help the quantification. one of the most powerful advantages of monte carlo simulation to help microscopists is to generate images. with high-speed computers we can now simulate images in a reasonable amount of time. in this paper we present images of spherical inclusion of mns in a fe matrix. it is shown that there exists a difference between the image dimension and the real dimension. also, it is shown that geometric effects can alter the resulting image. the monte carlo program used for low-energy simulation is described elsewhere . figures - show simulated image of mns inclusions (the dark center of the figures) in a fe matrix (lighter part). to build this image we need to simulate , primary electron trajectories and then calculate the associate backscattering coefficient for each pixel of the screen. the image will then need , simulations for a total time of approximately , cpu min on a risc workstation. because of the symmetry of the image we can use an better method. starting from the center of the inclusion we simulate points moving on a radius to the border. then we rotate this line on °and add random noise for the monte carlo simulation to obtain the full image. this image is coded in windows bitmap format and can then be converted to tiff or another format. for our simulation, we used a diameter of nm for the electron beam, at which value the incident beam current would be × − amps in our jeol sem. figure shows a spherical inclusion with a radius and at a depth of nm simulated with a beam energy of kev. figure shows the same inclusion but simulation at kev. figures and show the inclusion with a ratio depth/radius of . , simulated at and kev, respectively. in figure we can observe the discrepancy between the image dimension and the real dimension as a function of the ratio depth/radius. as was expected, the error increased with the depth/radius ratio. it is also interesting to note that increasing the energy of the incident electrons increases the image error. the geometry of the solids affect the backscattering coefficient. we can obseve a white border in figures - a similar problem occurs in the middle of the inclusion which appears lighter. it is interesting to see the same phenomena in figure for an image of a real inclusion. the photograph of the embedded particulates in a matrix taken by sem in backscattering mode should be analysed carefully because this method usually overestimates the dimension of such particulates. the measurement of strain, using lattice parameter changes, can be determined from channeling patterns in the scanning electron microscope (sem) using holz lines. the method used by kuzubowski, for example, in the [ ] orientation of silicon, utilizes the change of the height of the triangle from the intersections of the ( ) and two { } holz lines. more recently we have been investigating the channeling patterns for silicon to calibrate accurately the voltage in an sem and we have utilized a pin wheel pattern formed from the { } holz lines in [ ] pattern at . kev. at this voltage, the ( - - - ) and ( ) are very close to each other, within . degrees, and these two lines split as the voltage increases or decreases. the width of the splitting is quite sensitive to any voltage changes as are the { } intersections with these lines. an experimental electron channeling pattern (ecp) of these lines, along with a simulation are shown in figure . the voltage calibrated from the simulation is . kev as shown in figure c . these same holz lines are, of course, also appropriate for strain measurement since the variation in the voltage is equivalent to an isotropic strain field that would uniformly change the lattice spacings. the shifting of the holz lines in the electron channeling patterns due to strain are from both the change in the magnitude of bragg angle and the change in the orientation of the diffracting planes. the former corresponds to a change in the distance of reciprocal lattice point from the origin(i.e., d spacing) and the latter is due to the rotation of reciprocal lattice point about the origin. thus the total angular change can be written as where θ b is the bragg angle, ∆θ p is the rotation of the plane, g is the diffraction vector, λ is the electron wavelength, and | | represents the length of a vector. e is a matrix equal to ε + i where ε is the strain tensor and i a unit matrix. the approximation ~ is due to the small angle approximation for sin(∆θ b )~∆θ b and − ∆θ b ~ . the holz lines toward the center of the channeling patterns are more sensitive to lattice parameter variations because they have a larger value of g and the change of the bragg angle is in proportion to this magnitude. the angular iv- scanning vol. , supplement iv ( ) ∆θ = ∆θ b + ∆θ p~e g − g widths of these channeling lines are also smaller and so they are well defined. when the strain is not isotropic, the sensitivity is also determined by the orientation relationship between the strain ellipsoid and the g vector. thus the appropriate choice of the g vector can be used to maximize the second term in the equation. in figure a , for example, we show the simulation of a channeling pattern for a strain along only one direction (e.g., [ ] ) which breaks the four-fold symmetry of the [ ] pattern. it can be seen that the strain effects on the { } are all the same since all the planes in this family are symmetrical to the strain direction. however, the sensitivities to the strain in the { } are not all equal, ( ) being more sensitive than ( ). the comparison of the sensitivity can be seen from the simulation and from figure b , in which the angles of splitting are plotted as a function of the strain. for years lab has been the industry standard for thermionic emission cathode material. in , fei introduced ceb as an improved alternative to lab . ceb directly replaces lab and has certain distinct benefits. ceb has a lower volatility than lab , increasing the lifetime of the cathode. this reduces the frequency of cathode purchases and replacements. in addition, greater beam stability and faster startup are achievable from ceb 's greater resistance to contamination. however, to gain the benefits of ceb , it must be operated correctly. studies have shown that the operating characteristics of ceb , such as total emission current, are different from lab . proper operation of ceb comes from understanding the operating characteristics of ceb and how they interact with the system in which it is operating. to compare the operating characteristics of ceb and lab , we performed parametric studies in a jeol scanning electron microscope. the jeol is a self-biased system where the bias voltage (vb) is dependent upon the total emission current as vb = ie*rb, where rb is the bias resistor and ie is the total emission current. the results show that ceb operates at a lower total emission current than that of lab (fig. ) . therefore, for the same bias resistor settings, the bias voltage on ceb is less than that on lab (fig. ) . please note that the lower emission current of ceb does not imply a lower probe current. ceb has high transmission and has been shown to provide probe currents similar to lab . to optimize the operation of ceb in a self-biased system, the operating conditions must be optimized for a low emission current source. these operating conditions include the bias resistor value and wehnelt-to-tip spacing. we have found that increasing the bias resistor improves the performance and lifetime of ceb . other experiments show that adjustments to the wehnelt-to-crystal tip distance further improve the performance of ceb . the effects of adjusting the wehnelt-to-tip distance vary for different electron microscope systems. for independently biased systems, we recommend setting the filament temperature to k (the corresponding filament current is provided with the cathode) and subsequently adjusting the bias until the desired emission image is obtained. this is possible because the emission image of ceb is the same as the lab emission image. the wehnelt-to-tip distance should be set to the distance recommended for lab . in transmission electron microscopes (tems), the operating point is set by viewing the source image. because the emission image of ceb is similar to lab , the procedures for obtaining the lab operating point in tems still apply to ceb . however, the total emission current at the operating point will be lower than with lab . by following these guidelines, the operation of ceb can be optimized and with this optimized operation, the benefits from additional lifetime and increased stability over lab can be achieved. takeshi hatsuzawa, yoshihisa tanimura, kouji toyoda, makoto nara*, syuuji toyonaga*, shin-ya hara*, hirotaka iwasaki*, kazuhiko kondou* national research laboratory of metrology, miti tsukuba; *nikon corporation, tokyo, japan a compact laser interferometer with a piezo-driven scanner has been developed for metrological micro-linewidth measurement in regular scanning electron microscopes (sems). so far, special sems combined with various scanners and interferometers (postek , hatsuzawa et al. ) are necessary to perform absolute and precise measurements; however, this device solved the problem by miniaturizing a one-dimensional mechanical scanner and a multi-optical path interferometer. the arrangements of optical components are illustrated in figure . a mechanical scanner is constructed in the center of a mm diameter base disk. the square part, slit by using a electric discharging machine, is suspended by thin elastic suspensions at each corner. the table is driven by a piezo-electric actuator of a traveling length of micrometers. a he-ne laser beam is introduced on the disk by a single mode fiber through a collimator lens, and it is split and deflected by beam splitters and lenses so that two beams are facing each other and form a differential interferometer. at both ends of the table, right-angle prisms are facing each other so that the laser beam goes back and forth five times. by using the differential arrangement and optical-path multiplication technique, the resolution of the interferometer is improved ten times from the original michelson's arrangement. the reflected beams are surperimposed by a half-mirror to generate an interferometric signal, and it is detected by four photo diodes (pd -pd ) after changing its phase by ˚ through a half-and a quarter-wave plate arrangement. this detection method and an electrically operational processing enhance the resolution iv- scanning vol. , supplement iv ( ) times from its original. eventually, by using physical and electrical methods, the resolution of the compact interferometer is . nm (âλ/ ). to evaluate the performance of the compact interferometer, it was installed in the vacuum chamber of a regular sem (joel jsm- a) as shown in figure . the fiber and electric wires are introduced through two flanges next to the chamber. a software servosystem is constructed by using the interferometer and a piezo driver. according to the positional information read through the interferometer counter, a d/a converter commands the piezo driver to change table position precisely, allowing simultaneous sampling of the secondary electron intensity distribution. in the system, the resolution is improved to . nm (âλ/ ) by using the counter function. thus, a precise measurement system is realized for the absolute measurement of micro-linewidth in a regular sem. a maximum drift of the interferometer counter of nm/h was observed by fluctuations in room temperature, however, the influence of the drift can be neglected in actual measurements since a line-scan is finished within a dozen seconds. a comparison of measurements between the metrological sem (hatsuzawa et al. ) and the compact system was made by using silicon micro-line artifacts, ranging from . to . micrometer. the measured linewidths agree within a couple of nm in both measurements, although the measurement conditions are different in acceleration voltage, etc. the results show that the measurement system using the compact interferometer has the same performance as the metrological sem. this means that absolute and accurate measurements can be obtained everywhere by using the compact laser interferometer and a regular sem. this device can be applied to various types of scanning probing microscopes as well as to optical microscopes by improving the table mechanism and the arrangement of optics. institute of ecology and department of botany, university of georgia, athens, georgia recent studies of lake lanier, georgia, revealed a fungal epidemic on the planktonic alga, synedra acus. clonal isolates of the fungus were identified as zygorhizidium planktonicum (chytridiomycetes), an obligate parasite of freshwater diatoms. although frequently present in lakes and reservoirs of western europe, the occurrence of z. planktonicum in north america has not been previously confirmed. earlier studies have described the morphology of z. planktonicum on asterionella formosa; however, little is known of the fine structure and infection process on s. acus. as a basis for further investigations, morphology and mechanism of infection were characterized by scanning and transmission electron microscopy. these observations provided exceptional accounts of germinating spores and developing thalli. moreover, conjugation was characterized as the fusion of heterogametangia by means of an extended smooth-walled tube, emanated from the smaller "male" gametangium. upon attachment, the club-shaped conjugation tube adhered to a small region of the adjoining thallus. this point of contact became continuous with the maturing "female" gametangium which appeared smooth-walled and often highly vacuolate. ultrastructural examinations also illustrated a shared cytoplasm between conjugating gametangia and apparent migration of organelles; however, fusion of nuclei was not observed. the mechanism of infection on s. acus appeared identical to earlier descriptions of z. planktonicum on a. formosa (beakes et al. ) . following encystment, spores typically produced a single germ tube which grew over the frustule valve in an unwavering, linear fashion. penetration occurred between an overlapping region of the outer and inner frustule. at the point of intrusion, the rhizoid appeared slightly swollen, often further displacing the outer diatom wall. a program for monte carlo simulation of electron energy loss in nanostructures a monte carlo calculation of the backscattering coefficient for a multilayer sample monte carlo program for minicomputers using mott cross sections an improved method of measuring biological submicron motion and displacement using laser amplified motion detection and analysis friedlander sk: smoke, dust and haze, fundamentals of aerosol behavior subcommittee on airborne particles: airborne particles micromanipulators and micromanipulation moor h: recent progress in the freeze-etching technique optimization and application of jet-freezing platinum-iridium/carbon: a high-resolution shadowing material for tem, stm, and sem of biological macromolecular structures freeze-fracturing for conventional and field emission low-temperature scanning electron microscopy: the scanning cryo unit scu cryo-preparation and planar magnetron sputtering for low temperature scanning electron microscopy imaging of intramembranous particles in frozen hydrated cells (saccharomyces cerevisiae) vapor pressure data for some common gases. r.c.a. review an improved cryo-jet freezing method in vitro spinal cord trauma early post trauma changes in rat spinal cord: electron probe microanalysis surface studies by stm fabrication technique for tips with controlled geometry for scanning tunnelling microscopy scanning probe metrology low temperature thermal oxidation sharpening of microcast tips microfabrication of afm tips using focused ion and electron beam techniques dimensional metrology with scanning probe microscopes a rocking beam electrostatic balance for the measurement of small forces a scanning tunneling microscope with a capacitance-based position monitor scanning probe tips formed by focused ion beams envelope reconstruction of probe microscope images surface recovery in scanning probe microscopy probe characterization for scanning probe metrology comparison of diffraction techniques for the sem. scan electr microsc electron channelling in the sem a review of excimer laser projection lithography m: direct electron-beam patterning for nanolithography direct stem fabrication and characterization of selfsupporting carbon structures for nanoelectronics die entstehung einer vielzahl von kontaminationsfäden unter der electronen-mikrosonde transition from chemical etching to chemical polishing studied by the sem chemical preparation of dielectrics for studying their microtopography by the sem microprobe analysis in human pathology shelburne jd: preparation of biological tissue sections for correlative ion, electron and light microscopy negative staining: applications and methods detection and identification of viruses by electron microscopy diagnosis of viral infection by electron microscopy electron microscopy in diagnostic virology electron microscopy in viral diagnosis genetic identification of a hantavirus associated with an outbreak of acute respiratory illness isolation of muerto canyon virus, causative agent of hantavirus pulmonary syndrome distinction between bunyaviridae genera by surface structure and comparison with hantaan virus using negative stain electron microscopy anatomic complications of abdominal surgery with special reference to the ureter urological complications of renal transplantation can be prevented or controlled the microvasculature of the guinea pig ureter. a scanning electron microscopic investigation application of an naoh maceration method to a scanning electron microscopic observation of ito cells in the rat liver sem blood vessel cast-analysis microangioarchitecture of the islets of langerhans in the snakes, naja naja, vipera russelli and echis carinatus histochemical methods for acid phosphatase using hexazonium pararosanilin as coupler acid phosphatase activity in the inner ear the development of the stria vascularis in the mouse kimura rs: distribution, structure and function of dark cells in the vestibular labyrinth secretory epithelial linings in the ampullae of the guinea pig labyrinth la fosfatasi acida del labirinto membranoso dell'embrione di pollo durante lo sviluppo the development of human placental villous tree scanning electron microscopic observations on the surfaces of chorionic villi of young and mature placentas ultrastructure of the epithelium of the chorionic villi of the human placenta some new findings about hofbauer cells in the chorionic villi of the human placenta the fine structure of human placental villus as revealed by scanning electron microscopy monte carlo simulation with ebic a monte carlo calculation backscattering coefficients calculations of mott scattering cross section simulation of sem screen image by a monte carlo method quantitative x-ray microanalysis of spherical inclusions embedded in a matrix using a sem and monte carlo simulations a standard procedure for the modeling of the decrease in detection efficiency with time for low-energy eds spectra an empirical stopping power relationship for low-energy electrons measuring the backscattering coefficient and secondary electron yield inside a scanning electron microscope applications of a knock-on process monte carlo simulation based on the mott cross section to quantitative electron microprobe analysis x-ray production as a function of depth for low electron energies theoretical electron-atom elastic scattering cross section cross section for k-shell ionization by electron impact the use of polyacrylamide as an embedding medium for immunohistochemical studies or embryonic tissue immunocytochemical studies of cardiac myofibrillogenesis in early chick embryos i: presence of immunofluorescent titin spots in premyofibril stages novel applications of acrylamide for cryosectioning of isolated cells, tissues, and arthropods whole-mount analyses of cytoskeletal reorganization and function during oogenesis and early embryogenesis in xenopus confocal microscopy of thick sections from acrylamide gel embedded embryos resolution of subcellular detail in thick tissue sections: immunohistochemical preparation and fluorescence confocal microscopy the use of polyacrylamide as an embedding medium for immunohistochemical studies of embryonic tissues application of acrylamide as an embedding medium in studies of lectin and antibody binding in the vertebrate retina developmental angiogenesis: quail embryonic vasculature embryonic vascular development: immunohistochemical identification of the origin and subsequent morphogenesis of the major vessel primordia vasculogenesis and angiogenesis: two distinct morphogenetic mechanisms establish embryonic vascular pattern endothelial cell origin and migration in embryonic heart and cranial blood vessel development morphogenetic mechanisms in avian vascular development near-field optics: microscopy, spectroscopy and surface modification beyond the diffraction limit mechanical detection of magnetic resonance related scanning techniques morphology and diameters of crystallites in remineralized enamel the shape of enamel crystal within human enamel densitometric study of polarized light images from carious lesions conditions required for detection of specimen specific se- secondary electrons in an analytical sem a high resolution se- sem study of enamel crystal morphology high resolution topographic imaging of enamel crystal surfaces analysis of metal films suitable for high resolution se- microscopy antiviral activity of rnadye combinations microspectrophotometry and digestibility of alkali-treated walls in bermudagrass cell types simplified highly efficient apparatus for photographic transaxial x-ray tomography embryonic vascular development: immunohistochemical identification of the origin and subsequent morphogenesis of the major vessel primordia in quail embryos antibodies to b -integrins cause alterations of aortic vasculogenesis, in vivo capillary endothelial cell cultures: phenotypic modulation by matrix components in vitro rapid organization of endothelial cells into capillary-like networks is promoted by collagen matrices connective tissue morphogenesis by fibroblast traction. i. tissue culture observations reorganization of basement membrane matrices by cellular traction promotes the formation of cellular networks in vitro consumption of various process cheese products has been steadily increasing in the u.s.a. and worldwide. these products are manufactured in various styles depending on composition and physical properties sodium citrate (sc), trisodium phosphate (tsp), disodium phosphate (dsp), and sodium hexametaphosphate (shmp) were used at . % levels as melting salts. after cooking, the samples were held at °c for up to h. they were removed after , , . , and h, cooled at °c, and stored for days, after which they were analyzed for moisture, fat, protein, ph, firmness, and meltability. for transmission electron microscopy, small samples ( mm ) were fixed in . % glutaraldehyde and % osmium tetroxide, embedded in spurr's low-viscosity medium, sectioned ( nm thick sections), stained with uranyl acetate and lead citrate, and examined at kv accelerating voltage the data in figure were converted to a pseudo three-dimensional relief map of the embryonic vessels using digital image processing software. it can be argued that this digital rendering, which contains apparent overhead illumination (and shadows below), provides a more comfortable format for assessing visual information. references caric m, kaláb m: processed cheese products textural properties and microstructure of process cheese food rework microstructure of processed cheese products milk gel structure. vi. cheese texture and microstructure effect of draw ph on the development of curd structure during the manufacture of mozzarella cheese structure and rheology of string cheese mozzarella cheese: impact of coagulant type on functional properties encyclopedia of food science & technology food texture and microstructure electron microscopic observations on the casein micelles of buffalo milk: a preliminary study development of microstructure in raw, fried, and fried cooked paneer made from buffalo, cow and mixed milks the role of casein micelles in changes in the colour of milk microstructural evaluation of model starch systems containing different types of oils use of the bird-leider equation in food rheology starch gelatinization in the presence of emulsifiers. a morphological study of wheat starch fixation analysis of tetrahymena pyriformis ultrastructure distribution of polyphenol oxidase in organelles of hyphae of the wood-deteriorating fungus, coriolus versicolor. biodeterioration research a study of the bladder blood flow during distention in rabbits a vascular network closely linked to the epithelium of the urinary bladder of the rat the effects of acute overdistention of the rabbit bladder applications of an naoh maceration method to a scanning electron microscopic observation of ito cells in the rat liver light microscopy techniques for the demonstration of silicone synovial metaplasia of a periprosthetic breast capsule demonstration of silicon in sites of connective tissue disease in patients with silicone-gel breast implants biological specimen preparation for sem by a method other than critical point drying comparison of hexamethyldisilazane (hmds), peldri ii, and critical point drying methods for scanning electron microscopy of biological specimens acetonitrile as a substitute for ethanol/propylene oxide in tissue processing for transmission electron microscopy: comparison of fine structure and lipid solubility in mouse liver, kidney, and intestine optimum conditions may allow air drying of soft biological specimens with minimum cell shrinkage and maximum preservation of surface features a rapid method for cell drying for scanning electron microscopy nation jl: a new method using hexamethyldisilazane for preparation of soft insect tissue for scanning electron microscopy the use of a simple method to avoid cell shrinkage during sem preparation handbook of snow: principles, processes, management and use hobbs pv: ice physics snow crystals: natural and artificial interference reflection microscopy in cell biology: methodology and applications rekonstruktion des oberflächenreliefs von erythrozyten mit hilfe der leitz-reflexionskontrast-einrichtung. leitz-mitt wiss tech vii/ reflection contrast microscopy within chrome-alum haematoxylin stained thick tissueslides reflexionskontrastmikroskopie in der immunhistochemie lichtmikroskopische untersuchungen zum einfluss von atrialem natriuretischem peptid (anp) am nucleus supraopticus scanning electron microscopy of post-it ™ notes used for environmental sampling development of a monte carlo program for low energy work measurement of small elastic strains in silicon using electron channeling patterns electron channeling patterns in the scanning electron microscope a metrological electron microscope system for microfeatures of very large scale integrated circuits scanning electron microscope-based metrological electron microscope system and new prototype of scanning electron microscope magnification standards comparative ultrastructural ontogeny of zoosporangia of zygorhizidium affluens and z. planktonicum, chytrid parasites of the diatom asterionella formosa iv- scanning heterogametangia attached by a fully developed conjugation tube (arrowhead) infection sites of diatom host showing intruding germ tubes (arrowheads) and developing thalli. scale bar = µm the author is grateful to dr. david howell for critically reviewing this manuscript and to ms. lara muffley for technical assistance.this research is supported in part by nih grant nddk r dk - . the authors gratefully acknowledge dr. steven armstrong for his assistance in animal preparation. mic integrity and induced mitochondrial hypertrophy without altering microtubular ultrastructure (fig. a, b) . to localize exogenously administered cdz, a polyclonal antibody to the drug conjugated to keyhole limpet hemocyanin (pierce, rockford, ill.) was prepared by immunizing new zealand rabbits and subsequent bleeds. the antibody titer was both detected and quantified by an indirect elisa assay (pierce elisa starter kit). the detected antibody was separated from other serum proteins by immunoaffinity chromatography utilizing pharmacia's mab trap g and then tagged with nm immunogold particles. transmission immunoelectron microscopy employing tagged antibody and glutaraldehyde/ paraformaldehyde-fixed, dmf dehydrated, and lowicryl-embedded cdz-treated and nontreated tetrahymena revealed no immunogold association with either microtubules or any other cytoplasmic organelle. this suggested that intracellular cdz was cytosolic and leached out during em processing. thus, cdz appears to impair growth and motility through an effect on general metabolism, for example, protein synthesis and/or respiration, rather than a direct action upon microtubules. however, isolation, purification, and characterization of microtubular proteins from tetrahymena cultured with and without cdz are required to substantiate this tentative conclusion. support: nsf-rimi grant no. rii- . coriolus versicolor, a white-rot, wood-decay basidiomycetous fungus, elaborates extracellular ligno-cellulolytic enzymes which possess marked industrial and agricultural applications. thus, we have been attempting to overproduce and enhance/regulate the secretion of these enzymes employing polyphenol oxidase (ppo) as a model enzyme. it catalyzes the conversion of o-diphenols (tree-generated resistance factors) to o-diquinones and oligomerizes syringic acid, alignin derivative. previously, we (moore et al. ) employed biochemistry and immunoelectron microscopy to map the route of ppo secretion through intracellular endomembrane and possible wall-associated components for hyphae cultured in defined liquid (biochemistry) and solid (microscopy) media. here, the ultrastructures of c. versicolor hyphae cultured in kirk and kelman's defined liquid or solid media are compared. in addition, ultrastructural cytochemistry to define further the intracellular route of ppo secretion to the growth medium in liquid cultured hyphae is described. hyphae of various culture ages ( - days) were prefixed min in . - . % glutaraldehyde buffered with . m cacodylate/cacodylic acid, ph . and after washing with buffer postfixed for h in buffered % s . for cytochemistry, prefixed hyphae were washed and treated with either cacodylate buffer or buffered mg ml - tlc pure, dihydroxyphenylalanine (dopa) on ice, followed by h at °c and then s postfixed. the hyphae were dehydrated through a graded acetone series and embedded in spurr's low epoxy resin. the ultrastructures of hyphae cultured in defined medium containing or lacking agar were similar (fig. a , b) except that hyphae grown upon agar possessed a sheath (hs) external to the cell wall. comparisons of numerous micrographs of aldehyde-fixed hyphae treated with cacodylate christian h. rickert and timm j. filler institute of anatomy, westfälische wilhelms-universität, münster, germany until today, quantification in cytochemistry has mainly been performed on supracellular level and by subjective estimation which makes it difficult to compare results of different investigations, even for standardized cytochemical procedures. reasons for this are the lack either or of calibrations or of relative reference points. greyscale image analysis principally allows the quantification of dye-density, but in practice stain intensities depend on many technical circumstances, that is, slide thickness, density of materials, light conditions, or costaining effects, and do not allow automatic identification because of weak grey-contrast. thus, greyscale image analysis for cytochemical quantification on subcellular level necessitates four prerequisites: ( ) applicability within tissue-slides combined with high resolving power, ( ) thin optical tomolevels to avoid superimposition of stained structures, ( ) clear distinction of structures (specificity), and ( ) high contrast. the reflection contrast microscope (rcm; leica germany) meets the above mentioned requirements. it combines effective suppression of aspecific reflected light with epi-illumination. because of its confocal-like principle, the rcm can be applied on thick tissue slides to obtain distinct optical sections. , at a magnification of ×, the depth of these sections amounts to approximately - µm, circumventing an accumulative effect as seen in transillumination. some cytochemical stains show specific reflections in the rcm, increasing the detection sensitivity to a level of objects nm in size. , reflections are like a binary signal (all-or-nothing principle) and deliver an intense contrast against a dark background, thus facilitating image processing by substituting the common density measurements with field measurement corresponding to areas of reflections.we designed an analysis program on vidas . (zeiss/kontron germany) for quantification of rcm images. one of the main parts of this application handles the greyvalue manipulation. apart from standard routines for image optimization and automatic region selection, the mean greyvalue of the whole image was determined. this was used as a reference parameter for the identification threshold in order to minimize the deviation of the identified area from the real area of reflection caused by inconsistencies of the light intensity.applying the rcm with consecutive image analysis on gomori-stained neurons of the supraoptic nucleus (son), we verified the validity of our measuring routine by employing it on a recognized system. a linear correlation between the process of neurosecretion and nuclear volume of the son is well known. switching from rcm to bright field, it is possible to obtain topographically identical images of the chosen tissue region. thus, we photographed reflecting neurosecretory granules in the former while karyometry was performed on the latter. the nuclei were classified by area and matched with the corresponding nucleus area of the same cell. a linear regression of these parameters was computed within a % confidence interval. our results in general confirm former findings about the measurable relationship between nuclear size and specific cell activity, surpassing earlier methods by increasing the sensitivity and decreasing the duration.this paper introduces the rcm combined with digital processing as a useful tool for quantification in the investigative gap between light and electron microscopy. the reflection contrast microscope (rcm; leica germany) is a light microscopic instrument, making reflections along interfaces visible by means of centrally polarized epi-illumination. these reflections cause interference patterns that are suitable for the analysis of superficies or detection of contact zones . we present two properties of this widely unknown technique allowing three-dimensional ( -d) reconstructions in the following manners: . applying the rcm on the surface of air-dried unstained erythrocytes, we made use of the phenomenon that neighbouring zones of equal altitude are joined by closed interference fringes; these can be interpreted as isohypses. to quantify the angle of declivity within a period of the resulting dark-light pattern, it is necessary to know the mean wavelength. the difference of layer thickness between two interference lines of equal tone is about nm for monochromatic light of λ= nm. the advantage of this technique is the simultaneous presentation of the profile of all the isohypses in just one picture (fig. ) . key: cord- -ekgqdjlk authors: anand, shuchi; montez-rath, maria; han, jialin; bozeman, julie; kerschmann, russell; beyer, paul; parsonnet, julie; chertow, glenn m title: prevalence of sars-cov- antibodies in a large nationwide sample of patients on dialysis in the usa: a cross-sectional study date: - - journal: lancet doi: . /s - ( ) - sha: doc_id: cord_uid: ekgqdjlk background: many patients receiving dialysis in the usa share the socioeconomic characteristics of underserved communities, and undergo routine monthly laboratory testing, facilitating a practical, unbiased, and repeatable assessment of severe acute respiratory syndrome coronavirus (sars-cov- ) seroprevalence. methods: for this cross-sectional study, in partnership with a central laboratory that receives samples from approximately dialysis facilities across the usa, we tested the remainder plasma of randomly selected adult patients receiving dialysis in july, , using a spike protein receptor binding domain total antibody chemiluminescence assay ( % sensitivity, · % specificity). we extracted data on age, sex, race and ethnicity, and residence and facility zip codes from the anonymised electronic health records, linking patient-level residence data with cumulative and daily cases and deaths per population and with nasal swab test positivity rates. we standardised prevalence estimates according to the overall us dialysis and adult population, and present estimates for four prespecified strata (age, sex, region, and race and ethnicity). findings: the sampled population had similar age, sex, and race and ethnicity distribution to the us dialysis population, with a higher proportion of older people, men, and people living in majority black and hispanic neighbourhoods than in the us adult population. seroprevalence of sars-cov- was · % ( % ci · – · ) in the sample, · % ( · – · ) when standardised to the us dialysis population, and · % ( · – · ) when standardised to the us adult population. when standardised to the us dialysis population, seroprevalence ranged from · % ( · – · ) in the west to · % ( · – · ) in the northeast. comparing seroprevalent and case counts per population, we found that · % ( · – · ) of seropositive patients were diagnosed. when compared with other measures of sars-cov- spread, seroprevalence correlated best with deaths per population (spearman's ρ= · ). residents of non-hispanic black and hispanic neighbourhoods experienced higher odds of seropositivity (odds ratio · [ % ci · – · ] and · [ · – · ], respectively) compared with residents of predominantly non-hispanic white neighbourhoods. residents of neighbourhoods in the highest population density quintile experienced increased odds of seropositivity ( · [ · – · ]) compared with residents of the lowest density quintile. county mobility restrictions that reduced workplace visits by at least % in early march, , were associated with lower odds of seropositivity in july, ( · [ · – · ]) when compared with a reduction of less than %. interpretation: during the first wave of the covid- pandemic, fewer than % of the us adult population formed antibodies against sars-cov- , and fewer than % of those with antibodies were diagnosed. public health efforts to limit sars-cov- spread need to especially target racial and ethnic minority and densely populated communities. funding: ascend clinical laboratories. severe acute respiratory syndrome coronavirus (sars-cov- ) virus stimulates a rapid antibody response in people with symptomatic - and asymptomatic , , infection. seroprevalence of sars-cov- antibodies in a population thus serves as a reasonable measure of exposure and spread. seroprevalence surveys in the usa, however, have been restricted to single hotspots [ ] [ ] [ ] or under-represented high-risk or vulnerable populations. , moreover, these studies face challenges to timely repetition and longitudinal follow-up, limiting their utility for surveillance. [ ] [ ] [ ] patients receiving dialysis might be considered an ideal sentinel population in which to study the evolution of the covid- public health crisis. patients receiving dialysis in the usa undergo routine monthly laboratory studies to gauge the effectiveness of therapy and to screen for associated complications. in haemodialysis, regular access to the bloodstream abrogates the need for phlebotomy to acquire blood samples. risk factors for acquisition of sars-cov- and for severe covid- , including advanced age, non-white race, poverty, and diabetes, are the rule rather than the exception in the us dialysis population. testing remainder plasma from monthly samples obtained for routine care of patients on dialysis for sars-cov- antibodies therefore represents a practical approach to a population-representative surveillance strat egy, informing risks faced by a susceptible population while ensuring representation from racial and ethnic minorities. in addition, seroprevalence surveys in patients receiving dialysis can be linked to patient-level and community-level data to enable evaluation and quantification of differences in sars-cov- prevalence by demographic and neighbourhood strata, and thus facilitate effective mitigation strategies targeting the highest-risk individuals and communities. in partnership with a commercial clinical laboratory, we tested seroprevalence of sars-cov- antibodies in a randomly selected representative sample of patients. our goal was to provide a nationwide estimate of exposure to sars-cov- during the first wave of covid- in the usa, up to july, , with stratification by region, age, sex, and race and ethnicity. we also harnessed population data on sars-cov- cases and deaths and percentage testing positive using nasal swab testing to assess how seroprevalence estimates correlated with other epidemiological measures of covid- incidence. finally, to inform preventive strategies for the high-risk dialysis population as well as the general population, we investigated communitylevel correlates for seropositivity. we did a cross-sectional analysis of adult (≥ years) patients undergoing monthly laboratory testing at ascend clinical using samples obtained for routine clinical care that otherwise would have been discarded. ascend clinical is a commercial clinical laboratory based in redwood city, california, that receives samples from a nationwide network of around dialysis facilities, serving approximately patients. we randomly selected patients from the patient list on june , , for seroprevalence testing to be done in july, , using implicit stratification by region, age, sex, and race and ethnicity followed by systematic sampling with fractional polynomials. after sample selection and processing, ascend clinical sent anonymised data on patient age, sex, race and ethnicity, and residence and facility zip codes to stanford university investigators for analyses. stanford university investigators further linked patient geographical information (zip code) to census data and publicly avail able covid- burden and community mobility data. the study received expedited approval from the stanford university of medicine institutional review board; informed consent was waived. we used the us food and drug administration-approved siemens healthineers sars-cov- spike protein receptor evidence before this study measuring the seroprevalence of severe acute respiratory syndrome coronavirus (sars-cov- ) antibodies provides a comprehensive assessment of its community spread. community seroprevalence surveys require considerable infrastructure and expense, and face implementation challenges during the covid- pandemic due to restricted outreach in the worstaffected communities. of the two largest seroprevalence surveys in the usa, one was limited only to new york state (n= ) and used convenience sampling at grocery stores. a second survey used remainder plasma from people visiting commercial laboratories in six cities (n= ), but lacked details on race and ethnicity and other community-level risk factors. we tested the remainder plasma of patients receiving dialysis throughout the usa, using a chemiluminescence assay with high sensitivity and specificity. to our knowledge, we provide the first nationally representative estimate of sars-cov- seroprevalence in the us dialysis and us adult population, and estimates for differences in seroprevalence by neighbourhood race and ethnicity, poverty, population density, and mobility restriction. we also evaluate which of the existing measures of covid- incidence most closely correlate with seroprevalence. most importantly, we show that as patients receiving dialysis have monthly blood draws, without fail and without bias, and are a population with increased representation of racial and ethnic minorities, repeated crosssectional analyses of seroprevalence within this sentinel population can be implemented as a practical and unbiased surveillance strategy in the usa. similar to data from other highly affected countries and regions (eg, spain and wuhan, china), despite the intense strain on resources and unprecedented excess mortality being experienced in the usa during the covid- pandemic, fewer than % of us adults had formed antibodies to sars-cov- as of july, . there was significant regional variation from less than % prevalence in the west to more than % in the northeast. public health efforts to curb the spread of the virus need to continue, with focus on some of the highest-risk communities that we identified, such as majority black and hispanic neighbourhoods, poorer neighbourhoods, and densely populated metropolitan areas. a surveillance strategy relying on monthly testing of remainder plasma of patients receiving dialysis can produce unbiased estimates of sars-cov- spread inclusive of hard-toreach, disadvantaged populations in the usa. such surveillance can inform disease trends, resource allocation, and effectiveness of community interventions during the covid- pandemic. binding domain (s rbd) total antibody (immunoglobulin) chemiluminescence assay, which has % sensitivity (≥ days after a positive pcr test) and · % specificity. we chose this assay on the basis of its emergency use authorization in june, , in the context that s rbd is also the target of vaccine development efforts. sample processing is detailed in the appendix (p ). we linked patient-level resi dence data with cumulative and daily cases and deaths per population as compiled on a county level by the center for systems science and engineering at johns hopkins university and with nasal swab test positivity rates, as compiled on a state level by the covid tracking project. for utah, we followed the utah department of health groupings of several smaller counties and extracted data directly. new york city data are not available by county within the johns hopkins university dataset; therefore, we directly extracted data from the new york city dashboard. for county-level mobility restrictions, we used google mobility data that report an average percentage change in the number of workplace visits over the period march - , , before the implementation of shelterin-place restrictions in the majority of the country. percentage changes in the google mobility data are indexed to a corresponding weekday (eg, tuesdays are matched to tuesdays) from jan to feb , . we also linked patient-level residence data with zip code tabulation area (zcta) data from the american community survey (acs) -year estimates to ascertain patient neighbourhood proportion living below the poverty level and race and ethnicity mix, and with american census bureau estimates to ascertain population density. we defined zcta majority race and ethnicity as hispanic, non-hispanic black, or non-hispanic white if the population in the zcta was at least % hispanic, non-hispanic black, or non-hispanic white, respectively; where this was not the case, if the hispanic and black population combined was at least % of the population, the zcta majority was defined as hispanic and black, otherwise as other. for urban versus rural zcta status, we used the rural urban commuting area codes by census tract, categorising a zcta as dense urban, metropolitan, micropolitan, or small town or rural area if more than % of the population in the zcta was living in one of these area codes. we assumed a nationwide prevalence of sars-cov- antibody of %. , to generate prevalence estimates for patients on dialysis using preselected regional strata with precision within · %, a sample of was required (appendix p ). based on previous trends, we expected % of selected samples to be unavailable in july, , due to death, move to other facilities, or other reasons for missing laboratory data (eg, hospitalisation or non-adherence). accounting for this potential dropout, we randomly selected patients. we present prevalence estimates with % cis in our sample, standardised to the us adult dialysis population and to the us adult population. for the us adult dialysis based on the test sensitivity range obtained by schnurra and colleagues in their external validation, we also provide test characteristic-adjusted sample population estimates, ranging sensitivity from % to %. to compute the percentage of estimated sero prevalent cases that were likely to be diagnosed cases, , we compared the estimated seroprevalent cases per adult population with johns hopkins university esti mates of cumulative diagnosed cases per us adult popu lation as of june , . to standardise estimates, we assigned weights to each person based on their membership to each of strata of census regions (northeast, south, midwest, and west), age ( - , - , - , and ≥ years), and sex. we defined post-stratification weights as the proportion of each stratum represented in the us dialysis population or us adult population divided by the analogous proportion in the sample. [ ] [ ] [ ] we then computed weighted frequencies and % cis according to four prespecified strata (region, age, sex, and race and ethnicity) with differences evaluated using rao-scott χ² tests. , due to the missingness of race and ethnicity data in the electronic health records, we used the additional measure of zcta race and ethnicity distribution with categories adapted from moore and colleages. , next, we correlated five measures of covid- incidence-cumulative cases on june , (or first available date between june and june , ); cumulative deaths on june , (or last available date between june and june , ); -day averages of daily cases and daily deaths; and percentage testing positive on nasal swab tests between june and june , -with sars-cov- seroprevalence in patients on dialysis in july, . to do this, we first collapsed all measures to a state level and then assessed the spearman's correlation coefficient ρ for the association of each measure with seroprevalence. because of the high density of ascend clinical facilities in new york, texas, and california, we also chose those states to present county-level correlations. finally, using logistic regression, we determined the age-adjusted and sex-adjusted correlates of seropositivity for patient zcta race and ethnicity distribution, percentage living below poverty level, rural or urban classification, population density, and county mobility restriction. we assumed statistical significance at α< · . all statistical analyses were done with sas enterprise guide (version . ) and stata (version . ). ascend clinical laboratories supported the remainder plasma testing for sars-cov- antibodies. sa, mm-r, and jh had complete access to all data in the study and sa, mm-r, jh, jp, and gmc were responsible for the decision to submit for publication. of the people selected for testing on june , , were tested in july, (figure ), with ( · %) tested in the first weeks (appendix p ). the sampling was representative of the us dialysis patient distribution by age, sex, race and ethnicity (when excluding patients without race and ethnicity data), and region, except sampled patients were less likely to be non-hispanic black (table ) . compared with the us adult population, our sampled patient population was older, had more men, and was more likely to be non-hispanic black and living in non-white neighbourhoods seroprevalence ranged from · % ( · - · ) to · % ( · - · ) in our sampled population (appendix p ). when standardised to the us dialysis population, seroprevalence was · % ( · - · ), with high regional variation in seroprevalence (ranging from · % [ · - · ] in the west to · % [ · - · ] in the northeast; table ). seroprevalence was similar by sex and modestly lower in people aged years or older compared with those aged - years (table ) . differences in seroprevalence by race and ethnicity were similar using both our patient-level (electronic health record) and neighbourhood-level (zcta majority race and ethnicity) measures, with non-hispanic black patients having the highest seropositivity, followed by hispanic patients, and non-hispanic white patients having the lowest. we estimated the sars-cov- standardised seroprevalence in the us population to be · % ( % ci · - · ; table ). based on the johns hopkins university cumulative case data as of june , , the prevalence of (nasal swab) diagnosed cases was per us adult population, compared with our estimate of seropositive people per population, meaning that · % ( · - · ) of sero positive people were diagnosed. using data from our sampled population, variation by state was high, ranging from · % in seven states to · % ( · - · ) in new york, with the highest regional variation occurring in the northeast (figure ; appendix pp - ). when comparing state seropreva lence against cumulative cases and deaths per population, deaths correlated best (ρ= · for cases vs · for deaths; figure ). the percentage of people testing positive by nasal swab test and -day average of daily deaths in the latter half of june, , showed a weaker correlation (ρ= · and · , respectively), whereas -day average of daily cases did not correlate with seroprevalence (ρ=− · ). on a county level in california, new york, and texas, there was even more heterogeneity in the correlation between seroprevalence and other disease measures (ρ≤ · for all correlations for all three states' county-level data; appendix p ). likelihood of sars-cov- seropositivity was lower among older people (odds ratio · [ % ci · - · ] for people aged years or older vs people aged - years), but did not differ by sex ( · [ · - · ] for women vs men). in age-adjusted and sex-adjusted models, neighbour hood racial and ethnic distribution, poverty level, dense urbanisation, population density, and percentage change in workplace visits in early march, , were all strongly associated with seropositivity ( figure ). in our analysis of seroprevalence of sars-cov- spike protein receptor binding antibodies from a nationwide representative sample of patients receiving dialysis, we find that despite the usa contemporaneously leading the world in the numbers of diagnosed cases, overall, fewer than % of us adults had evidence of seroconversion in july, . a vast majority of us adults, including people receiving dialysis who are among the highest risk for mortality upon contracting sars-cov- , do not have evidence of exposure or immune response. furthermore, we find increased likelihood of sars-cov- seropositivity in residents of predominantly black and hispanic neighbour hoods (two to three times higher), poorer areas (two times higher), and the most densely populated areas (ten times higher). early reduction in community mobility in march, , was associated with % lower likelihood of individual-level seroconversion by july that year. unlike most published estimates of sars-cov- seroprevalence from the usa, , , patients included in our study sample had antibodies measured from blood collected as part of routine medical care. thus, our prevalence estimates should not be subject to selection bias due to presence versus absence of symptoms, availability of testing materials, local or regional testing strategies, geography, income, educational attainment, language proficiency, immigration status, mobility, anxiety, fear, or other factors. moreover, since end-stage kidney disease qualifies affected patients for medicare insurance, and since end-stage kidney disease disproportionately affects black, hispanic, and other disadvantaged populations, , , we are able to determine-with a high level of precision-differences in seroprevalence among patient groups within and across regions of the usa. of the two larger seroprevalence surveys published from the usa thus far, one was confined to new york state (n= ), employed a convenience sampling technique at grocery stores, and relied on a microsphere immunoassay with lower sensitivity. the second, the centers for disease control and prevention (cdc) six sites study (n= ), used remainder plasma from people getting testing for undefined clinical indications, and did not have detailed sociodemographic information about the tested people. uncertainty exists as to whether seroprevalence estimates in the dialysis population can be extrapolated to the us population more broadly. a recent analysis of sars-cov- igg antibodies in two dialysis units in london, uk, reported seroprevalence of %, higher than in healthy blood donors ( %) but lower than in healthcare workers ( %) sampled within a similar time frame. our data might overestimate overall seroprevalence in , , , states in white were not included in the sample. the general population since patients on dialysis are disproportionately from racial and ethnic minorities; , for example, black americans have a nearly four-times higher risk of end-stage kidney disease than white americans. moreover, the process of undergoing incentre haemodialysis might include the use of public or non-public shared transportation to and from the facility, and - h of care delivered in indoor facilities. conversely, these data might underestimate overall seroprevalence in the general population. patients receiving dialysis are less likely to be employed and more likely to restrict their mobility and social activity due to advanced age and frailty; therefore, they might have fewer opportunities to acquire the infection, par ticularly from asymptomatic individuals. extrapolating from multiple prospective hepatitis b immunisation studiesin which - % of vaccinated patients receiving dialysis mounted a response compared with % or more people from the general population-patients receiving dialysis might mount a weaker immune response and thus be less likely to seroconvert. finally, patients receiving dialysis might have been more likely to die or have been hospitalised due to complications of sars-cov- infection. if so, these patients would not have been present for testing in the dialysis facilities, creating a survival bias and yielding lower estimates of exposure. nonetheless, the ten-times difference we observed between diagnosed cases per population and our estimates of seropositive people per has been similarly reported in studies from new york, the cdc six sites study, and in a population-representative analysis from geneva. thus, our findings comport with other seroprevalence estimates. we confirm that as in other studies from covid- hotspots, , , a minority of the population has evidence of exposure and immune response, and a vast majority, including people at high risk for mortality (ie, the population on dialysis), remain vulnerable. in fact, even if the seroprevalence estimates derived from the us dialysis population overestimated true seroprevalence in the overall us adult population, our data nonetheless support that fewer than % of the us population has seroconverted as of july, , and all variables are at a neighbourhood (ie, zcta) level, except for reduction in workplace visits, which is at a county level, and are modelled separately, accounting for age and sex. poverty level is defined as percentage of people living below the federal poverty level in the zcta. population density quintiles are derived from the zcta (median people per square mile [iqr - ]). reductions in workplace visits were measured during the first weeks of march, , compared with a baseline in january-february, . or=odds ratio. sars-cov- =severe acute respiratory syndrome coronavirus . zcta=zip code tabulation area. · ( · - · ) age-sex-adjusted or ( % ci, log scale) herd immunity remains out of reach, as has been the conclusion from large international surveys from the uk and spain, where intense outbreaks of covid- occurred during the spring and summer of . furthermore, the seroprevalence differences captured by region, age, sex, and community-level risk factors (ie, internal comparisons) are expected to be similar in the us dialysis and us general adult population. our study provides convincing evidence that the covid- pandemic has dramatically amplified existing health disparities. data from the cdc highlighting sars-cov- health disparities evaluate hospitalisations and deaths by race and ethnicity, , calling into question whether black and hispanic populations are experiencing more severe illness versus facing higher likelihoods of exposure. some us state dashboards also report higher cumulative cases among black and hispanic people compared with non-hispanic white people, but none have as precisely quantified differences on a national level. neighbourhood poverty and population density were also highly correlated with seroprevalence, with a possible threshold effect for population density, such that there was a ten-times higher risk in the highest density zctas (> people per square mile). population density is recognised as a crucial factor, driving the spread in metropolitan areas, in confined spaces (eg, the diamond princess cruise ship), large gatherings (eg, the new orleans' mardi gras), , and in populous regions across the world. rocklöv and sjödin suggest that the basic reproduction number (r ) of sars-cov- increases linearly with population density. our data also show slightly lower likelihood of seropositivity among older people, as was seen in a recent report from geneva and attributed to better adherence to physical distancing measures by the authors. a higher competing risk from hospitalisations or mortality after sars-cov- exposure might be a larger contributing factor in the observed lower seroprevalence in older compared with younger age groups. in addition to providing an overall estimate of sars-cov- seroprevalence and quantifying differences by patient and community characteristics, our study puts forth a viable surveillance strategy for sars-cov- spread in the usa. who and other experts , advocate for repeated cross-sectional analyses of seroprevalence as a disease tracking system able to most completely measure the true incidence of sars-cov- , since these can more likely capture incidence of exposure in both symptomatic and asymptomatic individuals. in fact, we observed substantial heterogeneity in the correlation between seroprevalence and other measures of sars-cov- that are currently being used-with the exception of deaths per , which are a late outcome -supporting the use of rapidly instituted seroprevalence surveys as a complementary surveillance tool. additional public health implications of seroprevalence surveys include assessing testing adequacy. for example, in states where the difference between seropositive and diagnosed cases is decreasing over time, testing capacity is likely to be increasing. furthermore, following seroconversion rates over time can presage hospitalisations and intensive care unit stays, since the time between exposure and seroconversion is relatively short (median days), and can therefore facilitate resource allocation. finally, as we show by assessing community mobility restrictions, seroprevalence surveys can measure the effects of interventions to treat or prevent infection with sars-cov- . repeated serological surveys, if done in a community setting, would require extensive resources and yet remain subject to selection bias. however recurring monthly testing of remainder plasma of randomly selected sets of people-as is practically feasible in patients receiving dialysis-can serve as a representative surveillance system in the usa, with minimal phlebotomy or infrastructure requirement, and as our data show, include traditionally under-represented and socially disadvantaged groups. this analysis has numerous strengths. we used a highly specific and sensitive immunoassay, one which has been robustly linked to sars-cov- exposure. , , , the study sample was highly representative of the us dialysis population and, as noted, we used remainder plasma from specimens used in routine clinical care. the sample size and sampling scheme allowed us to estimate with precision prevalence across several patient characteristics. moreover, linking to us census and other publicly available data sources assembled during the pandemic provides valuable context when considering the implications of these data to the general population. there are also several important limitations. as noted previously, it is plausible that seroprevalence estimates from the us dialysis population overestimate seroprevalence in the us adult population. we do not have patient-level data on symptoms nor nasal swab testing results, and thus cannot test whether the likelihood of seroconversion differs in patients receiving dialysis from generally healthy adults, although preliminary data from london, uk, suggest no differences. we also do not have patient-level data on health status, employment status, income, household size, living space, and other sociodemographic factors, and so relied on neighbourhood proxies for some of these domains. dialysis units are more often located in urban areas, and thus we have under-representation of rural areas. finally, while large, our study was designed for precise regional, not state-level or county-level, estimates. in conclusion, we present sars-cov- seroprevalence data in a broadly representative sample of patients receiving dialysis across the usa and show striking differences in seroprevalence by several patient characteristics, with higher seroprevalence in younger patients, black and hispanic patients, and patients living in poorer and majority-minority neighbourhoods. these data can help to inform surveillance and management strategies during the next phase of the pandemic. serial sampling of dialysis remainder plasma should be used to determine trends in disease prevalence and the effect of various strategies being implemented around the usa to reduce the burden of covid- on the general population. sa assisted with data cleaning and analysis planning, and manuscript writing. mm-r developed the analysis plan, generated census data tables, supervised data analysis, and contributed to manuscript writing. jh undertook data cleaning and analysis, including linkage to external data and figure generation, and contributed to manuscript writing. jb undertook sample processing and data preparation and contributed to manuscript writing. rk selected seroprevalence testing, supervised sample processing, and contributed to manuscript writing. pb co-conceived the study, secured seroprevalence testing, and supervised sample processing and data preparation. jp supervised the study analysis plan, identified relevant external data, contributed to data interpretation, and supervised manuscript writing. gmc co-conceived the study, supervised the study analysis plan, and co-wrote the manuscript. jb, rk and pb are employed by ascend clinical laboratories. gmc is on the board of directors of satellite healthcare, a not-for-profit dialysis organisation. all remaining authors declare no competing interests. de-identified cross-sectional data from the analysis can be made available after authors' review of request and might require compilation of specific categories (eg, at the older age groups) to protect patient privacy. prevalence of sars-cov- in spain (ene-covid): a nationwide, populationbased seroepidemiological study sars-cov- shedding and seroconversion among passengers quarantined after disembarking a cruise ship: a case series antibody responses to sars-cov- in patients with covid- clinical validity of serum antibodies to sars-cov- : a case-control study eua authorized serology test performance natural history of asymptomatic sars-cov- infection sars-cov- infections and serologic responses from a sample of us navy service members-uss theodore roosevelt seroprevalence of sars-cov- -specific antibodies among adults in covid- antibody seroprevalence in cumulative incidence and diagnosis of sars-cov- infection in new york 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geneva, switzerland (serocov-pop): a population-based study post-stratification or non-response adjustment? post stratification analysis of health surveys the analysis of categorical data from complex surveys: chi-squared tests for goodness of fit and independence in two-way tables on chi-squared tests for multiway contingency tables with cell properties estimated from survey data availability of recreational resources in minority and low socioeconomic status areas the intersection of neighborhood racial segregation, poverty, and urbanicity and its impact on food store availability in the united states a report from the brescia renal covid task force on the clinical characteristics and shortterm outcome of hemodialysis patients with sars-cov- infection neighborhood poverty and racial differences in esrd incidence low income, community poverty and risk of end stage renal disease high prevalence of asymptomatic covid- infection in hemodialysis patients detected using serologic screening white/black racial differences in risk of end-stage renal disease and death racial differences in the progression from chronic renal insufficiency to end-stage renal disease in the united states employment among patients starting dialysis in the united states frailty, dialysis initiation, and mortality in end-stage renal disease review article: hepatitis b and dialysis seroprevalence of immunoglobulin m and g antibodies against sars-cov- in china antibody prevalence for sars-cov- in england following first peak of the pandemic: react study in , adults covidview: a weekly surveillance summary of us covid- activity comparison of weighted and unweighted population data to assess inequities in coronavirus disease deaths by race/ethnicity reported by the us centers for disease control and prevention covid- and african americans geographic differences in covid- cases, deaths, and incidence-united states population density and basic reproductive number of covid- across united states counties ethnic and regional variations in hospital mortality from covid- in brazil: a cross-sectional observational study high population densities catalyse the spread of covid- assessing the extent of sars-cov- circulation through serological studies risk factors associated with mortality among patients with covid- in intensive care units in evaluation of nucleocapsid and spike protein-based enzyme-linked immunosorbent assays for detecting antibodies against sars-cov- evaluation of sensitivity and specificity of commercially available sars-cov- antibody immunoassays ascend clinical laboratories supported the remainder plasma testing for sars-cov- antibodies. sa was supported by k dk . mm-r and gc are supported by national institutes of health niddk k dk . we thank martin gorfinkel (mountain view, ca, usa) for his feedback on sampling design. key: cord- - h pg b authors: klingelhöfer, doris; braun, markus; brüggmann, dörthe; groneberg, david a title: coronavirus: an insight into global research until outbreak of covid- and its implications for the future date: - - journal: journal of global health doi: . /jogh. . sha: doc_id: cord_uid: h pg b background: the currently prevailing global threat of covid- caused the publication numbers on coronaviruses to explode. the awareness of the scientific and public community is enormous. but what about the sense of all these undertakings and what can be learned about the future for a better understanding? these questions were answered with established bibliometric analyses of the time until the avalanche of publications unfolded. methods: chronological, geographical aspects of publication output on coronavirus were also evaluated under the influence of epidemiological and socio-economic parameters. results: the trend in publication and citation numbers shows the strong influence of the past pandemics sars and mers with an untypical decline afterward. research is becoming increasingly multidisciplinary over time. the usa and china, as the countries with the highest number of publications, are being displaced by other countries in the consideration of socio-economic and epidemiological aspects, which shows the effect of regional interest in corona research. a significant correlation was found between the number of sars cases per country and related publications, while no correlation was found for mers cases and articles. conclusions: the results underline the need for sustainable and forward-looking approaches that should not end with the containment of covid- . in line with the aphorism: "only those who know the past can understand the present and shape the future", the present study provides background information of research on coronaviruses (cov) as a basis for the scientific situation of the global pandemic covid- and a source for a better understanding of research patterns from the time before covid- . viewpoints research theme : were recognized as pathogens causing only mild infections such as common cold and their clinical significance was not recognized. by , sars had infected more than people in countries and killed people. the pandemic ended abruptly, and no cases occurred later [ ] . at an animal market in china, the sars-cov pandemic was found to be transmitted by palm civet cats [ ] . more than % of all cases worldwide occurred in china. the second cov pandemic mers (middle-east respiratory syndrome) occurred in , but unlike sars, mers did not end suddenly and cases continue to be recorded until now [ ] . mers affected more than people and caused deaths in countries by , according to heath authorities worldwide. more than % of the mers cases occurred in saudi arabia, transmitted by dromedary camels [ ] . later studies have shown that bats are reservoir hosts for both of these former cov diseases [ , ] . the current extremely rapid global spread of sars-cov- has led to the highly dangerous outbreak of the pandemic covid- with daily increasing numbers of new infections and deaths around the world. at this point in time, the peak of the infections has not yet been reached, so the final figures are not predictable -but it seems that they will reach devastating proportions and will certainly change world societies forever. there are no vaccines yet, and treatment options are still limited. only symptomatic treatment or support is possible [ ] . due to the enormous spread of infections and the severe course of many cases, the appearance of covid- is accelerating research, which is certainly unique in the history of science. almost real-time results provide new insights from all areas of science, from basic to applied research. the financing of cov research is also picking up speed. for the dissemination of this rapidly generated knowledge, international communication is obligatory, and the most common way of disseminating it is the publication of results [ ] . can we learn from previous research patterns regarding cov? what influence do they have on future research? how can we use past efforts, their intensification and the influences of research on cov positively to better understand the needs for sustainable and appropriate research? these are compelling questions in light of the currently exploding research output, which, in addition to interesting and meaningful approaches, appears in part excessive, arbitrary and not scientifically sound. although many scientists around the world are giving their best to solve and improve the pandemic, the current development also gives rise to the pressure to be the first to find the solution and not to be overtaken by colleagues. against this background, it is very important to know about previous research. therefore, we have analyzed the global research output on cov in the time before covid- . in the present study, established bibliometric parameters on chronological and geographical aspects were combined with state-of-the-art visualization techniques based on density equalization principles. additionally, socio-economic, scientific and epidemiological parameters were related to the publication numbers to obtain an even more meaningful picture of the global landscape of cov research. the results may help to find more adequate approaches for future research in the financing, planning, implementation and networking of research based on quality and sustainability. the present study belongs to the established bibliometric platform new quality and quantity indices in science (newqis) [ ] , which examines a broad range of biomedical questions with regard to their translational utility [ ] [ ] [ ] . the underlying methodology is constantly evolving and adapted to changing circumstances in terms of global scientific, political and socio-economic characteristics [ ] . standardized bibliometric parameters are combined with newly integrated indices and figures on the research topic in order to analyze and discuss the research landscape appropriately. in combination with state-of-the-art visualization, the results are provided convincingly. the default database for retrieving the needed metadata for all newqis studies is the core collection of web of science (wos). not only because of its status as one of the leading online databases for scientific literature, but also because of its listing requirements, which allow only quality work. moreover, the provision of the journal citation report (jcr) enables the analysis of citation-based parameters by specifying all citations received for each publication. viewpoints research theme : covid- pandemic the search was performed at / / . to record the respective articles dealing with cov, the elaboration of an adequate search term is mandatory. the aim is to include as many related entries as possible and to exclude the false entries from the analysis database. therefore, the term must be a combination of all variants or synonyms of the names of the virus or its transmitted diseases. in order to find as many entries as possible, the resulting term was: "*corona virus" or "*coronavirus" or "sars" or "mers" or "covid- " or "severe acute respiratory syndrome" or "middle east respiratory syndrome". wos offers different modes of search. we applied the title search mode here, because a lot of incorrect entries occur when searching with the topic search mode that also includes abstract and keywords. to further decimate unrelated entries, mostly due to different meanings of the abbreviations sars and mers, a specified topic search was added and combined with the boolean operator "and" searching for the terms: "virus" or "epidem*" or "cov" or "co-v" or "covid- " or "patient*" or "*coronavirus" or "severe acute respiratory syndrome" or "middle east respiratory syndrome". this strategy ensures content linkage by querying the occurrence of one of these terms either in the abstract or the keywords of the publication. the resulting entries were subsequently filtered by the document type "articles" to include only original papers in this study. the metadata of the articles found in the manner described above were downloaded and recorded in a database that provides a variety of parameters according to the key information encoded by the wos tags. their analyses refer to chronological and geographical parameters, using data on the number of articles in relation to, eg, publication year or country of origin. other advanced parameters include socio-economic [ ] [ ] [ ] and epidemiological data [ , ] . for the epidemiological evaluation, sub-analyses were performed using the same search term, with the only difference that it was reduced to either sars or mers. furthermore, institutions and research foci were analyzed and the international network was presented. it has to be noticed that the sum of all assigned subject areas of wos must be higher than the number of articles due to multidisciplinary journals assigned to more than one subject area. in this study an article with more than one subject area assignment to assigned to each area and therefore counts several times. citation-based analyses provide information on the recognition of the articles in the scientific community. a threshold was applied to all valuation ratios to reduce distortions of extremely low values, eg, citation rate, and socio-economic ratios. in this way, a concise picture of the global landscape of cov publications and their development could be created. the geographical results were partially visualized with density equalizing map projections (demp) that are distorted maps according to an algorithm developed by gastner & newman [ ] . depending on the value of the evaluation parameter, the countries were either enlarged or reduced in size according to the physical principle of density equalization. using the vosviewer application [ ] , the results of the keyword analysis to determine research priorities are visualized with a network diagram showing clustered nodes and connections for all terms that occurred at least times. every scientific methodology has some limitations that must be reported on. in this case, the first limitation to be mentioned is due to the characteristics of the data source, as wos does not list all publications in its core collection. wos requires special recognition for listed journals in order to ensure quality, but this led to the fact that some important articles on cov could not be included in the analysis as they are not provided by wos. the title search strategy applied in this study further reduces the database. the advantage, however, is that the included data represent a representative collection that can clearly be used for a valid evaluation. furthermore, the wos is said to give preference to english literature, so that the resulting dominance of english-speaking countries is supported by this fact. another point that should be mentioned is the manual correction method for metadata on institutions and authors. for this, a threshold had to be introduced to make the procedure feasible. as a result, the exact number of institutions and authors publishing on cov could not be given. however, the leading institutions and authors could be determined exactly in this way. viewpoints research theme : considering the limitations of citation analyses and their impact on meaningfulness in terms of the quality of publications, the use of several citation parameters is appropriate, which gives more weight to the importance of the analysis results for the resonance of the examined publications in the scientific community. in summary, the applied method has proven to be a valid strategy for the evaluation of bibliometric scientific questions. a total of articles (n) on cov could be added to the database and form the basis for all analyses except those valid for geographical analyses. analysis of the keywords used (threshold value: occurrences) revealed four thematic clusters of cov articles (figure , panel a) . first, the molecular and biological topics form a cluster (red). the second cluster (blue) outlines the articles dealing with the sars epidemic, and the third cluster (yellow) combines the articles dealing with the mers epidemic. the fourth cluster (green) forms an intermediate group that mainly focuses on the spike protein that is characteristic of cov, its pathogenesis, and its connection to the other clusters. in terms of subject areas, the most frequently assigned research fields are virology (n = ), infectious diseases (n = ), veterinary sciences (n = ), microbiology (n = ), and immunology (n = ). looking at the developments over time, it can be seen that the research has become increasingly multidisciplinary ( figure , panel b). especially since the beginning of the s, a change can be observed. veterinary sciences and virology were relatively declining. the relative frequency of articles assigned to infectious diseases increased from this time onwards. other areas of cov research also became more popular. the first article about cov found in this study was published in . from then on, articles were pub- it is obvious that the articles from deal with the new cov and the related disease called sars, which appeared year. working groups from the usa, china, southeast asian countries, germany, france and the netherlands identified and characterized the new virus and its association with sars [ ] [ ] [ ] [ ] . canadian scientists worked on the genomic sequence [ ] . viewpoints research theme : in , the mers coronavirus appeared, leading to this year' s high-ranking publication of dutch and saudi arabian scientists [ ] . the article, which ranks th , stated that bats are natural reservoirs for sars-covs [ ] . this was a collaboration between china, australia and the usa. all ten most frequently cited articles were published in renowned journals. the new england journal of medicine and science published four of them each. one each was published in the lancet and nature. the necessary information about the country of origin can be collected through affiliation data. before this information is not always available and the gaps are too large to be used. exactly n = articles could not be assigned to any country of origin, so that n = articles are included in the geographical evaluations. from onwards, the usa was the country with the highest number of publications for the entire evaluation period (n = ), followed by china (n = ). by a larger margin, germany ranked third (n = ), followed by canada (n = ) and the united kingdom (uk) (n = ) (figure , the leading countries were also represented by the leading publishing institutions ( table ). in terms of citation numbers (c), the landscape looks similar to that of article numbers (figure , when looking at the world map when socio-economic parameters are included in the analysis, the distortion of country sizes shifts again. regarding the ratio of the number of articles and population size in million inhabitants (rpop) of countries with at least articles on cov (threshold) the ranking another socio-economic parameter, the relation of the number of articles on cov to the gross domestic product (gdp) in billion us-dollars (rgdp) for countries with at least articles (threshold), shows a similar ranking. the rankings of both socio-economic analyses were compromised in table . the inclusion of parameters referring to the research infrastructure of the countries considers two values. first, the gross expenditure for research and development (gerd) in billion ppp$ (purchasing power parity in us-dollars) and the number of researchers in million fte (full time equivalents). again, the number of articles on cov for countries with at least articles (threshold) was set in relation to these parameter (rgerd, rres). table ). the socio-economic parameters gdp, gerd, and number of researchers were significantly correlated with the number of cov articles (p < . ), with correlation coefficients (spearman r) ranging from . to . . in order to show the publication performance of cov-affected countries in relation to the sars and mers epidemics, the relationship of articles on sars or mers to the respective cases per country was analyzed. according to who [ ] sars cases occurred until the epidemic came to an abrupt halt. the sub-analysis of the present study resulted in sars-related articles corresponding to the affected countries. table in total, the who reported cases of mers worldwide occurring with smaller boosts until today [ ] . articles could be clearly assigned to mers. table viewpoints research theme : of the total number of n = articles for the geographical analyses, n = articles were prepared in international cooperation. the maximum annual international partnerships for cov research took place in (n = , first publication peak) and in (n = , second publication peak) (figure , panel a). in principle, however, an upward trend can be observed. the usa was at the center of the international network and was involved in the five strongest international collaborations (figure ) . the most productive bilateral cooperation in cov research was between the usa and china with n = cooperation articles, followed by usa/canada (n = ), usa -uk (n = ) and usa -netherlands (n = ). the non-us partnership with the highest publication volume was between the netherlands and germany (n = ). especially since the beginning of the covid- pandemic, which is causing many serious cases and deaths in many countries, the importance of research on cov has become clear. basic research to date, the identification of novel viruses of the two pandemics sars and mers, has influenced global research efforts. as more is learned about the background, incentives and impact of research, the impact of more focused and improved focus, planning, implementation, collaboration and communication of scientific projects becomes clearer, for all parties involved. this is also shown by the identified research foci, which were analyzed by the keywords used in the articles on cov. however, the proportion of human-related research has increased significantly since the outbreak of sars, and multi-disciplinarity has also become increasingly widespread over time. the currently rampant covid- disease and its impact on economic, political and social spheres will provide new impetus for cov research in other scientific fields in the future. the development of publication output on the cov clearly followed a different trend than that of other biomedical topics in general, which usually increase exponentially over the evaluation period [ ] and could be shown in other studies on viral diseases [ , ] . instead, the increase in the number of articles and citations corresponds to the occurrence of the two pandemics sars and mers in the past with peaks at the beginning of each. in , the year in which sars emerged, publication numbers rose at an unprecedented rate in science, with an increase of about % within one year. chiu et al. also noted in a bibliometric study on sars publications that the high publication rate at the beginning of the pandemic resulted in high citation rates due to the immediate recognition in the scientific community [ ] . now, years later, the present study confirms this extremely high recognition by the scientific community at the time of the sars outbreak, which even shows a number of citations per publication year, even before the maximum peak in publication numbers is reached. in , the highest citation rate was achieved with cr = . , which means that each article on the cov was cited about times on average. this is certainly an extraordinary result. almost at the same pace, however, the number of publications and citations decreases thereafter, resulting in a value that is below the average biomedical topic. this shows the decline of the scientific interest with the abrupt end of sars. the awareness of the importance of cov research as a preventive measure for future virus infections does not seem to have been present at this time. the progression curves of both publication and citation numbers showed a minimum level at exactly the time when the cited half-life (chl) of publications in biomedical publications is generally reached. basically, the chl is given as - years that a publication needs to reach half of the expected citations, resulting in a maximum peak of citations up to that point [ ] . the outbreak of mers in caused a second increase, but this time it reached only about one third of the sars-related figures, due to the lower incidence rates compared to the sars pandemics. it also showed a declining trend, but only four years after the outbreak. the longer period during which mers cases occurred also led to a longer lasting interest, but at a significantly lower level. the timeframe of the evaluation was set until march , so that the first months of covid- were included in the analysis. the expected exponentially increasing numbers could even be shown in this small time-interval by a further significant increase in the number of publications. the short-term effect of research efforts of earlier cov research and the strengthening of international networking could also be shown with regard to another emerging virus epidemic that occurred in south america in : the zika virus infection. the publication patterns corresponded to those reported here and showed an unsustainable short-term effect of national and international efforts [ ] . this similar publication pattern was reinforced by the enormous research incentives of short-term funding and public recognition due to the acute threat. this is also evident in the case of cov research, and the same influences must be considered in future approaches. the most frequently cited publications highlight the outstanding impact of the two pandemics and the years of their outbreaks. most of the ten articles dealing with the identification and characterization of the novel sars virus were published in , which shows the importance of the first articles on sars in the scientific community. one article deals with the isolation of the mers virus, which was published in , and another with the function of bats as natural reservoirs. these articles were from the countries that are considered to be the most publishing countries throughout the assessment period. viewpoints research theme : what is also true for the publication output of most life science and biomedical topics is the leading position of the usa in terms of the absolute number of publications. the rapid catch-up trend and the now high numbers of chinese scientists is also not unique for cov research. other bibliometric studies confirmed the ranking of the leading countries in terms of absolute publication figures. bonilla-aldana et al. found the usa publishing . %, china . %, and germany . %. saudi arabia, as the country most affected by mers, contributed only . % to cov publications [ ] . in comparison, the percentages revealed in our study were similar: the usa ( . %), china ( . %), germany ( . %), and saudi arabia ( . %). in , kostoff et al. found in another bibliometric study on sars the declining share of chinese and the increasing trend of us-american studies. they stated a higher percentage of highly cited publication authored by china compared to other research fields [ ] . these findings have been confirmed by the present results showing also the percentage decrease of chinese articles. although, a relative downwards trend of us-american articles could also be shown. this is due not only to the decline in absolute figures but also to the efforts of an increasing number of countries worldwide. covid- will certainly continue to influence this trend enormously. although the study by chiu et al. showed a low level of international collaboration shortly after the outbreak of the sars pandemic in [ ] , the pace at which international collaboration on cov was established thereafter was remarkable [ ] and will certainly continue. as third most publishing country, germany has been involved in cov research from the very beginning in the s, when only the usa and canada was interested in this topic. although germany had its highest share at the beginning of the s, it has been able to maintain its position among the three leading countries to date. the most cited german article by drosten et al. from ranks second in the overall research on cov and identified a novel coronavirus in a patient with sars [ ] . this was a german-french-dutch partnership. the following ranking consists of canada, the uk, taiwan, and the netherlands. all are involved in prominent research on cov and have sufficient scientific resources and infrastructure not only for research on cov. the netherlands -ranked th in absolute numbers -took a leading position when it came to the results of citation-based, socio-economic and science-related parameters. it also participated in three of the most cited articles and thus also in the identification of sars-and mers-cov as a partner country of germany, the usa, and saudi arabia [ , , ] . with a relatively high proportion of articles in the field of molecular biology, the dutch scientists contributed the main research on the structure of the spike glycoprotein, eg, they uncovered the three-dimensional structure [ ] . nevertheless, other countries have led the way with respect to the inclusion of socio-economic and science-related. thailand received the highest citation rate, although with n = it was only slightly above the required threshold of articles. thailand' s participation in the most frequently quoted article is therefore responsible for this high value. the article by kziazek et al. [ ] , which demonstrates the etiological role of a novel cov in sars, is a joint effort of the sars working group: usa, vietnam, china, thailand, singapore and taiwan. scientists from the center for disease control and prevention (cdc) worked in addition to them from the international emerging infectious disease program in bangkok (thailand) as partners in this study. this program was founded by the ministry of public health (thailand) and the cdc (usa) to establish a prevention and detection system that responds to emerging public health threats. vietnam was also a partner country in this cooperation, but with n = it was just below the threshold and was therefore excluded from the analysis of citation rates. it should nevertheless be mentioned here. scientists from the vietnamese regional division of who were involved in the research. singapore, which was also involved in this successful cooperation via the singapore general hospital, could also be highlighted concerning the analysis of socio-economic and science-related parameters. it received the highest scores in terms of rpop, rgdp, rgerd, and rres. chahour et al. [ ] conducted a bibliometric analysis of the first covid- publications, which confirmed the leading position of singapore in terms of its demographic characteristics. without applying a threshold, this study ranked mauritius first in terms of economic strength. we could not find an article from mauritius until march , and the chahour study found one publication from mauritius. this shows how important threshold applied in our study is to avoid overestimating countries with such a low publication output. with sars cases, singapore was one of the most affected countries. the resulting scientific interest and the possible in-si-viewpoints research theme : covid- pandemic tu investigation of the cases caused the publication figures to rise at the beginning of the sars disease and to fall rapidly thereafter. at the time of the covid outbreak, however, the figures from singapore rose strongly again. tan tock seng hospital (ttsh) is at the heart of sars research in singapore, a place where secondary cases mainly affected health workers [ ] . the epidemiological impact and the incentive of in-situ research promoted research on both pandemics of the past and showed the clearly centered focus of the publishing countries. the exploding publication numbers in and the second peak starting after are clearly caused by the outbreaks of sars and mers. therefore, we defined both the number of sars-and mers-related articles and set them in relation to the number of disease cases of each country. in the case of sars, china had clearly the most cases worldwide and had also written the most articles about it. this was followed by the usa and taiwan. however, the ratio of sars articles from china and taiwan to cases was relatively low. in general, the countries with the most cases did not reach the top ranks, as case numbers varied widely among publishing countries, ranging from more than in china to only one case in eight countries. therefore, spain and switzerland reached the highest ratios. but with only one identified article, these high rates are not surprising, as both countries were among the most publishing countries. the contribution of literature from both countries to the cov literature was relatively stable and has fluctuated only slightly since the early s at an average level of about articles per year. therefore, the publication efforts of both countries could not be directly linked to the outbreak of sars or mers. with regard to mers, most of the contributions came from the usa, which was also found in a study [ ] . saudi arabia produced the second most articles on mers. as far as the relationship between mers publications and mers cases is concerned, the situation is similar to that of sars. saudi arabia also achieved a relatively low ratio with most mers cases. here, the usa and china are the highest-ranking countries, demonstrating their overall interest in cov research and also focusing on the mers pandemic, despite the relatively low case numbers. egypt also came into the focus of this analysis because of the occurrence of only one case and the associated high ratio. however, egypt' s interest is directly related to the outbreak of mers, as it has seen a significant annual increase in publication numbers from that point on. the occurrence of mers-cov in egyptian dromedary camels and its similarity to the human type influenced the regional research interest [ ] . another causal factor is the strong cooperation partnership with saudi arabia and usa in cov research. in addition, egypt ranked th in the inclusion of gerd as a marker of economic strength and was the only country that did not have a high-income status among the top countries. the analysis by chiu et al. from [ ] found no evidence of a correlation between the number of sars cases and the number of publications in the publishing countries [ ] , while our results showed a significant correlation. the reason for this discrepancy seems to be the timing of evaluation. in contrast to chiu et al. who consider the beginning of publication activity on sars, the present study shows the results of development years after the appearance of the pandemic. during this period, the affected nations seem to have created an awareness of the importance of sars research and the international interest in cov research that responds to it. however, with regard to mers, our study could not demonstrate a statistical correlation between the number of cases in countries and the number of publications. this could be due to the low number of mers-related cases and the resulting centered interest. taking into account the unusual patterns of previous cov research, the current situation of covid- -related publication output must be evaluated and discussed accordingly. a short note on the most recent publication figures, which include the output of , already shows publications on the cov that were found with the same search term (as of . . ). of these publications, only are articles ( . %), which means that almost more than two-thirds of the publication includes other types of documents, especially editorial materials and early access, which are almost invisible in the previous period. as a rule, articles make up the largest share of document types. the most frequently assigned subject area in was general and internal medicine, which pushed back the fields of virology and infectious diseases as the most frequently assigned areas of previous research, viewpoints research theme : thus providing an indication of publication in more interdisciplinary journals. the field of public, environmental, and occupational medicine moved on to third place. these figures imply the enormous interest of the scientific community and the enormous willingness to publish. but it leaves one questioning the priority given to quality and shows the attitude of publishers to value the rapid publication of articles related to the cov. china has the largest share of articles in , followed by the us and italy. this is due to the advance of chinese science and the high number of cases in both china and italy. the results of the present study show the need for sustainable, valid and high-quality research in global cooperation to address not only the current pandemic but also future threats. the increasing divergence of research areas allows for more interdisciplinary approaches, which should be completely free of any blinkered ambitions or dogmatic reservations about the big picture. what concrete effects the current covid- pandemic will have on the global landscape of cov research can currently only be estimated. funding is rising exorbitantly, and the first figures for show an equally exorbitant increase in publications. as a result, the development of vaccines and effective therapeutic methods can be expected in the near future. but the question arises whether this is a short-term effect which, once covid- is contained, will lead to an equally sharp decline in publication numbers as was observed with the earlier cov pandemics sars and mers? the need for continued interest and research efforts worldwide is characterized by the characteristics of the past, which lead to a lack of basic knowledge about cov, about advanced therapies and to difficulties in the search for vaccines. the present results show the development and incentives for research in the period before covid- and underpin the need for awareness and sustainable international relations for best possible strategies and the benefit of scientific projects. this is also important in view of the fact that the effects of the prevailing climate change will influence the incidence of zoonotic diseases caused by cov through human and animal accumulation. this will certainly lead to the need to combat new groups of viruses in the future. therefore, sustainable and climate-friendly approaches must also be pursued in science, especially if they focus on the newly emerging aspects of cov 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analysis of covid- research activity: a call for increased output the outbreak of sars at tan tock seng hospital -relating epidemiology to control global research trends of middle east respiratory syndrome coronavirus: a bibliometric analysis mers coronaviruses in dromedary camels key: cord- - t sshy authors: garcía-colón, ismael title: the covid- spring and the expendability of guestworkers date: - - journal: dialect anthropol doi: . /s - - - sha: doc_id: cord_uid: t sshy nan guestworker programs (Çağlar and schiller : ; kalb and tak ) . this article offers some notes of this history in order to elucidate the current agrarian labor regimes in the usa and western europe. in the twentieth century, dependence on guestworkers emerged mainly in western europe, south africa, and the usa as a result of nativism. employers' constant search for lower labor costs and disciplined workers led to their reliance on an ever-changing immigrant labor force facilitated by the unevenness of capitalism and segmentation. between the s and s, restrictions on immigration, such as the foran act's banning of contract immigrants in the usa, and the extension and growth of unemployment insurance and welfare benefits for citizens began to impose limits on immigration (baldoz : ; fahrmeir : - ; hahamovitch , : ) . local officials complained about immigrants becoming charges of government relief programs. labor unions represented a challenge to employers who sought to use new immigrant groups as strike breakers. guestwork became an alternative that appeased anti-immigrant sectors, as governments began to implement deportation and gatekeeping practices to restrict immigration. thus, employers could expand their profits by using temporary migrants, while governments ensured the rights and safety net of their citizens. in , the prussian government created one of the first guestworker programs with polish migrant farmworkers. in south africa, employers in the mining sector hired workers from overseas, mostly chinese migrants, and other african countries. in the usa, the first bracero program began during world war i with the recruitment of mexican workers and families (de genova and peutz ; garcía colón : - ; gonzalez ; hahamovitch : - ; jung ; lee ; ngai ; wolf : - ) . during the great depression, the us federal government's new deal policies attempted to smooth the unevenness of unemployment throughout the country by regulating the hiring and allocation of workers between regions. the wagner-peyser act of established a system to regulate employment by creating an interstate employment exchange system that was administered by the us employment service. when world war ii broke out, the federal government instituted a guestworker program whose main source was mexico, but which also recruited workers from canada and the british colonies in the caribbean. government officials and growers argued that wartime labor shortages made food production a matter of national security, although, in reality, most of the labor shortages were the result of the reluctance of war-related industries and growers to pay higher wages. after the war, the us government allowed the continuation of the mexican bracero program. the immigration and nationality act of expanded considerably the use of guestworkers under the h- visa program. the statute required employers to justify their requests to hire guestworkers by demonstrating that not enough local workers were available. employers filed job vacancies with the us department of labor, which would distribute them to state and local employment offices. if the supply of local workers available to work under the wage and conditions set forth by growers and government officials was insufficient, the us department of labor certified the use of guestworkers. when the mexican bracero program ended in , the h- visa program became the sole guestworker program. in , the immigration reform and control act (irca) transformed the hiring of guest farmworkers into the h- a visa program (cowan : - ; garcía colón : ; griffith : ; martin : - ) . the history of guestworker programs points to the entrenched use of labor-busting and deportation practices to maintain a docile and low-wage labor force. capital always looks to sustain these conditions no matter how, when, and where they find the workers they need (wolf : ) . in the spring of , the contradictions of using guestworker programs became visible when governments began to enact measures to contain covid- . the coming of march coincided with the beginning of the harvest and agricultural activities in the usa and western europe. as germany began to close its borders, the harvest began and the supply of farm labor stopped, risking the rotting of crops in the ground. in the uk, farmers were having difficulty finding workers for raspberry picking and harvesting potatoes. in italy, workers were needed to harvest more than a quarter of the strawberry, beans, and lettuce crops, but the lockdown excluded , guest farmworkers from romania, poland, india, and elsewhere from entering the country. growers and small farmers began to complain loudly that labor shortages in agriculture threatened food security. in fact, the us and western european governments declared food an issue of national security when millions of their citizens began hoarding food supplies in anticipation of shortages during the lockdown (alderman et al. ; bacon ; eddy ; horowitz ) . consequently, governments came to understand the need to keep the borders open for guest farmworkers and to designate agricultural jobs as essential. like the usa, western european countries are also experiencing problems with the hiring and transportation of guestworkers. germany, france, italy, and spain have recruited , , migrant workers from eastern europe to harvest asparagus, pick strawberries, and plant late-season crops. the german government is allowing farmers to airlift migrant workers from romania and bulgaria to ease labor shortages, and german farmers organized flights to transport , guest workers in april and may. florian bogensberger, a farmer in bavaria, paid approximately $ , to fly in romanian guestworkers. this measure has not solved the problem, but it has created concerns about the possibility of importing new covid- infections. friedrich ostendorff, a member of the german green party, denounced the importation of guest farmworkers as exploitation of people desperate to make a living during covid- (eddy ). in italy, unemployment has pushed citizens to find jobs in agriculture, though their numbers are not sufficient for a labor regime characterized by the mobilization of a temporary and mostly immigrant labor force. competition with other countries has led agricultural employers to rely on lowering labor costs in order to increase profits (horowitz ) . the argument from many government officials, growers, and labor advocates is that the need for guestworkers derives from the fact that only small numbers of local workers have applied for agricultural jobs, as most of the local population is unwilling to work under arduous agricultural labor conditions characterized by long hours and low wages (thompson ) . in france, people signed up for agricultural jobs on a website that matches workers with farms, while major supermarket conglomerates pledged to buy from local farms (alderman et al. ; eddy ) . some italians who had been working in restaurants, bakeries, and cafes are turning to farm labor after losing their jobs, and they find working outdoors safer (horowitz ) . however, the number of local workers willing to take agricultural jobs remains very low because of the low wages and paltry working and living conditions offered. meanwhile, in the usa, the trump administration is increasing the rhetoric of nativism and pandemic to implement aggressive immigration bans that affect other industries and arguing that these policies benefit us workers by reducing job competition from immigrants. large agribusiness interests and their allied government officials continue to promote policies facilitating labor control and guestworker programs because of their reliance on deportable, low-wage, and immigrant workers. in the usa, guest farmworkers are in a worse situation since their reliance on the h- a visas had rendered them virtually indentured because they depend almost totally on the employers who sponsor their visas, and they can lose their jobs anytime for complaining about their working and living conditions or for falling ill. employers can force guestworkers to leave the usa on a moment's notice, and workers' fear of losing their livelihoods makes them unlikely to confront and protest abuses. the us state department is also encouraging agricultural employers to bring guest farmworkers by citing growers' concerns about labor shortages and waiving visa interview requirements for h- a workers, thus making it easier for employers to re-hire current h- a workers (coleman ) . on april , , the us department of homeland and security, and its agency the us citizenship and immigration services, issued a temporary rule, valid until august , , extending h- a workers' stay in the usa beyond the -year maximum initially allowed. the reason given for this measure was "to provide agricultural employers with an orderly and timely flow of legal foreign workers, thereby protecting the integrity of the nation's food supply chain and decreasing possible reliance on unauthorized aliens" (homeland security ). the agency cited the disruptions and uncertainty caused by the covid- public health emergency in the us agriculture sector during the upcoming summer harvest season as the main reason for the revised policy (homeland security ). agricultural businesses expect , seasonal guest farmworkers from mexico and central america to enter the usa with h- a visas (leslie when government officials and growers talk of labor shortages, they are signaling their inability to attract domestic workers who would settle for low wages and still earn enough to sustain their families. even before the increase of covid- inflections among the nonguestworkers, us agriculture was experiencing a lack of workers, causing harvesting problems and higher prices. in may, two pounds of strawberries were being sold for % more than last year, and a pint of cherry tomatoes was % more costly (dorning and skerritt ) . labor shortages and the dependence on guestworkers are also a result of the working conditions in agriculture. workers toil the fields at high temperatures, soaked with sweat for h or more. most guestworkers experience violation of their legal rights, including substandard wages, unsafe housing, sexual harassment, and coercion. while most farmworkers are undocumented and therefore vulnerable to abuse and deportation, guestworkers tend to be isolated from their communities, subject to the inequities of contracts that are not the outcome of negotiations but rather an official expression of their unequal status. recruiters in countries like mexico extort fees from guestworkers who then arrive in the usa with debts. workers often are not paid the so-called adverse effect wage rate, the minimum wage required by the us department of labor for workers with h- a visas, because employers deduct transportation, housing, and other costs from their pay. aspiring guestworkers in rural areas of countries with high unemployment continue applying for h- a jobs because of the opportunity they offer to support their families (bacon ; coleman ) . agricultural labor has always been "essential work," though most people were unaware of this. food shortages caused by the covid- crisis, however, have made the public aware that they need farmworkers, even if they come from other countries (coleman ; thompson ) . nevertheless, designating guest farmworkers as essential workers has not stopped the efforts of secretary of agriculture sonny perdue to reduce their wages. many employers have lobbied against the h- a rules that require them to pay guestworkers the prevailing wage based on state-by-state surveys of prevailing wages. these efforts are led by white house chief of staff mark meadows and secretary perdue, both friends of big agribusiness interests. a reduction in guestworker wages would be concerning since migrants often struggle to feed their own families, and the pandemic has worsened their financial situation. farmworkers are paid about half of the wages of comparable workers in other industries (leslie ) . even as the american farm bureau supports these measures, the california farm bureau federation opposes them and prefers an increase in government paycheck protections and assistance in the delivery of crops to food banks as demand from restaurants and wholesale decreases. california growers experience particular difficulty in attracting farmworkers in areas where the high cost of living makes it impossible for them to subsist. reduction of guestworkers' wages is mostly intended to benefit employers in the southeast, where tobacco growers in north carolina could save $ . an hour and florida growers could save $ . an hour (leslie ) . secretary perdue has also been pushing for the us department of agriculture to administer the h- a visa program, currently under the us department of labor, in order to secure agricultural employers' control over the program. in addition, perdue has not earmarked any of the money from the cares act for farmworkers, instead providing farmers $ billion in direct aid and $ billion in purchases for food programs (coleman ) . the cdc's social distance recommendations under covid- also represent a problem for food production. employers need to transport farmworkers to the fields in crowded busses and trucks from housing-barracks with bunk beds-that tends to be crowded. working conditions in the fields make it difficult for farmworkers to wash their hands, and protective gear, when available, is often uncomfortable for them to wear when working under the high temperatures. in the usa, farmworkers generally lack health insurance and paid sick leave, so skipping a day work because of sickness is not an option for most of them (dorning and skerritt ; leslie ). many employers have not made changes to implement social distancing recommendations or provided farmworkers with protective equipment and information about covid- . the lack of information in spanish makes it even more difficult for many workers to protect themselves. a facebook live event held by the united farm workers attracted more than , farmworkers, most of who indicated that their employers did not provide information about covid- . some workers even heard from employers that, "there's no such thing as covid- . it's an invention of the government" (campbell ; coleman ) . instead of improving the working and living conditions in us agriculture, the trump administration insists on a pay cut for guest farmworkers, making their expendability, regardless of their working and living conditions, more evident (leslie ). many of these working and living conditions under covid- are not new, but the pandemic has made them more visible and increased the probability that they will worsen as time passes. in may and early june, reports pointed out that covid- is spreading among the farmworkers, with guestworkers infected at a farm in north carolina and farmworkers, including guestworkers, infected in washington state. in tennessee, workers at one farm were infected. new jersey reported farmworkers ill, and cases were confirmed in one greenhouse in new york (campbell ; coleman ; dorning and skerritt ) . many undocumented farmworkers also continue working in the fields because of their ineligibility to apply for unemployment and their fear of being deported if they use public services. the family first act requires us employers to provide workers weeks of paid leave, but farms with more than workers are exempt. at the same time, some us employers are telling workers not to get tested for covid- because they will get fired if they test positive, and if they get quarantined, they will not be paid, effectively forcing them to continue working in the fields (campbell ; dorning and skerritt ; thompson ) . a farmworker advocate in florida stated that, "when there is an economic reality that you need to work to feed your family, then you're going to be more likely to take a risk and not speak up about unsafe conditions, because you feel like you don't have a choice" (campbell ). the situation is very similar in europe, where a guest farmworker at a german farm remarked, "everybody feels a bit scared, but we also need to work" (eddy ) . this situation could be potentially catastrophic for guestworkers and all farmworkers in the usa since many workers travel between their countries of origin and the usa and across the country as they harvest a variety of crops during the different seasons. when workers finish in florida, they travel north to georgia, north carolina, indiana, new jersey, and new york, often making the journey from state to state in crowded school busses. these journeys could result in spreading the virus to other states, regions, and communities, including the workers' home communities when the harvest is over (dorning and skerritt ) . mobilizing workers from far distances to produce food is not impossible, as the ongoing history of guestworker programs and farm labor migration indicates. however, the political ecology of covid- challenges us to rethink contemporary labor relations in agriculture. securing an available farm labor force requires the immigration policing bureaucracy to maintain open borders, while employers need to sustain and expand their profits by lowering their labor costs. the extension of visas and lowering of guestworkers' wages and the opening of borders and airlifting of thousands of guestworkers are examples of the gamble that governments and growers take to secure their harvests. these policies not only reveal the contradictions within anti-immigrant and deportation policies, they are also impeding the containment of covid- by allowing travel and crowded living and working conditions. but lowering the standard of living of farmworkers who have been dispossessed and unemployed in their countries of origin seems not to be sufficient for large agribusinesses: governments continue to acquiesce to growers' preferences for controlling immigration and labor policies. contingencies like covid- affect government relations with capital and how the latter uses them to take advantage of and transform existing labor relations. as wolf points out, the power to deploy and allocate labor renders some behaviors possible and makes other less possible or impossible ( , ) . covid- containment is creating artificial labor shortages that cannot be resolved by hiring local workers because states rendered them unnecessary for agriculture a long time ago. the elimination of the local population from the production of food implied the elimination of a particular way of life and set of work skills. meanwhile, the need for a specific agrarian labor regime for temporary migrants continues, constituting guest farmworkers as racialized subjects who are available to fulfill the demands of capital as lowwage, docile, and disciplined workers because of their deportability status. undocumented and guestworkers are emerging as "essential" workers because of the pandemic, while their host countries keep cheating them out of decent living and working conditions. it is not by chance that u.s. farm labor is mostly constituted by undocumented workers, permanent authorized immigrants, colonial subjects, people of color, and guestworkers. during covid- times, guestworkers and other farmworkers have moved from being disposable to essential but expendable. as governments and capital across the globe keep deforesting large areas for agriculture and polluting, ravaging epidemics and climate change will make the problems of producing an adequate food acute. this moment presents us with another opportunity to reveal the consequences of power for the inequalities of agricultural production and the arbitrariness of its labor regimes. covid- made visible to all the continuous ruptures and unevenness of global capitalism that many people experience on a daily basis outside of the usa and western europe. migrant farmworkers whose harvests feed europe are blocked at borders guest workers on us farms are in the eye of the coronavirus storm the third asiatic invasion: empire and migration in filipino america migrants & city making: dispossession, displacement, & urban regeneration more covid- outbreaks are hitting farmworkers: it's not just the meat plants that we need to worry about as the trump administration restricts legal immigration, it's expanding a class of vulnerable guest workers apples and aliens: growers challenge feds over who does the picking the deportation regime: sovereignty, space and the freedom of movement every single worker has covid at one u.s. farm on eve of harvest farm workers airlifted into germany provide solutions and pose new risks citizenship: the rise and fall of a modern concept colonial migrants at the heart of empire: puerto rican workers on guest workers or colonized labor?: mexican labor migration to the united states peasants in reserve: temporary west indian labor in the u.s. farm labor market creating perfect immigrants: guestworkers of the world in historical perspective no man's land: jamaican guestworkers in america and the global history of deportable labor for some italians, the future of work looks like the past coolies and cane: race, labor, and sugar in the age of emancipation the covid- spring and the expendability of guestworkers introduction: critical junctions: recapturing anthropology and history introduction at america's gate: chinese immigration during the exclusion era op-ed: the white house wants to give farmworkers who put food on your table a dire pay cut the h- a program: evolution, impacts, and outlook. in (mis) managing migration: guestworkers' experiences with north american labor markets impossible subjects: illegal aliens and the making of modern america the sun is hot and you can't breathe in a mask'-life as an undocumented farmworker. the guardian temporary changes to requirements affecting h- a nonimmigrants due to the covid- national emergency europe and the people without history pathways of power: building an anthropology of the modern world publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord- -bd ba cp authors: johnstone, phil; mcleish, caitriona title: world wars and the age of oil: exploring directionality in deep energy transitions date: - - journal: energy res soc sci doi: . /j.erss. . sha: doc_id: cord_uid: bd ba cp this paper explores the role of the world wars in th century energy transitions, focusing on the growth of oil as a major energy source which accelerated after the second world war in north america and europe. we utilise the recently developed deep transitions framework which combines techno-economic paradigms and sociotechnical transitions approaches. the first deep transition entails the long running emergence of industrial modernity since the late th century which culminated in the post-second world war economic ‘golden age’ underpinned by rapid and stable growth and prosperity in north america and western europe. the deep transitions framework draws attention to the increasing role of fossil fuels over this long period, and how fossil fuel consumption accelerated in the th century taking on a particular direction where energy, mobility, and food systems became increasingly reliant on oil while the share of coal as a proportion of the energy mix decreased. this paper integrates sociotechnical, historical and geopolitical literatures to examine how the development of the age of oil was shaped by wartime demand pressures and logistical challenges and the search for new solutions to these challenges in the united states of america and the united kingdom. the post-world war ii era saw the share of oil in energy consumption rapidly increase with abundant supplies of oil products seen as key in underpinning the so-called 'golden age' of economic development [ ] . the 'golden age' constitutes a sustained period of stable economic growth, increased prosperity, and the rise of mass consumption in north america and europe, lasting until the oil crisis of . while energy transitions occurring during this post-world war ii era have often been a point of focus, until recently, as evenden [ ] points out, there has traditionally been a smaller proportion of studies focused on how wartime activities have influenced energy system change. in recent years however, there is a growing sub-set of literature that has focused on the importance of world war as a factor in energy transitions [ ] [ ] [ ] [ ] . elsewhere, in the fields of history and geopolitics, there has been significant attention on oil resource geographies, developments in oil technologies, its uses, industry, infrastructures, logistics, and patterns of import and export during wartime [ ] [ ] [ ] [ ] [ ] . this paper contributes to emerging literatures on war and energy transitions [ , , [ ] [ ] [ ] , integrating insights from historical and geopolitical literatures with sociotechnical perspectives to understand the role of world war in the th century development of the 'age of oil' [ ] . in doing so, this paper builds on the 'deep transitions framework' (here on in "dt") introduced by schot and kanger [ ] . the dt framework is relatively new and constituted by several key concepts which we discuss in more detail in section . , however we will now briefly outline some key features. the dt framework combines the techno economic paradigms (tep) approach developed by carlota perez [ ] , and sociotechnical transitions theories utilising the multi-level perspective (mlp) [ ] . both the tep approach [ , ] and the mlp [ , ] have been frequently drawn upon to understand energy transitions. the tep approach builds on long wave theory which has been widely used in economics for several decades [ ] , and identifies different phases of economic development referred to as 'great surges' of development. these are depicted as generally occurring over - - year periods, and are driven by the unleashing of particular technological innovations (such as the internal combustion engine); a paradigm of economic growth that coordinates activity (such as 'mass production'); particular patterns of financial investment; and a source of cheap energy which underpins each surge of economic growth. for the purposes of this study, the most relevant point to note is that tep framework specifically names the period from to as "the age of oil, the automobile, and mass production", where "the cheapening of oil-based fuels, electricity and road transport gave positive support to the very high growth rates of national mass markets" ( [ ] : ). the tep framework has generally focused at the 'macro' level of analysis entailing broad changes in entire economies driven by technological innovation and cycles of financial investment. analysis drawing on the mlp on the other hand, has focused at the level of 'sociotechnical systems' oriented around the provision of societal needs such as energy or mobility. different elements including technology & science, markets & users, industry, institutions & regulations, shape activities within a sociotechnical system [ ] . mlp approaches tend to focus on how novel technologies and practices operating in protective environments called 'niches' can, overtime, come to destabilise 'sociotechnical regimes', which represent the dominant technological and institutional configurations through which a particular societal need is delivered. rather than the 'macro' approach of tep, the mlp's focus on individual sociotechnical systems is understood as a 'meso' level approach. the framework has been used to examine the co-evolutionary dynamics of important historical technological changes including the transition from sailing ships to steam ships [ ] , as well as historical analysis focused on the long-term decline of previously dominant technologies such as the case of the uk coal industry in the th century [ ] . however, while world wars are mentioned as relevant exogenous factors in both tep and mlp literatures, activities during world war and the sociotechnical implications of wartime have generally not been a focal point of analysis. the dt framework addresses this gap, with one of its key propositions (proposition ) being that 'external shocks' and particularly world wars, are decisive 'turning points' in coordinating multiple sociotechnical systems (energy, food, and mobility) in a similar direction. the notion of 'directionality' is central to the dt framework. building on the work of stirling [ ] , schot & kanger outline their understanding of the term as follows: "socio-technical change has a direction, choices are made between directions and actors gradually become blind to alternatives, which is a central tenet of much of the innovation studies literature" ( [ ] : ). thus, a focus on 'directionality' recognises the plurality and diversity of potential energy transitions and is attentive to historically contingent factors whereby certain energy trajectories become dominant and alternatives are 'closed down'. the closing-down of alternative pathways occurs as technological market structures, regulations, political support, infrastructure, and user practices, align to form a particular technological trajectory that gains momentum and makes it challenging for new technologies and practices to break through [ ] . to take one example, electricity grids designed around constant 'baseload' production utilising increasingly large power plants often located far from where there is most demand, made it very challenging for variable renewables and energy conservation measures to break through in many countries without concerted political pressure over several decades [ ] . accordingly, energy transitions usually take several decades and are fraught with political struggles, resistance by existing energy incumbents, and usually require governmental intervention to overcome these barriers [ ] . however, a key distinction with regards to the understanding of directionality in the dt framework is the focus on the co-evolution and alignment of multiple sociotechnical systems, as compared to the tendency in sociotechnical approaches to focus on single systems [ ] . the notion of directionality is further refined in the dt framework through the concept of rules, which is a well-established concept used in institutional studies and a core concept of the mlp. schot and kanger define rules as "humanly devised constraints that structure human action, leading to regular patterns of practice" ( [ ] : ). an example of a rule given by schot and kanger is an "imperative to use fossil fuels". after the second world war this rule became dominant in energy, food, and mobility and therefore constitutes a 'meta-rule' because the rule structures human action and influences regular patterns of practice in multiple sociotechnical systems rather than a single system. another meta-rule relevant to this study identified elsewhere [ ] is the "imperative to maintain abundance and constancy of supply", which became particularly pronounced during the intense demand pressures of the first and second world wars, where the ability to maintain supplies of key materials to the front lines meant the difference between victory and defeat. historians have pointed out that after the second world war an era of "energy abundance" based on cheap oil emerged in the usa [ ] [ ] [ ] , and then in many european countries as "the years of surplus" from onwards [ ] . the expansion of oil and a system of energy abundance are closely linked [ ] . in this paper we combine geopolitical and historical literatures with sociotechnical approaches to understand how the heightened conditions of maintaining abundant and constant supply during the first and second world wars influenced the emergence of the 'age of oil' identified as being a key element in the fourth surge by perez which forms the culmination of the first deep transition. the overall research question is as follows: what was the role of world wars in shaping the emergence of the age of oil in the culmination of the first deep transition? building on diverse literatures from geopolitics and history, it is possible to build a plausible and evidence-based narrative to address this question. in undertaking this analysis, there are two central aims. the first is to contribute to the emerging literature on energy transitions and the role of world war. the second aim is to respond to proposition of the dt framework, concerning the key role of war in the coordination and alignment of sociotechnical systems. we proceed as follows: section situates this paper in the wider context of the broad field of 'energy transitions' research, and specifically the emerging interest in wars and the military as key factors in energy transitions. we then discuss the dt perspective in more detail, explaining key concepts and how discussions of deep transitions align with and can contribute to energy transitions literatures. in section , after discussing our approach, we outline an interpretive analysis drawing on extant literatures from history and geopolitics to build an interpretive evidence-based account of the role of the world wars in the emergence of the age of oil, focusing on world war i, the inter-war years, world war ii, and the post-war period of re-orientation and reconstruction. this study focuses on the united states (usa) and the united kingdom (uk). this focus is motivated by two observations. first is the fact that core to perez's theorisation of the age of "oil and mass production" is the lead role played by the usa and how oil-dependent forms of economic activity 'diffused' to europe. second, is that historic accounts of oil developments and geopolitical struggle highlight the central role played by relations between the usa and the uk -both as a point of friction and collaboration [ , ] -in th century oil developments. this focus is not to diminish the importance of oil developments in other countries, and -perhaps unsurprisingly given the nature of war and geopolitics -we do refer to the strategies of other belligerents in the analysis below. however, the main focus on usa-uk oil developments provides a necessary boundary (given the breadth of this topic) to focus more specifically on sociotechnical responses during war, and also explore international dynamics of friction and collaboration between the two countries identified as a significant factor in historical studies [ , , [ ] [ ] [ ] . however, as we discuss, the changing relations between these two countries over the course of the fourth surge and after the second world war also has important implications for understanding rapid oil transitions in western europe in the context of post-world war ii reconstruction. we are aware of the geographical limitations of the present study. our focus is on breadth rather than depth (covering a time period entailing two world wars), and so there will necessarily be gaps in the analysis. entire sub-fields of historical analysis are devoted to just one war, or even a war in a particular country. however, we seek to contribute to opening up for further elaboration and testing, propositions that can contribute to emerging discussions around energy transitions and world war, through answering the question of the role of world war in influencing the directionality of the first deep transition. while history is not a guide to the future, we argue that the historical imagination [ ] can contribute to enriched discussions in energy transitions on possible drivers and mobilising forces for sustainable energy transitions. we now situate the present study in the wider field of energy transitions research, and then go on to discuss the dt framework and our approach to the analysis that follows. 'energy transitions' is a broad and multi-disciplinary area of study. there is a plethora of different definitions and interpretations. in a discussion of the "emerging field of energy transitions", araujo outlines a broad definition of the term: "a shift in the nature or pattern of how energy is utilized within a system", where "an energy system is a constellation of energy inputs and outputs, involving suppliers, distributors, and end users along with institutions of regulation, conversion and trade" ([ ]: ). araujo continues that "change can occur at any level -from local systems to the global one -and is relevant for societal practices and preferences, infrastructure, as well as oversight" ([ ]: ). while some authors aim for more precision in defining energy transitions , sovacool concludes that "there is no standard or commonly accepted definition of an energy transition in the recent academic literature"( [ ] : ), but rather, there are different foci of analysis and conceptual tools which form the basis for differing perspectives. thus, rather than attempting to establish a strict definition, the key question is what perspectives are being used as an entry point to understanding energy transition phenomena and what particular discussions in energy transitions does the present analysis relate to. first, this paper relates to ongoing discussions on 'historic energy transitions' as fouquet identifies [ ] , which has seen more detailed examination of the historic factors influencing the speed and dynamics of transitions from the level of a single fuel or technology to the global scale [ ] [ ] [ ] . sovacool & geels highlight that interpretations of historic transitions vary widely because there are different points of focal interest in terms of the 'multiple dimensions' of energy transitions [ ] . this can include techno-economic analysis which primarily focuses on firms, sunk costs, investments, and the difficulties and long time-frames that energy transitions entail because of these economic factors, but also 'socioinstitutional analysis' which focus on political and policy interventions that influence past transitions and can trump these economic constraints leading to more rapid change. a perspective that tries to bridge these 'multiple dimensions', and that sovacool and geels identify as "a key conceptual approach" in energy studies, is 'sociotechnical transitions' [ ] . sociotechnical approaches focus on the co-evolutionary changes in energy systems and examine different elements including technology & science, markets & users, industry, institutions & regulations, where these different elements interact to form a 'seamless web' to deliver a particular societal function [ ] . as sovacool points out, many studies that take a sociotechnical perspective draw on the mlp which examines interactive developments between sociotechnical niches, regimes, and landscape pressures. a sociotechnical regime represents the dominant way of doing things, in terms of incumbent technologies and the rules and institutions that guide developments in an energy system. niches, on the other hand, represent the level of novelty, innovation and experimentation [ ] . however, despite a plethora of work drawing on a sociotechnical perspective and the mlp, the 'landscape' level often remains underexamined in such accounts [ ] . this includes wars, which in historic accounts drawing on the mlp are often used as temporal markers depicting discontinuity between different stages of development in energy transitions [ ] , rather than focal points of analysis in their own right. however, recently there has been increasing attention on world wars as a factor in energy transitions. for example, samaras et al. [ ] , in detailing the contemporary role of the us military in energy r&d around insulation and mini-grids, also highlight how strategic military decisions have shaped past energy developments such as the first world war transition of the british naval fleet to oil [ ] . matthew evenden offers a detailed analysis of the ways in which second world war mobilisation efforts in canada played a decisive role in accelerating transitions to hydroelectric power under emergency measures to maintain adequate electricity supply to fuel industrial efforts for the allied campaign [ , , ] . more recently, cohn, evenden, & landry conducted a historical analysis of canadian, german and american mobilisation during the second world war, and how this conflict played a crucial role in influencing the design of electricity systems in these countries [ ] . of particular relevance to this paper, recent work by ediger & bowlus [ ] moves beyond conventional commercial and technological factors outlining the importance of oil for military strategy in the build up to the first world war and its enactment, highlighting the central role of geopolitics as a driver of energy transitions. rubio-varas also focuses on the first world war, highlighting how disruptions caused by the conflict were decisive in influencing a rapid transition to oil that occurred in latin america, decades before europe [ ] . the present work builds on and contributes to this sub-field, through engaging with the dt framework which we now discuss. in this section, we discuss the dt framework, its constituent conceptual elements, what it is attempting to achieve and how it can be utilised to explore energy transitions. the framework builds an understanding of long-running developments in multiple sociotechnical systems across a -year period from the late th century and culminating in the post-war 'golden age' of economic growth and prosperity [ , ] . the first deep transition is theorised as entailing four different 'great surges' of development across this period which constitute the slow building up of industrial modernity. the focus of the present work is on the 'fourth surge' which for schot & kanger represents the culmination of the first deep transition (fig. ) . the red circle in fig. highlights the fourth surge. each surge constitutes a 'technological revolution' entailing a period of economic growth, based around a "…constellation of new inputs, products and industries, one or more new infrastructures -usually involving novel means of transport of goods, people and information-and alternative sources of energy or ways of getting access to it" ([ ]: ). the details of each surge are outlined in fig. . for perez, each surge follows a pattern entailing a period of 'irruption', where a number of new technological innovations emerge; a period of 'frenzy' entailing a battle between old and new technological configurations and economic paradigms; a period of 'synergy' where there is resolution and a particular new economic paradigm becomes stabilised; and then a period of 'maturity' where the particular paradigm and its constituent technologies and economic paradigms reaches saturation point, usually ending in crisis with the absence of new for example, smil outlines that an energy transition is the time that elapsed between the introduction of a new fuel and its rise to a % national or global market share [ ] . grubler, outlining 'grand transitions' as a new fuel reaching % market share [ ] . yet, these are not rigid definitions that should be adhered to, but particular definitions amongst a plethora of perspectives that constitute a varied and interdisciplinary field of research [ ] . growth opportunities. these stages are outlined in fig. : although building on the 'great surges' approach, the dt framework differs in several important ways. first, the framework emphasises continuity across these surges, where the first four surges represent a single deep transition. second, while perez identifies a fifth surge based around information communication technologies (ict) with renewable energy as the key energy input, schot & kanger propose that this period instead represents the beginnings of a second deep transition. as they outline, "through gradual accumulation and coordination, niches may contribute to a fundamental overhaul of existing socio-technical systems, introduce a new set of sustainable and just directionalities… and thereby give rise to the second deep transition"( [ ] : ). in this paper however, we are focused on the culmination of the first deep transition and more precisely, 'the age of oil' as depicted in the fourth surge. we do not discuss the second deep transition. second, the dt perspective posits that the drivers underpinning the emergence of particular surges can be understood from the perspective of the changing dynamics within sociotechnical systems. innovation in niches is driven by system builders responding to particular problems in delivering societal needs and the search for solutions and improvements. the concept of rules discussed in the introduction, is used to understand how routines and behavioural patterns within sociotechnical systems influence path dependency and form sociotechnical regimes. where the dt framework departs from most mlp-based analysis however, is it focuses on multi-system interactions and co-evolution, and how systems become increasingly aligned in a similar direction over time. that alignment process takes place when the same rule exists in multiple systems, referred to as a 'meta-rule'. an important point to also note, recognised by schot & kanger and perez, is that the time periods of surges strongly overlap. it is not the case that one surge disappears completely as another emerges. due to issues including sunk costs and resistance from incumbents, there is considerable cross-over between surges as the elements of each surge are reconfigured and compete against each other until there is a resolution at a 'turning point'. with respect to the fourth surge and the question of energy, this relates to understanding how oil rapidly gained a significantly larger share of energy consumption in the years after the second world war, in the usa, uk and europe more widely, while the share of coal (the dominant energy source of the previous two surges) decreased [ ] . the present study focuses on the role of war in shaping this oil transition and how the rules of using oil and maintaining abundance and constant supply evolved in the context of the world wars. we now briefly discuss the approach taken to build our interpretive analysis. in conducting our interpretive analysis, we seek to integrate geopolitical and historical accounts of oil developments in the twentieth century with a sociotechnical perspective. we focus on four different stages to frame our analysis based around perez's depiction of the age of oil as including the stages of irruption (covering the time period of the first world war), frenzy (the interwar years), synergy (that includes the second world war), and maturity (post world war ii). in doing so, we are attentive to historical literatures which outline the key differences between the world wars and peacetime in terms of the conditions of total war [ ] [ ] [ ] . in conditions of total war, as van creveld writes, "war itself extended its tentacles deep to the rear, spreading from the trenches into the fields, the mines, and the factories" ( [ ] , p. ). during periods of total war, entire economies and societies (rather than just a dedicated military as in other conflicts) are mobilised for the war effort [ ] . sociotechnical systems (including energy, food, mobility) become mobilised as part of a 'war machine' oriented around the single purpose of achieving wartime aims [ ] [ ] [ ] . multiple sociotechnical systems are reoriented from delivering civil societal functions to also supplying vast militaries. a key mechanism of total war identified by obinger et al is the immense demand-pressures that are placed on the economy entailing often unprecedented logistical challenges that can reorient patterns of production and consumption [ ] . building on the dt framework and the recognition of the key rule of maintaining abundant and constant supply during world wars [ ] , we build on diverse literatures to build an interpretive account of how the exceptional demand pressures of total war influenced dynamics in multiple sociotechnical systems and the relevance these changes had for oil transitions. as rip and kemp outline, world wars provided a particular 'selection environment' for particular innovations and developments to 'break through' [ ] . thus, we focus on examples of strategic openings afforded for oil as a 'solution' to a variety of demand side challenges, as well as being attentive to evidence of potential destabilising forces with regards to other technological pathways [ ] . obinger and petersen also draw attention to the importance of the demobilisation phase, where soldiers and workers employed for war production have to be reintegrated back into civil economies [ ] . this phase presents a particular economic challenge in warding off economic collapse once the demand-pull effects of war time cease and technological systems deployed in the war effort may also have to be reoriented back towards civil purposes. also relevant in this phase are the legacies of war as a catalyst for change, where policies can continue into peacetime. of particular relevance for our study and its focus on the usa and uk, are the legacies of policy diffusion and transfer which can be achieved in conditions of total war due to alliances between different countries and the effects of post-war reconstruction. these mechanisms of total war provide a guide to shape our narrative analysis. in doing so the analysis is interpreted around the core rule identified by the dt framework of the imperative to use fossil fuels. given our focus on the fourth surge and the recognition of the special importance of transitions from coal to oil during this period by schot & kanger [ ] , for the purposes of the present analysis, we have refined this as the imperative to use oil. in integrating broad trends, geopolitical developments, and a focus on demand pressures in multiple systems, we respond to our overarching research question: what was the role of world wars in shaping the emergence of the age of oil in the culmination of the first deep transition? in this section we outline our interpretive narrative of developments in multiple sociotechnical systems in four distinct periods, following the 'age of oil, automobile and mass production' identified by perez. first, we look at events in the first world war in the uk and usa and identify broad trends and geopolitical developments before discussing illustrative examples of relevant developments related to energy, food, and mobility for considering oil transitions. we then discuss geopolitical factors related to usa-uk oil relations and sociotechnical developments in the 'frenzy' period between the wars. in the third part of our analysis we focus on the 'turning point' of the second world war in the 'synergy' phase and in the fourth part, we look at the importance of post-war reconstruction efforts and the challenges of demobilisation. drawing on literatures that analyse the role of the marshall plan in influencing technological developments in europe, we critically assess the extent to which america's role in europe shaped the wider diffusion of oil technology, infrastructures and governance. we now discuss the first world war and developments relevant to oil transitions. we expand on the existing analysis of the role of the first world war on energy transitions [ , ] through a focus on the multisystemic demand-pull factors that influenced the enduring 'thirst' for oil and related geopolitical developments [ ] . we begin with a broad overview of trends and geopolitical developments before focusing on demand pressures and logistical challenges in energy, food, and mobility relevant to oil transitions. before the first world war the use of oil was limited with the main use being kerosene lighting [ ] . in coal was still king. oil provided under % of world energy supply while coal contributed to % [ ] . in , the value of the uk's coal trade surpassed £ billion, while the total value of all petroleum exports from the usa amounted to some £ million [ ] . yet the first world war was a significant rupture in this pattern of development as global use of petroleum grew by percent while coal production decreased in many european countries [ ] . in the century prior to the war, british geopolitical dominance was secured by its steam-powered navy, and at the turn of the th century, the uk was the world's leading energy exporter [ ] . however, the first world war interrupted this trend and concerns about access to oil would be key to these changing geopolitical dynamics [ ] . the uk faced a central challenge during world war i as it had limited reserves of oil. during the war, % of the oil required for the british as well as french war effort was supplied by the usa [ ] . the uk (along with france and italy) became dependent on the usa to fuel their military efforts during this increasingly oil-intense conflict. these dynamics intensified with the oil crisis of where, due to the everincreasing amounts of oil being consumed by militaries in europe and the disruption of oil supply routes by german submarine attack, there were significant risks of oil supply running out. influenced by these intense pressures, the usa, uk and other countries, including france and italy, were drawn closer together by the imperative to maintain constant supplies of oil. as yergin points out, the oil crisis caused by the first world war forced the united states and its european allies into tighter integration of supply activities [ ] . geopolitical tensions increased in the middle east driven in part by the search for oil, with britain attempting to gain control of important oil fields in iraq. the sykes-picot agreement signed in by the uk and france carved up mesopotamia between the two countries and is understood to have been partly influenced by desires to secure strategic oil resources [ , ] . as several authors point out, the first world war and the thirst for oil amplified the geopolitical significance of the middle east, leading to increased tensions between the usa and uk (along with france) after the war [ , , , ] . but these broad changes and geopolitical developments to secure oil resources were rooted in specific sociotechnical challenges driving the increased demand for oil in energy, food, and mobility. we now explore the demand pressures and logistical challenges caused by conditions of total war and the implications for oil transitions. in the years preceding world war i, the advantages of oil ships had been recognised by many navies around the world. the advantages of oil included the fuel being double the thermal content of coal meaning less space was required for storage; logistics of refuelling at sea were therefore made easier; reliance on docking stations for refuelling was reduced; and there was greater flexibility in changing speeds as well as less personnel required on ships [ ] . additionally, oil-based ships produced less smoke which was beneficial to gunnery operations and enabled more covert movements of naval fleets [ ] . an early adopter of oil-powered ships was the british navy. the share of oil-based vessels in the british navy rose from % at the start of the war to over % by the war's end [ ] . prior to world war i, most shipping was steampowered, and thus the application of oil to navies was a key niche development supported by the military and accelerated by the first world war. during the conflict, the us navy which had already converted several ships from coal to oil accelerated this trend [ ] . germany, on the other hand, did not use all-oil firing for surface fleets until after the war [ ] . internal combustion engine-based automobiles and trucks were a niche in the mobility system before the war. however, they became increasingly used for tactical advantage during the war by the uk and usa as significant problems were experienced with rail road transportation. the first world war has been described as the pinnacle of the 'rail age' [ ] dependent on coal, yet significant disruptions occurred with railroad bottlenecks on the western front disrupting the supply of weaponry and materials to troops. in allied campaigns such as the battle of verdun, the rapid construction of roads and use of motor vehicles for transportation further entrenched the importance of oil for military victory [ ] . an example used by historians to exemplify a general point about the greater military dependence on the automobile and trucks is that at the beginning of the war the uk military had automobiles and motorcycles. by the war's end they had , motorcars, , trucks, and , motorcycles [ ] . away from the front, in the usa, railroad arteries were also blocked and, under the auspices of the us high transport committee, caravans of trucks carrying important goods for the front line were initiated, "inaugurating the long-distance trucking of freight as an alternative to rail transportation and calling attention to the great need for a national system of interconnected, improved highways" ([ ]: ). during the conflict, a new reality of 'war from the air' emerged. the aeroplane, which was in its 'embryonic phase' as a niche technology at the start of the war, had consolidated as a major industry by the war's end increasing requirements for gasoline products [ ] . in britain, as edgerton outlines, this created a very large aircraft industry, with increases in output accelerating through the war. monthly output increased from about ten per month at the war's beginning to in and , in . the number of people working in the uk's aircraft industry rose from nearly , in october to , in november to , in october [ ] . in the usa, there was also considerable aeroplane construction with , constructed by the war's end [ ] . this created a surplus of aircraft after the war. as responses to demand pressures in the food system also had the decision had been made by winston churchill in to transition the entire british fleet from coal to oil and this is cited by historians to exemplify the general point concerning the greater dependence of militaries on oil emerging during world war i [ ] . implications for oil supply. food imports became significantly disrupted and blockading food supplies became a key strategy of both the uk and germany during this war. thus, a focus on 'self-sufficiency' intensified in the uk [ ] . there is evidence that the first world war also led to niche experimentation to maintain agricultural supply in response to these demand pressures. coal and steam power had not made significant inroads into the agricultural sector and farming was still mainly reliant on horse power. however, the first world war accelerated the development and marketing of tractors [ ] . in the uk in , the government ordered british saunderson tractors and $ . million was invested in us models such as the fordson. ford exported , tractors to britain in an effort to boost agricultural productivity [ ] . in the usa, tractor use also expanded under government loan schemes and propaganda campaigns with expos being used by government to further encourage their use [ ] . we now discuss both broad geopolitical developments and relevant sociotechnical developments with regards to the inter-war years and oil transitions. the first world war created an 'oil frenzy' which significantly influenced international relations after the war [ ] . the s can be considered as a period of international instability with respect to global oil supplies as the heightened recognition of the strategic importance of oil created tensions between different nations. as black notes, the first world war and the emergence of oil as a 'strategic resource' [ ] meant that "…new systems of negotiation and need had emerged that would eventually be referred to as "geopolitics." ( [ ] : ). before the first world war, britain controlled only % of the world's oil production, but by the war's end britain had acquired % of the world's known oil reserves [ ] . france and the uk struggled for control over middle eastern oil resources, and in april the san remo agreement was reached between the two countries. here, "britain conceded a share of the oil in exchange for a general agreement, effectively granting france a percent share of petroleum in exchange for mosul" ([ ]: ). this locked out foreign companies from being able to control oil production in the british empire. the usa retaliated with similar measures, using the mineral leasing act of to prevent any company from a nation that was excluding american oil companies in the middle east from gaining access to us oil fields. new oil discoveries in texas in eased this geopolitical tension. however, the new thirst for oil and wartime experiences of oil shortages had resulted in an 'exploration boom' [ , ] . this led to low prices which risked the stability of the global oil industry. the 'red line' agreement at achnacarry in sought to control global oil prices and form monopolies around oil access. the agreement saw the creation of the 'seven sisters' oil cartel where agreement was reached to cease independent prospecting for oil in the ottoman empire by individual oil companies and instead conduct coordinated explorations as a cartel [ ] . this agreement was an attempt to create stability in the global oil regime, yet tensions between the united kingdom and the usa continued as britain pursued a strategy of trying to secure its oil resource independently from america [ , ] . however, britain did not have the industrial capacities to exploit significant amounts of oil in the middle east, and the usa maintained its focus on exploiting indigenous oil reserves. it is worth pointing out that the uk's strategy for oil independence differed in the inter-war years from germany in significant ways which would ultimately have implications for world war ii. germany was economically and politically weakened after the first world war and could not acquire oil reserves. a key part of their strategy was to pursue petroleum synthesized from coal [ ] . germany established a synthetic fuels industry to meet the rising demand for oil as the country continued to industrialise during remilitarisation in the s. the energy intensive process of producing synthetic oil from coal would also ensure that territorial expansion to acquire resources of crude oil would be key to german military planning and strategy in war [ ] . the oil strategy of the uk also had its limitations during this period of 'frenzy' however. the uk put considerable effort into securing middle eastern oil supplies, and while in the usa produced more oil than any other country, % of oil production outside of the us was produced by british companies [ ] . however, this strategy proved flawed in the build up to the war as routes through the mediterranean were hampered by italy in the s. britain could not secure its supply routes and did not have adequate tanker capacity to transport the oil. the imperative to maintain a constant supply of oil amplified by world war i had accelerated production in the us oil industry and this presented the challenge of re-orienting to peacetime activities. as auzzanneau writes "unable to settle down and smoothly adapt its production to the new conditions of peace, the oil industry experienced an intense overproduction crisis after " ([ ]: ). yet, oil production and consumption continued to steadily increase in the usa, with the rise of car culture and mass consumption during the 'roaring twenties'. however, in the uk this expanse was more modest. despite being adversely affected by world war i and facing significant economic problems, rail in the uk was not challenged by road transportation to the same extent as in the usa and its 'clash of cultures' between the automobile and rail systems in the inter-war years [ ] . the significant wartime problems of bottlenecks in flows of truck freight resulting from poor road infrastructure experienced during the war gave impetus for mapping out and then funding improved road infrastructure in the usa. the federal highway act to construct a joined-up road network linking us cities was unveiled specifically citing military considerations as a reason to invest in highway construction [ ] . in the uk, the ministry of transport was established in and introduced a national system of road signage, a system of grants for local authorities to construct roads, and set up a research laboratory to investigate matters of road construction [ ] . several authors conclude that while ww had been an accelerating force for oil, coal had been negatively affected by the conflict, including through infrastructural damage, depleted labour supply and labour strikes [ , , [ ] [ ] [ ] [ ] . according to the official history of the british coal industry for example, british coal was an 'industry in decline' after the first world war [ ] . this is reflected in the writings of trade unionist ivor thomas, who in lamented this decline stating that "whether we like it or not, we are entering -or have indeed entered -an oil age" ([ ]: ). yet in the uk, the coal industry remained powerful and what was referred to at the time as the "back-to-coal" lobby still had considerable influence ([ ]: ). another impact of war relevant to oil transitions, was the emergence of new users capable of operating automobiles or aeroplanes as a result of their wartime experiences. the war popularised a product that had heretofore been viewed as elitist. automobiles, for example, moved from being niche vehicles to a more familiar form of transportation [ ] . the military surplus of aeroplanes created by world war i was also significant for post-war developments in the usa: some of the , planes that had been constructed were integrated into civil aviation which was being utilised for activities including mail delivery [ ] . while tractors continued to be used on both sides of the atlantic, growth was modest in both countries [ , , , ] , although in the usa, the use of tractors increased through support for agriculture under the new deal in the s. however, it would be during world war ii that the 'petrolification of agriculture' [ ] would take place, as well as acceleration and innovation in the oil industry leading to the further embedding of oil in the mobility system. we now focus on the second world war, again summarising broad trends and geopolitical developments before discussing relevant sociotechnical developments with regards to the conflict. the second world war was a global conflict with a marked increased dependence on oil compared to the first world war. for example, american forces in europe used one hundred times more gasoline in world war ii than in world war i [ ] . two years into the war, in , oil supplies to the uk became cut off as a result of german submarines attacks [ ] meaning that as the war progressed, a key challenge became sustaining constant supplies of oil to europe from the usa [ , , ] . as this challenge was met, the war transformed patterns of energy imports and exports across the transatlantic zone. before the second world war, the uk was still one of the world's main energy exporters. britain's increasing reliance on the united states for supplies of oil that emerged over the course of the first world war had dissipated, and instead the uk sourced much of its oil during the inter-war years from countries including venezuela, dutch west indies, iraq, and persia. however, shipping routes from these regions were disrupted during the second world war. as edgerton points out, even as late as the british were determined to maintain their oil independence from the usa [ ] . yet, as the war progressed the uk became reliant on the us for % of its oil supply, paying for it with assistance through the lend lease programme [ , ] . during the course of the war, the usa began turning attention towards securing future supplies of oil for future wars and projected increases in demand in post-war society [ ] . in american geologist everytt gower returned from saudi arabia to announce that the oil in the region was the "greatest single prize in all history" (quoted in yergin [ ] : ). negotiations between roosevelt and churchill took place regarding this future oil supply near the war's end. in secret meetings at the suez canal between roosevelt and abdul aziz ibn saud however, it was established that britain would not gain control over saudi's oil reserves and that saudi would exist in the american sphere of influence [ ] . this moment and subsequent agreements would be decisive in fundamentally altering international energy trade and geopolitics. it is also worth pointing out, that oil had a far stronger influence on war strategy during this conflict. due to its lack of indigenous oil resource and dependence on synthetic fuels derived from coal, germany sought to acquire oil resource through territorial expansion. the german quest for oil in the caucuses is identified as a motivating factor in the invasion of the soviet union for example [ ] . the allied oil campaign put significant resource into destroying german oil infrastructure and refining capacity [ , , ] . waging war was now dependent on oil resources with global networks of shipping routes and refineries extended as the allied campaign sought to acquire and deliver adequate oil supplies to multiple theatres of war. we now look from a sociotechnical systems perspective at the specific demand pressures and logistical challenges that were accentuating the imperative to use oil in the energy, food and mobility systems. during world war ii the construction of oil infrastructure was rapidly accelerated. in the usa, the 'little inch" and "big inch" pipelines were constructed in under a year, between and , as a means to transport oil across the us from the south to the north east [ , ] . at the start of the twentieth century, pipe size had been standardized at eight-inches, however this could only move , barrels a day even though a refinery could produce up to , barrels. as richard rhodes points out, solutions in the form of wider pipes and 'looping' techniques were already developed but private companies were not prepared to make the requisite investment. however, during the war these innovations in pipeline technologies were put to use. as johnson notes, "in the endeavour to meet the demand for crude oil and petroleum products on the east coast, government-financed pipelines embodied technological innovations -particularly with respect to diameter of pipe -that were to have a lasting impact." ([ ]: ). the "big inch" and "little inch" pipelines stretched for , miles across the usa with pumping stations every miles. the 'big inch' pipeline was capable of moving , barrels of crude daily and it was, at that time, the largest and longest pipeline ever built up [ ] . an industry pipeline management committee oversaw the expansions of pipelines under the 'tulsa plan' and over , miles of new pipeline was dug and laid during world war two in the us to enable meeting the vastly increased demand during wartime [ ] . in the uk, due to the threat of aerial bombardment of road infrastructure as a means to transport oil, construction began on the government pipelines and storage system (gpss) in , with the first phase completed by . the system connected key locations such as liverpool docks with avenmouth through underground pipelines stretching for km. in the pipeline system was extended to connect up various airfields in the east of england. as the gpss system was extended further, this network of pipelines would eventually supply aviation fuel to all of england's air fields [ ] . this was a significant technological breakthrough given that there was little knowledge of pipeline construction in the uk prior to the war [ , ] . innovation in the oil sector was stimulated by the changing demand pressures and logistical challenges of war. world war ii is referred by some historians as an 'air war' [ ] or 'the bombing war' [ ] and during this conflict the aircraft industry was transformed. innovations in oil production methods, including perfecting 'catalytic cracking techniques', made possible the production of large volumes of -octane gasoline [ ] . this fuel was in high demand from the once burgeoning and now mass-producing aircraft sector, with oil intensity increasing as jet engines and four engine bombers were developed. huge refineries were rapidly built to facilitate the increasing demand for different types of fuel [ ] . while -octane gasoline and associated innovations were developed before the war there had been no market for them prior to the conflict [ ] . with the 'mass mobilisation of science' during world war ii [ ] , a host of other scientific endeavours led to the rapid expansion of the aero-industry further entrenching a reliance on oil. this included advancements in radar technologies, air precision, electronics, computing, and cabin pressure. meanwhile the us auto industry was redirected towards the war effort and played a central role in the mass production of tanks, trucks, jeeps, aeroplanes, bombs, torpedoes, steel helmets and ammunition under huge contracts issued by government [ , , ] . the british car industry was similarly re-directed towards the production of war materiel [ ] . this mass mobilisation of the auto-industry for the war effort would see the sharing of innovation and collaboration around mass production techniques with significance for increased post-war production of automobiles [ ] [ ] [ ] in the food system, the immense pressures on agriculture to increase production in the usa and uk, accelerated the mechanization of agriculture, including the increased use of tractors and combine harvesters. for example, in there were , tractors in the uk, by this had risen to , [ ] . in the usa, the number of tractors on farms rose from almost . million tractors in to . million tractors in -an increase of two-thirds. innovations took place in the design of tractors as they were mass produced: tractors got smaller, hydraulic systems were introduced, and they became more powerful. the energy intensity of us agriculture also increased and new innovations such as 'hybrid corn' and larger farm sizes accentuated the need for pesticides and insecticides which were also being rapidly developed during the war to protect troops fighting in the pacific from disease [ ] . some plant protectants developed during the war were dependent on the petrochemicals industries that were consolidating as the refining of oil increased during the war [ ] . these changes in agricultural practices occuring during the war were wide-ranging and were rapidly adopted. as rasmussen writes, "higher prices and a seemingly unlimited demand for farm products, combined with a shortage of farm labor and appeals from the government to increase production, led farmers to adopt technological advances" ( [ ] : ). these advances entailed increased "input of mechanical power and machinery, fertilizer and lime, chemicals, feed and seed, and other items" ( [ ] : ). thus, an increasing thirst for oil in the food system was generated by these developments. the development of plastics from petrochemical sources was also accelerated during the second world war in the usa, and would have important implications for the food system in the post-war era. in , the first meeting of the society of plastics industry (spi) concluded that there was no market for these products [ ] . however, during the war, demand pressures meant there was an urgent need to conserve aluminium, copper, steel and zinc for war materiel. these pressures, combined with increased oil production during the war, meant that an opening was created for oil-derived products to act as substitute for scarce resources and were used in clothing as well as acting as a solution for food preservation and storage. between and production of plastics almost tripled in the usa [ ] . as black notes with regards to the burgeoning plastics industry, "petroleum became a primary component in allowing producers to overcome limits of supply and production" ([ ]: ). towards the war's end, the new condition of future us energy policy was stated by the state department's petroleum advisor: "[the central objective] …is not a rationing of scarcity, but the orderly development and orderly distribution of abundance." (quoted in yergin, [ ] : ). in oil surpassed coal as the main source of energy in the usa [ ] . the second world war had been key in transforming the infrastructure associated with oil, and much of this had been achieved through government spending. after the war, the us government set about transferring government holdings of pipelines refining capacity and tankers to the private sector [ ] . post-war lobbying by the us coal industry to close the 'little inch' and 'big inch' pipelines on the basis they put the coal industry at a competitive disadvantage failed [ ] , and these pipelines open up east coast america to increased supplies of oil as well as natural gas [ , ] . in , after the japanese surrender, oil rationing was suspended in the united states and the stage was set for consumers to "…design[…] their post-war lives around energy decadence" ( [ ] : ). in the usa, a key challenge for the post war period, according to bonneuil & fressoz, related to the immense productive forces that had been mobilised during total war and the "…problem of productive overcapacity and its reconversion into peacetime" ( [ ] : ). the automobile industry stabilised and set about producing cheap cars in record numbers, thereby sustaining the levels of high employment achieved in the war years and working with newly refined mass production techniques perfected during the war [ , ] . the highway expansion programmes of post-war america enabled a further rapid acceleration of the automobile culture and -similar to the programmes after world war i -were influenced by military as well as civil requirements building on wartime proposals set out by roosevelt in and the highways act . the second world war had also transformed air infrastructure: the wartime development of a network of runways equipped with new air traffic control systems and standardised aircraft paved the way for the rapid expansion of domestic and international aviation. oil flowed through the food system in different ways, with plastics increasingly used in food packaging and preservation [ ] . the mechanization of agriculture accelerated during the second world war continued to expand in peacetime and required increasing amounts of petroleum to power industrial farming [ ] . additionally, the transportation of agricultural produce by truck became more widespread as large highways systems were constructed [ ] . oil had seeped into every part of american life, forming what black refers to as a "ecology of oil" [ ] . here, "oil does not just fuel americans' vehicles. oil has changed their diet, their clothes, their neighbourhoods, their jobs, their fun-in fact, everything about u.s. society" ( [ ] : ) . the situation of extreme scarcity caused by the tragic destruction of war experienced by continental europe after world war ii was very different to that of the united states. production of coal had fallen in the uk and across western europe and coal infrastructure, especially in germany, had been damaged [ ] . traditional trade routes and supplies did not return to normal immediately after the war and the industry was in a state of crisis [ ] . although the uk had not been as badly affected as countries on the continent, there were still grave concerns about the depleted coal industry [ ] . the uk was now a net energy importer, and set about attempting to reconstruct its industries through nationalisation [ ] . yet, in the early s, the uk's share of oil began to rise markedly, reaching % in from a share of less than % at the war's end [ ] . this trend continued and the uk's imports of oil rose rapidly through the s. in the uk imported , thousand tonnes of oil. in the uk imported , thousand tonnes [ ] . but by this had increased over ten-fold to , thousand tonnes, increasing year upon year to a historic maximum of , thousand tonnes in . despite attempts to gain control of oil in the middle east after the second world war, the uk's oil imports were largely from middle eastern supplies under the sphere of influence of the usa [ ] . the outcomes of the second world war and the nature of reconstruction have been identified by several authors as important factors influencing a rapid transition to oil that occurred across western europe in the s [ , , ] . the 'black gold' of saudi arabia, controlled by the usa, was not used initially to fuel american abundance but rather to facilitate the reconstruction of europe. thus, the solution to the problem of european energy scarcity was, to some extent, american abundance. the european recovery programme (marshall plan) was initiated by the usa in and % of marshall plan loans related to the importation of oil to europe [ , ] . the share of petroleum products in the primary energy consumption mix of europe at the end of the war was around % [ ] ; by it was % and by it was % [ ] . in the s several pipelines were built connecting europe to mediterranean supply routes from the middle east and extensive networks of pipelines were built across europe including the tap line completed in , inter-provincial in , and the trans-mountain in [ ] . as historians of european infrastructure point out, these european pipeline projects drew on successful wartime experiences with constructing extensive pipeline networks such as the gpss in the uk [ ] . historians highlight that the european oil transition was fundamentally shaped by the usa's new-found global dominance and cold war concerns about europe becoming dependent on oil from the soviet union. as boon writes, after the war "neither a distinct western european oil regime nor an integrated [european economic community] governance framework for energy materialised", rather, "…the transition to oil evolved under a global regime dominated by multinational oil companies -then at the apex of their power and closely linked to the informal empires of the us and britain" ( [ ] : ). in short, the age of oil in europe was strongly facilitated by innovations and frameworks amplified during the war in the usa that diffused to the european context as oil companies constructed pipelines and refineries underpinning a rapid transition to oil. the oil dependence of europe grew as the restoration of europe's transport infrastructure took place, particularly evidenced by increases in road mobility, automobile ownership, and trucking. there is evidence of the usa's preference for exporting trucks to europe rather than rail freight after the war, and strong lobbying by the us to push for prioritisation of road construction in europe's infrastructural recovery [ ] . as seely highlights, "…the relative ease with which americanstyle highway and traffic engineering moved across the atlantic also was a product of the willingness-indeed eagerness-of american engineers, government officials, and industrial leaders to assist in this diffusion process" ( [ ] : - ). similarly, with regards to the food system, the marshall plan is also a relevant factor to consider. the technological transfer of tractors, industrial agricultural techniques, hybrid corns requiring fertilisers and other plant protectant innovations to the uk and other western european countries was faciliated through agricultural support mechanisms as part of the marshall plan [ ] . the emergence of the usa as a superpower during the second world war as the uk's geopolitical power diminished, therefore seems relevant to understanding the rapid diffusion of the imperative to use oil to europe after the conflict. the oil intensity of the us had increased markedly in multiple sociotechnical systems particularly as a result of rapid developments that had occurred in response to demand pressures in the second world war, and the embeddedness of oil in multiple sociotechnical systems underpinned a new society based on abundance, influencing the nature of the european recovery through the marshall plan. we now discuss the significance of our interpretive analysis in terms of the role of war in energy transitions, and the contribution the present work makes in responding to and developing the dt framework. we also discuss the value of the dt framework for energy transitions research. focusing mainly on the usa and uk, we have conducted an interpretive analysis of the world wars and the emergence of the oil age, integrating insights from geopolitics and historical literatures with a sociotechnical perspective on multi-system demand pressures. under the exceptional demand pressures of total war, the imperative to maintain abundance and constant supply saw the search for solutions to meeting demand challenges, and in both the first and second world wars this process deepened society's reliance on oil resources. the imperative to use oil was thus accentuated in multiple sociotechnical systems during war time. with regards to world war i, our analysis corroborates work in energy transitions that has interrogated the key role of the conflict in influencing a shift from coal to oil, where war can be seen as an extreme form of 'creative destruction' [ ] . the present work also chimes with studies of the role of war on energy transitions including hydro-electricity in canada, the usa, and germany [ , , , ] that highlight the importance of world wars as mobilising forces for energy transitions. given the limitations of our geographical focus, our broader conclusions on the role of war in energy transitions must necessarily be cautious. indeed, the pattern of the emergence of the 'oil age' at the global level does not necessarily fit the time period of the 'fourth surge' as depicted by perez. as rubio-varaz highlights in an analysis of latin american transitions to oil influenced by the first world war, these occurred at a far earlier stage than europe [ ] . however, the stages of the 'age of oil' in terms of irruption, frenzy, synergy, and maturity fit with the developmental processes with regards to the usa, uk and western europe. with the existing work on energy transitions and world wars in mind, the present analysis can be used to draw out propositions about the role of war in energy transitions and the mechanisms that influence energy transitions. these are not designed as definitive conclusions but rather insights from the present research that could be tested further with different case studies, national contexts, and international dynamics contributing to the emerging literature on world wars and energy transitions. we outline three propositions as follows: . world war amplifies both 'sides of the coin' of sociotechnical transitions. kivimaa and kern [ ] refer to the creative processes of sociotechnical niches being nurtured and the flip side of destabilisation of existing regimes as both being crucial in understanding transition processes. in this study, the demand pressures of war, and the search for solutions to these problems, saw a range of developments taking place during wartime that further embedded oil in multiple systems. this included the acceleration of automobiles and aeroplanes in the first world war, increased mechanisation and tractor use in response to labour shortages in the first world war, and petrochemical solutions to shortages of materials in the second world war. these technologies and innovations existed prior to the wars, however strategic opportunities for their use were opened up by the particular selection environment of wartime. at the same time, the coal industry, and associated industries in the mobility sector experienced destabilising consequences both in the first world war, which was compounded by the world war ii, due to severe disruption of export routes, infrastructural damage, and labour shortages. this at first seems counter-intuitive because war is of course divisive, and considerable literature has rightly drawn attention to the tendencies towards a push for greater national self-sufficiency and autarky in energy as a result of war. this is certainly true in some respects, however in the case of oil we see that the uk and other european countries became more tightly bound to the american oil regime during the first world war through the inter-allied petroleum conference. while in the period of 'frenzy' the uk attempted to forge its own path, the integration between the us and british energy systems intensified once again in the second world war. through re-orientation and reconstruction, the post-war period saw this integration continue as the uk, along with many western european countries, transitioned more rapidly to become embedded in a 'global' oil regime largely controlled by the united states. surplus and reconversion. the foundations of the oil-based society were laid during the second world war. extensive pipelines in the usa and the uk built to fuel the war effort were reconverted to supply oil and natural gas to the american east coast in the case of the usa, and aviation fuel to airports for civil aviation in the case of the uk's pipeline developments. runways, air traffic control systems, and streamlined global logistics networks shaped during world war ii for air transport were reoriented to civil aviation. the surplus of agricultural produce in the united states had to be maintained in order to ward off economic collapse and petroleumreliant industrial agricultural practices persisted and intensified to maintain this abundance. after the first world war, a surplus of planes in the us and uk were absorbed into civil airlines, then in their infancy. the first world war created hundreds of thousands of new users of oil-dependent automobiles and aeroplanes. in addition to these points, the role of war in energy transitions can be further explicated by returning to the implications of this analysis for the dt framework. through a focus on the demand pressures of world wars and the solutions that emerged to these challenges, our analysis has highlighted how in these wars the imperative to use oil was accelerated in the energy, food, and mobility systems by innovations and experimentation where reliance on oil was often a key solution. the three systemsenergy, food, and mobility -became more tightly integrated as a consequence of wartime activity. following schot & kanger, we have contributed to the dt framework in terms of "understanding how changes across multiple systems became connected and coordinated, developing a common directionality in the long run" ( [ ] : ). while the trend towards oil use accelerated faster in the mobility system after world war i and the growth of mechanised agriculture was modest, during world war ii these systems become even more tightly integrated. pipeline infrastructure, refinery capacity, petrochemicals, plastics, pesticides, increased mechanisation of agriculture, mass production of aeroplanes, construction of runways, and a host of other concurrent developments were accelerated by war. in the language of the dt framework [ ] , the role of war in deep transitions is the tighter integration and coordination of multiple sociotechnical systems in a similar direction. the world wars amplified the meta-rule of the imperative to use oil, and shaped the sociotechnical conditions to deliver abundant oil supply in numerous domains. more broadly, we argue that the dt framework can be a useful conceptual tool for energy transitions research. in focusing on multiple rather than single systems, the dt perspective analysis of the age of oil conducted in this paper, reveals the interdependencies between different systems and how they are tied together by oil infrastructure and use. these multi-systemic understandings can be of value in thinking about the shift away from an oil-based society, and the need for multiple policy interventions and policy mixes to enact a deep energy transition based around sustainable sources of energy. the focus on rules and meta-rules can assist in enhancing understandings of how broader transitions beyond one sociotechnical system occur and shine a light on the more deep-rooted unsustainable routines and patterns of behaviour associated with the age of oil that must be undone if more rapid sustainability transitions are to occur. with its focus on exogenous events like wars, the dt framework can also be utilised to explore mechanisms for deepening and accelerating renewable energy transitions beyond niche-regime dynamics, and the multi-system interactions that may need to be fostered if an 'ecology of renewable energy' rather than oil, is to be achieved. this highlights the value in further interrogating other 'landscape' events that could be influential in the directionality of renewable energy transitions. for example, the particular demand pressures of the covid- pandemic has had a significant impact on energy systems that could be decisive in shaping lasting change [ ] . additionally, the dt framework's attention to military and wars and their historic role in accelerating particular transitions, also emphasises the importance of considering mobilising forces that can also decisively and rapidly influence the directionality of energy systems but that are not reliant on the tragedy and violence of war. indeed, given the enduring links between militaries and unsustainability [ ] , a key question for sustainability transitions scholars is the extent to which rapid low carbon transition is feasible if current systems of defence, violence, and militarisation remain in place. this points to forms of 'deep incumbency' including the military that warrant more dedicated attention [ ] . in this paper we have focused on the emergence of the age of oil in the twentieth century which forms the culmination of the first deep transition. we have conducted an interpretive analysis drawing on geopolitical and historical literatures to build an understanding of how the exceptional wartime demand pressures and logistical challenges experienced in multiple sociotechnical systems influenced oil transitions. the role of the two world wars in shaping the emergence of the age of oil in the culmination of the first deep transition can be summarised as follows: the exceptional conditions of total war saw the imperative to use oil being accentuated in multiple sociotechnical systems. as a consequence of war time pressures, energy, food, and mobility were coordinated in a similar direction through the meta-rule of using oil, and became more closely integrated based around an increasing dependence on oil. wars saw the rapid development of the technological, infrastructural, logistical, scientific, and institutional conditions underpinning oil-intense societies of the post-war economic 'golden age'. thus, the two world wars were decisive in shaping the directionality of the culmination of the first deep transition. the present analysis suggests that war, often discussed in mlp approaches as a residual landscape factor rather than a focal point of analysis, has an important role in 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leer-buter, coretta title: the emergence of enterovirus d in a dutch university medical center and the necessity for routinely screening for respiratory viruses date: - - journal: j clin virol doi: . /j.jcv. . . sha: doc_id: cord_uid: f b l background: since august , an increase in infections caused by enterovirus d (ev-d ) was reported in the usa and canada, for the most part in children presenting with severe respiratory symptoms. objectives: to determine whether an increase in severe ev-d respiratory infections was observed in our region. study design: samples from patients with respiratory symptoms were screened for viral pathogens, including rhinovirus and enterovirus. subsequently, samples positive for rhinovirus and enterovirus were routinely sequenced for phylogenetic analysis. furthermore, an additional method was used to detect ev-d specifically. results: during the first three quarters of the year , respiratory samples were analyzed; ( %) of them tested positive for enterovirus. eighteen samples tested positive for ev-d , obtained from different patients admitted to our hospital. eleven were children below the age of , of whom five children needed intensive care treatment. the remaining five samples were from adults, who all had an underlying disease; three were transplant patients (heart, lung and renal transplantation), the other two had an underlying lung condition (copd, asthma). phylogenetic analysis showed a close relationship with the strains circulating currently in the usa, all belonging to the known ev-d genetic subtypes. conclusions: we observed an increase of ev-d infections in our population, both in children as well as in adult. in there have been cases so far, compared to none in and and a single case in . phylogenetic analysis identified two similar clusters as shown in the usa and canada. since august , an increase in infections caused by enterovirus d (ev-d ) has been reported in the usa and canada. in this outbreak, the majority of samples analyzed are from children with severe respiratory illness, many of whom have symptoms of wheezing. also, some fatalities have been documented [ ] . in the years and , our hospital in the northern part of the netherlands was confronted with an increase in respiratory illness, which was not only seen in our tertiary care hospital, but also regionally [ ] . an outbreak of ev-d was shown to be the cause of the increased incidence of respiratory infections. because ev-d was not detected by our routine respiratory panel at that time, we added an enterovirus pcr into our routine screening panel of respiratory viruses (influenza a, influenza b, parainfluenzavirus - , coronaviruses oc , nl and e, adenovirus, bocavirus, rsv-a, rsv-b and human metapneumovirus). moreover, we expanded our testing by routinely sequencing all enterovirus and rhinovirus strains to identify virus strains and to chart outbreaks and transmission patterns. adding to this sequencing strategy, we included noroviruses as well as parechoviruses. nationally, a structure called typened was available through the dutch national institute of health (rivm) [ ] . furthermore, by using our regional sequencing and epidemiological strategy, called regiotype, we were able to speed up the flow of information from our region, including hospitals and public health services covering the (not densely populated) northern part of the netherlands. the aim of this regiotype strategy is to provide a rapid sequencing http://dx.doi.org/ . /j.jcv. . . - /© the authors. published by elsevier b.v. this is an open access article under the cc by-nc-nd license (http://creativecommons.org/licenses/by-nc-nd/ . /). service for the whole region. this not only completes our diagnostics with sequencing data, but as clinical data are fed back into our database, we create a source of information which serves to obtain more insights in epidemiology patterns. recently, the european center for disease prevention and control (ecdc) concluded in a "rapid risk assessment" [ ] that no epidemic clusters of severe disease caused by ev-d in eu/eea countries were observed and that a moderate risk of ev-d was expected. the ecdc rapid risk assessment attributes the low level of reporting of ev-d to the low number of laboratories which routinely screen respiratory samples for ev-d . subsequently, only a small number of ev-d cases ( ) are observed in the netherlands during the enterovirus 'peak season' in , through both the entero-surveillance and the ili/ari surveillance networks [ ] . however, we are observing an increase in infections caused by ev-d in our tertiary care hospital since may , similar to the outbreak in the usa and canada. to improve the specific diagnostics for ev- , we developed a specific real-time pcr method. to determine the circulation of ev-d in our region, during a time when an outbreak of this virus is ongoing in north america, and to develop an ev-d specific pcr method. samples from patients with respiratory symptoms, are routinely screened for several viruses, including influenza a and b, rsv type a and b, metapneumovirus, parainfluenzavirus type - , coronavirus (oc , e and nl ), adenovirus, bocavirus, rhinovirus and enterovirus. during the first three quarters of the year , respiratory samples were analyzed. samples that tested positive for enterovirus were selected for sequencing. all primary patient materials sent to our laboratory for analysis of respiratory viruses, are routinely screened for rhinovirus and enterovirus by a laboratory developed real-time pcr that amplifies a bp fragment of the ntr region of both rhinovirus and enterovirus. differentiation between both viruses is done by the use of highly specific probes containing locked nucleic acids (lna) at the terminus (table ) . rna was extracted from l sample with the addition of l internal control, phocine distemper virus (pdv), using the nuclisense easymag (biomérieux, lyon, france). pcr was performed in a total reaction volume of l using l rna, xtaqman ® fast virus -step master mix (applied biosystems, foster city, ca, usa), nm forward and reverse primer for rhinovirus/enterovirus, nm rhinovirus probe, nm enterovirus probe each, nm forward and reverse primer for pdv, nm probe for pdv and dnase/rnase free water (sigma). reactions were run on an abi using the profile of min • c, s • c, followed by cycles of s • c and s • c. rhinovirus and enterovirus positive samples were routinely typed by sequencing. identification of the rhinovirus strains was done by amplification and sequencing of a nucleotide fragment spanning the hyper variable part of the ntr, the entire vp gene and the terminus of the vp gene with primers described by savolainen et al. [ ] . identification of the enterovirus strains was done by amplification and sequencing of - basepairs part of the vp gene as has been described by nix et al. [ ] . an automated dna sequencer was used (abi xl). sequence data were analyzed with sequencing analysis (version . ; abi) and subsequently phylogenetic analysis was performed using bionumerics software . (applied maths, sint-martens-latem, belgium). to support initiatives for the rapid and specific detection of ev-d , a real-time pcr was developed for fast detection of ev-d . primers and probes were developed using a clustalw . alignment of all currently available non coding region (ncr) sequences in the ncbi database and primer express . software (applied biosystems). twenty-two samples, spanning a period from to , previously detected by rt-pcr and identified with ev-d by sequencing, were tested for validation. for determining specificity, six currently circulating human rhinovirus (hrv) strains of the genogroups a, b and c (hrv-a , hrv-a , hrv-b , hrv-b , hrv-c and hrv-c ) and samples from containing different enterovirus species (cv-a , cv-a , cv-a , cv-a , cv-a , cv-b , e- , e- , e- , ev-c and ev-c ) were included in testing, as well as five reference strains provided by the dutch national institute of health (cv-a , cv-a , e- , e- and the ev-d reference fermon strain). sample types included were sputum, nasopharynx, flocked swab, stool and cerebrospinal fluid. rna was extracted as described previously. pcr was performed in a total reaction volume of l using l rna, xtaqman ® fast virus -step master mix (applied biosystems, foster city, ca, usa), nm forward and reverse primer for evd , nm ev-d probe each (table ) and dnase/rnase free water (sigma). reactions were run on a abi as follows: min • c, s • c, followed by cycles of s • c, s • c and s • c. since the beginning of , respiratory samples were tested, of which ( %) were positive for enterovirus. eighteen samples tested positive for ev-d , obtained from different patients admitted to our hospital. (table ). eleven cases were children of whom five presented with severe respiratory illness, requiring intubation and mechanical ventilation. of these five severely ill children, two (aged year and year/ months) had been previously healthy and two were ex-prematures (aged years/ months and month). one of the previously healthy children (patient ) was given resuscitation prior to admission to the pediatric intensive care unit. one child had sickle cell anemia as underlying condition ( years/ months). two children were admitted with a moderately severe bronchiolitis-type respiratory infection with wheezing and shortness of breath. four children had mild respiratory infections with common cold-symptoms only. these children all had underlying conditions for which they were admitted to our hospital. interestingly, two of these children (patient and ) acquired the infection several days after admission. the sequences of these isolates were identical with the sequence of patient (genbank accession numbers: km , km and km ). both patient and , were admitted to the same pediatric intensive care unit during the same period as the admission of patient , which suggests nosocomial transmission. five cases of ev-d infections were from adults. all adult patients had underlying conditions predisposing them to respiratory tract infections. one patient was a heart transplantation patient, one was a lung transplant patient, one was a kidney transplantation patient, one had asthma and one had chronic obstructive pulmonary disease (copd). all five adults had to be admitted to the hospital with symptoms of cough, shortness of breath and wheezing. the patient with a kidney transplant (patient ) had radiological signs compatible with pneumonia. in all cases, the diagnosis of ev-d infection was made following a positive pcr result for enterovirus and subsequent sequencing. ev-d partial vp sequences were submitted to genbank, accession numbers: km -km and km -km . we tested respiratory samples in our laboratory during the first three quarters in . ( %) samples were tested positive for enterovirus. different enterovirus types were isolated, of which samples were ev-d (table ) the ev-d test was highly specific, all strains were tested negative for types other than ev-d as described in section . the assay was designed in the ncr. all clinical samples of which tested positive in the standard ev assay and subsequently typed as ev-d , were also tested positive in the ev-d specific pcr. table enteroviruses detected from respiratory samples, umcg . number of samples total a nd, not defined. phylogenetic analysis shows that out of the sequences correspond with clade a and out of sequences with clade b, as described by tokarz et al. [ ] . none of our sequences corresponded with clade c. usa strains correspond with both, clade a and clade b, suggesting that similar strains are currently circulating in the usa and europe (fig. ) . in this paper, we presented cases of respiratory ev-d infections in the northern part of the netherlands in . these patients were in most cases admitted to the hospital because of a serious respiratory illness. of these cases, five were children with a life-threatening respiratory illness. we also noted five ev-d infections in adults, all with underlying conditions, predisposing to more severe respiratory illness. three were transplantation patients and two had pulmonary conditions. all of these five adults required hospital care for respiratory infections. this upsurge of respiratory infections caused by ev-d was similar to what was seen in our hospital in and [ ] . more importantly, the ev-d upsurge in our hospital occurred during a time period while a large ev-d outbreak took place in the usa and canada, involving around a thousand children. also in the usa, only the children admitted to the hospital were tested and reported. we expect that the number in north america could be higher if the adult population with severe respiratory illness were included. the incidence of ev-d observed in our hospital, which is located in the least densely populated part of the netherlands, is contrasted by the low number of reported ev-d infections in rest of the netherlands, while no data are known about the current situation in europe [ , ] . after all, it seems unlikely that ev-d is causing problems only in the usa, canada and the northern part of netherlands, without affecting other european regions. in the - upsurge, a number of children became severely ill. moreover, it was then shown that % of the cases were hospital acquired, which highlights the need for testing of not only patients with severe respiratory disease, but also patients with mild symptoms, who may spread the virus. similarly, in the current study, we possibly witnessed in-hospital transmission of ev-d from one severely ill child to two children who developed mild respiratory symptoms. the low number of reported ev-d infections in europe may be caused by the overall low circulation of ev-d . however, it could also be caused by the under-diagnosis of ev-d infections due to insufficient sampling of patients with respiratory illness, or an insufficient detection of this virus in routine screening panels, whether using laboratory developed tests (ldts) or commercial assays. from qcmd quality assurance data, it is known that the respiratory virus testing performed in a number of laboratories failed to detect ev-d (dr. paul wallace, personal communication, qcmd, glasgow, united kingdom). the recent ecdc report claims that the circulation of ev-d in europe is low, thus has to be viewed in the context of a near absent detection of this pathogen [ ] . in addition, the potential for undersampling has to be considered. although no exact data exists on underdiagnoses as yet, it is our experience that clinicians, at least in the netherlands, are discouraged from performing diagnostic tests for patients presenting with probable viral illnesses because of financial constraints. and if testing is done, only influenza virus and rsv are preferably tested. a recent initiative by the european society for clinical virology (escv) in collaboration with the ecdc was started to investigate the prevalence of ev-d in europe. further epidemiological information is needed on a larger scale than currently available to determine the circulation for this virus. the implicit risk of focusing on a limited number of viruses for diagnostic purposes, or not performing virological diagnostics at all, is contestable. patients with an underlying disease, whether children, transplant patients, or patient with a chronic respiratory condition, are not getting optimal care if the diagnostic options are limited or even absent. therefore, a diagnostic stewardship program should be discussed asides antimicrobial stewardship and an infection control programs, which are currently being introduced in many hospitals [ ] . we have documented upsurges of ev-d associated with sometimes severe morbidity twice in the last years, i.e. in / and in . it can be anticipated that the virus will reappear regularly and therefore diagnostic assays, whether ldts or commercial assays, should be able to detect this virus. the ev-d specific assay we describe in this paper, may be used for this purpose. all cases described in this paper were diagnosed as ev-d infections by following routine protocols of our laboratory, in which respiratory samples are tested for enterovirus along with other respiratory targets, and all enterovirus isolates are routinely sequenced. our sequencing data, compared to the usa sequence data, suggest that similar strains are circulating in the usa and europe. in conclusion, by following a routine protocol of screening respiratory samples for enterovirus and sequencing, we not only identified ev-d as the cause of respiratory infections in our hospital, but we also were able to show a potential link with the ongoing ev-d in north-america. ev-d is associated with at times severe respiratory illness. adequate surveillance for viruses like ev-d which can cause severe respiratory illnesses, is therefore desirable for university medical centers and reference centers. a collaborative approach effort between the diagnostic and reference laboratories could be taken to achieve this. the ev-d specific assay we present in this paper may aid in the assessment of the real prevalence of this virus in our population, both in children and adults. no funding was received for this study. upsurge of human enterovirus infections in patients with severe respiratory tract infections laboratory-based surveillance in the molecular era: the typened model, a joint data-sharing platform for clinical and public health laboratories european centre for disease prevention and control. enterovirus d detections in the usa and canada -first update continued seasonal circulation of enterovirus d in the netherlands genetic clustering of all human rhinovirus prototype strains: serotype is close to human enterovirus sensitive, seminested pcr amplification of vp sequences for direct identification of all enterovirus serotypes from original clinical specimens worldwide emergence of multiple clades of enterovirus navigating the web in search of resources on antimicrobial stewartship in healthcare institutions the development of a qualitative real-time rt-pcr assay for the detection of hepatitis c virus the authors have no competing interests to report. no ethical approval was required for this study. key: cord- -ww o kjr authors: al-anzi, bader s.; alenizi, mohammad; al dallal, jehad; abookleesh, frage lhadi; ullah, aman title: an overview of the world current and future assessment of novel covid- trajectory, impact, and potential preventive strategies at healthcare settings date: - - journal: int j environ res public health doi: . /ijerph sha: doc_id: cord_uid: ww o kjr this study is an overview of the current and future trajectory, as well as the impact of the novel coronavirus (covid- ) in the world and selected countries including the state of kuwait. the selected countries were divided into two groups: group a (china, switzerland, and ireland) and group b (usa, brazil, and india) based on their outbreak containment of this virus. then, the actual data for each country were fitted to a regression model utilizing the excel solver software to assess the current and future trajectory of novel covid- and its impact. in addition, the data were fitted using the susceptible–infected–recovered (sir) model. the group a trajectory showed an “s” shape trend that suited a logistic function with r( ) > . , which is an indication of the outbreak control. the sir models for the countries in this group showed that they passed the expected % end of pandemic dates. group b, however, exhibited a continuous increase of the total covid- new cases, that best suited an exponential growth model with r( ) > . , which meant that the outbreak is still uncontrolled. the sir models for the countries in this group showed that they are still relatively far away from reaching the expected % end of pandemic dates. the maximum death percentage varied from . % (india) to . % with usa recording the highest death percentage, which is virtually equal to the maximum death percentage of the world ( . %). the power of the exponential model determines the severity of the country’s trajectory that ranged from to with the usa and brazil having the highest values. the maximum impact of this covid- pandemic occurred during the uncontrolled stage ( ), which mainly depended on the deceptive stage ( ). further, some novel potential containment strategies are discussed. results from both models showed that the group a countries contained the outbreak, whereas the group b countries still have not reached this stage yet. early measures and containment strategies are imperative in suppressing the spread of covid- . today's world is changing rapidly at different levels during the current technological and medical renaissance due to research and development (r&d) that has resulted in innovative technologies and outcomes. sometimes such developments may lead to disastrous consequences causing adverse effects on the environment, which could be reflected on the health of living organisms at different levels such as global warming as a part of air pollution, water pollution, and even cancer. some of the advancements could be in the medical field, as well some new antibiotics and vaccines to fight new diseases and outbreaks. as a result, some microorganisms/pathogens have mutated to develop resistance to existing treatments causing many epidemics and outbreaks. disease and sickness have tormented humanity since the beginning of life on earth. human beings, along with plants and vegetables, have had to face challenges from microorganisms during their evolution. the mankind history has seen epidemics characterized by mortality and morbidity [ ] . however, the scale of these diseases has increased notably since the advent of globalization. with the rise in global trade, expansion of civilization, contact with populations across the globe, new opportunities for human interactions have become more common, resulting in the proliferation of such epidemics. the early years witnessed the onset of several diseases such as malaria, smallpox, tuberculosis, influenza, leprosy, whose cure has eventually been discovered [ ] . the scientific efforts made during the mid- th century has reduced the spread of epidemics in the world, mainly due to the advances in medical services, improvements in health care and the urban environment, and availability of vaccines and antibiotics [ , ] . however, the widespread outbreaks have again returned to the world in the st century, possibly due to pollution, overpopulation, global transportation network, and poverty in some parts of the world. hence, the recurrence and emergence of potential infectious diseases that may cause epidemics is most likely to continue [ ] . in fact, the world health organization (who) has claimed that approximately million deaths due to these infectious diseases takes place each year. in developing countries with the least economic resources, such diseases are the major causes of deaths. certain diseases such as tuberculosis and malaria, have reappeared due to the emergence of drug resistant microorganism strains. lastly, the perception of "deliberately emergent" pathogens (such as anthrax and smallpox), and the possibility of their use for bioterrorism in the contemporary world cannot be ruled out [ ] . recently, the world has gone through an intermittent communicable disease outbreak that led to unprecedented epidemics, which have significantly impacted humanity claiming many innocent lives and the economy. such epidemics are sever acute respiratory syndrome (sars), middle eastern respiratory syndrome (mers), and of course the current novel coronavirus that has been classified as a pandemic . table lists the historic and recent epidemics/pandemics [ , ] that have occurred in the world. no one can overlook what the world is going through these days as a result of the covid- pandemic that has already spread through the countries affecting , , people and, unfortunately, killing around , people over a short timeframe ( - months), still with an accelerated pace and notable upward trend [ ] . as aforementioned, this has affected the world economy, the humans' life, and has spread panic all around. on december , there was a cluster of pneumonia cases in wuhan, hubei province, china, whose investigation revealed that these cases were associated with the novel coronavirus or covid- , as it is called today [ ] . viruses are not from the plant or animal kingdom, and are neither bacteria, but are the typical parasites of the living kingdoms. viruses are not living organisms because they cannot live without a host cell. all viruses contain a core, made of a genetic material-nucleic acid, either dna or rna-and a protein shell, which encases the nucleic acid [ ] . coronaviruses are a large group of viruses [ ] surrounded by an envelope with protein spikes, which gives the appearance of a crown (or, in latin, corona) from where it derives its name. [ ] . there are different types of coronaviruses that cause respiratory and gastrointestinal problems [ ] . respiratory diseases can range from pneumonia and in most people the symptoms can generally cause a mild disease [ ] . however, there are some types of coronaviruses that can cause several diseases such as sars: the coronavirus sars-cov identified in table . historic and recent epidemics/pandemics [ , ] . the current novel coronavirus (covid- ) was first found through a group of chinese people who tested positive for pneumonia [ ] . this was at the end of year in wuhan city. the disease then spread to their family members and the surrounding people including their health care staff. the contagious nature of this disease resulted in its spread to other countries over few months [ ] . the coronaviruses circulate in a range of animals. this virus can "spill over" meaning they can jump from animal to human probably due to a range of factors such as mutation or increased contact between human and animals [ ] ; for example the mers-cov came from camels [ ] and the sars_cov from civet cats [ ] . the animal reservoir of coronavirus ( -ncov) is not known yet. in general, respiratory viruses are usually transmitted through droplets from an infected person's cough or sneeze or by touching a surface that has been contaminated with the virus [ ] . people at most risk of the coronavirus infection are those who work at an animal market, health care workers treating coronavirus patients, as well as family members caring for infected coronavirus members [ ] . the most common symptoms of this coronavirus ( -ncov) are fever, tiredness, respiratory symptoms such as cough, sore throat, and shortness of breath, and rare intestinal symptoms such as diarrhoea [ , ] . unluckily, this covid- virus has some features that contributed to its global spread at a relatively short time. the virus has spread all over asia and reached the united states (snohomish county, washington) on january [ ] and in germany on january [ ] through different routes from china. facts about this virus are still new, and what we know about this may change in the future. zhao et al. verified that the initial growth phase of the coronavirus in china was the exponential growth [ ] . they used the serial intervals (si) of infection caused by mers and sars as approximations for the si of the coronavirus and estimated the r [ ] . iwato et al. conducted simulations using the seir model to assess the impact of secondary outbreaks outside china, assuming that one infected patient travelled to an outside community [ ] . while applying the seir compartmental model, kuniya t. predicted the epidemic peak for the coronavirus in japan using real data from january to february [ ] . al qaness et al. developed a novel forecasting model that forecasted and estimated the covid- cases for the upcoming ten days using the adaptive neuro-fuzzy inference system (anfis), which uses the enhanced flower pollination algorithm (fpa) and the salp swarm algorithm (ssa) [ ] . roosa et al., in their study, generated forecasts based on two popular models used previously for forecasting infectious diseases outbreaks, i.e., richards growth model, and a sub-epidemic wave model [ ] . jung et al. modeled the epidemic growth using two methods, scenario- , from a single case recorded on december , and scenario- , using the growth rate fitted along with the other parameters based on data from exported cases reported by january [ ] . the current study aims to assess the trajectory of the recent pandemic due to the covid- outbreak utilizing a new splitting methodology of the selected countries into two groups and developing regression-based and sir-based statistical models and tools that depict the actual recorded data of covid- . different modeling techniques potentially provide different prediction results. we considered two modeling techniques that adopt two different prediction approaches to show that although the modeling techniques exhibit different detailed results, they lead to the same general conclusions. this study covers the entire world with emphasis on extreme cases based on the disease containment. such models will be useful in projecting the covid- trajectory to estimate the daily infection and death rates of the world and selected countries in advance. in addition, this study introduces new factors to be used directly to compare the countries' responses towards covid- . this will help the authorities take the necessary measures and proper action plans to minimize the covid- impact ahead of time. furthermore, the article also covers some novel potential strategies to contain the virus spread at healthcare settings. actual data (country wide population number, number of infected cases in a country, number of deaths in a country, number of new cases) for the entire world and a few selected countries were obtained from the worldometer website [ ] . according to the website, the data mentioned have been collected from the countries' health ministry, government institutions, or government authorities' social media accounts [ ] . the data were recorded daily where the day was reset after midnight gmt+ . all the countries recorded new cases for the current day while in progress except china who displayed the previous day cases. certain countries were chosen to conduct this study based on their outbreak containment and responses. then, a new splitting methodology was used in the current study to divide the selected countries into two groups (a) group a: that succeeded in containing the covid- outbreak, where its behavior was split into - subperiods and (b) group b: that failed and is still struggling in containing the outbreak, where its behavior was split into two subperiods. logistic and exponential growth statistical models were used to fit the actual model into regression equations utilizing the excel solver software. in addition, the susceptible-infected-recovered (sir) model [ ] was applied, which is a compartmental model that has been widely utilized in the literature to predict the spread of infectious diseases. in this model, the population (n) is categorized into three compartments including susceptible (s), infected (i), and recovered (r). the model assumes that the population and both infection and removal rates are constant during the whole epidemic period. in addition, it assumes that the population is well-mixed. during the epidemic period, susceptible cases become infected with a rate β and infected cases become recovered with a rate γ. the rate of change for the three compartments is estimated by the following three differential equations: this model is considered because it requires simple data that is available for the public. furthermore, its implementation is provided as an open source code. to obtain covid- prediction results for the selected countries, we applied an already existing matlab sir modeling tool [ , ] . the tool takes the daily new infection cases as an input and optimizes the model parameters by minimizing the difference between the actual and estimated number of cases and it considers the possibility of having multiple sub-waves. the number of daily new infection cases were collected from a publicly available repository [ ] on july . in this study, an assumption was made that all the data reported by the selected countries were accurate and up-to-date. it is important to note that the results of the prediction models might be inaccurate due to the fact that such models do not consider some affecting factors such as the containment strategies and other related governmental interventions. the considered pandemic prediction models assume that all such related factors remain the same. the prediction results are useful to assess whether more strict interventions must be applied to reduce the estimated number of infections and deaths and relieve the healthcare system. figure was generated to show the recorded daily total infected cases of the world over a certain timeframe ( january until present), which shows that the covid- disease started to increase slowly until about march and then accelerated at a faster speed afterwards. this means that the infected number of cases after march are significantly higher than the numbers before this date. to illustrate this further, the change in new infected cases for the first days (from january until march) is between to , cases, whereas the change for a similar period of time ( march until april)) varied between , to , , cases, which is a -fold increase in the new infected cases than that of the first interval. mathematically, this means that the change in the y over time (∆y/∆x) experienced a relatively drastic jump as opposed to the first interval. in other words, the slope of the second interval is steeper than the first interval ( figure ), which is also called the tangible line of the graph. this clearly means that the infection is spreading through the world in a faster change rate over a short timeframe. interventions. the considered pandemic prediction models assume that all such related factors remain the same. the prediction results are useful to assess whether more strict interventions must be applied to reduce the estimated number of infections and deaths and relieve the healthcare system. figure was generated to show the recorded daily total infected cases of the world over a certain timeframe ( january until present), which shows that the covid- disease started to increase slowly until about march and then accelerated at a faster speed afterwards. this means that the infected number of cases after march are significantly higher than the numbers before this date. to illustrate this further, the change in new infected cases for the first days (from january until march) is between to , cases, whereas the change for a similar period of time ( march until april)) varied between , to , , cases, which is a -fold increase in the new infected cases than that of the first interval. mathematically, this means that the change in the y over time (Δy/Δx) experienced a relatively drastic jump as opposed to the first interval. in other words, the slope of the second interval is steeper than the first interval ( figure ), which is also called the tangible line of the graph. this clearly means that the infection is spreading through the world in a faster change rate over a short timeframe. looking at the selected countries based on containment rates, the countries were classified into two groups: (a) countries that contained (had controlled) the covid- disease, and (b) those that failed to do so (uncontrolled), as follows ( table ) : china is taken as an example for group a where after the outbreak china managed to control the spread of the covid- virus, as shown in figure . china is the origin of covid- where the first cases were recorded in wuhan city on january . this virus started to spread in china with a short-term (interval in figure ) slow rate of infection for about days ( to january) followed looking at the selected countries based on containment rates, the countries were classified into two groups: (a) countries that contained (had controlled) the covid- disease, and (b) those that failed to do so (uncontrolled), as follows ( table ) : china is taken as an example for group a where after the outbreak china managed to control the spread of the covid- virus, as shown in figure . china is the origin of covid- where the first cases were recorded in wuhan city on january . this virus started to spread in china with a short-term (interval in figure ) slow rate of infection for about days ( to january) followed by a faster infection rate in the second interval that lasted for days ( january to february). then, the total infected cases continued to increase with a slow rate until it reached the maximum and levelled off thereafter (interval ). china went through three intervals where interval and represent the breakout stage, and interval the control stage. the slopes for intervals and increased from small to significant and then decreased until they became negligible in interval ( figure ). int. j. environ. res. public health , , x of by a faster infection rate in the second interval that lasted for days ( january to february). then, the total infected cases continued to increase with a slow rate until it reached the maximum and levelled off thereafter (interval ). china went through three intervals where interval and represent the breakout stage, and interval the control stage. the slopes for intervals and increased from small to significant and then decreased until they became negligible in interval ( figure ). an example of group b is brazil, where the covid- spreading rate went through two distinct intervals. interval where the slope of the curve was negligible indicating small increases in the new cases ( march to april) and interval represented by a sudden upward shift in the graph as a result of a massive increase in the newly infected cases each day from april onwards ( figure ). the daily increase rate for interval was from to new cases, whereas the same rate for interval was from to , , , which is significantly higher than that of interval with a notable upward trend. again, this is due to the drastic change in daily new cases as represented by the changing direction of the slope from a small slope to a steeper one. an example of group b is brazil, where the covid- spreading rate went through two distinct intervals. interval where the slope of the curve was negligible indicating small increases in the new cases ( march to april) and interval represented by a sudden upward shift in the graph as a result of a massive increase in the newly infected cases each day from april onwards ( figure ). the daily increase rate for interval was from to new cases, whereas the same rate for interval was from to , , , which is significantly higher than that of interval with a notable upward trend. again, this is due to the drastic change in daily new cases as represented by the changing direction of the slope from a small slope to a steeper one. by a faster infection rate in the second interval that lasted for days ( january to february). then, the total infected cases continued to increase with a slow rate until it reached the maximum and levelled off thereafter (interval ). china went through three intervals where interval and represent the breakout stage, and interval the control stage. the slopes for intervals and increased from small to significant and then decreased until they became negligible in interval ( figure ). an example of group b is brazil, where the covid- spreading rate went through two distinct intervals. interval where the slope of the curve was negligible indicating small increases in the new cases ( march to april) and interval represented by a sudden upward shift in the graph as a result of a massive increase in the newly infected cases each day from april onwards ( figure ). the daily increase rate for interval was from to new cases, whereas the same rate for interval was from to , , , which is significantly higher than that of interval with a notable upward trend. again, this is due to the drastic change in daily new cases as represented by the changing direction of the slope from a small slope to a steeper one. generally, the death rate is proportional to the infection rate but at a smaller scale that varies from country to country, depending on many factors such as population, awareness, health care system, hospital building capacity, demographics, and location. as shown in figures - , the death rates for the world and each selected country depict a similar trend as that of the infection rate. for example, figures and show the world and the usa death rates, respectively where they are still increasing continuously without reaching a maximum value. in switzerland, however, the death rate exhibited a similar trend as its infection rate behavior (figure ). generally, the death rate is proportional to the infection rate but at a smaller scale that varies from country to country, depending on many factors such as population, awareness, health care system, hospital building capacity, demographics, and location. as shown in figures - , the death rates for the world and each selected country depict a similar trend as that of the infection rate. for example, figures and show the world and the usa death rates, respectively where they are still increasing continuously without reaching a maximum value. in switzerland, however, the death rate exhibited a similar trend as its infection rate behavior (figure ) . figure is plotted to show the total deaths of the selected countries so far, which shows that usa recorded the highest total deaths due to the covid- infection. however, it would not be accurate to use such data to directly compare the death rates between the countries. therefore, the next section is dedicated to calculate the death percentage for the selected countries and the world. figure is plotted to show the total deaths of the selected countries so far, which shows that usa recorded the highest total deaths due to the covid- infection. however, it would not be accurate to use such data to directly compare the death rates between the countries. therefore, the next section is dedicated to calculate the death percentage for the selected countries and the world. figure is plotted to show the total deaths of the selected countries so far, which shows that usa recorded the highest total deaths due to the covid- infection. however, it would not be accurate to use such data to directly compare the death rates between the countries. therefore, the next section is dedicated to calculate the death percentage for the selected countries and the world. a simple equation ( ) is used to calculate the percentage of the deaths for each selected country for the sake of direct comparison. a simple equation ( ) is used to calculate the percentage of the deaths for each selected country for the sake of direct comparison. since population (p) is proportional to the number of infected cases (inf), then: where a is the infected cases per capita, which is the infection percentage of the population. similarly, another factor b that relates the total deaths (d) to the total infected cases is described below: although, china has the largest population in the world, it had the least infection percentage of population (a) compared to the other selected countries (figure ). furthermore, the death percentage (b) of the selected countries up to this date varies between . % (brazil) to . % (ireland). the world's death percentage of . % is within the foregoing values ( figure ). figure shows the accumulated daily death percentages of the foregoing countries in comparison with the world death percentage, which shows that the world experienced a sharp increase in the total death cases after march until it reached a maximum of . % on april and decreased afterwards until this moment with . %. group b showed a similar trend to that of the world with slightly less death percentages. this is due to the decline of the daily deaths in comparison figure shows the accumulated daily death percentages of the foregoing countries in comparison with the world death percentage, which shows that the world experienced a sharp increase in the total death cases after march until it reached a maximum of . % on april and decreased afterwards until this moment with . %. group b showed a similar trend to that of the world with slightly less death percentages. this is due to the decline of the daily deaths in comparison with the continuous daily increase in the infected cases. group a, on the other hand, exhibited a similar behavior at the beginning until it reached a maximum and then levelled off as an indication of containing the outbreak (negligible new infected cases with zero deaths). as stated in the previous sections, the trend of covid- outbreak varies between countries at different levels (e.g., death rates, total infected cases, and containment). this section focuses on fitting the actual data of the selected countries into regression-based equations/models that help in understanding the covid- trajectory for a better projection. once an accurate model is developed (r > . ) for each case, it was used to project the future behavior of covid- to provide potential statistics. this will help in developing proactive action plans and the necessary strategic measures to contain such pandemics in the future too. starting with group a, selected countries such as switzerland and ireland, a good regression fit for both countries was obtained from the logistic model with r > . ( figure ) to fit the "s" shaped trend. the fitted model of total covid- cases (tcov) for both countries is expressed by equation we have not carried out a sensitivity test in the current study to investigate the effect of each parameter of the logistic model (m and k) on the infection rate because it is not of our interest at this stage. given that, they are generally defined as follows, m is the amount after growth and k is the constant of proportionality (continuous growth). the values of the coefficients for each country are listed in table . as stated in the previous sections, the trend of covid- outbreak varies between countries at different levels (e.g., death rates, total infected cases, and containment). this section focuses on fitting the actual data of the selected countries into regression-based equations/models that help in understanding the covid- trajectory for a better projection. once an accurate model is developed (r > . ) for each case, it was used to project the future behavior of covid- to provide potential statistics. this will help in developing proactive action plans and the necessary strategic measures to contain such pandemics in the future too. starting with group a, selected countries such as switzerland and ireland, a good regression fit for both countries was obtained from the logistic model with r > . ( figure ) to fit the "s" shaped trend. the fitted model of total covid- cases (tcov) for both countries is expressed by equation ( ): we have not carried out a sensitivity test in the current study to investigate the effect of each parameter of the logistic model (m and k) on the infection rate because it is not of our interest at this stage. given that, they are generally defined as follows, m is the amount after growth and k is the constant of proportionality (continuous growth). the values of the coefficients for each country are listed in table . the trajectory projection of group a consistently suggests that the pandemic will continue to be contained for all of the group a countries. the group b countries' trajectory is different from the group a countries, and therefore a different model was sought to fit such trend. the regression fit that best described the group b behavior is an exponential growth model with r ≥ . as expressed below: = ( ) where the values of the coefficients for each country in this group are listed in table . generally, the exponential growth is deceptive because it starts off slowly and after a few days it jumps to enormous numbers. unfortunately, this is what exactly happened to some of the countries during the covid- pandemic. a set of graphs in figure shows the current and future trajectory of brazil and india in group b. over the same timeframe, all countries experienced a very slow increase on each day and continued to do so for a few days. this means that the change in y-axis was close to zero (slope of the curve). however, after a few doublings the total daily covid- infected cases was increasing with a sharp slope recording higher changes of new cases (Δy) until it reached enormous numbers. the group b countries' trajectory is different from the group a countries, and therefore a different model was sought to fit such trend. the regression fit that best described the group b behavior is an exponential growth model with r ≥ . as expressed below: where the values of the coefficients for each country in this group are listed in table . generally, the exponential growth is deceptive because it starts off slowly and after a few days it jumps to enormous numbers. unfortunately, this is what exactly happened to some of the countries during the covid- pandemic. a set of graphs in figure shows the current and future trajectory of brazil and india in group b. over the same timeframe, all countries experienced a very slow increase on each day and continued to do so for a few days. this means that the change in y-axis was close to zero (slope of the curve). however, after a few doublings the total daily covid- infected cases was increasing with a sharp slope recording higher changes of new cases (∆y) until it reached enormous numbers. if this is not controlled sooner, the total recorded new cases of covid- for these countries will further double claiming more precious lives. for example, the total predicted total covid- infected cases for brazil, india, and usa by august is expected to be around , , , , , , and , , with the number of deaths expected to be equal to , , , , and , , , respectively. despite the fact that the usa is leading the world in the total recorded infected cases, brazil will slightly pass the usa by august as predicted by the model. this is due to the fact that the coefficient of the exponential term for brazil is higher suggesting that the exponential growth stage of brazil is severer than that of usa. having said that, the total deaths in the usa will continue to be higher than the rest of the countries. actual observations proofed that the future actual statistics would be less than the predicted values due to the implementation of physical measures such as social distancing, wearing masks, lock-down, disinfection, and travel restrictions. currently, there are many countries exhibiting a similar trend to group b that caused the world trajectory to follow an exponential growth model ( figure ) with r = . . the world's total covid- infected cases trajectory was suited more for the exponential growth model (equation ( )) in the second interval (after march). if the pandemic is not contained, the model (tcovw) predicts that the world's total new infected cases would reach , , by august with total deaths equal to , , . if this is not controlled sooner, the total recorded new cases of covid- for these countries will further double claiming more precious lives. for example, the total predicted total covid- infected cases for brazil, india, and usa by august is expected to be around , , , , , , and , , with the number of deaths expected to be equal to , , , , and , , , respectively. despite the fact that the usa is leading the world in the total recorded infected cases, brazil will slightly pass the usa by august as predicted by the model. this is due to the fact that the coefficient of the exponential term for brazil is higher suggesting that the exponential growth stage of brazil is severer than that of usa. having said that, the total deaths in the usa will continue to be higher than the rest of the countries. actual observations proofed that the future actual statistics would be less than the predicted values due to the implementation of physical measures such as social distancing, wearing masks, lock-down, disinfection, and travel restrictions. currently, there are many countries exhibiting a similar trend to group b that caused the world trajectory to follow an exponential growth model ( figure ) with r = . . the world's total covid- infected cases trajectory was suited more for the exponential growth model (equation ( )) in the second interval (after march). if the pandemic is not contained, the model (tcov w ) predicts that the world's total new infected cases would reach , , by august with total deaths equal to , , . if this is not controlled sooner, the total recorded new cases of covid- for these countries will further double claiming more precious lives. for example, the total predicted total covid- infected cases for brazil, india, and usa by august is expected to be around , , , , , , and , , with the number of deaths expected to be equal to , , , , and , , , respectively. despite the fact that the usa is leading the world in the total recorded infected cases, brazil will slightly pass the usa by august as predicted by the model. this is due to the fact that the coefficient of the exponential term for brazil is higher suggesting that the exponential growth stage of brazil is severer than that of usa. having said that, the total deaths in the usa will continue to be higher than the rest of the countries. actual observations proofed that the future actual statistics would be less than the predicted values due to the implementation of physical measures such as social distancing, wearing masks, lock-down, disinfection, and travel restrictions. currently, there are many countries exhibiting a similar trend to group b that caused the world trajectory to follow an exponential growth model ( figure ) with r = . . the world's total covid- infected cases trajectory was suited more for the exponential growth model (equation ( )) in the second interval (after march). if the pandemic is not contained, the model (tcovw) predicts that the world's total new infected cases would reach , , by august with total deaths equal to , , . this section considers fitting the actual data for the infection cases of the selected countries using sir modeling and obtaining the prediction results in terms of total number of infection cases and end of pandemic dates. the results given in table and figures - show that the countries in group a are at the end state of the pandemic, which indicates an outbreak control. all these countries already passed the expected % end of pandemic dates. the prediction curves for the countries in group b show that they have just passed the inflection point of the curve and started the deacceleration phase. this indicates that the outbreak is still uncontrolled, and it is expected that these countries reach the outbreak control sometime in august. the prediction curve shows that some of the group b countries, especially usa, went through several sub-waves of the disease spread. this section considers fitting the actual data for the infection cases of the selected countries using sir modeling and obtaining the prediction results in terms of total number of infection cases and end of pandemic dates. the results given in table and figures - show that the countries in group a are at the end state of the pandemic, which indicates an outbreak control. all these countries already passed the expected % end of pandemic dates. the prediction curves for the countries in group b show that they have just passed the inflection point of the curve and started the deacceleration phase. this indicates that the outbreak is still uncontrolled, and it is expected that these countries reach the outbreak control sometime in august. the prediction curve shows that some of the group b countries, especially usa, went through several sub-waves of the disease spread. this section considers fitting the actual data for the infection cases of the selected countries using sir modeling and obtaining the prediction results in terms of total number of infection cases and end of pandemic dates. the results given in table and figures - show that the countries in group a are at the end state of the pandemic, which indicates an outbreak control. all these countries already passed the expected % end of pandemic dates. the prediction curves for the countries in group b show that they have just passed the inflection point of the curve and started the deacceleration phase. this indicates that the outbreak is still uncontrolled, and it is expected that these countries reach the outbreak control sometime in august. the prediction curve shows that some of the group b countries, especially usa, went through several sub-waves of the disease spread. applying the previous studies on local data for the state of kuwait resulted in the trajectory depicted in figure . the actual data was fitted to an exponential growth model with an r > . as expressed in equation ( ) . one can clearly visualize that the slope of the curve has changed since the end of april indicating the start of the exponential growth second stage/interval ( ) as those in group b and the world. this suggests that the state of kuwait has done a commendable job in reacting right away with this covid- outbreak and took all the necessary measures to control it, which resulted in delaying stage as much as possible. with the current trend, the model predicted that the total infected cases in the state of kuwait by august would be about , . applying the previous studies on local data for the state of kuwait resulted in the trajectory depicted in figure . the actual data was fitted to an exponential growth model with an r > . as expressed in equation ( ) . one can clearly visualize that the slope of the curve has changed since the end of april indicating the start of the exponential growth second stage/interval ( ) as those in group b and the world. this suggests that the state of kuwait has done a commendable job in reacting right away with this covid- outbreak and took all the necessary measures to control it, which resulted in delaying stage as much as possible. with the current trend, the model predicted that the total infected cases in the state of kuwait by august would be about , . as shown in figure , the results for sir-modeling for the infection cases in kuwait show that kuwait went through two main sub-waves of the disease spread and passed the inflection point of the second sub-wave by the beginning of july. the model predicts the pandemic to end % by july and % by august . the total infected cases are predicted to be , , which might be more realistic than the corresponding result of the regression model. as shown in figure , the results for sir-modeling for the infection cases in kuwait show that kuwait went through two main sub-waves of the disease spread and passed the inflection point of the second sub-wave by the beginning of july. the model predicts the pandemic to end % by july and % by august . the total infected cases are predicted to be , , which might be more realistic than the corresponding result of the regression model. applying the previous studies on local data for the state of kuwait resulted in the trajectory depicted in figure . the actual data was fitted to an exponential growth model with an r > . as expressed in equation ( ) . one can clearly visualize that the slope of the curve has changed since the end of april indicating the start of the exponential growth second stage/interval ( ) as those in group b and the world. this suggests that the state of kuwait has done a commendable job in reacting right away with this covid- outbreak and took all the necessary measures to control it, which resulted in delaying stage as much as possible. with the current trend, the model predicted that the total infected cases in the state of kuwait by august would be about , . as shown in figure , the results for sir-modeling for the infection cases in kuwait show that kuwait went through two main sub-waves of the disease spread and passed the inflection point of the second sub-wave by the beginning of july. the model predicts the pandemic to end % by july and % by august . the total infected cases are predicted to be , , which might be more realistic than the corresponding result of the regression model. the main pathways of covid- spread are through respiratory droplets either coughing, speaking or sneezing, body fluid contact, or touching contaminated surfaces [ ] . the prevention/containment of the virus at healthcare settings is more important because it will not only ensure the safety of healthcare workers but will prevent the transmission and spread of the virus. it has also been reported that conventional face masks and ordinary clothing do not provide % protection [ ] . therefore, development of new strategies to prevent virus transmission through common pathways is critical. below we describe current and potential strategies of prevention. the virus filtering capability of masks depends on the design and materials they are made up of and on the size of the particulates. the current masks have limited ability to protect against aerosol and smaller droplets with surgical and n masks having the best protection. therefore, development of masks with antiviral capabilities can substantially reduce transmission of the virus. furthermore, current masks from fossil fuel based polymeric materials do not degrade and will create a potential pollution threat to the environment. therefore, biodegradable antimicrobial masks could be a great potential option. the contaminated surfaces are another contributor to the spread and covid- is reported to remain present on surfaces for several hours to days [ ] . the current surface cleaning and disinfection methods are not highly effective where a single wiping of the surfaces becomes dry within min and recovery of the bacteria and viruses is reportedly high [ ] . therefore, efforts should be made to new preventive measures for surface decontaminations. one such method is nano-coating, which can enhance the effectiveness up to several folds compared to current technologies. the emerging self-cleaning nano-coatings have a great future potential to prevent surfaces against such microbial threats. other countries should learn from those who preceded them in the present covid- pandemic. in general, most of the developed countries went through a tough time in dealing with this outbreak. the trajectory of the covid- pandemic, for some countries, went through three critical stages depicting a logistic behavior, as shown in figure . the deceptive stage ( ) from the start of the outbreak that varied from one week (in china) to about a month (in usa). then, it was followed by an uncontrolled exponential increase stage that lasted in some countries for about days (in china, switzerland, and ireland) and the rest of the countries unfortunately still experiencing it (usa, brazil, spain, and india). the third stage (containment stage) applies to the countries in group a that controlled the outbreak. the maximum impact of this covid- pandemic occurred during the uncontrolled stage ( ), which mainly depended on the deceptive stage ( ). the shorter the deceptive stage, the shorter the controlling stage and hence the less damage occurred, and vice versa. that is why the countries performance of group a was better than that of group b because they took serious measures right from the beginning of the covid- infections (short deceptive stage ) that resulted in a shorter and less steep uncontrolled stage followed by a controlled stage. amongst the group b countries and based on regression modeling, brazil could potentially lead the world in the total infected cases in the next few weeks if the circumstances remain the same. however, the sir-modeling results predict that the usa will continue leading the world in terms of the total infected cases. the state of kuwait covid- trajectory is similar to that of group b (uncontrolled). both regression and sir-modeling results lead to the same general conclusion that countries in group a reached the controlling stage, whereas countries in group b are still far away from reaching this stage. the maximum impact of this covid- pandemic occurred during the uncontrolled stage ( ), which mainly depended on the deceptive stage ( ). the shorter the deceptive stage, the shorter the controlling stage and hence the less damage occurred, and vice versa. that is why the countries performance of group a was better than that of group b because they took serious measures right from the beginning of the covid- infections (short deceptive stage ) that resulted in a shorter and less steep uncontrolled stage followed by a controlled stage. amongst the group b countries and based on regression modeling, brazil could potentially lead the world in the total infected cases in the next few weeks if the circumstances remain the same. however, the sir-modeling results predict that the usa will continue leading the world in terms of the total infected cases. the state of kuwait covid- trajectory is similar to that of group b (uncontrolled). both regression and sir-modeling results lead to the same general conclusion that countries in group a reached the controlling stage, whereas countries in group b are still far away from reaching this stage. what needs to be done is to limit the spread of the disease as much as possible. this results in delaying the second stage, and the sharp increase would happen over a longer period instead of on a daily basis that results in reducing the slope of the curve to be less steep ( figure ). this will spread the new cases over a longer period enabling the health care system to accommodate the existing patients instead of having enormous new cases in a short timeframe. this can be achieved by taking the right measures at the government and individual levels, such as quarantine, personal hygiene, lock-down, curfew, etc. this is what is happening now in some of the countries. what needs to be done is to limit the spread of the disease as much as possible. this results in delaying the second stage, and the sharp increase would happen over a longer period instead of on a daily basis that results in reducing the slope of the curve to be less steep ( figure ). this will spread the new cases over a longer period enabling the health care system to accommodate the existing patients instead of having enormous new cases in a short timeframe. this can be achieved by taking the right measures at the government and individual levels, such as quarantine, personal hygiene, lock-down, curfew, etc. this is what is happening now in some of the countries. all the selected countries in both groups recorded lower death percentages than that of the world during the outbreak timeframe. the exponent value (b) of the exponential growth model determines the severity (slope) of the covid- trajectory. that is why the usa is leading the world in the total infected new cases now and brazil may surpass the usa in the next few weeks if the severity of the exponential remains the same. since the model did not consider effects such as containment strategies and other related governmental interventions taken by countries, this limitation could cause slight inaccuracies in the results of the prediction models. all the selected countries in both groups recorded lower death percentages than that of the world during the outbreak timeframe. the exponent value (b) of the exponential growth model determines the severity (slope) of the covid- trajectory. that is why the usa is leading the world in the total infected new cases now and brazil may surpass the usa in the next few weeks if the severity of the exponential remains the same. since the model did not consider effects such as containment strategies and other related governmental interventions taken by countries, this limitation could cause slight inaccuracies in the results of the prediction models. emerging and re-emerging viruses in the era of globalization visualizing the history of pandemics, visual capitalist epidemics: past, present and future-what are the risks? from pasteur to genomics: progress and challenges in infectious diseases as ebola death toll rises, remembering history's worst epidemics covid- . coronavirus outbreak. world meters. . available online biology of microorganisms-microbiology an overview of their replication and pathogenesis structure, function, and evolution of coronavirus spike proteins lung pathology of fatal severe acute respiratory syndrome characterization and 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prediction of the epidemic peak of coronavirus disease in japan optimization method for forecasting confirmed cases of covid- in china short-term forecasts of the covid- epidemic in guangdong and zhejiang real-time estimation of the risk of death from novel coronavirus (covid- ) infection: inference using exported cases the mathematics of infectious diseases matlab central file exchange estimation of the final size of the coronavirus epidemic by the sir model a familial cluster of pneumonia associated with the novel coronavirus indicating person-to-person transmission: a study of a family cluster protecting health-care workers from subclinical coronavirus infection aerosol and surface stability of sars-cov- as compared with sars-cov- residual viral and bacterial contamination of surfaces after cleaning and disinfection this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license funding: this research received no external funding. the authors declare no conflict of interest. key: cord- - x oop g authors: alola, andrew adewale; alola, uju violet; sarkodie, samuel asumadu title: the ncovid- and financial stress in the usa: health is wealth date: - - journal: environ dev sustain doi: . /s - - -w sha: doc_id: cord_uid: x oop g since its first report in the usa on january , the novel coronavirus (ncovid- ) pandemic like in other previous epicentres in india, brazil, china, italy, spain, uk, and france has until now hampered economic activities and financial markets. to offer one of the first empirical insights into the economic/financial effect of the covid- pandemic, especially in the usa, this study utilized the daily frequency data for the period february – march . by employing the empirical markov switching regression approach and the compliments of cointegration techniques, the study establishes a two-state (stable and distressing) financial stress situation resulting from the effects of covid- daily deaths, covid- daily recovery, and the usa’ economic policy uncertainty. from the result, it is assertive that daily recovery from covid- eases financial stress, while the reported daily deaths from covid- further hamper financial stress in the country. moreover, the uncertainty of the usa’ economic policy has also cost the americans more financial stress and other socio-economic challenges. while the cure for covid- remains elusive, as a policy instrument, the usa and similar countries with high severity of covid- causalities may intensify and sustain the concerted efforts targeted at attaining a landmark recovery rate. with , , cases (consisting of over countries and territories) and accounting for the death of , persons globally as reported by the world health organization (who ) on september , the ravaging effect of the novel coronavirus disease has remained a global emergency. as of august , there were , , reported confirmed cases, , deaths, and , , recovered casescorresponding to , daily change in confirmed cases, daily change in deaths, and , daily change in recovery cases [see fig. ] (lauren ) . so far, the dreadfulness of the covid- pandemic has further irked global reflection and the imagination of the world's most devastating epidemic, the influenza pandemic or 'spanish flu' of - (stanford university . since the january , declaration of the covid- as a global emergency by who and the subsequent global determination to 'flatten the curve', a few outlined measures have been implemented. some of the measures that are currently in place or now gradually have been suspended across the governments include the 'lockdown', 'social distancing', travel restriction/suspension between countries, quarantine, and other measures differ across states (sarkodie and owusu a) . giving the implementation of these measures, most nations continue to experience a spillover effect of the covid- pandemic to the socio-economic aspects: the primary sectors, secondary sectors, and the tertiary sectors of the world economy (kostova et al. ; gregori et al. ; khalatbari-soltani et al. ; mason-d'croz et al. ; nicola et al. ) . to safeguard livelihoods and sustain economic development due to the global pandemic shocks, several economic activities instituted across countries include fiscal policy cut, exchange rate, and monetary intervention (sarkodie and owusu b) . importantly, the aspects of the world's economy, especially that of the usa, and other leading economies such as china, uk, germany, france, italy, japan, and spain, are fast becoming the shadow of its image. for instance, the usa (as the largest economy by the measure of gross domestic product, gdp), has suffered the worst unemployment crisis since the great depression. with , , reported cases, , human deaths from covid- , a record high of . % unemployment rate by april , and the massive oil price slump in the usa (financial times ; johns hopkins university and medicine ; united states bureau of labour statistics ), the effect of covid- fig. global covid- pandemic overview. data: lauren ( ) pandemic continues to hamper every aspect of the country's economy. in response to the usa' west texas intermediate (wti), record oil price collapse (falling below $) amidst an increasing decline in demand arising from the covid- pandemic, the country's government has twice provided stimulus packages to alleviate the financial distress among the americans. the stimulus package as contained in the s. -cares act is expected to help businesses, families, and the individuals affected by the coronavirus pandemic with emergency assistance and health care response (united states congress ), thus alleviating financial stress. notwithstanding, these efforts of the us government are insufficient at addressing the argument that has consistently corroborated the link between high infection and mortality of covid- among the african american and latino population with the socio-economic disadvantage of these racial groups (abedi et al. ; de león-martínez et al. ). considering the above motivation, this concise study is billed at illustrating the financial stress consequence of the ravaging covid- pandemic in the usa. giving the almost unpredictability of the later cases of covid- deaths and recoveries as against that of the total covid- cases and deaths as illustrated in fig. , the current study seeks to establish a link between the health emergencies arising from covid- and financial stress. giving that only a sparse study such as nicola et al ( ) has explored the economic aspects of covid- , the novelty of the current study is expected to close the existing gap in the literature through the following approaches: (i) the examining of the impact of daily deaths from covid- , daily recoveries from covid- , and economic uncertainty on financial stress, (ii) illustrating with empirical evidence of the states of financial stress with the regime switching approach of markov-switch regression technique, and (iii) the use of daily frequency and quite up-to-date data spanning the period february - march . with the aforementioned novel approaches, this study is capable of further asserting that indeed 'health is wealth'. in the succeeding part of this study, the material employed, theoretical concept, the discussion of the results, and the conclusion of the study are all outlined orderly in sections two, section three, section four, and section five, respectively. in the usa and across major economies of the world, nothing else could be imagined possessing the potential of disrupting the normal economic activities and financial markets like the covid- pandemic. having caused the closure or suspension of international air travels, disruption of energy production and especially the oil prices, disruption of agricultural activities, closure of school activities at all levels and other disruptions, the daily deaths and recoveries from covid- in the usa have since compounded the americans financial woes. the resulting disruption of normal economic activities and financial markets is akin to the country's prevailing financial stress (hakkio and keeton ) . thus, in examining the probable financial stress experienced in the usa due to the covid- pandemic, the following materials are employed: • since the first reported (three) cases of ncovid- in the usa by the cdc ( ), the data for the daily deaths (dd) and recoveries (rc) from the ncovid- disease are provided by the johns hopkins university and medicine ( ). • the indexes of financial stress and economic policy uncertainty of the us economic policy uncertainty are used as proxies to capture financial stress (fs) and economic policy (eu), respectively. because of restricted data availability, the series is considered for the daily frequency period of february - march . consequently, the method employed in the study is based on the preliminary findings of the statistical evidence of correlation (see table ) among the aforementioned factors and the fig. the line plot for covid- for daily cases, daily deaths, total cases, total deaths, and daily recoveries in the usa. (data are computed from johns hopkins university and medicine and centers for diseases control and prevention (cdc ) individual statistical properties (see table ). importantly, the data show the high variance of death and recoveries from covid- as well as the economic uncertainty. nicola et al ( ) provided a foundation for the economic and financial perspective of ncovid- . however, park and mercado ( ) illustrated the empirical relationship between financial stress and economic uncertainty. by extending this concept, the financial stress (fs) effect of ncovid- amidst economic uncertainty according to nicola et al ( ) can be illustrated in an ordinary least square (ols) framework as: but, by incorporates the switching parameter as regressors, the approach of markov switching regression from the work of hamilton ( ) and reboredo ( ) is applied to the current concept through the following: given that for all ε i,t ~ n ( , σst ), the variance of the error is where i is the ( ) daily period starting from february to march for usa' covid- daily deaths (dd), covid- daily recoveries (rc), daily economic uncertainty (eu). therefore, the impact of dd, rc, and eu on financial stress (fs) is, respectively, ,i,rt , ,i,rt , ,i,rt . a latent unobserved state variable st is the two regimes and that represents a more stable and a distressing financial stress regime, respectively. in this context, the transition probability of the estimation matrix is given as: and, given the dynamic nature of the dd, rc, and eu, especially in the current circumstance of covid- in the usa, the aforementioned probabilities of the transitioning states are given as: from eq. , the parameters x and x , u and u in addition to v and v , respectively, determine the significant impact of dd, rc, and the eu. the changes in dd, rc, and eu make the financial stress more likely to remain in state (stability) and in state (unstable/ troubling) depending on the values of p and p in eq. a, b. other details regarding this approach are outlined in hamilton ( ) , but the estimation results and states' probability diagrams are illustrated in table and fig. , respectively. additionally, to further establish the relationship between coronavirus and financial stress in the usa, the dd, rc, eu, and fs are further explored through the empirical models of fmols (fully modified ordinary least square, see phillips and hansen ) , ccr (canonical cointegration regression, see park ) , and the ardl (autoregressive distributed lag). the use of these methodologies, especially the ardl (see pesaran et al. ) , is justified by the small sample size of the dataset. also, the step-to-step of the methods are not highlighted in this study. however, by employing eviews statistical software for the estimation, the series of output generated is summarized as results and diagnostics check are both presented in the lower part of table in addition to fig. . ( ) being a deadly disease and without a known cure or definite treatment, every recovery from covid- is a significant scientific/medical achievement as well as a life-saving experience. with about % recovery rate from covid- in the usa against the country's about % death rate, the news of recoveries is certainly encouraging. indicatively, the empirical results of both states (stable and distressing) financial stress in table show that the daily recoveries have a negative and significant impact of financial stress. this implies that a report of higher (increasing) recoveries from covid- is responsible for minimal financial stress. thus, the implication is that human lives are not only saved from the casualties of covid- , but the timely recovery of infected individuals also allays the potential burden of financial incapacitation of the sufferer and the vulnerable population. moreover, the results from the fmols, ccr, and ardl as implied in the lower part of table further affirm the desirable impact of recoveries on financial stress in the usa. interestingly, this evidence confirms the statistical inference that put covid- recovery rate as almost four ( ) times the covid- death rate in the usa. with , american deaths from the covid- pandemic, a new daily death record of as on may implies that death rate from the number of confirmed total cases is a little above % (johns hopkins university and medicine ). previous work reported that an increase in confirmed cases of covid- increases death rate by . - . % daily (owusu and asumadu ) . from the result, it is found that daily deaths from covid- have not only been a source of bereavement to the americans, but it has also remained a significant source of financial stress to the people. implicatively, for every increase in the number of deaths per day by , financial stress increases by . and . in a more stable financial stress situation and a more distressing financial stress, respectively. besides, the result from other estimation methods (the fmols, ccr, and ardl) further complements the aforementioned evidence. similar to the impact of covid- daily deaths on the americans' financial stress, the degree of uncertainty in the country's economic policy is a significant determinant of financial stress. as seen in table , for every unit increase in the level of economic uncertainty in the usa, there is a significant increase of . and . in the level of financial stress in a more stable state and distressing state, respectively. in a more assertive perspective, the results from the fmols, ccr, and ardl estimation techniques further corroborate that economic uncertainty has a negative influence on financial stress in the usa. this illustrated evidence of economic uncertainty-financial stress nexus is closely supported by the previous studies that have hinted on the economic impact of infectious diseases (keogh-brown et al. ; nicola et al. ). several diagnostic checks were performed to further provide an assertive stance for the estimated n-covid- model. regarding the situations of the different (stable and distressing) states, the result shows that a change from more stable to distressing state is likely to happen about every . days while it takes about . days before transitioning from a more distressing to a stable state. indicatively, there are . probability of having an enduring stable state and a . probability of switching from a stable state to distressing state. similarly, a distressing state will endure with a probability of . and a . chance of transitioning from a distressing to a stable state (illustrated in fig. ) . importantly, giving the ardl diagnostic results, the estimated model is free from experimental/ table ) that could have compromised the result in addition to the evidence of stability from fig. . until now, and since the end of the great depression of the s, the usa has not experienced the level of economic devastation arising from the ncovid- pandemic. with the current speculated unemployment rate heading to % and two stimulus packages already handed out to the americans, the impact of the virulent disease on the usa' economic activities and financial market cannot be less devastating. in this context, the current study examined how the daily deaths and daily recoveries from covid- in addition to the uncertainty in the us economic policy have impacted the americans' financial stress in a different dimension. the startling finding revealed that covid- daily recoveries will cause a significant relief to the us health care system and decline potential pressure on the financial and socio-economic means of the americans. however, daily deaths from covid- pandemic, reproductive rate of covid- cases and uncertainty fig. the stability evidence from the ardl approach: a cusum, b cusum of square of government's economic policy are two significant factors with the potential of plunging the usa into a state of financial stress. this study revealed the existence of two states of stable and more distressing financial stress with a certainty of . (lasting . days) and . (lasting . days). importantly, the result of this investigation has not provided an interesting perspective without an apparent policy direction. foremost, considering that no known cure has been proffered for covid- yet, a more concerted effort should be directed at improving the treatment of infected patients to overwhelming increase the recovery rate of covid- sufferers. thus, by increasing the recovery rate, the financial stress among americans will be significantly mitigated. moreso, the direction of the country's current economic policy can be further reviewed in the context of the existing covid- pandemic such that a more workable country-specific policy that is capable of further minimizing the americans' financial stress is deployed retrospectively. moreover, future study could consider the impact of covid- across racial divide, especially in the context of regime switching. funding open access funding provided by nord university. data availability data for analysis in this study are included in this published article. conflict of interest the authors declare that they have no conflict of interest. open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/ . /. racial, economic, 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cases of novel coronavirus disease (covid- ) in china using dynamic statistical techniques canonical cointegrating regressions determinants of financial stress in emerging market economies bounds testing approaches to the analysis of level relationships statistical inference in instrumental variables regression with i ( ) processes. the review of economic studies nonlinear effects of oil shocks on stock returns: a markov-switching approach global assessment of environment, health and economic impact of the novel coronavirus (covid- ). environment, development and sustainability impact of meteorological factors on covid- pandemic: evidence from top countries with confirmed cases the influenza pandemic of economic news release s. -cares act coronavirus disease (covid- ) situation reports key: cord- -fio cjj authors: nan title: peripheral nerve society meeting july – , sitges, barcelona, spain date: - - journal: j peripher nerv syst doi: . /jns. sha: doc_id: cord_uid: fio cjj nan the peripheral nerve society was founded in from two groups of academic investigators, peripheral nerve study group and peripheral neuropathy association of america, interested in the basic biology and function of the peripheral nervous system and its application to the clinic. their invite only biennial meetings involved - attendees in cloistered settings organized by shoestring and local initiative. from this, we have grown remarkably. we now have an annual meeting of over people including meetings within the meeting for the special interest groups in inflammatory, diabetic and hereditary neuropathy. with this substantial growth and the success of jpns, the journal of the peripheral nervous system the society continues to flourish. has proven to be a year full of exciting changes for the society. pns has transitioned from a biannual, to an annual meeting. next year, the meeting will be taking place at the renaissance baltimore harborplace hotel from - july in baltimore, maryland. the development of a new website has been completed, please visit www.pnsociety.com to see the new face of the society. finally, pns has adopted new executive staff. with their guidance and the leadership of an active and diverse board of prominent professionals in the field the peripheral nerve society continues to grow and anticipates more exciting changes in the year to come. the peripheral nerve society provides annual meetings, teaching courses, guidelines, and other resources to aid in the education of members. becoming a member of pns means collaborating with prominent global professionals in the field to develop and provide the best treatments for people with peripheral nerve diseases and setting standards of care within the field. please participate in our future by joining the pns, volunteering for a project aligned with your interests and sending your ideas for the future to the executive office, or board member. peles e . department of molecular cell biology, rehovot, israel. two schwann cell-dependent mechanisms control the presence of na + channels at the nodes of ranvier: i. clustering of the nodal complex by glia-derived proteins and ii. restriction of nodal proteins within the nodal gap by the paranodal junctions. these mechanisms depend on specific cell adhesion molecules that mediate the contact between myelinating glia and their underlying axons at the forming nodes and the paranodal junction. during myelination, na + channels initially clustered at heminodes that border each myelin segment. this process requires gliomedin, nrcam and neurofascin (nf ), three cell adhesion molecules (cams) that mediate the interaction between schwann cell microvilli and the axon. na + channels clustering activity of gliomedin is tightly regulated by two distinct and functionally opposing proteolytic events. while the clustering activity of gliomedin is enhanced by its shedding from the surface of schwann cells by a furin protease, its activity is negatively regulated by bone morphogenetic protein /tolloid-like (bmp /tld), and tolloid-like (tll ) metalloproteinase. cleavage by these enzymes restricts the activity of gliomedin to the nodal area and prevents the formation of ectopic clusters along axons that are devoid of myelin segments, as well as below the myelin internodes. hence, proteolytic processing of gliomedin facilitates, yet limits, the clustering of na + channels to specific sites along the axon in a timely manner. furthermore, axon-glial contact mediated by gliomedin and nf at the nodes, not only plays a role in na + channel clustering during development, but also contributes to the long-term maintenance of na + channels at nodes of ranvier. in addition to clustering by gliomedin, the distribution of na + channels is restricted between two growing myelin segments by the flanking paranodal junction. at this site, axon-glia contact is mediated by a distinct set of cell adhesion molecules (i.e., caspr, nf and contactin) that also delineate the underlying axonal and glial cytoskeleton. this paranodal junction-dependent restriction of na + channels to the nodes is mediated by the spectrin-based paranodal axonal cytoskeleton. illa i . neuromuscular unit, neurology department, hospital santa creu i sant pau, universitat autònoma de barcelona, barcelona, spain. chronic inflammatory demyelinating polyradiculoneuropathy (cidp) is an autoimmune disorder of the peripheral nerves with clinical and immunological heterogeneity. currently, the diagnosis of cidp is based on clinical and electrophysiological criteria and does not take into consideration the presence of immune biomarkers. several autoantibodies against proteins of the node of ranvier in patients with cidp have now been described. these antibodies define specific cidp subtypes sometimes referred to as nodopathies and can have diagnostic and prognostic implications. anti-contactin (cntn ) antibodies. we have described the presence of antibodies to cntn in a small subset of patients with cidp. these patients shared a phenotype and have poor response to ivig. the anti-cntn antibodies are predominantly igg . pathological studies from skin and sural nerve biopsies of patients show morphological changes in the paranodes. experimental data supporting the pathogenicity of anti-cntn igg antibodies include: a) demonstration in vitro that the antibodies disrupt the binding of the cntn -caspr complex to neurofascin- (nf ); b) intraneural injections of antibodies progressively and specifically disrupt the paranodal axo-glial junction; and c) chronic infusion of antibodies induced clinical and electrophysiological worsening in animals with experimental autoimmune neuritis (ean). anti-nf antibodies. antibodies to neurofascins were first reported in patients with guillain-barré (gbs) and cidp and subsequently, antibodies specific to the nf isoform were found in a small group (< %) of patients with cidp. studies by us and confirmed by others have demonstrated that patients with cidp and anti-nf antibodies have a distinct phenotype that often includes a low-frequency tremor and poor responses to ivig. the autoantibodies are predominantly of the igg subtype. the passive transfer of monoclonal anti-neurofascin antibodies (which recognize all neurofascin isoforms) to mice with ean strongly exacerbated the severity of the pathology, but no studies have yet demonstrated that patient-derived anti-nf igg antibodies are pathogenic. a pathogenic role of the antibodies is however supported by sural nerve biopsies from patients with cidp and anti-nf antibodies that showed paranodal demyelination in the absence of inflammation, the loss of septate-like junctions and, the interposition of cellular processes between the paranodal loops and the axolemma. these alterations are reminiscent of those found in nfasc-null mice suggesting that anti-nf antibodies may specifically disrupt the nf -cntn -caspr complex at the paranodes. antibodies to other nodal proteins. recently neurofascin- and neurofascin- were reported as the main targets of autoantibodies in five patients with igg reactivity against the nodes of ranvier; the antibodies were predominantly igg . these patients presented with clinical features distinct from those in patients with anti-nf igg antibodies. four of these patients had subacute onset of sensory ataxia without tremor. the presence of anti-caspr antibodies has been reported in two patients with inflammatory neuropathies, one classified as cidp, the other as gbs. both patients had intense neuropathic pain. the skin biopsy from both patients showed paranodal disruption. some patients whose sera show nodal or paranodal reactivity in teased nerve fiber preparations have antibodies against other nodal proteins, such as gliomedin or neuronal cell adhesion molecule (nrcam) . the skin is equipped with specialized mechanoreceptors that allow the perception of the slightest brush. indeed some mechanoreceptors can detect even nanometer-scale movements. movement is transformed into electrical signals via the gating of mechanically-activated ion channels at sensory endings in the skin. the sensitivity of piezo mechanically-gated ion channels are controlled by stomatin-like protein- (stoml ), which is required for normal mechanoreceptor function. under pathophysiological conditions following nerve injury or diabetic neuropathy the slightest touch can produce pain. it is at present unclear whether peripheral changes in sensory mechanotransduction may underlie hypersensitivity associated with neuropathic pain. here we have examined the role of the stoml modulation of piezo channels in mechanoreceptors and nociceptors to under pathophysiological conditions. we recently developed small molecules that act as inhibitors of stoml function. peripheral application of stoml inhibitors can alleviate hypersensitivity in models of neuropathic pain. our data strongly suggest that tactile evoked pain in models of peripheral neuropathy may be at least partly driven by sensitization of sensory mechanotransduction driven by stoml . coleman m . john van geest centre for brain repair, cambridge, uk. axons are lost early in many neurodegenerative disorders of peripheral and central nervous system. the degeneration of transected axons (wallerian degeneration) can be slowed tenfold by overexpression of a variety of nad-synthesizing enzymes, such as isoforms of nmnat or the related mutant fusion protein, wld s . wallerian degeneration is also delayed by deletion of tlr adapter protein sarm , a protein recently reported to promote nad degradation. it is important to understand fully the mechanism of wallerian degeneration because related mechanisms contribute to axon loss in a number of disease models, including models of peripheral neuropathies, parkinson's disease, multiple sclerosis and glaucoma. new data also suggest a role in hereditary spastic paraplegia. while depletion of nad is an attractive hypothesis for the mechanism of wallerian degeneration, especially as nad can be increased by dietary methods, it cannot explain a number of key observations. fk , an inhibitor of nampt, blocks the nad salvage pathway and strongly depletes nad, including within axons. however, instead of killing axons as the nad hypothesis would predict, it does precisely the opposite: it phenocopies the protective effect of wld s . moreover, ectopic expression of the bacterial enzyme nmn deamidase, a protein absent in mammals, protects injured axons both in transgenic mice and in primary neuronal cultures, but it has no effect on nad levels either under basal conditions or in degenerating nerves. these observations fit better with a proposed toxic role for the nad synthesis intermediate nmn, a model that can also explain the protective effect of wld s . a full understanding of the pathway should identify a number of points where intervention could be a treatment for multiple axonopathies. as with any medical discipline, expansoin of knowledge about the fundamental science behing a disorder of the human nervous system comes part and parcel with a change in our understanding of the epidemiology of any given disorder or groups of disorders. recent advances in our fundamental understanding of inflammatory neuropathies of the peripheral nervous system have been accompanied by drastic changes in our understanding of the neuroepidemiology of these disorders -the specific populationss affected by peripheral neuropathies, as well as the varying importance / contributions of select peripheral neuropathies to the overall burden of peripheral nervous system (pns) disease, and how this shift in epidemiological understanding influences the clinical approach to diagnosis and management of patients with pns disease. the past few decades have witnessed a paradigm shift in many aspects of pns disease diagnosis and treatment; from the association of human immunodeficiency virus (hiv)-associated neuropathies; to the increassing recognition of hereditary / familial peripheral neuropathies; to the increased recognition of specific neuropathies such as multifocal motor neuropathy with conduction block. in addition, timely events such as the recent, and increasingly irrefutalbe evidence for a link between zika virus and a guillain-barré syndrome, and the rather unexpected resurgence of peripheral neuropathies due to previously 'exotic' etiologies such as lepromatous neuropathy require prompt clinical attention. this plenary session aims to describe the evolving neuroepidemiology of peripheral nervous system disorders, and how these changes may influnece the clinical approach to the diagnosis, prognostication, and treatment of otherwise 'unusual' periphal nerve diseases. oxaliplatin chemotherapy for colorectal cancer is seriously limited by neurotoxic side effects which are not fully understood. oxaliplatin-induced peripheral neurotoxicity (oipn) comprises an acute syndrome and a chronic sensory neuropathy. the acute symptoms, notably cold hyperalgesia, have been attributed to transient ion channel dysfunction, and the worse they are the more severe the chronic neuropathy that ensues. we designed a combined in vitro and in vivo project, using neurophysiology to better understand the pathogenesis of oipn. in the in vitro study, differentiated f cells (rat drg neurons x mouse neuroblastoma n tg- cell line) were incubated for and hours in . m oxaliplatin, and their electrophysiological properties studied by patch-clamp. the treated f cells showed relatively depolarized resting membrane potentials, significantly decreased firing frequencies, and increased sodium current densities. moreover, a decrease in erg (ether-à-go-go-related gene) potassium current was also evident. in the in vivo study, we applied nerve excitability testing (net) to a wistar rat model of oipn. to investigate the acute syndrome, we compared behavioural and neurophysiological data of animal cohorts (controls and oipn rats, n= each) before and after oxaliplatin administration ( mg/kg, iv). twenty-four hours after the injection we observed differences between the groups in behaviour (cold plate test, p= . ) and in superxcitability of motor axons (p= . ). to investigate the chronic neuropathy, we compare a control group (n= ) with a treated group (n= , oxaliplatin mg/kg twice weekly x weeks, iv). both groups are studied with behavioural, neurophysiological (sensory and motor nerve conduction studies, net), and pathological (caudal and sciatic nerve, skin biopsy, drgs) methods. data are collected at baseline, end of treatment and weeks after treatment; to obtain a full net profile of all significant changes. in this highly translational approach to oipn, the in vivo net changes in the acute and chronic rat models can be matched on the one hand to findings from in vitro experiments, and on the other to clinical data, since net is also easily applied in humans. of these results. in denmark there is a unique situation to conduct epidemiological studies facilitated by the danish civil registration and the danish national hospital registry (dnhr). this enables us to identify all gbs patients in denmark in a given period. from the same period as the igos cohort was included we have identified all gbs patients admitted to or seen in outpatient's clinics of hospitals in denmark (september st to december st ). records from the population based danish cohort will be reviewed for demographic and clinical data and compared to the patients included in igos from denmark, as well as with the igos europe/america cohort. during this period patients from denmark have been included in igos. the danish group is comparable to the europe/america group not counting the danish patients (n= ) of the igos cohort in regard to sex and age at entry, gbs disability score at nadir, and percentage of patients needing mechanical ventilation. in the danish igos group % are males, the median age is ( - ) years, the mean(sd) gbs disability score at nadir . ( . ), and % of the danish group needed mechanical ventilation. in the europe/america group % are males, the median age is ( - ), mean(sd) gbs disability score at nadir . ( . ) and % of the group needed mechanical ventilation. at the meeting we will compare and present data from the danish population based cohort as well as epidemiological data. chronic inflammatory demyelinating polyneuropathy (cidp) is a common autoimmune disease of the peripheral nervous system (pns) that causes sensorimotor impairment. mice with a dominant autoimmune regulator gene (aire) g w mutation on the non-obese diabetic (nod) background (nod.aire gw/+ mice) develop spontaneous autoimmune peripheral polyneuropathy (sapp) resembling cidp. in sapp, demyelination is caused primarily by th t cells; however, the contributions of nerve-resident cells such as schwann cells are poorly understood. we identified a population of non-hematopoietic, integrin alpha + (itga +) cells in the pns that increases in frequency and number during sapp. these itga + cells coexpress numerous schwann cell markers including sox , p , s b, myelin protein zero, and peripheral myelin protein , suggesting that itga + cells are schwann cell-like. additionally, during sapp, these itga + cells upregulate the extracellular matrix protein periostin (postn), which has recently been shown to promote macrophage recruitment and activation in inflammatory disease and cancer. our data indicate that macrophages are pathogenic during sapp. therefore, we hypothesized that itg a+ cells promote macrophage recruitment during sapp via postn production. to test this hypothesis, we performed in vitro chemotaxis assays. conditioned media from nod.aire gw/+ nerve promoted significantly more macrophage chemotaxis than conditioned media from postn −/− nerve. furthermore, postn recombinant protein was sufficient to induce macrophage chemotaxis in vitro. our findings show that itg a+ schwann cell-like cells mediate macrophage chemotaxis by upregulating postn during sapp and suggests postn as a novel target for the treatment of cidp. "wear-off" frequency will be analyzed by assessing the proportion of subjects with any given degree of gs and rods intracycle fluctuation and the proportion of cycles in which gs and r-ods fluctuation occurs. to determine the extent of "wear-off" the degree of difference between maximum and minimum gs, r-ods, tugs, onls, and vas scores will be analyzed. currently subjects from different sites have been enrolled ( sites eligible for enrollment). this interim study report will provide preliminary representative data, demonstrating ivig "wear-off" effects on gs and other outcome measures. by better understanding the frequency and extent of ivig treatment-related fluctuations we expect that these results will help facilitate development of cidp treatment optimization strategies. we also expect that this information will be important in forming hypotheses to be tested in future studies (for example, comparing different dosage intervals, optimal ivig taper guidelines, or assessing the long-term outcome of short-term cycle to cycle clinical fluctuations). neuropathic pain is a frequent feature of peripheral neuropathy causing a significant impact on patients' quality of life and health care costs. resolving the genetic architecture of painful neuropathy will lead to better disease management strategies, risk stratification, and counselling. therefore, we aim to develop a reliable technique to rapidly and accurately re-sequence multiple genes in a large cohort of painful neuropathy patients at low cost. whole exome sequencing of thousands of samples remains expensive for clinical use. several targeted enrichment approaches are currently available to selectively enrich for genomic regions of interest. in this study, we compared the sensitivity, specificity, targeting efficiency, reproducibility of performance and cost effectiveness of truseq ® custom amplicon-next generation sequencing (tsca-ngs) and molecular inversion probes-next generation sequencing (mips-ngs) methods. for both methods, we constructed a targeted enrichment kit to capture the coding and exon-flanking intron sequences of nine sodium channel genes (scn a, scn a-scn a, and scn b- b) expressed in nociceptive neurons. probes were designed for the two methods using their respective informatics pipelines. in total, patients with diabetic and idiopathic neuropathy were tested by both methods. among the patients, patients were tested previously by sanger sequencing for scn a-scn a. approximately kb was captured and sequenced. % of the targeted regions showed an average coverage of ≥ x in tsca-ngs, and % in mips-ngs. we managed to identify potential pathogenic mutations and polymorphism variants by mips-ngs and tsca-ngs. moreover, we observed a perfect agreement ( %) between sanger sequencing data and those obtained using mips-ngs and tsca-ngs. both ngs approaches showed user-friendly software to design probes and exhibited a similar on-target efficiency. although the overall coverage per region varied across different dna samples, it was sufficient to detect any variant in these regions. mips-ngs has more versatile assay design, demonstrated a high degree of flexibility with probes re-placement and > x cheaper than tsca-ngs. mips-ngs is a reliable, flexible, and inexpensive method to detect genetic variations in thousands of patients. in our centers, this technology is currently implemented as a routine diagnostic tool for screening of sodium channel genes in painful neuropathy patients. alonso-jiménez a , , belvis-nieto r , diaz-manera j , , illa i , , querol l , . neuromuscular diseases unit, neurology department, hospital de la santa creu i sant pau, universitat autònoma de barcelona, spain; centro para la investigación biomédica en red para enfermedades raras, ciberer, madrid, spain; neurology department, hospital universitario dexeus, barcelona, spain. most acute demyelinating polyneuropathies have an immune-mediated pathogenesis and are included within the guillain-barré syndrome spectrum. occasionally, other mechanisms such as metabolic, infectious or toxic may lead to gbs-like presentations. thermatrim ® and pura alegria ® are different brands of the same illegal slimming product that is sold through online vendors and in which the exact composition is unknown. here we present a patient with an acute demyelinating polyneuropathy secondary to the intake of the slimming product "pura alegría". a year-old woman with no remarkable medical history reported days history of distal numbness in her feet that progressed in one week to her knees and her left hand. she had had an upper respiratory tract infection ten days prior to these symptoms. the neurological examination showed absent distal vibratory and arthrokinetic sensations and arreflexia in lower limbs, decreased vibratory sensation in her hands and a ataxic gate. the lumbar puncture showed . g/l of proteins with no cells. the emg fulfilled diagnostic criteria for acquired demyelination. intravenous immunoglobulin therapy was started but the symptoms kept worsening and corticosteroids were started. the patient mentioned then the slimming product. a brain mri showed diffuse leukoencephalopathy that was asymptomatic. steroids and the pura alegria slimming products were withdrawn and the patient recovered completely after one year of follow-up. "pura alegría", "thermatrim" and "thermatrim plus" are slimming products that were forbidden in spain after several cases of acute leukoencephalopathy and acute polyneuropathy. exact composition is unknown although the spanish drug agency detected the pesticide malonoben, a tyrosin kinase inhibitor, among the components. since nine cases of pura alegria/thermatrim neurological toxicity, including leukoencephalopathy and polyneuropathy have been described. all cases had good outcomes after treatment withdrawal, although recovery is slow and may be incomplete. our case highlights the need to carefully consider drug toxicity, including dietary supplements, in the differential diagnosis of gbs specially when evolution does not follow typical patterns. alvarez s , klein d , martini r , moldovan m , , krarup c , . center for neuroscience, university of copenhagen, denmark; department of neurology, developmental neurobiology, university hospital würzburg, germany; department of clinical neurophysiology, rigshospitalet, copenhagen, denmark. charcot-marie-tooth neuropathy type a (cmt a) resulting from peripheral myelin protein kda (pmp ) overexpression is the most common hereditary motor and sensory neuropathy in humans. the transgenic pmp (pmp tg) mouse line c carrying copies of the human pmp gene, has a slowly progressing neuropathy phenotypically like cmt a with thin and abnormally thick myelin profiles and supernumerary schwann cells. in addition, pmp tg nerves showed activated macrophages leading to axon-myelin compartment disruption and maldistribution of k+ channels (kohl b et al, am j pathol. ; : ) . the aim of the present study was to investigate the motor axon excitability in pmp tg versus wt littermates. multiple measures of motor axon excitability under anesthesia were carried out by stimulation of the tibial nerve at ankle and "threshold-tracking" the plantar compound muscle action potential (cmap). at age months, when the post-developmental maturation was nearly complete in the wt, the pmp tg cmap showed an increase in latency by %. the cmap amplitude was decreased by %, although the mean motor unit size (mscan method) appeared unchanged indicating a lack of collateral sprouting. furthermore, pmp tg showed abnormalities in both passive cable properties and voltage dependent parameters. at age month, the cmap latency of pmp tg was increased by % as compared to wt. in contrast to this marked conduction slowing along the tibial nerve from to months of age, the progression of excitability changes localized at ankle appeared modest. nevertheless, when pooling data from to months, the increase in pmp tg latency was correlated (spearman p< . ) with an increase in accommodation half-time during depolarizing electrotonus (+ % of threshold) from to ms and a reduction of the late subexcitable period of the recovery cycle from to % of threshold, both changes consistent with a redistribution of k+ currents consistent with the maldistribution of k+ channels. our data suggest that in the pmp tg cmt a model, a functional, thus potentially reversible abnormality in k+ channel distribution, accumulates along the nerve and aggravates the conduction impairment due to impaired myelin formation and maintenance. alvarez s , krarup c , , moldovan m , . center for neuroscience, university of copenhagen, denmark; department of clinical neurophysiology, rigshospitalet, copenhagen, denmark. mice heterozygously deficient of myelin protein p gene (p +/−) show a mild progressive dysmyelinating neuropathy, with conduction slowing and impaired excitability, phenotypically similar with charcot-marie-tooth disease type b (cmt b). we found that in p +/− the accumulating myelin abnormalities were paralleled by progressive changes in voltage-dependent motor axon function resulting in neurotoxic membrane depolarization (rosberg mr, et. al. neurobiol dis. : ) . the aim of this study was to investigate the relationship between demyelination and motor axon function in p +/−. demyelination of the right sciatic nerve by topic lysophosphatidylcholine (lpc) application was carried out in p +/− and wild-type (wt) mice, in year (mature) and years (aged) groups. multiple measures of motor axon excitability under anesthesia were carried out by stimulation of the tibial nerve at ankle (distal to lpc demyelination) and "threshold-tracking" the plantar cmap responses. live imaging studies by cellvizio (mauna kea technologies, paris, france) confocal laser endomicroscopy were carried out in transgenic mice expressing the fluorescent reporter yfp in peripheral nerve axons under the thy promoter. in mature wt the sciatic morphological and electrophysiological demyelinating features following lpc could be readily observed at hours but disappeared by weeks. no morphological changes could be observed at the tibial level. consistently, no conduction or excitability changes could be observed at the right tibial neve level as compared to the left tibial nerve in wt. in contrast, in p +/− the motor axon function was impaired at the tibial nerve level at weeks after sciatic lpc demyelination. in mature p +/−, although the cmap amplitude appeared preserved, the distal motor latency was prolonged whereas the excitability measures showed reduced deviations during threshold electrotonus and increased refractoriness at the expense of superexcitability of the recovery cycle, both consistent with membrane depolarization. furthermore, in aged p +/− the delayed tibial conduction was associated with a drop in cmap amplitude and a prolongation of the strength-duration time constant. taken together these data suggest that focal demyelination aggravates membrane dysfunction along the entire motor axon in p +/− providing a novel experimental model to explore the link between demyelination and axonal membrane dysfunction in cmt b. amass l , li h , gundapaneni b , schwartz j , keohane d . pfizer inc., new york, ny, usa; inventiv health inc., burlington, ma, usa. a number of factors can influence disease progression in transthyretin familial amyloid polyneuropathy (ttr-fap), a rare, fatal, hereditary amyloidosis. this analysis evaluated the specific role of baseline neurologic severity on neurologic disease progression in ttr-fap. a predictive model was created based on longitudinal data from val met patients who participated in the tafamidis (a selective ttr stabilizer) clinical development program. data from the intent-to-treat population of the double-blind, placebo-controlled registration study (tafamidis group, n= ; placebo group n= ) and its two consecutive open-label extension studies in which all patients received tafamidis were used. the second extension study is ongoing, but a formal, prospectively-planned interim analysis was conducted with the cut-off date of december , . this analysis focused on the first months of treatment for the overall study cohort analyzed. the neuropathy impairment score-lower limbs (nis-ll) was used to assess neurologic functioning at baseline and at subsequent study visits. a linear mixed-effects model for repeated-measures (mmrm) analysis, with baseline nis-ll, treatment, and their interactions with time as fixed effects, was used, and the slope and intercept for each patient were included as random effects. patients were primarily caucasian with early-stage neurologic disease (baseline nis-ll mean [standard deviation]: tafamidis, . [ . ]; placebo, . [ . ] ). across both groups, disease progression increased with increasing levels of baseline severity (nis-ll) (p< . ). however, the predicted magnitude of change from baseline to month for tafamidis was consistently less than that for placebo across a range of observed baseline nis-ll values, suggesting a disease-modifying effect of tafamidis. similar findings were observed for the nis-ll muscle weakness subscale. this mmrm analysis in patients with val met ttr-fap demonstrates that disease progression strongly depends on baseline neurologic impairment and highlights the disease-modifying effect of tafamidis across a range of baseline levels of neurologic severity. clinicaltrials.gov identifiers: nct , nct , nct . amino h , misawa s , sekiguchi y , shibuya k , watanabe k , suichi t , kuwabara s . department of neurology, chiba university, chiba, japan. guillain-barré syndrome (gbs) is a potential life threatening neurological disorder and respiratory insufficiency is one of the critical complications. eramus gbs respiratory insufficiency score (egris) is a method for predicting the chance of respiratory insufficiency in gbs. however, clinical characteristics and courses can vary for subtypes of gbs, whose occurrences differ for each region: acute inflammatory demyelinating polyneuropathy (aidp) is very common in european countries, whereas acute motor axonal neuropathy (aman) is frequently seen in asian countries. the aim of this study is to investigate the usefulness of egris in japan, where aman is more common than in the netherlands. clinical and electrophysiological profiles of consecutive gbs cases, who visited our hospital within days after symptoms onset between and , were reviewed. of the gbs patients, % were classified as aidp and % as aman according to the electrodiagnosis criteria by ho and colleagues. higher egris scores correspond to higher risk of respiratory insufficiency in total of the gbs patients, as well as in aidp patients. however, in patients with aman, egris scores did not always match the chances of respiratory insufficiency: up to % of the patients with low risk of egris showed respiratory failure, whereas only % of the patients with high risk of egris needs intubation/mechanical ventilation. in aman, associations with mechanical ventilation were seen for rapid progression (shorter duration between onset and hospital admission), more decreased vital capacity, and more frequent autonomic involvement. egris is useful also for japanese gbs patients. however, for aman patients, it should be used with discretion. another score to predict respiratory insufficiency might be required in asian countries. anandan c , litchy wj , laughlin rs , leep hunderfund an , naddaf e . mayo clinic, rochester, usa. in patients with suspected ulnar neuropathy, nerve conduction studies (ncs) are commonly requested to help with diagnosis and localization. however, routine ncs are often normal or not localizing. ulnar ncs recording from the first dorsal interosseous muscle (ncs-fdi) is thought to increase the diagnostic yield of electrodiagnostic testing, although not commonly considered. we developed a quality improvement strategy to routinely perform ulnar ncs recording from the abductor digiti minimi muscle (ncs-adm) as well as ulnar ncs-fdi in all patients referred for suspected ulnar neuropathy. we utilized the dmaic (define, measure, analyze, improve, control) model of process improvement to define our problem and create a map of the current process for ulnar neuropathy diagnosis in our electromyography laboratory. we determined baseline performance via review of distal sensorimotor polyneuropathy (dpn) is the most common complication of diabetes and risk factors beyond hyperglycemia have proven important particularly in type diabetes (t dm). only few prospective studies from early-stage t dm exist. we aimed to study the development of dpn during the first years after a screening-based diagnosis of t dm. from the addition-denmark study participants were eligible for this study. dpn was assessed by the michigan neuropathy screening instrument questionnaire (mnsi) at four time-points during follow-up. dpn was defined by a mnsi score ≥ . participants ( %) were positive in mnsi at baseline and thus excluded from this study. by kaplan-meier plot we evaluated the cumulative incidence of dpn and in cox proportional hazard models we calculated hazard ratios (hr) for the intervention groups in the addition trial and for various covariates proposed to influence the development of dpn. models were adjusted in steps for intervention group, age, sex, baseline mnsi, lipid-lowering and anti-hypertensive treatment. this study cohort consists of participants ( % men) with a median age of . years (p ;p : . ; . ) and median baseline hba c of . (p ;p : . ; . ). a cumulative incidence of % was seen during years of diabetes. there was no statistically significant difference in hr between the intervention groups or by sex but a significantly higher hr of . ( %ci: . ; . ) was seen for age (per year). the highest hr was found for a history of cardiovascular disease (myocardial infarction or stroke) up to ten years prior to the diabetes diagnosis with a hr of . ( . ; . ). weight, waist circumference, body-mass index and methylglyoxal (log transformed) showed modest but statistically significant associations with incident dpn with standardized hrs of . ( %ci: . ; . ), . ( %ci: . ; . ), . ( %ci: . ; . ) and . ( %ci: . ; . ) respectively. this study demonstrates a fairly low cumulative incidence of dpn in people with screen-detected t dm and provides evidence that macrovascular disease, obesity and oxidative stress are important risk factors for dpn even at the earliest stages of t dm. andermann syndrome, also known as agenesis of the corpus callosum and peripheral neuropathy (accpn), is an autosomal recessive disorder with a broad spectrum of mild to severe neuromuscular and psychiatric consequences. the gene variants causing disease were first identified in french-canadian families. in the present study, we intended to phenotype and genotype a series of non-french-canadian familial cases presenting with charcot marie tooth disorder associated with agenesis/dysgenesis of the corpus callosum. for this purpose, seven families, of consanguineous marriage, were studied. patients were clinically and para-clinically investigated using mri and electrophysiology (mncvs). for some, a sural nerve biopsy was taken. microsatellite markers around the accpn locus were used in two large families; followed by sanger sequencing of all the exons and intron-exons boundaries of the gene, in one patient from each of the families. the age at onset of the disease was at birth in the patients from the largest consanguineous family ( affected individuals). the biopsy from one patient showed a severe demyelinating neuropathy with many hypomyelinated fibres and mostly secondary axonal changes. these finding were compatible with electrophsiological data where the mncvs are of demyelinating range. dysgenesis of the corpus callosum in one patient and agenesis in another sib were revealed by mri. we identified one homozygous truncating mutation in this family. interestingly, no causative variant was found in a patient from another family and showing homozygous haplotype. two different heterozygous variants were identified at one hit in two patients from two non-consanguineous families. genetic investigations will be continued to identify the possible second hit. in the remaining families, no variant was found. the negative family cases will be subjected to ngs. at this stage, it is tempting to speculate on the genetic heterogeneity of accpn in our series. baba m , suzuki c , ogawa c , tomiyama m . department of neurology; diabetes center, aomori prefectural central hospital, aomori, japan. in we introduced a staging system of severity of diabetic polyneuropathy (dpn) by nerve conduction study (ncs) of the lower limb: sensory ncs of the sural nerve and motor ncs of the tibial nerve. the system consists of five stages; ncs- (normal): no abnormalities, ncs- (mildly abnormal): presence of delay of mcv, scv, minimal f-wave latency, or positive a-wave, ncs- (moderately abnormal): decrease in sural snap less than uv, ncs- (severely abnormal): decrease in plantar muscle-cmap to - mv, ncs- (ultimately abnormal): plantar muscle-cmap lost or less than mv with trace of sural-snap. to examine validity of the system, we conducted -year prospective observation on development of diabetic foot (df) by the ncs staging system. in addition, occurrence of ischemic heart disease (ihd) and stroke (is), and death of neuro-vascular events were also counted. n - , we carried out ncs in diabetics, and categorized them by the ncs staging system: % was ncs- , % was ncs- , another % was ncs- , % was ncs , and % was ncs- . we then followed them and prospectively counted the occurrence of df, ihd and is in patients (mean age ys). the occurrence of df during the following years was; ncs- : %, ncs- : %, ncs- : %, ncs- : %, ncs- : %. occurrence of any of df, ihd and/or is was as follows; ncs- : %, ncs- : %, ncs- : %, ncs- : %, ncs- %. there was no death from nsc- , − , and − groups (n= ), while two from ncs- group (n= ) were found dead in a bed or on a driver's seat, and other two from ncs- group (n= ) died of sudden cardiac arrest or infection after foot amputation. in summary, the present ncs grading system seems to work satisfactory not only for diagnosis of severity of the current dpn, but also for prognostic prediction of the dpn-related foot and vascular events. bacon c , feely sme , shy me . university of iowa hospital, iowa city, ia, usa. for patients with charcot-marie-tooth disease, also known as hereditary motor and sensory neuropathy, the rasch modified cmtnsv is a validated measurement of symptoms and impairment. the score is comprised of nine parameters of a clinical examination, including nerve conduction studies (ncs) . each parameter has an individual score ranging from zero to four, with the composite score having a maximum of prior to rasch modification. for patients who do not complete a ncs, the cmt exam score (cmtesv ) is used, with a maximum score of before rasch modification. one parameter of the cmtesv is the vibration sense. using a rydel tuning fork, a care-giver measures vibration sense in a patient's feet, ankles, and knees and a score is determined by the severity of reduced vibration sense. in cmt a patients, % received a score of out of , noted by a reduced vibration sense at the knee. in order to capture a more sensitive vibration measurement, we tested ways of taking the total raw score of the rydel tuning fork at each point of vibration sense, the toe, ankle, and knee bilaterally. scores range from zero to , with reflecting full vibration sense. in this modified measurement, vibration sense scores varied in wider distribution. for cmt a patients captured in this studied, a modified vibration sense score results in a potentially more sensitive total cmtesv score. bae dw , an jy . st. vincent's hospital, the catholic university of korea, suwon, korea. diabetic lumbosacral radiculoplexus neuropathies (dlrpn) are usually subacute painful, monophasic, asymmetrical lower limb neuropathies with incomplete recovery due to ischaemic injury and microvasculitis. the diagnosis relies mostly on clinical suspicion and characteristic electromyographic findings. however, in acute phase, neuroimaging has more important diagnostic significance than electrophysiological studies. here we describe mri and ultrasound findings in a -year-old woman with dlrpn who was diagnosed with diabetes three years ago, but has not received any other treatment. she had a -day history of acute-onset severe pain with weakness of muscles innervated by left femoral and obturator nerve and decreased sensation in left l -l dermatome. nerve conduction studies showed reduced amplitude in left femoral nerve and electromyography showed only increased insertional activity in left iliopsoas muscle and no volitional activity in muscles innervated by left femoral and obturator nerve. ultrasound revealed increased cross-sectional area (csa) of left femoral and lateral femoral cutaneous nerve. mri showed enhancement in left l , nerve roots and proximal femoral nerve and increased signal intensity in left iliacus and iliopsoas. we diagnosed her with dlrpn and started corticosteroid. nerve ultrasound has not been previously reported in a patient with cabin air in commercial airliners originates from aircraft engines or auxiliary power units. this bleed air may occasionally be contaminated with hydraulic fluids and engine oil that contains a number of potentially hazardous chemicals including tricresyl phosphate (tcp). over the last years reports are emerging about aircrew members that experience symptoms such as tingling or burning of extremities in addition to headache, and vertigo. this "aerotoxic syndrome" is controversially discussed in the literature and has been attributed to exposure to organophosphate contaminated cabin air. since tcp has been associated with peripheral neuropathy we aimed to determine the frequency of peripheral neuropathy among frequent flyers. civilian air crew members and frequent flying passengers (m:f = : , median age years, median exposition time in aircrafts of . hours) were examined at the university hospital of cologne or at the frankfurt airport (iata code fra) by a detailed questionnaire of past medical conditions, a standardized neurological examination and nerve conduction studies of sural, tibial, and ulnar nerves. we identified subjects with clinical and electrophysiological evidence for large fiber peripheral neuropathy. incidence of peripheral neuropathy was not correlated to exposition time in aircrafts. in addition subjects showed signs of ulnar neuropathy, subjects reported abnormal vibration sensation, subjects suffered from gait imbalance and individuals reported tingling of extremities. our study shows a . % prevalence of large fiber peripheral neuropathy among frequent flyers. comparison of these data with prevalence rate in an age-matched control group will reveal a possible association of chronic exposure to cabin air and risk for peripheral neuropathy. the high incidence of the symptom tingling in our cohort warrants further studies to determine the risk for small fiber neuropathy in this condition. intravenous immunoglobulin (ivig) are human igg derived from plasma pools of healthy donors. although there are studies in literature evaluating their effectiveness in different pathological animal models, there are no data about their possible role on bortezomib (btz)-induced peripheral neuropathies. female wistar rats were treated following a preventive schedule (btz and ivig co-treatment for and weeks) and a therapeutic schedule ( weeks of btz treatment followed by a -week ivig-btz co-treatment). caudal nerve conduction velocity (ncv), plantar and dynamic tests were performed at different time points. animals were sacrificed after ws (acute phase) or ws (chronic phase) and tissue samples (dorsal root ganglias -drg-, sciatic nerve, caudal nerve, skin) were collected for morphological, morphometrical and immunohistological analysis.in the preventive schedule, ivig was not able to rescue caudal ncv reduction caused by btz neither after nor after weeks of co-treatment. same results were observed in the therapeutic schedule. on the other hand, the evaluation of mechanical allodynia and cold hyperalgesia showed that ivig injection protected from btz effect in both treatment schedules. morphometric analysis evidenced that, even if not statistically significant only the preventive schedule has a tendency to protect the caudal nerve from btz damage. this result is consistent with the morphological evaluation of the nerve. also, intra-epidermal nerve fibers density was preserved in the preventive schedule but not in the therapeutic one. finally, sciatic nerve and drg macrophage infiltration levels tended to be reduced in the therapeutic schedule and were brought back to ctrl (rats not treated or injected with ivig alone) levels in the preventive one. in conclusion, we were able to demonstrate for the first time that ivig treatment especially used as preventive treatment option may reduce btz-induced neuropathic painful pointing out the possible role of inflammation in the pathogenesis of this invalidating pathology. this work was supported by kedrion spa. we studied the prevalence, the molecular cause and clinical presentation of hereditary motor neuropathies in a large cohort of patients from the north of england. detailed neurological and electrophysiological assessments and next generation panel testing or whole exome sequencing were performed in patients with clinical symptoms of distal hereditary motor neuropathy (dhmn, patients), axonal motor neuropathy (motor cmt , patients) or complex neurological disease predominantly affecting the motor nerves (hmn plus, patients) . the prevalence of dhmn is . affected individuals per . inhabitants ( % confidence interval: . - . ) in the north of england. causative mutations were identified in out of index patients ( . %). the diagnostic rate in the dhmn subgroup was . %, which is higher than previously reported ( %). we detected a defect of neuromuscular transmission in cases and identified potentially causative mutations in patients with demyelinating multifocal motor neuropathy. many of the genes were shared between dhmn and motor cmt , indicating identical disease mechanisms therefore we suggest changing the classification and include dhmn also as a subcategory of cmt. abnormal neuromuscular transmission in some genetic forms provides a treatable target to develop therapies. barohn rj , gajewski b , pasnoor m , brown l , herbelin l , kimminau k , jawdat o , parks c , shlemon p , dimachkie mm and the pain-controls study team . the university of kansas medical center, kansas city, ks, usa. cryptogenic sensory polyneuropathy (cspn) is a common slowly progressive neuropathy that affects adults and presents with significant neuropathic pain for which multiple medications have been tried including antiepileptics, antidepressants, topicals and narcotics. a web based survey among neuromuscular experts suggested pregabalin as being more effective than other medications, however there are presently no comparative studies to assess the most effective medication. the objective of this study was to determine which of the pharmaceutical therapies (pregabalin, duloxetine, nortriptyline or mexiletine) is most effective for neuropathic pain and best tolerated in cspn. to achieve this objective we performed a prospective randomized open labelled comparative effectiveness adaptive design study of cspn patients through the patient centered outcomes research institute (pcori). cspn patients who fulfilled the inclusion and exclusion criteria were enrolled into this study. patients underwent a baseline neurological evaluation and randomly assigned to one of the neuropathic medications for months. the primary outcome is the change in likert-like pain scale. the secondary outcomes included nih pain interference scale, nih fatigue interference scale, nih sleep disturbance scale, sf- and adverse events. the outcome measures are performed at baseline, month , and . statistical analysis using bayesian adaptive design developed by berry consultant software will be performed to determine winner and losers (winner = greater than point improvement in pain). total number of patients to be enrolled is . recruitment has been challenging and a number of recruitment techniques have been used. to date, there have been patients screened, patients randomized from us sites. anticipated completion of enrollment by june and end of final patient assessment by september . interim analysis performed after first patients completed their months as part of bayesian adaptive design analysis and occurs every weeks. the distribution of randomization of patients to the medications at last adaptive design randomization was . % to medication , % to medication , . % to medication and . % to the th medication. this study may give physicians and patients evidence for future management of cspn patients. transthyretin amyloidosis (attr), which encompasses a group of disorders with significant clinical variability, is caused by transthyretin (ttr) derived amyloid deposition. the clinical aspects of autonomic nervous system involvement in attr are only partially known. the ongoing, multinational, longitudinal, observational transthyretin amyloidosis outcomes survey (thaos) provides the opportunity to expand our understanding of dysautonomia in attr. data from all symptomatic subjects enrolled in the thaos registry with a diagnosis of attr (cut-off date: january , ) were assessed for the presence and temporal course of autonomic symptoms, genotype and phenotype associations, and clinical burden according to the frequency and severity of symptoms. of symptomatic subjects enrolled in thaos, ( . %) had autonomic symptoms at enrollment including: gastrointestinal ( subjects, . %), urinary ( , . %), erectile dysfunction ( , . %), orthostatic hypotension ( , . %), xerophthalmia ( , . %) and dyshydrosis ( , . %). subjects with autonomic manifestations, compared with those without, were younger (mean age [standard deviation, sd] of . [ . ] vs . [ . ] years), with a longer duration of attr symptoms ( . [ . ] vs . [ . ] years). autonomic dysfunction was less common with wild-type attr ( of subjects, . %) than in mutation groups: val met ( / , . %); non-val met/non-cardiac ( / , . %); and "cardiac mutations" (val ile, leu met, thr ala, or ile leu mutations; / , . %). similarly, time (mean [sd] , years) from first attr symptoms to onset of autonomic symptoms, was longest for wild-type attr ( . [ . ] ) followed by "cardiac mutations" ( . [ . ]), non-val met/non-cardiac ( . [ . ]), and val met ( . [ . ] ). autonomic symptoms were present at disease onset in over a third of subjects ( , . %) . autonomic dysfunction was less frequent in subjects with cardiac phenotype ( of subjects, . %), than with mixed ( / , . %) or neurologic ( / , . %) phenotypes. the burden of autonomic symptoms (mean [sd] ) varied by genotype, val met ( . [ . ] , non-val met/non-cardiac ( . [ . ], "cardiac mutations" ( . [ . ]), wild-type attr ( . [ . ]), and by phenotype, mixed ( . [ . ]), neurologic ( . [ . ]), cardiac ( . [ . ]). dysautonomia is common, and a significant burden, in subjects with hereditary forms of attr. its prevalence is higher in val met than in other genotypes, and in the neurologic or mixed phenotypes. the objective of this study is to assess the usefulness of motor unit number index (munix) technique in charcot-marie-tooth type a (cmt a) disease and to test correlation between munix and clinical impairment. munix technique was performed in abductor pollicis brevis (apb), abductor digiti minimi (adm) and tibialis anterior (ta) muscles in the non-dominant side. a munix sum score was calculated by adding munix of these muscles. muscle strength was measured using the mrc (medical research council) scale. disability was evaluated with several functional scales including cmt neuropathy score version (cmtnsv ) and overall neuropathy limitation scale (onls). cmt a patients with known pmp gene duplication were enrolled. the munix of the adm, apb and ta muscles were correlated with the mrc of the corresponding muscle (p< . ). munix sum score was correlated with clinical scales: cmtnsv (r=− . , p< . ), onls (r=− . , p< . ). in conclusion, munix correlates with muscle strength and clinical measurements of disability in cmt a patients. the munix technique evaluates motor axonal loss and correlates with disability. the munix sum score may be a useful outcome measure of disease progression in cmt a. the objective of this study was to assess the usefulness of mri in charcot-marie-tooth type a (cmt a) disease and to test correlation between muscle fat fraction and clinical impairment. mri was performed in the non-dominant lower limb of cmt a patients and healthy controls. fat fraction of tibialis anterior muscle, cross section area and volume of sciatic nerve were determined. muscle strength of dorsiflexion was measured using a dynamometer. disability was evaluated with cmt neuropathy score version (cmtnsv ). cmt a patients with known pmp gene duplication were enrolled. fat fraction of tibialis anterior muscle was significantly increased in patients compared to healthy controls. it was correlated with muscle strength (r=− . , p< . ) and cmtnsv score (r=− . , p< . ). cross section area and volume of sciatic nerve were significantly increased in patients compared to healthy controls. in conclusion mri fat fraction correlates with muscle strength and clinical measurement of disability in cmt a patients. it may thus be a useful outcome measure of disease progression in cmt a. basta i , peric s , cobeljic m , bjelica b , bozovic i , kacar a , nikolic a , rakocevic stojanovic v , stevic z , lavrnic d . neurology clinic, clinical center of serbia, school of medicine, university of belgrade, belgrade, serbia. there is a complete lack of data about epidemiological and clinical features of chronic inflammatory demyelinating polyneuropathy (cidp) in serbia and surrounding countries. furthermore, there is a striking scarcity of information about quality of life (qol) in cidp patients and all qol studies have been conducted in countries with high standards of health care. in august we have designed the iness (inflammatory neuropathy study of serbia) in order to comprise as many patients with cidp from serbia, republic of srpska (bosnia and herzegovina) and montenegro covering population of more than nine million people. our first aim is to analyze overall impact of cidp on physical, mental and social areas of life measured with generic, symptom specific and disease specific questionnaires -sf- , inqol and cap-pri, respectively. furthermore, we aim to analyze influence of the disease on patients' working status and presence of depressive mood measured by beck's inventory. following features of patients are included: sociodemographic data, clinical aspects of the disease, level of disability, severity of sensory symptoms, presence of comorbidities, electrophysiological characteristics, as well as fatigue, autonomic symptoms and neuropathic pain. we intend to define the most significant predictors of decreased qol in order to focus on patients with the highest risk and to improve care of cidp. we also want to see if cidp patients in complete remission as per clinical findings still have reduced quality of life. we have recruited patient so far and we expect to include around subjects overall. we will present the first data of the study at the pns meeting . beaudonnet g , prud'hon s , cauquil c , labeyrie c , not a , bouilleret v , adams d . neurophysiology chu bicêtre, le kremlin bicêtre, france; neurology chu bicêtre, le kremlin bicêtre, france. familial amyloid neuropathy (fap) is a life-threatening disease of autosomal dominant inheritance due to transthyretin (ttr) gene mutation, a liver-produced protein. current treatments slow down its natural course and are indicated from the very first objective symptoms. we aimed to evaluate two neurophysiological markers: sympathetic skin response (ssr) and heart rate variability (hrv) in the early detection of sympathetic damages due to fap. ssr and hrv were assessed in ttr gene mutated patients with neither clinic nor electroneuromyographic abnormalities and controls matched on gender and age. cases were recruited consecutively from current care in the french reference center for rare diseases of bicetre university hospital. ssr was recorded on the two palms and on the sole of the left foot with to stimulations between and ma. hrv was registered during three conditions of seconds each: normal breathing, deep breathing ( cycles of seconds of inspiration and seconds of expiration) and valsalva manoeuver during seconds. valsalva ratio, defined by the ratio between the longest and shortest rr intervals, was significantly higher in the control groups after bonferroni correction (means of . and . , respectively, p< . ). there was no significant difference between the two groups for any ssr parameter, although means of amplitudes were systematically higher in controls than among cases. our results confirm that autonomic nervous fibers are damaged early in both clinical and electroneuromyographic asymptomatic patients mutated on the ttr gene. valsalva ratio seemed to be the most discriminative marker. long-term follow-up with test repetition and confrontation with cardiologic assessment will help to precise how these tests could be used in current care. they might help to identify high risk patients to propose them an appropriate early treatment and could be used to follow treatment efficacy. familial amyloidotic polyneuropathy (fap) was originally characterized by andrade as an axonal neuropathy which subsequently was found to be associated with a number of mutations in the plasma protein transthyretin (previously named prealbumin). it is now recognized that cardiomyopathy may be a significant factor in a majority of patients with the hereditary form of transthyretin amyloidosis (fap) and many of the transthyretin (ttr) mutations are associated with cardiomyopathy with no or minimal signs of peripheral neuropathy. attr-wt also called senile cardiac amyloidosis and senile systemic amyloidosis is recognized as late-onset, usually in the th or th decade of life, and the fact that the majority of patients are males. transthyretin neuropathy proven by nerve biopsy has been rarely reported in this population. here we report our experience with patients having attr-wt characterized by cardiomyopathy but also with varying degrees of peripheral neuropathy. clinically, the neuropathy appears as typical axonal or mixed axonal/demyelinating neuropathy as is seen in fap. pathologically, two types of ttr deposition have been found, ( ) intraneural ttr amyloid deposits as seen in fap are present in some patients and ( ) other patients have extensive vascular deposition of amyloid in both perineural arteries and veins without deposits within nerve trunks. in conclusion, peripheral neuropathy may definitely be a part of the attr-wt clinical presentation and with the increase in numbers of attr-wt cardiomyopathy patients being identified, it is important to ascertain whether any evidence of peripheral neuropathy is due to the amyloidosis and not to compounding syndromes such as diabetes mellitus type ii. besora s , santos c , izquierdo c , martinez-villacampa m , simó m , bruna j , , velasco r , . neuro-oncology unit, hospital universitari de bellvitge-institut català d́oncologia, ĺhospitalet, barcelona, spain; medical oncology department, institut català d́oncologia, ĺhospitalet, barcelona, spain; department of cell biology, physiology and immunology, institute of neurosciences, universitat autònoma de barcelona and centro de investigación biomédica en red sobre enfermedades neurodegenerativas (ciberned), bellaterra, spain. oxaliplatin (oxa) is the first-line chemotherapy agent in the treatment of colorectal cancer (crc). oxa-induced peripheral neuropathy is the most frequent long-term side-effect. retreatment with oxa is frequently considered in patients as salvage treatment. patients receiving oxa-based chemotherapy regimen at least twice at our institution between and were reviewed. the aim of this study was to investigate whether retreatment with oxa increases the risk of developing or worsening previous oxa-induced peripheral neuropathy. the severity of neuropathy was measured by national cancer institute-common toxicity criteria (nci), total neuropathy score (tns) © and nerve conduction studies. one hundred twenty-five crc patients were included. median age was years. after first-line oxa-based chemotherapy, . % of patients developed neuropathy according nci, after a median of [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] cycles. severity of neuropathy was grade ( . %), grade ( %), and grade ( . %). median time to retreatment with oxa was months. frequencies of neuropathy before retreatment were as follows: . % grade , . % grade , and . % grade . after retreatment, severities of neuropathy were . % grade and . % grade . no patient developed grade . . % of patients did not develop neuropathy. peripheral neuropathy was the reason for stopping prematurely treatment after first-line and retreatment in . % and . % of patients, respectively. worsening of previous nci score was observed in . % of patients. the great majority of patients ( . %) remained within the same nci score than before retreatment after median [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] cycles. among those patients that did not develop neuropathy after first treatment (n= ), only and patients developed grade and , respectively, after a median of [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] cycles. among those patients who initially developed grade and neuropathy, no differences in tnsc © scores just before and after finishing retreatment with oxa were identified ( [ - ] vs [ - ], p= . ). retreatment with oxa in crc patients is a feasible option even in patients who developed moderate or severe neuropathy previously. lack of worsening of previous neuropathy is observed in the great majority of patients. neurological monitoring of patients candidates to retreatment with oxa should be considered. bessaguet f , sturtz f , magy l , , desmouliere a , bourthoumieu s , demiot c . ea -myelin maintenance & peripheral neuropathy, faculties of medicine and pharmacy, university of limoges, limoges, france; department of neurology, reference center for rare peripheral neuropathies, university hospital of limoges, limoges, france. prolonged pressure and the resulting local ischemia are widely accepted as the primary etiology of skin pressure ulcers (pus) but precise mechanisms of their formation remain unclear. in this study, we wanted to study the potential role of sensory small nerve fibers in regulation of inflammation during pus formation. to achieve this goal, we developed a mouse model of a purely sensory neuropathy and this was induced by resiniferatoxin (rtx). in this model, seven days after a single injection of rtx ( g/kg; ip), mice present a thermal and mechanical hypoalgesia associated with large substance p (sp) and calcitonin gene-related peptide (cgrp) depletion without neurodegeneration. this model mimic quite well what is observed in early stages of sensory nerve fiber defect. studies have shown that sp and cgrp are involved in cutaneous inflammation regulation. in fact, these neuropeptides are released by sensory fibers and are pro-inflammatory mainly through recruitment of immune cells and vasodilation. thus, we studied gene expression of pro and anti-inflammatory cytokines by a rna array approach during pus formation in control and rtx mice. seven days after a single injection of rtx, epidermis, dermis and subcutaneous tissue layer were pinched with magnetic plates during hours. pressure induced a stage pus. gene expression was evaluated in each compressed area h after pressure. results showed mainly a down-regulation of gene expression in pus of rtx mice compared to control mice. a decrease of cgrp/sp in skin sensory small fiber increased pus formation associated with an increase of interleukins (il)- , il- , il- and il- expression and a decrease of il- expression in rtx mice. supplementary experiments with rt-qpcr for each cytokine will be necessary to confirm these preliminary results. these observations suggest a cgrp/sp role in regulation of cytokines expression during pus formation. the new inflammatory profile exhibited in this study might help in the design of new treatments improving the quality of life of neuropathic patients prone to developing bedsores. bhattacharyya bj , jayaraj nd , belmadani a , ren d , rathwell ca , hackelberg s , miller rj , menichella dm . department of neurology, northwestern university, chicago, il, usa; department of pharmacology, northwestern university, chicago, il, usa. painful diabetic neuropathy (pdn) is one of the most common and intractable symptoms of diabetes, affecting % of diabetic patients. the hallmarks of pdn are neuropathic pain and small fiber degeneration, manifested by the loss of dorsal root ganglion (drg) nociceptor axons. neuropathic pain is associated with nociceptor hyper-excitability in the absence of physiologically appropriate stimuli. in states of neuropathic pain, drg nociceptors become increasingly responsive to a variety of excitatory influences, including inflammatory cytokines. in particular, we have shown that stromal cell derived factor- (sdf- ) and its receptor cxcr are necessary for the generation of neuropathic pain in mouse models of pdn. however, the molecular mechanisms leading to the hyper-excitability of drg nociceptors in pdn are unknown, as are the mechanisms leading to small fiber degeneration. this fundamental gap in our knowledge represents a critical barrier to progress in developing novel therapeutic approaches for pdn. the objective of this study is to identify the molecular cascade linking cxcr /sdf- chemokine signaling to drg nociceptor hyper-excitability, neuropathic pain, and small fiber degeneration in pdn. drg nociceptors can be identified by a series of molecular markers, including expression of the sodium channel na v . . indeed, > % of na v . -expressing drg neurons are nociceptors. feeding mice a high fat diet (hfd) for several weeks induces glucose intolerance, obesity, and mechanical allodynia, a particular pain hypersensitivity associated with pdn. using the hfd model combined with dreadd receptor technology, we have shown that reducing excitability of na v . -expressing neurons prevents and reverse neuropathic pain, neuronal calcium overload, mitochondrial dysfunction, and small fiber degeneration. furthermore, we have shown that cxcr receptors are necessary for neuropathic pain and small fiber degeneration in pdn. taken together these data demonstrate that na v . nociceptor hyperexcitability in pdn is driven through the activation of cxcr receptors. inhibition of hyperexcitability can prevent and reverse the development of pdn. furthermore, these observations will advance our understanding as to how changes in excitability, calcium influx, and mitochondrial dysfunction in nociceptors contribute to neuropathic pain and small fiber degeneration in pdn, which is a critical barrier to progression for effective and disease modifying treatment for pdn. bianco m , terenghi f , gallia f , nozza a , scarale a , fayoumi mz , giannotta c , morenghi e , nobile-orazio e . poems (polyneuropathy, organomegaly, endocrinopathy, m protein and skin changes) syndrome is an unusual multisystem disease with neurological disability due to a severe disabling polyneuropathy, with high mortality by multiorgan failure. peripheral blood stem cell transplantation (pbsct) is considered the treatment of choice for poems while lenalidomide is the most promising therapy for patients not eligible for pbsct. the aim of the present study was to compare the long-term effects on clinical, biological and neurophysiologic parameters in patients with poems treated with lenalidomide or pbsct. the clinical, biological and neurophysiologic data were reviewed in poems patients treated with pbsct (n: ) or lenalidomide (n: ). the mrc sumscore on muscles, onls scale, vegf serum levels and nerve conduction studies were assessed before (t ) and after (t ) and years (t ) of treatment and the differences were compared using anova. combining the two groups of patients, there was a significant improvement after treatment in the mean mrc sumscore (t = ± ; t = ± ; t = ± ; p = . ), in the mean onls score (t = . + . ; t = . + . ; t = . + ; p = . ), in the ulnar mean distal motor latency (t = . ± . msec; t = . ± . msec; t = . ± . msec; p = . ), distal compound muscle action potentials amplitude (t = . ± . mv; t = . ± . mv; t = . ± . mv; p = . ), motor conduction velocity (t = . ± . m/sec; t = ± m/sec; t = . ± . m/sec; p= . ) and serum vegf levels (t vs t : p = . ; t vs t : p = . ). the difference was also significant when we separately analyzed patients treated with lenalidomide and pbsct and there was no difference between the two groups in any of the analyzed parameters. treatment with pbsct and lenalidomide significantly and similarly improved clinical, biological and neurophysiologic parameters in patients with poems syndrome up to two years. since pbsct may not be suitable for all patients, lenalidomide may represent an effective and a valuable alternative in these patients or in those relapsing after pbsct inducing a prolonged clinical, biological and neurophysiologic improvement. bis d , tao f , abreu l , sleiman p , hakonarson h , zuchner s and inherited neuropathy consortium. dr. j.t. macdonald department for human genetics, hussman institute for human genomics, university of miami, miami, fl, usa; center for applied genomics, the children's hospital of philadelphia, philadelphia, pa, usa. inherited peripheral neuropathies are clinically and genetically heterogeneous diseases that can cause distal muscular atrophy and sensory loss. alleles in over one hundred different genes have been shown to cause peripheral neuropathies; yet, greater than % of axonal neuropathy patients do not receive a genetic diagnosis. large scale exome studies are now beginning to be sufficiently powered to perform mutational burden analysis. this approach compares damaging allele frequencies of cmt cases with a control group to identify additional causes for neuropathies. this approach will also identify genes that require an oligogenic inheritance to cause a phenotype. in a deviation from the classic linkage-based and heuristic variant filtering approaches to gene identification, we are performing burden analyses in a large cohort of cmt families. in known neuropathy genes, we saw that neuropathy cases carried on average . rare, non-synonymous variants, while unrelated non-neuropathy controls harbored . variants (p= . e- , mann-whiney u-test). enrichment of rare, non-synonymous variants in cmt disease genes within inherited peripheral neuropathy cases suggests the presence of multiple weaker alleles in individual patients. we also performed an unbiased exome-wide gene-based burden analysis and ranked genes after multiple testing correction. several new candidate genes were identified that need further follow up conformational studies. a number of known cmt and related genes were observed in the list of top candidates. we are currently analyzing additional aspects of this sample and are actively seeking more cmt exomes to enlarge our study. in summary, statistical methods traditionally reserved for more 'common' phenotypes' increasingly are becoming available for rare disease genetics. c bl/ mice were treated with bortezomib alone or in combination with monastrol. neuropathic changes were assessed by nerve conduction studies and histological analysis. analysis of axonal morphology was performed with light and electron microscopy. anti-neoplastic properties of monastrol alone and in combination with bortezomib were assessed in different blood cancer cell lines. prolonged treatment with bortezomib induced a sensory neuropathy in mice. significant changes in axonal morphology correlated with reduced function of peripheral nerves. the administration of monastrol substantially ameliorated morphological features of axonal alterations and sensory neuropathy. cytotoxicity studies in blood cancer cell lines showed no interference of monastrol with the cytostatic effects of bortezomib. our data indicate that monastrol may alleviate bortezomib induced neuropathy. the favorable cytotoxic profile of monastrol makes it an interesting candidate as neuroprotective agent to prevent bortezomib-induced neuropathy. boczonadi v , meyer k , gonczarowska-jorge h , , bartsakoulia m , roos a , , bansagi b , zahedi rp , talim b , bruni f , kaspar b , , lochmüller h , boycott km , müller js , horvath r . jwmdrc, wtcmr, igm, newcastle university, newcastle upon tyne, uk; rinch, columbus, oh, usa; leibniz-institute für analytische wissenschaften-isas-e.v., dortmund, germany; capes foundation, brazil; department of pediatrics, hacettepe university children's hospital, ankara, turkey; dbbb,university of bari aldo moro, bari, italy; department of neuroscience, the ohio state university, columbus, oh, usa; department of genetics, cheo, university of ottawa, ottawa, canada. while autosomal dominant mutations in gars, encoding the glycyl-trna synthetase, have been identified in patients with charcot-marie-tooth peripheral neuropathy (cmt d) and distal spinal muscular atrophy type v (dsma-v), autosomal recessive mutations cause mitochondrial disease affecting skeletal muscle and heart. gars is a bi-functional enzyme and it is responsible for normal protein translation both in mitochondria and the cytoplasm. in this study we have focused on the mitochondrial function of the gars by investigating a mouse model (gars c r) , human fibroblasts and induced neuronal progenitor cell lines (inpcs). mild mitochondrial abnormalities were detected in skeletal muscle of the gars c r mice while no other tissues were affected. control and patient fibroblasts harboring gars mutation were directly converted into inpcs. we identified tissue specific impairment of mitochondrial function in neuronal cells carrying not only recessive but also dominant gars mutations, suggesting neuron-specific effects of mitochondrial alterations.comparative proteomic analysis of inpcs showed significant changes in mitochondrial proteins. furthermore, the reduction of the vesicle-associated membrane protein-associated protein b (vapb) and its downstream pathways in gars-deficient inpcs suggests that altered mitochondria-associated endoplasmic reticulum (er) membranes (mam) may also contribute to the motor neuropathy. boczonadi v , king ms , bansagi b , roos a , , eyassu f , borchers c , lane m , ramesh v , lochmüller h , pyle a , griffin h , smith ac , chinnery pf , , alan j robinson aj , edmund rs kunji ers , horvath r . jwmdrc, wtcmr, igm, newcastle university, newcastle upon tyne, uk; mitochondrial biology unit, medical research council, cambridge; leibniz institute of analytic sciences (isas), dortmund, germany; uvic-genome bc proteomics centre, vancouver, canada; paediatric neurology, royal victoria infirmary, newcastle upon tyne foundation hospitals nhs trust, newcastle upon tyne, uk. members of the mitochondrial carrier family (slc ) transport nucleotides, keto acids, amino acids, fatty acids, co-factors and inorganic ions across the mitochondrial inner membrane. several inherited diseases with very variable clinical presentations are associated with dysfunctional mitochondrial carriers. we report a patient with childhood-onset spinal muscular atrophy and mitochondrial myopathy caused by a homozygous mutation in slc a , encoding the mitochondrial oxodicarboxylate carrier (odc). the mutation renders the carrier dysfunctional and, consequently, -oxoadipate cannot be imported into the mitochondrial matrix. computer modelling of the metabolic defect caused by the mutation predicted that the impaired transport leads to accumulation of -oxoadipate, pipecolic acid and the known neurotoxin quinolinic acid, which were precisely confirmed by targeted metabolomics in serum and urine. exposure of -oxoadipate and quinolinic acid reduced the level of mitochondrial complexes in sh-sy y cells in-vitro suggesting a possible pathomechanism. here we demonstrate that -oxoadipate and quinolinic acid are toxic for spinal motor neurons and their increased levels may contribute to neuropathy. and families have been reported. interestingly, most mutations target the same amino acid residue (k e, k m, k n, k t) in the highly conserved -crystallin domain of the hspb protein. the spectrum of diseases caused by mutations in the hspb gene was recently expanded to distal myopathy. hspb is ubiquitously expressed, but is highly expressed in motor neurons and muscles. the hspb is a chaperone that participates in clearing misfolded poly-q containing proteins such as mutant huntingtin and ataxin- involved in respectively huntington's disease and spino-cerebellar ataxia. hspb directly interacts with the co-chaperone bag and their role in chaperone-assisted selective autophagy is well described. to delineate the molecular deficits and functional consequences of hspb mutations we generated a knock-in (ki) mouse model for the k n missense mutation mimicking the neuropathy phenotype. we observed that homozygous mutant mice (hspb k n/k n ) develop a progressive axonopathy, with decreased compound motor action potential amplitudes, and loss of large and medium myelinated axons. this results in locomotor deficits with an impaired performance at the rotarod and grip strength tests. at the ultrastructural level, the hspb -ki model displays severe signs of axon degeneration and a clear myofibrillar myopathy, as observed in some patients with hspb mutations. interestingly, hspb positive aggregates were found in the sciatic nerve and gastrocnemius muscle of our mutant mice. additionally, our model allowed us to generate hspb knock-out (ko) mice using the same targeting vector. strikingly, the homozygous hspb -ko animals do not show any sign of axonopathy and display a much milder myopathy than the hspb -ki animals. these data suggest that part of the pathomechanisms is due to toxic gain-of-function of the mutant protein. boutahar n , reynaud e , nasser y , camdessanché jp , antoine jc . university hospital, saint-etienne, france. dysimmune sensory neuronopathies (snn) depend on neuron cell death induced by an inflammatory reaction in dorsal root ganglia. we have recently identified the intracellular tyrosine kinase (trk) domain of the fibroblast growth factor receptor- (fgfr ) as the target of antibodies in a subset of patients with non paraneoplastic snn. fgfr is one of the four fgfrs and has been involved in sensory neurons maintenance during development and cell death induction after axotomy. fgfrs ligand fixation results in the activation of several intracellular pathways through adaptator protein interactions with the trk domain. in particular ras activation may lead to cell proliferation or apoptosis through erk / or p map kinase signaling. the p mapk pathway is also involved in neuronal cell death induced by nmda and ampa receptor activation. as fgfr is a cell surface protein, human antibodies may interfere with the receptor functioning as a growing number of evidence has showed with other cell surface antibodies in neurological diseases. to test this hypothesis we developed an in vitro model using fvbn mice cortical neurons culture exposed to a rabbit polyclonal antibody reacting with the trk domain of fgfr . comparatively to normal rabbit iggs, the fgfr antibody induced neuron cell death in a dose dependent manner. neuron cells were exposed to fgfr antibody concentration leading to - % cell death in absence or presence of the p mapk inhibitor sb and the expression of fgfr , glur subunit of ampa receptors and nr subunit of nmda receptor was measured by quantitative rt-qpcr. the fgfr antibody induced an upregulation of fgfr while the glur and nr subunits were modulated. these changes were prevented in presence of sb . these preliminary results indicate that anti-fgfr iggs may interfere with the functioning of the intracellular domain of the protein and the expression of nmda and ampa receptors through the p map kinase pathway. this model may be used to test the effect of human anti-fgfr iggs in vitro. braathen gj , tveten k , holla Øl , busk Øl , hilmarsen ht , svendsen m , høyer h . section of medical genetics, department of laboratory medicine, telemark hospital, skien, norway. next-generation sequencing (ngs) has during the last years entered the clinical diagnostics. ngs has proven to be very efficient in the diagnostics of disorders where multiple genes can be involved. our ngs-based targeted gene panel consists of hereditary neuropathy genes, i.e. mostly charcot-marie-tooth genes. this study is a retrospective study of clinic samples received between may and february . we describe the identified novel likely pathogenic sequence variants, according to international guidelines. in this period we identified novel, not previously described, likely pathogenic sequence variants in the following genes: aars, fgd , gan, hint , litaf, lrsam , mme, mpz, nefl, pmp , sbf , sh tc and yars. there is now a large range of genes causing hereditary peripheral neuropathies and many likely pathogenic sequence variants. likely pathogenic sequence variants are not only identified in old well established neuropathy genes but also in the newer genes like mme. modelling brown-vialetto-van laere syndrome in c. elegans mutations in slc a and slc a , which encode the riboflavin transporters rfvt and rfvt . patients with rfvt deficiency exhibit proximal and distal limb weakness, sensory ataxia, diaphragmatic paralysis, optic atrophy, sensorineural deafness and bulbar palsy. riboflavin is critical for the biosynthesis of flavin mononucleotide and flavin adenine dinucleotide, essential cofactors for carbohydrate, amino-acid and lipid metabolism. mutations in slc a reduce or abolish rfvt expression resulting in impaired riboflavin uptake into sensory and motor neurons. high-dose riboflavin treatment can improve or stabilise a patient's condition, however the optimum dose and long term effects of riboflavin treatment, and disease pathomechanisms remains poorly understood. to further understand the pathophysiological consequences of slc a mutations, we propose developing an animal model for bvvl. caenorhabditis elegans (c. elegans) are small round transparent nematodes extensively used for studying the genetics and molecular biology of neurodegenerative diseases. there are two c. elegans riboflavin transporter genes, rft- and rft- . based on protein sequence homologies and expression profiles for both genes, rft- is the ortholog of rfvt . the expression of rft- is regulated by riboflavin availability and knock-down of the rft- gene by sirna perturbs c. elegans development. our aim is to develop a knock-in c. elegans model of bvvl. human rfvt and c. elegans rft- protein sequences were aligned with clustal omega to identify conserved amino acid residues associated with bvvl mutations. the amino acid involving the l p rfvt mutation is conserved in rft- (residue l ). to create our model, we will introduce the l p rft- mutation into the rft- locus in c. elegans genomic dna using crispr/cas- technology. this bvvl c. elegans model will allow us to explore the pathogenic consequences of rfvt deficiency underlying motor nerve degeneration and to evaluate drug therapy regimes by determining the optimal riboflavin dose and treatment initiation, and trialing other compounds that may improve benefits seen with riboflavin supplementation. chronic inflammatory demyelinating polyradiculoneuropathy (cidp) is an autoimmune-mediated inflammatory disease of the peripheral nervous system. cidp can present chronic progressive or relapsing-remitting courses and can predominantly affect motor but also sensory nerve fibers causing weakness of proximal and distal muscles. it represents the most common chronic autoimmune neuropathy and is pathologically characterized by focal inflammatory-mediated demyelination followed by axonal degeneration. recently, we have developed a new animal model for cidp, the chronic-ean, induced in lewis rats by active immunization with s-palmitoylated p ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) peptide. this model fulfills electrophysiological criteria of demyelination with axonal degeneration, a result confirmed by immunohistopathology. the late phase of the chronic disease is characterized by an accumulation of il- + cells and macrophages in sciatic nerves and as well as high serum il- levels. it is a reliable and reproducible animal model for cidp that can now be used for translational drug studies for chronic human autoimmune-mediated inflammatory diseases of the peripheral nervous system, particularly cidp, for which, there is a real need for new immunotherapies. the aim of this study was to test the therapeutic efficacy of ivig and a recombinant fc fragment (fcrec) in this new cidp animal model. treatments with ivig and fcrec proved effective in preventing further progression of cidp in rats. the therapeutic treatments not only decreased the maximal clinical scores of the cidp rats compared to albumin treatment but also abolished the disease chronicity. interestingly, a better efficacy of fcrec treatment compared to ivig was demonstrated at histological level, with the myelinated fibers well preserved and the greatly reduced accumulation of macrophages and il- + cells in sciatic nerves. ivig and fcrec therapeutic activities in this model can also be followed by measurement of antibodies in the serum and could therefore be used as biological markers. the current study provides for the first time direct evidence that ivig is effective in the treatment of cidp rats and suggests that a novel fcrec compound is more effective than ivig. it will contribute to the development of more effective and safer drugs for the treatment of autoimmune peripheral neuropathies like cidp. each time, one measurement with supramaximal stimulation according to the conventional ncs and another measurement with % enhanced stimulation intensity were conducted. we analyzed the results with special consideration of minimal and maximal fiber velocities and of the velocity with maximal fiber proportion. as a result, we detected statistically significant differences between cidp patients and controls in all these parameters. compared with controls, cidp patients had slower minimal and maximal fiber velocities and also the spectrum of motor conduction velocities was definitively shifted to slower velocities. slower minimal conduction velocities could be detected in some cidp patients by using the enhanced stimulation intensity. interestingly, in some cidp patients the conventional ulnar ncs were normal, but the collision technique showed fibers with a conduction velocity of less than m/s, indicating demyelination. to our knowledge, this is the first study using hopf's collision technique systematically in cidp patients. significant differences between the variation of ulnar motor nerve conduction velocity of cidp patients and controls could be detected. this method also showed signs of demyelination in some cidp patients with normal ulnar ncv. by enhancing stimulation intensity above the threshold of supramaximal stimulation in conventional ncs, the collision technique may be even more valuable. the clinical course of guillain-barré syndrome (gbs) is highly variable and some patients may develop treatment-related fluctuations (trfs) as an indication of ongoing disease activity and temporary treatment effect. other patients present as gbs, but subsequently develop repetitive relapses as an indication of acute-onset chronic inflammatory demyelinating polyneuropathy (a-cidp). this distinction is important because treatment may differ. we determined the frequency and clinical presentation of patients with gbs-trf and a-cidp in the first patients included in the international gbs outcome study (igos) with a follow-up of at least months. thirty-eight patients ( %) were excluded because of alternative diagnoses. of the remaining patients, ( %) had at least one trf, ( %) had a-cidp. preliminary analysis showed no significant differences between the groups (total-gbs, gbs-trf and a-cidp) for sex, age, sensory symptoms, cerebrospinal fluid results and mechanical ventilation. a-cidp patients had a median age of years (iqr - ), % was male, and all patients were treated in the acute phase of the disease (intravenous immunoglobulins (ivig) ( ), plasma-exchange (pe) ( ) or methylprednisolone (mp) ( )). gbs-trf patients has a median age of years (iqr - ), % was male and all patients received treatment (ivig ( ) and pe ( ). gbs-trf patients showed more antecedent events ( % versus %, p= . ), a higher gbs disability score (≥ ) at nadir ( % versus %, p= . ) and less frequently developed ataxia ( % versus %, p= . ) than patients with a-cidp. onset to nadir was longer in a-cidp than in gbs-trf ( days (iqr - ) versus days (iqr - ), p= . ) and the total gbs group ( days (iqr - ), p= . ). the time until the first clinical deterioration tends to be longer in the a-cidp patients (median days (iqr - ) versus median days in the gbs-trf group, not significant). the diagnosis a-cidp was made after a median of days . in conclusion, this first analysis identified distinctive characteristics of gbs-trf and a-cidp in support of a different pathogenesis that may help with early identification of these disorders in clinical practice. additional results will be presented at the conference. chronic inflammatory demyelinating polyneuropathy (cidp) is a disorder with a highly diverse clinical presentation, electrophysiological phenotype, response to treatment and outcome. this heterogeneity may indicate the presence of distinct subtypes of cidp, which may have a different pathogenesis and require more personalized treatment. the international cidp outcome study (icos) is a prospective, observational, international multi-center study that aims to describe this variation in clinical and electrophysiological subtypes and to define the clinical and biological determinants of these subtypes, disease activity, treatment response and outcome. in addition icos aims to provide an infrastructure for conducting new (therapeutic) studies in cidp, similar to the international gbs outcome study (igos). all patients fulfilling the efns/pns ( ) diagnostic criteria for cidp can be included in icos, independent of age, duration and severity of disease or treatment. we collect information on neurological deficits, diagnostic characteristics, various validated clinical outcome measures, previous and current treatment and we collect biomaterials (dna, cerebrospinal fluid, nerve biopsies and repeated serum samples). icos was started as a pilot study in dutch university centers. by february , patients were included in icos, patients recently diagnosed with cidp and previously diagnosed patients. included were ( %) males and ( %) females with a median age of years (iqr - ). the current cohort consists of classic (sensory-motor) cidp, madsam and pure motor variants. of the patients diagnosed in the past, patients ( %) were treated (intravenous immunoglobulins (ivig) ( ), prednisolone ( ), subcutaneous immunoglobulins (scig) ( ), dexamethasone ( ), plasma-exchange ( ) and ivig with methylprednisolone (mp) ( )). the recently diagnosed patients all received treatment (ivig ( ), dexamethasone ( ), plasma-exchange ( ) and patients were treated in a pilot study with ivig with methylprednisolone (mp)). the protocol has been evaluated and adjusted and will be shared with other researchers via the inflammatory neuropathy consortium and igos consortium. our aim is to include at least cidp patients worldwide with a minimum follow-up period of years. dominant mutations in glycyl trna synthetase (gars) cause inherited axonal neuropathy (charcot-marie-tooth type d). mutations in the mouse gars gene cause a similar phenotype, and represent valid disease models. we routinely use two mouse strains, one with a severe neuropathy, and one with a milder, later onset neuropathy. using these mouse models, we are exploring the mechanisms through which gars mutations cause peripheral axon degeneration. efforts include ribosome tagging to isolate ribosome-associated mrnas specifically from motor neurons, and non-canonical amino acid tagging to visualize and isolate newly synthesized proteins. taking advantage of the anatomy of motor neurons, we are able to analyze cell bodies separately from peripheral axons in both of these approaches. in addition, we are using these mouse models for preclinical studies testing gene therapy approaches to treat cmt d. consistent with our previous genetic studies in mice, knockdown of the mutant transcript, while preserving sufficient levels of the wild type, is a very successful approach when administered before the onset of neuropathy. we are now testing this approach in mice after the onset of symptoms, and in mice carrying a mutant gars allele associated with human disease. these studies are potentially translational, and also address mechanistic questions such as the timing and cell autonomy of the pathophysiology. burnor e , yang l , hao z , patterson k , quinn c , scherer ss , lancaster e . the university of pennsylvania, philadelphia, pa, usa; the second xiangya hospital, central south university, hunan, china. autoantibodies to two isoforms of neurofascin (nf and nf ) have been reported in patients with guillain-barre syndrome (gbs) or chronic inflammatory demyelinating neuropathy (cidp). it is not clear which of these responses reliably distinguish autoimmune neuropathies from other severe neuropathies, which responses are transient versus persistent, or which responses may target multiple isoforms of neurofascin. in addition, approximately % of neuropathy patients have no known cause, and it is unknown whether a subset of these patients may have autoantibodies to neurofascins. we have studied cohorts of patients with autoimmune neuropathy (n= ), genetic neuropathy (n= ), and idiopathic neuropathy (n= ) for igg and igm responses to neurofascins. neurofascin antibodies were found in ( %) of patients with autoimmune neuropathy, and ( . %) idiopathic neuropathy patients, but only % ( of ) in patients with genetic neuropathy. follow-up serum samples were available for positive cases. persistent responses were associated with chronic neuropathy while transient responses were seen in gbs or with remission of cidp. most patients had responses specific to either nf of nf . however, a particularly severe, treatment-resistant form of cidp, approaching a locked-in state, was seen in a patient with a unique response to all three isoforms of neurofascin (nf , nf , nf ). treatment of this patient with rituximab resulted in clinical improvement and resolution of the neurofascin antibody response. in conclusion, autoantibodies to neurofascins distinguish autoimmune neuropathies from severe genetic neuropathies, but the clinical phenotype may depend on the persistence and isoform specificity of the immune response. antibodies to the common domains shared by nf and nf may portend a severe but treatable neuropathy. a subset of idiopathic neuropathy patients may have an autoimmune mechanism. burns j , , sman ad , cornett kmd , wojciechowski e , , walker t , menezes mp , , mandarakas mr , rose kj , , bray p , , sampaio h , farrar m , , refshauge km , raymond j and the fast study group. university of sydney, new south wales, australia; sydney children's hospitals network (randwick and westmead), new south wales, australia; university of new south wales, sydney, australia. exercise has undisputed benefits for human health and potentially as a treatment for neuromuscular disease. but there is also a risk of harm due to overwork weakness. we report the results of a -month randomised, double-blind, sham-controlled trial evaluating progressive resistance exercise of foot dorsiflexor muscles in pediatric cmt. sixty patients ( cmt a, cmt e, cmt f, cmt a, cmt c, cmtx , cmtx ) aged - years were randomly assigned to undergo -weeks ( sessions) of moderate-intensity progressive resistance exercise or sham exercise. the primary endpoint was change in isometric dorsiflexion strength between groups assessed by hand-held dynamometry (expressed as a z-score based on age-and sex-matched normative reference values, positive values indicate an improvement in strength). the primary safety endpoint was change in muscle and fat volume of the muscles responsible for dorsiflexion by mri between groups. secondary outcomes were function (balance, long jump, -min walk test), walking ability ( d gait analysis), self-reported ankle instability, parent-reported quality of life (physical summary, psychosocial summary and global impression of change scores), and adverse events. fifty-five ( %) children completed the trial. adherence was comparable between exercise ( %) and sham ( %) groups. while patients experienced muscle soreness during training in the exercise group, adverse events did not differ between groups. the mean z-score for dorsiflexion strength increased in the exercise group by % at -months (from − . ± . to − . ± . ) and decreased in the sham group by %, mirroring the natural history of cmt (from − . ± . to − . ± . ). between-group ancova-adjusted difference at -months was . ( %ci, . to . ; p= . ). the mean scaled scores for mri muscle volume and fat volume were comparable between groups at months (p> . ). the global impression of change scores favoured the exercise group at -months (p= . ) and -months (p= . ). there was no other measurable effect of exercise. pre-specified subgroup analyses according to age ( - years vs. - years) showed a larger treatment effect with exercise in adolescents. targeted progressive resistance exercise was effective at halting progression of dorsiflexion weakness without detrimental effect on muscle morphology or other signs of overwork weakness in children with cmt. byung-nam y , yoon-ho h , suk-won a , seok-jin c , jung-joon s . inha university hospital, incheon, south korea; seoul metropolitan government boramae medical center, seoul national university, seoul, south korea; joong ang university hospital, seoul, south korea; seoul national university hospital, seoul, south korea. a years old female patient visited the hospital with weakness in both upper limbs from months under the suspicion of motor neuron disease with the finding of extensive denervation changes on electromyography. whole spine mri showed ventral nerve rootlet enhancement in the c and t bundles. the emg had confirmed active denervation changes in the muscles innervated by bilateral c ∼c roots. the cerebrospinal fluid culture test showed a protein (csf) elevation to mg / dl. she got a steroid pulse therapy. she had the symptoms of dry mouth and dry eyes during the recent years. a salivary gland scan test was performed for the possibility of sjögren's syndrome, and as a result, absorption of the contrast agent in both parotid and submandibular glands was decreased. the shammer test showed mm on the right side and mm on the left side. she was diagnosed of sjögren's syndrome. after weeks, overall strength improvement was observed, and bilateral shoulder abduction was improved to mrc grade or higher. the follow-up spine mri also showed that the initially seen ventral nerve root enhancement was disappeared. the case had visited the hospital with major symptoms of weakness and atrophy of the muscles, showing similar pattern to motor neuron disease, and was diagnosed as inflammatory polyradiculopathy and confirmed as primary ss during differential diagnosis. this case suggests that primary ss may induce inflammatory polyradiculopathy, which shows motor symptoms as major symptoms rather than sensory symptoms, and that a fast and accurate diagnosis is needed in terms that it can be treated with steroids and appropriate immune suppressive agents. traumatic injuries to peripheral nerves are frequent. however, effective pharmacological treatments are lacking. curcumin, a polyphenol found in rhizomes of curcuma longa, has been shown to develop antioxidant, anti-inflammatory, and neuroprotective properties. however, due to its poor hydrosolubility and its extensive metabolism, the use of large curcumin doses is required for therapeutic purpose. the aim of the present study was to investigate the effects of a local infusion of a low curcumin dose on nerve regeneration and functional recovery after sciatic nerve injury in rats. the experiments were conducted in g sd male rats submitted to unilateral sciatic nerve crush at d . curcumin was solubilized in polyethylene glycol and continuously administered using osmotic pumps ( . mg/day until d ) with a catheter delivering the drug near the lesion site. functional analyses using von frey, beam walking, static sciatic index (ssi) and grip strength tests were carried out at d (reference test) and every week after injury (d , d , d , d and d ). in addition, an evoked electromyogram was performed at d and d . after euthanasia (d ), nerve and muscle samples were collected and analyzed by light and electron microscopy. functionally, a significant improvement of the mechanical sensitivity (+ %) was observed at d in the curcumin-treated group (n= ) vs. vehicle group (n= ). in curcumin-treated group, skillful walking and finger spacing of the ipsilateral paw (ssi) were fully restored respectively at d and d contrary to vehicle group. furthermore, curcumin treatment improved the grip strength recovery (+ % at d ). the electrophysiological results indicated a full recovery of motor nerve conduction velocities (mncv) after days of curcumin treatment, while mncv remained altered in vehicle group ( % of the mncv at d ). morphometric analysis of nerve sections using g-ratio showed an improvement in the thickness of the myelin sheath in curcumin treated animals (+ % vs. vehicle group). histological investigation of gastrocnemius muscle indicated decreased neurogenic lesions in curcumin group. proteomic analysis is currently under investigation to understand the mechanisms involved in curcumin effects. our data could lead to the development of new therapeutic strategies in peripheral nerve regeneration using low doses of curcumin. previous studies suggest that the metabolic syndrome (mets) is associated with distal symmetrical polyneuropathy (dsp), and that diabetes, pre-diabetes, and obesity are the main metabolic drivers. the aim of this study is to investigate the association of mets components with retinal and cognitive function in a bariatric surgery cohort prior to surgery. patients were recruited from the adult bariatric surgery clinic at the university of michigan and lean controls from a research website (no mets components based on ncep/atpiii definition). participants underwent extensive metabolic phenotyping including a glucose tolerance test and fasting lipid profile. dsp was defined using the toronto consensus definition of probable clinical neuropathy. retinal function was measured with frequency doubling technology perimetry (average mean deviation), and cognitive function with the nih toolbox (composite score). univariate linear regression models were used to evaluate the association between mets components and retinal and cognitive function. to date, we have recruited bariatric surgery participants and lean controls. in the bariatric population, the mean (sd) age was ( . ) with % female compared with a mean age of . ( . ) with % female in the lean group. in the bariatric group, . % had diabetes, . % pre-diabetes, and . % normoglycemia. the dsp prevalence was % in lean controls, . % in normoglycemic, . % in pre-diabetic, and . % in diabetic bariatric participants (p< . for trend). retinal function was . ( . ), − . ( . ), − . ( . ), and − . ( . ) (p= . for trend), and cognitive function was . ( . ), . ( . ), . ( . ), . ( . ) (p= . for trend) in these same groups for lean controls, normoglycemics, pre-diabetics and diabetics, respectfully. pre-diabetes (− . , %ci: − . ,- . ) was the only mets component associated with retinal function, and waist circumference was the only one associated with cognitive function (− . , %ci − . ,- . ). dsp and retinal function, but not cognitive function decline with worsening glycemic status. similar to previous data for dsp, pre-diabetes and obesity are associated with retinal and cognitive function respectively. interestingly, while clinical dsp is common in this population, clinical retinopathy and dementia are not, indicating that dsp may be the first metabolic complication in the morbidly obese. and anti-cntn antibody-positive cases were confirmed by cba and were of igg subclass in half of them. by cba we identified additional / ( %) anti-caspr seropositive patients, whose isotype is currently being tested. sera of anti-nfasc and anti-cntn igg seropositive patients and patients with anti-caspr antibodies stained paranodes by indirect immunofluorescence on mouse teased nerve fibers. of note, seronegative patients for known antibodies showed reactivity against node and/or paranodes. compared to other seronegative cipd patients, seropositive patients had more frequently subacute onset of the neuropathy and a younger age at onset, particularly for nfasc or caspr antibodies. weakness was more severe and was often associated with proprioceptive loss, sensory ataxia and tremor. neuropathic pain was not a feature of caspr -seropositive patients. frequent findings were increased distal motor latencies and temporal dispersions on nerve conduction study and a higher protein level in csf. finally seropositive patients tended to have a higher disability and showed worst response to ivig. rituximab was effective in one patient with anti-nfasc antibodies and two patients with anti-cntn antibodies showed good and persistent recovery after cyclophosphamide. prevalence of antibodies was % in italian cidp patients and their presence was associated with distinctive clinical features. their determination, followed by characterization of igg subclass in positive cases, has clear clinical impact, by helping to guide therapeutic choices. the reactivity against nodal and paranodal components in sera from patients without known antibodies suggests that other targets could play a role in the autoimmune response in cidp and they still need to be identified. mutations in amynoacyl trna synthetases (arss), enzymes that catalyse the covalent attachment of amino acids to their cognate trna, are responsible for autosomal dominant cmt , intermediate cmt (cmt-i) and dhmn. we report the case of a male of italian ancestry who first presented with bilateral ankle clonus at three months, followed by toe walking and ankle instability. the ankle clonus subsided during adolescence. in the third decade he developed progressive walking difficulties followed by distal sensory loss. neurological examination at the age of revealed a steppage gait, distal lower limb weakness, decreased pinprick to the ankles, and reduced vibration sensation at the knees. reflexes were brisk in the upper limbs, reduced at the knees and absent at the ankles. muscle tone was increased in the lower limbs and plantar responses were extensor. nerve conduction studies revealed an axonal neuropathy. brain and spinal cord mri were normal. sanger sequencing of pmp , gjb , mpz , gdap and mfn were negative. sureselect focused exome sequencing (agilent technologies, santa clara ca, usa) demonstrated a c. g>a, p.arg his mutation in aars. the p.arg his mutation in aars has previously been reported in families with intermediate or axonal motor-sensory neuropathy, and in one case was associated with sensory-neuronal deafness. cns involvement has not previously been described with this mutation. mutations in aars have been associated with a range of phenotypes including cmti, cmt and dhmn with variable age on onset ranging from to years (mean years). of note, the aars p.gly arg mutation has been reported in a family with cmt and pyramidal signs. this study provides further evidence that pyramidal tract involvement can be an early feature of cmt n due to mutations in aars, further expanding the spectrum of arss-associated phenotypes. small fibres in the skin are vulnerable to damage in metabolic or toxic conditions such as diabetes mellitus or chemotherapy resulting in small fibre neuropathy and associated neuropathic pain (np). whether injury to the most distal portion of sensory small fibres due to a primary dermatological disorder can cause np is still unclear. recessive dystrophic epidermolysis bullosa, (rdeb) is a rare condition in which mutations of proteins of the dermo-epidermal junction lead to cycles of blistering followed by regeneration of the skin. damage is exclusive to the skin and mucous membranes, with no known direct compromise of the nervous system. it is increasingly recognised that most rdeb patients experience daily pain, the aetiology of which is unclear but may include inflammation (in the wounds), musculoskeletal (due to atrophy and retraction scars limiting movement) or np. in this study we investigated the incidence of np and examined the presence of nerve dysfunction in rdeb patients. around three quarters of patients presented with pain of neuropathic characteristics which had a length dependent distribution. quantitative sensory testing of the foot revealed striking impairments in thermal detection thresholds combined with an increased mechanical pain sensitivity and wind up ratio (temporal summation of noxious mechanical stimuli). nerve conduction studies showed normal large fibre sensory and motor nerve conduction however skin biopsy showed a significant decrease in intraepidermal nerve fibre density. autonomic nervous system testing revealed no abnormalities in heart rate and blood pressure variability however the sympathetic skin response of the foot was impaired and sweat gland innervation was reduced. we conclude that chronic cutaneous injury can lead to injury and dysfunction of the most distal part of small sensory fibres in a length dependant distribution resulting in disabling np. these neuropathic pain (np) following peripheral nerve injury is associated with hyperexcitability in damaged myelinated sensory axons which begins to normalise over time. we investigated the composition and distribution of shaker type potassium channels (kv channels) within the nodal complex of myelinated axons following injury. at the neuroma that forms after damage, expression of kv . and . (normally localised to the juxtaparanode) was markedly decreased. in contrast kv . and . , which were hardly detectable in the naïve state, showed increased expression within juxtaparanodes and paranodes following injury, both in the rat and in humans. within the dorsal root (a site remote from injury) we also noted a redistribution of kv channels towards the paranode. blockade of kv channels with dtx after injury reinstated hyperexcitability of a-fibre axons and enhanced mechanosensitivity. changes in the molecular composition and distribution of axonal kv channels, therefore represents a protective mechanism to suppress the hyperexcitability of myelinated sensory axons that follows nerve injury. members of the francophone anti-mag cohort group are listed in "appendix". appendix polyneuropathy with anti-mag igm antibodies is classically progressive, predominantly sensory, and distal with ataxia and sometimes postural-intention tremor. we assessed clinical, biological, electrophysiological, and histopathological features in patients with igm gammopathy and anti-mag antibody titres higher than btu. we focused on characteristics of patients according to the anti-mag antibody titres at diagnosis. we retrospectively and prospectively analysed standardized report forms of patients from fourteen french-speaking neuromuscular centres. mean age at onset was . years (range - . ). mean time between symptoms onset and last follow-up was . years ( . - . ). about . % of patients presented with a "variant" clinical phenotype independently of anti-mag titres (< or ≥ btu). this included acute or chronic sensorimotor polyradiculoneuropathies, paucisymptomatic sensory polyneuropathy and multifocal neuropathy. at the most severe disease stage, . % of patients were significantly disabled. anti-mag antibody titres at diagnosis were low (< btu), medium ( - ) or high (≥ ) in respectively , and % of patients. patients presented with mgus or waldenström's macroglobulinemia in respectively % and % of cases. sixteen percent of patients did not meet the electrodiagnostic criteria of definite demyelinating neuropathy, independently of anti-mag titres (< or ≥ btu). nerve biopsy, performed in nineteen patients, provided support to the diagnosis of anti-mag neuropathy in some particular issues (low titres of anti-mag, unusual clinical or electrophysiological phenotype). we assessed the degree of probability (probable, possible or uncertain) that patient neuropathies are directly related to anti-mag antibodies, according to anti-mag titre, electrophysiological data and nerve biopsy characteristics if available. it appears uncertain in patients with low anti-mag titres ( . % of the whole population). the clinical phenotype didn't appear to be different according to anti-mag antibody titre. many of the patients with low anti-mag titres presented "genuine" anti-mag neuropathy as demonstrated by edx studies, clinical presentation and sometimes nerve biopsies. for a small proportion of these patients, a direct relation between neuropathy and anti-mag antibodies is uncertain due to atypical clinical presentation, axonal neuropathy pattern or nerve biopsies, and positivity of antigangliosides antibodies. we assessed therapeutic management, response to immunotherapies and adverse events in a cohort of patients presenting with igm gammopathy and anti-mag antibody titres higher than btu. we retrospectively and prospectively analysed standardized report forms of patients from french speaking neuromuscular centres. mean age at onset was . years (range - . ). mean time between symptoms onset to diagnosis and last follow-up were respectively . ( - ) and . years ( . - . ). anti-mag antibody titres at diagnosis were low (< btu), medium ( - ) or high (≥ ) in , and % of patients. patients presented with mgus or waldenström's macroglobulinemia in respectively % and % of cases. seventy eight percent (n = ) of patients received immunotherapies. transient response to ivig or plasma exchanges at six month was observed in less than and % of patients respectively. chemoimmunotherapies and rituximab were more frequently administered in the group of patients with malignant hemopathies (n = ) compared to mgus (n = ) (mean lines of therapy = . , range - , sd . versus . , range - , sd . , p = . ). more than % of patients (n = ) received rituximab monotherapy. clinical worsening, mostly transient and reversible, was observed in / patients after rituximab. clinical response to rituximab at months and/or during - months follow-up period was observed in . % of patients, and correlated with anti-mag titre ≥ btu ( / responder versus / non-responder patients, p = . ). at - months follow-up, responder patients presented shorter symptom duration compared to non-responders, though not significant after logistic regression ( . years, range . - . , sd . in responder patients versus . years, range . - . , sd . , in non responder patients, p = . ). in some cases, electrodiagnostic studies were recorded during rituximab treatment follow-up and showed in responder patients a clear improvement of motor conduction velocities. these data may support another clinical trial to study benefice of rituximab in anti-mag neuropathies early in the disease. it raises issues about the value of incorporating electrodiagnostic parameters as end-points. schwann cells (scs) are essential for axon integrity and myelination in the uninjured pns. after pns injury, scs function as "first responders" , undergoing phenotypic re-programming and orchestrating many processes that lead to functional nerve repair. receptors in scs that contribute to sc repair programs remain incompletely understood. we identified a member of the ionotropic glutamate receptor (igr) family, n-methyl-d-aspartate receptor (nmda-r), in scs that is upregulated after nerve injury and acts as a co-receptor with ldl-receptor related protein (lrp ). lrp is a well-known regulator of the sc response to pns injury. herein, we used pcr to profile igr expression in cultured rat scs. obligate receptor subunits required for assembly of nmda-rs, ampa-rs, and kainate receptors were identified. treatment of rat scs with - microm glutamate or . - . microm nmda robustly activated akt and erk / . the response was transient and bimodal; glutamate concentrations greater than microm failed to activate cell-signaling. phosphoprotein profiling demonstrated other cell-signaling and transcription factors regulated by glutamate in rat and human scs, including p s -kinase, glycogen synthase kinase- , ribosomal s kinase, c-jun, and creb. activation of cell-signaling by glutamate in scs was blocked by eliminating calcium from the medium, by the selective nmda-r antagonist, mk , and by genetic silencing expression of the obligate nmda-r nr subunit. phosphoinositide -kinase/pi k functioned as an essential upstream activator of both akt and erk / in glutamate-treated scs. by activating pi k and erk / , glutamate promoted sc migration. glutamate ( microm) or nmda ( microm) injected into crush-injured sciatic nerves robustly activated p-erk / in both myelinating and non-myelinating scs in vivo. these results identify igrs as potentially important cell-signaling receptors in scs that may promote axon-glial interactions. understanding the function of sc nmda-r is important given current efforts to develop nmda-r-targeting drugs for patients with pain, depression, and alzheimer's disease. while frequently overlooked in a therapeutic context, scs are extremely important in the pathogenesis of chronic neuropathic pain. if these drugs modulate the activity of sc nmda-r and sc physiology, the response to pns injury may be altered and the possibility that neuropathic pain develops increased. candayan a , atkinson d , durmus tekce h , parman y , jordanova a , battaloglu e . department of molecular biology and genetics, bogazici university, istanbul, turkey; center for molecular neurology, antwerp university, antwerp, belgium; istanbul medical school, istanbul university, istanbul, turkey. charcot-marie-tooth (cmt) disease is a group of inherited peripheral neuropathies affecting one in individuals worldwide. the disease presents both clinical and genetic heterogeneity. so far, mutations in genes and loci are associated with cmt with autosomal dominant, autosomal recessive, x-linked and mitochondrial inheritance. despite the advances in genetic testing, approximately % of all cmt patients worldwide remain without a molecular diagnosis. we have investigated unrelated cmt patients of turkish descent, in all of which pmp duplication has been excluded previously. we used multiplex amplification of specific targets for resequencing (mastr) assay to sequence exonic regions of common cmt genes. recurrent mutations were identified in cases in mfn , gjb , mpz and hint genes. we have also identified novel variants in cases in mfn , pmp , gars, aars, ighmbp and gdap genes, all of which are very rare or not present in the variation databases and are predicted to be pathogenic by in silico tools. familial segregation analyses are ongoing for novel variations. mfn and gjb genes were the most commonly mutated causative genes in this cohort. cases without molecular diagnosis after the mastr testing are candidates for further analyses such as whole exome sequencing or whole genome sequencing. outcomes of the current study and our previous experience with turkish cmt patients suggest a high genetic heterogeneity. our insight is that different genetic strategies or larger panels are essential to determine the causes underlying cmt especially in regions where rare recessive types of the disease can be observed due to high frequency of consanguineous marriages. capoccitti g , giannini f , ginanneschi f , casali s , insana l , rossi a . university of siena, siena, italy. several efforts have been made to elaborate new electrophysiological criteria for early diagnosis of guillain-barré syndrome (gbs) and to differentiate demyelinating (aidp) from axonal (aman/amsan) forms. the aims were to verify the diagnostic power of total cmap (tcmap) duration, firstly applied in gbs field. this parameter was compared with commonly used neurophysiological measures, including negative phase cmap duration (ncmap), and was added to modified rajabally criteria. we reviewed the clinical and electrophysiological data of patients with gbs (level or of brighton clinical criteria). each patient underwent at least two neurophysiological studies, the first within weeks, the second between and weeks from symptom onset. at least four motor and three sensory nerve conduction studies were recorded for each test. regarding early diagnosis, the binary logistic regression model with multiple variables, including ncmap duration, showed that the features of predictive model presenting greater significance (p < . ) were tcmap duration, sural sparing and a-waves. among these, tcmap duration showed greater significance (p = . ). the tcmap was diffusely prolonged in aidp compared to aman/amsan, already in first examination and confirmed in the second one. roc analysis for tcmap duration in aidp vs. aman/amsan showed: cut-off . ms, auc . , ppv . %. we propose the tcmap duration as a new useful electrophysiological measure for early diagnosis of "generic" gbs and for early differentiation between aidp and aman/amsan. moreover, the prolongation of tcmap, the presence of a-waves and sural sparing represent a strongly diagnostic predictive triad of aidp. aifm (apoptosis-inducing factor, mitochondrionassociated- ) has captured great attention from biomedical researchers due its critical role in the regulation of cell apoptosis. this flavoprotein is typically located in the mitochondrial intermembrane space where it is associated with respiratory chain complex-i. upon a cell-death insult, aifm is cleaved into a kd protein that is released into the cytosol. the kd peptide may enter the nucleus to trigger chromosome condensation and fragmentation, initiating a caspase-independent pathway of apoptosis. however, this nuclear translocation may be blocked by cytosolic heat-shock protein- (hsp ) that binds with the fad domain (aa - ) of aifm . mutations in aifm gene have resulted in several clinical phenotypes, including a family with cmtx (glu val). clinical deficits in these patients usually involve multiple organs. in this study however, we identified a family with a novel missense mutation (phe leu) in aifm that developed a late-onset sensory motor axonal polyneuropathy by nerve conduction criteria. the proband and affected siblings exhibited distal muscle weakness and atrophy with normal cognitive and cranial nerve functions. there was no obvious phenotype from other organs. interestingly, this phe leu mutation affects a highly conserved amino acid at the center of the fad domain. we hypothesize that this mutation impairs the binding between aifm and hsp , leading to an enhancement of cell-death signaling. this family therefore provides a unique opportunity to explore how altered apoptotic signaling affects peripheral nerve system. supported by grants from ninds (r ns ) and the national center for advancing translational sciences (ul tr chronic inflammatory demyelinating polyneuropathy (cidp) can be the key symptom leading to diagnosis of associated lymphoma. patients with diagnosis of cidp according to efns/pns criteria associated with b cell lymphoproliferative disorders (bld), in one center, between november and november were included. demographical, clinical, nerve conduction, immunological, histological data and response to treatment were recorded retrospectively. eight patients ( men), median age yo were included. onset of polyradiculoneuropathy was either chronic (n= ) or acute (n= ). neurological condition led to diagnosis of bld in all but one case, because of onset (n= ) or worsening of neuropathy (n= ). clinical presentation was that of cidp in patients or pseudo-canomad in and plexopathy in one. lymphoma type was: lymphoplasmacytic (n= ), diffuse large b cell (n= ), small lymphocytic (n= ), marginal zone (n= ), unclassified small b cell (n= ). only patients presented with lymphadenopathies. bld was diagnosed in all patients on myelogram or bone marrow biopsy, performed because of cytopenia (n= ), atypical (n= ) or severe (n= ) neuropathy. monoclonal gammapathy was identified in / patients (igm n= , igg n= ). neuromuscular biopsy was performed in patients and disclosed endoneural infiltration in . anti-neuronal or antigangliosides antibodies were positive in patients. none of the patients presented a systemic autoimmune disease, hemolytic anemia associated with bld (n= ). immunomodulating treatment was administered in all patients (ivig n= , plasma exchange n= , steroids n= ) and immunosuppressants (n= ). immunochemotherapy for lymphoma was initiated because of lymphoma type or severity in cases, in cases because of the associated neuropathy. median follow-up was of months after treatment initiation. four out of patients treated within months of neurological onset improved as well as one out of patients whose preexisting neurological condition had worsened. two patients presented neurological relapse during progression of lymphoma. two patients died. unusual presentation of cidp -i.e., rapid progression or treatment failure -should lead to further testing for associated lymphoma. because general symptoms and lymphadenopathy often lack, diagnosis requires analysis of bone marrow with lymphocyte phenotyping. early treatment with immunochemotherapy was associated with better prognosis in our series. cardiac scintigraphy is a useful tool for the diagnosis, prognosis and pre-symptomatic early detection of familial amyloidosis-associated neuropathies three main types of familiar amyloidosis: transthyretin (ttr), apolipoprotein a and gelsolin. cardiac involvement is a leading cause of morbidity and mortality; one new described mutation strongly related with isolated cardiac amyloidosis is the ttr val ile. the discovery of tests that allow early diagnosis of cardiac involvement in amyloidosis and to infer about the etiology of the disease is of major importance. in a cohort of patients with different types of familiar ttr amyloidosis, we aimed to assess the role of mtc- , -diphosphono- , -propanodicarboxylic acid ( mtc-dpd) in detecting myocardial amyloid infiltration. we enrolled four patients diagnosed with late familiar amyloidosis, which mutations were documented at deoxyribonucleic acid analysis: three patients with ttr val met mutation and one patient with ttr val ile mutation. three patients were asymptomatic for cardiac involvement and one patient (val ile mutation) had a previous diagnosis of heart failure. myocardial uptake of mtc-dpd scintigraphy was semiquantitatively and visually assessed at five minutes and three hours.the uptake of mtc-dpd highly demonstrated amyloid in cardiac area in two out the three cases of ttr val met and in ttr val ile. ttr val ile case presented the highest uptake due to the exclusive deposition of amyloid in cardiac area resulting in severe heart failure. in hereditary transthyretin-related amyloidosis, including the mutations ttr val met and val ile, mtc-dpd cardiac scintigraphy can identify infiltration even in asymptomatic individuals, allowing an early diagnosis of cardiac compromise in this group of diseases. we can consider that this non-invasive test would be a tool with potential importance in the diagnosis, prognosis and pre-symptomatic early detection of cardiac amyloidosis, giving emphasis on its applicability in familial forms of amyloidosis. cervellini i , galino j , zhu n , birchmeier c , bennett dl . ndcn university of oxford, oxford, uk; max-delbrück-center for molecular medicine, berlin, germany. erk/mapk pathway has a critical role in pns development since its involvement in many physiological processes. sustained erk / mapk activation in schwann cells enhances myelin growth during development and overcomes signals ending myelination leading to a continuous myelin production. however, strong activation of erk has also been shown to cause schwann cells dedifferentiation and demyelination in vivo. our aim was to investigate whether a mild activation of this signalling pathway in adult schwann cells (scs), by expression of gain of function mek dd allele, could have a beneficial role in remyelination and regeneration after injury. erk/mapk activation in adult scs in plpcreer t ;mek dd mutant mice, did not affect myelination during development. following sciatic nerve injury, wallerian degeneration was enhanced in mutants pushing towards a dedifferentiation stage of scs as previously described. however, mapk activation was detrimental during regeneration with a delay in functional recovery and a negative impact in both myelinated and non-myelinated fibres compared to controls. one month after injury the total number of axons in mutant sciatic nerves was half of the controls. although no differences in g-ratio have been found in the two groups, mutants presented a higher number of myelinated axons showing myelin disruption with start of myelin decompaction, lack of cajal bands, abundant sc processes surrounding axons and a shorter sc elongation, as seen by decreased internodal distance. in addition, we found a negative effect of mapk activation also in small diameter axons with the presence of abnormal remak bundle structures, reduced number of c-fibres/remak bundle and a significant decrease in intra epidermal nerve fibres density in the skin. we concluded that mild mapk activation has a different role in development and remyelination where negatively affects axon survival, myelin stability, remak bundle formation and small fibres regeneration. cervellini i , galino j , zhu n , bao lu , bennett dl . ndcn, university of oxford, oxford, uk; harvard medical school, boston, ma, usa. neprilysin (nep) is an endopeptidase which has been of interest due to its potential role in neurodegeneration and pain as a consequence of its ability to degrade amyloid and substance-p respectively. nep expression is not limited to cns and it has been reported to be expressed in schwann cells, nodes and schmidt-lanterman incisures. our interest in this gene was related to recent findings that have associated homozygous and heterozygous nep mutations with charcot-marie-tooth type- . in old mice lacking nep subtle morphological changes have been reported. our aim was to determine whether nep expression was modulated by nerve injury and to investigate its role in axon regeneration and re-myelination. we find that nep gene expression was decreased after nerve crush and furthermore was dependent on the growth factor (and pro-myelin signal) neuregulin- . in control mice nep expression was transiently reduced and returned to baseline at day after injury, in neuregulin- knock-out (ko) mice, in which re-myelination was impaired, the expression was still decreased at day . in assessing behavioural measures of locomotor and sensory function one month after sciatic nerve crush, nep ko mice showed a functional regeneration comparable to wt, as seen by sciatic functional index measurement, beam and toe spreading tests. the only significant difference we observed between wt and ko was in the sensorial test, showing ko mice recovering faster in the pinch test by days after crush. the results for all the tests at baseline did not differ between the two groups. detailed histological analysis of nerve repair was undertaken using electron microscopy. there was no difference between wt and ko in total axon number, g-ratio, axon diameters and other myelin features one month post crush. in summary, although nep expression is regulated by nerve injury in a neuregulin- dependent fashion this endopeptidase is dispensable for axon regeneration and re-myelination after nerve injury in the rodent. diabetes neuropathy is a common complication of diabetes, and neuropathic pain has a detrimental impact on quality of life. this study investigated sensory nerve excitability properties to elucidate the axonal changes of diabetic neuropathy. a total of diabetes patients ( type ii, and type i) were enrolled in this study. clinical assessment, nerve conduction studies, and nerve excitability testing data were analyzed to determine axonal dysfunction in diabetic neuropathy. among those patients, seventeen subjects had complained of spontaneous painful sensation over feet or hands (painful cohort), and seventy-eight patients had no sensory symptoms or decreased the sensation over foot (non-painful cohort). sensory nerve excitability of the painful cohort showed reduced late subexcitability (p= . ), increased superexcitability (p= . ) in compared to the non-painful cohort. there is no difference in disease duration, blood glucose levels (hba c) between these two cohorts. these findings suggested the possible pathogenesis of painful sensory axons might be hyperpolarized or slow potassium channels dysfunction. these insights our further understanding of painful diabetic neuropathy, and may provide a basis for neuroprotective or therapeutic approaches for painful polyneuropathy. the main purpose of this study was to assess the clinical feasibility of diffusion tensor imaging (dti) for the diagnosis of chronic inflammatory demyelinating polyradiculoneuropathy (cidp). between march and december , we prospectively enrolled patients with definite cidp according to the efns criteria and two control groups: healthy volunteers matched on age and sex and patients with cmt- a. using a -t magnetic resonance imaging scanner, we obtained dti scans of brachial plexus of these groups and prepared fractional anisotropy (fa) maps, and compared these values between groups. adc values and cervical nerve roots diameters on stir sequences were evaluated too. two neuroradiologists, blinded to clinical informations, reviewed mri studies independently. in all patients with cidp, we also performed clinical evaluation and electroneuromyography. significantly decreased fa values (p< . ) and increased adc values were observed in cidp patients compared with healthy subjects. there is no significant difference between cidp and cmt group. inter-observer concordance was excellent for fa values ( c= . ; p< . ) and moderate for adc values ( c= . ; p< . ) and cervical nerve root diameters ( c= . ; p< . ). there is a significant correlation between fa and disease duration (r = − . , p < . ), inclusion mrc score (r = . , p < . ) and between fa mesured on c c and incat score at inclusion (r = − . , p < . ). no significant correlation is observed between fa and electrophysiological indices. compared with healthy subjects, cervical nerve root diameters were significantly increased (p< . ) in patients with cmt and cidp. contrary to fa values, moderate level of concordance was found between inter-observers measurements of diameters (cclin = . ). our preliminary data prove the clinical feasibility and reproductibility of dti for the evaluation of plexus and cervical nerve roots in patients with cidp. cheng yj , teng a , goh ejh , umapathi t . yong loo lin school of medicine, national university of singapore, singapore; department of neurology, national neuroscience institute, singapore. the sural-sparing pattern of the sensory nerve action potentials (snap) of guillain-barré syndrome (gbs) has been attributed to greater immunological injury of the blood-nerve barrier at its most vulnerable regions. we asked if entrapment sites, such as median nerve at the wrist, are more predisposed than the distal nerve endings to such injury. we compared the median snap with radial snap measured antidromically at digit in gbs patients whose nerve conduction study showed the sural-sparing pattern. the terminal nerves at digit are of similar length, but those of median nerve are prone to compression, often subclinically, at the carpal tunnel while those of radial nerve are not. we defined the sural-sparing pattern as a greater decrease in median and or ulnar snap than that of the sural, compared to age and height-matched normal controls. a total of gbs and miller fisher patients from our institution's database were studied. patients had the sural-sparing pattern, of whom had pre-existing carpal tunnel syndrome. of the remaining patients with sural-sparing, had abnormal median snap at digit , while had both abnormal median and radial snaps at digit . none had isolated abnormality of the radial digit snap. among the cases that had abnormal median and radial snaps at digit , the mean percentage decrease when compared to age and height matched norms was greater in median nerve compared to radial nerve ( % and % respectively). of the patients without sural-sparing pattern, had normal snaps; patient had inexcitable sensory nerves while the other had a length-dependent decrease in snap. in the latter patient, unlike those with sural-sparing, there was no differential decrease of median snap over radial snap at digit . our findings suggest that the disruption of blood nerve barrier at entrapment sites rather than the distal nerve endings may underlie the pathophysiology of the sural-sparing pattern seen in gbs. chiba a , uchibori a , gyohda a . kyorin university, tokyo, japan. serum igg anti-gq b antibody is the most specific biomarker for fisher syndrome and its related disorders (fs-rd), but approximately - % of the patients are seronegative for it in conventional assays (gq b-seronegative). some molecules need ca + cation to interact with their ligands, and antibodies with such a property (ca + -dependent antibodies) are reported. we have found that such a ca + -dependency is also present in igg anti-gq b antibody, and majority of gq b-seronegative patients with fs-rd have this type of antibodies. in patients with final clinical diagnoses as fs-rd (fisher syndrome, guillain-barré syndrome with ophthalmoplegia, bickerstaff brainstem encephalitis, and acute ophthalmoplegia), were seropositive for igg antibodies against gq b-related antigens (isolated gq b in , and gq b-conatinig complexes in two) in conventional elisa using phosphate-buffered saline. in the remaining patients, eight ( %) turned positive for igg antibody against gq b-related antigens (isolated gq b in seven and gq b-conatinig complexes in one) in elisa using ca + -added tris-buffered saline. the reaction strengths increased depending on ca + concentration, and reached to nearly maximum level in the physiological concentration. all the patients with the ca + -dependent antibodies were also positive for igg antibody against gt a-related antigens, suggesting that the terminal disialo residue common to both the gangliosides would be important as an epitope also for the ca + -dependent antibodies. in the patients with ca + -non-dependent antibodies, only two showed increased titers of igg anti-gq b antibody by adding ca + , and showed significantly decreased titers. this difference in the effect of ca + -addition between ca + -dependent and ca + -non-dependent antibodies suggests that ca + would not be just an enhancer of the antigen-antibody reaction. there are four single bonds between the two pyranose rings in the terminal disialo, and those rotatable bonds make it possible for the disialo structure to take various conformations. a molecular model shows that the distance between two minus-charged carboxy groups in the disialo could vary from nearly zero to approximately , pm and that the disialo would take specific conformations, if divalent ca + cation, which size is approximately pm in diameter, interacts with these two minus-charged groups. the ca + -dependent antibodies might recognize such particular conformations of gq b. charcot-marie-tooth disease (cmt) is a genetically and clinically heterogeneous disorder with variable inheritance modes. it is characterized by loss of muscle tissue and touch sensation, predominantly in the feet and legs but also in the hands and arms in the advanced stages of disease. as several molecules have been reported to have therapeutic effects on cmt, depending on the underlying genetic causes, exact genetic diagnostics have become important for executing personalized therapy. aminoacyl-trna synthetase (arss) genes encode enzymes responsible for charging trna with corresponding amino acids. arss are ubiquitously expressed, essential enzymes responsible for performing the first step of protein synthesis. specifically, arss attach amino acids to their cognate trna molecules in the cytoplasm and mitochondria. recent studies have demonstrated that mutations in genes encoding arss can result in neurodegeneration, raising many questions about the role of these enzymes in neuronal function. mutations in six cytoplasmic ars genes have been reported as the cause of cmt. this study was performed the whole exome sequencing to identify genetic defects in korean cmt patients from unrelated families. variants were sorted with cmt gene list that includes almost genes were related cmt neuropathy, and additionally sorted wes data as ars genes. capillary sequencing for family members and more than controls revealed five novel mutations, c. g>a (p.d n), c. c>t (p.s f), and c. c>a (p.p h) in gars; c. c>a (p.p t) in mars; _ ga>at (p.d i) in yars gene in each family. the mutation sites were well conserved between different species and each mutation were located in the well-conserved catalytic domain or between two catalytic domains or anticodon-binding domain. in silico analysis predicted all mutations may affect protein function. clinical features were similar to those reported in other countries, but differed in terms of age at onset and degree of disability. we believe that those novel ars mutations are the underlying causes of the each family. a -year-old man presented with a -year-history of weakness in biltearal upper limbs. he was complaining of intermittent fasciculation of upper and lower limbs with gradually worsening of paresthesia for years. dysphagia and dysarthria were also presented years ago. there was no patient affected muscle weakness and bulbar symptoms in his family members. in neurological examination, the patient had weakness in biltereal upper and lower limbs (mrc grade ) and prominent distal sensory loss were combined in length dependant pattern. deep tendon reflexes were absent on bilataral biceps and knee joints. in nerve conduction study, there was consistent with demyelinating sensorimotor polyneuropathy. molecular diagnostic analyses those spinobulbar muscular atrophy (sbma) and mutation related to peripheral myelin protein (pmp ) gene were performed and confirmed expansion of expansion of a polymorphic cag in androgen-receptor (ar) gene and deletion of pmp gene. smba, also known as kennedy disease, is an adult-onset, x-linked recessive trinucleotide, polyglutamine (poly-g) disorder caused by expansion of a polymorphic cag tandem-repeat in exon of ar gene on chromosome xq - . charcot-marie-tooth disease (cmt) is the most common hereditary neuropathies and cmt cases with motor conduction velocities(mcvs) of upper limb below m/s are defined as demyelinating (cmt ) and those with mcvs above m/s are defined as axonal (cmt ). most families with cmt linked to duplication of pmp gene on the short arm of chromosome ( p . ), called cmt a. the reciprocal deletion of pmp gene is a responsible genetic defect in % of hereditary neuropathy with liability to pressure palsy (hnpp). these "classical" phenotypes of cmt a and hnpp have been considered which are determined by different mutation mechanism of the same gene. however, an overlap of cmt a and hnpp due to pmp gene deletion was reported that suggestion the phenotype of hereditary neuropathies may differ variably. herein, we report a patient who simultaneously presented clinical and electrophysiologic features of smba and cmt a with genetical confirmation of cag expasion and deletion of pmp gene. charcot-marie-tooth type a disease (cmt a) is an inherited peripheral neuropathy stemming from overexpression of pmp protein in schwann cells due to the duplication of the pmp gene. this leads to abnormal schwann cell differentiation and dysmyelination, eventually leading to axonal loss and muscle wasting. no approved treatment is currently available for cmt a. we conducted a systems biology level analysis of the signaling network putatively underlying the processes driving cmt a pathology. based upon this, we identified and tested three repurposed drugs -baclofen, naltrexone and sorbitol -alone and in combination to determine their ability to rescue aberrant myelination in cultures derived from cmt a transgenic rats overexpressing pmp gene. to this end, we studied a validated in vitro co-culture model of sensory neurons and schwann cells adapted to -well culture plates. this model allows measurement of the appearance of myelin proteins as an index of the physiological process of in vivo myelination. total myelin length was quantified with an automatic image analyzer following pmp immunostaining. we first determined the full dose-response curves of single drugs, emphasizing their promyelinating activities. we then tested binary combinations of very low and inactive doses of each drug and compared these to the activity of the combination of the three, namely pxt . whereas combination of any two drugs was not significantly active at the doses tested, combination of all three produced a synergistic improvement in myelination. these findings clearly demonstrate the necessity of using pxt over its single components and highlight the value of pleiotropic combinational repurposing of drugs at low doses as a novel approach for rapid drug development in cmt a and other disorders. autonomic dysfunction is frequently observed in guillain-barré syndrome (gbs) and affects approximately % of the patients. it has been shown that the sweating function can be impaired in gbs. the aim of the present investigation is to summarize the current knowledge on sweating disturbances in gbs patients. we have used appropriate terms to systematically search for references published until and indexed in the following databases: medline, embase, lilacs and cochrane. the inclusion criteria were a diagnosis of gbs and a description of the methods used to test the sudomotor function. the search was limited to the english language. relevant information about study design, methods of assessing the deficit of sweating, patient's characteristics and main results were collected. we selected original references for the final analyses. the majority of the studies were cross-sectional in nature and there were two longitudinal studies. the severity of sweating impairment varied according to the applied method, ranging from normal to almost sympathetic nervous system failure. in seven research papers, the sympathetic skin response was used to evaluate the sudomotor function in patients, and approximately % demonstrated abnormal results. however, researchers used different stimulation protocols and parameters to interpret their results. regarding whole-body sweating test, four research papers applied the thermoregulatory sweating test in patients and they showed areas of anhidrosis on the lower limbs in all of them. eight patients presented sweating impairment on the upper limbs and abdominal wall. results on the sudomotor axon reflex test suggested a length-dependent pattern of sweating loss according to one case report. in another study, eight gbs patients were tested only on the distal leg and foot dorsum and the authors proposed an association between post-ganglionic sudomotor function and antiganglioside antibodies. the present literature review showed that the studies of sweating disturbances in gbs patients included only small cohorts. future studies with larger patients sample sizes are necessary to investigate the patterns of sweating loss in gbs and their changes along the follow up. funding: grants # / - and # / - , são paulo research foundation (fapesp). sensory neuronopathy (sn) represents a rare subgroup of peripheral neuropathies characterized by degeneration of primary sensory neurons at the dorsal root ganglia on the spinal cord. depending on the neuronal population affected, its clinical presentation may manifest as gait ataxia, proprioceptive sensory loss and positive and negative sensory symptoms. although a few reports have mentioned areas of anhidrosis in sn, we were not been able to find previous case series studies on the sweating function in sensory neuronopathies. the aim of the present investigation was to study the whole-body distribution of sweating on both anterior and posterior surfaces in patients diagnosed with sn. quantitative sensory testing for cold and warm sensation threshold (method of levels) was performed on the dorsum of the hands and feet in a randomized order. we tested the thermoregulatory sweating using a sweat chamber ( to ∘ c air temperature and % relative humidity). the oral and skin temperature was monitored and the test time did not exceed min. in order to study the sudomotor axon reflex we employed the q-sweat device on standardized body sites. the test was performed on both sides, simultaneously. we included seven patients (three male; mean age . years) with a mean disease duration of . months (range - ) and a confirmed diagnose of sn. patients presented an asymmetrical loss of cold and warm threshold on hands or feet compared with healthy control (p< , ). regarding the tst results, we found a striking variation of sweating disturbances, ranging from small areas of anhidrosis on the trunk to complete failure of the sympathetic nervous system. two patients underwent the axon reflex test and there was an asymmetrical and mostly distal pattern of sweating loss in one of them and a distal-symmetrical on the second one. our findings indicate a great variability of sweating losses in sn, not overlapped to the sensory loss areas. currently, we are testing more patients in order to confirm our results. funding: grants # / - , # / - and # / - , são paulo research foundation (fapesp). diet, exercise, and inflammation are established modulators of peripheral nervous system function, including pain. prior work examining exercise consistently demonstrates a benefit on heightened pain from a number of acute and chronic pain models. in the present work, we investigated several parameters of peripheral nerve function relevant to pain in rats bred for high (high capacity runners, hcr) or low running capacity (low capacity runners, lcr). the longtime selective breeding of these rat substrains has created divergent intrinsic aerobic capacities and predisposition of metabolic conditions between lcr and hcr rats. examination of the role of sex in the development of chronic pain has established key differences in males and females. to understand gender specific differences, this study focused on female rats to understand the role of metabolic status and peripheral nerve function in females. our analysis identified numerous parameters of peripheral nerve function relevant to pain and neuropathy that are different among lcr and hcr female rats. lcr female rats display reduced hind paw mechanical sensitivity, increased hind paw intraepidermal nerve fiber density and trka-positive epidermal axons, increased numbers of langerhans and mast cells in the hind paw dermis, and increased overall fat mass relative to body weight compared to female hcr rats. examination of sensory and motor nerve conduction velocities, thermal sensitivity, and mrna expression of selected genes relevant to peripheral sensation found no differences between hcr and lcr females. together these results suggest that a genetic component of aerobic capacity and metabolic status can influence sensory sensitivity and specific aspects of inflammation and immune responses in peripheral tissues, which may lead modify the animal's responses to tissue damage and painful stimuli. the lcr and hcr rat model will provide a useful model in the future to assess the involvement of metabolic status in the development of pain. ivig is often considered treatment of first choice in chronic inflammatory demyelinating polyradiculoneuropathy (cidp) because of its rapid onset of action and its relatively safe long-term adverse event profile. clinical trials published so far focused on a loading dose of . g/kg ivig and/or a standard maintenance dosage of . g/kg ivig once every weeks, but have not investigated different dosing options. this study is a prospective, double-blind, randomized, parallel group, multi-center phase iii efficacy study and will be conducted in centres in canada, eu, russia, ukraine and australia. adult patients with definite or probable cidp according to the efns/pns criteria will be enrolled and randomized : : to receive either . g/kg or . g/kg or . g/kg ivig (panzyga ® ) for seven maintenance infusions at -week intervals during the dose-evaluation phase. the starting loading dose will be . g/kg ivig (panzyga ® ) for all patients. primary objective: efficacy measured as percentage of responders (decrease in adjusted incat score by at least point) in the . g/kg ivig (panzyga ® ) arm (given every weeks) at week as this should corroborate the existing and published evidence on efficacy of ivig in cidp. secondary outcome: percentage of responders at week in the . g/kg and . g/kg ivig (panzyga ® ) arms relative to baseline and compared to the . g/kg arm. the procid study aims to confirm published clinical results obtained with the . g/kg standard dose and will in addition evaluate one higher and one lower maintenance dose, with the aim to offer cidp patients a more adequately dosed and effective treatment policy. * este medicamento no se encuentra comercializado en españa. understanding the rate of disease progression in patients with charcot-marie-tooth disease (cmt), both within and between subtypes is important for clinical prognosis and is crucial for clinical trial design. due to the progressive nature of cmt, intervening at the earliest stages of the disease is a priority. measuring progression of a disease with both motor and sensory deficits requires a multi-item composite scale. the cmt pediatric scale (cmtpeds) is a well-tolerated psychometrically robust -item scale measuring fine and gross motor function, strength, sensation and balance for children and adolescents aged - years with cmt. the aim of this study was to determine the rate of disease progression of children and adolescents within and between genetic subtypes of cmt. ( female) participants aged - years enrolled in the inherited neuropathies consortium were included in this study. demographic, anthropometric and diagnostic information were collected at baseline and -year follow-up. disease progression was measured with the cmtpeds. on average cmtpeds scores progressed at a rate of . points over -years ( % change from baseline, p< . ). there was no difference in rate of disease progression between males and females. of the most common genetic subtypes, participants with cmt a/pmp duplication progressed by . points ( % change from baseline, p< . ), nine participants with cmt b/mpz mutation progressed by . points ( % change), six participants with cmt a/mfn mutation progressed by . points ( % change) and seven participants with cmt c/sh tc mutation progressed by . points ( % change). participants with cmt a progressed faster than those with cmt a (p= . ). children with cmt a progressed consistently during childhood and adolescence while children with cmt b and cmt a progressed faster during childhood than adolescence. overall, children with cmt progress at a significant rate over -years according to the cmtpeds. understanding the rate at which affected children deteriorate is essential for adequately powering clinical trials of disease-modifying interventions. queen square, london, uk; department of neurology, university of iowa carver college of medicine, iowa city, ia, usa; department of neurogenetics, the national hospital for neurology and neurosurgery, ucl institute of neurology, london, uk; department of molecular neuroscience, ucl institute of neurology, london, uk and national hospital for neurology and neurosurgery, queen square, london, uk. in recent years, targeted ngs panels have changed the diagnostic work-up in patients with inherited neuropathies. however, there is limited data on the impact of targeted ngs panels on the diagnosis of cmt patients in everyday practice. the aim of this study was to investigate the impact of targeted ngs panels on the diagnosis of cmt across two tertiary referral centres in the united kingdom (london) and united states (iowa). in london, patients with a diagnosis of cmt (previous pmp duplication and common cmt genes excluded in appropriate cases) underwent targeted ngs panel sequencing covering genes associated with cmt and additional genes associated with hsp or als. a variable number of genes, ranging from to , were analysed depending on the clinical phenotype of the patients. a definite molecular diagnosis was achieved in cases ( %) including pathogenic and likely pathogenic mutations in sh tc ( cases), gjb ( cases, including cases with mutations in the promoter and 'utr regions), gdap ( cases), fgd ( cases), aars ( cases), ighmbp ( cases), mpz ( cases), nefl ( cases). vus were further identified in patients. the diagnostic rate was higher in demyelinating cmt cases ( / , %), compared to cases with axonal cmt ( / %), dhmn ( / , %) and hsn ( / , %). in iowa, patients were investigated by ngs panels covering to genes associated with cmt. a molecular diagnosis was reached in / ( %), and in particular / ( %) demyelinating and / ( %) axonal cmt cases. the most frequent genes identified were gjb ( cases), mfn ( cases), sh tc ( cases) and ighmbp ( cases). vus were identified in patients, including cases with novel variants in aars, warranting additional testing such as segregation of the variant in the family or functional validation studies. in clinical practise, targeted ngs panels represent an effective approach for the diagnosis of cmt. the lower diagnostic rate in london is likely to be due to prior sanger sequencing and exclusion of mutations in common cmt genes in this patient population. more than genes are known to cause cmt and an even larger number are known to cause peripheral neuropathy as part of a more complex neurological disorder. despite the use of custom panels, a significant proportion of patients with inherited neuropathy have no molecular diagnosis. the aim of this study was to investigate the diagnostic yield of a disease-associated gene exome (sureselect focused exome, agilent technologies, santa clara ca, usa) in the diagnosis of cmt and in cases with complex neurological syndromes associated with neuropathy. thirty-one patients with molecularly undiagnosed inherited neuropathy were analysed with sureselect focused exome sequencing. six patients had a more complex phenotype including learning difficulties, cerebral white matter changes, ataxia and pyramidal tract involvement. a genetic diagnosis was achieved in / ( %) of cases by detecting a mutation in cmt-associated genes mpz ( cases), aars, nefl, bscl , bicd and trpv . of note, six cases had mutations in genes which are not covered by currently available diagnostic targeted ngs panels, including kif a, polg, mme ( cases), dnajb , and a novel candidate gene. the average coverage was higher compared to the usual coverage of whole-exome sequencing; % of the targets were covered at x or more, and % of the targets were covered at x or more. this study provides evidence that the sureselect focused exome is a useful tool for the diagnosis of cmt and complex neurological disorders and provides further insight into the phenotypic spectrum of genes associated with inherited neuropathy. changes in the cis-regulatory sequences of a gene's untranslated regions (utr) are increasingly recognised as a significant cause of inherited disease in humans. for example, variants in the non-coding region of gjb account for % of all mutations in our cohort of cmtx patients. one of the biggest challenges in analysing the large number of non-coding variants in a gene is identifying those that are disease-causing and those which are polymorphisms. the aim of this study was to implement a reliable method for the in-vitro functional validation of non-coding variants in the promoter and 'utr regions of gjb . in our cohort of cmtx patients we have previously identified seven mutations (c.- _ inst, c.- g>a, c.- t>g, c.- t>c, c. t>c, c.- g>c, c.- c>t) in the promoter region and one novel mutation in the 'utr (c. c>t), which were considered likely to be pathogenic based on the clinical phenotype, segregation in affected family members and absence in control databases. we have now generated a luciferase-based reporter system and optimised it in a hela cell line. mutations in the promoter region were generated by site-directed mutagenesis using a commercially available gjb promoter clone (genecopoeia). our preliminary results show a reduction of luciferase activity for the c.- t>c and c.- _ inst mutations compared to the wild-type promoter. this difference was increased when transcription factors sox or egr were co-transfected with the t>c and c.- _ inst mutations respectively. validation of other variants is currently ongoing. if successful, our study will provide a useful tool for the validation of mutations in non-coding regions of gjb . moreover, it will constitute a proof-of-principle approach to the functional validation of non-coding variants in other cmt genes known to cause disease by a loss of function. recessive and dominant mutations in leucin-rich repeat and sterile alpha-motif-containing (lrsam ) have been associated with cmt p. lrsam is a ubiquitin e ligase containing a ring domain in its c-terminal, which is crucial for correct protein folding and ubiquitination activity. to date, the majority of dominant mutations reported have resulted in a frame shift disrupting a major portion of the ring-domain, although point mutations in this domain have also been described. the aim of this study was to report the prevalence, clinical features and genetic findings of patients with cmt p in our centre. we performed targeted next-generation sequencing in genetically undiagnosed cmt patients and identified cases with heterozygous mutations in lrsam ( . %) from unrelated families. the mutations identified included frameshift insertions and deletions, a non-frameshift deletion and non-sense and missense point mutations. all of the mutations were novel and were located in or flanking the ring domain. the average age of disease onset was in the rd decade but an earlier onset was reported in two cases. four had a positive family history in keeping with autosomal dominant inheritance. symptoms at presentation were heterogeneous and encompassed distal numbness, unsteadiness, distal weakness of upper or lower limbs and foot deformities. positive sensory symptoms, including tingling and shooting pains, and cramps were also frequently reported. neurological examination showed mild to moderate distal atrophy and weakness, with early ankle plantar flexion involvement in three patients. loss of vibration and reduced joint position sense were often prominent in the lower limbs and appeared to be disproportionate to the degree of weakness and impairment of pinprick sensation. ankle jerks were absent but knee and upper limb reflexes could be normal or brisk. after an average disease duration of years, all but one patient was able to walk independently. nerve conduction studies showed a sensory and motor axonal neuropathy with normal conduction velocities. our study highlights that mutations in lrsam are a relevant cause of cmt and are associated with prominent large fibre sensory loss. in recent years, the implementation of ngs panels for the molecular diagnosis of cmt has increased the number of patients with a genetic diagnosis. nevertheless the interpretation of a particular variant as disease causing can be challenging especially when multiple variants are identified in a single patient. we report two illustrative cases of such challenges. the first index case was born of non-consanguineous healthy parents. he presented with falls in early childhood. over the years he developed foot deformities and progressive length dependent weakness. he had multiple orthopaedic operations to his feet. the past history was also notable for kyphoscoliosis, sensorineural deafness from the age of and bilateral cataracts. nerve conduction studies at the age of revealed a demyelinating neuropathy consistent with the clinical phenotype of cmt . his younger brother had a similar, although more severe phenotype. a litaf (c. c>t,p.pro ser) mutation was identified by sanger sequencing and was present in both affected brothers but also in the unaffected sister. ngs for cmt -associated genes was therefore performed and identified two compound heterozygous pathogenic mutations detected in sh tc (c. c>t, p.arg *; delg, p.arg serfs* ), which segregated with the disease in the family. the second case describes a brother and sister with early onset demyelinating cmt associated with scoliosis and cranial nerve involvement. the male proband underwent ngs and a single previously reported pathogenic intronic splice-site mutation in sh tc (c. - a>c) was found. relative read-depth analysis of ngs was performed to look for possible copy number variants in sh tc , thus identifying a deletion of exon , which was confirmed by long pcr. cruz-velásquez gj , miramar-gallart md , alarcia-alejos r , roche-bueno jc , rodríguez-valle a , capablo-liesa jl . neurology service, university hospital miguel servet, zaragoza, spain; genetics unit, clinical biochemistry service, university hospital miguel servet, zaragoza, spain. charcot-marie-tooth disease (cmtd) defines a clinical and genetically heterogeneous group of inherited peripheral neuropathies characterized by chronic motor and sensory impairment. the type cmtd- also known as cowchock syndrome, is the product of the mutation in the apoptosis inducing factor mitochondria associated gene (aifm ). it is a slowly progressive, recessive, x-linked disease characterized by axonal neuropathy, deafness, and cognitive impairment. our purpose is to describe new cases, brothers, children of non-consanguineous parents, with a characteristic phenotype and a new mutation in the aifm gene. both siblings present from childhood, progressive weakness in lower limbs with diffuse amyotrophies. needing tenotomy before the year due to equinovarus foot. likewise they develops sensory deafness and one of them requires unilateral support at and the other wheelchair at , this one need a pacemaker for an atrioventricular block at . with brain functions and normal language, sensorial deafness, proximal and distal weakness in the four limbs with intense amyotrophies, predominantly distal. tactile and painful sensitivity decreased in glove and sock pattern. an extensive metabolic and biochemical study was normal. the electroneurography demonstrates an axonal neuropathy without response in most of the nerves explored. the electromyography shown a myogenic pattern with distal predominance. brain mri was normal in both cases. through a genetic study by exoma targeting genes associated with cmt and inherited related neuropathies was identified the homicigosis mutation in the aifm gene (p.glu lys), located in the chromosomal region xq . (cmtx ). cowchock syndrome is a rare entity, with few cases described in the literature. the in silico analysis indicates in of the predictors used (provean, sift, polyphen , lrt, mutationtaster, mutationassessor and condel) , that it is a deleterious variant. we audited all patients with peripheral neuropathy caused by a paraprotein, who received treatment and attended king's college hospital peripheral nerve service between - . patients were identified retrospectively from our database of patients attending the peripheral nerve outpatient clinic. clinical information was obtained retrospectively from hospital electronic patient records. we excluded patients with poems syndrome or in whom the neuropathy was not felt to be caused by the paraprotein. we identified patients with a diagnosis of paraproteinaemic neuropathy. we excluded four who did not fulfill the diagnostic criteria and eleven who had received no treatment or were under diagnostic study. we included patients in the final audit. ( %) had igm paraprotein. the haematological diagnosis was monoclonal gammopathy of undetermined significance (mgus) in %, waldenstroḿs macroglobulinaemia %, and lymphoma or plasmacytoma %. after treatment, overall ( %) patients improved neurologically, ( %) stabilised, and ( %) worsened. in the patients who received more than one type of treatment, we analysed outcomes according to the most powerful treatment received. patients received rituximab alone of which ( %) improved, ( %) stabilised and ( %) worsened. nine patients received rituximab combined with cyclophosphamide or bendamustine, of which ( %) improved, ( %) stabilized and ( %) worsened. eight patients received intravenous immunoglobulin, of which ( %) improved, ( %) stabilized and ( %) worsened. four patients received other chemotherapy, of which improved, stabilised, worsened. two patients received corticosteroids and both worsened. there was improvement in / ( %) with mag antibodies and / ( %) without. there was improvement in / ( %) with mgus and / ( %) with haematological malignancy. there was improvement in / ( %) with kappa light chains and / ( %) with lambda. factors associated with better outcome (by univariate analysis) were negative mag antibodies, kappa light chain, and haematological malignancy. there was no significant difference between treatments in the proportions who improved. cumberbatch m , cox a . addenbrooke's hospital, cambridge, uk. treatment of patients with autoimmune neuropathies such as chronic inflammatory demyelinating polyradiculoneuropathy (cidp) and multifocal motor neuropathy (mmn) has centred on the use of intravenous immunoglobulin (ivig). however, ivig therapy is associated with systemic side-effects, treatment wear-off effects and regular hospital attendance. subcutaneous immunoglobulin (scig) is an efficacious alternative that can be flexibly dosed, self-administered at home and avoids the 'peaks' and 'troughs' observed with ivig. these factors may combine to improve patient satisfaction and alleviate hospital capacity issues. here, we report on clinical and patient experience of switching from hospital-based ivig to home-based manual push scig for the treatment of cidp and mmn. this was a clinical case series of patients ( cidp, mmn; mean age . years) who were clinically stable on ivig and wished to switch to manual push scig. starting scig dose was equivalent to the final ivig dose for each patient (mean . g/kg week). clinical efficacy (medical research council sum score, -m walk, modified inflammatory neuropathy cause and treatment score, overall neuropathy limitations scale, romberg test) and patient-reported outcomes ( -item short form health survey , life quality index [lqi] ) were assessed at baseline and at regular intervals until the final visit ( - months after switching). at baseline, patients cited 'convenience' as their primary reason for switching to scig. eight patients completed the full assessment period and successfully undertook administrations at home (via hospital-at-home service in cases). dose adjustments, based on clinical need, were required in patients. treatment efficacy and patient quality of life, measured by sf- , were maintained after switching to scig; overall patient satisfaction, measured by lqi, increased from % to %. in the lqi, 'convenience', 'travel time/cost' and 'interference-work' were significantly improved (p< . ) after switching to scig therapy. adverse events included mild erythema and localised swelling, as expected for a ml subcutaneous injection. these findings suggest that manual push scig therapy is a viable alternative to ivig for patients with cidp and mmn, as it maintains disease stability, is more convenient for patients and may help ease hospital capacity concerns. cumberbatch m , soares regua d , cox a . addenbrooke's hospital, cambridge, uk. intravenous immunoglobulin (ivig) is used to treat a number of chronic autoimmune neurological diseases. in most centres, infusions are given at slow rates as there is a perception that this reduces the risk of adverse events (aes). this results in longer in-patient admissions, or frequent day case attendances, impacting on both the patients' quality of life and hospital capacity. however, there is little evidence to suggest that slow infusion rates are required. we used the manufacturer recommendations to optimise infusion rates and reduce the time patients spend in hospital. we report a retrospective audit which describes the impact of different ivig infusion rates on patients' clinical condition. the audit comprised three -month assessment periods: january-june (cohort ; infusion rates of . ± . ml/kg/hr, n = ); january-june (cohort ; . ± . ml/kg/hr, n = ) and july-december (cohort ; . ± . ml/kg/hr, n = ). clinical data were reviewed to determine: patient demographics, duration of infusion; time spent in hospital; and incidence of aes. the three cohorts were well matched in terms of patient demographics ( patients were treated in all treatment periods). cohorts and had significantly shorter treatment episodes than cohort ( . and . vs. . hours, p< . ), spent less time on the unit over the month period ( . and . vs. . hours, p< . ) and had fewer admissions/patient ( . and . vs. . , p< . ). the overall incidence of confirmed aes (mainly headaches) was similar across the cohorts (cohort : %; cohort : %; cohort : %). these findings indicate that increases in ivig infusion rate are well tolerated and significantly reduce treatment time, which benefits patients and offers potential cost savings and reduced pressures on hospital capacity for healthcare providers. cunningham me , yao d , meehan gr , barrie ja , willison hj . university of glasgow, glasgow, uk. one mechanism of injury in the acute motor axonal neuropathy (aman) form of guillain-barré syndrome (gbs) is the attack of peripheral nerve axons by anti-ganglioside antibodies (agabs). rodent models have demonstrated that that binding of these antibodies activates the complement cascade, resulting in the insertion of the terminal component, membrane attack complex (mac) into the axonal membrane. complement activation also results in the release of anaphylatoxins, which are known to recruit phagocytic immune cells to the site of injury. our current in vivo mouse model of agab and complement-mediated injury are acute and severe, resulting in respiratory distress over several hours of such magnitude to warrant termination of experimental procedures. to observe and potentially target immune cell infiltration following agab and complement-mediated injury, a subacute model extending over days is required. here, we demonstrate the development of such a model. to compare differences in immune cell infiltration subacutely under control and injury conditions, mice with endogenous expression of egfp in monocytes and macrophages underwent a modified agab and complement-mediated injury, resulting in a less severe phenotype than previously published models. six days following injury, immune cells in the diaphragm were compared by immunofluorescence and flow cytometry. flow cytometry found overall presence of neutrophils was significantly increased in the diaphragm. macrophages were also increased in injured mice, although did not achieve statistical significance at this timepoint. these results were reflected in immunofluorescent staining of the diaphragm where egfp+ macrophages were quantified surrounding the neuromuscular junction (the primary injury target in this model). the development of an extended mouse model of agab and complement-mediated injury is important, since acute models do not take into consideration either the late-term effects of complement-mediated activation at the nerve membrane, or the recovery phase. future studies will look at the effect of inhibiting complement activation on the presence of immune cells in distal motor nerves. family- is a large australian family with an autosomal dominant form of dhmn (dhmn : omim % ) -a group of length-dependent neurodegenerative disorders affecting the lower motor neurons leading to chronic disability. we recently reported a novel . mb chromosomal insertion within the dhmn locus which we hypothesise is likely to cause disease by dysregulating the expression of one or more nearby genes. studying gene dysregulation in peripheral nerve disease is challenging as the relevant tissues (spinal cord and peripheral nerve) are not easily accessible in patients. therefore, alternative strategies are needed to elucidate the disease mechanisms and pathways involved in peripheral nerve degeneration. to address this problem, we have devised a two-tiered strategy to assess dysregulation of candidate genes using patient lymphoblast and fibroblast cell lines. these cell lines can be easily established, are minimally invasive to obtain, and will harbour the natural mutation and genetic background of patients. our strategy firstly uses lymphoblast gene expression profiles as an initial screening tool to prioritise candidate genes for assessing altered expression. differentially expressed genes will then be modelled in c.elegans where behavioural and nerve morphology can be assessed. using rt-pcr, we have screened dhmn candidate genes in patient and control lymphoblast cell lines. eighteen candidate genes were expressed in lymphoblasts. twelve of the eighteen genes were prioritized for further analysis based on expression in both lymphoblast and neural tissues. quantitative analysis using qrt-pcr taqman assays revealed that ube c, was differentially expressed between patients and controls. it is important that patterns of differential expression can be recapitulated in neural cell-specific models. as part of our second strategy, we have generated patient and control induced pluripotent stem cell derived motor neurons (ipsc-mns) from reprogrammed fibroblasts. using this model, we will perform rna-seq and qpcr experiments to examine disease-relevant alterations in gene expression in neural tissue. we predict that utilization of these two strategies will shed light on the pathogenic mechanisms underlying the dhmn insertion and provide useful insights of pathways leading to peripheral nerve degeneration. the outcome of guillain-barré syndrome remains unchanged since plasma exchange and intravenous immunoglobulin were introduced over years ago. pathogenesis studies on gbs have identified the terminal component of complement cascade, the membrane attack complex, as a key disease mediator and thus a therapeutic target. the inhibition of complement in guillain-barré syndrome (ica-gbs) trial looked at the first use of c pathway inhibition with eculizumab in humans with gbs in a randomised, double-blind, placebo-controlled trial. its primary outcome was to look at safety and tolerability of administration concomitantly with ivig and in the context of severe (gbs disability score or greater) disease. participants were recruited for a month period, with regular follow up. subjects were screened, with ( %) being randomised. the two main causes for failure to proceed were participant concerns around eculizumab side effect profile, specifically the meningitis risk, and also intercurrent infection precluding treatment. five received eculizumab for four weeks, alongside standard intravenous immunoglobulin treatment, with receiving placebo. the safety outcomes, monitored via adverse events capture at each trial visit, showed eculizumab to be well tolerated and safe when administered in conjunction with ivig. the most common adverse events were mild derangement in transaminases or infection. there were no infusion reactions. primary and secondary efficacy outcomes were captured via gbs disability scores, mrc sum scores, rasch overall disability scores and overall neuropathy limitation scores. for the primary efficacy outcome at weeks after recruitment, of placebo and of eculizumab-treated subjects had improved by or more grades on the gbs disability score. all patients had improvements in other measured parameters. this trial highlights the challenges in recruiting acutely unwell patients, due to time constraints and intercurrent infection. although the small sample size precludes a statistically meaningful analysis, these pilot data indicate further studies on complement inhibition in gbs are warranted. charcot-marie-tooth disease (cmt) affects about one in . people. currently more than genes have been identified, with the most different phenotypes. the majority of cases in western countries are autosomal dominant and classified as demyelinating and axonal according to electroneuromyography (enmg). the clinical condition is characterized by weakness and predominant sensory changes in the feet and hands. sometimes there are different phenotypes. recently, variants in heterozygotes in the hars gene (histidil-trna synthetase) have been described associated with cmt called type w.to report a case of cmt-sensitive phenotype with a probable new mutation in hars gene (p.leu arg).a male patient, adopted son, caucasian, drug addict, for three years suffered pain in lower limbs, of great intensity, refractory to drug treatment. the examination showed retrognathism, abolition of patellar and achilleas reflexes, painful and thermal anaesthesia and apalesthesia in the feet.the enmg showed reduced sensory action potentials in sural and superficial fibular nerves. laboratory investigations for painful polyneuropathy of thick and fine fibres was normal. sural nerve biopsy revealed axonal predominance neuropathy. exome sequencing revealed a mutation in the hars gene with a pathogenic variant in heterozygosity, with replacement of the amino acid leucine at position by arginine. our patient, although we did not know the antecedents, presented painful polyneuropathy, whose genetic research, although not unequivocal, indicated a variant called cmt w. few cases of this variant were described, with several mutations. our case revealed mutation hitherto unknown (p.leu arg). we conclude by the importance of a thorough genetic evaluation, in cases of sensory polyneuropathy of unknown cause. small fiber neuropathy (sfn) is a condition that affects the small a -and c-fibers, leading to severe neuropathic pain and autonomic dysfunction. several sodium channel gene mutations have been found in patients with sfn, with scn a-gene mutations being the most frequent. because current available sodium channel blockers are not selective for na v . , these treatments often result in numerous side effects. lacosamide is an anticonvulsant that targets specific sodium channels with a slow-inactivation state, while sparing those with normal activity. several mutations of the scn a-gene with an impaired slow-inactivation of na v . have been found in patients with sfn. therefore, a positive effect of lacosamide on pain reduction in these patients is expected. the primary objective of this study was to determine the effect of lacosamide versus placebo on pain in subjects with scn a-associated sfn. secondary objectives were to determine the effect of lacosamide on autonomic symptoms, sleep interference, and quality of life, and to examine the safety and tolerability. the lacosamide-efficacy-'n'-safety in sfn (lenss) study was a randomized, placebo-controlled, double-blind, crossover-design study. subjects were randomized to start with lacosamide and end with placebo or vice versa. during both of the two phases of the study, the subjects were treated for a period of eight weeks of mg bid, preceded by a titration period, and ended by a tapering period. patients filled in a pain diary twice daily and scored a set of validated questionnaires on autonomic symptoms, sleep interference, and quality of life at multiple study visits. in total patients with scn a-associated sfn were included between november and february . the subjects had a median age of years, ranging from to years. sixty percent of the patients were female. the final results of the study, including the primary and secondary outcomes, will be presented. de la oliva n , del valle j , navarro x . department of cell biology, physiology and immunology, institute of neurosciences, universitat autònoma de barcelona and centro de investigación biomédica en red sobre enfermedades neurodegenerativas (ciberned), bellaterra, spain. intraneural interfaces must be in intimate contact with nerve fibres to have a proper function, but it has been shown that this is compromised due to the foreign body reaction (fbr). this fbr is the first response of the nonspecific immune system against an implanted device and is characterized by a first inflammatory phase followed by a second antiinflammatory and fibrotic phase. this process results in the formation of a tissue capsule around the interface causing function loss due to the physical separation between the active sites of the electrode and nerve axons. taking this into account, here we have tested several anti-inflammatory drugs such as dexamethasone, ibuprophen and maraviroc to reduce macrophage activation as well as clodronate liposomes to reduce monocyte/macrophage infiltration. moreover, sildenafil have been administered as an antifibrotic drug to reduce collagen deposition in a fbr model with longitudinal parylene c-based intraneural devices implanted in rat sciatic nerve. briefly, animals were systemically treated with dexamethasone, ibuprophen, sildenafil, maraviroc or clodronate liposomes for two weeks, and nerve damage, inflammatory reaction and matrix thickness around the implant were assessed. treatment with dexamethasone, ibuprophen or clodronate liposomes significantly reduced the inflammatory response in the nerve in comparison to saline group while sildenafil or maraviroc had no effect on iba positive cells infiltration in the nerve. however, only dexamethasone was able to significantly reduce the matrix deposition around the implant after two weeks of treatment. these results support the idea that inflammation triggers the foreign body response in peripheral nerves and a potent anti-inflammatory treatment with dexamethasone could have a beneficial effect on lengthening intraneural interfaces lifespan. de la oliva n , del valle j , navarro x . department of cell biology, physiology and immunology, institute of neurosciences, universitat autònoma de barcelona and centro de investigación biomédica en red sobre enfermedades neurodegenerativas (ciberned), bellaterra, spain. intraneural interfaces functionality decreases over time, among other factors, due to the foreign body response (fbr), which encapsulates the implanted devices and physically separates the active sites from the nervous tissue. here we have studied the fbr to parylene c or polyimide thin devices implanted in rat sciatic nerves, assessing thickness of the tissue capsule, signs of inflammation and nerve damage. we have characterized the responsible cells of this response and several molecular mediators over months of implant to find differences between the fbr to both materials. after weeks of implant, the inflammatory response due to the surgery was already decreased, whereas in the implanted nerves it reached its highest levels to then decrease at chronic time points. besides, the amount of foreign body giant cells (fbgc), as a result of macrophage fusion, found in the tissue capsule around the implant also increases progressively to reach a maximum after weeks. on the other hand, molecular analysis of the environment revealed a peak of inflammatory cytokines during the first day of implant to return to standard levels thereafter. however, an increase on ccls molecules was found at later time-points for both materials. with regard to the capsule thickness, all the devices were surrounded by a tissue deposition which appeared soon after the implantation. however, in the case of polyimide devices, the tissue capsule showed a peak weeks after the implant and signs of remodeling thereafter, while the parylene c devices showed a second increase from to weeks in comparison to polyimide devices. immunohistochemical and electron microscopy analysis revealed two different cell types implicated in the fbr in nerve to both materials: macrophages, in close contact with the interface, and fibroblasts which appear after weeks surrounding the tissue capsule. although further analyses are needed to elucidate the differences in the fbr to parylene c and polyimide polymers, these results can help to determine therapeutic targets in order to reduce this response and to improve the intraneural interfaces lifespan. delmont e , antoine jc , paul s , boucraut j , attarian s . referral centre for als and neuromuscular diseases, marseille, france; referral centre for neuromuscular diseases, saint etienne, france; immunology laboratory, saint etienne, france; immunology laboratory, marseille, france. peripheral neuropathies with antibodies against myelin associated glycoprotein (mag) are chronic sensory neuropathies characterized by the presence of an igm monoclonal gammopathy and high levels of anti-mag antibodies. these antibodies recognize a specific epitope called human natural killer (hnk ) shared by nk lymphocytes and several components of the peripheral nerve (mag, p , pmp , sgpg, phosphocan). recently an elisa test has been developed to detect antibodies against hnk epitope. our objectives were to determine the sensitivity and the specificity of anti-hnk antibodies in the diagnosis of anti-mag neuropathy and to know if these antibodies were correlated with the severity of the disease. anti-hnk antibodies were assessed in anti-mag neuropathies and in negative controls: chronic inflammatory demyelinating polyradiculoneuropathies (cidp), miller fisher syndromes, sensory neuronopathies, length-dependant axonal sensory polyneuropathies, healthy controls. in anti-mag neuropathies, were recorded age, disease duration, incat sensory sum score (iss), overall neuropathy limitation scale (onls), rasch-built overall disability scale (rods), mrc sum score, anti-mag antibodies titer, peak dosage of the igm monoclonal gammopathy. anti-hnk antibodies were measured with gangliocombi™ mag elisa test and anti-mag antibodies with anti-mag autoantibodies elisa test both from buhlmann company. anti-hnk antibodies were positive in / anti-mag neuropathies, and in / controls (sensitivity %, specificity %). in anti-mag neuropathies, anti-hnk titer was correlated with sensory deficiency evaluated with the iss score (r= . , p= . ) and with disability evaluated with the rods (r= − . , p= . ) and onls scales (r= . , p= . ). anti-hnk titers were not related to age, disease duration, mrc sum score, anti-mag antibodies titer, peak dosage of the paraproteinemia. anti-mag antibodies titers were associated with none of the characteristics of the patients with anti-mag neuropathy. anti-hnk antibodies have good sensitivity and specificity in the diagnosis of anti-mag neuropathy. compared to anti-mag antibodies, their value is that their titers are related to the disease severity. these results need to be confirmed in a larger prospective cohort. chronic inflammatory demyelinating polyradiculoneuropathy (cidp)is a heterogeneous and treatable immune-mediated disorder that critically lacks biomarkers to support diagnosis. recent evidences indicate that paranodal proteins (contactin- , contactin-associated protein- , and neurofascin- ) are the targets of autoantibodies in a subset of patients with cidp showing distinct clinical presentations. particularly, these biomarkers appear to have clinical relevance and help to orientate therapeutic choice. here, we examined five patients presenting an igg reactivity against the nodes of ranvier and the axon initial segment. using a proteomic approach, cell-based assays and elisa, we identified neurofascin- (nfasc ) and neurofascin- (nfasc ) as the main targets of autoantibodies at the nodes of ranvier. four patients displayed predominantly antibodies of the igg subclass, whereas one patient presented igg antibodies that activated the complement pathway in vitro. these antibodies recognized different epitopes than the previously described anti-neurofascin- igg suggesting different pathogenic functions. accordingly, patients with anti-nfasc / igg showed a distinctive clinical presentation. most patients had a severe phenotype associated with conduction block or decreased distal motor amplitude. tremors or neuropathic pain were not observed. four patients presented with a subacute-onset and sensory ataxia. of interest, the neuropathy occurred concomitantly with nephrotic syndromes in two patients and with an igg -related retroperitoneal fibrosis in one patient. this suggested that autoantibodies could be responsible for the occurrence of both disorders. intravenous immunoglobulin and corticosteroids were effective in three patients, and one patient improved following cyclophosphamide and rituximab treatment. clinical remission was found to correlate with the depletion of anti-nfasc / antibodies and the loss of igg reactivity toward the nodes of ranvier. in addition, recovery of conduction block and of distal motor amplitude were observed following remission and suggested a nodo-paranodopathy. our data demonstrate that nodal antigens are the target of autoantibodies in a subgroup of patients with cidp. this emphasizes that the pathogenic mechanisms involved in chronic immune-mediated demyelinating neuropathies are broad and may include dysfunctions of the nodes of ranvier. mutations in the neurofilament heavy (nefh) gene have been recently identified as a rare cause of autosomal dominant, axonal charcot-marie-tooth disease (cmt ). the clinical spectrum of this condition remains to be delineated. we report two french families with an axonal, predominantly motor, dominantly inherited form of cmt caused by two previously unreported mutations in the nefh gene. twelve patients belonging to two different families were included in the study. they displayed an axonal motor and sensory neuropathy, with no mutations in known axonal cmt genes. a remarkable feature in all patients was the early involvement of proximal muscles of the lower limbs, occurring approximately to years after the onset of motor deficit. proximal weakness affected predominantly the iliopsoas muscle, whereas quadriceps and hamstring muscles were relatively preserved. muscle weakness and muscle wasting progressed rapidly, with most of the patients requiring walking assistance after years of disease evolution. three patients in family had brisk reflexes. nerve-conduction velocity studies displayed evidence of a motor and sensory axonal neuropathy predominantly affecting the lower limbs. original deletions of nucleotides near the end of the coding sequence of nefh were identified: in family , c. _ del (p.lys argfs* ), and in family c. _ del (p.lys glyfs* ) causing a frameshift. interestingly, this frameshift leads to the loss of the terminating codon and to the translation of additional amino acids encoding a cryptic amyloidogenic element, suggesting that this type of mutations could induce protein aggregation. consistently, we showed that overexpression of the mutated forms of nefh in a human neuroblastoma cells induced the formation of protein aggregates. we also observed that it triggered caspase activation and apoptosis. using electroporation of chick embryo spinal cord, we confirmed in vivo that mutated nefh formed aggregates and triggered apoptosis of spinal cord neurons. altogether, this suggests that these mutations in nefh cause protein aggregation and neurotoxicity in neurons expressing nefh. progressive loss of such neurons would explain the early motor involvement and the pyramidal signs observed in some patients. our results provide a physiological explanation to the presence of cmt and als clinical features in affected patients. del valle j , , delgado-martínez i , righi m , santos d , , cutrone a , bossi s , d'amico s , micera s , , navarro x , . neuroprosthetic devices that are aimed to restore sensorimotor limb function of amputee patients require highly selective electrodes designed to establish a tight relationship with the nerve, allowing the bidirectional transduction of signals between nerve fibres and the interface and enabling close-loop control from the user. differently from extra-or intraneural interfaces, regenerative nerve electrodes are designed to enable electrical interface with regrowing axonal bundles of injured nerves, aiming to achieve high selectivity for recording and stimulation. however, most of the developed designs pose an obstacle to the regrowth mechanisms due to low transparency and cause an impairment of the nerve regeneration. in this work, we present a novel double-aisle planar regenerative electrode, a new type of highly transparent, non-obstructive regenerative electrode, which allows the selective stimulation and recording of separated nerve fascicles. the design consists of a thin and flexible double-sided electrode longitudinally inserted across a conduit thus creating two separated aisles in which regenerating fascicles can independently regrow after nerve transection. electrodes implanted in acutely transected nerves of rats showed the capability of selectively stimulating and recording different fascicles inserted in the aisles. moreover, chronic implantation of the electrode in a nerve gap of mm after sciatic nerve section allowed for fascicle regeneration and reinnervation of distal muscles as confirmed by the high number of myelinated axons inside each aisle, good biocompatibility, and adequate nerve conduction. in addition, three and six months after implantation, independent stimulation and recording of each separately regenerated fascicle were possible. our results demonstrate the potential contribution of the doubled-aisle regenerative electrode to selectively interface different fascicles of an injured nerve with no deleterious effects on nerve regeneration. therefore, this multi-aisle regenerative electrode may be suitable for neuroprosthetic applications, such as prostheses for the restoration of hand function after amputation or severe nerve injuries. demichelis c , garnero m , franciotta d , cortese a , callegari i , mancardi gl , schenone a , leonardi a , benedetti l . department of neuroscience, rehabilitation, ophthalmology, genetics, maternal and child health, university of genoa and irccs aou san martino-ist, genoa, italy; laboratory of neuroimmunology, irccs, "c. mondino" national neurological institute, university of pavia, pavia, italy; u.o. neurology, asl imperiese, imperia, italy. querol et al. showed that neurofascin (nf ) antibodies identify a chronic inflammatory demyelinating polyradiculoneuropathy (cidp) phenotype characterized by severe polyradiculoneuropathy, poor response to intravenous immunoglobulins (ivig), and disabling tremor. neurological improvement after therapy with rituximab has been previously reported in three patients with cidp with igg anti-nf antibodies. herein we describe the acute-onset of a case of cidp positive for nf igg antibodies resistant to conventional therapies and responsive to rituximab. the patient is a year-old woman who presented acute onset ataxia and gait disturbances; her symptoms progressed over two weeks and distal weakness, numbness and paresthesias appeared too. the nerve conduction study was suggestive for a motor-sensory polyradiculoneuropathy mainly demyelinating. the cerebrospinal fluid analysis showed elevated protein level and normal cellular count. the patient was initially diagnosed with guillain-barré syndrome (gbs) and treated with plasma exchange without improvement. an ivig cycle was started with a partial relief but at the time of admission to the rehabilitation center the patient still had a marked weakness in all four limbs. after six months she presented a further clinical deterioration and she was restricted to wheelchair. there was no response to additional treatment with ivig, while pulse corticosteroid treatment determined a significant clinical improvement. during the next months, despite the maintenance of steroid therapy, the patient presented a progressive deterioration and she was again restricted to wheelchair. postural and intention tremor appeared at upper limbs and became progressively more disabling. anti-nf ab dosage resulted positive. rituximab was administered at a dosage of mg/m /weekly for weeks. after three months the tremor improved, allowing her to eat independently and the patient was able to walk with bilateral support. antibodies anti-nf were negative. after six months she walked without support and she was able to stitch crochet. as previously reported, in this case a cidp positive for igg nf developed severe polyradiculoneuropathy with predominant distal weakness, ataxia, disabling tremor and resistance to conventional therapies. interestingly the onset was gbs-like. the correct identification of these cidp subtypes has diagnostic, prognostic and therapeutic implications. rituximab con be useful in these patients. demir Ö , yazıcı t . department of neurosurgery, university of gaziosmanpaşa school of medicine, tokat, turkey; department of neurosurgery, kent hospital, giresun, turkey. it is still challenging problem to maintain motor and sensory functions of peripheral nerve after nerve transection. after the nerve injury, calcium concentration in the damaged area increases. then the calcium ions act like cytotoxic agents in the damaged area. nifedipine is calcium channel blocker. we aimed to investigate the effects of nifedipine on nerve regeneration by modulating calcium in the damaged area. twenty-four swiss albino male rats were divided into two groups. left sciatic nerve transection surgery was performed to the all rats in both groups. then the all transected nerves were sutured primarily with epineural interfascicular method. in the experimental group, the anastomosis sites were wrapped with a piece of gel foam soaked into diluted nifedipine solution. in the control group, the anastomosis sites were wrapped with a piece of gel foam soaked into saline solution. we evaluated the effect of nifedipine by using functional, electro-physiological and histopathological studies after the surgeries. in the postoperative second week, walking test was performed and sciatic function index was calculated. in the postoperative third week electroneuronography (enog) was performed. there are significant differences between two groups. nifedipine improved nerve recovery functionally (p< . ) and electro-physiologically (p< . ). in the postoperative fourth week, we performed histopathological examination. in the experimental group with nifedipine there were more organized axons that reached the aim. we conclude from these results that nifedipine is an effective nerve protective agent when used locally at the anastomosis site after the transection of the nerve. the lack of effective, disease-modifying therapies for cmt highlights the need for novel preclinical models suitable for drug discovery. studies in rodent models of cmt tend to be time-consuming, and findings so far have translated poorly into clinical trials. primary and induced pluripotent stem cell (ipsc)-derived neuronal cultures are an established model of neurological diseases. however, due to the random distribution of neuronal bodies and neurites that happen when plating these cells, this system is not ideal to investigate axonal, length-dependent processes like peripheral neuropathies and particularly cmt. to optimize this well-established model system, we developed a robust human platform to study axonal morphology and physiology based on motor neuron neurospheres. we differentiated motor neurons from human induced pluripotent stem cells, purified them by magnetic sorting and cultured them in suspension until they formed neurospheres. floating neurospheres can be maintained in agitation for months as a reliable source of motor neurons. after neurospheres are platted, axons rapidly grow out of them in a radial fashion, resembling dorsal root ganglia cultures. this configuration allows for a better visualization of axons in imaging studies and for continued axonal growth over at least a -day period. axons grew at an average rate of micrometers/day and reached up to cm in length. neurospheres can be fixed and stained allowing for morphological analysis and investigation of protein distribution in axons. this system is also ideal for time-lapse imaging to study axonal transport of organelles and neurofilament kinetics. lastly, our motor neuron neurosphere system lends itself well for high content screening platform. neurospheres can be plated in -well plates where multiple compounds can be tested and the axons easily imaged by a high content screening microscope. in summary, we developed a new platform to investigate motor axons in vitro, which are particularly useful to study length-dependent processes such as inherited peripheral neuropathies and may facilitate the identification of new therapeutic compounds using high content screening systems. mutations in the neurofilament light chain (nfl) gene cause autosomal dominant axonal charcot-marie-tooth neuropathy (cmt e). nfl is a major component of the neuronal cytoskeleton, and is believed to function in conjunction with nfm and nfh to provide structural support for the axon and regulate axon diameter. despite the significant advances in understanding its biological basis, there is still no effective, disease-modifying therapy for cmt e, in part due to the paucity of preclinical models suitable for drug discovery. the development of novel preclinical platforms that can faithfully mimic mechanisms of axonal degeneration in vitro would be an essential and valuable resource to better understand the biology of cmt e and identify potential targets for therapy development. to address this, we generated control and cmt e patient-derived motor neurons and cultured them in suspension until they formed neurospheres. immunostaining of cmt e neurospheres with nfl and tubb antibodies revealed numerous areas of nfl accumulation in n s cmt e axons, resembling the accumulations of mutant nfl protein seen in the processes of catecholaminergic neuronal cell line cad overexpressing several nfl mutants. further analysis demonstrated that areas of nfl accumulation were also immunopositive for nfh, pnfh and nfm and that at least nfl and nfm co-localized in the same areas of deposits. taken together, these results demonstrate that abnormal axonal neurofilament distribution is a feature of cmt e ipsc-derived motor neurons and involve all three neurofilament subunits. we also developed an image analysis routine to allow for automatized quantification of neurofilament distribution. preliminary quantification of nfl signal intensity revealed that axons from patients have a weaker nfl signal compared to control axons, but present several signal peaks above the range observed in controls, which related to the areas of nfl accumulation. these results suggest that nfl accumulates in certain regions of cmt e axons but is reduced in the areas with no accumulation. these findings can be readily adapted into a high content screening platform and will be used to identify compounds able to reverse this axonal phenotype. in summary, we identified a strong axonal phenotype in human cmt e motor neurons with potential as a screening platform for drug discovery. nodal and paranodal proteins have been identified as antigens in peripheral inflammatory neuropathies, however the frequency and clinical relevance of antibody responses against these targets remain poorly investigated in gbs. patients with acute onset inflammatory neuropathies were identified by exploration of the local databases of the departments of neurology and the institute of neurology of the medical universities in innsbruck and vienna. patient data, electrophysiological classification and presence of anti-gangliosid antibodies were retrospectively retrieved by review of patient records. only patients with typical clinical presentation and electrophysiological results consistent with one of the subtypes of gbs were included in the study. among forty-nine patients, thirty-five were classified as aidp, six aman, three amsan, three mfs, and two pharyngo-cervico-brachial gbs. of the included patients had anti-ganglioside-antibodies. ten patients with the initial suspicion of aidp had a disease duration of more than months and were reclassified as cidp. all patient and twenty sera of control patients with non inflammatory polyneuropathy were screened by an optimized tissue based assay using rat brains for immune responses against surface antigens, and by cell-based assays with transfected hek cells for antibodies against contactin (cntn ), contactin (cntn ), contactin-associated-protein (caspr ) and neurofascin- (nf ). in the tissue based assay some of the patients showed a light neuropil staining. none of gbs patient's sera had antibody reaction to cntn , cntn , caspr or nf in cell-based assays. among the cidp patients, two patients demonstrated reactivity against cntn with similar clinical presentation as previously described. none of the control patients had any antibody reaction to the performed tests. our results suggest that antibody responses to cntn , cntn , caspr or nf are absent in austrian gbs patients, although more patients will be screened to substantiate these preliminary results. furthermore, it remains to be established whether antibodies against cntn may predict a chronic course in acute onset inflammatory neuropathies. a few variants of chronic inflammatory demyelinating polyradiculoneuropathy (cidp) have been described with a frequency of - %. their relation and possible evolution into typical-cidp remain unclear, as is their treatment response possibly because of differences in diagnostic criteria. we used the data from a web-based database on italian patients with cidp to determine the frequency and characteristic of these variants, the possible evolution into typical-cidp, and their treatment response. all the patients were assessed at study entry and the disease course before inclusion was analyzed. by february- , we included patients ( men, women), aged - years (median years) with a mean disease duration of . years (range . - years) and complete data available from . based on the clinical data and our revised diagnostic criteria, patients ( %) were classified to have atypical cidp at onset and for the following two years including with dads ( %), with motor cidp ( %), with sensory cidp and cisp ( . %), with lewis-sumner syndrome ( . %), and with recurrent cranial neuropathy. at study entry, patients ( %) had progressed into typical cidp after - years (median years) while ( %, % of total) still had atypical cidp after . - years (median years) with a similar proportion of progression ( - %) within each group. the diagnosis of atypical cidp at entry fulfilled efns/pns criteria in ( %). csf studies were diagnostic in / ( %) patients, nerve biopsy in / ( %), and nerve imaging in / ( %) tested patients. similarly to typical cidp, % of treated patients with atypical cidp improved after treatment with a proportion of response varying from % to % in the different forms. most patients with sensory or motor cidp had however an unsatisfactory response to steroids. this study shows that he proportion of patients with atypical cidp varies during the course of the disease with almost % of the patients evolving into typical cidp within years from onset. in addition, response to treatment is frequent in atypical cidp even if not all the forms respond to the same therapies. only few studies investigated the frequency of antecedent events and comorbidities in patients with chronic inflammatory demyelinating polyradiculoneuropathy (cidp), and little is known on the role of possible predisposing factors, dietary, and lifestyle habits, on the onset and progression of the disease. we used the data from a web-based database on italian patients with cidp to determine the frequency of antecedent events and comorbidities and the possible role of predisposing factors including lifestyle and dietary habits and exposure to toxic agents, using a structured questionnaire. partners of patients served as controls. impairment was evaluated using the mrc sumscore and disability with incat and r-ods scales. logistic regression was used to calculate odds ratio (or) with % confidence interval (ci) for the risk of cidp. sex and disease-duration were included as covariates. by february- , patients were enrolled, with complete data on patients for antecedent events and comorbidities and patients and controls for lifestyle habits. ninety-two patients ( %) reported an antecedent event, mostly infection or vaccination ( %). one or more comorbidity were present in % of the patients including hypertension ( . %), thyroid disorders ( %) and diabetes ( . %) and in % influenced the choice of initial therapy. exposure to toxic environmental agents (odds ratio [or] = . ; % ci, . - . ), cigarette smoke (or = . ; % ci, . - . ), and dietary supplements (or = . ; % ci, . - . ) were associated with a higher risk of cidp while rice consumption was associated with a reduced risk (or = . ; % ci, . - . ). concerning disease severity, more severely affected patients more frequently consumed raw-meat (or = . ; % ci, . - . ) and white meat (or = . ; % ci, . - . ), while rice (or = . ; % ci, . - . ) and soft drink consumption (or = . ; % ci, . - . ) and physical activity were associated with lower disability (or = . ; % ci, . - . ). this study confirms that comorbidities are frequent in patients with cidp and often influence the choice of initial therapy. in addition preliminary data show that toxic exposure and some lifestyle and dietary habits may influence the onset and progression of cidp. doppler k , schuster y , weishaupt a , sommer c . department of neurology, university hospital würzburg, würzburg, germany. autoantibodies against the paranodal protein contactin- have recently been described in patients with cidp. in most patients, autoantibodies of the igg subclass are predominant and are supposed to be pathogenic. the role of igg anti-contactin- is so far unclear. in the present study, igg of three different patients, one with igg anti-contactin- , one with a low titer of igg anti-contactin- and one with a high titer of igg anti-contactin- , and of controls were injected into the sciatic nerves of lewis rats. nerve conduction studies of the injected nerve and motor and sensory testing were performed before and after injection. conduction blocks and motor deficits were detectable in the two patients with high titers of igg and igg , not in the patient with low titers. the percentage of conduction blocks was . % in rats injected with igg of the igg patient and % in those injected with igg . motor deficits were detectable in both patients with conduction blocks but were most apparent in the patient injected with igg of the igg patient. no differences in sensory testing were observed. conduction blocks and motor deficits improved after five days and were normal after seven to eight days. our data give the first evidence of pathogenicity of igg anti-contactin- autoantibodies, not only igg . igg of the igg patient induced a more severe clinical and electrophysiological phenotype compared to the igg patient. remarkably, this reflected the clinical phenotype of the patients, as the igg patients showed an acute-onset of sensorimotor symptoms at the time of blood withdrawal whereas the igg patient presented with a more chronic course of disease. doppler k , frank f , koschker a-c , reiners k , sommer c . department of neurology, university hospital würzburg, würzburg, germany; endocrinology and diabetes unit, department of medicine i, university hospital würzburg, würzburg, germany. axoglial dysjunction and paranodal demyelination have been discussed as potential mechanisms of nerve fiber damage in diabetic neuropathy. studies on human tissue are limited, as nerve biopsies are invasive and only rarely performed in patients with confirmed diabetic neuropathy. skin biopsy has recently been suggested as a good tool to analyze paranodal and nodal changes of myelinated fibers. in the present study, we analyzed the paranodal and nodal region in myelinated fibers of skin biopsies of patients with diabetic neuropathy, patients with diabetes mellitus without neuropathy, and normal controls. immunofluorescence of skin sections with antibodies against caspr, neurofascin, sodium channels and myelin basic protein was performed to assess paranodal/nodal architecture, segmental demyelination and myelinated nerve fibers. staining with antibodies against protein gene product . was used to quantify unmyelinated nerve fibers. we found an increase of elongated ranvier nodes and a dispersion of neurofascin at the distal leg in patients with diabetes mellitus with and without neuropathy and at the finger in patients with diabetic neuropathy. an increased dispersion of caspr was only found in biopsies of the finger in patients with diabetic neuropathy. our data show that skin biopsy is an appropriate tool to analyze nodes of ranvier in patients with diabetes mellitus. structural nodal changes are detectable in diabetic neuropathy, and even in diabetic patients without neuropathy. dourado me , fernandes u , vital al , ramos e , urbano jc , sena a , queiroz jw , jeronimo smb . federal university of rio grande do norte, natal, brazil. the erasmus gbs outcome score (egos) is a validated prognostic model that uses acute phase and easy-to-obtain clinical characteristics to determine outcome at months in patients with gbs. this study aims to assess the validity of egos in rio grande do norte, brazil, and to compare with another european study. data collected prospectively from a cohort of patients with gbs of rio grande do norte, brazil, between june and august , was assessed. ninety patients were excluded for missing data or diagnoses of miller fisher syndrome and atypical forms of gbs. to calculate the egos, the gbs disability score was assessed in the second week of disease and at months. to compare this study with the european one in independent group proportions, we used the student's t-test, being considered statistically significant p< , . the patients included were divided in four groups based on egos. thus, patients had egos between and ; had egos between . and . ; had egos and had egos between . and . in the first, second, third and fourth group, ( %), ( . %), ( . %) and ( . %) of the patients were unable to walk independently after six months of the disease, respectively. overall, of the patients analyzed, ( . %) had poor outcomes in this study. in the european paper, based on the same group division, of ( . %), of ( %), of ( %) and of ( %) were unable to walk independently. comparing both studies, the patients of this study were younger, more seriously ill in the first weeks and with more sensitive deficits. there were no difference relative to sex, cranial nerves deficits and presence of anti-gangliosides antibodies. using the student's t-test for ability to walk after months according to egos stratification, we achieve in the first group p= . ; in the second p= . ; in the third p= . ; and in the fourth p< . .the egos did not have a good capacity to predict the ability to walk after months of gbs in rio grande do norte, brazil. historically, guillain-barré syndrome (gbs) epidemics are rarely seen. between and , we treated and followed cases of gbs in the state of rio grande do norte with a yearly incidence of . / , . no seasonality was observed. the mean age of the patients was years (range, - ), with % of the cases younger than years. demyelinating variant was the most frequent subtype of gbs. in march , the first report of autochthonous transmission of zika virus (zikv) was determined in natal, brazil. later that month, we documented an increase in incidence of gbs in natal, brazil. the incidence in was of . / . . of the cases of gbs diagnosed in , were diagnosed from march through may, which coincided with the outbreak of zikv in natal, brazil. eighteen patients ( % of the cases) had a history of rash and fever prior to onset of gbs symptoms, with the median age of years (range, - ) . the electroneuromyography studies of these patients indicated that ( . %) had acute inflammatory demyelinating polyneuropathy, ( . %) had acute motor axonal neuropathy, and ( . %) was inconclusive. the mean time from onset of zikv infection symptoms to onset of the gbs were days (range, - ). the mean time of nadir was days (ranged, - ) . cranial neuropathies were present in patients ( . %). nine patients were bedridden ( %) and ( . %) required mechanical ventilation. the mean protein content of the central spinal fluid was . g/l, with the white blood cell count below /mm in all patients. they were all treated with intravenous igev. they all improved quickly. anti-gm was negative in all patients. rt-pcr was negative for dengue, chikungunya and zika. serum mac-elisa igm for zika and dengue was made in patients and it had % of positivity. prnt for zika and dengue had % positivity. in summary, we report a geographically and temporally defined cluster of gbs associated with an outbreak of acute rash in the state of rio grande do norte, brazil. as the prevalence of diabetes mellitus continues to increase worldwide, diabetic complications represent a growing burden to patients and society. distal symmetrical polyneuropathy (dsp) is a common complication that affects up to % of diabetic patients. dsp reduces patient quality of life due to chronic pain, ulcerations, and may lead to lower extremity amputations. despite its high prevalence, the mechanisms underlying diabetic dsp are poorly understood and several mechanisms are believed to play a role. we hypothesize that diabetic dsp arises from microvascular complications characteristic to diabetes. specifically, capillary dysfunction -disturbances in capillary flow patterns -is a likely candidate to explain development of dsp, as it can limit oxygen and nutrient delivery to nervous tissue, causing nerve dysfunction and damage, and thus development of dsp. we will study this hypothesis utilizing the state-of-the-art blood flow imaging techniques to visualize and quantify endoneureal blood flow and then link these findings with measures of dsp (e.g. nerve conduction velocity; intra-epidermal nerve fibre density) in animal models. we will include several animal models of diabetic dsp caused by either type or type diabetes. two photon microscopy and optical coherence tomography allow visualisation and quantification of capillary transit times and blood flow within peripheral nerves at high resolution. we hypothesise that changes in blood flow patterns and subsequent impairment of nutrient and oxygen delivery to nervous tissue precede the onset of diabetic dsp. if our experiments support this prediction, we will attempt to develop interventions that improve capillary blood flow to prevent or delay the development of dsp. duman o , saracoglu m , haspolat s , bozkurt o . department of child neurology, akdeniz university hospital, antalya, turkey. axonal polyneuropathies are very heterogeneous group of diseases which are very rarely seen during infantile age. some of them may be accompanied by developmental retardation, severe muscle weakness and progressive course. we aimed to present two cases of axonal sensorymotor neuropathy with infantile onset and atypical course. our -year-old boy patient was admitted to our clinic for progressive gait loss since one month. he was the first offspring of consanguineous parents with normal prenatal and natal history. electromyography revealed axonal sensorymotor polyneuropathy. metabolic and cerebrospinal fluid (csf) examinations for etiology were all normal. brain and spinal magnetic resonance imaging (mri) were normal. he had partial benefit from oral steroid treatment. in the course of disease along with four neuropathy attacks he had significant benefit from serial intravenous immunoglobulin treatments in two years clinical course. a month-old girl who is the first offspring of nonconsanguineous parents was admitted to the clinic for acute tetraparesis. axial sensorymotor polyneuropathy was detected in the electromyography. metabolic and cerebrospinal fluid (csf) examinations were normal. she had three more acute polyneuropathy attacks during ivig cessation period. both patients revealed with serial immunoglobulin treatments but unresponsive to riboflavine treatment. we aimed to discuss our rarely seen and the pathogenesis is not completely understood cases' course. serial ivig treatment may be helpfull for such patients' treatment. transthyretin-related familial amyloid polyneuropathy (ttr-fap) is an autosomal dominantly inherited disorder caused by mutations of the transthyretin (ttr) gene. the mutant amyloidogenic transthyretin protein causes the systemic accumulation of amyloid fibrils that result in organ dysfunction. ttr-associated fap is a progressive and fatal disease, if left untreated, and should be considered in the differential diagnosis of any person presenting with a progressive polyneuropathy, particularly with accompanying autonomic involvement. the clinical, electrophysiological, histopathological, and genetic characteristics of patients from turkey ( female, male) from eleven families with polyneuropathy and mutations in ttr were evaluated. two patients had no family history of ttr-fap and were considered as sporadic cases, and the remainders were familial cases displaying an autosomal dominant inheritance pattern. sequence analysis of the ttr gene revealed five mutations (p.val met, p.glu gln, p.gly glu, p.glu gly and p.gly glu). most common mutation was p.val met (in unrelated families). mean age at disease onset was . ± . years (range - years). the most commonly reported initial complaint was paresthesia in the feet (asymmetric in three patients). four patients ( male) with the p.glu gln mutation presented with carpal tunnel syndrome. two patients with the p.gly glu mutation showed episodes of dysarthria and hemiparesis, consistent with this genotype. seven patients died during the follow-up period as a result of systemic involvement. this study suggests that our cohort of ttr-fap patients from turkey exhibits clinical and genetic heterogeneity. ebenezer gj , liu y , judge dp , cunningham k , truelove s , carter nd , sebastian b , byrnes k , polydefkis m . department of neurology, johns hopkins university, baltimore, md, usa; division of cardiology, johns hopkins university, baltimore, md, usa; department of epidemiology, johns hopkins bloomberg school of public health, baltimore, md, usa. effect of amyloid deposition on cutaneous nerves was assessed in subjects with pathogenic ttr variants and control subjects. three groups of subjects each including ttr-fap patients, age/gender-matched healthy subjects and disease controls as well as ttr mutation carriers without neuropathy (ttr-nopn) and with al-amyloid underwent neurological examination and mm skin biopsies. micron sections were stained with anti-pgp . , anti-ttr and congo red. amyloid burden with imagej, intraepidermal (ienfd) sweat gland (sgnfd) and pilomotor densities (pmnfd) measured and correlations between amyloid burden, fiber subtype, neuropathy impairment score-ll (nis-ll) and nis sensory subscore were evaluated. ienfd, sgnfd, and pmnfd were all significantly reduced in ttr-fap patients vs. healthy controls while mutation carriers had intermediate reductions. lower nerve fiber densities were associated with nis-ll (p< . ). congo red staining revealed brilliant red amyloid deposits with apple-green birefringence within dermal collagen, sweat glands, and arrector pili muscles. amyloid infiltration was observed in the endoneurium and perineurium of small fiber sensory and autonomic nerves that innervate sweat glands and arrector pili muscles. cutaneous amyloid deposition was detected in % of ttr-fap and not in healthy or disease controls subjects. both al and / ttr-nopn subjects were congo red positive. amyloid burden was inversely correlated with ienf (p< . , r=− . ) sgnf (p< . , r=− . ), pmnf (p= . , r=− . ) distal leg densities, and correlated with nis-ll (p= . , r= . ) and nis sensory subscore (p= . , r= . ). wild-type ttr staining was less prominent in pathogenic ttr carriers. the diagnostic sensitivity and specificity to detect amyloid in skin were % and % in ttr-fap. the repeat measurement of the amyloid burden from the same section with imagej was r = . , p< . and different sections from the same biopsy was r = . and p< . . we conclude that endoneurial amyloid contributes to sensory and autonomic nerve injury. amyloid burden correlated strongly with sensory/autonomic axon densities and nis-ll. skin punches offer a convenient alternative to establishing a tissue diagnosis. amyloid burden is an attractive biomarker marker for ttr-fap and treatment effect. the study was supported through a grant from pfizer. ebenezer gj , truelove s , polydefkis m . department of neurology, johns hopkins university, baltimore, md, usa; department of epidemiology, johns hopkins bloomberg school of public health, baltimore, md, usa. we investigated differences of unmyelinated sensory nerve fibers in the distal limb among healthy-weight subjects (bmi < kg/m ) and overweight/obese subjects (bmi ≥ kg/m ), aged - years. subjects underwent neurological examination and mm skin punches from distal leg (dl), thigh (dt), and proximal thigh (pt) sites, from which micron sections were stained with anti-pgp . antibody; intraepidermal nerve fiber density (ienfd; fibers/mm) and epidermal thickness were assessed. a second dl biopsy was processed for electron microscopic examination and both thick and thin sections were examined for ultrastructural changes. multivariable linear regression models were used to assess the effect of age, gender, height and weight. after controlling for height, age, and obesity, females were found to have lower distal leg ienfd (− . ; p= . ). increasing age and height were significantly associated with decreasing dl ienfd, with decreases of − . fibers/mm per years (p<. ) and − . fibers/mm per cm (p< . ), respectively. even after controlling for height, being overweight/obese was associated with reduced dl ienfd, with . fibers/mm lower dl enfd than healthy-weight individuals (p=. ). these findings remained consistent across distal thigh and proximal thigh enfd, though not all associations remained significant. the epidermis was thicker in obese subjects across the lower limb, most pronounced at the distal leg (μm, mean± sd, healthy-dl: ± . , dt: . ± . , pt: . ± . , obese-dl: . ± . , dt: ± . , pt: . ± . ). under em very few intact dermal nerve bundles were identified at the proximal thigh sites. the atrophic and degenerating axons were seen with perineurial infiltration by dense collagen in obsess/overweight subjects but not age/gender matched controls. obesity further accelerates attenuation of epidermal nerve fibers across the lower limb even after controlling for other associated factors. echaniz-laguna a , geuens t , petiot p , péréon y , adriaenssens e , haidar m , capponi s , maisonobe t , fournier e , dubourg o , degos b , salachas f , lenglet t , eymard b , delmont e , pouget j , juntas morales r , goizet c , latour p , timmerman v , stojkovic t . strasbourg university hospital, strasbourg, france; peripheral neuropathy group, vib department of molecular genetics and institute born bunge, university of antwerp, antwerpen, belgium; lyon university hospital, lyon, france; nantes university hospital, nantes, france; hôpital de la pitié-salpétrière, paris, france; nice university hospital, nice, france; marseille university hospital, marseille, france; montpellier university hospital, montpellier, france; bordeaux university hospital, bordeaux, france. in this study, we describe the phenotypic spectrum of distal hereditary motor neuropathy caused by mutations in the small heat shock proteins hspb and hspb and investigate the functional consequences of newly discovered variants. among unrelated patients with distal motor neuropathy, we identified mutations in hspb ( index patients/ ; . %) and hspb ( index patients/ ; . %) genes. patients have slowly progressive distal ( %) and proximal ( %) weakness in lower limbs, mild lower limbs sensory involvement ( %), foot deformities ( %), progressive distal upper limb weakness ( %), mildly raised serum creatine kinase levels ( %) and central nervous system involvement ( %). we found a broad range of disease onset with some patients presenting with foot drop at the age of years, and others presenting symptoms only after years. disease progression was slow in all patients, and even with a disease duration of more than years patients were still able to walk. none of our patients were wheelchair dependent. muscle pathology, nerve pathology and electrophysiology showed in all cases a slowly progressive, mostly symmetrical and predominantly distal motor axonal neuropathy. mild sensory involvement was observed upon nerve conduction studies, mostly the lower limbs, in % of cases. we identified hspb and hspb mutations, including respectively and not previously reported. transmission was either dominant ( %), recessive ( %) or de novo ( %). three missense mutations in hspb (pro ser, gly asp, gln arg) cause hyperphosphorylation of neurofilaments, while the c-terminal mutant ser leu triggers protein aggregation. two frameshift mutations (leu fs, ala fs) create a premature stop codon leading to proteasomal degradation. two mutations in hspb (lys met/asn) exhibited increased binding to bag . we demonstrate that hspb and hspb mutations are a major cause of inherited motor axonal neuropathy. mutations lead to diverse functional outcomes further demonstrating the pleotropic character of small heat shock proteins. eftimov f , querol l , rajabally ya and on behalf of all participants of the st enmc workshop. academic medical center, amsterdam, the netherlands; hospital de la santa creu i sant pau, universitat autònoma de barcelona, spain; aston university, birmingham, uk. although chronic inflammatory demyelinating polyneuropathy (cidp) is a treatable neuropathy further research is urgently needed to define the diagnostic clinical and electrophysiological boundaries of cidp and its subtypes, and to define the role of biomarkers in supporting the diagnosis, monitoring disease activity and predicting response to treatment and outcome. in recent years, several national registries and biobanks have been developed to enable systematic data collection in cidp. an international registry with large number of patients is needed to allow answering many important questions and develop validated prognostic models to predict outcome in individual patients with cidp. at the inflammatory neuropathy consortium (inc) meeting in , the inc members agreed that a european neuromuscular center (enmc) workshop would be the ideal setting to reach a consensus on the infrastructure of database and biobanks. the st enmc workshop will take place on may - , with participants representing different countries. primary objective of the workshop is to reach a consensus on inclusion and exclusion criteria, core sets and recommended sets of clinical data, diagnostic data and follow-up points and a manual of operations for collection of biomaterials. a secondary objective is to construct an infrastructure to allow sharing data between different databases and biomaterials. conclusions of the consensus meeting and outline of further perspectives will be presented at the peripheral nerve society meeting in . eichinger kj , burns j , cornett k , bacon c , shepherd m , mountain j , sowden j , shy r , shy me , herrmann dn . clinical outcome assessments that measure functional ability are important endpoints for clinical trials. dr. burns has led the development/validation of a functional outcome assessment (cmtpeds) for individuals with charcot marie tooth disease (cmt) ages - years in the inc rdcrn. the cmtpeds is reliable and sensitive to change. however a validated functional outcome measure (fom) for adults with cmt is needed. our data in - year-olds indicated that the cmtpeds could be modified for adult use. however, some items of the cmtpeds (e.g. balance beam and jumping) have floor effects in adults with cmt. we have developed an adult cmt-fom modeled on the cmtpeds, and refined based on literature review, patient interviews, a large-scale cmt patient survey and expert opinion. the cmt-fom is a performance-based scale comprising items that are combined to form a composite score to quantify functional ability of adults with cmt. the cmt-fom shares items with the cmtpeds. four items were added to measure functional abilities relevant to adults (sit to stand, meter walk/run, stair climb, and timed up and go test). the cmt-fom scoring mirrors the cmtpeds. to generate a score ranging from - , raw item scores are converted to z scores, based on age-and sex-matched normative reference values form the norms project and categorized to a - likert along a continuum of impairment levels. we have conducted a pilot study of the cmt-fom in adults with cmt a ( male, female, age . ± . yrs) of differing severity (cmt exam score (cmtes) range - ). the cmt-fom is feasible, individuals were able to complete all items, and takes minutes to perform. the mean cmt-fom score was . ± . (range - ). concurrent validity of the cmt-fom is supported by an association with the cmtes (r = . ). the overall score did not demonstrate floor or ceiling effects. in summary the adult cmt-fom is well-tolerated and captures upper and lower limb strength, dexterity, balance, speed, ambulation and endurance. the cmt-fom requires validation in a large longitudinal cohort, prior to application in clinical trials. estilow t , glanzman am , burns j , cornett kmd , menezes mp , shy r , moroni i , foscan m , pagliano e , pareyson d , laurà m , bhandari t , muntoni f , reilly mm , finkel rs , sowden j , eichinger k , herrmann dn , shy me , yum sw , ramchandren s and on behalf of the inherited neuropathies consortium. cmt is associated with progressive impairment of the hands. reducing this impairment by treating children who are in the early stages of the disease is crucial. studies assessing measures of cmt hand function in children and their associations with patient-reported outcomes are lacking. we analyzed the upper extremity items from the cmt pediatric scale (cmtpeds) and pediatric cmt quality of life scale (pcmt-qol) in children ages - years enrolled in the inherited neuropathies consortium, to explore the relationships between measures of hand function (impairment, activity, and activities of daily living), and patient-reported outcomes. weak grasp ( %), hand pain ( %), and tremor ( %) were prevalent impairments. performance on activity level tasks, the nine hole peg test ( hpt) and functional dexterity test (fdt), were impaired in % and % of the cases, respectively. patients reported difficulty "sometimes" to "always" in opening a jar/lid ( %), zipping/buttoning ( %), writing ( %), carrying a plate without spilling food ( %) and putting on shoes ( %). patients reporting tremor showed significant differences on the hpt (p=. ). grip strength was shown to have a moderately significant correlation with performance on the fdt (r=. ; p<. ). stepwise multiple linear regression showed that grip strength (beta=−. ; p<. ), hand pain (beta=. ; p<. ) and fdt (beta . ; p<. ) were predictive of ability to open a jar (adjusted r =. ; p<. . grip strength (beta=−. ; p<. ), and fdt (beta . ; p<. ) were predictive of ability to carry plate without spillage (adjusted r =. ; p<. ). grip strength (beta=−. ; p<. ), hpt (beta=. ; p<. ) and fdt (beta . ; p<. ) were predictive of ability to put on shoes (adjusted r =. ; p<. ). hand pain (beta=. ; p<. ) and fdt (beta . ; p<. ) were predictive of ability to zip/button (adjusted r =. ; p<. ). hand pain (beta=. ; p<. ) and hpt (beta . ; p<. ) were predictive of ability to use a pen/pencil (adjusted r =. ; p<. ). children with cmt present with frequent limitations in adl performance impacting qol. the upper limb measures of the cmtpeds are associated with hand performance and interventions to improve grip strength and reduce pain should be investigated further with respect to their impact on improving function, and ultimately qol. evans me , morrow jm , wastling s , sinclair cdj , fischmann a , shah s , emira ak , hanna mg , yousry ta , thornton js , reilly mm . mrc centre for neuromuscular diseases, ucl institute of neurology, london, uk; neuroradiological academic unit, ucl institute of neurology, london, uk; university of basel hospital, basel, switzerland. responsive outcome measures are needed in charcot-marie-tooth disease (cmt) to allow adequately powered clinical trials to test novel therapeutics. we have shown high responsiveness of mri quantified intramuscular fat accumulation in calf muscles of cmt a patients over months. the aim of the present study was to assess the responsiveness and longitudinal validity of quantitative mri over a year follow-up period. we undertook two further sets of quantitative mri, myometric and clinical assessments in the original mrc centre cmt a quantitative mri cohort. mri sequences included fat quantification using the point dixon fat-water separation method, t quantification and magnetisation transfer imaging. of the patients with genetically confirmed cmt a were assessed at baseline ( male, mean age . ± . years), underwent repeat assessments a median on months (data already published), underwent repeat assessments at a median of months, and underwent a final assessment at a median of months. the primary outcome measure currently being analysed is mean calf muscle fat fraction at a single axial slice a fixed distance distal to the knee joint. results of this analysis and correlation with clinical measures will be presented at the peripheral nerve society meeting. fainmesser y , dori a , , drory ve , . department of neurology and neuromuscular service, tel-aviv medical center, tel-aviv, israel; department of neurology, sheba medical center, ramat gan, israel; sackler faculty of medicine, tel aviv university, israel. the use of anabolic drugs by those who wish to increase lean body mass is widespread and not well supervised medically. a years old man was referred for evaluation of a slowly progressive sensory and motor disturbance in the distribution of the left ulnar nerve. his symptoms began months after repeated self-injections of anabolic steroids and vitamin e into the biceps and triceps brachii muscles bilaterally. his examination showed increased muscle mass of the injected muscles with a hard-rubbery consistency, atrophy and weakness of left interossei, mild weakness of the bilateral biceps brachii and sensory loss in an ulnar nerve distribution. nerve conduction studies showed a left ulnar neuropathy with reduced motor and sensory response amplitudes and denervation in the left first dorsal interosseus, as well as mild myopathic changes with significantly reduced insertional activity in both biceps muscles. mri of the soft tissues of the arms showed massive fibrosis and infiltration of fat in the arm muscles compressing the ulnar and median nerves on the left. neurolysis of the left ulnar nerve was performed, and the ulnar nerve was found enclosed in a fibrotic mass throughout the entire length of the upper arm. the brachial artery and the median nerve were similarly enclosed in fibrotic tissue and were released. a biopsy of the affected muscles showed muscle necrosis and fibrosis. the patient was treated with physiotherapy and losartan with only mild improvement in the consistency of the muscles, but without clinical improvement in ulnar nerve function, and worsening of nerve conduction. this case illustrates severe peripheral nerve damage due to entrapment in massive muscle fibrosis following improper intramuscular injection of anabolic steroids for cosmetic purposes. fabrizi gm , testi s , høyer h , braathen gj , squintani g , bertolasi l , ferrarini m , taioli f , cabrini i , pancheri e , cavallaro t , tonin p . department of neuroscience, biomedicine and movement, university of verona and department of neuroscience, aoui verona, italy; section of medical genetics, department of laboratory medicine, telemark hospital, skien, norway. inherited diseases of nerve and muscle may overlap phenotypically or coexists as facets of the same disorder. two families whose probands were initially diagnosed with a lower motor-neuron (lmn) syndrome and a hereditary distal motor neuropathy (dhmn) turned out to represent a vcp (valosin-containing protein)-related syndrome and a gne (udp-n-acetylglucosamine -epimerase/n-acetylmannosamine kinase)-related "distal myopathy" . both conditions shared the presence of rimmed vacuoles (rv) in muscle biopsies. in family , the year-old male proband had a lmn syndrome manifesting at age years (wasting/weakness, cramps, fasciculations of thigh muscles, later involving the distal upper limbs). his year-old brother had lower limb weakness and diffuse pain in bones/joints since age . the father was diagnosed with a "muscular dystrophy" thirteen years before dying a years; by age years he had developed a behavioural frontotemporal dementia (ftd). electrodiagnosis (edx) disclosed myopathic changes with ongoing denervation together with a mild sensory-motor axonal polyneuropathy in the proband, and a chronic sensory-motor axonal polyneuropathy in the brother. mri showed fatty replacement of weakened muscles in both siblings and diffuse changes of bones consistent with paget disease (pd) in the elder sibling. in family , a year-old african woman was affected by weakness and wasting starting at the distal lower-limb muscles at age years and soon progressed proximally; parents were not consanguineous and two sisters out of six siblings, deceased in their thirties for post-partum complications, had a similar disease. edx mainly disclosed a neurogenic process with ongoing denervation. muscle biopsies from the three family- patients and from the family- proband showed a myopathy with rv. next-generation sequencing demonstrated a heterozygous c. c>t change of vcp leading to a known pathogenic p.arg cys substitution in all family- patients, and two compound heterozygous c. g>a and c. g>a changes of gne in the family- proband leading to known p.ala thr and p.ala thr substitutions. vcp is known to cause autosomal dominant amyotrophic lateral sclerosis- , charcot-marie-tooth disease type y or inclusion body myopathy (ibm) with pd and ftd (ibmpfd); gne causes the autosomal recessive nonaka myopathy (alias ibm ). both cases emphasize the clinical and neurophysiological heterogeneity of those disorders. fargeot g , vandendries c , labeyrie c , viala k , theaudin m , adams d . neurologie, crmr nnerf, aphp, filnemus, chu bicêtre, le kremlin-bicêtre, france; neuroradiologie, crmr nnerf, aphp, filnemus, chu bicêtre, le kremlin-bicêtre, france; neurologie, groupe hospitalier universitaire pitié salpêtrière, paris, france. the diagnosis of cidp is often challenging, especially when electrophysiological signs of demyelination are lacking. plexus mri has documented nerve abnormalities in small series of typical cidp but its contribution in patients with no electrophysiological signs of demyelination remains to be proved. we report the results of plexus mri in a serie of patients suspect of having cidp without efns pns definite electrophysiological criteria. we did a retrospective study of patients consulting in kremlin bicetre and pitié salpétrière hospital. lumbar or brachial plexus mri (or both) were performed and we assessed nerve trophicity, t -stir signal intensity and gadolinium enhancement as well as the topography of abnormalities. a consensus diagnosis was made by a group of experts (based on clinical data and other supportive criteria) and allowed us to classified patients in "cidp" or "other diagnosis". the practical contribution of plexus mri to the diagnostic algorithm has been studied. diagnosis of cidp was made in patients. mri was abnormal in % of cidp patients and showed nerve roots hypersignal/hypertrophy/enhancement in respectively . / . / . % of patients. the pattern of abnormalities was often asymetrical ( . %), diffuse ( . %) or multifocal ( . %). after unblinding, the mri confirmed the diagnosis of experts in . % of patients and changed the diagnosis in % of patients. plexus mri has shown to be useful in our serie to confirm the diagnosis of experts or to modify it in patients ( %) . the "classical" pattern described in definite cidp (diffuse nerve root hypertrophy and hypersignal) was documented in % of our cidp patients whereas less typical pattern (focal or multifocal abnormalities, hypersignal without hypertrophy) was found in %. plexus mri seems usefull when facing patients suspected of having cidp when electrophysiological criteria are not met: both symetrical diffuse or asymetrical multifocal patterns can be found and should be always correlated to the clinical examination and other supportive criteria. the specificity of such abnormalities remains to be studied. feely sme , rebelo a , abreu l , tao f , bacon c , zuchner s , shy me . university of iowa, iowa city, ia, usa; dr. john t. macdonald department of human genetics and hussman institute for human genetics, miller school of medicine, university of miami, miami, fl, usa. mutations in bcle-associated athanogene (bag ) have been shown to cause a distal myofibrillar myopathy and cardiomyopathy that can severely affect children or only affect adults depending upon the particular mutation. children with severe cardiomyopathy and myopathy have also developed axonal peripheral neuropathy, consistent with the known localization of bag in neurons as well as in muscle. we have identified two large autosomal dominant families with adult onset charcot-marie-tooth disease (cmt ) with the identical novel missense mutation pro ser, a codon previously shown to cause severe or mild myopathy depending on the amino acid substitution. these families expand the phenotypes caused by mutations in bag to include cmt and provide an additional example of adult onset cmt that may previously have been diagnosed as chronic idiopathic axonal neuropathy (ciap). feely sme , saade d , shy me . carver college of medicine, university of iowa, iowa city, ia, usa. myelin protein zero (mpz), expressed only by myelinating schwann cells, has sequence alterations that have previously been reported to cause demyelinating, intermediate, and axonal forms of charcot marie tooth (cmt) disease. we describe a rare duplication in mpz which is causing an early onset, demyelinating form of cmt in our patient. patient was a product of a normal pregnancy and delivery. early milestones were delayed. she began walking at months of age. she was not able to keep up with her peers and was the slowest runner. she started wearing smos at years of age and afos by years of age. she was diagnosed with scoliosis at years of age and started wearing a brace at years. her examination at years of age showed that her fdi, apb, and adm were all / bilaterally. weakness in her lower extremities included / foot eversion and / great toe dorsi flexion. she had tight heel cords bilaterally. pinprick sensation was reduced throughout in her upper and lower extremities and absent at her toes bilaterally. vibration sensation with a rydel tuning fork was absent at her toes and ankles and reduced at her knees and fingers. nerve conduction studies were performed and revealed no responses in all sensory nerves tested with the exception of the radial nerve which had normal latency and mildly slowed conduction velocity ( m/s). prolonged latencies, demyelinating range slowing (between - m/s), and low cmap amplitudes in almost all segments of the median and ulnar motor nerves were also observed. these findings were consistent with a hereditary sensorimotor demyelinating polyneuropathy. she was diffusely areflexic and her total cmt pediatric score (cmtpeds) was / which is in the severe range. her cmt neuropathy score (cmtns) was / in the moderate range. the duplication of mpz has previously been identified as a rare cause of cmt b. inherited neuropathy consortium is part of the nih rare diseases clinical research network (grant# u ns - ). feely sme , saade d , shy me . carver college of medicine, university of iowa, iowa city, ia, usa. mutations in egr cause a severe, demyelinating form of cmt, cmt d. we describe a novel mutation in egr which led to extreme variability in severity in a family. proband was a year old girl who was the product of a normal pregnancy and delivery. early milestones were on time. problems with walking started at years of age. at . she was unable to use stairs, run, or jump. at she was wearing bilateral afos. by she was using a wheelchair and started breathing assist at night. she lost arm lift but could still hold a pen. by she could not ambulate independently and breathing assist was required day/night. she lost the ability to write and developed a head drop. by years she could not sit up. on exam her head and neck muscles were / . upper limbs, deltoids, biceps, wrist ext/flex, finger ext, fdi, and adm were / bilaterally. triceps were / ; finger flexors and apb were / bilaterally. lower limbs were / and she had contractures on the right. sensory examination was normal. she was diffusely areflexic. ncvs were absent. both the cmtns and cmt pediatric score were severe at / and / . exome sequencing revealed a r l variant in egr which was likely pathogenic. the probands' mother also had this mutation. she had no reported symptoms at the age of with strength at / throughout with the exception of left foot eversion which was +/ and great toe dorsi flexion which was -/ bilaterally. sensory exam showed a decrease of vibration and pinprick sensation at left toe. she had a normal gait, tandem gait and could toe walk, but not heel walk. she was diffusely areflexic. ncvs showed absent sensory responses. motor ncvs showed her latencies were prolonged and velocities reduced ( - m/s). cmtns was mild ( / ). additional family members who had the r l mutation were evaluated including maternal grandmother who was moderately impaired ( / ) and maternal aunt who was severe ( / ). no other mutations that could cause another known neuropathy or myopathy were identified and mitochondrial sequencing was normal. inherited neuropathy consortium is part of the nih rare diseases clinical research network (grant# u ns - ). fisgun a , luan x , hoke a . johns hopkins university, baltimore, md, usa. molecular mechanisms that underlie slow distal axonal degeneration seen in chemotherapy induced peripheral neuropathy (cipn) are unclear. however, several identified molecular targets suggest shared mechanisms with wallerian degeneration. since spontaneous mutation in wlds mice and genetic deletion of sarm gene lead to slow wallerian degeneration, we asked if wlds or sarm knockout (ko) mice are resistant to distal axonal degeneration induced by several chemotherapy agents. we chose chemotherapeutic drugs from different classes of agents, paclitaxel (taxane), cisplatinum (platin-based drugs) and bertozomib (proteasome inhibitor), to model cipn in mice. primary outcome measure was evaluation of epidermal nerve fibers in the hind paw plantar footpads. secondary outcome measures included thermal sensation and nerve conduction studies. sarm ko mice were almost % protected against development of sensory neuropathy but the protection in wlds mice was partial. this study confirms the pivotal role sarm plays in mediating axonal degeneration and identifies inhibition of sarm activity as a potential therapeutic target for prevention of cipn. florio f , scapin c , ferri c , feltri ml , wrabetz l , d'antonio m . myelin biology unit, san raffaele scientific institute, milan, italy; hjkri-university of buffalo, ny, usa. myelin protein zero is the most abundant structural protein in myelin of the pns. in humans, more than mutations in p are associated with hereditary neuropathies. deletion of serine causes charcot-marie-tooth (cmt) b disease in humans and a similar demyelinating neuropathy in mice (wrabetz et al., ) . p s del protein is misfolded and is retained in the er where it gives rise to a dose-dependent unfolded protein response (upr) (pennuto et al., ) . the upr results in the activation of transcriptional and translation control programs that reduce protein synthesis and increase the folding and degradative capacity of the cell. usually, when this first response is not sufficient the cells may activate apoptosis resulting in cell death. however, in p s del schwann cells there is no cell death suggesting that these cells may respond differently to chronic stress. transcriptomic analysis performed on p s del nerves showed increased expression of transcription factors normally present only in the early phases of differentiation such as c-jun, sox and id (d' antonio et al. ) . in order to understand the role of the expression of these factors in the peripheral nerve myelination we used ex vivo and in vivo approaches and we showed that sox and id act as negative regulators of myelination. these results suggest that the expression of sox and id may contribute to the hypomyelination observed in p s del mice. as such, we reasoned that their ablation could ameliorate the phenotype. surprisingly, the ablation of these factors in the p s del mouse severely worsens the neuropathy bursting schwann cell differentiation and increasing the expression of both p wild type and mutant allele with concomitant exacerbation of the upr. this suggests that the overexpression of early differentiation factors in schwann cell under chronic er-stress is an adaptive mechanism that limits differentiation and reduces the expression of toxic proteins. intravenous immunoglobulin (ivig) is the treatment of choice for the guillain-barré syndrome (gbs). the working mechanism of ivig in gbs is undefined, but most likely all potential effects are dose dependent. the pharmacokinetics (pk) and pharmacodynamics (pd) of ivig in gbs are highly variable between patients and a rapid consumption or clearance of ivig is associated with poor recovery. in the current study we developed a model to predict the pk of a standard dosage of ivig ( . g/kg for consecutive days) in individual patients with gbs. non-linear mixed-effects modelling (nonmem) was used to construct a model based on a cohort of gbs patients, with a total of sequential serum igg levels. the final model accurately describes the day to day increment in igg levels during the -day course and the initial rapid fall and graduate decline to steady-state levels thereafter. we explored several potential covariates that improved the predictive capabilities and decreased the between-subject variation in the model. the model including these covariates were evaluated successfully (bootstrap analysis) and through numerous simulation studies each based on (simulated) gbs patients. in conclusion, a first accurate and robust nonmem model for the pk/pd of standard ivig treatment in gbs was developed. the model can be used to predict the pk in individual patients applying a few simple baseline characteristics. in addition, the effect of different treatment regimens of ivig in gbs on a population pk/pd level can be simulated. this modeling technique is a new tool to optimize the pk in individual patients and the study design for new trials with ivig in gbs. forese mg , pellegatta m , rivellini c , podini p , quattrini a , previtali sc , taveggia c . axonal neuregulin (nrg ) type iii is an essential instructive signal for peripheral nervous system (pns) myelination, as its expression determines whether axons are myelinated as well as the thickness of the myelin sheath. we recently demonstrated that gamma-secretase cleavage of nrg type iii generates an axonal intracellular fragment, which translocates in the nucleus to upregulate the expression of the prostaglandin d synthase (l-pgds) gene in neurons. l-pgds catalyzes the conversion of prostaglandin h into prostaglandin d (pgd ). we also showed that specific inhibition of l-pgds activity impairs in vitro myelination. accordingly, myelin in l-pgds null mice is noticeably thinner, thus indicating that l-pgds is a new modulator of developmental pns myelination. previous studies have shown that prostaglandins are involved in the process of wallerian degeneration (wd) and axonal regeneration after injury. thus, to determine whether l-pgds and pgd could be important in pns regeneration and remyelination, we performed sciatic nerve crush injury in months old l-pgds null and wild type control mice and we analysed nerves by morphologic, biochemical, histological and molecular approaches at different time points (t) after crush. we focused on three phases: degeneration (t -t ), axon regeneration (t -t ) and remyelination (t ). our results indicate that in l-pgds null mice the amount of myelin proteins synthesized after crush as well as the number of remyelinated fibers do not change, suggesting that l-pgds might be dispensable for remyelination. however, we observed an increased number of macrophages in null nerves during regeneration (t ), possibly as a consequence of an increase in the blood-nerve barrier (bnb) permeability, indicating potential alteration in the regeneration process in l-pgds null mice. these results suggest that l-pgds could have a different role in developmental pns myelination and after injury. whether other prostaglandins and synthases might compensate for l-pgds activity is currently under investigation. fornasari be , , raimondo s , , ronchi g , , crosio a , budau ca , el soury m , muratori l , , tos p , battiston b , geuna s , , gambarotta g . department of clinical and biological sciences, university of turin, italy; neuroscience institute cavalieri ottolenghi, turin, italy; microsurgery unit, health and science city, cto, turin, italy; hand microsurgery and surgery, gaetano pini hospital, milan, italy. to repair nerve gaps following severe peripheral nerve injuries, chitosan tubes were proved to give good results, comparable with those obtained with nerve autografts, the gold standard technique. to further improve peripheral nerve regeneration using chitosan tubes, a conduit enrichment strategy was developed using longitudinal skeletal muscle fibres, which have been previously shown to be good fillers in the "muscle in vein" experimental paradigm, where they played a trophic and a structural role. to this aim, rat median nerve gaps were repaired using two different conduits: mm chitosan tubes filled with a longitudinal piece of pectoralis major muscle ("muscle in tube") and hollow chitosan tubes. samples were harvested at early time points ( , , , days) for biomolecular and morphological analysis, and later ( months) for stereological analysis. autologous nerve grafts were used as gold standard positive control in the early time points. biomolecular analysis carried out on in vitro degenerating muscle and on "muscle in tube" at early time points show that the muscle produces high levels of soluble isoforms of neuregulin , a key factor for schwann cell survival and activity, usually released by schwann cells after nerve injury. functional assay and stereological analysis carried out on the distal part of regenerated nerve months after nerve repair, show no significant differences in the regeneration outcome between hollow chitosan tube and "muscle in tube" groups. therefore, we conclude that for short gaps (≤ mm), both hollow chitosan tube and "muscle in tube" are good techniques to repair nerve defects and we suggest that the "muscle in tube", which spontaneously releases neureg-ulin , might be a promising strategy to promote regeneration when the gap is longer or the repair is delayed in time. foucquier j , bertrand v , jouve e , truillet r , mandel j , laffaire j , blin o , magy l , lehert p , , hajj r , guedj m , cohen d , attarian s . pharnext, issy-les-moulineaux, france; aix marseille université, aphm, marseille, france; hôpital dupuytren, limoges, france; university of melbourne, melbourne, vic , australia; faculty of economics, louvain, belgium. charcot-marie-tooth disease type a (cmt a) is a rare disease belonging to a group of inherited, progressive, chronic motor and sensory peripheral neuropathies. the charcot-marie-tooth neuropathy score (cmtns) (shy et al., ) and the overall neuropathy limitations scale (onls) (graham & hughes, ) are considered as the main clinical scales for evaluating progression of disability associated with cmt. as cmt a is a slowly progressive neurodegenerative disease, the choice of endpoints and their ability to monitor small changes over time remain a major concern for clinical drug development. with this in mind, we studied a cohort of french cmt a patients with a follow-up ranging from months to . years resulting from the merge of two multicentre clinical trials (micallef et al., ; attarian et al., ) and a non-interventional study (unpublished) . the sensitivity to change of both cmtns and onls were assessed using a mixed effect model estimating annual progression with time in years, cmtns or onls baseline value as covariates, study centre as a fixed factor and patients as a random effect to account for the repeated measures. disease progression was estimated to be + . points per year on the cmtns (p = . ) and + . points per year on the onls (p = . x − ), both corresponding to a deterioration of impairment and disability. while both endpoints have similar and favourable properties, our set of observations led us to conclude that the onls could be more promising to monitor disease progression in cmt a. frasquet m , , lupo v , mas f , , vílchez r , chumillas mj , , espinós c , sevilla t , , . hospital universitari i politècnic la fe, valencia, spain; instituto de investigación sanitaria la fe, valencia, spain; centro de investigación príncipe felipe, valencia, spain; eresa, valencia, spain; centro de investigación biomédica en enfermedades raras (ciberer), valencia, spain; departamento medicina, universitat de valencia, valencia, spain. mutations in the bicd gene are a cause of dominant spinal muscular atrophy, lower extremity predominant (smaled). we report six patients belonging to three spanish families who carry three different novel mutations in the bicd gene. we describe clinical, electrophysiological and magnetic resonance imaging (mri) data. we provide results of muscle biopsy of one patient and skin biopsy for the study of epidermal nerve fiber density (enfd) of other two patients. genetic diagnosed was reached using a gene panel for genetic testing of cmt and dhmn. three novel mutations in the bicd gene that segregated with the disease were detected: p.val gly; p.tyr his and p.s l. the most frequent clinical phenotype consisted of mild weakness in proximal muscles of lower limbs combined with foot deformities. one patient had prominent sensory symptoms and abnormalities on sensory examination. other two patients had minor sensory abnormalities on examination. in one patient sensory and motor nerve action potentials were reduced, in the rest of patients electrophysiological studies showed normal motor and sensory nerve responses, with chronic denervation predominantly in muscles of lower limbs. mri studies at the level of tight and calf were performed in all patients. the most affected muscles were rectus femoris, vastus lateralis and medial gastrocnemius. mri studies at the level of feet were obtained from five patients and showed that there was not fatty infiltration in intrinsic foot muscles. mri at the level of pelvis muscles performed in four patients showed marked fatty infiltration of gluteus medius muscle in two of them. muscle biopsy performed in one patient showed myopathic features. skin biopsy was performed in two patients of the same family. in the older patient, who had minor sensory abnormalities on examination, there was a marked reduction of enfd that followed a length-dependent pattern. in conclusion, we report three new pathogenic mutations in the bicd gene. in our study we include mri findings at the level of pelvis and feet, which allow us to better define the pattern of muscle involvement related with this gene. our results also raise the subject of a possible sensory involvement in the disease. hereditary neuropathy with liability to pressure palsies (hnpp) is an autosomal dominant disorder that usually results from deletions in the pmp- gene. the neuropathy is unique in that it manifests with recurrent mono-neuropathies at common sites of compression. while spontaneous recovery from episodes of nerve injury usually occurs, it is often incomplete, and over time patients may develop a length dependent polyneuropathy. given the relapsing/remitting nature of hnpp symptoms, standard clinical scores, such as the cmt neuropathy score, are not effective at capturing the severity or progression of the disease. a specific tool is therefore needed for measuring clinical severity of hnpp in preparation for emerging clinical trials. in the current study, we evaluate a new pilot measure, the hnpp severity score (hnpps). the score is composed of patient reported questions addressing current and prior sensory and motor symptoms, and the impact of symptoms on quality of life, followed by items based on a motor examination. total scores vary from - , with higher scores indicating increased disease severity. in this study, the hnpps was administered to patients with genetically confirmed hnpp at the ucl institute of neurology. subjects included males and females with a mean age of years (+/− , range - ). the mean hnpps was . points (+/− . , range - ) and the data did not demonstrate major skew. the cronbach alpha for the hnpps was . , and items based on the physical examination showed the least variability. a modest correlation was observed between the hnpps and the cmt examination scores (pearson correlation . , ci . - . ). we conclude that the hnpps may be a useful measure of clinical severity in hnpp, and should be refined in larger patient cohorts. fridman v , sillau s and on behalf of the inherited neuropathies consortium (inc) . university of colorado hospital, aurora, co, usa; university of iowa hospitals and clinics, iowa city, ia, usa. the most common of the hereditary neuropathies (hn) is cmt a, an autosomal dominant demyelinating neuropathy that results from duplications in the pmp- gene. recent advances in defining the pathomechanisms of the disease have led to an increasing number of potential therapies; however, the absence of reliable natural history data and the paucity of sensitive clinical outcome measures have been barriers to effective clinical trials. the charcot marie tooth neuropathy score (cmtns) was developed to quantify impairment and measure progression in hn. it was observed that while the score discriminates well between mildly and severely impaired patients, it tends to cluster together patients in the middle range of severity. to improve the score's sensitivity, rasch analysis-based weighted category responses were developed. we report a longitudinal study of weighted cmtns and cmt examination scores (cmtes) over a three-year time frame in patients with cmt a. baseline, one year, two year and three year wcmtnsv /wcmtesv scores were available for / , / , / and / patients respectively. mean wcmtns (sd)/wcmtes (sd) scores were as follows: . ( . )/ . ( . ) at baseline, . ( . )/ . ( . ) at one year, . ( . )/ . ( . ) at two years and . ( . )/ . ( . ) at three years. a mixed regression model showed significant change in wcmtns and wcmtes at years (mean change from baseline at years was . points (p=. ) for wcmtns and . points (p=. ) for wcmtes. significant change as compared to baseline was also seen at years (mean change from baseline . points (p= . ) for wcmtns and . points (p= . ) for wcmtes. we conclude that weighted cmtnsv scores show change over the first three years of the inherited neuropathies consortium natural history study and are a helpful measure of progression in cmt a. fridman v , novak p , david w , macklin ea , mckenna-yasek d , walsh k, oaklander al , brown r , hornemann t , eichler f . massachusetts general hospital, boston, ma, usa; university of massachusetts medical school, worcester, usa; university hospital zurich, zurich, switzerland. hsan is an autosomal dominant, severe sensory motor polyneuropathy caused by mutations to serine palmitoyl-coa transferase. mutations shift the substrate preference from serine to alanine leading to formation of neurotoxic -deoxysphingolipids ( -deoxysl). treatment with high-dose l-serine has been shown to reduce -deoxysl accumulation and improve neuropathy in transgenic hsan mice. we report a two-year, delayed-start, placebo-controlled clinical trial evaluating the safety and efficacy of oral l-serine in hsan . eighteen hsan subjects were equally randomized to l-serine ( mg/kg/d) or placebo for year. at -weeks, the placebo group crossed-over and all participants took open-label l-serine for one additional year. sixteen subjects completed their -week visit, and no serious adverse events related to l-serine were reported. participants randomized to l-serine experienced a decline in charcot marie tooth neuropathy scores (cmtns) over year relative to placebo (− . units, % ci − . to − . , p = . ). both groups improved in the second year of the study, with a diminished difference in cmtns at weeks (− . units, % ci − . to . , p= . ). skin biopsies from the distal leg site were largely devoid of intra-epidermal nerve fibers (ienf), but at year, a greater increase in ienf density was seen in participants on l-serine versus those on placebo (median change of vs. fibers/μm , p= . ). -deoxysl levels declined among participants on l-serine versus those on placebo after one year of treatment ( % decrease in deoxysphinganine vs. % increase on placebo, p < . ), and placebo participants experienced similar declines in -deoxysl levels after crossing over to l-serine. we conclude that l-serine is a safe and potentially efficacious treatment for hsan . fu liong h , yusuf r . regional neuromuscular clinic, queen elizabeth hospital, university hospitals of birmingham, birmingham, uk; school of life and health sciences, aston brain centre, aston university, birmingham, uk. the relationship between guillain-barré syndrome (gbs) and malignancy is uncertain. under the diagnostic criteria of paraneoplastic neurological syndrome (pns) by euronetwork, , neuropathy with no definite onconeural antibodies identified due to gbs has been classified as "non-classical" paraneoplastic disorder. we retrospectively analyzed data of consecutive patients admitted with gbs from birmingham, u.k. ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) to calculate the relative cancer risk using different definitions and determined characteristics of malignancy-associated gbs. patients were classified according to definitions: ) all cases of malignancies excluding preceding diagnoses > year before gbs and with no evidence of malignant disease activity at the time of gbs diagnosis, and ) malignancies diagnosed > years post-gbs onset as per pns criteria. total number of gbs patients with malignancy was ( . %). a total of patients ( . %) fulfilled requirements for inclusion as malignancy-associated gbs as per existing criteria for pns. associated malignancy consisted of angioimmunoblastic t-cell lymphoma ( ), poorly differentiated squamous cell carcinoma of nasal septum ( ), gastric adenocarcinoma ( ), hepatocellular carcinoma due to hepatitis b ( ), rectal carcinoma with liver metastasis ( ) and myelodysplastic syndrome ( ). malignancy was globally commoner in our gbs cohort compared to the general population (odds ratio: . ; ci: . - . ; p = . ). however, this was unconfirmed if paraneoplastic criteria were applied. gbs patients with cancer were significantly more likely to be older (p = . ), hyponatremic (p = . ) and demonstrate more electrophysiological axonal loss (p < . ). cerebrospinal fluid (csf) protein levels were lower in the malignancy group (p = . ) and neurological improvement was less likely (p = . ). in-patient mortality was significantly higher in patients with malignancy (p < . ). none of the patients in the malignancy group had positive anti-ganglioside antibodies or anti-neuronal antibodies (anti-hu, yo, ri, crmp ). we conclude global cancer risk is higher in gbs than in the general population, although definition-dependent. application of the strict published criteria for paraneoplastic syndrome reduced the number of cases and suggested absence of a link. malignancy requires consideration in elderly, hyponatremic subjects with normal csf protein, severe electrophysiological axonal loss who fail to improve post-treatment. fu liong h , aude-marie g , anne-catherine an , emilien d , julien c , shahram a , yusuf r . anti-myelin associated glycoprotein antibody (anti-mag) neuropathy is a slowly progressive neuropathy resulting in disability through distal sensory more than motor deficits and tremor. there are currently no evidence-based treatments for anti-mag neuropathy and the effect of the disease on quality of life (qol) in this patient population is unknown. our objective was to assess determinants of qol in patients with anti-mag neuropathy. the sf- questionnaire was assessed in patients, from marseille, angers (france) and birmingham (united kingdom). routine clinical evaluations included mrc sum score, incat sensory score, inflammatory rasch built overall disability score (i-rods), ataxia score, jamar grip dynamometry, timed meter-walk, neuropathic pain symptom inventory (npsi) score, and fatigue severity score (fss). there were males and females. mean age was . years (sd: . years). mean disease duration was . years (s.d.: . years). there were no significant differences between the french and u.k. cohorts in terms of gender distribution, age, disease duration, anti-mag antibody titre. all physical assessments, including mrc sum score (p= . ), jamar grip dynamometry (p= . ), onls (p= . ), irods (p= . ), incat sensory score (p= . ), -meter timed walk (p= . ), ataxia score (p= . ), tremor score (p= . ), were comparable. prevalence and/or severity of pain (p= . ), fatigue (p= . ), restless legs syndrome (p= ) and cramps (p= . ) were also similar. physical component summary (pcs) and mental component summary (mcs) of the sf questionnaire were significantly lower than in reported normal subjects of both countries (p< . ). all sf- qol domains correlated with i-rods, except mcs for which significance was however approached (p= . ). pcs correlated with mrc sum score, ataxia score, timed m-walk, tremor, jamar grip dynamometry, npsi pain score, fss and level of social support. mcs correlated exclusively with fss and level of social support. in multivariate regression, pcs was associated independently with i-rods (p< . ) and npsi pain score (p= . ), whereas mcs was associated independently with fss (p= . ). qol is accurately predicted in anti-mag neuropathy by the i-rods and fss, lending support to their use in clinical and research settings. effective measures to improve qol should include tremor and neuropathic pain treatment, fatigue management and improved social support. fu liong h , yusuf r . regional neuromuscular clinic, queen elizabeth hospital, university hospitals of birmingham, birmingham, uk; school of life and health sciences, aston brain centre, aston university, birmingham, uk. the electrophysiological diagnosis of guillain-barré syndrome (gbs) is dependent on a number of abnormalities affecting different parameters in peripheral nerves on nerve conduction studies (ncs). diagnostic sensitivity varies with different electrodiagnostic criteria, practices as well as extensiveness of nerve study. however, the most efficient method of performing electrophysiology for guillain-barré syndrome (gbs) is unknown. we retrospectively analyzed electrophysiological data of consecutive gbs patients from birmingham, uk ( - ), studied ≤ weeks post-onset. we first identify abnormal nerves from various regions which produced gbs electrodiagnosis using the recently described criteria by rajabally et al. we subsequently used pre-established hypothetical nerve conduction study protocols to determine the potential optimal method of achieving electrodiagnosis. we found the sensitivity of electrophysiology for each gbs subtype was dependent on the upper and lower limb nerves tested. in acute inflammatory demyelinating polyneuropathy (aidp), abnormalities were predominant in the arms, whereas leg abnormalities predominated in axonal gbs. in aidp, the most common abnormal parameters were distal motor latency ( . %) and conduction block ( . %), and the most frequently affected nerve was the median ( . %). prolonged f-waves were present in % and f-waves were absent in %. mcv was the least frequently abnormal ( . %) with demyelinating range slowing, significantly lower than cb or dml prolongation (p< . in both cases). in axonal gbs, reduced motor amplitudes ( . %) and conduction block ( . %) were the most common parameters, and the most frequently abnormal nerve was the tibial ( . %). f-waves were absent in . %. . % of all motor nerves were unexcitable, significantly more common in lower limb nerves compared to upper limb nerves (p= . ). on comparison of different hypothetical ncs protocols ( unilateral protocols with nerves, with nerves as well as the protocols with -nerves and with exclusive bilateral upper limb and lower limb testing), unilateral -nerve (median, ulnar, common peroneal and tibial) testing produced the highest diagnostic sensitivity for both aidp ( . %) and axonal ( . %) gbs. electrodiagnostic sensitivity in gbs is thus dependent on nerves tested and parameters considered. each subtype preferentially involves specific nerves and parameters. these findings may help per-procedure interpretation, improve electrodiagnostic sensitivity, and reduce patient discomfort. funakoshi k , nagashima t , kokubun n , hirata k , yuki n . department of neurology, dokkyo medical university, tochigi, japan; department of neurology, mishima hospital, nagaoka, japan. recently, igg anti-ganglioside complex (gscs) antibodies have been reported in patients with guillain-barré and miller fisher syndrome. some researchers (nobile-orazio et al., ) reported igm anti-gscs antibodies in multifocal motor neuropathy (mmn) and other chronic immune-mediated neuropathies. in ten of eleven mmn patients with anti-gm antibody, there was a decreased reactivity to gm /gd a compared to single antigen gm . similarly, in one of two chronic inflammatory degenerative polyradiculoneuropathy (cidp) patient with anti-gm antibody, there was a decreased reactivity to gm /gm , gm /gd a, gm /gd b, gm /gt b compared to gm . these relationships were defined negative interaction. in one cidp with anti-gd b antibody, anti-gm /gt b and -gm /gt b reactivity increased although gm and gm were negative (positive interaction). in japan, on the other hand, this correlation remains unclear. sera were investigated from one mmn patient ( y/o male) with anti-gm , and anti-gd a antibody, one cidp patient ( y/o female) with anti-gm , and one subacute sensory motor polyradiculoneuropathy ( y/o male) with anti-gd b, and anti-gq b. igm antibodies to gscs gm /gd a were tested with a mixture of gm and gd a (each pmol/well) as antigen. anti-gm /gd a antibodies were judged to have positive interactions when the optical density was . greater than the sum of the antibodies against individual gm and gd a, and negative interactions when the optical density was % or less compared to the antibodies against individual gm or gd a. antibodies to at least one combination of two of the six gangliosides (gm , gm , gd a, gd b, gt b, and gq b) were similarly tested and judged for positive or negative interactions. in one mmn, anti-gm /gd a had negative interactions. in one cidp, anti-gm /gd a, and anti-gm /gq b had negative interactions. in one subacute sensory motor polyradiculoneuropathy, various anti-gscs antibodies including gd b or gq b had negative interactions. in the cidp patient mentioned above, anti-gm /gt b or anti-gm /gt b had no positive interactions. the relationship between this attenuated reactivity presumably driven by adjacent gangliosides and the mechanism of chronic immune-mediated neuropathies needs to be clarified. galino j , cervellini i , zhu n , stöberl n , fricker fr , lee g , hütte m , lalli g , bennett dl . the nuffield department of clinical neurosciences, john radcliffe hospital, university of oxford, oxford, uk; wolfson card, king's college london, guy's campus, london, uk. schwann cells (scs) are the myelinating cells in peripheral nerve system and are important for normal peripheral nerve development and repair. rala and ralb are small gtpases that have been implicated in neural tube closure, neurite branching and have been described as upstream effectors of proteins involved in cell migration and membrane dynamics. due to their potential role in sc function here we investigated, by genetic ablation in transgenic animals of one or both gtpases, their importance in sc function in adulthood and in nerve repair. we showed that ral gtpases are dispensable for sc function in the naïve state. however ral signalling (provided by rala or ralb) is required for effective remyelination of axons following nerve injury. moreover, absent ral signalling produced defects in axon reinnervation of distal organs and a delay in motor function recovery after nerve injury. we also studied the ral dependent cellular mechanisms that may be responsible for impaired sc remyelination and noted abnormal sc lamellipodia formation that prevent normal axial and radial axon remyelination. this work demonstrates for the first time a novel mechanism for ralgtpases that controls sc lamellipodia formation and their importance in normal sc function during peripheral nerve repair. garcía-lareu b , , , ariza l , cobianchi s , , chillón m , , , , navarro x , , , bosch a , , . tumor necrosis factor (tnf) alpha has been implicated in the pathogenesis of diabetic peripheral neuropathy (dpn), among other inflammatory demyelinating diseases and neuropathic pain. tnf alpha is a pro-inflammatory cytokine that can act at several stages in the demyelination process. it is produced by schwann cells (scs) in the peripheral nervous system (pns) after nerve injury and released into the local environment to attract and activate macrophages at the site of injury, contributing to wallerian degeneration. in vivo studies demonstrated a local inflammation in the sciatic nerve of rats after injection of tnf alpha, followed by demyelination and axonal degeneration. furthermore, the application of tnf alpha resulted in acute mechanical hiperalgesia, a main characteristic of neuropathic pain and therefore tnf alpha is postulated as a biomarker for painful changes after nerve injury. with the aim to characterize tnf alpha effects in chronic neuropathic pain and in diabetic neuropathy, a transgenic mouse model overexpressing tnf alpha cdna under the peripheral myelin protein p promoter was generated. here we characterized the overexpression of tnf alpha in myelinated scs at different stages of myelination (postnatal days , and ) showing that high levels of tnf alpha in sciatic nerve leads to the downregulation of the major pns myelin proteins (p , mbp, pmp , mag) compared to wild type mice, correlating with the loss of structured myelin and an increase in p ntr, a marker for immature and non-myelinated scs in the sciatic nerve. iba staining showed high levels of macrophage infiltration in both sciatic nerve and spinal cord tissues, compared with wild type animals. stress conditions were induced by sciatic nerve crush surgery after which recovery and subsequent remyelination were delayed in the transgenic mice, as evaluated by the sciatic functional index (sfi) and electrophysiological tests. on the other hand, mechanical and thermal nociception seemed to be unaltered, with or without lesion. this model could be helpful in the characterization of the role tnf alpha in pain development, injury and dpn as well as in developing efficient therapeutic strategies to modulate such pathological conditions. garcía-sobrino t , , blanco-arias patricia , , vidal-lijó mp , quintáns bea , , sobrido mj , , pardo j , . department of neurology, hospital clínico, santiago de compostela, spain; neurogenetics research group, instituto de investigaciones sanitarias (idis), santiago de compostela, spain; genomic medicine group (u ), centre for biomedical network research on rare diseases (ciberer), spain; department of neurophysiology, hospital clínico, santiago de compostela, spain. charcot-marie-tooth (cmt) disease is a genetically heterogeneous group of hereditary motor and sensory neuropathies. more than genes were involved in the disease pathogenesis. the objective of this study was to assess the genetic distribution of cmt disease in galicia (northwestern spain). patients were diagnosed as cmt if they had a consistent neurological and/or neurophysiological examination or if they had sensory motor neuropathy with a positive family history. a total of cmt adult patients ( % females) were evaluated with a median age of [ - ] years. the molecular diagnosis was achieved in patients ( %) with a higher success in cmt ( %) than cmt ( %). globally, pmp duplication was the most frequent finding ( %), followed by mutations in mpz ( %), mfn ( %), gjb ( %) and gdap ( %). in cmt , with expception of the pmp duplication, pathogenic variants in egr , nefl and mpz gene were most common. pathogenic variants in mfn and gdap accounted for % of cmt patients. several patients referred to our institution as cmt were diagnosed as hereditary motor neuropathy (hmn) and pathogenic variants in the bscl gene were the most frequent. pathogenic variants not previously related to cmt were identified in mpz, mfn , gjb , egr , nefl and sh tc genes. sporadic or autosomal recessive (ar) cmt accounted for the % of all diagnoses. the genetic epidemiology of cmt in galicia follows a similar pattern to other populations, although some remarkable features are axonal gdap and demyalinating egr pathogenic variants as well a seemingly elevated proportion of ar cases. mutations in mpz gene are associated to a wide range of phenotypes. the r c, in particular, is associated to a severe and early onset disease. some cmt patients respond to immunosuppressive or immunomodulatory treatment, some of them harbouring mpz mutations. we present the case of a -year-old brazilian female with a severe cmt b (r c) that presented an acute episode of worsening after a febrile exanthematic disease. she complained of tingling, stopped walking without support, and could not raise her arms. her emg showed an asymmetrical reduction of motor cv, reduced amplitudes, severe temporal dispersion and possibly conduction block. csf protein was mg%, with normal cell count. diagnosed as gbs, she received ivig. curiously, she markedly improved, regaining the functional status of youth, recovering functions that she had long lost. on emg, motor amplitudes improved significantly, but cv remained the same. reviewing carefully her past history, we identified some motor fluctuations along her adult life. at age , while pregnant, she developed positive sensory symptoms, became unable to get up from the chair and could not walk without assistance. after delivery, she improved, but did not return to baseline. one year later, she presented a new transitory functional worsening after an influenza illness. her parents were healthy but two out her children have a severe cmt. her first daughter never walked and died at the age of six. six months after treatment with ivig, she faced a new relapse. we pulsed with corticosteroids, with great response. she remains stable to date, four months after treatment. the episodic fluctuations and the evident response to treatment clearly suggest an associated immunomediated process. the fast improvement of the amplitude with maintenance of cv suggests that another mechanism in addition to demyelination and axonal degeneration is involved. we propose the existence of an associated channelopathy. funded by cnpq, faepa, pronas (ministry of healthy) gbs is an acute, immune-mediated neuropathy that comprises three major subtypes: aidp, aman, and amsan. on clinical grounds, this distinction is based on nerve conduction studies (ncs): aidp is a demyelinating neuropathy, aman is an axonal motor neuropathy, and amsan, an axonal sensory and motor neuropathy. recently, it has been demonstrated that axonal gbs, in addition to axonal degeneration, may present conduction block (cb) that can progress to axonal regeneration or revert, characterising a reversible conduction failure (rcf), a finding usually associated to good prognosis. patients presenting axonal cb are frequently diagnosed as having aidp, a mistake that can be avoided with two sets of ncs, one as early as possible and the second after three weeks of disease. following these recommendations, we retrospectively classified a brazilian group with gbs followed in our institution that had been submitted to two ncs sets. from september to january , patients fulfilled clinical criteria for gbs at clinical hospital of ribeirão preto and had more than one ncs accomplished. at least four motor nerves (median, ulnar, peroneal, and posterior tibial) and five sensory nerves (medial, ulnar, radial, sural, and peroneal superficial) were evaluated in each examination, according to routine procedures. first ncs revealed patients with aidp ( %), cases of axonal gbs ( aman, amsan, %), and patients with equivocal results ( %). at follow-up study, patients ( %) had their classification changed. the main shifts were from aidp to axonal group and from equivocal results to aidp. aidp increased to % (n = ), axonal gbs increased to % (n = , aman, amsan), and there were no more equivocal patients. although the majority of studies have shown that aidp is more frequent in western countries while axonal gbs predominates in eastern countries, we found a different pattern of distribution in our population, with predominance of the axonal subtypes. the considerable increase of axonal gbs at follow-up studies reinforces that serial emg are mandatory for accurate diagnosis of gbs subtypes. prospective studies are now being carried out in order to confirm these results. in amyloid light chain (al) amyloidosis, misfolded light chain (lc) accumulates and causes progressive peripheral neuropathy (pn) and progressive failure of critical organs such as the heart and kidneys. progressive ascending sensorimotor neuropathy is often a related clinical finding. the deposition of toxic lc amyloid in peripheral nerves is associated with paresthesias, pain, muscle weaknesss, orthostatic hypotension, and diarrhea or constipation in approximately % of patients with al amyloidosis. there are no approved treatments for al amyloidosis. current therapeutic approaches for al amyloidosis are intended to limit lc production but do not directly target misfolded lc deposited as amyloid in organs. we report phase / trial results of neod , an investigational monoclonal antibody that targets misfolded lc and may neutralize circulating lc aggregates and clear insoluble deposits. trial inclusion criteria were one or more plasma cell-directed treatment completed before enrollment, partial or greater hematologic response to any previous therapy, and persistent organ dysfunction. neod was administered intravenously every days during a dose-escalation phase ( patients; . , , , , , , or mg/kg in a + study design) and an expansion phase ( patients; mg/kg). we assessed safety, tolerability, pharmacokinetics, immunogenicity, organ responses based on consensus criteria, and pn responses using the neuropathy impairment score-lower limb (nis-ll). neod treatment was not associated with dose-limiting toxicities or serious adverse events, drug-related discontinuations, or antidrug antibody development in any patient (n = ). of patients with measurable pn at baseline (n = ), % achieved pn response based on the nis-ll score after months of treatment, which resulted in a median % nis-ll score reduction (mean baseline nis-ll, . ). in a best response analysis, % of cardiac-evaluable patients (n = ) and % of renal-evaluable patients (n = ) met respective criteria for organ response. improvements in neuropathy have not been previously shown in al amyloidosis. these results demonstrated that monthly neod infusions were safe, well tolerated, and associated with responses across three different organ systems. gervais cbl , ross ma , goodman bp , khoury ja , muzyka i , smith be . mayo clinic in arizona, scottsdale, az, usa. this work describes clinical, examination and electrophysiologic findings in a cohort of patients with length dependent sensorimotor peripheral neuropathy (ldsmpn) with ventral abdominal sensory loss. ldsmpn affects the longest nerve fibers, namely those innervating structures in the feet and hands. what is less well appreciated is that length dependent involvement of sensory nerve fibers in ldsmpn from the thoracic segments gives rise to ventral abdominal sensory loss. consecutive patients seen for ldsmpn (n= ) were evaluated for the presence or absence of ventral abdominal sensory loss. demographic variables, symptoms and quantitative neurologic findings (neuropathy impairment score [nis]) were examined using descriptive statistics. final diagnoses were noted. ventral abdominal sensory loss to pinprick (which was asymptomatic in all patients tested) was documented in / ldsmpn patients ( . %), mean age was . years (range - ), m:f gender ratio was . ( : ), mean nis was . (range - ), ncs/emg abnormalities were found in / patients (the remaining showing objective evidence of small fiber sensory involvement). ldsmpn patients without ventral abdominal sensory loss (n= ) had a mean age of . (range - ), m:f of . ( : ), and mean nis of . (range - ). no patient ( / ) had dorsal torso sensory loss between the shoulder and buttock levels on either side. diagnoses of the ldsmpn patients with vs. without ventral abdominal sensory loss included charcot marie tooth or other hereditary neuropathy (n= vs. ), abnormal carbohydrate metabolism (n= vs. ), idiopathic (n= vs. ), hypothyroidism (n= vs. ), inflammatory (n= vs. ) and other (n= vs. ). ventral abdominal sensory loss appears to be common in patients diagnosed with ldsmpn of a variety of causes including inherited neuropathies; in addition to those innervating distal limb territories, distal sensory fibers from the thoracic region represent another category of length dependent involvement in ldsmpn; ) the clinical examination of ldsmpn should include the ventral abdomen. gibbons c , garcia j , casasola m , freeman r . beth israel deaconess medical center, harvard medical school, boston, usa. objective: to determine the relationship between measures of autonomic function, electrochemical sweat conductance (esc) and intra-epidermal (ienfd) and sudomotor nerve fiber density (sgnfd). background: structural and functional measures of small fiber neuropathy have been studied in patients with diabetes, but little information comparing these techniques exists. design/methods: we studied patients with diabetes (ages ± yrs, gender f) and healthy control subjects (age ± yrs, gender f). subjects underwent examination scores (nis-ll), quantitative sensory testing, autonomic testing (heart rate variability, valsalva maneuver, tilt test), esc, and millimeter punch skin biopsies at the distal leg and distal thigh for ienfd & sgnfd. results: there were strong correlations between exam scores (nis-ll) and biopsy ienfd (r=− . , p< . distal leg; r=− . , p< . distal thigh), and sgnfd (r=− . , p< . distal leg; r=− . , p< . distal thigh). moderate correlations were noted between exam scores and qst (r values . - . , p< . ), ienfd and qst (r= . - . ). modest correlations were noted between esc and parasympathetic function (r= . - . , p< . ). modest correlations were noted between esc and ienfd (r= . , p< . , but only at the distal leg) and esc and nis-ll (− . , p< . ). no correlations between exam scores and autonomic function were noted. no correlations were detected between esc and sgnfd (r=− . - . ) or esc and sympathetic adrenergic function (r= . ). conclusions: differences between tests are expected based on our understanding of the pathophysiology and natural history of diabetic neuropathy. exam scores, qst results and biopsy results do correlate, and are consistent with prior studies. in contrast, there was little relationship between tests of autonomic function and exam scores, similar to prior studies and our understanding of autonomic function and differences in p. esc had little correlation with either exam scores or biopsy ienfd or sgnfd. there was a modest correlation between esc and parasympathetic function. the exact relationship between esc and diabetic peripheral neuropathy is not clear, and further research studies are needed to determine the role this technique has in clinical practice. objective: to determine the optimal tissue thickness of skin biopsy sections for studies of cutaneous nerve fibers. background: although analysis of intra-epidermal nerve fiber density (ienfd) is routinely reported using μm thick tissue sections, many recent studies of peripheral alpha-synuclein deposition use or μm frozen sections, or μm paraffin embedded tissue sections. design/methods: we compared the results of biopsies from patients with parkinson's disease (pd), using tissue sections each of , and μm thickness. tissues were stained with pgp . and stained for phosphorylated alpha-synuclein (p-syn). the total number of dermal structures (hair follicles, sweat glands, pilomotor muscles) were quantified, nerve densities analyzed, and the frequency of p-syn positive results. we also studied μm paraffin embedded tissue samples from patients with pd. results: in the biopsies of patients with pd there were no differences in the number of sweat glands, hair follicles or pilomotor muscles in , or μm sections. there were significantly fewer blood vessels noted in and μm sections compared to μm sections (p< . ). ienfd and sgnfd declined with tissue thickness (p. . , all) and there was increased variability in results in thinner tissue sections. there was a highly significant reduction in p-syn positive sections in thinner tissue sections (p< . , all tissues compared to μm sections). paraffin embedded tissue sections had significantly lower nerve densities and positive p-syn results (p< . ) compared to all frozen tissue sections of , and μm thickness. thinner tissue sections carried a greater risk of false positive result or indeterminate results due to difficulty interpreting overlap with pgp . . conclusions: tissue sample thickness plays a critical role in interpretation of skin biopsy results. thinner tissue sections, or paraffin embedded tissue sections, do not provide equivalent data and significantly underestimate nerve densities and positive alpha-synuclein results with increase false positive results despite similar numbers of dermal tissue structures. ace- is an investigational protein therapeutic that acts as a localized ligand trap for myostatin and other negative regulators of muscle growth. local injection of ace- into the gastrocnemius muscle of wild-type, mdx, and sod mice produced dose-dependent increases in muscle mass and force without systemic effects. in a phase single-center, double-blind, placebo-controlled dose escalation study in post-menopausal women, unilateral injections of ace- into the rectus femoris (rf) or tibialis anterior (ta) muscle were generally safe and well tolerated. mean percent changes from baseline in muscle volume of the injected muscle were + . % in the rf and + . % in the ta at the highest dose administered with minimal changes observed in the contralateral side and placebo-treated subjects. frequent related aes (≥ %) included injection site pain, pain in extremity, injection site discomfort, and muscle twitching, with similar incidence in ace- and placebo-treated groups. all aes were grade - and reversible. together, these preclinical and clinical results support further studies of ace- in myogenic and/or neurogenic diseases with focal loss of muscle strength and function, including cmt. a phase study is ongoing in facioscapulohumeral muscular dystrophy (fshd). study a - is an ongoing multicenter, two-part, phase study to evaluate the safety, tolerability, pharmacodynamics, efficacy, and pharmacokinetics of ace- in patients with cmt and cmtx. part is open-label and will enroll up to dose-escalating cohorts ( patients per cohort); part is randomized, double-blind, and placebo-controlled, and will enroll an additional patients. ace- will be administered bilaterally to the ta muscle once every three weeks for up to five doses. a safety review team will meet periodically throughout the study to review safety data and make dosing recommendations, including the recommended dose level for part . eligible patients must have genetically confirmed cmt or cmtx with mild-moderate weakness in ankle dorsiflexion. safety and tolerability will serve as the primary outcome for part , muscle volume evaluated by mri for part . additional outcome measures of interest include strength by quantitative muscle testing, function by motor tests, and quality of life by the cmt-health index questionnaire. our study was aimed to evaluate the functional and morphological consequences of cellular and humoral responses in chronic inflammatory demyelinating neuropathy (cidp), using extensive standardized high-resolution sonography (hrus) and nerve conduction study (ncs) protocols in incident treatment-naive patients. we enrolled consecutive, newly diagnosed, treatment naive patients with cidp. in addition to all relevant clinical examinations, all patients underwent a standardized ncs and extensive hrus protocol, of median, ulnar, tibial, fibular and sural nerves. we assessed standard nerve and fascicle size, and echogenicity. we found focal sonographic enlargements in multiple nerves and nerve segments with and without ncs abnormalities. the degree of nerve hypertrophy was not associated with presence of ncs features of demyelination, i.e. / ( %) of median nerve segments showed enlargement without strong decrease in motor conduction velocity and / ( %) hypertrophic median nerve segments revealed no conduction block. a lower distal cmap of median nerve was related with lower mrc sums-scores (p < . ). we found no correlation between age, disease duration or mrc sum-score and nerve size. cellular and humoral responses in cidp may lead to nerve enlargement along the length of nerves, that can be detected by hrus, whereas ncs allows identification of its' specific focal disruption in nerve function. the most common complication of diabetes is peripheral neuropathy, which has prevalence as high as % and is characterized by damage to neurons, schwann cells and blood vessels within the nerve. the concept of schwannopathy as an integral factor in the pathogenesis of diabetic neuropathy is re-emerging, and it is now known that schwann cells cultured in hyperglycemic environments underproduce neurotrophins and exhibit loss of axonal associations, further indicating a non-optimal glial cell activation and function. furthermore, the increased expression of p ntr in myelin sheaths around fibers that are susceptible to axonal degeneration in diabetic neuropathy suggest an important role for this molecule in disease progression. with this project, it is our main goal to evaluate how disruption of p ntr signaling in the schwann cells affects the pathophysiology of diabetic neuropathy. by using a fluorescent live/death cell viability assay, our preliminary data indicate that wild-type schwann cell cultures present increased cell death rate h after stimulation with high levels of glucose. the p ntr has a highly recognized role in the activation of death signals and when absent, we observed that schwann cells are significantly more resistant to apoptosis in hyperglycemic conditions. the role of p ntr receptor signaling in neuron-schwann cell communication and myelination under in vitro diabetic conditions was investigated with primary schwann cell-sensory neuron co-cultures. after eight days of ascorbic acid stimulation, both under euglycemic and hyperglycemic conditions, myelination was assessed by confocal microscopy using specific markers for neurons and myelin. results highlight a compromised ability of wild-type schwann cells to myelinate axons when exposed to a hyperglycemic environment, which was even intensified in co-cultures with schwann cells lacking the p ntr . to complement the in vitro studies, we are modeling type diabetes in a p ntr schwann cell conditional ko mouse model and plan to investigate nerve mrna expression profile to disclose genetic regulation depending on this receptor signaling and its modulatory role in endoneurial hypoxia and neuroinflammation. the results from this project will provide an integrated vision of how impaired schwann cell activity guides neuropathy progression. gondim faa , barreira aa , cruz mw , cunha fmb , de freitas m , frança mc jr , marques w jr , nascimento ojm , oliveira asb , pereira rc , pupe c , rotta ft , schestatsky p . panelists on behalf of the scientific department of peripheral neuropathy, brazilian academy of neurology, brazil. neuropathy is one of the most common neurological manifestations of several diseases and sfn has been progressively receiving more attention in the medical literature. the aim of this study is to generate a set of recommendations to define and diagnose sfn in brazil. a group of neurologists, members of the scientific department of peripheral neuropathy from the brazilian academy of neurology reviewed a preliminary draft prepared by the first author that was distributed by email. the panelists got together on . . at the city of fortaleza, brazil, to discuss and finish the text for the first submission of the manuscript. sfn can be defined as a subtype of neuropathy characterized by selective involvement of unmyelinated or thinly myelinated sensory (sometimes also autonomic) fibers. it is usually characterized by sensory (pain/dysesthesias/pruritus) or combined sensory and autonomic complaints, associated with an almost entirely normal neurological examination (except for sensory changes). electromyography is normal. a growing list of medical conditions has been linked to sfn, although there is no evidence-based literature to support the use of any specific set of screening tests to diagnose the etiology of sfn (the panelists will suggest a basic screening panel). sfn may also serve as a fallacious but useful terminology to uncover discrepancies in the normal values from different neurophysiology laboratories. in brazil, skin biopsy is not usually performed and initial forms of leprosy may have predominant small fiber involvement. there are several tests to demonstrate involvement of small sensory and autonomic fibers. skin wrinkling test, sympathetic skin responses & heart rate variability (conducted on emg machines) and thermoregulatory sweat test may be low-cost screening alternatives. after the final meeting on . . , we finished the first draft for submission to arquivos de neuropsiquiatria (together with a translation to portuguese as supplementary material), the official journal of the brazilian academy of neurology to serve as a source for the definition and diagnosis of sfn in brazil. the final draft will be submitted after presentation at the pns meeting in barcelona on . . the lower cranial nerves (lcn) include the paired glossopharyngeal, the vagal, the accessory and the hypoglossal nerves. these are involved in the execution of swallowing, speech, phonation, tasting, as well as sensory and autonomic functions. lcn can be affected in central and peripheral nervous system diseases. this study investigates the use of ultrasound (us) to detect lesions of the peripheral course of lcn. in addition mri can be used in unilateral lcn local lesions to demonstrate indirect signs of nerve lesions such as muscle atrophy. the patients were examined in supine position with the neck in maximum extension with either a regular or a portable us system (logiq e and logiq e ge healthcare, milwaukee, wisconsin) and high frequency transducers ( to mhz). according to the detected pathology longitudinal or transversal images were recorded. a series of exemplary observations are demonstrated. in one case a paraganglioma in the glossopharyngeal nerve at the carotid sinus was found. in another patient an us of the thyroid gland revealed a nerve tumor, which was later identified to be a schwannoma. in another case multiple neurofibroma were identified in the vagal nerve during a routine neurofibromatosis screening. us has become also important in investigations of lesions of the accessory nerve, following damage by surgery. nerve continuity and scar formation can be distinguished. lesions of the hypoglossal nerve can be caused by tumor infiltration. in one patient an infiltration of the nerve by a squamous cell carcinoma, and in another case by a low grade sarcoma were detected. in addition mri demonstrated an atrophy of the tongue. identification and assessment of the lcn from the point of exit of the skull to their endpoint can be pursued by us techniques, which have been described for the individual nerves. us is easily applicable and -in addition to the "static" image of the mri -allows assessing nerve positions during different movements, as well as detecting muscle movements as fasciculations. mri can also be used to detect not only more proximal lesions, but also changes of the skeletal muscle by denervation. grümme l , hattenhauer st , wolffram k , kleinschnitz c , mausberg ak , stettner m . university hospital essen, essen, germany; heinrich-heine-university, düsseldorf, germany. in chronic inflammatory demyelinating polyneuropathy (cidp), t cells are suspected to play a crucial role in myelin destruction. cd and cd t cells contribute to the inflammatory process, while the exact mechanism of myelin damage is still under debate. to elucidate the molecular interaction of t cells and myelin sheets, we compared neuritogenic and control cells in a live imaging in vitro model. the myelinated fibres of rat dorsal root ganglia (drg) served as model for the peripheral nervous system. drgs of embryonic (e ) lewis rats were cultured; myelination was initiated after one week in vitro and continued for two additional weeks. lewis rats immunized with p and neuritogenic t cells of the lymph nodes were obtained ten days after immunization. control t cells were prepared from healthy rats. for vital tracking, neuritogenic t cells were stained with orange cell tracker, while the control cells were labelled with cfse. myelin detection in vital cultures was assessed by incorporation of c fatty acids conjugated with a fluorophore into the myelin layer. experiments were performed in a conditioned microscope chamber, the two t cell populations were added simultaneously to the drg culture and migration was tracked using live cell imaging. we observed differing migration patterns for neuritogenic and control t cells. the velocity as well as the directionality was altered. after initial contact, the non-neuriotgenic t cells in close proximity to myelin subsequently decreased over time, while the numbers of neuritogenic t cells close to myelin remained stable. long term incubation with neuritogenic t cells affected the myelin integrity in regard to the intermodal length as well as the myelin ratio. further experiments will elucidate the specific effects of neuritogenic t cells to decipher the role of t cells during inflammation in the pns, which could be useful in developing targeted therapies. gundapaneni b , sultan mb , keohane dj , schwartz j . inventiv health inc., burlington, ma, usa; pfizer inc., new york, ny, usa. transthyretin familial amyloid polyneuropathy (ttr-fap) is a rare, life-threatening disorder caused by protein destabilizing ttr gene mutations, broadly classified as val met (the most common mutation worldwide) and non-val met genotypes. tafamidis, a highly-specific ttr-stabilizer, is the only medicine approved to delay neurologic progression in ttr-fap. the objective of this post-hoc analysis was to compare the effects of tafamidis on neuropathy progression in patients with val met and non-val met genotypes. val met patients were participants in a randomized, double-blind, placebo-controlled clinical trial of tafamidis, while non-val met patients were participants in an open-label tafamidis study. patients were grouped into three cohorts: val met tafamidis (n= ); val met placebo (n= ); and non-val met tafamidis (n= ). baseline disease severity and change in disease severity from baseline to month was assessed using the neuropathy impairment score-lower limbs (nis-ll). the effect of tafamidis in the val met and non-val met cohorts versus the val met placebo cohort was determined using a mixed-effects model for repeated measures (mmrm). at baseline, patients in the non-val met cohort were older, had longer symptom duration, and more advanced neurologic impairment than the val met cohorts. at month , the baseline-adjusted mean ± standard error change in nis-ll was comparable between the val met tafamidis and non-val met tafamidis cohorts ( . ± . and . ± . , respectively). these changes were smaller than that observed in the val met placebo cohort ( . ± . ; p= . vs val met and p= . vs non-val met) indicating less disease progression. based on predicted values from the mmrm analysis, the size of the change in nis-ll across the full range of baseline nis-ll scores was remarkably similar in the val met tafamidis and non-val met tafamidis cohorts and was consistently smaller than that observed in the val met placebo cohort. moreover, in all three cohorts, as baseline nis-ll increased, the predicted level of disease progression also increased. in conclusion, while controlling for baseline disease severity, tafamidis delayed disease progression to a comparable extent in val met and non-val met patients. the similar trajectories of disease progression across val met and non-val met patients suggest that these two genotype groups may be more similar than previously considered. clin-icaltrials.gov identifiers: nct ; nct . gutmann l , shy m . university of iowa, iowa city, ia, usa. post tetanic potentiation (ptp) is a physiological phenonmenon seen with disorders of the neuromuscular junction (nmj). it is caused by the influx of ca++ into the terminal axon during the tetanus resulting in an increased number of acetylcholine (ach) vescicles released by each axonal action potential. in myasthenic syndromes it results in improved nmj function by increasing the probability of achieving a large enough end plate potential to generate a muscle action potential. ptp has different appearances depending on whether the nmj defect is pre-synaptic or post-synaptic. in pre-synaptic defects (the most common being lambert-eaton syndrome) there is an increased amplitude of the muscle action potential after a tetanus that persists up to minutes. prolonged ptp has now been reported in disorders, one on a genetically determined neuropathy/myasthenic basis and the other on an acquired toxic origin. prolonged ptp up to minutes was reported in families with a motor neuropathy (having leg weakness and foot deformites) and a congenital myasthenic syndrome caused by a heterozygous mutation of the synaptotamin ii (syt ) gene (c t>g p.pro [p.asp ala] and c g>a [p.pro leu]). electrophysiological testing showed features of a presynaptic defect with prolonged ptp persisting up to minutes. the same phenomenon has been noted in the acquired pre-synaptic defect caused by botululinum toxin. the ptp continued up to minutes. syt is the synaptic vescicle calcium sensor in the terminal axon, allowing for fusion of ach containing vescicles with the presynaptic membrane and the synchronous release of ach. the fusion requires a complex assembly process involving snare proteins. both the syt gene mutation and botulinum toxin affect normal syt function, the mutation by altering amino acids in the calcium-binding domain and the toxin by binding to syt as well as gangliosides gd a and gt b on the neural membrane. the mechanism for the ptp prolongation remains unknown. prolonged ptp appears to be a unique physiological abnormality resulting from altered syt . this phenomenon has been decribed to occur in syt mutations causing congenital motor neuropathy/myasthenic syndrome and botulism. the abnormality may represent a physiological marker for a presynaptic nmj defect involving altered syt . hachisuka a , , senger j , curran m , chan km , . division of physical medicine and rehabilitation, university of alberta, edmonton, canada; department of rehabilitation and medicine, university of occupational and environmental health, kitakyushu, japan; division of plastic surgery, university of alberta, edmonton, canada. background: motor unit number estimation (mune) techniques are valuable tools in neuromuscular disease. among them, the multiple point stimulation (mps) is one of the most common used. contamination by distant single motor unit potentials (smups) generated by neighboring muscles is a potential confounding factor. this is particularly problematic in ulnar neuropathy, one of the most common neuropathies in humans. reason being that the ulnar nerve innervates the majority of hand muscles. the goals of this study are to test the hypotheses that ) distant smups all have an initial positive deflection and ) elimination of smups generated by distal muscles will significantly lower the mune results in the hypothenar muscles. methods: to address the first hypothesis, we tested subjects by stimulating their median nerve while recording smups simultaneously over the hypothenar and thenar muscles. for the second hypothesis, we carried out mps mune of the hypothenar muscles using multi-channel recordings placed over ulnar innervated intrinsic muscles across the hand. when a smup with an initial positive deflection was detected at the hypothenar electrodes, its original was systemically tracked through all the recording channels. results: in the first series of experiments, in accordance with the dipole theory, all smups recorded at the hypothenar recording electrodes had an initial positive polarity. in the second series of experiments, of the studies carried out in subjects, distant smups generated by muscles other than those in the hypothenar eminence represented ± . % (mean±sd) of the overall sample. mune calculated using only smups generated by the hypothenar muscles was ± , compared to ± if all smups were included (p < . ). the extent of increase in mune was highly correlated with the proportion of distant smups found in each study (r = . , p < . ). conclusion: in contrary to some studies suggesting that smups from distant muscles could have an initial negative deflection, we found all smups from distant muscles had a positive deflection. exclusion of those smups from the sample had a significant impact on the mune results. hagiwara w , konno s , kihara inoue m , fujioka t . toho university, tokyo, japan. matrix metalloproteinase (mmp) plays crucial roles in developing immune-mediated neuritis as guillain-barré syndrome (gbs) and its animal model experimental autoimmune neuritis (ean). to investigate the intraneural expression of mmps during ean and the effect of a phosphodiesterase- inhibitor cilostazol (clz) on it, ean rats were treated with either mg/kg/day of clz or vehicle from one day post immunization (dpi). to induce ean female lewis rats were immunized with synthetic peptide from bovine p protein. cauda equina (ce) were removed in several time points, total rna was extracted and reverse-transcribed to obtain cdna that was subjected to real-time pcr analysis for expression of mmp- , mmp- and tissue inhibitor of matrix metalloproteinase- (timp- ) messages. mmp- and mmp- messages peaked at dpi, that is presymptomatic phase of ean. all rats developed motor paralysis at dpi, mmp- and mmp- messages subsided at this moment. however, timp- message reciprocally increased at dpi, persisted through dpi. treatment of clz suppressed motor paralysis of ean significantly. mmp- message peaked at dpi and mmp- message peaked at dpi. on the other hand, both messages at dpi were suppressed compared to untreated ean rats. timp- message in clz treated rats peaked at dpi coincided with motor paralysis peak. both mmp- and mmp- messages might result in subsequent upregulation of timp- that finally downregulates mmps activity and inflammatory process. clz treatment suppressed and delayed expression of mmps and facilitate timp- expression, resulting suppression of ean. the precise mechanism of expression of mmps and timp- remain unclear, however, that mmp- and mmp- messages peaked at dpi suggests involvement of pro-inflammatory cytokines such as tumor necrosis factor-alpha or interleukin- . clz might suppress these cytokines resulted in mmps down regulation. mmp- less affected by clz and thus stimulated timp- expression at dpi. clz might rational treatment for immune-mediated neuropathy via mmps modulation although further investigation especially in-vivo study is needed. haidar m , de winter v , asselbergh b , bouhy d , timmerman v . peripheral neuropathy research group, vib, university of antwerp, antwerp, belgium. the small heat shock protein hspb (hsp ) gene is ubiquitously expressed and encodes for a chaperone protein with essential cellular functions. our lab was the first to identify missense mutations in hspb responsible for axonal charcot-marie-tooth neuropathy (cmt f). since then we became interested in understanding the physiological functions of hspb and its association with cmt neuropathies. we demonstrated the involvement of hspb in microtubule stability. because of the link between autophagosome formation and its intracellular transport, and microtubules stability, we believed that the macro-autophagy process could be regulated by hspb . macro-autophagy is a cellular housekeeping process during which autophagosomes target, envelop and degrade aberrant protein aggregates and damaged organelles. there is strong evidence for an essential role for autophagy in the maintenance of neuronal homeostasis; hence its impairment can lead to a neuropathic condition. our data indicate that macro-autophagy is disrupted by hspb cmt-causing mutations. combining novel microscopy and interactomics techniques we unravelled the way different cmt-causing mutations in hspb impair the autophagic pathway. our data present the impairment of autophagy as a possible pathomechanism for cmt-causing hspb mutations. hajjar h , gautier b , berthelot j , gonzalez e , gess b , young p , tricaud n . institute of neurosciences of montpelllier, inserm, university of montpellier, montpellier, france; universitätsklinikum münster, klinik für schlafmedizin und neuromuskuläre erkrankungen, münster, germany. cmt a, the most common of charcot-marie-tooth diseases, results from the duplication of peripheral myelin protein (pmp ) gene. this gene encodes for a small protein of kda, pmp , mainly produced by schwann cells and the excess of pmp leads to demyelination. there is no cure for this disease but one approach for a treatment is gene therapy. a transgenic rat model exists for cmt a, which possesses copies of the mouse pmp gene. our goal is to provide a proof of principle for gene therapy in peripheral nerves using this rat model of cmt a. our strategy is to reduce the overexpression of mouse pmp protein in rats schwann cells using short hairpin rnas (shrnas). shrnas are small non-coding rnas that specifically bind to targeted mrnas resulting in their degradation. we tested for the efficiency of several shrnas targeting mouse pmp in vitro to find two shrnas that reduce pmp levels. the shrnas have been cloned in an adeno-associated serotype (aav ) viral vector together with green fluorescent protein in order to detect infected cells. aav was selected for its high transduction rate of myelinating schwann cells, for its good diffusion and low immunogenicity. we plan bilateral injections in the sciatic nerve of control and diseased rats. the efficiency of this gene therapy will be checked by assessing muscle strength (grip test), way of walking (catwalk), mobility (rotarod) and nerve conduction velocity of treated cmt a rats versus non-treated. the process of myelination and myelin maintenance in schwann cells will be analyzed by biochemistry and electron microscopy. biochemical tests include western blot for pmp protein expression in sciatic nerve, immunohistochemistry for pmp protein expression in myelinating schwann cells and pcr for mrna pmp expression. if the therapy is successful in rats, it could possibly be later on used in clinical trials. ultrasound is a widely-used tool in diagnosing carpal tunnel syndrome (cts). several different methods for sonographical evaluation of median nerve damage exist, such as calculating the ratio of cross sectional areas (csa) of median nerves in various sites. this enables detection of actual nerve swelling proximal to the carpal tunnel as an expression of median nerve damage inside the carpal tunnel (as seen in cts). in comparison to other diagnostic methods no data exist about the prevalence of cts like changes in an unselected population. a series of non-selected fresh cadavers were examined. arms of fresh, non-embalmed whole body cadavers were examined. the medical record did not allow to obtain conclusive information on the peripheral nerves. using a regular ultrasound system with a mhz transducer, median nerves were identified and tracked along their course in the forearms. csa measurements of the median nerves were performed at two sites in each arm: .) halfway between the elbow joint and wrist, .) directly proximal to the carpal tunnel. csa ratio was calculated with the following formula: csa ratio = csa wrist (cm ) csa forearm (cm ) csa ratio < . was found in ( . %) arms, csa ratio ≥ . in ( . %) arms and csa ratio ≥ in ( %) arms. csa ratio ≥ . was detected in . % of women and % of men. the overall mean (± sd) age was . ± . years. men ( . ± . ) were significantly younger than women ( . ± . ; p = . ). a weak but significant correlation between age and csa ratio was found in women (spearman -− . ; p= . ), but not in men (p= . ). the mean bmi for csa ratio ≥ . was . ± sd . . based on a csa ratio ≥ . as a criterion for cts, the present ultrasound results are consistent with the average cts prevalence reported in previous studies, which were obtained with electrophysiological methods. this study on a large unselected series of cadavers confirms the comparability of both methods. instrumental activities of daily living (badl and iadl), falls and gait patterns in the large, prospective, population based rotterdam study. in total, participants of this study (mean age years, % women) underwent a polyneuropathy screening involving a symptom questionnaire, neurological examination and nerve conduction studies. screening yielded four groups: no, possible, probable and definite polyneuropathy. participants were interviewed about badl (stanford health assessment questionnaire), iadl (instrumental activities of daily living scale) and frequency of falling in the previous year. in a random subset of participants, gait was assessed with an electronic walkway (gaitrite). associations of polyneuropathy with badl and iadl were analyzed continuously with linear regression, and dichotomously with logistic regression. history of falling was evaluated with logistic regression and gait changes were evaluated with linear regression. we found that participants with definite polyneuropathy had more difficulty in performing badl and iadl than participants without polyneuropathy. polyneuropathy related to worse scores of all badl (especially walking) and three iadl components (housekeeping, traveling, and shopping). participants with definite polyneuropathy were two times more likely to fall, and these falls more often resulted in injury. participants with polyneuropathy had worse gait parameters on the walkway, including lower walking speed and cadence, and more errors in tandem walking. in summary, chronic polyneuropathy is strongly associated with significant impairment in daily life. recognition of polyneuropathy and related disability is very important in order to inform, support and possibly treat patients, and to prevent future falls and dependence in daily functioning. patients with polyneuropathy often suffer from tingling sensations, numbness, weakness and pain. these symptoms are used in several screening questionnaires, most of which were developed for high-risk patient groups, such as individuals with diabetes mellitus. in most tools equal weights are applied to all symptoms, while some might be more informative than others. we evaluated the diagnostic value and frequency of occurrence of individual symptoms of chronic polyneuropathy and constructed and validated a simple screening questionnaire that can reliably help to diagnose polyneuropathy in low-risk patient groups. in a multi-step procedure, we initially compiled a twelve-item questionnaire concerning symptoms of polyneuropathy. the questionnaire was completed by polyneuropathy patients and controls (headache, transient ischemic attack, multiple sclerosis). we calculated the sensitivity, specificity and likelihood ratios of each individual symptom. next, stepwise multivariable logistic regression was used to create a compact model, able to discriminate cases from controls using only the most informative symptoms. a simple scoring system was subsequently developed based on the regression coefficients of this reduced model. external validation was subsequently conducted in a population of cases with chronic idiopathic axonal polyneuropathy and controls without polyneuropathy. performance was assessed with discrimination (area under the curve, auc), and calibration. numbness and tingling feet were most frequently reported by polyneuropathy patients and had the highest sensitivity. feeling as if walking on cotton wool and allodynia of the feet had the highest specificity. multivariable logistic regression yielded a model that contained these four symptoms, complemented with balance problems and tingling hands. based on this regression analysis, the erasmus polyneuropathy symptom score (e-pss) was created, a score ranging from to . this polyneuropathy symptom score had a good performance (auc . ) in de derivation set and proved to be valid in the external population (auc . ). in this study, we created a simple, validated polyneuropathy symptom score (e-pss) that takes both the individual value of only six different symptoms and its frequency into account. this tool can be helpful as screening instrument in clinical practice and for future studies on polyneuropathy. multiple sclerosis (ms) is traditionally viewed as a central nervous system disease. to date, there is no unequivocal evidence implicating involvement of the peripheral nervous system (pns). this study aims to prove whether the pns is additionally affected and if so, to detect, localize and quantify these peripheral nerve lesions in patients with multiple sclerosis (ms) by applying high-resolution mr-neurography (mrn) with large anatomical coverage in combination with standard electrophysiological and neurological tests. we prospectively enrolled patients with confirmed ms (> years), two patients with clinically isolated syndrome (cis), and age-/sex-matched healthy volunteers. any other potential causes for a concomitant polyneuropathy were excluded. all ms patients underwent detailed neurological and electrophysiological testing. tesla mrn with large anatomical coverage from lumbar plexus and spinal nerves down to ankle level was performed in all participants by using fat-saturated, t -weighted turbo-spin-echo (tse) sequences (tr/te / ms) and a dual echo tse sequence for t -relaxometry (tr ms; te /te / ms). a d t -weighted, fat-saturated space sequence (tr ms; effective te ms) was used for imaging of the lumbar plexus. manual segmentation of spinal/sciatic/tibial/peroneal nerves was performed on a total of , axial slices. besides evaluation of nerve t w-signal, detailed quantification of nerve lesions by analyzing morphometric (nerve caliber) and microstructural markers (proton-spin-density and t -relaxation-time) was conducted. mean lesion load at thigh level was higher in ms ( . ± . ) vs. controls ( . ± . ;p< . ). nerve proton-spin-density was also higher in ms (tibial/peroneal: . ± . / . ± . ) vs. controls (tibial/peroneal: . ± . / . ± . ;p< . ). in contrast, t -relaxation time was significantly higher in controls (tibial/peroneal: . ± . / . ± . ) vs. ms (tibial/peroneal: . ± . / . ± . ;p< . ). proximal tibial and fibular nerve caliber was also significantly higher in ms (tibial: p< . ; fibular: p= . ). for the first time, pns lesions in ms patients could be visualized and quantified in vivo by high-resolution mrn. lesions are indicated by an increase of proton-spin-density and a decrease of t -relaxation-time. nerve caliber as a morphometric criterion also significantly increased. this proof-of-concept study may offer new insights into the pathomechanism of ms and might have future implications on therapeutic approaches. immunoglobulin g (igg) fc-gammars confer diverse effector functions by linking the cellular and humoral arms of the immune system that has been involved in the pathogenesis of guillain-barré syndrome (gbs). in the post-polio era, the polymorphisms of fc-gammar and their relevant knowledge have become one of the main targets for new therapeutic strategies for the treatment of gbs patients. differences in severity and frequency of gbs subtypes found between south-asian and western populations can be attributed to their genetic susceptibility. therefore, we aimed to determine fc-gammar polymorphic alleles (fc-gammariia: h /r ; fc-gammariiia: v /f ; fc-gammariiib: na /na ) and their possible link with gbs on the currently available large gbs cohort in bangladesh. fc-gamma r polymorphisms of gbs patients and healthy controls were genotyped using sequence-specific pcr. for validation, we carried out the sequencing of some samples for fc-gammariia and fc-gammariiia alleles. no significant differences were found regarding the distribution of fc-gammar genotypes and allele frequencies in gbs patients and controls. fc-gammar-h/h- genotype was significantly predominant in patients with severe disease compared to patients with mild disease (p= . , or, . ; % ci, . - . ). no other significant associations were found in gbs patients for candidate alleles and disease severity. fc-gammariiia-f/f- was found to be significantly predominant in anti-gm antibody positive gbs patients compared to anti-gm antibody negative patients (p= . , or, . , % ci, . - . ). fc-gammariiia-v alleles were significantly higher in patients with poor prognosis when compared to patients with good outcome (p= . , or, . , % ci, . - . ). no significant association of fc-gammariiib genotypes and alleles were found with gbs patients, disease severity and disease outcome. extensive subgroup analysis revealed no significant association in genotype and allele frequencies between aman and aidp subtype. in conclusion, igg fc-gammar polymorphisms do not constitute significant risk markers for susceptibility to gbs, however homozygous fc-gammariia-h might be involved in the severe form of gbs. in addition, fc-gammariiia-f/f- might play an important role in the molecular mimicry against nerve gangliosides in gbs. further studies that enroll a large number of patients (e,g. igos) are required to confirm the present findings from different geographical areas. hayes jm , o'brien pd , backus c , feldman el . department of neurology, university of michigan, ann arbor, mi, usa. peripheral neuropathy (pn) is a common complication observed in patients with impaired glucose tolerance and type diabetes. male mice fed a high fat diet (hfd) develop metabolic impairments and pn serving as an appropriate animal model to study pn development and progression. it is well documented that female mice fed a hfd display a degree of protection against hfd-induced metabolic changes with mice retaining relatively normal insulin sensitivity. this protection is attributed to differences in fat accumulation and to the anti-diabetic effects of estrogen. based on these sex-dimorphisms we hypothesized that hfd-fed female mice would also exhibit resistance to developing pn. in the present study male and female c bl /j mice were fed either a standard diet ( % kcal fat; sd) or a high fat diet ( % kcal fat; hfd) from wk. at wk, wk and wk, neuropathy phenotyping was performed on all groups complemented with longitudinal metabolic assessments including insulin tolerance testing (itt). neuropathy phenotyping consisted of hindpaw latency to heat stimulus, motor and sensory nerve conduction velocities (ncvs), and terminal intraepidermal nerve fiber (ienf) counts. assessment of insulin resistance through itt demonstrated that during early hfd feeding, female hfd-fed mice exhibited relatively normal insulin responsiveness, while male hfd mice exhibited insulin resistance. despite this finding, wk female hfd mice displayed a similar pattern of pn to that of their male counterparts, with similar fold-changes in hindpaw latency and sensory and motor ncvs. therefore, although female hfd-fed mice exhibit resistance to hfd-induced metabolic changes, they display a pn comparable to male hfd-fed mice suggesting that systemic insulin resistance does not mediate pn. further studies are underway investigating the role of insulin signaling in the peripheral nerves of female hfd-fed mice. anti-mag (myelin-associated glycoprotein) neuropathy is a disabling autoimmune peripheral neuropathy caused by monoclonal immunoglobulin m (igm) autoantibodies that recognize the carbohydrate epitope hnk- (human natural killer- ). this glycoepitope is highly expressed on adhesion molecules, such as mag, present in myelinated nerve fibers. since the pathogenicity and demyelinating properties of anti-mag autoantibodies are well established, current treatments aim at a reduction of autoantibody levels. however, the therapies applied so far are primarily immunosuppressive and lack selectivity and efficacy. we therefore hypothesized that a significant improvement of the disease condition could be achieved by selectively neutralizing the pathogenic anti-mag antibodies with carbohydrate-based ligands mimicking the natural hnk- glycoepitope. in an inhibition assay, a mimetic (mimhnk- ) of the natural hnk- epitope inhibited mag-binding by pathogenic igm antibodies from patient sera, however only with micromolar affinity. therefore, considering the multivalent nature of the mag-igm interaction, polylysine polymers of different sizes were substituted with the mimetic. with the most promising polylysine glycopolymer pl (mimhnk- ) the inhibitory effect on patient sera was improved by a factor of up to , per epitope, consequently leading to a low nanomolar inhibitory potency. since clinical studies indicate a correlation between the reduction of anti-mag igm levels and clinical improvement, an immunological surrogate mouse model for anti-mag neuropathy, producing high levels of anti-mag igm, was developed. the observed efficient removal of these antibodies with the glycopolymer pl (mimhnk- ) represents a first step towards an antigen-specific therapy for anti-mag neuropathy. hinder lm , mendelson f , backus c , feldman el . university of michigan, ann arbor, mi, usa. in the united states % of children and young adults are obese and at risk of developing prediabetes. prediabetic patients largely develop the same macro-and microvascular complications as patients with type diabetes, including peripheral neuropathy (pn). moreover, recent clinical data suggest normoglycemic obese patients develop pn. central obesity, characterized by excess fat storage in visceral white adipose tissue, leads to systemic metabolic dysfunction largely due to an imbalance between pro-inflammatory/anti-inflammatory adipokine production. subcutaneous adipose tissue is considered 'benign', but adopts a visceral-like phenotype in response to metabolic stress, with reduced thermogenicity, reduced brown adipose identity, and increased pro-inflammatory gene expression. the popliteal adipose tissue (pat) depot, corresponding to subcutaneous adipose, is adjacent to the peripheral nerve affected in pn, contains the lymph node for lymphatic drainage of the hind limb, and expands following local, sterile hind paw inflammation. the aim of the current study was to characterize pat changes in the high fat diet (hfd) mouse model of obesity and pn, and consider its contribution to peripheral nerve dysfunction. we previously reported c bl /j mice fed % hfd from - wk develop obesity, 'prediabetes' and pn; and switching mice back to a standard diet from - wk improves metabolic and pn phenotypes. at and wk pat was bilaterally dissected and the lymph node removed. the left pat was processed for histomorphometry, and the right pat for rt-qpcr. at wk hfd was associated with a significant shift in adipocyte size-frequency distribution, with a greater number of larger adipocytes. switching the hfd mice back to standard chow from - wk restored the size-frequency distribution towards age-matched controls. rt-qpcr was performed to assess changes in thermogenicity (ucp ), brown adipose identify (cidea) and sterile inflammation (saa ). at and wk hfd pat had reduced thermogenicity and brown adipose identify, and increased sterile inflammation. this switch towards a visceral-like phenotype was reversed in the hfd mice switched back to standard chow. in summary, hfd-induced changes in pat histomorphometry and adipose identity closely associate with pn phenotype. these preliminary data suggest a potential role for pat-nerve signaling in pn. hinder lm , backus c , hayes jm , feldman el . university of michigan, ann arbor, mi, usa. peripheral neuropathy (pn) is a common and debilitating complication of obesity and diabetes that triggers pain and loss of sensation. substantial nerve damage occurs in many patients prior to noticeable symptoms and no treatments are currently available; therefore, there is a critical need to identify treatment strategies that impact the underlying disease pathogenesis. our in vivo fluxomics data in the bks-db/db mouse model of type diabetes (t dm) and pn suggest that 'metabolic reprogramming' occurs in the t dm nerve to downregulate mitochondrial oxidative phosphorylation of substrates derived from glycolysis and fatty acid beta-oxidation. therefore, we hypothesize that distinct systemic metabolic alterations occur in obesity and diabetes which induce tissue-specific metabolic reprogramming within the peripheral nerve, altering fuel utilization and ultimately leading to tissue dysfunction. we contend that identifying conserved bioenergetic profiles across mouse models of pn will provide insight into key pn mechanisms. the current study utilized two mouse models of pn: the % high fat diet (hfd) mouse model of obesity and prediabetes at wk of age ( wk hfd), and the leptin receptor-deficient bks-db/db model of t dm at wk of age. mitochondrial function was determined in primary dorsal root ganglia (drg) neurons and sural nerve tissue from both models using the seahorse xf analyzer. resting mitochondrial oxidative metabolism was upregulated in drg neurons from mice with pn, with increased resting atp production and maintained mitochondrial coupling. in contrast, resting atp generation was decreased in sural nerve from mice with pn, with decreased coupling efficiency. relative spare respiratory capacity was attenuated in both drg neurons and sural nerve from mice with pn, indicating that mitochondria were less able to increase respiration in response to an energetic challenge. moreover, mitochondrial copy number was unchanged in drg neurons, but decreased in sural nerve tissue of mice with pn compared with respective controls. these data suggest a change in absolute number and function of sural nerve mitochondria, and a conserved cross-model proximal-distal bioenergetic profile in pn. we are currently exploring the relationship between these changes and pn pathogenesis. hoang ttn , umapathi t . hospital, ho chi minh city, vietnam; national neuroscience institute, singapore. ho chi minh city (hcmc) is the biggest metropolitan city in southern vietnam. its population is more than ten million. adult patients with neurological disorders are seen at six city public hospitals, including , hospital. there has been no systematic study of guillain barré syndrome (gbs) at hcmc or in vietnam in general. we are in the process of starting a prospective gbs database that we hope to expand to the other public hospitals at hcmc. here we describe our experience from . we saw gbs patients at , hospital. most of the cases were admitted in the rainy season, from late april to november, when mosquito-borne flavivirus infections are more common. patients were seen in the first week of illness and reported antecedent fever. two patients had diarrhea. diagnoses were made largely on clinical features, cerebrospinal fluid analysis and nerve conduction study. clinical findings include limb weakness, numbness and vii cranial nerve palsy. extraocular eye movements were affected in one patient. none had respiratory involvement severe enough to require artificial ventilation or intensive care. there were no pure miller-fisher syndrome cases; we suspect this might be related to the mild deficits that did not prompt hospitalization. nerve conduction studies showed typical features such as loss of f waves and abnormal blink reflex. the electrophysiology of patients' was dominated by demyelinating changes, one case was largely axonal and the remaining patient had normal electrodiagnostic study. repeat nerve conduction studies were not feasible because of limited resources. neurologists use corticosteroids as the main treatment. intravenous immunoglobulin and plasma exchange are costly and not reimbursed by medical insurance. we are currently preparing to systematically study gbs in southern vietnam, specifically with regards to possible role of antecedent flavivirus infections. we are also exploring the possibility of using low volume plasma exchange as a feasible cost-effective therapeutic modality. about % of patients with guillain-barré syndrome (gbs) treated with intravenous immunoglobulin (ivig) or plasma exchange deteriorate after initial improvement or stabilization -a phenomenon that is termed treatment-related fluctuation (trf). it is important to distinguish acute onset cidp (a-cidp) from gbs-trf during early course of the disease, because their therapeutic strategies and prognoses are different. herein, we describe a patient with gbs-trf, but with an extended progression phase that exceeds weeks. a -year-old woman was admitted due to acute onset progressive leg weakness and diplopia that had developed weeks prior (onset, d ). on neurological examination, right facial palsy was also observed. lower extremity weakness was moderate in proximal and distal muscles (mrc grade iv) with absent knee and ankle jerks. sensory examination revealed no abnormality in all modalities. she denied any recent diarrhea or upper respiratory infection, and vaccination. albumino-cytologic dissociation was noted in csf analysis; white blood cell count of / l and protein level of . mg/dl. nerve conduction study revealed demyelinating sensorimotor polyneuropathy with prolonged distal latency and conduction blocks. anti-ganglioside antibodies (gm igm/g, gd b igm/g, and gq b igm/g) were all negative. following ivig treatment, she was discharged with considerable improvement (d ). days later, she was re-admitted due to deterioration of leg weakness and hand clumsiness (d ). after another ivig treatment, she was discharged with clinical improvement (d ). days later, however, she was admitted again (d ) due to another considerable deterioration with four extremity weakness being worst at this time (mrc grade ii-iv in upper extremity, grade ii in lower extremity). a-cidp was considered given the progression phase exceeding weeks, but, we decided to give another treatment with ivig instead of a switch to corticosteroids because of uncertainty regarding distinction between a-cidp and gbs-trf. she was significantly improved following ivig treatment, and finally discharged (d ). thereafter, there has been no further deterioration during long-term follow-up of year. conclusively, this is a rare case of gbs with extended progression phase and trf. we propose that this could be referred to as subacute inflammatory demyelinating polyradiculoneuropathy (sidp) with trf. hsieh s , chao c . national taiwan university hospital, taipei, taiwan. transthyretin (ttr)-related familial amyloid polyneuropathy (fap) constitutes a major etiology of adult-onset hereditary neuropathies worldwide, in particular, a mutant ttr of ala ser (ttr-a s) in taiwan, the most common cause of acquired genetic neuropathy with adult onset (> years of age) of taiwanese patients. fap is a pan-modality neuropathy involving motor, sensory, and autonomic components of the peripheral nervous system with early involvement of small fibers as a major symptom. the early symptoms of fap are sometimes minimal and difficult to ascertain, mainly related to the fact that conventional electrophysiological examinations were not sensitive enough to detect small fiber neuropathy. skin biopsy with quantification of intraepidermal nerve fibers (ienf) has become one of the standard approaches to diagnose small fiber sensory neuropathy based on pathological documentation of nociceptive nerve degeneration. to explore the issue of early biomarkers in fap, we performed skin biopsy and compared ienf density with parameters of nerve conduction studies (ncs) and quantitative sensory testing (qst) on subjects ( men, aged . ± . years) with genetic confirmation of ttr-a s: patients and carriers. the ienf densities were significantly reduced compared to the age-and gender-matched controls in carriers ( . ± . vs. . ± . fibers/mm, p = . ) and patients ( . ± . vs. . ± . fibers/mm, p = . ). the latter was consistent with our previous report (neurology, : - , ) . the abnormal rate of ienf density was significantly higher than that of ncs and qst, respectively. in conclusion, there was significant skin nerve degeneration in carriers with ttr-a s. compared with qst and ncs, ienf density assessment had the highest abnormal rate and highest sensitivity to detect neuropathic changes in the early stage of fap. charcot-marie-tooth type p (cmt p) has been associated with frame-shift mutations in the ring domain of lrsam (an e ligase). this study describes families with a novel missense mutation of lrsam gene and explores pathogenic mechanisms of cmt p. this american family with dominantly inherited axonal polyneuropathy reveals a phenotype similar to those in previously reported non-us families. the affected members in our family co-segregated with a novel missense mutation cys arg that alters a highly conserved cysteine in the ring domain. this mutation leads to axonal degeneration in the in vitro neuronal cell-line. moreover, using protein mass spectrometry, we identified a group of rna binding proteins (including fus, a protein critically involved in motor neuron degeneration) that interacted with lrsam . the interactions were disrupted by the cys arg mutation, which resulted in reduction of intranuclear rna-binding proteins. a knockin mouse of cys arg has been created for further explorations of cmt p mechanisms and therapeutic development. together, our findings suggest that the mutant lrsam may aberrantly affect the formation of transcription machinery. given a similar mechanism has been reported in motor neuron degeneration of amyotrophic lateral sclerosis, abnormalities of rna/rna-binding protein complex may play a role in the neuronal degeneration of cmt p. supported by grants from ninds (r ns ) and the national center for advancing translational sciences (ul tr ). this double-blind, multicenter, parallel-group trial randomized ( : ) adult participants (n= ) with chronic inflammatory demyelinating polyradiculoneuropathy being treated with intravenous immunoglobulin (ivig) or corticosteroids to . mg fingolimod (n= ) or placebo (n= ) once-daily. previous treatment was discontinued (ivig) or tapered (corticosteroids). in the total trial population, there was no significant difference between the groups in the primary outcome, time-to-first confirmed worsening (≥ -point increase on the adjusted inflammatory neuropathy cause and treatment [incat] scale vs. baseline), or time-to-first any worsening (confirmed on incat assessment or unconfirmed). no significant difference between the two treatment groups was shown on secondary outcomes; change from baseline in grip strength and rasch-built overall disability scale (r-ods) at six months or trial end. analyses of pre-specified subgroups were performed for primary and secondary outcomes. this trial evaluated the efficacy and safety of fingolimod in chronic inflammatory demyelinating polyradiculoneuropathy (cidp). corticosteroids, intravenous immunoglobulin (ivig) and plasma exchange are recognized treatment options but no other immunomodulators demonstrated efficacy in a controlled trial. fingolimod has been shown to be efficacious and is approved for the treatment of relapsing multiple sclerosis. results from experimental autoimmune neuritis in rats suggested that it might show an effect in cidp. in this double-blind, multicenter, parallel-group trial, cidp participants receiving ivig or corticosteroids were randomized to once-daily fingolimod . mg or placebo ( : ). participants were stratified by inflammatory neuropathy cause and treatment disability (incat) scores and prior treatment. previous ivig treatment was discontinued after one final course before randomization. previous corticosteroid treatment was tapered over weeks. the primary outcome was time-to-first confirmed worsening (≥ -point increase on the adjusted incat score versus baseline). secondary outcomes included change in grip strength and rasch-built overall disability scale (r-ods) score from baseline to month and at trial end. the trial was stopped for futility by an independent data monitoring committee after a pre-planned interim analysis based on pre-specified criteria. in all, participants received fingolimod (ivig: , corticosteroids: ; age: . ± . years [mean±standard deviation]; male: . %); received placebo (ivig: , corticosteroids: ; age: . ± . years; male: . %). the percentage ( % confidence interval) of participants free from confirmed worsening at the trial end was not significantly different between fingolimod ( . % [ . %- . %]) and placebo ( . % ( . %- . %); p= . ). in the first days, approximately % participants experienced worsening. at trial end, approximately % participants had no worsening. there was no significant difference after six months or at the trial end (whichever occurred earlier) in the secondary endpoints. adverse events were reported in / and / participants in the fingolimod and placebo group, respectively. there were no deaths. nine participants in the fingolimod group and in the placebo group had serious adverse events. adverse events leading to trial drug discontinuation occurred in ( %) participants on fingolimod and none on placebo. no new safety signals emerged in this trial. acknowledgment: the authors consulted for or were employed by the study sponsor novartis pharma ag, basel, switzerland. myelin sheath enwraps non-nociceptive mechanoselective abeta-afferents transmitting touch/vibration sense. a prominent reduction in the mechanical stimulus required to evoke a withdrawal response in rodents, a phenomena interpreted as mechanical allodynia, arises due to peripheral nerve/myelin damage. evidence has emerged that nerve injury-induced mechanical allodynia depends on the adaptive immune/t cell activity in female but not male rodents. having previously demonstrated both the release of the cryptic - peptide regions of myelin basic protein (mbp - ) following sciatic nerve chronic constriction injury (cci) and the direct, robust and t cell-dependent ability of the pure mbp - peptides to induce mechanical allodynia after injection into the intact sciatic nerve, we hypothesized that mbp - contributes to sexual dimorphism in mechanical allodynia. the pure mbp - wild-type (wt), its histidine (his) mutant or scramble peptides were administered into an intact sciatic nerve fascicle in male and female rats or mice, followed by von frey testing. intra-sciatic mbp - -wt peptide induced robust and lasting allodynia in females. in contrast, males responded with a brief and mild decline in mechanical sensitivity for one day post-injection of both wildtype and control peptides. the algesic ability of mbp - -wt was diminished in the his mutant. we here present the molecular changes in the sciatic nerve, drg and the spinal cord after the intra-sciatic mbp - injection in male and female animals. in addition, using the biotin-labeled mbp - peptide and the hrp-labeled goat anti-rat igg/igm antibodies, we developed an elisa to quantitatively assess seropositivity for the specific anti-mbp - peptide igm/igg autoantibodies in female and male rats post-cci. human serum from female patients with multiple sclerosis was used for control. our work corroborates the findings of sexual dimorphism of mechanical hyperpathia and suggests its potentially autoimmune nature in females. iijima m , nishi r , ikeda s , kawagashira y , koike h , sobue g , katsuno m . nagoya university, nagoya, japan. non-obesity diabetic (nod) b - knockout (ko) mice are characterized by chronic and progressive neuritis and expected as models of immune-mediated neuropathies, especially cidp. hindlimb-predominant weakness due to inflammatory demyelination followed by axonal degeneration begins from around twenty week-age in all female mice until thirty week-age. to clarify the efficacy of immunoglobulins as immune-regulating therapeutics and the similarity of pathogenesis of human cidp, we injected intraperitoneally human-derived immunoglobulins (ipig, mg/mg bw/week) and saline as a control to totally forty female mice. clinical and pathological estimations in sciatic nerves were performed in time series. as a result, the ipig-treated group was protected from weight loss which could be related to axon loss followed by muscle atrophy as well as inflammatory demyelination between twenty-five week-age and thirty week-age compared to the control. in addition, the pathological findings in sciatic nerves showed that ipig apparently suppressed inflammatory infiltrates. about the subsets of inflammatory infiltrates, while macrophages (cd +) and lymphocytes (cd +) highly existed and suggested to play a main role in the neuritis until thirty week-age, only macrophages naturally disappeared after thirty week-age without any therapeutic induction. immunoglobulins effectively suppressed only macrophages although that did not suppress cd + lymphocytes. in conclusion, nod b - ko mice respond to immunoglobulins in a similar manner to human cidp and this efficacy is due to the suppression of macrophage-dominant pathogenesis. therefore, macrophage-derived pathogenesis is for the main target of immunoglobulin therapy and we should focus on the lymphocyte-derived pathogenesis which might plays an important role in non-responders to immunoglobulins. ikeda s , nishi r , kawagashira y , iijima m , koike h , katsuno m , sobue g , . department of neurology, nagoya university graduate school of medicine, nagoya, japan; research division of dementia and neurodegenerative disease, nagoya university graduate school of medicine, nagoya, japan. chronic inflammatory demyelinating polyneuropathy (cidp) is an acquired immune-mediated polyradiculoneuropathy that is characterized by heterogeneous clinical manifestations. typical cidp is defined as neuropathy manifesting in a progressive manner, stepwise manner, or with recurrent symmetrical proximal and distal weakness and sensory impairment in all four limbs. although they occur at a lower proportion than the so-called typical cidp, atypical forms, such as multifocal acquired demyelinating sensory and motor (madsam), distal acquired demyelinating symmetric (dads), pure sensory, pure motor, and focal, are considered to cidp subtypes. thus far, pathological features characterizing each clinical subtype have not been fully elucidated. we analyzed clinical and pathological correlations in consecutive cidp patients who underwent sural nerve biopsy and fulfilled the definite or probable efns/pns criteria. there were male and female patients. the age at biopsy was . ± . (mean ± sd) years, and the duration from the onset of neuropathy to biopsy was ± months. fifty-five percent (n = ) of the patients were classified as having typical cidp. regarding atypical cidp, madsam (n = , %), dads (n = , %), and pure sensory (n = , %) subtypes were the major subtypes, while pure motor (n= , %) and focal (n= , %) subtypes were rare. no significant difference was found among these subtypes in terms of sex, age at biopsy, and disease duration. sural nerve biopsy specimens revealed that the densities of large myelinated fibers significantly decreased in the madsam subtype than in the other subtypes (p = . ). in addition, the variation in nerve fibers among fascicles was more conspicuous in the madsam subtype than in typical cidp (p= . ). patients with the dads subtype tended to show the formation of onion-bulbs. in conclusion, pathological findings of sural nerve biopsy specimens were different among the cidp subtypes. further studies are needed to clarify mechanisms leading to different pathological features. small volume plasma exchange (svpe) can be an affordable and potentially effective alternative form of plasma exchange. svpe is the repeated removal of small volumes of supernatant plasma over several days via sedimentation of patient whole blood. the aim of this study is to assess the clinical feasibility and safety of svpe in patients with gbs in low-income countries. twenty adult patients with gbs diagnosed as per the criteria for gbs of the national institute of neurological and communicative disorders and stroke (ninds) were enrolled for svpe at a centre in bangladesh. serious adverse events (sae) were defined as the number of patients developing severe sepsis associated with the central venous catheter (cvc) or deep venous thrombosis in the limb where the cvc is placed for svpe. the svpe procedure was considered safe if less than of svpe-treated gbs patients have a sae, and feasible if eight litres of plasma could be removed in at least of svpe-treated gbs patients. among the cases who received svpe, ( %) patients were male and the age range between to yrs. all the patients were quadriplegic and bedbound at enrolment for svpe with a median mrc score of (iqr, - ). cranial nerve involvement, autonomic dysfunction and requirement for assisted ventilation were observed in ( %), ( %) and ( %) patients respectively. electro physiologically ( %) patients were motor axonal and ( %) patients were sensory-motor demyelinating type. during the svpe none of our patients experienced sae and one patient experienced central line associated blood stream infection. common adverse effects were transient intravenous fluid responsive hypotension during the svpe sessions in ( %), cv catheter insertion site hemorrhage in ( %) and hypersensitivity reaction to fresh frozen plasma in ( %) patients. there was no hypo-albuminemia, anemia or electrolyte imbalance observed in most patients ( %) treated with svpe. improvement in one or more grade of the gbs disability score at four weeks after the onset of svpe was observed in ( %) patients. in conclusion svpe can be a safe, feasible and cost effective alternative to standard pe in the developing countries. guillain-barré syndrome (gbs) is a descriptive disease entity defined by a set of clinical, electrophysiological and laboratory criteria. various clinical phenotypes exist that may be triggered by different antecedent infectious events. although the disease appears to affect primarily the elderly in developed countries, but, scenario is different in developing countries. bangladesh has made an impressive progress towards the eradication of poliomyelitis, and no new cases have been reported since . gbs, an acute polyradiculoneuropathy, is the most frequent cause of acute flaccid paralyis. the crude incidence rate of gbs in < years of age reported here appears to be . × to × higher than that reported in the literature. we conducted a hospital based observational study including patients fulfilling the national institute of neurological disorders and stroke (ninds) criteria for gbs patients between and in dhaka medical college hospital, dhaka, bangladesh. detailed clinical, electrophysiological, serologic and microbiological data were obtained. gbs affected predominantly in young adults males (m/f= : ) living in rural areas. antecedent events were recorded in > % of patients; frequent events being gastroenteritis (> %) and upper respiratory tract infection ( %). more than % of the patients were bed-bound (gbs disability score ) at entry and about % patients required mechanical ventilator. about % patients did not receive specific treatment either intravenous immunoglobulin (ivig) or plasmapheresis due to high expensive treatment cost. % patients had died during hospitalization. % of patients had an axonal variant of gbs and evidence for a recent c. jejuni infection ( %). c. jejuni infection was significantly associated with serum antibodies to the gangliosides gm and gd a, axonal neuropathy, and greater disability. in conclusion, the majority of the patients do not receive standard treatment with ivig in view of its high price. therefore, we developed low-cost treatment strategies and conducted a safety and feasibility trials for small volume plasma exchange (svpe) on gbs patients in bangladesh. in future, it is essential to conduct a phase ii clinical trial to assess the efficacy of svpe for low-in-come countries. prognosis of guillain-barré syndrome (gbs) has not improved in last two decades. current therapies (intravenous immunoglobulin, ivig and plasma exchange, pe) had been proved to be effective on two third of patients in developed world. unpredictable and poorly understood clinical course of gbs hamper treatment development. in bangladesh, most patients affected by gbs cannot afford specific treatments with ivig or pe instead most of them receive only supportive care. therefore, we aimed to compare the outcome of ivig treated patients with supportive care patients in improvement of gbs disability score and mrc sum score by using world's largest gbs cohort in bangladesh. we conducted a prospective observational study enrolling gbs patients between and from dhaka medical college hospital and national institute of neuroscience and hospital, dhaka, bangladesh. only gbs patients ( %) received standard ivig treatment. in current analysis, ivig treated patients and age, sex and severity matched controls (supportive care only) were considered. outcome of both groups were compared using fisher's exact or chi square test and survival analysis were performed by kaplan meier method using log rank test. among patients (cases and controls), male/female ( / ), median age years, % patients were bed-bound, one-fourth patients required mechanical ventilation and % were axonal. we did not found any significant differences of treatment outcome in both cases and control groups in gbs disability score (week : p= . , months: p= . ) and mrc sum score (week : p= . , months: p= . ). survival analysis revealed, the differences of time required for independent locomotion, improvement of one gbs disability score and improvement of mrc score were not statistically significant between treatments (ivig) and supportive care patients. in conclusion, our analysis showed that standard dose of ivig use has no considerable advantage to improve specific outcome measures among gbs patients in bangladesh. as the phenotype of gbs in bangladesh is different from developed world; therefore, an efficacy trial for ivig is needed for developing countries like bangladesh or new targeted therapeutic strategies can append beneficial effects for gbs patients. isose s , , watanabe k , omori s , sekiguchi y , beppu m , shibuya k , amino h , suichi t , misawa s , kuwabara s . graduate school of medicine, chiba university, chiba, japan; national hospital organization chiba east national hospital, chiba, japan. diabetic neuropathy is a frequent cause of neuropathic pain, suggesting the small-fiber involvement. additionally, persistent peripheral pain-related inputs could cause neuronal hyperexcitability and complex interactions of the nociceptive pathways, i.e., central sensitization. to investigate the pathophysiology of neuropathic pain in diabetic neuropathy, we studied pain-related evoked potentials (preps) after selective intraepidermal electrical stimulation (ies) to a-deltaand c-fibers in diabetes patients with neuropathic pain (n= ) and without neuropathic pain (n= ). we also conducted a longitudinal study to assess changes in preps and pain profiles in patients with neuropathic pain months after the start of treatment with duloxetine. this study is registered with the umin clinical trials registry, umin . ies was applied in the hand and foot, and preps were recorded from the cz electrode referenced to the linked earlobes. we evaluated prep latencies, amplitudes, and amplitude ratios of preps after c/a-delta -fiber stimulation. in the conventional nerve conduction studies, patients with neuropathic pain significantly showed conduction slowing and decreased snap amplitudes in the median and sural nerves compared with those in patients without neuropathic pain. in pain-related sep studies, there were no significant differences in prep amplitudes and latencies after a-delta -or cfiber stimulation between the patients with neuropathic pain and without it. prep amplitude ratios after c/a-delta -fiber stimulation tended to increase in patients with neuropathic pain compared to patients without pain. after the treatment with duloxetine, c/a-delta -prep amplitude ratios were significantly decreased after both hand and foot stimulation, and as for numerical rating scale (nrs) scores as the intensity of pain. patients with less pain relief showed the tendency of higher c/a-delta prep amplitude ratios before treatment compared to patients with better pain relief. the correlation between reduction of c/a-delta prep amplitude ratios and nrs reduction did not reach statistical significance. this pain-related sep study demonstrated that abnormal cortical response in patients with neuropathic pain could improve after the treatment with duloxetine, this might reflect the cortical hyperexcitability as a central sensitization. this study is funded by shionogi & co., ltd. the sponsors played no role in the design and management of the study, collection and analysis of data, interpretation of the results, or the writing of the writing of the report. peripheral nerve injury is commonly associated with traumatic injury which is often amenable to surgery. despite improved methods in surgical repair, functional recovery remains a challenging clinical problem that often leads to significant morbidity in patients. alternative therapies that could augment surgical repair may be beneficial in functional outcomes. neuroinflammation is a complex pathway with different cellular components and cytokines that are activated following peripheral nerve injury. macrophages are responsible for the breakdown of debris following injury as well as promotion of regenerative signals. macrophage polarization is the process by which macrophages take on phenotypically distinct functions based on the local environment and signaling cues. exercise has been shown to drive macrophage polarization from a pro-inflammatory m phenotype towards an anti-inflammatory m phenotype in numerous tissues, but remains uninvestigated in the peripheral nervous system. the purpose of our study was to identify how exercise affects macrophage polarization, motor and sensory function, and neuroregeneration following sciatic nerve crush. c bl/ mice underwent sciatic nerve crush injury and were then given access to running wheels (exercised) or not given access to running wheels (sedentary) for weeks. exercised mice ran an average of . km per night. injured exercised mice were protected from the development of thermal hyperalgesia when compared to injured sedentary mice. exercised mice had fewer paw slips on beam walk testing compared to sedentary mice. no differences were measured in mechanical sensitivity or motor coordination and balance assessed by rotarod. while motor nerve conduction velocities were significantly reduced for injured mice compared to uninjured controls, motor nerve conduction velocities from injured exercised animals were significantly higher than injured sedentary animals suggesting improved nerve recovery with exercise. injured sciatic nerves from exercised mice demonstrated increased m macrophages compared to sciatic nerves from injured sedentary mice. the behavioral changes and altered macrophage polarization correlated with increased epidermal nerve fiber density, improved myelination, and increased in vitro neurite outgrowth from injured exercised animals. therefore, exercise alters macrophage polarization towards an anti-inflammatory phenotype which improves repair and recovery of the injured peripheral nerve. jack mm , shah k , everist b , reyna j , hylton p . university of kansas medical center, kansas city, ks, usa. diffusion tensor imaging (dti) has long been used to evaluate the location and integrity of white matter tracts in the brain. dti uses quantitative data and directionality of water diffusion to determine axonal connectivity of the nervous system. the technology has only recently been utilized in limited settings in the peripheral nervous system due to challenging technical factors and lack of widespread availability. magnetic resonance (mr) neurography or peripheral neurography is a technique which uses diffusion to differentiate between intraneural and perineural tissues. it allows for fascicle patterns to be visualized particularly in the setting of peripheral nerve sheath tumors. peripheral nerve sheath tumors of various pathologies cause surrounding nerves to be involved or displaced in a range of directions. this technique helps determine the anatomic location of these nerve fibers in relation to the mass, which is particularly helpful at distinguishing neuromas from schwannomas. this data is invaluable to the surgeon to ensure a safe and low morbidity operation. while this technology has benefit particularly with surgical planning, it has been underutilized due to the challenges of requiring complex software to produce fiber tracks and the inability to translate these images into the operating room. here, we utilized brainlab software that is commonly available and utilized in surgical suites to produce images of the radial nerve fiber tracts with an associated peripheral nerve sheath tumor prior to surgical resection. while the software is commonly used in the central nervous system, it has not been reported to have been used in the peripheral nervous system. this software offers a high usability and produces anatomically correct and reliable fiber tracts. this technique overcomes the reliance on highly specialized software and extensive training required for use that most other tractography software has. utilizing peripheral neurography in this case allowed for complete surgical resection without postoperative deficits. this data offers clinicians an option to investigate peripheral nerve fibers in various pathologic states, to plan appropriate operative trajectories to peripheral nerve pathology, and to improve surgical outcomes for patients with peripheral nerve sheath tumors. jacobsen b , parry g , allen j , walk d , muley s , ortega e . barrow neurological institute, phoenix, az, usa; university of minnesota, minneapolis, mn, usa. chronic inflammatory demyelinating polyneuropathy (cidp) is an immune mediated neuropathy that is responsive to immunomodulatory agents such as glucocorticoids, intravenous immunoglobulin (ivig) and plasma exchange (pe). the specific immunopathogenic mechanisms of cidp remain unclear but there is increasing interest in nodal proteins as a site of the immune attack. even though the majority of patients respond to one of the aforementioned immunomodulatory agents there are some who are unresponsive or incompletely responsive to these first line agents and other more aggressive treatments may be necessary. cyclophosphamide and stem cell transplantation may be effective but are associated with considerable morbidity. anecdotal reports suggest that rituximab may be beneficial for some patients that fail first-line therapy, especially if they have antibodies to nodal proteins. we present four patients with intractable cidp who responded to rituximab. one of the three patients had diabetes. disease duration prior to starting rituxan was short ( months) in two patients and longer and months in the other two. all patients had failed treatment with glucocorticoids and ivig, and in two, plasma exchange and ivig-pe were also ineffective. two of the four patients were quadriparetic and non-ambulatory. two gm doses two weeks apart of intravenous rituximab were instituted in all patients. all patient tolerated the treatments well without adverse effects. all patients responded within four weeks and continued to improve at six months. other immunomodulatory agents were successfully tapered but not totally discontinued. it remains unclear whether antibodies to nodal proteins were present in these patients. in conclusion, although rituximab efficacy remains uncertain on the basis of randomized controlled clinical trials, it may be beneficial in selected patients otherwise intractable to first-line treatments. further studies are necessary to better understand which patients may benefit most from rituximab and where in the treatment algorithm rituximab should be applied. neuropathic pain is a chronic condition seen in patients suffering a direct injury to the peripheral or central nervous system or an indirect injury due to, e.g., diabetes or multiple sclerosis. current treatment options fall short of preventing or completely relieving patients of their pain. for years, research has focused on understanding the role of neurons in neuropathic pain pathogenesis while overlooking the role of supportive cells in general and satellite glial cells (sgcs) in the dorsal root ganglion in particular. these cells not only buffer the neuronal microenvironment they are also involved in controlling the electrical activity flowing through the neurons and in neuropathic pain pathogenesis. the aim of this project is to understand the role of sgcs in neuropathic pain development and thereby aid the identification of new drug targets. to purify the sgcs from adult mice we optimized a fluorescently activated cell sorting (facs) protocol. the success of our purification method was confirmed using qrt-pcr and visual inspection of the sorted cells. finally, we are running rna sequencing on sgcs after peripheral nerve injury to compare their transcriptome to that of uninjured cells at different time points. the results from our study are likely to deepen our understanding of how sgcs contribute to the development and maintenance of neuropathic pain. guillain-barrésyndrome (gbs) is an immunemediated disorder in the peripheral nervous system (pns) triggered by molecular mimicry against nerve gangliosides. one of the cell surface receptors (fas)-ligand (fasl) interaction transmits apoptotic signal to eliminate the auto-reactive b and t-cells, which generates cross-reactive antibody against nerve cells. host genetic polymorphism of fas and fasl may alter their expression and induce aberrant apoptotic response to develop gbs. therefore, we determined the single nucleotide polymorphisms (snps) of both fas receptor (− g/a and - a/g) and fasl ligand (− c/t) in gbs patients (n= ) as well as healthy controls (n= ) using the lightcycler technique. serum level of soluble form of fas and fasl was measured using commercially available sandwich elisa kit. comparison of genotype, allele and haplotype frequencies was done with the gbs subgroups based on the clinical and serological data. ag heterozygote (p= . , or= . , % ci= . - . ) and polymorphic g-allele (p= . , or= . , % ci= . - . ) of fas receptor - a/g promoter snps were significantly associated with anti-ganglioside (gm ) antibody positive gbs patients. in addition, − g-allele (p= . , or= . , % ci= . - . ) and - g/- g haplotype (p= . , or= . , % ci= . - . ) were predominantly associated with the axonal variant of gbs patients. serum soluble form of sfas (median levels pg/ml vs. pg/ml, p= . ) and sfasl (median levels pg/ml vs. pg/ml, p= . ) were found to be elevated in anti-gm antibody positive gbs patients compared to anti-gm negative patients. no significant association was found in genotypic distribution between gbs patients and healthy controls. in conclusion, fas/fasl promoter snps are not a susceptible factor for gbs but could be one of the influencing factors to develop cross-reactive anti-ganglioside antibodies in gbs patients in bangladesh. furthermore, functional studies with a larger sample size (using cohort like international gbs outcome studies-igos) are required to explain the immune pathogenic role of these snps for gbs patients. jang sy , yoon ba , shin yk , yun sh , jo yr , park ji , shin kj , kim jk , park ht . dong-a university, busan, south korea; inje university, busan, south korea. myelination is essential for the proper function of the nervous system. schwann cells, which form the peripheral myelin sheath, have the unique ability to dedifferentiate and to destroy the myelin sheath under various demyelination conditions. during schwann cell dedifferentiation-associated demyelination in wallerian degeneration after axonal injury, schwann cells exhibit myelin and junctional instability, down-regulation of myelin gene expression and autophagic myelin decomposition. however, in inflammatory demyelinating neuropathy, it is still unclear how schwann cells react and contribute to segmental demyelination before myelin scavengers, macrophages, are activated for myelin clearance. here, we show that schwann cell dedifferentiation-associated demyelination is a mechanism involved in the initial demyelination observed in a mouse model of inflammatory demyelinating neuropathy using ultrastructural, biochemical and microarray analyses. myelin uncompaction and myelin membrane instability generated by dedifferentiated schwann cells lead to autophagolysosome-dependent cytoplasmic amputation between the axon-containing myelin sheath and the schwann cell body, resulting in the formation of the "myelin corpse", thereby allowing macrophages to phagocytose the myelin corpse in the end stage of segmental demyelination. we found myelin corpse formation in inflammatory demyelination to be a process similar to the myelin rejection during wallerian degeneration, which appeared to be dependent on schwann cell autophagolysosome activation since schwann cell-specific atg knockout mice exhibited delayed myelin rejection following nerve injury. finally, lysosome inhibition in schwann cells not only prevented segmental demyelination but also delayed the progression of clinical stages by suppressing the myelin corpse formation in inflammatory demyelinating neuropathy. thus, our findings indicate that demyelination by schwann cells and macrophages might be part of a process that includes sequential divisions of labor with respect to myelin rejection and digestion, respectively. in conjunction with previous studies showing schwann cell dedifferentiation and autophagy in toxic and hereditary neuropathies, the concept of "schwann cell dedifferentiation-associated demyelination" provides insight into the development of possible therapeutic strategies to prevent schwann cell demyelination in peripheral demyelinating neuropathies. methods: electrophysiological data, the charcot marie tooth exam score (cmtes), and bmi from patients with known cmt a were obtained and analyzed. results: when controlled for age, bmi does not affect studies of ulnar motor nerve conduction in ctm a patients, but rather specific components of the cmt exam scores (cmtes, loss of pinprick sensation and motor strength in the lower extremities). discussion: bmi and clinical components of the cmtes are correlated, but it is uncertain which is the primary effect -i.e., whether the reductions in pinprick sensation and motor strength in the lower extremities lead to a higher bmi, or higher bmi results in these signs. introduction: charcot-marie-tooth disease type c (cmt c) is a rare, dominantly inherited neuropathy caused by mutations in the lipopolysaccharide-induced tumor necrosis factor (litaf) or small integral membrane protein of the lysosome/late endosome (simple) gene. methods: we present a case series comprised of patients in whom cmt c is caused by a gly ser substitution in the encoded protein. we focus on clinical presentation, electrodiagnostic analyses, and our findings in the context of previously described cases. results: the gly ser mutation causing cmt c is a mild form of cmt, as patients walked on time, had less weakness than those with charcot-marie-tooth disease type a (cmt a), had a charcot marie tooth neuropathy score (cmtns) indicative of mild disease, and had faster ulnar and median motor nerve conduction velocities compared to those with cmt a. discussion: the g s mutation in litaf seems to be clinically indistinguishable from a mild presentation of cmt a. jha mk , russell k , lee y , rothstein jd , morrison bm . johns hopkins university, baltimore, md, usa. peripheral nerves are highly dependent on metabolic energy to maintain both basic cellular functions such as axon transport, na+/k+ ion gradients, and myelination, as well as to support regeneration following injury. though glucose certainly provides some metabolic support, our recent studies have shown that monocarboxylates, such as lactate, pyruvate, and ketone bodies, also contribute to recovery from peripheral nerve injury. monocarboxylate transporters, particularly mct , are the predominate transporters for monocarboxylates in the peripheral nerve. in a recent publication, we found that mct heterozygous null mice, which express % less mct in all cells, have slowed nerve regeneration and reduced myelination following sciatic nerve crush. this study was limited by the global reduction of mct , which is widely expressed in schwann cells (sc), dorsal root ganglia (drg) neurons, endothelial cells, macrophages, and perineurial cells within the regenerating peripheral nerve. to better understand the mechanism by which mct contributes to normal nerve function and nerve regeneration, we produced and validated conditional mct null (mct loxp) mice that allow selective deletion of mct from scs, drg neurons, endothelial cells, or macrophages through mating to cell-specific cre lines. we are currently quantifying peripheral nerve development, aging, and regeneration in each of these mouse lines. following sc-, but not drg-, specific mct knockdown, sensory peripheral nerves develop demyelination by months of age, manifest by reduced myelin thickness, increased g-ratio, and reduced conduction velocity. studies are ongoing in cultured scs to determine the mechanism for demyelination. neither sc nor drg knockdown of mct impairs nerve regeneration following sciatic nerve crush. these results suggest that sc-specific mct is critical for maintaining myelin in sensory, but not motor, peripheral nerves as they age. they also suggest that mct expression in peripheral nerve cell types, other than sc and drg, is important for nerve regeneration. ongoing studies are determining the contribution of mct in other peripheral nerve cell types, particularly endothelial cells and macrophages, to normal development and regeneration following injury. our results will clarify the role of lactate and its transporter, mct , in peripheral nerve function, potentially suggesting novel targets for demyelinating neuropathies or nerve injuries. small fiber neuropathy (sfn) is a condition in which the smallest nerve fibers are affected, characterized by neuropathic pain and autonomic dysfunction. according to international criteria, sfn diagnosis is based on clinical symptoms in combination with abnormal temperature threshold testing (ttt) and/or reduced intraepidermal nerve fiber density (ienfd) in skin biopsy. skin biopsy is moderately sensitive, invasive and the process is time consuming and expensive. ttt is a widely available diagnostic tool, but lacks specificity. previous studies introduced stimulated skin wrinkling (ssw) as an objective, non-invasive diagnostic tool to detect sympathetic nerve dysfunction in sfn by means of a categorical assessment. however, our unpublished data has shown that inter-observer reliability of categorically assessed ssw is quite low. in this current study we will use a new digital method for ssw quantification: the digit wrinkle scan© (dws©). the primary study objective is to define normative values for dws© expressed as total wrinkle length per fingertip surface (mm/mm ). subsequently we investigate the applicability of dws© in patients with definite sfn, based on abnormal iefnd and/or ttt, determining the dws© sensitivity and specificity, as well as its validity. for this cross-sectional study, we will include healthy participants and patients diagnosed with sfn. eligibility is based on meeting the inclusion and exclusion criteria and providing written informed consent. skin wrinkling is induced by emla (eutectic mixture of local anesthetics) cream© application and captured by taking pictures. the primary outcome measure is total length of wrinkles per mm as shown on the photographs, which will be calculated by a new software program. patients are stratified according to age and gender. based on the results of healthy participants, normative values will be defined. inter-and intra-observer reliability will be determined. in the sfn group, additional correlation analysis will be conducted to determine the correlation between dws© and different outcome measures (sfn-symptom inventory questionnaire, visual analogue pain scale, neuropathic pain scale, sfn-rasch-built overall disability scale, ienfd and ttt). we expect to provide digitally quantified ssw (dssw©) normative values that can be used in clinical practice in the diagnostic workup for sfn. cmt , characterized by axonal degeneration, is an inherited motor and sensory neuropathy accounting for about % of total cmt patients. the cmt subtype shows on its own, a vast genetic heterogeneity with more than mutations in known genes rendering the identification of relevant drug targets and therapies very challenging. so far, only hdac inhibitors have shown promising results in mouse models for hspb mediated axonal cmt (cmt f), albeit such a single gene approach may have a limited relevance at clinical levels owing to the limited number of patients per genotype. in this study, we investigated common causative molecular players of cmt associated axonal degeneration. for this, an itraq based proteome analysis was performed on five patient's derived lymphoblasts bearing different cmt causal mutations alongwith respective age and gender matched unaffected controls. software-assisted interpretations of the obtained data led us to identify two proteins which were significantly downregulated in cmt patients compared to controls. these two proteins were then validated using western blotting and qpcr on patient derived lymphoblasts and fibroblasts. our results prompted us to unveil whether these two proteins can be used as potential biomarkers for identifying cmt patients. therefore, through a europe-wide collaboration, we constructed a cohort of cmt patients and healthy controls. these two proteins exhibited significant downregulation in this cohort suggesting a potential new role of these proteins as cmt biomarkers. remarkably, we were also able to validate the significant decrease in ineurons (neurons differentiated from patient derived ipscs) strengthening the importance of our finding and also suggesting the relevance of these proteins in the pathogensis of axonal cmt. this will be the first study involving multiple cmt causal genes at once, thereby holding the potential to offer new drug targets and potential biomarkers with wider application both clinically and pharmaceutically. mesenchymal stem cells (mscs) represents a valuable source of stem cell therapy, can differentiate into various cell types. we investigated of the neuromuscular potential of human tonsil-derived mscs (t-mscs) for neuromuscular regeneration in trembler-j mice that is considered to be a model for charcot-marie-tooth disease type a (cmt a diseases), which is involving hereditary motor and sensory peripheral neuropathies. the t-mscs differentiated toward skeletal myocytes, as evidenced by increased expression of skeletal muscle-related markers (including troponin i type , and myogenin) and the formation of myotubes in vitro. in situ transplantation of t-msc-derived myocytes (t-myocytes) into gastrocnemius in trembler-j mice, a mouse model of cmt a, enhanced motor function, as identified with recovery by a compound muscle action potential (cmap) amplitude. and the regenerated shape of the sciatic nerve and skeletal muscle by immunochemistry, without the formation of teratomas. furthermore, the expression levels of nerve growth factor (ngf) and glial cell line-derived neurotrophic factor (gdnf) were significantly increased in t-myocyte compared with t-mscs in vitro. these results indicate that the transplantation of t-myocyte can be a therapeutic option of cell therapy for the neuromuscular regeneration in hereditary peripheral neuropathy, comprising cmt a disease. kagiava a , karaiskos c , richter j , tryfonos c , lapathitis g , sargiannidou i , christodoulou c , kleopa ka , . neuroscience laboratory, the cyprus institute of neurology and genetics, nicosia, cyprus; department of molecular virology, the cyprus institute of neurology and genetics, nicosia, cyprus; neurology clinics, cyprus school of molecular medicine, the cyprus institute of neurology and genetics, nicosia, cyprus. x-linked charcot-marie-tooth disease (cmt x) is a common form of inherited demyelinating peripheral neuropathy resulting from mutations affecting the gap junction protein connexin (cx ). using a cx knockout (ko) mouse model of the disease, we have shown that targeted expression of virally delivered cx results in morphological and functional improvement. since patients with cmt x express mutant forms of cx in schwann cells, that could potentially interact with virally delivered wild type (wt) cx through dominant-negative effects, we also treated mutant mice expressing the t i, r w and n d mutations associated with cmt x on a cx ko background. all three mutants were localized in the perinuclear compartment of myelinating schwann cells consistent with retention in the er (t i) or golgi (r w, n d) with loss of physiological expression in non-compact myelin areas. following intrathecal delivery of the human gjb gene we could detect the virally delivered wt cx correctly localized in the non-compact myelin areas only in t i/cx ko mutant mice, but not in the other two mutants, suggesting dominant effects of the r w and n d mutant but not of the t i mutant. gjb treated t i/cx ko mice showed improved motor performance, along with lower ratios of abnormally myelinated fibers and reduced numbers of inflammatory cells in all tissues examined compared to mock-treated animals. in contrast, gjb treated r w and n d mutant mice showed only slight but not statistically significant improvement. this study provides additional proof of principle for a clinically translatable gene therapy to treat cmt x even in the presence of endogenously expressed cx mutants, since at least one er-retained cx mutant did not interfere with the expression of virally delivered cx allowing a therapeutic benefit similar to cx ko mice. however, golgi-retained mutants may interfere with virally delivered wt cx and other approaches besides gene addition may be needed for effective treatment. funding: muscular dystrophy association (grant mda to kak). kaida k , kadoya m , koike h , iijima m , takazaki h , ogata h , moriguchi k , shimizu j , nagata e , takizawa s , chiba a , yamasaki r , kira j-i , sobue g , ikewaki k . national defense medical college, tokorozawa, japan; nagoya university graduate school of medicine, nagoya, japan; kyushu university, fukuoka, japan; university of tokyo, tokyo, japan; tokai university school of medicine, isehara, japan; kyorin university, tokyo, japan. antibodies to a glial protein, neurofascin (nf) have recently been identified in approximately % of patients with chronic inflammatory demyelinating polyneuropathy (cidp), which are igg -predominant. igg anti-nf -associated cidp may be a distinct subtype from typical cidp in terms of clinical features and response to immunotherapy. however, a diagnostic criterion of anti-nf -associated cidp has not established yet. to develop optimal criteria and design the best treatment plan for the anti-nf -associated cidp, procedures for determining anti-nf antibodies should be simplified, prevalent, and reproducible, as well as being accurate. cell-based assay (cba) has hitherto been utilized for determining antibodies to nf in sera from patients, results of which have been confirmed by immunohistochemistry (ihc) using teased nerve fibers from rodents. these methods are the most reliable techniques, while not necessarily easy-to-use and easy to maintain in most laboratories. in the present study, we aimed to validate the diagnostic utility of a conventional enzyme-linked immunosorbent assay (elisa) for determination of anti-nf antibodies and the igg subclass. sera from patients with efns/pns criteria-met cidp were examined with elisa using human recombinant nf . to verify elisa results, ihc on rat sciatic nerves, western blot (wb) and cba using nf -transfected and naive hek cells were conducted. the human nf -based elisa clearly distinguished between anti-nf antibody-positive and -negative sera. fifteen cidp patients ( %) were igg anti-nf antibody-positive, which were confirmed by wb, ihc and cba studies. none of disease controls or healthy subjects had positive results. twenty-five sera randomly selected from anti-nf -negative cidp sera were also negative on cba. the anti-nf activities on elisa were significantly positively-correlated with those on cba (p < . ). analyses of clinical and laboratory findings showed that anti-nf -associated cidp was characterized by younger onset, distal dominant phenotype, tremor, sensory ataxia, higher protein levels in cerebrospinal fluid, and poor response to ivig, which were consistent with those in previous studies. this elisa combined with determination of the igg subclass is a simple and reliable method for initial screening for anti-nf antibodies. the genetic abnormality responsible for x-linked charcot-marie-toothy neuropathy subtype cmtx was recently identified by whole genome sequencing to be a kb insertion into chromosome xq . . the clinical profile of cmtx in childhood is not well described. we reviewed the clinical characteristics, neurophysiological profile and cmt pediatric scale (cmtpeds) assessments of children with genetically confirmed cmtx . cmtx was characterized by early onset, and early and progressive hand weakness. most affected children were symptomatic within the first two years of life. the most common presentation was with equinovarus foot deformity in the first year of life. cmtpeds analysis in these children revealed that cmtx progressed more rapidly ( . ± . points/ year, n= ) than cmt a and cmtx . grip strength in the second decade of life in most affected males was two standard deviations below age-and sex-matched normative reference values. the most severely affected individual was wheelchair bound at years of age and two individuals had no movement in the small muscles of the hand in the second decade of life. there was only a single symptomatic female identified and she had mild signs. nerve conduction studies showed a demyelinating sensorimotor neuropathy with motor conduction velocity in eight children while one child had a length-dependent sensorimotor axonal neuropathy. understanding the unique phenotype of cmtx is essential for directing genetic testing, as the cmtx insertion will not be detected on the snp microarrays, multi-gene panels or whole-exome sequencing currently used for the diagnosis of cmt. the early onset of disease coupled with rapid progression means that many children with cmtx will have severe disability within the first two decades of life and hence early diagnosis is needed for early commencement of rehabilitation. kapoor m , catania s , sarri-gonzales s , lunn mp , manji h , reilly mm , carr as . centre for neuromuscular diseases, national hospital for neurology and neurosurgery, london, uk; department of neurophysiology, national hospital for neurology and neurosurgery, london, uk. the conventional dosing of ivig in cidp and mmn is based on treatment trials that used bolus and maintenance dosing of ivig between - . g/kg. there are rare published articles reporting the efficacy of higher maintenance ivig doses. we present three cases of inflammatory neuropathies, who are currently stabilized on ivig doses of mg- mg/kg of ivig per month, refractory to standard dose ivig and other immunosuppressants. the first case is a year-old-lady with cidp who presented with episodes of ascending sensory disturbance, weakness, and diplopia. she had activity related fluctuations and pre-dose deterioration on g/kg/month ivig. she then had an acute deterioration with mrc sum score dropping from to even with additional plasma exchange. her bilateral foot drop (mrc grade - ) and fluctuations persisted with an increase of ivig to . g/kg/month. she is now clinically stable (ankle dorsiflexion mrc grade - , mrc sum score ) on mycophenolate and g ivig weekly ( . g/kg/month). case is a -year-old male fitness instructor with mmn and sjogren's syndrome. he presented with recurrent proximal and distal weakness that responded to g/kg of ivig and deteriorated with iv methylprednisolone. he had peri-dose fluctuations, intermitted proximal weakness, and persistent foot drop (ankle dorsiflexion mrc grade - ) at . mg/kg/month, worsening to mrc grade - on . g/kg/month and fluctuating between mrc grade - on . g/kg/month. an increase of ivig to g weekly ( . g/kg/month), has resulted in mmn rods scores of / , improved distal power and return to full capacity at work. case is a -year-old man with predominantly upper limb cidp. he received g/kg ivig without any benefit, had no response to doses of plasma exchange, doses of cyclophosphamide or dose of rituximab between and . since , he has received g/kg/month ivig with improvement of mrc sum score from to . these cases highlight that some patients require a much higher than conventionally prescribed dose of ivig, and that these doses are tolerated over years without serious adverse events. idiopathic rapid eye movement sleep behaviour disorder (irbd) has been identified as a precursor of alpha-synucleinopathies, such as parkinson's disease, dementia with lewy bodies, multiple system atrophy. several studies linked changes in cutaneous innervation with central nervous system pathology in neurodegenerative disorders. recently small fiber neuropathy and alpha-synuclein deposition in the skin found to be a potential biomarker in parkinson's disease. we evaluated the epidermal innervation of irbd patients and age and sex-matched controls from skin punch biopsies from the distal leg using pgp . immunohistochemistry. furthermore, a battery of clinical examinations were performed on patients and controls alike, including structured interviews, clinical motor and non-motor questionnaires and rating scales (e.g. unified parkinson's disease rating scale [updrs], non-motor symptoms questionnaire [nms-quest] and beck depression inventory, epworth sleepiness scale, evaluation of cognitive and olfactory functioning as well as blood samples. irbd patients, compared to controls, showed a significant reduction in intraepidermal nerve fiber density (p = . ), whereas the axon swelling ratio, did not differ between groups. patients with irbd reported non-motor symptoms more frequently than controls (updrs i, nms-quest). olfaction and daytime sleepiness differed between both groups, whereas there were no differences regarding cognition. these in vivo findings demonstrate small fiber neuropathy in irbd patients that are associated with non-motor symptoms indicating that peripheral abnormalities may occur early in irbd. they warrant larger scale longitudinal studies in order to investigate their prognostic value. katz j , lewis r , spatafora d . california pacific med center, san francisco, usa; cedars-sinai medical center, los angeles, usa; neuropathy action foundation (naf), santa ana, usa. multifocal motor neuropathy (mmn) is a rare condition that affects . in every , individuals worldwide and is associated with motor dysfunction and moderate to severe disability. the neuropathy action foundation conducted a global survey to determine the impact of mmn on patient quality of life (qol) and gaps in patient/provider educational needs. the first global mmn qol survey was an item internet questionnaire available between january and july , . the survey focused on three primary areas: timely and accurate diagnosis, the efficacy of treatment, and the impact of the disease on patients qol. the survey was completed by patients from countries. the majority of respondents said they were diagnosed between the ages of and years ( . %), more than % reported that it took more than one year to be diagnosed and more than % reported that it took - years or longer to be accurately diagnosed. with respect to treatment options : . % reported receiving intravenous immune globulin and . % reported receiving subcutaneous immune globulin therapy. other therapies being used to treat mmn were gabapentin ( . %), and pregabalin ( %). almost half ( . %) said that mmn often impacts their overall schedule. half of the participants reported that mmn often or always interferes with their employment; % had difficulty typing on a computer or using a telephone, . % had trouble concentrating, and . % said they had to work really hard to pay attention or else they would make a mistake. this is the first assessment of mmn from a patient's perspective. the survey highlighted critical issues relating to the diagnosis, management, and impact on the qol of individuals with mmn. the data also identified gaps and insights in provider education relating to proper diagnoses and management of the condition from a patient's perspective. katz j , levine t , dimachke m , barohn r . forbes norris center, san francisco, ca, usa; phoenix neurological institute, phoenix, az, usa; kansas university medical center, kansas city, ks, usa. in the united states, cidp cases are submitted to insurance companies to determine whether ivig therapy is appropriate. this is done using specified diagnostic criteria, which reduce diagnosis to a boolean analysis, where a disease can only be present or absent. this leaves no room for uncertainty, even when it truly exists. boolean criteria are useful for clinical trials, but fall short where real decisions are made under uncertainty and based on perceived cost/benefit analysis. this project attempts to elucidate root causes of uncertainty and to find solutions to this dilemma. we asked cidp experts to select a single diagnosis in cases where ivig was approved using the submitted case records. while there was agreement on many cases, in the five most "uncertain" cases no more than reviewers agreed on a single condition, who chose up to four separate entities. among these, at least three reviewers diagnosed an immune neuropathy in all five. the root cause of the disagreement, to a large degree was unclear documentation (aunts) which consisted of pasted, missing, and disorganized data. reviewers missed useful information, admitting it was too difficult to fully parse records. to resolve uncertainty, reviewers admitted to discounting certain reported datum to help fit the entity they suspected, such as reported therapeutic responses or certain electrodiagnostic/exam findings. other disagreement, however, reflected the complexity of neuropathy diagnosis, such as knowing if improvement was due to natural history or treatment, unawareness of rare presentations, or analyzing a true uncle (complex case). reviewers used bayesian (select most likely diagnoses from a list) and fuzzy logic (compare best fits to base cases). when the "best" diagnosis did not fit the base case, they had to re-interpret the data. improving review procedures requires eliminating aunts by collecting all key information and simplifying how records are presented. it also needs more advanced data methods to analyze common and rare borderline presentations (uncles like mama v pma, cidp v cspn, etc..), developing diagnostic algorithms that address real uncertainty, educating prescribers and patients on process, and creating systems that measure outcomes longitudinally after induction or tapering of therapy. keisuke y , miyuki m , motoi k , susumu k . department of neurology, faculty of medicine, kinki university, osaka, japan. anti-ganglioside antibodies are closely associated with clinical phenotype and specific symptoms in acute immune-mediated neuropathies. igg anti-gq b antibodies are specifically associated with miller fisher syndrome (mfs), bickerstaff brainstem encephalitis (bbe) and guillain-barré syndrome (gbs) with opthalmoplegia (gbs-op). in addition, ganglioside complexes (gscs) containing gq b also can be targets in such diseases, and might be associated with the clinical features. however, factors regulating clinical phenotype in those gq b-associated antibodies-positive diseases have not yet been known. for investigating the differences of antibody reactivities among those diseases, we examined, using combinatorial glycoarray, igg antibodies to ten individual glycolipids [gm , gm , gm , gd a, gd b, gq b, galactocerebroside (gal-c), lactosylceramide (laccer), ga , sulfatide] and glycolipid complexes consisting of two of the glycolipids listed above in sera from patients with gbs-op who were positive for anti-gq b antibody by elisa (gbs-op-gq b), patients with mfs with the clinical triad (opthalmoplegia, ataxia, and areflexia), and patients with bbe. by combinatorial glycoarray, overall sensitivity of antibodies to gq b and gscs containing gq b was . % ( / ) in gbs-op-gq b, . % ( / ) in mfs, and . % ( / ) in bbe, respectively. there were no significant differences in antibody reactivities between mfs and bbe. it is notable that antibodies to gscs containing gd b were more frequently found in gbs-op-gq b patients than in mfs or bbe patients (e.g., gd b/sulfatide: p= . and p< . , respectively). presence of the antibody reactivities to gscs containing gd b may possibly be related with clinical features of gbs-op-gq b, including frequent need of artificial ventilation. kennedy r , , , carroll k , , paterson k , ryan mm , , , mcginley jl , . royal children's hospital, parkville, australia; university of melbourne, parkville, australia; murdoch childrens research institute, parkville, australia. problems with walking and footwear fit are often reported by children and adolescents with charcot-marie-tooth disease (cmt). a cross-sectional, case controlled study of gait was conducted in children with cmt and typically developing (td) children. gait was assessed barefoot and in two types of the participants' own typical footwear; optimal (e.g. athletic shoes) and suboptimal (e.g. slip-on footwear). the aims were to determine differences in spatio-temporal (s-t) gait variables between children with cmt and td children; and to investigate the effect of footwear choices. twenty-nine independently ambulant children aged - years with confirmed genetic or clinical diagnoses of cmt, and age and gender matched td children participated (mean age . years; males). exclusion criteria included developmental disorders, other neuromuscular conditions or musculo-skeletal diseases that could affect gait, and lower limb injury or surgery in the preceding months. assessment included s-t gait patterns, footwear characteristics, metre run, and cmtpeds. gait was assessed at self-selected speed with an electronic walkway (gaitrite™), with trials for each condition. the primary gait variable assessed was speed; other variables included step length, step time, cadence, base of support width and step-to-step gait variability. across all footwear conditions children with cmt walked more slowly ( regeneration of cutaneous unmyelinated axons is known to be slowed in dm-patients and after -months, the density of intraepidermal nerve fibers (ienf) does not return to baseline levels after chemical or mechanical axotomy. however, the long-term outcome of regeneration in dm or control subjects is not known. additionally, it is not clear if the regeneration of sensory distal axons is length-dependent. here we measured the rate of axonal regeneration -months after chemical denervation using a capsaicin model in dm patients (n= / dm /dm ) without neuropathy, and controls. dm skin punches were performed at distal thigh at baseline, -hours post-capsaicin, and at , , and days. blood glucose and hgba c were measured at baseline, , and days. healthy controls had skin punches at both distal leg and proximal thigh at baseline, after capsaicin chemical axotomy, and days , , and . regeneration rate was significantly higher at the thigh in healthy controls ( . fibers/mm/day ( % ci: . - . fibers/mm/day) compared to dm (p= . ), but no difference between dm ( . fibers/mm/day % ci: . - . fibers/mm/day) or dm ( . fibers/mm/day % ci: . - . fibers/mm/day) (p= . ). comparing regeneration rate at different time intervals, showed that regeneration was significantly slowed between day and dm patients, while it continued with the same rate in controls. blood glucose or hga c had no effect on regeneration rate. ienfd returned to baseline in controls by -months ( % of baseline) while it is did not in dm subjects, %/ % (dm /dm ) of ienfd baseline, (p= . dm vs. controls). there was no difference in regeneration rate ienfd %-baseline by -months at distal leg and proximal thigh in controls (p= . ). these results suggest that the rate and outcome of regeneration is independent of the length of the axon. additionally diabetic patient have incomplete nerve regeneration after months regardless of diabetes type or the level of glycemic control. regeneration of axons slowed down over time in patients with dm and reached a plateau after days. kiessling p , lledo-garcia r , watanabe s , langdon g , tran d , bari m , christodoulou l , price g , smith b , byrnes w , brock m , jolles s . ucb pharma, monheim, germany; ucb pharma, slough, uk; ucb pharma, braine-l' alleud, belgium; ptx solutions ltd, london, uk; ucb pharma, raleigh, durham, usa; university hospital of wales, cardiff, uk. ucb is a humanised high-affinity monoclonal igg antibody developed to bind human neonatal fc receptor (fcrn), selectively inhibiting igg salvage and recycling. conditions such as myasthenia gravis (mg) are characterised by pathogenic igg autoantibodies; inhibition of fcrn may provide a suitable therapeutic approach. this phase i, double-blind, dose-escalating, first-in-human study (nct ) evaluated the safety and pharmacology of ucb . forty-nine healthy adults (mean age years, range - ) were randomised and received a single dose of intravenous (iv) or subcutaneous (sc) placebo (n= and n= , respectively), or a single dose of iv or sc ucb ( , or mg/kg; n= per dose, per administration). subjects were followed up until day . one placebo iv subject did not complete the study. twenty-seven of subjects ( %) receiving ucb , and / ( %) receiving placebo, reported ≥ treatment-emergent adverse event (teae) of mild/moderate intensity. severe teaes occurred in four subjects, all in the ucb mg/kg iv group (headache [n= ], back pain [n= ]). no serious aes occurred. incidence of infections was similar with ucb and placebo. the most frequently reported infection was nasopharyngitis. treatment-related teaes were reported by % of subjects receiving ucb and % receiving placebo: the most common in the ucb -treated groups were headache ( / ; %) and vomiting ( / ; %); these occurred more frequently with the iv than sc route. non-linear increases in ucb plasma concentration-time profile with increasing dose were observed with ucb . serum igg was reduced in a dose-depended manner with ucb iv and sc: decreases from baseline to day with ucb iv were . %, . % and . % for , and mg/kg doses, respectively, and . %, . % and . %, with ucb sc doses, respectively. these data indicated that the fcrn inhibitor ucb effectively reduced serum igg, with sc administration generally better tolerated than iv. further to these observations, the efficacy, safety and tolerability of ucb sc for chronic-intermittent treatment of moderate-to-severe mg are being evaluated in an ongoing phase ii, multi-centre, randomised, double-blind, placebo-controlled study (eudract - - middle east respiratory syndrome (mers) has a high mortality rate and pandemic potential. however, very little information has become available on this syndrome since it first erupted in . this study aimed to evaluate the frequency of neurological complications and their clinical presentations in mers. we reviewed the medical records of all patients who were diagnosed with laboratory-confirmed mers coronavirus (cov) infections and subsequently admitted to a single reference center for mers treatment during the outbreak in korea. in total, patients ( . %) reported neurological symptoms during or after mers-cov infection. the potential diagnoses in these cases included bickerstaff's encephalitis overlapping with guillain-barré syndrome, critical illness polyneuropathy or other toxic or infectious neuropathies. neurological complications did not appear concomitantly with respiratory symptoms, but were instead delayed by - weeks. neuromuscular complications were not rare in mers-cov-infected patients, and they may have previously been underdiagnosed. understanding neurological manifestations is important in an infectious disease like mers, because evaluation is frequently limited during treatment, but it can interfere with prognosis and sometimes require modification of treatment. kim hj , hyun jk , kim tu . dankook university, cheonan, south korea. the diagnosis of carpal tunnel syndrome (cts) is based on clinical symptoms, physical examinations and supported by nerve conduction study (ncs). ultrasonographic examinations can be performed to assess peripheral nerves with less discomfort and the surrounding anatomic structures. while the usefulness of ultrasonography (usg) in the cts has been reported, no study to date has compared the diagnostic utility of various usg findings for cts. we investigated the correlation of various usg findings to the clinical symptoms/signs and ncs findings in patients with cts. twenty-eight hands ( patients) with cts based on electrodiagnostic criteria and clinical symptoms such as tingling sensation or pain in the first to third fingers, burning sensation, paresthesia and weakness of hand grip power. all subjects were examined with usg. cross-sectional area (csa) and flattening ratio (fr) of the median nerve was calculated at level of radio-ulnar joint, pisiform and hamate. swelling ratio of the median nerve and palmar displacement of the flexor retinaculum was also calculated. clinical assessment was conducted using the boston carpal tunnel questionnaire (bctq) scale and historical-objective (hi-ob) scale. the analysis of correlation between usg findings and clinical symptom scales/ncs findings was performed using correlation analysis. the csa of the median nerve at level of radio-ulnar joint was significant correlated with bctq scale, hi-ob scale, distal motor latency, and conduction velocity (cv). the csa of the median nerve at level of pisiform was significantly correlated with hi-ob scale, distal motor latency, and cv. the fr of the median nerve at level of radio-ulnar joint was significantly correlated with bctq scale, hi-ob scale, distal motor latency, and cv. the swelling ratio of the median nerve was also significantly correlated with distal motor latency and cv. in patients with cts, csa of the median nerve at level of radio-ulnar joint was most closely related to ncs findings and clinical symptoms. so, csa of the median nerve at radio-ulnar joint might be a complementary tool for the diagnosis of cts. kitaoji t , tsuji y , ashida s , yamada t , ishii r , tanaka a , mizuno t . department of neurology, graduate school of medical science, kyoto prefectural university of medicine, kyoto, japan. the case was a -year-old woman. first, she noticed paresthesia in the right plantar eight months before admission and in the left plantar four months before admission. three months before admission, she developed muscle weakness in her feet. the muscle weakness and paresthesia extended to the lower legs in a few months. twenty days before admission, she experience difficulty in walking. on admission, the muscle weakness was observed in the legs, especially in the right tibial anterior muscle (ta). there was severe sensory disturbance and loss of deep tendon reflex in the legs. she had trouble walking due to the weakness and sensory aphasia. in nerve conduction study (ncs), conduction block was observed between the ankle and popliteal in both tibial nerves. the blood level of angiotensin converting enzyme (ace) was elevated. cerebrospinal fluid analysis was normal. there was no enhancement in the lumbar nerve roots shown on mri. gallium- scintigraphy showed hot spots on bilateral hilar lymph nodes and mediastinal nodes and biopsy of mediastinal nodes showed non-caseating epithelioid granuloma. therefore, we diagnosed her with sarcoid peripheral neuropathy by sarcoidosis. by using the nerve ultrasound, partial spindle-shaped nerve enlargement was observed at the part of conduction block in the left tibial nerve. we started the treatment with methyl prednisolone ( mg, days) and oral prednisolone therapy ( mg/kg/day). after treatment, the paresthesia and muscle weakness in the legs had gradually improved. the partial enlargement in the left tibial nerve also improved on the -hospital day. in ncs, the conduction block improved, however, the compound muscle nerve potential of tibial nerve decreased because of axonal damage. this partial spindle-shaped nerve enlargement by using ultrasound has never been reported in sarcoid peripheral neuropathy before. the nerve ultrasound may be useful for evaluation of therapeutic effect of sarcoid peripheral neuropathy. peripheral nerve injuries are still debating problems in the world because of poor recovery. there is absolutely a need for new therapeutic agents to improve outcome by altering nerve regeneration. there are some studies in the literature about some therapeutic agents that used in the cases of peripheral nerve injuries. despite these studies, an agent with clinical use has not been presented yet. in this experimental study, we aimed to analyze the effects of curcumin (cur) in the cases of peripheral nerve injuries. forty rats were randomly and equally divided into four groups. the first group was control group. rats in this group were not operated. right sciatic nerve injuries were performed to the other groups. the second group was operation group with no therapeutic agent. the third group was operation and local cur applied group. the fourth group was operation and systemic cur applied group. electrophysiological evaluations were performed with electroneurography (enog) before and after the surgeries. systemic use of cur although caused improvement in the enog values but could not make a positive contribution to the nerve regeneration statistically. additionally local use of cur made negative effect to the nerve regeneration statistically. according to our statistical results we could not recommend cur as a nerve protective agent. klein cj , wu y , jentoft me , mer g , spinner rj , dyck pjb , dyck pj , mauermann ml . mayo clinic, rochester, usa. intraneural perineurioma is a hypertrophic peripheral nerve tumor having immunoreactivity to epithelial membrane-antigen, negative for s- . the origin of perineurial cells is debated to be similar to meningeal cells. ip does not metastasize, but motor deficits accumulate over time from tumor growth in nerve and plexus. after schwannomas and neurofibromas, perineuriomas are the most common nerve tumor of young adults. a chromosome q deletion has been reported in one patient. we identified ip cases from our previously published clinical cohort with available flash frozen ip tissue for dna isolation. wes with cnv analysis and cgh microarray analysis (agilent x k superprintg ) were performed on extracted dna; had available germline dna (lymphocytes and buccal tissue). we compared the exome data against online and in-house control data (∼ , ) examining variants less than . frequencies, predicted damaging or nonsynonymous. wes identified three novel, heterozygous, damaging mutations in tumor necrosis factor receptor-associated factor (traf ) in of ( %) cases; p.l p (n= ), p.h r (n= ) and p.s r (n= ). mutations were within the wd domain, p.l p, p.h r within exon and p.s r within exon , and mapped to a limited region of traf with protein structure modeling. two of cases ( . %) showed macroduplications/deletions on multiple chromosomes, including chromosome , confirmed with cgh microarray analysis and cnv results from exome data analysis. four of ( %) had no discovered mutation. age of onset or severity did not correlate with type of mutations. this study provides strong evidence that traf is a specific tumor driver of ip. mutations in traf are also linked to benign intracranial meningiomas suggesting a shared pathogenesis and close origins of perineurial and meningeal cells. study supported by: mayo foundation and the mayo center of individualized medicine. klein i , , bobylev i , , lehmann hc , . university hospital cologne, cologne, germany; center for molecular medicine cologne (cmmc), cologne, germany. peripheral neuropathy is a common side effect of paclitaxel. clinical evidence suggests that the delivery mechanism of paclitaxel formulations influence time course and severity of paclitaxel induced peripheral neuropathy. in a preclinical model we studied access, distribution and toxicity of two paclitaxel formulations (nanoparticle albumin-bound paclitaxel (nab) and solvent-based paclitaxel) in the peripheral nervous system (pns). c bl/ mice were treated with mg/kg or mg/kg of nab-paclitaxel or solvent based paclitaxel. kinetics of paclitaxel in neurons was assessed by a newly established immunostaining technique. neurotoxicity was evaluated by functional assays and nerve morphology. paclitaxel accumulated mostly in dorsal root ganglia, whereas distal nerve segments showed only low uptake of paclitaxel. treatment of mice with the two paclitaxel formulations resulted in paclitaxel uptake mostly in nf + larger fiber neurons. in ib +, and cgrp+ small fiber neurons, paclitaxel was less frequently detected. nab-paclitaxel was incorporated more rapidly compared to solvent-based paclitaxel but neurons also showed a faster clearance of nab-paclitaxel compared to solvent based paclitaxel. functional assays and nerve conduction studies indicated that nab-paclitaxel was less neurotoxic compared to solvent-based paclitaxel. this is the first study that characterizes in detail the access of nab-paclitaxel and solvent based paclitaxel into the pns. our findings have important implications to understand the pathomechanisms of paclitaxel induced neurotoxicity and to develop neuroprotective strategies by preventing access of paclitaxel to the pns. the aim of our study was to investigate the etiology of neuropathy in patients with rheumatoid arthritis (ra). subjects were neuropathy patients with ra admitted to our department. laboratory investigations, nerve conduction studies (ncs) and sural nerve biopsy were performed. mean patient age was . years (range, - years), and mean disease duration was . years (range, - years). clinical diagnosis for neuropathy was rheumatoid vasculitis (rv) in patients, rv with acute motor axonal neuropathy (rv-aman) in and chronic inflammatory demyelinating polyneuropathy with ra (ra-cidp) in . rheumatoid factor ( / ) and rheumatoid arthritis particle agglutination ( / ) was high and c tended to be lower in the rv group (rv and rv-aman). anti-ganglioside antibodies were examined in patients, with positive results in . an rv-aman case was diagnosed with motor-dominant clinical presentation and the presence of anti-galnac-gd a immunoglobulin (ig)g antibody. no other cases with rv were examined for anti-ganglioside antibodies. positive results for anti-gm and gm igg antibody were seen in one ra-cidp patient. we evaluated sural/median (s/m) ratio) for sensory nerve action potential (snap). s/m ratio was low in rv cases ( / ) and high ( / ) in immune-mediated cases, suggesting a so-called normal sural abnormal median pattern in immune-mediated neuropathies. the rv-aman case showed a moderate value in s/m ratio. nerve biopsy revealed thinly myelinated nerve fibers in ra-cidp cases compatible with demyelination, while the rv group showed the typical pathology for necrotizing vasculitis. rv cases were treated with prednisolone (psl), intravenous methylprednisolone, intravenous cyclophosphamide and increased psl dose. ra-cidp and rv-aman were treated with intravenous ig. in conclusion, neuropathy in ra can be divided into vasculitic and immune-mediated groups. ncs, and the s/m ratio of snap with some laboratory parameters in particular, may be of use in differential diagnosis and deciding treatment strategies. koh s , wong shj , loh kw , chng ysk , pawa c , ei ma , lee bjh , subramaniam t , umapathi t . national neuroscience institute, singapore, singapore; lee kong chian school of medicine, nanyang technological university, singapore; yong loo lin school of medicine, national university singapore, singapore; tan tock seng hospital, singapore; khoo teck puat hospital, singapore. treatment-induced neuropathy of diabetes mellitus (dm) (tind) is an acute painful autonomic neuropathy that develops with abrupt improvement in glycaemic control. typically, type or dm patients on insulin or oral hypoglycaemic agents (ohga), present with painful neuropathy and autonomic dysfunction within weeks of rapid improvement in glucose control. current emphasis to achieve good glycaemic control rapidly may inadvertently increase incidence of tind, hence the impetus to understand risk of over-zealous glycaemic control. we therefore set out to study the occurrence of tind in a dm cohort of a tertiary hospital. we screened all patients who had two hba c measurements between and . during this period, approximately patients were seen per year. we found patient-encounters that showed hba c decrease of ≥ % over months or ≥ % over months. we then used a structured checklist of tind symptoms to shortlist cases. these case-encounters were scrutinised and classified as; 'probable tind': acute painful neuropathy and acute dysautonomia with temporal relationship to the decrease in hba c; 'possible tind': acute painful neuropathy or acute dysautonomia or uncertain temporal relationship to decrease in hba c; unlikely tind: alternative explanation exists for symptoms. only one case was deemed 'probable tind'-a middle-aged man with newly diagnosed type dm who presented to emergency department with palpitations and worsening 'frozen feet' sensation that disturbed sleep. his hba c decreased by . % in weeks. his symptoms improved within a month with neuropathic pain medications and resolved months later. he also developed maculopathy and proliferative retinopathy. ten months later, he developed significant proteinuria. four other cases were classified as 'possible tind' while the remaining were unlikely tind. our study is limited by retrospective design and reliance on hospital records. nevertheless, our findings suggest that tind is uncommon in a general cohort of dm patients. on the other hand, the number of patients with painful neuropathy and acute dysautonomia symptoms contemporaneous with rapid decline in hba c raises the intriguing possibility that forme fruste of tind exists and one should interrogate the rate of hba c decline in dm patients with these symptoms. koike h , kadoya m , kaida k , ikeda s , kawagashira y , iijima m , kato d , ogata h , yamasaki r , matsukawa n , kira ji , katsuno m , sobue g . nagoya university graduate school of medicine, nagoya, japan; national defense medical college, tokorozawa, japan; nagoya city university graduate school of medical sciences, nagoya, japan; kyushu university, fukuoka, japan. we investigated the morphological features of chronic inflammatory demyelinating polyneuropathy (cidp) with autoantibodies directed against paranodal junctional molecules, particularly focusing on the fine structures of the paranodes. sural nerve biopsy specimens obtained from cidp patients with anti-neurofascin antibodies and patient with anti-contactin antibodies were assessed. these antibodies were examined using sera obtained from patients with cidp who fulfilled the criteria of the european federation of neurological societies/peripheral nerve society. thirteen cidp patients without these antibodies were also examined to compare pathological findings. characteristic light and electron microscopy findings in transverse sections from patients with anti-neurofascin and anti-contactin antibodies indicated a slight reduction in myelinated fiber density, with scattered myelin ovoids, and the absence of macrophage-mediated demyelination or onion bulbs. teased-fiber preparations revealed that segmental demyelination tended to be found in patients with relatively high frequencies of axonal degeneration and was tandemly found at consecutive nodes of ranvier in a single fiber. assessment of longitudinal sections by electron microscopy revealed that detachment of terminal myelin loops from the axolemma was frequently found at the paranode in both anti-neurofascin and anti-contactin antibody-positive cidp patients compared with antibody-negative cidp patients. patients with anti-neurofascin antibodies showed a positive correlation between the frequencies of axo-glial detachment at the paranode and axonal degeneration, as assessed by teased-fiber preparations (p < . ). in conclusion, paranodal dissection without classical macrophage-mediated demyelination is the characteristic feature of patients with cidp with autoantibodies to paranodal axo-glial junctional molecules. koike h , ikeda s , takahashi m , kawagashira y , iijima m , misumi y , ando y , ikeda si , katsuno m , sobue g . nagoya university graduate school of medicine, nagoya, japan; kumamoto university, kumamoto, japan; shinshu university hospital, matsumoto, japan. peripheral neuropathy is the cardinal feature of familial amyloid polyneuropathy (fap), but its mechanism has not been fully elucidated. we used electron microscopy to examine schwann cells and endoneurial microvessels. sural nerve biopsy specimens from fap patients with transthyretin val met mutation were assessed. patients were consisted of early onset cases from endemic foci and late onset cases from non-endemic areas. loss of nerve fibers with or without neighboring amyloid deposition was a common feature. the amount of amyloid deposition was greater relative to the extent of nerve fiber loss in early onset cases than in late onset cases. the atrophy of schwann cells, particularly nonmyelinating cells, that were apposed to amyloid fibrils was more conspicuous in early onset cases than in late onset cases. the numbers of endothelial cell nuclei, endothelial cell profiles, and occluded microvessels were significantly increased in the fap patients compared with patients with nutritional/alcoholic neuropathies (p < . , . , and . , respectively). findings suggestive of the disruption of blood-nerve barriers, such as the loss of tight junctions and the fenestration of endothelial cells, were also more frequently found in the fap patients (p < . ), irrespective of the presence or absence of amyloid deposition. in conclusion, these findings suggest that direct insult of amyloid fibrils causes schwann cell damage resulting in the predominant loss of small-fiber axons characteristic of early onset cases. in addition, vasculopathy may also participate in the pathogenesis of neuropathy, particularly in late onset cases. kolb n , smith ag , singleton jr , beck s , howard d , dittus k , karafiath s , mooney k . university of vermont, burlington, vt, usa; university of utah health, slc, ut, usa. chemotherapy induced peripheral neuropathy (cipn) is a major cause of morbidity due to numbness, pain, and gait instability. this prospective study compares the current standard care for cipn symptom management to a new care delivery model which utilizes an automated symptom tracking program paired with a nurse practitioner led intervention triggered by moderate to severe symptoms. all participants beginning taxane or platin based chemotherapy called a telephone based automated symptom tracking program daily (symptom care at home -sch) to report chemotherapy related numbness and tingling. sch tracked the presence and severity of neuropathic symptoms and their interference with activities of daily living (adls) on a - scale. participants were randomized to two groups. the usual care (uc) group was advised to call their oncology provider for recommendations on symptom management. in the nurse practitioner (np) group, when symptom severity was ≥ participants received automated self care strategies and a call from a nurse practitioner to provide treatment recommendations based on consensus guidelines. patients participated in the study. mean duration of follow up was . ± . days with . ± . calls. the np group had fewer days with any neuropathic symptom ( . % ± . vs. . % ± . , p= . ), with moderate to severe neuropathic symptoms ( . % ± . vs. . % ± . , p< . ) or days of distress from neuropathic symptoms ( . % ± . vs. . % ± . , p= . ). on days with moderate to severe symptoms participants also reported burning ( . ± . %), weakness ( . ± . %), balance problems, ( . ± . %), and tripping ( . ± . %). there was no significant difference between groups in the interference in adls (np . ± . vs. uc . ± . , p= . ). overall the automated telephone system effectively identified neuropathy symptoms and their severity. compared to usual care in which patients must independently reach out to their care team for symptom management, sch is effective in decreasing symptom prevalence, severity and distress. kouton l , kremer l , tard c , morales r , kuntzer t , attarian s , boucraut j , delmont e . referral centre for als and neuromuscular diseases, marseille, france; neurology department, strasbourg, france; neurology department, lille, france; neurology department, montpellier, france; neurology department, lausanne, switzerland; immunology laboratory, marseille, france. antibodies against proteins of the node of ranvier have been recently described in severe chronic inflammatory demyelinating polyradiculoneuropathies (cidp). they target paranodal proteins, namely contactin (cntn ) and neurofascin (nf ). cell-based assay and elisa are available in research, but no gold standard technic is admitted for the detection in routine of these antibodies. our objective was to evaluate if flow cytometry analysis is an efficient technic to detect antibodies against ctn and nf in a large cohort of cidp patients. flow cytometry analysis were performed on a bd facs-diva. human embryonic kidney (hek) cells were transfected either with nf or cntn . sera were diluted / . antibodies anti-cntn or nf were revealed using fitc conjugated anti human igg antibodies. delta mfi (mean fluorescence intensity) was calculated as mfi of transfected cells less mfi of non-transfected cells. measures were normalized using positive controls and negative controls from healthy blood donors. sera of cidp patients from different french neuromuscular referral centres were analysed with flow cytometry. respective delta mfi were (standard deviation ) and (standard deviation ) for antibodies against nf and cntn in cidp antibodies negative patients. antibodies against nf were found in patients (respective mfi , , ) and against cntn in two other patients (respective mfi and ). isotype of these antibodies was igg in patients and igg and igg in the remaining patient. all the patient had severe cidp. four patients had poor response to intravenous immunoglobulins (ivig) and have been treated with immunosuppressive drugs. as usually reported, the patient with anti-nf antibodies had postural tremor. flow cytometry seems effective to detect antibodies against nf and cntn . compared to other assays, benefits of flow cytometry are: to analyse a large number of sera in the same time and to give objective numerical results expressed in mfi that can be compared to the results of other samples. further studies are needed to confirm that flow cytometry can be the best test to assess antibodies against cntn and nf in routine. the mechanism by which intravenous immunoglobulins (ivig) improves peripheral nerve function in multifocal motor neuropathy (mmn) is unknown. the rapid clinical improvement following ivig could be related to blocking complement deposition on gm epitopes, change in ion-channel properties of affected motor axons, or both. the present study investigated median nerve motor excitability parameters at ∘ c just before ivig administration as well as at the peak of clinical improvement in patients with mmn. the investigated nerves were characterized either by conduction block (n= ), demyelinative slowing without block (n= ), or motor axon loss (n= ). the results of motor excitability testing in mmn showed no difference between pre-and post ivig recordings. clinical assessment of apb muscle showed increase in mrc score in patients and decrease in patient after ivig administration. in patients mrc score of the apb remained the same. those findings indicate that clinical changes following ivig administration are not related to excitability parameters of affected motor axons in mmn. where impulse conduction is blocked or markedly slowed in motor axons but is normal in sensory axons. sensory symptoms or signs are usually absent but have occasionally been reported in skin areas innervated by nerves with prominent motor axon loss. although the mechanism of selective motor involvement in mmn is unresolved, it may be related to differences in antigenic properties between motor and sensory axons or differences in biophysical properties. the objective of the present study was to compare ion-channel activity in both motor and sensory axons of nerves affected by mmn. affected nerves had to have motor conduction block, demyelinative slowing on motor ncs, or motor axon loss, whereas sensory ncs had to be normal. we performed excitability tests of motor and sensory axons in affected median nerves of mmn patients and healthy controls at ∘ c. conditioning and test stimuli were delivered at the median nerve at the wrist; cmaps were recorded from the thenar muscle and snaps from the rd digit. results of motor excitability testing in mmn showed fanning-out of threshold electrotonus, decreased i/v slope, and increased superexcitability, all compatible with persistent hyperpolarization of resting membrane potential in motor axons. sensory excitability testing in mmn showed decreased subexcitability but was otherwise normal. this may indicate minimal involvement of sensory axons in mmn. krarup c , , moldovan m , , alvarez s , ciano c , pisciotta c , pareyson d . university of copenhagen, copenhagen, denmark; rigshospitalet, copenhagen, denmark; fondazione irccs istituto neurologico c. besta, (incb), milan, italy. mutations in the gene coding for myelin protein zero (mpz, p ) are associated with different forms of charcot-marie-tooth (cmt) disease. we describe a family harboring a frameshift mutation (c. dela / p.asp thrfster ) in the p gene, predicted to result in a nonfunctional p truncated very early in the extracellular domain. this offered the rare opportunity to assess the consequences p deficiency in absence of the potential gain-of-function effects of the mutations itself. conventional conduction studies and multiple measures of nerve excitability by "threshold tracking" were carried out in heterozygote parents (aged and ) and their two homozygote sons (aged and ). in the homozygous patients, all distal limb cmaps and snaps were absent. for neurophysiological assessment, the spinal accessory nerve was stimulated at the neck and cmap was recorded over the upper trapezius muscle. eight normal subjects, mean age , were used as control. the two sibs showed a severe phenotype with early onset, severe scoliosis, complete loss of distal movements and relevant proximal weakness, cmt examination score (cmtes) - / ; both heterozygous parents had very mild adult-onset neuropathy with cmtes < / . control subjects had a trapezius cmap with a latency of . ms and an amplitude of . mv. heterozygotes had a mild cmt type b phenotype, with a cmap latency of . ms and an amplitude of . mv whereas the homozygotes had a severe neuropathy with a cmap latency of . ms and an amplitude of . mv. consistently, the homozygotes had a more severe impairment in excitability with a rheobase of . ma as compared to . ma in the heterozygote and . ma in controls. deviations in excitability measures were similar to our previous reports in p +/− and p −/− mice. mathematical modeling, indicated both altered passive cable properties due to dysmyelination and depolarizing features with increased na+ currents. our data suggest that p deficiency is associated with impaired axonal na+ channel function, arguing for the translational value of na+ channel blocker treatments as found in p null mouse models. krishnarajah s , divino v , mallick r , dekoven m . csl behring, king of prussia, pa, usa; quintilesims, fairfax, va, usa. chronic inflammatory demyelinating polyneuropathy (cidp) is a rare neurological disorder of the peripheral nervous system. the objectives of this retrospective real-world study were to compare demographic and clinical characteristics among cidp cases and matched controls and to assess cidp treatment utilization. adults newly diagnosed with cidp between / / and / / were identified in the quintilesims pharmetrics plus health plan claims database (first diagnosis date termed the index date). eligibility requirements were: confirmation of cidp (second cidp diagnosis or initiation of cidp therapy) within year of initial diagnosis, continuous health plan enrollment in the months prior to diagnosis (the pre-index) and the years following diagnosis (the follow-up), and no cidp diagnosis or use of cidp therapy in the pre-index. a total of , cidp cases met the study eligibility criteria. cases were direct-matched to controls based on age, gender, region, health plan, and payer type at index, and pre-index charlson comorbidity index score. the final sample consisted of cases matched to controls (both: mean [sd] age . [ . ]; . % male; . % commercially-insured). alternative pre-index diagnoses among cases included inherited neuropathies ( . %) and chronic acquired polyneuropathies ( . %). in the pre-index, neuropathic pain ( . % vs. . %), back pain ( . % vs. . %), and use of opioids ( . % vs. . %) and anti-convulsants ( . % vs. . %) were significantly higher among cases compared to controls (p< . for all). median total pre-index healthcare costs were . x higher for cases than controls ($ , vs. $ , , p< . ). over the follow-up, median total healthcare costs were . x higher for cases than controls ($ , vs. $ , , p< . [mean $ , and $ , ] ). cidp-related therapy costs accounted for . % of total healthcare costs for cases. the majority of cases ( . %) initiated cidp therapy over the follow-up, in a mean of . ( . ) days from initial diagnosis. half ( . %) of cases initiated treatment with corticosteroids only, while . % initiated ivig only. over the follow-up, . % of cases used any corticosteroid, while . % used any ivig. our findings suggest a substantial clinical and economic burden of cidp compared to matched controls. corticosteroids and ivig were most commonly used to treat cidp. kronlage m , baeumer p, pitarokoili k , schwarz d , schwehr v , godel t , heiland s , gold r , bendszus m , yoon ms . department of neuroradiology, heidelberg university hospital, germany; department of neurology, st. josef hospital, ruhr university of bochum, germany. objective: to evaluate large coverage magnetic resonance neurography (mrn) in chronic inflammatory demyelinating polyneuropathy (cidp). methods: in this prospective study patients with cidp and healthy controls were examined by a standardized mrn protocol at tesla. lumbosacral plexus was imaged by a t -weighted d-sequence ( mm isotropic voxel size); peripheral nerves of the upper and lower extremity by axial t -weighted turbo-spin-echo sequences ( . x . mm in-plane resolution). lesions were characterized by nerve cross sectional area (csa) and t -weighted signal (nt ). additionally, t -relaxometry of the sciatic nerve was performed using a multi-spin-echo sequence. all patients received a complementary electrophysiological exam. results: patients with cidp exhibited increased nerve csa and nt compared to controls (p < . ) in a proximally predominating pattern. roc analysis revealed best diagnostic accuracy for csa of the lumbosacral plexus (auc = . ) and nt of the sciatic nerve (auc = . ). csa correlated with multiple electrophysiological parameters of demyelinating neuropathy (f-wave latency, nerve conduction velocity) of sciatic and median nerve, while nt only correlated with f-wave latency of sciatic and not median nerve. t -relaxometry indicated that mr-signal increase in cidp was due to increase in proton-spin-density (p < . ), and not increase in t -relaxation time. conclusion: both nt and csa might aid in diagnosis of cidp, but csa correlates more robustly with electrophysiological parameters. since best diagnostic accuracy was shown for proximal nerve locations, mrn may be a useful complementary tool in select cidp cases. kühnemund j , wetzel c , bégay v , moshourab r , lewin gr . mdc & bih, berlin, germany; mdc, berlin, germany; charité, berlin, germany. damage of peripheral sensory nerves due to diabetes, herpes zoster infection, chemotherapy or trauma can cause chronic neuropathic pain. common symptoms include increased pain sensation (hyperalgesia), touch-induced pain (allodynia), paresthesia and spontaneous pain. we currently have poor understanding about the underlying molecular mechanisms at the peripheral level and treatment of patients suffering from neuropathic pain is inadequate. recent meta-analysis studies show that common first-line medications only yield nnts (numbers needed to treat) between . to . . it is still unclear to what extent allodynia can be attributed to changes in the physiological properties of intact sensory afferents. in this study, we aimed to elucidate whether changes occur in intact sensory afferents that innervate the plantar skin of the hind-paw following induction of neuropathic symptoms. this question was of particular interest considering that blocking mechanotransduction in the skin can alleviate mechanical hypersensitivity in neuropathic pain models (wetzel et al nature neuroscience ( ): - ). we used the chronic constriction injury (cci) model in mice which is behaviourally characterized by robust mechanical hypersensitivity. we made electrophysiological recordings from primary afferent neurons which had intact axons passing through the constriction to innervate the plantar skin using an ex-vivo skin-nerve preparation. we were able to record from myelinated afferent fibers and unmyelinated c-fibers with receptive fields in the control uninjured plantar skin as well as plantar skin of cci mice. we will present evidence that changes in the mechanosensitivity of sensory fibers innervating the glabrous skin may contribute to the symptoms of neuropathic pain. neuropathology, national hospital for neurology and neurosurgery, london, uk; irccs foundation "carlo besta" neurological institute, milan, italy; neurogenetics unit, national hospital for neurology and neurosurgery, london, uk; nuffield department of clinical neurosciences, oxford, uk; department of clinical neurophysiology, norfolk and norwich university hospital, uk. hsn secondary to sptlc / is a rare slowly progressive neuropathy resulting in marked sensory loss, especially nociception and significant motor deficit. despite most of the patients having the same c w mutation in sptlc , there is marked heterogeneity in the phenotype. l-serine oral supplementation has been suggested as potential therapeutic candidate however the lack of outcome measures is a major limiting factor in the initiation of a clinical trial. we undertook a natural history study to identify outcome measures that are responsive enough to be used in a clinical trial. the assessments used were cmt neuropathy score (cmtns version and cmtns version rasch modified), mri of calves and thighs, computerised myometry, quantitative sensory testing (qst), comprehensive neurophysiological assessment, proximal thigh skin biopsy for intra-epidermal nerve fibre density (ienfd), plasma dsl levels and patient based questionnaires (neuropathic pain symptom inventory and sf- v ). standardised response mean, srm (mean change/standard deviation of change) was used to compare responsiveness between tests. patients were recruited: with sptlc (c w) and with sptlc mutations. when analysed as a whole cohort, proximal calf mri fat fractions showed the most significant change over months. ienfd, plasma dsl levels, npsi and sf- v showed minimal change or the change was not in the clinically expected direction. for subsets of the remaining assessments which showed the highest responsiveness, the cohort was sub-divided into mild-moderate (cmtns ≤ ) and severe (cmtns> ) subgroups. in the mild-moderate subgroup, the greatest improvement in responsiveness was seen in computerised myometry (ankle plantarflexion: srm=− . and ankle eversion: srm=− . ). in the severe subgroup, qst (vibration detection thresholds on hands: srm=− . and face: srm=− . and pressure pain threshold on the face: srm= . ) and proximal calf mri fat fractions (srm range= . - . ) showed the greatest improvement. focusing on subgroups classified according to disease severity improved the responsiveness of some tests into the highly responsive range with mri still being the best outcome measure. this will reduce the number of participants required to power a clinical trial and might be a possible solution for designing a clinical trial for a rare, slowly progressive disease with a heterogeneous phenotype. kugathasan u , clark aj , suriyanarayanan s , laurá m , wilson e , , kalmar b , greensmith l , , hornemann t , reilly mm * , bennett dlh * . hsn secondary to sptlc / mutation is a slowly progressive sensory motor neuropathy leading to profound sensory loss with variable but often severe motor deficit. the genes sptlc and encode for the essential enzyme serine palmitoyltransferase (spt) which catalyses the rate limiting step in the sphingolipid de-novo biosynthesis. mutations in these two genes alter the substrate specificity of spt leading to the synthesis and accumulation of atypical metabolites called -deoxysphingolipids ( -deoxysl). plasma levels of -deoxysl are raised in hsn patients. deoxysphingolipids have been shown to be toxic in avian and by our group, in mammalian primary drg and motor neuron cultures. firstly, this study looked at the effects of -deoxysl on the survival and neurite integrity of human ipsc derived sensory neurons following exposure to different concentrations of deoxysphingolipids. later, we determined if there was autonomous -deoxysl production in hsn patient ipsc derived sensory neurons. sensory neurons were differentiated from human ipscs using a combination of small molecular inhibitors. deoxysphingolipids were found to be neurotoxic in this model after hours of treatment. in control lines, there is a significant reduction in neuronal survival following treatment with both -deoxysphinganine ( -deoxysa) and -deoxymethylsphinganine ( -doxmethsa). a clear dose-dependent increase in the expression of the axonal injury marker, atf , is seen with both -deoxysphngoid bases. in both instances, -deoxysa is more neurotoxic than -doxmethsa, which is similar to the findings in avian and mammalian primary neuronal cultures. autonomous -deoxysl production is seen in ipsc derived neurons obtained from three different hsn patients with the levels being significantly greater than that seen in multiple control lines. this is the first study to demonstrate that human ipsc derived sensory neurons can be used as an in-vitro model for hsn , providing a great opportunity both to probe the pathomechanisms mediating deoxysphingolipid toxicity and to test potential therapeutic agents. recent studies have demonstrated an association between autoantibodies directed against antineurofascin- (anti-nfasc ) and a subpopulation of cidp patients characterized by sensory ataxia, tremor and poor response to ivig treatment. here we report on the clinical features of three patients who developed acute changes in their phenotype during the course of their neuropathy, a potential clue to recognize a neuropathy with predominantly humoral dysimmunity. case reports: our index patient had developed sensory changes over weeks, followed by irregular locomotion, and muscle weakness with general areflexia. a first run of ivig improved the patient in a week, but he relapsed within days. a second ivig course with prednisone again normalized deficits within days. ivig runs and rituximab were still necessary to treat a nd , and then a rd relapse before obtaining complete improvement. the course of the neuropathy was months. two other patients were encountered with more chronic courses but in whom periods of worsening or improvement suggested a relapsing-remitting neuropathy, either spontaneously or following immunomodulating treatments. in all three, extensive work-ups were negative, anti-nfasc igg were positive, and detailed repeat nerve conduction studies demonstrated fluctuating conduction blocks. discussion: our report underscores that in chronic cidp patients a relapsing-remitting course could be encountered as a key feature of anti-nfasc neuropathy. this not yet described characteristic course could be of value when deciding using rituximab instead of immunomodulating treatments. kusunoki s , morikawa m , kuwahara m , ueno r , samukawa m , hamada y . faculty of medicine, kindai university, osaka-sayama, japan. anti-glycolipid antibodies are often detected in sera from patients with autoimmune neuropathies, such as guillain-barré syndrome (gbs), chronic inflammatory demyelinating polyradiculoneuropathy (cidp), and multifocal motor neuropathy (mmn). not only individual glycolipid antigens but also mixtures of two different glycolipids (glycolipid complexes) are sometimes recognized by serum antibodies. to investigate antibody activities against large number of glycolipid complexes in serum samples from patients with gbs, mmn, and cidp, we examined igm and igg antibodies against glycolipids [gm , gm , gd a, gd b, gq b, galnac-gd a, lm , galactocerebroside (gal-c), asialo-gm (ga ), and sulfatide] and glycolipid complexes consisting of two different glycolipids listed above, by using combinatorial glycoarray. serum was obtained from patients with gbs, patients with cidp and patients with mmn, all in the acute or relapsing phase. serum was also obtained from healthy controls and patients with other neurological diseases. we investigated the relationships between the clinical features and presence of those antibodies. high titers of igg antibodies were detected almost exclusively in gbs patients. in contrast, igm antibodies were frequently present in mmn and gbs. among the anti-glycolipid complex antibodies in gbs, anti-gm /sulfatide, anti-ga /sulfatide, anti-gm /gd a, and anti-gq b/sulfatide igg antibodies were common ( , , , and patients, respectively). igg antibodies against antigens containing gm were significantly correlated with pure motor gbs (p < . ) and those against antigens containing gq b were significantly correlated with gbs with ophthalmoplegia (p < . ). in seven of the patients with anti-gq b/sulfatide complex antibodies, the antibodies were specific to the gq b/sulfatide complex rather than the individual gq b and suldfatide antigens. moreover, four patients did not have antibodies other than those to the anti-gq b/sulfatide complex. in patients with mmn, igm antibodies to antigens containing gm or galnac-gd a were present in % and . %, respectively. glycoarray is efficient for detecting antibodies against numerous glycolipid complexes in immune-mediated neuropathies. we need further investigations on other immune-mediated diseases using larger number of antigens. kuwabara s , misawa s , sekiguchi y , susumu kusunoki and the jet-gbs study group . department of neurology, chiba university, chiba, japan; department of neurology, kindai university, osaka, japan; japanese eculizumab trial for guillain-barré syndrome, chiba, japan. guillain-barré syndrome is a monophasic immune-mediated neuropathy, but a substantial number of patients with severe disease have poor recovery, even if treated with immunoglobulin. recent studies suggest that complement activation plays a pivotal role in gbs-associated axonal degeneration, and eculizumab is a monoclonal antibody that specifically binds to complement component and inhibits complement activation. jet-gbs is an investigator-led, phase , randomized, placebo-controlled trial conducted in hospitals, this trial aims to investigate the safety and efficacy of eculizumab for treatment of severe gbs. patients were randomly assigned ( : ) to treatment with immunoglobulin plus either eculizumab ( mg/day; n= ) or placebo (n= ) once weekly for weeks. the primary outcome measures are safety and efficacy (the proportion of subjects who regain their ability to walk independently at week ). the secondary outcome measures included the proportion of subjects who were able to walk independently at week , and other measures such as mrc sum scores, nerve conduction parameters. enrollment for the trial began in august , and follow-up of the last patient was completed in october . analyses will be made in april , and the results will be presented at this meeting. this trial is registered with clinicaltrials.gov identifier: nct , and funded by the japanese agency for medical research and development, and alexion pharmaceuticals inc. neurofascin , a paranodal protein in peripheral nerve, is a target antigen for autoantibodies in a subset of chronic inflammatory demyelinating polyneuropathy (cidp). anti-neurofascin antibody-positive cidp is characterized by onset at younger age, tremor, and refractoriness to ivig. we have treated four patients with anti-neurofascin antibody-positive cidp at kindai university hospital. anti-neurofascin antibody was detected by both elisa and cell-based assay in those patients. igg subclass was predominantly igg in all four cases. the median age of the patients at admission was . years [range: - years]. among the four patients, three had tremor, and two had severe cerebellar ataxia. cerebrospinal fluid protein levels were remarkably increased [median: . mg/dl, range: - mg/dl]. although ivig treatment was administered in all four patients, the responses were poor or partial. in contrast, plasma exchange (pe) was performed in all four patients and the clinical symptoms dramatically improved in two of them. corticosteroids were also effective in those two patients. sural nerve biopsy was performed in all four patients. although sensory nerve action potentials of the sural nerves from those patients were not evoked, the transverse semithin sections of sural nerves from three patients revealed only slight or mild loss of myelinated fibers. partial paranodal demyelination was observed in teased-nerve fibers from three patients. in addition, abnormal paranodal lesions such as loss of the transvers bands were observed by electron microscope in all four patients. those electron microscopic findings were not observed in control patients with anti-neurofascin antibody-negative cidp. anti-neurofascin igg antibody-positive cidp shows distinctive clinical and pathological features. labeyrie c , besson f , vandendries c , cauquil c , beaudonnet g , not a , durand e , adams d . neurologie adulte, chu bicêtre, le kremlin bicêtre, france; médecine nucléaire, chu bicêtre, le kremlin bicêtre, france; clinique bizet, paris, france; unité de neurophysiologie clinique, chu bicêtre, le kremlin bicêtre, france. study of the proximal portion of the peripheral nervous system (pns) is difficult because less accessible to electrophysiological exploration and biopsy. the indications of mri are increasing in proximal neuropathies to analyse morphology of the roots, plexus and proximal nerves: integrity of the nerve bundle, inflammation or infiltration of these structures. pet-computed tomography (pet-ct) can detect infiltration of the proximal segments of the pns, with hypermetabolism of roots, plexuses and large nerve trunks. pet-ct is also useful in detection of solid neoplasm which can infiltrates pns and primary nerve sheath tumors. however, spatial resolution of pet -ct scan is limited to explore the roots and plexuses for moderate hypermetabolisms. we believe that a mild hypermetabolism could be highlighted in inflammatory neuropathies or in mild tumor infiltrations. we performed a fusion of whole body pet (performed because of suspected neoplasia) and plexus mri (pet-mri) images in ten patients with a peripheral neuropathy for which we suspected proximal involvement. the pet-ct and mri were first read separately, then with merge of the images by a nuclear physician and a neuroradiologist. five out of ten patients presented with hypermetabolism of pns (hmpn+) on pet-mri: root (n= ), and/or dorsal root ganglia (n= ). median sul of lesions was . . among the hmpn+ patients: hypermetabolism was already apparent on pet-ct in cases, the merge invalidated abnormalities seen on pet-ct in patients and mri detected additional lesions, not visible on pet-ct, in one patient. all hmpn+ patients had hypersignal and or hypertrophy of pns on mri either diffuse (n= ), or multifocal (n= ). among hmpn-(no hypermetabolism) out of ( %) had abnormal mri ( multifocal and diffuse). gadolinium enhancement was found in all patients receiving gadolinium in the hmpn+ (n= ) and only in / patients in the hmpngroup. final diagnoses of hmpn+ patients were neurolymphoma in one ( %), and idiopathic cidp in the others ( %) with histological proof on nervous root biopsy in one of them. final diagnosis in hmpn-patients was cidp in out of and sequelae of neuropathy in relation to lymphoma without relapse for the last one. cidp was more disabling (onls≥ ) in hmpn+ than in hmpn-group % vs. %. pet-mri could be helpful to detect a proximal inflammation of the pns, and to plan further testing. further studies are needed to evaluate the prognostic value of hmpn+. lancaster e , li j , liem r , scherer ss . perelman school of medicine, university of pennsylvania, philadelphia, pa, usa; columbia university school of medicine, new york, ny, usa. heterozygous nefl n s/+ mutant mice are the first animal model of a charcot-marie-tooth disease e (cmt e). people with this mutation have a severe, early onset axonal neuropathy. axons in the mutant mice have reduced number of neurofilaments and decreased diameters. they also show early onset of tremor and abnormal hindlimb clasping behavior. we measured the compound action potentials (caps) from tails every weeks from the same cohort of nefl n s/+ mutant mice and their wt littermates from to weeks. even at the age of weeks, the amplitude of mutant caps was only ∼ % of wt caps ( ± . microv, n= vs ± . microv, n= ), and the caps stayed substantially smaller than those in wt mice for weeks ( ± . microv, n= vs ± . microv, n= ). the conduction velocity and the duration of caps in mutants were slower and wider compared to those of wt. separate cohorts of mice (n= mutants and n= wt) were sacrificed at different time points for analysis by light microscopy. in caudal nerve of mutant mice, the number of axons was significantly reduced compared to that of wt at weeks and, by weeks, it was reduced more than %. this model system may be useful for preclinical studies of treatments for cmt e since the animals show progressive neuropathy over weeks, which can be objectively measured electrophysiologically and anatomically. despite more than cmt genes identified today, at least % of cmt patients do not carry a mutation in any of these genes. progress in gene identification in recent years suggests that there are still many more cmt disease genes to be discovered; however, it has become rare to gather support from multiple large families that allow for conclusive linkage analysis. we have studied multiple extended dominant cmt families with linkage support for a gene at chromosome p . . originally a czech family yielded a two point lod score of . at this locus and a family from southern italy showed a lod score of . . whole exome sequencing of multiple family members identified missense mutations in the gene atpase na+/k+ transporting subunit alpha (atp a ). atp a has not been associated with human diseases thus far. in expression studies on teased fiber preparations we already confirmed predominant expression in the nodes of ranvier of peripheral nerves. through collaborative efforts in the inherited neuropathy consortium and beyond we identified five additional multigenerational families via exome or sanger sequencing resulting in a total of seven unique segregating missense changes: leu arg, ile thr, ala thr, asp phe, pro ala, pro thr and asp ala. five of these mutations fall into a remarkably narrow motif associated with the sodium binding structure of atp a , flanking the flexible hinge motif. functional studies in different model systems (mammalian cell lines, xenopus oocytes, patient fibroblast lines) are underway to determine whether a loss or a dominant gain-of-function represents the disease mechanism. taken together, we show strong support for a major new dominant cmt gene, atp a . this finding represents a new pathway and an attractive new target for therapy development in axonal cmt. laurá m , ramdharry g , , singh d , kozyra d , skorupinska m , reilly mm . mrc centre for neuromuscular diseases, ucl institute of neurology, london, uk; school of rehabilitation sciences, st george's university of london/kingston university, uk; royal national orthopaedic hospital, stanmore, uk. charcot-marie-tooth (cmt) disease is the most common inherited peripheral neuropathy. foot deformities are frequent complications and orthopaedic surgery is often required. however there are no evidence based guidelines on the type or timing of the surgery. only few studies have described the long-term results of surgical procedures and evidence regarding optimal surgical management of these patients is lacking. we prospectively studied surgical management of cmt patients attending our centre. we collected data and assessed cmt patients before and after surgery. data included: history of ankle instability, pain, skin condition, details of physiotherapy and orthotic management, assessment of lower limb strength, charcot-marie-tooth examination score (cmtes), foot posture index, ankle dorsiflexion range of movement and specific questionnaires (foot index and manchester-oxford foot questionnaire, modified fatigue severity scale and modified falls efficacy scale), details of surgical procedures. patients were assessed yearly after surgery. so far patients ( males and females, age range - ) have been evaluated prior to surgery. all patients but one had genetically confirmed cmt ( cmt a, cmtx, cmt a). patients have been assessed after year, patients after years, patients after years and patient after years from surgery. a wide range of surgical procedures were performed by one dedicated orthopaedic surgeon. preliminary results showed reduction of number of falls in / ( %) patients and improvement of callosities in / ( %) patients at year follow up. there was also significant improvement of alignment of the operated foot (p= . ) and pain (p= . ). there were no significant changes in measures of strength and ankle range of motion. further analysis on a larger number of patients will be important to determine the long-term outcome of surgery. data acquired from this study will help develop orthopaedic intervention guidelines and identify areas for further research. lavigne-moreira c , oliveira mf , marques vd , onofre ptbn , dos santos acj , nascimento ojm , barreira a , marques w jr . division of neuromuscular diseases and neurogenetics, department of neurosciences and behaviour sciences, clinical hospital of ribeirão preto, university of são paulo, ribeirão preto, brazil; department of neurology, fluminense federal university, rio de janeiro, brazil. fap associated to ttr mutations is defined as a length-dependent axonal sensory and motor polyneuropathy that at early stages affects mainly the small nerve fibers, associated to autonomic and thermo-algesic sensations. the expected emg pattern was that of an axonal sensory and motor polyneuropathy, but in fact several unexpected patterns may be found. in this study we present the results found in a brazilian population with ttr mutation. patients were divided in three groups: ttr-met of early onset, ttr-met of late onset and ttr non-met . in the first group (ttr-met of early onset), ( . %) examinations were normal, ( . %) were axonal, were demyelinating fulfilling pidc criteria, one suggested a predominately motor polyneuropathy and the final one presented a lower motor neuron disease pattern. in the second group (ttr-met of late onset), ( . %) had an axonal pattern, ( . %) had an intermediate cv, ( . %) had a demyelinating pattern, had a lumbossacral pattern and the final one had no definite pattern. among the non-ttr met , two patients had the ttr -asp tyr, one presented an axonal pattern, while the second presented initially an axonal pattern, that changed to a demyelinating pattern in the second examination. patients with ile val mutation presented an axonal neuropathy associated to cts. most patients with demyelinating or intermediate pattern were treated with corticosteroids or ivig, with no satisfactory results. this small series of patients shows clearly that fap-ttr is associated to several emg patterns in addition to the expected sensory and motor axonal polyneuropathy. this variability is present in the same family and in the same patient in different occasions. clinicians should be alert to these possibilities to do not delay diagnosis and treatment. identifying the mechanisms involved in this variability could improve our knowledge of this intriguing disease. lavin tm . greater manchester neurosciences centre, salford royal hospital, manchester, uk. subcutaneous immunoglobulin (scig) has evidence from small trials for its use following intravenous igg (ivig) loading in chronic inflammatory demyelinating polyneuropathy (cidp). potential advantages of scig include stable igg levels and reduced complication rates. scig de novo can be considered as igg levels gradually rise with subcutaneous delivery and maybe beneficial for patients for whom complications are a concern. we present cases of cidp who were initiated on subcutaneous immunoglobulin g ( g/kg/month, % scig, hizentra, csl behring) following corticosteroids; without intravenous igg loading. patient is year old with a history of ihd with a progressive lower limb sensory changes and sensory ataxia over years. neurophysiology confirmed sensory demyelinating changes. he did not respond to corticosteroids. concerns were raised about the potential risk orf adverse cardiac events with iv immunoglobulin. scig therapy was introduced at g weekly. initial therapy has been well tolerated and objective measures (rods-cidp score, jamar grip strength, hole peg) have remained stable through initiation, and at months. patient is yr old male with a past history of branch retinal vein thrombosis. he developed a sensorimotor polyneuropathy over years with neurophysiology fitting efns criteria for cidp. given concerns regarding previous thrombosis, a trial of scig was given at g/week, with resolution of sensory ataxia and improvement in objective markers (jamar grip strength, rods-cidp score, hole peg) at months. unfortunately an adverse event occurred of an urticarial skin reaction. patient is a yr old male who presented with motor predominant cidp. there was no response to corticosteroids but improvement following plasma exchange. due to a past history of transient ischaemic attack, scig was started. there has been a positive response after months with improvement in walking distances and stable objective markers (rods-cidp score, grip and pinch strength). our cohort remained neurologically stable during initiation of scig, with adverse reaction. our experience would support further trials in this area regarding the efficacy of scig compared to ivig in both the short and long term. diabetic peripheral neuropathy is the most common and debilitating complication of diabetes and it is associated to neuropathic pain and non-traumatic amputations. despite the economic burden and human costs, nowadays there is not a specific treatment to cure diabetic peripheral neuropathy. hyperglycaemia and dyslipidemia-mediated oxidative and endoplasmic reticulum stress has been linked to diabetic peripheral neuropathy, among other altered pathways. in order to study in more detail the molecular mechanisms that lead to the development of diabetic peripheral neuropathy we set up in vitro models of the disease using nsc- (motoneurons) and med . (sensory neurons) cell lines or primary cultures of dorsal root ganglia and motoneurons exposed to saturated fatty acids. it has been reported that palmitic acid is able to induce endoplasmic reticulum stress and oxidative stress in multiple cell types including schwann cells and myenteric neurons, resulting in a good model of dyslipidemia-mediated stress. here we found that palmitate induces upregulation of the molecular chaperone bip/grp and the ccaat-enhancer-binding protein homologous protein (chop) mrnas and a dose-dependent downregulation of the apoptosis marker bcl- in primary cultures. moreover, palmitic acid induces loss of neuron dendrites and cell death at high doses and upregulation of heme oxygenase (ho- ) and chop at lower doses in the nsc- cell line. we are currently characterizing multiple molecules implicated in oxidative stress, endoplasmic reticulum stress and inflammation pathways in these cell types to find new therapeutic targets to treat type diabetic sensorimotor polyneuropathy, that will be subsequently validated in the db/db mouse model by pharmacological or gene therapy strategies. lee bjh , ohnmar o , wong j , koh sj , umapathi t . lee kong chian school of medicine, nanyang technological university, singapore; national neuroscience institute, singapore. treatment-induced neuropathy of diabetes mellitus (dm) (tind) is an acute painful peripheral neuropathy and autonomic dysfunction that occurs within weeks of tight glycaemic control. therapy with either insulin or oral hypoglycaemic agents (ohga) may result in tind. the quantum and rate of decline in hba c predicts development and severity of tind. both type i and ii dm patients are prone to this iatrogenic complication. besides the expected morbidity associated with painful somatic and autonomic neuropathy, tind patients also develop life-threatening eye complications such as maculopathy. with greater awareness, we picked up at least typical cases of tind in recent few months. however, there was a fourth possible tind case, whom we feel deserves special attention. the circumstances surrounding this case are common and may go under-recognised in acute hospitals. a year-old man with -year history of poorly controlled type ii dm was admitted with a partial left middle cerebral artery stroke. one month before admission, his hba c was . %. his hospitalization was prolonged because of his considerable disability that required rehabilitation. he stayed weeks. his blood sugar was difficult to control with hyperglycaemic episodes requiring ohgas and insulin. the highest capillary blood glucose recorded was mmol/l. he also had episodes of hypoglycaemia. three weeks into hospitalization, he developed severe orthostatic hypotension. he came to our attention two months later when he was sent for autonomic screening tests for severe postural hypotension, in spite of therapy with fludrocortisone and midodrine. his hba c was . %. he had marked orthostatic hypotension suggestive of sympathetic dysfunction. we were not able to discern if he had a painful neuropathy because of his aphasia. there was no recent ophthalmology review. we suggested a diagnosis of possible tind. he was discharged with lower doses of ohga. however, day after discharge, he attended the emergency department for a syncopal episode. this case raises intriguing questions on the safety of glucose control paradigms commonly employed in patients with acute stroke and myocardial infarction as well as the possible role of major fluctuations in blood glucose in the development of tind. lee hs , kim sm . presbyterian medical center, jeonju, korea; yonsei university college of medicine, seoul, korea. the occurrence of peripheral neuropathy by a tumor within or compressing a nerve in neurofibromatosis (nf) type and is relatively well known. however, nf presenting with demyelinating polyneuropathy unrelated to tumor masses is rarely reported. we report a rare presentation of an nf patient with demyelinating polyneuropathy of subacute onset. a -year-old woman was referred to our hospital with paresthesia on both limbs months before. she was healthy and had no family history. the symptoms worsened over the next months, tremor and weakness in both limbs occurred. although she treated as a chronic inflammatory demyelinating polyneuropathy (cidp) with intravenous steroid and immunosuppressant in another hospital, symptoms were getting worse. bifacial numbness and left eyeball pain occurred weeks ago and she visited our hospital. on neurologic examination, bilateral facial sensory hypoesthesia and symmetrical both limbs weakness (mrc grade iv+) were observed. decreased touch and pinprick sensation on both limbs were observed like stocking-glove distribution. the reflexes were sluggish. on nerve conduction study, sensorimotor demyelinating polyneuropathy with conduction blocks and temporal dispersions was observed. bilateral r , r responses were prolongation on facial blink test. mri of cervical spine and brain revealed contrast enhancing tumor-like enlargement of multiple nerve roots and cranial nerves. nerve biopsy was performed on right supraorbital nerve, neurofibromatous changes were observed. the symptom deterioration was stopped without treatment. gene test dose not revealed nf type and . on review of the literature, polyneuropathy in nf patient can result from tumor masses within the proximal nerve roots, or along the peripheral nerve, or in the extramedullary lesion affecting neighboring nerve roots. thus, axonal type polyneuropathy and focal amyotrophy have been reported in nf. demyelinating polyneuropathy has not been reported before in our knowledge. although the etiology of demyelinating polyneuropathy in nf requires further clarification, some authors claim that unknown local toxic or metabolic influences of the endoneurial pathological cells on adjacent nerve fibers. in this patient, atypical symptoms were observed in cidp such as cranial nerve involvement and stocking-glove sensory distribution. in this case, a nerve biopsy can be helpful the accurate diagnosis. lee jy , yoo jh , kang dk , bae js . department of neurology hallym university, seoul, korea. acute disseminated encephalomyelitis (adem) is an uncommon post-infectious inflammatory demyelinating disorder of central nerve system, while guillain-barre syndrome (gbs) is a prototype of acute post-infectious peripheral neuropathy. previous reports regarding the coexistence of these relatively rare diseases suggest that certain immunogenicity within central and peripheral nerves may share a common autoimmune process during the disease course. a previous healthy years old man was admitted because of fever, headache, nausea and myalgia in department of infectious disease. two weeks before admission, he suffered from watery diarrhea for days and spontaneously recovered. at initial presentation, he had high fever( ∘ c), headache and myalgia and intermittent horizontal diplopia. a few days after admission, he began to complain of drowsy mentality, bilateral extremities weakness, especially lower limbs, dysarthria, bilateral facial paralysis, urinary retention, dyspnea. on neurologic examination, he had mildly drowsy mentality, symmetric muscle weakness scoring of on bilateral hip, knee flexion and finger extension, but he had no sensory symptoms. he showed gazed evoked nystagmus with no extraocular muscle palsies. deep tendon reflexes were not present. pulmonary function test revealed a severe restrictive pattern. csf studies disclosed a dissociative increase of protein contents ( mg/dl) without pleocytosis. anti ganlioside antibody assay identified an anti-gt a igg positivity in his serum. nerve conduction study (ncs) showed prolonged motor terminal latencies and slow motor conduction velocity on multiple nerves. in contrast, sensory ncs revealed no abnormal findings. imaging studies unexpectedly revealed apparently symmetrical lesions across bilateral brainstem and basal ganglia suggesting a diagnosis of adem. after both ivig and high dose steroid treatment, he remarkably recovered from disturbed mental state and motor weakness. about month after the symptom onset, he could walk with assistant aid and discharge to other hospital for rehabilitation. our case suggest that certain component of autoimmunity simultaneously result in both cns and pns inflammation. specific immunological mechanism is remained to be elucidated. although we could not conclude whether cellular component or humoral component is dominant for our case, the presence of anti gt a antibody suggest a role of humoral mechanisms. the aim of this study is to evaluate whether peripheral neuropathies and headaches affect the same subgroups of patients with ibd. since , we have established a cohort study to evaluate the prevalence and incidence of neurological diseases in patients with ibd. over a period of years, all patients with ibd (either crohńs disease or ulcerative colitis) were invited to participate in a study designed to evaluate the risk factors for the presence of headaches and peripheral neuropathy in ibd. a separate group of control patients (age-matched relatives of ibd patients) was also formed. after a clinical interview and neurological examination, patients were invited to undergo skin wrinkling test (swt) to evaluate small fiber function and/or electromyography. headaches were present in . % of the patients with ibd, and were more common in patients with ulcerative colitis than in control patients (p< . ). migraine comprised . % of all cases of headache and was more prevalent in patients with crohńs disease than control patients (p< . ). tensional headaches were also common affecting . % of the ibd patients. electromyography was abnormal in . % of the ibd patients tested ( / ). swt was abnormal in . % of the ibd patients tested ( / ). . % of the ibd patients had abnormal swt but had no neuropathy symptoms. patients with abnormal swt or emg were not more likely to have headaches (p= . and . , respectively). overall, patients with symptomatic polyneuropathy were not more likely to have headache (p= . ). patients with abnormal swt or emg were also not more likely to have migraine (p= . and . , respectively). patients with abnormal swt or emg were also not more likely to have tension-type headache (p= . and . , respectively). in summary, although highly prevalent in this population of brazilian ibd patients, primary headaches and neuropathy do not affect the same subgroups of ibd patients. further studies are necessary to understand the mechanisms of both conditions in ibd patients. leitao amf , , araújo df , marques h , pamplona l , , souza mh , , nbraga ll , , gondim faa , . the aim of this study is to evaluate whether peripheral neuropathies and headaches affect the same subgroups of patients with ibd. since , we have established a cohort study to evaluate the prevalence and incidence of neurological diseases in patients with ibd. over a period of years, all patients with ibd (either crohńs disease or ulcerative colitis) were invited to participate in a study designed to evaluate the risk factors for the presence of headaches and peripheral neuropathy in ibd. a separate group of control patients (age-matched relatives of ibd patients) was also formed. after a clinical interview and neurological examination, patients were invited to undergo skin wrinkling test (swt) to evaluate small fiber function and/or electromyography. headaches were present in . % of the patients with ibd, and were more common in patients with ulcerative colitis than in control patients (p< . ). migraine comprised . % of all cases of headache and was more prevalent in patients with crohńs disease than control patients (p< . ). tensional headaches were also common affecting . % of the ibd patients. electromyography was abnormal in . % of the ibd patients tested ( / ). swt was abnormal in . % of the ibd patients tested ( / ). . % of the ibd patients had abnormal swt but had no neuropathy symptoms. patients with abnormal swt or emg were not more likely to have headaches (p= . and . , respectively). overall, patients with symptomatic polyneuropathy were not more likely to have headache (p= . ). patients with abnormal swt or emg were also not more likely to have migraine (p= . and . , respectively). patients with abnormal swt or emg were also not more likely to have tension-type headache (p= . and . , respectively). in summary, although highly prevalent in this population of brazilian ibd patients, primary headaches and neuropathy do not affect the same subgroups of ibd patients. further studies are necessary to understand the mechanisms of both conditions in ibd patients. the classic guillain barré syndrome (gbs) is characterized by motor weakness, hyporreflexia, but limited sensory deficits. sensory variants involving either small or large fibers or both are unusual and represent a diagnostic challenge. we described patients presenting with the sensory variant of gbs and retrospectively analyzed the clinical and electrophysiological findings of patients fulfilling the criteria for sensory gbs according to oh et al. criteria. six patients were identified (mean age years: range - years). four had a previous infection. they all consulted due to distal painful paresthesias and allodynia. on examination the patients presented normal strength and normal cranial nerves through the course of the disease with reduced knee and ankle reflexes in patients. distal hyperesthesia to pinprick was identified in and one of them additionally had hyperhidrosis and constipation. two additional patients presented hypoesthesia to pinprick and temperature. one patient had distal proprioceptive sensory loss with sensory ataxia. csf albumin cytological dissociation was present in patients. nerve conduction studies (ncs) identified a sensory motor demyelinating neuropathy in patients. among the with normal ncs, had abnormal cold and warm threshold in their qst evaluation. all patients received symptomatic treatment for the neuropathic pain and only two ivig therapies. longstanding pain, fatigue or both were persistent findings in patients after a mean follow up of months. in conclusion the sensory variant of gbs is both an infrequent presentation and a diagnostic challenge. longstanding pain and fatigue are common persisting findings. the epidemic of zika virus (zikv) throughout the americas and asia, and the subsequent rise in reported cases of guillain-barré syndrome (gbs) caused worldwide concern. as of january , countries have reported evidence of mosquito-borne zikv transmission and in of these countries, a sudden increase of gbs has been reported. moreover, case studies and a case-control study further indicate that zikv may trigger gbs. however, accurate diagnosis of both zikv and gbs in many of these studies is disputed, and a comprehensive description of the clinical phenotype of gbs related to zikv is lacking. the international gbs outcome study (igos) is a prospective observational study on the factors determining the onset, clinical course and outcome of gbs. at present, a research consortium of centers from countries has included patients in igos. our aim is to investigate zikv-related gbs in igos as is already occurring in colombia. in igos-zika, data on clinical features and ancillary investigations will be collected in zikv endemic areas according to the igos protocol with some modifications. first, igos-zika has a case-controlled study design to define the association between gbs and zikv and other arboviruses. second, urine samples will be collected and additional questions on preceding events will be asked, focusing on arbovirus infections. third, a more limited follow-up is required. our aim is to recruit additional centers via the inflammatory neuropathy consortium (inc) and centers in all arbovirus endemic regions that are willing to participate. the focus of igos-zika will be on the accuracy of the diagnosis of both gbs and zikv and on defining the associated clinical phenotype, course and outcome. igos-zika provides the opportunity to combine data and biobanks from various geographical regions using a standardized protocol and to compare these data with data and biosamples already collected in igos. studying these cases will help to optimize diagnostics and care for gbs patients in arbovirus endemic countries and provides a unique opportunity to further understand the pathogenesis of gbs. moreover, this study design and network can be used to adequately respond to other future viral epidemics related to gbs. lerat j , cintas p , dzugan h , , magdelaine c , , sturtz f , , lia as , . service de biochimie et génétique moléculaire -chu de limoges, limoges, france; service de neurologie et d'explorations fonctionnelles -chu de toulouse, toulouse, france; ea -université de limoges, limoges, france. pharc syndrome is an autosomal recessive neurodegenerative pathology leading to demyelinating polyneuropathy, hearing loss, cerebellar ataxia, retinis pigmentosa and early-onset cataract. these various symptoms can occur at different ages, so that pharc syndrome can be a differential diagnosis of charcot-marie-tooth disease (cmt) associated with deafness. only abhd mutations have been reported in patients. we described the th mutation and compared our results to the literature data. we analysed by next generation sequencing (ngs) strategy using a targeted cmt and associated neuropathies -gene panel the dna of a -year old male who has suffered from demyelinating sensory and motor polyneuropathy and ataxia since the age of . bilateral sensorineural deafness was diagnosed at the age of five. bilateral congenital cataracts were operated on at the age of . a new large complex homozygous mutation, with one deletion of seven base pairs and one insertion of base pairs, was detected. by analyzing our patient data and those of the literature, we evaluated that, in pharc syndrome, sensorineural deafness always occurs as the first feature in late teens. the ophthalmological symptoms are cataracts that occur at a mean age of yo and then retinis pigmentosa at a mean age of . demyelinating sensory-motor polyneuropathy is the most variable characteristics, which occurs in the thirties. we report the first large complex homozygous mutation in pharc syndrome, which is certainly under-diagnosed. therefore, it seems interesting to include abhd in the panels of the five symptoms, especially deafness ones. chronic inflammatory demyelinating polyradiculoneuropathy (cidp) is a chronic disabling disease that often improves with immune therapy. to date, the most reliable diagnostic criteria for cidp are the efns/pns revised criteria, with a reported sensitivity of % and specificity of %. we implemented a web-based database to collect data from patients with cidp followed by italian centers with expertise on cidp to determine the frequency and characteristic of cidp and it variants, the diagnostic criteria used for their diagnosis, the possible evolution into typical cidp, the association with specific anti-nerve antibodies, and their response to therapy. all the patients were evaluated at the time of inclusion and will be followed for two years to monitor their outcome and response to therapy. by february we included patients with cidp and variants ( men, women), aged - years (median ) with a mean disease duration of . years (range . - years). based on clinical symptoms, cidp was defined as typical in % and atypical in %. the diagnosis of typical cidp fulfilled efns/pns criteria in % of the patients while nerve conduction studies were not diagnostic in % (grouped as clinical cidp) or not available in %. we analyzed the frequency of supportive criteria for the diagnosis of cidp in patients with clinical cidp and found that increased csf proteins, demyelination or cell infiltration on nerve biopsy and imaging abnormalities consisting with cidp on us or nmr were present in %, % and % of the patients, respectively. a relapsing course was present in % of patients with clinical cidp, increasing the reliability of the diagnosis for cidp. in addition an improvement after one or more therapies was reported by % of the patients, with a positive response to ivig in %, steroids in % and plasma exchange in %, similarly to what observed in patients fulfilling efns/pns criteria. in % of the patients with clinical cidp two or more supplementary criteria for cidp were present. this study on a large population of patients is providing useful information that may help to revise the current diagnostic criteria for cidp. li j , cannell m , suragani r , pearsall r , kumar r . acceleron pharma inc, cambridge, usa. charcot-marie-tooth (cmt) is the most common hereditary peripheral neuropathy and is characterized by demyelination and/or axonal damage of peripheral nerves and muscle weakness. foot drop, steppage gait, and foot deformities are a typically seen in cmt patients. consequently, falls are commonly reported in these patients. improvement of dorsiflexor muscle function to prevent falls may improve quality of life and activities of daily living in patients with cmt. ace- , a locally-acting ligand trap that binds growth and differentiation factors (gdfs) and activins, has previously been shown to increase muscle mass and force in both duchene muscle dystrophy (dmd) and amyotrophic lateral sclerosis (als) mouse models. in the current study, we evaluated the therapeutic effects of ace- to improve muscle strength in the trembler (tr-j) mouse model of cmt a. these mice harbor a mutation in the peripheral myelin protein (pmp ) known to cause cmt a. seven-month old (b .d -pmp tr-j /j) mice were administered ace- ( g, twice weekly) intramuscularly to one of the unilaterally tibialis anterior (ta) muscle for weeks. the contractility of the ta muscle was evaluated during isometric contraction. all data were compared to the uninjected contralateral control hind-limb. after weeks of ace- treatment, ta muscle mass was increased by % (p< . ) and its physiological cross-sectional area was increased by % (p< . ). the increase in muscle mass correlated with an increase in strength, with maximum tetanic force and twitch force improved by % (p< . ) and % (p< . ), respectively. in addition, temporal properties during isometric contraction, such as maximum rate of contraction and relaxation, were accelerated by % and % respectively (p< . ) in the ace- -treated ta muscle compared to its contralateral hind-limb. pathological and biochemical assessment of ace- -treated mice showed enlarged myocyte area (+ %, p< . ) and reduced atrogin- mrna expression (− %, p< . ). together, these results demonstrated that ace- attenuates the degree of muscle atrophy and also improves muscle function in a mouse model of cmt a. the current study provides proof of concept for the use of ace- as a therapy for cmt to improve dorsiflexor muscle function and alleviate foot drop. we have generated a rat model of charcot-marie-tooth disease a (cmt a) harboring the p.arg trp mfn mutation, whose human counterpart results in a severe, early-onset axonal neuropathy. the mutation was made using zinc finger nuclease-mediated genome editing in fertilized rat eggs. a large cohort of mutant and wt littermates were characterized behaviorally and found to develop multiple motor deficits that worsened over time. nerve conductions of the tail (caudal nerve) was performed on a separate cohort of mutant (n= ) and wt littermates (n= ) every weeks from to weeks. mutant rats showed a progressively decline in the amplitude of the compound action potential after weeks, whereas the amplitude progressively increased in their wt littermates. separate cohorts of rats were sacrificed at , , and weeks and analyzed by light microscopy. in mutant rats, there was a reduced density of myelinated axons and active axonal degeneration in distal but not proximal nerves, and in the fasciculus gracilis of the cervical spinal cord at and weeks. these findings were not present in the -week-old cohort of mutant rats, or in wt rats at or weeks. a genetically authentic animal model of cmt a that develops a progressive, length-dependent axonal neuropathy will be a valuable tool for examining the pathogenesis and treatment of cmt a. lindborg ja , niemi, jp , defrancesco a , zigmond re . case western reserve university, cleveland, usa. traditionally the role of immune cells in nerve degeneration and regeneration has focused on the infiltration of inflammatory monocytes into the distal nerve after nerve injury and the phagocytosis by the resulting macrophages of myelin and axonal debris, thereby clearing a path for regenerating axons. therefore, it was surprising when we discovered that in ccr knockout (ko) animals, in which the entry of these inflammatory monocytes does not occur, that wallerian degeneration precedes normally. we now report that the reason for this is that neutrophils and schwann cells compensate for the decrease in macrophage accumulation. furthermore, nearly complete depletion of circulating neutrophils by systemic injection of an antibody to ly g leads to an inhibition of myelin clearance both in ccr ko and in wild type animals. on the other hand, we have demonstrated a second site of macrophage accumulation in wild type animals, namely around axotomized sensory neurons in dorsal root ganglia (drgs). blockade of that accumulation, for example as occurs in ccr ko animals, leads to a dramatic impairment of nerve regeneration. to examine the relationship between macrophages and regeneration further the monocyte chemokine ccl was overexpressed in drg neurons in intact animals by viral infection using an aav containing the ccl coding sequence. the resulting overexpression of ccl led to the accumulation of macrophages in drgs even though no injury had taken place and subsequently to an increase in the intrinsic growth capacity of the sensory neurons. examination of changes in gene expression in the drgs in these animals revealed increased expression of the cytokine leukemia inhibitory factor and an increase in its downstream signaling pathway that involves the phosphorylation and nuclear translocation of stat . strikingly, pharmacological blockade of stat activation inhibited the increase in the neurons' growth capacity produced by the virus. these results reveal unexpected interactions between immune cells and neurons facilitating nerve degeneration and regeneration and could lead to therapies to improve regeneration after injury or in disease. lin j , , qiao k , , huang j , , zhao cb , , lu jh , . institute of neurology, fudan university, shanghai, china; department of neurology, huashan hospital, fudan university, shanghai, china. we used terminal latency index (tli) as a tool in differentiation between poems syndrome and chronic inflammatory demyelinating polyradiculoneuropathy (cidp). comparison of median and ulnar nerve conduction studies including motor conduction velocity (mcv), distal motor latency (dml) and terminal latency index (tli) were studied in poems patients, matched cidp patients and normal controls. in this cohort, the average age at evaluation was . ± . years old in poems group and that of cidp patients was . ± . years old. except the ulnar terminal latency index in cidp group, poems and cidp patients demonstrated prolonged distal latencies, low conduction velocities and increased terminal latency indexes compared with the normal group. reduced conduction velocities and higher terminal latency indexes in poems group than in cidp group was found. increased tli was demonstrated in . %(median nerve) and . %(ulnar nerve) poems and that in cidp patients was . %(median nerve) and . %(ulnar nerve). decreased tli was found in . %(median) and . %(ulnar) cidp patients and none in poems. temporal dispersion (td) and conduction block (cb) were more often seen in cidp patients with increased tli than that in poems. compared with cidp and poems showed greater slowing of the intermediate nerve segments and relatively more uniform demyelination. about % cidp demonstrated more distal conduction slowing and more td and cb especially in those with increased tli. terminal latency index combined with td and cb may be helpful in differentiating poems from cidp. lin y , sung j , chang t , jowy t . department of neurology, taipei municipal wanfang hospital, taipei, taiwan. the purpose of our study is to exam whether electrophysiology changes could be detected in prediabetes patients and to discover the possible mechanism of nerve injury in prediabetes stage. we analysis and compare the nerve excitability test data between prediabetic patients and age-matched normal control subjects. prediabetes is defined by american diabetes association (ada) as one of the three following: hba c . % to . %, fasting glucose mg/dl to mg/dl, and hour oral glucose tolerance test to mg/dl. patients with radiculopathy, myelopathy, entrapment neuropathy such as carpel tunnel syndrome, and polyneuropathy were excluded. the strength-duration time constant (sdtc) and superexcitability showed significant difference (p< . ) between two groups. we also find increased threshold electrotonus in depolarization (ted) and reduced relative refractory period (rrp) and refractoriness in . msec. these early changes in prediabetic patient are similar in nerve excitability feature of diabetic patients. however, the above changes are not found in motor axonal excitability test. our data supports that nerve excitability test may be a useful, non-invasive, and less time dependent tool to detect peripheral nerve injury in prediabetic stage. the sensory axons are more vulnerable than motor axons. superexcitability is the most sensitive parameter in prediabetes. transthyretin-related familial amyloid polyneuropathy (ttr-fap) is an autosomal dominant disorder caused by mutations of ttr gene and is associated with variable penetrance. ttr-fap is rare, except for endemic areas. this is a retrospective study of ttr-fap patients diagnosed at our center between - . we identified four families with different ttr mutations. in one family with v a mutation, nine family members over four generations diagnosed with ttr-fap was followed since . in the other families the index cases with different mutations were identified between - . affected family members with v a mutation developed severe progressive polyneuropathy with cachexia, with onset of the disease between ages and . three patients presented with marked visual symptoms (one patient underwent vitrectomy). nine patients died to years after disease onset. two patients (sisters) underwent liver transplantation -one died after years of disease at age , second is years old and wheelchair-bound as her symptoms continue to progress. the ttr mutations diagnosed in the index cases of three other families are: d v, f l and v m. they all presented with similar clinical picture of late-onset ttr-fap with predominant progressive axonal sensory, motor and autonomic polyneuropathy. all three index cases were men, the onset of symptoms was between - years with numbness and paresthesia in the feet followed by weakness and autonomic dysfunction. all had excessive weight loss resulting in cachexia and were diagnosed with cardiomyopathy. no patient suffered from visual symptoms. all three patients progressed to stage ii of ttr-fap -walking with assistance. time to diagnosis was . - years. due to advanced stages of their disease these patients were not suitable for therapy with tafamidis or the liver transplantation. the ttr d v (p.d v) was confirmed as a de novo mutation, which is uncommon in ttr-fap. in the remaining families carriers of ttr mutations were identified and are followed up. pedigree analysis of the family with f l mutation revealed affected members who with high probability died from ttr-fap. our study suggests that patients with ttr-fap in poland exhibits clinical and genetic heterogeneity. liu x , fan d . department of neurology, peking university third hospital, beijing, china. objective: to identify the gene mutation of chinese charcot-marie-tooth pedigrees and investigate the correlation among the clinical manifestation, electrophysiology and mechanism of different genotype. methods: we included pedigrees with cmt enrolled in our hospital from january, to december . we recorded clinical features, cmtns and electrophysiological data at diagnosis. the patients underwent mutation analysis of pmp , cx , mpz, mfn , hspb , hspb using mlpa, dhplc and sanger gene sequencing. results: we found pmp duplication pedigrees ( . %), cx pedigrees ( . %), mfn pedigrees ( . %), mpz pedigrees ( . %) conclusions: in chinese han population, the proportion of pmp duplication is relatively low, the majority of clinical manifestation is classical cmt. axonal cmt can show isolated lower extremity injury, with central nervous system involvement. hmn may be an underestimated clinical types, the identification should be done with caution in differential diagnosis. liu y , liu b , sebastian b , wozniak km , wu y , slusher b , polydefkis m . johns hopkins school of medicine, baltimore, usa. chemotherapy-induced peripheral neuropathy (cipn) is a common dose-limiting toxicity in the treatment of many cancers. most cipn studies preferentially focus on sensory fiber loss and dysfunction. here, we compared the structural and functional recovery of autonomic fibers in sweat glands (sweat gland nerve fiber density, sgnfd) and sensory fibers (intra-epidermal fiber density, ienfd) in mouse footpads after exposure to a maximum tolerated dose (mtd) of several common chemotherapy agents. additionally, we assessed footpad sweat production as a functional correlate to sgnfd reductions. female balb-c mice ( -animals/group) were treated with a mtd of four anti-tubulin drugs: paclitaxel (pca, mg/kg), ixabepilone (ixa, mg/kg), eribuline (erib, . mg/kg), vinoelbine (vino, mg/kg), or corresponding placebo given intravenously, mwf for two weeks. recovery was assessed at -hours, , , , , and weeks following the last dose. footpads were processed to visualize epidermal nerve fibers using pgp . and autonomic nerve fibers with tyrosine hydroxylate and pgp . . ixabepilone-treated mice experienced significant reductions in sgnfd at hrs, while ienfd nadir occurred at a later time point, -weeks. the recovery to baseline levels occurred more quickly for ienfd ( -weeks) than sgnfd ( -weeks). in contrast, vinorelbine and eribuline treated mice experienced a maximum deficit in sgnfd and ienfd at hrs and sgnfd recovery was slower ( -weeks) compared to ienfd ( -weeks). pca-treated animals showed more severe ienfd and sgnfd deficits compared to the other agents with both ienfd and sgnfd not recovering completely until -months. reductions in th-sgnfd were comparable or more pronounced to decreases in pgp . -sgnfd for all agents and timepoints. pca-treated animals demonstrated reductions in footpad sweat droplet number thereby providing a functional correlate. together, these data indicate that in mouse models of cipn, autonomic nerve fibers are affected more severely than sensory nerve fibers, and also recover more slowly than intraepidermal nerve fibers. autonomic dysfunction may be an important and under-appreciated consequence of chemotherapy exposure. the pi -kinase vps (pik c ) synthesizes phosphatidylinositol -phosphate (pi p), a lipid critical for both endosomal membrane traffic and macroautophagy. human genetics have implicated pi p dysregulation, and endosomal trafficking in general, as a recurring cause of demyelinating charcot-marie-tooth (cmt) peripheral neuropathy. here, we investigated the role of vps , and pi p, in mouse schwann cells by selectively deleting vps in this cell type. vps -schwann cell knockout (vps scko ) mice show severe hypomyelination in peripheral nerves. vps −/− schwann cells interact abnormally with axons, and there is a delay in radial sorting, a process by which large axons are selected for myelination. upon reaching the promyelinating stage, vps −/− schwann cells are significantly impaired in the elaboration of myelin. nerves from vps scko mice contain elevated levels of the lc and p proteins, indicating impaired autophagy. however, in the light of recent demonstrations that autophagy is dispensable for myelination, it is unlikely that hypomyelination in vps scko mice is caused by impaired autophagy. endosomal membrane traffic is also disturbed in vps −/− schwann cells. we investigated the activation of the erbb / receptor tyrosine kinases in vps scko nerves, as these proteins, which play essential roles in schwann cell myelination, are known to traffic though endosomes. in vps scko nerves, erbb was hyperphosphorylated on a tyrosine known to be phosphorylated in response to nrg exposure. the overall level of erbb was also decreased during myelination. our findings suggest that the loss of vps alters the trafficking of erbb / through endosomes. abnormal erbb / signaling may contribute to the hypomyelination observed in vps scko mice. lombardi r , devaux j , cortese a , dacci p , benedetti l , demichelis c , lauria g . irccs foundation "carlo besta" neurological institute, milan, italy; aix-marseille université, marseille, france; irccs c. mondino national neurological institute, pavia, italy; university of genoa and irccs aou san martino-ist, genoa, italy. the recent identification of igg anti-neurofascin (nfascin) antibodies in a group of patients has widened the spectrum of presentation for chronic inflammatory demyelinating polyradiculoneuropathy (cidp). these patients can be distinguished by disabling tremor, poor response to intravenous immunoglobulin and distal and sensory disturbances. cell-adhesion molecule nfasc and cntn are expressed on the paranodal junction (pnj) of nodes of ranvier, and play key roles on sodium channel clustering and glia-axon interactions. quantification of unmyelinated intraepidermal nerve fibers (ienf) is a useful parameter employed in small nerve fiber pathology diagnosis. in addition the immunohistochemistry evaluation of dermal nerve fibers allows to examine morphological changes of myelin sheath and ranvier nodes structure. we performed immunofluorescent colocalization studies using antibodies to visualize axons (protein-gene-product . , neurofilament, tubulin), sheath of myelin (myelin-basic-protein) and specifically nodal/paranodal/juxtaparanodal structures (pannfascin, nfascin , nfascin , caspr, cntn , potassium and sodium channels) in skin tissues from seronegative and seropositive cidp patients. we analyzed axon and myelin sheath damage, abnormal nodal-paranodal-juxtaparanodal architecture and morphometric parameter as internodal length of ranvier nodes. our results on skin biopsies from three igg nfasc -positive cidp patients revealed complete loss of nfasc staining at the paranodes, asymmetrical paranodes and widening of the nodes of dermal myelinated nerve fibers. one igg cntn -positive cidp patient showed abnormal nodal/paranodal immunostaining with different features as compared with igg nfasc -positive patients suggesting specific changes. however, such alterations were not found in four seronegative cidp patients. our data support the hypothesis that examining specific axonal and myelin markers could provide diagnostic and prognostic clues on nodo-paranodopathies. the goal of this study is attempt a possible correlation between the presence of serum autoantibodies and structural changes in nodal/paranodal regions of dermal nerve fibers in cidp patients. knowledge of autoantibodies expression in the peripheral myelinated nerves of cidp patients could serve for stratifying patients and potentially guiding personalized treatments. lunati a , lerat j , dzugan h , , rego m , magdelaine c , , bieth e , calvas p , cintas p , gilbert-dussardier b , goizet c , journel h , magy l , toutain a , urtizberea j , sturtz f , , lia as , . charcot-marie-tooth disease is one of the most frequent inherited peripheral neuropathies ( / ). so far, mutations in more than genes have been identified causing either the demyelinating form (type ) or the axonal form (type ). duplication of pmp gene is the most frequent cause of autosomal dominant demyelinating form. autosomal recessive demyelinating form is often due to sh tc gene mutations. patients suffer then from early severe neuropathy starting in the first decade. scoliosis and deafness are often observed. we analysed patients suffering from peripheral neuropathy, by multiplex-ligation-dependant-probe-amplification (mlpa), followed by targeted next-generation-sequencing (ngs) using a -gene custom panel designed for the diagnosis of charcot-marie-tooth and associated neuropathies. mutations of interest were verified by sanger sequencing. diagnosis was positive for patients. as expected, the most frequent mutation was the pmp duplication detected in patients. deletion of pmp was observed in patients and pathogenic point mutations were detected in patients. sh tc gene appeared to be the most frequently mutated with nine patients diagnosed. associated with known mutations, four new mutations have been identified: two nonsense mutations and two missense mutations. all these patients presented deafness and/or scoliosis. sh tc appears to be an important gene involved in charcot-marie-tooth disease, often associated with deafness and /or scoliosis. it is important to pay attention to these associated symptoms in charcot-marie-tooth patients in order to guide their diagnosis and to improve their medical care. lupo v , , frasquet m , , sánchez-monteagudo a , , barreiro m , alberti ma , casasnovas c , quintáns b , , , camacho a , domínguez c , sedano mj , pelayo al , pardo j , sobrino t , sobrido mj , , , sevilla t , , espinós c , . mme (membrane metalloendopeptidase) mutations, inherited in an autosomal recessive fashion, have been recently identified in japanese probands (higuchi et al. ) . thus, mme has been included in the list of cmt genes as a new autosomal recessive axonal form, cmt t (mim ), and moreover, it is considered a strong candidate for the genetic diagnosis of unsolved late-onset cmt cases. in fact, few months later auer-grumbach et al. ( ) reported european probands with autosomal dominant late-onset cmt and mutations in mme. we have investigated a clinical series of patients diagnosed of motor or sensory-motor peripheral neuropathy using an updated version of our custom gene panel, which includes mme. in this study, we report probands with cmt , intermediate cmt or dhmn/cmt and homozygous or compound heterozygous mutations in mme, and proband with cmt and a heterozygous mutation in mme. we have identified different type of mutations: novel splice donor variant, frameshift, nonsense and missense mutations. the two nonsense changes, p.trp * and p.arg *, and the splice donor variant c. + g>a, were detected in homozygous or compound heterozygous state in patients, while the frameshift mutation, p.pro leufs* , was detected in homozygous or heterozygous in the remaining patients. strikingly, the two nonsense and the frameshift mutations had been previously reported as causative for autosomal-dominant cmt t (auer-grumbach et al. ) . out of three missense mutations, one is novel (p.his tyr), and two are reported in control databases (p.asn lys and p.arg trp). this study shows that the autosomal recessive cmt t is common in spanish population, and moreover, it suggests that screening of mme using gene panel testing could help to improve diagnosis of unclarified inherited peripheral neuropathies cases. funds: isciii (pi / , pi / ); fundació per amor a l' art. inherited peripheral neuropathies (ipns) encompass a group of disorders highly heterogeneous, clinically and genetically. charcot-marie-tooth (cmt) disease is closely related to distal hereditary motor neuropathy (dhmn) or distal spinal muscular atrophy (dsma), and some patients show additional signs associated with amyotrophic lateral sclerosis (als). targeted gene panel and exome sequencing are considered to be powerful and cost-effective tools for diagnosis of these disorders. we have investigated a clinical series of patients diagnosed of motor or sensory-motor peripheral neuropathy: families were investigated by exome sequencing and, cases were tested using different updated versions of a gene panel . each version comprises , or ipn genes, respectively and it shows a high coverage performance: percentage of analyzable target base with > coverage was , %. both exome and gene panel capture libraries were based on sureselect capture technologies (agilent technologies), and sequencing was performed in miseq or hiseq illumina equipment. we have identified novel genes and novel mutations in known genes, broadening the phenotypical spectrum associated with ipns. exome sequencing has allowed us to identify causative gene in % of familiar or sporadic cases: morc , aars, bscl , kif a, gars, egr , fig , dnajb , drp , ighmbp , dao, sod , fig . gene panel testing has been mostly performed in sporadic cases, and it has allowed us to identify either disease-causing or candidate mutations in % of cases: kif a and bicd were the most common genes mutated. update gene panels neuro and neuro have revealed novel mutations in genes recently associated to cmt and cmt disease: mme and pmp , respectively. in sum, both strategies have helped us to achieve a more accurate clinical and genetic reclassification of these disorders, an impossible challenge using conventional sequencing methods. our study expands the clinical phenotype previously associated to known ipn causing-gene, and emphasizes that gene panels should be considered as a first diagnosis method for unclarified ipn patients. funds: isciii (pi / , pi / , pi / ); fundació per amor a l' art. magy l , mathis s , goizet s , tazir m , vallat j-m . department and laboratory of neurology, national reference center for rare peripheral neuropathies, chu limoges, france; department of neurology, chu bordeaux, france; department of medical genetics, chu bordeaux, france; department of neurology, chu algiers, algeria. charcot-marie-tooth (cmt) disease is a hereditary neuropathy with a relatively homogeneous phenotype but is genetically heterogeneous. moreover, nerve conduction studies distinguish different forms, adding another level of complexity. the current classification of cmt being difficult to understand for physicians, scientists and patients, we presented and published a proposal for updating this classification, based on inheritance, nerve conduction findings and gene/mutation involved. inputs from colleagues prompted us to conduct a survey in order to try to reach some consensus about our proposals. we conducted an internet survey between october and december . the link to complete the survey was sent several times by email with an introduction to more than people. participants were contacted through the emailing list from the last cmt meeting in venice (september ) and additional physicians and scientists who are involved in cmt care and research were contacted as well. one hundred seven people from various countries (mainly france, italy and the usa) answered the survey. most ( %) of the participants were between and years of age, % being physicians and % being scientists. the vast majority ( %) considered the proposal constituted an improvement over the historical classification whereas % wanted to keep the old one. about the order of information, % of participants thought the mode of inheritance should come first, whereas % felt the phenotype should be placed at the beginning. ninety-one percent of people thought cmt should be kept as a generic name for hereditary sensory and motor neuropathy. for pure sensory neuropathy, % favoured hsn over hsan although % thought the opposite and % of participants felt dhmn should be kept for distal motor neuropathy. about nerve conduction findings, % of participants thought the intermediate phenotype has to be kept and % favoured our proposal to replace " " by "de" (for demyelinating) and " " by "ax" (for axonal). finally, % of responders thought that genetic information should be included in the classification of cmt. overall, our proposal of a new classification received a very good appreciation from physicians and scientists implicated in the care of patients with hereditary neuropathy. mallik r , hubsch a , gaida a , barnes d . csl behring, kop, usa; csl behring, bern, switzerland; csl behring, ottawa, canada. the risk of hemolytic events (hes) with intravenous immunoglobulin (ivig) therapy appears to be linked to the isoagglutinin (anti-a and anti-b) level of the specific ivig product. using published anti-a and anti-b titers for seven ivig products and corresponding he rates reported to the eudravigilance database, we developed a mathematical model to predict the risk of he to patients receiving ivig products of given anti-a and anti-b levels. modeling was performed separately for the risk to patients with blood groups a, b, ab and o and the overall population risk was estimated assuming a blood group distribution of % a, % b, % ab and % o. applying the prediction model, we calculated the he risk for an ivig product produced via a chromatographic process (privigen ® , csl behring) a) without any isoagglutinin reduction measures ( ) ( ) ( ) ( ) ( ) ( ) ( ) , b) with an anti-a donor screening program eliminating approximately % of donors with high anti-a titers ( ) ( ) ( ) , c) incorporating an anti-a/anti-b specific immunoaffinity chromatography (iac, igisolo tm ) step in the manufacturing process (since ) and d) with both measures (b and c) combined; as well as for an ivig product produced with a cohn-like cold ethanol fractionation process (carimune ® nf/sandoglobulin ® , csl behring). isoagglutinin titers in ivig products, measured by european pharmacopoeia direct assay, were provided by dr c bellac, swissmedic, bern, switzerland. the predicted risk was highest with the chromatographically purified ivig without isoagglutinin reduction ( . cases expected per kg ivig used). anti-a donor screening reduced the predicted risk to . cases/ kg. a greater risk reduction was predicted with the iac isoagglutinin reduction step ( . cases/ kg). the combination of both methods produced little benefit ( . cases/ kg) versus iac alone. the predicted hemolytic risk with ivig produced by cohn-like ethanol fractionation was low ( . cases/ kg). an observational cohort study to confirm these hemolytic risk reductions is in progress. at present, the observed hemolytic risk for anti-a donor screening appears consistent with the prediction calculated by the model; results for iac isoagglutinin reduction are expected in . the cmt infant scale (cmtinfs) is an outcome measure of functional ability for young infants and children aged < years. cmtinfs aligns with the cmt pediatric scale and cmt neuropathy score to measure disease severity across the lifespan. to measure gross motor and fine motor function, cmtinfs comprises of two subscales: gross motor (e.g. head control, crawling, walking, jumping and hopping) and fine motor function items (e.g. grasping, reaching, tearing paper and buttoning). overall and subscale-specific function is expressed as a z-score based on normative reference values (positive z-scores indicate poorer function). a total of controls aged - months (mean age , sd m) have been assessed across australia (n= ), thailand (n= ) and usa (n= ). total cmtinfs z-scores did not differ significantly between sites (australia vs thailand) (p= . ) or gender (p= . ). data collection is ongoing and infants aged < years are eligible for inclusion. to date, infants ( % male) aged - months (mean age , sd m) with a range of cmt subtypes ( cmt a, cmt d, cmt c, cmt x and unidentified gene) have been assessed with cmtinfs. mean total z-score for infants with cmt ( . , sd . , range: − . - . ) was significantly higher than controls ( . , sd . , range: − . - . , t=− . , p= . ). differences between affected infants and controls were larger in infants older than months. infants with cmt a (cmtinfs z-score . , sd . ) and cmt c (z-score . ) were less affected than cmtx (z-score . ) and cmt d (z-score . ). the gross motor function subscale differed significantly between cmt cases and controls ( . , sd . vs . , sd . ; p=. ) and a significant difference was also observed for the fine motor function subscale ( . , sd . vs . , sd . ; p= . ). reliability, factor and rasch analysis of the cmt-infs is underway to assess validity. initial results support the sensitivity of cmtinfs in distinguishing between infants with and without cmt. preliminary analyses also suggest the scale is sensitive to genetic subtype. with increased power, cmtinfs promises to become a useful outcome measure of disease severity and function in infants with cmt. manso c , querol l , mekaouche m , illa i , devaux j . aix-marseille université, marseille, france; universitat autónoma de barcelona, barcelona, spain. contactin- , contactin-associated-protein- (caspr ), and neurofascin- (nfasc ) are essential for the formation of paranodal axoglial junctions. igg autoantibodies to contactin- , caspr , and nfasc are associated with subsets of patients with chronic inflammatory demyelinating polyradiculoneuropathy (cidp) presenting with common clinical features. anti-contactin- igg autoantibodies have been shown to be pathogenic and to affect the paranodal axoglial junctions in vivo and in vitro. by contrast, the pathogenic effect of anti-nfasc igg have not been demonstrated. here, we purified anti-nfasc igg from cidp patients' plasma and investigated their effects after passive transfer. to determine whether these antibodies can pass the paranodal barrier, we performed intraneural injections of anti-nfasc igg autoantibody. by contrast to anti-contactin- igg , anti-nfasc did not penetrate the paranodal regions after intraneural injections, but bound to the surface of the schwann cell. to perform chronic exposure, lewis rats were implanted with intrathecal catheter and anti-nfasc igg were administrated in a daily manner during three weeks. igg to nfasc , but not control igg , induced progressive clinical deteriorations characterized by gait ataxia and hindlimb paraparesis. these deteriorations were associated with nerve activity loss in motor spinal nerves and with a selective loss of the paranodal specialization characterized by the disappearance of the caspr /contactin- /nfasc complex at paranodes. the passive transfer of anti-nfasc igg thus seem to induce similar pathogenic effects as the anti-contactin- igg . however, the pathogenic mechanisms leading to paranode disappearance appear different. our findings indicate that igg directed against nfasc are pathogenic and further show that these antibodies are reliable biomarkers of a specific subset of cidp patients. better fit the needs of the consortium and improve patient experience. these enhancements include creating mobile friendly webpages, updating enrollment form content, access to a customized dashboard, and the ability of registrants to explore their data in comparison to other registrants. we will review these enhancements in depth, and demonstrate their impact on the growth and development of the rdcrn inc contact registry. martinez c , hubsch a , watson dj , shebl a , wallenhorst c , simon tl . institute for epidemiology, statistics and informatics gmbh, frankfurt, germany; csl bering ag, bern, switzerland; csl behring llc, king of prussia, usa; csl bering gmbh, marburg, germany. hemolytic anemia (ha) is a complication of intravenous immunoglobulin (ivig) treatment, particularly in patients receiving high dose ivig for immune modulation, such as guillain-barré syndrome or chronic inflammatory demyelinating polyneuropathy. the primary mechanism for the increased risk is believed to be passive acquisition of anti-blood group a and b antibodies (isoagglutinins) from the ivig product. to reduce the quantity of isoagglutinins, an anti-a donor screening was implemented for the ivig privigen ® from - and donors with high titers were excluded from contribution to pooled plasma. anti-a donor screening was replaced since with an immunoaffinity chromatography step, which decreases isoagglutinins to a greater extent, but no data are available to test its clinical effectiveness. to test the effectiveness of the donor screening, two cohorts of patients treated with privigen ® before and after start of donor screening were identified from a hospital-based administrative database of us hospitals with in-and outpatient discharge diagnoses, procedures, drug utilization and laboratory tests between / and / (period ) and between / and / (period ). privigen ® dose per kg body weight was estimated from the daily quantity administered and age-and sex-specific us population body weight estimates. ha within days of privigen ® use was assessed from manual records review and the incidence rate of ha in the two periods compared. incidence rate ratios (irr) of ha were adjusted for sex, age, treatment setting, indication and dose per kg body weight using period as reference. the incidence rate of ha was . / , person-days ( % confidence interval: . - . ) in period ( has in , person-days) and . ( . - . ) in period ( has in , person-days). the adjusted irr was . ( . - . ). significantly less ha risk was found with high dose (≥ . g/kg body weight) privigen ® , irr . ( . - . , p= . ). we conclude that anti-a donor screening and exclusion of donors with high anti-a titers from plasma pools is associated with a decreased risk of ha with ivig. matsumoto a . department of neurology, keijinkai jozanki hospital, sapporo, japan. subacute myelo-optico neuropathy (smon) is the intoxication of clinoquinol with main clinical symptoms of paresthesia and spasticity of legs. these symptoms have been considered to be elicited by the disturbance of spinal cord and peripheral nerve as the intoxication of clinoquinol. however, as to the patients with smon who have been still living after the onset of disease, the examination of nerve conduction velocities are in normal ranges and the clinical symptoms of peripheral neuropathy are not observed now. in order to investigate whether the peripheral neuropathy were observed in the early stage of smon, we investigated the longtidunal changes of electrophysiological results in patients who could examine the nerve conduction studies from early stage of smon until the present time. as to the disturbance of pyramidal tract functions (myelopathy) in smon patients, the central motor conduction times were calculated by transmagnetic stimulation of motor cortex, cervical roots and lumbar roots. the peripheral nerve conduction velocities of sensory nerve were examined with the sural nerves. as the results, in patients with smon who could examine the electrophysiological examination from the early stages of smon until to years later, the central motor conduction times of leg muscles from motor cortex to lumbar roots were prolonged in the smon patients compared to the normal cases. these results suggest the presence of disturbances of conduction velocities of spinal cord. conduction velocities of sensory nerve velicities (sncvs) showed the delayed sncvs of sural nerves( - m/sec) at the first examination from the onset of - years. however from to years later, sncvs of these cases were covered to - m/sec. from these electrophysilogical examinations, it was suggested that the presenting main symptoms of smon were the disturbance of myelopathy, and the disturbance of peripheral nerve function had been recovered after onset of smon being elapsed a long time, natural killer (nk) cells are part of our innate immune system with regulatory and effector functions. they comprise the first line of defence in the recognition and destruction of virus-infected and pathologically altered cells. different studies suggest that the treatment with intravenous immunoglobulins (ivig) has an immunomodulatory effect on nk cells. ivig is a first-line treatment for various autoimmune diseases in particular in chronic inflammatory demyelinating polyneuropathy (cidp). the lack of a predictive marker for ivig responsiveness in cidp avoids the early preservation of non-responding patients. to better understand the effect of ivig in patients with cidp, we tested whether ivig treatment altered the nk cell status. additionally, we analysed if the alteration in the populations may serve as a surrogate marker in predicting the outcome of ivig treatment. using semi-quantitative pcr and flow cytometry in the peripheral blood of patients with cidp, we analysed the effects of ivig on the nk cell population before treatment initiation and h after first dose and correlated the changes with the reponsiveness to ivig. ivig administrations induced a reduction in the expression of several typical nk cell genes. interestingly, this ivig-induced reduction of nk cells was reversible four weeks after the ivig treatment. flow cytometry data revealed that ivig reduced the cytotoxic cd dim nk cell population, while regulatory cd bright nk cells remained almost unaffected or were even increased. interestingly, we found that the observed effects on nk cells almost exclusively occurred in cidp patients who responded to ivig therapy. correlation between the changes in the nk cell population and treatment efficiency suggests a crucial role for nk cells in the immunomodulatory mechanism of ivig. further studies are warranted to investigate whether the differences in the nk cell status of patients with cidp represent a reliable surrogate marker in predicting the outcome of ivig therapy. mccray b , sullivan j , woolums b , aisenberg w , lloyd t , sumner c . johns hopkins university, baltimore, usa. mutations in transient receptor potential vanilloid (trpv ), a calcium-permeable non-selective ion channel, cause charcot-marie-tooth disease type c (cmt c). trpv is unique in that it represents the only membrane-expressed ion channel in cmt and thus a potential therapeutic target. previous work has suggested that trpv mutations lead to gain of channel function and toxicity in cultured cells. neuropathy-causing mutations of trpv largely cluster in the cytosolic ankyrin repeat domain (ard) that is known to mediate protein-protein interactions, suggesting that pathogenesis may be related to disruption of such interactions. in order to identify trpv -interacting proteins, we performed two unbiased proteomics screens and identified multiple cytoskeletal-modifying proteins including syndapin- , a neuronal protein known to promote axonal outgrowth by influencing the actin cytoskeleton. in cultured cells, we have shown that trpv and syndapin- co-localize to highly dynamic actin-rich cellular processes and together stimulate robust neurite extension, but this facilitation of neuritogenesis is impaired by disease-causing mutations in trpv . we have also shown that over-expression of syndapin reduces trpv -mediated calcium influx in cultured cells and rescues toxicity of mutant trpv . in addition, syndapin over-expression suppresses mutant trpv phenotypes in a drosophila model of trpv -related neuropathy. further, we have demonstrated that treatment of drosophila with a specific trpv channel antagonist ameliorates trpv mutant toxicity. together, our data highlight the importance of trpv interaction with cytoskeletal proteins such as syndapin- in the pathogenesis of cmt c. specifically, our results suggest that mutations in trpv disrupt the normal role of trpv in regulation of cytoskeletal dynamics and that interactions with the cytoskeleton reciprocally modulate trpv channel function and influence toxicity of mutant trpv . mcgonigal r , yao d , barrie ja , crawford c , willison hj . university of glasgow, glasgow, uk. guillain-barré syndrome (gbs) is in part mediated by anti-gm ganglioside antibodies induced by preceding infections. anti-gm antibodies target plasma membrane gm that is extensively distributed in both glial and axonal membranes, particularly at the node of ranvier. antibodies deposited at this site in models of gbs are associated with complement deposition, conduction block, structural disruption of ion channels and macrophage infiltration. the wide distribution of the gm ganglioside target leads to unwanted complexity in ascribing pathological outcomes to injury of cell-specific membranes, in particular unravelling the consequence of paranodal schwann cell membrane injury on axonal function, and vice versa. to overcome this impasse, we have generated transgenic mice through glycosyltransferase manipulation that express gm exclusively in neurons or glia, thus allowing us to very specifically target and injure axonal or glial membranes with a single anti-gm ganglioside antibody. through this route we can create mouse models of both the axonal and demyelinating forms of gbs, induced by a single anti-gm antibody, thus creating otherwise highly comparable conditions. here, we show anti-gm antibody binding is restricted to the nodal axolemma in galnact −/− -tg(neuronal) mice and conversely to paranodal loops in galnact −/− -tg(glial) mice. when anti-gm antibody and a source of complement is added to a nerve-muscle ex vivo injury paradigm, there is a loss of axonal integrity (i.e. loss of neurofilament immunolabeling) when the neuronal membrane is targeted in galnact −/− -tg(neuronal). conversely, axonal integrity is maintained when the paranodal membranes are decorated by antibody and complement products ex vivo in galnact −/− -tg(glial) mice. in a passive immunisation model in vivo, galnact −/− -tg(neuronal) mice acutely develop weakness, respiratory dysfunction, associated complement deposition, and degenerative pathology in distal axons. in contrast, galnact −/− -tg(glial) mice have significantly fewer abnormalities under the same acute conditions. these data indicate the high vulnerability of axonal membranes to acute injury and underline the importance of developing specific axonal protection strategies. in summary, targeting the nodal axolemmal or glial membranes allows us to study associated nodal pathology, and determine the downstream consequences on function and axon fate, currently a major area in gbs clinical research. memon a , madani s , schultz l , grover k , arcila-londono x , sripathi n , ahmad bk . neuromuscular division, department of neurology, henry ford hospital, detroit, michigan, usa. objective: to differentiate sensory electrophysiology, tli and treatment response in patients with paraproteinemic cidp. background: low tli has been reported as a useful electrophysiological marker for mag-cidp. to our knowledge comparison of sensory electrophysiology and tli of paraproteinemic cidp subgroups have not been previously reported. methods: retrospective review(january -december ) of patients with cidp fulfilling electrophysiological criteria(aan ad hoc subcommittee and albers and colleagues).cidp patients with diabetes(n= ) were excluded. patients were divided into idiopathic (n= ) and paraproteinemic cidp(n= ). paraproteinemic cidp sub-groups: mag( ), non-mag( ) and igg( ) were compared to idiopathic cidp( ). these groups were compared for demographics, history of cancer, csf protein, sensory conductions, tli measurements and response to treatment using chi-square tests for binary and categorical variables and t-tests for continuous measures. results: there was a higher proportion of females in idiopathic-cidp compared to non-mag-cidp ( % vs %). idiopathic group having a higher proportion of patients on monotherapy( % vs %) and combination therapy( % vs %) compared to non-mag. higher mean csf protein compared to mag-cidp(p= . ) was seen in the idiopathic. the difference between idiopathic and igg-cidp was significant for overall rx response(p= . ) and rx response in patients with follow-up(p= . ). for both variables, patients in the idiopathic group had a higher proportion of patients on combination therapy and lower proportion of no treatment offered compared to patients in the igg-cidp. % of non-mag-cidp patients had a history of cancer vs % of mag-cidp. none of the other differences were significant. there were no group differences in sensory electrophysiology and tli. conclusions: sensory electrophysiology and tli may have no value in differentiating paraproteinemic cidp. csf protein is higher in idiopathic cidp compared to mag-cidp. idiopathic-cidp has a higher proportion of females compared to non-mag-cidp and a higher proportion of patients on combination therapy compared to igg-cidp. cancer screening should be considered in patients with non-mag-cidp. memon a , madani s , ahmad bk , schultz l , grover , arcila-londono x , sripathi n . department of neurology, henry ford hospital, detroit, michigan, usa. introduction: sensory electrophysiology and terminal latency index (tli) differences have been described in various cidp sub-groups. objective: evaluate electrophysiology, tli and treatment response in idiopathic and diabetic cidp. methods: retrospective review of patients with cidp who underwent electrodiagnostic evaluation (january -december . patients fulfilled electrophysiological criteria described by ad hoc subcommittee of american academy of neurology (aan) and albers et al. we excluded patients( ) with acute inflammatory demyelinating neuropathy, hereditary sensorimotor neuropathy, vasculitis and polyneuropathy with paraproteinemia. patients were divided into idiopathic( ) and diabetic( ) groups. these groups were compared for age, sex, history of cancer, csf protein, response to treatment, sensory response abnormalities and tli measurements using chi-square tests for binary and categorical variables and t-tests for continuous measures. all testing was at the alpha= . level. results: group differences for age, sex, history of cancer, csf protein and treatment response were not significant. comparing tli values in measurable responses, the difference between the two groups for tibial tli was significant (p= . ), with idiopathic group having a lower mean as compared to the diabetic. tli values differences for median, ulnar and peroneal nerves were not significant. the difference in abnormal rates of sensory responses was significant for the sural nerve with the idiopathic group having a lower rate compared to the diabetic group ( % vs %, p< . ). no differences were noted for the ulnar, median and radial nerves. tibial tli and sural sensory responses have some value in differentiating the two groups. larger prospective studies are needed to confirm our findings. ivig is an important treatment option for cidp. although recommended by treatment guidelines, little is known about the consequences of temporary ivig withdrawal to assess ongoing immunoglobulin need. path is a randomized, double-blind trial of the subcutaneous immunoglobulin (scig) igpro (hizentra ® , csl behring) in cidp. before scig randomization, subjects underwent periods of ivig withdrawal ( weeks or until pre-determined indications of clinical deterioration) and ivig restabilization (igpro ; privigen ® , csl behring). subjects not showing deterioration during withdrawal period were withdrawn from study. to proceed to scig randomization, subjects had to achieve "cidp stability" (no relevant change in incat score at last two restabilization visits and at least the same total score as at screening). subjects entered the ivig withdrawal period. ( %) of these qualified for igpro restabilization; subjects ( %) were not ivig dependent, and ( %) were withdrawn for other reasons. one subject withdrew consent before igpro dosing. at the end of the igpro restabilization period, cidp stability was achieved in % of subjects ( did not reach stability, were withdrawn for other reasons). post-study follow-up information was available for / subjects who did not reach stability: ( %) had improved to baseline clinical status and had not, meaning at least % of the subjects improved to their pre-study status. during the restabilization period, / subjects ( %) improved in at least one of the predefined outcome measures. on average, subjects improved by . points in incat total score, . points in i-rods centile score, kpa in mean grip strength (dominant hand), and . points in mrc sum score. improvement occurred with a median of days after the first igpro dose in one or more efficacy outcome measures and in % of cases after the third igpro maintenance infusion. headache and nasopharyngitis were the most frequently reported adverse events (aes) during restabilization. aes deemed causally related were mostly mild or moderate. no unexpected aes or laboratory or vital sign findings associated with igpro occurred during the study. in summary, igpro reversed neuromuscular disability and improved activity/participation after previous clinical deterioration during an ivig withdrawal period. pmp duplication is the most frequent cause of charcot-marie-tooth disease (cmt). since it discovery, more than genes have been identified to be potentially responsible for cmt disease. however only single nucleotide variations (snvs) or small indels have been described. this could be due to the new sequencing strategy (ngs), especially ngs by amplicon sequencing, for whose few convenient tools are available and easily usable to detect cnvs responsible for inherited disease. to overcome this problem, we designed "cov'cop", a user-friendly tool able to detect cnvs among amplicons sequencing data. using the run's coverage file provided by the sequencer, "cov'cop" simultaneously analyzes all the patients of the run using a two-stages algorithm containing correction and normalization levels and provides an easily understandable output, showing with various colors, potentially deleted and duplicated amplicons. we validated our method on several datasets, including those of our targeted ngs panel screening genes known to be involved in cmt and close pathologies. cov'cop detected easily pmp duplication and deletion in our patients, confirmed by mlpa. in addition, cov'cop permitted the detection of new cnvs different from the pmp duplication, in cmt patients. we confirmed these cnvs by quantitative pcr and cgh array. we present here one of these cnvs: the duplication of aars gene detected in cmt patients and we discuss the pathogenicity of this new cnv. additional cnvs responsible for cmt disease are probably still to be discovered and we believe that cov'cop will help molecular geneticists to rapidly identify them. poems (polyneuropathy, organomegaly, endocrinopathy, m-protein, and skin changes) syndrome is a rare cause of demyelinating neuropathy associated with plasma cell dyscraisia and vascular endotherial (vegf) overproduction. although number of therapeutic interventions for plasma cell dysorders have been applied to poems syndrome, there have been no randomized clinical trials. this phase / double blind, randomized, placebo comtrolled trial was performed to investigate the safety and efficacy of thalidomide for patients with poems syndrome who are not eligible for stem-cell transplantation. the primary endpoint was the reduction rate of serum vegf concentrations at weeks in intention to treat analysis. additional outcomes of long-term extension study included progression-free survival. twenty-five poems patients were randomly assigned to either thalidomide plus dexamethasone or placebo plus dexamethasone from nov , , to july , . one patient in the placebo group was excluded from analyses because of a protocol violation. the adjusted mean serum vegf reduction rate at weeks was . (sd, . ) in the thalidomide group compared with − . ( . ) in the placebo group (p= . ). the kaplan-meier rate of progression-free survival at months was . in the thalidomide group, as compared with . in the placebo group (hr, . ; % ci, . to . ). in the randomized study period, mild sinus bradycardia was more frequent in the thalidomide group than in the placebo group ( % vs %; p= . ). thalidomide suppresses serum vegf concentrations and lengthened pregression-free survival in poems patients who are ineligible for stem cell transplantation. although thalidomide treatment has a risk of bradycardia, the benefits would exceed the risk. this study is registered with the umin clinical trials registry, umin . montes-chinea ni , coutts m , vidal c , courel s , rebelo a , abreu l , zuchner s , saporta, ma , . department of neurology, university of miami, miami, usa; department of human genetics, university of miami, miami, usa. autosomal dominant mutations in synaptotagmin- (syt ), a synaptic vesicle protein that functions as a calcium sensor for neurotransmission, have been previously linked to presynaptic neuromuscular junction (nmj) dysfunction and motor neuropathy in two families. both pathogenic mutations (asp ala and pro leu) were located in the c b domain of syt , which is essential for neurotransmitter release at the nmjs. we report a family with a new missense mutation in the c b domain of syt and a similar phenotype characterized by a slowly progressive, predominantly motor neuropathy and evidence of presynaptic nmj dysfunction on nerve conduction studies. the index case is a year-old woman with gradually progressive weakness of her extremities. she had normal developmental milestones, but was found to have bilateral high arched feet and hammertoes and occasional falls around the age of . she gradually developed progressive leg weakness, worsening bilateral hand cramping, weak handgrip, and only mild paresthesias on distal extremities. family history is remarkable for similar symptoms reported by her maternal grandfather, two maternal uncles, her mother and a younger sister. her neurological exam revealed inability to walk on heels or toes, significant distal lower extremity weakness and absent ankle deep tendon reflexes. cranial nerve examination and coordination were normal and there were only non-specific sensory changes in the lower extremities. emg/ncs revealed normal sensory responses throughout; however, motor nerve evaluation demonstrated globally reduced amplitudes with a > % increment after brief isometric contraction. further electrophysiological evaluation with slow ( hz) repetitive nerve stimulation of the right ulnar motor nerve revealed a % decremental response in amplitude and a > % increase in amplitude immediately after a one-minute period of sustained muscle contraction, which rapidly extinguished after one minute. voltage-gated calcium channel (vgcc) antibodies and a chest ct were normal. targeted sanger sequencing revealed an ile lys mutation in syt , which is located in the c b domain and is predicted to impair protein function. syt -related neuropathy is a rare disease, but should be suspected in patients presenting with a combination of pre-synaptic nmj dysfunction (resembling lambert-eaton myasthenic syndrome) and a predominantly motor neuropathy, especially in the context of a positive family history. charcot-marie-tooth (cmt) disease affects roughly in , individuals and is described as an inherited peripheral neuropathy primarily affecting distal muscles. limited studies detail patient-reported impact of muscle weakness on functional activities. this anonymous survey was developed with input from clinical experts and patient interviews and aimed to better understand the prevalence and impact of various cmt clinical manifestations on patients' lives. the survey was administered online to the hereditary neuropathy foundation's (hnf) patient contact database and is ongoing through june . here we present preliminary data on patient characteristics and disease impact for cmt patients collected february - , . respondents were mostly female ( %) and mostly from the us ( %). median age (range) at symptom onset was years ( - years), at diagnosis was years ( - years), and at present was years ( - years). the sample was representative of all cmt types (cmt , , , ,and x). the most common physical and clinical manifestations of cmt were problems with balance ( %), ankle weakness/foot drop ( %), loss of feeling or abnormal sensation in the lower leg/foot ( %), and hand muscle weakness ( %). maintaining balance, walking long distances, and climbing up and down stairs were key challenges associated with ankle weakness/foot drop. foot drop was considered by % to be the primary factor contributing to falls, which averaged . falls to ground per month. of those with foot drop, % had bilateral weakness. a majority of respondents ( %) used some form of assistive device for mobility, including ankle-foot orthotics/below-the-knee leg braces ( %), canes/walking sticks ( %), and custom foot orthotics/inserts ( %). the most common drug therapy included pain and anti-inflammatory medications ( %). foot surgery was the most common surgical procedure received ( %) and toe surgery was the most common surgery considered ( %). key symptoms that affected quality-of-life "very much" included problems with balance ( %), ankle weakness (foot drop) ( %), and fatigue ( %). these data suggest a high prevalence of lower leg muscle weakness; therefore, therapies aimed at improving ankle weakness and the resulting foot drop and imbalance may be beneficial to patients' daily functioning and quality of life. morano m , , gambarotta g , ronchi g , , cillino m , fornasari be , , fregnan f , , tos p , cordova a , moschella f , geuna s , , raimondo s , . department of clinical and biological sciences, university of turin, orbassano (to), italy; neuroscience institute of the "cavalieri ottolenghi" foundation (nico), university of turin, orbassano (to), italy; plastic and reconstructive surgery. department of surgical, oncological and oral sciences, university of palermo, palermo, italy; hand microsurgery and surgery, gaetano pini hospital, milan, italy. nerve fiber regeneration and complete functional recovery after peripheral nerve injury do not always occur and can be influenced by many factors including patient age, gender, lesion site, injury severity, size of the gap between damaged nerve stumps and time interval that elapses before performing surgical repair. the poor outcome occurring after a long delay can be due to loss of the neuron ability to regenerate, loss of the schwann cell ability to support regeneration and, of course, progressive muscle atrophy. the aim of this study was to investigate the nerve regeneration after delayed repair and to study the degenerative processes of the denervated distal nerve stump and denervated muscle. in particular, the analyses were focused on the role of nrg/erbb system, that is expressed both in nerve and in muscle tissue, during degenerating and regenerating processes. functional recovery analysis performed after nerve repair showed that only the group repaired immediately and not the groups repaired with a delay of or months, recovered partially. nevertheless, morphological analyses demonstrated that, despite the delay, the nerve fibers are still able to regenerate, even if they are fewer and smaller than the immediate repaired group. moreover, the analysis of the nrg /erbb system showed a significant decrease of soluble nrg in both degenerating and delayed-repaired nerves. the poor outcome after delayed nerve regeneration might be explained by schwann cell impairment and the consequent ineffective support for nerve regeneration. as regards denervated muscle analysis, results showed that erbb receptors expression is related to the innervated state of the muscle, with an upregulation of erbb clearly associated with denervation state. interestingly, nrg isoforms are differently regulated depending on the type of nerve injury. future experiments will be needed to address the in vivo efficacy of different isoforms of nrg both in injured nerve and denervated muscle. we planned a multicenter, prospective, randomized, single blind, controlled study to evaluate the efficacy and safety of an innovative rehabilitation protocol based on the use of treadmill training in a cmt a population. the protocol required that subjects were blindly randomized into two treatment groups, spe (three months of respiratory, proprioceptive and stretching exercises) or trespe (the same treatment plus aerobic training at the treadmill). subjects were evaluated at baseline (t ), after three month of treatment (t ) and further three months of follow up free of therapy (t ). the full assessment included: -mwt (primary outcome measure), -mwt, walk- , short physical performance battery (sppb); lower limbs dynamometric strength evaluation; berg balance scale (bbs); cmt neuropathy score; medical outcomes study short form (sf ). a total of subjects (mean age of . ± . years) were recruited. at t we found a significant improvement in both groups in the -mwt (p< . ), -mwt (p< . ), bbs (p< . ) and sppb (p< . ), while at t only the -mwt was still significantly improved (p< . ) in the spe group. no significant differences between groups were observed for any of the outcome measures. performances on walk did not significantly change during follow-up (p= . ). concerning the sf , we did not observe consistent changes during follow-up or consistent differences comparing the two treatments. in conclusion, this multicenter, prospective, randomized, single blind, controlled study shows that the combination of respiratory, proprioceptive and stretching exercises has a positive impact on the performance of cmt patients, especially regarding walking tests. the aerobic exercise at the treadmill, is well tolerated but apparently does not add any further improvement to the conventional treatment. we speculate that the relatively low clinical severity of the patients, due to the selection criteria, may have prevented a positive effect of treadmill exercise. müller m , dohrn m , romanzetti s , reetz k , gess b . university rwth aachen, department of neurology, aachen, germany. muscle mri is increasingly used in neuromuscular patients to detect changes in muscle volume, muscle fat infiltration and edema. muscle mri are mostly analyzed by qualitative means as quantitative analysis is time-consuming and not well established. here, we developed a novel method for semi-automated segmentation of muscle mri data sets. based on axial t -weighted dixon mri stacks, muscle volumes were quantified by an adapted water-shed algorithm. muscle volumes of thighs and calves were determined separately and the ratio of thigh/calf was calculated. myopathy, neuropathy patients and healthy controls were included in the study. muscle volumes determined by semi-automated segmentation were very similar to manually segmented data sets, differences being < %. this was the case for patients as well as healthy controls. the time-saving effect of automated segmentation was very strong ( vs min. per patient). muscle volumes of the thigh and also of the calf of myopathy patients showed a highly significant difference (p< . ) compared to healthy subjects. in neuropathy patients there was a just significant difference (p< . ) of muscle volumes compared to healthy patients that did not sustain in bonferroni's multiple comparison test. the ratio of thigh/calf muscle volume was significantly different comparing patients with myopathy and neuropathy (p< . ). subgroup analyses of different groups of myopathy patients showed highly significant differences (p< . ) in myositis, limb-girdle-muscular dystrophy and metabolic myopathy, compared to healthy patients, but no significant differences in-between these groups. taken together, the data shows that automated segmentation of muscle mri allows for exact and fast quantification of muscle volumes in neuromuscular patients. higher patient numbers are necessary to test differences between specific disease groups. further studies should also address the possible use as a marker of disease progress for clinical studies or therapy monitoring. murakami t , nishimura h , nagai t , hemmi s , kutoku y , sunada y . department of neurology, kawasaki medical school, kurashiki, japan; department of pathology, kawasaki medical school, kurashiki, japan. familial amyloidotic polyneuropathy (fap) is an autosomal dominant hereditary systemic amyloidosis caused by mutation of transthyretin (ttr) gene, and usually shows sensory dominant polyneuropathy and autonomic neuropathy at the initial stage. the pathogenesis of neuropathy is not well understood, and explained by several mechanisms, including such as mechanical compression, vessel occlusion, ttr toxicity and schwann cell dysfunction. we describe a sporadic patient with late-onset fap due to ttr e k. she noticed dysesthesia first in the foot at age . the symptoms were slowly progressive, and abnormal sensations were extended up to the both upper arms and the both knees at age . distal muscle weakness and atrophy was also observed in the extremities. she noticed difficulties in walking and frequent diarrhea. echocardiogram revealed diffuse left ventricular hypertrophy, suggesting cardiac amyloidosis. amyloid deposits were not detected in the endoneurim or perineurium of the sural nerve years after the onset of the disease, but a marked loss of myelinaed and unmyelinated nerve fibers was observed in it. ttr-derived amyloid deposits were confirmed in the peroneous brevis muscle, salivary gland and heart tissue. dna analysis revealed the heterozygote mutation, p.e k (e k)/c. g>a, of ttr gene, and she was diagnosis as fap. these findings suggest that the proximal parts of peripheral nervous system might be strongly involved by ttr aggregates or amyloid fibrils. blood-nerve barrier in the distal part of peripheral nerves could be preserved until later in the patient. several biopsy sites other than nerve may be helpful and necessary for diagnosis of ttr amyloidosis in mild or late-onset fap as our case. amyloidgenic element (cae) encoded by the ' utr. this study also identified a de novo c. _ dup frameshift mutation predicting p.lys glnfs* in nefh from a cmt family with atypical clinical symptom of proximal dominant weakness. this mutation is located near the previously reported frameshift mutations, suggesting a mutational hot spot. these relatively frequent deletion/duplication events with this resign might be caused by the putative hairpin structure. patient's lower limb mri revealed a marked hyperintense signal changes in the hip muscles than those in the thigh or lower leg muscles. this study also observed an anticipation pattern of earlier onset ( yrs old for mother to yrs old for daughter) and more severe symptoms in later generation. therefore, this study suggests that the stop loss and translational elongations by the ' utr of the nefh mutations may be relatively a frequent genetic cause of axonal peripheral neuropathy with the specific characteristics of proximal dominant weakness and an anticipation pattern. neil j , haghi ashtiani b , choumet v , musset l , léger jm . department of immunology, pitié-salpêtrière hospital (ap-hp), paris, france; department of neurology, national referral center for rare neuromuscular diseases, pitié-salpêtrière hospital (ap-hp), paris, france; institut pasteur, emerging diseases epidemiology unit, paris, france. the myelin-associated glycoprotein (mag) is a transmembrane glycoprotein localized in periaxonal shwann cells and oligodendroglial membranes of myelin sheaths. mag contains a carbohydrate epitope (hnk- ) that is a target antigen in autoimmune peripheral neuropathy associated with monoclonal igm gammopathy. in these neuropathies, numerous studies report the absence of correlation between the titers of anti-mag antibodies and the disease course. anti-mag titers and igm level at diagnosis are not always associated with disease severity and there is not good correlation between pre-and post-treatment anti-mag titers in patients who respond clinically to immunomodulators. mag belongs to siglec- a family and the linkage of sialic acid to the underlying sugars is an important determinant of siglec binding. mag shows high affinity for alpha- , -linked sialic acid ( , -sa).moreover, human monoclonal igm possesses heavy chain glycosylation sites at asn , , , and with sialylated olidgosaccharides and high-mannose type oligosaccharides. igm may bind to mag via these glycan epitopes as an alternative and additional route of antigen binding other than through the fab v regions. this mag-glycans igm interaction may be clinically neutral but could lead to an overvaluation of the biological results. in this study, we analyzed sera from patients with igm reactivity against mag: of them had an anti-mag neuropathy with various degrees of severity, and the last one had igm monoclonal gammopathy, strong serum anti-mag reactivity but no neurological disease. igm were extracted and purified from these sera by affinity chromatography. for each batch, an aliquot was digested by jack bean alpha-mannosidase and anti-mag reactivity was performed by elisa and indirect immunofluorescence (iif), before and after demannosylation. these extracts, tested in an iso quantitative way with regard to the original serum, showed a decrease of activity (elisa) and intensity (iif) after demannosylation. furthermore, elisa anti-mag was carried out in sera from patients with igm monoclonal gammopathy without neurological impairment: of them ( . %) showed a significant biological response. taking into account the fact that anti-mag antibodies are pathogenic (in animals models), these results support the hypothesis of neutral intermolecular interactions between igm and mag. ng cjb , ng jph , tay lb, t , umapathi . yong loo lin school of medicine, national university of singapore, singapore; lee kong chian school of medicine, nanyang technological university, singapore; national neuroscience institute, singapore. recent developments have validated non-invasive means of measuring central arterial blood pressure (casp) and have shown that casp and peripheral blood pressure (pbp) are unidentical entities. patients with postural orthostatic tachycardia syndrome (pots) have marked tachycardia with no associated decrease in pbp. we asked if the reflex tachycardia, which corresponds to postural dizziness in these patients, could be a result of a decrease in casp. two male patients, and years of age, with clinical features typical of pots went through a complete battery of autonomic screening tests. the sympathetic and parasympathetic responses were normal other than a heart rate increase of and beats per minute, respectively, on standing. there was no significant decrease or increase in pbp on standing for and minutes. casp was measured non-invasively by a device bpro r that imputes the measured radial waveform onto the brachial blood pressure to generate a pressure waveform from which a numerical casp value is derived. the casp measurements for both patients did not decrease on standing for and minutes. our preliminary observation suggests that the basis of tachycardia in pots patients may not be a decrease in central blood pressure. we are proceeding to systematically study more pots patients to corroborate the above observation. we are also trying to compare the difference between pbp and casp in pots and age, gender-matched normal controls. the major limitation of the study is the model-based mathematical derivation, rather than direct measurement, of casp. we are proceeding to systematically stud y more pots patients to corroborate the above findings. ng jph , ng cjb , t umapathi . lee kong chian school of medicine, nanyang technological university, singapore; yong loo lin school of medicine, national university of singapore, singapore; national neuroscience institute, singapore. recent developments have validated non-invasive means of measuring central arterial blood pressure (casp) and have shown that casp and peripheral blood pressure (pbp) are unidentical entities. orthostatic hypotension (oh) is a prominent component of autonomic dysfunction (ad), the consequent hypoperfusion of vital organs responsible for considerable morbidity and mortality. these structures are exposed to casp rather than pbp. we sought to understand the relationship of casp to peripheral blood pressure (pbp) in patients with autonomic dysfunction exposed to orthostatic stress. we reviewed autonomic function tests of patients tested at our laboratory over a -year period. the patients were divided into cohorts: ( ) no ad and no oh ( ) no ad, with oh ( ) mixed ad ( ) parasympathetic dysfunction ( ) sympathetic dysfunction. casp was measured non-invasively by a device, bpro r , that imputes the measured radial waveform onto the brachial blood pressure to generate a pressure-wave form from which a numerical casp value is derived. the difference and ratio of casp to pbp was recorded at rest and after minutes of standing. out of patients had complete data and a definitive final diagnosis. cohorts - had , , , , patients respectively. mean casp-pbp difference in cohorts - were − . , − . , − . , − . , − . respectively at minutes, and − . , − . , − . , − . , − . respectively at minutes. mean casp/pbp ratio in cohorts - were . , . , . , . , . , . respectively at minutes, and . , . , . , . , . respectively at minutes. there was no significant difference in the response of casp and pbp to orthostatic stress across abnormal cohorts to and in comparison with the "normal" cohort (p= . to . ). there was also no relationship to symptoms, namely postural dizziness. in conclusion, autonomic dysfunction does not seem to affect the casp-pbp relationship as measured by non-invasive means. the absence of true normal controls, the exclusion of significant number of patients because of incomplete data and the model-based mathematical derivation rather than direct measurement of casp are limitations that we aim to address in follow-on studies. niu jw , guan hz , cui ly , guan yz , liu ms . the department of neurology, peking union medical college hospital, chinese academy of medical sciences, beijing, china. we aimed to explore the correlation between afterdischarges in motor nerve conduction studies and clinical motor hyperexcitability in patients with voltage-gated potassium channels (vgkc) antibodies. six patients with positive serum antibodies to contactin-associated protein-like (caspr ) or/and leucine-rich glioma-inactivated protein (lgi ) were recruited, including with autoimmune encephalitis, and with cramp-fasciculation syndrome. electromyography (emg), nerve conduction studies (ncs) and f waves were performed, and afterdischarges were assessed. one patient was followed up. five patients had clinical evidence of peripheral motor nerve hyperexcitability (myokymia or cramp), and four of them had abnormal spontaneous firing in concentric needle electromyography. prolonged afterdischarges following normal m waves were present in all six patients, including the two patients who had no emg evidence of peripheral nerve hyperexcitability (pnh). in the patient who was followed up, afterdischarges disappeared after treatment with intravenous immunoglobulin (ivig). afterdischarges in motor nerve conduction study might be more sensitive than needle electromyography for detecting peripheral motor nerve hyperexcitability in patients with vgkc antibodies, and could disappear gradually in accordance with clinical improvement and reduction of antibodies. in our research, multiple sites measurement of cross sectional areas (csa) by ultrasound was performed to differentiate charcot-marie-tooth type a (cmt a) and chronic inflammatory demyelinating polyradiculoneuropathy (cidp). twenty-eight patients with cidp, patients with cmt a, and healthy controls (hc) were recruited prospectively. consecutive ultrasonography scanning was performed from wrist to axilla on median and ulnar nerves. csas were measured at predetermined sites of each nerve. cmt a had significantly larger csas at all sites of median and ulnar nerves (all p< . ). in cmt a, csas increased gradually and homogeneously from distal to proximal along the nerve, except potential entrapment sites. cidp displayed three different morphological patterns, including mild enlargement in patients, prominent segmental enlargement in , and slight enlargement in one, among which different treatment responses were observed. all patients with mild nerve enlargement treated with intravenous immunoglobulin (ivig) were responsive ( / ), while less than half of those with prominent segmental enlargement ( / ) were responsive (p< . ). the patterns of csa enlargement were different in cmt a and cidp patients. consecutive scan along the nerve and multiple sites measurement by ultrasound could supply more detailed morphological feature of the nerve and help to differentiate cidp from cmt a. guillain-barre syndrome (gbs) mainly consists of acute inflammatory demyelinating polyradiculoneuropathy (aidp) and acute motor axonal neuropathy (aman). in aman, conduction block (cb) could be reversible or followed by axonal degeneration. we aimed to identify the correlation between existence of cb and the functional outcome for patients with gbs. gbs patients were prospectively recruited for serial electrophysiological tests and disability evaluation. all patients received treatment of intravenous immunoglobulin (ivig), and their disabilities were evaluated on the hughes functional grading scale before and month after treatment. patients were classified into aidp, aman, equivocal or normal according to electrodiagnostic criteria described by rajabally et al. aman patients who had follow-up nerve conduction studies were further classified into three groups. group was typical aman without conduction block, group had reversible conduction block, group had conduction block and subsequential axon degeneration. electrophysiological study results showed aidp, aman, equivocal and normal. probable or definite conduction block was observed in aidp patients and aman patients. aman with cb had higher reduction of hughes grade at one month ( . ± . vs . ± . ,p= . ), and lower percentage of patients with slow recovery (unable to walk independently at six months) ( % vs %, p= . ) compared with aman without cb. there were no significant differences between aidp with cb and without cb, in the reduction of hughes grade at one month. among the aman patients who were followed up, were typical aman without cb (type ), had reversible cb (type ), had cb and subsequential axon degeneration (type ). hughes grades at nadir were similar, while patients with reversible cb (type ) had the largest hughes grade reduction at one month (type - . vs type - . vs type - . ). none of the patients with axon degeneration (type ) showed rapid recovery, while % of those with reversible cb (type ) had rapid recovery (improvement by two or more hughes grades within four weeks after onset). electrodiagnosis of aman with conduction block, especially reversible conduction block, might be a marker of good recovery. we report a follow-up study of nerve ultrasound in a patient with primary neurolymphomatosis. a -year-old female presented with -months history of asymmetric limb pain, paresthesia, and weakness. electrodiagnostic studies and spinal cord mri showed an axonal neuropathy involving cervical and lumbosacral root, brachial plexus and left median nerve. detection of malignant b lymphocytes by cytology and flow cytometry of cerebral spinal fluid confirmed the diagnosis of b-cell non-hodgkin lymphoma. nerve ultrasound showed dramatic enlargement of upper, middle and lower trunks of left brachial plexus (cross sectional area-csas mm , mm , mm respectively), middle trunk of right brachial plexus (csa mm ), and proximal part of left median nerve (csa - mm ). five months later, after five chemotherapy of rituximab and high-dose methotrexate, and intrathecal injection of cytosine arabinoside and dexamethasone, the patient had clinical improvement. nerve ultrasound also showed alleviation of nerve enlargement. the csas of upper, middle and lower trunks of left brachial plexus were mm , mm , mm respectively; the csa of middle trunk of right brachial plexus was mm ; the csa of proximal part of left median nerve was - mm . peripheral nerve ultrasound could help locate the distribution of nerve involvement, and reveals disease progression. nolano m , provitera v , stancanelli a , caporaso g , saltalamacchia am , borreca i , lullo f , califano f , lanzillo b , iodice r , manganelli f , barone p , santoro l . irccs "salvatore maugeri" foundation, institute of telese terme (bn), italy; "maugeri" clinical and scientific institutes irccs, institute of telese terme (bn), italy; center for neurodegenerative diseases (cemand), department of medicine and surgery, neuroscience section, university of salerno, italy. a peripheral nerve involvement has been demonstrated in pd with the evidence of a small fiber pathology as possible intrinsic feature of the disease and a higher occurrence of large fiber neuropathy in patients longtime treated with l-dopa. however the role that disease itself and ldopa have on small and large fiber pathology in pd is still debated. we studied morphology and function of cutaneous innervation, in idiopathic pd patients ( male, aged . ± . ), including naïve and l-dopa treated subjects without electrophysiological signs of neuropathy, with the aim to assess and characterize small and large fiber involvement and the effect of l-dopa on it. all patients underwent a screening to rule out potentially neurotoxic conditions such as glucose intolerance, dysendocrinopathies, vitamin e, b and folic acid deficiency, hepatic or renal failure, hiv or connective tissue disorders. skin biopsies were obtained from thigh, leg and fingertip from the more affected side and bilaterally from thigh and leg in patients. samples were processed with indirect immunofluorescence technique using primary antibodies to mark different sensory and autonomic fiber populations. density of intrapapillary myelinated endings (ime), meissner's corpuscles (mc) and epidermal nerve fibers (enfs) was obtained as well as a semi-quantitative assessment of sudomotor, pilomotor and vasomotor innervation. further evaluation included sympathetic skin response, quantitative sensory testing and dynamic sweat test. morphological and functional findings were compared with data extracted from our age and sex stratified normative dataset. ienf, ime, mc densities were lower (p< . ) compared to controls in both naïve and l-dopa treated patients without differences between them except for mc density that was lower in l-dopa treated subjects ( . ± . vs . ± . /mm ). a loss of autonomic nerves was also found in both groups compared to controls. significant abnormalities (p< . ) of thermal sensory thresholds, tactile thresholds, mechanical pain perception and reduced sweating output were present and similar in both groups. our work confirms in pd an intrinsic peripheral nerve pathology involving both small and large fibers. small fiber pathology isn't affected by l-dopa treatment while sensory large fibers involvement, already present in naïve patients worsens with ldopa treatment. noto y , garg n , li t , timmins hc , park sb , shibuya k , kiernan mc . brain and mind centre, the university of sydney, sydney, australia. the diagnosis of amyotrophic lateral sclerosis (als), a progressive, fatal neurodegenerative disorder defined by combined upper and lower motor neuron involvement, remains clinically based. the purpose of this study was to determine the ultrasound appearance of peripheral nerves in als patients, and to investigate whether parameters such as distal/proximal ratios of nerve cross-sectional areas (csas) may effectively differentiate disease mimics from als. nerve ultrasound of the median, ulnar, and tibial nerves was performed in als patients compared to mimic patients ( patients with peripheral nerve hyperexcitability syndromes (pnhs) and patients with multifocal motor neuropathy (mmn)). comparison of nerve and the distal/proximal ratios was undertaken by ultrasound and compared across clinical and neurophysiological parameters.compared to normal controls, csa of the median nerve at the upper arm was decreased in als (p < . ). in comparison to als mimic disorders, csa at the proximal site of the median, ulnar and tibial nerve and the forearm/upper arm ratio of the median and ulnar nerves had diagnostic values. in addition to csa of the median, ulnar, and tibial nerves, the median and ulnar nerve forearm/upper ratios may provide a useful marker in for the diagnosis of als. approximately in , individuals are diagnosed with charcot marietooth (cmt) disease, making it the most common hereditary peripheral neuropathy. there is no documented cure for cmt, however, many of those affected, report difficulty with mobility, imbalance, and weakness of the feet and hands. in the general population, patients reporting difficulty walking, falls and/or fear of falling, and poor strength are often referred to physical (pt) and occupational therapists (ot) to skillfully address the impairments and help restore function and quality of life (qol). for patients diagnosed with cmt this is unfortunately not the norm, often leaving patients without any skilled guidance on managing their functional impairments. patients ( males) with a mean age of . years ( - ), went through a progressive and skilled pt intervention over the course of months. the program included: therapeutic activities and exercise, neuromuscular reeducation, and manual techniques to address the documented deficits. patients were progressed through the program based on the borg scale. each patient was assessed prior to and post the commencement of the program with the berg balance scale, minute walk test, timed up and go, sit to stand in seconds, foot self selected and fast gait speed, the lower extremity functional scale, activities balance confidence form, upper extremity functional index, oswestry, and the sf- qol measure. the patients were seen by the same skilled pt - a week for weeks. all but patients improved in all measures taken, indicating an improvement in function and overall quality of life. participants reported a total of falls in the months prior to the initiation of the study and only fall was reported during the month pt intervention. this study makes a strong case for the utilization of skilled pt to address deficits in patients with cmt. additionally, the utilization of objective, valid and reliable outcome measures in this population may help healthcare practitioners establish baseline function and response to change. a large randomized control trial is recommended to study the effects of a specific pt intervention on outcome measures in patients with cmt. ogata h , fujita a , yamasaki r , matsushita t , kira ji . department of neurology, neurological institute, graduate school of medical sciences, kyushu university, fukuoka, japan. clinical features of chronic inflammatory demyelinating polyneuropathy (cidp) patients with autoantibodies against neurofascin (nf) , one of the paranodal proteins, have been elucidated while the relation between anti-nf antibody levels and long-term clinical course in these patients still remains elusive. we retrospectively collected clinical, electrophysiological and immunological data of three anti-nf antibody-positive cidp patients. they were all males and their ages at onset were , , and years old. their clinical severity was evaluated by deep tendon reflexes (dtrs), grip strength and hughes functional scale. anti-nf antibody levels were measured by flow cytometry using hek cell lines stably expressing human nf . after immunotherapies of various combinations, including intravenous immunoglobulin, plasmapheresis, corticosteroids and other immunosuppressants, were introduced, their clinical parameters were gradually improved. decreased or absent dtrs were normalized and grip strength was increased by more than kg. hughes functional scale scores were decreased by at least one point compared with those at nadir. ncs findings of all three patients also showed obvious amelioration. for example, their f wave latencies in the right ulnar nerve were improved from to ms, from to ms, and from to ms, respectively. anti-nf antibody levels after treatment were decreased in two patients whose pre-and posttreatment sera were available. when dose of oral prednisolone was being tapered, they experienced re-exacerbation of clinical parameters, especially dtrs and grip strength. their ncs findings and serum anti-nf antibody levels were also deteriorated. exacerbation of these laboratory data in one patient preceded his clinical fluctuation, which suggests that ncs and serum anti-nf antibody levels could be used as early disease activity markers. in this case series, not only clinical but also laboratory data support a notion that anti-nf antibody-positive cidp patients were reactive to combined immunotherapies including corticosteroids. even though various treatments were administered to them, efficacy of oral corticosteroids seemed to be dose-dependent. optimal disease activity markers and immunotherapies for long-term maintenance of remission in anti-nf antibody-positive cidp should be identified. evaluated by deep tendon reflexes (dtrs), grip strength and hughes functional scale, after starting immunotherapies, ohnmar o , kamyw , ng lfp , t umapathi . university of medicine , yangon, myanmar; singapore immunology network, a*star, singapore; national neuroscience institute, singapore. singapore's zika virus (zikv) outbreak started in late august . over a period of months, we studied patients enrolled into our institution's prospective guillain-barré syndrome (gbs) database for relationship to zikv infection. we also studied gbs controls that were seen before the established outbreak and non-gbs controls. the index cases tested negative for zikv pcr in blood and urine. we proceeded to test zikv igg, igm, dengue virus (denv) igg and igm, and neutralization assays against zikv and denv. one patient with anti-gq b igg positive miller fisher syndrome had detectable zikv igm and zikv igg. the serum showed low titre denv igm and denv igg. follow-up serum at about months showed increase in zikv igg. we believe this patient has zikv-gbs. one patient with acute motor sensory axonal neuropathy and another with acute inflammatory demyelinating polyneuropathy had high zikv igm but low denv igm and igg. another patient with mfs showed high levels of zikv and denv igm but low igg. the latter two patients had gbs before the zikv outbreak in singapore. we suspect these patients could have zikv-gbs, but are awaiting convalescent sera for confirmation. two patients seen during the outbreak had detectable levels of zikv igg but serial testing showed a decline after a period of - months. the initial and follow-up sera showed raised denv igm and igg levels in one and raised igg levels in the other. in addition, both had stronger neutralizing capacity against denv than zikv suggesting that the initially detectable zikv igg levels was due to cross reactivity with previous denv infection. four patients, gbs control and non-gbs controls also showed serological response consistent with previous exposure to denv. one normal control showed nil exposure to both viruses. in summary, using various overlapping serological methods we diagnosed definite and suspect zikv gbs cases. our findings highlight ) insensitivity of blood and urine pcr to diagnose zikv-gbs ) the problems of interpreting zikv serology from cross-reaction with denv ) serial serology increases diagnostic accuracy. okar sv , ergunay k , bekircan-kurt ce , , erdem-ozdamar s , , tan e , . department of neurology, hacettepe university, ankara, turkey; virology unit, department of medical microbiology, hacettepe university, ankara, turkey; neuromuscular disease research laboratory hacettepe university, ankara turkey. guillain-barré syndrome (gbs) is an acute monophasic immune-mediated polyradiculoneuropathy. it is believed that acute inflammatory demyelinating poliradiculoneuropathy is caused by t-cell mediated autoimmune response targeting peripheral nerve myelin. molecular mimicry plays a role in the pathogenesis of some gbs cases. this mechanism has been well demonstrated in the acute motor axonal neuropathy (aman) variant, in which autoantibodies to camphylobacter jejuni share epitopes with peripheral nerve gangliosides. this molecular mimicry mechanism can be attributed to some cases with atypical triggers such as zika virus or west nile virus infections. moreover there are accumulating clinical data for vector borne viral infections triggering gbs. we evaluated vector-borne viral infections in our gbs and aman patients. eight patients with gbs, two patients with aman and as a control group, seven patients with normal pressure hydrocephalus were included. gbs and aman was diagnosed with clinical, electroneuromyograpic and cerebrospinal fluid (csf) findings. cerebrospinal fluid serum and urine samples were examined for vector borne viral infections via generic flavivirus and phlebovirus pcr. we also documented our patients prognostic scores such as modified erasmus gbs outcome score (megos) and modified erasmus gbs respiratory insufficiency score (megris). the mean age of the patients was , ( - years) , six of them were female. all csf, serum and urine samples of our patients and control patients were negative for flavivirus and phlebovirus families. the preliminary results of our study in this our small cohort did not show any correlation between the vector-borne viral infections and gbs. further studies with broad number of patients are needed for more suggestive results. neuromyotonia (nmt) consists of spontaneous motor unit activity that reflects increased peripheral nerve excitability, leading to involuntary, persistent muscle activity, visible as muscle twitching at rest, with generalized, easily provoked cramps. since the first electrophysiological (emg) description of nmt by denny-brown and foley ( ) , there has been discussion about the origin of the abnormal electrical activity recorded in needle emg studies. we studied two patients. patient 's nmt is aggravated by cold, and he has an associated non-progressive mild polyneuropathy with demyelinating features. he is now -year-old and has been followed in our centre for years. he is negative for anti-vgkc antibodies. firstly, he was treated with carbamazepine and phenylhydantoin with poor response, but he has shown major improvement on intravenous immunoglobulin (ivig) during the last years. patient is a year-old man with nmt, followed for year, with high titters of anti-vgkc antibodies ( pmol/l; normal< ). he improved on ivig during the last months. screening for neoplasia was unremarkable in both patients (negative anti-neuronal antibodies, in particular anti-hu, anti-yo and anti-ri antibodies; normal computerized tomography scan of the chest and abdomen). in addition to routine studies, we tested synchronicity to spontaneous discharges in different motor units in simultaneous recordings made with two needle electrodes in the first dorsal interosseus muscle. time-locked fasciculations in these double recordings would represent abnormal ectopic activity initiated in a nerve trunk with ephaptic stimulation of a nearby axon. in patient , this research protocol was applied once, years after regular ivig treatment. patient was investigated before and year after ivig. both patients improved after ivig, mirrored by a striking decrease in the amount of spontaneous activity on emg. moreover, our technique did not detect synchronous spontaneous activity (time-locked fasciculations) on the second assessment, although this was predominant before treatment in patient . in nmt, abnormal discharges originate both in distal axonal branches and in more proximal segments. it appears that ivig is more effective in blocking antibody activity in proximal axonal segments, perhaps related to factors such as blood-nerve barrier, temperature or differing ion channel distributions. celiac disease (cd) is a chronic, multisystem and immune-mediated disorder characterized by small-bowel sensitivity to dietary gluten in genetically predisposed individuals. neurological manifestations may occur in about % of patients, including peripheral nerve involvement. recent growing evidence strongly suggests that peripheral neuropathy in cd may be autoimmune and associated with anti-ganglioside antibodies. a -year-old woman presented with slowly progressive weakness of her right hand and fingers extensors. medical and family history were unremarkable. on examination, muscle strength (medical research council-mrc) was scored for right wrist and finger extensors, for right abductor pollicis longus and for right ankle dorsiflexion. right triceps brachii and brachioradialis reflexes were weak, but normal elsewhere. the remainder of the neurological examination was normal. neuroaxis magnetic resonance was unremarkable, specifically with no gadolinium-enhancing lesions. motor and sensory nerve conduction studies were normal. no conduction block or abnormal temporal dispersion was found. needle electromyography showed severe neurogenic changes with abnormal spontaneous activity in right radial-innervated muscles, and chronic neurogenic changes in homolateral tibialis anterior, peroneus longus and extensor digitorum brevis. ganglioside antibody testing was positive to igg anti-gm antibody, but negative to anti-gm and other anti-ganglioside antibodies. additional blood tests were unremarkable, in particular cryoglobulin testing was negative. intravenous immunoglobulin improved weakness, as right extensors of the wrist and fingers scored (mrc). a monthly treatment was initiated, which after months was changed to every weeks to preserve function. a diagnosis of mama was established. later, cd was diagnosed in her daughter due to chronic diarrhoea. our patient underwent anti-tissue transglutaminase antibodies determination and small-bowel biopsy after years disease' duration, and a diagnosis of cd was made. gluten-free diet was started, but her neurological picture did not change after six months. in our case the presence of igg anti-gm antibody may support a causal link between mama and biopsy-confirmed cd, and the lack of response to gluten-free diet may be explained by chronic axonal injury induced by memory t-cells. this case broadens our knowledge about neurological manifestations in cd, raising a probable association with purely axonal multifocal motor neuropathies and anti-ganglioside antibodies. Õunpuu s , , pogemiller k , acsadi g , pierz k , , . center for motion analysis, connecticut children's medical center, farmington, ct, usa; school of medicine, university of connecticut, farmington, ct, usa; division of neurology, connecticut children's medical center, farmington, ct, usa; division of orthopaedics, connecticut children's medical center, farmington, ct, usa. orthopaedic surgical intervention of the foot and ankle is performed in persons with charcot-marie-tooth (cmt) for improving foot pain, ankle instability, orthosis fitting/comfort issues and shoe wear. the impact of these surgeries on ankle function during gait is not known. therefore, the goal of this study was to measure gait changes in ankle function following surgical intervention by means of computerized motion analysis. fourteen patients with cmt ( ± years pre and ± years post-operative) with bilateral orthopaedic surgery were included. all patients had a plantar fascia release plus some combination of other soft-tissue (posterior tibialis and tendo-achilles lengthenings; extensor hallucis longus, peroneus longus and anterior tibialis transfers) and bony procedures (metatarsal and cuboid osteotomies). all patients completed two gait analyses (pre and on average . years post-surgery) during barefoot walking using a standardized d motion analysis protocol. the changes in ankle kinematics and kinetics and temporal-spatial parameters were analyzed in reference to a control group of patients with cmt without intervening surgeries with . years between gait analyses as well as normal reference data. the surgical group showed a significant increase in height between the pre and post-operative analyses and no changes in walking velocity (pre: ± , post: ± , normal: ± cm/sec). similarly, the ankle kinematics and kinetics showed no changes as a result of surgery. however, there was a trend for increased peak ankle dorsiflexion (pre: ± , post: ± , normal: ± degrees). as in the surgical group, the control group showed an increase in height but no simultaneous increase in walking velocity (test : ± , test : ± cm/sec). the control group also showed no changes in ankle kinematics and kinetics suggesting that the impact of the disease is, for the most part, not noticeable over . years. the results show that surgical intervention that primarily addresses foot alignment does not negatively impact ankle kinematics and kinetics during gait. the comparison with the control group supports this finding. however, there are some other findings such as the increase in peak dorsiflexion during stance in some patients that will need to be examined further in larger cohorts. Õunpuu s , , pogemiller k , pierz k , , , acsadi g , . center for motion analysis, connecticut children's medical center, farmington, ct, usa; school of medicine, university of connecticut, farmington, ct, usa; division of orthopaedics, connecticut children's medical center, farmington, ct, usa; division of neurology, connecticut children's medical center, farmington, ct, usa. ankle-foot-orthoses (afos) are commonly prescribed for patients with charcot-marie-tooth (cmt) to improve gait and reduce ankle instability and falling. there are no studies that examine bracing outcomes using objective evaluations. the purpose of this study was to examine the impact of various afo designs on gait parameters in children and adolescents with cmt. we predicted that the afos would improve excessive and delayed peak dorsiflexion in stance and equinus in swing. fifteen patients (mean age ± years) were analyzed barefoot and with their prescribed afos by means of a standardized motion analysis protocol. a full clinical examination was also completed including strength and passive range of motion measures. the afos included solid, hinged and posterior leaf spring (tapered, less supportive) designs. sagittal plane ankle kinematics and kinetics and temporal-spatial parameters were analyzed in comparison to normal controls. walking velocity improved from ± to ± cm/sec demonstrating the functional benefits of afos. ankle plantar flexion angle at initial contact improved from − ± to − ± degrees demonstrating improvement in drop foot in swing and at initial contact reducing the risk of tripping. however, the degree of peak ankle dorsiflexion in stance remained the same and excessive at ± degrees barefoot and ± degrees with afos suggesting that in many patients the afo design was not sufficiently supportive. peak ankle plantar flexor moment showed improvement from . ± . to . ± . nm/kg highlighting the improved base of support provided by the afos that compensate for ankle plantar flexor weakness. however, peak power generation was reduced from . ± . to . ± . w/kg indicating that some of the available ankle strength was impeded with the afos. the results of this study suggest that orthoses can provide improved gait outcomes which will improve overall function. however, there are individual differences in patient impairment (strength, range of motion and bony deformity) and associated gait presentation that need to be accounted for in afo design. afos do not always function as intended due to the complex interaction between patient impairment, orthosis stiffness and orthosis design. motion analysis can assist in identifying the specific afo needs for an individual with cmt. painous c , lópez-pérez ma , illa i , , querol l , . neuromuscular diseases unit, neurology department, hospital de la santa creu i sant pau, barcelona, spain; hospital san pedro, logroño, la rioja, spain; centro para la investigación biomédica en red en enfermedades raras (ciberer), madrid, spain. chronic inflammatory demyelinating polyradiculoneuropathy (cidp) is an autoimmune neuropathy with a heterogeneous clinical spectrum. specific autoantibodies define different clinical phenotypes. cidp with nf antibodies constitutes a specific cidp subset in which a high incidence of postural and intention tremor has been described and that responds poorly to immunoglobulins. we report a patient with an anti-nf positive cidp that presented head, voice and limb tremor that improved with immunotherapy. a -year-old man with unremarkable medical history, presented at the age of with progressive distal weakness and paraesthesia. numbness, gait ataxia and action tremor involving voice, head and limbs appeared sequentially. the emg showed features of acquired demyelination fulfilling cidp diagnostic criteria. a first course of intravenous immunoglobulins was ineffective. the weakness, ataxia and tremor worsened significantly, needing two walking aids first and becoming wheelchair bound later on. he received six plasmapheresis cycles that were also ineffective and oral corticosteroids ( mg/kg) were started with mild improvement. after corticosteroid tapering the patient developed a severe relapse and was referred to our centre. five plasma exchange cycles followed by rituximab ( mg/m , doses) were added to the corticosteroids. three months later the weakness, ataxia and tremor, including the head and voice tremor, started to improve significantly. six months later the patient presented a significant improvement and was able to walk unaided. the anti-nf antibody titres fell from / pre-rituximab to be undetectable. limb tremor is known to occur in patients with inflammatory neuropathies and, specifically in anti-nf + cidp patients but, to our knowledge, this is the first report of a cidp patient presenting with head and voice tremor ever reported. the improvement of tremor with immunotherapy strongly suggest that anti-nf autoantibodies are involved in its pathogenesis, expanding the phenotype of anti-nf specific clinical features. palaima p , peeters k , l. pelayo-negro a , garcía a , gallardo e , garcía-barredo r , de vriendt e , infante j , berciano j , jordanova a . vib and university of antwerp, center for molecular neurology, molecular neurogenomics group, antwerp, belgium; university hospital "marqués de valdecilla", santander, spain. charcot-marie-tooth disease type g is an autosomal dominant and slowly progressing inherited neuropathy which was first described over years ago. it has been attributed to a single spanish family consisting of individuals with affected members spanning four generation. initially, the genetic defect was linked to a . mb region in q -q . . however, extensive sequence and structural variant analyses using whole genome sequences (wgs) of three affected individuals did not reveal any known or novel genetic causes within the region. over the last decade, serial clinical re-evaluation of the entire pedigree was performed leading to changes to the affection status of seven individuals redefining the disease-linked region to chr q . -q . (z max = . at theta = ). additional family members were then submitted for whole exome sequencing. this has led to the identification of a novel missense variant in the e ubiquitin-protein ligase lrsam (p.cys tyr) that was previously not covered by wgs. the variants co-segregated with disease and were absent from controls. other mutations that are known to disrupt the ring domain of lrsam have been previously reported to cause both autosomal dominant and recessive cmt type p (cmt p). unlike prior reports, we demonstrated that the mutation does not influence overall protein levels of lrsam , nor of its ubiquitylation target tsg in patient derived lymphoblasts. transcriptomics analysis identified significant upregulation of another e ubiquitin-protein ligase (nedd l) and of a key regulator of axonal degeneration (tnfrsf ). notably, magnetic resonance imaging of lower-limb musculature systematically showed fatty atrophy in both clinical and subclinical mutation carriers emphasizing its use for the identification of mildly affected members. our findings demonstrate that the isolated genetic entity cmt g is caused by a missense mutation in lrsam and should be reclassified as cmt p. moreover, we reveal novel molecular players associated with lrsam dysfunction, and highlight pathways and therapeutic targets shared with amyotrophic lateral sclerosis and alzheimer disease. damage to peripheral nerves is a prerequisite for neuropathic pain. however, it remains unclear why some neuropathy patients have pain, but others with identical nerve conduction, qst and ienfd do not. we propose to examine the distribution of trpv and its co-localization with cgrp and sp in epidermal nerve fibers in patients with and without neuropathic pain. we aimed to define the expression pattern of trpv in normal healthy subjects first, and study the co-localization of trpv with cgrp and sp in normal controls and patients with painful neuropathy. skin biopsies from neuropathy patients and normal subjects were utilized. anti-trpv antibody generated in our university with support from ninds-funded dept. of neuroscience monoclonal core (ns ) was used. combined immunohistochemistry were performed to identify co-expression of trpv with cgrp, sp and pgp . . the distribution of trpv in controls revealed a proximal to distal gradient similar to that observed for ienf labeled by pgp . . trpv staining was more intense in nerve terminals in the epidermis. combined immunostaining revealed that % of pgp . labeled fibers in the epidermis were trpv +, while % of cgrp+ fibers trpv +. patients with pain had a higher density of trpv + fibers compared to that of patients with numbness. a greater proportion of cgrp+ fibers ( %) in the painful patients were trpv +. expression of trpv in controls exhibits a distal to proximal gradient. trpv expression and co-localization with cgrp were altered in neuropathic pain patients, suggesting that this receptor plays an important role in pathological states. activation of the nop-receptor (nop-r) by its endogenous ligand nociceptin/orphanin fq or non-peptide agonists modulates nociception and analgesia in neuropathic pain models. the wide distribution of nop-r and its endogenous ligand represents an attractive treatment target. since pain is the most prominent feature in pc, we defined expression of the nop-r in plantar skin biopsies and assessed whether alterations exist in pc-affected vs pc-unaffected and anatomically matched control skin. skin biopsies from k a pc and control subjects were immunohistochemically stained for nop-r. combined immunostaining for nop-r with pgp . , neurofilament h (nfh) and cgrp was used to define nop receptor expression in the epidermis and upper dermis. robust nop-r was detected in epidermal keratinocytes and a subset of pgp . + fibers in both epidermis and dermis. staining was inhibited through pre-incubation with a nop-r blocking peptide and western blot analysis using homogenized human skin tissue demonstrated a band at ∼ kd consistent with nop-r molecular weight. nop-r expression occurred in dermal nfh+ a beta-fibers in all groups though no cgrp+ fibers co-expressed nop-r. pc-affected skin had slightly lower nop-r expression than in pc-unaffected skin and a similar pattern in anatomically matched locations from healthy control subjects was observed. nop-r is expressed in human plantar skin epidermal keratinocytes as well as a subset of epidermal and dermal nerve fibers. these fibers are pgp . +, cgrp-and many co-express nfh. nop receptor is a viable pharmaceutical analgesic target in pc patients irrespective of its slight down-regulation as compared to pc-unaffected skin. this work was supported by grünenthal gmbh. pareyson d , stojkovic t , leonard-louis s , reilly mm , laurà m , parman y , battaloglu e , tazir m , bellatache m , bonello-palot n , sacconi s , guimarães-costa r , attarian s , latour p , megarbane a , schenone a , ursino g , sabatelli m , luigetti m , santoro l , manganelli f , quattrone a , valentino p , murakami t , scherer ss , dankwa l , shy me , bacon cj , herrmann dn , pisciotta c , previtali s , bolino a . milan, italy; paris, france; london, united kingdom; istanbul, turkey; algiers, algeria; marseille, france; nice, france; lyon, france; genoa, italy; rome, italy; naples, italy; catanzaro, italy; kurashiki, japan; philadelphia, usa; iowa city, usa; rochester, usa. cmt b and b are characterized by recessive inheritance, early onset, severe course, slowed nerve conduction, myelin outfoldings, association with loss-of-function mutations in myotubularin-related protein- and − (mtmr , mtmr /sbf ), respectively, involved in the metabolism of ptdins p and ptsins( , )p phosphoinositides, key regulators of membrane trafficking. in a multicentre retrospective study to better characterise cmt b, we collected a minimal dataset of information including cmtes/cmtns on cmt b patients, cmt b ( unrelated families) and cmt b ( families). cmt b patients were younger and with earlier onset than cmt b : last visit was performed at a mean age of years (sd . ; range - ) for cmt b and years ( . ; - ) for cmt b ; disease onset occurred at a mean age of . years ( . ; - ) in cmt b as compared to . years ( . ; - ) in cmt b ; delay in motor milestones occurred in / cmt b and / cmt b subjects. eleven cmt b patients became chair-bound, whereas all cmt b subjects but one are still ambulant, although with afos for patients and requiring unilateral support in two cases. both disease types are characterised by vocal cord involvement ( / cmt b and / cmt b ); respiratory involvement was seen almost exclusively in cmt b patients (n= , four requiring non-invasive ventilation and one tracheostomy, as compared to one cmt b patient on niv at age ); two cmt b subjects and an affected cmt b relative not included in the present study died of respiratory complications. glaucoma (n= ) and buphthalmos (n= ) occurred only in cmt b . cmtes/ cmtns scores were higher in cmt b patients in spite of their younger age, indicating more severe disease: cmt b = cmtes mean . (n= ; sd . ; range - / ), cmtns mean . (n= ; sd . ; range - / ,; cmt b = cmtes mean (n= ; sd . ; range - / ), cmtns mean (n= ; sd . ; range - / ). our data confirm that cmt b is more severe than cmt b . interestingly, mtmr interacts with mtmr . mtmr is a catalytically active phosphatase, whereas mtmr is a catalytically inactive protein, known to increase mtmr enzymatic activity. thus, in cmt b nerves a residual enzymatic activity of mtmr may result in a less severe clinical phenotype as compared to cmt b . the charcot-marie-tooth disease (cmt) national registry is fully operative (https:www.registronmd.it) with collection of clinical/genetic information (minimal dataset) and reporting of clinical scales; cmt patients have registered thus far and have chosen one of the reference centers; information collected during the ad hoc visit have been entered in the registry for of them. registered patients have the chance to participate to a study that requires filling online self-reported questionnaires related to five important issues: disease course and complications during pregnancy; use, efficacy and tolerability of orthotics and assistive devices; outcome of surgery for skeletal deformities; safety of anesthesia; occurrence of sleep disorders (including evaluation of fatigue, anxiety and depression). by february , patients and relatives/friends (as healthy controls) have filled the questionnaires. we are performing a first explorative analysis of results, but data collection on all questionnaires will be prolonged until novemebr , , to obtain a larger sample. pregnancy: / cmt women had at least one pregnancy; complications ranging from mild to severe occurred in / pregnancies vs / in controls. cmt worsened in pregnancies ( patients) with no recovery in instances. prenatal diagnosis was performed in / pregnancies. satisfaction related to surgical procedures for foot deformities, assessed with vas (score - ), was . ± . (n = ). repeat surgery was required in / instances. sleep: the epworth sleepiness scale questionnaire revealed abnormalities of sleep in / cmt patients ( %) and in / controls ( %). the vast majority of cmt subjects ( / ; mean . ± . ), but also of controls ( / ; . ± . ) were not good sleepers according to the pittsburgh sleep quality index (psqi) (range - , good sleep). fatigue: scores of modified-fatigue impact scale (range - , no fatigue) were higher for cmt (mean ± . ) than controls (mean . ± . ). hospital scale for anxiety and depression: / cmt subjects had mild-to-severe anxiety and / mild-to-severe depression as compared to / and / controls, respectively. data analysis on orthotics and anesthesia is ongoing. in conclusion, the first data analyses confirm that there are problems related to all the five domains explored, that will need to be specifically addressed in patients' care. supported by telethon-uildm grant gup . charcot-marie-tooth disease (cmt) is the most frequent motor and sensory peripheral neuropathy and is known to have the uniform demyelination. the traditional definition of conduction block is the reduction of negative peak of compound muscle action potential (cmap) of proximal segment relative to adjacent distal segment more than %. in this study, we tried to find the frequency of conduction block in cmt a patients and to investigate the differences of the clinical manifestation. we enrolled unrelated cmt a patients ( males and females) with pmp duplication and undertook nerve conduction studies from to . stimulation sites were wrist, elbow and axilla for median nerve study. eleven patients ( . % of all enrolled patients) had ncs features suggestive of conduction block. compared to cmt a patients without conduction block, functional disability scale was significantly higher in cmt a patients with conduction block (p < . ). however, onset age and disease duration were not different between cmt a patients with and without conduction block. in addition, conduction block was more frequently observed in distal segments than proximal segments. we suggest that the frequency of conduction block in cmt a patients is not low, and there is some heterogeneity of demyelination in cmt a patients. also, in cmt a patients, the conduction block might have relationship with clinical disability. park jg , park ms . yeungnam university college of medicine, daegu, korea. the subacute combined degeneration (scd) is diagnosed by a characteristic clinical manifestation, spinal mri, and decreased serum vitamin b levels. we report a case of scd with elevated homocysteine and methlymalonic acid level in the situation of spurious elevation of vitamin b concentration. an years old man presented with progressive gait disturbances and sensory disturbances. about a year ago, he felt both hands and feet paresthesia and gait disturbances. four months before the visit, nerve conduction tests were performed elsewhere due to symptoms. polyneuropathy was found. the laboratory test showed megaloblastic anemia. however, serum vitamin b concentration was increased to pg / ml (normal: - pg / ml). his symptoms progressed gradually. at presented, neurological examination showed spasticity of the lower limb without weakness, and vibration and position sensation were decreased in both lower limbs, and showed ataxic gait. spinal cord mri showed a lesion with a long t high signal intensity from c to t in the posterior column of the spinal cord. further laboratory test were added, then while folate was normal level but homocysteine at . umol/l (normal: - umol/l), and methylmalonic acid at . um / l (normal: - . um / l) were increased. finally, scd caused by vitamin b deficiency was diagnosed. in addition, gastric endoscopy was performed to find the cause of vitamin b deficiency and chronic atrophic gastritis was found. after cobalamine treatment for months, hemoglobin level was improved and his symptoms were all improving with a little gait disturbances. false-elevation of vitamin b level could lead to delayed diagnosis and cause irreversible changes in the nervous systems. one of the most commonly used methods to measure vitamin b concentrations is competitive-binding assay, which may result in normal levels even with vitamin b deficiency due to the effect of anti-intrinsic factor antibodies. when vitamin b deficiency is suspected, even if the vitamin b concentration is normal, additional tests of homocysteine and methylmalonic acid should be considered. parman y , durmus h , deymeer f , oflazer-serdaroglu p , battaloglu e . istanbul university, istanbul faculty of medicine, department of neurology, istanbul, turkey; bogazici university, istanbul, turkey. hereditary motor and sensory neuropathies, also called cmt neuropathies, are the most common group among the hereditary neuropathies. cmt subtypes are grouped by axonal, demyelinating or intermediate phenotype, or by autosomal-dominant (ad), autosomal-recessive (ar) or x-chromosomal inheritance. cmt- is considered axonal and characterized by distal muscle wasting, mild sensory loss and normal or near-normal nerve conduction velocities. mutations in more than genes are known to be able to cause autosomal dominant (ad) or recessive (ar) cmt- to date. the genetic heterogeneity in charcot-marie-tooth (cmt) is a challenge for genetic diagnostics. clinical clues and frequencies of mutations in cmt genes from large cohorts may help to develop strategies for efficient genetic testing. here, we present the clinical, electrophysiological and genetic features of patients from turkey, diagnosed as genetically confirmed cmt- in the department of neurology, istanbul faculty of medicine our clinic between and . genetic testing was performed for gdap by dna sequencing and samples from patients were exome sequenced (wes). twenty-one male and ten female patients from unrelated families were investigated. segregation was ar in eight, ad in six families and two were isolated cases. intraand interfamilial variability in the age of onset with a range of - (mean . ± . years) and disease progression rate were striking. slowly progressive weakness and atrophy of distal muscles in the feet and/or hands were the most common presenting symptoms. mfn was found in four unrelated ad kinship and in unrelated ar kinships and was most common gene mutated among whole cohort. gdap was the most commonly mutated gene among ar families ( / ). wes revealed further mutations in aars, nefl, kif b and hspb , however, the causative mutation could not be identified in known cmt genes in about % of patients. our data indicates the marked intra-and inter-familiar phenotype variability in cmt- as previously described in literature. many more genes causing arcmt- remain to be discovered. pasnoor m , veerapaneni k , murphy r , statland jm , kimple d , hamasaki a , glenn md , herbelin l , barohn rj , jawdat o , dimachkie mm . department of neurology, neuromuscular division, the university of kansas medical center, kansas city, ks, usa. electrodiagnostic (edx) studies involving electromyography/nerve conduction study (emg/ncs) are useful for diagnosis of neuromuscular disorders. although these are safe procedures, emg and ncs often evoke anticipated pain and anxiety. there are no published research studies to support the notion that providing detailed information to patients prior to these procedures would reduce procedural pain and ameliorate anxiety levels afterwards. the objective of this study was to perform a prospective survey to assess anticipated pain and anxiety in patients presenting for edx studies, design a detailed patient education form, and assess the change in perceived pain and anxiety pre and post procedure in the standard of care (soc) education versus the printed detailed education instrument group. after completing a brief pain/anxiety questionnaire, patients were randomly assigned to either soc verbal education group or to read the detailed education form. another brief questionnaire was completed post procedure. seventy-eight patients were enrolled at the time of abstract submission, in intervention and in soc groups. mean age of patients enrolled was ± years. pain was anticipated by % patients as a visual analog scale (vas) score mean of . ± . with no significant difference between both groups (p= . ). post-procedural pain was reported in % of patients with mean pain score of . ± . and no significant difference between the two groups (p= . ). anxiety pre-procedure was reported by % patients with a mean likert-like score of . ± . score in all cases (p= . between both groups). post-procedural anxiety frequency was reduced to % with mean level of . ± . (p= . between both groups). most patients reported pain with both emg and ncs ( %). we found that edx testing evokes moderate pain in most and in some cases moderate anxiety level. the detailed education intervention did not attenuate the frequency or severity of pain. post-procedural anxiety was expectedly reduced in severity and frequency in both groups with a suggestion that anxiety severity is lesser in the detailed education group but numbers were small. further studies with more detailed questionnaire and perhaps web-based education in larger population may be useful to reduce pain/anxiety in patients presenting for edx studies. pasnoor m , roach c , barohn rj , statland j , jawdat o , dick a , glenn m , dimachkie mm . department of neurology, neuromuscular division, the university of kansas medical center, kansas city, ks, usa. diagnostic criteria for chronic inflammatory demyelinating polyneuropathy (cidp) have been designed for use in clinical trials. there is limited data on the comparative diagnostic utility of these criteria in the clinic setting and it is unknown if some of these criteria are better for predicting treatment response. the objective of this study is to compare the sensitivity of various diagnostic criteria and review treatment response. after obtaining local irb approval, we performed a retrospective chart review of cidp patients seen at the university of kansas medical center between and . we abstracted the clinical, electrodiagnostic, and treatment information. we determined the frequency of patients fulfilling each of the following criteria: efns , aan, saperstein and incat. we defined treatment response based on treating physician's impression of change, patient-reported functional improvement or one point grade change in the mrc grade. fifty-three cidp patients charts were reviewed, were excluded due to missing data. mean age was . , and ( %) were female. elevated csf protein was seen in / ( %). twenty-eight ( %) fulfilled efns definite criteria, ( . %) efns probable, ( . %) aan, ( . %) incat, and ( . %) the saperstein criteria. treatment response in patients who fulfilled efns definite/probable criteria included / to ivig, / to iv or oral corticosteroids, / to mycophenolate mofetil; among patients who fulfilled aan criteria, these were respectively / , / and / ; among those meeting incat criteria / , / and / ; and for saperstein criteria it was / , / and / . half to two-thirds of patients responded to plex based on different criteria. while all cidp criteria can similarly predict ivig treatment response, the efns criteria are most sensitive for the clinical diagnosis of cidp. pellegatta m , canevazzi p , forese mg , podini p , quattrini a , taveggia c . division of neuroscience and inspe, axo-glia interaction unit, san raffaele scientific institute, milan, italy; division of neuroscience and inspe, experimental neuropathology unit, san raffaele scientific institute, milan, italy. axonal nrg type iii is an essential instructive signal for pns myelination, since its expression determines whether axons are myelinated and also the thickness of the myelin sheath. previous studies have shown that secretases' cleavage controls nrg type iii activity at post-transcriptional level. specifically, the beta-secretase bace- cleavage of nrg type iii promotes myelination, whereas the alpha-secretase tace inhibits pns myelination by inactivating nrg type iii. after a damage, the axon located distally to the site of injury degenerates and loses the myelin sheath, following a process termed as wallerian degeneration. unlike the cns, axons in the pns can regenerate and regrow. several studies have shown that regeneration is favored by the trans-differentiation of schwann cells, which start to create a permissive environment for axonal regrowth. in addition, it has been shown that nrg regulates the early stages of the dedifferentiation process and is essential for creating a permissive environment to regeneration. similarly, the beta secretase bace- promotes pns regeneration and remyelination. in the present study, we analyzed the role of the alpha-secretase tace during wallerian degeneration. our data indicate that tace is upregulated in sciatic nerves after injury, distally to the site of damage. since tace is expressed in schwann cells and axons, we analyzed the role of both glial and neuronal tace during degeneration and regeneration processes. thus, we performed crush injury in sciatic nerves of two different transgenic lines carrying a conditional deletion of tace either in schwann cells (p -cre//tace flox/flox ) or in neurons (chat-cre//tace flox/flox ). the analyses of the different phases of the wallerian degeneration in these animals suggest a role of glial tace during the early stage of the process. in fact, we observed an increased number of myelinated axons only in p -cre//tace flox/flox mice, while we did not detect substantial differences in mutant chatcre//tace flox/flox mice. these results suggest that even during regeneration tace has an inhibitory role, as deletion of tace in schwann cell likely induces the activation of a neuro-protective program that we are currently investigating. péréon y , nizon m , küry s , besnard t , quinquis d , boisseau p , magot a , mussini jp , mayrargue e , barbarot s , bézieau s , isidor b . reference centre for neuromuscular disorders; dept. of genetics; dept. of paediatric surgery; dept. of dermatology, university hospital, nantes, france. the hereditary sensory and autonomic neuropathies (hsan) constitute a clinically and genetically heterogeneous group. hsan are associated with sensory dysfunction, altered pain and temperature perception with varying degrees of autonomic dysfunction, and abnormal small fibers neurodevelopment. more than ten genes have been described so far in the hsan group. original classification encompassed four entities, but additional subgroups continue to be described. hereditary sensory neuropathies (hsan) type ii are characterized by autosomal recessive inheritance, onset at birth and self-mutilating behavior. until now, one homozygous frameshift variant, c.[ _ del], p.(lys phefs* ), was reported in arl ip (adprribosylation-like factor -interacting protein ) in a consanguineous family. patients presented with spastic paraplegia, diffuse sensory and motor polyneuropathy and acromutilation. the disorder was classified as autosomal recessive spastic paraplegia spg . here, we described a new patient with congenital insensitivity to pain, sensory neuropathy, self-mutilation, and spastic paraplegia. whole exome sequencing showed a homozygous frameshift variant c. [ _ del], p.(lys phefs* ) in arl ip . the protein harbours reticulon-like short hairpin transmembrane domains and has a role in endoplasmic reticulum shaping. the variant causes an additional c-terminus hydrophobic domain which could alter its function. arl ip interacts with atlastin- responsible for spg a and hsan type d. this report highlights the role of arl ip in pathophysiology of insensitivity to pain and spastic paraplegia. péréon y , nguyen a-l , leclair-visonneau l , fayet g , nguyen j-m , magot a . laboratoire d'explorations fonctionnelles, hôtel-dieu, nantes, france; département de statistiques médicales, hôtel-dieu, nantes, france. carpal tunnel syndrome (cts) is responsible for sensory and/or motor symptoms that may be increased by wrist flexion or extension (phalen sign). previous studies have shown that short duration (e.g. min or less) flexion or extension was not responsible for significant changes of median nerve conduction parameters. the objective is the study was to determine how prolonged extension (reverse phalen's test) affects median and ulnar nerve conduction parameters in normal subjects. after providing informed consent, normal subjects ( females, age rank - years) underwent motor and sensory testing of both median and ulnar nerves from the dominant side. edx testing was performed using standard techniques with wrist in neutral position, then during passive wrist extension ( ∘ ) for minutes, with sequential recording of both motor action potential (cmap) and sensory action potential (snap) latencies and amplitudes every minutes after extension onset. ulnar nerve conduction parameters remained unchanged in all the subjects. regarding median nerve, groups of subjects could be individualized: subjects with no changes; subjects with only significant decrease of snap amplitude (− %); subjects with both snap amplitude and velocity decrease; subjects with both cmap and snap significant changes. when present, changes did not appear before min, with snap decrease being the earliest observed abnormality. prolonged wrist extension was responsible for median nerve edx parameter modifications in / normal subjects. sensory fibers were firstly affected, corresponding to the chronology clinically encountered in cts. based upon these results, the same protocol will be used in patients presenting with mild clinical cts, in order to try and identify an additional edx technique being useful in the diagnosis of cts. one could also wonder whether the subjects with the most important changes would be more prone to present cts in the future. perez-siles g , , drew a , , ellis m , kidambi m , takata r i , speck-martins c e , hagerman k a , siskind c e , day j w , ginzberg m , nicholson g , , , kennerson m l , , . northcott neuroscience laboratory, anzac research institute, sydney, australia; sydney medical school, university of sydney, sydney, australia; molecular medicine laboratory, concord repatriation general hospital, sydney, australia; sarah network rehabilitation hospitals, brasilia, df, brazil; department of neurology, stanford health care, stanford, ca, usa; pediatric neuromuscular unit, wolfson medical center, holon, israel. mutations in the atp a gene cause of x-linked hereditary distal motor neuropathy (dhmnx). to date, missense mutations (t i and p s) in this copper transporting atpase are the only confirmed mutations causing dhmnx in two independent families. next generation sequencing (ngs), including whole exome and whole genome sequencing, is increasing the rapid detection of variants in genes known to cause disease, however the absence and size of additional families, make it challenging to determine the pathogenic or benign status of variants identified. we have recently identified new variants in atp a in patients with progressive peripheral neuropathy, suggesting further genetic heterogeneity of dhmnx. two of these new variants, pe v and pm v, are located at highly conserved amino acids within domains of atp a (a-domain and p-domain, respectively) that are critical for the catalytic cycle of the copper transporter. we have also identified a third variant (c. - a>g) located bases upstream exon that is predicted to abolish a conserved branch-site, a consensus intronic sequence necessary for the processing of immature rna during splicing. our recent investigations using both patient fibroblasts and an atp a conditional knock in mouse model for dhmnx expressing atp a t i , the orthologue of the human atp a t i mutation, showed reduced atp a protein levels and defective retrograde trafficking of atp a, as pathological hallmarks of dhmnx atp a causative mutations. to elucidate if the newly identified pe v, pm v and c. - a>g atp a variants are pathogenic, we have systematically assessed patient fibroblasts harbouring these mutations for altered parameters previously found in the pathogenic t i atp a mutation. we predict that functional characterisation of atp a using patient fibroblasts harbouring newly identified variants will provide the evidence to ascertain whether these variants are disease causing. establishing a systematic functional readout for atp a variants will improve accuracy of genetic counselling and patient management of dhmnx in those cases where genetic evidence is limited. this study represents a complementary and necessary approach to the use of ngs, for validating the pathogenic status of variants identified and for expanding the genetic heterogeneity of dhmnx. month of birth (mob) have been defined as a risk factor for more than diseases. for instance, susceptibility for multiple sclerosis (ms) seems to be associated with being born in may, while lower risk of ms is observed in those born in november. there are no studies that assessed potential association between mob and a risk for guillain-barré syndrome (gbs). we sought to determine if the risk of gbs is associated with the mob. study included gbs patients diagnosed in serbia, montenegro and republic of srpska in the period from january , until december , . mob of patients and of general population were compared. patients with gbs had tendency to be born in october ( % increase compared to general population, p= . ) and were less likely to be born in june ( % decrease, p= . ). when consider specific seasons, gbs patients were more likely to be born in winter months ( % increase, p< . ). no associations were found between month/season of birth and disease severity. results of this pilot study showed that gbs patients are more likely to be born in cold months, and less likely to be born in june. this might be explained by higher exposure to different pathogens during pregnancy in cold months. in accordance with this, it is well known that early exposure to infective agents reduces risk of allergic and autoimmune diseases. further studies are needed to test our findings in different cohorts and in regions with different climate. these results may shed new light on the disease pathogenesis. majority of patients with guillain-barré syndrome (gbs) tend to have a successful recovery, but in number of them significant long-term consequences may negatively affect their quality of life. aim of this research was to analyze the outcome of the disease one year after the acute episode of gbs. among patients diagnosed with gbs in seven tertiary centers in serbia, montenegro and republic of srpska during , subjects were retested after one year ( % males, mean age ± years). functional disability of patients was estimated based on the gbs disability scale (gds). severe form of the disease (gds score - ) was registered in % of patients at admission, % at nadir, and % on discharge. after one year follow-up period, % of patients had no symptoms of gbs, % had mild symptoms, % was able to walk but not to run, % needed unilateral support during walking, while % was wheelchair bound or bedridden. lethal outcome was registered in three patients during acute phase of gbs and in four more during one-year follow-up. paresthesias were present in % of gbs patients, musculoskeletal pain in %, and fatigue in % one year after acute phase of the disease. factors associated with the worse functional outcome (gds grade above ) after one year were: age above , preceding respiratory infection, and worse gds on discharge. one year after the onset of the disease, significant number of our gbs patients had neurological impairments including sensory symptoms, pain, fatigue and muscle weakness which may significantly affect patients' everyday functioning. phetteplace je , saade d , bacon c , shy me . university of iowa hospitals and clinics, iowa city, ia, usa. charcot-marie-tooth (cmt) disease, also known as hereditary motor and sensory neuropathy, is a heritable peripheral neuropathy caused by genetic mutations in many different genes. cmt type a is the most common form of cmt. individuals affected with cmt a commonly have distal muscle atrophy, foot deformities, abnormal gait, and reduced nerve conduction velocities caused by demyelination of the peripheral nerves. cmt a is known to be caused by a duplication of the pmp gene at p . . the proposed disease mechanism of cmt a is believed to be increased gene dosage of the pmp gene, although the exact function of pmp is not known. there have been previous reports in the literature of individuals who carry a homozygous duplication of pmp , therefore having four copies of the gene. however, many of these reports have focused on adult patients who symptoms may not vary significantly from individuals who harbor only a duplication of pmp . a month old male presented to the university of iowa hospital due to a family history of cmt a and concern for delay in independent ambulation. he was known to have both a maternal and paternal family history of cmt a, although the families were believed to be unrelated. on exam he was unable to walk independently, but could take a few steps when his hands were held. per report he began crawling and scooting at months of age. he was not found to have any structural abnormalities or atrophy in his upper or lower extremities. electrodiagnostic testing revealed a demyelinating neuropathy. only one nerve was tested, as the patient was unable to tolerate additional shocks. the peroneal nerve was found to have a nerve conduction velocity of m/s. based on the clinical presentation, electrodiagnostic examination and family history deletion and duplication testing for pmp was performed. this testing revealed a homozygous duplication of pmp , which has been previously reported to cause cmt a. this case supports the gene dosage disease mechanism, as our patient appears to be more severely affected than a typical infant or toddler affected with cmt a. piscosquito g , saveri p , provitera v , stancanelli a , ciano c , magri s , taroni mpz mutations are associated with the demyelinating early-onset charcot-marie-tooth (cmt) type b and with the axonal later onset cmt i/cmt j. paresthesias/dysesthesias and pain have been already reported in some cmt types and also in cmt i/j. we report patients ( from families, sporadic cases,) carrying the mpz c. dela mutation (p.asp thrfster ). this is predicted to be a loss-of-function mutation, leading to p haploinsufficiency. most of the subjects have genetic ancestry from puglia region (southern italy). two patients carried the mutation in homozygosity, showing a severe phenotype: onset at birth, severe scoliosis, proximal weakness, distal limb plegia, no autonomous walking, cmt examination score (cmtes) - / . all the other heterozygous patients had mild adult-onset (mean age , range - ) neuropathy, walked without aids or ankle-foot orthoses, had cmtes < / . five showed mild foot deformities, / had foot and / hand weakness (mrc never less than / ), / sensory loss in lower limbs (ll). paresthesias/dysesthesias (mostly tingling and burning in type) localized in hands and ll were reported by / subjects, neuropathic pain by / . in the homozygous patients, all cmaps and saps were absent. in all other subjects, motor conduction velocities (cv) were > m/s (range - m/s) and sensory cv > m/s (range - m/s). cmaps as well as saps were normal or moderately reduced in amplitude, indicating only slight axonal loss. we confirm that heterozygosity is associated with very mild cmt, whereas homozygosity causes a very severe neuropathy. our data suggest that paresthesias and neuropathic pain are very common and should be considered as part of the phenotype. although the clinical picture suggests a small fiber neuropathy, no such evidence is provided in the current literature. in the attempt to further investigate this field, we found in a -year-old male patient of our series that all thresholds were increased at quantitative sensory testing, and several abnormalities of small nerve fibers were present in skin biopsy. apparently, p aneuplody causes neuropathy and small fiber involvement through a mechanism different from other mpz missense mutations. poitras t , chandrasekhar a , singh v , martinez j , zochodne dw . neuroscience mental health institute, and division of neurology, department of medicine, university of alberta, edmonton, alberta, canada. a microarray mrna analysis of dorsal root ganglia examined in long-term diabetic mice yielded an unexpected upregulation of a unique set of proteins known as major urinary proteins (mups & ). originally, this family of barrel shaped proteins was described as including mediators of pheromone secretion in the urine of rodents, apparently unconnected to other functions. we identified its unanticipated expression in sensory neurons of adult mice and rats and explored its potential impact on the properties of these neurons. analysis of rat and mouse sensory neurons showed widespread and pan-neuronal cytosolic mup expression, not only in the cell body, but also in distal sensory projections located in the dermis of the footpad. to assess the role that mup might offer in the growth behaviour of adult neurons, we examined sirna-induced knockdown of mup on pre-injured dissociated drg primary cultures. mup knockdown in both species showed a significant increase in neurite outgrowth following mup sirna treatment when compared to a scrambled sequence control. mice treated with streptozotocin (stz) model features of human type diabetic polyneuropathy, including decreases in multi-fiber motor and sensory measurements, and changes in thermal sensation in the extremities. to explore the potential role of mup upregulation specifically in diabetes, we examined the impact of non-viral intranasal injection of mup sirna on indices of neuropathy in chronic type diabetic mice. following treatment, mice treated with mup sirna showed improvement in both motor and sensory nerve conduction velocities compared to scrambled controls. taken together, this data suggests that mup plays a growth-suppressive role in both diabetic and non-diabetic neurons and may also contribute to the electrophysiological and behavioral abnormalities associated with diabetic polyneuropathy. the repurposing of mups in sensory neurons identifies an interesting role for a supposed pheromone protein. [supported by cihr, cda and adi] enhancement of their outgrowth properties (singh et al, ) . here we asked whether similar activation, using low dose capsaicin, might similarly ramp up the regenerative activity of sensory neurons. using rat adult primary sensory neurons, we identified a dose dependent relationship between capsaicin exposure and their outgrowth with enhancement at lower doses and expected neurotoxicity at higher doses. doses that enhanced outgrowth were nonetheless associated with rises in intraneuronal calcium. we next tested whether a similar impact of trpv activation in vivo might exist. we used a high sciatic crush injury mouse model to assess the impact of capsaicin at low ( um) or high ( mm) doses applied directly at the site of axon outgrowth after injury. by days following the single application of capsaicin, low doses of capsaicin showed more rapid recovery of thermal, but not mechanical sensation, whereas high dose capsaicin showed no significant difference compared to vehicle treated mice. in parallel with the neurotoxicity observed in vitro, the high dose capsaicin resulted in a decrease in sensory nerve conduction velocity, while the low dose showed no significant difference when compared to the vehicle treated animals. there was no impact on motor axon recovery. taken together, subtoxic trpv activation with intraneuronal calcium mobilization, as applied to regenerating sensory axons, enhances their outgrowth after injury, and improves behavioural recovery similar to that following extracellular electrical stimulation. [supported by cihr] hereditary transthyretin (hattr) amyloidosis is a rapidly progressive, life-threatening disease caused by ttr gene variants, resulting in ttr misfolding and amyloid deposition in multiple organs, including peripheral nerves. hattr amyloid polyneuropathy has prominent small fiber involvement characterized by neuropathic pain and dysautonomia. measurement of intraepidermal and sweat gland nerve fiber density (ienfd, sgnfd) in skin biopsies has been useful in identifying small fiber abnormalities in hattr. we report the effect of patisiran, an investigational rnai therapeutic for the treatment of hattr, over a -month period on ienfd, sgnfd and dermal amyloid content from the phase open-label extension (ole) study. skin biopsies (distal leg, distal thigh) were obtained every months for up to years within the phase ole study (patisiran . mg/kg iv, q w, nct ) and analyzed in a blinded manner by a central laboratory. all analyses are exploratory; nominal p-values are reported without adjustment for multiple testing. twenty-seven patients enrolled; preliminary data indicated patisiran was generally well tolerated; resulted in sustained mean serum ttr lowering of ∼ % for > months and a mean . -point decrease in mnis+ (n= ). twenty-four patients underwent serial skin punch biopsies. at baseline, mean ienfd was . and . fibers/mm, while mean sgnfd was . and . m/mm for distal thigh and distal leg, respectively. amyloid was detected in ∼ % of skin biopsies, mean baseline dermal amyloid content . % and . % for distal thigh and distal leg, respectively, which inversely correlated with ienfd and sgnfd. overall, ienfd was stable throughout the -year treatment. sgnfd increased over time relative to baseline, reaching statistical significance at , , and months for distal thigh (p< . , all time points) and at months for distal leg (p= . ). dermal amyloid content decreased over time and reached statistical significance at , and months for distal thigh (p< . , all time points) and at , , , and months for distal leg (p< . , all time points). in summary, improvements in sgnfd and dermal amyloid content observed over a -year period suggest that these parameters have the potential to serve as biomarkers of response to patisiran treatment. polydefkis m , neuhaus s , doherty l , ebenezer gj . department of neurology, johns hopkins university, baltimore, md, usa. misdiagnosis and delayed identification of an etiology for sensory neuropathies hampers adequate management and delays initiation of therapy. we present several cases that illustrate variable presentations of amyloid neuropathy (an) and an complicated by amyloid arthropathy. a -year-old former college athlete developed painless difficulty walking. he was unable to keep up with family members during routine outings and subsequently was unable to navigate uneven ground during a hike, something he had previously excelled at. balance decreased, making bicycling difficult. neurological evaluation -months after symptom onset led to a diagnosis of peripheral neuropathy. a reversible neuropathy panel was normal though a c was elevated ( . %). over several months, his strength/sensation deteriorated despite diet improvement, losing lbs and a c improvement ( . %). upon referral, emg revealed a sensorimotor neuropathy with markedly reduced/absent sural, peroneal and tibial motor amplitudes. there was mild orthostasis. nis was . skin biopsies revealed severe ienfd, sgnfd reductions and positive congo red staining, confirmed with birefringence. a sural nerve biopsy confirmed amyloid deposits. genetic testing revealed the ttr-fap variant leu his. there was no history or suspicion for a family history of amyloid. a -year-old male with a history of controlled diabetes x years developed abrupt onset of rls months after a lymphoma diagnosis. treatment with rituximab led to radiographic resolution of the lymphoma though the patient developed progressive peripheral neuropathy confirmed by ncv/emg. new-onset early satiety, mild orthostasis and ed emerged. skin biopsies demonstrated a lichenoid pattern of amyloid deposition consistent with aa-amyloid. a -year old woman with leu his ttr-fap since developed progressive leg weakness, reduced ability to walk and low back pain. emg demonstrated progression of her sensorimotor neuropathy and superimposed multilevel radiculopathy. spine mri demonstrated severe lumbar stenosis requiring surgical decompression. postoperatively, she regained strength, walking more easily. congo red staining of vertebral bone demonstrated widespread positive staining with birefringence consistent with amyloid arthropathy. these cases illustrate to wide sprectrum of amyloid neuropathy presentation, an example of amyloid arthropathy-induced spinal stenosis and the utility of skin biopsy to diagnose and manage amyloid neuropathy. the differentiation between hereditary neuropathy and chronic inflammatory demyelinating polyneuropathy (cidp) in children is especially significant because of completely different treatment possibilities and prognosis in these conditions. the aim of the study was to compare electrophysiological abnormalities in a group of children and young adults with demyelinating neuropathy of chronic or subacute onset. retrospective analysis of clinical and nerve conduction study (ncs) data included children and young adults with charcot-marie-tooth neuropathy x type (cmtx ), children with charcot-marie-tooth neuropathy type a (cmt a) and children with cidp. in our study / cmtx had both axonal and demyelinating changes in ncs study. the aan and efns electrophysiological cidp criteria were fulfilled in / cmtx , / cmt a and / cidp patients. additionally patients with cmtx are classified with efns criteria as "probable/possible cidp". a distal compound muscle action potential (dcmap) was > ms in all cmt a and / cidp patients but none with cmtx . abnormal median/normal sural snap (amns) parameter was observed in // cidp and / ctmx patients but not any cmt a patient while abnormal sural/normal median snap (asnm) parameter was found in / patients with cidp. a difference between conduction velocities (cv) of two corresponding nerves > m/s was seen in / cmtx and / cidp patients but no one with ctm a. one patient with cmtx had especially conspicuous difference between nerve conduction in lower limbs, with axonal neuropathy and demyelinating features, and upper limbs with no changes. one -years-old patient with genetically confirmed cmtx had no abnormalities in ncs. electrophysiological data of cmtx , cmt a and cidp indicate diffuse demyelination, however in cmtx axonal changes coexist, seen in / patients, and asymmetrical abnormalities between corresponding upper and lower limb were observed in / patients. our study has showed that duration of dcmap is useful not only in diagnosing an inflammatory neuropathy but also in differentiating cmtx from cidp. however presence of the not homogenous abnormalities in ncs in patients with cmtx may mimic inflammatory neuropathy. prada v , mori l , francini l , accogli s , ursino g , gemelli c , schizzi s , grandis m , bellone e , mandich p , schenone a . department of neurosciences, rehabilitation, ophthalmology, genetics and maternal and child health, university of genoa, genoa, italy. the overwork weakness (ow) problem in cmt disease has been debated for long time. it has been reported that the non-dominant hand (ndh) of patients with cmt disease is stronger than the dominant hand (dh) as a result of ow and some authors verified this hypothesis using mrc on different muscles (van pomeren, ). more recently, piscosquito et al. ( ) found that the ow phenomenon does not exist using a dynamometer and the hole peg test, a dexterity test. we recruited cmt patients and healthy controls. we evaluated the strength of the -pinch and of the hand-grip with a dynamometer, the opposition ability with the thumb opposition test (tot) and applied an instrumental testing of hand function using the sensor engineered glove test (segt), which previously demonstrated its sensitiveness to measure severity of hands dysfunction in cmt patients. tot was significantly higher in the ndh compared to dh (ndh= , ± , ; dh= , ± , , p< . ) in cmt patients, instead there was no difference in dh and ndh in healthy controls. in the evaluation of -pinch and of the hand-grip, ndh performed slightly but not significantly better than the dh ( -pinch: dh= , ± , n; ndh= , ± , n; ± , n; ndh= , ± , n, p:ns) . in normal controls we confirmed the % rule (noguchi & demura, ). finally, segt results were similar between the ndh and dh in cmt patients but in normal controls there was a better performance in the dh. in conclusion, this study supports the existence of the overwork weakness in cmt, evident in every measurements. dexterity and overall ability of the hands impaired in the dh, probably because of compensating movements, compared with the healthy controls in the weaker hand. finally, our results support the importance of avoiding supramaximal exercises and educating the cmt patients to prevent incorrect movements, which may overload the hand muscles. prasad k , ohnmar o , teng a , cheng yj , umapathi t . national neuroscience institute, singapore; yangon general hospital, yangon, myanmar; yong loo lin school of medicine, national university of singapore, singapore. antecedent infections that have been linked to guillain-barré syndrome (gbs) include campylobacter jejuni, haemophilus influenza, epstein-barr virus, cytomegalovirus, mycoplasma pneumoniae and influenza a virus. in south-east asia, the burden of mosquito-borne flavivirus infections is considerable. even in a small country like singapore, with a population of only . million and in spite of excellent sanitation and public health measures, dengue virus (denv) infections can exceed , a year. both symptomatic and asymptomatic denv infections are associated with gbs. zika virus (zikv) infections were detected in singapore in late august . doctors working in south-east asia anecdotally report that gbs cases increase after rainy season when mosquitos breed. we are currently planning studies to estimate the hitherto unknown burden of gbs associated with denv, zikv and other mosquito-borne viruses in this region. as a preliminary step, we reviewed the gbs cases seen at our institution in for evidence of denv and zikv. three out of the tested cases were positive for denv serology. one of these was also positive for denv pcr. all these patients had symptomatic dengue. two had acute inflammatory demyelinating polyneuropathy (aidp) and acute motor axonal neuropathy (aman) subtype of gbs. none the patients tested were positive for zikv pcr in serum and urine. one patient, with miller fisher syndrome (mfs), had serological evidence of recent zikv. he did not have clinical zikv symptoms. in mfs cases, the initial detectable zikv igg was later proven to be due to cross reactivity with previous denv infection. in cases ( amsan, aidp, mfs) the initial zikv igm was raised. we await analysis of convalescent sera to decide if they indeed had zikv or the results were related to previous and co-infection with denv. our preliminary findings indicate that flavivirus infections may account for at least some of the gbs cases in singapore. the lack of symptoms in some cases and the interactions between zikv and denv antibodies make accurate diagnoses challenging. prior r , , benoy v , , vanden berghe p , van den bosch l , . ku leuven-university of leuven, department of neurosciences, experimental neurology and leuven research institute for neuroscience and disease (lind), leuven, belgium; vib-center for brain & disease research, laboratory of neurobiology, leuven, belgium; laboratory for enteric neuroscience, translational research center for gastrointestinal disorders, k.u. leuven, leuven, belgium. charcot-marie-tooth disease (cmt) is the most common inherited neurodegenerative disorder of the peripheral nervous system. it is divided into two main subtypes, a demyelinating subtype (cmt type ) and an axonal form (cmt type ). cmt is a length-dependent disease, in that it effects the longest neurons in the body, thus the muscles in the peripheral regions are affected first and foremost. moreover, the majority of cmt patients share a classical phenotype with shared pathological hallmarks, such as distal muscular atrophy, reduction in nerve conductions, etc. but also molecular pathological hallmarks, like the breakdown in the transport of organelles and vesicles in neurons in a process called axonal transport. currently, there is no cure or effective treatment available to cmt patients. histone deacetylase (hdac ) has been shown to be a key regulator in axonal transport. moreover, inhibiting hdac has been demonstrated to stabilize microtubules, which act as molecular tracks for motor proteins and facilitates axonal transport of cargos. our labs current focus is on cmt type a, the main cause of cmt, which is caused by a duplication of a segment of chromosome p . containing the gene encoding peripheral myelin protein (pmp ). pmp is mainly expressed by schwann cells, the cells that myelinate neurons in the peripheral nervous system. in the work presented, the commercial mouse schwann cell-line, sw cells, was transfected to overexpress pmp using a lentivirus vector system to mimic that of cmt a patient schwann cells. the overexpression of pmp was confirmed using immunofluorescence and western blot techniques. these transfected sw schwann cells were then co-cultured with primary mouse dorsal root ganglion neurons (drgs) isolated from adult mice. these co-cultures where then analysed after weeks in vitro for axonal transport. the investigated groups were: a co-culture of drgs + sw cells, a co-culture of drgs + sw cells transfected with pmp , and a co-culture of drgs + a sw cell-line transfected with green fluorescent protein using a lentivirus vector system, and a monoculture of drgs. furthermore, this work demonstrates that the overexpression of pmp in schwann cells can impair axonal transport in co-cultured drgs in comparison to the other co-culture groups. moreover, these axonal transport defects were able to be rescued by the treatment of a hdac inhibitor, tubstatin a. to conclude, selective hdac inhibitors have been shown to be a beneficial treatment for a number of cmt subtypes in preclinical studies in our lab and offer as a viable treatment for a currently, untreatable debilitating disease. provitera v , caporaso g , stancanelli a , piscosquito g , di caprio g , saltalamacchia am , santoro l , nolano m . "maugeri" clinical and scientific institutes irccs, institute of telese terme (bn), italy; department of neurosciences, reproductive and odontostomatological sciences, university 'federico ii' of naples, naples, italy. the observation of rate and patterns of cutaneous nerves regrowth after mechanical or pharmacological distal axonotmesis, has proven to be an excellent tool to study human nerve regeneration in normal and pathological conditions. most of the observations, however, are referred to small fibers, possibly due to the availability of excellent models of reversible distal cutaneous small fiber axonopathy. no such model is available to study the regeneration of myelinated fibers in human. we studied regrowth of cutaneous large fibers in fingertip of patients with carpal tunnel syndrome after surgical decompression. we recruited patients (m/f / ; age . ± . years) with carpal tunnel syndrome candidate to surgery. patients underwent clinical and electrophysiological evaluation, quantitative evaluation of discriminative threshold at rd fingertip. in the same site, patients also underwent mm punch skin biopsy. skin sections were stained by immunohistochemical techniques and cutaneous innervation was analyzed by confocal microscopy. meissner corpuscles and their myelinated afferences were assessed following previously published procedures. twelve months after surgery, patients repeated functional evaluation and underwent a second skin biopsy two mm apart from the first one. mean density of mcs/mm was . ± . at time and . ± . at follow-up, mean density of myelinated endings was . ± . at time and . ± . at follow-up. however, not in all patients regeneration occurred. based on the variation of mc density between time and follow-up we were able to identify patients in which active regeneration had occurred. in this subgroup mean density of mcs/mm was . ± . at time and . ± . at follow-up (p< . ) with a mean delta of + . . in the same group, mean density of myelinated endings was . ± . at time and . ± . at follow-up (p< . ) with a mean delta of + . . we describe morphological patterns associated to nerve regrowth in the biopsies of patients in which nerve regeneration occurs. we propose an in vivo model to study regeneration of large myelinated fiber endings in human skin. in addition to the count of nerve endings, the identification of patterns associated to nerve regeneration can increase the diagnostic yield of skin biopsy. the most common type of charcot-marie-tooth disease is caused by a duplication of pmp , leading to dysmyelination, axonal loss and progressive muscle weakness (cmt a). currently, no approved treatment for cmt a patients is available. among others, preclinical therapeutic approaches aim to correct the pmp overexpression in order to ameliorate axonal loss and muscle weakness. we previously reported that the novel polytherapeutic drug pxt , a low-dose combination of baclofen, naltrexone and sorbitol, improved dysmyelination and axonal loss after long-term application in adult pmp transgenic rats, a known animal model of cmt a. interestingly, after short-term application in young cmt a rats we observed a long-lasting prevention of muscle weakness as well but without obvious effects on dysmyelination and axonal loss. improved distal motor latencies in the electrophysiological recordings and a shift in the axonal diameter distribution raised the hypothesis that therapy may improve the neuromuscular endplate pathology as another therapeutic target for cmt a. here, we report a preclinical trial in adult cmt a rats treated from postnatal week for consecutive weeks as the most effective regimen in order to facilitate a deeper understanding of the mode-of-action of pxt . long-term therapy effects were confirmed by an ameliorated behavioral phenotype, improved distal motor latencies and also nerve conduction velocity in the electrophysiological recordings. histological analysis revealed an increased number of neuromuscular endplates, which were lowered to wildtype level after pxt treatment. further investigations include detailed analysis of peripheral nerve myelin thickness, internodal and nodal length, terminal axonal sprouting at the neuromuscular endplate and muscle fiber subtyping in treated cmt a rats. charcot-marie-tooth disease (cmt) is a rare hereditary neuropathy for which novel treatments are being developed and urgently needed. only few clinical trials and natural history studies have been performed. evaluation of intervention efficacy is hampered by slow progression and lack of sensitive outcome measures. the interindividual disease variability is high and prognostic disease measures are not established for cmt patients. previously, we have identified skin-derived disease and progression biomarkers in a large european and us-based cmt a cohort. within the german charcot-marie-tooth disease network (cmt-net, www.cmt-net.de) we aim to establish easier accessible biomarkers in blood of cmt a patients. blood analysis is less invasive and can be repeatedly performed during clinical trials and routine patient care. we have started clinically assessing approximately young, adolescent and adult cmt a patients at clinical centres (münster, münchen, göttingen) including a large set of clinical outcome measures (cmtnsv , onls, mwt, mwt, walk , hpt, sf- , fss, psqi, ess, bdi-ii). we will sample blood and skin tissue once a year at , and months for an observational period of years. tissue samples will be analyzed on the transcriptional, translational and epigenetic level. we envision that the diagnostic and progression biomarkers may be used to measure therapeutic effects within clinical trials and later in clinical routine monitoring. this is the first trial testing "circulating" biomarkers from blood in a prospective observational trial in cmt a patients. igm peripheral neuropathy is a slowly progressive and heterogeneous disease with symptoms ranging from mild foot numbness and minor imbalance to severe neuropathic pain and sensory and motor dysfunction. international consensus regarding assessment and treatment of patients with igm peripheral neuropathy is lacking. this might be caused by the repeated use of suboptimal outcome measures, the small trial sizes with low numbers of treated patients, the indolent disease course needing a longer follow-up period to capture relevant changes, or the possibility that administered treatments were not aggressive enough. the imagine study is an international, multi-centre, prospective, observational cohort study, which will result in a unique collection of a large number of prospectively collected and highly standardized clinical data, and a biobank from a large population of well-defined patients with igm peripheral neuropathy. the main objective is to describe in detail the variation in clinical subtypes, clinical disease course, past and current practice of treatment, antibody titres, and clinical picture at the various levels of assessing outcome. patients being at least years, and fulfilling the international criteria for igm peripheral neuropathy are eligible. exclusion is primarily based on concomitant diseases influencing peripheral nerve function. several study parameters measuring weakness, sensation, activity and participation, ataxia, pain, and quality of life are of interest. in february an enmc kick of meeting is organized, bringing together an igm peripheral neuropathy study group. the meeting will focus on the development of a core set of outcome measures to be used in future studies in igm peripheral neuropathy. the imagine study started in the netherlands in september , and to date patients were included. the imagine study starts in the united kingdom, italy, and france in the beginning of . centres recruiting at least patients with igm peripheral neuropathy are eligible to participate in the study. during the pns global recommendations from the enmc meeting, as well as the first results of the imagine study, will be presented. puget s , paolantonacci p , burlot l lfb biomédicaments, les ulis, france; lfb biotechnologies, les ulis, france. in dysimmune neuropathies patients (mmn, cidp, gbs), ivig have become the gold standard due to their efficacy and tolerability. acute renal impairment, a rare but serious adverse event, can be induced by all ivig. its incidence is not determined precisely for each ivig. it mostly occurs in patients at risk, with pre-existing conditions (prior renal insufficiency, diabetes, mellitus, arterial hypertension, elderly > years old, dehydratation, hypervolemia, sepsis, paraproteinemia or concomitant use of other nephrotoxic drugs) and treated by immunomodulatory doses. as the number of these patients increases over the years, the choice of ivig in association with the precautionary measures will be decisive to reduce or avoid the occurrence of this adverse event. from s to s, the nephrotoxicity of ivig has been explained by tubular toxicity related to osmotic nephrosis. sucrose, a stabilizer of some ivig, has been implicated as one of the causes of this. even if several new ivig without sucrose are used, renal impairment cases have been described and studies have highlighted that sucrose is not the only ivig stabilizer associated with tubular damage. however, in a recent study, renal impairment cases ( / ) have occurred in cidp elderly patients treated by free-sucrose ivig. the occurrence of renal failure seemed to be low (only / ) for patients treated with sucrose-ivig in association with precautionary measures (hydratation, adaptation of dosage according body mass index, rate infusion). otherwise, since s, cases of renal impairment secondary to hemolysis have been increased with some ivig but would be rare for full ethanolic fractionated products as they have been identified to generate very low occurrence of hemolysis. the occurrence of renal impairment related to ivig seems to be multifactorial, need to find out all causes (mechanisms of different stabilizers, hemolysis, and concentration of ivig). registries would be helpful for physicians to define the incidence, relative risk of ivig-related renal impairment and the right ivig in association with precautionary measures in these patients at risk. the published ultrasound (us) studies on chronic inflammatory demyelinating polyradiculoneuropathy (cidp) report diffusely increased cross-sectional area (csa) of nerves. these data have not been correlated with histological patterns. to date, no further information about us ultrastructural nerve modification has been provided because of limited frequency range ( - mhz) of the us probe. the aim of this preliminary study is to evaluate the correlation between histological findings from sural nerve biopsies and us patterns found with uhfus ( mhz) from sural nerve at the ankle. four patients with cidp underwent uhfus nerve evaluation of clinically affected sural nerve before undergoing a sural nerve biopsy. us findings included: cross-sectional area of the nerve, connective tissue depth and changes in echogenicity of fascicles. those data were then compared to the histological findings obtained in transversal and longitudinal sections. in all patients, uhfus nerve changes were as following: csa was increased; connective tissue was thickened and in two of the four patients hyperechogenicity of fascicles was observed. also, thanks to the ultra-high-resolution of the probe, a direct correlation of the histological and us images was possible, so as a direct measurement of internal microstructure of the nerve. uhfus may be of adjunctive diagnostic value in cidp assessment. more detailed images of sural nerve can be obtained compared with high-frequency us, allowing a better evaluation of the internal structure of the nerve. the increased csa observed in all our subjects seems to relate to an enlargement of connective tissue, as confirmed by the histological study. in particular, we have observed a hyperechogenicity of fascicles in most severe patients; in those cases, histology confirmed an increase of endoneurial depth. we speculate that those findings may explain a preliminary involvement of connective tissue in the pathogenesis of the cidp; our findings of nerve enlargement may be tool to monitor disease activity in cidp, and better understand disease pathogenesis. further studies are needed to confirm these findings and additional data are being processed and will be presented during the meeting. puma a , cambieri c , panicucci e , desnuelle c , raffaelli c , sacconi s . high-resolution ultrasound (hfus, mhz) of peripheral nerves is a valuable non-invasive, painless complement to neurophysiology, especially in the workup of cidp. nevertheless, the current spatial resolution of echographic images doesn't allow a detailed study of the nerves. ultra-high frequency ultrasound (uhfus, mhz) is a new tool with a - times higher spatial resolution than traditional hfus. the aim of this preliminary study is to evaluate sensory and motor nerves structural characteristics found with uhfus in patients with definite cidp. seven patients with cidp underwent uhfus nerve evaluation of median, ulnar, peroneal and sural nerves, bilaterally. us findings included: cross-sectional area of the nerve, connective tissue depth, nerve vascularization and changes in echogenicity of fascicles. patients also underwent electroneurography (eng) and plexus mri. in all patients, uhfus nerve changes were as following: csa was increased; connective tissue was thickened. two of the seven patients presented an epineural hypervascularization, observed at the doppler evaluation; none of them was treated by iv immunoglobulines. echographic changes were present even in the absence of mri abnormalities (root hypertrophy). eng characteristics correlated with the us patterns. uhfus may be of adjunctive diagnostic value in cidp assessment. more detailed images of nerves can be obtained compared with high-frequency us, allowing a better evaluation of the internal structure of the nerve. the increased csa observed in all our subjects seems to relate to an enlargement of connective tissue. in particular, uhfus allows a more detailed study of nerves, demonstrating that the structural abnormalities hit connective tissue, while -conversely to previous studies -fascicles anatomy seems to be spared. we did not show nerve vascularization except in non-treated patients. we speculate that those findings may explain a preliminary involvement of connective tissue in the pathogenesis of the cidp; our findings of nerve enlargement may be tool to monitor disease activity in cidp, and better understand disease pathogenesis. further studies are needed to confirm these findings and additional data are being processed. purcell l , , wojciechowski e , , gibbons p , , jamil k , , menezes, m , , burns j , . sydney children's hospitals network (randwick and westmead), new south wales, australia; university of sydney, new south wales, australia; universiti kebangsaan, kuala lumpur, malaysia. - % of children with cmt are reported to have hip dysplasia. we aimed to investigate the relationship between radiological hip dysplasia indicators and walking pattern in children with cmt. thirty children ( female; . ± . yrs; ± . cm; ± . kg cmt a, cmtx , cmtx , cmt f, cmt a) underwent d gait analysis ( dga), and had an anterior-posterior pelvis radiograph within months of assessment. radiographs were reviewed by two orthopaedic surgeons, and the reliability of measures of dominant limb hip health via radiograph was assessed. of the measures, measures had an intraclass correlation coefficient > . between raters, and the more experienced surgeon's measures were used for further analysis. the measures of acetabular index (ai), centre edge angle (cea), neck shaft angle (nsa), medial joint space, head width, lateral uncoverage, migration percentage and triradiate were used to investigate correlations with kinematic and kinetic dga variables of the pelvis and dominant limb hip, knee and ankle in planes, temporal spatial parameters and gait profile score. dga data were captured with an -camera vicon nexus motion capture system using the lower body plug-in-gait model. gait data were compared to typically developing children ( female; . ± . yrs, ± . cm, ± . kg). five of affected children had a migration percentage > ∘ , representing % of our sample. maximum hip abductor moment in terminal stance was significantly lower than normative reference values, and was moderately correlated with of the radiographic measures (ai r=− . , p= . ; nsa r=− . , p= . and medial joint space r=− . , p= . ). walking speed (normalised to leg length) was correlated with medial joint space (r= . , p= . ) head width (r=− . , p= . ) and triradiate (r=− . , p= . the ganglionopathies are a unique group of disorders with a varied etiology which includes autoimmune disorders, paraneoplastic syndromes and toxin induced causes. it has been observed that despite extensive investigations the cause of a sensory neuronopathy is often idiopathic in approximately - % cases. electrodiagnostic criteria to diagnose a possible ganglionopathy requires at least one sensory action potential absent or three sensory action potentials < % of the lower limit of normal in the upper limbs plus less than two nerves with abnormal motor nerve conduction study in the lower limbs. ulnar sensory-motor amplitude ratio values (usmar) lower than . is useful in differentiating ganglionopathy from axonal length-dependent neuropathy. we performed a retrospective analysis of patients who were either admitted or evaluated in the outpatient department in department of neurology, nizam's institute of medical sciences, with a possible ganglionopathy as per the diagnostic criteria proposed by camdessanchéet al. consecutive patients who sought treatment during a year period with a clinical pattern of sensory neuronopathy were analysed retrospectively. we reviewed the electrodiagnostic studies of patients and calculated the ulnar sensory-motor amplitude ratio values in comparison with patients with a clinical and electrophysiological diagnosis of diabetic axonal sensorimotor neuropathy. the clinical profile of a ganglionopathy from the indian subcontinent comprised of a prominent sensory ataxia as the initial presenting clinical manifestation with a predilection towards involvement of large-fiber sensory modalities. electrophysiologically a pattern of absent snaps was characteristically encountered with a significant value of usmar less than . , enabling differentiation from axonal length-dependent neuropathy. rahman im , jahan it , nahar s , khalid mm , jahan i , hayat s , , , islam z . guillain-barré syndrome (gbs) is a post-infectious autoimmune polyradiculoneuropathy where immune response is triggered by molecular mimicry between glycolipid antigens expressed by an infective agent such as campylobacter jejuni (c. jejuni) and human peripheral nerve gangliosides. cd molecules are mhc-like structures specialized in capturing and presenting a variety of glycolipids to antigen-specific t lymphocytes. the objective of this study was to investigate the possible role of coding region polymorphisms of cd genes in the pathogenesis of gbs in bangladeshi population. single nucleotide polymorphisms (snps) of exon of cd a (* /* ) and cd e (* /* ) in well defined gbs patients and healthy controls were studied to delineate their effect in developing gbs. we genotyped exon of both cd a (cys/tryp) and cd e (gln/arg) genes through pcr-rflp. to validate these findings, direct sequencing of pcr product was performed for at least samples for each position. we found no differences in genotypes and allele frequencies of both genes in gbs patients compared to controls. however, compared to control individuals with cd a* /cd e* haplotype were . times more likely to develop gbs, whereas individuals with cd a* /cd e* haplotype had a reduced relative risk by . times. a positive association of cd a* /cd e* haplotype was observed only in axonal form of gbs ( . % vs. . %, p = < . ). haplotype cd a* /cd e* was prevalent among anti-gm antibody positive gbs patient compared to anti-gm antibody negative patients ( . % vs. . %) though it was not statistically significant. snps in cd a and cd e were not associated with antecedent c. jejuni infection, disease severity and disease outcome at months of follow up. in conclusion, cd polymorphisms are not a susceptibility or disease causing factor in gbs. conversely, increasing knowledge of this field may offer new dimension for the research to elucidate better answer for disease pathogenesis and also contribute to conduct high power meta-analysis. extensive genetic testing was performed on a unique patient, who received national media attention due to her severe cmt phenotype. the index patient was never able to walk independently, had difficulty breathing and swallowing and demonstrated aspiration by a barium swallow study. nerve conduction velocity testing revealed velocities of m/s in her upper extremity. she required continuous positive airway pressure (cpap) for an upper respiratory infection and developed bilateral vocal cord paralysis. we used the combined brief assessment of motor function (bamf) scale to evaluate her disability. her fine motor scale was a / , her upper extremity gross motor scale was a / and her lower extremity gross motor scale was / . unfortunately, she died at age in her sleep, presumably from respiratory arrest. the proband's father was asymptomatic, however, his neurological exam showed pes cavus bilaterally and mild atrophy of the first dorsal interossei muscles in his hands. his nerve conduction velocities were slowed to m/s in the upper extremities and his cmt neuropathy score (v ) was / . we performed whole exome and subsequently whole genome sequencing in the trio. comprehensive structural variant analysis for copy number variations, large deletions, and recombinations was completed by a combination of software tools. known cmt genes were excluded as the underlying cause and the only viable candidate gene remaining was sh tc . we showed expression of sh tc in peripheral nerve and schwann cells. sh tc is a paralogue to sh tc , which causes the recessively inherited dysmyelinating form cmt c. sanger sequencing confirmed the variants in our family, p.v m (c. g>a, chr : ) and p. a p (c. g>c, chr : ) , and showed the segregation of the heterozygous variations. we hypothesize that the heterozygous paternal allele had a minimal effect and let to a very mild or subclinical form of peripheral neuropathy. of interest, the recessive sh tc gene has also been shown to cause mild aberrant forms of peripheral nerve degeneration in carriers of heterozygous disease alleles. in summary, this paper will present genetic and molecular evidence from an extensive n= study proposing a novel cmt gene. charcot-marie-tooth (cmt) is an important cause of morbidity worldwide. it is a heterogeneous disease manifesting as progressive weakness, wasting and loss of feeling in a length-dependent pattern. there are an increasing number of cmt-related genes in the literature. more recently, recessive mutations in the hint gene have been reported as causative of predominant motor axonal neuropathy associated with neuromyotonic discharges on emg. one of the characteristics of autosomal recessive cmt is it varying frequency in different populations and ethnic groups. we sought to evaluate the frequency of mutations in the hint gene in a brazilian cohort with axonal hereditary neuropathy. all patients included in this study born within south east area of brazil. the group consists of consecutive patients with axonal neuropathy screened for recessive or sporadic axonal neuropathy in the neurogenetics laboratory of clinical hospital of ribeirão preto. direct sequencing of the coding region of hint gene was done. among patients screened, were suspected of having recessive axonal neuropathy without neuromyothonic discharges, and two have axonal neuropathy associated with neuromyothonic discharges on emg. we did not find any disease causing mutations among our patients. some previously reported studies reported a high frequency of mutations in the hint gene among recessive axonal neuropathies in some european countries. our results demonstrated that frequencies of mutation underlying genetic hereditary neuropathies are different between different ethnic groups and have implications for the organization of services management of cmt, for genetic counseling, and for gene flow in different world populations. funded by cnpq, fapesp, faepa, pronas (ministry of health). rodriguez-menendez v , ballarini e , malacrida a , , ceresa c , semperboni s , , meregalli c , cavaletti g , nicolini g . school of medicine and surgery, experimental neurology unit, university of milano-bicocca, monza, italy; phd program in neuroscience, university of milano-bicocca, monza, italy. bortezomib (btz) is a proteasome inhibitor widely used for multiple myeloma treatment. btz induced peripheral neuropathy (bipn) is the most frequent adverse effect. bipn in humans, is a dose dependent painful sensory neuropathy characterized by nerve axonopathy and a tendency to recover in the follow-up period after btz withdrawal. bipn affects principally dorsal root ganglia (drg) and different rodent models have shown alterations in sensory neurons, small unmyelinated axons, large myelinated axons, axonal mitochondria and schwann cells. in this work, we evaluated the effects of btz in vitro on drg neurons isolated from adult mice. our interest focused on dystonin, a protein able to interact with all the three components of cytoskeleton (microtubules, microfilaments and intermediate filaments) and able to bind map b (a mt-associated protein) through which can influence golgi apparatus morphology. there are different neuronal isoforms of dystonin and in particular dystonin-a is able to modulate tubulin acetylation and stability through interaction with map b. it is noteworthy to underline that map b expression in the mature nervous system is restricted to sensory neurons. western blot analysis demonstrated that a treatment with nm btz induces a statistically significant decrease in tubulin acetylation. this result does not go along with our previous study where we observed a tubulin polymerization increase after btz treatment. therefore, we have decided to focus our interest on soma organelle organization that is well known to be dependent from cytoskeleton structure. immunofluorescence images showed an altered distribution of acetylated tubulin in soma cytoplasm after hour treatment. moreover, confocal analysis of cis golgi (gm ) demonstrated that in btz treated neurons the normal golgi structure is lost showing a spot-like non perinuclear labeling distribution. additionally, dystonin distribution seemed comparable to that of cis golgi apparatus suggesting that btz could induce a change in dystonin localization which in turn affects golgi organization, probably through map b.these data suggest that the cytoskeleton alterations, induced by btz, could probably cause wrong maturation and trafficking alteration of golgi vesicles consequently impairing the correct sensorial function. romão tt , aleixo bfl , wedemann d , herculano fgn , prado h , cal h , pupe c , bittar c , nascimento ojm . universidade federal fluminense (uff), rio de janeiro, brazil. acute motor and sensory axonal neuropathy (amsan) is a clinical variant of guillain-barré syndrome (gbs) which has rarely been reported in association with human immunodeficiency virus (hiv) and neurosyphilis. we describe a case of a -year-old woman who started with muscle weakness in the left leg followed by weakness in the right leg and in upper limbs in one month. motor symptoms were followed by urinary and fecal incontinence. no preceding symptoms, as diarrhea or fever were referred. neurological examination showed global areflexia with impairment of vibratory and tactile modalities, as well as loss of proprioception. pinprick sensation was moderately compromised. no impairment of higher mental functions was observed. other causes of neuropathies were ruled out. cerebrospinal fluid (csf) examination showed cell/mm , increased protein level, normal glucose level and negative vdrl test. blood analysis showed hiv and positive serology tests, vdrl : and positive t. pallidum hemagglutination assay. igm cytomegalovirus antibody was negative, cd was in normal range and hiv load was undetectable. cranial and spinal cord magnetic resonance imaging revealed extensive involvement of the posterior spinal cord tracts which supports the diagnosis of neurosyphilis. nerve conduction studies showed a significant reduction in compound motor action potentials amplitudes, particularly at lower limbs, and absent sural nerve responses. electromyography revealed positive waves and fibrillation at lower limbs. treatment with methylprednisolone, human intravenous immunoglobulin and ceftriaxone has been settled, resulting in improvement of upper limbs paresis and sphincters dysfunction. antiretroviral therapy was initiated. this amsan case is associated with central nervous system involvement, encompassing an encephalomyeloradiculopathyrelated to hiv/t. pallidum co-infection. c.jejuni is the main etiological agent associated with amsan, but there are some reports of hiv/cmv co-infected patients who presented this gbs variant. facing a patient with amsan, mainly when cns involvement is present, it is of crucial importance to undergo investigation of an ongoing infectious process, such as hiv and syphilis, considering the impact of early treatment on prognosis. roodbol j , , yiu e , doets a , , de wit mcy , monges ms , van nes s , catsman-berrevoets ce , van den berg b , , jacobs bc , and on behalf of the igos-kids consortium. only a few prospective cohort studies so far have investigated the clinical presentation and course of guillain-barré syndrome (gbs) in children using age-adjusted outcome scores. in we started a study based on the igos protocol but adjusted for children with gbs (igos-kids). (pediatric)neurologists from argentina, australia and the netherlands joined forces to determine the clinical and biological determinants of disease progression and recovery in gbs in children from different geographical areas. for each age category specific pain scales are used including the comfort score for children < years old, pain faces revised age - years old and numerical rating scale in children ≥ years old and outcome measures. a new strength score was developed: gbs kids score. this score will be used and validated in addition to the mrc-sum score. the onls, r-ods and fss are also be used in igos-kids. age-dependent quality of life questionnaires were added to the igos-kids protocol, namely the pedsql and the pedsql multidimensional fatigue scale. these scales are available for children ≥ years old. an activity score validated for neuromuscular disorders for children ≥ years old was also added. currently there are children included in igos-kids, eight children from australia and five from the netherlands. additional clinicians and researchers interested in gbs in children are most welcome to participate in igos-kids. prognostic models have been developed to predict the highly variable clinical course of guillain-barré syndrome (gbs) in adults. the clinical course of gbs is equally variable in children, but the current prognostic models have not been validated in children. in this study, we aimed to identify in children with gbs the characteristics at hospital admission that predict the clinical severity and outcome. the study was conducted in two patient cohorts from europe: one (largely) german cohort of children and one dutch cohort of children. clinical information was obtained regarding preceding infection, first symptoms, neurological deficits at admission and nadir, results of additional investigations (cerebrospinal fluid and nerve conduction studies). clinical severity, course and outcome was defined by the gbs disability score, especially the ability to walk unaided, at a follow-up of month, months, months and months after onset of symptoms. univariate and multivariate regression analyses were performed initially on the two separate cohorts. combined the cohorts consisted of children (age median years, range - years) including boys ( %). the median duration between onset of symptoms and hospital admission was days (range ). pain at admission was remarkably frequent and present in ( %) children. in the combined cohorts, children ( . %) developed respiratory failure and one child died. multivariate regression analysis showed that in both cohorts strong predictors of respiratory failure available at hospital admission were cranial nerve involvement, a higher gbs disability score and a shorter period in days between onset of symptoms and admission. this information was used to develop and validate a prognostic model for children with gbs that will be presented at the conference. in conclusion, based on the analysis of two independent cohorts of patients the predictors of respiratory failure and clinical recovery were identified and validated. similar factors were identified for adult patients, although the prognosis in children in general is better than in adults. this information will be used to develop a simple prognostic model for current clinical practice to predict the chance of respiratory failure and outcome in children with gbs. the negative trials of vitamin c in cmt a have highlighted the lack of sensitive outcome measures in charcot-marie-tooth disease (cmt). neurofilaments are abundant neuronal cytoskeletal proteins and their concentration in blood is likely to reflect axonal breakdown. we therefore examined plasma neurofilament light chain (nfl) concentration as a potential biomarker in cmt. blood samples were collected from patients with cmt and age matched healthy controls over a -year period. disease severity was measured using the weighted cmt examination and neuropathy scores. plasma nfl concentration was determined using an in house developed simoa assay based on the nfl antibodies from the nf-l light elisa kit (umandiagnostics). plasma nfl concentrations were significantly higher in cmt patients (median: . pg/ml; iqr: . - . ) than in controls (median: . pg/ml; iqr . - . , p< . ) and correlated with disease severity as estimated by the weighted cmt examination (n= , r= . , p< . ) and neuropathy scores (n= , r= . , p= . ) . concentrations were also significantly higher when subdividing cmt patients by genetic subtype; cmt a (n= , p< . ), sptlc (n= , p< . ) and gjb (n= , p= . ) or into demyelinating, cmt (n= , p< . ) or axonal, cmt (n= , p< . ) forms compared to healthy controls. there was no significant difference in the plasma nfl concentration after year in patients with cmt (n= , mean difference − . pg/ml, % confidence interval − . - . ) or healthy volunteers (n= , mean difference − . pg/ml, % confidence interval . - . ) which is unsurprising as cmt is a slowly progressive disease in which the rates of axonal degeneration are likely to be constant and elevated. in summary, we have shown that plasma nfl levels are significantly raised in patients with cmt and that they correlate with disease severity. this is of relevance not only for the field of cmt but for peripheral neuropathies in general, and suggests that plasma nfl holds promise as a biomarker of peripheral neuropathy in both routine practice and clinical trials. neurolymphomatosis (infiltration of the peripheral nervous system by lymphoma cells) is a rare and usually devastating condition belonging to the spectrum of lymphoma-associated neuropathies. cerebrospinal fluid examination with cytologic examination, flow cytometry and clonality testing by pcr may show malignant cells especially when nerve root involvement is prominent. however, nerve biopsy remains a useful tool to confirm the presence of malignant cells invading the nerve. neuropathological features are important and pcr-based immunoglobulin or t-cell receptor clonality testing on nerve fragments may add notable value for the diagnosis of neurolymphomatosis, although this has not been systematically investigated. we retrospectively studied clinicopathological data and clonality results of nerve biopsy samples in patients with nl from centres, performed between and . among patients with nl, we found % of b-cell lymphoma and one t-cell lymphoma. nl was the first manifestation in , % of patients. the main clinical pattern was symmetrical progressive sensorimotor polyneuropathy in % of patients and pain was a prominent feature in %. clonality testing showed a monoclonal rearrangement in ( %) cases, oligoclonal rearrangement and no amplification in ( %). the main histological pattern was perivascular infiltration of predominant b-cells in the epineurium, without signs of vasculitis. the extent of axon loss was highly variable between patients. various chemotherapeutic regimens were used and the median overall survival was months. only one case showed a monoclonal pattern among control nerve samples from patients with other types of neuropathy including vasculitis and cidp. overall, we found that clonality testing on nerve biopsy specimens may provide decisive information on the presence of neurolymphomatosis, with a sensitivity of . %, a specificity of . %, a positive predictive value of . % and a negative predictive value of . %. we confirm the utility of nerve biopsy for the diagnosis of nl and show the great yield of pcr-based clonality testing to assess the malignant feature of peripheral nervous system lymphoid infiltrates. despite an accurate diagnosis, neurolymphomatosis still remains a devastating disease with an overall poor prognosis. in the last years, cumulative data have shown that patients with amyotrophic lateral sclerosis (als) and mouse models of the disease present loss of small epidermal and dermal nerve fibers and sensory dysfunctions, in addition to the classical motor symptoms. our objective is to characterize this small fiber neuropathy and to clarify if axonal loss involves all sort of fibers equally, or if there is some specificity. for this purpose, we performed an immunohistochemical characterization of total intraepidermal nerve endings (protein gene product; pgp . ), peptidergic (calcitonin gene-related peptide; cgrp) and nonpeptidergical nerve epidermal endings (isolectin b ; ib ) of the sod g a mouse at different stages: presymptomatic stage ( weeks), disease onset ( weeks) and in symptomatic stage ( weeks). the sympathetic sweat gland innervation was immunolabeled for vasoactive intestinal peptide (vip) from very early stage ( weeks) to disease onset ( weeks). the results showed a marked reduction of the intraepidermal nerve fibers already in the presymptomatic stage compared to the wildtype littermates (p< . ). this axonal loss affected more markedly the nonpeptidergic axons from the disease onset stage ( % axonal loss, p< . ), whereas no significant differences were found in the cgrp positive fibers ( . % axonal loss). a reduction of the sympathetic innervation of the sweat glands was also found from the disease onset stage ( % axonal loss, p< . ). in summary, we have found that nonpeptidergic and sympathetic innervation of the skin are predominantly affected in the sod g a mouse model. these findings suggest that this specificity could be used as an accessible biomarker for the disease. hereditary and inflammatory neuropathies are peripheral nerve disorders of different pathophysiology whose identification is crucial for therapeutic approach. diagnosis of cmt is easy when there is a family history, disease course is slowly progressive, neurophysiological findings are homogeneously abnormal. cases of cmt patients with inflammatory-like phenotypes leading to a misdiagnosis of chronic inflammatory demyelinating polyradiculoneuropathy (cidp) have been described. the presence of "red flag signs", such as slowly progressive decline, mild sensory symptoms, minimal electrophysiological progression and no response to therapy should prompt re-evaluation of the diagnosis of cidp. the introduction of nerve neuroimaging has contributed to the diagnostic work-up. we describe cmt patients ( men, women, mean age . ± . yrs, mean neuropathy duration . ± . yrs) with genetically confirmed cmt, who were initially diagnosed with cidp. neurophysiology showed demyelinating features in patients, the remaining patients had axonal features. csf analysis showed albumin-cytological dissociation in patients, oligoclonal igg bands were present in patient. nerve ultrasound in patients with demyelinating neuropathy showed diffuse increased cross-sectional area. mr-neurography in one patient confirmed diffuse nerve hypertrophy and increased signal intensity, supporting the hypothesis of a overlap syndrome. all patients were treated with immunomodulatory therapies. among patients with demyelinating features, patients underwent ivig for months, without benefit; the remaining were treated with steroids, showing temporary improvement. the patients with axonal neuropathy complaining of a progressive history, underwent ex adjuvantibus plasma exchange and ivig, without benefit. given the lack of benefit from therapies, screening for hereditary neuropathies was performed. among patients with demyelinating neuropathy, had cmt a, cmt b, cmt d. among the patients with axonal neuropathy, one was diagnosed with cmt k. in patients an overlap syndrome was present. several clinical and laboratory features (csf protein elevation), might contribute to the misdiagnosis of cidp in cmt patients. refractoriness to immune-modulatory treatment should rise the suspicion of a hereditary neuropathy. an overlap cmt/cidp syndrome may be considered, in front of acute/subacute deterioration and/or proximal muscles involvement. two patient with an overlap syndrome showed benefit after either steroids or ivig. ruiz m , tripodi sm , campagnolo m , zara g , salvalaggio a , ruggero s , toffanin e , anglani m , briani c . neurology, department of neuroscience, university of padua, padua, italy; neuroradiology, padua hospital, padua, italy. facial diplegia is a rare regional subtype of guillain-barrè syndrome characterized by rapidly progressive bilateral facial palsy in absence of other cranial neuropathies, ataxia or limb weakness. the diagnosis is based on history, clinical examination, laboratory data. the outcome of this variant appears to be better than that of classical gbs. although about % of gbs patients clinically exhibit facial nerve involvement, there are only few reports that demonstrate mri gadolinium facial nerves enhancement. a -year-old previously healthy man sought neurological advice for the acute onset of right perioral paresthesias and hyposthenia at the extensor hallucis longus bilaterally. in the previous month, during a trip to china and japan, he had suffered from gastroenteritis. at neurological examination he presented with bilateral extensor hallucis longus hyposthenia. ankle jerk reflexes were absent. one week later, he developed bilateral facial palsy. neurophysiology disclosed absence of activity in the facial nerves innervated muscles bilaterally and signs of mild partial denervation at right l metamer. csf analysis showed increased protein levels ( mg%) and . leucocytes/ l. serological tests for infectious agents were negative and serum levels of angiotensin converting enzyme were normal. antibodies to monosialoganglioside gm of igg isotype were positive ( / ). t brain mri showed, after gadolinium administration, marked bilateral enhancement of the facial nerves in their extra-and intra-canalicular segments. t lumbosacral mri scan ruled out the presence of disc diseases as well as signal modifications within conus medullaris and cauda equine nerve roots. the patient was treated with iv immunoglobulins ( . g/kg/die for days) with benefit on facial weakness. at follow-up examination months later, the patient presented a further improvement of the facial diplegia and neurophysiology disclosed a partial recovery of activity in the facial nerves, with persistent axonopatic damage. bilateral extensor hallucis longus hyposthenia persisted. in conclusion, we report on a regional subtype of guillain-barrè syndrome with the curious association of facial diplegia and bilateral extensor hallucis longus hyposthenia, and igg anti-ganglioside antibodies, that are not commonly described in facial diplegia. moreover, we provide t brain mri evidence of facial nerve involvement. rumora ae , tabbey ma , lograsso g , dolkowski j , haidar j , lentz si , feldman el . department of neurology, university of michigan, ann arbor, usa; department of internal medicine, division on metabolism, endocrinology and diabetes, university of michigan, ann arbor, usa. diabetic neuropathy (dn) is the most common complication of diabetes affecting up to % of diabetic patients. the pathogenesis of dn in type diabetes is directly related to the metabolic syndrome associated with the western diet composed of elevated levels of long chain saturated fatty acids (sfas) and low levels of medium chain sfas. long chain sfas are associated with insulin resistance and dyslipidemia while medium chain sfas have been associated with decreased lipid accumulation and improved mitochondrial function. since dn is primarily a disorder of the sensory dorsal root ganglion (drg) neurons, we sought to evaluate the impact of sfa hydrocarbon chain length on mitochondrial trafficking mechanisms that are critical for distributing atp throughout drg axon to provide energy for synaptic transmission. we hypothesize that sfas with longer hydrocarbon chains will impair mitochondrial trafficking whereas medium length sfas will not impact mitochondrial movement along the drg axon. in this study, we examined the impact of sfa hydrocarbon chain length on mitochondrial trafficking, directionality and velocity in primary mouse drg neurons. drg neurons were treated with increasing concentrations of long chain sfas, stearate and palmitate, or medium chain sfa, laurate, ranging from . to micromolar for twenty-four hours. drg neurons treated with long-chain sfas, palmitate and stearate, showed a significant decrease in the percentage of motile mitochondria whereas medium chain sfa, laurate, does not alter mitochondrial motility. we next assessed whether motile mitochondria in drg neurons treated with palmitate or stearate exhibited altered directionality or velocity of mitochondrial trafficking. palmitate and stearate treatments resulted in a trending decrease in the number of mitochondria trafficking in both anterograde and retrograde directions. furthermore, . to micromolar palmitate and stearate induced a significant decrease in mitochondrial velocity. laurate treatment, on the other hand, retained directionality and velocity of mitochondrial trafficking. these results suggest that hydrocarbon chain length of saturated fatty acids plays an important role in regulating mitochondrial trafficking mechanisms in dorsal root ganglion neurons. impaired mitochondrial trafficking in drg neurons exposed to elevated levels of long-chain sfas may play a critical role in the progression of dn. dyslipidemia is a critical factor that contributes to the development of diabetic neuropathy (dn). the progressive nerve damage associated with dn correlates with the dyslipidemic state characterized by elevated circulating levels of harmful saturated fatty acids (fas) and low levels of beneficial unsaturated fas. in dn, primary sensory dorsal root ganglion (drg) neurons exhibit energy dyshomeostasis and mitochondrial dysfunction, however, little is known about the differential impact of saturated and unsaturated fas on mitochondrial mechanisms in drg neurons. mitochondrial trafficking is an essential mechanism for transporting mitochondria throughout drg axons to provide cellular atp for neuronal function and neurotransmission. since mitochondrial trafficking is regulated by metabolic flux, we sought to determine whether saturated fa, palmitate, and unsaturated fa, oleate, have differential effects on mitochondrial trafficking in drg neurons. we evaluated mitochondrial trafficking patterns and the mitochondrial membrane potential (mmp) in primary drg neurons treated with physiologically relevant concentrations of palmitate, oleate, and combinations of both fas. primary drg neurons treated with . to micromolar palmitate induced a significant and dose-dependent reduction in the percentage of motile mitochondria. these palmitate treatments also induced a dose-dependent reduction in mitochondrial velocity but did not impact the directionality of mitochondrial movement. alternatively, . to micromolar oleate treatments did not impair the percent of motile mitochondria, the direction of mitochondrial movement, or mitochondrial velocity. since palmitate and oleate have differential effects on mitochondrial motility, we next assessed whether oleate could counter the inhibitory effect of palmitate on mitochondrial trafficking. surprisingly, oleate/palmitate mixtures at ratios of : or : prevented palmitate-induced impairment of mitochondrial trafficking. we assessed the impact of palmitate and oleate on mmp by staining palmitate and oleate-treated drg neurons with tetramethylrhodamine methyl ester. drg neurons treated with micromolar palmitate exhibited an increase in the percentage of depolarized mitochondria while mitochondria in oleate-treated drg neurons retained mmp. interestingly, drg neurons treated with : oleate/palmitate mixtures also maintained polarized mitochondria. these results suggest that saturated and unsaturated fas have a distinct impact on mitochondrial trafficking mechanisms in drg neurons and that equimolar ratios of oleate/palmitate can prevent impairment of mitochondrial trafficking. of g alpha- in m r overexpressed sensory neurons significantly reversed the m r-induced reduction in relative levels of total neurite outgrowth (mean±sd: . ± . (control) vs . ± . (g alpha- knockdown), p< . , n= neurons). further, treatment of m r overexpressing neurons with mt or pirenzepine sequestered g alpha- / proteins at the m r and significantly reversed the impaired cytoskeleton-mediated reduction in total neurite outgrowth (mean±sd: . ± . (control), ± (+ nm mt ), ± . (+ micromolar pirenzepine); n= , and neurons, respectively; for treated groups p value= . vs control by one-way anova). mt and pirenzepine also significantly restored mitochondrial trafficking and abundance of mitochondria in the distal neurites. our findings suggest a novel mechanism in which modulation of m r influences tubulin polymerization, mitochondrial distribution in nerve terminals and controls axonal outgrowth and regeneration. funded by cihr and nih. muscarinic receptors are a group of five g-protein coupled receptors (gpcrs) that are targeted by drugs for the treatment of several human pathophysiological conditions. we have recently shown that selective (pirenzepine) and specific (muscarinic toxin : mt ) antagonists of the muscarinic acetylcholine type receptor (m r) induced elevated neurite outgrowth and protected from small and large fiber neuropathy in adult sensory neurons in various animal models (calcutt et al., ) . one of the major cellular effectors activated by gpcrs is extracellular signal-regulated kinase (erk). the erk signaling cascade regulates a variety of cellular processes including growth and proliferation. both g protein and beta-arrestin mediated signaling pathways can lead to erk activation by phosphorylation through different kinases. activated erk in turn can phosphorylate about cellular substrates, thereby mediating diverse functions. in this study, we have analyzed beta-arrestin recruitment, as part of the receptor internalization process induced by agonist/antagonist binding. in addition, we studied phosphorylation of erk by mt and pirenzepine using isoelectric focusing with phospho-erk specific antibodies and a variety of cell-based assays including beta-arrestin and g protein (gnas/gnasl/gnaq/gna /gna /gna ) knockout cell lines. our study revealed that beta-arrestin is recruited to the m r upon mt and pirenzepine treatment. treatment with nm mt and micromolar pirenzepine significantly increased the dual phosphorylation of tyr and tyr residues of erk / in primary rat sensory neurons (p< . ) in comparison to muscarinic agonist carbachol ( micromolar) and general antagonist atropine ( micromolar). we have identified multiple distinct phosphorylation events on the m r by isoelectric focusing that are specific to mt and pirenzepine induction. further, we have shown that mt and pirenzepine-mediated erk phosphorylation is dependent on both g protein and beta-arrestin recruitment to m r. finally, we reveal that increased erk phosphorylation by mt and pirenzepine significantly (p= . and . , respectively) increased phosphorylation of camp responsive element binding protein (creb) at ser . these results show for the first time that antagonists of the m r can activate the erk signaling pathway and possibly drive phenotypic change in adult sensory neurons. funded by cihr # mop- . charcot-marie-tooth disease (cmt) is representative of inherited neuropathies affecting an estimated in people. the immense advances in gene discovery gained from next-generation sequencing (ngs) projects have revealed the extent of cmt's genetic heterogeneity, with over loci already identified. this knowledge is rapidly translated into clinical comprehensive gene testing panels, often containing over genes. such a large genomic space will invariantly yield variants of uncertain clinical significance (vus) in nearly any person tested. this rise of the number of vus creates major challenges for genetic counseling. in addition, less individual mutations in already known genes are being published as the academic merit is decreasing, and most such testing now happens in clinical laboratories. we propose to capture more of this data in the cmt field to gain a more complete collection of alleles in cmt genes, ideally in conjunction with detailed phenotypic data. this represents a rational approach to eventually reduce the number of vus. thus, we have created a unique, community-driven variant database for cmt researchers and clinicians. the inherited neuropathy variant browser provides simple, user-friendly access to currently reported cmt variation, including patient-level genotypic and phenotypic information. we have also designed an interactive rating system of genetic variation to assist the community with interpretation of vus. for the initial release, we have collected genetic variation, along with genotypic and phenotypic data when available, from published literature, clinical lab reports, and our in-house database. we highly encourage new submissions of not only observed pathogenic variation, but also variation of unknown significance. the goal is to provide a platform for the cmt community to store, share, and discuss genetic data in order to resolve variation of uncertain significance as a joint-effort. with active participation, we aim to provide the community with a more complete mutational spectrum in cmt genes to assist allelic interpretation and patient diagnosis. neurofilaments are strictly neuron-specific intermediate filaments crucial for maintaining axonal architecture. pathogenic mutations in nefl, which encodes the light chain of neurofilament, cause dominantly and recessively inherited charcot-marie-tooth neuropathy (cmt). the nefl-associated neuropathy can be either axonal (type e) or demyelinating (type f). most pathogenic nefl variants are dominantly inherited missense mutations, which are thought to cause disease by inducing nefl aggregation, leading to the disruption of axonal transport. however, investigation of disease mechanisms caused by the mutations has been complicated by the neuronal specificity of nefl. we identified a homozygous nefl variant c. c>t predicting a nonsense change p.arg * in a patient with early-onset cmt. to elucidate the disease mechanism, we used pluripotent stem cells, reprogrammed from patient's skin fibroblasts, to differentiate patient-specific spinal motor neurons. the motor neurons revealed a near complete loss of nefl mrna, and absence of nefl protein. our results establish that nefl is not essential for the development of human nervous system but its absence causes progressive axonal neuropathy. we currently profile the transcriptomic alterations of the motor neurons lacking nefl using single cell rna sequencing to identify compensatory pathways. friedreich's ataxia (frda) is the most common autosomic recessive ataxia, due to a trinucleotide expansion within the frataxin gene. within the wide phenotype, frda patients present also with an axonal neuropathy whose pathological mechanisms are not completely known. eight patients ( women, mean age . yrs, range - ) with genetically confirmed frda underwent neurophysiological and nerve ultrasound evaluation at four limbs bilaterally. echogenicity and cross-sectional area (csa) of median, ulnar, radial, peroneal, tibial, and sural nerves were recorded. mr neurography and diffusion tensor imaging (dti) analysis were performed in one patient; fractional anisotropy (fa), radial (rd) and axial (ad) diffusivity of median, radial and ulnar nerve were calculated at proximal, intermediate and distal sites. all patients presented with sensory axonal neuropathy. seven patients ( %) presented with increased csa of median and ulnar nerves at arm and axilla. mean median nerve csa at mid-upper arm was . mm (normal values < mm ), mean ulnar nerve csa at mid-upper arm was . mm (normal values < mm ). mean median nerve csa at axilla was . mm (normal values < mm ), mean ulnar nerve csa at axilla was . mm (normal values < mm ). mr neurography (performed in one patient) confirmed diffuse swelling and signal hyperintensity of median and ulnar nerves at the arm and dti analysis showed abnormal values of fa, ad and rd along the whole course of evaluated nerves thus suggesting a wide alteration of nerves structure. frda patients presented with an axonal neuropathy characterized, at ultrasound, by a nerve enlargement strictly limited to mid-upper limbs in all patients, findings that cannot be solely explained by a dying-back axonopathy, as suggested by several authors. neither a dorsal root ganglia neural loss could explain by itself our findings, because a diffuse csa reduction would have been expected. on the whole, these findings represent a peculiar feature in frda, but its pathophysiologic meaning remains unclear. inherited peripheral neuropathies are an important health concern for which there is currently no disease-modifying therapy. dogs are affected by a variety of peripheral neuropathies that are breed-specific, indicating a strong genetic component. the use of in-bred populations, such as pure-bred dogs, is advantageous for genetic dissection of disease. acquired peripheral neuropathy (apn) is an inherited late-onset generalized polyneuropathy with high prevalence in labrador retrievers. the most prominent features of apn, laryngeal paralysis and pelvic limb weakness, are associated with the longest peripheral motor nerves in the dog. the pathologic features of apn are similar to human peripheral neuropathy. our aim is to understand the genetic and pathologic features of apn for development of this condition as a naturally occurring large animal model for human disease. we performed a genome-wide association study (gwas) and short-read high-depth whole genome sequencing (wgs) to investigate the genetic underpinning of apn in the labrador. our gwas data indicates that apn is an autosomal dominant disease. the initial analysis from the wgs study resulted in a potential causal variant with an autosomal dominant pattern; this variant is associated with an axonal gene. the neuropathologic progression and histologic features of apn are poorly defined. using genetic markers from our gwas study, we are able to confidently identify labradors with pre-clinical apn, from which nerve biopsies can be obtained. preliminary analysis of biopsies from labradors suggest apn is an axonopathy. further histologic data from preclinical and symptomatic dogs is being obtained to further define the pathogenesis in this model. mutations in morc lead to an axonal form of neuropathy (cmt z). to date, nine families have been published with mutations in the morc gene, showing that this gene is frequently involved in cmt. while the recent genetic data clearly established the causative role of morc in cmt z, its phenotypic consequences in patients and role in neuronal biology remains to be clarified. therefore, we aim to look for altered genetic and biochemical pathways with a transcriptomic approach in order to investigate the role of morc in hereditary peripheral neuropathy. we have performed transcriptomic analysis using a human gene expression microarray (v x k, agilent technologies), in three hek- t cell lines: control, morc knock-down (kd) and the overexpression of the most common morc mutation, the p.r w (np_ ) (kr). differential gene expression assessment was carried out using limma moderated t-statistics. standard analysis techniques perform one test for each gene. thus, for each gene, a t-test statistic is reported together with its corresponding p-value. in this analysis we have used the conventional multiple testing p-value correction procedures proposed by benjamini hochberg to derive adjusted p-values. the preliminary results reveals that kd shows up-regulated genes involved in transmission of nerve impulse and cilium metabolism, suggesting that morc might act at this level in the peripheral nervous system. otherwise, kr shows a major alteration of main axonal metabolic pathways, including the overexpression of genes related to the generation of neuronal action potential, transport through the axon and its targeting in synapse formation. kr also shows a marked alteration of gene expression related to organization, assembly and cilium movement and with the axonemal dynein complex assembly. this study provides an important step towards understanding the pathomechanism underlying to morc p.r w and its role in cmt z. aifm encodes a mitochondria associated apoptosis inducing factor. mutations in aifm lead to a wide spectrum of neurodegenerative disorders: cowchock syndrome; a combined oxidative phosphorylation deficiency (coxpd ) with severe encephalomyopathy; x-linked deafness with peripheral sensory neuropathy; spondyloepimetaphyseal dysplasia (semd) with mental retardation; and an infantile motor neuron disease. previous studies showed severe defects in mitochondrial metabolism, related to redox function, mitochondrial fragmentation, and respiratory deficiencies. in addition, some mutations impair the protein expression of aifm and cause an increase in caspase-independent apoptosis. by targeted next-generation sequencing, we detected the aifm c. c>t (p.phe ser), in a year-boy. this mutation was confirmed in his year-old affected brother. electromyography and nerve conduction velocities studies revealed an axonal polyneuropathy with exclusive involvement of motor fibers, with an early childhood-onset. both children currently show normal cognitive and cranial nerves functions. the in silico structural modeling of human aifm showed that the mutation of a phenylalanine to serine at position disrupts the hydrophobic interaction between phe and pro , and consequently, it destabilizes an alpha-loop domain. cartoon of protein superposition between two different human aifm structures suggest that a lack of constraints in this region could affect the interaction between - helix and the - -hairpin regions, a very important stage for the functional activity of the aifm protein. patient-derived fibroblasts were used to investigate the pathological effect of the p.phe ser mutation: fibroblasts from patients show a similar mrna but different protein expression of aifm compared to healthy control fibroblasts. however, they have an aberrant morphology, from fibroblastic to polygonal shape, and they are larger than control fibroblast; mitochondria from mutant fibroblasts are markedly fragmented compared to controls; the viability of the patient's fibroblasts is lower, but it does not correlate with an increase in apoptosis. instead, it seems to be caused by an increase in the expression of genes activating the senescent program, like p and p . our study confirms that variable effect of different mutations on the protein function may contribute to the clinical variability observed in aifm patients. funds: isciii (pi / ); fundació per amor a l' art. sango k , takaku s , niimi n , yako h . diabetic neuropathy project, tokyo metropolitan institute of medical science, tokyo, japan. coculture models of neurons and schwann cells have been utilized for the study of myelination and demyelination in the peripheral nervous system; in most of the previous studies, however, these cells were obtained from the primary culture with embryonic or neonatal animals. because it is recognized that some biological properties of both neurons and schwann cells change with maturation and aging, culture systems of adult animal cells appear to mimic peripheral nerve degeneration and regeneration better than those of immature animal cells. we have established spontaneously immortalized schwann cell lines from long-term cultures of adult fischer rat peripheral nerves. one of these cell lines, designated ifrs , has been shown to retain distinct schwann cell phenotypes, such as spindle-shaped morphology with expression of glial cell markers, synthesis and secretion of neurotrophic factors and cytokines, and fundamental ability to myelinate neurites in cocultures with adult rat dorsal root ganglion neurons and nerve growth factor-primed pc cells. our current investigation focuses on the establishment of the coculture system of ifrs cells and nsc- motor neuron-like cells. nsc- cells were seeded at a low density ( x /ml) and maintained for a week in serum-containing medium supplemented with non-essential amino acids and brain-derived neurotrophic factor (bdnf, ng/ml). after overnight exposure to mitomycin c (mmc, micro g/ml), nsc- cells were cocultured with ifrs cells ( x /ml) and maintained in serum-containing medium supplemented with bdnf ( ng/ml), ciliary neurotrophic factor (cntf, ng/ml) and coenzyme q ( micro m). under this culture condition, overgrowth of nsc- cells was prevented and gradual movement of ifrs cells toward the neurites emerging from nsc- cell bodies was observed. double-immunofluorescence staining carried out at day of the coculture showed myelin protein zero-immunoreactive ifrs cells surrounding the beta iii tubulin-immunoreactive neurites. this coculture system can be a beneficial tool to study the pathogenesis of motor neuron diseases (e.g. amyotrophic lateral sclerosis, charcot-marie-tooth diseases and immune-mediated demyelinating neuropathies) and novel therapeutic approaches against them. santos pp , torezani gs , maciero l , pagliarini lfd , romão tt , abunahman ms , ferreira is , bittar c , pupe c , nascimento ojm . universidade federal fluminense (uff), rio de janeiro, brazil. zika virus (zikv) is a flavivirus related to dengue, yellow fever and west nile viruses, and has been recently associated to the occurrence of neurological complications in children and adults. previous studies have linked zikv to the development of guillain-barré syndrome (gbs), myelitis, meningoencephalitis and ophthalmological manifestations in adults. guillain-barré syndrome (gbs) encompasses a spectrum of post-infectious neuropathies characterized by different distributions of weakness and sensory impairment. serum anti-ganglioside antibodies are often found and are related to different clinical patterns. recently we have encountered patients in rio de janeiro, brazil, with distal edema in lower limbs, acute weakness, pain and sensory disturbances during the acute stage of an acute febrile exanthematous illness. the symptoms persist for up to days, with complete resolution afterwards, without specific therapy. blood concentrations of muscle enzymes show normal values, and electromyography and nerve conduction studies (emg/ncs) are usually unremarkable. of note, all cases have had positive rt-pcr for zika virus, indicating an illness that occurred during the viremic phase of this arbovirus infection, and complete recovery within the expected timeframe for the resolution of the systemic viremia. there is a subset of patients who developed acute weakness very early after the initial viral symptoms, with clinical, laboratorial and electrophysiological findings that substantially differ from gbs. we describe three cases with similar features suggestive of an acute infective polyneuritis (aipn). one might hypothesize that zikv might lead to a direct neurotropic injury, significant enough to cause weakness and sensory complaints, but not severe enough to cause permanent damage, resolving in conjunction with decreasing blood viremia. we consider that these patients differ from classical gbs because their illness begins during the acute febrile stage of an infective illness and the clinical course is more rapid leading to complete resolution in a few days. savransky a , mozzoni j , massaro sanchez mp , reisin r , monges ms . department of neurology, hospital de pediatria j.p. garrahan, buenos aires, argentine; department of physical therapy, hospital de pediatria j. p garrahan, buenos aires, argentine; department of neurology, britain hospital, buenos aires, argentine. our objective is to describe a series of children with guillain barré syndrome (gbs) included in the igos protocol. as part of the igos multicenter protocol, pediatric patients meeting gbs diagnostic criteria, who consulted within the first weeks of symptom onset and had parental consent to participate in the study were included. patients were also offered to participate in the extended -year protocol. all patients were evaluated according to the igos protocol. patients were recruited between october and june . twenty-four patients, eight girls, participated. ages ranged from months to years (mean . years). all patients agreed to participate in the extended protocol, which was completed by of them. eight patients completed the -year follow-up and are still under evaluation. five patients were lost to follow-up.twenty-two had the classic variant of gbs and two miller fisher. radicular pain in the back or lower limbs was reported by %. twenty-three patients underwent lumbar puncture and albumin-cytological dissociation was found in . in all cases, csf was stored for proteomic studies. all patients underwent emg showing aidp in , and amsan and aman in each. one patient with aidp developed to cidp. in patients full-spine mri was performed and cauda equina enhancement was found in every case. three patients required ucip, two with invasive and one non-invasive ventilation. all patients were treated with gammaglobulin, with a second dose at weeks in cases with a poor response. all patients who followed the protocol were evaluated with the gbs disability score. median score was at baseline and between and at the -year assessment. we describe a series of children with gbs as a part of an international protocol including patients of different ages. pain was a frequent and early symptom and could be determined despite the young age of our patients. most patients fully recovered. we were invited to participate in igos kids to better assess this age group. saysavath k , somchit v , t. umapathi . mittaphab hospital, vientiane, lao pdr; setthathilath hospital vientiane; national neuroscience institute, singapore, singapore. laos people democratic republic is a country of . million people in south east asia. largely agricultural, its capital and metropolis is vientiane. adult neurological services are concentrated at mittaphab hospital, vientiane serviced by three neurologists. on the average about six cases of guillain-barré syndrome (gbs) are seen per year. it is believed that most patients do not seek medical attention. cases appear to cluster during the rainy season. most patients present late, often at the second week of illness when recovery is unapparent after seeking treatment from traditional medicine doctors and at district hospitals. a substantial number of patients seek treatment at hospitals across the border, in neighboring provinces of thailand. common antecedent symptoms are viral prodrome and diarrhea. miller fisher syndrome appears to be rare, possibly because of the mild deficits that do not prompt patients to seek medical attention. diagnosis is made largely from clinical features and from spinal fluid analysis. nerve conduction studies are not available. patients are often treated with steroids by internists. intravenous immunoglobulin and plasma exchange are not available. common complications include pneumonia, autonomic dysfunction (fluctuating blood pressure), pressure sores and depression. patients who develop respiratory failure are nursed at a twelve-bedded intensive care units. plans are afoot to set up a prospective gbs database, systematically study antecedent infections, including of flaviviruses, and develop low-volume plasma exchange as a feasible therapeutic modality. a gene therapy approach for treating cmt c neuropathy schiza n , markoullis k , richter j , tryfonos c , kagiava a , sargiannidou i , christodoulou c , kleopa ka , . neuroscience laboratory; department of molecular virology; neurology clinics, cyprus institute of neurology and genetics and cyprus school of molecular medicine, nicosia, cyprus. charcot-marie-tooth type c (cmt c) is the most frequent form among recessively inherited demyelinating neuropathies and results from mutations in the sh tc /kiaa gene. sh tc mutations cause loss of function of the sh tc protein suggesting that gene replacement therapy may be useful for treating cmt c. sh tc −/− mice develop all major aspects of the human pathology including early onset progressive peripheral neuropathy with hypo-and demyelination along with decreased motor and sensory nerve conduction velocities, offering a relevant model for testing treatments for cmt c. in order to develop a gene replacement strategy for cmt c, we generated a novel lentiviral vector, lv-mpz-sh tc .myc, to drive expression of the human sh tc cdna under the control of the myelin protein zero (mpz/ p ) promoter specifically in myelinating schwann cells. a c-terminus myc tag was included to facilitate expression analysis. a control vector (mock) was also produced in which the sh tc cdna was replaced by the egfp reporter gene. we first confirmed expression of hsh tc in hela cells transfected with the pcdna -cmv-sh tc .myc vector. immunofluorescence analysis confirmed a strong expression of sh tc specifically at the plasma membrane with additional localization in a dotted pattern intracellularly. for in vivo gene delivery we used both intraneural and intrathecal injections of the lv-mpz-sh tc .myc vector in -week to -month old sh tc −/− mice. expression of virally delivered hsh tc was assessed weeks after injection. immunofluorescence analysis showed hsh tc immunoreactivity in perinuclear schwann cell cytoplasm in sciatic nerve teased fibers of sh tc −/− mice following both intraneural and intrathecal delivery, while lumbar intrathecal gene delivery resulted additionally in expression of hsh tc in the lumbar roots. real time pcr analysis confirmed hsh tc mrna expression in both lumbar roots and sciatic nerves. thus, we have developed a novel lentiviral vector for schwann cell targeted gene delivery to treat cmt c and for testing possible therapeutic effects in the mouse model of the disease. hereditary neuropathies are a group of disorders which are characterised by the systemic impairment of peripheral nerves. more than neuropathies are associated with causative gene defects [ ]. charcot marie tooth (cmt a) neuropathy is the most frequent hereditary neuropathy, triggered by a mutation in the peripheral myelin protein gene (pmp ). cmt a leads to a primary loss of myelin sheath and afterwards to a degeneration of axons [ ] . symptoms appear with the degeneration of axons, whereas demyelination is thought to be largely asymptomatic. for that reason, we investigate in the mechanisms of axonal degeneration. for our analysis, we used purified axoplasma without detectable myelin proteins of the sciatic nerves of weeks old pmp -c mice. in this early stage of the axonal degeneration sarm was significantly increased. the nad + concentration in the axoplasma was dramatically reduced. this correlates to the previous finding that sarm promotes axonal degeneration by cleavage of nad + . additional studies showed an increase of the nad + -dependent axonal protective factor sirt in pmp -c sciatic nerves. nmnat- , known as the active component of the wallerian degeneration slow gene, was unaffected in axoplasma of pmp -c sciatic nerves. summarised, these results indicate that the pathway of sarm , nad + and sirt may play a critical role in axonal degeneration in neuropathy. we report two unrelated czech patients with cmt b , both sporadic cases in the family. patient is a year old man with congenital glaucoma after ophthalmological surgeries. his early motor development was normal. at the age of years parents noted gait problems, first neurological examination was at the age of years, when emg showed diffuse motor and sensory neuropathy with severely decreased ncv ( - m/s). he developed foot deformities (pes cavovarus) and underwent corrective orthopedic surgeries at the age of years. after several dna tests for demyelinating cmt the sfb gene was sanger sequenced and a novel missense mutation p.ile asn was found in homozygous state in the patient and in heterozygous state in both parents. the patient was later tested also by ngs of a panel of all genes to be causal for hereditary neuropathies and no other potentially causal variants were detected. patient is years old man, with normal early motor development. at the age of years parents noted gait problems with distal leg weakness which progressed into distal leg plegia at the age of years. hand weakness was noticed since the age of years. he has severe atrophies of distal muscles of all extremities, is self ambulant. at the age of , emg showed unrecordable responses from nerves of lower limbs and ncv was measurable only on ulnar nerve and was m/s. at the age of years increased intraocular pressure was diagnosed and he uses anti glaucoma eye drops. after many single gene tests for demyelinating cmt, we used ngs of a panel of genes known to be causal for hereditary neuropathies and two novel heterozygous, probably pathogenic variants affecting invariant splice sites were detected: c. - a>g and c. - _ del, both confirmed by sanger sequencing. the first variant is also in the father, but the second is probably de-novo (not detected in parents, despite correct parentity). sekiguchi y , kikuchi si , konno si , sekiguchi m . department of orthopaedic surgery, fukushima medical university school of medicine, fukushima, japan. carpal tunnel syndrome (cts) is the most common entrapment neuropathy. ultrasonography can be used to detect anatomic changes in cts. more recently, it has been shown that doppler ultrasonography can detect increased intraneural blood flow in cts. the purpose of this study was to determine the most suitable finding of pre-and postoperation in cts by ultrasonography. a total of wrists of patients with nerve conduction study (ncs) proven cts were evaluated with ultrasonography. we measured the median nerve's cross-sectional area (csa) and intraneural blood flow of median nerve by ultrasonography. the correlation between these ultrasonographic measurements, ncs severity and duration of clinical cts symptoms was analyzed. the csa (mean, mm ) was no significantly reduction after successful carpal tunnel release. morphologic median nerve changes may persist for a longer period regardless of successful surgery and clinical improvement. however, intraneural blood flow is increasing after successful carpal tunnel release. we conclude that doppler ultrasonography results strongly correlate with post operated cts improvement. hence doppler ultrasonography is a useful method for functional improvement of pre-and post cts operation. senger jl , chan km , olson jl , webber ca . university of alberta, edmonton, canada. the beneficial effects of a preinjury crush conditioning lesion (cl) on peripheral nerve regeneration is well-documented in animal models. no human studies have been attempted to date, given the ethical dilemma of deliberately injuring an intact nerve, and the difficulty in predicting the timing of a nerve injury. recent studies demonstrate that hour of electrical stimulation (es) produces effects similar to cl in neuronal cultures. this, coupled with a surgical environment favoring nerve transfers, in which an intact nerve is deliberately cut to reinnervated a denervated muscle, means that es may be clinically translatable to enhance regeneration. this study hypothesizes that es prior to nerve injury will enhance nerve regeneration. twelve sprague-dawley rats were divided into four groups based on conditioning-type to the mid-common peroneal (cp) nerve: es ( ), crush ( ), sham-es ( ), and naïve ( ). one week following conditioning, they underwent a cut/coaptation of the cp nerve at the sciatic trifurcation. post-cut day , nerves and dorsal root ganglia (drgs) were collected. axonal counts of nerves stained with nf- revealed similar regeneration between es and crush ( . vs. . mm, p= . ) that was superior to sham-stimulation ( . mm) or no-conditioning ( . mm, p< . ). a greater number of axons at the distal tip were present in animals that received either type of conditioning compared to the unconditioned cohorts. drgs were stained with neuronal injury marker growth associated factor- (gap- ), and satellite cell glial cells with glial fibrillary acidic protein (gfap). significant increase in gap- expression at three days was observed in es and crush cohorts compared to sham or naïve (p< . ) cohorts. the satellite glial cells of es and crush conditioning showed a significant increase in gfap expression ( . % and . % respectively) compared to sham ( . %) and naïve ( . %) drgs. by demonstrating similar improvements in axon regeneration, this proof of principle project suggests that es conditioning may produce regenerative outcomes comparable to the classical crush injury model. in turn, this suggests that es may be a promising method for delivering conditioning lesions in clinical trials for conditioning nerves prior to surgical intervention. we report a unique case of newly developed waldenstrom's macroglobulinemia (wm) in a patient with chronic inflammatory demyelinating polyneuropathy (cidp) with antibodies against myelin-associated glycoprotein (mag) and sulfatide who was undergoing treatment with intravenous immunoglobulines (iv-ig). subsequent rituximab infusions did not have a positive impact. patients with wm can develop demyelinating and axonal polyneuropathies and few patients have anti-mag and/or anti-sulfatide antibodies. anti-mag antibodies ( % of wm) are associated with sensorimotor axon loss and demyelination and anti-sulfatide ( % of wm) with sensory axonal loss. rarely, both antibodies can be present, with a more severe clinical phenotype. cidp associated with anti-mag and anti-sulfatide antibodies can represent independent entities, not associated with wm. there are no reports to date of patients with cidp associated with anti-mag and anti-sulfatide antibodies that developed wm during immunomodulatory therapy with iv-ig. in addition, subsequent rituximab infusions after the iv-ig were stopped have not been proven beneficial, as has been previously reported for anti-mag cidp patients. seventy-six year old right-handed gentleman presented with persistent numbness in his left foot, three months following artificial disc placement in his lumbar spine. gradually he developed sensory ataxia. no radicular signs were present on exam or impingement on serial spine mri's. ncs/emg studies were consistent with a cidp variant with severely prolonged distal motor latencies. serum anti-mag and anti-sulfatide antibodies were elevated. chronic therapy with iv-ig was able to partially stabilize the symptoms; however, six years later he newly developed wm. subsequent infusions with rituximab, after iv-ig was stopped, did not improve the clinical picture or the ncs/emg findings. wm can newly develop in an autoimmune setting, such as cidp associated with anti-mag and anti-sulfatide antibodies. in this particular case, there was an ongoing immunomodulatory therapy for our cidp patient, as he had monthly iv-ig infusions. this may reflect a possible induction of pathological b cell clone proliferation during the iv-ig treatment. subsequent rituximab infusions, after the iv-ig was stopped, did not improve the symptoms or the demyelination features on ncs/emg. he continues to be symptomatic despite efforts. charcot-marie-tooth disease (cmt) is an inherited neuropathy without known cure (prevalance: : ). duplication of the gene encoding the peripheral myelin protein of kda (pmp ) underlies the most common subtype cmt a. severely affected cmt patients suffer from sensory and motor symptoms with wheelchair-boundness. the clinical phenotype is highly variable and is determined by the amount of axonal loss, but the molecular mechanisms of the disturbed neuron-glia interaction are poorly understood. risk factors have not been investigated. therefore, cmt-net, a german network funded by the german ministry of education and research (bmbf, bonn, germany) includes interdisciplinary expertise from molecular biology, neurology, neuropathology and human genetics in order to identify genetic and non-genetic risk factors of disease severity of cmt by: (i) examining the mechanisms of the disturbed axon-glia-interaction and neuronal vulnerability, (ii) identification of genetic modifiers and (iii) novel therapeutic targets, (iv) validating outcome measures in children and adults, (v) establishing a biobank and (vi) exploring the disease burden via an internationally harmonised patient registry. cmt-net includes three service structures cmt-net will focus on cmt a, but also includes rarer subforms. we will provide the scientific basis for the development of translational approaches to therapy in patients. our approach bridges cutting edge molecular screening techniques, transgenic animal models of altered axon-glia interactions (fly, chick, mouse, rat), state-of-the-art genomic technologies and human patient trials in order to understand and treat the disease aggravation in cmt. sezer g , tekol y , sezer z , . erciyes university, betül ziya eren genome and stem cell centre, kayseri, turkey; erciyes university, school of medicine, pharmacology department, kayseri, turkey; erciyes university, good clinical practice and research centre, kayseri, turkey. analgesic effects of antidepressant drugs are well known for a long time, however, their systemic side effects limit their usage as an analgesics. venlafaxine is an antidepressant drug that has different structure. our purpose was to investigate whether systemic analgesic effect has been proved drug, venlafaxine, has local peripheral antinociceptive action. we applied vanlafaxine ( μl , , , μg) to male, sprague-dawley rats' paws by intraplantar injection and also by intraperitoneal route ( , , , mg/kg) in formalin test, a model for acute and tonic pain. we also pretreated another groups of rats with mg/kg naloxone (opioid receptor antagonist), mg/kg cpt (adenosine a receptor antagonist) or saline (ip.) before μg/paw venlafaxine injection. to check the effect is local or not, we determined the blood levels of venlafaxine in at different time points after both the local and systemic applications by gc-ms method. datas were expressed as number of flinches and total time for biting/licking of the injected paw over phase ( - min) or phase ( - min) and analyzed using the student's t-test. venlafaxine induced antinociception at , and μg concentrations by the local peripheral application and at , mg/kg doses by the systemic application in formalin test and the effects were comparable. pretreatment with naloxone diminished the effect of venlafaxine in the both phases, however, it was not statistically significant. pretreatment with cpt decreased venlafaxine induced antinociception only in phase . neither naloxone nor cpt changed formalin induced nociceptive behaviors alone. this is the first that determines the peripheral antinociceptive actions of venlafaxine in rat formalin test. with roles of opioid and adenosine a receptors in this action. our results suggest that venlafaxine has local peripheral antinociceptive effect and such an activity may led to trials for to use this drug as a cream-gel formulation for analgesia in clinics in the future. topical application might permit the attainment of higher and more efficacious concentrations in the region of the sensory nerve terminal, with limited systemic side effects. shah a , hoffman em , klein cj , staff np . mayo clinic, rochester, usa. cipn is a common dose-limiting complication for patients with cancer. the long-term disease burden of cipn is compounded by increasing cancer survivorship, yet there are minimal data on long term outcomes following onset of cipn, especially in population-based studies. we utilized the rochester epidemiology project to examine incidence and disease burden of cipn among individuals of olmsted county, minnesota with neurotoxic chemotherapy exposure between and . clinical records were queried for the presence of neuropathic signs, symptoms and icd- diagnostic codes as well as for patient provided information on impairment with activities of daily living and use of pain medications. a total of individuals with incident exposure to neurotoxic chemotherapy agents between and were identified. based on aan criteria for identifying peripheral neuropathy, ( . %) of these individuals were determined to have cipn, while ( . %) controls did not. the median time from incident exposure to reported symptom onset was days (iqr . - ). patients with cipn received a neuropathy icd- diagnosis in merely cases ( . %). median survival following incident chemotherapy exposure among all cases and controls was . years with a significantly longer mean survival in cases with cipn as compared to that of controls ( . years vs. . years, p< . ). in addition to acute effects in cipn, individuals surviving greater than years following exposure to neurotoxic chemotherapy continue to self-report increased symptoms of numbness (or . , % ci . - . ) and pain (or . , % ci . . . ) of the extremities. through utilization of patient provided information, our study was able to collect data on long-term impairment associated with previous history of exposure to neurotoxic chemotherapy. our results are consistent with previous reports of the high incidence of cipn in the first two years following incident exposure. additionally, our results provide evidence of high incidence of cipn independent of individual chemotherapeutic agent used. additionally, our results indicate icd- -cm diagnostic code attribution may dramatically underestimate the magnitude of cipn. increased survival following exposure to neurotoxic chemotherapy and its long-term disease burden necessitates further study of among survivors. the utility of quantitative muscle ultrasound as a marker of disease severity in charcot-marie-tooth (cmt) disease subtypes was investigated. muscle ultrasound was prospectively performed on individual muscles from cmt patients ( cmt a, cmtx , cmt a) and compared to muscles from age and gender-matched controls. muscle ultrasound recorded echogenicity and thickness in representative muscles including first dorsal interosseus (fdi) and tibialis anterior (ta charcot-marie-tooth (cmt) disease is the most frequent inherited peripheral neuropathy, and there is currently no available cure. the most common subtype of cmt, cmt a, is completely associated with duplication of the pmp gene, which encodes peripheral myelin protein of schwann cells. previous studies of cmt a mainly relied on rodent models, and it is not yet clear how pmp overexpression leads to the phenotype in patients. based on induced pluripotent stem cell (ipsc) technology, we herein developed a brand new in vitro cell model of cmt a, called cmt a-hipscs, in the hopes of simulating the developmental progress of the disease and gaining new insights into its pathogenesis. here, we efficiently derived ncscs from cmt a-ipscs and assessed the potential of the isolated cmt a-neural crest stem cells (ncscs) to differentiate into peripheral neurons and schwann cells using defined media. we found that, unlike normal control ncscs, cmt a-ncscs rarely generated schwann cells. instead, cmt a-ncscs produced numerous endoneurial fibroblasts in the schwann cell differentiation system. we further established a pmp -overexpressing ipsc model, and obtained similar results when pmp -ncscs were subjected to schwann cell differentiation. these results suggest that the development of schwann cells in cmt a patients is interrupted by the duplication of pmp . with the exception of the demyelination-remyelination process, developmental disabilities of schwann cells should be considered as an underlying cause of cmt a. shimoi t , yamada t . international university of health and welfare, tochigi, japan, cmt japan, tokyo, japan. charcot-marie-tooth (cmt) disease is the most common hereditary motor and sensory neuropathy. our preliminary report suggests that a certain cmt patient has the recruitment disorder of motor units during muscle fatigue and this disorder may be a factor of "super fatigability" in motor neuropathy patients. if the "super fatigability" occurs, we would expect patients with this characteristic to become slower in recovery from muscle fatigue than patients without this characteristic. in order to verify this hypothesis, we measured characteristic of recovery from muscle fatigue in charcot-marie-tooth patients with electromyographic study. twenty three participants were asked to maintain their % of maximal voluntary isometric contraction (mvc) of elbow flexor until exhaustion as the fatigue exercise. in addition, the participants asked to perform s of their % of mvc at , , , , , , , , , s after the fatigue exercise as recovery tasks. the surface emg (semg) signals of biceps brachii muscle were determined during the exercise and tasks. muscle force, median power frequency (mdpf) and the root mean square of semg amplitude (rms) were used as objective parameters of muscle fatigue. borg scale was used as a subjective parameter of muscle fatigue. six of twenty three participants showed significant decrease of rms during the fatigue exercise. in consideration of this result, we compared alteration of mdpf in recovery task between six participants with decrease of rms (abnormal group) and seventeen participants with increase of rms (normal group). as the result, the abnormal group had at least s as the recovery time from muscle fatigue in contrast with s of normal group. the recovery time from muscle fatigue in subjective parameter was shorter than the time in objective parameters in each group. our data support the "super fatigability" hypothesis. and that hypothesis may induce "hidden muscle fatigue". comparison between complex regional pain syndrome type and based on electrophysiologic, imaging and clinical findings shin jy , moon jy , sung jj . seoul national university hospital, seoul, republic of korea. complex regional pain syndrome (crps) is a constant regional neuropathic pain characterized by various kinds of motor, sensory, and autonomic changes. conventionally, the crps is divided into type i and ii according to the absence and presence of nerve injury. but the pathogenesis of crps is not fully understood yet. and there is still no systematic comparative study between crps type i and ii. we compared between crps type i and ii using multimodal approaches including electrophysiologic, imaging, and clinical findings. the patients ( type i and type ii) diagnosed with crps using the international association for the study of pain (iasp) diagnostic criteria were included. type i and ii were divided by electromyography and nerve conduction study. we obtained clinical information such as continuing pain, allodynia, hyperalgesia, edema, temperature, skin color, sweating, trophic change from patients. all patients were evaluated by bone scan, thermography, quantitative sudomotor axon reflex test (qsart), quantitative somatosensory test (qst). the ratio of qsart and temperature threshold abnormality in type ii was higher compared to type i ( . % vs . %, . % vs . % respectively, p = . and . ), among clinical symptoms, sweating change significantly high in type ii compared to type i ( . % vs . %, p = . ). other electrophysiologic and imaging, clinical findings were not significantly different in both type. in this study, we identified that crps type i and ii are distinguished not only by the nerve injury but also by the sudomotor function, and qsart can serve as a good technique to differentiate between crps type i and ii. it is estimated that there are two distinct pathogenesis in crps. our results may be helpful to diagnose crps correctly and understand the pathogenesis of crps. sjogren syndrome (ss) is an autoimmune inflammatory disorder of exocrine glands resulting in xerophthalmia and xerostomia. ross syndrome is a rare entity characterized by tonic pupil, hyporeflexia, and segmental anhidrosis. we present a -year-old hispanic woman with debilitating sensory and autonomic neuropathies, and persistently elevated anti-ss-a and anti-ss-b antibodies, without the classic sicca complex. she initially developed diarrhea and an ear infection, then felt toe and later leg numbness, which eventually spread to her cheeks and tongue, over few months. four years later, her thumbs and index fingers started tingling. also, she developed orthostatic lightheadedness, tachycardia, segmental hypohidrosis of the right abdomen, and hyperhidrosis of the remaining trunk, intermittent erythema, chronic diarrhea, and a -pound weight loss. her exam demonstrated orthostatic hypotension, bilateral tonic pupils and light-near dissociation, sectoral palsy of the right iris sphincter, stocking-distribution diminished sensation to all modalities, pseudoathetosis, areflexia, dysmetria, intention tremor, romberg, and sensory gait ataxia. mri of neuraxis demonstrated t -weighted hyperintensity in the dorsal spinal cord from c to the lower thoracic level, with mild atrophy. electrodiagnostic testing was consistent with moderate-to-severe, length-dependent, asymmetric, sensory polyganglionopathies. csf showed oligoclonal bands. serology showed elevated antinuclear antibody ( : , reference < : ), ss-a ( , reference < u), ss-b ( , reference < u), and rheumatoid factor ( , reference < iu/ml) titers. the remaining workup was negative for infection (syphilis, hiv, htlv, hepatitis, lyme disease), paraneoplastic syndrome (anti-hu and ganglionic nicotinic acetylcholine receptor antibodies), pyridoxine intoxication, malignancy (chest/abdomen/pelvis ct, breast ultrasound, axillary lymph node flow cytometry, colon and esophagus biopsy), celiac disease, vitamin deficiency, autoimmune disease (anti-aquaporin and anti-gq b antibodies), and adrenoleukodystrophy. the patient received ivig and steroid with some gait improvement and currently takes mycophenolate. midodrine and fludrocortisone resolved her dizziness. this case highlights an important, treatable sensory ganglionopathy and systemic autonomic neuropathy due to sjogren syndrome, and illustrates the overlapping clinical triad of ross syndrome, which may guide future management. siles am , , assylbekova d , , diaz-manera j , , rojas-garcia r , , cortes e , , gallardo e , , illa i , , querol l , . neuromuscular diseases unit, neurology department, hospital de la santa creu i sant pau, univeristat autònoma de barcelona, barcelona, spain; centro para la investigación biomédica en red en enfermedades raras (ciberer), madrid, spain. inflammatory neuropathies are a heterogeneous group of peripheral nerve diseases that respond to immune-therapies. chronic inflammatory polyradiculoneuropathy (cidp) and multifocal motor neuropathy (mmn) are two chronic inflammatory neuropathies responding to intravenous immunoglobulins (ivig). b lymphocytes are involved in their pathogenesis. while widely used in clinical practice, ivig's mechanism(s) remain not completely understood. ivig are reported to lead to b-cell anergy and to increase regulatory t-cell function and frequency. regulatory b cells (bregs) are a rare subset of b lymphocytes that suppress immunopathology acting upon several target cells in the immune system. impaired breg yields have been described in a plethora of autoimmune conditions. the presence of regulatory b cells in inflammatory neuropathies and the effect of ivig therapy on their frequencies has not been studied. the aim of this study is to describe the frequencies of bregs in cidp and mmn and the effect of ivig on their frequencies. patients fulfilling diagnostic criteria for cidp or mmn and matching controls were included. pbmcs were obtained by gradient centrifugation before ivig infusion and one week after treatment. b-cells were isolated with negative selection magnetic beads, cultured and activated with the tlr agonist odn and anti-human igg+iga+igm. il- secretion capacity was assessed by flow-cytometry. twenty-eight patients were included of whom where cidp and mmn. of all patients included, received ivig and were suitable for pre and post ivig breg frequency comparisons. breg frequencies did not differ in patients (before ivig treatment) and controls (p= . , mann whitney test, two-tailed). however, the frequencies of bregs significantly increased one-week after treatment with ivig (p= . , wilcoxon matched pairs test, two-tailed). when stratifying by disease subtype, breg frequencies increased in cidp patients after ivig (p= . , wilcoxon matched pairs test, two-tailed) and in mmn (p= . , wilcoxon matched pairs test, two-tailed) although results did not reach statistical significance in mmn. this is the first study that studies the breg frequencies in cidp and the first study that addresses the effect of ivig on breg frequencies. our study provides the proof of principle that bregs could become a biomarker for response to ivig but this would need a larger and prospective study. siles am , , martínez-hernández e , diaz-manera j , , rojas-garcia r , , gallardo e , , illa i , , graus f , querol l , . paraneoplastic neuropathies (pn) are rare, immune-mediated disorders of the peripheral nerve with important prognostic implications. ectopical expression of neural antigens in the tumor leads to the development of onconeural antibodies. several autoantibodies associate to pn, including anti-hu, anti-caspr or anti-cv antibodies but a significant proportion of pn lack identifiable antigens. adhesion molecules that are autoantigens in other neuropathies, like contactin- , are present in several types of tumors. our study proposes a systematic screening of autoantibodies against neural cell-adhesion molecules and neural structures to detect novel antigenic reactivies in pn. thirty-five patients followed in our centre and at the neuroimmunology-multiple sclerosis unit at hospital clínic de barcelona, with pn fulfilling diagnostic criteria of possible (n= ; . %) and definite (n= ; . %) paraneoplastic disease were included. serum samples were obtained and tested by immunocytochemistry against contactin- (cntnt ), neurofascin (nf ) and the cntn /caspr complex. primary cultures of dorsal-root ganglia (drg) and rat schwann cells were incubated with patients' sera to detect antibodies targeting neural structures. ten individuals ( . %) presented with a tumor and a neuropathy involving both sensory and motor symptoms. the remaining patients ( . %) presented with a tumor and a classical sensory neuronopathy without anti-hu or any other onconeuronal antibody. among the latter, ( . %) patients were diagnosed with small-cell lung carcinoma. the rest of the individuals ( . %) associated diverse malignancies. we did not detect any sera reacting against cntn , nf or the cntn /caspr complex. in igg antibody screening experiments, patients ( . %) reacted against drg neurons, of them ( . %) reacting strongly, and patients ( . %) reacted mildly against rat schwann cells. in igm experiments, patients ( . %) reacted slightly against drg neurons and patients ( . %) against rat schwann cells, of them ( . %) featuring strong staining. experiments screening antibodies against motor neurons and immunoprecipitation assays are ongoing. overall, % of patients reacted strongly against either neurons or schwann cells. our study did not detect antibodies against the neural adhesion molecules cntn , nf and the cntn /caspr complex in patients with pn. however, a significant proportion of pn patients harbour antibodies targeting neural structures, which suggests that novel neoplasm-associated antigens remain to be discovered. simmons m , tao f , abreu l , zuchner s , li j . department of neurology, vanderbilt university school of medicine, nashville, tennessee, usa; hussman institute for human genomics, university of miami, miami, florida, usa. objective: despite of a shared genetic mutation of the trisomy of chromosome p (c p ), patients with charcot-marie-tooth type- a (cmt a) present with a high variability of their disease severities. the underlying cause for the variability is still unclear. in this study, we tested a hypothesis whether a second genetic mutation known to damage the nervous system is also present in cmt a patients with early onset and severe phenotypes. methods: from a cohort of patients with cmt a mutation (chromosome p duplication), we identified patients with an early onset (< or = years of age) of the disease. four of the eleven also had dna testing for a panel of known cmt-related genes and sequencing of mitochondrial dna in addition to the dna testing for c p duplication. results: besides the c p duplication, we identified three additional mutations in the four patients with early onset. the mutations were a missense mutation of arg his in mpz gene, an a g mutation in mitochondrial trna for glycin and a homozygous mutation of c p duplication. three of the four had symptomatic onset at birth. one showed symptoms at years of age. conduction velocities were severely reduced in all four patients (from to m/s). interpretation: traditional approaches to identify genetic modifiers, including snp association, assume that those modifiers are clustered in a small region of human genome and shared by the studied patients. however, our study suggests that genetic modifiers in cmt a may be highly diverse and scattered throughout the genome, which could make the conventional approach via the genetic variants association difficult. supported by grants from ninds (r ns ) and the national center for advancing translational sciences (ul tr sjogren's syndrome(ss) is a systemic auto-immune disease that apart from exocrine glands may affect any organ. involvement of peripheral nervous system results in wide spectrum of neuropathic manifestations. the aim of our study was to evaluate the clinico-electrophysiological patterns and pathological characteristics of neuropathy in sjogren's syndrome (ss) patients presented to neuromuscular clinic in a tertiary hospital from south india. this is a retrospective study from the departments of neurology, rheumatology and pathology from nizam's institute of medical sciences. twenty one patients with diagnosis of ss and peripheral neuropathy between to were analysed. clinical records, conventional nerve conduction studies, lip and nerve biopsy reports were collected.in patients with ss associated neuropathy,male to female ratio was : . in ( . %) neuropathy was the initial manifestation,while in ( %)exocrinopathy preceded neuropathy. the patterns of neuropathy included mononeuropathy multiplex(mnm) in patients ( %),ganglionopathy in ( %),length dependant, trigeminal, autonomic neuropathy and cidp in ( %)and cranial neuropathy in ( %).eighteen( %) were seropositive..schirmer's test was positive in ( . %).nerve biopsy showed vasculitis in patients, demyelinating and axonopathy in patients each.we conclude that neuropathy is frequently the initial presentation of ss.mnm is the common pattern followed by ganglionopathy. pattern of neuropathy helps in arriving at the diagnosis of ss. confirmation of ss is not by mere serology. schirmer's test and lip biopsy are equally essential for the diagnosis especially in seronegative patients when clinical index of suspicion is high. siskind ce , tesi rocha c . stanford health care, palo alto, ca, usa; stanford university, stanford, ca, usa. here, we report the first case of a human found to have a homozygous, presumed disease causing variant in the arl ip gene, causing charcot marie tooth disease (cmt) with central nervous system findings. the proband was born at term with apgars of and at and minutes. he was found to have iugr and was hospitalized for days for weight gain. he developed respiratory distress during the admission and was intubated. due to inability to extubate, he was transferred to stanford children's hospital, where he remained for three months. noted during admission was hypotonia, areflexia, minimal voluntary movment, sinus tachycardia, and dysautonomia. brain mri found polymicrogyria and cerebral underdevelopment with generally normal-appearing brainstem, with moderate ventriculomegaly. ncs found length dependent polyneuropathy with axonal degeneration. follow up muscle and nerve biopsy found immature muscle and amyelinating neuropathy the patient had normal plasma amino acid, acylcarnitine, lactate, pyruvate, urine organic acid testing, opththalmology exam, newborn screen, and normal array cgh. genetics ordered whole exome sequencing through baylor genetics laboratory (houston, tx, usa), which found a homozygous nonsense variant in arl ip : c. c>t, p.r x. a second child had been identified by baylor with two variants in this gene. that child had hypotonia, respiratory distress and seizures, and a muscle biopsy consistent with sma. the parents of that child chose to withdraw care at months of age. our patient's parents continued with aggressive therapies, including tracheostomy and g-tube for feedings. he had several subsequent hospitalizations for respiratory distress, possible seizure activity, and buldging anterior fontenelle, but now, at two years of age, has made developmental progress and is living at home with his family. he is able to smile, reaching for toys and swatting objects. he has little voluntary movement, and no longer responds to light touch stimuli. overall, this is the first picture of a child affected with a severe amyelinating form of cmt that causes weakness, hypotonia, and possible seizures, with the main concerning feature being the severe respiratory distress that may be life threatening, but can be managed with extreme care. charcot marie tooth (cmt) disease is the most common inherited peripheral neuropathy. patients frequently ask whether pregnancy will affect their cmt, whether cmt will affect their pregnancy, the optimal delivery and whether they or their child will have a higher risk of complications during pregnancy or delivery. so far few studies address these questions. currently no guidelines exist for the management of pregnancy, delivery and postnatal care in cmt patients. the aim of the study is assess the impact of pregnancy on cmt and assess how cmt affects pregnancy, delivery and care of the new born baby. we designed a retrospective questionnaire with expert help from an obstetrician with a special interest in pregnancy in patients with medical conditions. the questionnaire is divided into four parts (prior, during, after pregnancy and delivery) and includes questions on impairment, falls, pain, fatigue and respiratory complications during those periods; type of delivery, possible complications, details of anaesthesia and difficulties looking after the baby in the first months postpartum. so far women ( pregnancies) with cmt and related disorders have answered the questionnaire. % of patients had cmt a, the remaining had various subtypes of cmt and related disorders. patients reported deterioration of cmt symptoms during pregnancy in % of pregnancies with resolution of symptoms after pregnancy in % of pregnancies. of symptoms questioned walking ( %), balance ( %), and hand function ( %) deteriorated the most. there was an increased use of orthoses and walking aids during pregnancy. the majority of women ( %) had natural delivery, % were assisted and % had caesarian sections which was similar to the uk population ( %). no complications with anaesthesia were reported. the survey is currently ongoing. we plan to survey consecutive patients. data acquired from this survey will provide valuable information on current practice and will inform future guidelines and standard of care in charcot marie tooth disease. multifocal motor neuropathy (mmn) is a slowly progressive disorder in adults, characterized by asymmetrical limb weakness, mainly affecting the arms. despite beneficial effect of immunoglobulins, weakness gradually progresses. a major determinant of muscle weakness is the degeneration of affected motor axons. treatments aiming to reduce loss of motor axons require objective tools to quantify such an effect. therefore, we applied the compound muscle action potential (cmap) scan, which is an electrophysiological method that, with increasing transcutaneous stimulus-currents, successively activates all motor units (mus) in a muscle. it captures the contribution of enlarged mus due to reinnervation by the presence of relative large discontinuities in the scan. the aim of the present study was to identify pathophysiological changes of mu-loss and reinnervation in mmn patients by means of the cmap-scan. recordings were obtained from mmn patients. cmap-scan recordings were performed in the median nerve at the wrist where motor responses were recorded from the thenar muscle. we determined the number of largest cmap-scan discontinuities by means of a novel marker, d , where a low number is indicative of mu-loss and enlarged mus. furthermore, we applied the recently developed method of professor hugh bostock for obtaining a mu-number estimate from the cmap-scan. the median peak cmap amplitude was . mv (range . - . mv) and median d was (range - ). in three mmn patients with a normal maximum cmap amplitude (> mv) a reduced d (< ) was found indicative of mu-loss and enlarged mus. furthermore, d and the estimate of mu number were significantly related (r = . , p < . , n = ). the findings suggest that the cmap-scan is a sensitive tool in detecting the underlying pathological changes of reinnervation and mu-loss in mmn, more so than standard maximum cmap amplitude. it is quick and easy to perform and has the potential to be useful for follow-up studies. smith ag , thurgood b , revere c , hauer p , aperghis a , singleton jr . university of utah, salt lake city, utah, usa. corneal confocal microscopy (ccm) directly and quantitatively assesses corneal innervation including nerve fiber length (nfl) and density (nfd). ccm has shown promise as a diagnostic test. we have previously demonstrated that ccm has a diagnostic performance for diabetic neuropathy (dpn) similar to skin biopsy with assessment of intraepidermal nerve fiber density (ienfd) and nerve conduction studies (ncs). the responsiveness of these surrogate measures to dpn progression and their relation to clinically meaningful outcomes has not been well explored. diabetic patients undergoing annual retinopathy examination were recruited. each underwent ccm, ienfd, ncs including sural sensory and peroneal motor responses, the utah early neuropathy score (uens), the norfolk quality of life -diabetic neuropathy (nqol-dn, a validated neuropathy specific qol scale), and a minute walk test ( mwt). with dpn based on symptoms ( %) or signs underwent repeat testing at months and at months. at baseline, nqol-dn correlated with sural sensory amplitude (ssa) (− . , p< . ), peroneal motor conduction velocity (pcv) (− . , p< . ) and ienfd (− . , p< . ). no ccm metric was related to qol. mwt distance correlated with ssa ( . , p< . ), nfl ( . , p< . ) and nfd ( . , p< . ). over months, there was a significant worsening in dpn signs assessed by the uens (increase . +/− . , p< . ). ssa declined . uv (p< . ) and ienfd . fibers/mm (p< . ). there was no change in any ccm metric, pcv or nqol-dn. these findings suggest measures of distal axonal integrity are most sensitive to neuropathy progression, with ienfd having the greatest responsiveness. in contrast, ccm was not responsive to dpn progression. both ncs and ienfd (but not ccm) were significantly correlated with neuropathy-specific qol, whereas ncs and ccm measures correlated with physical functioning. the responsiveness of ienfd and ssa, and their relationship to qol support their selection as endpoints in dpn clinical trials. chronic inflammatory demyelination polyradiculoneuropathy (cidp) affects in , people, and is marked by chronic autoimmune infiltration of peripheral nerves and destruction of the myelin sheath. with current therapies, only % of cidp patients achieve complete remission. to produce more effective, mechanism-based therapies, we study mice with a partial loss of function g w substitution in the autoimmune regulator (aire) gene on the non-obese diabetic (nod) background (nod.aire gw/+ ) that develop spontaneous autoimmune peripheral polyneuropathy (sapp) resembling cidp. autoimmunity can result from defective immunosuppression. the potent, immunosuppressive cytokine interleukin (il- ) is increased in the peripheral blood mononuclear cells (pbmcs) of active phase cidp patients relative to remission phase patients. further, pbmcs from cidp patients produce il- in response to the myelin protein p . despite these findings, whether il- is important for cidp pathogenesis is not known. thus, we sought to determine the role of il- in sapp. il- was highly upregulated in sciatic nerves of nod.aire gw/+ mice with sapp, suggesting it may play an immunosuppressive role in pathogenesis. however, genetic ablation of il- in nod.aire gw/+ mice lead to a paradoxical delay in disease development. age-matched il- -deficient nod.aire gw/+ mice exhibited no sciatic nerve infiltrate and no reduction in nerve conduction during electrodiagnostic studies. interestingly, the delay in sapp was specific, since the incidences of five other autoimmune manifestations in il- -deficient nod.aire gw/+ mice were unchanged relative to il- -sufficient nod.aire gw/+ controls. importantly, il- -deficient nod.aire gw/+ mice did not have colitis, which is consistent with previous studies of il- deficiency on the nod background. il- is known to perform effector functions in autoimmunity by promoting b cell secretion of immunoglobulins. however, genetic ablation of b cells did not affect neuropathy development in nod.aire gw/+ mice, suggesting b cells are dispensable for pathogenesis and unlikely to mediate the protective effect of il- deficiency. il- -deficient nod.aire gw/+ cd + t cells, which are sufficient to transfer sapp, exhibited increased activation, increased interferon gamma secretion, and preserved nerve-specific t cell activation. these data suggest t cell activation and priming are unperturbed and not the mechanism of protection. in summary, our data showed that il- was paradoxically an effector cytokine in sapp. long exercise test (let) has been used especially in myotonic syndromes and muscle channelopathies. marked decrement in compound muscle action potential (cmap) amplitude after prolonged exercise was previously reported in patients with paramyotonia congenita, hyperkalemic or hypokalemic periodic paralysis. we describe a patient with secondary hypokalemic paralysis who showed abnormal let results. a -year-old man presented with ascending flaccid paralysis which evolved in a hyperacute fashion. the patient became quadriplegic after two hours. initial laboratory evaluation revealed severe hypokalemia, with normal thyroid function. we performed electrodiagnostic studies including long exercise test as proposed by mcmanis et al. nerve conduction study was normal, but marked decrement in cmap amplitude (up to % decrease after minutes) was noted after prolonged exercise. despite oral and intravenous potassium replacement, serum potassium level was not corrected as expected. the unusual clinical course prompted for evaluation of secondary etiologies. abdomen computed tomography scan revealed a . x . cm-sized mass in the left adrenal gland. aldosterone to renin ratio was elevated, suggestive of primary hyperaldosteronism. genetic study for cacna s mutation turned negative. after receiving laparoscopic adrenalectomy, the patient experienced no further attacks, and also was able to stop his antihypertensive medication. let may show abnormal results in condition with reduced membrane excitability, even without true channelopathy. according to international criteria, the diagnosis small fiber neuropathy (sfn) is based on clinical symptoms in combination with a reduced intraepidermal nerve fiber density (ienfd) in skin biopsy and/or abnormal temperature threshold testing (ttt). the sensitivity of skin biopsy is moderate to good, although ienfd is normal in about % of patients with sfn complaints. furthermore, ttt is a widely available diagnostic tool, but lacks specificity. corneal confocal microscopy (ccm) has been described and is used in clinical practice as an objective, non-invasive diagnostic tool to detect small nerve fiber damage in patients with diabetes mellitus. this study examines the applicability of ccm in patients with sfn, and the value of ccm as an additional diagnostic tool in sfn. we will include healthy participants to compare the results with the recently published ccm normative values, and patients referred to the sfn center maastricht with the clinical picture of sfn. corneal nerve fiber density (cnfd), branch density (cnbd), fiber length (cnfl), and the tortuosity coefficient (cnft) will be determined in all participants. the results will be compared with the ienfd and ttt. preliminary results will be presented. southanalinh k , university of health sciences, vientiane capital, lao p.d.r. located in south east asia, lao pdr is a landlocked country with a population of about . million inhabitants. the health indicators are among the lowest in south east asia. the total health caregivers in consisted of , persons corresponding to a ratio of . health workers per inhabitants. the main network for health care service provision remains the public system. its health care facilities consist of four central teaching and referral hospitals; five regional hospitals, including one teaching hospital; provincial hospitals; district hospitals, and about health centers. only one in seven sick people receives modern health care treatment. most people rely on self-medication and/or reliance on self-healing. neurological care is a very new field. knowledge of common neurological disorders among both the lao population and medical staff is only beginning to be spread. there are three neurologists in the country. six neurology residents are currently being trained in a three-year program supported by the association pour la promotion des neuro-sciences au laos (association for the promotion of neuro-sciences in laos) and the asean neurological association. indeed, resources are scarce. in the peripheral nerve diseases domain for example, we have only one electromyography machine that was only temporary used when emg experts from france and from singapore came to teach residents. a significant mismatch between the provision of specialized neurologic services and the needs for them exists, especially in rural areas. also, health insurance is not available for the majority. as a consequence, patients have to bear the costs themselves, which constitutes a limit to the access of available healthcare facilities. neurologic training centers, laboratory facilities and equipments are limited. optimizing available human resources, integrating primary, secondary, and tertiary healthcare tiers and making medical treatment more affordable are need to improve neurologic care in the developing world. in certain low-income countries with limited human and financial resources, it may be difficult for governments to apply some of these recommendations on their own. in these circumstances, it is suggested that countries work with international agencies, nongovernmental organizations or other partners to put their plans into practice. spina e , topa a , iodice r , tozza s , dubbioso r , ruggiero l , santoro l , manganelli f . department of neuroscience, odontostomatological and reproductive sciences, university of naples "federico ii", naples, italy. chronic inflammatory demyelinating polyradiculoneuropathy (cidp) is a disabling disease and about % of patients may become persistently disabled over time. our aim was identify clinical prognostic factors of long-term disability in a large series of cidp patients. we collected data from cidp patients with definite diagnosis according efns/pns criteria and positive response to first-line therapies (immunoglobulin or corticosteroids) including sex, age of onset, phenotype, disease duration, course of disease (monophasic/relapsing-remitting or chronic progressive) and disability at the time of diagnosis assessed using the modified rankin scale (baseline mrs). all patients had clinical assessment of disability through mrs within the last months (last mrs). ordinal logistic regression model was applied to evaluate the relationship among the clinical parameters and last mrs, considered as ordinal outcome ( - ). anova test for repeated measures was applied to test the overall effects of different course on disability accumulation while t-test was performed to evaluate inter-group differences for parametric variables. we found a significant relationship between last mrs and the course of disease [p< . , z= . , or: . ]. disability accumulation was greater in patients with chronic progressive course than those with monophasic/relapsing-remitting course of disease [p= . ]. moreover, patients with progressive course were older [p= . ]. our data suggest that chronic progressive course of disease may be a major negative prognostic factor for long-term disability in cidp patients. to note that a chronic progressive course of disease is also associated with an older age from the beginning and a more pronounced worsening over the course of disease. sprenger a , lichtenstein t , henning t , lehmann hc . department of neurology, university hospital of cologne, cologne, germany; institute of diagnostic and interventional radiology, university hospital of cologne, cologne, germany; department of neurology, university hospital of cologne, cologne, germany. an unresolved problem in the treatment of inflammatory neuropathies is the lack of valid and reliable diagnostic biomarkers to evaluate axonal damage. we investigated if "diffusion tensor imaging" (dti) and mri t w multi echo dixon imaging are eligible methods to determine proximal nerve injury in chronic inflammatory demyelinating polyneuropathy (cidp). in this prospective observational cohort study the sciatic nerve of cidp patients and age matched healthy controls was investigated. all subjects underwent multimodal mri imaging to determine fractional anisotropy (fa) and muscle fat fraction of the biceps femoris and quadriceps femoris muscle. patients were evaluated by mri, clinical examination and nerve conduction studies at baseline and after six months. the mean fractional anisotropy (fa) value was significantly lower in the sciatic nerve from cidp patients compared to controls. fat fraction of the biceps femoris and quadriceps femoris muscle were significantly higher in cidp patients compared to controls. mri outcome parameters remained unchanged after six months. our study demonstrates the utility of mri imaging to differentiate between "healthy" and functional constricted proximal nerve segments. we postulate that dti and dixon mri might be eligible methods to assess proximal nerve damage in cidp. the presence of peripheral myelin protein (pmp ) has been known for decades, but its functional role was uncovered only recently. recent characterization of pmp -deficient mice revealed a role of pmp in the lipid homeostasis of myelinating schwann cells. in this study, we analyzed the functional impact of pmp on myelination. to decipher the role of pmp , experimental demyelination was performed in myelinating dorsal root ganglia cultures, and in vivo re-myelination was assessed after experimental peripheral nerve damage. we used the myelinating dorsal root ganglia (drg) model in pmp -deficient schwann cell cultures, combined with an established de-and remyelinating protocol in order to analyze myelination in vitro. we also performed experimental nerve crush in pmp -deficient mice. morphometric parameters were defined for the in-vitro experiments and functional parameters such as nerve conduction velocity and the clinical score were additionally measured for the in vivo experiments. structural analyses of the drg cultures revealed fibers expressing myelin basic protein (mbp) and pmp , as well as fibers positive for mbp alone. in contrast to our previous in vivo data, we were also able to detect myelin segments that stained positive for pmp , but were negative for mbp. pmp -deficient drg-cultures demonstrated slightly greater nodal lengths than the control cultures. this trend was significantly augmented after in vitro de-and remyelination, which also resulted in decreased internodal lengths only now, while conserving an intact myelin structure. concomitantly, in vivo nerve crush gives rise to a more severe phenotype in pmp -deficient mice than in wild-type controls. consistent with this, nerve conduction studies showed a delay in remyelination, and analysis of semi-thin sections demonstrated an altered fiber structure in the peripheral nerve biopsies. together, these data suggest that in addition to its role in glial cell lipid homeostasis, pmp also plays a role in remyelination of the injured peripheral nervous system. anti-mag neuropathy remains a difficult diagnosis to treat given its limited therapeutic options. of all interventions, rituximab has emerged as the most effective, although its effect has been with mixed results, especially in patients with advanced axonal loss. lenalidomide is another promising immune modulating therapy, whose effect has been well demonstrated in neuropathy associated with poems (polyneuropathy, endocrinopathy, organomegaly, m-spike protein, and skin changes) syndrome, a condition that has several striking parallels to anti-mag neuropathy. the use of lenalidomide has not been previously described in anti-mag neuropathy. herein, we describe a case of lenalidomide-responsive anti-mag neuropathy in a patient with advanced axonal loss. suichi t , misawa s , sato y , beppu m , sekiguchi y , shibuya k , watanabe k , amino h , kuwabara s . department of neurology, graduate school of medicine, chiba university, chiba, japan; clinical research center, chiba university hospital, chiba, japan. polyneuropathy, organomegaly, endocrinopathy, m-protein, and skin changes (poems) syndrome is a rare cause of demyelinating neuropathy associated with plasma cell dyscrasia and vegf overproduction. several diagnostic criteria for the disorder have been published, but sensitivity/specificity analyses, and their validation have never been performed. the aim of this study is to establish valid diagnostic criteria for poems syndrome. consecutive poems patients, seen at chiba university hospital since , were screened. of these, we have set a gold standard group of poems syndrome, based on treatment response and exclusion criteria during -year follow-up, and patients was diagnosed as having definite poems syndrome. we also collected patients with cidp (demyelinating neuropathy control) and with multiple myeloma, primary amyloidosis, or mgus (m-protein control). criteria for poems syndrome was defined as having two of the three major criteria (polyneuropathy, m-protein, and elevated serum vegf level) and at least two of the four minor criteria (extravascular volume overload, skin changes, sclerotic bone lesions, and thrombocytosis) which were determined by logistic regression analyses. according to the criteria the sensitivity was %, and the specificity was %. our results indicate that the proposed criteria have an excellent diagnostic accuracy, and are useful in clinical practice, presumably leading to early diagnosis and treatment. intraepidermal electrical stimulation (ies) is a new technique to that assesses the function of a-delta fibers in the epidermis. using this technique, we previously reported that the epidermal pain threshold was two-hold in asymptomatic diabetic patients than in normal subjects (muscle nerve : - , ). subsequently, we reported that the elevated pain threshold negatively correlated with intraepidermal nerve fiver density (jpns : s , ). empirically, it is known that lowering the skin temperature makes it less likely to feel pain. therefore, it is necessary to investigate whether the results of ies are affected by skin temperature. the aim of this study was to investigate the influence of a low skin temperature on pain threshold. we recruited subjects with a mean age of . years. for nociceptive stimulation, we used an ies method with a concentric micro-needle electrode that was developed specifically for the selective stimulation of cutaneous a-delta fibers. we placed the ies electrode onto the extensor digitorum brevis and began stimulation with intensity strong enough for the subject to feel a pricking sensation, then reduced the current in steps of . ma until no sensation was felt. we defined pain threshold as the minimum electrical intensity at which a subject felt a pricking sensation. firstly, we measured pain threshold at skin temperature above degrees celsius. then, we put an ice pack on the extensor digitorum brevis for min to lower the skin temperature, and measured pain threshold at skin temperatures below degrees. mean pain threshold values above degrees and below degrees of skin temperature were . and . ma (p< . ), respectively. our data indicated an elevated pain threshold in epidermis with a low skin temperature. one of most common methods for nociceptive stimulation is painful co laser stimulation. some co laser stimulation studies reported pain threshold increased with a low skin temperature. our result is similar to that of co laser stimulation. pain threshold using ies is very easy and non-invasive technique. it may be useful for the evaluation of small fiber neuropathy. svačina mkr , röth p , bobylev i , sprenger a , zhang g , sheikh ka , lehmann hc . department of neurology, university hospital cologne, cologne, germany; department of neurology, university of texas, houston, usa. intravenous immunoglobulins (ivig) are an effective treatment in guillain-barré-syndrome (gbs). in most patients, the optimal ivig dose and regime is unknown. in serum and ivig preparations, immunoglobulin (ig) g form igg dimers, which are assumed to consist of idiotypic/anti-idiotypic antibody pairs. however, data about kinetics of igg dimer formation in gbs are lacking. to study igg dimer formation, c bl/ mice were injected with ivig and anti-gd b antibody or pbs. blood sera were collected h, h and week post injection. a third cohort received an anti-gd a/gt b antibody and blood was collected h post injection. igg was extracted and subtyped into polymeric, dimeric and monomeric fractions using the Äkta fplc system. dialysed dimeric and monomeric igg fractions were examined for the presence of anti-ganglioside antibodies by anti-ganglioside antibody elisa. further, blood samples from gbs patients were collected before (pre-ivig) and after treatment with ivig (post ivig). serum samples were examined for igg dimers and monomers using the Äkta fplc system. in the mouse model, a maximum peak of igg dimer formation was observed h post injection. in gbs patients' samples, igg serum levels and igg dimer content was significantly higher after treatment with ivig. we demonstrate here the feasibility to assess igg dimer formation in an animal model and in gbs patients' samples after treatment with ivig. h after ivig treatment appears to be the optimal time point to assess igg dimer formation. further studies are warranted to determine the utility of igg dimer formation as surrogate marker for treatment response in gbs. svaren j , moran jj , wu x , gutmann l , shy m . university of wisconsin-madison, madison, wisconsin, usa; university of iowa, iowa city, iowa, usa. development of outcome measures for clinical trials in cmt a is a major challenge given the slowly progressive nature of the disease. outcome measures can be used to measure a) target engagement for a given therapy, as well as b) disease process and c) disease burden. several candidate therapies have been shown to reduce pmp levels in cmt a rodent models and thereby ameliorate the symptoms of pmp overexpression. measuring pmp mrna reduction in human trials has so far been limited to analysis of skin biopsies by qrt-pcr, which did not demonstrate clear elevations of pmp mrna during, nor a reduction following, ascorbic acid trials. the analysis of skin biopsies is hampered by variable amounts of schwann cells (sc) in skin biopsies, as well as the variable amount of pmp in sc as previously established by immuno-em in cmt a skin biopsies (katona et al., ) . therefore, it is important to develop optimal normalization criteria to address the variability inherent in skin biopsy analysis. ideally this will employ normalization to sc-specific genes that are not altered by cmt a status. to optimize normalization, we have performed rna-seq analysis of skin biopsies from patient and control skin biopsy samples. analysis of these data after normalization to read depth indicated that pmp levels were . fold higher in cmt a patient samples compared to control skin biopsies. however, there was significant variability in pmp levels particularly in cmt a samples, which may be due to variable amounts of schwann cells in cmt a skin. using a combination of sc-specific genes for normalization, we were able to reduce the apparent variability and optimize the differential levels between cmt a and control skin biopsy samples. we also identified other sc-specific genes that were apparently induced in cmt a skin biopsies relative to control. these studies provide a new framework for gene expression analysis in skin biopsies, enabling more precise evaluation of pmp levels in clinical trials for cmt a as a measure of target engagement. in addition, the normalization framework may also be applicable to other types of cmt. chronic inflammatory demyelinating polyneuropathy (cidp) is the most common chronic autoimmune neuropathy, with an estimated prevalence of between and per , people. it can cause temporary disability in the affected individuals and may eventually lead to permanent disability or death. cidp is commonly treated with intravenous immunoglobulin (ivig) therapy or corticosteroids. octagam ® % is licensed for cidp in france, while octagam ® % is licensed for cidp in germany and belgium. this analysis presents data from three open, multicenter, non-interventional, single-arm, non-controlled studies of a post-authorisation safety surveillance (pass) program for the subset of patients receiving octagam ® % or % for neurological indications, focusing on patients with cidp. briefly, data from in-and out-patients in austria, france, germany, and uk treated with octagam ® for neurological disorders were collected by physicians and analyzed to assess safety and tolerability of the treatment. of patients included in the three studies, patients ( . %) received octagam ® for neurological indications, of which patients ( . %; mean age . years [range - ]) had cidp. the mean dose of octagam ® per course was . g/kg bw for patients with cidp; for the other neurologic indications, the dose ranged from . (for multiple sclerosis) to . g/kg bw (for guillain-barré syndrome). premedication was not needed in . % of these patients. the development of clinical appearance since last observation (mean: every . months) was assessed for of the cidp patients by their treating physicians. the majority of observations ( . %) assessed the patients as stable and . % showed even an improved clinical appearance. only . % of the observation periods resulted in deteriorations. adverse drug reactions were rare: of the infusions received by patients with neurological disorders, . % of infusions were associated with an adr ( . % of infusions in cidp patients). overall, treatment with octagam ® was effective and well-tolerated in patients with cidp. these results are consistent with data for the overall patient population (including patients with primary and secondary immunodeficiencies, dermatological and other diseases). szepanowski f , szepanowski lp , kleinschnitz c , kieseier bc , stettner m . department of neurology, medical faculty, university duisburg-essen, essen, germany; department of neurology, medical faculty, heinrich-heine-university, düsseldorf, germany. lysophosphatidic acid (lpa) is a pleiotropic signaling lipid that acts as ligand for at least six specific g protein coupled receptors. schwann cells (sc) are known to mainly express the lpa receptor subtype. an emerging body of in vivo evidence has linked lpa with injury induced peripheral nerve demyelination as well as neuropathic pain. however, the molecular mechanism underlying its demyelinating effect has remained largely unclear. myelinated dorsal root ganglia (drg) cultures were treated either with lpa, lpa + am (lpa antagonist) or vehicle. we assessed myelin basic protein, tumor necrosis factor alpha (tnf-alpha) as well as the sc differentiation marker sox by immunocytochemistry. additionally, myelin was investigated by sudan black staining. to better understand the relevance of lpa signaling for demyelination in vivo, we performed sciatic nerve crush in c bl/ mice treated with am at mg/kg in order to study schwann cell expression of tnf-alpha, sox and sox , a marker for sc dedifferentiation, by immunohistochemistry. in drg cultures, lpa caused a significant reduction of myelin as demonstrated by both sudan black staining and immunocytochemical analysis of myelin basic protein. demyelination was paralleled by an upregulation of tnf-alpha as well as downregulation of sox . lpa mediated effects were found to be blocked by addition of the lpa receptor antagonist am . in c bl/ mice, am treatment prior to crush injury increased sox expression in scs in the distal nerve stump while reducing the number of cells expressing sox . these data indicate that lpa may be a critical factor to shift scs towards an injury-associated phenotype and contribute to the onset of wallerian degeneration. szepanowski lp , szepanowski f , kleinschnitz c , stettner m . department of neurology, university hospital essen, essen, germany. glyphosate-based formulations comprise the world's most commonly used herbicides. in non-resistant plants, glyphosate exerts toxic effects most likely via inhibition of aromatic amino acid synthesis by interfering with the shikimate pathway. while glyphosate is the active ingredient, herbicidal formulations contain several adjuvants, including polyethoxlated alkylamines (poeas). although glyphosate has long been considered safe for use in humans and animals, several studies have implicated glyphosate and/or the commonly used adjuvants in cytotoxicity, carcinogenicity and endocrine disruption. furthermore, glyphosate-based herbicide has been reported to mediate neurotoxicity in immature rat hippocampus involving glutamate excitotoxicity. however, it remains unclear whether glyphosate alone or in combination with its adjuvants may have detrimental effects on myelin integrity in the peripheral nervous system. myelinated dorsal root ganglia (drg) cultures were treated over the course of ten days with either pure glyphosate or a glyphosate-based herbicide at concentrations of . %, . % and . %. the concentration of the glyphosate-based herbicide was matched with regard to glyphosate content ( %). controls were treated with equal amounts of vehicle adjusted for the ph. subsequently, cultures were stained with sudan black and myelin content was assessed by determining the number of internodes per neurons. while glyphosate, regardless of its concentration, did not show any effect on myelin content, the glyphosate-based herbicide caused significant demyelination in a concentration-dependent manner. notably, at . %, drg cultures were completely devoid of myelin and appeared severely necrotic. these data raise the possibility that not glyphosate itself, but rather the adjuvants in glyphosate-based herbicide formulations may cause demyelination. the open question whether demyelination is a direct effect of the adjuvants or a consequence of increased cellular glyphosate uptake due to permeabilization warrants further investigation. tan cy , tan mp , yeoh ky , goh kj , shahrizaila n . department of medicine, university of malaya, kuala lumpur, malaysia. in guillain-barré syndrome (gbs), autonomic dysfunction is common and accounts for significant morbidity and mortality. there have been many studies investigating the electrodiagnosis of gbs but few have studied autonomic dysfunction in gbs. the current study comprehensively investigates quantitative autonomic function in patients with gbs and its variant. ten gbs patients were prospectively recruited and the results were compared to age-and gender-matched healthy controls. a series of autonomic function tests including computational (power spectrum analysis of heart rate variability (hrv) and baroreflex sensitivity (brs) at rest) and challenge tests (deep breathing, eyeball compression, active standing, valsalva manoeuvre, isometric exercise and ice-water hand immersion) were performed. parasympathetic function was represented by high frequency (hf) hrv, heart rate responses to deep breathing, eyeball compression, valsalva manoeuvre and active standing. sympathetic function was represented by low frequency (lf) hrv, blood pressure responses to active standing, sustained handgrip and ice-water hand immersion. in the frequency domain analysis of hrv, low frequency (lf: . ± . vs . ± . ; p= . ), high frequency (hf: . ± . vs . ± . ; p= . ) and total power spectral densities (psd: . ± . vs . ± . ; p= . ) were significantly reduced in patients compared to controls. the mean up slope ( . ± . vs . ± . ; p= . ), down slope ( . ± . vs . ± . ; p= . ) and total brs slope ( . ± . vs . ± . ; p= . ) were significantly lower in the gbs group. the diastolic rise in blood pressure upon ice-water hand immersion was significantly lower in gbs group compared to controls ( . ± . vs . ± . ; p= . ). our findings suggest that computation dependent tests (hrv and brs) were sensitive at detecting autonomic dysfunction and baroreceptor reflex insensitivity in gbs patients. in contrast, ice-water hand immersion was the only reliable challenge test making it useful as a bedside measure of autonomic function in gbs patients. sjögren's syndrome (ss) is an autoimmune disease that affects both east and west. nevertheless, we still have limited knowledge of how autoantibodies in ss affects the peripheral nervous system. in this study, we investigated the peripheral neuropathy in ss and sicca complex using the nerve excitability test, to elucidate how peripheral nerves are affected. we have enrolled a total of patients with ss or sicca complex. of these, two patients were excluded due to co-morbid carpal tunnel syndrome. each patient received clinical evaluation, examination for ssa/ssb antibodies titer, the nerve excitability test, conventional thermal quantitative sensory test, and conventional nerve conduction study. compared to normal control subjects, motor nerve excitability test of ss patients with positive ssa or ssb antibodies (n = ) were found to have increased rheobase (p< . ), increased relative refractory period (rrp) (p< . ), increased refractoriness at . ms (p< . ), increased accommodation toward depolarizing current in threshold electrotonus (te) (p< . ), and decreased superexcitability (p< . ). the sensory axonal study in seropositive ss also revealed increased rrp (p< . ), increased refractoriness at . ms (p< . ), and increased accommodation toward hyperpolarizing current in threshold electrotonus (te) (p< . ). meanwhile, in seronegative ss and sicca complex (n = ), we found no significant axonal properties changes. the present study revealed that peripheral nerves are affected differently in seropositive ss and in seronegative ss/sicca complex. in seropositive ss, motor axons tended to be depolarized, and both sensory and motor axons have increased refractoriness. the findings suggested that ssa and ssb antibodies might play a role in the inactivation of transient sodium channels. the effects of the antibodies on transient sodium channels might be the basis of peripheral neuropathies and even cardiac arrhythmias and heart block in ss. charcot-marie-tooth disease type a (cmt a), caused by the pmp duplication on chromosome p . , is the most common subtype of inherited peripheral neuropathies and affects in , individuals worldwide. while sharing the same genetic cause, cmt a patients often present great variability in their phenotypic presentation and disease severity. the cause of the phenotypic variability is largely unclear. in this study, we performed genome-wide association study (gwas) to identify novel genetic modifiers of various phenotypes in cmt a. dna samples from cmt a patients were genotyped on illumina omniexpress platform. after standard quality control, the dataset includes k markers in individuals ( individuals from european ancestry, and individuals from asian ancestry). we focused our analyses on the european population. logistic regression in plink was used to analyze the association between the clinical outcomes and patients' genotypes in an additive model. for cmt neuropathy score (cmtns), the analysis was performed using linear regression in plink, adjusting for patients' age. the analyses yielded several suggestive association signals. an association peak on chromosome was identified in difficulty with eating utensils (lead snp rs , chr : , p= . e- , odds ratio= . ). the peak is located within a non-coding gene linc . hearing loss showed an association peak on chromosome (lead snp rs , chr : , p= . e- , odds ratio= . ), located in an intergenic region near the megf gene. in foot plantar flexion, an association signal was identified in the dscam gene on chromosome (lead snp rs , chr : , p= . e- , odds ratio= . ). cmtns showed an association signal on chromosome (lead snp rs , chr : , p= . e- , beta= . ), located within an intergenic region close to dffb, c orf , and linc . while these suggestive signals require further validation, our study provides novel insights into the genetic architecture of cmt a. novel genetic modifiers may serve as potential targets for therapeutic interventions in the future. teng a , ohnmar , kalpana p , chai yh , umapathi t . yong loo lin school of medicine, national university of singapore, singapore; department of neurology, national neuroscience institute, singapore. we present an intriguing diagnostic puzzle, that was eventually cracked serendipitously. a -year-old man was seen for bilateral ptosis. evaluation for myasthenia gravis was negative. nevertheless, a diagnosis of ocular myasthenia gravis was made and he was put on pyridostigmine. he did not respond. the ptosis progressively worsened. he sought a second opinion. at this evaluation, he was noted to have complex ophthalmoplegia without diplopia, bilateral facial weakness and mild bulbar weakness. he had no sensory complaints. the limb examination was remarkable for slightly reduced reflexes, normal strength, and other than increased vibration threshold at the toes, intact sensory examination. repeat serological and electrodiagnostic work-up for myasthenia gravis was negative. a myopathic disorder such as chronic progressive external ophthalmoplegia was considered. serum creatine kinase and lactate were normal. he underwent biceps muscle biopsy which showed increased cox-negative and sdh positive fibers, supporting the then clinical impression of a mitochondrial cytopathy. at this point, he underwent blepharoplasty to improve his vision. routine histological examination of the levator palpebrae muscle showed amyloid deposits. this prompted a review of the earlier biceps biopsy, which revealed amyloid deposits that were not appreciated before. at this point, the significance of the patient and his son's history of lattice corneal dystrophy became apparent. he also reported that his late mother had similar facial appearance as his. the patient's nerve conduction study showed length-dependent sensory axonal polyneuropathy, right carpal tunnel syndrome and bilateral facial neuropathy. he had no definite symptoms of autonomic neuropathy. cardiac evaluation was unremarkable. the final diagnosis of familial gelsolin amyloid polyneuropathy was made. genetic confirmation for the patient and his family is being planned. we highlight the key clinical features of gelsolin neuropathy. the symmetric cranial neuropathy can resemble a muscle or neuromuscular junction disorder and the relative sparing of the cardiac muscle, somatic and autonomic nerves contrasts with transthyretin-related amyloid polyneuropathy. neuropathy is one of the most common long-term complications of diabetes. furthermore, % to % of diabetic neuropathy patients will develop neuropathic pain. the pathophysiology of neuropathic pain in diabetic peripheral neuropathy is complex and not fully understood. a potential mechanism is a change in voltage gated sodium channels, such as nav . . loss of function mutations in this channel cause insensitivity to pain, whereas gain of function mutations have been linked with different pain syndromes including small fiber neuropathy. in a cohort of patients with diabetic peripheral neuropathy we investigated whether mutations in nav . were associated with diabetic neuropathic pain. twelve nav . variants were identified in nine participants all within a cohort of participants with painful diabetic peripheral neuropathy. five of these variants were previously associated with pain disorders: v l, m l; w r, r h, l v. among the other variants two of them met the criteria of potential pathogenicity based on predictive algorithms and were further studied. functional analysis by whole cell patch clamp showed that one of these variants (m t) drastically impairs the inactivation of the channel by shifting the steady-state fast-inactivation towards more depolarizing potentials. there were no phenotypic difference between those participants with pathogenic variants and those participants without pathogenic variants. no rare nav . variants were found in participants with painless diabetic peripheral neuropathy. these observations suggest that mutations in nav . may contribute to painful diabetic peripheral neuropathy. tholance y , rosier c , f bouhour , psimaras d , kuntzer t , taieb g , créange a , delmont e , camdessanché jp , antoine jc . university hospital, saint-etienne, france; university hospital, lyon, france; university hospital, paris, france; university hospital, lausanne, switzerland; university hospital, montpellier, france; university hospital, creteil, france; university hospital, marseille, france. dysimmune sensory neuronopathies (snn) encompass paraneoplastic snn and snn associated with systemic autoimmune diseases such as sjögren syndrome, lupus or inflammatory bowel or rheumatic diseases but also a number of apparently idiopathic cases. biomarker antibodies are well-known in paraneoplastic snn but are lacking in non paraneoplastic cases. from a mono-center retrospective study we identified in anti-fgfr antibody as a potential biomarker of dysimmunity in patients with idiopathic or systemic autoimmune disease associated sensory neuropathy. the identified patients were more frequently women and had a non lenthg dependent neuropathy suggestive of snn. anti-fgfr antibody was the only immunological marker in / of cases at initial work-up although / of patients eventually developed with time systemic autoimmune disease. to confirm the incidence and the clinical pattern of patients with anti-fgfr antibodies we launched a prospective multicenter french study including patients with a sensory neuropathy suspected to be a snn of no paraneoplastic, genetic or metabolic origin. we present here the results on the first included patients compared to healthy blood donors. anti-fgcr antibodies were searched by elisa using the trk intracellular domain of the protein (invitrogen©). we found patients positive for anti-fgfr antibody ( . %). these patients were women and men aged . years as a mean ( - ). the neuropathy was acute and subacute in one patient respectively and progressive in the others. six patients fulfilled the diagnosis criteria of snn and the last one had a sensory neuropathy in the lower limb with abnormal sensory action potentials in the four limbs suggesting snn without reaching the requested criteria. one patient developed uveitis which is a new symptom with anti-fgfr ab. an unclassified dysimmune context was present at the initial work up in patients and one patient developed sjögren syndrome with follow-up. as a whole the clinical pattern of these patients is consistent with that of the initially published series. the lower prevalence of positive sera may be due to more stringent criteria used for elisa but needs to be confirmed on the complete prospective series. peripheral neuropathy research registry (pnrr) neurological assessment was scored using the total neuropathy score clinical version (tnsc), comprising pinprick and vibration sensibility, deep tendon reflexes, strength and patient symptom report. compound sensory action potential (csap) amplitudes were recorded antidromically at the lateral malleolus, stimulating the sural nerve at the mid-calf. of the total sample, % reported lower limb neuropathy, with % of patients reporting 'quite a bit' or 'very much' severity of tingling and numbness in their feet. the average sural csap amplitude was . ± . v and % of patients had sural amplitudes below the lower limit of normal for age. the total tnsc score correlated with the pro fact-gog ntx score (r = −. , p<. ) and sural amplitude (r = −. , p <. ). vibration sensibility correlated with the overall fact-gog ntx score (r =−. , p <. ), and sural amplitude (r = −. , p <. ). sural amplitude correlated with patient reported severity of numbness and tingling in the lower limbs (r =−. , p <. ) but not with the overall fact-gog ntx score. patient reports of neuropathic symptoms in the lower limb correlate with both objective neurophysiological and clinical measures of neuropathy severity. identifying links between objective neurophysiological markers and patient reported outcomes are critical to assess the impact of clinical interventions. tomaselli pj , gouvea sp , nyshyama kfs , nicolau n jr , lourenço cm , marques w jr , . division of neuromuscular diseases, department of neurosciences and behaviour sciences, clinical hospital of ribeirão preto, university of são paulo, ribeirão preto, brazil; neurogenetics, department of neurosciences and behaviour sciences, university of são paulo, ribeirão preto, brazil. mutations in the gab junction beta -protein gene (gjb ) are the second most frequent cause of charcot-marie-tooth disease (cmt), accounting for approximately % of cmt cases worldwide. the gjb codes for connexin protein (cx ). in the peripheral nervous system, the cx is expressed in the schwann cells and allows intercellular traffic of ions and small molecules between opposed cells. we analysed retrospectively detailed clinical and neurophysiological data of five families carrying novel gjb mutation submitted for testing at our neurogenetics laboratory. mutations were identified by bidirectional sanger sequence analysis of gjb coding region. we identified a total of subjects from five different kindreds with novel mutations (p.a v, p.l w, p.l q, p.f s, p.r l). these five novel mutations segregate with phenotype, are located in highly conserved amino acids among gjb and other gab junction protein sequences and among different species, are not present in any public database (exac, dbsnp and genome database), and were not found in normal brazilian controls. in silico analysis, predict these variants to be pathogenic, there was no male-to-male transmission; males were more severely affected than females. four out seven female have subclinical neuropathy and were only identified after clinical and electrophysiological evaluation. the conduction velocities were in the intermediated range in the males patients and higher in the females included in this study. we describe five new pathogenic mutations causing cmtx in a brazilian population and expand the number of causative mutations in the gjb gene. funded pure neural leprosy (pnl) is a slowly progressive, predominantly sensory patchy neuropathy presenting with positive and/or negative sensory symptoms, which are usually followed over time by distal asymmetrical weakness. despite rare, monomelic involvement in leprosy has already been reported. we sought to describe the clinical and electrophysiological patterns of an unusual leprosy neuropathy presentation. clinical data were retrospectively collected from nine patients who had monomelic involvement and were referred for further investigation to the emg lab. seven out nine patients were male. four patients had a brachial plexus like presentation and five have a lumbosacral plexus like presentation. the initial complaint was hypoesthesia in four patients, tingling in two patients and hypoesthesia with tingling in two patients. severe pain was observed in just one patient. all individuals from the group of patients with lumbosacral plexus-like presentation and three with brachial plexus-like presentation had no sensory nerve action potentials (snaps) for all nerves tested in the affected limb with or without motor involvement. one patient with brachial plexus-like presentation had focal slowing of conduction velocity with temporal dispersion of both median and ulnar nerves in the affected limb. one patient with plexus-like presentation had snaps with low amplitude of all nerves in the affect limb. the diagnosis of leprosy was confirmed by nerve biopsy findings, anti-pgl antibody, and positive response to specific treatment. nerve biopsy was performed in four patients, and the bacillus was found in two. the anti-pgl antibody was positive in four patients. plexus mri was performed in two patients and was normal. we found the distribution of motor and sensory symptoms were restricted to on limb in this group of patients. as a typically patchy disorder pnl may affect any nerve, although the reason why damage are restrict to only one limb has to be elucidated. the description of these cases increases the clinical spectrum of leprosy neuropathy. this possibility should be considered in the differential diagnosis of patients with plexopathy from endemic areas after excluding other causes. funded ataxia with oculomotor apraxia type (aoa ) is a very complex disorder characterized by an early-onset progressive cerebellar ataxia with cerebellar atrophy and peripheral neuropathy and it is caused by recessive mutations in the aprataxin gene (aptx ). when the neuropathy is present, it has been described in % of cases as primarily axonal. we describe a case of aoa due compound heterozygous mutations in aptx associated with demyelinating neuropathy. the patient was born from healthy and non-consanguineous parents and presented in the first decade with progressive cerebellar ataxia, multidirectional ophtalmoparesia, oculomotor apraxia, choreiform movements of limbs and peripheral neuropathy. he had normal cognition and stopped walking at age of . blood tests were unremarkable with normal levels of leucocytes, serum proteins, immunoglobulin, cholesterol, vitamin e, and alfa-feto protein. brain mri showed severe cerebellar atrophy. the motor conduction velocity in the upper limbs was slow with preserved amplitudes. the distal latencies and the minimal f wave latencies were prolonged. there was no evidence for superimposed acquired demyelinating neuropathy. direct sequencing of the aptx gene revealed two variants, c. - a>g, p? and c. g>a; p.w x. the first variant is novel and affects a highly conserved acceptor splice site of exon . the other variant is the most common portuguese variant, the nonsense mutation w x is located in exon . parental dna was tested and confirmed the variants were in different alleles. the presence of a demyelinating neuropathy in aoa suggests that phenotypic variability in this condition may be larger than previously considered. at the same time, it increases the differential diagnosis of inherited conditions with cerebellar ataxia and demyelinating neuropathy. finally, this finding opens the functional effects of the aptx gene. funded by: cnpq, fapesp, faepa, pronas (ministry of health). topa a , spina e , iodice r , tozza s , ruggiero l , dubbioso r , esposito m , santoro l , manganelli f . university of naples "federico ii", naples, italy. we report our -year experience of subcutaneous immunoglobulin (scig) in a cohort of patients with chronic inflammatory demyelinating polyneuropathy (cidp) from a tertiary care neuromuscular center. we analyzed data from cidp patients ( males and females, mean age: ± . years; mean age at onset: . ± . years; disease duration: . ± . years) treated with scig and with a follow-up period of months. all patients were previously responders to intravenous immunoglobulin (ivig). eight patients had a typical cidp and five patients had an atypical variant of cidp. five patients switched to weekly maintenance scig therapy (continuous regimen) because of short-lasting response to ivig therapy. eight patients with a longer lasting response to ivig received scig with a pulsed regimen similar to that used for ivig (from to cycles per year); seven of them because of difficulty in hospitalization and one for allergic reaction to ivig. changes in clinical status were assessed over the period of follow-up by using clinical evaluation of muscle strength, modified rankin scale, overall neuropathy scale and inflammatory neuropathy cause and treatmentsum score. in patients we evaluated also six minute walking test, hole-peg-test and meter walking test. all the five patients treated with a continuous regimen of scig remained clinically stable throughout the follow-up period. among the patients receiving pulsed scig treatment, out of ( %) responded to scig similarly to ivig, while three patients ( . %) worsened and needed to be treated again with ivig and the other one ( . %) stopped any therapy. subcutaneously administered immunoglobulin were well tolerated and no patients complained of adverse events. in conclusion, our findings confirm that continuous scig therapy is efficacious in maintaining clinical stability in patients with short-lasting response to ivig. moreover, our data suggest that pulsed therapy with scig may represent an alternative therapeutic option for the treatment of a subset of cidp patients. touvier t , ferri c , mastrangelo r , glimcher l , , wrabetz l , , , d'antonio m . myelin biology unit, division of genetics and cell biology, san raffaele scientific institute, dibit, milan, italy; department of cancer immunology and virology, dana-farber cancer institute, boston, usa; department of medicine, harvard medical school, boston, usa; hunter james kelly research institute, university at buffalo, buffalo, usa; department of biochemistry, university at buffalo, buffalo, usa; neurology, jacobs school of medicine and biomedical sciences, university at buffalo, buffalo, usa. mpz glycoprotein is an abundant product of terminal differentiation in myelinating schwann cells. the mutant mpzs del causes charcot-marie-tooth (cmt) b disease in humans and a similar demyelinating neuropathy in transgenic mice. mpzs del protein is retained in the endoplasmic reticulum (er) of schwann cells and induces an unfolded protein response (upr) characterized by activation of perk, atf and ire /xbp pathways. we have previously reported that activation of chop and gadd , two downstream mediators of perk, is pathogenetic in mpzs del mice (pennuto, ; d' antonio, ) but the role of the other upr branches remains to be investigated. in this study, we investigated the role of the er stress sensor enzyme ire and of xbp -a transcription factor specifically activated by ire -in mpzs del pathogenesis. we generated a new mouse model with schwann cells-specific ablation of xbp and in parallel we exploited mpzs del dorsal root ganglia (drg) explant cultures in which xbp signaling is modulated by gain/loss of function approaches. we observed that absence of xbp dramatically worsens hypomyelination and electrophysiological/locomotor parameters in young and adult mpzs del neuropathic animals. interestingly we observed that perk, atf and ire -mediated ridd signalings are upregulated in neuropathic animals lacking xbp . this suggests that activation of xbp targets have an essential role in limiting mpzs del toxicity, which cannot be compensated by other stress responses. moreover, we demonstrated in mpzs del drg cultures that inhibition of xbp splicing by u c (cross, ) decreases myelination whereas activation of xbp splicing by quercetin (wiseman, ) slightly ameliorates myelination. altogether, these data demonstrate that xbp pathway has a critical adaptive role in mpzs del neuropathy and suggest that activation of this pathway may be beneficial for cmt b and perhaps for a broad range of neuropathies characterized by upr activation. tozza s , bruzzese d , iodice r , esposito m , dubbioso r , ruggiero l , topa a , spina e , santoro l , manganelli f . department of neuroscience, reproductive sciences and odontostomatology, university of naples "federico ii", naples, italy; department of public health, university federico ii of naples, naples, italy. in cmt a patients, the clinical impairment progressively increases over time and correlates with the axonal loss. evidence has suggested that the decline of physical performance in cmt a patients may reflect a process of normal ageing. the aim of our study was to describe, by a case-control cross-sectional design, the progression of physical impairment with ageing in cmt a patients. we enrolled cmt a patients ( m; range - years) and sex-and age-matched healthy controls. to assess physical performance, all patients and controls underwent -meter walk test ( mwt), -minute walk test ( mwt) and -hole peg test ( hpt) of their dominant (d) and non-dominant (nd) sides. moreover, to assess clinical disability, impairment and quality of life in the cmt a group we used the charcot-marie-tooth neuropathy score (cmtns), the mrc sum score and the short form- (sf- ) questionnaire. the linear regression model was used to evaluate the changes over time of clinical measures in patients and controls. the chow test was used to determine whether the ageing had a different impact on clinical measures for the two groups. physical performance worsened with ageing in both patients and controls, but with a greater slope for cmt a patients [difference in slopes: mwt, . (c.i. . to . ), p< . ; mwt, − . (c.i. - . to − . ), p< . ; hpt-d, . (c.i. . to . ), p< . ; hpt-nd, . (c.i. . to . ), p< . ]. the rate of deterioration of physical performance was not different between patients and controls until the th year of age. after the th year of age the rate of deterioration became greater in cmt a group [difference in slopes: mwt, . (c.i. . to . ), p< . ; mwt, − . (c.i. - . to − . ), p< . ; hpt-d, . (c.i. . to . ), p< . ; hpt-nd, . (c.i. . to . ), p< . ]. moreover, in cmt a patients also cmtns, mrc sum score and sf- worsened with ageing and with a greater rate of deterioration after the th year of age. our study demonstrates that clinical decline in cmt a patients goes parallel to the normal ageing process until the th year of age, whereupon the clinical deterioration accelerates. tsouni p , devic p , moura b , planque e , bédat-millet al , devaux j , steck aj , delmont e , hottinger af , kuntzer t . dcn, chuv, lausanne, switzerland; centre de référence maladies neuromusculaires, hospices civils de lyon, lyon, france; cabinet médical, epinal, france; département de neurologie, chu de rouen, rouen, france; cnrs, crn m-umr , université aix-marseille, marseille, france; centre de référence maladies neuromusculaires et sla, hôpital la timone, marseille, france. new therapeutic options in immuno-oncology have allowed significant progress in the management of melanomas. treatment usually consists of a combination of two monoclonal antibodies targeting cytotoxic lymphocyte-associated protein and programmed cell death- . as a result, the immunologic barrier protecting tumor cells is overcome allowing an antitumor response. we report cancer patients with immune checkpoint inhibitor-induced neuropathies as a complication of this immunomodulating oncologic treatment. case reports: our index patient developed severe myalgia days after introduction of ipilimumab-nivolumab followed by painful paresthesias of the face and extremities day after the nd cycle of treatment. generalized areflexic quadriparesis (mrc ) with gowers'sign and distal loss of vibratory sensation were found. a -day course of ivig plus corticosteroids (cs) had no effect. three monthly ivig courses were necessary to improve the deficits at months. a survey of sfnp members revealed other patients with similar acute courses but with phenotypes varying from sensorimotor deficits with areflexia and myalgia to purely sensory ataxic forms following immunomodulating treatment. work-up including anti-nodal antibodies were negative in patients. from the other , had abnormal csf and had necrotizing myopathy. detailed repeat ncs demonstrated signs of nerve hyperexcitability and of demyelination or conduction blocks. evolution was slowly favorable following ivig and cs. discussion: our report underscores that atypical acute generalized demyelinating neuropathies are induced by these novel treatments. they may be associated with severe myalgia or other systemic toxic effects. discontinuation of the oncologic treatment depends on severity of symptoms. outcome was slowly favorable following ivig or cs, albeit slower than in the case of primary inflammatory neuropathies, probably given the long half-life of the monoclonal antibodies. gap junctions (gjs) are membrane channels found in most tissues connecting adjacent cells or different cell compartments as in schwann cells. they are involved in electrical connectivity and metabolic homeostasis allowing the passage of small molecules such as ions, second messengers, nucleotides and peptides. an important functional role of peripheral nerve connexins is suggested by their involvement in x-linked inherited neuropathy as well as in acquired neuropathy caused by oxaliplatin. although gjs play a role in electrical connectivity their specific role in the formation of the sciatic nerve compound action potential (cap) remains unclear. the aim of this study was to investigate the role of peripheral nerve connexins in the electrical responses of the mouse sciatic nerve under normal and stress conditions. for this purpose we used sciatic nerves of three different mouse models, the cx knockout (ko), cx ko and the cx /cx double knockout (dko) mice. using our ex vivo model for extracellular recordings we exposed sciatic nerves from different genotypes to three different gj blockers: octanol, -beta-glycyrrhetinic acid (gra) and octanoic acid (oa) and recorded the cap. amplitude and duration of the cap were used as an indication for the effects of the different blockers on the cap formation. all gj blockers caused a gradual decrease of the cap without any changes in the duration of the cap in all genotypes, suggesting progressive disturbance of axonal membrane excitability in the absence of one or two gj proteins. comparison of the three genotypes showed that cx may play a dominant role in the maintenance of the cap formation since nerves from cx ko mice proved to be more sensitive to the gj blockers compared to the cx ko nerves showing a faster decline of the cap amplitude. moreover the effect of gj blockers was similar in cx ko and dko nerves. finally, the effect of gj blockers on the dko nerves implies the presence of another gj protein. in conclusion, our results confirm the direct functional involvement of cx gj channels and cx hemichannels in the cap formation and indicate the existence of at least one more connexin in peripheral nerve. ca( +)-dependent anti-ganglioside antibody in seronegative guillain-barrÉ syndrome uchibori a , gyohda a , chiba a . kyorin university, tokyo, japan. we have reported ca + -dependent igg anti-ganglioside gq b antibodies in gq b-seronegative patients with fisher syndrome and its related disorders (fs-rd). in patients with fs-rd who were gq b-seronegative in conventional assays using phosphate-buffered saline without ca + , % turned seropositive for gq b-related antigens in assays using ca + -added tris-buffered saline. objective: we investigated whether ca + -dependent anti-ganglioside antibodies was present also in ganglioside-seronegative patients with other clinical disease types of guillain-barré syndrome (gbs) other than fs-rd. methods: the subjects were the following: patients with final clinical diagnosis as gbs (acute motor axonal neuropathy [aman], n = , and acute inflammatory demyelinating polyradiculoneuropathy [aidp], n = ), and patients with final clinical diagnosis as sensory ataxic neuropathy (san), n = . all subjects were ganglioside-seronegative in the conventional assays. we assayed serum igg antibodies against various gangliosides (including asialo-gm ) in elisa using tris-buffered saline as a basal buffer in both ca + -added and -non-added conditions. increase of the optical density (od) more than . in ca + -added condition compared with ca + -non-added one was taken significant, i.e. positive for ca + -dependent antibody. results: ca + -dependent antibody was negative in all aman and san patients. in aidp, the antibody titers (ods) against gainac-gd a were significantly increased in patients, and those against asialo-gm were increased in other patients in ca + -added condition. however, the titiers of those ca + -dependent antibodies were all at low level. conclusions: in clinical disease types of gbs other than fs-rd, ca + -dependent igg antibodies against ganglioside were detected in a few patients with aidp, but the positive rate and the antibody titers were low compared with case of gq b-seronegative fs-rd. ca + -dependent antibodies against ganglioside are considered to be more specific for gq b. the aim of the study was to investigate the relationship between median sensory conduction of median nerve and ulnar nerve in patients diagnosed with carpal tunnel syndrome. two hundred and eighty-six hands with carpal tunnel syndrome and hands in control group were investigated. patients were staged clinically and electrophysiologically. diagnosis of carpal tunnel syndrome was made according to the presence of paresthesia, pain in the innervation area of the median nerve, weakness and atrophy in the median nerve innervated muscles, positive phalen and tinel tests. median motor and sensorial nerve conduction study, including first, second, third finger and palm, and ulnar motor and sensorial nerve conduction of fifth finger studies were performed to all patients and control group. the ratio of distal latency and velocity of nerve conduction of first, second, third and palmar branches to fifth finger was calculated. distal latency of first, second, third finger and palm of patients with cts are longer and velocity is more slowly than controls. in addition to these findings, the velocity of fifth finger is also slower and distal latency of this one is longer than healthy subjects. the most sensitive method of classifying the carpal tunnel syndrome as normal, mild and moderate is the ratio of distal latency and velocity of second finger (p< . ). carpal tunnel syndrome is the most common encountered neuropathy. in nerve conduction studies can be used the ratio of distal latency and velocity of second finger as determine the degree of carpal tunnel syndrome. the most surprising finding of this report is the nerve conduction studies of fifth finger. the subclinical susceptibility of fifth finger can be explained by overusing of wrist. ursino g , gemelli c , grandis m , reni l , bellone e , geroldi a , gotta f , mandich p , ferrara m , schenone a dinogmi university of genoa, genoa, italy; irccs-aou san martino hospital genoa, genoa, italy. charcot-marie-tooth (cmt) neuropathy represents a clinically and genetically heterogeneous group of hereditary peripheral neuropathies characterized by chronic motor and sensory impairment. to date mutations in up to genes may cause cmt. the aim of this study is to describe our large population of cmt patients, and, within this, highlight specific phenotypes. the cmt clinic in genova, started in . during the years, patients underwent complete neurological, rehabilitative, neurophysiological examinations and genetic testing. the patients were routinely tested for common genes (as pmp , gjb , mpz, mfn ), while in specific cases we followed the candidate gene approach testing single genes based on the genotype-phenotype correlation. however, the ngs techniques were used when routine genetic testing was negative and a clear genotype-phenotype correlation could not be identified. cases are present to date in our database of cmt patients. in ( . %) patients, in spite of a clinical diagnosis of cmt, a genetic diagnosis is still lacking; ( . %) patients had alternative diagnosis (i.e hereditary spastic paraparesis etc.). instead, in patients ( . %) a defined genetic diagnosis was reached, of them being females ( . %) and males ( . %). among these, except for the more common cmt a, hnpp and cmt x phenotypes, we frequently observed patients affected by cmt b and cmt f. according to most literature data, we observed ( . %) patients with cmt b and patients ( . %) affected by cmt f. at the first visit, the cmt b phenotype was clearly length-dependent: . % patients showed impairment of the lower limbs and saving of the upper limbs; in terms of severity of the neuropathy, the mean cmtns was . and the mean age was . years. similarly, % of patients affected by cmt f, present with the same phenotype; the mean cmtns at the first visit was . and the mean age was . years. in conclusion, based on the experience of the genova cmt clinic, we describe a large population of cmt patients and a specific phenotype in cmt b and f patients, characterized by involvement of the lower limbs and selective sparing of the upper ones, which may help in addressing the diagnostic algorithm. non-freezing cold injury (nfci) develops following sustained exposure to cold temperatures, resulting in tissue cooling but not freezing. this can result in persistent sensory disturbance of the hands and feet including numbness, paraesthesia and chronic pain. both vascular and neurological aetiologies of this pain have been suggested but remain unproven. we prospectively approached patients referred for clinical assessment of chronic pain following non-freezing cold injury between february and november . of patients approached consented to undergo detailed neurological evaluations including: questionnaires to detail pain location and characteristics, structured neurological examination, quantitative sensory testing, nerve conduction studies and skin biopsy for intra-epidermal fibre assessment. of the study participants all had experienced nfci whilst serving in the united kingdom armed services and the majority were of african descent ( . %) and male ( . %). many patients reported multiple exposures to cold. the median time between initial injury and referral was . years. pain was principally localised to the hands and the feet, neuropathic in nature and in all study participants associated with cold hypersensitivity. clinical examination and quantitative sensory testing were consistent with a sensory neuropathy. in all cases large fibre nerve conduction studies were normal. the intra-epidermal nerve fibre density, however, was markedly reduced; ⋅ % of subjects having a count at or below the ⋅ centile of published normative controls. using the neuropathic pain special interest group (neupsig) of the international association for the study of pain (iasp) grading for neuropathic pain % had probable and ⋅ % definite neuropathic pain. chronic non-freezing cold injury is a disabling neuropathic pain disorder due to a sensory neuropathy. why some individuals develop an acute painful sensory neuropathy on sustained cold exposure is not yet known but individuals of african descent appear vulnerable. screening tools, such as the dn questionnaire, and treatment algorithms for neuropathic pain should now be used in the management of these patients. funded by the wellcome trust and the uk ministry of defence guillain-barré syndrome (gbs) is highly heterogeneous regarding clinical presentation, course, electrophysiological subtype and outcome. in part this variety is associated with differences between geographical regions, although this had not been investigated in a single comparative study. one aim of igos is to define the influence of the geographical origin on the heterogeneity of gbs. in igos all gbs patients within weeks of onset can participate, independent of age, variant, severity and treatment. in february , patients were included from countries. the first inclusions in igos were used in this analysis. seventy-five patients ( %) were excluded because of alternative diagnoses (n= , including a-cidp), protocol violations (n= ) or missing data (n= ). of the remaining patients, % were males and % female with a median age of years (iqr - ). at entry % presented with tetraparesis and % with paraparesis. during follow-up % needed mechanical ventilation and % died. of all gbs patients % received treatment ( % ivig, % pe). the remaining % received supportive care only (mild gbs or low social-economic status). antecedent events were reported in % of patients, including upper respiratory tract infection ( %) and gastro-enteritis ( %) as the most frequent events. the pure motor form was the predominant subtype in patients from bangladesh ( %). in europe/americas and asia (without bangladesh) the predominant subtype was the sensorimotor form ( % in europe/americas and % in asia). in asia (without bangladesh) there was a relatively larger proportion of patients with mfs/mfs-overlap syndrome ( %) than in other regions ( - %, p< . ). the proportion of patients able to walk unaided at months after follow-up was % in europe/americas, % in bangladesh and % in asia (without bangladesh). kaplan-meier analysis comparing electrophysiological subtypes of gbs (as reported by neurologist) showed that patients with inexcitable nerves or axonal neuropathy needed more time to regain the ability to walk unaided than the patients with demyelinating or equivocal result (log-rank test, p < . ). these findings demonstrate the extensive geographical differences in gbs. future igos studies will investigate the role of genetic and environmental factors that additionally could explain these differences. van den bergh pyk , attarian s , grapperon am , nicolas g , cassereau j , rajabally ya , delmont e , woodard jl , piéret f and the university of louvain gbs electrodiagnosis study group * . corticosteroids are considered as one of the first line treatments for chronic inflammatory demyelinating polyneuropathy (cidp). different types of corticosteroids are used and there are no comparative studies assessing the improvement rates, remission rates, tolerability and the side effect profiles of these treatment regimens. in addition, there are currently no reliable predictors of favorable treatment response to steroids, which would greatly ease the choice of first line treatment. in this retrospective study we will compare efficacy, tolerability and safety of three different corticosteroid regimens used as first line treatment in three large cidp centers in netherlands, serbia and italy. treatment naïve cidp patients who received either pulsed dexamethasone, pulsed methylprednisolone or daily prednisone will be included in the study. data will be extracted from patient charts. the primary outcome is the percentage of treatment responders at months after start of first treatment, in which treatment response is defined as subjects who improved after treatment and are either without treatment after six months or are still being treated with the first chosen therapy. secondary endpoints include the remission rates and in case of a relapse, the mean duration of remission to relapse; the discontinuation rate within months of treatment due to inefficacy, adverse events or intolerance; and the frequency of adverse events and serious adverse events (sae) during treatment or within month after stopping treatment. furthermore, we will explore the value of previously reported potential predictors of treatment response. results will be presented at the peripheral nerve society meeting . van lieverloo g , , musters a , adrichem m , esveldt r , doorenspleet m , klarenbeek p , van schaik i , de vries n , eftimov f . academic medical center, department of neurology, amsterdam, the netherlands; academic medical center/university of amsterdam, department of clinical rheumatology and immunology, amsterdam, the netherlands. following reports that pathogenic antibodies are present in a minority of patients with chronic demyelinating inflammatory neuropathy (cidp), we studied whether oligoclonal expansions of b-cell clones are present in patients with cidp. recently, we developed a new method for b-cell receptor (bcr) repertoire landscaping based on high throughput sequencing (hts) of rna extracted from blood. bcr repertoire was analyzed in patients with cidp: patients with active disease and starting treatment (group ), patients with stable disease using intravenous immunoglobulin (ivig) treatment in which treatment withdrawal was attempted (group ), and patients in remission (i.e. no treatment in the last months, group ). clinical parameters and sampling was performed at baseline (group , and ), at months after start of treatment (group ), at months or earlier in case of relapse in group and at baseline only in group . most cidp patients had highly expanded bcr clones, regardless of disease activity and response to treatment. however, in group , the most expanded b-cell clones at baseline showed no overlap with the expanded bcr clones after improvement. based on these preliminary data expanded bcr clones are observed in the peripheral blood of most cidp patients, regardless of disease activity (active, stable disease or remission/cure). functional characterization of these expanded clones remains to be performed. van rijs w , fokkink wjr , tio-gillen ap , brem md , jacobs bc , huizinga r . erasmus mc, university medical centre, rotterdam, the netherlands. myeloid cells, including monocytes, macrophages and dendritic cells, are critically involved in the induction of adaptive immune responses, clearance of pathogens and in the initiation of tissue repair. in the guillain-barré syndrome (gbs), macrophages are present in the peripheral nerve, where they phagocytose (damaged) myelin and axons. dendritic cells (dc) are increased in the cerebrospinal fluid of patients with gbs. however, the composition and phenotype of monocytes and dc subsets in the peripheral blood is unclear and it is unknown if these cells can be used as biomarker to monitor disease activity or response to treatment. here we investigated the frequency and phenotype of six myeloid subsets in the peripheral blood mononuclear cells (pbmc) using advanced -color flow cytometry. pbmc were isolated from patients with gbs, before and after immunomodulatory treatment, and age and gender-matched, healthy controls. the frequency of total monocytes, determined as percentage of cd + cells, was increased in gbs patients compared to controls (p< . ). the monocyte population was skewed towards more intermediate (cd +cd +; p< . ) and less non-classical (cd -cd +; p< . ) monocytes. classical (cd +cd -) and intermediate monocytes as well as cd c+ dc expressed significantly higher levels of cd compared to healthy controls. in contrast, the expression of cd and siglec- was significantly higher in the non-classical monocytes of gbs patients compared to controls. no differences were observed in the expression of cd , cd , cd and siglec- . immunomodulatory treatment strongly reduced the frequency of non-classical monocytes and all dc populations in cd + pbmc. the expression of cd , cd c and hla-dr was reduced in classical monocytes after treatment. in addition, siglec- expression was reduced in several monocyte and dc populations after treatment. in summary, our data identify significant changes in the monocyte compartment in gbs. the decrease in non-classical monocytes may suggest that these cells have migrated to peripheral tissues, promoting the differentiation of classical monocytes into intermediate monocytes. further analysis should reveal whether these changes are related to preceding infections, disease severity and response to treatment. in mme. the two mutations were absent from control databases (e.g. exac), affected highly conserved aminoacids and were predicted to have deleterious effects by in silico analysis. unfortunately, both parents were deceased and we were therefore unable to prove that the two mutations reside on separate alleles. mme encodes the metalloprotease neprilysin whose role in peripheral nervous system is still unclear. higuchi and colleagues have described japanese cmt families with late-onset sensory-motor axonal neuropathy and recessive loss-of-function mutations in mme. we report two novel missense mutations in mme in a case of late-onset cmt . we hypothesize an autosomal-recessive mode of inheritance as most likely given the clinical phenotype and the absence of a family history. anti-contactin- (cntn ) antibodies were recently identified in a subgroup of patients with chronic inflammatory demyelinating polyneuropathy (cidp) showing acute/subacute onset of severe sensory-motor neuropathy and poor response to intravenous immunoglobulin (ivig) and corticosteroids. these antibodies belong to the igg isotype and interact with cntn -neurofascin (nf ) complex at paranodes leading to loss of nodal integrity. a -year-old man presented with acute onset of distal weakness in the lower limbs, four limbs paraesthesias and sensory ataxia. at clinical examination ankle swelling was also observed. nerve conduction study showed a demyelinating polyneuropathy and cerebrospinal-fluid examination revealed cyto-albuminologic dissociation. sural nerve biopsy disclosed diffuse loss of myelinated fibres. at routine blood test serum albumin was reduced and proteinuria was gr/ hours, thus leading to the diagnosis of nephrotic syndrome. kidney biopsy showed changes consistent with membranous glomerulonephritis, together with sub-epithelial deposits of immune complexes and complement deposition. treatment with ivig and corticosteroids did not improve neurological status, while membranous glomerulonephritis showed moderate response to ivig. a six-month course of cyclophosphamide was started leading to normalization of renal function and muscle strength and partial improvement of sensory ataxia. the patient did not require any further treatment and after years his condition remains stable. cntn antibodies were tested on a recently collected patient's serum and resulted positive on both elisa and cell-based assay. the patient here reported showed the typical clinical features of anti cntn -associated cidp including older age, acute onset, severe motor impairment and sensory ataxia. the contemporary occurrence of membranous glomerulonephritis was reported in only one other case. contactin- is expressed at low levels in the kidney and a direct damage of anti-cntn antibodies could be hypothesized. alternatively, renal damage might have been secondary to unspecific immune complexes deposition. a good response to anti-cd rituximab was recently reported in patients with cidp associated with anti-cntn and anti-nf antibodies. notwithstanding this single-case observation, our report suggests that also cyclophosphamide may be considered an effective therapy in anti-cntn antibodies-associated cidp and membranous glomerulonephritis, leading to persistent clinical remission. velasco r , , besora s , santos c , sala r , izquierdo c , simó m , gil-gil m , , jiménez l , pardo b , calvo m , palmero r , clapés v , bruna j duloxetine is the only agent demonstrated effective in treating pain related with chemotherapy-induced peripheral neuropathy (cipn). patients with symptomatic cipn treated with duloxetine were retrospectively collected in a single-institution. aim of the study was to evaluate the drug's efficacy and rate of compliance. only patients with cipn with distressing positive symptoms (pain, numbness and/or paresthesia), and non-progressive disease were included. cipn was graded employing the total neuropathy score (tns © ) and national cancer institute-common toxicity criteria. response to duloxetine was assessed with patient global impression of change (pigc) scale ( : no benefit; : excellent response). consecutive first one-hundred cipn patients treated with duloxetine were analyzed. median age was ( - ). , , and received platinum, taxane, bortezomib and vincristine-based regimen, respectively. median tnsc © was ( - ). severity of neuropathy was grade ( %), grade ( %), and grade ( %). sixteen patients were on treatment with other analgesic agents. median time from finishing chemotherapy to duloxetine initiation was months [ - ]. median pigc score was [ - ]. among responders, . % and . % scored low ( - ) and high ( - ) benefit, respectively. fifty-seven ( %) patients discontinued early duloxetine due to intolerable side effects (n= ) the goal of this study was to determine whether predicted fork stalling and template switching (fostes) during mitosis deletes exon in peripheral myelin protein kd (pmp ) and causes a gain-of-function mutation associated with peripheral neuropathy in a family with charcot marie tooth disease type e. two siblings previously reported to have genomic re-arrangements predicted to involve exon of pmp were evaluated clinically and by electrophysiology. skin biopsies from the proband were studied by rt-pcr to determine the effects of the exon re-arrangements on exon mrna expression in myelinating schwann cells. transient transfection studies with wild type and mutant pmp were performed in cos and rt cells to determine the fate of the resultant mutant protein. both affected siblings had a length-dependent demyelinating neuropathy with severely slow nerve conduction velocities (< m/sec). rt-pcr studies of schwann cell rna from one of the siblings demonstrated a complete in frame deletion of pmp exon (pmp delta ). transfection studies demonstrated that pmp delta protein is retained within the endoplasmic reticulum and not transported to the plasma membrane. our results confirm that that fostes mediated genomic rearrangement produced a deletion of exon of pmp , resulting in expression of both pmp mrna and protein lacking this sequence. in addition, we provide direct experimental evidence for endoplasmic reticulum retention of the mutant protein suggesting a gain-of-function mutational mechanism consistent with the observed cmt e in this family. pmp delta is another example of a mutated myelin protein that is misfolded and thus likely to contribute to the pathogenesis of the neuropathy. in poems and cidp, distal limb nerve conduction studies are limited in identifying demyelination and detecting treatment effects in severely affected patients. blink reflex r latency may help to not only identify demyelination but also provide a meaningful treatment outcome measure especially in severely affected patients. poems and cidp patients having undergone routine nerve conductions and blink reflex testing were identified. correlation between r latency, limb nerve conduction studies and neuropathy impairment scores (nis) was calculated with treatment. blink reflexes were performed in patients ( poems, cidp; nis range: - points). overall, r latency prolongation occurred in . % of patients ( . % poems, . % cidp). patients with r prolongation (> ms) had more severely affected nerve conductions in both poems (ulnar cmap . mv vs . mv, p= . ) and cidp (ulnar cmap . mv vs . mv, p< . ). r latency correlated with nis severity in poems better than cidp (r = . vs . , p=< . vs. . ). follow-up nis and r latency evaluations after treatment were available in patients ( poems, cidp). the r latency changes were concordant with the nis changes in % of poems and % of cidp patients. in severely affected patients [ulnar cmap amplitude ≤ . mv ( . %: / )] except for one, all had prolonged r (> ms), allowing for treatment follow up and initial diagnosis. blink reflex r latencies are valuable in defining demyelination in severely affected poems and cidp patients, but also provide a sensitive, early treatment outcome measure among those same severely affected patients. watson dj , martinez c , wallenhorst c , hubsch a , shebl a , simon tl . csl behring llc, king of prussia, usa; institute for epidemiology, statistics and informatics gmbh, frankfurt, germany; csl behring ag, bern, switzerland; csl behring gmbh, marburg, germany. chronic inflammatory demyelinating polyneuropathy (cidp) rarely occurs in children. clinical trials in pediatric patients have not been performed and there are little data on therapy with intravenous immunoglobulin (ivig). we performed an observational trial to investigate the risk of adverse events of special interest (aesi) with ivig, i.e. hemolytic anemia, aseptic meningitis, acute renal failure, thromboembolic events and anaphylactic reactions. the study cohort was derived from the us premier perspective database and consisted of patients < years with a diagnosis of cidp (icd- cm diagnosis code . ) treated with the ivig privigen ® (csl behring, bern switzerland) between jan and dec . we identified pediatric cidp patients: preschool children (age - years at first treatment for cidp), children ( - years) and adolescents ( - years); females and males; white, one black and two allocated to "other race". six patients had a history of other ivig use for guillain-barré syndrome, one patient for myasthenia gravis and one for immunodeficiency before the diagnosis of cidp. the mean privigen ® dose calculated from the cumulative quantity of privigen ® per treatment episode and the corresponding age-and gender specific median of the us population body weight estimate was . g/kg body weight. the number of recorded treatment episodes per patient ranged from to . using an at-risk period of days for hemolytic anemia, and days for other aesi after each privigen ® administration, no aesi were observed in the patients with cidp with a total of person-days at-risk for ha and person-days at-risk for other aesi. this observational study shows that ivig (privigen ® ) is used for treatment of cidp in pediatric patients with or without concomitant conditions and revealed no particular safety issues in this patient group. hsn- is a peripheral neuropathy most frequently caused by missense mutations in the sptlc or sptlc genes, which code for two subunits of the enzyme serine palmitoyltransferase (spt). spt catalyzes the first and rate limiting step of de novo sphingolipid synthesis. it has been shown that mutations in spt cause a change in enzyme substrate specificity which results in the production of two atypical sphinganines, deoxysphinganine (dsp) and deoxymethylsphinganine (dmsp), rather than the normal enzyme product, sphinganine (sp). levels of deoxysphingolipids are elevated in the blood of hsn- patients and this has been shown to cause the peripheral nerve damage characteristic of the disease, which affects both sensory as well as motor axons. however, the underlying pathomechanism of how deoxysphingolipids damage neurons remains elusive. here, dsp and dmsp-mediated neurotoxicity was examined in primary mouse motor and sensory neurons, by assessing cell survival and neurite outgrowth following exposure to different concentrations of sp, dsp or dmsp. the abnormal enzyme products were found to have a rapid and dose-dependent neurotoxic effect in primary neurons. we also explored the potential mechanisms that underlie deoxysphingolipid neurotoxicity, by characterizing mitochondrial function and changes in calcium handling. we found that mitochondrial dysfunction and calcium handling deficits may be key mediators of abnormal sphingolipid neurotoxicity, in both motor and sensory cell models. specifically, we revealed mitochondrial abnormalities, signs of endoplasmic reticulum stress and dysfunction of store-operated calcium channels. we propose that early deficits in mitochondria and calcium handling may underlie deoxysphingolipid neurotoxicity and thus present potential therapeutic targets for hsn- . months. one patient was excluded because the decrease in hba c was not contemporaneous with weight loss. the mean and median decrease in bmi per month was . and . respectively. the mean and median interval between surgery and decrease in hba c was . and . days respectively. records of these patients were scrutinized and classified as: 'probable tind': acute painful neuropathy and acute dysautonomia with temporal relationship to the decrease in hba c; 'possible tind': acute painful neuropathy or acute dysautonomia or temporal relationship to decrease in hba c is uncertain; 'unlikely tind': when an alternative explanation exists for symptoms. only one patient was classified as 'possible tind': a -year-old woman who developed neuropathic pain in both lower limbs month after a rapid hba c decline of . % and about months after bariatric surgery. she had no documented autonomic symptoms. our study is limited by small cohort size, retrospective design and reliance on hospital records. nonetheless, it demonstrates that besides nutritional neuropathies, tind should also be considered in dm patients who develop peripheral neuropathy after bariatric surgery. in another study, we found tind is uncommon in a general cohort of diabetic patients. the occurrence of 'possible tind' in only dm patients corroborates earlier data that weight loss may act in tandem to increase the risk of tind. woolums bm , tabuchi m , sung h , sullivan jm , mamah c , yang m , blum id , wu mn , sumner cj , lloyd te . johns hopkins university, baltimore, usa. dominant missense mutations in the gene encoding the cation channel transient receptor vanilloid, family member (trpv ) cause inherited neuropathies including charcot-marie-tooth disease c (cmt c). in vitro, mutations in trpv that cause cmt c cause a gain of trpv channel function and increased intracellular calcium which subsequently leads to cellular toxicity. however, the mechanisms by which cmt c mutations in trpv lead to neuronal dysfunction in vivo remain poorly understood. we generated transgenic drosophila that express either wild-type or a cmt c causing trpv mutant (trpv r c ) to assess the effect of trpv r c on neuron function in vivo. selective expression of trpv r c in drosophila ccap neurons (n ccap ) results in a failure of drosophila wing expansion that is blocked by genetically inactivating the channel pore, demonstrating the requirement of channel function in mediating this phenotype. perforated patch clamp analysis of n ccap reveals that trpv r c causes a calcium-dependent increase in n ccap neuronal excitability. this hyperexcitability is restored to control levels by application of a trpv selective antagonist. high level expression of trpv r c causes synaptic and dendritic degeneration, both of which are rescued genetically by inactivating the channel pore or pharmacologically by feeding larvae a trpv selective antagonist. we conducted a genetic screen in n ccap and found that camkii knockdown potently suppresses the trpv r c mediated wing expansion phenotype and selectively rescues degeneration of synapses but not dendrites. we also find that trpv r c , but not controls, disrupts mitochondrial transport in axons by increasing the number of stationary mitochondria. our data demonstrate that trpv r c elevates neuronal intracellular calcium which disrupts mitochondrial transport and mediates neurodegeneration through compartment-specific calcium-mediated signaling pathways, and supports the further investigation of trpv antagonists as potential therapeutics for the treatment of cmt c. mood disorders, including anxiety and depression, are commonly observed among chronic pain patients with prevalence estimates ranging from to %. comorbidity between mood and chronic pain disorders has been linked to altered limbic regulation of the hypothalamic-pituitary-adrenal (hpa) axis. stress activates the hpa axis and can initiate and/or exacerbate symptoms related to both chronic pain and mood disorders. previous studies from our laboratory have investigated the influence of early life stress on mechanical pain hypersensitivity, visceral hypersensitivity and behavioral evidence of mood disorder later in life. here, we are testing the hypothesis that chronic stress exposure in adulthood can increase somatic and visceral sensitivity and anhedonic behaviors in a mouse strain with a genetic predisposition to anxiety. adult, female a/j mice were exposed to repeated foot shock stress for continuous days and tested for alterations in mechanical sensitivity, sucrose preference, visceromotor response (vmr) during urinary bladder distension, and serum corticosterone levels. mice that underwent shock stress had a significantly decreased mechanical withdrawal threshold in the hind paw compared to their baseline and sham group measurements. sucrose preference was measured prior to shock exposure and throughout the shock paradigm as an indicator of anhedonic behavior. mice that underwent shock stress displayed a trend toward decreased sucrose preference, indicating anhedonia, in comparison to mice in the sham group that did not display anhedonia. mice that underwent shock stress displayed significant increases in vmr during bladder distension compared to sham mice. finally, serum corticosterone levels were significantly higher in the mice that underwent shock stress compared to sham mice, indicating a stress-induced increase in hpa axis output. together these data suggest that chronic stress exposure can induce mechanical allodynia, visceral hypersensitivity, and depression-like behaviors in an anxiety-prone mouse strain. future studies will incorporate gene expression in the hypothalamus, amygdala, and hippocampus, as well as investigation of possible downstream peripheral neuroimmune modulation and neuronal morphology changes. guillain-barré syndrome (gbs) is an acute monophasic immune-mediated neuropathy. as prognostic markers of gbs, modified erasmus gbs outcome score (megos), erasmus gbs respiratory insufficiency score (egris), and Δigg have been reported. however, the proportions of subtypes of gbs are known to be different between the western countries and japan, it remains to be elucidated whether those markers can also be applied to gbs in japan or not. we here investigated retrospectively gbs patients and determined the megos and the egris of those cases. among them, Δigg could be obtained in cases. we evaluated the prognosis using gbs outcome score (functional grade: fg) at months; we called good prognosis when fg at months was less than and poor prognosis when that was or more. as a result, in cases with higher score than at megos on admission cases ( %) had poor prognosis and in cases with higher score than at megos at day of admission cases had poor prognosis. in cases with higher score than on egris cases ( %) needed the mechanical ventilator. patients with good prognosis had higher Δigg(average: mg/dl) than patients with poor prognosis(average: mg/dl). we calculated the cut-off value of Δigg in patients, which was mg/dl. patients with higher Δigg than mg/dl could significantly walk independently at six months (p< . ). patients ( %) had poor prognosis in patients with lower Δigg than mg/dl. ( %) of patients were treated with the single cycle of intravenous immunoglobulin (ivig). other patients ( %) were treated with the combined therapies, such as intra venous methylprednisolone and/or plasmapheresis or the second cycle of ivig in addition to ivig. although there was no difference in prognosis between patients with the single cycle of intravenous immunoglobulin (ivig) and patients with the combined therapies, in the patients who had fg> and megos > on admission, the combined therapies made better prognosis than the single course of ivig (p< . ). we found that megos, egris and Δigg were also available in japan. the efficacy of the combined therapies in severe gbs patients should be investigated in the future large scale prospective studies. yiu em , , , burns j , , , menezes mp , , ryan mm , , and for the paediatric cmt best practice guidelines consortium. royal children's hospital melbourne, melbourne, victoria, australia; murdoch childrens research institute, melbourne, victoria, australia; university of melbourne, melbourne, victoria, australia; university of sydney, new south wales, australia; sydney children's hospitals network (randwick and westmead), new south wales, australia. charcot-marie-tooth disease (cmt) often presents during childhood. common symptoms include weakness, limb pain and cramps, foot deformity, and balance impairment. guidelines for the optimal management of common problems experienced by children with cmt do not currently exist. development of these guidelines will provide an evidence base for the management and monitoring of children with cmt. a series of systematic literature reviews utilising the grade (grades of recommendation, assessment, development, and evaluation) approach were conducted to answer pre-specified key clinical questions related to the management of paediatric cmt. these included treatment recommendations for symptoms such as weakness, pain, balance impairment, joint deformity, and impaired upper limb function, and anticipatory monitoring for associated complications such as hip dysplasia. this yielded minimal to no evidence for the pre-specified clinical questions, and evidence-based management recommendations could not be made. consensus-based statements will therefore be formulated via a three-round delphi process, to be conducted in . the delphi panel will consist of local and international medical and allied health professionals who have experience in the management of children with cmt. efficacy of pxt in the treatment of adult patients cmt a (n= ) was shown in a multicenter, randomized, double-blind, placebo-controlled phase ii study (attarian et al. ) . pxt taken x/day, orally, for consecutive months was well tolerated and safe. significant improvement of disability was observed for the highest tested dose, thought indicative for an early, meaningful change in disease course (meta-analysis by mandel et al., ) . this formed the rationale to initiate a multicenter, randomized, double-blind, placebo-controlled pivotal phase iii study (clin-icaltrials.gov: nct ) of pxt in mildly to moderately affected cmt a patients in december . the primary objective is to assess the efficacy of doses of pxt compared to placebo on disability as measured by the mean change from baseline overall neurology limitations scale (onls) score at month and . furthermore, efficacy on the proportion of responders (i.e. improvement of onls), impairment (cmtns-v ), functional tests ( -mwt, qmt, -hpt), electrophysiological parameters (cmap, snap and ncv) and quality of life (eq- d, vas) are secondary endpoints. pursuant this study, patients will be eligible for a -month extension study, in which pxt assigned patients will continue with the previously assigned dose, whereas placebo patients will be randomized to one of the two pxt doses. the study is conducted in investigational sites in countries (eu, canada and us). in december patient randomization was completed (n= ). the screen failure rate was %, as expected ( patients were screened). the independent dsmb recommended to continue the study as planned following a safety analysis on the first patients treated for > months. preliminary baseline characteristics are based on patients (data not cleaned). the study population had a mean age of . ± . years (range - ; male . %) of which . % had a confirmed genetic diagnosis of cmt a. the mean cmtns-v was . ± . and the mean motor nerve conduction of the ulnar nerve was . ± . m/s. ten patients withdrew from the study, due to adverse events unrelated to study treatment. the last patient completing the study is expected in march . zhang g , ghosh p , lin j , ghauri a , sheikh ka . department of neurology, university of texas health science center at houston, houston, tx, usa. assessment of epidermal nerve fiber density and its structure integrity is critical for the diagnosis and evaluating the effectiveness of potential therapies in small fiber neuropathies. currently, skin biopsies, at multiple sites, are most commonly used to assess these diseases. these studies are expensive and time consuming due to cumbersome processing and quantification techniques and serial biopsies over time are often not feasible due to costs and patient acceptance. moreover, vast majority of normative data for skin biopsies in humans are available only for few distal sites and a significant proportion of patients with small fiber neuropathies have focal or regional symptoms not involving the commonly biopsied sites in the leg. live imaging could overcome these limitations and provide a noninvasive real time assessment of epidermal nerve fibers all over the body. we previously found that anti-ganglioside antibody (aga) is an effective neuronal delivery vector for transport of various cargos, such as fluorescent dyes, to peripheral nerves. in the current proof of concept study, we examined whether non-invasive multiphoton microscopy can be used to probe/image the epidermal nerve fibers in living animals after systemic and/or local delivery of fluorescently conjugated aga. we found that the individual nerve endings in skin and cornea are distinctly labeled, and visualized under two-photon microscope. the epidermal nerve fiber labelling by fluorescent-tagged aga was further validated using transgenic mice selectively expressing yellow fluorescent protein in their nervous system. in-vivo multiphoton imaging provide a tool with potential for dynamic longitudinal evaluation of small fiber neuropathies, including nerve degeneration and regeneration, without tissue removal. thus, the use of multiphoton microscopy in conjunction with fluorescently labeled aga as neuronal vector can have many research and clinical applications, such as labeling and live visualization of epidermal nerve fibers to assess small sensory nerve fibers in health and disease. zhou y , tavori h , lee s , al salihi m , fazio s , notterpek l . mcknight brain institute, university of florida, gainesville, florida, usa; knight cardiovascular institute, oregon health and science university, portland, oregon, usa. the majority of hereditary neuropathies are due to abnormalities in peripheral myelin protein (pmp ), whose genetic variants include increased expression (gene duplication), haploinsufficiency (gene deletion), or point mutations. phenotypic heterogeneity in clinical presentation is common among hereditary neuropathy patients even within the same family, the cause of which has not been determined. to investigate the role of pmp in the pathogenesis of the neuropathies we have generated and characterized pmp null (pmp −/− ) mice (amici et al., ) whose peripheral nerves show alterations in lipid metabolism (lee et al., ) . to examine the molecular changes underlying these abnormalities we determined the expression of cholesterol synthesis (srebp pathway), and cholesterol uptake, transport and efflux genes (lxr pathway). in affected nerves, we found the cholesterol synthesis pathway inhibited, while the lxr pathway, and particularly apoe and abca , upregulated at the mrna and protein levels. since pmp is expressed at low levels in the liver, the central organ for the regulation of cholesterol in the body, we studied liver tissue form pmp −/− mice. liver from pmp −/− mice showed significant hepatomegaly, clear features of microvesicular steatosis, as well as marked increase in lxr pathway genes and proteins (abca and apoe), as compared to wt. ultrastructural studies identified lipid droplets and significantly enlarged mitochondria in the liver of male pmp −/− mice, which is not due to mitochondria fusion, as the levels of mfn and remained similar to wt. as disturbed hepatic cholesterol homeostasis induces the activation of kupffer and stellate cells, we determined the extent of inflammation in nerve and liver tissues from pmp −/− mice with leukocyte (cd b) and macrophage markers (cd ). in nerve sections, we detected an increase in the number of cd b+ cells, which was confirmed by western blots. in the liver of pmp −/− mice we found a significant increase in cd -reactive kupffer cells and elevated levels of tnf-alpha. the severe dysregulation of cholesterol metabolism in nerve and liver, including neuroand hepatic inflammation in the absence of pmp − suggest that dysregulated cholesterol metabolism and inflammation may act as a disease modifier in pmp -dependent neuropathies. our aim was to ascertain frequency of and risk factors for tee, arterial (ate) and venous thromboembolic events (vte) in neuromuscular patients receiving regular ivig. we performed a retrospective case-note review of inflammatory neuropathy patients receiving regular ivig treatment. we collected the following data over a month study period this analysis suggests tee incidence is higher in ivig treated patients than comparable population-based rates. examination of tee occurrence in age and vascular risk factor matched ivig-treated and ivig-naïve individuals is required to appreciate the excess risk associated with ivig treatment. référence des maladies neuromusculaires et la sla, hôpital de la timone ha) and whether temporal dispersion (td) parameters are helpful. fifty-eight patients diagnosed according to asbury and cornblath ( ) were prospectively included between january and september . edx and were performed - (mean= ) and - days (mean= ) after disease onset, respectively. there were no differences in classification consistency between ho and ha at edx (p= . ) and edx (p= . ), but more patients were classified as aman when comparing ra with ho and ha at edx (p< . ). at edx , ra classified more patients as equivocal with ho (p< . ) and as aman with ha (p< . ) ) or edx (ho, fe p= ; ra, fe p= . ; ra with td, fe p= . ). gm , gd a, gd b and gq b igg antibodies were tested (willison at edx , only ho showed maybe more antibodies with aman compared to aidp (fe p= . , phi=− . ) and with aman compared to equivocal cases since correlation with factors associated with axonal gbs, in casu rcf and antibodies, is far from exclusive, the usefulness of edx subtype classification using specific criteria sets, remains doubtful. the frequency of rcf indicates that nodal/paranodal alterations may represent the main pathophysiology in more gbs patients than currently thought ruiz m , lessi f , cacciavillani m , riva m , salvalaggio a , campagnolo m , briani c . neurology, department of neuroscience, university of padua, padua, italy; hematology and clinical immunology unit, department of medicine, university of padua, padua, italy; cemes, data medica group, padua, italy.arsenic trioxide (ato) is highly effective in treatment of acute promyelocytic leukemia (apl). it is licensed in italy for treatment of relapsed-refractory apl and for first line chemotherapy in low risk patients. ato most frequent side effects are well described, but less is known on ato induced. we describe apl patients who were treated with all-trans retinoic acid (atra)/ato as first line therapy. the characteristics of ato induced neuropathy was prospectically analyzed by neurological evaluation using both the total neuropathy score, clinical version (tnsc) (a validated scale for chemotherapy induced peripheral neuropathy) and neurophysiological assessment. patients have been evaluated at baseline, at the end of the induction phase, at the end of ato/atra treatment and year after discontinuation of treatment. baseline neurophysiology was performed at the end of induction phase. both patients were men, respectively and -yr-old. none of the patients had previous history of neuropathy. baseline tnsc was (no clinical signs of neuropathy) in both patients. neurophysiological evaluation performed after the end of induction cycle did not reveal signs of peripheral neuropathy in both patients. patient received mg of ato during induction, total , mg. patient received mg of ato during induction, total , . both patients developed leg numbness during consolidation cycles and patient also hand numbness. tnsc at the end of therapy was in patient and in patient . neurophysiology at the end of therapy detected signs of sensitive axonal neuropathy in both patients. they received full doses of ato consolidation ( . mg/kg/day for days/week, on alternate months for total months and tretinoin weeks on weeks off). during the first year of follow-up both tnsc and neurophysiology year after the end of consolidation cycle were consistent with full recovery. our patients developed sensory axonal neuropathy during ato therapy, that clinically manifested during consolidation cycles and improved up to complete recovery during follow-up. published case reports show that outcomes may not be as good as in our patients. a multicenter prospective study evaluating the characteristics of ato-induced neuropathy in apl is ongoing. ruiz m , campagnolo m , salvalaggio a , cacciavillani m , taioli f , fabrizi gm , briani c . department of neuroscience, neurology unit, university of padua, padua, italy; data medica group, emg unit, cemes, padua, italy;mutations in the mitochondrial copper-binding protein sco , cytochrome c oxidase assembly protein, have been reported in several cases of fatal infantile cardioencephalomyopathy with cox deficiency. we identified compound heterozygous variants in sco in two unrelated patients with isolated length dependent axonal sensorimotor polyneuropathy of variable clinical severity (axonal autosomal recessive charcot-marie-tooth disease type , cmt ) by whole exome sequencing. although peripheral neuropathy has been described as a secondary feature in a few cases of fatal infantile cardioencephalomyopathy, the disease onset, clinical phenotype and survival in our patients differ significantly from the previously described cases. our patients developed predominantly motor neuropathy; moreover, they are still alive and they have not developed cardiomyopathy, which is the main phenotype and cause of death at early infancy in reported patients. both of our patients harbor mutations adjacent or near the conserved copper-binding motif (cxxxc), including the common reported pathogenic variant e k and the novel change d g. in addition, each patient carries a second mutation located in the same loop region of the protein, p t and r q. western blots from fibroblasts from these cmt patients showed reduced levels of cox , a subunit of complex iv, indicating cox deficiency. our findings demonstrate that cmt can be the predominant phenotype associated with sco mutations, pointing to a broader phenotypic heterogeneity. the mechanism linking mitochondrial respiratory chain dysfunction to isolated axonal loss of variable severity remains to be elucidated. the muscarinic acetylcholine (ach) type receptor (m r) is a metabotropic g protein-coupled receptor expressed by adult sensory neurons. cholinergic signaling through muscarinic receptors can modulate axonal plasticity in invertebrates and lower vertebrates. we have recently shown that selective (pirenzepine) and specific (muscarinic toxin : mt ) m r antagonists elevate neurite outgrowth and protect from small and large fiber neuropathy in adult sensory neurons in various animal models (calcutt, et al., ) . furthermore, we demonstrated that excessive cholinergic signaling due to m r overexpression caused a significant reduction in neurite outgrowth (calcutt, et al., ) . the mechanism of m r-antagonist driven neurite outgrowth remains poorly understood, however, we have proposed that ach constrains axonal outgrowth via m r activation. cholinergic signaling is mediated via recruitment of trimeric g proteins, of which g alpha- and g alpha- regulate cytoskeleton dynamics by control of tubulin polymerization. activated gtp-bound g-alpha proteins destabilize microtubules by increasing the intrinsic gtpase activity of tubulin. we have therefore tested the hypothesis that cholinergic signaling regulates neurite outgrowth via modulation of g protein mobilization and the dynamics of the tubulin cytoskeleton. we found that over-expression of m r in adult sensory neurons induced dissolution of the tubulin cytoskeleton in distal neurites. g alpha- expression in adult sensory neurons was significantly higher (p< . , -fold) than g alpha- . subsequent knockdown the peripheral neuropathy research registry (pnrr) is a multicenter collaborative research project sponsored by the foundation for peripheral neuropathy to advance the science in distal symmetrical polyneuropathies (dsp). the registry was designed to prospectively characterize clinical phenotype and natural history of patients with dsp and obtain biofluids to identify new causes and genetic modifiers of dsp with careful genotype/phenotype correlations, and to develop biomarkers. the enrollment in the registry is still ongoing but an interim analysis was carried at the end of december . eligible study participants were years or older with a diagnosis of idiopathic, diabetic, chemotherapy-or hiv-induced peripheral neuropathy. they were examined by a physician at one of the six consortium members (johns hopkins university school of medicine, icahn school of medicine at mount sinai medical center, beth israel deaconess medical center, northwestern university medical center, university of utah medical center and kansas university medical center). the collected data set included ( ) a detailed questionnaire, discussing their symptoms, medical history and family history, ( ) a standardized neurological examination form, ( ) electrodiagnostic evaluation and ( ) diagnostic laboratory testing. blood samples (whole blood, plasma and serum) were collected for future biomarker and genotyping evaluations. at the end of , complete data sets and blood samples were collected from patients. % had diabetic pn, % had hiv-associated pn, % had chemotherapy induced pn and % were diagnosed with idiopathic pn. detailed analysis of clinical presentation, examination findings and diagnostic investigations will be discussed at the presentation. standardized phenotyping with linked bio-specimen banking will help establish the minimum data set required for neuropathy diagnosis and support genotype-phenotype correlations with next generation sequencing technologies and development of novel biomarkers. pnrr data will improve our understanding of disease mechanisms paving the way for new therapeutic discoveries in painful and non-painful neuropathies. chemotherapy-induced peripheral neuropathy (cipn) is a major side effect of treatment, typically presenting as a sensory neuropathy. symptoms include pain, paraesthesia, and numbness in the extremities, resulting in functional impairment. increasingly, patient reported outcomes (pros) are utilized to accurately examine the impact of cipn symptoms on patient function. however, the links between objective neurological assessment and pro measures remain ill defined. this study aimed to identify links between neurophysiological measures and pros in patients treated with neurotoxic chemotherapy. assessments were conducted in patients (f= , mean age . ± . years) who had completed neurotoxic chemotherapy on average . ± months previously (platinum-based n= , taxane-based n= or taxane/platinum combination therapy n= ). patients reported the presence and severity of neuropathic symptoms via the fact-gog ntx , a validated patient questionnaire. clinical the most important neurological side effect of a large number of anti-cancer drugs is a painful peripheral neuropathy. mainly chemotherapeutics that interfere with microtubules, including the plant derived vinca-alkaloids such as vincristine, are well known to cause chemotherapy-induced peripheral neuropathies (cipn). to date, few treatments are available and focus on symptom alleviation and pain reduction rather than on preventing the neuropathy all together. for the first time, we highlight the potential of specific histone deacetylase (hdac ) inhibitors as a preventive therapy for cipn, using novel rodent models for vincristine-induced peripheral neuropathies (vipn), characterized by a sensory axonopathy. one reason so few therapies are available, is because the exact pathophysiological mechanisms are poorly understood. mounting evidence proposes axonal transport, a pathway frequently disturbed in neurological disorders, as a major player in the pathophysiology of vipn. proper axonal transport requires dynamic microtubules which are highly modulated by post-translational modifications. since vincristine interferes with the polymerization of microtubules, we reason disturbances in microtubule dynamics, and by extension axonal transport, could contribute to vipn. we illustrate that increasing acetylation of -tubulin after hdac -inhibition, can restore vincristine-induced defects in axonal transport in cultured dorsal root ganglion neurons. also in vivo, -tubulin acetylation was restored in the saphenous nerve and dorsal root ganglia, two sensory tissues that are affected by vincristine. ultimately, this correlates to a reduced severity of the neurological symptoms, both on the electrophysiological and on the behavioral level. moreover, we discovered that hdac -inhibition was not only protective against neurotoxicity, but also reduced tumor progression in a mouse model for acute lymphoblastic leukemia. taken together, our results show that hdac -inhibition is an ideal strategy to prevent vipn with beneficial effects both on the neurotoxicity as well as on tumor growth.approximately two-thirds of cidp subjects need long-term corticosteroids or intravenous immunoglobulins (ivig), with ivig being slightly preferred based on safety profiles. subcutaneous ig (scig) is an alternative option for ig delivery but has not previously been investigated in a large-scale clinical trial in cidp.we performed a randomized, double-blind trial in cidp investigating . and . g/kg weekly doses of scig igpro (hizentra ® , csl behring) versus placebo in subjects for maintenance treatment. ivig-dependent adults with definite or probable cidp according to efns/pns criteria were eligible. the primary outcome was the percentage of subjects with a cidp relapse ( -point deterioration on adjusted incat disability score) or who were withdrawn for any other reason during the -week scig-treatment period. multiple secondary endpoints were assessed. superiority of at least one igpro dose over placebo was tested one-sided using the cochran-armitage trend test for the primary outcome and the jonckheere-terpstra tests for secondary outcomes.the primary outcome occurred in % of high-dose scig, % of low-dose scig, and % of placebo subjects (p < . ); cidp relapse occurred in % of high-dose scig, % of low-dose scig and % of placebo subjects (p < . ), respectively. both scig doses were superior to placebo (low-dose vs placebo p = . ; high dose vs placebo p < . ). median incat score, mrc sum score, and grip strength remained stable in scig subjects. high-dose scig prevented the r-ods decline seen with low-dose scig and placebo (p < . ). all placebo subjects deteriorated on measures of strength and disability.causally related adverse events occurred in ( %) subjects ( % placebo, % low dose, and % high dose).scig igpro was efficacious and safe as maintenance treatment. mutations in metalloendopeptidase (mme) gene have been associated with autosomal-recessive late-onset charcot-marie-tooth type- (cmt ). to date, all patients have had at least one truncating mutation, either in homozygosity or in trans with a missense mutation. more recently, loss-of-function and missense heterozygous mutations were also identified in autosomal-dominant cmt. we report the case of a previously healthy caucasian woman, born to healthy unrelated parents, who presented at the age of thirty-nine with numbness and cold sensation in the lower limbs. subsequently she developed progressive gait disturbance and impaired hand dexterity. her homozygous twin presented at the same age with similar symptoms. the family history was otherwise uneventful, in particular neither neuropathy nor dementia were described. neurological examination at the age of fifty-three revealed a steppage gait, distal upper and lower limb atrophy and weakness, distal sensory loss and bilateral pes cavus. deep tendon reflexes were normal in the upper limbs and absent in the lower limbs. nerve conduction studies revealed an axonal sensory and motor neuropathy. a sural nerve biopsy revealed a reduction in myelinated nerve fibers and active axonal degeneration. targeted sanger sequencing of mpz , gjb , gdap , nefl, fkrp, bscl , hspb and mfn were negative. sureselect focused exome sequencing was therefore performed and identified two missense heterozygous mutations [c. g>a,p.c y;c. t>c,p.y h] or lack of efficacy (n= ). most frequently reported adverse events were cognitive ( %), gastrointestinal ( %) and genitourinary ( %). discontinuation due to perception of lack of efficacy was more frequently reported by men ( % vs % p= . ). women presented higher punctuations on pigc scale compared with men ( . ± . vs . ± . , p= . ). pigc scores were significantly higher in patients receiving taxane ( . ± . ) than platinum ( . ± . ) agents (p= . ). no significant differences according severity of neuropathy neither type of chemotherapy were observed in drop-out and retention rates. patients with long-lasting cipn (> months) reported lower pigc scores ( . ± . vs . ± . , p= . ) and higher frequency of suspension due to adverse events ( % vs %, p= . ) and less rate of continuation of duloxetine ( % vs %, p= . ). more than one-third of patients with disturbing cipn discontinued duloxetine prematurely due to intolerable side-effects. low tolerability, male gender and long-lasting cipn may limit duloxetine usefulness in the treatment of symptomatic cipn. verboon c , jacobs bc , and the igos consortium. department of neurology, erasmus medical centre, rotterdam, the netherlands; department of immunology, erasmus medical centre, rotterdam, the netherlands.the efficacy of intravenous immunoglobulin (ivig) in guillain-barré syndrome (gbs) has only been demonstrated in severely affected patients who are unable to walk independently. although there is no proof that ivig is effective in milder forms of gbs, some neurologists are treating these patients with ivig considering that even milder forms of gbs may result in poor recovery, residual deficits, fatigue or pain.we determined the effectiveness of a single course of ivig ( g/kg in - days) in relatively mild forms of gbs in the ongoing observational international gbs outcome study (igos). the gbs disability score, mrc sum score and patient reported outcome measures (prom) were compared at and weeks. ordinal logistic regression analysis was used to determine the effect of ivig on the gbs disability score, adjusted for previously identified prognostic factors.data were analyzed from the first patients enrolled in igos by december , including patients with mild gbs at entry, of which patients ( %) were treated with supportive care, while patients ( %) received ivig (start ivig after onset of symptoms in days, median , iqr - ). at baseline, patients in the ivig treated group compared to the untreated group less frequently had pure motor gbs ( % versus %, p< . ) and axonal damage or unresponsive nerves ( % versus %, p= . ), but a worse gbs disability scores at nadir (p= . ). the adjusted common odds ratio for a better gbs disability score at weeks was . ( % ci . - . ). at weeks, the median mrc sum scores and prom were not significantly different between treated and untreated patients. however, more patients in the ivig group showed complete recovery of muscle strength at weeks than patients in the control group ( % versus %) (p= . ) and more frequently showed full neurological recovery on the gbs disability scale ( % versus %, p= . ). additional results will be presented at the conference.based on the results of this interim analysis in observational data, we conclude that patients with a relatively mild form of gbs may benefit from a single course of ivig. despite treatment with intravenous immunoglobulin (ivig), many patients with guillain-barré syndrome (gbs) recover insufficiently. we primarily aimed to determine whether a second ivig course ( g/kg in - days) in patients with a poor prognosis improves outcome on the gbs disability scale after weeks. we included patients from the prospective, observational international gbs outcome study (igos) treated with ivig and who had a poor prognosis on the modified erasmus gbs outcome score (megos). of patients enrolled in igos, patients were eligible; patients ( %) were treated with one ivig course (control group); patients ( %) received an 'early' second ivig course ( - weeks after start first course) and patients ( %) a 'late' second ivig course (within - weeks). one week after study entry, patients receiving an 'early' or 'late' second ivig course had significantly worse gbs disability scores and mrc sum scores than controls, implying the need for adjustment of baseline characteristics. the adjusted common odds ratio for a better gbs disability score at weeks was . ( % ci . - . ) for the 'early' group, and . ( % ci . - . ) for the 'late' group, suggesting worse outcomes with a second course of ivig compared to controls. at months, patients ( %) in the 'early' second ivig group, patients ( %) in the control group and only ( %) in the 'late' second ivig group were able to walk unaided (p= . ). the adjusted common odds ratio for a better gbs disability score at weeks was . ( % ci . - . ) for the 'early' second ivig group and only . ( % ci . - . ) for the 'late' second ivig group. in gbs patients with a poor prognosis, we did not find a beneficial effect of a second course of ivig after weeks follow-up. our results suggest that an 'early' administered second ivig course might improve outcome at weeks. given the limitations of this observational study, a randomized controlled trial with a larger number of gbs patients with a poor prognosis being treated early in the disease course is needed to confirm or refute these results. vidal c , bhatt n , agudelo c , mahapatra a , saporta ma . department of neurology, university of miami miller school of medicine, miami, usa.multifocal motor neuropathy (mmn) is an inflammatory demyelinating chronic neuropathy characterized by progressive asymmetric weakness in the distribution of two or more peripheral nerves, without objective sensory loss or upper motor neuron signs. the cardinal neurophysiological finding is conduction block outside the usual sites of nerve compression. supportive clinical criteria include high titers of anti-gm antibodies, good response to ivig, increased cerebrospinal fluid (csf) protein (< g/dl) and magnetic resonance imaging (mri) with diffuse swelling of the brachial plexus. we report a case of a year old woman with a two month history of progressive muscular weakness which began in the right upper extremity, described as difficulty in gripping objects, writing and typing on the computer, followed by progressive lower extremity weakness with difficulty rising from a chair and left foot drop. she also complained of pain in her right trapezius and scapular area. she denied any tingling or numbness. on neurological examination there was right scapular winging and asymmetric weakness predominantly involving the right upper and left lower extremities. reflexes were absent throughout. lumbar puncture revealed albuminocytological dissociation (wbc and protein . g/dl). ncs revealed slow conduction velocities for both ulnar and median nerves and a median nerve conduction block at the forearm segment. sensory nerve action potentials were normal for all tested nerves. needle emg revealed acute denervation of right periscapular muscles. brachial plexus mri showed symmetric bilateral thickening from trunks to peripheral nerves. anti-gm igg/igm were negative. two days after lumbar puncture and the beginning of ivig, patient presented binocular diplopia on extreme left lateral gaze. brain mri with and without contrast showed no intracranial pathologies. after five days of ivig, patient was discharged with significant clinical improvement and recovery of the right scapular winging. diplopia improved a week after discharge. we report an atypical multifocal motor neuropathy case. although this patient fulfills all clinical criteria for mmn, we report some features usually not found in mmn such as scapular winging and a mild and transient left vi nerve palsy. mmn should be included in the differential diagnosis of scapular winging. vlckova e , , raputova j , , srotova i , , sommer c , Üçeyler n , birklein f , rebhorn c , rittner hl , kovalova e , , nekvapilova e , , belobradkova j , olsovsky j , weber p , dusek l , jarkovsky j , bednarik j , . despite many studies addressing biomarkers for pain in diabetic polyneuropathy (dpn), little is known about why it affects only a certain proportion of dpn patients. the somatosensory system plays a key role in the pathophysiology of neuropathic pain (neup) and subgroups with different sensory profiles might respond differently to pain treatment. we aimed to characterize sensory phenotypes of patients with painful and painless diabetic neuropathy and to assess demographic, clinical, metabolic, electrophysiological and psychological parameters related to the presence of neup in a large cohort of well-defined dpn subjects.this observational cross-sectional multi-centre cohort study (performed as part of the ncrnapain eu consortium) of subjects with dpn (non-painful, ndpn, n= ; painful, pdpn n= ) associated with diabetes mellitus of type and (median age years, range - years; women) comprised detailed history taking, neurological examination, laboratory tests, quantitative sensory testing, nerve conduction studies, neuropathy severity scores, and neuropsychological questionnaires. all parameters were analysed with regard to the presence and severity of neup. the presence and severity of neup were positively correlated with severity of neuropathy and thermal hyposensitivity (p< . ). a minority of pdpn patients ( . %) had a sensory profile indicating thermal hypersensitivity; this was associated with less severe neuropathy and better response to pain therapy. the presence of neup was also associated with female gender (p< . ) and with higher cognitive appraisal of pain as assessed by the pain catastrophizing scale (p< . ), while parameters related to diabetes (duration, hba c, microangiopathy) showed no influence on neup presence and severity. this study confirms the necessity of comprehensive dpn phenotyping and underlines the importance of the severity of neuropathy that should be taken into account in the stratification of patients with pdpn for analgesic treatment and drug trials.people with charcot marie tooth disease (cmt) experience slowly progressive muscle weakness, sensory loss and musculoskeletal changes over time. this leads to disability and risk of comorbidities due to inactivity. exercise is important to maintain general health but may also help to improve symptoms of cmt. we conducted a randomised controlled crossover trial of aerobic exercise to ascertain the effect of training on fitness levels, muscle strength, function and general well-being. in addition, we monitored the safety of training and feasibility of participation in this type of exercise in local community gyms. motivation, confidence and barriers to exercise were explored using qualitative interviews. the recruitment target was people. in total people with cmt a were approached to participate and were unable to commit to the trial or did not meet the study criteria on initial screening. thirty-one people underwent more detailed screening but three failed to meet the study criteria, five people withdrew before starting and three withdrew part way through the study. the data for the people who started the study were analysed using a random effects model. there was a % participation level in the training and it was well tolerated with no increases in pain or blood serum creatine kinase. an increase in vo peak (ml/min/kg) was observed in the cmt group with (pre training: n= , . ± . , % ci . to . ; post training: n= , . ± . , % ci . to . ; pre control: n= , . ± . % ci . to . ; post control: n= , . ± . , % ci . to . ) . there was wide between subject variation leading to a small overall effect size with cohen d of . ( % ci:- . to . ). a tentative regression model showed no effect of group or time point. there were no major changes in other measures of impairment, function or patient reported outcome measures. this pilot study showed that a community based model of training had a small effect on cardiopulmonary fitness, and was well tolerated with good participation. ankle-foot orthoses (afo) are commonly prescribed for children with charcot-marie-tooth disease (cmt) to manage foot drop, however the type and severity of functional impairment results in gait deviations that might require alternate orthotic designs. the aim of this study was to identify d gait patterns of children with cmt based on severity of functional weakness (based on heel walk, toe walk and foot drop items during gait using the cmt pediatric scale) to inform a design pipeline for d printed orthoses. d gait data were captured with an -camera vicon nexus motion capture system using the lower body plug-in-gait model in children with cmt ( male; ± . yrs, ± . cm, ± . kg), of various cmt types: cmt a, cmt e, cmt f, cmt a, cmt c, cmtx , cmtx and compared to typically developing children ( male; . ± . yrs, ± . cm, ± . kg). data were subdivided into three groups denoting increasing severity of dorsiflexion and plantarflexion weakness: no difficulty heel or toe walking (cmt nd ), difficulty heel walking (cmt dh ), difficulty toe and heel walking (cmt dth ). the cmt nd group showed a near-normal gait pattern. the only significant differences noted at the ankle were reduced peak dorsiflexion in stance (p< . ), indicating that an orthotic intervention may not be required. in addition to reduced peak dorsiflexion, the cmt dh group demonstrated significantly reduced dorsiflexion in swing (foot-drop) and a reduced dorsiflexor moment in loading response (p< . ). this suggest, the cmt dh group would require a flexible afo to allow activation of the plantarflexors during push-off, prevent foot-drop and restore a heel rocker in loading response. in contrast, the cmt dth group presented with significantly delayed and increased peak dorsiflexion in stance and reduced plantarflexion and power at push-off (p< . ). they also had significantly increased mean knee extensor moment (p< . ) revealing early signs of 'crouch gait'. therefore, the cmt dth group would require a rigid afo to limit the amount of dorsiflexion and assist movement of the ground reaction force anterior to the knee during stance. three distinct gait patterns at the ankle were identified in children with cmt, indicating patient-specific orthotic design pathways to target specific functional impairment. wojciechowski e , , chang a , cheng t , , little d , , menezes mp , , hogan s , burns j , . university of sydney, new south wales, australia; sydney children's hospitals network (randwick and westmead), new south wales, australia.children with cmt are often prescribed ankle-foot orthoses (afo) to manage lower limb impairment such as foot drop and foot deformities. they are handmade by plaster cast followed by thermoplastic moulding. this traditional approach provides limited design options, can be costly, with long outpatient wait times. d printing, also known as additive manufacturing, has the potential to transform the way orthoses are prescribed, designed and manufactured. the aim of this review was to evaluate the evidence of d printing afos compared to traditional manufacturing methods, for children with cmt. there are currently no studies evaluating the application of d printing afos for children with cmt. however, a small, but emerging evidence base exists for d printed afo's in adults from studies including healthy participants and populations with rheumatoid arthritis, post-polio syndrome, foot drop and ankle weakness secondary to injury. samples sizes ranged from - participants for studies related to in-shoe orthoses and from - for studies related to afos. the methods of d printing included sterolithography, selective laser sintering and fused deposition modelling using materials such as nylon , nylon , polylactide, polycarbonate and abs. d printed afos were comparable to traditional manufactured orthoses in terms of patient-perceived comfort, temporal-spatial parameters, plantar pressure measurements and d gait analysis. however, the effects of long-term usage and durability of d printed afos has not been investigated. d printing orthoses have potential advantages including increased design possibilities, improved productivity, higher compliance, and reduced labour needs. disadvantages include redesigning clinical pathways, limited evidence base for clinical effectiveness, limited biocompatible materials, occupational safety considerations and a high level of expertise required for software operation and fabrication of devices. further research is required to determine the feasibility of d printed afos for children with cmt, and the most appropriate and effective printing pathway, materials to improve health outcomes of affected patients. wong shj , koh sj , lee bjh , chng ysk , pawa c , subramaniam t , cheng ksa , umapathi t . lee kong chian school of medicine, nanyang technological university, singapore; national neuroscience institute, singapore; yong loo lin school of medicine, national university singapore, singapore; khoo teck puat hospital, singapore.treatment-induced neuropathy of diabetes mellitus (dm) (tind) is a complication of rapid glycaemic control. individuals present with neuropathic pain and autonomic dysfunction within weeks of improvement in glucose control. the use of both insulin and oral hypoglycaemic agents has been associated with tind. its severity is determined by the rate and quantum of hba c decline. other predisposing factors include anorexia and weight loss. we studied the incidence of tind in dm patients who have undergone massive weight loss and hba c decline after bariatric surgery. we screened electronic records of patients ( dm, non-dm) who underwent bariatric surgery between and . dm patients fulfilled the tind hba c criteria of a decrease of ≥ % over months or ≥ % over key: cord- -tdx ccj authors: bradley, benjamin t; maioli, heather; johnston, robert; chaudhry, irfan; fink, susan l; xu, haodong; najafian, behzad; deutsch, gail; lacy, j matthew; williams, timothy; yarid, nicole; marshall, desiree a title: histopathology and ultrastructural findings of fatal covid- infections in washington state: a case series date: - - journal: lancet doi: . /s - ( ) - sha: doc_id: cord_uid: tdx ccj background: severe acute respiratory syndrome coronavirus (sars-cov- ) is the cause of an ongoing pandemic, with increasing deaths worldwide. to date, documentation of the histopathological features in fatal cases of the disease caused by sars-cov- (covid- ) has been scarce due to sparse autopsy performance and incomplete organ sampling. we aimed to provide a clinicopathological report of severe covid- cases by documenting histopathological changes and evidence of sars-cov- tissue tropism. methods: in this case series, patients with a positive antemortem or post-mortem sars-cov- result were considered eligible for enrolment. post-mortem examinations were done on people who died with covid- at the king county medical examiner's office (seattle, wa, usa) and snohomish county medical examiner's office (everett, wa, usa) in negative-pressure isolation suites during february and march, . clinical and laboratory data were reviewed. tissue examination was done by light microscopy, immunohistochemistry, electron microscopy, and quantitative rt-pcr. findings: the median age of our cohort was · years (range – ; iqr · – · ). all patients had clinically significant comorbidities, the most common being hypertension, chronic kidney disease, obstructive sleep apnoea, and metabolic disease including diabetes and obesity. the major pulmonary finding was diffuse alveolar damage in the acute or organising phases, with five patients showing focal pulmonary microthrombi. coronavirus-like particles were detected in the respiratory system, kidney, and gastrointestinal tract. lymphocytic myocarditis was observed in one patient with viral rna detected in the tissue. interpretation: the primary pathology observed in our cohort was diffuse alveolar damage, with virus located in the pneumocytes and tracheal epithelium. microthrombi, where observed, were scarce and endotheliitis was not identified. although other non-pulmonary organs showed susceptibility to infection, their contribution to the pathogenesis of sars-cov- infection requires further examination. funding: none. in december, , severe acute respiratory syndrome coronavirus (sars-cov- ) was identified in wuhan, china from a cluster of severe pneumonia cases. the virus and the disease it causes (covid- ) have now spread globally and are responsible for an ongoing pandemic that has claimed hundreds of thousands of lives. in the months after its emergence, the community of health-care workers and researchers acted quickly to sequence the virus, establish transmission chains, elucidate the receptor, and test therapeutics. , these efforts have revealed similarities and differences between sars-cov- and the related virus, severe acute respiratory syndrome coronavirus (sars-cov). both viruses have similar clinical presentations, with the highest viral load identified in lower respiratory samples. , viral rna has also been detected in blood, stool, and urine samples, suggesting the potential for extrapulmonary spread and multiorgan involvement. , the viruses also share a common cellular entry receptor, angiotensin converting enzyme (ace ). despite their similarities, sars-cov was responsible for a restricted disease outbreak with high mortality, whereas sars-cov- has caused a greater number of infections with relatively lower mortality. post-mortem studies have shown pulmonary, renal, and small vessel injury, with particles resembling virus observed in the kidney by electron microscopy. [ ] [ ] [ ] [ ] one study described two complete autopsies without tissue-based methods for detection of sars-cov- . our study expands the literature by documenting a series of fatal covid- cases that occurred in washington state during february and march, . systematic evaluation of all major organs was done by a combina tion of light microscopy, immunohistochemistry, electron microscopy, and quantitative rt-pcr. our findings serve as a basis for generating further discussion related to the tropism, mechanisms of dissemination, and pathophysiology of severe sars-cov- infection. in this case series, patients with a positive antemortem or post-mortem sars-cov- result were considered eligible for enrolment. preliminary testing for sars-cov- was done at the washington state department of health public health laboratory (shoreline, wa, usa). confirmatory testing was done by the us centers for disease control and prevention (cdc) in atlanta (ga, usa). both locations used the cdc-designed ncov real-time rt-pcr assay for virus detection. autopsies were done at the king county medical examiner's office (seattle, wa, usa) and snohomish county medical examiner's office (everett, wa, usa) in negative-pressure isolation suites during february and march, . given safety concerns, in-situ dissection was done for patients , , , , , , and . patients , , , , , , and were examined by standard autopsy procedure. three to blocks were submitted per autopsy case. limited autopsies submitted two blocks containing sections from the left and right lung lobes, whereas complete autopsies submitted at least four blocks of lung-containing sections from central and peripheral locations of both lungs. resource limitations led to fresh tissue collection for patients , , and only. institutional review board approval was requested and waived for this study (study ). autopsy material was fixed in % neutral buffered formalin and submitted for standard processing with haematoxylin and eosin staining. select kidney sections were stained with periodic acid schiff and jones methenamine silver. evaluation of haematoxylin and eosin sections was done by consensus agreement by four board-certified forensic pathologists (ny, tw, jml, and dam) with expert guidance provided in cardiothoracic pathology (hx and gd) and renal pathology (bn). immunohistochemical staining was done on formalinfixed, paraffin-embedded -µm sections following citrate ph · antigen retrieval, endogenous biotin, and peroxidase block. immunohistochemistry for sars-cov- was done using a monoclonal antibody to the spike protein ( : ; genetex; irvine, ca, usa) on the ventana benchmark ultra ihc (roche diagnostics; basel, switzerland). images were visualised and captured with a digital camera mounted on a nikon eclipse i microscope using nis-elements advanced research software version . (nikon instruments; tokyo, japan). samples from patients and were placed in halfstrength karnovsky fixative. tissue was then post-fixed in % osmium tetroxide, processed according to standard transmission electron microscopy procedures, and embedded in polybed (polyscience; warrington pa, usa). suitable sections were identified by toluidine blue staining. thin sections were examined using a tecnai g spirit bio-twin transmission electron microscope (fei; hillsboro, or, usa) and digital images and measurements were acquired using amt image capture software (version . ). rna was extracted from · µg of tissue using the direct-zol rna miniprep plus kit (zymo research; irvine, ca, usa). complementary dna was synthesised using the iscript cdna kit (biorad; hercules, ca, usa). samples were tested in triplicate using itaq evidence before this study we searched pubmed and medline for peer-reviewed articles published between database inception and may , , that described the histopathological features of severe covid- infections, with the search terms "sars-cov- ", "covid- ", "autopsy", "postmortem", and "histology". our search was restricted to studies published in english. of the eight studies identified by our search, one documented complete histopathological findings from all major organs in two autopsies. other series showed evidence of diffuse alveolar damage, endothelial injury, and viral particles within renal cells. tissue quantitative rt-pcr (rt-qpcr) was used as an ancillary technique to identify virus in one study. this study provides crucial information related to the natural history of fatal covid- from early in the us outbreak. our analysis used multiple methods, including clinical chart review, histopathological evaluation, electron microscopy, immunohistochemistry, and quantitative rt-qpcr to examine all major organ systems. to our knowledge, no previous studies have used all these techniques simultaneously. our results support previous studies, which suggested that diffuse alveolar damage is the major source of pulmonary injury in covid- ; however, we found no evidence of widespread microvascular injury. additional investigations raised the possibility of extrapulmonary involvement in renal, intestinal, cardiac, and lymphoid tissues. in addition to the results of previous studies, our findings provide a histological explanation for physiological derangements observed by clinicians in patients who died with covid- . further investigation is required to characterise the extent of extrapulmonary injury caused by severe acute respiratory syndrome coronavirus infection. universal probes supermix (biorad) with the cdc ncov n and n primer/probe set and sarbeco e gene primer/probe set (idt; coralville, ia, usa). cycle threshold values less than were considered positive. the limit of detection was estimated to be copies per reaction based on serial dilutions of a positive control plasmid (cov ; exact diagnostics; fort worth, tx, usa). negative control reactions included on each plate repro ducibly showed no amplification. pcr was done on patients , , and . tissues examined included lung, trachea, subcarinal lymph node, heart, spleen, liver, large intestine, kidney, and whole blood. there was no funding source for this study. the median age of our cohort was · years (range to ; iqr · - · ). seven members of the cohort were part of a single cluster from a long-term care facility. all patients had clinically significant comorbidities, the most common being hypertension, chronic kidney disease, obstructive sleep apnoea, and metabolic disease, including diabetes and obesity (table ) . the most frequent presenting symptoms were respiratory distress, fever, and cough. less commonly encountered initial symptoms included altered mental status and gastrointestinal symptoms (nausea, vomiting, or diarrhoea). during hospital admission, six patients had acute kidney injury shown by elevated creatinine and three patients had elevated troponins. additional respiratory pathogens were isolated from two patientspatient had influenza a and meticillin-sensitive staphylococcus aureus and patient had sputum cultures positive for pseudomonas aeruginosa. excluding those who declined resuscitative measures (six patients), all patients were intubated. patient was electively extubated less than a day before death. the median time from symptom onset to intubation was · days (iqr - ; eight patients), with most intubations occurring at the time of admission. time to death after symptom onset varied from day to days, with a median time of days (iqr - ). for patients , , and , fresh tissue was collected at the time of autopsy for molecular and ultrastructural examination. patient was a -year-old woman who presented with cough and malaise for days after returning from international travel. on admission, the patient was hypoxic with elevated troponins and leukocytosis. her x-ray showed extensive bilateral opacities and testing was positive for sars-cov- , influenza a, and meticillin-sensitive s aureus. the patient had increasing oxygen demands and died days after admission. patient was a -year-old man with a complex cardiovascular history, end-stage renal disease, and chronic pulmonary fibrosis who presented with shortness of breath, delirium, and new onset atrioventricular node block. given his respiratory symptoms and lymphopenia, sars-cov- testing was done and returned a positive result. days after admission, the patient developed worsening hypoxia followed by sudden cardiac arrest and died. patient was a -year-old woman who presented after days of progressive shortness of breath while travelling abroad. on admission, she had clinically significant respiratory distress with hypoxia and signs of shock with elevated lactate, leukocytosis, and initial normal troponin. her chest x-ray showed diffuse lung disease and sars-cov- testing was positive. the patient's clinical course was complicated by multifactorial encephalopathy, new atrial fibrillation, and presumed ventilator-associated pneumonia and she died days after admission. all seven decedents examined by gross standard autopsy had heavy, oedematous lungs, with an average weight of g for the right lung and g for the left lung. intraparenchymal haemorrhage was observed in patient and pulmonary consolidation was present in patient . the volume of pleural fluid was highly variable and ranged from ml to ml per pleural space. patients and had evidence of central pulmonary emboli (figure ). splenomegaly was observed in patients and ( g and g, respectively), whereas splenic atrophy was observed in patient ( g). scattered punctate subarachnoid haemorrhages were observed in the brain of patient . additional gross findings showed changes including varying degrees of cardiac and atherosclerotic disease, hypertensive renal surface changes, and hepatic congestion in most patients. organ weights and gross obser vations are available in the appendix (p ). histopathological examination of the pulmonary system showed a spectrum of diffuse alveolar damage in ( %) of patients, which was evidenced by the presence of intra-alveolar fibrin, hyaline membranes, or loosely organising connective tissue in the alveolar septal walls. ( %) of patients with diffuse alveolar damage showed acute or acute and organising diffuse alveolar damage ( figure a, b) . for patient , only organising diffuse alveolar damage was observed. airways and alveolar spaces contained large, reactive multinucleated cells that stained positive for the epithelial marker ttf- and negative for the macrophage marker cd (figure c, d). microscopic haemorrhage was identified cardiac findings were mostly non-specific and associated with pre-existing comorbidities. the most common changes observed were fibrosis in ( %) patients and myocyte hypertrophy in ( %) patients. in patient , myocarditis was present with aggregates of lymphocytes surrounding necrotic myocytes ( figure c, d) . sars-cov- s protein immunohistochemistry was negative in patient . amyloid was identified in small vessels in patient and within the myocardium in patient . patients , , and had splenic white pulp depletion, which was confirmed by cd staining in patient (figure e). patient had a small sub-centimetre splenic infarction. subcarinal lymph nodes showed normal follicular architecture with haemophagocytosis observed in patients , , and . sars-cov- immunohistochemical staining was negative in the subcarinal lymph node from patient and spleen from patient . lymphoid tissue from additional patients was not available for testing. the kidneys showed mild to severe arterione phrosclerosis and diabetic nephropathy. although restricted by autolysis, features suggestive of acute tubular injury, including extensive tubular epithelial vacuolisation, were identified in patients. in patient , chronic inflammation of the renal parenchyma and focal segmental glomerulosclerosis were present. sars-cov- immunohistochemical testing in patients and showed patchy, granular cytoplasmic staining of the renal tubular epithelial cells (figure f). virus was not visualised in the endothelial cells or glomeruli by immunohistochemistry. pathological findings in the liver showed predominantly chronic changes associated with pre-existing comorbidities, with variable degrees of acute congestion. centrilobular necrosis consistent with hypoperfusion injury was identified in four patients (patients , , , and ). liver inflammation was not prominent, although some patients had mild periportal lymphocytic inflammation. weak sars-cov- immunohistochemical staining in the liver was indistinguishable from background. sam ples of the thyroid gland, pituitary gland, adrenal glands, and pancreas were largely unremarkable. post-mortem autolysis prevented thorough investigation of the gastro intestinal system and assessment by sars-cov- immunohistochemical staining. brain examination was done in five patients (patients , , , , and ). patient had acute pathology with scattered punctate subarachnoid haemorrhages and rare microhaemorrhage in the brainstem. neuropathological examinations were otherwise unremarkable. focal pulmonary micro thrombi were identified in five patients (patients , , , , ; figure ) . patient and patient also had microthrombi in the trachea, although in patient this was qualified by chronic tracheostomy. an organising thrombus was identified within a small renal vein in patient . microscopic involvement by grossly observed pulmonary emboli was seen in patient and patient . pathological findings for individual patients are provided in table . by electron microscopy, aggregates of uniform, round enveloped particles ranging in size from around nm to nm with peripheral spike-like projections consistent with the morphology described for sars-cov- were observed in the lung, trachea, kidney, and large intestine of patient and patient . , when confined to vesicles or in the extracellular space, structures with these characteristics were designated coronavirus-like particles. for simplicity, we refer to these coronavirus-like particles as viral particles. viral particles were present both inside tracheal epithelial cells and the extracellular space adjacent to the cell membrane or mixed with the luminal mucus ( figure a, b) . in the lung, extensive sloughing of pneumocytes into alveolar spaces was observed. some of these pneumocytes contained abundant autophagosomes, and occasionally viral particles were observed within the vesicles. viral particles were seen both in type and type pneumo cytes ( figure c, d) . aggregates of viral particles were found in enterocytes; however, the membranes of the surrounding vesicles were often disrupted and the tissue affected by post-mortem artefact (figure e, f). in the kidney, viral particles were observed in tubular epithelial cells, and more rarely in endothelial cells ( figure g, h) . some viral particles were associated with double membranes, and resembled double membrane vesicles (figure g). rare ill-defined round particles were also observed in podocytes. definitive viral particles were not observed in the other examined organs, including the heart, spleen, and liver. sars-cov- rna was detected in the lung, trachea, subcarinal lymph node, kidney, large intestine, and spleen of all three tested patients (patients , , and ) . viral rna was also detected in the liver, heart, and blood for patient and patient . in all three patients, the lungs and trachea had the lowest cycle threshold values. the spectrum of pathological findings in people who die from covid- is only beginning to emerge. [ ] [ ] [ ] [ ] we present a case series of autopsy findings in patients who died after sars-cov- infection. our results show the central role of lung damage and provide evidence that suggests extrapulmonary involvement of sars-cov- during severe infection. the major histopathological observation in our series of patients who died with covid- was diffuse alveolar damage-type lung injury in the acute or organising phases ( [ %] of patients). lung tissue from most decedents showed pulmonary oedema, prominent reactive type pneumocytes, intra-alveolar fibrin, and hyaline membranes. these findings are similar to those described during the - sars-cov outbreak and more recent covid- studies. , , [ ] [ ] [ ] in contrast to sars-cov, in which organising diffuse alveolar damage was predominantly observed in those with longer hospitalisations (> to days), we found evidence of organising diffuse alveolar damage in a patient with covid- who died days after symptom onset (patient ) and observed acute and organising diffuse alveolar damage in patients and , who died within a week of symptom onset. compared with the study by franks and colleagues, our patient cohort also had a shorter interval from symptom onset to death (median days vs · days). we hypothesise that there was a subclinical period during which lung injury was occurring in covid- patients with organising diffuse alveolar disease who died in the week after symptom onset. the hypothesis that lung injury might occur in covid- patients before symptom onset is supported by evidence of abnormal pulmonary ct findings in asymptomatic patients. these findings could have implications for screening patients at the time of admission to identify and aggressively manage those with pre-existing diffuse alveolar disease-type injury. additionally, we noted mostly focal sars-cov- immunohistochemical staining in the lungs of tested patients. in areas with less severe diffuse alveolar damage the virus was more readily visualised in pneumocytes. the absence of strong diffuse viral staining might indi cate that most cytotoxic damage caused by sars-cov- occurs early on in infection, with diffuse alveolar damage seen later as part of an exuberant host response. whether covid- patients are at increased risk for endothelial injury causing pulmonary microthrombi has become central to the discussion of patient management. this theory is based on the observed mismatch between lung compliance and oxygen saturation in ventilated patients. these results have led to the proposal that covid- acute respiratory distress syndrome (ards) might represent a novel type of lung injury. an early pathology report documenting three patients with covid- found active endotheliitis and endothelial cells containing coronavirus-like particles, supporting the claims of microvascular damage during infection. however, the observation of virally infected endothelial cells has been called into question. although our findings document coronavirus-like particles in the endothelial cells of the kidney, we did not observe endothelial cell infection in other organs surveyed by electron microscopy or immunohistochemistry. additionally, no histological evidence of endotheliitis was observed in our cohort. given the scarcity of these findings, we propose that the lung injury observed in our cohort presented the typical ards lung phenotype and not a novel type of injury. infection of endothelial cells by sars-cov- is hypothesised to cause dysregulation of the clotting system, which particularly affects small vessels and leads to pulmonary microthrombi and altered ventilatory patterns in intubated patients. , around a third of our cohort (five patients) had infrequent microthrombi. as no formal grading scale for the severity of microthrombi in tissue exists, we would classify the presence of microthrombi in our cohort as less than one per low power ( ×) field and within the scope of what is seen in other causes of diffuse alveolar damage. most microthrombi were seen when a full autopsy was done, and we suspect rare microthrombi might also be present in our cohort of limited autopsies. however, this factor is unlikely to have changed our overall findings or final cause of death. macroscopic pulmonary arterial thrombi were identified in two patients (patient and patient ). patient showed evidence of organisation with adherence to the pulmonary artery wall, indicating an event that probably predated covid- infection. an organising thrombus was also found in a small renal vein on microscopic evaluation of patient and was considered probably unrelated to covid infection. cardiac injury in covid- is common, although our results do not provide direct evidence of myocardial injury by sars-cov- . in a study that documented the clinical course of patients in the intensive care unit in kirkland, wa, % of patients had cardiomyopathy of unclear cause. previous post-mortem examinations have detected viral rna in cardiac tissue from a single patient, although histopathological evidence of myocarditis was not present. in our cohort, patients , , and had elevated troponin; however, only patient had histologically apparent lymphocytic myocarditis. myocardial tissue from this patient was positive for viral rna by pcr, but immunohistochemistry and electron microscopy results were negative. as the patient was also viraemic, the low rna level detected in the cardiac tissue might represent contamination by circulating virus rather than direct infection. patient also tested positive for influenza a, a known cause of viral myocarditis, before death. contamination by viral rna could also explain the results in the liver and spleen, where no definitive virus was identified by electron microscopy or immunohistochemistry. tubular epithelial cells, endothelial cells, and podocytes express ace , making kidneys a candidate target for sars-cov- infection. direct infection of kidney cells by the virus has been proposed as a mechanism for acute kidney injury observed during sars-cov- infection. two studies reported sars-cov- in tubular epithelial cells and podocytes. , these studies depended on ultrastructural findings, which carry a risk of confusion with cellular structures if used without immunolabelling. therefore, we labelled the observed structures as coronavirus-like particles, although positive pcr (three of three patients) and immunohistochemistry (two of four patients) data support our assertion that infection of renal cells occurs by covid- . multiple orthogonal approaches, including pcr, immunohistochemistry, and electron microscopy were used to help identify tissues that harboured viral particles. the pattern of virus distribution seen in our cohort raises questions about the mechanism of viral dissemination during severe infections, specifically whether sars-cov- can be transported by lymphocytes. current data support this hypothesis, with viral rna detected in blood samples and in vitro data showing pseudotyped sars-cov- capable of infecting lymphocytes. , in our study, two of three whole blood samples and three of three lymph nodes tested positive by pcr, in addition to sars-cov- immunohistochemistry highlighting lymphocytes in the tracheal submucosa of patient . if sars-cov- is capable of productive infection in lymphocytes, this could provide a mechanistic explanation for the poor survival and cytokine derangements of severe covid- cases with lymphopenia. the extent to which our findings of extrapulmonary involvement of sars-cov- are generalisable to nonsevere covid- infections is uncertain. as sars-cov- has been detected in urine and stool of non-severe covid- cases, there is evidence suggesting productive infection of non-pulmonary sites. , these questions raise concerns for susceptible groups, including those with chronic renal injury or inflammatory bowel disease. whether these patients are at increased risk for more serious complications during sars-cov- infection requires close monitoring. another patient group that requires close monitoring are those undergoing organ transplantation. although extrapulmonary infection might be less common in mild or subclinical disease, it is unclear whether active extrapulmonary infection can exist in a patient without concurrent respiratory infection. our study has several limitations. electron microscopy was limited to patients and , pcr to patients , , and , and immunohistochemical testing to patients , , , and . future studies will reveal whether the relatively consistent findings in this small number of patients are borne out in larger samples. as safety limitations in place during february and early march, , precluded complete autopsies of seven patients, our ability to detect rare findings was restricted for part of the cohort. in some situations, there was an extended postmortem interval before examination. as post-mortem autolysis reduces the sensitivity of electron microscopy and pcr, patient samples collected several days after death might generate false negative results. however, we could detect viral particles and viral rna from specimens up to h post mortem (patient ). extended postmortem intervals should not be an absolute criterion for exclusion in future studies. in conclusion, our findings show the central role of diffuse alveolar damage-type lung injury in patients with severe covid- . microthrombotic disease and endothelial injury were not as pronounced as reported in previous studies. we found broad tropism for sars-cov- with coronavirus-like particles identified in the pulmonary system, kidneys, and gastrointestinal tract. our results also raise the question as to whether sars-cov- can cause direct myocardial injury and whether direct infection of lymphocytes promotes viral dissemination and immune dysregulation. these findings provide histological context for clinical observations and help characterise the pathophysiology of sars-cov- , hopefully leading to novel treatment strategies. btb conceived and designed the study. hm, rj, and ic contributed to clinical data collection. hm, bn, and btb contributed to figure design. ny, tw, jml, dam, rj, ic, and btb did the post-mortem examinations. ny, tw, jml, dam, rj, ic, hm, hx, bn, gd, and btb contributed to histopathological evaluation of tissue. gd contributed to immunohistochemical studies. bn and btb contributed to electron microscopy images. btb and slf contributed to molecular testing. btb and hm wrote the manuscript. all authors contributed to data analysis, data interpretation, and editing the manuscript. we declare no competing interests. a novel coronavirus from patients with pneumonia in china genomic characterisation and epidemiology of novel coronavirus: implications for virus origins and receptor binding sars-cov- cell entry depends on ace and tmprss and is blocked by a clinically proven protease inhibitor characteristics of and important lessons from the coronavirus 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injury in covid- : emerging evidence of a distinct pathophysiology sars-cov- infects t lymphocytes through its spike protein-mediated membrane fusion longitudinal characteristics of lymphocyte responses and cytokine profiles in the peripheral blood of sars-cov- infected patients prolonged viral shedding in feces of pediatric patients with coronavirus disease we thank the technical staff of king county medical examiner's office (seattle, wa, usa) and snohomish medical examiner's office (everett, wa, usa) for their assistance with autopsy procedures. we thank jennifer swicord, gianni niolu, and the electron microscopy laboratory at the university of washington (seattle, wa, usa) for preparation of electron microscopy specimens. key: cord- -yf kt e authors: mejia, rojelio; hotez, peter; bottazzi, maria elena title: global covid- efforts as the platform to achieving the sustainable development goals date: - - journal: curr trop med rep doi: . /s - - -y sha: doc_id: cord_uid: yf kt e purpose of review: in this commentary, we summarize and put into perspective the recent information that highlights the associations between coronavirus disease and poverty. we also bring attention to another dimension that will most likely exacerbate the severity and long-term sequelae of covid- in impoverished populations, that is, the comorbidities and the presence of tropical infections. recent findings: during this first half of , the covid- pandemic has emerged as a poverty-related neglected disease on at least two fronts. first, is its significant impact in low-income neighborhoods in the usa, the epicenter of the pandemic. second, is its emergence in poor urban areas of south america, and now in asia and africa. in both fronts, the pandemic is contributing heavily towards the loss of public health gains that we managed to achieve globally during the last two decades. specifically, any advances made as part of the united nations millennium development goals (united nations, ) is eroding, and for the first time, the number of people entering extreme poverty is increasing. adding to this descent into poverty are new disruptions in ongoing disease control programs, routine vaccination strategies, and a reduction of capacity building efforts globally. therefore, and as highlighted by many others, we support the notion that a way forward to eliminate this coronavirus pandemic should include linking covid- control to other tropical or poverty-related diseases. summary: covid- is slowing or reversing global health and development gains. to be successful and achieve the global goals including the control of pandemics such as the one seen from the covid- , we must rely on strong leadership leading to impactful public policies and global collaborations, including global covid- vaccinations, and potentially linking them to programs for childhood and adult vaccinations and programs for malaria, tuberculosis, hiv/aids, and neglected tropical disease treatments. opportunities also include the creation of unique research opportunities and funding models and increase science engagement for international diplomacy. this can only be done with a better understanding of the relationships between coronavirus disease, poverty, and tropical diseases. the recent global pandemic caused by the novel severe acute respiratory syndrome coronavirus (sars-cov- ) and its associated coronavirus disease [ ] has affected almost every country in the world [ ] . the world health organization is closely tracking the rapid spread of covid- throughout the world, with daily situation reports [ ] . table shows a summary of the who's report of the total number of covid- cases and the total number of covid- deaths by region, taken from the last day of each month(march through july ). increasingly, there is a recognition that covid- is striking people living in poverty. this is happening on two fronts. beginning with new orleans in the spring of , covid- has caused significant devastation in low-income neighborhoods in us metro areas. african-american, latino, and native american populations living in these areas disproportionately account for the deaths and hospitalizations from covid- [ ] . in addition, now, covid- is affecting latin american nations, especially in poor urban areas-the favelas-of brazilian metro areas, and increasingly india and adjacent countries in south asia, and africa. covid- has spread into these regions and populations aided by weakened health systems with inadequate public health interventions leading to an increase in numbers of cases. we are already starting to see significant levels of mortality in brazilian cities, with the expectation that large metro areas and mega-cities in india and africa will follow. the initial entry of covid- into the southern hemisphere is happening in central and south america. overwhelmingly, brazil is the majorly affected country, especially in the favelas, the densely crowded slums of cities such as sao paulo, rio de janeiro, and salvador de bahia, but especially the northern tropical cities of belem, fortaleza, and manaus. urban areas of chile, peru, and ecuador are also affected, and covid- is expected to become one of the most widespread virus infections in latin america (table ) . we also see significant levels of illness in mesoamerica, referring to mexico and central america. currently, mexico has the highest case fatality rates in the americas [ ] . the caribbean is also beginning to see significant levels of infection with trinidad and tobago exceeded only by mexico for case fatality rates, according to the pan american health organization. for the past two decades, these countries within the americas have confronted many health challenges mixed together with other political and socioeconomic hurdles [ ] . beyond efforts to implement public health control, there are concerns that affected populations will experience long-term disabilities due to pulmonary scarring, cardiovascular complications, and neurologic deficits [ ] , as well as other clinical manifestations still being discovered [ ] . accordingly, these virus-related morbidities will add to the many already found in these populations already affected by non-communicable diseases functioning as major risk factors for covid- , as well as other infections, including the neglected tropical diseases (ntds). a recent article in the public library of sciences neglected tropical diseases journal provides a complete description and list of what are the ntds predominantly all found in these regions [ ] . now, covid- is accelerating in india and elsewhere in south asia. estimates indicate that india could eventually exceed the usa or brazil in terms of the total number of cases and deaths. there is an urgency to better understand the geographic distribution of india's covid- epidemic, especially in terms of specifically affected urban areas. covid- is also now affecting sub-saharan africa, beginning with south africa and egypt as the most affected countries. however, it is uncertain whether the relatively low number of cases across the region mostly reflects underreporting. for example, tanzania has made claims to be unaffected by covid- without external validation [ ] . the usa like many of the ntds, covid- has disproportionally targeted the most impoverished populations in high-income regions around the world [ ] . the concept of blue marble health refers to the "poorest of the rich" in nations such as the usa [ ] . a recent us study revealed that in the early stages of the pandemic, the populations living in the most underserved communities had higher rates of deaths due to covid- , compared with affluent locations [ ] . initial thoughts hypothesized that these findings could be primarily due to a lack of access to testing in these communities. but it also gave clarity that these underserved communities found themselves at a serious disadvantage since the start of the pandemic. social distancing is typically difficult in crowded households in low-income neighborhoods, where many do not have the opportunities to work from home. these poor populations generally have pre-existing health conditions due to inadequate access to high-quality healthcare. among them are high rates of obesity, diabetes, and cardiovascular disease. as is expected, of course, this also occurs in many areas around the world, especially in populations living in low-and middle-income countries (lmics). in brazil, for instance, a study evaluating the vulnerabilities and deprivations of their populations has identified several main factors contributing to this effect: hygiene, shelter, physical distance, and health recovery capacity [ ] . the basis by which poverty functions as a risk factor for covid- requires further investigation. sars-cov- uses a respiratory route of infection, associated with droplet contact and possibly airborne transmission, and therefore, population density is known to have large adverse effects [ ] . it is very common that those who fall within low socioeconomic strata and/or live below the poverty level [ ] generally have several generations living under a single household. therefore, living in close quarters with multiple inhabitants means direct contact with commonly used items; it can increase the risk of being exposed to the virus. in urban settings, additional factors that can lead to overcrowding may be the result of high rental costs, leading to families having to live in multi-family buildings and apartments [ ] . the economic downturn due to social distancing and decreased consumption of products and services has further disproportionally affected the poor. people living in poverty generally have less access to health communication on cable news networks or post-secondary education, working hourly positions that are more at the mercy of recessions. since they do not maintain a standard salary, many people in poverty need to continue working hourly jobs and place themselves at risk from public exposure with covid- . other factors can influence covid- morbidity and death, including age and non-communicable comorbidities such as diabetes, pulmonary, heart, or other organ-related diseases [ , ] . this, in turn, can feedback and influence the high levels of transmission of sars-cov- seen in these populations contributing to the observed epidemiology of covid- within these populations [ ] . individuals with low socioeconomics also have higher comorbidities that come from poor nutrition and the lack of access to primary medical services. health care is difficult to receive for people living below the poverty level. in both urban and rural settings, there is both a lack of accessible clinics for primary care and also a lack of insurance or the capacity to pay for preventive medical care. higher rates of smoking and alcohol consumption are also prevalent in both urban and rural people living in poverty. obesity, for instance, is associated with low-income populations, usually from consuming foods high in fats and carbohydrates that are still affordable. diabetes is also a significant problem that can be related to obesity. diabetes can severely impact the immune response of a person. all these health comorbidities can be the difference between asymptomatic covid- infection and intubated in the intensive care unit. there is increasing evidence that tropical diseases are also causative for propagating poverty in those infected [ ] . in the usa, an urban study in new york city reported a direct correlation with more toxocara parasite in city parks from poor neighborhoods compared with rich boroughs [ ] . another study in a clinic that serves low-income patients located in washington d.c. was found to have endemic levels of a potentially dangerous parasite called strongyloides stercoralis in this low-income population [ ] . a third study done in chicago found evidence of both toxocara and strongyloides in this urban population [ ] . major us cities had high levels of covid- infections and a higher prevalence in those living below the poverty line [ ] . for rural areas in the usa, there are studies in alabama that show hookworm (necator americanus), and strongyloides stercoralis is still present and transmitting in the population of poor rural alabama [ ] . in texas, a study detected . % of people infected with strongyloides stercoralis from a poor area outside of austin, tx, that has limited sanitation facilities [ ] . covid- has also been prevalent in the southern usa [ ] . given the dire public health crisis created by the emergence of covid- , it is anticipated that global resources will be committed to disease control and prevention. as for funds for the covid- vaccination scale, it will be interesting to look at opportunities to bundle these efforts for other tropical infections. for example, adult vaccinations for covid- could be linked to scale-up for other adult vaccines, including influenza. as new vaccines for schistosomiasis and chagas disease enter advanced development, there may be opportunities to link these as well [ ] . outside of vaccines, we will need to look at bundling covid- vaccines with mass treatments for malaria, tuberculosis, and hiv/aids, as well as largescale programs for neglected tropical diseases. until then, there are concerns that calls for social distancing might interrupt existing programs. there remains the possibility that we might see a resurgence in hiv/aids, tuberculosis, and malaria, and neglected tropical diseases. we have also seen how childhood vaccination programs for measles and other conditions were interrupted in the usa during the spring of [ ] . we may face similar interruptions in global child vaccination programs led by gavi, the vaccine alliance. the poor living in urban or rural areas of high-income countries and the most impoverished living in lmics have increased risk for both covid- and tropical diseases. these combinations disproportionally affect the working poor, both in health and economically, as to suffer long-term setbacks from missing work. the year is notable for the extraordinary global spread of covid- and the interruptions in key public health programs for both neglected diseases and vaccine-preventable diseases. we face the potential for a slowing, halting, or even reversal of all of the amazing gains since the launch of the millennium development goals. given the anticipated support for covid- vaccinations globally, it would seem wise to look at how some of the lapsed interventions could be resurrected and bundled. conflict of interest maria elena bottazzi and peter hotez are inventors of a low-cost covid- vaccine for global health which was recently licensed by baylor college of medicine to a commercial third party for scale up and production. human and animal rights and informed consent this article does not contain any studies with human or animal subjects performed by any of the authors. naming the coronavirus disease (covid- ) and the virus that causes it the impact of covid- and strategies for mitigation and suppression in low-and middle-income countries world health organization. coronavirus disease (covid- ) situation reports coronavirus disease (covid- ) situation report- coronavirus disease (covid- ) situation report- world health organization. coronavirus disease (covid- ) situation report- world health organization. coronavirus disease (covid- ) situation report- world health organization. coronavirus disease (covid- ) situation report- covid- in racial and ethnic minority groups americas region covid- dashboard central latin america: two decades of challenges in neglected tropical disease control will covid- become the next neglected tropical disease? tracing new clinical manifestations in patients with covid- in chile and its potential relationship with the sars-cov- divergence what constitutes a neglected tropical disease? tanzania' free of coronavirus'-president poverty and the impact of covid- : the blue-marble health approach poverty and covid- : rates of incidence and deaths in the united states during the first weeks of the pandemic vulnerability, poverty and covid- : risk factors and deprivations in brazil. researchgate racial, economic and health inequality and covid- infection in the united states office of the assistant secretary for planning and evaluation. poverty guidelines spatial disparities in coronavirus incidence and mortality in the united states: an ecological analysis as of comorbidities and multi-organ injuries in the treatment of covid- covid- )-united states human intestinal parasite burden and poor sanitation in rural alabama toxocara species environmental contamination of public spaces strongyloidiasis in latin american immigrants: a pilot study parasitic infections represent a significant health threat among recent immigrants in chicago clinical characteristics of covid- in new york city prevalence of intestinal parasites in a low-income texas community running the gauntlet": formidable challenges in advancing neglected tropical diseases vaccines from development through licensure, and a "call to action effects of the covid- pandemic on routine pediatric vaccine ordering and administration-united states about the sustainable development goals publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord- -cynpob b authors: godakova, svetlana a.; noskov, anatoly n.; vinogradova, irina d.; ugriumova, galina a.; solovyev, andrey i.; esmagambetov, ilias b.; tukhvatulin, amir i.; logunov, denis y.; naroditsky, boris s.; shcheblyakov, dmitry v.; gintsburg, aleksandr l. title: camelid vhhs fused to human fc fragments provide long term protection against botulinum neurotoxin a in mice date: - - journal: toxins (basel) doi: . /toxins sha: doc_id: cord_uid: cynpob b the bacterium clostridium botulinum is the causative agent of botulism—a severe intoxication caused by botulinum neurotoxin (bont) and characterized by damage to the nervous system. in an effort to develop novel c. botulinum immunotherapeutics, camelid single-domain antibodies (sdabs, vhhs, or nanobodies) could be used due to their unique structure and characteristics. in this study, vhhs were produced using phage display technology. a total of different monoclonal vhhs were selected based on their comlementarity-determining region (cdr ) sequences. different toxin lethal dose (ld( )) challenges with each selected phage clone were conducted in vivo to check their neutralizing potency. we demonstrated that modification of neutralizing vhhs with a human immunoglobulin g (igg) fc (fragment crystallizable) fragment (fusionbody, vhh-fc) significantly increased the circulation time in the blood (up to days). at the same time, vhh-fc showed the protective activity times higher than monomeric form when challenged with ld( ). moreover, vhh-fcs remained protective even days after antibody administration. these results indicate that this vhh-fc could be used as an effective long term antitoxin protection against botulinum type a. botulinum neurotoxin (bont) is the strongest organic poison for humans and animals. it is produced by the anaerobic, gram-positive, spore-forming, rod-shaped bacterium clostridium botulinum [ , ] . the estimated human lethal dose is about nanograms per kilogram of bodyweight if the toxin is inhaled and one microgram if it is taken orally [ , ] . the most common forms of natural botulism are food-borne, wound, and infant [ ] . food-borne botulism occurs through contaminated food ingestion, and the case fatality rate is now about - % in developed countries (as opposed to - % before ). wound botulism occurs when an open wound is exposed to c. botulinum spores, and the case fatality is approximately - % of patients even with aggressive treatment. infant intestinal botulism occurs with spores ingested and spread over the digestive tract, as infants lack the protective flora of adults, with case fatality rate estimated to be le,ss than - %. inhalation botulism does not occur naturally and may occur in the context of a bioterrorist attack [ , ] . among the four major human pathogenic bonts (bont/a, b, e, and f), bont/a poses the most serious challenge for medical treatment due to its extremely high potency and extraordinary persistence in human patients [ ] . according to the world health organization [ ] , an antitoxin, usually based on equine antitoxin, should be administered as soon as the diagnosis is made. however, side effects, such as allergic reactions, fever serum sickness, and anaphylactic shock, often occur. in the past, at-risk persons and populations have been vaccinated with a chemically inactivated penta-serotype bont/a-e toxoid [ ] ; however, its use has been discontinued due to declining potency [ ] . in other affected individuals, botulism treatment is mainly supportive, with mechanical ventilation being the only effective life-saving treatment [ , ] . a good alternative to antitoxin serum with minimal or no side effects is treatment with monoclonal antibodies (mabs) to bont, which can be prduced in vitro. along with conventional antibodies, single-domain antibodies (sdab), also referred to as vhhs (variable domains of heavy-chain only stibodies) or nanobodies, have been widely used since their discovery in camelids along with classical immunoglobulins (igg) [ ] [ ] [ ] . the absence of light chains in igg and a lack of constant domain (ch ) in the heavy chain are the key characteristics of heavy-chain antibodies (hc-abs). therefore, an antigen-binding site of hc-abs is formed only by a single domain, which is linked directly via a hinge region to the fc (fragment crystallizable) domain. hc-abs recognize the antigen with only one special variable domain, referred to as vhh. the structure of the vhh domain resembles the vh igg domain [ ] . the complementarity-determining region (cdr ) of these hc-abs possesses the extraordinary capacity to form long finger-like extensions, which can extend into cavities on antigens, as the cdr is often much longer than that of conventional vh domains [ , ] . despite their small size (~ kda), sdabs maintain affinities and antigen-binding specificities comparable to those of full-size mabs [ ] . clear advantages include the ability to recognize hidden antigenic sites that are inaccessible for conventional antibodies due to their structure; stability over a wide range of temperatures and ph; high solubility, as well as economical and facile expression and production in large quantities in microorganisms [ , ] . several studies were conducted to test the potency of vhhs as inhibitors of viral infections [ ] and different toxins, produced by such plants and microorganisms as ricinus communis [ ] , mycoplasma hominis [ ] , clostridium tetani [ ] , clostridium difficile [ ] , crotalus durissus terrificus [ ] , and shiga toxigenic escherichia coli (stec) [ ] . it has been demonstrated that the antigen-binding region of vhhs produced by camelids showed strong anti-bont activities in animal models [ , ] . due to their unique features and high efficacy, sdabs are currently in clinical phase trials for the treatment of a wide range of diseases, including cancer, inflammation, hematology, and respiratory diseases [ ] . for instance, cablivi is a nanobody-based medicine, which has recently been approved by the food and drug administration (fda) for acquired thrombotic thrombocytopenic purpura (attp) treatment [ ] . however, the advantages of vhhs may also become their impediments. their small size enables good penetration into tissue and rapid distribution, but their short half-life in the blood circulation limits the time for interaction with hard-to-reach epitopes and for crossing endothelial barriers in sufficient amounts [ , , ] . nevertheless, due to their relatively simple structure, vhhs can be optimized by genetic engineering to obtain desired properties, including extended half-lives [ ] . one approach was to add an albumin-binding peptide to the c-terminus of vhh anti-botulinum, which increases the serum half-life of the vhh to - days [ ] . another approach was to generate virus vectors encoding chimeric proteins with one or more vhhs fused in frame to a cdna encoding the red blood cell membrane proteins glycophorin a or kell. in vitro studies and stem cell transplantation research has demonstrated that the half-life of these vhhs could be extended to several weeks with retained functionality [ ] . the vhh could also be fused to the fragment crystallizable (fc) region of an igg. such modifications, termed fusionbodies, increase the total protein complex size, as well as vhh-fc interaction with neonatal fc-receptor (fcrn), making it impossible to clear by the renal filtration system [ ] . furthermore, the attached fc fragment can promote effector functions, such as phagocytosis and cytotoxicity, thereby enhancing the neutralization of pathogen entry and replication [ ] . in this study, we focused on screening specific bont/a neutralizing alpaca vhhs from a vhh antibody immune library by phage display using bont/a and bont/a treated with dithiothreitol (bont/a-dtt) as antigens. two clones (b and g ) with high neutralizing potency at lethal dose ( ld ) were obtained in phage forms. these clones were produced as vhhs, which were then modified as dimers or fused with human igg fc-fragments (vhh-fc, fusionbody). the vhh-fc modification greatly increased their neutralizing potency and serum half-life. alpaca immunization was performed using five sequential injections with an interval of days between the first and second immunizations and days between all subsequent immunizations to generate an immune library of single-domain antibodies (figure a ). after and days post-immunization, blood was collected, and the bont/a specific antibodies titer was measured by enzyme-linked immunosorbent assay (elisa). the immune serum showed a clear response to bont/a (figure b ). before immunization, all sera showed the background levels of antibody titers. the final titer of toxin-specific antibodies in the alpaca serum was more than / , as the result of the five-fold immunization scheme. to generate a panel of single-domain antibodies to bont/a, we constructed an alpaca immune vhh library for display on the bacteriophage surface by cloning the nucleotide sequences coding for vhh repertoires of the immunized alpaca into an expression phagemid vector. after the transformation of recombinant phagemids into competent e. coli cells, a library, with a size of × phagemids, was obtained. phage display and two rounds of selection were performed for acquiring phage libraries. purified bont/a was used as an antigen for the first round. two independent second rounds were performed separately, with bont/a and bont/a-dtt used as antigens. dtt reduction was used for fragmenting toxins into two constituent parts-the heavy chain (hc) and light chain (lc) [ ] -for more uniform absorption of chains on the immunoplate's surface and to evenly distribute the epitopes on both chains. the polyclonal phage library titer for all rounds was approximately cfu (colony-forming unit)/ml (figure a) . after two rounds of selection, the specificity of each library was determined by elisa ( figure b ). to test the neutralizing potency and specificity of the polyclonal phage libraries obtained in the second round of selection, an in vivo toxin neutralization assay was performed ( figure c ). balb/c female mice were divided into groups and received one intraperitoneal injection with ld or ld bont/a after h incubation with phage libraries. previously obtained phages specific to tetanus neurotoxin (tent) were used for controls as non-specific phages. all mice in the positive control group survived, while all mice in the negative control group and the tent group died. challenging two experimental groups with ld and ld provided full and partial protection, thus allowing further selection of monoclones. a total of clones showing elisa readouts higher than . ( figure a ) and negligible reactivity with bovine serum albumin (bsa) were selected for sequencing. a total of clones with different cdr amino acid sequences were selected for further research. to test the neutralizing potency of the selected clones, an in vivo monoclonal phage neutralization assay was performed in which mice were administered the corresponding phage clone (figure b ). each group received one intraperitoneal injection with ld bont/a previously mixed with the corresponding phage clone at cfu. only the four clones (b , c , b , g ) that fully protected the mice against a ld challenge were chosen to test their protectiveness with a ld challenge. mouse groups, which received clones b and c premixed with bont/a, were partially protected, while two other groups, which received clones b and g premixed with bont/a, showed % protection. thus, clones b and g were chosen for further research as the most protective. it should be noted that the clones obtained by selection on bont/a-dtt showed a greater diversity in the cdr sequence and demonstrated neutralizing activity, unlike the clones, which were selected on bont/a. the most protective clones, b and g , were titrated in series down to cfu and introduced intraperitoneally into mice simultaneously with ld of the toxin. the neutralizing potency of both clones was comparable, with the lower threshold being cfu (figure c ). finally, both clones were tested for cross-reactivity protectiveness with another toxin serotype, bont/b. each group of mice was challenged with ld of the toxin and administered cfu/ml phages. both clones failed to protect the mice, demonstrating that the two protective clones are specific to bont/a. to increase the protectiveness of the selected clones by increasing the antibody circulation time in the blood and/or their additional functionality, two modified constructions were synthesized. one construction was a dimer form of each clone (b -dimer and g -dimer) held by a glycine-serine linker (gly ser) and expressed in the pet plasmid. the second construction was a monomer of each clone linked to a human igg fc fragment (b -fc and g -fc) (figure a ). fc-modifications have been used to increase the antibody circulation time in blood. human fc-fragments cross-react with murine fc-receptors and are capable of binding mouse fcrn [ , ] . human fc was chosen for these constructions to be used in further research and clinics. the dimers were produced and purified from the bacterial periplasmic fraction and verified by sds-page to be~ kda. vhhs fused with fc fragments were produced in chinese hamster ovary (cho-s) cells and verified by sds-page to be~ kda under reducing conditions (figure b ). to determine the polypeptide chain of the toxin that bound specific antibodies, it was denatured by dtt into its hc and lc. sds-page was performed in denaturing conditions followed by western blot. b -fc and g -fc clones were tested, and anti-human igg (fc-specific)-peroxidase antibodies ( : ) were used as detection reagents ( figure c ). these antibodies were associated with the hc ( kda) of the toxin, which corresponds to the receptor domain. an immunoblot with bont/a and bont/a-dtt (under reducing conditions) were performed as control (figure d ). we investigated the affinity of clones b and g via surface plasmon resonance (spr). amine coupling was used to immobilize bont/a and bont/a-dtt on the sensor chip. the kinetic binding on-and off-rates between the antibodies and the toxin were determined from sensorgram analysis and used to calculate the equilibrium dissociation constant (kd) ( table ). table . kinetic parameters of antibody interactions with the toxin obtained by spr (surface plasmon resonance). association (on-rate, k a ), dissociation (off-rate, k d ), maximum analyte binding capacity (r max ), equilibrium association constants (k a ), equilibrium dissociation constants (k d ), and chi for the chosen vhhs (variable domains of heavy-chain only antibodies) binding to botulinum neurotoxin (bont)/a or bont/a-dtt (dithiothreitol). bont/a-dtt k a ( /ms) for the in vivo protection analysis, modified forms of vhhs, different amounts of monomers, dimers, and vhhs fused with fc fragments were premixed with ld bont/a and injected intraperitoneally into mice ( figure a ). monomers had only partial protectiveness even at the highest amount, µg, with protectiveness gradually decreasing and failing at µg. the dimers showed a better result on average, with full protectiveness of the b -dimer at µg and partial protectiveness at lower amounts, failing at µg, as well as partial protectiveness of the g -dimer at - µg amounts, failing at µg. the best results were demonstrated by vhhs fused with fc fragments, with g -fc failing to fully protect the mice at . µg and b -fc protecting the mice at amounts as low as . µg. all forms of vhhs partially protected the mice at high amounts, whereas vhhs fused with fc fragments could protect the mice even at the lowest amounts tested ( . - . µg). to assess the circulation time of various antibody modifications in the blood after a single injection, elisa was used to measure the concentrations of g , b , g -dimer, b -dimer, g -fc, or b -fc in mouse blood taken at different time points after injection. the concentrations of g , b , as well as b -dimer and g -dimer, detected at h post-injection were only about % of the initial level observed at h. however, b -fc and g -fc modification constructs had a relatively long serum half-life. the concentrations of b -fc and g -fc h post-injection were approximately % of the initial level. moreover, two weeks after injection, the concentrations of b -fc and g -fc antibodies were approximately % of the initial level. these data are in agreement with reports on other vhhs with monomeric and fusionbody formats [ ] (figure b ). vhhs fused with fc fragments demonstrated the best protection, and the mice treated with these preparations were alive two weeks after the end of the experiment. based on the analysis of b -fc and g -fc clones' circulation time in the serum (presence of antibodies days after injection), we decided to conduct an experiment on the survival of these mice, which previously received a single injection of the vhhs with the fc fragment, with a repeated administration of only the lethal toxin dose days after the original administration. these mice were challenged with bont/a ld to examine their protection over time (figure c ). all mice that previously received - µg of vhh-fc were still fully protected. the mice which previously received - µg of the vhh-fc were partially protected ( - %) (clone b -fc) or not (clone g -fc). after two weeks, µg of the preparation had no protectiveness. we also tested the prophylactic efficacy and possible treatment of the toxin by administering the two selected fc-fused clones one hour and three hours before and after toxin challenge with ld (figure d ). both clones fully protected the mice before the toxin challenge and one hour after. three hours after toxin administration, the clones lacked protectiveness. overall, we obtained numerous clones after two rounds of biopanning; we selected clones for initial analysis based on their cdr s, chose two clones (b and g ) with the best pre-mixed results in phage form in vivo, produced them in protein form, and modified their structure and characteristics by dimerization via a (gly ser) linker and fusion to a human igg fc fragment to enhance their protective activity. we demonstrated that modification of neutralizing vhhs with an fc fragment (fusionbody) significantly increased the circulation time of antibodies in the blood. at the same time, fc fusion significantly increased the protective activity of vhh clones to more than times compared to monomeric forms. moreover, clone b -fc increased the protective activity at least , times compared to the monomeric form, with % protectiveness even at . µg. the estimated molecular ratio of the ld to . µg pre-mix was : . bont is the most potent and lethal known toxin. therefore, the development of new therapeutic agents for exposure prevention and treatment is essential. therapeutic approaches have been summarized in previous publications [ ] . along with serum therapy, specific antibodies are a promising tool for neutralizing bonts. approaches for the generation, selection, combination, and modification of mabs against bont/a have been described and tested [ ] [ ] [ ] [ ] [ ] . camelid vhhs are a popular tool for constructing recombinant antibodies to detect and neutralize a range of targets [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in particular, it has been shown that hc-only antibodies or vhhs derived from hc-only antibodies, produced by camelids demonstrate strong anti-bont activities in animal models [ , , ] . the main differences of vhhs from conventional antibodies include specific conserved amino acid substitutions in framework region (fr ) that make contact with the lc of classical antibodies, as well as a longer length, more variable amino acid composition in the cdr s, and a folded back loop [ , , ] . specific vhhs can be isolated from vhh libraries. one type of such libraries-immune libraries-can be produced based on peripheral blood lymphocytes isolated from camelids that have been immunized with an antigen of interest in a prime-boost strategy [ ] . in our study, we used a toxoid consisting of bont/a toxin and small amounts of hemagglutinin (ha) as an antigen and screened for vhhs against bont/a and bont/a-dtt using phage display technology. the neutralizing clones in our work were selected on bont/a-dtt. it should be noted that the neutralizing clones were later found in the bont/a library as well. however, there were fewer of them, so a greater number of clones had to be analyzed. overall, approximately clones from bont/a-dtt and more than clones from bont/a libraries were examined. the approach of splitting the toxin into its hc and lc enriches the neutralizing clones in the library. based on their neutralizing activity when pre-mixed with different toxin ld doses, two clones were selected. interestingly, all clones with neutralizing potency did not have the highest elisa signals. therefore, the od (optical density) of the elisa signal does not always mean high neutralizing activity and in vivo protective activity of these vhhs. the next step was to produce and purify vhhs in protein form to test their neutralizing potency in vivo. however, their protection was weak. even when pre-mixing a µg dose of these vhhs with the toxin, they had a - % protection efficiency. administration of lower doses of the preparation was non-protective, with µg lacking effectiveness. therefore, two clones-b and g -were modified to increase their half-life and protection efficacy. increasing the vhh size by oligomerization-the coupling of two or more vhhs via a specific linker-increased their protectiveness; however, even bivalent constructs were rapidly cleared [ ] . another approach was vhh fusion to an fc region of the igg molecule. constructs with fused fc fragments were previously made to extend the antibody half-life to neutralize the middle east respiratory syndrome coronavirus (mers-cov) [ , ] , target the c-x-c chemokine receptor type (cxcr ) to prevent human immunodeficiency virus (hiv- ) strain entry and replication in vitro [ ] , and increase the fc effector functions to neutralize rotavirus [ ] as well as the influenza virus ha [ ] . a recent study of modified vhhs dimers and vhhs fused to fcs targeting unique epitopes on the immunogen, composed of a portion of the central delivery domain and the entire combined repetitive oligopeptides (crops) domain of clostridium difficile type b, showed modest and much greater toxin inhibitions, respectively [ ] . it should be noted that a combined approach for efficient serum clearance of bont/a has been developed before. sepulveda et al. [ ] used single-chain variable fragments (scfvs) as protein binding agents pre-complexed with one or two single anti-tag mabs with an fc domain and tested this construction in vivo for protection and pharmacokinetics. when three or four types of such constructions were given simultaneously, mice were protected at high ld s, and bont/a was rapidly cleared from the sera. protection against bont/a light chain was observed when scfvs fused with fc fragments (scfv-fc) obtained from a macaque immune library were tested ex vivo [ ] . furthermore, the substantial contribution of the homologous fc fragment to the potency of three individual anti-botulinum mabs in antibody preparations has been demonstrated [ ] . in our work, two chosen clones were dimerized via a (gly ser) linker or fusion to human fc fragments to stabilize the molecules and slow down their clearance. these modifications led to improved protectiveness of the preparations. the dimers demonstrated protectiveness at lower doses compared to vhhs, with - % protection efficiency at and µg. clone g -dimer failed to protect the mice at µg, like the vhhs, while clone b -dimer showed partial protectiveness and failed at µg. therefore, the effective dose (ed ) for clone b -dimer was µg, and for the clone g -dimer, was µg. the best results were obtained after the fusion of the vhhs to fc fragments, which is consistent with previous research. both clones fully protected the mice at doses as low as . µg. the protection provided by g -fc was half at . µg, and the clone failed to protect the mice at . µg. b -fc demonstrated full protection down to the lowest tested dose of . µg. the dose at which its protectiveness begins to decrease was not reached. therefore, both clones with fc fragments showed at least a -fold improvement in protectiveness compared to conventional vhhs and dimers. the ed dose for clone b -fc could not be determined, while it was . µg for clone g -fc. besides, the pharmacokinetics analysis of various antibody forms showed that clones b and g containing the igg fc fragment were detected in the mouse serum days after a single injection, which is confirmed by full or partial animal protection from the challenge with the toxin days after administration of various b -fc and g -fc doses. testing the prophylaxis and possible treatment of the toxin by administering b -fc and g -fc one hour and three hours before and after toxin challenge with ld showed that both clones fully protected the mice before the toxin challenge as well as one hour after, but failed to protect the mice three hours after toxin administration, suggesting that these clones could be used for prophylaxis and emergency therapy immediately after toxin administration. this corresponds with the results obtained previously by sepulveda et al. [ ] showing that mice receiving scfvs pre-complexed with anti-tagged mabs with fc fragments were protected only two hours after intoxication, while four hours after, toxin administration lethality was only delayed. the acquisition of antibodies against bont/a and confirmation of their neutralizing potency in vivo in phage forms when pre-mixed with the toxin has provided a new method to screen for neutralizing vhhs before obtaining them in protein form, which can efficiently reduce time and material costs for their production and testing. vhhs, along with their modifications as oligomers and fusions to fc fragments of the igg, increase the range of options for bont/a targeting and neutralization by improving the blood circulation time and therapeutic potential. alpaca immunization and blood collection were performed on one clinically healthy -year-old male alpaca (vicugna pacos) on the "russian alpacas" farm, private land located in pokhodkino, moscow region, russia. this sample collection did not involve endangered or protected species, and no specific permissions were required for these locations/activities. six-week-old female balb/c mice (weighing - g) were purchased from "pushchino breeding facility" (pushchino, moscow region, russia) accredited by association for assessment and accreditation of laboratory animal care (aaalac international) and maintained at the central animal facility at the gamaleya research center of epidemiology and microbiology. mice were kept at a constant temperature ( ± • c) and relative humidity ( %) with h of artificial light per day. they were housed in individual cages ( per cage). mice were fed with dried food and water ad libitum. mice were observed every two hours post-injection except during the night for one week. the animals with characteristic symptoms of botulism, including muscle paralysis and respiratory difficulty, were euthanized by cervical dislocation. bont/a from the c. botulinum strain a was obtained from the gamaleya research center, moscow, russia collection. bont/a is very toxic; therefore, appropriate safety precautions were taken during these experiments. the neurotoxin was handled at a class biosafety cabinet. the antigen was a toxoid consisting of bont/a toxin and small amounts of ha, and . % formaldehyde was used for toxoid preparation. the treatment was carried out for seven days at • c [ ] . mice received intraperitoneal injections to determine residual toxicity, which showed its absence. before use in alpacas, the antigen was purified through a . µm filter. the final concentration was µg/ml. before immunization, the neurotoxin was inactivated by . % formaldehyde at ph = during - days. bont/a preparations were used with freund's adjuvant without pre-adsorption. alpaca immunization was performed using five sequential injections with an interval of days between the first and second immunizations and days between all subsequent immunizations. for the first injection time, µg ( ml) of the antigen and complete freund's adjuvant (sigma, st. louis, mo, usa) at the v/v ratio of : were administered. the four subsequent immunizations were performed with µg ( . ml) of the antigen and incomplete freund's adjuvant (sigma, st. louis, mo, usa) at the v/v ratio of : . small blood samples ( - ml) were collected before immunization, as well as after the third and fifth immunizations as a control. five days after the final injection, ml blood sample was collected and placed into a sterile vacuum collection tube with lithium heparin to prevent blood clotting. mrna isolation, pcr amplification, and library construction were performed, as described elsewhere [ , ] . briefly, camelid vhhs from peripheral blood b-lymphocytes (about cells/ml) of the immunized alpaca were cloned into a phen expression phagemid vector [ ] . the primer set used for pcr amplification of antibody genes appended the sfii (neb) restriction site at the -end and the noti (neb) site at its -end (table ) . recombinant phagemids were introduced into freshly prepared competent suppressor tg e. coli cells (lucigen, middleton, wi, usa). using this method, a library of × individual clones for biopanning and isolation was obtained. the bacteria from the vhh library were added to × yt medium (sigma, st. louis, mo, usa) (with µg/ml ampicillin and % glucose) and incubated at • c in a culture shaker at rpm to an od = . . km helper phages (patrick chames "antibody therapeutics and immunotargeting team" of the cancer research center of marseille) were added to the bacteria (multiplicity of infection, moi = ) and left without shaking at • c for min. the culture was centrifuged at rpm for min at • c, and the cell pellets were resuspended in × yt medium (with µg/ml ampicillin and µg/ml kanamycin) and cultured overnight at • c, in a culture shaker at rpm. the next day, the culture was centrifuged, the supernatant was purified and concentrated by % polyethylene glycol (peg) , . m nacl precipitation, and the pellet was resuspended in phosphate-buffered saline (pbs) with % glycerol. bont/a-dtt was treated with mm dtt for at least min at • c. microtiter plate wells were coated with µg of bont/a for the first round and with bont/a and bont/a-dtt for the second round in . m nahco buffer (ph = . ) at • c overnight. after rinsing three times with pbs with . % tween (tpbs), the plate was blocked with blocking buffer (tpbs with % non-fat dried milk) at • c for h. a total of~ phages were added to each well and incubated at • c for h. unbound phages were removed by washing times with tpbs. the bound phages were eluted by trypsin with a final concentration of mg/ml. tg e. coli cells at od = . were infected with the eluted phages and incubated without shaking at • c for min. after culturing the mixture in × yt agar plates (with µg/ml ampicillin and % glucose) at • c overnight, the cells were scraped. recombinant phages were obtained by packaging with km helper phages, and their titers were determined. a total of two panning rounds were performed. for a polyclonal elisa, an immunoplate (maxisorp, nunc) was coated with ng of bont/a and bont/a-dtt with . m nahco buffer (ph = . ) at • c overnight. the plate was rinsed three times with tpbs, blocked with blocking buffer at • c for h, and a total of~ phages from the starting library or the first or second rounds were added to each well and incubated at • c for h. the unbound phages were removed by washing times with tpbs. the plate wells were detected by horseradish peroxidase (hrp)-conjugated anti-m antibodies ( : ) (abcam, cambridge, uk), followed by the addition of peroxidase substrate , , , -tetramethylbenzidine (tmb) (bio-rad, hercules, ca, usa). the reaction was stopped by m h so , and the absorbance at nm was read with an iems reader mf (thermo labsystems, waltham, ma, usa). a monoclonal elisa was performed following the same protocol, with particular clones bound to bont/a and bont/a-dtt being selected, and the phage plasmids were isolated for sequencing. antibody genes in phagemids were sequenced with a primer set used for pcr according to the protocol of big dye terminator . cycle sequencing kit for the genetic analyzer applied biosystems (waltham, ma, usa). the electrophoretic dna separation was performed in cm capillaries with pop polymer. the selected vhhs expressed in phen plasmid were transformed into e. coli bl cells (neb, ipswich, ma, usa) for expression and purification. an overnight culture was obtained at • c, in a culture shaker at rpm. the next day, the cells were harvested by centrifugation, lysed by bugbuster protein extraction reagent (novagen, madison, wi, usa), and vhhs were purified from the lysate by talon superflow containing co + agarose (ge healthcare bio-sciences ab, uppsala, sweden). the eluted fraction was subjected to dialysis with visking dialysis tubing (mwco - ) (serva, heidelberg, germany). vhhs were separated by sds-page (bio-rad, hercules, ca, usa) under denaturing conditions and had an expected molecular weight of~ kda. antibody affinity was determined by surface plasmon resonance (spr) using a biacore instrument (ge healthcare bio-sciences ab, uppsala, sweden). bont/a and bont/a-dtt were immobilized on the surface of cm sensor chips in the amount of µg each in mm sodium acetate buffer ph = . using the amine coupling kit recommended by the manufacturer (ge healthcare bio-sciences ab, uppsala, sweden). vhhs ( -fold dilutions from µg down to µg) were captured on the sensor chips and submitted at a constant flow rate of µl/min with hbs-ep ( . m hepes ph . , . m nacl, mm edta, . % v/v surfactant p ) as a running buffer at • c with an injection time of min and dissociation time of min. after each injection, the chip surface was regenerated with mm tris-hcl, ph = . for s at a flow rate of µl/min. calculations were performed using biaevaluation software (ge healthcare bio-sciences ab, uppsala, sweden) with reference-subtracted fitting. the selected vhhs were held by a glycine-serine linker (gly ser) and expressed in the pet plasmid. protein expression and purification were performed as for vhhs. nucleotide sequences of vhh genes fused to the human igg fc-fragment were synthesized and cloned into the plasmid pshuttle-cmvfuse (stratagene, la jolla, ca, usa). the cho-s cell culture (thermofisher, waltham, ma, usa, r ) was transiently transfected with pfuse plasmid using the cho gro system (mirus bio, madison, wi, usa), according to the manufacturer's protocol. a plasmid carrying the green fluorescent protein gene in a % amount was used as control. the efficiency of transfection was assessed using axio imager z (carl zeiss, oberkochen, germany) and determined by the number of fluorescence cells compared to their overall amount, which was %. cells were cultured in shake flasks at rpm, % co , % humidity, at • c during transfection and • c h after the transfection for days. starting from day three, cellboosts a ( %), b ( . %) (hyclone, san angeo, tx, usa), and % sigma bioreactor feed (sigma, st. louis, mo, usa) were added each day. after days of cultivation, the culture was clarified by centrifugation at × g and cleared through a . µm filter. the antibodies were purified using protein a affinity chromatography on an akta start chromatography system (ge healthcare bio-sciences ab, uppsala, sweden), with a mabselect sure ml column (ge healthcare bio-sciences ab, uppsala, sweden), according to the manufacturer's protocol. to determine which bont/a polypeptide chain binds vhhs, the toxin was denatured by dtt into its hc and lc. sds-page was performed using a mini-protean tgx stain-free precast gel (bio-rad, hercules, ca, usa) in denaturing conditions. the separated bands were transferred onto a nitrocellulose membrane using a trans-blot turbo system (bio-rad, hercules, ca, usa). the membrane was blocked with a blocking buffer at • c for h. vhhs were diluted in the blocking buffer, added to the membrane, and incubated at • c for h. after rinsing the membrane three times with tpbs, anti-his-tag-hrp-mouse antibodies (genscript, piscataway, nj, usa) diluted : in blocking buffer were added, and the membrane was then incubated at • c for h. the membrane was rinsed three times with tpbs, followed by the addition of clarity western ecl blotting substrates (bio-rad, hercules, ca, usa) for detection. the membrane was visualized on an amersham imager (ge healthcare, buckinghamshire, uk). the neurotoxin as a multi-oligomeric complex toxin with has and ntnha (non-toxic non-hemagglutinin) ( kda) was used for immunization. toxins were prepared and purified as follows. the strain was cultivated under anaerobic conditions for five days. bacterial cells were separated by centrifugation at × g for min at • c. the proteins from the culture filtrate were concentrated by acid precipitation at ph = . for min. the precipitate was separated by centrifugation at , × g for min at • c and dissolved in mm citrate-phosphate buffer with ph = . . gel filtration s and ion-exchange chromatography on akta start chromatography system (ge healthcare bio-sciences ab, uppsala, sweden) on de cellulose (pharmacia, uppsala, sweden) were then carried out. the toxin ( - %, kda) was purified by additional de cellulose chromatography (pharmacia, uppsala, sweden) in borate buffer ph = and eluted by mm nacl. non-sorbed material containing specific antigenic and biological activity was used as bont complex with ha. the specific antigenic activity was a positive reaction with monospecific antibodies to bont/a, ha, and ntnha (gamaleya laboratory of clostridiosis and commercial preparation of scientific centre for expert evaluation of medicinal products russian federation). phages with titers - cfu/ml were prepared, as described above, and mixed with standard saline solution. vhh proteins at different amounts ranging from µg to . µg were prepared, as described above, and mixed with standard saline solution. balb/c - g female mice were divided into groups of four, including the positive and negative control groups. phages or vhh proteins were premixed with the appropriate toxin ld ( ld~ pg bont/a) and incubated for h at • c. all mice received one intraperitoneal µl injection of phages or proteins premixed with various ld s of the toxin and were observed once a day for one week. a specific pathological pattern was observed in sick mice, which is the pathological pattern with dystonia's abdominal muscles (waistline increasing) and death at - pg that is neutralized by monospecific antibodies to bont/a. the positive control group received the rabbit antitoxin igg (gamaleya laboratory of clostridiosis), and the negative control group received a standard saline solution. a group of five six-week-old female balb/c mice was intravenously (i.v.) injected with µg g , b , g -dimer, b -dimer, g -fc, or b -fc into the tail vein. blood was collected from the facial vein at , , , , , , , , h time points. sera were separated and stored at − • c until further use. concentrations of the injected antibody molecules in the above-collected samples were measured by elisa. for elisa, bont/a was coated on microtiter plates (nunc) overnight at • c at ng/well in mm bicarbonate buffer. after washing three times with tpbs, plates were blocked with % dry milk in pbs for one hour at • c. then, × diluted sera were added to the wells, followed by a one-hour incubation. mouse anti-his-tag antibody [hrp] ( : ) (genscript, piscataway, nj, usa) was used to detect monomers and dimers of g and b antibodies in the mouse sera. goat anti-human igg bacterial toxins-a table of lethal amounts clostridial neurotoxins botulinum toxin as a biological weapon-medical and public health management a camelid single-domain antibody neutralizes botulinum neurotoxin a by blocking host receptor binding the effect of formalin on botulinum toxins a, b and c notice of cdc's discontinuation of investigational pentavalent (abcde) botulinum toxoid vaccine for workers at risk for occupational exposure to botulinum toxins clostridium botulinum-international programme on chemical safety-bacteria; world health organization current strategies for designing antidotes against botulinum neurotoxins neutralizing antibodies of botulinum neurotoxin serotype a screened from a fully synthetic human antibody phage display library the european antibotabe framework program and its update: development of innovative botulinum antibodies hammers, r. naturally-occurring antibodies devoid of light-chains properties, production, and applications of camelid single-domain antibody fragments single domain antibodies: promising experimental and therapeutic tools in infection and immunity nanobodies: natural single-domain antibodies single-domain antibodies as therapeutics against human viral diseases development of antiricin single domain antibodies toward detection and therapeutic reagents genetic passive immunization with adenoviral vector expressing chimeric nanobody-fc molecules as therapy for genital infection caused by mycoplasma hominis increasing the potency of neutralizing single-domain antibodies by functionalization with a cd b/cd binding domain selection of nanobodies that block the enzymatic and cytotoxic activities of the binary clostridium difficile toxin camelid single-domain antibodies (vhhs) against crotoxin: a basis for developing modular building blocks for the enhancement of treatment or diagnosis of crotalic envenoming structural basis for the specific neutralization of stx a with a camelid single domain antibody fragment a single-domain llama antibody potently inhibits the enzymatic activity of botulinum neurotoxin by binding to the non-catalytic α-exosite binding region botulinum neurotoxins and botulism: a novel therapeutic approach camelid single-domain antibodies: historical perspective and future outlook current approaches and future perspectives for nanobodies in stroke diagnostic and therapy fusion of higg -fc to in-anti-amyloid single domain antibody fragment vhh-pa h prolongs blood residential time in app/ps mice but does not increase brain uptake prolonged prophylactic protection from botulism with a single adenovirus treatment promoting serum expression of a vhh-based antitoxin protein genetically engineered red cells expressing single domain camelid antibodies confer long-term protection against botulinum neurotoxin beyond binding: antibody effector functions in infectious diseases botulinum neurotoxins: biology, pharmacology, and toxicology the neonatal fc receptor (fcrn) binds independently to both sites of the igg homodimer with identical affinity human igg subclass cross-species reactivity to mouse and cynomolgus monkey fcγ receptors differential tumor-targeting abilities of three single-domain antibody formats characterization of neutralizing antibodies and identification of neutralizing epitope mimics on the clostridium botulinum neurotoxin type a potent neutralization of botulinum neurotoxin by recombinant oligoclonal antibody characterization of botulinum neurotoxin type a neutralizing monoclonal antibodies and influence of their half-lives on therapeutic activity characterization of the monoclonal antibody response to botulinum neurotoxin type a in the complexed and uncomplexed forms development of germline-humanized antibodies neutralizing botulinum neurotoxin a and b llama-derived single-domain antibodies for the detection of botulinum a neurotoxin camelid single domain antibodies (vhhs) as neuronal cell intrabody binding agents and inhibitors of clostridium botulinum neurotoxin (bont) proteases antigen recognition by single-domain antibodies: structural latitudes and constraints single-domain antibodies and their formatting to combat viral infections engineered antibody fragments and the rise of single domains chimeric camel/human heavy-chain antibodies protect against mers-cov infection a novel nanobody targeting middle east respiratory syndrome coronavirus (mers-cov) receptor-binding domain has potent cross-neutralizing activity and protective efficacy against mers-cov nanobody-fc constructs targeting chemokine receptor cxcr potently inhibit signaling and cxcr -mediated hiv-entry and induce antibody effector functions intracellular antibody-bound pathogens stimulate immune signaling via the fc receptor trim universal protection against influenza infection by a multidomain antibody to influenza hemagglutinin neutralization of clostridium difficile toxin b with vhh-fc fusions targeting the delivery and crops domains efficient serum clearance of botulinum neurotoxin achieved using a pool of small antitoxin binding agents development of neutralizing scfv-fc against botulinum neurotoxin a light chain from a macaque immune library role of homologous fc fragment in the potency and efficacy of anti-botulinum antibody preparations monoclonal antibodies to type a, b, e and f botulinum neurotoxins selection and identification of single domain antibody fragments from camel heavy-chain antibodies basics of antibody phage display technology multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody (fab) heavy and light chains this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors declare no conflict of interest. key: cord- - klkojpo authors: harilal, divinlal; ramaswamy, sathishkumar; loney, tom; suwaidi, hanan al; khansaheb, hamda; alkhaja, abdulmajeed; varghese, rupa; deesi, zulfa; nowotny, norbert; alsheikh-ali, alawi; tayoun, ahmad abou title: sars-cov- whole genome amplification and sequencing for effective population-based surveillance and control of viral transmission date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: klkojpo background with the gradual reopening of economies and resumption of social life, robust surveillance mechanisms should be implemented to control the ongoing covid- pandemic. unlike rt-qpcr, sars-cov- whole genome sequencing (cwgs) has the added advantage of identifying cryptic origins of the virus, and the extent of community-based transmissions versus new viral introductions, which can in turn influence public health policy decisions. however, practical and cost considerations of cwgs should be addressed before it can be widely implemented. methods we performed shotgun transcriptome sequencing using rna extracted from nasopharyngeal swabs of patients with covid- , and compared it to targeted sars-cov- full genome amplification and sequencing with respect to virus detection, scalability, and cost-effectiveness. to track virus origin, we used open-source multiple sequence alignment and phylogenetic tools to compare the assembled sars-cov- genomes to publicly available sequences. results we show a significant improvement in whole genome sequencing data quality and viral detection using amplicon-based target enrichment of sars-cov- . with enrichment, more than % of the sequencing reads mapped to the viral genome compared to an average of . % without enrichment. consequently, a dramatic increase in genome coverage was obtained using significantly less sequencing data, enabling higher scalability and significant cost reductions. we also demonstrate how sars-cov- genome sequences can be used to determine their possible origin through phylogenetic analysis including other viral strains. conclusions sars-cov- whole genome sequencing is a practical, cost-effective, and powerful approach for population-based surveillance and control of viral transmission in the next phase of the covid- pandemic. the covid- pandemic continues to inflict devastating human life losses ( ), and has enforced significant social changes and global economic shut downs ( ) . with the accumulating financial burdens and unemployment rates, several governments are sketching out plans for slowly re-opening the economy and reviving social life and economic activity. however, robust population-based surveillance systems are essential to track viral transmission during the re-opening process. while rt-qpcr targeting sars-cov- can be effective in identifying infected individuals for isolation and contact tracing, it is not useful in determining which viral strains are circulating in the community: autochthonous versus imported ones, and -if imported -it is important to know the origin of the strains, which in turn influences public health policy decisions. in addition, it is vital to identify super-spreader events as they can be influenced by the virus strain ( ) . sars-cov- whole genome sequencing (cwgs), on the other hand, can detect the virus and can delineate its origins through phylogenetic analysis ( , ) in combination with other local and international viral strains, especially given the accumulation of thousands of viral sequences from countries all over the world (www.nextstrain.org) (figure ) . however, practical considerations, such as cost, scalability, and data storage, should first be investigated to assess the feasibility of implementing cwgs as a population-based surveillance tool. here we show that cwgs is cost-effective and is highly scalable when using a target enrichment sequencing method, and we also demonstrate its utility in tracking the origin of sars-cov- transmission. all patients had laboratory-confirmed covid- based on positive rt-pcr assay for sars-cov- in the centralized dubai health authority (dha) virology laboratory. this study was approved by the dubai scientific research ethics committee -dubai health authority (approval number #dsrec- / _ ). viral rna was extracted from nasopharyngeal swabs of patients with covid- using the ez dsp virus kit (qiagen, hilden, germany), optimized for viral and bacterial nucleic acids extractions from human specimens using magnetic bead technology. sars-cov- positive results were confirmed using a rt-pcr assay, originally designed by the us centres for disease control and prevention (cdc), and is currently provided by integrated dna technologies (idt, ia, usa). this assay consists of oligonucleotide primers and dual-labelled hydrolysis (taqman®) probes ( 'fam/ 'black hole quencher) specific for two regions (n and n ) of the virus nucleocapsid (n) gene. an additional primer/probe set is also included to detect the human rnase p gene (rp) as an extraction control. the reverse transcription and amplification steps are performed using the taqpath tm -step rt-qpcr master mix (thermofisher, ma, usa) following manufacturer's instructions. a sample was considered positive if the cycle threshold (ct) values were less than for each of the sars-cov- targets (n and n ) and the extraction control (rp). to estimate the viral load relative to human rna, we calculated the Δct value for each target as follows: Δct = ctnn -ctrp, where nn is either n or n . the average of the n and n target Δct values was then negated to reach a relative estimate of viral load which is inversely correlated with ct value. rna libraries from all samples were prepared for shotgun transcriptomic sequencing using the truseq stranded total rna library kit from illumina (san diego, ca, usa), following manufacturer's instructions. rna specific fluorescent dye is used to quantify extracted rna using the qubit rna xr assay kit and the qubit flourometer system (thermofisher, ma, usa). then, µg of input rna from each patient sample was depleted for human ribosomal rna, and the remaining rna underwent fragmentation, reverse transcription (using the superscript ii reverse transcriptase kit from invitrogen, carisbad, usa), adaptor ligation, and amplification. libraries were then sequenced using the novaseq sp reagent kit ( x cycles) from illumina (san diego, ca, usa). rna extracted (~ µg) from patient nasopharyngeal swabs was used for double stranded cdna synthesis using the quantitect reverse transcription kit (qiagen, hilden, germany) according to manufacturer's protocol. this cdna was then evenly distributed into pcr reactions for sars-cov- whole genome amplification using overlapping primer sets covering most of its genome (figure a and supplemental table ). the sars-cov- primer sets used in this study were modified from wu et al ( ) by adding m tails to enable sequencing by sanger, if needed (supplemental table ). pcr amplification was performed using the platinum tm superfi tm pcr master mix (thermofisher, ma, usa) and a thermal protocol consisting of an initial denaturation at °c for seconds, followed by cycles of denaturation ( °c for seconds), annealing ( °c for seconds), and extension ( °c for minute and seconds). a final extension at °c for minutes was applied before retrieving the final pcr products. amplification was confirmed by running µl from each reaction on a % agarose gel. all pcr products were then purified using agencourt ampure xp beads (beckman coulter, ca, usa), quantified by nanodrop (thermofisher, ma, usa), diluted to the same concentration, and then pooled into one tube for next steps. a minimum of - ng of the pooled pcr products in µl were then sheared by ultrasonication (covaris le -plus series, ma, usa) to generate a target fragment size of - bp using the following parameters: % duty factor, peak power of watts, cycles per burst, seconds treatment time, an average power of watts, and °c bath temperature. target fragmentation were confirmed by the tapestation automated electrophoresis system tapestation (agilent, ca, usa) (figure a) . subsequently, the fragmented product is purified and then processed to generate sequencing-ready libraries using the sureselectxt library preparation kit (agilent, ca, usa) following manufacturer instructions. indexed libraries from multiple patients were pooled and sequenced ( x cycles) using the miseq or the novaseq systems (illumina, san diego, ca, usa). a step-bystep sars-cov- target enrichment and sequencing protocol is provided in appendix i. demultiplexed fastq reads, obtained through shotgun or target enrichment sequencing, were generated from raw sequencing base call files using bcl fastq v . . , and then mapped to the reference wuhan genome (genbank accession number: nc_ . ) by burrow-wheeler aligner, bwa v . . . alignment statistics, such as coverage and mapped reads, were generated using picard . . . variant calling was performed by gatk v . - - , and was followed by sars-cov- genome assembly using bcftools v. . . ( figure b ). all tools used in this study are freely accessible. for laboratories without bioinformatics support, several publicly accessible, end-to-end bioinformatics pipelines (insaflu: https://insaflu.insa.pt/; genome detective: https://www.genomedetective.com/app/typingtool/virus/) ( , ) , composed of the above tools, can be used to generate viral sequences from raw fastq data. for downstream analysis, a general quality control metric was implemented to ensure assembled sars-cov- genomes have at least x average coverage (sequencing reads >q ) across most nucleotide positions ( - , ). for target enrichment and shotgun sequencing comparisons in table , we used data from samples (uae/p / , l , l , l , l , l , l ) generated using both methods. data from patient l were not included in this analysis since, unlike the above samples, significantly more sequencing data was allocated for the target enriched sample in this patient (supplemental table ) which can overestimate the efficiency of the target enrichment protocol. all new sars-cov- sequences (n= ) generated in this study were submitted to gisaid (global initiative on sharing all influenza data) under accession ids: epi_isl_ and epi_isl_ to epi_isl_ . we used nexstrain ( ), which consists of augur v . . pipeline for multiple sequence alignment (mafft v . ) ( ) and phylogenetic tree construction (iqtree v . . ) ( ) . tree topology was assessed using the fast bootstrapping function with , replicates. tree visualization and annotations were performed in figtree v . . ( ). shotgun transcriptome sequencing was used to fully sequence sars-cov- rna extracted from patients (n= ) who tested positive for the virus ( ). analysis of the sequencing data showed that this approach required, on average, . gb of data per sample yielding . million total reads, of which approximately . % of the reads (~ , reads) mapped to the sars-cov- genome with an average coverage of ~ x (table ) . this is attributed to the fact that most of the shotgun data (~ %) is allocated to the human transcriptome while a minority of the reads align to the sars-cov- genome (table ). in addition to cost and storage considerations discussed below, this approach is not highly sensitive for detecting sars-cov- genomes in general and specifically in samples with low viral abundance. in fact, despite high viral abundance relative to human rna, most samples had less than x sequencing coverage across the sars-cov- genome. samples with seemingly very low viral loads failed to yield full sars-cov- genome sequence using this approach (table and figure a ). to enrich viral sequences and minimize sequencing cost and data storage issues addressed below, we describe an alternative approach where the entire sars-cov- genome is first amplified using overlapping primer sets each yielding around . kb long inserts (figures and supplemental table ). all inserts were then pooled and fragmented to - bp inserts which were then prepared for short read next generation sequencing (figures a and c) . rna extracted from eight covid- patients, which were first sequenced by shotgun transcriptome (table and supplemental table ), were sequenced using the enrichment protocol. as expected, we observed significant enhancement in virus detection using this protocol where, on average, . % of the reads now mapped to the sars-cov- genome leading to tenfold increase in coverage relative to shotgun transcriptome (avg. x versus . x, respectively) despite generating a two hundred fold less sequencing data (avg. . gb versus . gb, respectively, table and figure d ). on average, x coverage per gb of sequencing data was generated using shotgun sequencing ( table ) compared to ~ , x per gb using target enrichment ( table ) suggesting the latter method is more cost effective and is highly scalable. we calculate the table ) . therefore, target enrichment sequencing is still more cost-effective and scalable than shotgun transcriptome sequencing even at higher sequencing throughputs. another factor impeding scalability of the shotgun approach is data storage. even with higher throughput sequencing (novaseq sp flowcell), shotgun sequencing requires an allocation of tb of data for ~ sequenced samples. on the other hand, with tb of data, a total of around , samples can be sequenced using the enrichment method and the miseq micro flowcell ( table ) . therefore, long term data storage allocations, and cost, are significantly higher, and perhaps formidable, when using the shotgun sequencing approach. to illustrate the utility of sars-cov- whole genome sequencing, we tracked the origin(s) of the virus in seven patients (uae/p / , l , l , l , l , l , l ) by comparing their assembled sequences to virus strains (n= ) identified during the early phase of the pandemic, between january and march , in the uae ( ). all seven patient samples were collected between march and april , and are therefore good candidates to determine whether transmissions were community-based due to the previously documented strains or were independent external introductions. multiple sequence alignment and phylogenetic analysis (figures b and ) showed that the new isolates from patients l , l , l , and l clustered with earlier strains of iranian origin (or clade a ), while that from patient l belonged to the early european (or clade a a) cluster (figure ) . this information suggests that transmissions for all those five patients were most likely community-based, which we then confirmed from patient medical records where no recent travel history was reported by any of those individuals. sars-cov- isolates from patients p /uae/ and l were, on the other hand, closer to the earliest asian strains, which are more diverse due to fewer but distinct mutations ( figure ) . hence, with the available sequencing data it is challenging to ascertain whether the p /uae/ and l transmissions were community-based or due to early independent introductions. however, patient l did not have any recent travel history before symptoms onset, suggesting the case for community-based transmission related to an asian strain. on the other hand, travel history in patient p /uae/ was not known and the corresponding isolate appeared to match closely to five other strains from the united states and taiwan (figure ) . therefore, transmission in patient p /uae/ was unlikely to be community-based from the early strains ( ), but rather due to an independent travelrelated introduction of the virus. genomics-based sars-cov- population-based surveillance is a powerful tool for controlling viral transmission during the next phase of the pandemic. therefore, it is important to devise efficient methods for sars-cov- genome sequencing for downstream phylogenetic analysis and virus origin tracking. towards this goal, we describe a cost-effective, robust, and highly scalable target enrichment sequencing approach, and provide an example to demonstrate its utility in characterizing transmission origin. our target enrichment protocol is amplicon-based for which oligonucleotide primers can be easily ordered by any molecular laboratory. next generation sequencing (ngs) has also become largely accessible to most labs, and in our protocol we show that highly affordable, low throughput sequencers, such as the illumina miseq system, can be used efficiently to sequence up to samples at x coverage each at a cost of $ per sample ( table ) . this cost is likely comparable to rt-pcr testing for the virus. one possible limitation is the use of ultra-sonication for fragmentation of pcr products after sars-cov- whole genome amplification. several labs might lack sonication systems due to accessibility and affordability issues. in such situations, our protocol can be easily modified to use enzymatic fragmentation instead provided by commercial kits, such as the agilent https://www.genomedetective.com/app/typingtool/virus/) ( , ) which can take raw sequencing (fastq) files to assemble viral genomes, and to perform multiple sequence alignment and phylogenetic analysis for virus origin tracking. in addition, the described approach does not require significant data storage or computational investment as shown by our cost, data, and scalability calculations ( table ) . our phylogenetic analysis demonstrates how sars-cov- genomic sequencing can be used to track origins of virus transmission. however, data should be carefully interpreted, and should be combined with other epidemiological information (such as travel history) to avoid inaccurate conclusions. the major limitation facing genomic-based sars-cov- surveillance includes the lack of virus sequencing data representing most strains in any country. nonetheless, sars-cov- strains are continuously being sequenced by government, private, and academic entities all over the world, and the sequencing data is being shared publicly. this proliferation of sequencing datasets will empower genomic-based surveillance of the virus, and the availability of cost effective sequencing options, like the one described in this study, will contribute to democratizing sars-cov- sequencing and data sharing. in summary, we show that sars-cov- whole genome sequencing is a highly feasible and effective tool for tracking virus transmission. genomic data can be used to determine community-based versus imported transmissions, which can then inform the most appropriate public health decisions to control the pandemic. disclosures. authors do not have any conflicts of interests to disclose. sars-cov- /covid- : viral genomics, epidemiology, vaccines, and therapeutic interventions identifying and interrupting superspreading events -implications for control of severe acute respiratory syndrome coronavirus genomic surveillance and phylogenetic analysis reveal multiple introductions of sars-cov- into a global travel hub in the middle east shotgun transcriptome and isothermal profiling of sars-cov- infection reveals unique host responses, viral diversification, and drug interactions a new coronavirus associated with human respiratory disease in china insaflu: an automated open web-based bioinformatics suite "from reads" for influenza whole-genomesequencing-based surveillance genome detective: an automated system for virus identification from highthroughput sequencing data nextstrain: real-time tracking of pathogen evolution mafft: a novel method for rapid multiple sequence alignment based on fast fourier transform terrace aware data structure for phylogenomic inference from supermatrices acknowledgements. authors would like to thank members of the microbiology laboratory, latifa women and children hospital, dubai health authority and al jalila children's specialty hospital genomics center for supporting sars-cov- diagnostic testing and for arranging samples used in this study. key: cord- -sbsg ix authors: zhan, choujun; tse, chi k.; lai, zhikang; chen, xiaoyun; mo, mingshen title: general model for covid- spreading with consideration of intercity migration, insufficient testing and active intervention: application to study of pandemic progression in japan and usa date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: sbsg ix a new susceptible-exposed-infected-confirmed-removed (seicr) model with consideration of intercity travel and active intervention is proposed for predicting the spreading progression of the new coronavirus disease (covid- ). the model takes into account the known or reported number of infected cases being fewer than the actual number of infected individuals due to insufficient testing. the model integrates intercity travel data to track the movement of exposed and infected individuals among cities, and allows different levels of active intervention to be considered so that realistic prediction of the number of infected individuals can be performed. the data of the covid- infection cases and the intercity travel data for japan (january to march , ) and the usa (february to march , ) are used to illustrate the prediction of the pandemic progression in regions of japan and states (plus a federal district) in the usa. by fitting the model with the data, we reveal that, as of march , , the number of infected individuals in japan and the usa could be twenty-fold and five-fold as many as the number of confirmed cases, respectively. moreover, the model generates future progression profiles for different levels of intervention by setting the parameters relative to the values found from the data fitting. results show that without tightening the implementation of active intervention, japan and the usa will see about . % and . % of the population eventually infected, and with drastic ten-fold elevated active intervention, the number of people eventually infected can be reduced by up to % in japan and % in the usa. finally, an assessment of the relative effectiveness of active intervention and personal protective measures is discussed. with a highly vigilant public maintaining personal hygiene and exercising strict protective measures, the percentage of population infected can be further reduced to . % in japan and . % in the usa. the global spread of the new coronavirus disease (covid- ) has shown no sign of subsiding since its emergence in wuhan, china, in december [ ] . as of march , , a total of , cases of covid- infection have been confirmed in over countries, with a death toll of , [ ] . different control strategies at different levels of stringency have been applied to slow the spread of the virus in different countries [ ] . while some countries have seen peaks of infected cases and observed significant reduction in the number of new infections in the local communities [ , ] , the spreading has continued in many countries, and surges in infected cases have been observed in europe, usa and australia. intercity travel has been found to be a contributing factor to the rapid spread of the virus [ , ] . thus, effective models for describing the pandemic progression in different cities should take into consideration the volume of intercity travels. furthermore, the rapid spread of the virus in a population has often been a result of delayed information or unawareness of the real situation in that population, despite the wide dissemination of information related to covid- outbreaks in other parts of the world. the most notable information latency lies in the number of confirmed cases reported, which depends on the ability of the particular country or city to perform tests as well as the possible bureaucracy in the local system of reporting. thus, the number of confirmed cases is almost certainly not the true number of infected individuals at any given time [ ] , and an improved model for predicting the spreading progression should incorporate the latency associated with the reporting system as well as the possible missing cases leading to delay and loss of information. the traditional susceptible-exposed-infectious-recovered (seir) model [ , ] thus has obvious shortfalls in describing the spreading dynamics of covid- pandemic. in this work, we attempt to fill the main gap between the number of confirmed cases and the actual number of infected cases. specifically, in the proposed model, an infected individual may become a confirmed case and then recovered/removed. moreover, an infected individual may also be recovered/removed without being confirmed as infected. in other words, the basic model proposed here is a susceptible-exposed-infectious-confirmed-recovered (seicr) model, which has an additional state corresponding to an individual having been confirmed by the authority as being infected. on the basis of an seicr model, we develop a model incorporating intercity travel data which accounts for any increase or decrease in the number of exposed and infected individuals in a city due to intercity migration. furthermore, the level of intervention in the form of travel restriction, regional lockdown or other active control measures would profoundly influence the rapidity of the spread of the virus and the eventual number of infected cases. the model should therefore allow the level of active intervention to be included as a control parameter and produce the appropriate progression profile. a specific parameter is used to adjust the level of active intervention in the simulation of future progression profiles, which corresponds quantitatively to the increase in the number of individuals eventually infected due to an additional infected individual at any given time. in this work, we apply the model to study the covid- spreading progression in japan and the usa. data of confirmed and recovered cases in japanese prefectures or regions (january to march , ) and states in the usa including washington dc (february to march , ) are used for fitting with the model and retrieval of parameter values. the parameters found are then adjusted to produce future progression trajectories corresponding to the implementation of different levels of active intervention. from the set of best-fit parameters, we reveal that the actual number of infected individuals could be up to -fold and -fold as many as the confirmed numbers in japan and the usa, respectively, as of march , . furthermore, if the level of active intervention is kept unchanged, the percentage of population eventually infected in osaka-fu and tokyo-to will reach around % and . % of the population (around , , and , people), respectively, and in total, japan will have about . % of its population eventually infected. however, implementing a four-fold elevated active intervention will improve the situation substantially, with the percentage of population infected in osaka-fu and tokyo-to reduced to . % and . %, respectively, and over % reduction in the overall number of infected cases in japan. our results for the usa also show a rather detrimental situation if the level of active intervention remains at the status quo in the coming months, with california and new york state having around % and . % of their population ( , , and , , people) eventually infected, respectively, and the overall infected population will reach . %. however, by implementing four-fold elevated active intervention, the percentage of infected population in the usa can be reduced to . %. furthermore, the effectiveness of the public in exercising protective measures can be assessed by varying the infection rates in the model, and it is found that active government intervention is more effective for japan, while exercising protective measures by the public is more important for the usa. with the public raising its level of vigilance in exercising strict protective measures and the government drastically elevating its active intervention, the percentage of population getting infected can be reduced to . % in japan and . % in the usa. the world health organization currently sets the alert level of covid- to the highest, and has made data related to the pandemic available to the public in a series of situation reports as well as other formats [ ] . our data include the number of confirmed infected cases, the cumulative number of confirmed infected cases, the number of recovered cases, and death tolls, for individual prefectures and regions in japan, from january to march , , and for states and a federal district (washington dc) in the usa, from february to march , . data organized in convenient formats are also available elsewhere [ , , ] . moreover, the monthly intercity migration data for february are available from official statistics provided by the japanese government [ ] , and are used as indicative migration strengths between prefectures or regions in japan. for the usa, annual data for the volume of inter-state travellers are available from the census bureau [ ] and the bureau of transportation statistics [ ] . in the proposed susceptible-exposed-infectious-confirmed-recovered (seicr) model, each individual would assume one of five possible states at any time, namely, susceptible (s), exposed (e), infected (i), confirmed (c), and recovered/removed (r). compared to the traditional seir model [ , ] , the new seicr model has an additional c state, corresponding an individual having been confirmed by the authority as infected. thus, not all infected individuals will become confirmed, and some infected individuals will transit to the recovered state without going through the confirmed state. for city or region j, the number of individuals in the five states are s j (t), e j (t), i j (t), c j (t) and r j (t), at time t. the transitions of the five states are illustrated in figure . in addition, p j (t) stands for the population of region j. furthermore, to account for intercity movement, we introduce a migration strength, m ij (t), which represents an indicative volume of people moving from region i to region j at time t [ ] . then, the augmented seicr model is given as follows: is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted march , . . , and ∆p j (t) = p j (t + ) − p j (t). the meaning of each parameter is given in table . also, we assume that the recovered and confirmed individuals would stay in region j. in this seicr model, the number of individuals eventually infected is set initially at n s j (t ) = δ j p j (δ j being constant), implying that some effective measures have been taken by the authorities to limit the upper bound of the susceptible population. moreover, in the case of inactive or less effective intervention, or even unchecked spread, the growth of the number of infected cases will add to the eventual infected number. hence, the number of eventually infected individuals should increase for each additional infected or exposed individual at time t. this is equivalent to adding an extra term (the boxed term below) to ∆s i (t) and ∆n s j (t). furthermore, as the number of infected cases increases and approaches a saturating percentage k h (such as a herd-immunity condition), the spreading is expected to slow down significantly, i.e., α j and β j will drop as n s approaches k h p j , where < k h < . thus, we have j is an inverse indicator of the level of active intervention implemented, and corresponds quantitatively to an increase in the number of eventual infected individuals for each additional infected or exposed individual in region j, and the added term in ∆s j and ∆n s j will approach zero as n s j → k h p j . the meanings of other parameters are given in table . again, the recovered and confirmed individuals are assumed to stay in region j. the model given in ( ) and ( ) is very general in the sense that it applies to populations with varied levels of effectiveness of active intervention during the outbreak. to further facilitate the assessment of control measures implemented in region j, we define the level of active intervention as thus, if ψ j > , the control measures are effective and the progression is limited such that k (c) j < . the total number of eventually infected individuals is equal to n j= p i (t )δ j . . cc-by-nc-nd . international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted march , . . α j : rate of infecting a susceptible individual by an exposed individual in region j β j : rate of infecting a susceptible individual by an infected individual in region j ρ j : rate of infecting a susceptible individual by a confirmed individual in region j λ j : confirmed rate of infected individuals in region j κ: rate of an exposed individual becoming infected γ (i) j : recovery rate of an infected but not confirmed individual in region j γ (q) j : recovery rate of a confirmed individual in region j k i : possibility of an infected individual moving from one region to another k (c) j : increase in number of individuals eventually infected for each additional infected or exposed individual in region j ψ j : level of active intervention, ψ j = /k (c) j k h : proportion of population infected achieving no further spreading, i.e., absolute upper bound for n s j for all j δ j : initial percentage of eventual infected individuals in region j i j, : initial number of infected individuals in region j e j, : initial number of individuals in region j c j, : initial number of confirmed infected individuals in region j however, in the case of less effective or ineffective control, i.e., ψ j < , infected and exposed individuals continue to spread the disease, and for each additional infected individual, there will be k (c) j more eventual infected individuals, and the pandemic progresses until the number of infected cases reaches k h p j . the model represented by ( ) and ( ) describes the dynamics of the pandemic propagation with consideration of human migration dynamics and the reality of insufficient testing that leads to confirmed infected cases being fewer than the actual infected cases. the parameters in ( ) and ( ) are unknown and to be estimated from historical data of c and r. we solve this parameter identification problem via constrained nonlinear programming (cnlp), with the objective of finding an estimated growth trajectory that fits the data. an estimated number of infected cases of each city can be generated from ( ) and ( ) with unknown set θ j given by where i j, = i j (t ) and e j, = e j (t ) are the initial numbers of infected and exposed individuals in region j, and {α j , β j , γ j , δ j , λ j , γ j } are the model parameters of region j. here, we assume that all confirmed cases are either quarantined or hospitalized, and hence not infectious, i.e., ρ j = . then, the unknown set is Θ = {θ , θ , · · · , θ k , κ, k i , k h }, which essentially has k+ unknowns, where k is the number of regions in the entire population . cc-by-nc-nd . international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted march , . . https://doi.org/ . / . . . doi: medrxiv preprint under study. the identification of unknown parameters would require a considerable effort of computation. specifically, the parameter estimation problem can be formulated as the following constrained nonlinear optimization problem: where f (·) represents the model given by ( ) and ( ), w is the set of estimated variables, with unknown set Θ being bounded between Θ l and Θ u . in this work, an inverse approach is taken to find the unknown parameters and states by solving ( ). the model parameters characterize the spreading dynamics, and once the set of parameters has been identified using the abovementioned optimization procedure, we may generate future progression profiles by using the same set of parameters. moreover, we may also adjust some of the parameters to examine different possible scenarios, corresponding to varying levels of active intervention ψ j = /k (c) j , including travel restriction, mandatory quarantine and other control measures. if the level of active intervention stays with the status quo, we will use the same value of k (c) j for generating future progression profiles. future paths under more active intervention can be predicted by reducing the value of k (c) j . in our study, we will consider three levels of active intervention, namely, . staying with the status quo, corresponding to the same value of ψ j or k (c) j ; . two-fold step-up of active intervention, corresponding to ψ j or . k (c) j ; . four-fold step-up of active intervention, corresponding to ψ j or . k (c) j . we will examine the results in the next section. the pandemic progression profiles of japanese prefectures or regions are examined. we perform data fitting of the model, described by ( ) and ( ), using historical daily data of confirmed and recovered cases [ , ] . a typical candidate set of parameter values that fit well wth the data from january , to march , is as follows: cc-by-nc-nd . international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted march , . . this set of parameters reflects an inadequate level of control to slow the spread of the disease, as indicated by the value of k (c) j being larger than . specifically, for each additional infected or exposed individual, the number of eventual infected individuals would increase by around . on the average. the number of individuals eventually infected will approach a saturating percentage k h . we have identified candidate sets of parameters which satisfy the fitting criteria, and for each set of parameters, we perform a separate simulation run. figure shows one particular simulation run of a well fitted candidate set of parameters for selected prefectures in japan. the averaged results of all simulation runs are consolidated in the charts shown in figure . based on the data up to march , , our model estimates that less than % of the infected cases are confirmed, with hokkaido having the highest percentage ( . %) and hyogo-ken the least ( . %), as shown in figure (a). in other words, the actual number of infected individuals could be times as many as the official confirmed number. statistics of percentages of the population confirmed and infected with the disease up to march , are shown in figure by extending each simulation run to the forthcoming days, we obtain a set of predicted progression profiles for each region in japan. moreover, different levels of active intervention can be assessed by adjusting parameter k (c) j relative to the values found in each candidate set. for instance, by reducing k (c) j and re-running the simulation, we may assess the effect of tightening the control measures. specifically, as described in section . , we examine three cases, corresponding to the level of active intervention being unchanged, two-fold elevated and four-fold elevated. the key results are summarized as follows: • staying with the status quo (k (c) j unchanged): if there is no further tightening of control aiming to slow the spread, all parameters of the candidate sets will remain unchanged. the total number of individuals eventually infected until september , in each region is shown in figure (c). in this case, the number of infected individuals in osaka-fu and tokyo-to will reach about , , and , ( % and . % of population), respectively, while most other regions will have around % of the population eventually infected by september , , as shown in figure (d). in total, about . % of the population in japan will be infected. • two-fold elevated active intervention (k figure (c) . specifically, the percentage of population eventually infected by september , in osaka-fu and tokyo-to would drop to about . % and . %, respectively, while most other regions would drop to less than %, as shown in figure (d) . in total, about . % of the population in japan will be infected. • four-fold elevated active intervention (k is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted march , . . https://doi.org/ . / . . . doi: medrxiv preprint jan jan feb feb feb mar mar jan jan feb feb feb mar mar jan jan feb feb feb mar mar (a) aichi-ken jan jan feb feb feb mar mar jan jan feb feb feb mar mar jan jan feb feb feb mar mar (b) chiba-ken jan jan feb feb feb mar mar jan jan feb feb feb mar mar jan jan feb feb feb mar mar (c) hokkaido jan jan feb feb feb mar mar jan jan feb feb feb mar mar jan jan feb feb feb mar mar (d) hyogo-ken jan jan feb feb feb mar mar jan jan feb feb feb mar mar jan jan feb feb feb mar mar (e) kanagawa-ken jan jan feb feb feb mar mar jan jan feb feb feb mar mar jan jan feb feb feb mar mar (f) osaka-fu jan jan feb feb feb mar mar jan jan feb feb feb mar mar jan jan feb feb feb mar mar (g) saitama-ken jan jan feb feb feb mar mar jan jan feb feb feb mar mar jan jan feb feb feb mar mar (h) tokyo-to is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted march , . . inal value in each simulation run, we observe a very drastic drop in the number of individuals eventually infected, as given in figure (c). specifically, the percentage of population eventually infected by september , in osaka-fu and tokyo-to would drop to about . % and . %, respectively, while most other regions would drop to less than %, as shown in figure (d). in total, about . % of the population in japan will be infected. in conclusion, the current level of control by the japanese government seems to be inadequate, and a significant step-up in the level of active intervention is necessary in order to curb the aggressive progression trend. in addition, our model estimates that the number is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted march , . . of infected individuals could be times as many as the currently confirmed number due to various reasons such as insufficient testing. based on the data collected so far and assuming no further tightening of control, our model estimates about . % of the population eventually infected, and a four-fold elevation in control efforts may bring it down to . % (about % reduction) and end the pandemic sooner. as will be shown in section . , a drastic -fold elevated active control may bring it further down to . %. the pandemic progression profiles of states and a federal district in the usa are examined. we again perform data fitting of the model, described by ( ) and ( ), using historical daily data of confirmed and recovered cases from february to march , [ , ] , and obtain candidate sets of parameters that satisfy the fitting criteria. for brevity of presentation, we show here the results for eight selected states having significant numbers of infected individuals as of march , . figure shows one typical simulation run, showing the number of confirmed cases, the estimated number of infected individuals (not confirmed), and the estimated number of exposed individuals. as of march , , our model shows that less than % of the infected cases are confirmed, with washington dc having the highest percentage ( %) and michigan state the least ( . %), as shown in figure (a) . in other words, the actual number of infected individuals in the usa could be times as many as the confirmed number. statistics of percentages of population confirmed and infected with the disease up to march , are shown in figure (b) . again, by extending each simulation run to the future days, we obtain a set of predicted progression profiles for each state and federal district in the usa. we also examine three cases corresponding to three different levels of active intervention, as described in section . . the key results are summarized as follows: • staying with the status quo (k (c) j unchanged): if there is no further tightening of control aiming to slow the spread, all parameters of the candidate sets will remain unchanged. the total number of individuals eventually infected until september , in each state is shown in figure (c). in this case, the number of infected individuals in california and new york state will reach about , , and , , ( % and . % of population), respectively, while most other states will have less than % of the population eventually infected by september , , as shown in figure (d) . in total, about . % of the population in the usa will be infected. • two-fold elevated active intervention (k (c) if active intervention is stepped up to twice the current level, i.e., the value of k (c) j set to half of the original value in each simulation run, we observe a significant drop in the number of individuals eventually infected, as given in figure (c) . specifically, the percentage of population eventually infected by september , in california and new york state would drop to about . % and . %, respectively, while most other states would drop to less than %, as shown in figure (d) . in total, about % of the population in the usa will be infected. . cc-by-nc-nd . international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted march , . . https://doi.org/ . / . . . doi: medrxiv preprint feb feb feb mar mar mar mar feb feb feb mar mar mar mar feb feb feb mar mar mar mar (a) california feb feb feb mar mar mar mar feb feb feb mar mar mar mar feb feb feb mar mar mar mar (b) illinois feb feb feb mar mar mar mar feb feb feb mar mar mar mar feb feb feb mar mar mar mar (c) louisiana feb feb feb mar mar mar mar feb feb feb mar mar mar mar feb feb feb mar mar mar mar (d) massachusetts feb feb feb mar mar mar mar feb feb feb mar mar mar mar feb feb feb mar mar mar mar (e) michigan feb feb feb mar mar mar mar feb feb feb mar mar mar mar feb feb feb mar mar mar mar (f) new jersey feb feb feb mar mar mar mar feb feb feb mar mar mar mar feb feb feb mar mar mar mar (g) new york feb feb feb mar mar mar mar feb feb feb mar mar mar mar feb feb feb mar mar mar mar is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted march , . • four-fold elevated active intervention (k (c) if active intervention is stepped up to four times the current level, i.e., the value of k (c) j set to a quarter of the original value in each simulation run, we observe further reduction in the number of individuals eventually infected, as given in figure (c) . specifically, the percentage of population eventually infected by september , in california and new york state would drop to about . % and %, respectively, while most other states would drop to less than %, as shown in figure (d) . in total, about . % of the population in the usa will be infected. . cc-by-nc-nd . international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted march , . . in summary, a significant step-up in the level of active intervention is necessary for us government to slow the spread of the virus. in addition, our model estimates that the number of infected individuals could be five times as many as the currently confirmed number due to insufficient testing and other reasons. based on the data collected so far and assuming no further tightening of government's control, our model estimates that about . % of population would eventually be infected, and a four-fold elevation in control efforts may bring it down to . %. as will be shown in section . , a drastic -fold elevated active control may bring it further down to . %. the results presented above have highlighted the ability of the model in assessing the impact of active intervention through adjusting one of the parameters, namely, ψ j = /k (c) j . moreover, it has been widely disseminated that maintaining personal hygiene is equally important in curbing the spread of the virus. the world health organization recommends several specific protective measures to be practiced by the public, including frequent hand washing, maintaining social distancing, avoiding touching one's eyes, nose and mouth, and practicing respiratory hygiene [ ] . recent studies also show that wearing surgical masks also help in some cases [ , ] . the level of vigilance of the public in exercising personal protective measures can also be incorporated in our model through adjusting infection rates α j and β j . we can therefore assess the combined effectiveness of active intervention and practicing protective measures in controling the pandemic. here, we vary α j , β j and k (c) j from % to % of the originally identified values in intervals, corresponding different levels of vigilance of the public and active intervention by the authorities. in particular, we assess α j and β j as one property and k (c) j as another, i.e., varying α j and β j in synchrony. specifically, for each candidate parameter set, we perform simulation runs for each combination of α j , β j and k our results have highlighted an interesting difference between the effectiveness of government's active intervention and maintaining personal hygiene by the public for japan and the usa. for japan, we observe a -fold reduction (from . % to . %) in the percentage of individuals eventually infected upon a drastic -fold step-up of active intervention, as given in the first column of figure (a), whereas less than -fold reduction (from . % is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted march , . to . %) is observed in the percentage of individuals eventually infected upon the same -fold improvement in personal hygiene, as shown in the first row of figure (a). thus, government's active intervention seems to be more important for japan. moreover, for the usa, we see the opposite. specifically, only about -fold reduction in the percentage of individuals eventually infected is observed upon a drastic -fold step-up of active intervention, as shown in the first column of figure (b), whereas a -fold reduction is observed upon a -fold improvement in maintaining personal hygiene by the public. thus, raising the level of vigilance of the public in exercising personal protective measures is comparatively more important for the usa. a plausible reason for the difference between japan and usa is that the model has captured higher infection rates for the usa compared to japan. reducing k j for the us case is thus less effective at such high infection rates, i.e., a fewer eventual infected number per additional infected individual would not help too much. in contrary, the parameter sets for japan already have relatively lower infection rates, and further improvement by reducing the infection rates would be limited. as a final remark, we observe from the charts given in figure that combining a very high level of vigilance of the public in exercising strict protective measures and a drastic step-up of government intervention, the percentage of the population getting infected can be reduced to . % in japan and . % in the usa. one of the key challenges in data-driven modeling and analysis is the delayed and missing information that makes fitting of models either difficult or unreliable, resulting in inconsistent or even erroneous dynamical profiles generated by a poorly parameterized model. the traditional susceptible-exposed-infectious-recovered (seir) model provides a general dy- is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted march , . . namical description of the spreading of a disease in a population, and involves a series of transitional processes that describe how a healthy individual becomes exposed, infected and eventually recovered or removed from the population. the model thus generally has four dynamical states. from the modeling point of view, the model parameters can be extracted from fitting with historical data consisting of the number of individuals in the infected state and recovered state, which are the usual practically observable data. outbreaks of the new coronavirus (covid- ) pandemic have occurred in over countries since the virus began to spread from china in january via an active global transportation network. the data of infected and recovered cases reported by different cities and regions have been found unreliable or incomplete, as they are subject to the availability of test facilities as well as other factors related to bureaucracy of reporting and the mode of operation of the medical systems. nonetheless, figures of "confirmed" cases can still be honest figures, though not necessarily the true figures of infected cases. in this work, we propose a new disease spreading model with consideration of the delayed and missing data of infected cases, intercity travel, and the level of active intervention. instead of matching the number of confirmed cases obtained from official sources directly with the number of infected cases in the model, we create a new state which is a delayed and contracted version of the original infected state of the seicr model, leading to a new seicr model. the model, which estimates the actual number of infected cases after identifying the best parameter sets, is applied to study the covid- pandemic progression in japan and the usa. results reveal that the actual number of infected individuals could be up to -fold and -fold as many as the confirmed numbers in japan and the usa, respectively, as of march , . our model also allows assessment of varying levels of active intervention implemented by the government, and results show that the current level of control by the japanese and us governments may be inadequate, and a significant step-up in the level of active intervention is necessary in order to slow the aggressive progression trend in both countries. for japan, based on the data collected so far and assuming no further tightening of control, our model estimates about . % of the population eventually infected, and a four-fold elevation in control efforts may bring it down to . %. for the usa, our model estimates about . % of population eventually infected if the government does not step up its control, and a four-fold elevation in active intervention may bring it down to . %. finally, adjusting the infection rates permits assessment of effectiveness of practicing protective measures and maintaining personal hygiene. simulations of various levels of implementation of combined active intervention and protective measures show that stepping up government's active intervention would be more effective for japan, while raising the level of vigilance of the public in maintaining personal hygiene and social distancing is comparatively more important for the usa. with the public raising its level of vigilance and the government drastically elevating its active intervention, the percentage of population getting infected can be reduced to . % in japan and . % in the usa. . cc-by-nc-nd . international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted march , . . https://doi.org/ . / . . . doi: medrxiv preprint early transmission dynamics in wuhan china of novel coronavirus-infected pneumonia coronavirus cases by country, territory, or conveyance coronavirus: the hammer and the dance modeling and prediction of the coronavirus disease spreading in china incorporating human migration data risk of transportation of novel coronavirus disease from wuhan to other cities in china the effect of travel restrictions on the spread of the novel coronavirus (covid- ) outbreak coronavirus testing: criteria and numbers by country mathematical tools for understanding infectious disease dynamics plausible models for propagation of the sars virus world health organization. coronavirus disease (covid- ) outbreak coronavirus pandemic in japan new coronavirus epidemic real-time tracking state-to-state migration bureau of transportation statistics world health organization. coronavirus disease (covid- ) advice for the public influenza virus aerosols in human exhaled breath: particle size, culturability, and effect of surgical masks does wearing a mask protect you from the flu and other viruses? this work was supported by national science foundation of china project , guangdong youth university innovative talents project kqncx , and city university of hong kong under special fund . key: cord- - l soxu authors: lee, jenny j.; haupt, john p. title: scientific globalism during a global crisis: research collaboration and open access publications on covid- date: - - journal: high educ (dordr) doi: . /s - - - sha: doc_id: cord_uid: l soxu this study sought to understand the nature of scientific globalism during a global crisis, particularly covid- . findings show that scientific globalism occurs differently when comparing covid- publications with non-covid- publications during as well as before the pandemic. despite the tense geopolitical climate, countries increased their proportion of international collaboration and open-access publications during the pandemic. however, not all countries engaged more globally. countries that have been more impacted by the crisis and those with relatively lower gdps tended to participate more in scientific globalism than their counterparts. matters, as demonstrated by the eu, for reasons having to do with size, geographic proximity, and political support (wagner, ; wagner & leydesdorff, ) . the drivers and benefits for countries can vary. for low-income countries, international research can develop their scientific capacity and output, while higher-income countries gain field access to special data and global exposure (engels & ruschenburg, ; leather, butterfield, peachey, silverman, & sheriff, ; iglic, doreian, kronegger, & ferligoj, ) . recent research has examined mixed patterns of international collaboration during global emergencies. similar to past trends, china and the usa lead in covid- scientific output (hook & porter, ; hossain, ; chahrour, assi, bejjani, et al., ) . based on a social network analysis of the current and past coronaviruses research over the past two decades, tao et al. ( ) found that while the usa has the highest level of degree centrality or has the highest impact on other countries (followed by france and the uk), research teams are relatively scattered, as demonstrated by the study's low-density global map. bibliometric studies further identified a positive relationship between covid- cases and publications; countries that were most affected by covid- also produced the most scientific documents (dehghanbanadaki, et al., ; zhang, zhao, sun, et al., ) , but this pattern was not always consistent. it was also observed that some of the countries especially hit by the current coronavirus (i.e., italy and iran) produced disproportionately fewer articles, suggesting a research shortage based on an overstrain of healthcare facilities and physicians, which took precedence over publishing observational studies and case reports (chahrour, assi, bejjani, et al., ) . although there are fewer publications on international collaboration patterns during covid- , there are some additional insights from the earlier coronavirus pandemic, known as severe acute respiratory syndrome (sars or sars-cov) in , affecting countries and resulting in known deaths. according to a bibliometric study of sars publications, international collaboration rates tended to be low, with english-speaking countries, particularly the usa and the uk dominating the production (chiu, huang, & ho, ) . the same study found that out of countries with sars cases, less than half did not produce a single sci publication. a broader bibliometric study based on publications of who's eight emerging pathogens, which included sars, found that international collaboration ranged from . to . % (sweileh, ) . the same study also found that among these topics, sars ranked first in quantity, followed by ebola and crimean-congo, suggesting a higher number of studies associated with higher case incidents. the countries most affected, however, do not produce the most articles; the countries with the highest number of publications in different areas were china (sars), the usa (ebola), and turkey (crimean-congo). the peaks in publication rates corresponded with the timeframe of the respective outbreaks, as was also found for covid- publications (zhang, l., zhao, w., sun, b. et al., ) . fewer studies have examined international research dynamics during a global crisis. based on a case study of sars research, lee ( ) observed the unavailability of modern technology, such as the availability of genome sequencing machines for some groups. it may be for this reason that less-resourced countries are more likely to be involved in international research collaboration than independent research (pouris & ho, ; iglic, doreian, kronegger, & ferligoj, ) . lee ( ) also identified covert competition for publication and patents between scientists and worries that genomic information would be withheld until intellectual property rights were secured. besides reasons having to do with rivalry, new knowledge might be suppressed due to political or cultural factors. from the perspective of a chinese research team, reasons having to do with "a lack of coordination and collaboration, stifling political influence, hesitation to challenge authorities, and isolation from the rest of the world" contributed towards a missed opportunity in sharing their major sars findings (enserink, , para ) . as demonstrated in the latter case, political factors can play a crucial role. while the world is facing an unprecedented global health crisis affecting almost every country, geopolitical tensions are rising, particularly around the source of the coronavirus and information sharing. as us leaders have referred to covid- as the "chinese virus" or "wuhan virus," chinese leaders have suggested that the sars-cov virus was brought to wuhan by the us army (crowley, wong, & jakes, ) . australia's call, with support from the usa, uk, and france, for a probe on covid- 's origins, has also added to diplomatic tensions with china (mercer, ) . as scientists throughout the world are racing to halt the current pandemic's spread, us and uk authorities issued a joint statement warning against malicious cyber activity, including intelligence on national healthcare policy and sensitive data on covid- -related research (national cyber security centre, ). more explicitly, the us director of the national counterintelligence and security center indicated that information on a possible vaccine would be a "huge prize," but "we have full expectation that china will do everything in their power to obtain any viable research that we are conducting here in the u.s." (myre, , para. ) . us policymakers have also suggested that any coordination with china is "a self-harming exercise in a zero-sum competition for global leadership" (hass & doug, , para ) . the us president also threatened to permanently cut funding to the world health organization (who) because of its alleged pro-china bias (restuccia, lubold, & hinshaw, ) . the effect of such a cut would be substantial given the usa is the world's largest sponsor to the who, contributing % of the organization's $ . billion budget (togoh, ) . meanwhile, the chinese government announced that funded studies on coronaviruses are to be published in local, over international, chinese journals (larivière, shu & sugimoto, ) . this move aligned with a broader research-evaluation reform initiative that prioritizes domestic needs and ultimately publishes in domestic over sci journals (huang, ; tao, ) . while most media attention related to such geopolitical tensions has involved china, strains are also occurring elsewhere. on april , two prominent french doctors suggested that africa should serve as the laboratory for vaccine testing, sparking a widespread public outcry and reminders of the continent's colonial past with europe (kossoff, ) . the following month, the usa shunned a global virtual meeting organized by europe in raising $ billion for vaccine research, noting that the usa is already investing billions of dollars in its own domestic efforts (stevis-gridneff & jakes, ). the conceptual framework for this study is scientific globalism, sometimes referred to as technoglobalism (cantwell & grimm, ) . according to sá and sabzalieva ( ) , scientific globalism's basic mission is for the advancement of knowledge and open science, which can be contrasted to scientific nationalism's focus on economic competitiveness and nation-building. scientific nationalism, on the other hand, views scientific and technological advancement as a way to ensure national security, strengthen economic advantage, and signal national prestige (sá & sabzalieva, ) . they found that both logics coexist, despite various tensions and contradictions in policy. cantwell and grimm ( ) similarly propose that academic science is both a global and cooperative enterprise as well as a nationalist endeavor in competition with state rivals. in the case of china, cantwell and grimm suggest that the country's rise is a consequence of savvy technonationalism, whereas the usa and uk's nationalism policies, in favor of right-wing populism, have limited their scientific enterprise. marginson ( ) further explains that china's successful "national/global synergy" over the past years can be explained as follows, "china's rise in science and higher education lies on one hand in the global conditions (the evolving global science system), on the other hand in the national conditions, and especially the conjunction between global and national drivers that national policy achieved" (p. ). in a study of co-publications between the aus and china over the past years, lee and haupt ( ) also found that scientific research can be both national and global pursuits as a positive sum endeavor. from a scientific nationalism view, the authors found that despite usa attempts to curb international research engagement with china, the usa benefits from china's contribution to us research productivity. from a scientific globalism lens, global science is advanced as demonstrated by the two country's increasing co-authored scientific papers, at least prior to covid- . in essence, scientific globalism (as well as scientific nationalism) will endure as long as there is international collaboration in research, but the concepts are not interchangeable. global research participation is uneven and will always be at least somewhat shaped by national, multinational, and commercial interests. based on their social network analysis of global science, wagner and leydesdorff ( ) found that the global research network is self-organizing, without any central authority directing science and that national interests alone do not explain the extent of international linkages being created. that is, international collaboration networks are "growing independently of and orthogonally to national systems of science. the national systems continue to operate, but they are affected by the emerging global system" (p. ). this emerging global system is largely shaped by highly resourced nations. as peters ( ) observes, global science has long been subject to western control and bias, making international collaborations an imperfect proxy to global science. center-periphery country relationships are thus not always equal or mutually beneficial. international collaborations may reflect competitive national agendas or private interests and thus are not always intended towards the global public good. likewise, domestically produced research has also benefitted global society. the scientific global system, thus, has to be understood in relation to the importance that international ties play in enabling resources to be pooled and maximally exploited in ways that national research might be limited. smaller national scientific systems tend not to allow for high levels of specialization, making international collaboration especially vital (iglic, doreian, kronegger, & ferligoj, ; engels & ruschenburg, ; luukkonen et al. ). collaboration benefits not only low-income but also high-income countries as well but for different reasons, often having more to do with accessing specimens and data, extending global networks and audiences and research labor (leather, butterfield, peachey, silverman, & sheriff, ; cantwell & lee, ) . thus, beyond the focus on public global goods, scientific globalism reflects cross-border networks towards the goal of extending science, regardless of the social impact. international collaboration becomes a key means of achieving this goal. as an extension of scientific globalism, this study also considered open access. while knowledge production, in the form of research publications, is synonymous to scientific progress, accessibility to knowledge tends to be either assumed or disregarded. lower-income countries in particular are on the scientific "periphery" as they have long been disadvantaged by the unaffordability of journal subscriptions, which chan, kirsop, and arunachalam ( ) refer to as "information famine" (para. ). their ability to fully participate and contribute to global science becomes hindered (salager-meyer, ; chan, kirsop, and arunachalam, ) as past research has well established that a country's economic status is highly linked to its research productivity (king; ; jaffe, ter horst, gunn, zambrano, & molina, ) . from a global view, open-access allows for unrestricted access to scientific knowledge in the form of free online publications. open-access is not, however, without its limitations, including authorpay models, sometimes with no quality control, giving rise to predatory publishers (haug, ) . despite such challenges, open access allows researchers in less represented parts of the world to participate in the international research community while also allowing researchers in higher-income countries to also access local research and knowledge towards the resolution of the world's major problems (chan et al., ) . in fact, kieńć ( ) found that researchers from peripheral countries preferred open-access journals for promotional as well as citation advantages. the average researcher from these countries published % of their works in gold open-access journals, referring to an academic publication made freely available on a publisher's website without embargo, compared with % for the average researcher from the socalled core countries. during the current pandemic, numerous efforts to make covid- research accessible went into effect. in the beginning of , publishers, funders, and scientific societies signed a statement committing to data sharing and making findings immediately available to address the health crisis (wellcome, a) . over major academic publishers, including elsevier, wiley, springer, taylor and francis, and others unlocked tens of thousands articles (change, ; wellcome, a). artificial-intelligence tools to extract specific findings and bridge fields to navigate the fast-rising number of research papers were being developed as well (hutson, ) . publications in machine-readable formats and open licenses were especially key for this purpose (wellcome b) . this study sought to understand patterns of scientific globalism during a global crisis. while previous research suggests international collaboration is heightened, the current pandemic is occurring in the midst of an unusually tense geopolitical climate. there is also little known about how the extent of scientific globalism compares with the pre-crisis. thus, this study specifically focused on internationally authored scientific articles on covid- published at the beginning of january to early may to observe the extent of international collaboration and open access compared with (a) the past five pre-pandemic years and (b) non-covid- articles published during the same time period. past research has also observed some discrepancies by countries, while this study also sought to more systematically understand the relative effects of past patterns, covid- cases, and country wealth on scientific globalism across all countries that produced covid- research. the study was based on bibliometrics, utilizing statistical methods to analyze publications. this approach is commonly used to systematically examine research collaborations given the limitations of surveys and observations (subramayan, ) . the data for this scientometric study was collected using scopus (elsevier, ) , the world's largest comprehensive abstract and citation indexing database of primary documents from serial publications (journal articles, conference papers, reviews, editorials, books, book chapters, letters, errata, notes, short surveys, and business articles), which also includes open-access publications. scopus and web of science (wos) are the most commonly used databases in bibliometric studies. while neither can perfectly account for all research studies and both tend to over-represent english language journals as well as under-represent social sciences, arts, and humanities fields, scopus offers a higher number of journals, including publications outside the usa and greater field coverage than wos (mongeon & paul-hus, ; tabacaru, ) . a strict criterion of "articles," which scopus defines as "original research or opinion," most commonly found in peer-reviewed journals (elsevier, , p. ) served as the primary data for this study. three datasets were created and compared: (a) all article publications from january , , to december , ( - ), (b) non-covid- articles from january , , to may , (non-covid- ), and (c) covid- article publications from january , , to may , (covid- ). to produce these data sets, country -specific publication information was downloaded from scopus that included a country's total number of publications and a country's total number of domestic-only publications. the latter measure was obtained for each country by excluding all other countries in the scopus advanced search window. a country's total number of international collaborations was calculated by subtracting its total number of publications by its total number of domestic-only publications. finally, articles were assigned to countries on a full count basis, so that an article was counted towards each co-authoring country's total number of publications. the latter two datasets were based on the inclusion (covid- ) or exclusion (non-covid- ) of articles containing at least one of the following phrases, "covid- ," " -ncov, "sars-cov- ," and "novel coronavirus," in the title, abstract, and keywords fields during this time frame. january , , was selected as the starting point that soon led up to covid- being declared as a pandemic by the world health organization (who). based on the who ( a) timeline, on december , , a novel coronavirus was identified, as the wuhan municipal health commission in china reported a cluster of pneumonia cases in wuhan, hubei province. since that date, covid- quickly accelerated and was declared a pandemic on march , . at the time of writing this study, june , scientists are still learning about the sars-cov virus and how to best contain it. given the particular interest in global science research during a global crisis, most of the analyses were based on the covid- bibliometric data, which was based on countries that produced at least one covid- article during january , , to may , . next, two of the three datasets, - articles and covid- articles, were merged together, along with information on each of the countries' economy size and covid- cases. gross domestic product (gdp), the most commonly used variable to measure the extent of economic activity with a country, was derived from the most recent available data from the world bank ( ). as it pertains to this study, a country's gdp represents a country's capacity to conduct scientific research. while research and development (r&d) expenditures would have provided a clearer measurement of scientific capacity, of the countries were missing this data in both the world bank and unesco databases. to have conducted the analyses with the limited r&d data would have eliminated these lower-income countries in the regression findings. the number of covid- cases per country as of may was obtained from the world health organization (who) reports ( b) and scaled by population. to examine scientific globalism, two areas were observed: international collaboration and open-access. international collaboration was measured by international co-authorship, a commonly accepted scientific indicator to study research collaboration patterns (laudel, ; lundberg, tomson, lundkvist, skår, & brommels, ) . for this study, international collaborations were identified as co-authored article publications that included authors from more than one country (lee & haupt, ) . open access was measured as a filter option within scopus, to identify that the article publication does not have a paywall or any other access restriction, regardless of the level of journal access (scopus, ) . scopus identifies open access on an article by article basis as opposed to journal by journal or publisher by publisher. for the - , non-covid , and covid- data, the percentage of international collaborations and the percentage of open access articles were calculated and compared. chi-square tests of independence were performed to compare the percentage of international collaborations as well as the percentage of open access for the following: (a) - articles with the covid- articles and (b) for the non-covid- articles with the covid- articles. the same tests and comparisons were also applied to each of the top covid- research-producing countries. although comparisons were not made between each of the countries, the results provided insight into increases or decreases in rates of collaboration for each of the top-producing countries. while these rates of collaboration by country demonstrate how global patterns are not uniform, they do not indicate country-level characteristics to explain such differences. thus, we used a generalized linear model (glim) to systematically understand the factors that contribute to the extent of a country's proportion of (a) international collaboration on covid- and (b) open access on covid- . the events variable was (a) the number of internationally authored covid- articles or (b) the number of open-access covid- articles. the trials' variable was the total number of covid- articles. the predictors included - international collaboration/open access, the number of covid- cases, and gdp. the - variables served as control measures to examine the unique effects of the global crisis. the number of covid- cases was used to examine the extent to which each country's covid- situation influenced the extent of global participation. finally, gdp was included to examine the extent to which country wealth played a role given that past research has demonstrated a positive relationship between gdp and scientific publication output statistics (samimi & roshan, ) . in total, articles were published on covid- from january , , to may , . as shown in fig. , countries published at least article on the topic and represented all major geographic regions of the world. the top five producers were china ( ), the usa ( ), italy ( ), the uk ( ), and india ( ). comparing covid- and pre-covid- time periods the extent of international collaboration on covid- is higher than recent years, prior to the global crisis, and higher than non-covid- research. table displays the extent of international collaboration during (a) - , (b) non-covid- , and (c) covid- . among the covid- publications, about a third were based on international collaborations. as shown in the last column, the percentage of international collaboration on covid- publications is higher compared with the past years ( . %; . %, respectively). according to the chi-square test, there is a significant difference on the percentage of international collaborations when comparing these two data points (χ ( ) = . , p < . ). furthermore, the percentage of international collaborations on covid- publications was also significantly higher than non-covid- publications in the same time frame (χ ( ) = . , p < . ). in sum, international collaborations accounted for . % more of the covid- publications than the non-covid- publications and % more than all international publications prior to the pandemic. while international collaboration on covid- has generally increased as measured by the percentage of international collaborations compared with the recent pre-pandemic period, there are varying patterns by countries. among the top top countries producing articles on covid- , the extent of international collaboration before and on covid- since varies (see appendix table ). among the countries with significant differences between these periods, most countries ( out of ) demonstrate a higher proportion of international collaboration on covid- compared with past international collaboration. on the other hand, china, the lead producer of covid- research, had a significantly lower percentage ( %) of international collaborations fig. map of the countries that published articles on covid- from january , , to may , on covid- research than on research over the past years ( . %). the usa, the second lead producer, shows slightly upward activity (from . to . %), but this difference is not significant. among the biggest significant differences from the proportion of international collaborations prior to and on covid- are singapore (− . %), austria ( . %), belgium ( . %), japan ( . %), and canada ( . %). in comparing the percent of international collaborations for covid- and non-covid- publications, similar to the comparison with the - data, the majority of countries ( out of ) that had significantly different rates of collaboration had a higher percent of international collaborations on covid- publications compared with non-covid- publications. additionally, china's percentage of international collaborations on covid- publications was significantly lower (− . %; p < . ) than their non-covid- publications, and there remained no significant difference for the usa between the two time periods. lastly, the countries with the largest significant differences changed slightly. singapore had the highest change of − . % followed by france (− . %), austria ( . %), canada ( . %), and the netherlands ( . %). results further indicate that among these countries combined covid- articles, three-fourths are open access (see table ). this percentage is significantly higher than the percentages for the - articles and the non-covid- articles. for - article publications, only . % of publications were labeled as open access compared with . % of publications on covid- (χ ( ) = . , p < . ). likewise, only . % of non-covid- publications were labeled as open access, which is . % less publications than for covid- publications (χ ( ) = . , p < . ). for each of the top covid- research-producing countries, there was a noticeably higher proportion of open-access articles on covid- than during the past years and on non-covid- publications during the same period, with all differences being significant except for one (see appendix table ). whereas the percentage of open-access articles on research during - ranged from . to . % and on non-covid- research from . to . %, the percentage of open-access articles on covid- research ranged from . to . %. among the biggest jumps in comparing the - publications with covid- publications were in east asia; the top difference was hong kong ( . %), followed by singapore ( . %), south korea ( . %), and taiwan ( . %), all noticeably higher than china ( . %). while the largest increases when comparing non-covid- publications with covid- publications were in east asia and north america, hong kong ( . %) and singapore ( . %) remained at the top of countries with the largest difference followed by canada ( . %), the united states ( . %), and south korea ( . %). the glim results indicate the relative country-level contributions of past patterns, covid- cases, and gdp on covid- international collaborations and covid- open-access articles. as shown in table , each of the independent variables significantly predicts covid- international collaboration. past international collaborations and the number of covid- cases increase the likelihood of international collaboration on a covid- publication [exp(b) = . ; . , respectively], whereas gdp has a negative impact [exp(b) = . ]. in essence, countries with a strong record of international collaboration have continued to collaborate on covid- , but this likelihood is also influenced by the extent of their cases and gdp. whereas the number of cases increases the probability, their relative wealth would lead them to collaborate less, likely given their already large domestic output. in examination of the countries with the highest international research collaboration on covid- (austria, belgium, netherlands, hong kong, and saudi arabia), their gdp is a much smaller fraction compared with the larger economies of the usa, china, japan, germany, and the uk. when examining the effects of producing covid- articles as open access, the number of covid- cases again had a positive effect [exp(b) = . ] and gdp had a slightly negative effect [exp(b) = . ] (see table ). pre-covid- open-access articles did not have a significant influence. based on the countries that produced covid- articles, the extent to which these countries were hit by covid- mattered, but the relatively higher wealth of top gdp countries suggest a greater likelihood of publishing covid- research as restricted articles. this study sought to explore the nature of scientific globalism, as measured by the extent of scientific research collaboration and open-access articles on covid- and how they compare to non-covid- research (a) during the pandemic as well as (b) the past five pre-pandemic years. the study also examined the degree to which the number of covid- cases and economic wealth influences these patterns. although geopolitical tensions are ongoing, the extent of scientific globalism is generally higher in times of a global crisis and on the global crisis issue. the findings support past research that research productivity increases during public health emergencies (zhang, zhao, sun, et al., ) and that most covid- research is produced within countries, with china leading in covid- publications (hook & porter, ; hossain, ; chahrour, assi, bejjani, et al., ) . further analyses showed variations by countries and that while a country's prior patterns mattered in collaborating on internationally authored publications, the extent to which the country was affected by covid- increases the likelihood they would participate in international collaboration. however, in observing open-access patterns, the country's past pattern of publishing in open access prior to the pandemic had no significant bearing on publishing in open access during this particular global crisis. similar to international collaborations, what did make a clear difference in open access was the extent to which a county was affected by covid- . the same analyses also showed that the higher the gdp, the lower the odds of international collaboration and open-access publishing during a global crisis. in other words countries with a higher gdp, already producing a high domestic output, which also tend to be in restricted journals, may not engage in as much scientific globalism (via international collaboration and open-access publishing) compared with their lower gdp counterparts. countries with a high gdp have resource concentrations (i.e., funds, equipment, staffing), which are necessary to solve problems and which makes them less dependent on outside collaborators. as demonstrated by china and the usa, there is an especially high volume and global proportion of domestic output. the two countries also have high covid- caseloads, indicating a lesser need to obtain data or conduct field research abroad, which also help to explain their relatively little change in international collaboration patterns. for countries with a lower gdp and predictably smaller-sized science systems, there is a greater dependency on international collaboration, particularly to mobilize resources (iglic, doreian, kronegger, & ferligoj, ) and on open-access outlets (kieńć; . previous research identified a positive relationship between resource concentration and researchers' collaboration propensity; scarce resources push researchers to work together (birnholtz, ) . another study found that a coauthor having special data or equipment motivated % of article collaborations (melin, ) . in the case of covid- , limited available specimens during the early part of and the costs to generate and analyze the data, combined with urgency of addressing the global health crisis helps to explain the significantly increased rate of international covid- research output worldwide and why the countries with higher cases were generally more inclined or sought after to collaborate. as a past study found that researchers in small countries and working in very specialized fields are more likely to collaborate internationally (iglic, doreian, kronegger, & ferligoj, ) , the unprecedented nature and novelty of covid- may further explain the overall international collaboration increase among lower gdp countries and those with higher covid- cases. in addition to these findings, the study more broadly extends how scientific globalism is understood and opens up several future lines of research. as this study argued upfront, scientific globalism reflects cross-border networks towards the goal of extending science, regardless of the impact. international collaboration becomes a key means of achieving this goal. additionally, scientific globalism should also require knowledge dissemination as a requisite to further scientific advancement given that scholarly publications are not universally accessible. beyond the covid- cases and country gdp results, the globally coordinated effort to make covid- research freely and widely available likely best explains the dramatic increase in open-access publications. while there are certainly other variables that might further operationalize scientific globalism, the data for this study was limited by the small dataset given the four-month covid- time period. as the new coronavirus continues to spread and research on it continues, further research based on a larger dataset could consider a broader array of country-level factors, including country-level networks, the availability of open-source data, as well as the influence of multinational organizations to promote global agendas. the next step for the current study will be a more focused examination among the leading international collaborators, the usa and china, as geopolitical tensions are increasing. despite the limitations in capturing all possible dimensions of scientific globalism, this study already demonstrates that there are varying levels of country participation during a global crisis, at least mediated by the country's wealth and the impact of the global crisis. obviously domestically produced research that has restricted access can still be a global good. the extent of global participation, however, is limited when research is conducted within a single country. thus as our framework suggests, scientific pursuits are not either/or as both scientific globalism and scientific nationalism can coexist. what remains unknown is whether or how much more international collaboration, in particular, would have taken place if there was less geopolitical rivalry. an important question for future research is whether one impedes the other, particularly during a global crisis, as finding scientific solutions remains urgent. the fourth age of research when do researchers collaborate? toward a model of collaboration propensity the geopolitics of academic science unseen workers in the academic factory: perceptions of neo-racism among international postdocs in the us and uk a bibliometric analysis of covid- research activity: a call for increased output open access archiving: the fast track to building research capacity in developing countries humanity wins: our fight to unlock , covid- articles for the world new approach to the visualization of international scientific collaboration bibliometric 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from to sharing research data and findings relevant to the novel coronavirus (covid- ) outbreak publishers make coronavirus (covid- ) content freely available and reusable gdp (current us$): all countries and economies. the world bank who timeline -covid- how scientific research reacts to international public health emergencies: a global analysis of response patterns publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations acknowledgements the authors would like to acknowledge mark bergstrom, university information technology support at the university of arizona for his assistance with the statistical analyses and the anonymous reviewers for their helpful comments. key: cord- -ungzlp b authors: mimkes, j.; janssen, r. title: test-adjusted results of mortality for covid- in germany, usa, uk date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: ungzlp b in a disease, where all infected persons show symptoms, it is reasonable to calculate mortality by case to fatality rate cfr. deaths follow infections by a certain time lag. however, in the covid- pandemic many infectious patients show no or hardly any symptoms. the reported infections and deaths do not run parallel, but diverge with the volume of tests. our investigations for germany, usa and uk indicate that deaths do not follow the number of infections, but the positive rate of tests, multiplied by a constant factor f and shifted by about two weeks. these test adjusted results of mortality allow for the estimation of the number of deaths of covid- about two weeks ahead, even in a sharply rising state of the pandemic. this gives medical authorities two weeks of time to plan for resources. mortality is the most important indicator of a pandemic and scientists all over the world are estimating mortality from covid- . a good introduction has been given in a scientific brief by the world health organization who [ ] . in a pandemic, two time series are generally reported: the time series of daily infections (i k), and the time series for daily deaths (d k), as provided by the johns-hopkins-university (jhu) in usa and the robert-koch-institute (rki) in germany. a wide range of values has been reported in the literature for the cfr, varying with time and location [ ] [ ] [ ] . in this paper we concentrate on mortality in germany, usa, and uk. the series of daily registered new infections (i k) and the series of daily deaths (d k) are given in figs. and . is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november , . ; https://doi.org/ . / . . . doi: medrxiv preprint normally, in mortality calculations the series of daily deaths (d k + l) follows the series of daily infections (i k) by a time lag (l) and is smaller by the case fatality ratio cfr, d k + l = cfr * i k. ( ) in fig. the maximum of deaths ( ) follows the maximum of infected ( ) in fig. about two weeks (l = ) later, and is smaller by the factor cfr = / = . . this leads to d k + = . * i k ( ) eq. ( ) has been plotted in fig. . data and calculation of cfr mortality in fig. agree for the first covid- wave from march to june, as would be expected. but from july on the calculations of daily deaths drifts away to higher numbers, whereas the actual death data stay close to zero between july and september, indicating only rising numbers in october. as the deaths in fig. do not follow is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november , . ; https://doi.org/ . / . . . doi: medrxiv preprint the infections, the cfr is not any more constant, but is time variant, cfr k. a similar observation has been made in many countries, as shown for usa and uk, below. in a recent paper the authors [ , ] proposed to explain the disagreement between calculations (eq. ) and mortal data in fig. by considering a change in test volume. indeed, the test volume in fig. in germany has been increased from july until september by a factor of . for a more detailed analysis, we divide the infection numbers i k by the test volume t k. then we scale the result by a mortality factor f = to obtain an optimal approximation to the deaths series of the pandemic in fig. . and we obtain a test depending mortality cfr k = / t k. . cc-by-nc-nd . international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november , . ; https://doi.org/ . / . . . doi: medrxiv preprint a test adjusted approach to mortality in order to explain the test depending mortality cfr k in eq. and fig. , we propose a test adjusted approach to mortality: in diseases where many infectious patients show no or hardly any symptoms, we do not know the total number of infections. but we can determine the daily number of infections i k, t by the positive rate p k of daily infections of a certain daily test volume t k, i k, t = p k * t k. in eq. we may now replace the ratio i k / t k by the positive rate of tests p k of eq. , in eq. and fig. the number of daily deaths d k + l follows the daily positive rate p k, and not the daily number of infections i k as in eq. . the daily numbers of infections and deaths are now represented in eq. and eq. by the daily positive rate p k and the daily test volume t k. the daily test volume t k and the daily positive rate of infections p k are given by the robert-koch-institute, in figs. and . the time lag l between infection and death is about days in germany. the relationship between the number of daily deaths and the number of daily infected in eq. ( ) is invalid, if the daily test volume is not constant. high numbers of infected do not necessarily mean high number of deaths. this statement does not diminish the danger of covid- , but puts it on a scientific basis. accordingly, the daily positive rate of tests should be published in addition to the presently dominating infection numbers, which are used for other purposes like tracking. the daily positive ratio of tests leads to a realistic estimation of the number of deaths two weeks in advance in eqs. or and in fig. . this gives medical authorities two weeks of time to plan ahead for the numbers of severely ill patients, which are closely related the number of deaths. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november , . ; https://doi.org/ . / . . . doi: medrxiv preprint jhu data for usa for usa, the johns-hopkins-university (jhu) provides the series of daily registered new infections (i k) and the series of daily deaths (d k) in figs. and . the daily test series is shown in fig. . is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november , . ; https://doi.org/ . / . . . doi: medrxiv preprint the mortality factor f calculates the number of deaths of a country at a certain positive rate of testing (p k), accordingly, the mortality factor will depend on the population of a country if the modalities of testing are comparable. since the population in usa ( million) is larger than the population in germany ( million) by a factor of about , we would estimate f usa = * f ger = , . this estimation is of the same order as the result above, and supports the test adjusted approach to mortality of covid- in germany and other countries. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november , . ; https://doi.org/ . / . . . doi: medrxiv preprint in uk, the test adjusted infection data (i k / t k) also lead to a realistic prediction of the death series of the covid- pandemic ( fig. ) . the time lag is about days and the mortality factor f uk about , , deviating slightly from those in usa and germany, if related to the population. fig. . daily deaths data by jhu (bars) in uk and calculation of d k + according to eq. (dashed line) and eq. (solid line). test data since . . [ ] . in a pandemic with symptomatic and asymptomatic courses of the disease like in covid- , the pure number of infections is not significant for the danger of the pandemic. the indicator for the number of deaths is the positive rate (p k) of tests. it is a scale for the number of deaths, and in addition, it is known about two weeks earlier. estimating mortality from covid- dianjun sun, real-time estimation and prediction of mortality caused by covid- with patient information-based algorithm systematic review modeling numbers of infected and deceased -adjustment by test volume, webinar "covid- forecast and prediction on the numbers of infected and deceased in the second corona wave covid- ) in the uk key: cord- -eqwxkwqa authors: kumar, roshan; verma, helianthous; singhvi, nirjara; sood, utkarsh; gupta, vipin; singh, mona; kumari, rashmi; hira, princy; nagar, shekhar; talwar, chandni; nayyar, namita; anand, shailly; rawat, charu dogra; verma, mansi; negi, ram krishan; singh, yogendra; lal, rup title: comparative genomic analysis of rapidly evolving sars-cov- viruses reveal mosaic pattern of phylogeographical distribution date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: eqwxkwqa the coronavirus disease- (covid- ) that started in wuhan, china in december has spread worldwide emerging as a global pandemic. the severe respiratory pneumonia caused by the novel sars-cov- has so far claimed more than , lives and has impacted human lives worldwide. however, as the novel sars-cov- displays high transmission rates, their underlying genomic severity is required to be fully understood. we studied the complete genomes of sars-cov- strains from different geographical regions worldwide to uncover the pattern of the spread of the virus. we show that there is no direct transmission pattern of the virus among neighboring countries suggesting that the outbreak is a result of travel of infected humans to different countries. we revealed unique single nucleotide polymorphisms (snps) in nsp - (orf b polyprotein) and s-protein within viral isolates from the usa. these viral proteins are involved in rna replication, indicating highly evolved viral strains circulating in the population of usa than other countries. furthermore, we found an amino acid addition in nsp (mrna cap- methyltransferase) of the usa isolate (mt ) leading to shift in amino acid frame from position onwards. through the construction of sars-cov- -human interactome, we further revealed that multiple host proteins (phb, ppp ca, tgf-β, socs , stat , jak / , smad , bcl , cav & specc ) are manipulated by the viral proteins (nsp , pl-pro, n-protein, orf a, m-s-orf a complex, nsp -nsp -nsp -rdrp complex) for mediating host immune evasion. thus, the replicative machinery of sars-cov- is fast evolving to evade host challenges which need to be considered for developing effective treatment strategies. downloaded on march , from ncbi database and based on quality assessment two genomes with multiple ns were removed from the study. further the genomes were annotated using prokka [ ] . a manually annotated reference database was generated using the genbank file of severe acute respiratory syndrome coronavirus isolate-sars-cov- /sh /human/ /chn (accession number: mt ) and open reading frames (orfs) were predicted against the formatted database using prokka (-gcode ) [ ] . further the gc content information was generated using quast standalone tool [ ] . to determine the evolutionary pressure on viral proteins, dn/ds values were calculated for orfs of all strains. the orthologous gene clusters were aligned using muscle v . team, ) . to infer the phylogeny, the core gene alignment was generated using mafft [ ] present within the roary package [ ] . further, the phylogeny was inferred using the maximum likelihood method based and tamura-nei model [ ] in megax [ ] and visualized in interactive tree of life (itol) [ ] and grapetree [ ] . to determine the single nucleotide polymorphism (snp), whole-genome alignments were made using libmuscle aligner. for this, we used annotated genbank of sars-cov- /sh /human/ /chn (accession no. mt ) as the reference in the parsnp tool of harvest suite [ ] . as only genomes within a specified mumi distance threshold are recruited, we used option -c to force include all the strains. for output, it produced a core-genome alignment, variant calls and a phylogeny based on single nucleotide polymorphisms. the snps were further visualized in gingr, a dynamic visual platform [ ] . further, the tree was visualized in interactive tree of life (itol) [ ] . sars-cov- protein annotation and host-pathogenic interactions updated in any protein database, we first annotated the genes using blastp tool [ ] . the similarity searches were performed against sars-cov isolate tor having accession no. ay selected from ncbi at default parameters. the annotated sars-cov- proteins were mapped against virusite [ ] and interaction databases such as virus.string v . [ ] and intact [ ] for predicting their interaction against host proteins. these proteins were either the direct targets of hcov proteins or were involved in critical pathways of hcov infection identified by multiple experimental sources. to build a comprehensive list of human ppis, we assembled data from a total of bioinformatics and systems biology databases with five types of experimental evidence: (i) binary ppis tested by high-throughput yeast two-hybrid (y h) systems; (ii) binary, physical ppis from protein d structures; (iii) kinase-substrate interactions by literature-derived low-throughput or high-throughput experiments; (iv) signaling network by literature-derived low-throughput experiments; and (v) literature-curated ppis identified by affinity purification followed by mass spectrometry (ap-ms), y h, or by literature-derived low [ , ] . filtered proteins (confidence value: . ) were mapped to their entrez id [ ] based on the ncbi database used for interactome analysis. hpi were stimulated using cytoscape v. . . [ ] . next, functional studies were performed using the kyoto encyclopedia of genes and genomes (kegg) [ , ] and gene ontology (go) enrichment analyses using uniprot database [ ] to evaluate the biological relevance and functional pathways of the hcov-associated proteins. all functional analyses were performed using string enrichment and stringify, plugin of cytoscape v. . . [ ] . network analysis was performed by tool networkanalyzer, plugin of cytoscape with the orthogonal layout. general genomic attributes of sars-cov- in this study, we analyzed a total of sars-cov- strains (available on march , ) isolated between december -march from different countries namely usa (n= ), china (n= ), japan (n= ), india (n= ), taiwan (n= ) and one each from australia, brazil, oronasopharynges or lungs, while two of them were isolated from faeces suggesting both respiratory and gastrointestinal connection of sars-cov- (table ) . no information of the source of isolation of the remaining isolates is available. the average genome size and gc content were found to be ± . bp and . ± . %, respectively. all these isolates were found to harbor open reading frames coding for orf a ( bp) and orf b ( bp) polyproteins, surface glycoprotein or s-protein ( bp), orf a protein ( bp our analysis revealed that strains of human infecting sars-cov- are novel and highly identical (> . %). a recent study established the closest neighbor of sars-cov- as sarsr- cov-ratg , a bat coronavirus [ ] . as covid transits from epidemic to pandemic due to extremely contagious nature of the sars-cov- , it was interesting to draw the relation between strains and their geographical locations. in this study, we employed two methods to delineate phylogenomic relatedness of the isolates: core genome ( figure a & c) and single nucleotide polymorphisms (snps) ( figure b ). phylogenies obtained were annotated with country of isolation of each strain ( figure a & b). the phylogenetic clustering was found majorly concordant by both core-genome ( figure a ) and snp based methods ( figure b) . the strains formed a monophyletic clade, in which mt . (south korea) and mt . (sweden) were most diverged. focusing on the edge-connection between the neighboring countries from where the transmission is more likely to occur, we noted a strain from taiwan (mt ) closely clustered with another from china (mt . ). with the exception of these two strains, we did not find any connection between strains of neighboring countries. thus, most strains belonging to the same country clustered distantly from each other and showed relatedness with strains isolated from distant geographical locations ( figure a & b). for instance, a sars-cov- strain isolated from nepal (mt ) clustered with a strain from usa (mt ). also, strains from wuhan (lr and lr ), where the virus was originated, showed highest identity with usa as well as china strains; strains from india, mt and mt clustered closely with china and usa strains, respectively ( figure a & b) . similarly, australian strain (mt ) showed close clustering with usa strain ( figure a & b) and one strain from taiwan (mt ) clustered nearly with chinese isolates ( figure b ). isolates from italy (mt ) and brazil (mt ) clustered with different usa strains ( figure a & b) . notably, isolates from same country or geographical location formed a mosaic pattern of phylogenetic placements of countries' isolates. for viral transmission, contact between the individuals is also an important factor, supposedly due to which the spread of identical strains across the border of neighboring countries is more likely. but we obtained a pattern where indian strains showed highest similarity with usa and china strains, australian strains with usa strains, italy and brazilian strains with strains isolated from usa among others. this depicts the viral spread across different communities. however, as genomes of sars-cov- were available mostly from usa and china, sampling biases is evident in analyzed dataset as available on ncbi. thus, it is plausible for strains from other countries to show most similarity with strains from these two countries. in the near future as more and more genome sequences will become available from different geographical locations; more accurate patterns of their relatedness across the globe will become available snps in all predicted orfs in each genome were analyzed using sars-cov- /sh /human/ /chn as a reference. snps were determined using maximum unique matches between the genomes of coronavirus, we observed that the strains isolated from usa although the primary mode of infection is human to human transmission through close contact, which occurs via spraying of nasal droplets from the infected person, yet the primary site of infection and pathogenesis of sars-cov- is still not clear and under investigation. to explore the role of sars-cov- proteins in host immune evasion, the sarscov- proteins were mapped over host proteome database ( figure b & table ). we identified a total of proteins from host proteome forming close association with viral proteins present in orfs of sars-cov- ( figure c ). the network was trimmed in cytoscape v . . where only interacting proteins were selected. only viral proteins were found to interact with host proteins ( figure a ). detailed analysis of interactome highlighted host proteins in direct association with viral proteins. further, the network was analyzed for identification of regulatory hubs based on degree analysis. we identified mitogen activated protein kinase similarly, the viral protein papain-like proteinase (pl-pro) which has deubiquitinase and deisgylating activity is responsible for cleaving viral polyprotein into mature proteins which are essential for viral replication [ ] . our study showed that pl-pro directly interacts with ppp ca which is a protein phosphatase that associates with over regulatory host proteins to form highly specific holoenzymes. pl-pro is also found to interact with tgfβ which is a beta transforming growth factor and promotes t-helper cells (th ) agreement with our prediction where we found il as an interacting partner. our study also showed jak / as an interacting partner which is known for ifnγ signaling. it is well known that tgfβ along with il and stat promotes th differentiation by inhibiting socs [ ]. th is a source of il , which is commonly found in serum samples of covid- patients [ , ] . hence, our interactome is supported from these findings where we found socs , stat , jak / as an interacting partner [ ] . the results suggested that proinflammatory cytokine storm is one of the reasons for sars-cov- mediated immunopathogenesis. in the next cycle of physical events the viral protein nc (nucleoprotein), which is a major structural part of sarv family associates with the genomic rna to form a flexible, helical nucleocapsid. interaction of this protein with smad leads to inhibition of apoptosis of sars- cov infected lung cells [ ] , which is a successful strategy of immune evasion by the virus. membranes. cav has been reported to be involved in viral replication, persistence, and the potential role in pathogenesis in hiv infection also [ ] . thus, orf a interactions will upregulate viral replication thus playing a very crucial role in pathogenesis. multiple structural proteins were observed to target the specc proteins and linked with cytokinesis and spindle formations during division. thus, major viral assembly also targets the proteins linked with immunity and cell division. taken together, we estimated that sars-cov- manipulate multiple host proteins for its survival while, their interaction is also a reason for immunopathogenesis. as covid- continues to impact virtually all human lives worldwide due to its extremely contagious nature, it has spiked the interest of scientific community all over the world to understand better the pathogenesis of the novel sars-cov- . in this study, the analysis was performed on the genomes of the novel sars-cov- isolates recently reported from different countries to understand viral pathogenesis. with the limited data available so far, we observed no direct transmission pattern of the novel sars-cov- in the neighboring countries through our analyses of the phylogenomic relatedness of geographical isolates. the isolates from same locations were phylogenetically distant, for instance, isolates from the usa and china. thus, there appears to be a mosaic pattern of transmission indicative of the result of infected human travel across different countries. as covid- transited from epidemic to pandemic within a short time, it does not look surprising from the genome structures of the viral isolates. the genomes of six isolates, specifically from the usa, were found to harbor unique amino acid snps and showed amino acid substitutions in orf b protein and s-protein, while one of them also harbored an amino-acid addition. this is suggestive of the severity of the mutating viral genomes within the population of the usa. these proteins are directly involved in the formation of viral replication-transcription complexes (rtc). therefore, we argue that the novel sars-cov- has fast evolving replicative machinery and that it is urgent to consider these mutants to develop strategies for covid- treatment. the orf ab polyprotein protein and s-protein were also found to have dn/ds values approaching and thus might confer a selective advantage to evade host responsive mechanisms. the construction of sars-cov- - human interactome revealed that its pathogenicity is mediated by a surge in pro-inflammatory cytokine. it is predicted that major immune-pathogenicity mechanism by sars-cov- includes the host cell environment alteration by disintegration by signal transduction pathways and immunity evasion by several protection mechanisms. the mode of entry of this virus by s-proteins inside the host cell is still unclear but it might be similar to sars cov- like viruses. lastly, we believe as more data accumulate for covid- the evolutionary pattern will become much clear. isolates. highly similar genomes of coronaviruses were taken as input by parsnp. whole- genome alignments were made using libmuscle aligner using the annotated genome of mt strain as reference. parsnp identifies the maximal unique matches (mums) among the query genomes provided in a single directory. as only genomes within a specified mumi distance threshold are recruited, option -c to force include all the strains was used. the output phylogeny based on single nucleotide polymorphisms was obtained following variant calling on core-genome alignment. c) the minimum spanning tree generated using maximum likelihood method and tamura-nei model showing the genetic relationships of sars-cov- isolates with their geographical distribution. table : general genomic attributes of sars-cov- strains. structural and functional basis of sars-cov- entry by using human ace structure, function, and antigenicity of the sars-cov- spike glycoprotein genome composition and divergence of the novel coronavirus ( -ncov) originating in china sars a/orf a sars b/orf b sars a/orf sars b/orf key: cord- -yowyy nr authors: balzano, francesca; garroni, giuseppe; cruciani, sara; bellu, emanuela; dei giudici, silvia; oggiano, annalisa; capobianco, giampiero; dessole, salvatore; ventura, carlo; maioli, margherita title: behavioral changes in stem-cell potency by hepg -exhausted medium date: - - journal: cells doi: . /cells sha: doc_id: cord_uid: yowyy nr wharton jelly mesenchymal stem cells (wj-mscs) are able to differentiate into different cell lineages upon stimulation. this ability is closely related to the perfect balance between the pluripotency-related genes, which control stem-cell proliferation, and genes able to orchestrate the appearance of a specific phenotype. here we studied the expression of stemness-related genes, epigenetic regulators (dnmt , sirt ), mirnas (mir- , mir- , and mir- ) related to stemness, exosomes, the cell-cycle regulators p (waf /cip ) and p , and the senescence-associated genes (p , p , and htert). cells were cultured in the presence or absence of the human hepatocarcinoma cell line hepg -exhausted medium, to evaluate changes in stemness, differentiation capability, and senescence sensibility. our results showed the overexpression of sirt and reduced levels of p mrna. moreover, we observed a downregulation of dnmt , and a simultaneous overexpression of oct- and c-myc. these findings suggest that wj-mscs are more likely to retain a stem phenotype and sometimes to switch to a highly undifferentiable proliferative-like behavior if treated with medium exhausted by human hepg cell lines. human mesenchymal stem (or stromal) cells are multipotent elements, capable of restoring tissue function after injuries [ ] . in particular, stem cells from wharton jelly (wj-mscs) represent a valuable model of these multipotent cells, and can be easily obtained without ethical issues [ , ] . the study included umbilical cords (n = ) retrieved from healthy full-term women between and years old, recruited according to the following criteria: spontaneous birth, and donors free from drugs, smoking, and diseases. all the experiments were performed twice (in three technical replicates), separately for each of the samples. fresh human umbilical cords (n = ) from both sexes were collected after natural childbirths at the gynecologic and obstetric clinic of the university of sassari. the patients gave written informed consent according to the approval of this study by the ethics committee (ethical clearance n. / , / / , commissione etica cnr). the umbilical cords were collected in phosphate-buffered saline (pbs) supplemented with u/ml penicillin (euroclone, milano, italy), mg/ml streptomycin (euroclone, milano, italy), and mg/ml amphotericin b (gibco life technologies) prior to storage at • c for further wj-msc isolation. tissues were dissected into small pieces and then digested with collagenase type i ( mg/ml) sigma at • c for - h with agitation. after neutralization of the enzyme with % fetal bovine serum (fbs) (life technologies, grand island, ny, usa) and filtering ( µm cell strainer) (euroclone, milano, italy), samples were centrifuged at × g for min and cultured in a basic medium (bm), dulbecco's modified eagle's medium (dmem) (life technologies grand island, ny, usa) supplemented with % fetal bovine serum (fbs) (life technologies, grand island, ny, usa), mm l-glutamine (euroclone, milano, italy), and u/ml penicillin- . mg/ml streptomycin (euroclone, milano, italy), and cultured in t flasks at • c with % co and saturated humidity for - days [ ] . after h of incubation, cultures were washed with pbs and kept in the fresh medium. the culture medium was changed every days. when cells reached - % confluence, they were harvested using . % trypsin edta (euroclone, milano, italy), counted and transferred into new flasks. wj-mscs were immunomagnetically sorted for c/kit using a monoclonal anti-c/kit (cd ) antibody (miltenyi biotech, minneapolis, mn, usa) directly conjugated to microbeads (miltenyi biotech). the wj-mscs used in this study were characterized by flow cytometry as previously described [ ] [ ] [ ] [ ] . hepg cells were cultured as previously described [ ] [ ] [ ] . cells secrete many plasma proteins, such as albumin and fibrinogen, acute-phase proteins, alpha -macroglobulin, alpha -antitrypsin, transferring plasminogen, insulin-like growth factor-binding protein , alpha-fetoprotein, and others [ ] [ ] [ ] . the hepg cells were seeded in a basic medium (bm), dulbecco's eagle's medium (dmem) (life technologies grand island, ny, usa) supplemented with % fetal bovine serum (fbs) (life technologies, grand island, ny, usa), mm l-glutamine (euroclone, italy), and u/ml of penicillin - . mg/ml of streptomycin (euroclone, milan, italy), and cultured in incubator at • c with % co and saturated humidity for days [ ] . when the cells reached - % confluence, the cells , , of waste medium was collected, after which ml of hepg [ ] [ ] [ ] waste medium was filtered and added to cm flasks containing wj-mscs. the wj-mscs were then expanded in a modified base medium (bm), dulbecco's eagle's medium (dmem) (life technologies grand island, ny, usa) integrated with ml of hepg waste medium [ ] [ ] [ ] . the flasks were placed in the culture incubator at • c with % co and saturated humidity for days. after days of incubation, the cultures were collected using . % of edta trypsin (euroclone, milan, italy), counted, and rna was extracted. after treatment, total rna was isolated using trizol®reagent and quantified by measuring the absorbance at / nm (nanodrop , spectrophotometer thermo scientific nd , thermo fisher scientific, waltham, ma, usa). approximately µg of total rna was reverse-transcribed to cdna by superscript®vilo™ cdna synthesis kit (life technologies, grand island, ny, usa). quantitative polymerase chain reactions were performed using a cfx thermal cycler (bio-rad) in triplicate (applied biosystems), incubated under standard qrt-pcr conditions ( • c for min, • c for min, and then cycled at • c for s, - • c for s, and • c for min, for cycles), according to the qrt-pcr protocol specified in the quantitative pcr master mix with power sybr®green. for each reaction, . µm of each primer, and µl cdna generated from µg of the total rna template were mixed in µl volumes and added. target ct values were normalized to hprt [ ] [ ] [ ] , considered as a reference gene, while the gene levels of stem cells were expressed as fold-change ( −∆∆ct) relative to the gene levels observed when stem cells reached % confluence before starting treatment. each experiment included a distilled water control. the qrt-pcr analysis was performed for the following set of genes: nanog, oct- , sox , c-myc, dnmt , p (waf / cip ), p (arf), p (ink a), p , and htert. all primers used (invitrogen) are described in table . exosomes were isolated from wj-msc waste medium using total exosome isolation reagent (invitrogen). the experiments were performed after and days of culture. samples were then cells , , of processed for hsa-mir- a- p, hsa-mir- - p, and hsa-mir- - p extraction and amplification with real-time pcr, as previously described [ ] . the concentration level of mature mirnas was tested by quantitative real-time pcr (qpcr) using taqman ® microrna reverse transcription kit, (life technologies) for the reverse transcription. taqman ® universal master mix ii, life technologies, was used for the pcr according to the manufacturer's instructions; amplification cycles were performed. mirna concentration levels were quantified using the iq biorad instrument, (milan, italy). the u snrna was used for data normalization [ ] . real-time pcr was done in duplicate. the sequences and the identification symbols were retrieved from mirbase and are reported in table . the kruskal-wallis test was applied to compare the groups (male and female) for each target. the statistical analysis was performed with the spss software version . . reverse transcription followed by polymerase chain reaction (rt-pcr) is the most suitable method for the detection and quantification of mrna. it provides high sensitivity, good reproducibility, and a wide range quantification. several mathematical algorithms have been developed to calculate a ratio of expression based on real-time pcr efficiency and the crossing point deviation of an unknown sample against a control. a software tool named rest© (relative expression software) [ ] was used, which compares two or more groups and different reference and target genes. the mathematical model used was based on the correction for exact pcr efficiencies and the mean crossing point deviation between sample group(s) and control group(s). subsequently the expression ratio results of the investigated transcripts were tested for significance using a pairwise fixed reallocation randomization test and plotted using standard error (se) estimation via a complex taylor algorithm. expression variation for each gene was visualized in a box-and-whisker plot. in this study, the relative expression of the mrnas was analyzed using rest software [ ] . the non-parametric bootstrapping test was used to evaluate expression differences between treated and untreated samples. the raw ct values for each mirna and u snrna were checked for normal distribution. the kruskal-wallis test was applied to compare the groups in each target. all the analyses were performed, and graphics generated using spss software version . . a software tool named rest © (relative expression software) [ ] was used to compare two groups, with a maximum of data points in a sample and in a control group, for reference and up to four target genes. the non-parametric bootstrapping test was used to evaluate concentration differences of mirnas between male and female samples. data were analyzed using statistical package for the social sciences version software (spss inc., chicago, il, usa). krustal-wallis rank sum and wilcoxon signed-rank tests were applied to evaluate the distributions of each group variance at different times of observation, assuming p-values < . as statistically significant. wj-mscs before and after treatment with hepg -exhausted medium were compared with untreated cells by statistical analysis. intrasexual analyzes were also cells , , of conducted to compare the results between control cells and cells under study. continuous parametric variables were analyzed using student's unpaired t-test. treated and control cells were seeded at the concentration of , cells/well in -well falcon culture slides (bd falcon, bd biosciences, bedford, ma, usa). once attached, cells were fixed with % paraformaldehyde (sigma aldrich chemie gmbh, münchen, germany) for min at room temperature. after permeabilization by . % triton x- (life technologies, usa)-pbs, cells were washed three times for min each in pbs and incubated with % bovine serum albumin (bsa)- . % triton x- in pbs (life technologies, usa) for min. cells were then exposed overnight at • c to the primary anti-rabbit anti-gapdh antibody (cell signaling technology, danvers, ma, usa) and anti-mouse anti-sirt antibody (cell signaling technology, danvers, ma, usa). finally, cells were washed two times in pbs for min each and stained for min at • c in the dark with the fluorescence-conjugated goat anti-rabbit and anti-mouse igg secondary antibodies (life technologies, carlsbad, ca, usa). nuclei were labeled with µg/ml , -diamidino- -phenylindole (dapi). all microscopy analyses were performed with a confocal microscope (tcs sp , leica, nussloch, germany). cells were cultured for days on well tissue culture plates (bd-falcon, bd biosciences, bedford, ma, usa), in the presence of stempro™ osteogenesis differentiation kit (trt) medium (life technologies, usa) or basic growing medium (ctrl). the positive control for osteogenic differentiation (ctrl+) was represented by wj-mscs cultured in osteogenic medium. after days, cells were fixed with % formalin for min at rt, washed three times in distilled water (ddh o), and then were stained with % alizarin red s solution (santa cruz biotechnology, dallas, texas, usa) for min at rt. cells were thoroughly washed several times in ddh o and observed by light microscopy to analyze calcium deposition. here, we investigated the relative expression levels of the c-myc, oct- , dnmt , nanog, sirt , p , sox , gapdh, p waf , p , p , and tert genes in wj-mscs before and after treatment with hepg -exhausted medium. the results of the analysis performed with the normfinder software suggested htrp as the normalizer (data not shown). we performed a comparison between the expression levels of these genes in wj-mscs exposed to hepg -exhausted medium and untreated wj-msc samples used as controls. figures and show that there are no significant differences in the expression levels of sox , nanog, gapdh, p , p , tert, and sox between treated wj-mscs as compared to untreated wj-mscs. on the other hand, the expression levels of c-myc, oct- , and sirt were significantly increased in treated wj-mscs as compared to untreated wj-mscs ( figure ). it is noteworthy that the expression levels of p , p , and dnmt were decreased in treated wj-mscs as compared to untreated wj-mscs (figures and ) . the expression levels of p , p , p , and tert did not show significant differences between the two groups of wj-mscs, exposed or not to hepg -exhausted medium ( figure ). statistical analysis showed that data were not normally distributed. we analyzed three mirnas-mir- - p, mir- a- p, and mir- - p-using u snrna as a normalizer. statistical analysis supported our decision to use u snrna as a normalizer for pcr analysis in real time, due to its greater stability between groups of samples [ ] . the kruskal-wallis test showed no significant cells , , of differences in wj-mscs before and after treatment with hepg -exhausted medium [ ] [ ] [ ] , for the expression of mir- and mir- ( figure ). on the other hand, the relative expression of mir- - p was significantly decreased in treated cells as compared to untreated cells ( figure ). statistical analysis showed that data were not normally distributed. we analyzed three mirnas-mir- - p, mir- a- p, and mir- - p-using u snrna as a normalizer. statistical analysis supported our decision to use u snrna as a normalizer for pcr analysis in real time, due to its greater stability between groups of samples [ ] . the kruskal-wallis test showed no significant differences in wj-mscs before and after treatment with hepg -exhausted medium [ ] [ ] [ ] , for the expression of mir- and mir- ( figure ). on the other hand, the relative expression of mir- - p was significantly decreased in treated cells as compared to untreated cells (figure ). statistical analysis showed that data were not normally distributed. we analyzed three mirnas-mir- - p, mir- a- p, and mir- - p-using u snrna as a normalizer. statistical analysis supported our decision to use u snrna as a normalizer for pcr analysis in real time, due to its greater stability between groups of samples [ ] . the kruskal-wallis test showed no significant differences in wj-mscs before and after treatment with hepg -exhausted medium [ ] [ ] [ ] , for the expression of mir- and mir- ( figure ). on the other hand, the relative expression of mir- - p was significantly decreased in treated cells as compared to untreated cells (figure ). figure show that mir- was less expressed in exosomes isolated from exhausted-mediumtreated wj-mscs as compared to untreated cells soon after one day of treatment. the same mirna could not be detected in the medium of treated wj-mscs after a week, while being still present in untreated control wj-mscs. the expression levels of mir- and mir- detected in the exosomes did not show any significant differences between treated and untreated wj-mscs. our results showed that mir- was not expressed in exosomes isolated from hepg -exhausted medium. (figures and ) . figure . expression of mirnas. the graph shows trends in mirna expression, displaying mir- - p, mir a- p, and mir- - p of wj-mscs treated with hepg -exhausted medium as compared to untreated wj-mscs. figure show that mir- was less expressed in exosomes isolated from exhausted-mediumtreated wj-mscs as compared to untreated cells soon after one day of treatment. the same mirna could not be detected in the medium of treated wj-mscs after a week, while being still present in untreated control wj-mscs. the expression levels of mir- and mir- detected in the exosomes did not show any significant differences between treated and untreated wj-mscs. our results showed that mir- was not expressed in exosomes isolated from hepg -exhausted medium. (figures and ) . the results showed the expression and localization of gapdh and sirt in control and treated cells. gapdh was detected both in the cytosol and in the nuclei of both cells. nevertheless, as shown in figure , it was clearly evident that in treated cells, gapdh was mainly located in the cytoplasm with a higher expression. sirt was found in the cytoplasm of treated cells, while in untreated control samples sirt was found mainly in the nucleus ( figure ). after days of differentiation, the morphology of treated wj-mscs (trt) was evaluated by light microscopy (leica, nussloch, germany). figure shows that trt cells cultured in the presence of the osteogenic conditioned medium did not acquire the typical morphology of mature osteocytes, as compared to positive control cells (ctrl+), in which intracellular calcium deposition was evident. the results showed the expression and localization of gapdh and sirt in control and treated cells. gapdh was detected both in the cytosol and in the nuclei of both cells. nevertheless, as shown in figure , it was clearly evident that in treated cells, gapdh was mainly located in the cytoplasm with a higher expression. sirt was found in the cytoplasm of treated cells, while in untreated control samples sirt was found mainly in the nucleus ( figure ). after days of differentiation, the morphology of treated wj-mscs (trt) was evaluated by light microscopy (leica, nussloch, germany). figure shows that trt cells cultured in the presence of the osteogenic conditioned medium did not acquire the typical morphology of mature osteocytes, as compared to positive control cells (ctrl+), in which intracellular calcium deposition was evident. after days of differentiation, the morphology of treated wj-mscs (trt) was evaluated by light microscopy (leica, nussloch, germany). figure shows that trt cells cultured in the presence of the osteogenic conditioned medium did not acquire the typical morphology of mature osteocytes, as compared to positive control cells (ctrl+), in which intracellular calcium deposition was evident. we observed progressive phenotypical changes after days in culture when stem cells were cultured with hepg -exhausted medium (figure ). cells , , x of we observed progressive phenotypical changes after days in culture when stem cells were cultured with hepg -exhausted medium (figure ). wj-mscs are becoming an interesting tool for allogenic transplantation in different kinds of disease, and also for covid- complications [ ] [ ] [ ] . therefore, understanding the behavior of mscs within the recipient tissues could be of great interest in assessing the safety of this kind of procedure. as reported by other authors, progenitor cells' microenvironments could influence their potential malignant transformation [ ] . here, we reveal important results on the potential influence of hepg hepatocarcinoma cells on wj-mscs' features and malignant transformation. other authors found that the overexpression of c-myc increases the expression of sirt . c-myc is able to induce sirt transcription by binding to its promoter [ ] [ ] [ ] [ ] [ ] [ ] . sirt is a class iii histone wj-mscs are becoming an interesting tool for allogenic transplantation in different kinds of disease, and also for covid- complications [ ] [ ] [ ] . therefore, understanding the behavior of mscs within the recipient tissues could be of great interest in assessing the safety of this kind of procedure. as reported by other authors, progenitor cells' microenvironments could influence their potential malignant transformation [ ] . here, we reveal important results on the potential influence of hepg hepatocarcinoma cells on wj-mscs' features and malignant transformation. other authors found that the overexpression of c-myc increases the expression of sirt . c-myc is able to induce sirt transcription by binding to its promoter [ ] [ ] [ ] [ ] [ ] [ ] . sirt is a class iii histone deacetylase (hdac) involved in gene regulation, maintaining genome stability, apoptosis, autophagy, senescence, aging, and tumorigenesis [ ] [ ] [ ] [ ] [ ] [ ] . several authors have highlighted a dual role of sirt as both a tumor suppressor and a tumor promoter [ ] [ ] [ ] [ ] [ ] [ ] . sirt contributes to cell survival through inhibition of gapdh translocation in the nucleus [ ] . gapdh is an enzyme of the glycolytic pathway, acting in the cytoplasm. it lacks any nuclear localization signal; nevertheless, it translocates to the nucleus via its interaction with the e ubiquitin ligase siah [ ] . sirt prevents the nuclear translocation of gapdh in response to apoptotic stress via a direct interaction [ ] . the nuclear translocation of gapdh is important for the cell signaling that activates apoptosis [ , ] . moreover, sirt inhibits the expression of p -regulated genes, such as p (waf /cip ) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , by inducing p deacetylation, thus preventing cell senescence and apoptosis [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . p is a cell-cycle inhibitor that promotes both cdk/cyclin inhibition and cell-cycle arrest during the g /s phase [ , ] . our results showed a path in which wj-mscs exposed to medium exhausted by hepg cells exhibited an increase in the expression levels of c-myc, sirt , and gapdh, and a simultaneous decrease in p and p gene expression ( figure ). these results confirmed that the overexpression of sirt is able to inhibit the p /p cip pathway and some related events, such as cell differentiation [ , ] . here, we highlight that p downregulation in treated wj-mscs (as compared to untreated controls) is related to an increase in cell proliferation, further implied by the greater expression of the stemness gene c-myc, able itself to inhibit p [ , ] . we also observed a downregulation of the expression of the epigenetic regulating gene dnmt (figure ). on the other hand, no significant differences in sox or nanog gene expression were observed between control and treated wj-mscs (figure ). these results revealed for the first time a pathway involving the capability of c-myc to potentiate sirt action, which in turns blocks gapdh in the cytoplasm ( figure ) [ ] [ ] [ ] [ ] [ ] [ ] [ ] . furthermore, sirt potentiates the expression of oct- [ , ] (figure ) , one of the crucial pluripotency-regulating factors in mesenchymal and embryonic stem cells [ , ] . considering previous findings described by other authors [ ] [ ] [ ] [ ] [ ] [ ] [ ] , we hypothesize that the downregulation of dnmt observed in this study (figure ) could induce the overexpression of oct- and c-myc, which, in turn, could enhance sirt gene expression. this event may be responsible for blocking gapdh in the cytoplasm (as is clearly shown in figure ). as shown by confocal microscopy analysis (tcs sp , leica) higher concentrations of sirt and gapdh were located in the cytoplasm of treated cells as compared to untreated control cells, where sirt and gapdh can be observed mainly in the nucleus. furthermore, in treated cells, an increased cell proliferation was observed; this could be related, as already known, to a greater availability of the glycolytic pathway, and to sirt -mediated downregulation of p /p gene expression (figure ), thus counteracting stem-cell senescence [ ] [ ] [ ] [ ] [ ] [ ] [ ] . these events were further supported by the upregulation of stemness-related genes such as c-myc (figure ). our results also showed that treated cells did not undergo differentiation toward the mesenchymal phenotype when stimulated with the conditioned differentiation medium (figure ) , indicating an attempt by the cell to maintain a prolonged "childhood" condition. sirt activation by c-myc, together with p deacetylation, provides a mechanism able to promote c-myc-induced cell proliferation by suppressing apoptosis and senescence. in the present paper, we also analyzed the expression levels of mir- , mir- , and mir- , three mirnas able to interfere with the expression of c-myc, oct- , and dnmt , which were also evaluated in our study. mir- may facilitate differentiation by repressing the core pluripotency factors oct- and sox , thus silencing the self-renewal program [ ] [ ] [ ] ] . the loss of mir- expression in most human tumors and cancer cell lines may provide a favorable environment for cell survival [ ] [ ] [ ] . our results showed that mir- -containing exosomes could be found in the culturing medium of wj-mscs, while progressively disappearing after cell exposure to hepg -exausted medium. all the mirnas analyzed in our study were mainly retained inside the cells (figures and ) , being able to regulate stemness genes, as it was demonstrated by us in previous studies [ ] [ ] [ ] ] . in particular, figures and show a progressive decrease of these mirnas in treated cells, along with a progressive loss of their ability to undergo differentiation toward the mesenchymal phenotype ( figure ), resembling the maintenance of a "childhood-like" signature. nevertheless, the upregulation of mir- can lead to cell-cycle arrest [ ] [ ] [ ] , stopping cell proliferation and leading to a decreased c-myc expression. our results demonstrated a downregulation of mir- after treatment with the hepg -exhausted medium (figure ) confirming the hypothesis of a permissive environment able to maintain a multipotent and a proliferative phenotype (figure ). our results seem to indicate that a condition required for cell transformation into a proliferative phenotype is the impairment of cell commitment and differentiation, and the establishment of a condition of prolonged stemness beyond the established biological time. we hypothesize that an unfavorable biological environment may contribute to the dysregulation of normal biological cell homeostasis, as observed by us. here, we clearly show that in treated wj-msc cultures, some cells seem to present a highly proliferative phenotype, resembling the behavior of hepg cells (figure ). following these considerations, we may assume the establishment of an autocrine/paracrine circuitry first triggered by hepg -exhausted medium, and then stably maintained. we observed the behavior of stem cells exposed to stimuli deriving from a tumor cell line, namely hepg cells. a greater expression of the stemness-related genes, together with a decrease of the mirnas regulating these genes, was clearly evident. we also observed a related increase in cell proliferation, a stop of differentiation, and a maintenance of stem cell potency. our aim was to understand the journey of stem cells towards the maintenance of stemness, which could lead to a dangerous proliferative phenotype. further studies are needed in order to understand if our experiments could be translated in future clinical applications, for example by introducing plasma cleaning after tumor removal. following these considerations, we may assume the establishment of an autocrine/paracrine circuitry first triggered by hepg -exhausted medium, and then stably maintained. we observed the behavior of stem cells exposed to stimuli deriving from a tumor cell line, namely hepg cells. a greater expression of the stemness-related genes, together with a decrease of the mirnas regulating these genes, was clearly evident. we also observed a related increase in cell proliferation, a stop of differentiation, and a maintenance of stem cell potency. our aim was to understand the journey of stem cells towards the maintenance of stemness, which could lead to a dangerous proliferative phenotype. further studies are needed in order to understand if our experiments could be translated in future clinical applications, for example by introducing plasma cleaning after tumor removal. funding: this research was funded by "fondo di ateneo per la ricerca ". osteogenesis from dental pulp derived stem cells: a novel conditioned medium including melatonin within a mixture of hyaluronic, butyric, and retinoic acids amniotic fluid stem cells morph into a cardiovascular 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pneumonia human adipose-derived mesenchymal stem cell-conditioned medium ameliorates polyneuropathy and foot ulceration in diabetic bks db/db mice induction of pluripotent stem cellsfrom mouse embryonic and adult fibroblast cultures by definedfactors highly efficientreprogramming to pluripotency and directed differentiation ofhuman cells with synthetic modified mrna deterministic directreprogramming of somatic cells to pluripotency a molecular roadmapof reprogramming somatic cells into ips cells microrna- rgulates oct , sox , and klf and represses pluripotency in human embryonic stem cells induced pluripotent stem cell generation using a single lentiviral stem cell cassette developmental context determines latency of myc-induced tumorigenesis an overview of myc and its interactome. cold spring harb targeting c-myc as a novel approach for hepatocellular carcinoma myc activation is a hallmark of cancer initiation and maintenance. cold spring harb in vivo control of cpg and non-cpg dna methylation by dna methyltransferases dna methylation in cancer: too much, but also too little microrna- binduces global dna hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly dnmt a and b and indirectly dnmt uhrf overexpression drives dna hypomethylation and hepatocellular carcinoma octamer binding proteins confer transcriptional activity in early mouse embryogenesis structure, expression and chromosomal location of the oct- gene regulatory networks in embryo-derived pluripotent stem cells formation of pluripotent stem cells in the mammalian embryo depends on the pou transcription factor oct octamania: the pou factors in murine development defining pluripotency derivation of novel human ground state naive pluripotent stem cells oct- controls cell-cycle progression of embryonic stem cells sirt deacetylates the dna methyltransferase (dnmt ) protein and alters its activities total phenols from grape leaves counteract cell proliferation and modulate apoptosis-related gene expression in mcf- and hepg human cancer cell lines the rna expression signature of the hepg cell line as determined by the integrated analysis of mirna and mrna expression profiles cellosaurus hep-g (cvcl_ ) identification of the secreted proteins originated from primary human hepatocytes and hepg cells the diversity and plasticity of adult hepatic progenitor cells and their niche downregulation of mir- - p in cancer cells and their derived exosomes may contribute to the development of ovarian cancer by targeting ct mir- inhibits non-small cell lung cancer cell proliferation and invasion through targeting of sox and regulation of wnt signaling mir- and mir- can induce cell cycle arrest in human non small cell lung cancer cell lines melatonin and vitamin d orchestrate adipose derived stem cell fate by modulating epigenetic regulatory genes stem cells, and autophagy: exploring a path involving mirna melatonin and vitamin d interfere with the adipogenic fate of adipose-derived stem cells regenerative treatment using a radioelectric asymmetric conveyor as a novel tool in antiaging medicine: an in vitro beta-galactosidase study lessons from human umbilical cord: gender differences in stem cells from wharton's jelly mirna stability in frozen plasma samples. molecules reac technology and hyaluron synthase , an interesting network to slow down stem cell senescence relative expression software tool (rest©) forgroup-wise comparison and statistical analysis of relative expression results in real-time pcr cytoplasm-localized sirt enhances apoptosis transcription regulation by class iii histone deacetylases (hdacs)-sirtuins nucleocytoplasmic shuttling of the nad+-dependent histone deacetylase sirt a c-myc-sirt feedback loop regulates cell growth and transformation sirt is highly expressed in brain metastasis tissues of non-small cell lung cancer (nsclc) and in positive regulation of nsclc cell migration sorting out functions of sirtuins in cancer sirt interacts with and protects glyceraldehyde- -phosphate dehydrogenase (gapdh) from nuclear translocation: implications for cell survival after irradiation reversible nuclear translocation of glyceraldehyde- -phosphate dehydrogenase upon serum depletion sirt alleviates senescence of degenerative human intervertebral disc cartilage endo-plate cells via the p /p pathway sirt knockdown up-regulates p and p /cip expression in renal adenocarcinoma cells but not in normal renal-derived cells in a deacetylase-independent manner dynamic signaling network for the specification of embryonic pancreas and liver progenitors p in cancer: intricate networks and multiple activities this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we would like to thank caterina serra, gabriele ibba, claudia piu and the staff of the gynecologic and obstetric clinic of sassari university for their kind technical support. the authors declare no conflict of interest. key: cord- -cuatqy u authors: wei, maryann title: social distancing and lockdown – an introvert’s paradise? an empirical investigation on the association between introversion and the psychological impact of covid -related circumstantial changes date: - - journal: front psychol doi: . /fpsyg. . sha: doc_id: cord_uid: cuatqy u the coronavirus disease (covid ) pandemic has effected the implementation of social distancing and lockdown measures across the globe, and the psychological impact of associated life changes is experienced more severely by some individuals than others. anecdotal evidence points to a common belief among the general public that introverts are faring better than their extraverted counterparts to this end. however, the claim lacks empirical research, and seems counterintuitive when the broader literature on the association between introversion and mental health is considered. the current study investigated whether the psychological impact of covid -related circumstantial changes was moderated by introversion, based on outcome measures across psychosocial, cognitive, and affective domains. the role of several demographic factors in determining covid -related mental health symptoms was also examined. one hundred and fourteen individuals ( usa residents) completed measures of introversion, and reported on the extent to which they experienced loneliness, anxiety, depression and cognitive impairments as a function of covid -related circumstantial changes. results showed that introversion predicted more severe loneliness, anxiety, and depression experienced as a function of covid -related circumstantial changes, but not cognitive impairments. among the range of demographic factors examined (age, gender, living condition, recent unemployment), living with others (vs. living alone) predicted more severe covid -related mental health symptoms. however, these effects were only observed on outcome measures pertaining to anxiety and cognitive impairments, but not loneliness and depression. current findings have implications for both consumers and disseminators of information on popular internet hubs. current findings also highlight the possibility that living with others (close human affiliation) may have protective and detrimental effects on different domains of mental health during the covid pandemic. the introversion-extraversion dimension is central to leading trait theories of human personality in psychology (e.g., myers, ; cattell, ; eysenck, ; hathaway, ; mccrae and costa, , although exact conceptualisations vary between theories), and the construct is ubiquitous in both academic and popular literature. commonly described in dichotomic terms, introverts and extraverts are often differentiated by the sources they draw energy from (internal vs. external, respectively). adjectives traditionally associated with introversion include "inhibited, " "reserved, " and "undemonstrative, " while those associated with extraversion include "outgoing, " "friendly, " and "enthusiastic" (eysenck, ) . the coronavirus disease (covid ) pandemic has effected the implementation of social distancing and lockdown measures across the globe, and the psychological impact of associated life changes is experienced more severely by some individuals than others (williams et al., ) . anecdotal evidence points to a common belief among the general public that introverts are faring better than their extraverted counterparts to this end. for example, a "how to survive social distancing as an extravert" guide on a popular psychology website begins with the following statements: "for introverts, being stuck at home without social interaction for long periods of time really isn't the worst thing at all. they are accustomed to this time spent alone and feel energized and recharged by it. when it comes to extroverts, the idea of social distancing can feel like somewhat of a death sentence" (personality growth, ) . in articles published on other widely-frequented non-psychology websites, introversion has been championed as an asset for thriving in covid -related social isolation (e.g., bloomberg, ; reuters, ; the conversation, ) . such beliefs are exemplified in the influx of user-generated pictorial content (more colloquially known as "memes") across the internet with similar sentiments (see supplementary material for exemplars (data sheet )). on that grounds that introverts prefer less stimulating environments (myers, ; cattell, ; eysenck, ; hathaway, ; mccrae and costa, ) , the assumption that introverts experience the psychological impact of covid related circumstantial changes less severely than extraverts seems plausible. however, the claim lacks empirical research, and there are several lines of work in light of which the claim appears counterintuitive. first, introversion has been linked to personality traits associated with the tendency to experience more intense emotions and more difficulties in regulating these emotions, namely the "feeling" dimension of the myer-briggs type indicator (janowsky, ) and neuroticism, respectively (janowsky, ; jylha et al., ; fadda and scalas, ) . additionally, and possibly resultantly, introversion has also been associated with more psychological problems in general (janowsky, ; jylha et al., ; fadda and scalas, ) , and adjustment problems in particular. specifically, studies have demonstrated that introverts struggle more than extraverts in adjusting to life events which entail changes in day-to-day life, including shifts between educational institutes (bauer and liang, ; davidson et al., ) , job relocation (pinder, ) , and retirement (löckenhoff et al., ; robinson et al., ) . although increased amounts of time alone should in theory be welcome by introverts, these findings raise questions on whether introverts necessarily have an advantage over their extraverted counterparts in adapting to covid related circumstantial changes. additionally, the psychological impact of covid -related circumstantial changes (and mental health in general) has psychosocial, cognitive, and affective aspects, which in turn represent functional domains which may be differentially moderated by personality traits (segel-karpas and lachman, ) . the primary aim of this study was to examine whether the psychological impact of covid -related circumstantial changes is moderated by introversion, based on outcome measures across psychosocial, cognitive, and affective domains. a second aim was to examine the unique role of several other demographic factors (which were also considered as control variables in fulfilling the primary aim) in determining covid related mental health symptoms. between late april and early may , a call for participants for a study on the psychological impact of covid was placed on the sub-reddit r/samplesize, an online platform designated to connect researchers and research volunteers. based on previous research (shatz, ; jamnik and lane, ) and the current author's own experience, recruitment using this platform reliably produces quality data from adult individuals dominantly residing in the united states of america (usa). the latter demographic profile seems appropriate for the current research, given the high incidence of covid in the usa and the strictness of lockdown/social distancing measures which ensued. one hundred and fourteen individuals responded to the call for participants (mean age = . , sd = . ; female). sixty two respondents were located in the usa. the other respondents were distributed across the following countries, including united kingdom (n = ), canada (n = ), australia (n = ), germany (n = ). usa and non-usa residents were compared on all outcome variables (described below) to identify cases where the current sample could not be considered as a whole. introversion-extraversion was measured as a continuous dimension using the introversion scale developed by richmond and mccroskey ( ) . this scale was developed based on the extraversion subscale in the eysenck personality questionnaire (eysenck et al., ) . to illustrate, items such as "can you usually let yourself go and enjoy yourself at a lively party?" and "do you tend to keep in the background on social occasions?" in the eysenck personality questionnaire have counterparts in richmond and mccroskey's introversion scale in "can you usually let yourself go and have a good time at a party?" and "are you inclined to keep in the background on social occasions?, " respectively . the introversion scale consists of such statements. respondents indicate whether each statement applied to them on a -point scale ranging from strongly disagree ( ) to strongly agree ( ). six statements serve as distractors and are not scored. alpha reliability estimates were above. in the initial validation study by richmond and mccroskey ( ) , and closely matched in the current study (cronbach's alpha = . ). scores range between and , with higher scores indicating higher introversion, and lower scores indicating lower introversion (i.e., higher extraversion). the following demographics were measured as predictor variables of interest: age, gender, living condition (alone/with others), recent unemployment due to covid (no/yes). the psychological impact of covid -related circumstantial changes was measured with a battery of established questionnaires, with instructions modified to elicit mental health ratings directly associated with the implementation of social distancing and lockdown measures. that is, instead of reporting on mental health symptoms based on a given retrospective timeframe, participants were asked to provide ratings as a function of covid -related circumstantial changes. the exact phrase of instructions participants received is detailed in context below. functional aspects in the psychosocial, cognitive, and affective domains were measured, with the affective domain further broken down into depressive and anxious sub-domains. participants completed the dejong gierveld loneliness scale (de jong gierveld and van tilburg, ) to provide an indicator of loneliness as a function of covid -related circumstantial changes. participants responded "no", "more or less, " or "yes" to six statements, headed by the question "how true are these statements for you, following the implementation of covid social distancing and lockdown measures?" on negativelyworded statements (e.g., "i miss having people around me"), "more or less" and "yes" responses are scored as while "no" responses are scored as . on positively-worded statements (e.g., "there are enough people i can trust completely"), "more or less" and "no" responses are scored as while "yes" responses are scored as . scores are summed across items (range - ), where higher scores indicate higher loneliness. reliability and validity of the scale has been demonstrated across the lifespan (de jong gierveld and van tilburg, ). cronbach's alpha for the de jong gierveld loneliness scale as presented in the current sample was slightly below the conventional acceptable benchmark of . at . ; however, cronbach's alphas bordering on . were observed in the initial validation study (de jong gierveld and van tilburg, ) , so that internal consistency estimates in a slightly lower tier are likely normative given the few number of items in the scale (tavakol and dennick, ) . cognitive impairments associated with covid -related circumstantial changes were assessed with the cognitive failures questionnaire (cfq; (broadbent et al., ) , a item inventory of self-reported day-to-day slips and errors in cognition. instructions were phrased as follows: "the following questions are about minor mistakes which everyone makes from time to time, but some of which happen more often than others. we would like to know how often these things have happened to you, following the implementation of covid social distancing and lockdown measures." respondents indicated on a scale ranging from (never) to (very often) how often they experience certain incidents (e.g., "do you find you forget what you came to the shops to buy?"). scores (range - ) are summed across all items, where higher scores indicate more extreme cognitive impairments. in the initial pilot study, internal consistency of . was demonstrated (broadbent et al., ) . cronbach's alpha for the cfq as presented in the current sample was . . depression. depressive symptoms associated with covid related circumstantial changes were assessed with the patient health questionnaire [phq- ; (kroenke and spitzer, ) ]. instructions were phrased as follows: "to what extent (frequency) have you experienced these symptoms, following the implementation of covid social distancing and lockdown measures?" participants report the frequency with which they experience nine depressive symptoms on a -point scale ranging from "not at all" ( ) to "nearly everyday" ( ) (e.g., little interest or pleasure in doing things). scores are summed across the nine items (range - ), where higher scores indicate higher depression severity. the phq- has been validated not only as a useful tool to recognize clinical depression but also subthreshold depressive symptoms in the general population (martin et al., ) . cronbach's alpha for the phq- as presented in the current sample was . . anxiety. anxious symptoms associated with covid related circumstantial changes were assessed with the generalized anxiety disorder screener (gad- ; (spitzer et al., ) . the response format for the gad- is identical to that of the phq- . instructions for the gad- were also identical to that which were presently used for the phq- . scores are summed across seven items (i.e., seven symptoms of anxiety; e.g., not being able to stop or control worrying), where higher scores indicate higher anxiety severity (range - ). similar to the phq- , the gad- has been demonstrated as a reliable and valid measure of anxiety in the general population (löwe et al., ) . cronbach's alpha for the gad- as presented in the current sample was . . all analyses described as follows (including the generation of descriptives) were processed with spss (version ). to evaluate whether introversion moderates the psychological impact of covid -related circumstantial changes, scores on the introversion scale were used to predict each outcome variable listed above. hierarchical regression analyses were used, with demographic factors entered in the first step as control variables. the predictive value of each demographic factor across the range of outcome variables was also of research interest (pertaining to the second aim). where significant differences were observed on outcome variables between usa and non-usa residents, regression analyses were performed separately for the two groups. for comprehensiveness, the two groups were also compared on all predictor variables. table gives means and correlations between all study variables for the full sample. usa and non-usa residents did not differ on any of the predictor variables (age, gender, living condition, recent unemployment, and introversion). however, pertaining to outcome variables, usa and non-usa residents differed on the psychosocial domain. specifically, usa residents reported experiencing higher loneliness as a function of covid related circumstantial changes compared to non-usa residents (m = . vs. m = . on the dejong gierveld loneliness scale, respectively), t( ) = . , p = . . thus, regression analyses predicting loneliness were performed separately for usa and non-usa residents. table gives standardized β coefficients for predictor variables and associated model statistics in hierarchical regression analyses predicting the psychological impact of covid related circumstantial changes, across psychosocial, cognitive, and affective domains. after controlling for age, gender, living condition and recent unemployment, higher introversion (higher introversion scale scores) uniquely predicted higher depression (phq- ) and anxiety (gad- ) experienced as a function of covid -related circumstantial changes, β = . , t = . , p = . and β = . , t = . , p = . , respectively. higher introversion also uniquely predicted loneliness (dejong gierveld loneliness scale) experienced as a function of covid -related circumstantial changes after controlling for demographic factors, although this effect was unique to usa residents (β = . , t = . , p = . ). introversion did not predict cognitive impairments (cfq) related to covid circumstantial changes after controlling for demographic variables (β = . , t = . , p = . ). in a model including introversion, recent unemployment predicted higher loneliness experienced as a function of covid -related circumstantial changes only for non-usa residents (β = . , t = . , p = . ). interestingly, after including introversion in the model, living with others (vs. alone) was associated with more severe cognitive impairments and anxiety experienced as a function of covid -related circumstantial changes (β = . , t = . , p = . and β = . , t = . , p = . , respectively). it is worth noting that the living condition did not have predictive value for loneliness and depressive symptoms experienced as a function of covid -related circumstantial changes (see table ). this study examined whether the psychological impact of covid -related circumstantial changes was moderated by introversion, based on outcome measures across psychosocial, cognitive, and affective domains. as a second aim of the current study, the role of several other demographic factors in determining covid -related mental health symptoms was also examined. overall, higher introversion (i.e., lower extraversion) was associated with higher loneliness, depression and anxiety experienced as a function of covid -related circumstantial changes. the finding that introverts experience the psychosocial and affective impact of social distancing and lockdown measures more severely than their extraverted counterparts converges and deviates from previous literature in several ways. first, the finding is in line with previous studies demonstrating that introversion is associated with more psychological problems in general (janowsky, ; jylha et al., ; fadda and scalas, ) , and adjustment problems specifically (pinder, ; bauer and liang, ; löckenhoff et al., ; robinson et al., ; davidson et al., ) , however, this finding appears to be in disagreement with the notion that introversion is associated with a preference for less stimulating environments (myers, ; cattell, ; eysenck, ; hathaway, ; mccrae and costa, ) such as that created in everyday life following the implementation of social distancing and lockdown measures. in turn, this assumption has fuelled the lay belief that introverts are coping better during the covid pandemic compared to extraverts (detailed in section "introduction"). current findings may be best understood without considering the two lines of thought as mutually exclusive. introversion has been linked to decreased help-seeking behavior (swickert et al., ; atik and yalçin, ; kakhnovets, ) , which may in part explain higher psychological problems among introverts at baseline (janowsky, ; jylha et al., ; fadda and scalas, ) . when experiencing negative emotions, introverts are similarly more likely turn inwardly to cope (shapiro and alexander, ) . while introspective behaviors can facilitate emotional self-regulation, such habits can also function as a double-edged sword in perpetuating internalization (bowker and rubin, ) , rumination (verhaeghen et al., ; cohen and ferrari, ) , and worry (philippi and koenigs, ) -key cognitive underpinnings of loneliness, depression, and anxiety, respectively (beck, ; newman et al., ; ypsilanti, ) . such an account of why individuals higher on introversion might experience the psychosocial and affective impact of covid -related circumstantial changes more severely is corroborated by other aspects of present findings. specifically, cognitive impairments experienced as a function of social distancing and lockdown measures were not moderated by introversion, suggesting that cognitive function and activity remains intact across the introversion-extraversion dimension through covid -related circumstantial changes. current findings are in keeping with previous research demonstrating that functional domains of mental health are differentially moderated by personality traits (segel-karpas and lachman, ) , and highlight the particular relevance of evaluating domain-specific effects in research on the association between introversion and mental health. crucially, these findings have implications for both consumers and disseminators of information on popular internet hubs -specifically, to keep in view that the notion of introverts thriving under lockdown and social distancing conditions may not necessarily be empirically supported. mental health professionals dealing with covid -related psychological issues should also be aware that introverts may risk being erroneously left out of the mental health system. one aspect of present observations is worth noting before proceeding to discuss findings pertaining to the second aim of the current study. namely, the psychosocial impact of covid related circumstantial changes (loneliness) was predicted by both introversion and demographic factors (specifically, recent unemployment), but these effects were unique to usa and non-usa residents, respectively. there are several possible explanations for this observation including: ( ) predictors of covid -related loneliness differ qualitatively at different levels of loneliness severity, given usa residents reported higher loneliness as a function of covid -related circumstantial changes in the current sample, and ( ) the psychosocial impact of covid -related circumstantial changes is predicted by qualitatively different factors in different cultures. while not within the scope of the present study's aims, these speculations represent testable hypotheses which may be of interest in future research. besides recent unemployment, other demographic predictors examined were age, gender, and living condition. only living condition made a unique contribution to covid -related mental health symptoms after accounting for introversion. specifically, living with others (vs. living alone) was associated with experiencing more cognitive impairments and anxiety as a function of covid -related circumstantial changes. adjacently, it was observed that covid -related loneliness and depressive symptoms were not predicted by living condition. interpreted together, it is possible that close human affiliation serves as a protective buffer against social disconnectedness and low mood during the covid pandemic, but works in the opposite direction for clarity of thought and keeping calm. further information on household dynamics would have helped in the development of this speculation, but was not obtained in the present study. other limitations of the current study include its crosssectional nature, so that pre-covid mental health issues may have been conflated with covid -related mental health symptoms as presently assessed. on a related note, the current study assumes that presently used outcome measures (worded with reference to covid social distancing and lockdown measures) captured psychological health as shaped specifically by social orders placed as preventative measures against covid . however, responses on these measures may also reflect psychological health as impacted by the globalscale pandemic more generally, so that responses may not be tied solely to increased amounts of solitary time. further, demographic variables were considered only in broad strokes in the present study. accounting for a wider range of demographic variables, including but not limited to income, would allow for a clearer picture of the association between introversion and the psychological impact of covid -related circumstantial changes to be drawn. next, the participant count in each non-usa country was small in the present sample, so that non-usa respondents had to be collapsed in a single "non-usa residents" group. although the covid outbreak is considered a global pandemic, there may still be subtle differences in the covid impact between countries. more targeted and selective recruitment according to location/residence should be considered in future research. finally, given present interests in multiple outcome variables, the current study would have benefited in terms of statistical power from a larger sample size. the datasets presented in this study can be found in online repositories. the names of the repository/repositories and accession number(s) can be found in the article/ supplementary material. ethical review and approval was not required for 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financial relationships that could be construed as a potential conflict of interest.copyright © wei. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -hl ln r authors: tulchinsky, theodore h.; varavikova, elena a. title: planning and managing health systems date: - - journal: the new public health doi: . /b - - - - . - sha: doc_id: cord_uid: hl ln r health systems are complex organizations. they are often the largest single employer in a country, with expenditures of public and private money of – percent of gross domestic product. overall and individual facility management requires mission statements, objectives, targets, budgets, activities planning, human interaction, services delivery, and quality assurance. health organization involves a vast complex of stakeholders and participants, suppliers and purchasers, regulators and direct providers, and individual patients, and their decision-making. these include pyramidal and network organizations and ethical decision-making based on public interest, resource allocations, priority selection, and assurance of certain codes of law and ethical conduct. this chapter discusses how complex organizations work, with potential for application in health, and the motivations of workers and of the population being served. organization theory helps in devising methods to integrate relevant factors to become more effective in defining and achieving goals and missions. health systems are complex organizations and their management is an important concept in the new public health. health is a major sector of any economy and often employs more people in the industrialized countries than any other industry. health has complex networks of services and provider agencies, including funding through public or private insurance or through national health service systems. whether insurance is provided by the state or through private and public sources combined, skilled management is required at the macro-or national and the micro-or local level, including the many institutions that make up the system. management training of public health professionals and clinical services personnel is a requisite and not a luxury. planning and management are changing in the era of the new public health with advances in prevention and treatment of disease, population health needs, innovative technologies such as genetic engineering, new immunizations that prevent cancers and infectious diseases, prevention of non-communicable diseases, environmental and nutritional health, and health promotion to reduce risk factors and improve healthful living for the individual and the community. modern and successful public health also must address social, economic, and community determinants of health and the promotion of public policies and individual behaviors for health and well-being. the social capital and norms that promote cooperation among people are the basis of a "civil society" (i.e., the totality of voluntary, civic, and social organizations and institutions of a functioning society alongside the structures of governmental and commercial institutions). health systems are ideally knowledge-and evidence-based in using technologies available in medicine and the environment to promote the health and well-being of a population, including security against the effects of threatened terrorism, growing social isolation, and inequities in health. management in health can learn much from concepts of business management that have evolved to address the economic and human resource aspects of a health system at the macrolevel or an individual unit of service at the microlevel. the new public health is not contained within one organization, but rather reflects the collective efforts of national, state, regional, and local governments, many organizations in the public and non-governmental sectors, and finally efforts of individual or group advocates and providers and the public itself. the political level is crucial for adequate funding, legislation, and promotion of health-oriented policy positions and in public health management. the responsibility for health management is shared across all parts of society, including individuals, communities, business, and all levels of government. the new public health identifies and addresses community health risks and needs. planning is critical to the process of keeping a health system sustainable and adaptable and in creating adequate responses to new health threats. monitoring, measurement, and documentation of health needs are vital to design and adapt an effective program and to measure impact. data on the targeted issues must be accessible while protecting individual privacy. health is a hugely expensive and expansive complex of services, facilities, and programs provided by a wide range of professional and support service personnel making up one of the largest employers of any sector in a developed country. services are increasingly delivered by organized groups of providers. but all health systems operate in an environment of economic constraints, imposing a need to seek efficiency in the use of resources. how organizations function is of great importance not only for their economic survival, but also, and equally important, for the well-being of the clients and providers of care. an organization is two or more people working together to achieve a common goal. management is the process of defining the goals and making effective use of an organization to attain those goals. even very small units of a human organization require management. management of human resources is vital to the success of an organization, whether in a production or service industry. health systems may chapter vary from a single structure to a network of many organizations. no matter how organizations are financed or operated, they require management. management in health care has much to learn from approaches to management in other industries. elements of theories and practices of profit-oriented sector management can be applied to health services even if they are operated as non-profit enterprises. physicians, nurses, and other health professionals will very likely be involved in the management of some part of the health care system, whether a hospital department, a managed care system, a clinic, or even a small health care team. at every level, management always means working with people, using resources, providing services, and working towards common objectives. health providers require preparation in the theory and practice of management. a management orientation can help providers to understand the wider implications of clinical decisions and their role in helping the health care system to achieve goals and targets. students and practitioners of public health need preparation in order to recognize that a health care system is more complex than the direct provision of individual services. similarly, policy and management personnel need to be familiar with both individual and population health needs and related care issues. health has evolved from an individual one-on-one service to complex systems organized within financing arrangements, mostly under government auspices. as a governmental priority, health may be influenced by political ideology, sometimes reflecting societal attitudes of the party in power and sometimes apparently at odds with its general social policy. following bismarck's introduction in germany in of national health insurance for workers and their families, funded by both workers and their employers, most countries in the industrialized world introduced variants of this national health plan. usually, this has been at the initiative of socialist or liberal political leadership, but conservative political parties have preserved national health programs once implemented. despite the new conservatism since the s with its pre-eminent ideology of market forces, the growing roles for national, state, and local authorities in health have led to a predominantly government role in financing and overall responsibility for health care, even where there is no universal national health system, as in the usa. the uk's national health service (nhs), initiated by a labour government in , has survived through many changes of government, including the conservative margaret thatcher period in which many national industries and services were privatized. health policy is a function of national (government) responsibility overall for health, but implementation is formulated and met at state, local, or institutional levels. the division of responsibilities is not always clear cut but needs to be addressed and revised both professionally and politically within constitutional, legal, and financial constraints. selection of the direction to be taken in organizing health services is usually based on a mix of factors, including the political view of the government, public opinion, and rational assessment of needs as indicated through epidemiological data, cost-benefit analysis, the experience of "good public health practice" from leading countries, and recommendations by expert groups. lobbying on the part of professional or lay groups for particular interests they wish to promote is part of the process of policy formulation and has an important role in the planning and management of health care systems. there are always competing interests for limited resources of funding, by personnel within the health field itself and in competition with other demands outside the health sector. the political level is vitally involved in health management in establishing and maintaining national health systems, and in determining the place of health care as a percentage of total governmental budgetary expenditures, in allocating funds among the competing priorities. these competing priorities for government expenditures include defense, roads, education, and many others, as well as those within the health sector itself. traditionally, there are competing priorities between the hospital and medical sector and the public health and community programs sector. a political commitment to health must be accompanied by allocation of resources adequate to the scope of the task. thus, health policy is largely determined by societal priorities and is not a prerogative of government, health care providers, or any institution alone. as a result of long struggles by trade unions, advocacy groups, and political action, well-developed market economies have come to accept health as a national obligation and essential to an economically successful and well-ordered society. this realization has led to the implementation of universal access systems in most of the industrialized countries. once initiated, national health systems require high levels of resources, because the health system is labor intensive with relatively high salaries for health care professionals. in these countries, health expenditures consume between and nearly percent of gross domestic product (gdp). some industrialized countries, notably those in the former soviet bloc, lacking mechanisms for advocacy, including consumer and professional opinion, tended to view health with a political objective of social benefits, and also as a "non-productive" consumer of resources rather than a producer of new wealth. as a result, budget allocations and total expenditures for health as a percentage of gdp were well below those of other industrialized countries (figure . ). salaries for health personnel in the semashko system were low compared to industrial workers in the "productive" sectors. furthermore, industrial policy did not promote modern health-related industries, compared to the military or heavy industrial sectors. the former socialist countries of eastern europe which have joined the european union (eu) have gradually increased allocation to health from . percent of gdp in to . percent in , while the pre- members of the eu increased their expenditures from . percent of gdp to . percent. the average spend in the commonwealth of independent states (russia, ukraine, and others) increased from . percent in to . percent in , and in the central asian republics (kazakhstan, uzbekistan, and others) from . percent in to . percent in (who health for all database, january ). however, russian health expenditure in was still only . percent of gdp and there is a lingering idea of health being a non-productive investment. the developing countries generally spend under percent of gnp on health, because health is addressed as a relatively low political priority, and they depend very much on international donors for even the most basic of public health programs such as immunization. financing of health care and resource allocation requires a balance among primary, secondary, and tertiary care. economic assessment, monitoring, and evaluation are part of determining the health needs of the population. regulatory agencies are responsible for defining goals, priorities, and objectives for resulting services. targets and methods of achieving them provide the basis for implementation and evaluation strategies. planning requires written plans that include a statement of vision, mission objectives, target strategies, methods, and coordination during the implementation. designation and evaluation of responsibilities, resources to be committed, and participants and partners in the procedure are part of the continuous process of management. the dangers of taking a "wrong" direction may be severe, not only in terms of financial costs, but also in terms of high levels of preventable morbidity and mortality. health policy is often as imprecise a science as medicine itself. the difference is that inappropriate policy can affect the lives and well-being of very large numbers of people, as opposed to an individual being harmed by the mistake of one doctor. there may be no "correct" answer, and there are numerous controversies along the path. health policy remains more an "art" than the more quantitative and seemingly precise field of health economics. societal, economic, and cultural factors as well as personal habits have long been accepted as having an important impact on vulnerability to coronary heart disease. but other factors such as the degree of control over one's life, as suggested in studies of british civil servants, religiosity, and the effects of migration on families left behind are part of the social gradients and inequalities seen in many disease entities, with consequent excess morbidity and mortality in some contexts, such as in russia and ukraine. health policy, planning, and management are interrelated and interdependent. any set goal should be accompanied by planning how to attain it. a policy should state the values on which it is based, as well as specify sources of funding, planning, and management arrangements for its implementation. examination of the costs and benefits of alternative forms of health care helps in making decisions as to the structure and the content of health care services, both internal structures (within one organization) and external linkages (intersectoral cooperation with other organizations). the methods chosen to attain the goals become the applied health policy. the world health organization's (who's) health for all strategy was directed at the political level and intended to increase governmental awareness of health as a key component of overall development. to some degree it succeeded despite its expansive aspirations, and even after nearly years, its objectives remain worthwhile even in well-developed health systems. within health, primary care was stressed as the most effective investment to improve the health status of the population. in , the world bank's world development report adopted the health for all strategy and promoted the view that health is an important investment sector for general economic and social development. however, economic policies promoting privatization and deregulation in the health sector threaten to undermine this larger goal in countries with national health systems. in the usa, major steps are being taken to increase coverage of health insurance for all as the number of uninsured americans declined from million people uninsured in to . million in , edging down from . to . percent of the total population. further decline in the uninsured population is expected as the patient protection and affordable care act (ppaca, or "obamacare") comes into effect in the coming years, bringing many millions of americans into health insurance and meeting federal standards of fair practices such as eliminating exclusion for preexisting conditions by private insurers. the ppaca comes into effect on january and will guarantee coverage for pre-existing conditions, and ensure that premiums cannot vary based on gender or medical history. it will subsidize the cost of coverage, and new state-based health insurance exchanges will help consumers to find suitable policies. it will introduce many preventive care measures into public and private insurance plans, and will promote efficiencies in the health systems including reduction in fraudulent claims and wasteful funding systems. all of this will require skilled management in the components of the health system (see chapters and ). in the new public health, health promotion, preventive care, and clinical care are all part of public health because the well-being of the individual and the community requires a coordinated effort from all elements of the health spectrum. establishing and achieving national health goals require planning, management, and coordination at all levels. the achievement of health advances depends on organizations and structured efforts to reach health goals such as those defined above, and more recently by the united nations (un) in the millennium development goals (mdgs) (see chapter ), and requires some understanding of organizations and how they work. the study of organizations developed within sociology, but has gradually become a multidisciplinary activity involving many other professional fields, such as economics, anthropology, individual and group psychology, political science, human resources management, and engineering. organizations, whether in the public or private sector, exist within an external environment, and utilize their own structure, participants, and technology to achieve goals. for an organization to survive and thrive, it must adapt to the physical, social, cultural, and economic environment. organizations participating in health care establish the connection between service providers and consumers, with the goal of better health for the individual and the community. the factors for this include legislation, regulation, professionalism, instrumentation, medications, vaccines, education, and other modalities of intervention for prevention and treatment. the social structure of an organization may be formal (structured stability), natural (groupings reflecting common interests), or open (loosely coupled, interacting, and self-adjusting systems to achieve goals). formal systems are deliberately structured for the purposes of the organization. natural systems are less formal structures where participants work together collaboratively to achieve common goals defined by the organization. open systems relate elements of the organization to coalitions of partners in the external environment to achieve mutually desirable goals. in the health system, structures should focus on prevention and treatment of disease and improvement in health and well-being of society. the social structure of an organization includes values, norms, and roles governing the behavior of its participants. government, business, or service organizations, including health systems, require organizational structures, with a defined mission and set of values, in order to function. an organizational structure needs to be tailored to the size and complexity of the entity and the goals it wishes to achieve. the structure of an organization is the way in which it divides its labor into distinct tasks and coordinates them. the major organizational models, which are not mutually exclusive and may indeed be complementary, are the pyramidal (bureaucratic) and network structures. the bureaucratic model is based on a hierarchical chain of command with clearly defined roles. in contrast, the matrix or network organization brings together professional or technical people to work on specific programs, projects, or tasks. both are vital to most organizations to meet ongoing responsibilities and to address special challenges. some classic organization theory concepts help to set the base for modern management ideas as applied to the health sector. scientific management was pioneered by frederick winslow taylor . his work was pragmatic and based on empirical engineering, developed in observational studies carried out for the purpose of increasing worker, and therefore system, efficiency. taylor's industrial engineering studies of scientific management were based on the concept that the best way to improve worker productivity was by designing improved techniques or methods used by workers. this theory viewed workers as instruments to be manipulated by management, and assumed that efficient, rationally planned methods would produce better industrial results and industrial peace as the tasks of managers and workers would be better defined. time and motion studies analyzed work tasks to seek more efficient methods of work in factories. motivation of workers was seen to be related to payment by piecework and economic self-interest to maximize productivity. taylor sought to improve the productivity of each worker and to make management more efficient in order to increase earnings of employers and workers. he found that the worker was more efficient and productive if the worker was goal oriented rather than task oriented. this approach dominated organization theory during the early decades of the twentieth century. resistance to taylor's ideas came from both management and labor; the former because it seemed to interfere with managerial prerogatives and the latter because it expected the worker to function at top efficiency at all times. however, taylor's work had a lasting influence on the theory of work and organizations. the traditional pyramidal bureaucratic organization is classically seen in the military and civil services, but also in large-scale industry, where discipline, obedience, and loyalty to the organization are demanded, and individuality is minimized. this form of organization was analyzed by sociologist max weber between and . leadership is assigned by higher authority, and is presumed to have greater knowledge than members lower down in the organization. this form of organization is effective when the external and internal environments, the technology, and functions are relatively well defined, routine, and stable. the pyramidal system (figure . ) has an apex of policy and executive functions, a middle level of management personnel and support staff, and a base of the people who produce the output of the organization. the flow of information is generally one way, from the bottom to the top level, where decisions are made for the detailed performance of duties at all levels. lateralizing the information systems so that essential data can be shared to help staff at the middle and field or factory-floor levels of management is generally discouraged because this may promote decentralized rather than centralized management. even these types of organization have increasingly come to emphasize small-group loyalty, leadership initiative, and self-reliance. the bureaucratic organization has the following characteristics: l there is a fixed division of labor with a clear jurisdiction and based on assignments, which are subject to change by the leader. l there is a hierarchy of offices, with each lower functionary controlled and supervised by a higher one. employment is viewed as a tenured career for officials, after an initial trial period. the bureaucratic system, based on formal rationality, structure, and discipline, is widely used in production, service, and governmental agencies, including military and civilian departments and agencies. health systems, like other organizations, are dynamic and require continuous management, adjustment, and systems control. continuous monitoring and feedback, evaluation, and revision help to meet individual and community needs. the input-process-output model (figure . ) depends on feedback systems to make the administrative or educational changes needed to keep moving towards the selected objectives and targets. organizations use resources or inputs that are processed to achieve desired results or outputs. the resource inputs are money, personnel, information, and supplies. process is the accumulation of all activities taken to achieve the results intended. output, or outcome, is the product, its marketing, its reputation and quality, and profit. in a service sector such as health, output or impact can be measured in terms of reduced morbidity and/or mortality, improved health, or number of successfully treated and satisfied patients at affordable costs. the management system provides the resources and organizes the process by which it hopes to achieve the established goals. program implementation requires systematic feedback for the process to work effectively. when targets are set and strategy is defined, resources, whether new or existing, are placed at the service of the new program. management is then responsible for using the resources to achieve the intended targets. the results are the outcome or output measures, which are evaluated and fed back to the input and process levels. health systems consist of many subsystems, each with an organization, leaders, goals, targets, and internal information systems. subsystems need to communicate within themselves, with peer organizations, and with the macro (health) system. leadership style is central to this process. the surgeon as the leader of the team in the operating room depends on the support and judgment of other crucial people on the team, such as anesthesiologists, operating room nurses, pathologists, radiologists, and laboratory services, all of whom lead their own teams. hospital and public health directors cannot function without a high degree of decentralized responsibility and a creative team approach to quality development of the facility. health systems management includes analysis of service policy, budget, decision-making in policy, as well as operation, regulation, supervision, provision, maintenance, ethical standards, and legislation. policy formulation involves a set of decisions made in pursuit of a course of action for achieving selected health targets, such as those in the mdgs or continuing to update healthy people health targets in the usa (see chapter ). cybernetics, a term coined by norbert wiener, refers to systems or organizations which are dependent on each other to function, and whose interdependence requires flexibility of response. cybernetics gained wide credence in engineering in the early s, and feedback systems became part of standard practice of all modern management systems. its later transformations appeared in operating service systems, as information for management. application of this concept is entering the health sector. rapid advances in computer technology, by which personal computers have access to internet systems and large amounts of data, have already enhanced this process. in mechanistic systems, the behavior of each unit or part is constrained and limited; in organic systems, there is more interaction between parts of the system. the example used in figure . is the use of a thermostat to control the temperature and function of a heater according to conditions in the room. this is also described as a feedback system. cybernetics opens up new vistas on the use of health information for managing the operation of health systems. a database for each health district would allow assessment of current epidemiological patterns, with appropriate comparisons to neighboring districts or regional, state, and national patterns. data would need to be processed at state or national levels in comparable forms for a broad range of health status indicators. furthermore, the data should be prepared for online availability to local districts in the form of current health profiles. thus, data can be aggregated and disaggregated to meet the management needs of the service, and may be used to generate real targets and measure progress towards meeting them. a geographic information system may demonstrate high rates of a disease in a region due to local population risk factors, and thus become the basis for an intervention program. in the health field, the development of reporting systems based on specific diseases or categories has been handicapped by a lack of integrative systems and a geographic reporting approach. the technology of computers and the internet should be used to process data systems in real time and in a more user-friendly manner. this would enable local health authorities and providers to respond to actual health problems of the communities. health is a knowledge-based service industry, so that knowledge management and information technology are extremely important parts of the new public health, not only in patient care systems in hospitals, but also in public health delivery systems in the community, school, place of work, and home. mobilization of evidence and experience of best practices for policies and management decisionmaking is a fundamental responsibility of health leaders. the gap between information and action is wide and presents an ethical as well as a political challenge. regions with the most severe health problems lack trained personnel in assessment and exploitation of current state-of-the-art practices and technology in many practical public health fields, including immunization policy and in management of risk factors for stroke. knowledge and evidence are continuously evolving, but the capacity to access and interpret information is commonly poorly implemented in many countries so that very large numbers of people die of preventable diseases even when there are, overall, sufficient resources to address the challenges. international guidelines are vital to help countries to adopt current standards and make use of the available knowledge for public policy. political support and openness to international norms are crucial to this process of technology diffusion and building the physical and human resource infrastructure needed to achieve better population health with current best practices. development of health standards in low-income countries is progressing but is seriously handicapped by low levels of funding, lack of emphasis on training sufficient and appropriate human resource personnel and administrative support to promote measures which can save millions of lives. in high-income countries, the slow adoption of best international health standards can have harsh effects on population health, such as in the long delay in adopting national health insurance in the usa. in the european context, the eu has failed to adopt a harmonized recommended immunization program, which is badly needed for the new and potential members, as well as the older member countries. in countries of the former socialist bloc, mortality rates from stroke and coronary heart disease are slowly declining but remain two to four times higher than in countries of western europe (see chapter ). systems management requires access to and the use of knowledge to bridge these gaps. adoption and adaptation of knowledge to address local problems are essential in a globalized world, if only to prevent the international spread of threatened pandemics or adoption of unhealthy lifestyles (diet, smoking, and lack of exercise) to middle-income countries, which are developing a growing middle class alongside massive poverty. the application of knowledge and experience that has been successful in leading countries can foster innovation and create experience that may generate a local renewal process. management is crucial to address the complex "strategy areas for improving performance of health organizations: standards and guidelines, organizational design, education and training, improved process, technology and tool development, incentives, organizational culture, and leadership and management" (bradley et al., ) . managing a knowledge-based service industry or facility relies on leadership, collaboration to realize the potential of technology, professional skills, and social capital to the address the health problems faced by all countries. the management of resources to achieve productivity and measurable success has been characterized and accompanied by the development of systems of organizing people to create solutions to problems or to innovate towards defined objectives. operations research is a concept developed by british scientists and military personnel in search of solutions for specific problems of warfare during world wars i and ii. the approach was based on the development of multidisciplinary teams of scientists and personnel. the development of the anti-submarine detection investigation committee for underwater detection of submarines during world war i characterized and pioneered this form of research. the famous bletchley park enigma code-breaking success in britain and the manhattan project, in which the usa assembled a powerful research and development team which produced the atomic bomb, are prime world war ii examples. team-and goal-oriented work was very effective in problem solving under the enormous pressure of wartime needs. it also influenced postwar approaches to developmental needs in terms of applied science in such areas as the aerospace and computer industries. the computer hardware and software industries are characterized by innovation conceived and developed through informal working groups with a high level of individual competence, peer group dynamism, and commitment to problem solving. thus, the "nerds" of macintosh and microsoft beat the "suits" of ibm in innovation and introduction of the personal computer. similar startup groups, such as google and facebook, successfully took the internet to startling new levels of global applications, showing the capacity of innovation from california's silicon valley and its counterparts in other places in the usa and worldwide. in the health field, innovation in organization developed prepaid group practice which became the health maintenance organization (hmo), and later the managed care organization (mco), now a major, if controversial, factor in health care provision in the usa. other examples may be found in multidisciplinary research teams working on vaccines or pharmaceutical research, and in the increasingly multidisciplinary function of hospital departments and especially highly interdependent intensive care or home care teams. the business concept of management by objectives (mbo), pioneered in the s, has become a common theme in health management. mbo is a process whereby managers of an enterprise jointly identify its goals, define each individual's areas of responsibility in terms of the results expected, and use these measures as guides for operating the unit and assessing the contributions of its members. the common goals and then the individual unit goals must be established, as well as the organizational structure developed to help achieve these goals. the goals may be established in terms of outcome variables, such as defined targets for reduction of infant or maternal mortality rates. goals may also be set in terms of intervening or process variables, such as achieving percent immunization coverage, prenatal care attendance, or screening for breast cancer and mammography. achievements are measured in terms of relevancy, efficiency, impact, and effectiveness. the mbo approach has been subject to criticism in the field of business management because of its stress on mechanical application of quantitative outcome measures and because it ignores the issue of quality. this approach had great influence on the adoption of the objective of "health for all" by the who, and on the us department of health and human services' health targets for the year , later as healthy people , and now, based on these experiences and new evidence, renewed as healthy people . targeting diseases for eradication may contribute to institution building by developing experience and technical competence to broaden the organizational capacity. however, categorical programs or target-oriented programs can detract from the development of more comprehensive systems approaches. addressing the mdgs of reducing child and maternal mortality is at odds to some extent with targeting poliomyelitis for eradication and reliance on national immunization days, which distract planning and resource allocation for the buildup of the essential public health infrastructure for the basic immunization system so fundamental to child health. immunization and human immunodeficiency virus (hiv) control draw the major part of donor resources in developing countries, while education for strengthening human resources and infrastructure draw less donor attention. a balance between comprehensive and categorical approaches requires very skilled management. the mdgs agreed to by the un in as targets for the year provide a set of measurable objectives and a formula for international aid and for national development planning to help the poorest nations, with the wealthy nations providing aid, education, debt relief, and economic development through fairer trade practices. they are now being reviewed for extension to based on experience to date, with successes and failures, and recognizing the vital importance of non-communicable diseases as central to the health burden of low-and middleincome countries. management is the activity of coordinating and integrating organizational resources, including people, money, materials, time, and space. the purpose is to achieve defined/ stated objectives as effectively and efficiently as possible. whether in terms of producing goods and profits or in delivering services effectively, management deals with human motivation and behavior because workers are the key to achieving goals. knowledge and motivation of the individual client and the community are also essential for achieving good health. thus, management must take into account the knowledge, attitudes, beliefs, and practices of the consumer as much as or more than those of the people working within the system, as well as the general cultural and knowledge level in the society, as reflected in the media, political opinions, and organizations addressing the issues. management, like medicine, is both a science and an art. the application of scientific knowledge and technology in medicine involves both theory and practice. similarly, management practice involves elements of organizational theory, which, in turn, draws on the behavioral and social sciences and quantitative methodologies. sociology, psychology, anthropology, political science, history, and ethics contribute to the understanding of psychosocial systems, motivation, status, group dynamics, influence, power, authority, and leadership. quantitative methods including statistics, epidemiology, survey methods, and economic theory are also basic to development of systems concepts. comparative institutional analysis helps principles of organization and management to develop, while philosophy, ethics, and law are part of understanding individual and group value systems. organizational theory, a relatively new discipline in health, as an academic study of organizations, addresses health-related issues using the methods of economics, sociology, political science, anthropology, and psychology. the application of organizational theory in health care has evolved and become an integral part of training for, and the practice of, health administration. related practical disciplines include human resources, and industrial and organizational psychology. translation of organizational theory into management practice requires knowledge, planning, organization, mobilization of professional and other staff support for evidence-based best practices, assembly of resources, motivation, monitoring and control. health organizations have become more complex and costly over time, especially in their mix of specializations in science, technology, and professional services. organization and management are particularly crucial for successful application of the principles of the new public health, as it involves integration of traditionally separate health services. delegation of responsibilities in health systems, such as in intensive care units, is fundamental to success in patient care, with nurses taking increasing responsibility for the management of the severely ill patient suffering from multiple system failure. delegation or devolution of health care responsibilities to non-medical practitioners has been an ongoing development affecting nurse practitioners, physician assistants, paramedics, community health workers and others, as discussed in chapter . it is a vital process to provide needs not met by physicians because of shortages and inappropriate location or specialty preferences that leave primary care or other medical specialties unable to meet community and patient needs. elton mayo of the harvard school of business carried out a series of observational studies at the hawthorne, illinois, plant of the western electric company between and . mayo and his industrial engineer, along with psychologist colleagues, made a major contribution to the development of management theory. mayo began with industrial engineering studies of the effect of increased lighting on production at an assembly line. this was followed by other improvements in working conditions, including reduced length of the working day, longer rest periods, better illumination, color schemes, background music, and other factors in the physical environment. these studies showed that production increased with each of these changes and improvements. however, the researchers discovered, to their surprise, that production continued to increase when the improvements were withdrawn. furthermore, in a control group where conditions remained the same, productivity also grew during the study period. these results led mayo to conclude that the performance of workers improved because of a sense that management was interested in them, and that worker participation contributes to improved production. traditionally, industrial management viewed employees as mechanistic components of a production system. previous theory was that productivity was a function of working conditions and monetary incentives. what came to be known as the hawthorne effect showed the importance of social and psychological factors on productivity. formal and informal social organizations among management and employees were recognized as key elements in productivity, now called industrial humanism. research methods adapted from the behavioral sciences contributed to scientific studies in industrial management. traditional theories of the bureaucratic model of organization and management were modified by the behavioral sciences. this led to the emergence of the systems approach, or scientific analysis to analyze complex structures or organizations, taking into account the mutually interdependent elements of activities, interactions, and interpersonal relationships between management and workers. some revisits to the hawthorne studies suggest that the data do not support the conclusions, and offer a different interpretation. one is that informal groups such as workers on a production line themselves set standards for work which assert an informal social control outside the authority system of the organization. the informal cohesive group can thus control the norms of the amount of work acceptable to the group, i.e., not "too much" and not "too little". others point out that the effects were temporary and that there were extraneous factors, but the added value of the hawthorne effect remains part of the history of and had a culturechanging effect on management theory. the hawthorne effect in management is in some ways comparable to the placebo effect in clinical research and health care practice. it is also applied to clinical practice, whereby medical care provided by doctors is measured for specific "tracer conditions" to assess completeness of care according to current clinical guidelines. review of clinical records has been shown to be a factor in improving performance by doctors in practice, such as in treatment of acute myocardial infarction, management of hypertension, or completeness of carrying out preventive procedures such as screening for cancer of the cervix, breast, or colon (see chapters and ). awareness of being studied is a factor in improved performance or response to an intervention. studies of clinical practice-based research or public health interventions need to consider whether different types of studies and outcomes are more or less susceptible to the hawthorne effect (fernald et al., ). abraham maslow's hierarchy of human needs made an important contribution to management theory. maslow was an american psychologist, considered "the father of humanism" in psychology. maslow defined a prioritization of human needs (figure . ), starting with those of basic physical survival; at higher levels, human needs include social affiliation, self-esteem, and self-fulfillment. others in the hierarchy include socialization and self-realization; later revisions include cognitive needs. the survival needs of an employee include a base salary and benefits, including health insurance and pension; the safety and security needs include protection from injury, toxic exposure or excess stress; social needs at work include an identity, pride, friendships, union solidarity, company social activities and benefits; esteem and recognition include job titles, awards, and financial rewards for achievement by individuals, groups, or all employees; and self-actualization includes promotion to more challenging jobs with benefits, both financial and in terms of recognition. this concept is important in terms of management because it identifies human needs beyond those of physical and economic well-being. it relates them to the social context of the work environment with needs of recognition, satisfaction, self-esteem, and self-fulfillment. maslow's conclusions opened many positive areas of management research, not only in the motivation of workers in production and service industries, but also in the motivation of consumers. maslow's hierarchy of human needs contributed to the idea that workers' sense of well-being is important to management. his theories played an important role in application of sociological theory to client behavior, just as the topic of personal lifestyle in health became a central part of public health and clinical management of many conditions, such as in risk factor reduction for cardiovascular diseases. this concept fits well with the epidemiological studies referred to in the introduction, such as those showing strong relationships with sociopolitical factors as well as socioeconomic conditions. theory x-theory y (table . ), developed by clinical psychologist and professor of management douglas mcgregor in the s, examined two extremes in management assumptions about human nature that ultimately affect the operations of organizations. organizations with centralized decision-making, a hierarchical pyramid, and external control are based on certain concepts of human nature and motivation. mcgregor's theory, drawing on maslow's hierarchy of needs, describes an alternative set of assumptions that credit most people with the capacity for self-direction. traditional approaches to organization and management stress direction and external control. theory x assumes that workers are lazy, unambitious, uncreative, and motivated only by basic physiological needs or fear. theory y places stress on integration and self-control. this model provides a more optimistic leadership model, emphasizing management development programs and promoting human potential, assuming that, if properly motivated, people can be self-directed and creative at work, and that the role of management is to unleash this potential in workers with performance appraisal. many other theories of motivation and management have been developed to explain human behavior and how to utilize inherent skills to produce a more creative work environment, reduce resistance to change, reduce unnecessary disputes, and ultimately create a more effective organization. variants of the human motivation approach in management carried the concept further by examining industrial organization to determine the effects of management practices on individual behavior and personal growth within the work environment. they describe two contrasting models of workforce motivation. theory x assumes that management produces immature responses on the part of the worker: passivity, dependence, erratically shallow interests, shortterm perspective, subordination, and lack of self-awareness. in contrast, at the other end of the immaturity-maturity spectrum was the mature worker, with an active approach, an independent mind capable of a broad range of responses, deeper and stronger interests, a long-term perspective, and a high level of awareness and self-control. this model has been tested in a variety of industrial settings, showing that giving workers the opportunity to grow and mature on the job helps them to satisfy more than basic survival needs and allows them to use more of their potential in accomplishing organizational goals. this model became widely influential in human resource management theory of organizational behavior, organizational communication, and organizational development, and in the practical management of business and service enterprises. in the motivation to work ( ) , us clinical psychologist frederick herzberg wrote of his motivationhygiene theory. he developed this theory after extensive studies of engineers and accountants, examining what he called hygiene factors (i.e., administrative, supervisory, monetary, security, and status issues in work settings). his motivating factors included achievement, recognition of accomplishment, challenging work, and increased responsibility with personal and collective growth and development. he proved that the motivating factors had a substantial positive effect on job satisfaction. these human resource theories of management helped to change industrial approaches to motivation from "job enrichment" to a more fundamental and deliberate upgrading of responsibility, scope, and challenge of work, by letting workers develop their own ways of achieving objectives. even when the theories were applied to apparently unskilled workers, such as plant janitors, the workers changed from an apathetic, poorly performing group into a cohesive, productive team, taking pride in their work and appearance. this approach gave members of the team the opportunity to meet their human self-actualization needs by taking greater responsibility for problem solving, and it resulted in less absenteeism, higher morale, and greater productivity with improved quality. rensis likert, with mcdougal and herzberg, helped to pioneer the "human relations school" in the s, applying human resource theory to management systems and styles. likert classified his theory into four different systems, as follows. l system -management has no confidence or trust in subordinates, and avoids involving them in decisions and goal setting, which are made from the top down. management is task oriented, highly structured, and authoritarian. fear, punishment, threats, and occasional rewards are the principal methods of motivation. worker-management interaction is based on fear and mistrust. informal organizations within the system often develop that lead to passive resistance of management and are destructive to the goals of the formal organization. l system -management has a condescending relationship with subordinates, with some degree of trust and confidence. most decisions are centralized, but some decentralization is permitted. rewards and punishments are used for motivation. informal organizations become more important in the overall structure. l system -management places a greater degree of trust and confidence in subordinates, who are given a greater degree of decision-making powers. broad policy remains a centralized function. l system -management is seen as having complete confidence in subordinates. decision-making is dispersed, and communication flows upward, downward, and laterally. economic rewards are associated with achieving goals and improving methods. relationships between management and subordinates are frequent and friendly, with a sense of teamwork and a high degree of mutual respect. case studies showed that a shift in management from likert system towards system radically changed the performance of production, cut manufacturing costs, reduced staff turnover, and increased staff morale. furthermore, workers and managers both shared a concern for the quality of the product or service and the competitiveness and success of their business. the health industry includes highly trained professionals and paraprofessional workers who function as a team with a high degree of cohesion, mutual dependence, and autonomy, such as a surgical or an emergency department team. the network, or task-oriented working group, is basically a more democratic and participatory form of organization meant to elicit free interchange of concerns and ideas. this is a more organic form of organization, best suited to be effective for adaptation when the environment is complex and dynamic, when the workforce is largely professional, and when the technology and system functions change rapidly. complexities and technological change require information, expertise, flexibility, and innovation, strengths best promoted in free exchange of ideas in a mutually stimulating environment. in a network organization, leadership may be formal or informal, assigned to a particular function, which may be temporary, medium term, or permanent, to achieve a single defined task or develop an intersectoral program. the task force is usually for a short-term specific assignment; a working group, often for a medium-term project, such as integrating services of a region; and a committee for permanent tasks such as monitoring an immunization program. significant advantages of this form of organization are the challenge and the sharing of information and responsibility, which give professionals responsibility and job satisfaction by providing the opportunity to demonstrate their creativity. members of the task force may each report within their own pyramidal structure, but as a group they work to achieve the assigned objective. they may also be interdisciplinary or interagency working groups to review the state of the art in this particular issue as documented in reports and professional literature, and to coordinate activities, review previous work, or plan common future activities. an ongoing network organization may be a government cabinet committee to coordinate government policy and the work of various government departments, or a joint chiefs of staff to coordinate the various armed services. this approach is commonly used for task groups wherein interdisciplinary teams of professionals meet to coordinate functions of a department in a hospital, or where a multidisciplinary group of experts is established with the specified task of a technical nature. network organizational activity is part of the regular functions of a health professional. informal networking is a day-to-day activity of a physician in consultations with colleagues and also a part of more formalized network groups. the hospital department must, to a large extent, function as a network organization with different professionals working as a team more effectively than would be possible in a strictly authoritarian pyramidal model. a ministry of health may need to develop a joint working group with the ministry of transport, the police, and those responsible for standards of motor vehicles to seek ways to reduce road accident deaths and injuries. if a measles eradication project is envisioned, a multidisciplinary and multiorganizational team, or a network, should be established to plan and carry out the complex of tasks needed to achieve the target (figure . ) . in a public health context, a task group to determine how to reduce obesity rates in school-aged children, or to eradicate measles locally, might be chaired by the deputy chief medical officer or senior health promotion person; if the project is reduction of obesity among school children, the lead agency may be the department of education, perhaps jointly with the local department of health; if reduction in road traffic deaths is the topic, the lead may be the police department with participation of emergency transportation and hospital emergency room lead personnel. members may include the chief district nurse, an administrative and budget officer, a pharmacist, the chief of the pediatric department of the district hospital, a primary school administrator, a health educator, a medical association representative, the director of laboratories, the director of the supply department, a representative of the department of education, representatives of voluntary organizations interested in the topic, and others as appropriate. most organizational structures are mixed, combining elements of both the formal pyramidal and the less structured network structure with a task-oriented mandate. it is often difficult for a rigid pyramidal structure to deal with parallel bodies in a structured way, so the network approach is necessary to establish working relations with outside bodies to achieve common goals. a network is a democratic functional grouping of those professionals and organizations needed to achieve a defined target, sometimes involving people from many different organizations. the terms of reference of the working group are crucial to its function as well as its composition, time-frame, and access to relevant information. the application of this concept is increasingly central in health care organization as multilevel health systems evolve in the form of managed care or district health systems. these are vertically integrated management systems involving highly professional teams and units whose interdependence for patient care and financial responsibility are central elements of the new public health. in the usa during world war ii, w. edwards deming, a physicist and statistician, developed a system of economic and statistical methods of quality control in production industries. following the war, deming was invited to teach in japan and moved from the university to the level of industrial management. japanese industrialists adopted his principles of management and introduced quality management into all industries, with astonishingly successful results within a decade. the concept, later called total quality management (tqm), has since been adopted widely in production and service industries. in the deming approach to company management, quality is the top priority and is the key responsibility of management, not of the workers. if management sets the tone and involves the workers, quality goes up, costs come down, and both customer satisfaction and loyalty increase. having their ideas listened to, and avoiding a punitive inspection approach, enhances the pride of the workers. it is the responsibility of leadership to remove fear and build mutual participation and common interest. training is one of the most important investments of the organization. the differences between traditional management and the tqm approach are shown in boxes . and . . in societies with growing economies, the role of an educated workforce becomes greater as information technology and services, such as health, become larger parts of the economy and require professionalism and self-motivating workers. the tqm approach integrates the scientific management and human relations approaches by giving workers credit for intellectual capacity and expects them to use it to analyze and improve the tasks they perform. even more, this approach expects workers at all levels to contribute to better quality in the process of design, manufacture, and even marketing of the product or the service. the tqm ideas were revolutionary and successful when applied in business management in production industries. the tqm concept is much in discussion in the service industries. the who has adapted tqm to a model called continuous quality improvement (cqi), with the stress on mutual responsibilities throughout a health system for quality of care. the application of tqm and cqi approaches is discussed in chapter , including the external regulatory and self-development tqm approaches. in the health sector, issues such as prevention of health facility-acquired infections require staff dedicated to promoting a culture of cleaning, frequent and thorough hand washing, sterilization, isolation techniques, intravenous and intratracheal catheter and tube care technique, and immunization of hospital personnel. these and many other crossdisciplinary measures promote patient safety and prevent the costly and frequently deadly effects of serious respiratory or urinary tract injection acquired in hospitals or other health care facilities. human behavior is individual but takes place in a social context. changes to individual behavior are needed to reduce risk factors for many diseases. change can be threatening; it requires alteration, substitution, transformation, or modification of purposes, procedures, methods, or style. the implementation of plans usually requires some change, which often meets resistance. the resistance to change may be professional, technical, psychological, political, emotional, or a mix of all of these. the manager of a health facility or service has to cope with change and gather the support of those involved to participate in creating or implementing the change effectively. the behavior of the worker in a production or service industry is vital to the success of the organization. equally important is the behavior of the purchaser or consumer of the product or service. diagnosing organizational problems is an important skill to bring to leadership in health systems. even more important is the ability to identify and alter the variables that require change and adaptation to improve the performance of the organization. high expectations are essential to produce high performance and improved standards of service or productivity. conversely, low expectations not only lead to low performance, but produce a downward spiraling effect. this applies not only within the organization, but to the individuals and community served, l judgment, punishment, and reward for above-or belowaverage performance destroy teamwork essential for quality production. l work with suppliers to improve quality and costs. l profits are generated by loyal customers -running a company for profit alone is like driving a car by looking in the rearview mirror. whether in terms of purchase of goods produced or in terms of health-related behavior. people often resist change because of fear of the unknown. participation in the process of defining problems, formulating objectives, and identifying alternatives is needed to bring about changes. change in organizational performance is complex, and this is the test of leadership. similarly, change at the individual level is essential to achieve the goals of the group, whether this is in terms of the functioning of a health care service unit, such as a hospital, or whether it is an individual's decision to change from smoking to non-smoking status. the health of both an individual and a population depends on the individual health team member's motivation and experience. the behavior of the individual is important to his or her personal and community health. even small steps in the direction of a desirable change in behavior should be rewarded as soon as possible (i.e., reinforcing positive performance in increments). behavior modification is based on the concept that change of behavior starts with the feelings and attitudes within the individual, but can be influenced by knowledge, peer pressure, media coverage, and legislative standards. change involves a number of elements to define a current or previous starting point: change in behavior is vital in the health field: in the organization, in the community, in individual behavior, and in societal regulation and norms. the health belief model (chapter ) is widely influential in psychology and health promotion. the belief intervention approach involves programs meant to reduce risk factors for a public health problem. it may require change in the law and in organizational behavior, with involvement and feedback to the people who determine policy, those who manage services, and the community being served. obesity in school-aged children is being fought by many measures including healthier menus and banning the sale of high sugar drinks on school property. high cholesterol is being fought on many fronts including dietary change and banning the use of transfats in food processing. deaths from bulimia are not uncommon and may stem from teenage identification of beauty with ultrathin body image. banning television and modeling agencies from using models with a very low body mass index is an intervention in advertising which encourages harmful practices that are a danger to health and life. banning cigarette advertising and smoking in public places promotes behavioral change, as does raising the taxes on cigarettes. gun control laws are meant to prevent disturbed individuals or political fanatics having easy access to firearms to commit mass murder. strict enforcement of drinking and driving laws can prevent drunk driving and reduce road traffic deaths (see chapter ). in the s, major industries in the usa were unable to compete successfully with the japanese in the consumer electronics and automobile industries. management theory began to place greater emphasis on empowerment as a management tool. the tqm approach stresses teamwork and involvement of the worker in order to achieve better quality of production. comparatively, empowerment went further to involve the worker in operation, quality assessment, and even planning of the design and production process. results in production industries were remarkable, with increased efficiency, less absenteeism, and greater searching for ideas to improve quality and quantity of production, with the worker as a participant in the management and production process. the concept of empowerment entered the service industries with the same rationale. the rationale is that improvements in quality and effectiveness of service require the active physical and emotional participation of the worker. participation in decision-making is the key to empowerment. this requires management to adopt new methods that allow the worker, whether professional or manual, to be an active participant. successful application of the empowerment principles in health care extends to the patient, the family, and the community, emphasizing patients' rights to informed participation in decisions affecting their medical care, and the protection of privacy and dignity. diffusion of powers occurs when management of services is decentralized. delegation of powers to professional groups, non-governmental organizations (ngos), and advocacy organizations is part of empowerment in health care organizations. governmental powers to govern or promote areas such as licensure, accreditation, training, research, and service can be devolved to local authorities or ngos by delegation of authority or transfer of funds. organizational change may involve decentralization. institutional changes such as amalgamation of hospitals, long-term care facilities, home care programs, day surgery, ambulatory care, and public health services are needed to produce a more effective use of resources. integration of services under community leadership and management should encourage transfer of funds within a district health network from institutional care to community-based care. such changes are a test of leadership skills to achieve cultural change within an organization, which requires behavioral change and involvement of health workers in policy and management of the change process. strategic management emphasizes the importance of positioning the organization in its environment in relation to its mission, resources, consumers, and competitors. it requires development of a plan of action or implementation of a strategy to achieve the mission or goal of the organization within acceptable ethical and legal guidelines. articulation of these is a key role of the management level of an organization. defining the mission and goals of the organization must take into account the external and internal environment, resources, and operational needs to implement and evaluate the adequacy of the outcomes. the strategy of the organization matches its internal approach with external factors, such as consumer attitudes and competing organizations. strategy is a set of methods and skills of the health care manager to attain the objectives of a health organization, including: policy is the formulation of objectives and priorities. strategy refers to long-range plans to achieve stated objectives, indicating the problems to be expected and how to deal with them. strategy does not identify all actions to be taken, but it includes evaluation of progress made towards a stated goal. while the term has traditionally been used in a military context, it has become an essential concept in management, whether of industry, business, or health care. tactics are the methods used to fulfill the strategy. thus, strategic mbo is applicable to the health system, incorporating definitions of goals and targets, and the methods to achieve them (box . ). change in health organizations may involve a substantial alteration in the size or relationships between existing, well-established facilities and programs (table . ). a strategic plan for health reform in response to the need for cost containment, redefined health targets, or dissatisfaction with the status quo requires a model or a vision for the future and a well-managed program. opposition to change may occur for psychological, social, and economic reasons, or because of fear of loss of jobs or changes in assignments, salary, authority, benefits, or status. downsizing in the hospital sector, with buildup of community health services, is one of the major issues in health reforms in many countries. it can be accomplished over time by naturally occurring vacancies or attrition due to retirement, or by retraining and reassignment, all of which require skilled leadership. the introduction of new categories of health workers in hospitals such as phlebotomists, hospitalist doctors, and technicians of all kinds has improved hospital efficiency and safety. community health has benefited from home care and in many situations community health workers to assist and supervise patient care in remote rural villages and in urban centers, even in high-income countries, with health guides trained to help people to function with chronic illnesses and dementias (see chapter ). the new public health is an integration or coordination of many participating health care facilities and health-promoting programs. it is evolving in various forms in different places as networks with administrative and financial interaction between participating elements. each organization provides its own specific services or groups of services. how they function internally and how they interact functionally and financially are important aspects of the management and outcomes of health systems. the health system functions as a network with formal and informal relationships; it may be very broad and loosely connected as in a highly decentralized system, with many lines of communication, payment, regulation, standards setting, and levels of authority. the relationship and interchange between different health care providers have functional and economic elements. as an example, an educated adult woman is more likely than an uneducated woman to prepare herself for the requirements of pregnancy by smoking and alcohol or drug cessation, folic acid intake, healthful diet, and attending professional antenatal care. a pregnant woman who is healthy and prepared for pregnancy physically and emotionally, and who receives comprehensive prenatal care, is less likely than a woman whose health is neglected to develop complications and require prolonged hospital care as a result of childbirth. the cost of good prenatal care is a fraction of the economic cost of treating the potential complications and damage to her health or that of the newborn. a health system is responsible for ensuring that a woman of reproductive age takes folic acid tablets orally before becoming pregnant, has had access to family planning services so that the pregnancy is a desired one, ensures that the space between pregnancies is adequate for her health and that of her baby, and receives adequate prenatal care. an obstetrics department should be involved in assuring or providing the prenatal care, especially for high-risk cases, and delivery should be in hygienic and professionally supervised settings. similarly, for children and elderly people, there is a wide range of public health and personal care services that make up an adequate and cost-effective set of services and programs. the economic burden of caring for the sick child falls on the hospital. when there is a per capita grant to a district, the hospital and the primary care service have a mutual interest in reducing morbidity and hence mortality. this is the principle of the hmos and district health systems discussed elsewhere. it is also a fundamental principle of the new public health. health care organizations differ according to size, complexity, ownership, affiliations, types of services, and location. traditionally, a health care organization provides a single type of service, such as an acute care hospital providing episodic inpatient care, or a home health care agency. in present-day health reforms, health care organizations, such as an hmo or a district health system, provide a populationbased, comprehensive service program. each organization must have or develop a structure suited to meet its goals, in both the internal and external environments. the common elements that each organization must deal with include governance of policy, production or service, maintenance, financing, relating to the external environment, and adapting to changing conditions. a functional model of an organization perhaps best suited to the smaller hospital is the division of labor into specific functional departments; for example, medical, nursing, administration, pharmacy, maintenance, and dietary, each reporting through a single chain of command to the chief executive officer (ceo) (figure . ) . the governing agency, which may be a local non-profit board or a national health system, has overall legal responsibility for the operation and financial status of the hospital, as well as raising capital for improvements. the medical staff may be in private practice and work in the hospital with their own patients by application for this right as "attending physician", according to their professional qualifications, or the medical staff may be employed by the hospital in a similar way to the rest of the staff. salaried medical staff may include physicians in administration, pathology, anesthesia, and radiology, so that even in a private practice market system many medical staff members are hospital employees. increasingly, hospitals are employing "hospitalists", who are full-or part-time physicians whose work is in the health facility, to provide continuity of inpatient and emergency department services, augmenting the services of senior or attending staff or private practice physicians. this shift is in part related to the increasing numbers of female physicians who run their homes and families as well as practice medicine and who find this mode of work more attractive than full-time private practice. this model is the common arrangement in north american hospitals. the governing board of a "voluntary", nongovernmental, not-for-profit organization with municipal and community representatives may be appointed by a sponsoring religious, municipal, or fraternal organization. the corporate model in health care organization (figure . ) is often used in larger hospitals or where mergers with other hospitals or health facilities are taking place. the ceo delegates responsibility to other members of the senior management team who have operational responsibility for major sectors of the hospital's functioning. a variation of the corporate model is the divisional model of a health care organization based on the individual service divisions allowing middle management a high degree of autonomy (figure . ). there is often departmental budgeting for each service, which operates as an economic unit; that is, balancing income and expenditures. each division is responsible for its own performance, with powers of strategic and operational decision-making authority. this model is used widely in private corporations, and in many hospitals in the usa. with increasing complexity of services, it is also employed in corporate health systems in the usa, with regional divisions. the matrix model of a health care organization is based on a combination of pyramidal and network organization. this model is suited to a public health department in a state, county, or city. individual staff people report in the pyramidal chain of command, but also function in multidisciplinary teams to work on specific programs or projects. a nutritionist in the geriatric department is responsible to the chief of nutrition services but is functionally a member of the team on the geriatric unit. in a laterally integrated health maintenance organization or district health system, specialized staff may serve in both institutional (i.e., hospital) and community health roles (figure . ) . the organizational structure appropriate to one set of circumstances may not be suitable for all. whether the payment system is by norm (i.e., by predetermined numbers of staff, their salaries, and fixed costs for all services), per diem (i.e., payment of a daily rate times the number of days of stay), historical budget, or per capita in a regional or district health system structure (see chapters and ), the internal operation of a hospital will require a model of organization appropriate to it. hospitals need to modify their organizational structure as they evolve, and as the economics of health care change. leadership in an organization requires the ability to define the goals or mission of the organization and to develop a strategy and define steps needed to achieve these goals. it requires an ability to motivate and engender enthusiasm for this vision by working with others to gain their ideas, their support, and their participation in the effort. in health care as in other organizations, it is easier to formulate plans than to implement them. change requires the ability not only to formulate the concept of change, but also to modify the organizational structure, the budgeted resources, the operational policies and, perhaps most importantly, the corporate culture of the organization. management involves skills that are not automatically part of a health professional's training. skilled clinicians often move into positions requiring management skills in order to build and develop the health care infrastructure. in some countries, hospital managers must be physicians, often senior surgeons. clinical capability does not transfer automatically into management skills to deal with personnel, budgets, and resources. therefore, training in management is vital for the health professional. the manager needs training for investigations and factfinding as well as the ability to evaluate personnel, programs, and issues, and set priorities for dealing with the short-and long-term issues. negotiating with staff and outside agencies is a constant activity of the manager, ranging from the trivial to major decisions with wide implications. perhaps the most crucial skill of the manager is communication: the ability to convey verbal, written, or unwritten messages that are received and understood and to assess the responses as an equal part of the exchange. interpersonal skills are a part of management practice. the capable manager can relate to personnel at all levels in an open and equal manner. this skill is essential to help foster a sense of pride and involvement of all personnel in working towards the same goals and objectives, and to show that each member of the team is important to meeting the objectives of the organization. at the same time, the manager needs to communicate information, especially as to how the organization is doing in achieving its objectives. the manager is responsible for organizing, planning, controlling, directing, and motivating. managers assume multiple roles. a role is an organized set of behaviors. henry mintzberg described the roles needed by all managers: informational, interpersonal, and decisional roles. robert katz ( ) identified three managerial skills that are essential to successful management: technical, human, and conceptual: "technical skill involves process or technique knowledge and proficiency. managers use the processes, techniques and tools of a specific area. human skill involves the ability to interact effectively with people. managers interact and cooperate with employees. conceptual skill involves the formulation of ideas. managers understand abstract relationships, develop ideas, and solve problems creatively". technical skill deals with things, human skill concerns people, and conceptual skill has to do with ideas. the distribution of these skills between the levels of management is shown in figure . . hospital directors in the past were often senior physicians, often called superintendents, without training in health management. the business manager ceo has become common in hospital management in the usa. during the s, the ceo was called an administrator, and worked under the direction of a board of trustees who raised funds, set policies, and were often involved in internal administration. where the ceo was a non-physician, the usual case in north american hospitals, a conflict often existed with the clinical staff of the hospital. in some settings, this led to appointment of a parallel structure with a full-time chief of medical staff with a focus on clinical and qualitative matters. in european hospitals, the ceo is usually a physician, often by law, and the integration of the management function with the role of clinical chief is the prevalent model. over time, as the cost and complexity of the health system have increased, the ceo role has changed to one of a "coordinator". the ceo is now more involved in external relations and less in the day-to-day operation of the facility. the ceo is a leader/partner but primus inter pares, or first among equals, in a management team that shares information and works to define objectives and solve problems. this de-emphasizes the authoritarian role and stresses the integrative function. the ceo is responsible for the financial management of operational and capital budgets of the facility, which is integral to the planning and future development of the facility. budgets include four main factors: income, fixed or regular overhead, variable or unpredictable overhead, and capital or development costs, all essential to the survival and development of the organization. the key role of top management is to develop a vision, goals, and targets for the institution, to maintain an atmosphere and systems to promote the quality of care, financial solidity, and to represent the institution to the public. the overall responsibility for the function and well-being of the program is with the ceo and the governing board of directors. community participation in management of health facilities has a long-standing and constructive tradition. the traditional hospital board has served as a mechanism for community participation and leadership in promoting health facility development and management at the community level. the role of hospital boards evolved from primarily a philanthropic and fund-raising one to a greater overall responsibility for policy and planning function working closely with management and senior professional staff. this change occurred as operational costs increased rapidly, as government insurance schemes were implemented, and as court decisions defined the liability of hospitals and reinforced the broadened role of governing boards in malpractice cases and quality assurance. centrally developed health systems such as the uk's nhs have promoted district and county health systems with high degrees of community participation and management, both at the district level and for services or facilities. the role of local authorities, as well as state and national governments, is crucial to the functioning of public health in its traditional issues such as safe water supply, sanitation, business licensing, social welfare, and many others, as discussed in chapter . these functions have not diminished with the greater roles of state and federal or national governments in health. in healthful living environments the local authority functions are of continuing and indeed expanding importance, as in urban planning and transportation, promoting easy access to commercial facilities for shopping and healthy food sources for poorer sections as well as those available to prosperous members of the community. advocacy has always been an important part of public health. an illustration of this is seen in box . in changing the law banning birth control in massachusetts in the s. the issue of birth control still casts a heavy burden on women globally owing to religious objections, so this example from the s is still relevant as a political issue both in the usa and in many other countries. community participation can be crucial to the success of an intervention to promote community health. sensitivity to local, religious, or ethnic concerns is part of planning any study or intervention in public health. this does not mean that the national, state, and local health authorities must continuously canvass public opinion, but there is advantage in holding referenda on some issues compared to governmental fiat. the usa has higher rates of fluoridation than most countries, and this is implemented after referenda in each municipality (see chapter ). in portland, oregon, the city council profluoridation vote in (new york times, september ) was later rejected in the public referendum. portland is the only major american city without fluoridation (portland tribune, may ). rationalization of health facilities increasingly means organizational linkages between previously independent facilities. mergers of health facilities are common events in many health systems. in the usa, there are frequent mergers between hospitals, or between facilities linked to hmos or managed care systems. health reform in many countries is based on similar linkages. governmental approval and alteration to financing systems are needed to promote linkages between services to achieve greater efficiency and improve patient care (see chapters and ). lateral integration is the term used for amalgamation among similar facilities. like a chain of hotels, in health care this involves two or more hospitals, usually meant to achieve cost savings, improve financing and efficiency, and reduce duplication of services. urban hospitals, both notfor-profit as well as for-profit, often respond to competition by purchasing or amalgamating with other hospitals to increase market share in competitive environments. this is often easier for hospital-oriented ceos and staff to comprehend and manage, but it avoids the issues of downsizing and integration with community-based services. vertical integration describes organizational linkages between different kinds of health care facilities to form integrated, comprehensive health service networks. this permits a shift of emphasis and resources from inpatient care to long-term, home, and ambulatory care, and is known as the managed care or district health system model. community interest is a factor in promoting change to integrate services, which can be a major change for the management culture, especially of the hospital. the survival of a health care facility may depend on integration with appropriate changes in concepts of management. in the s, a large majority of california residents moved to managed care programs because of the high cost of fee-for-service indemnity health insurance and because of federal waivers to promote managed care for medicare and medicaid beneficiaries. independent community hospitals without a strong connection to managed care organizations (mcos) were in danger of losing their financial base. hospital bed supplies were reduced in the usa however, the article served to stimulate the legislature to revisit the law, leading to its repeal in , thus allowing use of all methods of birth control. the controversy subsided and women were free to control their own fertility as a result of this advocacy. by diagnosis-related group (drg), rather than on a per diem basis. similar trends are seen in european countries, although in the commonwealth of independent states the number of hospital beds declined between and - but stabilized at high and inefficient levels ( beds per population) compared to the number in western europe, which fell from beds per in to . in , and in some countries to per population despite increased longevity and aging of the population. there was a shift to stronger ambulatory care, as occurred throughout the industrialized countries despite an aging of the population. these trends were largely due to greater emphasis on ambulatory surgery and other care, and major medical centers responded with strategic plans to purchase community hospitals and develop affiliated medical groups and contract relationships with managed care organizations to strengthen their "market share" service population base for the future. the new payment environment and managed care also promoted hospital mergers (lateral integration) and linkages between different levels of service, such as teaching hospitals with community hospitals and primary community care services (vertical integration). vertical integration not only is important in urban areas, but can serve as a basis for developing rural health care in both developed and developing countries. the district hospital and primary care center operating as an integrated program can provide a high-quality program. hospitalcentered health care, common in industrialized countries, has traditionally channeled a high percentage of total health expenditures into hospital services. over recent years, there has been a reduction in hospital bed supply in most industrialized countries, with shorter length of stay, more emphasis on ambulatory care, improved diagnostic facilities, and improved outcomes of care (see chapter ). expenditures on the hospital component of care have come down to between and percent of total health expenditures in many countries, with a growing proportion going to ambulatory and primary care, and increased percentages to public health. this shift in priorities has been an evolutionary process that will continue, but requires skilled management leadership, grounded in health systems management training and epidemiological knowledge, and skilled negotiating skills to foster primary care and health promotion approaches both within the organization and in relation to outside services, especially preventive services. this shift in policy direction will be fostered in implementation of the ppaca (obamacare), discussed in chapters and . managed care systems or accountable care organizations (acos) will integrate hospital and community care and try to limit hospital care by strengthening ambulatory and primary care, and especially preventive care. this will have both economic and epidemiological benefits, but will depend on skilled management to understand and lead in their implementation. much of the rationale for these changes is discussed in the literature and summarized in a report from the us institute of medicine, entitled "best care at lower cost". this report calls for overhauling the health system in a continuous evolution based on evidence and lessons learned from decades of innovative care systems and research into their workings. the health system needs to relate to other community services with a shared population orientation (institute of medicine, ). norms are useful to promote efficient use of resources and promote high standards of care, if based on empirical standards proved by experience, trial and error, and scientific observation. norms may be needed even without adequate evidence, but should be tested in the reality of observation, experience, and experiment. this process requires data for selected health indicators and trained observers free to examine, report, and publish their findings for open discussion among colleagues and peers in proceedings open to the media and the general public. normative standards of planning are the determination of a number per unit of population that is deemed to be suitable for population needs; for example, the number of beds or doctors per population or length of stay in hospital. many organizations based on the bureaucratic model used norms as the basis for planning and allocation of resources including funding (see chapter ). this led to payment systems which encouraged greater use of that resource. if a factory is paid by the number of workers and not the number and quality of the cars produced, then management will have no incentive to introduce efficiency or quality improvement measures. if a district or a hospital is paid by the number of beds, or by days of care in the hospital, there is no incentive to introduce alternative services such as same-day or outpatient surgery and home care. performance indicators are measures of completion of specific functions of preventive care such as immunization, mammography, pap smears, and diabetes and hypertension screening. they are indirect measures of economy, efficiency, and effectiveness of a service and are being adopted as better methods of monitoring and paying for a service, such as by paying a premium. general practitioners in the uk receive additional payments for full immunization coverage of the children registered in their practices. a block grant or per capita sum may be tied to indicators that reflect good standards of care or prevention, such as low infant, child, and maternal mortality. incentive payments to hospitals can promote ambulatory services as alternatives to admissions and reduce lengths of stay. limitations of financial resources in the industrialized countries and even more so in the developing countries make the use of appropriate performance indicators of great importance in the management of resources. pay-for-performance is a system of paying for health services developed in the uk for paying general practitioners, with apparently satisfactory results. it is now widely used in the usa. it is defined as "a strategy to improve health care delivery that relies on the use of market or purchaser power. agency for healthcare research and quality (ahrq) resources on pay for performance (p p), depending on the context, refers to financial incentives that reward providers for the achievement of a range of payer objectives, including delivery efficiencies, submission of data and measures to payer, and improved quality and patient safety" (agency for healthcare research and quality, ) . more than half of commercial hmos are using pay-for-performance. recent legislation requires the medicare and medicaid programs to adopt this approach for beneficiaries and providers. as commercial programs have evolved during the past years, the categories of providers (clinicians, hospitals, and other health care facilities), number of measures, and dollar amounts at risk have increased. this method of payment is likely to be promoted in the affordable care act implementation to improve quality and control cost increases in us health care (see chapters , , and ). payfor-performance has also been adopted in other countries trying to improve quality of care, such as macedonia (lazarevik and kasapinov, ) . social marketing is the systematic application of marketing alongside other concepts and techniques to achieve specific behavioral goals for a social good. initially focused on commercial goals in the s, the concept became part of health promotion activities to address health issues where there was no current biomedical approach, such as in smoking reduction and in safe sex practices to prevent the spread of hiv. social marketing was based initially on commercial marketing techniques but now integrates a full range of social sciences and social policy approaches using the strong customer understanding and insight approach to inform and guide effective policy and strategy development. it has become part of public health practice and policy setting to achieve both strategic and operational targets. a classic example of the success is seen with tobacco reduction strategies in many countries using education, taxation, and legislative restrictions. other challenges in this field include risk behavior such as alcohol abuse through binge drinking, unsafe sex practices, and dietary practices harmful to health. philanthropy and volunteerism have long been important elements of health systems through building hospitals, mission houses, and food provision, and other prototype initiatives on a demonstration basis. this approach has been instrumental in such areas as improved care and prevention of hiv, immunization in underdeveloped countries, global health strategies, and maternal and child health services. during the late twentieth and early twenty-first centuries, a new "social entrepreneurship" was initiated and developed by prominent reform-minded former us president bill clinton, microsoft's bill gates, and the open society institute of george soros. the rotary club international has been a major factor in funding and promoting the global campaign to eradicate poliomyelitis. this has promoted integration and consortia for the promotion of acquired immunodeficiency syndrome (aids) prevention and malaria control in many developing countries. the global alliance for vaccine and immunization (gavi) is a us-based organization which links international public and private organizations and resources to extend access to immunization globally. it includes the united nations children's fund (unicef), who, bilateral donor countries, the vaccine industry, the gates foundation, and other major donors. gavi has made an important contribution to advancing vaccine coverage and adding important new vaccines in many developing countries and regions. these organizations focus funds and activities on promoting improved care and prevention of hiv, tuberculosis, and malaria, along with improved vaccination for children, reproductive health, global health strategies, technologies, and advocacy. these programs generate publicity and raise consciousness at political levels where resource allocations are made. a central feature of these programs is the promotion of "civil society" as active partners in a globalized world of free trade, democracy, and peace. specific initiatives included promoting improved largescale marketing of antiretroviral drugs for the treatment of hiv infection, including price reduction so that developing countries can offer antiretroviral treatment, especially to reduce mother-to-infant transmission. programs have branched out into the distribution of malaria-preventing bed nets, provision of low-cost pharmaceuticals, marketing drugs for the poor, desalination plants, solar roof units, lowcost small loans, and cell phones, mainly in africa. another form of social entrepreneurship that has gained support in the private sector is proactiveness in environmental consciousness to address issues raised by the environmental movement, and public interest for environmental accountability. the automobile industry is facing both public concern and federal legal mandates for improved gas mileage as opposed to public demand for larger cars. hybrid cars using less fuel have been successfully introduced into the market for low-emission, fuel-efficient cars, and electric cars are gradually entering the field. public opinion is showing signs of moving towards promoting environmentally friendly design, marketing, and purchasing practices in energy consumption, conservation practices, and public policy. public opinion and the price of fuel will play a major part in driving governments to legislate energy and conservation policies to address global warming and damage to the environment, with their many negative health consequences. however, such changes must work with public opinion because of the sensitivity of consumers to the price of fuel. in addition, when food crops, such as corn, are used to produce ethanol for energy to replace oil, then food prices rise and consumers suffer and respond vigorously. corporations adopt policies of environmental responsibility in part because of public relations and partly because of potential liability claims. much of the planning and financial costs of offshore petroleum and gas drilling is spent on safety measures to protect the environment. the explosion in at a british petroleum site in the gulf of mexico, off the coast of texas and louisiana, caused massive pollution and environmental damage, and resulted in the us government being awarded us$ . billion against bp for cleanup and damages. the reputation of the corporation suffered and some executive officers lost their positions. thus, corporate social responsibility can be seen as self-interest. new models of health care organization are emerging and developing rapidly in many countries. this is partly a result of a search for more economical methods of delivering health care and partly the result of the target-oriented approach to health planning that seeks the best way to define and achieve health objectives. the developed countries seek ways to restrain cost increases, and the developing countries seek effective ways to quickly and inexpensively raise health standards for their populations. new organizational models that try to meet these objectives include district health systems, managed care organizations (mcos) and accountable care organizations (acos), described in greater detail in chapter . critical and basic elements of a health system organization are shown in figure . . new initiatives are part of the growth and development of any organization or health service system, as needs, technologies, resources, and public demand change. identification of issues and decisions to launch new endeavors or projects to advance the state of the art, to address unmet needs, or to meet competition are part of organizational responsibility, in the public sector to meet needs, and in the private sector to remain competitive. in developing and developed countries, many ngos provide funding from abroad for essential services that a government may be unable to provide. such projects focus on issues directed from the head offices in the usa or europe of the funding source or management offices for specific vertical programs which are often not fully integrated with national priorities and programs. however, these need coordination and approval by the local national government agency responsible for that sector of public service. new projects run by ngos may run in parallel to each other, or to state health services as uncoordinated activities. governmental public health agencies have responsibility for oversight of health systems and can play a leadership and regulatory role in coordinating activities and directing new programs to areas of greatest national need. the public health agency may also seek funding to launch new pilot or specific needs programs. the agency may introduce a new vaccine into a routine immunization program in phases, pending government approval and funding to incorporate it as a routine immunization program based on evaluation of the initial phase. an example is the introduction of haemophilus influenzae type b vaccine in albania in , which was funded by gavi for years based on a study and proposal including a cost-effectiveness study (bino s, ginsberg g, personal communication, ) . proposals for health projects by ngos or private agencies need to be prepared in keeping with the vision, mission, and objectives of the responsible governmental agency, with ethics review and community participation. a project proposal should include why the project is important, its specific goals and objectives, available or new resources, and the time-frame required to achieve success (box . ). it should describe the means proposed to accomplish the goals, and how the proposed program will impact the community, providing recommendations for follow-up and/or further action. the introduction of the project proposal outlines the current state of the problem and the case for action. it should describe existing programs which address that issue, with proposed collaboration, and expansion or improvement of programs, but avoiding duplication of services. background information needs to relate the project to the priorities of the prospective funding organization. the objectives should follow the acronym "smart": specific, measurable, achievable, relevant, and time-based. this term, originally used for computer disc self-management, has been adapted as a current form of mbo from the s and s. the project objectives should be feasible and the expected results of the project should be based on the stated objectives. organizations: behavior, structure, process. new york: mcgraw-hill/ irwin; . the proposed funding agency expects convincing evidence of how this program will be effective, efficient, practical, and realistic. this information is presented in the activities section, which also needs to address the resources that will be needed to implement the program such as the budget for staff, supervison, training, management, materials (vaccines, syringes, equipment, ongoing supplies and others), transportation, and costs of premises. after completing the activities section, a realistic and achievable work plan and time-frame are required. well-planned projects have monitoring and evaluation criteria. monitoring follows the performance of the program, documenting successes, failures, and lessons learned, as well as expenditures. evaluation guidelines of the program define the methods used to assess the impact of the project and whether the project was carried out in an effective and efficient manner, and may be required periodically throughout the life of the project. the most difficult issue is sustainability. a project funded by an ngo is usually time limited to - years and the survival of the program usually depends on its acceptability and the capacity of government to continue it. thus, evaluation becomes even more crucial for the follow-up of even successful short-term projects. harm reduction programs include tackling hiv in drug users, reducing maternal-child hiv transmission, tobacco control programs, and reducing levels of obesity in schoolchildren. sustainability and diffusion of positive findings to wider application are important challenges, especially to global health. even in high-income countries, diffusion of best practices is often slow and fraught with controversy and inertia. examples of this slow or non-diffusion of evidence-based public health include the failure of most european countries to harmonize salt fortification with iodine or total indifference to flour fortification with folic acid to prevent neural tube defects (see chapters and ). public health work within departments or ministries of health or local health authorities operates at a disadvantage in comparison with other health activities, especially hospitals, pharmaceuticals, diagnostics, and medical care. the competition for resources in a centrally funded system is intense, and the political and bureaucratic battles for funds may pit new immunization agents or health promotion programs against new cancer treatment drugs or scanners, and this is very often a difficult struggle. the presentation of program proposals for new public health interventions requires skill, professionalism, good timing, and the help of informed public and professional opinion. allocation of resources is decided at the political level in a tax-based universal system, while even in a social security (bismarckian) system where funding is through an employee-employer payroll deduction, additional funding from government is essential to keep up with the continuing flow of new modalities of treatment or prevention. public health is handicapped in portraying the costs and benefits of important interventions, leaving new programs with insufficient resources, including the staffing and administrative costs (e.g., office space, phone service, transportation costs), which are essential parts of any public health program. portraying the cost of the new proposed program should be based on the total population served, not just the specific target population for a new program; that is, it should be represented as a per capita cost. similarly, projected benefits should extrapolate the results from other areas, such as pandemic or avian flu or severe acute respiratory syndrome (sars), and the likely impact on the target geographic area and its population. public health has prime responsibility for monitoring the health status of the population as well as in preventing infectious and non-communicable diseases and injuries, preparing for disasters, and many other functions. this role requires an adequate multidisciplinary workforce with high levels of competencies. this topic is discussed extensively in chapter . canada's experience with the sars epidemic in led to a reappraisal of public health preparedness and standards. this, in turn, led to the establishment of the national public health agency of canada, which is mandated to develop standards and practices to raise the quality of public health in the country and especially to prepare for possible pandemics. the agency issued standards of competency for public health personnel and fostered the development of regional laboratories, and schools of public health were developed across canada. core competencies for program planning implementation and evaluation are seen in box . . health care systems throughout the world are being scrutinized because of their growing costs in relation to national wealth. at the same time, techniques for evaluating health care with respect to appropriateness, quality, and resource allocation are being developed. these techniques are multifactorial since they must relate to all aspects of health care, including the characteristics of the population being served; available health care resources; measures of the process and utilization of care; measures of health care outcomes; peer review, including quality assessment of health care providers; consumer attitudes, knowledge, and compliance; care provided for "tracer" or sample conditions; and economic cost-benefit studies. evaluation in health care assumes that a health care system and the providers of health care within that system are responsible and accountable for the health status of the population. it must, however, recognize that health services are not the sole determinants of health status; social, economic, and cultural factors also play key roles. a comprehensive approach to evaluation in health care is described in chapter . many of the components that are available in health care systems exist, while others that remain to be developed are discussed. evaluation is an integral part of a comprehensive health care system, in that the components of evaluation must be built into any national system. as long as rationality is expected of health care, evaluation is an essential element of the overall system (tulchinsky, ) (see chapter ). the purpose of management in health is the improvement of health, and not merely the maintenance of an institution. separate management of a variety of health facilities serving a community has derived from different historical development and funding systems. in competition for public attention and political support, public health suffers in comparison to hospitals, new technology and drugs, and other competitors for limited resources. the experience of successes in reducing mortality from both non-infectious and infectious conditions comes largely from public health interventions. medical care is also an essential part of public health, so that management and resource allocation within the total health sector are interactive and mutually dependent. the new public health looks at all services as part of a network of interdependent services, each contributing to health needs, whether in hospital care or in enforcing public health law regarding; for example, motor vehicle safety and smoking restriction in public places. separate management and budgeting of a complex of services results in disproportionate funds, staff, and attention being directed towards high-cost services such as hospitals, and fails to redirect resources to more cost-effective and patient-sensitive kinds of services, such as home and preventive care. however, reducing the supply of hospital beds and implementing payment systems with resources for early diagnosis and incentives for short stays have changed this situation quite dramatically in recent decades. the effects of incentives and disincentives built into funding systems are central issues in determining how management approaches problem solving and program planning, and are therefore important considerations in promoting health. the management approach to resolving this dilemma is professional vision and leadership to promote the broader new public health. thus, managers of hospitals and other health facilities need broad-based training in a new public health in order to understand the interrelationships of services, funding, and population health. managers who continue to work with an obsolescent paradigm with the traditional emphasis, regardless of the larger picture, may find the hospital non-competitive in a new climate where economic incentives promote downsizing institutions and upgrading health promotion. defensive, internalized management will become obsolete, while forward-looking management will be the pioneers of the new public health. this may be seen as a systems approach to improve population and individual health, based on strategic planning for immediate needs and adaptation of health systems in the longer term issues in health. examples of national planning that cut across health and social services include national insurance policies and the provision of new services to meet rising needs, as shown for alzheimer's disease, in france since (box . ) and in the usa since (box . ). health care is one of the largest and most important industries in any country, consuming anywhere from to nearly percent of gnp, and still growing. it is a service, not a production industry, and is vital to the health and well-being box . core competencies for program planning, implementation and evaluation management, from policy to operational management of a production or a service system. creative management of health systems is vital to the functioning of the system at the macrolevel, as well as in the individual department or service. this implies effective use of resources to achieve objectives, and community, provider, and consumer satisfaction. these are formidable challenges, not only when money is available in abundance, but even more so when resources are limited and difficult choices need to be made. modern management includes knowledge and skills in identifying and measuring community health needs and health risks. critical needs are addressed in strategic planning with measurable impacts and targets. public health managers should have skills gained in marketing, networking, data management, managing human resources and finance, engaging community partners, and communicating public health messages. many of the methods of management and organization theory developed as part of the business world have become part of public health. these include defining the mission, values and objectives of the organization, strategic planning and management, mbo, human resource management (recognizing individual and professional values), incentives-disincentives, regulation, education, and economic resources. the ultimate mission of public health is the saving of human life and improving its quality, and achieving this efficiently with high standards of professionalism and community involvement. the scope of the new public health is broad. it includes the traditional public health programs, but equally must concern itself with managing and planning comprehensive service systems and measuring their function. the selection of targets and priorities is often determined by the feasible rather than the ideal. the health manager, either at the macrolevel of health or managing a local clinic, needs to be able to conceptualize the possibilities of improving the health of individuals and the population in his or her service responsibility with current and appropriate methods. good management means designing objectives based on a balance between the feasible and the desirable. public health has benefited greatly from its work with the social sciences and assistance from management and systems sciences to adapt and absorb the new challenges and technologies in applied public health. the new public health is not only a concept; it is a management approach to improve the health of individuals and the population. for a complete bibliography and guidance for student reviews and expected competencies please see companion web site at http://booksite.elsevier.com/ bibliography electronic resources glossary of managed care terms national association of public hospitals and health systems world health organization, the health manager's website pay for performance (p p): ahrq resources dr. deming: the american who taught the japanese about quality achieving a high performance health care system with universal access: what the united states can learn from other countries improving the effectiveness of health care and public health: a multi-scale complex systems analysis framework for program evaluation in public health biological and chemical terrorism: strategic plan for preparedness and response. recommendations of the cdc strategic planning workgroup a framework for program evaluation. office of the associate director for program -program evaluation developing leadership skills patterns of ambulatory health care in five different delivery systems capacity planning in health care: a review of the international experience. who, on behalf of the european observatory on health systems and policies strategic management of health care organizations social media engagement and public health communication: implications for public health organizations being truly "social crossing the quality chasm: a new health system for the twenty-first century future of the public's health in the st century best care at lower cost: the path to continuously learning health care in america behavioral interventions to reduce risk for sexual transmission of hiv among men who have sex with men cd . available at the wisdom of teams: creating the high-performance organization social marketing: influencing behaviors for good the interaction of public health and primary care: functional roles and organizational models that bridge individual and population perspectives the powers and pitfalls of the payment for performance three skills every st-century manager needs total quality management as competitive advantage: a review and empirical study making the best of hospital pay for performance the public health approach to eliminating disparities in health public health systems and services research: building the evidence base to improve public health practice united states innovations in healthcare delivery a call to action: lowering the cost of health care reduced mortality with hospital pay for performance in england practical challenges of systems thinking and modeling in public health public health: essentials of public health health united states the funding organization will want to know what will be the expected product of the program in measurable process (e.g., immunization coverage) or outcome indicators (e.g., reduced child mortality). projections will be based on the intended activities and known outcomes of other past programs with similar goals in the same or other countries (environmental scan), and should be supported by a review of local and international literature on the topic. the activities section of a proposal should include a timeline of the intended actions and a description of activities based on best practices. the expected outcomes, monitoring and evaluation, and justification are all part of the presentation (box . ). the following utility standards ensure that an evaluation will serve the information needs of intended users: l identify and engage stakeholders, including relevant government agencies, people or communities involved in or affected by the evaluation, so that their needs and concerns can be addressed. l develop and describe the program. l focus the evaluation design with ethical standards and review requirements respected.l gather credible evidence -the people conducting the evaluation should be trustworthy and competent in performing the evaluation for findings to achieve maximum credibility and acceptance. information collected should address pertinent questions regarding the program and be responsive to the needs and interests of clients and other specified stakeholders.l justify the conclusions -the perspectives, procedures, and rationale used to interpret the findings should be carefully described so that the bases for value judgments are clear. l ensure sharing and use of information and lessons learned -evaluation reports should clearly describe the program being evaluated, including its context and the purposes, procedures, and findings of the evaluation so that essential information is provided and easily understood. substantial interim findings and evaluation reports should be disseminated to intended users so that they can be used in a timely fashion to encourage follow-through by stakeholders, to increase the likelihood of the evaluation being used.l standards of a project should focus on scientific justification, utility, feasibility, propriety, and accuracy. l a program in this context includes: -direct service interventions -community mobilization efforts -research initiatives -surveillance systems -policy development activities -outbreak investigations -laboratory diagnostics -communication campaigns -infrastructure building projects -training and education services -administrative systems and others. title page -name of project; principal people and implementing organizations; contact person(s); timeframe; country (state, region); target group of project; estimated project cost; date of submission.l introduction -provides project background including the health issue(s) to be addressed, a situational analysis of the health problem, the at-risk and target populations, and existing programs in the community; includes an international and national literature review of the topic with references. budget -estimated cost of expenditures, including human resources, activities, running costs, and overheads for project and evaluation. l monitoring and evaluation -what evidence will be used to indicate how the program has performed? what plan is recommended for periodic follow-up of project activities (including timeline and measures) to implement lessons learned from positive or negative outcomes, and use of resources? how efficient and effective is the project? l conclusions -what conclusions regarding program performance may be drawn? what conclusions regarding program performance are justified by comparing the available evidence to the selected standards? l reporting -report the project to the key stakeholders and public bodies; publication in peer-reviewed journal if possible.l justification -why is this project important and timely, and how will implementation benefit health of the community?core competencies are essential knowledge, skills, and attitudes necessary for the practice of public health. they transcend the boundaries of specific disciplines and are independent of program and topic. they are the building blocks for effective public health practice, and the use of an overall public health approach.generic core competencies provide a baseline for what is required to fulfill public health system core functions. these include population health assessment, surveillance, disease and injury prevention, health promotion, and health protection.the core competencies are needed to effectively choose options, and to plan, implement, and evaluate policies and/ or programs in public health, including the management of incidents such as outbreaks and emergencies.a public health practitioner is able to: l describe selected policy and program options to address a specific public health issue l describe the implications of each option, especially as they apply to the determinants of health and recommend or decide on a course of action l develop a plan to implement a course of action taking into account relevant evidence, legislation, emergency planning procedures, regulations, and policies l implement a policy or program and/or take appropriate action to address a specific public health issue l demonstrate the ability to implement effective practice guidelines l evaluate an action, a policy, or a program l demonstrate an ability to set and follow priorities, to maximize outcomes based on available resources l demonstrate the ability to fulfill functional roles in response to a public health emergency. of the individual, the population, and the economy. because health care employs large numbers of skilled professionals and many unskilled people, it is often vital to the economic survival of small communities, as well as for a sense of community well-being.management includes planning, leading, controlling, organizing, motivating, and decision-making. it is the application of resources and personnel towards achieving targets. therefore, it involves the study of the use of resources, and the motivation and function of the people involved, including the producer or provider of service, and the customer, client, or patient. this cannot take place in a vacuum, but is based on the continuous monitoring of information and its communication to all parties involved. these functions are applicable at all levels of an estimated , french people lived with dementia; half were diagnosed and one-third were receiving treatment; percent of people with alzheimer's disease were living at home; percent of all nursing home residents lived with some form of dementia; a day's care cost € while full-time residency in a nursing home ranged between € and € . l identify the early symptoms of dementia and refer people to specialists. l create a network of "memory centers" to enable earlier diagnosis. "for millions of americans, the heartbreak of watching a loved one struggle with alzheimer's disease is a pain they know all too well. alzheimer's disease burdens an increasing number of our nation's elders and their families, and it is essential that we confront the challenge it poses to our public health. " on january , president barack obama signed into law the national alzheimer's project act (napa), requiring the secretary of the us department of health and human services (hhs) to establish the national alzheimer's project to: l create and maintain an integrated national plan to overcome alzheimer's disease (ad). l coordinate alzheimer's disease research and services across all federal agencies. l accelerate the development of treatments to prevent, halt, or reverse the course of ad. l improve early diagnosis and coordination of care and treatment of ad. l improve outcomes for ethnic and racial minority populations that are at higher risk for ad. l coordinate with international bodies to fight ad globally. the law also establishes the advisory council on alzheimer's research, care, and services and requires the secretary of hhs, in collaboration with the advisory council, to create and maintain a national plan to overcome ad. research funds are being allocated towards that end. education for health providers, strengthening of the workforce, for direct care and for public health guidelines for management of ad, education and support for caring families, addressing special housing needs for ad patients and many other initiatives are proposed in this comprehensive approach to a growing public health problem. enhancing public awareness is crucial to achieve the goals set out in this plan. key: cord- -qmu mrmx authors: velásquez, ricardo manuel arias; lara, jennifer vanessa mejia title: forecast and evaluation of covid- spreading in usa with reduced-space gaussian process regression date: - - journal: chaos solitons fractals doi: . /j.chaos. . sha: doc_id: cord_uid: qmu mrmx in this report, we analyze historical and forecast infections for covid- death based on reduced-space gaussian process regression associated to chaotic dynamical systems with information obtained in days with continuous learning, day by day, from january (th), to april (th). according last results, covid- could be predicted with gaussian models mean-field models can be meaning- fully used to gather a quantitative picture of the epidemic spreading, with infections, fatality and recovery rate. the forecast places the peak in usa around july (th) , with a peak number of , death with infected individuals of about , , and a number of deaths at the end of the epidemics of about , . late on january, usa confirmed the first patient with covid- , who had recently traveled to china, however, an evaluation of states in usa have demonstrated a fatality rate in china ( %) is lower than new york ( . %), but lower than michigan ( . %). mean estimates and uncertainty bounds for both usa and his cities and other provinces have increased in the last three months, with focus on new york, new jersey, michigan, california, massachusetts,... (january e april (th)). besides, we propose a reduced-space gaussian process regression model predicts that the epidemic will reach saturation in usa on july . our findings suggest, new quarantine actions with more restrictions for containment strategies implemented in usa could be successfully, but in a late period, it could generate critical rate infections and death for the next month. able on the center for systems science and engineering at johns hopkins university [ ] , the available data analyzed is considered between january th and april th , included, with a feedback process in a neural network applied; it allows to examined the information in real time in each state, at fig. • . with covid- , the spatial representation of the disease by using gis plat- form allows to verified the "material, population and social psychology at three scales: individual,group and regional" [ ] ; in this case, with gis technology is necessary to implement big data techniques, for cross validations and analysis. • associated to gpr the idea is to "utilize nonlinear diffusion map coordinates and formulate a deterministic dynamical system on the system manifold" [ ] . a interesting approach has a reduced-space data-driven dynamical system with an evaluation, with efficiency with low intrinsic dimensionality. • an "advantage of employing gpr to reconstruct the reduced-order dynamics is the simultaneous estimation of the dynamics and the associated uncertaint" with an input xn ∈ r and "noisy scalar output yn" [ ] . for infection population, where: where: θ : is a hyper-parameter with maximum covariance in chaotic systems. in eq. ( ), bayes rule is written with a normalized process to find (f, f * ) in eq. ( ). in eq. ( ), associates to "conditioning the joint gaussian prior distribution on the observations, resulting in the closed-form gaussian distribution" [ ] . with eq. ( ), f * , the mean and covariance should be directly added to obtain eq. ( ). finally, in eq.( ) makes feasible to use up to more than twelve thousands of training x ∈ r in the eq. ( ), individually node in the hidden layer has "linear combination" [ ] with a chain events. the hidden layers are composed as following: in eq. ( ) describes a matrix of weights, it is analogous to regression coefficient in where: h: it is a description of the hidden layer, due to restriction of the "linearly trans- formed and passed to the output layer". however, china has implemented robust strategies for distancing control and quar- antine around this country, therefore, the recovery rate is %. far away from new york, with recovery rate of . % and on the other hand, cities as texas is . %. besides, hawaii has different rate of . % for the restrictions initiated with more influence in their population. combining a mathematical model with multiple datasets, fig. and fig. , we found that the median daily rt of covid- in usa probably varied between . and . in april, , before weeks of travel restrictions were introduced. we in traditional methods, we detect mistakes in the forecast associated to old pan- demic researches, and as we can see, the covid- has behaved in unpredictable and changing ways, according to the factors used in its treatment. therefore, we think that the pandemic behaves as a dynamic-chaotic system, which sets new start- ing points that do not intersect and changes its behavior according to the way it is treated. consequently, this study could be a real contribution for understanding this problem with a good tool of processing a systematic review. as we can see in the fig. , the example started with three countries, with three initial states. hence they have been evolving together until some days, but after a month, they grants/financial support. none. credit authorship contribution statement. conflict of interest. there is no conflict of interest in this work. isolation, quarantine, social distancing and community containment: pivotal role for old-style public health measures in the novel coronavirus ( -ncov) outbreak the covid- pandemic in the usa: what might we expect? potential impact of seasonal forcing on a sars-cov- pandemic real-time forecasts of the covid- epidemic in china from february analysis and forecast of covid- spreading in china an interactive web-based dashboard to track covid- in real time real-time estimation and prediction of mortality caused by covid- with patient information based algorithm propagation analysis and prediction of the covid- , infectious disease mod- elling covid- : challenges to gis with big data why is it difficult to accurately predict the covid- epidemic? gaussian processes in machine learning. the mit reduced-space gaussian process re- gression for data-driven probabilistic forecast of chaotic dynamical systems, mit industrial liaison program nonparametric forecasting of low- dimensional dynamical systems converting data into knowledge for preventing failures in power transformers corrosive sul- phur effect in power and distribution transformers failures and treatments, engi- neering failure analysis pattern recognition and image preprocessing removal of impulse noise in color images based on convolutional neural network gradient-based learning applied to document recognition an overview of deep learning in medical imag- harmonic failure in the filter of static var compensator automatic diagnosis of skin diseases acknowledgments. key: cord- -vre bq authors: groneberg, david a.; braumann, hannah; rolle, stefan; quarcoo, david; klingelhöfer, doris; fischer, axel; nienhaus, albert; brüggmann, dörthe title: needlestick injuries: a density-equalizing mapping and socioeconomic analysis of the global research date: - - journal: int arch occup environ health doi: . /s - - - sha: doc_id: cord_uid: vre bq background: needlestick injuries have caused a deleterious effect on the physical and mental health of millions of health-care workers over the past decades, being responsible for occupational infections with viruses such as hiv or hepatis c. despite this heavy burden of disease, no concise studies have been published on the global research landscape so far. methods: we used the new quality and quantity indices in science platform to analyze global nsi research (n = articles) over the past years using the web of science and parameters such as global versus country-specific research activities, semi-qualitative issues, and socioeconomic figures. results: density-equalizing mapping showed that although a total of n = countries participated in nsi research, large parts of africa and south america were almost invisible regarding global participation in nsi research. average citation rate (cr) analysis indicated a high rate for switzerland (cr = . ), italy (cr = . ), and japan (cr = . ). socioeconomic analysis revealed that the uk had the highest quotient q(gdp) of . nsi-specific publications per bill. us-$ gross domestic product (gdp), followed by south africa (q(gdp) = . ). temporal analysis of hiv versus hepatitis research indicated that nsi-hiv research culminated in the early s, whereas nsi-hepatitis research increased over the observed period from the s until the last decade. conclusion: albeit nsi research activity is generally increasing, the growth is asymmetrical from a global viewpoint. international strategies should be followed that put a focus on nsi in non-industrialized areas of the world. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. background as elegantly stated by recent reviews, the risks of needlestick injuries (nsi) may have fallen over the past three decades, but even in the presence of guidelines, they still occur frequently (king and strony ) . health-care workers have the highest risk of experiencing needlestick injuries, but also other occupations are endangered (king and strony ; mona et al. ) . when injured, the individual clinical, psychological and economic burden can be substantial (cooke and stephens ) . as previously stated, the rates of nsis have been estimated to vary between . and . % in health-care workers, depending on different settings, countries, and methodologies (cooke and stephens ) . hence, research in this area is crucial and knowledge about previous research is important to plan future strategies. since there are no data existing on the international architecture of nsi research available, the present study used the newqis project platform (groneberg-kloft et al. a) to construct the first picture of global nsi research strategies. we aimed to ( ) perform an in-depth analysis of the global nsi research activities and to ( ) visualize a variety of figures related to nsi research by the use of density-equalizing procedures proposed by gastner and newman (gastner and newman ) . to analyze nsi research, we used the new quality and quantity indices in science (newqis) platform termed newqis (groneberg-kloft et al. a, b) , ranging from public and global health issues trost et al. ) to fields such as internal medicine (schoffel et al. ) , surgery (schoffel et al. (schoffel et al. , or gynecology and obstetrics (bruggmann et al. a, b) . since its foundation, about peer-reviewed studies have been published using newqis. as data source, we used the web of science (clarivate) since this database enabled us to calculate various citation parameters ). to cover studies related to occupational needlestick injuries, we constructed a detailed search algorithm, illustrated in fig. . using this term, we performed a topic search in the period - . as in earlier studies, the period after - - was excluded to avoid incomplete data acquisition. as earlier stated, newqis enables to analyze and visualize a wide range of different parameters (i.e., the time of publication, the originating countries, the attributed subject categories, the citations). with these raw data, further parameters such as country-specific h-indices (hi) of the present set of publications were calculated. these hi are not related to single authors, but to the countries. also, country-specific citation rates of nsi-related research of each country were calculated (cr, total country-specific citations numbers per total numbers of country-specific nsi publications) (hirsch ) . in a next step, the numbers of collaborating countries in collaborating articles were also analyzed. absolute research activity is important to assess the overall contribution of a single country toward the global research efforts in the areas of nsi. however, there are vast socioeconomic differences between single countries. therefore, the research activity data should also be related to socioeconomic figures of each country to be able to interpret the relative contributions. we approached this issue by relating nsi research activity to the ( ) total economic power index "gross domestic product" (gdp) per billion us-$, ( ) gdp per capita, and ( ) country population sizes (world factbook (world economic outlook database )). we here used the approach of density-equalizing map projections (demp) established by gastner and newman in (gastner and newman ) . to construct the demps, we introduced several nsi-specific parameters to the algorithm including total numbers of nsi-related publications, country-specific citation rates of nsi-related research and others. as stated earlier, newqis also assesses global collaborative networks. using a methodological approach which was previously described, all affiliations of nsi-related articles were analyzed for their originating countries. then, chart diagrams were generated to visualize the network (mund et al. ). in two separate analyses, we assessed the research activities for the most prominent nsi-related acquired diseases-hiv and hepatitis. in specific, the search term listed in fig. was separated into two different, hiv-and hepatitis-specific terms as listed in online supplement . the metadata was analyzed for chronologic development and countries of research origin. the first publication on nsi was identified for the year , but until , only a moderate activity was present with less than articles being published annually. in the s, an increase was present and beginning of the s, more than annual articles on nsi were published for the first time. in total, articles were identified. more than % of all articles were published after the year . a regression analysis of the investigated research interest over time reveals a high coefficient of determination (r = . ) since (fig. ) . in total, countries published nsi-related articles. the nation with the highest level of nsi research activity was the usa with n = , articles. next, great britain showed an activity of nsi-related articles, followed by canada (n = ), australia (n = ), germany (n = ) and france (n = ). when all data are transferred to a demp algorithm, a world map distorted toward north america and western europe is present. in the eastern hemisphere, japan and australia are prominent. australia is the only country south of the equator with an activity over nsi articlesindicating a strong north-south slope (fig. ) . a similar landscape is present, when the number of nsi publishing institutions/affiliations per country is analyzed (fig. ) : the usa is the leading country with i = different affiliations, followed by great britain (i = ), france (i = ), italy (i = ), germany (i = ), and japan (i = ) (fig. ). the analysis of the crude parameter of the total citation count followed the picture of the total publication activities with only small variations being present: the usa was at the lead position with c = , citations. it was followed by great britain (c = citations), italy (c = ), canada (c = ), japan (c = ), australia (c = ), and germany (c = ) (fig. a) . as a second citation readout, the country-specific hirsch indices (cohi) were assessed for the presently identified nsi-related publications. in this assessment, demp calculations led to a global architecture similar to the total citation count projections with the usa at the lead position with a cohi of publications being at least cited times. the usa was followed by the uk (cohi = ), canada (cohi = ), australia (cohi = ), france and italy (both a cohi of ), germany, japan and switzerland (all a cohi of ) and the netherlands (cohi = ). the only african country with a cohi more than is south africa (cohi = ) (fig. b) . a complete different global architecture was found when the nsi publications were analyzed for the average citation rates with a threshold of at least publications: here, switzerland was ranked first (cr = . ), followed by italy (cr = . ), japan (cr = . ), usa (cr = . ), and spain (cr = . ) (fig. c ). countries with a high level of economic prosperity are able to allocate more funding toward nsi research from a financial point of view. therefore, we also assessed nsi research in relation to socioeconomic figures. we first assessed the correlation between the country-specific number of nsirelated articles and the countries' gross domestic product in bill. us-$ (gdp , table ). when applied for all countries with at least specific articles related to nsi, the uk is ranked highest with a quotient q gdp of . nsispecific publications per bill. us-$ gdp. the uk is followed by south africa with a quotient of q gdp = . , and iran (q gdp = . ). next, pakistan (q gdp = . ), switzerland (q gdp = . ), australia, belgium, canada, and israel follow with a q gdp of . . the usa, turkey and the netherlands have a q gdp of . ), followed by sweden and poland (q gdp = . ), and spain (q bip = . ). in a next step, we assessed the correlation between the country-specific number of nsi-related articles and the countries' gross domestic product per capita (table ): in this ranking with a threshold of at least specific articles, india is ranked first with . nsi-specific articles per gdp/capita, followed by the usa and pakistan. in a final assessment, nsi research was related to population sizes. here, switzerland was ranked first with a calculated number of . nsi-related articles per million of inhabitants, followed by australia, the uk, canada and the usa (table ). in total, there were collaborative articles (n coop ) present. about % were bilateral cooperations (n coop = ). trilateral publications were present in with n coop = cases, followed by collaborations of four countries (n coop = and five countries (n coop = ). there were , us collaboration articles, followed by uk (n coop = ), french (n coop = ), chinese (n coop = ), german (n coop = ), and canadian (n coop = ) collaborations (fig. ) . the leading nation was the usa with a total of collaboration publications. the most prominent collaboration was between us-american and canadian scientists (fig. ) , followed by us-swiss and us-uk collaborations. subject category analysis of published nsi research showed an increase in public, environmental and occupational health-topics related to nsi over the past decades (fig. a) . also, the field of nursing gained research activity in the period of - . when single countries were analyzed for differences in their research focuses, a heterogeneous picture was present (fig. b) . the most apparent difference was found for japan with an emphasis on the field of gastroenterology and hepatology. besides, turkey was the country with the highest proportion of nsi research related to the field of nursing. a temporal analysis and comparison of research activities focused on nsi and hiv or nsi and hepatitis shows that there are major differences present. whereas nsi research, specifically on hiv, culminated in the beginning of the s, nsi-research focused on hepatitis increased over the observed period from the s until the last decade (fig. ) . when a sub-analysis of countries' research activities was performed (table ) , the usa dominated both nsi research on both acquired diseases, followed by the uk. it is noteworthy that south africa also appeared in the top ten most active countries concerning nsi and hiv. by contrast, the only asian country which is present in the overall activity ranking, japan, does not belong to the most active nsi-hiv-focusing countries. however, it is present in the nsi-hepatitis top ten ranking countries. nsis are omnipresent around the globe and endanger the health and life of hundreds of thousands of health-care workers. as stated before, they generate a large amount of indirect, direct, potential, and intangible costs, which why? probably, because nsis are not just a single entity such as a disease or a pathogen, but an accident, resulting from a series of failures, which can be analyzed from different perspectives: determinants, characteristics, distribution, consequences, prevention of accidents, and prevention of consequences. therefore, the present results need to be interpreted with caution. our approach aimed to visualize global research activities in the field of nsi and related areas of science. to approach this aim, metadata were used from the web of science between and and , publications were identified which were specifically related to nsi. interestingly, almost all nsi-related articles ( . %) were published between the years and , indicating only a very minimal activity in the decades before. the reason for this increase after is based upon two findings: observations that (a) hepatitis b (first articles appearing in - ), and then (b) hiv (first documented case in literature in ) could be acquired through a needlestick. one might argue that due to the minimal activities more than three decades ago, the analysis should have been limited to, i.e., the past decades. however, including a time span the usa plays a dominant role in country-specific analyses with the highest number of articles published, the largest number of institutions of higher education as well as the highest modified h-index. furthermore, the usa possesses the most national and international collaborations among all nations. how can these data be interpreted? first of all, it is important to discuss these results with regard to the overall global research performance and general trends. to achieve this, the presently observed increase in nsi research activities needs to be related to the increase in global research activities, in specific, biomedical research activities. it is wrong to take the growth of databases such as the presently used web of science as a precise tool for increase in research activities (since no existing database captures everything) (noorden ). however, it may serve as a proxy (bornmann and mutz ) . by analyzing more than million cited references in million publications from to , bornmann and mutz estimated the growth of research for between to % (bornmann and mutz ) . although only restricted to wos data, our analysis shows for nsi a growth rate which is not constantly increasing, but varying between years, especially in the s to the s. this is an uncommon picture, because usually the growth is more or less linear, as previously demonstrated (noorden ; bornmann and mutz ) . how is the global architecture of nsi research? as stated above, the usa is by far dominating the international efforts in nsi research. is this a common picture-yes, there is a multitude of other bibliometric studies that demonstrated a unique leading role of the usa in almost every area of science. but how is the pattern of leading countries apart from the usa? does this ranking follow any previously known pattern? our ranking with the uk at position , followed by canada (n = ), australia (n = ), germany (n = ) and france (n = ) shows a global architecture that is similar to those of many other fields of biomedicine. but is this pattern specific for research conducted in the area of viral diseases? there are a number of studies that addressed global research activities on infectious diseases so far: both nsirelevant infectious diseases/agents such as hiv or hepatitis virus and other diseases such as ebola or influenza. in general, there are two global patterns present: a pattern for infectious diseases with a large burden of disease in industrialized countries and/or infectious diseases with the possibility of vaccination such as hepatitis (schmidt et al. ) , hpv , or influenza (fricke et al. ) and a pattern for diseases that are more common in non-industrialized or tropical countries such as leishmaniasis (al-mutawakel et al. ) . the present nsi research activity pattern is more or less similar to the ones reported for hiv or hepatitis (schmidt et al. ) . it differs from the patterns of diseases linked to viruses such as yellow fever ) and mers-cov (zyoud ) . here, countries such as brazil and india appear in the top ten ranking most active countries. next to these continental differences, one might also focus future studies upon further variations, i.e., between countries of the european union, which enjoy shared health and safety legislation, and other regions of the northern globe. in this respect, it has been recently demonstrated with regard to psychosocial risks and violence in the workplace that there are important differences between these regions (chirico et al. ) . a similar study set up using databases such as legosh" of the international labour office (ilo) might add important new insights into the understanding of global and regional nsi research differences. we also related overall nsi research figures to socioeconomic features including gdp or population sizes. a limitation of this approach is the constant change in these socioeconomic parameters. however, they are commonly used as proxies to relate research productiveness to socioeconomic features (chong et al. ; wang and zhao ) . apart from the country-specific analysis of research activities, it is also of major importance to analyze the subject areas in which nsi research is performed and published. in contrast to global research activities, e.g., on hepatitis (schmidt et al. ) , or hpv ), we here found for nsi that the field of public, environmental and occupational health gained importance in the past decades. in the final -year period of analysis, this field was among the most active areas of nsi research. this points to the importance of nsi for this area of biomedicine. obviously, nsi cases among health-care workers have also occurred in countries where the hiv epidemic was widespread (i.e., african countries), but they were less likely to be identified due to the ongoing epidemic in the general population, and the lack of specific surveillance at the beginning of the epidemic. in our sub-analysis, south africa was detected to belong to the top ten most active countries concerning hiv-specific nsi research. other countries, such as japan, with a significant prevalence of hcv in the general population, observed cases of hepatitis c linked to nsis after the identification of a specific antibody test to detect seroconversion in the early s, and this is why papers appear in gastroenterology and hepatology journals. this is visualized by our nsi research area analysis in which japan is very prominent in the field of gastroenterology and hepatology. also, in our sub-analysis, japan appeared in the top ten activity ranking of specific nsi-hepatitis research. the different waves of publications are in relationship with the different subjects related to nsis (and countries) as demonstrated here. the evolution of these areas with regard to nsi occurred over time, i.e., pathogens acquired, effects of the first preventive measures (universal precautions, banning of recapping, sharps containers, hepatitis b vaccination), post-exposure prophylaxis against hiv, introduction of safety devices, and impact of laws and regulations supporting preventive measures. we here present data on activity of nsi research in the twentieth and twenty-first centuries. our data clearly show that there is a heavy global asymmetry with the southern globe being underrepresented to a major extent. although the area of public and occupational health-being underpresented concerning research activity in the majority of other medical areas-is omnipresent in nsi research. this study points to the need to foster nsi research activities in non-industrialized countries in which health-care personnel are crucial for public health. substantial contributions to the interpretation of the data. they all have been involved in drafting and revising the manuscript. all authors have read and approved the final manuscript. funding this study was supported by bgw (institution for statutory accident insurance and prevention in the health and welfare services), germany. the bibliometric data are owned by and have been obtained from the web of science database. any researcher with access to the web of science database can obtain the data using the methods described in the paper. readers who do not have access to web of science should contact clarivate analytics to obtain a license. as per clarivate analytics terms of use, the authors may also be able to provide limited access to data, subject to their agreement. conflict of interest the authors declare that they have no conflict of interest. open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit 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density-equalizing maps silicosis: geographic changes in research: an analysis employing densityequalizing mapping snakebite envenoming-a combined density equalizing mapping and scientometric analysis of the publication history quality and quantity indices in science: use of visualization tools new quality and quantity indices in science (newqis): the study protocol of an international project an index to quantify an individual's scientific research output how much do needlestick injuries cost? a systematic review of the economic evaluations of needlestick and sharps injuries among healthcare personnel a systematic review on occupational hazards, injuries and diseases among police officers worldwide: policy implications for the south african police service global research on smoking and pregnancy-a scientometric and gender analysis pancreatic cancer-critical examination of the global research architecture and recent scientific developments hirschsprung disease: critical evaluation of the global research architecture employing scientometrics and density-equalizing mapping crohn's disease: a critical approach to publication procedures and citation behavior of the global research network statistisches jahrbuch deutschland und internationales immigration: analysis, trends and outlook on the global research activity global scientific output doubles every nine years worldwide research productivity in the field of back pain: a bibliometric analysis world economic outlook databa www global research trends of middle east respiratory syndrome coronavirus: a bibliometric analysis publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations acknowledgements open access funding provided by projekt deal. we thank c. scutaru and b. kloft for their pioneering work concerning newqis. this study is part of a md/phd thesis project (hb). we also thank the two peer referees for excellently helping us to improve the quality of the data.author contributions dag, hb, dk, and db have made substantial contributions to the conception and design of the study, acquisition of the data and interpretation. sr, dq, af, and an have made key: cord- -d n aokl authors: natesan, mohan; ulrich, robert g. title: protein microarrays and biomarkers of infectious disease date: - - journal: int j mol sci doi: . /ijms sha: doc_id: cord_uid: d n aokl protein microarrays are powerful tools that are widely used in systems biology research. for infectious diseases, proteome microarrays assembled from proteins of pathogens will play an increasingly important role in discovery of diagnostic markers, vaccines, and therapeutics. distinct formats of protein microarrays have been developed for different applications, including abundance-based and function-based methods. depending on the application, design issues should be considered, such as the need for multiplexing and label or label free detection methods. new developments, challenges, and future demands in infectious disease research will impact the application of protein microarrays for discovery and validation of biomarkers. large-scale genome sequencing projects first advanced knowledge of the theoretical composition of proteomes and led to the development of dna microarrays for studying gene transcription at the organism-scale. using genome sequence data to guide the direct examination of proteomes then enabled the study of host-pathogen interactions occurring beyond the level of gene transcription. it soon became evident that direct correlations between gene expression and protein abundance were rare [ , ] , driving the development of new approaches to study complex proteomes. protein microarrays are ideally suited to serve this purpose and have enormous potential applications in biomarker discovery, diagnosis, vaccine development, and drug discovery for infectious diseases. fluorescence- based detection of protein interactions is similar to gene array methods, and data analysis often employs approaches previously developed for genome and transcription studies. the number of proteins that can be printed on a single microarray surface also approaches the same upper limits as nucleic-acid based systems. multiplexing of fluorescent probes is limited by the ability to separate signals of overlapping emission spectra. generally, pair-wise comparisons of - differentially labeled, experimental and control samples can be analyzed. for example, serum igg and igm binding to arrayed proteins can be independently probed by fluorescently labeled secondary antibodies, and detected by a confocal laser scanner (such as genepix, molecular devices, sunnyvale, ca, usa). a complete high-throughput screening of thousands of interactions can be performed accurately and rapidly ( figure ). direct labeling of probes may also be used, though structure and function may be adversely affected. most label-free techniques [ ] provide real-time measurements and in some cases yield kinetics (figure ), providing further insight into molecular interactions. surface plasmon resonance (spr) [ ] , nanowire surfaces [ ] , and mass spectrometry [ ] are examples of label-free methods that are applied in the field of protein microarrays. of these, only a limited number of spr-based instruments are currently available for analysis of complex protein microarrays. spr imaging (spri) is a more recent and promising development [ , ] . while planar arrays that use slides or chips for immobilizing capture molecules are most common, suspension arrays based on microbeads have many important applications. the xmap technology developed by luminex corporation (austin, tx, usa) uses . micrometer polymer beads infused with two fluorescent dyes at different ratios to yield up to distinct bead sets. the binding measurements are performed by flow cytometry with two lasers, one for the identification of the bead and the other for the sample. bead-based arrays are very useful for standardizing assays, with an upper limit of independent immobilized probes. protein microarrays are increasingly used in infectious disease research to identify new biomarkers that are involved in the disease process or that are targets of immune responses [ ] [ ] [ ] [ ] [ ] [ ] . in contrast with other proteomic techniques such as -d gel electrophoresis and mass spectrometry, the arrayed probes are known, conforming easily to high-throughput applications that require examination of the entire proteome of an infectious agent. a variety of formats have been developed for the study of pathogen proteomes. capture and reverse-phase are examples of abundance-based microarrays produced by spotting either antibodies [ ] or antigens [ ] on a substrate. the arrays can be probed with serum, plasma, and other biological samples to detect binding interactions. microarrays based on wellcharacterized antibodies are specifically useful for identifying biomarkers and quantification of proteins. aptamers [ ] , affibodies [ ] , or engineered antibody fragments [ ] are alternative capture molecules. antigen arrays find applications in serodiagnosis of disease, antibody-response profiling, and evaluation of immunity after vaccination. in reverse-phase protein microarrays (rppms), small amounts of biological samples from cells, tissues, sera, or plasmas are directly printed on the slide [ , ] . these arrays are probed with a single protein of interest that is detected by labeled antibodies. rppms were successfully used for the screening of cell-signaling pathways and posttranslational modifications of proteins [ ] [ ] [ ] . the biochemical activities of arrayed proteins have also been analyzed [ ] by enzyme activity or substrate activity, and protein interaction with other proteins, nucleic acids, or drugs [ ] [ ] [ ] . the host antibody response provides a signature of pathogen proteins displayed by infectious diseases that can be captured and detected by microarrays. our studies with vaccinia virus [ ] and the bacterium yersinia pestis [ ] exemplify the application of antigen microarrays for the analysis of immune responses ( figure ). vaccinia is a large dna virus (orthopoxviridae) that replicates in the cytoplasm of host cells from genomes encoding - proteins. the virion is comprised of approximately proteins. most phenotypic variability occurs in proteins encoded in the terminal regions of the genome that are associated with host virulence or immune evasion. some of these terminal-region proteins are secreted during cell infection and interfere with host immunity by binding complement factors, cytokines, and chemokines, while others interfere with signaling pathways regulating host gene expression and apoptosis. to construct a proteome microarray, genomic dna of the copenhagen (nc_ . ) vaccine strain (dryvax) was used as the template for pcr amplification of the open reading frames (orf). gateway recombination cloning (invitrogen, carlsbad, ca, usa) was employed to facilitate high-throughput production of proteins from all orf clones. all dna clones were sequence-verified through the entire length of their inserts. baculovirusbased expression was used to produce the recombinant viral proteins as gst-tagged fusions to ensure high yield of properly folded proteins with posttranslational modifications that are similar to those encountered in the human host. the gst-tagged proteins from cell lysates were affinity purified to % homogeneity in a single step by using glutathione-agarose in -well plates and analyzed for correct size and abundance by western blots. virus and control proteins were printed onto glass slides coated with a thin layer of nitrocellulose. ultimately, % of the proteome was successfully expressed, purified, and arrayed. a standard assay for measuring antibody interactions with proteins of the vaccinia proteome microarray was first developed with a pool of therapeutic human sera collected from vaccinia-immune individuals (vig) and this data was compared to results obtained from individuals vaccinated against smallpox using dryvax. the assay consisted of incubating a dilution of serum on the microarray surface, washes, and finally detection with a fluorescently labeled anti-ig antibody. fluorescent images were captured by scanning with a confocal laser (genepix b; molecular devices). because only a very small sample volume ( - l) is required, this approach is ideal for limiting the amount of sample consumed, often necessary with clinical material. a high level of reproducibility with a very low background was apparent in repetitive assays that confirmed previously reported antigens and identified new proteins that may be important for neutralizing viral infection. incubation of the microarray with vig identified nine proteins that consistently bound antibody, while antibody interactions with all other proteins were insignificant, requiring no further treatment to suppress non-specific signals. these antigens were also recognized by antibodies from individual subjects following primary smallpox vaccination and were diverse in function, consisting of regulatory, surface, core, and secreted proteins. the identical vaccinia proteins clustered into proteins recognized by primary and secondary vaccinated subjects based solely on signal intensities. the vaccinia proteins c l and i l were not previously reported as antibody-recognized antigens. the nine antigenic proteins we identified did not bind antibody from non-vaccinated sera, confirming the specificity of these antibody-antigen interactions. however, o l and h r were reactive with antibodies from both vig and non-vaccinated control sera, suggesting that these were cross-reactive or non-specific interactions. these results indicated that only a small subset of proteins present within the complex orthopoxvirus proteome was associated with antibody responses. in addition, the human response to individual proteins was variable as sera from more than half of the subjects contained igg that recognized > vaccinia proteins, while the remaining samples recognized one to three proteins. further, antibodies from most individuals recognized a greater number of viral proteins after a boost vaccination compared to a single vaccination, suggesting that repeated infection expands the total number of proteins recognized by igg. a previous report [ ] similar to our study on vaccinia [ ] , analyzed the serological response to vaccinia and smallpox, observing that a significant amount of the antibody response to vaccinia was not involved in the virus neutralization, but could be used as potential markers for diagnostic and vaccine development. a later report [ ] evaluated an attenuated smallpox modified vaccinia virus ankara (mva) an alternative to dryvax vaccine by comparing antibody profiles in the sera of humans and monkeys. the overall immunogenicity of mva was reported to be comparable to that of the dryvax vaccine and the study concluded that mva may be a useful alternative to dryvax. our vaccinia virus microarray was expanded to allow analysis of additional poxviruses. monkeypox is a zoonotic viral disease that occurs primarily in central and west africa. immunity to monkeypox is provided by vaccination against smallpox caused by the closely related variola virus. to differentiate antibody responses to monkeypox virus infection from human smallpox vaccination, we developed a protein microarray covering - % ( - proteins) of representative proteomes from monkeypox viral clades of central and west africa, including % coverage ( proteins) of the vaccinia virus proteome as a reference orthopox vaccine. serum igg of cynomolgus macaques that recovered from monkeypox recognized at least separate proteins within the orthopox meta-proteome, while only of these proteins were recognized by igg from vaccinated humans. there were of antigens detected by sera of human vaccinees that were also recognized by igg from convalescent macaques. the greatest level of igg binding for macaques occurred with the structural proteins f l and a r, and the membrane scaffold protein d l. significant igm responses directed towards b r, f l, and a r of monkeypox virus were detected before onset of clinical symptoms in macaques. results from this study suggested that antibodies from vaccination recognized a small number of proteins shared with pathogenic viral strains, while recovery from infection also involved humoral immunity to antigens uniquely recognized within the monkeypox virus proteome (unpublished data). to identify antibody biomarkers that could distinguish plague from infections caused by other bacterial pathogens [ ] , we developed a microarray comprised of the proteome from the bacterium yersinia pestis. the chromosome of y. pestis co encodes approximately proteins, while an additional are episomally expressed by pcd , pmt , and ppcp . for comparison, the proteome of y. pestis kim contains individual proteins, % in common with co , and the closely related enteric pathogen y. pseudotuberculosis contains approximately proteins (chromosome plus plasmids). the microarray was comprised of proteins representative of over % of the orfs present within the y. pestis genome. in a manner similar to the vaccinia microarray, the y. pestis proteins were produced as full-length polypeptides fused to gst as an affinity isolation tag. an in vitro translation method, based on e. coli lysates, was used to express the proteins in a gram-negative background. the orf clones were fully sequenced to confirm quality and identity. the affinity-purified proteins were characterized by sds gels and western blots probed with an anti-gst antibody. different approaches for studying the antibody repertoire for plague in rabbits and non-human primates were investigated. based on results from experiments using the y. pestis proteome microarray, we identified new candidates for antibody biomarkers of bacterial infections and patterns of cross-reactivity with a panel of other gram-negative pathogens [ ] . we were able to cluster the proteins recognized by the antibodies into three groups: common proteins of all gram-negative bacteria, combinations of proteins unique to one pathogen, and proteins unique to one pathogen. this example demonstrates the power of a high-density proteome microarray to dissect the complex immunological relationships among a group of related human pathogens. convalescence sera from vaccinated non-human primates that survived an otherwise lethal aerosol challenge with y. pestis co (plague) or bacillus anthracis ames spores (anthrax) were also examined. in addition to the caf and lcrv proteins comprising the vaccine components, a subset of proteins from the y. pestis proteome were recognized by antibodies from plague survivors and none of these were detected with antibodies from anthrax survivors or non-challenged controls. signature patterns of antibody recognition were identified that reflected the orthologous relationships among proteomes of these bacteria. a high degree of cross-reactivity in the antibody response to burkholderia and related bacteria was also previously reported [ ] . further, a protein microarray consisting of % of y. pestis proteins was used by li et al. to profile antibody responses to live plague vaccine in rabbits [ ] , sentinel animals [ ] , and plague patients [ ] . predominant responses were observed for proteins in addition to f and v antigens in rabbits. in plague patients, they identified proteins that were potential serodiagnostic biomarker candidates and found antibody responses from different plague foci responsible for different clinical symptoms. additional microarray studies of antibody responses to bacterial pathogens were reported. sundaresh et al. [ ] developed a protein microarray with of the most reactive francisella tularensis proteins derived from a larger chip. in this study, they identified a set of immunodominant antigens from f. tularensis patients suitable for diagnosis development. in another study [ ] , a similar protein microarray approach was employed to find immunodominant antigens in mice vaccinated with killed f. tularensis vaccine and challenged with a virulent f. tularensis strain. the study found antigens in addition to known immunodominant antigens of f. tularensis discovered by other methods. this was also the first published report to use protein microarrays for studying protective immune responses to an infectious agent. immunoreactive antigens of coxiella burnetti, the causative agent of q fever, were identified using a protein microarray consisting of % of the c. burnetti proteome assembled by using a cell free protein expression system [ ] . serum from q fever patients and individuals vaccinated with q-vax vaccine strongly reacted to antigens of c. burnetti that were confirmed by elisa including antigens isolated from an e. coli expression system. in yet another study, human and goat antibody responses to brucella melitensis infection were analyzed [ ] , finding that there was differential recognition of antigens by the two host species. the serodiagnostic proteins were of potential value for diagnostic purposes as brucellosis is diagnosed by measuring the antibodies to lipopolysaccharide (lps), which cannot distinguish between past and present infection in endemic areas. antibody response to lyme borreliosis disease in humans and mice was studied by barbour et al. [ ] , using a protein microarray comprising % of the proteins from borrelia burgdorferi. a comprehensive analysis of the sera from b. burgdorferi patients and rodents revealed that only a small set of antigens elicited antibody responses in both hosts, while the majority of b. burgdorferi proteins did not induce antibodies. it is also possible to focus on specific subsets of antigens identified by bioinformatics. for example, a protein array produced from the outer membrane proteins of pseudomonas aeruginosa was constructed [ ] to study the immune response in patients, and several antibody-binding antigens were identified by this group as potential diagnostic markers. in another example, a recombinant niesseria meningitidis protein array was used for screening serum from meningitis patients [ ] for immune responses to phase-variable expressed proteins. the development of new proteomics methods has been driven in part by the need to identify new types of infection biomarkers. though studies of antibody responses dominate the field, protein microarrays should be considered as an alternative approach to examine other catagories of host-pathogen interactions. research areas with well-established cell proteomics data are prime targets. for example, because many of the host factors associated with phagosomes have been identified [ ] , protein microarrays may provide a convenient model system to examine interactions with pathogen proteomes associated with this portal of entry. in a similar manner, targeted microarrays comprised of pathogen proteins may be used to examine host interactions. the few published examples from this developing field of study should be mentioned. in one report, rppm along with other techniques [ ] were used to examine phosphoprotein signaling pathway in primary human small airway epithelial cells infected with bacillus anthracis, identifying reduced akt (protein kinase b) phosphorylation as a contributor to increased bacterial survival and pathogenicity. in another study [ ] , proteins from group a and b streptococci were arrayed on a chip and probed with human fibronectin, fibrinogen, and c binding protein, all well-known targets of gram-positive bacteria. the use of purified and well-characterized proteins to assemble the microarray will insure the highest quality and most reproducible results. however, the high level of quality control required to sequence, purify, and characterize elements of a proteome microarray in the examples we described may not be as important for provisional screening purposes. a coupled transcription/translation reaction was previously reported as a rapid method to develop protein microarrays against a number of infectious organisms [ , ] , bypassing sequencing and purification steps. in this technique, both transcription and translation occur simultaneously to synthesize proteins in parallel from pcr products, directly on the chip. presumably, results obtained by these methods will require follow-up studies with more rigorous controls for confirmation. the protein in situ array (pisa) developed by he and taussig [ ] first produces dna constructs from a pcr of the protein of interest and a t promoter for translational initiation followed by immobilization using a n or c terminal tag sequence. another approach [ , ] called nucleic acid programmable protein array (nappa) included slides coated with avidin to capture biotinylated plasmid dna for synthesizing proteins. proteins with fused tags were synthesized and immobilized on the chips coated with molecules to bind the tags [ ] . he et al. [ ] described a technique called dna array to protein array (dapa) where they utilized a single dna array to make multiple copies of the same protein array. in one example, a self-assembled nappa-based on a cell-free expression system was used for constructing a protein microarray of all proteins produced by varicella zoster virus (vzv), the cause of chickenpox [ ] . three sets of antigens in the vzv proteome were identified from human sera that may be useful in defining the clinical status of the infection and diagnostics. another vzv protein array produced from an e. coli expression system was later used to detect antibodies in human sera reactive to vzv viral proteins [ ] . the orf (ge) antigen identified in the study showed high-confidence for determination of the serological response to vzv. although these alternative in situ synthesis methods are rapid, they are not without significant shortcomings. it is difficult to characterize proteins expressed on the chip in contrast to direct spotting of purified proteins. two studies investigated the antibody response to vzv by protein arrays made from either nappa cell-free expression system [ ] or e. coli expression system [ ] . antibodies from the sera of patients recognized three microarrayed vzv proteins produced recombinantly with e. coli, whereas these identical proteins were not recognized on a nappa protein expression-based protein microarray. interestingly, these proteins were abundantly expressed in the nappa protein microarrays, suggesting that they did not fold correctly to expose their linear epitopes for binding. despite these limitations, microarrays based on in situ synthesis offer simplicity, efficiency, and highthroughput capabilities. the relatively small proteomes of most viruses increase in diversity by considering variations in clinical isolates. for example, while human papilloma virus produces only a few proteins, sequences for these may diverge in the more than strains of the virus. a protein microarray developed with human papilloma virus types [ ] was used to examine patient sera compared to asymptomatic subjects. the study identified e proteins as the most reactive antigens in the patient group. however, the e proteins were not useful as a diagnostic biomarker because the differences between patients and asymptomatic subjects were not significant. in another report, a bead suspension multiplex array (luminex xmap) was used to study serological responses to antigens of human papilloma virus [ ] . the data correlated well with elisa results and in addition the bead array was able to measure weak antibody responses. a protein array constructed from six coronaviruses including severe acute respiratory syndrome (sars) was used for screening serum samples for virus-specific antibodies [ ] . the microarray data predicted sars infection in % of the patients correctly with a specificity of %. the high efficiency, low cost, and high-throughput nature of chemically synthesized peptides are significant advantages compared to production of recombinant polypeptides. therefore, peptide microarrays have been explored as another alternative to recombinant proteins, with a limited degree of success. a chemically synthesized peptide microarray representing the major antigens of hepatitis b and c viruses, human immunodeficiency virus, epstein-barr virus, and syphilis was constructed on a glass slide and antibody responses were simultaneously detected [ ] . the assay showed very high sensitivity and specificity for the diagnostic identification of these viruses. another proof of principle study [ ] was reported to identify diagnostically relevant peptides using a peptide array deduced from bioinformatics data for echinococcus granulosa (tapeworm) infection, achieving only % sensitivity and % specificity compared to elisa. as an alternative to full-length polypeptides, expression and purification of protein domains may improve yield of stable products for use in microarrays. in one reported study, a total of protein domains (sh , sh , pdz etc.) were immobilized on nitrocellulose and screened with peptides [ ] . the results proved that the immobilized domains were stable and could be used for binding studies. kaushansky et al. [ ] constructed an array based on protein interaction domains such as src homology, phosphotyrosine binding domains, and mouse pdz domains produced from recombinant e. coli. these arrays required low sample consumption and may reduce false positive rates. advanced knowledge of the specific recognition events will be required to substitute peptides or single protein domains for full-length proteins to ensure that important epitopes are not missed for the purpose of studying antibody interactions. protein stability is an important and particularly challenging factor in using protein microarrays. the high-throughput nature of construction tends to sacrifice detailed knowledge about individual proteins. there are few methods available to assess folding and functionality of all arrayed proteins immediately before use. recombinant proteins constructed with fusion tags to facilitate folding during translation offer a partial solution, especially if proper folding of the fusion protein can be readily assessed. for example, unfolded glutathione-s-transferase (gst) will not bind to glutathione, the tripeptide ligand of gst, hence purification schemes employing gst fusions will favor proteins with stable structures. fusion tags also provide a convenient marker for monitoring quality control of protein spotting (figure ) . however, the fusion tag may hinder protein accessibility or alter function in other ways. because proteins may denature during printing and storage, enzymes or other denaturing-sensitive proteins that can be assessed after printing should be included in the microarray. antibodies are a natural choice to be used as capture molecules due to their specificity, affinity, ease of production, and potential for engineering. for probe development, the analysis of antibody specificity and cross-reactivity [ ] [ ] [ ] can be simplified by screening against target or off-target proteins printed as microarrays. antibody arrays are routinely used in the field of biomarker discovery and validation. the detection of binding molecules to an antibody array is usually done by direct labeling of proteins in the sample (serum, cell, or tissue) or by a sandwich format similar to elisa. for example, protein abundance in cells was determined by haab et al. [ ] , using antigen and antibody reactions. this was the first published report to use a two-color differential labeling of samples in a protein microarray. the microarray format is also convenient for standardizing the routine analysis of analytes present in screening assays, such as cytokines released by cell cultures [ ] . an antibody-based microarray for the multiplexed detection of cholera, diphtheria, staphylococcal enterotoxin b, tetanus toxins, anthrax protective antigen, and lethal factor was reported [ ] . a competition assay between labeled and unlabeled toxins in serum was used to simultaneously detect all the toxins in the antibody microarray. in another report, a sandwich fluorescence immunoassay based on an antibody microarray format was developed for the capture and detection of e. coli o .h [ ] , and later to screen large number of food samples in a high-throughput manner to simultaneously detect e coli o :h and salmonella typhimurium [ ] . the major drawback of capture microarrays is that few well-defined and high-quality antibodies against microorganisms are available. engineering antibody fragments is a potential alternate approach to accelerate the development of antigen-capture molecules. antibody fragments such as the fab portion of igg [ ] , phage display libraries or recombinant single chain variable fragment (scfv) containing the antigen-binding motif can be used in place of antibodies in protein microarray development [ , ] . a decreased cross-reactivity was also observed with fragments as compared to full-length antibodies. thin-layered nitrocellulose or chemically modified -d surfaces (glass, silicon, gold, and polymer) are generally used for immobilization of microarrayed proteins. buffer additives like glycerol, polyethylene glycol, or sugars that prevent drying of the proteins and choice of buffers should be carefully selected to match the type of surfaces used for arraying. alternatively, gel-based -d surfaces made from polyacrylamide or agarose provide a hydrophilic environment [ ] . regardless of the printing substrate selected, microarrayed proteins must retain functionality and stability during preparation and use. though it may not be possible to directly examine all elements of an array, it is important to include printing controls of interacting proteins that can be tested for functionality before use. there are several methods by which proteins are spotted onto substrates. the choice of technology used impacts spotting consistency, speed, spot diameter, and ease of use. in turn, the final spot quality required for printed proteins will depend on the method of detection. control of the laboratory environment to maintain constant temperature, humidity, and clean-room conditions will vastly improve printing consistency. printing with solid pins (for example stealth pin, arrayit corportion, sunnyvale, ca, usa) relies on capillary forces to release spots on contact with the surface, resulting in - m diameter spots depending on buffer composition and pin diameter. dip-pen lithography (dpn) uses atomic force microscopy microcantilevers to deposit spots in the range of - m diameter (nano enabler, bioforce nanosciences, ames, ia, usa) and inkjet printers (for example arrayjet, roslin, scotland) use piezoelectric elements to transfer the protein solution in the form of droplets to the target surface, resulting in spot diameters of - m. both pin and inkjet spotting methods deposit a very small amount of protein, requiring highly concentrated samples. unfortunately, highly concentrated samples may fail to adsorb completely on the surface and tend to spread during hydration. a new continuous flow microspotting method (wasatch microfluidics, taylorsville, ut, usa) uses microfluidic channel networks to continuously circulate protein samples over a spot to achieve uniform and maximum protein adsorption [ ] . this technique may be especially useful for dilute and crude protein sample arraying. fluorescence-based labeling of probes is most commonly used to detect binding events, due primarily to simplicity and sensitivity. examples are amine-reactive dyes such as cyanines (cy and cy ) or labeling probes with biotin followed by detection with an avidin-fluorescent dye conjugate. ccd cameras or fluorescent slide scanners are used for detection of the labeled probes within defined grid areas. sandwich immunoassay methods provide increased sensitivity and at the same time reduce non-specific binding, but their use is limited by the availability of paired antibodies. using a rolling circle amplification (rca) method can dramatically increase fluorescent signal intensity [ ] , thus increasing sensitivity in protein microarray detection methods. in this scheme, proteins are labeled with biotin and recognized by an antibody conjugated with a primer to which a circular dna is hybridized. fluorescently labeled oligonucleotides are used for elongation of dna and detection. a universal rca signal amplification scheme was used [ ] for measuring cytokines in a multiplexed format. a total of cytokines were simultaneously measured on an antibody microarray, demonstrating that even larger number of analytes can be measured by this technology. thus, rca is a powerful tool for improving the detection limits of protein microarrays by signal amplification. current fluorescence-based detection in protein microarrays is limited to two to three independent probes because broad emission profiles exhibited by the dyes used for detection often overlap with each other and with the background fluorescence emitted by substrates, such as the commonly used nitrocellulose. also, multiple excitation and emission channels are required to detect combinations of fluorescent markers. quantum dot (qds) nanoparticles are one alternative to fluorescent dyes. the qds are semiconducting fluorophores that are extremely bright, resist photobleaching, and have multiplexing capability. each contains an inorganic fluorescent core, commonly cdse, coated with a shell of another semiconductor such as cds or zns. the most important property of qds with reference to protein microarray detection is that they absorb light at broad wavelengths, from uv to visible range and emit light at a very narrow bandwidth, in contrast to fluorescent dyes. additionally, the qd surfaces may be modified with suitable chemistries for immobilizing biomolecules. streptavidin-coated qds in a protein microarray format were used to detect six different cytokines at picomolar concentrations [ ] , currently the practical limit for multiplexing with qds. additional multiplexing strategies have been previously described in detail [ ] . bead-based microarrays offer one solution to multiplexing. most commercially available methods were developed by luminex (xmap platform). the xmap technology is based on polymer beads incorporated with fluorescent dyes that produce unique signatures. these beads can be conjugated to different molecules to perform multiplexed assays. up to discrete interactions can be monitored simultaneously using this technology [ ] , perhaps extended to bead sets in the near future. antigens or peptides have been attached to the luminex beads and antibody responses to papillomavirus [ ] , epstein-barr virus [ , ] , and mycobacterium tuberculosis [ ] were reported. becton dickinson (franklin lakes, nj, usa) markets a cytometric bead array (cba) system, which is another multiplexed bead-based assay that includes flow cytometry for cytokine, chemokine, mouse isotyping, and signaling molecule analysis. label-free detection methods are used for real-time monitoring of binding events and to minimize artifacts caused by using labeled probes. spr imaging technology is widely used in a microarray format for many applications. spr is label-free, allows real-time monitoring, and has the additional ability to measure kinetics of the molecular interactions ( figure ). spr imaging was developed to simultaneously measure spr dip changes in an array format, recorded with a ccd camera. grating-coupled (flexchip, ge/biacore, piscataway, nj, usa) and prism-coupled (spri-plex, horiba scientific, edison, nj, usa, proteon xpr , bio-rad, hercules, ca, usa) spri systems are some examples of commercially available spri microarray systems. a model protein-protein interaction system was reported [ ] , consisting of the e protein of human papillomavirus complexing with the proteins e ap and p using a spri system. in another example, an array-based spectral spr system was used for measuring antibody response to mumps virus infection [ ] . in this system the reflected light from the array was collected into a fiber optic spectrometer for analysis. our laboratory routinely uses spri microarrays to validate interactions that were detected during primary microarray screens from a fluorescent probe read-out. because spot sizes must be large enough for resolution by the commonly used ccd imaging, practical array densities are currently less than independent immobilized probes. additional label-free detection methods have been described. surface-enhanced laser desorption/ionization time of flight mass spectrometry (seldi-tof-ms) has been used as a low throughput method for on-chip purification of proteins, ionization, and detection [ ] . however, this approach was not very successful in identifying biomarkers in parasitic diseases, and most markers found in the study were intact host proteins [ , ] . matrix-assisted laser desorption/ionization (maldi)-ms in combination with -d gel surfaces [ ] and silicon nanoporous nanovials [ ] were also used for biomarker discovery. the ms-based detection systems are important for specific applications, but there have been only limited advancements in the development of microarray formats, currently limiting use as a primary screening tool. nanowire-based systems detect changes in conductivity as a result of molecular binding [ , ] . it is possible to make an array of nanowires such that each nanowire is coated with a specific capture molecule. gantelius et al. [ ] reported a magnetic bead-based multiplexing format for the detection of auto-antibodies to antigens, comparing the assay results to fluorescence-based detection system. although the magnetic bead-based system was rapid, it was not sufficiently sensitive for routine use. despite the rapid progress in protein microarray development standardized methods for data handling and analysis are not universally accepted. fluorescence scanners made by different commercial vendors use stand-alone software to analyze data, while assay protocols used by investigators may vary widely. hence, it is often difficult to compare studies reported by different groups, and there is a need to formulate standardized methods for protein microarray data handling and analysis. a number of reports have outlined ways to normalize protein microarray data, mainly for different approaches to antibody microarrays. these normalization methods include concentrationdependant analysis [ , ] , spiked internal standards [ ] , and algorithm-based [ ] , and all have significant pros and cons. invitrogen/life technologies (carlsbad, ca, usa) developed a data analysis package (protoarray prospector) specifically designed for protein antigen microarrays, including a suite of statistical methods. bioarray software environment (base), a software package for microarray data management and analysis (http://base.thep.lu.se) developed by lund university, sweden, also addresses some of the issues we have outlined. however, it remains to be seen how rapidly standards will be adopted by the research community. antibodies are primary biomarkers of infection that are commonly used for diagnosis, measuring vaccine efficacy, and discovery of disease interventions. protein microarrays are important tools for measuring these complex antigen-antibody interactions and other host-pathogen interactions at different stages of the disease. the major impediments to development of protein microarrays are the inherent complexity of proteins themselves, availability of well-characterized antibodies to infectious agents, and standardized statistical methods for analysis of data. however, we expect many of these issues to be resolved in the coming years as these tools are used more frequently in infectious disease research. quantifying e. coli proteome and transcriptome with single-molecule sensitivity in single cells correlation between protein and mrna abundance in yeast label-free detection methods for protein microarrays present and future of surface plasmon resonance biosensors multiplexed electrical detection of cancer markers with nanowire sensor arrays high-speed biomarker identification utilizing porous silicon nanovial arrays and maldi-tof mass spectrometry protein and antibody microarrays: clues towards biomarker discovery. front. drug des protein-protein interactions and selection: array-based techniques for screening disease-associated biomarkers in predictive/early diagnosis defining the humoral immune response to infectious agents using high-density protein microarrays design of high-density antibody microarrays for disease proteomics: key technological issues extensive antibody cross-reactivity among infectious gram-negative bacteria revealed by proteome microarray analysis analysis of the human immune response to vacccina by use of novel protein microarray suggests that antibodies recognize less than % of the total viral proteome applications of protein microarrays for biomarker discovery protein microarrays: high-throughput tools for proteomics advances and perspectives in aptamer arrays affibody molecules in protein capture microarrays: evaluation of multidomain ligands and different detection formats use of a single-chain antibody library for ovarian cancer biomarker discovery reversephase protein microarrays: application to biomarker discovery and translational medicine development of reverse phase protein microarrays for clinical applications and patient-tailored therapy reverse phase protein microarrays which capture disease progression show activation of pro-survival pathways at the cancer invasion front analysis of signaling pathways in cancer cell lines by protein lysate array reverse-phase protein lysate microarrays for cell signaling analysis multiplex approaches in protein microarray technology printing proteins as microarrays for high-throughput function determination self-assembling protein microarrays global analysis of protein activities using proteome chips proteome-wide analysis of the serological response to vaccinia and smallpox antibody profiling by proteome microarray reveals the immunogenicity of the attenuated smallpox vaccine modified vaccinia virus ankara is comparable to that of dryvax a burkholderia pseudomallei protein microarray reveals serodiagnostic and cross-reactive antigens protein microarray for profiling antibody responses to yersinia pestis live vaccine serologic survey of the sentinel animals for plague surveillance and screening for complementary diagnostic markers to f antigen by protein microarray antibody profiling in plague patients by protein microarray from protein microarrays to diagnostic antigen discovery: a study of the pathogen francisella tularensis immunodominant francisella tularensis antigens identified using proteome microarray candidate antigens for q fever serodiagnosis revealed by immunoscreening of a coxiella burnetii protein microarray large scale immune profiling of infected humans and goats reveals differential recognition of brucella melitensis antigens a genome-wide proteome array reveals a limited set of immunogens in natural infections of humans and white-footed mice with borrelia burgdorferi genome-wide study of pseudomonas aeruginosa outer membrane protein immunogenicity using self-assembling protein microarrays bacterial protein microarrays for identification of new potential diagnostic markers for neisseria meningitidis infections analysis of phagosomal proteomes: from latex-bead to bacterial phagosomes anthrax infection inhibits the akt signaling involved in the e-cadherin-mediated adhesion of lung epithelial cells capturing host-pathogen interactions by protein microarrays: identification of novel streptococcal proteins binding to human fibronectin, fibrinogen, and c bp profiling the humoral immune response to infection by using proteome microarrays: high-throughput vaccine and diagnostic antigen discovery identification of humoral immune responses in protein microarrays using dna microarray data analysis techniques single step generation of protein arrays from dna by cell-free expression and in situ immobilisation (pisa method) next-generation high-density self-assembling functional protein arrays printing protein arrays from dna arrays systematic analysis of the igg antibody immune response against varicella zoster virus (vzv) using a self-assembled protein microarray a systematic approach for the identification of novel, serologically reactive recombinant varicella-zoster virus (vzv) antigens high-throughput profiling of the humoral immune responses against thirteen human papillomavirus types by proteome microarrays multiplex human papillomavirus serology based on in situ-purified glutathione s-transferase fusion proteins severe acute respiratory syndrome diagnostics using a coronavirus protein microarray peptide-protein microarrays for the simultaneous detection of pathogen infections felger, i. serodiagnosis of echinococcus spp. infection: explorative selection of diagnostic antigens by peptide microarray a protein-domain microarray identifies novel protein-protein interactions quantifying protein-protein interactions in high throughput using protein domain microarrays protein arrays as tools for serum autoantibody marker discovery in cancer protein microarrays: a new tool for profiling antibody cross-reactivity analyzing antibody specificity with whole proteome microarrays protein microarrays for highly parallel detection and quantitation of specific proteins and antibodies in complex solutions inhibition of toxic shock by human monoclonal antibodies against staphylococcal enterotoxin b antibody microarrays for native toxin detection antibody microarray detection of escherichia coli o :h : quantification, assay limitations, and capture efficiency an antibody microarray, in multiwell plate format, for multiplex screening of foodborne pathogenic bacteria and biomolecules protein microarrays for antibody profiling: specificity and affinity determination on a chip evaluation of antibodies and microarray coatings as a prerequisite for the generation of optimized antibody microarrays microarrays based on affinity-tagged single-chain fv antibodies: sensitive detection of analyte in complex proteomes why -d? gel-based microarrays in proteomics continuous-flow microfluidic printing of proteins for array-based applications including surface plasmon resonance imaging mutation detection and single-molecule counting using isothermal rolling-circle amplification multiplexed protein profiling on microarrays by rolling-circle amplification protein microarrays and quantum dot probes for early cancer detection antibody suspension bead arrays within serum proteomics serologic response to oncogenic human papillomavirus types in male and female university students in evaluation of three different assays for the assessment of epstein barr-virus immunological status evaluation of a multiplexed bead assay for assessment of epstein-barr virus immunologic status profiling antibodies to mycobacterium tuberculosis by multiplex microbead suspension arrays for serodiagnosis of tuberculosis surface plasmon resonance imaging protein arrays for analysis of triple protein interactions of hpv, e , e ap, and p array-based spectral spr biosensor: analysis of mumps virus infection is seldi-tof a valid tool for diagnostic biomarkers? identification of novel diagnostic serum biomarkers for chagas' disease in asymptomatic subjects by mass spectrometric profiling use of proteinchip technology for identifying biomarkers of parasitic diseases: the example of porcine cysticercosis (taenia solium) analysis of protein interaction and function with a -dimensional maldi-ms protein array nanowire nanosensors for highly sensitive and selective detection of biological and chemical species protein recognition via surface molecularly imprinted polymer nanowires magnetic bead-based detection of autoimmune responses using protein microarrays a concentration-dependent analysis method for high density protein microarrays development of an internally controlled antibody microarray optimized normalization for antibody microarrays and application to serum-protein profiling determination of relative protein abundance by internally normalized ratio algorithm with antibody arrays the authors appreciate the many contributions of sarah keasey, christine pugh, emily cisney, stefan fernandez, and other members of the riid protein interactions team, the contributions of barry schweitzer and our other collaborations at life technologies for pioneering studies in protein microarray technology, and justin rodrigo for critical reading of the manuscript. opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the united states army. key: cord- -vcys q t authors: kawachi, ichiro title: covid- and the ‘rediscovery’ of health inequities date: - - journal: int j epidemiol doi: . /ije/dyaa sha: doc_id: cord_uid: vcys q t nan in the wake of the brutal police killing of george floyd in minneapolis on may , cities and counties across the usa came out to declare that racism is a 'public health emergency'. needless to point out, systemic racism has existed for over years in america. the crisis sparked by george floyd's murder illustrates the point that it takes a shock to the system to bring about broader acknowledgment of the daily realities lived by a whole segment of the population. indeed, ignorance of the existence of systemic racism (a.k.a. white privilege) is what enables stark inequalities to fester. likewise, health inequalities have been evident since the beginning of public record keeping-q.v. villermé's tabulations of mortality rates by income ( - ). the covid- pandemic just happens to be latest crisis that has brought renewed attention to the existence of health inequalities. throughout history, people's experiences of pandemics have differed according to their access to power, privilege and resources. in th century plague-stricken florence, wealthy patricians fled the city to their secluded villas in the tuscan hills, where they amused themselves in the evenings by drinking fine wine and recounting stories to each other. unfortunately, there are few surviving records of the suffering of those left behind in the city, as the majority of the population was illiterate. a future historian writing about the covid- pandemic will note the dramatically different ways in which people experienced the pandemic according to their race, social class, gender and immigrant status. it is as if people inhabited alternate realities. the historian will note how high-speed internet and zoom enabled the comfortably well off to escape crowded urban centres and wait out the pandemic in their second homes in the countryside, while silicon valley billionaires at the apex of the economic pyramid fled on their private jets to their bunker-equipped estates on waiheke island, new zealand. meantime, for millions of others, the stark reality of life under covid consisted of losing their jobs, falling behind on the rent and ending up either evicted or doubling up with other family members, thereby piling on their risk of infection. pandemics disrupt everybody's lives, but not in the same way. a basic tenet of social epidemiology is that the probabilities of exposure and outcomes (conditional on exposure) are not random, but socially patterned. almost always the odds are stacked against the socio-economically disadvantaged. consider the case of other major disasters. media stories often portray these events (earthquakes, hurricanes or the sinking of the titanic) as if they were random acts of god in which everyone, rich and poor alike, are vulnerable. this is seldom the case. the social epidemiology of disaster shows that socio-economically disadvantaged groups are both more likely to be exposed to disaster (because they live in disaster-prone areas or live in structurally unsound houses) and more likely to suffer the consequences of exposure (because they suffer disproportionately from preexisting morbidity, making them vulnerable to problems such as the interruption of services that follow inevitably in the wake of disaster). by the time covid- has run its course in - years, we should not be surprised if the toll of infection and mortality turn out to be the highest in the most unequal societies in the world-the usa, brazil, india, russia, and among african countries, south africa. the usa is a textbook case of longstanding inequalities in income, working conditions and access to healthcare, leaving large swaths of the population vulnerable to the effects of crises. persistent segregation of the workforce by race/ethnicity all but ensured that coronavirus infection would be concentrated in communities of colour. black americans are over-represented in jobs involving close contact with people, including in the health care support sector (nursing and home aides), personal care and service (e.g. barbers, hair salons) and food preparation/serving occupations. in turn, higher risk of exposure in the workplace is compounded by the persistent residential segregation of neighbourhoods, where unequal exposure to air pollution (see also the analysis of the uk biobank data by chadeau-hyam et al. featured in this issue) and overcrowded housing conditions amplify the spread of infection. besides residential neighborhoods and workplaces, some of the biggest outbreaks have been recorded in prisons, where some . million americans are incarcerated, half of whom are black or hispanic. in short, it was predictable that the devastating toll of covid- would be starkly patterned by race, social class and geography. landing like a hurricane, the coronavirus tore the cover off decades of disinvestment and neglect of communities of colour. wearing masks can only go so far in protecting vulnerable members of society. compounding the misery of covid- morbidity and mortality, the public health response to the pandemic has come at the cost of a sharp global economic contraction. an unfortunate (but all too predictable) consequence of 'bending the infectious curve' is that business and school closures, as well as directives to shelter in place, disproportionately afflict the already disadvantaged. small business closures in the usa have hit black-owned businesses the hardest, because a higher share of them are in the restaurant, personal services and retail sector. many of these businesses may never come back. during economic contractions that accompany pandemics, workers with the least skills and lowest pay are usually the first to be let go. but we have always known this. writing about the great plague of london in , daniel defoe remarked: 'all families retrenched their living as much as possible, as well those that fled as those that stayed; so that an innumerable multitude of footmen, serving-men, shopkeepers, journeymen, merchants' bookkeepers, and such sort of people, and especially poor maid-servants, were turned off, and left friendless and helpless, without employment and without habitation, and this was really a dismal article.' (p. ) some londoners might have considered themselves even lucky to have escaped with just being out of work. others who were less fortunate were pressed into compulsory service as watchmen to guard over 'infected houses' to make sure that people in them did not break quarantine rules, or forcibly put to work by parishes as 'searchers'generally 'women of honest reputation, and of the best sort as can be got'-whose job it was to search for dead bodies. when the haves and have nots inhabit separate worlds and experience divergent realities, it hamstrings society's ability to mount a coherent response to a common threat. it is characteristic of a divided society that people question the need for universal public health measures. they ask: 'why am i being asked to shelter in place, when i don't see a problem in my community?' a society lacking the social basis of solidarity means that slogans such as 'together we can beat the virus' fail to mobilize unified action. the problem is compounded (as in the usa) when the policy response is devolved to each locality-states, cities and counties-resulting in a patchwork of directives that are inconsistently enforced. in many localities it is left to each individual's choice to decide whether to comply with recommendations such as wearing masks or practicing social distancing. it does not help that politicians have exploited existing fracture lines by encouraging their supporters to flout public health advice as an expression of fealty. 'lockdown fatigue' is cited as the reason why governors in several us states are rushing to reopen businesses despite rising caseloads. their rallying call is that the cure cannot be worse than the disease. but the voices of frontline workers are being drowned out by the clamour of business owners to reopen. when employers call them back to work, workers are being thrust into the position of choosing whether to stay at home (and lose their unemployment benefits), or to return to work and risk losing their health. the 'frontline' is a metaphor borrowed from war, and echoes past conflicts when young men lacking the wherewithal to defer the military draft were dispatched to the frontlines. in the context of the pandemic, 'essential' workers are being sent back to work in order to resuscitate the economy (and to improve the re-election chances of incumbent politicians). in the usa, frontline workers are even being equated with 'warriors' (as when president trump declared during a visit to a face mask factory in arizona in may , 'the people of our country should think of themselves as warriors. our country has to open.'). meanwhile, far away from the frontlines, conversations about when businesses should reopen seemingly revolve around an entirely different set of concerns, such as how long one can go without a haircut. for those with the ability to work remotely, the pandemic might even produce a tiny health dividend. for them, tele-work has resulted in less commuting stress, more discretionary time to engage in daily physical activity (as evidenced by booming sales of stationary exercise bikes) and more home cooked meals. (needless to add, these theoretical benefits apply mainly to families who do not have young children at home.) when future historians look back on the dismal performance of unequal societies during the covid- pandemic, they will conclude that their leaders were too slow to implement public health measures, and too hasty in loosening them. a polarized society in which those who feel protected from the virus do not share the same lived reality as the vulnerable is a recipe for flawed decision making. a health equity framework is needed to guide debates about how societies should respond to the ongoing covid- crisis. yet the information systems upon which we base our forecasting models are sorely lacking in socio-economic data. electronic health records in the usa omit information such as occupation, educational attainment and household income, resulting in the resort to imperfect proxies such as medicaid status or neighbourhood socioeconomic status. in turn, the absence of relevant socioeconomic data means that the current tools we use to guide decisions lack an explicit equity focus. for example, infectious disease models do not formally incorporate social inequalities in transmission dynamics and infection severity. compartmental epidemiological models, such as susceptible-infected-recovered (sir) models, typically do not factor in the influence of social determinants when estimating the transition probabilities between different states (e.g. exposed ! infected, or infected ! recovered). the r-nought is discussed in the aggregate but not broken down by race, social class or other axes of social stratification. as a result the health equity impact of policy choices are presented in qualitative terms, but never quantified. similarly, mathematical forecasting of the covid- pandemic only speaks of aggregate numbers. modelling has suggested that one in five people around the globe are at risk of severe covid infection, due to underlying conditions like diabetes, respiratory and cardiovascular disease. yet these projections are silent with regard to the social distribution of vulnerability. the reason for this lacuna is because the global burden of disease project (upon which the projections are based) is silent with regard to the social distributions of underlying diseases that elevate the risk of severe covid infection. aggregate projections stripped of their socio-economic context serve as an insufficient basis to guide momentous decisions, such as who should receive priority when a vaccine eventually becomes available. what will be the basis to debate whether a white-collar professional with pre-existing conditions (working from home) deserves higher priority access to a vaccine compared with an otherwise healthy black frontline worker? economic models are equally silent with regard to distributive effects of policy choices. cost-benefit and costutility analyses of border closures and lockdowns inform us about costs to society in the aggregate, but typically they do not consider who bears the costs of job losses and lost productivity vs loss of life and morbidity. it may turn out to be the case that lockdowns produce a net health equity gain by saving the lives of the most vulnerable segments of the workforce (especially if adequate social protections are in place to mitigate the consequences of job loss). but we cannot know the true answer unless formal modelling is undertaken to incorporate equity considerations. for example, recent quasi-experimental evidence from the national lockdown in italy showed a proportionally sharper reduction in the mobility of the less well off, suggesting that both poverty and income inequality will be exacerbated as a result. forecasting models do not set out, by design, to ignore socio-economic status; the problem is that the raw data are not being collected at source (e.g. hospital records, covid registries). epidemiologic forecasting models need to advance beyond making aggregate projections. the goal of an equity-informed forecasting exercise should be to inform decision makers about how to anticipate and mitigate the inevitable health equity consequences of policy debates about when to reopen borders, schools, workplaces and public spaces, as well as how to prioritize the global distribution of a vaccine (when it materializes). the moment seems ripe when the international epidemiological community should be calling upon hospitals, health authorities and governments to begin collecting socio-economic data. ), a pioneer in social epidemiology: re-analysis of his data on comparative mortality in paris in the early th century the decameron rich americans activate pandemic escape plans. interest in new zealand bunkers has surged. blacks in the labor force exposure to air pollution and covid- mortality in the united states: a nationwide cross-sectional study a journal of the plague year ( ). london: penguin books trump drafts everyday americans to adopt his battlefield rhetoric incorporation of socioeconomic status indicators into policies for the meaningful use of electronic health records global, regional, and national estimates of the population at increased risk of severe covid- due to underlying health conditions in : a modelling study economic evaluation of border closure for a generic severe pandemic threat using new zealand treasury methods economic and social consequences of human mobility restrictions under covid- key: cord- -ytcwt qk authors: smith, k. m.; zambrana-torrelio, c.; white, a.; asmussen, m.; machalaba, c.; kennedy, s.; lopez, k.; wolf, t. m.; daszak, p.; travis, d. a.; karesh, w. b. title: summarizing us wildlife trade with an eye toward assessing the risk of infectious disease introduction date: - - journal: ecohealth doi: . /s - - - sha: doc_id: cord_uid: ytcwt qk the aim of this study was to characterize the role of the usa in the global exchange of wildlife and describe high volume trade with an eye toward prioritizing health risk assessment questions for further analysis. here we summarize nearly years ( – ) of the most comprehensive data available (usfws lemis system), involving billion individual specimens and an additional million kilograms of wildlife. the majority of shipments contained mammals ( %), while the majority of specimens imported were shells ( %) and tropical fish ( %). most imports were facilitated by the aquatic and pet industry, resulting in one-third of all shipments containing live animals. the importer reported origin of wildlife was . % wild-caught and . % captive-reared. indonesia was the leading exporter of legal shipments, while mexico was the leading source reported for illegal shipments. at the specimen level, china was the leading exporter of legal and illegal wildlife imports. the number of annual declared shipments doubled during the period examined, illustrating continually increasing demand, which reinforces the need to scale up capacity for border inspections, risk management protocols and disease surveillance. most regulatory oversight of wildlife trade is aimed at conservation, rather than prevention of disease introduction. major drivers of human-animal contact allowing pathogen exchange include animal domestication for companionship and food production, anthropocentric alteration of the environment and the global movement of animals and goods. approximately one-quarter of human deaths are caused by infectious disease and nearly % of infectious diseases are considered zoonotic (pathogens transmissible between animals and humans); most of these (> %) are caused by pathogens of wildlife origin (taylor et al. ; jones et al. ; drexler ) . whereas historically disease spillover events were likely to remain local, even undetected due to natural, cultural and geographic barriers, modern transportation allows emerging diseases to spread along various globally connected networks in a matter of days. in the past decade alone, we have witnessed several novel disease threats to global health, food security and economic stability as a result of one of these networks-the trade of live wild animals and/or their products (karesh et al. (karesh et al. , . anthropogenic movement and manipulation of domestic and wild animals, including globalized trade, were proposed as ''the'' biggest potential trigger drivers for disease emergence and spread since the advent of agriculture [who, food and agriculture organization (fao) and world organisation for animal health (oie) joint report ]. lack of wildlife trade surveillance and proper systematic management of the data available represents a major gap to understand and determine high-risk pathways of potentially adverse organisms' introduction. in order to properly assess this threat to the usa, we must ( ) better understand the scope of the trade in terms of species, volume, condition and origin; ( ) determine high-risk pathways of introduction for further assessment; and ( ) understand the regulatory framework that exists to manage these threats. the goal of this study is to characterize the wildlife trade entering the usa as a baseline for understanding the magnitude of the potential threat these activities may pose to the environment, animals and humans. although reports exist in the literature, to our knowledge this is the broadest summary (in terms of time scale and detail) aimed at supporting risk assessments surrounding us wildlife trade importation. wildlife trade is one of the largest and most complex commerce exchanges in the world. the legal global trade in wildlife and wildlife products involves the movement of billions of plants and animals comprising an economic value estimated at us $ billion per annum (ahlenius ; wwf/dalberg ) . the illegal aspect of wildlife trade is estimated to be a $ - billion-dollar industry, comparable to the international trade of narcotics and weapons (wyler and sheikh ; haken ; wwf/ dalberg ) . there are no adequate estimates of the full scale of wildlife traded throughout the world given its diversity, scope and partial underground existence. uncertainty surrounding this issue is enhanced by lack of international data standards and varying commitments to data collection infrastructure within and between countries. fundamental terms such as ''legal'' and ''illegal,'' ''formal'' and ''informal'' may be subjective and based upon which regulations are applied and the context of the trade (e.g., national laws vary by country for trade in a particular species, certain species can be traded for particular purposes but not for others). in some cases, legal trade is well recorded by border officials while in other cases it is largely ignored. confiscated illegal trade is often reported but undetected illegal shipments regularly go unrecorded. legality of wildlife trade in most instances does not correlate with disease risk posed, as the majority of wildlife trade regulations (e.g., the convention on international trade in endangered species of wild fauna and flora; cites) are in place to conserve certain species or regulate economies rather than protect health. although wildlife trade is often lumped into a single entity, this enterprise is comprised of a multitude of products such as food, trophies, pets, fashion, medicine, artifacts and aphrodisiacs. within each category exist a range of specialty market value chains that vary in motivating economics, cultural and societal origins, geographic source and destination, transportation type and route, trader and consumer identities, behavioral practices, species volume and condition, local and international legality. this results in vastly variable threats including loss of biodiversity, invasive alien species, food security and emergence of both high-and low-consequence pathogens. thus, threats can only be quantified in response to specific questions (i.e., examining unique traits of specific market chains/pathways). timber and plants are estimated to comprise nearly % of the known (broadly defined) wildlife global trade value, leaving non-aquaculture fisheries products responsible for % and ornamental fish, mammals, herpetofauna and other species responsible for roughly % (us $ . billion) (engler and parry-jones ; ahlenius ) . the majority of live wild animal trade is comprised of aquatic animals and herpetofauna traded mainly for the pet industry. china and southeast asian countries are the top global exporters, while the usa and european union (eu) are the top importing consumers (altherr et al. ) of aquatic and herpetofauna wildlife. a portion of this trade is recorded by weight only, leaving the total number of individual animals involved unquantified. approximately million live fish are imported to the usa annually, % of which are freshwater taxa (smith et al. ) . live turtles and frogs are also commonly imported as pets as well as food items. the usa imports on average tons of frog legs in addition to tons of live frogs for consumption each year (altherr et al. ) . birds and mammals are also highly represented among a myriad of known global trade routes for exotic pets (bush et al. ) . a review of this trade found it to be an expanding, yet fluid and dynamic industry with reasons for its growth including human population expansion, increasing affluence in south america and east asia (resulting in a larger market for exotic pets), use of the internet and a broadening interface with wildlife habitat (bush et al. ). given global variability in laws and difficulty in distinguishing between legal and illegal transactions (e.g., false declarations of geographic origin, captive vs. wild-caught, misrepresentation of purpose of import or final destination), monitoring legality of wildlife trade is comparable in complexity to weapons trade. in the majority of instances, the legality of trade of wildlife at the international and national level is determined by authorities tasked with conservation rather than public or animal health protection. specifically, unpermitted trade of cites-listed species across international borders comprises the bulk of what is considered and/or reported as global illegal wildlife trade. as with legal trade of wildlife, species are traded illegally as exotic pets, specialty foods, traditional medicines, trophies and fashion items. drivers of this illicit trade vary from financial to cultural to relic. because the drivers and components of illegal wildlife trade are highly variable, the perpetrators do not fit any one category nor does their trade behavior follow a single pathway. diverging networks include local village hunters, criminal groups engaged in drugs or terrorism, government officials and other economically driven sellers and consumers (hayman and brack ; warchol et al. ; wyler and sheikh ; wwf/dalberg ). cites maintains a database of reported trade of citeslisted species only. the database is managed by the united nations environment programme-world conservation management center (unep-wcmc) and currently holds million records of trade involving , scientific names of taxa listed by cites. currently, more than , trade records are reported annually (http://www.cites.org/eng/re sources/trade.shtml). there are also trade data held by the united nations statistics division comtrade. these data are maintained in broader categories such as ''live animal'' or ''reptile skins.'' although some of these data are more specific, species level detailed information is generally not available (chan et al. ). the comtrade data are self-reported by trading partners, and as a result, there are inconsistencies and may also be variable reporting even within the broader categories. additionally, there are data held by national governments that vary widely in their format and scope, and rely largely upon efforts of authorities given national laws and priorities. these data are often not available to the public but some summaries may be found in gray literature reports. all importers of wildlife to the usa are mandated to submit a - (www.fws.gov/le/pdf/ _ .pdf) request to the us fish and wildlife service (usfws) which in turn records details of the imports into the law enforcement management information system (lemis) database. this database includes both cites and non-cites species considered to be wildlife per the usfws definition ( cfr . ). this database therefore holds records of all declared wildlife imports to the usa and, theoretically, details of illegal imports confiscated by authorities at us ports of entry. although some wildlife species are regulated by other us agencies such as the united states department of agriculture (usda), centers for disease control and prevention (cdc), and food and drug administration (fda), imports of wildlife as defined by usfws are also tracked in lemis despite this overlap in jurisdiction. therefore, the lemis system represents a comprehensive data source for incoming wildlife to the usa (with few exceptions such as bushmeat items that are not determined to be of cites origin). lemis data are maintained by usfws for years. since , lemis data spanning january , -august , , have been collected, standardized, cleaned (e.g., misspellings) and entered into a propriety database structure curated by ecohealth alliance (eha). the dataset, entitled ''wildb,'' includes taxonomical, geographic and count information for each shipment that entered the usa and its territories, allowing for pathway analyses to be conducted on a global level for wildlife that entered the usa. the results presented here are a preliminary analysis of this comprehensive database of wildlife trade into the usa. the dataset continues to be periodically updated. as of january , wildb included a total of , , individually identifiable wildlife shipments entering the usa between january , , and august , . the number of annually declared wildlife shipments doubled during the period examined (figure ), reaching approximately , declared shipments imported in . these shipments included a total of , , , individual specimens/animals, plus an additional , , kg of specimens/animals measured only in weight. of these, , , , ( . %) individuals plus , , ( . %) kg were recorded as live upon entry. thus, for the period - , there was an annual average of . million (s = . million; median = . million) live animals plus an additional . million kilograms of live animals imported into the usa as recorded in the lemis database. we selected the top ten categories represented by the data for illustration of the most frequent wildlife taxa imported to the usa. the majority of wildlife shipments (by taxon) contained mammal products (most of which were non-live; figure a) , while the majority of total specimens imported were shells and tropical fish (figure b ). this is due to the fact that shipments of mammals and their products contain fewer individuals or items while large volumes of aquatic species can be transported in a single shipment. nearly one-third of all wildlife shipments entering the usa contained live animals, the vast majority of which were imported by the aquatic and pet industry. aquatic, amphibian and invertebrate species accounted for approximately % of recorded shipments of live animals to the usa. top specimens involved in such live shipments are represented in figure . once the aforementioned subset is removed, reptile, rodent and bird species destined for the exotic pet trade make up the majority of remaining live imports. while roughly % of incoming live wildlife shipments contained mammals, this taxon only represented % of overall number of specimens imported (approximately , , individual mammals, plus additional mammals documented only by weight vs. number of ani-mals). excluding those recorded only by weight, , , live mammals were imported. the data contained reported both ''country of origin'' and ''country of export.'' this reporting is by the importer/exporter, and therefore, country of origin may be falsely reported without means for authorities to verify source. the stated origin of imported live wildlife from to was roughly . % wild and . % captive ( . % listed as ranched or other). since many species traded may be wild-caught or captive-raised, it remains difficult for authorities to identify false reporting of wild versus captive and true country of origin, despite visual inspection and means of import. the reported countries of origin from to for all declared us wildlife imports by shipment are shown in figure , with indonesia as the leading exporter. however, at the specimen level (i.e., number of individual animals/products imported), china was the leading exporter. many imports were not identified at the species level. china and southeast asia was a primary region of origin for us wildlife imports. the vast majority of both live and non-live wildlife imported from this area were aquatic, invertebrate and herpetofauna species. indonesia was responsible for exporting the most live wildlife ship- ments to the usa during the period examined, comprised mainly of these aforementioned species. however, live mammals and birds were also imported. examples of identified live species imported from china included over , kg of live american bullfrogs (rana catesbiana)-a conservatively estimated , frogs-imported mainly for food, nearly million live pheasants (phasianus colchicus spp.), approximately , live macaques, in addition to live bats for research and asiatic chipmunks for pets in fewer numbers. vietnam was the exporter of shipments of live macaques to the usa during the period examined. taiwan exported approximately , finchlike live pet birds including canaries and goldfinches, and indonesia exported over , kg of ''edible-nest swiftlet'' nests to the usa. the primary origins of mammal imports specifically were canada and south africa based on number of shipments, and the usa and china for number of specimens/ animals imported. more than , live bison were imported from canada annually, and additional amounts recorded only by weight. likewise hoofstock made up the majority of mammal imports from south africa. wildlife imports that were listed as having the usa as country of origin included deer, squirrel, bear, alligator, avian products and aquatic species such as squid. nearly half of all declared wildlife imports to the usa came through the ports of new york, los angeles and miami (usfws regions , and , respectively) ( figure ). ninety-nine percent of recorded imports were legally declared. this percentage is a reflection of the vast amount of declared trade recorded dwarfing the number of confiscations at us borders. commonly refused imports (shipments deemed illegally imported to the usa by usfws or other us agency regulations and thus refused entry) included sturgeon (caviar), baby harp seal pelts, indian peafowl (peacock) feathers, white tailed deer products such as antler, elephant ivory (e.g., décor, trophies, jewelry), sea turtle products (e.g., leather), crocodilians (e.g., leather), musk deer (traditional medicine products), and reptile and ostrich products (e.g., leather). while most illegal shipments presented at the mexican border, the majority of illegal specimens (number of animals) that presented at ports of entry originated in china. such items included deer and bear medicinal items, macaque scientific specimens, live aquatic species and reptiles. documented origins of illegally imported live wildlife to the usa by specimen are illustrated in figure , with indonesia as the leading country of origin. the most common of these live refused specimens were comprised of corals, fish and herpetofauna from southeast asia, as well as birds and corals from the caribbean. the most common origins of live, non-aquatic confiscations (by specimen) included herpetofauna and bird species from africa, asia and south america. the usa is a top global consumer at the national level of legal wildlife and wildlife products according to records, along with china, and the eu as a whole (asmussen et al., unpubl. data). to our knowledge, this is the most comprehensive report of us wildlife trade importation for this time period and of this scale. the most remarkable finding of this review is that the number of declared wildlife shipments into the usa has doubled since . the economic value of wildlife imports paralleled this increase in shipments, rising % from to (ferrier ). over billion specimens and an additional million kilograms of wildlife were imported during the period examined, with one-third of shipments containing live animals, mostly for the aquatic and pet trade. with this volume of live wildlife entering the usa for commercial purposes, concerns have been raised regarding the unwanted side effect of invasive alien species and their pathogens. the , recorded invasive alien species imported to the usa have cost the government an estimated us $ billion per year (pimentel et al. ) in damage or control efforts. over species of imported fish have resulted in introductions to the wild in the usa with nearly half establishing breeding populations at least for some time (smith et al. ) . beyond the environmental impacts, translocation of such live wildlife has resulted in pathogen pollution (the introduction of viruses, bacteria, fungi and parasites into new environments) with consequences to native wildlife health and us fisheries, for example (springborn et al. ) . while in some cases wild-caught specimens are more likely to harbor pathogens due to previous exposure, poorly captive-reared species also may serve as a source of pathogens. it is suspected that there is a high frequency of false reporting regarding wild versus captive origin given that many species are legal to trade if captive-raised but not if wild-caught; many species are easier and cheaper to catch than to breed; and it is nearly impossible for officials to tell the difference between wild-caught or captive-reared specimens. this is the case in seafood trade where genetic testing data have identified many examples of mislabeling of species and origin (warner et al. ) . it is not surprising that the vast majority of imports (by specimen number) consist of aquatic species and herpetofauna; given that these animals are often shipped together in large numbers, they are in high demand by consumers and a number of other factors (e.g., small size, lack of requirement for individual health certificates, less likelihood of being protected). the fact that these species are likely to be shipped live and in large numbers means that a significant number could survive even given long distance shipment from asia. the survival of large numbers of highly stressed live animals entering the usa increases the overall risk of disease introduction. the majority of individual specimens entering the usa, most of which were aquatic species, were from china and southeast asia. the degree to which such shipments pose a risk to us natural resources in terms of aquatic pathogen introduction is likely dependent upon their final destination and disposition, as well as how water used in shipments is disposed. mammal and bird species were also imported from this region, with several notable imports such as swiftlet nests, over half of which were imported since the emergence of h n highly pathogenic avian influenza (hpai) (swiftlets were proven host species of avian influenza in vietnam, although to our knowledge no nests have tested positive; fao empres/glews, april ). canada and south africa were responsible for importing significant numbers of mammals, including live hoofstock and their products. interestingly, the usa is a primary country of origin of its own imports. this may occur if an item passes through another country such as takes place when importing wildlife from alaska to the lower states via canada or if wildlife species are exported for processing and then re-imported, as we do with some agricultural species. the majority of illegal shipments of live non-aquatic wildlife were confiscated at the mexican border, especially those containing reptiles and birds imported for the pet trade (ferrier ). overall, most non-aquatic confiscations were from african and asian countries and were comprised largely of reptiles as well as birds targeted for the pet trade. nearly half of all declared wildlife imports to the usa came through the ports of new york, los angeles and miami, thus providing opportunities for targeted strengthening of monitoring and law enforcement efforts. the vast majority of wildlife imports through new york are commercial, and % of declared wildlife imports come via air cargo (us fish and wildlife service ). reasons for high traffic through new york include the fact that it is a fashion capital, the home of many scientific and educational facilities, and a top port of entry for tropical fish importers (united states fish and wildlife service (usfws) ( ). accordingly, the top live imports to new york are medicinal leeches and fish, while top commodities include caviar, shell products, furs and skins (united states fish and wildlife service (usfws) ( ). los angeles is also a predominantly commercial port when it pertains to wildlife imports. over % of imports arrived via air and most remaining imports via ocean cargo. main imports include live aquatic species and reptiles as well as shell products, jewelry, eggs and skin/hair products (us fish and wildlife service ). miami, the largest port of entry from central and south america, showed similar trends, receiving over % of its imports by air, comprised mainly of fish and reptile species. the purpose of international illegal wildlife trade varies by region. for example, in china illicit imports are primarily for exotic foods, traditional medicines and trophies; caviar, fashion and exotic pets are in demand in the eu; and exotic pets, souvenir items and hunting products comprise most illegal imports to the usa (wyler and sheikh ; ferrier ; karesh et al. ; bush et al. ) . given existing trade and travel routes, much of the trade that enters north america passes via flight patterns from africa and asia via the eu (asmussen et al., unpubl. data) , itself a significant global consumer of illegal wildlife. from to , the eu executed over seizures including . million cites-listed items (engler and parry-jones ) . the annual seizure rate in the usa is similar to this number based on our analyses. a review of the us lemis database found that only % of all commercial wildlife shipments, and . % by value, were refused entry by usfws (ferrier ). this finding is in alignment with our review of the wildb database from to . however, this percentage is based on assessment of refused shipments in the lemis database and therefore does not take into account smuggled shipments not detected; detected by department of homeland security customs and border protection (dhs cbp) but not reported to usfws; detected by usfws but not entered into lemis after the fact; or detected by cbp but reported only to another regulating agency of the same item such as the cdc, usda or fda. as previously noted, the usfws lemis database is mainly a reflection of approved or rejected wildlife imports that are declared to usfws by the importer and that non-declared (and thus illegal) imports that are successfully detected are done so through the dhs cbp. these confiscations should be reported to usfws and entered into lemis; however, confiscation data housed in cbp databases are not readily available to the public in a significant level of detail for comparison. thus, the amount of illegally imported wildlife is more than likely an underestimate. as with all big data, there is uncertainty in this dataset. for example, non-cites-listed species imports often lacked detail in several areas of the usfws lemis database, suggesting such shipments were less scrutinized. typically, ''species'' was recorded by usfws using a four-letter ''species code.'' however, codes exist for several taxonomic levels (species, genus and more general ''non-cites'' or ''na'' descriptors), and a large portion of the data did not include species level identification. further, codes often overlapped and/or several different codes were used to describe a single species. currently, wildb contains , unique species codes. eha was able to ascertain some level of taxonomic information for over % of the data entries despite the fact that the majority of these did not provide specific species identification. this study was further limited to wildlife imports that were either accepted or rejected by authorities and did not include illegal shipments that evaded authorities as those go inherently unrecorded and unrecognized. given the sheer volume of live wildlife and wildlife product imports to the usa, and the fact that most refused shipments were due to cites status and not based upon the risk of disease introduction, we believe it is prudent to further assess risk of pathogen introduction via wildlife trade. the current regulatory atmosphere for this goal is highly fragmented. the usfws currently does not focus primarily on disease prevention, but on conservation status; the cdc currently focuses on specific health risks associated with non-human primates, african rodents and bats; and the usda regulates non-domestic hoofstock, birds and few other specific mammals that originate in countries positive for reportable diseases. these species are regulated for specific diseases and thus may be approved entry if deemed safe. many countries of origin for legal and illegal wildlife imports to the usa include ''hotspots'' of emerging and reemerging infectious and zoonotic pathogens (jones et al. ; smith et al. ) such as hpai, middle east respiratory syndrome (mers) coronavirus, nipah virus and brucella ssp., as well as economically important livestock diseases. introducing disease purposefully or accidentally need not utilize illegal trade since regulations concerned with pathogen introduction via trade are focused mainly on domestic species (regulated by cdc and usda) and not enforced by the agency primarily monitoring wildlife trade into the usa (usfws). since the majority of regulatory oversight of the wildlife trade is not specifically aimed at prevention of disease introduction, it remains a challenge to prioritize collection of the relevant information or risk mitigation measures. the congressional research service notes that while the usa is involved in cites, and contributes to the coalition against wildlife trafficking and association of southeast asian nations (asean) wildlife law enforcement network, the usa ''does not participate in international efforts to regulate international wildlife trade to prevent disease transmission or invasive species, as no such international organization currently exists'' (wyler and sheikh ). in , president obama issued the national strategy for combating wildlife trafficking to guide federal agencies in the global fight against wildlife trade. yet even after the recent ebola outbreaks in africa, disease has not been a priority in this fight. the usa does adhere to the world trade organization's (wto) sanitary and phytosanitary (sps) agreement, which regulates the international trade in animals, animal products and plants, and is a member of the oie, which sets international health standards for animals and animal products, recently including wildlife. in an attempt to support this effort, eha recently worked with the oie to develop a comprehensive list of proven wildlife hosts of oie-listed diseases in order to inform member countries of the broad range of potential carriers of diseases of importance and to raise awareness surrounding potential wildlife trade health risks (smith et al., unpubl. data) . we do not yet have a comprehensive picture of the scope and associated health risks posed by the international trade of wildlife. however, it is clear that the usa is a global leader in legal and illegal wildlife consumption. the demand for wild animals for use as companion animals/ pets has been responsible for the majority of the live animal trade in the western hemisphere. this market involves billions of individual live animals, ranging from invertebrates and corals to non-human primates, originating from all over the globe. the demand for trophies, fashion, traditional medicines and exotic foods are some of the main drivers of the importation of wildlife products. the import process provides an opportunity to reinforce ''critical control points'' prior to entry through us borders. this is especially pertinent given that there is very limited traceability of wildlife species once entry has been gained into the usa. the overarching goal of this work is to mitigate risk of pathogen introduction to us agriculture via wildlife trade. to accomplish this, we must first understand and characterize trade pathways as described herein. given the large volume of imports, limited enforcement resources and lack of surveillance tools and infrastructure for many wildlife spp., the authors believe there is great opportunity for both regulated and non-regulated diseases of importance to public, agricultural or wildlife health to enter the usa. thus, there should be an emphasis within the us government and wildlife disease communities on filling gaps in the data for high priority pathways in order to better characterize risk. specifically, threats posed by ( ) large volumes of live aquatic species, ( ) wild animal host species not currently regulated (e.g., some rodents) and ( ) species closely related to domestic agriculture (e.g., hoofstock/camels) that may enter the usa for multiple purposes were prioritized by this working group for further assessment. the economy of legal wildlife trade canapés to extinction: the international trade in frogs' legs and its ecological impact. pro wildlife, defenders of wildlife and animal welfare institute global trade in exotic pets improve customs systems to monitor global wildlife trade institute of medicine (us) what you need to know about infectious disease opportunity or threat: the role of the european union in global wildlife trade the economics of agricultural and wildlife smuggling transnational crime in the developing world. global financial integrity international environmental crime: the nature and control of environmental black markets. the royal institute of international affairs implications of wildlife trade on the movement of avian influenza and other infectious diseases the unregulated and informal trade in wildlife: implications for biodiversity and health. animal health and biodiversity: preparing for the future update on the environmental and economic costs associated with alien-invasive species in the united states reducing the risks of the wildlife trade us drowning in unidentified fishes: scope, implications, and regulation of live fish import integrating invasion and disease in the risk assessment of live bird trade risk factors for human disease emergence us wildlife trade: an overview for - . usfws united states fish and wildlife service (usfws) ( ) us wildlife trade: an overview for - . office of law enforcement intelligence unit oceana study reveals seafood fraud nationwide transnational criminality: an analysis of the illegal wildlife market in southern africa fighting illicit wildlife trafficking: a consultation with governments. gland: wwf international wyler ls, sheikh pa ( ) international illegal trade in wildlife: threats and us policy acknowledgement this project is supported by the us department of homeland security s&t through a grant awarded by the food protection and defense institute. this study was made possible by the generous support of the american people through the united states agency for international development (usaid) emerging pandemic threats pre-dict- project. the contents are the responsibility of the authors and do not necessarily reflect the views of usaid or the united states government. the authors thank the united states fish and wildlife service for their contribution of lemis data. this article is distributed under the terms of the creative commons attribution . international license (http:// creativecommons.org/licenses/by/ . /), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. key: cord- -q dqsl n authors: milani, fabio title: covid- outbreak, social response, and early economic effects: a global var analysis of cross-country interdependencies date: - - journal: j popul econ doi: . /s - - - sha: doc_id: cord_uid: q dqsl n this paper studies the social and economic responses to the covid- pandemic in a large sample of countries. i stress, in particular, the importance of countries’ interconnections to understand the spread of the virus. i estimate a global var model and exploit a dataset on existing social connections across country borders. i show that social networks help explain not only the spread of the disease but also cross-country spillovers in perceptions about coronavirus risk and in social distancing behavior. in the early phases of the pandemic, perceptions of coronavirus risk in most countries are affected by pandemic shocks originating in italy. later, the usa, spain, and the uk play sizable roles. social distancing responses to domestic and global health shocks are heterogeneous; however, they almost always exhibit delays and sluggish adjustments. unemployment responses vary widely across countries. unemployment is particularly responsive to health shocks in the usa and spain, while unemployment fluctufations are attenuated almost everywhere else. after being identified in december in wuhan, china, the novel coronavirus (sars-cov- ) initially spread in the hubei region and later across mainland china. although the rest of the world soon learned about the first publicly known cases, several countries did not perceive an immediate risk for their populations. starting in january , the epidemic spread outside china, first in thailand, south korea, japan, and in the usa, and in many cases it was connected to recent travelers to the country. in europe, italy reported its first official community-based case on february , and, very quickly, clusters of cases developed in the lombardy region. it was later discovered that the virus had been circulating in lombardy since at least early january (cereda et al. ) and, possibly, since december. by mid-march, the vast majority of countries in the world had multiple cases, with the centers of the outbreak moving first to europe and later to the usa. most countries responded by requiring their populations to adhere to some form of social distancing to reduce the rate of infection and lessen the strain on healthcare providers. responses, however, have been widely heterogeneous. italy reacted with a few-days delay after the outbreak and then implemented restrictive stay-athome policies. a minority of countries initially experimented with laxer restrictions, either based on a misguided attempt to have their populations achieve herd immunity on their own (the uk, which soon moved away from the policy), or because of an unwritten "social contract" with citizens rather than enforcement from policymakers (sweden). others acted quickly and decisively to attempt to eradicate the disease before it became widespread (new zealand). the spread of coronavirus has highlighted the importance of interdependencies across different regions. depending on business links and other existing relationships, the virus rapidly moved across borders. perceptions about the crisis and social behavior responded generally with lags, but they were also likely affected by observed experiences abroad. countries had the opportunity of learning from others about social adjustments that were more or less effective in containing the disease. the main objective of this work is to study these global interrelationships in the early response to covid- shocks. in particular, this paper exploits information about social networks across countries to study interdependencies in the number of disease cases, in the perceptions of their citizens about coronavirus risk, and in their social responses. i also provide some preliminary evidence on the early economic effects of the pandemic by looking at a potential leading indicator of unemployment. i include in my sample countries and use a variety of data sources. to capture the extent of pairwise country social connections, i use data obtained from facebook, which measure the total number of friendships across pairs of countries as a fraction of the total number of combined users in the two countries. this social connectedness indicator allows me to have a measure that can account for different types of relationships: regular friendships, business links, family ties, relations based on older and more recent patterns of immigration, and tourism flows. social networks can help explain the transmission of covid- cases across borders, and they are likely to represent a superior measure compared with the use of geographic distance alone. other contemporaneous papers make a similar observation (e.g., kuchler et al. ) . at the same time, social networks not only can potentially explain patterns of disease contagion, but they can also help account for spillovers in ideas and behavior. controlling for the country-specific dynamics of covid- cases, people's risk perceptions may respond differently and also be affected by the experience and perceptions of individuals in their networks of social connections, including those residing abroad. the same is true for responses in terms of social distancing: individuals who had large connections to countries where the virus outbreak and the social distancing responses were already happening may have learned from their early experiences, taken the epidemic more seriously, and responded similarly. to measure the actual social distancing response in each country, i exploit a novel dataset made available by google through its country-specific social mobility reports. finally, i use internet data from google trends to measure coronavirus risk perceptions and to have a real-time, daily indicator of unemployment. i estimate a global var model to study the transmission of pandemic health shocks both domestically and globally. in my global framework, for each country, covid- cases can affect risk perceptions about the virus, which can trigger a social distancing response. as a result of social distancing or general uncertainty, unemployment may increase. the model allows me to treat all these variables as endogenous. this is necessary since social distancing, for example, is likely implemented in response to rising numbers of covid- cases, but it also itself has an impact on the future number of cases. moreover, domestic variables in the model are also allowed to respond to foreign aggregates. the foreign variables enter each domestic model with weights that depend on the matrix of social connections. the relevant foreign aggregate for each country is different, since the patterns of connections are unique to the country. in the gvar literature, the domestic models can be estimated separately as conditional vars. all endogenous variables can then be stacked together to form a large-scale global var; it is then possible to track the responses of all variables to each shock in each country. through the use of a connectivity matrix (my social connection matrix), the global var model offers a relatively simple and parsimonious way to deal with potentially complex interactions across different variables and countries. main results my estimates highlight the importance of interdependencies and social networks in the transmission of coronavirus cases, in the increase of risk perceptions, and in social distancing behavior. domestic variables, for the vast majority of countries, are significantly affected by foreign aggregates, constructed with weights based on the strength of social connections across countries. for example, as documented in brynildsrud and eldholm ( ) , the first cases in nordic countries (in their case, norway, but likely similar in neighboring countries) were due to travelers returning from vacations in lombardy. to the extent that some of these tourism patterns increase the probability of facebook links as well, which i believe reasonable, my measure will allow me to track likely routes for the spread of the disease. the gvar model has been proposed by pesaran et al. ( ) and is surveyed in chudik and pesaran ( ) . given the role played by italy and the usa as centers of the outbreak in different phases of the epidemic, i study how variables in the rest of the world respond to coronavirus shocks originating in these countries. i document strong and significant responses of risk perceptions and social distancing to the italy covid shock almost everywhere in the world. countries also respond to the subsequent us shock, although with a smaller magnitude. spillovers from spain and the uk also play a sizable role. the countries' responses to foreign and their own domestic coronavirus shocks are heterogeneous. i can, however, reveal some common patterns. the countries that respond with social distancing do so with a delayed and sluggish adjustment. they seem to learn from the experience of other countries, but they display an adaptive behavior: they do not adjust their habits instantly; instead, they gradually reduce their social mobility, which reaches a negative peak almost a week after the shock. in the opposite direction of causality, changes in social distancing lead to a decline in the growth rate of covid- cases. the implications of the pandemic for unemployment also vary significantly by country. labor markets in the usa and spain are the most negatively affected, with large expected increases in unemployment rates. but large spikes in unemployment are not inevitable since most other countries seem to experience much more contained fluctuations. the results suggest that different institutional features can partly insulate the corresponding populations from the worse effects of large exogenous shocks. due to the historical importance of the covid- pandemic, research related to the disease and its effects has been growing swiftly. many papers use the leading model in epidemiology, the sir (or, alternatively, the extended seir) model based on kermack and mckendrick ( ) , to simulate the evolution of the disease (e.g., ferguson n and et al ( ) ). in economics, a number of recent papers have adopted a similar framework and developed the theory further by adding relevant trade-offs between health and economic costs (e.g., eichenbaum et al. ( ) , alvarez et al. ( ) , jones et al. ( ) ). this paper, instead, takes a different route by providing empirical evidence related to the social response to the outbreak, and using an alternative framework. in contrast to studies using the sir model, i do not aim to predict the evolution of the number of infected individuals in a population; my focus lies more on explaining the social responses to the original health shocks around the world. other recent works investigate the determinants of different approaches to social distancing. gupta and et al. ( ) find that social distancing responses do not necessarily correspond to policies mandated by state and local governments. painter and qiu ( ) and adolph et al. ( ) find that political beliefs affect compliance with social distancing indications in the usa. andersen ( ) finds evidence of substantial voluntary social distancing, and he also shows that it is affected by partisanship and media exposure. in light of these results, my approach does not use data on mandates, but it exploits, instead, the actual decline in mobility, as measured using location tracking technologies. qiu et al. ( ) focus on the early months of the pandemic. they provide empirical evidence on the transmission of coronavirus cases across cities in china between january and february. they estimate how the number of new daily cases in a city is affected by the number of cases that occurred in nearby cities and in wuhan, over the previous weeks. they show that social distancing measures reduced the spread of the virus, whereas population flows out of wuhan increased the risk of transmission. my paper stresses the importance of modeling cross-country interrelationships to understand the evolution of the next phase of the pandemic. a recent work by zimmermann et al. ( ) shares a similar goal. they find that countries that are more globalized are affected by the pandemic earlier and to a larger extent. therefore, they discuss how early measures that temporarily reduce inter-country mobility would be beneficial. outside of the recent covid- literature, my paper also provides a contribution to the literature on gvar models (see chudik and pesaran , for a survey) . most papers in the literature consider macroeconomic applications and study the global spillovers of policy and other shocks (e.g., pesaran et al. ; chudik and fratzscher ; dees et al. ) . others have studied interdependencies in housing markets (holly et al. ) , firm-level returns (smith and yamagata ) , and a variety of other applications (di mauro and pesaran ; pesaran et al. ). the effect of foreign variables is usually assumed to depend on trade balances across countries. my framework, instead, introduces a different connectivity matrix, based on social networks, which can be promising for a different set of applications. therefore, my paper is also connected to recent papers that propose the use of facebook connections to measure social networks across locations (bailey et al. ) . finally, i measure risk perceptions and fears of unemployment using google trends data. this approach has become more and more popular and is now exploited in different fields to measure people's attention (da et al. ) , in forecasting and nowcasting economic variables (see the various examples discussed in choi and varian ) , and to track the spread of diseases (e.g., ginsberg et al. ; brownstein et al. ) in the absence of easily observable private information. askitas and zimmermann ( ) discuss how internet data can be useful for empirical research in a variety of social science applications and, in particular, for research about human resource issues (askitas and zimmermann ( ) and simionescu and zimmermann ( ) provide evidence directly related to the unemployment rate). the paper exploits a variety of newly available datasets to study the interrelationship between health shocks originating from the covid- pandemic, people's real-time perceptions about coronavirus risk, the extent of their social distancing response, and unemployment. i investigate the connections among these variables both within countries, and across borders, by studying contagion and spillovers internationally. the data are collected on a sample of countries. those include current oecd member countries, candidate countries that applied for membership, and the countries that the oecd defines as key partners (brazil, india, indonesia, south africa). the countries account for % of global gdp (besides china, the main omission is russia, which accounts for about %) and % of global population; they also account for % of coronavirus cases in the sample period. data on novel covid- cases each day for each country are made available by johns hopkins university's center for systems science and engineering (csse). the estimations use either the growth rate or, as a robustness check, the number of daily cases. the epidemiology literature stresses the importance of social distancing to contain the spread of the virus, by reducing the basic reproduction number r (the expected number of secondary infections produced by a single infection in a population where everybody is susceptible) and flattening the curve of infected individuals. the response has been different across countries, either in terms of policies, enforcement, or voluntary reductions in mobility. therefore, it is important to have accurate data on actual social distancing by different populations to track the implied health and economic effects. to this scope, i use daily time series indicators on social mobility made available by google. the indicators are obtained using aggregated, anonymized data from gps tracking of mobile devices, for users who opted in to "google location history." the data measure the change in the number of visits and length of stay at different places compared with a baseline. for each day of the week, mobility numbers are compared with an historical baseline value, given by the median value for the corresponding day of the week, calculated during the -week period between january and february , . the data are reported for five place categories: grocery and pharmacies, parks and beaches, transit stations, retail and recreation, and residential. in addition to the official number of covid- cases, which may be an imperfect measure of the pervasiveness of the virus in the population, i also measure the population's risk perception about coronavirus. the risk perception is measured using daily data on web searches from google trends. i use the search results for the whole "topic" category; therefore, the indicator also includes all related search terms, such as "coronavirus symptoms," "coronavirus treatment," "coronavirus vs. flu," and so forth. finally, i similarly use an indicator of unemployment to measure the initial economic effects of the outbreak. given that actual unemployment data are typically available only at monthly frequency and that their release is lagged by more than a the full list of countries is as follows: australia, austria, belgium, brazil, canada, chile, colombia, costa rica, czech republic, denmark, estonia, finland, france, germany, greece, hungary, india, indonesia, ireland, israel, italy, japan, south korea, latvia, lithuania, luxembourg, mexico, netherlands, new zealand, norway, poland, portugal, slovak republic, slovenia, south africa, spain, sweden, switzerland, turkey, uk, the usa. the only country that has been removed from the oecd list is iceland, since google mobility data were not available. for non-oecd key partners, i exclude china, since for my sample the numbers of cases had already declined (google mobility data would also be unavailable for the country). google llc "google covid- community mobility reports." https://www.google.com/covid / mobility/ month, i also exploit google trends data about unemployment as a variable that can be used to have early and real-time indications of the official variable. as before, i use google searches about the unemployment topic (again, including all searches related to unemployment, such as "unemployment benefits," "unemployment insurance," "how to apply for unemployment," "losing my job," and so forth). askitas and zimmermann ( ) and choi and varian ( ) , among others, show that unemployment searches can help predict initial unemployment claims and the unemployment rate. more recently, askitas and zimmermann ( ) and simionescu and zimmermann ( ) document how internet data can be useful for nowcasting and forecasting the unemployment rate in a diverse set of countries. my unemployment variable can, therefore, be interpreted as a real-time signal for unemployment, or, alternatively, as a measure of people's perceptions, attention, or fears, about unemployment over the time period that i study. finally, i measure international social connections using facebook's social connectedness data. the index uses active facebook users and their friendship networks to measure the intensity of connectedness between each pair of locations. the measure of social connectedness between two locations i and j is given by: where fb connections i,j denotes the number of friendship connections between region i and j , and fb users i , fb users j denote the number of facebook users in i and j . the social connectedness index, therefore, measures the relative probability of a facebook connection between any individual in location i and any individual in location j . the data used in this paper refer to the measure calculated for march . bailey et al. ( ) proposed the measure to study the effects of social networks across us counties. other current papers are uncovering the link between social networks and the diffusion of covid- (e.g., kuchler et al. ) . the measure can be preferred to alternatives based simply on inverse geographic distance, since it can provide a more accurate account of business relations, tourism patterns, and family or friendship ties, across different areas. i argue here that the strength of social connections can also affect information about the outbreak and social distancing responses. as bailey et al. ( ) show, facebook friendship links between the usa and other countries, for example, are strongly correlated both with bilateral migration patterns and trade flows. they regress social connectedness on geographic distance, the number of residents with ancestry in the foreign country (as an indicator of past migration), and on the number of residents born in the foreign country (indicating current migration), and show that all three are strongly significant. friendship connections also lead to statistically significant increases in both exports and imports between the usa and the foreign country. for italy, the strongest social connections are with switzerland and slovenia, followed by austria, germany, spain, belgium, and the uk. distance is clearly a determinant of social networks, but not the only determinant. social connections are stronger between italy and australia, italy and the usa, and italy and canada, than between italy and turkey, although the latter is geographically much closer. for the usa, as expected, the most socially connected countries are mexico and canada, followed, at lower levels, by ireland and israel. the usa have strong connections with australia and new zealand, which would be downplayed based on a pure measure of distance. figure shows, instead, the social distancing response across a sample of major countries in the sample (for easiness of exposition, i show the experiences of out of countries in the figure). mobility declined by % or more in italy, france, spain, and new zealand. while in some countries, the adjustment was abrupt (e.g., new zealand, france, spain), it was slower and more gradual in others, such as the uk (where the response starts a few days later) and the usa; their overall declines in mobility were also more modest. sweden is an outlier in europe, as it maintained only small fluctuations of mobility around the historical mean. japan and korea observed their first cases earlier; therefore, their social distancing responses during this period appear more limited. in many european countries and in the usa, mobility returns to its historical average by the beginning of june. to model global interdependencies in the spread of the disease and countries' responses, i follow the gvar approach proposed in pesaran et al. ( ) and surveyed in chudik and pesaran ( ) . assume that there are n units, representing countries in this case, and for each unit, the dynamics is captured by k i domestic variables. for each country i, the k i × vector x i,t of endogenous variables includes four domestic series: the growth rate of covid- cases, the risk perception about covid- , the change in social mobility, and the perception about unemployment. the vector of domestic variables is modeled as: for i = , , ..., n, and where il , i , and il denote matrices of coefficients of size k i × k i and k i × k * i , where k * i denotes the number of "foreign" variables included in the vector x * i,t , and ε i,t is a k i × vector of error terms. in the empirical analysis, i select the optimal number of lags p i and q i for each country using schwartz's bayesian information criterion (bic). for each country i, therefore, domestic variables are a function of their p i lagged values, possibly of the contemporaneous values and q i lagged values of foreign, or global, variables. the foreign variables x * i,t are k * i × cross-section averages of foreign variables and they are country-specific: the matrixw i has size k × k * i and contains countryspecific weights, with diagonal elements w ii = . my approach uses the extent of social connections across country borders from the facebook social connectedness index dataset to measure the weights. gvar models assume that the variables x * i,t are weakly exogenous. this corresponds to the popular assumption in open-economy macro models that the domestic country is treated as "small" in relation to the world economy, i.e, it does not affect global variables. this assumption can be easily tested for all the variables. for cases in which a domestic variable has an unduly large effect on global variables, weak exogeneity will not be invoked there and the foreign variable, instead, will not be included in that var. the estimation works in two steps. first, varx* (that is, vars with weakly exogenous foreign variables) models can be estimated for each country separately. since i study the social responses to the covid- outbreak, i include in the domestic var also the risk perception variable, in addition to the number of cases. i believe that changes in the number of confirmed coronavirus cases may lead to different risk perceptions in the different countries, which, in turn, can affect people's willingness to adhere to stay-at-home orders or to voluntarily engage in social distancing. as a measure of economic consequences, i choose an indicator of unemployment. other options that are available at daily frequencies include stock returns, interest rates, and electricity data. stock returns and interest rates are inferior indicators of economic activity in this period as they were largely influenced by government and central banks' emergency interventions. electricity data would be appropriate, but they have been made available only for a small selection of european countries (mcwilliams and zachmann ). in the analysis, the number k * i is also equal to , as the vector x * i,t contains the country-specific global counterparts for the same variables in x i,t , i.e., the growth rate of covid- cases, coronavirus risk perceptions, social mobility, and unemployment. second, the estimated country models are stacked to form a large gvar system, which can be solved simultaneously. domestic and foreign variables are stacked in the the model in eq. can be rewritten as: where gives: substituting into eq. and stacking all the unit-specific models yield: where with g invertible, as it is in this case, the gvar is given by: with f l = g − g l . the gvar solution can be used to trace the impact of shocks on the variables of interest, both domestically and globally. to find the impulse response to shocks, i adopt the generalized impulse response function (girf) approach, proposed by koop et al. ( ) , pesaran and shin ( ) , and also used in pesaran et al. ( ) . the vector of girfs is given by: where j indexes the different shocks, h denotes the horizon for the impulse response function, i t = x t , x t− , ... denotes the available information set at time t, and where √ σ jj indicates that the magnitude of the shock is set at one standard deviation of the corresponding ε j,t . the gvar specification can be seen in relation to a number of econometric alternatives: spatial vars, panel vars, and dynamic factor models. spatial vars are very strongly connected. they also assume a connectivity matrix, which is usually based on geographic distance. the main difference between the two approaches lies with the structure of correlations: as discussed at length in elhorst et al. ( ) , spatial vars may be preferred when correlations across units are extremely sparse, for example, when a unit is only affected by few bordering units ("weak," or local, crosssectional dependence). the gvar is meant to capture stronger interrelationships, with dense connectivity matrices, where each country unit is affected, in different ways, by several other countries, or by an aggregate measure ("strong" crosssectional dependence). spatial vars can also be seen as a particularly restricted case of a gvar model. the approach can similarly be seen as a particular form of panel var. the main advantage here is that, through the weight matrix w i , this approach exploits knowledge about social networks and uses that knowledge to inform the magnitude of cross-country interdependencies. panel vars often impose the same coefficients for each unit, shutting down static and dynamic heterogeneity, as well as neglecting cross-country interdependencies. an exception is provided by canova and ciccarelli ( ) : they introduce a factor structure in the coefficients to solve the curse of dimensionality. their approach is particularly useful when there is no a priori knowledge that can be exploited about the spillovers. in this case, the extent of social networks can be, instead, exploited to provide some information about the relative strength of interdependencies. finally, the gvar has relations with dynamic factor models. as chudik and pesaran ( ) show, the gvar specification approximates a common factor across units, and it extracts common factors using structural knowledge. the model is particularly suited to account for potentially complex patterns of interdependencies across countries. at the same time, the gvar specification does so while maintaining simplicity and parsimony. the dimensionality issue is resolved by decomposing a large-scale var into a number of smaller scale vars for each unit, which can be estimated separately, conditional on the dynamics of weakly exogenous foreign variables. the interdependencies are not left entirely unrestricted, since it would be unfeasible to estimate all the parameters, but they are given a structure based on knowledge of the data. in the benchmark analysis, i estimate the gvar model using daily data from february to april , . the dates are chosen based on availability of google social mobility data at the time the paper was written. the exogeneity assumption is relaxed where it appears unlikely: for covid cases, i do not include foreign variables in the model for the usa, italy, and spain, since they may be endogenous. those countries, at different times, have accounted for a large share of global cases. i allow the covid variable for all other countries to be affected by foreign series. i also allow risk perceptions in each country, as well as social distancing outcomes, to be affected even contemporaneously by corresponding variables in different countries. finally, i assume that domestic unemployment perceptions are affected by foreign unemployment perceptions, but not within the same day. this assumption is not important for the results (which are robust), but it is motivated by the idea that the unemployment data are driven more by country-specific, than across-the-border, factors. i test the weak exogeneity assumptions for all foreign variables, and they are never rejected in the data. recently, some studies have emphasized the importance of superspreaders in the transmission of the virus (e.g., adam et al. , who study clusters in hong kong). beldomenico ( ) discusses how sars-cov- appears to start by spreading gradually in a region, until transmission is triggered by a possible cascade of superspreader events, and cases explode. as a result, the pattern of transmission can become highly heterogeneous. here, i focus on numbers of cases aggregated at the country level. my framework can account for heterogeneous responses across countries. however, even if the weak-exogeneity tests suggest that domestic countries do not affect global variables in a statistical sense, it is conceivable that, with superspreaders, covid infections can transmit very quickly, and do so even between country pairs with a limited degree of social connections. my identification assumption, however, requires that the impact of a superspreader from country i on the total number of global cases remains small enough. first, to study the magnitude of global interdependencies, table shows the contemporaneous effects of foreign variables on domestic variables, for each country. the table reports the estimated coefficients, alongside the associated standard errors. domestic variables are significantly affected by the country-specific foreign aggregates, computed using the matrix of social connections as country-by-country weights. the results indicate that the international spread of covid- cases can be, in part, explained by existing social networks across country borders. moreover, the contagion not only relates to the number of cases and the spread of the disease, but it also affects the spread of perceptions and social behavior. both the measure of risk perceptions about coronavirus and the social distancing responses are significantly influenced by developments in the rest of the world. only few countries do not show a statistically significant response to global conditions. risk perceptions do not rise in response to increasing international distress only in brazil, south africa, and turkey. it is likely that their populations initially underestimated the likelihood of the pandemic reaching them, as they were farther from the epicenters. most countries also gradually learn from each others' social distancing responses. among the few exceptions, japan and korea are not significantly affected by foreign experiences: they implemented social distancing earlier than other countries, but they already relaxed many of the restrictions before the period that i consider. these results highlight the importance of considering global interrelationships and social connections in understanding the transmission of the virus and societal responses. my results add to those in zimmermann et al. ( ) , who investigate the role of globalization during the pandemic. countries with a higher index of globalization had faster transmission speed and higher infection rates, although they responded better to the challenges by achieving lower fatality rates. international travel and migration play key roles in the transmission. their paper, therefore, discusses the benefits of inter-country distancing, based on the imposition of temporary travel restrictions. my empirical results point to similar policy implications: since the table reports the estimated gvar coefficients with the associated standard error shown below in parentheses. significance at the % level is denoted by ***, at the % level by **, and at the % level by * coronavirus cases spread internationally as a result of existing social networks, early border closures and travel restrictions can be effective. i study the global responses to shocks from italy and the usa since these countries played outsized roles in different phases of the pandemic. figures and show the impulse response functions for all countries in the sample for the risk perception and social distancing variables to a one-standard-deviation covid shock originating in italy. risk perceptions increase, with some sluggish impulse responses across countries of coronavirus risk perception to a covid- growth rate shock originating in italy adjustment, almost everywhere in the world in response to the initial shock from italy. the responses typically reach their peak about - days after the original shock. the response is more delayed in brazil, india, and south africa. as seen in the previous section, these countries are less influenced by global variables in this period. populations in neighboring european countries, as well as in the usa, australia, and canada, instead significantly update their perceptions. the overall effect is much smaller in sweden, finland, turkey, israel, and lithuania. again, japan and south korea do not seem to significantly respond, as they experienced their outbreaks earlier than the rest of countries. similarly, most countries respond with reductions in social mobility. the social distancing response, however, is already delayed and sluggish in italy, with a negative impulse responses across countries of social mobility to a covid- growth rate shock originating in italy peak in mobility occurring days after the shock. other european countries, such as france, switzerland, and the uk, do not seem to adjust at all for the initial - days, after which they gradually reduce their social mobility as well. the patterns are similar everywhere: after the situations worsen in one country, the others do not immediately learn from its experience and change their behavior. instead, they appear to behave more adaptively, by only gradually altering their habits in response to the evolving situation. one issue to consider is whether the joint declines in social mobility are driven by policies that happened at the same time. my measure of actual mobility captures both the effects of mandates and those of voluntary distancing. i use data on the government response index made available for different countries through the oxford covid- government response tracker (oxcgrt)'s website and discussed in thomas hale et al. ( ) . i regress the google mobility indicator on a constant and on the government response index for each country. figure shows the estimated coefficients for the sensitivity of mobility to the government response, and the resulting r for each country's regression. the results clarify that measures based on actual mobility carry additional information that goes beyond what can be captured by looking only at the implemented policies. for many countries, mobility responds negatively to policy restrictions, with r coefficients falling in the . - . range. the explanatory power is particularly strong in mexico and new zealand. but simply using policy responses would miss the extent of social responses in many other countries, where the explanatory power is closer to (as in korea, netherlands, and scandinavian and baltic countries). the focal point of the pandemic later moved to the usa, at least starting from mid-march. figure displays the effects on coronavirus risk perceptions in the rest of the world to a us coronavirus risk shock. i consider the risk perception shock for the usa, rather than the one based on the number of cases, since testing was initially fig. relation between voluntary social distancing and government lockdown policies. the results are based on the regression social mobility t = β + β govt. response t + ε t for each country. the top panel shows the estimated coefficient β , the second the regression r the spillovers in risk perceptions are again statistically significant, but much smaller in magnitude than those observed in response to the corresponding italian shock. the same is true for responses of social mobility to a us coronavirus risk shock, shown in fig. . for many countries, i observe a slight increase in social distancing, including for the usa themselves. in terms of policy implications, the results highlight the importance of rapid interchanges of information: the rest of the world can learn from policies and behaviors that seemed to work in the countries that were reached early by the virus. the results show that perceptions about the pandemic spread to different countries. the resulting the responses to the pandemic have been heterogeneous across countries. figure overlaps, for a selection of countries, the impulse responses of social distancing and unemployment to the country's own coronavirus risk shock. i single out the responses for italy, spain, the uk, the usa, sweden, and japan, since they characterize somewhat different approaches to the crisis. the populations of italy and spain sharply decreased their social mobility after the domestic coronavirus shock. the responses reach their maximum effects after - days, and they last for weeks. their behavior suggests that even in the countries that were most affected by the virus, their social distancing responses, while substantial, have been unnecessarily delayed. japan displays a smaller, and more sluggish, response. the usa and the uk are also characterized by negative and statistically significant adjustments in mobility, but their responses are many order of magnitudes smaller than the ones observed in italy and spain. finally, it is well documented that sweden adopted a more permissive approach, by letting its citizens adjust their ita spa uk usa swe jap fig. impulse response functions of coronavirus risk perceptions and unemployment to the country's own coronavirus risk shock behavior without the same strict enforcement that was observed in other countries. the response for sweden, accordingly, does not show any significant decrease in mobility to the country-specific risk shock. turning to the early estimates about potential economic effects, i show the responses of the real-time unemployment indicator to each country-specific coronavirus shock. the figure shows that unemployment does not necessarily need to skyrocket in response to health shocks. unemployment insurance claims have reached record levels in the weeks after the outbreak in the usa. the impulse responses are consistent with the behavior of unemployment claims, revealing an extremely large response of the google unemployment indicator. unemployment is also set to considerably increase in spain. the country has a large share of workers on temporary contracts, who are more likely to become unemployed due to the uncertainty generated by the pandemic. other countries in the sample, however, as well as the vast majority of countries not shown in the figure, appear more successful in insulating their labor forces from the crisis. even if the recessionary effects on output are likely to be large almost everywhere, for most countries, early indicators of unemployment suggest that local labor markets are not going to experience the same turbulence as those in the usa. so far, the analysis has focused on the direction of causality that goes from covid cases to social and economic responses. here, i provide evidence on the opposite direction: the effects of social distancing on new covid cases. figure shows the impulse responses of the growth rate of covid- cases in different countries to a social distancing shock, measured as a one-standard-deviation decline in social mobility. social distancing leads to declines in the growth rate of coronavirus cases in the days after the shock. the only country in the figure that does not show a negative response is the uk, for which social distancing has, in fact, been much slower to start. the results reaffirm the importance of social distancing, whether through mandatory policy or voluntary behavior, in reducing the spread of the virus. while in epidemiology, the benefits of social distancing are usually modeled as changes in the parameters of a sir model, here i show that the effects can be uncovered also in a simpler linear framework. moreover, the results regarding unemployment, presented in the previous section, suggest that social distancing does not necessarily have to translate into high unemployment rates. a prompt social distancing response, coupled with labor institutions that attenuate the impact of business cycles, can successfully limit the health shocks from the pandemic, without causing extensive economic damage. the empirical analysis, so far, has been based on data up to mid-april. i now update the dataset to include the most recent months. after april, the social distancing efforts were successful in most of europe: the number of daily cases in italy, spain, germany, france, and most neighboring countries, declined; as a result, the countries started to relax most restrictions on mobility. the global centers of the virus moved instead to the americas, with us cases still remaining high, and with brazil's situation rapidly deteriorating. the situation also worsened considerably in india. to incorporate data for this second phase, i re-estimate the gvar model for the more recent sample between april and june (the last day of availability of google mobility data at the time of writing). the results are reported in tables and . table shows the values of the peak responses for the impulse response functions of coronavirus risk perceptions in each country in the sample to corresponding coronavirus risk shocks from seven countries: italy, the usa, spain, uk, brazil, chile, and india. these countries are selected as they had large number of cases at different times, during the sample. table reports similar information (in this case, the size of the largest negative responses across horizons) for the social distancing responses, instead. to compare the role played by the different countries, i show the results for both the first phase, starting in mid-february and ending in mid-april, and for the second phase, from mid-april to mid-june. most countries were significantly affected by italy's shocks during the first phase. risk perceptions particularly increased in spain, the uk, and the usa. higher risk perceptions led to a much larger decline in social mobility in spain (− . ), though, than in the other two countries (− . and − . , respectively). in the second phase, italy's role diminished, and many countries reacted instead to shocks from the usa, spain, and the uk. although cases exploded in brazil, chile, and india, between april and june, the spillovers from these countries to the rest of the world have remained more limited. the largest effects may be detected in neighboring countries: for example, the largest increase in risk perceptions in response to shocks in brazil and chile is observed in colombia. the effects on social mobility are somewhat larger, but far from the values obtained in response to shocks from spain and the uk, for example. the results suggest that, in most countries, public perceptions and behavior respond to global, not only to domestic, variables. the impact of individual countries, instead, varies over different phases of the pandemic and depending on the extent of social connections. overall, this paper's results highlight the importance of interconnections to understand not only the spread of the virus, but also adaptation and gradual learning in importing ideas and behavior from other countries. risk perceptions and the willingness to engage in social distancing by the populations of most countries significantly respond to the corresponding variables in socially connected countries. i stress the role of existing social networks across borders in the transmission of health shocks, perceptions about the risk of the disease, and ideas regarding the merit of social distancing. the results reveal heterogeneous responses across countries to their own domestic coronavirus shocks. a common feature in all responses is that individuals responded with a lag and only gradually reduced their social mobility. this observation is consistent with epidemiological models that include adaptive human behavior, such as the model presented in fenichel and et al ( ) . that research stresses the role of public policies based on informing and motivating people to reduce person-to-person contacts. this may be particularly important for countries in which citizens have weaker social connections to the rest of the world, and in which, policymakers may delay in implementing mitigation policies. institutional differences among the countries' labor markets are likely responsible for substantially different increases in unemployment. the lower degree of employee protections in the usa and the large share of temporary workers in the spanish economy are likely to account for the far worse outcomes in these countries. everywhere else, fluctuations in unemployment have remained more subdued. there are some possible limitations related to the data series used in the analysis. unemployment indicators based on internet data may be more or less accurate depending on the country: as discussed in simionescu and zimmermann ( ) , their predictive power for actual unemployment may depend on the internet penetration in the country, and on demographic variables, such as the age composition of internet users. internet use may also vary across the income distribution, particularly in less economically developed countries. perceptions about coronavirus risk may not be captured equally well in all countries in the sample. the matrix of social connections based on facebook friendships may be subject to similar problems: facebook users may have different average income and age than the population as a whole, and such friendships may capture to a larger extent personal, rather than business, links. my sample of countries necessarily excludes others (for example, china), which may be important in terms of social connections. their omission may potentially lead to an omitted variable bias in the var regressions. this section assesses the sensitivity of our estimates to alternative data and econometric choices. the benchmark estimation used data on covid- cases transformed into daily growth rates. i can examine the sensitivity of the results to using the number of new daily cases instead. table reports the estimated interdependencies corresponding to those previously shown in table . to save space, the results are shown for a subset of six countries. the estimates remain similar, with the exception of a smaller spillover of global risk into the domestic italian risk perception variable. also, in the benchmark estimation, the conditional country-specific models corresponded to vars with the addition of weighted foreign aggregates. another option sensitivity check i) repeats the estimation using the level of new daily covid- cases rather than their growth rate; case ii) estimates conditional vector-error-correction models rather than a var for each country; case iii) computes changes in social mobility excluding the series related to residential mobility often used in the gvar literature is to allow for long-run relationships and estimate vector error correction (vecm) models instead. the results shown in table , as well as all the main findings, remain in line with those discussed so far. finally, the google mobility indicator was computed by taking the average of mobility changes across all available categories. it can be argued that the relevant social distancing measure that matters for health outcomes should exclude residential mobility. therefore, i repeat the analysis by constructing social mobility, but now excluding the residential component. again, the results remain substantially unchanged. i estimated a global model of countries to examine the interconnections in coronavirus cases and in social and economic responses during the first months of the covid- pandemic. the results suggest that social connections across borders are helpful to understand not only the spread of the disease, but also the spread in perceptions and social behavior across countries. initial shocks from italy affected risk perceptions about coronavirus in most countries in the world. many of them responded by significantly reducing their mobility. populations in most countries, however, displayed a degree of behavioral adaption: they did not change their habits instantly, but only gradually over time. shocks from the usa, spain, and the uk also had significant effects later on. a subset of countries did not respond much through social distancing to global or domestic shocks. as a result, they do not show the same reduction in the growth rate of covid- cases in response to social distancing that is observed in other countries. the original health shocks, either directly, or through increased uncertainty and social distancing, have economic effects. while i do not have data at high frequency on realizations of the unemployment rate, i exploit daily data on an indicator that has been shown to predict actual unemployment quite accurately: unemployment from google searches. the response of unemployment across countries is very heterogeneous. in 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in linear multivariate models impacts of social and economic factors on the transmission of coronavirus disease (covid- ) in china big data and unemployment analysis, glo discussion paper series , global labor organization (glo) firm-level return-volatility analysis using dynamic panels inter-country distancing, globalisation and the coronavirus pandemic acknowledgments i would like to thank the editor, klaus f. zimmermann, and three anonymous referees for comments and suggestions that substantially improved the paper. i outline here the steps for the estimation of the gvar model (see also smith and galesi ): . first, the connectivity matrix w (of size × in this case) is constructed using facebook's social connectedness index data. for each country i, i fix w i,i = (the domestic country is not used for the construction of the foreign variable) and i calculate the weights w i,j , i = j , as the social connectedness between countries i and j as a fraction of the sum of connectedness between country i and each country in the sample, sci i,j / n j = sci i,j . therefore, the resulting connectivity matrix has columns that sum to . . country-specific foreign variables are then constructed as x * i,t = n j = w i,j x j,t , using the weights w i,j , for each reference country i. . i estimate conditional varx* (that is, a var with a foreign, weakly exogenous, variable) models, as specified in expression eq. . the models can be estimated separately for each country by ols. i choose lag length also separately for each of them based on schwartz's bayesian information criterion (bic). in most cases, the data select short lag lengths (p and q equal to or ) as optimal. i did not find consistent patterns of seasonality in the data. therefore, we do not perform any seasonal adjustment before the estimation. the benchmark estimation considers varx* models. the robustness section experiments with vecmx* specifications, which allow for cointegrating relationships both within the variables in x i,t and between variables x i,t and x * i,t . in that case, for each domestic vecmx*, the cointegration rank is selected based on johansen's trace statistics. . after being estimated independently, the domestic vars are stacked together as shown in eq. . the global var is "solved" for all the k = n i= k i endogenous variables, as shown in ( )-( ). . i check the moduli for the system eigenvalues and confirm that they are all within the unit circle. . i compute generalized impulse response functions following koop et al. ( ) , as shown in expression ( ) as. the response to a one standard-deviation shock is given, for each, where e j is a selection vector, composed of zeros, except for an element equal to to select the shock of choice. the matrix r h is the matrix of coefficients in the gvar's moving average representation: x t = ε t + r ε t− + r ε t− + .... i use bootstrapping to compute the impulse response error bands. key: cord- -idjkqcee authors: costa, kauê m.; sengupta, ayesha title: short-term scholar visas are essential for science date: - - journal: neuropsychopharmacology doi: . /s - - - sha: doc_id: cord_uid: idjkqcee nan the recent (june nd) presidential proclamation suspending short-term work visas, including the h- b and certain categories of j- programs [ ] , has created significant distress among the community of visiting scholars in the usa. as participants in the j- program, we would like to voice our concerns about what we believe to be a misguided policy. there are legitimate issues regarding the exploitation of short-term work visas and their recipients that should be addressed specifically, and while j- research scholars (the category encompassing most us postdoctoral researchers) are currently exempt from the proclamation, many scientists will still be affected. the usa is ranked first in the world in scientific output but is closely followed by countries like china, germany, the uk and japan. suspending temporary visa programs for scientists severely affects scientific enterprise in the usa, where it is estimated that over half of post-doctoral scientists, the cornerstone of the research workforce, are temporary visa holders [ ] . the problems that this proclamation creates are manifold. ( ) scientific work is highly specialized, and sometimes there are only a handful of people in the world that have the expertise to apply certain experimental or theoretical methods. sometimes these people are not in the us and bringing them in as visiting scholars allows for the transfer of crucial knowledge to american laboratories. limiting this exchange of expertise severely restricts the capacity of american work groups to absorb new methods developed abroad. ( ) visiting scholars often bring their own funding from their countries of origin, and therefore also represent a direct net financial gain for the country. this is in addition to the indirect gain obtained by absorbing the expertise of people who have received several thousands of dollars' worth of education and training in other countries. ( ) there is empirical evidence that the impact of research by foreign-born scientists is on average higher than that of locals who have no international mobility experience, even when accounting for differences in relative career positions in their countries of origin [ ] . also, studies show that diverse research groups, which are often characterized by international members, tend to publish more frequently and are cited more often [ ] . these groups benefit from the range of ideas and perspectives that are essential for the innovative thinking that drives scientific advancement. this indicates that there is a gain in research quality to be obtained from international cooperation that cannot be replaced by quickly substituting visiting scientists with locals. ( ) moving to another country takes a substantial financial, logistic and emotional toll on visiting scholars. scientists who choose to do this often must relocate their families, be separated from friends and loved ones, learn a new language and deal with varying levels of bureaucratic hurdles. due to emergencies, they may also need to travel abroad without much warning. adding another barrier to this process just increases the burden inherent to these circumstances. even though the new policy is temporary and does not immediately affect visiting researchers currently in the us, there is now a growing concern that harsher measures might be implemented in the near future. the lack of security this creates will likely dissuade many foreign scholars from uprooting their lives to work in the usa. instead, many scientists are already choosing to pursue positions elsewhere. other countries, such as germany, france and china, have recently increased their efforts to attract foreign scientists [ , ] , for instance by offering permanent residency status for visitors with phds who secure certain positions. the june nd proclamation therefore reduces the ability of us labs to attract highly qualified personnel who can otherwise secure positions in other countries. ( ) the proclamation causes unnecessary distress not only for the visiting scholar community but also for their american colleagues and group leaders who rely on their work for the continuation of their research, which is only exacerbated by the current disruption imposed by the covid- pandemic. in closing, we urge the current administration and all relevant policy makers to reconsider this course of action, and we request that all members of the academic community express the importance of these visa programs to the general public and to their government representatives. though temporary, the enacted suspensions may lead to a long-term disruption of scientific research in the us, much of which is crucial for public health and technological advancement. the resulting exodus of scientific talent to other industrialized nations could directly damage the nation's leading scientific research and higher education institutions for years to come. no significant funding was received for work on this manuscript. the authors declare no competing financial interests in relation to the work described proclamation suspending entry of aliens who present a risk to the u.s. labor market following the coronavirus outbreak. the white house table - b -ncses survey of graduate students and postdoctorates in science and engineering a mobility boost for research the power of diversity several countries launch campaigns to recruit research talent from u.s. and elsewhere kmc and as conceptualized and wrote the manuscript. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -m m zgfy authors: pharo, elizabeth a.; williams, sinéad m.; boyd, victoria; sundaramoorthy, vinod; durr, peter a.; baker, michelle l. title: host–pathogen responses to pandemic influenza h n pdm in a human respiratory airway model date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: m m zgfy the respiratory influenza a viruses (iavs) and emerging zoonotic viruses such as severe acute respiratory syndrome-coronavirus- (sars-cov- ) pose a significant threat to human health. to accelerate our understanding of the host–pathogen response to respiratory viruses, the use of more complex in vitro systems such as normal human bronchial epithelial (nhbe) cell culture models has gained prominence as an alternative to animal models. nhbe cells were differentiated under air-liquid interface (ali) conditions to form an in vitro pseudostratified epithelium. the responses of well-differentiated (wd) nhbe cells were examined following infection with the pandemic influenza a/h n pdm strain or following challenge with the dsrna mimic, poly(i:c). at h postinfection with h n pdm , the integrity of the airway epithelium was severely impaired and apical junction complex damage was exhibited by the disassembly of zona occludens- (zo- ) from the cell cytoskeleton. wdnhbe cells produced an innate immune response to iav-infection with increased transcription of pro- and anti-inflammatory cytokines and chemokines and the antiviral viperin but reduced expression of the mucin-encoding muc b, which may impair mucociliary clearance. poly(i:c) produced similar responses to iav, with the exception of muc b expression which was more than -fold higher than for control cells. this study demonstrates that wdnhbe cells are an appropriate ex-vivo model system to investigate the pathogenesis of respiratory viruses. over the past years, there has been a rapid rise in emerging infectious diseases both of zoonotic and human origin [ ] . the recent emergence and worldwide spread of the severe acute respiratory syndrome-coronavirus- (sars-cov- ) virus which produces the coronavirus disease (covid- ) respiratory illness demonstrates the risk to human health and the economy posed by respiratory pathogens [ ] . for novel viruses, often no diagnostics or therapeutics exist, hence they present an enormous risk of a catastrophic global pandemic [ , ] . respiratory rna viruses, particularly influenza a viruses (iav) of the orthomyxoviridae family are a major pandemic risk as they replicate rapidly, lack a proof-reading mechanism, have a high mutation rate and are readily transmissible [ , [ ] [ ] [ ] . over years ago, the h n "spanish flu" pandemic killed up to million people worldwide [ ] [ ] [ ] . three global influenza pandemics have occurred since, "asian flu" h n undifferentiated nhbe and wdnhbe cells were fixed in % neutral buffered formalin and embedded in paraffin, sectioned ( µm; microtome), mounted and stained with hematoxylin and eosin. for immunofluorescence assays, airlifted nhbe cells were fixed in % w/v paraformaldehyde for at least min, rinsed with and then stored in dpbs at • c. fixed cells were permeabilized for min at rt with % v/v triton x- /dpbs (sigma-aldrich, st. louis, mo, usa), blocked for h in % w/v bovine serum albumin (bsa; sigma-aldrich, st. louis, mo, usa)/ . % v/v triton x- /dpbs. apoptotic dna fragmentation which occurs in the last phase of apoptosis (programmed cell death) was detected by terminal deoxynucleotidyl transferase dutp nick end labelling (tunel) staining using the in situ cell death detection kit, tmr red (cat# ; roche, mannheim, germany) according to the manufacturer's instructions. cells were then stained with primary antibody overnight at • c, washed three times with dpbs and then incubated with the appropriate species-specific secondary antibody diluted at : in % w/v bsa/ . % v/v triton x- /dpbs for at least h at rt. transwell membranes were rinsed three times with dpbs, stained with phalloidin (a , alexa fluor phalloidin, thermofisher scientific, usa) which stains f-actin filaments for min at rt, washed three times with dpbs, stained with the nuclear stain ', -diamidino- -phenylindole (dapi, d ; thermofisher scientific, usa) for min at rt. transwells were washed twice with sterile water, excised and mounted on glass slides with prolong gold antifade mountant (p , thermofisher scientific, usa). primary antibodies were used at the following dilutions : mucin ac (muc ac, ma - , thermofisher scientific, usa), : mucin b (muc b, hpa , sigma-aldrich, usa), : zo- (zo- - a ; - , thermofisher scientific, usa), : acetylated tubulin (tubac, t , sigma-aldrich, usa) and : claudin (cldn , ab , abcam, cambridge, uk). a monoclonal antibody specific to the nucleoprotein (np) of type a specific influenza (iav np) was produced using hybridoma technology in mice, as previously described [ ] . the hybridoma iav np monoclonal antibody h -l - used at : dilution was kindly provided by paul selleck (australian centre for disease preparedness, csiro, geelong, australia). secondary antibodies were purchased from thermofisher scientific, usa and included goat anti-mouse igg (h+l), alexa fluor and alexa fluor (a and a respectively), goat anti-rabbit igg (h+l), alexa fluor (a ) and donkey anti-rabbit igg (h+l) alexa fluor (a ). cells were imaged using the zeiss lsm confocal microscope (zeiss, oberkochen, bw, germany) using a × oil immersion objective unless specified otherwise. images were captured as z-stacks and maximum intensity projections generated. images were captured and processed using zen . blue software (zeiss, germany). the presence of α - and α - -linked sialic acids (sas) on the surface of wdnhbe cells was analyzed by fluorescence activated cell sorting (facs). wdnhbe cells were detached from transwell membranes by two the pandemic strain of influenza virus a/california/ / (h n )pdm , kindly provided by the who collaborating centre for reference and research, melbourne, vic, australia was propagated by allantoic cavity inoculation of day old embryonated chicken eggs at • c for h. the virus was passaged once in mdck cells in the presence of µg/ml l- -tosylamide- -phenylethyl chloromethyl ketone (tpck)-treated trypsin (sigma-aldrich, usa) at • c for h or until cytopathic effect was observed. virus was aliquoted and titrated to determine the median tissue culture infectious dose (tcid ) on mdck cells in the presence of µg/ml tpck-treated trypsin. briefly, virus was serially diluted -fold and applied in quadruplicate to cell monolayers. cytopathic effect was assessed five days post infection. viral titres were calculated according to the method of reed-muench as tcid /ml [ ] . . . immune challenge of wdnhbe cells with influenza a virus and poly (i:c) wdnhbe cells were washed three times with ali media to remove excess mucus from the upper surface and inoculated with influenza a/h n pdm ( µl) at a multiplicity of infection (moi) of . for h at • c. the virus inoculum was removed, the cells washed three times with ali media to remove unbound virus and the cells incubated at • c for h postinfection (experiment endpoint). at each time point ( , , and h postinfection), the virus was harvested by the addition of ali media to the upper surface for min at • c, with both apical and basolateral media collected and stored at − • c for endpoint analyses. in addition to iav challenge, wdnhbe cells were inoculated with varying concentrations of poly(i:c) dsrna viral mimic [poly(i:c) high molecular weight, cat: tlrl-pic, invivogen, san diego, ca, usa]. briefly, airlifted cells were washed in prewarmed dpbs for min at • c to remove excess mucus. poly(i:c) ( µg and µg) in ali media was added to the apical surface of wdnhbe cells and ali media added to the basolateral compartment. cells were incubated for h at • c, then washed three times with ali media to remove excess poly (i:c). basolateral media was replaced and the cells incubated for an additional h at • c. transepithelial electrical resistance (teer) across the epithelium of undifferentiated and wdnhbe cells was measured using an epithelial voltohmmeter (evom ) with an stx chopstick electrode (world precision instruments, sarasota, fl, usa) [ ] . ali media was added to the apical and basolateral compartments, equilibrated for at least - min at rt and the teer measured. teer readings were membrane-corrected by subtraction of measurements from control transwells (no cells) and expressed in units of Ω or Ω × cm . dextran conjugated to fluorescein isothiocyanate (fitc-dextran, kda; sigma-aldrich, usa) was used to determine the paracellular permeability of wdnhbe cells, i.e., the transport of fitc-dextran from the apical to the basolateral compartment of the transwell via extracellular space in the pseudostratified airway epithelium [ ] . fitc-dextran diluted in ali media ( . mg/ml) was added to the upper transwell compartment and ali media alone added to the basolateral compartment and the cells incubated for h at • c. basolateral media was mixed, sampled in triplicate in a microtiter plate and fitc-dextran analyzed on a biotek synergy ht microtitre plate reader. assay standards and sample solutions were prepared concurrently. lactate dehydrogenase (ldh) release was quantified using the cytotox non-radioactive cytotoxicity assay (g , promega corporation, madison, wi, usa) according to the manufacturer's instructions. controls including no cells, untreated cells and maximum ldh release (triton x- -treated cells, % cytotoxicity) were analyzed together with the apical samples using a biotek synergy ht microtitre plate reader. cytotoxicity percentage was expressed as experimental ldh release compared to maximum ldh release for triton x- treated cells. quantitative reverse transcription pcr (rt-qpcr) was performed on total rna extracted from cells. total rna was isolated from wdnhbe cells using the rneasy mini kit (qiagen, hilden, nw, germany) with on-column dnase i digestion. total rna was quantified using the ds- fx spectrophotometer (denovix, wilmington, de, usa) and first strand cdna made using the superscript iii first-strand synthesis system (thermofisher scientific, usa), total rna ( ng) and oligo(dt) . gene expression was determined by rt-qpcr using first strand cdna ( : dilution), taqman gene expression master mix and taqman inventoried probes (applied biosystems, foster city, ca, usa), all of which were exon-spanning apart from the single exon interferon beta (ifnb ) probe (table s ). rt-qpcr reactions for the iav h n pdm and poly(i:c) experiments were performed on the quantstudio system and quantstudio flex real-time pcr system (applied biosystems, usa) respectively with the appropriate no template controls included on each plate. samples were assayed in triplicate. conditions used were • c for min, denaturation at • c for min followed by cycles of • c for s, • c for min. a cycle threshold (ct) of . was applied to all gene probes. gene expression levels were normalized to the human glyceraldehyde -phosphate dehydrogenase viruses , , of (gapdh) housekeeping gene and expressed as fold over detectable (fod), as described [ ] . minimum detectable ct was set at cycles. to assess the effect of iav h n pdm infection in wdnhbe cells on the transcription of twelve innate immune genes and two mucin-encoding genes quantified by rt-qpcr, we constructed a gene coexpression network [ ] . we used the normalized gene copy number in n = transwells at h postinfection with iav h n pdm , i.e., the number of gene copies per copies of gapdh (see above) to produce the network. for each pair of genes, the pearson correlation coefficient (pcc) was calculated to estimate the strength of the linear relationship between gene expression levels. a pcc value exceeding ± . was used as the coexpression threshold. for each gene-pair, a network was constructed with the expressed genes forming the network's nodes and the pcc values exceeding the . threshold forming an edge. the pcc values were estimated using the "corr" function in the r stats package, and the network was constructed and visualized using the igraph package [ ] . statistical analyses for most experiments were performed in graphpad prizm . . (graphpad software, inc., ca, usa). data from three independent experiments for each of the iav and poly(i:c) challenges was pooled and analyzed as described below. for iav h n pdm experiments, growth kinetics of iav h n pdm (tcid /ml) were analyzed by one-way anova using tukey's multiple comparison test; percentage of preinfection teer, fitc-dextran transported across the epithelium and percentage cytotoxicity were analyzed by multiple t-tests using the two-stage linear step-up procedure of benjamini, krieger and yekutieli, with a false discovery rate (fdr) of %. teer resistance in iav h n pdm -inoculated and mock-inoculated wells at h postinfection was also analyzed with r using a mixed effects model using the "aov" and the "lme" functions in the r stats and nlme packages respectively [ ] . poly(i:c) teers were analyzed by one-way anova with brown-forsythe and welch anova tests and dunnett's t multiple comparison test. poly(i:c) fitc-dextran transported and cell cytotoxicity were analyzed by one-way anova and tukey's multiple comparisons test. iav muc ac and muc b gene expression was analyzed by unpaired t-tests and for poly(i:c), by one-way anova using tukey's multiple comparison test. cytokines secreted apically form iav-infected cells were analyzed by unpaired t-tests. the use of human cells in all experiments was approved by the csiro health and medical human research ethics committee (ethics approval # _ _lr, th may, ). prior to innate immune system challenge studies, we characterized our commercially sourced nhbe cells. cells cultured at ali on collagen i-coated transwells formed a polarized, pseudostratified epithelium around four weeks postairlift. wdnhbe cells exhibited a mucociliary phenotype, characterized by the secretion of mucus on the apical surface and the coordinated beating of cilia on the surface of ciliated cells (video s ). histological staining confirmed that wdnhbe cells formed a columnar epithelium - cells thick, consisting of basal, goblet, club and ciliated cells, representative of the human airway epithelium in vivo (figure a ). immunofluorescence assays confirmed the expression of muc ac, a goblet cell marker and muc b, produced in secretory cells and the partial colocalization of muc ac and muc b (figure b) . immunostaining of wdnhbe cells also confirmed the presence of ciliated cells, characterized by the expression of acetylated tubulin on cell-surface cilia ( figure c ). in order to confirm that airlifted cells formed an intact epithelial barrier, we measured the teer of undifferentiated and differentiated nhbe cells. the teer indicates the degree of polarization and differentiation of nhbe cells and hence the formation of an intact airway epithelial barrier [ , ] . for undifferentiated nhbe cells, the average teer was < Ω × cm . in contrast, teer values ≥ Ω × cm were recorded for a majority of wdnhbe cells with average values consistently ≥ - Ω × cm ( figure s ; table s ; table s ). therefore, our cells provide an excellent model to study the integrity of the airway epithelium in vitro before, during and after immune system challenges. iav infects cells by binding to cell-surface sialic acid receptors. therefore, we characterized the iav receptors present on the exterior of wdnhbe cells by facs using the lectins sna i and maa ii which bind to α - -linked sialic acids (sa) and α - -linked sas respectively. while hemagglutinins of human iavs preferentially bind α - -sa, those from avian iavs recognize α - sa [ , ] . almost all ( . %) wdnhbe cells had α - -linked sialic acids on the cell surface, while . % had cell-surface α - -linked sialic acids ( figure s ), demonstrating the susceptibility of wdnhbe cells to iav infection in this study. wdnhbe cells were infected with h n pdm at moi of and apical virus titre monitored over a h time period postinfection for three independent replicate experiments ( figure a ). h n pdm replicated efficiently in wdnhbe cells exhibiting peak viral titre of tcid /ml at h postinfection. the titre decreased by . logs at h postinfection ( . × ; p < . ). the virus titre at h and h postinfection ( tcid /ml) when the virus binds and penetrates the cells is slightly higher than expected, which may be due to incomplete removal of unbound virus, despite numerous washes, and also the binding of the virus to mucus on the transwell surface. this was consistent across all three experiments. in preliminary experiments, no virus was detected in basolateral media at h postinfection of wdnhbe cells with h n pdm . this indicated that the virus is shed on the apical surface of polarized epithelial cells, consistent with previous studies [ , ] . immunofluorescence assays confirmed that at h postinfection, the majority of wdnhbe cells were infected with h n pdm , as indicated by staining with the influenza a nucleoprotein (iav np, figure b ). teer indicates the degree of polarization and differentiation of nhbe cells and hence the formation of an intact airway epithelial barrier [ , ] . for undifferentiated nhbe cells, the average teer was < Ω x cm . in contrast, teer values ≥ Ω x cm were recorded for a majority of wdnhbe cells with average values consistently ≥ - Ω x cm ( figure s ; table s ; table s ). therefore, our cells provide an excellent model to study the integrity of the airway epithelium in vitro before, during and after immune system challenges. to understand the effect of h n pdm on the integrity of the barrier formed by wdnhbe cells, the effect of the virus on the airway epithelium was determined by the change in teer, paracellular permeability and ldh release (percent cytotoxicity) (figure ). at - h postinfection, there was no difference in percentage teer values. during this period, the virus is undergoing virus assembly and early release of virus particles. however, at h and h postinfection, dramatic reductions in teer values of . and . % respectively occurred, significantly lower values than the teers of mock-infected cells (p < . ; refer to figure s for a graph of the individual teer values (Ω x cm ) for all three independent h n pdm experiments). this significant decrease in teer resistance was also confirmed by mixed effects modelling (p < . ). from - h postinfection, higher viral replication (observed in figure a ) correlates with a reduction in barrier integrity of wdnhbe cells. in contrast to virus-infected cells, teer values for to understand the effect of h n pdm on the integrity of the barrier formed by wdnhbe cells, the effect of the virus on the airway epithelium was determined by the change in teer, paracellular permeability and ldh release (percent cytotoxicity) (figure ). at - h postinfection, there was no difference in percentage teer values. during this period, the virus is undergoing virus assembly and early release of virus particles. however, at h and h postinfection, dramatic reductions in teer values of . and . % respectively occurred, significantly lower values than the teers of mock-infected cells (p < . ; refer to figure s for a graph of the individual teer values (Ω × cm ) for all three independent h n pdm experiments). this significant decrease in teer resistance was also confirmed by mixed effects modelling (p < . ). influenza a viruses damage apical junctional complexes and alter the cell cytoskeleton and morphology of airway epithelial cells [ , , ] . to investigate these characteristics in our model we performed immunofluorescence assays on mock and h n pdm -infected wdnhbe cells h postinfection ( figure ) . we characterized the distribution of the zo- adapter protein that links tight junctions and adherens junctions to the cell cytoskeleton [ ] . phalloidin was used to stain f-actin filaments of the cytoskeleton. in mock infected wdnhbe cells, zo- forms an intact apical to further characterize airway barrier integrity in response to iav infection, the paracellular transport of fitc-dextran ( kda) across the epithelium was measured. paracellular transport is regulated by ajcs, hence increased fitc-dextran transport from the apical to basolateral compartment of the transwell is an indicator of "leaky" or damaged junctions [ ] . at h postinfection, fitc-dextran transported across iav-infected cells was . -fold higher than for mock-treated cells (p < . ; figure b ), indicative of increased epithelium permeability. influenza a virus induces cell death [ , ] . hence, we determined the cytotoxicity of h n pdm to wdnhbe cells by measuring the apical release of ldh h postinfection. ldh is a soluble cytoplasmic enzyme that is released from the cytoplasm upon damage to the plasma membrane and is directly proportional to the number of cells undergoing apoptosis or necrosis [ , ] . at h postinfection, cell death due to h n pdm was . -fold greater for iav-infected cells compared to cells treated with media alone (p < . ; figure c ). tunel staining also showed a greater number of apoptotic cells in wdnhbe cells infected with pandemic influenza compared to mock-infected cells ( figure s ). therefore, h n pdm damage to the in vitro airway epithelium is characterized by a reduced teer, increased paracellular flux and increased cell death. in order to compare the immune response of wdnhbe cells to different immunostimulants, we challenged our cells with poly(i:c) at µg and µg and evaluated barrier integrity as for iav-infected cells. at h poststimulation, teer values were significantly reduced in a dose-dependent manner in comparison to mock-treated cells (p < . ; figure d ). similar to the h n pdm treatment, a . % reduction in teer was recorded for ug poly(i:c) and a . % reduction for the µg treatment. in contrast, the teer of mock-treated cells increased by . %. a graph of the individual teer values (Ω × cm ) for all three, independent poly(i:c) experiments is also provided ( figure s ). the effect of poly(i:c) on paracellular flux was not as great as for h n pdm . at µg and µg poly (i:c), fitc-dextran transported across the airway epithelium was -fold and -fold greater respectively compared to control cells (p < . and p < . respectively; figure e ). similarly, poly(i:c) was not as cytotoxic as h n pdm . at the higher, µg poly(i:c) dose, cell death was -fold higher than for control cells (p < . ; figure f ). however, there was no difference in cytotoxicity between the µg poly(i:c) and mock treatments. in summary, both h n pdm and poly(i:c) had a detrimental effect on the integrity of the in vitro pseudostratified airway epithelium. influenza a viruses damage apical junctional complexes and alter the cell cytoskeleton and morphology of airway epithelial cells [ , , ] . to investigate these characteristics in our model we performed immunofluorescence assays on mock and h n pdm -infected wdnhbe cells h postinfection ( figure ) . we characterized the distribution of the zo- adapter protein that links tight junctions and adherens junctions to the cell cytoskeleton [ ] . phalloidin was used to stain f-actin filaments of the cytoskeleton. in mock infected wdnhbe cells, zo- forms an intact apical circumferential belt (also known as the perijunctional actomyosin ring) around the plasma membrane of each cell (figure a ). in sharp contrast, zo- was disrupted and discontinuous in many iav h n pdm -infected cells, suggestive of damage to ajcs and the epithelium (figure b) . these results were consistent with phalloidin staining of f-actin filaments of the cell cytoskeleton. f-actin filaments are intact in mock-infected cells (figure a ) but disrupted in iav-infected cells (figure b) . merged images show that the disruption of zo- and f-actin of the cytoskeleton occurs in the same cells in the in vitro airway epithelium (merge, figure b) . furthermore, iav-infected cells show evidence of cell distension. while h n pdm disrupted zo- and f-actin in wdnhbe cells, this was not observed in cells treated with µg poly(i:c) ( figure s ). results were consistent with phalloidin staining of f-actin filaments of the cell cytoskeleton. f-actin filaments are intact in mock-infected cells (figure a ) but disrupted in iav-infected cells (figure b) . merged images show that the disruption of zo- and f-actin of the cytoskeleton occurs in the same cells in the in vitro airway epithelium (merge, figure b) . furthermore, iav-infected cells show evidence of cell distension. while h n pdm disrupted zo- and f-actin in wdnhbe cells, this was not observed in cells treated with µg poly(i:c) ( figure s ). the mucosal barrier is a major component of the innate immune system in the lungs [ , ] . muc ac and muc b are the major secreted mucins and play a critical, though poorly understood role in airway defense and mucociliary clearance [ , ] . therefore, we analyzed the effect of h n pdm on the expression of these genes by rt-qpcr (figure a) . iav had no effect on the expression of the muc ac goblet cell marker in wdnhbe cells (figure a ). in contrast, muc b was suppressed more than -fold in h n pdm -inoculated cells (p < . ; figure a ). poly(i:c) treatment also had no effect on muc ac expression when compared to mock-treated cells (ns, figure b) , consistent with pandemic iav. in contrast, poly(i:c) had a dose-dependent effect the mucosal barrier is a major component of the innate immune system in the lungs [ , ] . muc ac and muc b are the major secreted mucins and play a critical, though poorly understood role in airway defense and mucociliary clearance [ , ] . therefore, we analyzed the effect of h n pdm on the expression of these genes by rt-qpcr (figure a) . iav had no effect on the expression of the muc ac goblet cell marker in wdnhbe cells (figure a ). in contrast, muc b was suppressed more than -fold in h n pdm -inoculated cells (p < . ; figure a ). mock, n = ; iav, n = for muc b; poly(i:c) data n = biological replicates per treatment. statistical significance: ns, not significant; *, p < . ; **, p < . ; ***, p < . ; ****, p < . . we used rt-qpcr to determine whether wdnhbe cells produced an innate immune response to iav h n pdm in vitro. genes were selected based on their response to iav infection with poly(i:c) treatment also had no effect on muc ac expression when compared to mock-treated cells (ns, figure b) , consistent with pandemic iav. in contrast, poly(i:c) had a dose-dependent effect on muc b expression with -fold and -fold higher expression in wdnhbe cells treated with µg and µg poly(i:c) respectively compared to mock-treated cells ( µg poly(i:c): p < . ; µg: p < . ; figure b ). we used rt-qpcr to determine whether wdnhbe cells produced an innate immune response to iav h n pdm in vitro. genes were selected based on their response to iav infection with h n pdm and/or h n subtypes as reported for in vitro and/or in vivo studies [ , , [ ] [ ] [ ] [ ] [ ] [ ] . genes assayed included: the chemokine-encoding c-x-c motif chemokine ligand (cxcl ), also known as interferon γ-induced protein (ip ), c-c motif chemokine ligand (ccl ), alias regulated on activation, normal t cell expressed and secreted (rantes), c-c motif chemokine ligand (ccl ), also known as monocyte chemoattractant protein- (mcp ), c-c motif chemokine ligand (ccl ), alias macrophage inflammatory protein -α (mip α), and c-x-c motif chemokine ligand (cxcl ), also known as interleukin (il ). proinflammatory cytokine-encoding genes characterized included: tumor necrosis factor (tnf), known as tnf-α, interleukin β (il b), colony stimulating factor (csf ), also referred to as granulocyte-macrophage colony stimulating factor (gmcsf) and the potent proinflammatory interleukin (il ). expression of the anti-inflammatory-encoding interleukin (il ) and antiviral-encoding interferon β (ifnb ) and radical s-adenosyl methionine domain containing (rsad ), commonly known as virus inhibitory protein, endoplasmic reticulum-associated, ifn-inducible; viperin [ ] was also characterized. all genes assayed were upregulated in h n pdm -infected versus mock-inoculated cells (table ; figure s ). muc ac and muc b values ( figure ) have also been included in table . notably, there was a difference in the magnitude of the response to pandemic influenza with four different levels of fold-change observed. the highest fold-increases in expression were recorded for cxcl (> , -fold) and the antiviral-encoding ifnb (> -fold). ccl and rsad expression were also significantly upregulated in response to iav (> and > fold respectively). il , tnf, mip α and il exhibited a - -fold increase in expression compared to mock-inoculated cells. in contrast, the magnitude of response to iav-infection was lower for the ccl , cxcl , csf and il b genes ( - -fold increases). the increased expression of these cytokine, chemokine and antiviral genes suggests that innate immune responses are activated in our wdnhbe cells in response to pandemic influenza. this relationship was explored by the generation of a gene coexpression network ( figure ) based on gene copy numbers (table s ). the relationship between pro-and anti-inflammatory cytokines and chemokines genes is highlighted by the network connectivity. similarly, association between mucin gene expression is shown. in contrast, the expression of the antiviral ifnb and rsad were not linked to other genes. the induction of innate immune response genes in wdnhbe cells was also investigated by the stimulation of wdnhbe cells with µg or µg poly(i:c) ( figure s ). in contrast to iav, poly(i:c) had no effect on csf expression. while the lower, µg dose of poly(i:c) was sufficient to upregulate il and cxcl expression, tnf and il b were only induced by µg of poly(i:c) compared to mock-treated cells. to determine whether gene expression was correlated with protein secretion, multiplex immunoassays for il- , tnf-α, cxcl (il- ) and csf (gm-csf) were performed on media harvested from the apical (figure ) and basolateral compartments ( figure s ) at h postinfection of wdnhbe cells with h n pdm . apical secretion levels of il- and tnf-α after a min period were > - -fold higher in iav-infected versus mock cells (figure a, b) , with il- more than -fold higher (figure c ). in contrast, while csf trended lower in iav-infected cells, it was not significantly different to mock levels (figure d ). il- β was secreted at the minimum limit of detection (data not shown). cytokine and chemokine secretion into the lower compartment of transwells over a h period from to h postinfection exhibited similar trends to apical supernatants, although protein levels were generally reduced apart from il- ( figure s ). multiplex immunoassays for il- , tnf-α, cxcl , csf and il- β were also performed for apical and basolateral media from the µg and µg poly(i:c) treatments ( figure s ). trends were analogous to the response to iav, apart from csf , which was stimulated, rather than suppressed by poly(i:c). the apical secretion of il- β, il- and csf was only higher for µg poly(i:c) compared to mock-treated cells. in contrast, tnf-α and cxcl levels were greater than mock cells for both poly(i:c) doses. cytokine and chemokine secretion in basolateral media over a h period the induction of innate immune response genes in wdnhbe cells was also investigated by the stimulation of wdnhbe cells with µg or µg poly(i:c) ( figure s ). in contrast to iav, poly(i:c) had no effect on csf expression. while the lower, µg dose of poly(i:c) was sufficient to upregulate il and cxcl expression, tnf and il b were only induced by µg of poly(i:c) compared to mock-treated cells. to determine whether gene expression was correlated with protein secretion, multiplex immunoassays for il- , tnf-α, cxcl (il- ) and csf (gm-csf) were performed on media harvested from the apical (figure ) and basolateral compartments ( figure s ) at h postinfection of wdnhbe cells with h n pdm . apical secretion levels of il- and tnf-α after a min period were > - -fold higher in iav-infected versus mock cells (figure a,b) , with il- more than -fold higher (figure c ). in contrast, while csf trended lower in iav-infected cells, it was not significantly different to mock levels ( figure d ). il- β was secreted at the minimum limit of detection (data not shown). cytokine and chemokine secretion into the lower compartment of transwells over a h period from to h postinfection exhibited similar trends to apical supernatants, although protein levels were generally reduced apart from il- ( figure s ). multiplex immunoassays for il- , tnf-α, cxcl , csf and il- β were also performed for apical and basolateral media from the µg and µg poly(i:c) treatments ( figure s ). trends were analogous to the response to iav, apart from csf , which was stimulated, rather than suppressed by poly(i:c). the apical secretion of il- β, il- and csf was only higher for µg poly(i:c) compared to mock-treated cells. in contrast, tnf-α and cxcl levels were greater than mock cells for both poly(i:c) doses. cytokine and chemokine secretion in basolateral media over a h period exhibited similar trends to apical media. therefore, airlifted nhbe cells secrete cytokines and chemokines in response to both h n pdm and poly(i:c) immune system challenges. however, under experimental conditions used and properties measured, iav elicits a much stronger inflammatory response than poly(i:c) in wdnhbe cells. exhibited similar trends to apical media. therefore, airlifted nhbe cells secrete cytokines and chemokines in response to both h n pdm and poly(i:c) immune system challenges. however, under experimental conditions used and properties measured, iav elicits a much stronger inflammatory response than poly(i:c) in wdnhbe cells. and mock csf (n = ); ns, not significant; *, p < . ; **, p < . . we show that well-differentiated nhbe cells grown at the air-liquid interface are an anatomically and physiologically relevant in vitro model to investigate changes in the airway epithelium in response to h n pdm and the dsrna viral analogue, poly(i:c). our model we show that well-differentiated nhbe cells grown at the air-liquid interface are an anatomically and physiologically relevant in vitro model to investigate changes in the airway epithelium in response to h n pdm and the dsrna viral analogue, poly(i:c). our model recapitulated the in vivo response of the human airway epithelium to influenza in vitro. the cytopathic effect of h n pdm included damage to the airway epithelium, the induction of innate immune responses including the expression of pro-and anti-inflammatory cytokines and chemokines and antiviral genes and proteins, consistent with pulmonary host defense. to our knowledge, this is the first study that has shown zo- damage in wdnhbe cells treated with h n pdm . additionally, the downregulation of muc b, which plays an important role in mucociliary clearance suggests another mechanism of pathogenesis induced by the virus. these results form the basis for using the ali model for studying host-pathogen interactions and for testing therapeutics against newly emerged and re-emerging respiratory pathogens. the striking effect of h n pdm on zo- and the disruption of the apical perijunctional actomyosin ring of the cytoskeleton highlights iav damage to the d architecture of cells within the pseudostratified epithelium and is consistent with reduced teer, increased paracellular flux across the epithelium and increased apoptosis. the pattern of zo- damage in our wdnhbe cells infected with pandemic iav is similar to that of the immortalized human bronchial epithelial cell line ( hbe o-) infected with tissue culture adapted influenza virus a (h n ) [ ] . it also mimics zo- dissociation from tjs caused by rhinovirus infection of hbe o-cells, human airway epithelial cells grown at ali and in mice in vivo [ , ] . zo- dissociation is also consistent with the in vivo airway epithelium of asthmatic patients [ ] . while poly(i:c) disrupted zo- in cell membranes of immortalized hbe ocells [ ] , this was not observed in our primary wdnhbe cells. this may reflect differences between primary and immortalized cells and/or different concentrations and preparations of poly(i:c). airway protection is provided by a broad-spectrum of antimicrobial factors in mucus, e.g., lysozyme, β-defensins, cathelicidin, lactoferrin, elafin, secretory leukocyte peptidase inhibitor (slpi) and mucins [ ] [ ] [ ] . however, the precise roles of mucins, in particular, muc ac and muc b, the major secretory mucins in the human airway epithelium are still to be determined. optimal airway defense requires a balance between mucus production and clearance [ ] . in our wdnhbe model, mucus removal does not occur due to the closed nature of the transwell system. however, differences in mucin gene expression for h n pdm and poly(i:c) compared to mock-inoculated cells demonstrated that wdnhbe cells respond to these immune system challenges. differences in mucus composition and the relative abundance of muc ac and muc b can alter mucus gel viscosity, promoting pathogen growth, rather than their removal [ , ] . hence, the significant increase in muc b expression in response to poly(i:c) but decrease in response to h n pdm and unchanged muc ac for both immune system stimulants was intriguing. whilst the increase in muc b in response to poly(i:c) in our study was consistent with lever and colleagues, we did not observe an increase in muc ac expression [ ] . additionally, different iav strains (seasonal and pandemic) and different subtypes (h n and h n ) induced different levels of muc ac expression in an nci-h human pulmonary mucoepidermoid carcinoma cell line [ ] . seasonal strains were stronger inducers of muc ac expression than pandemic strains and h n generally had a greater effect on expression than h n [ ] . the differences observed in muc ac expression in our study may reflect the use of normal primary cells versus immortalized cancer lines, the use of different virus preparations and mois, different concentrations and/or preparations of poly(i:c) and a difference between cell donors. once the mucus layer of the airway epithelium has been penetrated, pathogens such as iav attach to cell-surface receptors (α , -and/or α , sialic acids), enter and replicate in epithelial cells. the virus egresses and spreads to other nonimmune and immune cells [ ] . although our model lacks immune cells, wdnhbe cells mount an innate immune response to both iav and poly(i:c), consistent with separate studies of these stimulants [ , ] . the body's natural defense mechanism of inflammation which promotes cell repair and healing [ ] was mimicked in our wdnhbe model, with the increased expression of proinflammatory cytokines and chemokines such as tnf, il b, il , cxcl and cxcl . this suggests that wdnhbe cells recognize iav and poly(i:c) through binding to pattern recognition receptors (prrs) such as the toll-like receptors (tlr , tlr , and tlr ), retinoic acid-inducible gene i (rig-i) and melanoma differentiation-associated protein- (mda- ), triggering innate immune response signaling cascades as occurs in vivo [ , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . antiviral, pro-and anti-inflammatory cytokines and chemokines are then upregulated in the host [ , , ] . key genes upregulated in our h n pdm in vitro challenge model, mimic the innate immune and inflammatory response in human patients in vivo infected with the pandemic iav. these include ifnb , cxcl , il , tnf and rsad . the more than -fold increase in ifnb , which encodes the antiviral type i interferon, ifn-β, confirms the host recognition of iav and the host-defense response to the virus in the airway epithelium in vitro, as observed by chan et al., [ ] . interestingly, the kinetics and magnitude of ifn-β secretion and hence the early innate and antiviral responses can vary for different influenza a subtypes [ ] . ifn-α/β stimulates the activation of hundreds of ifn-stimulated genes (isgs, e.g., cxcl , rsad , etc.), the first line of defence against viral infection [ ] [ ] [ ] [ ] [ ] . this is observed in our model with the dramatic, , -fold increase in cxcl expression in response to h n pdm infection, consistent with differentiated human airway epithelial cells infected with a hong kong isolate of the a/h n pandemic strain [ ] , wdnhbe cells infected with pandemic iav strains from a fatal (a/ky/ / ) and nonfatal (a/ky/ / ) case [ ] and in patients infected with pandemic iav [ , [ ] [ ] [ ] . upregulation of cxcl also occurs in vitro in the a human lung alveolar adenocarcinoma response to iavs [ ] . cxcl is a key chemokine responsible for early response to viral infection, is associated inflammation, e.g., asthma [ ] and is a biomarker that predicts disease severity and pathogenesis [ ] [ ] [ ] . the impact of cxcl is reflected in the gene coexpression network, with the increased expression of a number of other chemokines, cytokines and the anti-inflammatory il gene. cxcl is upregulated in response to other respiratory viruses, including rsv, rhinovirus and h n [ , , ] . however, while cxcl is protective in sars-cov infection [ ] , its precise role in h n pdm infection is unclear. increased levels of cytokines and chemokines are detected in the sera of patients with pandemic influenza [ ] , consistent with our model. in particular, increased levels of the proinflammatory il- and the anti-inflammatory il- which protects host tissue from damage during acute inflammatory responses are observed [ , , , [ ] [ ] [ ] . elevated levels of tnf and il b [ ] are also detected [ , , ] . tnf-α stimulates il- β which exacerbates lung injury during severe influenza but may also have a vital role in lung repair after infection [ ] . other cytokines and chemokines elevated in sera of patients include the chemoattractants cxcl [ , ] and ccl [ ] [ ] [ ] . increased expression and/or secretion of these and other chemoattractants such as ccl and ccl were observed in our wdnhbe model, consistent with other in vitro lung cell culture studies [ , , ] . hence, cell-mediated immunity is triggered in wdnhbe cells, as occurs in vivo [ , ] . the more than -fold induction of the antiviral-encoding rsad (viperin) gene in our pandemic iav-infected wdnhbe cells confirms the antiviral response of the airway epithelium in vitro. it is consistent with increased rsad expression in wdnhbe cells infected with the pandemic influenza strains a/ky/ / and a/ky/ / [ ] and in immortalized nci-h [ ] and a cells [ ] . rsad has multiple modes of antiviral activity. it catalyzes the conversion of cytidine triphosphate (ctp) to -deoxy- , -didehydro-ctp (ddhctp) which prematurely terminates rna-dependent rna polymerase (rdrp) of selected viruses but does not interfere with host rna and dna polymerases [ ] . hence, it reduces the replication of a range of rna and dna viruses including iavs, rabies virus, hiv, west nile virus, zika virus, dengue virus and hepatitis c virus [ ] [ ] [ ] . rsad also restricts the release (budding) of iav h n and other viruses by disrupting lipid raft microdomains on the plasma membrane of host cells [ , , ] . rsad also plays a role in the activation of t-cells and t-cell-receptor-mediated activation of nf-κb and activating protein (ap- ), key transcription factors in the expression of proinflammatory cytokines [ ] . hence, rsad is a multifunction antiviral that has a vital role in combatting viruses such as iav. airlifted primary nhbe cells grown on transwells at the air-liquid interface are the gold standard for bronchial epithelial cell culture [ ] . this model provided an excellent system to investigate innate immune responses to h n pdm and poly(i:c) in the airway epithelium in vitro. future studies could investigate the addition of other cell types including fibroblasts, endothelial smooth muscle cells and immune cells, e.g., neutrophils, to better reproduce the human airway in vitro and will undoubtably provide further information on host-pathogen interactions in the lung. airlifted nhbe cells recapitulated the pseudostratified airway epithelium in vivo, with the formation of ajcs, mucus secretion and the coordinated beating of ciliated cells. the disruption of the airway epithelium by iav h n pdm and poly(i:c), plus the induction of the innate immune response and antiviral, and pro-and anti-inflammatory genes demonstrated the viability of this model to investigate pandemic influenza. the disassembly of the ajc by h n pdm as shown by damage to zo- suggests that the virus can penetrate the epithelium and hence produce systemic infection. the use of multiple immune system challenges enabled the identification of differential responses of wdnhbe cells to h n pdm and poly(i:c). the reduction of muc b in response to iav is indicative of impaired mucociliary clearance of iav which may contribute to the severity of pandemic influenza in vivo. future studies will focus of the use of this model to investigate zoonotic, emerging infectious viruses with pandemic potential including the sars-cov- coronavirus currently sweeping the world today. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s , table s : taqman probes used for rt-qpcr analyses, video s : beating of cilia on the surface of ciliated cells of the wdnhbe epithelium, figure s :'transepithelial electrical resistance (teer) readings of wdnhbe cells, table s : teer readings (Ω & Ω × cm ) wdnhbe cells -iav h n pdm , table s : teer readings (Ω & Ω × cm ) wdnhbe cells -poly(ic), figure s : facs analysis of α- - and α- - -linked sialic acids on the surface of wdnhbe cells, figure s : transepithelial electrical resistance (teer) readings for three independent experiments of mock vs iav h n pdm inoculated wdnhbe cells, figure s : apoptosis in wdnhbe cells infected with pandemic iav h n pdm , figure s : transepithelial electrical resistance (teer) readings for three independent experiments of wdnhbe cells treated with µg and µg poly(i:c) 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for assistance with immunofluorescence assays, matt bruce for facs analysis, leanne davis for assistance with rt-qpcr and ryan farr for advice on rt-qpcr analysis. we acknowledge the insightful advice and assistance from dr kirsty short in establishing the transwell models at acdp. we thank microscopy australia for supporting the confocal microscopy capability utilized in this study through national collaborative research infrastructure strategy (ncris) funding. the authors declare no conflict of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. key: cord- -rz r sj authors: nan title: abstracts for the th annual meeting of the japan neuroscience society (neuroscience ) date: - - journal: neuroscience research doi: . /j.neures. . . sha: doc_id: cord_uid: rz r sj nan the brain basis of conscious experience is one of the great unsolved mysteries of science. how can a material object became aware of the world around it and of its very own awareness? many scholars think this question is unanswerable. new approaches to this age-old mind-body problem have recently been encouraged by the development of pet and mri and the powerful tools of modern neuroscience. we are now witnessing the explosive growth of a new field, called cognitive neuroscience which focuses on behavior and uses classical reaction-time paradigms together with new computer-based technology. a parallel approach is to cross-correlate formal aspects of conscious experience as the brain spontaneously pursues its regular trajectory through the objectively defined states of waking nrem and rem sleep. at the level of the brain it is possible to record pet and mri and by using an animal model to analyse cellular and molecular mechanisms. the advantage of this approach, which i call the conscious state paradigm, is that it, is quantitative, integrative, and holistic (in the rigorous sense of that word). the brain basis of activation (a) input-output gaining (i) and modulation (m) can now be described in relation to the changes in consciousness associated with them. the data can be used to create a three-dimensional model (aim) which describes the brain-mind trajectory in its state space. neural circuits in many parts of the developing nervous system exhibit highly rhythmic episodes of electrical activity. such activity has generally been considered to fine tune connections that are initially made by axons responding to a complex array of molecular guidance cues. however, chick and mouse spinal cords exhibit activity at early stages as motoneurons are still migrating and beginning to extend their axons. modest alterations in the frequency of such episodes produced changes in the expression of several guidance/recognition molecules and caused motor axon pathfinding errors. interestingly the type of pathfinding decision, the binary dorsal-ventral choice or the subsequent pool specific fasciculation and projection of axons to specific muscles, was differentially affected by decreasing or increasing the frequency respectively. thus, activity generated by developing circuits interacts at early stages with the molecular signaling pathways that govern the formation of precise circuits and any drugs that alter this activity could adversely affect circuit formation. supported by nih grant ns . sl - - frontal cortex and higher-order motor control: preferential use of multiple premotor and prefrontal areas dependent on behavioral context jun tanji brain science research center, tamagawa university, machida, tokyo, japan in the cerebral cortex of primates, there exist a number of motor areas rostral to the primary motor area and caudal to the prefrontal cortex. although each area has been defined based on anatomical and physiological criteria, functional roles played by each of them have been the matter of much debate. in fact, in a recent trend of research reports, it is popular to stress commonalities rather than specificities in the use of multiple cortical areas in motor control. for instance, the involvement of the primary motor cortex in cognitive aspects of motor behavior has been an eye-catching subject of recent reports. the involvement of the prefrontal cortex in movement planning has also been inferred. up to a certain extent, it is true that the use of different areas have some factors in common. however, it is a mistake to ignore profound differences in the use of each area, depending on specific aspects of motor behavior. in this lecture, i will describe such differences that could be clarified only when neural activities are examined properly, by studies designed to reveal individual aspects of functional significance of each area. sl - - neuronal functions and molecular motor, kinesin superfamily proteins, kifs: from transport of synaptic proteins and mrnas, to brain wiring, neuronal survival and higher brain function nobutaka hirokawa department of cell biology and anatomy, graduate school of medicine, university of tokyo, japan the intracellular transports are fundamental for neuronal morphogenesis, functioning and survival. to elucidate this mechanism we have identified and characterized kinesin superfamily proteins, kifs, using molecular cell biology, molecular genetics, biophysics, and structural biology. kifs play essential roles on neuronal function and survival by transporting synaptic vesicle precursors (kif a/kif bbeta), nmda type(kif ) and ampa type(kif s) glutamate receptors and mrnas(kif s) such as camkii ␤ mrna and arc mrna with a large rna-transporting protein complex containing at least rna related proteins such as hn rnp-u, staufen, and fmrps. kif a is fundamental for correct brain wiring by suppressing elongation of axon collaterals through depolymerizing microtubules in growth cones. kif suppresses tumorigenesis by transporting ncadherin/beta catenin containing vesicles from golgi to plasma membrane in the neuroepithelium. kif controls the activity-dependent survival of postmitotic neurons by regulating parp- activity in brain development. thus, kifs play significant roles not only on various neuronal functions but also on brain wiring, development and higher brain functions such as memory and learning. tsukahara award - what we can learn from the functional recovery after brain injury tadashi isa national institute for physiological sciences, okazaki, japan it is believed that when a part of a neuronal system is damaged, some of the lost functions can be taken over by residual systems through training. such concept is considered as the basis of neurorehabilitation, however, the mechanism of the "take-over" is not well understood. in this talk, i will present our recent progress in two lines of studies using non-human primate models related to this issue. the first topic is on the recovery of dexterous finger movements after lesion of the lateral corticospinal tract at the cervical spinal cord. after the virtually complete lesion, relatively independent finger movements can be recovered in - months. we have found that bilateral primary motor and ventral premotor cortices are involved at various stages of the recovery process by combining the pet imaging and reversible local inactivation technique. in the second, i will talk about the visuo-motor processing in monkeys with unilateral lesion of the primary visual cortex (v ). after complete ablation of v , the monkeys recover performance of visually guided saccades toward the blind field in months. saccades to the blind field have low sensitivity and less accurate. however, the monkeys can perform surprisingly cognitively demanding tasks in the blind field. i will discuss on our hypothesis on the bottom-up and top-down control of learning during recovery. takuji iwasato riken brain science center (bsi), wako-shi, saitama, japan in the rodent primary somatosensory (barrel) cortex, the configuration of the whiskers on the face is topographically represented as "barrels", discrete modules of layer iv neurons and thalamocortical afferent (tca) terminals. while barrel formation is an important model of the establishment of patterned topographic connections between the sensory periphery and the brain, the molecular mechanisms underlying this process are poorly understood. we developed and applied mouse reverse genetic technologies to examine these molecular mechanisms. we have focused on the nmda-type glutamate receptor (nmdar) and calcium-stimulated adenylyl cyclases, as nmdar-and camp-cascades are central to various types of neuronal plasticity, both in adulthood and during development. series of global and region-specific knockout mice have revealed the roles of these molecules in the patterning of barrel cortex and differentiated the specific mechanisms at presynaptic tca terminals compared to those at postsynaptic cortical neurons. research funds: presto (jst), kakenhi , kakenhi sy - - - distinct roles of two -pass transmembrane cadherins in neurite growth control tadashi uemura , , yasuyuki shima , , keisuke sehara , manabu nakayama , shinya kawaguchi , mikio hoshino , yoichi nabeshima , tomoo hirano , graduate school of biostudies, kyoto university, kyoto, japan; school of science, japan; kazusa dna research center at chiba, japan; graduate school of science, japan; graduate school of medicine, japan; crest, jst, japan drosophila flamingo (fmi) and mammalian celsr - , which are pass transmembrane cadherins, have been considered to mediate the regulation of neuron contact-dependent neurite growth. we show that mammalian -pass transmembrane cadherins celsr and celsr are activated by their homophilic interactions and regulate neurite growth in a distinct manner. both gene-silencing and co-culture assay showed that celsr enhanced neurite growth whereas celsr suppressed it. our result suggested that celsr had a stronger activity as g-protein coupled receptor than celsr did, most likely due to a difference of a single amino acid residue in the transmembrane domain, and this functional difference resulted in distinct effects in neurite growth regulation. thus, neuron-neuron interactions modulate neurite growth differentially through this couple of -pass transmembrane cadherins. masatoshi takeichi riken center for developmental biology, kobe, japan for synapse formation, axons need to recognize their specific partners, and subsequently stabilize their contacts. while a number of cell surface molecules should be involved in such processes, cadherin adhesion molecules play a role. when cadherin activities are blocked, synaptic contacts become destabilized in cultured neurons. this is also the case in vivo; e.g., in the neural retina whose cadherin activities are impaired without perturbing their overall architecture, a certain class of synaptic contacts does not normally form. another series of our study demonstrate that the cadherins cooperate with nectins, a subfamily of ig-domain molecules, for establishment of axon-dendritic contacts: in early hippocampal pyramidal neurons, nectin- is preferentially localized in axons; and nectin- , in both axons and dendrites. we present evidence that the heterophilic binding between axonal nectin- and dendritic nectin- is important for facilitating the axon-dendritic attachment; and cadherins seem to be required to stabilize the nectin-initiated contacts. thus, multiple classes of adhesion molecules work together to ensure the correct linking between axons and dendrites. hitoshi sakano department of biophysics and biochemistry, university of tokyo, tokyo, japan we have studied how the olfactory sensory neurons (osns) expressing the same odorant receptor (or) gene converge their axons to a specific set of glomeruli in the olfactory bulb (ob). retrogradestaining of osn axons indicated that the dorsal/ventral (d/v) arrangement of glomeruli in the ob is correlated with the expression areas of corresponding ors along the dorsomedial/ventrolateral axis in the oe. in contrast, the anterior/posterior (a/p) arrangement of glomeruli appears to be independent of the epithelial locations of osns and more dependent on the expressed ors. it was found that g proteinmediated camp signals regulate the positioning of glomeruli along the a/p axis in the ob. we also found that multiple sets of cell adhesion molecules, e.g., ephrin-as and eph-as, are expressed in a complementary manner, whose transcription levels are uniquely correlated with the expressing or species. we propose that differential levels of repulsive/adhesive interactions of axon termini may regulate the sorting of like-axons during the process of osn projection. research funds: crest, jst, kakenhi sy - - - lamina-restricted guidance of hippocampal mossy fibers hajime fujisawa , fumikazu suto nagoya university, nagoya, japan; institute of genetics, mishima, japan axons from different sources terminate at particular dendritic segments of target neurons in a laminal fashion. one important issue to be addressed is how individual axons are instructed to invade and arborize in particular laminae. projection of hippocampal mossy fibers is one of good experimental models to analyze molecular mechanisms that govern lamina-restricted termination of axons, because the fibers project to the proximal dendritic segment of ca pyramidal cells. we here report the following three mechanisms that provide lamina-restricted projection of mossy fibers. first, a neural repellent sema a is expressed in ca pyramidal cells and principally suppresses invasion of mossy fibers to ca . second, the repulsive signal of sema a is mediated by plexin-a expressing in mossy fibers. third, the repulsive activities of sema a are attenuated by plexin-a in the proximal dendritic segments of ca pyramidal cells, resulting in the segments permissive for mossy fibers to invade. over the course of development, children become increasingly able to control their thoughts and actions (i.e., cognitive control). the term cognitive control is an umbrella term for a set of putative control processes. these control processes may reach adult levels at different rates, depending on the rate of functional development of the specific brain structures involved. the structure most closely associated with cognitive control is prefrontal cortex (pfc). pfc is composed of what are believed to be functionally distinct subregions, including ventrolateral, dorsolateral, rostrolateral, and medial pfc. i will discuss the control processes associated with each of these regions, and how the functionality of these regions differs between school-aged children, adolescents, and young adults. three fmri studies will be presented, focusing on ( ) working memory maintenance and manipulation, ( ) rule representation and task-switching, and ( ) relational reasoning. based on these data, i will discuss some general points about neurodevelopment changes in cognitive control, and outline the approach that our laboratory has taken in our developmental cognitive neuroscientific research. sy - - - towards manipulative neuroscience based on non-invasive brain decoding in atr computational neuroscience laboratories, we proposed several computational models such as cerebellar internal models, mosaic, modular and hierarchical reinforcement-learning model. some of these models can quantitatively reproduce subject behaviors given sensory inputs and reward and action sequences which subjects received and generated. these computational models possess putative information representation such as error signals for internal models, action stimulus dependent reward prediction, and they can be used as explanatory variables in neuroimaging and neurophysiology experiments. we named this approach as computationalmodel-based neuroimaging, as well as computational-model-based neurophysiology. this new approach is very attractive and appealing since this is probably the only method with which we can explore neural representations remote from either sensory or motor interfaces. but, sometimes limitation of mere temporal correlation between the theory and data became so apparent, and we started to develop a new paradigm 'manipulative neuroscience' where physical causality is guaranteed. research funds: nict, karc sy - - - neural mechanisms in williams syndrome-insights from neuroimaging andreas meyer-lindenberg nimh, bethesda, md, usa williams syndrome (ws), a rare disorder caused by hemizygous microdeletion of − genes on chromosome q . , has long intrigued neuroscientists with its unique profile of striking behavioural abnormalities, such as hypersociability, combined with a differential impact on cognitive functions, with some types of abilities only mildly affected while others are severely impaired. ws, thus, raises fundamental questions about the neural mechanisms of social behaviour, the modularity of mind, and brain plasticity in development, and provides a privileged setting to understand genetic influences on complex brain function in a bottom-uph way. recent months have seen dramatic advances in uncovering the functional and structural neural substrates of ws and a beginning understanding of how these are related to dissociable genetic contributions characterized both in special participant populations and animal models. we will review neuroimaging work indicating abnormal function and structure in subsystems of visual processing, long term memory, and emotional regulation and social cognition, and discuss advances in relating them to the underlying molecular biology of this unique syndrome. daisuke yamamoto tohoku university graduate school of life sciences, japan the fruitless locus of drosophila was originally recognized by its mutants, the males of which preferentially court males rather than females. the fruitless primary transcript is subject to sexually dimorphic splicing mediated by transformer, and encodes a group of proteins that are putative transcription factors of the btb-zn finger protein family. the male-specific fruitless protein is expressed in small groups of cns neurons of males, but not of females. fruitless masculinizes these neurons thereby establishing the neural substrates for male-typical behavior. by experiments that label individually the neurons that express fruitless, we have identified a subset of brain interneurons that display marked sexual dimorphism in their number and projection pattern. fruitless supports the development of those neurons with male-specific dendritic fields, which are programmed to die during development in females as a result of the absence of fruitless. thus, the fruitless protein expression can produce a male-specific neural circuit likely used for heterosexual courtship by preventing cell death in identifiable neurons. hitoshi okamoto riken brain science institute, wako, saitama, japan the emotional behavior depends on the evolutionarily most conserved neural circuits. especially the fear behaviors involve the basal telencephalic nuclei such as the amygdala and the nucleus accumbens. thanks to the progress in understanding of the telencephalic development among different species, we can determine the correspondence of the parts between the teleost and mammalian telencephalons. with these in mind, we initiated the characterization of the emotional neural circuits in the zebrafish brain which are amenable to various modern technology. we already reported the asymmetric axonal projection from the left and right habenulae which act as the relay station to conduct the emotional information from the telencephalon to the monoaminergic neurons in the midbrain and the hindbrain. to investigate whether such asymmetric neural circuits cause the laterality for emotional behaviors, we are now in the process of establishing the paradigms for combining the behavioral assay with genetic manipulations to control the activity of the emotional neural circuit in zebrafish. research funds: kakenhi ( ) sy - - - a molecular biological approach for songbirds to study learned vocal communication kazuhiro wada, erich jarvis duke university, usa songbirds possess one of the most accessible neural systems for the study of brain mechanisms of behavior, particularly that for learned vocal communication. however, neuroethological studies in songbirds have been limited by the lack of high-throughput molecular resources and gene manipulation tools. to overcome this limitation, we generated a resource of full-length cdnas for gene expression analyses and functional gene manipulation in songbirds. we constructed total full-length cdna libraries from brains in different behavioral and developmental conditions. with these cdnas, we created a novel database and k songbird cdna array. we used the arrays to reveal a set of genes regulated by singing behavior. their molecular functions spanned most cellular and molecular categories, including signal transduction, structural, and synaptically released molecules. with the full-length cdnas, we were able to express proteins of singing-regulated genes in targeted brain area, using a lentiviral system. this resource now opens to more thoroughly study molecular neuroethological mechanisms of behavior. research funds: uehara memorial fellowship to k.w. and nih grant to e.j. - - - stepping pattern learning using mice: histochemical identification of activated neuronal circuits takashi kitsukawa graduate school of frontier biosciences, osaka university, osaka, japan identifying brain areas and neuronal circuits activated in a behavior is a critical step in understanding how the brain works in that behavior. also, identifying neuronal types involved in a behavior is a key step toward connecting behavioral approaches with molecular and genetical approaches. an efficient method of clarifying neuronal types activated by behavior is histochemical identification of neuronal types combined with c-fos staining. i would like to introduce our work as an example of using this method. in order to understand the neural processing involved in sequential motor skill learning we built a wheel running system in which a mouse learns sequential stepping patterns. we double-stained brain sections from mice which performed this task with c-fos and a neuronal marker such as enkephalin, substance p or nitric oxide synthase, each of which denotes a particular neuronal type. our results indicate that particular types of stiriatal neurons are activated during this learning, suggesting that cortico-striatal circuits are involved. synaptic plasticity that is dependent on precise timing of spikes between pre-and postsynaptic neurons plays important role in development and plasticity of brain functions. such spike-timingdependent plasticity (stdp) has attracted wide attentions because of its high computational power and physiologically plausible induction. we previously demonstrated that long-term potentiation was closely associated with structural plasticity of dendritic spines. however, how stdp is associated with structural changes has not been elucidated. we here report that paired two-photon uncaging of a caged-glutamate compound at a single spine and postsynaptic spike of whole-cell clamped neuron rapidly induced long-lasting bidirectional structural plasticity of spines in hippocampal ca pyramidal neurons. our results indicate that stdp is intimately associated with bidirectional structural plasticity at the level of single spines. research funds: kakenhi sy - - - role of camkii as a structural protein that stabilizes actin cytoskeleton in dendritic spines kenichi okamoto, radhakrishnan narayanan, yasunori hayashi massachusetts institute of technology, usa actin serves as a major cytoskeleton which maintains spine structure and exists in a equilibrium between f-actin and g-actin. tetanic stimulation causes a persistent shift of actin equilibrium towards f-actin which enlarges dendritic spines. but the mechanisms which maintain these changes remain elusive. we propose that camkii ␤ acts as an actin stabilizing molecule to maintain spine structure. camkii ␤ is not only an abundant f-actin binding protein, it can also make oligomers. we found that camkii ␤ oligomer crosslinks f-actin and stabilizes actin depolymerization kinetics. in spines, camkii ␤ oligomer slows down actin dynamics and camkii ␤ is enriched in spines by actin polymerization. the suppression of endogenous camkii ␤ alters spine shape to filopodia-like structures. these experiments suggest that camkii ␤ plays a role as a major stabilizer of the actin cytoskeleton to maintain spine structure. we also found that camkii ␤ detaches from f-actin in an activity dependent manner. we will discuss how camkii ␤ maintains actin equilibrium in activity dependent dendritic plasticity. ryohei yasuda duke university medical center, usa the small gtpase protein ras plays central roles in calcium signaling important for many forms of synaptic plasticity and regulation of neuronal excitability. using -photon fluorescence lifetime imaging microscopy in combination with a fret-based ras activity sensor, we visualized the activity of ras signaling with high spatiotemporal resolution. our studies indicate that calcium entry due to action potentials causes ras to activate in a supra-linear manner (yasuda et al., ) . furthermore, in response to single spine stimulation using -photon glutamate uncaging, ras activation initially occurs at the stimulated spine, subsequently spreading into its parent dendrites and nearby spines. these results suggest that nonlinear filtering by ras regulators as well as the spatial spreading of ras and ras regulators shape spatiotemporal patterns of ras signaling. hiroshi shibasaki takeda general hospital, kyoto, japan involuntary movements are unintended, generalized or focal, movements of abnormal nature, and include tremor, myoclonus, dystonia, chorea/ballism, athetosis and dyskinesia. myoclonus is characterized by abrupt, shock-like movements caused by brief muscle contraction (positive myoclonus) or abrupt cessation of on-going muscle contraction (negative myoclonus), or their combination. depending on the estimated origin, it is classified into cortical, brain stem, and spinal myoclonus. cortical myoclonus is short in duration ( ms). by back averaging eeg or meg time-locked to spontaneous myoclonus, a cortical activity is demonstrated in the corresponding area of the contralateral primary motor cortex immediately preceding the myoclonus (by ms for hand). it is mediated by fact-conducting corticospinal pathway. cortical myoclonus is often stimulus-sensitive (cortical reflex myoclonus), showing extremely enhanced cortical responses to somatosensory or visual stimulus, and enhanced longloop transcortical reflexes. these findings, together with transcranial magnetic stimulation, suggest increased excitability of sensorimotor cortex in cortical myoclonus. mark hallett ninds, bethesda, md, usa there have been many recent advances in the understanding of the physiology of focal dystonia. three main avenues of research have shown abnormalities in cortical inhibition, sensory processing including sensorimotor integration, and plasticity. this lecture will emphasize the abnormal inhibition. abnormal inhibition appears to be the most direct cause of unwanted muscle contractions that make up both the involuntary spasms and the overflow movements also seen in this condition. a loss of inhibition is seen in spinal and brainstem reflexes, but these changes are likely secondary to cortical abnormalities. cortical inhibition is also diminished as demonstrated most clearly with transcranial magnetic stimulation. gaba content may be decreased as shown with magnetic resonance spectroscopy. a particular type of defective inhibition is surround inhibition, the inhibition that normally operates to sharpen fine skilled movements. studies are now in progress to determine the synaptic mechanisms of surround inhibition and how this becomes abnormal in dystonia. understanding about inhibition in dystonia has led to some new treatments including some non-invasive cortical stimulation methods. research funds: nih intramural program sy - - - basal ganglia-cortical systems reinforcing tonic motor activity in health and disease peter brown sobell department, institute of neurology, uk the synchronisation of neuronal activity in the beta frequency (∼ hz) band has been noted in healthy primates, including humans, at both striatal, putamenal and cortical levels. it is most obvious in the motor cortex during tonic motor activity and is suppressed by voluntary movement. in this talk i will develop the idea that beta band synchronisation in the basal ganglia-cortical loop promotes tonic/postural contraction at the expense of new movements. thus, spontaneous phasic increases in beta activity in healthy subjects can be shown to be associated with a slowing of voluntary movements and a reinforcement of transcortical stretch reflexes. beta synchrony is also greatly exaggerated in untreated parkinson disease, where it may bias against new movement and contribute to bradykinesia and rigidity. excessive dopaminergic stimulation, either during treatment for parkinson disease, or in conditions such as dystonia, may overly suppress beta activity in bg-cortical loops leading to excessive movement. recordings of local field potentials in the basal ganglia of patients with movement disorders will be described that support this schema. research funds: mrc sy - - - coding of reward value of actions and valuebased action selection in the basal ganglia a damage of the nigrostriate dopamine system results in severe impairments of voluntary movements as well as involuntary behavioral states like rigidity, akinesia and tremor as typically observed in parkinson disease. recent studies revealed that long-term potentiation of corticostriatal synaptic transmission occurs dopaminedependent manner, and that neuronal firing related to external stimuli and body movements are modulated by whether the stimuli and movements are associated with reward or not. we recorded striatal neurons of monkeys who chose between left-and right-handle-turns based on the estimated reward probabilities of the actions. during a delay period before the choices, activity of more than one-third of striatal projection neurons was selective to the values of one of the two actions. during handle-turns, another subset of neurons was activated. these results suggest representation of action values in the striatum, which can guide action selection in the basal ganglia circuit. roles of the basal ganglia circuit in voluntary and involuntary action selection will be discussed. in vivo reporter gene imaging is expected to be a powerful tool in gene and cell therapy monitoring. we designed a new pet reporter gene system with f- fluoroestradiol (fes) and human estrogen receptor ligand binding domain (herl), which would work in various tissues with little physiological effect. we have been evaluating its potential in gene therapy monitoring constructing a plasmid co-expressing thymidine phosphorylase (htp), a factor works for revascularization, as therapeutic gene and herl. cos cells transfected with the plasmid expressed the both proteins and, when the plasmid was in vivo electroporated into mouse calf muscle, the electroporated muscle accumulated significantly higher amount of fes that the control side. this system was successfully applied to es cell transplantation monitoring also. inducible herl expression system was stably transfected into mouse es cells and viable es cells could be detected in vivo using fes. these data support the prospect that our in vivo reporter gene system would be useful in gene/cell transplantation therapy monitoring. tetsuya suhara molecular imaging center, national institute of radiological sciences, chiba, japan the molecular imaging using positron emission tomography enables to visualize various brain molecules with radio labeled ligand. neurons and glias express various receptors and transporters and those can be a specific target of the imaging. the functions of those molecules can be examined in various types of pharmacologically or genetically modified animal models. amyloid precursor protein transgenic mice provide the target of in vivo imaging of amyloid protein and glial reaction. because pronounced neuronal death is frequently heralded by microgliosis, in vivo analysis of glial activation in a quantitative manner could be a powerful means for assessing neuroglial degeneration. on the other hand, clinical finding of molecular imaging can also provide important cues for the basic research targets. since there is no ideal animal model for psychiatric disorders, the abnormal dopamine d receptor found in clinical research indicates a possible therapeutic target of negative symptoms of schizophrenia. the bidirectional interaction between basic research and clinical research using the molecular imaging technique can expand our knowledge in brain disease. sumiko mochida department of physiology, tokyo medical university, tokyo, japan in presynaptic terminals, packages of neurotransmitters, synaptic vesicles (svs), are localized at specific sites in different stages by regulation of proteins complexes. the recent outstanding studies have revealed molecular mechanisms of presynaptic structure and function. for svs, new proteins were found and the anatomy of vesicle structure was clarified. readiness for transmitter release, svs are docked and primed at the cytomatrix at the active zone where proteins complex formation is regulated by phoshorylation. new kinase sad- found at the sv and active zone or pka phosphorylates specific proteins. sv exocytosis is triggered by conformational changes in the fusion proteins complex when ca + sensing protein was activated. synchronies of sv fusion are maintained by a ca + sensing protein, synaptotagmin i. after transmitter release svs are recycled: surprisingly, recycled svs are shared between synaptic boutons regulated by cytoskeletal and motor proteins. these new findings suggest fine mechanisms in presynatic terminals that regulates transmitter release. shigeo takamori st century coe program, department of neuorology and neurological science, tokyo medical and dental university, tokyo, japan synaptic vesicles are storage organelles for neurotransmitters that recycle in the presynaptic terminals. to achieve their functions, i.e. neurotransmitter uptake and membrane fusion, they have to be equipped with specified proteins that play essential roles for each process. since decades ago, we have witnessed major advances in our knowledge about the molecular constituents on synaptic vesicles and we've functionally characterized many key players on membrane fusion machinery such as snare proteins and the rab gtpases and on neurotransmitter uptake such as vesicular transporters and vacuolar h + -atpase. however, a detailed picture of a vesicle membrane with all of its constituents is not yet available. in the present study, we have applied a combination of biochemical and biophysical approaches on purified synaptic vesicles from rat brains in order to arrive at a comprehensive and quantitative description of synaptic vesicles. in particular, with a newly developed counting method for synaptic vesicles in solution, we estimated the copy number of each molecule in a single synaptic vesicle. sy - - - sad: a novel kinase implicated in phosphoproteome at the presynaptic active zone toshihisa ohtsuka department of clinical and molecular pathology, faculty of medicine/graduate school of medicine, university of toyama, toyama, japan sad is a serine/threonine kianse, which has been shown to regulate various neuronal functions during development, including clustering synaptic vesicles, maturation of synapses, and axon/dendrite polarization: these have recently been revealed by genetic studies in c. elegans and mice. to test if sad is also involved in synaptic functions at mature neurons such as neurotransmitter release, we have recently isolated and characterized human orthologues of sad. interestingly, sad localizes both on synaptic vesicles and at the presynaptic active zones. moreover, sad, together with prominent active zone proteins cast and bassoon, is tightly associated with the active zone cytomatrix. in accord with its unique localization at the nerve terminals, sad appears to be involved in a late step of neurotransmitter release, via direct phosphorylation of another active zone protein rim . thus, these results suggest that sad regulates not only neural polarization during development but also neurotransmitter release at mature synapses. toshiaki sakisaka , takeshi baba , sumiko mochida , yoshimi takai department of molecular biology and biochemistry, osaka university graduate school of medicine, osaka, japan; depatment of physiology, tokyo medical university, tokyo, japan two types of factors are involved in ca + -dependent neurotransmitter release: the snare system and its regulators. the regulators of the snare system include many factors, such as the rab system, munc- , munc- , doc- , and tomosyn. we have previously reported that tomosyn, a syntaxin- -binding protein, works as a molecular clamp that controls free syntaxin- availability for the formation of the snare complex and thereby regulates synaptic vesicle exocytosis. here we show that pka-catalyzed phosphorylation of tomosyn decreases its binding to syntaxin- , resulting in enhanced the formation of the snare complex. conversely, rock phosphorylates syntaxin- , which increases the affinity of syntaxin- for tomosyn and forms a stable complex with tomosyn, resulting in inhibition of the formation of the snare complex. thus, tomosyn is likely to be an upstream regulator of the snare system, whose activity is regulated via well known signal transduction pathways. tei-ichi nishiki department of physiology, okayama university, okayama, japan a synaptic vesicle membrane protein, synaptotagmin i is thought to be a ca + sensor for neurotransmitter release. however, the physiological contributions of its ca + -binding domains (c a and c b) are still unclear. we have studied the roles of aspartate (asp) residues in the c b ca + -binding sites. in synaptotagmin i deficient neurons, although synchronous release was abolished as previously reported (cell , p. ) , asynchronous release was significantly increased. this defect was completely rescued by expressing wild-type synaptotagmin i, indicating the crucial roles of synaptotagmin i for triggering synchronous release and suppressing asynchronous release. synaptotagmin i with mutations in the second or third asp inhibited synchronous release, but still could suppress asynchronous release. thus, we conclude that synaptotagmin i maintains the synchrony of transmitter release in two ways. ca + binding to the c b is essential for synchronizing release. suppressing of asynchronous release seems not to require ca + binding to the c b because mutation in the second asp inhibits ca + binding, yet still allows the protein to suppress asynchronous release. yukiko goda, kevin j. darcy, kevin staras, lucy m. collinson mrc cell biology unit and lmcb, university college london, uk it has been assumed that vesicle replenishment at central synapses operates autonomously at individual presynaptic terminals. we tested the classical model of a compartmentalized synaptic vesicle cycle using fluorescent styryl dyes in combination with methods of fluorescence recovery after photobleaching and correlative light and electron microscopy in cultured hippocampal neurons. we found that endocytosed, recycling synaptic vesicles travel along axons and incorporate into non-native synapses by an actin-dependent mechanism. these newly-incorporated vesicles underwent exocytosis upon stimulation, demonstrating that they form part of the functional recycling pool at their host synapses. our findings indicate that synaptic vesicle recycling is not confined to individual presynaptic terminals but rather a substantial proportion of synaptic vesicles are shared constitutively between synapses. research funds: mrc, nih and narsad hiroshi kunugi department of mental disorder research, national institute of neuroscience, ncnp, japan neurotrophins such as brain-derived neurotrophic factor (bdnf) and neurotrophin- (nt- ) have been implicated in the phathogenesis of several neuropsychiatric diseases including schizophrenia, mood disorders, and neurodegenerative diseases. in the past decade, we have systematically screened bdnf, nt- and its low-affinity receptor p genes for polymorphisms and their possible association with neuropsychiatric diseases. as a result, three polymorphisms of the bdnf gene (c t in the noncoding region, bdnf-linked polymorphic region, and val met), three polymorphisms of the nt- gene (g- a, microsatellite in intron , and gly- glu) and a missene polymorphism of the p gene (ser leu) have been found to be associated with susceptibility to schizophrenia, bipolar disorder, depressive disorder, or alzheimer's disease, although some contradictive negative results have also been reported. here i summarize these findings, review the relevant literature, and discuss future directions of the promising role of the genetic variations of neurotrophins and p in neuropsychiatric diseases. recently, a single nucleotide polymorphism (val met) in the bdnf gene resulting in a prodomain substitution at position from a valine (val) to methionine (met) has been shown to lead in humans to altered hippocampal size and function, and susceptibility to neuropsychiatric disorders. we have recently determined in vitro that bdnf met aberrantly engages a specific vps protein, sortilin, that is part of a highly specialized sorting machinery that regulate bdnf trafficking to secretory pathways. in order to determine whether these trafficking defects are responsible for impaired hippocampal functioning, we have developed a transgenic knock-in mice containing the genetic variant bdnf (bdnf met/met ). we have determined that there is a regulated secretion defect for bdnf met , as well as altered hippocampal structure and function in these bdnf met mice, in a manner similar to that reported in humans with this variant bdnf. thus, this bdnf met/met mouse may provide an in vivo model system to inform human studies focused on associations of this variant bdnf with clinical disorders. research funds: nih grant# ns sy - - - processing of bdnf and brain disorders masami kojima , aist, osaka, japan; sorst, jst, saitama, japan the fact that pro-and mature neurotrophins elicit opposite effects through p neurotrophin receptor (p ntr) and trks, respectively suggests that proteolytic cleavage of proneurotrophins is an important mechanism that controls the direction of neurotrophin actions. here we examined the effects of two rare single nucleotide polymorphisms (snps); (t/g) and (t/g) of the human bdnf gene, causing amino acid substitution (r m and r l) near the cleavage site. western blot analysis and two-side elisa demonstrated that these snps prevented the cleavage, resulting in secretion of probdnf, but not mature bdnf. these snps did not affect intracellular distribution or mode of secretion of the protein. application of the uncleavable probdnf (probdnfml) elicited apoptosis of cerebellar granule neurons, but inhibited dendritic growth of basal forebrain cholinergic neurons. together, these results reveal structural determinants for the cleavage of probdnf, and demonstrate distinct functions of probdnf for different populations of neurons. we have now analyzed the brain functions of the mice expressing this form of bdnf. sy - - - pleiotropic effects of gdnf in regulation of enteric nervous system development hideki enomoto laboratory for neuronal differentiation and regeneration, riken center for developmental biology, kobe, japan formation of the enteric nervous system (ens) is governed by multiple extracellular signals at a given time during development. inactivation of the gene encoding gdnf, ret or gfr␣ leads to nearly complete absence of enteric neurons during early development. although this finding establishes gdnf as an essential extracellular signal acting at the initial stage in ens development, little is known about whether and how enccs continue to depend on gdnf later in development. we have generated mice in which function of gfr␣ , the high affinity receptor for gdnf, is conditionally inactivated in a time-specific fashion. we will show how gdnf signal influences cell migration, differentiation, proliferation and survival of developing enteric neurons, and discuss the biological significance of the findings in development and regeneration of the nervous system in general. bone marrow stromal cells (mscs) including the primitive pluriopotent mesenchymal stem cells and the multipotent adult progenitor cells, are attractive targets for cell and gene therapy for the range of central nervous system disorders. we present using replicationincompetent hsv- vector that msc population can be efficiently engineered to secrete a series of various cytokines in the large quantities and in long term in vivo to be able to treat the ischemic stroke of the brain potentially. three kinds of gene-transferred mscs, hgf, il ss+fgf- , and vegf were prepared and directly transplanted into the lesioned brain of rat transient middle cerebral artery occlusion model. each growth factor gene-transferred mscs achieved the remarkable amelioration of neurological symptoms and apparent decreasing of infarct volume comparison with native research funds: kakenhi ( ) sy - - - hgf gene therapy for the treatment of spinal cord injury masaya nakamura , akio iwanami , kazuya kitamura , , yoshiaki toyama , hideyuki okano department of ophthalmology surgery, keio university, tokyo, japan; department of physiology, keio university, japan hepatocyte growth factor (hgf) has recently been reported to exhibit neurotrophic activity and to play a role in angiogenesis. in this study, we demonstrated the validity of hgf for treatment of spinal cord injury (sci) in adult rats. first, we analyzed temporal expression of hgf and c-met in the injured spinal cord. hgf-mrna expression was relatively low in the acute phase of sci compared with c-met mrna expression. hypothesizing that hgf is insufficient immediately after sci, we induced sci at th level in adult rat days after injecting herpes vector-mediated gene transfer of hgf (hgf group) or lacz (control) into spinal cord. motor function was evaluated by bbb score. or weeks after injury, histological analyses were performed. there were significant decreases in apoptotic cell number and significant enhancements of angiogenesis and gap +fibers in hgf group compared to the control group. animals of the hgf group showed better functional recovery than the controls. these findings suggest that hgf could have therapeutic effects for sci. hiroshi funakoshi, toshikazu nakamura division of molecular regenerative medicine, osaka university graduate school of medicine, osaka, japan hgf was initially identified and molecularly cloned as a mitogen for primary hepatocytes (nakamura et al., ) . recently, hgf is found to be a novel neurotrophic factor for various types of neurons, such as hippocampal, cerebral cortex, cerebral granular, motor, and sensory neurons. mutant c-met/hgf receptor knock-in mouse reveals that hgf decreases neuronal survival and axonal elongation of several types neurons, including motor, sympathetic and cerebral granular neurons, during development (maina et al., ) . therefore, it is possible to speculate that hgf could play an important role in the retardation or regeneration of neurons in neurodegenerative diseases. here we show the examples of beneficial effects of hgf on model animals of different neural and neurodegenerative diseases, using several delivery methods for hgf including gene therapy approach. we also present the possible application of hgf in modifying the neurogenesis for the disease. references maina et al., . nature neuroscience. nakamura et al., . nature. hiroyuki kato center for clinical medicine and research, international university of health and welfare, nasushiobara, japan we examined stroke patients using fmri at acute/subacute and chronic stages, and visualized areas of brain activation during paretic hand movements. normal hand movement activated the contralateral primary sensorimotor cortex, supplementary motor areas, and ipsilateral cerebellum. at the acute/subacute stages, we observed reductions of these activations and/or addition of activation in ipsilateral cortex or contralateral adjacent cortex during paretic hand movements. at the chronic stages, recovery of activation and/or persistent addition of activation were observed. thus, motor functional recovery was accompanied by restoration of brain activation and/or appearance of additional activation within the motor network of the brain. the findings suggest that cortical motor reorganization as well as recovery from reversible injury plays a role in the restoration of motor function. interestingly, the time period during which reorganization occurred was limited to first - months after stroke, suggesting the presence of a critical period. in the cat, illert et al. ( ) first demonstrated that disynaptic pyramidal excitation in forelimb motoneurons can be mediated via propriospinal neurons located in the c -c segments. in contrast, recently it has been shown that polysynaptic pyramidal epsps are only rarely observed in forelimb motoneurons of macaque monkeys and humans. we reexamined the indirect corticomotoneuronal inputs in the primates, and obtained the following evidence for the pathway. ( ) in the macaque, recordings from forelimb motoneurons showed polysynaptic pyramidal epsps after blockade of glycinergic inhibition by strychnine. moreover, we recently identified c -c propriospinal neurons, which receive pyramidal inputs and project to forelimb motor nuclei. ( ) in human arm motor units, magnetic stimulation of the motor cortex produced multiple peaks at short latency in the post-stimulus time histogram, whose total duration was longer than the corresponding value of a finger muscle. stimulation of the pyramidal tract in the medulla could also produce multiple peaks, though in a lower frequency. functions of the pathway both in physiological and pathological conditions will be discussed. bisphenol-a (bpa) has been extensively evaluated for toxicity in a variety of tests as the most common environmental endocrine disruptors. we previously reported that prenatal and neonatal exposure to bpa potentiated central dopaminergic neurotransmission, resulting in supersensitivity to psychostimulant-induced pharmacological actions. many recent findings have supported the idea that astrocytes, which are a subpopulation of glial cells, play a critical role in neuronal transmission in the central nervous system. we found that in vitro treatment with bpa caused the activation of astrocytes, as detected by a stellate morphology and an increase in levels of gfap. a low concentration of bpa significantly enhanced the ca + responses to dopamine in both neurons and astrocytes. these findings provide evidence that bpa induces dopaminergic changes in neurons and astrocytes. this phenomenon may, at least in part, contribute to the enhancement by bpa of the development of psychological dependence on drugs of abuse. mami yamasaki, yonehiro kanemura the department of neurosurgery, clinical research institute, osaka national hospital, national hospital organization, osaka, japan l cam(l ) is a member of the immunoglobulin superfamily of cell adhesion molecules. x-linked hydrocephalus, masa syndrome and certain forms of x-linked spastic paraplegia are now known to be due to mutations in the gene for l . therefore, these syndromes have been reclassified as l syndrome. we performed a nation-wide l gene analysis and identified li gene mutations in families with l syndrome. all the patients showed developmental delay in various degree. we discussed genotype and phenotype correlations, a striking correlation between the mutation class and the severity of symptoms and molecular basis of severity of developmental delay. the loss of extracellular domain functions like l -mediated cell adhesion and cell migration is considered to be responsible for molecular genesis of ventricular dilatation and disturbance of the functions of cytoplasmic domain would cause symptoms related axon growth in l syndrome. research funds: kakenhi ( ), ( ) sy - - - rett syndrome and developing brain yoshiko nomura segawa neurological clinic for children, japan rett syndrome (rtt) is a neurodevelopmental disorder with mental retardation, autistic feature, and stereotyped hand movements. hypofunction of the brainstem monoaminergic neurons is suggested. pathology showed no degeneration. methyl-cpg-binding protein gene (mecp ) located at xq is the causative gene. types of mutation at different functional domains are correlated to clinical severities. x-inactivation also influences phenotypic variability. mecp was thought as a global transcriptional repressor, but finding of bdnf as a target gene suggest its role in the neuronal activity-dependent gene regulation. genetic heterogeneities have been suggested and the mutation of cyclin dependent kinase-like gene (cdkl ) manifest as atypical rtt. the mutations of mecp are found in other clinical conditions, such as x-linked mental retardation, angelman syndrome, autism, and severe neonatal encephalopathy. thus, the evaluation of rtt gives the clue to study the clinical, pathophysiological, biological and molecular correlation of not only rtt but also other neurodevelopmental disorders. in our previous studies, we have proposed that ros and/or ros-mediated signal play(s) an essential role in -ohda-induced, caspase-dependent apoptosis. in contrast, mpp+-mediated death is not blocked by caspase inhibition and is accompanied by an increase in intracellular free calcium. subsequently, we have demonstrated that mpp+ induces release of cytochrome c but not activation of caspase and proposed that depletion of atp and/or calcium-activated calpain-mediated degradation of procaspase- are responsible for the absence of subsequent activation of caspases. furthermore, we have identified that degradation of several important proteins by activated calpain and proteasome system is linked to mpp+-mediated dopaminergic neuronal death. as such, we have found that one of onconeural proteins seems to play a role as a potential survival factor, degradation of which is involved in mpp+-induced cell death. taken together, we reason that distinct set of proteases activation is involved in experimental models of pd. therefore, novel strategies interfering activation of these proteases may contribute to prevention of dopaminergic neuronal death. satoshi ogawa department of neuroanatimy, kanazawa university of medical school, ishikawa, japan we discuss the role of er-stress in neuronal cell death in snpc by introducing two models. upregulation of pael-receptor in the substantia nigra pars (snpc) of mice induces endoplasmic reticulum (er) stress leading to a decrease in tyrosine hydroxylase and death of dopaminergic neurons. the role of er stress in dopaminergic neuronal vulnerability was highlighted by their enhanced death in mice deficient in the ubiquitin-protein ligase parkin and the er chaperone orp , suggesting parkin dysfunction result in er-stress mediated neuronal cell death. conversely, transgenic rats overexpressing megsin (tg meg), a newly identified serine protease inhibitor (serpin), demonstrated intraneuronal periodic-acid schiff (pas) positive inclusions, which distributed throughout the deeper layers of cerebral cortex, hippocampal ca , and substantia nigrta. enhanced er stress was observed in dopamine neurons in snpc, accompanied with loss of neuronal viability and motor coordination. in both subregions, pas-positive inclusions were also positive with megsin. these data suggest that enhanced er stress causes selective vulnerability in a set of neuronal populations. noradrenaline (na) transmission modulates synaptic excitability and plasticity through distinct receptor subtypes. accumulating evidence has suggested that the central na system modulates consolidation and reconsolidation of long-term emotional memory. here we show that the na system is particularly important for retrieval of reconsolidated emotional memory. the mutation of the gene encoding tyrosine hydroxylase causes a deficit in conditioned taste memory after its reactivation. this memory deficit is restored by pharmacological stimulation of na activity before the test and is also restored by intra-amygdala na stimulation through ␣ or ␤-adrenergic receptors. moreover, intra-amygdala na stimulation in the wild type animals increases their susceptibility to recall reconsolidated memory. our findings indicate that the amygdalar na system, primarily through ␣ and ␤-adrenergic receptors, acts to improve the retrieval of reconsolidated memory trace. shigeru morinobu, shigeto yamamoto, shigeto yamawaki departmnet of psychiatry and neurosciences, hiroshima university, hiroshima, japan as psychophysiological reactivity on exposure to cues resembling an aspect of the trauma is the major symptom in ptsd, it is hypothesized that impaired extinction may be involved in ptsd. rats subjected to single prolonged stress (sps) exhibit the enhanced negative feedback of the hpa axis, exaggerated startle response, and analgesia. thus, sps is a good model of ptsd. we examined whether extinction of fear memory was impaired in sps rats, using the contextual fear conditioning. sps rats exhibited the significant longer freezing during re-exposure to the context - days after the conditioning. furthermore, repeated administration of d-cycloserine markedly inhibited the development of enhanced freezing in sps rats. we measured the levels of nmda receptor subunits (nr , nr a, b, c), glycine transporter , and eaac , by real-time pcr. no significant changes were found in the hippocampus. based on these findings, it is speculated that the increase in other types of glutamate transporters or nmda receptor modification may play a role in impaired extinction in sps rats. ichiro masai masai initiative research unit, riken, wako, japan in human, there are hereditary retinal diseases such as retinitis pigmentosa. to understand these molecular mechanisms, we performed a large-scale mutagenesis using zebrafish as an animal model. here we report two zebrafish mutants, twilight (tli) and eclipse (els), both of which show no normal erg and okr response. in the tli mutant, photoreceptors initially differentiate but degenerate later. electron-microscopic analyses revealed that photoreceptive membranes are severely disorganized in the tli mutants, suggesting that tli is required for the formation of photoreceptive membranes. in the els mutant, photoreceptors seem normal in morphology, suggesting that phototransduction is compromised. we found that the els gene encodes cgmp phosphodiesterase ␣ -subunit (pde c), a component of cone-type pde. since genetic mutations of pde c have not been reported in human, the els mutant provides a good model for studying roles of cone-pde in visual functions. shinichi nakagawa , masatoshi takeichi , , fumi kubo , nakagawa initiative research unit, riken, wako, japan; riken cdb, kobe, japan; department of biostudies, kyoto university, kyoto, japan the marginal region of the optic vesicle contains retinal stem cells that remain undifferentiated and proliferate for a much longer period compared to other progenitor cells in the central retina. we have previously shown that wnt b, a signaling molecule expressed in a region neighboring the stem cell area, functions as a putative stem cell factor that endows undifferentiated retinal cells with the characteristics of the stem cells. interestingly, wnt b inhibits cellular differentiation in the absence of notch activity, a well-known signaling receptor that inhibits neuronal differentiation. wnt b antagonizes proneural gene functions independent of the notch signaling pathway, presumably through unidentified transcriptional repressors. we have isolated several candidate genes that are upregulated upon an activation of the wnt signaling pathway, and some of them are expressed in the stem cell containing region. physiological roles of those genes will be discussed. research funds: kakenhi ( ) sy - - - identification of cell lineage of retinal progenitor cells by cell surface markers sumiko watanabe, hideto koso, shinya satoh department of molecular and developmental biology, university of tokyo, tokyo, japan i would like to discuss about early cellular developmental stages of retina, which we identified by examination of the expression pattern of cell surface markers. we found c-kit and ssea- to be spatiotemporal markers of distinct populations of retinal progenitor cells, and these cells dramatically changed their expression profiles of c-kit and ssea- during development. c-kit-positive cells expressed various immature retina specific genes; and later onset of rhodopsin expression and stronger proliferation activities were observed. c-kit/ssea- double-positive cells showed stronger proliferation activities than ckit single-positive ones. although the number of ssea- -positive cells was augmented by beta-catenin signal, c-kit-positive cells were positively regulated by notch signaling, suggesting that c-kit and ssea- have intrinsically distinct characters. prolonged expression of c-kit by a retrovirus resulted in promotion of proliferation and the appearance of nestin-positive cells in response to scf, suggesting a role for c-kit in retinal development. the retinal photoreceptor cells play a primal and central role in the phototransduction system. they are susceptible to deterioration in human retinal diseases, which lead to severe visual impairment. we have been demonstrated that transcription factors, otx and crx play critical roles in retinal photoreceptor development. while otx is a key molecule for retinal photoreceptor cell fate determination, crx is essential for the terminal differentiation and maturation of photoreceptors. meanwhile, the photoreceptor cell is a highly polarized neuron and also has epithelial characteristics such as adherens junctions. our investigation of a role of apkc, which has been proposed to play a critical role in the establishment of epithelial and neuronal polarity, in differentiating photoreceptors has shown that apkc is required for the formation of outer & inner segments and ribbon synapse. in addition, we also found that photoreceptor polarity formation has important roles in proper retinal lamination. we would like to present our recent analysis of photoreceptor development. research funds: kakenhi ( , , ) raj ladher riken center for developmental biology, kobe, japan the inner ear translates mechanical energy into neural signals that the appropriate centers of the brain can decode into balance or sound information. the inner ear forms from bilateral thickened discs of ectoderm located on either side of the hindbrain, early during development. induction of the inner ear is mediated by localized signals emanating from the paraxial mesoderm. in the chick, the inner ear is induced by localized fgf found in the mesoderm. we find that although fgf can induce the inner ear, it is unable to support differentiation of the inner ear. differentiation, that is the development of the chick inner ear hair cells, is triggered by another family member fgf and is actually inhibited by fgf . for full functionality, the inner ear needs to be integrated into the larger auditory complex, made up of the middle ear, the external ear and the auditory centers in the hindbrain. these components develop from diverse origins but are intimately linked during development. we have been trying to understand how integration occurs and present one model by which this could occur. research funds: center support grant, mext leading projects grant sy - - - how is olfactory receptor-dependent axonal wiring conducted? shou serizawa, kazunari miyamichi, haruki takeuchi, yuya yamagishi, tokiko tsubokawa, hitoshi sakano department of biophysics and biochemistry, crest jst, university of tokyo, tokyo, japan in the olfactory system, termini of primary axon segregate depending on the type of olfactory receptor (or) expressed, forming the olfactory sensory map. to study how the or-dependent axonal wiring is conducted, we analyzed the gene expression profile in the olfactory epithelium of the transgenic mouse in which the majority of olfactory sensory neurons (osns) express a particular or gene. we found that the expression of the immunoglobulin superfamily gene kirrel , encoding homophillic adhesion molecule, is down-regulated in the transgenic mouse compared to the wild type control. the expression level of kirrel in each osn is found to be correlated with the type of or species expressed in the osn. moreover, kirrel promoted fasciculation of osn axon termini in the mosaic gain-of-function experiment. here, we propose that the information of which type of or is expressed in the osn is converted to the expression level of kirrel which determines the adhesiveness of axon termini, contributing to or-dependent segregation of osn axons. in spite of its morphological similarity to the other species in the melanogaster species subgroup, drosophila sechellia has evolved distinctive physiological and behavioral characters adapting to its host plant morinda citrifolia, known as the tahitian noni fruit. the ripe fruit of m. citriforia contains hexianoic acid and octanoic acid, the main components of the odor from the fruit. d. sechellia is attracted to these two fatty acids, while the other species are repelled by them. using inter-species hybrid between d. melanogaster deficiency mutants and d. sechellia, odorant binding protein e was identified as the gene responsible for this behavioral difference among the species. obp e forms a gene cluster with obp d, and these two genes are expressed in the same cells associated with the chemosensory organ. the history of dynamic obp d/e-cluster evolution was revealed by comparison of the genomic sequences of the obp d/e region obtained from species phylogenetically located between d. melanogaster and d. pseudoobscula. sy - - - an approach of dissociating complex traits into fine genetic elements using consomic strains of mouse aki takahashi , , akinori nishi , , toshihiko shiroishi , , tsuyoshi koide , sokendai, kanagawa, japan; national institute of genetics, shizuoka, japan much of the genetic variation that underlies most behavioral traits is complex and is regulated by loci that have quantitative effect on the phenotype. we have previously shown that laboratory strain c bl/ (b ) and wild-derived strain msm/ms have great differences in many behavioral traits. consomic strains were established by natural mating between b and msm, and those strains have the same genetic background as b except for one chromosome from msm. by examining bunch of consomic strains on many behavioral trait, such as spontaneous activity, anxiety-like behavior, pain sensitivity, and social behavior, we were able to map which chromosome have a locus or loci affecting those phenotype. one strain b - msm, which have msm chromosome , showed increased fear responses and riskassessment behavior, and thus it is thought that there is a locus/loci related to the emotionality. to identify the gene in the loci, we have made congenic strains, and successfully narrowed the locus down in the telomeric region. research funds: kakenhi ( . ) sy - - - cloning of the major quantitative trait locus underlying capsaicin resistance in mice capsaicin is the main compound of hot chili peppers, and induces sensations of heat and pain. however, sensitivity to capsaicin differs among individuals. a genetic approach using a mouse model reveals some quantitative trait loci for this sensitivity. capsaicin resistance linked on chromosome (capsq ) is the major locus affecting reduced taste sensation in kjr mice. here we show that intracellular recycling of capsaicin receptor (trpv ) was impaired in kjr neurons in contrast to that of c bl/ j mice. by searching the candidate genes, eh domain-containing four (ehd ), a trp-binding scaffold protein encoding gene was found. ehd binds to c-terminal of trpv . three mutations were found in ehd of kjr, which remarkably diminished the binding, leading to changes in the intracellular distribution of trpv . this study is the first genetic dissection associated with capsaicin/heat resistance in a nature strain and shows a novel binding protein to trpvs. sy - - - comprehensive behavioral analysis of genetically-engineered mice tsuyoshi miyakawa hmro, kyoto university, kyoto, japan one of the major challenges in the life sciences of the post-genome era is to elucidate the functions of the genes at the level of individual animals. final output level of the functions of the genes expressed in the brain is behavior, indicating a need for systematic investigations of the behavioral significance of the genes. in our laboratory, we use a "comprehensive behavioral test battery" for genetically-engineered mice to reveal causal relationships between genes and behaviors. the battery covers broad areas of behaviors, from simple reflexes to highly cognitive functions. so far, we have assessed more than different strains of mutant mice with the battery. surprisingly, more than % of the strains showed at least one significant behavioral phenotype, suggesting that a large part of the genes expressed in the brain may have some functions. representative results for a few strains of mutant mice and the meta-analytic results of the combined data will be presented. also, a potential impact of our approach to "large-scale neuroscience" will be discussed. research funds: kakenhi ( , , , ) , jst bird hiroshi takashima department of neurology and geriatrics, kagoshima university, kagoshima, japan inherited neuropathies are clinically and genetically heterogeneous. at least genes and loci have been associated with charcot-marie-tooth disease (cmt) and related inherited neuropathies. most causes of inherited neuropathy have been discovered by positional cloning technique and in the past two years, the pace of cmt gene discovery has accelerated. these recently discovered cmt causing genes/proteins include those which, although showing unpredictable correlations with the peripheral nervous system, are definitely important for the peripheral nerve. their discovery should pave the way for dramatic progress in the understanding of peripheral nerve biology. on the other hand, genotype-phenotype correlations of these genes are also important in order to understand the pathomechanisms of inherited neuropathy since, based on mutation studies, a large number of genes associated with both the demyelinating and axonal forms of cmt have been identified. to clarify the specific features and molecular mechanisms, we reviewed recent progress in cmt research, especially cmt f caused by prx, and scan caused by tdp . sy - - - gangliosides are important for the maintenance of the nodes of ranvier nobuhiro yuki, keiichiro susuki department of neurology, dokkyo university school of medicine, tochigi, japan gangliosides are abundant in vertebrate nervous system, but the function has yet to be elucidated. some patients with guillain-barre syndrome have autoantibodies to gangliosides such as gm , who show failure of peripheral motor nerve conduction. sensitization of rabbits with gm can produce the disease model. in ventral roots from the paralyzed rabbits, igg and complements deposited on the nodes of ranvier, and sodium channel clusters were disrupted. in ganglioside-deficient mice with disrupted gm /gd synthase gene, motor nerve conduction velocities were reduced in the sciatic nerves. some myelin loops failed to contact the paranodal axolemma, and potassium channels were aberrantly localized at the paranodes. the abnormality became prominent with age. these findings using different models showed that gangliosides are important for the maintenance of the node of ranvier and saltatory conduction along the myelinated nerve fibers. hiroshi ueda division of molecular pharmacology and neuroscience, nagasaki university graduate school of biomedical sciences, nagasaki, japan neuropathic pain caused following nerve injury is one of important issues in neuroscience as well as clinics, since its pain pathway is apparently distinct from that in healthy humans and naive experimental animals. this is clearly evidenced by the finding that the tactile information is converted to noxious one in allodynia characterized in neuropathic pain. in our recent paper (nature medicine, ) , we firstly demonstrated that lysophosphatidic acid (lpa) and its receptor (lpa ) activation initiate the neuropathic pain. in this and following studies we proposed that the demyelination of nociceptive fibers reorganizes the nociceptive spinal inputs through sprouting and electrical synapses (ephapses). i will discuss four issues, lpa-induced demyelination of dorsal root fibers using in vivo and ex vivo culture models, the signal transduction of underlying lpa-mediated downregulation of myelin proteins, evidence for sprouting and ephapses following demyelination and the origin of injury-specific lpa production in terms of demyelination and allodynia. research funds: kakenhi ( ) toshihide yamashita department of neurobiology, graduate school of medicine, chiba university, chiba, japan axons of adult central nervous system are capable of only a limited amount of regrowth after injury, and an unfavorable environment plays major roles in the lack of regeneration. some of the axon growth inhibitory effects are associated with myelin. three myelin-derived proteins have been identified to inhibit neurite outgrowth in vitro. these proteins induce activation of rho in some neruons. inhibition of rho or rho-kinase promotes axon regeneration in vivo. these findings establish rho and rho-kinase as key players in inhibiting regeneration of the central nervous system. i will review recent findings regarding the signaling mechanism of axon growth inhibitors. our experiments suggest that several new candidate proteins may be axon growth inhibitors. these proteins activate not only rho/rhokinase but also other signals to inhibit neurite outgrowth from some neurons in vitro. these findings suggest that agents that block the multiple signals elicited by these axon growth inhibitors may provide efficient tools that produce functional regeneration following injuries to the central nervous system. sha mi biogen idec, usa lingo- is a cns-specific protein expressed in both neurons and oligodendrocytes. in neurons, lingo- mediates the inhibition of axonal growth as a component of the ngr /p /lingo- and ngr /troy/lingo- signaling complex. inhibition of endogenous lingo- by soluble lingo- or dominant negative lingo- can reverse the inhibition of neurite outgrowth by myelin components. soluble lingo- treatment significantly improves functional recovery of spinal cord injured rats as determined by bbb scores. soluble lingo- treatment promotes axonal regeneration and reduced axon dieback in the corticospinal tract, rubrospinal tract, and optic nerve. in oligodendrocytes, lingo- mediates the inhibition of differentiation and myelination. loss of lingo- function using dominant negative lingo- , lingo- rnai, or soluble lingo- or lingo- knockout increased oligodendrocyte differentiation and myelination, whereas over-expression of lingo- led to inhibition of oligodendrocyte differentiation and myelination, in vitro and in vivo. the discovery of a significant role for lingo- in neurons and oligodendrocyte biology are an invaluable step for understanding cns axon regeneration and myelination. alex reyes new york university, usa neurons in the auditory cortex exhibit a wide range of firing patterns. to elucidate the cellular properties and circuitry that give rise to these responses, a d sheet of excitatory and inhibitory neurons were reconstructed in vitro using an iteratively-constructed network (icn) modified to contain both feedback and feedforward circuits. a disc of neurons was stimulated and the resultant firing pattern and spread was documented. simultaneous whole-cell recordings were performed from pyramidal and interneurons in a slice preparation of the mouse auditory cortex. a computer simulated the activities of thalamic neurons and calculated the net synaptic conductance that would be generated by their firing. this waveform was converted to current, injected into the recorded neurons via a dynamic clamp circuit, and the resultant firing documented. using the icn method, we reproduced the firing of a realistic network of excitatory and inhibitory neurons. we replicated many of the responses recorded in vivo. morever, the firing patterns of neurons depend substantially on their distance from the stimulus center and on the identity of the local interneurons. research funds: nih dc - a sy - - - neuronal avalanches reveal neuronal wirings of layer / cell assemblies jun-nosuke teramae, tomoki fukai brain science institute, riken, japan how cortical neurons process information crucially depends on how their local circuits are organized. spontaneous synchronous neuronal activity propagating through neocortical slices displays highly diverse, yet repeatable, activity patterns called 'neuronal avalanches'. they obey power-law distributions of the event sizes and lifetimes, presumably reflecting the structure of local cortical networks. however, the explicit network structure underlying the power-law statistics remains unclear. here, we present a neuronal network model of pyramidal and inhibitory neurons that enables stable propagation of avalanche-like spiking activity. we demonstrate a neuronal wiring rule that governs the formation of mutually overlapping cell assemblies during the development of this network. the resultant network comprises a mixture of feedforward chains and recurrent circuits, in which neuronal avalanches are stable if the former structure is predominant. we investigate how the resultant power laws depend on the details of the cell-assembly formation as well as on the inhibitory feedback. research funds: kakenhi ( ) sy - - - spike-timing dependent and homeostatic plasticity from an optimality viewpoint taro toyoizumi , jean-pascal pfister , kazuyuki aihara , , wulfram gerstner department of complexity science and engineering, university of tokyo, japan; school of computer and communication science & bmi, epfl, japan; aihara complexity modelling project, erato, jst, japan maximization of information transmission by a spiking neuron model predicts changes of synaptic connections that depend on timing of pre-and postsynaptic spikes as well as on the postsynaptic membrane potential. under the assumption of poisson firing statistics, the synaptic update rule exhibits all the features of the bienenstock-cooper-munro rule, in particular regimes of synaptic potentiation and depression separated by a sliding threshold. the learning rule is found by maximizing the mutual information between presynaptic and postsynaptic spike trains under the constraint that the postsynaptic firing rate stays close to some target firing rate. an interpretation of the synaptic update rule in terms of homeostatic synaptic processes and spike-timing dependent plasticity is discussed. research funds: grant-in-aid for jsps fellows j and sci. res. on priority areas from mext of japan, and swiss natl. sci. found. - / sy - - - timing computations in the auditory brain stem john rinzel center for neural science and courant institute of mathematical sciences, new york university, usa sound localization involves precise temporal processing by neurons in the auditory brain stem. the first neurons in the auditory pathway to receive input from both ears can distinguish interaural time differences (itds) in the sub-millisecond range. these cells in the mammalian medial superior olive have specialized biophysical features: two dendrites, each receiving input from only one side; very short membrane time constant; specialized ionic channel properties, including a low-voltage activated k+ current, i-klt. this i-klt contributes to phasic firing (one spike in response to a step of current), precise phase-locking, and extremely timing-sensitive coincidence detection. we will describe the temporal feature-selecting properties of mso cells based on biophysical (hh-like) modeling, in vitro electrophysiology and application of concepts from dynamical systems theory and coding theory. neuronal information is often inferred by counting spike numbers over tens to hundreds of milliseconds. however, if relative spike timings at the scale of milliseconds would carry information, neuronal circuits could have large information capacity. in response to various visual inputs, the retina fires spike bursts separated by hundreds of milliseconds of silent periods. onsets and spike numbers of these bursts are highly reproducible. we asked if spike patterns, i.e., combinations of interspike intervals within single bursts, carry information. using the retinas of salamanders and mice, we found that bursts have various spike patterns, which are unique to the preceding inputs. differences in spike patterns at the scale of milliseconds encode differences in the input as long as - ms. when single bursts contain three or more spikes, the multiple interspike intervals combinatorially encode multiple features of the input. this suggests the spike patters are not determined sorely by slowly modulating instantaneous firing rates. we propose that the retina encodes multiple features in hundreds of milliseconds of input into burst spike patterns at the scale of milliseconds. accumulating evidence reveals that the generalized seizure activity can produce regenerative, in addition to degenerative, structural changes in the hippocampus, including the enhancement of progenitor cell division of dentate granule cells. although the regulatory mechanisms underlying such neurogenesis are unknown, we hypothesized that newly generated granule cells may contribute to the reorganization of the hippocampal formation in the early course of seizures, constituting a possible substrate for epileptogenicity. to address this issue, we examined the division of dentate granule cell progenitors in rats after kainic acid administration, or perforant path kindling. the results indicate that initial limbic seizures trigger the enhancement of dentate progenitor cell division, but progenitor cells may become unreactive to prolonged generalized seizures. the degenerative process is not necessary for triggering the upregulation. it is also suggested that newly generated granule cells may play a role in the network reorganization that occurs during epileptogenesis. the molecular basis underlying such neurogenesis will be discussed. keiichi itoi , ikue otaki , saya suzuki , yasunobu yasoshima , kazuto kobayashi laboratory of informational biology, graduate school of informational science, tohoku university, sendai, japan; institute of biomedical science, fukushima medical university, japan in order to examine functional roles of the noradrenergic (na) neurons in the locus coeruleus (lc) we developed a novel method to ablate specifically the na neurons in the lc, and examined the behavioral and stress responses using the animal model. a transgenic mouse line was used in which human interleukin- receptor ␣ subunit (hil- r␣) was expressed under the control of dopamine ␤-hydroxylase gene promoter. anti-hil- r␣ antibody fused to pseudomonas exotoxin was microinjected into bilateral lc of a transgenic mouse stereotaxically to destroy specifically the na neurons. as behavioral paradigms, elevated plus maze and open field test were used. plasma adrenocorticotropin levels were measured following lipopolysaccharide injection intraperitoneally, as an immune stress. thus, the effect of lc ablation how it affects the behavioral and stress responses will be elucidated. - - - integrated circuits controlling the stress response james p. herman department of psychiatry, university of cincinnati, oh, usa the hypothalamo-pituitary-adrenocortical (hpa) axis is a primary stress-response system in all vertebrates. the end-product of hpa activation, glucocorticoids, serve the general function of redirecting bodily resources to meet a real or perceived challenge. however, prolonged glucocorticoid secretion has deleterious effects on metabolism, immune function and behavior, making control of hpa activity a priority for the organism. this control is exerted in large part by limbic structures in the brain. our studies indicate that the amygdala, hippocampus and prefrontal cortex play major roles hpa axis regulation. the amygdala is primarily stress excitatory, whereas the hippocampus has an inhibitory influence on hpa activity. the role of the prefrontal cortex is considerably more complex; its prelimbic region is primarily stress inhibitory, whereas the infralimbic region may participate in stress activation. all of these regions exert their influence via subcortical relays to hypothalamic paraventricular nucleus (pvn) neurons controlling the hpa response, allowing convergence of information from multiple limbic sources prior to the pvn. sy - - - molecular mechanism for the inverse incidence of parkinson's disease and cancer: synuclein as stimulator of tumour differentiation makoto hashimoto department of chemistry and metabolism, tokyo metropolitan institute for neuroscience, tokyo, japan neurodegenerative disease and cancer are major age-associated disorders. however, the pathogenesis of these diseases may be in sharp contrast, since the former is featured by cell death, whereas, the latter is associated with immortalization. in parkinson's disease (pd) research, smoking, the risk factor for a variety of cancers, had been known to reduce the risk of pd. furthermore, epidemiological studies described that the incidence of cancer was reduced in pd patients. recent study provides evidences of the inverse relationship of pd and some cancers at the molecular level. for example, loss of neuroprotection of dj- is causative for familial pd, while increased expression of this molecule stimulates oncogenesis. in this context, we show that proteasomal inhibition by ␣-synuclein, which has been thought as one major pathogenic mechanism for pd, may induce differentiation of cancer cells. thus, unifying approach on the basis of the opposite pathogenic mechanism to neurodegenerative disease and cancer might uncover unexpected findings in both fields. kiyomitsu oyanagi department of neuropathology, tokyo metropolitan institute for neuroscience, tokyo, japan neurodegenerative diseases and malignant tumors develop symptoms usually at middle or old-age in humans. however, it is well known that critical periods of some malignancies are in fetal period, which are ( ) leukemia in patients exposed with atomic bomb during the iind world war, and ( ) brain tumors in rats with ethylnitrosourea administration. as to neurodegenerative diseases, ( ) many genetic/familial diseases show clinical symptoms at the middle or old age. ( ) epidemiological study revealed that emigrants from guam to the main land of usa show relatively high incidence of amyotrophic lateral sclerosis, and the critical period of exposure to some environmental noxiousness was considered to be childhood/adolescence. ( ) relating to parkinson disease, low magnesium intake over generations induced selective degeneration of the dopaminergic neurons in the substantia nigra in rats [oyanagi et al., in press] . these findings indicate that not only certain malignant tumors but also some sporadic neurodegenerative diseases may be induced originally by the insults in embryonic stage/childhood. to understand the role of synuclein, the major component of pathological inclusions, we examine the expression of synuclein in the embryonic mouse cerebral cortex. we found that a-synuclein and b-synuclein were predominantly detected in the subplate neurons, which are known to enter programmed cell death at a postnatal stage. in another line of inquiry, we are interested in a zinc finger protein containing poz domain, rp , which functions as a sequence specific transcriptional repressor and involved in cortical layer formation. when the rp gene is disrupted, apoptosis is enhanced, and a-synuclein, but not b-synclein, is upregulated in the mutant cortex, suggesting that a-synuclein is involved in the cell death. interestingly, in the mutant cortex the expression of s-phase marker, pcna increased, suggesting that rp mutant mice are useful to analyze the relation among neurodegeration, synuclein and cell cycle. minoru saitoe, junjiro horiuchi, daisuke yamazaki tokyo metropolitan institute for neuroscience, tokyo, japan age-related memory impairment (ami) is a striking feature of ageassociated neuronal dysfunction. to identify gene mutations that affect ami, we screened ∼ drosophila lines and found that heterozygous mutants for the pka catalytic subunit (dc /+) exhibit robust suppression of ami without affecting memory at young ages. this result suggests a causal relationship between pka and ami. of particular interest, igf/pi k/akt signaling, which results in decreased gsk activity, has also been shown to ameliorate ami. both pka and gsk phosphorylate the microtubule-associated protein tau, causing tau aggregation and neurodegeneration. while igf signaling suppresses activity of gsk at young ages, declining igf levels during aging may increase gsk activity in aged animals. in support of this idea, we found suppression of ami in flies fed gsk inhibitors. we hypothesize that similar to the mechanisms occurring in neurodegenerative diseases, tau phosphorylation by pka and gsk causes neuronal dysfunction during normal aging. research funds: kakenhi sy - - - molecular mechanism of cancer progression by gamma-synuclein koji okamoto radiobiology division, national cancer center research institute, tokyo, japan synucleins, a family of small proteins consisting of three known members, are implicated in both neurodegenerative disorder and tumorigenesis. ␣synuclein is involved in the formation of pathologically insoluble deposits characteristic of neurodegenerative diseases such as alzheimer disease and parkinson disease, whereas overexpression of ␥synuclein is associated with progression of breast and ovarian cancer. however, the normal cellular function of synucleins remains largely unknown. in order to get an insight into biological function of synucleins, we focus on cancer progression induced by ␥synuclein. we introduced ␥synuclein into breast cancer cells in order to recapitulate malignant transformation of breast cancer. using such cells, the attempt to elucidate the biochemical function of ␥synuclein is underway. the impact of synuclein over-expression, especially on known tumor suppressor pathways such as the p pathway, will be discussed. research funds: kakenhi ryuichi sakai growth factor division, national cancer center research institute, - - tsukiji, chuo-ku, tokyo - , japan numbers of growth factors and their membrane receptors which possess tyrosine kinase activity are involved in proliferation and differentiation of the neural system. shc family docking molecules conduct signals directly downstream of various growth factor receptors as substrates and binding partners of these tyrosine kinases. in the neural systems, two unique shc family molecules, shcb and shcc, are found to be specifically expressed and analysis of mice lacking these proteins revealed that they have redundant functions during mammalian neural development as mediators of ngf/trka signaling. it was recently found that tyrosine phosphorylation of shcc is frequently detected in majority of neuroblastoma cell lines. we showed that hyperphosphorylated shcc detected in some of neuroblastoma cell lines is associated with constitutively activated anaplastic lymphoma kinase (alk) caused by the gene amplification. identification of binding partners of shcc and expression of mutant shcc in several cancer cell lines revealed novel roles of shcc as a regulator of differentiation and proliferation of neuroblastic tumors. research funds: kakenhi sy - - - identification of estrogen receptor target genes and role of their gene products in cancer and nervous system satoshi inoue , department of geriatric medicine, university of tokyo hospital, tokyo, japan; research center for genomic medicine, saitama medical school, saitama, japan estrogen has crucial roles in the cancer growth and in the neural function. here, we have isolated and characterized novel estrogenresponsive genes to clarify the molecular mechanism of the estrogen action in target cells using genomic binding-site cloning (gbsc) method. one of the first identified genes is the estrogen-responsive ring finger protein (efp). efp expression was observed in uterus, mammary gland and certain regions of the brain where er is also expressed and positively regulated by estrogen. we revealed that efp targets proteolysis of - - sigma, a negative cell cycle regulator that causes g arrest and that efp is an essential oncogenic factor in breast cancer growth. on the other hand, another gene identified by gbsc is nr d, an nmda receptor. this gene was regulated by estrogen in the hypothalamus, together with er, pr and efp. these estrogen responsive genes could mediate roles of estrogen action in specific organs, utilizing differential mechanisms as well as sharing common mechanisms. keiji tanaka , hossein esteky , kiani roozbeh , tadashi sugihara , gang wang riken brain science institute, wako, saitama, japan; institute for studies in theoretical physics and mathematics, tehran, iran; kagoshima university, kagoshima, japan individual cells in the monkey inferotemporal cortex, which is the final unimodal stage along the ventral visual pathway, respond to moderately complex features, but not to objects nor to object categories. then, questions arise where and how view-general objects and object categories are represented. a possibility is the representation by a population of inferotemporal cells. to examine it, we recorded responses of inferotemporal cells to object images in a fixation task. we also conducted psychophysical experiments with monkeys to determine conditions for view-invariant object recognition. the results suggest that a population of inferotemporal cells represent object categories and their relational structure, and that the representation is common to nearby views of objects with up to • rotation. research funds: kakenhi alexander thiele , gene stoner , louise s. delicato , mark roberts university of newcastle upon tyne, uk; the salk institute, japan a variety of different roles of synchronized activity for sensation and perception have been proposed, ranging from object binding, through attentional enhancement, to mechanisms of learning. we have employed different paradigms to investigate the role of neural synchrony in visual perception and attentional selection in the awake macaque monkey. using two different tasks and stimulus conditions, well suited to probe the role of feature binding in the motion domain, we found no support for the idea that neuronal synchrony in macaque area mt underlies the binding of an object's component features. recent reports have focused on the role of synchrony in the mediation of attention. we will discuss the role of synchronized activity in primate v while attentional load was varied, and how it could be mediated by cholinergic mechanisms. research funds: hfsp, wellcome trust, bbsrc sy - - - context-dependent changes in noise correlation in mt william newsome, marlene r. cohen stanford university and howard hughes medical institute, usa changes in the correlated firing of a pair of neurons may provide a metric of changes in functional circuitry within the nervous system during ongoing behavior. we studied dynamic changes in functional circuitry by analyzing the noise correlations of simultaneously recorded mt neurons in two behavioral contexts: one that promotes cooperative interactions between the two neurons and another that promotes competitive interactions. we created cooperative or competitive contexts by changing the axis of motion of the discrimination task from trial to trial. we found that identical visual stimuli indeed give rise to differences in noise correlation in the two behavioral contexts. specifically, noise correlations were higher in the cooperative than in the competitive context. this result suggests that mt neurons receive inputs of central origin whose strength changes with the task structure. the changes in correlation appear to reflect differences in how mt neurons are pooled for the purpose of perceptual discrimination, and may derive from higher-level cognitive processes such as feature-based attention. research funds: howard hughes medical institute sy - - - effects of task demands on color processing in area te of the monkey hidehiko komatsu , , kowa koida national institute for physiological sciences, okazaki, japan; sokendai, okazaki, japan color vision has two different functions, namely, categorization and discrimination, and the same color stimulus can be processed according to these two functions depending on task demands. lesion studies suggested that inferior temporal (it) cortex of the monkey plays a key role in color vision, and we have recently found that color selective neurons are concentrated in a small region in area te of it cortex. to study how the color selective responses in this region are affected by the task demands, we trained monkeys a color categorization task and a color discrimination task using the same set of color stimuli, and analyzed how the responses are affected. we found response magnitudes of many neurons changed between two tasks while the color tuning is well reserved. in several extreme cases, large gain change almost completely eliminated the responses in one task. these results suggest that color signals are gated by the top-down signal representing task demands in area te and color channels specific to different tasks are formed at this level of the visual cortex. yoichi sugita neuroscience research institute, aist, tsukuba, japan early visual experience is indispensable to shape the maturation of cortical circuits during development . monocular deprivation in infancy, for instance, leads to an irreversible reduction of visually driven activity in the visual cortex through the deprived eye and a loss of binocular depth perception - . here, i show exposure only to monochromatic illumination in infancy results in the disruption of color perception. infant monkeys were reared for nearly a year in a room where the illumination came from only monochromatic lights. after extensive training, they were able to perform color matching. but, their judgment of color similarity was quite different from that of normal animals. furthermore, they had deficits in color constancy; they could not compensate for the changes in wavelength composition. these results indicate that early visual experience is also indispensable for color perception. research funds: crest sy - - - dendritic growth, spinogenesis and synaptogenesis in response to neurosteroids in the developing purkinje cell kazuyoshi tsutsui , hirotaka sakamoto , katsunori sasahara , hanako shikimi , kazuyoshi ukena , mitsuhiro kawata laboratory of brain science, faculty of integrated arts and sciences, hiroshima university, higashi-hiroshima, japan; department of anatomy and neurobiology, kyoto prefectural university of medicine, kyoto, japan new findings over the past decade have established that the brain synthesizes steroids de novo from cholesterol. such steroids synthesized de novo in the brain are called neurosteroids. recently we have identified the purkinje cell as a major site for neurosteroid formation in the brain. this is the first demonstration of de novo neuronal neurosteroidogenesis in the brain. in mammals, the purkinje cell actively synthesizes progesterone and estradiol de novo from cholesterol during neonatal life, when cerebellar cortical formation occurs. subsequently, our recent studies on mammals using the purkinje cell have demonstrated organizing actions of neurosteroids. both progesterone and estradiol promote dendritic growth, spinogenesis and synaptogenesis via each cognate nuclear receptor in purkinje cells. research funds: kakenhi ( and to kt) sy - - - roles of estrogen receptors in the regulation of socio-sexual and emotional behaviors-studies with knockout mice and rnai sonoko ogawa kansei, behavioral and brain sciences, university of tsukuba, tsukuba, japan the gonadal steroid estrogen plays a major role in the regulation not only of female reproductive behavior but also an array of social and emotional behaviors in both sexes, by acting through intracellular estrogen receptors (ers), ligand dependent transcription factors. a series of studies using single and double knockout mice for er-␣ and/or er-␤ genes have revealed that activation of er-␣ and er-␤ differentially regulate a number of behaviors as well as neuroendocrine functions. our studies have suggested a unique role of activation of er-␤ in the hypothalamic and limbic brain areas, dorsal raphe nuclei and locus coeruleus in the regulation of socio-sexual and emotional behaviors. in this talk, our findings from behavioral studies using er-␣ and er-␤ knockout mice along with possible brain mechanisms underlying the behavioral effects will be first overviewed. our most recent studies on brain site-specific manipulation of er gene expression with the use of small interference rna combined with adeno-associated virus will then be presented. research funds: kakenhi ( , ) sy - - - sex steroid receptor function in sexual behavior shigeaki kato , , takashi sato , takahiro matsumoto , imcb, university of tokyo, tokyo, japan; erato, jst, saitama, japan androgen actions are believed to mediate nuclear androgen receptor (ar)-mediated gene regulations. ar is a member of nuclear receptor, and acts as a hormone-induced transcription factor to control of target genes through chromatin remodeling/histone modification. we generated the floxed ar mice to avoid testicular feminization mutant (tfm) abnormalities with infertility, and then crossed with female ar(−/+) heterozygoutes expressing cre to generate ar(−/−) female mice. the ar(−/y) ko males grew healthy with typical features of tfm abnormalities, and genital organs were atrophic with a marked decrease in the serum testosterone level, but with normal estrogen level (kawano et al., ) . no sexual behaviors and reduced aggressive behaviors were seen in ar(−/y) male mice (sato et al., ) . female ar ko mice were normal in sexual behavior but exhibited premature ovarian phenotype (shiina et al., ) . together with these results, the ar function will be discussed in terms of ar function as a transcription factor. references kawano, h., et al., . pnas usa , . sato, t., et al., . pnas, usa , . shiina, h., et al., research funds: probrain sy - - - annexin : a mediator of cell-cell communication in the neuroendocrine system julia buckingham , helen christian , john morris imperial college london, uk; department of human anatomy and genetics, university of oxford, uk annexin (anxa ) plays an important part in mediating the regulatory effects of glucocorticoids (gcs) on neuroendocrine function, particularly within the hpa axis. it is expressed by folliculostellate (fs) cells in the pituitary gland and by ependymal cells and activated glia in the hypothalamus but not by classical secretory cells. gcs act on cells expressing anxa to cause the translocation of the protein to the plasma membrane at points with particular accumulation at points where the cells make contact with endocrine cells. this process is effected via a non-genomic mechanism and is dependent upon phosphorylation, lipidation and a transport protein, possibly abca . the released protein then acts, via cell surface receptors on the endocrine cells to suppress stimulus-evoked peptide release. the nature of the anxa receptor is unclear but, increasing data suggest that members of the formal peptide receptor family may be important in this regard. katsuhiko nishimori , yuki takayanagi , masahide yoshida , yoshiyuki kasahara , masaki kawamata graduate school of agricultural science, tohoku university, sendai, japan; department of physiology, jichi medical university, minamikawachi-machi, japan we examined the behaviors of mice lacking oxtr gene and discovered that oxtr null females displayed impaired nurturing behavior, and their pups showed defect in ultrasonic vocalization, instead, increased locomotor activity by social isolation test. those are implying impaired mother-infant relationship. oxtr null males also showed more aggressive and having social amnesia as well as the phenotype of oxt null mice. in addition, oxtr null mice failed to maintain their body temperature after acute cold exposure. their rectal temperature rapidly dropped in comparison of that of wildtype animals at • c ambient temperature. our studies demonstrate that oxtr plays a critical role in regulating several aspects of social behavior and the other physiological function, and may have important implications for developmental psychiatric disorders, such as autism. research funds: grant-in-aid for scientific research (b) ( ) sy - - - cortical mechanisms mediating visuomotor control of primate grasp roger n. lemon ucl institute of neurology, uk primates demonstrate an exquisite ability to precisely shape their hand when grasping an object. a network of parietal and frontal motor areas is thought to play a key role in this behaviour. our work shows that: hand shape can be unambiguously determined from emg activity of hand and digit muscles. information about grasp is represented by neuronal populations in the ventral premotor cortex (area f ); f activity shows graspspecific discharge soon after an object becomes visible, well in advance of activity in primary motor cortex (m ). local field potential activity in f and m is also tuned to grasp, and there is strong beta coherence between f and m , indicating reciprocal transmission of information. this is also seen in synaptic responses of m neurones to stimulation of f (and vice-versa). single pulse stimulation in f strongly modulates corticospinal outputs from m through corticocortical pathways between these two areas. paired-pulse tms can probe the excitability of these pathways in humans. facilitation of meps is both object and muscle specific and indicates that activity in these pathways is selectively enhanced during object grasp. research funds: wellcome trust, bbsrc sy - - - where tactile signals are ordered in time shigeru kitazawa , department of neurophysiology, juntendo university graduate school of medicine, tokyo, japan; crest, japan science and technology agency, saitama, japan how does the brain order successive events? it is generally accepted that the brain can resolve the order of two stimuli that are separated in time by ms. this applies to temporal order judgment of two tactile stimuli, delivered one to each hand, as long as the arms are uncrossed. however, crossing the arms caused misreporting (that is, inverting) of the temporal order. the reversal was not due to simple confusion of hands, because correct judgment was recovered at longer intervals (e.g., . s). when the stimuli were delivered to the tips of sticks held in each hand, the judgment was altered by crossing the sticks without changing the spatial locations of the hands. we recently found that temporal order judgments of tactile stimuli are strongly affected by visual distractors and/or eye movements. the results suggest that tactile stimuli are ordered in time only after they are referred to relevant locations in space, where multiple modalities of sensory signals converge. results from functional imaging support this idea. sy - - - decision making and underlying neural mechanisms-auditory-visual ambiguity solving and preference shinsuke shimojo , biology/cns, california institute of technology, pasadena, ca, usa; jst.erato shimojo implicit brain function project, atsugi, japan we explore mechanisms underlying crossmodal ambiguity solving (passive decision), and preference (active decision). we've employed the illusory flash effect, where a single flash appears to be doubled when accompanied by two sounds. -channel meg was employed, while the observer reported number of flashes. partial directed coherence was applied to see if there was a causal influence by the auditory on the visual cortices. the results indicate a strong causal influence in a-v direction in alpha ( ) ( ) ( ) ( ) ( ) and ranges only in the illusion-reported trials, while stimulus parameters were identical. no such difference was found in v-a direction. for preference, the observer's gaze shifted towards the to-be-chosen stimulus (face) before conscious decision. our fmri study shows activity specific to preference task in the ventral amygdala and the ventromedial prefrontal. while such results enable the same causality analysis, it also raises a question as to what determines active/passive nature of decision. research funds: jst.erato, hfsp sy - - - why look there? insights from spatial neglect and the medial frontal cortex masud husain ucl institute of neurology, uk why do we look where we do? studies in humans show that when we look at a scene, our initial fixation patterns can be predicted to a high degree of accuracy. our eyes go to the most salient locations where local feature contrast is greatest. these findings have led to the concept of a salience map which directs attention and gaze bottomup. in humans, damage to the right posterior parietal cortex often leads to dramatic neglect of the left side of space. recent research has begun to unravel the components of this syndrome, demonstrating several underlying mechanisms. these include a disturbance of the salience representation, a failure to keep track of spatial locations across saccades and difficulty in sustaining attention over time. gaze is directed not only bottom-up by but also top-down by voluntary mechanisms. our recent investigations of human medial frontal regions reveal important roles for the supplementary eye field and the pre-supplementary motor areas in the control of competing eye movement plans and deciding where to look. parietal and medial frontal gaze regions appear to play different, complementary roles in controlling why we look where we do. research funds: wellcome trust ( ) sy - - - recognizing self actions through externalized eyes atsushi iriki , symbolic cognitive development, riken brain science institute, saitama, japan; cognitive neurobiology, tokyo medical and dental university, tokyo, japan we can recognize ourselves and our own actions through the mirror or video images. thus, human can use such apparatus as externalized eyes (sensory tools), while non-human animals can normally use tools as extension of their effectors (motor tools) at most. human fmri studies revealed that the right temporo-parietal junction region and the mesial superior frontal gyrus are involved in perceiving and manipulating the representation of the self actions under different third person perspectives. japanese monkeys could be trained to use a hand-held video camera as a manipulable extension of their eyes only when their own vision was gradually transferred to the distant cues via motor-tools to extend their body images. the emergence of novel cortico-cortical projections between temporo-parietal junction and the intra-parietal cortex was described in monkeys that were trained to use motor tools, therefore, integrate the tool in their own body image. thus, presence of a self-objectification mechanism is suggested for acquisition of sensory tools as externalized eyes to recognize self actions. yoshiyuki kubota division of cerebral circuitry, nips, okazaki, japan gabaergic nonpyramidal cells in the neocortex are composed of several different subtypes. we found that most of gabaergic cell types, including fs basket and somatostatin martinotti cells, that innervate dendritic spines in addition to the somata and dendritic shafts. most postsynaptic spines also received an asymmetrical input, called double innervated (di) spines. to better characterize the other asymmetrical input on the di spines, excitatory presynaptic terminals were stained by immunohistochemistry for two types of vesicular glutamate transporters (vgluts): vglut , existing mostly in cortical pyramidal cells, and vglut , found in thalamocortical fibers. gabaergic inputs were rarely observed in spines innervated by vglut -expressing terminals (n = ), but were found in- % of spines innervated also by vglut -expressing terminals (n = ). symmetrical synapses on di spines were positive for gaba, as shown by postembedding immunohistochemistry. these results indicated that some thalamocortical inputs are likely selectively inhibited at the spine level by gabaergic synapses from cortical nonpyramidal cells. research funds: kakenhi sy - - - gabaergic recruitment of excitation by cortical axo-axonic cells gabor tamas, csaba varga, gabor molnar, szabolcs olah, pal barzo, janos szabadics university of szeged, hungary the axon has the lowest threshold for action potential generation and axons in the cerebral cortex receive input only at the axon initial segment exclusively from axo-axonic cells (aacs), which use the dominant inhibitory neurotransmitter, gamma-aminobutyric acid (gaba). thus, aacs are considered as strategically placed inhibitory neurons controlling cortical information flow. we applied multiple patch clamp recordings in slices of rat and human neocortex and found that single spikes in aacs can trigger action potentials in pyramidal cells and initiate stereotyped series of multiple synaptic events in the cortical network. the excitatory action of aacs is based on a depolarized reversal potential for axonal relative to perisomatic gabaergic inputs as determined in paired perforated patch recordings. powerful axo-axonic depolarization from the resting membrane potential is supported by a ∼ -fold decrease in the potassium-chloride co-transporter (kcc ) expression from somatic to axon initial segment membranes detected by quantitative immunogold labeling. in my talk i will describe the integrative and plasticity properties of thin basal dendrites of cortical pyramidal neurons. these dendrites receive the majority of the cells' synaptic inputs, so determining their integrative and plasticity properties is of prime importance. previous studies have most often reported global linear or sublinear summation in these dendrites. using confocal imaging and dual-site focal synaptic stimulation of identified thin dendrites in rat neocortical pyramidal neurons we show that thin dendrites provide a layer of independent computational "subunits" that sigmoidally modulate their inputs prior to global summation. next i will describe the plasticity rules used by these fine basal dendrites putting a special emphasis on the role of nmda-spike in local synaptic plasticity processes. yumiko yoshimura department of visual neuroscience, research institute environmental medicine, nagoya university, nagoya, japan neocortical circuits contain fine-scale networks of excitatory neurons interconnected precisely. we previously showed that layer / pyramidal cells in visual cortex share common excitatory inputs from layer and from within layer / , when they are directly connected. here, we tested whether inhibitory cells are incorporated into the fine-scale specificity of excitatory connections. we recorded photostimulation-evoked synaptic currents from pairs of layer / cells, consisting of one inhibitory cell and one pyramidal cell in rat visual cortex slices, and measured the extent of common inputs to the pairs based on cross-correlation analysis. fast spiking inhibitory cells shared extensive common excitatory inputs with neighboring pyramids only when the pairs of cells were reciprocally connected. adapting inhibitory cells shared little or no common input with neighboring pyramids, regardless of their direct connectivity. therefore, fine-scale specificity depends on the type of inhibitory cell and on the direct connectivity between neighboring pyramidal-inhibitory cell pairs. research funds: kakenhi ( , ) sy - - - local circuitry of cortical inhibitory neurons edward callaway, takuma mori, xiangmin xu the salk institute, usa we used laser scanning photostimulation to map local input to inhibitory neurons in layer of rat visual cortex and layer / of mouse barrel cortex. mouse studies used transgenic animals with gfp expressed in subsets of inhibitory neurons. in layer , axondescending cells receive excitatory input predominantly from layer / while neurogliaform cells receive stronger input from deeper layers. layer / neurons also receive inputs that vary systematically by cell type. two subtypes of martinotti cells, distinguished by calretinin (cr) expression, also differ in morphology and intrinsic physiology. cr+ martinotti cells receive excitatory input predominantly from layer / , while the cr− martinotti cells also receive strong excitation from layer . irregular-spiking basket cells also receive strong excitatory input from layers / and , but they often have a gap at the top of layer , with little or no input. fast-spiking basket cells and pyramidal cells in mouse barrel cortex receive input indistinguishable from cells in rat visual cortex, with strong input from layers / and , and only weak input from deeper layers. research funds: nih sy - - - physiological genomics of cortical microcircuits sacha b. nelson brandeis university, czech republic cortical microcircuits are comprised of highly diverse neuronal cell types that differ in their morphology, synaptic connectivity and intrinsic electrophysiology. presumably, these phenotypic differences are orchestrated and maintained by unique transcriptional programs. in order to begin to reveal those programs we have recently developed methods for measuring genome-wide gene expression from small numbers ( - ) of fluorescently labeled, hand-sorted neurons. subsets of pyramidal neurons and gabaergic interneurons were labeled genetically with gfp or anatomically with fluorescent microspheres. labeled neurons were characterized electrophysiologically and sorted for expression analysis. the resulting expression profiles revealed highly diverse expression of molecules involved in cell-cell signaling and cell-cell adhesion, as well as transcription factors. based on this diversity of expression we constructed a taxonomic tree using an unsupervised clustering algorithm, that correctly reflects known relationships between cortical cell types. research funds: r ey , mcknight neuroscience of brain disorders award sy - - - axon guidance mediated by localized ca + signals in the growth cone hiroyuki kamiguchi laboratory for neuronal growth mechanisms, riken brain science institute, wako, japan axonal growth cones migrate along the correct paths, not only directed by guidance cues but also contacted by local environment via cell adhesion molecules (cams). many guidance cues attract or repel the growth cone via asymmetric ca + signals. its turning direction depends on the occurrence of ca + -induced ca + release (cicr) through the ryanodine receptor type (ryr ). the activity of ryr is controlled by cams via camp and pka. in this way, axon-guiding and cam-derived signals are integrated by ryr , that serves as a key regulator of axon guidance. attractive ca + signals facilitate intracellular membrane transport to the leading front and subsequent vamp -mediated exocytosis on the side with elevated ca + . in contrast, repulsive ca + signals do not trigger such membrane dynamics. growth cone attraction, but not repulsion, is prevented by inhibition of vamp -mediated exocytosis. the results indicate that growth cone attraction is driven by asymmetric membrane dynamics and that growth cone repulsion is driven by different mechanisms, not simply by changing the left/right polarity of the same molecular machinery. sy - - - molecular mechanisms for signaling through plexin-a hitoshi kikutani, atsushi kumanogoh, toshihiko toyofuku research institute for microbial diseases, osaka university, suita, japan sema a acts as a guidance cue for axons through a receptor complex comprising neuropilin- as the ligand-binding subunit and plexin-a as the signal-transducing subunit. the ferm domain-containing gef, farp , associates directly with plexin-a . sema a induces the dissociation of farp from plexin-a , resulting in activation of farp 's rac gef activity, rnd recruitment to plexin-a and down regulation of r-ras. simultaneously, the ferm domain of farp sequesters pipki␥ from talin, thereby inhibiting its kinase activity. these activities are necessary for sema a-mediated repulsion of outgrowing axons. plexin-a also functions as a ligand binding receptor of a transmembrane semaphorin, sema d and contributes to cardiac morphogenesis. sema d exerts a migration-inhibitory activity on cells from the ventricle and a migration-promoting activity on those from the conotruncal segment. plexin-a forms a receptor complex with off-track in the ventricle or with vegf receptor type in the conotruncal segment, which are responsible for the effects of sema d on the respective regions. research funds: crest sy - - - axonal transport elicited by axon guidance molecules yoshio goshima department of molecular pharmacology & neurobiology, yokohama city university graduate school of medicine, yokohama, japan for the wiring of individual neurons together into an orderly network, control by axon guidance molecules of navigation to their targets is a critical event, and molecular components destined for specific subcellular domains of neuron must be targeted correctly. we previously reported that semaphorins a (sema a), a repulsive axon guidance cue, increases the rate of bi-directional axonal transport in dorsal root ganglia (drg) . to address if the individual molecules rides on the sema a-facilitated cargo, we used time-lapse imaging of several egfp-fusion proteins expressed in drg. sema a stimulated the transport of neuropilin- ::egfp, plexin-a ::egfp, and fyn::egfp, which are the components of sema a signaling, but not neuropilin- ::egfp. interestingly, sema a accelerated the anterograde transport of trka::egfp. these facts suggest that sema a selectively facilitates the transport of its own signaling components and that sema a may modulate ngf-sensitivity of neurites by accelerating the transport of trka. kozo kaibuchi department of cell pharmacology, nagoya university graduate school of medicine, japan neurons are one of the most highly polarized cells, comprised of two structurally and functionally distinct parts, axon and dendrites. however, it remains largely unknown how neuronal polarity is established. we previously showed that collapsin response mediator protein- (crmp- ) is enriched in growing axon, and play a crucial role in axon specification. crmp- interacts with tubulin dimers to promote microtubule-assembly, and binds to sra- , an effector of rac to regulate wave-dependent reorganization of actin filaments. crmp- links kinesin- to tubulin dimmers and sra- , and participates in the kinesin- -dependent transport of tubulin dimmers and the sra- /wave complex to growing axons. we also found that the par- /par- complex and the ras/pi -kinase/akt/gsk- b pathway are involved in neuronal polarization. akt appears to phosphorylate gsk- b and inactivates its kinase activity downstream of ras/pi -kinase, thereby increasing non-phosphorylated active crmp- which promotes axon growth. this time, i summarize and discuss functions of these polarity molecules in regulation of neuronal polarity. research funds: grant-in-aid for creative scientific research sy - - - regulation of actin dynamics during neurite initiation and axon guidance frank gertler, adam kwiatkowski, doug rubinson, erik dent, leslie mebane mit, usa nervous system development requires extensive cell migration and elaboration of neurites that become axons and dendrites. axons are guided to their targets by motile growth cones. both whole cell and growth cone migration involve dynamic remodeling of the actin and microtubule cytoskeleton in response to environmental cues. the ena/vasp protein family regulates cell migration and axon guidance. ena/vasp proteins modulate actin remodeling and promote the formation of long, sparsely branched actin networks, such as those found in filopodia. results of recent work on phenotypes arising in mice lacking all three ena/vasp proteins (mena, vasp, evl) will be presented. such animals exhibit a "cobblestone cortex" in which groups of neurons migrate beyond the pial membrane. the mutants also contain little if any cortical axonal fiber tracts. analysis of primary cells from the mutants indicates ena/vasp function is required for neurite initiation. mutant neurons express differentiation markers but form few, if any filopodia and exhibit alterations in actin-microtubule interactions. kimitaka anami department of psychiatry, national center hospital for mental, nervous and muscular disorders, ncnp, tokyo, japan recent years, studies using eeg and fmri in simultaneous manner has become flourished. this is because the feasibility that any eeg events is, in principle, able to be mapped on the mri tomographic view has attracted many researchers. applications of this methodology are to basic eeg researches including event-related potentials and background activities, and as clinical aspect, localization of epileptic foci. and applications of this methodology is not matured yet but still developing. in this presentation, we will introduce our study using this method to epilepsy and to other eeg events. masaya misaki , , takashi abe , , , shigeyuki kan , , satoru miyauchi , national institute of information and communications technology, kobe, japan; japan society for the promotion of science, tokyo, japan; graduate school of biosphere sciences, hiroshima university, higashi-hiroshim, japan; kyushu institute of technology, kitakyushu, japan; crest, japan science and technology agency, tokyo, japan recording fmri and an eeg simultaneously is effective for studying spontaneous brain activities. we used this method to examine the relationship between an eeg rhythm and a bold signal. some studies have hypothesized that the hemodynamic response for a change in power of certain eeg frequency bands, such as alpha waves, is canonical in shape. however, the appropriate response shape for a change in the rhythmic eeg has not yet been determined. we measured the eeg and fmri simultaneously while subjects were in a resting or sleeping state. we applied nonlinear regression analysis using an artificial neural network to detect correlations between the changes in rhythmic eeg waves and fmri signals without a priori hypothesis of the response shape. research funds: crest of jst and grant-in-aid for jsps fellows norihiro sadato, hiroshi toyoda department of cerebral research, national institute for physiological sciences, okazaki, japan previous studies have demonstrated a nonlinear relationship between blood oxygenation level dependent (bold) response and stimulus parameters. however the origin of this nonlinearity still remains unclear. to investigate the origin for the nonlinearity of bold response, we underwent simultaneous measurement of fmri and near infrared spectroscopy (nirs) . temporal dynamics of the responses in oxy-, deoxy-and total hemoglobin concentrations as well as bold signal were simultaneously measured during a visual stimulation with various durations. to quantify the degree of the nonlinearity of responses, we introduced a model using an impulse response function modified with additional nonlinearity scaling. this model was applied to the nirs measures as well as bold responses. the nonlinearity of the response in oxygen extraction fraction (oef) was also estimated from nirs measures. the non-linearity of bold was almost identical to oef across the wide range of nonlinearity of the neuronal responses. and hence we conclude that the non-linearity of bold responses to the neural activity is mainly due to the nonlinear response of oef. the bold-fmri signal is ambiguous regarding the underlying neurophysiology. in our work we attempt a) to better understand the neurophysiological basis of fmri and b) to improve on the information obtained by functional brain imaging by adding additional information, e.g. obtained by electrophysiological measurements. in one series of experiments, we combined transcranial magnetic stimulation with near-infrared imaging in order to clarify how changes in deoxy-hb concentration (the inverse of bold) is related to neuronal inhibition. in another series of experiments, we combine eeg with fmri in order to identify bold correlates of neuronal background rhythms such as alpha rhythm, mu rhythm, etc. in a third series of experiments, we combine fmri with the measurement of high-frequency oscillations in eeg. the latter is an expression of evoked spike burst in the somatosensory cortex, i.e. this kind of measurements adds the information about action potentials to fmri haruhiko akiyama tokyo institute of psychiatry, japan activation of microglia is a part of host defense mechanisms in the brain. microglia remove invading microorganisms as well as cell debris that contains hazardous substances such as lysosomal proteases. brain is particularly vulnerable to the immune and inflammatory attacks and, therefore, has multiple mechanisms that regulate the immune and inflammatory responses more strictly than other organs. nevertheless, many neurodegenerative lesions are associated with activated microglia and low-grade, but sustained, inflammation. neuroinflammation is a term that refers to such conditions. in alzheimer's disease (ad), microglia play a central role for phagocytic removal of amyloid beta-protein (abeta) from the brain. the process is enhanced by complement activation. however, these cellular and humoral responses to abeta may be toxic to neurons in ad. neuroinflammation could be a double-edged sword in the brain. in patients with neurodegenerative diseases, complication of systemic inflammatory diseases, depletion of some neurotransmitters such as catecholamines, and the presence of brain lesions may adjunctly upregulate neuroinflammation, which further accelerates neuronal degeneration. makoto sawada department of brain life science, riem, nagoya university, japan microglia, macrophage-like cells in the cns, are multi-functional cells; they play an important role in removal of dead cells or their remnants by phagocytosis in the cns degeneration as well as are one of important cells in the cns cytokine network. they are thought to be originated from mesoderm, and to be similar cells to other tissue-resident macrophages. activated microglia have been shown to remove potentially deleterious debris and promote tissue repair by secreting neurotrophic factors at the neuronal injury sites, however, they can release potentially cytotoxic substances in vitro, and at least so-called fully activated form of microglia which are observed at the injury site in aids dementia is completely neurotoxic. these suggest that some factor(s) may contribute to change microglial phenotype from protective to toxic, but the detail is not clear. recently we generated hiv-derived nef protein tranduced microglia. they are found to increase both the potential to produce o -and mpo-like peroxidase activity resulting in the neurotoxicity. therefore, the target protein(s) of nef might to be involved in the control of microglial neurotoxicity. there is abundant evidence that extracellular atp have an important role in pain signaling. the focus of attention now is on the possibility that atp receptor of microglia might be involved in neuropathic pain. neuropathic pain is often a consequence of nerve injury through surgery, bone compression, diabetes or infection. this type of pain state is generally resistant to currently available treatments. we recently reported that the expression of p x receptors in the spinal cord is enhanced in spinal microglia after peripheral nerve injury, and blocking pharmacologically and suppressing molecularly p x receptors produce a reduction of the neuropathic pain behaviour ( . nature , - ) . more recently, we have reported that brain-derived neurotrophic factor (bdnf) released from microglia by the stimulation of p x causes the depolarizing shift in reversal potential of anion in li neurons of rats with nerve injury ( ( . nature , ( - . understanding the key roles of these atp receptors may lead to new strategies for the management of neuropathic pain. research funds: kakenhi ( ) sy - - - pet imaging of microglia using peripheral benzodiazepine ligands richard b. banati university of sydney, australia brain disease often results in significant changes in the functional state of microglia, the brain's resident tissue macrophages. the response is thought to be an important step in the pathophysiology of traumatic, inflammatory, neoplastic and degenerative brain disease. part of the structural and functional plasticity of microglia is the de novo expression of the kda transposor protein or "peripheral benzodiazepine binding site" (pbbs). the pbbs is bound by the isoquinoline pk , which labeled with carbon- can be used for positron emission tomography (pet). using c-(r)-pk pet in inflammatory and neurodegenerative brain disease as well as receptor autoradiography, we have shown that distributed regional pbbs up-regulation correlates with clinical deficit and mirrors the histologically described activation of microglia in the penumbra of focal lesions, but importantly also in the distant, anterograde and retrograde projection areas of the lesioned neural pathway. sy - - - application of simulation of light propagation in tissue to nirs imaging of brain function eiji okada department of electronics and electrical engineering, keio university, japan in nirs imaging, the functional image is obtained from the variation in intensity of detected light caused by concentration change in haemoglobin in cortical tissue. the serious problem of nirs imaging is ambiguity in light propagation in the head caused by the scattering of tissue. this poses results in poor spatial resolution and contrast in the image. the development of simulation model to calculate light propagation in the head to deduce the path length in the brain and the spatial sensitivity profile is very important to improve the nirs imaging. in this study, simulation of light propagation in the head model for nirs imaging is briefly reviewed. the heterogeneity of the tissues in the head, especially low-scattering cerebrospinal fluid (csf), has a strong effect on the light propagation in the brain. the sensitivity to concentration change in haemoglobin in the cortical tissue is improved by the effect of the csf. the simulation of nirs imaging indicates that the intensity and size of activated region in the functional image depend on the relative position of activated region to fibre pairs. yoko hoshi integrated neuroscience research team, tokyo institute of psychiatry, tokyo, japan quantification of near-infrared spectroscopy (nirs) data has been a central issue in the nirs field. over the past years, many approaches to quantification have been tried, and in the case that hb concentration changes are global within the tissue, the quantitative accuracy of time-resolved spectroscopy (trs) and phase-resolved spectroscopy (prs) has been established. when the changes are localized, however, as with functional brain activation, the difficulty of quantification has not yet been fully overcome because elimination of the effects of hemodynamic changes in the extracerebral tissue is also challenging. the temporal profile of detected light intensity measured by trs carries information about depth-dependent attenuation, because light that penetrated into deeper positions in the head is detected later. thus, several time-domain methods to determine absorption changes with depth resolution have been proposed. diffuse optical tomography (dot) is also a potential technique for quantitative detection of focal changes in cerebral hemodynamics. in this symposium, i will outline these approaches. sy - - - brain functional imaging in cerebral ischemic disorders: comparison of nirs and fmri kaoru sakatani department of neurological surgery, nihon university school of medicine, tokyo, japan we compared the evoked cerebral blood oxygenation (cbo) responses measured by nirs and activation maps of bold-fmri in stroke patients with mild and severe (misery perfusion) cerebral ischemia (ci). in the age-matched controls, deoxyhemoglobin concentrations decreased with increases in oxyhemoglobin and total hemoglobin in the primary sensorimotor cortex (psmc) during contralateral motor tasks. the psmc on the non-lesion side exhibits normal cbo response pattern. on the lesion side, the mild ci did not affect the cbo response pattern, but the severe ci caused an increase of deoxyhemoglobin during the task associated with increases of oxyhemoglobin and total hemoglobin. the mild ci caused only slight reduction of the activation volumes of bold imaging on the lesion side; however, the severe ci, caused markedly reduction of the activation volumes on the lesion side. misery perfusion caused marked reductions of activation volumes of bold imaging associated with an increase of deoxyhemoglobin during activation. bold-fmri should be performed, giving consideration to the baseline circulatory conditions. masato fukuda, toru uehara, masahiko mikuni department of psychiatry and human behavior, gunma university graduate school of medicine, gunma, japan near-infrared spectroscopy (nirs) has been increasingly employed in psychiatry researches such as personality ( . neuropsychobiology , ), aging ( . neuroimage , , and psychiatric disorders ("progress in schizophrenia research", nova science publishers, ) . frontal lobe reactivity was investigated using multichannel nirs machines in unipolar depression, bipolar depression, and schizophrenia ( . biol. psychiatry , ; . neuroimage , ) by monitoring changes of oxy-hemoglobin concentration ([oxy-hb]) every . s during a verbal fluency task. the unipolar depression was characterized by smaller [oxy-hb] increase, the bipolar depression by comparable but delayed [oxy-hb] increase, and the schizophrenia by reduced [oxy-hb] increase during the task period followed by [oxy-hb] re-increase during the post-task period, suggesting reduced, preserved but delayed, and inefficient frontal lobe reactivity, respectively. collaborators: itsuro ida, akihiko oshima, makoto ito, tomohiro suto, masaki kameyama, yutaka yamagishi, toshimasa sato, masashi suda sy - - - clinical application of nirs to neurorehabilitation optical imaging using near-infrared spectroscopy (nirs) is suitable for assessing cortical activation during human gait because of its flexibility and portability. in healthy subjects, walking induced increase of oxygenated hemoglobin levels that centered in the medial sensorimotor cortex and supplementary motor area. in hemiparetic patients with stroke, cortical activation was characterized by asymmetrical activation in the sensorimotor cortex and recruitment of the premotor and prefrontal cortex. a longitudinal study revealed that locomotor recovery was associated with improvement of asymmetrical activation in the sensorimotor cortex as well as enhanced activation in the premotor cortex. sensorimotor stimulation by facilitation technique induced enhanced activation in the motor related areas, particularly in the premotor cortex. partial body weight support and speed-dependent exercise decreased sensorimotor activation, suggesting relative shift of locomotor control to the hierarchically lower structures including the spinal cord. thus nirs may contribute to establishing brain-based strategies for neurorehabilitation. research funds: funds for comprehensive research of aging and health sy - - - measurement of language related brain activities during recovery from aphasia eiju watanabe , yumiko muroi , chizuru nakajima department of neurosurgery, jichi medical university, tochigi, japan; department of neurosurgery, tokyo metropolitan police hospital, tokyo mechanism which supports the recovery of language after aphasia is not well understood. it has long been discussed that language related areas including the regions surrounding the language area and compatible cortex on non-dominant side could be candidates which support the recovery. several studies suggest the compensation by these areas using fmri and pet. we used optical topography (ot) to know the participation of these areas during the recovery from the aphasia. we measured aphasics who showed recovery from the aphasia after apoplexy. word generation task was used. in seven cases ot was measured more that twice. seven cases showed the activity on the non-dominant frontal lobe. they all showed activities on the dominant frontal lobe in the follow-up measurements along with the deactivation of non-dominant side. these findings showed dynamic participation of non-dominant frontal lobe during the recovery phase suggesting that the rehabilitation protocol should be changed according to the area activated in each phase. tamami fukushi research institute of science and technology for society (ristex), japan science and technology agency (jst), tokyo, japan recent development of neuroscience has provided remarkable scientific discoveries, as well many newer philosophical, ethical, legal and social issues. for example, the consequences of the progress of non-invasive neuroimaging technologies, such as functional magnetic resonance imaging (fmri) and near infrared spectroscopy (nirs) show the possibility to read the mind of others, which may lead the criminal and commercial applications. brain-machine interface (bmi) technology and pharmacological manipulation of the human brain can cause the unpredictable enhancement of human ability. in advance to the expansion of "applied neuroscience", neuroscientists should consider "what the ethical problem is in the current neuroscience" and "how we learn and interact with the ethics". in this symposium, the panels will talk about the history of neuroethics then provide the ethical aspects of basic research. we will also discuss the future perspective of the neuroethics in japan and world in terms of sharing the problems across neuroscientists, ethicists, mass media, and public. judy illes stanford university, usa akin to genetic testing in the s, the translation of neuroimaging capabilities from the laboratory to the clinical setting has raised ethical questions about how new diagnostic and predictive information will be managed in the absence of effective treatments for certain diseases, about the timing of technology transfer and handling of technology that falls in the regulatory gray zone between research and clinical use, and what impact increasing opportunities for selfreferral to health care will have on patient-physician relationships, medicine, and society overall. potential off-label uses of advanced neuroimaging outside the health care setting -in law, education, employment and even for national security -are already being tested and debated. we will discuss how these issues converge in st century neuroethics, the presence of neuroethics in the international domain, and the critical role of ethics in neuroscience in the future. sy - - - neuroethics from primate's eyes naotaka fujii bsi, laboratory for symbolic cognitive development, japan neurophysiologists working on monkeys have been trying to understanding how their brains are working. the aim of the studies was not merely revealing functions of primate's brain but also trying to extrapolate the findings in primates onto human brain functions. this was true but not really true due to technical limitations which prevented us from expanding the findings in primates directly to the human brain function. however, recent advancement of technology has changed the world. findings in primates can be directly applied to human studies with or without researcher's intention. technologies invented in primate physiology are now readily transferred into human without much discussion. brain machine interface is one example. now, monkey's brains are forcing us to think about social impact of our research from ethical view, which we have not discussed before. as an experienced primate neurophysiologist but with little ethical training, i will discuss 'what is ethically correct primate research' and 'how our scientific contribution has to be made' from very practical and ground level of neuroethics. sy - - - neuroethics of nurturing the brain takao k. hensch critical period mechanisms group, riken brain science institute, japan at no time in life is the brain so easily shaped by experience than in infancy and in early childhood. it is during these critical periods that neural circuits acquire language and other basic brain functions. unraveling mechanisms that limit such dramatic plasticity to early life would pave the way for novel paradigms or therapeutic agents for rehabilitation, recovery from injury or improved learning in adulthood. conversely, a deeper insight into early postnatal brain development will provide valuable inspiration for effective brain-based education methods for our children-perhaps the greatest potential contribution of neuroscience to society. this raises urgent and important ethical questions for our consideration: is there an "ideal" brain we should be nurturing? to what extent can/should drugs be used not merely to correct developmental disorders but also to enhance performance? how do we determine what is good or bad for the maturing brain? research funds: riken bsi sy - - - neuroethics beyond laboratories and across cultures daofen chen national institute of neurological disorders and stroke, usa recent progress in systems and cognitive neuroscience poses new ethical challenges to both investigators and to the funding agencies that support scientific investigations. potential uses of many of these recent advances go beyond their intended medical applications. a growing array of neurotechnologies capable of monitoring or even intervening in human cognition makes it imperative to consider the social, ethical, and legal implications of these scientific advances. while it once might had been possible to conduct research with naive ignorance of its societal implications, this is no longer the case. moreover, we need to be cognizant that modern brain science is profoundly influenced by the distinct cultural and social values held by different sectors of the world population. we will discuss what can be done from the perspective of funding agencies to facilitate intercultural dialogue, foster mutual understanding, and develop a framework and strategies to address emerging neuroethical issues and prioritize our future efforts in neuroscience research. sy - - - bridging neuroscience and public: neuroethics in cultural contexts osamu sakura , interfaculty initiative in information studies, university of tokyo, tokyo, japan; research institute of science and technology for society (ristex), jst, japan to bridge between neurosciences and public society-it should be one of the important aims of neuroethics. for this purpose we need to draw the outline of neuroscience within the cultural context. the method and the result of natural sciences are universal, of course, but its social consequences are highly variable among cultures. although the systematic comparative researches are open to the future, we should discuss how the neurosciences could create the healthy relationship between the public society, especially focusing on the method for public participation and on the previous successful cases. mitsuru kawamura , , akira midorikawa , yoshiki kaneoke , shinichi koyama , masato taira , argye hillis showa university school of medicine, japan; crest, jst, saitama, japan; national institute of neuroscience, ncnp, tokyo, japan; national institute for physiological sciences, okazaki, japan; nihon university, tokyo, japan; johns hopkins university, baltimore, usa this symposium aims to provide an opportunity to talk between clinical neuropsychologists and neuroscientists. focusing on the visual system, we will discuss up-to-date studies from neuropsychological and neuroscientific viewpoints. the topics include motion perception in brain-damaged patients, neuroimaging of motion perception, surface and depth perception in brain-damaged patients, and neuroimaging of surface and depth perception, and neuropsychological and neuroimaging studies of visual attention. we will discuss consistency and inconsistency of our findings, and discuss what to do in order to produce synergy between clinical neuropsychology and neuroscience. research funds: kakenhi ( ), crest sy - - - impairment of surface perception in patients with ventral occipital damages shinichi koyama , mitsuru kawamura , akira midorikawa , yoshiki kaneoke , masato taira , argye hillis showa university, tokyo, japan; national institute of neuroscience, ncnp, tokyo, japan; national institute for physiological sciences, okazaki, japan; nihon university, tokyo, japan; johns hopkins university, baltimore, usa we examined the perception of faces and objects in two patients with ventral occipital damages, using psychophysical techniques. patient was a -year-old woman with bilateral damage in the fusiform and parahippocampal gyri. although she could recognize pictures of famous people, she frequently failed to decide the races of unfamiliar faces and occasionally failed to see the hollow-face illusion (gregory ) . in addition, her performance in object identification task became poorer when the objects were presented in inverted (negative) pictures. patient was a -year-old men with bilateral damage in the lingual and fusiform gyri. his recognition of faces and objects became poorer when they were presented in inverted pictures. based on the above results, the role of the ventral visual cortex in the perception of faces and objects will be discussed. research funds: grant-in-aid for jsps fellows sy - - - how do pictorial cues influence d information processing in the parietal association cortex? masato taira , arish, nihon university, tokyo, japan; department of applied system neuroscience, nihon university graduate school of medical science, tokyo, japan pictorial cues are one of the most influenced cues for d perception. basically, it is thought that the parietal association cortex processes d visual information by binocular disparity cues and the temporal association cortex processes that by pictorial cues in the concept of two visual information processing systems in the brain. however, recent studies have suggested that there are many crosstalk of d information between these association areas. our recent studies have shown that a group of neurons in the parietal cortex (area cip) is involved in perception of d surface orientation and used both disparity and pictorial cues. functional mri data in human also suggest that pictorial cues, such as attached and cast shadow, are processed in the parietal cortex in d perception. furthermore, human psychophysical data gives us some insights of how the pictorial cues influence d information processing in the parietal association cortex. research funds: kakenhi sy - - - case report of a patient with posterior cortial atrophy who relatively preserved perception of moving objects akira midorikawa national center of neuroscience, national center of neurology and psychiatry, tokyo, japan it has been presented that severe type of bálint syndrome behaves like a blind person; however, it also reported that there are some cases who behave like a blind person but could walk without collision. up to today several cases have been reported, but the underlying mechanism of the phenomenon has not been mentioned. in this report, i will talk about a patient with bálint syndrome due to degenerative disease known as posterior cortical atrophy (pca), who could not only walk around without collision but also play catch very well, nevertheless having blind like behavior. the crucial visual information underlying these phenomenons was assumed to be motion parallax induced by not only objects movement but also self movement. in addition, discrepancy between the patients who could walk and not walk will be discussed. research funds: crest, japan science and technology agency sy - - - neural mechanism underlying visual perception of motion as revealed by non-invasive human study yoshiki kaneoke department of integrative physiology, national institute for physiological sciences magnetoencephalography (meg) measures the neural activity representing the synchronized inputs to the millions of pyramidal neurons in the localized cerebral cortex. thus, it will show us another aspect of the neural activity related to the specific brain function that would not be revealed by the recording of single neuronal activity. our meg studies have revealed several important findings in the human visual motion detection system. first, the response properties for the apparent motions indicate the importance of the human mt/v + for the perception and the existence of the parallel processing for the motion and light blinking. second, the existence of the spatiotemporal filtering mechanism for the perception of motion speed is shown by the various motion stimuli. third, we present the evidence that the spatial integration of the speed without direction information occurs in our visual system. based on the results, scalar fields theory for the spatial integration of motion is proposed to explain various complex motion perception. research funds: kakenhi ( ) sy - - - neural correlates of visual attention argye hillis , mitsuru kawamura , akira midorikawa , yoshiki kaneoke , shinichi koyama , masato taira johns hopkins university, usa; showa university, tokyo, japan; national institute of neuroscience, ncnp, tokyo, japan; national institute for physiological sciences, okazaki, japan; nihon university, tokyo, japan in this paper i report a series of studies of unilateral spatial neglect (usn) in acute stroke, demonstrating a frequent double dissociation between stimulus-centered neglect and viewer-centered neglect, and showing that these types of neglect can be modality-specific. other data reveal that different types of usn are observed when there is hypoperfusion of temporal cortex versus parietal cortex. still other data provide evidence that severity of neglect depends on the volume of hypoperfused tissue in acute stroke, and that reperfusion results in early recovery of neglect. finally, i will review new evidence that right usn is common after left cortical infarcts or hypoperfusion in acute stroke, but the distribution of types of usn is very different from the distribution of types of usn after right hemisphere stroke. takeo kubota, takae hirasawa, kaoru nagai department of epigenetic medicine, university of yamanashi faculty of medicine, chuo, yamanashi, japan how are brains controlled molecularly? this is one of fundamental questions in neuroscience. several lines of evidences have suggested that genes are more strictly controlled in the brain than in other organs. epigenetics is one of such systems to control expression of the genes not based on dna sequence, but based on 'beyond the dna sequence' (chromatin modifications and small rnas). the failure of this system is known to result in neurodevelopmental diseases, such as an autistic rett syndrome. it has recently been demonstrated that the epigenetic system is affected by an environmental stress after birth, and that the system is associated with neural differentiation and cell fate determination and human brain diversity. these findings imply that epigenetics is an important research field to understand mechanisms of neural development and mental diseases. topics from update epigenetic researches in neuroscience will be discussed in this symposium. as one of epigenetic disorders, we introduce studies of rett syndrome (rtt) and its model mouse. rtt is a neurodevelopmental disorder, characterized by mental retardation and peculiar behavior. mutations of the mecp gene, encoding methyl-cpg binding protein , cause rtt. mecp acts as a transcriptional silencer. abnormal expression of some genes due to mecp dysfunction is thought to be the first step of rtt pathophysiology. to understand how mecp mutation makes rtt, we have two approaches that are morphological investigation of brain and discovery of mecp -downstream genes. using mecp -null mice (mecp −/y ), we revealed small number of dendritic spines and premature postsynaptic density at presymptomatic period. premature synaptogenesis may be the initial neuronal changes of rtt. we also found that mecp directly regulates expression of insulin-like growth factor binding protein (igfbp ) gene in human and mouse brains. pathological features of mecp −/y have the similarity of igfbp transgenic mice, which show reduction of early postnatal brain growth. over-expression of igfbp due to lack of mecp may lead to delayed brain maturation. growing evidence suggests that alterations in the epigenetic status such as dna methylation and histone modifications in brain are involved in the behavioral response to environmental factors and the pathogenesis of psychiatric diseases. however, in contrast to mrna profiling, there are few established methods for profiling the genomewide epigenetic status to date. we developed a method for profiling the genome-wide dna methylation pattern using tiling arrays, and focused our analysis on human brain samples derived from psychiatric patients and control subjects. in this symposium, general picture of the genes that are heavily methylated or non-methylated in human brain, and the relationship between dna methylation and mrna expression patterns will be presented. understanding what produces neuronal diversification has been a longstanding challenge for neuroscientists. the recent finding that long interspersed nucleotide elements- or l (line- ) retroelements are active in somatic neuronal progenitor cells provided an additional mechanism for neuronal diversification. together with their mutated relatives, retroelements sequences constitute % of the mammalian genome with l elements alone representing %. the fact that l can retrotranspose in a defined window of neuronal differentiation, changing the genetic information in single neurons in an arbitrary fashion, allows the brain to develop in distinctly different ways. this characteristic of variety and flexibility may contribute to the uniqueness of an individual brain. however, the extent of the impact of l on the neuronal genome is unknown. the characterization of somatic neuronal diversification will not only be relevant for the understanding of brain complexity and neuronal organization in mammals but may also shed light on the differences in cognitive abilities, personality traits and many psychiatric conditions observed in humans. sy - - - notch-induced acquisition of astrocyte differentiation potential of neural stem cells kinichi nakashima , jun kohyma , tetsuya taga , masakazu namihira grad. sch. biol. sci., naist, ikoma, japan; inst. mol. embryol. genet., kumamoto univ., kumamoto, japan neurons and astrocytes are generated from common neural stem/precursor cells, however, neurogenesis precedes astrocytogenesis during brain development. we have previously reported that gfap-positive astrocyte differentiation is dependent on the transcriptional activity of stat . a cpg dinucleotide in the stat recognition sequence within the gfap gene promoter is highly methylated at midgestation which becomes demethylated as the brain develops, enabling stat to induce gfap expression. thus, it is conceivable that the epigenetic modification such as dna methylation of cell type-specific gene promoter controls the switch from neurogenesis to astrocytogenesis in the developing telencephalon. here we report that neurons, which are generated earlier than astrocytes can potentiate neural precursors at midgenstation to differentiate into astrocyte through the activation of notch-signaling. the activated notch-signaling accelerates demethylation of stat binding element in the gfap gene promoter. sy - - - neurogenesis and stem cell supply as therapeutic approach to overcome ischemic stroke masayasu matsumoto department of clinical neuroscience and therapeutics, hiroshima university graduate school of biomedical sciences, japan in order to overcome the brain damage caused by ischemic stroke, several strategies have been so far applied. in the present symposium, i will address the following points to be considered prior to clinical application of neurogenesis and/or stem cell supply to repair the damaged brain function. ( ) which type of brain infarction will be a future target of this therapeutic approach? ( ) which phase of brain infarction (i.e., acute or chronic phase) will be selected as a future timing of therapeutic intervention? ( ) which will be the best way to be applied in the clinical settings, neurogenesis control, stem cell supply or both? the present research status and future directions will be presented and fully discussed. research funds: kakenhi sy - - - gene therapy for cerebral ischemia setsuro ibayashi, hiroaki ooboshi department of medicine and clinical science, graduate school of medical sciences, kyushu university, fukuoka, japan cerebrovascular disease is the leading cause of the disabled people in japan and western countries. gene transfer technique may be applicable to the treatment of serious types of cerebrovascular disease. cerebral blood vessels have been targeted by gene transfer with intravascular or perivascular approaches. several experimental studies have revealed potential usefulness of gene therapy for prevention of vasospasm after subarachnoid hemorrhage. as for cerebral infarction, studies using various brain ischemia models have shown effectiveness of gene transfer in reduction of infarct size and functional recovery. our recent studies of post-ischemic gene transfer have provided promising results in attenuation of ischemic damages by inhibiting apoptosis, inflammation and vascular permeability. approaches to cerebral ischemia using gene transfer for angiogenesis and neurogenesis appear to be novel and promising strategies. thus, gene therapy has a potential for the future therapy against cerebral ischemia. isao date department of neurological surgery, okayama university, okayama, japan cerebral ischemia is one of the neurological disorders that cell transplantation is expected to be applied. in this presentation, the author will summarize our recent basic research and clinical application reported in the literature. it is now possible to make several types of neurotrophic factor secreting cell line by genetic manipulation. in order to prevent immunological reaction and tumor formation, we have been using encapsulated cell grafting technique. we transplanted several types of neurotrophic factor secreting cell line into the middle cerebral artery occlusion model and could confirm the histological and behavioral efficacy. we have also been using adultderived neural stem cells as donor cells because they have merits to make autografitng possible. as donor tissue, neural protection can be expected similar to fetus-derived neural stem cells. the effect of neural protection increases when neurotrophic factor secreting genes such as gdnf were inserted into neural stem cells. cell transplantation is considered a new therapeutic approach for cerebral ischemia and clinical application is expected. we now know that ( ) motor function may recover after minor injury to the primary motor cortex, ( ) this recovery is, at least in part, associated with reorganization of cortical motor representation, ( ) the molecular mechanism for synaptic plasticity and axonal regrowth is being elucidated, and ( ) recent clinical experience revealed that the motor function in patients with spinal cord injury is improved after transplantation of her/his own olfactory mucosa. furthermore, recent neuroimaging techniques can display the cortical functions as we as the specific fiber connections in individual brain. virtually any part of the brain can be approached with the accuracy of millimeters by the current image-guided neurosurgery. those theoretical and technical backgrounds suggest we might be ready for the reconstruction of brain function. nobuyuki nukina laboratory for structural neuropathology, riken brain science institute, japan a major hallmark of the polyglutamine (pq) diseases is the formation of pq inclusions. recently, misfolding has come to be considered one of the primary factors for pq protein aggregation, although, the nature of misfolding is not yet well known. the protein misfolding induced by pq expansion was investigated with our molecular model system using mutant myoglobin which is inserted different size of pq. expanded polyglutamine stretches form intramolecular and intermolecular beta sheets and amyloid fibrils. the surface of the mutant myoglobin with expanded pq was partially unfolded and destabilized. we also investigated the early phase of fibrillization by small-angle x-ray scattering and electron microscopic studies, revealing that the expansion of pq to repeats induced the formation of quasi-aggregate in the earliest stage of the protein fibrillization. this structure could be closely involved in recruitment of various functional proteins into aggregates, leading to the cellular dysfunction that causes pq diseases. furthermore using cellular model system we also studied the aggregates interacting proteins (aips) by analyzing the purified polyglutamine inclusions and the lists of aip including chaperones, proteasome subunits, ubiquitin interacting proteins and others suggest the pathological role of aips in the disease cascades. sy - - - neuronal dysfunctions in dentatorubralpallidoluysian atrophy (drpla) shoji tsuji , toshiya sato , mitsunori yamada department of neurology, the university of tokyo, tokyo, japan; center for bioresource-based researches, japan; department of pathology, brain research institute, niigata university, niigata, japan to investigate molecular mechanisms of neurodegeneration in drpla, a polyglutamine disease caused by expansions of cag repeats of drpla gene, we have established transgenic mice harboring a single copy of the full-length human mutant drpla gene with cag repeats. the q mice exhibited neurological phenotypes similar to juvenile type of drpla characterized by ataxia, myoclonus and epilepsy. electrophysiological studies disclosed age-dependent abnormalities in the globus pallidus and cerebellum. neuropathological studies revealed progressive brain atrophy without obvious neuronal loss and an age-dependent increase in neuronal intranuclear accumulation of mutant proteins with the regional distribution vulnerable to drpla. expression profiling analyses revealed down-regulated genes including camp responsive genes. these results suggest that "neuronal dysfunction", but not the "neuronal cell death", is the essential mechanism of neurodegeneration in drpla. huntington's disease (hd) is caused by an expansion of a cag repeat encoding polyglutamine in the huntingtin protein and involves progressive motor, cognitive and psychiatric symptoms. using a transgenic mouse model of hd, we have shown that environmental factors can dramatically modify the disease process and delay the onset and progression of motor and cognitive symptoms. further, we have attempted to correlate these behavioural findings with changes in gene expression, neuronal morphology, neurogenesis, and cortical plasticity, in an attempt to elucidate cellular and molecular mediators in hd, and understand how gene-environment interactions can modulate these pathogenic pathways. our findings indicate that the modulatory effects of environmental manipulations are mediated by amelioration of specific molecular and cellular deficits, and provide experimental paradigms for the identification of novel therapeutic targets for hd and related brain disorders. sy - - - control of neural organization in the developing cerebral cortex yasuto tanabe mitsubishi kagaku institute of life sciences, tokyo, japan in the developing cerebral cortex, the generation of neurons with distinct identities and patterns of connectivity is controlled by a hierarchical series of cellular interactions that culminate in the laminar organization of distinct cortical areas. over the past three years we have begun to examine cerebral cortical development by focusing on three distinct major neuronal subtypes, namely, cajal-retzius cells, cortical projection neurons, and cortical interneurons. the analyses of these distinct neuronal subtypes allowed us to identify several candidate molecules and cellular interactions that might contribute to the laminar and areal organization of the cerebral cortex. in the first part of my talk, i would like to deal with the issue of ontogeny of cajal-retzius cells, and present the way cajal-retzius cells are generated, migrate and finally distribute in the developing cerebral cortex. then, i would like focus my talk on the issue of the way the acquisition of radial migration and axonal trajectory patterns of distinct cortical projection neurons is controlled during the development of the cerebral cortex. research funds: kakenhi , sy - - - mechanisms of the regulation of neuronal migration and corticogenesis kazunori nakajima , dept. of anat., keio univ. sch. of med., tokyo, japan; inst. of dna med., jikei univ. sch. of med., tokyo, japan mammalian cerebral cortex has a six-layered structure where the neurons are aligned depending on their birth-date. to determine whether the migration from the ventricular zone (vz) to beneath the marginal zone (mz) is essential for neuronal segregation into layers, we investigated whether migrating neurons have different cell aggregation properties in vitro depending on their birth-dates, even before they arrive beneath the mz. we analyzed vz cells and cells from the intermediate zone (imz) mainly composed of migrating cells, and found that the cells had acquired a birth-date-dependent preferential segregation mechanism in a reelin-independent manner. these findings suggest that cortical neurons acquire a birth-date-dependent segregation property (or fate) before their somas reach the mz. in silico experiments of the reaggregation culture supported that this mechanism might indeed contribute to the layer formation in the developing cerebral cortex in concert with other mechanisms such as reelin signaling. kenji shimamura division of morphogenesis, institute of molecular embryology and genetics, kumamoto university, japan neurons of the thalamus originate in restricted regions of the proliferative zone of the diencephalic compartment before settling in their final locations in the nuclei. to investigate cellular and molecular mechanisms underlying nucleus formation, we analyzed the sequence and pattern of expression of specific markers that distinguish the subsets of neuronal precursors during development of the thalamus. we found that a morphogen-like activity of sonic hedgehog (shh) precisely defines positions of neurons with distinct properties, and that some gabaergic interneurons migrate from their birth place to distant nuclei in a highly organized manner. we also provide evidence that shh produced by the zona limitans intrathalamica (zli), which abuts the prethalamus and thalamus, is likely to be a cue for this directed migration. our results suggest that local production of prespecified neurons coupled with distinct migration properties and local guidance cues such as compartment boundaries could be principle elements for the nucleus formation. layers and nuclei are important functional units in the vertebrate cns. neurons in these structures have common physiological and anatomical features. despite their importance, mechanisms for nucleogenesis are poorly understood. we focused on the lower rhombic lip (lrl)-derived precerebellar neurons, and utilized exo utero electroporation with an enhanced yellow fluorescent protein (eyfp) gene, to study the process of nucleogenesis. after the unilateral transfer of eyfp to the lrl of embryonic day . mice, eyfp-labelled neurons migrate tangentially from the lrl in two distinct streams, one toward the ventral metencephalon and the other toward the ventral myelencephalon. the former formed the pontine grey nucleus and reticulotegmental nucleus and the latter the external cuneate nucleus and lateral reticular nucleus. before forming the clusters, the labelled neurons begin to migrate toward the ventricle along the radial fibres, and aggregate as they detach from the fibres. perturbation experiments such as introduction of dominant negative constructs and sirna suggested involvement of several molecules in the migration of these neurons. the brains of fetal alcohol syndrome patients exhibit impaired neuronal migration, but little is known about the mechanisms underlying this abnormality. here we show that ca + signaling and cyclic nucleotide signaling are the central targets of alcohol action in neuronal cell migration. an acute administration of ethanol reduced the frequency of transient ca + elevations in migrating neurons and cgmp levels, and increased camp levels. experimental manipulations of these second messenger pathways, through stimulating ca + and cgmp signaling or inhibiting camp signaling, completely reversed the action of ethanol on neuronal migration in vitro as well as in vivo. each second-messenger has multiple but distinct downstream targets, including camkii, calcineurin, pp , rho gtpase, mapk and pi k. these results demonstrate that the aberrant migration of immature neurons in the fetal brain caused by maternal alcohol consumption may be corrected by controlling the activity of these second-messenger pathways. sy - - - membranes, water and diffusion denis j. le bihan shfj/cea, france among einstein papers is one which unexpectedly gave birth to a powerful method to explore the brain. molecular diffusion was explained by einstein on the basis of the thermal random translational motion of molecules. in the mid s it was shown that water diffusion in the brain could be imaged using mri. a dramatic application of diffusion mri has been brain ischemia, following the discovery that water diffusion drops immediately after the onset of an ischemic event, when brain cells undergo swelling through cytotoxic edema. also, water diffusion is anisotropic in white matter, because axon membranes limit molecular movement perpendicularly to the fibers. this feature can be exploited to map out the orientation in space of the white matter tracks and image brain connections. more recently, it was discovered that diffusion mri could detect transient swelling of activated cortical cells. this represents a significant breakthrough, allowing non invasive access to a fast and direct physiological marker of brain activation. this approach will bridge the gap between invasive optical imaging techniques and current functional neuroimaging approaches in humans, which are based on indirect and remote blood flow changes. sy - - - diffusion tensor fiber tractography using a tesla mr system yukio miki department of diagnostic imaging and nuclear medicine, kyoto university, kyoto, japan diffusion tensor imaging (dti) is an mr imaging technique that is sensitive to orientation of mobility in water molecules. dti reveals two specific characteristics: diffusion anisotropy; and directional distribution of water diffusivity. white matter shows high diffusion anisotropy, because diffusion is faster in parallel to fiber direction than in other directions. dti of the brain can be reconstructed to display d macroscopic fiber tract architecture, in a process known as fiber tractography. with recent advances in actively shielded -t magnets and parallel imaging techniques, high-field mr imaging has become practical in clinical settings. we have demonstrated that depiction of most fiber tracts was improved on -t tractography compared to . t. we have also established an integration of tractography and intraoperative subcortical motor-evoked potential, and demonstrated that diffusion tensor tractography of the corticospinal tract using -t mr was able to provide interactive information on fiber tracts, depicting the course of eloquent fiber tracts during an operation. to test whether mr tractography is reproducible and reliable, we used this technique to assess acute tiny infarcts located in the supratentorial brain. we analyzed the data of patients who presented to our institute with sensorimotor symptoms. there was an excellent correlation between the location of the infarct as assessed by tractography and clinical symptoms. next, we applied the technique to patients with evolving symptoms after admission to hospital. we specifically assessed the change in the tract-infarct relationship over time. the data showed that, in most cases when there was symptomatic progression, the distance between the tract and the infarct border depicted on dwi diminished. finally, we studied whether the use of tractography could help predict a patient's prognosis. to simplify the analysis, we specifically focused on patients with lenticulostriate artery (lsa) infarcts. we analyzed the correlation between the extent of cst involvement within the infarcts and the severity of motor deficits. the data indicated that the tractographic technique could be useful to predict a patient's outcome. sy - - - anatomical and functional tractography: a combined approach with diffusion tractography and corticocortical evoked potential riki matsumoto department of neurology, kyoto university graduate school of medicine, japan recent advances in diffusion-weighted imaging have raised the possibility of in vivo investigations of brain circuitry in humans. the probabilistic tractography provides estimates of the likelihood of a pathway between two brain regions without tensor estimation and thus could trace the fiber pathways beyond regions of low diffusion anisotrophy into the grey matter. however, the results depend merely on anisotropic movement of water molecules and need validation. for presurgical evaluation of epilepsy patients, we developed an in vivo tracking method, cortico-cortical evoked potential, to electrically track the cortico-cortical connections by stimulating a part of the brain through epicortical electrodes and recording the cortical evoked potentials that emanate from a distant region of the cortex via projections. combined with preoperative diffusion analysis, this invasive evaluation provides a unique opportunity to study the cortico-cortical connectivity both functionally and anatomically. results of the combined approach will be presented. parkinson disease (pd) is the second commonest neurodegenerative disorder after alzheimer disease characterized by tremor, rigidity, bradykinesia, and postural instability. pathologically, the most outstanding change is the neurodegeneration of the nigral dopaminergic neurons. although familial forms of pd can be encountered up to % of the patients, the remaining cases are sporadic. it has been postulated that nigral neurodegeneration in pd is induced by the interaction of genetic risk factors and environmental factors. epidemiological studies revealed numbers of environmental factors that are positively correlated with increased risk of pd; such factors include pesticide, herbicides, rural living, well water drinking, metals such as manganese and iron, fuel oil, industrial chemicals, and hydrocarbon solvents. in addition, certain employments were reported to be associated with increased risk of pd; these include steel/alloy industry, wood/pulp plant, farming, carpentry, cleaning, orchard, mining, and welding. these studies suggest importance of environmental factors in the pathogenesis of pd. recent progress in these areas will be discussed. masami ishido national institute for environmental studies, tsukuba, japan there are getting much public concerns about children health since environmental factors such as industrial chemicals cause deficit in developing brains. it has been suggested that they may be incident of attention deficit hyperactivity disorder or autism. epidemiologic studies also suggested that parkinson's disease was found in the peoples who were exposed to pesticides in their childhood. thus, we examined the effects of industrial chemicals, called endocrine disruptors, on rat neurodevelopment. oral administration of an endocrine disruptor ( - mg/kg) into male wistar rats (from days to weeks of age) significantly caused hyperactivity at - weeks old. immunohistochemical analyses of the brain tissues at weeks of age revealed a large reduction of immunoreactivity for tyrosine hydroxylase, but not for glutamic acid decarboxylase, both of which are localized in the substantia nigra, suggesting the specific degeneration by the chemical of dopaminergic neurons. tunel-positive cells were seen in the substantia nigra. thus, environmental insults in early life may be of particular etiologic importance. sy - - - non-thermal effects of mobile phones upon the rat brain leif g. salford, b. persson, j. eberhardt, g. grafstrom, l. malmgren, a. brun dept of neurosurgery and the rausing laboratory, lund university, sweden we have shown that rf electromagnetic fields can cause significant leakage of albumin through the bbb of exposed rats as compared to non-exposed animals. one remarkable observation is that sar values around mw/kg give rise to a more pronounced albumin leakage than higher sar values -all at non-thermal levels. if the reversed situation were at hand, we feel that the risk of cellular telephones, base-stations and other rf emitting sources could be managed by reduction of their emitted energy. the sar value of around mw/kg is exists at a distance of more than one meter away from the mobile phone antenna and at a distance of about - meters from a base station. another remarkable observation in our studies is the fact that a significant (p < . ) neuronal damage is seen in rat brains days after a hour exposure to gsm at sar values , and mw/kg. we have followed up this observation in a study where animals were sacrificed and days respectively after an exposure for hours to gsm mobile phone electromagnetic fields at sar values , , , . and (controls) mw/kg. significant neuronal damage is seen after days and albumin leakage after . our findings may support the hypothesis that albumin leakage into the brain is the cause for the neuronal damage observed after and days. sy - - - the mitochondrial toxin -nitropropionic acid: an environmental toxin to study striatal degeneration in huntington disease emmanuel brouillet neuronal death laboratory, ura cea-cnrs , france huntington disease is a neurodegenerative disorder caused by a mutation in the gene encoding huntingtin. the mechanisms underlying the preferential degeneration of the striatum, the most striking neuropathological change in huntington disease, are unknown. the behavioral and anatomical similarities found between huntington disease and animal models of striatal degeneration using the environmental toxin -nitropropionic acid ( np) support the hypothesis that mitochondrial defects could play a role in huntington disease. we will discuss the mechanisms of np toxicity and show that np and mutated huntingtin have certain mechanisms of toxicity in common. in particular, we show that mutated huntingtin can alter the expression of mitochondrial complex ii, the respiratory chain enzyme specifically inhibited by np. in summary, the np story is a good example showing how the study of environmental toxins can greatly help to elucidate the complex mechanisms underlying chronic neurodegenerative disorders. we recently demonstrated that rats received intrecisternal injection of -ohda or environmental chemicals, such as bisphenol a, nonylphenol, p-octylphenol, diethylhexylphthalate or dibutylphthalate, at days of age showed behavioral hyperactivity at - weeks of age. immunohistochemical studies revealed a deficit in the development of dopamine (da) neurons. adult rats received these chemicals showed degeneration in nigro-striatal da neurons similarly to parkinson's disease. in this study, we investigated the mechanism of -ohda-induced neurotoxicity, using pc cells as an in vitro model system. we observed the generation of reactive oxygen species (ros) and p-quinone via auto-oxidation of -ohda. we also characterized the oxidation of cellular proteins by -ohda and the protective effect of antioxidants such as catalase, glutathione, and n-acetylcysteine with different manner. we will discuss about apoptotic cell death pathway including cytochrome c release and caspase activation induced by -ohda, ros and p-quinone. sy - - - environmental factors in the pathogenesis of alzheimer's disease joanna l. jankowsky california institute of technology, usa epidemiological studies indicate that environmental factors significantly influence the risk of developing alzheimer's dementia. foremost among those factors are education, occupation, and leisure activities. although not universal, most studies have found that individuals with greater education, more challenging occupation, or active leisure hobbies show relative protection against dementia. animal models for alzheimer's disease have recently been used to explore the mechanism of this effect. transgenic mice designed to recapitulate alzheimer's amyloid pathology are protected from functional decline by enriched housing designed to provide cognitive stimulation. both enriched housing and exercise modify the level of amyloid-beta in the brains of transgenic mice, demonstrating that environmental factors can significantly influence brain biochemistry. intriguingly, traditional environmental enrichment can improve cognitive behavior while paradoxically elevating amyloid-beta levels in transgenic mice, suggesting that environmental stimulation may alter amyloid metabolism and cognitive function by competing mechanisms. the efficacy of synaptic inhibition depends on the number of gaba a receptors expressed on the neuron surfaces. in the present study, we have elucidated the role of prip (plc-related inactive protein) in trafficking of the receptors by analyzing prip knockout (ko) mice; the sensitivity to diazepam was reduced as assessed by biochemical, electrophysiological and behavioral analyses of ko mice, suggesting the dysfunction of the ␥ subunit-containing receptors. we then examined the mechanisms by which prip molecule regulates cellsurface expression of ␥ subunit-containing receptors. disruption of the direct interaction between prip and the ␤ subunit of receptors by prip-binding peptide inhibited cell-surface expression of ␥ subunit-containing receptors in gh and hek cells. constitutive internalization of the receptors was also modified by the peptide. collectively, prip molecules are involved in trafficking of ␥ subunit containing gaba a receptors to/from cell-surface membrane. research funds: kakenhi ( ) sy - - - involvement of bdnf in the induction of ltp at visual cortical inhibitory synapses yukio komatsu dept. visual neurosci., res. inst. environ. med., nagoya univ., nagoya, japan high-frequency stimulation (hfs) induces long-term potentiation (ltp) at inhibitory synapses of layer pyramidal cells in rat visual cortex. this ltp requires a postsynaptic ca + rise for induction and spike firing of presynaptic cells for maintenance, although the necessary frequency is low, suggesting that ltp is expressed presynaptically and some information must be sent backwards from the post-to presynaptic cells during induction. in this study, we investigated whether bdnf could act as such retrograde messengers. ltp did not occur when hfs was applied in the presence of k a at nm, inhibiting trk receptor tyrosine kinases selectively at that dose. hfs induced ltp when k a application was started soon after hfs or when k a was loaded into postsynaptic cells. ltp did not occur in the presence of trkb-igg or anti-bdnf antibodies. in cells loaded with bapta, the addition of bdnf to the medium enabled hfs to induce ltp without affecting basal synaptic transmission. these results suggest that bdnf released from postsynaptic cells activates presynaptic trkb, enabling the induction of ltp. research funds: kakenhi ( ) sy - - - autocrine mglur activation in cerebellar purkinje cells regulates gaba-mediated synaptic inhibition trevor smart, ian c. duguid university college london, uk in the cerebellum, retrograde signalling is important for the induction of short-and long-term changes to synaptic inhibition at interneuron-purkinje cell (in-pc) synapses. endocannabinoids, via cb receptors, mediate a short-term decrease in synaptic efficacy, while glutamate, via presynaptic nmda receptors, induces a sustained increase in gaba release. we now report that dendritically released glutamate also acts as an autocrine messenger, activating mglur on pcs to enhance synaptic inhibition via the release of endocannabinoids. this process was triggered by repetitive pc stimulation and blocked by uncoupling the mglur -gq/ transduction pathway as well as being initiated by direct mglur activation during pc depolarisation. glutamate uptake by excitatory amino acid transporters controlled the extent of autocrine mglur activation, whilst basal glutamate levels were unable to enhance endocannabinoid release. our study suggests that autocrine mglur activation provides a powerful homeostatic mechanism to dynamically regulate inhibitory synaptic transmission. sy - - - regulatory mechanism of inhibitory synaptic transmission in the cerebellum shin-ya kawaguchi , , tomoo hirano , dept. biophys., grad. sch. sci., kyoto univ., kyoto, japan; crest, jst, kawaguchi, japan at the gabaergic synapses between inhibitory interneurons and a purkinje neuron in the cerebellum, postsynaptic depolarization induces long-term potentiation of transmission efficacy mediated by gaba a receptors (rebound potentiation: rp). the signaling cascades regulating the induction of rp has been clarified. the balance of activities of protein kinases and phosphatases determines whether rp is induced or not. here we show another molecular mechanism involved in the rp induction. using both electrophysiological experiments and computational kinetic simulation of biochemical reactions, we demonstrate how the long-term potentiation of gaba a receptormediated responses is brought about. rp induction was impaired by inhibition of ca + -activated protease calpain or by disturbance of association of gaba a receptor ␥ subunit with gabarap (gaba a receptor associated protein). binding of gabarap to microtubule was also involved in the regulation of rp. our results suggest that structural alteration of gabarap caused by calpain activity is critical for establishment of rp. sy - - - contribution of hebb's "organization of behavior" to the development of brain science masataka watanabe tokyo metropolitan institute for neuroscience, tokyo metropolitan organization for medical research, tokyo, japan more than years have passed since the publication of "organization of behavior". this book has been one of the most influential books in neuroscience. around the time of the th anniversary of this book, special issues and articles concerned with this book appeared in several journals. his idea, which is a general framework for relating behavior to synaptic organization through the dynamics of neural networks, has stimulated variety of neuroscience researches in relation to, for example, environmental effects on development, naturenurture interaction, memory consolidation and sensory deprivation. however, he also made some mistakes, for example he advocated frontal lobotomy. in this symposium, i will briefly review how influential this book has been on basic and practical neurosciences, and will re-consider the importance and limitation of studying mental processes, such as emotion, memory and thought by exploring brain mechanisms, in reference to the idea of cell assembly, phase sequence and hebb synapse. sy - - - detection of cell assembly in neuroscience experiments and brain-machine interfaces yoshio sakurai , , susumu takahashi department of psychology, kyoto university, kyoto, japan; crest, japan science & technology agency, japan the reality of cell-assembly coding in the working brain depends on how we could detect specific properties of cell assembly from multi-neuronal activities in behaving animals. first in the present paper, we show experimental results indicating some of the properties, i.e., functional overlapping of individual neurons and connection dynamics among multiple neurons, that depend on tasks and events being processed and on the distance among the neurons. second, we demonstrate a newly developed method, brain-machine interface (bmi), to test the reality of cell assembly as neural information and the plastic formation of cell assemblies during learning processes. we introduce our recent bmi system with independent component analysis (ica) and specific multi-electrodes and show some neuronal and behavioral data obtained by the bmi system. hebb postulated that coincident activities of pre-and postsynaptic neurons trigger input-specific plasticity. how relevant is it in protein synthesis-dependent late-phase plasticity (lp)? synaptic tagging hypothesis explains how new proteins reach the activated synapses to establish input-specific lp. using live-imaging techniques, we measured entry of vesl- s-egfp into dendritic spines (ve trapping) of rat hippocampal neurons in culture, and found that ve trapping activity serves as synaptic tag in many criteria. ve trapping conforms to the hebb's rule in a sense that it required both presynaptic activity and postsynaptic no-pkg pathway, but their coincident time window was far wider (∼h) than that of early-phase plasticity, suggesting an involvement of persistently synchronized rather than transiently coincident activity. no spreading from the activated synapses may persistently prime the postsynaptic tag components at the surrounding synapses, during which brief inputs to these synapses will establish associative and heterosynaptic tags. thus, tagging one synapse would lead surrounding synapses to multiple metaplastic states. tomoki fukai laboratory for neural circuit theory, riken brain science institute, saitama, japan in the cell-assembly hypothesis, cortical neurons are considered to form functional subnetworks depending on a particular demand of information processing. such cell assemblies may be organized through synchronous firing of the constituent neurons and synapses modifiable by hebbian learning. in this talk, i will overview recent in vivo and in vitro experimental findings that provide new evidence for synchronous or precisely timed neuronal activity. i propose a hardwired structure of local cortical networks, "entangled synfire chains", on the basis of the experimental observations of cortical activity. in this model, multiple cell assemblies can be defined by the pattern of neuronal wiring. however, the same experimental findings can lead us to a different type of cortical network models. in this type of models, cortical networks may self-organize to develop a critical dynamical state, which may be useful for realizing a hypothetical "liquid-state machine". i will discuss the characteristic properties of both types of models and the possible implications in cortical computations. research funds: kakenhi ( ) sy - - - impact of hebbian hypothesis on neuroscience keisuke toyama shimadzu institute of basic technology, seikacho, hikaridai, kyoto - , japan hebb has seeded two major concepts in the modern neuroscience, i.e., cell assembly hypothesis for perception, and hebbian synapses to construct that cell assembly. the former concept stimulated extensive searches for the response selectivity extending from the primary visual cortex to the inferotemporal cortex and even to the hippocampal cortex, while the later concept triggered neuroscience studies of the learning and memory in the developing and adult brains. recently, these concepts refreshed the impact with new dressing of 'dynamics'. cell assembly that was originally assumed to be static, became dynamic and opened a new possibility for the neural computation, combined with dynamic hebbian synapses conceptualized as the spike-timing dependent plasticity (stdp). i would like to discuss about speaker's talks in this context. sy - - - tonic gaba a receptor mediated conductances: properties, functions and plasticity alexey semyanov riken brain science institute (bsi), japan communications mediated by non-synaptic receptors are important for information processing in the brain. high affinity extrasynaptic gaba a receptors mediate a persistent "tonic conductance" which reflects their activation by ambient concentrations of gaba. this phenomenon is found in different brain regions, shows cell-type specific differences in magnitude and pharmacology, and changes during brain development. our findings have revealed functional significance of gaba a receptors mediated tonic conductance in the hippocampus. we have shown that it modulates rate-coded information processing by individual neurons, and acts in a cell-type specific manner to regulate the excitability of the local neuronal circuit. the magnitude of the conductance is regulated by efficiency of gaba uptake and membrane potential. gaba a receptor mediated tonic conductance undergoes adaptive plasticity. it is up-regulated in hippocampal pyramidal cells in a model of pilocarpine status epileptics in rats. in mice lacking gad the amount of the tonic inhibition is reduced in ca hippocampal interneurons, while unchanged in pyramidal cells. sy - - - modulatory effects of peri-interneuronal glial cells on neuronal activities in hippocampal ca region yoshihiko yamazaki , yasukazu hozumi , kenya kaneko , satoshi fujii , hiroshi kato dept. of neurophysiol., yamagata univ. sch. of med., yamagata, japan; dept. of anat. & cell biol., yamagata univ. sch. of med., yamagata, japan glial cells, in addition to their supportive roles in the nervous system, make up a functional unit with neurons and have been suggested to play novel roles in neuronal activities. we focused on interneuron/peri-interneuronal glial cell (pg) pairs in the hippocampal ca region and performed dual whole-cell recordings to investigate the modulatory effect of glial cells on neuronal activities. direct depolarization of pg suppressed the excitatory postsynaptic currents in an adjacent interneuron. this suppression was inhibited by adenosine a receptor antagonist. moreover, pg activation modulated the firing pattern of the interneuron. since interneurons in the hippocampus are mainly inhibitory and the terminals of a single interneuron make a large number of synapses on a group of pyramidal cells, direct inhibitory regulation via pg would have marked effects on the information processing of neurons in the ca region. research funds: kakenhi ( ) sy - - - carbachol-induced beta oscillations in rat hippocampal slices kiyohisa natsume, jun arai graduate school of life science and systems engineering, kyushu institute of technology, fukuoka, japan rat hippocampus has the cholinergic input from medial septum and diagonal band in vivo. the input involves the generation of hippocampal rhythm, theta, gamma rhythm. to mimic the system, we applied carbachol, a cholinergic agent, to rat hippocampal slices. carbachol can induce beta oscillation as well while carbachol can induce theta, and gamma oscillations in the slices. in the present paper, we introduce the beta oscillations. the application of m carbachol induces beta oscillations which occur intermittently with the interval of - s. during the intervals, gamma oscillations are induced. the mean frequency of the oscillations is . ± . hz (mean ± s.e.m.). the oscillations are induced via muscarinic m , , receptors. the frequencies of them are significantly decreased by the application of bicuculline, a gaba a antagonist. they are sensitive to bicuculline, while theta oscillations are not. it is indicated that the character of beta oscillations are different from those of theta oscillations. the neocortex and the hippocampus are connected by way of the entorhinal cortex and the subiculum. to examine ongoing network interactions among these distinct cortices during neocortical slow oscillations ( - hz), we recorded intracellular potentials in single neocortical, entorhinal, subicular, and hippocampal neurons, together with hippocampal field and multi-unit activities in adult anesthetized rats. we have found that ( ) most entorhinal and subicular neurons displayed bimodal active (up) and quiet (down) states of membrane potential, in synchrony with neocortical slow oscillations, ( ) no bimodal up-down transition was present in hippocampal neurons. hippocampal granule cells were directly driven by entorhinal up-state activity, while ca and ca neurons discharged during both up and down states, ( ) gamma and fast (ripple) oscillations were observed in hippocampal ca area irrespective of up-down transition. these observations suggest that entorhinal and subicular regions are "neocortex-like" and hippocampal networks can generate self-organized activity independent of neocortical slow oscillations. the cholinergic neurons in the mesopontine reticular formation (mprf) seem to control sleep-wake cycle and hippocampal activity, because stimulation of the mprf elicits rem sleep and hippocampal theta wave. in this study, we recorded neuronal activity in the mprf and pontine and hippocampal eeg during rem sleep and investigated time-relationship between them. our results are summarized as follows: ( ) most of the mprf neurons were active during rem sleep; ( ) the mprf activity increased over ten seconds before transition from nrem to rem sleep, i.e. from non-theta to theta period; ( ) the theta wave was instantaneously accelerated concomitant with activation of the mprf neurons. these results suggest that cholinergic neuron in the mprf is important in generation and maintenance of rem sleep and theta wave. because hippocampal theta waves are involved in memory consolidation during rem sleep, our findings might help to clarify this mechanism. research funds: kakenhi ( ) sy - - - clock mechanisms of the scn involving in the entrainment to the morning and evening light sato honma, natsuko inagaki, nobuko tokumaru, ken-ichi honma dept. physiology, hokkaido univ. grad. sch. med., sapporo, japan the circadian clock, by entraining to the light-dark cycle of different day length, controls seasonality in biological functions. the mechanism is currently explained by morning and evening oscillators which change their coupling intensity depending on the day-length. by using clock gene expression as a marker of clock functions, we examined the localization and molecular bases of the two oscillators. rats and mice were housed under light-dark (ld) : h and ld : h. clock gene expression patterns in the entire scn were examined by in situ hybridization on the first day of constant darkness. the phase relations of per and per rhythms suggest that light-on resets per rhythms in both light conditions, while per rhythm also relates to the light-off. in cultured scn of transgenic mice expressing luciferase under the control of per promoter, we observed two bioluminescent peaks a day only in the anterior scn from the mice kept in ld : . the finding suggests that two distinct oscillators, which respond to the day-length, reside in the anterior scn. the suprachiasmatic nucleus (scn) is the center of the mammalian circadian clock. tissue transplantation of the scn restores the behavioral circadian rhythm in scn-lesioned mice in spite of the impaired neural connection with the host brain. we have investigated whether grafted scn regulates the circadian oscillator in peripheral organs using the scn-transplanted mice that have a limited time information transmission paths. as a result, the grafted scn restored not only circadian behavior rhythm but also the circadian rhythms of peripheral organs. many of clock genes showed dynamic oscillations with identical phase relationship as shown in intact animals, however, per and per showed low amplitude of oscillation. the findings suggest that diffusible signal molecules released from the transplanted scn entrain the circadian clock in peripheral organs and that they differentially modulate the expression of clock genes. sy - - - genome-wide analysis of adrenal-dependent and independent circadian regulation of mouse hepatic genes norio ishida clock cell biology group, national institute of advanced industrial science and technology (aist), ibaraki, japan recent progress in genome-wide expression analysis has identified hundreds of circadian regulated genes in the suprachiasmatic nucleus as well as in peripheral tissues of mammals. adrenal gland is important for circadian regulation for mammalian peripheral clocks. to identify circadian expressed genes regulated by adrenal glands pathways, we performed dna microarray analysis using hepatic rna from adrenalectomized (adx) and sham-operated mice. we identified genes that fluctuated between day and night in the livers, lost circadian rhythmicity in adx mice. these included the genes for key enzymes of liver metabolic functions such as glucokinase, hmg-coa reductase, and glucose- -phosphatase. the present study showed that the circadian expression of mouse liver genes is governed by core components of the circadian clock such as clock, and the other genes depend on adrenal glands pathway such as glucocorticoids. hitoshi okamura kobe university graduate school of medicine, japan light is a powerful synchronizer of the circadian rhythms, and bright light therapy is known to improve metabolic and hormonal status of circadian rhythm sleep disorders, although its mechanism is poorly understood. in the present study, we revealed that light induces gene expression in the adrenal gland via the suprachiasmatic nucleus (scn)-sympathetic nervous system. moreover, this gene expression accompanies the surge of plasma and brain corticosterone levels without accompanying activation of the hypothalamoadenohypophysial axis. the abolishment after scn-lesioning, and the day-night difference of light-induced adrenal gene expression and corticosterone release, clearly indicate that this phenomenon is closely linked to the circadian clock. the surge of plasma corticosterone after light exposure indicates that environmental light signals are instantly converted to glucocorticoid signals in the blood and csf. the light-induced clock-dependent secretion of glucocorticoids adjusts cellular metabolisms to the new light-on environment. sy - - - neuronal and hormonal control of peripheral clock function through suprachiasmatic nucleus shigenobu shibata, naomi hayasaka, takashi kudo, tsuyoshi yaita department of pharmacology, school of science and engineering, waseda university, tokyo, japan the clock genes are expressed not only in the suprachiasmatic nucleus (scn) of the hypothalamus where the master clock exists, but also in other brain regions and various peripheral tissues. in the liver and lung, clock genes are abundantly expressed and show clear circadian rhythm. although oscillation of clock genes in the liver and lung is controlled under the circadian clock mechanism in the scn, we do not know the resetting signals on peripheral clock function. communication between the scn and peripheral tissues occurs through various systems involving the sympathetic, nicotinic and glucocorticoide functions. this symposium mainly describes both anatomical and physiological experiments to reveal the sympathetic and glucocorticoid control over peripheral clock function. sy - - - a to z of gene transfer with adenoviral vector-application to neuronal birth date-specific gene transfer using replication-defective adenoviral vectors, we successfully performed 'pulse gene transfer' into progenitor cells in a neuronal birth date-specific manner. when adenoviral vectors were injected into the midbrain ventricle of mouse embryos between embryonic days (e) . to e . , the adenoviral vectors introduced a foreign gene into a specific cohort of birth date-related progenitor cells. this technique allows us to distinguish a cohort of birth date-related progenitor cells from other progenitor cells with different birth dates and to introduce a foreign gene into specific subsets of neurons by performing adenoviral injection at specific times. this adenovirus-meditated gene transfer technique will enable us to examine the properties of each subset of progenitor cells that share the same neuronal birth date. i will explain directions how to use an adenoviral vector and application of an adenoviral vector in my talk. research funds: crest sy - - - live imaging in the specific neuronal cells by the combination of transgenic mice and viral vectors kaori kashiwagi, naoaki saito lab. mol. pharmacol. biosig. res. ctr, kobe univ, kobe, japan live imaging analysis has revealed that each protein kinase c (pkc) subtype shows spatio-temporally distinct targeting in response to various stimuli. we demonstrated that the trans-synaptic stimulation induced translocation of ␥pkc-gfp in cerebellar slices from bitransgenic mice (nse-tta/tetop-␥pkc-gfp) which express ␥pkc-gfp in time and region-specific manner. this translocation was not restricted, but propagated from the distal to the proximal dendrites close to the soma of purkinje cells. in order to gain further insight in to the molecular mechanisms of pkc translocation, we introduced viral vectors to primary cultured purkinje cells. the propagative ␥pkc-gfp translocation was also observed in cultured purkinje cells derived from nse-tta mice. the molecular mechanisms of pkc translocation in purkinje cells were analyzed by live imaging with various kinds of viral vectors. the combination of tg mice and viral vectors is useful to understand the physiological role of pkc in the specific neuronal cells. research funds: kakenhi ( ) sy - - - visualization and manipulation of the signaling systems in the cns using sindbis viral vectors sho kakizawa dept. of pharmacol., grad. sch. of med., the university of tokyo, tokyo, japan virus vectors can efficiently deliver genes to neurons and other cells in the nervous systems in vitro and in vivo. because many viral vectors are in common use, it is important to select the best viral vector for each specific application, and a number of factors must be considered when making a decision. sindbis virus is an enveloped plus-strand rna virus belonging to the alphavirus genus of the togaviridae family. the sindbis viral vector is characterized by its effective, rapid, high-level and preferential transduction of neurons. these facts indicate that the vector is a powerful tool for the robust expression of target genes in the specific population of neurons. in this symposium, we will introduce our recent topics on the synaptic functions in the cerebellar systems revealed by visualization and manipulation of signaling molecules, such as nitric oxide and inositol , , -trisphosphate, in the cerebellar purkinje cells. research funds: grant-in-aid for scientific research on priority areas-molecular brain science-from the ministry of education, culture, sports, science and technology of japan sy - - - rescue of phenotypes of null-mutant mice by virus vector-mediated gene transfer kazuhisa kohda, wataru kakegawa, kyoichi emi, michisuke yuzaki dept. of physiol., keio univ. sch. of med., tokyo, japan even after the completion of genome project in major species, functions of many molecules remain uncharacterized. a transgene-based rescue approach is one of the powerful methods to decipher the mechanisms of actions of an orphan receptor; however, it is quite labor intensive and time consuming. here, we have developed a virus vector-based rescue approach and applied to investigate the mechanisms of action of the orphan glutamate receptor ␦ (glur␦ ) in the cerebellum. by introducing a sindbis virus carrying a wild-type glur␦ into glur␦ -null cerebellum in vivo, we could rescue abnormal phenotypes, such as impaired long-term depression at parallel fiber-purkinje cell synapses. by examining whether a mutant glur␦ lacking a specific domain could similarly rescue the phenotypes, we could evaluate functional importance of the domain. alternatively, by introducing a partial sequence of the gene of interest into wild-type brain and examining its dominant-negative effect, we will be able to identify the region of the gene product that is functionally important. research funds: kakenhi sy - - - gene transfer into in vivo cerebellar purkinje cells by hiv-derived lentiviral vectors hirokazu hirai advanced science research center, kanazawa university, ishikawa, japan cerebellar purkinje cells are key elements regulating motor coordination and motor learning. gene transfer into purkinje cells is an effective approach for the study of cerebellar function and treatment against cerebellar disorders. although adenoviral vectors or sindbis vectors are frequently used for gene delivery into neurons, the former has extremely low affinity for purkinje cells, while the latter causes substantial damage to the infected cells. to achieve effective gene transfer into purkinje cells, we used human immunodeficiency virus (hiv)-derived lentiviral vectors. purkinje cells were efficiently transduced without significant influence on the cell viability and synaptic functions. gene expression was also detected, though less efficiently, in other cortical cells, whereas no transduced cells were observed outside of the cerebellar cortex. these results suggest that hiv-derived lentiviral vectors are useful for the study of gene function in purkinje cells as well as for application as a gene therapy tool for the treatment of diseases that affect purkinje cells. research funds: jst/presto, kakenhi ( ) sy - - - physiological basis for stereotaxic surgery in basal ganglia atsushi nambu division of system neurophysiology, national institute for physiological sciences, and school of life science, the graduate university for advanced studies, okazaki, japan stereotaxic surgery in movement disorders such as parkinson's disease dramatically improves the symptoms of such diseases. i assume the physiological basis for the treatment is to disrupt abnormally increased information flow through the basal ganglia. i will discuss the pathophysiology of basal ganglia disorders and the effect of stereotaxic surgery in light of the three major pathways in the cortico-basal ganglia loop, i.e., hyperdirect (cortico-stn-gpi), direct (cortico-striato-gpi) and indirect (cortico-striato-gpe-stn-gpi) pathways, that dynamically control the activity of the thalamus and cortex to perform correct motor programs in correct timing. research funds: kakenhi ( ) sy - - - deep brain stimulation of subthalamic nucleus on parkinson's disease fusako yokochi department of neurology, tokyo metropolitan neurological hospital, tokyo, japan parkinson's disease is a progressive and degenerative disease caused by dopamine deficiency attributed to the degeneration of neurons in the substansia nigra. consequently, various symptoms appear, such as cardinal symptoms, those in the advanced stage and those as the side effects of long-term levodopa therapy. many antiparkinsonian drugs have been developed, but all of the symptoms are not improved by these drugs. stereotaxic surgery was started for treating severe tremor and rigidity in the mid- th century. stimulation by chronically implanted electrodes in the brain, that is, deep brain stimulation (dbs), has recently been applied and has been shown to have marked effects on the symptoms resisted to conventional treatments. in particular, dbs of the subthalamic nucleus (stn) is an effective treatment for the symptoms. much basic research on the function of stn has been reported, but stn function is still unclear. clinical outcomes including the side effects of stn dbs, the neural activities recorded from stn and the localization of clinical effects are reported in this paper. sy - - - firing patterns of basal ganglia neurons and effects of deep brain stimulation in parkinson's disease takao hashimoto center for neurological diseases, aizawa hospital, matsumoto, japan high-frequency electrical stimulation of the subthalamic nucleus (stn), internal segment of the globus pallidus (gpi) and thalamus can improve motor signs in patients with parkinson's disease, however, its mechanism of action remains unclear. a leading hypothesis regarding the development of movement disorders of basal ganglia origin suggests that hyperkinetic and hypokinetic disorders occur as a result of changes in the mean discharge rate in the gpi and substantia nigra, which in turn suppress thalamocortical output. on the other hand, altered firing patterns in the basal ganglia have been reported in mptp-induced parkinsonian animals: increases in bursting activity and periodic oscillatory activity in the gpi and stn, and synchronization of gpi, or gpi and striatal neurons. synchronous oscillation in the basal ganglia may break down independent processing in the motor circuit and disrupt signal processing at the cortical level. kaoru takakusaki department of physiology, asahikawa medical college, asahikawa, japan locomotion is composed of volitional and automatic processes. particular attention is required to perform volitional processes such as avoiding obstacles and accurate limb placements during locomotion. however, we are largely unaware of the automatic control processes of rhythmic limb movements, muscle tone and postural reflexes that accompany locomotion. because each process is seriously impaired in parkinsonian patients, the basal ganglia must play a crucial role in integrating the volitional and automatic processes of locomotion. the basal ganglia contribute to the planning and execution of voluntary movements via basal ganglia thalamocortical loops. on the other hand, recent our findings suggest the importance of the direct basal ganglia outflow to the brainstem where fundamental neuronal networks for controlling postural muscle tone and locomotion are located. in this presentation we discuss the role of the basal ganglia in the integration of volitional and automatic movements during locomotion, which has been less understood aspect of gait control. research funds: grant-in-aid for scientific research (c) and priority area (area no. ) sy - - - characteristics of neuronal activity within the globus pallidus interna (gpi) in patients with dystonia yoichi katayama , department of neurological surgery, nihon university school of medicine, tokyo, japan; division of applied system neuroscience, nihon university graduate school of medical science, tokyo, japan dystonia represents disordered muscular tonicity of the trunk and/or extremities, and is often dramatically controlled by chronic gpistimulation in humans, indicating that dystonia is attributable to certain abnormal activity of gpi neurons. little is yet known, however, regarding characteristics of neuronal activity within the gpi underlying dystonia. we analyzed activity of gpi-neurons in patients with dystonia or parkinson's disease, which were recorded during surgery for chronic gpi-stimulation. as compared to gpi neurons in patients with parkinson's disease, gpi neurons in patients with dystonia were distinctive with following three characteristics; firing rate ( . ± . hz, n = ) was low, firing pattern was often composed of irregular pauses and bursts, and many were responsive to body movement with wide receptive fields. these findings suggest that dystonia may be related to unstable movement-related sensory processing within the gpi. it has been considered that dystonia, which is generally characterized by postural abnormalities and involuntary movements including torsion, is caused by dysfunction of the basal ganglia. the purpose of the present work is to clarify the neural mechanisms underlying the onset of dystonia by analyzing the pathophysiology of a model for torsion dystonia, wriggle mouse sagami (wms). the genomic mutation of wms occurs in the gene encoding plasma membrane ca + -atpase isoform that is located on the th chromosome. recent immunohistochemical and electrophysiological investigations on wms have shown that ( ) d -type dopamine receptors are downregulated presynaptically in the striatum, and ( ) a large number of purkinje cells in the cerebellum express tyrosine hydroxylase (the synthesizing enzyme for dopamine) and their excitability is greatly reduced. in this symposium, the possible correlation between these data and dystonic phenotypes will be discussed. kei-ichiro maeda reproductive science, graduate school of bioagricultural sciences, nagoya university, nagoya, japan gnrh has been well established to regulate reproduction through two modes of secretion: surge and pulse. the surge mode is female-specific and induces lh surge and then ovulation through positive feedback action of estrogen. the pulse mode tonically activates gonads in both sexes with being negatively regulated by gonadal steroids. environmental cues, such as photoperiod or nutrition, are considered to affect reproductive activity through altering pulse mode of gnrh release. pulse mode seems much more robust than surge, because estrogen can induce a surge under a certain condition when the pulse is suppressed. the following four papers aim to unveil the physiological mechanism underlying two modes of gnrh secretion in various experimental models. sy - - - metastin: a neuropeptide playing a central role in the regulation of ovulatory cycle hiroko tsukamura, kei-ichiro maeda graduate school of bioagricultural sciences, nagoya university, japan estrous cyclicity is regulated by a sequence of neuroendocrine events consisting of hypothalamus-pituitary-gonadal axis. gonadotropinreleasing hormone (gnrh)/luteinizing hormone (lh) surge is induced by positive feedback action of estrogen secreted by mature ovarian follicles. the central mechanism of positive feedback action of estrogen on gnrh/lh secretion, however, is not fully understood yet. metastin was first isolated as a natural ligand for a g-proteincoupled receptor, gpr ( . nature , ) . recent studies reported that a genetic alteration leading to homozygous loss of function of gpr impairs pubertal development in mice and human. we have first demonstrated that endogenous metastin plays a physiological role in inducing ovulation through stimulating gnrh/lh surges to control estrous cyclicity in the female rat ( . endocrinology , ) . the present paper focuses on the role of metastin in regulating gnrh/lh surge based on our recent study in rats and discusses possible mechanism underlying positive feedback action of estrogen. suzanne moenter, catherine christian university of virginia, usa a surge in gonadotropin-releasing hormone (gnrh) secretion is the cns signal that triggers the luteinizing hormone (lh) surge, which causes ovulation. the gnrh surge depends on a switch in estradiol (e) feedback from negative to positive and in rodents on time of day, occurring in the pm. treating ovariectomized (ovx) mice with a constant release e capsule (ovx+e) elicits daily pm lh surges; there is no diurnal change in ovx controls. likewise, extracellular recordings of firing activity of gfp-identified gnrh neurons showed no diurnal changes in cells from ovx mice. in contrast, e increased firing in the pm compared to am. gabaergic neurons form a major input to gnrh neurons, and activation of the gabaa receptor can be excitatory in these cells. whole-cell patch-clamp recordings of synaptic activation of gabaa receptors on gnrh neurons revealed e-dependent decreases in transmission during the am (negative feedback) and increases in transmission near surge onset that persisted for some populations of afferents thru the surge peak. together these data indicate one mechanism by which e induces the gnrh surge is by altering gaba transmission to gnrh neurons. yoshitaka oka grad. sch. sci., univ. of tokyo, tokyo, japan the gonadotropin-releasing hormone (gnrh) peptidergic neuronal systems consist of hypothalamic neuroendocrine and extrahypothalamic neuromodulatory gnrh neurons. here, i introduce our recent studies on the physiological properties of neuroendocrine preoptic (poa) gnrh neurons in comparison with the neuromodulatory terminal nerve (tn) gnrh neurons. to study the both types of gnrh neurons, we use a fish model system in which we can easily identify both of them in intact brain preparations in vitro, which is a great advantage over most other vertebrates. the poa-gnrh neurons had quite different basic electrophysiological membrane properties from those of tn-gnrh neurons and showed alternating active and silent phases of firing activities, in contrast to the regular pacemaker activities of tn-gnrh neurons. now that we have various experimental approaches (electrochemical measurement of gnrh release, ca + imaging after single-cell electroporation, single-cell rt-pcr, double patch clamp recording, etc.) in hand, simultaneous multidisciplinary approaches should be possible to study the physiology of gnrh neurons. research funds: kakenhi sy - - - metabolic regulation of puberty onset using the prepubertal rat model helen i'anson biology department and neuroscience, washington and lee university, usa we hypothesize that glucose availability determines the timing of puberty onset in rats. abdominal fat stores are low in dieted, prepubertal female rats with delayed puberty, suggesting that such rats may be particularly sensitive to dietary fuel changes. such rats are fed a single daily meal and demonstrate decreased blood glucose levels between meals. we hypothesized that such daily hypoglycemic bouts delay onset of puberty during dieting. when glucose supplement was given to prepubertal dieted rats, and they exhibited first estrus with similar timing to previously dieted re-fed rats. conversely, when dieted rats were re-fed ad libitum and simultaneously glucose-deprived, first estrus was delayed. blood glucose levels during glucose-supplementation which induced first estrus, and during glucoprivation and re-feeding which delayed first estrus, were similarly elevated, suggesting that central, rather than peripheral, glucose levels are monitored in the prepubertal animal. in summary, central glucose availability may be an important signal timing puberty onset. research funds: jeffress memorial trust and washington and lee university sy - - - molecular mechanisms of thyroid hormone action in developing brain toshiharu iwasaki, noriyuki koibuchi department of integrative physiology, gunma university graduate school of medicine, maebashi, japan thyroid hormone (th) plays an important role in the developing brain. the action is mainly exerted by controlling gene expression through binding to its specific receptor, th receptors (trs). trs are ligand-regulated transcription factors that are expressed in many organs, including brain. trs bind to target dna sequences known as th-response element (tre). coactivators and corepressors are involved in the tr-mediated gene regulation through histone modification. we characterized the coactivator and the corepressor that were expressed in embryo brain. although precise mechanism of the th action for brain development has not been fully clarified, these cofactors may be involved in these actions. th affects brain development only during a limited period, which is referred as the critical period of th action. recently, it has been speculated that environmental chemicals may cause the abnormal brain development. possible mechanism of action of such chemicals on tr system will be discussed. research funds: kakenhi , sy - - - thyroid hormone and organic anion transporters in brain hiroyuki kusuhara, yuichi sugiyama graduate school of pharmaceutical sciences, the university of tokyo, tokyo, japan organic anion transporting polypeptides (oatps/slco) currently consist of isoforms in human and rodents. they are initially identified as part of detoxification system in the body, and involved in the tissue uptake of xenobiotics, especially amphipathic organic anions. some members accept a variety of structurally unrelated compounds as substrate. oatp c is characterized by its unique substrate specificity, highly selective for thyroid hormones, particularly for t and reverse t , but the transport activity of t is quite low. it is predominantly expressed in the brain capillaries and choroid plexus, acting as barrier of central nervous system, where oatp a , the transport activities of t and t are similar, is also expressed. the uptake of t and t by the brain determined using in situ brain perfusion technique was saturable and inhibited by oatps substrates and inhibitors. these two transporters may play a role in regulation of brain levels of t and t . research funds: the advanced and innovational research program in life sciences from the ministry of education, culture, science and technology sy - - - alterations of gene expression profiles in the developing brain by chemicals disrupting thyroid hormonedependent signals takayuki negishi , masaki takahashi , yasuhiro yoshikawa , tomoko tashiro department of chemistry and biological science, aoyama gakuin university, kanagawa, japan; department of biomedical science, the university of tokyo, tokyo, japan there is increasing concern about the possibility that environmental chemicals such as polychlorinated biphenyl (pcb) and its hydroxylated metabolites interfere with normal brain development through acting as thyroid hormone disrupting agents. in this presentation, based on comprehensive dna microarray analysis, we demonstrate alterations in gene expression in the brain of neonatal rats perinatally exposed to -hydroxy- , , , , pentachlorobiphenyl (anti-thyroid hormone-like), as well as in primary cultured rat hippocampal neurons exposed to -hydroxy- , , , , , , -heptachlorobiphenyl (thyroid hormone-like). among genes whose mrna expression levels were affected by these compounds, a number of genes essential for the establishment of synaptic networks were detected, suggesting that long-lasting effects on higher brain functions may result from exposure of the developing brain to these compounds. hydroxylated metabolites of pcbs (oh-pcbs) have chemical structures similar to thyroid hormones (ths). we reported that low doses of two types of oh-pcb inhibited th-dependent extension of purkinje cell dendrites ( . dev. brain res. , ) . koibuchi et al. ( ) clarified that they interfere with th-dependent gene expressions in reporter gene assays. further, takasuga et al. ( ) detected some pcb and oh-pcb congeners in human csf, of which oh-cb is the highest concentration. to determine its effects on developing neurons, we examined oh-cb in rodent cerebellar culture cells. interestingly, oh-cb promoted dendritic development of purkinje cells in the absence of th and increased significantly their survival numbers. these results indicate that oh-pcb congeners may disrupt normal brain development by different mechanisms depending on their chemical structures. we have reported that rat pups exposed to an antithyroid agent, propylthiouraci l (ptu), via maternal milk exhibit hyperactivity, impairment in spatial learning and social interaction, and audiogenic hypersensitivity extending to audiogenic seizures, thus this ptu rat can be a possible candidate of animal model for autism. in ptu rats, the delay in migration of the extragranular cells of cerebellum, and in innervation from inferior olive nuclei to purkinje cells were shown. these neurons transiently expressed serotonin-ir, therefore we treated ssri or -htp to examine the relevance of serotoninergic function. the treatment of -htp but not ssri recovered the delay of cell migration. these effects of serotonin manipulation in ptu rats on the behavioral impairment and the development of cns will be discussed. it is hoped that embryonic stem (es) cells will be used in transplantation therapy for neurological diseases. however, because grafts of neural stem cells derived from es cells may contain residual undifferentiated cells, there would be a risk for teratomas. to reduce this risk, we applied to es cells herpes simplex virus thymidine kinase (hsv-tk) gene and ganciclovir (gcv) treatment. stable mouse and cynomolgus monkey es cell lines expressing hsv-tk were obtained. gcv sensitivity was higher in undifferentiated es cells than in es cell-derived neural stem cells. es cell-derived neurons were resistant to gcv treatment. nude mice with transplants of undifferentiated es cells expressing hsv-tk formed teratomas, but the tumor growth was suppressed after the gcv treatment. suicide gene delivery might increase the safety of the use of es cells in cell replacement therapy. enzymatic degradation of chondroitin sulfate is known to promote axonal regeneration in the central nervous system. the physiological role of chondroitin sulfate up-regulated after injury was examined in the nigrostriatal dopaminergic system which was unilaterally transected and treated with chondroitinase abc. in transected mice, dopaminergic axons did not extend across the lesion. chondroitin sulfate was up-regulated around the lesion and a fibrotic scar containing type iv collagen deposits were developed in the lesion center. in chondroitinase abc-treated mice, numerous dopaminergic axons were regenerated across the lesion. in these animals, chondroitin sulfate immunoreactivity was remarkably decreased and the formation of a fibrotic scar was unexpectedly prevented. these results support our previous supposition that chondroitin sulfate does not act as an obstacle to regenerating axons, but involved in the repair process of the brain injury including the formation of the fibrotic scar (kawano et al., ) . reference kawano et al., . j. neurosci. res. , - . research funds: kakenhi os a- - neurotransmitters that maintain and suppress the tonic firing of the serotonergic neurons in the dorsal raphe during sleep waking cycles yoshimasa koyama , kazumi takahashi , yukihiko kayama cluster of science and technology, fukushima university, fukushima, japan; department of physiology, fukushima medical university, fukushima, japan the present experiment was done to examine, under unanesthetized natural sleep-waking condition, which neural systems were involved to regulate the firing of the serotonergic ( ht) neurons in the dorsal raphe (dr) during sleep waking cycles. using head restrained, unanesthetized rats, single neuronal activity was recorded and each drug was applied iontophoretically or by pressure close to the recording neurons. spontaneous firing of the ht neurons in dr were excited by glutamate and orexin a or b. they were inhibited by noradrenaline. an ␣ receptor agonist (phenylephrine or methoxamine) increased the firing rate during sws or ps, but had no effect when applied during w. in ps-off type dr neurons, cessation of firing during ps was recovered by bicuculline, however in the dr neurons that did not stop firing during ps, bicuculline had almost no effect. os a- - correlation between regional grey matter volume and proficiency increase in second language: a vbm study arihito nauchi , , kazuyoshi hirano , , yukimasa muraishi , , kuniyoshi l. sakai , department of basic science, university of tokyo, tokyo, japan; crest, jst, japan; secondary education school, university of tokyo, japan although neuroimaging studies have contributed to clarify the brain function, the neural basis of individual variation in cognitive abilities such as language still remains unknown. in the present study using voxel-based morphometry (vbm), a whole-brain unbiased technique for detecting regional differences in mr images, we examined the relationship between the proficiency increase in second language (l ) and grey matter volume among students aged - , who received special classroom training in the use of english verbs. in specific regions including the left lateral premotor cortex and the left inferior frontal gyrus (the grammar center), we found a positive correlation between the regional grey matter volume and improved performance for the grammaticality of english sentences. these results suggest an anatomical basis for the language faculty, such that the capacity of a specific region is related to proficiency increases in l . os a- - grammar center activation in honorification judgment of japanese sentences kanako momo , , kuniyoshi l. sakai , department of basic science, university of tokyo, tokyo, japan; crest, jst, japan one linguistic theory proposes that japanese honorification is a syntactic feature, because syntactic agreement is required between subject/object and honorific forms. to investigate whether such syntactic processing is actually realized in the brain, we examined cortical activation using fmri under several types of normal/anomalous judgment for japanese sentences including honorification. when activation in a honorification task was contrasted with that in a spelling task, we observed significant increase in the left including grammar center (ifg) as well as the bilateral cerebellum for all tested participants. moreover, the lower performance group showed greater activation in the left f op/f t and the bilateral cerebellum. these results suggest that syntactic process is required for japanese honorification and that activation in these regions shows modulation according to performance level, even in native language. research funds: crest, jst os a- - top-down modulation for melody-related activity in the right auditory areas: an meg study takuya yasui , , , kimitaka kaga , kuniyoshi l. sakai , dept. of basic science, univ. of tokyo, tokyo, japan; dept. of otolaryngology, univ. of tokyo school of medicine, japan; crest, jst, japan we previously reported right-hemisphere dominance for melody error-induced fields (m ) (neurosci. res. , s ). in a subsequent study, we confirmed that m was independent from mismatch negativity usually induced by oddballs. in the present study, we examined whether m was induced by deviation from a memorized melody. we used four pairs of unfamiliar songs, each pair consisting of an original song and a modified song in which the third note deviated from that of the original. subjects learned these songs and judged whether there were one or two deviations in notes. there was no significant difference in dipole amplitudes between m elicited by the original songs and that by the modified ones. however, while m without the deviation showed no significant effect of lateralization, m with the deviation resulted in significant enhancement in the right hemisphere. these results suggest the existence of memory-induced, i.e., top-down modulation for melody-related activity in the right auditory areas. research funds: crest, jst os a- - cortical plasticity in adulthood for learning phonics rules for english orthography and phonology makiko muto , , kuniyoshi l. sakai , department of basic science, university of tokyo, tokyo, japan; crest, jst, tokyo, japan although matching english orthography with correct pronunciation is difficult for second language learners, learning phonics rules may rapidly improve their performance. in the present fmri study, we tested an english matching task during the course of phonics training in sessions, in which infrequent words were visually shown, while matched/unmatched speech sounds were simultaneously presented. comparing the first half of the sessions with the latter half, the left posterior inferior temporal gyrus (the letter center) and a part of the left lateral premotor cortex (the grammar center) showed activation decreases, when the performance was significantly improved. these results suggest that the plasticity of functional systems involving these critical regions is essential for establishing phonics rules and for forming a new link between orthography and phonology. research funds: crest, jst os a- - hierarchical syntactic processing in the left frontal region: an meg study kazuki iijima , , naoki fukui , kuniyoshi l. sakai , dept. of basic science, univ. of tokyo, komaba, japan; crest, jst; dept. of linguistics, sophia univ., yotsuya, japan previous erp studies have shown word-related activation based on semantic association or context. however, it remains unclear how syntactic information of preceding words is integrated into the ongoing sentence processing. in the present meg study, we measured brain activity during each of four tasks: a syntactic task, a semantic task, a memory task, and an evaluation task. sentence stimuli consisted of one noun phrase and one verb, where the noun phrase had either an objective or nominative case particle. the first peak of the activity for a verb presentation was observed at the left frontal region as early as ms after the onset. in the objective-case condition, this activity was enhanced only for the syntactic task, while in the nominative-case condition no such task-selectivity was observed. these results are consistent with the current linguistic theory (the minimalist program), which holds that a noun phrase with an objective case particle is directly merged with a verb, to form a new hierarchical level. research funds: crest, jst os a- - individual difference of brain activity in medial prefrontal cortex and superior temporal sulcus during social cognition koji jimura, seiki konishi, tomoki asari, junichi chikazoe, yasushi miyashita dept. physiol. univ. tokyo sch. med., japan previous neuroimaging studies have reported brain activity in the medial prefrontal cortex (mpfc) and the superior temporal sulcus (sts) during performance of theory of mind tasks. the present fmri study explored individual difference of the mpfc and sts activity by employing false belief paradigms. the task consists of two sessions, study and test. during the study session, subject studied a brief story in which two characters have false beliefs. then, the subject answered questions about the false belief and the fact that constitutes the false belief during the test session. consistent with previous studies, significant activity was observed in the mpfc and the sts during representing the false belief. the individual differences of the mpfc and the sts activity were correlated with psychodiagnostic indices that represent controlled and automatic idealization, respectively. these results suggest that the two indices represent distinct neural mechanisms participating in social cognition. research funds: grant-in-aid from mext ( ), jsps research fellowship ( ) os a- - brain activity of happy facial recognition in mother-daughter relationship jun shinozaki , nobukatsu sawamoto , toshiya murai , takashi hanakawa , hidenao fukuyama hbrc, kyoto univ. grad. sch. of med. kyoto, japan; neuropsychiatry, kyoto univ. grad. sch. of med. kyoto, japan relationship between parents and children is special, and affective facial recognition between them should evoke specific neural activity not shared by other personal relationships. eleven healthy females participated in this fmri experiment. the subjects saw happy and neutral faces of their own mothers, and newly learned other subjects' mothers during the scan. when happy face recognition was compared with neutral face recognition, the mother-daughter combination induced greater activity than the non-familial combination in the following areas; the lateral prefrontal cortex, anterior cingulate cortex, middle temporal cortex, striatum, and anterior insula. it has been shown that the lateral prefrontal and anterior cingulate cortices are associated with familial facial recognition, whereas the middle temporal cortex is related to happy facial recognition. the activity in the striatum and anterior insula might be related to positive affection and empathy, respectively. os a- - anatomical connections among functionally identified brain regions for sentence processing yukari yamamoto , , atsushi maki , , kuniyoshi l. sakai , advanced research laboratory, hitachi, ltd., tokyo, japan; crest, japan science and technology agency, saitama, japan; dept. of basic science, univ. of tokyo, tokyo, japan we have functionally identified the left dorsal inferior frontal gyrus (ifg), the left lateral premotor cortex, and the triangular/orbital part of the left ifg (f t/f o) as regions associated with sentence and discourse level processing. in the present study, we examined whether there are direct anatomical connections among these regions by using diffusion tensor tractography. f t and f o of the left ifg were chosen as seed areas for fiber tracking. fiber bundles that went through two spherical regions were extracted from the tracking data. the central coordinates of these regions were (− , , ) , (− , , ) , and (− , , − ) in the standard brain, which are associated with syntactic processing (the first and second coordinates) or sentence comprehension (the third coordinate). direct connections among these regions were consistently observed among the subjects. this result suggests a critical network among multiple regions that are associated with sentence processing. os a- - effect of the incongruity controlled by semantic distance on visually evoked magnetic fields nobuyoshi harada , sunao iwaki , mitsuo tonoike aist, osaka, japan; chiba university, chiba, japan visual incongruities of heads changed on animal pictures, which were controlled by the semantic distance of the word of the animal, were investigated on visually evoked magnetic fields. the semantic distance was decided by the numbers of links of the semantic network of the taxonomic layer in a japanese thesaurus. the words for mammalians were grouped into five semantic categories in the thesaurus. the heads of the animals were changed with those from another semantic category (deviant, d = ), and with those from an inner semantic category (middle, d = ), while others were not changed (normal, d = ). peak amplitudes of waveforms of the root mean square values on the components of ms (f ( / ) = . , p = . ) and ms (f ( / ) = . , p = . ) were significantly decreased with increments of the semantic distance in left occipital sensors. the gradient of the decreasing line of the amplitudes of the and ms components indicated the capability of extracting the structure of a typical prototype of the form of the animal. we call this capability, the structural sensitivity for prototype (ssp). ryohei yasuda duke university medical center, usa calcium signaling in dendritic spines is important for many forms of synaptic plasticity. however, the quantitative mechanisms of how calcium elevations are translated into spatial and temporal patterns of biochemical reactions leading to modifications of synaptic strength are unclear. identifying and following the spatiotemporal activation of molecules necessary for synaptic plasticity is crucial for a better understanding of this complex process. to visualize the activity of signaling pathways in neurons deep in brain tissues, we have combined fluorescence lifetime measurements and two-photon microscopy. this technique allowed us to measure spatiotemporal aspects of the activity of signaling proteins including ras gtpase proteins in response to physiologically relevant stimuli with single spine resolution. research funds: burroughs wellcome fund, dana foundation os p- - mechanisms of p y purinoceptor-mediated long-term enhancement of inhibitory transmission examined by multiple-probability fluctuation analysis at cerebellar gabaergic synapses yumie ono , xiaoming zhu , takashi tominaga , fumihito saitow , shiro konishi , waseda-olympus bioscience research institute, singapore, singapore; department of neurophysiology, tokushima bunri university, kagawa, japan; department of pharmacology, nippon medical school, tokyo, japan postsynaptic p y receptor activation by atp enhances ipscs at cerebellar interneuron-purkinje cell (pc) synapses. to investigate the underlying mechanisms, we here employed the non-stationary fluctuation analysis to estimate the number (n) and single channel conductance (i) of gaba a receptors in pcs using whole-cell recordings of evoked ipscs in pcs of rat cerebellar slices before and - min after application of atp. the atp-induced enhancement of the ipsc amplitude was associated with a significant increase in the single channel conductance, but not the number, of gaba a receptors in pcs: i and n after atp treatment were ± . % and ± . % of the controls, respectively. pretreatment with the protein kinase a inhibitor h- , but not the calmodulin kinase ii inhibitor kn- , completely abolished the atp-induced ipsc enhancement. os p- - activin induces long-lasting nmda receptor activation via scaffolding pdz protein arip isao inoue, akira kurisaki, hiromu sugino institute for enzyme research, tokushima university, tokushima, japan calcium entry into the postsynaptic neuron through nmda type glutamate receptors (nmdars) triggers the induction of long-term potentiation (ltp). the ca + permeability of nmdar is regulated by phosphorylation of its tyrosine residues. we report here that activin, a member of the transforming growth factor-b (tgf-b) superfamily, and one of proteins synthesised after ltp, promotes phosphorylation of nmdars and increases the ca + influx through those receptors in primary cultured rat hippocampal neurons. this signal transduction occurs in a functional complex of activin receptors, nmdars, and src family tyrosine kinase, fyn formed on a multimer of postsynaptic scaffolding pdz protein, activin receptor interacting protein (arip ). activin-induced nmdar activation persists more than h, which is complimentary to the transient activation of nmdars by brain derived neurotropic factor (bdnf). our results show that activin is a long-lasting potentiator involved in synaptic plasticity regulatory mechanisms. os p- - roles of cam kinase i in the hippocampal longterm potentiation kohji fukunaga, takashi komori, shigeki moriguchi department of pharmacology, graduate school of pharmaceutical sciences, tohoku university, sendai, japan cam kinase i (camki) family members are highly expressed in the adult rat hippocampus and camki-alpha is predominantly localized in the cytosol. camki activation requires phosphorylation of thr by camkk as an upstream kinase. we here documented a marked increase in camki-alpha-thr phosphorylation following ltp induction in rat hippocampal ca region. like camkii activation following ltp (fukunaga et al., ) , the increased camki-thr phosphorylation remained elevated at least for min after ltp induction. the increased camki-thr phosphorylation was closely associated with prolonged increases in phosphorylation of creb and myosin light chains in the ca region. this is in contrast with transient increases in camkiv and erk phosphorylation. treatment with camkk inhibitor, sto- significantly inhibited both creb and mlc phosphorylation with concomitant reduction of ltp in the ca region. taken together, camki likely mediates the late phase of creb phosphorylation and an increased mlc phosphorylation in the hippocampal ltp. to investigate how the excitatory postsynaptic inputs of the proximal dendrite effect the information processing of synaptic inputs at the distal dendrite, stimulation was applied to induce bap and epsp at the alveus and the proximal dendrite, respectively. the resulting coincidence of magnitude of bap and epsp at the distal dendrite was enhanced when the bap was delivered at a timing ( ms) to induce ltp. furthermore, the magnitude of bap at the distal dendrite was attenuated by the input from the proximal dendrite at a timing ( ms) to induce ltd. these results suggest that the magnitude of bap delivered to the distal dendrite can be amplified or attenuated depending on the relative timing between proximal input and bap. this may be due to an effect on the coding process at the distal dendrite and could support the basis for a novel learning rule in the brain. research funds: kakenhi ( ) os p- - mouse brains deficient in neuronal pdgf receptor-␤ develop normally but are vulnerable to injury yoko ishii, takeshi oya, lianshun zheng, masakiyo sasahara department of pathology, faculty of medicine, university of toyama, japan the platelet-derived growth factors (pdgfs) and pdgf receptors (pdgfrs) are widely expressed in the mammalian cns. here, we developed novel mutant mice in which pdgfr-␤ subunit gene was genetically deleted in the neurons of cns to elucidate the role of pdgfr-␤. our mutant mice reached adulthood without apparent anatomical defects. the cerebral damage after cryogenic injury was severely exacerbated in the mutants compared with the controls. furthermore, this exacerbated lesion formation was suggested to be, at least partly, due to the enhanced excitotoxicity after injury, because nmda-induced lesion formation was also extensively enhanced in the cerebral cortex of the mutants without altered nmda receptor expression. this is the first known report to address the postnatal function of pdgfr-␤ expressed in cns neurons, using genetically engineered mutant. it was clearly demonstrated that pdgfr-␤ expressed in neuron protects cns neurons from cryogenic injury and nmda-induced excitotoxicity. early postnatal days (especially the first three weeks in the rat) are the critical period for newborn hippocampal granule cells (gcs) to dynamically migrate from the dentate hilus and form the gc layer. to investigate the mechanism that regulates newborn gc migration, we developed a new slice coculture system. the hilar parts of entorhino-hippocampal slices prepared from postnatal six-day-old (p ) rats that had received a single brdu injection at p were substituted with the corresponding region of entorhino-hippocampal slices from p rats. after five days in vitro, newborn gcs, detected by brdu and prox , migrated out of the hilar graft and reached the host gc layer. chronic application of picrotoxin, a gaba a receptor antagonist, facilitated the migration of newborn gcs into the gc layer. these results indicate that gaba a receptors regulate the migration of newborn gcs in early postnatal days. os p- - cdk is required for neuroblast migration in the adult mouse brain yuki hirota , , , toshio ohshima , takuji iwasato , ashok b. kulkarni , hideyuki okano , , kazunobu sawamoto , , bridgestone lab. keio univ., tokyo, japan; dept. physiol., keio univ., tokyo, japan; dev. neurobiol. riken, tokyo, japan; behavioral gen. riken, tokyo, japan; nih, bethesda, usa; sorst, jst, saitama, japan neuroblasts generated in the subventricular zone (svz) of the lateral ventricles migrate into the olfactory bulb (ob) through the pathway called rostral migratory stream (rms). molecular mechanisms regulating the directional long-distance migration remain largely unknown. here we studied adult function of cyclin-dependent kinase (cdk ) that has been revealed to play a role in neuronal migration in the embryonic brain. crossing the floxed-cdk mice to emx cre mice resulted in decreased size of ob and abnormal distribution of neuroblasts. svz explants from these mice cultured in matrigel showed decreased migration distance. leading process of neuroblasts infected with cre-encoding retrovirus were found in random orientations and frequently failed to migrate out of the svz compared to control cells. these results indicate that cdk has a cell autonomous function in neuroblast migration in the adult brain. os p- - colocaliztion of neuron markers and glial markers in gabaergic neuron progenitors as revealed by singlecell microarray analysis shigeyuki esumi , wu sheng-xi , yuchio yanagawa , , kunihiko obata , nobuaki tamamaki kumamoto univ., kumamoto, japan; fourth military medical univ., xi'an, people's republic of china; gunma univ., maebashi, japan; sokendai, hayama, japan; riken, wako, japan gabaergic neurons and oligodendroglia share many characters in the murine forebrain. both of the cell types has been reported to originate in the medial ganglionic eminence and migrate to the neocortex. in addition, it is reported that they share several glial markers, such as ng , plp, and cnp at their prematured stages. in order to investigate its nature, we have established a single-cell microarray analysis method. single gfp-positive gabaergic neuron progenitors were corrected from the subventricular zone of the gad -gfp knock-in mouse neocortex at e -p by dissociation and picking. complemental dna from the single cells was amplified by universal pcr amplification and converted into biotin-labeled crna using t rna polymerase. after these procedures, crna sufficient for a microarray analysis was obtained. as the result we found, mbp and s -␤ expression in the gabaergic neuron progenitors. os p- - role of ␤-catenin signaling in regulating proliferation of transit-amplifying cells in the adult mouse subventricular zone kazuhide adachi , , , masanori sakaguchi , toru yamashita , , , yuko fujita , yukiko gotoh , arturo alvarez-buylla , takeshi kawase , hideyuki okano , kazunobu sawamoto , neurosurgery, keio univ. sch. med., tokyo, japan; bridgestone lab. dev. regenerative neurobiol., keio univ. sch. med., tokyo, japan; physiol., keio univ. sch. med., tokyo, japan; inst. mol. cell biosciences, univ. tokyo, tokyo, japan; neurosurgery, ucsf, san francisco, usa; neurol, okayama univ, med, dentistry and pharmaceutical sci, okayama, japan the subventricular zone (svz) continuously produces olfactory bulb neurons in the adult rodent brain. neural stem cells generate migratory neuroblasts via highly proliferative transit-amplifying cells in this region. here, we studied the role of ␤-catenin signaling in the adult mouse svz. ␤-catenin accumulated in the nucleus of only the transitamplifying cells in the svz. activated ␤-catenin signaling promoted the proliferation of transit-amplifying cells, resulting in an increased number of new neurons in the olfactory bulbs. these results suggest that ␤-catenin signaling plays a role in the proliferation of transitamplifying cells in the adult mouse svz. the ciliary marginal zone (cmz) is a region between the neural retina and ciliary epithelium, and contains retinal progenitor cells that give rise to neuron and glia. wnt b is expressed in cmz, and has been shown to control the differentiation of the retinal progenitor cells. we have isolated a novel bmp antagonist, chick tsukushi (c-tsk), which belongs to the small leucine-rich proteoglycan family. in the eye, the expression of c-tsk is observed in the cmz which is similar with that of wnt b. to examine the molecular interactions between c-tsk and wnt b, we co-electroporated them into the optic vesicle at stage - chick embryo and observed the proliferation of the retinal progenitor cells. we found that c-tsk inhibited the wnt b activity that sustains prolonged proliferation of retinal progenitor cells. our result suggests that c-tsk controls the proliferation of retinal progenitor cells interacting with wnt b. to reveal the role of epigenetic gene regulation in neuronal differentiation, we studied subcellular distributions of histone deacetylase (hdac) in developing cortical neurons. an expression vector of gfp-tagged hdac was transfected to dissociated cortical cell cultures as well as cortical neurons in vivo. hdac was primarily localized in nuclear until week in vitro, but was translocated to cytoplasm in the later stages. such translocation was found in a similar time course after birth in vivo. to examine a possibility that neural activity is involved in the translocation, firing activity of cultured neurons was examined using multi-electrode dishes. as a result, spontaneous firing activity was prominent in the late stages when cytoplasmic translocation occurred. however, ttx addition to the culture medium produced the inverse translocation. these results suggest that activity-dependent intracellular localization of hdac contributes to neuronal differentiation in cortical development. research funds: kakenhi ( ) dept. of physiology, fujita health univ. sch. of med., japan we described electrical synapses in alpha retinal ganglion cells (␣-gcs). precise temporal synchronization of spikes is generated from ␣-gcs (hidaka et al., ) . the fraction of open channels in gap junctions were evaluated with techniques of dual patch-clamp, connexin immunocytochemistry, and high-voltage electron microscopy. junction conductance (maximum . ns) was measured. in high-voltage electron microscopy (hitachi m, nips, , gap junctions (average size . m long) were present in contacts. in confocal laser-scanning imaging, connexin localization at contacts counted gap junctions (seven sites in a pair on average). assuming that the density of connexons would be /m and a single channel conductance is ps, the conductance of each junction would be ns. the presence of seven junctions between a pair will lead to estimate a total junction of ns. the measured conductance could allow to estimate a fraction of open channels as . %. the open fraction is small, when we consider whether electronic transmission acts to synchronize the spikes in the intercellular network. the visual system separates different types of information into parallel, anatomically distinct processing streams. despite their significance for visual processing, the molecular mechanism underlying the physiological stream formation is largely unknown, partially because these physiological streams have not been reported in mice. to identify molecular correlates of functionally distinct streams, we fabricated a custom cdna microarray of higher mammal ferrets. we successfully identified molecules whose unique distribution and developmental profiles define the lgn itself, its constituent layers, or identify cells comprising one of the physiological streams in the lgn. using these molecules as temporal and spatial markers, we investigated mechanisms of the physiological stream formation in the ferret lgn. research funds: kakenhi ( ), coe research akira muto, herwig baier department of physiology, university of california san francisco (ucsf), usa the visual system operates over a broad range of luminances. this is accomplished by adjustment of photosensitivity, called light adaptation. to study the molecular mechanisms of light adaptation, we screened for zebrafish mutants that showed compromised optokinetic responses (reflexive eye movements to large field motion) after an abrupt dark-to-light transition. in this experimental paradigm, wildtype fish larvae recover their full optokinetic response within about two minutes after being brought back to light. in a screen of almost genomes, we identified five mutants all of which showed substantially delayed recovery of the okr. positional cloning of one of the loci revealed a mutation in the dna-binding domain of glucocorticoid receptor (gr). gr is known for its role in the stress response, but its function in the visual system is unexplored. we propose that gr is regulating genes essential for light adaptation in the retina. os p- - multisite recording of the signal propagation pattern in the visual cortex makoto osanai, yusuke takeno, satoshi tanaka, tetsuya yagi graduate school of engineering, osaka university, suita, japan recently, the visual prosthesis systems with implanted stimulus electrode in the visual cortex are developed. but the signal propagation pattern induced by electrical stimuli in the visual cortex is not fully investigated. therefore, we studied the signal propagation pattern induced by electrical stimuli in the mouse visual cortex slice, using a channel multielectrode array and a calcium imaging system. in the electrophysiological study, the responses conducted vertically against the layer of the cortex with layer stimuli and propagated horizontally in the layer / . in the calcium imaging study, the area of the higher calcium concentration region spread vertically with layer stimuli. signal propagation was restricted within several tens m around the stimulus electrode by ap + cnqx administration and was completely blocked by ttx administration. administration of bicuculline increased the area of the signal propagation in a dose-dependent manner. we concluded that these restricted patterns of the signal propagation in the visual cortex were due to the inhibitory system. os p- - presence of two phases in the sensitive period of orientation plasticity shigeru tanaka , toshiki tani , kazunori o'hashi , , jerome ribot brain science institute, riken, saitama, japan; graduate school of life science and systems engineering, kyushu institute of technology, kita-kyushu, japan recently we have revealed that orientation-restricted visual experience induces drastic reorganization of orientation maps in the cat visual cortex. in this study, we examined the effect of release from single orientation exposure on once reorganized orientation maps during the sensitive period using intrinsic signal optical imaging. when kittens were returned to the normal visual environment by removing the goggles after weeks of goggle rearing starting around the age of weeks, the over-representation of the exposed orientation was preserved. on the contrary, when the goggle rearing started around the age of weeks and then the animals were returned to the normal visual environment, orientation maps rapidly changed to represent orientations equally. these findings indicate that the sensitive period of orientation plasticity consists of two phases: orientation map reorganization is irreversible in an early phase and reversible in a late phase. os p- - residual visuomotor processing in the animal model of blindsight: comparison with normal, near-threshold vision masatoshi yoshida , , , tadashi isa , , dept. dev. physiol., nat'l inst. physiol. sci., okazaki, japan; sch. life sci., grad. univ. adv. stud., hayama, japan; crest, jst, kawaguchi, japan in two macaque monkeys with unilateral v lesion performing a visually guided saccade task, saccadic parameters were compared between the saccades to the affected hemifield and those to the intact hemifield. the luminance contrast of the target presented in the intact hemifield was reduced so that the detectability was comparable to that in the affected hemifield ( - % correct). in the saccades to the affected hemifield, the curvature of the trajectories was smaller and the deviation of the saccadic end points from the target was larger than those to the intact hemifield. these results suggest that without geniculo-striate pathway, online compensation for the variation of the initial saccadic command is not fully functional, thus leading to inaccurate saccades. we propose that the residual visuomotor processing of monkeys with v lesion is unlike normal, near-threshold vision. research funds: kakenhi , kakenhi and crest, jst os p- - comparison of the angle representation in macaque visual areas v and v minami ito , , hidehiko komatsu , national institute for physiological sciences, okazaki, japan; the graduate university for advanced studies, hayama, japan previously, we have reported that fairly large number of area v neurons has angle selectivity. here, we studied the angle selectivity of area v , which is the major source of inputs to area v . we conducted single-unit recordings from the superficial layer of area v , while animals performed a fixation task. for comparison, we used a similar stimulus set. the stimuli were much larger than the size of the classical receptive fields. area v neurons responded mainly to sharp angles ( • ), straight lines ( • ) or right corners ( • ), but not to intermediate angles ( • or • angle width). this contrasted with area v , where neurons showed a variety of the optimal angle width including intermediate angles. we also observed several v neurons showed fine orientation tuning to short line segments, while weak or no responses were induced by a set of large angle stimuli. we suggest that area v neurons largely contribute to representing line components (lines and line-ends) and to sending such information to area v . os p- - firing rates and dynamic spatiotemporal patterns of ganglion cells both contribute to retinal information processing xin jin, ying-ying zhang, xue liu, hai-qing gong, pei-ji liang department of biomedical engineering, shanghai jiao tong university, shanghai, china population activities of retinal ganglion cells (rgcs) were recorded using a multi-electrode recording system. single unit analysis showed that firing rate of individual neuron was strongly dependent on the luminance intensity of stimulation. however, population activity of ganglion cells usually showed particular spatiotemporal pattern, in response to a specific velocity of the moving bar. differing from single direction-selective ganglion cell (dsgc), which responds to its preferred direction of movement by firing at its maximal rate, population activity of non-direction-selective ganglion cells may encode the motion information in a temporally ranked manner, independent to their individual firing rates. these results suggest that an efficient and economical coding mechanism may be employed by the retina, where the firing rate of individual neurons and spatiotemporal pattern of population neuronal responses could act in parallel to encode different aspects of visual information. yasuto tanaka, satoru miyauchi, masaya misaki brain information group, nict, kobe, japan visual long-range interaction was reported to be limited in space. here, we show the evidence of long-range interaction extending to an order magnitude larger using the right-left symmetrical configuration. two horizontally collinear gabor signals, one defined as probe and the other cue, were presented at the left and right side of the visual field at mirror symmetrical regions. detection threshold of gs probe reduced with cue-probe separations up to • . the facilitation was highly tuned to the symmetrical locus. furthermore, the facilitation was substantially longer at upper visual field than the lower visual field. the reduction was specific to orientation, phase, and horizontal direction, the results indicate long-range mirror symmetrical interaction across vertical meridian, suggesting symmetrical neuronal communication between early visual cortices. the anisotropy between left-right hemifield (symmetry) and upper-lower hemifield (upper-field advantage) signifies hemifield inhomogenity in human vision. os p- - integrity of visuospatial attention in a split brain patient noudoost behrad, seyed reza afraz, maryam vaziri, hossein esteky school of cognitive sciences (scs), iran transfer of visual information between hemispheres is severely impaired following transection of posterior part of the corpus callosum. we investigated whether attentive visual object tracking across vertical meridian of the visual field is possible for a posterior callosotomized patient (md). we asked md to track one bouncing ball among four identical distracters while fixating at the center of the screen. target crossed the vertical midline in half of the trials. her performance in crossed conditions was significantly above chance level. also, we asked her to make decision about horizontal alignment of two balls presented simultaneously in one of three conditions: both in right or left hemifield, or each in one hemifield. in this alignment task md was able to compare location of the two bilaterally presented stimuli well above chance level. our data suggest that inter-hemispheric transfer of position information required for spatial attention is preserved without posterior corpus callosum. pei sun, justin l. gardner, mauro costagli, kenichi ueno, r. allen waggoner, keiji tanaka, kang cheng laboratory for cognitive brain mapping, riken brain science institute, wako-shi, japan although the preference for stimulus orientations in human visual cortex has been inferred indirectly in a few studies using fmri, tuning to particular stimulus orientations has not been directly demonstrated using this technique. in an effort towards revealing orientation selectivity and its spatial arrangement in human v , we have conducted an fmri study with a novel stimulation paradigm and a differential mapping method. we found that responses of the majority of activated voxels were modulated by the grating orientation and individual voxels were sharply tuned to particular orientations. our results provide the first demonstration that orientation selectivity in humans can be directly studied using fmri. os p- - probing the spatial scale of classifier performance with high spatial resolution fmri justin l. gardner , , pei sun , keiji tanaka , david j. heeger , kang cheng department of psychology and center for neural science, new york university, usa; laboratory for cognitive brain mapping, riken brain science institute, japan recently, classifier analysis with conventional resolution fmri has been used to decode the orientation of a grating stimulus from the fmri responses of early visual cortex. it has been proposed that classifier analysis exploits small but robust orientation biases in voxels that are created by local inhomogeneities in the columnar organization. we have examined this proposal by using classifier analysis to decode stimulus orientation using high spatial resolution fmri ( . mm × . mm × mm voxels) in human v . we found that many voxels that are weighted heavily in the classifier analysis and carry similar orientation biases closely follow draining veins that are visible on t *-weighted venograms. we suggest that large draining veins with orientation specific responses, rather than local inhomogeneities in orientation maps, may provide a basis for classifier performance using large voxels. research funds: nrsa fellowship from the nih ( f ey - ) os p- - relationship between horizontal connections and functional structure in macaque anterior inferotemporal cortex (area te) hisashi tanigawa, kathleen s. rockland, manabu tanifuji riken brain science institute, wako, japan we have studied the relationship between horizontal connections and functional structure in te using a combination of optical imaging, unit recording, and anatomical tracing. intrinsic signal imaging was performed in exposed te, under anesthesia, during presentations of visual object stimuli. this resulted in multiple optical spots evoked by each stimulus. in some animals, subsequently, unit recording was carried out at multiple sites within the imaged region. then, an anterograde tracer was injected into one of the spots. both optical imaging and unit recording revealed regions with stimulus preference similar to that at the injection site. however, these regions and the injection site were not always connected by horizontal axons. some regions sharing a preference to particular stimuli were connected, even though they showed different preferences to the other stimuli. these results suggest that horizontal axons can connect regions with different stimulus preferences in te, in contrast to like-to-like connectivity, as understood in early visual cortices. we recorded single cell responses from the inferotemporal cortex of a fixating monkey while visual stimuli with various durations ( - ms; isi = s) were presented. presentation of visual stimuli at all of the tested durations resulted in prolonged responses. the brief presentations evoked multiple phasic responses while the long presentations evoked sustained activities. there was a significant difference in average firing rate of late phase ( - ms) of response to optimal stimulus across presentation durations. but no such differences were found for the first phase ( - ms). in addition, the optimal stimulus evoked significantly different response magnitudes in the first and second phase particularly in the short presentation durations. but the suboptimal stimulus (∼ % of max response) evoked similar response magnitudes in the first and second phase. these results suggest that stimulus selectivity of inferotemporal cells depends on the stimulus presentation duration and the time window that is used to measure the firing rate. os p- - the perceptual learning effect in myopes by the lateral masking procedure keiko mizobe , kazuto terai , osamu hieda , shigeru kinoshita dept. of ophthal., kyoto second red cross hospital, kyoto, japan; dept. of ophthal., kyoto pref. univ. of med., kyoto, japan; baptist eye clinic hospital, kyoto, japan purpose: the study of the visual cortex revealed the lateral masking collinear configuration modulated the neuronal responses and psychophysical studies also showed perceptual learning improved the visual detection. we asked whether perceptual learning could improve the myopic blurred vision, using the new instrument of the lateral masking technology, neurovision. method: nine low myopes were studied. non-corrected digital visual acuities (va) ranged from . to . . the logmar average was . . eight sessions of neurovision treatment were performed to each individual. the estimation was done by comparison of va before and after the treatment. results: four eyes showed more than one octave improvement of va. the logmar average of the four was . , improved from . . the residual five eyes showed less or no improvement. the change of logmar average was from . to . . conclusion: some myopes showed the perceptual learning effects by new treatment, using the lateral masking technology. os p- - the hindbrain neuroepithelial cells exclude the migrating facial motor neurons by expression of planar cell polarity (pcp) genes hironori wada , hideomi tanaka , , satomi nakayama , miki iwasaki , , hitoshi okamoto , laboratory for developmental gene regulation, bsi, riken, japan; crest, jst, japan many neurons migrate tangentially through one cell layer at a specific depth within the brain. in the developing zebrafish hindbrain, the facial (nvii) motor neurons originate in rhombomere (r) and migrate tangentially to r near the pial surface of the hindbrain. in this study, we demonstrate that expression of the planar cell polarity (pcp) genes celsr and frizzled a in neuroepithelial cells maintain the nvii motor neurons near the pial surface during their caudal migration in the zebrafish hindbrain. mosaic analyses show that expression of the frizzled a gene in the surrounding neuroepithelial cells prevented the entry of the nvii motor neurons in the neuroepithelial layer. the demonstration of a role for neuroepithelial cells in excluding differentiated neurons from the neuroepithelial layer may provide new insights into the general mechanisms underlying formation of the layered structures in the mammalian brain, such as in the cerebral cortex. os p- - disrupted-in-schizophrenia (disc ) regulates the transport of the nudel/lis complex to axons via direct interaction with kinesin- shinichiro taya, kozo kaibuchi department of cell pharmacology, nagoya university, nagoya, japan disc is a candidate gene for susceptibility to schizophrenia. in a scottish family, the chromosome translocation interrupts the coding sequence of disc . disc is reported to interact with nudel, which forms a complex with lis . although the functional significance of this complex in axon growth and neuronal development has been reported, the transport mechanism of the complex into axons and the functions of disc remain largely unknown. here we report that disc interacted with kinesin- , a motor protein of anterograde axonal transport. kinesin- interacted with the nudel/lis complex through disc , and these molecules accumulated at the distal part of axons. the knockdown of disc by rnai of disc induced the delocalization of nudel and lis from the axons and reduced axonal growth. the knockdown of kinesin- induced the delocalization of disc from the axons. taken together, these results indicate that disc links kinesin- to the nudel/lis complex and regulates its transport as a cargo receptor for axon elongation. research funds: mext os p- - role of a novel collapsin response mediator protein- interacting molecule, synaptotagmin-like protein in hippocampal neuron nariko arimura, saeko kawabata, atsushi hattori, kozo kaibuchi department of cell pharmacology, graduate school of medicine, nagoya university, nagoya, japan during the development, neurons recognize the extracellular signals and extend the axons to proper directions. certain kinds of receptors are transported from the nerve cell body to the axon terminal, and participate in the recognition of extracellular environments. however, the mechanism of controlled recruitment of receptors remains unsolved. here, we report that synaptotagmin-like protein (slp ) can mediate the vesicle transport. slp is known to associate with rab . we found that slp associates with collapsin response mediator protein- (crmp- ), which is a key regulator of axon formation. slp could form the trimeric complex with rab b and crmp- , and also associate with kinesin- through crmp- . slp is accumulated on microtubules at the axonal growth cones, and is co-localized with a receptor of growth factor. these findings suggest that slp functions as a mediator of recruitment of certain receptor depending on crmp- and kinesin- . os p- - absolute quantification of mdr a, mrp , mrp and bcrp proteins at the mouse brain blood barrier by lc-ms/ms junichi kamiie , , yuki katsukura , , sumio ohtsuki , , xiao-kun cai , , tetsuya terasaki , graduate school of pharmaceutical sciences, tohoku university, sendai, japan; sorst, jst, japan the abc transporter proteins are thought to limit permeability across the blood-brain barrier as the efflux transporters. however, contribution of each transporter to the bbb function is not clarified. the purpose of this study was to clarify the protein amounts of mdr a, mrp , mrp , and bcrp in the brain capillaries of mouse by newly developed membrane protein quantification method using lc-ms/ms. by this method, the standard curve showed linearity between and fmol, and amino acid sequence of the detected fragment was confirmed by ms/ms spectrum. in the brain capillaries, the protein amounts of mdr a, mrp , bcrp were . , . and . fmol/g, respectively, while it of mrp was under detection limit of standard curve. this quantitative profile suggests that mrp and bcrp function as the efflux transporter at mouse blood-brain barrier as well as mdr a. os p- - dominant expression of claudin- in highly purified brain capillary endothelial cells sumio ohtsuki , , hirofumi yamaguchi , saori sato , tmoko asashima , , tetsuya terasaki , graduate school of pharmaceutical sciences, tohoku university, sendai, japan; sorst, jst, japan claudins are major constituents of tight junctions (tjs). the purpose of this study was to clarify the expression levels of each claudin subtype in brain capillary endothelial cells (bcecs), which form the blood-brain barrier. mouse bcecs were highly purified using endothelial surface antigen (pecam- ) and magnetic cell sorting. mrna expression of caludin- - was measured by real-time rt-pcr. claudin- showed the highest mrna expression in the purified mouse bcecs. mrna levels of claudin- and - were . % and . % of that of claudin- . claudin- mrna was concentrated in the purified bcecs, while claudin- and - mrna in the purified bcecs were lower than that in the whole brain. rat claudin- mrna was also concentrated in rat brain capillary fraction, but claudin- mrna did not. these results suggest that claudin- is a dominant tjs protein in bcecs, and expression of claudin- and - , which was reported as tj protein in bcecs, are not restricted in bcecs. os p- - effects of hydrogen peroxide towards gap junction communication in astrocytes and permeability of blood brain barrier f. ahmad , a. pauzi m. yusof , p.d. mourad , m. bainbridge , s. ab ghani universiti sains malaysia; university of washington, seattle, usa; brody school of medicine, east carolina university, nc, usa h o is the main peroxides produced in mammalian cells that consume o . the main source of h o in the brain, produced in large amount, was from the superoxide dismutase catalyzed reaction in mitochondria. therefore, we look into the effects of h o towards the gap junction communication in astrocytes and permeability of blood brain barrier. in this study, by using a h o microsensor, we investigated the level of h o in the brain that altered the permeability of bbb. the microsensor was implanted in the rat's brain and operated amperometrically. we measured h o level from the current generated by the electron transfer at the electrode. we observed a change in permeability when external h o was injected into the brain. fatality occurs when the injected h o exceeds m. these finding showed that the altered paracellular permeability in the presence of h o is caused by a series of events that happen one after another. research funds: short term grants pkimia and pfar-masi os p- - somato-ovarian sympathetic reflex discharges in anesthetized rats sae uchida, fusako kagitani, harumi hotta dept. auton. nerv. syst., tokyo metropol. inst. gerontol., tokyo, japan ovarian sympathetic efferent reflex discharges caused by single electrical shock stimulation of spinal (t - ) afferent nerves or limb (tibial) afferent nerves were studied in urethane anesthetized rats. in central nervous system (cns) intact rats, stimulation of the t - spinal afferent nerve produced early and late a-reflex discharges, and a late c-reflex discharge. after spinalization at the third thoracic level, stimulation of the same spinal afferent nerve produced an a-reflex with the same latency as the early a-reflex in cns-intact rats and an early c-reflex discharge with the similar latency as the late a-reflex in cns-intact rats. on the other hand, stimulation of the tibial afferent nerve produced late a-reflex and c-reflex discharges in cns intact rats, those were not observed after spinalization. it was concluded that ovarian sympathetic aand c-reflex discharges evoked by stimulation of a segmental spinal afferent nerve in cns-intact rats are of spinal and supraspinal origin, and those evoked by tibial nerve stimulation are of supraspinal origin. os p- - responses of renal sympathetic nerve activity and sodium excretion to days sodium loading in rats misa yoshimoto, nozomi iinuma, rie itokawa, eri hayashi, kenju miki integrative physiol. grad. sch. humanities and sci. nara-women's univ., nara, japan in the present study, a month recording of renal sympathetic nerve activity (rsna) in freely moving rats was made to explore the long-term regulation of rsna and sodium excretion. wistar male rats were instrumented chronically with electrodes for the measurements of rsna and electrocardiogram. after the days recovery period, rsna, heart rate and sodium balance were measured over three weeks. animals were allowed to drink four different concentration of sodium chloride solutions ( , , , meq./l nacl) over days. the sodium loading with meq./l nacl suppressed rsna significantly and then it gradually recovered while either meq./l nacl or meq./l nacl loading had no effects on rsna. sodium excretion changed significantly in proportion to the each sodium loading levels. these results indicated that the changes in rsna were not always correlated with the changes in sodium excretion in rats. os p- - cross correlation analysis of respiratoryrelated optical imaging signals yoshitaka oku , haruko masumiya , yasumasa okada dept. physiol., hyogo col. med., nishinomiya, japan; dept. med. keio univ. tsukigase rehab. ctr. shizuoka, japan we aimed to establish an objective method to identify the distribution of respiratory-related regions and the timing when these regions are activated relative to the inspiratory activity from optical imaging signals. optical signals were recorded from the ventral medullary surface of neonatal rats in vitro using a voltage-sensitive dye. cross correlation between integrated c ventral root (c vr) activity and each pixel was calculated after cycle-triggered averaging and detrending. the maximum of cross correlation coefficients and the lag at which the cross correlation became maximal (lagmax) were displayed as d pseudocolor maps. in all preparations, two respiratory-related regions were consistently identified: ( ) a continuous column extending from the para-facial region to the pre-bötzinger complex, and ( ) a region corresponding to the ventral horn. pixels where lagmax were negative (meaning that the activity preceded the c vr activity) tended to be distributed in the para-facial region, and this tendency was more evident when superfusate ph was lowered. os p- - slow afterhyperpolarization determines the firing pattern of action potentials in rat gnrh neurons masakatsu kato, yasuo sakuma department of physiology, nippon medical school, tokyo, japan gonadotropin-releasing hormone (gnrh) neurons play a pivotal role in the hypothalamo-pituitary-gonadal axis. gnrh neurons must be able to continuously fire in response to depolarizing stimuli. for this type of firing, gnrh neurons may have a certain intrinsic property. to address this issue, we investigated the ca + -activated voltage-independent k + currents underlying afterhyperpolarization. dispersed gnrh neurons from adult gnrh-egfp transgenic rats were cultured overnight and used for an electrophysiological experiment with perforated patch clamp configuration. the gnrh neurons showed a slow afterhyperpolarization current (i sahp ). in contrast to previous reports, the i sahp observed in rat gnrh neurons was potently blocked by an sk channel blocker apamin. in current clamp condition, gnrh neurons evoked a train of action potentials to depolarizing current pulse. apamin increased the susceptibility to spike failure. the results indicate that rat gnrh neurons exhibit an apaminsensitive i sahp , which regulates the firing pattern. research funds: kakenhi , os p- - the effect of music to sex hormones of elderly person hajime fukui , kumiko toyoshima , kiyoto kuda , katsuhiko iguchi nara univ. of edu., nara, japan; grad. school of human sciences, osaka univ. japan; nara city medical clinic, japan it has been known that testosterone or estrogen protects nervous system and regulates cell death in a brain. also, it is pointed out that the decline of t and est accelerates depression. therefore the treatment such as hormone replacement therapy (hrt) has been tried to cure depression and alzheimer's disease. however, it has been pointed out that hrt has serious side effects. on the other hand, there are reports that music influences on a steroid hormone. in addition, it is known that music has certain therapeutic gain toward ad and dementia. in this study, from a point of view of the prevention of ad and dementia, we examined the effect of music to sex hormones of normal elderly person. four males and females participated music session and t and est were evaluated. as a result, in female, in the high hormone group, the values decreased after the session, and in the low hormone group, the values increased. from above, there might be possibility that through a steroid hormone music participates in protection and improvement of function on brain. tuberculous meningitis (tbm) is the most common form of chronic infection of the central nervous system. despite the magnitude of the problem, the general diagnostic outlook is discouraging. this study identifies a specific protein marker in csf, which will be useful in early diagnosis of tbm. we have demonstrated the presence of a -kda protein band in csf of % (n = ) of confirmed and % (n = ) of suspected tbm patients out of tbm patients. the -kda protein band was analyzed by lc-ms/ms analysis. in the present study we have identified two mycobacterial proteins rv c (ag a) and rv c (ag b) and one host derived protein as the components of the tbm specific -kda protein. involvement of mitochondrial extrinsic and intrinsic apoptotic pathways in dopaminergic neurodegeneration was tested in rotenone-and mpp + -induced rat models of parkinsonǐs disease (pd). hplc-ec, patch clamp, fluorimetry, immunoblot and rt-pcr were used for measuring neurotransmitters/free radicals, membrane currents, caspases activities, levels of proteins and mrna of mitochondria-linked signaling in brain. we report here a retrograde mode of neuronal death via mitochondrial intrinsic pathway in mpp + -, but an extrinsic mode of cell death in rotenone-induced model. drug screening in these models (l-deprenyl as positive control) indicated that quercetin, coenzyme q , vitamin d and melatonin act via interfering the signaling events in neurons. loss of complex-i and -iv activities and changes in some of the protein subunits in pd postmortem brains were confirmed in pd and control cybrids. results from the present study provide evidences for a direct involvement of mitochondria and are suggestive of existence of both intrinsic and extrinsic apoptotic pathways in dopaminergic neuronal death. os p- - involvement of thioredoxin on the neuroprotective effect of (−)-deprenyl tsugunobu andoh , boon chock , dennis l. murphy , chuang c. chiueh dept. applied pharmacol., univ. toayama, toyama, japan; lab. bioch., nhlbi, nih, md, usa; lab. clin. sci., nimh, nih, md, usa; cent, brain diseases and aging, taipei med. univ., taipei, taiwan the present study investigated whether the induction of thioredoxin (trx) involves in the cytoprotective mechanisms of (−)-deprenyl which is known as the inhibitor of mao-b. after confirming (−)-deprenyl protects against mpp + -induced cytotoxicity in human sh-sy y cells, we observed further that (−)-deprenyl induced trx for protection against oxidative injury caused by mpp+. the induction of trx was blocked by pka inhibitor through a pka-sensitive phosphoactivation of map kinase erk / and the transcription factor c-myc. (−)-deprenyl-induced trx and associated neuroprotection were concomitantly blocked by the antisense against trx mrna in human sh-sy y cells. consistently, trx increased the expression of mnsod and bcl- supporting cell survival. in conclusion, (−)-deprenyl augments the gene induction of trx leading to elevated expression of antioxidative mnsod and antiapoptotic bcl- proteins for protecting against mpp + -induced neurotoxicity. os p- - pgd induces neuronal apoptosis via d- , -pgj tatsurou yagami , noboru okamura , toshiyuki sakaeda facul. health care sci., himeji dokkyo univ., himeji, japan; kobe univ. grad. sch. med., japan prostaglandin d (pgd ) is abundant in the brain, but its neuropathologic role has been unclear. here, we found that pgd induced neuronal apoptosis in rat cortical cultures. however, a pgd receptor blocker did not suppress neurotoxicity of pgd . little pgd receptor was detected, suggesting an involvement of pgd metabolites in the apoptosis. among pgd metabolites, -deoxy- , -prostaglandin j ( d- , -pgj ) caused neuronal apoptosis most potently and rapidly. although d- , -pgj is an endogenous ligand for peroxysome proliferator-activated receptor ␥ (ppar␥), ppar␥ activators did not kill neurons, suggesting that d- , -pgj induces apoptosis independently of ppar␥ activation. we found specific binding sites of [ h] d- , -pgj (jbs) in plasma membranes. there was a close correlation between the neurotoxicity of various eicosanoids and their affinity for jbs. in conclusion, we demonstrated that pgd induced apoptosis via d- , -pgj in rat cortical neurons, and suggested that jbs in the plasma membrane was involved in the d- , -pgj -induced apoptosis. yoshiki iwamoto, daisuke umetsu, shigeru ozaki, naohito terui department of physiology, university of tsukuba, tsukuba, japan stability of a driver's head is crucial for clear vision and consistent, smooth operation of a vehicle. we reported last year that bilateral sternocleidomastoid muscles (scm) of drivers showed a symmetrical increase in activity during forward acceleration of a vehicle. in the present study, we analyzed the relationship between scm activity and vehicle acceleration. emgs of the right and left scm of drivers were recorded during rapid forward acceleration. the time course of the rectified, smoothed emgs did not match that of vehicle acceleration. for a given acceleration, emg was larger when acceleration was increasing than when it was decreasing. we compared emgs and a linear sum of acceleration and its time derivative, jerk. with optimal weights for the two variables and a proper time lag, the linear sum reproduced the emg profile. the optimal weight and lag varied across subjects and vehicles. we suggest that the jerk-related muscle activity may be necessary to quickly restore proper head position after sudden acceleration. grasping is a highly developed movement in primate including human. in contrast to the well-known involvement of cerebral cortex, role of spinal neurons in controlling this behavior has never been examined. here, we show the first direct evidence suggesting the significant contribution of spinal neurons. we trained japanese monkeys to perform the precision grip task, pinching the two springloaded levers with their index finger and thumb, and recorded neural activities through an oval recording chamber implanted over the cervical spinal cord (c to t ). majority of the recorded neurons showed movement-related modulation of firing rate, and the modulation sometimes started before movement onset. spike-triggered averaging of muscle activities revealed some neurons had post-spike effects to hand muscles, suggesting that spinal neurons were capable to generate and modulate muscle force during precision grip. we suggest that primate spinal neurons have a significant role in preparation and execution of grasping movement. research funds: kakenhi os p- - compartmentalization of the cerebellar nuclei: aldolase c expression and the olivonuclear projection pattern izumi sugihara, yoshikazu shinoda dept. systems neurophysiol., tokyo med. & dental univ., tokyo, japan the cerebellar cortex is compartmentalized into more than longitudinal stripes by the aldolase c (=zebrin) expression pattern, which is tightly correlated with the topographic olivocortical projection. however, no equivalent compartmentalization has been known in the cerebellar nuclei. we mapped aldolase c labeling of terminals of purkinje cell axons and anterograde labeling of collaterals of olivocerebellar axons in the rat cerebellar nuclei. the cerebellar nuclei were divided into the caudoventral aldolase c-positive and rostrodorsal negative parts, indicating purkinje cells in the positive and negative stripes in the cortex project to the caudoventral and rostrodorsal parts in the nuclei, respectively. olivonuclear projections showed clear topography within these parts, which was completely congruent with the olivocortical topography. these results clarified the compartmentalization of the cerebellar nuclei and supported that the aldolase c expression is tightly related with the functional organization of the cerebellum. we examined a context dependency of neuronal activity of the pedunculopontine tegmental nucleus (pptn) in monkeys during visually guided saccade tasks. about half of movement-related activities occurred for only the saccades to the saccade target in the task, but they did not occur for the saccades outside the task. on the other hand, for the other half of neurons, movement-related activities occurred for every saccade regardless of the task condition. for visual responses, some neurons responded either the initial fixation point or saccade target, and others responded equally to both stimuli. we further analyzed mutual relationship among modulation timing, preferred direction, effect of reward expectation and this context dependency of the activities, and discussed the visuo-motor processing of pptn. in the reinforcement learning theory, the midbrain dopamine (da) neurons send reward prediction error signal to the striatum. the cholinergic pedunculopontine tegmental nucleus (pptn) is one of the strongest excitatory input sources to da neurons. we hypothesized that pptn may play an important role for relaying necessary components of reward prediction error signals to da neurons. during recording of pptn neurons, we utilized reward predictable visually guided saccade tasks where a shape of fixation point indicated a reward volume. for more than half of the neurons, which showed cue related responses, the cue responses were dependent on association of cue feature and reward size. from another population, we recorded reward related activity. in conclusion, pptn neurons may relay both reward and reward prediction signals, sufficient for computation of reward prediction error. research funds: kakenhi ( ) os p- - timing activity in supplementary eye field during a saccadic eye movement task shogo ohmae , xiaofeng lu , , yusuke uchida , toshimitsu takahashi , , shigeru kitazawa , dept. of neurophysiol., juntendo univ. grad. sch. of med., tokyo, japan; crest, jst, tokyo, japan to act properly in our daily life, the ability to detect and predict timing of events is always required. how do we deal with timing in the brain? to address this question, we trained two japanese monkeys to perform a visually guided saccadic eye movement task in which the monkeys made saccades to each of targets following a gosignal given at a random timing between and ms after the appearance of the target. we recorded neuronal activity from the supplementary eye field (sef) during the task. we found a group of cells that showed activity related to the length of the delay period from target-on to the go-signal. these cells were classified into two types: ( ) those that showed buildup activity during the delay period until the go-signal, and ( ) those that displayed changed activity after the go-signal in relation to the length of the delay period. the results suggest that sef is involved in timing the onset of the go-signal during the saccadic eye movement task. in reaching, a spatial visuomotor transformation should occur in our brain. we can make the transformation not only when the relationship between visual and motor coordinates is default, but also when a gain for the relationship is changed, for example, in a microsurgery. we trained monkeys to make reaching movements when visuospatially identical targets were presented on a computer display by aligning a cursor that indicated their hand position, while the gain was systematically changed. we recorded and analyzed movement-related neuronal activity in the ventral premotor cortex (pmv) and the primary motor cortex (mi) during reaction time. it was revealed that a majority of the mi neurons and a part of the pmv neurons showed activity changes depending on executed movement direction, amplitude, and velocity, whereas a number of the pmv neurons exhibited activity consistent to the visual location of the targets, but not to motor parameters such as amplitude and velocity. the results indicate that the pmv contributes to gain control of reaching during visuomotor transformation. local oscillatory changes in the human sensorimotor cortex induced by simple motor tasks were investigated using supragyral and intrasulcal surface electrodes which was temporarily implanted for the treatment of intractable deafferentation pain. time frequency spectrogram and coherence between electrodes revealed that, before and after several hundred milliseconds of the motor execution, the coherence in the premotor cortex increased cooperatively between neighboring electrodes but that the coherence in the intrasulcal primary sensorimotor cortex decreased exclusively. this result reflects that the premotor cortex plays a role in motor planning with diffuse network while the primary motor cortex plays a role in selective motor execution with local motor output unit. the human sensorimotor processing may be hierarchical and similar to an artificial neural computer. we have shown that the trigeminal oral nucleus (vor) neurons with the receptive field in the intraoral structures project bilaterally to either the jaw-closing (jc) or jaw-opening (jo) motor nucleus in the cat. it is known that neurons in the somatosensory cortex project to the trigeminal sensory nuclei in the rat. thus, we conducted this study to reveal whether there are vor neurons that receive cortical projections and project to the jc or jo nucleus in the rat. we injected a retrograde tracer, fluorogold (fg), in the vor, and found many retrogradely labeled neurons in the contralateral rostral primary somatosensory cortex (si). thus, we injected an anterograde tracer, biotinylated dextranamine (bda), in the rostral si, and also fg in the jc or jo nucleus in the same animals. we found a considerable number of fg-labeled vor neurons made contact with bda-labeled axon terminals. these results suggest that si neurons control jawreflexes through vor neurons. tsunehiko kohashi, yoichi oda grad. sch. science, nagoya univ., nagoya, japan the mauthner (m) cells, paired large reticulospinal neurons in teleost hindbrain, are known to initiate fast escape from sudden aversive stimuli. to investigate how the fast escape is established during early developmental stages, we examined motor performance of the escape in zebrafish embryos or larvae, and the contribution of mcell activity on the behavior. the rostral portion of the zebrafish, - h post fertilization (hpf), was embedded in agar and the tail flip in response to water pulse applied to the head was examined. thirty hpf embryos, in which m-cell has already received trigeminal nerve innervation and is still extending its axon in the spinal cord, showed tail flips contralateral to the stimulated side with longer latency (> ms) than larvae (> hpf, ms). m-cell activity monitored with confocal ca + imaging during the tail flip (> hpf) tightly correlated with the initiation of fast escape, whereas delayed escapes without m-cell firing appeared in some cases (< %) after hpf. thus, the development of the escape behavior coincided with that of m-cell circuit. junctophilins (jps) expressed in the er/sr interacts with plasma membrane thereby constructing junctional membrane complexes (jmc). we here report that lacking neural jps subtypes exhibit an irregular hindlimb reflex and impaired memory. to define neural mechanism of memory deficit in jp-dko mice, we performed whole-cell patch clamp recording of hippocampal neurons. in wild mice, an obvious afterhyperpolarization (ahp) was observed and its ahp was totally blocked by apamin. by contrast, ahp was absent in the jp-dko mice and was insensitive to apamin treatment. the er ca + release through ryanodine receptors, triggered by glutamate receptor-mediated ca + influx, is essential for the activation of sk channels toward ahp generation in the hippocampal neurons. therefore, jp-mediated jmc formation likely plays an essential role in neural excitability underlying neural plasticity and memory. os p- - distribution of voltage-gated calcium channel ␣ ␦- mrna in mouse central nervous system takeshi houtani, satoru sakuma, masahiko kase, tetsuo sugimoto department of anatomy and brain science, kansai medical university, moriguchi, osaka - , japan the ␣ ␦ subunits are the auxiliary subunit of voltage-gated calcium channels and modulate the biophysical properties of the pore-forming ␣ subunits. these auxiliary subunits are composed of four genetically different molecules, ␣ ␦- to ␣ ␦- . the distributions of ␣ ␦- , - , - mrna have been intensively investigated in the rat central nervous system by in situ hybridization, but that of ␣ ␦- remains to be determined. we cloned ␣ ␦- cdna fragment from mouse brain by rt-pcr and examined the distribution of ␣ ␦- mrna-expressing cells in the mouse central nervous system by in situ hybridization using digoxigenin-labeled crna probe. while the ␣ ␦- mrna was found to be broadly expressed, some neuronal types or sites such as piriform cortex, hippocampal pyramidal cells, paraventricular hypothalamic nucleus, facial nucleus and motor neurons of the ventral horn had intense mrna expression. our results suggest that ␣ ␦- subunit may play an important role in learning and memory, neuroendocrine secretion and somatic motor control. the mushroom bodies of insect brains are essential in associative olfactory learning. here we show that the drosophila larval mushroom body calyx, the dendritic region, is organized in about glomeruli, which we have mapped. individual glomeruli receive specific innervation from second order olfactory neurons. by contrast, they contain dendrites from hundreds of mushroom body neurons (kenyon cells), which show low specificity for individual glomeruli. glomeruli therefore potentially transmit specific sensory inputs to a large fraction of kenyon cells. quantitative analysis of dendritic termini of single larval-born kenyon cells suggests that they arborize in about glomeruli in an apparently random manner. this pattern of connectivity is consistent with a model in which kenyon cell dendrites process olfactory input by a combinatorial mechanism that allows the discrimination of a large number of odors. withdrawn os p- - hypothalamic defense reaction involves purkinje cells in the flocculus folium p via orexin and gaba in anesthetized rabbits, electric stimulation in the hypothalamic defense area either excited or inhibited "simple spike" discharges in purkinje cells located in folium p of the flocculus. iontophoretic application of an orexin antagonist (sb ) depressed the excitation, while bicuculline depressed the inhibition. h or h histamine antagonist had no effect. labeling orexin fibers by immunocytochemistry showed that they were most numerous in folium p as compared with other folia of the flocculus. stimulation of the hypothalamic defense area produced little field potentials in the folium p unlike those evoked by mossy fibers. these observations suggest that the excitation and inhibition are mediated by orexin-containing fibers, which contact purkinje cells directly and also indirectly via other gabaergic neurons. os a- - activity-dependent development of corticispinal synapse in mouse slice co-culture takae ohno, masaki sakurai dept. physiol., teikyo univ. sch. med., tokyo, japan we showed nmda-dependent synapse elimination of corticospinal (cs) tract in vitro in rat. in order to use the genetically modified mice to study the underlying molecular mechanisms of this developmental plasticity, we studied development of cs synapses in c bl/ mice. by recording field epsp (fepsp) along m interval lattice in the spinal gray matter in response to the stimulation of deep cortical layer, we evaluated spatial distribution of synapse formation quantitatively. fepsps were recorded diffusely throughout the spinal gray matter at - div, then the amplitudes of fepsps in the ventral side began to decrease at - div, and dominated in the dorsal area at div. cs axon terminals labeled anterogradely with biocytin distributed diffusely throughout the spinal gray matter at - div but the axons terminals in the ventral area were eliminated until div. this synapse elimination from the ventral side was blocked by apv application from div, indicating that this process is also nmda-dependent. in slice coculture study, we showed that corticospinal (cs) axons grow rapidly and reach the ventral spinal gray until div. the number of those ventral axons is reduced before div. to study the behavior of the cs axons at the single axonal level, we transfected a small number of cortical neurons with eyfp expression vector pcag-eyfp by way of electroporation to visualize them and took the time-lapse images of their axons under the confocal microscope equipped with an on-stage co incubator. some axons showed rapid growth, reaching the ventral most part of the spinal gray matter already at div. some axons had collaterals at the dorsal part and retracted the ventral branch while extending the dorsal branch during - div. some ventral axons showed a fragmented tip during retraction, which was indicative of axonal pruning. these observations provide direct evidence that there are early cs axons that once reach the ventral spinal gray and then retract to stay dorsally. we identified click-iii/camki␥ as a novel brain-enriched isoform of the camk-i family that was lipid-anchored by multiple lipid modifications, prenylation and palmitoylation, resulting in enrichment of click-iii into lipid rafts fractions. in situ hybridization revealed the abundant presence of click-iii transcript throughout the central nervous system in mouse embryos. to test the role of click-iii during early neuritogenesis, a shrna vector specific for click-iii was delivered into dissociated cortical culture. we found that knock-down of click-iii resulted in significant decrease in the number and total length of dendrites. results from introduction of click-iii into a click-iii-null context confirmed this finding. surprisingly, lipid modifications of click-iii seemed to contribute to fully elicit such an effect. we thus uncovered a novel signaling mechanism by which lipid raft insertion and local activation of a camk can be efficiently coupled to actin cytoskeletal signaling during dendritogenesis. os a- - transcription factor control of dendrite arbor ultrastructure adrian moore, reiko amikura, shiho nakao, andrew liu, emi kinameri riken brain science institute, japan the different functions of neurons in a complex nervous system are reflected in a large diversity of dendrite arbor morphologies. the drosophila larva dendritic arbourization (da) neurons consist of four classes (i-iv) with increasing levels of arbor complexity. these diverse arbor shapes develop due to class specific mechanisms of dendrite branching and outgrowth. here we show that these class specific differences in dendrite arbor morphology are controlled by a combinatorial code of transcription factors. we have developed a system to label individual dendrite arbors then subsequently identify them in electron microscopic sections. using this method we illustrate that the dendrites of class i neurons, with a simple arbor, contain a high density parallel array of microtubules; on the other hand class iv neurons, with a complex arbor, contain a low density meshwork of microtubules. we are presently investigating how these differences in ultrastructure are controlled by the transcription factors making up the combinatorial code. os a- - segmental and hox related cues are involved in the establishment of the somatotopy yasunori murakami igbmc, strasbourg, france in the rodent, trigeminal sensory inputs are topographically relayed, and mapped in the somatosensory cortex. little is known about the mechanism underlying the development of the somatotopic organization. by fate mapping of specific rhombomeres (r), we found that principal sensory (prv) neurons derived from r receive predominantly inputs from the maxillary branch of the trigeminal nerve and uniquely contribute to the whisker map. by conditional inactivation, we found that early expression of hoxa in r is required for pathfinding and positioning of trigeminal nerve afferents. at later stages, hoxa expression in prv neurons provides instructive cues for topographic arborization of maxillary axons. moreover, while prv neurons appeared normally specified, loss of hoxa function resulted in selective loss of eph expression, and altered axonal projections from prv to the ventral posterior medial (vpm) nucleus of the thalamus, and absence of a postnatal whisker map at any level of the neuraxis. thus, hoxa dependent cues are required to determine the territory for whisker representation in r and the assembly of a somatosensory circuit. os a- - the second wave of corticospinal innervation after synapse elimination of the first wave tsutomu kamiyama, masaki sakurai dept. physiol, teikyo univ. sch. med., tokyo, japan in the previous study we showed that the rat corticospinal (cs) terminals and synapses were widely distributed at p and those in the venrtolateral (vl) area were eliminated from p to p and that the number of terminals in the dorsomedial (dm) and vl area began to increase again from p and further increased thereafter. in the present study we further studied the subsequent developmental time course of cs terminal distribution. cs axons were anterogradely labeled by injection of biotin dextrane (bda) into the sensorimotor cortex. the number of the terminals began to increase from p , reaching peak around the third postnatal week. labeling of single or a few by microinjection axons revealed that at p some additional cs axon branches appeared within the dorsal column of the target spinal segment and further ramified after entering the gray matter. however, the number of axons did not increase in the brainstem and the upper cervical cord. these suggest that the second wave of innervation is explained mainly by branching of cs axons just before and after entering the spinal gray matter. os a- - proteomics of the growth cone: i. protein profiling of the growth cone the growth cone is a motile tip formed at the developing neuronal processes, and functions for the accurate determination of the axon pathway and the synaptogenesis. in higher organisms, however, the molecular basis of the growth cone is poorly understood for the present, since the information on the protein localization there is insufficient to explain the growth cone functions. proteomics is a powerful strategy for identifying the protein composition in a given cell or a subcellular compartment, and the application of this method to the growth cone should help us solve the above question. we obtained the whole growth cone (gcp) obtained from neonatal rat forebrain and the membrane subfraction of the gcp (gcm), and then those fractions were analyzed using proteomics. we have identified several hundreds of the distinct proteins of these fractions. here, we show the profiling of gcp and gcm, and will discuss the overview of these protein profiles in relation to the growth cone functions. axonal branching is thought to be regulated by not only genetically specified molecules but also neuronal activity. however, the interplay between these two mechanisms remains largely unknown. to study this issue, we analyzed the role of electrical activity in layer-specific thalamocortical (tc) axon branching by using organotypic cocultures. during the second week in vitro, yellow fluorescent protein-labeled tc axons formed branches primarily in the target layer. spontaneous firing was found to increase when branches were formed abundantly. pharmacological blockade of synaptic transmission diminished layer-specific branching considerably. moreover, time-lapse imaging showed that branching was generated dynamically by elimination as well as addition in the target layer and that blockade of synaptic activity reduced this remodeling. these findings suggest that synaptic activity modifies layer-specific tc axon branching by regulating the remodeling process with molecular cues expressed in the target layer. research funds: kakenhi ( ), kakenhi ( ) os a- - the application of navigation-guided repetitive transcranial magnetic stimulation for intractable deafferentation pain naoki tani, yoichi saitoh, haruhiko k., satoru oshino, masayuki hirata, amami katoh, toshiki yoshimine department of physiology, university of osaka, osaka, japan repetitive transcranial magnetic stimulation (rtms) has been applied to control intractable deafferentation pain (dp). but nobody has investigated which cortical area is the most effective target for pain relief. therefore, we stimulated m , s , sma, premotor accurately with a navigation-guided rtms and compared their effects of pain relief. at the same time, rtms ( , , hz, stimulations) was compared in dp patients. the pain relief was evaluated with visual analogue scale. high frequency ( , hz) rtms of m was the only effective stimulation for treating intractable pain in of patients ( %). the pain relief continued for h significantly. we would like to discuss the mechanism of pain relief with high frequency rtms of m . os a- - involvement of atp on nociceptive modulation in rat model of masseter muscle pain yasuo sugiura, noriyuki ozaki, masamichi shinoda department of functional anatomy and neuroscience, nagoya university graduate school of medicine, nagoya, japan we determined the role of p x r on pressure pain and mechanical hyperalgesia in a newly developed rat model of pain in masseter muscle (mm) . the pain in the mm was assessed by the pressure pain threshold (ppt) defined as the amount of pressure required to induce head flinching. the mm injection of ␣,␤-meatp (p x , , / rspecific agonist) significantly enhanced the behavioral response to the pressure. this enhanced response was completely blocked by the co-application of ␣,␤-meatp with ppads (p x , , , , / , / r-specific antagonist). excessive muscular contraction of mm produced by the electrical stimulation significantly decreased the ppt indicating mechanical hyperalgesia of the mm. administration of ppads to the exerted mm produced a complete recovery of decreased ppt. p x rpositive neurons innervating the exerted mm increased in trigeminal ganglia. our results suggest that p x r plays an important role in pressure pain, and mechanical hyperalgesia caused by excessive muscular contraction of mm. the present study was undertaken to investigate the change in the activation of the nociceptive neuronal circuit under a neuropathic pain-like state. here we found sciatic nerve ligation (snl) produced a marked increase in the number of c-fos-positive cells in the periaqueductal gray (pag). using the fluoro-gold (fg) microinjection into the pag, numerous fg-labeled cells were detected in the hypothalamus. in the arcuate nucleus (arc) of the hypothalamus, the immunoreactivity (ir) for an excitatory neuronal maker, fosb was increased, whereas the ␤-endorphin (␤-ep)-ir was decreased days after snl. furthermore, the subpopulations of ␤-ep-positive cells were co-labeled with fosb in the arc. the present data suggest that the hypothalamus can be received by snl-induced concomitant nociceptive signals, leading to continuous activation of neurons projecting to the pag. this phenomenon, in turn, indirectly controls pain transmission in the dorsal horn through the descending antinociceptive pathway. os a- - the cantor-like patterns in rat hippocampal ca pyramidal neurons tsuda and kuroda proposed a mathematical model for the cantor coding in the hippocampal ca . this prediction includes an attractor dynamics expected in the associative network, which was proposed by many authors, since marr's theory of simple memory in the hippocampus. however, our mathematical model is too abstract to describe physiological feature of neurons. then, we have tried to find cantor-like patterns experimentally from the ca pyramidal neurons. temporally associated and non-associated electrical stimulations were delivered to schaffer collaterals, and membrane potentials were recorded by patch-clamp recording method. in our results, cantor-like patterns were observed in hippocampal ca pyramidal neurons. young songbirds shape their songs using memorized tutor songs and auditory-vocal feedback. we prevented zebra finches from hearing their own vocalizations by exposure to loud noise after days of age, before which they had been reared with song tutors from birth. when the noise stopped at - days of age, the birds sang unstable and noisy song syllables that did not resemble the tutor syllables. the similarity to the tutor syllables steadily increased until the time of song crystallization ( days later). these findings show that the memory of tutor syllables still exists well beyond the normal age of song crystallization (d of age) and that zebra finches can develop songs using the memory well after the normal period of song development. the temporal order of syllables resembled the tutor model only in birds released from the noise before days of age. thus, different schedules and processes may govern the learning of syllable phonology and syntax. in addition to well-characterized areas, a novel adult neurogenic region; the temporal germinal layer (tgl) was identified in rats (takemura, ) . a tracer study revealed that there is an interconnection between the dorsal part of the tgl and the lateral nucleus of the amygdala, suggesting a functional implementation of tgl neurogenesis in amygdala-dependent emotional memory processing. to investigate this possibility, we performed a tgl region-specific low-dose irradiation, which can selectively kill proliferating cells and hence can reduce neurogenesis, using a gamma knife. the tgl-irradiated rats expressed a significantly increased tone-related long-term fear memory, indicating a functional significance of the tgl neurogenesis for aversive memory reduction. we (tsukada and pan, ) systematically examine the functional difference between spatio-temporal learning rule (stlr) proposed by tsukada ( ) and hebbian learning rules in a single-layered neural network, computing their ability to differentiate spatiotemporal sequence. in this paper, we tested physiologically the cooperative plasticity without a postsynaptic spike in the ca hippocampal network. tsuda and kuroda proposed a mathematical model for the cantor coding in the hippocampal ca . they also predicted chaotically transitory dynamic behavior called chaotic itinerancy in the hippocampal ca . this prediction includes an attractor dynamics expected in the associative network, which was proposed by marr and others. the time series of events, which could be output from ca , may be encoded in ca in an efficient way. the proposed cantor coding is effective, because the topology of time series is naturally measured on the cantor set since each element of cantor set represents a single time series. however, our mathematical model is too abstract to describe physiological feature of neurons. then, we have tried to make more realistic model of ca , using -compartment model of neuron, and we found the cantor coding of information of time series in the model ca . it is known that neurons can propagate action potentials with high temporal precision. however, it is unclear how precisely closely neighbouring neurons synchronize and whether they can code information. here we show that sub-millisecond synchronization can code information as well as the discharge rate modulation. we found that closely neighbouring pyramidal neurons in the ca region of the hippocampus synchronize with sub-millisecond precision. the optimal frequency bands for transmitting these synchronizations matched the beta, gamma and fast-ripple oscillations. moreover, we found that the synchronizations were commonly coupled with rate modulations in relation to both internal (retention and comparison) and external (stimulus and motor) events. the synchronization often occurred in relation to stimulus inputs even when rate modulation was clearly absent. therefore, our results suggest that sub-millisecond synchronization plays an important role in propagating information in the hippocampus. the alterations of cerebral motor function by chronic ischemia are poorly understood, since no motor symptoms are noticeable in most of the cases. we evaluated spatial distribution and intensity of eventrelated desynchronization of beta band (beta-erd) evoked in motor area using synthetic aperture magnetometry in patients with chronic ischemia due to diverse vascular occlusive diseases (n = ) and moyamoya disease (n = ). contrary to the normal motor activation, ipsilateral beta-erd was dominant during grasping task of affected hand in patients. this abnormal activation was obscured by self-paced finger tapping requiring more selective hand motor programming. and it was more frequently observed in the atherosclerotic hypoperfusion (with white matter change) than in other pathogenesis. ipsilateral beta-erd may be a new indicator of subclinical functional alteration in motor cortices caused by chronic ischemia. os a- - hypothermia protects against cerebral ischemia by suppressing ␦pkc activation takayoshi shimohata , , heng zhao , gary steinberg department of neurology, brain research institute, niigata university, niigata, japan; department of neurosurgery, stanford university, stanford, usa hypothermia protects the brain from ischemia, but the underlying mechanisms of this effect are not fully elucidated. ␦pkc is reported to induce apoptosis upon activation. its activity is modulated by phosphorylation, translocation and proteolytic cleavage. we investigated effects of hypothermia on ␦pkc activation using a rat permanent distal mca occlusion model. mild hypothermia ( • c) reduced infarct size by %. western blots indicated that ␦pkc cleavage increased markedly in ischemic core but moderately in penumbra after stroke, which is suppressed by hypothermia (p < . ). p-␦pkc (t ) dephosphorylated after stroke; this effect is blocked by hypothermia. full-length and cleaved form ␦pkc as well as p-␦pkc (s ) translocate from the cytoplasm to the mitochondria and nucleus, which is suppressed by hypothermia. ␦pkc activator suppressed the protective effect of hypothermia. taken together, hypothermia blocks ␦pkc activation after focal ischemia. this effect might contribute to hypothermic neuroprotection. calcium responses in situ following ischemia remain unclear. we sought to determine, in rats, the calcium changes following transient forebrain ischemia. in anesthetized adult rats, -vessle occlusion was induced. fluo- /am was microinjected, and the fiber-coupled confocal microscope [imaging fiber bundle coupled to the microlensattached nipkow-disk scanner (csu- , yokogawa, japan) equipped with × objective lens] was inserted into the brain. -vessle occlusion induced comparable ischemia in both hippocampus and frontal cortex. fluorescence intensity of fluo- increased up to %, and persistently increased up to % during -min reperfusion, indicating the long-lasting ca + increase in the ca region. in contrast, in the frontal cortex, -min ischemia increased fluorescence intensity during ischemia but not reperfusion. in the ca region but not in the frontal cortex, transient forebrain ischemia induces long-lasting increase in ca + in situ. research funds: kakenhi # , # os a- - reevalution of classical view on resident microglia: neutrophils may play more critical roles than resident microglia at acute phase of ischemic and traumatic brain insults hiroaki matsumoto , h. watanabe , y. kumon , t. ohnishi , chi ii , y. imai , j. tanaka dept. neurosurgery, ehime university, japan; dept. molecular and cellular physiology, ehime university, japan resident quiescent microglia (mg) are thought to respond quickly to a variety of pathologic events in the brain, by proliferating and producing a number of bioactive substances including proinflammatory cytokines and nitric oxide (no). in the present study, however, we found that the majority of resident mg died through apoptosis within h after the onset of ischemic and traumatic brain insults. we further noticed that traditional mg markers isolectin b and cd b recognized with ox antibody histochemically stained neutrophils, which were identified by neutrophil-specific elastase, rather than iba + mg or macrophages. accumulation of neutrophils was observed at the very early phase of the insults, while they expressed proinflammatory cytokines and inducible no synthase. iba + amoeboid-shaped mg started to accumulate days after the insults. the data prompted us to reevaluate the roles and the fate of resident mg in the brain. os a- - insulin regulates the hepatic clearance of amyloid ␤ peptide tetsuya terasaki , , chihiro tamaki , sumio ohtsuki , graduate school of pharmaceutical sciences, tohoku university, sendai, japan; sorst, jst, japan the liver is the major organ that eliminates amyloid ␤-peptide (a␤) from the circulation, and we have revealed that low-density lipoprotein receptor-related protein (lrp- ) is a molecule responsible for the hepatic clearance. since epidemiologic investigations suggest the high incidence of alzheimer's disease in diabetes mellitus, the purpose of this study was to clarify the effect of insulin on the hepatic clearance of a␤ . insulin infusion into the rat portal vein increased lrp- expression in plasma membrane fraction of liver, but did not affect the expression in whole lysate. insulin treatment also increased the hepatic uptake of a␤( - ), which reached . -fold greater uptake than non-treated control after min treatment. increase of the hepatic uptake of a␤( - ) by insulin was concentration dependent (ec = pm), and was completely suppressed by rap ( m), an lrp inhibitor. these results suggest that insulin induces translocation of lrp- to the plasma membrane of hepatocytes, leading to increase of a␤ hepatic clearance from the circulation. research funds: sorst, jst os a- - mr images of intra-arterially administered microglia surrounding ␤-amyloid deposit in the rat brain the therapeutic use of microglial cells has recently received some attention for the treatment of alzheimer disease (ad), but few noninvasive techniques exist for monitoring cells. here we present a magnetic resonance imaging (mri) technology to track micrgolia cells injected intra-arterially in a rat model of ad. we labeled microglia expressing gfp with resovist using the hvj-e vector. we administered labeled microglia into the carotid artery of the rats. mri revealed clear signal changes attributable to resovist-containing microglia in a␤-injected areas. this study demonstrates the usefulness of mri for non-invasive monitoring of exogenous microglia, and suggests a promising future for microglia as therapeutic tools for ad. extravasation of protease-activated receptor (par) activators, such as thrombin, into brain parenchyma can occur after blood-brain barrier breakdown in a number of cns disorders, which causes pathophysiological changes in neurons and glial cells. to elucidate the mechanism of thrombin-induced activation of astroglial cells, we used n astrocytomas that show a characteristic retraction of bipolar protrusions after activation of pars with thrombin. the thrombin-induced morphological change of n cells was inhibited by an inhibitor of ip receptors, -aminoethoxydiphenyl borate ( -apb) or an endoplasmic reticulum ca + -atpase inhibitor, cyclopiazonic acid (cpa). in parallel, thrombin-induced mobilization of ca + was inhibited by -apb and cpa. moreover, removal of external ca + accelerated the reversal of thrombin effects. these results suggest that refilling of ca + store by ca + entry play an important role in the cytoskeletal dynamics of astroglial cells. to clarify the occurrence range of neurofibrillary tangles (nft), we reexamined an autopsied alzheimer patient with the onset at age and a -year-clinical course. the brain showed severe atrophy ( g). microscopic examination disclosed that all telencephalic neocortices had nft of more than and sp of more than . all limbic cortices and nuclei had nft of more than and sp of more than . although there was no sp, various numbers of nft were observed in the following structures: claustrum , caudate , globus pallidus , hypothalamus , meynert's nucleus , thalamus , substantia nigra , central gray , locus ceruleus , purkinje cells , posterior root ganglion , adrenal medulla . this study revealed that there exist nft-rich neurons and free neurons. the latter includes purkinje cells and posterior root ganglion cells. considering the pathogenesis of nft, it must be valuable to clarify qualitative/quntitative differences between nft-rich neurons and free neurons. os a- - transcriptional regulation of androgen receptor in aging mouse brain androgen receptor (ar) mediates action of androgen, which is involved in memory, behavior and other brain functions that deteriorate with advancing age. in aging mice brain, ar mrna expression was measured by rt-pcr, ar promoter methylation by southern hybridization, and proteins binding to promoter by emsa. ar mrna level was significantly higher in male than female, and it was downregulated by testosterone, but upregulated by estradiol in adult mice. female mice exhibited higher methylation of ar promoter than males. methylation was increased by testosterone, but decreased by estradiol. furthermore, dnasei accessibility to ar promoter was reduced in males, increased by gonadectomy but reduced by sex steroids in adult male. incubation of brain nuclear extract with plabeled ar promoter yielded three specific complexes. the intensity of these complexes varied with age and sex. these findings show that ar mrna expression and promoter methylation are inversely regulated by sex steroids in the adult mice cerebral cortex. such regulation of ar expression might influence androgen action and consequently brain function during aging. reliability of synaptic transmission depends on the efficiency of transmitter removal from the synaptic cleft, as well as on the release machinery and the postsynaptic response mechanism. it has been shown in various synapses that postsynaptic and glial excitatory amino acid transporters (eaats) contribute to glutamate removal. however, the role of presynaptic eaats remains unclear. using mouse retinal slices, we examined the contribution of eaats at the rod to rod bipolar cell (rbc) synapse. the kinetics of the rbc current evoked by electrical stimulation of rods was slowed by pharmacological blockade of eaats. recordings of the evoked rbc currents from eaat subtype-deficient mice and the eaat-coupled anion current revealed that functional eaats are localized to rod terminals but not to postsynaptic or glial cells. model simulations suggest that rod eaats are densely packed near the release site, and that rods are equipped with an almost self-sufficient glutamate recollecting system. trpv is a thermosensitive trp channel, and activated by body temperature. we found functional-trpv was expressed in soma, dendrites and synapses in the neurons. since trpv was firstly cloned as an osmotically activated channel, we hypothesized trpv might be involved in volume regulation of the spines. therefore, we quantified the spine volume changes by glutamate stimulation, and confirmed trpv expression related to the volume increase of spines. next, we compared the resting membrane potential (rmp) between wild type and trpv -deficient neurons at • c, and found rmp in wild type was more depolarized by approximately mv than rmp in trpv -deficient neurons. we also performed current-injection experiments in both neurons, and found that trpv -deficient neurons required much bigger currents to get their firing. thus, we conclude that trpv is involved in regulation of both neural activity and spine motility in hippocampus. os p- - a system for rapid uncaging in defined patterns and its application hiroshi kojima department of intelligent information systems, tamagawa university, tokyo, japan neurons integrate many sysnaptic signals at dendrite. understanding these information processes is a central topics in experimental and computational neuroscience. the use of focused laser beam for uncaging can provide fine spatial resolution to analysis of neural function. however, most experiments were carried out either at spatial locations or in a very simple scanning patterns. we developed a system for performing uncaging in arbitrary pattern in order to emulate realistic neural activity. our system is capable of patterned photorelease of caged neurotransmitters at locations per ms with submicron resolution. ultraviolet laser light is steered by galvano-mirrors and projected onto the surface of preparations for uncaging the caged chemicals. simultaneously, imaging of neurons are obtained by -photon microscopy and electrophysiological experiments can be done. we briefly report the present system for rapid uncaging and its application to neurophysiological research. os p- - d -like receptors selectively block p/q-type calcium channels to glutamate release onto cholinergic neurons in the rat basal forebrain a number of molecules have been identified in the sensory ganglia including those involved in the signal transmission to the brain. their functions, however, remain largely unknown. we tried to develop a method enabling to inhibit gene expression in the sensory ganglia in vivo by rnai and to evaluate its effect on the synaptic transmission in the brain slices. for this purpose, we selected the nodose ganglion (ng), in which the neurons sending glutamatergic projections to the nucleus tractus solitarii in the brainstem, are located. in anesthetized young wistar rats, synthetic sirna against the genes coding adenosine a receptors (adora ) was introduced to the ng by electroporation. one to five days after sirna delivery, the expression level of adora in the ng decreased by > % of that in the non-treated ng, being not accompanied by a change in mrna level for a a receptors. this technique might be promising in analyzing the function of specific molecules involved in transmitter release regulation at the brain synapses. nmda-receptors are specific constituents of glutamatergic system in brain responsible for molecular mechanisms of recognition and learning. activation of neurons by nmda results in intracellular generation of reactive oxygen species (ros) and reorganization of cell metabolism. exposure of rodent and human lymphocytes with nmda results in ros increase within the cells which is suppressed by nmda antagonists. moreover we have demonstrated by rt-pcr technique and by using anti-nmda-antibodies the expression of nmdareceptors on lymphocyte membranes. in addition, we shown that nmda receptor dependent signal from lymphocyte membrane is transformed into specific intracellular reactions controlling caspase- activity and interferon-␥ synthesis. in the presentation, properties of nmda-receptors and their functional role in immunnocompetent system are discussed. small molecule g-protein arf in combination with phospholipase d (pld) is essential for intracellular trafficking of the proteins from endoplasmic reticulum to golgi apparatus. however, it is recently reported that it also regulate ionic channel activity at the cytoplasmic membrane. to examine possible involvement of arf and subsequent pld in regulation of receptor-induced responses in neurons, we recorded k + -current response to dopamine (da) in the ganglion cells of aplysia under conventional two-electrode voltage clamp. intracellular application of arf blockers such as brefeldin a, exo , and arf n-terminal peptide, markedly suppressed the da-induced response. furthermore, intracellular application of ␣-synuclein, a specific blocker of pld, significantly depressed the k + -current response to da. these results suggest that arf and subsequent pld may regulate the k + -current response induced by da. os p- - p gap, a brain-enriched rhogap, is involved in the nmdar-mediated signaling takanobu nakazawa , toshihiko kuriu , ayako m. watabe , toshiya manabe , shigeo okabe , tadashi yamamoto div. of oncology, inst. med. sci., univ. of tokyo, tokyo, japan; dept. of cell biol., tokyo medical and dental univ., tokyo, japan; div. of neuronal network, inst. med. sci., univ. of tokyo, tokyo, japan nmdar regulates structural plasticity by modulating actin organization within spines. however, the signaling pathways that link nmdar activity to the postsynaptic actin cytoskeleton are poorly understood. we identified a brain-enriched rhogap, p gap, which interacts with the nr b subunit of nmdar. within neurons, p gap was highly concentrated in the postsynaptic density and co-localized with nr b and an actin-binding protein, cortactin. p gap promoted gtp hydrolysis of cdc and rhoa in vitro and in vivo. nmdar stimulation led to de-phosphorylation and redistribution of p gap. when over-expressed in dissociated neuron, p gap suppressed the activities of rho gtpases, which resulted in spine elongation. taken together, the results suggest that p gap is likely to be involved in nmdar activity-dependent actin re-organization in spines. os p- - non-static method to directly quantify the transfer of firing correlation from one neural population to another: fokker-planck method hideyuki cateau riken brain science institute, saitama, japan firings of only a few neurons are too weak to be transmitted safely, to activate other neurons to fire, or to contract muscles. therefore, we implicitly assume that brain function is exerted by macroscopic population of neurons. to characterize how a macroscopic neural population behave, the simulation method provide an indirect approach. many single neuron simulation runs need to be performed first before extracting macroscopic features by statistically averaging. unlike this method, the fokker-planck (fp) method directly evaluates the macroscopic features, thereby giving a clearer insight into function achievable with neuronal population. despite the lasting interests in firing correlation in coding and conveying information, theoretical studies on it have been largely confined to complicated simulation studies. here, we provide a first non-static fp analysis to directly calculate how correlation and population rates are transferred from one population to another and elaborate a dynamical interplay between these macroscopic quantities at work in time. os p- - spatial frequency tuning of disparity-selective neurons in macaque v hironori kumano , seiji tanabe , ichiro fujita grad. sch. of engineering science, osaka univ., osaka, japan; grad. sch. of frontier biosciences, osaka univ., osaka, japan to examine whether convergence across spatial frequency channels contribute to stereoscopic processing, we recorded single neuron activity from area v of awake, fixating monkeys. for each neuron tested, we first measured the spatial frequency tuning with sinusoidal gratings or two-dimensional ( -d) filtered noise images, and then examined the disparity tuning with both correlated and anticorrelated dynamic random-dot stereograms (rdss). neurons with broader spatial frequency tuning had more attenuated disparity tuning for anti-correlated rdss. in a subset of v neurons, we analyzed responses to various combinations of binocular disparity and spatial frequency by using -d filtered noise stereograms. the disparity tuning of most v neurons was consistent across a range of spatial frequencies to which they were sensitive. we suggest that v neurons pool disparity signals across spatial frequency channels to create an unambiguous representation of stereoscopic depth. os p- - predicting the monkey's behavioral choice in a stereoacuity task from neuronal responses in area v hiroshi shiozaki, seiji tanabe, ichiro fujita lab. cognitive neurosci., grad. sch. frontier biosciences, osaka univ., japan many neurons in visual area v of macaque monkeys are selective for binocular disparity. most disparity-selective neurons in v are sensitive to small changes in disparity near zero, suggesting that they might contribute to stereoacuity. however, the role of these neurons in stereoscopic depth discrimination has not been directly addressed. we recorded single unit activity from v while a monkey was engaged in a fine stereoscopic depth discrimination or stereoacuity task. the monkey was trained to report by saccadic eye movement whether the center region of a random-dot stereogram was nearer or farther than its immediate surround. trial-to-trial fluctuation of visual responses of v neurons was correlated with the monkey's subsequent behavioral choice. given the cell's disparity preference, an ideal observer can predict the monkey's upcoming behavioral response from the visual response of v neurons. the results suggest that v neurons are involved in mediating stereoacuity. os p- - the role of disparity energy and binocular matching processes in stereopsis takahiro doi, seiji tanabe, ichiro fujita lab. cognitive neurosci., osaka univ., japan the early visual system computes disparity energy of stereo images. some of the next stages retain this information, while other stages perform further computation to solve the stereo correspondence problem. we addressed how the energy and correspondence computations underlie stereopsis. we asked human subjects to discriminate depth of random-dot stereograms with various amounts of disparity. at each disparity level, we manipulated the proportion of dots with the same luminance contrast between the two eyes by reversing the contrast of some dots in one eye. at small disparities, the proportion of correct choices increased monotonically from chance to perfect as the proportion of the same-contrast dots was increased. at large disparities, the subjects perceived reversed depth when contrastreversed dots dominated, and the proportion of correct choices reached only chance level when the two types of dots were balanced. the results suggest that the correspondence and energy computations underlie fine and coarse stereopsis, respectively. we introduce a novel receptive field (rf) analysis, lsrc, which can reveal various aspects of visual receptive fields that were undetectable previously in a single measurement. the visual stimuli are standard wide-field -d ternary dynamic random noise, generally refreshed every - ms. unlike the conventional reverse correlation which computes a spike-triggered average (sta) of the stimuli themselves, lsrc computes the sta of the spectra of localized regions of the stimuli. both simulations and recordings from cat v /v neurons demonstrate that lsrc is capable of revealing details of complex cell rfs, cross-orientation suppression, variations of orientation tuning within rfs that might lead to shape selectivites. since the stimuli can cover a wide visual field area, and few assumptions are made regarding specific shapes or features in stimuli, lsrc is highly suitable for multi-neuron, multi-area studies spanning retina, v , and especially areas beyond. research funds: mext( ), jsps( ), coe os p- - analysis of center-surround organization of v neurons as a high-order receptive field hiroki tanaka, izumi ohzawa graduate school of frontier biosciences, osaka, japan responses of area (v ) neurons are influenced by stimuli not only in their classical receptive field (rf) center, but also in its surround. such a center-surround organization may be considered as a unified higher-order rf. we have sought to obtain detailed structures of such a rf by harmonic analyses of responses to drifting contrast-modulated sinusoidal gratings that cover both the center and surround regions. of cells analyzed, % showed spatial frequency tuning curves that were well fitted with gaussian. by taking the inverse fourier transform of these curves, spatial center-surround rf was obtained as gabor functions with spatial phases between ± degrees. highly asymmetric structures were observed for cells with strong surround suppression. estimated sizes of center and surround were well correlated with those from size tuning curves. moreover, there was no space-time tilt in the center-surround rf. the results suggest that neurons with surround suppression are capable of coding various spatial forms of higher-order features (figure-ground borders), but are insensitive to motion of such stimuli. os p- - spatial organization of receptive fields of complex cells in the early visual cortex kota sasaki , izumi ohzawa , grad. school of eng. sci., osaka univ., japan; grad. school of frontier biosci., osaka univ., japan little is known about the quantitative internal structure of the receptive fields (rf) of complex cells, although this is crucial for understanding how a complex cell acquires its function by collecting inputs from neurons in the preceding stage. therefore, we have analyzed the relationship between the spatial nd-order interaction kernels and the rf envelopes of complex cells. extracellular single unit recordings were performed in anesthetized and paralyzed adult cats. threevalued (i.e. gray, dark, and bright) dynamic white noise stimulus with × dots was presented over an area to times larger than the rf of a complex cell. for each dot location, a nd-order kernel and its envelope (by hilbert transform) were calculated. the rf envelope of the neuron was determined by summing the envelopes of nd-order kernels at all locations. nd-order kernels had roughly comparable extent as the rf, and contained . subregions on average (n = ). among complex cells, whose rf envelopes were elongated, cells exhibited the horizontal elongation. research funds: mext( ), jsps( ), coe os p- - orientation tuning of neuron in cat lateral geniculate nucleus tomoyuki naito , osamu sadakane , masahiro okamoto , hironobu osaki , hiromichi sato grad. sch. med., osaka univ., osaka, japan; grad. sch. front. biosci., osaka univ., osaka, japan; med. sch., osaka univ., osaka, japan we examined the orientation selectivity of lgn neurons of anesthetized cats and found that although about % lgn neurons showed significantly orientation-biased response to the grating with optimal size and spatial frequency (sf), and that % of lgn neurons exhibited significant orientation selectivity to gratings with diameter larger than its classical receptive field (crf) and sf higher than the optimal for crf response. two stimulus-size tuning curves measured for responses to stimulation with the optimally-or null-orientated grating exhibited profile similar to each other under the optimal sf condition. however, high sf grating caused stronger surround suppression for response to the orthogonally oriented stimulus than that to the optimally orientated stimulus. our results suggested that elliptic crf center produces orientation-biased response of lgn neurons. furthermore, surround suppression of lgn neurons tuned to particular stimulus orientations enhances orientation selectivity of lgn neurons. os p- - temporal dynamics of suppressive receptive field surround in cat v satoshi shimegi, hiroyuki kida, ayako ishikawa, hiroshi sakamoto, hiromichi sato graduate school of medicine, osaka university, toyonaka, japan in the primary visual cortex (v ), a neuronal response to stimulation of the classical receptive field (crf) is suppressively modulated by the stimulus presented at the receptive field surround (srf). using stationary flashes ( ms) of sinusoidal grating with optimal parameters and varying radii as stimuli, we examined the temporal dynamics of the surround suppression in v cells of anesthetized cats. stimulus slightly larger than the crf caused suppression in early response (< ms) but not in middle ( - ms) and late responses ( - ms). as stimulus size was further enlarged, the middle and late responses were remarkably suppressed while the early response was only moderately or weakly suppressed. radius of surround suppressive field progressively expanded in temporal sequence from . deg (early response) to deg (middle response) and . deg (late response). thus, modulation of early response seems to reflect whether stimulus is larger than crf size or not, and late response to reflect how wide area is stimulated. research funds: kakenhi ( ) os p- - spatial-frequency dependent surround suppression in cat v ayako ishikawa , satoshi shimegi , hiroyuki kida , hiromichi sato grad. sch. front. biosci., osaka univ., osaka, japan; grad. sch. med., osaka univ., japan; grad. sch. eng. sci., osaka univ., japan we examined the temporal dynamics of the surround suppression of visual response in terms of spatial-frequency (sf) tuning of neurons in cat v . we used a stationary flash (duration, ms) of a circular sinusoidal grating patch with optimal orientation and sf as crf stimulus, and that of an annulus ( ms) with optimal orientation but varying sf as srf stimulus. first, we stimulated crf and srf simultaneously (stimulus-onset-asynchrony (soa) = ) and analyzed time course of surround suppression. sf tuning of the surround suppression changed along time course of response, and effective sf of surround suppression shifted from the sf lower than that optimal for crf response (c-sf) to that near c-sf. next, changing soa, we examined surround suppression on different temporal phases of crf response. soa-dependency of surround suppression changed according to the temporal phase of response. these results suggest that multiple mechanisms with different sf-and temporal characteristics are involved in the surround suppression. os p- - contrast-dependency of spatial summation property in cat v and lgn masahiro okamoto , tomoyuki naito , osamu sadakane , hiromichi sato grad. sch. front. biosci., osaka univ., japan; grad. sch. med., osaka univ., toyonaka, japan we examined contrast-dependent change in a receptive field (rf) size and strength of surround suppression of neurons in the primary visual cortex (v ) and the lateral geniculate nucleus (lgn) of anesthetized cats. rf structure was modeled by spatial interactions of excitatory and inhibitory gaussians. both in v and lgn, ratio of gaussians (rog) model captured size-tuning curves of responses better than difference of gaussians (dog) model. under the high contrast stimulus condition, the peak of size tuning curve shrank by . and . times in v and lgn, respectively. in lgn, surround suppression was strengthened under high contrast stimulus condition, but in v , the strength of surround suppression did not affected by stimulus contrast on average. we conclude that ) rog model describes the surround suppression better than dog model both in v and lgn, ) under high contrast stimulus condition, there is a reduction of rf size with a shrinking of excitatory gaussian, which is confirmed with rog model. hiroyuki kida , satoshi shimegi , ayako ishikawa , hiroshi sakamoto , hiromichi sato grad. sch. eng. sci., osaka univ., japan; grad. sch. med. sci., osaka univ., japan; grad. sch. front. biosci., osaka univ., japan in the primary visual cortex (v ), neuronal responses to stimulation of the classical receptive field (crf) were suppressed by the presence of stimuli at surround receptive field (srf). we examined whether the suppression varied according to spatial configuration of srf stimuli in v neurons of anesthetized cat. the crf stimulus was a circular patch of sinusoidal grating with optimal stimulus parameter. srf was divided into flanks ( • step), and stationary stimulated with an annulus, oppositely-faced flanks ( -fk) or a flank ( -fk) stimulus. the durations of stimulus presentation were ms for crf and ms for srf stimulation. localized srf stimulation with either -fk or -fk exerted significant suppression on crf responses. according to the analysis of spatiotemporal change in srf effects, there was no particularly suppressive srf area for -fk stimulation throughout the crf response. however, -fk stimulation of end position to crf had strong and long-lasting suppression on responses during - ms after onset. os p- - temporal-frequency dependency of receptive field size and surround suppression in lgn and v osamu sadakane , tomoyuki naito , hironobu osaki , masahiro okamoto , hiromichi sato grad. sch. med., osaka univ., japan; med. sch., osaka univ., japan; grad. sch. front. biosci., osaka univ., osaka, japan spatial summation property of neurons in the primary visual cortex (v ) varies depending on stimulus parameters (e.g., stimulus contrast). in this study, we examined how temporal frequency (tf) of grating stimulus affects size-tuning properties of cat v neurons. our results showed that, when the tf was higher than the optimal, the strength of surround suppression became weak and receptive field size became larger, suggesting that v neurons change their spatial property according to tf in such a way that neurons integrate wide visual field for fast moving stimulus, whereas localized field for slow stimulus. we also tested the effect of changing stimulus size on tf tuning curve. consistent with above-mentioned results, large grating made the peak and the high cut-off of tf-tuning curve higher than those for small grating. in the lateral geniculate nucleus (lgn), we obtained basically similar results to those of v neurons, suggesting that the subcortical tf tuning property contributes to that in v . ryo sasaki, takanori uka department of physiology (i), juntendo university, tokyo, japan a few studies have shown that basic tuning functions in early visual cortex change during visual perceptual learning (schoups et al. ; yang and maunsell ) . the change in neuronal sensitivity in these studies, however, is small compared to the improvement in behavioral sensitivity. here we hypothesized that the read out of information from sensitive neurons was modified by learning. to test this hypothesis, we investigated whether learning modifies neuronal sensitivity or read out of middle temporal (mt) neurons during learning of a depth discrimination task. two monkeys were trained to report the depth of moving dots (near or far), and we recorded from isolated mt neurons during the course of training. the monkeys showed improvement in discrimination thresholds across daily sessions. in contrast, the sensitivity of mt neurons did not change, whereas the correlation between neuronal activity and the monkey's behavioral choice increased during the course of training. these results suggest that plasticity due to perceptual learning occurs within the neural pathway following area mt. we developed an in vivo method to localize the fine tip of a glassinsulated tungsten microelectrode for chronic recording using . t mri. the scan conditions were first optimized by imaging a microelectrode that was sunk into copper sulfate solution. the microelectrode tip was precisely localized up to a resolution of m under particular geometrical scan condition. we then examined the applicability of the method in vivo under this optimized scan condition in the temporal cortices of three monkeys. the microelectrode was penetrated into the dorsal or ventral bank of the superior temporal sulcus and the tip was localized by the high-resolution mri. the accuracy of this method was validated by comparing the localized positions of the microelectrode tips with the corresponding electrolytic lesion marks in histological sections. a transient signal change in diffusion-weighted image of the brain has been detected in human visual cortex. the time course of this signal was ahead of the bold signal and characterized by a steep onset. diffusion-mri thus represents a new exciting mechanism for fmri. in order to increase its efficiency we aimed at defining a diffusion response function (drf) as a counterpart of the hemodynamic response function (hrf). an volume of interest was defined using spm with a boxcar function. gamma-variate functions were used to model the steep onset. the parameters of the drf were estimated by fitting the time-course with the drf convolved with a boxcar. although the magnitude of the signal change (around %) was smaller than that of bold (> %), the temporal profile showed a constant precedence of the diffusion signal by . s. os p- - new insights on normal and pathological brain function from tomographic analysis of magnetoencephalographic signals laboratory for human brain dynamics, brain science institute (bsi), riken, wako-shi, japan tomographic analysis of magnetoencephalography (meg) data combines exceptional temporal resolution with accurate localization, at least for places a few centimeters away from the center of the head [moradi, et al., neuroimage; ioannides et al., cerebral cortex] . this unique capability of probing brain function across the entire cortex and deep brain structures from milliseconds to minutes in the same experiment has already provided new insights about normal [ioannides et al., cerebral cortex;ioannides et al., neuroimage] and pathological [ioannides et al., j. neurosc.] brain function. novel ways of analyzing meg data provide direct measures of regional brain activity over much longer timescales. these new methods are used in ongoing studies to probe the nature of global brain activity in different states of awareness (e.g. different stages of sleep) and explore the relationship between estimates of electrophysiological activity derived from meg with hemodynamic measures of brain activity. os p- - spatial registration of stand-alone fnirs data to mni space ippeita dan, archana singh, masako okamoto national food research institute, japan the registration of functional brain data to the common brain space offers great advantages for inter-modal data integration and sharing. however, this is difficult to achieve in functional near-infrared spectroscopy (fnirs) because fnirs data is primary obtained from the head surface and lacks structural information of the measured brain. therefore, we present a method for probabilistic registration of fnirs data to the standard montreal neurological institute (mni) template through international - system without using the subject's magnetic resonance image (mri). the standard deviation in probabilistic registration thus performed for given head surface points is approximately within cm. this means that if the spatial registration error is within an acceptable tolerance limit, it is possible to perform multisubject fnirs analysis to make inference at the population level and to provide information on positional variability in the population, even when subjects' mris are not available. stochastic perturbation in scale is a basic property of biological systems and generates scale-independent structuration and functional dynamics in spatial and temporal patterns, which can be characterized by fractal dimensionality. it allows a user-independent evaluation and does not rely on subjective evaluation in image assessment. we have used a box-counting algorithm in scale-space segmented images to determine the mass fractal dimension of ventricles in different neurological disorders. three groups of subjects [alzheimer disease (ad), obstructive hydrocephalus (oh) and normal controls] were examined. mass fractal dimension is high for ad ( . ), approaching unity (∼ . ) for oh, and in between for control ( . ). statistical analysis was performed and significant differences were observed for these groups (p < . ). the observations are accounted by a flow dynamics heterogeneity model. the implications are that stochastic structuration and fractal dimension may be useful to track temporal progression of disease and assess therapeutic management. thrombin, a serine protease essential for blood coagulation, also plays an important role in injury associated with intracerebral hemorrhage. in this study, we revealed that mitogen-activated protein kinase (mapk) pathways contribute to thrombin-induced brain injury in two experimental models. firstly, we employed organotypic cortico-striatal slice cultures. application of thrombin to slice cultures resulted in cortical neuronal injury and striatal shrinkage. the cortical neuronal injury was ameliorated by inhibition of extracellular-signal regulated kinase (erk) but not p mapk, while the striatal shrinkage was prevented by both of them. secondly, thrombin was injected into rat striatum. thrombin-induced brain injury determined by immunoreactivity of neuronal marker was reduced by inhibition of erk and p mapk. these results suggest that mapk pathways play important roles in thrombin-induced brain injury and they should be therapeutic targets against neurodegeneration associated with blood-brain barrier destruction. positron emission tomography was used to study brain activations during motor imagery of standing and during performance of standing posture in parkinson's disease (pd). eight pd patients performed mental and motor tasks: ( ) resting, ( ) staring at a standing human object, ( ) thinking of standing, ( ) standing with eyes open, ( ) standing with eyes closed. regional cbf data analyzed by spm were compared with normal counterparts. the cerebellar vermis was more activated during imagination of standing in the pd group than in healthy group. as seen in healthy subjects, standing also activated the primary sensorimotor foot area and cerebellar vermis in pd patients, but the between-group comparison generated greater activations in the vermis and prevuneus in pd. the cerebellar vermis engages in postural balance both in mind and reality, and the precuneus may play a more important role in postural control in pd. os p- - potentiation of nmda receptor-mediated current by metabolic failures through glycine release facilitation in the hypoglossal motoneurons of the rat yu kono , , eiji shigetomi , kiyoharu inoue , fusao kato dept. neurol., jikei univ., sch. med., tokyo, japan; lab. neurophysiol., jikei univ., sch. med., tokyo, japan to elucidate the mechanism underlying the selective vulnerability of motoneurons (mns) to metabolic failures (mfs), we compared the membrane current responses of mns and non-mns to mfs. experiments were performed on neurons in the hypoglossal nucleus (xii) and dorsal motor nucleus of the vagus nerve (dmx) in the young rat brainstem in the presence of ttx. mfs were induced by nacn or oxygen deprivation. in xii neurons, mfs induced large persistent inward currents accompanied by marked increase in strychnine-sensitive synaptic inputs, indicating facilitation of glycine release onto xii neurons. furthermore, nmda receptor-mediated current evoked by exogenous nmda was increased by nacn. in dmx neurons, mfs evoked outward currents without affecting synaptic inputs. these pre-and postsynaptic responses to mfs in mns might play a role in their selective vulnerability in various neurodegenerative diseases including the amyotrophic lateral sclerosis. os p- - effects of mdma on serotonergic neurons in rat organotypic mesencephalic slice culture including the raphe nuclei yuichi suzuki, megumi higuchi, takayuki nakagawa, shuji kaneko dept. mol. pharmacol., grad. sch. pharmaceu. sci., kyoto univ., kyoto, japan , -methylenedioxymethamphetamine (mdma) is a recreational drug of abused which has been shown to increase serotonin ( -ht) release and cause degeneration of -htergic nerve terminals via -ht transporter, although the mechanisms are unclear. in this study, we developed rat organotypic mesencephalic slice culture including the -htergic raphe nuclei, and examined the effects of mdma and methamphetamine (meth) on -ht release and -htergic neurotoxicity. immunohistochemical studies for tryptophan hydroxylase revealed abundant -htergic neurons around the raphe nuclei. treatment with a -htergic neurotoxin , -dihydroxytryptamine dramatically reduced the tissue contents of -ht and its metabolite, which was blocked by a selective -ht reuptake inhibitor. mdma and meth ( . - m) increased -ht release, and reduced the tissue contents of -ht and its metabolite at higher doses. the mesencephalic slice culture including the -htergic raphe nuclei may be useful to examine the mechanisms underlying -htergic neurotoxic effect of mdma in vitro. os p- - studies on drug dependence (rept. ): involvement of platelet-derived growth factor (pdgf) receptor in the morphine-induced rewarding effect masami suzuki, minoru narita, michiko narita, tomoko takeuchi, yasuyuki nagumo, keiichi niikura, tsutomu suzuki dept. of toxicol., hoshi univ. sch. pharm. pharmaceut. sci., tokyo, japan the present study was undertaken to investigate the involvement of platelet-derived growth factor (pdgf) receptor in the morphineinduced rewarding effect in rodents. extensive coexpression of tyrosine hydroxylase with pdgf receptor was apparently observed in the rat ventral tegmental area (vta). the levels of dopamine and its major metabolites in the nucleus accumbens (n.acc.) were markedly increased by the microinjection of pdgf into the rat vta. the morphine-induced rewarding effect was suppressed by intra-vta microinjection of pdgf receptor fc chimera. the increased level of dialysate dopamine produced by morphine in the rat n.acc. was significantly decreased by intra-vta injection of pdgf receptor fc chimera. these findings suggest that the stimulation of -opioid receptors in the vta by morphine leads to the activation of pdgf receptor, which may be directly responsible for the morphine-induced rewarding effect in rodents. os p- - prostaglandin d is a strong mediator of neuroinflammation in genetic demyelinating mouse model prostaglandin (pg) d , an inflammatory mediator, mainly produced by hematopoietic pgd synthase (hpgds). microglial activation and gliosis are commonly observed during the neuroinflammation. in twitcher (galct wi/twi ), a genetic demyelinating mouse model, we found that hpgds expression was upregulated in activated microglia accompanied by the dp receptor induction in hypertrophic astrocytes. using primary culture of glial cells, we demonstrated that activated microglia produced large amount of pgd by hpgds and that astrocytes expressed both dp and dp receptors and were activated by pgd . we found that gliosis and demyelination were well suppressed in hpgds-or dp -null twitcher and twitcher treated with an hpgds-inhibitor. these results suggest that pgd is a key molecule of neuroinflammation involved in the demyelination. research funds: , os p- - on a sodium channel distribution enabling high frequency signal processing go ashida , , kousuke abe , kazuo funabiki grad. sch. medicine, kyoto univ., kyoto, japan; grad. sch. informatics, kyoto univ., kyoto, japan some auditory neurons, such as the owl's nucleus laminaris (nl) cells, can sense very high frequency signals (up to khz). from the theoretical point of view, it seems exceptionally difficult to handle these high frequency signals because the membrane time constant is far longer. first, we discuss a biophysical mechanism of shifting the membrane time constant by connecting the large cell body (soma) with the small node of ranvier. next, we discuss the effect of sodium channel distribution on the impedance function of the membrane. sodium conductance in the soma amplifies low frequency signal components below khz, while that in the node does up to khz. last, as a typical example, we discuss the capability of high frequency signal processing in the owl's nl neuron. some biological evidences indicate that sodium channels in the nl neuron are distributed mainly in the nodes but less in the soma. by using an nl neuron model, we show that a neuron with low somatic sodium conductance and high nodal sodium conductance can achieve fine sensitivity to high frequency signals. interaural time difference (itd) is calulated using axonal delay lines and coincidence detector neurons (nucleus laminaris:nl). however, little is known about the cellular mechanisms of coincidence detection. here, we report the results of in vivo intracellular recordings from the barn owl's nl. we used coaxial glass electrodes in which one (microelectrode) was inserted into a patch-electrode type capillary. the inner sharp electrode was protected by the outer one during penetration of the cerebellum. we isolated nl cells from owls and achieved intracellular recordings in of them, as judged by a sudden dc potential drop and the resting membrane potential (mean rp = ± mv). nl neurons produced small spikes and oscillatory potentials whose waveform closely resembled the superposition of the tones delivered to the two ears (sound analogue psps:sap). the amplitude of saps varied as a function of itd. spike rates changed in linear proportion to the amplitude of sap. we evaluated sound localization ability of vision impaired and sighted persons by using a 'two-sound sources discrimination test' in a semianechoic darkroom. in total, vision impaired ( blind and low vision) and sighted persons participated. the stimuli were pure tone pulses. for each trial, the same single sound pulse was emitted consecutively from a pair of speakers with the same angle either left or right from the midline of the subject. localization ability was assessed whether the subjects are able to discriminate two sound sources or not in each trial. the discriminability of the blind subjects slightly exceeded that of sighted subjects but the difference was not significant. the discriminability of the low vision subjects, on the other hand, was significantly lower than that of blind or sighted. it was suggested that a peculiar 'object perception' of blind persons is not able to measure by means of 'two-sound sources discrimination test.' os p- - autocrine bmp signaling in astroglia sensitizes the glial scarring masahisa yamada , runa araya , naoto kitamura , yuji mishinsa yamada unit, riken bsi, saitama, japan; nihs, nieh, nc, usa bone morphogenetic proteins (bmps) affect growth of glial cells however, contribution of bmps during glial scar formation is unknown. to study the role of bmp signaling in vivo, we disrupted bmpr a, one of the type i receptors for bmps, in a telencephalic neuronal stem cell-specific manner. we found that aberrant architecture of microvessels that led to a failure in maintaining the blood-brainbarrier in the mutant mice. although mutant mice showed inflammation around the cortical microvessels, proliferation of hypertrophic reactive-astrocytes in the mutant mice was attenuated. disruption of astroglial bmpr a expression by cre-adenovirus recapitulates the same phenomena. bmps were upregulated in reactive astrocytes in after brain injury. knocking down of bmpr a by small interfering rna in primary astrocytic culture negatively affected their astrocytic growth injured by scratch, which reinforced the importance of autocrine bmp signaling in astrocytes. this result opens up the understanding of novel mechanisms underlying the autocrine bmp signaling on glial scarring after cns injury. the present study was undertaken to evaluate the functional role of the glial cells in the induction of stress. here, we found that aging mice promoted anxiety-like behaviors as characterized by both the light-dark and elevated plus-maze tests, and they exhibit an increase in astrocytes in the cingulate cortex. a robust increase in gfap-positive astrocytes was noted in the cingulate cortex of nerve-ligated mice that exhibited the anxiety-like behavior. in contrast, iba -positive microglial cells were dramatically increased as compared to that in control mice and some of them were co-localized with brdu-like immunoreactivity in the hippocampus of mice exposed to chronic psychological stress. our results indicate that the increase in astrocyte or microglia in the cingulate cortex or hippocampus may lead to emotional disorders including aggravated anxiety under aging, chronic pain-like state or exposure to chronic psychological stress. withdrawn os p- - fucosylation prevents overshooting of the migration by the vagus motor neuron precursors shigeharu kinoshita , , hideomi tanaka , , sachiko tsuruoka , hironori wada , , hitoshi okamoto , riken bsi, wako, japan; jst crest, kawaguchi, japan; riken rrc, wako, japan the vagus motor nuclei are important as the autonomic center for the maintenance of homeostasis. aberrant positioning of nuclei is implicated in the etiology of the sudden infant death syndrome (sids). therefore, control of precursor cell migration into the right position may be crucially important. the zebrafish embryo has two vagus motor nuclei, the dorso-laterally and medially located nuclei (dmx and mmx). the dmx precursors are born near the floor plate, migrate dorso-laterally and then are accumulated at the defined position. in the towhead mutant embryos, ectopic neurons are distributed between bilateral dmx where precursors aberrantly migrate in dorsal direction and fail to stop at the right position. positional cloning and mrna rescue analysis identified towhead as a gdp-mannose , dehydratase (gmds), a key enzyme for de novo synthesis of a gdp-fucose. as a result, the mutant embryos showed exclusive reduction of fucosylated glycans. our findings represent that fucosylation is responsible for maturation of these neurons. in development of the drosophila visual center, photoreceptor cells extend their axons (r axons) to the lamina ganglion layer and trigger proliferation and differentiation of synaptic partners (lamina neurons) by delivering the inductive signal, hedgehog (hh). this mechanism helps to establish an orderly arrangement of connections between the r axons and lamina neurons, termed a retinotopic map because it results in positioning the lamina neurons in close vicinity to the corresponding r axons. it is found that the bhlh-pas transcription factor single-minded (sim) is induced by hh in the lamina neurons and is required for the association of lamina neurons with r axons. in sim mutant brains, lamina neurons undergo the first step of differentiation but fail to associate with r axons. as a result, lamina neurons are set aside from r axons. the data reveal a novel mechanism for regulation of the interaction between axons and neuronal cell bodies that establishes precise neuronal networks. research funds: kakenhi ( ) os p- - initial molecular steps in synaptogenesis in vivo: trans-synaptic interaction of cell adhesion molecule is involved in postsynaptic assembly of psd -homolog dlg hiroshi kohsaka, etsuko takasu, akinao nose department of physics, university of tokyo, tokyo, japan trans-synaptic interaction via cell adhesion molecules (cam) is essential in constructing synapse structures. although this notion has been supported by various studies in vitro, evidence in vivo has been lacking. here we used live-imaging and genetic analysis to show that a drosophila cam fasciculin (fas ) mediates early interaction between pre-and postsynaptic cells in synaptogenesis in vivo. by visualizing gfp-tagged fas genetically expressed on a muscle, we found fas accumulated at postsynaptic site just after the contact between growth cones and its target muscle. genetic and deletion analysis implied that trans-synaptic interaction with presynaptic fas is crucial for the postsynaptic localization of fas . in addition, postsynaptic localization of a scaffolding protein dlg, psd -homolog, and glutamate receptors was impaired in fas mutants. these results provide the first in vivo evidence that trans-synaptic cell adhesion molecule has a role in inducing the assembly of synapses. gaudilliere brice harvard medical school, usa postsynaptic differentiation of dendrites is an essential step in synapse formation. we report here a requirement for the transcription factor myocyte enhancer factor a (mef a) in the morphogenesis of postsynaptic granule neuron dendritic claws in the cerebellar cortex. a transcriptional repressor form of mef a that is sumoylated at lys promoted dendritic claw differentiation. activity-dependent calcium signaling induced a calcineurin-mediated dephosphorylation of mef a at ser and thereby promoted a switch from sumoylation to acetylation at lys , leading to inhibition of dendritic claw differentiation. our findings define a mechanism underlying postsynaptic differentiation that may modulate activity-dependent synapse development and plasticity in the brain. research funds: ns , ag ps a-a characterization of mrna species that are associated with postsynaptic density fraction by gene chip microarray analysis we previously reported the partial identification by random sequencing of mrna species that are associated with the postsynaptic density (psd) fraction (tian et al., ) . we report here further characterization by gene chip analysis of the psd fraction-associated mrnas, which were prepared in the presence of rnase inhibitor. we confirmed that a large number of mrna species are associated with the psd fraction and found that mrnas encoding various postsynaptic proteins were highly concentrated in the psd fraction. we identified some mrna species that were highly concentrated in the psd fraction. we also constructed a cdna library using the psd fraction-associated mrnas as templates, and identified randomly selected clones by sequencing. our data suggested that the psd fraction-associated mrnas are a very useful resource, in which as yet uncharacterized genes are concentrated. tian et al., . mol. brain res., , - . research funds: kakenhi ( ) ps a-a the distribution of snap- protein is regulated in isofom-specific manner makoto itakura, saori yamamori, kouta takano, masami takahashi department of biochemistry, kitasato university school of medicine, sagamihara, japan two isoforms of snap- derived from exon splicing are expressed in brain. we generated two specific antibodies for snap- a and b, and studied the distribution in rodent brain. there was a sticking difference in expression of snap- a and b during early postnatal period. snap- b was low at the birth and increased remarkably thereafter. by the contrast, snap- a increased transiently and attained a maximum level around seven day after birth. furthermore, there seemed to be a difference in their distributions in plasma membrane, since a substantial amount of snap- b but not snap- a was recovered in raft-enriched fractions of triton x- -treated lp membrane after sucrose density gradient centrifugation. immunohistochemistry demonstrated that snap- b was widely distributed throughout brain, whereas, snap- a was restricted to some particular regions of brain. these results indicate that expression and distribution of snap- protein are regulated differently in isoformspecific manners, and snap- a and snap- b play different functional roles in brain. ps a-a erc(elks/rab ip /cast) regulates syaptic short-term plasticity by recruiting bmunc - to the active zone camkii in the postsynaptic sites is localized as a psd-anchored or a cytoplasmic form. camkii in the two sites is interchangeable by its translocation. translocation and targeting of this kinase to appropriate subcellular compartments are crucial for its physiological function. we have previously suggested that postsynaptic camkii is also localized in lipid raft microdomain ( . mol. brain res. , - ) . in this report, we proved the lipid raft localization of camkii by detergent-treatment and successive sucrose floatation assay of spm or cos cells expressing camkii, and by cholesterol depletion from membrane using mbcd. we also investigated the mechanism and properties of camkii targeting to lipid raft. camkii targeted to lipid raft microdomain possibly through protein-protein interaction. our data suggest that lipid raft microdomain is a major site of camkii distribution, as well as postsynaptic density and cytoplasmic region, at the postsynaptic site. glial glutamate transporters, glast and glt- , are co-localized in processes of bergmann glia wrapping excitatory synapses on purkinje cells (pcs). although glast is expressed six-fold more abundantly than glt- , the decay kinetics of climbing fiber (cf)mediated excitatory postsynaptic currents (cf-epscs) in pcs in glast(-/-) mice are not significantly different from those in wildtype mice. here we attempted to clarify the roles of glial glutamate transporters in cf-pc synapses using glast(-/-) and glt- (-/-) mice, and a novel antagonist of glial glutamate transporters, ( s, s)- -[ -( -methoxybenzoylamino)benzyloxy]aspartate. our results indicate that glial glutamate transporters can retain the fast decay kinetics of cf-epscs in the normal range if a small proportion (approximately %) of functional transporters, glast and/or glt- , is preserved. glutamate is well known as an essential neurotransmitter in nervous system. how glutamate-mediated synaptic transmission is controlled in neural circuit of live animal, however, remains to be poorly understood. we found that the loss-of-function mutations in vglut (vesicular glutamate transporter) encoded by eat- gene led to abnormal sensory behaviors including thermotaxis in c. elegans. thermotaxis defect of eat- mutant was caused by malfunction of both thermosensory neuron afd and its downstream interneuron ria, suggesting that thermal signals from afd or ria to their downstream neurons are transmitted by glutamate through eat- vglut. a mutation in avr- glutamate receptor also led to abnormal thermotaxis. we are trying to investigate whether avr- functions in the downstream neurons of afd or ria, and to identify other glutamate receptors involved in thermotaxis. through the analysis of thermotaxis neural circuit, we are hoping to reveal the mechanisms of glutamate-mediated synaptic transmission at neural circuit level. ps a-a biochemical characterisation of the vesicular glutamate transporter stephan schenck, shigeo takamori department of neurology and neurological science, st century coe program, tokyo medical & dental university, tokyo, japan vesicular glutamate transporters (vgluts) load synaptic vesicles with glutamate, the major excitatory transmitter in the brain, thus making these transporters of outstanding importance for the function of the central nervous system. the three known isoforms of these secondary active transporters have been characterised in terms of tissue distribution, developmental expression patterns and some pharmacological features. while the third isoform constitutes only a minor fraction, vglut and vglut are abundantly expressed in the brain with a complementary distribution pattern and divergences in the expression profile during ontogeny. so far, no clear difference in the function of vglut and vglut has been found. to further characterise the specific properties of the transporters we make use of the vglut -ko mouse which gives us the opportunity to investigate a brain devoid of vglutl. we focus on synaptic vesicle fractions from ko-mice to study the vglut-associated transport biochemically. in recent years, three isoforms of vesicular glutamate transporters (vgluts) have been molecularly identified in mammals. histological investigations have revealed that the distribution of three vglut isoforms in the cns is largely complementary with limited overlap, suggesting that differential expression of vglut isoforms may contribute to functional diversity in glutamatergic synapses. however, functional differences among the isoforms remained poorly understood. to get insights into their isoform-specific property, we searched for interacting protein(s) to the c-terminus of vglut by yeast two-hybrid screening and found endophilin a . as expected for the interacting molecule to vglut , endophilin a was typically localized to vglut -positive synaptic terminals in cultured hippocampal neurons. we are currently investigating physiological significance underlying their direct interaction and co-localization. the aim of this study is to investigate the molecular basis for lactate utilization. hippocampal neuronal culture was continuously superfused with glucose or lactate solution and spontaneous excitatory postsynaptic currents (sepscs) were recorded from a voltage-clamped pyramidal neuron. in lactate solution, amplitude of epscs was decreased in ∼ min, followed by spontaneously recovered after min, while epsc in glucose medium remained unchanged. application of apv+ni in lactate medium, spontaneous recovery was not observed. in neuron cultures, incorporation of c-lactate was gradually increased, which was suppressed by applications of inhibitors for calcium calcium channels or protein kinase c. in glial cell cultures, incorpotation of lactate was initially maintained. increased expression of monocarboxylate transporter (mct) was demonstrated in the lactate medium. results suggested that increased mct expression of neurons may lead to utilization of lactate to sustain synaptic function via calcium-dependent manner. yumei wu , kazuhito tomizawa , shuang liang , iori ohmori , teiichi nishiki , kohji takei , hideki matsui dept. of physiol., okayama univ., okayama, japan; dept. of neurosci., okayama univ., okayama, japan synaptic vesicle endocytosis is regulated by phosphorylation of endocytotic proteins, such as amphiphysin (amph) i and dynamin i. here, we show a novel type of regulation of vesicle endocytosis by proteolysis. in mouse hippocampal slices, amph i was found to be cleaved by a ca + -activated protease, calpain during prolonged depolarization or stimulus trains. the calpain-cleaved n-terminal amph i fragment lost its ability to bind dynamin and inhibited transferrin uptake as overexpressed in cos- cells, indicating that the calpain cleavage of amph i inhibits endocytosis. amph i in hippocampus was also cleaved by calpain in vivo after kainate seizure. although the second administration of kainate caused less severe seizure activity than the first one, this relieved second seizure was not observed in pre-treatment with a calpain inhibitor, allm during the first seizure. thus, the proteolytic activity of calpain could protect neurons from excitotoxicity by inhibiting vesicle recycling. synaptic vesicles (svs) are effectively recycled by endocytosis for continuous synaptic transmission. previously, we have suggested that a high level of synaptic transmission is maintained by recycling of svs through two types of endocytosis operating coordinately ( th this meeting). in the present study, we labeled endocytosed svs at nerve terminals of drosophila with fluorescence dyes, fm - and fm - , and also measured quantatively exocytosis and endocytosis of svs, using these dyes. egfp-labeled cacophony ca + channels and anti hrp stained the active zone and non-active zone at synapse, respectively. imaging analysis revealed that two distinct types of endocytosis of svs occurred at the active zone and the non-active zone of motor nerve terminals. we have previously shown that baclofen, a gaba b receptor agonist, inhibits exocytosis in synapses of mouse hippocampal neurons. syntaxin a is also known to modulate exocytosis. to characterize the molecular mechanisms involved, the inhibitory effects of baclofen in neurons transfected with antisense oligonucleotide to syntaxin a were investigated by patch-clamp recording and counting the number of release sites. transfected neurons showed higher frequency of miniature epscs and stronger inhibition by baclofen than controls, but no change in number of sites. increased exocytosis is thus induced by increases in transmitter release per site, rather than by more sites due to neurite sprouting. these results suggest that gaba b receptor shares part of the mechanism involved in modulation of exocytosis with syntaxin a in mouse hippocampal neurons. we have previously shown a transient localization of tubulin (tub) during synaptic vesicle (sv) cycling in drosophila nerve terminals. the tub localization is detected during sv recycling, while microtuble (mt)-loop is observed throughout sv cycle. in this study, we characterized the two distinct tub localizations and showed their relation with sv pool formation. axonal mts and mt-loops abounded in acetylated (acetyl) tub. the transient localization was either polymerized or depolymerized, and organized by non-acetyl tub. taxol decreased the non-acetyl tub localization but not mt-loops, and inhibited exo/endo cycling pool (ecp) formation. in boutons containing mt-loops, ecp formation was also inhibited. acetyl mt-loops tend to be stable whereas presynaptic non-acetyl tubs are either free dimers or dynamic mts. these results suggest that presynaptic dynamic tub, especially non-acetyl tub, controls ecp formation. presynaptic tub dynamics may regulate functional presynaptic plasticity by controlling sv pools. research funds: grant-in-aid for jsps fellows the mechanism by which pregnenolone sulfate (pregs) enhances synaptic transmission was studied at the rat calyx of held. pregs increased the amplitude of evoked epscs, without affecting that of spontaneous miniature epscs, indicating that the site of its action is presynaptic. pregs facilitated presynaptic voltage-gated ca + channel (vgcc) currents via accelerating their activation kinetics, but had no effect on k + currents, resting conductance, or action potential waveforms. in simultaneous pre-and postsynaptic recordings pregs did not change the relationship between presynaptic ca + influx and epscs, suggesting that exocytotic machinery downstream of ca + influx was not involved in the pregs effect. neither bapta nor gtp␥s loaded into presynaptic terminals blocked the effect of pregs. we conclude that pregs enhances transmitter release via facilitating vgccs by a novel mechanism, which is independent of intracellular ca + or g-proteins. ps a-b spine targeting of endocannabinoid synthesizing enzyme, diacylglycerol lipase-␣ in the cerebellum and hippocampus endocannabinoids are neuromodulator that is released from postsynaptic neurons, acts retrogradely on presynaptic cb cannabinoid receptor, and induce suppression of transmitter release. to understand the retrograde signaling mechanisms, we investigated subcellular localization of a major endocannabinoid biosynthetic enzyme, diacylglycerol lipase-␣ (dagl␣), in the mouse brain. in the cerebellum, dagl␣ was predominantly expressed in somatodendritic membrane of purkinje cells, and highly concentrated at the base of spine neck. however, dagl␣ was excluded from the main body of spine neck and head. in hippocampal pyramidal cells, dagl␣ was selective to spines, but widely distributed within spines. these results indicate that dagl␣ is essentially targeted to postsynaptic spines in cerebellar and hippocampal neurons, but its fine distribution within and around spines is differently regulated between the two cell types. synprint site of voltage-gated ca + channels interacts with synaptotagmin. however, its physiological role is not entirely clear. here we report that ap- subunit can directly bind with synprint site. this interaction was ca + -dependent, being weaker at concentrations higher than nm. in contrast, the interaction of synaptotagmin with synprint was optimal at m ca + , being weaker at lower or higher concentrations. the binding domain of synprint for ap- and synaptotagmin was indistinguishable, and these proteins competed with each other for the synprint site. to assess physiological role of these interactions, we made a peptide containing synprint site, and loaded it directly into the nerve terminal at the calyx of held. this peptide blocked endocytosis measured with capacitance, and gradually diminished exocytosis upon repetitive presynaptic activations. we conclude that ca + channel synprint site makes ca + -dependent interactions with ap- and synaptotagmin thereby contributing to vesicular endocytosis. ps a-b acl- , an evolutionarily conserved acyltransferase like gene is required for normal synaptic transmission in c. elegans naoko hara, takao inoue, yasukazu takanezawa, hiroyuki arai department of health chemistry, graduate school of pharmaceutical sciences, university of tokyo, tokyo, japan it is generally accepted that various phospholipid molecular species are formed by phospholipids acyltransferase reactions. however, the physiological significance and the molecular mechanism of the remodeling are largely unknown. to address these questions, we focused on evolutionarily conserved acyltransferase like genes in c.elegans acl- ˜ , and generated their deletion mutants. the mutants of acl- gene, which is predominantly expressed in neurons and muscles, showed no apparent phenotype. however, the mutants exhibited severe movement abnormalities in fat- mutant background in which long chain polyunsaturated fatty acids are depleted. pharmacological analysis revealed that these mutants showed presynaptic defects in synaptic transmission. these abnormalities were rescued by neuron specific acl- expression, suggesting that certain phospholipid species produced by acl- are involved in maintaining normal synaptic transmission and motility of c.elegans. daisaku yokomaku , hussam jourdi , akiyoshi kakita , tadasato nagano , hitoshi takahashi , nobuyuki takei , hiroyuki nawa dept. mol. neurobiol., brain res. inst., niigata univ., japan; brain resource center, brain res. inst., niigata univ., japan; dept. pathology, brain res. inst., niigata univ., japan scaffolding proteins containing pdz domains interact with synaptic receptors and cytoskeletal components and are therefore implicated in synaptic development and plasticity. little is known, however, about what regulates the expression of the pdz proteins and how the levels of these proteins influence synaptic development. here, we show that ligands for epidermal growth factor (egf) receptors (erbb ) decrease a particular set of pdz proteins and negatively influence synaptic formation or maturation. in neocortical cultures, egf decreased the expression of grip and sap . moreover, egf treatment resulted in a decrease in the frequency of pan-pdzimmunoreactive aggregates on dendritic processes. these findings revealed a novel negative effects of erbb receptor ligands that attenuates the expression of the pdz proteins and inhibits postsynaptic maturation in developing neocortex. takatoshi iijima , eriko miura , keiko matsuda , tetsuro kondo , , masahiko watanabe , michisuke yuzaki dept. physiol., sch. med., keio univ., tokyo, japan; dept. anatomy, hokkaido univ., sch. med., sapporo, japan; mol. neurophysiol., aist, tsukuba, japan cbln is a member of the c q and tumor necrosis factor families predominantly produced in cerebellar granule cells. recently, we have shown that cbln is secreted as a glycoprotein and plays crucial roles in synaptic plasticity and synaptic integrity of purkinje cells. although other members of the cbln family, cbln - , are known to be expressed in the brain, their precise expression patterns and biochemical properties remained unclear. here, we show that each cbln member is expressed in various regions of developing and mature brains. all cbln family members could form both homomeric and heteromeric complexes each other in heterologous cells. like cbln , cbln and cbln were secreted as glycoproteins, whereas cbln was retained in the endoplasmic reticulum. these results suggest that each cbln member is potentially involved in synapse development and plasticity in various brain regions. s-scam is a synaptic membrane-associated protein with pdz domains, a guanylate kinase domain and ww domains. it interacts with various synaptic components including nmda receptor subunits, psd- and neuroligin. as we previously reported, s-scam is recruited to excitatory synapses by ␤-catenin. s-scam forms a ternary complex with neuroligin and psd- . more importantly, s-scam is involved in synaptic accumulation of neuroligin and subsequently affects the localization of psd- at excitatory synapses. in the course of these studies, we observed signals detected by anti-s-scam antibody at inhibitory synapses. we have here examined whether s-scam is indeed localized at inhibitory synapses in hippocampal neurons. we have raised questions which molecules s-scam interacts with at inhibitory synapses and which role s-scam plays in the assembly of inhibitory synapses. eriko fujita, yuko tanabe, takashi momoi division of differentiation and development, department of inherited metabolic disorder, national institute of neuroscience, ncnp, oawahigashi, tokyo, japan igsf /ra (ra ), which is a member of immunoglobulin superfamily having pdz binding domain at c-terminals, has ca +independent homophilic trans-cell adhesion activity. ra participates in synaptic junction and epithelial junctions in various tissues including testis. homozygous null (ra -/-) male is infertile and shows the defective elongating spermatids and fails to mature further. ra interacted with par- being involved in the polarity of epithelial cells via pdz binding domain at c-terminals. par- was colocalized in the cell adherent region of p embryonal teratocarcinoma cells during ra-induced differentiation into epithelial-like cells and mainly localized in the spermatid of ra +/+ testis, whereas it was undetectable in the spermatid of the ra −/− testis. ra and jam-c were localized around the head portion of spermatid and ra deficiency provided the abnormal polarization of the jam-c, which is necessary for the differentiation of round to elongated spermatid. jam-c inhibited the interaction between ra and par- . research funds: izumi kawabata, shigeo okabe department of cell biology, tokyo medical and dental university, tokyo, japan coordinated development of excitatory and inhibitory synapses is critical for both stability and temporal fidelity of neuron network in the hippocampus. however, there have been few analyses on postsynaptic molecular assembly in interneurons during development. to address this question, we examined dynamic properties of psd- clusters in cultured hippocampal interneurons. higher density of dendritic psd- clusters was observed in interneurons at div. at div, this difference was less prominent, mainly due to > -fold increase of psds in excitatory neurons. psd- -gfp imaging revealed lower rate of cluster appearance/disappearance in interneurons at div. the higher rate of cluster turnover in excitatory neurons, together with their higher rate of net cluster increase, may explain the delayed boost of cluster density. photobleaching of psd- -gfp revealed similar kinetics in two neuron types, suggesting additional determinants of cluster dynamics apart from the steady-state assembly rate. possible involvement of other postsynaptic molecules in interneuron psd dynamics is now being investigated. ps a-c two-photon imaging of immature dendritic protrusions and astroglial processes in hippocampal slice cultures hideko nishida, shigeo okabe department of cell biology, tokyo medical and dental university, tokyo, japan several lines of evidences indicate roles of astroglia in synaptogenesis, possibly mediated by either cell adhesion or diffusible factors. however, structural evidences supporting this claim are virtually lacking, mainly due to technical limitations in simultaneous imaging of neuronal and astroglial structures. here we visualized astroglia and pyramidal neurons in hippocampal slice cultures by combining adenovirus-mediated, cre-dependent expression of gfp with electroporation of rhodamine-dextran. two-photon time-lapse imaging of immature dendritic protrusions and astroglial processes in - div slice cultures revealed longer lifetime of dendritic protrusions having experienced astroglial contacts than those without contacts. dendritic protrusions with astroglial contacts also showed higher tendency to form spines. furthermore, expression of mutant rac in astroglial cells induced significantly longer, non-spiny protrusions than control. these findings suggest an involvement of direct astroglia-filopodia contacts in subsequent maturation of dendritic protrusions. taiko imura, fusao kato lab. neurophysiol., jikei univ. sch. med., tokyo, japan application of p x receptor agonists to the neurons in the nucleus of the solitary tract (nts) results in glutamate release facilitation (kato & shigetomi, ; shigetomi & kato, ) . recently accumulated evidence indicates that astrocytes affect the neuronal excitability by releasing gliotransmitters such as atp. this study was performed to determine whether such astrocyte-neuron interaction takes place in the nts. first, we analyzed the spatial localization of these cells by immunohistochemistry. a large number of gfap-positive cells with processes in the close apposition to the neun-positive neurons were found. second, we analyzed the effect on synaptic activity of localized application of atp using laser-based photolysis of caged atp in brainstem slices. uncaging of atp at neuronal dendrites ( s, -micrometer diameter) resulted in an immediate rise in mepsc frequency, in a manner sensitive to p x receptor antagonists. these results provide supports for the possible interaction between astrocytes and neuronal presynaptic terminals. research funds: kakenhi ( ) ps a-c ealy synapsin i accumulation in a granule cell axon at the filopodial attachment site of developing rodent purkinje cell dendrites in vitro isao nagata, junko kimura-kuroda department of brain structure, tokyo metropolitan institute for neuroscience, tokyo, japan synapse formation between the parallel fibers (pf) and dendrites of purkinje cells (pc) occurs at an early stage in the developing cerebellar cortex of the neonatal rodent. however, the precise spatio-temporal pf-pc interaction has not been elucidated. we have found that growth of pc dendrites was initiated by the attachment of axonal neurite bundles of granule cells orienting at right angles in several types of d-and d-cerebellar cultures. here, we investigated the expression of a synaptic vesicle marker, synapsin i, in granule cell axons by multiple immunofluorescence labelings in these cultures. synapsin i was first expressed at the filopodial attachment site of a pc dendrite as a cluster of faint punctate deposits in a long axon, then they appeared to gather into a slender and finally into a small round deposit. thus, the filopodial attachment of the juvenile pc dendrites to the axons of granule cells may induce rapid formation of presynaptic terminals via local clustering of synaptic vesicles. ps a-c integrative spike dynamics of rat ca neurons: an in situ multineuronal imaging study takuya sasaki, rie kimura, norio matsuki, yuji ikegaya department of pharmacology, university of tokyo, tokyo, japan the brain operates through a coordinated interplay of numerous neurons. our new technique with large-scale optical recordings reveals the diversity of synaptic integration in hundreds of neurons. in hippocampal slices bolus-loaded with calcium fluorophores, we stimulated the schaffer collaterals and monitored the bulk presynaptic activity from the stratum radiatum and individual postsynaptic spikes from the ca stratum pyramidale. single neurons responded to varying synaptic inputs with unreliable spikes, but at the population level, the networks output a linear sum of synaptic inputs. the network activity varied from trial to trial, even though given constant stimuli. this variation emerged through time-varying recruitment of different neuron subsets, which were shaped by correlated background noise. our imaging approach enables linking single-cell behaviors to their communal dynamics, and we discovered that, even in a relatively simple ca circuit, neurons could collectively be engaged in complex information processing. it is assumed based on previous in vitro experiments by other researchers that mglur connects with syntenin at the dendrites and mglur with pick at the axon terminal in on cone bipolar cells. to prove this possibility, we investigated wild-type mouse retinas immunohistochemically and confirmed their co-localized immunopositive labels at the respective places. next, we examined which scaffold protein would connect with mglur that was known to be ectopicly expressed in the dendrites of mglur -deficient on cone bipolar cells. we observed no pick but only syntenin at the mglur -deficient dendrites, and also the syntenin immunopositivity was co-localized with mglur immunopositivity. these findings suggest that mglur connects with syntenin in place of mglur that was knockout from the on cone bipolar dendrites. noriko trpv family is identified as thermosensitive, ca + -permeable channels. trpv , expressed in sensory neurons, is activated by noxious heat above • c, whereas trpv , expressed in keratinocytes, is sensitive to moderate temperatures (> • c). here we examined the role of trpv and in regulation of body temperature (bt) by using infrared laser as a heat stimulus. in wild type mouse, though the laser irradiation which caused the increase in skin temperature up to • c did not induce the change in bt, desensitization of trpv with capsaicin resulted in the increase in bt. on the other hand, in trpv -knockout mouse, moderate thermal radiation (> • c) caused the increase in the bt. the processing of noxious and moderate thermal radiation stimuli may depend on the trpv and respectively. research funds: kakenhi ( ) ps a-c generation and biochemical analysis of a glur␣ knockout mouse hirotsugu azechi , manabu abe , rie natsume , , kenji sakimura , department of cellular neurobiology, brain research institute, niigata university, niigata, japan; sorst/jst, saitama, japan glur␣ (glur ) is a key subunit of ampa receptors, since it is a critical determinant of their calcium permeability. to clarify the molecular function of glur␣ , we generated a conditional glur␣ knockout mouse using the cre/loxp recombination system. we first established a "floxed" mutant line gra f using c bl/ (b ) es cell line renka. the homozygous floxed mutants showed no significant abnormalities, thus our gra f was used as a target of glur␣ line. by crossing gra f and tlcn-cre that expressed cre in germ line cells, glur␣ null ko mice were produced, but most of them died within days after birth. to overcome the lethality, the glur␣ mutation was transferred onto b / or b /cd- genetic background. subcellular fractionation and quantitative immunoblot showed changes in the amount of ampa receptor subunits. these results indicated a significant role of glur␣ in the distribution of functional ampa receptors in vivo. ps a-c gisp: a novel brain specific protein that binds to the gaba b subunit and promotes its surface expression here we report the identification and characterisation of a novel brain specific kda protein, gaba b r interacting scaffolding protein (gisp), that interacts directly with the gaba b subunit via a coiledcoil domain. gisp coimmunoprecipitates with gaba b and gaba b from rat brain. in cultured hippocampal neurons gisp displays a punctate dendritic distribution and colocalises with gaba b receptors. when co-expressed with gaba b rs gisp increases the amount of gaba b protein and also promotes gaba b surface expression in the heterologous cells. furthermore, gisp increases surface expression of gaba b /gaba b complexes. these results suggest that gisp is involved in the forward trafficking and stabilisation of gaba b rs. thus gisp is an novel gaba b -binding protein potentially involved in the cell surface and/or synaptic targeting of the gaba b rs. three distinct isoforms of vesicular glutamate transporters (vglut - ) have been cloned and shown to exhibit differential distribution patterns in the brain. recent work shows the presence of vgluts in synaptic-like microvesicles (slmvs) of endocrine cells. mammalian pineal melatonin-secreting cells, pinealocytes, contain numerous slmvs which likely accumulate glutamate to inhibit melatonin synthesis. vglut and vglut seem to participate in this glutamate accumulation. in the present study, we found that vglut mrna is also expressed in the adult rat pinealocytes. vglut immunoreactivity (ir) was distributed throughout the pineal gland, and was co-localized with vglut -ir or vglut -ir in many, but not all, processes of pinealocyte. these data indicate that there are some subpopulations of slmvs which differ in the kind of vglut isoforms contained and/or in their combinations, suggesting vglut isoform-dependent sorting of slmvs to pinealocyte processes. kenzi saito , , , kenji nakamura , toshikazu kakizaki , , satoe ebihara , masakazu uematsu , shigeo takamori , minesuke yokoyama , shiro konishi , masayoshi mishina , , jun-ichi miyazaki , kunihiko obata , yuchio yanagawa , gunma univ., maebashi, japan; sokendai, hayama, japan; sorst, kawaguchi, japan; mitsubishi kagaku inst. life sci., machida, japan; kumamoto univ., kumamoto, japan; toyohashi univ. tech., toyohashi, japan; tokyo med. den. univ., tokyo, japan; univ. tokyo, tokyo, japan; osaka univ., suita, japan; riken, wako, japan the vesicular gaba transporter (vgat) loads gaba from neuronal cytoplasm into synaptic vesicles and is selectively expressed in inhibitory neurons containing gaba and/or glycine. to assess the functional role of vgat in development, we have disrupted the gene encoding vgat using cre-loxp system. western-blot analysis showed that vgat protein was absent in the homozygous embryos, indicating that the mutation had generated in a null allele. vgat knockout mice died around birth. all vgat knockout mice displayed cleft palate and omphalocele. our results suggest that vgat plays essential roles in both palate formation and ventral body wall development. research funds: kakenhi ( ) ps a-c postnatal changes in the colocalization of vglut and vglut immunoreactivities at single axon terminals of the mouse neocortex kouichi nakamura , , akiya watakabe , hiroyuki hioki , fumino fujiyama , yasuyo tanaka , tetsuo yamamori , takeshi kaneko , dept. morphol brain sci., grad. sch. med., kyoto univ., japan; crest, jst, japan; div. brain biol., nat. inst. basic biol., okazaki, japan vesicular glutamate transporter (vglut) and vglut accumulate transmitter glutamate into synaptic vesicles. the vgluts show a complementary expression pattern in the brain, but colocalize at single axon terminals in some synapses. here we quantitatively evaluated postnatal changes in the colocalization of vgluts at single axon terminals of the developing mouse neocortex by using a pixel-based correlation coefficient (cc) as an index of the colocalization. the cc was calculated from pixel values for vglut and vglut in each pixel of confocal micrographs of double immunofluorescence-labeled brain sections. in the barrels, the cc showed a prominent increase transiently around p . the cc was higher in area s than areas m and area v throughout postnatal development. our results indicate that the colocalization of vgluts in the neocortex is regulated in an age-, area-and layer-specific manner. gaba b receptors mediate slow and prolonged synaptic inhibition in the brain, and are members of the g protein-coupled receptors. here we have investigated the role of amp-activated protein kinase (ampk), as an endogenous regulator of gaba b receptor function. site-specific mutagenesis identified multiple phosphorylation sites for ampk within the cytoplasmic tails of both gaba b r and r . the activation of ampk regulated stability of gaba b receptors coupling with k + channels. together highlights a novel role for ampk in regulating the functional properties of gaba b receptors, by direct phosphorylation. given the role of ampk as a sensor of cellular stress this potential mechanism may be relevant in regulating the efficacy of synaptic inhibition under anoxic conditions and during periods of high synaptic activity. takao hirai, hiroaki nishio department of molecular pharmacology, faculty of pharmacy and pharmaceutical sciences, fukuyama university, hiroshima, japan serotonin ( -hydroxytryptamine, -ht) is a central neurotransmitter that is widely implicated in the regulation of mood and cognition, and is a peripheral signaling molecule that affects hemostasis, immune function, intestinal physiology, and other systems. there is increasing evidence for contribution of neuronal system to regulation of bone metabolism. this study was thus aimed at elucidation of possible functional expression of serotonergic system in mouse osteoblasts. rt-pcr analysis revealed constitutive expression of mrna for several -ht receptor subtypes, -ht transporter ( -htt) and vesicular monoamine transporter (vmat ) in primary cultured mouse osteoblasts and mc t -e osteoblastic cells. sustained exposure to fluoxetine, a selective -ht reuptake inhibitor, significantly prevented increase in alkaline phosphatase activities and mineralization in mc t -e . these results suggest that serotonergic system may be functionally expressed to regulate mechanisms underlying cellular differentiation and maturation in mouse osteoblasts. junko motohashi department of physiology, keio university school of medicine, tokyo, japan hotfoot mice are spontaneous mutants with ataxic phenotype. most hotfoot alleles identified so far have deletions of one or more exons coding for portions of the n-terminal domain of the ␦ glutamate receptor (glur␦ ). however, because only genomic dna was available for most hotfoot mutants, it was unclear whether truncated forms of glur␦ were actually translated and involved in the ataxic phenotype. here, we report that a newly identified hotfoot mutant, ho j, was caused by a new type of intragenic deletion of the grid gene, which was indeed translated as glur␦ lacking -amino acids in the n-terminus. mutant glur␦ proteins were retained in the soma of purkinje cells and degraded. as a result, ho j mice exhibited a severe motor discoordination on rotarod tests. furthermore, these mice exhibited sustained innervation of purkinje cells by multiple climbing fibers, and impaired long term depression, which is thought to underlie motor learning. these results indicate the importance of the n-terminal domain in glur␦ signaling and cerebellar functions. research funds: kakenhi ( ) ps a-d role of the dry motif in melanin-concentrating hormone receptor in signaling yumiko saito , yoshimi aizaki , mituse nakano , kei maruyama dept. pharamacol., saitama med. sch., saitama, japan; international university of health and welfare, tochigi, japan considerable attention has been focused on the functional importance of the highly conserved dry triplet in class a g protein-coupled receptors (gpcr). here we investigated the role of asp , arg and tyr in the dry of rat melanin-concentrating hormone receptor (mch r). in transfected cells, mutation of asp (d/a) resulted in nonfunctional receptor despite of showing moderate level of cell surface expression and an apparent affinity to mch. d/a mutation occurred with no increase in basal signaling pathway, suggesting no indication for constitutive activity. y/a mutation also yielded a loss of function phenotype that is similar to d/a mutation. mutation of the arg (r/a) showed higher ec value in signaling with a decrease in mch binding, while the level of cell surface expression exhibited only moderate decrease. these data suggest that a function for dry motif different from that widely accepted for class a gpcrs in regulating mch r-mediated signal pathway. in this study we confirmed functional heteromultimerization between a r and p y r electrophysiologically using xenopus oocyte expression system. when a r and p y r were coexpressed, application of non-hydrolyzable atp analogue induced g i/o response, showing formation of functional heteromultimers with a unique phenotype. it was also observed that the heteromultimers can activate g q/ pathway by atp analogue and also g i/o pathway by adenosine analogue, maintaining the features of the original subunits. ps a-d dual signaling via metabotropic glutamate receptor ␣ is regulated by a cytoskeletal protein . g michihiro tateyama , , yoshihiro kubo , department of biophysics and neurology, nips, aichi, japan; sorst, jst, saitama, japan the g protein-coupled metabotropic glutamate receptor ␣ (mglur ) is known to functionally couple to different types of g proteins. recently we have reported that the signaling pathways through mglur are differentially regulated by different types of ligands, glutamate and gd + . on the other hand, several cytoskeletal proteins have been reported to interact with the c-terminal cytoplasmic tail of mglur . these proteins, such as homer and . g, are also known to change the membrane expression of and modulate the function of mglur . here we investigated whether or not these cytoskeletal proteins regulate the multi path signaling of mglur . interestingly, the functional couplings of mglur to gq and gs pathways were altered by co-expression of . g, but not by homer. deletion of the c-terminal tail abolished the effect of . g, indicating that the interaction of . g with the c-terminal tail of mglur regulates the multi path signaling. ps a-d modulation of the eaac -mediated glutamate uptake by the addicsin mutant mitsushi j. ikemoto , , saori akiduki , age dimension research center, aist, ibaraki, japan; graduate school of science, toho university, chiba, japan addicsin is a murine homologue of rat glutamate-transporterassociated protein - (gtrap - ), an inhibitory modulator of neural glutamate-transporter excitatory amino acid carrier (eaac ). it contains two potential pkc phosphorylation motifs at positions - and - . however, its physiological function remains almost unknown. to clarify a significance of these pkc phosphorylation motifs, we investigated eaac -mediated glutamate transport activity in c bu- cells provided with a mifepristone-inducible expression of addicsin (wt), its mutants mutated at serine into alanine (s a) or at serine into alanine (s a). as compared with wt, s a had no inhibitory effect on glutamate transport activity under exposure to nm pma, and had increased glutamate transport activity under normal condition. by contrast, s a had the same glutamate transport activity as that of wt. thus, the eaac -mediated glutamate transport activity may be regulated by a pkc-dependent phosphorylation at serine in addicsin. kaori akashi , manabu abe , toshikazu kakizaki , rie natsume , , kenji sakimura , department of cellular neurobiology, brain research institute, niigata university, japan; sorst-jst, saitama, japan kainate type glutamate receptors are composed of various combinations of glur␤ - (glur - ) and glur␥ - (ka - ) subunits. although their physiological functions and subunit compositions have been inferred from various studies, they are still not clear. to clarify the functions and subunit dynamics of kainate receptors, we generated glur␤ ko mice from c bl/ es cell line. the glur␤ ko mice were viable, fertile, and displayed no overt phenotype. on the other hand, the amounts of glur␥ and glur␥ proteins were significantly decreased in the crude fraction of ca region of glur␤ ko. furthermore, subcellular localizations of both subunits were also changed in glur␤ ko. these results suggested that native kainate receptors might function as heteromeric channels (glur␤/␥) and the glur␤ subunit might determine subcellular localization of the glur␥ subunits, similar to the roles of nmda receptor glur subunits determining stability and distribution of the glur subunits. ps a-d sema d/plexin-b activates gsk- ␤ via r-ras gap activity, inducing growth cone collapse yuri ito, izumi oinuma, hironori katoh, manabu negishi laboratory of molecular neurobiology, graduate school of biostudies, kyoto university, kyoto, japan plexins are receptors for repulsive axonal guidance molecules semaphorins. we have recently reported that semaphorin d (sema d) receptor plexin-b induces growth cone collapse by functioning as an r-ras gap. here we characterized the downstream signaling of plexin-b -mediated r-ras gap activity, leading to growth cone collapse. sema d suppressed the endogenous r-ras activity in hippocampal neurons, in parallel with dephosphorylation of akt and activation of gsk- ␤. ectopic expression of the constitutively active mutant of akt, myr-akt, or treatment with gsk- ␤ antagonist suppressed the sema d-induced growth cone collapse. the r-ras gap activity was necessary for plexin-b -induced dephosphorylation of akt and gsk- ␤. plexin-a also induced dephosphorylation of akt and gsk- ␤ through its r-ras gap activity. thus, we conclude that plexin-b dephosphorylates akt and gsk- ␤ through r-rasgap activity, inducing growth cone collapse. to find proteins having relations in receptor trafficking, we searched human genome database and selected hepatocyte odd protein shuttling (hops) as a candidate gene. hops had three transmembrane domains, and expressed abundantly on a brain tissue. hops was detected in membranous regions from subcellular fractionation and immunohistochemistry. hops was recruited to membranous structures when overexpressed in cos cells. when expressed in hippocampal cultures, hops enhanced the amplitude of mepsc. from antibody feeding assay, we discovered that hops enhanced the recycling of glur . hops was co-immunoprecipitated with grip (glutamate receptor interacting protein ) when they were co-transfected to hek cells. thus, it was suggested that hops had roles in synaptic transmission enhancement by stabilization of surface glur via grip binding. serine must be taken up into neurons for their survival, because neurons lack serine biosynthetic enzyme. we have recently identified a serine transporter asc- . a neural amino acid transporter snat /ata also transports serine. we investigated their roles as serine transporters by comparing the localization of these serine transporters in the rat brain. the asc- immunoreactivity (asc- -ir) was detected in dendrites and somata of pyramidal neurons. the snat -ir was widely detected in neurons, whose intracellular localization was similar to that of asc- -ir. deferent from asc- -ir, snat -ir was also located in astrocytes and ependymal cells, especially around capillary blood vessels and ventricles. these results suggest the significant contribution of asc- and snat to the neuronal uptake of l-serine. snat might also accumulate l-serine in astrocytes from the extracellular spaces including blood and csf. ps a-d neuronal glutamate transporter eaat controls climbing fiber-mediated presynaptic inhibition of gabaergic transmission at cerebellar interneuron-purkinje cell synapses shin'ichiro satake , si-young song , shiro konishi , keiji imoto natl. inst. physiol. sci. (nips), okazaki, japan; mitsubishi kagaku inst life sci, tokyo, japan; tokushima bunri univ., sanuki, japan through extrasynaptic diffusion and activation of presynaptic ampa receptors in bc terminals. we here examined possible roles of glutamate transporters in this cf action. the eaat /glt- blocker threo- -methylglutamate, but not the glt- blocker dihydrokainate, augmented the cf-induced inhibition. cf stimulation obviously inhibited gabaergic transmission onto pcs in the lobule iii, where eaat expression was low, whereas the cf-induced inhibition was minimal in the lobule x, where eaat was abundant. the results suggest that eaat plays a major role in regulating the concentration of cf transmitters, possibly glutamate, in the route of its extrasynaptic diffusion, and determining the degree of cf-induced inhibition of gaba release from bcs depending on the regional difference of eaat expression in postsynaptic pcs. chitoshi takayama , yoshiro inoue department of molecular neuroanatomy, hokkaido university school of medicine, sapporo, japan gaba mediates inhibitory transmission in the adult central nervous system (cns). in contrast, gaba induces depolarization in the immature cns. this developmental shift from depolarization to hyperpolarization may be caused by decreasing of the intracellular chloride ion concentration regulated by two chloride ion co-transporters, na-k- cl co-transporter (nkcc ) and k-cl co-transporter (kcc ). in this study, we focused on kcc , which lowers the intracellular chloride ion concentration, and examined the developmental localization of the kcc with special reference to the neuronal development in the cerebellum. kcc was negative in the proliferating and migrating neurons. post-migratory neurons, which formed synapses, expressed the kcc . the kcc -protein was localized at the membrane of dendrites and cell bodies, whereas growth cones, axons and terminals were negative. these results suggested that formation of synapses might induce kcc -expression and localization, and gabaergic transmission might shift from excitation to inhibition after synapse formation. akinori nakajima , hisashi mori molecular neuroscience, university of toyama, toyama, japan the actions of many neurotransmitters are mediated by the members of a superfamily of receptors coupled to heterotrimeric guanine nucleotide binding proteins (g-proteins). the dopamine receptors are classified into two categories, d -like and d -like according to their pharmacological properties. the d -like receptors consist of d and d receptor, and are coupled to the adenylyl cyclase activating g proteins (gs). in the present study, we have generated a series of d receptor mutants and examined the effect on the gs coupled receptor signaling. we found that the expression of the third intracellular loop ( i loop) domain of d r fused with egfp effectively reduce camp production mediated by d and d receptors. interestingly, we also identified that the i loop domain of d r interfere with gs coupled beta adrenergic receptor signaling. these results suggest that the third intracellular loop of the d receptor is a primary determinant in its coupling to gs signaling. the activation of phosphatidylinositol-linked d -like dopamine receptor profoundly suppresses the exaitatory transmission in the developing hippocampus yoshinobu noriyama , yoichi ogawa , hiroki yoshino , masayuki yamashita , toshifumi kishimto dept. psychiatr.; dept. physiol i, nara med. univ., kashihara, japan we studied the effect of dopamine (da) on gabaergic and glutamatergic transmission in neonatal rat hippocampus from the early period of synapse formation by whole-cell patch-clamp recordings from ca pyramidal cells. da ( m) profoundly decreased gaba a receptor-mediated postsynaptic currents to % in the first postnatal week, when gaba provides excitatory drive. da also decreased ampa receptor-mediated excitatory post synaptic currents to % in the second postnatal week, when glutamate responses first appear. the da-induced inhibition declined after these periods. the receptor subtype involved in the da-induced inhibition was phosphatidylinositol (pi)-linked d -like receptor, since skf , a selective agonist for pi-linked d -like receptor, clearly mimicked the action of da. these results suggest that the activation of pi-linked d -like receptor profoundly suppresses the excitatory transmission during the early period of synapse formation in the developing hippocampus. ayuka ina , jinko konno , sachine yoshida , hideki ohmomo , hitoshi kawano , fumihiro shutoh , haruo nogami , setsuji hisano graduate sch., comprehensive human sci, univ. tsukuba, tsukuba, japan; tokyo metro inst neurosci, tokyo, japan supporting critical neurobiological roles of glutamate in mouse corticogenesis, we recently reported that cortical cells express vglut or - mrna at early fetal ages. to know roles of fetal vglut in cortical development, we studied expressions of vglut proteins in mouse fetuses by immunohistochemistry. on embryonic day (e ), vglut immunoreactivity (ir) was first detected in the marginal zone (mz), subplate (sp) and intermediate zone (imz). on e , vglut -ir was seen as puncta close to l -ir thalamocortical fiber tracts in the sp and also localized to fiber tracts expressing l or tag -ir in the imz, whereas vglut -ir was first observed in the sp and upper imz where l -ir existed. these results show that vglut ir corticofugal fibers appose to elongating vglut -ir thalamocortical fibers, suggesting that vglut may play a crucial role in glutamatemediated axon guidance to determine thalamic innervation patterns in the developing cortex. ps a-d developmental changes in the mechanism underlying activity-dependent swelling of the hippocampal ca regions michie kon , yoichi avil , hiroshi tsubokawa , dept. of information engineering, tohoku univ., sendai, japan; grad. schl. of information sciences, tohoku univ. to investigate the mechanisms underlying swelling of brain cells in association with neuronal activity, we analyzed interactions between changes in cell volume and synaptic activities in mouse hippocampal slices. swelling of several areas within the ca region were detected as increases in transmittance of near infrared light (irt). field epsps (feps) were recorded simultaneously from the stratum radiatum of ca region. in adult mice, repetitive stimulation of afferent fibers induced transient increases in irt at both somatic and dendritic regions in a frequency-dependent manner, which was temporally associated with feps. application of the bicuculline, a gaba-a receptor antagonist, reduced these optical signals. however, in mice under days old, the optical signals did not follow by high-frequency stimulation of inputs, and were not affected by an application of bicuculline. these results suggested that gaba-dependency in the mechanisms of cell volume regulation developmentally changes in the hippocampal ca region. in the present study, the effects of bilateral injections of glutamatergic agents into the hippocampal ca region on morphine-induced conditioned place preference (cpp) were investigated in rats. subcutaneous administration of different doses of morphine ( . - mg/kg) produced a dose-dependent cpp. using a -day schedule of conditioning, it was found that intra-ca administration of nmda receptor antagonist, mk- ( and g/rat) significantly attenuated the morphine ( . mg/kg)-induced cpp. moreover, nmda receptor agonist, nmda ( . , . and g/rat) significantly potentiated the morphine ( . mg/kg)-induced cpp. these results suggest that the development of morphine-induced cpp may be related to nmda and mk- receptors in that the glutamatergic system can modulate opiate reward. takahiro sonomura , kouichi nakamura , , hiroyuki hioki , masanori uemura , takeshi kaneko , dept. anatomy for oral sciences, grad. sch. med. and dent., kagoshima univ., kagoshima, japan; dept. morphological brain sciense, grad. sch. med., kyoto univ., kyoto, japan; crest, jst the majority of neostriatal neurons are medium-sized projection neurons with spiny dendrites and have so far been classified into three groups: striatonigral neurons producing ppd, and striatopallidal neurons producing ppe, and striatoinnominatal neurons producing pptb. these projection neurons are regulated in part by dopaminergic input from the substantia nigra pars compacta. it has been assumed that d receptor are expressed in striatonigral neurons and d receptor are expressed in striatopallidal neurons. in recent years, molecular cloning work has shown that there are at least five dopamine receptor genes (d , d , d , d , d ). in this study, the double-labeling method combining in situ hybridization and immunocytochemistry revealed how these five dopamine receptor subtypes are distributed among three projection neuron groups. the cerebellar tissue is good model system for the analysis of neuronal development, since dynamic neuronal development such as migration and axonal and dendritic outgrowth after birth. in the present study, we examined the localization of chondroitn sulfate proteoglycans (cspgs) by cspg-specific antibodies and lectins. cspgs are mainly observed at molecular layer in developing cerebellum (p - ) but they scarcely seen at external granular layer. electron microscopic observation demonstrated that phosphacan, one of cspgs, is localized at axonal membrane of parallel fibers. moreover, phosphacan inhibited adhesion and axonal extension of cerebellar granular neurons, while it promoted axonal fasciculation of their aggregated cultures. thus, cspgs, inhibitory molecules for axonal extension, are participated in axonal guidance cue in developing cerebellum. the vasopressin neurons are well knwon to show structural plasticity during chronic physiological stimulation such as salt loading. in the present study, salt loading significantly diminished the levels of chondroitin sulfate proteoglycans (cspgs) in vasopressin neurons. this downregulation is possibly due to proteolysis by tpa, since ( ) tpa immunoreactivity was observed at neurosecretory granules of vasopressin dendrites and terminals, ( ) salt loading increased protein and mrna levels of tpa in the somata and dendrites in the supraoptic nucleus but reduced protein levels of it in the terminals of the neurohypophysis, ( ) depolarizing agent released tpa from isolated neurosecretosomes, ( ) tpa knockout mice revealed lower ability of osmotic homeostasis and vasopressin release. thus, it is probable that tpa is participated in regulating structural plasticity of vasopressin neurons by degrading cspgs. chondroitin sulfate (cs) proteoglycans are essential for neuronal morphogenesis, including neural migration, survival and neurite formation in the developing brain. cs chains are modified by various sulfotransferases generating diverse sulfation patterns, which are assumed to be involved in the selective binding to various proteins such as growth factors. in this study, we analyzed the expression patterns of several cs sulfotransferases in the developing mouse cerebrum. using in situ hybridization analysis, it was revealed that cs sulfotransferase mrnas (u st, galnac - st, d st) were expressed in various types of cells, especially in the ventricular zone, and the cortical plate neurons just below the marginal zone. immunohistochemical analysis with anti-cs antibodies revealed that cs were highly expressed in the ventricular zone and the marginal zone. these results suggest that the cs structural domains generated by these cs sulfotransferases are involved in the regulation of the proliferation of neural progenitor cells and neuronal migration. nobuaki maeda , maki ishii , isao nagata , yumiko shimazaki dept. of dev. neurosci., tokyo metro. inst. for neurosci., tokyo, japan; dept. of brain structure, tokyo metro. inst. for neurosci. chondroitin sulfate (cs) is a long polysaccharide with enormous heterogeneity that binds with various proteins in a structure-dependent manner. previously, we revealed that cs is involved in the morphogenesis of the purkinje cell dendrites. in this study, we analyzed the expression of cs in the postnatally developing cerebellum using monoclonal antibodies that recognize specific structural motifs in cs. among the epitopes recognized by these antibodies, the expression of mo- epitopes, glca( s)␤ - galnac( s) (d unit)containing structures, remarkably increased during development. detailed immunohistochemical analysis indicated that d unit-rich cs was deposited between purkinje cell surface and the processes of bergmann glia. furthermore, it was found that pleiotrophin bound to d unit-rich cs on phosphacan distributed around purkinje cells. these observations suggest that d-type structure in cs is important for the signaling of pleiotrophin, which play roles in purkinje cell-bergmann glia interaction. nobuna fukazawa , mineko kengaku , nobuaki maeda dept. of dev. neurosci., tokyo metro. inst. for neurosci., tokyo, japan; lab. for neuronal cell polarity, riken bsi, wako, japan ptp is a receptor-type protein tyrosine phosphatase, which is synthesized as a chondroitin sulfate proteoglycan that pleiotrophin-ptp signaling regulates the morphogenesis of purkinje cell (pc) dendrites. we previously revealed that ptp associated with delta/notchlike egf-related receptor (dner), which mediates the pc-bergmann glia (bg) interaction and regulates morphological differentiation of these cells. here, we found that ptp was expressed by both pcs and bgs and the expression by pc occurred at relatively late developmental stage. ptp showed patchy distribution in the dendritic shafts of pcs, which partially overlapped with the localization of dner. furthermore, we revealed that multiple tyrosine residues in the cytoplasmic domain of dner were phosphorylated and that these tyrosine phosphorylated residues were efficiently dephosphorylated by the ptp catalytic domain. these results suggested that ptp participate in the pc-bg interaction by regulating tyrosine phosphorylation level of dner. masahiko tanaka , tohru marunouchi division of cell biology, institute for comprehensive medical science, fujita health university, toyoake, aichi, japan cerebellar purkinje cells have the most elaborate dendritic trees among the neurons in the cns. to investigate the cellular and molecular mechanisms of dendrite development of purkinje cells, we cocultured purkinje cells on a coverslip with other cerebellar cells such as granule cells and astrocytes on the cell culture insert of m pore size. when purkinje cells were co-cultured with granule cells, dendrite development of purkinje cells was promoted in comparison with that in control conditions. this co-culture effect was abolished by addition of a glutamate antagonist in the cultures. in contrast, dendrite development of purkinje cells was inhibited when purkinje cells were co-cultured with astrocytes. we propose that (i) glutamate secreted by granule cells and diffused through the porous membrane of the cell culture insert promotes the dendrite development of purkinje cells and (ii) astrocytes inhibit the effect of glutamate through their glutamate transporting activity. heparan sulfate (hs) proteoglycans regulate neural development through the interaction with cell surface proteins and extracellular matrix molecules. an extracellular endosulfatase, sulffp , has been implicated in the regulation of growth factor/morphogen signaling through hs remodeling in vitro, but its physiological roles remain unknown. here we generated knockout mice lacking the sulffp gene, and examined the motor control. homozygotes appeared to be normal, showing no sign of ataxia. performances of the rotarod and beam-walking tests were normal compared with the control mice. both short-term and long-term adaptations in the optokinetic response were normal, while the gains in optokinetic response and vestibulocular reflex were significantly reduced. heparan sulfate (hs) proteoglycans regulate a number of developmental signaling through interactions with cell surface proteins and extracellular matrix molecules. these interactions are mediated by the specific sulfation patterns in hs, but the mechanism generating such modifications has not been fully elucidated. here we show that a new class of hs endosulfatases plays an important role in brain development. the mice deficient in either sulffp or sulffp appeared to be normal, while most of the double knockout mice died soon after birth. mutant brains had higher content of -o-sulfated disaccharide units in hs, suggesting a role of sulffps in heparan sulfate remodeling in vivo. the double mutant brains were smaller than the controls and showed some axon guidance defects. these data demonstrate that specific hs modification generated by sulffps is important for normal brain development. recent studies have suggested monoamine affects neural development, but it is unclear which receptor subtypes mediate actions of monoamine. here, we examined roles of -hydroxytryptoamine ( -ht), noradrenaline (na) and dopamine (da) in the formation of dendrites and synapses by dissociation culture. embryonic day or rat cerebral cortex was cultured in the presence of -ht, na or da. after days, we analyzed dendrite formation using anti-map antibody. after - days, we analyzed synaptogenesis with anti-psd- , anti-synaptophysin, and anti-map antibodies. the addition of -ht ( - nm), na ( - nm) or da ( - nm) increased dendritic length of pyramidal neurons. -ht ( - nm) also increased the synaptic density. by using receptor agonists and antagonists, it was suggested that dendritic outgrowth may be promoted by -ht a receptor, ␣ a receptor and d receptor, while inhibited by -ht a and ␤receptors. in addition, synaptogenesis was promoted by -ht a and -ht c receptors, whereas inhibited by -ht a receptor. tatsuya mori, tomoe wada, takahiro suzuki, naoyuki inagaki department of cell biology, nara institute of science and technology, nara, japan most neurons have polarized shape consisting of a single long axon and multiple dendrites. several proteins have been implicated in the establishment of neuronal polarity; however, the mechanism for neuronal polarization is not well understood. in this study, with proteomic approach, we identified a novel protein, singar, as one of the proteins which are up-regulated during neuronal polarization of rat cultured hippocampal neuron. singar was expressed specifically in brain and developmentally up-regulated during neuronal polarization in vitro and in vivo. in t cell, singar associated with p and p , the subunits of pi kinase which is considered as one of the key molecules in neuronal polarization. moreover, inhibition of singar by rna interference induced the formation of multiple axon-like neurites. these data suggest that singar ensures the formation and maintenance of neuronal polarity by suppressing the formation of surplus axons. ps a-e lrfn , a neuronal leucine-rich repeatcontaining transmembrane protain can interact with psd- naoko morimura, takashi inoue, kei-ichi katayama, jun aruga laboratory for comparative neurogenesis, riken bsi, saitama, japan in a variety of organisms, proteins with leucine-rich repeat domain (lrr) function significantly in neural development. lrfn, a neuronal lrr transmembrane family, was expressed in the brain specifically. expression of lrfn was low in embryonic brain, and increased dramatically after birth. in the rat dissociated hippocampal neurons, lrfn protein was detected predominantly at mature dendrites, where it was accumulated at spines and colocalized with psd- , a postsynaptic scaffold protein. we examined the physical interaction between lrfn and psd- by immunocrecipitation and pull-down assay, since lrfn contains class i pdz domain-binding motif at its c-terminal tail. we revealed that lrfn associated with psd- /nmda receptor complex in the brain extracts and lrfn directly bound to psd- via its pdz domain-binding motif. in this study, we suggest that lrfn may play an important role in the regulation of synaptic functions. tsuya taneda, shingo miyata, hiroaki okuda, masaya tohyama department of anatomy and neuroscience, graduate school of medicine, osaka university, osaka, japan protein arginine methylation is a common post-translational modification catalyzed by a family of protein arginine n-methyltransferases (prmt - ). among the prmt proteins, the prmt has some characteristic motifs in the n-terminal tract which follows its active methyltransferase site. although little attention has been paid to protein methylation in the nervous system. first of all, we have examined the distribution of the prmt in the rat brain. the prmt was expressed in the cell bodies and dendrites in the hippocampal neurons. further, the ontogenetic analysis revealed the prmt expression increased from the perinatal stages to the adulthood. these findings suggest that the prmt relates to the neural function in the young and adult brain. furthermore in order to study the role of the prmt in the brain, we tried to identify novel interacting proteins with the prmt in rat hippocampal neurons using tandem affinity purification assay coupled with mass spectrometry. ps a-e proteomics of the growth cone: ii. the systematic immunostaining analysis of the growth cone proteins identified by the proteomic research motohiro nozumi , , michihiro igarashi , div mol cell biol, grad. sch. med dent sci; trans-diciplinary res program, niigata univ., niigata, japan proteomics is a powerful method to understand the molecular composition of a given cell or a compartment of the cell. in the accompanying paper, we applied this method to the growth cone from the rat forebrain, and we identified more than several hundred proteins there. although the proteins have been determined using the powerful methods, the localization of each protein in the neuron should be confirmed; thus, we checked the immunostaining in the cultured rat cortical neurons. currently, we have already performed the immunocytochemistry concerning more than identified proteins including cytoskeletal components, signaling molecules, receptors, and cell adhesion molecules. by quantitative analyzing the fluorescent intensity using the digital imaging, we classified the growth cone proteins into several groups. we have found more than twenty proteins specifically localized in the growth cone by this analysis. research funds: kakenhi ; project-promoting grant from niigata univ ps a-e netrin- is involved in the sensory axonal projection toward the spinal cord as a repulsive guidance cue tomoyuki masuda , keisuke watanabe , kazuhiro ikenaka , katsuhiko ono , hiroyuki yaginuma dept. anat., fukushima med univ. sch. of med., fukushima, japan; div neurobiol bioinfo, nat inst physiol sci, aichi, japan in higher vertebrate embryos, the ventral spinal cord exerts chemorepulsion for dorsal root ganglion (drg) axons to orient them toward their targets. netrin- is known to be a chemorepellent for a subset of axons, the role of netrin- for ventral spinal cord-derived repulsion is, however, unknown. by employing culture assays, we report here the involvement of netrin- in this repulsion. in the mouse embryo at e , netrin- is expressed in the floor plate and the dermamyotome, and the netrin- receptor unc c is expressed in drg neurons. we show that hek-cell aggregates secreting netrin- repelled chick e drg axons. moreover, using function-blocking antibody against netrin- , we revealed the fact that netrin- plays an important role in ventral spinal cord-derived repulsion. together, these findings suggest that the ventral spinal cord repels drg axons by secreting netrin- to shape the initial trajectories of drg axons. research funds: grants-in-aid on priority area (c) (mecst to t.m.) hitoshi maeda, masaki sakurai department of physiology, teikyo university school of medicine, tokyo, japan in the previous reports, we showed that in the early development, corticospinal synapses (cs) were formed widely in the spinal gray matter but those in the ventral side were eliminated later in an activity dependent manner. however, the property of postsynaptic cells to cs input is poorly understood. in the present study, we investigated the electrophysiological and morphological properties of the neurons that receive cs synapses in the acute spinal cord slices of neonatal rat. the postsynaptic neurons that were confirmed by the stimulation of the posterior funiculus, where the cs tract is located in rodents, were whole cell patch clamped and labeled by neurobiotin tm . responsive neurons are widely distributed in the p neonates, but the ventral neurons became unresponsive after p . the majority of the ventral neurons are of multipolar type with large somata showing "repetitive" or "phasic" firing patterns; on the other hand, most of dorsal neurons have smaller somata and multipolar branches with "single" or "phasic" patterns. keisuke watanabe , hirohide takebayashi , , kazuhiro ikenaka , katsuhiko ono div. neurobiol. bioinfo., natl. inst. physiol. sci., okazaki, japan; dev stem cell biol. program, ucsf, usa netrin- is a long-range diffusible factor that exerts chemoattractive or chemorepulsive effects on developing axons growing to or away from the neural midline. however, it is not known whether netrin- also exerts chemoattractive effect on ventral-ward migrating dorsal interneurons in the developing spinal cord. to test this hypothesis, we examined dorsal interneuron migration in netrin- −/− background, using olig -lacz knockin allele, which marks most of ventral-ward migrating dorsal interneurons. in the embryonic spinal cord of olig +/lacz ;netrin- −/− mice, ventral migration of olig cells was significantly impaired. furthermore, a netrin receptor, dcc was expressed in olig -positive cells. these results suggest that netrin- exerts chemoattractive effects on ventral-ward migrating dorsal interneurons in vivo. netrin-g and netrin-g are vertebrate-specific membrane-anchored members of the unc- /netrin family that have no affinity to classic netrin receptors and their function is unknown. here we show that netrin-g and netrin-g proteins are selectively distributed on axons of distinct pathways, and each interacts with a specific receptor on target dendrites. netrin-g and netrin-g differentially bind to lrrcontaining proteins, ngl- and a related molecule nag , in vitro. ngl- and nag in the mouse brain are concentrated in distinct dendritic segments, corresponding to lamina-specific termination of axons expressing netrin-g and netrin-g , respectively. furthermore, in netrin-g and netrin-g deficient mice, in which axonal pathfinding is normal, there is selective mislocation of individual receptors within dendrites. together, these results suggest that axonal netrin-g proteins transneuronally regulate the localization of distinct receptors on dendrites, and thereby determine the properties of subdendritic segments. jinhong huang , ryuichi sakai , teiichi furuichi lab. molecular neurogenesis, brain science institute of riken, saitama, japan; division of cell growth factor, national cancer center of japan, tokyo, japan cas is a tyrosine-phosphorylated docking protein that is indispensable for the regulation of actin cytoskeletal organization and cell migration in fibroblasts. the neuronal function of cas, however, is poorly understood. here we report that cas is dominantly enriched in the brain, especially the cerebellum, of postnatal mice. during cerebellar development, cas is highly tyrosine phosphorylated and is concentrated in the neurites and growth cones of granule cells. cas coimmunoprecipitates with src family protein tyrosine kinases, crk, and cell adhesion molecules. the axon extension of granule cells is inhibited by either rna interference knockdown of cas or overexpression of the cas mutant lacking the crk binding motifs. these results demonstrate that cas acts as a key scaffold to link the proteins associated with tyrosine phosphorylation signaling pathways to the granule cell axon elongation. research funds: huang was a postdoctoral fellowship recipient of jsps in in vitro cerebellum-pons-medulla block preparations isolated from neonatal rats on p -p , stimulation of parallel fibers produces excitation of purkinje cells lasting for - ms. this unusually prolonged response is observed in the lateral region of the cerebellum (paraflocculus/flocculus), where purkinje cells develop primary dendrites on p -p . since -agatoxin iva and -conotoxin mviic abolished the prolonged response, we suggest the involvement of p/q type ca channels. immunohistochemical labeling revealed that p/q type ca channels emerged in paraflocculus/flocculus and uvula/nodulus lobules on p and that they then locate in purkinje cells, in cell body on p and in primary dendrites on p -p . the parallel development of p/q type channels, primary dendrites, and the occurrence of prolonged parallel fiber-purkinje cell transmission suggests their causal relationships. naoya ichikawa, yasuo kitagawa, tatsuhiko kadowaki graduate school of bioagricultural sciences, nagoya university, aichi, japan we have recently identified a novel gene, mahya, which is specifically conserved between hymenoptera and deuterostome. mahya encodes a secretory protein with a follistatin-like domain, two immunoglobulin domains, and a c-terminal novel domain. mouse mahya genes (mmahya- and mmahya- ) are expressed in the olfactory bulb, hippocampus, and cerebellum of the adult brain. we have found that mmahya- protein is specifically synthesized in the pre-migratory granule cells and localized at the molecular layer of the postnatal cerebellum. these results suggest that mmahya- is involved in either the migration of granule cells or the dendritic maturation of purkinje cells. we will further report the analysis of the functions of mmahya- for the early cerebellum development. toshitaka morishima , erina fukushi , kazuto kobayashi , naohiro hozumi , sachiko yoshida toyohashi university of technology, toyohashi, japan; honda electronics co. ltd., toyohashi, japan in cerebellar development, granule cells migrate with elongation their axon, called parallel fibers, and form neuronal circuit in molecular layer. although density and thickness of parallel fibers are important information for cerebellar development, few were simple and useful methods. we have proposed a new method for two-dimensional acoustic impedance imaging for developing cerebellar slices. an acoustic impedance microscopy was obtained by mechanically scanning the transducer and the reflection intensity was interpreted into local acoustic impedance of no treated acute slices with no invasion. the developing parallel fibers were clearly observed as the contrast in acoustic impedance, whereas they were cloudy in immature egl from neonatal rat. the reflection from molecular layer enlarged and floated to deep layer, so that its spatial pattern was changed during cerebellar development. this imaging method is believed to be a powerful tool for observation of neuronal development, as neither fixation nor staining is required. tatsuro yamamoto , hideyuki dekimoto , tomiyoshi setsu , masahiko watanabe , mikio hoshino , yo-ichi nabeshima , toshio terashima dept. of anat., kobe univ. grad. sch. of med., kobe, japan; dept. of anat., hokkaido univ. grad. sch. of med., sapporo, japan; dept. of pathol and tumor biol, grad. sch. of med., kyoto univ., kyoto, japan a mutant mouse, cerebelles (cbll), lacks the entire cerebellar cortex but survives into the adult. the responsible gene for this mutation is ptf a, whose expression is lost in this mutant. in the present study, we examined cerebellar afferent and efferent systems of this mutant mouse by neural tracing methods with a combination of immunohistochemistry. the injection of fluoro-gold (fg) into the cbll thalamus resulted in retrograde labeling of neurons in the contralateral cerebellar nuclei. these fg-labeled neurons were glutaminase-positive. after the injection of bda into the cbll lumbar cord, spinocerebellar terminals projecting to the deep cerebellar nuclei were anterogradely labeled in spite of absence of the cerebellar cortex. these findings suggest that afferent and efferent systems of the cerebellar nuclei of the cbll are preserved in spite of absence of the cerebellar cortex. kumiko ishida , tomoko nishiyama , hitoshi tatsumi , masahiro sokabe , department of physiology, nagoya university graduate school of medicine, nagoya, japan; icorp, cell mechanosensing project, japan science and technology corporation sprouting and synaptic reorganization of the mossy fiber (mf) are commonly found in the hippocampus of temporal lobe epilepsy patients. as the muscarinic agonist, pilocarpine, can induce similar morphological changes, hippocampal slices treated with this drug have been widely used as a model of epilepsy. we found that pilocarpine induced a transient retraction and subsequent elongation of the neurites of granule cells in the slice cultures; the retraction was peaked approximately h and the elongation started at approximately h after the drug application. tetrodotoxin strongly inhibited both the retraction and elongation, while the bdnf sequestering protein, trkb/fc, retarded only the elongation. this result suggests that na + channel dependent neuronal excitation and following activitydependent bdnf releases are essential in the biphasic morphological changes induced by pilocarpine in hippocampal slices. rieko muramatsu, yuji ikegaya, maki k. yamada, norio matsuki, ryuta koyama laboratory of chemical pharmacology, graduate school of pharmaceutical sciences, the university of tokyo hippocampal granule cells extend their axons, i.e. the mossy fibers (mfs), from the dentate gyrus (dg) to the area ca . once this oneway projection is disrupted, the mfs retrogradely innervate granule cell dendrites and make excitatory synapses that induce epileptic neural activities in the dg. to clarify the mechanism that regulates normal, anterograde mf projections, we used a co-culture system of hippocampal slices. when a dg slice from a gfp(+) rat was juxtaposed to the ca region of a host hippocampal slice from a wild type rat, the gfp(+) mfs ran through the host ca toward the host dg but failed to invade it even after ten days in vitro. thus the dg seemed to serve as a barrier that blocks retrograde projections of mfs. however, the mfs extended into the dg when forskolin, an activator of adenylate cyclase, was chronically applied. these results suggest that the dg has a mechanism supporting anterograde mf projections to ca , which is regulated by the levels of adenylate cyclase activation. calcitonin gene-related peptide (cgrp) is a amino acid neuropeptide that is widely distributed in central and peripheral nervous systems. cgrp is expressed from early developmental stage in rat brain, suggesting that cgrp may be involved in not only neurotransmission but also neural development. but roles of cgrp in neuronal development of cerebral cortex and hippocampus remain unclear. in the present study, we made dissociation culture of cerebral cortex and hippocampus of embryonic day (e) or e rat. dendritic outgrowth of pyramidal neurons was analyzed after days using anti-map antibody. synapse formation was analyzed after - weeks, using anti-psd- and anti-synaptophysin antibodies. in the presence of cgrp ( - nm), both dendritic length and synaptic density were increased. however, the number of dendritic branching was not affected. these results suggest that cgrp promotes dendritic outgrowth and synapse formation. chisako kanamaru, kazunori suda, kouji senzaki, takashi shiga university of tsukuba, graduate school of comprehensive human sciences, tsukuba, japan recent studies have suggested monoamine affects neural development, but it is unclear which receptor subtypes mediate actions of monoamine. in this study, we examined roles of hydroxytryptoamine ( -ht) and noradrenaline (na) in the formation of dendrites and synapses using dissociation culture of rat hippocampus. embryonic day rat hippocampus was cultured in the presence of -ht or na. after days, we analyzed formation of dendrites using anti-map antibody. after days, we analyzed formation of synapses using anti-psd- , anti-synaptophysin, and anti-map antibodies. the addition of -ht ( nm) or na ( nm) increased dendritic length and number of branches of pyramidal neurons, whereas decreased number of primary dendrites -ht ( - nm) and na ( - nm) also increased the synaptic density. by using receptor agonists and antagonists, it was suggested that ␣ a receptor promotes dendritic outgrowth, while ␤ receptor suppress dendritic outgrowth and branching. in addition, -ht a receptor and ␣ a receptor promote synapse formation. kenji amano down syndrome cell adhesion molecule (dscam) knock-out (ko) mouse died within h after the birth. to investigate possible etiology of the neonatal death, we examined the respiratory activity using whole body plethysmography and the c inspiratory activity using brainstem-spinal cord preparation. the respiratory activity of dscam-ko mice using plethysmography was irregular frequency and small amplitude accompanied with apnea. furthermore, c inspiratory activity also showed irregular frequency and narrow duration of the bursting. we then analyzed spatio-temporal pattern of the respiratory neuronal activity using combination of the voltage-sensitive dye (di- anepeq) and the imaging system (micam ). in dscam-ko mice, the optical signal which precedes c inspiratory activity was depressed. these results suggest that pre-inspiratory neuronal network, which determines respiratory rhythm, does not develop normally in dscam-ko mice and causes lethal respiratory dysfunction. ps a-f hippocampal cells cultured on d collagen substrate secrete a dense extracellular matrix, supporting neuritic outgrowth shantanu sur, thomas launey, masao ito brain sc. inst., riken, japan the brain extracellular matrix (ecm) influences neuronal migration and morphogenesis. we explored how hippocampal cells modify their extracellular environment when seeded onto collagen gel, a major component of the ecm. after weeks in vitro, neurons formed a dense layer, > . mm below the gel surface, with neurite outgrowth toward the surface, within the top gel layer (tgl). initially, we thought that hippocampal cells were penetrating the gel, following partial degradation of the collagen matrix. however, ( ) collagenasespecific inhibitor did not affect cell depth, ( ) limiting gliosis by antimitotics reduced the thickness of the tgl by %, ( ), neither glial nor neuronal cell body were found in the tgl by gfap/map detection, ( ) neurite outgrowth was observed only within this tgl, but not toward the bottom of the gel. to see whether the tgl is the remains of the initial collagen substrate, we embedded fluorescent beads in the collagen gel before cell seeding. the tgl was completely devoid of beads after weeks, suggesting that the tgl is newly formed by ecm material, largely secreted by glial cells. emi kumamaru, tadahiro numakawa, yuki yagasaki, hiroshi kunugi disorder research, national institute of neuroscience, ncnp, tokyo, japan the level of glucocorticoid is regulated through hpa axis, and glucocorticoid itself has a negative feedback effect on hpa axis. however, under the intense stress, the glucocorticoid level is increased, and the high level of it is suggested to induce neuronal damage and to cause the mood disorder. on the other hand, it is possible that the reduction of neuronal function mediated by bdnf is partly related to the cause of the disorder. therefore, in the present study, we investigated the effect of glucocorticoid (dexamethasone, dex) on synaptic maturation and function enhanced by bdnf in early developing hippocampal neurons. we found that bdnf increased the expression of synaptic proteins including glutamate receptor and presynaptic protein, however, pretreatment with dex significantly inhibited the up-regulation of these proteins by bdnf. further, increase in release of glutamate and in intracellular ca + by bdnf was suppressed after dex pretreatment, suggesting that dex inhibits the maturation of synaptic function mediated by bdnf. takashi ueyama , kazuto kujira , tetsuya kawabe , takao ito , yoshihiro tsuruo department of cell biology and anatomy, wakayama medical university, wakayama, japan; department of cardiovascular medicine, wakayama medical university, wakayama, japan in this study, we investigated the effect of castration on the emotional stress response in the brain by comparing the c-fos expression in response to immobilization stress (imo) between castrated rats (cast) and sham-operated rats (sham). increased c-fos immunoreactive cells in response to imo were observed in septum, thalamus, hypothalamus, midbrain, pons and medulla oblongata in accordance with previous findings. in cast compared with sham, the numbers of c-fos-ir cells were significantly lower in the medial parvocellular part of paraventricular hypothalamic nucleus, while they were significantly higher in the supraoptic nucleus and medial amygdaloid nucleus. these data suggest that neuronal activity in these areas is influenced by systemic androgen level. this may underlie the pathophysiology of partial androgen deficiency in aged men (padam). research funds: grant-in-aid for scientific research (c) ( ) ps a-f metabolic and glucagon response of a genetically heat-tolerant rat to ambient heat and cold fujiya furuyama , hitoo nishino , takehiro yahata nagoya city university graduate school of medical sciences, nagoya, japan; nayoro city college, nayoro, japan the inbred fok rat was developed by us using heat selection and inbreeding for generations. fok rats avoided serious multisystem disorders caused by heat stroke and by extreme dehydration. saliva spreads widely over the whole ventral body surface in fok rats. however, no strain difference was not found in vitro in the salivation rate, suggesting exsisting of a negative feedback loop between the central thermoregulation system and evaporation system. on the other hand, body temperature of the fok rats did not decreased in a extream cold environment as those in control rat strain. thermogenesis induced by cold in fok rats was larger than those in control rat strains. the larger increase in thermogenesis was partly attributable to glucagon-induced thermogenesis in brown adipose tissue. blood levels of triglryceride was lower, but polyunsaturated fatty acids were higher in fok rats than those in control rat strains. these changes can be considered to be results of genetically acquired heat-tolerance. oxidative stress is involved in the degeneration of nigrostriatal dopaminergic system in parkinson s disease (pd). vitamin e is a potent antioxidant, and its retention and secretion are regulated by alpha-tocopherol transfer protein (ttp) in brain. dysfunction of ttp has been shown to result in systemic deficiency of vitamin e in human and mice. in the present study, we using the ttp knockout mice, investigated the effect of vitamin e deficiency in pd development by generating mptp mouse model of pd. we confirmed that vitamin e depleted in the brain of ttp knockout mice completely. while the mptp treatment decreased striatal dopamine in the all three ttp genotypic groups, there were no significant differences among them. our results suggest that vitamin e does not play a major protective role in mptp-induced nigrostriatal dopaminergic neurodegeneration in the brain. priyanka dikshit , anand goswami , nobuyuki nukina , nihar ranjan jana national brain research centre, india; laboratory for structural neuropathology, riken brain science institute, - hirosawa, wakoshi, saitama - , japan a major pathological hallmark of the polyglutamine diseases is the formation of neuronal intranuclear inclusions (niis) of the disease proteins, often associated with various chaperones and proteasome components. but, how the polyglutamine proteins are ubiquitinated and degraded by the proteasome is not known. here, we demonstrate that the expanded polyglutamine proteins that are misfolded, become ubiquitinated. secondly, we identified chip ubiquitin ligase that is able to target polyglutamine expanded huntingtin and ataxin- for the misfolding-dependent ubiquitination and degradation by the proteasome. the over expression of chip reduces the aggregate formation and cell death mediated by expanded polyglutamine proteins and the suppressive effect is more prominent when chip is over expressed along with hsc . finally, we show that the expression of chip is increased in the expanded polyglutamine protein expressing cells. hypothalamic-pituitary-adrenal axis is central to the regulation of stress response. for the comprehensive detection of genes responsive to stress, we identified and catalogued the entire partial complementary dna sequences (expressed sequence tags (ests)) from rat hypothalamus. we have identified the total of , ests ( , non-redundant sequences). of them matched known genes of rodents in the genbank databases, but remained unknown. now we classified a full set of hypothalamic ests on the basis of their functional domains. complete profile of them will be presented in the meeting. these ests will also be applied to a cdna microarray for stress experiments. the present study will provide a refined genomic resource for molecular studies of animal models of stress-related disorder. research funds: grants-in-aid from the ministry of health, labor and welfare shinya yanagita, seiichiro amemiya, satoko suzuki, ichiro kita graduate school of science, tokyo metropolitan university, japan our previous study suggests that acute running is one stressor activating corticotropin-releasing hormone (crh) neurons in the hypothalamic paraventricular nucleus (pvn). many studies have reported that several weeks of voluntary running improved stress tolerance during non-exercise stress. it is, thus, possible that housing in cages attached running wheel can alter activation of stress-related neurons during acute running. in this study, we examine the effects of , , or weeks prior wheel running (i.e. housing in the cages attached running wheel) on activation of stress-related neurons, such as pvn, central nucleus of amygdala (cea), locus coeruleus, dorsal raphe, ventral tegmental area (vta), and prefrontal cortex during acute running using immunohistological methods in rats. prior wheel running altered activation of various stress-related neurons during acute running, especially markedly decreased activation of cea, and increased that of vta. these results suggest that prior wheel running influences stress-related neuronal activity during acute running. ps a-f transforming growth factor-␤ in the brain regulates fat metabolism during exercise kazuo inoue, toma ishikawa, wataru mizunoya, tetsuro shibakusa, tohru fushiki division of food science and biotechnology, graduate school of agriculture, kyoto university, kyoto, japan we have previously reported that the concentration of transforming growth factor-␤ (tgf-␤) increases in the cerebrospinal fluid of rats during exercise and that an increase in fat oxidation was observed following intracisternal administration of tgf-␤. these results led us to postulate that tgf-␤ in the brain regulates the enhancement of fatty acid oxidation during exercise. to test this hypothesis, we carried out respiratory gas analysis during exercise while inhibiting the effect of tgf-␤ in the brain using intracisternal administration of anti-tgf-␤ antibody or sb- , an inhibitor of the type tgf-␤ receptor (t␤r ). we found that each reagent blocked the increase in fatty acid oxidation. these results suggest that brain tgf-␤ has a role in enhancing fatty acid oxidation in peripheral tissues during endurance exercise, and this regulation is executed at partly via the t␤r signal transduction system. yoshii takanobu it has been demonstrated that vasopressin (avp) might play a role in anxiety-related behavior. we hypothesized that traumatic stress changes avp activity and avp contribute to the symptom of ptsd. we carried out in situ hybridization (ish) for avp mrna expression and avp immunohistochemistry (ihc) with an experimental paradigm of single prolonged stress (sps) as ptsd model. sd male rats were exposed to sps ( h restraint; min forced-swimming; ether anesthesia) then they were put in untouchable situation for days. avp mrna expression significantly decreased in the son. ihc showed no significant change in avp-ir, but after additive stress (forced swimming min), avp-ir in the son was significantly diminished. we considered that the stress decrease avp synthesis, but has little effect to the storage of avp. mumeko tsuda, takaaki ozawa, aosa fukushi, sonoko ogawa kansei, behavioral and brain sciences, university of tsukuba, tsukuba, japan neonatal maternal separation (ms) is known to affect anxiety and fear responses in adult whereas its effect on socio-sexual behaviors is not fully understood. in the present study, we examined the effect of ms on an array of emotional and socio-sexual behaviors in both sexes of c bl/ j mice. pups were separated from mothers daily ( h) on postnatal days through . starting at weeks of age they were tested for ( ) emotionality and anxiety levels in open field (oft), light-dark transition (ldt), and elevated plus maze tests; ( ) responses to social stimuli in social investigation (sit) and social preference tests; and ( ) socio-sexual behaviors in aggressive and sexual behavior tests. overall, there was no apparent effect of ms on behaviors measured in the oft and ldt except for higher levels of exploration in the ms group compared to the non-stressed (ns) group in both sexes. during the sit, social investigation time and general activity in ms females were much lower than those in ns females suggesting ms females may be more fearful to social stimuli. in the present study, we investigated the effect of environmental stress applied during perinatal period on spatial learning activity of mouse evaluated by morris water maze test. mice were exposed to the noise of db (so), or were forced to swim (sw). these manipulations were performed for min once a day at weeks after birth (from postnatal days to ) or weeks after birth (from postnatal day to ). normal mice were left undisturbed (no). the spatial learning activity was tested at the age of weeks. it was found that the spatial learning activity of both so and sw mice manipulated weeks after birth was impaired as compared to no mice. so mice manipulated weeks after birth exhibited the same learning behavior as no mice, while that of sw mice manipulated weeks after birth was impaired. present results indicated that the effect of the environmental stress on the learning activity of the adolescent mice might be dependent on the period of the stress manipulation. kin-ya kubo , yukiko yamada , mitsuo iinuma , yasuo tamura , fumihiko iwaku , kazuko watanabe , minoru onozuka dept. oral anat., asahi univ. sch. dent., japan; dept. ped. dent., asahi univ. sch. dent.; dept. physiol., gifu univ. sch. med., japan; dept. physiol. and neurosci., kanagawa dent. coll., japan recent studies have suggested that occlusal disharmony is related to temporomandibular arthorosis and braxism, which may come from a hypothalamic-pituitary-adrenal (hpa) axis. in addition, aged mice with masticatory dysfunction show deficits in spatial memory, being due to various pathological changes in the hippocampus, suggesting the link between malocclusion induced by abnormal occlusion and hippocampal pathology. in this study, to prove this hypothesis, we examined the effect of this malocclusion on plasma corticosterone levels, the numbers of hippocampal neurons and spatial performance in water maze in samp mice. this treatment age-dependently advanced a decline in spatial memory, an increase in plasma corticosterone levels, and a decrease in neuron density in the hippocampal ca region. the results suggest that abnormal occlusion may progress hippocampal neuron loss via stress, thereby leading to senile deficits in memory. yurie nakamoto, go mugishima, mitsuko sato, masako miwa, mitsunobu yoshii division of psychobiology, tokyo institute of psychiatry, tokyo, japan it has been shown that pbr are increased after acute stress and decreased under chronic stressful conditions. in our previous studies, expression of pbr was significantly correlated with trait anxiety in normal human subjects, which might reflect polymorphism of the pbr gene. in addition, males appeared to have higher pbr densities than females in their prime lives. the present study was designed to analyze these sexual differences in rats. blood samples were obtained from adult male and female slc wistar rats immediately after acute random electrical footshock and also from these animals after chronic social isolation (for weeks after weaning). in naïve, male rats expressed higher densities of platelet pbr than females. chronic social isolation caused a marked increase in platelet pbr in male rats compared to female. the results indicate that pbr responses to environmentally induced stress are much less in female, probably under the influence of estrogen. kanako tambara , yayoi kitamura , junichi tanaka , yukio hattori , yasushi hayashi department of human nutrition, notre dame seishin university, okayama, japan; department of curriculum, teaching and memory, naruto university of education, tokushima, japan we investigated the effects of exogenous putrescine on stressinduced hyperthermia (sih) in male c bl/ j mice after systemic injection of putrescine to clarify the role of brain putrescine in stressful conditions. in addition, we examined the effects of spermidine, spermine, and the anxiolytic diazepam on sih. the rectal temperature of singly housed mice was measured twice at a -min interval, to measure the basal temperature (t ) and stress-enhanced temperature (t ), respectively. the difference ( t = t − t ) gives the sih. in control mice, t was approximately • c. pretreatment with diazepam caused dose-dependent inhibition of the sih. similarly, putrescine reduced t, although it caused a dose-dependent decrease in t . furthermore, spermidine and spermine also lowered t and t at doses lower than that of putrescine. these results suggest that endogenous brain putrescine and other polyamines have an anxiolytic-like effect in stressful conditions. eriko iguchi, yasuhiro tanaka, toshiyuki matsuoka, shuh narumiya department of pharmacology, kyoto university, kyoto, japan prostaglandins (pgs) are synthesized in many organs including the brain. of their synthesis, the rate limiting step depends on cyclooxygenase (cox), which has two subtypes, cox- and cox- . it has been known that, under some stressful conditions, cox- is induced in some neurons and increases pgs production. but the roles of the increased pgs under stress are not fully elucidated. in this study, we restrained mice in small tubes individually for h and subjected them to the elevated plus maze task h later. these mice showed more anxiety. immunohistochemistry showed significant induction of cox- by restraint in some parts of the brain, such as cerebral cortices and amygdala. next, we examined the effect of indomethacin on this stress-induced anxiety. indomethacin is expected to reduce pgs production. mice treated with indomethacin stayed on open arms longer than control mice. these data suggest that pgs synthesized during stress may have anxiety-increasing effect. ps a-g imaging brain and immune association accompanying cognitive appraisal of acute stressor to investigate association between brain and immune systems accompanying cognitive appraisal of an acute stressor, we recorded o-water positron emission tomography, cardiovascular, neuroendocrine, and immune indices, when male subjects conducted a mental arithmetic task in a high controllability (hc) condition and a low controllability (lc) condition. activation in the orbitofrontal (ofc) and medial prefrontal (mpfc) cortices was observed in the lc compared to the hc. furthermore, significant correlations between brain activation and hr, hrv, bp, and nk cells were found commonly in the ofc in the lc, but not in the hc. thus, the ofc is a pivotal region for top-down regulation over immune activity accompanying cognitive appraisal on a stressor. wei zhang , takesi sakurai , yasuitirou fukuda , tomoyuki kuwaki , dept. molec. integ. physiol., chiba univ., japan; dept. pharmacol., univ. tsukuba, japan; dept. autonom. physiol., chiba univ., japan we have previously proposed that orexin plays as a master switch to elicit multiple efferent pathways of the defense response. it is still open question, however, how information of stressor activates the orexinergic neurons. in this study, we examined possible afferent nuclei to activate orexinergic neurons. in urethane-anesthetized mice, a gaba-a receptor antagonist, bicuculline, was microinjected into the amygdala or the bed nucleus of stria terminalis (bnst), of which electrical stimulation induced simultaneous increases in blood pressure, heart rate, and respiration. bicuculline dose-dependently induced cardiorespiratory excitation in both orexin neuron-ablated and wild-type mice. however, dose-response curve was rightward shifted in the former. we conclude that the amygdala and bnst constitute one of the afferent pathways to the orexinergic neurons that involved in the defense response against stressor. in this study, developmental changes of anxiety behavior as well as myelin formation were investigated in male balb/c mice. the early-weaned mice had lower number of entries to the open arms of elevated plus maze at the age of - weeks, indicating persistent higher anxiety. high performance thin layer chromatography analysis was conducted for amygdaloid galactosyl ceramide, which is a typical lipid of myelin. the early-weaned mice had higher levels of galactosyl ceramide at the age of weeks, and an electron microscopic study suggested increased number of myelinated axon and reduced diameter of myelinated axon in the basolateral amyglaloid nucleus. these results suggest that the early weaning induces precocious myelin formation in the amygdale between and weeks of age, which would be related to higher anxiety state in the early-weaned mice. research funds: sasakawa sci. res. grant takefumi kikusui, yuji mori veterinary ethology, university of tokyo, tokyo, japan we previously reported that early-weaned mice developed persistent increase in anxiety as well as aggression. in this study, developmental changes of brain derived neurotrophic factor (bdnf) protein levels were investigated in early-weaned icr mice. the early-weaned male and female mice had lower number of entries to the open arms of elevated plus maze at the age of weeks, and this change was persistently observed in males. concurrently, the early-weaned males showed decrease of bdnf in the prefrontal cortex between and weeks of age, and in the hippocampus at the age of weeks. however, there was no difference of bdnf expression in females. in addition, the early-weaned males, but not females, showed reduced brdu immunoreactivity in the dentate gyrus. these results suggest that the deprivation of mother-infant interaction during the late lactating period augments the anxiety in the adulthood by decreasing the level of bdnf in the pre-limbic system, and that these stress responses are sexually dimorphic, i.e., male is more vulnerable to early weaning stress. research funds: kakenhi # ps a-g a systematic analysis of genetic factors associated with behavioral diversity between msm and c bl/ koide tsuyoshi , , aki takahashi , , toshihiko shiroishi , , akinori nishi mgrl, national institute of genetics, mishima, japan; sokendai, hayama, japan; mammalian genetics lab, nig, mishima, japan in the previous study conducting a multi-phenotype behavioral tests, we observed a great difference of the behavioral phenotype between mouse strains, msm and c bl/ . in order to elucidate a genetic factors underlying the behavioral difference, we analyzed a series of consomic strains which are made by replacing one of the chromosomes with that of msm strain. the behavioral data clearly indicated involvement of multiple genetic factors for each behavioral phenotype. one of the consomic strains, b - cmsm, which carries chromosome of msm, showed extreme behavioral differences from c bl/ . the strain showed lower activity in home cage and novel cage, and showed decreased number of transition in the light dark box test. by conducting analyses of composite interval mapping and a series of sub-consomic strains, we successfully identified genetic loci for the behavioral phenotype. tomoko soga , yu kajiyama , shigenobu shibata , hiroshi kunugi department of mental disorder research, national institute of neuroscience, center of neurology and psychiatry, tokyo, japan; department of electrical engineering and bioscience, waseda university the hypothalamus-pituitary-adrenal (hpa) axis plays an important role in the pathophysiology of depression. alterations of brain derived neuronal factors (bdnf) have been implicated in depression. we examined the effects of synthetic glucocorticoid (dexamethasone; dex) on emotional behavior and gene expression of hpa-related molecules and bdnf in mice. dex treatment for days after birth showed a significant decrease in locomotor activity and a significant rise in the time of immobility during forced swimming test. dex treatment to mature mice resulted in significant decrease in the number of entries into the open arm during elevated plus maze test. there was no change in gene expression of hpa-related molecules in dex-treated group. bdnf gene expression decreased significantly in dex-treated group, which showed behavioral abnormalities. our results lend further support for the involvement of glucocorticoid and bdnf in depression-related behavior. sachiko chikahisa, hiroyoshi sei, atsuko sano, kazuyoshi kitaoka, yusuke morita department of integrative physiology, the university of tokushima graduate school, tokushima, japan music is known to be able to elicit emotional changes including anxiolytic effect. the gonadal steroid hormone estrogen (e ) has been associated with anxiety levels. in this study, we examine whether the effect of music on anxiety is related with ovarian steroid in female mice. behavioral paradigms measuring anxiety (open field, elevated plus maze, dark-light transition and marble burying test) were tested in gonadally intact (sham-operated) and ovariectomized (ovx) female mice treated with placebo (ovx + placebo) or chronic estradiol (ovx + e ) replacement. in three behavioral tests except for open field, sham-operated mice exposed to music showed less anxiety than those exposed to white-noise and silence, while ovx + placebo mice did not show these effects at all. ovx + e mice showed the anxiolytic effect of music only in the marble burying test. these results suggest that exposure to music reduce anxiety levels, and ovarian steroids may be, at least partially, involved in the anxiolytic effects of music observed in female mice. tatsuhiro yasuda free, tokyo, japan strength and periodicity of periodical air pressure ascent around ones' ears induced by others' respiration may impact upon ones' awaken level, i.e. cognition. the air vibration acts upon tympanic membrane and then cochlear receptor stereocilia transforms it to neural signals which are sent via cochlear nucleus to inspiration nucleus in the medulla, and inspiration is induced. simultaneously afferent signals generated by external intercostals contraction are forward to medulla, thalamus and cortical areas. the stimuli with larger strength and periodicity compared to ones body size yields to auditory startle reflex. continuation of this may induce hyper ventilation or tension. if the input may be lasting with smaller strength and periodicity, insufficient diaphragm activity after hypoxia and gasping fade-out may induce afferent signal shortage that shrinks various cortical neural activity. lasting this situation may fall into depression. suitable timing of inspiration inducing may keep good mood, strong motivation and effective cognition. body system is suggested to own inherent observer that detects alerting or safe state so called homunculus. kenichi sasaguri , takero in general, it has been proposed that the mandibular retrusive position resulted from either malocclusion or inadequate occlusal reconstruction is one of the causes of indefinite complaint. we determined whether the malocclusion model influences brain activities by using fmri study. the results indicated that in some of volunteers, significantly bold signals in the hypothalamus and the amygdala, being associated with emotion and/or stress increased during clenching. it is, therefore, suggested that malocclusion influences the whole body through emotional system, thereby causing the indefinite complains. ps a-g synaptic organization between the amygdaloid axon terminals and the parvicellular reticular formationprojecting neurons in the retrorubral field of the rat toshiko tsumori, yi qin, shigefumi yokota, tatsuro oka, yukihiko yasui dept. anat. & morphol. neurosci., shimane univ. sch. med., izumo, japan the retrorubral field (rrf) is known as one of the areas containing numerous dopaminergic neurons in the midbrain. in the present study, we showed that the axon terminals from the central amygdaloid nucleus (ace) made synaptic contacts with non-dopaminergic rrf neurons sending their axons to the parvicellular reticular formation (rfp), where many premotor neurons projecting to the orofacial motor nuclei have been well known to exist. the ace axon terminals, which usually contain small pleomorphic vesicles and occasionally contain both small pleomorphic vesicles and large dense-cored vesicles, formed symmetrical synapses with cell bodies and dendrites of the rfp-projecting rrf neurons. moreover, most of these axon terminals showed glutamic acid decarboxylase immunoreactivity. the present study suggests that the ace exerts inhibitory influences upon the non-dopaminergic rfp-projecting rrf neurons to control orofacial movements closely related to emotional behavior. research funds: kakenhi ( ) ps a-g involvement of nr b tyrosine-phosphorylation in emotional responses mediated at the amygdala mina delawary , takanobu nakazawa , yuji kiyama , toshiya manabe , tadashi yamamoto div. of oncology, ims, univ. of tokyo, tokyo, japan; div. of neuronal network, ims, univ. of tokyo, tokyo, japan nr b is tyrosine-phosphorylated, with tyr- being its major phosphorylation site. to investigate the role of tyr- phosphorylation, we generated mice with a tyr phe knock-in mutation (yf/yf mice). in the elevated plus-maze test, time spent in open arm was reduced in yf/yf mice as compared to that in wild-type mice. similar phenotype was seen in the corticotropin-releasing factor (crf) overexpressing mice. this phenotype of yf/yf mice was canceled by the administration of crf receptor antagonist. as expected, in yf/yf mice, the expression level of crf in the amygdala was increased compared with that in wild-type mice. in the slice of amygdala from wild-type mice, nmda application induced de-phosphorylation of tyr- and up-regulation of crf mrna level. given that crf is important in emotional responses, these data strongly argue that phosphorylation of nr b is involved in the control of emotional responses by regulating crf content. ps a-g increase in anxiety in transgenic mice overexpressing camkii in forebrain previous studies have shown that ␣calcium/calmodulin dependent protein kinase ii (␣camkii) plays important roles in aggressive and fear response in mice. to understand roles of alpha camkii in emotional behaviors, we have generated transgenic mice overexpressing ␣camkii in forebrain. because these mutant mice showed increase in anxiety in open field and elevated zero maze tests, we here examined effects of administration of selective serotonin reuptake inhibitor (ssri) on anxiety-related behavior of these mutant mice. treatment with ssri suppressed anxiety-related behavior of camkii mutant mice, suggesting that camkii mutant mouse is a mouse model of anxiety disorder. to investigate the mechanisms for increase in anxiety led by overexpression of camkii, we next compared the expression profiles between wild and mutant mice using dna micro array. these mutant mice showed abnormal changes in expression levels of genes related to ca + signal transduction in hippocampus. yumiko ikeda, katsunori kobayashi, hidenori suzuki department of pharmacology, nippon medical school, tokyo, japan environment is known to influence behavior of animals. however, cellular and synaptic mechanisms underlying behavioral changes by environment remain largely unknown. we examined effects of changes in environment on locomotor activity and mossy fiber (mf) synaptic transmission in hippocampal slices. in mice housed in enriched condition for weeks, locomotor activity and longinterval ( , and ms) paired-pulse facilitation (ppf) at mf synapses were reduced. in contrast, in mice housed in isolated condition for weeks, there was no detectable change in either the total ambulation distance or the magnitude of ppf. we compared properties of the mf synaptic transmission with the locomotor activity in individual mice used in all experiments and found that the magnitude of synaptic potentiation induced by dopamine was negatively correlated with the ambulation distance. our results suggest that the modification of the hippocampal mossy fiber synaptic transmission could be involved in the environmental regulation of locomotor activity. yilong cui , , yosky kataoka , , yasuhisa tamura , yasuyoshi watanabe , , hisao yamada department of anatomy and cell science, kansai medical university, osaka, japan; department of physiology, osaka city university graduate school of medicine, osaka, japan; molecular imaging research program, riken frontier research system, saitama, japan during long-term intracranial self-stimulation (icss; electrical stimulations to the hemi-lateral medial forebrain bundle of rats by their lever pressing behavior at - times/min), inhibition periods (less than times/min) were often observed h after start of icss. we have been demonstrated that the inhibition was not induced by thermal effect on the neural function or by muscular fatigue. furthermore, the inhibition period was significantly decreased by pre-treatment with ns- , a selective cox- inhibitor. these observations indicate that the arachidonic acid cascade is involved in inhibition of long-term icss and would be in weariness or fatigue sensation. male bluegill, lepomis macrochirus, is known to display alternative reproductive tactics. "parental" males defend nests and provide parental care, and "satellites" or "sneakers" are non-nesting, attempting to achieve parasitic fertilizations via sperm competition. in teleost and other non-mammals, arginine vasotocin (avt), the homologue of mammalian avp, is known as an important hypothalamic peptide involved in the alteration of reproductive behavior. behavioral evaluation and immunohistochemical study in preoptic area (poa) were conducted in parental and satellite bluegills to clear the role of avt in teleost reproductive tactics. parentals displayed more aggressive and courtship behavior than satellites and satellite males had significantly more cells than parentals, while the size of avt cells showed no difference between the male morphs. these results suggested that hypothalamic avt might play some part in the central control of reproductive behavior in teleost. ps a-g impulsive choice in domestic chicks: context dependence and dissociation between delay and handling cost toshiya matsushima , naoya aoki , andras csillag biology, hokkaido univ., sapporo, japan; agriculture, nagoya univ., nagoya, japan; anatomy, semmelweis univ., budapest, hungary choice between small/immediate reward and large/delayed reward has been widely used as a behavioral measure of impulsiveness. to study how ecological factors shaped underlying neural processes, we examined week-old chicks in four different tasks with identical economical consequences. in task , chicks chose between small reward (one pellet) delivered immediately and large reward (six pellets) after a delay up to s. in task , chicks chose between small reward located at cm and large reward at − cm, where cues signaled the distance of invisible food. task was similar to the task , except that cues signaled the food quantity. in task , total handling time differed due to lowered food accessibility, while the delay was kept identical. lesion experiments revealed that ventral striatum was specifically involved in choices based on anticipated proximity (but not quantity), whereas arcopallium (association cortex analogue) in choices based on anticipated handling cost. research funds: kakenhi ( , ) ps a-g analysis of the brain regions associated with the dance language of the honeybees taketoshi kiya, takekazu kunieda, takeo kubo dep. biol. sci., univ. tokyo, tokyo, japan social animals have highly developed communicative abilities. the worker honeybees (apis mellifera l.) can transmit location of food sources by the dance language. in spite of the simple structure of the honeybee brain and the stereotyped dance behavior, its neural mechanisms remain totally unknown. previously, we found active brain regions in the dancing workers (dancers) by using a novel immediate early gene, kakusei, as a marker for neural activities and found its prominent expression in the small-type kenyon cells (skcs) of the mushroom bodies. here, we report that kakusei was similarly expressed in the skcs of the foraging workers (foragers), which do not always show the dance behavior. in contrast, the skcspreferential kakusei expression was not observed in the brains of the orienting workers, which were flying to learn the hive location. these results imply that the activities of the skcs in the dancer brain are neither due to dance presentation itself nor sensory inputs during foraging, but complex information processing accompanying the foraging behavior. c. elegans wild type animals are usually attracted to nacl, but show avoidance behaviors after being conditioned with nacl and starvation (food−/nacl+). this behavioral plasticity is not induced under the food−/nacl− or food+/nacl+ conditions. we isolated learning-defective mutants including pe , which had a missense mutation in the casy- gene. several casy- deletion mutants also showed learning defects. casy- has an extensive similarity to human calsyntenin/alcadein, which is a single-pass transmembrane protein with cadherin-like repeats localized to the postsynaptic membrane of cns synapses. alcadein forms a stable tripartite complex with app and x l/mint . however, after dissociation of x l, alcadein is susceptible to cleavage by protease(s). we found that casy- was expressed mainly in neurons and functioned at the adult stage. we are now investigating the localization pattern of the gfp-tagged protein, and whether casy- can also be proteolytically cleaved. ps a-g insulin-like signaling is required for association between temperature and feeding state in c. elegans eiji kodama , atsushi kuhara , akiko mohri , , kotaro kimura , , masatoshi okumura , masahiro tomioka , yuichi iino , ikue mori , div. of biol. sci., nagoya univ., japan; present address: univ. of texas, health sci. cent., usa; present address: natl. inst. of genet., japan; mol. genet. res. lab., univ. of tokyo, japan; inst. for advanced res., nagoya univ., japan c. elegans can associate cultivation temperature with feeding state. mutations in ins- encoding insulin homologue caused defective associative learning, mutations in daf- and age- encoding the homologues of insulin receptor and pi -kinase, respectively, suppressed the defect of ins- , and the mutation in daf- encoding forkhead transcriptional factor caused the learning defect. this suggests that ins- antagonizes daf- insulin-like signaling for associative learning. interestingly, age- animals associate their cultivation temperature with feeding-state quicker than wild type. this defect was rescued by expressing age- in some head interneurons. in addition, the activity of these interneurons were down-regulated by starvation through ins- . we suggest that insulin-like signaling modulates the neuronal activity of interneurons essential for associative learning. ps a-g analysis of ttx- : novel thermotaxis gene conserved among various organisms akiko miyara, akane ohta, yoshifumi okochi, masatoshi okumura, ikue mori laboratory of molecular neurobiology and institute for advanced research, nagoya university, nagoya, japan c. elegans can memorize the food condition in relation to the cultivation temperature and migrate to the cultivation temperature when looking for the food. this response to temperature is called thermotaxis. several neurons and genes required for thermotaxis have been identified, but molecular mechanism of thermotaxis is still poorly understood. the ttx- (nj ) and ttx- (nj ) mutants are obviously defective in thermotaxis and partially defective in chemotaxis. we revealed that ttx- encodes novel protein and is expressed in many neurons and functions in several neurons responsible for the thermotaxis behavior. the predicted protein structure of ttx- is similar to ric- , identified in c. elegans at first and conserved among several species (halevi et al., (halevi et al., , . ric- is thought to be required for the maturation of acetylcoline receptor (halevi et al., ) , so ttx- may play a similar role such as folding, assembly, transmission or anchoring of some kind of membrane protein. ps a-g analysis of aho- mutant that cannot associate cultivation temperature with feeding state in c. elegans nana nishio , akiko mohri , , eiji kodama , atsushi kuhara , mizuho koike , kotaro kimura , , ikue mori , div. of biol. sci., nagoya univ., japan; present address: univ. of texas, health sci. cent., usa; present address: natl. inst. of genet., japan; inst. for advanced res., nagoya univ., japan the nematode c. elegans can associate cultivation temperature with feeding state: well-fed animals migrate to and starved animals avoid from the cultivation temperature on a temperature gradient. to identify genes required for this associative learning, we screened mutants that are defective in starvation-induced cultivation temperature avoidance. we isolated aho- (nj ) mutants that were normal in thermotactic migration after cultivated well-fed state and normal in response to food in locomotion assay (sawin et al., ) , indicating that they are normal in temperature and food recognition and may be defective in the associative learning. aho- gene encoded a predicted hydrolase and the molecular properties have not been characterized yet, although aho- is a highly conserved protein throughout yeast to human. currently, we are trying to dissect the molecular and cellular analysis of aho- gene further. we have demonstrated aversive conditioning in lymnaea using mm sucrose presentation as the appetitive stimulus (cs) and mechanical tactile stimulation to the head as the noxious stimulus (ucs). we measured the feeding response before and after pairing with the aversive stimulus to determine whether learning alters the innate preference for sucrose. we also measured the neuronal activity of b , located in the buccal ganglion. an associative memory, lasting h, was produced with pairings of cs and ucs. the learning was characterized by a shift in the response to the ucs from a whole body withdrawal response to the cessation of feeding behavior. b neuron responded with repetitive impulse discharge regularly as fictive feeding patterns to a sucrose application in naive animals, on the other hand cs application failed to generate regular impulse activity rather it resulted in generation of epsps in the conditioned animal. this can interpret that the conditioning decreased the excitability of b neuron activity thus to decrease the fictive feeding behavior. yasutaka nomura , dai hatakeyama , tetsuro horikoshi , etsuro ito , manabu sakakibara lab. neurobiol. engr, sch. high-tech, tokai univ., numazu, japan; cris, hokkaido univ., sapporo, japan calexcitin, low molecular weight gtp-binding protein is found to be phosphorylated in the visuo-vestibular conditioned hermissenda at the type b photoreceptor. we found positively stained neurons to anti-calexcitin antibody (gift from dr. kuzirian) at the cerebral and pedal ganglion in the circumesophageal nervous system of conditioned lymnaea with two different ways. one was the same conditioning paradigm as hermissenda and the other was taste aversion conditioning. both of these conditioning response is the whole-body withdrawal. no positive neuron was found in naïve animal. neurons in cb cluster and pea cluster showed both positivity to calexcitin and serotonin. this suggested the functional role in conditioning. ken honjo, katsuo furukubo-tokunaga graduate school of life and environmental sciences, university of tsukuba, ibaraki, japan the fruit fly drosophila melanogaster has been utilized as a successful model to study underlying mechanisms of learning and memory. we have established a novel larval olfactory paradigm and found that appetitive and aversive memories are considerably different in their stability whereas both are localized to the mushroom bodies (mbs). we found that larval memory induced by sucrose lasts six times longer than that induced by quinine although the initial learning performances are comparable. by expressing shi ts in larval mbs, we demonstrate that disruption of neural output from mbs abolishes both appetitive and aversive memory indicating that both memories are stored before the mb output synapses. moreover, we show that disruption of either creb or amnesiac functions abolishes appetitive but not aversive memory. thus these data suggest that appetitive and aversive reinforcements stimulate different intracellular and/or intercellular signaling pathways generating distinct memory components in mbs. motomi matsuno , minoru saitoe , , tim tully tokyo metropolitan institute for neuroscience, tokyo, japan; department of biology, tokyo metropolitan university, japan; cold spring harbor laboratory, usa we identified ruslan as a novel memory mutant, and found that it encodes a cell adhesion molecule, klingon (klg). klg belongs to the immunoglobulin superfamily and was originally identified as an essential gene for the development of photoreceptor neurons. we report here that klg is necessary for long-term memory as well as early-phase memory. we show that klg expression is dependent on neural activity and functions as a downstream of both the transcription factor, creb and the cell surface receptor notch, both of which are well known to function in ltm formation. transgenic expression of klg improves memory of a klg mutant. since klg protein localizes along the surface between neuropil and neuropil glia, we propose that klg mediates an interaction between neurons and glia that is required for memory formation. we have investigated the ability of context-dependent olfactory learning in the cockroach, periplaneta americana. we trained one group of cockroaches to associate peppermint odor (conditioned stimulus, cs, p) with sucrose solution (appetitive unconditioned stimulus, us+), and vanilla odor (cs, v) with saline solution (aversive us, us−) under illumination (l), and to associate p with us− and v with us+ in the dark (d). another group received training with opposite stimulus setup (l: v+/p−, d: v−/p+). before training, cockroaches preferred v over p. day after training, the former group significantly preferred p over v under illumination but preferred v over p in the dark, and the latter group displayed the invert odor preference. result of the control experiment excluded the possibilities that conditioning hours of the day or its order was used as cues to disambiguate the meaning of css. thus cockroaches are capable of disambiguating the meaning of cs odors according to the visual context. hidehiro watanabe, makoto mizunami graduate school of life sciences, tohoku university, sendai, japan a century had passed since pavlov reported classical conditioning of salivation in dogs. however, the cellular mechanisms underlying this conditioning remain obscure. in insects, salivation is regulated by salivary neurons of the subesophageal ganglion which innervate the salivary grand. here, we established antennal classical conditioning of salivation and that of activities of salivary neurons in cockroaches, periplaneta americana. in insects, antennae are elaborate sense organ that processes many sensory modalities including odor and taste. we found that responses of salivary neurons to an odor was increased after repetitive pairing of the odor with sucrose or saline solution presented to an antenna, but those to an odor paired with water or tactile stimulus presented to an antenna did not changed. the level of salivation to sucrose-associated odor was significantly greater than that to non-associated odor. these results are the first to suggest the classical conditioning of salivation in non-mammalian species. these results are useful to study neural mechanisms underlying classical conditioning of salivation. research funds: kakenhi ke zhang , jian z. guo , ai k. guo , institute of neuroscience, chinese academy of sciences, china; institute of biophysics, chinese academy of sciences, beijing, china the cooperation of dopamine system and other brain cortices is essential for decision-making in mammal. drosophila can make clearout choice in visual flight simulator when facing conflicting visual cues based on the saliency of the cues previously trained to follow. here we show this behaviour is impaired when the transmission of dopaminergic neurons or mushroom bodies (mb), was genetically silenced by gal /uas-shi ts system, suggesting that this behaviour is mediated by dopaminergic system acting through mb, a structure shown to be densely innervated by dopaminergic fibers. however, the dopaminergic and mb synaptic activities were required only during the early choice period (< min), but not for the sustenance of the chosen flight path. thus the dopaminergic system and mb are specifically devoted to the cognitive function exemplified by the flyǐs choice behaviour and further studies of the circuit in drosophila may help to understand the neural basis of higher cognitive functions. sae unoki, yukihisa matsumoto, makoto mizunami graduate school of life sciences, tohoku university, sendai, japan in mammals, the dopaminergic reward system plays ubiquitous roles in reward learning. previous studies in insects suggested that octopamine (oa) and dopamine (da) mediate various kinds of reward and punishment signals in olfactory learning. however, whether such roles can be generalized to learning of sensory signals other than odors remained unknown. we pharmacologically studied the roles of oa and da in appetitive and aversive forms of visual pattern learning in crickets. crickets injected with oa receptor antagonists exhibited no significant levels of appetitive visual learning, but aversive one was unaffected. the opposite influences were observed by injection of da receptor antagonists. our finding that oa and da participate in reward and punishment conditioning in visual learning, together with results of previous studies in olfactory learning, suggests ubiquitous roles of the octopaminergic reward system and dopaminergic punishment system in insect learning. this suggests conserved roles of aminergic reinforcing systems among different phyla. aiko watanabe, neal a. hessler laboratory for vocal behavior mechanisms, riken brain science institute, saitama, japan in adult songbirds, neural turnover occurs in hvc, a forebrain motor control nucleus. cells labeled by bromodeoxyuridine (brdu), a cell birth marker, appear in the ventricular zone, migrate into hvc, and some of them mature into projection neuron. to assess the role of neurogenesis in adult song plasticity, we deafened adult bengalese finches, whose songs are disorganized and become plastic within the first month after deafening, and then stabilize. deafened birds had more brdu-labeled cells in hvc than control birds within the first month. more tunel-stained apoptotic cells also tended to be seen in hvc of deafened birds. however, number of the brdu-labeled cells decreased months after deafening, when the songs had stabilized. most of the brdu-labeled cells in hvc of deafened birds were immunoreactive for a neuron-specific marker, hu. additionally, amount of singing in deafened birds, which may affect amount of neurogenesis, did not significantly differ from that in control birds. these results suggest that the amount of neurogenesis is related to adult song plasticity. yasko tobari , , kazuo okanoya , , lab. for biolinguistics, riken-bsi, wako, japan; grad. sch. of sci. and tech., chiba university, chiba, japan; presto, jst. kawaguchi, japan a set of brain nuclei controls song production in songbirds. among these nuclei, the robust nucleus of arcopallium (ra) is the telencephalic site of direct projections onto vocal motor neurons and respiratory premotor neurons. the projections of ra to the mudulla included the tracheosyrigeal part of the hypoglossal nucleus (xiits), which innervates the syrinx, the birds , vocal organ, and respiratoryrelated nucleus, retroambigualis (ram) were present in bengalese finches. in this study, we have focused our attention on the descending projections of ra, with a view to the presence of contralateral projections to xiits and ram, using in vivo tract-tracing technique. the results indicated that ipsilateral and contralateral projections of ra to respiratory-vocal nuclei in the brainstem were defined in adult male bengalese finches. birdsong is composed of various song elements that have typical frequency modulation. each element is aligned in own sequential rule. especially in bengalese finches, the sequential rule obeys finite state grammar. it has been focused what neural mechanism enables such a complex sequential rule. in order to learn and maintain their own song, they have auditory neural representation of their own song in the forebrain area hvc. we collectively recorded the activities of hvc neurons driven by all possible element pair stimuli. the results show that most of neurons in hvc respond not only the sequence included in their own song but also the sequence not included. each neuron has typical response distribution toward the whole element sequence. in addition, the distribution property is different among neurons in same individual. taken together, information of the entire song element sequence would be stored in the neural ensemble of these neurons as a population coding. hironobu sakaguchi department of physiology and biological information, dokkyo university, school of medicine, japan avian vocal learning provides a good model for human speech learning. young male songbirds learn to imitate their tutor's song during a specific time in development, which is referred to as a sensitive period. many behavioral studies have shown that vocal learning is affected by a song template and social factors. if a young bird is raised without a tutor's song template (father) and/or social contacts with other birds, including its mother and siblings, it produces an abnormal isolated song, meaning that isolation delays the sensitive period for song learning. here, we investigated for the delayed song learning of socially isolated zebra finches from new tutors. consequently, isolated birds, exposed to new tutors from day , developed the zebra finch-typical song (song syntax), similar to song acquisition in young birds during the sensitive period of song learning. however, they were not able to imitate the syllable phonology from new tutors. the differences between two aspects of song organization suggest that the schedules and processes of the learning of phonology may be different from those of song syntax. ps a-h facilitatory effects of oxytocin on synaptic plasticity in the olfactory bulb and olfactory learning in young rats fumino okutani, jing-ji zhang, guang-zhe huang, hideto kaba department of integrative physiology, kochi medical school, nankoku, japan oxytocin (ot) within the olfactory bulb (ob) has been reported to be important for the induction of maternal behavior and recognition of offspring. the activity of mitral cells, olfactory relay neurons in the ob is inhibited by granule cells via reciprocal dendrodendritic synapses. electrophysiological studies have revealed that ot modulates mitral cell activity by acting on mitral and granule cells. in a classical conditioning paradigm, young rats show aversion to the odor that has been paired with foot shock. our studies have shown that plasticity in the ob is critical for this olfactory learning. pups that received ot infusion into the ob in the presence of citral odor developed an aversion to the odor without shock, suggesting that ot infusion has a facilitatory effect on olfactory learning. using ob slices, long-term potentiation (ltp) was induced in field epsps recorded in the granule cell layer. ot administration also facilitated ltp. these results demonstrate that ot is involved in olfactory learning in young rats. research funds: kakenhi ps a-h the gaba a receptors in the ventral pallidum are involved in the retrieval of conditioned taste aversion in rats tadashi inui, tsuyoshi shimura, takashi yamamoto div. behav. physiol., dept. behav. sci., grad. sch. human sci., osaka univ., japan we examined the effects of microinjections of gaba a receptors antagonist bicuculline into the ventral pallidum (vp) on the retrieval of conditioned taste aversion (cta). in experiment , rats received a pairing of saccharin or quinine hydrochloride (cs) with an i.p. injection of . m lithium chloride (us). after this conditioning, vehicle or bicuculline was bilaterally infused into the vp just before the re-exposure to the cs. the microinjections of bicuculline significantly increased the intake of saccharin cs, but not quinine hydrochloride. in experiment , rats were presented with saccharin as cs via an intraoral cannula. the microinjections of bicuculline significantly increased ingestive responses and decreased aversive responses. these results suggest that the gaba a receptors in the vp play an important role in the expression of ingestive and/or aversive responses to saccharin cs during the retrieval of cta so that the microinjection of bicuculline might increase the intake of saccharin cs. research funds: kakenhi ( ) ps a-h transient blockade, but not genetic deficiency, of c-fos gene expression impairs long-term memory in taste aversion learning roles of c-fos gene expression and its protein product, fos, in conditioned taste aversion (cta) learning were examined using the antisense oligodeoxynucleotide (odn) method in rats and in mice carrying c-fos gene deficiency. infusion of antisense odn (as-odn) directed against c-fos mrna into the parabrachial nucleus (pbn), but not into the amygdala or insular cortex (ic), impaired the acquisition, while infusion of randomized and inverted control odns had no effect. suppression of fos synthesis in the amygdala or ic impaired the retention. retrieval of an acquired cta was not impaired by as-odn infusion into the pbn or amygdala. in contrast, mice carrying c-fos gene deficiency showed normal acquisition and retention. the present results suggest that the fos-mediated signals in the pbn, amygdala or, ic plays key roles in the acquisition and/or consolidation, but not the retrieval, of long-term cta memory. ps a-h gaba receptors in the deep cerebellar nuclei are essential for mouse eyeblink conditioning classical eyeblink conditioning is a useful experimental system to analyze the neuronal substrate underlying learning and memory. the knowledge on the mouse eyeblink conditioning is far less compared with rabbit's. we examined the role of the deep cerebellar nuclei (dcn) during delay eyeblink conditioning in c bl/ mice by using gaba a receptors agonist and antagonist. in the acquisition tests, in which muscimol (msc) or picrotoxin (ptx) was injected from beginning of training, acsf-injected control mice learned this task, but both msc-and ptx-injected mice showed a significant impairment in acquisition of conditioned response (cr). in the retention tests, in which the drug was injected after acquisition of training, cr % in acsf-injected mice were kept over %, while those in the mscand ptx-injected mice decreased to %. these results revealed that gabaa receptors in the dcn play important roles in acquisition and retention of mouse eyeblink conditioning. various forms of synaptic plasticity are found in cerebellar circuits, but their significance in motor leaning remains unknown. in the cerebellum, delphilin is expressed selectively in purkinje cells (pcs) and localized exclusively at parallel fiber (pf) synapses, where it interacts with glutamate receptor ␦ that is essential for long-term depression (ltd) and motor learning. here, we showed that ablation of delphilin proteins facilitated ltd induction at pf-pc synapses and enhanced optokinetic response adaptation without affecting histology. this finding suggests that threshold regulation of ltd at pf-pc synapses is a limiting step for motor learning efficiency. ps a-h post-training cerebellar cortical activities are necessary for transfer of memory trace of motor learning from cortex to nuclei soichi nagao , , takehito okamoto , fumihiro shutoh , lab for motor learning control, riken bsi, saitama, japan; sorst, jst, saitama, japan; dept. anat., grad. univ. tsukuba, ibaraki, japan one-hour optokinetic training induces short-term adaptation of horizontal optokinetic response (hokr) gains in mice. succession of h daily training for week induces long-term adaptation. we recently reported that the memory trace of adaptation of hokr is initially acquired within the cerebellar flocculus through long-term depression (ltd), and later transferred to the vestibular nuclei for consolidation. in order to reveal the neural mechanisms underlying the memory transfer, we reversibly inactivated the neural activities of flocculus bilaterally by local application of muscimol immediately after the end of daily training. mice treated with muscimol showed depressed long-term adaptations, while the short-term adaptations were intact, suggesting that the neural activities of cerebellar cortex in a certain period after training are necessary for the transfer of memory trace from flocculus to vestibular nuclei. research funds: kakenhi ( ) ps a-h modification of gene expression in the cerebellar cortical neurons related with long-term motor learning yuji t. katagiri , , takehito okamoto , shin-ichi nishimura , fumihiro shutoh , , soichi nagao , lab. for motor learning control, riken bsi, saitama, japan; univ of the air, chiba, japan; dept. of anatomy, human comprehensive science, grad. univ. tsukba, japan; sorst, jst, japan we recently reported that the cerebellar ltd plays a crucial role for both acquisition and consolidation of memory trace of long-term motor learning using the adaptation paradigm of mouse horizontal optokinetic eye movements (shutoh et al., ) . in order to listup the molecules involved in the motor learning, we sampled total rna from the cerebellar flocculus of short-and long-term adapted mice, and quantified amounts of gene expression by the microarray methods. we found that the number of genes modulated by longterm motor learning much exceeded that modulated by short-term motor learning, and the number of down-regulated genes were larger than that of up-regulated genes. we furthermore examined the gene expression of purkinje cells by the laser micro-dissection and quantitative rt-pcr methods. ps a-h influence of spatial cues on hippocampal neuronal activity in spatial navigation tasks in mice hippocampal neurons were recorded while mice performing spatial tasks of searching for unpredictable and predictable rewards. the influence of spatial cues, including distal and proximal cues, on the response of hippocampal cells that exhibited place-related activity was examined. place cells predominantly shifted their fields accordingly by changes of visual and auditory distal cues, and fewer cells shifted their fields by changes of proximal cues. these results provide evidence that hippocampal neurons of mice can use flexibly information of spatial cues to represent the environment, and this ability is important for spatial learning. son ho , , t kobayashi , , e hori , , k umeno , , t ono , h nishijo , system emotional science, univ. of toyama, toyama, japan; crest, tokyo, japan we investigated a role of the hippocampal formation (hf) in encoding of a moving object in an open field. rats acquired icss rewards if they moved freely. then, a remote-controlled car was placed inside the open field. the rats could receive icss if it chased and approached the car. of a total of place cells recorded, activity of was significantly modulated by the car speed and/or distance between the car and rat; , and cells displayed distance-dependent, car speeddependent, and distance and car speed-dependent firing, respectively. furthermore, six cells, which did not show the place field in reference to rat position, but showed the place fields in reference to car position. in a control experiment, the same car was introduced, but the rats could receive icss rewards without relation to relative distance between the rat and car. so far, place cells were recorded in this experiment. of these, six and three place cells displayed distancedependent and car speed-dependent firing, respectively. the results suggest that hf encodes not only spatial information of own location, but also that of other moving object in an environment. hisae gemba, kazuko nakao, ryuiti matsuzaki, yusaku amaya department of physiology, kansai medical university, moriguchi, japan cortical field potentials were recorded by electrodes implanted on the surface and at a . - . mm depth in the cortex of monkeys in the process of learning somatosensory-initiated hand movements and then analyzed. it was found that an s-n, d-p potential, at about ms latency from stimulus, in the caudal bank of the left arcuate sulcus (homolog of broca's area) was related to recognition learning (association of stimulus with movement), and that an s-n, d-p potential in the motor and somatosensory cortices, and areas and , contralateral to the operating hand, was related to skill learning (making movements quicker and more appropriate). in visuo-initiated hand movements, the left prefrontal cortex was related to recognition learning; the motor and somatosensory cortices, and area to skill learning, as previously reported. this indicates that motor programming for somatosensory-initiated and visuo-initiated hand movement differs. computational studies of hippocampal function generally assume that ca performs a match-mismatch comparison of memory retrieval with sensory input. here we investigated this comparator model using an ensemble recording during task behaviors in the rat. we employed directional memory-guided alternation and visual cue discrimination tasks for the same animal. after training, the animals tended to predict a next direction according to the alternation paradigm even in the visual cue discrimination task. during this task, we found that some ca neurons showed specific bursts when a predicted event did not occur or along the trajectories of their corrective movements from a wrong cite to a correct cued one. these data suggest that ca plays an important role in the mismatch detecting and correcting process of behavior. n-methyl-d-aspartate (nmda) receptor has high permeability to ca + but is blocked by mg + in a voltage-dependent manner. this property is a molecular basis of nmda receptor-dependent long-term potentiation, which is thought to play a central role in learning and memory. we have generated the genetically engineered mice in which mutated nmda receptors defecting in mg + binding ability are expressed specifically in the granule cells of the dentate gyrus, the entry point to the hippocampal trisynaptic circuit. the mutant mice showed a variety of behavioral abnormalities including hyperactivity, impaired prepulse inhibition. to elucidate the effect of mutation on the information processing in the hippocampus, we recorded the place-related activity from hippocampal ca cells, the output stage of hippocampal circuit. the link between the behavioral anomaly and the hippocampal activity is discussed. mikako sakurai, ko zushida, masayuki sekiguchi, keiji wada department of neurodegenerative diseases, national institute of neuroscience, ncnp, tokyo, japan uch-l is a component of the ubiquitin system. uch-l is expressed at high levels in the hippocampal neurons. however, the functional role of uch-l in synaptic plasticity and behavior is not understood. we examined behavior and synaptic plasticity in gad mouse which is an autosomal recessive spontaneous mutant carrying an intragenic deletion in the gene encoding uchl . gad mice have significantly impaired performance in the open field and one-trial passive avoidance tests. theta burst stimulation (tbs) of shaffer collateral in hippocampal slices from gad mice elicited decremental long-term potentiation (ltp) in the area ca . in contrast, non-decremental ltp was induced in control wild-type mice. the maintenance of tbsinduced ltp in the wild-type mice was impaired by actinomycin d, an inhibitor of transcription, whereas tbs-induced ltp in gad mice was insensitive to actinomycin d. these results suggest that uch-l is a molecule participating in the synaptic plasticity elicited by tbs and the memory function. ps a-i learning stages in rat operant reversal task and cross-correlation between hippocampal and prefrontal local field potential powers yoshinori izaki, tatsuo akema department of physiology, st. marianna university school of medicine, japan to investigate whether the relationship between hippocampus (hip) and prefrontal cortex (pfc) spontaneous local field potentials changes with leaning stages, we analyzed cross-correlation (cc) of these local field potential powers during operant reversal training sessions. rats were trained with initial discrimination task until a stable discriminative performance was achieved (learning stage ). then the rats received the reversal training. learning stages examined were as following: the first training session (stage i), leaning stage for s+ (stage ii) and for s− (stage iii). different changes of the cc in some frequency-band powers with learning stages were observed. the cc in higher gamma-band ( - hz) was strong at stage and changed with leaning stages. particularly, the cc decreased to almost zero at stage ii. these results suggest that functional connection between hip and pfc is reflected in this frequency-band and changes with learning stages. ps a-i longitudinal fiber systems in the dentate gyrus of the rat norio ishizuka, yoshitomo umitsu department of brain structure, tokyo metropolitan institute for neuroscience, tokyo, japan longitudinal fiber systems in the dentate gyrus of the rat were investigated by anterograde labeling method of pha-l and retrograde labeling method with fluorescent dyes. the flattened hippocampal formation allowed sections to be cut perpendicular to the full septotemporal axis of the dentate gyrus. injection of pha-l into the hilar region elucidated that two longitudinal fiber systems existed in the dentate gyrus. the first fiber system gives rise to projections to the superficial portion of the dentate molecular layer, and the longitudinal axonal trajectory of this system ceased within the range of about . mm from the injection level. in the second fiber system, axonal terminations began to appear at the level of mm apart from the injection level and were distributed in further full septotemporal extent of the dentate molecular layer. the axonal arborizations of the second system were found in the deepest portion of the dentate molecular layer immediately above the granular cell layer. in the experiment of fluorescent dye injection, several kinds of cells in the hilus were retrogradely labeled. ryoichi moki, ryang kim, hisahiro umeeda, akinobu suzuki, satoshi kida department of bioscience, tokyo university of agriculture, tokyo, japan recent studies have shown that when conditioned fear memory is retrieved, fear memory becomes labile and requires gene expressiondependent reconsolidation for the re-storage. in addition, previous our study using conditional creb mutant mice indicated that creb is required for reconsolidation of conditioned fear memory. we also observed protein synthesis-dependent reconsolidation of spatial memory using morris water maze. in this study, to understand the mechanisms of reconsolidation of spatial memory, we examined a role of creb in reconsolidation of spatial memory. using conditional creb mutant mice that enable to induce the inhibition of creb activity in a tamoxifen-dependent manner, we found that inhibition of creb activity leads to disruption of spatial memory after the retrieval. this result indicates that creb is required for reconsolidation of spatial memory. shunsuke hasegawa, hirosi hosoda, satoshi kida department of bioscience, tokyo university of agriculture bhlh-pas transcription factor bmal ubiquitously expresses in brain. bmal functions by forming a heterodimer with either clock or npas , which has been known to play important roles in control of circadian rhythm and memory formation, respectively. to understand roles of bmal in forebrain function, we generated conditional mutant mice that enable to induce the inhibition of bmal function in a forebrain. using a dominant negative mutant of bmal (bmal r a) that forms a heterodimer with clock but loses the binding activity with e-box (hosoda et al., ), we generated transgenic mice expressing this mutant under the control of tetracycline-dependent promoter. these mutant mice were crossed to transgenic lines expressing tetracycline-dependent transcription factors (tta) under the control of alpha camkii promoter. we observed the expression of bmal r a in several double transgenic lines in a tta-dependent manner. behavioral analyses showed that these mutant mice showed an impairment of memory formation, indicating crucial roles of bmal in learning and memory. stress sometimes causes memory deficits. and chewing has been shown to reduce stress. however, the chewing-related mechanism in stress-induced memory deficits is unclear. we thus examined the effects of chewing on spatial memory using morris water maze and fos induction in the hippocampus and amygdala in stressed mice. when mice were exposed to restraint stress, reduction in learning ability and density of fos-positive cells in the dg and the bla was seen, but not in the mice chewing a thin wooden bar during stress exposure. the results suggest involvement of the amygdaloidmechanism by which chewing may prevent the stress-induced impairment of hippocampus-dependent memory. seiichiro amemiya, shinya yanagita, satoko suzuki, ichiro kita graduate school of science, tokyo metropolitan university, tokyo, japan we examined the effect of background noise (bgn) on spatial learning and its neuronal activity related to arousal and stress using maze task and c-fos immunostaining. rats performed maze task under different intensity of bgn ( , , or db; intermittent white noise). db bgn induced significant decreases in number of error and time to goal in maze compared with and db. although bgn increased fos positive acetylcholinergic neurons (fos-chat) in mesopontine tegmentum (mt) regardless of the intensity, fos-chat in basal forebrain (bf) increased intensity-dependently. in locus coeruleus (lc) and cortex, fos positive cell increased intensitydependently. furthermore, db bgn remarkably enhanced fos expression in stress-related nuclei, such as paraventricular nucleus and central nucleus of the amygdala. these results suggest that bgn improve spatial performance by enhancing arousal following activation of cholinergic neurons in mt and bf, and lc neurons. however, higher bgn intensity could evoke over-arousal and stress responses, thereby prevent the maze task. siriporn chattipakorn , , anucha pongpanparadorn , wasana pratchayasakul , anchalee pongchaidacha , nipon chattipakorn fac. dent. cmu, chiang mai, thailand; cert, cmu, chiang mai, thailand current pharmacotherapy of ad is the use of ache inhibitors. previous in vitro study showed that tde inhibited ache activity. this is the first study investigating the effects of tde on cortical ache activity and neuronal activity in in vivo. we used fos immunohistochemistry to determine the neuronal activity and the colorimetric method to investigate cortical ache activity following the single injection of various tde doses. mean fos-positive neurons in cortex were ± , ± and ± in the groups administered , and mg/kg tde, respectively. cortical fos-positive neurons in all three tde-treated groups were greater than those in the control group. percent inhibition of cortical ache activity was . ± . , . ± . and . ± . for , and mg/kg tde, respectively. these ache inhibitory effects were significantly different from the control. these findings suggest that tde could be beneficial as a possible novel therapeutic agent for ad. we showed the effects of a -h tde administration in animals on the inhibition of cortical ache activity and the enhancement of cortical neuronal activity. ache activity in circulation following a -h tde administration was not different compared to the control. this study investigated that the effects of tde on circulating ache activity (cache) in animal models was time-dependent. we used the colorimetric method to investigate cache activity in rats following the single administration of tde at various doses at different time courses ( , and min). percentage inhibition of cache activity following a single tde injection at doses and mg/kg significantly decreased at and min after tde injection, but not at min. cache inhibitory effects among two doses of tde administrated groups at various time courses were not significantly different. these findings suggest that tde may be a short-acting ache inhibitor. donepezil, galanthamine and tacrine are acetylcholinesterase (ache) inhibitors used for treatment of alzheimer's disease. we examined the neuroprotective mechanisms of ache inhibitors against apoptotic glutamate neurotoxicity using cortical neurons. we show that they protect neurons through mechanisms other than ache inhibition. the protective effects are mediated through nicotinic receptors (nachrs). donepezil and galanthamine protect neurons through ␣ and ␣ -nachr and kinases involved in pi k-akt pathway, and increase the levels of phosphorylated akt and bcl- . these results suggest that these ache inhibitors express their neuroprotective effects against glutamate neurotoxicity through nachrs and that donepezil and galanthamine protect neurons through pi k-akt pathway via ␣ and ␣ -nachrs. ps a-i arachidonic acid preserves hippocampal neuron membrane fluidity in senescent rats yasuto kashiyae , yoshiyuki ishikura , shigeaki fujikawa , yoshinobu kiso , manabu sakakibara lab. neurobiol. engr., tokai univ., numazu, japan; inst. health care sci. suntory, shimamoto, japan previous studies indicate that long-term dietary supplementation with arachidonic acid (aa) in -month-old rats (oa) effectively restores performance in a memory task and induction of long-term potentiation in the hippocampus to the level of young control animals (yc). this study examined fluorescent recovery after photobleaching (frap) in yc, old control (oc), and oa neurons in hippocampal slice preparations. three measures: mobile fraction (mf), diffusion constant (d), and time constant (τ), were estimated among yc, oc, and oa. each of these parameters was significantly different between oc and yc, suggesting that membrane fluidity is lower in oc than in yc. in contrast, d and τ were almost comparable in oa and yc, indicating that hippocampal neuronal membranes supplemented with aa were more fluid than those in oc, whereas the fraction of available molecules remained smaller than in yc. long-term administration of aa to senescent rats might help to preserve membrane fluidity and maintain hippocampal plasticity. ps a-i thimet oligopeptidase co-exists in gfap-and cd b-positive glia in rat pc/rsc treated with mk- takeshi kato , mohammad arif , michiyuki yamada , toshiyuki chikuma , md. mahiuddin ahmed lab. natural info. sci., grad. sch. integr. sci., yokohama city univ., yokohama, japan; grad. sch. integr. sci., yokohama city univ., yokohama, japan; dept. hygien. chem., showa pharmaceut. univ., machida, japan; dept. r&d, bioelectro. anal. sci., japan thimet oligopeptidase (ep . ) hydrolyzes not only neuropeptides but also the peptides generated by proteasomes. in the present immunohistochem study we found that mk- activated gfapand cd b-positive glia cells in rat posterior cingulate/retrosplenial cortex (pc/rsc) day after the treatment. mk- also increased ep . and prolyl oligopeptidase. immunohistochem data showed that ep . co-localized with gfap and cd b positive glial cells. since mk- causes schizophrenia-like psychosis and produces neurotoxicity in adult rodent brain, we further examined the pretreated effect of neuroleptics. clozapine co-administration suppressed the increased ep . in the pc/rsc. these data suggest that ep . in the astroglia and microglia cells of rodent brain might in part control positive and/or negative schizophrenia symptoms. ps a-i effect of age and sex steroids on the expression of alzheimer's disease presenilin (ps) and in the mouse brain soumi ghosh, m.k. thakur banaras hindu university, india alzheimerǐs disease is a neurodegenerative disorder characterized by the impairment of cognition and memory. these functions are improved by supplementation of sex steroids. the genes causing lateonset of ad, presenilin (ps) and , code for highly homologous integral membrane proteins. the proteolytic fragments of these proteins are main biological components. we have analysed the effect of age, sex and gonadal hormone supplementation on ps expression at protein level by western blotting. ps shows a significant decrease with aging in both males and females. however, there is no significant variation in expression of ps and ps with sex. gonadectomy also lowers the level of presenilin proteins in old age. ps protein shows increase in expression with gonadal hormone treatment in both ages, but estrogen supplementation to old mice lowers ps level. these modulatory effects of age, sex and gonadal hormones on ps proteins may explain the therapeutic interventions of hormone replacement therapy. research funds: ministry of science and technology, india ps a-i effects of the monomeric, oligomeric and fibrillar beta-amyloid peptides on the proliferation and differentiation of adult neural stem cells from svz dept. of pharmacol., seoul natl. univ., south korea the subventricular zone is the largest neurogenic area of the adult brain. in this region, neural stem cells (nsc) serve to produce newly generated neurons and glia cells throughout adulthood. however, the common neurogenesis of nsc cannot replace neuronal loss in alzheimerǐs disease (ad) induced by amyloid deposits composed mainly of amyloid␤proteins. in vitro, we examined the effects of various form of a␤peptide on the proliferation and differentiation of nsc from svz of -week-old adult mice. in this study, a␤ peptide was prepared three forms of aggregating stage, monomeric, oligomeric and fibrillar a␤ peptide. we found that treatment of nsc with oligomeric form of a␤ peptides remarkably increased the number of neurospheres during proliferation and neurons during differentiation in-vitro. we also found that these neurogenesis was accompanied by morphological change of neuron. the number of secondary and tertiary neurites increased at submicromolar concentrations of oligomeric a␤ peptide without shrinkage of axonal length. in alzheimer's disease (ad) brain, the formation of senile plaque with accumulated microglia is observed. although the role of microglia in ad is not clarified, their involvement in a␤ clearance is noted. high mobility group box protein- (hmgb ) is a non-histone chromosomal protein. here, hmgb was associated with senile plaques and protein level was increased in ad brain. diffuse hmgb immunoreactivity was observed around dying neurons in the kainic acid-and a␤ - (a␤ )-injected rat hippocampi. hmgb was not co-localized with a␤ in transgenic mice which show massive a␤ production without neuronal loss. furthermore, co-injection of hmgb delayed the clearance of a␤ and accelerated neurodegeneration in a␤ -injected rats. these results suggest that hmgb released from dying neurons may inhibit microglial a␤ clearance and enhance the neurotoxicity of a␤. perineuronal nets consisting of chondroitin sulfate proteoglycan (cspg) and hyaluronic acid (ha) are associated with distinct populations in mammalian brain. in the present study, we observed perineuronal net-like structure by rat cortical neurons in dissociated culture using wisteria floribunda lectin, ha binding proteins, and cspgspecific antibodies. this perineuronal net-like structure was observed often at parvalbumin-positive neurons, indicating gabaergic ones. it is well known that perineuornal nets-containing neurons are survive against alzheimer disease in human. to elucidate significance of perineuronal nets in alzheimer disease, we applied beta-amyloid peptide into cultured cortical neurons. perineuronal nets-containing neurons were resistant against beta-amyloid peptide, while negative neurons were often dead. these results indicate that perineuronal nets are participated in protecting neurons from cytotoxic substances such as beta-amyloid. ps a-i x -like protein regulates metabolism of app in the mouse brain yoshitake sano , , tadashi nakaya , shigeyoshi itohara , toshiharu suzuki riken bsi, saitama, japan; hokkaido university, neuroscience, sapporo, japan abnormal metabolism of amyloid beta precursor protein (app) results in the accumulation of beta amyloid (a␤) in the brain, and contributes to the pathogenesis of alzheimer's disease. app has a functional sequence in its cytoplasmic domain, the yenpty motif, which is involved in trafficking, internalization, and metabolism of app. x -like protein (x l) was identified as a molecule that interacts with the motif and regulates app metabolism in cultured cells ( . j. biol. chem. , . j. biol. chem. , ) . there is no evidence, however, that endogenous x l suppresses app metabolism and a␤ generation in vivo. to examine the physiologic role of x l in app metabolism in the brain, we generated x l null mutant mice. the mutant mice developed normally without gross anatomic brain abnormalities. there were increased amounts of cterminal fractions cleaved at the ␤-site and a␤, but the amount of total app was unaltered in the mutant mouse brain. these results suggest that x l suppresses the production of a␤ by inhibiting ␤secretase-induced proteolysis of app. it is still unknown how human's central nervous system (cns) controls its body system to keep the body balanced. this study aims to analyze the characteristics of spectral response of body sway in eyes open and in eyes closed during static upright stance based on a pid control model. in this model, body sway in medial-lateral direction is considered, and the body is simply modelled as a multi-link inverted pendulum system. spectral response analysis showed the gain varied with input frequency and time lag. peaks of the gain were intensively influenced by controller's parameters (kp, kd and ki). parameters identification showed that kd is decreased in eye-closed. by simulation, the spectral responses of the pid model quite agreed with the experimental data. the results proved that the spectral characteristics of body sway is determined by the dynamics of body system and its controller's parameters, suggest the balance-keeping control in cns can be modelled as a pid controller. nuclear dysfunction is a critical element of the pathology of polyglutamine (polyq) diseases. proteome analysis of soluble nuclear proteins in the nuclear matrix of neurons expressing normal or mutant huntingtin or ataxin- protein by d-electrophoresis and tof-mass delineate that mutant at and htt proteins similarly reduce transcriptional factor x and x . immunoprecipitation and pull-down assays support interaction between polyq and factor x and x . immunohistochemistry of hela cells and primary neurons reveal sequestration of factor x and x into inclusion bodies and reduction of them in the nuclear matrix. compensatory expression of factor x and x ameliorates poly-q pathology in htt-/at -expressing neurons and transgenic drosophila. these results suggest that factor x and x are critical regulators of polyglutamine disease pathology and could be a target for developing therapeutics. ps a-j ba - was reduced in rat brains fed with coconut juice n. radenahmad, p. subhadhirasakul psu, thailand young coconut juice (ycj), cocos nucifera (arecaceae), believed to contain phytoestrogen and other sex hormone-like substances, was investigated for its possible beneficial effects on halting dementia in ovariectomized (ovx) rats, a model system for the postmenopausal condition. sixty ovx rats were divided into six groups, rats/group (g). group received e at . g/kg per day; groups and received ycj at ml, and ml/kg day, respectively, once everyday. group received ycj ml/kg plus e at . g/kg day twice a week, all for weeks. the other two were ovx and sham-operated controls. using a chemiluminescent immunoassay, circulating e in group was insignificantly different from the control groups. after rats were sacrificed, brains were removed, fixed and paraffin embedded for ihc staining. using anti-␤-amyloid - antibody, this alzheimer pathology was found in cytoplasm and dendrites, but not in nuclei or axons, of pyramidal cells both in hippocampus and in layer and layer of cerebral cortex. it was found that amyloid deposition in frontal, temporal and hippocampus of rat brains in group was lesser than ovx and control groups. amyloid deposition was correlated with e serum at r = − . . ps a-j correlation between semantic memory and regional gray matter volume of anterior aspect of right temporal lobe in normal elderly subjects. a voxel-based morphometry yasuyuki taki , shigeo kinomura , kazunori sato , shinya uchida , ryoi goto , kentaro inoue , ichiro tsuji , hiroyuki arai , ryuta kawashima , hiroshi fukuda department of radiology and nuclear medicine, institute of development, aging and cancer, tohoku university, sendai, japan; tohoku univ. grad. school of med., sendai, japan; niche, tohoku univ., sendai, japan the purpose of this study was to determine whether there is a correlation between semantic memory and regional gray matter volume in community-dwelling normal elderly people by voxel-based morphometry. we collected brain magnetic resonance images of community-dwelling normal elderly subjects. we performed multiple regression analysis of raw score in the wais-r information subtest, gender, and regional gray matter volume. the volumes of the right superior and middle temporal gyri showed significant positive correlations with raw score in the information subtest. our study indicated that normal elderly individuals show a significant correlation between regional gray matter volume and semantic memory. research funds: (h -kenko- ), (h -choju- , h -choju- ) ps a-j effects of fluoxetine on the cognition of patients with mild cognitive impairments arash mowla, azadeh pani shiraz university of medical sciences, iran recent researches suggest a role for monoaminergic hypofunction in age related cognitive decline. in several studies selective serotonin reuptake inhibitors demonstrated neurogenesis in hippocampus. we studied the effects of fluoxetine on cognition of patients with mild cognitive impairment (mci). fifty-two non-depressed patients with mci were randomly assigned to take fluoxetine or placebo. the patients were administered mini-mental status examination (mmse) and wechsler memory scale iii (wmsiii) pre intervention. twenty-six patients completed the weeks trial. treatment response was defined as a final mmse and wms-iii scores. the patients in the fluoxetine group showed improvement in mmse and immediate and delayed logical memory scores of wms-iii. the placebo group had not significant changes in the cognitive measurements. fluoxetine enhanced memory and cognition in the patients. this was consistent with pervious studies that emphasized on the role of fluoxetine in improving memory and promoting neurogenrsis in the hypocampus. however, this conclusion should be tempered by the small sample size. lisa l. cook , d.g. goodenowe , y. yamazaki , j. flax phenomenome discoveries inc., saskatoon, canada; precisionmed inc., san diego, ca, usa dementia affects about % of the population over the age of and can result from various neuropathological conditions. currently, there is no way to differentiate specific forms of dementia (alzheimer's disease (ad), vascular dementia, etc.) prior to autopsy. pdi has discovered an -metabolite biomarker panel within the serum of patients with ad, non-ad dementia and healthy non-demented controls that can simultaneously differentiate the type of dementia and identify cognitive impairment using a non-targeted metabolomics technology based a fourier transform ion cyclotron resonance mass spectrometry (fticr-ms). the accurate measurement of the metabolite mass is sufficient to elucidate its molecular formula, thereby leading to metabolite identification, explication of biological significance and efficient biomarker validation. the -metabolite biomarker panel could provide a non-invasive method to aid in the diagnosis of specific subtypes of dementia. the development of a high throughput assay for these markers will also be presented. neurons become photosensitive by genetically introducing one of green algae-derived protein, channelrhodopsin- (chr ). in this study, we quantitatively investigated the rapidness of the light-gated current of chr expressed in pc cells using blue led light. the light-gated current consists of two components, inactivating and noninactivating. the magnitude of inactivating component was almost linearly related to the light intensity. the non-inactivating component showed the tendency to saturate at high illumination. we also found that the activation rate is about -fold faster than the inactivating rate, but both are linearly dependent on the light intensity. since the photoactivated current was very rapid in both onset and offset, the neuronal firings were phase-locked to short light pulses in an acute slice of hippocampus. it is suggested that the genetic expression of chr is one of the most ideal photostimulation methods of a genetically identified neuron with defined activity patterns in intact nervous system. yujiro hattori , shigeki ohta , kenji hamada , naofumi yamada-okabe , yonehiro kanemura , hideyuki okano , yutaka kawakami , masahiro toda , neuroimmnology research group, keio univ., tokyo, japan; chugai co. ltd., kanagawa, japan; inst. cli. res., onh, japan; physiology, keio univ., tokyo, japan; cellular signaling, institute for advanced medical research, keio univ., tokyo, japan; neurosurgery, keio univ., tokyo, japan to identify neuron specific genes, we performed two gene profiling techniques, dna microarray and est analysis. in this study, we focused on genes expressed specifically in the normal brain tissues but not in glioma tissues and identified the human kiaa gene which was a homologue of rat synarfgef (po). rt-pcr analysis revealed that the human kiaa homologue was expressed only in adult brain tissue. western blot and immunocytochemical analyses showed the kiaa protein was expressed in adult brain tissues and differentiated neuronal cells but not in fetal brain tissues nor neural stem/progenitor cells. in conclusion, we identified an adult neural-specific gene using the combined gene profiling method and our results suggest the usefulness of this method to identify tissue specific genes. ritsuko the objective of this study was to find the proteins related to sexual differentiation and to elucidate its molecular mechanism. methods: developing hypothalamic and cortical cells from fetuses on embryonic day were dissociated. after the cells were treated with nm estradiol- beta (e ) or ethanol for days, proteins were extracted and labeled with cydyes. two-dimensional difference gel electrophoresis ( d dige) was then performed. the differential protein spots were analyzed by software analysis, subject to in-gel digestion, and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (maldi-tof-ms). results: more than spots were detected from d dige. compared with ethanol treatment, e increased the expression of spots in the hypothalamic cells and spots in the cortical cells (p < . , difference > . ). proteomics analysis showed different effects of e for hypothalamic and cortical cells. in order to relate cellular brain structure to function, it is necessary to manipulate neural circuits at the level of individual cell types. genetic methods for neuronal inactivation combined with cell-typespecific promoters will achieve this goal. here, we have developed a genetic method for quickly and reversibly inactivating in vivo mammalian neurons using allatostatin receptor (alstr), which causes neuronal hyperpolarization when treated with peptide ligand allatostatin (al). in rat barrel cortex neurons expressing alstr, al reversibly inactivated neuronal activity evoked by electrical stimulation of the whisker pad. both inactivation and recovery were seen within several minutes. we also confirmed the effectiveness of the al/alstr system in ferret visual cortex, lateral geniculate nucleus (lgn), and monkey lgn. therefore, the al/alstr system will be a powerful tool to investigate neuronal circuits and function. prospective purification of neural stem cells (nsc) through the specific cell surface marker is crucial for functional recovery of the damaged brain. in the last meeting, we showed that living nsc were enriched from mouse whole brain as positive cells for erythro-phytohemagglutinin (e-pha), which binds to complex type asparagine-linked oligosaccharide (n-glycans). in this study, by using facs system, we found that high selective affinity of e-pha binding to the brain cells; e-pha negative cells were neurons, mid-positive cells were nsc, and highly positive cells were endothelial cells, respectively. ligand blot analysis revealed the existence of the e-pha binding proteins different from the known selective nsc markers in e days brain homogenate, suggesting that n-glycosylated proteins could be distinctive markers for nsc. masahiro waza, hiroaki adachi, masahisa katsuno, makoto minamiyama, fumiaki tanaka, manabu doyu, gen sobue department of neurology, nagoya university, nagoya, japan the pathogenic gene product of spinal and bulbar muscular atrophy (sbma) is polyglutamine (polyq)-expanded androgen receptor (ar), which belongs to hsp client protein family. -allylamino- -demethoxygeldanamycin ( -aag) is a new derivative of geldanamycin that shares its important biological activities but shows less toxicity. -aag is now in phase ii as a potential anti-cancer agent because of its ability to selectively degrade several cancer-related client proteins. we examined the efficacy and safety of -aag in a mouse model of sbma. administration of -aag significantly ameliorated polyq-mediated motor neuron degeneration by preferential proteasome degradation of mutant ar. the ability of -aag to preferentially degrade mutant protein would be directly applicable to sbma and other neurodegenerative diseases. modulation of hsp function by -aag has emerged as a candidate of molecular targeted therapy for neurodegenerative diseases. the avian embryo has long been a popular and an excellent model for studying vertebrate development because of its classical manipulative advantages. in the present study, we tried to develop regulated gene transfer by using the tet regulatory system in the chick. the reverse tetracycline-controlled transactivator was expressed under the control of the motor neuron (mn) specific hb promoter. tetracycline responsive elements were used for inducible gfp expression. after these constructs were introduced into neural tube by in ovo electroporation, gfp expression was induced in spinal mns in the presence of doxycycline. approximately - % of mn express gfp very intensely whereas the remaining mns never express gfp, suggesting that, although the transgene is induced in limited numbers of mns, once activated, cells express a large amount of the protein product of the experimental gene. thus, this strategy can be applicable for a variety of experiments that require specially and temporally regulated gene expression in the chicken embryo. ps a-k selective collection of catecholaminergic (ca) neurons in the brain and its application to gene expression analyses hiroaki nakamura , yoshiyuki ishii , kazuto kobayashi , yasufumi sato , keiichi itoi lab. info. biol., grad. sch. info. sci., tohoku univ., sendai, japan; dept. mol. genet., fukushima med. univ., japan; inst. develop., aging, cancer, tohoku univ., japan ca neurons are involved in a wide spectrum of physiological functions in the brain. most ca neurons are localized in the brainstem and hypothalamic regions and typically make clusters of cells, among which the noradrenergic (a , a and a ) and dopaminergic (a , a and a ) neurons predominate. in order to explore functional roles of these neurons, we collected ca neurons selectively using tyrosine-hydroxylase (th)-green fluorescent protein (gfp) transgenic mice in which gfp was expressed under the control of th gene promoter. in fetal mice, most gfp-positive neurons expressed th-immunoreactivity in limited brain regions including the locus coeruleus (lc). therefore, lc-containing region was dissected under fluorescent microscopy, and neurons were dispersed by treating with trypsin, then gfp-positive cells were sorted out by flow-cytometry (facs). rna was extracted from the gfp-positive (th) neurons, reverse-transcribed, and analyzed by pcr. atg b has been shown to play an important role in the processing of lc , a mammalian homologue of yeast atg , but the tissue distribution of atg b remains unknown. to understand the role of atg b in rat tissue cells, we prepared an antibody to atg b and pc cells in which atg b expression was knocked down by rnai. in rnaitreated pc cells where atg b expression was % of that in the wild-type pc cells, the expression of cytosolic lc -i was similar to that in wild-type cells. the knockdown cell lysates, however, suppressed cleavage of prolc to lc -i. moreover, the expression of atg b mrna was high in the cerebellum and olfactory bulb, while its protein was evenly distributed in the brain. immunostaining for atg b was intense in neurons, especially in the cerebellum. these results suggest that atg b plays a major role in the processing of lc , while autophagy is deeply associated with the metabolism in neurons, especially in the cerebellum. ps a-k spatial and time-dependent transneuronal propagation of swine coronavirus (hemagglutinating encephalomyelitis virus, hev) in the rat central nervous system after its hind footpad inoculation transneuronal propagation of hev n strain into the central nervous system was examined after its subcutaneous inoculation ( pfu) in the rat hind footpad. on day post-inoculation (p.i.), antigenpositive neurons were detected in cell groups of the ipsilateral spinal cord of lumber segments. on day p.i., they increased in number, and higher-order transneuronally infected neurons were observed in restricted brain areas that project to the spinal cord. on day p.i., the viral infection became more extensive and complex, and neurological signs appeared from this period. in this model the th day would be critical for the analysis of the long-distance connections. hev can be used as a novel tracing probe, being equivalent to other reported virus probes. hiroyuki hioki , hiroshi kameda , hisashi nakamura , taro okunomiya , koji ohira , kouichi nakamura , , takahiro furuta , takeshi kaneko , dept. of morphol. brain sci., grad. sch. of med., kyoto univ., kyoto, japan; crest, japan vesicular stomatitis virus g-protein (vsv-g) pseudotyped lentiviral vectors are useful vectors for gene transfer into the central nervous system. vsv-g achieves a broad transduction spectrum and lentiviral vectors provide an efficient vehicle to integrate transgenes into dividing and non-dividing cells. thus, vsv-g pseudotyped lentiviral vectors with ubiquitous promoters, such as human cytomegalovirus (hcmv) promoter, infect and express transgenes in neuronal and glial cells. the purpose in this study is to explore neuron-specific promoters and to quantitatively examine their characteristics. at first, we used five kinds of well-known neuron-specific promoters; hsyn , rta , mcamkii, rnse and hpdgf promoters. then, we developed new hybrid promoters by a combination sequence of hcmv enhancer and neuron-specific promoters listed above. all of the new hybrid promoters dramatically improved expression of reporter gene (gfp), but the specificity deteriorated in the rat striatum, thalamus and neocortex. although green fluorescent protein (gfp) is a useful tool to label living neurons, neuronal processes are not completely labeled with gfp. in the present study, we tried to develop dendritic membrane-targeted gfp using non-prolilferative lentivirus vector with human synapsin i promoter. palmitoylation site of gap n-terminal and myristoylation site of fyn n-terminal were first tested for membrane targeting of gfp. myristoylated gfp (myrgfp) was efficiently localized at the plasma membrane of infected neurons, but not palmitoylated gfp. since myrgfp was located at both dendritic and axonal membranes, we further added the putative dendrite-targeting or basolateral targeting signals, such as c-terminals of telencephalin (tlc), fc ␥ ii ␤ receptor (fcr), polymeric immunoglobulin receptor (pigr), and low density lipoprotein receptor (ldlr), to c-terminal of myrgfp, and compared their efficiency on dendrite targeting. recently, we developed recombinant rabies virus vectors which were expected to act as a potential neurotracing tool. the vectors infected neurons specifically from axon terminals and were transported to the downstream neurons trans-synaptically. by using two different recombinant vectors, each of which expresses reporter protein of different kind, we attempted double labeling of a neuron. it was expected that we could detect and visualize the divergence or convergence of a neurocircuit. in the study, we could demonstrate the efficiency of double labeling in vivo. in the present study, we examined the potential of this technique particularly in terms of the quantitative detection of double labeled neurons in complicated neurocircuit. the experiments were performed in the hippocampus and the neighboring cortices of rats. we could show that this technique is also useful for the quantitative analysis of neurons which forms projections to different region of the brain. shuchen lee, lihao ge institute of neurobiology, institute of brain science, fudan university, shanghai, china glycine receptors on bullfrog retinal cone photoreceptors were characterized by immunocytochemical and whole-cell patch clamp techniques. cone terminals were both gly␣ and gly␤ immunoreactive. in freshly dissociated cones, an inward current could be induced while glycine was focally applied to the terminal. the glycine-induced current was strychnine-sensitive and reversed in polarity at a membrane potential, close to the equilibrium potential of chloride ions. these results suggest that glycine, which may be released by glycinergic inplexiform cells, could modulate functions of cone photoreceptors. ␦-catenin has armadillo motifs and a carboxyl terminal type i pdz ligand. in neurons, ␦-catenin is enriched in the postsynaptic density, where it serves as a link between the adherens junction and the post-synaptic protein complex including the nmda and ampa receptors. electrophysiological recordings from ca hippocampal neurons overexpressing ␦-catenin demonstrated that ␦catenin increased the ampa receptor-mediated epsc but had no significant effect on the nmda receptor-medicated epsc. the effect of ␦-catenin on the ampar-epsc was medicated by its pdz ligand. in cos cells, co-transfection of ␦-catenin/grip showed that ␦-catenin regulated the membrane localization of grip through its pdz ligand. co-transfection of ␦-catenin/grip/gulr increased the surface expression of glur in cos cells compared with grip/glur or ␦-catenin/glur transfection. this study points to ␦-catenin as a regulator of glur receptor trafficking. inseon song, kunihiko obata, alexey semyanov bsi, riken, japan gaba a receptor mediated tonic conductance is a major component of membrane conductance which determines the way how neuron integrates incoming synaptic signals as well as input-output characteristics of the cell. we measured density of picrotoxin (gaba a receptor antagonist) sensitive holding current (which reflects gaba a receptor mediated conductance) in interneurons of hippocampal ca area in wild type (wt) and gad knockout (ko) mice. this parameter was twice lower in gad ko mice (wt: . ± . pa/pf, n = ; gad ko: . ± . pa/pf, n = ; p = . ). the total membrane conductance was similar in both types of animals suggesting adaptive compensation. application of gaba ( m) increased tonic current in both type of mice by the same amount. no significant difference in amplitude or frequency of spontaneous ipscs was detected, although their decay time was shorter in gad ko animals (wt: . ± . ms, n = ; gad ko: . ± . ms, n = ; p = . ). the changes in inhibition which we have found may explain previously reported behavioral abnormalities in gad ko. research funds: bsi, riken hiroki mutoh, thomas knopfel lab. for neuronal circuit dynamics, bsi, riken, wako, japan olfactory glomeruli constitute the first stage of central odor processing. yet, their role in integration of odor information is only partially understood. we previously discovered that hz olfactory nerve (on) stimulation induces long-term depression (ltd) in young (p to p ) mice. the present experiments were designed to understand in more detail the molecular mechanisms underlying on ltd. bath application of dhpg, a selective group i mglur agonist, induced on ltd and occluded subsequent hz stimulation-induced ltd. on ltd was not induced by activation of group ii or iii mglur agonists. the dhpg-induced on ltd was mediated by mglur but not by mglur because it was antagonized by the mglur antagonist ly but not by the mglur antagonist mpep. expression of dhpg-induced on ltd was accompanied by an increase in paired-pulse ratio suggesting that on ltd is caused by a decrease of release probability. we propose that mglur is expressed at the on. on ltd may be important for establishment and maintenance of odor maps in the olfactory bulb but may also involve in the regulation of the sensitivity for specific odorants. ps p-a involvement of dopamine system in long-term potentiation of thalamo-prefrontal cortex pathway masatoshi takita , michiko ohtomi cognition and action group, national institute of advanced industrial science and technology (aist), ibaraki, japan; department of biomolecular science, faculty of science, toho university, chiba, japan a mesocortical dopaminergic (da) input to prefrontal cortex (pfc) with the d receptor is necessary for long-term potentiation (ltp) to occur at hippocampal-pfc synapses, which is involved by working memory (wm) in rats. here the da system was investigated in another wm-involved pathway from mediodorsal nucleus of the thalamus (md) to pfc. preliminarily, local perfusion of the d antagonist sch into pfc by using a microdialysis method impaired md-pfc ltp but the d antagonist sulpiride did not. extracellular da levels in the pfc robustly increased after the tetanus of md (by - %). as a result both excitatory synaptic inputs to the pfc involved the wm-related da profile, implying da system enables a contrast-emphasis for cooperative crosstalk among several neuroplasticities in the pfc to selectively store intersectional information of multiple brain areas. neuronal activity is necessary for postnatal maturation of synaptic connections only grossly laid out in the neonatal brain. in sensory cortices, synaptic maturation involves strengthening of sensory-evoked responses and development of receptive field (rf) maps with defined rf size and shape. evoked activity is thought to shape synaptic maturation in sensory cortices by mechanisms of competitive hebbian plasticity. dendritic excitability, mediated by voltage-gated na + channels, is required for active backpropagation of axosomatic action potentials (aps) and initiation of dendritic spikes; backpropagating aps and dendritic spikes enable forms of synaptic hebbian plasticity, such as spike-timing dependent plasticity (stdp). here we examined the role of dendritic excitability in synaptic maturation of layer / pyramidal neurons in the rat somatosensory barrel cortex. in the present study we compared ltp induction in neocortex of captreated and normal rats in present of gaba antagonist, picrotoxin (ptx). the result of present experiment showed that ptx plays an important facilitatory role in the induction of ltp in both normal and cap-treated group. in cap-treated group, in present of ptx, the ltp responses significantly were higher than normal group. we conclude that the enhancement of ltp by ptx can be explained by product of competition between excitatory and inhibitory pathways or synapses. these results suggest that gabaergic system has an important role in synaptic plasticity. also, these results indicated that gabaergic inhibition has been increased in cap-treated group. tohru kurotani, komatsu yukio department of visual neuroscience, research institute of environmental medicine, nagoya university, nagoya - , japan we showed in previous study that somatic inhibitory synapses of neocortical layer pyramidal neurons undergo long-lasting depression and potentiation depending on the intrinsic firing pattern of the cell that mimics slow wave sleep (sws) and arousal states. in the present study, using a minimal stimulation method, we recorded somatic ipscs from layer pyramidal cells in visual cortical slices prepared from rats at sws like state under urethane anesthesia and in those prepared from rats at arousal state. the average amplitude of somatic ipscs recorded in slices from the former group was significantly larger than that recorded in slices from the latter group. the mean rise time, decay time constant of ipscs and the mean input resistance of the cells were not significantly different between these two groups. the present results further confirmed that the somatic inhibition in neocortical layer pyramidal neurons is bidirectionally modified in accordance with behavioral state. corticothalamic fibers (ct), originated from cerebral layer pyramidal cells, make excitatory synapses with both thalamic relay neurons and reticular neurons. since these pyramidal cells abundantly express kainate receptors (kars) mrna, we studied the effect of kainate on the presynaptic function of the two ct synapses in mouse thalamic vb nucleus. bath application of kainate ( nm) depressed ct-epscs and increased the paired pulse ratio in relay neurons. in contrast, kainate at the same concentration facilitated ct-epscs and decreased the paired pulse ratio in reticular neurons. these results suggested that kars differentially regulated release at the two ct synapses. furthermore, high frequency stimulation of ct depressed relay cell synapses but facilitated reticular cell synapses. blocking endogenous kars abolished these effects. because reticular cells are the main source of inhibitory input to relay neurons, we suggested that endogenous kars presynaptically regulate the balance of excitatory and inhibitory inputs to thalamic relay neurons. to examine the involvement of ntr in the regulatory mechanisms for ltp in the amygdala, we utilized ntr -knockout (ko) mice. we performed whole-cell patch-clamp recordings from the pyramidal neurons in the basolateral amygdala (bla), where da-nt neurons project. we found that the bla-ltp, induced by la stimulation, was significantly greater in ntr -ko mice than in wild-type mice. the bla-ltp in ntr -ko mice was attenuated by sulpiride, a d receptor antagonist. these results suggest that d -ntr interaction regulates the extent of ltp in the mouse la-bla synapses. ps p-a facilitation of axonal plasticity in recovery from traumatic brain injury and the role of tnf␣ in mouse model recent studies suggest axonal plasticity as possible mechanism of recovery from brain injury. apart from that, tnf␣, an inflammatory cytokine, has also been suggested to serve neuroprotective roles. the present study evaluated motor function recovery after controlled cortical impact (cci) brain injury, and also the facilitation of plasticity by biotin dextran amine (bda) axonal tracing in tnf␣ko mice and wild type (wt) mice. mice were subjected to left sided cci or served as sham controls, and were evaluated by composite neuroscore and rotarod over -day period. bda was injected in right cerebral cortex to observe new axonal connections. so far, we observed recovery of motor function in wt mice, whereas tnf␣ko mice showed continuous functional deficit. we also observed greater number of new axonal connections in wt mice. our results suggest that tnf␣ is necessary for functional recovery after brain injury, and axonal plasticity may be the mechanism involved. disuse of synaptic activity causes homeostatic adaptation presynaptically and/or postsynaptically. here we show that in hippocampal autaptic cultured neurons tetrodotoxin-induced chronic inactivity increases the fraction of high vesicular release probability pool with the entire readily releasable vesicle pool size remained intact. kinetics of short-term plasticity and unchanged apparent ca + sensitivity indicate that ttx-induced presynaptic modification is unlikely due to an increase in the fusion rate crucial for the ca + at the final fusion step. in addition, analysis of neurons genetically lacked the synaptic vesicle protein synaptotagmin- , and timing-dependent rescues using two different viruses provide a novel conception, namely, vesicle machinery requires prolonged period so that the fast burst vesicle pool orchestrates presynaptic homeostasis system underlying "vesicle mobilization". ps p-a inhibitory modulation of the hippocampal ca transmission and plasticity by glucagon-like peptide- jun-ichiro oka, takashi iwai lab. pharmacol., fac. pharm. sci., tokyo univ. sci., japan glucagon-like peptode- (glp- ) is a proglucagon-derived peptidehormone in the intestine and brain. we reported that glp- (i.c.v.) improved the concussive brain injury-or scopolamine-induced amnesia in mice. however, the mechanisms of glp- effects on hippocampal neurons are unclear. in this study, we investigated the effects of glp- on the synaptic function of neurons in the acute hippocampal slices. hippocampal slices ( m) were prepared from to days wistar rats of both sexes. patch-clamp recordings were made from pyramidal cells of the ca in the whole-cell mode using glass microelectrodes (resistance: - m ). in extracellular recordings, field excitatory postsynaptic potentials (fepsp) were evoked with a bipolar tungsten electrode, placed in the mossy fibers. glp- ( nm- m) inhibited spontaneous excitatory postsynaptic current. glp- ( nm) did not affect fepsp amplitude or the paired-pulse ratio, but attenuated the long-term potentiation. these results suggest that glp- may play an inhibitory role in the dg-ca transmission. ps p-b quantitative imaging of exo-endocytosis at mossy fiber presynaptic terminals of hippocampus by genetically expressed fluorescent probe takuya hkima, rikita araki, toru ishizuka, hiromu yawo dept. of dev. biol. and neurosci., tohoku univ. grad. sch. of life sci., japan both presynaptic and postsynaptic mechanisms are proposed for the synaptic plasticity. however, the presynaptic mechanisms have been analyzed indirectly on the postsynaptic responses. it has been difficult to quantify the exocytosis at the presynaptic terminals, particularly those in vivo or in acute slices. to measure exocytosis directly, we applied the synaptophluorin (sph) method to the individual presynaptic terminals in hippocampal slices of a mouse genetically expressing a conjugate protein of vamp- and superecliptic phluorin selectively in the mossy fiber terminals. the sph fluorescence at individual mossy fiber terminal was increased by nerve stimulation and was followed by its reduction which is blocked by bafilomycin a , a vesicular h+-atpase inhibitor. therefore, the rising phase of sph fluorescence corresponds to exocytosis whereas the decreasing phase to endocytosis and subsequent re-acidification of vesicles. this method would enable us to evaluate the presynaptic contribution to synaptic plasticity. jyoti parkash, gurcharan kaur gndu amritsar, india we have earlier reported that gnrh nerve terminals in the me continue to express high levels of polysialylated form of neural cell adhesion molecule (psa-ncam) in a cyclic fashion and psa-ncam covers both the gnrh axon surfaces and the associated glial cells in the proestrous phase rats indicating that psa plays important role in the neurosecretory activity in hypothalamus. to further establish the functional significance of psa-ncam molecule, we have studied the expression of psa-ncam on gnrh axon terminals and glial cells in the proestrous phase of cycling rats as well as gaba and pbz treated proestrous rats by using dual immunohistofluorescent staining. both gnrh and psa-ncam immunostaining was much higher in proestrous phase rats, whereas, gaba and pbz treatments significantly reduced their expression. the expression of pst has been studied within gnrh cell bodies as well as at their terminals by combining in situ hybridization with immunohistofluorescent in poa and me-arc regions of cycling female rats as well as in gaba and pbz treated proestrous rats. cortical plasticity has important roles in the development of neural circuits in sensory cortices. however, the roles and mechanisms for various types of ltp and ltd are not clear. we investigated supragranular ltp and two types of supragranular ltd in the slices obtained from the rat auditory cortex, and compared their properties. frontal cortical slices were prepared from male wister rats. supragranular field potentials elicited by the stimulation applied to layer vi were recorded. ltp was induced by tetanic stimulation (ts, hz for s) applied to layer vi. ltd was induced by low-frequency stimulation (lfs, hz for s) applied to layer vi. ltd was also induced by ts applied to supragranular layers near the recording site. lfs-induced ltd and ts-induced ltd were completely abolished in the presence of m apv, m bicuculline, but not m mcpg. lfs-induced ltd and ts-induced ltd occluded each other, suggesting that that both types of ltd share cellular and molecular mechanisms. kazuyoshi kawa department of neurophysiology, tohoku university, graduate school of medicine, sendai, japan using slice-patch techniques, synaptic transmission in neurons of the area postrema (ap) of the rat was studied. when mm kcl was applied from a "y tube" to ap neurons (whole-cell clamped at − mv), massive inhibitory postsynaptic currents (ipscs) were induced. most of the evoked ipscs were blocked by bicuculline confirming gabaergic identity. when nicotine ( - m) or capsaicin ( . - m) was applied to ap neurons, robust appearance of ipscs with gabaergic identity was induced. after blocking action potential generation in the slice with tetrodotoxin ( m), nicotine and capsaicin could still induce gabaergic ipscs. interestingly, responses to capsaicin of the synaptic facilitation showed marked desensitization even after min of rigorous washout. it is concluded that nicotinic receptors, as well as capsaicin receptors (presumably, trpv ), are expressed at gabaergic presynaptic terminals in area postrema neurons and play a distinctive role in controlling autonomic neural functions. research funds: grant from the smoking research foundation (japan) takako morimoto-tanifuji , akira komatu , akinao nose dept. phys., univ. tokyo, tokyo, japan; dept. physiol., sch. med., tokyo women's med. univ., tokyo, japan the molecular mechanisms that target neurotransmitter receptors to the postsynaptic membrane and keep them clustered remain unknown. we investigated how the localization of glutamate receptors (glurs) is regulated in neuromuscular junctions (nmjs) of drosophila rd instar larvae. there are mainly two classes of glurs, containing either gluriia or iib. gluriia has a sequence predicted as ca + -permeable site. when camkii was inhibited by the expression of inhibitory peptide, ala, the content of gluriia in synapses was dramatically increased and the mean amplitude of extrajunctional potential (ejp) was enhanced. the expression of constitutively active form of camkii (t d) resulted in decreased gluriia content and enhanced gluriib content. although miniature ejp amplitude was reduced, ejp amplitude was normal in t d expressing larvae, suggesting the existence of some homeostatic mechanisms. taken together, camkii regulates the localization of glurs in a subunitspecific manner and modulates synaptic function in nmjs. ) . notably, neuronal dnrs from dnr * flies did not show mg + blockade, and dnr * flies displayed significant impairment in transcription-dependent long-term memory (ltm) but not in transcription-independent acquisition and short-term memory. we identified salient increases in genes involved in l-ltp formation, e.g. homer, and activin, as well as the increase in genes involved in ltm, e.g. staufen, upon ltm formation. however, such increases were absent in dnr* flies. transcription for ltm is mediated, at least, by transcription factors such as creb, adf- , and notch. we examined how mg + blockade of dnr links to these transcription factors. research funds: kakenhi ps p-b response properties of wind-sensitive giant interneurons in the th-instar nymphs of the cricket tetsuya matsuura , masamichi kanou dept. of welfare eng., iwate univ., morioka, japan; dept. of biology, ehime univ., matsuyama, japan the response properties of four wind-sensitive giant interneurons (gis) - , - , - and - in the th-instar nymphs of the cricket gryllus bimaculatus were investigated. air current was presented to the animal from different directions in the horizontal plane. the intensity-response curves showed that the response magnitudes of gi - increased with stimulus velocity up to mm/s regardless of the stimulus direction. the response magnitudes of gi - reached a plateau at a stimulus velocity of mm/s in most stimulus directions. the response magnitudes of gis - and - increased with stimulus velocity up to mm/s regardless of the stimulus direction. the directional sensitivity curves revealed that the preferential directions of the gis in nymphs were the ipsilateral-side in gi - , the ipsilateralfront and contralateral-rear in gi - , the ipsilateral-rear in gi - and the ipsilateral-front in gi - , designated with respect to the side of the ventral nerve cord containing the axons, which were basically the same with those of adults. yasuyuki ishikawa, sadao shiosaka division of structural cellular biology, nara institute of science and technology, nara, japan long-term potentiation (ltp) is an enhancement of synaptic strength that may contribute to information storage in the mammalian brain. ltp expression can be regulated by previous synaptic activity, a process known as "metaplasticity." we report a novel form of cellwide metaplasticity in hippocampal area ca . serine protease, neuropsin, is involved in the regulation of synaptic plasticity. neuropsin increased the stability of ltp induced later at the same inputs via l-type vdcc. moreover, neuropsin-deficient mice impaired l-ltp induction by l-tbs. our findings have revealed the effects of neuropsin on the conversion of e-ltp to l-ltp. ptp, a form of presynaptic short-term plasticity, is mediated by a transient increase in transmitter release probability caused by tetanic stimulation. although it has been known that ptp is induced by the elevation of presynaptic ca + , the molecular mechanism of ptp is poorly understood. in order to elucidate the specific role of presynaptic trkb receptors in ptp, we analyzed ptp using hippocampal slices from conditionally gene-targeted mice in which the knockout of the trkb gene is restricted to presynaptic sites in the ca region. we found that ptp induced by the -hz tetanus was reduced in mutant mice, and that ptp in control mice was partially reduced by an n-type ca + channel blocker, while ptp in mutant mice was unaltered by the blocker. thus, these data suggest that the n-type ca + channel-dependent component of ptp requires trkb receptor activation. research funds: jsps and mext of japan kiyoshi ohnuma , kunihiko kaneko , , makoto asashima , grad. sch. of arts & sci., univ. of tokyo, tokyo, japan; jst, tokyo, japan measuring fluctuations or population distributions of a system can be used to understand the dynamics of the system. we have used this approach to study intercellular interaction between neuronal cells. here we show that the shape of the population distribution of intracellular ca + concentration ([ca + ] i ) may change because of nonsynaptic communication. we loaded pc cells with a ca + indicator, indo- am, and the [ca + ] i of more than , cells was measured using flowcytometry. the [ca + ] i distribution of unstimulated single cells had a long right tail, suggesting that [ca + ] i is usually low but sometimes becomes high. on the other hand, the distributions of cell clumps and depolarized single cells were bell shaped, suggesting that many ca + -related mechanisms such as channels and pumps were activated by nonsynaptic communication or by depolarization to change the shape into a normal distribution according to the central limit theorem. our results suggest that measuring the distributions is useful in researching intercellular interaction. na x is a sodium channel involved in sensing the sodium level of the body fluid. our recent studies showed that na x is specifically localized to perineuronal lamellate processes of specialized glial cells in the circumventricular organs, the cns organs involved in the sodium reception. however, molecular and cellular mechanisms underlying the sodium reception of the glial cells has not been elucidated. to address this issue, we developed a functional expression system of the channel protein in cultured glial cells, and found that na x enhances glucose uptake and lactate release in an extracellular sodium-dependent manner. these results suggest that na x alters the state of energy metabolism of the glial cells by sensing a physiological increase of the sodium level. the state of inexcitable glial cells thus play a key role for the control of excitable neural cells in the circumventricular organs. we have isolated spinesin/tmprss from human and mouse cns. in mouse cns, four isoforms (types - ) were expressed. subcellular localization analysis revealed that type (full length) spinesin was predominantly localized to the er, golgi apparatus and plasma membrane, whereas type variant was localized to the cytoplasm. furthermore, we performed expression analysis of m-spinesin in some cell lines. nsc and nb a derived from neuronal cell express only type , whereas os and kt- derived from astrocyte express both type and type . interestingly, it was observed that the level of spinesin mrna was increased by a dibutyryl cyclic amp treatment only in os and kt- . we analyzed promoter region of m-spinesin gene, and identified that -flanking region from − to − bp was essential for m-spinesin gene expression. however, this region did not involve camp-dependent regulation of m-spinesin expression. these results indicate that cell-specific expression and regulation of spinesin gene may play multifunctional roles in cns. it has recently been elucidated that l-serine (l-ser) is one of the glia-derived neurotrophic factors in the brain and its biosynthetic enzyme -phosphoglycerate dehydrogenase (phgdh), which is the first committed enzyme of l-ser biosynthesis in the phosphorylation pathway, is selectively expressed in glial cells, but not in neurons. since l-ser seems to be important in retinal functions as well, we investigated in the present study the cellular distribution of phgdh in the mouse retina. phgdh immunoreactivity was detected in müller cell soma in internal nuclear layer, being close to internal plexiform layer. immunopositive profiles were cellular processes surrounding rod spherules and retinal neurons in internal nuclear layer through nerve fiber layer. it was suggested that müller cells contribute in l-ser synthesis and its transportation to neurons in the retina. astrocytes frequently show spontaneous intracellular ca + signals, such as intra-and intercellular ca + waves; however, their physiological roles remain elusive. the overexpression of an ip -hydrolyzing enzyme, ip -phosphatase, suppressed the spontaneous ca + signals in rat hippocampal astrocytes in culture without noticeable effects on their viability. hippocampal neurons were cultured on a monolayer of astrocytes, and their neurite outgrowth was analyzed. the total neurite length and the number of proximal dendrites and branches decreased significantly when neurons were cultured on the monolayer of ca + -signal-deficient astrocytes. moreover, time-lapse imaging revealed that the extension speed of growing neurites was markedly reduced on ca + -signal-deficient astrocytes. these results indicate that spontaneous ca + signals in astrocytes are essential for glial cells to promote neurite outgrowth. katsuyasu sakurai , noriko osumi , tohoku univ. sch. med., japan; crest, jst, japan astrocytes are the most numerous cells in mammalian brain tissues, although factors regulating their structure and function are still poorly understood. we have previously reported that pax transcription factor is expressed in gfap positive cells in the rat hippocampus. in the present study, we first investigated the expression patterns of pax in postnatal mouse brain and found that pax was expressed in almost all astrocytes in the cerebral cortex. to address the role of pax in the astrocytes, we examined the morphology of the astrocytes in the wild type (wt) and pax heterozygote mutant (sey/+) mice at weeks. confocal imaging revealed that arborization and extension of the astrocytes were poor in sey/+ mice as compared with the wt. in primary culture, the astrocytes isolated from sey/sey cortex showed no morphological difference. however, and h after dibutyryl-camp treatment, the majority of the wt astrocytes had undergone the conversion from a polygonal to stellate shape, while sey/sey astrocytes rarely showed this response. these results suggest that pax regulates the morphology of astrocytes, thereby being involved in astroglial functions. we raised mouse monoclonal antibody (mab) dim to study its distribution and function in cell membrane and found not only its preferential reaction with ptdglc on tlc, but also its labeling in rodent cns (yamazaki et al., ) . we previously reported a unique expression of dim ag in developing mouse brain, especially in cell membranes of embryonic radial glia (kinoshita et al., ) . we show here that mab dim also recognizes adult neural stem cells and glial cells at postnatal period. dim , brdu and gfap co-expressed in cells of mouse neurogenic subventricular zone. we discuss a possibility that the dim ag may be expressed in the radial glia/astrocyte lineage cells. the bone morphogenetic protein (bmp) receptors are thought to have a role in neural patterning of early neuronal development. the bmp receptor is widely expressed throughout the central nervous system (cns) including cerebellum. however, the physiological roles of bmp signaling in mature brain remains obscure. to understand bmp function in cns, we generated a transgenic mouse line that conditionally overexpresses bmp signaling through the type i receptor alk (alternatively known as avcri) in a purkinje cell-specific manner using a cre-loxp system. we bred this mouse line with the cre transgenic mouse line of which expression was driven by l promoter. tissue specificity of cre recombination was monitored by a bicistronically expressed egfp following a constitutively active alk cdna. increased bmp signaling was confirmed by ectopic phosphorylation of smad / / (p-smads) in purkinje cells. we will discuss functional changes of the purkinje cells which receive excess amount of bmp signaling through alk . lipopolysaccharide component of the cell wall of certain bacteria is pyrogenic whose administration to spinal cord injured animals was found to inhibit glial scar formation. glial scar being considered as an impediment for axonal growth, it had been proposed in s and s that sub-febrile doses of pyrogen could be considered for spinal cord injury repair research. we tested this ignored hypothesis in paraplegic bonnet monkeys and found that such sub-febrile doses of bacterial pyrogen derived from salmonella typhi was indeed effective in preventing the glial scar formation in short-term and at least prolong the formation of such scar in long term. therefore, pyrogen therapy may be considered as an adjunct to other strategies such as transplantation approaches to treat spinal cord injury. kavita seth, r.k. chaturvedi, s. shukla, a.k. agrawal dev. tox. div., industrial toxicology reserch center, lucknow, india crosstalk between neurons and glial cells (astrocyte and microglia) in neurodegenerative conditions such as parkinson's disease has gained attention of more than supportive interaction. here contribution of glial cells in -ohda induced degeneration of dopaminergic neurons was investigated. glial cultures showed significant loss in cell viability after h ( and %) and h ( and %) exposure to − and − m -ohda respectively. it was accompanied by morphological changes and induction of gfap, s- and ox . -ohda ( − m, h) was found to cause a significant impairment in h glutamic acid uptake ( %) and gsh levels ( %). further neurons (in coculture with -ohda pre exposed glial cells) on exposure to -ohda ( − m), showed loss of th expression and significant neuronal cell death ( %). the results of the present study suggest that -ohda may impair glial cell functioning, which eventually affect neuronal fate making them more vulnerable toward toxic insults. nestin is an embryonic intermediate filament component, which is transiently expressed by the immediate precursors to neurons and glia during brain development. we studied nestin distribution in the olfactory system after injection of diethyldithiocarbamate in adult rats to cause reversible lesion of the olfactory epithelium (oe). the oe presented a near-complete destruction at day after injection, then started to repair at days and returned to the normal levels at weeks. nestin was expressed in olfactory ensheathing cells (oecs) of the olfactory mucosa at ∼ days, but not in those of the olfactory bulb (ob). simultaneously strong expression of nestin was detected in certain population of astrocytes in glomeruli. the reversion of astrocytes in glomeruli to immature phenotype may reflect their involvement in reinnervation of glomeruli. (ng ) is currently considered a marker of multipotent progenitor cells in the brain. in the present study, most iba + cells accumulated in stab wounds and ischemic lesions were found to express ng , of which molecular weight of its core protein was higher by kda than that of ng expressed in contralateral brain region. this was due to the lack of shedding of ng in the brain lesions. we found that iba + cells accumulated in stab wounds and ischemic core lesion, most of which were ng +/pdgfra+. furthermore, some of these cells expressed gfap, nestin, cd and von willebrand factor. ng + mg isolated from stab wounds often formed cell aggregates bearing alkaline phosphatase activity turned into cells with neuroectodermal phenotypes in serumfree culture medium. these variety of antigens expressed by ng + mg in brain lesions may be related to their multipotentiality to regenerate damaged brain tissue. saroj sharma, l.k. singh, b. ray, t.s. roy all india institute of medical sciences, india oculomotor nerve (on) supplies most of the extra-and intraocular muscles. it shows changes with normal ageing, metabolic and degenerative diseases. though there are various studies on the on, no definitive data regarding the morphometry and the fine structure is available. so, in the present study, neural and the connective tissue organization of the extradural part of the on from cadavers were studied. light microscopy revealed multi-fascicular nerve with myelinated fibers of various calibers. small sized myelinated fibers were noted at the junction of the central and the paracentral zone of most of the nerves. using unbiased stereology techniques the size of myelinated fiber axonal areas showed a multi-modal distribution and presented range from < to m . most of the fibers were myelinated and counts produced a mean of , ( , - , ). ultrastructurally, difference in the compactness of arrangement of connective tissue was observed with advancing age. the cell junctions of the perineurial cells and the endoneurial capillaries were observed. myelin thickness ranged from . to . m (from fetal age to years age). during the development of the drosophila visual system, retinal axons project to the optic lobe through the optic stalk. the optic stalk is composed of glial cells and adopts tube-like structure. fak is a non-receptor protein tyrosine kinase involved in many aspects of cell behavior including cell migration through the regulation of actin or microtubule dynamics. in drosophila fak (dfak) mutant animals, the optic stalk was abnormally broadened and retinal axons were defasciculated. cdgapr encodes one of gaps that regulate rho-family gtpases. putative cdgapr mutants showed dfak-like phenotype. since dfak and cdgapr interacted genetically, they are likely to act in the same signaling pathway to regulate cytoskeletal rearrangement via rho-gtpases. tissue specific rescue experiment showed that dfak autonomously acts in the glial cells. our results suggest that dfak and cdgapr regulate glial cell rearrangement to establish precise tubelike structure of the optic stalk and organized retinal axon projection. astrocytes are thought to be active participants in synaptic plasticity in the developing nervous system. spontaneous gabaergic postsynaptic activity is reported to be decreased in small neurons of the caudal nts at the end of the first postnatal week. to investigate whether astrocytes might be involved in this phenomenon, we examined developmental expression of gfap, an astrocytic marker. gfap began to be immunohistochemically expressed in the caudal nts at p - . costaining with calbindin, a marker for a certain type of small neurons, showed that gfap positive processes were thereafter closely apposed to soma of small calbindin neurons. electron microscopy showed that some astrocytic processes were interposed between orphan gabaergic varicosities and soma of small neurons at the specific developmental stages. these findings indicate that astrocytes may participate actively in regulating the postnatal differentiation of local neural network of the caudal nts. hitoshi ozawa, naoyuki yamamoto, nobuhiko sawai, hao-gang xue department of anatomy and neurobiology, nippon medical school, tokyo, japan it is well known that the hypothalamo-pituitary-adrenal (hpa) axis is an important system for responding and mediating the stress. in addition, hippocampus is also an important area for the stress response. in the hippocampus, the expression of glucocorticoid receptor (gr) has been reported in the ca , ca pyramidal neurons and the dentate gyrus neurons. on the other hand, while astroglia around the hippocampus also expresses gr, the morphological and functional changes under different corticosteroid condition have not been well elucidated. in the present study, we investigated morphological changes of astroglia around pyramidal neurons. under the lack of corticosteroids, astroglia showed well developed morphology with the spread fibrous processes, however the changes recovered to the control level with corticosterone replacement. these suggested that the astroglia were directly regulated by glucocorticoids as associated with the changes of hippocampal neurons. ps p-d impact of s b on hippocampal spontaneous activities in anesthetized and epileptic conditions seiichi sakatani , akiko seto-ohshima , shigeyoshi itohara , hajime hirase neuronal circuit mechanisms research group, japan; lab. for behavioral genetics, bsi, riken, wako, japan s b is a calcium binding protein mainly expressed in astrocytes and has a role in synaptic plasticity and learning. in order to assess the physiological roles of s b, we have recorded hippocampal spontaneous activities from urethane anesthetized s b ko and wt mice. typical eeg patterns including theta ( - hz) and sharp wave associated fast ripple ( - hz) oscillations were observed in both populations and these patterns were indistinguishable between the wt and ko. when epileptic activity was induced by kainic acid (i.p.), a difference appeared in ca radiatum, where ictal event was characterized by hyper-synchronous gamma band ( - hz) activity. while both populations developed ictal event within min, mean power during the development was significantly smaller in ko mice. our results suggest that deficiency of s b does not have a profound impact on neural activity in normal conditions. however, when neural activity was raised, activation of s b related pathways could potentially be activated. yoshiko takagishi , erina okabe , xiaoyang sun , sen-ichi oda , yoshiharu murata riem, nagoya univ, nagoya, japan; grad sch bio-agricult sci, nagoya univ, nagoya, japan shambling (shm) is a spontaneous mouse mutation that causes neurological and motor deficits, characterized by ataxia and the hind limb paralysis. we have recently identified the shm gene that encodes caspr, which constitutes paranodal junction of myelinated nerves. to determine whether the mutation alters the node of ranvier, we performed morphological analysis of myelinated nerves in shambling mice. by electron microscopy, we found that paranodal loops were disorganized and septate-like transverse bands were absent in mutant mice. immunohistochemistry revealed that caspr was diffusely located at the paranodal region, though the staining was extremely weak in mutant sciatic nerves. contactin, a component of the paranodal junction, was distributed similar pattern to that of capsr. further, k + channels were mislocalized to the paranode, while na + channels were normally restricted to the node. these findings suggest that the mutation disrupts the paranodal structure and may disturb salutatory conduction of myelinated nerves in shambling mice. ps p-d regulation of hippocampal neurocircuit activity by glutamate transporter glt- noriko koganezawa, shinsuke muraoka, ken-ichiro tsutsui, toshio iijima div. systems neurosci. tohoku univ. grad. school of life science, japan glial cells are now recognized as an essential functional element in synapses. in order to investigate their function, we focused on the activity of glt- , the glutamate transporter which is expressed in the astrocytes of hippocampus, in the rat brain slice preparations. response to an electrical stimulation of the schaffer collaterals was recorded using the optical imaging technique. by combining the application of the glutamate receptor blockers (nbqx, ap ) and the glt- blocker (dhk) with the signal subtraction, we could visualize the activity of glt- as a slow, tonic rise of the optic signal following electrical stimulation. then we evaluated the function of glt- by applying its blocker dhk. an obvious reduction of neural activity was observed in the hippocampal neurocircuit after application of dhk. furthermore, the blocking of glt- function in the ca region was elicited by much lower concentration of dhk than that in the ca region. ps p-d monoclonal antibody rip specifically recognizes , -cyclic nucleotide -phosphodiesterase in oligodendrocytes the antigen recognized with monoclonal antibody (mab)-rip has been used as marker for oligodendrocytes and myelin sheaths. however, the rip-antigen has been unknown yet. to identify the rip-antigen, we performed immunopurification with mab-rip using the differentiated cg- cells lysate. maldi-qit/tof ms n analyses revealed that one of molecules was , -cyclic nucleotide phosphodiesterase (cnp). immunocytochemical and immunohistochemical studies showed that rip-antigen colocalized with cnp in rat cerebellum, cultured rat oligodendrocytes and cg- cells. moreover, the same localization was also observed in rat cnp transfected hek t cells. overall we first demonstrated that the antigen labeled with mab-rip is cnp in oligodendrocytes. the expression of bdnf gene is regulated by four promoters (pi-piv), and is under activity-dependent control. until now, it has been established that bdnf pi is activated by ca + signal via cre. on the other hand, neuron-restrictive silencer cis-element (nrse), located in bdnf pii, represses bdnf gene expression through binding nrsf and recruiting hdac in non-neural cells. here, we found that nrse repressed the activity of bdnf pi in neuron. using rt-pcr and chip assay, the bdnf exon i expression and the histone acetylation of bdnf pi were increased by the administration of ca + signals or hdac inhibitor. in addition, nrsf bound to bdnf pii in neurons but was detached by ca + signals. these results suggest that bdnf pi activity is regulated by creb and nrsf through an alteration of chromatin structure. since creb and nrsf are playing an important role in neuronal differentiation, it is considered that the bdnf pi is deeply involved in the regulation of neurogenesis. singo suzuki , , hisatsugu koshimizu , megumi kashihara , tomoko hara , masami kojima , research institute for cell engineering; sorst, jst, japan brain-derived neurotrophic factor (bdnf) plays a crucial role in synapse development, especially, in the central nervous system (cns) . although this concept is now accepted extensively, the underlying molecular mechanisms are poorly understood. here we show that -day treatment with bdnf leads to a significant increase in cholesterol content in primary neuron. this change was in its dose-dependent manner and blocked by co-application of a cholesterol synthesis inhibitors. to understand the molecular relationship between cholesterol content and synapse development, we estimated the amount of cholesterol and sv proteins in lipid raft fractions prepared from cultured cortical neurons. the results indicated that bdnf treatment increased the amount of cholesterol and sv proteins in lipid rafts, but not in non-rafts fraction. these data suggested a possibility that bdnf regulated synapse development by increasing the amount of cholesterol and sv proteins in synaptic rafts. (p ) is known to be expressed in the cells of the central nervous system, and supposed to be involved in the control of cell proliferation, differentiation and survival. in this study, we found that p expressed in the neural progenitor cells at embryonic days (e ) mice brain. to ascertain the function of p on these cells, we treated the cultured e cells with the selective chemical inhibitor for p (sb ) for days, and determined the number of neural progenitor cells. the inhibitor specifically enhanced the number of neural progenitors compared to the control cells. this result suggests the involvement of p in the proliferation and/or survival of neural progenitor cells in developing mouse brain. hemragul sabit , takashi yamazaki , , takeshi oya , yoko ishii , masakiyo sasahara pathology ii, university of toyama, toyama, japan; oral and maxillofacial surgery, university of toyama, toyama, japan purpose: we had reported the increase of pdgf-b and active src in rat peripheral nerve regeneration. here examined activation of pdgf receptors (pdgfrs) and signals in the peripheral nerve regeneration. method and result: crushed sciatic nerve was removed on to days after injury, and activation of pdgfrs, mapks, akt and p were examined by phosphoprotein purification kit (qiagen) and western blot. expression of pdgfrs increased from to days after injury. p-tyr was highly detected from to days after injury, and activation of pdgfrs also increased during this period. activation of erk and jnk increased up to days after injury and then gradually decreased. activation of akt and p continuously increased from to day after injury. conclusion: pdgfrs and their signals were activated in rat peripheral nerve regeneration. autocrine signal of pdgf may contribute to the regenerative processes, such as proliferation and differentiation of schwann cells and axonal extension. cbln is a cerebellum-specific protein structurally related to the c q and tumor necrosis factor families. recently, we have shown that cbln is secreted from cerebellar granule cells (gc) and controls synaptic structure and plasticity of gc-purkinje cell (pc) synapses. however, because cbln was previously shown to serve as a precursor of a pc-specific peptide cerebellin, it remains unclear whether cbln needs to be processed before it trans-synaptically activates signaling pathways in pc. here, we show that purified recombinant cbln proteins, which formed a hexamer, preferentially bound to spines on pc dendrites. furthermore, cbln mutants that did not form a hexamer lost the binding affinity to pc spines. although cerebellin peptide may also contribute to different aspects of signaling, these results indicate that cbln released from gc directly bind to postsynaptic pc as a hexamer and activates signaling pathways in pc. activity-dependent gene expression in neurons contributes to expressing a variety of neuronal functions including a long-lasting neuronal plasticity. recently, we found that specific kinds of mrna can be stabilized in an activity-dependent manner. to elucidate the mechanisms for activity-dependent mrna stabilization, we have focused on bdnf, which is a member of neurotrophin family and plays an important role in exerting neuronal functions. we constructed firefly luciferase gene fused to -untranslated region (utr) of bdnf mrna to investigate the effect of the utr on the calcium signal-mediated mrna stabilization. in cultured neurons, we found that the degradation of firefly luciferase-bdnf utr mrna induced by the treatment with actinomycin d was prevented by calcium signals evoked via l-type voltage-dependent calcium channels (l-vdccs) and nmda receptors. we are now investigating to identify the cis-regulatory elements involved in the calcium signal-mediated stabilization of bdnf mrna using a series of mutant bdnf utr. recently, it has been established that bdnf and pacap regulate the expression of a group of genes which encode proteins involved in expressing neuronal functions. in this study, we found that the treatment of cultured rat cortical neurons with bdnf or pacap acutely induced the mrna expression of the activityregulated cytoskeleton-associated protein (arc) and homer a, whose products are necessary for the synaptic plasticity. bdnf induced arc mrna expression through the activation of trkb-erk/mapk pathway, whereas pacap induced it partly through the activation of nmda-receptors. using affymetrix genechips, we are now investigating a comprehensive profile of gene expression controlled by bdnf or pacap in cultured rat cortical neurons. ps p-e bmp expression in the adult rat brain bone morphogenetic protein- (bmp ) is a member of the transforming growth factor ␤ (tgf-␤) superfamily and plays important roles in multiple biological event. although bmp expression has been well described in the early development of central nervous system (cns), little information is available for its expression in the adult cns. we, thus, investigated bmp expression in the adult rat cns using immunohistochemistry. bmp is intensely expressed in most neurons and their dendrites. in addition, intense bmp expression was also observed in the neuropil of the gray matters where high plasticity is reported, such as the molecular layer of the cerebellum and the superficial layer of the superior colliculus. furthermore, we found that astrocytes also express bmp protein. these data indicate that bmp is more widely expressed throughout the adult cns than previously reported, and its continued abundant expression in the adult brain strongly supports the idea that bmp plays pivotal roles also in the adult brain. ps p-e hgf as a target-derived trophic factor for rat nigro-striatal dopaminergic (da) system during post natal development wakana ooya, hiroshi funakoshi, toshikazu nakamura div. molecular regenerative medicine, osaka univ. grad. sch. med., osaka, japan hgf is a novel neurotrophic factor in vitro on da neurons. however, little is known about expression and biological activities of hgf in nigral-striatal system in vivo. here we show that hgf is a targetderived trophic factor for rat da system. real-time rt-pcr revealed that c-met mrna was expressed in substantia nigra (sn) and striatum (str), while hgf mrna was expressed in str but not in sn in programmed cell death period. hgf, c-met, phospho-c-met, th, da transporter immunostaining revealed the presence of concentration gradient of hgf from sn to str and c-met was phosphorylated in da nerve end during early postnatal development. phospho-c-metpositive da neurons decreased at later developmental stage, while it became prominent in oligodendrocytic leanage. hgf application into str increased da neuronal number and neurites and modified oligodendrocyte maturation, while opposite effects were observed by the application of blocking antibody for hgf. therefore, hgf may be a critical trophic factor for nigro-striatal da system development. the neuroprotective effects of g-csf were reported in neurological disease models. in the present study, we examined whether g-csf can protect dopaminergic neurons from mptp-induced cell death in a pd. the mice were intraperitoneal injected with mptp for five consecutive days, g-csf is intraperitoneal administered two days and one day before first mptp injection, and min before each mptp injection. in our results, g-csf significantly prevented mptpinduced loss of th-positive neurons, and increased bcl- protein, decreased bax protein expression. these findings suggest that g-csf has therapeutic potentiality to protect mptp-induced cell death through increasing the level of bcl- expression, decreasing the level of bax expression in c bl/ mice. kazue takahata has been shown to increase the expression of brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor, and the activity of superoxide dismutase . here, we evaluated the effects of (−)-bpap on the phosphorylation of mitogen-activated protein kinase (mapk) and akt in slice cultures as well as in an in vivo model. (−)-bpap significantly increased phosphorylation levels of mapk, but not those of akt. (−)-bpap attenuated the decrease in nigrostriatal tyrosine hydroxylase immunoreactivity of -methyl- -phenyl- , , , tetrahydropyridine-treated mice. (−)-bpap may exert antiparkinsonian activity through neuroprotective effects on dopaminergic cells in addition to catecholaminergic enhancement in parkinsonian substantia nigra. shyuichi maeda , yoko tohyama , shinichi kohsaka , tadashi kurihara , kazuyuki nakajima , soka university, hachioji, tokyo, japan; dept. of neurochem., national institute of neuroscience, kodaira, tokyo, japan astrocytes (ast) are a cell type that supports cns not only nutritionally but also neurotrophically, by supplying neurotrophic factors (ntf) required in the neuronal survival, maturation and protection. however, the ability of ast to produce/secrete ntf has not been accurately known. thus, in the present study, we investigate the capacity of ast to produce/secrete various ntf in vitro. ast were prepared from the mother culture of neonatal rat brain. the ntf in the conditioned medium (cm) were detected by immunoblotting. the analysis of neurotrophins in the cm revealed that ast produce/secrete ngf, bdnf and nt- , and promote the production of them by stimulation with lipopolysaccharide (lps). furthermore, tgf␤ among tgf␤ family was detected in the cm of ast, and the production was enhanced by stimulation with lps. these profiles of ast were different from those of microglia, suggesting the differential regulation of ntf by glial cells. ritsuko katoh-semba , chiaki nakagawa , masako tsuzuki , motoko matsuda , satoshi ichisaka , yoshio hata inst. dev. res., aichi human ser. ctr., kasugai, japan; div. neurobiol., tottori univ., sch. med., yonago, japan; div. integrative biosci., tottori univ. grad. sch. med., yonago, japan the sleep-awake rhythm is formed during the early period after birth. the rhythm is very important for the functional development of brain. when the rhythm formation is disturbed, autism-like behaviors are often observed. brain-derived neurotrophic factor (bdnf) is known to be one of the factors forming circadian rhythms. we have found increases in levels of bdnf in the entorhinal cortex as well as the visual cortex from adult male rats h after beginning an -h phase advance of the light-dark cycle. here we planned to reveal the effects of the phase advance on neurotrophins in juvenile rats. we first examined circadian changes in the concentrations of bdnf and neurotrophin- (nt- ) in selected brain regions from -day-old male rats and compared to those from adults. the changes in levels of bdnf and nt- were observed in the neocortex and hippocampus. objective: this study was aimed to investigate the possible beneficial effects of granulocyte colony stimulating factor (g-csf) compared to methylprednisolone (mp) in experimental spinal cord injury (sci). materials and methods: (in vivo) adult female sprague-dawley rats had moderate sci ( kdyne, ih injury device) at t / and were assigned to three groups; a (placebo), b (mp treated; mg/kg i.v. immediately after injury), c (g-csf treated; g/kg i.v. for days after injury). animals were assessed with the bbb locomotor rating scale for w post injury and then killed for assessment of tissue sparing around the lesion. result: the behavioural recovery rates of the group c was as good as group b and significantly better than that of group a. morphological assessment showed better tissue sparing in group b and c compared to group a. these results suggest that g-csf is a possible neuroprotective agent in sci. research funds: kakenhi ( ) ps p-e glutamate signals enhance the expression of rnase-l in primary cultured cortical neurons of mice chie sugiyama, kiyokazu ogita dept. pharmacol., setsunan univ., osaka, japan mitochondrial dysfunction results from a decline in the mitochondrial rna (mtrna) transcripts and mitochondrial enzyme activity, as well as from mitochondrial dna (mtdna) damage. to evaluate involvement of mtdna expression in glutamate-induced neuronal death, in this study, we examined the effects of glutamate exposure on mtrna level in cultured cortical neurons of mice. cultured neurons ( div) exposed to glutamate for min at m. glutamate exposure led to a decrease in mrna of nd and nd , which are subunits of nadhubiquinone oxidoreductase, before cell death. since mtrnas level is regulated at least in part by rna degradation mediated by rnase-l, we next examined the effect of glutamate on expression of rnase-l. rt-pcr analysis revealed that glutamate was effective in increasing the level of rnase-l mrna at least - h after treatment. the increase in the expression of rnase-l was abolished by the nmda receptor antagonist mk- . these results suggest that the activation of nmda receptor by glutamate reduces mtrna level probably through enhanced expression of rnase-l in cultured cortical neurons. yuka gotoh, kiyokazu ogita dept. pharmacol, setsunan univ, osaka, japan expression of dj- is enhanced by oxidative stresses. although exact functional significance of dj- has still unknown, it is thus proposed that dj- is protective against neural damage under oxidative stresses. in this study, we tested expression of dj- in the hippocampus damaged by trimethyltin (tmt) treatment in mice. tmt was systemically injected into mice to cause neural damage in the dentate gyrus selectively. immunohistochemical analysis indicated that dj- was markedly increased in the molecular layer of the dentate gyrus on days and post tmt injection. on day post tmt injection, enhanced expression of dj- was observed in the stratum lucidum of the ca . in glutathione-depleted mice, tmt was more effective in enhancing expression of dj- , compared with that in untreated mice. furthermore, double staining of dj- and gfap demonstrated that most of cells highly immunoreactive to dj- were co-localized with gfap in the dentate gyrus of tmt-treated animals, but not of untreated animals. these results suggest that dj- is enhanced in the dentate astrocytes activated by tmt treatment. kei higuchi, kiyokazu ogita dept. pharmacol, setsunan univ., osaka, japan the systemic administration of trimethyltin (tmt) is known to induce granule cell death in the dentate gyrus of mice. we have previously shown that an injection of tmt ( . mg/kg, i.p.) led to significantly reduction of granule cells in the dentate gyrus days later, with visually apparent recovery of the granule cell layer days afterward. in this study, we examined the effects of glucocorticoids on tmt-induced damage in the dentate granule neurons of mice. tmtinduced neuronal cell damage was assessed by the immunohistochemical analysis using an antibody against single-stranded dna. the systemic injection of dexamethasone ( . - mg/kg) led to a significant reduction in neuronal damage induced by tmt in the dentate gyrus. the neuronal damage induced by tmt at the dose of . mg/kg was enhanced by adrenalectomy. dexamethasone was effective in completely preventing this neuronal damage in adrenalectomized animals. taken together, these results suggest that glucocorticoid released from adrenal cortex may be capable of protection against tmt-induced dentate granule cell death in mice. masami ishido national institute for environmental studies, tsukuba, japan melatonin, a secretory product of the pineal gland, has antitumor activities and is involved in the regulation of circadian, seasonal rhythms and in inducing osteoblast differentiation. furthermore, melatonin is reported to be a scavenger of a number of reactive oxygen and reactive nitrogen species both in vitro and in vivo. in this chapter, antioxidant nature of melatonin was demonstrated to prevent the cultured neural cells from apoptosis induced by endocrine disrupting chemicals, maneb. neurotoxicity of maneb ( g/ml) on the pc cells was elicited through apoptotic cell death. activation of caspase- / was associated with this process. a fluorescence rationing technique using mitochondrial dye revealed that maneb altered mitochondrial membrane potential of the neural cells. however, melatonin ( nm) could largely prevent the neural cells from the neural toxicant by inhibition of both caspase- / activation and disruption of the mitochondrial transmembrane potential. thus, melatonin could be a powerful free radical scavenger against manebcaused mitochondrial dysfunction in pc cells. ps p-e kinesin superfamily protein (kif ) regulates activity-dependent neuronal survival by suppressing parp- enzymatic activity ryosuke midorikawa, yosuke takei, nobutaka hirokawa department of cell biology and anatomy, university of tokyo, tokyo, japan in brain development, apoptosis is a physiological process that controls the final numbers of neurons. here we report that the activitydependent prevention of apoptosis in juvenile neurons is regulated by kinesin superfamily protein (kif ), a microtubule-based molecular motor. the c-terminal domain of kif is a module that suppresses the activity of poly (adp-ribose) polymerase- (parp- ), a nuclear enzyme known to maintain cell homeostasis by repairing dna and serving as a transcriptional regulator. when neurons are stimulated by membrane depolarization, calcium signaling mediated by camkii induces dissociation of kif from parp- , resulting in upregulation of parp- activity, which supports neuron survival. after dissociation from parp- , kif enters into the cytoplasm from the nucleus, and moves to the distal part of neurites in a microtubule-dependent manner. we suggested that kif controls the activity-dependent survival of postmitotic neurons by regulating parp- activity in brain development. research funds: ps p-e expression of hsp , apg- , and apg- in the hippocampal neural cells by trimethyltin masanari orita, kiyokazu ogita dept. pharmacol, setsunan univ, osaka., japan we tested changes in expression of high-molecular-weight heat shock proteins (hsps) in the hippocampal dentate gyrus in vivo and in the cultured cortical neurons in vitro after trimethyltin (tmt) treatment, which caused neuronal damage in the dentate gyrus and cultured hippocampal neurons. tmt ( . mg/kg) was systemically injected into mice, and then an immunohistchemical analysis was performed to identify cells immunoreactive to antibodies against hsps, neun, and gfap in coronal sections of hippocampus. tmt was effective in enhancing the expression of hsp , apg- , and apg- in the granule cell layer of the dentate gyrus, but not in ca -ca pyramidal cell layer, h to days later. double staining of neun and these hsps revealed that these hsps expressed by tmt almost co-localized with neun in granule cells of the dentate gyrus. whereas hsp highly expressed in survival neurons in the culture, apg- and apg- highly expressed in damaged neurons with nuclear condensation. taken together, the high-molecular-weight hsps may be involved in neuronal survival and damage caused by tmt. brain irradiation is often performed in patients with brain tumors. however, little has been known about radiosensitivity of neurons, especially in the developmental stages. in this study, we investigated the effect of irradiation on immature neurons with that on mature neurons. primary neuronal cultures were prepared from fetal rat hippocampi at embryonic day . thirty gray of x-irradiations were performed on the cultured cells at or days in vitro (div). then the cells were fixed at h after the irradiation with dapi. at -div, irradiation significantly increased the number of nuclear pyknosis of neurons. in contrast, radiation did not induce any nuclear pyknosis of neurons at -div. this indicates that the radiosensitivity of -div immature neurons is higher than that of -div mature neurons. glutamate receptors are believed to be involved in various neurological disorders via its excitotoxicity. ataxic mice lurcher (lc) are caused by a mutation in the ␦ glutamate receptor (glur␦ ), which shows constitutive channel activities in purkinje cells and leads to the cell death. thus, lc is the first example of neurodegeneration caused by chronic excitotoxicty. interestingly, glur␦ is also suggested to regulate autophagy via its association with beclin. however, it is unclear how excitation caused by constitutive channel activities is related to the autophagic pathway and cell death. here, using heterologous cells in vitro, we show that continuous influx of na + , but not ca + , was necessary and sufficient to induce autophagic cell death. in addition, we found that intracellular atp levels and subsequent activation of map kinase are involved in this process. junko taniguchi a major pathological hallmark of the polyglutamine diseases is the formation of neuronal intranuclear inclusions (niis) of the disease proteins that are ubiquitinated and often associated with various transcription factors, chaperones and proteasome components. but, how the expanded polyglutamine proteins or their aggregates elicit a complex pathogenic responses in the neuronal cells are not fully understood. here, we demonstrate that the expression of expanded polyglutamine proteins down-regulates the nf-kb-dependent transcriptional activity. expression of expanded polyglutamine proteins increases the stability and the levels of ikb-a and its phosphorylated form. we have also found that various nf-kb subunits and ikb-a aberrantly interacts with the expanded polyglutamine proteins and associates with their aggregates. finally, we have shown several nf-kb-dependent genes are down-regulated in the expanded polyglutamine protein expressing cells. molecular mechanisms for selective neuronal death in polyglutamine diseases remain to be clarified. by microarray analysis, we compared gene expression profiles in cerebellar granular cells under expression of normal and mutant ataxin- and found a novel gene down-regulated in response to mutant ataxin- in cerebellar granular neurons. we named the novel gene maxcell (mutant ataxin-affected gene in the cerebellum). nothern blot shows that maxcell mrna in human brain is expressed in cerebellum and cerebral cortex. immunohistochemistry with anti-maxcell antibody shows cytoplasmic stains of neurons but not glial cells in mouse brain. confocal microscopy shows that maxell-egfp is colocalized with ribosomal protein s as a ribosomal marker. we are analyzing the function of maxcell protein that might relate to sca molecular pathology. ps p-f permeability transition in mitochondria isolated from cold perfused brain and spinal cord-a detailed comparison of calcium sensitivity the purpose of this study was to compare the sensitivity of isolated brain and spinal cord mitochondria to ca + -induced permeability transition (mpt). the spinal cord is more traumatized than the brain during the extraction because it takes more time. in order to minimize confounding factors, we induced severe hypothermia in animals prior to removal of tissue and isolation of mitochondria. sensitivity to ca + -induced mpt was evaluated in brain and spinal mitochondria in energized and de-energized model of swelling with or without mpt inhibitor, cyclosporin a (csa). the present findings imply that the general features of mpt are similar in brain and spinal cord mitochondria and that mpt may be an important pharmacological target in disorders affecting the spinal cord. the role of cyclophosphamide monohydrate (cp), which is known as an immunosuppression drug, in the central nervous system (cns) has not been elucidated. in the present study, we found that treatment with cp prevented the cultured cortical neurons from cell death induced by serum deprivation. furthermore, cp exposure induced the activation of both the map kinase (mapk) and pi kinase (pi k) pathways. interestingly the up-regulation of bcl- , a survival promoting molecule was observed after cp treatment. these observations suggest that cp protects cns neurons from neuronal damage through intracellular signaling pathways. the research of cell death mechanisms has rapidly progressed. however, cell death is an inherently difficult process to measure. to investigate the roles of cell death in vivo, we introduced scat probe. scat is an indicator protein for caspase- activation that uses fret between two types of fluorescent protein, ecfp and venus, linked by a peptide containing the caspase- cleavage sequence. using this probe, we could monitor the activation of caspase- at the singleneuron level in culture. we will discuss about neural cell death through the detection of caspase activity. keiichi seko, koichi kawada, chie sugiyama, masanori yoneyama, kiyokazu ogita dept. pharmacol., setsunan univ., osaka, japan trimethyltin chloride (tmt) is a kind of organotin derivates that are known to induce neuronal damage in human and rodent. in this study, we examined tmt-induced neuronal death in mouse primary cultured cortical neurons in vitro and the frontal cortex in vivo of mice. in vivo analysis using mice revealed that injection of tmt ( . mg/kg, i.p.) led to an increase in single-stranded dna-positive cells, as well as in dnase ii-positive cells, in the frontal cortex days later. in cortical neurons, tmt exposure for h led to a marked decrease in the cell viability, as well as to an increase in nuclear condensation and ldh released. tmt exposure was effective in activating dnase ii in the nucleus. in addition, caspases and , but not caspase , were significantly activated by tmt treatment. cytochrome c release was not affected by tmt treatment. the caspase inhibitor zvad-fmk completely prevented tmt-induced neuronal death. these results suggest that tmt-induced neuronal death is involved in caspases and dnase ii activated by mitochondria-independent pathway in cortical neurons. we investigated the pattern of hippocampal damage and the levels of brain polyamines after systemic injections of trimethyltin (tmt) chloride ( . mg/kg, i.p.) in -( w) and -week-old ( w) icr mice. in addition, we measured the brain tin level following tmt injection. tmt induced marked, localized cell death in granule neurons of the dentate gyrus in w mice. by contrast, slight, diffuse neuronal damage was found in the ca and ca subfields and dentate gyrus of w mice. the hippocampal putrescine level was elevated markedly in w mice on tmt administration, whereas a minor putrescine increase was detected in w mice. there was no difference in the brain tin level between these two age groups. these results revealed the age-dependent vulnerability of mice hippocampal neurons to tmt administration, and suggest that massive activation of polyamine metabolism is associated with tmt-induced neurodegeneration. withdrawn ps p-f establishment of memory guided actions of taking food with tweezers in monkeys naoki hirai , toshinori hongo , kimisato naito , shigeto sasaki department of physiology, kyorin university, school of medicine, tokyo, japan; tokyo metropolitan institute for neuroscience, tokyo, japan monkeys learned a task of taking food with tweezers (twz) under the visual guidance. the task consisted of sequential actions of looking at twz, grasping it by hand simultaneously shifting gaze to food, bringing twz to food, and picking it up with twz. brief interruption of vision for . - . s during any actions by a liquid crystal shutter disrupted the ongoing actions, indicating that each action needed visual information as guidance. this contrasted with the task of taking directly with hand, which was done without vision. with repeated practice, they developed a mode of using more memory and somatosensory cue as guidance. they directed their gaze to invisible food in advance, and when vision of . s became available, they grasped twz, brought the twz to memorized location of food and grasped the food without vision. these results show that they acquired food taking actions using twz based on memory and somatosensory cues, the latter allowing monkeys to use twz as an extension of the hand. masahiko nishimura, yoshihiko yoshii university of ryukyus, okinawa, japan we have experienced that the patients with arm impairments by brain disorder were difficult to manipulate the tools with the paralyzed arm, and healthy arm. lt. parietal lobe and ifg are commonly recognized tools-semantic neuro-system. however, nobody knows a neural network contributed to suitability for a purpose of toolsmanipulation. we examined an fmri to evaluate the brain activation of tools-manipulation in volunteers. experiment was performed by three tasks, control task is a forearm rotation, task is simulation of tools-manipulation, task is execution of tools-manipulation. we found different brain regions by this experiment. task to investigate the role of synchronous firing in the prefrontal cortex (pfc), we performed cross-corelational analysis of the pfc neurons, while monkeys performed a path-planning task, which required multiple steps of actions to reach goals. first, we analyzed synchrony among pfc neurons during the execution period in comparison with that during the preparatory period. we found that neuronal synchrony was enhanced transiently for each step of movement during the execution period. next, we examined relationship between neuronal synchrony and task-related activities. we found that the relationship between neuronal synchrony and response selectivity of pfc neurons was more distinct during the preparatory period than during the execution period. we would discuss dynamical roles in neuronal synchrony in planning multiple steps of actions. the functional significance of primate medial prefrontal cortex in the selection of action has been unclear. we studied neuronal activity in this region while monkeys were performing a variant of conflict solving task in which visual cues instructed them to push either the left or right. the location of the cue was either compatible (congruent) or incompatible (incongruent) with the target's location. we found a focus of reaching-related neurons in the medial prefrontal cortex rostral to the pre-sma. the activity of neurons in this newly identified area was dependent on conflict. intracortical microstimulation in this area did not evoke eye movements, distinguishing this area from the sef. we found that the local field potential in this area, but not in other areas, differed when congruent and incongruent trials were intermixed, and when only the congruent trials were presented repeatedly, suggesting the involvement of this area in the selection of actions is dependent on the task demand. masaki maruyama, peter fenwick, andreas a. ioannides laboratory for human brain dynamics, riken-brain science institute, saitama, japan we used infrared corneal reflection, sampling at khz, to record simultaneously and independently the • horizontal saccades of each eye for subjects. two paradigms were used, in go-only sessions saccade direction with the cue to move, and in go/no go sessions, saccade execution, direction and move. mutual information (mi) analysis showed the two eyes were most consistently yoked for position than for velocity, but both provided adequate signals. mi showed coupling between the start and end of saccades and the importance of velocity signals in their ballistic nature. surprisingly leftward movement latency was longer to peak-velocity and showed more complex mi interactions. comparing go-only to go/no go saccades, significant differences were longer onset latencies and a higher eye velocity before the end of saccades. a recent meg study using this protocol, found just before and during the saccade the additional go/no go difficulty led to more interaction between left and right brainstem and cerebellum. these could be related to eye velocity changes with the higher cognitive loading. wriggle mouse sagami (wms) has been presented as a mouse model for dystonia, as it is characterized by postural and motor impairments, such as sever tremor, sustained muscle contractions of the limbs, and wriggling of the neck and trunk without coordination. by extracellular unit recordings under awake conditions, we analyzed neuronal activity in the basal ganglia and the cerebellum of this mutant mouse. in the basal ganglia (globus pallidus, entopeduncular nucleus, substantia nigra pars reticulata), neither rates nor patterns of spike discharges were significantly different as compared to normal mice. on the other hand, the discharge rate of cerebellar purkinje cells in wms was markedly decreased. these results suggest that the decreased activity of purkinje cells may be responsible for movement disorders in wms. hiromi hirata division of biological science, nagoya university, nagoya, japan wild-type zebrafish respond to mechanosensory stimulation with multiple fast alternating trunk contractions at day, whereas bandoneon (beo) mutants contract trunk muscles on both sides simultaneously. muscle voltage recordings confirmed that muscles on both sides of the trunk in beo are likely to receive simultaneous synaptic input from the cns. recordings from motor neurons revealed that glycinergic synaptic transmission was missing in beo mutants. furthermore, immunostaining with glyr antibody failed to show clusters in beo neurons. these data suggest that clustering defect of glyrs at synapse causes the impairment of glycinergic transmission and abnormal behavior in beo. indeed, mutations in the glycine receptor beta subunit were identified in beo. this is the first direct demonstration that glyr␤ is essential for physiologically relevant clustering of glyrs in vivo. since glycine receptor mutations in humans lead to hyperekplexia, a motor disorder characterized by startle responses, zebrafish bandoneon mutant should be a useful animal model for this condition. medium-sized spiny projection neurons in the striatum receive inputs from gabaergic and cholinergic interneurons as well as from extrinsic sources, including the cerebral cortex. in the present study, the effect of gabaergic modulation on striatal projection neuron activity was investigated by infusion of the gaba a receptor blocker gabazine in the vicinity of the recorded neurons in monkeys who performed a memory-guided reaching task. the gabazine infusion enhanced the activity of striatal projection neurons in response to both cortical stimulation and task events, while the neuronal activity specific to the task events was decreased. these results suggest that local gabaergic input may play an important role in fine tuning of striatal projection neuron activity. research funds: kakenhi ( ) ps p-f dependence of synchrony in the subthalamic network on temporal characteristics of afferent inputs katsunori kitano, fumito kosuga department of human and computer intelligence, ritsumeikan university, japan the subthalamic nucleus (stn) and the external segment of global pallidus (gpe) constitute the indirect pathway of the basal ganglia and highly modulate the basal ganglia functions. the evidence that the emergence of synchronized oscillatory activity in the network of the two nuclei is relevant to movement disorders such as parkinson's disease shows temporal structures of the neuronal firings play an important role for the functions. among possible underlying mechanisms for the abnormal activity, the characteristic membrane properties of stn neurons is likely to be one of the most crucial origins. to clarify the detailed mechanism, we focus on and investigate the dynamical properties of the neuron theoretically and numerically with the model neuron. we apply the phase reduction method to the dynamics of the neuron to analyze the stability of synchronous activity. in particular, how the stability depends on temporal characteristics of afferent inputs to the neurons as well as the intrinsic membrane properties are investigated. during phasic voluntary movement, electromagnetic oscillatory activities of ∼ hz around the central sulcus show decrement and increment (erd/ers, respectively), that are assumed to reflect the cortical activation and inhibitory/recovery process respectively. we investigated the correlation between personality and these oscillatory changes. from healthy subjects, high and low scorers (n = each) of novelty seeking dimension on the psychometry were selected. magnetic fields were recorded while they performed selfpaced movements of their right index fingers, and frequency analysis was carried through the beta band ( - hz). high ns group showed less amount of erd in the left hemisphere, smaller magnitude, larger latency of ers in the right hemisphere and smaller amount of baseline activity in both hemispheres than low ns group. it was suggested that individuals with high ns trait may have less inhibition after the movement and higher readiness during resting state. despite the emerging methodology of combined fmri and tms, the quantitative relationship between tms intensity and bold signals is poorly understood. eight healthy subjects were scanned on a -t scanner, with an mri-compatible figure-of-eight tms coil attached for eliciting right hand movement. bold measurement was performed with the stepping stone sequence (tr = . s) with online monitoring of meps. the intensity of tms pulses was varied from % to % at a % step (frequency at ∼ . hz). bold signal changes were assessed in the primary motor cortex. a sharp increase in bold signals was observed above % stimulation. bold signals were weakly but significantly correlated with tms intensity adjusted by the resting motor threshold (r = . , p = . ). this finding gives a theoretical background for the application of fmri with tms to cognitive brain regions. ps p-f shift of activation areas induced by hand movement during recovery from post-stroke hemiparesis: an nirs study kotaro takeda , , yukihiro gomi , itsuki imai , nobuaki shimoda , , hiroyuki kato , international university of health and welfare, ohtawara, japan; crest, jst, kawaguchi, japan; nasu neurosurgical center, nasushiobara, japan we investigated the cerebral hemoglobin (hb) changes in hemiparetic stroke patients under voluntary hand grasping task from acute to chronic phases by using near infrared spectroscopy (nirs). fortyfour channels ( channels on each side) were placed on the scalp overlying both sensorimotor cortices, and the cerebral hb changes were observed during four to six cycles of s task and s resting periods while sitting on a chair. the amounts of oxy-hb change were significantly increased in the bilateral sensorimotor areas during hemiparetic hand grasping at the acute phase, though the significant increase was mainly observed in the contralateral sensorimotor area during hemiparetic hand grasping at the chronic phase and during normal hand grasping at all phases. this result suggests that the functional recovery from post-stroke hemiparesis may be attributed to neuronal reorganization of sensorimotor areas via recruiting ipsilateral cortex. research funds: crest, jst todd pataky , , rieko osu , hiroshi imamizu , mitsuo kawato , computational brain project, icorp, jst, japan; atr cns laboratories, japan movement direction encoding in primate single cortical cells has been widely documented. this study was designed to test whether this directional tuning is observable at the voxel level in human fmri. three subjects performed hz isometric force pulses to seven targets separated by • in the shoulder/elbow flexion-extension plane. while in mri, online force feedback was provided by a d strain gauge. twenty repetitions of each condition were performed in s blocks (tr = s). all subjects showed broad activation over the contralateral motor area, and from functional rois an average of voxel time series were extracted. many voxels exhibited continuous cosine-like tuning with movement direction. decoding using linear svm revealed that while correct classification rate was only . % (chance: . %), errors were distributed normally about the target such that . % (chance: . %) of the data was classified correctly to within • . these data demonstrate that non-invasive neuroimaging is sufficiently sensitive to study the problem of coordinate system representation. the behavioral thermoregulation is important for living in various temperature environments. however, the neural mechanism of behavioral thermoregulation is poorly understood. in this study, we aimed to establish a new model to analyze the neural mechanism of behavioral thermoregulation using zebrafish. we investigated whether zebrafish perform behavioral thermoregulation against heating. when water temperature was changed from • c, fish showed repetition of short time swimming in the range . - • c. the frequency of the heat induced escape behavior was increased with temperature dependant. these results suggest that the heat induced escape behavior is a part of behavioral thermoregulation. the heat induced escape behavior was observed stably in - days past fertilization, indicating that the neural mechanism which control behavioral thermoregulation is matured in days. in conclusion, we established an effective new model to analyze behavioral thermoregulation. ps p-f to learn with one limb or two: limited transfer between unimanual and bimanual skills recent studies on neural activity in primary motor cortex of nonhuman primates suggest that unimanual and bimanual movements are controlled by partially overlapping neural processes. here we demonstrate that unimanual and bimanual motor learning also reflect a partially overlapping process. first, motor adaptations to reach with a novel force field applied to a limb could not be fully transferred to the same limb across unimanual to bimanual conditions, and vice versa. second, learning acquired during unimanual reaching could not be fully eliminated by repeated bimanual reaching with no loads, and vice versa. rather, some learning remained intact (but invisible) until the original context was again performed. lastly, two conflicting force fields can be learned simultaneously if they are separately associated with unimanual and bimanual reaching. these results support the view of partially overlapping neuronal processes and illustrate the intimate relationship between neural control and motor learning. research funds: jsps and nserc rieko osu , ken-ich morishige , jun nakanishi , , hiroyuki miyamoto , mitsuo kawato atr computational neuroscience labs, kyoto, japan; kyushu institute of technology, japan; jst-icorp, japan human can execute multiple motor tasks by using the same limbs, which makes human different from industrial robots. recently the optimal feedback control hypothesis has given a significant impact on the motor control community because it produces an optimal behavior for a given task by avoiding offline computation of optimal desired trajectory that would result in suboptimal behavior in the presence of noise. it, however, requires considerable amount of resources and learning to realize multiple tasks on nonlinear system. on the other hand, a desired trajectory enables the brain to share resources with multiple tasks and save learning time by dividing a difficult problem into easier sub-problems of plan and execution. considering the modularity of the brain and viability for nonlinear system, the hierarchical implementation is a better solution for global optimality as a versatile creature. here, we experimentally demonstrate that the hand variance modulation during multiple via-point tasks supports the existence of a desired trajectory. the purpose of this study is to computationally predict arm-reaching movements and posture controls from neuronal activity of premotor (pm) and primary motor area (mi). the activity was collected with single-unit recording method during the animal performing a visually guided arm-reaching task. electromyograms (emgs) and kinematics were also measured. we reconstructed the emgs from the neuronal data using a linear regression model, and then we estimated the kinematics from the reconstructed emgs with an artificial neural network model and proportional derivative controller. as a result, these serial processes allowed us to accurately predict the kinematics during both moving and maintaining her posture from the activity. the advantage of our bmi system is to estimate not only the kinematics but also the muscle tension from the neuronal activity. we have recently reported essential role of the tongue in breastfeeding in the hypoglossal (xii) nerve-injured newborn rats. of particular interest were the findings that the rates of the amounts of milk intake in the unilateral xii nerve-injured p pups of the surviving cases increased greatly between p ( % of the control value) and p ( %), suggesting adaptive tongue movement during development. this study was undertaken to reveal underlying basic mechanisms for such adaptation focusing on neural plasticity allowing effective suckling. after resection of the ipsilateral xii nerve on p , dii, a postmortem neuronal tracer, was applied to the contralateral uninjured xii nerve of p and p pups. dii-labeled neuronal fibers were successfully traced within the tongue and were found to extend over the xii nerve-injured side with gradual increase from p to p . we show evidence for functional neural plasticity that allows effective suckling in the xii nerve-injured newborns with suckling disturbance. previously we reported that decorticated rats showed abnormal righting movements in the air when dropped from the supine position, while the air righting reflex (arr) could be evoked purposefully ( • turn around the body axis) in decerebrated ones. thus, the basal ganglia might send interference signals to the arr center via the midbrain tegmentum. to clarify its functional roles in arr control, we examined arr movements in rats with the midbrain lesioned. wistar, male rats were prepared; after the posterior cerebral cortex was removed by sucking, the superior colliculus and surrounding structures were ablated in various degrees. arr movements were examined post-operative , , and days. in rats with the superior colliculus lesioned extensively on both sides, arr onset were delayed and body turn around the longitudinal axis was weakened, so that either insufficient or no rotation occurred in the air. furthermore, coordination between the body and tail rotations was lost in many cases. the ablated region may relay cortical signals that give a top priority to the arr center. ps p-g role of plateau potentials in feeding system of aplysia kurodai aiko kinugawa , tatsumi nagahama dept. of life sci., grad. sch. of sci. & technol., kobe univ., kobe, japan; fac. phar. sci., toho univ., funabashi, japan rhythmic motor activities seen in the animal behaviors can be generated by specific neural circuits termed the central pattern generator (cpg). in the feeding system of aplysia kurodai, the le neuron we identified produces the long-lasting plateau potentials and may be a cpg element. during the feeding-like responses duration of the depolarization of the follower neurons was shortened by hyperpolarization of the le. in this study we found that the le plateau potentials had refractory periods and they were turned to activation periods by application of large depolarizing currents. and various depolarizing pulses tended to produce the stable plateau potentials with almost constant depolarizing size and duration, suggesting that the le can supply the constant long-lasting depolarizing outputs to the follower neurons even when it receives various length and intensity of excitatory inputs from the presynaptic neurons. the le may be an important cpg element to determine the size and duration of the basic depolarization of many buccal neurons. withdrawn ps p-g neural organizations for vocal control in a social rodent, the deguneural organizations for vocal control in a social rodent, the degu naoko tokimoto , sayaka hihara , kazuo okanoya , atsushi iriki lab for symbolic cognitive development, bsi, riken, japan; lab for biolinguisutics, bsi, riken, japan vocalizations of most animals are innate, the region for the direct control of such sound is known to be localized in the pag. on the other hand, a few animals with the cortico-medullary projection path can learn a new sound. in this research, we investigated about vocal control in pag of social rodent, the degu (octodon degu). it is known that degus have fifteen kinds of vocal repertoires, and that their courtship song has a complex structure. we verified the hypothesis by electrical stimulation of the pag that the neural mechanism of degus that enables complex vocalization differs from that of guinea pigs with simple vocalization. guinea pig is near relation with degu. as a result, in guinea pigs, each sound is controlled in different area of the pag. in degus, however, multiple sounds are controlled by the same area, and the different sound was occasionally evoked by the different kind of stimulation. the sound with the time-series specific to the spontaneous vocalization of degu was not emitted. the effects of a heat-and steam-generating sheet (hsg sheet) on autonomic nerve activity and bowel movement were examined in women suffering irregular defecation, the hsg sheet was applied to the lumbar or abdominal regions, causing the temperature between the sheet and skin to increase to about . • c. application of the hsg sheet to either the lumbar or abdominal region significantly increased the rate of miosis in the pupillary light reflex. as for changes in r-r, the hf increased after application, suggesting that the parasympathetic nerve system had become dominant. bowel movement assessed by electrogastrography increased in amplitude. based on the above findings, we concluded that the application of an hsg sheet to the lumbar or abdominal region may lead to dominant parasympathetic nerve activity and improve gastrointestinal motility. ps p-g prostaglandin e -induced thermogenesis involves a gaba-receptive mechanism in the preoptic area toshimasa osaka national institute of health and nutrition, - - toyama, shinjuku, - , japan unilateral microinjection of pge into the region around the rostroventral wall of the third ventricle (av v) elicited thermogenic, tachycardic, vasoconstrictive, and hyperthermic responses simultaneously in urethane-chloralose anesthetized rats. the magnitude of these responses increased dose-dependently in a range of - pg, except for the vasoconstrictive response. next, the effects of pretreatment with a gaba a receptor antagonist, bicuculline methiodide ( . mm, nl), microinjected into the preoptic area (poa) ipsilateral or contralateral to the pge injection site was examined. this treatment alone had no effect on the o consumption rate and temperatures of colon and skin but elicited a bradycardic response. however, all pge -induced responses were blocked min after the pretreatment with bicuculline, and recovered at ∼ min. pretreatment with vehicle saline had no effect on the pge -induced responses. these results suggest that the gaba-receptive mechanism in the poa is required for the pge -induced thermogenesis. tetsufumi ito , hiroyuki hioki , kouichi nakamura , takeshi kaneko , yoshiaki nojyo dept. anat., univ. of fukui, fukui, japan; dept. of morphological brain sci., kyoto univ., kyoto, japan although gaba-immunoreactive (ir) fibers in the rat superior cervical ganglion (scg) were thought to originate in small cells located in the cervical sympathetic trunk (cst), almost all gaba-ir axon terminals showed markers for sympathetic preganglionic neurons (spns) in our recent report. in this study, we performed series of experiments to confirm the origin of gaba-containing fibers. gad -ir fibers were not found in dorsal roots (drs), but in ventral roots (vrs), stellate ganglion, cst, and scg. gad -positive somata were not found in dr ganglia and cst, but in intermediolateral (iml) nucleus of thoracic spinal cord (tsc). after intraperitoneal injection of fluorogold (fg), to label the entire spns, some fg-ir neurons were also positive for gad . we injected sindbis virus, an anterograde tracer, in iml, and some labeled terminals in scg showed gad -ir. after cutting t -t vrs and drs, almost all gad -ir fibers were abolished in scg. these results indicate that gaba-containing fibers in scg originate from spns in iml of tsc. masato nagahama , ning ma , reiji semba , satoru naruse dept. of anat. ii, mie univ. school of med., tsu, japan; inst. for developmental research, kasugai, japan; dept. of int. med., nagoya univ. graduate school of med., nagoya, japan aquaporin (aqp ) is first found as a water-transporting protein and has been demonstrated in various organs and tissues. in the present study, we have demonstrated the presence of aqp immunoreactivity in a particular neuronal subtype in the enteric nervous system (ens) of the rat ileum. aqp -immunoreactive (ir) neurons simultaneously expressed a neuronal marker huc/d. moderate numbers of aqp -ir neuronal somata were found in the myenteric plexus, and a very few were found in the submucosal plexus. aqp -ir neurons can be classified as dogiel type i cells, which have several short processes and a single long process. many aqp -ir fibers were found both in the myenteric and submucosal plexi. many aqp -ir varicose fibers were closely associated with neuronal somata in the ganglia, whereas other aqp -ir fibers penetrated into the muscle layers. these results suggest that aqp -ir neurons probably play a significant role within the ens to control gut functions. research funds: kakenhi ( ) ps p-g abdominal expiratory nerve activity in the decerebrate neonatal rat center for medical sciences, ibaraki prefectural university of health sciences, ibaraki, japan the abdominal expiratory activity was recorded from the iliohypogastric nerve in the decerebrate, vagotomized, paralyzed, ventilated neonatal rat at postnatal days - . the increase in the volume and frequency setting of the artificial ventilator (fio = %, fico = %) failed to make the rat apnea. under this condition, the phrenic nerve showed unstable rhythmic inspiratory bursts, and the tail pinch increased the respiratory frequency. although the iliohypogastric nerve showed expiratory discharges, their amplitudes and shapes were not consistent. when fico was increased, the cycle period was prolonged and the abdominal expiratory activity was enhanced. in many rats, the iliohypogastric nerve showed biphasic discharges that consisted from the pre-and post-inspiratory discharges. the preinspiratory discharge has larger amplitude and shorter duration than the post-inspiratory discharge. since the post-inspiratory discharge was usually small or indistinguishable in the adult rat, the present results suggest that the pattern of abdominal expiratory activity will change during the postnatal development. we investigated the role of gabaergic neurons in the rostral ventrolateral medulla (rvm) in central respiratory control. we used gad -gfp knock-in mice in which we could identify gabaergic (i.e., gfp-positive) neurons in a living condition. we recorded gabaergic neuron activities (n = ) in medullary transverse slices. about % of gabaergic neurons were inspiratory, and all of the remaining neurons were non-respiratory. about % of gabaergic neurons recorded in the superficial rvm were co inhibitory, and all of the remaining neurons were co insensitive. we suggest that gabaergic inhibition in the rvm respiratory neuron network is mediated mainly by inspiratory neurons. gabaergic neurons are also involved in central chemosensitivity. we investigated two groups of people with a different initial level of an emotional tension before intellectual loading (il). first group had the initially increased emotional level, stressed group (sg), and the second group had not, calm group (cg). reaction time (rt) of simple visual sensorymotor reaction and asymmetry of skin potential level (spla) in two facial zones: forehead and nasal were measured. from research groups, subgroups with and mv spla were extracted. thus the distinction in the greater rt gain after il in subgroups with mv forehead spla, in comparison to subgroups mv forehead spla. such law was common for both for sg and for cg. in two subgroups of and mv nasal zone spla in sg the greater rt gain after il was in mv nasal spla subgroup, and for cg in mv subgroup. it was shown, that individual features of il performance are related to spl lateralization. but the low of the relationships of spl asymmetry in nasal zones depends on the level of emotional tension of investigated groups. mitsuko kanamaru, ikuo homma department of physiology, showa university school of medicine, tokyo, japan we have reported that serotonin ( ht) in the dorsomedial medulla oblongata in mice increases tidal volume and minute ventilation via ht receptors. peripheral administration of p-chlorophenylalanine (pcpa) reduces the whole brain ht level. the present study examined whether peripheral pcpa pretreatment affects hypercapnic ventilatory responses in mice. adult male mice (c bl/ n) were pretreated with pcpa ( . g/ ml/kg body weight) or saline intraperitoneally for consecutive days. on the next day, each mouse was placed into a double chamber plethysmograph to obtain respiratory flow curves. one hundred percent o inhalation was changed to stepwise , and % co in o inhalation every min. hypercapniainduced increases in tidal volume and minute ventilation during % co inhalation were reduced by pcpa pretreatment; these results suggest that ht may increase tidal volume in hypercapnic ventilation. saori nishijima, kimio sugaya, minoru miyazato division of urology, department of organ-oriented medicine, faculty of medicine, university of the ryukyus, okinawa, japan we investigated the effect of gosha-jinki-gan on bladder activity and the autonomic nervous system in rats. forty-two female rats were divided into a control diet group and a . % gosha-jinki-gan diet group. after weeks, continuous cystometry with physiological saline or . % acetic acid solution and biochemical analysis were done. the amplitude of bladder contraction with physiological saline was lower in the gosha-jinki-gan diet group than in the control diet group, and plasma dopamine and serotonin levels were also lower in the gosha-jinki-gan diet group. when cystometry was done with . % acetic acid, the interval between bladder contractions was shortened in the both groups. however, the interval and duration of bladder contractions were longer in the gosha-jinki-gan diet group than in the control diet group. therefore, it is suggested that gosha-jinki-gan inhibits bladder activity by maintaining the balance of the sympathetic nervous system and the parasympathetic nervous system at a low level. satoko suzuki, shinya yanagita, seiichiro amemiya, ichiro kita graduate school of science, tokyo metropolitan university, japan we examined the effects of negative air ions (nai) on physiological responses and neuronal activity with c-fos immunohistochemistry. in addition, we investigated the effect of vagotomy to reveal afferent pathways of nai stimulation. we analyzed neuronal activity of the paraventricular nucleus of hypothalamus (pvn), the locus ceruleus (lc), the nucleus ambiggus (na), and the nucleus of solitary tract (nts). nai significantly decreased blood pressure, heart rate, and respiratory rate, and increased hf component which is an index of parasympathetic nervous activity. nai decreased c-fos expression in the pvn and lc, and enhanced in na significantly. after vagotomy, the physiological responses and changes of c-fos expression in pvn, lc, and na was disappeared. furthermore, increase of c-fos expression in nts induced by nai was also disappeared. these results suggest that effect of nai on sympathetic and parasympathetic nervous activity was induced by reducing the activity of the pvn and lc, whereas enhancing the na activity, and that these effects of nai was caused through vagus nerve. yusuf o. cakmak, umit suleyman sehirli school of medicine, university of marmara, turkey previous assessments of the autonomic nerve supply of testis from vagus and brainstem nuclei were conflicting in the literature. we challenged this consensus by using neuronal tracer fluorogold in rats. fluorogold dye solutions was injected unilaterally under the capsule of rat testis. rats were sacrificed by transcardiac perfusion-fixation in the fifth and seventh days after injection. brainstem of the control group, subdiaphragmatic vagotomy group and main group rats were dissected. in the main group the fluoroscent-labelling were dense in area postrema. dorsal vagal nucleus, nucleus of solitary tract and nucleus ambiguous were also labelled. these preliminary data provide an evidence of testicular innervation by vagus nerve. taking into account that brainstem structures could be labelled from the testis, it can be assumed that the areas detected might be involved in the neural control of testicular functions. the results of this study cautioned that innervation of the testis may not be fully explained by innervation from pelvic and paraaortic ganglia. research funds: scientific researches comittee of medical school of marmara university ps p-g differential control of renal and lumbar sympathetic nerve activity during freezing behaviour in conscious rats yoshimi tahara, misa yoshimoto, keiko nagata, kenju miki integrative physiol. grad. sch. humanities and sci. nara-women's univ., nara, japan the present study was designed to examine sympathetic and hemodynamic responses to loud noise exposure, which induced freezing behaviour, in chronically instrumented rats. wistar male rats were instrumented with electrodes for measurements of renal (rsna) and lumbar (lsna) sympathetic nerve activity and catheters for measurements of systemic arterial and central venous pressure. rats were exposed to db white-noise for min. db noise exposure resulted in an immediate and significant increase in rsna while lsna did not change significantly during the exposure in sham-operated (so) rats. there was a significant difference in the response between rsna and lsna during the db noise exposure in so rats. sinoaortic denervation attenuated the magnitude of the increase in rsna while it had no influence on the changes in lsna observed in so rats. these data suggest that arterial baroreceptor significantly contribute to the differential control of rsna and lsna during freezing behaviour in conscious rats. here, we first demonstrated that in both the kolliker-fuse nucleus (kf) and the rostral ventral respiratory group (rvrg) region, phrenic nucleus (phn)-projecting neurons were embedded in the plexus of axons originating from the ventrolateral subnucleus of the nucleus of the solitary tract (vlnst) and that the vlnst axon terminals made synaptic contacts with somata and dendrites of the phnprojecting neurons, using a combined anterograde and retrograde tracing technique. secondly, we indicated that some of the vlnst neurons innervate both the kf and the rvrg by way of axon collaterals, using the double-labeling method. using retrograde tracing combined with in situ hybridization for mrna encoding glutamic acid decarboxylase (gad ), we finally showed that most of the kf/rvrg-projecting vlnst neurons expressed gad mrna. these results suggest that vlnst neurons may exert inhibitory influences upon the phn-projecting kf/rvrg neurons for inspiratory control. we have examined whether the neurons of the dmv have direct synaptic contacts on the myenteric ganglia using wga-hrp. the myenteric ganglia of the stomach were composed of four types of neurons. the average numbers of axosomatic terminals per profile were . on the small neurons, . on the medium-sized neurons, . on the large neurons, and . on the elongated neuron. most of the terminals contained round vesicles and formed asymmetric synaptic contacts on the small, medium-sized and large neurons. about % of the axosomatic terminals on the elongated neurons contained pleomorphic vesicles and formed asymmetric synaptic contacts. when wga-hrp was injected into the dmv, many anterogradely labeled terminals were found around the myenteric neurons. the labeled terminals were large ( . m), and contacted exclusively the somata. most of them contained round vesicles and formed asymmetric synaptic contacts. serial ultrathin sections revealed that almost all neurons in a ganglion received projections from the dmv. ps p-h neuronal mechanisms of respiratory rhythm modulation induced by external k + concentration change in the newborn rat brainstem-spinal cord preparation hiroshi onimaru, ikuo homma dept. physiol., showa univ. school of med., tokyo, japan it has been suggested that two distinct rhythm generators (pfrg-pre-i and pre-bötzinger insp) for respiration in the medulla possess different sensitivity to various neuromodulators. we hypothesize that the dominancy of these rhythm generators to determine basic respiratory rhythm depends on the back ground stimulation level. to verify this hypothesis, we studied neuronal mechanisms of respiratory rhythm modulation induced by external [k + ] change. we recorded membrane potential of pre-i neurons, c nerve and facial nerve activities. addition of mm k + to the standard superfusate decreased burst rate of c activity. addition of or mm k + caused initial inhibition of c burst and subsequent high frequency c burst. the facial nerve burst was depressed. pre-i neuron was depolarized strongly by application of high k + , and the burst activity was disturbed and action potentials were inactivated. results suggest that pfrg-pre-i or pre-bötzinger insp rhythm generator is dominant in low or high back ground stimulation level, respectively. research funds: kakenhi ( ) ps p-h regulation of synaptic transmission in the reticular formation of medulla oblongata by substance p we have examined the response of neurons in the reticular formation near the nucleus ambiguous (na) to the administration of substance p (sp). whole-cell recording was applied to the postsynaptic neurons in coronal slice preparations of medulla oblongata isolated from infant rat. bath application of sp ( m) increased or decreased the frequency of spontaneous activities. several neurons were clamped at − mv and recorded epscs evoked by electrical stimulation to dorsoventral adjacent area from recording neurons. in several neurons, evoked inward epscs were augmented by sp perfusion. i-v curve suggested that voltage dependent current was both augmented and not changed by sp. our previous studies have shown that administration of neurokinin receptor (nk r) antagonist near the na inhibited gastric and respiratory movement in anesthetized rat. these results indicated that sp affect to both post and presynaptic nk r and regulate the transmittance efficiency to generate the output signal of certain autonomic reactions. ps p-h effects of local warming in the back or abdominal region by means of a heat-and steam-generating sheets on physiological response in the low temperature room to investigate effects of local warming of the body on physiological functions as well as subjective feeling, eegs, ecg, respirometer, bis (bispectrum) index, blood pressure (bp), and local skin temperature of the body were monitored while a steaming heat pack was put on the lower lumber or abdominal region of the subjects for h in the cold room. in the control experiment without the heat pack, lf/hf of hr variability (lf/hf-hr) and lf of bp variability (lf-bp) increased, while hf of hr variability (hf-hr) and skin temperature decreased, suggesting elevation of sympathetic nervous activity. in the warming experiment with the heat pack, an increase in lf/hf-hr and/or lf-bp was suppressed and hf-hr increased. we will discuss these autonomic data in relation to subjective unpleasant or pleasant feeling, eeg and bis data. junichi arai, yasuhisa endo, ryouichi yoshimura, huan wang kyoto institute of technology, japan in the ventromedial hypothalamic-lesioned animals, the abnormal cell proliferation in liver and pancreas are thought to be due to the vagus hyperactivity and/or the sympathetic repression. we conducted the co-culture system of several cell lines and demonstrated that the proliferation of hepatocytes and min- cells (a cell line of pancreatic b cells) were stimulated by the administration of carbachol, when they were co-cultured with cell lines of endothelial cells or smooth muscle cells. these effects were also found in the filter-insert coculture system, but never seen in the culture using single cell line. we discuss the possible mechanism of their intracellular signal transduction. research funds: kakenhi to study the correlation between the trans-cranial oxy-and deoxyhemoglobin (hb) dynamics and sbp, we measured hb dynamics (f-nirs ® , omm- , shimadzu corp. japan) over the frontal area and sbp (finapres ® , bp monitor, ohmeda , usa) at the right middle finger from volunteers ( . ± . years). mild thermal stimuli ( ± • or ± • ) were administered every min alternatively to the left hand. some area showed positive correlation between the oxy-hb and sbp, the other showed negative correlation between them. hb dynamics over the frontal area have any correlation to sbp to some extent. so, trans-cranial nirs should be discussed carefully for neural activation. we thank shimadzu corp. for the use of omm- . we examined the effects of color environments on cognitive function in healthy subjects and patients after traumatic head injury using p components and loreta analyses. the examination was performed in color environments of red, green, or black using visual oddball tasks with photographs of a crying baby face as the target stimuli. the p latency in the red environment was significantly shorter in controls than in patients. the p amplitude in the red environment was significantly larger in controls than in patients. loreta analysis demonstrated that the neurological activities in the occipital lobes, left tonsillar nucleus, anterior cingulated gyrus, and brodmann area in the red environment were significantly higher in controls than in patients. hironori nakatani, cees van leeuwen riken brain science institute, saitama, japan some figures, such as rubin's vase/face and the necker cube, have two or more distinct interpretations and are, therefore, called 'ambiguous'. when an ambiguous figure is presented continuously for a period of time, we experience spontaneous switching between the alternative interpretations. as this occurs without any changes in the figures themselves, perceptual switching phenomena are eminently suitable to study how perceptual processes are influenced by the intrinsic dynamics of neural activity. we analyzed eye-movement and eeg during perceptual switching in the necker cube. blink probability showed a peak about ms before the button press responses. we found that only blinks that appeared around the peak time led to a characteristic spatiotemporal pattern of eeg. our results indicate that some, but not all, blinks play an active role in perceptual switching processes. ps p-h neural basis of social cognition investigated by functional near infrared spectroscopy and electroencephalograms recorded from the whole brain tsuneyuki kobayashi , , mikinobu takeuchi , , takahiro omote , naoyuki yosimura , etsuro hori , , kazuo sasaki , taketoshi ono , , hisao nishijo , system emotional science, univ. toyama, toyama, japan; crest, japan science and technology agency, japan; bio-information engineering, univ. toyama, toyama, japan; molcul. & integ. emotional neurosci., univ. toyama, toyama, japan neural basis of social cognition was investigated by functional near infrared spectroscopy (fnirs) and electroencephalograms (eegs). a head cap for recording fnirs and eegs was set on heads of subjects. the probes of the fnirs imaging systems ( channels) and/or electrodes of the eeg system were attached on the heads of the subjects. the subjects were required to perform social cognition tasks to discriminate ( ) human facial stimuli with different gaze directions and ( ) simple animation videos representing social interaction. whole brain hemodynamic images were superimposed on the d reconstructed mri images of the brains. now we are analyzing hemodynamic images and eeg data related to social cognition, and the results indicated some heterogeneity of the cortex in social cognition. hiroshige takeichi , sachiko koyama , ayumu matani , andrzej cichocki riken, wako, japan; hokkaido university, sapporo, japan; university of tokyo, kashiwa, japan to evaluate the level of spoken sentence comprehension objectively and quickly, electroencephalograms (eeg) were recorded from five japanese adults, while they were listening to fifty-second spoken sentences. natural japanese (native) and spanish (foreign) sentences were modulated in amplitude by an eleventh-order m-sequence at hz, and played twice: forward and backward. evoked responses to the modulation were analyzed as follows: ( ) circular cross correlation functions were calculated between the eeg data and the m-sequence for each subject. ( ) the functions were averaged across subjects. ( ) independent component analysis (ica) was applied to the averaged functions and independent eeg components were estimated for each stimulus for each subject. ( ) phase-locked component responses to the modulation were inspected. as a result, two components showed differential responses to the comprehensible forward japanese and the other incomprehensible stimuli. research funds: jst and kakenhi ( ) perceptual rivalry, such as ambiguous figure perception and binocular rivalry, reflects the flexibility of our brain, because it produces fluctuating perception though an unchanging stimulus. in this study, we carried on meg recordings of healthy subjects while they reported perceptual alternation of bistable apparent motion. we investigated power and phase synchronization analyses of meg signals and compared the spatiotemporal patterns during spontaneous perceptual alternation (rivalry condition) with the externally-triggered alternation (replay condition) to extract the inherent dynamics of perceptual alternation. as results, we detected transient anterior-posterior synchronizations in advance of subjects' reports of perceptual alternation in the rivalry condition. these results suggest that these synchronized activities are involved in a higher-order process inducing spontaneous alternations in perceptual rivalry. ps p-h the reflection of category perception of sound in the auditory evoked n m magnetic responses to periodic complex sounds with equivalent acoustic parameters except for different fundamental frequencies (f ) and different spectral envelopes of vocal, instrumental and linear shapes were recorded to clarify the cortical representation of timbre categorization. responses to vocal and instrumental (nonlinear) sounds were localized significantly anterior to linear sound responses. n m source strength for nonlinear sounds was significantly larger than that for linear sounds. n m peak latency only for vocal sounds was not affected by f . these results suggest that perceptual categorization was reflected in n m source strength and location (linear or nonlinear), and in n m latency (vocal or nonvocal). sunao iwaki , hiroko kou , kouichi sutani , mitsuo tonoike national institute of advanced industrial science and technology, osaka, japan; chiba univ., chiba, japan interactions between neural activities detected at multiple brain regions involved in the visual target detection processing were assessed using meg and the causal modeling. meg signals were measured during subjects performing a visual infrequent target detection task. distributed source model was used to infer the dynamic neural activities at the multiple regions and the structural equation modeling (sem) was then used to compare two possible causal models underlying the generation of major event-related components, namely p , related to the target detection. we used akaike information criteria (aic) and goodness-of-fit index (gfi) as measures of the goodness of the models. the results of the comparison of two possible sem models, whose major difference was on the contribution of the activities in the parieto-temporal region to the generation of p components, suggested the involvement of frontal and anterior cingulate cortex in the early p component (p a) and the contribution of the parietal and temporal regions to the later component (p b in our study, we investigate whether or not bilinguals use distinct neural substrates to recognize words in their first and second languages (l and l , respectively). we compared the brain activity of chinese learners of japanese as l with that of japanese natives studied in our previous study. we obtained written informed consent from each subjects. in data analysis, we used spm . while natives showed specifically greater activation in the left middle temporal gyrus than learners, learners showed specifically greater activation in the bilateral parieto-occipital and left occipito-temporal junction than natives. these results indicate that there are distinct neural substrates for word recognition of l and l . neural activations for lexical processes were measured using noun, vowel, and pseudo-character decision tasks with magnetoencephalography (meg) and functional magnetic resonance imaging (fmri) on ten right-handed subjects, and their time courses were analyzed with an fmri-constrained meg-multi-dipole method. the average activations rose at latencies around ms in the occipital gyrus or cuneus (og/cuneus) and ventral occipito-temporal areas (vot), and at latencies around ms in the posterior superior temporal and inferior parietal areas (pst/ipl), anterior temporal area (at), and posterior inferior frontal gyrus (pifg). the differences in activation between tasks are considered to reflect visual-form process in the og/cuneus and r.vot, phonological process in the l.pst/ipl and l.pifg, and semantic process in the l.at. the decay of activation for these areas was found to be well fitted to exponential functions with time constants around ms. the effectiveness of a habituation/dishabituation paradigm for determining the cerebral dominance for language was examined using a . t fmri. healthy right-handed adult volunteers with prior written informed consent were instructed to listen to analysis-synthesized words. after habituated to a single word presented repeatedly, the subject was presented with contrastive words which comprised comparison and habituation words in a pseudo-random order. the two blocks were repeated alternately for times. comparison words were phonemic or intonational derivative of the habituation word, and presented in respective sessions. the results showed that the left auditory cortex responded more to the phonemic contrast, and the right to the intonational contrast, which is in line with other paradigms/techniques for determining cerebral dominance, while the present paradigm demands little effort on the subject. the issue that whether meaning of kanji words is accessed from orthography, or from both orthography and phonology representations is still debated. the present fmri study investigated brain areas underlying the use of orthography and/or phonology in kanji reading by engaging subjects in semantic categorization task with homophone and orthographic similarity effects. fifteen native japanese volunteers participated. stimuli were pairs of definitions and their target words, including correct words and foils. the subjects were asked to decide the correct target words of definitions. the results showed that homophone versus non-homophone foils increased activation of the left fusiform and middle frontal gyri. orthographically similar versus dissimilar foils increased activation of the left middle and inferior frontal gyri. these findings reflected the roles of both orthography and phonology in kanji reading. moreover, homophone versus non-homophone minus orthographically similar versus dissimilar foils revealed activation of the left fusiform gyrus. this might suggest the role of this area in character-to-sound conversion of kanji words. chieko takamiya , mie matsui , , tsuneyuki kobayashi , , hisao nishijo , , michio suzuki , , yasuhiro kawasaki , , masayoshi kurachi , , jun nakazawa , kyo noguchi , hikaru seto neuropsychiatry, univ. toyama, toyama, japan; crest, japan science and technology corporation, japan; psychology, univ. toyama, toyama, japan; system emotional science, univ. toyama, toyama, japan; neuropsychiatry, univ. toyama, toyama, japan; developmental psychology, univ. chiba, chiba, japan; radiology, univ. toyama, toyama, japan an individual has a theory of mind (tom) if he imputes mental states to himself and others. this ability is necessary for our well-rounded social communication. we used functional magnetic resonance imaging (fmri) in ten healthy subjects to study the neural mechanisms underlying tom. we adopted the picture sequencing tasks which demanded inferring mental states to self and others as tom task. as a result, there were significant brain activations in the medial frontal cortex and middle frontal gyrus. these activations coverged with a part of results in previous neuro-imaging studies on tom and social cognitive functions. objective: the purpose of this study was to investigate the neural bases of evaluation of ambiguous facial expression using whole brain functional magnetic resonance imaging (fmri). methods: participants underwent fmri scanning during which they performed a task evaluating facial expression of human (happy or sad). the task consisted of three conditions: ambiguous, middle, and high intensity of facial expression. pictures were chosen from atr facial expression image database. results: subtraction between ambiguous and other conditions revealed the activation of anterior cingulate cortex and prefrontal cortex in evaluation of ambiguous expression. the present results suggest that these area may be involved in evaluation of ambiguously expressed emotions. motoaki sugiura , atsushi sekiguchi , keisuke wakusawa , , yuko sassa , , hyeonjeong jeong , , kaoru horie , , shigeru sato , , ryuta kawashima , miyagi university of education, sendai, japan; niche, tohoku univ., japan; dep. pediatrics, tohoku univ. school of medicine, japan; ristex, jst, japan; gsics, tohoku univ., japan; lbcrc, tohoku univ., japan using an fmri, we examined the cortical mechanisms for risk perception during observation of risky tool usage. normal subjects were presented with a picture of a naturalistic situation involving two actors, in which risks related to a tool and the direction of action were modulated in a two-factorial design. after the fmri, each subject self-evaluated the degree of risk in each picture. main effects of object-and direction-related risks were observed in the left ventromedial prefrontal cortex, and dorsolateral parieto-frontal network, respectively, suggesting that the object-and direction-related risk signals are separately processed in these networks. significant positive correlation between self-evaluated risk and cortical activation was observed in the anterior part of the left superior frontal sulcus, suggesting an involvement of this region in phenomenal risk-perception. in this fmri study, we identified cortical areas where activation during experience of risky situation is correlated with the harm avoidance (ha) scores, subscale of temperament and character inventory (tci). forty-six healthy subjects performed a rule speculation task in risky, normal, and safe situations in fmri. each situation was arranged for subjects to gain , , points or lose , , points, respectively. cortical activation induced by experience of risky situation was estimated. a significant positive correlation with the ha scores, was observed in activated areas in the right anterior insula in risky versus safe comparison. the results suggested that activation in this region predicts the individual difference in behavioral response to risky situation. this finding indicates that the right insula underlies individual difference in response to risky situation. ps p-h brain activation related to the evaluation of absolute and relative value of outcome juri fujiwara , masato taira , , toshio iijima , ken-ichiro tsutsui div. sys. neurosci., tohoku univ. grad. sch. life sci., sendai, japan; arish, nihon university, tokyo, japan; appl. sys. neurosci., nihon univ. grad. sch. med. sci., tokyo, japan one way to evaluate the behavioral outcome is in terms of absolute gain or loss (absolute value), but the evaluation can also be achieved by comparing the outcome with the possible outcomes of unchosen options (relative value). here we attempted to disentangle the brain processes involved in the absolute and relative value evaluation by using event-related fmri. subjects were instructed to compete with a computer to maximize the income in a task, in which they had to choose one option out of two, each of which were associated with either yen or a gain or loss of , , or yen. in each trial, a choice period was followed by a serial presentation of the outcomes of the chosen and unchosen options. we analyzed the brain activity during the presentation of each outcome. the activation changes related to the evaluation of absolute and relative value were observed mainly in the basal ganglia and in the cerebral cortex, respectively. ps p-i neural activation during experience-based reasoning chisato suzuki , , takashi tsukiura , hiroko mochizuki-kawai , yayoi shigemune , , toshio iijima neurosci. res. inst., aist, japan; div. systems neurosci., tohoku univ., japan the aim of this study is to investigate neural activations when reasoning future events based on experienced events. before fmri, subjects encoded two kinds of four-scene comics; the complete version with four scenes and incomplete one without the last scene. after encoding, subjects performed three tasks during fmri. in the first task, subjects chose a last scene associated with the first scene encoded in the incomplete version (memory-based reasoning: mr), whereas in the second task, subjects recognized a last scene encoded in the complete version (memory: m). in the third task, subjects chose a last scene appropriate to the first scene in the new comics (reasoning: r). activations specific to mr was found in a relatively anterior part of the left pfc and right pfc. the common activations between mr and m were identified in the right mtl, whereas a relatively posterior part of the left pfc was activated commonly between mr and r. the findings suggest that the network including bilateral pfc and right mtl may contribute to the experience-based reasoning of future events. to assess neural responses to reciprocal mindreading in socially strained human relationships, we performed an fmri study in healthy subjects who participated in the chicken game. statistical parametric mapping showed that the counterpart effect (human versus computer) activated the anterior paracingulate cortex (pcc) and the posterior superior temporal sulcus (sts). when we analyzed the data to evaluate whether the subjects made aggressive or reconciliatory choices, the posterior sts showed that the counterpart had a reliable effect regardless of risky or safe decisions. in contrast, a significant opponent x selection interaction was revealed in the anterior pcc. it could be inferred that the posterior sts and the anterior pcc play differential roles in mentalizing; the former serves as a general mechanism for mentalizing, while the latter is exclusively involved in socially risky decisions. creativity is the ability to generate new and original ideas. the most of studies of creativity used linguistic tasks which involve multiple aspects oflinguistic information processing in addition to creativity. we used new artistic creativity task such as designing new tools, in which we could quantitatively evaluated the creativity by the originality (os: originality score) of the products. using fmri, we observed bold signal change during designing task in art students (trained) and non-art students (untrained). we observed clear difference between two groups; in the trained highly creative group, the os is correlated with the interhemispheric difference of neural activities of the prefrontal cortex with right hemisphere dominance. in the untrained group we saw no such correlation. thus, our result supports the notion that both right prefrontal dominance and the increase of interhemispheric cooperativity could be the source of the artistic creativity. ps p-i the difference of brain activity elicited by different styles of art hiromi yamamura , yasuyuki kowatari , , shigeru yamane , miyuki yamamoto , comprehensive human sciences, university of tsukuba, tsukuba, japan; system brain science division, aist, tsukuba, japan artworks are categorized according to time and place where they were produced (cultural effects). surrealistic art is one of those categories and it gives uneasy impression to our mind. we investigated brain activity during viewing pictures of different art styles using functional magnetic resonance imaging (fmri). works of several artists who are well-known as representatives of renaissance, impressionism and surrealism were used as stimuli and results were analyzed by spm . while renaissance arts or impressionism arts elicited a similar activation pattern in the occipital and inferior temporal areas, surrealisms showed deactivation in parietal with the activation in the right dorsal prefrontal cortex (ba , ba ). these results suggest that a particular style of artwork may have commonly activated brain regions. research funds: coe(j- ) ps p-i effects of chewing on the activity of the prefrontal cortex in working memory processing: an fmri study in general, it has been proposed that chewing produces holding or enhancing effect on attention. furthermore, recent studies have shown that chewing causes activation of various brain regions, including prefrontal cortex. we therefore examined the influence of chewing on brain activities using fmri. the subjects used were - aged healthy adults, being conducted continuously to two-back task with intermittent gum-chewing. gum without odors and taste component was used to remove effects other than chewing. the results indicated that chewing tended to increase the bold signals in the prefrontal area including the dorsolateral prefrontal cortex during two-back task. this suggests the possibility that chewing may accelerate the process of working memory. research funds: kakenhi , ps p-i the tip-of-the-tongue with an emotional reaction caused by recall of celebrities' names hirohito m. kondo , michio nomura , jun kawaguchi ntt commun. sci. labs., ntt corp., atsugi, japan; dept. psychol., tokai women's univ., kakamigahara, japan; dept. psychol., grad. sch. environ. studies, nagoya univ., nagoya, japan the tip-of-the-tongue (tot) phenomenon is a mental state where you cannot recall something though you have every confidence that you know it. the tot state generates emotional reactions, but it is not clear what neural mechanisms are involved in the awareness of frustration. participants were instructed to recall the full names of celebrities when their faces were presented. event-related fmri analysis demonstrated that the anterior cingulate cortex (acc), anterior insular cortex (aic), inferior frontal cortex, intraparietal sulcus, and fusiform gyrus were activated during the tot state with frustration. activity of the acc and right aic was positively correlated with the degree of frustration in unsuccessful retrieval. roi analysis indicated that the acc and right aic were sensitive to retrieval demands and awareness of frustration, respectively. we suggest that the cinguloinsular circuit regulates the self-monitoring processes during the tot state. noriko kudo , , , yulri nonaka , katsumi mizuno , kazuo okanoya , riken, bsi, biolinguistics, saitama, japan; chiba university, chiba, japan; jsps, japan; department of pediatrics, showa university, tokyo, japan; presto, jst, japan segmentation of speech stream is a prerequisite for language acquisition. language learners use the transitional probability between vocal tokens to segment continuous auditory stream into distinctive words. we consider that the ability for statistical learning is not specific to language, but more general cognitive competence. and we ask whether this ability could be considered as innate. in this study, we measured erps for neonates within days, in order to examine whether neonates can learn transitional probabilities and statistically segment words. four three-tonal-words were presented in random order without intervals during recording of the eeg. as a result, only the first tone of each word evoked a significant positive component in the frontal area. since this potential is not evident during the first session, this is likely to be due to statistical learning. these results suggest that the ability to distinguish words based on statistical information is innately prepared in humans. using near-infrared spectroscopy (nirs), changes in concentration of oxygenated hemoglobin (oxy-hb) in the prefrontal cortex were evaluated while eleven human subjects performed the paintings appreciation task. in this task, subjects were required to appreciate abstract and representational paintings that appear one after another on a computer monitor. subjects were then required to judge the degrees of interest, beauty, and desirability immediately after the appreciation. it was shown that the peak of averaged oxy-hb change was higher while subjects appreciated abstract paintings. average differentiation for each oxy-hb change revealed that the changes while the appreciation of representational paintings were more accelerated than that while the appreciation of representational paintings. these results suggest the different cortical activity dependent on appreciation of abstract and representational paintings. we used meg to investigate the spatiotemporal cortical activities during mental calculations and their modulation by arithmetic complexity. eleven healthy subjects have participated in the study. three conditions were considered: easy: add three ( ) to a two-digits number without carry-over; difficult: stimuli were the same as easy, but with carry-over; nocalc: add zero to the two digits number. probe stimuli were presented s after the presentation of task stimuli (a pair of two-digit and one-digit number), and the subjects were required to respond by lifting the right index or middle finger. root-mean-square values for different meg sensor groups covering entire cortical area were calculated to evaluate local signal power in each condition. increased neural activities in the bilateral frontal/prefrontal and the parietal regions during both calculation conditions were observed in the latencies around - ms. the activities in the bilateral prefrontal and the left parietal areas in the same latencies were found to be complexity-dependent, i.e., increased activities in these regions were observed in difficult condition compared to easy condition. we investigated an effect of auditory feedback on self-produced speech in children with and without autism by measuring the lombard effect. ten children with autism ( : - : ) and agematched typically developing children ( : - : ) were instructed to name pictures of objects aloud in control and masking conditions. in masking, weighted-white noise was continuously delivered through a headset. the subjects' speech responses were recorded from a microphone. in typically developed children, the enhancement (masking/control) in masking was significantly greater (duration = . ± . , loudness = . ± . ) than in the children with autism (duration = . ± . , loudness = . ± . ) (p < . ). the present findings suggest that deficits in speech audio feedback in autistic children and this could be one of the reasons for their delay in speech development. since the mechanism underlying the effect of low power laser irradiation on the soft tissue is still unknown, we examined whether it can influence the muscle contraction as well as its fatigue in the frog (xenopus laevis) gastrocnemius or not. muscle tension continuously induced by a supramaximal stimulus to the sciatic nerve at . /s chronologically attenuated and showed a simple fatigue curve. direct irradiation of laser ( nm, mw) to the muscle surface ( . mm ) significantly delayed its attenuation (p < . ). when the rest period was set between stimulating sessions and the laser irradiation was applied during the rest period, averaged muscle tension during stimulating period for min decreased according to the session sequence. however, comparing with no or cooling application during the rest periods, such laser irradiation case significantly delayed the muscle fatigue (p < . ). it is suggested that laser irradiation has a potential to more activate atp synthesis during as well as after muscle contraction. ps p-i nedl , a novel e ubiquitin ligase for dishevelled- , targets mutant superoxide dismutase- and interacts with p yuanyuan li , , , kou miyazaki , toshinori ozaki , akira nakagawara division of biochemistry, chiba cancer center research institute, chiba, japan; production technology development center, the furukawa electric co., ltd., ichihara, japan; hisamitsu pharmaceutical co., ltd., tokyo, japan we have cloned a novel hect-type e ubiquitin ligase gene termed nedl . previous study has shown that nedl is exclusively expressed in neuronal tissues and its expression level is high in favorable neuroblastomas and undetectable in unfavorable ones. dishevelled- , a regulatory molecule in the wnt signaling pathway, was identified as the physiological target of nedl for uniquitination and proteasome-mediated degradation. on the other hand, nedl bound and ubiquitinated mutant (but not wild-type) sod in a mutant sod type-dependent manner, which is proportionally related with the fals severity. in the present study, we show that nedl physically bound p , and induced apoptosis in a p -dpendent manner. taken together, our results suggest that nedl may play a critical role in neuronal cell death occurring in fals through interacting with mutant sod and p . spinal and bulbar muscular atrophy (sbma) is an inherited motor neuron disease caused by the expansion of polyglutamine tract within the androgen receptor (ar). chip (carboxyl terminus of hsc interacting protein), u-box type e ubiquitin ligase, has been shown to interact with hsp or hsp and ubiquitylates unfolded proteins trapped by molecular chaperones and degrade them. we demonstrated in a neuronal cell model that transient over-expression of chip reduced the monomeric mutant ar more than the wild-type, suggesting that the mutant ar is more sensitive to chip than is the wild-type. we also demonstrated high expression of chip ameliorated motor impairments in the sbma transgenic mouse model. these findings suggest that chip over-expression ameliorates sbma phenotypes in mice by reducing nuclear-localized mutant ar, which probably due to enhanced mutant ar degradation. we performed an electrophysiological study demonstrating inhibition of spontaneous muscle action potentials within a co-culture of rat muscle and spinal cord by exposure to patients with guillain-barré syndrome (gbs) serum, as well as purified igg, from selected patients with gbs. using a whole-cell recording technique, we then investigated the effects of serum and purified igg from patients with gbs on voltage-dependent calcium currents (vdcc) in ngf-differentiated pc cells and cerebellar purkinje cells. serum from selected patients with gbs and purified igg from some serum of patients with gbs inhibited ca + current in both cells. these results suggest that muscle weakness in some patients with gbs might be induced by changes in p/q-type calcium channel function within motor nerve terminals. the aim of the present study was to explore the possible role of cox- inhibitor, rofecoxib in pentylenetetrazol (ptz, mg/kg, i.p.)induced kindling. rofecoxib was administered orally daily min before either ptz or vehicle. seizure severity was measured according to a prevalidated scoring scale. biochemical estimations were performed on the th day of ptz treatment. chronic treatment with rofecoxib ( . and . mg/kg, p.o.) for days showed significant decrease in ptz-induced kindling score. chronic treatment with ptz significantly increased lipid peroxidation, nitrite levels (no levels), and myeloperoxidase levels and decreased the reduced glutathione (gsh) levels in brain homogenate, which was reversed with rofecoxib treatment. research funds: university supportted study ashish dhir, shrinivas kulkarni uips, panjab university, chandigarh, india the objective of the present study was to elucidate the effect of cyclooxygenase inhibitors on pentylenetetrazol (ptz)-induced ( mg/kg) convulsions in mice with possible mechanism of action. various cox-inhibitors were administered min prior to the ptz administration. onset, duration of clonic convulsions and percentage mortality/recovery were recorded. pretreatment with cox-inhibitors aspirin ( and mg/kg, p.o.), naproxen ( and mg/kg, p.o.), nimesulide ( - mg/kg, p.o.) or rofecoxib ( - mg/kg, p.o.) dose dependently showed protection against ptz-induced convulsions. rofecoxib ( mg/kg) or nimesulide ( mg/kg) also enhanced the subprotective effect of diazepam or muscimol showing gabaergic modulation of cox- inhibitors. cox- inhibitors also antagonized the effect of flumazenil ( mg/kg) against ptz-induced convulsions further confirming the gabaergic mechanism. ps p-j cell proliferation after domoic acid-induced neuronal damage in adult rats domoic acid (da) is structurally related to kainic acid, which is a rigid analogue of the putative neurotransmitter l-glutamate that causes neuronal excitation. da-induced convulsions affects limbic structures such as hippocampus and entorhinal cortex. in this study we examined the neuronal damage after intraperitoneal da administration and cell proliferation in the adult rat brain. the most extensive neuronal cell damage was observed in ca subfield as evaluated by he staining, while tunel positive cells were mainly observed in the granular cells of cerebellum and dentate gyrus (dg) of the hippocampus. to elucidate the relations between damage and cell proliferation, we examined bromodeoxyuridine (brdu) labeled cells. brdu labeled cells were detected in dg and the granular cells of cerebellum. the cell proliferation was not associated with damage. ps p-j a-type potassium channel truncation mutation in temporal lobe epilepsy the role of voltage dependent calcium channels on the pentylenetetrazol (ptz) kindling induced learning deficits was investigated in rats. in this study animals were divided into three groups. in the test group verapamile were injected in the hippocampus ( mg/ min). after min kindling was established in rats with ptz. the control animals were the same age and undergone the same treatment in term of acsf injections and post-kindling waiting time as the kindled animals. and in sham group the animals received saline. one month after induction of kindling spatial learning and memory was tested by morris water maze. results showed that intra-hippocampal injection of verapamil significantly decreased spatial learning, suggesting that only working memory impaired but reference memory remain intact. the results with this study suggest that intera-hippcampal injection of verapamil significantly impaired spatial learning in rats. we showed that -oxoguanine ( -oxog) in mitochondrial (mt) dna and cellular rna increased significantly in the ca subregion of the mouse hippocampus after kainate administration. laser scanning confocal microscopy revealed that -oxog accumulated greatly in mtdna of the ca microglia. wild-type and mth -null mice, the latter lacking an ability to hydrolyze -oxo-dgtp and -oxo-gtp to the monophosphates to avoid their misincorporation into dna or rna, exhibited similar degree of the ca neuron loss after kainate administration, however, levels of -oxog accumulated in mtdna and cellular rna in the ca microglia were significantly increased in mth null mice in comparison to wild-type mice. we thus demonstrated that mth efficiently suppresses the accumulation of -oxog in both cellular dna and rna in the hippocampus, especially in microglia, caused by excitotoxicity. ps p-j transcription factor nrf regulates brain response to kainate-induced excitotoxicity yukihiko dan , kosuke kajitani , noriko yutsudo , ken itoh , masayuki yamamoto , yusaku nakabeppu kyushu univ., med. inst. bioreg., div. neurofunc. genomics, japan; univ. tsukuba, grad. sch. comp. hum. sci., japan nf-e related factor (nrf ) is the key transcription factor that serves to transmit the inducer signal to an antioxidant response element (are), a cis-acting element required for gene expression of a battery of proteins acting on anti-oxidative stress and detoxification of electrophiles. since loss of nrf has been reported to increase neuronal death under increased oxidative stress, nrf seems to play a role for neuroprotection. administration of kainite, a potent agonist of an excitatory neurotransmitter glutamate, to rodents produces epileptiform seizures followed by a delayed loss of pyramidal cells in the ca subregion of hippocampus. to unveil the functional significance of nrf in the brain, we compared seizure responses between wild-type and nrf -null mice after systemic kainate administration. we found that nrf -null mice exhibited an increased susceptibility to the kainate-induced seizure, and their loss of the pyramidal cells and gene expression profiles are now under investigation. ps p-j synaptic plasticity and -aminopyridineinduced epileptic discharges in rat hippocampal slices makoto otani, tetsuo furukawa, kiyohisa natsume department of brain science and engineering, kyushu institute of technology, kitakyushu, japan four-aminopyridine ( -ap) at the concentration below . mm suppresses k d channel and induces the epileptic discharges in rat hippocampal slices. in the present study, the involvement of the activation of nmda receptor on the ictogenesis of the -ap induced discharges in ca region was studied. ten m -ap induced the epileptic discharges with the frequency of . ± . hz (mean ± s.e.m.; n = ) and the amplitude of . ± . mv. when ap- , an nmda receptor blocker, was applied to the pre-established epileptic discharges, the frequency and the amplitude of the discharges did not change significantly. on the other hand, when ap- was applied from the ictogenesis period of the discharges, the discharges did not appear. these results suggest that the nmda receptor-dependent synaptic plasticity involves in the ictogenesis of -ap-induced epileptic discharges. chronic exposure of cultured astrocytes to morphine is reported to induce differentiation of the cells. using primary astrocyte cultures, we observed that under thyroid hormone (th) deficient conditions, morphine significantly decreased cell viability. further studies showed that the loss of cell viability was due to apoptosis of the cells. the effect is attenuated by th supplementation to the culture medium. the observed effect of morphine appears to be mediated through the opioid receptor since the opioid antagonist, naloxone, inhibited the decline in cell viability. ni, a specific inhibitor of nnos, completely blocked loss of cell viability suggesting that morphine induced intracellular no production, leads to cell death. studies suggested that no acts through a cgmp independent pathway. the involvement of no induced cgmp independent pathway in morphineinduced apoptosis during th deficiency has been investigated. collectively, the present study demonstrates that morphine mediated cytotoxicity of astrocyte is critically influence by the level of thyroid hormone in cultured medium. ps a-a influence of conductance-input signal and prior activation history on spike generation in rat somatosensory cortical neurons takashi tateno , hugh p.c. robinson engineering science, osaka university, osaka, japan; university of cambridge, uk in the cortex, a profusion of electrophysiological cell types, which form specific synaptic connections, is becoming apparent. a quantitative understanding of the dynamics of different cell types when responding to complex, natural inputs, is an important prerequisite for understanding the cortical network. neurons compute by transforming excitatory and inhibitory synaptic conductance inputs into a spike train output. we have examined the properties of synaptic conductance inputs which are most effective in evoking spikes, by injecting broad-band excitatory and inhibitory conductance inputs, and using spike-triggered reverse correlation and wiener-kernel estimation to calculate the average conductance input trajectory (acit) preceding spikes. the time course of the acit provides a general description of a neuron's response to dynamic conductance stimuli. our analysis showed that the acit reflects both previous stimulus history and previous discharge history, and that the relative influences of these two factors depend on the cell type. amyotrophic lateral sclerosis (als) is a rapidly progressive neuromuscular disease caused by the destruction of motor neurons. our study has investigated the effects of als-csf on voltage-gated calcium p/q-type channel (␣ a) expression in pre synaptic terminals of rat spinal motor neurons. csf from als and non-als (neurological patients) was injected into the -day-old rat pup spinal subarachnoid space at the rate of l/ . min. the rats were sacrificed h after csf injection and spinal cord sections were processed for immunocytochemistry with p/q-type channel ␣ a antibody and also for cytochrome oxidase labeling. als-csf significantly increased p/qtype channel expression compared to csf from non als patients. als-csf significantly decreased cytochrome oxidase activity in the rat spinal motor neurons, which may be a sign of degeneration. it is probable that, toxic factors present in the als patients csf might induce the expression of p/q-type channel observed in pre synaptic terminals synapsing on the spinal motor neurons. ps a-a on the membrane potential profile of ca pyramidal cells recorded with voltage sensitive dye imaging in rat hippocampal slices takashi tominaga , , yoko tominaga dept. neurophysiol., kagawa sch. pharmaceutical sci., tokushima-bunri univ., kagawa, japan; lab. for dynamics of emergent intelligence, riken bsi, hirosawa - , wako, saitama, japan integration of membrane potential response in a single neuron is a basis of neuronal calculation. we have been aiming to visualize this with voltage sensitive dye (vsd). hippocampal slices, with its unique laminar structure, allow us to assign optical signals to particular membrane fractions. but, it has not been clear whether the profile of optical signal could be a measure of membrane potential profile. to solve this, we visualized rather steady membrane potential change caused by perfusion of high potassium medium. a steep peak in optical signal was seen along stratum pyramidale. an application of ttx diminished this peak, and made the optical signal profile flat along the cell. thus, we concluded that the specificity of the vsd is small. with "neuron", by assuming a population nature to the optical signal, the membrane potential profile in a response to stimulation was successfully simulated. ps a-a overexpression of inwardly rectifying k + channel . in hippocampal slice culture masayoshi okada, hiroko matsuda department of physiology, kansai medical university, japan the expressions of mrnas for the inwardly rectifying k + channel (kir) . have been reported in mammalian central nervous system, but regulation of expression or its role in synaptic transmission remains unknown. in our rat hippocampal slice cultures, the endogenous kir current was hardly detected with whole cell recordings in the ca pyramidal neurons. then, egfp and kir . expressing virus vectors were constructed, and infected to the neurons in the slices. the vectors succeed to express the kir current, and the translocation of the fusion protein to the plasma membrane was also observed. furthermore, the overexpression significantly reduced the raise in whole-cell membrane potential evoked by depolarizing current injection, suggesting that kir plays a role of noise-filter for synaptic input in central neurons. takeshi otsuka, mieko morishima, yasuo kawaguchi div. cerebral circuitry & structure, nips, okazaki, japan layer pyramidal cells are heterogeneous in morphological and physiological properties, and project to multiple subcortical areas. although recent studies have addressed anatomical features of pyramidal cells identified projection regions, little is known about intrinsic membrane properties of these subtypes. here, we obtained whole cell recordings from rat frontal layer pyramidal cells that project to the striatum (ccs) or pontine nucleus (cpn), identified by injection of fluorescent retrograde tracer to these regions. firing properties of pyramidal cells had similarity depending on the projection regions. ccs cells showed strong adaptation of successive spike intervals in response to the depolarizing current injection. however, cpn cells exhibited very little spike frequency adaptation during current injection. we also examined synaptic inputs from layer / neurons to these subtypes by single cell stimulation, and detected excitatory inputs in both subtypes. our results suggest that physiological properties of layer pyramidal cells are correlated with their subcortical target. this study aimed to clarify expressional changes in types and of ryanodine receptors (ryr and ryr ) in the cerebellum of a ca + channel ␣ a subunit mutant, rolling mouse nagoya. semi-quantitative rt-pcr revealed altered mrna signal levels of ryr but not ryr in the rmn cerebellum: a less ryr mrna signals than in the control cerebellum. well consistent with the semi-quantitative rt-pcr results, ryr immunostaining in soma and primary dendrites of purkinje cells was less intense in rmn than in control mice. in contrast, ryr immunostaining was detected in cerebellar glomeruli but the staining intensity was not different between rmn and controls. the present study suggests that somatodendritic ryr expression in purkinje cells was decreased in the cerebellum of rmn. this may suffer ryr -mediated ca + release, contributing altered ca + homeostasis in the rmn purkinje cells. ps a-a dopamine-based modulation of lateral amygdala neuron excitability: a possible involvement of potassium current ryo yamamoto, yoshifumi ueta, noubuo kato integrative brain sci. med., kyoto univ., kyoto, japan the amygdala and dopaminergic innervation thereonto are considered to cooperatively regulate emotional states and behaviors. in the present slice experiments, we investigated the effects of dopamine (da) on lateral amygdala (la) neurons by whole cell recordings. application of da depolarized la neurons, reduced the action potential threshold, and induced slow afterdepolarization (sadp). this sadp was induced voltage dependently, and lasted for more than s. d receptor agonists induced the same sadp. previous reports have repeatedly suggested that sadp is triggered by calcium influx. consistently, calcium channel blockers or chelating intracellular calcium inhibited the present da-induced sadp. a membrane conductance decreased at the peak of sadp current (i sadp ). also, i sadp was suppressed by including cesium in the pipette solution. these results suggest that the present da-induced modulation of la neuron excitability may depend on a potassium current that can be masked by calcium influx. toru aonishi , , hiroyoshi miyakawa , masashi inoue , masato okada , tokyo institute of technology, japan; brain science institute, riken, japan; tokyo university of pharmacy and life science, japan; the university of tokyo, japan it has been reported that amplification of ap paired with epsp boosts the induction of ltp. there are two alternative hypotheses of such amplification mechanisms; one is activation of the na channel and other is inactivation of the a-type k channel. which is essential? in this talk, by mathematical analyses and the neuron simulator, we demonstrate that the balance of inward and outward currents, which can be controlled by down/up-regulation of the a-type k channel induces a divergence of the membrane input resistance, i.e. a singularity, and such super-sensitivity is the fundamental mechanism for boosting amplification of ap paired with epsp. the balance of na and a currents is essential for controlling dendritic integration manners. we also show that the down-regulation of the a-type k channel, which modifies the ratio between the inward and outward currents, leads to a drastic change from amplifying ap mode to shunting epsp mode. miharu komai , maya yamazaki , , mika tsujita , manabu abe , rie natsume , , kenji sakimura , department of cellular neurobiology, brain research institute, niigata university, niigata, japan; sorst/jst, saitama, japan we previously reported that stargazin family (␥ , ␥ , ␥ , and ␥ ) not only promoted ampa receptor surface expression but also modulated receptor activity and channel property (yamazaki et al., ) . therefore, we assumed these family proteins were auxiliary subunits of ampa receptors. to prove this hypothesis, we generated ␥ subunit knockout (ko) mice using the cre/loxp recombination system and analyzed their phenotypes. the ␥ subunit ko mice were viable, fertile, and displayed no overt phenotype. on the other hand, on western blot analysis, protein expression levels of ampa receptor subunits were reduced in ko mice compared with those in wild-type at postnatal day , while the reduction was not so significant in adult brain. these results suggested that ␥ might regulate dynamics of ampa receptor subunits during early development. in the cns, neural damages, such as hypoxia, ischemia and degenerating diseases, are often accompanied by disturbances in the ph environments. ambient ph plays as a significant signal for neural functions. microglia (brain phagocytes) express abundant voltagegated proton (hv) channels which have extremely high selectivity for h + and potent h + efflux ability. exposure to na-lactate (ph . ) induced cell acidosis and activation of the hv channels. the channel activation was characterized by increased conductance, facilitation of activation kinetics, prolongation of deactivation kinetics and a shift of the activation voltages to negative potentials. consequently, the hv channel could open more easily over a wide range of the membrane potential during lactic acidosis, and may contribute to a quick relief of the cell acidosis. mari sasaki, masahiro takagi, yasushi okamura okazaki institute for integrative bioscience, aichi, japan here we report a novel four transmembrane protein similar to the voltage sensor domain (vsd) of the voltage-gated channels that exhibits activities of a voltage-gated proton channel. voltage-gated proton channel currents have classically been described in snail neurons and recently in mammalian blood cells. however, the molecular basis underlying this channel has been elusive. here we identify a novel cdna clone named as mouse voltage-sensor domain only protein (mvsop ). cells overexpressing this protein showed depolarization-induced outward currents accompanied by tail currents during repolarization, which reversed at equilibrium potentials for protons. imaging analysis demonstrated that phin recovers rapidly after an acid load in mvsop -transfected cells. mvsop induced currents exhibited two key features of native voltage-gated proton channels: ph-dependent gating and zn + sensitivity. neutralization of a positive charge in the s -like segment caused shift of the voltage-conductance relationship, suggesting that it plays important role in gating. oscillatory extracellular electric fields have been observed in mammalian brains. the electric fields modulate neuronal excitability and synaptic events. to investigate the effect of the oscillatory electric fields on the ca pyramidal neuron, we applied sinusoidal electric fields to the rat hippocampal slice and recorded voltage responses with a voltage sensitive dye (rh ). application of sinusoidal electric fields induced transmembrane voltage oscillations in all the layers of the ca region. in the pyramidal layer, the amplitudes of the responses to the -hz field were the largest. the amplitudes were decreased monotonically when the frequency of the fields became higher. however, in the stratum radiatum, the amplitudes of the responses to the - -hz fields were larger than those to the other frequencies. the frequency preference in the dendritic region may be an underlying mechanism for the synchronization of the membrane potentials among large population of neurons within the theta frequency range. acid sensing ion channels (asic ) have proposed to constitute mechanoreceptors and nociceptors. we examined the localization and characterization of asic -expressing cells in rat central nervous system (e -p ) using immunohistochemical techniques. asic positive fiber first appeared in brain stem and spinal cord at e - stage. asic -expresseing cells appeared in white matter of brain stem and spinal cord at e stage. in early postnatal stages asic expressing cells appeared in corpus callosum, cerebellar medulla and dorsal horn of spinal cord at p stage. these cells were identified as an oligodendroglia by oligodendrocyte specific antibody and immunoelectron microscopy. these results are suggested the hypothesis that the function of asic mediate the myelin formation in the developmental stages of central and peripheral nervous system. masato shino, seiji ozawa, yasuhiko saito department of neurophysiology, gunma university graduate school of medicine, maebashi, gunma, japan nucleus prepositus hypoglossi (nph) is involved in horizontal eye movement. previously, we found nph neurons exhibiting a characteristic firing pattern in response to depolarizing current pulses (fil neurons). fil neurons exhibited a spike train with a long first interspike interval ( st isi) that is attributed to a large, slow hyperpolarization (ahp) after the first spike. in this study, we investigated ionic conductances underlying the long st isi by whole-cell recordings in rat slices. application of m apamin, an sk-type ca + -activated k + (kca) channel blocker, shortened the st isi and decreased the amplitude of the slow ahp. the shortening of the st isi was observed when membrane potentials were depolarized. moreover, application of m mibefradil, a t-type ca + channel blocker, shortened the st isi. these suggest that the firing pattern of fil neurons arises from activation of sk-type kca channels induced by ca + influx through t-type ca + channels. research funds: kakenhi (c) ( ) jafar vatanparast , , mahyar janahmadi , houri sepehri , ali haeri-rohani , ali reza asgari neuroscience research center, shaheed beheshti medical sciences university, tehran, iran; dept. of biology, university of tehran, tehran, iran the roles of the ionic channels and muscarinic receptors in paraoxon (px) induced burst firing in snail neurons were studied using current clamp method. px ( . m, within min) increased the frequency of spikes and shortened ahp. slow waves of depolarization with superimposed bursts were recorded within min. atropine blocked the depolarization shift but not the other effects of px. px was able to reversibly decrease the duration of calcium spikes elicited in a na + free ringer. this effect observed in the presence of atropine and was along with a decrease in the duration of ca + spike ahp and an increase in the spike frequency. the px blockade of ca + channels may decrease the activation of ca + dependent k + channels that underlies ahp. blockade of these channels possibly makes the neurons susceptible for burst induction, while activation of metabotropic muscarinic receptor by px underlies the depolarization shift with associated bursts. dendritic membrane properties are reported to be non-uniformly distributed in a single neuron and the non-uniformity could be important for synaptic integration. however their distribution is still unclear. we estimated distribution of membrane resistance by fitting a compartment model to voltage imaging data of membrane response in hippocampal ca slices to perturbation, such as propagating epsp induced by synaptic inputs and biphasic response to extracellular electric field. by numerical simulations, we found that these imaging data were consistently reproduced if we assume a step function as distribution of membrane resistance. this implies that a steep decrease of membrane resistance exists in distal dendrite of hippocampal ca pyramidal neuron. it is known that cooling-induced desensitization of cold receptors, however, its intracellular mechanism has remained unresolved. in this study, we analyzed molecular mechanism of desensitization of cold/menthol receptors (trpm ). repeated menthol application induced trpm desensitization. this desensitization was depended on extracellular ca + , indicating that involvement with ca + -dependent kinase. pkc activator (pma) desensitized trpm and go (pkc inhibitor) abolished pma-induced trpm desensitization. pma similarly desensitized wild type trpm and mutant trpm , in which serine or threonine residues in some putative pkc phosphorylation sites were replaced by alanine. pma treatment did not induce internalization of trpm . as the basis of cooling-induced desensitization of cold receptors, we conclude that cooling-activated trpm causes pkc to desensitize trpm itself. yosuke sawada , hiroshi hosokawa , kiyoshi matsumura , shigeo kobayashi dept. of int. sci. and tech., grad. sch. of info., kyoto univ., kyoto, japan; dept. of info. sci. and tech., osaka institute of technology, osaka, japan cooling below • c evokes cold pain sensation. however, the molecular basis of the cold pain sensation is still unknown. trpa is activated by pungent compounds stimuli. if trpa responded to cooling to noxious cold range, it could be candidate for evoking cold pain sensation. however, whether trpa is activated by cooling or not is still controversial. here, we investigated that trpa -expressing hek cells responded to noxious cold stimuli. whole-cell recording demonstrated that cooling below threshold evoked inward current. threshold temperature was . ± . • c. in inside-out singlechannel recording, cooling activated trpa directly. single channel conductance was . ± . ps. single channel currents showed inword rectification. in conclusion, trpa is the cooling activated cation channel. yoshiki matsuda, foong yen ang, jinsun yoon, noriko ebisu, satoshi takahashi, shinichi kogure dept. bioengin., soka university, tokyo, japan hyperplarization-activated and cyclic-nucleotide-gated nonselective cation channels (hcn - ) have been demonstrated in the cns. since they contribute to various physiological functions including neuronal pacemaking activity, setting of resting membrane potential and generation of paroxysmal discharge, we examined their expressions as well as functions in the pns using the frog (xenopus laevis) sciatic nerves. western blot analyses for hcn - demonstrated that samples from the nerve and the heart showed an hcn band whereas those from the dorsal part of skin and the gastrocnemius did not, and that immunoreactivities for hcn , hcn and hcn could not be found in those samples. when an hcn channel blocker, zd was applied on the stimulus portion of sciatic nerve and the nerve was elicited at . /s by a duration of ms pulse with supramaximal intensity, the generation of anode-break-excitation rather than cathode-makeexcitation was significantly blocked (p < . ). it is suggested that hcn channels exist in the pns and they contribute to the burst or recurrent discharges. ifenprodil, a clinically used cerebral vasodilator, interacts with several receptors, such as ␣ adrenergic, n-methyl-d-aspartate, serotonin and receptors. however, the molecular mechanisms underlying the various effects of ifenprodil remain to be clarified. here, we show that ifenprodil inhibited g protein-activated inwardly rectifying k + (girk; kir ) channels, which play an important role in the inhibitory regulation of neuronal excitability in most brain regions and the heart rate, expressed in xenopus oocytes. in contrast, kir . and kir . channels in other kir channel subfamilies were insensitive to ifenprodil. the girk currents induced by -opioid receptors or ethanol were also attenuated in the presence of ifenprodil. the inhibitory effects of ifenprodil were not observed when ifenprodil was applied intracellularly. our results suggest that inhibition of girk channels by ifenprodil, at submicromolar concentrations or more, may contribute to some of its therapeutic effects and adverse side effects. ps a-b proliferation of rat c glioma cells is controlled by the concentration-sensitive na + channel (na c ) shigeru yoshida, hiroyuki yamaguchi, takashi takeuchi, hokuto tanaka, yoshiyuki morimoto, teruki hagiwara department of life science, kinki university, higashi-osaka, japan the role of na + as a regulator of cell growth was studied using the tumor cell line (c ), which has a large quantity of concentrationsensitive na + channels (na c ; c = concentration). cell proliferation was suppressed when [na + ] o was raised from control ( mm) to or mm. an increase in [na + ] i was revealed by an image processor in c cells loaded with a na + indicator (sbfi), under high [na + ] o conditions. [na + ] i elevation was augmented by ouabain or by bumetanide (na + /k + /cl − cotransporter blocker), while it was decreased when na c expression was inhibited by rnai techniques. the real-time pcr method revealed that the expression level of the immediate early gene egr- , which is involved in cell growth, was concomitantly reduced. it is to be noted that similar alterations in cell growth, egr- level and [na + ] i were induced by a na + ionophore (monensin) without raising [na + ] o . these data indicate that na + enters through na c upon [na + ] o increase, and [na + ] i elevation itself is responsible for these phenomena. hiroshi kuba, takahiro ishii, harunori ohmori dept. physiol., univ. kyoto, kyoto, japan na + channels are concentrated in the axon to generate action potentials. however, little is known about how distribution of na + channels contributes to the activity and function of single neurons. in avian nucleus laminaris (nl), neurons act as coincidence detectors for sound source localization, and are tuned to both characteristic frequency (cf) and interaural time difference (itd) of sounds. we show here in the chick that nl neurons have distinct distribution of na + channels along the axon and optimize the itd sensitivity depending on their cf. neurons of high and middle cf (higher than khz) had small action potentials, and had no na + currents in the somatic membrane, but clustered only in the axon at some distance from the soma ( - m). while, neurons of low cf generated large overshooting spikes, and na + channels were clustered closer to the soma ( m) in the axon. thus, nl neurons had a spike generator on the axon, at a greater distance from the soma with the increase of cf. by computer simulation, these unique distributions of na + channels were found essential to enhance the coincidence detection. research funds: kakenhi ( ) il-sung jang , in-sun choi , eun-ju park , jin-wha cho , man-gee lee , byung-ju choi kyungpook national university, school of dentistry, south korea; kyungpook national university, school of medicine, south korea bisphenol a (bpa), an endocrine disrupter, is contained in cans, polycarbonate bottles and some dental sealants. here we report the effect of bpa on gaba a receptors using a conventional whole-cell patch clamp technique from acutely isolated rat ca pyramidal neurons. bpa itself elicited a postsynaptic current, which is highly sensitive to bicuculline, in a dose-dependent manner. bpa increased postsynaptic currents induced by gaba at lower concentrations (< m), but decreased those induced by gaba at higher concentrations (> m). in addition, bpa decreased both the current amplitude and decay time constant of gabaergic mipscs. finally, mechanisms underlying bpa-induced modulation of gaba a receptors will be discussed. we recently generated nav . -deficient mice and showed that these mutant mice developed epileptic seizures and died prematurely. we have now used these nav . -deficient mice as negative controls to examine nav . distribution in the mouse brain using rna in situ hybridization histochemistry and immunohistochemistry. at low magnification, nav . expression was higher in the thalamus, superior colliculus, inferior colliculus, pons, medulla and cerebellar nuclei relative to other brain regions. contrary to previous studies indicating a somato-dendritic nav . distribution, in the present study, higher magnification analysis revealed that nav . is predominantly distributed to axons in some brain parts. this apparent discrepancy may reflect the lack of specificity of anti-nav . antibodies used in these previous studies, none of which utilized nav . -deficient mice. based on our findings, we propose that nav . might be involved in propagating action potential to presynapses. keiji miura , , masato okada , , , shun-ichi amari department of physics, kyoto university, kyoto, japan; "intelligent cooperation and control", presto, jst, japan; department of complexity science and engineering, university of tokyo, chiba, japan; brain science institute, riken, saitama, japan we considered a gamma distribution of inter-spike intervals as a statistical model for neuronal spike generation. a gamma distribution is a natural extension of poisson process and it can generate spike trains with various irregularities. the model parameters consist of a time-dependent firing rate and a time-independent spiking irregularity. because the environment changes over time, the firing rate varies for each interspike interval. we used a novel method of information geometry to estimate the spiking irregularity whatever the functional form of the firing rate is. our estimator is simple and easily applicable to experimental data. the estimator is useful for characterizing spiking irregularity which varies among neuron types. it may be possible to classify neurons into functional groups according to their spiking irregularities. research funds: grant-in-aid for scientific research (nos. and ) mitsuyo watanabe, yuko ishimaru, taketo nakadai, tomoyuki kanamatsu graduate school of bioengineering, soka university, tokyo, japan we examined the effect of colchicine, inhibitor of axonal flow, on cerebral amino acid metabolism in the rat. the rats were injected with [ - c] glucose intravenously ( g/kg) or h after the intraventricular injection of colchicine ( g/ l) and the amino acid fractions were extracted from the brains at , or min after the glucose injection. the amount of [ - c] glucose in the cerebra was increased, however, the c incorporation into glutamine, glutamate, gaba and aspartate from [ - c] glucose were decreased. only glutamine concentration in all amino acids was increased in the cerebra of the colchicine group, compared to those values in the control group. the microdialysis analysis showed that the amount of gln in the dialysate was increased by three times in the colchicine group compared with the control group. these data may suggest that the glycolysis of glucose is decreased and that the influx of glutamine from blood to brain occurs with neuronal dysfunction induced by colchicine. these results indicate that a ␤ alters the bhlh gene expression in neural stem cells toward cell death. ken kojima, akiko nishida, shinji takebayashi, jyuichi ito department of otolaryngology-head and neck surgery, graduate school of medicine, kyoto university, japan basic helix-loop-helix (bhlh) transcription factors play crucial roles in development of the central and peripheral nervous systems. to visualize expression of hes or hes gene, phes -and phes -egfp transgenic (tg) mice were generated (ohtsuka et al., ) . in each transgenic mouse, a promoter of hes or hes gene drives enhanced green fluorescent protein (egfp) gene. in the inner ear, it is suggested that hes or hes regulate cell division and differentiation of sensory and supporting cell progenitors via notch signaling pathway. by use of immunohistochemical technique, we examined distribution of gfp expressing cells in the inner ear of the transgenic mice from embryonic day (e ) to postnatal day (p ). in the phes -egfp tg mouse inner ear, gfp immunoreactive (gfp-ir) cells were detected from e to p . in the phes -egfp tg mouse inner ear, gfp-ir cells were observed from e . to p . gfp-ir cells in phes -gfp tg mouse are candidates of sensory cell progenitors in mature mammalian inner ear. ohtsuka et al., . mol. cell neurosci. ps a-c expression of zfh- in the developing mouse brain: mrna, antisense rna and protein expression yuriko komine , kenji nakamura , motoya katsuki , tetsuo yamamori national institute for basic biology, okazaki, japan; mitsubishi kagaku institute of life science, machida, japan zfh- is a transcription factor containing three homeodomains and zn fingers and expressed in differentiating neurons. we have reported that the level of zfh- mrna is negatively regulated by antisense transcripts of the zfh- gene. in several types of neurons, including pyramidal cells in the hippocampus and granule cells in the cerebellum, the zfh- antisense rna is expressed prior to the mrna; as the level of the antisense rna gradually decreases, zfh- mrna starts to be expressed. recently, we have raised an antibody against mouse zfh- and examined the expression profile of the zfh- protein. in the most regions of the brain, the protein expression pattern consisted with that of mrna. however, in the several types neurons mentioned above, zfh- protein was not detected even when the zfh- mrna was already expressed. this observation together with other data suggested that the zfh- protein level is regulated by several mechanisms including suppression by the antisense rna and translational control. takashi inoue , maya ota , katsuhiko mikoshiba , jun aruga laboratory for comparative neurogenesis, riken bsi, saitama, japan; laboratory for developmental neurobiology, riken bsi, saitama, japan zic family zinc-finger proteins play various roles in animal development. in mice, five zic genes (zic - ) have been reported. despite their partially overlapping expression profiles, mouse mutants for each zic gene show distinct phenotypes, suggesting the functional redundancy of zic proteins. it is expected that the common and specific roles of mouse zic proteins can be clarified by studying compound mutant mice. in the present study, we characterized zic /zic compound mutant mice. mice carrying homozygous zic mutant allele together with zic null allele showed defects in midline structures, including abnormalities in forebrain and thalamus. especially, the compound mutants showed severe anatomical abnormalities in the dorsal and ventral telencephalon and olfactory system, which are not obvious in either zic -or zic -single mutant. these observations indicate that zic , in cooperation with zic , have an essential role in controlling proliferation and differentiation of the neuronal projenitors in the medial telencephalon. chiaki maruyama, haruo okado department of molecular physiology, tokyo metropolitan institute for neuroscience, japan rp , a novel zinc finger protein containing a poz domain, functions as a sequence specific transcriptional repressor. rp gene disrupted mice show severe abnormalities in brain cortical layer formation, suggesting that rp has a crucial role in cerebral development. to understand the role of this protein in brain development, we examined rp gene expression in mouse embryo and adult brain by in situ hybridization. as a result, we found that rp transcripts are first detected at embryonic day in the neuroepithelium of the spinal cord and telencephalic vesicle. in the day - embryos, rp transcripts are predominantly observed in the preplate region but not in outside the nervous system. at e , rp transcripts were detected throughout the neocortex and hippocampus, but not in the thalamus and striatum. in the cortex, the transcripts were detected primarily in cortical neurons, but not in the marginal zones and ventricular zone. in adult mice, rp is expressed in neocortical and hippocampal neurons and granule cells in the cerebellar cortex toshiki kameyama, fumio matsushita, yuzo kadokawa, tohru marunouchi division of cell biology, fujita health university, toyoake, japan neural zinc finger (nzf) proteins are transcription factors with dnabinding domains of c hc-type zinc finger motifs. using p cells, we demonstrated that nzfs were expressed transiently during neuronal differentiation, and forced expression of nzf cdnas resulted in neuronal differentiation. these results suggest that nzf family have a function regulate neuronal differentiation. to elucidate in vivo functions of nzf family in detail, we generate knockout mice of nzf- and nzf- respectively. nzf- null mice are born alive, but die within min after birth with cyanosis. on the other hand, nzf- null mice are viable, fertile and appear normal. these mice look normal morphologically. then we generate double knockout mice of nzf- and nzf- by intercrossing. double knockout mice have a forelimb posture abnormalities like arthrogryposis multiplex congenita. and we find out that the spinal nerves projecting forelimb and trunk are decrease dramatically in the double knockout mice embryo. , ) . in the present study, to examine the role of runx in the development of drg in more detail, we examined the development of drg neurons in runx -deficient mice from the early embryonic stages to birth, using various markers for subpopulation of drg neurons. in newborn runx −/− mice, parvalbumin-positive drg neurons were greatly reduced in number, whereas calretinin-positive neurons were slightly decreased. similar decreases were observed in embryonic days . and . . shin hisahara , , susumu chiba , hiroyuki matsumoto , yoshiyuki horio department of pharmacology, sapporo medical university, sapporo, japan; department of neurology, sapporo medical university, sapporo, japan in mammalian cns, the function of histone deacetylase sirt is still unclear. recent studies indicated that sirt interacts with nuclear receptor co-repressor (n-cor) and n-cor represses intracellular domain of notch-icd activation of the hes promoter. we performed overexpression of sirt and n-cor in neurosphere by nucleofection, then induced differentiation. we found remarkable promotion for neural differentiation by overexpression of sirt and n-cor in the sirt with n-cor. sirt and n-cor suppressed hes transcription by notch-icd in the luciferase assay. hes transcription was suppressed in overexpression of sirt and n-cor, suggesting that interaction between sirt and n-cor represses hes transcription. consistent with this, chip assays revealed that not only n-cor but also sirt bind to the promoter of hes gene. taken together, these results indicate that sirt and n-cor accelerate neural differentiation of the undifferentiated cells via binding hes promoter site and repressing hes transcription. yasushi maruyama , mitsuhiko kurusu , masataka okabe , katsuo furukubo-tokunaga grad. school life and envir. sci., univ. tsukuba, japan; natl. inst. genetics, mishima, japan; inst. dna medicine, jikei univ. school of medicine, japan during brain development, a large number of neurons are generated by proliferation of neural stem cells. with a characteristic proliferation mode that persists through development, the neuroblasts of drosophila mushroom bodies (mb) provide an attractive model system to study mechanisms of neural stem cell proliferation. here we show that tailless (tll), a member of the orphan nuclear receptor super family, has a crucial function in maintaining cell cycle progression of the mb neuroblasts. mosaic analysis demonstrates that cell autonomous activity of tll is crucial for maintenance of the mb neuroblast cell cycles. moreover, gain-of-function analyses confirm instructive functions of tll in maintaining neuroblast activity. we propose that tll plays pivotal roles in proliferation of the mb neuroblasts and suggest a conserved mechanism of neural stem cell control with the tll/tlx homologs in both drosophila and vertebrate brains. kouji senzaki, masaaki yoshikawa, shigeru ozaki, takashi shiga graduate school of comprehensive human science, university of tsukuba, ibaraki, japan runx family transcription factor is an important component of tgf-␤ and bmp signaling. we reported previously that runx mrna is expressed in the dorsal root ganglion (drg) from the early developmental stages, and that runx regulates axonal projection of trkcexpressing proprioceptive drg neurons (inoue et al., ) . furthermore, we announced previously that runx mrna is expressed in cranial ganglia of v, vii, viii, ix and x in mouse developmental stages. the expression was restricted to subset of neurons in each ganglion. to examine the influence of runx on the differentiation of trigeminal ganglion neurons, we investigated the expression of neurotrophin receptors, calcium binding proteins and neuropeptides in trigeminal ganglia of runx knockout mice using immunohisitochemical staining. we found the decrease of trkc-expressing neurons in trigeminal ganglia of neonatal runx knockout mice, however, we observe little change in the proportions of nuen-expressing neurons. kouko tatsumi , hirohide takebayashi , takayuki manabe , kazuhiro ikenaka , akio wanaka dept. anatomy, nara med. univ., kashihara, nara, japan; division of neurobiology and bioinformatics, nips, nins, okazaki, aichi, japan our previous study with double labeling of brdu and cell lineage markers suggested that a number of astrocytes were differentiated from resident oligodendrocyte progenitor (opcs)-like cells in the injured adult brain. and we found out that these opcs expressed ng proteoglycan and olig at early phase after injury. to directly trace the lineages of these opcs, we employed double transgenic mice that express tamoxifen-sensitive creer under the control of the olig promoter together with rosa-egfp reporter. the gfp positive cells were detected around the injured region, and the almost all of these cells co-expressed gfap at late phase after injury. furthermore, we confirmed that the morphological characteristics of these cells were those of the astrocyte by immunoelectron microscopy. our results clarified that dormant opcs in vivo differentiate into astrocytes in adult injured brain, and suggested that these cells participate in glia scar formation after brain injury. olig is a bhlh transcription factor, essential for oligodendrocytes (ols) and motoneurons differentiation in the spinal cord. however, differentiation of olig lineage cells in the forebrain is largely unknown. here we examined fates of olig expressing cells in the fetal forebrain by tamoxifen (tm)-inducible cre-loxp system. olig -creer knockin mice were mated with reporter mice, and tm was injected at embryonic day (e) . or . , when most of olig + cells are observed in the basal forebrain. the olig + cells at e . gave rise to more neuron than glia that included both ols and astrocytes. majority of neuronal olig lineage cells differentiated into gabaergic neurons, and a lesser number, into cholinergic neurons. the olig + cells at e . generated more glial cells than neurons. these results indicate that olig lineage cells generate three major types of neural cells with a stage dependent manner, and may have multiple functional roles on neural differentiation in the fetal forebrain. mana igarashi , , masato yano , , satoru hayashi , , hirotaka j. okano , , hideyuki okano , dept. physiol., keio univ. sch. med, tokyo, japan; sorst jst, japan the mammalian neuronal hu rna binding protein family is homolog of drosophila elav protein which is essential for differentiation and maintenance of the nervous system. in mammals, neuronal hu expresses in both early postmitotic and mature neurons and has ability to induce neuronal differentiation by binding to the utrs of specific target mrnas. to understand the molecular mechanism of hu induced neuronal differentiation, we purified hub associated complexes. among them, nf family, a double strand rna binding protein which is one of hu associated proteins, is known to bind to utrs of p , p and tau mrna known as hu targets. we generated rabbit polyclonal antibodies against nf and nf , binding partner of nf , respectively. in mouse embryonic brains, we found that nf / expressed highly in postmitotic neurons where neuronal hu proteins are highly distributed. moreover, we found that hu and nf / formed mrnp complexes in mouse brain extracts. we will discuss the role of hu binding partners in neuronal differentiation through post-transcriptional regulation. sachiko the pallium is specified as a homologous field in the vertebrate telencephalon. however, little is known about how species-specific pallial structures are generated during embryogenesis. to address this issue, we compared several neuronal subtypes and their migration patterns in the developing pallium of the mouse and quail. cell tracing analysis revealed that neurons born at the dorsal pallium tangentially migrated in the developing quail telencephalon, as in the mammalian cortex. next we investigated distribution of later-born neurons in the quail telencephalon using laminar specific genes (er and brn ) in the cerebral cortex. in situ hybridization and immunohisitochemical studies indicated that these neuronal markers were expressed in discrete regions of the developing quail telencephalon. our data suggest that early stages of cortex/pallium development are comparable between the mammalian and avian embryos, whereas neuronal specification in later stages is regulated by distinct mechanisms in each species. research funds: kakenhi ( ) ps a-d protein expression in hippocampal cells dissociated and re-cultured from organotypic slice cultures we established a re-cultivation technique of hippocampal cells dissociated from long-term cultured organotypic slices. protein phenotype of the cells was analyzed using immunocytochemical technique. antinestin immunoreactivity was observed in cells with short processes days in the re-cultivation. the anti-nestin immunoreactivity was progressively declined, whereas number of cells expressing anti-␤iii tubulin immunoreactivity increased through the re-cultivation for - weeks. presence of neurons, astrocytes and oligodenderocytes was examined using anti-␤iii tubulin, anti-glial acidic fibrillary protein and rip antibody, respectively. apart from the cells expressing one of the markers, the cells marked with multiple sets of antibodies were observed. these results suggest that protein expression was changed backward in normal differentiation course in hippocampal cells once matured in organotypic slices. we have shown that perineuronal ng + cells are major populations of proliferating cells in the cerebral cortex of rats. in the adult cortex, ng is known as a marker for oligodendrocyte progenitor cells (opcs) that retain ability to proliferate and differentiate into new oligodendrocytes. however, it is still unclear whether all ng + cells in the neocortex are the opcs. we investigated about subtypes of ng + cells found in the perineuronal regions of the cerebral cortex using cell markers. two subtypes of perineuronal ng + cells could be distinguished by the subcellular localization of gst-protein. one is nuclear type, the other is cytoplasmic type. only the nuclear gst-+ cells have the proliferative activity. these data suggest that the nuclear gst-+ /ng + cells in the perineuronal territory are progenitor cells engaging in reproduction of cortical cells. muguruma keiko, su hong-lin, matsuo-takasaki mami, watanabe kiichi, sasai yoshiki neurogenesis and organogenesis group, riken center for developmental biology, kobe, japan in this study, we report in vitro generation of math + cerebellar granule cell precursors and purkinje cells from es cells by using soluble patterning signals. when neural progenitors induced from es cells in a serum-free suspension culture are subsequently treated with bmp and wnt a, a significant proportion of these neural cells become math + . the induced math + cells mitotically active and express markers characteristic of granule cells precursors (pax , zic , and zipro ). after purification by facs and coculture with postnatal cerebellar neurons, es cell-derived math + cells exhibit typical features of neurons of the external granule cells layer, including extensive motility and a t-shaped morphology. interestingly, differentiation of l + /calbindin-d k + neurons (characteristic of purkinje cells) is induced under similar culture conditions but exhibits a higher degree of enhancement by fgf rather than by wnt a. this is the first report of in vitro recapitulation of cerebellar neurons by using the es cell system. sachiyo misumi, kim hye-jung, hideki hida, hitoo nishino department of neurophysiology and brain science, nagoya city university graduate school of medical science, nagoya, japan regulation of the cell cycle plays an important role in cell proliferation, differentiation, and apoptosis. we have shown that pretreatment with cell cycle blocker increase the number of neurons from neural stem or progenitor cells (npcs) without influencing apoptosis after differentiation. in this study, we investigate the molecular mechanism of neuronal differentiation by cell cycle arrest. in rt-pcr, the expression of p cip , p kip and p kip mrnas were elevated during differentiation to neuron from npcs. especially, prolonged enhancement of p kip mrna was shown. transfection of p kip into npcs induced activation of neurod promoter and increase of number of ␤tublin iii-positive cells. treatment with deferoxamine to npcs from e . rat midbrain and hb .f cell line did not activate erk and akt phosphorylation during the treatment. date suggest that prolonged p kip elevation is related to enhanced production of neuron from npcs, and that cell cycle regulation in g /s phase did not activate mapk and pi -k signaling. yuichi tanaka , yusuke tozuka , dai muramatsu , kin-ichi nakashima , tatsuhiro hisatsune departement of integrated biosciences, graduate school of frontier sciences, university of tokyo, kashiwa, japan; graduate school of biological sciences, nara institute of science and technology, ikoma, japan we previously reported no definite evidence for in vivo neurogenesis in adult neocortex. however, we also confirmed dividing cells in this area. in this study, we analyzed the characteristics of adult cortical nestin+ cells. in vivo, they belonged to ng + and olig + cells, showed slowly proliferating ability compared to those in adult dentate gyrus. for in vitro analysis, we precisely isolated progenitor cells by percoll gradient procedure. they differentiated into tuj- + or map- + neuronal cells by adding retinoic acid or bdnf. more than % of newborn neurons expressed gabaergic neuronal markers, gaba, gad or calretinin. we also purified nestin-gfp+ cells from nestin-gfp transgenic mice using the facs system, and confirmed their neuronal potential. moreover, integration of a neural bhlh transcription factor neurod significantly promoted this neurogenesis. we demonstrated neurogenic potential of adult cortical nestin+ cells. mie gangi , michiko imanishi , teiko kuroda , masao tachibana , masahiko takada department of psychology, graduate school of humanities & sociology, university of tokyo, tokyo, japan; tokyo metropolitan institute for neuroscience, tokyo metropolitan organization for medical research, tokyo, japan a kv subfamily of voltage-gated k + channels is thought to play an important role in high-frequency repetitive firings. it is unknown which subtype of kv channels is expressed in the frog retina where ␥-range oscillatory spikes are evoked presynaptically by light stimulation. we found immunohistochemically that kv . b and kv . were expressed both in the mouse and frog retinas. however, a laminar pattern with two bands in the inner plexiform layer was displayed by kv . in the frog retina and by kv . b in the mouse retina. it has been shown that mammalian cholinergic amacrine cells express kv . b. thus, the differential expression of kv channels may reflect their functional diversity between the frog and mouse retinas. hiroshi jouhou , , kazunori yamamoto , masayuki hara , akinori homma , akimichi kaneko , masahiro yamada tokyo metropolitan univ., hino, tokyo, japan; astellas pharma. inc., osaka, japan; sch. rehabili., seijoh univ., aichi, japan in order to interpret the formation of receptive field surrounds in the retinal neurons, hirasawa and kaneko ( ) proposed a phmediated mechanism to substitute for the gaba-mediated feedback hypothesis from horizontal cells (hcs) to cone photoreceptors. to verify the idea that the depolarized hcs release protons we measured, by a fluorescent ratio imaging technique, the ph of the immediate external surface (ph o ) of hcs isolated from carp or goldfish retina. when hcs stained by -hexadecanoylaminofluorescein, a phsensitive lipophilic dye, were depolarized by application of kainate or by high extracellular k + , ph o acidified. the amount of ph o acidification was monotonically dependent on the amount of depolarization, as much as . ± . ph unit by mm k + . acidification of pho was suppressed by . m bafilomycin a , a specific inhibitor of v-atpase, suggesting that the hc depolarization enhanced an outward proton movement by the outward electrogenic h + pump. ps a-e analysis of spread of activity in the local circuit of superior colliculus by using multi-channel field potential recording system penphimon phongphanphanee, katsuyuki kaneda, tadashi isa national institute for physiological sciences, japan to study how the visual signal is processed in the local circuit of superior colliculus (sc) from the superficial layers (ssc) to the deeper layers (dsc), we analyzed the propagation of excitation following the electrical stimulation of the ssc by using a planar -channel field potential recording system in slice preparations obtained from to days old mice. stimulation at ssc induced negative field potential with short latency and short duration ( - ms) at the recording site in ssc adjacent to the stimulating site. after application of bath containing m bicuculline, the same stimulus induced a large negative field response with long duration ( - ms) that spreads laterally in ssc and ventrally to dsc. these responses disappeared after application of m apv, when only short latency response remained. the results suggest that when gaba a receptormediated inhibition is reduced, visual signal in the ssc propagates to the dsc as large response with long duration and nmda receptors contribute to propagation of the response. osamu hosoya , ken tsutsui , kimiko tsutsui dept. of neurobiol. and neuroanat., okayama univ. grad. sch. of med., dent., and pharm. sci., japan; dept. of genomics and proteomics, okayama univ. grad. sch. of med., dent., and pharm. sci., japan amphiphysin ir (amph ir) is alternatively spliced variants of amphiphysin i which is specifically expressed in retina. amph ir is composed of conserved domains including the n-terminal bar, the central clathrin/ap- binding, and the c-terminal sh domains and the variant specific two novel insertions (a and b). insert a may be a determinant for the retina-specific expression. insert b has no significant homology to known proteins and two shorter transcripts with -truncations in the insert were also expressed. recently, we found that a human retinal pigment epithelia cell line, arpe- , also expresses amph ir. arpe- thus can be a useful tool for investigating the cellular function of amph ir in retina. immunofluorescence analyses with arpe- cells revealed that amph ir occasionally colocalized with mitochondria, raising the possibility that amph ir may participate in structural or functional organization of mitochondria. further characterization of the variant is under investigation. hironori takamura , satoshi ichisaka , chihiro hayashi , hirotoshi maki , yoshio hata div. integrative biosci., tottori univ. grad. sch. med. sci., yonago, japan; div. neurobiol., sch. life sci., fac. med., tottori univ., yonago, japan monocular deprivation (md) induces significant plasticity in the primary visual cortex (v ) during critical period. it was reported that inhibition of extracellular signal-regulated kinase (erk) activity in the visual cortex suppressed the ocular dominance plasticity. if erk is involved in the mechanism of this plasticity, visual deprivation would change the activity of erk in v and such change might be induced only in the critical period. to test this possibility, we examined effects of md on the amount of phosphorylated (activated) erk (perk) in the rat visual cortex. by md, we found a significant decrease in the amount of perk in v receiving deprived eye inputs in both young and adult rats. as to the subcellular localization of erk, we found a significant increase of the nuclear perk only after md in young rats. these results suggest that erk signaling might be regulated by different mechanism between young and adult rats. research funds: kakenhi ( ) ps a-e rapid pre-synaptic weakening by experiencedependent competition in mouse visual cortex nobuko mataga, yoko mizuguchi, takao hensch neuronal circuit development, riken brain science institute, saitama, japan in the binocular zone (bz) of mouse visual cortex, critical period (cp) plasticity is accompanied by a transient loss of spines on pyramidal cell dendrites. to explore a correspondingly rapid and local pre-synaptic refinement by sensory deprivation, excitatory intracortical or thalamocortical axon terminals were visualized in the bz by vesicular glutamate transporters (vglut) and vglut , respectively. a complementary distribution of vglut and vglut was established by postnatal day (p) and both signal intensities increased further by p - (peak cp). the immunoreactivity for vglut decreased around layer iv after brief monocular deprivation ( dmd) during the cp. interestingly, both signals in all layers were lower in the bz contralateral to an eye injected with ttx than in the ipsilateral bz, consistent with the stronger functional plasticity and rapid dendritic refinement as compared to dmd. these results suggest that rapid and local weakening of excitatory inputs corresponds to dendritic spine pruning during experience-dependent competition. reiko meguro, masao norita department of sensory and integrative medicine, niigata university, graduate school of medical and dental sciences, niigata, japan we investigated how the geniculate and the extra-geniculate visual systems reorganize by monocular deprivation at birth. using anterograde/retrograde tracer, biotinylated dextran amine (bda), we made a small injection into the dorsal lateral geniculate nucleus (dlgn) or the lateral posterior nucleus (lp) of the degenerated side of the monocular deprived rat. the geniculate projection terminated mainly in layer iv of area , with a small projection to layer vi of areas and a. cells in layer vi of area projected to dlgn. in addition, cells in layer v of area projected to dlgn, which is not observed in normal rats. in area a, cells in layers v and vi projected to dlgn. the projection from lp terminated mainly in layer iv of a. cells in layers v and vi of area a projected to lp. smaller number of cells in layer v of area also projected to lp. these findings suggest that major parts of visual system developed normally, but some developed cross talk between geniculate and extra-geniculate systems. ps a-e activity dependent plasticity of feedback projection from the primary visual cortex to the dorsal lateral geniculate nucleus miho yoshida , takemasa satoh , yoshio hata div. integrative biosci., tottori univ. grad. sch. med. sci., yonago, japan; div. neurobiol., sch. life sci., fac. med., tottori univ., yonago, japan the projection from the lateral geniculate nucleus (lgn) to the primary visual cortex (v ) shows significant morphological plasticity responding to visual experiences in early life. such experiencedependent plasticity enables the geniculocortical projection to form functionally specific connections. it is not clear whether similar plasticity operates in other neural connections in the visual system. therefore, we tried to investigate the plasticity of feedback projection from v to the lgn. we focused on the density of type metabotropic glutamate receptor ␣ (mglur ␣) in the lgn which locate postsynaptically at synapses of feedback projection. immunohistochemical signal for mglur ␣ in the lgn decreased after elimination of v , showing that this signal reflects density of functional feedback synapses. to explore the activity dependent plasticity, we examined the effect of cortical activity blockade on the mglur ␣ signal in the lgn of young rats. yu morishima , hiroshi sakamoto , takahumi akasaki , yoshio hata div. integrative biosci., tottori univ. grad. sch. med. sci., japan; div. neurobiol., sch. life sci., fac. med., tottori univ., japan monocular deprivation (md) during postnatal development reduces cortical response to the deprived eye and input axons serving the deprived eye retract. however, when md is combined with continuous inactivation of the visual cortex by muscimol, cortical neurons lose their response to the open eye and the open eye axons retract. to clarify mechanisms underlying the two forms of ocular dominance (od) plasticity in different direction, we examined other characteristics of them, ( ) how rapidly the reverse od shift proceeds and ( ) whether the shift is induced only in young animals. we infused the cortex with muscimol in -week-old kittens and in adults. the reverse od shift was observed after days md, but not significant after days md. in adults, od distribution remained unchanged. morphological change of individual input axons was also examined after days md. the reverse od shift might reflect a mechanism of developmental plasticity that has a slower time course than the normal od shift. ps a-e experience-dependent plasticity in the absence of ampa receptor subunits in mouse visual cortex youichi iwai , nafiseh atapour , john renger , john roder , peter seeburg , takao hensch neuronal circuit dev., riken, bsi, wako, japan; mount sinai hospital, toronto, canada; max planck inst. med. res., heidelberg, germany two ampa receptor subunits (glur , ) play prominent roles in hippocampal models of homosynaptic plasticity (ltp/ltd). brief monocular deprivation ( d md) rapidly alters both the phosphorylation state and surface expression of glur in visual cortex. here, we addressed whether these coincidental events are essential for subsequent ocular dominance (od) plasticity. mice lacking glur (ko) displayed little ltd in visual cortical slices, while baseline transmission was normal. they also exhibited normal visual receptive field properties in vivo and shifted responsiveness toward the open eye after d md during a typical critical period. the rate of plasticity appeared somewhat slowed, as d md eventually led to full od shifts. in glur ko mice, even d md robustly activated od plasticity. thus, experience-dependent modification of ampa receptors is not essential for plasticity in vivo, although glur may contribute to the very earliest stages. shigeyoshi higo, nobuaki tamamaki department of morphological neural science, kumamoto university, kumamoto, japan virus-assisted transduction with reporter genes is a useful technique to investigate morphology of neurons in the central nervous system. however, the mechanisms to induce reporter expression in vivo often depend on gene-manipulated mice. since mice are not the best experimental animal for the study of mental disorder, we developed an adenovirus in which gfp expression is driven by dlx promotor and dlx / enhancer. this virus labels gabaergic neurons and oligodendrocyte in the wild-type mouse neocortex and allows us to trace gfp-labeled axons of gabaergic neurons in serial brain sections. we used this virus to investigate gabaergic neurons with long projecting axon branches beyond a functional area. the virus was injected into the stratum oriens of the mouse hippocampal field ca and revealed a nonpyramidal neuron projecting to ca and fimbria. further we shall introduce this virus to the cat brain and investigate axon branches of gabaergic projection neurons in the neocortex. akiko yamashita , takao oishi , motoharu hayashi div. appl. system neurosci., nihon univ. grad. sch. med. sci., tokyo, japan; dept. cell. and mol. biol., primate res. inst., kyoto univ., inuyama, japan gabaergic cells in the cerebral cortex are divided into subgroups: parvalbumin (pv)-, somatostatin (som)-, calretinin (cr)-, and calbindin-containing types. to clarify inhibitory system in primates, we determined coexistence of these molecules and proportions of these subtypes within gabaergic cells in the various cortical areas. pv, som or cr did not coexist with each other in primates as observed in rodents. more than % of gabaergic cells contained pv; showing that pv cells are more abundant in primates than in rodents. proportion of som cells in gabaergic cells was smaller in the primary visual area ( . %) than in other areas, such as the prefrontal ( . %), primary motor ( . %), somatosensory ( . %) and secondary visual areas ( . %), indicating cortical differentiation in gabaergic system of the primate cerebrum. our recent retrograde labeling studies in mice and cats showed that the neocortical areas are connected not only by excitatory neurons but also by gabaergic projection neurons. in order to address the importance of the gabaergic projection neurons in the neocortical information processing, we need to know the branching pattern and postsynaptic elements of the gabaergic projection axons. since more than % of the gabaergic projection neurons showed npy immunoreactivity, we used npy-cre transgenic mouse that express cre in npy neurons and adenovirus that encodes gfp in the downstream of floxed stop to label the gabeergic projection axons. after injection of the adenovirus into deep layers of the npy-cre mouse neocortex and immunoperoxidase staining of gfp in the brain section, we could reveal gabaergic neurons in a golgi-like image with their axons. also this method seemed to allow us to label gabaergic projection neurons retrogradely. koji ikezoe , guy n. elston , , tomofumi oga , hiroshi tamura , , ichiro fujita , osaka univ., japan; univ. queensland, australia; crest, jst, japan layer iii pyramidal cells in adult monkeys exhibit systematic differences in their dendritic morphology among cortical areas. basal dendrites of cells in visual association cortex such as inferior temporal area te spread more extensively and are more branched than those in the primary visual cortex (v ). pyramidal cells in prefrontal cortex, such as area , have even more dendritic branches than those in area te. here, we investigated whether a similar regional difference in the dendritic morphology was present in infant monkeys. we stained individual layer iii pyramidal cells in v (n = ), area te (n = ), and area (n = ) of a -week old monkey (macaca fascicularis) using intracellular dye-injection techniques in lightly fixed tissues. the number of branches and the tangential extent of dendrites was greatest in area , followed by area te, and v . thus, considerable heterogeneity in pyramidal cell structure already exists -weeks after birth. hiroaki matsushita , mahito ohkuma , masami watanabe , ei-ichi miyachi department of physiology, fujita health university, aichi, japan; department of perinatology, institute developmental research, aichi, japan acetylcholine (ach) receptors are believed to be expressed in developmental and regenerative process of retinal neurons. we performed the patch-clamp recording and fura- based calcium imaging in cat retinal ganglion cells (rgcs). under whole cell clamp conditions, transient sodium currents and action potentials were observed in all of normal or axonal regenerated rgcs. however, these currents and spikes were not observed in the % of axotomized rgcs. bath application of m carbachol, an ach receptor agonist, rose [ca + ] i in % of normal rgcs. although the % of rgcs responded to carbachol at days after axotomy, no responsive rgcs appeared during - days. ach responsiveness recovered in axonal regenerated rgcs ( %). since pycnotic cells were observed few days after axotomy, ach may modulate neurotrophic effect in survived rgcs. these results suggest that ach is an important marker for neuronal degeneration and regeneration in cat rgcs. research funds: kakenhi ( to em, to mw) kenichiro miura, masakatsu taki, hiromitsu tabata, kenji kawano dept. integrative brain sci., grad. schl. of med., kyoto univ., kyoto, japan the initiation of smooth pursuit eye movements is facilitated by the bottom-up attention to the target (hashimoto et al., ) . to study the effects of the attention on the processing of second-order motion stimuli, we recorded smooth pursuits in three humans with a dualpurkinje-image eye tracker. the pursuit target, presented on a crt monitor ( hz), was a gaussian patch of texture displayed on a neutral gray background. the gaussian envelope moved at deg/s, while the texture consisting of black and white random-noise pixel blocks remained stationary (drift-balanced stimulus). the number of the frames displaying the target before the motion onset was selected to manipulate the attention to the target, either eight frames ( ms) or only one frame ( ms). the initial tracking responses were larger when the target became visible eight frames before the motion onset. the result suggests that the second motion processing underlying the smooth pursuit initiation is facilitated by the attention to the target. ps a-e motion picture effects on eye movements and blood flow in the frontal area atsuhiko iijima , , tohru kiryu , kazuhiko ukai , takeshiko bando div. integrative physiol., grad. sch. med., niigata univ., niigata, japan; div. inform. sci., grad. sch. & tech., niigata univ., niigata, japan; dept. appl. phys., sch. sci. & tech., waseda univ., tokyo, japan motion pictures taken by rider's view of motocross bike elicited horizontal eye movements coherent to the motion vectors in some subjects, and not coherent in the other subjects, while those taken by passenger's view of roller coaster evoked similar eye movements in all of the subjects. subjects watched the two-dimensional motion pictures in random order. eye movements were measured by a binocular video oculography (newopto), and head movements were measured by a magnetic motion sensor (polhemus). blood flow in the frontal area was simultaneously monitored with a near infrared spectroscopy (hamamatsu). the patterns of eye movements and the blood flow variation during movie presentation changed in relation to motion components of the movie. possible mechanisms of the differences will be discussed. kiyoto matsuura , kenichiro miura , masakatsu taki , hiromitsu tabata , naoko inaba , kenji kawano , frederick a. miles grad. schl. med., kyoto univ., kyoto, japan; lab. sensorimotor res., nei, nih, bethesda, md, usa human ofrs show winner-take-all behavior when elicited by moving grating patterns composed of two sinusoids (sheliga et al., sfn ) . we recorded the ofrs to the motion of vertical grating patterns composed of two sinusoids of spatial frequency f and f, which created a repeating pattern with beat frequency, f, in two monkeys. motion consisted of successive steps ( hz), each one-fourth of the wavelength of the beat, so that with each step the two components shifted one-fourth of their wavelengths and had opposite directions, the f forwards and the f backwards. the contrast of the f was fixed at , , or %, while the contrast of the f was varied from one-fourth to four times the contrast of the f. when the contrast of the f ( f) was less than about half that of the f ( f), the f ( f) dominated initial ofr: winner-take-all. thus, the motion processing underlying the ofr in monkeys, like that in humans, includes nonlinear interactions. masazumi katayama, takahiro fujita department of human and artificial intelligence systems, faculty of engineering, university of fukui, fukuki, japan when executing prehension movement to grasp an object such as a tool, we plans the hand shape and grasping position to grasp a target object. while, goodale proposes the hypothesis that the roles of two visual streams (dorsal and ventral streams) are "vision for action" and "vision for perception", respectively. from the above points of view, we investigated independence of visual estimation and motor execution for grasping position of a target object. in this experiment, grasping positions were measured under the following four conditions: visual estimation without grasping, grasping without lift-up movement, grasping and lift-up movement and visual estimation without grasping. as a result, we found that grasping positions of visual estimation are significantly different from grasping positions of motor execution in the second and third conditions (p < . ). we concluded that grasping positions of visual estimation and motor execution are independent and these results support the goodale's hypothesis. ryuichi hishida, masaharu kudoh, katsuei shibuki dept. neurophysiol., brain res. inst. niigata univ., niigata, japan cortical sensory areas are divided into modality-specific domains such as the visual and auditory cortices, in which sensory neurons are driven by modality-specific inputs. recently, wallace et al. found that multimodal neurons clustered in deep layers are present near the borders between sensory cortices. multimodal properties of these neurons may be explained by three types of inputs: overlapped projections from the thalamus, projections from multi-modal sites, or overlapped horizontal projections from the modality-specific sensory cortices. in this study, we tested the third possibility. we prepared the mouse cortical slices including the visual and auditory cortices. the horizontal activity propagation elicited by local electrical stimulation were visualized using flavoprotein fluorescence imaging. these results indicate that cortical areas between the visual and auditory cortices receive horizontal projections originated in the visual and auditory cortices, suggesting that multimodal horizontal connections are important for the multimodal properties of sensory neurons. jumpei naito , yaoxing chen , yukiko tanada dept. animal sci., teikyo univ. sci. & tech., uenohara, japan; china agricul. univ., beijing, china twenty white-leghorn chicks (p - ) were perfused with % paraformaldehyde through the heart under deep anesthesia of nembutal ( mg/kg bw). two to three small crystals of dii were implanted into the optic tectum, thalamus, or hypothalamus under a dissecting microscope. a total of rgcs were classified into six groups according to the somal area and dendritic field (naito and chen, ). group ic projected dominantly to the tectum. group is and iiis showed high hypothalamic-and thalamic-dominance, respectively. group iic was non-specific in the central projections. group ivc was tectal-dominant. patterns of the dendritic stratification were counted to in tec-rgcs, in tha-rgcs, and in hyo-rgcs. of these stratification patterns, many patterns were common among tec-, tha-, and hyp-rgcs. in contrast, the rgcs that showed a same dendritic pattern were consisted of a single rgc in most of the non-common rgcs, and their dendrites extended mainly to the superficial inner plexiform layer (sublayers - ). yasuro atoji, shouichiro saito laboratory of veterinary anatomy, gifu university, gifu, japan the present study was examined afferent and efferent fiber connections of the intermediate part of the caudal nidopallium (nci) in the pigeon by a tract-tracing method. in the present study we define nci an area which is located lateral to the field l complex and ventral to ncl. following a ctb injection into nci, a large number of neurons was labeled in nci, the mesopallium, and intermediate arcopallium (ai) and in the thalamic posterior dorsointermediate and posterior dorsolateral nuclei. contralateral ai contained a small number of labeled neurons. a few labeled neurons were found in lst. few labeled cells were found in ncl, field l, piriform cortex, or hippocampal formation. following a bda injection into nci, a large number of labeled fibers extended in nci, mesopallium, and ai. lst contained a small number of labeled fibers. few labeled fibers were located in ncv and limbic regions. the diencephalon contained very few labeled fibers. in summary, nci has strong reciprocal connections within nci itself and with the mesopallium and ai, and little connections with the limbic system. hidenori horie , kenji yuda , eiichi okawa , katsuyoshi maruyama , hiroshi uozato , hiroko horie , satomi nakajima , kenkichi tanioka , yuji ohkawa , tomoki matsubara , wolfram tetzlaff advanced res. centr. biological sci., waseda univ., tokyo, japan; technomaster co. ltd., yokohama, japan; kikuna yuda eye clinic, yokohama, japan; healthcare business co., matsushita electric ind. co. ltd., yokohama, japan; dept. ophthalmol. & visual sci., kitasato univ., kanagawa, japan; nhk sci. technical res. labo., tokyo, japan; icord, univ. british columbia, vancouver, canada we describe here a highly effective method to improve visual acuity of children with myopia and adult with presbyopia by repeatedly offering a visual object at variable distances while keeping the apparent retinal projection size of the object constant using a novel electronic device. in our experiments on human subjects, we used an lcd screen that was rhythmically moved between and cm toward and away in a high speed (top speed: mm/s) from the subjects. the device significantly improved visual performances in over % of the school-aged children with myopia and % of adults with presbyopia. hiroyuki miyamoto , toral s. surti , takao k hensch laboratory for neuronal circuit development, riken brain science institute, wako, japan; san francisco, usa competitive plasticity of binocular response following monocular deprivation (md) is prominent in the primary visual cortex (v ) during an early critical period. recently, md has been shown to enhance head-tracking behavior induced by slow rotation of grating stimuli in adult mice and is critically dependent upon the integrity of v . here, we addressed to what extent these two types of plasticity induced by the same md share common mechanisms. adult mice lacking a gaba-synthetic enzyme (gad ko), which do not exhibit ocular dominance (od) plasticity by brief md during the critical period, showed normal optomotor acuity and enhancement with day md. od shifts did not correlate with optomotor enhancement in these mice. finally, early md spanning the entire critical period had no effect on optomotor acuity through the deprived-eye. these observations support the view that adult perceptual learning and classical od plasticity are independent. junya hirokawa , miquel bosch , shuzo sakata , yoshio sakurai , tetsuo yamamori division of brain biology, national institute for basic biology, okazaki, japan; mit, ma, usa; rutgers university, nj, usa; department of psychology, kyoto university, japan the brain is able to integrate information from different sensory sources to enhance behavioral responses. to identify the neuronal populations responsible for multisensory enhancement in rats, we have mapped the activation of neurons during an audiovisual integration paradigm (sakata, et al., ) by the expression of c-fos. a pronounced c-fos upregulation was found in superior colliculus and in layer iv and deep layer of latero-medial secondary visual area (v lm). local injection of gaba agonist muscimol into this region selectively suppressed the behavioral enhancement related to multisensory integration, while no suppression was found by the injection into primary auditory and visual areas. these results suggest a key role of v lm in integration of auditory and visual information to facilitate the behavioral reaction for bimodal stimuli. takashi shinozaki , youichi miyawaki , tsunehiro takeda department of complexity science and engineering, university of tokyo, chiba, japan; mathematical neuroscience, riken bsi, saitama, japan drifting grating patterns with different motion directions independently presented to the two eyes induce two sets of perceptual rivalries: interocular rivalry (left or right eye's image) and motiontype rivalry (pattern or component motion). we studied this double rivalry process based on psychophysical and magnetoencephalography (meg) measurements. pattern-motion percept exclusively arose and persisted for a long duration whereas component-motion percept was soon followed by percept of either of left or right eye's single motion direction. reaction time (rt) measurement showed that the pattern-motion was perceived faster than left or right eye's motion direction. we then compared meg signals among those perceptual conditions and found a meg response of interocular rivalry in the latency range expected from the result of rt measurement. these results suggest that the double rivalry process has a hierarchical structure in which motion-type rivalry is resolved before interocular rivalry. visual stimuli evoke several brain potentials with relatively precise time courses. the role of these brain potentials in visual object categorization is not clear. in this study we recorded event related brain potentials (erp) while subjects participated in a face/non-face categorization task. gray face and non-face natural object images were presented briefly ( ms) followed by a noise mask with pseudo randomly selected stimulus onset asynchrony (soa = - ms). subjects reported presentation of face or non-face images by pushing one of the two assigned keys. we found that the face category discrimination performance significantly declined only in short soa ( and ms) with a larger impact of masking on non-face discrimination. in erp, the peak amplitude and latency of p , n and area under curve of a late positive potential expanding from to ms were correlated with the subjects behavioral performance. the effect of backward masking on early erp components may be due to altering sensory processing of visual stimuli while the effect on late erp potential could be related to its impact on decision making processes. yasushi naruse , ayumu matani , , tomoe hayakawa , , norio fujimaki university of tokyo, kashiwa, japan; nict, kobe, japan; teikyo university, tokyo, japan to study the process of alpha rhythm resetting, we investigated the relationship between visual evoked potential and the seamlessness: how much the phase angle of prestimulus alpha rhythm and the backward-extrapolated phase angle from poststimulus alpha ringing synchronize. alpha ringing is an evoked potential in alpha frequency band around ms in latency. eight clinically normal adult volunteers participated in the experiment, in which the subjects passively viewed a series of flash stimuli with their eyelids closed throughout the experiment. eeg was simultaneously recorded during the experiment. we classified the trials into four subsets owing to the seamlessness, and then averaged the trials in each subset. the result showed that the larger the amount of the alpha rhythm resetting is, the larger the p amplitude becomes. this suggested that a factor of the variability of the p amplitude is the amount of the prestimulus alpha rhythm resetting. research funds: a grant-in-aid from the ministry of education, culture, sports, science and technology (no. ) hitoshi sasaki , takuya ishida , masayoshi todorokihara , junichiro miyachi , tahei kitamura , ryozo aoki dept. physiol. & biosignal., osaka univ. grad. sch. med., suita, japan; dept. phys. & elec., osaka pref. univ. grad. sch. eng., sakai, japan; dept. elec. eng. & elec., col. industri. tech., amagasaki, japan recently it has been shown that noise can improve detection of sensory stimuli in several modalities. here we investigated whether visual contrast detection sensitivity can be improved by adding a certain amount of noise. contrast detection thresholds of a light changing brightness periodically were measured with or without overlapping noise in five normal participants. the contrast detection threshold, measured by using the psychophysical method (up-anddown method), decreased at around the threshold level of the noise intensity. these findings are consistent with our previous findings obtained by using another psychophysical method and confirm that noise can improve signal detection in human visual perception. narumi katsuyama , nobuo usui , izuru nose , , masato taira , department of applied system neuroscience, nihon university school of medical science, tokyo; faculty of human science, bunkyo university, saitama, japan; arish, nihon university, tokyo, japan when an object is moving, perception of its d trajectory in depth can be strongly influenced by the trajectory of its cast shadow. for example, a ball moving in a diagonal trajectory can appear to rise in a frontal plane when the shadow moves along the horizontal trajectory (rising configuration) or to roll in depth when the shadow follows the same trajectory as the ball, while the trajectory of ball is identical. using fmri, we found that several visual areas, including human mt and the posterior sts and the posterior parietal cortex, are activated in the comparison between rising configuration and ball only condition. additional correlation analysis by modifying the slope of the shadow' s trajectory also showed activation in the posterior part of sts and the posterior parietal cortex, including precuneus. these results suggest that cortical areas in the temporal and parietal cortex might be involved in the processing of apparent motion of ball induced by the moving cast shadow. ps a-f local area network in the gerbil's auditory cortex: reversible focal inactivation with infrared laser irradiation akira yamamoto, hiroshi riquimaroux gratuate school of engineering, doshisha university, scnrl, japan this study investigated local area networks in the primary auditory cortex (a ) and the anterior auditory field (aaf) by blocking neural activities with the near-infrared laser irradiation (wave length = nm). in previous in vivo studies, the laser irradiation could focally inactivate neural activities in a few minutes after the irradiation started, while the activities recovered in a few minutes after its cessation. by using this technique, the present study examined corticocotical relationships in the auditory cortex of the mongolian gerbils (meriones unguiculatus). cf (constant frequency) and fm (frequency modulated) tones were presented to anesthetized animals, and neural responses were extracellularly recorded contralaterally to the ear of stimulation. when irradiated aaf area and recorded neural responses from ai, the irradiation changed phasic responses into tonic responses, and vice versa. these results indicate that there are functional connections within ai or aaf, and between ai and aaf. takashi doi, hiroshi riquimaroux department of knowledge and engineering and computer sciences, doshisha university, kyoto, japan in a previous behavioral study, ablation of right auditory cortex (ac) made the discrimination between ascending and descending frequency modulated (fm) tones by mongolian gerbil (meriones unguiculatus) difficult (wetzel et al., ) . this result indicates that some neurons in gerbil's right ac represent the directions of fm sweeps. actually, we could find direction-dependent neurons and these neurons were mainly in anterior auditory field (aaf). in aaf, bfs are gradually shifted along the rostrocaudal direction, and the same bfs are arranged in dorsoventral direction (thomas et al., ) . moreover, aaf has dense synaptic connections within the area (budinger et al., ) . we made network models based on this structure of aaf and could gain similar responses to the actual responses of directiondependent neurons. this result suggests that aaf in gerbil's ac has good structure to process fm tones. research funds: a grant to rcast at doshisha univ. from mext, innovative cluster creation project by mext it has been demonstrated that the auditory space, namely the direction of a sound source, is represented topographically in the mammalian superior colliculus (sc). however, it is unclear as to how this auditory space map of the mammalian sc is formed in the auditory pathway. the present study investigated the topographical representation of auditory space in the external nucleus of the inferior colliculus (icx) of anesthetized gerbils. the icx is the major auditory nucleus that has projections to the sc. the stimuli were -ms noise bursts whose azimuths varied on the horizontal plane in the virtual acoustic space. single-unit responses were recorded from the icx. the majority of units exhibited some degree of spatial selectivity and preference for the azimuth contralateral to the recorded side. for supra-threshold stimulus only, there were topographical gradients of preferred azimuths in the icx. however, the spatial tuning width and preferred azimuth of the units depended markedly on stimulus level. the results indicate that in mammals, the formation of a rigorous auditory space map is incomplete at the icx level. manabu toyoshima , yasuo takeda , yasushi shimoda , kazutada watanabe department of bioengineering, nagaoka university of technology, nagaoka, japan; department of clinical pharmacy and pharmacology, kagoshima university, kagoshima, japan nb- that we isolated and identified is a neural cell recognition molecule belonging to contactin subgroup. we reported previously that nb- expression is prominent in the auditory system. nb- knockout mice exhibit impaired neural function in the auditory system. these findings indicate that nb- is indispensable for the function of auditory system. here we report the detailed analysis of the nb- localization using anti-nb- monoclonal antibody that we produced recently. immunohistochemical analysis of the rat brain showed that nb- was detected not only in all brain regions of the auditory pathway, but also in substantia nigra (sn), caudate putamen (cpu) and fibers projecting from sn to cpu. the nb- immunopositive cells in sn are restricted to gabaergic neurons. since gabaergic neurons play essential roles in the development and function of the auditory system, it is highly likely that nb- regulates the development and/or function of gabaergic neuron in the auditory pathway. reiko nagashima, kiyokazu ogita department of pharmacology, setsunan university faculty of pharmaceutical sciences, osaka, japan sensorineural hearing loss can be caused by a variety of insults, including acoustic trauma. there is compelling evidence that reactive oxygen species (ros) are formed in the cochlea during acoustic stimulation. glutathione (gsh) protects against the hearing loss through scavenging ros generated by noise. in this study, we investigated the changes in expression of gamma-glutamylcysteine synthetase (gcs) gene, which is the rate-limiting enzyme in de novo gsh synthesis, in the cochlea following acoustic stimulation. nuclear extracts were prepared from the cochlea at various time points after acoustic stimulation ( khz octave band, db, h), and then subjected to electrophoretic mobility shift assay to determine activator protein- (ap- ) dna binding. ap- binding was increased - h after the exposure. rt-pcr and immunostaining revealed that noise exposure was effective in elevating the expression of gcs in the cochlea h later. taken together, ap- may participate in the expression of gcs gene in the cochlea after acoustic stimulation. masaharu kudoh, ryuichi hishida, katsuei shibuki department of neurophysiology, brain research institute, niigata university, niigata, japan multiple formants compose vowels. we have previously reported that bilateral lesions including in the auditory cortex (ac) of rats impaired discrimination learning between synthesized vowel-like sounds with multiple formants, while discrimination between stimuli of a single formant or pure tones was not significantly impaired. in the present study, we determined the responsible auditory fields, which were required for the discrimination leaning between vowel-like sounds. water-deprived rats were trained to discriminate between two sounds including four different formants. licking a spout during presentation of one sound was rewarded with water while the other was not. surprisingly, local lesions in the primary ac or the ventral association cortex had no clear effect on the discrimination learning. in contrast, the dorsal association areas impaired the discrimination learning. these findings indicate that the dorsal auditory association cortex plays a critical role in discrimination learning of vowel-like sounds with multiple formants. hiroaki tsukano, yamato kubota, manavu tohmi, masaharu kudoh, katsuei shibuki department of neurophysiol, brain research institute, niigata university, niigata, japan we used transcranial flavoprotein fluorescence imaging for visualizing cortical responses to missing fundamentals in mice. c bl/ mice were anesthetized with urethane. the skull on the auditory cortex was exposed and covered with liquid paraffin to keep the skull transparent. cortical images of green fluorescence in blue light were recorded by a cooled ccd camera. responses in the auditory cortex elicited by sound stimuli ( - khz for ms) exhibited mirrorsymmetrical tonotopic maps in the primary auditory cortex (ai) and anterior auditory field. the activity patterns in ai elicited by khz were different from those elicited by or khz. however, the areas activated by khz were also activated by the mixture of plus khz but not by that of plus khz, suggesting that cortical responses to missing fundamentals in ai were visualized using flavoprotein fluorescence imaging. hiroko kosaki national priting bureau, tokyo, japan we constructed a functional scheme of macaque auditory by distribution of calcium binding protein, parvalbumin (pv). auditory cortex is consisted of one core (primary cortex), and five surrounding rings, which correspond with secondary, tertiary, quaric, and quintic cortices. parvabumin showed a graduation, that is, inner core is most pv-rich, and outer rings showed the decrease of pv concentration. comparing with pv staining in visual cortex, these six-levels suggested similar hierarchic and reciprocal structure, which are proposed by deyoe and vanessen by analysis of feed-forward and feedback connections. akihisa kimura, tomohiro donishi, keiichiro okamoto, yasuhiko tamai department of physiology, wakayama medical university, wakayama, japan tonotopically comparable subfields of the primary and non-primary auditory areas in the rat cortex have similar topographies in the projection to the medial geniculate body but reverse topographies in the projection to the thalamic reticular nucleus (trn). in the present study, we determined how cortical and thalamic afferents intersect in the trn with regard to tonotopic organization. in light of the fact that a subset of auditory cells in the trn responds to visual or somatosensory stimulus, we also explored the potential sources of cortical and thalamic afferents that would set up polymodal sensory interaction in the trn. small injections of biocytin into the trn, which were made with guidance of electrophysiological recording of auditory response, resulted in retrograde labeling. retrogradely labeled cortical and thalamic cells exhibited distinctive patterns of distribution depending on the injection sites. the results indicate anatomical nodes in the auditory trn that would implement selective relay of auditory and/or polymodal sensory inputs. ps a-f functional connections between the core and belt fields of the guinea-pig auditory cortex observed by optical recording and partial cortical inhibition using muscimol junsei horikawa, daisuke uchiyama, tatsunori matsui, shunji sugimoto department of knowledge-based information engineering, toyohashi university of technology, toyohashi, japan guinea pigs were anesthetized with ketamine and responses to puretones in the auditory cortex stained with a voltage-sensitive dye rh were recorded with a photodiode array. after determining the core (ai and dc) and belt fields, ai or dc was inhibited by putting a muscimol-containing agar piece on each field. the inhibition of ai resulted in reduction of responses in the belt fields by - %, whereas the inhibition of dc resulted in reduction only by - %. the reduction by ai inhibition was larger in the anterior and ventral belt fields and that by dc inhibition was larger in the posterior belt fields than in the other fields. further inhibition of dc after the ai inhibition or vice versa resulted in suppression of the responses in all the fields. these results suggest that the responses of the belt fields are elicited mostly via connections from the core to the belt fields and the belt fields receive differential connections from ai and dc. masataka nishimura, hiroyuki kaizo, wen-jie song department of electrical, electronic, and information engineering, graduate school of engineering, osaka university, suita, japan the auditory cortex of many mammals has a core area and surrounding belt regions. in guinea pigs, the primary auditory area, the secondary auditory area, and many belt regions have been reported. however, the activity of the belt regions has not been fully examined. using a high-resolution optical imaging system, we examined cortical responses to tone stimulations in anesthetized guinea pigs. the auditory cortex of six guinea pigs was exposed to the ventral end and stained with the voltage-sensitive dye rh- . a novel field in the ventro-anterior region was identified based on its isolated responses to a pure tone stimulation and the relatively long latency of the responses. the field was located ventro-caudal to the primary auditory area, and was close to the ventral edge of the auditory cortex. we thus named the field as ventro-caudal field (vc). smooth frequency gradient was observed in vc in rostro-caudal direction, with the frequency axis in opposite direction to that of the primary auditory area. yoko kato , , kazuo okanoya laboratory for biolinguistics, riken bsi, wako, saitama; graduate school of humanities, university of chiba, chiba, japan bengalese finches sing complex courtship songs. to sing complex sequences, they require auditory feedback during singing. song nucleus nif has a projection from primary auditory area field l and then it projects to sensory/motor nucleus hvc. moreover, bilateral lesion of nif cause song deterioration on complex sequences (hosino and okanoya, ) . we recorded auditory responses by multiunit activity from nif and field l. auditory responses of nif showed selectivity to bird's own song (bos) than its reversed song (rev). comparing selectivity of nif and field l, nif showed stronger selectivity than field l. however, nif did not show sequence dependent selectivity. these results suggest that nif relays auditory information and enhances bos selectivity. however, we did not observe a direct evidence that nif related to generation of complex sequences. we started to record responses extracellularly from ac neurons of guinea pigs. in general, animals show a stereotyped pattern of behaviors; they have a quiet, almost-motionless period, usually for tens of min. during this period, animals do not appear to sleep but be sensitive to the environmental disturbance. thereafter they usually fall asleep with their eyes closed. during this presleeping period, the best frequency tone was repeatedly presented - times at a fixed interval, through a speaker at - db spl. responses to such repetitive tones are apparently irregular, with the occurrence of spikes in most trials but no spikes in some trials. however, if all the trials are accumulated, there was global phase alternation every a few to tens of seconds. one phase constitutes relatively high rates of spike occurrence, while the other very low rates of spike occurrence. we suppose that, unlike a machine, the brain has a unique mechanism that automatically turns on and off the cortical processing of the redundant sensory stimulus. masashi sakai, sohei chimoto, ling qin, yu sato department of physiology, university of yamanashi, japan a periodic click train produces a continuum of several perceptual qualities: (i) at low repetition rate (< hz), the individual clicks are clearly heard as discrete events so that the entire train produces "rhythm" percept, (ii) at high repetition rate (> hz), the entire train is heard as a single continuous event leading to a strong "pitch" percept, and (iii) in the transition range, the periodicity can still be detected as "roughness". we physiologically explored how those perceptual qualities are represented in the primary auditory cortex in awake cats. we found that distinct population of cells conducted two coding modes: (i) representing low-rate stimuli through stimuluslocking activity (i.e., temporal code) and high-rate stimuli as only onset responses or (ii) exhibiting sustained responses with generating larger amount of discharges at higher repetition rate (i.e., rate code). in addition, pure-tone stimuli elicited onset responses or sustained responses in each of these cell populations, respectively. we will discuss functional consequences and spike evocation mechanisms of each population. atsuhito toyomaki , , , , , hokkaido university, sapporo, japan; hokkaido university, sapporo, japan; sakushin gakuin university, utsunomiya, japan; kobe shoin women's university, kobe, japan; riken, wako, japan; sakushin gakuin university, utsunomiya, japan gaps in a continuous sound play important roles for perception of voiceless consonants (i.e./k/,/p/,/t/) and japanese special mora (sokuon). we recorded auditory evoked responses to short gaps and tones from children ( - years old, n = ) and adults ( - years old, n = ). there were six gap conditions with durations of , , , , and ms embedded in a continuous tone and six tone conditions with the same durations. the frequency of all the tone was hz. the responses elicited by the onset of gaps differed between the children and the adults: the responses in children were significantly larger and more sustained than those in adults for all the durations. in contrast, an n and p complex followed the onset of all the tones in all the subjects. thus development time course of neural process is conceivably different between gaps and tones. ps a-f an fmri study on pitch control of voice using transformed auditory feedback method akira toyomrua , tamaki miyamoto , atsushi terao , sachiko koyama , takashi omori , harumitsu murohashi , shinya kuriki jst, saitama, japan; hokkaido university, sapporo, japan auditory feedback plays an important role in natural speech production. in the present study, we conducted an fmri experiment while subjects performed a transformed auditory feedback (taf) task to delineate the neural mechanism for control of pitch. the subjects were required to vocalize a and to hold the pitch of a feedback voice constant. in taf condition, the pitch was altered suddenly two or three times, whereas in non-taf condition the pitch was not modulated. under the taf condition, auditory feedback control is selectively expected to work more strongly than the non-taf condition. thus, a comparison between these conditions could neatly extract brain regions involved in auditory feedback control of pitch. as a result, right supramarginal gyrus, right frontal lobe (ba ), right anterior insula, left premotor area and right superior temporal gyrus showed greater activation ( subjects, p < . corrected). this result suggests that auditory feedback of pitch is mainly controlled by the right hemisphere. sachiko koyama-takeichi , yuko toyosawa , fumiya takeuchi , michinao matsui , shinya kuriki research institute for elecronic science, hokkaido university, sappro, japan; jst, saitama, japan; hokkaido university, school of medicine, japan; kobe shoin institute for linguistic science, kobe, japan sounds with relatively long duration elicit a sustained component (slow field, sf). in the present study, we recorded cortical magnetic responses elicited by vowels and examined whether sf differs between native and non-native vowels (n = ). four synthesized vowels were used as stimuli (stimulus duration ms). two of the vowels (a, o) are native for japanese and the other vowels (ae, schwa) are not. two inter-stimulus intervals were used ( / ms). for the native vowels, an early sf ( - ms) was larger for the long than for the short interval session in both hemispheres. for the non-native vowels, the early sf was larger for the long than the short interval session only in the right hemisphere. neither an effect of interval nor hemisphere was significant for a late part of sf ( - ms) regardless of stimulus types. research funds: japan science and technology agent (brain sciences and education), kakenhi ( ) ps a-f spatio-temporal representation of frequencymodulated sounds in the auditory cortex revealed by optical imaging shunji sugimoto , , junsei horikawa department of knowledge-based information engineering, toyohashi university of technology, toyohashi, japan; riken brain science institute, wako, japan optical imaging (voltage-sensitive dye, rh ) showed spatiotemporal response patterns for frequency-modulated (fm) sounds in the multiple fields of the guinea pig auditory cortex. an fm sound evoked a strong onset response spreading widely over the cortex, which was followed by a later response moving across the iso-frequency contours in the core fields. the location of the later response was corresponding roughly to the instantaneous frequency input of each fm sweep. on the other hand, a pure tone evoked a wide-spreading onset response followed by strong and long-lasting inhibitory effects. the later response to an fm sound appeared clearly when the frequency of the fm sweep was modulated over a wider range of frequencies, while it was diminished when the sound frequency was less modulated. these results imply that the cortical representation of such a later response contributes to a detection of frequency modulations in sounds. yamato kubota, kuniyuki takahashi, ryuichi hishida, masaharu kudoh, katsuei shibuki department of neurophysiology, brain research institute, niigata university, niigata, japan mitochondrial flavoprotein fluorescence is intimately coupled with energy metabolism. if the flavoprotein fluorescence is photobleached, energy metabolisms and neural activities can be inactivated. we applied this photo-inactivation technique to demonstrate auditory signal transmission from the anterior auditory field (aaf) to the primary auditory cortex (ai). cortical responses in aaf and ai after sound stimuli ( - khz) were visualized using transcranial flavoprotein fluorescence imaging in mice anesthetized with urethane. after determination of tonotopic maps, the auditory cortex was irradiated with strong blue light derived from a xenon lamp for min, while the surface either aaf or ai was covered with a piece of carbon paper for preventing photo-inactivation. although photoinactivation of ai had almost no effect on the responses in aaf, photo-inactivation of aaf significantly reduced the responses in ai. these results suggest the presence of auditory signal transmission from aaf to ai. kousuke abe , go ashida , , kazuo funabiki graduate school of informatics, kyoto university, kyoto, japan; hmro, faculty of medicine, kyoto university, kyoto, japan sound signals are translated to dispersed sporadic firing of the auditory nerves, and are converged to the third auditory station called the nucleus laminaris (nl) in birds. in vivo intracellular recording from owl's nl cells revealed that sound waveforms are observed in the postsynaptic membrane potentials (sound analogue potential; sap). we simulated synaptic inputs to the owl's nl neurons by recruiting the convergence of phase-locked excitatory inputs. several parameters such as the degree of phase-locking, the number of convergence and the time course of a unitary synaptic input affected the amplitude of sap, the amplitude of dc depolarization and the spectral features of synaptic noise in a complex manner. biophysical mechanisms for recreating sound waveforms by synaptic potentials will be discussed. takashi nihashi , shigenori takebayashi , masahiko bundo , masazumi fujii , toshihiko wakabayashi , jun yoshida , hiroyuki fujisawa , kazunori ando , kazumasa hayasaka department of radiology, national hospital for geriatric medicine, obu, japan; department of neurosurgery, national hospital for geriatric medicine, obu, japan; department of neurosurgery, nagoya university school of medicine, nagoya, japan we identify si, using fmri in a routine scan for the patient who need a surgical approach. the activation of the brain with a tumor is complicated. we considered the pattern of the response. twelve patients were participated in this study. using . tesla mr imager, tactile stimulation was applied to bilateral palm, respectively. the statistical threshold was set for individual. contralateral activation on si was found in out of patients in the affected hemisphere. when region is near central sulcus, the multiple sites were activated. on the other hand, when the tumor is from central sulcus, the activation is simple: contralateral si. this method is useful to decide si in affected hemisphere in a short time. however, there are great inter-individual differences due to the locations of the tumor. takayuki iwano, shinya yamamoto national institute of advanced industrial science and technology (aist), neuroscience research institute, tsukuba, japan to examine how body surface with low spatial resolution is represented in the brain, we conducted a tactile identification task on toes. subjects (n = ) lay on their backs with their eyes closed, and one of their toes was touched with a toothpick. the subjects were required to identify the toe by verbal response. the subjects responded correctly when the great or fifth toe was touched (cf. fein, ) . surprisingly, subjects tended to misidentify the second toe as the third ( . %), and the third toe as the fourth ( . %), while the reverse misidentification rarely occurred (third as second, . %; fourth as third, . %). this unidirectionality suggests that misidentification arises not only from large overlapping receptive fields associated with the toes, but from some additional factors such as a lack of experience with visuotactile integration, which could be used to reshape the toe receptive fields. ps a-g effects of saccades on subjective temporal order of somatosensory signals toshimitsu takahashi , , shunjiro moizumi , ayami okuzumi , humine saito , shigeru kitazawa , department of neurophysiology, juntendo university graduate school of medicine, tokyo, japan; crest, jst, saitama, japan morrone et al. ( ) recently reported that subjective temporal order of two successive visual stimuli was reversed when the stimuli were delivered just prior to the onset of a saccade. in this study, we examined whether saccades affect temporal order judgments of tactile stimuli. right-handed subjects were required to make a visually guided rightward saccade ( • ), and to judge the order of successive tactile stimuli that were delivered one to each hand at various timing relative to the onset of the saccade. with a stimulation interval of ms, subjects generally judged the order correctly as long as the stimuli were delivered after the saccade. however, they often misreported (i.e., inverted) the order when the stimuli were delivered just prior to the onset of the saccade (within ms). the results show that the reversal effect of saccades is multimodal and further suggest that multimodal brain areas are involved in ordering sensory events in time. ps a-g function-directed organization of the postcentral somatosensory cortex representing oral structures takashi toda , , miki taoka , department neuroscience oral physiology, osaka university graduate school of dental sciences, suita, japan; secondrary cognitives neurobiology, tokyo medical & dental university, tokyo, japan; department physiology, toho university school of medicine, tokyo, japan the representation of oral structures in areas b and of four conscious macaque monkeys was studied by recording single-neuron activities. a total of electrode penetrations were made in areas b and . in penetrations, pairs of adjacent neurons along the track had receptive fields (rfs) on continuous oral portions with or without overlapping, or otherwise on the same portion. in the remaining penetrations, however, % of adjacent pairs ( / ) had rfs on discrete but functionally-related sets of oral portions, e.g., the lip and tongue tip, the cheek mucosa and lateral margin of the tongue, the corresponding portions of the upper and lower lips, the corresponding portions of the palate and tongue, etc. we speculated that such an organization in areas b and might be responsible for forming composite rfs of area neurons. those composite rfs often covered discrete but functionally-related oral portions as reported earlier. research funds: kakenhi ( , ) miki taoka , michio tanaka , hisayuki ojima , atsushi iriki secondrary cognitives neurobiology, tokyo medical and dental university, tokyo, japan; laboratory of symbolic cognitive development, riken brain science institute, wako, japan we previously reported neuronal projections from the hand region of the second somatosensroy cortex (sii) to higher motor cortices (vetral premotor cortex etc.) suggesting that sii may be related to motor control of the hand movement. in the present study, we investigated the activities of sii hand neurons during voluntary movements. we recorded neurons from two animals that were active when animals took small pieces of food by hands and put them into the mouth. among them ( % contra-, % bi-and % ipsilateral hand movements), we could determine receptive fields for only neurons ( %). most of activities ( neurons) were related to a certain phase of movements such as reaching, pinching a food piece, and putting it into the mouth. we found neurons showing phasic activities just before/after a certain phase, for example, just before pinching the object, or just after putting it into the mouth. those results suggest that sii hand neurons code the start or end of a certain act of hand. takahiro furuta , , kouichi nakamura , takeshi kaneko department of morphological brain science, graduate school of medicine, kyoto university, kyoto, japan; crulrg, laval university, canada we investigated response properties of whisker-responsive neurons in the nucleus interpolaris (spvi) combining juxtacellular recording and a piezo-stimurator. the spvi is one of the first relay stations in the vibrissal system. rostral part of the spvi sends axons to the posterior thalamic nuclear group, whereas the caudal part of the spvi projects to the ventral lateral part of the ventral posterior medial nucleus. in the rostral part of the spvi, the vast majority of recorded neurons were multi-whisker responsive neurons, which are considered as projection neurons. in the caudal part of the spvi, about a half of neurons were mono-whisker-responsive neurons, which are thought as local circuit neurons. almost all neurons had angular tunings to upward deflection of whiskers in the rostral part of the spvi, while neurons in the caudal part of the spvi exhibited various angular tunings to all directions. these results indicate that the spvi could be divided into two sectors by response properties. seiji komagata , , shanlin chen , hiroki kitaura , masaharu kudoh , minoru shibata , katsuei shibuki department of neurophysiology, brain research institute, niigata university; department plastic surgery, school of medicine, niigata university, japan we visualized neural responses in the mouse somatosensory cortex using transcranial flavoprotein fluorescence imaging. mice were anaesthetized with urethane ( . g/kg, i.p.), and somatosensory responses were elicited by vibratory brush stimulation ( hz for s) applied to the left plantar forepaw. changes in green fluorescence in blue light were observed in the right somatosensory cortex. immediately after cutting the left median and ulnar nerves, the somatosensory responses were almost completely abolished. however, the responses appeared again within a few hours after the partial denervation, and almost complete recovery was observed a few weeks after the partial denervation. the recovered responses were eliminated by cutting the remaining radial and muscle cutaneous nerves. the rapid recovery of the responses observed in the present study may explain the mechanisms for allodynia and cortical plasticity in the somatotopic maps. shin-ya nakamura, takaaki narumi, ken-ichiro tsutsui, toshio iijima division system neuroscience, tohoku university graduate school of life science, sendai, japan in the rat somatosensory pathway, information received with a whisker is conveyed to the barrel cortex via trigeminal and thalamic nucleus mainly by two parallel pathways, the lemniscal and paralemniscal. the former includes the nucleus of trigeminal prv and thalamic vpm, which are known to contain neurons selective to the direction of whisker stimulation, and the latter includes trigeminal spvi and thalamic pom. in this study, we examined the specific involvement of the lemniscal pathway to the discriminative perception of whisker stimulus direction. rats were trained to perform a single-whisker directional discrimination task, and the task performance was evaluated before and after the selective electrolytic lesion or muscimol inactivation of each trigeminal and thalamic nucleus. the lesion or inactivation of prv or vpm significantly impaired the task performance, whereas those of spvi or pom did not. this result suggests the specific involvement of the lemniscal pathway in the single-whisker directional discrimination. kumiko yokouchi , nanae fukushima , tetsuhiro fukuyama , akira kakegawa , tetsuji moriizumi department of anatomy, shinshu university school of medicine, matsumoto, japan; department of pediatrics, shinshu university school of medicine, matsumoto, japan to know sensory cues for suckling behavior, rat pups of the suckling period received unilateral or bilateral resection of the infraorbital (io), mental (m) or lingual (l) nerves responsible for sensation of the upper and lower lips, and the tongue. for comparison, unilateral or bilateral removal of the olfactory bulb was done in these pups. the control, unilaterally io or m nerve-injured, and unilaterally bulbectomized pups showed successful suckling by their access to the mother's nipple, oral contact to it and long-lasting sucking. the bilaterally bulbectomized pups could not have access to the nipple. the bilaterally io nerve-injured pups could have access to the nipple with no oral contact, while the bilaterally m nerve-injured pups showed successful suckling. suckling behavior of the bilaterally l nerve-injured pups was characterized by frequent oral contacts for a very short duration, resulting in ineffective milk-intake. the results show fundamental roles of olfaction and sensation of the upper lip and the tongue in suckling. takahiro kawashima , takeshi kawano , hidekuni takao , , , kazuaki sawada , , , hidekazu kaneko , , makoto ishida , , department electrical & electronic engineering, toyohashi university of technology, aichi, japan; issrc, toyohashi university of technology, aichi, japan; aist, hsbe, ibaraki, japan; jst, crest, japan a si microprobe array (probe: au-coated recording site at the tip, m in diameter, m in length; array: -m pitch) to record neuronal activities has been developed by using selective si probe growth technique. to examine electrical properties of the array, single motor unit action potentials evoked by the electrical stimulation of the sciatic nerve of a rat were recorded in the left tibialis anterior muscle. signal-to-noise ratio of observed signals decreased with probe impedance, suggesting that lower impedance is better for recording small action potentials. however, lower impedance makes more difficult to record local signals, because signals observed at probes with low impedance were highly correlated (r = . ). to record local signals, it is necessary to decrease the area of the recording site of each probe at the expense of an increase in the impedance. research funds: kakenhi ( ), the st century coe program "intelligent human sensing" ps a-g a microelectrode positioning system for longterm extracellular recording of multiple neuronal activities hidekazu kaneko , hiroshi tamura , shinya s. suzuki , takahiro kawashima aist, hsbe, ibaraki, japan; laboratory of cognitive neuroscience, graduate school of frontal bioscience, osaka university, osaka, japan; department electrical & electronic engineering, toyohashi university of technology, aichi, japan a novel microelectrode-positioning system was devised that tracks a target neuron by countermoving a microelectrode against uncontrollable movements of brain tissue. the system automatically adjusted the position of a seven-core microelectrode such that the amplitude of each spike of a target neuron at the tip was the same as that at a lateral recording site (differential mode). the differential mode was compared with a conventional (peak-search) mode in which the spike amplitude of a target neuron at the tip was continually maximized. the differential mode was more stable to forced electrode movements and more sensitive to small displacements than the peak-search mode. furthermore, the differential mode enabled stable recording of not only spikes of a target neuron but also those of non-target neurons for over h. seiji matsuda , takehiro terashita , tetsuya shimokawa , kyoujy miyawaki , yuji miguchi , takuya doihara , jie chen , shuang-yan gao , chun-yu li , min wang , zhong wang , bing xue , naoto kobayashi , , kazuhiro shigemoto department anatomy, ehime university of medicine, ehime, japan; medical education c, ehime university of medicine, ehime, japan; department of hygiene, ehime university of medicine, ehime, japan this study shows the phylogenetic development of cajal's initial glomeruli (ig) and dogiel's pericellular nests (pcns) in the dorsal root ganglion of the healthy adult frog, chick, rat, and rabbit. the three-dimensional architecture of the neurons was observed in ganglia by scanning electron microscopy, after removal of the connective tissue. the proportion of neurons having ig or pcns increased with increasing phylogenetic complexity in the species examined here. in the chicks, the stem processes were longer and sometimes tortuous. typical ig were observed not in frogs or chicks, but in rats and rabbits. dogiel's pcns also have been observed in the drg of rats and rabbits. the nerve fibers in the pcns were less than . m in diameter and had some varicosities. some pcns contain tyrosine hydroxylase-positive nerve fibers and varicosities. masayo okumura, eiji kondo matsumoto dental university, institute for oral science, shiojiri, japan we established a rat nerve injury model using axotomy of the inferior alveolar nerve (ian), and investigated its effect on gene expression in the trigeminal ganglion. microarray analysis three days after surgery showed the up-regulation of some genes which are regulated by transcription factor stat , whereas other genes known to be regulated by stat were not detected. stat is expressed in many tissues and plays various roles. however, there have been few reports about the role of stat in the peripheral nervous system, despite its welldocumented activation in the central nervous system after injury or stress. the aim of this study is to elucidate the role of stat in gene expression in the trigeminal ganglion after ian injury. at various time points, we analyzed and investigated changes of gene expression which are known to be influenced by stat and stat phosphorylation, which indicates transcriptional activity, as well as cell types in which the genes and stat are expressed. these results should help us understand injury-induced change mechanisms of the peripheral nerve. hirofumi hashimoto , susumu hyodo , makoto kawasaki , minori shibata , takeshi saito , hiroaki fujihara , takashi higuchi , yoshio takei , yoichi ueta department of physiology, school of medicine, university of occupational and environmental health, kitakyushu, japan; laboratory of physiology, department of marine bioscience, ocean research institute, university of tokyo, japan; department of integrative physiology, university of fukui, japan adrenomedullin (am ) (identical to intermedin) belongs to the super family of am. centrally administered am and am activated oxytocin (oxt)-secreting neurons and increased plasma oxt level in rats. in the present study, we examined the effects of central administration of am on oxt-secreting neurons and sympathetic outflow in comparison with that of am in conscious rats. effects of central administration of am was stronger than those of am and the effects of am on oxt secreting neurons could not be blocked completely by pretreatment with cgrp or/and am receptor antagonists. these data suggested that am would have unknown receptor except cgrp and am receptor. arata oh-nishi , makoto saji , taku uchida , sen-ichi furudate , nobuyuki suzuki division of brain science, kitasato university, graduate school of medical science, kanagawa, japan; division of reproduction and fetal development, kitasato university, graduate school of medical science, kanagawa, japan the mechanism whereby neonatal hypothyroidism impairs cognitive function has not been well studied. in this respect, nmda receptors are thought to be crucially involved in cognitive and memory function. we have examined the effect of neonatal hypothyroidism and hyperthyroidism on the nmda receptor function, using rats treated with methylmercaptoimidazole (mmi), which specifically blocks the biosynthesis of thyroid hormone and mmi-treated rats injected with thyroxine, respectively. dose-response curves indicated that the sensitivity to nmda of the nmda receptors was significantly reduced in the hippocampus of the hyperthyroid rats, compared to that of normal and the hypothyroid rats. concomitant with this observation, western blot analysis showed that the nmda receptor subunit nr expression significantly decreased in the hippocampus of the hyperthyroid rats, compared to that of normal and the hypothyroid rats. our lab demonstrated that estradiol is endogenously synthesized within hippocampal neurons in the adult male rat (pnas, ) . here we report that the density and morphology of spines of pyramidal neurons in ca region are rapidly altered by treatments with nm estradiol and bisphenol a (xenoestrogen). hippocampal slices are incubated with estradiol or bisphenol a for h, and then neurons were injected iontophoretically with lucifer yellow. three-dimensional imaging of neurons is performed by confocal laser microscopy, and the analysis of individual spines is performed by neurolucida software. the results showed that in ca , both estradiol and bisphenol a induce a significant increase in the total spine density, especially the density of thin spine. synaptic plasticity of hippocampal neurons is demonstrated to be rapidly modulated by estrogen and xenoestrogen. we investigated the effects of stress on enhanced green fluorescent protein (egfp) expression in the arginine vasopressin (avp)-egfp transgenic (tg) rats. after bilateral adrenalectomy and intraperitoneal administration of lipopolysaccharide egfp fluorescences were increased in the parvocellular division of the paraventricular nucleus and the external layer of the median eminence. this tg rat is a convenient tool to study dynamic changes of avp expression in the hypothalamus under stressful condition. chitose orikasa, yasuhiko kondo, yasuo sakuma department of physiology, nippon medical school, tokyo, japan we report here a sex difference expression of somatostain mrna within the sexually dimorphic nucleus of the preoptic area (sdn-poa), the volume of which depends on gonadal hormones during the ontogeny. in infant rats aged day - , the volume of somatostain mrna-positive region within the poa was significantly larger in males than in females and overlapped the sdn-poa in both sexes. the sdn-poa visualized by nissl staining in adjacent sections agreed precisely with the extent of somatostain mrna-positive cellular distribution. orchidectomy of males neonates and estrogen treatment of female pups reverse brain phenotypes when examines on day . the staining of somatostain mrna in individual neurons was diminished when examined on day or , albeit that the sex difference of the volume of somatostain mrna-positive region persisted thoughtout the observed period. somatostatin may play a role in the establishment of the sdn-poa, which lacks classic nuclear receptor for estrogen. ps a-g short chain sugar acid, -buten- -olide, activates oxytocin-secreting neurons in the hypothalamus of rats makoto kawasaki , tatsushi onaka , hirofumi hashimoto , hiroaki fujihara , yoichi ueta department of physiology, school of medicine, university of occupational and environmental health, kitakyushu, japan; department of physiology, jichi medical school, tochigi, japan -buten- -olide ( -b o), an endogenous sugar acid, which may be involved in the regulation of feeding. we examined the effects of -b o on the hypothalamo-neurohypophyseal system in rats. the plasma oxytocin (oxt) levels were significantly increased at - min after intraperitoneal (i.p.) administration of -b o ( mg/kg), whereas plasma arginine vasopressin (avp) levels did not change. dual immunostaining revealed that fos-like immunoreactivity (li) was predominantly observed in oxt-secreting neurons in the paraventricular and the supraoptic nuclei min after i.p. administration of -b o. in addition, many fos-li neurons were also observed in the nucleus of the tractus solitarius (nts) after i.p. administration of -b o. these results suggest that peripherally administered high dose of -b o activates oxt-secreting neurons in the hypothalamus through the activation of the nts neurons. ps a-g estrogen receptor ␣ gene promoter activity is a marker for the sexually dimorphic nucleus of the preoptic area tomohiro hamada, yasuo sakuma department of physiology, nippon medical school, tokyo, japan the volume of the sexually dimorphic nucleus in the preoptic area (sdn-poa) is two to four times larger in male rat than in female, however function of this nucleus has not well known. in contrast, estrogen causes the sexually dimorphism by acting in perinatal periods. recently, transgenic rats expressing enhanced green fluorescent protein (egfp) under the control of an estrogen receptor (er) ␣ promoter were generated to tag er␣-positive neurons in the brain. in the present study, we examined gfp expression could be used a marker for the sdn-poa. gfp labeled cells were distributed in the core of sdn-poa of male and female transgenic rats and in the majority of these cells included er␣, immunohistochemically. both area and number of gfp expressed cells in the sdn-poa were larger in male than in female, however, female gfp cells in the sdn-poa showed concentrated distribution than male. these results suggest that gfp labeled cells in sdn-poa could be useful marker to make clear the function of the sdn-poa. recent studies on gonadal steroids imply that testosterone and estradiol are involved in learning and memory with modification of excitatory synapses in the hippocampus. although previous in vivo studies have demonstrated that these steroids increase the number of dendritic spines in neurons, it is still unclear whether each steroid has a direct effect on the modulation of the spatio-temporal patterns of dendritic morphogenesis. in the present study, we investigated steroid-induced morphological changes using cultured hippocampal neurons derived from neonatal or embryonic mice. the neurons were transfected with venus-actin. time-lapse images were taken by laser scanning confocal microscope during steroid treatment. testosterone but not estradiol increased the number of spines/filopodia of the dendrites within h. these results obtained from in vitro studies suggested that testosterone affects dendritic morphogenesis of hippocampal neurons in short term. tetsuya kimoto , , shinpei higo , , yasushi hojo , , kouhei nakajima , , hironori nakanishi , , hirotaka ishii , , suguru kawato , department of biophysics & life science, university of tokyo, tokyo, japan; crest, jst, japan hippocampus is one of the main target of sex steroids (androgen and estrogen) and stress steroids (corticosteroids). neuronal signal transmission in the hippocampus is modulated acutely by these steroids, and we recently demonstrated that the hippocampus of the adult male rat contained enzymes required for the synthesis of these steroids. however, the full diagram of hippocampal neurosteroid synthesis has not been obtained yet. in the present study, we therefore investigated the synthesis of sex steroids and corticosteroids in the hippocampus of adult male rats, by monitoring the metabolism of tritiated steroids with hplc system. ps a-g gaba depolarizes gnrh neurons isolated from adult gnrh-egfp transgenic rats chengzhu yin, nobuyuki tanaka, masakatsu kato, yasuo sakuma nippon medical school, department of physiology, tokyo, japan gnrh neurons are essential in the reproductive neuroendocrine system. in regulation of gnrh neurons, gaba may be one of the major players, especially in relation to gnrh/lh surge. we, therefore, performed a cell-physiological analysis of gaba action on rat gnrh neurons. cells were dispersed from adult gnrh-egfp transgenic rats and cultured overnight. gnrh neurons, were applied to the perforated patch-clamp configuration with gramicidin d. gaba evoked cl − conductance, which was almost completely blocked by either picrotoxin or biccuculin. the reversal potential of the response was ranged from − to - mv in identified gnrh neurons in both sexes. there was no difference in the reversal potential among the stages of estrous cycle. in unidentified neurons, however, the reversal potential was more negative than - mv and most of them were ∼− mv. in conclusion, gnrh neurons isolated from adult rats express gabaa receptor and its reversal potential is more positive than the resting potential. although the neural activation in the subfornical organ (sfo) by angiotensin ii (angii) is widely regarded for the increments of angii-induced water intake and vasopressin release, galanin (gal) have been reported to inhibit them. therefore, gal may inhibit neural activity of angii-sensitive sfo neurons. rt-pcr analysis demonstrated existences of all mrnas of gal receptor subtypes, galr , galr and galr , in the sfo. in extracellular recording on sfo slice preparation, gal dose-dependently led to inhibition of neural activity. all gal sensitive neurons showed excitatory response by angii. galr selective agonist m induced inhibitory responses, as well as gal. in patch-clamp recordings, gal induced outward current in some neurons. these results suggest that gal inhibits neural activity in sfo neurons through, at least partially, outward current following activation of galr . hiroaki fujihara , tomoki fujio , david murphy , yoichi ueta department of physiology, school of medicine, university of occupational and environmental health, kitakyushu, japan; molecular neuroendocrinology research group, the henry wellcome laboratories for integrative neuroscience and endocrinology, university of bristol, bristol, uk we have generated transgenic (tg) rats expressing an arginine vasopressin (avp)-enhanced green fluorescent protein (egfp) fusion gene. in this study, we investigated the amount of drinking and food intake, the urinary output, the urine osmotic pressure, the urine sodium concentration and body weight after drinking % saline for days in , , and months old tg rats. in and months, there were no difference between tg rats and tg(−) rats about the amount of drinking and food intake, the urinary output, the urine osmotic pressure, the urine sodium concentration and body weight under normal condition and salt loading. in aged tg rats ( and months old), there were no obvious changes in water balance. these results suggest that the expression of avp-egfp transgene does not disturb body fluid homeostasis in tg rats. ps a-h prolactin-releasing peptide is a potent mediator of stress response in the brain through the hypothalamic paraventricular nucleus takashi mera , hiroaki fujihara , hirofumi hashimoto , makoto kawasaki , tatsushi onaka , takakazu oka , sadatoshi tsuji , yoichi ueta department of neurology (division of psychosomatic medicine), school of medicine, university of occupational and environmental health, japan; department of physiology, school of medicine, university of occupational and environmental health, japan; department of physiology, jichi medical school we examined the effects of restraint stress (rts), nociceptive stimulus and acute inflammatory stress on the prolactin-releasing peptide (prrp) gene expression in the hypothalamus and brainstem. moreover, we examined the effects of pretreatment with an anti-prrp antibody on nociceptive stimulus-induced c-fos gene expression in the hypothalamic paraventricular nucleus (pvn). rts, nociceptive stimulus and acute inflammatory stress upregulated the prrp gene expression in the brainstem. pretreatment with anti-prrp antibody significantly attenuated nociceptive stimulus-induced c-fos gene expression in the pvn. these results suggested that prrp is a potent and important mediator of stress response in the brain through the hypothalamic pvn. taieb bousejin , afsaneh eliassi , nasser naghdi , ali ghanbari ghsemi; pastor institute, tehran, iran the purpose of this study was to consider the role of the ventromedial hypothalamus (vmh) d receptors on histamine-induced gastric acid secretion (gas). the animals were anasthetized and guide cannulas were implanted unilaterally above ( . mm) vmh. animals were anasthetized and two polyethylene tubes were introduced into the stomach through esophagus and pylorododenal junction. iv infusion of histamine in sham grup induced marked increase in gas with a peak response that started from min up to the end of experiments ( min). at the peak acid response, the vmh microinjection skf ( . , ) significantly reduced the amount of gas (p < . ). there was no any effect by microinjected sch ( . ) into the vmh. injecting skf into the vmh, min after sch , had no effect on gas in compare with control. but, the acid suppressant effect of skf was completely removed by peripheral injection of sch (p < . ). our results show that the vmh d dopamine receptors have regulatory mechanisms of gas by interaction with h receptors through an inhibitory neural pathways. zhilin song, celia d. sladek department of physiology, uchsc, aurora, co, usa although prior studies demonstrated expression of p x purinoceptors in supraoptic neurons (son) and indicated their importance in atp stimulated vasopressin release, in studies monitoring the effect of atp on intracellular ca ++ ([ca ++ ] i ), we have obtained evidence that p y purinoceptors (p y r) are important in the response to atp. atp stimulated [ca ++ ] i increase was maintained in ca ++ -free medium and reduced by pretreatment with thapsigargin to deplete [ca ++ ] i stores. p y r agonists increased [ca ++ ] i in son, with p y r agonist being the most effective. the possibility that p y r mediates atp induced [ca ++ ] i increase in son was further evaluated using a p y r antagonist, mrs . atp stimulated increase in [ca ++ ] i was greatly attenuated by mrs ( m) in mm ca ++ medium. in ca ++ -free medium, there was no significant response to atp in the presence of mrs . furthermore, combined treatment with mrs and ppads ( m, a p x r antagonist) also abolished the [ca ++ ] i response to atp. these results demonstrated that p y r mediates a large portion of the [ca ++ ] i response to atp challenge in son. ps a-h analysis of the ontogenic expression of enzymes for brain neurosteroids in the male rat hippocampus hirotaka ishii , , yasuhiro sonoki , , aizo furukawa , , yasushi hojo , tetsuya kimoto , , suguru kawato , department of biophysics and life science, university of tokyo, tokyo, japan; crest, jst, japan; kurihama national hospital, japan brain neurosteroids are steroids synthesized endogenously in the brain. our recent studies have demonstrated that the adult male rat hippocampus is equipped with a complete machinery for the synthesis of androgen and estrogen. to define the physiological role of brain neurosteroids in the hippocampal development and function, detailed information about the expression profiles of enzymes for brain neurosteroids in the hippocampus is essential. this study have comprehensively investigated the temporal patterns of enzymes for brain neurosteroids in the male rat hippocampus from postnatal day (pd ) to the adult stage using rt-pcr/southern blotting. enzymes required for the synthesis of estradiol from cholesterol were expressed form pd to pd with a higher level than in the adulthood. these results indicate that the rat hippocampus synthesizes estradiol more vigorously during the postnatal stage than in the adulthood, which may play an important role in the hippocampal development and function. hideo mukai , , gen murakami , shirou kominami , john h morrison , william g.m janssen , tetsuya kimoto , , suguru kawato , department of biophysics and life sciences, graduate school of arts and sciences, the university of tokyo, meguro, tokyo - , japan; crest project, jst, japan; faculty of integrated arts and sciences, hiroshima university, higashi-hiroshima , japan; kastor neurobiology of aging laboratories, fishberg research center for neurobiology, usa estrogens elicit rapid non-genomic effects on the synaptic transmission, and spinogenesis in the hippocampus. however, the existence of estrogen receptor alpha (er␣) still remains elusive. with highly purified antibody rc- , mass spectrometric analysis identified er␣ in the hippocampus and immunohistochemistry showed er␣ localization in principal neurons of ca , ca , and granule cells in dentate gyrus. further, western blot revealed that er␣ is contained in psd fraction, confirming the observation with immunoelectron microscopy. these results imply that the synaptic er␣ mediates the effects of estrogen in hippocampal neurons. ken takumi gnrh neuron is the key modulator of reproductive systems, directly regulating the synthesis and secretion of gonadotropins from anterior pituitary gland. gnrh neurons have been reported to be contacted by various neuronal systems, suggesting that the biosynthesis and release of gnrh is controlled by a complex of excitatory and inhibitory inputs. however, anatomical studies which quantified the direct input on gnrh neuron are few. in this study, we quantitatively analysed glutamatergic and gabaergic input onto gnrh neurons of the rhesus monkey by immunofluorescence method and confocal laser scanning microscopy; the close appositions between gnrh neuron and axon terminals immunoreactive for either vgluts or vgat were counted and the densities of the appositions on the dendrites and soma were calculated. sabine gouraud , song t. yao , jing qiu , julian fr paton , david murphy university of bristol, hw-line, uk; department of physiology, bristol heart institute, university of bristol, united kingdom the neuropeptide hormone vasopressin (vp) is produced in the magnocellular neurons of the hypothalamic supraoptic (son) and paraventricular (pvn) nuclei and stored in the posterior pituitary (pp). dehydration evokes an increased expression of the vp gene in magnocellular neurons and a massive release of vp from the pp in the circulation to promote the water conservation at the kidney level. in parallel, a functional remodelling of the hypothalamo-neurohypophyseal system (hns) is observed but poorly understood. we investigated this activity dependent plasticity of the hns using proteomic ( d fluorescence difference gel electrophoresis (dige)) combined with maldi mass spectrometry approaches to identify proteins that change in abundance in the son and the pp from days dehydrated rats. a truncated form of prosaas, a granin-like neuroendocrine peptide precursor known as a potent inhibitor of the prohormone convertase , has been found decreased in the pp and increased in the son. ichiro nishimura, masakatsu kato, yasuo sakuma department of physiology, nippon medical school, tokyo, japan function of gonadotropin-releasing hormone (gnrh) neurons is regulated by gonadal steroid estrogen. however, the precise mechanism of estrogen action upon these cells has not been clarified. we investigated a direct action of estrogen on the regulation of potassium current in gnrh neuronal cell line gt - . delayed rectifier potassium current (i k ) and large-conductance calcium-activated potassium (bk) current were recorded by patch clamp configuration in gt - cells cultured in dmem supplemented with % fbs for days. bk current was increased by addition of ␤-estradiol (e ) in culture medium in a physiological concentration range. this action of e was blocked by ici- , , a potent estrogen receptor (er) antagonist. we further examined whether e acted through er ␣ or er ␤ by using selective agonists ppt and dpn, respectively. the dpn augmented the bk currents similar to the effect of e but ppt had no effect. e had no effect on the i k . these results indicate that e increases the bk current by activating er␤ without affecting the i k . research funds: kakenhi , ps a-h myelin protein zero is one of the components of the detergent-resistant membrane microdomain fraction derived from rat pituitary katsutoshi taguchi , haruko kumanogoh , shun nakamura , seiji miyata , shohei maekawa department of biosystems science, kobe-university, kobe, japan; division of biochemistry and cellular biology, national institute of neuroscience, ncnp, tokyo, japan; department of applied biology, kyoto institute of technology, kyoto, japan a membrane microdomain enriched in cholesterol and glycosphingolipids was found to contain many signal transducing and cell adhesion molecules. here, we studied the components of the membrane microdomain fraction derived from rat pituitary, and found specific enrichment of several proteins in this fraction. one of them, kda protein, was identified as myelin protein zero (p ) from mass analysis and this result was confirmed by western blotting that a specific antibody to kda band reacted to an authentic p prepared from rat sciatic nerve myelin. p is a type i transmembrane glycoprotein and a member of the immunoglobulin superfamily. the expression of p has been believed to be restricted to the peripheral myelin in mammals. our result, however, indicates that p expresses more widely and participates in cell communications. mari ogiue-ikeda, norio takata, suguru kawato department of biophysics and life sciences, graduate school of arts and sciences, university of tokyo, tokyo, japan ␤-estradiol (e ) has a rapid effect on synaptic transmission. recently, we found that hippocampal neurons synthesize e (hojo et al., ) , and express estrogen receptor ␣ (er␣) at synapses. endocrine disrupters are representative estrogenic industrial compounds. while their disrupting effects on reproductive organs are well documented, their effects in the central nervous system are almost unknown. in this study, we investigated the effects of e and endocrine disrupters (des, bpa, np, op and tbt) on nmda-induced ltd in the rat hippocampal ca , ca and dg with a custom made multi-electrode measuring system (med ). ltd was enhanced by e dose-dependently in ca , ca and dg. des, bpa, np, op and tbt had similar or different effects on ltd dose-dependently. our results suggest that estradiol and endocrine disrupters rapidly modulate synaptic plasticity in the hippocampus and that the action of endocrine disrupters can be quantitatively analyzed by measuring the modulation of ltd of the hippocampal neurons. we examined the effects of chronic salt loading on the hypothalamic expressions of the green fluorescent protein (gfp), arginine vasopressin (avp) and oxytocin (oxt) genes and body fluid balance in avp-enhanced (e) gfp transgenic rats. chronic salt loading caused marked increase of the egfp fluorescence in the hypothalamoneurohypophyseal system in transgenic rats. there were no differences of the avp and oxt gene expressions in the hypothalamus, plasma avp and oxt levels and water balance between nontransgenic and transgenic rats under normal condition and after salt loading. humoral responses to chronic salt loading were maintained in avp-egfp transgenic rats. takeshi saito , takushi x. watanabe , tomoko urabe , hirofumi hashimoto , hiroaki fujihara , yukio hirata , yoichi ueta department of physiology, school of medicine, university of occupational and environmental health, kitakyushu, japan; peptide institute, inc., osaka, japan; department of clinical and molecular endocrinology, tokyo medical and dental university, tokyo, japan salusin-␤ was newly discovered as a bioactive endogenous peptide. low concentration of salusin-␤ stimulates the secretion of arginine vasopressin (avp) from perifused rat hypophysis. salusin-␤ coexists with avp, but not oxytocin, in the rat magnocellular supraoptic (son) and paraventricular nuclei (pvn). to further investigate the physiological role of salusin-␤ in body fluid homeostasis, we examined the effects of salt loding for days on salusin-␤-like immunoreactivity (li) in the son and pvn of rats by immunohistochemistry. the marked increase of salusin-␤-li in the son and pvn were observed in the salt loaded rats. the result suggests that salusin-␤ may play a role of body fluid balance by regulating avp release. naoyuki yamamoto , hao-gang xue , yuji ishikawa , yoshitaka oka , hitoshi ozawa department of anatomy and neurobiology, nippon medical school, tokyo, japan; national institute of radiological sciences, chiba, japan; department of biological sciences, graduate school of science, the university of tokyo, tokyo, japan the terminal nerve gnrh (gonadotropin-releasing hormone) system, an extrahypothalamic peptidergic system, is thought to modulate neural circuitries involved in the control of motivational status for certain behaviors in teleosts. the major afferent source to the gnrh neurons is a midbrain nucleus, the nucleus tegmento-terminalis in percomorph teleosts, while a comparable nucleus appears to be missing in cyprinids teleosts. here, we examined the presence of such an afferent pathway in medaka oryzias latipes. injections of a dii crystal into the cluster of gnrh neurons resulted in labeled cells in the midbrain tegmentum, and injections to the midbrain tegmentum resulted in labeled terminals close to the gnrh neurons. these results suggest that the afferent pathway to the gnrh neurons is a character shared by "advanced" teleosts like medaka and percomorphs. takeshi yamazaki , eiji munetsuna , asuka kamogawa , suguru kawato , shiro kominami graduate school of integrated arts science, hiroshima university of higashihiroshima, japan; graduate school of arts and science, tokyou university of tokyo, japan tributyltin, tbt, an endocrine disruptor, induced increases in estradiol content in rat hippocampal slice culture. to analyze molecular mechanism of stimulation of estrogen synthesis, we determined mrna contents of estrogen biosynthetic enzymes and activity of p arom in the hippocampus. method: the cultured hippocampus slices from days male rat were treated with - nm of tbt for h. after the treatment, total rna was extracted and the levels of mrna of estrogen synthetic enzymes were quantified by real-time rt-pcr. activity of p arom was determined by quantification of [ h]estradiol from [ h]testosterone. result: forty-eight hours treatment of hippocampal slices with nm tbt induced increases in mrna contents of p arom, and with nm tbt induced that of ␤-hsd. estradiol content was increased by the treatment with nm tbt, but not affected by nm tbt. tbt may modulate estradiol synthesis by alteration of expression of p arom. the medial preoptic area (mpoa) is an important neural site for regulation and maintenance of sleep. studies have indicated that gabaergic neurons and terminals at the mpoa are active during sleep. present study was carried out to elucidate the contribution of gaba-a receptor at the mpoa in sleep-wakefulness (sw) in male wistar rats. the sw was assessed by chronically implanted electrodes for eeg, eog, and emg. a bilateral guide cannula was also implanted for drug injection into the mpoa. after recovery, three baseline sleep recordings were taken for h on different days. bicuculline methoiodide (gaba-a receptor antagonist) at a dose of , , and ng in nl was injected bilaterally into the mpoa in different groups of rats and their sw was studied for subsequent h. the ng dose of bicuculline methoiodide had minimum effect whereas and ng produced arousal. maximal wakefulness was observed at dose of ng with no further increase in wakefulness at higher dose of ng. the results suggest the involvement of gaba-a receptors at the mpoa in sw. yoshiaki isobe , hiroyuki tsuda department of neuro-physiology and brain science, nagoya city university, graduate school of medical sciences, nagoya, japan; department of molecular toxicology, nagoya city university graduate school of medical sciences, japan locomotor activity in rodents shows free-running circadian rhythms even under the constant light. constant light exerts a promoting effect on hepatic carcinogenesis. after the partial hepatectomy, hepatic cell proliferation is regulated by circadian rhythm information (via wee ). to know the relation of proliferating factor (cell cycle) with circadian rhythmicity, locomotor activity against a diethylnitrosamine (den), widely used to initiate the hepatic neoplastic foci, is analyzed in preliminary. den was injected (i.p., mg/kg) on rats during the free-running condition under the constant dim light (dd) and constant light (ll). the effects of den were gentle under the dd. however, under the ll, phase delay accompanying the elongation of circadian period () was observed. decrement of an amount of activity in h after the den administration was obvious under the ll compared with that under the dd. this study was designed to investigate the central regulating system of hypothermia during maintenance phase of hibernation. although intracerebroventricular (icv) injection of naloxone (non-selective opioid receptor antagonist) and naloxonazine, ( antagonist) were effective, naltrindole (␦ antagonist) and nor-bni ( antagonist) did not interrupt the hibernation. the increment of c-fos expression was observed in arcuate nucleus (arc) at h after from hibernation onset compared with before hibernation. in addition, a localized ␤-endorphin-like immunoreactivity (␤-end ir) was observed in neuronal perikarya in arc at h after from hibernation onset. although ␤-end ir in arc got weak, the ␤-end ir of nerve fibers in preoptic nucleus (pon) got strong with progression of hibernation. these results suggest that the ␤-endorphin was transported to pon from arc by axonal flow and then played an important role in maintenance of hypothermia via -opioid receptors in hibernation. wei-min qu, zhi-li huang, naomi eguchi, yoshihiro urade, osamu hayaishi department of molecular and behavioural biology, osaka bioscience institute, osaka, japan prostaglandin (pg) d is a potent somnogenic substance, and isomerized from pgh through the action of pgd synthase (pgds). pgds has two distinct types, the lipocalin-type pgds (l-pgds) and hematopoietic pgds (h-pgds). selenium compounds have been reported to decrease sleep by inhibiting pgds in rats. to clarify what type of pgds inhibition is involved in sleep reduction by selenium or whether selenium intoxication decreases sleep, we intraperitoneally injected secl into l-pgds and h-pgds knockout (ko), and their wild-type (wt) mice. in wt mice, secl decreased rapid eye movement (rem) and non-rem sleep for h after injection and, concomitantly, increased wakefulness. similar results were observed in h-pgds ko mice. in contrast, l-pgds ko mice did not exhibit any significant changes in sleep-wake profiles after secl administrations. these findings indicate that pgd plays an essential role in the maintenance of the sleep state under physiological conditions, and l-pgds is a key enzyme for the production of pgd involved in sleep-wake regulation. under baseline conditions, h r ko mice showed essentially identical sleep-wakefulness cycles to those of wild-type (wt) mice but with fewer incidents of brief awakening (< s epoch), prolonged duration of non-rapid eye movement (nrem) sleep episodes, a decrease in the number of state transitions between nrem sleep and wakefulness, and a shorter latency for initiating nrem sleep after an intraperitoneal injection of saline. the h r antagonist pyrilamine mimicked these effects in wt mice. these results indicate that h r is involved in the regulation of behavioral state transitions from nrem sleep to wakefulness (huang et al., ) . ps a-h dissociation of responsibility in firing activity to dim light between the optic nerve and the suprachiasmatic nucleus neuron of mice koichi fujimura, ai fukushima, takahiro nakamura, toshihiro jogamoto, kazuyuki shinohara division of neurobiology & behaviour, nagasaki university, graduate school of biomedical science, nagasaki, japan the involvement of light response in the optic nerve to the firing activity of suprachiasmatic nucleus (scn) neuron was investigated by extracellular single unit recordings from the optic chiasma and the scn in mice. recordings were carried out during the early night in a light:dark cycle, and the illuminations were applied to a contralateral retina with a high-power led (λ = nm). the scn neurons responded to the light in intensities above photons/cm /s and were activated maximally at around photons/cm /s, they were about . log units less sensitive than optic fibers with high sensitivity. a sustained illumination in the intensity range between suprathreshold for the optic fibers and subthreshold for the scn neuron did not suppress the subsequent light response in the scn neurons, except in a few neurons. these results suggest that the most of the light responsive scn neurons are driven by any inputs independent of the high sensitive optic fibers. masayuki ikeda, tomoyoshi kojiya department of biology, faculty of science, toyama university, japan the hypothalamic suprachiasmatic nucleus (scn) has a pivotal role in the mammalian circadian clock. scn neurons generate circadian rhythms in action potential firings and neurotransmitter releases, and the core oscillation is thought to be driven by clock gene transcription-translation feedback loops. we have found robust circadian rhythms in the cytoplasmic concentration of ca + in scn neurons. since cytosolic ca + regulates diverse cellular systems, we have hypothesized that the cytosolic ca + rhythms may mediate the cellular output from the clock gene oscillations. here, to address the clock gene functions on the ca + rhythms, mouse bmal and its dominant negative sequence (mbmf r ) are transfected into the organotypic culture of scn with a yellow cameleon ca + sensor by the gene gun. the results demonstrated that over-expression of bmal or mbmf r significantly inhibited the circadian ca + rhythms and thus we concluded that the native bmal rhythm is essential for cellular output processes of the murine clock system. ps a-h the activation of ␣ adrenergic receptor increases the frequency of carbachol-induced ␤ oscillation in rat hippocampal slices masafumi nakano, jun arai, kiyohisa natsume kyushu institute of technology, kyushu, japan recently it is found that locus ceruleus (lc) activation suppresses ␤ rhythm in hippocampus in vivo. noradrenergic fibers derived from lc project to hippocampus. carbachol, a cholinergic agent, can induce ␤ oscillation in rat hippocampal slices like ␤ rhythm in vivo. in the present study, the effect of epinephrine on the generation of carbachol-induced ␤ oscillation in ca region of rat hippocampal slices. carbachol ( m) induced ␤ oscillation with the frequency and the amplitude of . ± . hz, and . ± . mv, respectively (mean ± s.e.m.; n = ). epinephrine ( m) significantly increased the frequency of . ± . hz (**p < . ), not change the amplitude. clonidine ( m), an ␣ receptor agonist, alone significantly increased the frequency at the concentration of m (*p < . ). yohimbine, an ␣ receptor antagonist, suppressed the oscillation. these results suggest that the application of adrenaline will increase the frequency of hippocampal ␤ rhythm via ␣ receptor. attractor dynamics of recurrent neural network are believed to play an important role in information processing in the brain. we recorded transient activities of two neuron groups by two tetrodes apart . mm from each other in the hippocampal ca region in vitro and applied micro-iontophoresis of glutamic-acid near the tetrodes to activate the neurons selectively. it was found that number of spikes during twosite (pairing) stimuli is fewer than the total number of spikes during the single-site stimuli, suggesting synaptic interaction in the network. peri-stimulus time histogram (psth) of ensemble as well as individual neuronal activity in response to the single-site stimuli applied far from the recording site composed of transient (latency - ms), oscillatory ( - hz) and sustained responses. following the pairing stimuli, the psth showed change in transient response properties ( / slices). these results suggest the pairing stimuli would change attractor dynamics of the neural network in the ca region. ryozo aoki , hiroshi wake , hitoshi sasaki , kiyokazu agata dept. physiol. & biosignal. osaka univ. grad. sch. med., suita, japan; dept. elec. eng. & elec. col. industri. tech., amagasaki, japan; dept. biophys., kyoto univ. grad. sch. sci., kyoto, japan by insertion of a stainless-steel monopolar electrode to the head of planarian, continuous waveform of electrical potential could be first observed in microvolts. the frequency spectrum showed an almost monotonously decreasing distribution likely as /f, ranging from − to + hz. during the eeg recordings the planarian was kept still by cooling in several degrees. when it was cooled down to lower temperatures the amplitude of eeg was suppressed, and by warming again restored with spikes provably due to motions. this eeg active state continued beyond min after the electrode insertion but the amplitude gradually decreased, and became natural noise at the time up to min. by observing the sample it turns out the sticked head was degraded. strong photo stimulation suppressed this eeg signals and recovered after over min. however little response to light pulse stimuli was observed on the eeg spectrum. mariko uchida , hiroki sato , , naoki tanaka , atsushi maki , japan science and technology agency, crest, saitama, japan; advanced research laboratory, hitachi, ltd., saitama, japan previous studies about electroencephalography (eeg) described that alpha-wave power (the frequency band from to hz) decreases and the sleep spindle power (from to hz) increases in falling asleep. the purpose of this study is to analyze the crosscorrelation between the eeg power changes (eegpc) of each band and the cortical hemoglobin concentration changes (hbcc) during sleep. we measured optical topography (ot) and eeg simultaneously. the hbcc was measured at eighty-eight positions covering whole head of subject by ot probes. five females and eight males participated in this measurement. the results showed the high correlation between eegpc and hbcc at the location of dorsolateral prefrontal area, both in the period of (i) dominance of alpha-wave and (ii) dominance of sleep spindle. the time lag from eegpc to hbcc was from to s in (i), and from to s in (ii). we examine these differences between (i) and (ii) in detail. carnitine deficiency disturbs fatty acid oxidation under the fasting condition (fc). we show herein that nocturnal locomotor activity (la) was reduced under fc and recovered to normal by carnitine injection in jvs −/− mice, a model of systemic carnitine deficiency. as judged from eeg/emg profiles, jvs +/+ mice showed prolonged wakefulness under fc, but jvs −/− mice revealed disruption of the prolonged wakefulness with a high frequency of non-rem sleep. as the orexinergic arousal system plays an important role in la, we determined orexin neuronal activity in the fasted mice. fasted jvs −/− mice had fewer c-fos + orexin neurons in their lateral hypothalamus and a reduced orexin-a content in their csf, suggesting that the fasted jvs −/− mice exhibited reduced la and fragmented of wakefulness due to suppressed orexin neuronal activity. juhyon kim , kazuki nakajima , yutaka oomura , kazuo sasaki div. of bio-information eng., univ. of toyama, toyama, japan; dept. of integrat. physiol., kyushu univ., fukuoka, japan novel peptide, orexin, identified in the lateral hypothalamus (lh) participates in the regulation of sleep-wakefulness. orexin-containing neurons in the lh project to the pedunculopontine tegmental nucleus (ppt). the ppt is one of brain sites which control sleepwakefulness. thus, we examined effects of orexin on ppt neurons electrophysiologically using brain slice preparations in rats. applications of orexin depolarized the membrane potential of ppt neurons dose-dependently, and the depolarization was associated with the increase in membrane resistance. when extracellular k + concentration was increased, the magnitude of the depolarization significantly decreased. when extracellular na + was replaced by n-methyl-dglucamine, the magnitude of the depolarization also decreased significantly. these results suggest that the ionic mechanism for orexininduced depolarization includes k + channel, non-selective cation channel and/or na + /ca + exchanger, and that orexin participates in the regulation of sleep-wakefulness via the excitatory effect on ppt neurons. ben-shiang deng , wei zhang , akira nakamura , masashi yanagisawa , yasuichiro fukuda , tomoyuki kuwaki , dept. molec. integ. physiol., chiba univ., japan; dept. autonom. physiol., chiba univ., japan; dept. molec. genet., univ. texas, usa we examined whether the respiratory chemoreceptor reflex in prepro-orexin knockout mice (ko) was blunted or not, and if so, whether supplementation of orexin restored the abnormality. we also studied whether pharmacological blockade of orexin in the wildtype mice (wt) resulted in a similar abnormality. ventilation was recorded by whole body plethysmography before and after intracerebroventricular injection of orexin-a, -b, sb- (an orexin receptor antagonist), or vehicle. data were examined for only awake periods because sleeping distorts the chemoreflex. hypercapnic ventilatory responses but not hypoxic responses were attenuated in ko. similar abnormality was reproduced in wt treated with sb- . icv injection of orexin partially restored the hypercapnic chemoreflex in ko. our findings suggest that orexin plays a crucial role for co -sensitivity at least during waking periods. research funds: kakenhi , junko hara , taizo matsuki , katsutoshi goto , masashi yanagisawa , takeshi sakurai , department of pharmacology, basic medical science (coe), university of tsukuba, ibaraki, japan; yanagisawa orphan receptor project, erato, jst, tokyo, japan; howard hughes medical institute and department of molecular genetics, university of texas, dallas, texas, usa when the production of inflammatory cytokines is stimulated by acute inflammatory, the nonrem-sleep amount of animals increases. this is possibly due to changes in the biological activity of the tnfalpha system. besides their important function in sleep regulation during acute immune response, cytokines also seem to be involved in physiological sleep regulation. orexins (hypocretins) are recently identified neuropeptides that are derived from a common precursor peptide. recent studies suggest that specific degeneration of orexincontaining neurons occurs in brains of human narcolepsy patients, suggesting critical roles of these neurons in the regulation of vigilance states. here, we examined the effects of inflammatory cytokines on the activity of orexin neurons, by means of patch-clamp recording. these effects might also possibly be involved in the pathophysiology of narcolepsy. ps a-i prenatal exposure to bisphenol a enhances avoidance response to predator odor and impairs sexual differentiation of olfactory response of medial amygdala neurons tetsuya fujimoto , kazuhiko kubo , shuji aou dept. brain sci. eng., kyushu inst. technol., kitakyushu, japan; dept. otorhinolaryngol., chidoribashi hospital, fukuoka, japan prenatal exposure to bisphenol a (bpa) impairs the sexual differentiation of exploratory behavior and enhances depressive behavior (fujimoto et al. ) . in this study, the effects of bpa on general motor activity and avoidance response to predator odor and olfactory responses in medial amygdala neurons were examined. the smell of fox predominantly suppressed locomotor activity and enhanced avoidance response by bpa. in the electrophysiological study, male medial amygdala neurons showed selective excitatory responses to predator odors. this type of neurons did not respond to plant odors. in contrast female amygdala neurons did not show such selectivity. the sex difference in this neuronal response pattern was attenuated by bpa exposure. these findings suggest that bpa impairs sexual differentiation of medial amygdala neurons which affect emotional responses to the olfactory cues of predators. research funds: grants-in-aid for scientific research (no. , s.a.) shuji aou , tetsuya fujimoto , yumi ichihara , kimiya narikiyo , toru ishidao , hajime hori , yukiko fueta dept. brain sci. eng., kyushu inst. technol., kitakyushu, japan; dept. environm. manage., sch. health sci., univ. occup. environm. health, kitakyushu, japan -bromopropane ( -bp), an ozone-depleting substance replacement, has neurotoxicity and exhibited reproductive toxicity in adult animals. in this study, we investigated the effects of prenatal exposure to -bp on sexual differentiation of reproductive and non-reproductive behaviors. pregnant rats were exposed to ppm of -bp during prenatal period. the open-field test, lashley iii maze test and sexual behavior were evaluated at adult age. -bp significantly reduced the locomotor activity and the number of entries into the center area in female rats but not in males in the open-field test. in sexual behavior, the number of ear wiggles, an index of proceptive behavior, was decreased and the rejection score was increased in female rats. these results suggest that -bp is the potential candidate of endocrine disruptors which affect brain development. ( ) ps a-i changes in hippocampal excitability of rats prenatally exposed to -bromopropane yukiko fueta , toru ishidao , susumu ueno , yasuhiro yoshida , hajime hori department of environmental management, school of health sciences; department of pharmacology; department of immunology, school of medicine, university of occupational and environmental health, kitakyushu, japan inhalation exposure to -bromopropane ( -bp), a substitute for ozone depleting compounds, alters the function of gabaergic system in the hippocampus of adult male rats. but the neurotoxcitiy induced by prenatal exposure has not been well investigated. in this study pregnant rats were exposed to -bp ( ppm) during gestational day - ( h/day), and the hippocampal excitability in pregnant rats and their offspring was examined. basic excitability was enhanced and disinhibition was observed in the hippocampus of pregnant rats. offspring, however, exhibited an enhancement of averaged s/r curve of ps in the ca at the pnd - . conversely, s/r curves of fepsp as well as ps in the ca were inhibited at the age of - weeks. our results suggest that -bp causes hyperexcitability in pregnant rats, and disrupts basic excitability in the ca of the offspring during development. research funds: grant-in-aid for exploratory research ( ) ps a-i effects of endocrine disrupting chemical bisphenol a on the development of mouse cerebral cortex keiko nakamura , , kyoko itoh , takeshi yaoi , tohru sugimoto , shinji fushiki dept. pathol. appl. neurobiol., kyoto pref. univ. med, kyoto, japan; dept. pediatr., kyoto pref. univ. med, kyoto, japan bisphenol a (bpa), a widely distributed xenoestrogen, has been shown to disrupt thyroid hormone function. we have thus studied whether prenatal exposure to low-doses of bpa affects morphology and the expression of thyroid hormone-dependent genes in murine fetal neocortex. pregnant mice were injected subcutaneously g/kg of bpa daily from embryonic day (e ). control animals were injected vehicle alone. for evaluating cell proliferation, neuronal differentiation and migration, bromodeoxyuridine (brdu) was given to pregnant mice and processed for immunohistochemistry. the total rna was extracted from embryonic telencephalons at different embryonic period. brdu-labeled cells were decreased in the ventricular zone at e . and e . , whereas those cells increased in the cortical plate at e . , as compared with control mice. some of the genes associated with neurogenesis and thyroid hormone function were upregulated in bpa-treated group. research funds: jsps grant keiko ikemoto , teruko uwano , hisao nishijo , taketoshi ono , masayuki ito , ikuko nagatsu , katsuji nishi , shin-ichi niwa dept. neuropsychiat., fukushima med. univ. sch. med.; toyama med. pharm. univ.; faculty med., mie univ., mie, japan; fujita health univ. sch. med., toyoake, japan; dept. leg med., shiga univ. med. sci., japan we examined the effect of maternal repeated cold stress (rcs) on development of catecholamine neurons of offsprings using by tyrosine hydroxylase (th) immunohistochemistry. rcs was loaded to pregnant rats between day and after fertilization. pups were perfused at postnatal day . in the frontal cortex, the number of largesized (more than m in diameter) th-immunoreactive (-ir) varicosities was significantly smaller in prenatally rcs rats than controls. in the locus coeruleus of prenatally rcs rats, th immunoreactivity was less than that of controls. in the medullary c /a catecholaminergic field, the size of th-ir neurons was smaller and the quantity of thir fibers were less in prenatally rcs rats, although there were not significant differences. it was suggested that prenatal rcs impaired development of catecholaminergic neurons, especially noradrenergic neurons of neonates. ps a-i developmental exposure to pentachlorophenol affects thyroid hormone responsive gene in the brain but not stress response maiko kawaguchi , , , kaori morohoshi , , rie yanagisawa , erina saita , gen watanabe , , masatoshi morita , kazuyoshi taya , , hirohisa takano , , toshiyuki himi , , hideki imai , dept. toxicol and pharmacol., facul. pharmacy, musashino univ., tokyo, japan; res. inst. pharmaceut. sci., musashino univ., tokyo, japan; nation. inst. for environ. stud., ibaraki, japan; grad. sch. environ. sci., univ. tsukuba, ibaraki, japan; wildlife rescue veterinarian associ., tokyo, japan; facul. agriculture, tokyo univ. agriculture & technol., tokyo, japan; the united grad. sch. veterinary sci., gifu univ., gifu, japan; div. environ. health sci., dep. social med., facul. med., miyazaki univ., miyazaki, japan antiseptic pentachlorophenol (pcp) treatment to rats affects thyroid hormone (th) system, which is essential for normal development of central nervous system. in this study, we show the exposure to pcp during gestation and lactation suppressed plasma th level, and induces gene expression of neurogranin and th receptor ␤, which play a role in neural formation. the present data suggest that pcp may affect central nervous system development, though stress response was not affected by pcp exposure. ps a-i the effect of psychological stress during pregnancy on the open-filed behavior, the forced swim test, the fos expression in the brain, and the level of plasma corticosterone in offspring rat hiroshi abe, noriko hidaka, kei odagiri, yuko watanabe, yasushi ishida dept. of psychiatry, miyazaki med. coll., univ. of miyazaki, japan one group (psy) was born from the dams which observed, during their pregnancy, that another rat was exposed to the foot-shock stress in a communication box. the other group (c) was born from the dams not exposed to such stress. psy, comparing to c, showed decreased activities in the open-field test and prolonged immobility time in the forced swim test. on the other hand, there were no significant differences between the number of fos immunopositive cells in various regions of the brain in two groups before and after the foot-shock. however, plasma corticosterone was elevated in psy compared with c. these results suggest that the prenatal psychological stress might enhance reactivity to novel environment and depressive behavior induced by forced swim, and chronically elevated level of corticosterone might be involved in this neurobiological substrate. akane nakasato, yasushi nakatani, yoshinari seki, hideho arita department of physiology, toho university school of medicine, tokyo, japan to evaluate roles of da and serotonergic ( -ht) systems in stressinduced anxiety, we measured brain da and -ht levels before, during and after a forced swimming test (fst) in autistic model of the rat. the model rat was made by exposing a pregnant rat to valproic acid (vpa). our previous study demonstrated that the autistic model exhibited abnormality of -ht system and behavioral impairments related to autism. in the present experiment, we gave a prolonged fst for min in the model rat, which frequently experienced to be drowned after the immobility time during fst. brain da and -ht levels were measured from samples collected from the prefrontal cortex (pfc). we found a gradual and steady increase in pfc da level during fst, although -ht level showed only transient augmentation. behavioral alteration after fst was characterized by an increased appetite during light phase (sleep) of circadian cycle. we suggest that the feeding abnormality may be caused by the stress-induced anxiety mediated by mesocortical da system. shigeo masaki, eiko aoki, satoshi yonezawa, atsuo nakayama dept. embryology, inst. developmental res., kasugai, aichi, japan neuroligin (nl - ) is a family of neuronal cell-surface proteins to be involved in intercellular junctional formation and signalling. recently, several studies have implicated nl and nl in autistic disorders. nls have a relative identical structure (∼ %); nl and nl localize in the glutamatergic excitatory, and inhibitory synapses, respectively, while nl seems express in the olfactory glia, but nl distribution is unknown. here we have generated antibody against human nl , and explored its distribution in the post mortem human tissues. in the central and peripheral nervous system, nl was expressed exclusively in the neurons, and was especially abundant in particular subsets of neurons, including neurons producing nonapeptides. nl was observed in paraneurons and some endocrine cells outside the cns. these results suggested that nl is important for neuroendocrine function. nl cdna was transfected to in neuroblastoma. formed spine-like structures on the cells expressing nl were rough and thicker than those of nl or nl transformants. it suggested the unique activity of nl for synapse formation. motopsin is a serine protease secreted from pyramidal neurons of cerebral cortex and hippocampus. recently, the truncation of motopsin gene has been reported to cause non-syndromic mental retardation. however, the underlying mechanisms are yet to be elucidated. we report here that the knockout (ko) mice deficient in the expression of motopsin exhibit morphological abnormality. golgi-cox staining revealed that spine density on both apical and basal dendrites of hippocampal ca pyramidal neurons in the ko mice significantly decreased than in the wild type mice. similarly, spine density tended to decrease at cingulate cortex of the ko mice than wild type. our results suggest that motopsin affects dendritic spine formation and/or stabilization. mental retardation is a frequent disorder affecting - % of the population. recently the truncation of motopsin/neurotrypsin gene has been identified in algerian family in which four out of eight children affected by a severe impairment of cognitive functions with an iq below . here we report that knockout (ko) mice lacking motopsin gene mildly impaired water maze performance and social behavior. the ko mice significantly delayed the latency to the platform area on a probe test of hidden version of morris water maze although they showed the similar performance to wild-type mice during training session. in a social memory test, the ko mice showed significant elongation of sniffing time to an intruder, despite of normal performance of social memory. our results suggest that the ko mice provide insights into the molecular mechanisms important for development of cognitive functions. natsue yoshimura , daisuke horiuchi , tomoyuki miyashita , minoru saitoe , hitoshi okazawa department of neuropathology, medical research institute, tokyo medical and dental university, tokyo, japan; tokyo metropolitan institute for neuroscience, tokyo, japan polyglutamine tract binding protein- (pqbp- ) was originally isolated as one of the candidates for polyglutamine disease related protein. recently, several groups has reported about pqbp- disease that pqbp- mutant causes x-linked mental retardation (xlmr). to investigate the function of pqbp- in xlmr pathology, we produced two kinds of flies, human pqbp- overexpression flies (hpqbp flies) and drosophila pqbp- knock-down flies (dpqi flies), and examined olfactory learning and memory to analyze their memory consolidation process from short-term memory (stm) to long-term memory (ltm). the hpqbp flies showed memory impairment in ltm. in current study, we analyze memory abilities of the dpqi flies to observe detailed function of pqbp- in memory formation. seiji hayashizaki, masahiko takada tokyo metropolitan institute for neuroscience, usa when two alternatives are available in instrumental behavior, animalǐs behavior is biased toward responding on one lever with which each behavioral response results in delayed large reward delivery, and against responding on the other lever with which each response results in immediate but small reward delivery. this has been used as an index of impulsive behavior and is known to be susceptible to lesions of brain structures such as the basolateral amygdala (bla) and the nucleus accumbens (na). it has been shown that the bla and na are involved in maintaining reward seeking behavior with a secondary reward when a secondary reinforcer is available. thus, a question arises as to how the behavioral response on the delayed lever is maintained through functions exerted by these structures when no secondary reinforcer is available. to this end, we implanted cannulae bilaterally and electrodes into the bla and na to identify neuronal substances and activities involved in the mediation of 'putative secondary reward' without secondary reinforcer. xue-zhi sun , sentaro takahashi , yoshihisa kubota , rui zhang , chun cui , yoshihiro fukui natl. inst. radiol. sci., chiba, japan; sch. med. tokushima univ., tokushima, japan heavy ion irradiation has the feature to administer a large radiation dose in the vicinity of the endpoint in the beam range, and its irradiation system and biophysical characteristics are different from ordinary irradiation instruments like x-or gamma-rays. using this special feature, heavy ion irradiation has been applied for cancer treatment. the safety and efficacy of heavy ion irradiator have been demonstrated to a great extent. for instance, brain tumors treated by heavy-ion beams became smaller or disappearance. however, fundamental research related to such clinical phenotypes and their underlying mechanisms are little known. in order to clarify characteristic effects of heavy ion irradiation on the brain, we developed an experimental system for irradiating a restricted region of the rat brain using heavy ion beams. the characteristics of the heavy ion beams, histological, behavioral and elemental changes were studied in the rat following heavy ion irradiation. yukio imamura department of psychiatry, university of ottawa, on, canada nmdars contain two nr subunits paired with two nr subunits. nr and nr (a-d) subunits harbor the glycine and glutamate binding sites, respectively. nmdars are localized in both synaptic and extra-synaptic areas, but they are found at higher density within the synapse. after the peak of synaptogenesis, the nr /nr a complex, characterized by rapid offset kinetics, dominates at the synapse, while the nr /nr b complex, characterized by slow kinetics, predominates in the extra-synaptic area. the activation of extrasynaptic nmdars by glutamate escaping from the synaptic cleft during episodes of high synaptic activity suggests that they may have a different role. using whole-cell voltage-clamp recordings from ca pyramidal neurons from mice (at weeks of age), we found that following induction of ischemia, ifenprodil, a selective nmdar-nr b antagonist, reduced the inward current of the isolated nmdar at extra-synaptic site while it had less effect at the synaptic nmdar. the molecular mechanisms involved are currently under investigation and these new data will be also presented at the meeting. in the present study, we observed expression and changes of mineralocorticoid receptor (mr) and glucocorticoid receptor (gr) in the gerbil hippocampal ca region after ischemia. in blood, corticosterone levels increased biphasically at min and h after ischemia, and thereafter its levels decreased. in the sham group, mr and gr immunoreactivities were weakly detected in the ca region. by days after ischemia, mr and gr were not significantly altered in the ca region. from days after ischemia, mr and gr immunoreactivities were detected in astrocytes and microglia in the ca region, and at days after ischemia. the specific distribution of corticosteroid receptors in glia may be associated with the differences of mr and gr functions against ischemic damage. the present study was investigated the effects of early treadmill training after cerebral infarction in rats. we determined whether treadmill exercise changes cellular expression of caspase- and midkine in the mca area. stroke was induced by a -min mca occlusion using an intraluminal filament. rats were exercised for min each every day on a treadmill. brain damage in ischemic rats was evaluated by infarct volume. exercised and non-exercised rat brains were processed for immunocytochemistry to quantify the areas of caspase-and mkimmunoreactive calls. no significant differences in infarct volume were found between rats trained with treadmill and non-exercised controls. cellular expressions of mk were significantly increased in striatum (glia) of the exercised rats. treadmill exercise was shown to suppress the decrease in caspase- expression in the penumbra. the present study showed the exercise after cerebral infarction might have important implication for post-ischemic recovery. ps a-j reversed astrocytic glutamate transporter glt- crucial to the ca + paradox-like insult-induced neuronal death in neuron/astrocyte co-cultures tatsuro kosugi, koichi kawahara, takeshi yamada, motoki tanaka lab. of cellular cybernetics, graduate school of information science and technology, hokkaido univ., sapporo, japan "ca + paradox" is the phenomenon whereby the intracellular concentration of ca + paradoxically increases during reperfusion with normal ca + -containing media after brief exposure to a low ca + solution. the present study aims to characterize the ca + paradoxinduced cell injury in neuron/astrocyte co-cultures. prior exposure of the cultures to a low ca + solution for min significantly injured only neurons after reperfusion with a normal ca + medium for h, but astrocytes remained intact. after the onset of reperfusion, the intracellular concentration of na + in astrocytes increased significantly during the reperfusion episode, resulting in a reversal of the operation of the astrocytic glt- . the present findings suggested that ca + paradox-induced accumulation of na + in astrocytes was involved in the reperfusion-induced excitotoxic neuronal injury resulting from the reversed operation of astrocytic glt- during the reperfusion episode. common genetic mutation in cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (cadasil) has been associated with missense mutations of notch concerning cysteine residues within the extracellular amino-terminal region. we report new mutations of two japanese cadasil families, which did not directly involve a cysteine residue. exons of the notch were amplified by pcr and subsequently analysed for dhplc and direct sequence. the first patient carried the missense mutation c t, which results in pro ser. the second patient carried the missense mutation c g, which results in arg pro. new mutations had not changed the number of cysteine residues, but coding the extracellular amino-terminal region of the notch receptor which may involve an alteration in the ligand binding or putative dimerisation properties. ps a-j mci - , a radical scavenger, protected cortical neurons from cell death through the activation of mitogen-activated protein kinase and phosphatidylinositol kinase madinyet niyaz , tadahiro numakawa , yoshinori matsuki , emi kumamaru , yuki yagazaki , harumi kitazawa , hiroshi kunugi , motoshige kudo pathology department of tokyo medical university, tokyo, japan; department of mental disorder research, national institute of neuroscience, ncnp, tokyo, japan the role of mci - , a radical scavenger, in the central nervous system (cns) has not been fully elucidated. in the present study, we found that treatment with mci - prevented the cultured cortical neurons from cell death induced by serum deprivation. furthermore, we found that mci - exposure induced the activation of both the map kinase (mapk) and pi kinase (pi k) pathways and that the mci - -dependent survival effect was blocked by the inhibitors, u (an mapk pathway inhibitor) or ly (a pi k pathway inhibitor). these results suggested that mci - exerts a protective effect on cns neurons via enhancing survival-signaling pathways in addition to a role such as a radical scavenger. osamu tokumaru , noriko yoshimura , tetsuro sakamoto , takaaki kitano , naoko nisimaru , isao yokoi dept. physiol., sch. med., oita univ., japan; med. edu. ctr., sch. med., oita. univ., japan protective effects of ethyl pyruvate (ep) on energy metabolism of rat brain exposed to ischemia were investigated by p-nuclear magnetic resonance ( • c). brain slices were incubated in standard artificial cerebrospinal fluid (acsf) with mm ep (ep- ), acsf replaced by acsf with mm ep after ischemia (ep- ), or acsf only (control). the brain slices were exposed to ischemia by stopping the perfusion for h. high-energy phosphate, creatine phosphate (pcr) and ␥-atp, levels were measured. decrease in pcr level was not different among the three groups when exposed to ischemia. but increase in pcr level after the reperfusion was significantly larger in ep- than in control (p < . ). these results indicate that ep is effective in the reperfusion period and is more protective when administered before ischemic exposure. the importance of timing of administration of ep in clinical use was suggested. research funds: grant-in-aid for scientific research (c) # from mext to t.k. hideaki tamai , kuniko shimazaki , norimasa seo department of anesthesiology and critical care medicine, jichi medical university, graduate school, tochigi, japan; department of physiology, jichi medical university, tochigi, japan we investigated the effects of acupuncture on cell proliferation in the dentate gyrus (dg) and the lateral ventricle (lv) of adult rats. in this study, acupuncture was performed at the acupoints neiguan (pc ), yintang (ex-hn ) and sanyinjiao (sp ), which have been used for the enhancement of conscious and functional recovery in stroke patients. eight weeks old male wistar rats were used in the experiment. through -bromo- ,-deoxyuridine (brdu) immunohistochemistry, a significant increase in cell proliferation in the dg of the acupunctured group was observed. however, the cell proliferation in the lv was not affected with the acupoints pc , ex-hn and sp . the present findings indicate that the sensitivity on cell proliferation in the dg by acupuncture stimulation is higher than in the lv. yukio ago , keiko takahashi , shigeo nakamura , akemichi baba , toshio matsuda laboratory of medicinal pharmacology, graduate school of pharmaceutical sciences, osaka university, osaka, japan; laboratory of molecular neuropharmacology, graduate school of pharmaceutical sciences, osaka university, osaka, japan this study examined the effect of isolation rearing on anxiety-related behavior of mice in the staircase test, an animal model of anxiety. the staircase test consisted of placing an experimentally naive mouse in an enclosed staircase with five steps. in group-reared mice, an anxiolytic diazepam increased the number of steps climbed to the top step of the staircase, but did not affect the frequency of rearing behavior. the anxiogenic drug ␤-cca increased the number of rearing, but did not affect the number of steps climbed. on the other hand, methamphetamine increased the number of steps climbed to the second step. in these circumstances, isolation-reared mice showed an increase in the numbers of steps climbed to the top step and rearing in the staircase. these findings suggest that isolation rearing increases in exploratory and anxiety-like behaviors in mice. tomonori fujiwara , tatsuya mishima , takefumi kofuji , kimio akagawa department of cell physiology, kyorin university school of medicine, mitaka, tokyo, japan; radio isotope laboratory, kyorin university school of medincine, mitaka, tokyo, japan hpc- /syntaxin a is believed to regulate the exocytosis of synaptic vesicles. in order to examine the neurophysiological function in vivo, we have produced hpc- /syntaxin a knock-out mice. surprisingly, the null mutant mice revealed normal development and basal synaptic transmission in cultured hippocampal neurons appeared to be normal. however, in conditioned fear memory test, consolidation of the memory was impaired in homozygous mutant mice but not in heterozygote. however, once memory consolidation was acquired, the extinction process was disturbed in homozygote. we further examined latent inhibition of cued fear memory (li) to access behavioral property. interestingly, li was suppressed both in heterozygous and homozygous mutant mice unlike the case of conventional conditioned fear memory test. implication of these behavioral abnormalities in hpc- /syntaxin a knock-out mouse will be discussed. research funds: kakenhi ( ) ps a-k effects of local administration of the gaba agonists into the hippocampus ca area on active avoidance learning and serotonergic systems in the administration area in rats satoko hatakenaka , hiroko miyakubo , junichi tanaka , yasushi hayashi , yukio hattori , masahiko nomura department of curriculum, teaching and memory, naruto university of education, tokushima, japan; department of human nutrition, notre dame seishin university, okayama, japan; department of physiology, saitama medical school, saitama, japan in fischer male rats, bilateral injections of the ␥-aminobutyric acid (gaba) a agonist muscimol into the ca area slightly decreased the avoidance rate in an active avoidance task. similar injections of the gaba b agonist baclofen enhanced the avoidance rate. there are significant differences between the muscimol-and baclofen-treated groups in the avoidance rate, implying that gaba a and gaba b receptors have the opposite action on the performance of avoidance learning. perfusion with muscimol through the microdialysis probe decreased the serotonin metabolite -hydroxytryptamine ( -hiaa) concentration in the ca area, whereas baclofen perfusion had no effect, suggesting that the gabaergic system may exert to inhibit the serotonin release in the ca area through gaba a receptors. sawako arai, taku nagai, kenji takahashi, hiroyuki kamei, kazuhiro takuma, kiyofumi yamada lab. neuropsychopharmacol, kanazawa univ., kanazawa, japan we performed immunohistochemical c-fos mapping after a prepulse inhibition (ppi) test of the startle reflex in mice. startle stimulus increased the number of c-fos-positive cells in the somatosensory cortex, nucleus accumbens shell and the caudal pontine reticular nucleus (pnc), while prepulse trials without startle stimulus increased c-fos expression in the lateral globus pallidus (lgp). in mice subjected to startle stimulus with prepulses, most of the startle stimulus-induced c-fos expression was diminished but c-fos expression remained in the lgp. prepulse-induced c-fos expression in the lgp was colocalized with gad- . fluoro-gold infusion into the pnc and the pedunculopontine tegmental nucleus (pptg) retrogradely labeled neurons in the pptg and lgp, respectively. microinjections of phaclofen, but not picrotoxin, into the pptg impaired ppi of the startle reflex. these results suggest that gabaergic neurons in the lgp which project to the pptg play a crucial role through the activation of gaba b receptors in the ppi of the startle reflex. shiho kitaoka , sho koyasu , akinori nishi , tomoyuki furuyashiki , toshiyuki matsuoka , shuh narumiya department of pharmacology, university of kyoto, kyoto, japan; department of physiology, university of kurume, kurume, japan prostaglandins e (pge ) exert their actions in various organs through specific receptor, ep to . the previous study suggests that ep modulates da system. to investigate the roles of ep in da system, we examined ep ko mice with behavioral sensitization induced by cocaine. the administration of cocaine elevated da concentration in the nucleus accumbens up to ∼ % in both wild-type and ep ko mice. however, increase of locomotor activity in ep ko mice was significantly lower than that in wild-type mice. because locomotor activity is closely related to dopamine d receptor (d r) signaling, we tested the density of d r and d r signaling with phosphorylation of darpp- . there were no differences in d r binding. d r signaling was significantly attenuated in the striatal slices from ep ko mice. the effect of d r agonist on locomotor activity was also attenuated in ep ko mice. these results indicate that pge has enhancing effects on locomotor activity via ep by potentiating the d r signaling. central serotonin ( -ht) function has been implicated in impulsivity. the present study examined rats with -ht depletion by parachloroamphetamine (pca) in simple and reversal go/no-go visual discrimination tasks, and analyzed the relationships between learning performance and focal concentrations of -ht and its metabolites ( -hiaa) in the brain. for both tasks, significant negative correlations between learning performance and -ht and -hiaa concentrations were observed in the medial prefrontal cortex and nucleus accumbens. in contrast, for reversal task only, significant correlations between learning performance and -ht and -hiaa concentrations were observed in the orbitofrontal cortex and amygdala. these data suggest the regional difference of -ht roles on selective indices of impulsivity. yuki sato , , , tatsushi onaka , norimasa seo , eiji kobayashi dept. anesthesiol., jichi med. univ., tochigi, japan; dept. physiol., jichi med. univ., tochigi, japan; div. organ replacement research, center for mol. med., jichi med. univ., tochigi, japan cyclosporine is widely used for preventing allograft rejection. however, in a considerable number of transplant recipients, cyclosporine causes neuropsychological side effects such as confusion, depression, and anxiety. cyclosporine inhibits calcineurin activity and forebrain-specific calcineurin knockout mice exhibit deficits in social behaviour. it is thus possible that cyclosporine causes psychological side effects via disturbing social interactions. here, we examined effects of cyclosporine upon anxiety and social behaviour in mice. calcineurin did not significantly change percent entries into open arms and time spent on open arms in the elevated plus maze test. on the other hand, in the social interaction test in home cage, cyclosporine increased the number of particles in home cage, an index of social activity. all these data suggest that impaired social interaction is a cause of psychological side effects of cyclosporine. to investigate the distribution of functionally activated vestibularrelated brainstem neurons during postnatal development, ombined immuno-/hybridization histochemistry of c-fos expression was performed in sprague-dawley rats (p - ; adult). conscious animals were subjected to rotational or translational stimulus which activates hair cells of the horizontal semicircular canals or utricle, respectively. neuronal activation within brainstem nuclei was defined by the expression of c-fos. labyrinthectomized controls and normal stationary controls showed only a few sporadically scattered fos-expressing neurons. with rotational stimulation that comprised cycles of constant angular acceleration and deceleration, fos-labeled neurons were observed by p in the vestibular nucleus and downstream relay stations of vestibular pathways, such as the prepositus hypoglossal nucleus and inferior olive (subnuclei dmcc, ioa, ioc, iok). a later maturation time was evidenced for the utricular system. fos-labeled neurons were only identifiable in the vestibular nucleus by p ; in the prepositus hypoglossal nucleus and inferior olive (subnuclei dmcc and io␤) by p . within the vestibular nucleus of p - rats, neurons activated by canal or utricular inputs were intermingled throughout its rostro-caudal length. in p and adult rats, neurons activated by canal or utricular inputs were intermingled in localized regions of the medial and spinal vestibular nuclei. however, neurons in the rostral half of spinal vestibular nucleus were activated only by utricular inputs. taken together, we have demonstrated that canal-and otolithrelated brainstem neurons that encode rotational and translational movements in the horizontal plane are histologically segregated and exhibit different developmental time frame. to determine whether perineuronal nets (pn) within the vestibular nuclei contribute to plasticity of central connectivity, we studied the presentation of pn within the vestibular nuclei during development (rats, p to adult) and after unilateral labyrinthectomy (ul) in the adult. histochemistry with the lectin wisteria floribunda agglutinin was used to map pn about neun-immunopositive neurons within the vestibular nuclei. in normal postnatal rats, pn was detectable by p in the vestibular nucleus as fuzziness about neuronal cell bodies. from p onwards, the fuzzy pn progressively consolidated into a network organization. the fuzziness was no longer observable after p . during postnatal development, the number of neurons showing pn increased with age, reaching the adult level by p . with ul, the pn network on the lesioned side remained compact until days post-lesion when the fuzziness reminiscent of that in early postnatal rats became evident. by days after ul, the pn of some neurons resumed the network pattern as was observed in normal adult rats. this phenomenon was found in the pn of the remaining neurons by days after ul. the pn on the labyrinth-intact side showed the compact network of uninjured age-matched rats. taken together, our findings indicate pn changes that suggest possible correlation with vestibular nuclear neuronal function both during postnatal development of normal rats as well as in adult rats following destruction of the ipsilateral inner ear. minori ueda, takayuki suzuki, hiroyoshi miyakawa laboratory of cellular neurobiology, tokyo university of pharmacy and life science, tokyo, japan dynamics of transmitters in the synaptic cleft depends on many processes such as transmitter release, uptake and diffusion. to better understand these processes, we analyzed ampar-and nmdarmediated epscs and synaptically induced transporter currents (stcs) elicited with high-frequency stimuli. recordings were made from pyramidal cells and astrocytes in the ca region of rat hippocampal slices, hz/ pulse tetanic stimulations were delivered to schaffer-collaterals, and the evoked currents during the course of tetanic stimulation were isolated. the decay time course of the last isolated stc during the tetanic stimulation was not significantly different from that of the first. while the amplitude of the ampar-mediated epscs showed significant decay in the presence of cyclothiazide, there was no marked decay of the amplitude of the nmdar-mediated epscs. these findings imply that synaptic fatigue and saturation of glutamate transporters do not take place during the course of high-frequency stimulation at hz. ikuko yao , hiroshi takagi , hiroshi ageta , tomoaki kahyo , ken hatanaka , kaoru inokuchi , mitsutoshi setou , mitsubishi kagaku institute of life sciences, tokyo, japan; university of tokyo, tokyo, japan; okazaki institute for integrative bioscience, national institute for physiological sciences, okazaki, japan we identified and characterized a novel ubiquitin ligase named scrapper. scrapper is an f-box protein which has leucine rich repeat and c-terminal membrane localization sequence, highly expressed in neurons throughout the brain. to investigate the physiological role of scrapper in the neuron, we recorded mepscs from the neuron over-expressed the egfp-tagged full-length scrapper construct or truncated form of scrapper constructs. they exhibited a strong suppression or enhancement in the frequency of mepscs while showing a non-significant change in mepsc amplitude, rise, and decay time compared with neurons expressing egfp. the passive membrane properties of neurons such as membrane resistance (rm), series resistance (rs), and membrane capacitance (cm) were not statistically different from those of control. these data suggests a presynaptic effect of scrapper protein. ps p-a presynaptic membrane potential-dependent regulatory mechanism of transmitter release tetsuya hori, tomoyuki takahashi department of neurophysiology, university of tokyo graduate school of medicine, tokyo, japan in simultaneous pre-and postsynaptic recordings at the calyx of held, we addressed the mechanism underlying presynaptic membrane potential-dependent changes of transmitter release. a weak sustained depolarization (e.g., mv, s) of calyceal nerve terminal potentiated epscs despite that it diminished presynaptic action potential (a.p.) amplitude. as we further depolarized the terminal epscs became eventually depressed concomitantly with a marked reduction in the a.p. amplitude. when presynaptic ca + currents (i pca ), induced by an a.p.-waveform command pulse, were used to evoke epscs, a weak sustained depolarization enhanced i pca and epscs in parallel. this epsc facilitation was robust at the calyx of held both in rats and mice, but was almost absent in p/q-type ca + channel knockout mice. we conclude that the p/q-type specific ca + channel facilitation plays an essential role in the facilitation of transmitter release following presynaptic depolarization. hiroshi takagi , koji ikegami , ken hatanaka , , , yoko fujiwara-tsukamoto , mineo matsumoto , ikuko yao , mitsutoshi setou , , mitsubishi kagaku institute of life sciences, japan; presto, japan; school of pharmaceutical sciences, the university of tokyo, japan; okazaki institute for integrative bioscience, japan a variety of post-translational modifications to the exposed cterminal tails of tubulin, such as detyrosination/tyrosination, polyglycylation and polyglutamylation would play a crucial role in the neuron. however, evidence for the implication of these modifications in regulating the translocation of channels and receptors is currently unavailable. of the modifications, polyglutamylation is highly abundant in the mammalian brain, thus, this modification might account for the translocation of channels and receptors in the mammalian brain. in the rosa (−/−) mouse, which shows a gross loss of polyglutamylated ␣-tubulin, transient a-type currents were largely suppressed in hippocampal pyramidal neurons in vitro. we provide herein, using rosa mice, the evidence for the implication of ␣tubulin polyglutamylation in the regulation mediated a-type k current. satoshi kawasaki , shingo kimura , reiko fujita , shuji watanabe , kazuhiko sasaki dept. of physiol., sch. of med., iwate medical univ., morioka, japan; dept. of chem., sch. of lib. arts & sci., iwate medical univ., morioka, japan application of dopamine (da) induces a slow na + -current response in the identified neurons of aplysia ganglia under voltage clamp. this type of response is produced by the activation of trimeric g-protein sensitive to cholera toxin (ctx) as previously reported. the na +current response to da was gradually and irreversibly depressed after intracellular injection of clostridium difficile toxin b, which is known to inactivate all types of rho family g-proteins. intracellular application of clostridium botulinum exoenzyme c , a specific toxin to rhoa-c, also depressed the da-induced response irreversibly. furthermore, the da-induced current response was significantly depressed by gap domain of p rhogap applied intracellulary. in contrast, gef domain of rhogef dbs had a tendency to increase the response. these results suggest that the da-induced na + -current response may be regulated by the activation of rho family g-protein. the ␦ glutamate receptor (␦ r) plays a crucial role in cerebellar functions. although ␦ r has a putative channel pore domain, and ␦ r displayed ca + -permeable channel activities in lurcher mutant mice, it has been unclear whether wild-type ␦ r functions as a channel. here we introduced a ␦ r transgene, which had a mutation (gln arg) in the putative channel pore conserved in ca + -permeable glutamate receptors, into ␦ −/− mice. surprisingly, a mutant ␦ r transgene, as well as a wild-type transgene, rescued all abnormal phenotypes of ␦ −/− mice, such as ataxia and loss of long-term depression. these results indicate that ca + influx through ␦ r is not required for its function in the cerebellum in vivo, and that wild-type ␦ r may not function as a ca + -permeable ion channel. research funds: kakenhi ( ) and takeda science foundation ps p-a distribution of tarp - on hippocampal neurons and its key role in synaptic and extrasynaptic expression for ampa receptors masahiro fukaya , mika tsujita , maya yamazaki , etsuko kushiya , manabu abe , kaori akashi , masanobu kano , haruyuki kamiya , kenji sakimura , masahiko watanabe department of anatomy, hokkaido university school of medicine, sapporo, japan; department of cellular neurobiology, brain research institute, niigata, japan; department of cellular neuroscience, graduate school of medical science, osaka university, suita, japan; department of molecular anatomy, hokkaido university school of medicine, sapporo, japan the - is one of four transmembrane ampar regulatory proteins (tarps). pre-and post-embeding immunogold visualized - on excitatory synaptic and extrasynaptic membrane. in - -ko mice, ampars were reduced in hippocampal homogenates ( % of control) and psd fraction ( %). immunogold labeling also exhibited reduction of extrasynpatic ( %) and synaptic ( %) ampars in ca pyramidal cells. the reduction of extrasynaptic receptors was particularly severe on dendrites ( %) and spines ( %). ampar-mediated responses were reduced at ca synapses ( %). therefore, - is the major auxiliary subunit of hippocampal ampars. etsuko tarusawa , yugo fukazawa , elek molnar , masahiko watanabe , ryuichi shigemoto , div. cerebral structure, nips, okazaki, japan; mrc, univ. of bristol, bristol, uk; hokkaido univ., sapporo, japan; sorst, jst, kawaguchi, japan relay cells in the dorsal lateral geniculate nucleus receive two types of glutamatergic inputs; retinogeniculate (rg) and corticogeniculate (cg) synapses. it has been shown that the synaptic transmission at both rg and cg synapses is mediated via ampa and nmda receptors. however, how ampa and nmda receptors are expressed in these two types of synapses have not been elucidated. we examined the expression pattern of ampa and nmda receptors in rg and cg synapses using sds-digested freeze-fracture replica labeling (sds-frl). the sds-frl revealed that synaptic size of individual rg synapses was significantly smaller than that of cg synapses. rg synapses expressed . to times higher density of ampa receptors than cg synapses. on the other hand, cg synapses expressed . to times more nmda receptors than rg synapses. these results indicate differential effects on the relay cell by the retino-and cortico-geniculate inputs through ampa and nmda receptors. katsuyuki kaneda , , , hitoshi kita dept. of anat. & neurobiol., univ. of tennessee, memphis, tn, usa; japan society for promotion of science, tokyo, japan; dept. of developmental physiology, nips, okazaki, japan to investigate the properties of synaptically induced slow responses in globus pallidus (gp) neurons, whole-cell recordings were performed using rat brain slice preparations. repetitive stimulation of the gp and internal capsule induced mixed fast epsps/ipsps followed by a slow ipsp (sipsp), and a long-lasting slow depolarization (sdepo). bath application of nbqx, cpp, and gabazine blocked the mixed epsps/ipsps. the gaba b receptor antagonist cgp abolished the sipsp. an mglur antagonist, but not an mglur antagonist, partially blocked the sdepo. in addition, cgp enlarged the amplitude of fast ipscs, but not of epscs, that were evoked during the repetitive stimulation, suggesting an involvement of presynaptic gaba b receptors in gaba release. these results indicate that synaptically released gaba and glutamate can evoke gaba b receptor-and mglur -mediated responses in the gp. contribution of these responses to the control of gp activity will be discussed. research funds: nih and the jsps ps p-a essential contribution of glutamate to gaba depolarization involved in hippocampal seizure-like activity yoko tsukamoto , yoshikazu isomura , , michiko imanishi , tomoki fukai , masahiko takada system neurosci., tokyo met. inst. neurosci., tokyo, japan; neural circuit theory, riken bsi, saitama, japan we have previously shown that neuronal synchronization is achieved by excitatory gabaergic and glutamatergic inputs during a hippocampal seizure-like afterdischarge. however, it still remains unclear how the gaba response is converted from inhibitory to excitatory in the process of afterdischarge induction. here we traced the time-course of amplitude and reversal potential of gabaergic transmission in pyramidal cells and interneurons entraining the afterdischarge, and examined influence of glutamate on the conversion of gaba response. the gaba reversal potential in pyramidal cells rose to spike-threshold levels for > s after the induction. gaba amediated cl-influx lasted for . s, and then glutamate enhanced the conversion effectively in a gaba a -independent manner, which was dependent on an extracellular k increase. coapplication of gaba and glutamate caused a similar oscillatory activity. the results show gaba and glutamate may cooperatively induce as well as maintain seizure-like activity. michiko nakamura , yuko sekino , , toshiya manabe , division of neuronal network, department of basic medical sciences, institute of medical science, university of tokyo, tokyo, japan; crest, jst, japan profound activity-dependent facilitation of synaptic transmission at hippocampal mossy fiber synapses is a unique and functionally important property. in the present study, we found that this synaptic strengthening was partially mediated by presynaptic gaba a receptor activation during the developmental period (p < ), using electrophysiological methods and optical imaging. in immature animals (p ), fiber volley amplitudes were activity-dependently increased during short-train stimulation of mossy fibers. this fiber volley facilitation was significantly decreased by either inhibition of gaba a receptors or suppression of gaba release from interneurons. these results suggest that gaba released from inhibitory interneurons and gaba a receptors on mossy fibers contribute to activity-dependent facilitation of the excitatory synaptic transmission during development. takuya nishimaki, il-sung jang, jyunichi nabekura dep. dev. physiol., nips, sokendai, okazaki, crest, jst, japan lateral superior olive (lso) is the first auditory center. during the early postnatal period, the inhibitory synaptic inputs to lso neurons from medial nucleus of the trapezoid body (mntb) change from predominantly gabaergic to glycinergic. we focused on metabotropic gaba b receptor (gaba b r) as the key molecule of difference between gaba and glycine. in immature lso neurons postsynaptic gaba b r could activate k + channels, but this effect ceased by the third postnatal week. baclofen, a gaba b r agonist, reduced ipsc amplitude at mntb-lso synapses in neonate (<p ) with a significant change in the paired-pulse ratio. the presynaptic effect of baclofen was gradually decreased with development (p - ). in situ hybridization and immunohistochemistry experiment revealed gaba b r expression in mntb neurons was also gradually decreased. the property of mntb-lso synapses in gaba b r knock out mouse remained immature at p - compared with control mouse. thus, gaba b receptor seems to play an important role in development of synaptic property. pyramidal cells in the hippocampal ca area receive gabaergic input from interneurons, which make synapses on the soma, dendrites and the axon initial segment (ais). here, we used the sdsdigested freeze-fracture replica labeling method to visualize the cell surface distribution of the ␣ , ␣ , and ␤ / subunits quantitatively on distinct subcellular compartments of pyramidal cells. labelings for these subunits were accumulated over clusters of intramembrane particles (imp) on the protoplasmic face (p-face) of the plasma membrane, indicating that many imp clusters of a certain size represent gabaergic synapses. the synaptic labelling densities for gaba-a receptor subunits were not significantly different on the tested subcellular compartments. double labelling experiments showed that the majority of synapses contains ␣ , ␣ , and ␤ / subunits. in the cerebellar cortex, the effects of ␣-adrenoceptor activation on inhibitory synaptic transmissions have not yet been fully understood. therefore, we investigated the effects of the ␣ -or ␣ -adrenoceptor agonist on firing rates of presynaptic interneurons (ins). presynaptic cell-attached recordings showed that spontaneous activity of gabaergic ins was enhanced by the ␣ -adrenoceptor agonist phenylephrine, while reduced by the ␣ -adrenoceptor agonist clonidine. immunohistochemical studies showed that ␣ -adrenoceptors were expressed in the molecular layer and the purkinje cell (pc) layer, while ␣ -adrenoceptors were observed in cell bodies of pcs and ins. these results suggest that noradrenaline enhances inhibitory neurotransmitter release via ␣ -adrenoceptors, which were expressed in presynaptic terminals and somatodendritic domains, whereas suppresses the excitability of ins via ␣ -adrenoceptors, which were located in the presynaptic in soma. thus, cerebellar ␣-adrenoceptors play roles in a presynaptic dual modulation of gabaergic inputs from ins to pcs. research funds: kakenhi ( ) ps p-b involvement of basal pkc and erk / activities in constitutiveinternalization of ampa receptors in cerebellar purkinje cells tetsuya tatsukawa , takahiko chimura , azumi matsumoto , kazuhiko yamaguchi lab. for motor learning control, riken, bsi, japan; lab. for memory & learning, japan ampa receptor (ampar) internalization provides a mechanism for cerebellar ltd, which is the underlying basis of motor learning and motor coordination. however, the relationship between cerebellar ltd and constitutive ampar internalization in parallel fiber (pf)-purkinje cell (pc) synapses remains unclarified. in the present study, we demonstrated that tetanus toxin (tetx, a blocker of exocytosis mediated by vamp) infusion into pcs caused the rundown of pf-epsc amplitude through the constitutive ampar internalization, and then addressed question whether intracellular signals involved in cerebellar ltd induction modify the constitutive ampar internalization at pf-pc synapses using electrophysiological and immunocytochemical techniques. our results suggest that the regulation of actin polymerization is involved in the surface expression of ampars and the surface expressing ampars are constitutively internalized depending on both basal pkc and mek-erk / activities at pf-pc synapses. taisuke miyazaki , kohichi tanaka , masahiko watanabe department of anatomy, school of medicine, hokkaido university, sapporo, japan; department of molecular neuroscience, tokyo medical and dental university, tokyo, japan glutamate released to the synaptic cleft has to be removed by glutamate transporter. in the cerebellum, glutamate transporter glast is richly expressed in bergmann glial cells (bg) that enwrap synapses, dendrites and somata of purkinje cells (pcs). in this study, anatomical analysis was applied to glast-deficient mice. in the mutant mice, lamellate processes from bg fibers became atrophic, resulting in incomplete sealing of synaptic cleft. furthermore, the distribution of climbing fiber (cf) terminals was decreased proximally. by anterogradely labeling, multiple cf innervation was demonstrated to be formed by ectopic innervation to and from nearby proximal dendrites, particularly at their crossing point. these results suggest that glast is essential for complete ensheathment of pc synapses and for the establishment of cf mono-innervation by suppressing local ectopic innervation between nearby pc dendrites. miwako yamasaki , , kouichi hashimoto , taisuke miyazaki , masahiko watanabe , masanobu kano dept. of anatomy, grad. sch. of med., hokkaido univ., sapporo, japan; dept. of cellular neuroscience, grad. sch. of med., osaka univ., suita, japan the ␥ isoform of protein kinase c (pkc␥) is highly expressed in cerebellar purkinje cells (pcs), and its deficiency impairs the late phase of climbing fiber (cf) synapse elimination. in the present study, we analyzed multiple cf innervation in pkc␥-deficient mouse by immunohistochemistry combined with anterograde tracer labeling of cfs. in general, cf innervation regressed proximally in the pkc␥-deficient mouse. in some mutant pcs, a considerable part of the proximal dendrites lost association with cf terminals. in the majority of multiply innervated pcs, single main cf innervated broad region of proximal dendrites, whereas surplus cfs innervated the somata and basal portion of the proximal dendrites. moreover, immunoelectron microscopy revealed synaptic contact of cf terminals to somatic spines. these results suggest that pkc␥ is crucial for strengthening innervation by a main cf and elimination of surplus cfs from proximal somatodendritc compartments of pcs. keiji imoto , , takashi kodama , , ryuichi shigemoto , , , yugo fukazawa , , yasuo mori division of neural signaling, nips, okazaki, japan; school of life science, graduate university for advanced studies (sokendai), okazaki, japan; division of cerebral structure, nips, okazaki, japan; sorst, japan science and technology agency, kawaguchi, japan; department of synthetic chemistry and biological chemistry, kyoto university, kyoto, japan a murine ataxic mutant, rocker, has a point mutation in the ca v . (p/q-type) ␣ subunit gene and shows the mildest phenotype among the series of ca v . mutants. although functional defects of the mutant channel were relatively mild, we found that synaptic transmission in parallel fiber-purkinje cell synapses was considerably impaired. we demonstrated that this impairment was mainly attributable to postsynaptic changes, which included reduction of the number of ampa receptors in psd, whereas the presynaptic function remained normal. we also found the dendrites of purkinje cells showed poor arborization in rocker mice. these lines of evidence indicate that ca v . exerts its strong influence on postsynaptic maturation and dendritic structure. bai-chuang shyu, chia-ming lee, wei-chih chang institute of biomedical sciences, academia sinica, taipei, taiwan roc the aim of the present study was to investigate synaptic transmission and organization of thalamus evoked activities in the anterior cingulate cortex (acc) using a novel mouse brain slice preparation protocol. the mono-and poly synaptic responses in the acc were excited by direct thalamic activation. the synaptic transmission involves both ampa and nmda glutamate receptors. several thalamus-evoked responses were identified: the early negative component (n ) emerged in layer vi near cingulum. subsequently a positive component (p) appeared in layer vi. the second negative component (n ) became apparent in layers ii/iii and v. then the activities spread downward to layer vi and developed as n component in a medial-to-lateral direction. we confirmed that functional thalamocingular activities were preserved in our newly developed brain slice preparation. the thalamus-evoked responses were activated and progressed along a lateral-medial-lateral trajectory loop in the acc and this process was modulated extensively by glutamatergic synapses. ps p-b target-dependent extracellular ca-dependence of the short-term plasticity of the solitary complex synapses in the rat lab. neurophysiol., dept. neurosci., jikei univ. sch. med., tokyo, japan the discharge frequency-dependence of the transmission efficiency at the synapses from the vagal primary afferents to the second-order neurons in the solitary complex (sc) forms the primary filter of the visceral information. such frequency filtering of the synaptic transmissions from the solitary tract (ts) to neurons in the sc depends on their short-term plasticity as evidenced by strong correlation between frequency-dependence and the paired-pulse ratio (ppr). here we analyzed the mechanism underlying distinct pprs among distinct types of synapses. in synapses from ts to the neurons in nucleus of the solitary tract, which show prominent low-pass filtering, ppr significantly increased from . ± . to . ± . (n = ; mean ± s.d.) by reducing [ca + ] o from to . mm, whereas that in low-pass synapses from ts to the neurons in dorsal motor nucleus of the vagus was affected only slightly. these results suggest that the synaptic frequency filtering in the sc is under distinct control depending on the function of the postsynaptic target systems. ps p-b response of spinal monosynaptic reflex to high frequency stimulation in newborn rat the susceptibility of synaptic transmission to the stimulation frequency is a characteristic feature of immature animals, and has been attributed to the incompleteness of transmitter release mechanisms. in an isolated spinal cord preparation of newborn rat, monosynaptic reflexes (msrs) evoked by dorsal root stimulation, were mediated by both nmda and non-nmda glutamate receptors. msrs were constant in amplitude at / s, and were depressed completely by cnqx. when stimulus rate was increased to /s, msr amplitudes were greatly reduced initially, and recovered later. this recovery of msr was eliminated by apv. non-nmda component of msrs was eliminated completely at /s, but appeared in constant amplitudes in the presence of cyclothiazide. from these results, non-nmda glutamate receptor mediated almost most of msrs at / s, but not at /s due to desensitization. in contrast, nmda glutamate receptor does not mediate msrs, but activated at /sec, in place of non-nmda receptor. in conclusion, incompleteness of transmitter release mechanisms could not be solely attributed to the feature of immature synaptic transmission. kenichi ono , keisuke shiba , ken nakazawa , ichiro shimoyama department of otolaryngology, chiba university, chiba, japan; department of otolaryngology, kimitsu central hospital, chiba, japan; department of integrative neurophysiology, chiba university, chiba, japan; section for human neurophysiology, research center for frontier medical engineering, chiba university, chiba, japan we determined synaptic source of the respiratory-related activity of laryngeal motoneurons (lms) using spike-triggered averaging of lm membrane potentials triggered by spikes of medullary respiratory neurons in decerebrate, paralyzed cats. in inspiratory (i) lms, monosynaptic epsps were evoked by spikes of i neurons with augmenting firing patterns, and monosynaptic ipsps were evoked by spikes of expiratory (e) neurons with decrementing firing patterns and of i neurons with decrementing firing patterns. in elms, monosynaptic ipsps were evoked by spikes of i neurons with decrementing firing patterns and of e neurons with augmenting firing patterns. we conclude that various synaptic inputs from respiratory neurons contribute to shaping the respiratory-related trajectory of lm membrane potentials. ps p-b in-vivo gene transfer of exogenous protein to the primary afferent neurons chiaki yamada , , eiji shigetomi , , fusao kato lab. neurophysiol., jikei univ. sch. med., tokyo, japan; dept. basic biol. sci., kyoritsu univ. of pharm., tokyo, japan; jsps, japan in order to understand the mechanism how sensory information is transmitted to the brain, it is indispensable to identify the functional roles of already-identified molecules related to synaptic transmission between the afferent fibers and the central second-order neurons. here we developed a method for efficient in-vivo gene transfer into the neurons in the sensory ganglia and evaluated expression of their gene products. in the anesthetized young wistar rats, we injected pcaggs-egfp plasmid vector (through courtesy of drs. j. miyazaki and k. nakajima) into the nodose ganglion (ng) at the neck and transferred it by electroporation. two days after transfection, the ng was extirpated and fixed to observe egfp signals. a large portion of somata in the ng as well as the centrally and peripherally projecting afferent fibers expressed high levels of egfp with few damaged cells. this technique might enable to analyze the function of specific molecules in the transmitter release from the sensory afferent termini in the brain. research funds: kakenhi ( , ) ps p-b laminar sources of synaptic input to layer neurons in rat visual cortex takuma mori, edward m. callaway salk institute, usa layer of the mammalian neocortex consists of interneurons, including late-spiking neurogliaform cells and non-late-spiking axondescending cells. very little is known about connectivity of these cells with cells in other cortical layers. we examined the laminar sources of local excitatory and inhibitory functional synaptic inputs onto individual layer neurons of rat visual cortex, using scanning laser photostimulation. we find that axon descending cells get excitatory synaptic inputs primarily from layer / . neurogliaform cells receive more excitatory inputs from layers and . the dominant inhibitory synaptic inputs onto both types of cells originate from layer / . these neurons also get inhibitory inputs from layers and - . the presence of convergent excitatory and inhibitory synaptic inputs from multiple cortical layers suggests that the computations in layer are highly integrative. takako nishi , tsukasa gotow , shiro nakagawa ins. nat. sci., senshu univ., kawasaki, japan; dept. neurol., kagoshima univ. grad. sch. med.-den. sci., kagoshima, japan four extra-ocular photoreceptors, the photoresponsive neurons, a-p- , es- , ip- and ip- which respond directly to light, exist in the abdominal ganglion of the mollusc onchidium. a-p- /es- of these neurons respond to light with a depolarizing receptor potential, whereas light hyperpolarizes the other ip- /ip- . the present study was conducted to examine the functional significance of the above ip- /ip- , having a peak sensitivity at nm light. the body wall of the animal transmitted enough nm to hyperpolarize ip- /ip- in the ganglion covered by the body wall. ip- /ip- were coupled with weak electrical synapses and involved in a spontaneous beating or bursting spike activity. ip- /ip- were not coupled to a-p- /es- one another. finally, ip- /ip- , the first order photosensory cells were also the second order interneurons or motoneurons relaying various sensory inputs and innervating the pneumostome and viscera. ps p-c cellular mechanism of interaural level difference calculation in the auditory brainstem of the chicken tatsuo sato, iwao fukui, harunori ohmori department of physiology, faculty of medicine, kyoto university interaural level difference (ild) and timing difference (itd) are the major two cues to localize the sound source, and are processed separately. although the details of itd processing are well investigated, not much is known about ild. in the barn owl, it is proposed that ild is calculated in lldp (posterior part of the dorsal lateral lemniscal nucleus). we confirmed already that in vivo lldp calculates ild in the chicken as well. in this work using the patch clamp recording with the brainstem slice preparation, we found that lldp neurons fire tonically under the regulation of two k + channels activated at low and high voltage, and receive both excitatory and inhibitory inputs representing the contralateral and the ipsilateral sound intensity, respectively. we are intending to clarify the cellular mechanism of ild calculation as the interaction of both excitatory and inhibitory synaptic inputs. to understand the functional organization of the cns, it is essential to know how sensory information is processed within the cns. we have been approaching this topic by following the ontogenetic patterning of neural circuit formation related to the cranial and spinal sensory inputs using optical imaging. in this study, we surveyed developmental organization of neural networks related to the olfactory nerve in the embryonic chick forebrain. stimulation applied to the olfactory nerve elicited epsp-related optical signals in the olfactory bulb from the -day old embryonic stage. the epsp was mediated by glutamate, and nmda-and non-nmda-receptor components were identified. in more developed stages, in addition to the responses in the olfactory bulb, another response area was discriminated within the cerebrum, which seemed to correspond to the higher-ordered nucleus of the olfactory pathway. the results suggest that the olfactory pathway is functionally generated at early stages of development when neural networks related to other visceral and general somatic sensory inputs are also in the process of developing. yoko momose-sato, katsushige sato dept. physiol., tokyo med. dent. univ. sch. med., tokyo, japan in an accompanying study, using a voltage-sensitive dye recording technique, we examined the developmental organization of the olfactory pathway in the embryonic chick forebrain, and showed that functional synaptic transmission in the olfactory bulb was expressed at around e . it is known that odor stimuli elicit oscillatory events in the olfactory bulb in various species. we found that oscillatory activity was also generated in the chick olfactory bulb during embryogenesis. at early stages of development (e -e ), postsynaptic responserelated optical signals evoked by olfactory nerve stimulation exhibited a simple monophasic waveform that lasted a few seconds. as development proceeded, the pattern of the optical signal became complicated, and oscillatory activity was observed in a later phase of the postsynaptic response. the oscillation was restricted to the olfactory bulb, and this spatial pattern was different from that of the propagating wave activity termed the depolarization wave. we examined spatio-temporal patterns of the oscillatory activity in different stages, and studied its developmental dynamics. akiyoshi shimada , nyberg tobias , nahoko kasai , yuriko furukawa , keiichi torimitsu , , kunihiko obata , yuchio yanagawa , , tadaharu tsumoto , ntt basic research laboratories, ntt corporation, kanagawa, japan; sorst, jst, saitama, japan; neuronal circuit mechanisms research group, brain science institute, riken, saitama, japan; department of genetic and behavioral neuroscience, graduate school of medicine, gunma university, gunma, japan in the immature stage of cerebral cortex, gaba a receptor activation depolarizes rather than hyperpolarizes the membrane potential of neurons. cortical neurons of glutamic acid decarboxylase -green fluorescence protein (gad -gfp) knock-in mice at postnatal day - were cultured on a microelectrode array. spontaneous firing of cortical neurons appeared within the first days in vitro (div). when a gaba a receptor antagonist, bicuculline, was added, four types of modulation of firing pattern were observed until at least div: increase, decrease, disappearance and no change. these results and an additional analysis suggest that a portion of the spontaneous activity emerged through an activation of gaba a receptors. ps p-c pathway specific signal modulation by purinergic receptors in the parallel processing system makoto kaneda , toshiyuki ishii , , yasuhide shigematsu , toshihiko hosoya , yukio shimoda dept. physiol., keio univ. sch. med., tokyo, japan; bsi, riken, wako, japan; mri, tokyo womens' medical univ., tokyo, japan; dept. biomolecular science, univ. toho, funabashi, japan the retina divides visual information into on-and off signals, and processes them with independent subsets of neurons. so far no difference between these pathways has been found for neurotransmitter functions. we have previously found that p x -purinoceptors are selectively present on the off-type, but not on the on-type cholinergic amacrine cells of the mouse retina. we therefore asked if atp has different functions in the two pathways. as expected, only the off-type, but not the on-type, cholinergic amacrine cells responded to atp. this response was mediated by the p x -purinoceptors. furthermore, a p x-purinergic receptor antagonist activated the tonic phase firing of the off ganglion cells, but not of the on cells. thus atp has off-pathway specific modulatory functions mediated by p x-purinergic receptors. bhupesh mehta, gulnaz begum, preeti g. joshi department of biophysics, national institute of mental health and neuro science, bangalore, india cultured hippocampal neurons in network exhibit synchronized oscillations of cytosolic ca + . these oscillations are an inherent property of various cell types and are essential for the maintenance and development of neuronal plasticity, and for normal brain functioning. the synchronized ca + oscillations are primarily controlled by glutamate receptor activation, but may be modulated by a variety of other factors. these spontaneous oscillations are inhibited by activation of purinergic receptors. we observed a dose dependent inhibition of the oscillatory activity by purinergic receptor agonists atp and utp, but an enhanced oscillatory pattern was observed when the action of mesatp was observed. the inhibition of mesatp response by p y receptor specific antagonist mrs indicates that different subtypes of purinergic receptors play different role in the regulation of synchronized ca + oscillations. data will be presented on the p receptor mediated regulation of synchronized ca + oscillations in cultured hippocampal neurons. mahomi kuroiwa, akinori nishi department of pharmacology, kurume university school of medicine, kurume, japan presence of additional d -like dopamine receptors that selectively couple to gq/phospholipase c (plc) has been proposed. in recent studies, skf was shown to selectively stimulate plc-linked d -like dopamine receptors. darpp- is a phosphoprotein that regulates the efficacy of d receptor signaling. activation of d receptors that couple to golf/pka signaling leads to darpp- phosphorylation at thr . we investigated the effect of skf on darpp- phosphorylation at thr by using mouse neostriatal slices. treatment of neostriatal slices with skf increased thr phosphorylation. the effect of skf was enhanced by a plc inhibitor, u . in contrast, the effect of skf was partially blocked by a d receptor antagonist, sch , and the sch -insensitive increase in thr phosphorylation was completely abolished by an adenosine a a receptor antagonist, zm . thus, analysis of darpp- phosphorylation revealed that skf activates at least three signaling cascades in the neostriatum: ( ) plc, ( ) d receptor/golf/pka, ( ) adenosine a a receptors. ps p-c thiamylal may more effectively reduce neuronal excitability in cerebral cortex than that in hippocampus naoshi fujiwara division of medical technology, niigata university school of health sciences, japan effects of thiamylal (a barbital derivative anesthetic) on excitation propagation in the cerebral cortex and hippocampus were analyzed using membrane potential imaging. cortical and hippocampal slices of the c bl mouse were dyed with a voltage-sensitive dye rh . in cortical slices, electrical single-pulse stimulation to the layer v evoked transient depolarization in the vicinity of stimulated site, and then this excitatory response propagated to the layers ii-iii and widely expanded in these layers. the excitation propagation in the layers ii-iii was significantly inhibited by thiamylal ( mol/l). in hippocampal slices, excitation propagation elicited by the same stimulation to the ca stratum radiatum remained without significant changes in the presence of thiamylal at the same dose. on the other hand, an ampa/kainate receptor antagonist cnqx ( mol/l) inhibited excitation propagation in both cortical and hippocampal slices. the results suggest that thiamylal more effectively reduce neuronal excitation in cortex than that in hippocampus. research funds: grant-in-aid from jsps no. ps p-c brain-derived neurotrophic factor regulating ampa receptor translocation to postsynaptic density through ip r and trpc calcium signaling hiroko nakata, shun nakamura national institute of neuroscience, tokyo, japan the change in the number of postsynaptic ampa-type glutamatergic receptors (ampars) by neuronal activity is implicated in the excitatory synaptic plasticity. here, we showed that bdnf induced ampar translocation to postsynaptic site of the dissociated cortical pyramidal neurons. using calcium imaging, we located the spines that were activated by bdnf. with thus identified spines, we analyzed the ampar subunit glur localization to postsynaptic density (psd) by immunostaining. we found that bdnf increased the ratio of surface glur at psd within min. bdnf selectively translocated ampar, but not nmdar. the essential signaling was ca + transients evoked by the activation of trkb/plc␥ pathway. both the intracellular ca + rise, that is, ca + release from ip r and ca + influx through storeoperated cation channel trpc contributed to the increase of the surface expression of glur at psd. these results suggest an important role of bdnf in the regulation of ampar trafficking by spatial and temporal ca + signaling regulation. research funds: health sciences research grant of nano- hiroyuki konishi , kazuhiko namikawa , , keiji shikata , yuji kobatake , taro tachibana , hiroshi kiyama dept. anat. & neurobiol., osaka city univ., grad. sch. med., osaka, japan; dept. anat., asahikawa med. col., hokkaido, japan; dept. bioeng., osaka city univ., grad. sch. eng., osaka, japan activation of akt-mediated signaling pathways is crucial for injured neurons to survive and regenerate. in this study, we attempted to identify novel substrates for akt by using an antibody, which recognized phosphorylated consensus motif by akt. using pc cells, which overexpressed constitutively active akt, we screened protein spots that exhibited increased immunostaining by the antibody, and consequently identified the neuronal intermediate filament protein, peripherin. using some mutant forms of the recombinant proteins, the phosphorylation site was determined in vitro. we then generated an antibody against phosphorylated peripherin, and demonstrated that peripherin was phosphorylated not only in akt-activated cultured cells but also in nerve-injured hypoglossal motor neurons. these results suggest that peripherin is a novel substrate for akt in vivo and that its phosphorylation may play a role in motor nerve regeneration. the truncated trkb receptor, t , is involved in the control of cell morphology via the regulation of rho proteins via rho gdi . however, it is unclear whether t regulates the downstream of rho signaling and the actin cytoskeleton. in this study, we investigated this question using c rat glioma cells, which express t endogenously. rho gdi was dissociated from t in a bdnf-dependent manner, which also causes decreases in the activities of rho-signaling molecules such as rhoa, rock, pak and erk. moreover, bdnf resulted in the disappearance of stress fibers in the cells treated with lpa, an activator of rhoa, and in morphological changes in cells. furthermore, a competitive assay with cfp fusion proteins of t -specific sequences reduced the effects of bdnf. these results suggest that t regulates the rho signaling pathways and the actin cytoskeleton. anjana kalita eukaryotic gene expression laboratory, national institute of immunology, new delhi, india jak and stat are activated in response to many cytokines and growth factors. this pathway has been primarily studied in non-neuronal origin cells. it has been shown that stat activated cellular transformation, occurs through transcription regulation of specific genes like c-myc, cyclin d , bcl . the present study was undertaken to identify target genes of jak/stat pathway in response to tnf-alpha & igf- in neuronal origin cells. we also looked at the mechanism of activation of these target genes. we saw that jak/stat pathway was involved in upregulation of cyclind in response to igf- both at protein & rna levels but no change was observed in c-myc, bcl levels. interestingly, mapk-erk, which is also known to activate cd in other systems, had no effect on the cd level in neuronal cells. on the other hand, pi kinase not only activated jak/stat pathway but also affected on cyclind level. thus it appears that jak/stat is activated by pi k, which in turn upregulates cyclind level. pi k also activated erk without changing cd level, thus we show that jak/stat pathway is important for survival of neurons, activated by pi k. cd is a one of the major downstream targets of jak/stat. hiroko inoue , haruhisa kawasaki , ritsuko fujii , tohru yoshioka , kazunori sango , toshihiko kadoya , hidenori horie sch. of sci. and eng., waseda univ., tokyo, japan; advanced res. center for biol. sci., waseda univ., tokyo, japan; tokyo metropolitan inst. for neurosci., tokyo, japan; pharmaceut. res. lab. kirin brewery co., ltd., gumma, japan oxidized galectin- (gal- /ox) stimulates macrophages to promote axonal regeneration in peripheral nerves after nerve injury. but, the mechanism how gal- /ox stimulates macrophages remains to be clarified. in the present study with rt-pcr and western blotting, we searched for the molecules contributing to the mechanism. rt-pcr analysis with cultured rat peritoneal macrophages revealed that the application of gal- /ox enhanced mrna expression of nerve regeneration-related factors such as il- , il- ␤. however, those of gdnf, ngf, bdnf, nt- , nt- , igf-i, and igf-ii scarcely detected in control condition were not changed by the application of gal- /ox. since il - , il- , and lif can promote axonal regeneration in the in vitro axonal regeneration model, the up-regulation of these cytokines by the treatment with gal- /ox may enhance nerve regeneration after nerve injury. kennichi katou, taku iwamoto, satoshi kida dep. of bioscience, tokyo univ. of agriculture, japan ␣camkii has been known to play essential roles in synaptic plasticity. in response to increase in ca + concentration, ␣camkii is activated by the interaction with ca + /cam. prolonged increase in ca + level leads to further activation of ␣camkii through autophosphorylation at t . to understand the molecular dynamics of ␣camkii activation in vivo, we have tried to visualize and monitor the ␣camkii activity in various types of cells using fret. in hela cells, increase in ca + level induces continuous interaction between ␣camkii and cam and conformational change of ␣camkii, indicating that the conformational change of ␣camkii mainly reflects the interaction with cam. in sh-sy y cells, the increase in ca + level induces only transient interaction between ␣camkii and cam but the conformational change of ␣camkii is maintained following the termination of the interaction with cam, suggesting that conformational change of ␣camkii is induced by the interaction with cam and then maintained by autophosphorylation. thus, mechanisms for regulation of ␣camkii activation is cell-type dependent. akifumi kamata, yusuke takeuchi, kohji fukunaga dept. pharmacol., grad. sch. pharmaceu. sci., tohoku univ., sendai, japan ca + /calmodulin-dependent protein kinase ii (camkii) is highly expressed in brain including dopaminergic neurons, however, the role of camkii in the dopaminergic neurons remains to be unclear. camkii consist of four isoforms, ␣, ␤, ␥ and ␦, and differential isoforms are implicated in the different neural functions. we have examined the isoforms of camkii expressed in rat substantia nigra (sn). rt-pcr and immunoblot analyses revealed that the ␥ and ␦ isoform mrnas including ␦ isoform, a nuclear isoform of camkii, were predominantly expressed in sn. the immunohistochemical study also confirmed the preferential localization of the ␥ and ␦ isoforms in the sn dopaminergic neurons, and immunoreactivity against anti-camkii␦ - antibody was detected in both nucleus and cytoplasm. furthermore, we defined expression of bdnf mrnas having the exon ii and iv in sn. taken together with our previous observation, the camkii␦ isoform was involved in the expression of bdnf in sn dopaminergic neurons. ps p-d the role of ca + /calmodulin-dependent protein kinase ii in neuronal functions revealed by inactivated camkii␣ knock-in mouse yoko yamagata , , hiroyuki sakagami , keiji imoto , , kunihiko obata , yuchio yanagawa okazaki, japan; sokendai, okazaki, japan; tohoku univ., sendai, japan; riken, wako, japan; gunma univ., maebashi, japan ca + /calmodulin-dependent protein kinase ii (camkii) is one of the major protein kinase in the central nervous system and proposed to play important roles in various neuronal functions. we engineered knock-in mice with the inactivated ␣ subunit of camkii by replacing k with r to study functional significance of camkii activity in intact animals. as expected, camkii activity was selectively reduced, while camkii subunit protein levels were comparable to those of wild type controls in homozygous mutants. in situ hybridization revealed normal expression patterns of camkii subunit mrnas, including dendritic localization of camkii␣ mrna. further analyses of these mice will be presented in the meeting. research funds: a grant-in-aid for scientific research from japan society for the promotion of science (kakenhi , # ) ps p-d characterization of ca + -dependent membrane binding proteins of rat brain shohei maekawa , katsutoshi taguchi , haruko kumanogoh , shun nakamura division of biosystems science, grad. sch. of sci/tech, kobe-u kobe - , japan; division of biochemistry and cellular biology, national inst. neurosci. kodaira, tokyo - , japan ca + -dependent membrane binding proteins were purified through a ca + -dependent membrane binding and egta-induced membrane dissociation protocol from a crude triton solubilized membrane fraction. molecular characterization of these ca + -dependent membrane binding proteins through lc-ms/ms identified annexin vii, annexin xi, and copin isoforms, in addition to annexin vi and neurocalcin␣. since the membrane fraction contains phosphatidylethanolamine (pe), cholesterol, and phosphatidylcholine (pc), the membrane binding activity of annexin vi, a major protein in this fraction, was studied using artificial liposomes. annexin vi showed a pe-dependent, but not cholesterol-dependent liposome binding activity. immunostaining of annexin vi in cultured neurons showed a punctuate staining of neuronal cell membrane and this spots increased during maturation. these results suggest the participation of annexin vi in the membrane cycling at the presynaptic region. ps p-d generation of dominant negative mutants of transcription factor crest takuzou simo, kenichi kato, hiroshi hosoda, satoshi kida department of bioscience, tokyo university of agriculture, tokyo, japan transcription factor calcium-responsive transactivator (crest) has been shown to be required for dendritic growth of neuron. previous studies have shown that crest contains two functional domains; multifunctional domain (mfd; aa ∼ ) required for transactivation, nuclear localization and homodimerization and c-terminal transactivation domain (ctd; aa ∼ ). to further understand the function of crest on brain function, we tried to generate the dominant negative mutant of crest to use mouse genetics study. indeed, we generated fusion proteins of gal -dbd with mutants of crest; crest c ( ∼ ) lacking c-terminal transactivation domain, mfd and mfd-nls that is a fusion protein of mfd with nuclear localization signal (nls). from the experiments using gal one hybrid system, we found that mfd-nls shows the strong dominant negative effects on transcriptional activation by crest. ps p-d characterization of iq-arfgef, a novel exchange factor for arf , in mouse brain hiroyuki sakagami, hisatake kondo department of cell biology, tohoku university graduate school of medicine, sendai, japan adp-ribosylation factor (arf ) regulates cytoskeletal dynamics and membrane trafficking that are critical processes for synaptic plasticity. to understand its neuronal functions, we have focused on guanine nucleotide exchange factors (gef) for arf . in this study, we determined the entire sequence of a partially identified cdna (mkiaa ) encoding a novel sec domain. the deduced protein contained coiled coil, iq, sec , plekstrin homology domains. interestingly, it contained a type i pdz domain-binding motif at its cterminal tail. arf pull-down assay demonstrated its ability to active arf more selectively than arf . thus, we named this protein as iq-arfgef. in situ hybridization analysis demonstrated the preferential expression of iq-arfgef in the forebrain and cerebellar granule cells. furthermore, iq-arfgef mrna was localized not only in the hippocampal neuronal layers but also in their dendritic fields. our present findings suggested that iq-arfgef may play important roles in dendrites through activation of arf and interaction with various pdz proteins. eriko miura , masahiro fukaya , tokiharu sato , kazushi sugihara , masahide asano , katsuji yoshioka , masahiko watanabe dept. anat., hokkaido univ. sch. med., sapporo, japan; dept. mol. cell. biol., cancer res. inst., kanazawa univ., kanazawa, japan; adv. sci. res. center, kanazawa univ., kanazawa, japan the c-jun n-terminal kinase (jnk) is one of the major mitogenactivated protein kinases (mapks), and jsap (or jip ) is a jnkassociated scaffold that controls the specificity and efficiency of jnk signaling cascades. here we studied its expression and distribution in mouse brains. jsap mrna was expressed in developing and adult brains, showing spatial patterns similar to jnk ∼ mrnas. in embryos, jsap immunolabeling was intense for progenitor cells in the ventricular zone and external granular layer, and for neurons and glial cells differentiating in the mantle zone. in adults, jsap was distributed in spines, dendrites, perikarya, and axons of various neurons, and often associated with ser and cell membrane. this wide spatiotemporal expression suggests its fundamental role in mediating external stimuli to jnk mapk pathway and other intracellular machineries. yasukazu hozumi, kaoru goto department of anatomy and cell biology, yamagata university school of medicine, japan diacylglycerol kinase (dgk) is involved in intracellular signal transduction as a regulator of a second messenger, diacylglycerol, and consists of several isozymes. to address the functional implications of dgk isozymes in the brain, we have raised specific antibodies against the isozymes. by immunoblot analysis, we found that the immunoreactive bands for dgk␤, -␥ and -were detected in the light membrane/microsome enriched fraction of rat brains, suggesting that these isozymes localize to the internal membrane system. immunohistochemical examination on rat brain revealed that dgk␤ was detected as dot-like structure in the cell membrane, and dgk␥ as round-shaped structure in the juxtanuclear region of neurons. dgk was detected in the perinuclear region and in the dendrites of purkinje cells. these data show that dgk isozymes localize differentially to the internal membrane system in neurons, suggesting that dgk isozymes play unique roles in distinct internal membrane system. kotaro hattori , shigeo uchino , tomoko isosaka , , shinichi kohsaka , takeshi yagi , shigeki yuasa dept. ultrastractural res., nat. inst. neurosci., ncnp, kodaira, japan; dep. neurochem., nat. inst. neurosci., ncnp, kodaira, japan; kokoro biology group, fbs, osaka univ., suita, japan recently, we have found that fyn-mediated tyrosine-phosphorylation of nmda-r subunits is required for haloperidol-induced catalepsy. haloperidol induced catalepsy in the control mice, but such response was significantly reduced in fyn-deficient mice. fyn activation and enhanced tyrosine-phosphorylation of the nmda-r nr b subunit were induced after haloperidol injection to the control mice, but both responses were significantly reduced in fyn-deficient mice. both nr b phosphorylation and the nmda-induced calcium responses increased after dopamine d -r blockade in cultured striatal neurons of the control, but not in fyn-deficient neurons. accordingly, d -r antagonist activates striatal fyn and the subsequent tyrosine phosphorylation of nr b alters striatal neuronal activity, thereby inducing catalepsy. we also report further proteomics analysis on this molecular pathway. hiroaki okuda, shingo miyata, tsuya taneda, atsushi yamaguchi, shinsuke matsuzaki, natsuko kumamoto, masaya tohyama department of anatomy & neuroscience, graduate school of medicine, osaka university, osaka, japan the schizophrenia is a well-known debilitating psychiatric disorder. recent studies focused on genetic contributions to schizophrenia and have revealed several susceptibility genes, including dysbindin (dtnbp : dystrobrevin binding protein ) at p . . however it is difficult to explain the relationship between the onset of the schizophrenia and only single genetic factor. since, there is an important another factor, an environmental factor, for the schizophrenia. although little attention has been paid to the importance of both factors for the schizophrenia in the nervous system. therefore, to investigate the mechanism of the onset of the schizophrenia, firstly we performed a yeast two-hybrid screening, using the n-terminal region of dys-bindin as a bait. as a result, we detected several transcriptional factors. thus, we further examine that these interactions regulate the expression level of particular genes in the neurons. ryota adachi , naoko oda , ryuzo shingai , st coe program, iwate university, morioka, japan; department of welfare engineering, faculty of engineering, iwate university, morioka, japan the response to environmental stimuli is important to live for organisms. the soil nematode caenorhabditis elegans responds well to chemical and thermal cues known as chemotaxis and thermotaxis behavior. c. elegans is attracted to some ions, amino acids and alcohol that are byproducts of bacteria, and migrates to the temperature of cultivation with food on a temperature gradient. the sensory neurons detected these stimuli are in the head region. the neural network and genes required in each neuron are well known. however, it is under investigation about the integration of chemical and thermal stimuli. to understand the mechanism of this integration, we investigated the chemotaxis behavior under each temperature using the worms cultivated under each temperature. wild type worms cultivated at • c showed decreased chemotaxis response to water-soluble chemicals and alcohol. alteration of assay temperature did not affect response to cl − but that to na + . to discuss more detail, we used thermotaxis abnormal mutants for chemotaxis assay. takashi takekawa , masaki nomura , , toshio aoyagi , tomoki fukai riken brain science institute, saitama, japan; graduate school of informatics, kyoto university, kyoto, japan; crest, japan science and technology agency, kawaguchi, japan the neuron model proposed by izhikevich can exhibit a variety of firing patterns of cortical neurons and gives an efficient mean to conduct large-scale network simulations. however, simplified models may lose essential properties of real neurons, and hence their properties must be compared with those of more realistic models. in fact, our previous studies have shown that two neuron models with almost identical membrane potential profiles sometimes have quite different phase responses and synchronization properties. here, we study the phase responses of the izhikevich model showing fast rhythmic bursting and demonstrate that it exhibits synchronization properties similar to those shown by complex conductance based models. furthermore, its synchronization properties are consistent with those of realistic models in other firing patterns. therefore, we may conclude that the model with tuned parameters is suitable for simulating the dynamic behavior of large-scale networks. honghai cui, hirotaka sakamoto, mitsuhiro kawata department of anatomy and neurobiology, kyoto prefectural university of medicine, kyoto, japan the amygdala plays a critical role in stress responses, especially for modulation of anxiety and fear. in the present study, we investigated the effects on the neuronal morphology for severe stress in the amygdala by using single prolonged stress (sps) paradigm as ptsd (post-traumatic stress disorder) model. rats were perfused days after sps, and intracellular injections of lucifer yellow were carried out in neurons of central (cea) and basolateral amygdala (bla). at the cellular level, we observed a significant increase of dendritic arborization in bla pyramidal neurons but no effect on the cea neurons. these results suggest that sps-induced structural plasticity of the bla provides a cellular substrate for affective disorders that are triggered in ptsd. takahiko kawasaki , , tatsumi hirata division of brain function, national institute of genetics, sok-endai, mishima, japan; prest, kawaguchi, japan in macrosmatic mammals such as mice, olfactory information is detected by two distinct sensory organs, the olfactory epithelium and the vomeronasal organ, and their signals are integrated into the main olfactory bulb (mob) and the accessory olfactory bulb (aob), respectively. the secondary projections from the mob and aob first run parallel in the lateral olfactory tract and eventually diverge into the discrete targets in the telencephalon. although recent studies have revealed general principles in the primary projections, it has been largely unknown how the secondary projections from the olfactory bulbs are constructed. therefore, we investigated the secondary olfactory projections in mutant mouse lines for several axon guidance molecules and their receptors using a specific dye labeling technique, especially focusing on the distinctive pathways of mob and aob neurons. ps p-d an essential role for click-iii/camki␥ in regulation of neurite extension natsumi ishihara, sayaka takemoto-kimura, hiroyuki okuno, haruhiko bito dept. of neurochem., univ. of tokyo, tokyo, japan click-iii/camki␥, a membrane-anchored camk, is a novel member of the camki family that acts downstream of camkk. in order to uncover the biological function of this kinase, we first examined its subcellular distribution and found that tagged click-iii was concentrated at the tips of filopodia-like processes in cultured hippocampal neurons, in close vicinity of actin cytoskeleton. phenotypic studies in pc- cells expressing various mutants of camkk and click-iii revealed that depolarization-induced activation of camkk-click-iii signaling is sufficient to robustly induce neuritogenesis. interestingly, click-iii localization in membrane fractions was regulated via sequential lipidification in an activity-dependent manner. furthermore, lipidification mutants of click-iii were impaired in their ability to facilitate activity-dependent neuritogenesis. our results thus indicate a novel role of click-iii in activity-dependent neurite formation and extension. takuro maruyama , masahiro matsuura , nobuhiko yamamoto grad. sch. of frontier biosci., osaka univ., suita japan; riso kagaku corp., tsukuba japan during development, thalamocortical (tc) axons invade into the cortex and stop growing in layer . to study tc axonal targeting, we examined the effect of the membrane proteins that are expressed in the developing cortex. the dorsal thalamus dissected from embryonic rat brain was dissociated and plated on the dishes that were coated with preclustered fc-tagged extracellular domains of several candidate molecules. after four days in vitro, axonal growth was enhanced on the substrata with low concentrations of ephrin-a , sema a or kit ligand, while a growth-inhibitory effect was found in higher concentrations. a tendency for tc axons to prefer or avoid the region containing these proteins was observed in a new culture method, in which purified proteins were printed on filter membranes in a real laminar size. these results suggest that ephrin-a , sema a and kit ligand may contribute to regulating tc axonal growth in the developing cortex. research funds: kakenhi , kakenhi ps p-e expression pattern of the guidance molecules and their receptors during gnrh neuron migration from the nose to the ventral forebrain shizuko murakami , katsuhiko ono dept. anat., juntendo univ. sch. of med., tokyo, japan; div. of neurobiol. and bioinfo., nat. ins. for physiol. sci., okazaki, japan using in situ hybridization, we examined the expression pattern of attractive and repulsive guidance molecules and their receptors in the migration pathway of gnrh neurons of chick embryos. netrin- expression was seen in the ventral hypothalamus, whereas was absent in the nasal region and the rostral forebrain. a few gnrh neurons expressed netrin- receptor neogenin. slit- was expressed in the forebrain, but its receptor robo- was not expressed in gnrh neurons. semaphorin (sema) a expression was detected in the olfactory-forebrain region. within the forebrain, sema a was absent in the restricted region of the medial forebrain where gnrh neurons migrated in association with a subset of olfactory fibers. at the end of this pathway, sema a expression was observed in the caudal forebrain, and then gnrh neurons changed their course ventrally. many gnrh neurons expressed sema a receptor neuropilin- , suggesting that sema a may act as a repellent during the migration of gnrh neurons. kazuto fujishima , , qing-hua jin , junko kurisu , tomoo hirano , mineko kengaku riken, bsi, lab. for neural cell polarity, saitama, japan; department of biophysics, graduate school of science, kyoto university, kyoto, japan during development, cortical pyramidal neurons elaborate their dendrites in correct pattern and orientation in response to various environmental factors such as neurotrophins or guidance molecules. dendrite morphology is also regulated by cell-cell contacts with neighboring cells. recent studies have implicated notch signal as a candidate for such a contact-dependent regulator of dendrite patterning in postmitotic cortical neurons. we previously showed that delta/notch-like egf-related receptor (dner), neuron-specific transmembrane protein, acts as a notch ligand and mediates neuron-glia interaction during cerebellar development (eiraku et al., ) . dner is expressed in the dendrite of developing cortical pyramidal neurons. here, we analyzed the role of dner in the development of pyramidal neuron dendrites. we quantitatively compared the dendritic morphology of cortical pyramidal neurons in wild-type and dner knock out mice. reelin signal controls neuronal migration in the developing brain. because the binding of reelin to apoer /vldlr induces dab phosphorylation, dab is a critical component of reelin signal. recent studies suggested that reelin signal is involved not only in neuronal migration but in other aspects of neural development. we investigated a possible function of the reelin signal in the filopodia and neurite formation. administration of reelin protein increased number of filopodia in a lesser extent than bdnf treatment. pp suppressed effects of reelin and bdnf on filopodia formation, suggesting that both signal pathways involve src kinase function. in spite of these similarities of reelin and bdnf functions in the filopodia formation, bdnf did not phosphorylate dab . reelin treatment of the neurons derived from dab deficient mice did not induce significant increase in the number of filopodia. these results suggest that the filopodia and neurite formation involves the reelin-dab signal pathways. hidenobu mizuno , , tomoo hirano , , yoshiaki tagawa , dept. biophys., kyoto univ. grad. sch. sci., kyoto, japan; crest, jst, kawaguchi, japan the left and right hemispheres are interconnected via the corpus callosum. in mouse visual cortex, callosal axons from one hemisphere make dense projections to a restricted region at the area / border of the other hemisphere, where they terminate in layers / and . we used in utero electroporation-mediated gene transfer of gfp to assess distribution of callosal projections during development. gfp-labeled callosal axons arrived at the / border and started to invade the cortical plate around p . they generated exuberant axonal arborizations by p then underwent remodeling to form the adult pattern by p . neonatal enucleation did not affect the development, suggesting no apparent role of retinal activity. exogenous expression of a potassium channel kir . in callosal projection neurons, a manipulation known to reduce neuronal firing, attenuated axonal arborizations in layers / . area-specific targeting to the / border was not affected. results suggest that certain stages of callosal axon development require neuronal activity in cortex. ps p-e identification of a novel tetraspan membrane protein, nplp, that is up-regulated after the facial nerve axotomy chihiro akazawa, yoh sasaki, masato hoshi, yasuko nakamura, shinichi kohsaka department of neurochemistry, national institute of neuroscience, ncnp, japan facial nerve axotomy of adult rats, with its extensive ability to regenerate, provides us of a useful model in which to study the molecular mechanisms of nerve regeneration. to identify genes up-regulated during regeneration processes, we constructed a subtractive library enriched for cdnas expressed in the injured facial nucleus. we identified a novel putative tetraspan protein designated as neuronal pmp like protein (nplp) that has a distant homology to pmp /claudin family proteins. the mrna expression of nplp was detected on the cell bodies of neurons meanwhile mrna of pmp resided on the cell bodies of schwann cells. the nplp mrna level significantly increased in motor neurons of axotomized facial nerve, hypoglossal nerve or sciatic nerve. nplp has a large extracellular domain that may interact with other membrane molecules. overexpression of nplp showed the microspike formation in pc cells. our results suggest that nplp plays an important role in the regeneration of injured motor neurons. shinsuke shibata , shin-ichi sakakibara , hirotaka j okano , hideyuki okano , dept. physiol., keio univ., sch. med., japan; dept. histol. neurobiol., dokkyo med. univ., japan ; crest-jst, japan musashi (msi) is an evolutionarily conserved rna-binding protein. drosophila-msi regulates asymmetric cell division in neural development. in mammalian cns we identified two members, msi and msi , which are co-expressed in neural precursor cells including neural stem cells, suggesting msi proteins regulate the immaturity of cns stem cells. to reveal the roles of msi family in vivo, we generated msi and msi deficient mice. msi −/− mice frequently died of obstructive hydrocephalus with aberrant cell proliferation of cerebral aqueduct. msi −/− mice survive to adult show poor weight gain, low spontaneous movement and temperature insensitivity, with the malformation of pns. msi −/− msi −/− mice die in a few hours after birth with severe defects in the brain, including malformation of hippocampal dentate gyrus. in vitro and in vivo analysis show msi family proteins control the expression of several downstream target molecules post-transcriptionally, and play important roles cooperatively in mammalian cns and pns development. youhei koshimizu , satoshi yamagoe , kazuo suzuki , michiko ohtomi department of biomolecular science, graduate school of science, toho university, chiba, japan; department of bioactive molecules, national institute of infections diseases, tokyo, japan we have previously suggested that lect (leukocyte cell-derived chemotaxin ) is expressed in neurons and may play a role in the regulation of both dendritic extension and morphology of astrocytes in the mouse brain. however, the mechanisms of these regulations have not yet been clarified. in this study, we carried out multi-immunostaining against lect and tau or map in neuronal cell cultures and in tissues of adult mouse brain to examine the functional sites of lect in neurons. at the early stages in cultured neurons, lect was remarkably localized in the vicinity of membrane of soma, and was detected around root and end of neurites. in tissues of adult mouse brain, lect was localized to soma, axons and dendrites in neurons. these results suggest the possibility that lect influences the extension of dendrites and axons from the early stage of neuritic development. furthermore, it is thought that lect may regulate the dendritic and axonal morphology in mature neurons. mitsuru noda , , takahiro moriya , hideyuki terazono , suguru kudo , masahiro yamaguchi , kenji yasuda , izumi nagata , kazuyuki shinohara div. neurobiol. & behav., nagasaki univ. grad. sch. biomed. sci., nagasaki, japan; dpt. neurosurg. radiol. nagasaki univ. nagasaki, japan; dpt. life sci., grad. sch. arts & sci., univ. tokyo, tokyo, japan; dpt. physiol., grad. sch. med., univ. tokyo, tokyo, japan; special division for human life technology, national institute of advanced industrial science and technology (aist), japan neural stem cells (nscs) are defined as self-renewing, multipotent progenitor cells that give rise to neurons, astrocytes and oligodendrocytes. using single-cell-based on-chip cell-cultivation system, we analyzed the process of neural differentiation and examined the effects of bdnf. the individual nscs from e . nestin-promoter gfp transgenic mice were placed into pairs of agar microchambers connected by microchannels and were differentiated in the presence or absence of bdnf. we observed that a part of nscs exhibited neuron-like morphology and extended some neurites. the application of bdnf increased the rate of neurite outgrowth. thus we addressed the process of neural differentiation in a single-cell-based level and demonstrated the effect of bdnf. ayumi kyuka, ryoichi yoshimura, yasuhisa endo department of applied biology, kyoto institute of technology, kyoto, japan in order to investigate the relationship between neurite outgrowth and target cells, ng cells were co-cultured with vascular smooth muscle cells (sm- ). sm- cells promoted the neurite outgrowth, but repelled the contact of their growth cones. the dna microarray analysis indicated that neuropilin- mrna was specifically up regulated in ng cells. but the western blotting analysis did not show the significant increase of neuropilin- . sema a, a candidate of ligands for neuropilin- , was found in ng cells rather than in sm- cells. the immunocytochemical analysis revealed that neuropilin- was increased in the surface of ng cells co-cultured with sm- cells or cultured with the conditioned medium of sm- cells. these results suggest that neuropilin- may be transferred from cytoplasmic pool to surface of ng cells by some soluble factors released by sm- cells. we will discuss about kinetics of neuropilin and chemorepellents. yoshikuni edagawa , j. nakanishi , h. hamada , y. yokomachi , k. yamaguchi , n. takeda inst. biomed. eng., waseda univ., tokyo, japan; riken, japan; kanagawa univ., japan we show a novel method for controlling neurite outgrowth with a photochemical micropatterning technology, which is based on the photoreactive molecule switching its character by irradiation of uv light. this molecule formed a self-assembled monolayer on a glass coverslip, which was used for cell-culture surface. for the neurite outgrowth, pc cell was recruited as a model of neuron. by irradiating uv light at small spots on the cell-culture surface, single cells were specifically attached to the corresponding spots with intermediated ecm. to draw narrow paths for neurite outgrowth, new regions were irradiated in adjacent to the cell bodies attached on the spots. ngf stimulated each pc cell to extend a neurite along the narrow path: the elongation required both photo-activated path and ngf. the successive irradiation in front of developing neurite achieved bending or branching of the neurite. these results indicate that the timing and direction of neurite outgrowth can be controlled with this functional culture system. tetsuhiro kakimoto, hironori katoh, manabu negishi lab. of molecular neurobiology, grad. sch. of biostudies, kyoto univ., kyoto, japan n-wasp is important for actin polymerization and is strongly expressed in brain. toca- was shown to be required for cdc to activate n-wasp in vitro, although its physiological role is unknown. here we studied neural function of toca- . toca- is strongly expressed in developing brain. knockdown of toca- in pc cells significantly enhances neurite elongation. consistently, overexpression of toca- suppresses neurite elongation through its amino terminus including the f-bar/efc domain, which induces plasma membrane invagination. in addition, knockdown of n-wasp also enhances neurite elongation in pc cells, in clear contrast to the previous report using dominant negative mutants. on the other hand, knockdown of toca- in rat hippocampal neurons enhances axon branching a little but not axon elongation, while knockdown of n-wasp enhances both axon elongation and branching. these results suggest that a vesicle trafficking regulating protein toca- regulates different aspects of neuronal morphology from n-wasp. research funds: kakenhi ( ) ps p-e pragmin, a novel effector of rnd gtpase, stimulates rhoa activity and regulates neurite outgrowth hironori katoh, hiroko tanaka, manabu negishi kyoto university, japan the rho family small gtpase rnd is specifically expressed in brain. although rnd and rnd display an antagonistic action for rhoa, signaling pathways of rnd are not well known. here we have performed a yeast two-hybrid screen using rnd as bait and identified a novel rnd -effector protein, pragmin (pragma of rnd ). pragmin is predominantly expressed in neurons, including cortical and hippocampal neurons. in in vitro and in vivo binding assays, pragmin specifically bound to rnd among the rho family gtpases in a gtp-dependent manner. rnd -bound pragmin significantly stimulated rhoa activity and induced cell contraction through rhoa-and rho-kinase-dependent pathway in hela cells. in pc cells, expression of pragmin inhibited the ngf-induced neurite outgrowth in response to rnd , and knockdown of pragmin by a pragmin-specific small interfering rna enhanced the neurite elongation. therefore, these results suggest that rnd controls neurite outgrowth by regulating rhoa activity through pragmin. ps p-e regulation of growth cone motility by collapsin response mediator protein- (crmp- ) masumi iketani , yanai shigeki , fumio nakamura , yoshio goshima , , kohtaro takei , dept. of mol. pharmacol. & neurobiol., yokohama city univ. grad. sch. of med., yokohama, japan; crest, japan sci. & tech. agency, kawaguchi, japan nerve growth cone located at a distal tip of a neurite is known to be the site governing axonal growth and guidance. collapsin response mediator protein- (crmp- ) is enriched in the distal part of axon and participated in axonal formation and growth as well as growth cone collapse by semaphorin signaling. the local functions of crmp- within growth cones, however, remains unclear. chromophoreassisted laser inactivation (cali) that can inactivate selectively a target protein with high spatial and temporal resolutions. acute localized loss of function of crmp- in the entire growth cone region resulted in temporal growth arrest of neurite in chick dorsal ganglion neurons. asymmetric inactivation of crmp- in a half region of growth cone caused growth cones to turn temporally toward the inactivated region. these findings suggest that a spatial distribution of crmp- activity within the growth cone can regulate growth cone motility and direct neurite outgrowth. research funds: crest, jst ps p-e eph receptors are negatively controlled by protein tyrosine phosphatase receptor type o takafumi shintani , , masaru ihara , , hiraki sakuta , , hiroo takahashi , , ikuko watakabe , masaharu noda , division of molecular neurobiology, national institute for basic biology, okazaki, japan; crest, jst, kawaguchi, japan eph receptors are involved in numerous events such as cell movements, axonal guidance, the formation of synapses, and the maintenance of synaptic plasticity. eph receptors are activated by autophosphorylation of tyrosine residues upon the binding of their ligands, ephrins, however, the protein tyrosine phosphatases (ptps) responsible for the negative regulation of eph receptors have not been elucidated. here, we identified protein tyrosine phosphatase receptor type o (ptpro) as a specific ptp that efficiently dephosphorylates both epha and ephb receptors as substrates. using the retinotectal projection system, we show that ptpro controls the sensitivity of retinal axons to ephrins, and thereby plays a crucial role in the establishment of topographic projections. ps p-e neogenin acts as a displacement factor that releases rho from rho-gdi katsuhiko hata, toshihide yamashita department of neurobiology, graduate school of medicine, chiba university, chiba, japan repulsive gudiance molecule (rgma) is a potent inhibitor of axon regeneration in the adult central nervous system. rgma inhibits mammalian cns neurite outgrowth by a mechanism dependent on activation of the rho/rho-kinase pathway. here we show that direct interaction of the rho gdp dissociation inhibitor (rho-gdi) with neogenin which is a receptor of rgma initiates the activation of rhoa, and this interaction between neogenin and rho-gdi is strengthened by rgma. we also found that neogenin facilitates the release of prenylated rhoa from rho-gdi. thus, understanding of the molecular mechanisms of rgma may be useful for developing methods to overcome the inhibitory signals. nobuyuki fukushima, masumi nakashima, ryou tokunaga, ryutaro moriyama department of life science, kinki university, higashiosaka, japan lysophosphatidic acid is an extracellular signaling lipid that regulates neurite morphology in neuronal cells through g protein-coupled lpa receptors, including lpa and lpa . here we examine how lpa , third lpa receptor subtype, is involved in neuronal development by using pheochromocytoma (pc ) cells. pc cells expressed low levels of endogenous lpa mrna. when pc cells were infected with retrovirus expressing lpa and treated with nerve growth factor under serum-free conditions for days, neurite formation was observed in % of total infected cells, which was similar to that in control virus-infected pc cells. however, the mean length of neurites was increased in pc cells expressing lpa , compared with that in control pc cells. interestingly, multiple neurite sprouts were frequently observed in lpa-treated lpa -expressing cells. indeed, the numbers of neurites and neurite branching points per cell were increased in these cells. these results indicated that lpa was involved in neuritogenesis and branch formation. masao sakurai , tomoko aoki , kyoko ishikawa , shingo yoshikawa , john b. thomas , chihiro hama cdb, riken, hyogo, japan; salk institute, usa in drosophila, odor information is represented as a combination of activated glomeruli in the antennal lobe (al). each glomerulus is formed by specific synaptic connections between olfactory receptor neurons (orns) and projection neurons (pns). in an attempt to address the question of how these glomeruli are stereotypically organized during development, we identified wnt as a regulatory factor for glomerular patterning. in the wnt mutant, several glomeruli were shifted to ectopic positions, and this phenotype was partially rescued by wnt expression in orns. previous studies in embryos have shown that wnt repels the axons expressing derailed (drl). in the als of drl mutants, however, some glomeruli were located at incorrect positions, with the pattern different from that in the wnt mutant. this and other genetic data suggest that drl is an antagonist for wnt signaling. we propose that wnt signal transmitted from orns to pns provide one of mechanisms for glomerular patterning in drosophila. ps p-e a homeodomain transcription factor, homothorax, controls precise dendritic and axonal targeting of the antennal lobe projection neurons in the drosophila brain mai ando, yoko totani, katsuo furukubo-tokunaga grad. sch. life envir. sci., univ. tsukuba, japan the precise neuronal connectivity in the nervous system depends on specific axonal and dendritic targeting of individual neurons. temporal and spatial regulation of gene expression by transcription factors is though to play seminal functions in determining wiring specificity. we are interested in the identification of the genes that control specification and connectivity of antennal lobe projection neurons, which convey olfactory information from the primary olfactory center to the higher order structures such as mushroom bodies and lateral horns. here, we show that homothorax (hth), a homeodomain protein, is expressed in most of the projection neurons. marcm mosaic analyses showed profound targeting defects in both dendritic and axonal projection patterns. currently, we are looking for genes that are either downstream or work cooperatively with hth. based on these data, we will discuss the functional roles of hth in the specification of antennal lobe projection neurons. ps p-e differential microarray analysis of mushroom body transcripts using chemical ablation ayako ino , masatomo kobayasi , taiki nakajima , lydia michaut , indrayani ghangrekar , kuniaki takahashi , ryu ueda , kaoru saigo , walter j. gehring , katsuo furukubo-tokunaga grad. sch. life envir. sci., univ. tsukuba, japan; biozentrum, univ. of basel, switzerland; genet. strains res. ctr., natl. inst. genet.; dept. biophys. biochem. grad. sch. sci., univ. tokyo, japan mushroom bodies (mbs) are the centers for olfactory associative learning in the drosophila brain. we have surveyed mb transcripts using a drosophila whole-genome microarray and identified , genes that exhibited reduced expression levels with pharmacological mb ablation. among the identified genes, we have further selected genes that exhibited a most consistent reduction of expression levels in the ablated brains. in situ hybridization analyses confirmed preferential mb expression patterns for the majority of the identified genes. moreover, we also demonstrate that rnai of many of the identified genes causes mild to strong mb defects. these results provide novel and genome-wide insights into the repertoire of the genes that control differentiation and function of the mb neurons in brain development and plasticity. mikiko inaki , yoshie suzuki , hiroyuki aburatani , akinao nose dept. phys., univ. tokyo, tokyo, japan; rcast, univ. tokyo, tokyo, japan in drosophila neuromuscular junction, the axons of individual motoneurons precisely recognize and project to specific muscle cells. to understand the molecular logic of target specificity, we tried to identify systematically target recognition molecules expressed on individual muscles. we focused on two neighboring muscles, and , which are innervated by distinct motoneurons, and searched for genes that are differentially expressed between them by using wholegenome microarrays. by comparing gene expression data from the two muscles, we identified ∼ genes with more than two-fold difference in expression level. for candidate genes out of ( %), the differential expression was confirmed by in situ hybridization and/or quantitative pcr. we focused our functional analyses on those encoding transmembrane or secreted proteins, with confirmed differential expression pattern. ectopic expression and/or knockdown of some of them altered the target specificity of muscles or , implicating them in neuronal target recognition. makoto aoki, hiroshi segawa, mayumi naito, hitoshi okamoto laboratory for developmental gene regulation, brain science institute, riken, saitama, japan the sensory neurons have two axons, the central and peripheral axon. previously, we have demonstrated that peripheral axons of sensory neurons are completely abolished in the zebrafish embryos overexpressing the lim domains of islet- . to find the molecules which regulate the formation of peripheral axons of sensory neurons in the islet- signaling cascade, we have created cdna library from trigeminal sensory neurons of zebrafish larvae. most of clones were known genes which are implicated in signal transduction, transcription and cytoskeltal formation, but several clones show no similarity with any known genes. most of them show cns or sensory neuron-specific expression patterns and the expression level of these genes were affected by the overexpression of the lim domains of islet- . loss of function and gain of function studies of these clones, whose function are unknown, showed that these clones might be involved in the development of peripheral axons or the formation of neural circuitry. hiroshi yamamoto, kiyokazu agata development of biophysics, graduate school of science, kyoto university, kyoto, japan highly conserved netrins are known as bi-functional guidance molecules, attracting some axons and repelling others. they act through receptors of the deleted in colorecal cancer (dcc) and unc receptor families. freshwater planarians can proliferate by fission and decapitation, the anterior piece regenerates a tail, and the posterior piece regenerates a head. the planarian central nerve system (cns) can be used as a model for studying neural regeneration and understanding how we can rebuild nervous system after injury. we have identified eight netrin related genes from planarians, dugesiajaponica, including four different netrin homologues and three unc homologuesand one dcc homologue. we analyzed expression patterns of eight genes in both intact and regenerating planarians, and then conducted rnai experiments to investigating their functions in the process of regeneration of visual neurons. here we will summarize these results and speculate their molecular actions during eye regeneration in planarians. ps p-e analysis of the significance and mechanism of neurite elimination using c. elegans as a model yu hayashi , takaaki hirotsu , eriko kage , hideaki takeuchi , hirofumi kunitomo , yuichi iino , takeo kubo dept. biol. sci., grad. sch. sci., univ. tokyo, tokyo, japan; dept. biol., fac. sci., kyushu univ. grad. sch., japan; mol. genet. res. lab., univ. tokyo, japan during brain development, a large amount of neural connections once formed undergo elimination, the mechanism and physiological significance of which are poorly understood. we previously showed that elimination of neurites occurs in the nematode c. elegans and that a novel transcription factor mbr- is involved in this process. in the present study, we attempted to clarify the significance of this phenomenon. the mbr- mutant is defective in odor adaptation, and we therefore hypothesized that neurite elimination is critical for maturation of the olfactory circuit. by executing cell-specific rescue experiments, we suggest that mbr- functions in a set of interneurons involved in olfactory processing. we are now examining whether neurite elimination occurs in these neurons using gfp reporters. we expect that our research provides a useful model for genetic dissection of neurite elimination. taro asakura , ken-ichi ogura , yoshio goshima , dept. of mol. pharmacol. and neurobiol., yokohama city univ., grad. sch. of med., yokohama, japan; crest, japan sci. and tech. agency, kawaguchi, japan netrin is an evolutionary conserved axon guidance molecule. in c. elegans, netrin/unc- is secreted from ventral cells, and attracts some axons ventrally and repels dorsally. one of the neurons guided by netrin/unc- is the hsn neuron. the hsn neuron of c. elegans has interesting axon guidance. netrin/unc- is required for the first ventral growth of the hsn neurons. however, what molecules participate in the axon guidance is largely unknown. in this study, we found that the vulval precursor cells strongly expressed netrin/unc- during the axonal guidance of the hsn neurons. silencing of netrin/unc- only at the vulval precursor cells resulted in abnormal axon guidance of hsn. in addition, expression of netrin/unc- only at the vulval precursor cells in unc- null mutants partially restored the hsn guidance defects. these results suggest that netrin/unc- expressed in vulval precursor cells plays essential roles on axon guidance of the hsn neuron. tomomi sato, takanori hamaoka, hidenori aizawa, hitoshi okamoto brain science institute, riken, saitama, japan the optic tectum is a visual center in vertebrates. it receives topographically ordered visual input from the retina in the superficial layers and then sends motor outputs from the deeper layers to the premotor reticulospinal system in the hindbrain. although it is well known how the retinal axons project to the tectum, it has not yet been well understood how the tectal axons project to the hindbrain. here, we established a stable transgenic line tg(brn a-hsp :gfp) in zebrafish to visualize the retinotectal and the tectobulbar projections. to explore the question, we developed an efficient single-neuron labeling system in combination with cre/loxp and gal /uas systems. using the system, we found that the tectum projected to each hindbrain segment with a two-dimensionally ordered pattern. in addition, we showed that ephrinb a was involved in the patterned projection from the posterior tectum to the hindbrain segment rhombomere . these results suggest a neuroanatomical and a molecular basis for the motor map in the tectum. research funds: grant-in-aid for young scientists (b) masahiko taniguchi , yoshinori mikami , tomoyuki masuda , tomoyuki yoshida , naoto matsuda , masayoshi mishina , takao shimizu department of biochemistry, cancer research institute, sapporo medical university, sapporo, japan; department of molecular neurobiology and pharmacology, graduate school of medicine, university of tokyo, japan; department of anatomy, fukushima medical university, japan; dapartment of biochemistry and molecular biology, faculty of medicine, university of tokyo, japan semaphorin gene family contains a large number of secreted or transmembrane proteins, and some of them function as the repulsive and attractive cues of axon guidance during development. here we report the identification of zebrafish semaphorin d (sema d). the orf of sema d is bp ( aa). zebrafish sema d protein showed a . % identity with mouse sema d protein. to clarify the expression pattern of sema d, in situ hybridization was performed. in the embryos, sema d is expressed in the lens and hindbrain. we also found that sema d has the repulsive activity. these results indicate that sema d repels specific types of the axons and functions in the nervous system development. research funds: kakenhi on priority areas from the mext ( ) mayumi yamada , shohei maekawa , seiji miyata department of applied biology, kyoto institute of technology, kyoto, japan; division of bioscience, graduate school of science and technology, kobe university, kobe, japan obcam belongs to the immunoglobulin superfamily cell adhesion molecule (cam) and was synapse-specific cam in cortical and hippocampal neurons in vivo. however, it is uncertain how obcam is involved in synaptic functions. in this study, we showed that obcam was highly concentrated on spines of cultured mature neurons. the inhibition of obcam function with its specific antibody resulted in a decrease in the number of presynaptic terminals and postsynaptic spines. the suppression of obcam mrna expression with its specific antisense oligonucleotide also resulted in impairment of synapse formation. in contrast, overexpression of obcam augmented the formation of synapses. moreover, obcam was internalized via a raft-dependent pathway and the internalization was promoted by increased neuronal activity. these results suggest that obcam, a spine-specific cam, is a critical modulator of synaptogenesis and its surface expression is dynamically regulated in response to neuronal activity. ps p-f semaphorin d inhibits ␤ integrin activity through the r-rasgap activity of plexin-b izumi oinuma, hironori katoh, manabu negishi lab. mol. neurobiol., grad. sch. biost., kyoto univ., kyoto, japan plexins are cell surface receptors for semaphorins and regulate cell migration in many cell types. we have recently reported that the semaphorin d (sema d) receptor, plexin-b functions as a gap for r-ras, a member of ras family gtpases implicated in regulation of integrin activity and cell migration. here we characterized the role of r-ras downstream of sema d/plexin-b in cell migration. activation of plexin-b by sema d suppressed the ecm-dependent r-ras activation, r-ras-mediated pi -k activation, and ␤ integrin activation through its r-ras gap domain, leading to inhibition of cell migration. in addition, inactivation of r-ras by overexpression of the r-ras-specific gap or knockdown of r-ras by rna interference was sufficient for suppressing ␤ integrin activation and cell migration in response to the ecm stimulation. thus, we conclude that r-ras activity is critical for ecm-mediated ␤ integrin activation and cell migration, and that inactivation of r-ras by sema d/plexin-b -mediated r-ras gap activity control cell migration by modulating the activity of ␤ integrins. shun hamada , emi fukuda , , shota katori , , takeshi yagi , dept. nutrition health sci., fukuoka women's univ., fukuoka, japan; grad. sch. frontier biosci., osaka univ., osaka japan, crest, jst, japan cnr/protocadherin (pcdh)␣ is a subfamily of the clustered pcdhs that comprise > cadherin-related molecules and are encoded by three gene clusters (␣, ␤ and ␥). the molecular features and synaptic localization of the clustered pcdhs have raised the possibility that they are synaptic recognition molecules. we have demonstrated that overexpressed pcdh␣ family proteins alone in several cell lines are rarely transported into the plasma membrane. furthermore, we found that a stretch of about fifty amino acids located at the c-terminus of pcdh␣s interfered the trafficking to the cell surface. in the present study, we compared the transport properties of a series of the cytoplasmic region truncation mutants and found that truncation mutants lacking or more c-terminal residues were detectable at the cellular surface suggesting a role for lysine-rich motif in the c-terminus of pcdh␣s in the intracellular retention. mdga is a novel cell surface glycoprotein similar to ig-containing cell adhesion molecules (igcams) with functions in migration and process outgrowth. mdga is expressed by layer / neurons throughout the neocortex at p mice, but is absent in adults. between e . and late p , stages that span the generation and radial migration of layer / neurons, mdga is expressed in patterns consistent with its expression by migrating layer / neurons, suggesting a role for mdga in controlling their migration and settling in the superficial cortical plate. we performed loss-of-function studies using rna interference (rnai) with in utero electroporation into the lateral ventricle at e . to transfect progenitors of superficial layer neurons. we found that an rnai suppressing mdga protein blocks proper migration of superficial layer neurons to the superficial cortical layer. we conclude that mdga acts cell autonomously to control the migration of superficial layer cortical neurons. in various pathological conditions, activated microglia mediate immune responses to injured cns neurons. however, it is not clear whether and how activated microglia affect neurons via direct contacts. this study aimed at examining whether direct contacts between microglia and hippocampal neurons increase following cns injury and whether telencephalin (tlcn), a dendrite specific adhesion molecule, which potentially binds to immune cells, mediates the direct contact. hippocampal neurons were damaged by local injection of excitotoxin, kainic acid (ka). compared to control animals, ka-injected mice showed higher density of contacts between activated microglia and dendrites of ca pyramidal neurons. contacts with longer interface appeared in ka-injected mice. these results suggest the importance of direct contacts for the immune response of microglia to injured neurons. similar contact formation was also observed in tlcn-deficient mice, indicating that the direct contacts are mediated by other molecules than tlcn. kilon is belonging to immunoglobulin superfamily of cell adhesion molecules and contains three igg-like domains. western analysis revealed that the expression levels of kilon is low at early neuronal culture and increased with progress of culture days. immunocytochemical observation showed that kilon was localized at elongating axon and growth cones but not at dendrites on days in vitro (div), while kilon was observed at synapses, mainly at presynaptic terminals on div. similar tendency was observed in kilon immunohistochemistry of brain sections in vivo. kilon was observed at axonal fibers of the cerebral cortex on postnatal day , but it was seen at synapses in adult brains. these results suggest that kilon is axonal cell adhesion molecule to control axonal guidance and/or extension. ps p-f analysis of mice that show abnormal expression of neuroglycan c, a central nervous system-specific transmembrane proteoglycan sachiko aono , yoshiyuki kuroda , fumiko matsui , yoshihito tokita , keiko nakanishi , michiru ida , masahito ikawa , masaru okabe , katsuhiko ono , atsuhiko oohira institute for developmental research, aichi human service ctr., kasugai, japan; research institute for microbial diseases, osaka university, suita, japan; national institute for physiological sciences, okazaki, japan neuroglycan c (ngc) is a membrane-spanning chondroitin sulfate proteoglycan that is exclusively expressed in the central nervous system. to study the role of ngc in the brain, we produced two strains of ngc-mutant mouse by gene-targeting; a mouse strain with no ngcexpression and a strain with low expression (knockdown mice). both mice were viable and fertile. they did not show obvious abnormalities in gross brain anatomy. to examine their behavioral phenotype precisely, the ngc-knockdown mice were subjected to several kinds of behavioral tests sequentially. they displayed obvious abnormalities in morris water maze and passive avoidance tests, suggesting that ngc is involved in learning and memory. we are now carrying out the same experiments using the ngc-knockout mice. research funds: kakenhi ( ) ps p-f phosphorylation of extracellular signal-regulated kinase in aged rats with acute face inflammation koichi iwata , tatsuhisa watanabe , ikuko suzuki , junichi kitagawa , akiko ogawa , kenro kanda , kazunao kuramoto dept. of physiol., sch. of dent., nihon univ., tokyo, japan; dept. of oral and maxillofacial surgery, sch. of dent., nihon univ., tokyo, japan; dept. of oral diagnosis, sch. of dent, nihon univ., tokyo, japan; shinjuku vocational school of acupuncture, moxibustion and judo therapy, tokyo, japan; division of research animal center, tokyo metropolitan institute of gerontology, tokyo, japan the capsaicin-induced perk expression was studied in the aged rats ( - months) following noxious face stimulation. a large number of perk-li cells were expressed in the superficial laminae of the trigminal spinal nucleus in adult and aged rats following subcutaneous capsainsin injection into the whisker pad region. the larger number of perk-li cells was expressed in adult rats than aged rats following intravenous administration of naloxone before capsaicin treatment. the present results suggest that the descending modulation system was impaired in the aged rats, resulting in the abnormal pain sensation advancing age. hirokazu katsura , koichi obata , masafumi sakagami , koichi noguchi department of anatomy and neuroscience, hyogo college of medicine, hyogo, japan; department of otorhinolaryngology, hyogo college of medicine, hyogo, japan recent studies demonstrated that the activation of extracellular signal-regulated protein kinase (erk) / and p mitogen-activated protein kinase (mapk) in dorsal root ganglion (drg) neurons contributes to the development of inflammatory and neuropathic pain. in the present study, we examined whether the newest member of the mapk family of proteins, erk (also known as big mapk or bmk ) is activated in the drg and participate in pain-related behaviors in the complete freund's adjuvant (cfa) model. peripheral inflammation induced an increase in the phosphorylation of erk , mainly in tyrosine kinase a-containing small-to-medium-diameter drg neurons at days and after cfa injection. furthermore, time course of phosphorylated-erk level in the drg matched the emergence of cfa-induced pain hypersensitivity. our data suggest that activation of erk in drg neurons may contribute to the development of inflammatory pain. ps p-f activation of erk in drg neurons contributes to acute pain toshiyuki mizushima , , koichi obata , takashi mashimo , koichi noguchi department of anatomy and neuroscience, hyogo college of medicine, hyogo, japan; department of anesthesiology, osaka univ. recently, we have reported that phosphorylation of extracellular signal-regulated protein kinase (erk) / and p mitogen-activated protein kinase (mapk) occurred in primary sensory neurons in response to natural noxious stimulation of the peripheral tissue, i.e., activity-dependent activation of erk and p in dorsal root ganglion (drg) neurons. however, there has been no study examining erk (also known as big mapk or bmk ) activation in drg neurons after noxious stimulation of normal tissue. here, we report intensity-dependent erk phosphorylation in drg neurons by painful stimulation. noxious stimulation induced phosphorylated-erk in small-to-medium diameter sensory neurons with a peak at min after stimulation. furthermore, we found a stimulus intensitydependent increase in the number of activated neurons. our data suggest that activation of erk in drg neurons may contribute to acute pain induced by noxious stimulation. koichi obata, koichi noguchi department of anatomy and neuroscience, hyogo college of medicine, japan there is compelling evidence indicating that the activation of extracellular signal-regulated protein kinase (erk) / and p mitogenactivated protein kinase (mapk) in the dorsal root ganglion (drg) and spinal cord contributes to the development of inflammatory and neuropathic pain. in the present study, we examined whether the newest member of the mapk family of proteins, erk (also known as big mapk or bmk ) is activated in the drg and spinal cord and participate in pain-related behaviors in the l spinal nerve ligation (snl) model. l snl induced an increase in the phosphorylation of erk not only in the injured l drg, but also in the spared l drg at day after surgery. furthermore, l snl induced a striking increase in erk phosphorylation in glial cells in the ipsilateral dorsal horn. our data suggest that activation of erk in the drg and spinal cord may contribute to the development of neuropathic pain. atsushi sakai , minoru asada , naoki seno , hidenori suzuki department of pharmacology, nippon medical school, tokyo, japan; pharmaceutical research center, kyowa hakko kogyo co., shizuoka, japan glial cell line-derived neurotrophic factor (gdnf) has been known to alleviate the neuropathic pain. however, the mechanisms of gdnfinduced analgesia remain almost unclear. gdnf binds to gfr␣- , which forms receptor complex and signals intracellularly through ret. recently, neural cell adhesion molecule (ncam) has been found to be an alternative signal-transducing receptor for gdnf. here, we report that ncam is involved in gdnf-induced analgesia in a rat model of the neuropathic pain. ncam mrna expression was decreased in the ipsilateral dorsal horn of the spinal cord after the nerve injury, but gdnf treatment returned its expression to the normal level. treatment with ncam antisense oligodeoxynucleotide blocked the analgesic effect of gdnf without affecting ret phosphorylation. these results suggest that activation of ncam signaling may provide a new strategy to relieve intractable chronic pain. ps p-f interleukin- ␤ enhanced the excitability of trigeminal root ganglion neurons via activation of satellite glia following inflammation mamoru takeda , jun kadoi , msanori nasu , masayuki takahashi , shigeji matsumoto dep. physiol and res. cent. for odont. nippon dent. univ., japan the present study was investigated whether activation of satellite glial cells modulates the excitability of trigeminal root ganglion (trg) neuronal activity via the il- ␤ paracrine mechanism following inflammation. two days after cfa into the whisker pad area, the mean number of trg neurons that were encircled by glial fibrillary acidic protein and il- ␤-immunoreative satellite cells were significantly increased compared with those in the control. fg labeling was used to identify the trg neurons innervating the site of inflammation. in the fg-labeled small trg neurons, the occurrence of il- ␤ induced depolarization in inflamed rats was larger than that in control rats. il- ␤ application significantly increased the firing rate evoked by depolarizing pulses in the inflamed neurons compared with the control neurons. these results suggest that activation of trg satellite glial cells modulates the excitability of trg neuronal activity via the il- ␤ paracrine mechanism following peripheral inflammation. junichi kitagawa , mamoru takeda , jun kadoi , yoshiyuki tsuboi , shigeji matsumoto , koichi iwata dept. of physiol., sch. of dent., nihon univ., tokyo, japan; dept. of physiol, sch. of dent. at tokyo, japan, nippon dental univ., tokyo, japan the present study was designed to elucidate an involvement of the primary afferent neurons in the trigeminal neuropathic pain using the rats model with chronic constriction nerve injury of the infraorbital nerve (ion-cci). the mechanical escape threshold was significantly lower in ion-cci rats at day after ion treatment and the threshold decrement was lasting more than day . single unit activities of ion were recorded from the ion-cci rats. the firing frequency was significantly higher in a␦ fibers in ion-cci rats as compared with naive at day - after ion-cci. whole cell patch clamp recording was performed from the middle trg neurons. ik and ia currents were significantly smaller and ih current was larger in ion-cci rats than that of naive rats. the present results suggest that ik, ia and ih currents are involved in abnormal firing of trg neurons in the rats with ion-cci, resulting in neuropathic pain in trigeminal region following peripheral nerve injury. hisako urai, munehiro uda, katsuya kami graduate schools of sport and exercise science, osaka university of health and sport sciences, osaka, japan mechanical hyperalgesia of skeletal muscles has been known to occur following intense eccentric contraction such as downhill running (dhr). the present study examined the number of c-fos-positive neurons in spinal dorsal horn to determine peak of mechanical hyperalgesia following dhr in rats. furthermore, we investigated whether glial cells are activated in dorsal horn with excitation of secondary afferent neurons (san). rats performed an intermittent bout of dhr for min. at , , , and h post-dhr, the rats were applied a weight on the right triceps surae muscle. immunohistochemical staining for c-fos and gfap on spinal cords was performed by freefloating abc method. the number of c-fos-positive neurons detected in superficial dorsal horn were increased at h, peaked at h and then decreased. intense gfap immunoreactivities were also detected at and h post-dhr. these results suggest that dhr generates mechanical hyperalgesia by increasing responsiveness of san, and moreover astrocytes may regulate excitability of san. katsuya kami, hisako urai, munehiro uda department of health science, osaka university of health and sport sciences, osaka, japan a production of inflammatory cytokines is increased in injured skeletal muscles. the present study examined relationship between production of inflammatory cytokines in skeletal muscles and fospositive neurons in spinal dorsal horn following downhill running in rats. the rats performed the downhill treadmill running for min at m/min. after the running, rats were applied the weight on the gastrocnemius muscles for min, and then spinal cord and soleus muscles were removed from the rats. productions of il- beta, il- and tnf-alpha in soleus muscles and expression of fos protein in dorsal horn were examined using immunohistochemical approach. at h post-running, number of fos-positive neurons was increased, peaked at h and then decreased to control level at h post-running. vigorous inflammatory reactions with necrotic myofibers in soleus muscles were observed at days post-running. these results indicated that increased numbers of fos-positive neurons in dorsal horn are induced prior to vigorous inflammation of skeletal muscles. shinichi sugiyo , yusuke sakai , aya masawaki , takashi shimoda , masayuki moritani , motohide takemura dept. oral anatomy and neurobiology, osaka university grad. sch. of dentistry, osaka, japan; dept. of fixed prosthodontics, osaka university grad. sch. of dentistry, osaka, japan; dept. of dental anesthesiology, osaka university grad. sch. of dentistry, osaka, japan diabetes mellitus is among the most common causes of painful peripheral neuropathy, worldwide. we examined if there exist the diabetic rat (dm)-specific difference in nociceptive behavioral and c-fos immunoreactivity (ir) by formalin test. injection of formalin into the upper lip weeks before streptozotocin injection induced biphasic specific pain related behavior (prb) for min. first phase was greater in dm than in the control rat (ctrl). in dm, second phase was much greater than ctrl. c-fos ir in the trigeminal caudal nucleus was also greater in dm than in ctrl. these results indicate that dm induced greater prb and c-fos expression following formalin injection into the rat upper lip. yasuko kozaki, satoshi hurune, fukushi kambe, hisao seo, kazue mizumura res. inst. environ. med., nagoya university, nagoya, japan we have reported that prostaglandin ep receptor (ep r) activation attenuates the desensitization of bradykinin (bk)-induced increase of intracellular calcium ([ca + ] i ) in a ptx-sensitive manner in cho cells expressing canine ep r and mouse bk b receptor (b r). in this study, we examined the involvement of protein kinase a (pka) in the desensitization of the bk response. when bk ( nm) was applied twice with a -min interval to the cells expressing b r, the second [ca + ] i increase by bk was markedly attenuated. however, the pretreatment with a specific inhibitor of pka, h- ( m) restored the second response. to further confirm camp increase by bk, the expression of a camp responsive reporter gene was examined. bk ( pm) treatment for h significantly increased the reporter gene expression. it is likely that bk increases the level of intracellular camp, and thus activates pka, resulting in the desensitization of the bk response. these results suggest that the desensitization of bkinduced increase in [ca + ] i was at least in part mediated by pka. ps p-f contribution of peripheral ht a or ht receptors to fos expression in the trigeminal spinal nucleus (vsp) produced by the masseter muscle injury of rats keiichiro okamoto, akihisa kimura, tomohiro donishi, yasuhiko tamai dept. physiology, wakayama med univ., japan we have recently reported that orofacial nocifensive behavior evoked by the masseter muscle (mm) injury is attenuated by blocking peripheral ht a or ht /r in male rats with tmj inflammation. here we tested if these two ht/r subtypes contribute to fos responses in vsp after mm injury. formalin injection into mm produced fos-like immunoreactivity (li) in several areas of vsp and c . fos-li was distributed mainly in the ventrolateral trigeminal subnucleus interpolaris/caudalis transition (vl-vi/vc) and vc/c transition regions. the number of fos-li induced by mm injury was increased in these areas in cfa-evoked tmj-inflamed rats for days compared to naive rats. we tested if local ht a or ht /r antagonist affects fos expression in both groups. the number of fos-li in the vc/c but not vl-vi/vc region was reduced when drugs were injected locally prior to formalin injection in tmj-inflamed rats. these data suggest that peripheral ht a and ht /rs play critical roles in mediating mm nociception during tmj inflammation. keiko abe, hidemasa furue, kohei kga, go kato, toshiharu yasaka, akihiro tamae, toshihiko katafuchi, megumu yoshimura department of integrative physiology, graduate school of medical sciences, kyushu university, japan we examined the postsynaptic effects of -ht on substantia gelatinosa (sg) neurons in slice preparations of rat spinal cord and their relationship to the morphological features. in ∼ % of sg neurons examined, -ht induced an outward current. the outward current was mimicked and suppressed by a -ht a agonist and -ht a antagonist, respectively. in ∼ % of sg neurons, -ht evoked an inward current which was mimicked by a -ht agonist. the outward current was observed mostly in excitatory neurons such as vertical cell, while the inward current was induced in an inhibitory neuron, islet cell. these findings suggest that -ht inhibits excitatory neurons and excites inhibitory neurons in the sg through activation of -ht a and -ht receptors, respectively. the reciprocal postsynaptic actions of -ht on sg neurons in addition to presynaptic inhibitory effects on primary afferents might play an important role in descending control of nociceptive transmission by -ht. we examined effects of levobupivacaine, ropivacaine, bupivacaine and r-bupivacaine on epscs in substantia gelatinosa (sg) neurons of the spinal dorsal horn evoked by dorsal root stimulation, and on action potentials in dorsal root ganglion neurons generated by the dorsal root stimulation. in sg neurons, levobupivacaine reversibly suppressed the amplitude of monosynaptic a␦ and c fiber-evoked epscs. however, a␤ fiber-evoked epscs were slightly inhibited in amplitude. on the other hand, bupivacaine equally suppressed those three fiber-evoked epscs. in drg neurons, ic of bupivacaine and r-bupivacaine were almost equal on a␤, a␦ and c neurons. however, ic of levobupivacaine and ropivacaine on a␦ and c neurons were lower than that on a␤ neurons. the present results suggest that pure s (−) enantiomers especially levobupivacaine effectively inhibits noxious transmission to the spinal dorsal horn by the blockade of ap conduction through a␦ and c fibers. ps p-g nitric oxide-dependent long-term potentiation revealed by real time imaging of nitric oxide production and neuronal excitation in spinal dorsal horn hiroshi ikeda, kei kusudo, kazuyuki murase dept. human & artificial intelligence systems, univ. fukui, fukui, japan no plays an important role in the induction of long-term potentiation (ltp) in spinal dorsal horn, which is believed to underlie hyperalgesia and allodynia. in this study, to elucidate the relationship of no to ltp, we measured the spatiotemporal distribution of no signal with the no-sensitive dye, and neuronal excitation with the voltagesensitive dye, in rat spinal cord slices. in superficial dorsal horn, neuronal excitation evoked by dorsal root stimulation was potentiated for more than h after low-frequency conditioning stimulation (lfs). in the same slices that exhibited ltp, no was produced and distributed in the superficial dorsal horn during lfs. ltp and production of no were inhibited in the presence of no synthase inhibitors and an inhibitor of heme oxygenase, the synthetic enzyme for carbon monoxide (co). research funds: kakenhi to hi ( ) and km ( ) and grants from novartis foundation and promotion of science and sumitomo foundation to hi tao liu, tsugumi fujita, akiko koga, masafumi kosugi, terumasa nakatsuka, eiichi kumamoto dept. physiol., facult. med., saga univ., saga, japan in order to know the effect of a pla activator melittin on inhibitory transmission in the substantia gelatinosa (sg; lamina ii of rexed), we applied the blind whole-cell patch-clamp technique to sg neurons in adult rat spinal cord slices. in about % of neurons examined, melittin ( m) superfused for min gradually increased the frequency and amplitude of spontaneous glycinergic inhibitory postsynaptic currents which were recorded at mv in the presence of bicuculline. this action was visible about min after the beginning of its superfusion and subsided within min after washout. these melittin actions were reduced in extent by a pla inhibitor -bromophenacryl bromide, while being unaffected by tetrodotoxin, and also by inhibitors of cyclooxygenase (cox) and lipooxygenase (lox). it is concluded that pla activation pre-and postsynaptically enhances glycinergic transmission in sg neurons, possibly not through metabolites of cox and lox; this action would contribute to a modulation of nociceptive transmission. research funds: kakenhi ( ) ps p-g presynaptic p y receptor-mediated enhancement of inhibitory synaptic transmission in the rat spinal dorsal horn terumasa nakatsuka, shugo koga, tsugumi fujita, tao liu, masafumi kosugi, eiichi kumamoto department of physiology, faculty of medicine, saga university, saga, japan using whole-cell patch-clamp recordings, we examined whether the activation of p y receptors can modulate synaptic transmission in dorsal horn (dh) neurons of adult rat spinal cord slices. bath applied -methylthio adp ( mesadp, m), a p y receptor agonist, did not change excitatory transmission, but clearly increased the frequency and amplitude of spontaneous inhibitory postsynaptic currents (ipscs) in about % of dh neurons recorded. miniature ipsc in the presence of ttx was increased in frequency by mesadp with no change in the amplitude. the mesadp-induced increase in miniature ipsc frequency was attenuated in extent by mrs ( m), a selective p y receptor antagonist. these results indicate that the activation of presynaptic p y receptors enhances inhibitory but not excitatory synaptic transmission in a subpopulation of dh neurons. thus, spinal p y receptors can be involved in an inhibitory effect on pain transmission. research funds: kakenhi ( ), the japanese health sciences foundation (kh ) ps p-g presynaptic enhancement by proteinaseactivated receptor- agonist peptide of glutamatergic excitatory transmission in rat substantia gelatinosa neurons tsugumi fujita, terumasa nakatsuka, akiko koga, tao liu, masafumi kosugi, eiichi kumamoto dept. physiol., facult. med., saga univ., saga, japan we have previously reported that proteinase-activated receptor (par)- but not par- agonist (each m) enhances glutamatergic excitatory transmission in substantia gelatinosa (sg) neurons. the present study examined a detail of the par- mediated enhancement by applying the whole-cell patch-clamp technique to sg neurons in adult rat spinal cord slices. par- agonist (sfllrn, m) reversibly increased the frequency of spontaneous epsc without a change in the amplitude and also in holding current at - mv. this facilitatory action was resistant to tetrodotoxin, and was not seen in the presence of par- antagonist (yfllrnp, m). these results indicate that the activation of par- s existing in nerve terminals in the sg results in an increase in the spontaneous release of l-glutamate from there. it is suggested that par- activation in glutamatergic neuron terminals in the sg may be involved in the modulation of nociceptive transmission from the periphery. research funds: kakenhi ( ) ps p-g effect of tramadol metabolite m on glutamatergic excitatory transmission in rat spinal dorsal horn neurons akiko koga, tsugumi fujita, tao liu, terumasa nakatsuka, eiichi kumamoto dept. physiol., facult. med., saga univ., saga, japan in order to know the antinociceptive effect of tramadol, we examined the effect of m , which is one of its metabolites, at mm on glutamatergic excitatory transmission in substantia gelatinosa (sg) neurons of an adult rat spinal cord slice by using the whole-cell patchclamp technique. bath-applied m reduced the frequency but not amplitude of spontaneous excitatory postsynaptic currents (epscs) at − mv. this action was not seen in the presence of a -opioid receptor antagonist ctap ( m). m also reduced the peak amplitudes of epscs which were monosynaptically evoked at − mv by stimulating primary-afferent a␦-and/or c-fibers in a spinal cord slice with an attached dorsal root. we conclude that m inhibits the quantal release of l-glutamate from nerve terminals in the sg through the activation of -opioid receptors; this action is not distinct in extent between primary-afferent a␦-fiber and c-fiber transmission. this effect of m would give a cellular basis for the antinociceptive effect of systemically-administered tramadol. narihito iwashita, natsu koyama department of physiology, shiga university of medical science, otsu, japan in our previous study, subcutaneous injection of glutamate into the human forearm evoked pain and produced skin temperature increase around the injection site. these results suggest peripheral glutamate receptors contribute to nociceptive signaling and neurogenic inflammation. in order to further investigate which subtype of glutamate receptors is involved in neurogenic inflammation, effect of nmda receptor antagonist mk- or non-nmda receptor antagonist cnqx was evaluated in hindpaws of pentobarbital-anesthetized rats. attenuation of skin temperature increase induced by simultaneous mk- injection with glutamate was larger than that of skin temperature increase induced by simultaneous cnqx injection with glutamate at the same concentration. on the other hand, inhibition of paw edema formation by cnqx was stronger than by mk- . these data demonstrate that peripheral nmda receptor predominantly contributes to vasodilatation, while peripheral ampa/ka receptor predominantly contributes to increase of vascular permeability in glutamate-induced neurogenic inflammation. ps p-g studies on pain control system (rept. ): changes in phosphorylation of nr b-contained nmda receptor in the spinal cord obtained from rats with painful neuropathy following chronic ethanol consumption kan miyoshi, minoru narita, michiko narita, tsutomu suzuki dept. toxicol., hoshi univ. sch. pharm. pharmaceut. sci., tokyo, japan chronic ethanol consumption produces a painful peripheral neuropathy. mechanical hyperalgesia was clearly observed during ethanol consumption and even after ethanol withdrawal in rats, and it lasted for weeks. under these conditions, the immunoreactivities of phosphorylated-ser- nr b (p-ser- nr b) subunit and phosphorylated-conventional protein kinase c (p-cpkc) were significantly increased in the spinal cord following chronic ethanol consumption, whereas p-tyr- nr b subunit immunoreactivity was not changed in this region. the hyperalgesia induced by chronic ethanol consumption was significantly attenuated by repeated i.p. injection of ifenprodil, a selective nr b-containing nmda receptor antagonist. these findings provide evidence for a substantial role of the phosphorylation of cpkc-dependent nr b-contained nmda receptor in the development or/and maintenance of ethanoldependent neuropathic pain-like state in rats. ps p-g prolonged depression of nociceptive response in the prefrontal cortex with high frequency stimulation of the amygdala yumi izawa, yoriko kawakami dept. physiol. tokyo women's medical university, tokyo, japan high frequency stimuli (hfs, hz, a, s) delivered to the basolateral nucleus of the amygdala (bl) induced prolonged depression of the nociceptive specific response in the prefrontal cortex (pfc). we examined the receptor mechanism underlying this depression of pfc neuron activity. extracellular neural activities, induced by nociceptive stimulation applied peripherally, were recorded in the rat pfc. inhibitory effects of hfs delivered to the bl on nociceptive responses were blocked by specific antagonists of a metabotropic glutamate receptor (mglur) or nmda receptor microinjected locally into the pfc. dopamine depletion, produced by -ohda injected into the substantia nigra, also reduced the inhibitory effects of hfs. the mglur and dopamine receptor mediated prolonged depressions of nociceptive responses were induced by hfs of the amygdala. our results suggest that emotional condition modulates pain sensation. ps p-g the nav . sodium channel pathologically reconfigures the thalamic pain amplifier-generator after spinal cord injury bryan c. hains, stephen g. waxman yale university school of medicine, usa spinal cord injury (sci) induces pain-related phenomena associated with the aberrant expression of nav . , a rapidly repriming voltage-gated sodium channel. in this study we hypothesized that, following sci, neurons in the thalamus undergo similar electrophysiological changes linked to nav . . four weeks post-sci, nav . protein was upregulated within thalamic neurons, where unit recordings revealed increased spontaneous discharge, afterdischarge, hyperresponsiveness to innocuous and noxious peripheral stimuli, expansion of peripheral rfs, and bursting. these properties persisted after interruption of ascending spinal barrage. lumbar intrathecal administration of specific antisense oligodeoxynucleotides against nav . caused a significant reduction in nav . expression and reversed electrophysiological alterations. these results show, for the first time, a change in sodium channel expression within neurons in the thalamus after injury to the spinal cord, and suggest that these changes contribute to altered processing of somatosensory information after sci. tomoki fukuda , hiroyuki ichikawa , ryuji terayama , tomosada sugimoto department of oral maxillofacial rehabilitation, okayama university, okayama, japan; department of oral function and anatomy, okayama university, okayama, japan ib -sap is a neurotoxin designed for targeting primary nociceptors with ib binding sites on the cell surface. however, the exact cell spectrum that is affected by the toxin has not been thoroughly investigated. we, therefore, unilaterally injected ib -sap ( . l of . % solution for each ganglion) directly into the th and th lumbar (l and ) dorsal root ganglia (drgs). three weeks later, the rats were killed and drg sections were stained for ib -binding. after counterstain, the cell body size of neurons were measured. ib -sap reduced the total number of drg neurons in l and ganglia combined by % ( ± on untreated side versus ± on treated side). small neurons (< m ) were reduced by % whereas large ones (≥ m ) were not affected. ib -binding neurons were mostly small (≥ %) and were reduced by %. the number of small neurons, that were not stained for ib -binding, increased by % ( ± versus ± ). schuichi koizumi , kaoru nasu-tada , makoto tsuda , emiko kunifusa , , kazuhide inoue div. pharmacol., natl. inst. hlth. sci., tokyo, japan; dept. mol. system pharmacol., grad. sch. pharmaceut. sci., kyushu univ., fukuoka, japan although microglial p x receptor, a key molecule for the mechanical allodynia, is increased after peripheral nerve injury, the molecular mechanisms underlying its upregulation remain unknown. here, we describe the influence of fibronectin on p x receptor expression in microglia. microglia that were cultured on fibronectin-coated dishes showed a marked increase in p x receptor expression. western blot examination of the spinal cord from rat with spinal nerve injury indicated that fibronectin was upregulated on the ipsilateral side. interestingly, intrathecal injection of atp-stimulated microglia revealed that microglia cultured on fibronectin-coated dishes was more effective in the induction of allodynia than microglia cultured on control dishes. taken together, our results suggest that spinal fibronectin is elevated after the peripheral nerve injury and it may be involved in the upregulation of the p x receptor in microglia, leading to neuropathic pain. research funds: mf , kakenhi ( ) ryousuke fujita, hiroshi ueda div. mol. pharmacol. & neurosci., nagasaki univ. grad. sch. of biomed. sci., nagasaki, japan we have reported that intrathecally administered lpa or endogenous lpa generated upon sciatic nerve injury causes demyelination of dorsal root (dr), which is supposed to be one of key molecular mechanisms underlying neuropathic pain (nat. med. ). however it remained whether lpa has direct actions on myelinated schwann cells (sc). in the present study we examined the direct effects of lpa on dr fibers in ex vivo culture system. scanning electron microscopy (sem) study revealed that lpa caused a swelling and disruption of myelinated fibers at h. in transmission em analysis, the addition of lpa caused a disruption of myelin sheath of a␦-and a␤-fibers. on the other hand, it was found that c-fibers were separated to each other by scs in naive fibers. following the addition of lpa, c-fibers showed direct contacts and some of them were uncovered. all these effects were also observed either with or without dr ganglion. thus, it is suggested that lpa has direct actions on myelinated and unmyelinated scs to cause demyelination of a-fibers and to uncover c-fibers. research funds: kakenhi ps p-g lysophosphatidic acid (lpa) down-regulates myelin associated proteins in cultured dorsal root fibers norikazu kiguchi, ryousuke fujita, hiroshi ueda div. mol. pharmacol. & neurosci., nagasaki univ. grad. sch. biomed. sci. nagasaki, japan we have reported that intrathecally administered lpa or endogenous lpa generated upon sciatic nerve injury causes demyelination of dorsal root, which is supposed to be one of key molecular mechanisms underlying neuropathic pain (nat. med. ). these treatments also caused a decrease in myelin protein and their gene expression levels. here we report the biochemical evidence underlying this demyelination in cultured fibers. the addition of lpa at mm decreased the protein levels of myelin basic protein (mbp) at h. this action was significantly inhibited by botulinum neurotoxin/c (bont/c ). on the other hand, lpa also caused a decrease in gene expression of various myelin proteins, such as mbp, pmp , mag, p in cultured fibers. the maximal decrease was observed all at as early as h after the addition of lpa. bont/c and y abolished the lpainduced down-regulation of mbp gene. all these findings suggest that the down-regulation of gene expression of myelin proteins is through rhoa-rock pathway underlying lpa-induced demyelination. neuropathic pain arise from peripheral never injury. the purpose of this study was to explore behavioral characteristics and investigate the involvement of nmda receptors and opioid receptors in the behavioural responses following spared nerve injury (sni). the hind paw withdrawal threshold to cold-and mechano allodynia and heatyperalgesia were tested at and , , , , days after operation. pre-emptive co-administration of mk- and morphine were tested. sni produces mechanical and cold allodynia and heat hyperalgesia. co-injection of morphine and mk- decreased cold-and mechanoallodynia, but had slightly effect on heat-hyperalgesia. the present data demonstrate that the sni procedure result in severe changes in behavioral responses in whether hyperalgesia or allodynia. coadministration of both drugs seems to be more effective to alleviate induced neuropathic pain. satoshi deyama , , naomi akiyama , mikie hirata , takayuki nakagawa , shuji kaneko , masabumi minami dept. of pharmacol., grad. sch. of pharm. sci., hokkaido univ., sapporo, japan; dept. of mol. pharmacol., grad. sch. of pharm. sci., kyoto univ., kyoto, japan the bed nucleus of the stria terminalis (bst) is involved in the regulation of negative affective states such as anxiety and fear. in this study, we examined the role of the noradrenergic (na) transmission within the bst in the negative affective component of pain in rats. we found that excitotoxic lesion of the bst attenuated intraperitoneal acetic acid-or intraplantar formalin-induced conditioned place aversion (cpa) without reducing nociceptive behaviors. we showed that na release within the bst was significantly elevated by these noxious stimuli. intra-bst injection of a ␤-adrenoceptor antagonist timolol significantly suppressed these noxious stimuli-induced cpa without affecting nociceptive behaviors. these results suggest that visceral and somatic noxious stimuli-induced na release within the bst contributes to the negative affective, but not sensory, component of pain. noriyuki ozaki, mariko kawai, yasuo sugiura department of functional anatomy and neuroscience, nagoya university, graduate school of medicine, nagoya, japan neonatal maternal separation induces visceral hyperalgesia in colon. this study compares the effects of maternal separation on response sensitivity to gastric and colorectal distension in long-evans rats. maternal separation was performed for h per day between postnatal day and . visceral sensitivities were assessed in stomach and colon at weeks of age by visceromotor responses induced by either gastric or colorectal distension. somatic pain sensitivities were also assessed by von frey filaments and radiant heat. in contrast to the response to colorectal distension, maternal separation induced decreased response to gastric distension, especially in male rats. no difference was found between control and separated rats in somatic pain sensitivities. these results indicate that maternal separation differentially modulates visceral pain sensation in stomach and colon. research funds: grant-in-aid for scientific research ps p-g change by aging in muscular mechanical hyperalgesia after lenghtening contraction k. mizumura, t. taguchi, t. matsuda, t. nasu res. inst. environ. med., nagoya univ., nagoya, japan our previous experiments have shown that the mechanical threshold of the edl muscle underwent lengthening contraction (lec) lowered to days after exercise in rats ( w old). c-fos expression in the superficial dorsal horn increased in l spinal segment when the edl muscle was compressed days after exercise. from these results we have concluded that the muscle became hyperalgesic after lec. in the present experiment, we examined whether this hyperalgesia after lec changes along aging. male sd rats , ( - ) and ( - ) w old were used. the basal mechanical threshold (randall-selitto method) of edl muscle tended to be higher in w old rats, but not significant. after lec, the threshold started to decrease day after lec in all three age groups. it returned to the pre-lec level days after lec in and w old rats only. recovery of w old rats delayed up to days after lec. increased c-fos expression in the superficial dorsal horn was observed in l as well as in l in w old rats. these results suggest that hyperalgesia occurs in larger areas and lasts longer in aged animals. tong liu, hong p. wei, chun y. yuan, ai k. guo institute of neuroscience, chinese academy of sciences, china drosophila can display complex courtship behavior. male-male courtship behavior shown in some fly mutants, but here we report for the first time that the male-male courtship behavior can be induced by disturbance of dopamine level. to up-regulate dopamine level, uas-th/th-gal males were used, which showed high level of dopamine and performed active male-male courtship behavior. this behavior was attenuated by decreasing dopamine level either through drug breeding or genetic method. the increased courtship behavior in uas-th/th-gal males is specific to male partners, because the males courted females normally. to down-regulate dopamine level, pale ts , th temperature sensitive mutant was used. when raised at restrictive temperature, pale ts showed obvious attraction to wild type males. our study shows that the high level or low level of dopamine can induce male-male courtship behavior in active or passive manner. athushi yokoyama , masaharu akita kanagawa life-science res., japan; kamkura, kanagawa, japan we have developed the screening system for drug and chemical compounds of food by the used of ratwhole embryo culture. the advantages of whole embryo culture are to examine the direct effects of l-calnitin (lcal) on embryo and also to find the non-teratogenic agent (d-calnitin:dcal). as the testing agent, lcal was examined in this study using the rat embryo cultured from day to of gestation. in treated embryos of lcal, the embryonic heart beat, the crown-rump length, the embryo weight and the total embryonic somites were not decreased. on the other hand, the malformation (the defects of neural tube) and the short size of head length were observed in the embryos cultured with lcal. in treated embryos of d-calnitin (dcal), there parameter was not decreased. the observed malformation of lcal was not observed in the embryos cultured with dcal. these results might be due to the differences between lcal and dcal in the embryo toxicity. yoshihisa uenoyama, kenji takase, junya hirata, hiroko tsukamura, kei-ichiro maeda laboratory of reproductive science, nagoya university, nagoya, japan the mechanisms underlying the pubertal increase in gonadotropinreleasing hormone (gnrh) secretion are poorly understood. recently, metastin was found to stimulate gnrh secretion and mutations of its receptor are associated with lack of puberty. effect of immunoneutralization of endogenous metastin in the brain on the onset of puberty was examined to clarify the physiological significance of metastin in timing the puberty. when wistar-imamichi strain female rats received an infusion of anti-metastin antibody into the third ventricle during days - of age, they did not show the first estrus before days of age with mean age of . ± . day. in contrast, most of normal mouse igg-treated controls showed the first estrus by days of age with mean age of . ± . day. the age of vaginal opening was also delayed in the anti-metastin-treated rats. thus, the present study demonstrates that the puberty onset was delayed by immunoneutralizing central metastin. central metastin may be involved in timing the onset of puberty in female rats. kenji takase, yoshihisa uenoyama, shunji yamada, hiroko tsukamura, kei-ichiro maeda lab. of reproductive science, nagoya university, aichi, japan metastin has been considered to be involved in triggering pulsatile gonadotropin-releasing hormone (gnrh)/luteinizimg hormone (lh) secretion to time the onset of puberty. the present study aimed to determine expression of metastin, a novel kiss- gene product, in the rat brain during peripubertal period. wistar-imamichi strain female rat shows vaginal opening on around days of age (d ), and first estrus on around d . brain tissues were obtained on d , , , and . kiss- mrna expression in the arcuate nucleus-median eminence region (arc-me) and anteroventral periventricular nucleus (avpv) increased significantly from d to and was kept at a high level thereafter. gpr mrna expression in the medial preoptic area increased significantly from d to . metastin-immunoreactive cells were not found on d but were apparent in the arc-me on d onward. these results indicate that metastin expression increases in the arc-me and avpv before vaginal opening, suggesting that metastin triggers the onset of gnrh/lh secretion in female rats. toshiyuki saito , sei-etsu fujiwara , kenjiro konno , takashi yamaguchi , tetsu nemoto , etsuko kasuya , ryosuke sakumoto anim. neurophysiol. lab., natl. inst. agrobiol. sci., tsukuba, japan; inst. exp. anim. res., fac. med., gunma univ., maebashi, japan; grad. sch. sci. & eng., yamagata univ., yonezawa, japan; sch. health sci., fac. med., kanazawa univ., kanazawa, japan in the present study, we recorded and examined local field potentials (lfps) in the hippocampus of piglets performing an operant task by a radio-telemetry system. under halothane anesthesia, a pair of tungsten electrodes was implanted into the hippocampus and fixed on the surface of the skull with a transmitter using dental cement. after recovery from surgical procedures, the piglets were moved to a training cage. in the lfps, spike-shaped waves were frequently found just before the piglets pushed a switch with their noses. these waves may represent some of the hippocampal neural activities associated with switch manipulation for getting a food reward. yasuo osawa department of bioscience, tokyo university of agriculture, tokyo, japan memory extinction is an inhibitory learning rather than forgetting or erase of conditioned memory. from the view of treatment of phobia and post traumatic stress disease (ptsd) caused by fear memory, it is important to find the drugs to facilitate extinction of fear memory. importantly, previous studies using pavlovian fear conditioning have shown that d-cycloserine, a nmda receptor agonist, facilitates memory extinction. in this study, to examine whether d-cycloserine is applicable for the treatment with another type of fear memory, we investigated effects of d-cycloserine on extinction of aversive memory in mice. indeed, we performed conditioned taste aversion (cta) task, where the ingestion of a novel taste is paired with transient sickness. our results indicated the injection of d-cycloserine before but not after the re-exposure to cs facilitates extinction of cta. ps p-g hippocampal neural responses during a conditional delayed stimulus-response task in awake mice nobuhide kitabayashi , teruko uwano , , anh tran , , eturou hori , , taketoshi ono , , hisao nishijo system emotional science, univ. of toyama, japan; integrative neurosci, molecular and integrative emotional neurosci., univ. of toyama, japan; crest, japan to investigate a hippocampal (hf) involvement in the representation of temporal sequence in mice, neural responses were recorded during performance of a conditional delayed stimulus-response association task. a trial was initiated by one of two different conditioned tones. after a s delay, two serial reinforcements with an intervening delay was presented; aversive air puff-delay-tube protrusion to evoke licking sucrose solution and the opposite order of the same reinforcements. of hf neurons, responded to the tones, the reinforcements, and during the delay. some neurons responded to a presentation of a sensory stimulus, and other responded differentially during the delay depending on the reinforcement sequence. the results suggest a crucial role of the hf in representation of serial events in episodic memory in mice as well as in rats and primates. further studies will be conducted using genetic modified-mice to clarify the neural substrate in episodic memory. naoko inoue , atsu aiba , kaoru inokuchi mitsubishi kagaku inst. life sci. (mitils), tokyo, japan; grad. sch. med., kobe univ., kobe, japan vesl- s/homer- a and vesl- l/homer- c are splicing isoforms encoded by the vesl- gene. vesl proteins bind and regulate mglur / , ip receptor, ryanodine receptor, and trp channel at the postsynapse. the expression of vesl- s is upregulated by tetanic stimulation that elicits l-ltp. vesl- s is thought to play a critical role in the conversion from short-term to long-term memory (ltm). in this study we generated vesl- s gene-targeting mice (ko) and examined whether vesl- s plays a role in the ltm formation. analysis with the contextual fear conditioning revealed a defective in consolidation process, reconsolidation process, and remote memory formation in ko. ko further showed an enhanced freezing decrement within a test session, indicating faster within-session extinction. in contrast, consolidation process of the extinction was normal in ko. these results demonstrate that the vesl- s protein plays critical roles in various processes of the ltm formation. we now examine the signaling pathways important for ltm formation that are altered in ko. hiroshi ageta , r. migishima , s. kida , k. inokuchi , mitsubishi kagaku inst. life sci (mitils), japan, tokyo univ. agricul., japan; crest, jst, japan memory process consists of at least four distinct phases, acquisition, consolidation, maintenance, and retrieval. activin ␤a mrna increases following l-ltp induction in the hippocampus, suggesting that activin plays a role in the memory formation. here, we generated activin and follistatin (antagonizes activin function) transgenic mice in which the transgene expression was tightly regulated by dox in a forebrain-specific manner (tet off system). transgene expression was turned off or on within d by (+/−) dox. contextual fear conditioning with these mice revealed that activin function is required during maintenance phase of fear memory for one week retention. furthermore, activin plays a role in the re-consolidation process. thus, fear memory that was once acquired and consolidated tightly could be "erased" by inhibiting the activin function during maintenance phase. these mice are useful for the study of ptsd. ps p-h sex differences in the effects of chronic estrogen treatment on fear conditioning in c bl/ j mice takaaki ozawa, mumeko tsuda, sonoko ogawa kansei, behavioral and brain sciences, university of tsukuba, tsukuba, japan it has been suggested that estrogen may play a role in the regulation of learning and cognitive functions. although most of previous studies have focused on elucidating facilitatory effects of estrogen on learning in females, estrogen is also known to affect various behaviors in males. in the present study, we investigated the effects of different doses of estrogen on fear conditioning (fc) learning in both sexes of mice. gonadectomized c bl/ j mice were implanted with a silastic capsule containing , , or g of estradiol benzoate. since it is possible that estrogen may indirectly modify learning by affecting general activity, emotionality and anxiety levels, we tested the mice in open-field and light dark transition paradigms prior to fc. mice were then conditioned for fear responses (freezing) to tone stimulus and tested for both contextual and cued fc responses. we found that estrogen facilitated both types of fc learning in females, whereas it inhibited them in males especially at a higher dose, with a small effect on emotional behaviors. ps p-h analysis of brain regions activated during memory consolidation in passive avoidance task zhang yue department of bioscience, tokyo university of agriculture, tokyo, japan short-term memory (stm) is labile. to generate long-term memory (ltm), stm is stabilized through a process known as memory consolidation. importantly, previous studies have shown that memory consolidation requires the function of transcription factor creb whose activation induces c-fos expression. in this study, we tried to understand molecular mechanisms of consolidation of passive avoidance memory that has been known to be amygdala and hipocampusdependent. indeed, we investigated brain regions that are activated following the learning by analyzing the expression level of c-fos using immunocytochemistry. consistent with previous study, we observed increase in c-fos expression in amygdala and hippocampus. more interestingly, we also found this increase in prefrontal cortex, indicating that prefrontal cortex plays critical roles in memory consolidation in light-dark passive avoidance task. hiroshi nomura, norio matsuki laboratory of chemical pharmacology, graduate school of pharmaceutical sciences, university of tokyo, tokyo, japan we have demonstrated the effect of ethanol on reactivated fear memory for the first time, using contextual fear conditioning. rats were conditioned with mild footshock, reexposed to the training context, immediately injected with ethanol or saline, and finally tested h after reexposure. ethanol-treated groups expressed longer freezing and the effect lasted for weeks. reactivation was necessary for the effect. the injection of ethanol itself did not induce a fearful response. as memory retrieval triggers memory extinction and reconsolidation, we investigated whether extinction process is involved in this ethanol effect. increasing retrieval time did not enhance freezing by ethanol, suggesting that ethanol had no effect on memory extinction. post-reactivation injections of anisomycin revealed that retrieval triggered reconsolidation. moreover, picrotoxin inhibited the memory enhancement by ethanol. these studies demonstrate that ethanol enhances reactivated contextual fear memories via activation of gaba a receptors. ps p-h analyses of brain regions activated in reconsolidation and extinction phases of contextual fear memory nori mamiya, akinobu suzuki, satoshi kida department of bioscience, tokyo university of agriculture, tokyo, japan the retrieval of conditioned fear memory by conditioned stimulus (cs) initiates two processes; reconsolidation or extinction. we previously found that the change in memory stability after retrieval (reconsolidation) associates with memory extinction. to understand the regulatory mechanisms of memory stability after the retrieval at the anatomical level, we here investigated the brain regions that are activated in reconsolidation and extinction phases. we measured the levels of phospho-creb inducing changes in neural plasticity following the re-exposure to cs. short re-exposure to cs inducing reconsolidation increased in phospho-creb in amygdala and hippocampus. in contrast, longer re-exposure inducing extinction increased in phospho-creb in amygdala and prefrontal cortex. these results indicate that distinct brain areas are activated in response to short or long re-exposure to cs and suggests that amygdala plays crucial roles in the interaction between reconsolidation and extinction. ps p-h analysis of molecular mechanism for the destabilization of retrieved contextual fear memory akinobu suzuki, satoshi kida department of bioscience, tokyo university of agriculture, tokyo, japan reconsolidation acts to stabilize, whereas extinction tends to weaken the expression of the original memory. to understand the mechanisms for the regulation of memory stability after the retrieval, we have investigated the relationship between reconsolidation and extinction using contextual fear conditioning. we previously found that memory extinction is associated with regulation of fear memory stability, indicating the interaction between memory reconsolidation and extinction phases. in this study, we compared molecular signatures of reconoslidation and extinction using mice. pharmacological experiments using antagonists for cannabinoid receptor (cb ) and l-type voltage-gated calcium channels (lvgccs) indicated that both cb and lvgccs are required for memory extinction but not consolidation and reconsolidation. more interestingly, blockade of either cb or lvgccs function prevents the disruption of the original memory by protein synthesis inhibition. these results suggest that cb and lvgccs are required for not only memory extinction but also the destabilization of reactivated memory. hotaka fukushima, akinobu suzuki, satoshi kida department of bioscience, tokyo university of agriculture, tokyo, japan previous our studies using contextual fear conditioning revealed three distinct time-dependent phases following memory retrieval: stable, reconsolidation, extinction phases. to understand the nature of memory processing following retrieval, we examined the effects of reexposure on memory reconsolidation and extinction using light-dark passive avoidance task. this task is thought to allow us to discriminate between reconsolidation and extinction phases at the time point when mice enter dark box from light box. brief re-exposure to light box did not affect the stability of fear memory (stable phase). further extending re-exposure to light box triggered the requirement of protein synthesis for re-storage of fear memory (reconsolidation phase). in contrast, entry from light into dark box initiated extinction of fear memory (extinction phase). additionally, using pharmacological blockade of cb and lvgccs, we also found that cb is required for only memory extinction but that lvgccs are required for memory extinction and reconsolidation. wakoto matsuda , takahiro furuta , kouichi nakamura , , takeshi kaneko , ps p-h difference in organization of corticostriatal and thalamostriatal synapses between patch and matrix compartments of rat neostriatum fumino fujiyama , tomo unzai , kouichi nakamura , , sakashi nomura , takeshi kaneko , department of morphological brain science, kyoto university, kyoto, japan; department of physical therapy, kyoto university, kyoto, japan; crest, japan the striatum, which has patch/matrix compartments, receives glutamatergic inputs from cortex and thalamus. in the present study, the differences in synaptology of these inputs between both compartments were examined. axon terminals positive for vesicular glutamate transporter (vglut) , thalamostriatal inputs, were less dense in patch region, whereas vglut -positive corticostriatal inputs were evenly distributed. quantitative analysis revealed % of vglut positive synapses in patch region were formed with spines, whereas % in matrix region were made with dendritic shafts. in contrast, the targets of vglut -positive inputs were mainly spines in both regions. moreover, vglut -positive axospinous synapses in patch region were larger than vglut -positive ones. the present observation suggests that thalamostriatal connection is more plastic in patch region. research funds: kakenhi ( , , , ( ), ) ps p-h single cell tracing of thalamostriatal projection neurons with reference to patch and matrix compartments of rat striatum tomo unzai , fumino fujiyama , takeshi kaneko , department of morphological brain science, university of kyoto, kyoto, japan; crest, japan the striatum consists of patch and matrix compartments, and receives glutamatergic inputs mainly from the cerebral cortex and thalamus. thalamic intralaminar nuclei are known to project exclusively to matrix compartment. on the other hand, it has not been clarified which thalamic nuclei project to patch compartment. in the present study, we combined single cell tracing with immunohistochemistry for mu opioid receptor, which is specifically expressed by patch neurons, to reveal the distribution of thalamostriatal axon terminals in relation to striatal compartments. recombinant sindbis virus expressing membrane-targeted green fluorescent protein (palgfp) was injected into the rat thalamus. a single neuron in the thalamic paraventricular nucleus extensively projected to the striatum and preferentially to patch compartment compared with matrix compartment. the axons were also distributed in the thalamic reticular nucleus, accumbens nucleus, amygdala, and cerebral cortex. research funds: kakenhi ( , , , ( ) , ) ps p-h lesion of the nucleus accumbens dopamine system shortens the lever pressing interresponse time and delays the response initiation in mice yuji tsutsui , kayo nishizawa , nobuyuki kai , kazuto kobayashi , dept. of psychology, fukushima univ., japan; dept. mol. genet., fukushima medi. univ., japan; crest, jst, kawaguchi, japan dopamine transmission is thought to be important for rodents to perform operant behaviors such as lever pressing. the lever pressing experiment was conducted to examine the effects of -ohda injections into the nucleus accumbens (acb) in c bl/ j mice. all mice were trained to press the lever for a food pellet using a fixed ratio (fr ) schedule. the mice were injected with ascorbate vehicle or -ohda into the acb, and then tested post-surgically using the fr schedule again. the -ohda-injected mice showed the acceleration of response speed, which was revealed by the shortening of interresponse time between each of the five lever pressings, and the suppression of the initiation of the response to the next step. this suppression of initiation was revealed by the increase of time from the last presentation of food to the next initiation. these results suggest that the acb dopamine system is important for the initiation and control of the operant behaviors in rodents. hideshi shibata laboratory of veterinary anatomy, tokyo university of agriculture and technology, fuchu, tokyo, japan retrosplenial area is one of the important structures for spatial memory and behavior in the rat. to understand more fully the functional roles played by area , it is essential to clarify the neural circuitry subserving these functions. in the present study, we analyzed the organization of frontal cortical projections to area in the rat, using retrograde transport of cholera toxin b subunit (ctb). ctb injections into area d retrogradely labeled cells in the orbital cortex and the caudal parts of the anterior cingulate and primary and secondary motor cortices. ctb injections into area c labeled cells in similar cortical regions, except for the orbital cortex. ctb injections involving areas a and b labeled cells in the caudal part of the anterior cingulate cortex. the results show that the orbital, anterior cingulate, and primary and secondary motor cortices have a different pattern of projections to each subdivision of area , suggesting different functional roles played by each subdivision of area in spatial memory and behavior. eiichi jodo , yoshiaki suzuki , tadahiro katayama , ken-yo hoshino , yukihiko kayama dep. of physiol., fukushima med. univ., fukushima, japan; dep. of neuropsy., fukushima med. univ., japan it has been shown previously that the dopaminergic neurons in the ventral tegmental area (vta) selectively respond to a stimulus repeatedly paired with reward stimuli in a classical conditioning paradigm. since the vta receives dense projection from the medial prefrontal cortex (mpfc), such response selectivity of vta neurons may in part be produced by inputs from the mpfc. however, few studies have compared the firing pattern between these two regions. our present experiment was designed to make such a comparison in freely moving rats. two different tones were sequentially presented, one of which (target, %) was paired with intracranial simulation of the reward area. the unit activity was recorded from the mpfc and/or the vta. pfc and vta neurons exhibited phasic excitation with the peak latency of about . s to both tones, while only the target tone induced sustained activation of firing activity lasting until presentation of the reward. masato inoue, akichika mikami primate research institute, kyoto university, inuyama, aichi, japan to investigate the neuronal mechanism in the ventrolateral prefrontal cortex (vlpfc) and inferotemporal cortex (it) for holding information for object and their order of presentation, we examined single neuronal activities in the vlpfc and it while monkeys were performing a serial probe reproduction task. in the task, two sequentially presented objects were memorized and then a target object was selected from memorized objects based on a color stimulus. in % out of vlpfc neurons, the delay-period activity showed objectselectivity and order-selectivity. in only % out of it neurons, the delay-period activity showed object-selectivity and order-selectivity. the starting time of the order-selective activity was earlier in the vlpfc. these results suggest that the vlpfc plays a role in holding information for object and their order of presentation and the it receives information for object and their order of presentation from the vlpfc. masao yukie, yasutaka oosawa department of behavioral physiology, tokyo metropolitan institute for neuroscience, fuchu, japan relational memory theory (eichenbaum et al., ) has been proposed from evidence that the hippocampal damage in rats impairs learning of transverse patterning task (a+ versus b−; b+ versus c−; c+ versus a−). very recent monkey study (alvarado et al., ) demonstrated that lesion of the hippocampus produced a significant impairment in that task and supported such a theory. in our study, however, ischemic damage of the hippocampus has not impaired learning of such a transverse patterning task (yukie et al., ) . in the present study, we examined effects of lesion of the monkey perirhinal cortex on transverse patterning task using two sets of d visual stimuli presented in a wgta. our three monkeys with perirhinal lesions failed to attain a learning criterion within a training limit of sessions in phase , although they learned easily the four problems in phases and . our results suggest that the perirhinal cortex, but not the hippocampus, is important for learning of transverse patterning task, that is, for formation of relational memory. yasuko sugase-miyamoto , noriyuki higo , munetaka shidara neuroscience research inst., aist, tsukuba, japan; grad. sch. of tsukuba univ., tsukuba, japan a recent dopamine d receptor study using antisense cdna showed that d receptor in rhinal cortex is crucial for learning associations between visual stimuli and reward schedules. neuronal responses in the perirhinal cortex differentiate the visual cues only when the cues are associated with the schedule states, while those in area te are related to physical attributes of the cue independently of the schedule states. to investigate the cellular substrate for d mediated associative learning, we examined monkey temporal lobe immunohistochemically with a d receptor antipeptide antiserum. d receptor immunoreactivity was observed in the pyramidal cells in layers ii-vi of the rhinal cortex and area te. the signal was mainly observed in cell bodies, and also in both apical and basal dendrites for some cells. the signal in layers v-vi was stronger in area of the perirhinal cortex than in area teav. the differential localization between area and te suggests the differential roles of the two areas in associative learning process. by using axonal transport of fast blue, diamidino yellow and tritiated amino acids, we determined the afferent and efferent connections of the retrosplenial cortex (rsp) in the macaque monkey. the rsp receives heavy projections from the subiculum, presubiculum and the caudal entorhinal areas (ec-ecl), and projects back to the presubiculum and the ec-ecl. the supracallosal portion of the rsp has connections primarily with the caudal half of the subiculum and presubiculum, as well as the lateral zone of the ec-ecl. the caudoventral portion of the rsp is, in contrast, mainly connected with the rostral half of the subiculum and presubiculum as well as the medial zone of the ec-ecl. the two portions of the rsp, thus, have access to different portions of the medial temporal lobe. these results indicate that there are two distinct neural systems in the retrosplenial-medial temporal network. hideko nakano , natsuko yoshida , kiyohisa natsume kyushu kyoritsu university, fukuoka, japan; kyushu institute of technology, fukuoka, japan; kyushu institute of technology, fukuoka, japan eeg activity was examined in english rhythm acquisition of japanese students who learn english as a foreign language (efl). we measured theta, alpha and beta rhythms of five subjects while they were reading aloud the materials and listening to the audio-recording, using eight electrodes attached to their skulls. the result shows that the increase of theta power at f and f was the highest and suggests that the theta rhythm at f and f may have a relationship to the process of english rhythm acquisition. moreover we found the highest increase in theta power when the subjects began to orally reproduce every line of the rhythm materials. this finding was observed in three right handlers except a left handler and a right handler who had just returned after -month english study experience in australia. these results suggest that the change of theta power at frontal areas may be more closely related to the japanese efl learners' english rhythm acquisition. research funds: kakenhi ( ) ps p-h neural correlates of music retrieval: an eventrelated fmri study using sparse temporal sampling takamitsu watanabe , sho yagishita , hideyuki kikyo , department of physiology, the university of tokyo school of medicine, tokyo, japan; department of molecular neuroimaging, national institute of radiological sciences, chiba, japan we investigated neural correlates of music memory using eventrelated functional magnetic resonance imaging and sparse temporal sampling technique with originally composed musical materials. written informed consent was obtained from all the subjects in accordance with the declaration of helsinki, and the experimental procedure was approved by the institutional review board of the university of tokyo school of medicine. a . t scanner system was used (te = ms; tr = s; acquisition time = . s). we demonstrated that the right hippocampus, bilateral lateral temporal cortices, left prefrontal cortex and left precuneus are involved in music retrieval. in addition, performance-based analysis suggested that the right hippocampus is associated with the accuracy of music memory. in this fmri study, we determined the neural correlates of the intellectual excitement. sentences describing facts in natural and human science were visually presented, and subjects judged whether they know the fact or not. after the fmri, each subject self-evaluates subjective "intellectual excitement" of each sentence. positive correlation with the self-evaluated intellectual excitement for known facts and novel facts were analyzed. significant correlation between cortical activation and self-evaluated intellectual excitement for novel facts was observed in the left and the right parahippocampal gyrus and for known facts was in the left orbital part of inferior frontal gyrus. it suggests the cortical areas related to self-evaluated intellectual excitement are different between getting of novel knowledge and recognition of existing knowledge. hyeonjeong jeong , motoaki sugiura , yuko sassa , keisuke wakusawa , , kaoru horie , , shigeru sato , , ryuta kawashima , gsics, tohoku university, sendai, japan; niche, tohoku university, japan; miyagi university of education, sendai, japan; department of pediatrics, school of medicine, tohoku university, japan; the lbc research center, tohoku university, japan a foreign language word is learned and retrieved either in daily situations (situation) or written text (text), and memory transfer is required when the learning and retrieval modes are different. in this experiment, normal japanese subjects learned korean words in the situation and text modes in video clips. during a subsequent fmri session, subjects were presented with the learned words in different movie clips; half of the learned words was presented in the same mode as in the learning session (match), and the rest was presented in a different mode (mismatch). comparison of the mismatch with match condition revealed significant activation in the orbital part of the left inferior frontal gyrus. the results suggest that this area plays a role in the memory transfer of foreign language words when the learning and retrieval modes are different. georgina e. cruz , christie l. sahley , kenneth j. muller physio. & biophys., univ. of miami, miami, fl, usa; biol. sci., purdue univ., west lafayette, in, usa in some animals much is known at the level of single synapses about mechanisms underlying behavioral sensitization, but in no system is the involvement of interactions at the network level well understood. the s-cell network of the medicinal leech is a chain of electrically coupled interneurons spanning the nerve cord with distributed sensory input and motor output and is crucial for sensitization of reflex shortening. its firing increases with sensitization although few additional s-cells initiate impulses during the reflex. we tested the hypothesis that the initial burst of impulses from the s-cell in the stimulated segment suppresses initiations in adjacent segments. hyperpolarizing the central s-cell to reduce its firing during skin stimulation markedly increased the number of initiations in adjacent s-cells, which corroborated the limited expansion of initiation sites seen in the behaving animal. a computational model of s-cell refractoriness further supported the idea of interaction among s-cells during sensitization. research funds: nih, u.s.a. ps p-h sensory/motor modules regulating the development of peer social relationship mamiko koshiba , , shun nakamura jst, crest, japan; ncnp social intelligence is indispensable for animal's survival and could have evolved to language capability. further, as a recent problem in japan, 'fewer children' supposedly causes the more tight interaction of child-parent, reciprocally the less between siblings or friends. in order to study the genetic and epigenetic development of peer social relationship after birth, we controlled peer interaction through limiting a particular sensory/motor modality as social deprivation and examined the effect on the active attachment behavior of domestic chick to conspecific mates. the chick has a merit of being precocial and unique in higher animal with no need of parent-care. comparing to the chicks reared as a group, the isolated chicks didn't develop their active attachment to peers. meanwhile, the behavior study with the chicks deprived not sensory/motor function itself, but only social interaction in auditory, visual, olfactory or tactile system, suggested that vocal communication at least must play a key role for the development. dna-chip study along the different social context brought candidates of social genes. shogo sakata , minoru hattori department of behavioral sciences, hiroshima university, higashi-hiroshima, japan; graduate school of biosphere science, hiroshima university, higashi-hiroshima, japan peak interval (pi) -s procedure is a very good method to investigate for timing. six male wistar rats were trained for five days a week in pi -s procedure over days. the -s bin of lever press responses on probe trials showed a clear peak point. the temporal distributions had the peak time of regression curve fitting with the gaussian function. the peak time corresponded to near the -s with reinforcement durations. then nicotine was administrated to the rat by intraperitoneal injection before daily pi -s session. results showed that the peak time in the nicotine administration was slightly leftward shift compared to the saline injection. however the pattern of temporal distribution of responses was not changed by the nicotine treatment as well as control condition. it suggests that the nicotine administration affects on the time perception that was reflected by the peak durations of responses. ps p-i the effect of random practice schedule on arbitrary stimulus-response association learning satoshi tanaka , , ritz oshio , , norihiro sadato , , manabu honda , nips, okazaki, japan; jsps, tokyo, japan; nagoya univ., nagoya, japan; ristex, jst, tokyo, japan; sorst, jst, tokyo, japan; ncnp, tokyo, japan previous studies suggest that randomly ordered practice facilitates retention and transfer of motor skills compared to blocked or regularly ordered practices. it remains unclear, however, whether the advantageous effects of random practice can be expended to cognitive skill learning in humans. we examined the simultaneous learning of multiple arbitrary stimulus-response (s-r) associations under three different practice schedules: blocked, random and regularly ordered. behavioral data indicate that subjects performing the random practice showed better performance of the retention and transfer of learning compared to those performing the blocked or regularly ordered practice. the present result indicates that random practice schedule is effective also for s-r association learning, which are considered as a bridge between motor control and cognitive control. ps p-i sports rats show increased level of bdnf in the cerebellum, possibly learning and memorizing well masaki morishima, sayuri hara, yutaka nakaya dept. nutrition and metabolism, univ. of tokushima, japan previously, we reported that the activation of hippocampal norepinephrine neurotransmission following a decrease in monoamine oxidase a was observed in sports, a novel hyper-running rat on wheel. this study assessed whether sports show increased bdnf levels and better learning and memory. compared to control, both protein and mrna levels of bdnf in cerebellum were significantly elevated in sports even without wheel running, and slightly increased in hippocampus. in the cerebellum of sports, trkb/pi k pathway was activated, whereas mapk pathway was activated in the hippocampus. locomotor activity assessed by the open field test showed that the sports were significantly more active in center coat than control. in the passive avoidance test, sports did not enter a dark area at next time indicating that sports showed better passive avoidance learning. these results suggest that bdnf signaling of sports were activated from trkb to mapk and pi k in the hippocampus and cerebellum, respectively, and that these signaling pathways might play an important role in learning and memory. research funds: kakenhi ( ) ps p-i selective manipulation of working memory through d and d receptors: computer simulation shoji tanaka, hiroki yata dept. of electrical & electronics eng., tokyo, japan though a number of experimental results suggest that working memory processes are controlled by the dopaminergic system, its mechanism is still unclear. to elucidate the mechanism, we have constructed a model of the prefrontal cortical neural circuit for working memory. the neurons in the model are leaky integrate-and-fire model with ampa, nmda, gaba, and leak conductances and have dopamine d and d receptors. the computer simulation with this model shows that d receptor activation mainly affect working memory activity itself, while d receptor activation affect the termination of working memory, being consistent with the experimental result. the simulation also mimics the hyper-and hypo-dopaminergic states. under such conditions, like schizophrenia, simulated pharmacological treatments using agonists and antagonists of d and d receptors indicate efficacies of some these treatments for the restoration of working memory. in conclusion, this kind of simulation shows how dopamine controls working memory by using the synergism of the actions of dopamine, glutamate, and gaba. kozo sugioka, tomiyoshi setsu, tatsuro yamamoto, toshio terashima div. anat. & dev. neurobiol., dept. neurosci., kobe univ. grad. sch. med., kobe, japan we examined activity and habituation in rats with experimentallyinduced abnormal morphogenesis of the hippocampus. pregnant rat (jcl:wistar) was injected with saline or mg/kg mam on the th day of gestation. the activity of male and female offspring was measured for each h light and dark period, and the habituation to the visual stimulation was observed by measuring the activity with every min interval for h under min dark/light alternative schedule during weaning and adult periods. activity was measured using infra-red sensor in a home-cage placed in the experimental room. the mam-treated rat showed hyperactivity for dark-period during both weaning and adult periods, and showed retarded habituation during weaning period. sex difference of behavioral alteration was evident during adult period in both groups. these behavioral disorders were discussed in relation to the mam-treated rat showing abnormal hippocampus (disruption of the ca pyramidal layer and ectopic neuron mass). ps p-i long-lasting tagging of functionally activated neurons in the mouse brain naoki matsuo, leon reijmers, mark mayford the scripps research institute, la jolla, ca, usa immediate-early genes (iegs) have been widely used as activity markers for mapping neurons involved in specific animal behaviors including learning and memory. however, conventional ieg approaches that use immunohistochemistry or in situ hybridization allow to detect neurons only shortly after their activation and does not enable genetic manipulations. here we have developed transgenic mice that allow selective and long-lasting tagging of neurons that were activated in a given brain region at a given time point. the mice consist of two components; c-fos promoter driven tetracycline-controlled transactivator (tta) and teto promoter regulated feedback loop. when strong neuronal activity occurs in the absence of tetracycline analogs such as doxycycline (dox), c-fos promoter driven tta initiates the teto-linked expression of mutant tta (tta*) that is not inhibited by dox. this teto-linked gene expression is then maintained indefinitely by feedback activation via the tta* even in the presence of dox. using this system, we have examined the expression of bicistronic teto promoter driven tau-lacz and egfp-glur . hamid gholamipour , shirin babri , khameneh saied department of physiology, university of tabriz, tabriz, iran; university of tabriz, iran; university of tabriz, iran diabetes mellitus is one of the most prevalent diseases in the world. because hippocampus is an important area for memory formation, the present study is scheduled to investigate the effect of insulin injection in ca region of hippocampus on memory formation. fifty male rats were divided into five groups. ( ) control ( ) sham operation ( ) test ( ) diabetic/saline ( ) diabetic/insulin. groups and were made diabetic by treatment with stz ( mg/kg, i.p.). in all but the control group, two canula were stereotaxically implanted in ca region of hippocampus. learning was tested and compared between groups through passive avoidance test. results showed that in the test group the latency increased as compared to control and sham groups (p < . ). compared to sham group diabetic/insulin group showed increased latency (p < . ) but no significant difference was found between diabetic/saline and diabetic/insulin groups. in conclusion, according to the results obtained in this study, insulin facilitates memory in intact rats but not in diabetic sex differences in hippocampus-dependent memory formation are well documented, but the mechanisms are poorly understood. the ca + /calmodulin (cam) kinase cascade regulates gene transcription in the hippocampus, which is required for long-term memory (ltm) formation. we hypothesized that sex differences in transcriptional regulation may account for the sexual dimorphisms in memory formation. we tested this idea by studying the role of cam kinase kinases (camkks). using mouse molecular genetics we found that camkk␤ is required for spatial, but not contextual ltm. consistent with the impaired spatial memory formation, camkk␤ null mutants lacked spatial training-induced creb activation and had impaired late ltp. in contrast to camkk␤, camkk␣ is required for contextual, but not for spatial ltm. furthermore, female camkk mutants had normal spatial and contextual ltm. thus, we show that there are malespecific mechanisms to regulate gene transcription that may explain sex differences in hippocampus-dependent memory formation. akshay anand, sudesh prabhakar, monika bhatia, c.p. das department of neurology, post graduate institute of medical education and research, chandigarh, india background: parkinson's disease has a prevalence rate of per , in india. we studied the park polymorphism in north indian population and parkin expression in early parkinson's disease (n = ) and sporadic parkinson's disease (n = ). methods: pcr, sscp, rflp and direct sequencing analysis were used to screen mutations. results: our results revealed homozygous exonic mutations in exon- , and in early pd and exon- and in sporadic pd, heterozygous mutations in exon and in five early pd and one sporadic pd patient. frequency of s/n polymorphism was significantly high suggesting that exchange of serine to asparagine at position of protein affects the secondary structure or hydrophobicity of the protein resulting in pathogenicity. our facs analysis of these samples indicates reduced parkin expression correlating with severity of mutations. conclusions: we conclude that high frequency of parkin mutations in pd population in india affect parkin expression resulting in pd. wanida tripanichkul , kittisak sripanichkulchai , david finkelstein faculty of medicine, srinakharinwirot university, thailand; faculty of medicine, khon kaen university, thailand; university of melbourne, australia emerging data suggests beneficial effect of estrogen for parkinson's disease (pd), yet the exact mechanisms implicated remain obscured. activated glia observed in mptp mouse model and in pd may participate in the cascade of deleterious events that ultimately leads to dopaminergic nigral neuronal death. estrogen can modify glial expression of inflammatory mediator, such as cytokines and chemokines implicated in neurodegeneration. to determine whether estrogen-elicited neuroprotection in pd is mediated through glia, adult male c bl/ mice were pretreated with beta-estradiol (e ), injected with mptp on the day and brains were collected on day . e pretreatment decreased nigral neuronal loss and diminished striatal fibers deficit induced by mptp. the neuroprotective effect of e was coincident with an attenuation of a glial response within the snpc and striatum. these findings propose that e neuroprotection in mptp mouse model may mediate through reactive glia inhibition. ps p-i effect of angiotensin-converting enzyme inhibitor perindopril in mptp-treated mice; immunohistochemistry and in vivo electron spin resonance (esr) study rumiko kurosaki , fumihiko yoshino , masaichi chang-il lee dept. of food sci. and nutrition, showa women's univ., tokyo, japan; clin. care and med. div. of pharmaco., kanagawa dental college, japan we investigated the effects of perindopril on the dopaminergic system and the oxidative stress in mice after mptp treatment. administration of perindopril showed dose-dependent neuroprotective effects against striatal dopamine and its metabolites depletion after mptp treatment. we have reported that th, gfap, pv, nnos and cu, zn sod positive cells in the substantia nigra was changed after mptp treatment in our immunohistochemical study. the administration of perindopril significantly attenuated mptp induced changes of these immunopositive nigral cells. we could measure increased oxidative stress in the brain of mptp and perindopril treatment mice using by in vivo esr technique. our results provide further evidence that the ace inhibitor perindopril may offer a novel therapeutic strategy for parkinsonǐs disease. research funds: kakenhi ( ) ps p-i recruitment of calbindin into substantia nigra dopamine neurons suppresses the onset of parkinsonian motor signs shigehiro miyachi , kaori sawada , haruo okado , atsushi nambu , masahiko takada tokyo met. inst. neurosci., fuchu, tokyo, japan; natl. inst. for physiological sci., okazaki, japan there is a consensus that dopaminergic neurons in the substantia nigra that express calbindin, a calcium-binding protein, are selectively invulnerable to parkinsonian insults. based on this notion, an attempt was made to test the hypothesis that parkinsonism may be suppressed by recruitment of calbindin into a subpopulation of nigral dopamine neurons that does not normally contain calbindin. an adenoviral vector expressing calbindin was injected unilaterally into the striatum of macaque monkeys, to let calbindin express in the dopaminergic neurons via retrograde transport. two to three weeks later, the parkinsonism-inducing drug mptp was systemically administered several times. parkinsonian motor signs, such as muscular rigidity and flexed posture, appeared only on the side ipsilateral to the calbindin recruitment when cumulative doses of mptp exceeded threshold for their bilateral onset. toru yasuda, hideki mochizuki, yoshikuni mizuno department of neurology, juntendo university school of medicine, tokyo, japan using the serotype- raav vectors, we have recently reported the protective effect of parkin on the ␣-synuclein (␣s)-induced nigral dopaminergic neurodegeneration in a rat model. here we investigated the neuronal specificity of ␣s toxicity and the effect of parkin co-expression in a primate model. another serotype (type- ) of raav (raav ) carrying αs cdna (raav -␣s), and a cocktail of raav -␣s and raav carrying parkin cdna were unilaterally injected into the striatum of macaque monkeys, resulting in protein expression in striatonigral gabaergic and nigrostriatal dopaminergic neurons. the injection of raav -␣s alone caused a decrease of th-immunoreactivity in the striatum, while there was no effect on gabaergic neurons. in the presence of overexpressed parkin, ␣s seemed to be less accumulated and/or phosphorylated at ser residue in gabaergic neurons. these suggest that the ␣s toxicity is not expressed in non-dopaminergic neurons but the ␣s-ablating effect of parkin is exerted in all neurons introduced in primates. tomokazu oshima, yohsuke narabayashi narabayashi memorial laboratory of neurology, neurological clinic, tokyo, japan rigidity in aged parkinsonians is often intractable against surgeries. we investigated how their rigidity scored by updrs was related with ␤-band local field potentials (␤-waves) of the surgical targets in thalamic ventrolateral nucleus (vl) and posteroventral pallidal internal segment (pvp). forty patients aged - s gave informed consent for thalamotomy and/or pallidotomy. we divided the patients into groups with rigidity of . - (i), . - (ii), . - (iii), and . - (iv). the ␤waves were rated with total periods (%) of - hz wavelets in -s sample records. rigidity was re-scored after the surgeries. the vl ␤-waves were rated - % in groups i-iii with a slightly increasing tendency for increasing rigidity, but declined to about % in group iv. the pvp ␤-waves were - %, but with a decreasing tendency for increasing rigidity. the surgeries alleviated rigidity in all the groups, but were least effective in group iv with least vl and pvp ␤-waves. the results suggest that the pathology of aged parkinsonian rigidity develops beyond the pallido-thalamic pathway. yoshihisa tachibana , , hirokazu iwamuro , , masahiko takada , atsushi nambu , div. syst. neurophysiol., natl. inst. physiol. sci., okazaki, japan; sokendai; dept. neurosurg., univ. tokyo, tokyo, japan; dept. syst. neurosci., tokyo met. inst. neurosci., fuchu, tokyo, japan to approach a new therapy for parkinson's disease, extracellular unit recordings combined with microinjections of glutamate-related drugs were performed in the external and internal segments of the globus pallidus (gpe/gpi) of mptp-treated parkinsonian monkeys (macaca cyclopus). compared with the normal state, spontaneous oscillatory discharges were so often observed in the gpe/gpi and the subthalamic nucleus (stn) of the parkinsonian monkeys. microinjections of ionotropic glutamate receptor antagonists into the vicinity of recorded gpe/gpi neurons reduced their abnormal oscillations. these results suggest that glutamatergic excitatory input from the stn contributes to the oscillatory activity of gpe/gpi neurons, and that intrapallidal injections of ionotropic glutamate receptor antagonists may ameliorate some of parkinsonian symptoms. one of the pathological features of parkinsonǐs disease (pd) is loss of dopaminergic neurons in the substantia nigra pars comapacta (snpc). and it has been known that ␣-synuclein is involved in the neuronal loss. during the dopaminergic neuronal loss, activated microglia were centered in snpc. we hypothesize that ␣-synuclein may play a role in microglial activation to migrate to the pathological regions and to perform the neuronal cytotoxicity. we demonstrated that ␣-synuclein induced the cd expression on microglia and also enhanced the mt -mmp expression to shed off cd at the cell surface and degrade surrounding ecm to open the migratory way. a t mutant ␣-synuclein showed greater level of cd shedding and cell migration. extracellular treated ␣-synuclein also increased cd and mt -mmp expressions dose-dependently. among the multiple signaling pathways, erk pathway was involved in ␣-synuclein induced cell migration. these induced cell migration were also confirmed in human pd patients. research funds: national creative research initiative grant ( grant ( - ps p-j serotonergic fibers are involved in the conversion of l-dopa to dopamine in the striatum and the substantia nigra pars reticulata of parkinsonian model rats ryohachi arai , hiromasa yamada , yoshinari aimi , ikuko nagatsu department of anatomy, shiga university of medical science, otsu, japan; fujita health university school of medicine, japan dopaminergic neurons in the substantia nigra pars compacta (snc) project their axons to the striatum (st) and their dendrites to the substantia nigra pars reticulata (snr). dopamine released from these axons and dendrites is important in the regulation of motor activity. in parkinson's disease, dopaminergic neurons in the snc degenerate. l-dopa is the most effective drug for this disease. we hypothesize that, in parkinson's disease, a part of administered l-dopa is converted to dopamine in serotonergic fibers of the st and snr. here we produced parkinsonian model rats by the unilateral injection of hydroxydopamine into the snc, and found that serotonergic fibers in the st and snr were immunohistochemically positive for dopamine after l-dopa administration in the rats. therefore, it is possible that serotonergic neurons may be involved in the therapeutic effects of l-dopa for parkinson's disease. in mptp-induced pd monkey, reactive microglia are observed around neurons in nigra several years after mptp treatment and may be related to the progression of pd. to evaluate if reactive microglia in striatum and/or nigra of mptp-induced pd mice are present for a long time after mptp administration, like pd monkey. iba -and tb distribution in microglia were immunohistochemically investigated at h and days after twice mptp-treatments (one treatment comprised of intraperitoneal injections of mg/kg mptp at h interval) to c bl/ and balb/c at months (mo) interval. the recognizable change of iba -and tb -distibution in microglia of both mice strains was observed even mo after the first treatment. the twice mptp treatments tended to aggravate the symptoms in both mice strains, compared with once treatment. these results suggest that reactive microglia are present for a long time after the treatment by mptp and must play a role in the chronic progression of pd. ps p-j activated microglia affect the nigro-striatal dopamine neurons differently in neonatal and aged mice treated with mptp hirohide sawada , ryohei hishida , yoko hirata , kenji ono , hiromi suzuki , shin-ichi muramatsu , imaharu nakano , kunihiro tsuchida , toshiharu nagatsu , , makoto sawada school of medicine, fujita health university, aichi, japan; division of neurology, jichi medical university, japan; department of biomolecular science, gifu university, japan; research institute of environmental medicine, nagoya university, japan microglia play an important role in inflammatory process of parkinson's disease. we examined the effects between neonatal and aged microglia activated with lps on the nigro-striatal dopamine (da) neurons in mice treated with mptp. by mptp administration to neonatal mice, the number of da neurons in the substantia nigra was significantly decreased, whereas that in mice treated with lps and mptp was recovered. on the contrary, the number of da neurons of the week-old mice treated with mptp was significantly decreased with lps treatment. these results suggest that activated microglia in neonatal mice have neurotrophic potential, in contrast to the neurotoxic effect in aged mice. hyposmia is one of the most characteristic symptoms of parkinson's disease (pd). it may occur even before the motor symptoms start. in the olfactory bulb (ob), dopaminergic cells were present at glomerular layer. furthermore, it has been reported that ob contains neural stem cells. thus, ob has attracted attention because of its unique regenerative potential. in the present study, we established isolation of neurosphere forming cells (nsfcs) derived from adult mice ob, and examined proliferation potential in ob after dopaminergic neuronal loss induced by mptp, a selective toxin for dopaminergic neurons, utilized frequently as pd model. the number of neurospheres derived from adult ob was not decreased with mptp administration, rather significantly increased. we also evaluated nsfcs differentiation into neural subtypes. the isolation of neural stem cells has helped to establish the cellular basis of neurogenesis and the exciting potential for transplant-mediated treatment of degenerative cns disease like pd. ps p-j phosphorylation of erp in adult rat brain with neonatal -ohda treatment qinghua li, yasuyoshi watanabe department of physiology, osaka city university graduate school of medicine, osaka, japan dopaminergic neuron degeneration occurs in sporadic parkinson's disease (pd), but the mechanism of sporadic pd is not clarified. we prepared neonatal dopamine depleted rats, by i.c.v. injection of -ohda at (p ) and days after birth, to investigate the mechanism of dopaminergic neuron degeneration. at p , tyrosine hydroxylase (th) immunostaining cells were significantly reduced in the substantia nigra, and th immunostaining fibers were significantly reduced in the striatum, thus this model mimics the selective dopaminergic neuron degeneraion in sporadic pd. by two-dimensional electrophoresis we found that a certain protein was phosphorylated in the -ohda lesioned rats at p , and it was identified as disulfide-isomerase a precursor (erp ) a kind of molecular chaperone of the endoplasmic reticulum (er) by maldi-tof ms. the result suggests that the phosphorylation of erp may have the key function to induce dopaminergic neuron degeneration and somehow relates to the pathogenesis of sporadic pd. katsunori nishi department of neurology, tokyo metropolitan institute for neuroscience, tokyo, japan regrowth of survived dopaminergic (da) neurons after the administration of psi, a potent proteasome inhibitor, was examined in vitro. dissociated cell co-culture was prepared from embryonic rat mesencephalon and striatum. psi ( or nm, h) was applied to cultures at days in vitro and succeeding changes of da neurons were investigated up to days. more than % of da neurons reduced in number after the administration of psi, and a few truncated da neurons, devoid of neurites, being observed. non-da neurons were less severely affected at these concentrations of psi. regrowth of da neurites was observed approximately weeks after the administration of psi and continued during the observation period. in most of the regrowing da neurons, one of the processes extended far longer than the rest, suggesting that severely injured neurons retain the capacity to reextend axons. regrowth was less remarkable in mesencephalic culture lacking striatum indicating that target cells are necessary for this effect. in conclusion, psi-damaged da neuron has strong regrowth potential in vitro. ps p-j specific expression of proapoptotic factor pag on motor neurons in spinal cords of l-dopatreated parkinsonian models ikuko miyazaki, masako shimizu, francisco j. diaz-corrales, maria f. esraba-alba, masato asanuma dept. of brain sci., okayama univ. grad. sch. of med., dent. and pharmaceut. sci., japan we previously identified a proapoptotic gene, p -activated gene (pag ), as a dopa-induced gene in the striatum of l-dopa-treated parkinsonian models, which increased p expression to promote apoptosis by its nuclear translocation. last year, we also reported specific induction of pag in the internal capsule of l-dopatreated and constitutive expression in the smi- -immunopositive motor neurons in the pontine nucleus and motor nuclei of trigeminal nerve and facial nerve. in the present study, we examined distribution of pag in the spinal cords of l-dopa-injected parkinsonian rats by immunohistochemistry. l-dopa treatment showed inducing tendency of pag expression on the motor neurons in the anterior corn and lateral corticospinal tract of spinal cords. the expression of pag in the motor nuclei of cranial nerves and its induction in the spinal cords suggests its possible involvement in motor dysfunction such as dyskinesia. ps p-j an approach to the generation of ar-jp mouse model: crossbreeding of pael-r transgenic mice with parkin knockout mice hua-qin wang , , yuzuru imai , haruhisa inoue , , ayane kataoka , sachiko iita , nobuyuki nukina , ryosuke takahashi , neurology, university of kyoto, kyoto, japan; bsi, riken, saitama, japan since loss of parkin e activity appears to be causal of ar-jp, accumulation of potentially toxic parkin substrates should result in degeneration of da neurons. however, parkin knockout mice show no different da neuronal loss even at old ages, presumably due to relative short lifespan of mice. pael-r is one of the best characterized parkin substrates. we generated pael-r transgenic mice and crossbred it with parkin knockout mice. pael-r transgenic mice showed modest alterations in dopamine metabolism and behavioral deficits without displaying obvious dopaminergic neuronal loss at the age of one year. however, when pael-r transgenic mice were crossbred with parkin knockout mice, the da neuronal loss was induced in a pael-r gene dosage-dependant manner. these results strongly support that pael-r accumulation substantially contributes to dopaminergic neurodegeneration in ar-jp. parkinson's disease, a common motor disorder, is caused by a degeneration of dopaminergic neurons in the substantia nigra. after dopamine denervation, an over-activity of glutamatergic pathways has been found and that is implicated in the neuropathology of parkinson's disease. previous study (lai et al., ) have found that application of an antisense oligodeoxynucleotide specific for nr have successfully knockdown the expression of nr gene expression in the striatum of -hydroxydopamine-lesioned rats. in the present study, modulation of gene expression of nr was re-addressed using a small interfering rna (sirna) specific for nr . in pc cells, reductions of nr proteins after a single application of nr sirna were found by western blot experiments. and after one single application of nr sirna in the striatum of the lesioned rats, a significant reduction in apomorphine-induced rotation was found. slight reductions in the levels of nr immunofluorescence were found in the striatum after the sirna treatments. lai et al., . neurochem. int. , - . research funds: faculty research grant, frg/ - /ii- , hong kong baptist university ps p-j homocysteine and parkinson's disease: effects of acute intranigral administration on dopaminergic system g. chandra, k.p. mohanakumar indian institute of chemical biology, kolkata, india homocysteine (hcy) is implicated in a number of geriatric multisystem disorders and patients with hyperhomocysteinemia exhibit profound neuropsychological abnormalities. parkinsonǐs disease (pd) patients receiving long-term l-dopa therapy are reported to have elevated plasma hcy levels. we studied whether hcy is neurotoxic to the nigrostriatal dopamine (da)-ergic system in sd rats. animals infused unilaterally in substantia nigra pers compacta (snpc) with hcy ( . - mol in l) showed dose dependent loss of da and its metabolites, in the ipsilateral striatum on th day. animals with mol hcy exhibited significant motor disabilities and spontaneous and da-ergic drug-induced turning behaviors. in these animals a clear loss of neurons was visible in snpc, which were shown to be daergic by tyrosine hydroxylase immunoreactivity. intra-raphe infusion of hcy did not alter the neurotransmitter levels in the serotonergic perikarya or terminals. these results indicate the toxic potential of hcy to the da-ergic system and suggest that chronic l-dopa therapy in pd patients may further deteriorate the disease. ps p-j ubiquitin proteasome system was impaired by the aggregate formation of mutant ␥pkc found in sca takahiro seki , takayuki shimahara , naoko adachi , naoaki saito , norio sakai dept. mol. pharmacol. neurosci., grad. sch. biomed. sci., hiroshima univ., hiroshima, japan; lab. mol. pharmacol., biosig. res. ctr., kobe univ., kobe, japan we have previously demonstrated that several mutant protein kinase c gamma (␥pkc), found in several families of spinocerebellar ataxia type (sca ), are susceptible to cytoplasmic aggregation and cause cell death in cho cells, indicating that this property is involved in the etiology of sca . however, the relationship between the aggregate formation of mutant ␥pkc and cell death remains unclear. accumulating evidences indicate that the impairment of the ubiquitin proteasome system (ups) is related to the pathogenesis of many neurodegenerative disorders. therefore, we examined whether the aggregate formation of mutant ␥pkc affects ups function. the immunoreactivities for ubiquitin and proteasome were intensely accumulated in the aggregates of mutant ␥pkc. decreased proteasome activities were also observed in cells having aggregated mutant ␥pkc. these results indicate that the aggregation of mutant ␥pkc exert cytotoxic effect via the impairment of ups. it is well known that oxidant stress is involved in many pathologic conditions including brain ischemia and neurodegenerative diseases. recently, however, another type of stress, endoplasmic reticulum (er) stress has also been reported to be associated with such diseases. er stress is characterized by accumulation of unfolded proteins in the er that is caused by inhibition of protein modification, disturbance of ca + homeostasis or oxygen deprivation. we recently reported that targeting disruption of herp, a novel er stress-related gene, caused f cells vulnerable to er stress. using these cells, we developed a screening system for molecules that suppress er stress. approximately compounds have been screened, and we found some molecules that protect human neuroblastoma cells against er stress and oxidative stress. we speculate that this system could provide novel therapeutic targets to the er stress and oxidative stressrelated diseases. toshiyuki araki , yo sasaki department of peripheral nervous system research, national institute of neuroscience, ncnp, tokyo, japan; washington university school of medicine, st. louis, missouri, usa axonal degeneration which is observed in a variety of neuropathological conditions or physical damage to axons is a self-destructive program that is independent from programmed cell death. we previously reported that increased nicotinamide adenine dinucleotide (nad) production by the overexpression of nicotinamide mononucleotide adenylyltransferase (nmnat ) or exogenously applied nad can protect neurites from degeneration caused by mechanical or neurotoxic injury of neuronal cells. the mammalian nad biosynthesis is mediated by at least different kinds of enzymes and each enzyme converts different substrate to nad or its precursors. here we investigated whether overexpression of these enzymes or exogenous application of nad precursors protects neurites from degeneration through increased supply of nad. cocaine is considered to affect spine morphology and the composition of postsynaptic density (psd) of medium spiny neurons in nucleus accumbens (nac). we examined the accumulation of several proteins altered by cocaine challenge after withdrawal of repeated cocaine administration in psd fraction of rat nac at different time points. total psd protein yield was decreased at min, but next increased at h and returned to basal at h after cocaine challenge. actin showed a similar pattern but was maintained at high level at h. both psd- and glur were increased between h and h like actin. by contrast, some proteins such as drebrin were decreased after the peak at h. interestingly, the s proteasome subunit demonstrated a dramatic upregulation at h. these data suggest that the composition of psd proteins is regulated by proteasome activity as well as actin cycling. it is possible that some proteins may be removed from psd by proteasome following transient requirement for organizing psd in the nac of chronic cocaine-administrated animals. ps p-k effects of mdma and -meo-dipt on serotonin transporter and dopamine transporter yosuke yamauchi, takaya izumi, takayuki nakagawa, shuji kaneko dept. mol. pharmacol., grad. sch. pharm. sci., kyoto univ., kyoto, japan by two electrode voltage-clamp recordings from xenopus oocytes heterologously expressing serotonin transporter (sert) or dopamine transporter (dat), the effects of two addictive agents, , methylenedioxymethamphetamine (mdma) and -methoxy-n,ndiisopropyltryptamine ( -meo-dipt), on sert and dat were examined. as previously reported, mdma ( . - m) dose-dependently induced transport-associated, inward current response in the sertexpressing cells. interestingly, mdma-induced current response was also observed in dat-expressing cells. on the other hand, -meo-dipt ( . - nm) evoked an outward current response in sertexpressing cells similarly to that of selective -ht reuptake inhibitors. no current response was observed when -meo-dipt was applied to dat-expressing cells. these results suggest that mdma is transported not only by sert but also by dat, and that -meo-dipt suppresses the spontaneous transport activity of sert. junichi kitanaka , nobue kitanaka , tomohiro tatsuta , , yoshio morita , motohiko takemura department of pharmacology, hyogo college of medicine, nishinomiya, japan; department of neuropsychiatry, hyogo college of medicine, nishinomiya, japan we examined the effects of pretreatment with clorgyline on morphine-induced behavioral changes and antinociception. a single administration of morphine ( mg/kg, i.p.) to male icr mice induced a hyperlocomotion. the anova analysis revealed statistical significance of a morphine effect (hyperlocomotion) and of a clorgyline pretreatment x morphine interaction effect (inhibition), but not of an effect of clorgyline pretreatment. clorgyline pretreatment itself did not affect the spontaneous locomotion. clorgyline at a dose of . mg/kg but not other doses tested significantly potentiated morphine-induced antinociception evaluated by tail flick but not hot plate test. clorgyline at the doses of and mg/kg significantly inhibited dopamine and serotonin metabolism. these results suggest that clorgyline showed its inhibitory effect on morphine-induced hyperlocomotion, but not antinociception, through mao inhibition. recent studies in our laboratory have shown that methamphetamine (meth)-induced hyperlocomotion and behavioral sensitization in mice were inhibited by clorgyline, an irreversible monoamine oxidase inhibitor. in this presentation, the effect of clorgyline pretreatment on meth reward was assessed by conditioned place preference (cpp) paradigm, using an apparatus developed with supermex ® sensors. although intact male icr mice showed a significant cpp for meth ( . mg/kg, i.p.), pretreatment with subchronic clorgyline ( . - mg/kg, s.c.) did not affect the magnitude of cpp. pretreatment with clorgyline significantly decreased apparent dopamine and serotonin turnovers in the striatum in a dose-dependent manner. these results indicated that clorgyline pretreatment did not influence meth reward in mice. of lobeline pretreatment on methamphetamine-induced stereotypy and monoamine metabolism in mice motohiko takemura , nobue kitanaka , tomohiro tatsuta , , yoshio morita , junichi kitanaka department of pharmacology, hyogo college of medicine, nishinomiya, japan; department of neuropsychiatry, hyogo college of medicine, nishinomiya, japan the effects of lobeline, an alkaloid constituent of indian tobacco, on methamphetamine (meth)-induced stereotypy and monoamine metabolism were investigated in male icr mice. pretreatment with lobeline ( . - mg/kg, i.p.) min prior to drug challenge significantly decreased an intensity of stereotypies and increased its latency to onset in a dose-dependent manner. in saline challenge groups, doses of lobeline examined did not affect the spontaneous locomotion nor induce any stereotyped behaviors. the range of lobeline doses examined except mg/kg did not affect apparent monoamine turnovers in the brain regions including striatum min after drug challenge. these results suggested that the inhibitory effect of lobeline ( . - mg/kg) on meth-induced stereotypy did not attribute to the change in the brain monoamine metabolism. kazuto sakoori, niall murphy riken bsi, wako-shi, japan previously we showed that endogenous nociceptin suppresses drug reward. here, we examined the effect of blockade of nop receptors on methamphetamine (meth) induced behavioral sensitization in order to understand the role of endogenous nociceptin in the chronic response to addictive drugs. first, nop receptor ko and wt mice were treated with mg/kg meth and locomotor activity measured daily for days. wt mice showed gradually increasing sensitivity to meth with repeated treatment of meth, whereas nop receptor knockout mice did not. next, nmol ufp- (a nop receptor antagonist) and mg/kg meth were co-administrated to mice and locomotor activity measured daily for eight days. ufp- strongly suppressed locomotor activity. thus, it was unclear if ufp- suppressed behavioral sensitization to meth during chronic drug treatment. however, when challenged with meth after four or more days without treatment, ufp- co-administrated mice showed a lower locomotor response. these results suggest that endogenous nociceptin facilitates the plastic changes induced by chronic treatment with addictive drugs. the influence of olanzapine (a d dopamine receptor antagonist) on the morphine-induced conditioned place preference (cpp) in male and female mice was investigated in the present study. subcutaneous (s.c.) injection of morphine ( - mg/kg, three drug sessions) induced place preference both in male and female mice. intraperitoneal (i.p.) administration of olanzapine ( . - mg/kg) induced place aversion (cpa) in female mice but not in male mice. administration of olanzapine ( , . and mg/kg, i.p.) reduced both the acquisition and expression of morphine-induced cpp in male and female mice. however, olanzapine ( mg/kg, i.p.) caused more than % mortality in female but not male mice. the effects of olanzapine were reversed by l-arginine ( mg/kg, i.p.) pre-administration. in conclusion, it seems that olanzapine reduced morphine effects in part via a nitric oxide (no) mechanism. feed-forward associative learning (ffal) theory of cerebellar motor learning proposed by the author presumes that higher motor centers have place-coding systems and the same systems are shared by the cerebellum. when a new motor learning proceeds with respect to a certain movement, previous learning results of the movement will turn out to be modified or erased. ffal theory presumes that transferred memory from the cerebellar cortex to nucleus will serve as the maintenance of the previous learning. from this line, many aspects of saccadic adaptation are successfully demonstrated by computer simulation based on the theory. another theoretical issue is the credit assignment problem of motor error. a motor error is generally an integrated result of maladjusted multiple learning elements, and is to be decomposed to each element credit. this problem naively leads to an idea of a dual redundant system for movement, one for execution and the other for error decomposition. ffal theory naturally and simply resolves the credit assignment problem and demonstrates a computer simulation of motor learning of multi joint movement system, using the place-coding hypothesis. ps p-d regulation of camp responsive element binding protein to stress in rat amygdala and hippocampal formation the department of anatomy and histology, shanghai medical school, fudan university, china amygdala (am) and hippocampal formation (hf) are important structures relating with emotional learning and memory. transcription factor, camp-responsive element binding protein (creb) in am and hf plays important roles in memory modulating processes. creb is a nuclear protein and is wldely accepted as prototypical stimulusinducible transcription factor. creb is activated in response to a vast array of physiological stimuli and then becames phosphorylated creb (pcreb). neurophysiological and neuropharmacological studies said that creb may regulate gene transcription and protein synthesis to maintain the long term and sustaining changing of synaptic efficiency during the long-term process of synaptic plasticity. but we cannot tell exactly via what kind of neurons in am and hf creb regulate these processes. we used the animal model, forced swimming (fs) as emotional stimuli and the experiment methods such as, immunocytochemistry, western-blotting with anti-pcreb antibody. the distributing profiles and changing rules of pcreb immunoreactive nuclei in amygdala and hippocampal formation of both control and experiment groups were investigated. the neuronal types of pcreb immunoreactive nuclei were analyzed by double-labelling immunocytochemistry with anti-pcreb, anti-glu and anti-pv antibodies. the results were: ( ) the number of pcreb immunoreactive neuclei and total amount of pcreb in the subnuclei of rat amygdala, dentate gyrus (dg) and cornu ammonis (ca ) were increased after fs. the rule of this kind of changing was of region-and time-specific. ( ) pcreb immunoreactive neuclei were expressed in glutamate immunoreactive neurons and were devoid in interneurons. these results suggested that pcreb in limbic system regulated the fs process and the regulation was finished via exciting neurons, glutamate neurons. hideto takahashi , , tomoaki shirao dept of neurobiol & behav.; ercgsm, gunma univ. grad. sch. of med., maebashi, japan dendritic spines are developmentally-regulated and activitydependent polymorphic structures based on actin cytoskeleton. drebrin is a spine-rich actin-binding protein regulating spine morphogenesis during development. here we find that chronic blockade of ampa receptors (ampar) inhibits synaptic drebrin clustering during development of hippocampal neurons, but not that of nmdar. further, the analysis of fluorescence recovery after photobleaching for egfp-drebrin a reveals that only . ± . % of drebrin in the spine is stable, with a turnover time of . ± . min. blockade of ampar by m cnqx reduces the population of stable drebrin ( . ± . %), and has no effect on a turnover time. on the other hand, blockade of nmdar by m ap has no effect on the population of stable drebrin, whereas shortens a turnover time ( . ± . min). these data suggest that ampar activities increase the binding capacity of drebrin in spines, and therefore promote drebrin clustering at spine synapses. instead, nmdar activities regulate spine-shaft shuttling of drebrin. itsuko nihonmatsu , yoshito saitoh , kaoru inokuchi , mitsubishi kagaku inst. life sci. (mitils), tokyo, japan; crest, jst, tokyo, japan dendritic protein synthesis requires dendritic localization of mrnas in neurons. however, ultrastructural localization of these mrnas have not been well described. here we employed in situ electronmicroscopic technique to examine the precise localization of ␣camkii mrna in dendrites. ␣camkii mrna was located at the specific sites of dendritic shafts of pyramidal neurons, close to the spines, rather than in a diffused manner. we observed an increase in the ␣camkii mrna signals at the synaptic layer undergone l-ltp in the hippocampal dentate gyrus in unanesthetized freely moving rats. the increase was transient and returned to the basal level at h. the alteration in the ␣ camkii mrna localization in dendrites may reflect a functional change in the translational apparatus along with synaptic plasticity. reiko okubo-suzuki , , daisuke okada , kaoru inokuchi , , mitsubishi kagaku inst. life sci. (mitils), tokyo, japan; yokohama natl. univ. environment information sci., kanagawa, japan; crest, jst, japan late-phase long-term potentiation (l-ltp) depends on de novo protein synthesis. synaptopodin (synpo), an f-actin-associated protein, increases in the activated synapses following l-ltp induction. spine volume and f-actin content in the spines also increase during l-ltp. to reveal the roles synpo plays in the regulation of spine volume and f-actin content, we examined synpo-egfp (se) localization and spine volume in the hippocampal neurons using time-lapse confocal imaging techniques. se-overexpression did not alter spine volume, but the amount of se in spines positively correlated with the spine volume. pharmacological activation of the nmda receptors increased both spine volume and synpo content in spines. furthermore, experiments with ptk cells indicated that synpo stabilizes f-actin. these results suggest that synpo synthesized in soma and transported into the activated spines following l-ltp induction stabilizes spine f-actin that may lead to the maintenance of increased spine volume. mineo matsumoto , mitsutoshi setou , , kaoru inokuchi laboratory for molecular gerontology, mitils, japan; laboratory for nano-structure physiology, nips, japan subcellular localization of rna is an efficient way to localize proteins to a specific region of a cell. a requirement for dendritic rna localization and subsequent local translation has been demonstrated in several forms of experience-dependent synaptic plasticity. in spite of several attempts to identify these rnas, the population of rna species present in dendrites as a whole has not been well described. here we show the results of microarray analyses with rnas isolated from rna granule or synaptosome fractions prepared from the rat brain. these analyses revealed the complex nature of the dendritic rna population, which included rnas that were not expected to be in the dendrites. neural activity caused by an electroconvulsive shock triggered a redistribution of the dendritic transcriptome towards the synaptosome, a translationally active region. our results suggest that the redistribution of dendritic rnas is one of the mechanisms regulating local translation in response to synaptic inputs. ps a-a an activity that traps vesl- s protein into spines serves as synaptic tag synaptic tagging hypothesis explains how new proteins reach the activated synapses to establish input-specific late-phase plasticity, but it has not yet been substantiated. original idea of synaptic tagging is supposed to regulate protein entry into synaptic region including spines. using live-imaging techniques, we measured entry of vesl- s-egfp into spines (ve trapping) of rat hippocampal neurons in culture, and found that ve trapping activity serves as the synaptic tag in many criteria. ve trapping required synergistic activation of postsynaptic no-pkg pathway and an activity abolished by ttx at m, but not nm. because nm ttx is supposed to suppress na channels only postsynaptically, we concluded that ve trapping is a hebbian-like process that requires both pre-and postsynaptic activities. however, their coincidence time window was far wider (hrs) than that of early-phase plasticity, suggesting a requirement of persistently synchronized, rather than transiently coincident, activities, and a possibility of metaplastic states for late-phase plasticity. ps a-a acute effects of dehydroepiandrosterone sulfate (dheas) on the synaptic transmission and plasticity in rat hippocampal slices yuxia xu , ling chen , masahiro sokabe , , dept. physiol., nagoya univ., grad. sch. med., nagoya, japan; dept. physiol., nanjing med. univ., nanjing, china; sorst cell mechanosening, jst, nagoya, japan; dept. mol. physiol., nips, okazaki, japan the neurosteroid dehydroepiandrosterone sulfate (dheas) is known to improve memory and learning in mammals. recently we report that chronic administration of dheas facilitates the induction of ltp in the rat hippocampus. to elucidate the underlying synaptic mechanism of the dheas effects, we examined in this study the acute effects of dheas on the synaptic transmission and plasticity at the ca region in rat hippocampal slices. an application of . dheas for min to the slice augmented instantly the epsp, which was terminated within min. however, even h after the drug application, a subthreshold tetanus could induce ltp without alteration of ppf. this facilitating effect of dheas on ltp induction was blocked by a coapplication of a nmda receptor antagonist with dheas for min, suggesting that the dheas effect involves a sustained modulation of the postsynaptic signaling mediated by nmda receptor. xiaoniu dai , ling chen , masahiro sokabe , , dept. physiol., nagoya univ., grad. sch. med., nagoya, japan; dept. physiol., nanjing med. univ., nanjing, china; sorst cell mechanosensing, jst, nagoya, japan; dept. mol. physiol., nips, okazaki, japan to know whether ␤-estradiol (e ) can protect ca neurons from functional deficit due to ischemia, adult male wistar rats were subjected four-vessel occlusion ( vo) for min, and the effect of e against this ischemic injury was examined. the electrophysiological properties of ca -ca synapses were examined by a real-time optical recording method days after ischemia. the ischemic brain showed a decreased synaptic transmission and an impairment of ltp induction but no alteration in paired-pulse facilitation. administration of e ( mg/kg) h before vo was able to protect ca neurons from these ischemic synaptic dysfunctions. the estrogen receptor-␣ selective agonist ppt ( mg/kg) produced a similar protective effect, but the estrogen receptor-␤ agonist dpn ( mg/kg) did not. above results suggest that e can protect neurons not only from cell death but also from functional damages caused by cerebral ischemia. ps a-a non-genomic rapid effects of estradiol on hippocampal synapses: multi-electrode dish analysis kohei nakajima , mari ogiue-ikeda , yuki oishi , suguru kawato , department of biophysics and life sciences, graduate school of arts and sciences, university of tokyo at komaba, tokyo, japan; department of physics, university of tokyo, tokyo, japan estradiol has a non-genomic, rapid effect on synaptic transmission, which is manifested within seconds to minutes. recently, hippocampal neurons were shown to synthesize estradiol de novo, and to express estrogen receptor ␣ (er␣) at synapses. although these results imply that estradiol rapidly modulates synaptic plasticity through synaptic er␣, there are few electrophysiological evidence about it. here we investigated effects of estradiol on ltd by using wild type, er␣ hetero and er␤ hetero mouse hippocampal slices with a multi-electrode dish (med, panasonic). med enabled us to measure epsps in ca , ca , and dentate gyrus simultaneously. hippocampal slices were perfused with estradiol before nmda-induced ltd. we found that estradiol enhanced ltd both in wild type and er␤ hetero mouse, but not in er␣ hetero mouse. our data suggested non-genomic rapid action of estradiol through synaptic er␣. withdrawn ps a-a morphological changes of dendritic spines mediated by glucocorticoid receptor (gr) in rat hippocampus yoshimasa komatsuzaki , gen murakami , , tetsuya kimoto , , suguru kawato , college of humanities and sciences, nihon university, tokyo, japan; department of biophysics and life sciences, university of tokyo, tokyo, japan; crest, jst, japan modulation of hippocampal synaptic plasticity by glucocorticoids has been attracting much attention, due to its importance in stress responses. dendritic spines are essential for memory storage processes. here we investigated the effect of dexamethasone (dex), a specific agonist of glucocorticoid receptor (gr), on density and morphology of dendritic spines in adult male rat hippocampus by imaging of lucifer yellow-injected spines in slices. the application of nm dex induced rapid modulation of the density and morphology of dendritic spines in ca pyramidal neurons within h. the total spine density increased from . spines/m to . spines/m. dex significantly increased the density of thin and mushroom type spines, however only a slight increase was observed for stubby and filopodium type spines. because the presence of m cycloheximide, an inhibitor of protein synthesis, did not suppress the dex effect, these responses are probably non-genomic. hideki tamura , yuji ikegaya , sadao shiosaka division of structural cell biology, naist, nara, japan; laboratory of chemical pharmacology, university of tokyo, tokyo, japan the capacity of activity-dependent synaptic modification is essential in processing and storing information, yet little is known about how synaptic plasticity alters the input-output (i-o) conversion efficiency at the synapses. in the adult mouse hippocampus in vivo, we carefully compared the i-o relationship, in terms of presynaptic activity levels versus postsynaptic potentials, before and after the induction of synaptic plasticity and found that synaptic plasticity led synapses to respond more robustly to inputs, that is, synaptic gain was increased as a function of synaptic activity with an expansive, power-law nonlinearity, i.e., conforming to the so-called gamma curve. in extreme cases, long-term potentiation (ltp) and depression (ltd) coexist in the same synaptic pathway with ltp dominating over ltd at higher levels of presynaptic activity. these findings predict a novel function of synaptic plasticity, i.e., a contrast-enhancing filtering of neural information through a gamma correction-like process. research funds: st century coe research ps a-a actin organizations within single dendritic spines in ca pyramidal neurons studies with two-photon photoactivation naoki honkura, masanori matsuzaki, haruo kasai center for disease biology and integrative medicine, faculty of medicine, the university of tokyo, japan the major cytoskeleton of dendritic spines is filamentous actin (factin). we have here investigated sub-spine actin organizations using two-photon photoactivation of pa-gfp fused with ␤-actin in rat ca pyramidal neurons. we found segregated and discontinuous organizations of two pools of f-actin, dynamic and stable pools, which turned over with time constants of . min and min, respectively. fractions of the stable f-actin pool were greater in larger spines, therefore, the entire f-actin pool was more stable in larger spines. we succeeded in visualizing a retrograde flow of f-actin in the dynamic pool from the apex to the base of spine, and found that both the speeds ( . - . um/min) and lengths ( . - . um) of the f-actin flow were greater in spines with larger head volumes. moreover, spine heads rapidly shrank when actin polymerization was blocked by latrunculin a, suggesting that the rate of actin polymerization in each spine actively and continuously determines the volume of spine head via the length of f-actin. tomoharu nakamori , katsushige sato , kohichi tanaka , hiroko hamazaki mol. neurosci., tmdu, tokyo, japan; physiology, tmdu, tokyo, japan the visual wulst (vw) in the thalamofugal pathway in chicks is known to have a critical role in the visual learning. to understand the function of the vw in the learning process of imprinting, we investigated the neuronal activity of vw region in chick brain. the slice stained with a voltage-sensitive dye was prepared for a multiple-site optical recording. when chicks were reared in quasi-dark condition, the extent and amplitude of response induced by electrical stimulation were different between at or days post-hatching (p or p ), and at p . this corresponds to behavioral data showing that chicks have high ability of visual learning in imprinting behavior until p , but they lose this ability at p . in addition, the light-exposed chick showed larger optical response than the dark-rearing one. the optical response in the vw was partly inhibited by the glutamate-and gaba-receptor antagonists. these results suggest that the glutamatergic as well as gabaergic neurons are active in the area including vw and that the neuronal activity of vw affects the learning ability for imprinting. withdrawn ps a-b effect of estrogen on hippocampus in male and female mice takanori sugawara , shinji hayashi , victoria luine graduated school of integrated sience, yokohama city university, yokohama, japan; department of psychology, hunter college, city university of new york, new york, usa we examined structural difference in the hippocampal neurons with golgi stain among the male, the female and the female treated with estrogen neonatally. the mice were gonadectomized and received ␤-estradiol (e ) or oil-vehicle injections at adult before golgi impregnation. spine densities m of apical dendrites of the pyramidal neurons in the hippocampus ca region were calculated with categorization into three shapes, i.e., mushroom type with large head, thin type and filopodia-like type. as a result, only in the female not estrogen treated neonatally, the mushroom type and total spine densities were increased but the thin type spine density was decreased by e treatment in adult. the present results indicate that estrogen given at adult induces an enlargement of spine to mushroom type and generates new spines only in the female mice not treated with estrogen neonatally. thus, dendritic spine formation seems sexually dimorphic and depends on the sex steroid environment during the neonatal period. jun-ichi goto , , takafumi inoue , , akinori kuruma , katsuhiko mikoshiba , , lab. developmental neurobiology, brain science inst., riken, saitama, japan; div. molecular neurobiology, inst. medical science, univ. tokyo, tokyo, japan; calcium oscillation project, icorp-sorst, jst, tokyo, japan changes in synaptic efficacy at the parallel fiber (pf)-purkinje cell (pc) synapse are postulated to be a cellular basis for motor learning. although long-term efficacy changes lasting more than an hour at this synapse, i.e., long-term potentiation and depression, have been extensively studied, relatively short lasting synaptic efficacy changes, namely short-term potentiation (stp) lasting for tens of minutes, have not been discussed to date. here we report that this synapse shows an apparent stp reliably by a periodic burst pattern of homo synaptic stimulation. this stp is presynaptically expressed, since it accompanies with a reduced paired-pulse facilitation and is resistant to postsynaptic ca + reduction by bapta injection or in p/q-type ca channel knockout cerebella. this novel type of synaptic plasticity at the pf-pc synapse would be a clue for understanding the presynaptic mechanisms of plasticity at this synapse. aya ishida, wataru kakegawa, michisuke yuzaki department of physiology, keio university, tokyo, japan mitogen-activated protein kinase (mapk) cascade is thought to be essential for the synaptic plasticity and learning. in the hippocampus, three different mapk subfamilies, including extracellular signalregulated kinase (erk), p mapk and c-jun nh -terminal protein kinase (jnk), have been shown to selectively regulate different forms of synaptic plasticity -long-term potentiation (ltp), longterm depression (ltd), and depotentiation after ltp, respectively. although erk was previously shown to play a role in cerebellar ltd in cultured purkinje cells, the role of mapks has not been systemically studied. here, we examined the effect of specific inhibitors of three different mapks on ltd by patch-clamp recordings from cerebellar slices. we found that u , a specific inhibitor for erk activation, significantly inhibited ltd induction, whereas sb and sp , antagonists for p mapk and jnk, respectively, had no effect. therefore, unlike hippocampal ltd, cerebellar ltd was dependent on erk, suggesting involvement of different intracellular downstream pathways. ps a-b regulation of ampa receptor trafficking by aaa atpases in cerebellar purkinje cells: are nsf and vcp playing complementary or antagonistic roles? thomas launey , chou-chi li , yumiko motoyama , junko yamaoka , masao ito riken brain sci. inst., japan; national cancer institute, nih, ma, usa the number of postsynaptic ampa receptors (ampar) is regulated by interactions with multiple protein complexes, throughout its synthesis, maturation, transport, synaptic insertion and degradation. aaa atpases influence several of these stages, the most extensively studied being nsf's contribution to ampar trafficking. in cerebellar purkinje cell (pc), we show that valosin containing protein (vcp), an atpase with high homology to nsf, is bound to ampa receptors in pc's dendritic compartment. following glur co-ip from molecular layer, vcp was detected by ms/ms and by monoclonal anti-vcp. pull-down assay showed a direct interaction between vcp and glur c-term domain, requiring vcp n-term domain and both the nsf and pdz binding domains of glur . glur phospho-ser promotes vcp complex dissociation, suggesting a relation with synaptic plasticity. further, pep m-related peptides, thought to interfere specifically with nsf-regulated ampar trafficking, also blocked the glur -vcp interaction. yuichi kitagawa , , shin-ya kawaguchi , , tomoo hirano , dept. biophys., grad. sch. sci., kyoto univ., kyoto, japan; crest, jst, kawaguchi, japan at inhibitory synapses on a cerebellar purkinje neuron (pn), postsynaptic depolarization induces long-lasting potentiation of the gaba a receptor (gaba a r) responsiveness (rebound potentiation: rp). previous studies have clarified the molecular mechanism regulating rp induction. whether rp is induced or not is determined by the balance of activities of protein kinases (camkii and pka) and phosphatases (pp- and calcineurin). to understand the complex behavior of biochemical reactions systematically, a kinetic simulation model to analyze the behaviors of signaling network was developed. computer simulation reproduced the bistable states of gaba a r phospholyration according to stimulation patterns, which apparently corresponded to whether rp was induced or not. we further studied the systematic property of the molecular network, and obtained several experimental predictions. these possibilities were evaluated by experiments such as immunocytochemistry using cultured pns. ps a-b long-term depression of synaptic transmission in a songbird motor nucleus essential for song learning yuki haruta, yachun huang, neal hessler vocal behavior mechanisms riken brain science institute, japan in order to fully understand the neural basis of song learning, it is critical to characterize forms of synaptic plasticity that could be involved in this process. we previously reported that, in synapses of the song motor nucleus ra, participation of postsynaptic nmda receptor nr b subunits and presynaptic transmitter release both decrease from young birds to adults. here, we tested whether synaptic function could be modified in a similar way by acute stimulation. after pairing slight postsynaptic depolarization with presynaptic stimulation, ltd was reliably induced at both hvc and lman inputs in juvenile birds from to days old. this depression required activation of postsynaptic nmda receptors, and was expressed by decreased transmitter release, which required activation of cannabinoid receptors. no ltd could be induced in normal birds over days old, when song learning is nearly complete, but ltd remained possible in birds over days old who had been isolated from song tutors, and thus retained the capacity for learning. ps a-b involvement of ca + -permeable ampar in the repetitive-ltp induced synaptic enhancement (rise) yukiko ueno, keiko tominaga-yoshino, akihiko ogura graduate school of frontier biosciences, university of osaka, osaka, japan we showed previously that exposures to glu of cultured rat hippocampal slices at h intervals produced a long-lasting enhancement in synaptic strength accompanied by synaptogenesis (rise). we examined here whether the conversion of ampar subunits occurred during the development of rise. immunochemical staining for ampar subunits, glur and glur , showed that the number of glur -positive puncta increased transiently after the repeated glu exposures, whereas the number of glur -positive puncta increased gradually and persistently. jstx (a ca + -permeable ampar blocker) suppressed fepsp amplitude recorded at ca -ca synapses by - % in the period corresponding to the transient increase of glur -positive puncta. this transient increase should represent the delivery of ca + -permeable (glur -lacking/glur -including) ampar to synaptic sites. furthermore, jstx application at that period blocked the rise production. these results suggest that the transient delivery of ca + -permeable ampar to synaptic sites is involved in the rise production. yoshihiro egashira, tsunehiro tanaka, yuji kamikubo, yo shinoda, keiko tominaga-yoshino, akihiko ogura osaka univ. grad. sch. frontier biosciences, toyonaka - , japan long-lasting synaptic plasticity, the cellular basis of long-term memory, is assumed to be associated with protein synthesis. using cultured rat hippocampal slices, we previously found that a long-lasting synaptic enhancement coupled with an increase in the number of synaptic structures was established after inductions of ltp, not after its single induction. this synaptic enhancement required protein synthesis for its establishment. we recently found an apparently mirror-image phenomenon; inductions of ltd led to a long-lasting synaptic decrement coupled with a decrease in the numbers of synaptic structures. to know whether this synaptic decrement also requires protein synthesis, we induced ltd times ( h intervals) by applications of dhpg (a type i mglur agonist), during or after which anisomycin (a protein translation blocker) was applied. we found that anisomycin did not block the induction of ltd but blocked the establishment of the long-lasting synaptic decrement. haruo mizutani, tetsuya hori, tomoyuki takahashi department of neurophysiology, graduate school of medicine university of tokyo, tokyo, japan bath-application of -ht ( m) attenuated the amplitude of evoked epscs and facilitated paired-pulse ratio without affecting the miniature epsc amplitude, suggesting that its site of action is presynaptic. the -ht b receptor agonist cp mimicked the presynaptic inhibitory effect of -ht. -ht b receptor antagonist nas- reversed the -ht inhibitory effect, indicating that the -ht induced inhibitory effect occurs by mediating -ht b receptors. the presynaptic inhibitory effect of -ht became weaker as animals matured. in whole-cell recordings from calyceal presynaptic terminals, -ht attenuated voltage-dependent calcium currents, but had no effect on potassium currents. this -ht effect was characterized with a marked desensitization, but sustained under the fast calcium chelating agents, bapta. these results suggest that -ht, upon activating -ht b receptors, inhibits presynaptic calcium channels thereby inhibiting transmitter release and induces receptor desensitization by calcium influx at the immature calyceal synapse. takako ohno-shosaku , masato ano , yuki hashimotodani , tadasato nagano , masanobu kano dept. impair. study, grad. sch. med. sci., kanazawa univ., kanazawa, japan; dept. neurophysiol., grad. sch. med., osaka univ., osaka, japan; dept. cell. neurosci., grad. sch. med., osaka univ., osaka, japan retrograde endocannabinoid signal contributes to activitydependent modulation of synaptic transmissions in various brain regions. endocannabinoid release is triggered by depolarizationinduced elevation of intracellular calcium level or activation of gq-coupled receptors. here we report that nmda receptors can also contribute to generation of endocannabinoid signal. inhibitory postsynaptic currents (ipscs) were recorded in cultured hippocampal neurons prepared from newborn rats. application of nmda induced a transient suppression of cannabinoid-sensitive ipscs but not cannabinoid-insensitive ipscs. the nmda-induced suppression of ipsc was blocked by a cannabinoid receptor antagonist. these results indicate that activation of nmda receptors induces the endocannabinoid release, and suppresses the inhibitory synaptic transmission through activation of presynaptic cannabinoid receptors. the most caudal region of the rat spinal cord, the conus medullaris has a simple anatomical feature, which lacks ventral as well as dorsal root fibers and somatic motor neurons in the ventral horn. a small number of neurons distribute around the central canal, and some of them are nitric oxide synthase (nos) positive. a dense distribution of nerve fibers immunoreactive to cgrp, sp, and npy was found in dorsal part of the conus medullaris similarly to that of other spinal cord levels. in addition, enk-, -ht-, and th-immunoreactive varicose fibers were richly distributed throughout the sectional plane. to analyze this unique structure may provide valuable information on the basic neural cytoarchitecture and fiber connections of the spinal cord, particularly for the intraspinal circuitry. for this purpose, we made an electron microscopic study using nadph-diaphorase histochemistry combined with immunohistochemistry for neuronal markers. adenosine has been known to be a neuro-modulator in the nervous systems and four types of adenosine receptor are identified (a , a a, a b and a ). adenosine a and a receptors have been reported to inhibit high-threshold ca channel currents in neurons. to investigate the interaction between adenosine a and a receptors in rat striatum neurons in culture, l-type ca channel currents were recorded by whole-cell clamp method before and after administration of a agonist (cpa) and a agonist ( -cl-ib-meca). ca currents were decreased after administration of low concentration of cpa and -cl-ib-meca as reported previously. although ca currents were decreased by -cl-ib-meca in the presence of cpa, ca currents applied with cpa were not decreased on cells in the presence of -cl-ib-meca. at administration of cpa and -cl-ib-meca on cells simultaneously, ca currents were not decreased. these results suggested that adenosine a receptor may inhibit adenosine a receptor throughout a intracellular pathway in neurons. ps a-c influence of extracellular gaba and taurine to gaba a receptor-mediated actions in radially migrating cortical plate cells with identified by in utero electroporation t. furukawa , j. yamada , k. inoue , y. yanagawa , a. fukuda , dept. physiol., hamamatsu univ. sch. med., japan; dept. biol. info. process, grad. sch. elec. sci. & tech., shizuoka univ., hamamatsu, japan; dept. developmental and integrative neurosci., gunma univ. sch. med., gunma, japan it is well known that role of gaba a -r mediated actions is important for early cns development. the radially migrating cells may affected by the actions. gaba content in the brain of gad -gfp knock-in mouse decrease compared with the wild type mice. therefore, we investigate the influence of the circumferential gaba concentration to radially migrating cells. furthermore, as it was known that gaba a -r is affected by taurine, the influence of taurine to radially migrating cells was also investigated. there was no significant difference in distribution of radially migrating cells that was labeled by means of electroporation. evoked gaba a -r mediated currents of labeled cells had dose-dependent manner and had no differences among genotypes. therefore, we have examined the influence of circumferential taurine to gaba a -r mediate actions. takashi hayakawa , hiroyuki hioki , kouichi nakamura , , hisashi nakamura , takeshi kaneko , dept. morphol. brain sci., grad. sch. med., kyoto univ., kyoto, japan; crest, jst, kawaguchi, japan we previously reported that almost all vesicular glutamate transporter (vglut )-immunoreactive (ir) cells were also gabair in neocortex and choline acetyl transferase (chat)-ir in caudate-putamen in rat. although, in dorsal and median raphe nuclei, many vglut -positive cells showed immunoreactivity for -hydroxytryptamine ( ht), a significant proportion ( . %) of vglut -postive cells was ht-negative. in this study, triple immunofluorescence staining was performed for vglut , ht and one of the following proteins: neuronal nuclear antigen (neun), glial fibrillary acidic protein (gfap), glutamic acid decarboxylase (gad ) and tyrosine hydroxylase (th). our results showed that all of the vglut -positive/ ht-negative cells were immunoreactive for neun but not for gfap. furthermore, we found that these vglut positive/ ht-negative neurons didn't show any immunoreactivities for gad nor th, and thus it is indicated that there is a group of exclusively glutamatergic vglut -positive neurons in these nuclei. research funds: kakenhi , , ps a-c cortico-striatal and fast-spiking cell activity in the rat frontal cortex during cortical oscillations in vivo: modulation by serotonin m victoria puig , mika ushimaru , yoshiyuki kubota , akiya watakabe , tetsuo yamamori , yuchio yanagawa , yasuo kawaguchi div. cerebral circuitry, nips, okazaki, japan; div. brain biology, nibb, okazaki, japan; dept. genetic and behavioral neurosci., gunma univ. graduate school of med., japan we studied how cortico-striatal (cs) and fast-spiking (fs) cells are modulated by slow-wave-sleep (sws) oscillations and by serotonin ( -ht). cs and fs cells were recorded simultaneously with the electrocorticogram in the secondary motor area of anesthetized rats that expressed a gfp in gabaergic interneurons. fs displayed a highsuccess excitation to striatal stimulation, suggesting a control of cs over fs. during sws, both cs and fs fired during the up-states though with different patterns. the stimulation of the dorsal raphe promoted longer up-states. moreover, % of the cs were inhibited by -ht through -ht a r and % were excited through -ht a r. however, % of the fs cells were inhibited and % excited. these results show that cs cells are more inhibited by -ht than fs. the expression of -htr was confirmed by in situ hybridization. research funds: jsps pe and ryohei tomioka, kathleen rockland laboratory for cortical organization and systematics, riken brain science institute, saitama, japan in small mammals, gabaergic neurons have been shown to contribute to ipsi-and contralateral cortical projections. here, we report in monkey as well that some gabaergic neurons send long-distance projections. identification was partly based on golgi-like labeling of the dendritic tree, achieved by injecting adenovirus as a retrograde tracer in areas v , teo, or tep. aspiny or sparsely spinous nonpyramidal neurons were clearly visualized in the white matter or, less frequently, in cortical gray matter, in mainly layer but also in layer and/or . in each of the cases, about - gabaergiclike neurons were scored, with a preferential location anterior to the injection sites. in addition to their characteristic dendritic morphology, the neurons were identified as positive for gabaergic neuronal markers; namely, gad , somatostatin, or nos. thus, we conclude that gabaergic projection neurons are phylogenetically conserved; but more work is needed to determine ( ) their other features, ( ) possible species variability, ( ) their functional significance. supported by riken bsi. withdrawn ps a-c regional, cell type, and layer-specific differences in cholinergic modulation of neocortical neurons allan gulledge , , susanna b. park , greg j. stuart , yasuo kawaguchi national institute for physiological sciences, japan; div. neurosci., jcsmr, australian national university, canberra, australia we examined cholinergic modulation of pyramidal and nonpyramidal neurons in neocortical areas (prefrontal, somatosensory, and visual cortex). transient ach exposure ( m) inhibited layer pyramidal neurons in all areas via activation of an sk-type potassium conductance. pyramidal neurons in layers / were generally less responsive to ach, but ach inhibited layer cells in visual cortex. prefrontal layer pyramidal neurons were more responsive to ach than were layer cells in other areas of cortex. fast spiking (fs) nonpyramidal neurons were completely non-responsive to ach, even at very high concentrations ( mm). on the contrary, ach generated fast, nicotinic receptor-mediated responses in % of non-fs interneurons ( of cells). laminar or regional differences in ach responses were not observed in nonpyramidal neurons. these data suggest that ach may act to inhibit the output of cortical projection neurons while preserving information processing in superficial neurons. toshikazu kakizaki , , kenzi saito , , yuchio yanagawa , department of genetic and behavioral neuroscience, gunma university graduate school of medicine, maebashi, japan; sorst, jst, kawaguchi, japan; sokendai, hayama, japan a major inhibitory neurotransmitter gaba is synthesized by glutamate decarboxylase (gad), and is accumulated into synaptic vesicles by vesicular gaba transporter (vgat). another inhibitory neurotransmitter glycine could be transported into synaptic vesicles by vgat, and be co-released with gaba. several molecules related to gabaergic or glycinergic neurotransmission are expressed in nonneural tissues, suggesting that gabaergic and glycinergic systems exert their activities outside the cns. vgat-deficient mice die in the perinatal period, and display omphalocele, defect in ventral body wall closure, suggesting that gaba and/or glycine are involved in body wall formation. to further investigate whether gaba is essential for the ventral body wall formation or not, we have been examining how the body wall developed in the gad -deficient mouse fetus. ps a-c gaba mediated glutamate release from developing cerebellar cortex and ca sensitivity sachiko yoshida, miyuki ohshita, masakazu uematsu, shoichiro hirano, shinya tanaka, naohiro hozumi toyohashi university of technology, toyohashi, japan gaba (␥-amino butyric acid) and glutamate are known to play important roles as modulators in the survival and development of cerebellar neurons. during cerebellar development, gaba-mediated responses, gaba excitations, become depolarized inducing an increase in intracellular calcium concentrations, and are thought to have important trophic effects. many observations of gaba excitations using cultured cells have been reported, whereas few using acute slices. we recently reported the spatial nature of glutamate and gaba releases from acute slice with an enzyme-linked assay system and ccd imaging technology. in the present study, we evolved this measurement system to allow observations of spontaneous or gaba-mediated glutamate release from developing postnatal acute cerebellar slices. glutamate was released spontaneously, but gaba-mediated glutamate release appeared from postnatal to day in egl. its release, especially from premigratory zone, was inhibited by ni + , but cd + couldn't. we suggest that gaba excitation induces granule cell migration. ps a-c gabaergic fiber in the rat trigeminal motor nucleus reorganized following masseter nerve transection hiroyuki hayashi , hiroaki wake , junichi nabekura , osamu takahashi department of histology, kanagawa dental college, yokosuka, japan; national institute of physiological science, okazaki, japan it has been reported that gabaergic nerve terminals are seen in the trigeminal motor nucleus (vm) of the rat, and that there are primary afferent inputs from the muscle spindle of masticatory muscles to the vm cell bodies. we recently found that the number of these gabaergic fibers projecting to vm is markedly reduced in postnatal development. in this study, to elucidate the possibility that the re-arrangement of gabaergic circuits could be reproduced after neuronal injury, we examined the effect of axonal injury of the masseter axon on the gabaergic circuits in the vm. two to eight weeks after unilateral surgical transection of the masseter nerve of rats, gabalike immunoreactive (gaba-ir) varicosities were examined using immunofruorescence technique. the significant increase in number of gaba-ir varicosities were seen after eight weeks of the operation. this result suggest that gabaergic inputs may play one of important role for reorganization of afferent inputs in the vm. akiko arata , kunihiko obata , jonathan davies , mark bellingham , peter g. noakes lab. for memory & learning, riken-bsi, wako, japan; obata res. unit, riken-bsi, wako, japan; sch. biomed. sci., univ. queensland, queensland, , australia during embryonic development, approximately half of the motoneurons (mns) undergo programmed cell death. this process depends also on glycinergic and/or gabaergic synaptic activity, as suggested by increased mn number in gephyrin-deficient mice (banks et al., ) . we investigated the involvement of gaba alone in the mn death using gad -deficient mice, in which cerebral gaba is reduced to less than % of the wild-type. mn numbers at embryonic day (e) were counted by the method of banks et al. brainstemupper spinal cord blocks were prepared from e embryos and subjected to electrical recording from the c and c ventral roots and also gaba measurement. in gad -deficient embryos, increase in number of brachial mns ( %) and decrease in both spontaneous discharges in the c , c roots and gaba content (less than %) were observed, compared with those of the wild-type littermates. gaba might control cell death in developing network. abolghasem esmaeili, joe lynch, pankaj sah queensland brain institute, the university of queensland, australia the amygdala has key role in processing emotional information. distribution of gaba a receptor subunits is crucial for understanding physiology and pharmacology properties of these receptors in the amygdala. we examined the pharmacology of gaba a receptors by expressing different subunit combinations in hek cells and comparing the pharmacology with specific gabaergic inputs in the amygdala. dmcm blocked the actions of gaba at expressed ␣ ␤ ␥ and ␣ ␤ ␥ combinations ( % reduction) but had no effect at ␣ ␤ ␥ or ␣ ␤ ␥ . in slice recordings dmcm blocked ipscs by % in the lateral amygdala and had variable effects in the central amygdala. diazepam and zolpidem enhanced ipscs in the lateral whereas the response in the central amygdala was either reduction or enhancement. real time pcr and western blotting revealed differences in the distribution of gaba a receptor subunits between the lateral and central amygdala. we conclude that in the lateral amygdala all inputs have ␥ subunits whereas in the central amygdala some inputs contain ␥ while others contain ␥ subunits. masayuki kobayashi department of pharmacology, nihon university school of dentistry, tokyo, japan noradrenergic agonists have different effects on the excitatory neural transmission according to their subtypes in rat cerebral cortex. the present study aimed to explore what kind of second messengers and the precise site of synaptic membrane, pre-or postsynaptic, is involved in these noradrenergic modulation. the suppressive effect by activation of ␣ -adrenoceptors was mediated by protein kinase c, and excitatory effect by activation of ␤-adrenoceptors was mediated by camp/protein kinase a cascade. phenylephrine suppressed inward currents evoked by puff application of glutamate, and it decreased mepsc amplitude and increased mipsc frequency. isoproterenol increased mepsc frequency and decreased mipsc amplitude. gaba-induced postsynaptic currents were suppressed by isoproterenol. these results suggest that phenylephrine may decrease postsynaptic currents through glutamate receptors and increase the release probability of gaba from presynaptic terminals. on the other hand, isoproterenol may facilitate glutamate release and suppress gaba a receptor-mediated postsynaptic currents. ps a-d hydrogen sulfide modulates synaptic transmission in rat hippocampal neurons mamiko tsugane , takashi iwai , yasuo nagai , junichiro oka , hideo kimura dept. mol. genetics, nat'l. inst. neurosci., ncnp, tokyo, japan; lab. pharmacol., fac. pharm. sci., tokyo univ. sci., chiba, japan hydrogen sulfide (h s), which is a well-known toxic gas and facilitates the induction of hippocampal long-term potentiation, has been proposed as a neuromodulator in the brain. the aim of this study is to understand the mechanism of regulation on synaptic transmission by h s. we examined the effect of h s on spontaneous excitatory postsynaptic currents (sepsc) as well as paired-pulse facilitations using both whole-cell and field potential recordings from rat hippocampal slices. sodium sulfide (na s), a donor of h s, reduced the amplitude of field excitatory postsynaptic potentials and increased the ratio of paired-pulse facilitation. the frequency and the amplitude of sepsc were initially reduced by na s then gradually increased, while the inward currents elicited by glutamate were not significantly suppressed by na s. these observations suggest that h s may modulate glutamatergic synaptic transmission by suppressing the release of a transmitter. several studies show that activation of locus coeruleus (lc) play an important role in the symptoms of opiate withdrawal. in this study the effects of lc inactivation on self-administration of morphine and on morphine withdrawal syndrome in rats has been investigated. male rats were anaesthetized and implanted with silastic catheters inserted in to the right jugular vein. after days animals were fitted and the external end of the catheter was connected with a syringedriven pump, then were placed in the self-administration apparatus. lc was inactivated by ( l) lidocaein ( %) min before training. animals were allowed to self administer morphine ( mg/kg per inf.) ten consecutive daily -h session. during all morphine self administration session lever pressing was measured. our results show that: ( ) lc inactivation produced a significant decrease in the initiation of morphine self administration during all session. after the last test session morphine withdrawal symptom signs (mws) precipitated by naloxone were measured. ( ) most of mws were decreased by lc inactivation in comparison with morphine group. these results suggest that extracellular atp plays a dual role in astrocytic ca + wave propagation with activation of distinct purinergic receptors in the hippocampus of the rats. the electrophysiological analysis of the rescue effect of ␤ estradiol from glucocorticoid activity yuki oishi , suguru kawato department of physics, graduate school of science, university of tokyo, tokyo, japan; graduate school of arts and sciences, university of tokyo, tokyo, japan it is well known that stress reduces several activity of brain. especially, hippocampus is the largest target of stress. these phenomena are caused by glucocorticoids which are synthesized at adrenal when suffering stress. on the other hand, ␤ estradiol is one of the neuro protective factors and rescues neural death caused by several neurotoxins, such as ␤-amyloid, glutamate, glucocorticoids. in this study, we focused attention on the acute effects of steroid hormones and researched the effects of glucocorticoids and estradiol on rat hippocampal long term potentiation (ltp), which is the index of learning and memory. the results was that corticosterone (glucocorticoid of rat) acutely reduced ltp via glucocorticoid receptor. ␤ estradiol rescued this reduction via estrogen receptor ␣ and ␤. so we found that ␤ estradiol affected not only neuro protection but synaptic protection from stress-induced suppression of synaptic transmission acutely. ps a-d the hypothalamic neuropeptide y neuron system of rats after long-term, high-dose dexamethasone treatment jinko konno, ayuka ina, sachine yoshida, hideki ohmomo, fumihiro shutoh, setsuji hisano lab. neuroendocrinol., graduate sch. comprehensive human sci., univ. tsukuba, ibaraki, japan effects of dexamethasone (dex) on hypothalamic neuropeptide y (npy) expression were evaluated with semi-quantitative in situ hybridization and immunohistochemistry. adult male wistar rats received an injection of dex ( . mg/ g b.w., sc) or sesame oil (vehicle control) everyday for - days. the two and intact rats (intact control) were decapitated, and the hypothalamus was dissected out, fixed and cut into paraffin sections. npy-immunoreactive axonal varicosities in the external zone of the median eminence were apparently more frequent in the dex-treated rat than in controls. npy hybridization signals in the arcuate nucleus were significantly higher in the treated-rat than in controls. no difference was found between both control animals. these results indicate stimulatory effects of dex on hypothalamic npy production and suggest enhanced npy influences on pituitary function. akiko shingo, idumi yamashita, shozo kito lab. of neuroscience, hyogo university, hyogo, japan we examined estrogen-like actions of isoflavones in the cerebral cortex and hippocampus on the basis of our previous data that estradiol induces igf- mrna expression, upregulates estrogen receptors and facilitates ere binding in these brain areas. materials are ovxed and non-ovxed rats. each group of rats were divided into the following groups. a: rats fed with phytoestrogen-free control diet, b: rats fed with diet with soy bean-derived estrogen and c: rats fed with control diet combined with chronic intraperitoneal injections of minimum dose of ␤-estradiol. after feeding, rats were sacrificed to remove the cerebral cortex and hippocampus. expressions of mrnas of igf- , estrogen receptors ␣ and ␤, and ere binding were analysed. as the results, it was revealed that isoflavones induced increased expression of mrnas of igf- and estrogen receptors in both ovxed and non-ovxed rats. difference between estrogen receptor ␣ and ␤ in responses to isoflavones were analysed. isoflavones feeding increased ere binding as much as chronic injections of estrogen did in the ovxed rats. research funds: kampo science foundation, japan ps a-d mechanism of central metabolic control by tgf-beta in the rat brain: using the rat with depletion of hypothalamic noradrenaline teppei fujikawa, kazuo inoue, tohru fushiki division of food science and biotechnology, graduate school of agriculture, university of kyoto, kyoto, japan we have previously reported that activated transforming growth factor-beta (tgf-beta) increase in the rat brain during exercise. intracranial administration of tgf-beta induced an increase in fat oxidation, free fatty acid and keton body in the blood. these results suggest that activated tgf-beta in the rat brain participates in metabolic control of peripheral tissue by cns. it is, however, not known how tgf-beta increases in specifically fat oxidation. many investigations suggest that hypothalamus is essential for central metabolic control. in addition, some reports suggest that noradrenergic system in the hypothalamus may play important role for fat oxidation. in this study we measured concentration of extracellular noradrenaline (na) in the hypothalamus by using microdialysis after injection of tgf-beta. then, we measured respiratory exchange ratio and serum samples, after administration of tgf-beta in the rat with depletion of hypothalamic na by injection of -hydroxydopamine. ps a-d the effect of brain-derived neurotrophic factor (bdnf) on neuropeptide y (npy) neurons in the mouse corpus callosum: an examination using organotypic brain slice culture ryoichi yoshimura, kazuto ito, yasuhisa endo department of applied biology, faculty of textile science, kyoto institute of technology, japan the morphology of neuropeptide y (npy) neurons existing in the corpus callosum (cc) and the effects of brain-derived neurotrophic factor (bdnf) on the npy neurons were examined by using organotypic slice culture system. bdnf treatment significantly increased the number of the npy-immunopositive cell bodies and fibers in cc assessed with immunocytochemistry. electron microscopy demonstrated that the npy immunoreactivities were mainly localized in the regions associated with accumulating synaptic or cored vesicles in cc nerve fibers. the sectional area of npy-positive fibers was larger in the bdnf-treated culture than in the control culture. the number of nerve fibers adjacent to the npy-positive fibers was also larger in the bdnf-treated culture than the control. these results suggest that npy may play a key role in the neuronal regeneration, and bdnf takes part in the development of npy neuron fibers as well as the increase of the number of npy neurons in cc. reiji semba , kimi watanabe , munekazu komada institute for developmental research, aichi human service center, aichi, japan; graduate school of medicine, kyoto university, kyoto, japan d-serine is hypothesized to be a glia-derived neurotransmitter activating the nmda receptor because d-serine was reported to be formed and localized exclusively in astrocytes. however, we reported strong immunoreactivity of d-serine in some axons. to reveal which cells are producing d-serine in the brain, an in situ hybridization study of serine racemase, the enzyme producing d-serine from l-serine, was performed. using antibodies against neun, a neuronal marker, gfap, an astrocyte marker, and cnpase, an oligodendrocyte marker, type of the cells containing the mrna was examined. coincidentally with our immunohistochemical study of d-serine, strong signals for serine racemase mrna were found in some neurons while weak signals were found in astrocytes. present results suggest that d-serine will be a neurotransmitter activating the nmda receptors produced in a specific type of neurons. takatoshi hikida , , asif k mustafa , kenji hashimoto , kumiko fujii , , kazuhisa maeda , , hiroshi ujike , richard l. huganir , solomon h. snyder , akira sawa dept. of systems biology, obi, suita, japan; depts of neurosci. & psychiat, johns hopkins univ. med., baltimore, maryland, usa; chiba univ. forensic mental health, chiba, japan; dept. of psychiat, shiga univ. med. sci., shiga, japan; div. of neuropsychiat, tottori univ., yonago, japan; dept. of neuropsychiat, okayama univ., okayama, japan accumulating evidence from both genetic and clinical studies suggests a critical role of d-serine in schizophrenia (sz). we identified and characterized pick as a protein interactor of the d-serine synthesizing enzyme, serine racemase (sr). d-serine levels in the hippocampus and frontal cortex of pick knockout mice were significantly lower than those of their wildtype littermates at age of p , but not in adults, suggesting regulation of pick on sr at developing stage. in case-control association study, we observed an association of the pick gene with sz, which is more prominent in disorganized sz. our findings suggest that pick contributes to sr activity, d-serine production, and nmda neurotransmission in the pathophysiology of sz. ps a-d epileptiform activity is inhibited by taurine which can activate glycine and gaba a receptors in immature rat hippocampus akihito okabe , , werner kilb , ileana l. hanganu , taizhe qian , daiichiro nakahara , atsuo fukuda , heiko j. luhmann dept. of physiol., hamamatsu, japan; inst. of physiol., mainz, germany; dept. of psychol., hamamatsu, japan many studies indicate that the underlying mechanism of epileptic seizures differ between children and adults. the depolarizing gabaergic responses in immature neurons may contribute to higher epilepsy susceptibility. to investigate whether taurine, a neurotransmitter found in high concentrations in the immature cns, modulates epileptiform activity in immature hippocampus, we performed field-potential recordings in neonatal rat hippocampal ca region of an intact preparation. mm taurine blocked epileptiform activity induced by mg + free acsf and m -ap. this taurine effect was prevented by the glycinergic antagonist strychnine and the gaba a antagonist gabazine. inhibition of taurine uptake by ges also suppressed epileptiform activity in strychnine and gabazine sensitive manner. these results suggest that taurine mediates an inhibition in immature hippocampus via glycine and gaba a receptors that suppresses epileptiform activity. ps a-d responses of pge in undifferentiated and differentiated ng - cells kayoko matsushima , takashi imanishi , akinori kawaguchi , tetsuyuki wada , shigeru yoshida , seiji ichida school of pharm. sci., kinki univ., osaka, japan; school of pharm. sci., kinki univ., osaka, japan; school of pharm. sci., kinki univ., osaka, japan; school of pharm. sci., kinki univ., osaka, japan; school of sci. & eng., kinki univ., osaka, japan; school of pharm. sci., kinki univ., osaka, japan our previous findings showed that -ht-and bk-induced [ca + ] i increases were enlarged in differentiated ng - cells. for the next stage, we investigated the effect of pge , an inflammatory mediator for -ht and bk, on the cells. ng - cells were loaded with fura- /am, and the change in [ca + ] i was monitored by an image processor. the results showed: ( ) pge -induced response was decreased when ng - cells were differentiated by bt camp, ( ) − m ah and sc irreversibly inhibited pge -induced response by about % and %, respectively, while − m ah and sulprostone had no effect, and ( ) pge -induced response was abolished under ca +free conditions in about % of both ng - cells. these results indicate that the response to pge , via ep and ep receptors, significantly decreased during differentiation. mitsumasa murano, fumihito saitow, hidenori suzuki department of pharmacology, nippon medical school, tokyo, japan the most of cerebellar outputs are generated as a result of synaptic interaction in the deep cerebellar nuclei (dcn) and by the electrical membrane properties of dcn neurons themselves. this study aimed at examining mechanisms underlying the serotonergic modulations of both the gabaergic transmission at the purkinje-to-nuclear cell synapses and the membrane properties of dcn neurons using cerebellar slices prepared from -to -day-old rats. bath application of serotonin ( -ht) decreased the amplitude of stimulation-evoked ipscs in dcn neurons in a dose-dependent manner. furthermore, slow inward currents ware observed in dcn neurons during -ht application. under the current-clamp recording, -ht markedly depolarized and increased action potential discharges of dcn neurons. taken together, these results suggest that -ht facilitates the voluntary activity in dcn neurons by both pre-and post-synaptic mechanisms. ps a-d searching for endogenous ligands of trace amine receptors in mammals ( ) akira komatsu , airi yamaguchi , noriko makikusa , osamu koizumi dept. physiol., tokyo women's med. univ., sch. med., tokyo, japan; neurosci. lab., fukuoka women's univ. fukuoka, japan trace amine receptors were discovered in mammals, but their endogenous ligands have not yet been found. to search for them, we developed a new method to make antibodies against monoamines for immunohistochemistry (ihc). monoamines, phenylethylamine (pea), tyramine (ta) and histamine (ha), were conjugated to a hemocyanine, klh, using an imidoester cross-linker, dimethyl suberimidate (dms). rabbits were immunized by the conjugated macromolecule. the obtained antibodies were assayed by elisa and competitive elisa technique to check their antibody titer and specificity respectively. the antibodies recognized specifically the monoamine-dms part within the complex. for ihc, the rat brain was perfused by % dms, post-fixed by % formaldehyde and then frozen-sectioned. the antibody against ha revealed the immunoreactive neurons in the hypothalamus, showing that this method is effective to demonstrate the presence and localization of monoamines. the antibodies against pea and ta failed to reveal immunoreactive neurons in the rat brain. ps a-d effects of mg + on neural activity of cultured cortical neurons of the rat and mouse yuriko furukawa , , nahoko kasai , akiyoshi shimada , keiichi torimitsu , , kunihiko obata , yuchio yanagawa , , tadaharu tsumoto , ntt basic research laboratories, kanagawa, japan; sorst/jst, saitama, japan; neuronal circuit mechanisms research group, brain science institute, riken, saitama, japan; dept. of genetic and behavioral neurosci., grad. sch. of med., gunma university, gunma, japan it is well known that mg + plays an important role not only in energy metabolism, but also in neural information processing. however, the mechanism of such a role in cns is not well understood. previously we reported that neural activity and the intracellular ca + concentration are largely affected by mg + removal in cultured cortical neurons of the rat. transient glutamate release was also detected. in the present study, we investigated effects of the mg + removal on neural activity in cultured cortical and hippocampal neurons. in particular, we measured the intra-and extracellular mg + concentration and their actions on neural activity using a mg + indicator, kmg- -am together with fluo -am. we observed different effects of the mg + removal on gabaergic and non-gabaergic neurons by using gad -gfp knock-in mice. research funds: jst/sorst ps a-e transient zinc-positive terminations in the developing rat somatosensory cortical system noritaka ichinohe, daniel potapov, kathleen s. rockland lab. for cortical organization and systematics, bsi, riken, usa synaptic zinc (zn) is a neuromodulator used by a subset of nonthalamic glutamatergic connections, and associated with both experiencedependent and developmental plasticity. during development, transiently high levels of synaptic zn occur in both sensory and nonsensory cortical areas. by injecting the retrograde tracer sodium selenite into barrel cortex, we demonstrated a transient subset of zn + thalamocortical neurons from p -p . zn + cortical neurons were also labeled, intrinsic and extrinsic, from p . unlike in the adult, these were in layer , instead of layers , , and . at p , neurons occurred in layers , , and and, in some areas, layer . at p , zn + neurons first appeared in layer ; and at p , there is the adult lamination. as whisking and exploratory behavior commences in the second postnatal week, these transient zn + terminations may play a role in experience-dependent adjustments in cortical circuitry. research funds: bsi, riken and kakenhi no. ps a-e systematic comparison of the structure of the serotonin immunoreactive neurons between insect species masaaki iwano , , ryohei kanzaki , kei ito , center for bioinform., imcb, univ. of tokyo, tokyo, japan; dept. of mechano-inform., grad. sch. of inform, sci. and tech., univ. of tokyo, tokyo, japan; bird, jst, saitama, japan in the vertebrate central nervous system, the distribution of the serotonin immunoreactive neurons (sirns) is known to be preserved remarkably during evolution. systematic comparison of the invertebrate sirns has not been performed, on the other hand. in the current study we analyzed the morphology of the sirns in the brains of holoand hemi-metabolous insects including flies, bees, moths, beetles, crickets, dragonflies and cicadas. in spite of the large variation in the size and cell numbers of the brain, the number and distribution of the sirns were highly consistent between species. for example, we observed either one or two pairs of bilateral sirns with similar morphology that connect specific subregions of the lateral accessory lobe, a candidate pattern generator of the zigzag locomotion of the insect. variation was greater in the antennal lobe, the insect primary olfactory center, where sirns project either ipsil-or contra-laterally depending on the species. maki kagohashi , , taizo nakazato , shigeru kitazawa neurol, juntendo univ., tokyo, japan; physiol, juntendo univ., tokyo, japan in vivo voltammetry has been used for measuring neurotransmitter releases in the brain of behaving rats (e.g. nakazato, ) . however, task freedom was restricted by cables connecting the head and the measurement system. to overcome the difficulty we developed a wireless voltammetry system and examined its sensitivity in vitro (kagohashi et al., jns ). the system consisted of a wireless transmitter with a potentiostat and a signal receiver. in the present study, we reduced the size and weight and measured dopamine (da) currents in vivo with the wireless system mounted on the back of the rat. a single-step voltage pulse ( to mv for da; to mv for ht) was applied at hz through a carbon electrode that was chronically implanted in the striatum. after administration of l-dopa, da currents showed a gradual increase in good agreement with the data measured with conventional systems. the present wireless system would be applicable to measurement of neurotransmitters in various situations (e.g. social interaction). research funds: scientific research on priority areas (mobiligence) hiroyuki yamazaki, tomoaki shirao department of neurobiology and behavior, gunma university graduate school of medicine, maebashi, japan dendritic spines are multiple functional units that receive most of excitatory inputs in central nervous system. in the purpose of finding a novel molecule that is involved in regulation of dendritic spines, we have done a screening of a novel drebrin binding protein. yeast twohybrid system was conducted with drebrin as bait, and a novel drebrin binding protein was isolated. in neurons, this protein was localized primarily in nucleus and dendritic spines. hence, we named it spikar for its unique intracellular localization in spine and karyoplasm. we studied the role of spikar in spine formation. hippocampal neurons were transfected with shrna expression vector for spikar at several developmental stages. in early stage, spikar knock down (kd) did not affect the density of dendritic protrusions that were mostly filopodia. in contrast, spikar kd reduced spine density at the stage of synapse formation. these results suggest that spikar plays a role in the formation of dendritic spines, without affecting the filopodia formation. ps a-e time-lapse analysis of the translocation of drebrin-actin complex from dendritic spines to dendritic shafts by glutamate stimulation toshiyuki mizui , , yuko sekino , , tomoaki sirao dept. of neurobiol. & behav., gunma univ. grad. sch. of med., maebashi, japan; div. of neural network, inst. med. sci. univ. of tokyo, tokyo; crest, jst, kawaguchi, japan; jsps, japan we have shown that nmda receptor activation induced translocation of drebrin, with retaining its binding to f-actin, from dendritic spines to their parent dendrites. in the present study, we analyzed the time course of gfp-tagged drebrin a (gfp-da) dynamics after glutamate receptor activation. we prepared primary hippocampal cultured neurons, transfected them with gfp-drebrin a expression vector using microinjection methods at days in vitro (div), and analyzed the dynamic localization of gfp-da at div. glutamate stimulation started gfp-da translocating within s and completed in min. after washout of glutamate, gfp-da gradually re-accumulated in the spine, and the fluorescence intensity of gfp-da is fully recovered in min. these data suggest that translocation mechanism of drebrin from spines to shafts is different from that from shafts to spines. research funds: grant-in-aid for jsps fellows ps a-e distribution of the srf co-activator mal in developing mouse brain mitsuru ishikawa , jun shiota , hiroyuki tsutsumishita , hiroyuki sakagami , masaaki tsuda , akiko tabuchi dept. biol. chem., fac. pharm. sci., univ. toyama, toyama, japan; dept. cell biol., tohoku univ., grad. sch. medicine, sendai, japan the srf co-activator mal (megakaryocytic acute leukemia) plays an important role in controlling srf-dependent gene, whose expression is regulated by rearrangement of actin cytoskeleton. recent studies with conditional deletion of srf gene demonstrated that srf was required for inducing genes such as egr- , c-fos,␤-actin but also for neuronal migration and plasticity. in this study, we investigated the expression of mal in developing mouse brain and the role of mal for dendritic morphology. the in situ hybridization analysis revealed that mal mrna was highly and developmentally expressed in hippocampus and broadly expressed in cortex, olfactory bulb. staining of mal displayed cytoplasmic localization at cell bodies and apical dendrites. furthermore, dominant negative mal mutants and rnai led to a reduction of dendritic number, as well as a decrease of srf transcription. these findings indicate that mal is involved in the formation or the stability of dendrites. research funds: kakenhi ( ) to a.t. shoko shimizu , shinsuke matsuzaki , tsuyoshi hattori , ko miyoshi , masaya tohyama department of anatomy and neuroscience, graduate school of medicine, osaka university, japan; department of brain science, graduate school of medicine and dentistry, okayama university, japan disrupted-in-schizophrenia (disc ) was identified as a novel gene disrupted by a ( ; ) (q . ;q . ) translocation segregating with schizophrenia and affective disorders in a scottish family. kendrin was identified as a protein which interacts with disc at centrosome and residues - of disc (kendrin-binding region: kbr) were essential for the interaction with kendrin. in this study, we show that c-terminal of disc downstream of kbr is indispensable structure for kbr to interact with kendrin and also essential for disc to target to the centrosome. furthermore, we have shown that inhibition of the disc -kendrin interaction perturbs the tubulin network formation. these results suggest that the c-terminal region of the disc is important to the disc -kendrin interaction and that a truncated form of disc lacking the c-terminal downstream of the translocation breakpoint might affect the microtubule organization. tatsuro kumada, yasuhiko nakanishi, atsushi fukuda department of physiology, hamamatsu university school of medicine migratory cells exhibit dynamic morphological changes in the cell soma and process in both normal developmental program and tumor growth. the morphological changes in the cells are correlated with the rate of cell migration and ion transfer such as ca + or cl − . although the highly invasive migration of glioblastoma in the brain is known to be influenced by a variety of ion channels, there were a little evidence about the relationships among the morphological changes and ion homeostasis. to clarify it, we have developed a glioma cell culture system for the simultaneous observation of the cell movement and ca + and cl − imaging. we found that the relatively low density a glioma cells actively moved on the substrate. the movement has the correlation with intracellular ca + oscillation in the cells. the relationship between cell movement and intracellular ion levels is further studied. ps a-e involvement of ca + influx in the unpolarized non-vesicular release of fgf- hayato matsunaga, hiroshi ueda division of molecular pharmacology and neuroscience, nagasaki university graduate school of biomedical sciences, nagasaki, japan little is known of molecular basis mechanisms for the er-golgiindependent or non-vesicular release of fgf- lacking a conventional signal peptide sequence. we found that fgf- is co-released with s a , a ca + binding protein from cultured rat astrocytes upon the serum-deprivation stress. here, we report that fgf- is co-released with s a from the axon and dendrites in cultured rat hippocampal neurons upon depolarization stimulation, but serum-deprivation stress leads to release, which is seen in neurites as well as in soma. the interaction between fgf- and s a required ca + . the overexpression of s a - mutant lacking an ability of interaction with fgf- inhibited the their release, suggesting that s a is a cargo molecule. the release of fgf- upon either stimulation was abolished by voltage-dependent n-type ca + channel blocker. these findings suggest that ca + influx may be involved in the unpolarized non-vesicular release of fgf- . in neuron, intracellular calcium involves a large number of physiological phenomena, including cell migration, differentiation, and neurite outgrowth. pc cell is a useful model of neural differentiation and neurite outgrowth, and recently we demonstrated that -ht has an effect on neurite outgrowth via the increase in intracellular calcium concentration ([ca + ] i ) in pc cells. however, it is unclear how [ca + ] i regulates neurite outgrowth via actin cytoskeleton. in this study, we investigated effects of [ca + ] i on actin dynamics in pc cells transfected with yfp-actin. filopodial growth speed and actin retrograde flow were increased by treatment with calcium ionophore, a . treatment with calcineurin inhibitors decreased the filopodial growth speed, while treatment with camk inhibitor did not. these effects could contribute to -ht induced enhancement of neurite elongation. the actin cytoskeleton is a complex protein network that not only provides cellular structure but is fundamental for cellular dynamics. on stimulation of pc cells by ngf, proteins that directly interact with f-actin such as actinin rapidly translocate to the f-actin-rich cytoskeleton. clp is a pdz-lim protein which was originally identified as an actinin-interacting protein in skeletal muscles. here, we show that clp is endogenously expressed in pc cells and plays an important role in actin dynamics during ngf-induced neurite outgrowth. immunofluorescent studies showed that clp is accumulated in irregular cell surface and membrane extrusion soon after ngf-stimulation, where colocalized with actin filaments. we next performed rnai experiments to explore the role of clp in actin dynamics in growth cones and found that knockdown of clp expression lead to the suppression of ngf-mediated neurite outgrowth. in addition, we revealed using clp deletion mutants that both of pdz and lim domains are necessary for the proper function of clp . ps a-e screening of genes expressed preferentially in migrating gabaergic neurons of developing cerebral cortex toshiya kimura , tsuyoshi kobayashi , yuchio yanagawa , kunihiko obata , fujio murakami , grad. sch. of frontier biosci., osaka univ., osaka, japan; grad. sch. of medicine, gunma univ., maebashi, japan; bsi, riken, wako, japan; sorst, jst, japan neuronal migration plays a critical role in constructing brain architecture organization. however, molecular mechanisms underlying this process still remain elusive. in an attempt to identify molecules that regulate the motility of migrating neurons, we focused on migrating cortical interneurons, and performed subtractive hybridization, differential screening and in situ hybridization. subtraction was done between the embryonic and postnatal interneurons, because they robustly migrate prenatally but not postnatally. among the clones tested, two genes, neuronatin and seizure related gene (sez- ) attracted our attention. they were expressed in the subventricular zone of the embryonic cortex, implicating that these molecules are expressed in interneuron subpopulations. postnatally, mrna signals were hardly detectable. these results raise the possibility that they are expressed preferentially in subpopulations migrating cortical interneurons. yan zhu , , tomoko matsumoto , , sakae mikami , takashi nagasawa , fujio murakami , grad. sch. of frontier biosci., osaka univ., japan; sorst, jst, japan; inst for frontier med. sci., kyoto univ., japan long distance neuronal migration takes place typically along the tangential plane of the developing neural tube. the migratory behaviour and the underlying molecular mechanisms of tangential migration are poorly understood. we address these issues using the hindbrain precerebellar system as model system. precerebellar neurons, born dorsally in the lower rhombic lip, migrate in close association with the pial membrane (except inferior olive neurons) ventrally or rostroventrally. we therefore studied the role of pia-secreted chemokine sdf- and its receptor cxcr in the precerebellar migration. we show that cxcr is expressed in the migrating precerebellar neurons, and its expression is down-regulated towards the end of migration. in cxcr and sdf- knock out mice, migrating precerebellar neurons are less confined to the pial surface. more strikingly, the rostrally-directed migration of pontine precerebellar neurons is severely disrupted, leading to a caudalized ectopic pontine-like cluster. ps a-e involvement of an immunoglobulin superfamily molecule, neph /mkirre in the migration of precerebellar neurons kazuhiko nishida , , kazuhide nakayama , saori yoshimura , , fujio murakami , grad. sch. of frontier biosci., osaka univ., osaka, japan; sorst, jst, saitama, japan neural cell migration plays a crucial role in central nervous system development. in this study, we analyze the involvement of neph family transmembrane proteins of the immunoglobulin superfamily in the migration of precerebellar neurons (pcns). postmitotic pcns derived from the rhombic lip in the hindbrain first migrate tangentially along the pial surface, followed by radial migration to settle at their final positions (kawauchi, d., taniguchi, h., watanabe, h., saito, t., and murakami, f., development, in press ). in situ hybridization analysis showed that among neph family members including neph , neph /mkirre, and neph , only neph /mkirre was strongly expressed in pcns. expression of neph /mkirre was detected from e . when pcns migrate tangentially. the expression level became weaker at p , when pcns stop the radial migration, raising the possibility that neph /mkirre might be involved in the migration of pcns. we are currently analyzing the function of neph /mkirre in the migration of pcns. hiroki umeshima , , toshio ohshima , tomoo hirano , mineko kengaku lab. for neural cell polarity, riken bsi, wako, japan; department of biophysics, kyoto university, kyoto, japan; lab. for developmental neurobiology, riken, bsi, wako, japan during lamination of the cerebellar cortex, granule cells exit their final mitiosis at the external granular layer and migrate to the internal granular layer. we analyzed the molecular mechanisms regulating migration of granule cells. using an in vivo electroporation system followed by time-lapse confocal microscopy of a slice culture, we found a dominant negative form of cdk (cdk -dn) disrupted the morphology of granule cells during radial migration. recently, centrosome positioning is thought to be one of the important factors for neuronal migration. double-labeling of the centrosome and the whole-cell images by transfecting centrin -gfp and rfp enabled us to record dynamic movement of the centrosome during radial migration. we found that the motion kinetics of the centrosome was disrupted by cdk -dn. based on these results, we will discuss the role of centrosome during neuronal migration. keisuke ito , , takahiko kawasaki , , tatsumi hirata , division of brain function, national institute of genetics, mishima, shizuoka, japan; department of genetics, school of bioscience, sokendai newly generated neurons migrate through proper pathways toward their own targets, where they are integrated into specific neuronal circuits. we have analyzed a unique tangential migratory stream of early-generated cortical neurons designated as lot cells, and performed pharmacological perturbations to characterize the intracellular mechanism of the migration. among various drugs, we found that a protein kinase inhibitor, k a has the most interesting effect on the lot cell migration. during the normal migration, leading processes and cell bodies of lot cells move forward in a coordinated manner, but k a blocks the migratory movement of cell bodies without inhibiting the extension of leading processes. we also found that k a has a similar effect on cerebellar granule cells. these phenomena are quite intriguing because the drug seemed to switch the neurons from "whole cell migration" to "neurite extension" mode. we are now analyzing possible targets of k a, aiming for dissection of these phenomena. the conserved ser/thr kinase unc functions with unc- to regulate axonal transport in drosophila hiroaki mochizuki , hirofumi toda , , emiko suzuki , joseph gindhart , toshifumi tomoda , katsuo furukubo-tokunaga grad. school life and envir. sci., univ. tsukuba, tsukuba, japan; gene net. lab., natl. inst. genet. mishima; beckman res. inst., city of hope, ca, usa; dep. biol., univ. richmond, va, usa neural network develops through regulated guidance of axons and interconnection among them. despite intensive researches in the past years, genetic mechanisms of axonal development still remain unclear. we have identified the drosophila homolog of unc , which encodes a ser/thr kinase and is required for axonal formation in c. elegans and mouse. we found that unc is essential for neural development in drosophila. loss of function of drosophila unc results in reduced locomotion and axonal transport defects reminiscent of the phenotypes observed in kinesin mutants. we also found that unc genetically interacts with unc- , an evolutionarily conserved cytoplasmic protein that binds to kinesin heavy chain. in unc mutants, unc- was separated from synaptotagmin vesicles. these results suggest that unc coordinates kinesin-cargo interaction via unc- to regulate dynamic axonal transport. ps a-e change in microtubule polarity during the conversion of dendrites into axons kensuke hayashi , daisuke takahashi life science inst. sophia university, tokyo, japan; waseda university, tokyo, japan axons and dendrites of neurons differ in the polarity of their microtubules. the mechanism for the difference, however, is not well understood. we found previously that dendrites convert into axons in cultured neurons isolated from rat cerebral cortex. in this study, we examined whether microtubule polarity changes during the conversion. in dendrites of neurons before culture, microtubule polarity was nonuniform. after h of culture, we found that most of microtubules in the original dendrites had their plus ends oriented distal. this indicates that microtubules with their minus-ends distal disappeared during the culture. microtubule movement along actin filaments is a candidate for this mechanism among several types of microtubule movement reported in neuronal processes so far. however, the change of microtubule polarity within dendrites was observed even in the presence of actin polymerization inhibitors. our results suggest a rearrangement of microtubules by a yet-unreported movement in neuronal processes. research funds: kakenhi ( ) and kakenhi on priority areas ( ) ps a-e generation and analysis of region-specific rac -deficient mice hidetoshi kassai , masahiro fukaya , eriko miura , mizuho sakahara , masahiko watanabe , , atsu aiba div. cell biol., kobe univ. grad. sch. med., kobe, japan; div. physiol. sci., hokkaido univ. grad. sch. med., japan rac is a member of the rho family of small gtpases, and assumed to be involved in regulation of neuronal development through actin cytoskeletal reorganization. nevertheless, physiological role of rac in the cns is poorly understood because of the embryonic lethality of rac knockout mice. in this study, we generated and analyzed region-specific rac -deficient mice (emx -rac ko mice) by the cre-loxp system, in which a promoter for emx homeobox gene induces expression of cre recombinase exclusively in the dorsal telencephalon, including cerebral cortex, hippocampus and olfactory bulb. emx -rac ko mice showed partially abnormal layering of cerebral cortex, indicating impaired migration of neuronal cells during cortical development. furthermore, emx -rac ko mice lacked corpus callosum and anterior commissure, both of which connect the left and right cerebral hemispheres. these results suggest that rac regulates neuronal cell migration and axonal growth in cerebral cortex. previously we reported overexpression of map b containing nterminal amino acids promoted neuronal death. to reveal the mechanism of map b n-terminal induced neuronal death, we searched for the proteins that interact with n-terminal of map b by two-hybrid system. alpha-tubulin was found to interact with map b n-terminal and their in vitro interaction was proved with pull-down assay. the interaction of tubulin and map b n-terminal has not yet been reported. beta-tubulin was also found to interact with map b n-terminal. when ␤ tubulin was divided in fragment at between amino acid and , there was no interaction between ␤ tubulin fragments and map b n-terminal. interaction needs the continuous region over aa and . there were much proportion of round formed cos cells in n-terminal containing map b transfected cells than in n-terminal lacking map b transfected cells. there might be some interference in interaction between map b and tubulin in cells express map b containing n-terminal. otone endo , , masaaki mizuno , yasukazu kajita , jun yoshida department of neurosurgery, ja kainan hospital, aichi, japan; department of neurosurgery, nagoya university, nagoya, japan; department of molecular neurosurgery, nagoya university, nagoya, japan primate es cells have rather different character from rodent ones, but it is inevitable to elucidate mechanism for stable culture, purification and induction into object-oriented differentiation, because human es cells might show wide similarity to cynomolgus ones. we refined the way of large scale culture maintaining totipotency without contacting feeder cells indispensable for primate es cells. our super selective induction method for dopaminergic neurons is also refined, and induced neurons transplanted in vivo which survive without forming tumor such as teratoma for long period, are evaluated not only immunohistologically but eletrophysiologically and ethologically suggesting its enough stability, activity, ability to make neural network system and potentiality to improve clinical symptom of parkinsonism. differentiation of other types of neurons and development of fully functional neural network must be established. shigeki ohta , masae yaguchi , yumi matsuzaki , yoshiaki toyama , yutaka kawakami , hideyuki okano , masahiro toda , neuroimmunology research group, keio univ., tokyo, japan; physiology, keio univ., tokyo, japan; orthopaedic surgery, keio univ., tokyo, japan; institute for advanced medical research, keio univ., tokyo, japan; neurosurgery, keio univ., tokyo, japan we have shown that mouse dendritic cells (dcs) have the ability to induce the proliferation and survival of neural stem cells/progenitor cells (nspcs) in vitro. implantation of dcs into injured mouse spinal cord could improve the motor function through activation of endogenous nspcs in vivo. in this study, to identify an effective dc subtype for the treatment of spinal cord injury (sci), we analyzed the effects of different mouse dc subtypes on the proliferation of nspcs in vitro. among mouse splenic cd c + dcs, cd ␣ + dcs increased the number of neurospheres most effectively in vitro. furthermore, a significant functional recovery after mouse sci was induced by implantation of cd ␣ + dcs compared to cd c + dcs. these results suggest that cd ␣ + dcs can be an effective subtype of mouse dcs for the treatment of sci. ps a-e von hippel-lindau protein regulate the neurogenesis in skin-derived precursor cells atsuhiko kubo , hiroshi kanno , takaakira yokoyama , shuichi nakano , naoki sugimoto , nahoko kobayashi , tetsuhiko yoshida , isao yamamoto dept. of neurosurgery, yokohama city university graduate school of medicine, yokohama, japan; fiber and dept. of chemistry, konan university, kobe, japan; toagosei co., ltd. corporate research laboratory, nagoya, japan skin-derived precursors (skps), multipotent somatic stem cells, are preferred cell source for autologus cns cell replacement therapy. they are proliferated by the mitogens of egf and bfgf. to investigate the effects of von hippel-lidau (vhl) protein in the neural cell fate commitment, skps were inoculated with hsv vector expressing vhl protein. skps showed promotion of neurogenesis and inhibition of gliogenesis. to detect the intrinsic factors that control lineage commitment, vhl peptides fused with the protein transduction domain (ptd) were synthesized. the ptd-vhl peptides showed rapid cell internalization in nearly %, and peptide with the elongin c binding site (residues - ) showed a high ability of inducing neuronal differentiation by interacting with jak/stat pathway. these findings are important in its application to the cns cell grafting. ps a-e the effect of pueraria mirifica on erk / and s- following sciatic nerve injury in rats pornpen chaiworakul, supin chompoopong department of anatomy, mahidol university, bangkok, thailand to investigate the effects of pueraria mirifica (pm) compared with genistein (g) and estrogen (e ) on the expression of erk / and s- following sciatic nerve crush and transection in rats. protein levels of perk / and s- in distal segments of nerve at day were determined by western blot analysis. it was demonstrated that pm and g treatments, similar to e , caused a significant decrease in the expression of perk / levels in both nerve crush and transection injuries. however, transected nerves showed high and sustained levels of erk / phosphorylation. following treatments, levels of s- were significantly decreased both in crushed and transected nerves with respect to control group at p < . . this estrogenic effect was blocked by ici , . because of their structural similarity to e , pm may have therapeutic potential in nerve injuries which as previously reported to enhance sfi following sciatic nerve crush in rats after day . this study suggested that pm as well as g could enhance nerve regeneration like e by interfering with the injury-induced erk signaling pathway. yasuhiro kato , takafumi suzuki , kunihiko mabuchi department of advanced interdisciplinary studies, graduate school of engineering, the university of tokyo, japan; department of information physics and computing, graduate school of information science and technology, the university of tokyo, japan mems technologies have been established to fabricate a multichannel neural probe for interfacing with the nervous system. there is, however, no suitable probe for long-term neural recording and stimulation. one main reason is the death of brain tissues damaged by the probe insertion and implantation. thus, a new skeleton-like multichannel flexible neural probe coated with hybrid biodegradable polymer was fabricated. the skeleton-like probe was designed to minimize the volume of the flexible probe and buffer injurious micromotion between the probe and the tissues in a post-implantation. the probe was coated with mixed polyethylene glycol and microspheres with nerve growth factor (ngf) to improve the stiffness for the probe insertion, and deliver ngf for an optimal period to promote regrowth of damaged neural tissues around the probe. damage-induced neuronal endopeptidase (dine) is a newly identified nerve regeneration-associated molecule. it encodes neuronspecific membrane-spanning metalloprotease and belongs to nep/ece family which degrades/processes neuropeptides. although the precise mechanism of dine including substrate is still unclear, dine seems to play a protective role in damaged neurons. the most marked property of dine is a striking response to various kinds of nerve injury in both central nervous system and peripheral nervous system. to clarify the transcriptional regulation of dine after nerve injury, we analyzed untranslated region of dine gene. previously, we found that lif treatment and ngf deprivation additively increased dine mrna. in this study, promoter analysis showed that dine promoter activity was cooperatively up-regulated by atf- and stat , which were induced after nerve injury and activated at the downstream of lif treatment and ngf deprivation. this combination of transcription factors may be pivotal to promote gene expression, which is responsible for nerve regeneration. tomohiro miyashita, takekazu kubo, masashi fujitani, katsuhiko hata, toshihide yamashita department of neurobiology, graduate school of medicine, chiba university, chiba, japan wnt proteins are known as those concerning with formation of central nervous system. we tested whether they play a role in inhibition of axon regeneration after spinal cord injury. cerebral granule neurons from p - wistar rats were cultured. wnt proteins were added into the culture medium. twenty-four hours after culture, neurite length of each neuron was measured. immunohistochemistry was done employing anti-wnts antibody and anti-ryk (wnt receptor) antibody. anti-ryk antibody was injected continuously for two weeks into the subarachnoid space of contused rat spinal cord. locomotor behaviour was evaluated up to six weeks after injury. immunohistochemistry showed that several wnt proteins and ryk were upregulated after spinal cord injury. wnt proteins inhibited neurite outgrowth of cultured cerebral granule neurons. and this effect was abolished by y , a rho-kinase inhibitor, and anti-ryk antibody. suppression of wnt proteins may promote axon regeneration and improve locomotor behaviour after spinal cord injury. akihito takeda, richard goris, kengo funakoshi department of neuroanatomy, yokohama city university graduate school of medicine, yokohama, japan in contrast to mammals, spontaneous nerve regeneration after lesion of the spinal cord occurs in fishes. we examined tissue remodeling and axon regeneration after spinal hemisection in the goldfish. in the lesioned spinal cord, neurogenesis reached the maximum level days after the hemisection. glial cells positive for glial fibrillary acid protein (gfap) temporarily increased at the lesion site one day after. many gfap positive cells expressed somatostatin. serotonin ( ht) positive cells increased in number progressively from day to weeks after. six weeks after, the regenerated axons with glial fibers invaded fibrotic scar centered about the lesion site, and ht cells surrounded the axons and glia. thus, ht may promote these neural elements to invade the fibrotic scar. six weeks after the hemisection, projections from locomotion center in midbrain to spinal motoneurons were restored, and swimming ability was also recovered. these results suggest that the goldfish have ability to reestablish correct projections after the spinal injury. masao koda , yukio someya , ryo kadota , chikato mannoji , tomohiro miyashita , atsushi murata , masashi yamazaki department of orthopaedic surgery, togane hospital, department of ortopaedic surgery, graduate school of medicine, chiba university, japan; division of rehabilitation medicine, chiba university hospital, japan objective: anoikis is a type of apoptosis due to the detatchment from the extracellular matrix. preparation of graft cells for cell therapy includes dissociation of cultured cells, which may cause anoikis. here we tested the effect of bdnf for anoikis of schwann cell. methods: (in vitro) schwann cells were cultured from sciatic nerves of neonatal rats. schwann cells were transferred to suspension culture. bdnf was added into the culture medium. cell death was detected h after suspension culture. (in vivo) schwann cells were transplanted with or without bdnf treatment into contused rat spinal cord. immunohistochemistry was performed to detect survival of grafted cells. the olfactory bulb and its caudal extension are unique forebrain regions with the residence of neural stem cells and the ability of persistent neurogenesis. however, evidence for active functional involvement of neural stem cells is still very limited. this study was undertaken to know whether or not newly generated neurons are integrated in olfactory neuronal circuits in the neonatally bulbectomized rats that had been proved to show olfactory discriminative abilities. for this purpose, retroviral vector, a very useful tool to trace neural stem cells, was applied to the anterior part of the subventricular zone of the rats of which olfactory bulbs had been unilaterally ablated at the neonatal stage. we will show cell dynamics of newly generated neurons in the neonatally bulbectomized olfactory nervous system, with special reference to their neuronal circuits. koichi kawada, masanori yonayama, kiyokazu ogita dept. pharmacol., setsunan univ., osaka the subventricular zone (svz) contains undifferentiated cells, which proliferate and generate the olfactory bulb (ob) interneurons. throughout life, these cells leave the svz and migrate to the ob via the rostal migratory stream, where they differentiate. we have shown that trimethyltin (tmt) causes neuronal damage in the hippocampal dentate gyrus. in this study, we examined neuronal degeneration and regeneration in the ob after tmt treatment in mice. ddy mice were given tmt ( . mg/kg) to prepare slices for an immunohistochemical analysis using antibodies against single-stranded dna (ssdna), -bromo- -deoxyuridine- -monophosphate (brdu), neuronal nuclei (neun) and nestin. positive cells immunoreactive to ssdna markedly increased in the ob on days after tmt treatment. positive cells immunoreactive to brdu markedly increased in the ob on days after tmt treatment. double staining of brdu and neun in the ob revealed that almost brdu was not incorporated into mature neurons on day after the treatment. these results suggest possible enhancement of neurogenesis in the ob following tmt treatment. ps a-f early migration of human umbilical cord blood neural stem cells transplanted into rat brain miroslaw janowski , hanna kozlowska , marcin jurga , aleksandra habich , elzbieta wanacka , barbara lukomska, krystyna domanska-janik department of neurorepair, medical research center, warsaw, poland many neurological disorders result from progressive cell loss or rapid cell damage. as stem cell technology appeared there is an arising hope for cell replacement therapy and definitive cure. recently, in our laboratory human umbilical cord blood neural stem cell line (hucb nsc) was established. the aim of the study was to analyze the migratory potential of hucb nsc transplanted into intact rat brain. hucb nsc transfected with gfp gene was stereotactically transplanted (tx) into intact brain of csa immunosuppressed adult wistar rats. cell detection was performed h, h, h and days after transplantation using abs anti gfp, hla class i and numa. analysis of rat brains revealed viable gfp positive hucb nsc cells migrating from tx site and dispersed through the host brain tissue , , and days after grafting. immunohistochemical studies confirmed that these cells were of human origin: hla class i or numa. in future we plan to study their lesion directed migratory potential. heparan sulfate proteoglycans (hspgs) are considered to play roles in cns development, such as axonal guidance. however, little is known about the function of hspgs during nerve regeneration. in this study, we examined the expression of ext , one of the enzymes for heparan sulfate biosynthesis, after hypoglossal nerve injury. the upregulation of ext mrna was detected using in situ hybridization in injured hypoglossal motoneurons, and heparan sulfate glycosaminoglycan was also upregulated in the injured hypoglossal nucleus. we also examined the expression of mrna for hspg core protein. the mrnas for glypican- and syndecan- were upregulated in injured motoneurons. these results indicate that the synthesis of hspg is upregulated in injured motoneuron and hspg might be involved in nerve regeneration. masami watanabe , hiroe sagawa , masahiro ichikawa , yoshihito tokita dept. perinatol., int. dev. res., kasugai, japan; dept. ophthalmol., nagoya univ. sch. med., nagoya, japan; dept. neurosurg., nagoya univ. sch. med., nagoya, japan we examined whether rho/rock inhibitor, y , can make injured rgc axons regenerate into the crushed optic nerve (opn) of cats. methods: culture; retinal pieces were cultured in dmem for d. after fixation, the neurites were stained with anti-tuj antibody to obtain number and length of tuj neurites. crush; after an intravitreal injection of drug, the left opn was crushed with thread. on day , wga-hrp was injected into the vitreous. sections of opn were reacted for hrp with tmb reaction. masanori yoneyama, kiyokazu ogita dept. pharmacol, setsunan univ., osaka, japan in this study, we evaluated the effects of glutathione depletion on proliferative activity in neural progenitor cells of -days-old embryonic mice. neural progenitor cells were prepared from the hippocampus of -days-old embryonic mice by culturing in dmem/f medium for days in vitro (div). marked round spheres were formed from cells adhered to each other under the culture conditions in the presence of bfgf and egf, and then subsequently proliferated to form large neurospheres in proportion to the duration of cultivation. to evaluate the effects of glutathione depletion on proliferation in the neural progenitor cells, buthionine sulfoximine (bso) were exposed into cultured neural progenitor cells for a period of - div. treatment with bso resulted in a marked reduction in endogenous glutathione in the cells. mtt assay revealed that the deletion of glutathione led to a marked decrease in surviving neurospheres cultured for - div. these results suggest that glutathione would positively regulate proliferative activity and/or survival in neural progenitor cells of murine hippocampus. michio hashimoto, eisuke kawakita, masanori katakura, osamu shido dept. of environ. physiol., sch. of med., shimane univ., japan docosahexaenoic acid (dha), one of the main lipids in brain, plays crucial roles in the development and function of brain neurons. we examined the effect of dha on neuronal differentiation of neural stem cells (nscs) in vitro and in vivo. nscs obtained from rat embryos were propagated as neurospheres and cultured with or without dha for days. dha increased the number of tuj (+) neurons compared with the control, and the newborn neurons in the dha group were morphologically more mature than in the control. dha decreased the incorporation ratio of brdu, the mitotic division marker, during the first h period. thus, dha promotes the differentiation of nscs into neurons by promoting cell cycle exit. furthermore, dietary administration of dha significantly increased the number of brdu(+)/neun(+) newborn neurons in the granule cell layer of the dentate gyrus in adult rats. these results demonstrate that dha effectively promotes neurogenesis both in vitro and in vivo, suggesting that it has the new property of modulating hippocampal function regulated by neurogenesis. research funds: kakenhi ( ) ps a-f interaction among cues for visual depth motion perception tomokazu shimizu, akitoshi hanazawa kyushu institute of technology, fukuoka, japan when an object surface approaches or leaves us, we perceive visual depth motion. cues for this motion are change in binocular disparity, change in spatial frequency and optical flow. we investigated interactions among these cues by using visual stimuli in which the cues provides opposite depth motion direction. for the stimulus without optical flow component, spatial frequency was changed continuously by presenting uncorrelated random dot patterns filtered by different band-pass filters. when binocular disparity and spatial frequency was oppositely changed, subjects perceived depth motion corresponding to the change in binocular disparity or special frequency. for the stimulus with optical flow component, a random dot pattern filtered by a band-pass filter was expanded or contracted. when binocular disparity and the other two cues were oppositely changed, subjects perceived depth motion corresponding to the change in the other two cues. when depth motion was perceived from binocular disparity, stimulus image was perceived as changing its size. when from the other cues, depth perception from binocular disparity was suppressed. research funds: coe-j kazuyuki takahashi, akitoshi hanazawa kyushu institute of technology, japan in phenomena such as biological motion and structure from motion, a global structure is perceived by an integration of local motion signals. to clarify the fundamental mechanism of this motion integration process, we psychophysically examined the influence of directional motion coherency on motion grouping. moving dots were presented in three apertures that were aligned horizontally. before presenting these stimuli, subjects were instructed to detect a dot moving in a direction among noise dots presented in the central aperture. the dots moving in the same as or different from the instructed direction were presented in the side apertures. the performance of the subjects was the best when the dots in the side apertures moved in the same direction as the instructed one. the performance kept high when the directional difference was up to ± • and declined as the difference increased. the high performance would be due to the grouping of the dots presented in the central and side apertures that have the same or similar motion direction. the underlying motion grouping mechanism was suggested to integrate motion signals that have a certain directional variation. ps a-f effect of spatial context on structure-frommotion perception koshi makino, akitoshi hanazawa kyushu institute of technology, japan when viewing an orthographic projection of dots on the surface of a rotating cylinder, one perceives a transparent rotating d cylinder. this phenomenon is called structure-from-motion (sfm). the direction of the rotation is ambiguous. we investigated the influence of spatial context on the perceived direction of the rotation. three spatially separated stimuli were horizontally aligned. subjects reported in which direction the central random-dot sfm cylinder rotated. they perceived the same direction of rotation as the side stimuli when the side stimuli were corotating cylinders whose direction of rotation was disambiguated by binocular disparity. this effect was strong when the stimuli consisted of a small number of dots, and was attenuated as the number of dots increased. the perception was also influenced by translational motion stimuli that had front and back planes comprising oppositely moving random-dots whose depth was specified by binocular disparity. these results suggest that the neural mechanism determining the rotation direction of bistable sfm is strongly influenced by the d structure of surrounding stimuli defined by binocular disparity. ps a-f rotational motion aftereffect in positive direction for -dimensional random-dot pattern masako ono, akitoshi hanazawa kyushu institute of technology, kitakyushu, japan when viewing a unidirectionally moving pattern followed by a stationary pattern, we will see the stationary pattern moving in the direction opposite to the preceding movement. this phenomenon is well known as motion aftereffect (mae). this mae can be perceived for -dimensional motion such as rotating cylinders. we found that adaptation to the rotation of a stereoscopic random-dot cylinder generate mae like phenomenon in the same positive direction as the rotation of the adaptation stimulus (positive mae). this positive mae was strong when cylindrical random-dot was used as a stationary test stimulus. this aftereffect could not be perceived for uniformly distributed non-cylindrical random-dot. although ordinary mae declined in a few seconds, this positive mae remained for a few minutes. this is a new phenomenon that is different from known dimensional mae. this finding suggests that the visual system has a mechanism that detect -dimensional rotation direction specifically, and this mechanism has a property that gives a bias to the perception of stereoscopic rotation direction in an adapted direction. research funds: coe-j takanori uka, ryo sasaki department of physiology , juntendo university school of medicine, tokyo, japan crowding refers to a subjectǐs difficulty in identifying a target in the presence of distracters. as a first attempt towards identifying the neural mechanism of crowding, we investigated perceptual crowding using a random-dot kinematogram. human subjects were required to report the direction of moving dots within a center patch ( deg) of a center/surround display presented degrees to the left of fixation, and to ignore the dots in the surround. motion coherence of the dots in the center patch, as well as surround size varied randomly across trials. motion coherence of the surround was always percent. for each of subjects, we calculated direction discrimination thresholds (at % correct) at each surround size. consistent with crowding, thresholds increased when surround size was . and degrees, compared to those with no surround. surprisingly, however, thresholds decreased when surround size was and degrees, relative to degrees. our results show that the spatial resolution of motion direction discrimination improves when the area we have to ignore exceeds a defined size. ps a-f generation of receptive fields in higher visual areas based on v columnar structure: a model study yoshitaka toyoda, yoshiyuki shimizu, izumi ohzawa graduate school of frontier biosciences, osaka university, osaka, japan neurons in higher cortical areas of the visual pathway, such as v and v , respond to stimuli with complex shapes. how do these neurons integrate signals from v ? in particular, does the well-known columnar organization of v play a role in determining the shape selectivity of higher-order neurons? to explore these questions, we devised a feed-forward hierarchical model. in our model, higherorder neurons sum the activities of v neurons linearly according to a neural receptive field (nrf), a weighting function defined over the cortical surface. the manner a nrf sums over multiple columns determines its shape selectivity. since there is no physiological data regarding possible forms for nrf, we have tested simple functional prototypes, gaussians and gabor functions. reponses of these model neurons are examined using non-cartesian gratings and other stimuli, and compared to published physiological data. about % of model neurons exhibit responses similar to those of v and v neurons. odd-symmetric gabor nrfs tend to generate more of these neurons. taihei ninomiya, takahisa m. sanada, izumi ohzawa graduate school of frontier biosciences, osaka university, osaka, japan when images with different spatial frequencies (sfs) are projected onto the two retinae, a -d surface slant is perceived (blakemore, ) . the relationship between the binocular receptive fields (brfs) and sf tuning properties indicate that the early cortical neurons can signal slant-in-depth (sanada and ohzawa ) . however, their measurements of brf were conducted in the spatial domain, and the sf tunings were tested monocularly. in this study, interactions are examined directly in the sf domain between grating stimuli presented to the left and right eyes. frequency-domain brfs were measured by a reverse correlation technique. both binocular and monocular sf profiles were obtained by this method. we predicted binocular sf (bsf) maps from monocular sf profiles, and compared the prediction and the actual bsf maps to assess the binocular interactions. with this method, neural response properties which previous studies couldn't access were revealed. research funds: mext( ), jsps( ), coe ps a-f consistency of simple cell receptive fields: space and spatial frequency domain measurements yuka tabuchi , kota sasaki , izumi ohzawa , grad. school of frontier biosci., osaka univ., japan; grad. school of eng. sci., osaka univ., japan frequency-domain subspace reverse correlation and -d spacedomain dynamic dense noise have become increasingly popular for mapping receptive fields (rf) of early visual cortical neurons. however, it is not known whether results from these methods are mutually consistent. to examine this issue, we compared an rf in the space domain measured by -d noise stimuli and an rf reconstructed from the response in the spatial frequency (sf) domain measured by flash grating stimuli of various orientation (or), sf and spatial phase presented in rapid succession. we fitted these two rfs by gabor functions, and examined the consistency of their parameters. all parameters including sf, or, spatial phase, and size of the rf agreed well when an expansive nonlinearity is considered for each cell. the optimal sf obtained in the space domain increased over time to the same extent as that obtained in the sf domain. therefore, responses of a simple cell can be encapsulated in a concise framework of a linear filter followed by expansive nonlinearity. research funds: mext( ), jsps( ), coe ps a-f firing statistics and stimulus selectivity of inferior temporal cortical neurons in the monkey shunta tate , , hiroshi tamura , , ichiro fujita , graduate school of frontier biosciences, osaka university, osaka, japan; jsps, japan; crest, jst, japan inferior temporal (it) cortical cells are selective for visual shape, and vary in their spontaneous firing pattern among them. cluster analysis indicated that it cells were classified into five groups based on inter-spike interval (isi) histograms of their spontaneous firing. the first two groups showed a single peak at a long or a short isi in isi histograms. the other three had multiple peaks, whose positions and relative heights varied among the groups. principal component analysis and other analyses of visual responses showed that the five groups differed in their stimulus selectivity for a predetermined set of visual stimuli. stimulus selectivity was sharper in the single-peak groups than in the multiple-peak groups. one of the single-peak groups was modulated by natural images more strongly than the other groups. the results suggest that cells with different firing patterns carry different aspects of visual information, and may perform different functions in the coding of visual object images. supported by jsps and crest. research funds: kakenhi - ps a-f spatial-frequency dependency of receptive field size and surround suppression in lgn and v hironobu osaki , tomoyuki naito , osamu sadakane , masahiro okamoto , hiromichi sato , med. sch., osaka univ.; grad. sch. med., osaka univ.; grad. sch. front. biosci., osaka univ., osaka, japan in the primary visual cortex (v ), neurons change their responses depending on stimulus parameters such as orientation, size, spatial frequency (sf). we investigated how sf of stimulus affects on stimulus-size tuning property of responses of neurons in v (n = ) and lateral geniculate nucleus (lgn) (n = ) in anesthetized cats. first, we found that v neurons exhibited shifts of their sf tuning from high to low according to a change in stimulus size from small to large. second, we measured stimulus-area summation curve of responses and found that a higher sf stimulus caused a reduction of the receptive field (rf) size and an increase of the surround suppression. similar results were obtained for lgn neurons implying that the relationship between sf and area summation properties observed in v has its origin in lgn. these results suggest that the sf tuning of rf surround is broader than that of rf center and this center-surround mechanism reduces redundancy in visual information processing. hiroyuki nakamura , akichika mikami , kazuo itoh department of morphological neuroscience, gifu university graduate school of medicine, gifu, japan; department of behavioral and brain sciences, section of neurophysiology, primate research institute, kyoto university, inuyama, japan an extrastriate visual area v a is considered to be involved in the dorsal stream visual areas, however, its connections are not understood. to demonstrate the cortico-cortical connections of v a, we injected a bi-directional tracer biotinylated dextran amine into the v a. our results indicated that the v a has connections with the occipital, parietal and temporal cortices. the v a may thus be involved in the visual information processing of both the dorsal and the ventral stream visual areas. in addition to these connections, we found that v a has commissural connections with the v , the v a, the parieto-occipital area, the dorsal parietal area, and the ventral intraparietal area, and receives commissural projections from the dorsal and ventral aspect of secondary visual area v . these commissural connections may convey ipsilateral visual information near the vertical meridian representations. ps a-g activity of neurons in the isthmo-optic nucleus and its relationship with head movements hiroshi ohno, hiroyuki uchiyama department of information and computer science, faculty of engineering, kagoshima university, kagoshima, japan retinopetal neurons in the isthmo-optic nucleus (ion) send their axons to the contralateral retina in birds. the centrifugal visual projection is thought to be involved in attentional modulation of retinal output. we recorded activity of neurons in the ion in awake, headunrestrained japanese quails using an implanted electrode assembly. head movements were videotaped with a high-speed video camera ( fps), and were also monitored with a d or d accelerometer. we found two distinct types of activity pattern: phasic and tonic. the majority of neurons in the ion discharge in a phasic manner. phasic and tonic cells are also different one from another in relation to head movements. phasic cells show phasic elevation of activity - ms after end of head movements, while tonic cells show tonic suppression during head movements. we will discuss the activity profiles of neurons in the ion in terms of their possible role in visually guided behaviors. ps a-g timing of face specificity in fusiform gyrus responses to stimuli in different parts of the visual field yuka okazaki , , arman abrahamyan , catherine stevens , andreas a. ioannides , brain science institute, riken, saitama, japan; graduate school of life science and systems engineering, kyushu institute of technology, fukuoka, japan; school of psychology, university of western sydney, sydney, australia neuroimaging techniques have demonstrated the preferential responses to faces in the fusiform gyrus (fug). event related potential (erp) and magnetoencephalography (meg) studies have shown that such the responses specificity to faces occurs approximately ms (n ) after stimulus onset by comparing with the other objects. in the present study, we examined whether these and earlier fug activities, which have been already identified by our team (within ms), were selective for face. we achieved this by analyzing meg data elicited by static human faces, hands and shoes stimuli placed in fovea and four quadrants. we found robust statistically significant activities for faces in fug about ms after stimulus onset which depended on the stimulus location in the visual field. narihisa matsumoto , shoutaro akaho , kenji fujikumi , yasuko sugase-miyamoto , masato okada aist, ibaraki, japan; ism, tokyo, japan; university of tokyo, chiba, japan to understand the temporal aspects of information encoded at a population level in the inferior-temporal (it) cortex, we applied a cluster analysis method to the responses of neurons. each response was recorded while one of the visual stimuli that consisted of geometric shapes and faces of humans and monkeys was presented. population activity vectors of neurons for visual stimuli were clustered by a mixture of gaussian model. we estimated the number of clusters by using variational bayes algorithm. we assumed that the probability of the number of clusters depended on the one at one time step before. in the early period, the population vectors formed three clusters corresponding to global categories (human versus monkey versus shape). in the subsequent period, each cluster expanded to form sub-clusters corresponding to detailed categories. moreover, the number of clusters changed smoothly over time. these results suggest that the responses of it neurons represent different levels of categorical signals separated along the time axis. ps a-g relationship between color and shape selectivity in area teo of the monkey masaharu yasuda , , hidehiko komatsu , national institute for physiological science, okazaki, japan; sokendai, okazkaki, japan visual objects typically consist of multiple features such as color, shape, texture etc. it is reported that neurons selective for these object features exist in the inferior temporal (it) cortex of the monkey and some of them are selective for more than one of these features. however, little is known about the relationship between the selectivity for different features. last year, we have reported that there exist many neurons in the posterior part of it cortex (area teo) that are selective for both color and shape. to study the relationship between the color selectivity and shape selectivity, we tested the responses of each neuron using all combinations of the sets of colors and shapes, and conducted svd (singular value decomposition) analysis. we found that some teo neurons exhibited selectivities for color and shape that were independent (separable) each other, whereas in some other neurons they were not independent (nonseparable). these results suggest a possibility that color and shape informations interact at cellular level in this area. ps a-g neural correlates of stimulus shape detection in monkey inferior temporal cortex taijiro doi lab. cogn., neurosci., osaka univ., japan we searched for a neural "correlate" of conscious perception of shape by recording neuronal activities from inferior temporal (it) cortex while a monkey performed a -choice shape detection task. the monkey was required to judge whether or not a sample stimulus was presented immediately after a forward masking stimulus. when there was, the monkey was required to select the stimulus identical to the sample from three targets, two shapes and one small dot. trial-totrial variation of firing rates of many it neurons correlated with the monkey's seen versus not-seen choices. the mean choice probability (cp) of it neurons was . , a value significantly larger than the chance level. neurons with stronger visual responses exhibited larger cps. we also searched for temporal firing patterns within the spike train from a single neuron or across - simultaneously recorded neurons, but failed to find any temporal structure related to the monkey's behavioral choice. the results indicate a link between the firing rates of it neurons with conscious perception of stimulus shape. research funds: mext grant ( ) ps a-g behavioral visual performance of the zebrafish mutant, eclipse yuko nishiwaki , atsuko komori , tomonori manabe , toshihiko hosoya , hiroshi sagara , emiko suzuki , hitoshi okamoto , ichiro masai masai initiative research unit, riken, wako, japan; riken bsi, wako, japan; ims, university of tokyo, minato-ku, japan eclipse was identified as a visual zebrafish mutant that does not show both electroretinogram and optokinetic response. in the last meeting, we reported that the els gene encodes the ␣ subunit of cgmp phosphodiesterase (pde c), which functions in phototransduction in cone photoreceptors. since genetic mutations of pde c have not been reported in human patients of hereditary eye diseases, the els mutant is a good model for studying physiological roles of pde c. here we investigated whether the structural integrity of photoreceptors and visual sensitivity are affected in the els mutants. our electron-microscopic analyses revealed that photoreceptors do not undergo degeneration and are maintained in the els mutant until day-post-fertilization. however, we found that visual response to the contrast is slightly affected in larvae heterozygous for the els mutation. these data suggest that the level of pde c activity is important for the sensitivity of vision. ps a-g localisation of two markers of oxidative phosphorylation in the ageing human retina: an immunohistochemical study tapas nag, shashi wadhwa aiims, india the enzymes of oxidative phosphorylation are known to be affected by reactive oxygen species, which cause mutations in them, leading to reduced energy production. we examined the distribution of two markers of oxidative phosphorylation (nadh-ubiquinol oxidoreductase and cytochrome c oxidase) in the human retina at different ages. eyeballs of donors (age: - years) were fixed in paraformaldehyde, frozen retinal sections from macular to midperipheral regions cut and immunolabelled for nadh-ubiquinol oxidoreductase (complex i) and cytochrome c oxidase (complex iv; molecular probe, usa). complex i-immunoreactivity (ir) was moderately present in photoreceptors, outer plexiform layer and few ganglion cells from to years of age, and showed a decline and lack of ir in older retinas ( - years). complex iv-ir was intensely present in most ganglion cells, outer plexiform layer and photoreceptors from to years of age, and absent at years of age. thus, complex i and iv-ir decline with age, with the former showing an earlier reduction in its ir. the data signify a reduced mitochondrial activity in the retina with ageing. research funds: aiims ps a-g temporal characteristics of neural activity related to target detection during visual search tomoe hayakawa , norio fujimaki , toshihide imaruoka nict, kobe, japan; kit, kanazawa, japan meg and fmri experiments were conducted during the orientation singleton search task, and moment magnitudes of dipoles were estimated with an fmri-constrained meg-multi-dipole method to obtain differences between target-present and -absent conditions in each brain region for the whole time course. activity around the cas consisted of a prominent and a subsequent smaller but still obvious peak ( , ms); the first peak showed no difference between conditions while the second peak was significantly larger in the target-present. activity around the pfug had a prominent peak and subsequent small activity ( , ms), whereas the target's presence or not had no influence on either activity. the activity of the right intraparietal sulcus (ips) was significantly larger than that for the left ips at latencies around ms irrespective of the target's presence or not. the results demonstrate that neural activities of multiple regions had different temporal characteristics and the later activity around the cas was related to the target segregation from its surroundings. kaoru amano , , derek arnold , alan johnston , tsunehiro takeda univ. tokyo, chiba, japan; ntt cs lab., kanagawa, japan; univ. sydney, sydney, australia; ucl, london, uk when a moving border defined by small luminance changes (or by color changes) is shown in close proximity to moving borders defined by large changes in luminance, the low contrast border can appear to jitter at a characteristic frequency -a phenomenon we refer to as misc (arnold & johnston, ) . in order to reveal the neurophysiological substrates of this illusion, brain activities measured using magnetoenceohalography (meg) were compared with the perceived rate of illusory jitter measured psychophysically. the result showed that the perceived rate was around hz and matched with the alpha frequency of meg. as hz meg responses were enhanced in the presence of illusory jitter relative to the presence of isoluminant motion and physical hz jitter, we believe that the activity is related to illusory jitter generation rather than to jitter perception or to isoluminant motion per se. these results support our hypothesis that misc is generated within cortex by the dynamic characteristics of a cortical feedback circuit rather than by any physical stimulus properties. ps a-g the internal structure and the visual neuron projection patterns of the ventrolateral protocerebrum (vlpr) in the drosophila central brain kazunori shinomiya , , kei ito , , center for bioinform., imcb, univ. of tokyo, tokyo, japan; dept. comput. biol., grad. sch. frontier sci., univ. of tokyo, kashiwa, japan; bird, jst visual information processing in the insect brain has so far been analyzed mainly within the optic lobe. many visual pathways are known to project from the optic lobe to a central brain area called the ventrolateral protocerebrum (vlpr). the vlpr is therefore expected to be one of the major higher-order visual centers. the neural circuits in this area, however, remain essentially unknown. our study is to reveal the detailed internal structure of the vlpr, for the first time, using the drosophila brain as a model system. we have identified discrete glomerulus-like structures (gls) in the vlpr, among which at least five are innervated by the visual projection neurons from the optic lobe. we analyzed the detailed internal structure of these gls by visualizing single cells in each visual pathway using the combination of the gal enhancer-trap and the flp-out systems, and revealed the directionality of each pathway by specifically labeling the pre-and post-synaptic terminals. ps a-g dynamic reorganization of orientation maps in a late phase of the sensitive period kazunori o'hashi , , toshiki tani , shigeru tanaka , graduate school of life science & systems engineering, kyushu institute of technology, japan; laboratory for visual neurocomputing, brain science institute, riken, japan we have found that there are two phases in the sensitive period of orientation plasticity: an early irreversible phase and a late reversible phase. in this study, we attempted to elucidate how orientation maps are reorganized in the late reversible phase, performing intrinsic signal optical imaging several times from the same kittens. we observed the over-representation of the exposed orientation even one day after the onset of goggle rearing around the age of weeks. we also found that when the goggles were removed after or weeks of goggle rearing, drastically reorganized orientation maps returned to regular orientation maps that had been established before goggle rearing. these results suggest that once established orientation maps in an early phase serve as template maps to which later rapidly reorganized orientation maps are restored by the release of single orientation exposure. manavu tohmi, seij komagata, yamato kubota, masaharu kudoh, katsuei shibuki department of neurophysiology, brain research institute, niigata university, niigata, japan fourier analysis of intrinsic signals produced by periodic visual stimuli has been applied for constructing retinotopic maps (kalatsky and stryker, ) . in the present study, we used fourier analysis of flavoprotein fluorescence signals for constructing retinotopic maps in the mouse visual cortex. periodic bar stimuli that moved across the visual fields produced periodic fluorescence signals in the visual cortex of anesthetized mice. the fourier components of the signals locked with the periodic stimuli were calculated in each pixel regarding the magnitude and phase. retinotopic maps were constructed based on these components. vascular artifacts could be removed when the stimulus frequency was higher than . hz, since fluorescence signals but not vascular responses could follow up to these frequencies. combination of flavoprotein fluorescence imaging and fourier analysis is a powerful tool for investigating high-resolution retinotopic maps with short acquisition time in the mouse visual cortex. yoshitake kohei, manavu tohmi, masaharu kudoh, katsuei shibuki dept. neurophysiol., brain res. inst, niigata univ., nigata, japan we have reported that ocular dominance plasticity induced by monocular deprivation can be visualized in mice using transcranial flavoprotein fluorescence imaging. another condition for producing ocular dominance plasticity is strabismus, which causes an increase in the proportion of monocular cells in the visual cortex. however, this possibility has not been tested in mice, mainly because surgical operations for producing large and stable shifts in eye position are difficult in mice. in the present study, we designed a new prism goggle for mice. this goggle was attached on the skull of mice during the critical period. the neural responses in the visual cortex of these mice were investigated using transcranial flavoprotein fluorescence imaging. preliminary experiments suggested that the responses in the monocular zone of the visual cortex were not affected in the strabismic mice. however, binocular interaction, which was additive in the binocular zone of normal mice, turned to be more repulsive in the strabismic mice. ps a-g retinotopy-based morphing of brain activity hiroshi ban, hiroki yamamoto, jun saiki graduate school of human & environmental studies, kyoto university, kyoto, japan the topographic visual field map is a fundamental property of the primate early visual cortex. we propose a new method to represent and sample topographic activities in the space of visual field by extending our previous study (maeda et al., . neurosci. res.) . the procedure was as follows. first, eccentricity and visual angle representations were measured for each subject using standard phase-encoding stimuli. second, individual cortical surfaces were reconstructed. third, the transformation between the position in the visual field and that on the cortical surface was established. finally, by using this transformation, brain activities were sampled and then displayed as an image spanning visual field dimensions, each pixel of which represents the activity of neurons representing a given position in the visual field. this retinotopy-based morphing is useful to analyze brain activity related to spatial and form vision and is more reasonable to integrate individual data than normalizing methods based on stereotaxic coordinates and anatomical structures. masahiro yamada , yasuhiro enami , hiroshi jouhou , takehiko saito , kaj djupsund tokyo metropol. univ., hino, tokyo; astellas pharma. inc., osaka, japan; suny upstate med. univ., center for vision and ophthal., ny, usa; univ. kuopio, dept. neurobiol., kuopio, finland on-off type amacrine cells are intensely connected with each other by gap junctions (gjs), forming a syncytium with a wide receptive field. we studied effects of external ph (ph ) on the control of cell functions. photoresponses of the cells were recorded intracellularly. slits of light stimuli simplified the estimation of the current flow in the cellular network into a one-dimensional problem. by lowering ph only . units from the baseline of . , we found a remarkable reduction of the conduction velocity by - %, an increase of the length constant and a hyperpolarisation of the resting potential. based on our theoretical model, combined with measurements of conduction velocity and length constants of the receptive field, we could estimate both gj and plasmamembrane conductances of the cell. thus, we suggest that protons could contribute to the reduction of conductances, especially at the plasmamembrane but also at gjs. ps a-g analysis of the band-pass filtering of the retinal rod by the ionic current model it is known that the rod network behaves like a band-pass filter. it was found that the time to peak of the response was shorter in rods further away from a slit of light. the band-pass filtering behavior has been attributed to an inductance element, i h , or i k(ca) . however, biophysical mechanism underlying the band-pass filter is not fully understood. to analyze the functional roles of ionic currents in the band-pass properties of rods, a model of the rod network was developed. the model incorporates much of the known parameters in rods, i.e., the phototransduction cascade, ionic currents (i ca , i kv , i k(ca) , i h , i cl(ca) ), calcium system and gap junctions between rods. in simulation, the band-pass properties of the rod was analyzed. it was found that single rod itself behaves as a band-pass filter. the mechanism underlying the band-pass filter was examined by changing model parameters. the result suggests that i k(ca) , i cl(ca) and i h are responsible for the bandpass filtering. research funds: kakenhi ( ) ps a-g stimulus selectivity and correlated spontaneous activity of distant neurons in monkey inferior temporal cortex go uchida, mitsuhiro fukuda, manabu tanifuji bsi, riken, wako, japan in inferior temporal (it) cortices of anesthetized macaque monkeys, we have previously shown that spontaneous spike activities (sas) of % ( of ) of neuron pairs (inter-neuronal distance > m) are significantly correlated. in the present study, to investigate how the correlated sas relate to functional structure in it cortex, we measured stimulus selectivity for each neuron of the pairs and explored similarity of stimulus selectivity by calculating correlation coefficients of responses to visual stimuli. this analysis revealed that the pairs with correlated sas tended to show more similar selectivity than the pairs lacking correlated sas. in addition, model analysis showed that in % ( / ) of the pairs the correlation of sas reflect synchronous transition between two activity states: periods with high and low mean firing rates. these results suggest that a network underlying the synchronous state transition provides circuitry that functionally connects distant it neurons showing similar stimulus selectivity. toshiyuki ishii , , toshihiko hosoya bsi, riken, japan; dept. biomolecular science, toho univ., japan understanding the significance of single spikes can be of critical importance in the analysis of neuronal information coding. it is often assumed that the firing rate is the sole carrier of information. however, if fine temporal patterns of spikes would carry information, the system could have large encoding efficiency. the vertebrate retinal ganglion cells fire burst spikes, separated by hundreds of milliseconds of silent periods. here we show that temporal patterns of spikes within these bursts carry visual information. when three or more spikes are fired, the multiple interspike intervals encode the input in a cooperative, non-redundant manner. this suggests that the spike patterns are not sorely determined by slowly modulating instantaneous firing rates. we also found that millisecond-scale structures in the spike patterns encode light intensity waveforms over ms. we propose that the retina compresses hundreds of milliseconds of light sequences into spike patterns at the scale of milliseconds. kazuhiro shimonomura, takayuki kushima, tetsuya yagi osaka university, osaka, japan purpose of this study is to design a neuromorphic hardware model that emulates fundamental architecture and function in the primary visual cortex (v ). we have constructed a binocular vision system consisting of two silicon retinas and simple cell chips and fpga circuits. the silicon retina has a concentric center-surround laplacian-gaussian-like receptive field. the output image of the silicon retina is transferred to the simple cell chips. the simple cell chip aggregates analog pixel outputs of the silicon retina to generate an orientationselective response similar to the simple cell response in v . this architecture mimics the feed-forward model proposed by hubel and wiesel, and computes physically a two-dimensional gabor-like receptive field. the fpga circuits compute complex cell responses based on the disparity energy model. the system can emulate the neural image of the binocular complex cells responding to natural scene in real-time and is useful to verify computational models of v neurons. masayoshi tsuruoka , masako maeda , bunsho hayashi , ikuko nagasawa , tomio inoue dept. physiol. showa univ. sch. dent. tokyo, japan; dept. anestesiol. showa, univ. sch. dent. tokyo, japan the present study investigated the involvement of ventral root looping afferent fibers in visceromotor function. under halothane anesthesia, the t -l dorsal roots were cut bilaterally to eliminate thoracolumbar influences. an electromyogram (emg) of the external abdominal oblique muscle evoked by colorectal distention was measured. colorectal distention ( mmhg) was produced by inflating a balloon inside the descending colon and rectum. emg activity evoked by colorectal distention significantly increased when the colon was inflamed with mustard oil ( %, ml). the increased emg activity significantly reduced following bilateral l -s ventral rhizotomies. a baseline emg did not significantly alter when the l -s ventral roots were cut bilaterally prior to inflammation. following the development of inflammation, there was less of an increase in emg activities. these results suggest that looping afferent fibers in the ventral root are involved in visceromotor function during colon inflammation. ps a-g hypnotic modulation of the cerebral processing of human visceral sensation using positron emission tomography using positron emission tomography (pet), we examined cerebral processing to visceral perception during neutral, hyperalgesic or analgesic suggestion with standard hypnosis. activation within right dorsolateral prefrontal cortex (dlpfc) and right inferior parietal cortex (ba ) was significantly greater (p < . , uncorrected) during rectal distention with analgesic suggestion than with neutral suggestion. on the other hand, activation within right medial frontal cortex (mpfc) was significantly greater (p < . , uncorrected) during rectal distention with hyperalgesic suggestion than with neutral suggestion. this is the first evidence with pet for a modulation of cerebral processing during visceral stimulation by hypnotic suggestion. these results suggest a role of dlpfc and mpfc in the cognitive control of the interoception. the participation of bladder receptors sensitive to cold temperature has been proposed in overactive bladder for decades. bladder cooling reflex (bcr) which consists of immediate sense of urgency and detrusor contraction in response to ice water infusion may be a neuropathic cause of detrusor overactivity (do). recently, urothelial cells display a number of properties similar to sensory neurons and have many sensors including gene for transient receptor potential (trp). we detected cold sensitive receptor trpm in the urothelial cell by immunofluorescence in an animal model for boo. intravesical administration of trpm agonist (l-menthol: . - mm) in freely moving rats, increased the micturition pressure (mp) in either normal (n = ) or boo rat (n = ). the micturition interval (mi) did not change in normal rat, but decreased in boo that have do. the results suggest that bcr is enhanced in boo by increasing trpm on the urothelium cell of the urinary bladder. ps a-g caudate projection from the vagal responsive site in the thalamic parafascicular nucleus in monkeys shin-ichi ito , a.d. craig dept. physiol, shimane univ. sch. med., izumo, japan; atkinson res. lab., barrow neurol inst, phoenix, usa we investigated efferent projections to the forebrain, from the vagal afferent activation focus in the thalamic lateral parafascicular nucleus (pf) (ito & craig, j neurophysiol ) . evoked potentials were mapped in the right thalamus from stimulation of the left cervical vagus nerve, and fluorescent dextrans were iontophoretically injected at the response focus. the injection sites were all located in the ventrolateral part of caudal pf, lateral to the habenulointerpeduncular tract, medial to the basal ventromedial nucleus, and ventromedial to the centre median. labeled terminals were found in the caudate nucleus (cd) in all cases. terminal patches extended longitudinally in the head of cd, concentrated in its ventral aspect. dense terminal patches also occurred throughout the tail of cd. these results suggest that visceral information modulates the portion of the striatum that has been implicated in cognitive function, and they implicate the caudate nucleus in the control of heart rate and respiration. research funds: nih grant ns ps a-g ascending general visceral sensory pathways to the telencephalon via the medial inferior lobe in a percomorph teleost, tilapia masami yoshimoto, naoyuki yamamoto, chun-ying yang, hironobu ito, hitoshi ozawa department of anatomy and neurobiology, nippon medical school, tokyo, japan general visceral sense is relayed to the telencephalon via thalamic and hypothalamic centers in mammals and birds. in teleosts, an ascending connection that corresponds to the thalamo-telencephalic pathway is present. however, it remained unclear whether or not a hypothalamo-telencephalic pathway exists in teleosts. the medial inferior lobe (mil), which corresponds to part of the hypothalamus of other vertebrates, is known to receive general visceral sensory inputs from the rhombencephalon in a percomorph teleost tilapia. hence, telencephalic connections of the mil were studied in this study. tracer injection experiments into the mil revealed that this hypothalamic zone projects to the preoptic area, the ventral telencephalon (i.e., vs, vd, and vv), and the dorsal telencephalon (i.e., dm, rdc, and dl). these findings suggest that the mil corresponds to hypothalamic relay zones in mammals (e.g. ventromedial hypothalamic nucleus). tatsushi onaka, yuki takayanagi department of physiology, jichi medical university, tochigi, japan administration of prolactin releasing peptide (prrp) decreases food intake. we have previously shown that an icv injection of anti-prrp antibodies increases food intake. neurones producing prrp are activated after peripheral administration of cholecystokinin octapeptide, a satiety factor. it is thus possible that prrp may mediate satiety signals in the brain. here we examined effects of anti-prrp antibodies upon total amounts of food intake and meal patterns. an icv injection of anti-prrp antibodies increased the total amounts of food intake and amounts of food intake during a meal but did not significantly change meal frequency. these data suggest that prrp may play an important role in the short-term control of food intake and are consistent with a hypothesis that prrp is a satiety signal within the brain. research funds: grant-in-aid for scientific research (c) ps a-h fasting induced long-chain fatty acid receptor gpr expression in the anterior pituitary of mouse ryutaro moriyama, shingo imoto, shinya shano, nobuyuki fukushima department of life science, kinki university, higashiosaka, japan g-protein-coupled receptor (gpr ) is known as a receptor for unsaturated long-chain fatty acids. the present study investigated the effect of h fasting on gpr expression in several regions of male mouse by real-time quantitative pcr, in situ hybridization and immunohistochemical method. gpr mrna expression was highly observed in the anterior pituitary, lung, colon, rectum, skeletal muscle, adipose tissue and testis in normal fed animals. h fasting induced gpr mrna expression increase in the anterior pituitary, lung and rectum. in the anterior pituitary, gpr -like immunoreactive cells were only observed in fasting animals. these results suggest that long-chin fatty acid regulates endocrine function in the anterior pituitary via gpr at least fasting period. ps a-h ketone body sensing cells in the lower brain stem to regulate food intake and reproductive functions kinuyo iwata, mika kinoshita, hiroaki sato, hiroko tsukamura, keiichiro maeda laboratory of reproductive science, graduate school of bioagricultural sciences, nagoya university, nagoya, japan ketone bodies are used for energy in the brain under malnutrition, such as prolonged fasting. we have previously revealed that hydroxybutylate ( hb), one of ketone bodies, sensed by the ependymocytes lining the fourth ventricular walls ( v) in the rat brain to regulate reproductive functions and feeding behavior. the present study was aims to determine if the ependymocytes located on the wall of v respond to the change in hb. change in the intracellular calcium concentration ([ca + ] i ) in vitro was measured in dispersed ependymocytes taken from the v in rats. the present results showed that the [ca + ] i increased in response to hb, but the increase was blocked by ␣-cyano- -hydroxycinnamic acid, which is a monocarboxylate transporter (mct ) inhibitor. immunohistochemistry showed that mct -immunoreactivities were located on the v ependymocytes. these results indicate that the ependymocytes may sense hb through a mct -dependent mechanism. research funds: kakenhi ps a-h comparison of hypothalamic histamine release by leptin in normal mice and high fat diet-induced obese mice tomoko ishizuka, kouta hatano, atsushi yamatodani dept. med. sci. and technol, grad. sch. allied hlth sci., fac med., osaka univ., osaka, japan leptin is a satiety factor which is produced by the white adipose tissue. peripheral administration of leptin decreases body weight and food intake acting on the hypothalamus. circulating concentration of leptin is in proportion to body fat mass, however, in obese humans, elevated concentrations of endogenous leptin cannot prevent the accumulation of the adipose tissue. we previously reported that leptin decreases food intake via the activation of the histaminergic system. in the present study, the effect of leptin on hypothalamic histamine release was compared in normal and high fat diet-induced obese (dio) mice. leptin ( . mg/kg, ip) reduced food intake in normal mice but not in dio mice, suggesting that dio mice have resistance for exogenous leptin like obese humans. the same dose of leptin increased hypothalamic histamine release in normal mice, while it had no effect in dio mice. these results suggest that the lack of the activation of the histaminergic system partly contributes to obesity in leptin-resistant dio mice. tomoya kitayama, yuri onitsuka, katsuya morita, toshihiro dohi department of dental pharmacology, hiroshima university, hiroshima, japan parkinson disease (pd) is neurodegenerative disorder of the substantia nigra accompanied by depletion of dopamine levels. symptoms of pd include disorder of aspiration and mastication, and dysphagia. in this study, rats injected with -hydroxydopamine ( -ohda) resulted in an extension of feeding time and a marked increase in the amount of feed powder on cage floor after clump feeding at weeks after -ohda without affect on number of neuron in solitary tract. these rats were transplanted with neural progenitor cells at mm; anteroposterior, + mm; lateral and − and − mm; dorsoventral from bregma at weeks after -ohda injection. the treatment shortened feeding time and decreased the leavings on the cage floor, as well as achieving decrease of neuronal death in substantia nigra. however, neural progenitor cells were not detected in substantia nigra. these results suggest that transplantation of neural progenitor cells may better -ohda-induced eating disorders via protection of neurons. research funds: grant-in-aid for young scientists b most tools used by nonhuman animals are extension of their effectors (motor-tools), while humans can use a kind of tools as substitute for their sensory organs (sensory-tools). to understand biological bases of using such tools, we trained japanese monkeys to use a tool as an extension of the eyes, and analyzed its learning processes to proceed as follows: ( ) retrieving the food with a rake (a motortool), ( ) retrieving the hidden food with a mirror-attached rake, ( ) using the reflected image of the food on a mirror separated from the rake, placed stationally beyond hidden food, ( ) moving a mirror hung along the rail by hand to find the food, ( ) using a rake with a small camera mounted inside, with which the monkeys searched for the food using the live video image captured by the camera on the monitor. finally, they could use a hand-held camera (a sensory-tool) as a manipulable extension of their eyes. thus, acquisition of using the externalized eyes can be achieved by gradual transfer of their own vision to the distant visual cues via motor-tools to extend their body image. kaori sawada , , shigehiro miyachi , michiko imanishi , masato taira , masahiko takada div. applied system neurosci., nihon univ. sch. med., tokyo, japan; dept. system neurosci., tokyo metropol. inst. neurosci., tokyo, japan to investigate the outflow of information from the temporal lobe to the prefrontal cortex, we injected rabies virus into three prefrontal regions: medial area ( m), dorsal area ( d), and ventral area ( v). the retrograde transsynaptic labeling was examined in the temporal lobe cortex days after prefrontal injections when the second-order neurons were labeled. the labeled neurons were observed in the lateral and medial aspects of the temporal lobe. in the lateral temporal lobe, neuronal labeling from m, d, and v was arranged topographically in and around the superior temporal sulcus. the labeing in the medial temporal cortex was also topographically arranged, such that m, v, and d receive multisynaptic projections from the entorhinal cortex, area , and both, respectively. these results suggest that there are parallel streams of information flow from the temporal lobe to the prefrontal cortex. research funds: crest, japan science and technology agency ps a-h new neural activities of reward anticipation and task errors h. ogawa , h. ifuku , t. nakamura , s. hirata kumamoto kinoh hosp, kumamoto, japan; fac educ, kumamoto univ., kumamoto, japan; nat kikuchi hosp, kumamoto, japan; dept. psych, kumamoto univ. hosp, kumamoto, japan neural activities at reward phase were recorded from the primary (pgc: areas g, & - ) and higher-order (hgc: prco & ofc) gustatory cortices of a monkey engaged in a taste discrimination go/nogo task. a lever had to be pressed after led onset when nacl was delivered, but not to water delivery. reward was given ca s after led offset at correct trials. relations between cues and responses were reversed. of reward-related neurons found, . % showed on type responses and the rest usual expectation responses. three types of on responses were noticed; c-type (n = ) only at correct trial, i-type (n = ) at around possible reward onset only at incorrect trials, and c-i type (n = ) at both. two classes of the c-i type were found; class i increased discharges at correct trials but decreased them at incorrect, but class ii increased them at both. all types were found in both cortices, but most class i were found in pgc and most class ii in hgc. i-type and class ii c-i type may represent error signals and reward anticipation. hiroaki ishida, masahiko inase, akira murata department of physiology, school of medicine, kinki university, japan in the macaque monkey, the ventral intraparietal area (area vip) integrated visual-tactile information in the body centered reference frame. the receptive fields of these neurons mapped on the same body parts in each sensory modality, so this area contributes to own body representation often referred to as body image. recent psychological studies implied that shared body representation of self and other might be required in the brain for social interaction. this means other¸s body image is mapped on own body image in the same neuron. in our experiments, we studied visual-tactile receptive field of the bimodal neuron in vip, then recorded activity during observing the experimenter being touched. some of neurons that had receptive fields anchored on the monkey¸s body showed visual response while the experimenter was being touched on corresponding body parts. the results suggested that bimodal neurons in vip may be related to matching mechanism between own body image and others, then we discussed that this area may contribute to the human social ability such as imitation. daichi hirai , takayuki hosokawa , masato inoue , akichika mikami section of brain sciences, primate research institute, kyoto university, inuyama, japan; department of psychology, tokyo metropolitan institute for neuroscience, tokyo, japan amygdala is involved in stimulus-reinforcement association learning, and have neural responses related to prediction of rewarding and aversive outcomes. however, it remains unclear whether representation of reinforcement value in the amygdala depends on other available outcomes in a given trial block. to elucidate how rewarding and aversive infomation are coded in the amygdala, we recorded single neuronal activity in monkey amygdala during delayed color matching task. we compared the neural responses to cue that rewarding outcome in two different stimulus-outcome conditions; one included electrical stimulus as aversive outcome, and the other included only rewarding outcomes. we found amygdala neurons to code the relative preference of available outcomes in a given trial block. ps a-h neuronal correlates of expectation-evaluation based on previous and ongoing contextual memories in the monkey prefrontal cortex kyoko matsuda, toshiyuki sawaguchi lab. cogn neurobiol, hokkaido univ. grad. sch. med., sapporo, japan to expect future events based on the ongoing context and to evaluate it are important for flexible control of goal-directed behavior. to examine a possible involvement of the lateral prefrontal cortex (lpfc) in such functions, we recorded neuronal activity from the lpfc of monkeys that performed an oculomotor task. in this task, the target of a saccade was indicated by combinations of successively presented two cues; symmetrically allocated two objects (cue ), and centrally allocated one of the objects presented in cue (cue ). the frequency of which object was presented as cue , i.e., task context, was manipulated across blocks. we focused on cue period and found that a subset of neurons showed object preference depending on current task context (cc type) or previous task context (pc type). cc type and pc type activities may be neuronal correlates of expectation-evaluation based on current and previous contexts, respectively. thus, neuronal processes for expectation-evaluation based on previous and ongoing "contextual memories" may progress in the lpfc. ps a-h anterior insular cortex neurons in monkey are activated when reward might be delivered, such as occurs in gambling takashi mizuhiki , barry j. richmond , munetaka shidara , grad. sch. of tsukuba univ., ibaraki, japan; neurosci. ri., aist, tsukuba, japan; lab. neuropsychol., nimh, bethesda, usa the human insular cortex has attracted interest because it is activated during risk-taking or decision-making tasks in fmri studies. to identify related neuronal signals, we recorded single insular neurons while two monkeys worked in a reward schedule task in conditions: ( ) a cue is picked at random so it is uncertain whether a correctly performed trial will be rewarded [uncertain condition], ( ) a cue indicates whether the current trial will be rewarded or not [certain condition]. in the uncertain condition / neurons responded in all trials. in the certain condition / neurons responded in the rewarded trials only. of these showed significant differences in firing rate between in the first trials after reward and other trials. these insular neuron responses seem related to reward expectancy and recent reward delivery. these neuronal responses might underlie the activation identified in imaging studies during gambling and decision-making tasks. research funds: kakenhi (priority areas ), aist masamichi sakagami , , kosuke sawa , xiaochuan pan , bsrc, tamagawa university, tokyo, japan; senshu university, kanagawa, japan; presto, jst, japan reward prediction behavior based on integration of associative information was investigated. monkeys were trained to perform a sequential association task with symmetric reward by symbolic delayed matching-to-sample procedure. at first, they learned two sequences of stimuli: a -b -c and a -b -c . after monkeys could acquire the sequences, new pairs of stimuli (i.e., d and d , e and e , etc) were introduced to associated with b or b (d -b , d -b , etc). the asymmetric reward rule was instructed by pairing c (c or c ) with the reward. after this instruction, reward predictive behavior was tested by using trained sequences and new stimuli. monkeys could show reward predictive behavior for not only a and a , which were associated with c and c in trained sequences, but also new pairs of stimuli, which were not directly associated either with c or reward. these results suggested that monkeys could use reward predicting information by integration of association among trained sequences, c-reward association, and new stimuli. research funds: kakenhi ( ), hsfp, presto, jst ps a-h reward predicting activity of prefrontal neuron based on group of stimuli xiaochuan pan , , kosuke sawa , , masamichi sakagami , bsrc, research institute, tamagawa university, japan; presto, jst, japan; department of psychology, senshu university, japan ability to anticipate a reward based on grouped events is important for guiding appropriate behavior. the main purpose of this study is to examine the pfc neuronal mechanism involved in predicting reward using learned associations among groups of stimuli. monkeys performed a sequential association task with symmetric reward. at first, they learned two sequences of stimuli: a -b -c and a -b -c . the asymmetric reward rule was instructed by pairing c (c or c ) with the reward block by block. monkeys were also trained with two different orders of stimuli (b-c-a and c-a-b). out of neurons from the lateral pfc, % showed reward-related activity in the first cue period. and one third of them (sr type) predicted reward only when a preferred stimulus was presented as a first cue. interestingly, the preference was not based on visual properties of stimulus, but on stimulus-group. the results suggest that about % of lateral prefrontal neurons predict reward based on stimulus-groups that were formed through the associative learning. attention evoked by novel stimuli is important for behavioral adaptation to new environment. however, it remains unknown whether the novelty is processed in a specific region of the prefrontal cortex. we trained two monkeys on a pavlovian conditioning task interleaved with an instrumental conditioning task and recorded cell activity from the lateral and medial prefrontal cortex (lpfc and mpfc). in a block of the pavlovian task (pv block), a visual stimulus (cs) was paired with a liquid reward and the trial repeated times. in a following block of the instrumental task, the monkey searched a correct action to obtain the cs as positive feedback. the cs was alternated every pv blocks. in many lpfc cells, responses to the cs were enhanced immediately after the change of cs, while such enhancement was less popular in mpfc. this result suggests that lpfc more contributes to coding of stimulus-novelty than does mpfc. when an outcome of action is uncertain, a top-down attention is directed to the coming outcome. to clarify the neural mechanisms, we trained two monkeys on a task with secondary reinforcers and recorded single cell activity of the medial and lateral prefrontal cortex (mpfc and lpfc). in a pavlovian block (pv block), a visual stimulus was paired with a liquid reward. in a following instrumental block (inst block), the monkey searched a correct action based on the visual feedback. the same visual stimulus as the one presented in the preceding pv block followed a correct action, whereas another visual stimulus followed a wrong action. when the monkey made more than consecutive correct trials, a new pv block started. both mpfc and lpfc cells gradually increased their firing toward the visual feedback when the outcome was uncertain, while the onset of the activity was significantly earlier in mpfc than in lpfc. these results suggest that the top-down attention first occurs in mpfc and propagates to lpfc in individual trials. ps a-h neuronal activity in the presupplementary motor area during a bimanual sequential motor task toshi nakajima , hajime mushiake , jun tanji department of physiology, tohoku university school of medicine, sendai, japan; brain science research center, tamagawa university, machida, japan to investigate the involvement of the pre-supplementary motor area (pre-sma) in organizing bimanual sequential movements, we recorded neuronal activity while a monkey was performing a motor task consisting of pronation or supination of either arm, with an intervening delay. in this report, we focus on neuronal activity during a period when the monkey was preparing to start the -sequence movements in a memorized order. we made regression analysis of neuronal activity in this period. we found that neuronal activity in the pre-sma rarely reflected muscle activity. instead, we found neuronal activity representing forthcoming actions such as supination, regardless of the arm to be used. we also found neuronal activity that reflected the second movement in a preparatory period before the execution of the first movement. we would demonstrate typical examples of pre-sma neurons and discuss their functional implications. ps a-h neuronal activity in the putamen and cm thalamus during response bias and its complementary process yukiko hori, takafumi minamimoto, minoru kimura dept. of physiol., kyoto prefect univ. med., japan we showed previously that cm thalamus participates specifically in complementary process to response bias (minamimoto et al. ) . to study the roles of the putamen and cm in response bias and it complementary processes, we recorded activity of cm and putamen projection neurons from two macaque monkeys performing asymmetrically rewarded go-nogo button press task. instruction of go or nogo activated cm neurons (n = ) preferentially when the instruction was associated with small reward. the instructions activated groups of putamen neurons preferring small reward-(n = ), large reward-action (n = ) and both types of action (n = ). onset latencies of these putamen neurons and rts in large-reward-go trials were shorter than those in small-reward-go trials by - and - ms, respectively. putamen neuron activation lead that of cm neurons by - ms. these results suggested that the putamen plays a major roles in both response bias and its complementary process while cm participates in the complementary process in concert with the putamen. research funds: kakenhi ( ) ps a-h encoding expected total rewards and their errors through a series of action choices by dopamine neurons naoyuki matsumoto, kazuki enomoto, minoru kimura dept. physiol. kyoto pref univ. med., japan to examine how dopamine (da) neurons represent reward expectation and its error through a series of action choices, we recorded activity of da neurons in two japanese monkeys making trial-anderror and repetition choices to find a correct, rewarding target among three alternatives. there are trials of first (t ), second (t ) and third (t ) choices with reward probabilities of about , and %, respectively. monkeys got reward after they hit a correct target, and got one more time by choosing the same target in the next trial (r , %). most da neurons ( / ) responded to the start cue of each trial and reinforcer beep after the choices. magnitude of the start cue responses progressively increased from t to t and to t trials, then decreased in r trial. in another task with two repetition trials (r and r , %), magnitude of start-cue responses decreased gradually from t to r and to r trials. thus, the start cue responses may reflect expected total rewards through a series of action choices for a goal, while reinforcer beep responses may reflect their errors. research funds: kakenhi ( ) ps a-h striatal neuron activity during decisions and action selections for probabilistic, scheduled rewards hiroshi yamada , , hitoshi inokawa , minoru kimura dept. of physiol. kyoto prefect univ. med., kyoto, japan; jsps, japan to study roles of the striatum in decision and selection of actions for probabilistic, multiple rewards, we recorded striatal projection neurons from a monkey. after depressing a start button, the monkey chose of target buttons with correct rates at st, nd, rd and repetition trials of , , and %, respectively. correct choices were followed by reward water. neuronal firing rates at starting each trial were related either to expected reward probability or to schedule states to obtain reward twice ( / ) rather than to upcoming choice of target ( / ). during the target choice, another subset of neurons showed firings selective to choosing particular target ( / ) rather than to expected reward probability ( / ). after the target choices, another group of neurons fired related to expected reward ( / ) rather than to chosen action ( / ). our results suggested that striatal neurons encode expected reward probability, schedule states to obtain multiple rewards and choice of actions during decision and action choices for a goal. research funds: kakenhi ( ), jsps fellows ps a-h encoding of reinforcement after rewardbased action selection by tonically active neurons in the striatum hitoshi inokawa, hiroshi yamada, minoru kimura department of physiology, kyoto pref. univ. of med., japan to study the signals encoded by tonically active neurons (tans) in the striatum, presumed cholinergic interneurons, reward-based decision and action selection, activity of tans was recorded from the putamen and caudate nucleus of a japanese monkey. after depressing a start button, the monkey chose of target buttons at average correct rates of (first), (second), (third) and % (repetition choices). correct and incorrect choices were followed by high-tone beep, reward water and low-tone beep, respectively. about a half of tans ( / ) responded differentially to the high and low tone beep respectively. number of responsive tans and magnitudes of the responses to high-tone beep was highest at the first choices, then, decreased gradually at second, third and repetition choices. these results suggested that the tans may encode reinforcement after reward-based action choices which is modified by reward expectation errors and motivation. research funds: kakenhi ( ) ps a-i representation of value of action, action and its outcome in sub-populations of striate neurons y. ueda , k. samejima , k. doya , m. kimura dept. physiol., kyoto pref. univ. med.; brain sci. res. center, tamagawa univ.; irp, oist to know the mechanisms of reward-based action selection in the basal ganglia, we recorded activity of striatal projection neurons of two macaque monkeys performing a free choice task with probabilistic reward. after a s delay, monkeys chose between left-and right-handle turn, followed by water reward at probability of , or %. a linear regression of neuronal discharge rates showed: neurons encoded reward values of either action during delay period before go signal, with most ( %) of them not having the action value signal in other task epochs. another subset of neurons encoded action signal selectively during action selection after go signal (n = ), while other neurons encoded presence or absence of reward at reinforcer epoch after the action selection. neurons encoding action values were in more anterior part of putamen than the neurons encoding actions. these findings suggested that sub-populations of striate neurons process action values and selection of actions during rewardbased decision and action selection. research funds: kakenhi ( ) ps a-i delay period activity of the monkey striatum in duration discrimination task atsushi chiba, ken-ichi oshio, masahiko inase dept. physiol., kinki univ. sch. med., osaka sayama, japan neuronal activity was recorded from the striatum of a monkey during a duration discrimination task. two visual cues (a blue or red square) were presented consecutively followed by delay periods, and the subject then chose the cue presented for the longer duration. durations of both cues, order of cue duration (long-short or short-long), and order of cue color (blue-red or red-blue) were randomized on a trial-by-trial basis. striatal neurons phasically responded during the first cue (c ), first delay (d ), second cue (c ), second delay (d ), and response periods. activity during the d and d periods was analyzed in this study. firing rates during the d period linearly depended on c durations. on the other hand, d period activity depended on trial types (ls and sl), but not on the variety of c durations in each trial type. our results suggest that striatal neurons encode, in the delay periods, not only temporal information with monotonic dependence on cue durations to prepare a comparison to a forthcoming cue duration, but also encode discrimination results between two cue durations. research funds: kakenhi ( ) ps a-i neuronal activities in the anterior inferior temporal cortex of monkeys during an asymmetrical pair association task based on facial identity satoshi eifuku , ryoi tamura , teruko uwano , taketoshi ono dept. integrative neurosci., univ. toyama, toyama, japan; dept. molecular integrative emotional neuroscience, univ. toyama, toyama, japan to elucidate neuronal basis of face memory, neuronal activities in the area teav of monkeys were recorded during a pair association paradigm that involves recognition of facial identity (i-apa task). in the i-apa task, monkeys were required to memorize paired associates of patterns and facial identity. each association has a particular direction, either the 'face to pattern' direction in which a cue stimulus which is a face is associated with a test stimulus which is a pattern, or the 'pattern to face' direction in which a cue stimulus which is a pattern is associated with a test stimulus which is a face. during the i-apa task, neuronal responses to a particular paired associate were identified. many of these neurons showed asymmetrical activities during the delay periods which were dominant in the 'face to pattern' trials. this asymmetrical delay activity are indicative of the crucial role of the teav area in face memory. research funds: kakenhi ( ) ps a-i reflexive social attention elicited by biological motion in monkeys and humans yoshiya mori , mikio inagaki , wu lisa , taijiro doi , eishi hirasaki , hiroo kumakura , ichiro fujita osaka univ., japan; massachusetts institute of technology, usa determining where another individual is attending and preparing for his/her upcoming action is crucial for members of a social group. here we report that the walking direction of another individual elicits a reflexive shift of visuospatial attention in monkeys and humans. we examined how the reaction time to peripheral visual targets was affected by a prior, brief presentation of a walking biological motion (bm) stimulus. during the task, subjects responded to a target point after the disappearance of the bm stimulus and fixation point. the walking direction of the bm stimulus was not predictive of the target direction, and was irrelevant for performing the task. we found that the reaction times in congruent trials, where the walking direction of the bm stimulus and the direction of the target appearance were the same, were significantly shorter than those of incongruent trials. we believe the attention mechanisms driven by bm may be part of the intentionality inference system. research funds: grants from and takeda science foundation ps a-i response properties of posterior parietal neurons during a multidimensional visual search task tadashi ogawa, hidehiko komatsu natl. inst. physiol. sci., aichi, japan the posterior parietal cortex (ppc) is thought to be one of crucial areas to direct spatial attention toward the target in visual search. visual sensory information (e.g. stimulus features) might be integrated in ppc to form a saliency map that controls spatial attention. to examine this hypothesis, we recorded the neural activity from the lateral intraparietal (lip) and a areas of monkeys performing a multidimensional visual search task. the monkeys had to make a saccade to either shape or color singletons in a stimulus array depending on the instructed search dimension. ppc neurons increased their activity when the receptive field stimulus became the target. some neurons showed target enhancement depending on the stimulus condition (singleton type and stimulus features), whereas others exhibited it irrespective of the stimulus condition. the mixed existence of these two distinct types of activities suggests that ppc is one of critical stages that integrate feature-dependent signals to produce featureindependent signals identifying the target location toward which spatial attention should be directed. monkeys utilize visual information in social communication. to elucidate visual function to categorize sexes, ( ) performance of visually guided sex discrimination task and ( ) neuronal activity during the task in orbitofrontal cortex (obf), the region could be related to sex recognition and vision processes, were investigated. monkeys were trained to discriminate the sex of a monkey shown in a picture that was presented on the display. the monkeys pressed the right bar for pictures of males and the left for females to get water reward. as a result, the monkeys were able to discriminate the sexes of monkeys shown in pictures. extracellular recordings of neurons in obf during the task showed that some cells responded to the pictures in a sexspecific manner. the present results suggest that visual information alone sufficiently contribute to discriminate sex in monkeys. obf could be involved in visual categorization of sex. research funds: kakenhi (a) ( ) (sa) and coe program in kit from the mext ps a-i activities of bursting neurons during color discrimination task in the monkey prefrontal cortex naoki ishikawa , satoshi katai , masanori saruwatari , masato inoue , akichika mikami section of brain sciences, primate research institute, kyoto university, inuyama, japan; third department of internal medicine, shinshu university, school of medicine, matsumoto, japan the neurons in the prefrontal cortex of monkeys are involved in the behavioral control of saccadic eye movements. on the other hand, cerebral cortex consists of different types of neurons. in this study, we trained macaque monkeys to perform a delayed matching to sample task with saccadic eye movement. and we classified neurons whether they had burst episode or not, and then classified bursting neurons into fast spiking (fs), fast rhythmic bursting (frb), and intrinsic bursting (ib) neurons (katai et al. neuro ) . most of bursting neurons activated during the target presentation or during the saccade period were selective to the target location or saccade direction. these results suggest that the bursting neurons have the significant role in the target selection and decision-making of the eye movement toward the specific direction. atsushi matsumoto , tetsuya iidaka department of psychology, nagoya university, nagoya, japan; department of psychiatry, nagoya university, nagoya, japan several studies indicated that gamma band activity (gba: - hz) reflects the process to form mental representation of objects or information. we investigated whether the gba is observed during subliminal visual word processing as well as supraliminal word processing. gba were observed both in masked and unmasked condition. at the - ms time window, gba was significantly higher in the word condition compared to the nonword condition in the unmasked condition. similarly, in the masked condition, gba of the word condition was significantly higher than that of the nonword condition at that time window. these results indicate that the unconscious lexical processing was reflected in the gba at that time window. furthermore, at the - ms time window, gba induced by word was significantly higher than that induced by nonword. this effect was not observed in the masked condition. in addition we found the significant semantic priming effect, indicating that the information of briefly presented words was processed unconsciously. wakayo yamashita, junichi hayashi, tomoki murakami, gang wang department of bioengineering, kagoshima university, kagoshima, japan the purpose of this study was to investigate the dependency of view association learning on the separation of the views. each stimulus set included images ( objects × views). novel objects were generated by deforming a prototype in four directions. for deg-interval object sets, views were obtained by rotating each object with the interval of deg, deg-interval set and deg-interval set were with deg and deg interval respectively. task performances were evaluated while the subjects performed an object matching task, in which the subjects had to recognize one object from others regardless of the viewpoint. the performance across deg separated views was significantly higher in the trials with deg-interval sets than those with deg-interval sets. similarly, the difference was also found in the performances across deg separated views between those with deg-interval sets and deg-interval sets. the results suggest that the exposure of interpolated views significantly improved the association learning of the views. ps a-i brain regional activity during attention task ( the kana pick-out test, treated as inspecting higher brain function, has been proposed to be suitable for screening dementia, which is widely used among public health nurses in japan. however, few fmri studies while demonstrating the test have reported. we therefore assessed the effect of brain regional activity with computerized kana pick-out test projected on the screen with clicking a mouse button to pick kana out under fmri running. executing the test resulted in significant increases in bold signals in right prefrontal area, bilateral hippocampus and broca's area. the results indicate the existence of the attention pathway from and/or to prefrontal area as association mechanisms for execution of kana pick-out test, suggesting that this test is useful in screening dementia. ps a-i obsessive compulsive symptoms in middle school students and its association with tic disorder, body dysmorphic disorder and trichotilomnia in shiraz, iran, ashkan mowla, arash mowla shiraz university of medical sciences, iran aim: the aim of this study is to evaluate ocd symptoms, tic disorder, body dismorphic disorder (bdd) and trichotilomnia (ttm) among middle school students of shiraz, iran. methods: middle school students were selected in a cluster random sampling from the four educational regions of shiraz, iran.persion standardized moci was used to assess obsessional symptoms. for evaluating bdd, tic disorder and ttm symptoms, a semi-structured interview was done according to dsm-iv-tr criteria. results: students with more obsessional symptoms were more girls and demonstrated more positive family history.they were more likely to be from lower socioeconomic class and with lower school average. they also showed more association with body dysmorphic disorder and tic disorder. conclusion: girls especially those from lower socioeconomic class demonstrated more obsessional symptoms. this study, like pervious ones, confirmed bdd symptoms and tics to be more in individuals with ocd symptoms. it was seen that ocd symptoms would affect school performance. ps a-i sirna-induced nr knockdown causes hypofunction of nmda-r and cognitive deficit m. saji , , t. utida , a. ohnishi , k. noda , m. ogata , h. akita , n. suzuki , physiol, health sci. sch. kitasato univ., sagamihara, japan; brain sci., graduate sch. kitasato univ., sagamihara, japan blockade of nmda-r by antagonists causes psychomimetic effects, suggesting involvement of nmda-r dysfunction in mental disorders like schizophrenia. however, the relationship between mental disorders and molecular abnormality has not been cleared. to identify the role of nmda-r in brain function, we performed sirnainduced knockdown of nmda-nr using hvj-envelope vectors. we confirmed that marked down-regulation ( %) of nr expression occurred only in the hippocampus among various brain regions - days after intra-ventricular injection of sirna-vector complex. in the hippocampal slice from rats with the nr knockdown, the nr down-regulation prevented depressive effects of nmda on fepsps, while the treatment did not affect ltp or ltd. in rats with the nr knockdown, the nr down-regulation caused disruption of prepulse inhibition, while the same treatment did not affect locomotor activity. these results suggest that hypofunction of hippocampal nmda-r by sirna-treatment causes a deficit of cognition. ken hatanaka , , hiroshi ageta , ikuko yao , kaoru inokuchi , yutaka kirino , mitsutoshi setou , graduate school of pharmaceutical sciences, the university of tokyo, tokyo, japan; mitsubishi kagaku institute for life sciences, tokyo, japan; okazaki institute for integrative bioscience, national institute for physiological sciences, okazaki, japan schizophrenia is a severe psychiatric disorder that characterized by psychotic symptoms in particular delusions and hallucinations, reduced interest and drive, altered emotional reactivity and disorganized behavior. to know the molecular mechanisms of the disease, we screened altered gene expression on the brain of schizophrenic patients by using microarray analysis, and found that the expression of ubl mrna was significantly decreased in the enthorinal cortex, whose size is known to be reduced in some schizophrenic patients. ubl is a highly conserved protein, which has a ubiquitin-like domain (ubl domain) and caax motif which is a membrane localization signal. we found that ubl mrna was expressed in the hippocampus, and purkingie cells of the cerebellum. the putative molecular function of ubl wil be discussed. research funds: grant-in-aid for young scientists (b), presto ps a-i decreased interneurons in the pax mutant mouse limbic system hasumi haba , tadashi nomura , yoshinobu hara , , noriko osumi , div. dev. neurosci., ctaar, tohoku univ. sch. med., sendai, japan; crest, jst, japan core features of schizophrenia are impairments in certain cognitive functions such as working memory, in which a number of brain regions in the corticolimbic system are involved. recent studies have revealed abnormality in distribution of interneurons in these regions. we have previously found that pax heterozygous mutant rats show behavioral abnormalities including impairment in fearconditioned memory and sensorimotor gating. in the present study, we thus analyzed distribution of interneurons in several regions of pax heterozygous mutant mouse (sey/+) brain. we focused on three subpopulations of interneurons: parvalbumin (pv)-, calretinin-, and somatostatin-posive interneurons. immunohistochemical studies indicated marked decrease in pv-positive interneurons in two brain regions of sey/+ mice, i.e., the olfactory bulb and the amygdala. reduced number of pv-positive interneurons was observed in the sey/+ amygdala at weeks, but not at weeks. our results suggest that age-dependent decrease of pv-positive interneurons might underlie behavioral abnormalities in sey/+ mice. schizophrenia is a complex genetic disorder, characterized by multiple susceptibility genes. dysbindin (dtnbp ) is a susceptibility gene for schizophrenia. genetic evidence for the association between the disorder and the dysbindin gene has repeatedly been reported in various populations world wide. recently, decreased expression levels of dysbindin mrna and protein have been reported in postmortem brain in patients with schizophrenia. thus, we performed behavioral analysis in sandy mouse, which has a deletion in dysbindin gene and expresses no protein. sandy mouse showed decreased locomotor activity and time in the center in the open field test. and an acute treatment of atypical antipsychotic, olanzapine ( . mg/kg, i.p.), improved the decrease in time in the center. moreover, subtle behavioral abnormality was observed in elevated plus maze test and social interaction test in sandy mouse. our results suggest that dysbindin might be involved in anxiety-related behavior in novel environment. research funds: , ps a-i gene expression analysis of dysbindin mrna in peripheral blood in schizophrenia sachie chiba , , satoko hattori , hiroaki hori , tetsuo nakabayashi , hiroshi kunugi , ryota hashimoto department of mental disorder research, national institute of neuroscience; tokyo university of agriculture and technology department of biotechnology and life science, koganei, japan; musashi hospital, ncnp, kodaira, japan although many efforts have been spent to discover a biological marker of schizophrenia, no biological marker has been established. as genetic evidence suggested that dysbindin (dtnbp ) is a susceptibility gene for schizophrenia, we measured dysbindin mrna expression level in peripheral blood samples of patients with schizophrenia and age-sex matched healthy controls by a quantitative real time rt-pcr method. we quantified the expression levels of two major dysbindin transcripts among several known splicing variants. no significant difference in the expression levels of examined dysbindin transcripts was observed between control and schizophrenia. further examination measuring other dysbindin transcripts should be warranted to find a biological marker for schizophrenia. research funds: , ps a-i genetic variation in dysbindin influences memory and general cognitive ability ryota hashimoto , hiroko noguchi , hiroaki hori , tetsuo nakabayashi , satoko hattori , sachie chiba , seiichi harada , osamu saito , hiroshi kunugi department of mental disorder research, national institute of neuroscience, national center of neurology and psychiatry, kodaira, japan; musashi hospital, ncnp, kodaira, japan; tokyo university of agriculture and technology department of biotechnology and life science, koganei, japan dysbindin (dtnbp ) is a susceptibility gene for schizophrenia, a neuropsychiatric disorder characterized by cognitive dysfunction. we examined the possible association between genetic variants in the dysbindin gene and memory and iq in healthy volunteers and patients with schizophrenia. individuals who did not carry a protective haplotype had lower performance in several memory domains wms-r, although this haplotype did not affect iq measured by wais-r. a risk independent polymorphism for schizophrenia influences both memory and iq in the opposite direction. these data suggest that dysbindin gene may have impact on the cognitive function such as memory and iq and that memory might be an intermediate phenotype of dysbindin on risk for schizophrenia. research funds: , ps a-i detection of f-dopa signal in brainstem monoaminergic nuclei in schizophrenia yuri kitamura , nicola bright , toshio yanagida , masatoshi takeda , paul grasby department of physiology, osaka university, japan; department of psychiatry, osaka university, japan; cyclotron unit, imperial college, hammersmith hospital, uk we used f-dopa pet to investigate presynaptic dopamine dysfunction in schizophrenic patients. the object of this study was to test that a schizophrenic cohort would show elevated aadc activity in the substantia nigra, midbrain raphe and locus coeruleus compared to normal controls. all subjects and f-dopa scans were obtained from a database of scans published in mcgowan et al. , archives general psychiatry. the schizophrenic patients all met dsm-iv criteria on medication and healthy volunteers were compared. we attempted to improve the quality of the f-dopa signal by implementing a fbf-realignment movement correction method. significant increases in f-dopa uptake were found in the striatum, substantia nigra and raphe nuclei of schizophrenic patients (p > . ). our result suggests that an elevated presynaptic dopamine function is present in dopaminergic neurons that innervate striatal areas associated with enhanced dopamine activity in schizophrenia. in this study, we analyzed the p component of the visual eventrelated potential in patients with schizophrenia and healthy controls, and also performed loreta analysis. the ethics committee of kurume university approved this study. the p amplitude for the crying face was significantly smaller in patients than in controls. in controls, the p amplitude was significantly larger for the crying face than for the laughing face, while in patients, there was no significant difference in the p amplitude between the faces. loreta analysis demonstrated that there were significant differences in the activity in brodmann area between the faces in controls, while in patients, there was no significant activity difference between the faces. stimulation with crying face induced higher activities in the and right areas in controls than in the patients. these results indicated that the cognitive function was influenced by affective stimulus. ps a-j inappropriate input produces schizophrenialike working memory deficits in a simulated neural circuit kensuke nomura , shoji tanaka , koki yamashita , motoichiro kato , haruo kashima department of neuropsychiatry, school of medicine, keio university; department of electrical and electronics engineering, sophia university a number of studies indicate that the prefrontal cortex (pfc) is intrinsically linked to working memory (wm) and that dopamine critically modulates wm activity. according to the hypothesis proposed by goldman-rakic and her colleagues, we constructed an electrophysiological circuit model for wm which represents eight directions. the computer simulation with this model shows that the working memory activity is dampened by cue-irrelevant inputs and greater noise inputs lose the directional selectivity of the representation. a lot of studies suggested that increase of noise was related to schizophrenia, especially in wm disturbance. our study indicates that noise inputs cause wm impairment in patients with schizophrenia and that working memory performance is not always positively correlated with the neuronal activity of the pfc. ps a-j pericentrin is localized to the base of neuronal primary cilia in the developing cerebral cortex ko miyoshi, ikuko miyazaki, masato asanuma department of brain science, okayama university, okayama, japan we previously identified pericentrin, a mammalian centrosomal protein, as a binding partner of the product of disc , a candidate gene for schizophrenia. in this study, we analyzed in vivo expression of pericentrin in the mouse embryo. in the developing cerebral cortex, pericentrin mrna was highly expressed in migrating cells of the intermediate zone, though proliferating neuroepithelial cells and mature neurons revealed a low expression level of pericentrin. the pericentrin protein was shown to be localized to the base of primary cilia in the pre-plate of the developing cerebral cortex, in agreement with a recent study demonstrating the involvement of pericentrin in primary cilia formation. specific subtypes of receptors such as -ht are known to be localized to the plasma membrane of neuronal primary cilia in certain regions of the brain, and then our results raise the possibility that pericentrin dysfunction may result in perturbed chemosensory function of neuronal primary cilia and increased vulnerability to psychiatric disorders. dysregulation of gr has been thought to play an important role in the pathophysiology of mood disorders. two isoforms of human gr-alpha and -beta arise from alternative splicing of the pre-mrna primary transcripts. previously, we evaluated these two isoforms mrna level in the peripheral white blood cells of the patients with mood disorders. we found that the reduced gr-alpha mrna level in the patients with both bipolar and major depressive disorders, while gr-beta mrna level was not altered. these results suggest that dysregulation of alternative splicing play an important role in the pathophysiology of mood disorders. to test this, we evaluated mrna level of alternative splicing-related sr protein family, which regulate alternative splicing in several genes including gr, in the peripheral white blood cells of the patients with mood disorders. we did not find any differences in of the sr protein mrnas level in the patients compared to healthy controls and now, we are examining other sr family mrnas level. ps a-j alteration of neocortical long-term depression following electroconvulsive shock yoshifumi ueta , ryo yamamoto , shigeki sugiura , kaoru inokuchi , nobuo kato dept. integrat. brain sci., grad. sch. med., kyoto univ.; nara med. univ.; mitsubishi kagaku inst. life sci. electroconvulsive therapy is useful in treating drug-resistant depressive disorders, though its mechanism remains unclear. there have been a few reports that studied effects of electroconvulsive shock (ecs) on long-term potentiation. however, its effects on long-term depression (ltd) have not been investigated to date. the present experiments examined roles of ecs in inducing ltd at a variety of corticocortical synapses in rat cortex slices by using whole-cell patch clamp. following ecs, ltd magnitude at layer ii/iii-to-vi pyramidal cell synapses was significantly reduced in comparison to no-ecs subjects. as described in recent microarray studies, homer a/vesl- s was identified as one of the most up-regulated molecules after ecs. we therefore injected homer a protein by diffusion from patch pipettes. homer a injection, as well as with ecs treatments, reduced ltd magnitude only at layer ii/iii-to-vi pyramidal cell synapses, implicating that homer a may be a biological mediator of ecs effects. masanori kasai , nozomi miyagi , norio kawashiro , daisuke torizuka dept. of chem. & biosci., faculty of sci., kagoshima univ., kagoshima, japan; sanko shokuhin co., ltd., tokyo, japan it is well known that zinc is an essential mineral necessary for a multitude of body functions, including acuity of taste. to know a change of serum level in adjuvant-induced inflammation, we measured a zinc level in serum from male lewis rats received a suspension of complete freund's adjuvant ( . mg), injected intradermally into the tail. body weight, food intake and water intake were also measured. all rats showed signs of systemic inflammation (weight loss, hind paw swelling, nodules around eyes and penis) after the th day. the rats were sacrificed to measure the serum mineral contents (zn, na, cl, p, ca, k, mg) on the nd, th, th, st, th and th days. the serum zinc level was decreased on all of the measurement and the average of serum zinc ( . ± . g/dl, n = ) on the the day was significantly lower than that in intact rats ( . ± . g/dl, n = ). this decrease of zinc was correlated with weight loss but not hind paw swelling. other minerals did not show any significant changes throughout the measurement period. ps a-j molecular cloning of a novel candidate for ethanol-responsive genes, yy ap-related protein (yarp), in rat brain in order to elucidate the molecular mechanisms of etoh action on the cns, we investigated changes in gene expression in the adult rat brain after chronic etoh treatment. by means of cdna subtraction, we identified a candidate for etoh-responsive genes in the hippocampus. cdna cloning and sequence analysis revealed that this gene encodes a novel homolog of yy ap (yy -associated protein) and is well conserved in rats and humans. homology search for functional domains predicted that the yarp polypeptide contains nlss', a dnabinding motif, and a chromatin decondensation domain, as well as yy -binding and transactivation domains previously demonstrated in yy ap. in the brain, neurons such as hippocampal pyramidal cells were stained by in situ hybridization, and co-expression of yarp and yy genes was demonstrated in the same neurons. analogous to yy ap as a co-activator of transcription factor yy , it is postulated that yarp can regulate cerebral gene expression in response to etoh treatment. ps a-j excitotoxic degeneration of hypothalamic orexin neurons: involvement of nr b-containing nmda receptors and rescue by gaba a receptor stimulation hiroshi katsuki, shinsuke kurosu, toshiaki kume, akinori akaike department of pharmacology, graduate school of pharmaceutical sciences, kyoto university, kyoto, japan selective degeneration of orexin neurons, a pathological hallmark of narcolepy, is in part reproduced in hypothalamic slice cultures by application of quinolinic acid (qa), an endogenous nmda receptor agonist. we report here that nr b-selective nmda antagonists ifenprodil ( and m) and ro - ( . and m) markedly inhibited degeneration of orexin neurons induced by h application of nmda ( m) or qa ( . mm). we also show that stimulation of gaba a receptors by muscimol ( and m) or isoguvacine ( and m) potently inhibited qa cytotoxicity. in addition, the protective effect of gaba ( m) plus a gaba uptake blocker nipecotic acid ( mm) was abolished by a gaba a antagonist picrotoxin ( m). norepinephrine and serotonin did not provide a neuroprotective effect. thus, gabaergic inhibition may be decisive on survival of orexin neurons under excitotoxic stimuli mediated by nr b-containing nmda receptors. yoshika kurokawa, shinji tsukahara, hidekazu fujimaki national institute for environmental studies, tsukuba, japan to evaluate neurotoxicological influence of volatile organic chemicals (vocs), such as toluene, on hippocampal function, we attempted to develop an in vivo optical imaging technique for the hippocampus of mice with or without receiving voc inhalation. we dissected out the cerebral cortex in mice anesthetized with pentobarbital in order to prepare an optical window for monitoring the dorsal surface of the hippocampus, and stained the hippocampus with voltage-sensitive dye (rh ). we then monitored optical signals responding to electrical single-pulse stimulation to the parahippocampal region or hippocampal formation with a time resolution of ms. we also examined optical signals in the hippocampus during toluene inhalation. as a result, neural excitation of the superficial layer was observed in the hippocampal formation after electrical stimulation. on the other hand, acute perinasal exposure of toluene gas did not alter any signal pattern in the hippocampal formation. we will discuss the usefulness of this technique for examination of the neurotoxicological influence of vocs. ps a-j a simple method for fabricating electrodes array for multichannel neural recording -investigation of the alignment of the array and the measurement system-noriyuki taniguchi , osamu fukayama , takashi sato , takafumi suzuki , kunihiko mabuchi , dept. biomed. eng., univ. tokyo, tokyo, japan; dept. info. physi. comp., univ. tokyo, tokyo, japan various types of electrodes have been developed for use as brain-machine interface (bmi) to record signals from neurons. electrode arrays can be purchased from vendors. however, economic considerations and the adjustment of the array alignment for experimental design still make it worthwhile to develop fabrication methods inhouse. thus we developed a low-cost multichannel microwire array electrodes for recording from the cerebral cortex of conscious rats. the electrodes were able to align for the experimental paradigms. the effectiveness of the arrangement of the array as a bmi device was investigated. the electrodes were implanted in the primary motor cortex of wistar rats. we used a wheel-formed rat exercising kit to measure the walking speed of a rat. the neural signal of the rat and the rotating speed of the wheels were simultaneously recorded. and we evaluated the estimation of the walking speed by multiple electrodes with different alignments. ps a-j on-chip electrophysiological measurement of artificially constructed single-cell based neuronal networks ikurou suzuki , yasuhiko jimbo , kenji yasuda department of life sciences, graduate school of arts and sciences, university of tokyo, tokyo, japan; department of precision engineering, graduate school of engineering, university of tokyo, tokyo, japan we have developed a single-cell-based on-chip um-diameter multielectrode arrays with an agarose microchambers (amc) for topographical control of the network patterns of living neurons. this system enables flexible and precise control of the cell positions and the pattern of connections through photo-thermal etching. and sampling rates of measurement are khz in ch electrodes simultaneously. using this system, we formed a single-cell-based neural network pattern of rat hippocampal cells within the amc array and controlled the growth direction of axon/dendrite selectively using photo-thermal etching methods during cultivation, and recorded the spontaneous firings and evoked responses. moreover, we identified propagation along patterned neural network and found the effects of tetanic stimulation within this neural network. in the meeting we will present the results in detail and will discuss the potential of our method. yuichi yamashita , tetsu okumura , kazuo okanoya , jun tani lab. for behavior & dynamic cognition, riken-bsi, japan; lab. for biolinguistics, riken-bsi, japan how the brain generates and learns temporal sequences is a fundamental issue in neuroscience. the production of birdsongs, a process which involves complex learned sequences, provides researchers with a good biological model to study this phenomenon. bengalese finches (bf) learn highly complex songs that have grammatical structure. the underlying neural mechanisms that allow the birds to learn these songs are however not fully understood. to address this issue, we developed a neural network model of bf's songs that might explain how different regions of the brain work together. to test the model, we also conducted empirical experiments on the brains of bf. the model shows that complex grammatical songs can be replicated by simple interactions between deterministic dynamics of a recurrent neural network and random noise. moreover, comparison between the model and the empirical data on real birds shows similar trends. this work is a part of an integrated research project combining model simulations and empirical study. please see also the empirical component of this project as reported by okumura. ps a-k local administrations of muscimol into the nif alter song grammar of the bengalese finches (bf) tetsu okumura , yuichi yamashita , kazuo okanoya , jun tani behav & dynamic cognition, riken-bsi, saitama, japan; biolinguistics, riken-bsi, japan songs of passerines are learned behavior which used by males to attract females. their songs consist of several song notes, and these notes are produced in a fixed temporal order. among the passerines, however, bfs sing complex song which follows finite state syntax. the song control system of bf consists of a set of discrete nuclei including the hvc and nif. previous study showed that nif lesioned bfs sung simpler songs, with less phrases to phrases branching. therefore, nif-hvc connection may play important role in generating song grammar. in this study, we perfused nif with muscimol via microdialysis probes as a perturbation on nif-hvc system. following a local perfusion, song grammar was modified. some of chunks in their grammar were disappeared and introductorily notesǐ duration was elongated. nif is also known as one of auditory relay nucleus to hvc. part of the effects is possibly caused by disruption of auditory feedback. we also developed a neural network model of nif-hvc system. please refer yamashitaǐs poster for details of this model. the reason for the emergence of reward expectancy neurons suggested by a model using reinforcement learning and an artificial neural network katsunari shibata , shinya ishii , munetaka shidara dept. of e&e engineering, oita univ., oita, japan; grad. sch. of comprehensive human sci., univ. of tsukuba, tsukuba, japan in the experiment of multi-trial schedule task to obtain a reward, reward expectancy neurons, which respond only in the non-reward trials prior to the reward trial, have been observed in the anterior cingulate cortex of monkeys. it is difficult to explain directly by reinforcement learning why they do not respond in the reward trial. here, we interprets that such neurons emerge as an intermediate representation to generate appropriate value and actions in reinforcement learning by simulation analysis using a model that consists of an artificial recurrent neural network trained by reinforcement learning. the simulation result suggests that the reward expectancy neurons emerge to realize smooth temporal increase of the state value by complementing the neurons that respond only in the reward trial. [ ] s. ishii, et al., "a model to explain the emergence of reward expectancy neurons using reinforcement learning and neural network", neurocomputing, behavior is adjusted by outcomes of actions. to examine the neural mechanisms of the behavioral adjustment, we recorded single cell activity of the medial prefrontal cortex (mpfc) of two monkeys performing a behavioral adjustment task. the monkey searched a correct action (left or right lever press) on the basis of the two kinds of visual feedback, one (cs+) paired with a liquid reward and the other (cs−) that did not appear in a preceding pavlovian conditioning. cs+ followed a correct action and cs− followed a wrong action. when the monkey made more than consecutive correct trials, a new block of pavlovian conditioning started. we calculated the prediction errors provided by cs+ and cs− on the basis of a reinforcement learning model of action selection. we found that the neuronal activity corresponds to the prediction error of value of the selected action. this result suggests that mpfc contributes to behavioral adjustment by providing prediction errors of action values. makoto miyazaki , shinya yamamoto , sunao uchida , shigeru kitazawa , faculty of hum sci., waseda univ., tokorozawa, japan; neurosci. res. inst, aist, tsukuba, japan; faculty of sport sci., waseda univ., tokorozawa, japan; dept. of neurophysiol, juntendo univ. grad. sch. med., tokyo, japan; crest, jst, saitama, japan our judgment of temporal order of two sensory signals is not always fixed but subject to changes due to prior experiences, such as repeated exposure to a constant stimulus sequence. to date, such perceptual changes occurred so that signals in the order of the most frequent sequence are judged as simultaneous. in this study, we examined temporal order judgment of two tactile stimuli, delivered one to each hand, using stimulation intervals sampled from biased gaussian distributions (mean = ± ms, s.d. = ms). previous studies predict that the point of simultaneity would be shifted toward the peak of the gaussian, i.e. toward the most frequent interval. however, the point of simultaneity was shifted away from the peak by about ms. our results disagree with the previous studies, but conforms to a contrasting prediction from a bayesian integration theory. research funds: kakenhi ( ) ps a-k single measurement of oxy-and deoxyhemoglobin for a functional near infra-red spectroscopy ichiro shimoyama , fumiko sato , ken nakazawa , kenichi ono chiba university, japan; field of home economics, faculty of education, chiba univ., japan; department of integrative neurophysiology, graduate school of med. chiba univ., japan to study single dynamics for oxy-and deoxy-hemoglobin to a single task, we measured near infra-red spectroscopy (omm- , shi-madzu) over the frontal area ( channels) for volunteers ( - y). thirty tasks were presented visually every s, the subjects were asked to think about the question immediately following the sentences and asked not to think moreover if the question was difficult (e.g., how to cook curried rice? or how to fold paper into a turtle? etc). a comprehension-test was done just after the record. easy/difficult serial tasks were selected, and the oxy-and deoxy-hemoglobin differences between tasks were calculated to obtain correlation coefficients between the oxy-and deoxy-hemoglobin. grand averaged correlation coefficient was − . +/− . between the dynamics of the oxy-and deoxy-hemoglobin. the correlation should be considered in discussing neural activation for nirs. we thank shimadzu corp. for providing the nir station. kazuya ishibashi , , kosuke hamaguchi , masato okada , , department of complexity science and engineering, graduate school of frontier sciences, university of tokyo, kashiwa, japan; jst, japan; riken bsi, wako, japan a synfire chain is one of the networks which generate stable synchronous pulse packets. although the networks with a single stable synfire state is intensively analyzed by using several neuron models, the networks with several stable synfire states have not yet been investigated so thoroughly. by using leaky integrate-and-fire neuron model we construct a layered associative feedforward network embedded with several memory patterns. we analyse the network dynamics with the fokker-planck equation. first, we analyze the activity of the network when we activated one memory pattern of the first layer. we show that the layered associative network has stable synfire state. second, we investigate the activity when we activated different memory patterns. then we observe several characteristic phenomena, which are not observed in the conventional homogenous synfire chain. we will report the details of those phenomena. research funds: kakenhi ( ) and ( ) ps a-k auditory erps can be identified as corresponding stimuli by classifier with naive bayes method akitoshi ogawa , , sachiko koyama , , takashi omori , takashi morotomi research institue for electronic science, hokkaido university, sapporo, japan; japan science and technology agency, saitama, japan; graduate school of information science and technology, hokkaido university, sapporo, japan; sakushin gakuin university, utsunomiya, japan in an attempt to reversely estimate the input stimulus from measured erps, we developed computational classifier using naive bayes method. correct classification rates could be index values of the erp characteristic. in this study, we applied the classifier to identify auditory erps (n = ). the erps were elicited by tones ( hz) with different durations ( , , , , , ms) and gaps ( , , , , , ms) embedded in a continuous pure tone ( hz). to confirm the generality of the method, we used leave-one-out cross validation. erps of each subject were identified by the classifier which was constructed from the others' erps. as a result, the correct rates for and ms were high both for the tones ( ms, %; ms, %) and the gaps ( ms, %; ms, %). ps a-k determination of channel parameters for construction of a neural model of caenorhabditis elegans kazumi sakaa, akane andoh, taro ogurusu laboratory of bioscience, faculty of engineering, iwate university, iwate, morioka, japan caenorhabditis elegans (c. elegans) is one of the most suitable model animal for investigation of the relationship between the connection and the function of the neural network because its connection was revealed with the electronmicroscopy. on the other hand, it has been difficult to build a precise model neuron because the neuronal electrophysiological data of c. elegans has not been sufficient. we have been developing a precise neural model by extracting parameters required for model of voltage dependent channels from the electrophysiological data by the genetic algorithm with a neural simulator genesis and parallel genesis. using these simulation softwares, not only the optimum parameter set was determined for each channel but also the ratio of the conductances of several channles were determined. we report validity of obtained parameters and the possibility of the existence of unknown channel. supported by grant from jsps. ps a-k theoretical consideration about nmda current change and its effect on synaptic plasticity shigeru kubota, tatsuo kitajima department of bio-system engineering, yamagata university, yonezawa, japan it is well known that nmdar plays an important role in learning and memory. several experiments have shown that the property of nmdar epsc can change within a few weeks after birth, leading to the shortening of its decay time course. since the calcium current through nmdar is involved in ltp and ltd induction, it is possible that such change can work as the modulation of the plasticity rule or higher-order plasticity. here we show by the biophysical compartmental model that the alteration of nmdar property can modulate the calcium influx into the spine, which finally switches plasticity rule. we also show that this type of plasticity switch can promote synaptic competition and separate postnatal synapses rapidly into two groups of either strong or week ones. our results suggest that changing nmdar time course is very useful for the developing animals in order to promote fast and stable formation of the polysynaptic circuit. manish kumar jain department of psychiatry, r.d. gardi medical college, india introduction: i want to inform you regarding the some of challenges coming across my practice with the person with the psychiatric disorder in social rehabilitation like education and training, work and employment, family, groups, social, sexual, environmental and regional, coordination with the other health group and care giver, insurance problems, medical, physical, occipital vocational, languages problems mostly how to give oppurtinies with in the society and many more to be come in future. method: i keep the records with me since i join the medical college and my during practice but this is really challenging to calm down for question with their relatives and care givers. results: it is always to see the experience of the other people including self help groups in this regards and most challenging with near by perfect action and required more interaction with the rehabilitation groups because some are social problems in psychiatric disorder. conclusions: there is big challenge in the for social rehabilitation for the persons with psychiatric disorder as multifactor involvement s are there in this groups with early intervention and long term rehabilitation so that we can produced many working induals with in the society among the person with psychiatric disorder the more interaction among the society and care giver working in this field as well as neuroseiencents working in this field so that we will able to achieve almost complete social rehabilitation as till today we are not able to achieve social rehabilitation up to % till now. hepatic encephalopathy (he) refers to acute neuropsychiatric changes accompanying fulminant hepatic failure (fhf). in the present study we investigated changes in lipid composition of membranes isolated from cerebral cortex of rats treated with thioacetamide (taa), a hepatotoxin which induces fhf and thereon he. estimation of phospholipid fatty acid content in cerebral cortex membranes from taa treated rats revealed a decrease in monounsaturated fatty acid namely oleic acid and the poly unsaturated fatty acids ␥-linolenic acid, decosa hexanoic acid and arachidonic acid compared to controls. assesment of membrane fluidity with pyrene, , -diphenyl- , , -hexatriene, and -[ (trimethylammonio)phenyl]- -phenyl- , , -hexatriene revealed a decrease in annular membrane fluidity while the global fluidity was unaffected. the level of thiobarbituric acid reactive species-marker for lipid peroxidation also increased in membranes from taa treated rats indicating prevalence of oxidative stress. results from the present study demonstrate gross alterations in cerebral cortical membrane fatty acid composition and fluidity during taa induced he and their possible implications in the pathogenesis of this condition are also discussed. nagatoki kinoshita, shigenobu yonemura cellular morphogenesis, cdb, riken, kobe, japan rho-gtpases are well known as regulators of cytoskeletal reorganization and many cellular morphogenetic movements. however, little is known about their distributions and their physiological functions in vertebrates. immunohistology of chick embryos revealed apical accumulation of rho, rac and cdc in neural plate cells, especially in bending hinge points. after neural tube closure, the apical accumulation decreased. coordinately, activities of rho-gtpases and myosin ii in neural plate cells were higher during neurulation than after neural tube closure. inhibitions of actin filament formation, myosin ii-mediated contraction or rho-associated kinase activity affected neural tube formation. inhibition of rho activity induced the disruption of its apical accumulation and the defects of neural tube formation. these results suggest that rho-gtpases in an active form accumulate in the apical surface of neural plate cells and play important roles in neurulation. furthermore, we are screening regulators and effecters of rho-gtpases transiently expressed in neural plate cells during neurulation. setsuko sahara, dennis dm o'leary mnl-o, the salk institute, usa gradients of morphogens are postulated to establish the initial patterning of the mammalian forebrain, but little is known about their downstream targets and the mechanisms of patterning. here we report mouse buttonhead homogoues, the sp gene family, as candidates of downstream of those morphogens: sp expression correlates with wnts/bmps in the cortical hem, sp with fgfs in the cop, and sp with shh in the ventral midline and mge. by using in utero electroporation, we show that sp regulates anterior-posterior patterning of the cortex into areas by controlling distinct fgfs that having opposing effects. sp and fgf exhibit reciprocal induction, indicating that sp is a positive feedback regulator of fgf . surprisingly, though, ectopic expression of both sp and its dominant active form shift cortical areas in the opposite manner to fgf , suggesting that sp activates additional targets that overcome fgf function. our results indicate that fgf is an additional target of sp , showing effect on patterning similar to sp . these findings indicate that sp balance the proper cortical arealization through fgf and fgf . research funds: nihr ns ps p-c fyn-fak signal transduction is involved in the radial migration of late-generated neocortical neurons eiko nakahira , kotaro hattori , takeshi yagi , shigeki yuasa dept. ultrastructural res., nat. inst. neurosci., ncnp, tokyo, japan; kokoro biology group, fbs, osaka univ., suita, japan fyn tyrosine kinase posphorylates focal adhesion kinase (fak) that is involved in cell migration. taking into account the defective formation of neocortical layers ii-iii in fyn-deficient mice, fyn-fak signal transduction might be involved in the control of the migration of neocortical neurons. accordingly, we analyzed the neuronal migration in the mutant neocortex and compared the phenotypes to the changes induced by fak gene-knock down by foreign gene transfer by means of in utero electroporation. late-generated neocortical neurons exhibited defective radial migration in the mutant and this defect was rescued by the transfer of fyn-expression vector to the neocortical primordium. fyn and fak were colocalized in the migratory neurons, and fak sirna transfer into neocortical primordium induced migration defect similar to that in fyn deficiency. these findings strongly suggest that the coordination of fyn and fak is essential for the radial migration of late-generated neocortical neurons. noriyo ishibashi , kazuko keino-masu , tatsuyuki ohto , satoshi kunita , satoru takahashi , masayuki masu dept. of mol. neurobiol., grad. sch. of comprehensive human sci., univ. of tsukuba, tsukuba, japan; laboratory animal resource center, univ. of tsukuba, tsukuba, japan heparan sulfate (hs) proteoglycans regulate developmental patterning through the interactions with cell surface proteins and extracellular matrix molecules. these interactions are mediated by the specific hs structures generated by sulfation and epimerization. a recently identified extracellular sulfatase, sulffp , has been implicated in the regulation of growth factor/morphogen signaling through hs remodeling in vitro, but its physiological roles remain unknown. here we generated knockout mice lacking the sulffp gene, and examined the brain development. a previous study showed that the brain-specific disruption of the ext gene, which encode a hs synthesizing enzyme, led to severe brain defects including hypoplasia of the cerebral cortex and cerebellum. in this study, we thus examined the morphological changes of the cerebellum in the neonatal and adult sulffp -deficient mice. heparan sulfate (hs) proteoglycans play a crucial role in mediating important signaling by wnt, hedgehog and fgf. recently, novel sulfatases, sulffp /sulfatase- and sulffp /sulfatase- , which have hs -o-endosulfatase activity have been isolated. since these sulffps are detected in the extracellular space, sulffps are thought to regulate cell surface signaling through hs remodeling. in order to examine the function of sulffp genes in zebrafish, we isolated zebrafish sulffp and sulffp . here we report the isolation and the characterization of the third homologue, sulffp . sulffp has about % and % overall amino acid homology with sulffp and sulffp , respectively. at h postfertilization, sulffp is expressed in the ventral region of spinal cord, whereas sulffp is expressed only in the floor plate and sulffp is expressed in the lateral floor plate and ventral regions of spinal cord. detailed expression patterns of sulffp will be presented. masahiko ajiro, kenichi arai, mika maeda-sato, masuo obinata, wataru shoji dept. of cell biology, idac tohoku univ., japan collapsin response mediator proteins (crmps) are cytosolic proteins involved in neuronal differentiation and axonal guidance. a member of this family, crmp was shown to mediate the repulsive effect of sema a on axons. crmps appear to play more complex roles in axonal differentiation, elongation and branching during development. since less is known about their in vivo function, we studied their roles during development using transparent zebrafish embryos. at early axogenesis stage, zebrafish crmps are expressed in specific patterns. in trigeminal sensory ganglia, crmp , , , and are highly expressed. knocking down of these gene results in disorganization of the ganglia, separating into several clusters. however, their axonal patterns including direction, extension, and branching appears normal. same defects were observed in the knockdown of neuropilins, receptor component for class semaphorins. these results suggest that crmps may functionin keeping trigeminal neurons as a ganglia by mediating semaphorin-neuropilin signals. ps p-c developmental origin of diencephalic sensory relay nucley in teleosts y. ishikawa , n. yamamoto , m. yoshimoto , t. yasuda , k. maruyama , t. kage , h. takeda , h. ito nat. inst. rad. sci., chiba, japan; nippon med. sch., japan; tokyo univ., japan we propose a novel interpretation of the embryonic origin of cells of diencephalic sensory relay nuclei in teleosts, based on our studies in the medaka embryonic brain. it has been proposed that the relay system in teleosts is unique among vertebrates. teleost relay nuclei, the preglomerular complex (pg), have been assumed to originate from the basal plate (posterior tuberculum, pt) of the diencephalon, whereas relay nuclei in mammals are derived from the alar plate. our results show, however, that many pax -or dlx -positive cells migrate laterally and ventrocaudally from the diencephalic alar plate to the basal plate during development. massive clusters of the migrated alar cells become localize in the mantle layer lateral to the pt neuroepithelium, from which the pg appear to differentiate. we therefore consider most neurons in the pg are be of alar, not basal origin. thus, the teleost pg can be regarded as migrated alar nuclei. the organization of the diencephalic sensory relay system may have been conserved across vertebrates. hideyuki dekimoto, yoshihiro oomiya, satoshi kikkawa, toshio terashima, yu katsuyama department anatomy and developental neurobiology, kobe university graduate school of medicine laminaiton is one of features unique to the brain of vertebrates. to understand the evolution of layer formation in the vertebrate brains, we are studying genes which exhibit layer-specific expression. since one of ets family transcription factors, er is expressed specifically in the layer v of the mouse neocortex, we selected this gene for the purpose of our study. here we cloned zebrafish er homologue (zfer ), and found that the amino acid seuqence of the putative protein is highly conserved throughout the entire length. expression of zfer was observed in multiple sites of developing brain. the expression disappears sequentially in some sites, whereas it persisted in other sites until adult stage. er expressing sites in the brain was basically conserved between mouse and zebrafish, whereas expression pattern in each site (i.e. telencephalon, tectum) was different. based on these observations, evolution of the gene expression in the brain lamination will be discussed. hiroyuki koizumi, teruyuki tanaka, joseph g. gleeson university of california, san diego, usa doublecortin (dcx), encoding a microtubule-associated protein, is critical for neuronal migration, as mutations result in x-linked lissencephaly in hemizygous males and subcortical band heterotopia in heterozygous females, whereas in mouse, rnai-mediated knockdown but not germline knockout shows abnormal positioning of cortical neurons. dclk (doublecortin-like kinase) is one of the homologous genes of dcx, encodes for protein with an n-terminus that is % identical to dcx, but also additional c-terminal protein kinase domain. here, we report that the dclk functions in a partially redundant pathway with dcx in the formation of axonal projections across the midline and migration of cortical neurons in mouse. dosagedependent genetic effects were observed in both interhemispheric connectivity and migration of cortically and subcortically derived neurons. rnai-mediated knockdown of either gene results in similar migration defects. these results indicate the dcx microtubuleassociated protein family is required for proper neuronal migration and axonal wiring. hiraki sakuta , , hiroo takahashi , , takafumi shintani , , kazuma etani , masaharu noda , div. of mol. neurobiol., nibb, okazaki, japan; crest, jst, japan in the developing chick retina, the expression of bmp is relieved by that of bmp at around e with a change from a dorsal high to dorsotemporal high pattern, complementary to that of ventroptin, a bmp antagonist. we previously demonstrated that misexpression of ventroptin altered the retinotectal projection along both the dv and ap axes. here, we show that topographic molecules along the dv axis, together with ephrina , are expressed in a double-gradient fashion from e on like ventroptin and bmp . when bmp expression is manipulated by using the gene-specific knockdown and the reagent-inducible gene expression techniques, the expression patterns of these double-gradient molecules are all changed. moreover, in the bmp knockdown and ephrina -misexpressing embryos, the retinotectal projection is altered along the two axes. the expressional switching from bmp to bmp thus appears to play a key role in retinal patterning and consequently in topographic retinotectal projection, by changing the direction of the dv axis toward the posterior side during retinal development. noriyuki morita, teiichi furuichi lab. for molecular neurogenesis, riken-bsi, wako, japan the mammalian cerebellum is anteroposteriorly and mediolaterally compartmentalized at the level of neuroanatomy and also at the level of gene expression. to elucidate the molecular mechanisms underlying the establishment and the maintenance of functional cerebellar compartment, genes responsible for mouse cereballar development transcriptome were examined for patterned expression in cerebellum by whole-mount in situ hybridization. not a few known and novel genes were found to be expressed in parasagittal band pattern in the embryonic mouse cerebellum, which could be categorized as "early-onset-genes". parasagittally expressed genes were classified in comparison with the band pattern of en , wnt b and pcp /l gene expression in declival vermal lobule, to investigate the correlation between spatial expression profiles and transcriptional regulatory elements. our accumulating data suggest that not only patterning genes like engrailed and wnts, also genes related in later events in neural development such as synaptogenesis are expressed as earlyonset-genes. yasufumi tanaka, tomiyoshi setsu, hideyuki dekimoto, yu katsuyama, toshio terashima kobe university graduate school of medicine, japan the nissl staining of the brains of the adult reeler and normal mice showed that the size of the pontine nuclei (pn) was reduced in the reeler compared with the normal counterpart. the injections of dii and di- asp into the left and right hemicerebellum, respectively, resulted in that only a few pn neurons were doubly labeled in the control, but in the reeler most of pn neurons were doubly labeled. the placements of solutions of dii and di- asp into the left and right cerebellar peduncles of paraformaldehyde-fixed brains resulted in that dii-labeled or di- asp-labeled pontocerebellar fibers made a fascicular formation in the cerebellum of the normal mouse, but such a fascicular formation was not recognized in the reeler and labeled terminals of mossy fibers were randomly arranged along the course of the pontocerebellar projection. reelin mrna and reelin were both expressed in the pn of the normal mice. these data elucidate that the reelin may play a key role in fasciculuation and collateral formation of pontocerebellar projections in addition to cell positioning or migration of pn neurons. kudoh suguru , , takahisa taguchi aist, ikeda, japan; presto, jst the spatiotemporal patterns of spontaneous action potential were analyzed, using the multi-site recording system for extracellular potentials of neurons and the living neuronal network cultured on a -dimensional electrode array. the map of functional connections between neurons revealed that each culture contained some hublike neurons and the distribution of the number of functionalconnections approximated a power-law distribution. we confirmed that the spatiotemporal pattern of spontaneous action potentials became more complex pattern along with developmental stage, and the constant pattern of stimulation promote this developmental change. in addition, the spatiotemporal pattern and the functional connections between neurons were drastically re-organized by real-time feedback stimulation. these results strongly suggest that the network structure of the cultured hippocampal neurons is neither stable nor random, but is functionally dynamic and is suitable for certain types of information processing. research funds: presto, jst ps p-d laterality of the human cerebral hemisphere taiko kitamura, jinzo yamada department of anatomy, tokyo medical university, tokyo, japan it has been reported that some functional predominance is located in the right or left hemisphere of the human brain. especially, the speech center and the center related to thought and emotion are located in the left and in the right hemisphere, respectively. in this study, the laterality between the right and the left human hemisphere was investigated macro-anatomically. we measured the weight, the medial-lateral width (m-l), the anterior-posterior lenght (a-p), and the width of the medial surface in the right and the left human hemisphere using in anatomical practice for medical students. the weight of each hemisphere was roughly equal. the m-l was wider in the right side than the left side. the a-p was longer and the width of the medial surface was larger in the left side than in the right side. because of the longer a-p and the larger width of the medial surface in the left hemisphere, it appeared that the left hemisphere overspreads the medial-dorsal marginal surface of the right hemisphere by the naked eye. such overspreading suspects that the left hemisphere develops earlier and faster than the right hemisphere. ps p-d synchrony-induced transition behaviors organized under spike-timing dependent plasticity for retrieving the memorized patterns takaaki aoki , toshio aoyagi department of physics, kyoto university, kyoto, japan; graduate school of informatics, kyoto university, kyoto, japan temporally correlated spikes, such as spike synchrony, have been observed in relation to behaviors or cognitions. however, it is unclear how the neurons read out the incoming spike synchronization in the dynamical behavior of network. in this modeling study, considering a network of excitatory and inhibitory neurons organized under spiketiming dependent plasticity, we present a type of network model in which incoming spike synchrony causes a transition between learned activity patterns in the order they were experienced in the learning process. furthermore, using appropriate training patterns, this network exhibits a context-dependent transition, in which the network switches to multiple patterns from a single pattern depending on the temporal structure of neuronal activity at the onset of incoming spike synchrony. this ability of the network may provide one of mechanisms by which a neuronal system can be trained to carry out tasks in a context-dependent manner. shozo kito, maiko kitagawa, akiko shingo lab. of neurosci., hyogo univ., kakogawa japan in our previous studies, we showed that a part of nicotine's beneficial effects on hippocampal and cortical neurons were due to increased igf- mrna expressions. nevertheless, the situation may be somewhat different as far as nicotine's effects on the neuronal progenitor cell, which is still on the way of differentiation are concerned. to clarify this problem, nicotine was intraperitoneally injected into weekold wistar strain rats in several doses followed by successive injections of brdu for the next days. then rats were sacrificed and vertical sections of the hippocampus formation were offered for double immunohistochemical staining of brdu/psa-ncam, brdu/neun or brdu/gfap. as the results, numbers of both brdu(+)/psa-ncam(+) cells and brdu(+)/neun(+) cells were much decreased nicotine-dose dependently. on the other hand, as much as mg/kg was needed for nicotine to exert its effect on the number of brdu(+)/gfap(+) cells. these results reveal that nicotine inhibits neurogenesis and plasticity in the hippocampus of adult rats. ps p-d the establishment of the organotypic slice culture of postnatal rat forebrain involving egfp-labeled neural progenitors kaoru sato , james e. goldman division of pharmacology, national institute of health sciences, tokyo, japan; department of pathology, columbia university, new york, usa after injecting egfp-encoding retrovirus into p rat svz, sagital sections of forebrain were made at p and cultured for days. the migration pattern of the egfp-labeled neural progenitors in the cultured slices is almost same as that at the corresponding age. the expression patterns of the glial differentiation-markers were also in accordance with those at the corresponding age. when slices were cultured with anti-␣ integrin antibody, the migration of the neural progenitors inside svz was significantly enhanced along the rostrocaudal extent. these results suggest that the organotypic slice culture of postnatal rat forebrain is an efficient experimental system for pharmacological studies about migration and differentiation of neural progenitors. radial glia is involved in the contact guidance of neuronal migration and also the neuronal and astroglial precursors. to make clearer the role of radial glia, we developed a method for the selective ablation of a subset of radial glia. it has been reported that tenascin-c (tn-c) is one of the markers for radial glia. accordingly, diphtheria toxin (dt)gene and enhanced green fluorescence protein (egfp)-gene both driven by tn-c gene promoter were co-transferred into the ventricular zone cells of the mouse neocortical primordium by means of in utero electroporation. the numbers of egfp-labeled cells in that tn-c gene promoter and subsequently dt gene are activated selectively decreased by this approach. using this method, the examination of radial glial morphology and neuronal migration following selective ablation is in progress. takayuki manabe, kouko tatsumi, eri makinodan, manabu makinodan, takahira yamauchi department of nd anatomy, nara medical university, kashihara, nara, japan it has been well documented that neurogenesis persists at the subventricular zone and the subgranular layer of the dentate gyrus in the adult mammalian brain. in the adult mice, we demonstrated that cells around a cryo-injured cortical lesion had a proliferative activity (labeled with brdu in vivo) and formed neurosphere-like aggregates in the sphere-forming culture condition. significantly lager number of spheres was observed in the culture from the injured hemisphere, which excluded the neurogenic regions (i.e. the svz and hippocampus), than those cultured from the control (contralateral and intact) hemisphere. furthermore, the sphere-forming cells differentiated to neuronal-and glial-marker positive cells in vitro. these results suggest that the cells forming sphere-like aggregates in vitro may function as a kind of progenitor cells in the injured brain. if this is a case, it would be tempting to transplant these sphere-forming cells to cure brain injury or disease. further characterization of the cells is underway. ps p-d localization of neurotrophin receptors trka in pc cells: d reconstruction analysis of membrane proteins tomoki nishida , hiroshi jinnai , tatuo arii , akio takaoka , ryoichi yoshimura , yasuhisa endo department of applied biology, kyoto institute of technology, kyoto, japan; department of polymer science and engineering, kyoto institute of technology, kyoto, japan; national institute for physiological sciences, myodaiji, okazaki, japan; osaka university, mihogaoka, ibaraki, osaka, japan it was previously reported that trka (ngf receptor) was associated with caveolae, small invaginations on the cell membrane, but its subcellular localization is not clarified in detail. we performed immunocytochemistry of trka and caveolin- in pc cells, analyzed by high-voltage electron microscopy, and reconstructed d structure of their subcellular distribution by imod. our results indicated that localization of caveolin- , known as an integral membrane protein of caveolae, was never found in the invagination structure in pc cells, but trka and caveolin- immunoreactivities were mainly found as a mesh-like structure in the cytoplasmic matrix. kensuke shiomi, kazuko keino-masu, masayuki masu department of molecular neurobiology, graduate school of comprehensive human sciences, university of tsukuba, tsukuba, japan the wnt signaling plays important roles in cell growth, differentiation, polarity formation, and neural development. previously we identified ccd , a third-type of the dix domain-possessing protein, as a positive regulator of the wnt/␤-catenin pathway. ccd mrna was mainly detected in the neural crest derivatives and differentiated neurons in mouse embryos, suggesting the importance of ccd in the wnt-mediated neuronal development. there are three subtypes of mouse ccd gene products, ccd a, ccd b and ccd c, which are generated by different promotor usage. mouse ccd a as well as zebrafish ccd a has a calponin homology domain which can mediate the interaction with the actin cytoskelton. we found that in the ccdtransfected hela cells, only the type a ccd proteins co-localized with the actin filament. in order to examine the function of the type a ccd proteins, we are now doing overexpression and functional blocking experiments using zebrafish embryos and cell culture. research funds: kakenhi ( , ) ps p-d analysis of a role of r-spondin on proliferation of the cortical neuroepithelium yumiko hatanaka , masahiro yamaguchi , fujio murakami , masayuki masu grad. school of comprehensive human sci., univ. of tsukuba, japan; grad. school of med., univ. of tokyo; grad. school of frontier biosci., osaka univ r-spondin (rspo ) is a secreted activator of wnt/␤-catenin signaling (kazanskaya et al. ) . rspo is expressed in the developing medial cerebral wall and transgenic mice expressing rspo in the entire neuroepithelium show enlarged lateral ventricle with a slight increase of brain size (hatanaka et al. ) . since wnt a has a role for expansion of caudomedial cortical progenitor cells (lee et al. ) , these findings lead us to the idea that rspo may synergistically promote proliferation of cortical neuroepithlial cells together with wnt a. to clarify their role on proliferation of cortical neuroepithelial cells, we first introduced a ␤-catenin/tcf reporter gene into these cells of embryonic day . mouse. an application of wnt a on these cells increased level of the reporter expression, and an addition of rspo further increased its level. we are now monitoring incorporation of brdu in neuroepithelial cells to know whether wnt a and rspo directly promote their proliferation. tae sun kim, hideki hida, tomoko narita, sachiyo misumi, hitoo nisino department of neurophysiology & brain science, nagoya city university graduate school medical sciences, nagoya, japan to investigate whether physiological low oxygen during development and cytokines expressed in the dopamine (da)-depleted striatum increase the number of da neurons from es-derived neural progenitor cells (npcs), npcs were treated with cytokine cocktail (il- ␤, il- , lif, gdnf) or lowered o ( . %), followed by tyrosine hydroxylase (th) immunostaining. low oxygen increased total number of th (+) cells ( . -fold) as compared to normal o . cytokine cocktail significantly increased th (+) cells ( . -fold) compared to nontreated control. treatment of lif and il- ␤ to npcs exhibited major contribution in the effect of cytokine cocktail. data suggest that physiologically relevant low oxygen in development and cytokines and trophic factors that were enhanced in da-depleted striatum cause in the increase of daergic neurons from es-derived npcs. ps p-d structural basis for reelin signaling: determination of receptor-binding site and its three-dimensional structure norihisa yasui , terukazu nogi , mitsuharu hattori , kenji iwasaki , , junichi takagi research center for structural and functional proteomics, inst for protein res., osaka univ., suita, japan; dept. of biomed. sci., grad. sch. of pharm. sci., nagoya city univ., nagoya, japan; core research for evolution and technology (crest) a large secreted glycoprotein reelin acts on target neurons through its receptors (apoer and vldlr), resulting in tyrosine phosphorylation of dab . in the present study, we have carried out structural and functional studies on the reelin signaling. first, we determined the structure of a single reelin repeat by x-ray crystallography. it had a horseshoe-like globular structure with some similarities to carbohydrate binding modules from many enzymes. moreover, electron micrographic d reconstruction of four-domain reelin fragment (i.e. r - ) revealed an elongated rod-like structure. next we determined minimum active unit within reelin. a fragment containing both the fifth and sixth reelin repeats (r - ) was capable of binding to the receptor (apoer ), and was also able to induce tyrosine phospholylation of dab in primary neuronal culture. ps p-d effects of astrocyte-derived factor and cell-cell communication on uni-directional differentiation from mouse embryonic stem cells into neural cells embryonic stem (es) cells uni-directly differentiate into neurons via neuroectoderm and neural stem cells by neural stem sphere (nss) method. cultured with astrocyte-derived factor, colonies of es cells give rise to nsss. we analyzed structure and gene expression of cell spheres formed under various culture conditions, in order to elucidate mechanisms of the uni-directional differentiation into neurons. quantitative real-time rt-pcr analysis demonstrated that the neuronal differentiation did not occur in the cell spheres. these results suggest that astrocyte-derived factor and cell-cell communication are necessary for the differentiation. we have previously established es cell differentiation system, by which we can derive neurospheres containing neural stem/progenitor cells (ns/pcs) with the identity of early caudal neural tube. taking advantage of this culture system, we have recently found conditioned medium of a stromal cell line (cmsc) has the activity to support the formation of neurospheres. this activity was more prominent when cultured at low cell density than when cultured at high cell density, suggesting that it supports the survival of ns/pcs. moreover, rt-pcr analysis of regional identities of the cmsc treated neurospheres revealed elevated expression of pax and pax compared with those of untreated neurospheres, indicating that cmsc promotes dorsalization of ns/pcs or selective proliferation of dorsal ns/pcs. elucidation of underlying mechanisms may provide important tools to derive early ns/pcs which can generate variety of projection neurons and be applicable to regenerative medicine. research funds: sorst jst ps p-d neudesin, a secreted factor, promotes neural cell proliferation and neuronal differentiation in mouse neural precursor cells neudesin expressed in adult mouse brain encodes a secreted signal with neurotrophic activity in neurons (j neurosci res : , ) . most neurotrophic factors are involved in neural cell proliferation and/or differentiation. however, the role of neudesin in neural development remains to be elucidated. neudesin mrna was expressed in the neural precursor cells before the appearance of neurons. therefore, roles of neudesin in neural development were examined using the neural precursor cells. neudesin significantly promoted neuronal differentiation. in addition, neudesin transiently promoted neural cell proliferation early in the developmental process. the differentiation was mediated though activation of the pka and pi- k pathways. in contrast, the proliferation was mediated through the mapk and pka pathways. the expression profile and activity indicate that neudesin plays unique roles in neural development. ps p-d fabp is required for maintenance of neural stem/progenitor cells in the postnatal hippocampus motoko maekawa , miho matsumata , , yuji owada , shigeki yuasa , noriko osumi , natl. inst. of neurosci., ncnp, tokyo, japan; tohoku univ. sch. of med., sendai, japan; crest, jst pax transcription factor is a key player for brain patterning and embryonic neurogenesis, and also expressed in the postnatal brain. we have previously shown that pax is necessary for keeping neural stem/progenitor cells in the hippocampus. in this study we have focused on a fatty-acid binding protein fabp , a downstream of pax , regulating maintenance of embryonic neural stem/progenitor cells (arai et al., ) . fabp was expressed in neural stem/progenitor cells in the hippocampal dentate gyrus (dg). % of fabp -expressing cells co-expressed gfap (a marker for early progenitors), and % of them co-expressed psa-ncam (a marker for late progenitors). fabp expression was also overlapped with pax , and expression of fabp was down-regulated in the dg of pax deficient rats and mice. finally, brdu-labeling analysis revealed decreased cell proliferation in the dg of fabp knockout mice. taking all together, it is concluded that fabp is required for maintenance of neural stem/progenitor cells in dg. ps p-d involvement of the psa-ncam expressing cells in early development of the vascular system of the forebrain momoko miyakawa, tatsunori seki department of anatomy, juntendo university school of medicine, tokyo, japan early development of the vascular system of the forebrain were studied in the chick embryo. staining of vascular endothelial cells by fitctomato lectin and immunohistochemical staining of the surrounding cells were performed on the same cryostat sections of embryos of embryonic day - . sections were examined under a confocal laser scanning microscope. capillaries were found in the lateral pallium and seemed to grow from psa-ncam-positive outer zone to negative inner zone of the pallium. psa-ncam is thought to be expressed in the immature neurons. the rims of capillaries were immunoreactive with psa-ncam in both zones. immunoreaction of doublecortin (neuronal marker) and punctate immunostaining of laminin also were observed on rims of capillaries. by immuno-electron-microscopy it appeared that the endothelium were covered with very thin processes of cells of which outer surface was immunoreactive with psa-ncam. psa-ncam expressing cells may be involved in the development of the vascular system of the forebrain by supporting or guiding the growing capillaries. masaharu kotani , , shiki okamoto , masato imada , kouichi itoh , atsushi irie , hitoshi sakuraba , hideo kubo department of molecular biologu, ohu univ., koriyama, japan; dept. deve. physiol., natl. inst. physiol. sci., okazaki, japan; dept. anatomy, nihon univ. shl. med., tokyo, japan; dept. mol. pharma., univ. tokushima bunri, sanuki, japan; dept. biochem. cell res., tokyo metro. inst. med. sci., tokyo, japan; department of clin. genet, tokyo metro. inst. med. sci., tokyo, japan; dept. med. biol, tokyo metro. inst. med. sci., tokyo, japan as randam- shows the highest expression level with the proliferating stage of neural stem cells (nscs), it is thought that the isolation of nscs based on the expression level of randam- is possible. in the present, we show that the isolated randam- high+ cells enrich nscs. the randam- high+ cells had the characteristics as the highly self-renewal capability and potential for multilineage differentiation into neural cells. in contrast, almost all of the randam- low+/− cells exhibited not only the extremely low self-renewability but the differentiation capability restricted to neurons. the results demonstrate that randam- is a usefule marker for the isolation of nscs by facs. yasuharu takamori , yasuhisa tamura , , yosky kataoka , , yilong cui , , hisao yamada department of anatomy and cell science, kansai medical university, osaka, japan; department of physiology, osaka city university graduate school of medicine, osaka, japan; morecular imaging reserch program, riken frs, saitama, japan lamins are major structural proteins of nuclear envelope. three lamin subtypes, a/c, b and b are mainly present in mammalian somatic cells. to investigate the pattern of lamin expression during neuronal differentiation, we immunohistochemically analyzed the existence of lamins in two neurogenic regions of rat brain; subgranular zone of dentate gyrus and subventricular zone, with confocal microscopy. gfap-positive primary progenitor cells possess lamin a/c (++), b (++), b (++), psa-ncam-positive subsequent progenitor cells possess lamin a/c (−), b (+++), b (+), and mature neurons possess lamin a/c (++), b (+), b (+++), in both neurogenic regions. these observation showed that the composition of lamin subtypes was distinct in particular differentiation stages during adult neurogenesis. yusuke tozuka , yuichi tanaka , tatsuhiro hisatsune department of integrated biosciences, university of tokyo, chiba, japan recent work has shown that nestin + neural progenitor cells exist in the adult brain, and suggested that neural activity itself could act directly on these progenitor cells. it has been unclear, however, how do adult progenitor cells sense activity signals from surrounding neural circuit. in the hippocampus where new neurons are continuously produced throughout life, nestin + adult progenitor cells received gabaergic inputs. the gabaergic activity depolarized these progenitor cells, and then promoted their neuronal differentiations. although neuronal production does not readily occur in the adult neocortex, nestin + neural progenitor cells exist in this area too. interestingly, these progenitor cells also received excitatory gabaergic inputs. this gabaergic inputs inhibited their cell proliferations. from these results, we here propose that adult progenitor cells are a direct target of gabaergic neuronal networks, and that this networkto-progenitor cell interaction influences progenitors development by regulating their cell proliferations and/or neuronal differentiations. ps p-e new migration pattern in the postnatal neurogenesis of the dentate gyrus takashi namba , , hideo namiki , tatsunori seki dept. of anat, juntendo univ. sch. of med., tokyo, japan; integrative biosci. and biomed. eng, sch. of sci. and eng, waseda univ., tokyo, japan in the hippocampus, granule cells continue to be generated from embryonic to adult stages. the early postnatal neurogenesis is a transitional state between the embryonic and adult neurogenesis. previously, we have suggested that the postnatal hilus contains astrocytic neural progenitors that divide and differentiate into neuroblasts, and that finally the neuroblasts settle in the granule cell layer (gcl). however, the questions remain how astrocytic progenitors divide and differentiate into neurons, and how the neuroblasts migrate to the gcl. to observe them, we developed a time-lapse imaging system. retrovirus-gfp was injected into the rat hippocampus at p . three days after the injection, the hippocampal slices were prepared for the time-lapse imaging. the present data show that neuroblasts migrate from the hilus to the gcl, changing the direction of their movement. this is inconsistent with the previous report suggesting simple radial migration (rickmann, et al., ) . the dividing pattern is currently under investigation. akiya watakabe , noritaka ichinohe , sonoko ohsawa , tsutomu hashikawa , kathleen s. rockland , tetsuo yamamori div. of brain biol, nibb, okazaki, japan; lab. for cortical organization and systematics, bsi, riken, wako, japan, lab. for neural architecture, bsi, riken, wako, japan by using gene expression profiles, we have tried to classify layer neurons in several areas of monkey neocortex. we previously reported that nurr , ctgf and sema e mrnas are specifically expressed in subsets of layer neurons. we further show here that cholecystokinin (cck) mrna is expressed in a subset of excitatory neurons in layer . by double ish, layer neurons in monkeys are roughly divisible into cck(+) and sema e(+) subgroups. each subgroup was further subdivided by other markers. tracer experiments showed that cck and sema e mrna expression correlate well with corticocortical and corticothalamic connectivity, respectively, but the correlation was only partial. from this, we infer that subtypes defined by gene expression may not directly correspond to classical neuronal types. the implication of our findings will be discussed in terms of constancy of laminar structure across areas and species. research funds: kakenhi ps p-e rbp-j regulates the cortical laminar formation kenji tanigaki , kazue muraki , norio yamamoto , tasuku honjo shiga medical center, research institute, shiga, japan; department of medical chemistry, kyoto university, kyoto, japan precise patterns of cell cycle exit and migration of neural progenitors are crucial for the formation of cortical layer structure. to examine involvement of notch-rbp-j signaling in the cortex laminar formation, we deleted rbp-j from neural progenitors in anatomically restricted areas by in vivo electroporation of cre-expressing plasmids. such studies revealed that rbp-j deficiency caused transformation of glutamatergic pyramidal neurons in layer ii/iii to layer iv neurons with concomitant loss of astrocytes. the loss of rbp-j accelerated neuronal differentiation and changed their laminar fates. in addition, time-lapse studies indicated the migration defect of rbp-j-deficient neurons. the results showed that notch-rbp-j signaling regulates migration of differentiated neurons as well as the timing of the cell cycle exit of neuronal progenitors to determine the laminar and cellular fates of neural progenitors. ps p-e search for the genes that define mammalian cortical progenitor cells using single-cell gene expression profiles ayano kawaguchi , tomoko ikawa , yuya kasukawa , hironori ueda , , kazuki kurimoto , michinori saitou , fumio matsuzaki , lab. for asymmetric cell division, cdb, riken, kobe, japan; functional genomics subunit, cdb, riken, kobe, japan; lab. for systems biology, cdb, riken, kobe, japan; lab. for mammalian germ cell biology, cdb, riken, kobe, japan; crest, jst, japan in the mammalian brain, cellular heterogeneity of the progenitor cells has largely hindered the molecular analysis of neuronal diversity. to overcome this problem, we randomly picked individual vz/svz cells of mouse embryos, and constructed cdnas from each of them by global pcr amplification method. we could classify these "single cell derived cdnas" into several groups retrospectively based on the expression of marker genes, including cell cycle related genes, transcription factors, and regional marker genes. samples that showed typical marker gene expression pattern of the groups were applied for genechip analysis. the obtained data were confirmed by quantitative pcr and in situ hybridization. by this strategy, we identified nine genes that were specifically expressed in the svz progenitor cells. research funds: kakenhi ( ) ryosuke tatsuno , tomoaki sai , , masahiro otsu , kuniko akama , takashi nakayama , tosifusa toda grad. sch. of sci. and tech., chiba univ., chiba, japan; lab. regener neurosci., tokyo metropol. univ. fac. health sci., tokyo, japan; dept. orthop. surg., jikei univ. sch. med., tokyo. japan; dept. biochem., yokohama city univ. sch. med., yokohama, japan; proteomics collab. res., tokyo metropol. inst. of gerontol., tokyo, japan embryonic stem (es) cells possess pluripotency and self-renewal. however, the proteomic analysis of neural stem cells and neurons differentiated in vitro from es cells has not so proceeded yet. we investigated the expression levels of proteins during in vitro differentiation of mouse es cells into neurons via neural stem cells by neural stem sphere (nss) method, using -d gel electrophoresis and maldi-tof ms. we identified vimentin, creatine kinase, atp synthase beta subunit, and some proteins with no annotation in murine brain the database, which were up-regulated in neural stem cells, and down-regulated in es cells and neurons. these results suggest that the neural stem cells have characteristic protein expression profile. ps p-e identification of se , a novel gene expressed in the nural progenitor cells shin-ichi sakakibara, kazuhiko nakadate, shiichi ueda department of histology and neurobiology, dokkyo university school of medicine, tochigi, japan identification of the genes regulating neural progenitor or neural stem cell functions is critical to understand the mechanisms of the adult neurogenesis and neurodegenerative disease. we compared the gene expression profile of proliferating neural stem cell cultures with those of differentiated cells. a subtractive library was constructed by using the suppression subtractive hybridization and the differential screening was performed. among two thousand of the differentially expressed subtracted clones, we identified genes that significantly upregulated in neural stem cell culture. these included several novel genes, in addition to the known genes involving in the cell cycle and signal transduction. in situ hybridization and the developmental northern analysis demonstrated that these mrnas were enriched in the germinal neuroepithelium, embryonic ventricular zone and the postnatal subventricular zone surrounding the lateral ventricles. we further analyzed the expression pattern of the novel gene se in developing and matured cns. teiichi furuichi , akira sto , , yukiko sekine , noriyuki morita , tetsushi sadakata , satoshi shoji , jin-hong huang , toshio kojima laboratory for molecular neurogenesis, riken brain science institute, japan; comparative systems biology team, riken genome sciences center, yokohama - , japan mouse cerebellum develops through a series of cytogenetic and morphogenetic events that are genetically coded within the first three weeks of life. we have extensively investigated the spatio-temporal gene expression profiles during the postnatal development of mouse cerebellum by differential display, rt-pcr, genechip, cdna microarray, and in situ hybridization. we have informatively systematized all the profiles in an online neuroinformatics database cdt-db (http://www.cdtdb.brain.riken.jp) with various search functions. we have demonstrated that the postnatal development of mouse cerebellum is genetically programmed by thousands of genes that exhibit differential expression patterns in time and space. further studies on a scale that includes the underlying expression of all genes and more detailed studies on their transcriptional regulation will shed light on the genetic basis for cerebellar development. miwako ozaki , makoto mizuno , kazuhisa sakai , yoshimoto kiyohara , kazuhiko yamaguchi , tsutomu hashikawa , hiroyuki nawa institute of biomedical engineering, waseda university, tokyo, japan; department of molecular neurobiology, brain research institute, niigata university, niigata, japan; laboratory for memory and learning, bsi, riken, saitama, japan; laboratory for neural architecture, bsi, riken, saitama, japan neuregulin (nrg), a neurotrophic factor, involved in the development, differentiation and repair of the nervous system, regulates the activation of ion channels and neurotransmitter receptors. in order to examine the molecular mechanism on the relationships between network, synapse formations and higher orders functions, we prepared ig-nrg knock out mice (nrg type i and iv were disrupted). the mutant mice showed motor disco-ordination and abnormality of synaptic structure in related areas in cerebellar nuclei and cortex. in addition, the number of vesicles in presynaptic neurons decreased in their synapses. the study on cerebellum that is very clear in the network input information would give some suggestions to the relationship between synaptic functions and behaviors. ps p-e psd- protein expression in rat oromaxillofacial motoneurons during postnatal development kohji ishihama , , satoshi wakisaka , shiho honma , akira ito , , kei azuma , , mikihiko kogo department of oral anatomy and developmental biology, osaka university graduate school of dentistry, osaka, japan; first department of oral and maxillofacial surgery, osaka university graduate school of dentistry, osaka, japan postsynaptic density (psd), which is composed of diverse proteins, involved in synaptic structure, neurotransmission and signal transduction. psd- implicates in formation and maturation of excitatory synapses. psd- regulates the localization of the nmda receptor by means of binding with nr . rhythmical oro-maxillofacial activities, such as suckling and chewing, are generated in the brainstem, and we showed that nmda receptors played critical role for the rhythm and pattern generation and signal transmission around the trigeminal motor nucleus during prenatal and early postnatal development. here we examined the temporal distributions of psd- protein using with immunohistochemical study, in developing rat brainstem from suckling to mature chewing stage. there was early emergence of psd- expression in the interneurons located at medial of the trigeminal motor nucleus. masami miura, masao masuda, toshihiko aosaki neural circuits dynamics research group, tokyo metropolitan institute of gerontology, japan the striatum, an input stage of the basal ganglia, contributes to habit formation as well as motor functions. recent studies suggest that striatal interneurons play an important role in processing of cortical input. we investigated the synaptic connections between interneurons using paired whole-cell recordings and immunohistochemical techniques. we found that fast-spiking (fs) interneurons sent gabaergic inhibitory input to cholinergic interneurons, which were gaba a receptor-mediated and suppressed by gaba b receptor agonist skf . in turn, cholinergic interneurons sent cholinergic excitatory input to fs interneurons. because the excitatory postsypnatic potentials (psps) were blocked by hexamethonium and dihydro-␤-erythroidine, the psps were nicotinic acetylcholine receptor-mediated. these results suggest that gabaergic interneurons and cholinergic interneurons mutually influence their excitability and might modulate the activity of striatal local circuits. ps p-e ocular following responses (ofrs) to a brief background motin are modulated in relation to preparation for upcoming pursuit hiromitsu tabata, kenichiro miura, kenji kawano dept. integ brain sci., grad. schl of med., kyoto univ., kyoto, japan recently, our group reported that the ocular responses to a brief perturbation of a small target during fixation increased when subjects (humans, monkeys) were preparing for upcoming smooth pursuit eye movements (spems) rather than preparing for saccades or stationary fixation. here, we report that the increase in ocular responses based on the anticipation of spems was also observed in monkeys when a large-field visual stimulus (background) was moved briefly prior to pursuit. the result indicates that the visual region where the gain of the visuomotor transmission increased is not limited to a small region near the target but spreads to a larger field. in other words, the anticipation of upcoming spems could affect the generation of ofrs. furthermore, directionally biased ocular responses to the brief background motion were observed when the animals repeatedly performed spems toward one direction, implying that the prediction of the upcoming spem direction might cause the directional asymmetry of the visuomotor transmission gain. ps p-e comprehensive characterization of motor neurons related with locomotory central pattern generator in the earthworm by imaging toshinobu shimoi , kenji mizutani , hiroto ogawa , kohji hotta , kotaro oka ctr. for biosci. and info, keio univ., yokohama, japan; neuro, karolinska inst, stockholm, sweden; bio, saitama med. sch., saitama, japan in this study, we comprehensively identified and characterized motor neurons concerning with locomotory central pattern generator (cpg) in the earthworm by calcium imaging as multiple recording. the candidates of motor neurons were stained with dextran conjugated calcium indicators using retrograde labeling from projection nerves. we obtained the responses of up to cell bodies of motor neurons and sensory neurons on the ventral surface of the segmental ganglion ( % or less for all neurons on the ventral surface). we analyzed the activity patterns of the candidates of motor neurons using pattern matching method comparing between calcium responses or between calcium responses and locomotory motor pattern. as a result, we detected motor neurons as pairs of neurons having strong synchrony to each other neuron or to motor pattern. these results were great progress to identify motor neurons related with locomotory cpg in the earthworm. ps p-e three dimensional ( d) pursuit eye movement signals in cerebellar dorsal vermis takuya nitta, teppei akao, sergei kurkin, kikuro fukushima department of physiology, hokkaido university school of medicine, sapporo, japan for pursuit of a target moving in d space, signals for frontal and vergence-pursuit must be synthesized. studies in our laboratory have demonstrated that d pursuit signals are generated in the frontal eye fields, and also present in cerebellar floccular region. however, the majority of floccular purkinje (p-) cells discharged after onset of vergence-pursuit. cerebellar dorsal vermis is another cerebellar area for frontal pursuit. to examine whether d pursuit signals are present in this area, we examined simple-spike discharge of vermal pursuit p-cells in monkeys. of a total of p-cells that were examined during both frontal and vergence-pursuit, % discharged for both, % only for vergence, and % only for frontal pursuit. these results indicate that most of vermal pursuit p-cells discharged for vergence and that about half of them had d pursuit signals. majority ( %) of these p-cells discharged before onset of vergence eye movements with the typical lead time of ms, suggesting their involvement in the initiation of vergence-pursuit. research funds: kakenhi ( ) ps p-e information processing in fef-rnrtp pathway for smooth pursuit seiji ono, michael j. mustari division of sensory-motor systems, yerkes national primate research center, emory university, atlanta ga, usa the frontal eye field (fef) cortex is known to play a role in smooth pursuit (sp). this role is supported by fef projections to the rostral nucleus reticularis tegmenti pontis (rnrtp) which projects heavily to the vermis. using multiple linear-regression modeling, we have shown that sp neurons in rnrtp were biased towards eye acceleration. however, the functional characteristics of sp related fef neurons that project to rnrtp have never been described. therefore, we used micro-electrical stimulation to deliver single pulses in rnrtp to antidromically activate fef neurons. the majority of sp related fef neurons that we identified as projecting to rnrtp were most sensitive to eye acceleration and much less sensitive to eye velocity. the neurons in fef-rnrtp pathway carry signals that could play a primary role in sp initiation. our antidromic studies may help address a fundamental question regarding whether basilar pontine nuclei integrate signals from multiple cortical areas or mostly relay signals with little transformation to cerebellum. research funds: nih grants ey , rr aya takemura , yumi murata , , kenji kawano , neurosci. res. insti, aist, tsukuba, japan; dept. integ brain sci., grad. sch. med., kyoto univ., japan; grad. sch. compreh hum sci., univ. tsukuba, japan previous studies in monkeys suggest that the medial superior temporal (mst) area is involved in visual motion processing. to understand the role of the mst in optokinetic nystagmus (okn) and afternystagmus (okan), we examined the effects of bilateral chemical lesions in the mst in two monkeys. when each monkey was injected with ibotenic acid ( mg/ml, - l total), the initial rapid rise in okn was reduced. consequently, it took longer for the eye velocity to reach a steady state (i.e., an eye velocity close to the stimulus velocity). by contrast, the steady state okn was not affected and the okan persisted. the initial amplitude and falling time constant of the okan increased. the results suggest that the mst is part of the direct pathway for the initial rapid rise in the okn, but is not involved in the velocity storage mechanism for the steady state okn and okan. smooth pursuit is performed by coordination of eye and head movements. we have reported that the majority of fef pursuit neurons in monkeys with their head free to rotate about a vertical axis were modulated not only during eye-and gaze-pursuit but also head-pursuit to a moving reward feeder while the monkeys fixated an earth-stationary spot without gaze movement. to examine the origin of head-pursuit modulation, we moved the reward feeder in a ramp trajectory at • /s with random intervals. the majority of pursuit neurons discharged before the onset of head movements with the mean lead time of ms. discharge modulation during head-pursuit and passive whole body rotation was not correlated in most neurons. these results suggest that proprioceptive neck inputs or vestibular inputs are not the main origin of head-pursuit modulation. rather, our results suggest that the main origin reflects pursuit commands. ps p-e the local feedback loop of the saccadic system: an analysis of the eye movements induced by pdb stimulation rikako kato department of developmental physiology, national institute for physiological sciences, okazaki, japan saccadic amplitude are controlled by a comparator that calculates dynamic motor error. some models place the comparator in the superior colliculus while others assign this role to the reticular formation. to decide between the two hypotheses one would need to stimulate pathways in between their putative comparators. we stimulated collicular axons descending in the pdb. our data demonstrate that electrical stimulation of the pdb evokes saccades and they always terminate before the end of the stimulus train. the characteristics of evoked saccades are comparable to those spontaneously generated by the cat. our data clearly demonstrate that the feedback path of the local loop of the saccadic system closes downstream of the superior colliculus. katsuo fujiwara , kenji kunita , kaoru maeda , takeo kiyota department of human movement and health, graduate school of medical science, kanazawa university, kanazawa, japan; institute for health and sport sciences, osaka city university, osaka, japan we investigated changes in visual evoked potential (vep) during postural adaptation process while subjects maintaining standing posture on an oscillation floor with periodic vision shut. the subjects were undergraduate students. a shutter goggle was used as a vep stimulator which was opened periodically for ms with -ms intervals. the oscillation trial ( . -hz frequency and . -cm amplitude) ( - s) was repeated times. postural steadiness was evaluated by mean fluctuation speed of the center of foot pressure. the mean speed decreased as trial was repeated, and reached a plateau before the th trial. a significant correlation was shown between th- st trial differences in mean speed and vep amplitude (r = . ). this indicates that the role of visual information is different among subjects with various adaptation processes of postural control. ps p-e primary motor cortex contributes to generating manual following response toshitaka kimura , naoki saijo , hiroaki gomi , ntt cs labs, kanagawa, japan; erato shimojo implicit brain function proj, jst, saitama, japan a large-field visual motion during arm movements induces a shortlatency, involuntary arm response called as manual following response (mfr). the mfr exhibits similar features to the ocular following response (ofr) elicited by the similar visual stimulus, with respect to the stimulus-response directional characteristics and the spatiotemporal frequency tuning property. this suggests that computational mechanism is shared for both responses. however, the neural basis of the mfr motor command generation remains unclear, while ofr is known to be generated subcortically. here we show, by using transcranial magnetic (tms) and electrical (tes) stimulation over the primary motor cortex (m ), that ( ) an emg response evoked by tms was facilitated during mfr, while that by tes was not, and ( ) intracortical inhibition within m assessed by paired-pulses tms was reduced during mfr. these results suggest that mfr is generated through activity of interneuronal networks within m . such cortical mechanisms for mfr generation are distinct from the subcortical processes for ofr generation. naoki saijo , hiroaki gomi , ntt cs labs., kanagawa, japan; erato shimojo implicit brain function proj, jst, saitama, japan when a visual target is suddenly shifted during a reaching movement, we can quickly adjust the arm movement. however, the computational mechanism to generate quick adjustment is still unclear. here we investigated this mechanism from the viewpoint of visuomotor coordinate transformation. we observed the hand responses to the target shifts in radial directions applied during reaching. the data show that the direction of the initial phase ( - ms) of hand response acceleration was slightly biased from the corresponding target shift direction, whereas the direction of the late phase ( - ms) was little biased. additionally, when we use a target shift having less-motion energy, the response latency greatly increased and the directional bias significantly decreased. these results suggest that the on-line reaching adjustment would be generated by two different mechanisms: a reflexive controller which is induced by visual motion with short latency and generates spatially inaccurate response, and voluntary controller which generates spatially accurate response with long latency. ps p-e spatial relationship between gaze and reaching-target modulates manual following response naotoshi abekawa , hiroaki gomi , ntt cs labs., kanagawa, japan; erato shimojo implicit brain function proj, jst, saitama, japan to explore the functional mechanism of the manual following response (mfr) induced by a large-field visual motion during arm movement, we examine its modulation caused by the spatial relationships between gaze, target, and background. on a large vertical screen placed in front of the subject, full field checker pattern, two markers (upper and lower), and a gray mask around one of the markers, were displayed. in the first condition, subjects kept watching the upper marker, and pointed the upper (congruent) or lower (incongruent) marker instructed before every reaching. the checker pattern suddenly moved either rightward or leftward brief after reaching start. in the second condition, subjects did the same task with watching the lower marker. in both conditions, the mfr amplitude was significantly grater in the congruent condition than in the incongruent condition, whereas the mask location did not significantly affect the mfr amplitude. this suggests that the spatial relationship between gaze and target is important in modulating mfr. misako komatsu, eizo miyashita dept. compu. intelligence & systems sci., tokyo tech., yokohama, japan when a subject performed pointing to a remembered target under eyes fixated, we have reported that endpoints tended to sift closer to the fixation point. moreover, we have noted that the greater the distance between a target and the fixation point, the larger the errors. the result was consistent even when the position of the fixation point was changed. the above tendency was considered to occur in eye-or gaze-centered coordinates. it is open question, however, if the brain correctly compensates the difference of the relative position of eyes to the head? to answer this question, we investigated the dependency of the endpoint errors on the positions of a monitor and the fixation point. the subjects, sitting in front of the monitor, were asked to point a remembered target as accurately as possible using a computer mouse. all the results were consistent with the previous ones regardless of the position of monitor or the fixation point. these results suggest either the eye-position doesn't affect how we recognize the target position, or the brain correctly compensates the eye-position with a fixed head position. ps p-f influence of the coupling of muscle activity on rhythmic movements of ipsilateral hand and foot tetsuro muraoka , takashi obu , kazuyuki kanosue asmew, waseda university, saitama, japan; graduate school of human sciences, waseda university, saitama, japan; faculty of sports sciences, waseda university, saitama, japan the aim of this study was to investigate the influence of the coupling of muscle activity on rhythmic movements of ipsilateral hand and foot. the subjects (n = ) were supine, and their hand was prone. they performed cyclical flexion-extension coordinations of the hand and foot in the iso-(iso) or opposite-(oppo) directions, and those with an elastic load against wrist flexion (el-iso and el-oppo) at . , . , and . hz. over % success rate was observed in all tasks except oppo ( - %). the in-phase muscle activity of wrist and foot muscles was obserbed in all tasks except oppo. it was suggested that the in-phase muscle activity might be an important factor in a coordinated movement of ipsilateral hand and foot. research funds: the special coordination funds for promoting science and technology, mext, japan ps p-f simultaneous muscle activity stabilizes the coordinated movement of ipsilateral hand and foot takashi obu , tetsuro muraoka , kazuyuki kanosue , , faculty of human sciences, waseda university, saitama, japan; faculty of sport sciences, waseda university, saitama, japan; asmew, waseda university, saitama, japan in human, voluntary opposite-directional movement (antiphase) of ipsilateral hand and foot is more difficult than iso-directional movement (inphase). the purpose of the present study was to investigate the influence of the coupling of muscle activity on these movements. eight normal subjects lay in supine position with hand prone and their foot was forcedly moved by a dynamometer cyclically at , . , and . hz. they were asked to perform tasks, concentric/eccentric contraction of ankle dorsiflexors with in-phase/antiphase wrist extension/flexion. all tasks were performed successfully. muscle activity of hand flexors was observed in concentric-antiphase and eccentric-inphase tasks, indicating simultaneous muscle activity of hand and foot. it may be suggested that simultaneous muscle activity would make the movement easier regardless of the direction of movement. ps p-f activities of erector spinae muscles during jaw clenching in man kayoko yasunaga , , tadachika yabushita , kazuo toda , kunimichi soma orthodontic science, tokyo med. & dent. univ., tokyo, japan; div. integrative sensory physiology, nagasaki univ., nagasaki, japan recent studies focused the functional relationships between the masticatory and the posture system. the hypothesis of our present study is an existence of functional connections between the masticatory system and the spinal muscles which maintain the posture. therefore, we investigated the effect of the maximum jaw clenching on the spinal muscle activities. bipolar needle electrodes were inserted into erector spinae muscles to record the motor unit activities when the sitting subjects relaxed and performed maximal jaw clenching. as a result, the instantaneous frequencies of the spinal muscles decreased with clenching, compared with relaxed jaw position. our results suggested that there were some relationship between spinal muscle activities and jaw clenching. the effects of bipedal walking on the central nervous systems-influence of bipedal walking on the spinal reflex-naomi wada , sachiko motoyama , futoshi mori , shigemi mori department of veterinary physiology, yamaguchi university, yamaguchi, japan; national institute for physiological science, okazaki, japan the one of the biggest questions in the vertebrate evolution is how human got the highly developed brain. many investigators suggest that upright posture and bipedal walking caused remarkable development of brain and produced the human being. the purpose of our experiments is to show the influences of bipedal habits on central nervous systems. we have established the bipedal walking model using rats (rbm) by amputation of forelimbs and training of upright posture and bipedal walking. after training of upright posture and bipedal walking for - weeks, rats got abilities of the stable upright posture and bipedal walking with symmetrical hindlimb movements between left and right side. in the present experiments, we studied about the effects of bipedal habits on the lumbar spinal reflex. the results of out experiments showed that bipedal habits inhibit the spinal reflex pathways. ps p-f neuronal activity in primary motor cortex during quadrupedal locomotion of the japanese monkey katsumi nakajima , futoshi mori , akira murata , masahiko inase dept. of physiol., kinki univ. schl. of med., osakasayama, japan; dept. of vet. physiol., facult. of agr., yamaguchi univ., yamaguchi, japan to elucidate cortical mechanisms related to the control of primate locomotion, we recorded neuronal activity in m of the monkey walking quadrupedally on the treadmill. tungsten microelectrodes were inserted into m hindlimb region using a custom-made micromanipulator. we found that all neurons recorded in m modulated their discharge phasically time-locked to the step cycle or increased their discharge frequency tonically during simple locomotion. the neuron exhibiting phasic modulation peaked once or twice per step. the peak activity occurred at widely different times during the step cycle in different recorded neurons. as the treadmill speed increased, most of recorded neurons increased their discharge frequency. all these results suggest that m output in monkeys directly and/or indirectly acts on spinal circuitries generating a basic pattern of rhythmic activity during simple locomotion in a manner different from that in subprimates. research funds: kakenhi ( ) ps p-f activity of putaminal neurons receiving inputs from motor cortical areas in behaving monkeys sayuki takara , , nobuhiko hatanaka , , masahiko takada , atsushi nambu , school of life science, the graduate university for advanced studies, japan; division of system neurophysiology, national institute for physiological sciences, japan; tokyo metropolitan institute for neuroscience, japan the putaminal (put) neurons receive motor cortical inputs and change their activity in relation to movements. to investigate how these inputs contribute to put neuron activity in behaving monkeys, extracellular unit activity was recorded from identified put neurons during the performance of a memory-guided reaching task. based on orthodromic spikes evoked by cortical stimulation, individual put neurons were defined in terms of whether they receive input from the primary motor cortex (mi), the supplementary motor area (sma), or both. the results showed that mi-recipient neuron activity was responsive to the movement, while sma-recipient neuron activity was responsive to the cue stimuli and/or the delay period. the activity of neurons receiving convergent inputs was related to both the movement and the delay period. we previously reported that electrical stimulation of cerebrofugal fibers induced short latency facilitation and succeeding suppression on phrenic activities, while train pulse stimulation of caudal raphe nuclei (raphe magnus, rm, and raphe pallidus, rp) induced suppression or facilitation on respiratory neural activities in cats and rats. in this study, in order to analyze the cerebral and raphe projections to the respiratory neuron network, we examined the effects of stimulation of cerebrofugal fibers and caudal raphe nuclei on activities of ventral respiratory group neurons (vrgs) in the medulla and upper cervical inspiratory neurons (ucins). animals were anesthetized, immobilized and artificially ventilated. stimulation of cerebral peduncle (cp) induced short latency facilitation and succeeding suppression on activities of ucins. stimulation of rm or cp evoked inhibitory postsynaptic potentials in the caudal vrgs. these results suggest that rm and cerebral cortex directly inhibit main respiratory output neurons in vrg. ken muramatsu , sei-ichi sasaki , yuichiro cho , kenji sato anatomy and physiological science, tokyo medical and dental university, tokyo, japan; department of physiology, ibaraki prefectural university of health sciences, ibaraki, japan distribution of average diameters of external anal sphincter (eas) motoneurons and peripheral motor fibers were examined in cats. to identify eas motoneurons, horseradish peroxidase was applied to the central cut end of the anal branches of the pudendal nerve. eas motoneurons were found in the onuf's nucleus of s and s spinal levels. to examine size of peripheral motor fibers, ganglionectomy was performed onl -s spinal segments which contain afferent fibers of eas muscles. after weeks survival period, anal branches of the pudendal nerve was examined. histograms of the distribution of average diameters of cell body and motor fiber shows unimodal distri bution. also, distribution of muscle spindles of eas muscle were examined by serially sectioning the distal colon and staining with mayer's haemotoxylin and eosin. no muscle spindles were found. these results suggest that eas muscle is controlled without gamma loop. mariko miura, yoshiki iwamoto, kaoru yoshida neurophysiol., univ. tsukuba, tsukuba, japan saccade accuracy is ensured by an adaptation mechanism. the speed and magnitude of adaptation vary greatly across experiments even for the same subject. one factor that might cause this variability is adaptation history. the present study aims to clarify whether preceding adaptation influences subsequent adaptation over several days. gain decrease adaptation was induced in a monkey by stepping the target backward during saccades. adaptation experiments were repeated for consecutive days. we compared adaptation in day and that in day . the gain decrease for the first saccades in day ( . ± . ) was larger than that in day ( . ± . ) (p = . , n = , paired-t test). the rate of adaptation in day ( . ± . × − /sac) was higher than that in day ( . ± . × − /sac) (p = . ). the overall gain change ( saccades) in day ( . ± . ) was larger than that in day ( . ± . ) (p = . ). thus, both the speed and magnitude of adaptation were increased by preceding adaptation. the present study suggests that the memory of saccadic adaptation is retained for days and facilitates following adaptation. research funds: kakenhi ( ) ps p-f asymmetry of the anticipatory convergence eye movement haruo toda, takehiko bando div. integr. physiol., grad. sch. med. sci., niigata univ., niigata, japan typically, convergence eye movement is known as symmetric adduction of the both eyes. but asymmetrical convergence also found in the natural condition. these asymmetrical convergence may reflect asymmetries of central control of convergence eye movement. the lateral suprasylvian (ls) areas are extrastriate cortices which receive visual information from v . the ls has contralateral dominant receptive fields and convergence eye movements evoked from the long latency regions were asymmetrical. cats (n = ) were trained to start convergence by an alarm signal (buzzer sound or combination of buzz and blinking of led), preceding target movement by s. after training, ocular convergence was elicited by the alarm signal before target movement (predictive open-loop convergence) in % of trials. in three cats, we used training with obliquely approaching target. after training, asymmetrical anticipatory eye movements were observed. based on these findings, related ls neuronal activities and results from lesion study, we will discuss the role of ls in asymmetry of anticipatory and visually-evoked convergence eye movement. yusuke uchida , xiaofeng lu , , shogo ohmae , toshimitsu takahashi , , shigeru kitazawa , dept. of neurophysiol., juntendo univ. grad. sch. of med., tokyo, japan; crest, jst, tokyo, japan we examined reward related neural activity in the supplementary eye field (sef). for this purpose, two monkeys were rewarded after each visually guided saccade from a central fixation point to one of targets that were arranged in a radial pattern. a target appeared while the monkeys were fixating on the central point, and the monkeys made a saccade to the target when the fixation point disappeared and held on the target until the target turned off. reward was delivered during or after target-hold period. we found that many sef cells became active during the period of reward delivery (r-cell). more than half of r-cells showed enhancement of the neural discharge in the specific target directions but not other directions in which the same amount of reward was given (rd-cell). interestingly, most of rd-cells displayed activity with the clear directional tuning. these results demonstrate reward dependent activity specific to spatial direction in the sef, and further suggest that sef cells provide reinforcement mechanism. research funds: kakenhi ps p-f frontal pursuit area is involved in the retinalslip dependent adaptation of monkey post-saccadic pursuit eye velocity hiromasa kitazawa , soichi nagao , lab. for motor learning control, riken bsi, saitama, japan; sorst, jst, saitama, japan smooth pursuit is under learning control by several brain areas including cerebrum and cerebellum. smooth pursuit velocity is modifiable by repetition of target velocity for a brief period at its onsets. role of cerebellar vermis and hemisphere in the adaptive control of smooth pursuit is suggested by lesion experiments, but the role of frontal pursuit area (fpa) is not known. to reveal possible involvement of fpa in the adaptation of smooth pursuit, we identifying fpa by unit recording and microstimulation, and reversibly inactivated it by local injection of muscimol. we found that inactivation of fpa not only reduced of the velocities of pursuit in the ipsi-and contra-versive directions to the inactivated fpa, but also appreciably depressed its adaptation, suggesting that fpa is involved in the adaptation of smooth pursuit. shinji matsutani department of functional morphology, kitasato university school of nursing, kanagawa, japan distribution of terminals on individual centrifugal axons in the main olfactory bulb was studied using an anterograde tracer to elucidate function of the centrifugal system. the tracer was injected into olfactory cortical areas, and individual labeled axons were traced from serial sections. as already reported in the last meeting, the centrifugal axons had multiple terminals with discrete locations. distribution of these terminals was examined in reconstructed maps in which localization of the terminals was projected onto a sagittal plain. in most axons, the terminals were clustered to form a patch that was stretched in a rostrocaudal direction. it was also common that patches belonging to the same axon were found in distant locations and in both sides of the single bulb. while most of the terminals were seen in the granule cell layer, those located in the glomerular layer and in the external plexiform layer were found following injections into the anterior olfactory nucleus. the centrifugal fibers may couple the activity of discrete and distant subsets of bulbar neurons. ps p-f projection targets of the drosophila taste receptor neurons in the primary gustatory center of the brain takaaki miyazaki , , kei ito , , dept. of comput. biol., grad. sch. of frontier sci., univ. of tokyo, japan; center for bioinform., imcb, univ. of tokyo, japan; bird, jst, japan in order to figure out the way of information processing linking gustatory stimulus and taste-associated behavior, systematic knowledge about the underlying neural networks is required. drosophila melanogaster is an attractive model organism for this task, thanks to its relatively simple brain structure and a wide variety of molecular and genetic tools available. gustatory sensory neurons in the labellum of the mouth project their axons via the labial nerve to the suboesophageal ganglion (sog) of the brain. to understand the entire neural circuits of these first-order neurons in the primary gustatory center, we searched for the gal enhancer-trap strains that visualize specific neural fibers in the sog and the labial nerve. screening , strains, we identified about candidate lines. the projection targets of the labeled neurons were classified into seven areas. the terminals of the already identified sensory neurons appear to fall into specific subsets of these areas. research funds: bird, jst ps p-f immunoreactivity and voltage-gated channels of mouse taste bud cells kennji kimura , yoshitaka ohtubo , takashi kumazawa , kiyonori yoshii graduate school of life science and systems engineering, kyushu institute of technology, kitakyushu, japan; department of applied chemistry, saitama institute of technology, fukaya, japan mammalian taste buds comprise four heterogeneous cell types, type i to iv, and their collaboration seems to generate taste sensation. we investigated the electrophysiological properties of these cell types except type iv with taste buds preserved in mouse lingual epithelia. type i cells elicited smaller ttx-sensitive, tea-sensitive, and teainsensitive currents in magnitude than other cell types. type ii cells elicited a smaller tea-sensitive current and a larger tea-insensitive current than type iii cells. these results suggest that type ii and iii cells elicit action potentials with different ionic mechanisms, and that the difference results from the functional differences of these cell types. research funds: kakenhi ( ) and the st coe program (center # ) granted by mext of japan ps p-f inositol monophosphatase maintains synapse localization and regulates behavior in the mature nervous system of c. elegans yoshinori tanizawa , atsushi kuhara , hitoshi inada , eiji kodama , takafumi mizuno , ikue mori , lab. of mol. neurobiol., nagoya univ., japan; institute for advanced research, nagoya univ., japan inositol monophosphatase (impase) is suggested to be relevant to bipolar disorder. although lithium is believed to exert therapeutic effect by inhibiting impase in patients, the mechanism underlying lithium therapy is largely unknown. here we show that the loss of impase causes defects in behavior and localization of synapses in c. elegans. mutations in ttx- gene encoding impase exhibit defective thermotaxis behavior, which is attributable to the loss of impase activity in the most essential integrative interneuron ria in the nervous system. the ttx- mutations also cause mislocalization of synaptic proteins in ria. both behavioral and synaptic defects in ttx- mutants were rescued by expression of impase at adult stage and inositol application, and were mimicked by lithium application in wild type animals. these results suggest that impase is required in the mature nervous system for maintaining synapses of the central interneurons in order for animals to behave properly. research funds: kakenhi ps p-f postnatal alterations in expression of vesicular glutamate transporters in the main olfactory bulb (ob) of rats h ohmomo, f shutoh, a. ina, s. yoshida, h. nogami, s. hisano lab. neuroendocr., graduate sch., univ. tsukuba, tsukuba, japan olfactory information is conveyed to the brain by transmission from primary olfactory neurons to mitral or tufted cells. however, little is known about development of these ob glutamatergic neurons in early postnatal life. vesicular glutamate transporters (vglut) have been used as the best histological markers to identify glutamatergic neurons. we here studied expressions of two vglut isoforms (vglut and - ) during rat ob development from postnatal day (p ) to p by in situ hybridization and immunohistochemistry. at p vglut immunoreactivity (ir) was detected in all layers except the olfactory nerve layer, and thereafter its localization expanded and intensity increased. vglut mrna signals were detectable in the mitral cell layer from p to p . in contrast, vglut ir was prominent in the glomerulus at all days examined, and only at p and p in mitral cells. despite mitral vglut ir disappeared at p , the mrna signals were still detectable. these results suggest that glutametergic neurons in the rat ob continue to develop even after birth. ps p-f v r genes multiplied in amphibian and expressed in the main olfactory system atsuko date-ito , , masumi ichikawa , yuji mori , kimiko hagino-yamagishi tokyo metrop. inst. med. sci., tokyo, japan; the univ. of tokyo, tokyo, japan, tokyo metrop. inst. neurosci., tokyo, japan in rodent, v r gene family is expressed specifically in the vomeronasal organ (vno) and is thought to be responsible for pheromone reception. however, teleost fishes lacking for the vno have a single v r gene, which is expressed in the olfactory epithelium (oe). to examine when the v rs function as pheromone receptors in the course of evolution, we analyzed the amphibian xenopus tropicalis genome, and identified v r sequences. these v rs were not expressed in the vno, but most of them were expressed in the oe of the middle cavity, which is considered for reception of water-soluble odorants. from these results, we speculate that the amphibian v rs get a chance to receive diverse odorants such as pheromones by gene multiplication and sequence diversification. our results raise the possibility that pheromonal information is transmitted via the main olfactory system. ps p-f analyses of ligand binding sites and snps on sweet taste receptor system in human noriatsu shigemura, a.a. islam, yuki nakamura, shinya shirosaki, yuzo ninomiya sect. oral neurosci., grad. sch. dent science, kyushu univ., japan recent studies have shown that t r /t r heterodimer plays a role as a sweet taste receptor. but, mice lacking t r showed diminished but not abolished behavioral and nerve responses to sugars, suggesting t r -independent sweetener binding site also exist in mice. in this study, to predict binding sites on t r /t r and/or other sweet receptor in human, we measured sensitivity thresholds to various sweet compounds and examined the qualitative similarities. we also used gymnemic acid and ␥-cyclodextrin, which selectively inhibits sweet responses and reduces the inhibitory action of it. the ten sweet compounds were classified into five groups [( ) sucrose, glcose, fructose, ( ) saccharin, aspartame, acesulfame-k, glycine, ( ) d-phenylalanine, ( ) d-tryptophan, ( ) l-proline]. in sequencing analysis, four and two snps with amino acid substitution were revealed in t r and t r , respectively. these results suggest that there may be at least five binding sites in human sweet receptor system. the individual differences in sweet sensitivities may be due to these snps. keiko yasumatsu , sachiko saito , yuko murata , ding ming , tatsu kobayakawa , robert f. margolskee , yuzo ninomiya sect. oral neurosci., grad. sch. dent. sci., kyushu univ., fukuoka, japan; saito sachiko taste and smell research institution, ibaraki, japan; national res. institute of fisheries sci., kanagawa, japan; dept. of physiol. & biophys., mount sinai sch. med., new york, usa; national institute of advanced industrial science and technology, ibaraka, japan the effect of unsaturated fatty acids on taste responses was examined by measuring perceived taste intensity in human, behavioral short-term lick responses and electrophysiological taste responses recorded from the chorda tympani and glossopharyngeal nerves in mice. the results showed that dha and other polyunsaturated fatty acids inhibit responses to bitter taste compounds without affecting other taste stimuli. we also found fatty-acid inhibition on bitter responses in an in vitro g-protein activation assay using bovine taste membrane, but lack of the bitter taste inhibition in ggustducin ko mice. these results suggest that fatty acids specifically inhibit responses to bitter stimuli by suppression of activation of t r receptors which coupled with ggustducin. ps p-f newborn infant body odor attenuates their mother's postpartum moods shota nishitani , mayumi kokuryo , tsunetake miyamura , kazuyuki shinohara div. neurobiol. & behav., nagasaki university, japan; obstet. & gynecol. of miyamura hospital, japan mothers are attracted to the body odor of newborn infants, but little is known about its reason. in the present study, we examined whether the body odor of newborn infants exert effects on moods in postpartum mothers. the body odors of newborn infants were collected from their undershirts. postpartum mothers were exposed to odors of a part of the undershirt with control odors, their own infant body odors or other infant body odors. we used the poms to assess the effects of infant body odors on postpartum moods. this study was approved by the ethics committee of nagasaki university. the infant body odors significantly increased hedonics and friendliness scores, and significantly decreased anxiety, depression and fatigue scores, whether infant odors may be originated from their own infants or other infants. these results suggest that body odors of newborn infants attract their mothers because they have calming effects on postpartum mothers. research funds: japan science and technology agency (jst), research institute of science and technology for society (ristex) ps p-f human prefrontal activity in taste encoding: an fnirs study masako okamoto , mari matsunami , haruka dan , tomoko kohata , kaoru kohyama , ippeita dan national food research institute, tsukuba, japan; nippon suisan kaisha, ltd., japan taste remains one of the least-explored human senses. using multichannel functional near-infrared spectroscopy (fnirs), we examined the lateral prefrontal cortex (lpfc) of healthy volunteers (n = ) while they tasted and encoded the quaternary taste mixtures. the contrast between the cortical activation under encoding conditions and that under control conditions without memory requirement revealed activation in the bilateral ventro-lpfc and the right posterior portion of the lpfc. the activation pattern, which was in line with those that have been associated with intentional encoding of non-verbal materials of other senses, supported an amodal role of lpfc in intentional encoding, at least at a macro structural level. this study also demonstrates that, by using fnirs, lpfc functions on taste can be examined with experimental paradigms comparable to those used for other senses. recently, we performed simultaneous respiration and electroencephalographic recordings during odor stimulation. we sought to identify changes in respiratory pattern, inspiratory phase-locked alpha oscillation (i-␣) and location of dipoles estimated from the potentials. electroencephalographic dipole tracing identified the location of dipoles from the i-␣ in the limbic area and the cortex; the entorhinal cortex, hippocampus, amygdala, premotor area and orbitofrontal cortex. in this study, we compared the respiratory pattern during odor stimulations, i-␣, dipole localizations without habituation with those with habituation of odors. onset of inspiration was used as a trigger for averaging, and potentials were averaged before and after the habituation period. habituation of odor caused to return to the normal respiratory pattern, decrease of amplitudes of ␣, and entorhinal cortex, hippocampus, amygdala were less active. akio tsuboi, takaaki miyazaki, takeshi imai dept. of biophys. & biochem., univ. of tokyo, tokyo, japan vertebrate odorant receptor (or) genes are divided phylogenetically into two distinct classes, the fish-like class i and the terrestrialspecific class ii. in the present study, we systematically analyzed mouse class i or genes ( subfamilies) to elucidate the expression profiles in the olfactory epithelium (oe) and the projection sites of their olfactory sensory neurons (osns) in the olfactory bulb (ob). in situ hybridization (ish) revealed that most class i or genes ( subfamilies) were expressed in the dorso-medial zone (zone ) of the oe. furthermore, there appeared to be no significant differences in the distributions of osns expressing class i genes within zone . these results indicate that there is a clear boundary between zone and non-zone areas in the oe. some class i ors are known to possess ligand specificity for aliphatic acids, aldehydes and alcohols. our ish analysis has revealed that osns expressing the class i ors in zone tend to converge their axons on a cluster of glomeruli in an antero-dorsal domain that is assumed to be involved in responses to the aliphatic compounds on the ob. research funds: kakenhi ( ) ps p-g taste response characteristics of putative interneurons in the rat gustatory cortex tatsuko yokota, kunihiro eguchi, katsunari hiraba department of physiology, school of dentistry, aichi-gakuin university, nagoya, japan previous studies have indicated that the extracellular spike waveforms and discharge rate properties of cortical neurons differed between pyramidal cells and interneurons, the latter tending to have narrower spike-widths and higher discharge rates. taste-sensitive neurons in the rat gustatory cortex were classified according to ( ) best-taste profiles and ( ) spike-widths which were found to form a bimodal distribution (narrow and broad). narrow-spike neurons had a significantly larger response to nacl than broad-spike neurons, but no differences were found to other tastants. the proportion of narrow-spike neurons in the n-best neurons was higher than that in the h or nh-best neurons. these results indicate that putative interneurons may play an important role in the coding of salt taste information. research funds: kakenhi ( ) of japan to t.y. yuki sato, nobuhiko miyasaka, yoshihiro yoshihara laboratory for neurobiology of synapse, riken bsi, wako, japan in the fish olfactory system, individual olfactory sensory neurons (osns) are thought to express only one or at most a few different odorant receptors (ors) from the large or family consisting of ∼ members. here, we investigated the mechanisms underlying or gene choice by using transgenic zebrafish that carried a modified bac containing a zebrafish or gene cluster. replacement of the or coding regions in the bac transgene with reporter genes allowed the reporters to be expressed in a small population of osns in the transgenic fish. in situ hybridization analysis using or-specific probes revealed that or genes expressed in reporter-positive cells were mostly restricted within the same or subfamilies to which the replaced ors belonged. additionally, the reporter-expressing osns projected their axons to a topographically fixed cluster of glomeruli in the olfactory bulb. these findings suggest the hierarchical regulation of or gene choice, whereby an individual osn may express one or gene from a limited subpopulation that is chosen from the entire repertoire in advance. research funds: kakenhi ( ) ps p-g identification of perisomatic-targeting granule cells in the mouse olfactory bulb hiromi naritsuka , kazuhisa sakai , tsutomu hashikawa , kensaku mori , masahiro yamaguchi dep. physiol. grad. sch. med., univ. of tokyo, tokyo, japan; laboratory for neural architecture, bsi, riken, saitama, japan in the olfactory bulb (ob), odor information is processed by the local circuit that includes inhibitory interneurons. granule cells (gcs) are major interneurons in the ob, but their diversity is not well understood. in the ob of adult transgenic mice expressing gfp under the control of nestin gene regulatory regions, we observed gcs with strong gfp expression (referred to as type s cells). their dendrites branched and formed spines within the granule cell layer, internal plexiform layer and mitral cell layer but did not reach the external plexiform layer, where typical gcs make synapses with dendrites of mitral and tufted cells. type s cells had huge protrusions at their dendritic ends, which formed contact with mitral cell somata. electron microscopic analysis revealed the existence of reciprocal synapses between type s cell protrusions and mitral cell somata. characteristic morphology of perisomatic-targeting gcs indicates that they have functions distinct from typical gcs in the ob. keiko moriya-ito, kentaroh endoh, yuuki ishimatsu, masumi ichikawa department of neuroscience basic technology, tokyo metropolitan institute for neuroscience, fuchu, tokyo, japan a coculture system of accessory olfactory bulb (aob) neurons and vomeronasal neurons was established for studying the functional roles of aob neurons in pheromonal signal processing. in this study, the effect of vomeronasal neurons on the development of aob neurons was examined in a coculture system. the densities of dendritic spines were lower in the coculture than in single culture. the ratio of the density of synaptophysin-immunopositive spine/total spine density was larger in the coculture than in the single culture. the volume of spine head was larger in the coculture than in single culture. by electron microscopic observation, the synapses on dendritic shafts were decreased and the synapses on dendritic spines were increased in the coculture. the synapses between aob neurons and vomeronasal neurons were recognized in the coculture. these observations suggest that synapse formation of aob neurons is modified by synaptic contact with vomeronasal neurons. ps p-g nacl induced responses of mouse fungiform taste cells: existence of amiloride sensitive and insensitive taste cells ryusuke yoshida, tadahiro ohkuri, keiko yasumatsu, noriatsu shigemura, yuzo ninomiya sect. of oral neurosci., grad. sch. of dental sci., kyushu univ., fukuoka, japan previous electrophysiological studies showed that the chorda tympani nerve contains two types of nacl-responsive fibers, amiloride sensitive (n-type) and insensitive (e-or h-type) fibers, suggesting the existence of amiloride sensitive and insensitive taste receptor cells in fungiform papillae. in this study, we examined nacl responses of mouse fungiform taste cells in isolated taste bud and amiloride sensitivity of them. some taste cells respond to apical restricted nacl stimulation with increase in firing frequency and their responses were concentration dependent. amiloride mixed with apical nacl solution inhibited nacl responses in some taste cells [amiloride sensitive (as) cells] but not in others [amiloride insensitive (ai) cells]. ai cells responded to other electrolytes such as kcl and hcl. these results suggest the existence of at least two types of nacl sensitive cells, as and ai cells. n-or e-type fiber may selectively innervate as or ai cells respectively. research funds: kakenhi ( ), kakenhi ( ) ps p-g integration of olfactory and oral sensory input in the rat insular cortex hideki kashiwadani, kensaku mori department of physiology, university of tokyo, tokyo, japan axonal connections between olfactory cortex and insular cortex suggest that insular cortex integrates olfactory information and information originated from the oral cavity (taste, tactile, temperature). however cellular mechanisms underlying the integration of multimodality are poorly understood yet. in this study, we examined single-unit spike responses of insular cortical neurons to odor stimulation and intraoral water stimulation in urethane-anesthetized rat. we found that more than % of recorded neurons in the insular cortex responded to odors. about half of the odor-responsive neurons were activated by intraoral water stimulation, indicating the convergence of olfactory and oral sensory information onto individual neurons in the insular cortex. when odor stimulation and intraoral water stimulation were simultaneously applied, some neurons showed spike responses larger than the responses evoked by each stimulus. the integration of olfactory and oral sensory information in the insular cortex might contribute to form the flavor sensation. research funds: kakenhi ( ) ps p-g odor combination selectivity of the rat piriform cortex neurons ikue yoshida, kensaku mori dept. physiol. grad. sch. med., univ. of tokyo, tokyo, japan olfactory cortex is thought to integrate signals from different odorant receptors to form the olfactory image of objects. however, the manner of integration at the level of individual cortical neurons is not well understood yet. using single-unit recording method, we examined the response selectivity of individual neurons in a dorsocaudal part of the anterior piriform cortex (apc) to classes of odorous compounds, each class being present in odors from many different vegetables and fruits. individual neurons typically responded to more than classes of odorants. each neuron was uniquely tuned to a specific combination of odorant classes, and different neurons typically showed different odor combination selectivity. single-unit responses to odor mixtures showed mixture facilitation and mixture suppression. these results suggest that individual neurons in the apc can be characterized by the odor combination selectivity and that the apc neurons may integrate signals from different odorant classes. research funds: kakenhi ( gs ) ps p-g odor-driven activity in the anterior piriform cortex of an in vitro isolated whole brain with the olfactory epithelium takahiro ishikawa , takaaki sato , akira shimizu , ken-ichiro tsutsui , toshio iijima div. of systems neuroscience, grad. sch. of life sciences, univ. of tohoku, sendai, japan; res. inst. for cell engineering, aist, amagasaki, japan to examine the neural mechanisms underlying odor-induced response in the anterior piriform cortex (apc), we analyzed odorinduced local field potential (lfp) and multiunit activity in an in vitro preparation, isolated guinea-pig whole brain with the olfactory epithelium. in apc, odor-induced lfps consisted of a phasic initial component followed by a fast oscillatory activity in the beta range ( hz). by comparison a result of current source-density analysis with unit activity data, we confirmed that the initial component of odor-induced response has a characteristic temporal pattern, generated by a relatively weak direct afferent input, followed by an intracortical associative response, which was associated with a phasic inhibition. the beta oscillation might be generated by the repetition of these network activities. these electrophysiological data were consistent with the results of previous studies that used slice or anesthetized in vivo preparations. ps p-g chemotaxis of c. elegans to concentration gradient of an attractant superimposed on a uniformly distributed attractant lin lin, hiroyuki oikawa, miyako sasaki, tokumitsu wakabayashi, ryuzo shingai department of welfare engineering, iwate university, morioka, japan to investigate the informational interaction between pathways from different sensory inputs to the behavior in the nervous system of c. elegans, chemotaxis toward the concentration gradient of an attractant spotted on a uniformly distributed another attractant was investigated. lysine and chloride ions are water soluble chemoattractants. when m lysine was spotted on ammonium chloride background, . - . m and . m background did not influence lysine chemotaxis, while . m background augmented and . - . m background suppressed the chemotaxis. in contrast, when . m ammonium chloride was spotted on the lysine background, the background did not alter or suppressed the chemotaxis. interaction between informational pathways from different sensory inputs could be seen also in the presentation of an odorant spotted on chemoattractant background, and vice versa. ps p-g glutamate receptors are regulated by the ras-mapk pathway in neural circuit-dependent odor adaptation in c. elegans takaaki hirotsu , , , takeshi ishihara , eisuke nishida , yuichi iino dept. biol., fac. sci., kyushu univ., japan; mol. genet. res. lab., univ. of tokyo, japan; grad. sch. biostudies., kyoto univ., japan c. elegans shows a decrease in chemotaxis to odorants after exposure to the odorant for min. this plasticity, called early adaptation, requires aiy interneurons, which receive synaptic inputs from olfactory neurons, indicating that early adaptation depends on neural circuit. the ras-mapk pathway is activated by odorant exposure in aiy and plays essential roles for early adaptation. the function of glr- , a non-nmda type glutamate receptor, in aiy is also important for early adaptation. glr- appears to localize at postsynaptic sites in aiy. this localization was changed by odorant exposure in early adaptation. mutation of the ras-mapk pathway impaired localization of glr- . in vitro kinase analyses revealed the possibility that mapk directly phosphorylates glr- . these results suggest that the ras-mapk pathway controls odor adaptation by directly regulating glr- localization in aiy neurons. kohei ueno , yoshiaki kidokoro dept. behav. sci., grad. sch. med., gunma univ., maebashi, japan; inst. mol. cel. reg., gunma univ., maebashi, japan sodium chloride (nacl) is the major substance that induces nacl taste. in rodents, some strains prefer nacl solutions (∼ %), but others do not or even avoid them. although it is reported that the difference is based on the genetic background, the molecular information involved in the difference is not known. in the th ns annual meeting, we have shown that nacl preference in several wild-type strains of drosophila melanogaster is variable and p-element insertion in a single gene suppressed nacl preference. here, we carried out the sequencing analysis and found eight single-nucleotide polymorphisms (snps) in the gene. moreover, we found that one of the snps was correlated with nacl preference among wild-type strains. we generated transgenic flies and rescued the low preference phenotype of p-element insertion strain using the gal /uas system. finally, we examined the expression pattern of the gene and found the gene is expressed in taste organs. taken together, we suggest that the gene is a novel nacl receptor gene. ps p-g spatial and temporal organization of odor representation by moth antennal lobe output neurons shigehiro namiki , graduate school of life and environmental sciences, university of tsukuba, ibaraki, japan; department of mechano-informatics, graduate school of information science and technology, university of tokyo, tokyo, japan the antennal lobe (al) is the first relay station for olfactory information in the insect brain and is the anatomical equivalent of the mammalian olfactory bulb. both systems have common structures called glomeruli, functional units of olfactory processing. odor-evoked spatial and temporal patterns by an array of glomeruli are both important in olfactory coding. but the details of olfactory coding mechanisms are still unclear. we confirmed that projection neurons (pns, al output cells) innervating the same glomerulus had similar olfactory responses in the silkmoth. by pooling data from many pns that innervate identified glomeruli i reconstructed odor representations. i found that olfactory information is encoded by distributed spatiotemporal activity of a pn population and that there are no clear correlation between the similarity of slow temporal patterns of pns and spatial distances of innervating glomeruli. research funds: brain ps p-g medial nucleus amygdala neurons have morphologically and electrophysiologically heterogeneous properties makoto yokosuka , yoshinori sahara , shinichiro horie , masumi ichikawa , shun nakamura st. marianna univ. schl. med., kawasaki, japan; ntl. inst. neurosci., ncnp, tokyo, japan; tokyo metropol. inst. neurosci., tokyo, japan we characterize the electrophysiological and morphological properties of the medial nucleus amygdala (mea) neurons using whole-cell recordings in mice slice preparations. most mea neurons showed either tonic-bursting or adapting burst of action potentials to deporalizing currents. biocytin labeling showed that mea neurons possessed bipolar to multipolar cell bodies and dendritic fields covering projection areas from the accessory olfactory bulb. norepinephrine increased the frequency of spontaneous ipscs in some neurons, while serotonin increased spontaneous epscs in others. morphologically and physiologically heterogeneous mea neurons seem likely to produce multiplex outputs of many instinct behaviors. hideyuki matsumoto, kensaku mori department of physiology, graduate school of medicine, university of tokyo, tokyo, japan olfactory sensation sometimes lasts even after odorant stimulation has ceased. neuronal mechanisms for the olfactory afterimage are not well understood yet. single unit recordings from mitral/tufted cells in the mouse olfactory bulb (ob) showed that some neurons continued to discharge for more than s even after the cessation of odorant stimulation. the induction of the sustained spike discharge depended on the intensity of odorant stimulation, and showed an allor-none behavior. spike discharges during the sustained discharge mode phase-locked to the respiration cycle and the phase-locking pattern during the sustained discharge mode differed from that during odor stimulation. these results suggest that neuronal mechanism in the ob may be responsible for the induction of the post-stimulus sustained discharges. the respiratory-phase-locked sustained discharges were recorded from juxta-glomerular cells. this implies that neuronal interactions within the glomeruli are involved in the induction of the sustained spike activity of mitral/tufted cells. ps p-g synaptic transmission shows state-dependent change in the urethane-anesthetized rat olfactory bulb yusuke tsuno, hideki kashiwadani, kensaku mori department of physiology, graduate school of medicine, the university of tokyo, tokyo, japan olfactory cortex (oc) shows a state-dependent sensory gating that is controlled under the modulatory inputs from the basal forebrain and brainstem. since the olfactory bulb (ob) receives the modulatory inputs heavily, neuronal activity in the ob might change in a state-dependent manner. in the present study, we demonstrate a clear state-dependent change in the magnitude of the transmission of granule-to-mitral dendrodendritic inhibitory synapses and olfactory cortex-to-granule excitatory synapses. transmission of granule-tomitral synapses and olfactory cortex-to-granule synapses was facilitated during slow-wave state and suppressed during fast-wave state. in addition, we observed synchronous slow oscillations (about hz) in the granule cell layer of the ob, layer iii of the oc, and the occipital cortex. thus the ob shows state-dependent synaptic modulation and presumably receives top-down periodic signals from the cortex. research funds: kakenhi ( ) ps p-g rem sleep deprivation decreases na-k atpase phosphorylation gitanjali das, birendra n. mallick school of life sciences, jawaharlal nehru university, new delhi, india it has been hypothesized that "one of the functions of rem sleep is to maintain brain excitability" rem sleep deprivation increases noradrenaline in the brain that increases the na-k atpase activity causing increased brain excitability. however, the molecular mechanism of such increased na-k atpase activity was unknown; although it was known that dephosphorylated state is the active form of na-k atpase. rats were rem sleep deprived by flower-pot method; large platform and recovery from lost rem sleep were carried out as controls. at the end of experiment, brains were quickly removed by cervical dislocation and synaptosomes prepared, which were used for western blotting against phosphoserine and phosphothreonine antibodies as well as for na-k atpase activity. after rem sleep deprivation the activity increased, while the level of phosphorylated form of na-k atpase decreased in the same sample. this confirms our hypothesis that rem sleep deprivation induced increased activity is due to dephosphorylation of na-k atpase. research funds: icmr (govt. of india) and upoe (govt of india) takeshi fujii , , ken yoshikawa , yuki takatori , koichiro kawashima dept. of pharmacol., fac. of pharmaceut sci., doshisha women's coll., japan; dept. of pharmacol., kyoritsu univ. of pharmacy, japan stimulation of muscarinic (machr) and nicotinic (nachr) receptors with respective agonists induces ca + signals in t cells. in the present study, using rna interference approach, we investigated roles of machr and nachr subtypes in ca + signals in ccrf-cem (cem) cells, a human t cell line, as a model of t cells. cem cells express m , m , m and m machr subtypes, and ␣ , ␣ , ␣ , ␣ , ␣ , ␣ and ␤ nachr subunits. transfection of anti-m , anti-m and anti-␣ small interfering rna (sirna) significantly down-regulated respective mrna expression, while no changes were observed in gene expression of other machr subtypes or nachr subunits. ca + signals evoked by oxotremorine-m, a non-selective machr agonist, were reduced by anti-m or anti-m sirna. ca + signals evoked by nicotine were reduced by anti-␣ sirna. these findings indicate that m , m machr and ␣ nachr subtypes play major roles in ca + signals to acetylcholine in t cells, and suggest that these receptors are involved in regulation of immune function. research funds: kakenhi ( ) ps p-g is "seronegative" mg explained by autoantibodies to musk? kazuhiro shigemoto , sachiho kubo , seiji matsuda , naoki maruyama dept. of preventive medicine, ehime univ. schl. of med., ehime, japan; dept. of mol. path., tokyo metro inst. for gerontology, tokyo, japan; dept. of integrated basic medical science, ehime univ. schl. of med., ehime, japan muscle-specific kinase (musk) is critical for the synaptic clustering of nicotinic acetylcholine receptors (achr). musk is activated by agrin, which is released from motoneurons, and induces achr clustering at the postsynaptic membrane. although autoantibodies against the ectodomain of musk have been found in a proportion of patients with generalized myasthenia gravis (mg), it is unclear whether musk autoantibodies are the causative agent of generalized mg. in the present study, rabbits immunized with musk ectodomain protein manifested mg-like muscle weakness with a reduction of achr clustering at the nmj. the autoantibodies activated musk and blocked achr clustering induced by agrin or by mediators that do not activate musk. thus, musk autoantibodies rigorously inhibit achr clustering mediated by multiple pathways, an outcome that broadens our general comprehension of the pathogenesis of mg. (shigemoto et al., j. clinical investigation, ) research funds: kakenhi ( ) ps p-g dynamic changes in the thalamo-cortical system associated with thalamic neurodegeneration shin-ichi kyuhou, hisae gemba department of physiology, kansai medical university, japan in purkinje cell degeneration (pcd) mice, degenerating thalamic neurons were found morphologically in the particular thalamic nuclei including the ventral medial geniculate nucleus around postnatal day . electrophysiologically, auditory evoked potentials in the primary auditory cortex began to decrease gradually in amplitude from postnatal day . analysis of spontaneous cortical field potentials by fast fourier transform, revealed that high frequency oscillation (hfo) of around hz appeared prominently in the auditory cortex. local injection of kynurenic acid, a glutamate receptor blocker, into the thalamus suppressed the hfo in the auditory cortex, indicating that the thalamus is involved in the generation of the hfo. the real time polymerase chain reaction analysis demonstrated the upregulation of the mrna of nmda receptors in the auditory cortex. these results suggested dynamic changes occurred in the thalamo-cortical system after thalamic neurodegeneration in pcd mice. research funds: grant c from kansai medical university ps p-h unusually folded sod species sequester specific motor molecules and inhibit the axonal transport of their cargos minako tateno , yumiko simazaki , fuminori saitoh , ryosuke takahashi , toshiyuki araki national institute of neuroscience (ncnp), tokyo, japan; dept. of neurology, kyoto university, kyoto, japan misfolding of mutant sod protein is thought to be responsible for the selective loss of motoneurons in sod -related familial amyotrophic lateral sclerosis (als), although the molecular mechanisms underlying the toxicity of such unusually folded sod species are not yet clarified. since we have detected accumulation of unusual sod species in motoneuronal axons from g a sod -tg mice, we fractionated the ventral white matter of spinal cords to isolate the unusual sod species. immunoprecipitation analyses revealed specific interaction of unusual sod species with certain kinds of motor molecules. moreover, the axonal transport of cargos mediated by those molecules was found to be significantly reduced in symptomatic mutant sod -tg compared with wt sod -tg mice. these data strongly suggest that the toxic property of unusual sod proteins is partially ascribable to the transport inhibition of specific cargos. research funds: grant-in-aid for scientific research c ( ) ps p-h relationship between the amount of the cathepsin d expression and the symptomatic manifestation of neuronal ceroid-lipofuscinosis in a mouse model masahiro shibata, masato koike, yasuo uchiyama department of cell biology and neuroscience, osaka university graduate school of medicine, japan mice deficient in cathepsin d (cd), a representative lysosomal aspartic proteinase, have been shown to be an excellent model of neuronal ceroid-lipofuscinosis (ncl). here we report that the phenotype of mice in which cd is partially expressed is decided depending on the amount of the protein expression of cd. the proteolytic activity and protein expression of cd in the mutant mice were approximately % of those in the wild-type mice, while the growth of the mice appeared intact until postnatal day . the mice started to show ncl symptoms on p , and their life span was prolonged for one to three days, compared to that of the cd-null mice. the protein expression of cd in the heterozygous mice was approximately half of that in the wildtype mice and the mice showed no pathological finding. these results indicate that a threshold of the cd expression required for the manifestation of ncl symptoms in the mice may be present in the range from % to % of that in the wild-type mice. research funds: kakenhi ( ) ps p-h neuronal toxicity of expanded polyglutamine depends on intracellular distribution among cells with similar expression levels mamoru satoh, atsuyoshi shimada, noriko kawamura, yoichi chiba, yuko saitoh, hiromi keino, masanori hosokawa dept. pathol., inst. develop. res., aichi human service center, aichi, japan we previously reported that expanded polyglutamine (polyq) tracts induced cellular toxicity of neuro a cells in the form of massive cytoplasmic aggregates but not of intranuclear inclusion. however, we did not rule out the possibility that such toxicity depends on the level of intracellular expression of polyq. in this report, we compared the toxicity of polyq among cells expressing polyq tracts with a variety of intracellular distribution but at similar expression levels. damages were most remarkable in cells with cytoplasmic massive aggregate in terms of shrunken cellular and nuclear sizes. cells with cytoplasmic homogeneous distribution, cytoplasmic punctate distribution and intranuclear inclusion of polyq tracts were relatively spared. these data suggest that the severity of cell damages depends on the type of intracellular distribution of polyq tracts in cells expressing polyq tracts at similar level. ayumi takamura , katsumi higaki , junichiro matsuda , yoshiyuki suzuki , eiji nanba division of functional genomics, research center for bioscience and technology, tottori university, tottori, japan; national institute of biomedical innovation, osaka, japan; clinical research center, international university of health and welfare, tochigi, japan g m -gangliodisosis is an autosomal recessive lipid storage neurodegenerative disorder. due to a deficiency of lysosomal ␤-galactosidase, excessive lysosomal accumulation of gm is observed in patients and animal model brains. however pathogenesisi of this disease is still unclear. since gm is known to be a major sialoglycolipid constituent of plasma membrane (pm) in neuron, we examined the analysis of brain of mouse model. cerebellar granule cells from this mouse showed gm accumulation of lysosome and pm and the membrane fluidity was also reduced. gm -bound phosphorylated trka was markedly decreased in cultured neuron and brain tissues. subsequent plc␥, known as a downstream signal of trka, was also impaired. these results suggest that dysfunction of neurotrophin signaling may cause the onset of neurodegeneration in g m -gangliosidosis. katsuya inoue , , katsuaki endo , takamitsu fujikawa , seijyun fukuda , tatsuo nakamura department of physical therapy, university of aino, osaka, japan; institute for frontier medical science, kyoto university, kyoto, japan regeneration of spinal cord injury is an important thema in rehabilitation science as well as basic one. the experiment was designed to reveal the process after spinal cord injury by asphyxia. to establish the animal model of spinal cord injury produced by asphyxia, we used adult cats with aorta occulusion under deep pentabarbital anesthesia. twenty minutes after occulusion electrical reflex activity of spinal cord disappeared. after min occulusion, irreversible functional changes were observed, long term depression of reflex activities and disorders of motorsensory function. we also traced time course of electrical and functional changes after min occulusion. ps p-h development of a rodent behavioral model to study the direct interactions of reward and learning adam weitemier, niall p. murphy riken brain science institute, japan cognitive and reward processes often occur simultaneously, and perhaps interdependently. learning is a necessary condition in many experimental models aimed at assessing the rewarding value of a given stimulus. conversely, reward is often used as an experimental tool to engage mnemonic processes in studies aimed at investigating learning and memory. recent studies have demonstrated shared neurobiology between memory and reward. a direct behavioral interaction between reward and memory has never been studied. cognitive impairments observed in psychiatric conditions of dysregulated reward, such as drug abuse and depression, make this issue important, particularly in light of ongoing efforts to investigate higher brain functions. we are developing a rodent behavioral model with which to directly assess the influence of reward processes on learning and memory. we will introduce our recent progress with this new model, including two variations of the procedure designed to study the influence of reward on memory acquisition and memory recall. tetsuya ando , yuya kawanaka , minoru saito , hiroaki mochizuki , ken honjo , hirofumi toda , , toshifumi tomoda , akira sawa , katsuo furukubo-tokunaga grad. school of life & envir. sci., univ. tsukuba, japan; molecular physiol., tokyo metropolitan inst. neurosci. tokyo, japan; beckman res. inst., city of hope. california, usa; dept. of psych. & neurosci. johns hopkins univ. school of medicine. baltimore, usa the disrupted-in-schizophrenia- (disc ) gene, originally identified at the breakpoint of a chromosome ( ; ) (q . ; q . ) translocation in a scottish schizophrenia pedigree, is a promising candidate gene for schizophrenia and affective disorder. however, cellular and molecular mechanisms underlying cognitive impairments are yet to be elucidated. to address disc functions in vivo, we expressed disc in drosophila and examined developmental and behavioral phenotypes. overexpression of disc resulted in marked suppression of olfactory associative learning in flies whereas it caused no symptoms of neural degeneration even in aged animals. we anticipate that the drosophila system will serve as a novel model system amenable to a variety of genetic manipulations for the study of schizophrenia. ps p-h effect of hypothermia on discrepancy between memory learning ability and anatomical brain damages in rats with neonatal hypoxic ischemic encephalopathy yuji miyatake , ayumi kamo , kenji minato , hitoshi haruna , hiritsugu fukuda , yuji murata , takayoshi hosono department of bomedical engineering, osaka electro-communication university, japan; graduate school of medicine, osaka university, japan we investigated the effect of brain hypothermia on neonatal hypoxic ischemic encephalopathy (hie) in hie-model rats using olton t-maze and anatomy. the common carotid artery of of -day-old rats was ligated and cut under anesthesia. after the operation the rats were put in a box containing % oxygen at • c for min. after the insult, of the rats were put in a box at • c for h (hypothermia, h-group). the other rats were returned to their mother without hypothermia (normothermia, n-group). sham operations were performed on three rats (s-group). eight weeks after the operation, their learning and memory ability was assessed by olton t-maze, and no statistical difference was observed in either the working or reference memory in the three groups although the anatomical brain size in the n-group was significantly smaller than in the h-group and s-group. withdrawn ps p-h tau hyperphosphorylation in ts cje, a partial trisomy mouse model for down syndrome ebrahim abdul , a. shimohata , w. yu , m. yamaguchi , m. murayama , d. chui , t. akagi , t. takeuchi , k. amano , h.s. karthik , t. hashikawa , h. sago , c.j. epstein , a. takashima , k. yamakawa research scientist; lab. for neural arch.; lab. for alzheimers disease; div. of fetal med. ncchd; ucsf, usa although down syndrome (ds) or trisomy is the most common genetic cause of mental retardation, its neuropathology remains unclear. ts cje, a ds mouse model partially trisomic for chromosome , shows learning and behavioral abnormalities mimicking ds mental retardation. the trisomic segment, corresponding to parts of human chromosome q , has about genes. importantly, sod and app, which may contribute to the ds phenotype, are excluded from the ts cje trisomic segment. here we report that ts cje brains show hyperphosphorylation of tau in the absence of nft formation, as well as increased gsk ␤ and jnk/sapk activities without alterations in a␤pp metabolism. our results suggest that genes on the trisomic ts cje segment other than app and sod can cause hyperphosphorylation of tau, which in turn may be critical in the pathogenesis of ds mental retardation. research funds: kakenhi number: ps p-h increased oxidative stress and mitochondrial dysfunction in ts cje, a down syndrome mouse model atsushi shimohata , ebrahim a. s. , m. yamaguchi , w. yu , h. sago , c.j. epstein , k. yamakawa lab. for neurogenetics, riken-bsi, japan; div. of fetal med. ncchd, japan; dept. pediatrics, ucsf, usa down's syndrome (ds), caused by chromosome (hsa ) trisomy, is the most common genetic cause of mental retardation and affects every major organ in the body. ts cje is one of a number of segmentally trisomic ds mouse models, and is triplicated for a region of mouse chromosome extending from sod to znf , containing genes syntenic with hsa . since these mice show learning and behavioral abnormalities mimicking ds mental retardation, ts cjespecific trisomic segment genes may be involved in the ds phenotype. in the present study, we observed increased levels of reactive oxygen species (ros), mitochondrial function impairment in primary cultured astrocytes and hippocampal neurons, and increased cabonylated proteins in ts cje brains. collectively, our results implicate dosage imbalanced genes other than sod and app in both ros generation and mitochondrial dysfunction, which in turn possibly contribute to the ts cje ds mental retardation-like phenotype. ps p-h polyinosinic-polycytidylic acid injection in early pregnancy causes the hypomyelination in the hippocampus, but not in the cortex manabu makinodan , , kouko tatsumi , takayuki manabe , takahira yamauchi , , eri makinodan , juro shimoda , toshifumi kishimoto , akio wanaka department of psychiatry, nara medical university, kashihara, japan; department of nd anatomy, nara medical university, kashihara, japan polyinosinic-polycytidylic acid (poly i:c) elicits maternal immune response similar to anti-viral ones. recent studies demonstrated that poly i:c injection into pregnant mice resulted in behavioral changes including deficits in prepulse inhibition in the offspring, rendering this system an animal model of schizophrenia. in the present study, we observed such behavioral abnormalities reproducibly in the experimental group born from poly i:c-injected mice, but not in the control group born from pbs-injected mice. they showed decreased myelination in the hippocampus at juvenile period with unaltered number of oligodendrocytes. on the other hand, myelination in the cerebral cortex did not significantly differ between the experimental and control mice. the hypomyelinaton in the hippocampus at the juvenile period may be a possible cause for the behavioral changes in later periods. joanna doumanis, ritsuko kazama, adrian moore, nobuyuki nukina riken brain science institute, japan the fruitfly drosophila melanogaster is well established as a model system in the study of human neurodegenerative diseases. to model the polyglutamine expansion disease, huntington disease (hd), we have established stable, inducible cell lines expressing n-terminal truncated huntingtin fused to egfp with an expanded ( q) polyglutamine repeat in a drosophila larval central nervous system-derived cell line. induction of expression results in the formation of protein aggregates, characteristic of hd. utilising rnai, we have carried out a high-throughput screen for modifiers of aggregate formation in these cells. genes, encompassing around % of the drosophila genome, were screened, resulting in the identification of candidates that either suppress or enhance aggregation. most candidates identified have mammalian orthologues, validating the use of drosophila to screen for genes relevant to human disease. we established in vivo models of hd by expressing polyq-egfp in the drosophila nervous system and are further characterising selected candidates in our model. the rodent model of harmaline-induced tremor has been used as an animal model of essential tremor. the present study investigated effects of harmaline on olivocerebellar systems of mice and rats. systemic administration of harmaline produced generalized tremors in both types of rodents. immunohistochemical studies revealed significant degeneration of purkinje cells that was associated with activated microgliosis in the cerebellar cortex, following administration of harmaline in rats but not in mice. however, in mice but not rats, microgliosis was induced following administration of harmaline in the inferior olivary nucleus (ion). numbers of neurons in the mouse ion did not decrease, suggesting the possibility that microgliosis in ion might not be a simple neurotoxic effect. presumably, differences in sensitivity of purkinje cells between rats and mice may be related to differences in functional alterations in their respective olivocerebellar systems induced by harmaline. recognition of these species-specific differences is an important consideration for experimental analysis of the rodent model of tremors. ps p-h analysis of ␣-synuclein expression in young mouse model of multiple system atrophy kimiko nakayama, yasuyo suzuki, ikuru yazawa laboratory of research resources, national institute for longevity sciences, aichi, japan multiple system atrophy (msa) is a sporadic neurodegenerative disease that affects oligodendrocytes and neurons in human central nervous system. glial cytoplasmic inclusions (gcis) are diagnostics of msa. gcis are shown to be abnormal accumulation of filamentous ␣-synuclein. yazawa et al. ( ) generated a transgenic (tg) mice overexpressing human wild-type ␣-synuclein in oligodendrocytes under the control of the , ,-cyclic nucleotide -phosphodiesterase (cnp) promoter. tg mouse study demonstrated that formation of gci-like ␣-synuclein inclusions in the oligodendrocyte leads directly to neuronal degeneration, as shown by motor impairment and novel accumulation of mouse ␣-synuclein in neuron. to elucidate the mechanisms of neurodegeneration in tg mice, we prepared primary cultures of neurons and glial cells from tg mice. the cells are examined the effects of ␣-synuclein accumulation. ps p-h dysregulation of sodium channel ␤ subunit by expanded polyglutamine in huntington disease transgenic mice fumitaka oyama, haruko miyazaki, kazumasa okamura, yoko machida, kurosawa masaru, takashi sakurai, nobuyuki nukina laboratory for structural neuropathology, riken bsi, wako-shi, japan sodium channel ␤ (␤ ) is a very recently identified auxiliary subunit of the voltage gated-sodium channels. we have identified ␤ as an est that was significantly downregulated in the striatum of hd model mice and found that reduction in ␤ started at a presymptomatic stage of the hd model mice. in contrast, spinal cord neurons, which generate only negligible levels of expanded polyq aggregates, maintained normal levels of ␤ expression even at the symptomatic stage. expanded polyq with nls expression suppressed the promoter activity of ␤ gene in pc cells. forskolin, an activator of the camp/pka pathway, did not affect b promoter activity, indicating that ␤ is not camp-responsive gene. these findings strongly suggest that sodium channel ␤ subunit is a novel molecule, which is an upstream non-camp-responsive gene in hd pathogenesis. ps p-h repeat length-and age-dependent changes in behavioral phenotypes of drpla transgenic mice harboring a single copy of a full-length human drpla gene kazushi suzuki , yuji takahashi , jun goto , mutsuo oyake , toshiya sato , shoji tsuji department of neurology, the university of tokyo, tokyo, japan; department of neurology, brain research institute, niigata university, niigata, japan; center for bioresource-based research, brain research institute, niigata university, niigata, japan we carried out detailed analyses of the behavioral phenotypes of drpla transgenic mice carrying an expanded cag repeat of (q ), (q ), (q ), or (q ). in the accelerating rotarod ( w), the latencies of q , q , q and q were %, %, % and %, respectively. in the open field, moving distances of q , q , and q were decreased to %, %, and %, respectively, while that of q was increased to %. home cage activity was decreased depending on the repeat length. the q mice, however, showed increased ratios of the activity during the light time to that during the total day at weeks ( %) and weeks ( %), suggesting that drpla mice display not only impaired motor coordination, but also changes in emotional behavior, and disrupted night and day activity patterns. ps p-h the mice lacking schnurri- show multiple behavioral abnormalities related to psychiatric disorders keizo takao , nobuyuki yamasaki , keiko toyama , tsuyoshi takagi , shunsuke ishii , tsuyoshi miyakawa hmro, kyoto university graduate school of medicine, kyoto, japan; riken, tsukuba, japan schnurri- (shn- ) is a zinc finger transcription factor, a mouse homologue of human hiv-ep , that binds to nuclear factor kappa b-binding site in the hiv long terminal repeat. shn- is known to play important roles in the mammalian immune systems. however, the role of shn- in the central nervous system (cns) is still unknown. to investigate the functional significance of shn- in mammalian brain, we analyzed the shn- knockout (ko) mice using a comprehensive behavioral test battery. shn- ko mice were dramatically hyperactive under novel environment and in their home cage. they also showed increased acoustic startle response and impaired prepulse inhibition, indicating their impairment in sensorimotor gating. anxiety-like behavior and depression-like behavior were also significantly reduced in shn- mice. our results demonstrate a critical role of shn- in cns and suggest that shn- ko mice may serve as an animal model of psychiatric disorders. research funds: kakenhi ( , , , ) , jst bird ps p-h comprehensive brain-behavior phenotyping of camkii␣ heterozygous knockout mice nobuyuki yamasaki, koichi tanda, keiko toyama, yasuyuki fukui, keizo takao, tsuyoshi miyakawa hmro, kyoto university graduate school of medicine, kyoto, japan ca + /calmodulin-dependent protein kinase ii (camkii) is a ubiquitous serine/threonine protein kinase that is abundant in brain as a major constituent of the postsynaptic density and critically involved in synaptic plasticity, learning and memory. several behavioral abnormalities of camkii␣ mutant mice were reported, but systematic assessments of behaviors of camkii␣ mutant mice have not been conducted. to analyze the behavioral effects of camkii␣ deficiency, we subjected camkii␣ heterozygous knockout mice to a comprehensive behavioral test battery. the mutant mice showed hyperactivity, decreased anxiety, decreased depression-related behavior, increased offensiveness, selective spatial working memory deficit, and dramatic periodic change of locomotor activity in home cage. to identify the mechanism underlying these behavioral abnormalities, gene expression analysis was conducted. the potential involvement of camkii␣ in pathogenesis/pathophysiology of psychiatric disorders will be discussed. research funds: kakenhi ( , , , ) , jst bird ps p-h effects of various factors on the results of a comprehensive behavioral test battery for genetically engineered mice: a factor analytic study hiroshi ougino, nobuyuki yamasaki, koichi tanda, keiko toyama, keizo takao, tsuyoshi miyakawa hmro, kyoto university graduate school of medicine, kyoto, japan we have been using a behavioral test battery to reveal unknown phenotypes of genetically engineered mice. for the adequate experimental design and interpretation of data, it is essential to know experimental variables which may potentially influence results, and various kinds of factors which underlie many indices measured in the tests. in this study, we investigated the effects of background strains (c bl/ j, c bl/ n, c bl/ c, svev, balb/c), body weight, age at test, and start time of test on the results of each test, by analyzing data of more than mice (, including wild type and mutant mice from strains of genetically engineered mice), which had been tested in our laboratory. also, we conducted factor analyses of a large set of data to examine the relationship between behavioral indices. the potential implications of our findings for the improvement of the behavioral test battery will be discussed. calcium-and calmodulin-dependent protein kinase iv (camkiv) is a protein kinase that activates the transcription factor, camp responseelement binding protein (creb). camkiv has been hypothesized to play a significant role in synaptic plasticity and in learning and memory. however, functions of camkiv in a variety of behaviors, e.g., motor function, nociception, fear, anxiety, depression, learning and so on, have not yet been fully elucidated. to gain more insight into behavioral significance of camkiv, we subjected camkiv−/− mice to a battery of behavioral tests. camkiv−/− mice did not display any deficit in spatial reference memory and working memory tests, but had mild performance deficit in fear conditioning tests. these results indicated selective and specific involvement of camkiv in regulating emotional behavior. research funds: kakenhi ( , , , ) , jst bird ps p-h comprehensive behaivoral analysis of ryanodine receptor type knockout mouse suzuko ohsako , koichi tanda , , nobuyuki yamasaki , keiko toyama , hiroshi takeshima , tsuyoshi miyakawa kyoto university graduate school of medicine, kyoto, japan; dep. of pediatrics, kyoto prefectural univ. of medicine, kyoto, japan; dep. of biochem. and mol biol., tohoku univ. graduate school of medicine, miyagi, japan ca + signaling is essential for the regulation of neuronal processes including synaptic transmission and transmitter release. ryanodine receptors (ryrs) are family of intracellular calcium channels and mediate calcium-induced calcium release from the endoplasmic reticulum. ryr is highly expressed in the hippocampus, caudate putamen, and thalamus. to investigate the behavioral effects of ryr deficiency, we subjected ryr knockoout mice to a battery of behavioral tests. ryr knockout mice exhibited hyperactivity and abnormal behavior in social interaction test, while they did not show any deficit in motor function, depression, attention, and working memory tests. these results suggest a role of ryr in regulating general locomotor activity and social behavior. research funds: kakenhi ( , , , ) , jst bird ps p-h comprehensive behavioral analysis of neuronal nitric oxide synthase knockout mouse keiko toyama , koichi tanda , , nobuyuki yamasaki , tsuyoshi miyakawa hmro, kyoto university graduate school of medicine, kyoto, japan; dept. of pediatrics, kyoto prefectural univ. of medicine, kyoto, japan nitric oxide (no) plays several important roles in the brain, including in regulation of synaptic signaling and plasticity. no is synthesized from the amino acid l-arginine by the enzyme nitric oxide synthase (nos). in neurons, no is produced by neuronal nitric oxide synthase (nnos), representing one of three nos isoforms expressed in most tissues. to elucidate function of nnos/no in a variety of behaviors, e.g., activity, motor function, nociception, attention, anxiety, depression, social interaction, learning and so on, we subjected nnos knockout mice to a battery of behavioral tests. nnos knockout mice exhibited increased locomotor activity and decreased depressionrelated behavior. furthermore, they displayed increased social contacts in novel environment and homecage. these results indicate that nnos/no is involved in regulation of their behaviors. research funds: kakenhi ( , , , ) , jst bird ps p-h primate model of attention-deficit/hyperactivity-disorders (adhd) shintaro funahashi , keiko shimizu grad. sch. human and environmental std, kyoto univ., kyoto, japan; primate res. inst., kyoto univ., inuyama, japan adhd is one of the prevalent childhood psychiatric disorders. children with adhd show hyperactive behavior and attention problems, suggesting prefrontal (pfc) contribution to adhd. adhd is also known as dopamine (da) related dysfunctions, because methylphenidate is the most effective drug for the treatment of adhd. pfc is the cortical area where the strongest da innervation is observed. injection of da-related drugs to pfc produces behavioral deficits in cognitive tasks. these suggest that da-related dysfunction in pfc could be a candidate of biological causes of adhd. to prove this notion, we injected -ohda into bilateral pfc to destroy da innervation in infant monkeys and examined whether these monkeys exhibited hyperactivity. -ohda injected monkeys showed significant increase of spontaneous activity in test cages. oral administration of methylphenidate reduced spontaneous activity in -ohda injected monkeys. these results suggest that monkeys injected -ohda into pfc are good candidates of the primate model of adhd. research funds: kakenhi ( ) ps p-i training-induced recovery of precision grip after primary motor cortex damage in the adult monkey yumi murata , , , noriyuki higo , , takao oishi , , , akiko yamashita , keiji matsuda , motoharu hayashi neurosci. res. inst, aist, tsukuba, japan; grad. sch. compreh. hum. sci., univ. of tsukuba, tsukuba, japan; crest, jst, kawaguchi, japan; dept. cell mol. biol., primate res. inst., kyoto univ., inuyama, japan; div. applied sys. neurosci., nihon univ. sch. med., tokyo, japan in the present study, we compared the motor recovery between monkeys that received daily training and that did not receive any training after lesion of the primary motor cortex (mi), in order to investigate the effects of postlesion training on motor recovery. we derived a hand representation map in mi, and ibotenic acid was then injected to destroy the digit region, which resulted in hand paralysis. after one or two months of postlesion training, skilled use of the affected hand including a precision grip was recovered. untrained monkeys also became able to grasp objects with their affected hand, but they couldn't use a precision grip. this suggests that recovery of precision grip requires postlesion training. research funds: a grant-in-aid for scientific research on priority areas from mext ( ) mouse mutants with behavioral abnormality are indispensable tools to elucidate molecular pathways underlying behavior. in order to develop numbers of novel behavioral mutants, we have been carrying out dominant behavioral screening in potential mouse mutants that was randomly induced point mutations by a chemical mutagen enu (n-ethyl-n-nitrosourea). we screened about , g animals (dba/ j × enu-treated c bl/ j) for home-cage activity, open-field activity, and passive avoidance response, and obtained lines of dominant behavioral mutants. by linkage analysis, the causative genes were mapped in of mutant lines. hyperactivity was predominant phenotype, and of mutants showed hyperactivity in home-cage and/or open-field. we will report the recent results of initial characterization and the progress of fine mapping in these enuinduced mutants. ps p-i ubiquitin signal in neurons of cathepsin ddeficient mouse brains with special reference to the autophagic process masato koike, masahiro shibata, yasuo uchiyama dept. of cell biol. and neurosci., osaka univ. grad. sch. of med., suita, japan we have shown that autophagy contributes to the accumulation of vacuolar structures in neurons obtained from cd−/− and cb−/−cl−/− mice, murine models for neuronal ceroid lipofuscinoses (ncls) (koike et al., ) . until recently, it remains unknown what signaling is essential for autophagosome formation. interestingly, in the conditional atg -knock-out mice where autophagy is absent specifically in the liver, numerous ubiquitinated aggregates are detected in the cytosol of hepatocytes (komatsu et al., ) , suggesting that protein ubiquitination may serve as a signal to the autophagic process. we therefore examined the immunohisto/cytochemical localization of ubiquitin and lc , and found that in our ncl model mice, positive signals for ubiquitin and lc were co-localized on the membranes of granular structures in the neuronal perikarya. these results suggest that protein ubiquitination may be involved in signaling for autophagosome formation in ncls. research funds: grant-in-aid for young scientists (b)( ) and creative scientific research ( gs ) ps p-i activation of medial prefrontal cortex neurons by systemic phencyclidine is primarily mediated via ampa/kainate glutamate receptors tadahiro katayama , eiichi jodo , yoshiaki suzuki , ken-yo hoshino , yukihiko kayama dept. of physiology, fukushima medical university, fukushima, japan; dept. of neuropsychiatry, fukushima medical university, fukushima, japan it has been shown that tonic activation of the medial prefrontal cortex (mpfc) plays a pivotal role in development of behavioral abnormalities induced by systemic phencyclidine (pcp). however, receptors mediating such activation are not clearly specified, though several studies indicate the increase of extracellular acetylcholine, dopamine, and glutamate in the mpfc. here, we examined effects of local application of those antagonists on increased firing activity of mpfc neurons by systemic pcp in anesthetized rats. after tonic activation of mpfc neurons by pcp had been established, cnqx, sch , mecamylamine or scopolamine was locally applied with iontophoresis or gas pressure on the recorded neuron. cnqx reduced pcp-induced augmentation of firing activity to the baseline level, while others gave little changes. these results suggest that pcpinduced activation of mpfc neurons be mediated primarily via ampa/kainate receptors. ps p-i increased depressiveness and decreased sensitivity to antidepressants in calcium/calmodulin-dependent protein kinase iv (camkiv)-knockout mice jiro kasahara , hiroyuki sakagami , hisatake kondoh , kohji fukunaga department of pharmacology, graduate school of pharmaceutical sciences, tohoku university, sendai, japan; department of histology, graduate school of medicine, tohoku university, sendai, japan calcium/calmodulin-dependent protein kinase iv (camkiv) is expressed abundantly in the nuclei of neurons and thought to regulate ca-dependent gene expressions mediated by the transcriptional factors such as creb. recently, we found that chronic treatments of the rats with antidepressants increased camkiv activity and creb phosphorylation in the prefrontal cortex, suggesting the importance of camkiv in the effects of antidepressants. this result led us to perform the behavioral assessments of depressiveness and the sensitivity to antidepressants in camkiv-knockout mice by some experimental paradigms. from the experiments, the increased depressiveness and decreased sensitivity to antidepressants were observed in the mice, suggesting the importance of camkiv for the regulation of depressiveness and the effects of antidepressants. ps p-i severity of audiogenic seizures is influenced by multiple factors in vlgr -mutated mice hideshi yagi , , makoto sato , division of cell biology and neuroscience, department of morphological and functional sciences, faculty of medical sciences, university of fukui, fukui, japan; research and education program for life science, university of fukui, fukui, japan epilepsy is a highly prevailed disorder and reports are accumulating that demonstrate that single gene mutation causes such disorders. we made vlgr -mutated mice and found that they showed high susceptibility to audiogenic seizure, one of the reflex seizures provoked by loud noise. to evaluate whether the genetic backgrounds influence on phenotype of the audiogenic seizure in our mice, we made c bl/ backcrossed vlgr -mutated mice and /svs backcrossed vlgr -mutated mice. these two backcrossed lines showed different susceptible periods and severity of audiogenic seizure from the original line. furthermore, phenotype of audiogenic seizure was altered by restraining mice from free moving while being exposed to loud noise. these observations suggest that genetic factors and environmental factors may modify the phenotype of seizures and our vlgr -mutated mice are good model of reflex epilepsies that are evoked by multifactors. ps p-i reduction in the density of parvalbumin-positive cells in the medial frontal cortex of rats behaviorally sensitized to methamphetamine tomoko kadota , ken kadota , department of bioenvironmental medicine, university of chiba, chiba, japan; chiba institute of psychiatry, chiba, japan our previous study demonstrated that the development of behavioral sensitization of rats to methamphetamine (map) corresponded in time with the progress of neurotoxic changes induced in the medial prefrontal cortex (mfc). the present study further examined morpholological changes of rats that were administered a daily dose of mg/kg of map i.p. for days (d d ) and then withdrawn from the drug for days (wd wd ). the regimen reduced the densities of parvalbumin positive cells (pac); these were probably gabaergic cells and distributed in the strata covering layers ii, iii and v in the anterior cingulate cortex (cg ) and mfc. the decrease in the density of pac was first observed in cg and then in mfc. the reduction began on d and advanced to higher levels on d and subsequently wd . these findings suggest that the behavioral sensitization regimen leads to the deterioration of inhibitory processes in the neural circuits in cg and mfc, particularly in layers ii and iii. ps p-i up-regulation of ␤ -adrenergic receptor immunoreactivity in astrocytes in the spinal cord after dorsal rhizotomy teruyoshi kondo, yoshihiro ishibashi, kei-ichiro nakamura department of anatomy, division of microscopic and developmental anatomy, kurume university school of medicine, kurume, japan stimulation of ␤ -adrenergic receptor (␤ -ar) induces astroglial proliferation and activation after brain injury, but little is known concerning the potential role of adrenergic receptors in the spinal cord. present study demonstrated that rhizotomy induced a marked and prolonged up-regulation of ␤ -ar-immunoreactivity (ir) in the regions of the dorsal root entry zone and dorsal funiculus containing the central processes of the injured primary sensory neurons. ␤ -arimmunoreactive cells coexpressed gfap-ir and were positive for nestin which is characteristic of reactive astrocytes. a population of ␤ -ar-immunoreactive cells were labeled with ki- , a marker of cell proliferation, indicating some of them went into cell mitotic state. interestingly, a major population of ␤ -ar-immunoreactive cells also exhibited fgf- -ir. these findings suggest that ␤ -ar may play important roles in astrocytic activation and neuroprotection associated with induction of synthesis of growth factor such as fgf- . ps p-i effects of lateral fluid percussion injury (fpi) on the optical signals in dentate gyrus of the rat brain slice preparations shin yamashita , norihiro muraoka , hiroshi hasuo , takashi akasu , minoru shigemori dept. of physiology, kurume univ. sch. of med., kurume, japan; dept. of neurosurgery, kurume univ. sch. of med., kurume, japan we investigated the effects of experimental traumatic brain injury on the neuronal function in dentate gyrus (dg) using optical recording techniques with voltage-sensitive dye (rh ). horizontal hippocampal slices were obtained from the control and the fpi rats (one week after the single moderate impact). electrical stimulation of perforant path (pp) produced the optical signal spread in the molecular layer of dg. temporal change in the optical signal, obtained from an area on the propagation pathway, had two peaks (fast and slow peaks). increment of stimulus intensity ( - v) increased the amplitude of both fast and slow peaks. the intensity for producing the maximal response was - v. the amplitude of slow peak in fpi group was about % larger than that in control group, while the amplitudes of fast peak were not different in the two groups. these data suggest that the excitatory pp synapse onto granule cells of dg is facilitated after fpi. ps p-i comparative study of neural activities in mouse hippocampal slices by flavoprotein autofluorescence and ca + imaging chikafusa bessho, yasuharu mitsushima, ryo matsumoto department of physics, kyoto sangyo university, kyoto, japan recently k. shibuki et al. have succeeded in flavoprotein autofluorescence imaging of neural activities in the rat brain. we examined neural activities in mouse brain (hippocampal) slices by the modified method and ca + imaging. the slices ( m) were prepared from the block in an ice cold acsf medium using microslicer and incubated for h in the oxygenated medium at room temperature. a slice was placed on a recording chamber perfused with the medium at a flow rate of ml/min. green autofluorescence (> nm) of the slices illuminated by blue light ( - nm) was observed by an inverted microscope. images of the autofluorescence were recorded using a calcium imaging system. ca + imaging was also performed in the slices. slices were incubated in acsf medium containing m of fluo / am for h at • c. the ca + image was recorded with an excitation wavelength of - nmand an emission wave length of > nm. the autofluorescence and ca + responses wereobserved in slices perfused with l-glutamate ( mm). takuya hayashi , hiroshi sato , shinichi abe , takashi hanakawa , hiroshi watabe , hidenao fukuyama , babak aldekani , hidehiro iida department of investigative radiology, national cardiovascular center research institute, osaka, japan; human brain research center, kyoto university, kyoto, japan; nathan kline institute for psychiatric research, ny, usa we show connectivity pattern between cortex and striatum in macaque and human by using the non-invasive method of diffusionweighted magnetic resonance imaging (dwi). in macaque, the dwibased striatal connectivity of brodmann's area corresponded to that revealed by the tracer (mncl ) tractography. the dwi-based connectivity pattern also isolated a part of the ventral striatum corresponding to the histochemically-specific 'shell' region in both human and macaque. in addition, we confirmed the species-homology in intra-striatal topography of cortical connection by quantitatively analyzing the connectivity; however, we found that human striatum was more intensively connected to prefrontal cortex and less connected to extra-frontal cortices. these results suggest that human striatum has a dominant and specific role in processing prefrontal information. research funds: h -kokoro- ps p-i optical analysis of synaptic transmission by a fluorescent glutamate probe shigeyuki namiki, hirokazu sakamoto, sho iinuma, kenzo hirose department of cell physiology, nagoya university graduate school of medicine, nagoya, japan glutamate is an essential excitatory neurotransmitter in the central nervous systems. for optical analysis of glutamatergic synaptic transmission, we have developed a fluorescent glutamate probe called eos. by imaging with eos, we successfully detected the synaptically released glutamate following axon firings in cultured hippocampal neurons; the spatial distribution of the glutamate release was non-uniforml along dendrites. we also succeeded in monitoring the phorbol ester-induced potentiation of the glutamate release. furthermore, we found spontaneous and stochastic glutamate release which was confined to small regions. neither application of tetrodotoxin nor removal of extracellular calcium blocked the release. high concentrations of sucrose increased the frequency of the release. these features are reminiscent of those of miniature epsc in electrophysiological recordings and thus suggest that the spontaneous release is quantal vesicular release. in conclusion, our probe directly visualizes the presynaptic release. shingo miyata , , yasutake mori , , tsuya taneda , , hiroaki okuda , , masaya tohyama , department of anatomy and neuroscience, graduate school of medicine, osaka university, osaka, japan; st coe program, tokyo, japan local protein synthesis in neuronal dendrites is one of the mechanisms that may mediate a rapid and synapse-specific mobilization of proteins from the resident mrnas. a great deal of effort has been made in analyzing the dynamic state of protein synthesis in the living cells, chiefly by quantifying protein level. however, the protein level cannot mirror the spatio-temporal alteration of translation, because it cannot be affected only by protein synthesis but also by other factors like degradation. therefore, it is problematic to visualize the dynamic state of translation by the present methods. to solve the problem, we applied fret (fluorescence resonance energy transfer) technique to in situ detection of the assembly and disassembly cycle among a pair of translation initiation factors (eifs), thereby showing that bdnf and ephrin could potentiate local protein synthesis in the dendrites of hippocampal neurons. ps p-i a model selection of glm applied to fmri data using aic jobu watanabe , fumikazu miwakeichi , andreas galka , , ryuta kawashima , tohru ozaki , sunao uchida , institute for biomedical engineering, consolidated research institute for advanced science and medical care, waseda university, japan; department of medical system engineering, faculty of engineering, chiba university, chiba, japan; institute for statistical mathematics, tokyo, japan; institute of experimental and applied physics, university of kiel, keil, germany; new industry creation hatchery center, tohoku university, sendai, japan; faculty of sport sciences, waseda university, tokorozawa, japan in the general linear model (glm) that is widely used in analyses of functional neuroimaging data, several combinations of explanatory variables are possible. the akaike information criterion was applied as a basis of comparison and selection among several glms that analyze block-designed functional magnetic resonance imaging (fmri) data. the glms with/without a resting condition, head motion covariates, time derivatives and dispersion of hemodynamic response function were compared. we demonstrate that a combination of these explanatory variables can effectively improve the model and that aic is a useful tool for model selection in fmri studies. ryuzo shingai, katsunori hoshi, tokumitsu wakabayashi department of welfare engineering, iwateuniversity, morioka, japan to investigate the relationship between the behavior and function of the nervous system of caenorhabditis elegans, quantitative analysis of behavior that indirectly represents the internal states of the worm is necessary. we devised an automated analysis system of c. elegans locomotion. the system is well suited for detecting four locomotion states: forward or backward movement, curl and rest. the system was applied to a phenotype that when a worm is transferred from a seeded plate to a bacteria-free plate, the worm shows frequent backing and short duration of forward movement for - min and then a gradual increase in the duration of forward movement. accuracy of the state identification for wild type and several mutants was sufficiently high, indicating the system is robust in studies of locomotion. ps p-j flavoprotein fluorescence responses elicited by thalamic stimulation in slices obtained from the mouse barrel cortex daiki kamatani, ryuichi hishida, masaharu kudoh, katsuei shibuki dept. neurophysiol., brain res. inst., niigata univ., niigata - , japan we have reported that whisker trimming induced activity-dependent changes in the barrel cortex of rat cortical slices using flavoprotein fluorescence imaging. however, contribution of thalamo-cortical afferents in this plasticity was not clear, since specific stimulation of thalamo-cortical afferents was not possible in the coronal cortical slices obtained from rats. in the present study, we used the mouse cortical slices that kept thalamocortical connections to the barrel cortex intact. the cortical activities in layer iv were observed as fluorescence responses after thalamic stimulation. the magnitude of the fluorescence responses was increased as the amplitude of cortical field potentials was increased. these cortical responses were suppressed by antagonists of glutamate receptors such as cnqx and apv, and almost completely abolished in the presence of cnqx plus apv. in preliminary experiments, we confirmed that whisker trimming induced activity dependent changes in the barrel cortex of mice. ps p-j effects of implicit emotional processes on encoding-related activations of episodic memory: an eventrelated fmri study yayoi shigemune , , takashi tsukiura , hiroko mochizuki-kawai , chisato suzuki , , toshio iijima neurosci. res. inst., aist, tsukuba, japan; div. systems neurosci., tohoku univ. grad. sch. life sci., sendai, japan in this study, we investigated the effects of implicit emotional processes on encoding-related activations of episodic memory using fmri. nineteen healthy right-handed male participated in this study. we prepared emotional pictures with three kinds of emotional valence (negative: nega, neutral: neu and positive: posi) and line drawings for encoding. in the fmri scanning, subjects memorized line drawings, which were presented after the emotional pictures. after the scanning, subjects were presented with the names of line drawings, and were required to judge whether or not line drawings with the names were learned. we found significant activations of the right anterior cingulate gyrus specifically in the nega condition, the right lingual gyrus in the neu condition and the right amygdala and pulvinar in the posi condition. these results suggest that encodingrelated activations of episodic memory may be modulated by the implicit primer with emotional valence. ps p-j different neural correlates of stimulus-actiondependent and stimulus-dependent reward predictions revealed by fmri masahiko haruno , kenji kansaku , yu aramaki , mitsuo kawato atr cns, kyoto, japan; institute of physiology, okozoki, japan efficient decision making requires multiple reward predictions in switching different contexts and learning. we conducted a human fmri experiment (n = ) to examine stimulus-action-dependent and stimulus-dependent reward predictions. in condition a, each of two fractal figures specifies a monetary reward associated with a button push (left or right). if the button is pressed correctly, or yen is provided with a probability of . , but only with . if pressed wrongly. the key difference in condition b is that a fractal determines the reward but not the action. subjects had learned the two conditions fully before scanning. at the fractal onset, the putamen, lateral ventral and medial dorsal prefrontal cortices showed stronger activity correlated with the predicted reward (p < . ) in a, while it was more prominent in the caudate, dorsolateral prefrontal cortex and cerebellum in b. the striatum also showed a similar difference correlated with the reward prediction error at reward feedback, suggesting the different neural substrates for different reward predictions. research funds: nict ps p-j brain networks for communicative speech production: feeling inference and speech content production yuko sassa , , motoaki sugiura , hyeonjeong jeong , , keisuke wakusawa , , kaoru horie , shigeru sato , ryuta kawashima niche, tohoku university, sendai, japan; ristex, jst, tokyo, japan; miyagi university of education, sendai, japan; gsics, tohoku university, sendai, japan; department of pediatrics, tohoku university, sendai, japan; the lbc research center, tohoku university, japan communicative speech production often accompany inference of the targetperson's feeling. in this fmri study, we segregated the brain networks forthe feeling inference and speech content production processes incommunicative speech production. during presentation of a picture showingan actor's utterance in a balloon, normal subjects covertly talked to theactor (speech), inferred feeling (feeling), or described the action (des). greater activation in the contrasts speech-feeling was observed in themedial prefrontal cortex, and that in the contrast feeling-des wasobserved in the right superior temporal sulcus extending to the temporalpole. the results suggest that these two regions play roles in the speechcontent production and feeling inference, respectively. research funds: the st coe program ps p-j the construction of a brain-computer interface using the brain activity measured by near infrared spectroscopy takafumi miyoshi , yasuhisa fujibayashi , yoshiharu yonekura , tatsuya asai department of human and intelligence systems, university of fukui, fukui, japan; biomedical imaging research center, university of fukui, fukui, japan people with severe motor disabilities can increase the quality of life if they can communicate with the external world. a brain-computer interface using brain activity is one of the ways to provide such communication without depending on muscular controls. brain activity was measured non-invasively by multi-channel near infrared spectroscopy (nirs) during various motor tasks from healthy subjects. these spatial brain activities were fed to neural networks, and pattern learning was carried out by matching the tasks and the brain activities. we propose that nirs signals may be used to construct a brain-computer interface. ps p-j imaging of brain activity by near infrared spectroscopy in response to various sounds tatsuya asai , kuniyoshi shinya , tetsuo araki , masahiro kusakabe , yasuhisa fujibayashi , yoshiharu yonekura department of nuclear power and energy safety engineering, university of fukui, fukui, japan; department of human and intelligence systems, university of fukui, fukui, japan; biomedical imaging research center, university of fukui, fukui, japan brain activity can be monitored non-invasively by near infrared spectroscopy (nirs). in the present study, we measured changes in cerebral hemoglobin concentrations during a listening task using multi-channel nirs from healthy right-handed subjects, and hemispheric dominance for various sounds including verbal sounds was assessed. we have found asymmetrical brain activity when subjects listened to sounds with their left or right ear. these results suggest that hemispheric sound dominance may exist in addition to language dominance in healthy humans. kazuo kitamura , , winfried denk , michael hausser department of cellular neuroscience, graduate school of medicine, osaka university, osaka, japan; university college london, london, uk; max-plank institute, heidelberg, germany we describe a new approach for making targeted patch-clamp recordings from single neurons in vivo visualized using two-photon microscopy. the method involves using a patch electrode to perfuse the extracellular space surrounding the neuron of interest with a fluorescent dye, thus allowing the neuron to be visualized as a negative image and identified on the basis of its somatodendritic structure. the same electrode can then be placed on the neuron under visual control to allow gigaseal formation. we demonstrate the reliability and versatility of the method using recordings from principal neurons and interneurons in mouse and rat barrel cortex and cerebellum. we also show that the method can be used for in vivo juxtacellular labelling in identified cell types. this approach thus offers the prospect of targeted recording and labelling of single neurons in the intact native mammalian brain without the need to pre-label neuronal populations. research funds: wellcome trust, gatsby foundation, jsps and uehara foundation ps p-k analysis on viability of gabaergic neurons in cerebral cortical slices of adult mice yasuyo tanaka , yasuhiro tanaka , takeshi kaneko , dept. of morphological brain science, kyoto univ., kyoto, japan; crest, jst, kawaguchi, japan whole cell clamp recording and intracellular staining in adult brain slices are technically difficult because of their low viability. we analyzed the effect of slice cutting and incubation conditions on viability of cortical gabaergic neurons, using gad -gfp knock-in mice. we considered gfp positive cells as having survived. we observed more gfp-positive cells in the slices when nacl in cutting solution was replaced with n-methyl-d-glucamine (nmdg) chloride, choline chloride or sucrose. however, the viability was lower after h incubation in nmdg-based solution than in nacl-based solution. cutting at • c did not reduce the number of gfp-positive cells, but decreased gfp fluorescence in single neurons as compared with cutting at • c. the viability after h incubation was better kept at • c than at or • c. we thus recommend that slices be cut at • c in na-free solution, and incubated at • c in nacl-based solution. we thank dr yanagawa for his generous gift of knock-in mice. research funds: kakenhi ( , , ) ps p-k contribution of reduced and oxidized glutathione to signals detected by magnetic resonance spectroscopy as indicators of local brain redox state takumi satoh , yoshichika yoshioka faculty of engineering, iwate university, morioka, japan; iwate medical university, takizawa, japan we evaluated gsh signals by the mega-press (a frequencyselective refocusing technique) signals assessed by magnetic resonance spectroscopy (mrs). gsh gave a single positive signal ( . ppm) by mega-press. in contrast, gssg gave a multiplet of reversed signals ( . , . , and . ppm). a phantom solution mimicking the normal condition (gsh:gssg = : ) gave a single positive peak. gsh was prominent and gssg signals were minimal. thus, the signals originated from gsh, not from gssg. in the phantom solution (creatine: gsh: aspartate: gaba = : : : ), the creatine signal overshadowed the other signals. through mega-press, a single peak of gsh stood out over other signals. in vivo, the brains of healthy volunteers gave similar signals as the in vitro phantom solution, indicating that the signal originated from gsh. the estimated concentration of gsh in the human brain was . mm. in conclusion, mega-press allowed us to assess gsh levels in vivo non-invasionally. hiroshi kadota, hirofumi sekiguchi, yasoichi nakajima, yutaka kohno, makoto miyazaki department of sensory and communicative disorders, research institute, national rehabilitation center for persons with disabilities, tokorozawa, japan we investigated the brain regions related to the inhibition of habitual responses by using functional mri. we used the rock-paper-scissors game as an example of a familiar habitual behavior. it is considered that making positive attempts to lose when presented with the gesture of a rock, paper, or scissors is associated with the inhibition of habitual responses. in this study, the subjects were randomly assigned to one of the following two groups: the "win group" and the "lose group." a comparison between these groups showed that the lose group displayed activation of multiple cortical areas in the brain. with regard to the prefrontal cortex, the comparison revealed a higher activation in the left middle frontal gyrus (brodmann area ) and the right superior and middle frontal gyri (brodmann area ) in the lose group. these findings suggest that these regions play a role in the inhibition of habitual responses. ps p-k cortical commissural connection in macaque and human callosum using diffusion mri rishu piao , takuya hayashi , hiroshi sato , shinichi abe , , takashi hanakawa , hidenao fukuyama , hidehiro iida national cardiovascular center, osaka, japan; human brain research center, kyoto university, kyoto, japan we investigated the cortical commissural connection in human and macaque using the non-invasive diffusion-weighted magnetic resonance imaging (dwi). we used the probabilistic algorithm to track connection paths between a pair of the left and right homologous in subcortical areas. in macaque, the classification of callosum based on the highest interhemispheric connections paralleled with the results of tracer studies. however, the territory corresponding to the interfrontal connectivity extended more posteriorly than suggested in the tracer studies. the human interhemispheric connectivity showed similar topography in callosum as in the current macaque study, except that the connectivity territory of the frontal areas extended more posteriorly than in macaque. this study revealed that the commissural connectivity of the two species has a common intra-callosal topography. ps p-k optimal resolution of eeg/meg source imaging by spatial filtering wan xiaohong , niche, department of qutantum science and energy engineering, tohoku university, sendai, japan, niche, tohoku university, sendai, japan nowadays, electro-and magnetoencephalography (eeg/meg) is the sole invasive technique which is able to directly measure the human brain neural cortical dynamics. although we are well aware that it is impossible to accurately estimate the -d neural cortical activity using the -d eeg/meg surface potential topography, the upper limit of these techniques is not well described. during the past decades, various inverse approaches based on different criteria have been proposed, from the single dipole or multiple dipoles to the distributed current dipoles. however, it is difficult to systematically evaluate their efficiencies due to the different criteria and regularizations adopted in these methods. in this paper, we ask the question whether there exists an optimal approach based on a systematical criterion. this motivation firstly seems to be conflicted with the primary knowledge that there is no unique solution for the bioelectromagnetic inverse problem. essentially, here we are trying to find an optimal inverse solution that is closest to the real current distribution. ps a-c sensitivity of serotonin synthesis to synthesis inhibitor gtp cyclohydrolase i in senescence-accelerated mouse-prone inbred strain (samp ) nobuyuki karasawa , kazuko watanabe , keiki yamada seijoh universitry, tokai, japan, dept. physio., sch. med., gifu univ., gifu, japan, dept. anat., sch. health sci., fujita health univ., toyoake, japan to study the relationship between aging and levels of monoaminergic neurons, , -diamino- -hydroxypyrimidine (dahp), an inhibitor of monoamine synthesis, was intraperitoneally administered to senescence-accelerated mouse-prone (samp ) mice. time course of immunoreactive intensity for serotonergic ( -ht) neurons in the dorsal raphe nucleus, which were stained using laboratory-raised serotonin-specific antibody, was quantitatively evaluated using an image analysis system. results showed that -ht neruons are not highly sensitive to a synthesis inhibitor common to both catecholaminergic and -ht neurons. katsuya yamada , , , yoshihiro matsumura , takashi miki , makoto wakui ␣-smooth muscle actin + arterioles were fewer in kir . (−/−) barrel cortex than in wild-type one. in addition, whisker stimulation-induced increase in local cerebral blood flow was much smaller in kir . (−/−) barrel cortex than in wild-type one for short ( s, hz) but not long ( s) stimulation, suggesting crtical involvement of thin arterioles in a short-time neuro ps a-h learning to use sensory-tools by japanese monkeys yumiko yamazaki , hiromi namba support by public health research foundation (japan). acknowledgement supported by hkrgc. we wish to thank professor miyashita for valuable advice. ps p-h mechanisms for processing of intellectual excitement kazuhiko yanai, hongmei dai dept. pharmacol tohoku grad. univ. sch. med., sendai, japanthe aim of this study was to investigate the role of histamine h receptor (h r) in cognition in physiological and pathological conditions by using h r mutant (h −/−) mice. in normal condition, several behavioral studies indicated h −/− mice show impaired object recognition and spatial memory, improved conditioned fear memory. moreover, hippocampal long-term potentiation was reduced in h −/− mice. these results indicate h receptor is involved in memory process for which the frontal cortex, amygdala and hippocampus interact. in pathological condition, both h −/− and control mice were subjected to social isolation, an animal model of schizophrenia. social isolation impaired locomotion, prepulse inhibition of startle response and water maze performance in control mice, but not in h −/− mice. mutation of h receptor decreases isolation-induced hyperactivity of cortical dopaminergic neurons. these findings indicate blockage of h r attenuates social isolation-induced behavioral changes. in conclusion, blockage of h r impairs cognition in normal conditions, whereas h r blocking inversely improves cognition in disease models of schizophrenia.research funds: kakenhi ( ; ) ps a-h -ht a receptor gene polymorphism modulates activation in the human ventrolateral frontal lobe during go/no-go task michio nomura , , hirohito-m. kondo , makio kashino , department of psychology, tokai women's university, kakamigahara, japan; ntt communication science laboratories, ntt corporation, atsugi, japan; shimojo implicit brain function project, erato, jst, kawaguchi, japan impulsive behavior has been suggested to be due to a dysfunction of -ht neurotransmission. we examined whether this -ht a receptor gene polymorphism is involved in impulsive aggression by evaluating a reward-punishment go/no-go task using fmri. participants were required to learn to respond to active stimuli and inhibit their response to passive stimuli both under the reward-only (r) condition and the punishment-only (p) condition. the r condition, compared with the p condition caused right prefrontal activation mainly seen in ventrolateral regions. it has been reported that the possible involvement of the -ht a receptor gene polymorphism in impulsive behavior (nomura et al., ) , together with the present findings, this observation indicates the involvement of -ht a receptor gene polymorphisms in ventrolateral frontal lobe.research funds: shimojo implicit brain function project, erato, jst ps a-h role of cortical thin arterioles in neurovascular coupling; analyses of kir . -containing atpsensitive potassium channel-deficient mice ps p-g rem sleep deprivation increases serum ceruloplasmin level manoj jaiswal , chinmay k. mukhopadhyay , birendra n. mallick school of life sciences, jawaharlal nehru university, new delhi, india; special centre of molecular medicine, jawaharlal nehru university, new delhi, india rapid eye movement sleep (rems) is present across higher species and is essential for life. its loss predisposes one to several pathophysiological conditions. continuous loss of rems leads to several diseases and extreme loss may be fatal. rems loss is reported to increase metabolism and food intake though associated with hypothermia. hence, we proposed that the rems deprivation would affect acute phase response protein. in this study rats were rems deprived by platform method. free moving normal, large platform and recovery from rems loss were used as controls. blood was collected from the same rat before and after experimental as well as control periods. level of serum cruloplasmin, a positive acute phase response protein, was detected using western-blot analysis. the results showed that rems deprivation increased the serum ceruloplasmin level suggesting that the rems deprivation triggers an acute phase response at least in rats.research funds: council of scientific and industrial research, india and dst, india the indirect cytopathic effect in hiv- and the direct infection of hsv- are critical in their pathogenesis. we established murine neurosphere and evaluated with cocultivation of hiv- jrfl-infected macrophages or with hsv- . the generation of primary neurospheres did not suppressed by hiv- -infection or by hsv- infection at no more that moi . in the secondary neurospheres, cd + neural stem cells were intact in these infections, although beta- -tubulin + cells were decreased in hiv- infection and intact in hsv- -infection. in the differentiation assay, neun + nfp + neurons in hiv- -infection and gfap + s + astrocytes in hsv- infection were significantly decreased. the migration capacity of the neurosphere cells was suppressed in hiv- infection and in hsv- infection. we conclude that neural stem cells in vitro are resistant to cytopathic effect by hiv- and hsv- infection and their differentiation capacities are different in these infections. our assay will be one of the significant methods in neurovirological research.research funds: kakenhi grant-in-aid for young scientists(b) ps p-g effects of attraction to favorite opposite gender on nervous, endocrine, and immune systems masahiro matsunaga , , taeko yamauchi , toshihiro konagaya , hideki ohira department of psychology, nagoya university, japan; department of internal medicine, aichi medical university school of medicine, japan everybody can "fall in love". thus everybody knows that attraction to favorite opposite gender invokes positive feelings and often makes us energetic. to investigate effects of this positive emotion on the biological systems, we recorded various parameters, namely mood states, heart rate, skin conductance level (scl), serum levels of several hormones, and proportions of t cells and natural killer (nk) cells in the lymphocytes simultaneously when subjects viewed the video films of their favorite opposite genders. when the subjects were evoked their attraction to favorite opposite gender, they became more vigorous and felt better. as for the biological systems, scl and the proportion of nk cells in the lymphocytes significantly increased. these results suggest the possibility that attraction to favorite opposite gender may have a role in activating nk cell-related innate immune system by means of the activation of scl-related sympathetic nervous system. hiroko ikeshima-kataoka , shen jin-song , saburo saito , shigeki yuasa dept. mol. immunol., inst. dna med., jikei. univ. sch. med., tokyo., japan; dept. gene ther., inst. dna med., jikei univ. sch. med., tokyo, japan; dept. ultrastruc. res., natl. inst. neurosci., ncnp, tokyo, japanto investigate the role of tenascin (tn)-expressing astrocytes played in the injured brain, we analyzed tn knockout (tn/ko) mouse. we have previously reported that tn is one of the essential molecules for proliferation of the primary culture of astrocytes. from injured mouse brain model with stab wound, gfap expression was down regulated sharply at earlier stages in tn/ko mouse than in the wt mouse. some of the inflammatory cytokines are known to be expressed in injured cns, and also those receptors are expressed in the primary culture of astrocytes. to evaluate immune responses in the cns, some of the inflammatory cytokine production was determined in the lesioned mouse brain compared with tn/ko and wt mouse. from rt-pcr method, tn seemed to have the possible roles for some of the cytokine prodution at the cns lesion sites. we are currently investigating the function of tenascin for the cytokine production around the lesion site. aiko hori , tomoko yamamoto , kiyoshi matsumura , hiroshi hosokawa , shigeo kobayashi dept. of intelligence science and technology, grad. sch. of informatics, kyoto university, kyoto, japan; dept. of information science and technology, osaka institute of technology, osaka, japan intracerebroventricular (i.c.v.) injection of arachidonic acid (aa) evokes fever. this response has been thought to occur simply because aa is converted to prostaglandin e (pge ), the final mediator of fever. however, our recent study suggested that aa might not only be the precursor of pge but also induce an enzyme cyclooxygenase- (cox- ) that catalyses aa to form prostaglandins. we here examined in rats whether i.c.v. injection of aa induces cox- , and whether cox- is involved in aa-induced fever. two hours after i.c.v. injection of aa, cox- was expressed in the perinuclear region of brain endothelial cells. aa-induced fever was partly suppressed with a cox- specific inhibitor, ns- . these results indicate that aa itself or its metabolites induces cox- that accelerates the formation of pge from aa, and, hence, enhances fever. mitsunari abe , tatsuya mima , shinichi urayama , toshihiko aso , nobukatsu sawamoto , hidenao fukuyama human brain research center, kyoto university graduate school of medicine, japan; nano-medicine merger education unit, kyoto university, japanrepetitive transcranial magnetic stimulation (rtms) can induce lasting changes in the cortical excitability. however, its cellular mechanism remains unknown. diffusion weighted imaging (dwi) is a useful tool for measuring microscopic states of the brain tissue by probing water diffusion.we examined changes of dwi following rtms to further understand its effects. four healthy volunteers received rtms at . hz ( min; % of the rest motor threshold) applied over the left primary motor area (m ). we scanned sets of dwi (before, and min, min and min after rtms) using -t mr scanner, and calculated apparent diffusion coefficient (adc). in out of subjects, the adc decreased (mean . × − /mm s − ) in the left m just after the rtms, which recovered at min. it is possible that the rtms-induced change of adc might occur as the cellular response. further examination is needed for confirming this point. vahe poghosyan, andreas a. ioannides laboratory for human brain dynamics, brain science institute riken, wako-shi, japanretinotopic areas v and v a in macaques occupy almost the entire extend of the anterior bank of parieto-occipital sulcus (pos). v a located more dorsal has a larger receptive field size then v . both areas lack a foveal magnification. we used meg to record brain activity while human subjects were viewing stimuli presented at two different eccentricities in each quadrant of visual fields. to verify the reliability of results, for each subject, the experiment was repeated on three different days. tomographic analysis of meg signal, in each subject, identified highly reproducible activations throughout visual cortex in accord with the known organization. two new areas along the pos with a similar retinotopy to that of macaques v and v a were identified. in the ventral one, activations in response to each stimulus were spatially separated. the foveal magnification was much reduced compared to v in both areas and in the more dorsal area activations elicited by stimuli in the same quadrant could not be separated. given the above finding we suggest these areas as possible homologues of macaques v and v a.ps p-i measurement of magnetic evoked field of ratmeasurement of magnetic evoked field of rat using micro squid naohiro tsuyuguchi department of neurosurgery, osaka city university graduate school of medicine, japanthe study of neural activity in rodents would be enhanced by the stimulation of neuronal function in vivo. magnetoencephalography (meg) is used to study brain function in humans, but the limited resolution and sensitivity of conventional instruments have precluded the use of meg to study neuronal function in rodents. we demonstrate that micro meg developed for use with small animals, can be used to detect assess neuronal activity in conscious rodent brain. we used a micro -channel magnetometer consisting of a × matrix of superconducting quantum interference device (squid) with its integrated base of . × . mm to measure the visual evoked magnetic field (vef) and auditory evoked field (aef) of rats. we obtained the vef wave with - ms peak by the white led flashing stimulation and the aef wave with - ms peak by the tone and burst stimulation. this study demonstrate that micro meg can be used for serial assessment of neuronal function of individual, live animals with a minimal degree of invasiveness, has the potential for use in the study of brain function and plasticity. kentaroh takagaki, michael t. lippert, jian-young wu department of physiology and biophysics, georgetown university, washington, dc, usa voltage-sensitive dye (vsd) imaging is used to study visually evoked responses in rat visual cortex, in single trials without averaging. the signal is small, and a diode array with an effective dynamic range of bits was used, along with a "blue dye" (rh ) with small heartbeat artifact. a subtraction algorithm was used to further remove heartbeat artifact in the data. with the combination of the array, the blue dye and the algorithm, we were able to visualize sensory evoked wave activity with a high signal-to-noise ratio in single trials. the signals were . - . % of the resting fluorescence intensity. spatiotemporally, the evoked response manifested as propagating waves in the visual areas. there were large trial-to-trial variations in the propagating velocity and directions of the waves. the evoked wave apparently interacted with spontaneous waves in the cortex, and varied greatly according to anesthetic regimen. visualizing evoked waves may contribute to the understanding of cortical dynamics underlying sensory processing. masahito nemoto , yoko hoshi , chie sato , susumu terakawa tokyo institute of psychiatry, tokyo, japan; photon medical research centre, hamamatsu university school of medicine, hamamatsu, japanwe investigated interhemispheric interactions and neurovascular coupling by simultaneous recordings of neuronal and hemodynamic signals in rats. bilateral somatosensory cortices were activated with a stimulus time lag between test stimuli (electrical pulses to contralateral hindpaw) and conditioning stimuli (to a homologous somatosensory region of the contralateral hemisphere). we measured electrophysiological signals (local field potentials and multiunit activity) and optical intrinsic signals ( and nm, indicators of cbv and oxygenation), and analyzed the dependence of the signals on the time lag. the results showed that both neuronal and hemodynamic signals were suppressed around -ms time lag. average and trial-by-trial correlation analyses suggested that the hemodynamic signals reflected a balance of neuronal excitation and inhibition via callosal connections. we can infer some parts of underlying neural interactions by imaging of the hemodynamic signals. ps p-j multiple-site optical detection of spontaneous activity in the rat sensorimotor cortex akihiko hirota, shin-ichi ito department of physiology, shimane university school of medicine, izumo, japan multiple-site optical recording provides a powerful tool for the cerebral cortical neurophysiology, but its application has largely been restricted to reproducible, stimulus-evoked activation. we have developed the recording system with longer continuous recording capacity and larger signal-to-noise ratio to detect spontaneous activity in a single sweep. we applied this system to the sensorimotor cortex of rats anesthetized with a mixture of urethane and ␣-chloralose. the hindlimb region was exposed and stained with rh , a voltage sensitive dye. optical records, after compensation for pulsation artifacts, contained deflections time-locked to the high amplitude transient waves, characteristic to ␣-chloralose anesthesia, recorded with a wire electrode placed in the optically sampling area. as the transient in the electrocorticogram fluctuated, the optical signal also varied. this signal was distributed over a broad region, whose latency, amplitude or shape varied systematically within the region, probably reflecting the regional differences in the transient activity. yuko tanaka, r. allen waggoner, kenichi ueno, keiji tanaka, kang cheng bsi, riken, saitama, japanobjective: in this fmri study, we attempted to identify the brain regions involved in the process completing objects with degraded image information.method: fourteen healthy subjects were studied using a t mri scanner while performing a matching-to-sample task with three task conditions. the main condition required the subject to judge in a -s trial if a trial-unique, degraded animal image matched a contour image. in the comparison condition, we reversed the order presenting intact and degraded images (id epoch).results: comparing images acquired in di epochs with those acquired in id epochs, significant activation was found in the left parietooccipital cortex spanning the cuneus (ba ), superior occipital gyrus around the parieto-occipital sulcus (ba ) and superior parietal lobule (ba ). other activated foci include the left anterior cingulated cortex, left dorsal frontal gyrus and right middle frontal gyrus.conclusion: these results indicate that the parieto-occipital cortex is critically involved in the object completion with degraded images.ps p-j influence of task difficulty during meter inspection: an fmri study naoki miura , , makoto takahashi , jobu watanabe , , shinya uchida , , shigeru sato , kaoru horie , masaharu kitamura , toshio wakabayashi , katsuki nakamura , , ryuta kawashima crest, jst, kawaguchi, japan; niche, tohoku univ. sendai, japan; graduate school of engineering, tohoku univ. sendai, japan; bme institute, waseda univ., tokyo, japan; idac, tohoku univ., sendai, japan; lbc research center, tohoku univ., sendai, japan; department of animal models for human disease, ncnp, tokyo, japanthe purpose of the study was to analyze the cognitive process of a subject facing a human-machine interface (hmi) using fmri. we compared brain activation during meter inspection tasks with different task difficulty. during the meter inspection tasks, the subjects were instructed to inspect the three meters, and to press the button, if the subject found abnormal state. the task difficulty was devised by controlling the rate of change for the value to be displayed. in the right occipitotemporal area and the left cerebellar posterior lobule, activation during analog meter inspection was greater when the task difficulty was higher case. the results suggest that these regions are related to attention and perception of visual appearance of hmi.ps p-j neural connectivity among brain areas related to language function shinya uchida , , , naoki miura , , jobu watanabe , shigeo kinomura , kazunori sato , yasuyuki taki , kentaro inoue , ryoi goto , ai fukushima , kaoru horie , shigeru sato , katsuki nakamura , hiroshi fukuda , ryuta kawashima department of nuclear medicine and radiology, idac, tohoku university, sendai, japan; niche, tohoku university, sendai, japan; national institute of neuroscience, ncnp, kodaira, japan; japan science and technology agency, kawaguchi, japan; bme institute, asmew, waseda university, tokyo, japan; graduate school of international cultural studies, tohoku university, sendai, japanthe present study examined the neural connectivity of languagerelated regions using functional mri (fmri) and diffusion tensor imaging tractography (dtt). twenty subjects were participated. functional region of interest (roi) in the left inferior frontal gyrus (lifg) defined by fmri during speech production task was used as a seed point for dtt. in more than % of subjects, tracts between the roi and the left thalamus (lth) were estimated. post hoc fmri analysis showed activation in the lth during speech production tasks. therefore, cortical connectivity between the lifg and lth may have certain functional roles in speech production. keisuke wakusawa , , motoaki sugiura , yuko sassa , , hyeonjeong jeong , , kaoru horie , shigeru sato , hiroyuki yokoyama , kazuie inuma , ryuta kawashima niche, tohoku univ., sendai, japan; department of pediatrics, tohoku univ. graduate school of medicine, sendai, japan; miyagi univ. of education, sendai, japan; ristex, jst, tohoku univ., sendai, japan; gsics, tohoku univ., sendai, japan; lbc rc, tohoku univ., sendai, japanthis study examines the cortical mechanisms of comprehension of implicit social meanings such as irony and metaphor. healthy subjects judged whether the utterance in a picture such as irony, metaphor, or control expressions was situationally appropriate (s), or literally correct (l). greater activation during s than l task was analyzed to identify the activation for implicit meanings and neural responses to irony or metaphor were analyzed. the left medial prefrontal cortex showed higher activity during the s than l task. the medial orbitofrontal cortex and the right temporal pole showed responses selective to the irony; the responses in the former were observed during s task only, while the latter in both tasks. no selective response to metaphor was observed. keiichi onoda , yasumasa okamoto , kazuhiro shishida , akiko kinoshita , shigeru toki , kazutaka ueda , hidehisa yamashita , shigeto yamawaki department of psychiatry and neuroscience, hiroshima university, hiroshima, japan; training and research center for clinical psychology, hiroshima university, higashi-hiroshima, japan anticipation of emotional events may affect perceptual and cognitive processes when the events actually happen. we studied the effects of anticipation of positive and negative affective images on neural processes estimated with meg and event-related fmri. participants were presented emotionally positive or negative images with cue stimuli. the cue stimulus indicated the emotional valence of the image which followed a few seconds later. in meg study, visual evoked field (vef) was smaller for the anticipatable negative image than the anticipatable positive image. this result suggests that when the presentation of a negative image is anticipated before the event, neural processing for the image is depressed compared to when a positive image is anticipated. furthermore, we report the difference of brain activation between anticipation of positive images and that of negative images in event-related fmri study. makoto wada , , , kenji yoshimi , , noriyuki higo , yong-ri ren , hideki mochizuki , yoshikuni mizuno , shigeru kitazawa , , dept. of physiol, juntendo univ. schl. of medicine, tokyo, japan; dept. of neurol., juntendo univ. schl. of medicine, tokyo, japan; crest, jst, tokyo, japan; neurosci. res. inst., aist, tsukuba, japanwe developed a new method for comparing immunopositive cell densities across groups of animals and creating statistical parametric maps on standardized sections. as an example, we compared iba- positive glial cell densities in rats with and without unilateral injection of mpp+. immunopositive cell density map was automatically created in each animal over a coronal section in the midbrain (bregma − . mm). after the map was normalized to a template section, positive cell densities of the two groups were compared in each pixel and a statistical parameter was mapped on each pixel. we were able to detect significant increases of microglias in the side of the injection not only in the substantia nigra pars compacta but also in the white matters. the new method was proven to be useful for detecting significant changes of cell densities over the entire area of immunostained sections. key: cord- -ilhr iu authors: nan title: isev abstract book date: - - journal: nan doi: . / . . sha: doc_id: cord_uid: ilhr iu nan introduction: primary tumours secrete large amounts of extracellular vesicles (evs), which play critical roles in preparing distant sites for a pre-metastatic niche formation, thereby promoting metastasis and even determining metastatic organotropism. whether biogenesis, secretion rates and organotropism of evs are linked remains unknown. we have recently shown that ral gtpases control evs secretion in nematodes as well as in mouse mammary tumour cells (hyenne et al. jcb ) . since both rala and ralb are overexpressed or over-activated in various human cancers, we aimed to investigate the mechanisms by which these two gtpases control evs secretion and to determine how this affects metastatic progression, with a focus on breast cancer. methods: we used t mouse mammary carcinoma cells knocked down for either rala or ralb and determined their ability to induce orthotopic tumours and metastasis in a syngeneic mouse model. in vitro, we investigated ev secretion mechanisms using confocal and electron microscopy (em). evs were isolated either by uc or sec and characterized by nta, em, rna sequencing and mass spectrometry. the function of evs was assessed using a transwell assay. finally, we tracked the organotropism of fluorescently labelled evs and their capacity to induce pre-metastatic niches in mice. results: we show that rala and ralb promote lung metastasis of breast cancer cells in mice without affecting their invasive behaviours. we found that rala and ralb control the biogenesis of exosomes, by acting on the formation of multi-vesicular bodies though the phospholipase pld . as a consequence, knock down of rala or ralb reduces the levels of secreted evs and modifies their rna and protein contents. these differences alter the pro-tumoural function of evs, as demonstrated with an in vitro permeability test. importantly, we show in vivo that evs from rala or ralb depleted cells have a decreased lung organotropism and, as a consequence, are less efficient in priming lung metastasis. finally, we show that high expression of rala or ralb is associated with a bad prognosis in human breast cancer patients. summary/conclusion: altogether, our study identifies ral gtpases as central molecules linking the mechanisms of evs secretion, their dissemination and their capacity to promote metastasis. nuclear proteins are recruited into tumour-derived extracellular vesicles upon expression of tetraspanin tspan introduction: tetraspanin tspan is a transmembrane protein that exhibits a unique expression pattern, being overexpressed in many cancer types, but undetectable in most healthy tissues. although there is increasing evidence of an effect of tspan in invasion, metastasis, and regulation of extracellular vesicle cargo, the molecular mechanisms of tspan are yet not fully understood. methods: to study the function of tspan , we have established a fibrosarcoma model consisting of the parental cell line (ht ) and its derivatives expressing tspan (ht -tspan ) either fused with different fluorescent tags or tag-free. life imaging, sted and storm microscopy were used to determine the intracellular localization of tspan . co-immunoprecipitation from nuclei lysates was performed to detect direct and indirect interacting partners of tspan . small evs were purified from cell-conditioned media using sec and subjected to mass spectroscopy and ngs for a comprehensive comparative analysis of the proteome and transcriptome of the evs. results: the results of the proteome analysis showed a strong effect in the protein cargo of evs upon tspan expression. remarkably, among of the most regulated targets, several histones and ribosomal proteins were enriched in the evs derived from ht -tspan cells. in line with this finding, life imaging and super-resolution microscopy revealed that, while a majority of the intracellular tspan is located on the cell membrane or intracellular membranes, -as it is known for other tetraspanins-, a portion of tspan is located on the nuclear envelope. in fact, several histones co-immunoprecipitated with tspan , indicating their interaction. summary/conclusion: our data show that the expression of tspan in the tumour cells greatly impacts ev cargo. moreover, localization of tspan on the nuclear envelope, together with the enhanced recruitment of nuclear and ribosomal proteins to the evs, suggests a new mechanism of action of tspan . introduction: extracellular vesicles (evs) modulate tissue development, regeneration and disease through the transfer of proteins, nucleic acids and lipids between cells. currently, the mechanism of cytosolic delivery of ev cargo is largely unknown. here, we unravel how evs release their cargo in recipient cells. methods: evs were isolated from gfp-cd and cd -rfp expressing hek t cells by ultracentrifugation. gfp-cd and cd -rfp evs were added to hek t cells stably expressing anti-gfp fluobody and fluorescently tagged galectin- , respectively. clem and fluorescence microscopy were employed to visualize fluorescent markers in recipient cells. bafilomycin a and u a were used to inhibit endosomal acidification and cholesterol export from lysosomes, respectively. results: fluorescent galectin- which binds to betagalactosides present at the luminal side of endosomes was used to detect endosomal permeabilization. the absence of galectin- recruitment to endosomes in presence of cd -rfp evs showed that endosome permeabilization is not the mechanism behind ev cargo release. gfp-cd ev addition to cells expressing anti-gfp fluobodies resulted in the formation of fluobody punctae, reflecting cytosolic exposure of ev cargo. subsequent clem of the fluobody punctae revealed endosomes as the underlying cellular compartments from where cargo release takes place. neutralization of endosomal ph and accumulation of endosomal cholesterol blocked cargo release, showing that ev cargo release is dependent on endosomal ph and cholesterol level. summary/conclusion: we show that genetically encoded cytosolic probes and clem offer an excellent approach to study both the mechanism and efficiency of ev cargo release in cells. we provide experimental evidence that ev cargo release occurs from endosomes. funding: the research was supported by dutch technology foundation ttw and netherlands organization for scientific research nwo, de cock-hadders stichting, and erasmus mundus namaste scholarship. healthy humans. to determine cut-off values for diagnoses, reference intervals of evs in plasma are needed. to establish such reference intervals, ( ) a significant number of healthy donors should be included, ( ) the presence of non-ev particles, residual platelets, lipoproteins, and haemolysis should be quantified, ( ) flow cytometry signals should be in si units. the long-term aim of this study is to determine reference intervals of ev concentrations in human plasma within known dynamic ranges of the detectors. methods: ( ) to establish a clinical reference, we collected blood from healthy volunteers and prepared platelet-free plasma. ( ) we performed quality control measurements including residual platelet count, serum index, and lipid spectrum. ( ) we measured all samples by flow cytometry (apogee a -micro) and used custom software (matlab r b) to automate calibration of all signals and data processing. scatter signals were calibrated in comparable units of scattering crosssection (nm ) and diameter (nm). fluorescence signals were calibrated in units of molecules of equivalent soluble fluorophores (mesf). results: the quality controls showed that most residual platelet concentrations ranged from ^ to ^ per ml except for one outlier, while the serum index and lipid spectrum were normally distributed. preliminary results of the first donors analysed, show that within the ev size range of - , nm, the median concentration of cd + evs is . • ^ per ml (apc> mesf), cd p+ evs is . • ^ per ml (pe> mesf), cd a+ evs is . • ^ per ml (pe> mesf), and cd + evs is . • ^ per ml (apc> mesf). summary/conclusion: we have developed reliable procedures for establishing reference intervals of ev concentrations, within a well-defined size and fluorescence intensity range, in human plasma by flow cytometry. we are currently applying these procedures to samples to obtain, for the first time, ev reference intervals for human plasma. funding: pol, e. van der is supported by the netherlands organisation for scientific research -domain applied and engineering sciences (nwo-ttw), research programmes veni . introduction: the use of extracellular vesicles for diagnostic and therapeutic applications has seen a major interest increase in recent years because of their capacity to exchange components such as nucleic acids, lipids and proteins between cells. isolation of a pure population of evs is the first step in studying their physiological functions since contamination of ev preparations with non-ev proteins can lead to incorrect conclusions about their biological activities. we have developed a new method termed tangential flow for analyte capture (tfac) using ultrathin nanomembranes to purify extracellular vesicles from pure, highly complex biological fluids such as blood plasma, resulting in a new method for extracellular vesicle purification. methods: the tff microfluidic devices are assembled through a layer stack process using patterned polydimethylsiloxane (pdms) sheets with the membranes sandwiched between top and bottom channels. undiluted plasma was tested in both normal flow filtration (nff) and tangential flow filtration (tff) modes on ultrathin nanomembranes. we have utilized a pore patterning technique called nanosphere lithography (nsl) that uses close-packing of nanoscale beads to pattern pores in an ultrathin membrane. results: nff of undiluted plasma resulted in a protein cake of~ μm on the membrane, which prevented further transport across the membrane and evs were buried in the formed cake that were impossible to identify. however, tfac as a modified version of tff, led to capturing cd positive evs on the pores of the membrane with little evidence of protein fouling. nsl allows us to fabricate nanopockets (bowls with a single pore at the base) with various diameter, depth and pore diameter. using nsl, we further utilize nanopocket membranes to purify ev samples in tfac devices. this nanomanufacturing technology will allow us to pattern nanopockets with various diameter, depth and pore diameter which increases the efficiency of capturing of evs. furthermore, nanopockets can be modified and coated by specific ev markers to capture different subpopulation of evs based on size and affinity and further allows identifying the phenotypic subsets of evs by combining both size and affinity-based techniques. summary/conclusion: we have developed a method for the capture and release of nanoparticles such as evs called tfac using ultrathin nanomembranes. nsl technology can be applied to fabricate nanopockets with different physical and biochemical properties. utilizing nanopocket membranes in tfac system will allow us to separate different subpopulations of evs based on size and affinity. funding: this project was supported in part by the national science foundation (iip ) to j.l.m and t.r.g., department of defence (ca ) to j. l.m., and the national institutes of health (r gm ) to t.r.g. the addition of a size exclusion chromatography step to various urinary extracellular vesicle concentrating methods reveals differences in the small rna profile introduction: urinary extracellular vesicles (evs) and their rna cargo are a novel source of biomarkers for various diseases, however non-vesicular rna (e.g. associated with proteins) is also present within urine. this study aimed to identify the optimal method for isolating and enriching evs from human urine prior to small rna analysis. methods: three ev concentration methods, ultracentrifugation (uc); a precipitation-based kit (pk); and ultrafiltration (uf), were compared using ml aliquots of pooled healthy volunteer urine. evs were then separated from protein contaminants by size-exclusion chromatography (sec). presence of evs was confirmed by transmission electron microscopy and western blotting, and evs were quantified using nanoparticle tracking analysis (nta). small rna content of concentrated urine and fractions obtained by sec (evs and proteins) were evaluated with the agilent bioanalyzer small rna chip. results: ev recovery following sec of concentrated samples was - %, however particle: protein ratio (indicating ev purity) was approximately x greater after sec, regardless of the concentrating method used. uf+sec yielded the highest number of evs (per ml of urine) compared with pk+sec and uc+sec. small rna analysis from uf-concentrated urine (prior to sec treatment) identified peaks at nucleotides (nt) and nt. following sec, rna analysis indicated that ev fractions contained mostly small rna of~ nt, whereas the protein factions contained small rna of nt in size (consistent with mirnas). summary/conclusion: uf+sec provided the best balance between ev recovery (per ml urine) and particle: protein ratio. these data indicate that most of the nt sized rnas, presumably mirnas, are not within evs in urine. ev preparations obtained after uc, pk and sec (regardless of concentrating method) contain pre-dominantly~ nt sized small rna. these data outline the importance of removing non-vesicular proteins and rna from urine ev preparations prior to small rna analysis. funding: this research has been funded by petplan charitable trust. the use of rev for the optimization of ev separation and characterization by af introduction: the reproducibility of extracellular vesicle (ev) research has been hampered by the infinite number of separation and measurement techniques and the lack of appropriate reference materials (van deun et al., nat methods, ) . recombinant extracellular vesicles (rev) were developed as a biological reference material to overcome these limitations (geeurickx et al. nat comm ) . since rev have ev-like physical an biochemical characteristics and as they are trackable and distinguishable from sample ev they can be used as a spike-in material for data normalization and method development, and as a quality control. we used rev to optimize ev separation by asymmetrical flow field-flow fractionation (af ). methods: an af long channel column with a frit inlet driven by the eclipse system (wyatt) was coupled to a uv detector (shimadzu), mals dawn helios-ii (wyatt) and fluorescent detector (agilent). a spacer of µm and a regenerated cellulose membrane of kda were used. pbs supplemented with . % nan was used as a running buffer. light scatter profiles and uv profiles were analysed as well as the fluorescent emission spectrum as the rev are gfp positive. fractions were collected and analysed by nanoparticle tracking analysis (nta) and western blot. we also estimated the repeatability and reproducibility of the af technique by light scatter and fluorescence profiles as well as the recovery efficiency by nta. results: in a first step * ^ rev isolated from conditioned medium by a velocity gradient were injected in the af system to optimize the ev characterization protocol. later concentrated conditioned medium was spiked with * ^ rev and injected in the af column to optimize ev separation from non-ev contaminants. the most optimal separation protocol was obtained by varying detector and cross-flow settings. this protocol shows elution of monodisperse particles at each time point and size distribution estimations by af correspond to size determination by nta and electron microscopy. summary/conclusion: we were able to optimize the af protocol for characterization of ev by af as well as for separation of ev from crude conditioned medium samples by using rev. we demonstrate that rev are suitable for method development and that af has high potential as an ev separation technique. comparative evaluation of ev isolation methods for ev subpopulation analysis in human urine, plasma and cell culture media liang dong, richard zieren, kengo horie, sarah amend and kenneth pienta the brady urological institute, johns hopkins university school of medicine, baltimore, usa introduction: extracellular vesicles (evs) are membrane-enclosed particles of variable sizes that are released by any cell types to the extracellular space and are identified in all body fluids. a shortcoming in ev research is the lack of standardized isolation protocol for various sample types, resulting in heterogeneous outcomes in downstream analyses. in this study, we compared the ev isolation purity and efficiency among ultracentrifugation (uc), precipitation, sizeexclusion chromatography (sec) and a microfluidic tangential flow filtration device (exodisc) in human plasma, urine and cell culture media (ccm). methods: all evs were isolated by different isolation methods and characterized per misev guidelines. single-particle interferometric reflectance imaging senor (sp-iris) with optional fluorescence and nanoflow (nfcm) were used for single particle analysis. results: in ccm, total particle yield of exodisc was about times higher than those of the rest three methods. size distribution differed per sample, but the ranges were comparable between the different isolation methods. the total protein amount of sec, precipitation and exodisc were similar which were - times higher than that of uc. uc had the highest particle-toprotein ratio followed by exodisc. precipitation and sec had low ratios. when loading ug of total protein for western blot, cd , cd , cd and flot could only be detected in uc and exodisc samples, but not precipitation or sec. sp-iris and nfcm demonstrated consistent purity findings. in urine, total particle yields of exodisc and sec were about times higher than those of the rest two methods. the total protein amount of precipitation was times higher than exodisc and sec, times higher than uc. sec had the highest particle-to-protein ratio followed by uc and exodisc. precipitation had low ratios. in plasma, total particle yields of exodisc and precipitation were times higher than those of the rest two methods. and so were the total protein amount. sec had the highest particle-to-protein ratio followed by uc. exodisc and precipitation had low ratios. western blot, sp-iris and nfcm demonstrated consistent purity findings in urine and plasma. to evaluate particle capture efficiency, we spiked a known number of density-gradient uc purified evs to each method and the recovery rate of uc, precipitation, exodisc and sec was . %, %, . % and %, respectively. summary/conclusion: the order of ev isolation purity in ccm is uc, exodisc, sec and precipitation. in urine it's sec, exodisc, uc and precipitation. and in plasma, this order is sec, uc, exodisc and precipitation. exodisc and sec have similar high isolation efficiency followed by precipitation. uc has low efficiency for ev capture. a capillary-channelled polymer (c-cp) fibre spin-down tip approach for the isolation and biomarker characterization of extracellular vesicles of ovarian cancer origin kaylan d. kelsey, rhonda r. powell, terri f. bruce and r. kenneth marcus clemson university, clemson, usa introduction: extracellular vesicle (evs) profiling has shown promise for disease detection through less invasive sampling (liquid biopsies). current diagnostic tools for ovarian cancer are invasive or only semiinformative. thus, use of evs could prove useful in early disease detection. demonstrated is a hydrophobic interaction chromatography (hic)-based capillarychannelled polymer (c-cp) fibre tip spin-down process for the isolation of ovarian cancer evs for use in diagnostics. methods: polyester c-cp fibre micropipette tips are employed in the isolation of evs from biological matrices including cell culture media, urine, and blood plasma in a spin-down solid-phase extraction (spe) approach. evs were isolated from standards of healthy urine origin and from skov cells (human ovary adenocarcinoma). the c-cp fibre isolation method (taking less than mins and μl sample volumes) preserves the morphology and functionality of evs as confirmed by sem, tem, and confocal fluorescence microscopy. results: the dynamic binding capacity of ev standards on a cm pet c-cp fibre tip was found to be~ e particles ( %). the release of evs was confirmed using dot blot analysis for cd , cd , and cd tetraspanin proteins. immobilized evs were subjected to immunolabeling to allow the positive identification of a profile of ovarian cancer biomarker proteins (her , cd , egfr, epcam, ca ). summary/conclusion: this new ev isolation method introduces a simple capture mode, allowing for direct immuno-characterization and imaging on the fibre surface. this offers a unique and cost-effective opportunity for clinical analyses related to early detection and diagnosis of ovarian cancers (and others). the longterm goal is the creation of a rapid ev isolation and biomarker detection platform. funding: support from the national science foundation, eppley foundation for scientific research, gibson foundation, prisma health system and itor biorepository are gratefully acknowledged. development and optimization of purification method of exosomes by tangential flow filtration and ion-exchange chromatography approach tek lamichhane, ali navaei, sandeep choudhary, yonatan levinson and senthil ramaswamy cell & gene therapy r&d, lonza inc, rockville, usa introduction: extracellular vesicles (evs) such as exosomes have significant therapeutic potential, however, translation of ev-based therapies has been slowed down because of the biomanufacturing challenges. the isolation of evs, especially exosomes, is inherently challenging due to their small size, and heterogeneity in the mixture. the current isolation methods either have low recovery rate, aggregation, damaging the structure, time consuming or co-precipitation of contaminants. specially, it is difficult to process larger sized samples by centrifugation-based or immunoaffinity based methods because of the time and cost associated with these methods. methods: to overcome these roadblocks, we developed and optimized alternative purification techniques to isolate evs with higher purity and yield by using tangential flow filtration (tff) coupled with ion-exchange chromatography. we used bioreactor platform to produce evs from serum-free medium using bm-msc and hek s cells. bm-mscs were cultured on stirred tank bioreactors using microcarriers which provide a high surface area to volume ratio for the optimal cell growth and evs production. impellers were used to enhance mixing and maintain homogeneous culture conditions that can be easily monitored and controlled. results: depth filtration was applied for clarification of conditioned medium. we screened different types of filters during depth filtration for the best recovery of evs. tff membranes with different pore sizes were used to optimize the purity and yield of evs. because of the negatively charged nature of evs, anion exchange chromatography was chosen to capture and separate tff purified vesicles by their surface charge characteristics. we compared monolith based and membrane-based anion exchange columns to remove contaminants and purify exosomal fractions. the purity, size and presence of exosomal markers in isolated evs at each step of purification was evaluated by f-nta, nano-fcm and tetraspanins based elisa kits. summary/conclusion: in summary, our optimized methods improved the speed of isolation and purity of evs to the clinical grade. the production and isolation methods of exosomes that we developed here will be easily expandable to support large-scale and cgmp compatible bio-manufacturing in the future. use of an alternating current electrokinetic microelectrode chip to positively identify oncology, neurology, and infectious disease samples through plasma extracellular vesicle analysis juan pablo hinestrosa, jean lewis, david searson, orlando perrera, alfred kinana, heath balcer and rajaram krishnan biological dynamics, inc., san diego, usa introduction: cancer, neurological, and infectious diseases are leading causes of death, with early detection needed to improve outcomes. extracellular vesicles (evs) in the blood contain disease biomarkers, but current methods do not allow rapid analysis, and are often limited to one biomarker type. methods: we developed methods using alternating current electrokinetics (ace) to isolate evs from bloodbased samples and analyse the evs in situ with downstream assays for protein and nucleic acid biomarkers. we investigated if we could identify tuberculosis (tb) donor samples, protein and nucleic acid biomarkers in evs derived from cancer cell lines, and alzheimer's disease (ad) protein biomarker levels. results: ev isolation was confirmed by positive identification of the proteins cd , cd , and cd and measurement of ev mrnas using a direct rt-ddpcr assay. different disease models were analysed following method development. tb was used as a model for infectious disease, with tb positive and tb negative samples isolated on ace chips and analysed for levels of lipoarabinomannan and ag . using a cut-off above the negatives, the auc of roc curves were . and . , respectively. for oncology, cancer cell lines were cultured and evs isolated from supernatants were spiked into human plasma for analysis. levels of pd-l or glypican- on evs were able to be measured following ace capture. additionally, dna and rna mutations known to be present in the cell lines were able to be detected using ngs and qrt-pcr, respectively. using ad samples as a neurological disease model, tau and phospho-tau t (p-tau t ) in human donor plasma were detected. in ad and healthy donor samples, p-tau t signal increased % in diseased versus healthy donors. summary/conclusion: ace chips are an innovative ev isolation and analysis platform that allow rapid disease sample detection in a wide range of studies with high sensitivity and specificity. introduction: colorectal cancer (crc) is one of the most frequent causes of cancer-related death. in the majority of crc patients, mutation in the apc gene is among the first genetic events. it leads to uncontrolled activation of the wnt pathway, and thus, to adenoma formation. some of these adenomas may then further progress to crc with the accumulation of other mutations. the d organoids maintain the cellular and genetic heterogeneity of in vivo tissues and haves proved to be so far the best ex vivo model of human cancers. here we analysed the ev-based communication between cancer cells and fibroblasts by i) identifying factors that substantially increase ev release from intestinal cancer cells and ii) by determining cargo components of evs that enhance tumour cell proliferation. methods: we used commercially available and patientderived fibroblasts and crc organoids. the medical research council of hungary approved all experiments with human samples and informed consent was obtained from patients. evs were studied by using antibody-coated beads, trps, nta, tem and western-blotting. we introduced apc mutation into wild type murine small intestinal organoids by crispr-cas . results: we found that in crc patient-derived organoid cultures, small evs were preferentially secreted. we observed that apc mutation and the accumulation of the extracellular matrix component collagen critically enhanced ev secretion in intestinal organoids. furthermore, we showed that amphiregulin, present on fibroblast-derived ev, contributed to the maintenance of the intestinal stem cell pool and to cell proliferation in epidermal growth factor-dependent crc organoids. summary/conclusion: by proving the key role of mutations, collagen deposition and ev-bound amphiregulin in the release intensity and functions of the evs, we identified novel mechanisms in the progression if crc. funding: this work was funded by otka-nn , by the national competitiveness and excellence program nvkp_ - (national research, development and innovation office, hungary) and by the national excellence program in higher education (ministry of human resources, hungary). prostate cancer-derived evs induce a pro-inflammatory phenotype in the stroma blandine f. victor a , dolores di vizio b , andrew chin c , tatyana vagner b , javier mariscal b , mandana zandian a , catherine grasso a , roberta gottlieb a and helen goodridge a a cedars-sinai medical center, los angeles, usa; b cedars-sinai medical center, west hollywood, usa; c cedars-sinai, los angeles, usa introduction: since % of patients with metastatic prostate cancer (pc) develop bone metastasis, identifying the mechanism that drives this process is essential. most ev research has been focused on the role of exosomes in mediating the pre-metastatic niche formation. however, most of these studies do not separate exosomes from large evs. our preliminary studies have demonstrated that a subclass of evs known as large oncosomes (lo) can reprogram prostate fibroblasts, at the primary tumour site, promoting angiogenesis and enhancing the migration and invasion of pc cells in vitro and tumour growth in vivo. the bone marrow is the initial site of entry into the bone microenvironment for disseminating tumour cells (dtcs) and is a rich source of nutrients that houses various cells types including bone marrow derived mesenchymal stem cells (bm-msc) and immune cells such as neutrophils, which have been implicated in metastasis. here we investigate the role of lo in reprogramming bm-mscs and driving bone metastasis in pc. methods: differential centrifugation, density gradient centrifugation, trps, rna sequencing, qpcr, migration assay, invasion assay, chemotaxis assay. results: we report that pc-derived evs induce distinct gene expressions changes in bm-mscs. rna-seq analysis identified inflammatory and immune regulating cytokines as top differentially expressed genes (deg) in bm-msc. moreover, lo induced a more potent response in bm-msc in comparison to exo and to non-treated controls. the genes enriched in lo treated bm-msc were associated with tumour cell motility. in agreement with the gene expression data, lo-treated bm-msc attracted migration and invasion of significantly more pc cells than exo -treated bm-mscs. in addition, the top deg expressed in ev treated bm-msc were identified as potent neutrophil chemoattractant proteins. in line with the rnaseq findings, the lotreated bm-msc demonstrated enhanced chemotaxis of neutrophils towards them in comparison with exo or vehicle-treated bm-msc. finally, we show that the observed differences in bm-msc's response to lo and exo may be mediated by distinct molecular pathways. summary/conclusion: the results from this study provide novel insight into how tumour derived evs alter the bone marrow microenvironment and how they may drive bone metastasis in prostate cancer. the αvβ integrin in cancer cell-derived small extracellular vesicles enhances angiogenesis introduction: prostate cancer (prca) cells crosstalk with the tumour microenvironment by releasing small extracellular vesicles (sevs). sevs isolated from prca cell media, express the epithelial-specific αvβ integrin, a surface receptor for fibronectin and vitronectin. the αvβ integrin is not detectable in healthy prostate tissues but is highly expressed in prca. in this study, we hypothesized that αvβ in cancer sevs plays a crucial role in angiogenesis. methods: the sevs isolated from prca cell media were characterized by nanoparticle tracking analysis, iodixanol density gradients and expression of sev markers. the αvβ -negative endothelial cells (hmec ) were incubated with αvβ -positive sevs from prca cells to evaluate the transfer of αvβ by immunoblotting (ib) and facs. the effect of αvβ -positive sevs on motility, tube formation and angiogenic signalling were assessed by boyden chamber, angiogenesis assays and ib in hmec . results: we demonstrate for the first time that the αvβ is de novo expressed on endothelial cell surface by sevmediated protein transfer. prca cell-derived αvβ -positive sevs, significantly promote the motility and the formation of nodes, junctions and tubules by hmec . mechanistically, we demonstrate that hmec treatment with sevs from pc cells that endogenously express αvβ , decreases pstat (y ), a negative regulator of angiogenesis, while upregulating survivin, an inducer of angiogenesis. hmec treatment with sevs isolated from pc cells harbouring crispr/cas -mediated downregulation of β , or shrna-mediated downregulation of β , results in increased levels of pstat (y ). this sev treatment also results in a decrease of survivin in sevs and hmec . summary/conclusion: overall, our findings show that αvβ in prostate cancer sevs regulates a novel proangiogenic signalling pathway. funding: this study was supported by nci r - (lrl); p - (lrl and dca). introduction: advanced prostate cancer (pca) is asso-introduction: extracellular vesicles (evs) are secreted from cells, and carry bioactive proteins and rna cargoes. increasing numbers of studies have identified key roles for exosomes in driving aggressive tumour behaviours, including metastasis. however, the detailed mechanisms and responsible factors in the ev cargo are still unclear. recently, immune system has been considered as an important factor in establishing and maintaining metastasis. our goal is to identify the role of head and neck squamous cell carcinoma (hnscc) derived small evs (sevs) in tumour metastasis from the study analysing the effects of sevs on metastasis and tumour immunity. methods: sevs were collected from the conditioned media of hnsccs and purified through cushioned density gradient ultracentrifugation. an orthotopic mouse model was used for the assessment of tumour angiogenesis and metastasis. moc (inflammationinducing rarely metastasizing murine hnscc line) and moc (highly metastasizing murine hnscc line) were used for this study. moc and moc cells were transplanted into mice tongues orthotopically, and moc /moc derived sevs or pbs were injected into the tumour twice in a week. two weeks after tumour transplantation, mice were sacrificed and tumours were sectioned for pathological analysis and facs analysis. in facs analysis, the number and species of tumour-infiltrated immune cells were measured. results: injection of sevs from moc into moc tumours suppressed frequency of lymph node metastasis. on the other hand, injection of sevs from moc into moc tumours didn't promote metastasis. cd positive t-cell distribution in moc tumour was significantly changed by moc sev injection. t-cell deprivation treatment using anti-cd antibody increased the frequency of metastasis in moc -sev treated moc tumours. from the result of proteomics analysis on moc and moc sevs, immune-regulated proteins and metastasis-suppressing proteins were observed in moc sevs. summary/conclusion: we find that low aggressive hnscc sevs affect metastasis of highly metastasized hnscc, and also find that changing immune cell distribution may be related to the result. this mechanism and finding contributes to understanding the possible role of hnscc sevs on metastasis as well as on the tumour immune microenvironment. funding: this work was supported by the nih under award numbers r ca and r ca to aw. desmoglein enhances squamous cell carcinoma tumour development through extracellular vesicles in an il- /mir- a-dependent mechanism introduction: the cadherin dsg is a stem cell marker that is upregulated in many different cancers, including sccs, and its expression correlates with poor prognosis. dsg activates mitogenic signalling and plays a key role in cell proliferation, migration, and survival. we recently showed that dsg enhances ev release, but the mechanism by which these evs modulate tumorigenesis is not fully understood. methods: we established scc cell lines stably expressing wildtype dsg or a palmitoylation deficient mutant, dsg cacs. evs were isolated by sequential ultracentrifugation, iodixanol gradient separation, or qev izon column, and analysed by nta and bca. tumour xenografts were established by subcutaneous injection of cells in scid mice and monitored up to weeks. cytokine profiling was determined by antibody array. mirna expression was analysed by rnaseq and confirmed by qpcr. results: dsg enhanced ev release by % and promoted a~fivefold increase in tumour size in xenograft models. tumour growth was increased when control cells were treated with a single µg dose of evs. loss of palmitoylation, which altered membrane trafficking of dsg , reduced ev release (~ %) as well as tumour development. plasma evs from xenograft mice reflected in vitro particle counts from scc cell lines. a cytokine array analysis was performed revealing that dsg -evs were enriched with pro-inflammatory cytokines including il- , a potent chemotactic and angiogenic factor. most importantly, il- was surface-bound on evs. furthermore, rnaseq revealed mir- a, a negative regulator of il- , to be significantly downregulated in response to dsg . treatment with mir- a mimic or mir- a inhibitor decreased or increased, il- expression in scc cells, respectively. summary/conclusion: in summary, dsg plays a key role in scc tumour development by increasing ev biogenesis and downregulating mir- a, which in turn upregulates il- synthesis and release which can promote invasion, angiogenesis and metastasis. funding: nih r introduction: stem-and progenitor cell transplantation therapy holds great promise for regenerating damaged heart tissue. several lines of evidence suggest that its efficacy is mainly caused by secreted extracellular vesicles (evs). indeed, cardiac progenitor cell (cpc)-derived evs have been shown to protect the myocardium against ischaemia/reperfusion injury in several preclinical models. however, the underlying mechanisms for cpc-ev-mediated cardioprotection remain elusive. here, we utilized the proteomic composition of cpc-evs released during different culture conditions, to unravel protein-mediated effects of cpc-evs on the endothelium. methods: cpcs were stimulated with calcium ionophore (ca ion-evs) or vehicle (control-evs) for hours and evs were isolated from serum-free conditioned medium using size exclusion chromatography. ev concentration and size was assessed using nta. evs were functionally characterized based on endothelial cell activation by western blotting and an endothelial cell scratch assay. the proteomic composition of both ev conditions was profiled using mass spectrometry. cpc-ev knockouts for specific proteins were generated using crispr/cas technology. results: we found enhanced phosphorylation of erk / and akt in endothelial cells and increased wound closure after stimulation with control-evs, but not after stimulation with ca ion-evs. proteomic analysis identified a total of ev-associated proteins, with proteins uniquely expressed in control-evs. another proteins were revealed as candidate proteins, based on their relative enrichment in control-evs compared with ca ion-evs. go analysis demonstrated that differentially expressed proteins were involved in vascular endothelial growth factor signalling, extracellular matrix organization and angiogenesis. to investigate the involvement of the individual candidate proteins on endothelial cell activation, knockout evs of multiple proteins were generated and functionally characterized. summary/conclusion: a specific set of ev proteins is identified that may be functionally responsible for the activation of endothelial cells upon exposure to cpc-evs. generating knockout evs for each of these proteins will help to investigate their individual roles. this may lead to a better mechanistic understanding of the use of cpc-evs as therapeutics for cardiac repair. funding: erc- -cog- evicare grant. hypoxia enhances the therapeutic potential of human cd + stem cell exosomes in ischaemic hindlimb repair ischaemic cardiovascular disease. we have previously shown that human cd + cell-derived exosomes (cd exo) improve perfusion and function of the ischaemic tissues. hypoxia is shown to modulate the secretion and content of exosomes in both cardiovascular and cancer research. therefore, we hypothesized that hypoxia can modulate the content and regenerative efficacy of human cd exo. methods: cd exo were isolated from primary human cd + stem cells cultured under hypoxia ( . % o , or normoxia ( % o , n-cd exo) using density gradient ultracentrifugation. cd exo size was measured using trps, nta, and dls and surface protein expression was determined using imaging flow cytometry. function of cd exo was assessed using cell viability, migration and matrigel tube formation assays in vitro and a mouse hind limb ischaemia model (hli) in vivo. protein content of hypoxic or normoxic cd exo was evaluated via lc-ms/ms and -d -dige followed by lc-ms/ms. results: we did not observe any significant differences in size or in quantity of exosomes secreted from h-or n-cd cells. both h-and n-cd exo expressed cd , cd and cd surface markers. interestingly, h-cd exo significantly improved cell viability, migration and tube formation of huvecs in vitro compared to n-cd exo. in the same line, h-cd exo also significantly improved perfusion (ratio: . ± . v . ± . ) and prevented ischaemic limb amputation ( % v . %) as compared to n-exo (p < . ; n = - ) in a murine (balbc nude) model of hind limb ischaemia. flow cytometry and confocal microscopy indicated that h-exo was uptaken by endothelial cells in the ischaemic limb. remarkably, we detected several proteins (including a fragment of hemopexin) and mirnas (mir- ) that could be responsible for the proangiogenic and beneficial function of h-cd exo. we have also demonstrated that removal of surface proteins diminished the pro-angiogenic function of cd exo. summary/conclusion: hypoxia enhanced the proangiogenic and regenerative potential of cd exo, and thus, may represent a more efficient clinical strategy for cd exo therapy. our research is clinically important to improve therapeutic angiogenesis in diabetic and cardiovascular patients with compromised stem cell populations. hyun-ji park a , jessica r. hoffman b and michael davis b a emory university, decatur, usa; b emory university, atlanta, usa introduction: exosomes, a subset of membrane nanovesicles, transfer cellular information by passing proteins and nucleic acids between cells. exosomes have been implicated as the mechanistic unit in stem cell therapy, as inhibition of exosome synthesis abrogates the effects of cell therapy following cardiac injury. more importantly, increasing evidence indicates that mirnas (mirs) within exosomes serve as important signalling molecules to regulate inflammation, recruit stem cells, and repair diseased tissue. among exosomal mirs, mir- and − are known to decrease angiogenesis, cell migration, and increase inflammation in various types of cells. here, we investigated the inhibition of these negative mirs as a means to improve the reparative capacity of c-kit+ progenitor cell (cpcs) exosomes. methods: cpcs were isolated from three paediatric patients using magnetic-bead sorting. ʹ-o-methylated rna duplexes inhibited mir- and − expressions in cpcs. exosomes (inhexos) were isolated from mirinhibited cpc conditioned medium. mir expression in exosomes and cpc was quantified by qrt-pcr. migration and proliferation of mesenchymal stem cells (mscs) were assessed two days post-exosome treatment. for inflammation analysis, thp cells with/without tnfα exposure were treated with exosomes and the expression of il- , − , and − was quantified by qrt-pcr. finally, the angiogenic potential of inhexos was tested by tube formation of cardiac endothelial cells. results: inhibitor treatment of cpcs decreased exosomal mir- and − expression. treatment with inhexos enhanced msc migration and proliferation compared with normal cpc exosome (norexo). moreover, inhexos showed promising results for immune regulation, as tnfα-induced inflammation was decreased in thp exposed to inhexos for h. however, tube formation capacity is slightly decreased (~ %) by inhexo compared to norexo. summary/conclusion: exosomes from mir- and − -depleted cpcs may be a promising strategy for the treatment of various cardiac diseases, as they enhanced stem cell recruitment and proliferation, and regulated inflammation and angiogenesis. while other studies focus on boosting the reparative potential of exosomes by increasing positive mir and mrna cargo, the inhibition of negative mir in exosomes could be an overlooked strategy for the treatment of cardiac disease. endo-lysosomes as an alternative intracellular location for ev cargo delivery with disease relevance introduction: extracellular vesicles (ev) are lipidbilayer nanovesicles that carry macromolecules and act as paracrine vectors for cell-to-cell communication. the processes regulating ev biogenesis are largely known, whereas how ev cargo is delivered to recipient cells remains poorly understood. a simple mechanism proposed is direct ev fusion with the cell membrane that liberate cargo into the cytosol. in this study, we observed that cargo release occurs also at an alternative intracellular location and that this acquires a disease relevance. methods: ev were isolated by serial centrifugation and characterized. for uptake studies, ev were traced by labelling donor cells with a lipophilic dye or by overexpressing gfp-cd . uptake was assessed by cytofluorimetry or by live confocal imaging. co-localization studies were performed with ectopic marker expression or by immune staining. protein-protein interaction was analysed by bi-molecular fluorescence complementation (bifc). prion-like transmission was studied using a pro-fibrillogenic tau fragment in donor cells and full-length tau in recipient cells. for quantification of subcellular localization, an automated algorithm based on machine learning was developed. lysosomal stress was monitored by nuclear translocation of tfe and lysotracker staining. antibodies directed against pathogenic epitopes of tau were employed to assess prionlike transmission. results: ev were taken up by recipient cells through an endocytic process and accumulated in endo-lysosomes (el). when cells were exposed to ev carrying a profibrillogenic tau, recipient cells accumulated tau within el by an autophagic process. direct interaction of ev-tau and cellular tau in el favoured the appearance of pathological epitopes. cells displaying this condition showed an increased el stress and cytotoxicity. summary/conclusion: in this study, for the first time we report that el represent a critical subcellular location where transcellular prion-like transmission mediated by ev of a neurodegeneration-associated protein occurs. thus, the degradative pathway most likely involved in the recycle of ev and endogenous proteins is highjacked in disease. these findings represent a novel mechanism for ev acting as vector for transcellular propagation of tau, which opens up new therapeutic interventions trying to halt the disease. funding: supported by gelu foundation. anti-human fab fragment of cd antibody prevents the endocytosis of melanoma and colon cancer-derived extracellular membrane vesicles and nuclear transfer of their cargos introduction: interfering with the mechanisms regulating intercellular communication mediated by extracellular membrane vesicles (evs) may find relevance especially in oncology where cancer cell-derived evs have an implication in the malignant transformation of tumour microenvironment. our laboratories recently demonstrated a novel intracellular pathway in which a fraction of endocytosed ev-associated proteins is transported into the nucleoplasm of the host cell via a subpopulation of rab + late endosomes entering into the nucleoplasmic reticulum. here, we have investigated the effect of a monovalent fab antibody against the tetraspanin cd (referred hereafter as cd fab), on the internalization of evs and nuclear transfer of their cargo proteins. chair: david r f. carter -oxford brookes university chair: neta regev-rudzi -weizmann institute of science methods: to monitor the intracellular transport of ev-associated proteins, we used bioengineered fluorescent evs containing cd -gfp fusion protein from femx-i melanoma, sw colorectal cancer and bone marrow-derived mesenchymal stromal cells (msc) as donors and the same cell types as recipients. evs were enriched by differential centrifugation from h serum-free conditioned media and characterized by zetaview nanoparticle tracking analysis, zetapotential and immunoblotting. cd fab was prepared from h hybridoma cells using the pierce fab purification kit. results: we previously demonstrated that silencing cd both in evs and recipient cells strongly decreased the endocytosis of evs and abolished the nuclear transfer of their cargos. here we show that cd fab significantly reduced the cellular uptake of cd -gfp+ evs and the nuclear transfer of their proteins in melanoma, colorectal cancer and msc used as receptor cells in a dose-dependent manner. the effect on the nuclear transfer is probably a direct consequence of the endocytosis inhibition of evs. in contrast, the divalent, intact cd antibody stimulated both events. summary/conclusion: the effect of cd fab appears independent of the used ev-donor cell types or receptor cells, probably due to the widespread expression of cd both at plasma membrane and ev surface. in conclusion, by impeding intercellular communication in the tumour microenvironment, cd fab-mediated inhibition of ev uptake, combined with direct targeting of cancerous cells could lead to the development of novel anti-cancer therapeutic strategies. a bright, versatile reporter for multivesicular body trafficking and exosome secretion and uptake bong hwan sung, ariana von lersner, jorje guerrero, evan krystofiak, david inmann, roxanne pelletier, andries zijlstra, suzanne ponik and alissa weaver vanderbilt university, nashville, usa introduction: live imaging of exosomes is one of the required tools to understand the function of exosomes. our previous live-cell reporter, phluorin-cd allows dynamic subcellular monitoring of exosome secretion in migrating and spreading cells. however, there were some caveats to its use, including dim fluorescence and the inability to make cell lines that stably express the protein. methods: a stabilizing mutation, m r is incorporated in the phluorin moiety and now exhibits stable expression in cells and superior monitoring of exosome secretion. a dual-tag reporter was created by incorporating a further ph-insensitive red fluorescent protein, mscarlet to the c-terminus of phluo_m r-cd . cancer cells stably expressing the constructs were imaged using a variety of microscopy techniques in vitro as well as in vivo. purified small evs labelled with phuo_m r-cd were imaged using immunogold transmission electron microscopy (tem) and quantitated for the half-life in the blood circulation using flow cytometry. results: phluo_m r-cd and phluo_m r-cd -mscarlet are exclusively detected in exosomeenrich small ev preparations. immunogold tem visualizes the phluo_m r tag is located on the surface of small evs. live cell imaging reveals phluo_m r-cd -positive puncta left behind migrating cells suggesting the deposition consists of exosomes. those puncta and trails are not only positive for exosome markers such as cd , alix, and tsg but also correspond to small evs observed by a scanning electron microscopy. the dual-tag reporter allows visualization of the exosome lifecycle, including multivesicular body (mvb) trafficking, mvb fusion, exosome uptake and endosome acidification. summary/conclusion: using phluo_m r-cd construct, we demonstrate superior visualization of exosome secretion in multiple contexts and a role of exosomes in promoting leader-follower behaviour in collective migration by observing that exosomes are secreted at the front of migrating cells and left behind in exosome trails. the dual-tag reporter allows visualization of the entire exosome lifecycle. we anticipate that these reporters will be broadly useful to investigate regulation and functions of exosome secretion and uptake in diverse physiological conditions. funding: r gm , r ca , u ca - s , r ca . uncovering novel genes regulating ev-mediated functional rna transfer using a crispr/cas -based reporter system introduction: extracellular vesicles (evs) play a pivotal role in intercellular communication through functional transfer of bioactive cargo, including rna molecules. despite increasing interest in ev-mediated rna transfer, our understanding of the pathways and mechanisms regulating ev-mediated rna delivery and processing is limited due to a lack of suitable readout systems. we recently developed a novel crispr/cas -based reporter system that allows study of ev-mediated rna transfer at single-cell resolution. here, we further validate this system by studying the role of known targets involved in ev uptake and intracellular membrane trafficking, and subsequently employ this system to uncover various novel genes that play a regulatory role in functional rna transfer. methods: we employed a novel crispr/cas -based stoplight reporter system, in which egfp expression is activated upon functional delivery of targeting single guide rnas (sgrnas) stably expressed by donor cells. intercellular functional rna transfer was assessed by measuring egfp expression in acceptor cells using fluorescence microscopy and flow cytometry after direct co-culture, transwell co-culture, and upon addition of isolated evs. potential roles of various genes in intercellular rna transfer were assessed by rnaimediated target knockdown in acceptor cells, prior to co-culture experiments. rnai knockdown was confirmed by qpcr analysis. results: a significant activation of egfp expression was observed in acceptor cells after direct co-culture and transwell co-culture with donor cells expressing sgrnas, as well as after addition of evs from cells expressing sgrnas. reporter activation was substantially decreased after knockdown of multiple targets involved in ev uptake through endocytosis and/or intracellular membrane trafficking. based on these results, a potential role of various novel genes in intercellular rna transfer was studied in acceptor cells. these experiments uncovered various novel targets involved in ecm binding, endocytosis, intracellular membrane trafficking, as well as various rho gtpase interactors. summary/conclusion: we previously demonstrated a crispr/cas -based reporter system that allows the study of functional delivery of small non-coding rnas with single-cell resolution. here, we show that this novel approach allows the study of specific genetic targets and pathways in ev-mediated functional rna delivery, and unravel the regulatory pathways that dictate the underlying processes. quantitative characterization of extracellular vesicle uptake and content delivery within mammalians cells gregory lavieu a , emeline bonsergent b , eleonora grisard c and clotilde théry d introduction: extracellular vesicles (evs), including exosomes, are thought to mediate intercellular communication through the transfer of biomolecules from donor to acceptor cells. occurrence of ev-content delivery within acceptor cells has not been unambiguously demonstrated, let alone quantified, and remains debated. methods: we developed a cell-based assay in which evs containing luciferase-tagged cytosolic cargo are loaded on unlabelled acceptor cells. measurement of luciferase activity associated with acceptor cells revealed ev uptake efficacy. additional cell fractionation procedure that separates membranes from cytosol revealed the occurrence of ev-content release within the cytosol of acceptor cells. results: results from dose-responses, kinetics, and temperature-block experiments suggest that ev-uptake is limited ( % spontaneous rate at h), does not depend on bona-fide ev-receptor, at least for the tested acceptor hela cells. yet, further characterization of this limited ev-uptake, through cell fractionation that separates membranes from cytosol, revealed the occurrence of ev-content release within the cytosol of acceptor cells. cytosolic release is inhibited by bafilomycin-a and overexpression of ifitm proteins, which prevent virus content delivery. summary/conclusion: our results show that ev-content release requires endosomal acidification and suggest the involvement of membrane fusion. funding: anr -ce - - and arc pja and pga rf . introduction: glioblastoma is a highly malignant brain tumour with a poor prognosis. its ability to develop therapeutic resistance result in devastating clinical outcomes. to solve the intractable problem, we need highly sensitive diagnostics that can detect the molecular changes during treatments. extracellular vesicles (evs) can be a potential biomarker to monitor treatments and the host cell ev mapping can better reflect molecular changes in the tumour immune microenvironment. we have developed a droplet-based single ev protein sequencing platform that overcomes limitations of current bulk measurement technologies, which make it difficult to discover a rare ev population in the presence of high background. methods: we multiplex protein measurements to profile hundreds of proteins at a time by using an antibody-dna conjugate and sequencing. we barcode each ev in droplets and make amplicons that are comprised of both ev barcodes and antibody barcodes for sequencing. barcoded antibodies are made using tco-tetrazine click reaction and evs are labelled with these barcoded antibodies. the labelled evs are encapsulated into droplets with barcoded beads that serve as a template for ev barcodes. we then perform extension to make amplicons that contain both ev barcodes and antibody barcodes for sequencing. results: we successfully fabricated barcoded beads using a split-pool approach and validated by observing a fluorescence decrease of the sybr green after dna strand denaturation. we used a -channel droplet maker to encapsulate barcoded beads, single ev, and master mix into droplets. close packing of barcoded beads allowed > % encapsulation into droplets. both droplet and tube-based methods achieved a similar high amplification efficiency (ct < for evs). we confirmed the amplicon size by running a gel, which showed the right amplicon size (~ bp) from the droplet and tube prepared samples and no signal from the negative control. summary/conclusion: the droplet-based single ev profiling platform has the ability to identify rare immune ev subtypes in the peripheral blood, which would otherwise be impossible to detect due to the copresence of abundant normal evs. this cutting-edge technique has the potential to revolutionize treatment monitoring of high-cost immunotherapies, avoid unnecessary toxicities, and enhance personalized medicine capabilities. funding: schmidt science fellows, in partnership with the rhodes trust po ca , ro ca , r ca quantbio graduate student award at harvard university. introduction: in this study, we compared four orthogonal technologies for sizing, counting, and phenotyping of evs. the platforms were: single-particle interferometric reflectance imaging senor (sp-iris) with optional fluorescence, nanofcm nanoflow (nf), nanoparticle tracking analysis (nta) with fluorescence, and microfluidic resistive pulse sensing (mrps) . results from these platforms were compared with results from standard ev characterization techniques such as transmission electron microscopy (tem) and western blot (wb). methods: human t lymphocyte h (high cd , low cd ) and promonocytic u (low cd , high cd ) cells were chosen for their distinct tetraspanin profiles without abnormalities that might result from genetic manipulation. evs were isolated from culture conditioned medium (ccm) by differential ultracentrifugation (duc) and size exclusion chromatography (sec) and characterized per misev guidelines. synthetic particles (silica and polystyrene spheres) with known concentrations and mixed size distributions were also tested. results: particle counts from nf and mrps were consistent, while nta detected approximately one order of magnitude lower for ccm derived evs, but not for synthetic particles. sp-iris events could not be used to estimate particle concentrations. for sizing, nf, mrps, and sp-iris returned similar size profiles, with smaller sizes predominating (per power law distribution), but with sensitivity typically dropping off below diameters of nm. nta detected a population of particles with a mode diameter above nm. additionally, sp-iris, nf, and mrps were able to identify at least three of four distinct size populations in a mixture of silica or polystyrene nanoparticles. finally, for tetraspanin phenotyping, the sp-iris platform in fluorescence mode and nf were able to detect at least two markers on the same particle. summary/conclusion: based on the results of the study, we can draw conclusions about existing singleparticle analysis capabilities that may be useful for ev biomarker development and mechanistic studies. funding: this project is funded by mh and ug ca . importance to ev organotropism. yet, most techniques rely on bulk characterization, or are severely restricted by the diffraction limit. the exoview r (nanoview biosciences) combines interferometry, immunocapture, and immunofluorescence, introduced as an alternative technique to multiplex protein detection on single evs below the limit of diffraction. here, we use this technique to characterize tetraspanin multiplexing on evs and to identify spatial patterning of tetraspanins using steric hindrance of antibodies (abs). methods: evs were isolated from conditioned media from skov- cell culture or human serum. evs were incubated overnight on chips to allow immunocapture by anti-cd , anti-cd , or anti-cd . chips were then incubated with three fluorescent abs against the same epitopes and imaged on the exoview r . following concentration optimization, evs were tested after preincubating with carboxy-fluorescein diacetate succinimidyl ester (cfse) or fluorescent abs against tetraspanins. results: using different concentrations of evs, binding curves could be fit to characterize binding kinetics of abs. maximum concentration of evs could be identified that minimized fluorescent overlap. bright-field interferometry (detection limit~ nm) distinguished x fewer bound evs than fluorescent detection, while pre-labelling evs with cfse produced x more detectable evs than immunofluorescence. interestingly, evs captured by one tetraspanin did not necessarily show high fluorescent detection of the same tetraspanin. upon pre-incubating evs with a single ab, vastly different expression profiles were identified, indicating significant steric hindrance between abs. furthermore, pre-incubating evs with anti-cd ab significantly decreased detection of cd with less impact on cd . this discrepancy indicated possible spatial patterning of tetraspanins with cd and cd closely colocalizing on the ev surface. summary/conclusion: this combination of interferometry, immunocapture, and immunofluorescence produces unique information about size distribution of evs and single ev protein profile. this data corroborates that evs have distinct subpopulations of tetraspanins and indicates that tetraspanins may be spatially patterned. regulation of liver homoeostasis, regeneration and diseases by mesenchymal stem cell-derived apoptotic extracellular vesicles university of pennsylvania, philadelphia, usa introduction: billions of cells undergo apoptosis and produce apoptotic extracellular vesicles (apopevs) each day, whereas the roles of apopevs in regulating the organismal health and disease remain poorly understood. mesenchymal stem cells (mscs) emerge as critical contributors to tissue homoeostasis, while mscs suffer from apoptosis in regenerative transplantation. in this study, we investigated the function and mechanisms of msc-derived apopevs in regulating the organismal homoeostasis. methods: fas mutant (fasmut) and caspase knockout (casp -/-) mice were applied for apoptotic and apopev deficiency. mouse bone marrow mscs were cultured and apoptosis was induced by staurosporine (sts). msc-derived apopevs were collected by serial centrifuges and were infused into mouse circulation via caudal vein. tracing of apopevs were performed by radioisotope or fluorescent labelling. liver homoeostasis was evaluated at the histological and functional aspects. liver regeneration was induced by partial hepatectomy (phx). acetaminophen (apap) was used to establish acute liver drug injury. high-fat diet (hfd) was used to establish type diabetes (t d) and non-alcoholic fatty liver disease (nafld). results: after systemic injection, msc-derived apopevs migrate to liver and can be uptaken by liver macrophages and hepatocytes. fasmut and casp -/mice develop hepatomegaly with structural disorders, which particularly reveals hepatocyte polyploidization. furthermore, fasmut and casp -/-mice demonstrate liver glucose and lipid metabolic disorders. importantly, msc-derived apopev infusion significantly rescues structural and metabolic dysfunction in fasmut and casp -/-mice. mechanistically, apopevs use the soluble n-ethylmaleimide-sensitive fusion protein attachment protein receptor (snare) protein for interactions with recipient organelles thus transferring signalling molecules. moreover, msc-derived apopev infusion promotes liver regeneration after phx, prevents apap-induced liver injury, and ameliorates nafld in t d. summary/conclusion: msc-derived apopevs serve as crucial regulators of liver homoeostasis, regeneration and diseases. these findings indicate potential significant roles of apopevs in maintaining the organismal health and in developing therapeutics for diseases. (msc-sevs) mediate osteochondral regeneration in rats. however, the therapeutic effects of these msc-sevs/exosomes in restoring the mechanical competence of the repaired cartilage for joint function in a clinically relevant animal model remain to be addressed. to investigate this, we compared the structural and mechanical properties of the repaired cartilage in a rabbit model after intraarticular administration of msc-sevs and hyaluronic acid (ha) with that of ha alone, which is widely used as visco-supplementation. methods: bilateral osteochondral defects were surgically created on rabbits. immediately after surgery and at days and post-surgery, rabbits received ml injections of µg msc-sevs and ha in both knees, and rabbits received -ml injections of ha in both knees. at and weeks, macroscopic evaluation, histological scoring and compressive testing at different points on the repaired cartilage were performed. results: defects treated with msc-sev/ha showed improvements with time in macroscopic and histological scores and mechanical properties than defects treated with ha alone. in contrast, ha treated defects showed some repair at weeks, but this was not sustained, as evidenced by significant deterioration of histological scores and a plateau in mechanical properties from to weeks. by weeks, the msc-sev/ha repaired tissues demonstrated significantly better macroscopic score ( . vs . ; p < . ) and histological score ( . vs . ; p < . ). mechanical strength as measured by the young's modulus was significantly higher in the msc-sev/ha repaired cartilage than that in ha repaired tissues [defect centre ( . vs . mpa; p = . ) and overall periphery ( . vs . mpa; p = . ], and approximated that of the adjacent native cartilage. summary/conclusion: our findings demonstrated that msc-sevs and ha not only improved tissue morphology of the repaired cartilage but also promoted functional mechanical competence. this study establishes a clinically translatable protocol for use of msc-sevs for cartilage repair. introduction: mesenchymal stromal/stem cell (msc)exosome (mex) treatment has shown considerable promise in experimental models of bronchopulmonary dysplasia (bpd) and pulmonary hypertension (ph). mechanisms by which mex afford their beneficial effects remain incompletely understood and here, we embark into investigating them through assessment of mex biodistribution and impact on immune cell heterogeneity. methods: newborn fvb mice were exposed to hyperoxia (hyrx, % o ) at birth and returned to room air at postnatal day (pn) . mice received a bolus mex dose at pn . adoptive transfer studies were used to determine the role of mex-educated myeloid cells in vivo. mice were harvested at pn , , , or to characterize mex biodistribution and for assessment of pulmonary parameters. results: mex therapy effectively ameliorated core features of hyrx-induced neonatal lung injury, improving alveolar simplification, pulmonary fibrosis, vascular remodelling and blood vessel loss. exercise capacity testing and assessment of ph showed functional improvements following mex therapy. biodistribution studies demonstrated that mex localize in the lung, where they interact with lung monocytes/macrophages. whole lung mass cytometry (cytof) revealed that mex treatment promotes a pro-homoeostatic shift in lung immune cell apportion, replenishing the early hyrx-induced depletion in pulmonary cd + immune cells, restoring alveolar monocyte and macrophage populations and suppressing cellular inflammation. ex vivo and in vivo analysis showed that mex promotes a "pro-resolving" ccr -monocyte phenotype. notably, adoptive transfer of mex-educated bone marrow-derived myeloid cells (bmdmy), but not naïve bmdmy, restored alveolar architecture, blunted fibrosis, improved vascular remodelling and pulmonary blood vessel loss. summary/conclusion: mex treatment ameliorates core features of experimental bpd, restoring lung architecture, decreasing pulmonary fibrosis and vascular muscularization, ameliorating ph and improving exercise capacity. the beneficial actions of mex are associated with modulation of immune cell phenotypes, arising from mex-monocyte interaction. furthermore, adoptive transfer of mex-educated bmdmy rescued, at least in part, alveolar architecture, reduce fibrosis, improve vascular remodelling and pulmonary blood vessel loss. funding: this work was supported in part by an american thoracic society foundation grant (grw); the little giraffe foundation (grw); charles h. hood foundation major grants initiative (sk), nih r hl (sk) and united therapeutics research grant (sk and sam). immunomodulatory small extracellular vesicles derived from mesenchymal stem cells: a potential cell-free therapy for acute and chronic pulmonary vascular diseases introduction: vascular inflammation plays a critical role in acute respiratory distress syndrome (ards) and pulmonary arterial hypertension (pah). despite decades of research, there is no curative therapy for either condition. mesenchymal stem cells (mscs) have shown preclinical efficacy, mediated by release of extracellular vesicles. hence, msc-derived small extracellular vesicles (sevs) can harness the benefits of mscs with advantages in cost and safety. this study aims to evaluate the immunomodulatory effects of sevs in preclinical ards and pah. methods: msc-sevs were characterized by nanoparticle tracking analysis, electron microscopy and western blot. live fluorescence imaging measured in vitro and in vivo distribution of sevs. using a lipopolysaccharide (lps)-induced mouse model of acute lung injury (ali), a time course study of inflammatory response guided endpoint analyses. cell count and cytokines were measured in bronchoalveolar lavage fluid (balf) and histological lung injury was assessed. in ali mice, saline, mscs, msc conditioned media or sevs were administered . h post-lps. using a monocrotaline (mct)-induced rat model of pah, animals received saline or sevs at day . haemodynamic changes and right ventricular hypertrophy were evaluated at weeks. results: msc-sevs were nm in size with cd / expression. pkh -labelled sevs were taken up by endothelial cells. in the ali time course study, cell count and il b in balf peaked at h post-lps, whereas il peaked at h. histology showed significant intra-alveolar cell infiltrate at h. msc conditioned media attenuated il b in balf, whereas a trend towards reductions in il b and cell count were seen from delivery of mscs and sevs. using fluorescence imaging, lung accumulation of dir-labelled sevs was highest when administered h post-lps as compared to h, h or h. for pah rats, sevs reduced right ventricular systolic pressure ( . ± . mmhg) as compared to control ( . ± . mmhg; p = . ), whereas no changes were observed for right ventricular remodelling. summary/conclusion: these findings demonstrate the potential of msc-sevs to be used as a cell-free immunomodulatory therapy for acute and chronic lung vascular diseases. additional live and ex-vivo biodistribution studies will determine optimal timing of sev administration, tissue distribution and clearance in both ali and pah. changes in extracellular vesicle protein cargo after pro-inflammatory priming of umbilical cord mesenchymal stem cells (ucmscs) have been shown to suppress inflammatory responses in studies of autoimmune diseases. these therapeutic effects can be attributed to paracrine signalling, by which extracellular vesicles (evs) are one of the essential components. this study looks at how the culture conditions of ucmscs affects the type of evs they secrete. it also aims to identify an ev population with an anti-inflammatory potential for the treatment of autoimmune diseases. methods: ucmscs were isolated and culture expanded in a quantum® cell expansion system, then grown at %o , %o and primed with a pro-inflammatory cocktail. evs were isolated from ucmsc conditioned media by differential ultracentrifugation using a sucrose cushion and characterised by transmission electron microscopy and nanoparticle tracking analysis. ev markers were analysed using a europium-based immunoassay, macsplex exosome detection kit and immunoblotting. a proximity-based extension assay was used to identify inflammatory proteins in the evs. results: there was no difference in evs cultured at %o , %o or with pro-inflammatory conditions when analysed for size and morphology. all evs displayed the tetraspanin markers (cd / / ) and internal proteins (alix, hsp ). evs from primed cells showed a > twofold increase of cc chemokines and a > sixfold increase in cxcl and csf- . protein cargo did not differ in evs from %o and %o . summary/conclusion: this study showed that proinflammatory culture conditions alter ev protein cargo, evidenced by the increased production of chemotactic and angiogenesis associated proteins. upcoming rnaseq analysis will show if ucmsc culture conditions also affect mirna expression in evs. ongoing functional studies will determine how changes in ev cargo correlates with changes in t-cell proliferation and polarisation. funding: this work is fund by the orthopaedic institute ltd, keele university and the rjah orthopaedic hospital charity. alzheimer's disease biomarkers in plasma extracellular vesicles of neuronal origin correlate with brain pathology in mice introduction: multiple studies have shown that neuronal-derived extracellular vesicles (ndes) in blood contain alzheimer's disease (ad) biomarkers, especially tau. however, the convergent validity of tau in blood ndes in relation to brain pathology is yet to be determined. to address this, we measured total and phosphorylated tau levels in matched nde and brain tissue samples from ad mouse models. methods: we collected the cortex, hippocampus and plasma of xtg-ad, xtg-ad, and wild type (total of mice; female, male; age: mean = . , sd = . , - months) . plasma samples were collected retro-orbitally for weeks and at euthanasia via heart puncture. ndes from the pooled serial blood collections (nde ) and the single endpoint (nde ) were immunocaptured by targeting the neuronal marker l cam. we measured human total tau and pthr -tau (p-tau) in ndes and cortex and hippocampus homogenates using a luminex multiarray. results: overall, there were strong positive correlations for both total tau and p-tau between ndes and brain tissues across mice types. total tau in ndes showed positive correlations with levels in the cortex and hippocampus (r = . and . , p < . , cortex vs nde and nde ; r = . , p = . , hippocampus vs nde ; r = . , p = . , hippocampus vs nde ). levels of p-tau in nde showed positive correlations with levels in the cortex (r = . , p = . ) and hippocampus (r = . , p = . ); however correlations were not observed for nde (r = . , p = . vs cortex; r = . , p = . vs hippocampus). summary/conclusion: tau levels in circulating ndes reflect levels in cortex and hippocampus across ad model mice, supporting their convergent validity as "liquid biopsy" biomarkers for ad. funding: this research was supported in part by the intramural research program of the national institute on ageing, national institutes of health. exosomal ceramide mediates neurotoxicity of amyloid beta (aβ) in alzheimer's disease. ahmed elsherbini a , simone crivelli b , alexander kirov c , michael dinkins d , zhihui zhu a , haiyan qin a , sanjib karki a , priyanka tripathi a and erhard bieberich a a university of kentucky, lexington, usa; b university of kentucky, lexington, usa; c augusta university, augusta, usa; d augusta university, augusta, usa introduction: amyloid beta is a pathologic hallmark of alzheimer's disease (ad), however, the mechanism of aβ neurotoxicity is not fully understood. it has been reported that exosomes associate with aβ, but it is not clear how this association affects aβ neurotoxicity. methods: here we utilized several techniques to isolate exosomes from the sera of wild type (wt) and ad transgenic mouse model. ( xfad) as well as alzheimer's patients and healthy controls. we used exoquick, exoeasy, sequential ultracentrifugation, and size exclusion chromatography. particles' size and number were characterized by nanoparticle tracking analysis (zetaview). results: we report that the sphingolipid ceramide mediates neurotoxicity of aβ. we show that sera from ad transgenic mouse model ( xfad) and ad patients, but not the wt or healthy controls, contain a subpopulation of astrocyte-derived exosomes that are enriched with ceramide and are prone to aggregation (termed astrosomes) as confirmed by nanoparticle tracking and cluster analyses. when taken up by introduction: multiple sclerosis is the most common chronic inflammatory demyelinating disease of the central nervous system, affecting more than million people worldwide. ms is a multifactorial, immunemediated disease caused by complex genetic and environmental interactions. in recent years, extracellular vesicles (evs) have been described as powerful mediators of the modulation of biological processes (e.g. inflammatory and immune response) following environmental exposures such as particulate matter (pm), and have been described altered in ms. we characterized evs in patients with ms and healthy subjects matched for age and gender and evaluated the effects of pm exposure on ev release patients with ms compared with controls. methods: evs isolated from blood samples were characterized by nanotracking analysis and by flow cytometry after labelling with the following markers: cd + (monocyte), cd + (platelet), cd + (neutrophil), cd + (t-reg), and cd + (endothelium). pm and pm . concentrations at the residency of each subject were obtained from the regional air quality monitoring network. results: we observed decreased concentrations of cd + (p < . ), cd + (p < . ), cd + (p < . ), cd + (p < . ), and cd + (p < . ) in patients compared with controls. in cases, pm was inversely associated with cd + evs (pm . , β = − . ; p < . ), cd + evs (pm . β = − . ; p < . ), and cd + evs (pm β = − . ; p < . ; pm . , β = − . ; p < . ). on the contrary, in controls pm was positively associated with cd + evs (pm β = . ; p < . ; pm . , β = . ; p < . ). summary/conclusion: our findings showed a different composition of blood-derived ev subpopulations in patients compared with controls. moreover, we observed that patients and controls react differently to pm exposure in terms of blood-derived ev release, suggesting the involvement of this mechanism in the modulation of both inflammatory and immune responses, and thus in ms pathogenesis. plasma neuronal and astrocyte-derived exosomes serve as biomarkers of neurodegeneration and systemic bioenergetic effects in male cynomolgus monkeys self-administrating oxycodone ashish kumar a , yixin su a , david soto-pantoja a , jingyun lee a , ravi singh a , cristina furdui a , michael nader b and gagan deep a a wake forest baptist medical center, winston-salem, usa; b wake forest baptist medical center, winston-salem, usa introduction: opioid use disorder (oud) is currently a health emergency in the usa affecting millions of people. oud is a complex issue requiring a multipronged strategy. at the biological level, there is an urgent need to understand the dynamic molecular changes and adverse effects associated with opioid addiction. here, we aimed at identifying the biosignature of brain cells-derived exosomes associated with opioid addiction in a non-human primate (nhp) model of oud in which cynomolgus monkeys perform cognitive tasks and self-administer (sa) intravenous oxycodone daily. we also characterized the systemic adverse effects of the brain cells-derived exosomes from drug-naïve and oxycodone sa monkeys. methods: we isolated total exosomes (te) by ultracentrifugation and exoquick methods from the plasma of male monkeys self-administrating oxycodone for years and naive monkeys. subsequently, from the te population, we isolated neuron-derived exosomes (nde) and astrocytes-derived exosomes (ade) using surface biomarkers l cam (l cell adhesion molecule) and glast (glutamate aspartate transporter), respectively. this novel method involved streptavidin coated magnetic beads and photo-cleavable (pc) biotin, providing us biologically intact exosomes useful for co-culture studies. we characterized the exosomes by nanoparticle tracking analyses (nta), western blotting, flow cytometry, immunogold labelling, transmission electron microscopy (tem), elisas and mass spectrometry. respirometric profiling in cardiac myoblasts and monocytes following exosomes treatment was performed by seahorse xf. results: the quality of isolated exosomes (te, nde, and ade) was confirmed by nta (size distribution and concentration), western blotting (e.g. cd ) and tem (size and shape). nta did not show any significant difference in exosomes size and concentration (number per ml) between control and oxycodone sa groups. flow cytometry (e.g. l cam and glast) and immunogold labelling (cd , cd and l cam) confirmed the purity of nde and ade isolated from te. proteomics analyses of te, nde and ade identified several unique proteins present in exosomes from the oxycodone sa group. interestingly, we observed significantly higher expression of neurodegeneration markers neurofilament light protein (nfl) and alpha-synuclein in nde and ade of oxycodone sa group compared to controls. furthermore, te treatment of h c cardiac myoblasts and raw . monocytes significantly compromised their mitochondrial metabolism (basal and maximum respiratory capacity). summary/conclusion: these results suggest the utility of plasma exosomes as biomarkers for better understanding of the neurodegenerative and systemic effects of oxycodone addiction. funding: da , da . vesicles released during mycobacterium tuberculosis infection: immunomodulatory (glyco)lipids and role in host-pathogen interactions emilie layre, pierre boyer and jerome nigou cnrs-université paul sabatier, toulouse, france introduction: the tuberculosis disease remains one of the top causes of death worldwide. mycobacterium tuberculosis (mtb) has evolved strategies to evade immune responses and to persist within the hostile intracellular environment of alveolar macrophages. the current lack of efficient anti-tuberculosis strategies is largely due to our incomplete understanding of the host-pathogen interactions of mtb infection. vesicles released by the bacillus itself (bacterial membrane vesicles, bmv) and by infected cells (host extracellular vesicles, hev) have immunomodulatory properties in vitro and when administered to animals. if vesicles likely play key role in host-pathogen interactions of the tuberculosis infection, their content in bacterial factors, their uptake, trafficking and interaction with host cells receptors remain incompletely deciphered. methods: bmv and hev have been purified by combining differential centrifugation, density gradient and exclusion chromatography. after characterization by microscopy, nanosight and western blot, their content in bacterial (glyco)lipids has been characterized by the use of high sensitivity mass spectrometry-based lipidomic approach. bmv have been tested for their capacity to activate reporter cell lines of pattern recognition receptors. in addition, fluorescent-labelled bmv have been used to study their uptake by host cells thank to super-resolution microscopy. results: we have undertaken to characterize the content, the trafficking and interaction with pattern recognition receptors of bmv and hev released during infection by mycobacteria of variable virulence. we have importantly optimized the purification of bmv showing that lipoproteins aggregates are co-purified with vesicles on density gradient. sfc-ms lipidomic analyses allowed the characterization of the repertoire of immunomodulatory bacterial lipids released by bmv and hev, which excluded a continuum between these two release pathways. preliminary, assays have shown that these vesicles are capable to interact with different pattern recognition receptors including tlr and lectins. finally, we have been able to visualize fluorescent-labelled vesicles uptake by macrophages using superresolution microscopy. summary/conclusion: during m. tuberculosis infection, the bacillus as well as infected cells release vesicles that harbour different content in immunomodulatory bacterial (glyco)lipids, including strain-specific lipids. these vesicles likely play important role in host-pathogen interactions by modulating immune response beyond the infected cells, in part through their interaction with different pattern recognition receptors. funding: fondation pour la recherche medicale, fondation fonroga. introduction: conventional diagnoses of mycobacterium tuberculosis (mtb) rely on quantifying bacteria in sputum samples, which make it incapable of measuring the body's total bacterial load and diagnosing patients that have difficulty producing sputumsuch as children and those that are hiv-positive. nanoscale ( - nm) outer membrane vesicles (omvs), which are shed from their bacterial cells of origin and circulate in the bloodstream, have been found to contain rich molecular information from their mother cells. despite the diagnostic potential, their nanoscale size in the presence of high background has complicated the use of these promising biomarkers for clinical diagnosis of tuberculosis. chair: amy buck -the university of edinburgh chair: cherie blenkiron -the university of auckland methods: here we report two complementary approaches to systematically discover and clinically detect mtb-derived omvs using protein and rna biomarkers. first, we employ a digital droplet elisa on whole, unprocessed samples to detect and quantify the presence of these omvs using surface protein markers. second, we have developed a platform to specifically enrich for mtb-derived omvs using our previously developed magnetic nanopore platform, wherein millions of nanofluidic devices are operated in parallel, increasing throughput relative to a single nanofluidic device by a million-fold. using this approach, we identify rnas that are specifically enriched in mtb-derived omvs and can be used to identify tb strain, infectious activity, and total body burden. results: using these platforms, we enriched for mtbderived omvs from plasma and profiled their cargo, both proteins and rna. we first determined a panel of protein biomarkers for multiplexed detection of omvs through a digital droplet sandwich elisa. we then tested our protein markers on spiked plasma samples as models for clinical tb samples. simultaneously, we performed rna sequencing and discovered a panel of rna biomarkers that are preferentially enriched in omvs. we picked ten of the most highly-expressed rna biomarkers and also tested for them on spiked plasma samples using our magnetic nanopore platform. summary/conclusion: these results demonstrate the capability of omv biomarkers in the development of novel liquid biopsy based mtb diagnostics. building on this work, we are working with clinical collaborators to test our assays on clinical samples from philadelphia and west africa. funding: nih ot . introduction: a dearth of knowledge exists regarding the molecular mechanisms by which host exosomes regulate immune response to infections caused by gram-negative pathogens. to address this gap in knowledge, our laboratory has been using two wellestablished model organisms; yersinia pestis (yp), and burkholderia pseudomallei (bp). yp and bp cause the emerging human diseases plague and melioidosis respectively. currently, no licenced vaccines or highly effective therapeutics are available for either disease. methods: ex were purified from naïve u monocytes (exu) and infected u (exi) by serial centrifugation followed by sucrose density gradient purification, and characterized by tem, zetaview nanoparticle tracking, and exosome markers (cd , tsg , . immune responses of naïve u cells and response mechanisms were analysed following treatment with equivalent amounts of exi or exu (as control). these included macrophage differentiation assays, multiplex measurements of inflammatory cytokines, bacterial clearance assays, quantitative protein microarray analysis of host signalling proteins, sirna knockdown of exi-induced cytokines in recipient cells, and mass spectrometry (ms) analysis of exi contents. for all assays, at least four biological replicates were performed. results: exi induce monocyte differentiation to macrophages and dramatic release of il- , il- and il- cytokines, effects that are also seen when monocytes are infected with the bacteria. the exi also induce a substantial increase in the capacity of the recipient monocytes to clear bacteria in an il- -dependent manner. specific host signalling molecules are strongly modulated by the exi, including p , jak and alk for exi-yp, influencing the observed phenotypes. ms analysis showed lack of lps in exi-yp and demonstrated the presence of specific bacterial proteins that have antigenic properties. summary/conclusion: we have identified some of the molecular mechanisms by which exi assist the host in clearing infection. exi prime distant naïve monocytes through modulation of distinct pathways such as p to mount immune responses similar to when they become infected. these include differentiation to macrophages and migration to infection site for increased il- -dependent bacterial clearance. introduction: recruitment of monocytes to sites of infection is important in restricting growth and invasion of various microorganisms such as pathogenic fungi c. albicans. beside complement supported phagocytosis and extracellular trap formation, human monocytes secrete extracellular vesicles which are crucial in cellular communication in physiology and pathophysiology as they transfer proteins, lipid, and nucleic acids. the current study attempts to shed light on immune evasion mechanisms by c. albicans via extracellular vesicles. methods: human monocytes were isolated by magnetic beads technique and extracellular vesicles were isolated using polymer precipitation or ultracentrifugation or size exclusion chromatography. vesicles were characterized by elisa, lc/ms-based proteomics, confocal laser scanning microscopy (clsm) as well as electron-and dynamic light scattering microscopy, etc. crispr-cas based genome editing was performed to knockout cd b in human monocytic thp- cells. effect of isolated vesicles were determined by using proximity ligation assay (pla), elisa, western blot, next generation rna sequencing, qpcr, immunohistochemistry, etc. results: here we show for the first time that human blood derived monocytes alone and in a whole blood model strongly induced and released extracellular vesicles in response to the pathogenic fungus c. albicans. one induced population carried the anti-inflammatory cytokine tgfβ- . release of these vesicles is triggered by binding of soluble β-glucan from c. albicans to the cr receptor on monocytes as demonstrated by crispr-cas based cr genome editing in thp- cells, and by using cr knock out mice. isolated tgf-β -transporting vesicles reduced the inflammatory response in human m macrophages and in a whole blood model. the anti-inflammatory effect by tgf-β transporting vesicles is investigated in detail and results in inhibition of il- β gene transcription. summary/conclusion: showing that human apoptotic bodies similarly induced tgf-β -transporting vesicles from human monocytes we hypothesize that c. albicans hijacks this new cr -dependent anti-inflammatory vesicle pathway for immune escape. funding: this work was supported by the "deutsche forschungsgemeinschaft" transregio funginet projects c , c , c and z . introduction: to date, most research involving extracellular rnas has focused in rnas encapsulated inside extracellular vesicles (evs) or in total unfractionated biofluids. it is known that exrnas also exist outside vesicles or in lipoprotein particles. however, nonvesicular exrnas remain widely uncharacterized despite being a feasible source of contaminants in ev preparations. our interest in nonvesicular exrnas arises from the observation that some small rnas, such as specific trna-derived fragments, have much higher relative representation in this extracellular fraction. at least in part, this enrichment seems to be a consequence of their differential extracellular stability. methods: to get a representative picture of the whole set of rnas released to the extracellular nonvesicular space by cultured human cells, we inhibited extracellular degradation by adding recombinant ribonuclease inhibitor to the cell-conditioned media and studied the kinetics of rna release and degradation. high-resolution iodixanol gradients were used to separate evs from extracellular rnps or vesicle-free rna. the conversion rate between parental ncrnas and their fragments was studied by high-throughput sequencing and northern blot. results: the inhibition of extracellular rnase activity revealed the presence of full-length trnas and ribosomes in the extracellular space of a variety of malignant and non-malignant cell lines. extracellular ribosomes co-isolate with evs purified by ultracentrifugation or size-exclusion chromatography, but not with evs purified by density gradients.these ncrnas are substrates of extracellular rnases, demonstrating an extracellular biogenesis route for the formation of ncrna-derived fragments, some of which achieve remarkable stability and can be detected in biofluids. we also highlight the immunoregulatory potential of purified rna-containing extracellular complexes. summary/conclusion: in conclusion, ribonuclease inhibition dramatically shapes extracellular rna profiles and uncovers a population of extracellular ribosomes, trnas and other coding and noncoding rnas which exists outside evs. although these rnas are prone to degradation, some of their fragments can accumulate in cell culture media and in biofluids. this dynamic view of exrnas impacts our understanding of rna secretion mechanisms and may offer a window to new molecules with biomarker potential. in contrast, evs confer an rnase-protected environment and contain more full-length ncrnas (trnas, yrnas, sl rnas, rrnas depending on vesicle size) than their fragments. introduction: cd is a ubiquitously expressed membrane protein that functions as a receptor for thrombospondin- and the counter receptor for signal regulatory protein-α in phagocytes. high expression of cd is associated with a poor prognosis for some cancers. conversely, cd blocking agents are in clinical trials for enhancing innate and adaptive antitumor immunity in cancer patients. these studies suggest utility of cd as a diagnostic and prognostic biomarker and as a therapeutic target. cd is also expressed on extracellular vesicles (evs), and we reported that cd expression identifies a distinct population of evs from those that express the traditional ev markers cd or mhc . cd -, cd -and mhc -enriched vesicles contain distinct small rna populations (pmid: ), and these differ in rna content from evs that lack any of these markers. the mechanisms by which cd directly or indirectly regulates which rnas are packaged into ev remain unknown. methods: to elucidate the mechanism by which cd regulates ev rna composition and function, we performed global mirna microarray analysis between evs produced by wild type and cd -deficient t cells. results were further validated using real-time pcr and rna-immunoprecipitation. interactions between cd and exportin- /ran complex was identified by mass spectrometry and confirmed by using co-immunoprecipitation, subcellular localization, flow cytometry, and confocal and electron microscopy. results: ev released from cd -deficient human t cells and in cd -/-mouse plasma were enriched in ʹ- -methylguanosine-capped micrornas and mrnas that depend on the exportin- /rangtp pathway. knockdown of cd in wild type cells or thrombospondin- treatment correspondingly enhanced levels of capped-rnas released in ev and re-expressing cd in null cells decreased their levels. mass spectrometry and co-immunoprecipitation identified specific interactions of cd with components of the exportin- / ran nuclear export complex and its known cargos and between the cd cytoplasmic adapter ubiquilin- and the exportin- /ran complex. interaction of cd with exportin- was inhibited by leptomycin b, which inactivates exportin- and increased levels of cap-dependent rnas in ev released from wild type but not cd -deficient cells. summary/conclusion: these findings indicate that cd -dependent thrombospondin- signalling regulates cytoplasmic levels of cap-dependent rnas in t cells at least in part through ubiquilin- -and gtpdependent physical interactions with the exportin- / ran transport complex, which regulate levels of specific pre-mirnas and mrnas available for sorting into evs. funding: this work was supported by the intramural research program of the nih/national cancer institute (zia sc ). role of membrane protein palmitoylation in extracellular vesicle biogenesis in squamous cell carcinoma introduction: desmoglein (dsg ), is a palmitoylated cadherin that is involved in cell-cell adhesion. interestingly, dsg promotes mitogenic cell signalling and is upregulated in many cancers, including scc, contributing to poor prognosis and survivability. we recently demonstrated that dsg promotes ev release, but the mechanism by which dsg enhances ev biogenesis and role of palmitoylation is poorly understood. methods: pharmacological drug inhibitors -bromopalmitate, gw , and bafilomycin a were used. stable scc cell lines were established by retrovirus infection expressing gfp, wild type dsg /gfp, or palmitoylation deficient dsg cacs/gfp. evs were isolated by sequential ultracentrifugation and iodixanol gradient separation and analysed by nta. proteins associated with the endocytic pathway were analysed by immunofluorescence and imaged by confocal microscopy or immunoblotting and signals were quantitated using imagestudio. results: here we demonstrate that the effect of dsg on ev release was reduced by the palmitoylation inhibitor -bromopalmitate. furthermore, mutations that prevented palmitoylation (dsg cacs) dramatically abrogated ev release by targeting of un-palmitoylated dsg to the lysosomes for degradation. dsg increased expression and subcellular localization of flot , a membrane lipid raft protein critical for membrane invagination. dsg also altered membrane localization of several early (eps and eea ), but not late (rab , rab , and hrs), endocytic pathway proteins. loss of palmitoylation in the dsg cacs mutants abrogated these effects. finally, dsg -induced ev release was abrogated by the sphingomyelinase inhibitor gw or augmented by the v-atpase inhibitor bafilomycin a . summary/conclusion: the combined results of the drug treatments and functional mutations of dsg suggest that dsg plays a critical role in ev biogenesis by modulating proteins involved in early endosome sorting and is dependent on post-translational palmitoylation. introduction: the translation initiation factor eif e ( e) is an oncogenic protein that is upregulated in % of cancers including a subgroup of acute myeloid leukaemia (aml) patients. eif e regulates post-transcriptional rna processing including the nuclear export and/or translation of mrna transcripts. in particular, it selectively increases the expression of genes that have a prominent role in cancer progression such as myc, cyclin d , and mcl . furthermore, our lab pioneered studies demonstrating that a subset of ehigh aml patients is clinically responsive to treatment with a e inhibitor (ribavirin) indicating the importance of e in aml progression and its relevance as a therapeutic target. we investigated an as yet unexplored perspective of e-whether the oncogenic role of e is in part mediated by its function as a master regulator of vesiculation. methods: to assess mrna export and identify ebound mrna targets that correspond to vesiculationrelated genes and associated cargo, we used cellular fractionation and rna immunoprecipitation techniques. to determine whether e regulates the number of extracellular vesicles (evs) released as well as their protein and rna cargo we used nanoparticle tracking analysis (nta) as well as mass spectrometry, antibody microarrays, and rna sequencing technologies. results: eif e upregulates cellular protein levels of the vesiculation marker cd by increasing its nuclear export. in addition to increased cellular expression, cd , cd , cd , and flotillin- proteins are elevated in evs released from e-high cells. this is also associated with an increased release of vesicles that are - nm in size. currently, we have validated the upregulation of several receptors and cytosolic proteins in evs isolated from e-overexpressing cells that function in cell growth, migration, invasion, and stemness. the most abundant rnas in our ev preparations are micrornas (mirs) and we have confirmed downregulation of several of these. summary/conclusion: our work shows that e reprograms the vesiculation of cancer cells changing the release and cargo of evs. this may impact cellular communication and tumour biology, which we are currently addressing in functional studies. we hope that these studies will highlight novel therapeutic strategies for aml patients. intranasal administration of neural stem cells-derived extracellular vesicles promotes neurogenesis and reduces neuroinflammation and amyloid plaques in a mouse model of alzheimer's disease introduction: cognitive and memory impairments worsen with time in alzheimer's disease (ad), likely due to a progressive loss of hippocampal neurogenesis, and escalation of neuroinflammation. these changes are also accompanied by increased deposition of amyloid plaques in the brain. methods: in this study, using the xfad mouse model, we examined the efficacy of extracellular vesicles (evs) shed from the rat subventricular zone neural stem cells (svz-nscs) for disease modification. we first purified evs from the rat svz-nsc cultures through ion-exchange chromatography and then administered intranasally to -months old xfad mice (~ billion/week for two weeks). two months later, the functional effects of ev treatment were quantified through a series of behavioural tests, and animals were euthanized for quantification of hippocampal neurogenesis, oxidative stress, neuroinflammation, and amyloid plaque deposition. results: in comparison to ad mice receiving vehicle, ad mice receiving nsc-evs displayed improved cognitive function to discern minor changes in the environment in an object location test, better spatial recognition memory in an object-in-place test, and improved pattern separation ability in a pattern separation test. besides, ev-treated ad mice displayed no anhedonia in a sucrose preference test. analyses of neurogenesis using the birth-dating marker ʹ-bromodeoxyuridine and the newly born neuron marker doublecortin revealed maintenance of a higher level of hippocampal neurogenesis in ad mice receiving evs, in comparison to vehicle-treated ad mice. moreover, analyses of brain tissues from ev-treated ad mice revealed decreased concentrations of oxidative stress markers malondialdehyde and protein carbonyls and elevated levels of antioxidants catalase and superoxide dismutase. also, the concentration of proinflammatory cytokines tumour necrosis factor-alpha and interleukin- beta and the extent of amyloid plaques were significantly reduced in ev treated ad mice. immunohistochemical analysis showed reduced hypertrophy of astrocytes. summary/conclusion: intranasal administration of nsc-derived evs restrains the deterioration of cognitive and mood dysfunction of ad by maintaining higher levels of neurogenesis and curtailing the progression of neuroinflammation. funding: supported by a grant from the national institute of neurological disorders and stroke ( r ns - to a.k.s.) ot . the case of mesenchymal stromal cells, opening new perspectives in the use of ipsc in regenerative medicine. the aim of this study is to evaluate the potential of ipsc-ev in the treatment of kidney disease. methods: the ipsc were generated from skin fibroblast after informed consent of healthy donors (cytotune®-ips . sendai reprogramming kit -protocol: clementino fraga filho uh . . . / . ). the ev were isolated from ipsc supernatants (cultured h in mtesr- medium) by ultracentrifugation ( , g for h at °c). characterization of ipsc-ev was performed using zetaview, tem, exoview™ tetraspanin kit and macsplex exosome kit. for in vitro injury, renal epithelial cells were cultured under hypoxia ( % o ). for in vivo injury, male wistar rats were submitted to bilateral renal arterial clamping ( min) followed by reperfusion without or with injection of ipsc-ev (protocol approval: federal university of rio de janeiro - / ). kidney damage was assessed by histological and immunohistochemistry analyses (pcna, tunel and ed- ). modulation of rna levels was assessed by rt profiler pcr array. results: the results show that ipsc-ev reduce renal cell death, tissue damage, macrophage infiltration, promote mitochondria protection and ameliorate renal function. the ipsc-ev mechanism of action is related to the regulation of key genes known to prevent damage caused by oxidative stress like gstk , sod , sod , txn and txnrd . characterization of ipsc-ev showed that ipsc-ev can carry important molecules that can support renal recovery as epcam and prominin- . summary/conclusion: ipsc-ev presents renoprotective properties, acting on different aspects of aki. this presents a new relevant application of ipsc as a source of ev for therapeutic purpose in kidney diseases. the hospital for sick children, toronto, canada introduction: incomplete lung development, also known as pulmonary hypoplasia (ph), is a recognized cause of neonatal death. we have previously shown that experimental ph can be rescued by the administration of extracellular vesicles derived from amniotic fluid stem cells (afsc-evs) through an rna-mediated mechanism. this effect was not observed with evs derived from mesenchymal stromal cells (msc-evs) . the aim of this study was to ) evaluate which rna species were responsible for ph rescue, and ) to define the mechanism behind this effect. methods: evs were isolated and characterized from conditioned medium of rat afscs and rat mscs (control group) using ultracentrifugation. evs were assessed for size (nanoparticle tracking analysis), morphology (tem), and expression of cd , hsp , flo- , and tsg (western). to identify the mediators of afsc-evs, we used deseq (fdr< . ) to differentially analyse rna from: a) asfc-ev and msc-ev cargo, isolated with seramir and sequenced with nextseq. b) lung epithelial cells from rat ph lungs treated with vehicle or afsc-evs. epithelial cell rna was isolated with mirvana and sequenced with nextseq. we correlated afsc-ev cargo mirna with validated mrna targets that were downregulated after ev conditioning in lung epithelial cells. results: of the rna species contained in asfc-ev and msc-ev cargo, mirnas were the most proportionally different between the two ev populations. afsc-evs were enriched for mirnas that are critical for lung development, such as mir ~ and their paralogues that control lung branching morphogenesis. afsc-ev administration to ph lung cells significantly downregulated genes, which formed mirna-mrna reported interactions. summary/conclusion: afsc-evs contain many rna species in their cargo, but mirnas are the main effectors of their ability to rescue underdeveloped foetal lungs. we have identified for the first time that afsc-ev biological effect on underdeveloped foetal lungs is in part due to the release of mir ~ cluster. funding: cihr-sickkids foundation grant. bottom-up assembly of fully-synthetic extracellular vesicles oskar staufer a , franziska dietrich a , jochen hernandez a , martin schröter a , sebastian fabritz b , heike böhm a , ilia platzman a and joachim spatz a a max planck institute for medical research, department for cellular biophysics, jahnstraße , heidelberg, germany, heidelberg, germany; b department for chemical biology, max planck institute for medical research, jahnstraße , heidelberg, germany, germany, germany introduction: extracellular vesicles (evs) are considered as key elements for future therapeutic and diagnostic procedures. however, despite enormous research efforts to understand their physiological relevance and several greatly successful clinical trials, evs are currently not authorized for clinical routines by american or european regulation and approval agencies. this is especially because therapeutic evs are produced or isolated from cell cultures or biofluids, both of which are subjected to batch-to-batch variations and ill-defined contaminations. therefore, complementary technologies that produce evs as reproducible and defined as state of the art nanotherapeutics, would revolutionize the application of evs in clinical settings and provide the scientific community with a holistic understanding of ev-mediated signalling processes. in our study, we achieve de novo bottom-up assembly of fully synthetic evs (fsevs) that comprise identical physiological and therapeutic functionalities to natural evs. methods: we applied droplet-based microfluidic synthesis to sequentially amalgamate synthetic lipids, proteins and nucleic acids into defined vesicles that display analogous therapeutic capabilities to natural evs. fsevs were characterized by electron and confocal microscopy, dynamic light scattering and mass spectrometry and tested on organotypic models or in vivo. results: using previously described evs as "naturegiven" blueprints, we assembled several fsevs in their exact molecular composition. in particular, we produced wound-healing promoting evs composed of several exosomal proteins, lipids and micrornas and showed that their therapeutic performance on human skin wounds is equivalent to that of natural evs. besides their high molecular complexity, being composed of dozens of different molecular building-blocks, the presented fsevs are completely defined on a quantitative level. based on this, we achieved a stoichiometric understanding of cell-vesicle interactions. summary/conclusion: by applying bottom-up synthesis of fsevs for quantitative studies on ev signalling, we not only provide innovative and safe compounds for ev-therapeutics but also a vastly new perspective on the application spectrum of extracellular vesicles in fundamental research. introduction: small extracellular vesicles (sevs) contain functional molecules from their cell of origin and can enter recipient cells for intercellular communication. ifnβ has been shown to induce some lncrnas to regulate host immune response and play a major role in the positive regulation of the activity of natural killer (nk) cells. here, we aim to clarify whether ifnβ induced sevs can regulate the cytotoxicity of nk cells by transferring specific lncrnas into nk cells. methods: evs were purified from a with/without ifnβ treatment by serial centrifugation followed by sucrose density gradient purification. elisa assay were performed to demonstrate the cytotoxicity of nk cells. qpcr and western blot were used to verify the expression of nkp . results: surprisingly, ifnβ induced sevs can strengthen the cytotoxicity of nk cells. through human transcriptome array (hta) we found the expression levels of lncrnas were significantly changed within sevs isolated from a cells following ifnβ treatment. additionally we found a specific sev cargo, linc-epha - , acted as a competing endogenous rna (cerna) for hsa-mir- which subsequently up-regulate the natural cytotoxicity receptor (nkp ) expression. furthermore, we verified over-expression of linc-epha - significantly enhance the cytotoxicity of nk cells against zika virus-infected a cells. summary/conclusion: our results demonstrated that ifnβ-induced linc-epha - wrapped in sevs can regulate the cytotoxicity of nk cells. our study provides a novel link between type i ifn and nk cells, which are two major players for the host innate immunity against pathogen infections. introduction: hiv-infected t cells release simultaneously viral particles and small extracellular vesicles (sevs) including mvb-derived exosomes and plasma membrane-derived evs. sevs and hiv share many physical and chemical characteristics, which makes their separation difficult. although several approaches have been used to obtain sevs free of virus they leave a majority of sevs within hiv preparations. for this reason, the function of sevs during hiv infection remains unclear. methods: we have developed a novel un-biased proteomic profiling approach to identify specific markers of the virus or sev subtypes released by a human t lymphoma cell line. our approach was to combine differential centrifugation of medium/small evs contained in the ccm with quantitative mass spectrometry to generate protein abundance profiles across the different sub-fractions. we generated an interactive database to define groups of proteins with similar profiles, suggesting their release in the same evs. results: we thus identified different categories of evs, which bear different surface proteins, e.g. different combinations of t cell surface markers, integrins or tetraspanins. in evs released by infected cells, we identified cellular proteins behaving like hiv proteins, and several that changed behaviour after infection, either moving towards or away from the hiv cluster. we identified two cell-derived proteins that are included in the viral particles and one that is specific of non-viral sevs that are modified by infection, and analysed their respective roles in controlling ev composition or virus infectivity. summary/conclusion: our approach presents a powerful tool for identification of common cargoes of given ev subtypes, and could be now used to identify modifications of ev composition in any given physiological or pathological situation. the encephalomyocarditis virus leader modulates autophagic pathways to promote the release of virions inside extracellular vesicles introduction: recent data indicate that naked viruses belonging to the picornaviridae family can be released from host cells via enclosure in extracellular vesicles (ev). ev cloak virus particles in a host-derived "envelope" and can thereby affect antiviral immune responses and disease severity. a better understanding of the formation and function of ev-enclosed viruses is therefore required. previously, we showed the presence of the autophagosome marker lc in ev isolates from encephalomyocarditis virus (emcv) infected cells, suggesting the involvement of a secretory autophagy pathway in ev-mediated virus release. however, little is known about the viral and host factors that regulate this process. here, we have assessed the role of the emcv leader, a viral protein that is dispensable for replication but is required for symptomatic disease. methods: cells were infected with wildtype virus or a mutant carrying an inactive leader. ev produced during the infection were isolated using differential ultracentrifugation and density gradient purification. ev were characterized by high resolution flow cytometry and their infectivity determined using end-point dilution assay. in addition, the fate of autophagosomes in infected cells was monitored using a reporter assay for autophagosome-lysosome fusion and analysis of the secretion of autophagosomal proteins. results: inactivation of the emcv leader strongly reduced the release of ev-enclosed virus. whereas autophagosomes are typically degraded, we show this is blocked by the leader. instead, autophagosomes fuse with the plasma membrane, as indicated by the secretion of autophagy marker lc during infection with wildtype but not the mutant virus. pharmacological reactivation of degradative autophagy in infected cells resulted in a strong reduction in the release of ev and ev-enclosed virus. similarly, the reduction in evenclosed virus release in the absence of the leader could be partially reversed by drugs that promote the secretion of autophagosomes. summary/conclusion: our data supports a role for secretory autophagy in the release of viruses in ev, a pathway that is regulated by the emcv leader. these findings highlight an unconventional route for ev formation that intersects with autophagosomal compartments and contributes to viral pathogenesis. introduction: zika virus (zikv) causes a public health emergency of international concern because of its correlation with microcephaly. during viral infection, the innate immune response quickly to produce some endogenous functional molecules which can prevent viral invasion or replication. extracellular vesicles (evs) contain molecules from their cell of origin under virus infection and can enter recipient cells for intercellular communication. here, we aim to clarify whether zikv induced evs can regulate viral pathogenicity by transferring specific rna. methods: evs were purified from a with/without zikv infection by serial centrifugation followed by sucrose density gradient purification. human transcriptome array (hta) was used to found rna expression within evs. flow cytometry was used to determine cell cycles. zikv replication was assayed by qpcr and western blot. flow cytometry was used to determine cell cycles. results: through hta we found the defensin alpha b (defa b) expression was significantly increased within evs isolated from zikv infected a cells. additionally, we found that the extracellular defa b but not the intracellular defa b exerts anti-zikv effect mainly before entry step. surprisingly, up-regulate defa b can retard cell cycles of host cell. we verified defa b could bind with the origin recognition complex (orc ) which is required to start dna replication during the cell cycle. furthermore, up-regulate defa b decreased the orc level in nuclear. interestingly, evs with defa b can internalize into recipient cells and inhibit their cell cycles. summary/conclusion: together, our results demonstrated that zikv infection can induce defa b wrapped in evs, and defa b not only exerts anti-zikv effect but also regulate cell cycles which may affect neurodevelopment. our study provides a novel viewpoint that defa b act as first-line anti-viral molecules during zikv infection also correlate with neurodevelopment by retarding cell cycles. extracellular vesicles mediate bacterial-immune cell interactions during respiratory viral-bacterial co-infections sidney w. lane a , matthew hendricks b and jennifer bomberger a a university of pittsburgh, pittsburgh, usa; b university of washington, seattle, usa introduction: respiratory infections are a major cause of morbidity and mortality worldwide and host-derived extracellular vesicles (evs) play important roles in mediating these infections. during respiratory infection, evs are shown to have a modulatory effect: promoting or suppressing infection dependent on the pathogen and cell type. in the age of next-generation sequencing, we now appreciate that many respiratory infections are polymicrobial in nature, with viral-bacterial co-infections correlating with worse disease outcomes. epidemiological studies correlate acute viral infections with the increased likelihood and severity of both acute and chronic secondary bacterial infections; however, the exact mechanisms of these interactions remain poorly understood. evs have been understudied in the context of respiratory viral-bacterial coinfections; thus, their role in mediating these infections is relatively unknown. unpublished data from the lab shows that in airway epithelial cells (aecs), viral infection induces the release of evs that associate with pseudomonas aeruginosa (pa) and promote biofilm growth. here, we aim to expand upon these findings and determine how aec evs mediate pa-immune cell interactions during respiratory viral-bacterial co-infection. methods: to determine how exposure to evs impacts pa-immune cell interactions, evs were isolated from the apical secretions of aecs and co-cultured with pa. ev-treated pa was then co-cultured with macrophages to evaluate ev impact on pa uptake and survival. results: in preliminary experiments using control evs, we observed that evs associate with pa. interestingly, during co-culture with macrophages, ev-treated pa are more susceptible to phagocytosis in comparison to non-treated pa. however, after hours of co-culture with macrophages, ev-treated pa are able to survive and replicate, while nontreated pa are effectively controlled by the macrophages. summary/conclusion: these findings suggest that while pa-ev association promotes pa uptake, it may ultimately enhance pa immune evasion and survival. ongoing experiments in the lab are evaluating the mechanism of pa-ev association and how evs from virus-infected aecs affect the phenotypes observed with control evs. notably, this is one of few reports of a mammalian ev influencing the pathogenesis of a bacterium; thus, results from these experiments will define the function of aec evs in regulating bacterial-immune cell interactions during respiratory co-infections. using machine learning with neuronal ev target proteins and clinical data to predict cognitive impairment in hiv infection lynn pulliam a , michael liston b , bing sun c and jared narvid d a university of california, san francisco, san francisco, usa; b veteran affairs, san francisco, usa; c ncire, san francisco, usa; d ucsf, san francisco, usa introduction: objective biomarkers are needed to assess and predict neuronal function and cognitive impairment. in people ageing with chronic infections, such as hiv, determining the mechanism of impairment will be important when therapies are available. methods: sixty plasma samples from hiv-infected people were obtained from nih-sponsored aids banks. clinical and epidemiological data were collected. all underwent neuropsychological testing and were considered impaired. neuronal extracellular vesicles (nevs) were isolated from plasma and assayed for high-mobility group box (hmgb ), neurofilament (nf-l) and phosphorylated tau- (p-tau) proteins. results: using different algorithms, support vector machines (svm) performed the best with an area under the curve (auc) value of . ± . . using different combinations of clinical data and the nev protein targets, selected clinical data and hmgb best predicted cognitive impairment (auc = . ). the most important features included cd count, hmgb , nf-l and education. summary/conclusion: specific clinical features plus nev hmgb , an inflammatory marker, were the best predictors of cognitive impairment. previous published data showed nev p-tau- elevated in alzheimer's disease and in this study p-tau had no importance in assessing hiv-associated cognitive impairment. nev target discovery can be improved to better identify neuronal damage, possibly to differentiate other neurodegenerative diseases and hopefully recovery after therapies are identified. in recent years, we have been able to separate and characterize extracellular vesicles (evs) from several different viruses including hiv- , htlv- , rift valley fever virus and ebolavirus. however, to date it is not clear whether there is a timing difference between ev and virus release from infected cells. methods: ev isolation by nanoparticle capture and differential centrifugation, ev quantification by nanoparticle tracking analysis, western blot, rt-qpcr, virus rescue assay. results: we have attempted to address the kinetics of ev and virus release from multiple-infected cells using serum starvation experiments from infected ( %) cells. these infected cells were initially put in g quiescent stage using serum starvation. both supernatants and cell pellets were collected postinduction release ( % fbs + pma/pha) at , , , , and hours and examined for the presence of ev, autophagy and viral proteins as well as viral rna expression. results from supernatants of uninfected cells showed a peak of tetraspanin proteins (cd , cd , and cd ) at hours and a gradual decrease of all ev associated proteins by hours. however, the ev from hiv- infected cells showed all three tetraspanins present at hours and expression gradually increased up to hours. when compared to htlv- infected cells, the three tetraspanin proteins peaked at hours and expression continued to decrease up to hours. htlv- infected cells also showed a unique pattern of cd expression. autophagy associated proteins (lc a, lc b and p ) from uninfected cells and htlv- infected cells plateaued at hours, whereas in hiv- infected cells their expression continued to increase and peaked at hours. hiv- viral proteins (p , gp , nef) expression was present at hours and continued to increase and peaked at hours. htlv- proteins (p and gp / ) peaked at hours and gradually decreased overtime. hiv- and htlv- rna gene expression analysis was performed, and data correlated with viral protein expression. additionally, evs release was quantified and showed significant increase of ev concentration overtime in both uninfected and infected samples. finally, experiments of infectivity from -and hour supernatants were performed on three naive cells. hiv- supernatant -hour sample was found not to be infectious. however, hiv- was successfully rescued from -hour sample. introduction: urinary extracellular vesicles (uevs) are important intercellular communicators. by systems biology integration, uevs prove to be relevant in genitourinary disease detection. however, it has recently been shown that labelled evs administered to the circulation can be detected in the urinary system, as well. thus, this pilot study aimed at phenotyping haematopoietic surface markers on uevs to create enough plausibility for future non-invasive biomarker studies of circulation and immune disorders that may translate into urine but are not yet timely recognized. methods: urine was obtained from healthy men signing a written informed consent (n = ). sampling was approved by the local ethics committee and in compliance with the declaration of helsinki. cell-free urine was obtained by serial centrifugation and ml, each, were utilized for the macsplex exosome kit, human (miltenyi biotec). the manufacturer's recommendations were followed to examine distinct uev surface markers of cd +/cd +/cd + vesicles in a multiplexed bead-based manner including respective controls. the accuri c (bd) was utilized for data acquisition. for further misev -compliant characterization, cd +/cd +/cd + uevs were isolated by immunoaffinity and analysed by fluorescence nanoparticle tracking (f-nta), transmission electron microscopy (tem) and western blotting (wb). urinary creatinine (ucrea) was determined to control for variances in urinary dilutions and used for data normalization. results: except cd , all other surface markers could be identified. the most abundant markers were cd and cd , which were detected in % of samples, followed by cd / ( %), cd ( %), cd and cd ( %, each). cd ( %), cd , cd ( %), cd e ( %) and cd showed similar relative median fluorescent intensities (rmfi), while cd yielded significantly higher (p = . ) and all other markers significantly lower rmfi (p < . ). tem and f-nta revealed cup-shaped vesicles ( ± nm) with . ± . e + particles/g ucrea. wb indicated uev isolates that were positive for alix, syntenin, tsg , cd , cd and cd without any uromodulin or calnexin contamination. summary/conclusion: our results imply that considerable quantities of circulatory evs are, indeed, filtered into urine and could serve as valuable non-invasive biomarkers for systemic dysfunctions. cardiovascular risk markers are strongly related to numbers of circulating extracellular vesicles ruihan zhou a ,esra bozbas a , plinio ferreira b and parveen yaqoob a a university of reading, reading, uk; b imperial college london, london, uk introduction: extracellular vesicles (evs) are small plasma membrane-derived vesicles released from various cells, which potentially affect many physiological and pathophysiological processes, and are emerging as a potential novel biomarker in cardiovascular diseases (cvds). however, there is little information about the association of circulating ev levels with traditional cardiovascular risk markers and cvd risk score. methods: • subjects (n = ) aged - yrs with moderate risk of cvds were recruited and assessed for body mass index (bmi), blood pressure (bp) and plasma lipid profile (triacylglycerol, total cholesterol and high-density lipoprotein). • evs were isolated from platelet-free plasma by size exclusion chromatography and analysed by both nanoparticle tracking analysis (nta) and flow cytometry (fcm). nta was used to measure the concentration and size distribution of evs population, and evs were phenotyped by fcm via a -colour panel, which included annexin v (for the majority of circulating evs), cd (for platelet-derived evs) and cd (for endothelial-derived evs). • the association between risk markers and ev numbers was examined by pearson's correlation coefficient and stepwise multivariate regression model. analysis of covariance (ancova) was performed after adjustment for various variables to determine the correlation between the quartile range of ev numbers and -yr cvd risk detected by qrisk . results: ev numbers, as determined by nta, were strongly associated with bmi (r = . , p < . ), blood pressure (systolic bp: r = . , p = . ; diastolic bp: r = . , p < . ) and plasma triacylglycerol levels (r = . , p < . ). plasma total cholesterol level was positively associated with platelet-derived evs, determined by fcm (r = . , p = . ). a multivariate regression model demonstrated that plasma triacylglycerol and diastolic bp independently predicted total ev numbers, with plasma triacylglycerol concentrations explaining . % of the variance for total ev numbers. an additional . % of the variance in total ev numbers was predicted by diastolic bp. ancova of the -yr cvd risk score in the quartile range of total ev numbers were positively and independently associated. summary/conclusion: bmi, blood pressure, plasma triacylglycerol and total cholesterol levels are strongly associated with ev numbers. plasma triacylglycerol and diastolic bp independently predict circulating ev numbers. elevated numbers of evs are independently associated with -yr cvd risk. introduction: extracellular vesicles from cardiospherederived cells (cdc-evs) are known to be anti-inflammatory in various disease models. to further dissect the mechanism, we examined the effects of cdc-evs on t lymphocytes. methods: naïve cd + t cells were isolated from secondary lymphoid organs of foxp -rfp reporter mice, using magnetic-activated and fluorescence-activated cell sorting. cells were subsequently polarized into effector subtypes (th , th , and th ), as well as regulatory t cells (tregs), and the effects of exposure to human-derived cdc-evs on proliferation and cytokine production were assessed. cdc-evs were isolated from serum-free, -day conditioned medium, using ultrafiltration by centrifugation. results: after polarization and culture for days, cdc-evs resulted in dose-dependent and cell-specific proliferative responses. effector t cells (th , th , th ) showed either no change in proliferation (th ) or decrease in proliferation (th , th ), compared to the vehicle control. in contrast, tregs proliferated much more than control (p < . ). next, we sought to characterize the changes in cytokine production by each effector t cell and tregs. compared to the vehicle control, exposure of polarized effector t cells to cdc-evs had little effect on the expression of characteristic cytokine genes, including ifnγ and tnfα (th ), il and il (th ), or il a and il f (th ). in contrast, exposure of tregs to cdc-evs resulted in~ -fold increase in expression of il , a key paracrine agent utilized by tregs in suppression of inflammation. this response was specific to cdc-evs insofar as it was not recapitulated with dermal fibroblast exosomes. concentrations of il- in the culture media of cdc-ev-conditioned tregs mirrored the increases in gene expression. summary/conclusion: cdc-evs potentiate tregs by increasing their proliferation and enhancing production of il- . this offers an attractive therapeutic approach to inflammatory diseases that relies on harnessing an endogenous mechanism of immunosuppression. funding: nih t hl . prostanoids impair platelet reactivity, thrombus formation and platelet extracellular vesicle release in patients with pulmonary arterial hypertension aleksandra gąsecka a , marta banaszkiewicz b , rienk nieuwland c , edwin van der pol d , najat hajji e , hubert mutwil f , sylwester rogula a , wiktoria rutkowska a , szymon darocha g , grzegorz opolski a , krzysztof j. filipiak f , adam torbicki g and marcin kurzyna g introduction: prostanoids (epoprostenol, treprostinil and iloprost) induce vasodilation in advanced pulmonary arterial hypertension (pah) but also inhibit platelet activation, thereby increasing the risk of bleeding. therefore, the platelet function and extracellular vesicle (ev) concentrations were measured in pah patients treated with prostanoids and compared to patients with pah not receiving prostanoids. methods: venous blood was collected from patients treated with prostanoids (study group; n = , ± years, % female) and patients not treated with prostanoids (control group; n = , ± years, % female). platelet reactivity was analysed in whole blood by impedance aggregometry using arachidonic acid (aa; . mm), adenosine diphosphate (adp; . µm) and thrombin receptor-activating peptide (trap; µm) as agonists. in a subset of patients, concentrations of evs from platelets (cd + and cd p+; pevs), leukocytes (cd +, levs) and endothelial cells (cd +, eevs) were measured in plateletdepleted plasma by flow cytometry (a -micro). platelet-rich thrombus formation was measured using a whole blood perfusion system. results: compared to the control group, patients treated with prostanoids had lower platelet reactivity in response to aa and adp (p = . ) and lower concentrations of pevs and levs (p ≤ . ). furthermore, thrombus formation was delayed (p ≤ . ) and thrombus size was decreased (p = . ) on prostanoids. epoprostenol did not affect platelet reactivity in vitro, but decreased the concentrations of cd + pevs (p = . ). in contrast, treprostinil and iloprost decreased both platelet reactivity in response to aa and adp (p ≤ . ) and the concentrations of pevs (p ≤ . ). all prostanoids delayed thrombus formation and decreased thrombus size (p ≤ . ). introduction: progressive lung disease is the leading cause of mortality in cystic fibrosis (cf), a chronic condition characterized by recruitment of polymorphonuclear neutrophils (pmns) into the airways. newly arrived pmns are exposed to extracellular vesicles (evs) from the airway epithelium and pmns recruited before them. in controlled experiments, these evs were necessary and sufficient to induce pathological changes including reduced bacterial killing and immunosuppressive activities towards macrophages and t-cells. however, children with cf do not always show a high pmn presence in their airways, which suggests that the balance between pmn recruitment and the activity of other cells is still in flux in early stage disease. methods: we utilized spectral nanoflow cytometry to profile the single ev content of the bronchoalveolar lavage fluid (balf) from cf children (< years of age). for nanoflow cytometry, evs were stained with di- -anepps, and with epcam, cd b and cd (to ascertain epithelial, pmn, and macrophage origins, respectively). violet side scatter and/or fluorescence threshold triggering were used for ev detection. results: the ratio of neutrophil-to epithelial-derived evs in cf balf correlated positively with the percentage of pmns that are present in the airways (p = . , spearman's rho = . ). this ratio also correlated with the pragma disease score, which quantifies airway damage by chest computed tomography (p = . , rho = . ). summary/conclusion: using a method to quantify evs from specific cell types in vivo, we demonstrated that the ratio of pmn-and epithelial cell-derived evs tracks with airway damage and neutrophil influx, suggesting a critical interplay between these cells in early cf disease. this ev-focused method can be applied to other diseases in which sampling cells is difficult. future experiments will use cf balf biobanks to strengthen data presented here. funding: cf foundation (tirouv a ), emory pediatrics flow core. the potential of crude extracellular vesicle micrornas for the diagnosis of community-acquired pneumonia and for the detection of pneumoniarelated sepsis as a severe secondary complication introduction: circulating cell-free micrornas (mirnas), often associated to extracellular vesicles (evs), are essential for cell-cell communication in the pathogenesis of infectious pulmonary disorders. as early pneumonia diagnosis is often clinically challenging, advances in disease detection could improve outcomes. we characterized crude ev mirnas as potential biomarkers for community-acquired pneumonia and sepsis as a severe secondary complication. methods: individuals were enrolled into our study, subdivided into a training (volunteer n = , pneumonia n = , sepsis n = ) and testing cohort (volunteer n = , pneumonia n = , sepsis n = ). after precipitating crude evs from sera (mircury exosome isolation kit-serum and plasma) and extracting total rna, small rna sequencing was performed. mirnas were selected as biomarker candidates by differential gene expression analysis (deseq ) and sparse partial-least-squares discriminant analysis (mixomics). technical and biological validation was performed by reverse transcription quantitative realtime pcr. group classification was predicted by partial-least-squares discriminant analysis. gene targets and causal networks were identified by ingenuity pathway analysis. results: differential gene expression analysis revealed significantly regulated mirnas in pneumonia compared to volunteers, and mirnas in pneumonia related to sepsis. based on sparse-partial least discriminant analysis, group separation was achieved by mirnas as discriminators with high sensitivity and specificity (area under the curve of the receiver operated curve: volunteer: . , pneumonia: . , sepsis: . ). mir- a- p (log fc = . , padj = . e- ) and mir- - p (log fc = . , padj = . e- ) differentiated between pneumonia and volunteers and mir- (log fc = − . , padj = . e- ) between pneumonia and sepsis. expression levels of mir- a- p and mir- were related to disease severity. mir- - p was higher expressed in pneumonia compared to volunteers and had equal expression in patient groups. prediction of group classification in the testing cohort was . %. signalling networks were constructed for "cellular and humoral immune response", "antimicrobial response" and "pathogen influenced signaling" involving the significantly regulated mirnas. summary/conclusion: crude ev mirnas are potentially novel biomarkers for community-acquired pneumonia and may help to identify patients at risk for progress to sepsis allowing early intervention and treatment. introduction: it remains unclear the specific mechanisms that lead to airways inflammation in asthma and the subsequent remodelling of the airways. exosomes, small extracellular vesicles, has become in an important mechanism of cell-to-cell communication and participate in diverse biological processes including inflammation. in this study, we hypothesize that exosomes and their mirna cargo play an important role in the proinflammatory status of the upper airway of asthma patients, especially in those patients with severe asthma. methods: in a pilot study, healthy subjects had induced sputum using standard methods. after several centrifugation steps, we were able to isolate exosomes from sputum supernatant by both precipitation and size exclusion cromatography (sec). exosome size was observed with transmission electron microscopy (tem) and the protein markers cd and cd were analysed by western blot (wb). then, total rnas were isolated from sputum exosomes and mirnas (mir- a-p, mir- - p, mir- a, mir- b- p, mir- - p, mir- - p, mir- - p, mir- - p, let- g- p) , were evaluated by rt-qpcr. after the optimization of the methodology, healthy adults subjects and patients with persistent moderatesevere asthma, matched by age and sex were selected and induced sputum was collected. results: exosomes isolated with both methodologies (precipitation and sec) were observe under the tem with a correct range of size. furthermore, wb assay displayed a coherent protein profile for the exosome markers cd and cd . however, sec displayed low signal and the variability of between subjects was to higher. using the optimized method of precipitation, we observed that after normalization, mirna- a showed a significant increased (p = . ) in asthma patients compared to control. this mirna has been linked with an active proinflammatory status. summary/conclusion: our results confirm the presence of exosomes in induced sputum with promising applications in the field of asthma. the upregulation of exosomal mir- a, which is related with inflammation, suggest that exosomes could play a crucial role in the chronic inflammation of airway described in asthma patients. human nrf -active multipotent stromal cell exosomes reverse pathologic delay in the healing of cutaneous diabetic wounds joseph kuhn a , absara hassan b , sonali sharma b , jennifer kwong b , montaha rahman b , salma adam b , jasmine lee b , alvaro villarreal ponce b and piul rabbani b a nyu langone health, new york, usa; b nyu langone health, new york, usa introduction: multipotent stromal cells (mscs) have attracted much attention for their capacity to accelerate wound healing. exosomes, nanosized extracellular vesicles, may be key to translating msc therapy. we previously found that nuclear factor erythroid -related factor (nrf ) regulates msc promotion of diabetic tissue repair. here, we explore a novel role of nrf in exosome biogenesis and investigate whether exosome treatment recapitulates the effects mscs have on healing. methods: exosomes were harvested by differential ultracentrifugation of conditioned bone marrow derived msc media. for nrf -active exosomes, mscs were incubated with potent nrf activator, cddo-im. exosomes and mscs were vigorously characterized. full-thickness humanized-stented wounds were created on adult leprdb/db diabetic mice (db/db). exosomes were injected intradermally and circumferentially to the wound margin. results: mscs adopt an adherent fibroblast morphology, demonstrate robust osteogenic, chondrogenic, and adipogenic differentiation, express > % positive msc markers (cd , cd , cd , and cd ) and < % express negative markers (cd , cd , cd , cd , or hla-dr). immunoblotting of msc exosomes shows enrichment for positive exosomal markers cd , cd and tsg . nanoparticle tracking analysis (nta) shows a nanoparticle population with mean diameter of . ± . nm. transmission electron microscopy of exosomes reveals flattened cup-like spheres. nta demonstrates that nrf -active human mscs increase exosome secretion by %, compared to nrf -baseline mscs (p < . ). both nrf -baseline and nrf -active exosome treatment significantly reduced closure time to . and days respectively, compared to . days for vehicle-treated wounds (p < . ). this reduction eliminated the delay in closure time compared to wounds of c /b mice. nrf -active exosome treatment of db/db wounds reduced closure time by a further . days compared to untreated c /b wounds. at day , exosometreated db/db wounds have significant decreases in epithelial gap, expanded granulation tissue, and greater density of cd + vessels compared to vehicle-treated wounds. introduction: obesity increases prostate cancer aggressiveness and adipose tissue (at) is a rich source of extracellular vesicles (ev) that have been shown to contribute to pro-oncogenic effects in various malignancies. twist is a key mediator of tumour cell metastasis.. the goal of this study was to determine molecular and phenotypic changes of prostate cancer cells in response to evs from obese human at and the role of different levels of endogenous twist . methods: ev were harvested from human at (atev) obtained from bariatric subjects or from at endothelial cells treated with proinflammatory cytokines (pic-ev) to mimic the obese at environment. evs were isolated by ultracentrifugation and characterized by electron microscopy, nta and protein markers. we determined the effect of atev and pic-ev on pc -ml prostate cancer cells proliferation and invasion. ev mirna cargo and transcriptome of pc -ml cells treated with atev or pic-ev were assessed using nanostring. to establish the contribution of twist to the ev-related phenotypic and molecular changes in recipient cells, we used pc -ml lines stably overexpressing or deficient in twist . results: atev from obese subjects and ev-pic from at endothelial cells both reduced invasion and increased proliferation in wild-type pc -ml cells. a molecular signature showing decreased expression of genes mediating invasion, adhesion and metabolism supported these functional effects. also atev and ev-pic shared a subset of mirna that target multiple mmps, inhibit glycolytic genes and target cell cycle inhibitory genes. pc -ml overexpressing twist showed an increase in both proliferation and invasiveness and this phenotype was supported by the transcriptomic analysis following ev treatment. summary/conclusion: ev produced by obese at or by at endothelial cells share a subset of mirna that in conjunction with increased twist expression contribute to tumorigenesis and metastasis of prostate cancer cells in vitro. funding: american heart association of symposium introduction: as researchers continue to explore the therapeutic potentials of extracellular vesicles (evs) for the treatment of many diseases, there is a growing unmet need for real-time in vivo monitoring of these therapeutic evs after they are injected into a subject to understand their safety, targeting, and effectiveness. while current optical imaging solutions like bioluminescence and fluorescence are useful for ev tracking studies in animal models, there is limited utility in clinical applications. here we present a novel ev tracking solution utilizing clinically applicable mri technology. methods: to generate trackable evs, cells were labelled with a clinically applicable novel magnetic agent. evs secreted by the labelled neural stem cells and amniotic fluid stem cells (afscs) were isolated by differential ultracentrifugation. the viability and morphology of labelled-cells were evaluated, and the in vitro mr properties of their derived evs were analysed by magnetometer. a proof of concept in vivo biodistribution study was conducted by injecting labelled evs into wt and alport mice (a model of chronic kidney disease) via retro-orbital and intra-cardiac routes and tracking them via mri at min and hr postinjection. results: the magnetic label did not affect the physiological characteristics of the cells. the mr detectability of labelled-evs was confirmed by in vitro/ex vivo mri phantoms. mri studies showed that homing of afsc evs to the kidney injected intra-cardiacally into alport mice were more efficient versus the retro-orbital route, and prussian blue staining of kidney sections confirmed the mr findings. introduction: a central question in ev biology is the fate of circulating ev. this can be evaluated by developing non-invasive ev bioimaging techniques in mice in order to benefit from transgenic and knock-out models. recent reports described ev biodistribution in vivo using optical (fluorescence) and nuclear imaging. but the physicochemical properties of the probes impact ev integrity, labelling efficiency, background signals and observation timecourse. methods: we developed the radiolabeling of red blood cells (rbc) and ev with [ f]fluorodeoxyglucose ( f-fdg). we used rbc-derived ev in their native, intact form, without pre-experimental processing (no centrifugation or filtration). we tracked f-fdg in vivo by pet-scan, within seconds of ev, rbc or free f-fdg injection, and during their dissemination in blood and recruitment by organs over one hour. ev and rbc biodistribution were confronted to the kinetics of free f-fdg. results: we collected images of the biodistribution of rbc, and rbc-derived ev. nuclear imaging was well suited for accurate studies of ev organotropism, with high sensitivity, excellent signal-to-noise ratio, very low signal absorption by tissues and an inherent quantitative tomographic nature. ev-specific signals were mostly accumulated within minutes of injection (tail vein), in the spleen and liver, with a small part in the bone marrow (femurs). signals in other compartments were largely transient and linked to tissue perfusion and blood volume. we selected the most drastic control conditions to secure a correct interpretation of the data. this made kidneys, hearts and brains unavailable for analysis. hence the new approach came with limitations, but we describe how "free" f-fdg signals can be used to draw sound conclusions for ev. summary/conclusion: we propose that three types of compartments coexist in control mice at rest: active ev-capturing organs with high capacity and specificity including the spleen, and to a lesser degree the bone marrow; passive ev-retaining organs with high capacity, including the liver; and ev-neutral organs where transient signals only mirror tissue perfusion. we also report how ev biodistribution patterns are altered in ageing animals, as an example. we hope that this novel, non-invasive, quantitative, dynamic wholebody imaging approach will help characterize native cell-derived ev and help set standards for the reproducibility of ev bioimaging in mice. funding: frm grant "biface", inserm copoc, cnrs. introduction: extracellular vesicles (ev) are important mediators of intercellular communication; however, basic principles of ev biogenesis and loading remain largely unknown. a limited repertoire of tools has thus far made these processes challenging to research. the development of an ev-transfer reporter in a genetically tractable organism such as drosophila has allowed us to study mechanisms of cargo loading in vivo and has provided us with a platform to explore fundamental aspects of ev biology. methods: we have developed a bioinformatic pipeline to analyse the properties embedded in the ʹutr of mrnas enriched in evs released by drosophila cells. in parallel, we have adapted a cre-loxp system for use in fruit flies that appears to be proficient to reveal the exchange of bioactive molecules between secretory and recipient cells. results: taking advantage of computational methods, we uncovered sequence motifs that preferentially appear in combinations along the ʹutr. these sequence motifs occur within characteristic secondary structures, in a way that is more variable and motif dependent than previously reported. identified motifs also show similarities to known binding sites for rna binding proteins; a feature potentially important for ev-loading. in parallel, we developed a drosophila in vivo system to detect cell communication in complex tissues and between different cell types. using this system, we studied the biological significance of specific sequence motifs and identified their ability to modulate mrna ev-transfer in a context dependent and evolutionarily conserved manner. summary/conclusion: in summary, we have developed a novel tool to study cell communication in complex tissues, and shown its effectiveness to study principles of ev biogenesis and loading. beyond improving our understanding of ev biology and providing a novel tool to the scientific community, we hope this knowledge will pave the way to harnessing evs as a means of remotely manipulating cell communication in many biological contexts. introduction: the idea of cross-kingdom, species and inter-individual transfer of bioactive compounds via extracellular vesicles (evs) is a recent avenue. however, the bioactivity and bioavailability of these dietary compounds upon consumption is highly debated. it has been proposed that evs from diet can absorbed by consuming organisms, be bioavailable in various organs and exert phenotypic changes. milk is the most vastly consumed beverage and is an abundant source of evs that may act as signalosomes. whether these milk-derived evs can serve as cross-species messengers and have a biological effect on host organism has been poorly understood. methods: bovine milk-derived evs were isolated by ultracentrifugation and optiprep density gradient centrifugation. the evs were characterised by tem, nta, quantitative proteomics and rna-seq. evs were orally administered to various mice models of colorectal, breast and pancreatic cancer. primary tumour burden was monitored, and the rate of metastases was measured by imaging and qpcr. immune cells were analysed by facs. mechanistic insights were obtained using quantitative proteomics, confocal microscopy and biochemical experiments. results: we demonstrated that upon oral administration, bovine milk-derived evs were able to survive the harsh degrading conditions of the gut and be bioavailable in peripheral tissues. interestingly, oral administration of milk-derived evs reduced the primary tumour burden in various cancer models and attenuated cancer cachexia. intriguingly, despite the reduction in primary tumour growth, milk-derived evs accelerated metastasis in breast and pancreatic cancer mice models. timing of ev administration was critical as oral administration after resection of the primary tumour reversed the pro-metastatic effects of milkderived evs in breast cancer. biochemical and quantitative proteomics analysis highlighted the induction of epithelial-to-mesenchymal transition and senescence upon treatment with milk-derived evs. summary/conclusion: taken together, we were able to demonstrate the capacity of bovine milk-derived evs in mediating cross-species communication and regulating cancer progression in a context-dependent manner. bacterial membrane vesicles (mvs)a bacterial innate defence system against viral infection xiaomei yan, qian niu and ye tian xiamen university, xiamen, china (people's republic) introduction: in order to survive the constant onslaught of phage, bacteria have evolved diverse defence mechanisms that act at every stage of the phage life cycle. it has been suggested that bacterial membrane vesicles (mvs) may play a key role in innate bacterial defence against phage infection by acting as a decoy to prevent phage adsorption. nearly a decade has passed since mvs were first proposed as a decoy, but details of how bacteria utilize mvs to defend against phages remain poorly understood. here we use the laboratory-built nano-flow cytometer (nfcm) to reveal details of the interaction between mvs and phages at the single-particle level, and to provide new insights into innate defence mechanisms of mvs. methods: s. typhimurium was used as the model system. differential ultracentrifugation and density gradient centrifugation were used to isolate and purify mvs and bacteriophage p . cryo-tem was used to determine the morphologies of mvs and phage p . the purity of mv isolates was validated by measuring the particle concentration before and after triton x- treatment. monodisperse silica nanoparticles were used as the size reference standards to measure the size distribution of mvs via single-particle light scattering detection. the purity of phage p was verified by concurrent detection of side scatter and fluorescence signals of single phages upon nucleic acid staining by syto . results: by incubating mvs and af -labelled p , the number of phages adsorbed on single mvs were accurately quantified. we found that s. typhimurium and mvs it secretes express different affinity for phage p attachment. the binding ability of p to mvs is greater than that of bacteria. we confirmed that p can inject their nucleic acids into mvs, and these nucleic acids can be degraded by non-specific nucleases inside mvs for the first time. besides, by labelling the nucleic acids of mvs with syto , we were able to distinguish three different subpopulations of mvs. summary/conclusion: taking advantage of the superior sensitivity of nfcm in single-particle analysis, we developed a novel approach to the characterization of the interaction between mvs and phages. our study revealed that bacteria produce mvs as bait to attract viral adsorption and nucleic acids injection. funding: this research was supported by the national natural science foundation of china (grants , and ). introduction: the development of evs for therapeutic applications requires an in-depth understanding of their in vivo biodistribution and pharmacokinetic profile. in this study, we have made a comprehensive comparison of nuclear, fluorescent, and bioluminescent imaging technologies to identify the most suitable in vivo ev tracking method. methods: evs were purified from expi f cell supernatant by differential centrifugation followed by iodixanol density gradient separation and further characterized following misev guidelines. engineered expi f cells were used to generate evs carrying mcherry or nanoluc (nluc) proteins. the membrane of naïve ev was labelled with indium (in )-dtpa or xenolight dir post-ev isolation. ct tumour-bearing balb/c mice were intravenously dosed with × evs followed by imaging at h, h and h using spec/ct and ivis systems. tissue distribution and blood circulation profile of evs were analysed from ex vivo samples up to h post-injection. results: xenolight dir and (in )-dtpa were the most suitable ev labels for live whole-body animal imaging, ex vivo organ imaging, and tissue lysate quantification. nluc was appropriate for ex vivo imaging and tissue lysates quantification, but suboptimal for live imaging with limited sensitivity. mcherry evs were found not suitable for in vivo tracking studies due to high background signal fluorescence. ex vivo organ quantification of in -dtpa and dir showed that naïve evs mainly accumulate in liver, followed by spleen, kidneys, and lungs at h post-dose, with less than % ev exposure to the tumours. interestingly, nluc-evs accumulated mainly in the lungs, regardless of the small size of the particles injected and the absence of aggregation. blood circulation profile of in -dtpa and nluc evs showed rapid clearance of vesicles from circulation, with % of injected dose detected in blood after min and less than % after h. summary/conclusion: radionuclide imaging is an excellent technology to detect evs in vivo and ex vivo with high resolution and sensitivity but requires advanced infrastructure for radiolabeling. the optical methods have limited tissue penetration and sensitivity but can be improved with the right selection of the dye. these results contribute to the understanding of the biodistribution and pharmacokinetics of evs and are highly relevant to exploiting their potential for targeted delivery to diseased tissues in vivo. symposium introduction: new methods for quantifying extracellular vesicles (evs) in complex biofluids are critically needed. we report the development of a new technology combining size exclusion chromatography (sec), a commonly used ev purification technique, with fluorescence detection of specifically labelled evs (flu-sec). methods: flu-sec was validated using red blood cell derived evs (revs). size and concentration measurements were performed by microfluidic resistive pulse sensing (mrps) using the ncs instrument (spectradyne llc, usa). pe-cd a (anti-glycophorin a) and alexa -wga (wheat germ agglutinin) were used to label revs. flu-sec experiments were performed on a liquid chromatography system using a tricorn / glass column filled with sepharose cl- b gel (ge healthcare). results: a log-normal size distribution was obtained for revs with a mean diameter of . ± . nm and standard deviation of . ± . nm. the concentration of revs measured by mrps was . * e particles/ ml. the fluorescence chromatograms of the rev samples labelled with pe-cd a and with alexa -wga show the typical features of the separation of evs from soluble proteins with sec and enables the determination of the labelling efficiency of the markers. the linear range for quantification of evs in our experiments spans over two orders of magnitude ranging from e particles/ml to e particles/ml. the lod depends on the type of the label. in our experiments the lowest lod was e particles/ml for alexa -wga. summary/conclusion: the results indicate that flu-sec is a quantitative technique with very good linearity over a wide range of concentrations, though the limit of detection depends largely on the employed label (sci. rep. , , ) . moreover, the ratio of ev-bound and free-antibody molecules can be also determined by flu-sec, which can be used to calculate the labelling efficiency of the used marker. funding: this work was supported by the national research, development and innovation office (hungary) under grant numbers pd and nvkp_ - - - . zv was supported by the janos bolyai research fellowship. the conan assay: purity grade and concentration of ev microlitre formulations by colloidal nanoplasmonics. (evs). control over such properties is constantly experienced by researchers to be critical for ev proper manipulation, engineering and translation. however, the need for characterization methods that strike the balance between robustness, working volume, cost and accessibility remains unmet. methods: the colorimetric nanoplasmonic (conan) assay we developed consists of a solution of gold nanoparticles (aunps) into which - μl of the ev formulation is added. the solution turns blue if the formulation is pure, while stays red if soluble exogenous single and aggregated proteins (saps) are present. the colour shift is visible by the naked eye and can be quantified by conventional uv-vis spectroscopy, providing a quantitative index of purity and an estimation the ev molar concentration (particle number). results: the assay specifically targets saps, and not the ev-related proteins, with a detection limit < ng/μl (an order of magnitude higher resolution than the bradford protein assay). for pure solutions, the assay also allows for determining the ev number, as the colour shift is linearly dependent to the aunp/ev molar ratio. instead, it automatically reports if the solution bears sap contaminants, thus avoiding counting artefacts. experiments, conducted on ev separated from milk and ascaris suum culture medium, are repeatable, with an error below %. summary/conclusion: conan proves to be robust and reliable, while displaying appealing performances in terms of cost (inexpensive reagents, run by standard microplate reader), working volumes ( - μl) and time (the procedure takes less than one hour). the ability to assign a quantitative purity grade is, up to date, a unique peculiarity of this assay. finally, the assay is potentially extendable to all classes of natural and artificial lipid micro-and nanoparticles. funding: evfoundry project, horizon -future and emerging technologies (h -fetopen), id: . marina cretich a , roberto frigerio b , alessandro strada b , greta bergamaschi b , marcella chiari c and alessandro gori c a consiglio nazionale delle ricerche (cnr), istituto di scienze e tecnologie chimiche (scitec), milano, italy; b consiglio nazionale delle ricerche (cnr); istituto di scienze e tecnologie chimiche (scitec), milano, italy; c consiglio nazionale delle ricerche (cnr); istituto di scienze e tecnologie chimiche (scitec), milano, italy introduction: small extracellular vesicles (sev) present fairly distinctive lipid membrane features in the extracellular environment. these include high curvature, lipid packing defects and a relative abundance in lipids such as phosphatidylserine and ceramide. sev membrane could be then considered as a "universal" marker, alternative or complementary to traditional characteristic surface-associated proteins. here we introduce the use of membrane sensing peptides as new, highly efficient ligands to directly integrate sev capturing and analysis on a microarray platform. methods: we designed and synthesized membranesensing peptide ligands as molecular baits for small ev and we demonstrate their use in a microarray platform as valuable alternative/complement to antibodies. evs from blood serum and plasma were isolated by ultracentrifugation, characterized by tem, nta, wb. samples were analysed by label-free, single particle counting and sizing on peptide microarrays coupled to fluorescence co-localization immune staining with fluorescent anti-cd /anti-cd /anti-cd antibodies. results: peptide microarrays were realized using a click-chemistry strategy for optimal peptide surface orientation and used to analyse evs from human blood. membrane sensing peptides showed a capturing capacity higher than anti-tetraspanin antibodies. in addition to purified vesicles, peptide ligands were tested with pure serum showing capacity to capture evs even from complex samples. in order to get insights into the ev-peptide binding mechanism and verify whether it is directly mediated by the lipid membrane, trypsin-treated evs were captured on peptide microarrays demonstrating that binding is not directly mediated by surface associated proteins. summary/conclusion: we introduced the use of membrane sensing peptides as a novel class of molecular ligands for integrated sev isolation and analysis, reporting for the first time on peptide microarrays for extracellular vesicles. given their affinity to the membrane of small ev, these molecules can serve as general baits, enabling vesicles capturing unbiased by differential surface protein expression. these new class of molecular probes may be integrated with the use of protein markers towards improved small ev isolation and characterization. compared to proteins and antibodies, peptides are characterized by low cost of preparation, remarkable stability and ease of chemical manipulation, offering virtually unlimited possibilities for experimental design. we anticipate that this new class of ligands, may greatly enrich the molecular toolbox for ev analysis. funding: hydrogex (regione lombardia&fondazione cariplo, grant n. - ) and index (european union's horizon research and innovation programme under grant agreement n° ) projects are acknowledged for partial financial support. high-resolution size-based profiling and morphological analysis of extracellular vesicles by scanning electron microscopy sara cavallaro a , federico pevere b , petra hååg c , kristina viktorsson c , rolf lewensohn c , jan linnros a and apurba dev b a kth royal institute of technology, stockholm, sweden; b uppsala university, uppsala, sweden; c karolinska institute, stockholm, sweden introduction: extracellular vesicles (evs) have been found to mediate intercellular communication in physiological and pathological processes. nevertheless, the understanding of evs bio-functionality remains elusive, mainly because of their high heterogeneity in molecular content, but also in size ( - nm) . therefore, accurate size measurements of evs are highly desired, particularly for exploiting their full diagnostic/therapeutic potential. currently available techniques, such as nanoparticle tracking analysis (nta), cannot accurately measure evs smaller than nm and are not capable to distinguish them from protein aggregates. on the contrary, electron microscopy (em) techniques allow high-resolution size-profiling and morphological analysis of evs over their whole size range. however, their low throughput combined with several long preparatory steps have prevented em from being routinely used for ev size profiling. methods: we shall present a method improvement in throughput and reproducibility of ev size-analysis by scanning em (sem). the technique is based on covalent ev capture onto a silicon wafer, using the protocol reported by cavallaro et al. up to the glutaraldehyde step. after immobilization, critical point drying (cpd) is performed to dehydrate evs before sem, while preserving their shapes. results: sem images, showing the comparison in densities of evs prepared by covalent and non-covalent coupling to substrate, indicated a good capture efficiency of our covalent protocol. the size distribution analysis showed good agreement between nta and sem for evs > nm. for smaller evs, sem is more sensitive than nta, thus more suitable to check the purity of ev-isolation techniques. last, atomic-force microscopy (afm) measurements was also used to validate our measurements. introduction: extracellular vesicles (evs) are membrane vesicles secreted into extracellular space, by almost all cellular populations, playing a major role in cell-to-cell communication. it has been already demonstrated that changes in luminal or surface protein cargos of these vesicles, may reflect the status of producing cells. for this reason, evs are considered as potential biomarkers in several types of diseases ranging from cancer diagnosis to heart rejection. periostin (postn) is a matricellular protein associated with evs, and its level is considered a possible biomarker, which indicate malignancy and poor clinical outcome in different types of cancer. here we extensively characterize the presence of postn associated on evs, showing how different isolation methods can drastically affect the amount of postn content in extracellular vesicles fraction. methods: serial ultracentrifugation steps or size exclusion chromatography were used to isolate evs from primary culture of cardiac progenitor cells. evs were characterize, according to misev guidelines, by western blot, nta, facs and cryotem analysis methods. postn amount, associated with evs, was analyses by western blot and elisa. furthermore, functional tests were performed on h c cardiomyoblast cell line, treated with the same amount of evs from different isolation methods; cells response were analysed by western blot. results: evs, from both the isolation methods, showed tsg , syntenin , cd positivity while grp was absent. nta showed no differences, in terms of amount and size of particles. by facs analysis evs resulted enriched with cd , cd and cd . cryotem showed a similar morphology in the two preparations with presence of protein contaminant in the ultracentrifuge pellet. in vitro, h c treated with evs showed activation of pfak after ʹ of treatment, this induction was . times higher in cells treated with evs isolated with ultracentrifuge compared to evs isolated with sec, confirming a drastic effect of postn protein contamination. furthermore, by phospholipase-c treatment, we found that postn is bound to evs surface through a gpi anchor. summary/conclusion: these results suggest that selection of a proper isolation method is critically relevant in evs studies, in particular when protein analysis is considered. different isolation methods dramatically influence protein amount in extracellular vesicles and consequentially their function. furthermore, in this study we show for the first time, that postn is actually bound to evs surface and not carried in their lumen as previously believed. members of the y-rna family have been detected in ev from various cell types and are among the most abundant non-coding rna types in plasma. we previously showed that shuttling of full-length y-rna into ev is modulated by tlr-activation of ev-producing immune cells. this suggested that y-rnas may have potential as biomarker for immune-related diseases. methods: we separated rna-containing structures in plasma based on differences in size, density, and resistance to protease/rnase treatment. using rt-qpcr, we quantified full-length y-rna subtypes (y , y , y ) in ev from various blood-related cell types cultured with or without lps-stimulation. inflammationinduced changes in y-rna were assessed in plasma samples from a human endotoxemia study. results: full-length y-rna in plasma was mainly found in ev (early sec-fractions, density . - . g/ml). in contrast, specific mirnas were either enriched in lpp (e.g. mir- ), in both ev and lpp (e.g. mir- and mir- ), or in ev (e.g. mir- ). evenclosed full-length y-rna was resistant to enzymatic degradation, while lpp-bound mirnas were degradation sensitive. we discovered that ev released by different blood cell types varied in y-rna subtype ratios. these ratios remained stable upon lps-stimulation of the ev-producing cells. in endotoxemia plasma samples, the neutrophil-specific y /y ratios and pbmcspecific y /y ratios changed significantly during systemic inflammation. importantly, the plasma y-rna ratios strongly correlated with the number and type of circulating immune cells during the inflammation process. summary/conclusion: cell type specific "y-rna signatures" in plasma ev can be determined without prior ev-enrichment, and may be further explored as biomarkers to diagnose inflammatory responses or other immune-related diseases. mining public ev small rna-seq data with mirev -insights into potential reference transcripts and abundant mirnas recently, extracellular membrane vesicle (mv) production has been proposed as a general secretion mechanism that could facilitate the delivery of functional bacterial nucleic acids into host cells. s. aureus produce membrane-bound, spherical, nano-sized, mvs packaged with a select array of bioactive macromolecules and they have been shown to play important roles in bacterial virulence and in immune modulation through the transmission of biologic signals to host cells. the present study sought to examine the nature of the association between nucleic acids and mvs produced by s. aureus. we also sought to analyse the immunostimulatory potential of mvassociated rna and dna, and to evaluate receptormediated recognition of mv-associated rna and dna molecules by innate immune cells. methods: by following a stringent purification protocol, we characterized the rna and dna content of mvs produced by actively growing s. aureus. nuclease protection assays were performed to determine whether mv-associated nucleic acids are protected from degradation. we assessed the immunomodulatory potential of mv-associated rna and dna by treating cultured mouse macrophages with mvs and measuring the induction of interferon-β mrna using qpcr. introduction: urinary extracellular vesicle (uev) transcriptome could potentially reflect the kidney gene expression profile and serve as virtual/liquid biopsy. in order to explore this possibility, we performed mrna sequencing of uevs from individuals with type diabetes to assess whether it can capture a "kidney enriched genes" expression signature that could lead to novel biomarker discovery for diabetic kidney disease. methods: the study included type diabetic individuals ( normoalbuminuric, microalbuminuric and macroalbuminuric). urine samples were collected either overnight (n = ) or during -hours (n = ) and uevs were isolated from - ml of urine by differential centrifugation. the evs quality was ensured by electron microscopy (em), western blotting and ev-rnasprofiling with the bioanalyzer. isolated rnas were subjected to rna sequencing after cdna library preparation (ultra-low amount protocols) using hiseq (illumina) pair-end protocol. the association between kidney specific gene expression levels (> fold higher compared to other tissues, n = ) and degree of albuminuria or glomerular filtration rate was explored. results: isolated ev quality appeared good by em and western blotting. rna quantity and quality were sufficient for sequencing of all samples with > million pair end reads. we detected on average expression of , genes. principal component analysis (pc) of the expression of all genes did not reveal any systematic batch differences between the overnight and -hour urine collections. comprehensive look-up of kidney-enriched genes revealed expression of > % (total ) of these genes in urine evs with high expression of five kidney-specific genes (slc a , slc a , nphs , aqp and slc a ). pc analysis combining the impact of kidney-enriched genes revealed that most macroalbuminuric patients clustered together along the pc axis, and the axis also correlated with the albumin-to-creatinine ratio (p = . ) explaining % of the variance (p = . ) in the whole data set. the pc axis also showed correlation with hba c (p = . ), but not with diabetes duration, bmi, age and egfr. introduction: due to their safety profile, tissue tropism and long-term transgene expression, adeno-associated viruses (aavs) have become the vector of choice for human gene therapy. however, pre-existing neutralizing antibodies (nabs) to many aav serotypes pose a critical challenge for the translation of gene therapies to clinic. here, we describe the use of exosomal aavs (eaav) as a robust cardiac gene delivery system that enhance transduction efficiency while shielding from pre-existing humoral immunity to the viral capsid. methods: we developed an ultracentrifugation-based purification strategy to obtain eaav specimens from aav-producing hek- t cells, and used electron microscopy-based visualization, confocal microscopybased colocalization studies, qpcr, immunoblotting, dynamic lights scattering, exoview technology and protease assays to characterize eaav morphology, contents and mechanism of action. we then evaluated efficiency of heart targeting for eaav or eaav and standard aav or aav encoding for egfp, mcherry or firefly luciferase in different human cell lines in vitro, in black mouse and in passive immunity nude mouse model in vivo using flow cytometry, confocal microscopy, langendorff perfusion system and methods: hlhs patients (n = ) after glenn procedure and swine (n = ) after pab were given rv injections of allogeneic/xenogeneic mscs. donor-specific, hla-i+, exosomes were isolated from plasma. in swine, exosomes were collected and rv fractional area change (fac) was measured post-msc-injection. in the elpis patients, exosomes were collected and outcome measurements (fac, stroke volume (sv), rv mass) were recorded and -months post-injection. exosomal mrna, microrna (mirna), and proteins were quantified and partial least squares regression (plsr) reduced the dimensionality of the datasets to build a swine model, upon which elpis outcome predictions were made. results: multiomics analysis of swine exosome cargo revealed mirna to be the largest contributor to overall variance. in swine and elpis patients, mirnas were similarly expressed ( %, fold-change< ). plsr reduced the dimensionality of the swine mirna dataset to mirnas with the highest weighted coefficients for changes in fac. pathway analysis of mirna targets revealed links to smooth muscle cell proliferation and cardiac chamber development. importantly, the swine mirna plsr model predicted elpis patient improvements in fac, sv, and rv mass with strong correlation (r > . ). summary/conclusion: these findings support the use of: ( ) swine pab model for rv failure in hlhs, ( ) circulating donor-specific msc-exosomal mirna as a novel, non-invasive biomarker of patient outcomes, and ( ) introduction: evs have been shown promising potential as a drug delivery vehicle, especially nucleic acid therapeutics. however, the overall short of specificity to target cancer cells has led to low therapeutic efficacy and potential toxicity. rna nanotechnology is the bottom-up self-assembly of nanometre-scale rna architectures. we previously discovered a stable phi prna three-way junction ( wj) motif and used it to construct multivalent rna nanoparticles with high chemical and thermodynamic stability. the resulting arrow-shape rna nanoparticles are homogenous, uniform in size and shape, and can harbour different functionalities while retaining their tertiary folding and independent functionalities both in vitro and in vivo. this flexible platform using rna nanotechnology to achieve tumour-specific targeting has been demonstrated over the last decade. here we introduce a strategy to take advantage of both evs and rna nanotechnology to develop a versatile platform for efficient target-specific delivery of sirnas for cancer treatment. methods: we design membrane-anchoring arrowtail wj rna nanoparticles to display tumour targeting ligand (psma rna aptamer or egfr rna aptamer or folate) on birc sirnas loaded evs (fig. ). nanoparticles were characterized by nanoparticles tracking analysis (nta), transmission electron microscopy (tem), dynamic light scattering (dls) and atomic force microscopy (afm). evs were produced by hollowfiber bioreactor and purify by tangential flow filtration (tff) follow by ultracentrifugation. cell binding were evaluated by flowcytometry and confocal microscopy and gene knockdown effect were assay by quantity reverse transcription-pcr (qrt-pcr). formulated evs were introduced to tumour (prostate, triple negative breast cancer, colon pdx) xenograft mice by tail-vein injection and evaluate in vivo tumour inhibition. results: ) we found the orientation of arrow-shaped rna can be used to control ligand display on evs membranes for specific cell targeting. ) by placing membrane-anchoring cholesterol at the tail of the arrow results in display of rna aptamer or folate on the outer surface of the evs and enhance cancer cell binding and uptake. ) taking advantage of the rna ligand for specific targeting and evs for efficient cytosolic delivery, the resulting ligand-displaying evs or plant derived evs-like nanovesicles were capable of specific delivery of sirna to cells, and efficiently blocked tumour growth in three cancer models. summary/conclusion: we developed an rna-evs based nanoparticles platform and shown the flexibility for different cancer type treatment. related publications: pi f et.al. nature nanotechnology. , : . li z et.al. sci rep. introduction: extracellular vesicles (evs) contain plasma membrane surface markers that provide insights into their cell source. until now, our understanding of the circulating ev-biome has been limited by the lack of celland size-specific ev quantitation methods. we have developed and validated a multiplex nanoscale flow cytometry approach to image cell-and size-specific ev populations using a novel human "ev-lyoplate" with differently coloured monoclonal antibodies per well in a well plate format (n = separate antibodies with isotype, stained pbs, unstained plasma, and quant-beads controls per plate). we hypothesized that platelet poor plasma samples from patients diagnosed with pancreatic cancer would have significantly different ev-biome profiles than screen negative study subjects. methods: study subjects were enrolled and sampled before clinically scheduled endoscopic ultrasoundguided biopsy (eus-fna) procedures to screen patients with symptoms of pancreatic duct obstruction who later had at least two years of clinical follow up, including surgical resection in cases of pancreatic neoplasia (n = ) or at least one follow up clinic visit to confirm resolution of symptoms. blood samples were uniformly collected, processed, and banked per isev recommended guidelines. uniform machine (facsymphony) settings to standardize light scatter and fluorescence detection were based on commercially available beads (eg. megamix). samples were coded and randomized for testing and results were reported as the mean cell-and size-specific ev events/ul of plasma. results: clinical outcomes confirmed cases of cancer and screen negative controls. principle component analysis suggested that a number of different celland size-specific evs were significantly more common in the cancer cases (adjusted p-value < . , with aucs > . ), including epcam+/cd + events likely from cancer cells and cd +/cd p+/cd + microvesicles from platelets, among others. summary/conclusion: in this proof of principle study employing an ev-lyoplate design and nanoscale flow cytometry, we could reliably discriminate the ev-biomes in patients with cancer from negative controls. ongoing studies will determine whether these discriminators will be validated in larger cohorts and provide at least noninferior predictive value compared with the current gold standard clinical testing assay (eus-fna introduction: small cell lung cancer (sclc) is an aggressive tumour type, usually metastatic at diagnostic leading to poor overall survival. interestingly, sclc tumours are composed by distinct subpopulations of cells that cooperate as an ecosystem to drive tumour survival. since the subtype of sclc may have prognostic significance, the aim of this study was to identify surface marker proteins as biomarkers of sclc. methods: a linear discriminant analysis (lda) model, implemented in python via sci-kit learn, was used to choose the best markers for distinguishing subtypes. this analysis was based on rna-seq data from a previous study. in order to identify ev-based biomarkers that would identify sclc evs and not normal evs, we excluded from this analysis proteins without a verified transmembrane domain and proteins associated with evs expected to be present in white and red blood cells, and endothelial cells (according to exocarta and vesiclepedia databases). we also prioritized proteins that could be pan markers for sclc and that might have prognostic significance. to validate our findings, we performed western blotting and flow cytometry in sclc cell lines from different subtypes. results: our rna analysis indicated that the best surface markers to distinguish sclc subtypes were ceacam , fam a, lrfn , epha . immunoblot analysis validated ceacam and epha but not fam a or lrfn . we also found that ncam , a commonly used sclc marker, only marks some of the subtypes. for further analysis, we chose proteins with antibodies validated for flow cytometry as our chosen biomarker platform. flow cytometry analysis of cd is suitable as a pan-sclc marker. however, the expression of non-ne cell lines was decreased compared to rna-seq data. summary/conclusion: protein analysis of ceacam and epha corresponded to rna-seq data. ncam was not detected as a pan marker for all sclc subtypes. however, we could see cd expression in all sclc subtypes, indicating it may be a useful pan marker for sclc. future studies will be performed to validate the expression of other surface markers in cells, purified evs, and plasma of sclc patients. funding: nih u ca and nih u ca . leukobiopsyexploiting extracellular vesicle-mediated leukocyte sequestration of cancer-specific signatures introduction: in cancer, extracellular vesicles (evs) act as a unique exit mechanism for mutant and oncogenic macromolecules (proteins, rna and dna) en route from malignant cells to blood . while this process has inspired major liquid biopsy efforts, the biology of circulating evs that carry oncogenic mutations (oncosomes) is still poorly characterized. it is also unclear what part (if any) of the tumour-related cell free dna (ctdna) , , a major liquid biopsy analyte, is linked to circulating evs and what is their fate, receptacles and biological activity. methods: we employed as series of cancer cell lines carrying mutations in major oncogenes (hras, her , egfrviii). ev-dna was analysed by digital droplet pcr (ddpcr), along with nuclear anomalies in donor cells (dapi, electron microscopy) and transfer of dna to recipient cells of endothelial (huvec, mmbec), astrocytic (nha) or myeloid (hl ) origin. blood underwent fractionation into red blood cells (rbc), white blood cells (wbc), platelets (plt), evs ( , g ultracentrifugation) and soluble plasma (sup) . results: hras-mediated cellular transformation (in ras- cells) triggers profound changes in the structure of nuclear chromatin, which is driven into the cytoplasm and released as cargo of evs. oncogenic dna is detectable in blood fractions of tumour bearing mice. while evs, ctdna and plts contain intermediate levels of mutant dna, rbcs contain only traces of this material. the highest hras copy number per ml of blood is found in wbcs (monocytes and neutrophils), which contain more cancer dna/cell than liver, spleen and bone marrow. depletion of neutrophils using anti-ly g antibody results in an increase in ev-and ctdna-associated mutant dna in blood, suggesting the role of these cells in regulating the circulating levels of cancer cell-derived particles. uptake of dna-containing evs impacts the phenotype of myeloid cells, which adopt thrombo-inflammatory properties. these cells also retain cancer-specific transcripts and other cargo. finally, normal astrocytes treated with oncogenic evs also exhibit phenotypic changes and signs of genomic instability including formation of micronuclei. summary/conclusion: we propose that the process of leukocyte sequestration of circulating particles containing tumour-related nucleic acids renders these cells potentially usable as a novel liquid biopsy platform (leukobiopsy) in cancer. introduction: early diagnosis of colorectal cancer (crc) and precancerous adenoma patients is of vital importance. previously we profiled small extracellular vesicles (sevs) derived mirnas isolated from plasma, proposed a new promising biomarker category of crc patients. here we further gave a full landscape of circulating sevs derived rnas to explore and evaluate sevs based rna biomarkers for early detection of both crc and adenoma patients. methods: plasma sevs were isolated from participants, including early-stage crc patients, adenoma patients, and normal controls (nc), and characterized according to misv guideline. the total sevs derived rna expression profile of all participants was investigated by next-generation sequencing (ngs). weighted gene coexpression network analysis (wgcna) was performed to categorize differentially expressed rnas, and t-distributed stochastic neighbour embedding (tsne) was adopted to distinguish crc, adenoma, from nc samples with the top-ranked genes in wgcna modules. rt-qpcr validation was performed in a cohort of additional participants. results: a total of rna species (including mirnas, mrnas, and lncrnas) were found differentially expressed between plasma sevs in crc and nc participants. additionally, rna species were differentially expressed between plasma sevs in adenoma and nc participants. rna species were differentially expressed between plasma sevs in crc and adenoma participants. wgcna categorized all rnas into modules, which exhibited different expression trends during the carcinogenesis of crc. a -gene combined tsne model consists of the top genes in each module could perfectly classify crc, adenoma, and nc samples. a -gene combined tsne model consists of the top gene in each module could roughly distinguish crc and adenoma from nc, with only sample misclassified. rt-qpcr assays also confirmed the potential classification ability of those genes in another validation cohort of participants. introduction: although the concept of systematic "liquid biopsy" using bodily fluids is simple and elegant, the path of clinical reality has been challenging. recently, numerous tissue-specific biomarkers have been discovered in evs derived from blood, urine, cerebrospinal fluid, cell culture media, and a variety of other fluids. however, tracing the lineage of evs to their tissue of origin remains challenging due to their minute amount of cargo and unavailability of matching biopsied tissue and bodily fluids from the same patient. we recently demonstrated in three separate publications (dogra et. al; smith et. al; murillo et. al) , a new device (nanodld) for ev isolations, it's comparison with current technologies, bioengineered vesicles, and a detailed study of rna types present in small/ large vesicles, lipoproteins, and ago protein in different biofluids. in the present study, we aim to investigate the lineage of prostate derived evs in biofluids. methods: using our chip technology, we have isolated exosomes from prostate cancer cell lines and patient tissue, blood and urine samples. after exosome isolation, small rna libraries were prepared, and sequencing is carried out at icahn school of medicine and new york genome center using illumine sequencer hiseq . our nanofluidic pillar array is manufactured in an sio mask using optical contact lithography and deep ultraviolet lithography. results: our study revealed i) rna markers, which are exclusive to their prostate tissue of origin and are secreted in evs; ii) approximately - % of prostate tissue-specific rna were discovered in evs; iii) over % ( of rna) of literature curated prostatespecific rna signatures were detectable in serum and urine evs from pca patients; iv) evs contained over - % of noncoding rna ( - % was mirna), while tissue predominantly yielded rrna (> %); v) finally, gene set analyses generated that over % of evs rna were enriched for signalling pathways, yielding mirna-associated, non-canonical wnt signalling, and androgen receptor pathways. this study enables us to noninvasively monitor prostate tissue-specific biomarkers, identify tumour-specific rna, and potentially may benefit in liquid biopsy by avoiding unnecessary surgical procedures. summary/conclusion: in summary, we have investigated patient matched tissue, serum, and urine derived evs in prostate cancer. we present a set of prostatic rna in evs, which are enriched in noncanonical wnt signalling, and androgen receptor pathways. this study enables us to noninvasively track prostatic biomarkers, identify tumour specific rna, and potentially may benefit in liquid biopsy by avoiding unnecessary surgical procedures. a multi-model, liquid biopsy approach for diagnosing and staging pancreatic adenocarcinoma introduction: pancreatic ductal adenocarcinoma (pdac) is the third largest contributor to cancerrelated death in the usa. since there is not yet a feasible technology to diagnose pdac early in the disease, % of patients are diagnosed at an advanced stage. moreover, for patients with confirmed pdac, standard imaging method has low sensitivity to detect early metastatic disease, which complicates the selection of therapy. to address these challenges, there has been great interest in developing minimally-invasive, extracellular vesicle (ev) based blood tests for pdac. to this end, we have integrated measurements of tumour derivedev rna cargo with circulating proteins and cell free dna (cfdna), and use machine learning algorithms to distill this multiplexed diagnostic to . diagnose pdac patients from healthy and disease controls and . distinguish pdac patients with distance sites of metastasis to guide their treatment. we make use of our lab's magnetic nanopore isolation technique to specifically enrich for tumour derived evs directly from patient plasma. methods: we have developed a high throughput nanofluidic sorting platform, which immunomagnetically isolates individual evs from plasma using magnetic nanostructures. however, our architectures is uniquely designed for massive parallelization allowing high throughput, robust processing of ml of plasma in minutes. we performed sequencing on a discovery set of patients and controls (n = ). subsequently, we trained our panel of biomarkers using a training set of n = . finally, we validated the performance of our platform using an independent blinded test set of n = . results: the results of a blinded test set achieved an accuracy = % and an auc = . on binary classification of pdac patients versus those that were healthy or disease controls. in addition, we achieved an auc = . and accuracy = % with sensitivity of % and specificity of % on detecting occult metastasist. summary/conclusion: we developed a highly sensitive pancreatic cancer diagnostics by combining our nanomagnetic isolation platform for tumourderived ev isolation, rna sequencing, and machine learning. we isolated tumour-derived evs and profiled their rna cargo, combined with cfdna and ca - for pancreatic cancer diagnosis. the predictive panels successfully distinguished non-cancer patients from pdac patients, and nodistant metastasis patients(m ) from distant metastasis patients(m ) for appropriate treatment. the resulting auc and accuracy from the independent blinded test set outperformed any individual biomarker, showing both the benefits and the robustness of combining multiple orthogonal biomarkers for pdac diagnosis. introduction: both hypertension and diabetes exhibit significant molecular changes to the vasculature that are associated with increased cardiovascular risk. here we examined the protein composition of large evs (l-evs) isolated from the plasma of hypertensive, diabetic and healthy mice to identify common and diseasespecific molecular changes. methods: we examined circulating l-evs isolated from transgenic mice expressing active human renin in the liver (ttrhren, a model of hypertension), ove type diabetic mice, and their wild-type (wt) littermates. at weeks of age mice were sacrificed and blood samples were obtained by cardiac puncture. l-evs were isolated from platelet-free plasma via differential centrifugation and protein content was assessed via mass spectrometry (ms). results: ttrhren mice exhibited increased blood pressure compared with ove mice or their wt littermates. ( . ± . vs . ± . [ove ] vs. . ± . mmhg [wt], p < . ). ms identified independent proteins with at least peptides per protein. of these, proteins were found in all groups studied, were exclusive to wt mice, were exclusive to ove mice and were exclusive to ttrhren mice. in addition, proteins were observed with > . fold change (fc) compared to wild-type mice, and proteins were reduced by > %. amongst the top ten differentially expressed proteins, fibrinogen was upregulated in both ove and ttrhren mice compared with wild-type controls. similarly trem-like transcript , sarcoplasmic/endoplasmic reticulum calcium atpase and junction plakoglobin were all downregulated in both ove mice and ttrhren mice suggesting molecular changes common to both conditions. conversely, arginase was up-regulated in diabetic, but not hypertensive mice while carboxypeptidase was upregulated in hypertensive but not diabetic mice. summary/conclusion: taken together, these results show that the protein composition of circulating l-evs is altered in diabetes and hypertension and that both common and disease-specific changes may be detected. further analysis of these changes may lead to the identification of novel pathways associated with the pathogenesis of vascular injury in hypertension and diabetes. funding: this study was supported by grants (to db) from the canadian institutes of health research, an ontario early researcher award, and the canada foundation for innovation. understanding the role of endothelial cell-derived apoptotic bodies in inflammatory signalling and cell clearance in an atherosclerosis model of inflammation. introduction: apoptotic bodies (apobds) are a class of large (~ - um) evs formed during apoptotic cell disassembly, that are becoming increasingly recognized as potential mediators of intercellular communication, e.g. via the transfer of proteins and other cargoes to target cells. during the inflammatory vascular disease atherosclerosis, endothelial cell (ec) apoptosis contributes to loss of barrier function and promotes the formation of plaques in regions of ec damage. although, experimentally, ecs generate an abundance of apobds, a specific role for ec-derived apobds (ec-apobds) in the progression of atherosclerosis remains poorly defined. methods: in the present study, a detailed in vitro characterization of ec disassembly was performed via flow cytometry, confocal live cell imaging and cytokine profiling, followed by function analyses of ec-apobds using a murine in vivo model of dead cell clearance. results: characterization of ec disassembly revealed that apobd formation in ecs is regulated by rho-associated, coiled-coil-containing protein kinase (rock ), a process that can be pharmacologically inhibited using a rock- inhibitor, thereby providing tools for functional in vivo studies. the specific cargo and role in clearance of ec-apobds were then investigated. profiling of ec-apobds was performed via cytokine antibody array to reveal that ec-apobds generated under inflammatory conditions contain high levels of pro-inflammatory cytokines including mcp- and il- , suggesting a potential role for ec-apobds in the propagation of inflammation during vascular disease. furthermore, the ability of ec-apobds to be cleared from the vasculature via phagocytosis was investigated, revealing that ec-apobds can travel to distal organs to undergo clearance. summary/conclusion: these findings provide important insights into the potential functions of ec-apobds generated under both non-inflammatory and inflammatory conditions and may contribute to future studies involving the therapeutic targeting of ec disassembly for the treatment of atherosclerosis. funding: this work was supported by grants from the national health & medical research council of australia (gnt , gnt ) adipose mesenchymal stromal cell derived evs foster cardio-renal protection in the doca-salt hypertensive rat model introduction: cardio-renal syndromes (crs) are disorders of the heart and kidneys whereby "acute or chronic dysfunction in one organ may induce dysfunction of the other". stem cell-derived extracellular vesicles (evs) mediates the protection of the kidney from development of chronic kidney disease (ckd). we here investigated the potential of adipose-mesenchymal stromal cells derived evs (asc-evs) as therapeutic tools for the treatment of crs. methods: adult wistar rats were uninephrectomized and treated with a high-na+ diet and deoxycorticosterone-acetate (doca-salt) for -weeks ( / ; a / - - ). evs were isolated by ultracentrifugation method. ev dimension, concentration and surface markers were characterized by nta, cytofluorimetric analysis and transmission electron microscopy. to characterize the role of evs in crs, doca-salt rats were injected weekly with asc-evs. systolic blood pressure was measured by the tail-cuff method. plasma creatinine and urinary protein excretion were determined by colorimetric assays and microalbuminuria by immune turbidimetric assay. qrt-pcr and western blot were conducted to evaluate fibrosis and inflammatory-related genes and proteins in the kidney and heart of doca-salt rats. immunohistochemistry was used to confirm matrix accumulation (a-sma) and immune infiltrate (cd + cells). results: multiple administration of asc-evs in doca-salt rats induced a protective effect on the kidney, by reducing tubular and vascular damage. kidney function was also conserved by ev treatment as detected by the normal glomerular filtration rate and the absence of proteinuria with respect to doca-salt untreated rats. ev administration significantly decreases the pro-inflammatory molecules mcp- and pai and reduce the recruitment of macrophages in the kidney. the mitigation of the inflammatory response by asc-ev infusion consequentially affected the development of fibrosis, as detected by the decrease in collagens (col a , col a ) and fibronectin (fn) expression in respect to doca-salt animals. asc-evs were able to act in multiple organs, preventing fibrosis and inflammation also in the heart, therefore alleviating blood pressure rise during the -weeks of treatment in doca-salt rats. summary/conclusion: our results indicate that asc-ev administrations in hypertensive-induced ckd rats promote protection from renal damage, reduction of the inflammatory response and prevention of interstitial fibrosis in the kidney. asc-evs are also able to protect the cardiac tissue and to control blood pressure increase, displaying complex and multiorgan beneficial effects. introduction: alveolar macrophages (ams) tonically secrete extracellular vesicles (evs) containing suppressor of cytokine signalling (socs ) protein. uptake of socs -containing evs by alveolar epithelial cells is critical for restraint of cytokine-induced janus kinasesignal transducer and activator of transcription (jak-stat ) signalling to promote homoeostasis in the distal lung. at steady state, ams exhibit suppressed glycolytic activity, a metabolic phenotype that promotes homoeostatic function. whether this glycolytic restraint is critical for am secretion of socs is unknown. in fact, to our knowledge, metabolic control over release of any ev cargo has never been explored in any cellular context. methods: immortalized mouse ams (mh-s) were treated with various doses of -deoxy-d-glucose ( -dg) and oligomycin, inhibitors of glycolysis and oxidative phosphorylation, respectively. primary rat ams collected by lung lavage were treated with an aqueous extract of cigarette smoke (cse) with or without -dg. metabolic activity was measured by seahorse assay, evs were quantified by nanoparticle tracking analysis, and vesicular (> -kda) socs secretion was determined by western blot of conditioned medium. additionally, ams collected from wild-type (wt) and lsl-krasg d mice bearing lung tumours weeks after intrapulmonary ad-cre were cultured ex vivo in the presence or absence of -dg. vesicular (> -kda) socs secretion was measured by elisa. results: in a dose-dependent manner, oligomycin inhibited, whereas -dg enhanced, socs and ev release by mh-s cells. treatment of rat ams with cse ( %) attenuated secretion of socs , an effect that coincided with increases in glycolytic activity, and co-treatment of ams with -dg abrogated the inhibitory effect of cse on socs release. finally, ams collected from lsl-krasg d mice exhibited a deficiency in socs secretion relative to wt ams, an effect that was reversible by overnight culture in the presence of -dg. summary/conclusion: in tandem, our data generated using in vitro and in vivo approaches demonstrate that am secretion of vesicular socs is down-regulated by glycolysis. we speculate that metabolic control over release of ev cargoes is a phenomenon of broad biologic relevance within and outside of the lung. introduction: bacterial extracellular vesicles (ev) are described to play roles in defence and resistance, pathogenesis and stress responses. cyanobacteria pioneered oxygenic photosynthesis, and are the ancestors of modern chloroplasts. we previously described that by deleting the gene encoding tolc (Δtolc) in the model cyanobacterium synechocystis sp pcc (s ), a key player in protein-mediated secretion systems, a hyper-vesiculating phenotype could be obtained. the goal of this work was to understand why Δtolc hyper-vesiculates. methods: isobaric tag for relative and absolute quantitation (itraq) was used for quantitative proteomic analyses of total cell extracts. ev were isolated as follows: cells were separated from the extracellular medium (em) by centrifugation ( g, min) and filtration ( . µm pore-size filters). cell-free em was concentrated using centrifugal filters (mwco of kda), and later ultracentrifuged for h at g. the final ev fraction was suspended in growth medium. ev characterization was performed using tem, dls, nanosight, and by the detection and quantification of lps (lipopolysaccharides). detection of specific proteins in ev was carried out by western blot. copper (cu) levels were quantified by atomic absorption spectrometry (aas). results: a large-scale quantitative proteomic analysis was performed, resulting in the identification of several metal-related proteins with differential regulation in s Δtolc. both wild-type (wt) and Δtolc cells were then challenged with different metals. compared to the wt, Δtolc showed impaired growth only when exposed to cu, a co-factor for several proteins with roles in primary metabolism. the intracellular cu levels were quantified and Δtolc accumulates threefold more cu than wt cells. we then asked whether the hyper-vesiculating phenotype observed could be linked to the stress induced by cu accumulation. in ev isolated from Δtolc we detected the metallochaperone copm, a periplasmic cu-binding protein involved in cu-resistance mechanisms in s . in addition, cu could also be detected in isolated Δtolc-ev. in addition, more ev were detected when s wt cells were challenged with cu, in a cu-concentration dependent manner. summary/conclusion: these results support the idea that bacterial ev represent an alternative cu-secretion mechanism to deal with cu-induced stress. funding: fct phd grant sfrh/bd/ / ; feder-compete -poci-fct project: poci- - -feder- . juan wang and maureen barr rutgers university, human genetics institute of nj, piscataway, usa introduction: extracellular vesicles (evs) function in intercellular communication. despite their physiological importance and biomedical relevance, knowledge of ev fundamental biology is not well understood, in part due to a lack of tractable animal systems. our analysis of environmentally-released c. elegans ciliary evs provides strong evidence that nematodes package cargo in evs that mediate inter-organismal communication, in analogy to intercellular signalling in mammals. we predict that conserved mechanisms underlie ev cargo sorting, biogenesis and signalling. cilia act as cell towers to both receive extracellular signals and to send information via ciliary evs. ciliary defects result in human ciliopathies including autosomal dominant polycystic kidney disease (adpkd). adpkd is a life-threatening disease that affects / and is caused by mutations in pkd and pkd , which encode polycystin- and − . in c. elegans and humans, the polycystins are architecturally similar, act in the same genetic pathway, function in a sensory capacity, localize to cilia, and are shed in evs, suggesting ancient conservation. moreover, ciliary ev biogenesis and shedding is an evolutionary conserved process from algae to worms to humans. by studying how cilia make and receive evs, we aim to uncover fundamental principles of how cells communicate using evs. methods: to study ciliary ev cargo sorting and biogenesis, we use genetically-encoded fluorescent-tagged ev cargo and superresolution zeiss airyscan confocal microscopy in living animals. results: we find that cargoes are sorted into distinct populations. in cilia, kinesin- motors and kinesin- klp- /kif transport different ev cargoes to the ciliary tip and generate an ev cargo enrichment zone. from here, evs are shed and released into environment in a spatially and temporally regulated manner. ciliary ev biogenesis and release is regulated by mechanical pressure and ph. our work revealsat the single cell levelthat different evs are made in response to environmental stimuli, which may be important for ev signalling properties. summary/conclusion: cells exploit the spatiallyrestricted cilium and its sophisticated transport system to generate distinct populations of ciliary evs. how these ciliary ev communicate cellular messages awaits decoding. introduction: we recently demonstrated that recycling endosomes marked by rab a generate exosome subtypes distinct in cargos and functions from late endosomes, which we collectively term rab -exosomes. these exosomes are preferentially released from cancer cells in response to metabolic stress and promote adaptive changes in a xenograft model. here we use comparative ev proteomics in hct colorectal and hela cervical cancer cell lines to identify rab -exosome signature proteins and screen for functional effects. methods: we analysed ev preparations by mass spectrometry using tandem mass tag® labelling to identify changes in ev protein cargo in response to glutamine depletion. candidate genes were subsequently knocked down in drosophila secondary cells, which permit visualisation of rab -exosome biogenesis using fluorescence microscopy, and in human cancer cell lines. results: we show that accessory escrt-iii proteins, chmp , chmp and ist , are enriched on glutamine-depletion-induced evs and play a selective and conserved role in generating rab -exosomes. they are, however, not required to traffic ubiquitinated cargos into late endosomes and lysosomes. escrt- components, thought to regulate trafficking of ubiquitinated cargos into intraluminal vesicles, are also required to make rab -exosomes. in flies the escrt- , hrs, localises to the limiting membrane of rab -endosomes. comparative proteomics reveals other proteins enriched in rab -exosomes, which also appear to be needed to mediate this novel exosome formation mechanism. summary/conclusion: we conclude that rab -exosome subtypes are formed via a distinct mechanism requiring accessory escrt-iii components, suggesting a route to selectively target these exosomes. introduction: the tumour microenvironment consists of a complex network of host cells embedded within extracellular matrix. communication between these cellular compartments is critical for tumour progression and exosomes have emerged as important regulators of intercellular communication. while a number of studies have implicated exosomes in cancer progression, mechanisms controlling exosome transfer are not well understood. we developed three-dimensional ( d) culture models to evaluate the role of cues provided by the extracellular matrix in exosome release and uptake. methods: exosomes were isolated from cells in two-and three-dimensional culture via ultracentrifugation and characterized by nanosight, qubit protein quantification, and flow cytometry analysis of exosome markers. exosomes were labelled with fluorescent lipophilic dyes and uptake in recipient cells quantified by flow cytometry. results: cells cultured in d display decreased exosome release and increased uptake compared to d cultured cells. exosome release in d culture was inhibited with the exosome release inhibitors brefeldin a and gw , but was not significantly altered by knockout of rab b. in addition, disruption of polarity signals provided by d culture did not impact exosome release or uptake in d, but induction of oncogenic hras increased both secretion and uptake of exosomes through activation of pi k signalling. summary/conclusion: release and uptake of exosomes is altered in d environments. these studies help provide insight into exosome production and uptake in vivo and have potential implications for therapeutically targeting exosome release and the development of exosome based therapeutic delivery vehicles. introduction: previous studies in our lab found that expression of r w-fibulin- induces rpe to undergo emt. the purpose of current study was to characterize the extracellular vesicles (evs) in rpe cells expressing wt-fibulin- versus rpe cells expressing r w-fibulin- and investigate the effects of these evs on rpe cell differentiation. methods: arpe- cells were infected with lentivirus with luciferase-tagged wild-type (wt)-fibulin- or luciferase-tagged r w-fibulin- . evs were isolated from the media of arpe- cells by conventional ultracentrifugation or density gradient ultracentrifugation. transmission electron microscopy (tem) and cryogenic electron microscopy (cryo-em) were performed to study the morphology of the evs. the amount and size distribution of evs were analysed by nanosight tracking analysis (nta). ev protein concentrations were quantified using the dctm protein assay (bio-rad). ev cargo were analysed by unbiased proteomics using lc-ms/ms with subsequent pathway analysis (advaita). migration ability was evaluated in arpe- cells with or without the exposure of evs by conducting scratch assays. results: morphologically, tem imaging showed concave-appearing vesicles and cryo-em imaging showed spherical vesicles with two subpopulations of evs: a small group with diameters around nm and a large group with diameters around nm. moreover, tem and cryo-em showed an increased amount of small evs (~ nm) in the mutant group compared to the wt group. this result was further confirmed by nta showing that, in the mutant group, the particle size distributions were smaller than the wt evs. no significant differences were shown in ev protein concentrations per particle between wt and mutant groups. our previous data suggest that the expression of r w-fibulin- causes rpe cells to undergo emt as evidenced by upregulated emt drivers and an increased migration ability. proteomic studies showed that evs derived from arpe- cells overexpressing wt-fibulin- contain critical members of sonic hedgehog signalling (shh) and ciliary tip components, whereas evs derived from rpe cells overexpressing r w-fibulin- contain emt mediators, indicating that ev cargo reflects the phenotypic status of their parental cells. ev transplant studies showed that exposing native rpe cells to mutant rpe cell-derived evs containing emt drivers, including tgf-β-induced protein (tgfbi), vim, and smad , leads to an enhanced migration ability of rpe cells in a dosedependent manner. introduction: despite of high expectations, mesenchymal stromal cell (msc)-based therapies still lack efficacy, partially due to loss of cell viability and function upon administration. msc-derived extracellular vesicles (msc-ev) emulate the regenerative potential of msc, shifting the field towards cell-free therapies. clinical applications require the establishment of a scalable and gmp-compliant processes for the production and isolation of msc-ev, combined with robust characterization platforms. methods: to develop a well-established process for the production of therapeutic msc-ev, we compared different msc sources (bone marrow, adipose tissue, umbilical cord matrix), culture media compositions (dmem supplemented with foetal bovine serum (thermo fisher scientific), dmem supplemented with human platelet lysate (aventacell biomedical) and stempro msc sfm xeno free medium (thermo fisher scientific)) and culture parameters (oxygen tension and shear stress) in two different culture platforms ( d static tissue culture flask vs d dynamic spinner vessels). subsequently, msc-ev were isolated by ultracentrifugation or a commercially available isolation kit and characterized according to isev guidelines. results: msc derived from different sources/donors were able to grow under normoxia and hypoxia in d t-flasks and d spinner vessel culture systems, while maintaining their immunophenotype and differentiation potential, according to the minimal criteria defined by the isct. the time point for pre-conditioning and collection of conditioned medium for msc-ev isolation was also optimized for both d and d culture systems. introduction: extracellular vesicles (evs) have great potential in prostate cancer (pca) diagnosis and progression monitoring to complement the inaccurate prostate specific antigen (psa) screening and invasiveness of tissue biopsy. however, current methods cannot isolate pure evs and therefor evs characteristics remain largely unknown. in order to develop an accurate approach for ev isolation, we aimed to compare three emerging methods with different characteristics of small evs (sevs) from human pca plasma samples and to choose the best one for diagnostic and functional studies methods: pca patients and age-matched healthy controls (hc) plasma (n = in each group) were used to isolate sevs with different isolation methods including commercial exoquick ultra kit, qev and qev size exclusion chromatography (sec). isolated sev were characterized by nanoparticle tracking analysis, immunoblotting, cyrogenic electron microscopy, flow cytometry (fc) and proteomics analysis. for fc characterizing surface marker expression, the sevs were further purified by cd and cd commercial immunoaffinity magnetic beads . lipoprotein was captured by streptavidin biotinylated apob magnetic beads to measuring the lipoprotein contamination results: the sev size, morphology, surface protein and protein cargo with proteomics were analysed between the three isolation methods. sevs isolated from sec methods had a lower particle size, protein amount, protein/sev marker ratio and apob+/sev marker ratio than those from exoquick ultra method. in addition, sevs isolated from qev demonstrated a significantly higher sev content, more up-regulated and down-regulated pca proteins from proteomics but lower sev marker/protein ratio and a higher protein contamination than those from qev . furthermore, sev marker signal also showed a good correlation with particle numbers instead of protein content in all the methods summary/conclusion: qev method demonstrated better performance in isolating relatively pure sevs from human plasma; qev has the better performance in isolating samples with higher sev content; exoquick ultra isolated samples with closely sev content to the qev but with the highest non-sev protein contaminations. introduction: extracellular vesicles (evs) are released to biological fluids from different tissues and organs and they contain molecules proposed as biomarkers for multiple pathological conditions. however, most ev biomarkers have not been validated due to the lack of sensitive techniques compatible with high-throughput analysis required for routine screenings. using immunocapture techniques, combining antibodies against tetraspanins and candidate tumour-specific markers we have recently optimized several assays that greatly facilitate ev characterization. methods: we have improved flow cytometry and elisa assays, increasing substantially the sensitivity for ev detection. using dls, em and analytical ultracentrifugation, we have characterised the biophysical basis of this enhancement. the final methodology can be performed in any laboratory with access to conventional flow cytometry or elisa reader. results: using combinations of antibodies specific for the tetraspanins cd , cd and cd , it is possible to detect evs in minimal volumes of urine and plasma samples without previous enrichment. additionally antibodies against other less abundant markers, like the epithelial marker epcam, have been used to capture and identify evs directly in minimal volumes of urine or plasma with sensitivity higher than western blot analysis of isolated evs. furthermore, we demonstrate that additives altering the biophysical properties of an ev suspension, increased detection of tumour antigens in these immune-assays. summary/conclusion: the development of sensitive, high-throughput methods, easily translatable to clinical settings, as elisa and flow cytometry described here, opens a new avenue for the systematic identification of any surface marker on evs, even scarce proteins, using very small volumes of minimally processed biological samples. these methods will allow the validation of ev biomarkers in routine liquid biopsy tests. introduction: when ev subpopulations are enriched on antibody microarrays and probed for their surface proteins, the detection signal is biased towards abundant subpopulations as it is dependent on both the protein expression level and the number of evs captured. to address this challenge, we developed a novel normalization approach allowing: ) the estimation of a target signal independent of ev subpopulation size through dye-based ev quantification, and ) the assessment of subpopulation target enrichment relative to the population average by leveraging tim as an unbiased, lipidbased ev capture. here, we investigated the expression of cancer-associated proteins, particularly metastasisassociated integrins (itgs), in breast cancer evs with varying metastatic potential and organotropism. methods: the relative protein enrichment profiles for various ev subpopulations were established from evs of skbr (her +), t d and mcf- (er+pr+), bt and mda-mb- (triple negative) breast cancer cell lines, as well as five mda-mb- -derived cell lines of four different organotropisms (brain, bone, lung, liver) using our custom antibody microarrays with our normalization approach. results: as expected, her was broadly detected in her + skbr evs. interestingly, her -t d and mcf- evs also expressed her where it was highly enriched in its epcam+ subpopulations. itg α , β and β were only found in triple negative and organotropic evs with itg β and β differentially enriched based on the organotropism. the population average of mda-mb- and lung-tropic evs had high expression of itg β , where subpopulations of cd + evs showed positive enrichment while cd + and cd + evs showed negative enrichment. itg α , β and β were absent in the bone-tropic cd + ev subpopulation, a profile atypical in other organotropisms. lastly, egfr was negatively enriched in tetraspanin+ subpopulations in mda-mb- evs, but positively enriched in these subpopulations in organotropic evs, especially for brain-tropism. summary/conclusion: following normalization, we were able to quantify specific protein associations, uncovering a multitude of co-enrichment profiles that characterize specific metastatic and organotropic cell lines. notably, we found enrichment signatures that distinguish between different organotropisms derived from the same parental cancer line. op . = pf . heparan sulphate proteoglycans are required for ev-mediated delivery of multiple growth factors sara veiga, alex shephard, alex cocks, aled clayton and jason webber cardiff university, cardiff, uk introduction: the tissue microenvironment surrounding tumours is complex and the cross-talk between cancer and non-cancer cells is essential for tumour growth and progression. we have previously shown that heparan sulphate proteoglycans (hspgs), on the surface of prostate cancer evs, are required for delivery of tgfβ and initiation of a disease-supporting fibroblast phenotype. however, hspgs are known to bind numerous growth factors, so here we have explored the repertoire of such proteins tethered to evs by hspgs. methods: evs were isolated from du prostate cancer cell conditioned media by ultra-centrifugation onto a sucrose cushion. vesicular hspgs were modified either by removal of heparan sulphate (hs) glycosaminoglycan (gag) chains using the enzyme heparinase iii (hepiii), or attenuation of hspg core protein expression using shrnas to knockdown specific hspgs within the parent cell. differences in proteins present in control vs modified evs were identified by a sensitive protein array, based on proximity-ligation technology, and selected targets validated by elisa. functional delivery of growth factors by ev-associated hspgs to recipient fibroblasts is being explored using a variety of in vitro techniques. results: proteome analysis identified targets that bind to hs-gag chains, and also different proteins that showed altered expression following the loss of one or more hspgs from evs. using elisa, we have been able to quantify selected candidates on wild type vesicles, some of these are lost following hsdigestion. we were also able to validate proteins on hspg-deficient vesicles. gene ontology analysis suggests that ev hspg-mediated delivery of growth factors is important for control of processes such as angiogenesis, tumour invasion and immune regulation. functional validation of proteins identified is ongoing. summary/conclusion: here we demonstrate that hspgs play a key role in loading of evs with a complex assortment of growth factors, and therefore subsequent ev-mediated growth factor delivery. we anticipate that loss or damage of ev- introduction: methamphetamine (ma) and related amphetamine compounds, which are potent psychostimulants, are among the most commonly used illicit drugs. neuroimaging studies have revealed that chronic ma abuse can indeed cause neurodegenerative changes in the brains of human ma abusers including prominent microglial activation throughout the brain. it is still unclear how chronic inflammation caused by ma abuse leads to long-term damage to the brain. with this in mind, we are particularly interested in studying the role of extracellular vesicles (evs) in eliciting chronic inflammation in ma exposed brains. in the present study, we focus on the role of a mirna, mir- a- p (mir- a) in chronic ma exposure. here, we present novel data that shows for the first time how chronic ma impacts not only the biogenesis but also the ev associated mirna cargo thereby affecting the overall health of the neurons and glial cells in the brain. methods: -density gradient centrifugation for isolation of brain-derived vesicles -characterization of bdes by western blotting, nanoparticle tracking analysis and transmission electron microscopy -quantitative rt-pcr -digital droplet pcr -confocal imaging of dendritic spines and synapses results: in the present study, we show from both in vivo and in vitro studies that chronic methamphetamine (ma) treatment alters ev biogenesis and microrna (mirna) cargo. brain-derived evs (bde) isolated from frontal grey tissue of rhesus macaques that were administered ma in a chronic regimen revealed a significant increase in both number and size. further analysis revealed increase in biogenesis genes and increased levels of mirna, mir- a- p (mir- a). in situ hybridization of the frontal brain area revealed that mir- a was exclusively expressed in microglia and neurons. further, in vitro studies revealed that ev associated mir- a elicited not only neuronal damage but also was able to activate microglia to release pro-inflammatory cytokines thereby inducing a chronic inflammatory cycle. finally, we show that an anti-inflammatory drug was able to rescue inflammation, mir- a levels and synaptodendritic injury. summary/conclusion: in summary, our results present for the first time show that chronic ma exposure in the brain affects ev biogenesis and mirna expression. we further confirm that mir- a can serve as potential marker to diagnose synaptic deficits for chronic ma addiction in humans. finally, we reveal that anti-inflammatory drug could rescue the ev biogenesis and reduces the secretion of mir- a, thereby rescues synaptodendritic injury. our data further supports the use of the anti-inflammatory drugs as therapeutic interventions for ma addiction. funding: nida funding # r da blood-borne and brain-derived ectosomes/microparticles in morphineinduced anti-nociceptive tolerance deepa ruhela, veena bhopale, ming yang, kevin yu, eric weintraub, aaron greenblatt and stephen r. thom university of maryland school of medicine, baltimore, usa introduction: opioid pain treatment is impeded because chronic administration decreases analgesia, a condition called tolerance that prompts dose escalation contributing to morbidity and mortality. inflammatory interleukin (il)- β is required for tolerance development, so we hypothesized that pro-inflammatory extracellular vesicles (evs) play a role. methods: evs with opioid administration were assayed in mice and humans. annexin v-positive, . - µm diameter microparticles (mps) were assessed by flow cytometry in murine and human blood and in murine deep cervical lymph nodes that drain brain glymphatics. blood-borne exosomes (< nm) were assayed by tunable-resistance pulse sensing (trps). anti-nociceptive tolerance following morphine administration to mice was assessed by speed of tail removal from warm water. results: repetitive morphine dosing of mice to induce anti-nociceptive tolerance increased blood-borne mps by eightfold, and by tenfold in cervical lymph nodes. mps expressed proteins specific to neutrophils, microglia, astrocytes, neurons and oligodendrocytes. il- β content of mps increased -fold. administration of an il- β antagonist to mice diminished blood and glymphatic mps elevations and abrogated tolerance induction. intravenous polyethylene glycol telomer b that lyses mps and intraperitoneal methylnaltrexone that binds peripheral opioid-mu receptors and myeloid differentiation factor- to inhibit toll-like receptors, inhibited mps elevations and tolerance. neutropenic mice did not develop anti-nociceptive tolerance, elevations of blood-borne mps or cervical node mps expressing microglial proteins. elevations of blood-borne exosomes were not identified based on trps analysis. patients entering treatment for opioid use disorder exhibited similar mps elevations as do tolerant mice. summary/conclusion: neutrophil-derived mps containing il- β are required for morphine-induced antinociceptive tolerance. funding: this project was supported by grant n - - - from the office of naval research and an unrestricted grant from the national foundation of emergency medicine. evs are a conveyor of toxic dipeptide repeat proteins in c orf als/ ftd models thomas jefferson university, philadelphia, usa introduction: amyotrophic lateral sclerosis (als) is a neurodegenerative disease characterized by loss of motor neurons. in als, motor symptoms initiate focally and then progress gradually, distal from the initial focus. abnormal forms of als-associated proteins are physically exchanged between neuronal cells. pathogenic als proteins like sod , fus and tdp are transmitted between cells by assisted mechanisms, mainly extracellular vesicles (evs), spreading toxicity and misfolding of native proteins within the recipient cells. an intronic g c aberrant nucleotide repeat expansion in c orf gene is the most common genetic cause of als. translation of this expanded region occurs by a process called repeat associated non-aug (ran) translation that produces five dipeptide repeats proteins (dprs), polyga, polygp, polygr, polypa and polyga. polyga, polygr and polypr are associated with toxicity in neurons. in this work we study the recruitment of these aberrant proteins into extracellular vesicles (evs) and the potential role of these evs in spreading toxicity between cells of the central nervous system. methods: to isolate the evs from cell culture media we isolated by ultracentrifugation the larger vesicles at , xg and the smaller evs at , xg. number, size and fluorescence of the vesicles were analysed by fluorescent nanotrack analysis (f-nta) and by cytoflex. the protein content of the vesicles was analysed by western blot (wb). to evaluate the potential toxicity of the evs, a transwell system (tw) was employed. neuron viability was assessed using live imaging techniques. results: nsc were transfected with reporter constructs expressing dprs tagged with gfp protein. by f-nta, cytoflex and wb analysis we assessed that all the five dprs were loaded in both the large and the small vesicles isolated from cell culture medium. by tw, nsc transfected with the dprs were put in contact with primary cortical neurons (cns) transfected with synapsin driven td-tomato for live imaging purposes. we observed that polygr+ nsc were able to cause a significant decrease in cns viability. we also observed that polygr+ evs associated toxicity was directly dependent on polygr length. this effect was reverted reducing the number of polygr+ evs treating nsc with gw . to understand the downstream effect of polygr+ evs in recipient cells we studied tdp mislocalization, ran-translation and activation of the integrated stress response, finding a dysregulation of all these potentially toxic pathways in neurons treated with polygr+ vesicles. summary/conclusion: concluding, dprs are actively secreted in evs and polygr+ vesicles cause the activation of toxic mechanisms in the recipient cells, possibly contributing to the spreading of als introduction: pregnancy is the a condition that profoundly mitigates symptoms of multiple sclerosis (ms) a complex disease characterized by immune dysfunction and neurodegeneration affecting . million people worldwide. serum exosomes, released by specific cells during pregnancy, modulate the immune and central nervous system function and contribute to pregnancy-associated suppression of experimental autoimmune encephalomyelitis (eae), an induced preclinical model of ms. extracellular vesicles (evs) are the new means for communication among cells. the aim of our study was to characterize the ability of human amniotic fluid stem cells-derived evs (hasc-evs) to antigen presenting cell function thus correcting immune dysfunction in eae. methods: amniotic fluids were obtained from human - -week pregnant women. hasc-evs were collected by ultra-centrifugation. evs were characterized for their specific proteins, lipids and nucleic acids expression. the ability of evs to modulate immune responses was performed in vitro, testing the ability of evs to induce a tolerogenic phenotype in mouse bone marrow derived dendritic cells, and in vivo for their potential to suppress eae, induced by immunization c /b female mice with mog - peptide. results: we found that hasc-evs expressed high levels of galectin- and promoted a significant increase of the immunoregulatory enzyme indoleamine , dioxygenase- enzyme in dcs. moreover in in vivo experiments administration of hasc-evs significantly reduced disease severity in eae. such effect was associated with reduced neurological deficits and suppression of pathogenic t helper (th ) cells, and increased percentage of regulatory t cells (treg-foxp +) cells. summary/conclusion: our findings unravel immunoregulatory effects of evs secreted by hascs. evs may represent a novel cell-free immune regulatory and regenerative therapeutic approach that can potentially mitigate immune dysfunction and promote remyelination. association of neuronal-derived extracellular vesicles cargo with cognitive decline in late middle life introduction: alzheimer's disease (ad) is characterized by a long preclinical stage during which phosphorylated tau pathology spreads in the brain leading to clinical symptoms. pathogenic tau spreads, in part, via extracellular vesicles (evs). we and others have demonstrated that tau cargoes of neuronal-derived evs (nevs) from blood can serve as biomarkers for ad. we aimed to examine whether nev tau cargo can predict cognitive decline in late middle age by leveraging samples from participants in the wisconsin registry for alzheimer's prevention (wrap) study. methods: we blindly immunoprecipitated nevs using antibody against neuronal l cell adhesion molecule (l cam) from serum samples of wrap participants who were cognitively unimpaired at baseline (mean age . ± . years old; . % females; . % apoe carriers), of whom half subsequently developed cognitive decline. we measured phosphorylated (p and p ) and total tau in nevs using electrochemiluminescence assays. we used linear regression models to identify differences between cognitive status groups including age, sex apoe status and the cognitive status*age interaction in the model. results: at baseline, we found trends for higher p -(p = . ) and p -tau (p = . ) levels in future decliners compared to stable participants. further, there were significant cognitive status*age interactions for ptau (p < . ), total tau (p < . ) and ptau (p < . ) with higher levels with increasing age in future decliners summary/conclusion: nev tau cargo differs between late middle-aged individuals at risk for ad with and without future cognitively decline even before decline occurs, presumably due to subclinical spread of tau pathology. further nev biomarker development may allow preclinical ad diagnosis. introduction: in the brain, circulating extracellular vesicles (evs) in the cerebrospinal fluid (csf) contain a variety of signalling factors, including proteins, enzymes, and rna transcripts. while evs have been implicated in many cell-to-cell signalling contexts, the vast majority of these studies are based on findings derived from cell culture conditions. thus, the ability to identify cell typespecific ev release from cellular subpopulations within the brain represents a critical barrier in the field. methods: to address this knowledge gap, we utilized a novel transgenic mouse model to determine the release of cell-type specific evs. here we report the exomap- mouse, which is designed to express an exosomal green fluorescent protein in response to expression of cre recombinase. specifically, the exomap- transgene was inserted at the mouse h locus and consists of (i) a broadly expressed cag promoter/enhancer, (ii) a floxed orf encoding mts-tdtomato, (iii) an orf encoding the exosomal protein acyltya fused to mneongreen (mng), and (iv) a ʹ utr containing the wpre element and polyadenylation signal from the bovine growth hormone gene. results: intracranial ventricular injections of the viral vector aav-ttr-cre, which drives cre recombinase expression from the choroid plexus-specific promotor of the transthyretin gene, leads to acyltya-mng expression in the choroid plexus. moreover, we observed that these mice released mneongreen-positive evs into the cerebrospinal fluid and also visualized the vesicles in the blood. furthermore, these mice displayed an accumulation of acyltya-mng fluorescence in the medial habenula. summary/conclusion: the results indicate that choroid plexus-derived evs are trafficked to the csf and the medial habenula, and more generally, that the exomap- mouse can be used to follow the trafficking of tissuespecific evs into biofluids and between tissues in vivo. introduction: large-scale colorectal cancer (crc) sequencing studies have shown that % of all tumours had at least one mutation in proteins implicated in the wnt signalling pathway. mutations in β-catenin have often been associated with the constitutive activation of wnt signalling pathway and has been established as a major driver of crc. one of the proposed mechanisms of activating wnt signalling involves extracellular vesicles (evs) as cellular couriers to transfer wnt ligands from one cell to another. however, the association of oncogenic mutant β-catenin with evs has not been studied. subpopulations of cancer cells with different mutational loads and behavioural variations lead to intra-tumour heterogeneity methods: integrative proteogenomic analysis showed the secretion of mutant β-catenin via evs. evs were isolated by ultracentrifugation and optiprep density gradient centrifugation. silac-based quantitative proteomics analysis, immunofluorescence, biochemical analysis, qpcr and xenograft models were employed to unveiling the role of evs carrying mutant βcatenin. results: an integrative proteogenomic analysis identified the presence of mutated β-catenin in evs secreted by colorectal cancer (crc) cells. follow up experiments established that evs released from lim crc cells stimulated wnt signalling pathway in the recipient cells with wild type β-catenin. silac-based quantitative proteomics analysis confirmed the transfer of mutant β-catenin to the nucleus of the recipient (rko crc) cells. in vivo tracking of dir labelled evs in mouse implanted with rko crc cells revealed its bio distribution, confirmed the activation of wnt signalling pathway in tumour cells and increased the tumour burden. introduction: there has been a significant increase in incidence of human papillomavirus (hpv ) driven oropharyngeal cancer (opc) in developed countries. there is evidence that hpv alters the molecular cargo of exosomes released by opc. emerging evidence suggests that hpv integration within the human genome is associated with both genomic and transcriptomic alterations. consistent with previous studies, the genomic viral-cellular junctions were identified using dips-pcr method in ( %) saliva samples collected from hpv -driven opc. methods: morphology and molecular features of exosomes derived from three different saliva sampling methods: unstimulated saliva; acid-stimulated saliva; and salivary oral rinses were examined using transmission electron microscopy (tem), nanoparticle tracking (nta) and western blot analysis. hpv- dna detection in salivary exosome was determined by using qpcr method. proteome profile of salivary exosomes derived from both cancer-free controls and hpv -driven opc patients was characterized using liquid chromatography-electrospray ionization-tandem mass spectrometry (lc-ms/ms). results: we demonstrate that unstimulated saliva had greater abundance of exosomes when compared to the other sampling methods. three common exosome markers (cd , cd and cd ) were higher in unstimulated saliva. only salivary exosomes derived from hpv-driven opc patients had a detectable level of hpv- dna. the proteomic signature of salivary exosome was significantly (p < . ) different between cancer-free controls and hpv-driven opc. we found elevated protein abundance of five main glycolytic enzymes (i.e. phosphoglycerate kinase (pgk ), glyceraldehye- -phosphate dehydrogenase (gapdh), aldolase (aldoa) and lactate dehydrogenase a (ldha) in salivary exosomes derived from opc patients, suggesting a functional role of salivary exosome in the reciprocal interplay between hpv-driven opc and glucose metabolism. summary/conclusion: our data suggest that the development of a low-cost non-invasive saliva-based test using both salivary exosomal dna and protein may offer an opportunity to detect hpv-driven opc, that may be clinically useful in managing these patients. continuous in vivo release of mast cell derived extracellular vesicles from an implanted device spreads pro-inflammatory response in mice introduction: mast cells are important players of the immune system and they secrete a wide range of mediators during bacterial infections. mast cells are also able to release extracellular vesicles (evs). here, we report that mast cells communicate with each other in vivo by evs. methods: we isolated bone marrow-derived and peritoneal mast cells from gfp-transgenic and wild type mice. evs were separated from the conditioned media of these cells cultured in the presence or absence of lipopolysaccharide (lps). evs were characterised according to the misev guidelines by flow cytometry, electron and fluorescent microscopy, trps, the spv lipid and the bca protein assays. separated ev-s were cultured with naïve mast cells, and tumour necrosis factor (tnf)-α production was tested by elisa and intracellular flow cytometry. gfp+ mast cells were seeded in diffusion chambers which were implanted into the peritoneal cavities of mice enabling us to investigate the continuous in vivo release of evs. uptake of gfp+ evs and tnf-α expression of peritoneal mast cells were tested by flow cytometry and fluorescent microscopy. results: here, we showed that bacterial lps-sensing mast cells release evs that in turn, induce tnf-α expression in resting mcs in vitro. moreover, we confirmed that evs are transmitted to other peritoneal mast cells in vivo spreading the pro-inflammatory response by inducing tnf-α secretion in peritoneal mast cells. summary/conclusion: ev communication between members of the mast cell network, play an important role in spreading and escalating pro-inflammatory responses to immune stimuli. our data may provide an explanation how the relatively rare tissue resident mast cells can play key roles in diseases such as autoimmune arthritis. the ability of small extracellular vesicles (sevs) to reprogramme cancer cells is known. integrins, receptors for extracellular matrix proteins, are major players in mediating sev functions. previously, we have reported that the αvβ integrin is detected in sevs of prostate adenocarcinoma (prca) cells and transferred into recipient cells in a paracrine fashion; however, its role and expression have never been explored in the most aggressive forms of prca, such as neuroendocrine prca (neprca). neprca does not express androgen receptor (ar) but does express neuron-specific proteins, such as aurora kinase a, synaptophysin and neuron specific enolase, that activate pro-tumorigenic pathways independently from the ar. methods: we isolated sevs from prca c - b cells using iodixanol density gradients and characterized them by immunoblotting and exoview. the experiments were performed in vivo by injecting subcutaneously, in nude mice, du cells treated with sevs expressing or lacking the αvβ integrin, and in vitro, by testing anchorage-independent growth of different cell lines treated with the same sevs. discarded human tissues from prca metastasis were analysed by immunohistochemistry (ihc). results: we demonstrate that a single treatment of prca cells with sevs significantly stimulates tumour growth and anchorage-independent growth. moreover, we show that one treatment with sevs, shed from c - b cells that express αvβ , but not from the control cells that lack αvβ , induces differentiation of prca cells towards a neuroendocrine phenotype and downregulates ar. finally, our ihc analysis shows coexpression of αvβ integrin and synaptophysin in neprca metastatic lesions. summary/conclusion: in conclusion, our current study shows, for the first time, that αvβ integrin expression in donor cells generates sevs that reprogramme recipient cells towards an aggressive tumour phenotype. funding: this study was supported by nci-p - , r - to lrl. introduction: exosomes are small extracellular vesicles (sevs) that carry a variety of cargoes and have been shown to promote tumour cell motility and metastasis. cell motility is influenced by dynamic formation and stability of filopodia: actin-rich protrusions that extend from the leading edge and perform directional sensing. filopodia regulators such as fascin are upregulated in multiple epithelial cancers and can promote invasive phenotypes. however, how filopodia are induced and controlled by extracellular factors is poorly understood. here, we describe a role for sevs in regulating filopodia formation and tumour cell motility. we utilized b f melanoma cells and ht fibrosarcoma cells for fixed-and live-cell imaging to quantify filopodia numbers and dynamics in control and exosome-deplete conditions. itraq proteomics was used to identify sev protein cargoes that contribute to filopodia formation. in vivo experiments were performed using a chick embryo model for metastasis. results: inhibition of exosome secretion in cancer cell lines, via rab a or hrs knockdown, led to decreased filopodia numbers. specificity to sevs was demonstrated by rescue experiments in which purified sevs but not large evs rescued the filopodia phenotypes of exosome-inhibited cells. live imaging of hrs-kd cells revealed that exosome secretion regulates formation and stability of filopodia. proteomics data and molecular validation experiments identified the tgf-beta coreceptor endoglin as a key sev cargo regulating filopodia formation, cancer cell motility, and metastasis. summary/conclusion: in this study, we identified exosomal endoglin as a regulator of filopodia formation and in vivo metastasis. these data are relevant to cancer as endoglin expression is altered in many cancers. in addition, endoglin is the disease gene for hereditary haemorrhagic telangiectasia, and may influence angiogenesis. overall, our data implicate sev-carried endoglin as a key cargo regulating filopodia. astrocyte-derived ev-mediated blood-brain barrier disruption shilpa buch, ke liao, susmita sil, fang niu and guoku hu university of nebraska medical center, omaha, usa introduction: the breach of the blood-brain barrier (bbb), resulting in ensuing neuroinflammation, is a key feature of hiv-associated neurological disorders (hands). while combination antiretroviral therapy (cart) has successfully suppressed peripheral viraemia, cytotoxicity associated with the presence of viral tat protein in tissues such as the brain, remains a significant concern. our previous study has demonstrated that hiv- tat can induce disruption of bbb by downregulation of tight junction (tj) proteins in human brain microvascular endothelial cells (hbmecs) and that this is regulated by the autophagic pathways. methods: evs were isolated from hiv tat-stimulated mouse/human primary astrocytes using the standard differential ultracentrifugation method and characterized by transmission electron microscopy, nanosight & western blot analyses. among the various mirs dysregulated in hiv tat -stimulated astrocyte ev cargo, mir- was found to be upregulated by realtime pcr. confocal microscopy identified uptake of astrocytic evs by hbmecs. functional assessment of astrocytic ev uptake by hbmecs involved cell permeability using transepithelial electrical resistance as well as trans-well endothelial cell monolayer permeability assays. results: hiv- protein tat-mediated induction of micrornas (mirs) in astrocyte-derived extracellular vesicles (adevs) regulated the permeability of bbb by targeting the expression of tj proteins in the hbmecs. exposure of hbmecs to tat-adevs resulted in down-regulation of the tight junction protein claudin , resulting in increased endothelial cell monolayer paracellular permeability. microarray data of tat-adevs demonstrated upregulation of several mirs compared to that of controls, among which upregulated mir- was identified to target the tj proteins using ingenuity pathways analysis. increased expression of mir- was validated in tat exposed astrocytes and tat-adevs. adevs loaded with mir- oligos showed similar effects as that observed with tat-adevs in inducing permeability in hbmecs. increased expression of mir- with downregulation of claudin- was also recapitulated in microvessels isolated from the brains of doxycycline-inducible hiv- tat transgenic mice (itat) mice and in lysates isolated from the frontal cortices of siv+ macaques/hiv+ autopsied brains. summary/conclusion: our findings demonstrated that tat-adevs containing mir- as an important mediator underlying tat-mediated disruption of the bbb. introduction: endogenous exosomes and related extracellular vesicles (evs) are potent nanoparticles released by all cells tested to date. the exploitation of their unique scaffolding for engineering next-generation drug delivery systems represents a major area of academic and commercial interest. the lag in exploiting this potential is in part due to our inability to measured extent and efficiency of modification, e.g., composition and drug loading. here we report a robust pipeline of optical tweezing combined with raman spectroscopy to molecularly characterize engineered evs and quantitatively assess extent of drug loading at single particle resolution. methods: evs derived from cell culture and isolated by ultracentrifugation were fused with synthetic liposomes to create engineered evs (eevs). these eevs were formed via well-established vesicle fusion techniques, namely ( ) mechanical extrusion, ( ) freeze-thawing, or ( ) probe-tip sonication. prior to formation, calcein was encapsulated in the liposomes and used as a surrogate for soluble drug loading. laser trapping raman spectroscopy (ltrs) was used to optically trap single evs, before and after synthetic manipulation. raman spectral analysis was used to assess trapped eevs compared to pure standards to quantify ratiometric variation in chemical composition. results: raman laser trapping experiments confirmed that each formation method results in largely varying ( ) extent of fusion between evs and synthetic calceinloaded liposomes, ( ) efficiency of calcein loading, and ( ) particle size. we could also quantify the molar amounts of liposome vs. ev molecules for single particles, revealing a great amount of variation from particle to particle. functional membrane proteins we left intact to varying degree across fusion methods. summary/conclusion: given the rising importance of analytical tools able to characterize extent of molecular loading for engineered evs, we believe this technology will be very useful, thus warrants further investigation for eev characterization across a variety of clinical applications. funding: randy carney, phd was supported by a research scholar grant, rsg- - - -cdd, from the american cancer society. extracellular vesicles containing host restrictive factor ifitm inhibited zika virus infection of foetuses in pregnant mice through trans-placenta delivery allen z. wu nanjing university, nanjing, china (people's republic) introduction: zika virus (zikv) infection can lead to neurological complications and foetal defects, and has attracted global public health concerns. effective treatment for zikv infection remains elusive and a preventative vaccine is not available yet. therapeutics for foetus need to overcome blood brain barriers to reach placenta and require higher safety standard. methods: in the present study, we engineered mammalian extracellular vesicles (evs) to deliver a host restrictive factor, interferon-induced transmembrane protein (ifitm ), for the treatment of zikv infection. results: our results demonstrated that the engineered ifitm -containing evs (ifitm -exos) were overall safe to the animals and suppressed zikv viraemia by log s in the pregnant mice. moreover, the engineered evs effectively delivered ifitm protein across placental barrier and suppressed overall zikv viraemia in the foetuses to the basal level with significant reduction of viraemia in key foetal organs as measured by q-pcr. mechanistic study showed that ifitm was delivered to the endosomes/lysosomes where it inhibits viral entry to the host cells. summary/conclusion: our study demonstrates that exosomes can act as a cross placenta drug delivery vehicle to foetus and ifitm , an endogenous restriction factor that is highly expressed in placenta, is a potential treatment for zikv infection during pregnancy. introduction: extracellular vesicles (ev) are natural and abundant nanoparticles capable of transferring complex molecules between neighbouring and distant cell types. translational research efforts have focused on co-opting this communication mechanism to deliver exogenous payloads to treat a variety of diseases. important strategies to maximize the therapeutic potential of evs include payload loading, functionalization of the ev surface with pharmacologically active proteins, and delivery to target cells of interest. methods: through comparative proteomic analysis (lc/ms) of purified evs, we identified several highly enriched and ev-specific proteins, including a transmembrane glycoprotein (ptgfrn) belonging to the immunoglobulin superfamily. leveraging ptgfrn as a scaffold for surface display, we generated evs with functional targeting ligands, including single domain antibodies (sdabs), single chain variable fragments (scfvs), single chain fabs (scfabs), and receptor ligands, on the surface to direct ev uptake to cell types of interest. biological activity of these engineered evs was assessed in an array of in vitro and in vivo assays and compared to untargeted controls. results: we engineered evs displaying anti-clec a scfabs to target conventional type dendritic cells (cdc s), anti-cd scfabs to target t cells, and cd ligand to target b cells. in mice, systemic administration of anti-clec a evs resulted in a % increase in the percentage of cdc cells that take up evs over controls. anti-cd evs resulted in both an increase in the percentage of ev positive t cells ( . and -fold for cd + and cd +) and the number of evs per cell ( and -fold for cd + and cd +) in the blood. furthermore, in primary mouse dendritic cells, anti-clec a evs loaded with sting agonist achieved a fold greater pathway induction compared to untargeted controls. preliminary in vivo data suggest that anti-clec a evs reduce the required sting agonist dose -fold to achieve efficacy and induce anti-tumour responses, compared to control evs. summary/conclusion: these results demonstrate the potential of our ev engineering platform to generate novel ev therapeutics targeted to cell types of interest for pharmacologic payload delivery. a novel method for the delivery of cell-free therapy to foetuses with congenital anomalies: a proof of principle study lina antounians, louise montalva, gabriele raffler, maria sole gaffi and augusto zani the hospital for sick children, toronto, canada introduction: antenatal cell-based therapies are currently considered invasive for the foetus. a promising cell-free strategy that holds great regenerative potential for several organs is the administration of stem cell derived evs, whose cargo contains bioactive molecules that epigenetically regulate target cells. herein, we aimed to ) assess the ability of evs to reach foetal organs when administered to the mother intravenously or intra-amniotically; ) compare these administration routes on normal foetuses and foetuses with a congenital anomaly. methods: evs were isolated from rat amniotic fluid stem cell conditioned medium using ultracentrifugation. evs were assessed for size (nanoparticle tracking analysis), morphology (tem), and expression of cd , hsp , flo- , and tsg (western). we injected rat dams with evs stained by exoglow™-vivo or saline (control) via maternal tail vein (iv) or intra-amniotically (ia) at e . . ia and iv injections were performed on dams carrying normal foetuses or foetuses exposed to nitrofen to induce congenital diaphragmatic hernia. after h, dams and pups were sacrificed. d high-sensitivity optical reconstructions of whole foetuses or micro-dissected foetal organs were imaged using the ivis® spectrum imaging system. ev fluorescence signal was compared between normal (n = ) and nitrofen-exposed (n = ) foetuses. results: both iv and ia injection routes were successful in delivering evs to foetal organs. no fluorescent signal was detected in saline only control. ia injections yielded higher signal than iv, and evs reached more organs with ia than iv injections. ia injected evs were detected in the lungs, gastrointestinal, and urinary tract of normal and nitrofen-exposed foetuses. nitrofen exposed foetuses had higher signal than normal foetuses. summary/conclusion: this proof of concept study shows that antenatal administration of stem cell evs is feasible with different routes. although maternally administered evs cross the placenta, ia injection is more effective at reaching foetal organs. further studies are underway to reproduce these findings in experimental models of various congenital anomalies. funding: cihr-sickkids foundation grant os . introduction: safe, efficient and specific nano-delivery systems are essential to the current cosmetic, nutraceutical and therapeutic medicine sectors. the ability to optimise the bioavailability, stability, and targeted cellular uptake of bioactive molecules while mitigating toxicity, immunogenicity and off-target/side effects is of the utmost priority. ves us is a european project, which aims to develop an innovative platform for the efficient production of extracellular vesicles (evs) from microalgae, which constitute a promising renewable bioresource (www.ves us.eu). here we present characteristics of evs from several microalgal lineages, which offer the opportunity for a potentially developing a new and scalable tailor-made biogenic nanotechnology. methods: we cultivated a number of ev-producing microalgal species and developed protocols for ev isolation both at laboratory (differential ultracentrifugation) and pilot scales (tangential flow filtration). the physico-chemical characterization of microalgal evs was carried out according to the minimal information for studies of extracellular vesicles (misev- guidelines): biochemical methods to verify the presence of specific ev-biomarkers, tuned for microalgal evs; dynamic light scattering (dls) and nanoparticle tracking analysis (nta) to assess the particles number and size distribution; electronic scanning microscopy (sem), atomic force microscopy (afm), and cryo transmission electron microscopy (cryo-tem) for imaging analyses; bilayer-specific fluorescence staining (f-nta) to test the purity of ev preparation. results: we identified microalgae as a novel natural source of evs that could constitute a cost-effective and sustainable way of mass-producing them. we screened strains of microalgae and generated an "ev identity card" for each, which contained a variety of ev features relating to their biophysical, biochemical and biological characteristics in line with the misev- . our approach will next focus on the scalable production, surface functionalization and bio-engineering of selected microalgal evs. at the same time, their bioactivity will be explored using both in vitro and in vivo biological models. summary/conclusion: the ves us consortium is investigating the potential of microalgae as novel ev bioresources. this research will attempt to bioengineer novel naturally-derived nanocarriers, microalgal evs, suitable for the development of future cosmetics, nutraceutical or therapeutic formulations. funding: this project has received funding from the european union's horizon research and innovation programme under grant agreement no . sequence-specific rna trafficking to extracellular vesicles is conserved across cell types several sequences have been identified that act as a zipcode for preferential rna targeting into ev (evtropic) or for retention in parental cells (cell-tropic) . in this work, we aimed to compare the ev-tropic capacity of specific rna sequence motifs in promoting loading into ev, across different cell models representing the main cell types found in the body. methods: immune, epithelial and mesenchymal cell lines were transiently transfected with xenogeneic c. elegans micrornas (mirnas) containing ev-tropic or cell-tropic sequences and grown in culture. ev were isolated from the supernatant by differential (ultra)centrifugation. rna was extracted from both cell pellets and isolated ev fraction, and target mirnas were quantified by digital droplet pcr. distribution of cargo mirna across cells and ev was also analysed for chimeras of ev-and cell-tropic sequences. results: the mirnas containing an ev-tropic sequence were highly enriched on the ev fraction, with - , higher levels than in parental cells. contrarily, cell-tropic mirnas were only - times higher in ev. no significant differences were observed in the ev loading efficiency for the various ev-tropic motifs tested. mutations in the ev-sorting motif resulted in reduced ev loading. ev-tropic sequences consistently promoted mirna loading into ev across all the cell models evaluated, suggesting conserved biological mechanisms. summary/conclusion: we showed that rna loading into ev is dependent on the presence of defined evtropic rna motifs, and that sorting mechanisms are conserved across the major cell types tested. the highest loading efficiencies resulted in . mirna copies per particle on average, suggesting a limited scope for ev-tropic motifs for therapeutic rna loading into ev. funding: as, os and eli are fellows of the astrazeneca postdoc programme. introduction: coordinated activity between pancreatic islet cells is critical for the regulation of glucose homoeostasis. chronic exposure to diabetogenic factors such as pro-inflammatory cytokines, perturb islet cell crosstalk and β-cell function in diabetes. extracellular vesicles (evs) derived from cytokine-exposed β-cells modulate physiological and pathological responses to β-cell stress. however, the mechanisms governing this process remain largely unknown. we set out to test the hypothesis that β-cell failure in diabetes is mediated in part through β-cell autocrine release of pro-inflammatory evs which promote inflammation and inhibit βcell function. methods: pro-inflammatory cytokine-exposed evs (cytoevs) were generated using conditioned media from mouse min β-cell line treated with diabetogenic cytokines (tnfα, il- β, ifnγ, h). evs were also isolated from human type diabetic (t dm) and lean non-diabetics (lnd) plasma. gw (n-smase inhibitor) was used in the presence of cytokines to determine the effect of reduced ev concentrations on the restoration of β-cell function. proteomic and rna-seq analysis was conducted on min β-cell cytoev (vs. control ev) and cytoev treated mouse islets, respectively. results: assessment of ev concentrations from cytoev and human t dm plasma revealed a~twofold increase (p < . , vs. control (ctl) and lnd ev). immunofluorescence staining of cd and cd expression was significantly elevated in human t dm pancreas (p < . , vs. lnd). while acute inhibition of ev formation with gw ( µm) showed significant restoration in β-cell function (glucose stimulated insulin secretion assay, gsis) in cytokine-exposed mouse and human islets (~ and fold vs. cytokines alone, p < . ). moreover, functional assessment of mouse islets exposed to cytoev ( h) resulted in suppression of gsis (~ %, vs. untreated, p < . ). identification of cytoev content through proteomic analysis revealed a significant upregulation of the chemokine, cxcl (~ fold vs. ctlev) and rna-seq analysis of cytoev treated mouse islets depicted a marked upregulation of transcripts associated with cxcl -cxcr signalling (p < . ) and downstream pathways (e.g. nfκb; p = . and jak/stat; p = . ). furthermore, inhibition of cytoev (gw ) with cytokines markedly decreased cxcl (~ %) and cxcr receptor (~ %) expression in min β-cells. summary/conclusion: these data suggests that cytokines elevate cxcl expression in β-cell ev to enhance inflammation-induced diabetes. this is mediated through ev-autocrine release of cxcl consequently activating cxcr signalling and downstream pathways to impair β-cell function in diabetes. synergy between -lipoxygenase and secreted pla promotes inflammation by formation of tlr agonists from extracellular vesicles introduction: damage associated molecular patterns (damps) are endogenous ligands that induce innate immune response, thus promoting sterile inflammation. during oxidative stress, stress-derived evs (stressevs) were found to activate toll-like receptor (tlr ), but the activating ligands were not fully determined. additionally, several enzymes, among them -lipoxygenase ( -lo) and secreted phospholipase a (spla ) are induced during inflammation and were suggested to promote damp formation. methods: stressevs were produced from hek cells exposed to um a and isolated with ultracentrifugation. : lysopi was oxidized for min with -lo. additionally, synevs were prepared from phospholipids (pls), oxidized with -lo and hydrolysed with spla . activity was measured by qpcr and elisa on wt and tlr -ko macrophages. -lo oxidized : lysopi was analysed by mass spectrometry. spla activity was measured in synovial fluid from rheumatoid and gout patients using fluorometric assay. k/bxn serum transfer induced arthritis model on wt and tlr ko mice (c bl/ mice) with spla -iia injection was used (approval no. u - / / by mkgp of slovenia). results: stressevs released after oxidative stress were found to activate tlr with a gene profile different from bacterial lipopolysaccharide (lps). stressevs, -lo oxidized synevs, but only -lo oxidized lysopls activated cytokine expression through tlr /md- . hydroxy, hydroperoxy and keto products of : lysopi oxidation were determined by ms and they activated the same gene pattern as stressevs. furthermore, spla activity, which we detected in the synovial fluid from patients, promoted formation of tlr agonists after -lo oxidation. injection of spla -iia into mice promoted k/bxn serum induced arthritis in tlr -dependent manner. summary/conclusion: both -lo and spla are induced during inflammation, therefore these results imply the role of oxidized lysopls in stressevs in promoting sterile inflammation through tlr signalling. the formation of tlr agonists is enzyme driven so it provides an opportunity for therapy without compromising innate immunity against pathogens. funding: h -msca-itn project tollerant (grant no. ), slovenian research agency (project no. j - to mmk, research core no. p - to rj). monocytes traffic extracellular vesicles to damaged muscle and adopt a novel immunophenotype to support muscle regeneration russell g. rogers, akbarshakh akhmerov, weixin liu, lizbeth sanchez and eduardo marbán smidt heart institute, cedars-sinai medical center, los angeles, usa introduction: extracellular vesicles (evs) are secreted membrane vesicles that carry bioactive molecules such as mirnas, mrnas, proteins, and lipids to modify recipient cell behaviour. we recently demonstrated evs secreted by cardiosphere-derived cells (cdc-evs) augment endogenous muscle regeneration in mdx mice, a model of duchenne muscular dystrophy, when delivered intravenously. in parallel, macrophages preferentially accumulate surrounding small regenerating myofibers in cdc-ev treated mdx muscle. however, it is currently unclear how intravenous cdc-evs home to dystrophic muscle and exert their therapeutic bioactivity. methods: fluorescently-labelled and unlabelled cdc-evs were infused into the contralateral femoral vein of wild-type mice with unilateral muscle injury induced by bacl . injured and uninjured muscles were dissected h following infusion and subjected to optical imaging, immunohistochemistry, and confocal microscopy. this experiment was repeated using clodronate liposomes to deplete endogenous monocytes/macrophages. next, rna-seq was preformed on bone marrow-derived m , m , and cdc-ev (mcdc-ev) polarized macrophages from mdx mice. conditioned media (cm) from these macrophages were tested in an in vitro model of myogenesis. lastly, small rna-seq was performed on evs secreted by m , m , and mcdc-ev macrophages. results: when delivered intravenously, cdc-evs naturally home to injured, but not uninjured, skeletal muscle. cdc-evs were detected in the interstitium adjacent to non-muscle cells, macrophages, and within surviving myofibers. after depletion of monocytes/ macrophages by clodronate liposomes, the presence of cdc-evs in the injured muscle was attenuated. bioinformatic analyses indicate cdc-evs confer a novel immunophenotype to mdx macrophages with features of both m and m . indeed, mcdc-ev cm promotes myoblast proliferation and supports myogenic differentiation. interestingly, mcdc-ev evs have a unique mirna signature and contain several mirnas with known roles in myogenesis. summary/conclusion: these data indicate circulating monocytes traffic cdc-evs to damaged muscle where they adopt a novel immunophenotype to support muscle regeneration. we propose mcdc-ev macrophages mediate their pleiotropic effects via paracrine factors, possibly including evs. introduction: microglia, the immunocompetent cells of the cns, play an important role in maintaining cellular homoeostasis in the cns. these cells secrete immunomodulatory factors including nanovesicles and participate in the removal of cellular debris by phagocytosis or autophagy. the contribution of microglial-derived extracellular vesicles (m-evs) to the maintenance of cns homoeostasis is unclear. in addition, knowledge of canonical signalling pathways of inflammation and immunity gene expression patterns in human microglia exposed to m-evs is scarce. methods: here, we analysed the effects of m-evs produced in vitro by either tnfα-activated or non-stimulated microglia bv cells. we showed that m-evs are internalized by both mouse bv and human c microglia and that the uptake of m-evs in microglia induced autophagic vesicles at various stages of degradation including autophagosomes and autolysosomes. consistently, exposure of microglia to m-evs increased the protein expression of the autophagy marker, lc b-ii, and promoted autophagic flux in live cells. to elucidate the biological activities occurring at the transcriptional level in c microglia exposed to m-evs, the gene expression profiles, potential upstream regulators, and enrichment pathways were characterized using targeted rna sequencing. results: inflammation and immunity transcriptome gene panel sequencing of both activated and normal microglia exposed to m-evs showed involvement of several canonical pathways and reduced expression of key genes involved in neuroinflammation, inflammasome and apoptosis signalling pathways compared to control cells. summary/conclusion: we demonstrate that in vitro produced microglial evs are able to influence multiple biological pathways and promote activation of autophagy in order to maintain microglia survival and homoeostasis. funding: this work was financed by hasselt university and by efro through the interreg v grensregio vlaanderen nederland project trans tech diagnostics. evaluation of plasma extracellular vesicles as biomarkers for longevity xin zhang a and virginia kraus b a laboratory medicine center, nanfang hospital, southern medical university, guangzhou, guangdong, , p. r. china, guangzhou, china (people's republic); b division of rheumatology, duke molecular physiology institute, duke university school of medicine, durham, usa introduction: extracellular vesicles (evs) have emerged as key indicators and effectors of ageing. although plasma concentrations of evs decline with age, the ev biomarkers associated with ageing and longevity are not fully understood. recently, our group found an age-related decline of plasma evs associated with immune cells during normal human ageing. our study aims to evaluate the association of plasma evs with longevity. methods: plasma samples were selected from the established populations for epidemiologic studies of the elderly study subjects (n = ): half dying within years (short-lived group) and half surviving ≥ years (long-lived group) after the blood draw; all matched for age (median age . ± . years, range - ), gender ( % female), and race ( % white/ % black). the samples were acquired under donor consent and irb approval of duke university. evs were separated from the plasma samples, and profiled based on the surface markers of haematopoietic stem cells (hscs), mesenchymal stem cells, immune cells, skeletal muscles, cardiac muscles and adipocytes (cd , cd , cd , cd , cd , cd , cd , cd , cd , cd , cd , cd a, cd a, cd , cd , hla-abc, hla-g, hla-drdpdq, cd , cd , cd , m cadherin, ryr , ryr , fabp , dlk ). the percentages of evs expressing each tested molecule were determined using a high-resolution multicolour bd lsr fortessa x- flow cytometer as we recently reported. graphpad prism . software was used for statistical analysis. results: we found significantly increased percentages of cd +, hla-abc+, cd + and cd a+ large evs ( - nm) in the long-lived compared to the short-lived group. none of the tested surface marker expressing medium ( - nm) or small (< nm) evs showed differential percentages between the shortand long-lived groups. summary/conclusion: evs carry surface markers from their parent cells. cd is expressed by hscs and immune cells. cd regulates homing of human cord blood cd + hscs, and delivers a potent cd independent costimulatory signal to activate t cells. hla-abc, the key human immunogen, is expressed by nucleated cells and platelets. cd is expressed by hscs, immune cells and epithelial cells, and cd + plasma evs declined with age in healthy people. cd a is expressed by hscs, megakaryocytes and platelets, and is functionally relevant for hsc maintenance and haematopoietic homoeostasis. our preliminary data suggest that hscs and immune cell associated plasma evs (cd +, hla-abc+, cd +, cd a+ large evs) inform on health status related to longevity. introduction: it is anticipated that stem/progenitor cells-derived extracellular vesicles (spc-evs) will rapidly progress towards clinical studies, and the development of reproducible, efficient, scalable and costeffective process for their production is expected to boost the therapeutic applications of evs-based products. in addition, the use of defined serum-/xenogeneic(xeno)-free culture medium formulations could result in substantial improvements for spc-evs production in terms of reproducibility, stability and quality, while ensuring the approval of regulatory agencies. the main goal of this work is to develop a full-controlled manufacturing platform for the spc-evs production. methods: human mesenchymal stromal cells (msc) were expanded in a xeno-free microcarrier-based bioreactor culture system operating in fed-batch feeding mode and after days the conditioned medium was collected. different methods for spc-ev isolation/purification from the msc-derived conditioned medium, including chromatography were compared and the the quality of the final product obtained was characterized by different methods according to misev, including nanoparticle tracking analysis, lipidomics and western blot. moreover fourier-transform infrared (ftir) spectroscopy was evaluated in terms of its implementation as a standard technique for the identification and characterization of evs. results: after days of msc expansion under dynamic conditions, we collected . l of conditioned medium with approximately . million evs/msc. a combination of a pretreatment with a nuclease for the digestion of dna/chromatin with a purification using strong anion exchange chromatography led to the best results so far in terms of evs isolation. of notice, by ftir spectroscopy, it was possible to define ratios of spectral bands, that can be used as biomarkers, enabling the discrimination of evs chemical fingerprint in function of the culture conditions tested. summary/conclusion: the platform established herein could be applied to the production of wellcharacterized spc-evs targeting their biomedical use in different settings (e.g. as drug delivery systems), as well as evs from other parental cells lines (i.e. dendritic cells) in therapeutic settings as cancer. ultrasensitive protein detection for quantification of extracellular vesicles in human biofluids enables comparison of isolation techniques dmitry ter-ovanesyan, maia norman, wendy trieu, roey lazarovits, george church and david walt wyss institute, boston, usa introduction: extracellular vesicles (evs) are released by all cells into biofluids and hold great promise as reservoirs of disease biomarkers. one of the main challenges in studying evs and using them in diagnostics is a lack of suitable methods to quantify evs that are sensitive enough and can differentiate evs from similarly sized lipoproteins and protein aggregates. we propose using ultrasensitive single molecule array (simoa) assays to quantify evs by immunoisolating and detecting ev transmembrane proteins in microwell arrays. we developed single molecule array (simoa) assays using the quanterix hd-x analyser for the quantification of evs using the tetraspanins cd , cd , and cd . simoa allows for the detection of single proteins using arrays of femtoliter wells, turning elisa into a digital immunoassay. we then used these assays, together with an additional assay for albumin, to compare commonly used ev isolation methods from plasma and cerebrospinal fluid (csf): ultracentrifugation, precipitation (exoquick), and size exclusion chromatography (sec) using the izon qev columns. we further used these assays to rapidly optimize and improve sec by comparing different sec resins and column dimensions in both plasma and csf. results: in comparing our simoa assays to traditional elisa with the same antibodies, we found that the simoa assays were more than times more sensitive, detecting the tetraspanins in samples where the proteins were undetectable by elisa. given the high dynamic range and high-throughput capabilities of simoa, we were able to comprehensively compare relative ev yields and ev purity for different isolation methods of evs from plasma and csf. we provide average tetraspanin and albumin levels to directly compare the methods. we also tested different sec resins and provide data for custom sec columns that outperform izon qev and allow for fine tuning of different ratios of evs to albumin. summary/conclusion: our results highlight the utility of quantifying evs using ultrasensitive simoa assays for tetraspanins. we were able to rapidly simoa to rapidly evaluate different ev isolation methods in csf and plasma. in general, the experimental framework we present could be easily applied to evaluate new ev isolation methods, or applied to any other biological fluid. thus, we think simoa is a powerful new tool for relative ev quantitation. introduction: the protein profile of extracellular vesicle (ev) subpopulations has been shown to contain valuable disease information, notably in cancer. currently, techniques aiming to find ev proteins that associate together mainly focus on transmembrane proteins, while methods that also probe cytosolic proteins generally resort to a combination of affinity capture, elution, and lysis, which limits throughput. to allow the high-throughput analysis of both membrane and cytosolic ev proteins, we optimized a total extracellular vesicle antibody microarray (tevam) incorporating fixation and heat-induced epitope retrieval (hier), then leveraged it to perform combinatorial protein profiling of evs from colorectal cancer (crc) cell lines ht and sw . methods: arrays of iggs targeting surface protein markers were incubated overnight with evs purified from cancer cell line supernatants. hier optimization was carried out through variation of buffer contents, presence or absence of prior permeabilization, as well as incubation time and temperature, for a total of conditions. a evs, previously profiled with other methods, were used as a model during the optimization. cytosolic protein hsp and membrane marker egfr, both with high expression in a evs, were probed and the results used to compare hier conditions. following hier treatment, protein targets were detected through incubation with primary antibodies and fluorescent secondary antibodies or streptavidin. the resulting optimized tevam workflow was used to phenotype ht and sw evs through probing of trios of surface ( ) and internal ( ) protein targets. results: the selected tevam protocol successfully maximized hsp signal while minimally affecting egfr detection, enabling simultaneous analysis of surface and internal proteins. profiles of more than combinations, featuring integrins, claudins, cytokines, and other key actors of cancer-relevant pathways, were obtained for ht and sw evs, revealing coexpression patterns that highlight the biomolecular heterogeneity both within and between crc cell line evs. summary/conclusion: using tevam, intra-and extravesicular proteins can be detected simultaneously in evs immobilized based on surface protein content, yielding extensive combinatorial protein profiles with significance for health and biomarker research. characterization of evs using orthogonal techniques identifies discrete ev populations from a mouse dendritic cell line bryce killingsworth a , timothy traynor b , joshua a. welsh c , aleksandra dakic a , jason savage a , kevin camphausen d , kenneth aldape a and jennifer jones a a laboratory of pathology, national cancer institute, national institutes of health, bethesda, usa; b laboratory of pathology, national cancer institute, national institutes of health, gaithersburg, usa; c laboratory of pathology, national cancer institute, national institute of health, bethesda, usa; d radiation oncology branch, national cancer institute, national institutes of health, bethesda, usa introduction: extracellular vesicles (evs) have the potential to serve as valuable biomarkers for patient response to cancer therapy. however, development of robust ev-based clinical assays relies on knowledge of ev concentration and diameter distribution. many different methods exist to measure the size and concentration of evs, and each method exhibits strengths and limitations. it is important to use orthogonal methods for determination of these important properties of ev preparations. here, we use dendritic cellderived evs to demonstrate that some ev analysis methods can give a biased interpretation of both diameter and concentration. through comparison, we highlight why orthogonal assays are essential in providing measurement reliability. methods: dc . mouse dendritic cells were cultured in flasks containing a total of . l of ev-depleted media ( % fbs, centrifuged hr. x , g.) when cells reached % confluency, conditioned media was collected, depleted of debris with two min. x , g spins, and concentrated down to~ ml using a pall jumbosep kda mwco filter. the ev concentrate was purified from protein using an izon qev- column, with ml fractions collected. the protein content of the ev-containing fractions was analysed by a , pierce bca, and bioanalyzer. the diameter distribution of the evs was determined by nanoparticle tracking analysis (nta), resistive pulse sensing (rps), flow cytometry (fcm), and electron microscopy (em.) concentration was compared using nta, rps, and fcm. evs were further analysed by protein mass spectrometry and rna sequencing. results: we have identified two distinct populations of evs with our dc . preparation, one highly abundant population with a power-law distribution, whose peak diameter is below nm, and a second, less abundant population with a peak diameter at approximately nm. these two distinct populations and their relative concentration were not detectable with all analysis techniques. based on cross-platform measurements, these populations appear to have distinct compositions that warrant further investigation. summary/conclusion: the use of orthogonal methods allowed the detection of two discrete populations of evs which was not possible on some platforms and would have resulted in a biased perspective of the sample composition. this work has highlighted the need for orthogonal measurements to be conducted by pairing techniques that do not have the same biases. introduction: extracellular vesicles (evs) are nanosized vesicles shed by all cells that serve vital roles in cell-to-cell communication. tumour-associated ev subpopulations vary in molecular content (lipids, proteins, nucleic acids, small molecules), enabling minimally invasive spectroscopic analysis for a wide variety of cancers. here, we use surface-enhanced raman spectroscopy (sers) in combination with a novel plasmonic substrate for global chemical composition analysis of cancerous and non-cancerous populations of evs to determine distinguishing surface characteristics. methods: evs were isolated from ovarian cancer (ovca) patient serum samples by differential ultracentrifugation. a new hybrid nanoplasmonic scaffold comprised of a microscale biosilicate diatoms embedded with silver nanoparticles (agnps) was used for sers measurements. the substrate was incubated with cysteamine to positively-charge the agnps (responsible for the sers enhancement) so that evs could attach (evs are naturally anionic). in a typical experiment, μl of~ particles/ml evs per sample were incubated with the porous substrate surface, which was inverted on a glass cover slip for raman interrogation. principle component analysis (pca) was used to compare the spectra and determine distinguishing characteristics between populations from tumour and non-tumour sources. we also trypsinized evs before sers analysis to see the extent of influence the surface molecules play in localizing the evs to the agnp "hot spots." results: a total of clinical samples ( ovca and non-malignant control) were tested in combination with ovca skov- cell line evs. simple pca was able to separate clinical samples according to disease subtype and major peaks were identified to provide chemical content analysis. each sample exhibited inherent heterogeneity but clustered together in a distinguishable way from the others. summary/conclusion: despite innate heterogeneity within single samples (i.e., evs isolated from a single patient sample), evs isolated from clinical samples could be easily distinguished from each other using our hybrid sers substrate, with minimal sample processing, a label-free approach, and only a few microlitres of sample. our study using this novel plasmonic material demonstrates its potential for use as a component in next-generation diagnostic platforms. introduction: single-particle analysis is critical for understanding extracellular vesicle (ev) heterogeneity. yet such techniques remain technically challenging due to low detection sensitivity and presence of variable amounts of "contaminants," including lipoproteins. the high degree of structural similarity between evs and lipoproteins in size, density, and chemical composition, results in their co-isolation using any of the standard ev isolation techniques. here we introduce laser trapping raman spectroscopy (ltrs) as a wellsuited, label-free, and non-destructive tool to distinguish evs from various lipoprotein species at single particle resolution. methods: ev samples were isolated from skov- cell culture supernatant by differential ultracentrifugation and their raman spectra measured. as the most abundant lipoproteins in ev isolations from human biofluids are sub-micron low density lipoprotein (ldl), very low density lipoprotein (vldl), and high density lipoprotein (hdl) particles, these were purchased as pure components and also measured by ltrs. ldl and vldl were then spiked-in to isolated evs to mimic "contaminated" post-isolation ev samples. raman spectra were analysed by principal component analysis (pca) using a custom matlab script. results: ldl and vldl have been observed to adhere to ev surfaces in vitro after standard isolation techniques. we could readily distinguish pure vldl, ldl, and hdl standards according to their raman spectra. pca revealed distinction of skov- evs from both ldl and vldl. pca also differentiated skov- evs incubated with ldl from skov- evs incubated with vldl. extent of ldl and vldl adherence to evs could be observed and quantified. summary/conclusion: through raman and pca, classes of lipoprotein and evs can be identified and quantified when co-incubated. ltrs is a quantitative single-ev analysis technique that can be used to differentiate between lipoprotein classes and evs when incubated together. this technique allows for analysis of evs where standard isolation methods fall short. introduction: extracellular vesicles (evs) are endogenous membrane-derived vesicles that shuttle lipids, proteins or nucleic acids between glia and neurons, thereby promoting neuronal survival and plasticity in the cns and contributing to neurodegenerative conditions. although evs hold great potential as cns theranostic nanocarriers, the specific molecular factors that regulate neuronal ev uptake and release are currently unknown. methods: we used a combination of patch-clamp electrophysiology and ph-sensitive dye imaging to examine stimulus-evoked ev release in individual neurons in real time. results: whereas spontaneous electrical activity and the application of a high-frequency stimulus (hfs) induced a slow and prolonged fusion of multivesicular bodies (mvbs) with the plasma membrane (pm) in a subset of cells, the neurotrophic factor bfgf (basic fibroblast growth factor) greatly increased the rate of stimulusevoked mvb-pm fusion events and, consequently, the abundance of evs in the culture medium. proteomic analysis of neuronal evs demonstrated bfgf to increase the abundance of the v-snare vesicle-associated membrane protein (vamp , cellubrevin) on evs. conversely, knocking-down vamp in cultured neurons attenuated the effect of bfgf on ev release. introduction: heat shock proteins (hsps) function as chaperones under both normal and pathologic conditions. as chaperones they assist in protein folding, in holding protein complexes for current or future activation, and in degradation of senescent proteins for recycling of components and display for immune surveillance. during stressful situations, hsp quantities and/or activities increase as cells and tissues seek protection from insults. these insults can result in the cell surface display of hsps, which can then lead to the surface display of hsps on extracellular vesicles (evs). hsps present on the cell surface or in the extracellular space are regarded as "danger signals" in an ancient biologic paradigm. hsp-accessorized evs may act as "danger boli", carrying not only the hsps, but hundreds of components of the stressed parental cell, capable of prompting differential responses depending on the status of the recipient cell. methods: clarified/filtered plasma from patients suffering from neurologic maladies (cancer, brain injury, multiple sclerosis) was incubated with peptides designed to bind hsps. the evs congeal under these conditions and are pelleted (microfuge) and washed with increasing-stringency buffers. we lysed the evs and subjected them to metabolomic analyses (focused on lipids) or assayed them on phosphokinase arrays. results: we show that evs from the blood of patients suffering from brain tumours, or from tbi, or from ms, possess distinct metabolomes compared to blood evs from healthy donors. we found hundreds of differentially-expressed lipids amongst the patients vs the healthy donors. the levels of annotation and identification for these compounds ranges from level (low, no matches in databases) to level (high, annotation matches to known database components). in addition, we found differences in phosphorylated kinases as cargo in these evs between patients with matched primary vs recurrent gliomas, and among tbi/stroke patients compared to healthy donors. summary/conclusion: hsp-accessorized evs present different metabolomic and phosphokinase content which may serve as biomarkers in a "liquid biopsy" setting, but may also play roles in the pathobiology of neurologic diseases. introduction: methamphetamine (ma) has deleterious effects to both peripheral organs and the central nervous system. the rewarding properties and addictive potential of ma are correlated with increased synaptic dopamine availability following alterations in dopamine and vesicular monoamine transporter function. in rodents, ma alters brain mirna expression and the mirna content of serum extracellular vesicles (ev). here we examined plasma evs isolated from human subjects actively using ma (ma-act) for size, concentration, protein markers, and mirna content. methods: plasma samples from ma-act, and controls (ctl) were obtained from the methamphetamine abuse research center. plasma evs were evaluated by vesicle flow cytometry (vfc) for size, concentration, and surface protein markers. vfc antibodies included markers for a pool of tetraspanins (cd , cd , and cd ), platelet evs (cd ), pro-coagulant evs (annv), and red blood cell evs (cd ). next plasma ev isolated by size exclusion chromatography were analysed by qpcr on taqman® array human microrna a + b card set v . . fold change was calculated by ΔΔcq between ma-act and ctl for mirna expressed in ≥ % of samples in at least group. we identified the top % of ranked mirna by fstatistic; of these, the mirna of interest for ma-act were identified by at least a (i) . fold change in expression, (ii) area under the receiver operating characteristic curve of . , and (iii) glass's Δ of . for mirna of interest correlations to additional ma variables were conducted, along with ingenuity pathway analysis of predicted gene targets. tobacco use was controlled for. results: vfc data show that the size (~ nm) and concentration (~ . x particles/ml) of all plasma evs is comparable between ma-act and ctl groups. in addition, the plasma evs primarily consist of tetraspa-nin+, annv+, or cd + evs, and to a much lesser extent cd + evs. of the mirna expressed in ma-act and/or ctl plasma evs, there were mirna that have at least a . fold increase or decrease in ma-act. mirna were identified to be of interest in ma-act based on fold change, effect size and diagnostic potential, compared to ctl. further, of the mirna correlate with a ma associated variable, including frequency of use and age of first use. together the mirna best cluster subjects based on ma-act status, not tobacco use. finally, the predicted gene targets of the mirna are associated with canonical pathways linked to ma. summary/conclusion: ev mirna expression in ma-act subjects was unique to ctl participants, suggesting that ma may affect ev communication among cells. the differential mirna expression also implicates a role for evs in behavioural and physiological effects specific to ma, and suggests that there may be changes in expression of mirna that are relevant to specific drugs of addiction, as well as to a spectrum of drug-mediated addiction disorders. bone marrow-derived extracellular vesicles may alter the ageing phenotype of murine haematopoietic stem cells. sicheng wen a , jill kreiling b , mark dooner c , elaine papa c , michael del tatto c , yan cheng c , mandy pereira c , peter quesenberry c and laura r. goldberg d in natural ageing of haematopoietic stem cells (hscs) is unclear. we tested the hypothesis that bone marrowderived evs (bm-evs) can modulate the ageing hsc phenotype. methods: we flushed bone marrow from old ( - month old) and young ( - -week old) c /bl mice and collected bm-evs by differential centrifugation ( × g for min, supernatant collected and centrifuged , × g for hour, bm-ev pellet collected and quantified by nanoparticle tracking analysis). we injected old mice with x ^ young bm-evs via tail vein, daily x days. control mice were injected with age-matched bm-evs or vehicle alone. we euthanized the mice one month post-injection, harvested whole bone marrow (wbm) and highly purified hscs (lineage negative/c-kit+/sca- +/cd +; lsk-slam) and tested stem cell function in competitive bone marrow transplants ( - recipients/group). results: at months post-transplant, wbm from old mice exposed to young bm-evs exhibited a statistically significant decrease in engraftment when compared to wbm exposed to age-matched bm-evs (percent average donor chimerism ± sem: % ± % (young evs) vs. % ± % (old evs)). for lsk-slam from old mice, we observed a trend towards decreased engraftment when exposed to young bm-evs and a trend towards increased engraftment potential when exposed to old bm-evs (percent average donor chimerism ± sem: % ± % (young evs), % ± % (old evs), % ± % (vehicle)). these findings are consistent with our previous data showing that, in contrast to highly purified hscs which develop impaired stem cell function with ageing, total un-separated old wbm actually has increased engraftment capacity when compared to young wbm. of note, we found that the classic myeloid skewing by old lsk-slam was partially reversed by exposure to both young and old bm-evs. finally, consistent with the known increase in highly purified hscs with age, our preliminary data showed that old mice exposed to young bm-evs had an approximately -fold decrease in the number of lsk-slam cells in marrow, indicating that bm-evs may influence agerelated changes in hsc number. summary/conclusion: these preliminary data suggest bm-evs may play a role in modulating hsc ageing phenotypes, potentially altering engraftment capacity, lineage fate, and lsk-slam population size. future studies delineating the molecular mechanisms underlying these ev-mediated effects could provide key insights into normal haematopoietic ageing. funding: this work was supported by the nih grants p gm , dk - a and by the savit foundation. oral with poster introduction: brain extracellular vesicles (evs) are heterogenous and include previously described microvesicles and exosomes. herein we characterized a formerly unappreciated population of mitochondriaderived evs that we term "mitovesicles". mitochondrial dysfunction is a well-established hallmark of ageing and neurodegenerative disorders as down syndrome (ds). hence, we examined mitovesicle levels and cargo under these conditions to characterize in vivo mitovesicle biology and responsiveness to mitochondrial stressors. methods: employing a high-resolution density gradient, distinct and novel populations of evs were isolated from murine and human ds and diploid control postmortem brains or from cell media. morphometric ev features were analysed by nanoparticle tracking analysis and cryogenic electron microscopy, while ev constituents were characterized by western blotting, mass spectrometry, lipid profiling and mitochondrial rna qpcr. results: we identified a population of double-membrane, electron-dense brain evs containing multiple mitochondrial markers ("mitovesicles") that are highly distinct from microvesicles and exosomes. proteomic data show that mitovesicles contain a unique subset of mitochondrial proteins while lacking others, such as tom . mitovesicles have a lipid composition that is unlike that of previously described evs and is consistent with mitochondrial origin. functionally, the complex-iii inhibitor antimycin-a stimulated in vitro mitovesicle release into the cell media, suggesting an interrelationship between mitochondrial dysfunction and mitovesicle biology. in mouse brains, mitovesicle levels increased with age and were found to be higher in ds compared to diploid controls. mitochondrial rna and protein levels were also altered in ds compared to diploid controls. summary/conclusion: we describe a previously unidentified type of metabolically competent evs of mitochondrial origin that we designate mitovesicles. our data demonstrate that brain mitovesicle levels and cargo are tightly regulated in normal conditions and are modified during pathophysiological processes in which mitochondrial dysfunction occurs, suggesting that mitovesicles are a previously unrecognized player in mitochondria quality control and may have a role in the trans-cellular tissue response to oxidative stress. introduction: alzheimer's disease (ad) is a devastating neurodegenerative disease leading to progressive memory loss and ultimately death with limited therapeutic options. growing evidence supports the theory that toxic proteins, like tau and amyloid, may propagate from diseased cells by packaging toxic proteins into extracellular vesicles (evs) and releasing them to infect other cells. one enzyme involved in the biogenesis of evs is neutral sphingomyelinase (nsmase ), which catalyzes the hydrolysis of sphingomyelin to produce phosphorylcholine and ceramide. several groups have reported improved cognition and reduced tau propagation when nsmase is pharmacologically inhibited or genetically knocked down in ad mouse models. unfortunately, current nsmase inhibitors are not suitable for clinical development due to poor solubility and inadequate pharmacokinetic profiles. methods: our group carried out a high-throughput screening campaign followed by extensive medicinal chemistry efforts leading to the discovery of phenyl (r)-( -( -( , -dimethoxyphenyl)- , -dimethylimidazo [ , -b] pyridazin- -yl) pyrrolidin- -yl) carbamate (pddc), an orally active, nm potent inhibitor with excellent selectivity and brain penetration. we tested pddc's ability to inhibit exosome release in cultured primary glial cells as well as an in vivo model of acute ev release. we then treated xfad mice with mg/ kg of pddc daily for six months and monitored their behaviour in the fear conditioning assay. results: pddc dose dependently reduced ev release from cultured primary glial cells and significantly reduced plasma ev numbers in an in vivo model. following chronic treatment with pddc, xfad mice demonstrated significantly improved cognitive function in the fear conditioning assay. summary/conclusion: these promising findings are currently being expanded using mouse models of tau propagation. if successful, these data would support pddc as a novel compound for targeting the pathological spread of tau as a therapeutic for ad. profiling evs in the anterior cingulate cortex of individuals with major depressive disorder introduction: major depressive disorder (mdd) is one of the leading causes of disability worldwide, affecting % of the population. the environment has been thought to play a role in the disease development, resulting in biological changes mediated by epigenetic mechanisms. microrna's (mirna) are well known epigenetic regulators that are disrupted in the depressed brain, and they are packaged into extracellular vesicles (evs). evs have emerged as means of intercellular communication, a process that is also disrupted in mdd. they are thought to transfer mirna between cells, which can alter gene expression in recipient cells. therefore, we hypothesize that ev cargo is altered in mdd subjects compared to healthy controls (hc). the aim is to extract evs from human post-mortem anterior cingulate cortex, a region previously associated with depression, and profile the mirna cargo and compare it between mdd subjects and hc. methods: post-mortem human brain tissue from the anterior cingulate cortex of mdd subjects and hc was mildly dissociated in the presence of collagenase type iii. residual tissue, cells, and large vesicles were eliminated, and evs were isolated using size exclusion chromatography. the quality was assessed by western blots and transmission electron microscopy (tem). rna was extracted and a small-rna library was constructed and sequenced using the illumina platform. differential expression analysis was then performed. results: western blots showed little to no endoplasmic reticulum (calnexin), golgi (bip), or mitochondrial (vdac) contamination, along with enrichment of the exosomal marker cd . tem images showed the typical cup-shaped morphology with sizes mostly between and nm. preliminary sequencing results revealed that mir- a- p, which is predicted to target glutamate receptors, is downregulated in evs from mdd subjects. summary/conclusion: high quality ev extractions can be obtained from post-mortem brain tissue using our method. this will be the first study to profile brainderived ev mirna in the context of depression. future studies will be needed to determine the effect of the different levels of mir- a- p. this could provide novel mechanistic insights into the pathophysiology of mdd and will serve as a starting point to examine the potential role of evs in mdd pathology. op . = ps . combining nanomagnetic isolation and artificial intelligence to guide the treatment of traumatic brain injury zijian yang a , kryshawna beard b , david meaney b , danielle sandsmark b , ramon diaz-arrastia a and david issadore c a university of pennsylvania, philadelphia, usa; b university of pennsylvania, philadelphia, usa; c department of bioengineering, university of pennsylvania, philadelphia, usa introduction: traumatic brain injury (tbi) is characterized by diverse primary mechanisms of injury that lead to the development of secondary pathological cascades that drive neurological deficit post-tbi. inability to separate patients based on the presence of these different endophenotypes represents a major challenge for diagnosis and treatment of tbi. extracellular vesicles including exosomes isolated from patient plasma have emerged as promising potential biomarkers for tbi due to their ability to cross the bbb into systemic circulation with molecular cargo intact for analysis. we have developed a novel microfluidic platform for rapid isolation of brain-derived evs providing a tool with which the biochemical state of neurons and glia can be directly assessed post-tbi. we used the ultra-sensitive, single molecule array (simoa) to quantify concentrations of protein biomarkers from the plasma and brain derived evs from mild tbi patients and controls. by combining multiple protein biomarkers, we could discriminate mtbi patients from controls in both the training and the blinded test set. building on this work, we are also characterizing single ev heterogeneity of neuron derived evs by developing novel droplet based digital assay for single ev quantification at ultra-low concentration. droplet based assay for single ev analysis would potentially be very informative for early disease diagnosis and therapy decision. methods: our microfluidic platform for ev isolation consists of tracked-etched membranes with millions of nanopores ( nm), coated with a magnetic film (nife) to precisely capture immunomagnetically labelled brain-specific evs from plasma. single molecule array (simoa) was used to quantify concentrations of the protein biomarkers (tau, uchl- , nfl, gfap, il , il , and tnf) in the plasma and brainderived exosomes of mild tbi (mtbi) patients and controls. to identify single ev, we applied droplet based enzyme-linked immunosorbent assay and encoded the fluorescent signal for single ev quantification within parallelized microfluidic platform. results: we report that concentrations of plasma and exosome gfap, nfl, and uchl were elevated in mtbi patients compared to controls (p < . ), and that each of these biomarkers are uncorrelated with one another. discrimination of mtbi patients from controls was most accurate when machine learning algorithms on the panel of biomarkers. specifically, combining plasma nfl, gfap, il and tnf-with tau from glur + evs showed % accuracy with % sensitivity and % specificity. summary/conclusion: this data suggests that neuronderived exosomes contain information that characterizes the injured and recovering brain. it also suggests that analysis of a panel of biomarkers from a combination of both blood and exosomal compartments could lead to more accurate diagnosis of mtbis. l cam is not associated with extracellular vesicles in cerebrospinal fluid or plasma wyss institute, boston, usa introduction: neurons in living psychiatric and neurological patients are inaccessible for cell type specific analysis of rna and protein. our understanding of these diseases instead relies upon imperfect sources of biochemical information such as post-mortem brain tissue analysis and animal models. furthermore, there is a paucity of biochemical assays available to diagnose and manage brain diseases. extracellular vesicles (evs) present an opportunity to noninvasively sample the contents of neurons in cerebrospinal fluid (csf) and plasma. in order to isolate neuron-derived evs (ndevs), a cell type specific transmembrane protein is necessary for immunocapture. l cam, a protein abundant on the surface of neurons, has been used extensively in the literature for ndev isolation. however, l cam exists in humans in several isoforms without a transmembrane domain, and as such it can be secreted as a free protein. additionally, the ectodomain of l cam can be cleaved off of the cell surface in physiological processes. it remains to be demonstrated whether the l cam found in csf and plasma is ev associated, or if it is instead a spliced or cleaved isoform behaving as a free protein. methods: using single molecule arrays (simoa), a digital form of elisa, as well as western blotting, we quantify ev markers (cd , cd and cd ) as well as l cam and albumin. we use these assays to determine in which fractions of size exclusion chromatography (sec) and density gradient the l cam appears. we also immunocapture l cam from csf and plasma and perform western blots for the internal and external domains of l cam. results: simoa and western blot analysis of sec and density gradient fractions demonstrated that while the ev markers peaked all together, l cam eluted in the free protein fractions along with albumin in both csf and plasma. when immunoprecipitation was performed, western blotting revealed different isoforms of l cam in csf and plasma. summary/conclusion: our data utilize a multitude of distinct techniques that converge to demonstrate that l cam is not associated with evs in csf or plasma. furthermore, our data suggest that the isoforms present in csf and plasma are distinct, which indicates that the l cam in plasma is likely not coming from the brain. this data call into question the utility of l cam as a ndev marker and point to the need to find novel candidates for immunoprecipitation of ndevs. introduction: in parkinson's disease (pd), α-synuclein (α-syn) aggregates known as lewy bodies (lb) are present in both the central and peripheral nervous system. furthermore, data showing that α-syn can spread from pd patients to transplanted tissue has led to a new theory postulating that pathological forms of α-syn can drive disease by "infecting" healthy cells and corrupting normal proteins. the exact routes and mechanisms involved in such spreading are yet to be fully understood but it is known that α-syn can be secreted from cells and transported via extracellular vesicles (ev). ev derived from erythrocytes (eev) are of particular interest in this regard as they have been shown to contain α-syn. methods: we first optimized a protocol for the isolation of fluorescently labelled human eev. the capacity of these eev to cross the blood-brain barrier (bbb) was then evaluated in vitro using a boyden chamber composed of primary human brain endothelial cells. next, eev were added to a more complex and physiologically relevant d human bbb model including ipsc-derived brain microvascular endothelial cells. in both in vitro protocols, flow cytometry was performed on media collect from each compartment to determine the number of eev. immunofluorescence was performed to assess the localization of fluorophore tagged eev. we are also using an in vivo paradigm for the extraction and testing of eev spread and an in situ cerebral perfusion (isbp) model in wt mice to investigate if and how eev cross the bbb using confocal microscopy. results: in both in vitro models, flow cytometry analyses showed that fluorescently tagged eev added to the luminal side traversed the endothelial cell barrier. confocal analysis revealed that some eev could also be found within endothelial cells themselves. ongoing experiments are being conducted in our newly developed d bbb to further confirm these results. our preliminary in vivo experiments showed that fluorescently labelled beads, similar in size to eev, used in the isbp experiments are detectable in the brain parenchyma of injected wt mice using confocal microscopy. preliminary work also includes isbp injections of eev in -month-old wt mice, (n = /groups) derived from pd patients (at different stage of the disease) and a healthy individual as a control. summary/conclusion: our preliminary data suggests that eev can indeed move across the bbb in both in vitro and in vivo experimental setups. ongoing experiments will determine the dynamics and processes involved in this transport and whether eev can precipitate and/or exacerbate disease-related features. introduction: neuroblastoma accounts for % of childhood cancer mortality. amplification of the oncogene n-myc is a well-established poor prognostic marker for neuroblastoma. whilst n-myc amplification status strongly correlates with higher tumour aggression and resistance to treatment, the role of n-myc in the aggressiveness of the disease is poorly understood. exosomes are released by many cell types including cancer cells and are implicated as key mediators in cell-cell communication via the transfer of molecular cargo. hence, characterising the exosomal protein components from n-myc amplified and non-amplified neuroblastoma cells will improve our understanding on their role in the progression of neuroblastoma. methods: in this study, comparative proteomic analysis, nanoparticle tracking analysis, transmission electron microscopy, rnai-based knockdown, migration and cellular survivability assays were performed to understand the role of exosomes isolated from cells with varying n-myc amplification status. results: label-free quantitative proteomic profiling revealed proteins that are differentially abundant in exosomes released by the n-myc amplified and nonamplified neuroblastoma cells. gene ontology-based analysis highlighted the enrichment of proteins involved in cell communication and signal transduction in n-myc amplified exosomes. treatment of less aggressive sh-sy y cells with n-myc amplified sk-n-be cell-derived exosomes increased the migratory potential, colony forming abilities and conferred resistance to doxorubicin induced apoptosis. incubation of exosomes from n-myc knocked down sk-n-be cells abolished the transfer of resistance to doxorubicin induced apoptosis. summary/conclusion: these findings suggest that exosomes could play a pivotal role in n-myc-driven aggressive neuroblastoma and transfer of chemoresistance between cells. op . = ps . introduction: quantification and characterization of single extracellular vesicles (sevs) based on surface markers can aid in dissecting the heterogeneous landscape of ev subpopulations. we and others have demonstrated the potential of imaging flow cytometry (ifc) to perform sev characterization. we recently showed release of protoporphyrin (ppix) positive sevs by -aminolevulinic acid ( -ala) dosed glioma cells, in vitro and in vivo. rickfels et al. also used ifc to demonstrate the enrichment of cd +/cd + evs in the plasma of glioma patients. herein, we performed in vitro studies to characterize ev subfractions using -ala as well as ev and cns specific surface markers. methods: we use ifc to characterize evs released by glioma using -ala, fluorescently labelled ev (cfda-se, cd ) and glioma specific (tenascin c and epidermal growth factor receptor viii, egfrviii) markers. furthermore, we characterized evs released by egfrviii positive glioma cells treated with dexamethasone, a steroid commonly used in glioma patients, to determine the effect of steroids on ev release. evs were quantified by ifc and results were confirmed by qpcr for the levels of egfrviii mrna. results: firstly, we optimized protocols to label glioma sevs using fluorescently labelled ev markers (cfda-se, cd ) and tumour specific markers (tenascin c and egfrviii). of the total evs (cfda-se), we demonstrate that % are tenascin c positive, . % are egfrviii positive and . % are -ala positive. there was only a minor overlap (< %) between the sub-populations. finally, we show that dexamethasone treated glioma cells release lower total evs ( . -fold), tumour specific evs ( . -fold; egfrviii), egfrviii mrna compared to mock treated cells. summary/conclusion: we demonstrate the potential of ifc to monitor sevs released by glioma cells exposed to different stimuli. this allows the characterization of ev sub-populations providing a working model to understand the dynamics of tumour evs at a single vesicle level. introduction: f. graminearum (fgr) and f. oxysporum f. sp. vasinfectum (fov) are severe fungal pathogens of cereals and cotton, respectively. fgr and fov cause economic losses and threaten food and fibre supplies worldwide. understanding host-pathogen interactions is crucial for developing new strategies for disease control. we are determining whether extracellular vesicles (evs) have a role in the interaction between fungal pathogens and their host plant. methods: we isolated evs from fgr and fov by sizeexclusion chromatography and characterized them by nta and tem. evs from fgr and fov are between - nm and have morphology similar to evs reported for other fungi. we performed label-free quantitative proteomics to describe the protein cargo of evs from fgr and fov, including a comparative study of evs from fov grown on different media: czapek dox (cd) and saboraud's dextrose broth (sdb). results: a total of proteins were detected in fgr evs and, according to prediction software effectorp, . % of these were potential effectors. similarly, % of ev proteins do not contain signal peptide indicating that packaging into evs is a novel mechanism of secretion for these proteins. notable fgr ev proteins include lipases, proteases and synthases for toxins and chitin. fov produced evs in similar quantities in both growth media tested, but ev protein cargo differed between them. there was a % overlap in proteins identified in the cd and the sbd ev proteins. in general, ev proteins were involved in metabolism, cell wall architecture and oxidoreduction, with . % and . % of potential effectors, respectively. polyketide and toxin synthases, proteases and effectors were present in both types of fov evs. summary/conclusion: this new fungal ev isolation method was rapid, yielded high-quality evs, and did not submit particles to high centrifugal forces. our data revealed that both fgr and fov produce evs enriched with proteins that could alter host immune responses or facilitate fungal infection. furthermore, the protein composition of fov evs was dependant on culture conditions. this supports a potential role for fungal evs in disease progression in plants and provides the foundations to pursue the role of evs in plant-fungal interactions with the potential to identify new targets for disease control. introduction: extracellular vesicles (ev) released by infective forms of trypanosoma cruzi, the agent of chagas' disease, modulate inflammatory response of macrophages through the activation of toll receptor (tlr ) via mitogen-activated protein kinase pathway. this induces the production of nitric oxide (no) and expression of the cytokines tnf-α, il- and il- , which could explain the inflammation observed in experimental chagas' disease, and eventually in the progression of human disease. evs released by the parasite are heterogeneous and it is unknown which factor, or factors present in the different vesicle populations act during the interaction with host cells. objectives. the goal of the present work was to characterize and isolate the different populations of evs released by t. cruzi and test their effects on macrophages. methods: ev released by trypomastigotes forms of t. cruzi (y strain) were purified by asymmetric flow fieldflow fractionation (af ) and characterized by nanoparticles tracking analysis (nta). the different populations of evs were incubated with host human monocytes cells (thp- ) and cytokines production determined by elisa and qpcr. the different ev populations were also incubated with llcmk- epithelial cells and the infection by t. cruzi determined. results: we found two distinct populations of evs. a population with to nm (ev ) and another with to nm (ev ). ev induced more tnf-alpha, il- , ip- and ccl than ev . it was also more effective in promoting t. cruzi infection in epithelial cells. summary/conclusion: t. cruzi released two ev populations that affects differently host cells. identification of these evs composition might help to better understand the role of evs in the modulation of t. cruzi infection funding: fapesp, cnpq and capes op . = ps . commensal bacterial extracellular vesicles act as carriers for norovirus sutonuka bhar, melissa jones, annalise galbraith and mariola edelmann university of florida, gainesville, usa introduction: human norovirus (hunov) are one of the most common causes of gastroenteritis and, along with inducing morbidity and mortality by diarrhoea, have a massive economic impact resulting in approximately usd billion each year in healthcare costs and missed worker productivity. development of anti-viral therapies for hunov has been hampered by the lack of robust in vitro cultivation systems. several cell types support viral replication but only produce modest amounts of virus due to unknown reasons, making these systems insufficient for use in drug development and infectivity assays. noroviruses are known to attach to gram-negative enteric bacteria and this facilitates infection in vitro. however, the microbiome-norovirus-host communication link is missing. noroviruses infect immune cells present in lamina propria during acute infection, but bacteria themselves are large enough to cross the mucosal and the tight epithelial barrier which separates gut lumen from lamina propria. we hypothesized that binding of noroviruses to bacteria enhances extracellular vesicles (ev) production. because commensal bacterial evs by themselves do not have any detrimental effects on host cells, we believe using evs in in vitro culture will enhance norovirus infection, thus producing higher titre of viruses for vaccine and anti-viral drug development. methods: attachment assay: purified norovirus was incubated with enterobacter cloacae, lactobacillus acidophilus and bacteroides thethiotaomicron, and grown to produce evs. the attachment was confirmed via qpcr. isolation of evs: clarified media supernatants were subjected to ultracentrifugation at varying speeds and . um filtration. co-purification of norovirus with the evs was checked. ev quantification and characterization: ev total protein content was measured by microbca. the number of vesicles were quantified by nanoparticle tracking analysis. scanning and transmission electron microscopy was performed to check quality of ev preparation and determine if virus was attached to the vesicles. internal ev protein content was evaluated using ms-hplc. the evs were also check for infectivity via tcid assay. results: incubation of noroviruses with commensal bacteria resulted in significant increases in production of evs compared to uninfected controls. murine norovirus (mnv), used as a surrogate, was found to be associated with evs. em analysis determine association of viruses with the bacteria as well as the mvs, while also showing certain surface structural changes in virus attached bacteria compared to mock bacteria. the evs were found to cause infection in naive macrophages. summary/conclusion: changes in ev production and content by bacteria exposed to noroviruses will provide insight into its pathogenesis and possible solutions to the low viral output from hunov culture systems. detection of bacterial extracellular vesicles in blood from healthy volunteers kylie krohmaly a , claire hoptay b , andrea hahn a and robert freishtat a a children's national hospital, washington, usa; b childrens national hospital, washington, usa introduction: bacteria constitutively produce biologically active extracellular vesicles (evs), which contain rna, dna, and/or proteins. bacteria use these evs for communication with other bacteria and recent research suggests bacterial evs can also affect host cells. given these findings, it is necessary to examine the role of bacterial evs in human disease. current methods of bacterial ev isolation from human specimens cannot distinguish between bacterial species. however, there is utility in examining evs from specific species, as bacterial species and their evs may have unique contributions to human disease. our objective was to isolate circulating evs specifically from escherichia coli (eevs) and haemophilus influenzae (hevs), two known colonizers and pathogens in the gut and airway, respectively. methods: total evs were isolated from the blood of six healthy volunteers via precipitation and size exclusion chromatography. evs were then selected via a novel latex bead-based fluorescent antibody construct targeting species-specific outer membrane proteins. we used flow cytometry to evaluate the isolated evs. results: the constructs were saturated with eevs at an antibody concentration of . µg/ml of plasma, as geometric means ≥ . µg/ml were nearly equal. hevs were detected at µg/ml of plasma, but saturation is yet to be determined. eevs were imaged by a fei talos f x electron microscope and measured between - nm, and hevs were between - nm. both types of evs were spherical. summary/conclusion: using this novel technique, we were able to isolate, detect, and visualize eevs and hevs. this technique enables the study of specific bacterial evs. in the future, ev contents will be assayed. furthermore, this technique will be modified so that specific bacterial evs from body fluids can be used for downstream functional applications. this is the first time that bacterial evs from targeted bacterial species have been detected in blood from healthy humans. introduction: new zealand (nz) has a population of just . million people with a remote geographical location in the pacific ocean. its unique culture, food-based industries and ethnic population make nz an invaluable place for extracellular vesicle research into all areas. however, as for many places in the world, standardization of methodologies, training and access to appropriate equipment is challenging. methods: the hub for extracellular vesicle investigations (hevi) is a virtual research centre established in with three-year seed funding from a university of auckland strategic research initiatives fund. two staff members are employed to support training, education and optimization of methods. the hevi is guided by a governance group providing valuable input from australasian experts in evs. results: since the hevi has organized research symposia, hands-on training days, hosted international students as well as providing one-on-one training for individuals from universities and institutes across nz. training is provided on multiple isolation and characterization methods and tailored to individuals access to essential equipment without bias towards individual manufacturers or techniques. travel funding has supported people to attend conferences and workshops for the purposes of education, networking and research dissemination. the hevi also provides support for project design with grants awarded to hevi members and a number of manuscripts in submission for publication. the embo practical course "extracellular vesicles: from biology to biomedical applications" is organized each year by a group of researchers active in the ev field in collaboration with the embl advanced training center in heidelberg. the course focuses on training phd students and postdoctoral researchers who enter or are already active in the field of ev research. given the large number of methods and protocols that is being used by researchers in the ev field, the organizers aim to provide practical guidance to new researchers and teach them appropriate skills. methods: participants obtain theoretical knowledge and hands-on experience on different ev purification and characterization techniques, such as fluorescent labelling, density gradient centrifugation, size exclusion chromatography, electron microscopy, flow cytometry and nanoparticle tracking analysis and on databases like ev-track and funrich. in addition, the organizers and invited lecturers from several different research areas explain which strategies are used to understand the role of ev in biomedical applications and give an overview of the current state of the field of ev research. results: the course therefore covers a broad range of topics important in the ev field, including heterogeneity in ev subpopulations, mechanisms of ev cargo selection, ev biogenesis, pre-analytical variables, therapeutic and diagnostic use of ev, and in vivo functions of ev. group discussions are facilitated and stimulated via assignments to analyse data obtained during the practicals and to critically evaluate literature. participants also have the opportunity to present their own research during poster presentations and ask for feedback from organizers and invited lecturers. summary/conclusion: among the participants selected for the course, a large geographical distribution is reached, including researchers from the newer eu member states and from outside of europe, to ensure a broad geographical distribution of the knowledge gained during this course. introduction: on october , we organized the st lugano exoday, first initiative in the southern switzerland to bring together resident researchers and european experts in the field of extracellular vesicles (evs). the workshop, centred on "technical challenges of extracellular vesicle research" aimed to highlight technical requirements and advances in the evs area, focusing on isolation, characterization and tracking. methods: the workshop started with a lecture by dr. cecilia lässer, from the university of gothenburg. the rest of the workshop was divided in two working groups (wg), each introduced by a keynote lecture followed by presentations by young researchers and a round-table discussion. wg , introduced by dr. mercedes tkach, from the institute curie in paris, focused on recent advances on evs characterization and isolation. wg was centred on evs tracking and introduced by dr. frédérik verweij, from the institute of psychiatry and neuroscience of paris. results: dr. lässer opened the workshop with a comprehensive review and introduced recent developments in the evs field. the first wg discussed different isolation methods, focusing on ultracentrifugation, size exclusion chromatography and immunoprecipitation-based techniques. supported by the keynote speakers, the participants agreed that the best approach to optimize the isolation process consists in the combination of different techniques. wg shared insights about new strategies to visualize and tracking evs, focusing on how to improve the routinely approaches used, defining optimal criteria for evs labelling and imaging. all the participants had an in-depth overview on the requirements and the state-of-the-art techniques currently in use for the isolation, characterization and tracking of evs. summary/conclusion: the transferable knowledge acquired during the workshop ensures participants to remain up-to-date with the advances in the field of evs. as our ultimate goal is to create a competence centre in southern switzerland around the field of evs, the workshop was an invaluable opportunity to intensify collaborations between resident laboratories and broaden the scientific exchange with laboratories of the experts hosted during the event. given the success of this first workshop we are already working to prepare the second edition and make the event a recurring appointment. funding: supported by the swiss national science foundation the role of core facilities and emerging technologies in maximizing rigour and reproducibility of ev quantification and characterization and following misev guidelines rachel derita a and andrew hoffman b a thomas jefferson university, philadelphia, usa; b university of pennsylvania school of veterinary medicine, philadelphia, usa introduction: it remains very clear in the field of extracellular vesicle (ev) research that the rapid rate of increase in publications and expansion of interdisciplinary clinical ev interest has created the need for increased standardization and access to the appropriate technologies to uphold these standards. as the first core facility in the usa with the sole intention of creating a space where users can both isolate and characterize evs, we provide a central location for the facilitation of ev research via access to multiple technologies (both established and emerging) such as resistive pulse sensing, nanoparticle tracking analysis, ultracentrifugation, high-performance liquid chromatography, flow cytometric analysis of evs and additional immune or fluorescence-based ev characteri zation techniques. methods: we surveyed a group of leading scientific investigators and researchers in varying stages of their scientific careers in the mid-atlantic region of the us. the survey data demonstrate applications of greatest current and future interest to be employed in a shared lab resource. results: the current demand is highest for isolation services, ultracentrifugation and nta, with a gradually increasing demand for immunophenotying analyses such as the exoview chip array, fluorescent nta and flow cytometry. we additionally present strategies and data-based examples of how shared resource facilities can facilitate multifactorial and rigorous ev characterization in accordance with misev guidelines, and encourage collaboration among ev researchers. summary/conclusion: in order to answer the larger remaining questions in the ev field such as the isolation of specific ev subsets, ev tracking between cells and the use of evs for biomarker discovery and drug delivery, it is essential that shared resource facilities interact not only with investigators, but with each other to integrate the necessary resources to progress. programme to assess the rigour and reproducibility of extracellular vesicle-derived analytes for cancer detection national cancer institute, rockville, usa introduction: cancer cells release more evs than normal cells and evs secreted from tumour cells can promote tumour progression, survival, invasion and angiogenesis. the ev cargo may mirror the altered molecular state of the cell of origin. therefore, evs have potential for the development of non-invasive markers for early detection of cancers. evs and their cargo also have the potential to be multiplexed with other molecular markers or screening modalities (e.g., imaging) to develop integrated molecular-based computational tools for the early detection of cancer. one challenge with using evs as a biomarker is the lack of robust and reproducible methods for the isolation of a pure vesicular population. there is a lack of clear consensus for an optimal method of isolation of a pure ev population that is devoid of contamination with similar-sized vesicles of different origins. there is also a lack of standards to ensure rigour reproducibility. methods: the current funding opportunity announcement (foa), par - , is promoting research on the isolation and characterization of extracellular vesicles (evs) and their cargo for the discovery of biomarkers to predict cancer and cancer risk. results: the previous cycle of this foa, par - / , successfully funded r and r grants. these awards are focused on proteomics profiling of evs, effect of methodological and biological variability, asymmetric-flow field-flow technology, therapeutic monitoring, lss and sers lab on a chip optical spectroscopic, evs in obesity-driven hepatocellular carcinoma, nanoscale structure and bio-molecular heterogeneity, urinary ev dna, and ev markers in paediatric cancers. progress from these awardees have shown separation of two discernible exosome subpopulations and identified a distinct nanoparticle, the exomere (nature cell biology, ); and have shown that large-evs contain the entire genome of the cell of origin, including cancer-specific genomic alterations (journal of extracellular vesicles, ). protocols that critically evaluate and refine the existing methodologies to improve utilization of evs in clinical use have been shared (nature protocols, ). summary/conclusion: drs. sudhir srivastava and matthew young are the program directors for the par which began accepting applications on january . this and other ev funding opportunities will be discussed. funding: this is a funding opportunity announcement offered by the national cancer institute introduction: early detection of cancer as well as monitoring cancer treatment are important to improve cancer care. diagnostics for cancer are mainly based on tissue biopsies and re-biopsy during treatment is challenging. moreover, current diagnostics are expensive, time-consuming and have low-throughput. therefore, liquid biopsies are expected to bring the next breakthrough in cancer diagnostics. in liquid biopsies tumour-secreted material is isolated from body fluids and subsequent analyses thereof allow for non-invasive diagnostics. one type of tumour-secreted materials are extracellular vesicles (evs), which are schedded from tumour cells. evs are surrounded by a lipid bilayer, whichs composition resembles the plasma membrane of their parental cell. as many tumours are driven by over-expression or upregulation of transmembrane proteins e.g. growth factor receptors, detection of the later on evs holds promise for early tumour detection and treatment monitoring. methods: for the immuno-pcr evs were first affinitycaptured on magnetic beads, allowing immobilization of purified evs as well as evs secreted into cell culture medium or spiked into plasma. afterwards each sample was divided and affibody-dna-conjugates directed against different targets were added. affibodies are small affinity proteins, which often are developed as high affinity binders for tumour imaging, making them suitable probes in the presented assay. after washing, the bead-ev-affibody-dna-complexes were analysed for the immobilized dna-amount via qpcr. results: via the presented immuno-pcr evs secreted from the non-small cell lung cancer cell line h as well as the ovarian cancer cell line skov were analysed. the immuno-pcr method allowed the detection of the tumour-associated membrane receptors epidermal growth factor receptor (egfr), receptor tyrosineprotein kinase erbb /her and insulin-like growth factor receptor (igf r). different levels of membrane receptors depending on the ev source and concentration were detected. summary/conclusion: the presented immuno-pcr showed to be a comparably fast and robust method for detection of tumour-associated membrane receptors on evs derived from cancer cell lines with medium through-put and is currently further developed into a method for liquid biopsy for non-small cell lung cancer patients. introduction: introduction. evs produced by cells can originate from different cellular compartments and evs in complex biofluids may originate from many different cell types. traditional biochemical analysis, which reports on the total composition of all evs in a sample can't adequately resolve this heterogeneity. single vesicle analysis methods can, if they have the necessary specificity, sensitivity and speed. flow cytometry (fc) is capable of rapid and quantitative analysis of individual particles, but conventional fc-based assays lack the specificity and sensitivity to measure individual evs. assays that combine sensitive instruments with ev-selective sample staining can measure individual evs with accuracy and precision. to better understand the nature and origins of ev diversity, we used single vesicle fc (vfc) to quantitatively measure vesicle number, size, and surface cargo expression on individual evs. methods: methods. evs in culture supernatants ( t, a , u , thp- , sh-sy y) were used neat or enriched by standard methods including differential centrifugation or ultrafiltration. evs from platelets (plt) and red blood cells (rbc) were induced by ionophore treatment of washed cells, and measured in diluted supernatant. evs were stained with a membrane-selective dye and fluorescence-labelled antibodies using a commercial vfc assay kit (cellarcus biosciences), measured using a commercial flow cytometer (cytoflexs, beckman coulter), and data analysed using fcs express v (de novo software). vesicle size, fluorescence intensity, and antibody binding were calibrated using appropriate vesicle and beadbased standards and essential controls performed as recommended by the miflowcyt-ev reporting guidelines. results: results. to assess the compositional heterogeneity of evs, we first characterized the expression of tetraspanins (tss; cd , cd , cd , cd , cd , cd , cd ) on evs released from cultured cell line and primary cell cultures. we find quantitative differences in the expression of ts on evs from different cell types that generally reflected the expression on the cell of origin, with most ev types expressing detectable amounts at least one of the common ts molecules (cd , cd or cd ) but generally not all three. in evs from some cell types, ts expression was uniform across the ev population (cd on evs), but evs from other cell types differentially expressed tss, with some evs expressing no detectable ts (rbc evs). intracellular cargo labelled genetically using fluorescent proteins (egfp or mneongreen) or fluorogenic enzyme substrates (cfse) were measured in individual evs and revealed distinctive associations between ev surface and internal cargo. summary/conclusion: conclusions. high resolution measurement of cargo on/in individual evs can help interpret ev heterogeneity in terms of cell of origin, signals carried, and effects on target cells. integrated omics reveal conserved and divergent modulation of cardiovascular disease by tissue-entrapped extracellular vesicles introduction: fewer than % of patients develop both vascular and valvular calcification, implying differential pathogenesis. while circulating extracellular vesicles (evs) act as biomarkers of cardiovascular diseases, tissue-entrapped evs are implicated in early mineralization but their contents and function are unstudied. we developed an innovative method to isolate and study evs from fibrocalcific tissue and investigated entrapped ev cargoes in human cardiovascular diseases. methods: human carotid artery endarterectomies and stenotic aortic valves were obtained from donors under irb-approved informed consent. tissues underwent enzymatic digestion, ultracentrifugation, and a -fraction optiprep density gradient. global proteomics was performed on intact tissue, each optiprep fraction, and ev-enriched pooled fractions; the latter also underwent mirna-seq. fractionated samples were also studied by cd immunogold electron microscopy (tem) and nanoparticle tracking analysis (nta). high confidence mir targets were predicted by targetscan, pathway analyses utilized the biocarta/kegg/reactome databases, and protein-protein interaction networks were built in string. results: vesicle-associated pathways were increased . x (p < . ; / vesicle-related top go terms) in proteins common to intact arteries and valves (n = , ). proteomics found ev markers to be highly enriched in the four least-dense optiprep fractions of arteries and valves, while extracellular matrix and mitochondria were predominant in denser fractions, as confirmed by tem/nta. proteomics and mirna-seq of tissue evs quantified , proteins and mir cargoes linked to , target genes. pathway networks of proteins and mir targets common to artery and valve tissue evs revealed a shared regulation of rho gtpase and mapk intracellular signalling cascades. proteins and mirs were significantly altered between artery and valve evs (q < . ); multi-omics integration found that evs differentially modulated cellular contraction and p mediated transcriptional regulation in vascular and valvular tissue. summary/conclusion: our findings delineate a novel strategy for studying tissue-entrapped ev protein and mir cargoes and identify critical roles that tissue-resident evs play in mediating cardiovascular disease. funding: this study was supported by a research grant from kowa company (ma) and nih grants r hl , r hl and r hl (ea). mir- a regulates exogenous cd expression on proliferation, invasion, migration and angiogenesis of gastric cancer zhengzhou university, zhengzhou, china (people's republic) introduction: to investigate the possible mechanism of mir- a regulating the expression of exosome cd on proliferation, invasion, migration and angiogenesis of gastric cancer, and to study the application value of cd in the early diagnosis and prognosis of gastric cancer. methods: the gastric cancer cell line mgc- was used as the research object. the exosomes were extracted from the culture supernatant of mgc- by exosome extraction kit. the extracted exosomes were identified by transmission electron microscopy and western blotting. the expression of cd in exosomes was detected by elisa. the expression of cd in exosomes and cd in whole blood and serum were detected by western blot. they were randomly divided into blank group (mock) and mir- a lentivirus experimental group (mir- a group). the lentivirus control group (mir- a/con) was transfected into cells. qrt-pcr was used to verify the status of mir- a after transfection; western-blot was used to detect the expression of cd and downstream erk / , akt and m tor proteins; mtt assay, cell colony formation assay, transwell migration assay for cell proliferation, invasion, and migration. a nude mouse xenograft model was constructed to observe the growth of transplanted tumours,microvessel density (mvd) was detected by immunofluorescence, and distant metastasis was recorded. results: the expression of cd in exosomes was detected by elisa and western blot. the expressions of cd , akt, erk / and m tor in mir- a group were significantly lower than those in mir- a/con and mock groups. cd protein is positively correlated with downstream protein levels.the growth rate and cell invasion ability of mir- a group were significantly lower than those of mir- a/con group and mock group. the weight of the nude mice in the mock group and the mir- a/con group decreased, while the weight loss in the mir- a group was not significant. the tumours in the mir- a/con group and the mock group showed invasive growth accompanied by abundant microvessels, while the mir- a group had smaller tumour volume and uniform cell distribution. only a small amount of microangiogenesis was observed, and no obvious necrotic area was observed. summary/conclusion: mir- a affects the proliferation, invasion, migration and angiogenesis of gastric cancer mediated by akt/erk/m tor signalling pathway by regulating the expression of exosome cd . streamlined detection and quantification of plasma extracellular vesicles and their protein cargo by high-performance nanoscale flow cytometry and label-free mass spectrometry introduction: nanoscale flow cytometry (fc) and mass spectrometry (ms) are useful for profiling ev surface proteins and performing bulk ev proteomics, respectively. this study sought to develop pre-analytical and analytical pipelines for ev protein profiling that are applicable to clinical studies. methods: to optimize plasma ev detection and quantification by fc, modifications of instrument settings and serial dilutions of platelet-free plasma (pfp) and antibodies were tested for improved separation of signal from noise and reduction of event coincidence and swarming. the high-performance flow cytometry (hpfc) platform was used to assess the effect of time ( , , , , , , or hrs) between blood draw (into acd, nacit, edta or heparin) and blood processing, on ex-vivo release of evs from blood cells. label-free ms was used to examine the intensity and breadth of identified proteins in plasma evs purified using several density and size separation methods, either manually or automated, along with various buffer conditions. results: ev event aborts were minimized at a pfp dilution, prior to staining, of : and by using a narrow cytometer window extension. target ev signals were distinct from noise and were triton x- labile. the most significant changes in plasma evs were associated with platelet-derived fractions, use of heparin and > -hour delay before blood processing. yet, platelet ev numbers did not significantly change for up to hrs in citrated and edta plasma. higher overall coverage of known ev proteins and a fivefold increase in number of uniquely identified proteins were observed in ms profiling of evs prepared by a combination of ultracentrifugation (uc) and manual size-exclusion chromatography (sec) compared to preparation by fplc on capto core /superose resins. uc/sec was better than direct sec at reducing contamination by excipient plasma proteins. column buffers with trehalose increased ev protein recovery while adding protease inhibitors had minimal effect. summary/conclusion: with our optimized hpfc protocol, we established that blood ev numbers remain stable for up to hrs in acd or edta and that uc+sec with trehalose-containing buffer result in high canonical ev protein recovery. we are applying these workflows to investigate cancer-associated changes in plasma ev protein cargo. the value of exosomes as a potential biomarker for devil facial tumour disease. university of tasmania, hobart, australia introduction: the tasmanian devil (sarcophilus harrisii), the largest living carnivorous marsupial is endangered because of two transmissible cancers: devil facial tumour disease (dftd) one and two. current efforts to manage dftd are hindered by the lack of a preclinical diagnostic test for dftd. detecting dftd infection is only possible once tumours are noticed, too late to stop dftd progression. a preclinal test could tell us about unknown components of dftd pathogenesis, such as latent period and host-tumour dynamics. exosomes are extracellular vesicles released by most types of cells under both physiological and pathological conditions. exosomes have utility as diagnosis and prognosis biomarkers in a range of diseases, including cancers. the aim of this study is to investigate exosomes-based approaches towards a preclinical and progression biomarker for dftd and in tasmanian devils. methods: exosomes were isolated from three different dftd- , dftd- and devil fibroblast cell lines by sizeexclusion chromatography. likewise, exosomes were isolated from plasma of healthy and diseased devils. to determine the size and morphology of exosomes, samples were imaged with transmission electron microscopy. exosomes isolated from cell lines and devil plasma were analysed with mass spectrometry to characterise proteins and determine their differential expression between the cell origins, and healthy and diseased animals. results: this study identified the presence of myelin proteins in exosomes from dftd cells relative to fibroblasts, which are diagnostic of dftd. additionally, we found that exosomes derived from dftd- abundantly express the inhibitory checkpoint molecule cd relative to exosomes from dftd- cells and devil fibroblasts, indicating a potential candidate for a differential diagnosis between tumours. moreover, exosomes from dftd cells present a greater amount of proteins related with metastasis in comparison with fibroblast exosomes, such as integrins. finally, we report the protein expression profile of exosomes from healthy and diseased devils, showing clear differences between them and the presence of immunosuppressive and metastasis proteins in animals in late stages of the disease. summary/conclusion: dftd-exosomes may provide a non-invasive diagnosis tool to detect early stages of dftd in tasmanian devils to facilitate the prevention of the disease. furthermore, dftd-exosomes may have utility as a prognosis biomarker, determining late stages of the disease using a simple a blood test, which would facilitate monitoring of wild populations. this project will provide long-term benefits for the future of the devils and encourage exosome-based solutions for other future wildlife disease outbreaks. introduction: despite the increased understanding of evs, from involvement in disease pathophysiology to therapeutic delivery, improved molecular tools to track biodistribution are largely lacking. current approaches used for ev labelling lacks sensitivity and specificity. here, we have explored bioluminescent labelling of evs to achieve a highly sensitive system for absolute in vivo quantification and tracking of exogenous evs at low cost and in a high throughput manner. methods: ev-producing cells were genetically engineered to express various tetraspanin-luciferase fusion proteins. evs purified by uf-sec from these cells were characterized by nta, multiplex bead-based array, tem and wb, followed by luciferase assay to determine the labelling efficiency. for in vitro applications cell lysate from treated cells or the conditioned medium were subjected to luciferase assay. for in vivo applications two different methodologies were applied to determine biodistribution; either by non-invasive real time in vivo imaging using ivis or by luciferase assay on harvested tissues for absolute quantification of injected evs. results: we initially performed a systematic comparison of five different luciferases for endogenous labelling of evs and identified nanoluc and thermoluc as lead candidates. we applied this technology to monitor in vitro cellular uptake and observed cell type differences in cellular uptake of engineered evs. in addition, we also observed an effect of different culturing conditions on exocytosis kinetics. for in vivo application, we applied the nanoluc labelling strategy to determine the pharmacokinetics and effect of different routes of injection on ev distribution. our results indicated a rapid uptake profile of administered evs in different tissues with liver, spleen, and lungs being the primary recipients. we also observed similar results upon tracking in vivo biodistribution in real time immediately after administration. finally, we show how different subpopulations of evs differ in their in vivo biodistribution. summary/conclusion: overall, nanoluc and thermoluc labelling of evs holds great potential for various in vivo and in vitro applications. in addition, it can enable the simultaneous detection of different subpopulations of evs in vivo, which may aid in our understanding of different sub-populations and their behaviour in vivo. apart from monitoring therapeutic evs, with one simple modification this platform offers great potential for tracking tumour derived evs both in vivo and in vitro and thus could aid in the development of anti-tumour therapies. biofunctional peptide-modified extracellular vesicles for cell targeting, macropinocytosis induction, and effective intracellular delivery ikuhiko nakase department of biological science, graduate school of science, osaka prefecture university, sakai-shi, japan introduction: [introduction] in our research group, developing therapeutic techniques based on extracellular vesicles (exosomes, evs) by effective usage of peptide chemistry to deliver therapeutic/diagnostic molecules into targeted cells has been focused. in this presentation, modification techniques using biofunctional peptides such as arginine-rich cell-penetrating peptides [ ] , artificial coiled-coil peptides with receptor targeting [ ] , and cell-penetrating sc peptides [ ] derived from cationic antimicrobial protein, cap for cancer targeting with macropinocytosis induction, on the ev membranes will be introduced. i will also show effects of lyophilization of the peptidemodified evs on their biological activity [ ] . methods: [methods] cd (ev marker)-gfpfusion protein expressed evs were used for cellular evs uptake assessments. all biofunctional peptides were synthesized by fmoc solid-phase method. results: [results] macropinocytosis with actin reorganization has been shown to be crucial for cellular ev uptake [ ] . we developed the methods for modification of arginine-rich cpps or sc peptides on ev membranes using chemical linker techniques, and for example, arginine-rich cpps modification can induce proteoglycan-clustering (e.g. syndecan- ) and macropinocytosis signal transduction [ ] . the artificial leucine zipper peptide-modified evs recognize the peptide-tagged epidermal growth factor receptor (egfr) on targeted cells, leading to macropinocytotic cellular ev uptake [ ] . in addition, lyophilization is a useful technique for long term storage, however, we found that lyophilization negatively affected biological functions of encapsulated proteins in the evs after their cellular uptake [ ] . summary/conclusion: [conclusion] these techniques and findings will contribute to development for the ev-based intracellular delivery systems. reference: [ ] sci. rep. , ( ), [ ] chem. commun. , ( ), [ ] chrmmedchem. , ( ) [ ] anticancer res. , ( ), [ ] sci. rep. , ( ) os . multi-compartmented microvesicles: novel extracellular secretory organelles that release exosomes and extracellular vesicles introduction: extracellular vesicles (ev) bud from the plasma membrane (pm) as microvesicles (mv) or arise from the fusion of multivesicular bodies (mvb) with the pm to release intralumenal vesicles (ilv) as exosomes. the variety of bioactive molecules carried by ev imparts diverse functionality to ev in intercellular signalling. the biogenesis and extracellular release of these specialized messenger organelles is not well understood. to investigate, we studied endothelial cells that line the inside of blood vessels, known to release ev that support angiogenesis. methods: cultured human umbilical vein endothelial cells (huvec) were examined by thin-section electron microscopy (em), serial sectioning and immunogold labelling to study the structure and composition ev release sites. to obtain optimal views of cellular ultrastructure, cells were preserved by fast-freezing and a freeze-substitution. results: a potential release site was identified in em thin sections as a discrete domain, up to several microns long, on the otherwise smooth huvec pm, where numerous bulbous membrane protrusions with thin necks were clustered. the cytoplasm in these protrusions was enriched with mvb and other vesicles and appeared to be on the verge of pinching off to release multi-compartmented mv (mcmv). consistent with this notion, in the neighbouring extracellular space, a plethora of mcmv of - nm with ultrastructural features matching the bulbous protrusions were observed, supporting the concept that mcmv bud from the release site. serial sections confirmed that these extracellular mcmv were independent of cells and not linked by nanotubes or other processes. remarkably, fusion of mvb with the mcmv membrane was directly observed, presumably caught in the act of releasing ilv (exosomes) from the mcmv. immunogold labelling for ev markers is being used to identify proteins enriched at release sites and on released mcmv. summary/conclusion: in summary, ) mcmv bud from localized sites on the endothelial pm, ) mcmv contain mvb, and ) fusion of mvb to mcmv to release exosomes occurs extracellularly. mcmv can now be evaluated as a potential source of exosome and ev release that occurs after budding from the cell of origin, adding new layers of regulation to when, where and how ev are assembled and released. funding: this work was supported by the division of intramural research of the nih. one size does not fit all: overcoming barriers to successful discovery and scaled manufacturing of therapeutic extracellular vesicles jieun lee a , wei guo b , hal sternberg c , mike west d and dana larocca d a stem cell team, seoul, republic of korea; b university of pennsylvania, philadelphia, usa; c agex therapeutics inc, alameda, usa; d agex therapeutics inc., alameda, usa introduction: extracellular vesicles have tremendous intrinsic therapeutic potential. however, the limited availability of production cell lines presents a barrier to scaled ev production and novel ev discovery. indeed, ev sources have been largely confined to a handful of cell types with the vast majority consisting of msc evs. to overcome this limitation, we developed a diverse library of hundreds of clonally pure and scalable progenitor cell lines that provides an alternative resource for ev drug discovery and production. methods: we harnessed the capacity of human pluripotent stem cells (hpsc) to differentiate into virtually any cell type by subjecting hpsc to a wide variety of media and culture conditions to maximize the diversity of partially differentiated cells. the resulting heterogeneous "candidate cultures" were plated at clonal density and further selected for self renewing and scalable clones. transcriptomic analysis indicated > distinct progenitor lines. cell fate potentials were mapped by screening for cell type specific marker expression in various differentiation conditions. evs were produced using cgmp methods (tff and sec) and characterized by nta, trps, surface marker analysis, rna and protein content. bioactivity assays included proliferation, migration, vascular tube network formation, senolysis, and oxidative stress. results: the progenitor library contained > distinct lines with diverse lineage fates including various types of bone, cartilage, muscle,and fat cells, as well as all blood vessel cell types. the lines displayed much longer replicative lifespans ( - pd) than primary cell lines like msc. clonal purity minimized phenotypic drift resulting in maintenance of cell identity, genome integrity, differentiation capacity and bioactive ev production over extended culture. evs were highly diverse in their rna and protein cargo and bioactivity displaying various degrees of migratory, proliferative, angiogenic and senolytic activity. library screening identified evs with higher angiogenic potency than primary adult stem cell evs. summary/conclusion: we demonstrated scalable and stable production of bioactive evs from a large progenitor cell library. library screening resulted in discovery of novel angiogenic and senolytic evs having diverse rna and protein cargo. we are currently creating a corresponding library of progenitor cell evs to accelerate discovery of novel evs and their production cell lines. funding: the initial establishment of the cell library was funded in part by grants from the california institute of regenerative medicine and national institutes of health. introduction: besides extreme potential in biomedical applications, extracellular vesicles (evs) are also promising candidates to expand biophysical understanding of membrane active biomolecules. their complex bilayer composition allows the better understanding of adsorbed proteins and protein coronas as well, which sets of macromolecules will likely be key for advanced ev targeted delivery. considering cargo, membrane active peptides are interesting as these can be both drugs to be delivered, but can also facilitate cargo insertion through lipid bilayers. however, at present very little is understood regarding interactions between the peptides and the ev lipid bilayer, and between peptides and membrane associated proteins on evs. methods: we have recently demonstrated, that ev membrane adsorbed proteins and their interactions can be studied by techniques such as polarized light spectroscopy, microfluidic resistive pulse sensing measurements and freeze-fraction transmission electron microscopy [ ] . furthermore, initially we studied several peptides with known antimicrobial properties and found that these strongly interact with the ev surface proteins, resulting in efficient removal of some from the lipid bilayer [ ] . results: here we present investigation of further evpeptide interactions also focusing on anticancer peptides, which may be promising drug candidates for targeted delivery. these studies allowed to gain insight to novel functions of several peptides, such as melittin, magainin, buforin, lasioglossin, temporin, but also provide a more detailed understanding on how ev protein coronas, or ev bilayers are affected, to such extent that they cannot exert their potential function as delivery systems. summary/conclusion: the above interactions are expected to be interesting both for applicability, i.e. for selecting suitable compounds for ev processing, and also for curiosity-driven understanding of peptide functions, and ev-biomolecule interactions. based on these we promote that peptide -ev interactions will receive increased focus in ev-engineering. introduction: our late-breaking finding is the identification of a non-coding rna (ncrna) in extracellular vesicles (evs) from neuronal cells that is a natural antisense transcript for the dbh gene and associated with epigenetic changes and gene silencing. dna methylation in neurons is involved in memory and neurological disorders (science ( )). earlier work found that during chronic brain infection with toxoplasma gondii induced a decrease in norepinephrine levels and expression of the host dbh gene; and the decrease is correlated with behaviours linked to noradrenergic signalling (infect immun. ( ); infect immun. ( )). dbh catalyzes the production of norepinephrine from dopamine in noradrenergic neurons. we found that evs from infected cultures suppress transcription of the dbh gene and hypermethylation of the gene in noradrenergic cells in vitro. in this study, we identify a ncrna in the evs from infected neuronal cells. methods: neuronal cells were induced by infection with toxoplasma gondii and evs purified on sucrose gradients. evs were characterised by electron microscopy and used to treat rat and human neuronal cells and expression levels of dbh mrna and nascent dbh gene transcription were measured. induced evs were injected into the locus coeruleus of rats and dbh gene expression was monitored. rna purified from evs was screened for natural antisense transcripts (nats) by strand-specific rt-pcr. results: we found that evs purified from infected neuronal cultures induced transcriptional gene silencing (tgs) and dna methylation of dbh in recipient neuronal cells. the induced evs down-regulated dbh gene expression > -fold and induced dna hypermethylation of the dbh gene. this could be induced in the brains of recipient rats by intracerebral injection of evs. using a panel of strand-specific primers, antisense transcripts for the dbh gene were identified in infected cells. this permitted us to examine the rna in purified evs and identify a lncrna in evs selective for evs from infected cultures. summary/conclusion: this is the first study to find a specific neurotransmitter antisense lncrna in evs associated with transcriptional gene silencing and epigenetic changes in the gene. this represents a different type of neuron-to-neuron signalling than the classic chemical and electrical neurotransmission. the findings will enhance our understanding of neurological disorders (ie. schizophrenia, epilepsy, drug addiction) and how memory works. human cd + t regulatory-derived extracellular vesicles and associated micrornas: role in cell-to-cell communication and involvement in the loss of immune tolerance during multiple sclerosis introduction: an impairment of immune tolerance is a determining factor in multiple sclerosis (ms) and dysregulation of cd + t regulatory (treg) cell function is believed to be a major pathogenic factor. micrornas (mirnas) released by treg cells in association with extracellular vesicles (evs) have been shown to participate in the block of pathological immune responses by inhibiting the growth and cytokine production of cd + t conventional (tconv) cells, but the molecular mechanism is still poorly characterized. aim of the present work was to evaluate whether treg cell-derived ev-associated mirna signature is dysregulated in ms and whether this defect may play a role in the development of autoimmunity. methods: human treg cells isolated from blood of naïve to treatment relapsing-remitting ms patients and healthy controls were in vitro stimulated and released evs were isolated by size exclusion chromatography and characterized by nanoparticle tracking analysis, electron microscopy and flow cytometry. evassociated mirnas were quantified by traditional rt-qpcr and droplet digital pcr for absolute quantification. the actual ev-mediated passage of rna molecules from cell to cell was followed through rnaspecific fluorescent staining and treg-derived ev effect on tconv cell transcriptome was evaluated by rnaseq. results: in healthy conditions, the treatment of tconv cells with treg-derived evs was shown to cause the specific repression of genes involved in the proteasome-dependent proteolytic process, known to be crucial for t cell activation. in ms, treg-derived evs may have lost this capability as a direct consequence of a significantly decreased expression of mir- - p, able to target key factors of the proteasome system. summary/conclusion: our results unveil a novel molecular mechanism for treg-mediated maintenance of self-tolerance based on ev-associated mir- - p and its potential alteration in human autoimmunity. funding: fondazione italiana sclerosi multipla, fism, # /r/ and # /r/ revealing the proteome of brain derived extracellular vesicles isolated from human amyotrophic lateral sclerosis post-mortem tissues. introduction: amyotrophic lateral sclerosis (als) is a neurodegenerative disease characterised by the deposition of misfolded proteins in the motor cortex and motor neurons. although a multitude of als-associated proteins have been identified, few have been associated with extracellular vesicle (ev) trafficking, a form of inter-cellular communication. additionally, the role of evs in als is undetermined, specifically in relation to pathogenic stress granule formation, a response to cellular stress involving aggregation of non-coding rnas and their rna binding proteins. therefore, this study aimed to determine the proteome of brain derived small extracellular vesicles (bdevs) isolated from als subjects and identify novel alsassociated deregulated proteins and their potential contributions to pathogenic pathways in als. methods: bdevs were isolated from human post-mortem als (n = ) and control (n = ) motor cortex brain tissues through an ultracentrifugation protocol (vella et al., ) . following thorough characterisation, bdevs that successfully met the minimum criteria required by the international society for extracellular vesicles were classified as evs. the bdevs subsequently underwent mass spectrometry analysis on the thermo scientific q-exactive hf with ultimate rslcnano. proteins identified to be statistically significant differentially expressed then underwent validation by western blotting. results: a panel of statistically significant differentially packaged proteins were identified in the als bdevs. this included several up-regulated rna binding proteins and a down-regulated cell adhesion molecule; dhx , stau and vcam , respectively. pathway analysis revealed that the bdevs were enriched in proteins associated with stress granule dynamics, exosomal and lysosomal pathways. summary/conclusion: the identification of the rna binding proteins in the als bdevs suggests there may be a relationship between als-associated stress granules and als bdev packaging. the packaging of stress granule associated rna binding proteins into als bdevs may be an attempt by the cells to compensate for lysosomal dysfunction caused by stress granule accumulation, a feature of als. thus, these results highlight a potentially novel role for evs in the pathogenesis of als for long-term cultivation . the whole cultivation process of tissue preparation, cultivation, and cryopreservation has been established using strict serum-free conditions under a good manufacturing practice. long-term-cultivated hmnpcs retained stemness and hmnpcs have excellent differentiation efficiency into dopaminergic neurons. hmnpcs reversed impaired motor function in a rodent model of parkinson's disease (pd). based on the promising results in animal experiments, the clinical trial is under way (nct ). multiple-system atrophy (msa) is one of fatal neurodegenerative diseases with a combination of progressive autonomic nervous system disorders, parkinson's syndrome, and cerebellar pyramid syndrome. there are three types of msa such as msa-a, msa-c, and msa-p. in case of a msa-p type, it is difficult to diagnose due to the similarity of symptoms with parkinson's disease (pd). methods: in vitro and in vivo animal msa model were established and rotational behavioural was performed. npc cells were isolated and cultured based on moon et al. mirna sequencing (bgi) was performed and several bioinformatics analyses were done. results: based on the finding that hmnpcs exhibited therapeutic effects on pd, we hypothesize that hmnpcs will have a therapeutic effect on msa-p, where sympotoms are largely common with pd. as expected, transplanted hmnpcs survived, integrated, and differentiated in to dopamine neurons in the host brain, consequently leading to the functional recovery in the msa-p model. to further investigate the therapeutic key factors of hmnpcs in msa-p, mirna sequencing of the extracellular vesicles (evs) secreted from hmnpcs was performed. we found that mir- a highly expressed in the npc-derived evs is one of key regulators of inflammatory response via nfkb pathway. we further experimentally demonstrated that mir- a had anti-inflammatory effect on cells of msa-p condition such that the level of cx cl expression and its receptor, cx cr were both decreased in the msa-p modelled cells and in severe inflammatory environment in msa brain. summary/conclusion: our study first showed that mir- a in hmnpcs-evs is one of key therapeutic factors for the recovery of brain damage through immuno-modulation in msa-p. introduction: oxidative insults are known to be involved in the pathophysiology of alzheimer's disease (ad). we have previously demonstrated that some blood-based redox-signature were associated to the cognitive scores in mild cognitive impairment patients and in ad (perrotte et al., ) . the aim of this study was ( ) to evidence the presence of some oxidative markers in circulating extracellular vesicles (evs), and ( ) to compare to their plasma levels. methods: plasma samples from healthy, mci and ad patients were from the memory clinic of sherbrooke (québec, canada). ad patients were stratified in three groups (moderate, mild and severe) according to the mmse and moca scores. total plasma extracellular vesicles (pevs) were isolated from plasma with the total exosome isolation reagent (invitrogen™ by life technologies inc.). pevs were then characterized by electronic microscopy, nta, dls and western blot. antioxidants apolipoprotein j, d (apo j, apod), the glyoxalase- and protein carbonyls were determined by western blot. results: in pevs, we found that apo d levels were higher in mci patients but not in ad patients. protein carbonyls levels were higher later, in pevs from moderate and severe ad while apo j levels were not different in pevs from the five groups of patients. in plasma, the pattern of apo j and apo d was different. the levels of apo d was not different in the five groups of patients while apo j levels were elevated in mci and in all ad groups. protein carbonyls were higher earlier from mild ad group, earlier than in pevs. the levels of the detoxifying enzyme glyoxalase- were higher in pevs than in plasma and were significantly decreased in early ad as compared to control subjects and mci summary/conclusion: these results demonstrate a differential regulation of redox homoeostasis in plasma and in pevs from ad patients. funding: acknowledgements: this work was supported by the chaire louise & andré on alzheimer's disease, foundation armand-frappier (cr) and cihr grant (tf). carlos j. nogueras-ortiz a , pavan bhargava b , sol kim b , francheska delgado-peraza a , peter calabresi b and dimitrios kapogiannis a a laboratory of clinical investigation, national institutes of ageing, baltimore, usa; b department of neurology, johns hopkins university school of medicine, baltimore, usa introduction: multiple sclerosis (ms) is a neurological disorder characterized by white matter demyelination and extensive synaptic pathology. recent studies have shown synaptic loss in the grey matter of ms brains in the absence of demyelinating lesions which could account for disease progression independent of demyelinating episodes. opsonization of synapses with complement components is a mechanism by which phagocytic cells normally prune synapses, but, when occurring in excess, it may underlie pathologic synapse loss. we sought to identify blood-borne biomarkers of hypothesized complement-mediated synaptic loss in ms using circulating neuronal-enriched and astrocytic-enriched extracellular vesicles (nevs and aevs). methods: nevs and aevs were immunocaptured in parallel from the plasma of ms patients ( with relapsing remitting, with progressive ms) and healthy controls, targeting the neuronal-specific marker l cam and the astrocyte-specific marker glast, respectively. we measured the protein levels of preand post-synaptic proteins synaptopodin and synaptophysin in nevs using elisas and multiple complement cascade components (c q, c , c b/ic b, c , c , c a, c , factor b, factor h) in aevs using a luminex array. results: synaptopodin and synaptophysin protein levels in nevs of ms patients compared to controls were markedly reduced ( . -fold; p < . for both), whereas multiple complement components in ms aevs were markedly increased (c q: . -fold change; c : . -fold change; c b/ic b: twofold change; c : . -fold change; c a: . -fold change; factor: . -fold change; p < . ); differences were not observed in total circulating evs or neat plasma. strikingly, we found the nev-associated synaptopodin/synaptophysin and the aev-associated complement levels to be negatively correlated in people with ms (synaptopodin vs: c q, r = − . and p < . ; c , r = − . and p < . ; factor h, r = − . and p < . /synaptophysin vs: c q, r = − . and p < . ; c , r = − . and p < . ; factor h, r = − . and p < . ), but not in controls. summary/conclusion: circulating evs provide markers of synaptic loss and complement activation in ms and suggest a link between astrocytic complement production and synaptic decline. funding: this research was supported in part by the intramural research program of the national institute on ageing, national institutes of health. methylglyoxal and glyoxal affect the protein cargoes in neuronal-derived extracellular vesicles introduction: advanced glycation end-products (ages) and their receptor rages are known to be involved in the pathogenesis of alzheimer's disease (ad). methylglyoxal (mg) or glyoxal (go) are the precursors of ages and particularly n-( -carboxymethyl)-l-lysine (cml), the most abundant ages. mg induced tau hyperphosphorylation and causes hippocampal damage and memory impairment in mice. the aim of our study was to analyse the effects of mg and go on the neuroprotective, neurotrophic factors, inflammatory and neurodegenerative markers in the human cell line sk-n-sh and their release into the neuronal derived-evs. methods: briefly, sk-n-sh cells were incubated in fbs free media with mg and go ( . mm) for hours. neuronal derived-evs (nevs) from culture media were isolated as previously described (haddad et al. ). nevs were characterized by electronic microscopy, nta and by western blot. cellular and nevs concentrations of bdnf, prgn, nse, app, mmp , angptl- , lcn , ptx , s b, rage, dj- and alpha synuclein were determined by a luminex assay from r&d systems, inc. aβ - , aβ - , ptau t and total tau levels were measured also with luminex assay from emd millipore corp. results: we found that both ages precursors, at non toxic concentration, reduced the neuronal levels of nse with no effect on bdnf, ptrx- , lcn- , dj- , on neurodegenerative markers and on cml. go decreased the levels of prgn, app, angpl- while the expressions of mmp- and angpl- were, respectively lower and higher in the presence of mg. mg and go greatly reduced the release of lcn- by neuronal cells in nevs. bdnf and prgn in nevs were reduced in the presence of go. both mg and go did not modify the release of nse, app, mmp , agntl- , ptx- , dj- , aβ, ptau and cml in nevs. summary/conclusion: our data demonstrated that mg and go differently affect the content of some protein cargoes in nevs and suggest that targeting mg and go may be a promising therapeutic strategy to prevent neurodegeneration. introduction: peripherally circulating brain-derived extracellular vesicles (evs) and their encapsulated rnas may serve as biomarkers for hiv-associated neurocognitive disorders (hand). however, rates of cigarette smoking are significantly higher in hiv+ individuals than the general population, and smoking can modulate the expression of these markers. to better understand how cigarette smoke might modulate rna expression and ev release, we examined several cnsderived cell lines, representing astrocytes (u mg), microglia (sv ), and oligodendrocytes (hog). methods: cigarette smoke extract (cse) was prepared by bubbling through culture medium using a standardized and published method. all cell types were exposed to either % or % cse for hours. cell viability was assessed by musetm cell analyser, and evs were isolated from culture conditioned media (ccm) by size exclusion chromatography. the void (fractions - ), ev ( - ), and protein ( - ) enriched fractions were pooled and concentrated. evs were characterized by transmission electron microscopy (tem), microfluidic resistive pulse sensing, and western blotting. total rna was isolated from cells and circular rna (circrna) expression was assessed with a circrna microarray. results: in response to cse exposure, cell viability was only slightly reduced for all cell types. tem images validate the presence of vesicles in the ev fractions, and their absence in the void and protein fractions. spectradyne particle counts indicated cse exposure substantially increased the ccm particle count in the ev fraction when compared with control. the presence of expected ev markers (cd , cd , and tsg ) in the ev fractions, and their absence in the void and protein fractions was observed via western blot. intracellular circrna expression was significantly altered in all three cell lines. summary/conclusion: cns cells display physiologic responses to cse that include vesiculation pathways and significant alterations in circrna expression. we are now studying the effects of cse exposure on circrna expression in released evs. funding: this work is supported by da , da , and ai . a method for exosomal rna extraction from paired human brain and blood specimens emily n. moya a , lillian wilkins a , esther cheng a , lisa linares b , brian kopell b , navneet dogra c , bojan losic a and alexander charney a a icahn school of medicine at mount sinai, new york, usa; b mount sinai hospital, new york, usa; c department of genetics and genomic sciences, department of pathology, icahn school of medicine, mount sinai, new york, usa introduction: diagnosis and treatment of neuropsychiatric disorders has made little progress in the last half-century likely in large part due to the absence of a scalable technique to profile the complex biological activity of the brain in a living person. exosomes are nanovesicles - nm in size that mediate intercellular communication and contain proteins, lipids, and nucleic acids. it has been shown that brain derived exosomes can be found in peripheral blood, but determining whether peripheral exosomes truly reflect ongoing brain processes has to date not been possible due to the absence of paired living brain and blood specimens. here, we present a novel method for paired sampling of the dorsolateral prefrontal cortex (dlpfc) and peripheral blood from living human subjects for exosomal rna profiling. methods: informed consent, approved by the irb at the icahn school of medicine at mount sinai, was obtained for patients undergoing deep brain stimulation (dbs). paired brain and blood specimens were collected from patients at two deep brain stimulation (dbs) electrode implantation procedures: left hemisphere followed by right hemisphere (total of samples). we developed protocols to profile rna from exosomes of brain tissue extracellular matrix (ecm) and peripheral blood. exosomes were isolated via our in-house protocol using ultracentrifugation. rna was then extracted from the exosomes using the qiagen mirneasy mini kit protocol. quality control (qc) was performed to determine whether rna obtained was sufficient for next-generation sequencing. results: we demonstrate the safety of a novel procedure to sample the brain in living human subjects. bioanalyzer traces and qc data show a mean total rna of . ng (range . - . ng) and no samples fell below the threshold required for library preparation and sequencing ( pg) determined by inhouse optimization on the smart-seq v ultra low input kit. summary/conclusion: to our knowledge, we have performed the first study to sample pairs of dlpfc and blood from living human subjects for exosomal rna for subsequent next-generation sequencing. ongoing analyses by our group promise to establish peripheral exosomal rna transcripts reflective of brain activity. this non-invasive approach to probing neurobiology in the living human brain may facilitate the development of exosome-based diagnostics for neuropsychiatric disorders. introduction: the relationship between obesity and dementia is complex. while obesity in middle age triples the risk of dementia years later, many patients with alzheimer's disease (ad) are cachectic, and a decline in adiposity portends progression of dementia. this suggests adipose-derived factors are important to nervous system homoeostasis. we previously showed that adipocyte-derived small extracellular vesicles (ad-sevs) induce pathologies critical to developing obesity-related diseases and may provide a mechanistic link between adiposity and dementia. we hypothesized that altered expression of ad-sev micrornas involved in neurodegenerative pathways is associated with more severe cognitive impairment methods: we studied serum and cerebrospinal fluid (csf) from participants with ad and non-ad controls. ad-sevs were isolated from samples by precipitation and immunoselection. ad-sev microrna expression was profiled in both biofluids and compared. results: serum and csf microrna expression correlated strongly (r = . ). in serum, micrornas were differentially expressed by a fold change ≥| . | in the ad and control groups (p ≤ . ) and micrornas were differentially expressed in csf. using ingenuity pathways analysis, we identified mrnas expressed in nervous system tissue that are targeted by the differentially expressed micrornas. the mrnas map to diseases and functions; neuronal cell death, neurodegeneration, and neuronal growth and developmental pathways are highly represented. of the differentially expressed micrornas in serum, were moderately correlated with participants' score on the mini-mental state exam, a test of cognitive function (rs = | . - . |). as validation, rencell cx cortical derived neuronal stem cells had decreased doubling time when exposed to ad-sevs from obese adipose tissue in vitro. summary/conclusion: these findings support our hypothesis that altered expression of circulating ad-sev micrornas are involved in neurodegenerative pathways associated with cognitive impairment. these findings support using serum ad-sevs as a surrogate for csf ad-sevs. functional validation is underway to define the connection between ad-sevs and ad. understanding the link between obesity and ad is crucial as the population ages and the global obesity epidemic grows. funding: supported by uw adrc (nih:p ag ) expression of extracellular vesicles after acute traumatic brain injury: an exploratory flow-cytometry study introduction: coagulation derangements related to disseminated intravascular coagulation (dic) are common after tbi and contribute to secondary neural injury. extracellular vesicles (evs) are released from all cell types, including platelets, endothelium, and lymphocytes, which are responsible for dic. we hypothesized that specialized flow cytometry techniques could identify a unique ev signature of dic in acute tbi. methods: using a modified flow cytometry instrument for detection of small particles, fluorescence panels were created to assess for evs from endothelial cells (cd , cd ), platelets (cd , cd p, cd a, cd b), and erythrocytes (cd ) as well as brain biomarkers (s b, uchl- , gfap, tau and nse) and t-lymphocytes (cd , cd , cd , cd ). samples were prepared in trucount tubes to determine volume and treated with triton to confirm presence of evs. results: / study patients and / controls were male. % of study patients presented with a glasgow coma scale of . in the hypercoagulability panel, of the subsets with statistically significant differential expression, involved s b+ and were elevated in patients. platelet-derived cd a evs and uch-l evs were significantly elevated in controls in ev subsets identified in the brain-specific panel. finally, cd +/ + evs, derived from t-cells and identified in the endothelial/t cell panel, are significantly lower in patients suggesting cns recruitment. summary/conclusion: endothelial and platelet/erythrocyte evs may be elevated early after tbi. s bcarrying evs are significantly elevated in circulation of tbi patients; if reproducible, this signature profile may be informative for diagnosis and risk stratification. further study is warranted to evaluate whether this expression correlates with secondary microvascular brain injury. funding: intramural award from the university of pennsylvania enrichment of mir- a in cns extracellular vesicles following impairment of the blood brain barrier nasser nassiri koopaei a , ekram-ahmed chowdhury b , lais da silva a , jinmai jiang a , behnam noorani b , ulrich bickel b and thomas d. schmittgen a a university of florida, gainesville, usa; b texas tech university, amarilo, usa introduction: extracellular rnas (exrnas) are present in essentially all biofluids and include all types of rna including mirna. to enhance their stability outside of the cell, exrnas are bound within ribonucleoprotein complexes or packaged into extracellular vesicles (evs). the blood brain barrier (bbb) is a dynamic interface between the systemic circulation and the cns and is responsible for maintaining a stable extracellular environment for cns cells. the intent of this study was to determine if evs and their contents are transferred from the peripheral circulation to the cns under conditions of an impaired bbb. methods: the bbb of mice was disrupted by hyperosmolar mannitol injections. to validate that the bbb has been disrupted with mannitol, intravenously-dosed [ c]-sucrose was increased in the forebrain by fold with mannitol compared to sham treated mice. evs were isolated from the forebrain, hindbrain and spinal cord following gentle tissue lysis and differential ultracentrifugation. evs were validated by nta, tem and western blotting. mir- a, a mirna that is highly abundant in erythrocytes, was measured in the evs by qpcr. results: qpcr showed that mir- a in cns tissue evs increased with mannitol treatment in the forebrain, hindbrain and spinal cord by -, . -and twofold respectively. qpcr analysis of mrna from reported mir- a target genes showed reduced target gene expression with mannitol. summary/conclusion: we demonstrate that evs containing mir- a, a highly abundant mirna present within erythrocytes and erythrocyte evs, is enhanced in the cns upon bbb disruption. astrocyte-derived extracellular vesicles in morphine tolerance guoku hu, rong ma, naseer kutchy, yuetong zhao, susmita sil and shilpa buch university of nebraska medical center, omaha, usa introduction: opiates, such as morphine are used extensively in the clinical setting owing to their beneficial effects. paradoxically, however, the prolonged use of morphine often results in the development of tolerance, drug addiction, and ultimately leading to various comorbidities associated with drug abuse. although great efforts have been made, at present there is no treatment. the sonic hedgehog (shh) plays a key role in brain development, and brain cells fine-tuning processes such as their proliferation, patterning, and fate specification recent findings have demonstrated that inhibition of the shh signalling prevents morphine tolerance in rodent models. we thus hypothesize that extracellular vesicles (evs) derived from morphine exposed astrocytes and their cargo such as shh are critical for the development of morphine tolerance. methods: mice were received either saline or chronic morphine injection with escalating doses of morphine for days (subcutaneously; mg/kg, day , mg/kg days - , and mg/kg days [ ] [ ] . the development of tolerance was assessed by measuring the tail-flick latency using tail flick analgesia metre (le , harvard apparatus). evs were isolated using either differential ultracentrifugation from astrocyte conditioned media or gradient ultracentrifugation from brain tissues. western blotting and qpcr were performed to determine the expression/activation of shh signalling pathway components. results: our data showed that the levels of shh protein were upregulated in morphine exposed astrocytederived extracellular vesicles (morphine-adevs). furthermore, shh containing morphine-adevs activated shh signalling in astrocytes. our in vivo study further demonstrated the upregulation of shh, as well as the activation of shh signalling, in astrocytes of morphine-administered mice. summary/conclusion: these findings thus demonstrated an autocrine mechanism for shh pathway activation in astrocytes associated with morphine tolerance. these findings could pave the way for the development of shh signalling pathway targeted strategies in the prevention and treatment for substance use disorders. biophotonics-based platforms for the evaluation of circulating extracellular vesicles as biomarkers of neurodegeneration in alzheimer's disease silvia picciolini a , cristiano carlomagno a , alice gualerzi a , monia cabinio a , francesca baglio a and marzi bedoni b a irccs fondazione don carlo gnocchi, milan, italy; b irccs fondazione don carlo gnocchi, milano, italy introduction: in the search for novel and non-invasive biomarkers of alzheimer's disease (ad), both circulating brain-derived extracellular vesicles (evs) and whole serum represent a valuable integration of the currently used classification system. to face the technological challenge of evs and serum analysis, we propose the use of biophotonics techniques as reliable, sensitive, fast and label free methods, potentially useful in tailoring pharmacological and rehabilitation treatments. methods: circulating evs, isolated by sec, and serum samples were collected from healthy subjects (hc) and ad patients. all subjects were asked to complete montreal cognitive assessment scale and mri examination. surface plasmon resonance (spr) was performed in order to detect evs coming from neurons, astrocytes, oligodendrocytes and microglia and to characterize each of them for the amount of ganglioside m (gm ), aβ and tspo expressed on their surface. serum analysis was performed using a raman microscope through the surface enhanced raman spectroscopy (sers) effect by mixing serum with ag nanoparticles. the pearson's correlation index was used to assess the linear correlation between spri data and clinical, mri data and data obtained from multivariate analysis (mva) of sers spectra. results: the spr analysis of evs showed that the selected bioactive molecules are differently loaded on neural ev populations and that their amount is increased on total evs in ad patients compared to hc. we observed a significant correlation between mva data from sers and the presence of aβ on neuronal and microglial evs and of tspo on neural evs, measured with the spr array. summary/conclusion: thanks to our methodological innovation we have verified the potentiality of evs as ad biomarkers, correlating biophotonics blood-based analysis with clinical data. this platform could provide a powerful tool for the evaluation of ad neurodegeneration. funding: the study was supported by the italian ministry of health (ricerca corrente - to irccs fondazione don carlo gnocchi). raman profiling of extracellular vesicles as new blood-based biomarker for brain disorders: focus on parkinson's disease introduction: extracellular vesicles (evs) play a pivotal role in brain homoeostasis and intercellular communication in both physiological and pathological conditions. in parkinson's disease (pd), evs are key players in the transfer of α-synuclein, with blood evs reported to undergo proteomic modifications. nonetheless, the detection and characterization of the ev cargo is technologically challenging, limiting the use of evs as biomarkers so far. herein, we propose raman spectroscopy for the label-free, bulk characterization of blood evs in pd patients. methods: evs were isolated by sec and ultracentrifugation from the serum of healthy subjects (hc) and pd patients. in all patients, the severity of pd was evaluated with the unified parkinson's disease rating scale (updrs) part iii and with hoehn and yahr scores (hy). after proper ev characterization following misev guidelines, raman analysis was performed. the raman microspectroscope was used with a nm laser in the spectral ranges - cm- and - cm- . data from hc and pd patients were compared by multivariate statistical analysis (pca-lda). results: the raman analysis of evs highlighted differences in the biochemical profile of the two groups, with the main variations in the spectral regions related to proteins, lipids and saccharides. a preliminary estimate of the accuracy of raman profiling of blood evs for pd diagnosis was obtained, demonstrating an accuracy of %. even more interestingly, we demonstrated the correlation between the raman spectra and the clinical scales (updrs and hy) used to stratify pd patients. summary/conclusion: in conclusion, the biochemical signature of blood evs can be detected by raman spectroscopy in pd patients and the ev spectral modifications can be related to their clinical status. these data suggest the possibility to use the raman profile of circulating evs as a biomarker for brain disorders, complementary to other specific molecular markers. funding: the study was supported by the italian ministry of health (ricerca corrente to irccs fondazione don carlo gnocchi) impact of circulating extracellular vesicles on brain functions and behaviours introduction: peripheral immune alterations have been described in psychiatric disorders such as schizophrenia, depression, and autistic spectrum disorders. in addition, behavioural changes have been observed in various immunodeficient animal models. however, the mechanisms by which peripheral immune system influences brain development and function are not well understood. in this study, we explored the mechanisms by which circulating extracellular vesicles (evs) mediate immune-brain communication and influence mouse behaviours. methods: mice deficient for rag or rag gene (rag ko mice) were used as a model to study the effects of loss of adaptive immune cells (t and b cells) on brain cellular phenotypes and behaviours. circulating evs were collected from their sera and analysed by using electron microscopy, nanoparticle tracking assay, and western blotting. brain cellular phenotypes were assessed by immunofluorescent staining and gene expression analysis. behavioural phenotypes of rag ko and wt mice were examined in social interaction test. in vivo transfer of evs was performed to see its effects on behavioural alterations of rag ko mice. results: rag ko mice displayed social behavioural deficits, accompanying by enhance c-fos immunoreactivity and altered microglia morphology in the medial prefrontal cortex (mpfc). circulating evs were also affected in these mice and lacked the expression of markers for t cells. a set of micrornas (mirnas) in circulating evs were diminished in rag ko mice. in vivo transfer of circulating evs rescues the social behavioural deficits of rag ko mice and ameliorate the c-fos immunoreactivities in mpfc of rag ko mice. summary/conclusion: our data showed that circulating ev profiles were altered in mice lacking adaptive immune cells and, accordingly, showing social behavioural deficits. notably, our in vivo experiments suggest that circulating evs may contribute to social behaviours. further study will provide a novel biological insight into the mechanisms underlying peripheral-to-brain immune communication via evs. introduction: the involvement of neuroinflammation on ageing process is widely recognized. extracellular vesicles (evs), such as exosomes, are able to cross the blood-brain barrier and were related to neuroinflammation. in this context, evs have been considered a potential mechanism of spreading molecules, including micrornas (mirnas) that can promote mrna degradation or inhibit translation of their targets. our aim was to investigate the mirna profile of circulating total evs during ageing process and their impact on canonical pathways. methods: the local ethics committee (comissão de Ética no uso de animais -ufrgs; n ) approved all animal procedures and experimental conditions. plasma was obtained from wistar rats ( and months-old) and total evs were isolated. ev microrna isolation and microarray expression analysis was performed to determine the predicted regulation of targeted mrnas. results: the analysis of global microrna expression revealed differentially expressed micrornas (p < . ; fold change of ≥ | . |); mirnas were up-regulated and were down-regulated in circulating total evs from aged animals compared to youngadult ones. a conservative filter was applied on ingenuity pathway analysis (ipa) and only experimentally validated and highly conserved predicted mrna targets were used. ipa showed that neuroinflammation signalling is ranked among the top canonical pathway impacted by differentially expressed micrornas and is upregulated in aged animals (p < . ; z-score: . ). the differentially expressed mirnas impacted molecules in the neuroinflammation pathway. interestingly, the ion channel grin b is predicted to be up regulated and is a target of many evs mirnas; in accordance with our results grin b was previously related to neurodegenerative diseases. moreover, let- a- p is predicted to be downregulated and target all the molecules of the neuroinflammation signalling pathway. previous studies have correlated let- a- p and neurodegenerative diseases. summary/conclusion: our data suggest that circulating total evs cargo, specifically mirnas, are altered by ageing and impact neuroinflammation pathway, suggesting the involvement evs mirna on ageinginduced susceptibility of neurodegenerative diseases. introduction: bidirectional cell-cell communication via paracrine mechanisms is critical for wound healing. a new paradigm involving exosome-borne distinctive repertoire of cargo such as mirnas has emerged as a predominant mechanism of cellular communication at the site of injury. unlike other shedding vesicles of similar size, exosomes selectively package mirna by sumoylation of heterogeneous nuclear ribonucleoprotein (hnrnp). methods: keratinocyte-derived exosomes (exoκ) were genetically labelled with fluorescent reporter (gfp) using tissue nanotransfection. purified, gfp-labelled exoκ were isolated from dorsal murine skin and wound-edge tissue by differential ultracentrifugation followed by affinity selection using magnetic beads. distributions of intact exosome were analysed using a prototype jarrold-geometry charge-detection mass spectrometer to directly measure differences in particle mass and charge distributions. complementary ms and ion mobility spectrometry (ims)-ms experiments have been used to characterize surface glycans and glycopeptides. to selectively inhibit mirna packaging within the exoκ in vivo, ph-responsive targeted sirna functionalized lipid nanocarriers (tlnκ) were designed using materials that have prior history of fda approval for human use. results: an increase in mass/charge ratio with glycan binding sites on the surface of wound-edge exoκ were observed compared to dorsal skin exoκ. wound-edge exoκ were selectively taken up by the macrophages in the granulation tissue (n = ). keratinocyte targeting sirnahnrnp functionalized lipid nanocarriers (tlnκ) were designed with encapsulation efficiency of . %. application of tlnκ encapsulating sirna of hnrnp (tlnκ/si-hnrnp) to murine dorsal woundedge significantly inhibited the expression of hnrnp by % in epidermis compared to control (tlnκ/sicontrol)(n = ). moreover, mice treated with tlnκ/si-hnrnp showed impaired barrier function, with significant presence of macrophage in granulation tissue at day , suggesting impaired conversion of macrophage in the granulation tissue. summary/conclusion: this work provides a novel insight wherein exosomes of keratinocyte lineage are recognized as a major contributor that directs macrophage conversion in granulation tissue for wound healing. multifaced effects of milk-exosome (mi-exo) as modulator of scar-free wound healing gna ahn, hyo-won yoon, yang-hoon kim and ji-young ahn chungbuk national university, cheong-ju, republic of korea introduction: recently, milk exosome (mi-exo) has been focused particularly on the possibility of oral distribution for therapeutic agents. however, studies related to the cosmeceutical effects associated with mi-exo are fairly limited. the purpose of this study is to suggest the anti-oxidant and antiinflammatory effect of mi-exo and possibility that can be induced by scar free healing by micro rna in mi-exo. methods: the characteristics of the extracted mi-exo were verified by size measurement, morphological characteristics through cryo-em and western blot. for antioxidant experiments, an abts assay was performed. next, mrna expression through four major cytokines (tnfα, il- , cox- , inos) was used to evaluate anti-inflammatory effects. finally, cell migration assay was performed to confirm the effect of scar-free healing and the detection of mir- b in mi-exo and vegf mrna expression confirmed. results: mi-exo using % acetic acid extraction showed the highest yield. the average size of the exosomes is approximately nm, confirmed the typical double membrane vesicle. as a result of antioxidant experiments, it was confirmed that the treatment of exosomes of ^ particles showed about % antioxidant activity. when ^ particles were treated, rna expression of cytokines showed about times more inhibitory effect than control. elisa test results also confirmed that the concentration-dependent decrease. the activation of the raw cell less proceeded as the treated mi-exo increased. the cell scratch assay cells did not close the cells as the number of milk exosomes increased (wound closing % of ^ particle = . %). and mir- b in milk exosomes was detected at ct value = . summary/conclusion: the antioxidant and antiinflammatory effects of mi-exo showed the greatest efficacy when ^ particles were treated. in addition, it induced to scar free healing rather than wound healing. mi-exo has great potential as a superior natural material in the future cosmeceutical field. extracellular vesicles in human milk expose tissue factor and promote coagulation introduction: tissue factor (tf), a transmembrane protein, initiates coagulation by binding and activating coagulation factor vii (fvii). tf is associated with extracellular vesicles (evs) in saliva and urine, but it is unknown whether also human milk (hm) contains evs exposing coagulant tf. methods: hm was collected from six healthy nursing women with informed consent. evs were isolated by ultracentrifugation and size exclusion chromatography (sec). the presence of tf antigen exposing evs was studied by western blot, flow cytometry, cryo-electron microscopy (cryo-em), and surface plasmon resonance imaging (spri). the ability of tf exposing evs to trigger coagulation was investigated with a plasma fibrin generation test (fgt), performed in the absence or presence of antibodies against tf or fvii(a). results: addition of hm to plasma shortened the plasma clotting time, even when hm was highly diluted. after ultracentrifugation of hm, both tf antigen and tf activity were detected in the ev-containing pellet. after sec, tf antigen and tf activity were present in the ev-containing fractions and . the presence of tf-exposing evs in these sec fractions was confirmed by western blot (cd , cd and tf), flow cytometry, spri, and fgt. in addition, the presence of evs in hm was confirmed by cryo-em. scalable isolation of evs from different probiotic strains with potential as cosmetic ingredients laura soriano-romaní, joaquin espí and begoña ruiz ainia, paterna, spain introduction: extracellular vesicles (evs) are increasing their application in a number of fields. recently, it has been shown that skin health may be affected not only by commensal skin bacteria, but also by the evs that they secrete. however, because most of the efforts have been directed to the characterization and evaluation of evs, the scaling up of the production process remains a bottleneck at the industrial level. in this work, the goal was to evaluate the potential applications of evs produced by different probiotic strains commonly used in the cosmetic field, considering the economic and technical viability of the process. methods: to meet our goal, a standardized workflow was defined to isolate evs from probiotic strains such as lactobacillus and bifidobacterium species, that have demonstrated cutaneous immuno-regulatory effects. the different bacterial strains were produced under standard culture conditions. to isolate the secreted bacterial evs, different chromatographic techniques were performed starting from clarified growth medium. then, evs were evaluated in vitro for a number of biological effects related with skin health. results: the ev yields obtained after downstream processing were calculated for each strain and isolation technique by means of nanoparticle tracking analysis (nta) and total protein content. moreover, evs were visualized by electron microscopy. the in vitro evaluation of isolated evs was based on changes in the expression of five biomarkers related with anti-ageing, anti-inflammatory and whitening effects using distinct skin cell types to identify possible cosmetic claims that could be associated to each probiotic source. summary/conclusion: the potential of evs obtained from probiotic strains as cosmetic active cell-free ingredients was preliminarily assessed with this work, where the process yield and cosmetic function were evaluated. however, additional experiments will be needed in order to increase and optimize the productivity of each step of the ev manufacturing process. acerola derived exosome-like nanovesicles enhances the repair of ultraviolet b-induced dna damage in cultured skin fibroblasts tomohiro umezu, masakatsu takanashi, yoshiki murakami and masahiko kuroda tokyo medical university, shinjyuku, japan introduction: acerola (melpighia emarginata dc.) is a fruit is known to contain not only high amounts of ascorbic acid but also various nutritional components such as carotenoids and polyphenols. previous reports showed the acerola juices are able to confer protection against ultraviolet radiation b (uvb), to improve barrier function of skin. uvb is the main cause of dna damage in epidermal cells, generating several types of pro-mutagenic lesions, like cyclobutene prymidine dimers (cpds) and prymidine ( - ) prymidinone photoproducts ( - pps): if not repaired, this dna damage leads to skin cancer. in this study, we investigated the biological property of the acerola derived exosome-like nanovesicles (adens), aiming to clarify the involvement of adens in repair of uv-induced dna damage. methods: normal human dermal fibroblasts (nhdfs) were purchased from lonza inc. the exosome-like nanovesicles were isolated from acerola juices using exoeasy maxi kit (qiagen). the morphology and size distribution of adens were checked by transmission electron microscopy (tem) and nanoparticle tracking analysis (nta, nanosight lm , malvern). nhdfs were exposed to uvb ( mj/cm ) with pre-or post-adens. effect of uvb was assessed by examining cell viability, cell morphology, and dna damage levels through biochemical assays, microscopy and protein expression studies. results: purified adens were compatible with nta or tem for assessing the nanovesicle size range and concentration ( - nm). when nhdfs were added with adens and incubated at °c for h, there was no effect of adens on cell proliferation of nhdfs. we found that adens treatment to uvb exposed nhdfs significantly reduced cpds and - pps dna adduct formation. present results showed that aden treatment prevented uvb induced dna damage in nhdfs. summary/conclusion: we confirm that adens have the effect of repairing dna damage caused by uvb. these results provide that adens can be a new source to protect human skin from uv-induced skin cancer. introduction: introduction: despite the development of a variety of therapies, complex wounds resulting from disease, surgical intervention, or trauma remain a major source of morbidity. extracellular vesicles (evs) derived from mesenchymal stem/stromal cells (mscs) have been shown to improve wound healing, especially via enhanced wound angiogenesis. however, despite their clearly established potential, evs have limitations that may limit clinical relevancy, such as low potency. hypothesis: increased expression of pro-angiogenic lncrna hotair within msc evs enhances their proangiogenic effects and thus their wound healing properties. methods: methods: hotair was overexpressed in human dermal microvascular endothelial cells (hdmecs) to determine any molecular or functional pro-angiogenic effects. anti-angiogenic mirnas and angiogenic mrna levels were quantified by rt-qpcr. effects of hotair on proliferation of hdmecs was also determined. hotair was then loaded into msc evs by delivering a cmv-based hotair plasmid to mscs for endogenous loading via a concentration gradient. evs were collected by differential centrifugation. hotair content within evs was confirmed by gel electrophoresis and rt-qpcr. effects on migration of hdmecs by hotair-loaded msc evs were determined using a scratch assay. results: results: overexpression of hotair decreased mir- c and mir- , while increasing vegf and hif- a. hdmec proliferation was also increased in hdmecs overexpressing hotair (p < . ). hotair was visually confirmed in hotair-loaded msc evs by gel electrophoresis, but was undetectable in unmodified msc evs. rt-qpcr confirmed a -fold increase of hotair compared to control msc evs. hdmecs showed a more statistically significant rate of gap closure when treated with hotair-loaded evs (p < . ) than compared to control msc evs (p < . ). summary/conclusion: summary: loading lncrna hotair into msc evs is achievable by a concentration gradient-dependent method and offers potential to enhance the angiogenic properties of msc evs. nanomaterial labelling of exosomes for cell biology introduction: exosomes are vesicles secreted by many, if not all, cell types and have been known about for decades. among larger micro vesicles that are produced directly from the cell membrane, the small ( - nm), exosomes are similar in size to a virus surrounded by a lipid bilayer. we and others have demonstrated that exosomes contain proteins, lipids, rna, and dna, making them promising materials for diagnosing and treating diseases, including many cancers such as brain cancer. in addition, exosomes from neurons and glial cells represent a novel type of intercellular communication. however, their size makes them hard to track with traditional fluorescence microscopy. to address this, we developed photothermal microscopy (ptm), which uses gold nanomaterial labelling to track exosomes' interaction with and effect on cells/tissue. methods: exosomes secreted by tumour cells and general exosomes found in the blood were isolated using differential ultracentrifugation or a commercially available kit (invitrogen). next, the exosomes were characterized by (tem), (nta), and western blotting to determine shape, size, morphology and the protein profile in the exosomal membrane. after characterization, the exosomes were labelled with gold nanoparticles via sonication. next, the samples were washed, and the exosomes were labelled with fluorescence dye to stain the membrane. after staining and labelling, the exosomes were added to u cells in culture and incubated for h. they were then fixed by % paraformldehyde and imaged by ptm. results: ptm found that exosome-cell interactions are exosome-type dependent, as u cells took up exosomes from other u cells but not human serum exosomes. this suggests that exosome uptake is a selective process and depends on the source of the donor cells. summary/conclusion: exosomes can be labelled with gold nanoparticles via sonication then successfully tracked by ptm to study the effect of exosome source on exosome-cell interactions and communication. cells incubated with u exosomes took the vesicles up rapidly, while cells incubated with serum exosomes had little uptake. ptm will help us design selective exosome-based strategies to treat different conditions, including brain cancer and cns damage. funding: nsf epscor riii award . loading of goat´s whey extracellular vesicles with spiked microrna and curcumin as an strategy for developing new nanocarriers for acellular therapies introduction: extracellular vesicles (ev) are involved in cell signalling and are present in a variety of cell secretions such as milk, from which enormous amount of ev can be purified, thus milk is an attractive raw material for scaling up ev production for therapeutic, cosmetic or other uses. here we isolated evs from the whey fraction of goat´s milk and demonstrated that such evs can be loaded with molecules like polyphenols and mirna. methods: to achieve this, milk was collected from lactating goats and fractionated by acidification and centrifugation into whey and caseins. evs were purified from the former fraction by serial centrifugation and precipitation with commercial kit (total isolation/ thermo fisher) and characterized by electron transmission microscopy (tem), western blot to identify surface markers and measurement of size through nanotracking analysis. once isolated, evs were loaded with different concentration of a spiked synthetic mir or with the polyphenol curcumin. mirna or curcumin were co-incubated over night with evs at oc, precipitated and purified as described above, with an additional washing and precipitation for curcumin. concentration of mirna uploaded by evs was quantified using mir specific qpcr. curcumin was measured using a spectrophotometer at nm. results: evs isolated from whey had an average size of nm, were positive for hsp , cd and alix. in tem, evs were identified with their natural conformation and corresponding size to exosomes. qpcr showed a significant difference of expression of mir in relation to control (loaded with shame) and the negative control (p < . ). curcumin presence was also confirmed after washing and precipitacion. summary/conclusion: in conclusion, milk evs and exosomes can be loaded with mirna and a polyphenol and can be used as alternative nanocarrier for acellular therapies. introduction: extracellular vesicles (evs) are cellderived lipid membrane nanoparticles that serve as messengers of intercellular communication, transferring bioactive molecules to recipient cells. evs have a natural therapeutic potential with high flexibility and biosafety for employing natural and synthetic biomolecules as therapeutic delivery vehicles. considering the importance of evs, their isolation methods are still a bottleneck. to get insights into the tissue-specific cargo in vivo for complete exploitation of evs as therapeutic, biomarker and diagnostic tools, ev purification methods are critical. the aim of the study was brought about to develop an efficient ev purification method both in vitro and in vivo and to further investigate function of evs in cellular senescence. methods: to isolate tissue-specific evs in vivo we developed recombinant evs by genetically fusing snorkel-tag to the cd . the snorkel-tag enables on-column protease treatment for purifying evs which does not rely on traditional immunoaffinity purification protocols using low ph or high salts solutions. results: we systematically evaluated the purification of evs harbouring snorkel-tag by employing different methodologies. our findings suggest that evs harbouring snorkel-tag indeed can be purified at high purity without altering ev biophysical properties. furthermore, we expressed cd -snorkel-tag under p ink a promoter and were able to purify evs derived from senescent cells. summary/conclusion: finally, we are developing an in vivo model with cd -snorkel-tag under p ink a promoter. this will provide us detail insights into the ev cargo secreted from senescent derived cells, by purifying evs harbouring snorkel-tag under pathophysiological conditions, allowing us to develop biomarkers and therapeutic tools. summarized, we have here developed novel tool for studying content and function of evs in the context of ageing and disease. this tool will now pave the way for studying the molecular mechanisms underlying these ev functions in vivo. funding: this work was funded by the austrian science fund phd program biotopebiomolecular technolgy of proteins (w ). engineering exosomes with gata- jie xu, christian paul, yi-gang wang and meifeng xu university of cincinnati, cincinnati, usa introduction: exosomes, are small vesicles ( - nm) secreted from cells that can transport and deliver of their components such as lipids, proteins, dna, mrna, and mirna to target cells. gata- , a cardiac transcription factor, has been shown to regulate differentiation, proliferation, and survival of a wide range of cell types. delivering gata- protein into ischaemic tissues may be one of the most straightforward approaches to improve cardiac function following myocardial infarction. here, exosomes were engineered with gata- by infusing gata- with exosome targeting peptide. methods: the open reading frame of mouse gata- cdna was ligated to xpack lentivirus vector (xpack-gata- ) and plvx-ef -ires-pouro lentivirus vector (plvx-gata- ), respectively. hek cells were transduced by lentivirus, then exosomes were isolated from conditioned medium of hek cells using ultracentrifugation. exosomes were identified using transmission electronic microscope (tem), and the expression of gata- was semi-quantified using western blot. the internalization of exosomes was tracked via treating bend cells with exosomes pre-labelled with pkh . introduction: chinese hamster ovary (cho) cells have dominated as the mammalian cell host for the manufacture of humanized biologics, in part owing to their genomic plasticity and robust growth in suspension culture. there is great interest surrounding the use of extracellular vesicles (evs) as novel therapeutics owing to their capacity to deliver bioactive molecules. however, much remains unknown about the mechanisms involved in ev cargo loading, limiting their development as novel biologics. to this end, we have engineered cho cells to stably express constructs enabling loading of gfp into evs. methods: tetraspanins are established markers of ev identity. accordingly, cd was selected as a tethering point to generate evs with gfp cargo and constructs were generated via golden gate assembly. cho cells were stably transfected by electroporation and expression was verified with fluorescence microscopy and western blotting. growth in batch culture was monitored to establish maximum viable cell densities for ev harvest and recovered evs were characterized by nanoparticle tracking analysis (nta). finally, uptake of gfp-evs was studied using time-lapse fluorescence imaging in co-culture experiments. results: strong localization of cd -gfp was observed at the cell membrane and blotting confirmed intact tetraspanin fusion present at the expected molecular weight. additionally, cells were confirmed to retain high gfp expression post-cryopreservation. stable cell pools were able to reach viable densities greater than million cells/ml in batch culture and nta allowed for detection of gfp cargo even prior to ev isolation. evmediated transfer of functional gfp to recipient cells was found to occur over a period of hours. introduction: extracellular vesicles (evs) are considered promising for therapeutic applications. evs resemble the cell membrane, allowing high biocompatibility to target cells, while their small size makes them ideal candidates to cross biological barriers. despite the promising potential of evs for therapeutic applications, robust manufacturing processes that would increase the scalability and consistency of ev production are still lacking. methods: in this work, evs were produced by mesenchymal stromal cells (msc), isolated from different human tissue sources (bone marrow, umbilical cord matrix and adipose tissue). msc were selected as these cells allow for a scalable production of evs, while displaying low immunogenicity. a vertical-wheel™ bioreactor system was implemented for the production of msc-derived evs and compared with traditional static systems. the obtained ev products were characterized by nanoparticle tracking analysis, atomic force microscopy, zeta potential and western blot. results: the bioreactor system allowed to obtain evs at higher concentration and productivity, as well as more homogeneous size distribution profiles, when compared to traditional static culture systems. functional studies were performed using breast cancer and lung cancer cell lines. proliferation assays allowed to determine the dose-response profiles of these cell lines when exposed to msc-derived evs. a bell-shaped profile was observed for most cases, since raising the ev concentration lead to increased cell proliferation until a certain point ( - µg/ml), after which cell proliferation was attenuated with increasing ev concentrations. summary/conclusion: the bioreactor culture system allowed a substantial improvement in the production of msc-derived evs, while the obtained dose-response profiles will be valuable to determine the most appropriate ev concentrations for anticancer drug delivery. overall, we demonstrate that this culture system is able to robustly manufacture human msc-derived evs in a scalable manner towards the development of novel therapeutic products such as anticancer drug delivery systems. biodistribution and cellular location of inhaled exosomes and liposomes in the lung introduction: increasing evidence reveals the potential role of extracellular vesicles, such as exosomes and liposomes, in lung regenerative medicine for the treatment of lung diseases. encapsulation and delivery of potential rna and microrna targets into liposomes and exosomes are attractive drug delivery methods, but remain difficult to deliver to the pulmonary parenchyma to reach target lung cell types. here, we demonstrate effective delivery and cellular uptake of exosomes and liposomes to the pulmonary parenchyma via inhalation treatment in a murine model of idiopathic pulmonary fibrosis. methods: human lung stem cells (lscs) were generated and expanded from healthy whole lung donors. lsc-exosomes were purified via ultrafiltration and diilabelled using vybrant☐ labelling solution according to the manufacturer's instructions. dsred-labelled liposomes were generated using lipofectamine™ rnaimax transfection reagent and block-it™ alexa fluor™ red fluorescent control according to the manufacturer's instructions. lsc-exosomes and liposomes were delivered via nebulization to cd mice with bleomycin-induced pulmonary fibrosis. exosome and liposome delivery and biodistribution were visualized -and -hours post-treatment through histological analysis. the study was approved by the institutional animal care and use committee of north carolina state university and complied with all national and state ethical standards. results: exosome and liposome delivery to the pulmonary parenchyma was confirmed by the presence of dii and dsred fluorescence in lung histological sections that penetrated the mucus-lined respiratory epithelium. more exosomes and liposomes surpass mucus-lined surfaces -hours post-treatment compared to -hours post-treatment. fluorescent colocalization of exosomes and liposomes with alveolar type i cells, alveolar type ii cells, basal lung cells, and cd + macrophages was observed through immunohistochemistry analysis. more exosomes and liposomes colocalize with these cell types -hours post-treatment compared to -hours post-treatment. summary/conclusion: lsc-exosomes and liposomes penetrate the mucus-lined respiratory epithelium and reach the pulmonary parenchyma through inhalation treatment. lsc-exosomes and liposomes are uptaken by alveolar epithelial cells, basal cells, and interstitial macrophages with improved biodistribution -hours post-treatment. funding: this study was supported by the nc state chancellor's innovation fund. transfection reagent artefact accounts for some reports of extracellular vesicle function codiak biosciences, cambridge, usa introduction: extracellular vesicle (ev) functions are frequently investigated by transiently transfecting cells with plasmid dna to produce evs modified with protein(s) or nucleic acid(s) of interest. however, evs and the dna-complexes used to transduce cells are physically similar, raising the possibility that they may co-purify during differential ultracentrifugation, the most common ev isolation procedure. activities attributed to evs may therefore be due to contaminating dna -transfection reagent complex. methods: ev producing cells were transiently transfected with plasmid dna encoding gene-editing or split enzymes fused to ev-targeting protein sequences. differential and density gradient ultracentrifugation were used to purify evs from cell culture supernatant or dna lipoplexes from cell-free culture media. protein expression and localization to evs was confirmed by western blot. cell lines stably expressing fluorescent or luminescent reporters were used to assess functional enzyme delivery in recipient reporter cells. results: reporter cells treated with ultracentrifuge pellet material (ucp) from media of transiently transfected cells showed robust and reproducible signal, however fractionating the ucp with an iodixanol density gradient revealed that reporter activity was associated with high-density fractions that were depleted in evs. ucp isolated from identical transfection conditions, but lacking cells (and exosomes), showed identical biological activity levels and distribution in iodixanol gradients, suggesting that the activity was due to contaminating transfection reagent complexes and not evs. serial media changes on ev producing cells post-transfection did not significantly reduce ucp activity on reporter cells. treatment with nucleases did not digest complexed dna, did not significantly reduce dna levels in the ucp as measured by qpcr, and did not decrease activity in reporter cells treated with ucp from either transfected cells or no-cell controls. summary/conclusion: we find that dna-transfection reagent complexes are not separated from evs using differential ultracentrifugation and that common approaches to remove such complexes, including media exchanges and nuclease treatment, are ineffective. due to the pernicious nature of the dna-complex in these cellular assays, it is likely that some reports of ev function are likely artefacts produced by contaminating dna-complexes. we find that density gradient centrifugation can effectively separate evs and dnacomplexes, highlighting the importance of validating elimination of contaminating transfection reagent complexes when using transient transfection to interrogate ev function. chair: suresh mathivanan -la trobe university cancer stem cell-derived exosomes: potential biomarkers for early diagnosis and prognosis in pancreatic cancer introduction: pancreatic cancer (paca) is the most deadly manlignancy, due to late daignosis and early metastatic spread, which prohibits surgery. it is urgently for relaible, early detection. research shows that tumour-derived exosomes, which had been present in the blood in the early stage of tumour formation and before metastasis, is the vanguard forces of tumour formation and metastasis; cancer stem cell-derived exosomes (csc-exos) has stronger migration ability, so the detection of blood csc-exos for early diagnosis and monitoring of progress for paca has great research potential and the value of application. methods: protein markers were selected according to expression in exosomes of paca cell line culture supernatants, but not healthy donors' serum-exosomes. according to these preselections, serum-exosomes were tested by flow cytometry for the pancreatic cancer stem cell marker cd v and tspan . results: the majority ( %) of patients with paca and patients with nonpa-malignancies reacted with anti-cd v and anti-tspan . serum-exosomes of healthy donors' and patients with non-malignant diseases were not reactive. recovery was tumour grading and staging independent including early stages. introduction: chronic traumatic encephalopathy (cte) is a tauopathy that affects individuals with a history of mild repetitive brain injury frequently seen in contact sports. initial neuropathologic change of cte include perivascular deposition of phosphorylated tau (p-tau) in cortical neurons and, in later stages, the formation of neurofibrillary tangles in neurons throughout the brain. extracellular vesicles (ev) are known to carry neuropathogenic molecules in neurodegenerative disease and able to cross the blood brain barrier. we therefore examined the protein composition of ev separated from cerebrospinal fluid (csf) and plasma in former national football league (nfl) players with cognitive dysfunction, and an agematched control group with no history of contact sports. methods: evs were separated from csf and plasma from former nfl players (n = , ) and controls (n = , ) by affinity separation method or size exclusion chromatography, respectively. the ev protein profiling was characterized by simoa for tau and ptau and mass spectrometry. the protein data was analysed for ev enrichment, differentially expressed proteins, pathway analysis and correlation with cognitive function, head impact and tau/p-tau levels by biostatistics and bioinformatics. results: the level of total tau and p-tau in csf evs was not significantly changed, but significantly elevated in plasma evs from former nfl players. the proteins were commonly identified between the paired plasma-csf from the same patients, but there was no significant correlation with disease status. collagen alpha- (vi) chain (col a ), − (vi) chain (col a ) and reelin (reln) were differentially expressed in former nfl players' plasma evs. a combination of these proteins in plasma ev can distinguish former nfl players from controls with % accuracy by machine learning. summary/conclusion: the interacting plasma-csf ev proteomes provide an original resource to ev biomarker development for neurodegenerative disease, and col a , reln and col a in plasma evs can be potential biomarker for monitoring the cte development. density-based fractionation of urine to unravel the proteome landscape of extracellular vesicles in prostate cancer introduction: current diagnostic tests are unable to discriminate indolent from aggressive prostate cancer (pca), leading to overdiagnosis and overtreatment, and an intense interest in biomarkers to improve clinical decision making. urine is considered an ideal proximal fluid for biomarker identification in pca due to its direct contact with the urogenital system. the discovery and translation of extracellular vesicle (ev) content into pca biomarkers remains challenging due to the difficulty of obtaining urinary ev (uev) with high specificity. methods: we developed a step-by-step protocol to separate uev by orthogonal implementation of ultrafiltration and bottom-up density gradient centrifugation (bu-odg). we implemented complementary particle and protein measurements to identify uev (lower density) and protein rich fractions (higher density) and assess the performance of bu-odg (specificity, efficiency and reproducibility). using mass spectrometry-based proteomics we interrogated uev and protein rich fractions from matched urine and radical prostatectomy tissue samples from pca patients (n = ), and urine from men with pca prior to (n = ) and after local treatment (n = ), benign prostatic hyperplasia (n = ) and other urological cancers (n = ). results: bu-odg separated uev from soluble proteins and tamm-horsfall protein (thp) complexes with high specificity and reproducibility, outperforming differential ultracentrifugation, exoquick and size-exclusion chromatography. comparison of the uev proteome from men with benign or malignant prostate disease, allowed us to expand the known human uev proteome and identify a pca specific uev proteome not uncovered by the analysis of the protein rich fraction. proteomic analysis of ev separated from prostate tumour interstitial fluid and matched uev confirmed pca specificity of the uev proteome. analysis of the uev proteome from patients with bladder and renal cancer provided additional evidence of the selective enrichment of protein signatures in uev reflecting their respective cancer tissues of origin. summary/conclusion: we identified hundreds of previously undetected proteins in uev of pca patients and developed a powerful toolbox to map uev and protein rich fractions, ultimately supporting biomarker discovery for urological cancers. immunoglobulin a coating of faeces-derived bacterial vesicles as a marker of inflammatory bowel disease in humans nader kameli a , frank stassen b , heike becker c , john penders c , daisy jonkers d and paul savelkoul b introduction: iga is the most abundant antibody in mucosal secretions and plays a crucial role in maintaining the balance between the host and the gastrointestinal microbiome. recent studies suggested that pronounced iga coating is especially prominent among inflammatory commensals which drive intestinal disease. membrane vesicles (mvs, nano-sized particles released by bacteria) have also been found to interact with the host and modulate development and function of the immune system. however, their interaction with iga has not been studied yet. here we developed a method to isolate and characterize the mvs from faecal samples and checked for possible differences in iga coating patterns of mvs in health and disease. methods: mvs were isolated by using a combination of ultrafiltration and size exclusion chromatography from faecal samples of healthy controls (hc), patients with active crohn disease (cd) and cd patients in a remissive state. quantification and verification have been done with tunable resistive pulse sensing (trpsbased analysis) bead-based flow-cytometer (bbfc) and transmission electron microscope (tem). mvs were selected with specific antibodies for capturing (gram +: lta, gram-: ompa) followed by pe-conjugated anti-human iga antibodies as detection. results: we could successfully isolate * - * particles/ml from mg of faeces. bbfc in combination with trps provide a valuable method for (semi-)quantitative measurements of mixed populations. intriguingly, remarkable differences were found between iga coating mvs derived from healthy controls and active and remissive cd patients as mvs derived from healthy controls were significantly more coated compare to both cd patient groups. in details, for selected g-ve derived mvs: % of the total population of mvs derived from hc were coated, % from remissive cd patients, and < % of active cd patients; and for selected g+ ve derived mvs: % of the total population of mvs derived from hc were coated, % from remissive cd patients, and % of active cd patients. (data are represented as the mean). summary/conclusion: here we demonstrate for the first time that mv isolated from the faecal samples are also coated with iga, and surprisingly mvs from healthy volunteers were more densely coated than mvs from diseased patients. the possible consequence of this difference remains to be determined in future studies. monitoring altered tetraspanin and psma expression in prostate cancer derived extracellular vesicles via advanced image flow cytometry (isx) lukas w. prause a , christopher millan b , natalie hensky c , tullio sulser c and daniel eberli c a universityhospital zurich, zurich, switzerland; b university of zurich hospital, schlieren, switzerland; c university of zurich hospital, zurich, switzerland introduction: new diagnostic and therapeutic options for patients with prostate cancer are urgently needed. prostate-specific membrane antigen (psma)-based imaging and therapy are increasingly used for prostate cancer management. unfortunately, as a membrane protein, psma is not found as a soluble protein in the blood and therefore has limited utility as a diagnostic biomarker. however, psma has reportedly been observed as a cargo protein of prostate cancer-derived extracellular vesicles (evs). we demonstrate altered psma expression on evs derived from prostate cancer cell cultures (c - , lncap) in response to novel next-generation androgen receptor inhibitor (enzalutamide), a standard chemotherapy agent (docetaxel), a novel experimental nonsteroidal antiandrogen (epi- ) that binds covalently to the n-terminal domain of the androgen receptor and dihydrotestosterone (dht). additionally, evs were isolated from the plasma of prostate cancer patients who participated in the prococ biobank campaign at the usz. plasma was taken and stored from patients both pre-and post-prostatectomy. results: transmission electron microscopy, nanoparticle tracking analysis and simple western (wes) analysis show stable size distribution and amount of evs produced by treated and non-treated cells. using advanced image-based flow cytometry, altered tatraspanin and psma expression could be detected in evs isolated from cell culture supernatants of lncap and c - prostate cancer cells following their treatment. summary/conclusion: measuring psma expression on extracellular vesicles might pave the way to use image flow cytometry of evs to develop a blood based diagnostic test for prostate cancer patients with a wide range of possible applications including: ) monitoring response to therapy and, ) early indications of potential relapse. funding: vontobel fondation. proteomic profiling of human neural cells derived extracellular vesicles to identify human brain cell-type specific markers introduction: alzheimer's disease (ad) is a common neurodegenerative brain disease which affects appropriately million patients worldwide. one of the major challenges in ad is to develop reliable biomarkers for early diagnosis and disease-modifying therapies, especially before the clinical symptoms. extracellular vesicles (evs) carry cargos of proteins, lipids and nucleic acids. there was no comprehensive characterization of evs isolated from specific brain cell types, which may be useful for cell type-specific biomarkers. the purpose of this study is to isolate evs from human induced pluripotent stem cell (ipsc)derived brain cells for proteomic profiling and characterization of cell type-specific molecules. methods: human ipscs-derived neurons, microglia and primary cultured astrocytes were differentiated in ev-depleted media. the evs were isolated by differential centrifugation combined with size exclusion chromatography, followed by characterization using nanoparticle tracking analysis and mass spectrometry. the proteomic data were subjected to bioinformatics analysis results: we identified proteins from neuronderived ev (nde), proteins from microgliaderived ev (mde) and proteins from astrocytederived ev (ade) by proteomics. gene ontology analysis indicated that most of these proteins are associated with evs. furthermore, , and proteins are present individually in ndes, mdes and ades. among them, high levels of atp a and syt in ndes, itgam and cd a in mdes, and eaat and gfap in ades were found, all of which are typically and highly expressed in the original cells. summary/conclusion: our results provide us the potential candidates for cell-type specific ev markers, which will be helpful to develop non-invasive tools to enrich ev originating from specific brain cells and may lead to the development of new biomarkers for neurodegenerative disorders. ) are a tremendous resource for extracellular vesicle (ev) research, but they are heavily focussed on mammalian evs, i.e. evs from humans and laboratory animals, where protein cargoes are well characterised, and a wide selection of antibodies are commercially available. protein markers can be used to identify and define the types of mammalian ev and to determine the presence of any contaminants that might confound functional studies. similar resources are not as readily available for bacterial evs as these are not as well characterised, commercially available antibodies are much less abundant and immunological variation between different bacterial species (and there are trillion bacterial species on planet earth!) means that each species, strain, or group of related species may require different antibodies. methods: to identify quality markers for bacterial evs, we have characterised the proteome of cells, crude evs (ultracentrifuge pellet from cell free culture supernatant) and size exclusion chromatography or density gradient centrifugation purified evs from two different (pathogenic vs probiotic) strains of escherichia coli grown under two different environmental conditions, and one strain of mycobacterium marinum grown in one medium. results: our results identify a selection of proteins enriched in purified ev preparations, and proteins that are depleted after purification steps. summary/conclusion: our results allow the identification of potential markers for ev purity and non-ev contaminants, but also highlight the variability in bacterial ev preparations and suggest potential targets that can be used to investigate the heterogeneity of bacterial ev populations. introduction: recent findings indicate an increase in mid-life mortality rates in the usa and persistent, significant race-related health disparities exemplified by differential mortality rates. this suggests that exploring new molecular markers that may be linked to mortality could provide novel insights into factors that are driving mortality rates. accumulating data suggests that extracellular vesicles (evs) circulating in blood may be potential biomarkers of age-related disease. evs are nano-sized membranous vesicles that bear molecular cargo and mediate intercellular communication between different cells and tissues. little is known about whether ev characteristics differ by race or whether evs are associated with clinically relevant mortality risk factors. methods: in this cross-sectional study, plasma evs were isolated from middle-aged african american (aa) and white males and females. results: we report no significant differences in ev size or concentration with race or sex. there were significantly higher ev levels of phospho-p , total p , cleaved caspase , erk / and phospho-akt in whites compared to aas. higher ev levels of phospho-igf- r were found in females compared to males. we examined ev characteristics and protein cargo in the context of well-established clinical mortality risk factors. ev concentration was significantly, and positively, associated with several mortality markers including, high-sensitivity c-reactive protein (hscrp), homoeostatic model assessment of insulin resistance (homa-ir), alkaline phosphatase, pulse pressure, body mass index, and waist circumference. the relationship of ev concentration and cargo with mortality markers differs by race. summary/conclusion: our data show that ev-associated proteins can differ by race and sex and are associated with mortality risk factors. this study provides insight into the characterization of evs in middle-aged aas and whites, which may aid in the development of ev-based diagnostics. funding: this study was supported by the national institute on ageing intramural research program of the national institutes of health. repurposing specialised cell-free dna blood collection tubes for extracellular vesicle isolation introduction: liquid biopsies offer a minimally invasive approach to patient disease diagnosis and monitoring. however, many plasma processing protocols have been designed with a single biomarker in mind. here we investigate whether specialised dna blood stabiliser tubes could be repurposed for the analysis of extracellular vesicles (evs). methods: peripheral blood (n = ) was collected into k -edta, roche or streck cell-free dna (cfdna) blood collection tubes and processed using sequential centrifugation immediately or after storage for days. microev were collected from platelet poor plasma by , g centrifugation and nanoevs isolated using size exclusion chromatography. particle size and counts were assessed by nanoparticle tracking analysis, protein by bca assay and dot blotting for blood cell surface proteins. results: major variations in micro and nanoevs were seen with delayed time to processing. nanoev counts did not change with processing delay or tube collection type but the associated protein amount increased, indicative of cell lysis or activation. the protein was predominantly derived from from platelets (cd ) and red blood cells (cd a). the increase in associated protein was seen more in the k -edta and streck tubes indicating that the roche tubes may offer improved cell stability. conversely, microevs increased in both quantity and protein content with delay to processing indicative of both lysis and cell activation, irrespective of tube type. epithelial cell surface marker epcam abundance remained the same across conditions in both micro and nanoevs demonstrating that epcam+ evs were stable. summary/conclusion: specialised cfdna collection tubes can be repurposed for micro and nanoev analysis, however simple counting or using protein quantity as a surrogate of ev number may be confounded by pre-analytical processing. the evs would be suitable for disease selective ev subtype analysis if the molecular target of interest is not present in blood cells. introduction: nutrigenomics and nutrigenetics have been defined as the effect of nutrients on gene expression and genetic variation on dietary response, respectively. here, we propose the isolation and characterization of exosomes from donors carrying different alleles of hla-dqa and hla-dqb , to investigate their involvement in coeliac disease (cd) management. methods: a chilean population (n = ) was investigated for snps mutations in hla class ii alleles associated to cd predisposition (as well as other mutations related to other food intolerances), using the genochip food technology. exosomes have been isolated from donors' serum by ultracentrifugation and characterized by sds-page, western blotting (cd and cd ), and transmission electron microscopy. exosomes were also studied for their interleukins (il- and il- ra) content. results: among the studied population, % present at least one of the alleles leading to cd development and % carry alleles encoding for αand β-chains heterodimers associated with very high risk to develop cd. in parallel, isolated exosomes from donors with low to extremely high risk for cd showed high il- ra content ( . ± . to . ± . ), as the persons were not following any treatment. however, values of il- ra decrease in exosomes isolated form persons receiving treatment for cd. a relationship between exosomes' content and genetic susceptibility for cd has been observed, which may suggest their possible use as biomarkers for cd as the diagnostic of this disease is still a big issue. summary/conclusion: until this point of this underway project, we demonstrate the existence of a relationship between the exosomes' content in il- ra and genetic susceptibility for cd. furthermore, the genetic predisposition to cd could also modulate the gut colonization process, another important player in intestinal homoeostasis. in the next step, extracellular vesicles from gut microbiota will be isolated and analysed to determine their role in cd management. nasibeh karimi a , razieh dalir fardouei a , jan lötvall a and cecilia lässer b a krefting research centre, institute of medicine, sahlgrenska academy at university of gothenburg, göteborg, sweden; b krefting research centre, institute of medicine, sahlgrenska academy at university of gothenburg, gothenburg, sweden introduction: the ability to isolate extracellular vesicles (evs) from blood is vital in the development of evs as disease biomarkers. both serum and plasma can be used but few studies have compared them in terms of amount and type of evs. we have previously developed a method to isolate evs from plasma with minimal contamination of lipoprotein particles (karimi et al ) . the aim of this study was to compare the presence of different subpopulations of evs in plasma and serum. methods: blood was collected from healthy subjects, from which plasma and serum were isolated. evs were isolated using a combination of density cushion and size exclusion chromatography (sec) (protocol ) or a combination of density cushion and density gradient (protocol ) or immune-capturing (anti-cd , anti-cd and anti-cd beads) (protocol ). purity and yield of evs were determined by nanoparticle tracking analysis (nta), western blot, electron microscopy (em), exoview, flow cytometry and mass spectrometry (lc-ms/ms). results: as determined by nta and protein measurement more evs could be isolated from plasma with protocol and the majority of the vesicles were cd / cd a positive as determined with exoview and western blot. additionally, flow cytometry and western blot showed that more cd /cd a positive evs where also identified with protocol . furthermore, western blot showed increased amount of cd a in plasma samples in protocol . when labelled evs were spiked in freshly collected blood, no difference in recovery was seen for plasma and serum. summary/conclusion: this study shows that a larger amount of evs could be isolated from plasma compared to serum when three different isolation methods were used. firstly, this suggests that more evs are present in plasma. secondly, it suggests that these vesicles are probably released by platelets and that evs are not trapped in the clot during serum formation. future studies are needed to answer how this affects the use of blood-derived evs as biomarkers from serum and plasma. tumour-derived extracellular vesicles contain distinct integrin proteins stephanie n. hurwitz a and david g. meckes b a university of pennsylvania, philadelphia, usa; b florida state university, tallahassee, usa introduction: cargo profiling, including proteomic analyses, of tumour cell-derived extracellular vesicles (evs) may provide ripe opportunities for further understanding cancer growth, drug resistance, and metastatic behaviour. accumulating data suggest that cancer-derived evs contain membrane-bound integrin proteins which may aid in cell detachment, migration, and homing to future metastatic niches. we have previously published an extensive proteomic profile of secreted vesicles from the nci- panel of human cancer cells. methods: here, we further examine the distinct integrin components in these cancer-derived evs, and additionally profile evs released from benign epithelial cells by liquid chromatography and tandem mass spectrometry for comparison. results: we demonstrate the enrichment of integrin receptors in cancer evs compared to vesicles secreted from benign epithelial cells. total ev integrin levels, including the quantity of integrins α , αv, and β correlate with tumour stage across a variety of epithelial cancer cells. in particular, integrin α also largely reflects breast and ovarian progenitor cell expression, highlighting the utility of this integrin protein as a potential circulating biomarker of certain primary tumours. other integrins including α , αl, and β are enriched in vesicles derived from leukaemia cells, and may provide a means to distinguish haematopoietic cell-derived evs. summary/conclusion: this study provides preliminary evidence of the value of vesicle-associated integrin proteins in detecting the presence of cancer cells and prediction of tumour stage. differential expression and selective packaging of integrins into evs may contribute to further understanding the development and progression of tumour growth and metastasis across a variety of cancer types. effect of nicotine and menthol on cytochrome p and antioxidant enzymes in rat plasma-derived extracellular vesicles introduction: tobacco products such as e-cigarettes pose potential adverse health effects caused by direct exposure to aerosolized nicotine, flavorants such as menthol, and other particulates. here, we aimed to study the hypothesis that whether nicotine and menthol modulate nicotine-metabolizing cytochrome p a (cyp a ), antioxidant enzymes (aoes), sod and catalase in plasma extracellular vesicles (evs). modulation of these enzymes would eventually lead to nicotine-induced toxicity and hiv- pathogenesis via evs-based cell-cell interactions. methods: we isolated and characterized evs from rat plasma before and after nicotine self-administration (nic) with audiovisual cue (av) and menthol and characterized using ev markers according to the isev guidelines. protein associated with cyp a , sod , and catalase were quantified by western blot. results: we measured size, total protein, and acetylcholine esterase activity of evs and found no significance difference in these characteristics before and after nic. to investigate the effect av, menthol alone or in combination in the absence and presence of nic, first we evaluated the expression of ev markers cd and cd . the results showed menthol and av together increased the levels of cd (p ≤ . ), the marker of small vesicles, in the presence of nic. the nic with menthol and av showed a pattern of increased levels of small vesicle but could not reach to significance. next, we demonstrated that the nic with av increased the level of sod (p ≤ . ), which showed a pattern of increased levels of catalase and cypa , though statistically non-significant. the expression of nicotine receptor did not change under any conditions used. the results showed an increased level of cyp a (p ≤ . ), sod (p ≤ . ), and catalase (p ≤ . ) in plasma evs in the menthol-nic group compared to menthol group only. nic group with a combined av and menthol, showed further increase in the levels of cyp a (p ≤ . ), and catalase (p ≤ . ). further analysis of plasma evs on inflammatory cytokines/chemokines in these groups, and the effect of plasma evs on nicotine-induced toxicity and hiv pathogenesis are underway. summary/conclusion: nicotine administration increased, though not statistically significant, the levels of circulatory evs. moreover, the study provided evidence that nicotine in the presence of menthol, av, and/or menthol+av increased nicotine-metabolizing cyp a in all the groups and aoes in specific groups. funding: we thank the national institute on drug abuse (grant #da , da- ) for supporting our work. introduction: biomarker discovery in breast cancer (bc) is a clinical need for therapeutics and non-invasive diagnostics. tumour exosomes are involved in premetastatic niche formation and drug resistance and represent a source of non-invasive biomarkers. the identification of tumour exosomal biomarkers provides, not only, the possibility to discriminate patient groups also potential targets to control cancer progression that could be exploited to develop innovate bc therapeutic strategies. methods: we have performed a comparative differenti. al proteomic profile of four bc cell lines and their derived-exosomes, representative of the most relevant bc subtypes in clinic to search non-invasive biomarker candidates. then, we have carried on two bioinformatics approaches: ) protein association network analysis interaction (string) and ) pathway inference analysis (hipathia), to characterize the functional profiling for each bc subtype. results: we have found differentially-expressed proteins, in both cells and exosomes, that include indicators of invasion, metastasis, angiogenesis and drug resistance. exosome proteome profile reflects their different bc cell origin suggesting potential indicators of bc subtype. further, bioinformatics analysis reveals a differential role of exosomes in bc signalling pathways in recipient cells, according to their protein cargo and cell origin. summary/conclusion: our results show a set of cells and exosome proteins that highly discriminate bc subtypes and may significantly contribute to further studies for the design of bc biomarker predictor to stratify bc patients and the development of novel therapeutic strategies. funding: a set of potential biomarkers to discriminate breast cancer subtypes. circulatory evs as potential biomarkers of hiv-drug abuse interactions and neurological dysfunction in hiv-infected subjects and alcohol/ tobacco users sunitha kodidela a , kelli gerth a , namita sinha a , asit kumar b , prashant kumar a and santosh kumar a a uthsc, memphis, usa; b university of tennessee health science center, memphis, usa introduction: abuse of alcohol and tobacco can exacerbate hiv pathogenesis and its associated complications. further, the diagnosis of neurocognitive disorders associated with hiv infection and drug abuse using csf or neuroimaging are invasive or expensive methods, respectively. therefore, extracellular vesicles (evs) can serve as reliable non-invasive markers due to their bidirectional transport of cargo from the brain to the systemic circulation. hence, we aimed to study the specific evs proteins, which are altered in both hiv and drug abusers to identify a physiological marker to indicate the immune status and neuronal dysfunction of hiv-positive drug abusers. methods: evs were isolated from plasma of the following subjects: a) healthy b) hiv c) alcohol drinkers d) cigarette smokers e) hiv+alcohol drinkers f) hiv +cigarette smokers. quantitative proteomic profiling of evs was performed by mass spectrometry and potential ev proteins associated with neuronal dysfunction were quantified by westernblot. results: the evs were characterized according to the isev guidelines. a total of proteins were detected in evs of all the study groups. comparison of proteins among all the study groups revealed that hemopexin was significantly altered in hiv+drinkers compared to drinkers and hiv subjects. further, our study is the first to show properdin expression in plasma evs, which was decreased in hiv+smokers and hiv+drinkers compared to hiv patients. though we couldn't identify the few other cns-specific proteins, g-fap and l -cam, associated with neuronal dysfunction in plasma evs by mass spectrometry, we could detect those by westernblot. the protein expression of gfap (p < . ) was significantly enhanced in plasma evs obtained from hiv-positive subjects and drinkers compared to healthy subjects, suggesting enhanced activation of astrocytes in those subjects. the l cam expression was found to be significantly elevated in smokers (p < . ). both gfap and l cam levels were not further elevated in hiv+smokers compared to hiv+nonsubstance users. summary/conclusion: the present findings suggest that hemopexin, and properdin show potential as markers for hiv-drug abuse interactions. further, astrocytic and neuronal-specific markers (gfap and l cam) can be packaged in evs and circulate in plasma, which is further elevated in the presence of hiv infection, alcohol, and/or tobacco and thus may represent as potential biomarkers for neurological dysfunction in those subjects. funding: we thank the national institute on drug abuse (da ) for supporting our work. . electrochemical detection of mirna- - p introduction: micrornas (mirnas) are small, single-stranded, non-coding rna species that regulate gene expression post-transcriptionally, and are transported by extracellular vesicles (evs). they play an essential role in biological processes, such as development, cell proliferation, apoptosis, stress response and tumorigenesis. thus, mirnas are considered relevant biomarkers in health. more particularly, mirna- - p is expressed in neurons after traumatic brain injury, being expectably transported to peripheral fluids by brain evs that cross the blood-brain barrier. the main goal of this work is to develop an electrochemical biosensor for the detection of mirna- - p in serum. methods: overall, the experimental assembly of the biosensor was made in three stages. the first one consisted in the electrodeposition of aunps, the second one in the incubation of anti-mirna - p on the carbon screen-s printed electrodes and the final stage in the incubation of mercaptosuccinic acid for blocking unspecific bindings. the probe was hybridized with the target mirna - p by a consecutive incubation of several standard solutions. each modification was evaluated with cyclic voltammetry (cv), electrochemical impedance spectroscopy (eis) and square wave voltammetry (swv). the electrochemical behaviour of the biosensor was followed in all steps by monitoring the electron transfer features of a standard redox system. the redox probe selected for this purpose was [fe (cn) ] -/[fe(cn) ] -. results: the results indicated that the electrodeposition of gold was more effective for − . v for s and could lead to better signals upon anti-mirna- - p hybridization. summary/conclusion: in general, the experiments showed increasing charged transfer resistance upon the incubation of higher concentrations of mirna- - p. in these experiments, ev concentration is a critical variable that must be carefully controlled to ensure scientific rigour and reproducibility: without controlling for concentration (dose), experimental outcomes will exhibit excess variability that could mask important biological discoveries. in this study, three orthogonal methods are compared for accuracy in ev quantification: microfluidic resistive pulse sensing (mrps) and nanoparticle tracking analysis (nta) were compared to each other and relative to the gold standard method, transmission electron microscopy (tem). the ability of nta to accurately measure particle concentration is shown to depend on the polydispersity of the sample itself. results validate the accuracy of mrps and emphasize the importance of using orthogonal techniques to quantify evs. methods: reference urinary vesicles were prepared and analysed with the three methods and the relative concentration accuracy of nta and mrps were compared as a function of particle size. the hypothesis that nta concentration accuracy was impeded by sample polydispersity was tested using polystyrene bead mixtures having a range of polydispersity. a theoretical argument based on fundamental physics explains the experimental observations. results: tem and mrps measurements of the evs were in excellent agreement and showed a broad, polydisperse particle size distribution with no peak on the measured size range ( nm - nm diameter). nta differed significantly from tem and mrps by reporting a steep decrease in measured concentration below about nm that resulted in a peak in the reported particle size distribution. bead measurements confirmed the hypothesis to be tested: sample polydispersity significantly affects the ability of the nta method to accurately measure concentration, even for particles as large as nm diameter. summary/conclusion: these experiments validate mrps as an accurate method for quantifying evs and highlight the importance of using orthogonal measurement methods in accordance with misev guidelines. clinically relevant synthetic reference materials to standardize concentration measurements of extracellular vesicles: state-of-the-art and future prospects introduction: there is an unmet need to standardize concentration measurements of extracellular vesicles (evs). flow cytometry is the clinically most applicable method, but the currently available reference materials for calibration are insufficient. for example, the refractive index (ri) between standard particles and evs substantially differs, whereas concentration and fluorescence calibration particles are too bright. the goal of this study is to ascertain the most desired properties of reference materials to standardise ev measurements. methods: an online survey was prepared within the meves ii project to measure the desired size, concentration range, optical properties, choice of fluorochromes, and stability of synthetic ev reference materials for flow cytometry (fcm) measurements. besides the desired properties of ev reference particles, also the available instrumentation was assessed in the survey, which was sent to the members of the stakeholder committee of metves ii project and members of the ev flow cytometry working group. results: the most desired size, concentration, and ri range for ev reference particles is nm to nm, e to e /ml, and . - . , respectively. based on mie-theory evaluation of the sensitivity of the available instruments, none of the respondents would be able to detect nm particles with ri = . with their current instruments. regarding fluorescence intensity, the most desired range according to the responses is from molecules of equivalent soluble fluorochromes (mesf) to mesf. considering the sizes of evs and fluorescent labels, the maximal mesf that can be obtained for ev reference particles with nm diameter and high molecular mass fluorescent dyes is in the range of several hundreds. typical antigen densities on evs fall below copies per ev with nm diameter, i.e. mesf values above are probably not physiologically relevant in this size range. summary/conclusion: a part of the desired properties of ev reference materials precludes either their physical feasibility of production or their detection at most currently available fcms, meaning that the intended reference materials will be future-proofed. funding: this work was supported under hlt metves ii project by the european metrology programme for innovation and research (empir). the empir initiative is co-funded by the european union's horizon research and innovation programme and the empir participating states. comparison of production and activity of amniotic fluid stem cell extracellular vesicles from d hollow fibre bioreactor and d culture. culture conditions may affect ev composition and potency. here we compare production, potency, identity and therapeutic potential of evs collected from cells grown in culture dish ( d) versus hfbr ( d). methods: human clonal afsc were derived from patient-consented amniotic fluids. x e hafsc were seeded in d ( cm ), and . x e hafsc on a small kd mwco hfbr (fibercell-c d, cm ) with fibronectin coating; both cultured in chang medium with % of es-fbs, starved for hr and then evs collected. the effect of harvest frequency was tested ( hrs, hr, hrs, wk). d-evs and d-evs were compared by nanosight, potency assay (by wb), identity (by exoview analysis) and therapeutic effect (in vivo in an animal model of kidney disease, alport syndrome). results: d production was~ . x e ev/ml/ hrs while d was~ . x e ev/ml (first four hrs) and . x e ev/ml (two days of hourly harvests). very little difference in ev concentration and very similar size distribution (~ nm) were observed during harvest intervals; possibly indicating either significant ev re-uptake or inhibition of ev secretion dependent upon free ev in the supernatant. d-evs trapped vegf (an in vitro established potency assay) as efficiently as d-evs, and expressed cd , cd , cd , cd , cd and vegfr as d-evs. summary/conclusion: d-evs had comparable properties and bio-activity to d-evs, but the hfbr produced x more evs. hfbr cell culture conditions for hafsc still need optimization, however an available . m cartridge provides a x scale up potential. the hfbr, a cgmp closed system, can produce sufficient numbers of ev to support pre-clinical and clinical applications with at least similar properties to evs produced by conventional d methods. funding: -intramural funding -intramural ev core pilot funding demonstration of high gain mode in combination with imaging flow cytometry for improved ev analysis luminex corporation, seattle, usa introduction: extracellular vesicles (evs) are membrane-derived structures that include exosomes, microvesicles, and apoptotic bodies. in recent years, the importance of evs has become apparent, as they are key mediators of intercellular communication. however, quantifying and characterizing evs in a reproducible and reliable manner is challenging due to their small sizeexosomes range from to nm in diameter. it is well-known that flow cytometers were originally designed to measure and detect cells, and due to the quantitative power flow cytometry offers, there has been a push to quantify and characterize evs using flow cytometric methods. however, these systems have not been designed to measure objects smaller than a cell. methods: here, we describe the use of high gain mode on the amnis® imagestream® imaging flow cytometer to address the challenges of measuring small particles. in this new high gain mode, the charge-coupled device (ccd)-camera is manually adjusted to higher gain settings, increasing the signal obtained from the ev. object thresholds and masking have also been adjusted to better identify and detect small particles. results: preliminary results using murine leukaemia virus-sfgfp reference particles have shown up to a fivefold increase in the number of gfp-positive objects collected in high gain mode, when compared to standard gain on the imagestream system. summary/conclusion: in this study, we demonstrate improved small particle detection, including evs, using this new high gain mode on the imagestream imaging flow cytometer. distance-controlled accelerated catalysed hairpin dna circuit for multiple and sensitive detection of exosomes-associated mirnas introduction: sensitive and simultaneous monitoring of multiplexed exosome-associated rnas is of great value for early cancer diagnosis remains a challenge. methods: here, we report a simple, multiple and sensitive exosomes-associated multiplex mirnas detection method that uses distance-controlled accelerated catalysed hairpin dna circuit (chdc) system without any complex operation or enzymatic amplification. the distance-controlled accelerated chdc can directly enter the plasma exosomes to generate fluorescent signal quantitatively by specifically targeting mirnas without any transfection means. results: we show that distance-controlled accelerated chdc strategy with signal amplification capability could selectively and sensitively identify low level rnas in serum evs, distinguishing patients with early-and late-stage breast cancer from healthy donors and patients with benign breast disease. summary/conclusion: this simple, accurate, sensitive, and cost-effective liquid biopsy by the distance-controlled accelerated chdc method is potent to be developed as a non-invasive breast cancer diagnostic assay for clinical applications. impact of isolation methods on biophysical heterogeneity of single extracellular vesicles university of california los angeles, ca, los angeles, usa introduction: current biophysical analysis of extracellular vesicles (evs) typically encompasses particle density and size distribution determinations using various techniques. however, variabilities in ev isolation methods and the structural complexity of these biological-nanoparticles (sub- nm) necessitate more rigorous nanoscale biophysical characterization of single evs to facilitate more reliable and comparable evbased assays. methods: combining atomic force microscopy (afm), super-resolution optical and conventional particle sizing light scatter and microfluidic techniques, we compared the unique sub-nanometre scale biophysical properties of breast cancer cell-derived ev isolates obtained using different isolation methods. results: afm and dstorm particle size distributions showed coherent unimodal and bimodal particle size populations in centrifugation and immune-affinity isolates respectively. more importantly, afm imaging revealed striking differences in nanoscale morphology, surface undulations, and vesicle-to-non-vesicle ratios among ev isolates from different isolation methods. our findings demonstrate the effectiveness of orthogonal high-resolution biophysical characteristics of single evs, not discernable via particle size distributions and counts alone. summary/conclusion: the identified nanoscale biophysical characteristics of ev isolates represent a strategic and complementary framework to resolve differences in the heterogeneity and purity of evs from introduction: extracellular vesicle (ev) concentrations measured by flow cytometry are incomparable. to improve comparability, the metves ii consortium is developing traceable reference materials and procedures, which require validation by test samples. in previous interlaboratory comparison studies, however, a main source of variation was introduced by pre-analytical variables and measurement artefacts introduced by test samples. to minimize variation introduced by test samples, our aim is to develop off-the-shelf biological test samples containing pre-labelled evs. methods: human urine and plasma were collected from healthy donors. evs were labelled with lactadherin-fitc, isolated by size-exclusion chromatography to remove free dye and minimize swarm detection, and mixed with dimethyl sulphoxide (dmso), exocap or trehalose, frozen in liquid nitrogen and stored at − °c . after thawing, ev concentrations were measured by a calibrated flow cytometer (apogee a -micro). results: compared to the ev concentrations measured in fresh plasma and urine, the concentrations decreased % in plasma (p = . ; mean of the cryopreservation agents) and % in urine (p = . ) after one day of storage. after months of cryopreservation, the concentration of plasma evs decreased % (dmso and exocap) and . % (trehalose) compared to one day of storage, whereas the concentration of urine evs decreased % (exocap) and % (dmso and trehalose). summary/conclusion: we have developed ready-touse, pre-labelled human evs that are stable up to months and dedicated for use in interlaboratory comparison studies. to further increase stability, other cryopreservation agents will be tested. our biological test samples will be key to validate the new reference materials and procedures developed by metves ii in . funding: this project has received funding from the empir program co-financed by the participating states and from the european union's horizon research and innovation program. understanding intracellular fate of ev-delivered content introduction: despite much work performed on evaluating the potential effects of extracellular vesicles (evs), the functional uptake of their cargo is still controversial. this project aimed to demonstrate that ev content (protein and mrna) is protected and can be subsequently transferred with functional activity into recipient cells, while also developing a tool to assess and quantify functional ev uptake. methods: fusion proteins used were mitochondrial localized coxviii-cfp-nanoluc(cox) and nuclear localized h b-rfp-nanoluc(h b). results: hek t cell-derived evs protected cox proteins from proteinase k digestion while demonstrating significantly improved efficiency of uptake when compared to free protein, as measured by bioluminescence that was still detectable in recipient cells hrs post-ev-exposure. to confirm functional uptake, recipient cells exposed to evs containing h b for hrs were imaged and some recipient cells manifested fluorescent red nuclei. to demonstrate the presence of functional mrna within evs, producer cells were transfected for such a duration as not to have detectable levels of protein in the evs while still containing detectable levels of mrna (qpcr) even after rnasea treatment. transfer of these evs to hela cells showed an increase in expression of h b which was blocked by cyclohexamide, confirming translation of the mrna ( . kb). to determine if recycling of ev delivered proteins occurs, recipient hela cells were exposed to evs containing cox for hrs. all extracellular evs were removed and cells were trypsinized ( . % for min) to remove any non-internalized cox protein. hrs later, evs (cd + and cd +) released from cells contained cox suggesting recycling of protein or possibly recycling of entire evs. lastly, an assay was developed to measure functional ev uptake. nanoluc protein was split in two and fused to mturquoise (n ) or mscarlet-i( c). expression of each fragment alone exhibited non-detectable levels of luminescence while expressing both together had a significantly increased signal. delivery of either fragment within an ev to a cell expressing the corresponding fragment worked as confirmation and quantification of ev uptake (hek , u , hela cells). summary/conclusion: this study robustly demonstrates ev delivery of functional mrna and protein to cells, while also establishing a simple assay to quantify and validate functional ev uptake. theoretical model of ev losses due to adsorption on the tube walls. application for immunomagnetic detection of the vesicles introduction: short-term storage of unfrozen samples of vesicles, mainly at °c, overnight or during a couple of days is rather common laboratory practice. however, it was found to lead to significant losses of vesicle concentration supposedly due to adsorption on the walls of the tube. the present work develops a theoretical model intended to describe the vesicle adsorption process. the experimental validation of the model was made using method of immunomagnetic precipitation. methods: the theoretical model considers the "diffusion-limited" case of vesicles storage. the maximal adsorption capacity of the surface of contact between the tube and the solution is given as the number of vesicles in hexagonally packed monolayer. for experiment, the vesicles were purified from ht cell culture supernatant by differential centrifugation, aliquoted and kept at − c. further the aliquots were consequently unfrozen, and placed into the tubes with different surface treatment and kept at + c. the kinetics of vesicles loss was measured by anti cd immunomagnetic capturing followed by cd , epcam and cd staining and flow cytometry. results: the model allows the estimation of the adsorption-associated losses as dependent on initial vesicles concentration, volume of the solution, tube geometry, the storage temperature and duration case of quiet vesicles storage (without mixing) and also accounts an expected effect of active agitation of the solution (ev-beads complexes formation). theoretical calculations were illustrated by analysis of ev at different storage conditions and during reaction of immunomagnetic precipitation of the vesicles. summary/conclusion: it was demonstrated that application of tubes surface treatment allows increasing sensitivity of immunomagnetic precipitation method to x ^ for cd +, x ^ for epcam+ and x ^ for cd + vesicles. introduction: it is now largely accepted that the intestinal microbiota plays a key role in intestinal bowel diseases (ibd). an imbalance in the composition and diversity of the intestinal microbiota (i.e. dysbiosis) of patients has been repeatedly pointed out by several teams. there are also indications that extracellular vesicles produced by bacteria and exosomes produced by epithelial cells might be increased in this family of diseases. methods: in order to differentiate healthy and ibd faecal samples on the basis of their vesicle profiles, we want to develop a means to enumerate rapidly particles in faecal samples, based on interferometric microscopy. the videodrop technology, developed by myriade, relies on the creation of single beam interferences between two signals from the same light path by nanoparticles such as small vesicles. it will permit to compare on large scales the viral load of healthy subjects and ibd patients. results: this fast and easy-to-use device was compared to the nta on several types of eukaryotic and prokaryotic vesicles and our preliminary results are encouraging. introduction: small extracellular vesicles (sevs) produced by mesenchymal stromal cells (msc-sevs) may be useful in cell-free therapies for immunomodulation and tissue regeneration. methods: to characterize msc-sevs produced ex vivo, human bone marrow mscs were cultured in mesencult-acf plus (macfp), an ev-free and animal component-free culture medium for days and spent medium collected to isolate sevs by ultracentrifugation (uc). analyses of sevs were performed by nanoparticle-tracking analysis (nta), western blot (wb), and human umbilical vein endothelial cell (huvec) tube formation assay. results: analysis of fresh uncultured macfp by uc, nta and wb for cd , cd , and cd confirmed the absence of sevs. msc-sevs isolated from spent macfp by uc ranged from - nm in size and were positive for cd , cd , and cd proteins. these sevs could be stored at − °c for > months in solution or lyophilized with minimal loss based on nta and wb analysis. the msc-sevs contained the msc-associated micrornas let a, mir , and mir a as per qpcr analysis. the biological function of ex vivo isolated msc-sevs was assessed using a human umbilical vein endothelial cell (huvec) tube formation assay. huvecs treated with msc-sevs generated tubes as early as h after seeding, which were not observed in control huvec cultures until h. moreover, the number of branch points present in such tube structures was >fourfold higher in huvec cultures (n = ) supplemented with msc-sevs versus control, with the former lasting > h and the latter lasting < h in culture. direct comparison of the performance of macfp medium to media containing non-depleted or ev-depleted foetal bovine serum demonstrated that only mscs cultured in macfp (n = ) were able to expand robustly with a doubling time of . , . and . days in these media, respectively. lastly, methods for isolating sevs using newly developed easysep-ev™ magnetic separation kits and size exclusion columns will be presented. summary/conclusion: taken together, these data demonstrate that msc-sevs can be produced in high yield in macfp medium and that these possess similar physical, phenotypic and functional characteristics as sevs in vivo. funding: this work was privately funded by stemcell technologies inc. introduction: extracellular vesicles (evs) are heterogeneous group of small vesicular structures released by different types of cells, including stem cells (scs). as recent studies demonstrate that they may enclose bioactive content and transfer it into the target cells, growing interest is placed on the utilization of evs in the field of biomedical research. however, there is still lack of standardized methods of evs characterization. as an example, typical flow cytometry-based protocols, commonly used for cells phenotyping, may be inadequate for the characterization of evs as particles with size close to the detection limit of conventional cytometers. thus, the aim of this study was to optimize and compare the use of different flow cytometry platforms for the multiparameter analysis of evs isolated from different types of scs populations. methods: ev samples were obtained by ultracentrifugation of conditioned media collected from selected scs types, including human induced pluripotent scs (ips) and mesenchymal scs (mscs). next, several high resolution flow cytometry systems: cytoflex, apogee (a and a micro-plus) and image stream mk ii were employed to compare their sensitivity and resolution, as well as influence of "swarm" effect. furthermore, we examined evs phenotype, including expression of tetraspanins and other surface markers. results: our results have revealed that tested flow cytometry systems may be utilized for the phenotypic characterization of evs secreted by scs populations. however, the conventional staining and gating strategy protocols have to be thoroughly optimized. additionally, depending on a type of tested cytometer, we have demonstrated the difference in a "swarm" effect and its influence on obtained results regarding evs phenotype. finally, imaging flow cytometry platform was also employed to visualize evs on the single particle level. summary/conclusion: in conclusion, we have demonstrated that tested high-resolution flow cytometry platforms are convenient methods for the multiparameter characterization of evs produced by different types of scs populations. however, careful selection of particular measurement parameters should be performed depending on a type of employed system. funding: this study was funded by ncbr grant strategmed iii (strategmed / / / ncbr/ ) to ezs. evaluation of atcc's exosomes from cell culture supernatant as reference standards in research and development. introduction: exosomes are subcellular particles - nm in size released from cells through a fusion of multicellular bodies with the plasma membrane. exosomes are stable carriers of cell-free cargo in the form of dna, rna, and proteins, thereby making them an attractive candidate for diagnostic and therapeutic applications. however, isolating a consistent population of exosomes can be challenging and there is an unmet need for highly characterized exosomes for use as reference standards in extracellular vesicle research (ev). methods: exosomes were isolated from cell culture supernatants of different atcc cell lines including stem cells and cancer cell lines representing the most prevalent cancer types -prostate, colorectal, breast, lung, cervical and glioblastoma, using tangential flow filtration (tff). these exosomes underwent sterility and mycoplasma tests as a part of their quality control. the morphology and size distribution of these exosomes were evaluated through multiple strategies including nanoparticle tracking analysis (nta), asymmetrical flow field-flow fractionation (af ), cryo-electron microscopy (cryo-em) and spectra dynetm particle analyser. exosome surface markers were also analysed through multiple strategies such as electro chemiluminescent elisa, flow cytometry and western blotting. also, stem cell exosomes and cancer exosomes were further evaluated for functionality through in vitro functional assays including migration assay, angiogenesis and anchorage independent growth assay. results: our optimized tff method resulted in high yields of > × exosomes/ml and average protein equivalent of more than mg/ml. more than % of the exosomes population had an average size distribution of - nm and median size of nm confirmed through a number of different size distribution instruments. although cell line dependent, we were able to obtain similar expression levels of different cell surface markers including tetraspanins (cd , cd , cd ) when evaluated through different methods. our functional data demonstrated stem cell exosomes were functionally active in promoting cell migration and tubule formation. additionally, cancer cell exosomes were found to promote a malignant phenotype in an anchorage independent growth assay. summary/conclusion: collectively, we demonstrated our ability to reproducibly manufacture production-scale batches of exosomes from multiple different cell types. our purified exosomes are of high yield, meet well-established quality control specifications, and are robust in maintaining size distribution, surface marker expression, and functionality in vitro. therefore, they can serve as ideal reference materials that can support different ev-based research applications. exo-cise: extracellular vesicles enriched from plasma post-exercise promotes myogenesis and neurogenesis bianca paris a , yaomeng liu a , vicente pagalday-vergara a , julie davies b , priya samuel a , ayman abu seer a , johnny collett a , laura gathercole a , ken howells a , karl j. morten b , zhidao xia a , daniel anthony b , david r f. carter a , helen dawes a and ryan c. pink a a oxford brookes university, oxford, uk; b university of oxford, oxford, uk introduction: physical activity brings about a widespread physiological response and elicits the beneficial adaptation of several tissues and organs. furthermore, regular participation in physical activity reduces the risk of developing major non-communicable diseases such as cardiovascular disease, diabetes, cancer, osteoporosis, and dementia. two important processes known to occur following physical activity are myogenesis and neurogenesis; both of which involve the activation and proliferation of specialised tissue-resident stem cells. the molecular mechanisms regulating these processes following exercise are poorly understood to date. here, we investigated the contribution of extracellular vesicles, which are released into the circulation after exercise, to benefit adult myogenesis and neurogenesis. methods: small extracellular vesicles were enriched from the blood of healthy participants before and following maximum and moderate intensity exercise. differentiation and proliferation using a range of methods was measured following vesicle treatment onto primary myoblasts and neuronal primary exvivo stem cells. activation of key cellular pathways were measured. results: we show significant proliferation and differentiation changes of both stem cell types. this is independent of extraction method, extracellular vesicle depleted fractions and is interestingly conserved across mammalian species. remarkably, we see an age-related effect. summary/conclusion: this advocates that short single bouts of exercise may promote myogenesis and neurogenesis via systemic signalling of extracellular vesicles which opens an interesting field in endogenous ev therapies. show promise as a cell-based therapy for retinal degeneration. while clinical trials are ongoing, the potential of extracellular vesicles (evs) as biomarkers for monitoring eye health and disease is not well studied. this study characterized the ev surface profile and cargo of hipsc-rpe to offer a baseline assessment in normal and disease conditions. moreover, we evaluated the importance of pnpla , a gene involved in membrane integrity and when mutated causes retinal degeneration, in ev biogenesis and secretion. methods: evs were isolated from serum-free culture medium of hips-rpe and identified with nanoparticle tracking analysis, transmission electron microscopy, and immunoblot analysis of exosomal markers, including alix, tsg , and cd . surface marker detection and proteomic profiling were completed using an ev surface marker kit and mass spectrometry, respectively. small interfering rna targeting pnpla was used to knockdown the expression in hipsc-rpe and evs were characterized. results: nanoparticle tracking analysis confirmed the presence of both microvesicles (> nm) and exosomes (< nm) by size distribution and the concentration of evs ( x particles/ml) from rpe. tem displayed typical morphological characteristics of evs. the presence of known ev markers, alix, tsg , and cd was confirmed via immunoblot and flow cytometry. surveillance of ev surface markers revealed enrichment of epithelial markers (cd ) and stem cell markers (cd / ) that depict donor cell origin and functional proteins including integrin-binding (cd ) and tgf-beta receptors (cd ). in addition, proteomic analysis revealed regulators of inflammation and rpe function, including hemopexin, clusterin, complement factor i, and pigment epithelium-derived factor. furthermore, reduction in pnpla expression reduced vesicle secretion and vesicle size compared to non-targeting controls. introduction: vascular endothelial growth factor (vegf) is a potent angiogenic factor and was first described as an essential growth factor for vascular endothelial cells. vegf plays a role in normal physiological functions such as bone formation, haematopoiesis, wound healing, and development. mesenchymal stem cell (msc) was found to secretes potential growth factors such as vegf when cultured in vitro. however there are some beliefs that foetal bovine serum (fbs) which usually used as serum in cell culture content vegf. methods: msc seeded in in -well plate in with concentration of , cell/well. cells were incubated for hours and fasted for another hours using only dmem. cells were treated with complete medium consist of dmem and % fbs. culture medium were collected after , , and hours after treatment. cell were culture in ºc dan % co . vegf concentration was detected using elisa technique. results: vegf concentration was not found in fbs which do not contact with msc. an increasing of vegf concentration in time-dependent manner was shown when culture medium was used in msc cell culture in normoxic condition. the result of vegf concentration when culture , , and hours were . pg/ml, . pg/ml, and . pg/ml, respectively. the mechanism of msc release growth factor is still under investigated. however, the classic growth factors and cytokines serves paracrine control molecules which were important in regenerative medicine. vegf was found to be an important molecules in angiogenesis process and determine the fate of cells. summary/conclusion: msc secreted vegf and concentration increased in time-dependent manner. isolation and characterization of exosomes from canine stem cells introduction: unlike induced disease models using laboratory animals, naturally occurring disease models display pathophysiologic attributes that are more similar to human diseases. unfortunately these models are underutilized in translational regenerative medicine research. this is partly due to the slow development of species-specific experimental therapeutics to investigate comparative efficacy. thus, we set out to isolate and characterize exosomes from canine adipose-derived mesenchymal stem cells (cad-msc) to use as a comparative therapeutic in dogs. to accomplish this, we optimized an isolation and purification strategy and characterized their molecular properties. methods: exosomes were isolated by sequential centrifugation and subsequent ultrafiltration. the proteome was characterized by tandem mass tag (tmt) mass spectrometry and the mirna cargo was identified using a canine specific pcr array with subsequent target and enrichment analysis using targetscan and the panther platform, respectively. also, nanoparticle tracking analysis and transmission electron microscopy were used to determine exosome size and structure. to investigate bioactivity, we measured the ability of exosomes to inhibit collagen production in an in vitro model of fibrosis. results: exosomes were purified by ultrafiltration using a kda cut-off. proteomic analysis by tmt mass spectrometry identified unique proteins. % of the exocarta top were identified from this list. additionally, we identified the mirna cargo within exosomes and found highly expressed mirnas. enrichment analysis identified multiple pathways of probable regulation including angiogenesis (fold enrichment = . ; p < . ) and transforming growth factor-beta (tgfb) signalling (fold enrichment = . ; p < . ). exosome size was quantified to be . ± . nm with a modal average of nm. lastly, in the presence of exosomes, tgfb stimulated fibroblasts deposited . % less collagen than vehicle controls (p = . ). summary/conclusion: in summary, cad-mscs exosomes display structural and functional features comparable to stem cell derived exosomes from other species. use of these exosomes in naturally occurring disease canine models may provide superior predictive value for human clinical trials. funding: support provided by the ccah, school of veterinary medicine, uc davis. mesenchymal stem cells-derived exosomes promote in vitro the progression of triple negative breast cancer cells introduction: mesenchymal stem cells (mscs) are multipotent stromal cells and have been described as key regulators of different aspects of tumour physiology. in tumour pathogenesis, mscs can integrate the tumour microenvironment after recruitment and are able to interact with cancer cells to promote tumour modifications by affecting epithelial-tomesenchymal transition (emt). it was revealed that exosomes derived from mscs are critical players in the tumour niche. exosomes are a novel way of cellto-cell communication and play crucial roles in the majority of pathways that contribute and affect response to therapy, cell-adhesion molecules and the progression of tumour cells. because of the known importance of this communication we decided to investigate the implication of mscs with triple negative breast cancer (tnbc) cell lines as well as exosomal profiles between the experimental conditions. methods: the interactions of mscs with triple negative breast cancer cell lines (mda-mb- and hs t) was performed by coculturing mscs (or tnbc cell lines) with exosomes derived from tnbc cell lines (or mscs). physical characterization of isolated exosomes was performed followed by their molecular investigations. cell proliferation was detected by mtt assay and migration was analysed by wound healing assay using d cultures. moreover, we also used d culture to assess the exosomes uptake and to observe their capability of internalization into a d structure. the alterations in expression level of some transcripts (mrnas and mirnas) and protein profile were investigated by qrt-pcr, western blot and immunofluorescence staining. results: we found that mscs-derived exosomes are actively incorporated by triple negative breast cancer cell lines ( d culture). in coculture, in tnbc cells the expression level of mesenchymal markers and emt markers (e-cadherin, vimentin) at mrna and at protein levels, as well as mirna-derived exosomes targeting mesenchymal genes were significantly affected. using bioinformatics tools, we highlighted the important biological processes which were activated by promoting tumour modifications. in addition, using d culture we provided a comprehensive understanding regarding exosomes internalization in d structures, which closely mimics in vivo conditions, compared to d culture. summary/conclusion: in this work, we focus on the investigation of mscs-derived exosomes in order to highlight their implication in several biological processes, including tumour proliferation and progression of triple negative breast cancer cells. all these alterations affect the response to therapy and should be considered for developing efficient therapeutic strategies. natural killer cell-derived extracellular vesicles have a potent anti-leukaemic effect and selectively target the cancer stem cell subpopulation introduction: natural killer (nk) cells of the immune system recognize and kill tumour cells. extracellular vesicles (evs) secreted from nk cells are capable of killing tumour cells independent of the cell to cell contact required for nk cell activation. cancer is a leading cause of death, primarily due to metastasis and recurrence. cancer stem cells (csc) within tumours are resistant to chemotherapy and immune attack, and cause metastasis and relapse. identification of the cancer types killed by nk evs is limited, and the effect of nk evs on cscs has not been described. here we determine whether nk-derived evs kill a myeloid leukaemia cell line and its csc subpopulation. methods: nk evs were isolated from our nk cell line, nk . , derived from normal human lymphocytes. nk . evs were characterized by immunoblotting, proteomics, and next generation rna sequencing. human k leukaemia cells were treated with nk . evs in vitro and analysed for proliferation and markers of cell death. results: nk . evs contain ev-associated proteins alix, cd , hsp , and tsg , nk effector molecules perforin, granzymes a and b, granulysin and nklam/ rnf b, an e ubiquitin ligase required for maximal nk cytotoxicity, and tumour suppressor mir- . nk . ev treatment of k significantly decreased its expression of proliferation markers cd and ki , and increased the frequency of apoptotic and necrotic cells, paralleled by elevated levels of active caspases − and − . non-tumorigenic cells were unaffected by nk ev treatment. most notably, nk . ev treatment significantly reduced the frequency of k cells highly expressing aldh, a csc marker. summary/conclusion: nk . -derived evs have a robust anti-tumour effect on k myeloid leukaemia cells and selectively target the csc population, suggesting they may circumvent the evasion and resistance mechanisms used by cscs. nk . evs therefore have introduction: due to their potential as a key bioactive agent in regenerative medicine applications, mscderived extracellular vesicles (msc-evs) are increasingly being investigated as a clinical therapy. manufacturing that generates enough evs for product development and clinical doses is currently a limitation in the field and clearly a scalable manufacturing solution will be necessary for successful translation. moreover, a complementary approach that increases the ev productivity, i.e. the number of evs produced per cell, could further help to accelerate the development of msc-evs as a therapy. methods: we developed a process that leverages a series of new cell culture reagents to couple to our established cell-media system for scalable manufacturing of msc-evs. briefly, human bone marrow-or umbilical cord-derived mscs were rapidly expanded under xeno-free conditions (i.e. > x expansion within days). cultures were then switched to our proprietary ev collection medium and evs were harvested for up to three additional days. at the end of culture, the evs in the conditioned media were concentrated using a tangential flow filtration (tff) system. to increase the productivity of mscs, two medium supplements were developed that increased ev yield by either increasing the number of evs generated per cell in a shortened culture process or increasing the number of collected evs by lengthening the ev collection culture period. results: this scalable msc-ev manufacturing method was implemented in both d flask and d bioreactor culture and generated over , particles per cell in d and over , particles per cell in d. with the addition of a medium supplement to increase evs produced per cell, the ev productivity was increased > x after hrs. alternatively, ev productivity was also increased > x by addition of the medium supplement that extended ev collection culture period. summary/conclusion: msc-ev success in clinical translation will be reliant on a manufacturing method that can scalably and reliably generate large amounts of evs. these results present one such solution. furthermore, increasing ev productivity, for instance by medium supplements that increase evs per cell or lengthen culture times could further address the limitation of generating the evs required for development and translation of clinical therapies. simplifying scalable msc ev production in a microcarrier-based bioreactor system divya patel, josephine lembong, katrina adlerz, jon rowley and taby ahsan roosterbio inc, frederick, usa introduction: the growpt ing numbers of msc-ev clinical applications drives the need for a scalable msc-ev production platform. while most msc-evs are generated while cells are attached to tissue culture plastic, such d cultures cannot be scaled up to meet the yields necessary for commercialization of ev-based therapeutics. we have shown that d bioreactors can be used to generate msc-evs and that paradigm can be scaled directly in terms of yield from the to l scales. the technical expertise of seeding cells onto microcarriers for expansion in bioreactors, however, requires technical expertise not available to all those in the ev field. therefore, our goal here is to simplify and expedite the ev collection process in bioreactors by cryopreserving cells on microcarriers, such that end users can merely thaw and then collect msc-evs. methods: mscs were expanded in d and then seeded on three different microcarriers and cultured in a bioreactor for days. when confluent, cells on microcarriers were cryopreserved. to evaluate the microcarriers and the cryopreservation protocol, the cells-microcarriers were thawed, cultured in a bioreactor in growth media for hours, then in ev collection media for additional days. cell recovery and ev production upon thaw was evaluated and compared to ev collection from fresh, non-cryopreserved cells. results: total cell counts hrs post thaw were comparable to those before cryopreservation and to fresh samples prior to ev collection. following -day ev collection, concentration of particles collected from cryopreserved cells on microcarriers were similar to those collected from the fresh cells ( e particles/ml). this process was validated for two different microcarriers using two separate cryopreservation solutions. summary/conclusion: our results show that cryopreserved hmscs on microcarriers can support ev collection in a d bioreactor process with a particle yield that is comparable to those collected from fresh cells. this cryopreserved product can simplify ev production, reducing cost and time by removing process steps associated with the hmsc expansion, with in a paradigm suitable for scale-up. the whitening, anti-wrinkle, and wound-healing effects of extracellular vesicles from orbicularis oculi muscle-derived stem cells. introduction: skeletal muscle-derived stem cells possess potent therapeutic activities in the treatment of muscle-related disorders. in our study, we tried to isolate and characterize orbicularis oculi muscle (orm)-derived stem cells (orm-scs) from the discarded human tissues which were obtained from the ocular surgery-subjected patients. we also prepared the natural extracellular vesicles (evs) from the cultured orm-scs and assessed the their therapeutic actitities including the skin whitening, anti-wrinkle, and wound healing effects. methods: we isolated the orm-scs from the patients subjected to ocular surgery and characterized the orm-scs by analysing cell morphology, proliferation, expression levels of the cell surface and stemness-associated markers, and tri-lineage differentiation and colony-forming capacities, confirming the stemness properties of the orm-scs. then, we prepared the natural evs from the orm-scs via the centrifugation and filtration of the media supernatants and their therapeutic activity was investigated. results: the isolated orm-scs showed spindle-like morphology and positive expression of cd , cd , and cd , but they were negative in expression of cd and cd . the orm-scs showed the capacity of osteogenic, adipogenic, and chondrogenic differentiations. the evs from orm-scs (orm-sc-evs) possessed the apparent inhibitory effect on the melanin synthesis in b f cells by blocking the tyrosinase activity, although orm-sc-evs treatment did not dramatically change the expression level of melanogenesisrelated genes, such as microphthalmia-associated transcription factors (mitf), tyrosinase (tyr), tyrosinaserelated protein (tyrp- ), and tyrp- . in addition, we confirmed that orm-sc-evs could stimulate skin cell migration and increase the expression level of antiwrinkle related genes and wound-healing properties. summary/conclusion: this study revealed the stem cell property of orm-scs and the whitening, antiwrinkle, and wound healing effects of orm-sc-evs, suggesting that orm-scs and orm-sc-evs can be successfully used for stem cell-based ev therapy and cosmetics, by regulation the melanogenesis, wrinkle, and wound. funding: this work was supported by grants from the national research foundation (nrf) funded by the korean government ( m a h ). use of stem cell extracellular vesicles as a holistic approach towards cns repair introduction: neurological diseases and disorders are leading causes of death and disability worldwide. many of these pathologies are associated with high levels of neuroinflammation and irreparable tissue damage. we have previously shown that extracellular vesicles (evs) from infected cells contain viral by products (noncoding rnas and proteins) and that these evs can exert deleterious effects on recipient cells - . therefore, in the context of neurotrophic viruses evs may contribute to or perpetuate processes relating to neuroinflammation and neurodegeneration. due to their multipotent properties, stem cells have broad applications for tissue repair; additionally, stem cells have been shown to possess both immunomodulatory and neuroprotective properties. in recent years it has been well-established that stem cell evs play a critical role in the functionality associated with stem cells. the diverse biological cargo contained within these vesicles are proposed to mediate their effects and, to date, the reparative and regenerative effects of stem cell evs have been demonstrated in a wide range of cell types. while a high potential for their therapeutic use exists, there is a gap of knowledge surrounding their characterization, mechanisms of action, and how they may regulate cells of the central nervous system (cns). methods: we have isolated and recovered high yields of evs from large scale cultures of both induced pluripotent stem cells (ipscs) and mesenchymal stem cells (mscs) using tangential flow filtration. our ev characterization includes both phenotypic (size, tetraspanin expression) and biochemical assays. ev functionality has also been assessed in vitro utilizing several cellbased assays related to cellular viability, migration, angiogenesis, and immunomodulation in both healthy and damaged recipient cells with relevance to the cns. results: our data suggests that evs from different sources of stem cells display unique phenotypes, exhibit differential association with various cytokines, proteins, and long non-coding rnas, and have the ability to significantly enhance processes that are critical for cellular repair . lastly, utilizing an ipsc-derived neurosphere model, we have observed a robust uptake of stem cell evs and have found that these evs are able to effectively penetrate these d structures. summary/conclusion: collectively, these results highlight the "holistic" properties of stem cell evs by demonstrating their ability to partially reverse or reduce damage in various cell types. funding: this work was supported by national institutes of health (nih) grants ai , ai , ai - , ai , and ns to fk and r ca and r ar to lal. the effect of cell culture media on extracellular vesicle secretion from mesenchymal stem cells and human pluripotent stem cell-derived neurons introduction: cell culture media and its supplements are known to affect the secretion and isolation of extracellular vesicles (evs) from cell cultures. identification of these effects is crucial especially when planning to use evs as therapeutic agents. here, we investigated the effect of cell culture media on ev yield from human mesenchymal stem cells (mscs) and human pluripotent stem cell (hpsc)derived neurons. methods: evs were collected from cell-conditioned media (ccm) and no cell control (ncc) media using size-exclusion chromatography (sec). mscs were cultured in dmem/f :neurobasal medium or in opti-mem reduced serum medium, both supplemented with exosome-depleted foetal bovine serum (fbs). the ev yield from hpsc-derived neurons was compared at two maturation time points (day and ), in dmem/f :neurobasal or in opti-mem, with and without -hour kcl stimulation. sec fractions were analysed by nanoparticle tracking analysis (nta), protein concentration assay and blinded transmission electron microscopy (tem). results: ccm samples had a clear peak of evs in sec fractions - , which was not detected with ncc. interestingly, a second population of evs eluted in sec fractions - in both ccm and ncc, indicating presence of evs in exosome-depleted fbs. moreover, this second population differed largely between used media batches. culture medium had no significant effect on msc ev yield (dmem: . e+ particles/ ml, opti-mem: . e+ particles/ml). with neuronal cultures, no significant differences in ev yield were found between culture media or cell maturation time points. in contrast to earlier findings, -hour stimulation of neurons by kcl resulted in significantly smaller ev yield compared to non-stimulated controls (stimulated: . e+ particles/ml, non-stimulated: . e+ particles/ml, p < . ). summary/conclusion: our results indicate that exosome depleted-media are not entirely devoid of vesicles, which can cause bias in downstream analyses. however, sec is a good method to separate cellsecreted evs from the contaminating medium-derived evs. culture medium did not affect the number of evs secreted by mscs or neurons; instead, we observed larger differences between media batches. this data emphasizes the importance of analysing the ncc as negative control in all cell culture experiments. mouse mesoangioblast stem cell extracellular vesicles are able to influence macrophage cell activity maria magdalena barreca a and fabiana geraci b a dept stebicef university of palermo, palermo, italy, palermo, italy; b dept stebicef, university of palermo, italy, palermo, italy introduction: it is largely demonstrated that stem cells release extracellular vesicles (evs) that are able to modify target cell behaviour. interestingly, there is a bidirectional signalling exchange between stem cell evs and damaged cells. moreover, it is well known that macrophages, could also play a role in wound repair and tissue regeneration. it was also demonstrated that stem cell evs are involved in immune cell regulation. for this reason, today takes hold the idea that evs could replace stem cells in regenerative medicine. the aim of our work was to evaluate if evs released by mouse mesoangioblast stem cells (a ) could have a role in immune cell regulation. specifically, we have investigated the possible a ev effect on murine macrophages (raw . ) in terms of cell proliferation, migration and phagocytic ability, and cytokines/chemokine release. methods: a evs were collected from conditioned milieu by ultracentrifugation. raw . cell proliferation with or without a evs was evaluated via cfse assay. scratch test was performed to assay their migration ability. to study raw . cell phagocytosis they were treated with μm beads. finally, cytokine array was used to monitor their secretion after ev treatment. results: we have found that a evs inhibited macrophage proliferation as proved by a proliferation index significantly reduced after ev treatment. simultaneously, we have noticed that evs increases raw . migration ability. furthermore, a evs are able to increase macrophage phagocytic activity. as it is known that hsp is involved in for macrophagic activity increase and a evs express hsp on their surface, we performed phagocytosis assays assay by blocking the protein or its receptor tlr , tlr and cd . our data demonstrated that a evs increase phagocytosis through hsp and its receptors. we have also proved that a evs modify the expression pattern of cytokines/chemokines released in the extracellular milieu by raw . cells. in particular, we observed an increase in anti inflammatory cytokines, and a decrease in some inflammatory ones, suggesting that evs could polarize macrophages towards an anti inflammatory m phenotype. summary/conclusion: in conclusions, our data show that a evs influence macrophage activity and additional studies could provide a new insight into understanding the underlying potential of evs in tissue regeneration. and ) . in a healthy kidney, the polycystins localize to renal cilia. mutations that abrogate ciliary localization of pkd (yet preserve its channel function) also cause cysts. besides cilia, pkd is also found in other subcellular locations including extracellular vesicles (evs) of human urine. how dysfunction of pkd trafficking and localization leads to the kidney pathology remains unknown. pkd is evolutionarily conserved across all members of eumetazoa. in c. elegans, pkd- is exclusively expressed in ciliated male-specific neurons, where it is trafficked to cilia and evs. gfp-tagged pkd- -containing evs play a signalling role in inter-organismal communication between animals. conservation of polycystin- cellular localization between worm and human suggests that their network of molecular interactions may also be conserved. we propose that pkd- plays distinct roles in cilia versus ciliary evs. methods: to understand the role of evs in c. elegans inter-organismal signalling, we aim to identify the pkd- -associated ev proteome, transcriptome, and metabolome. we established a pipeline for fluorescent labelling and tracking specific ev cargoes in a living animal using super-resolution microscopy. we used fluorescence of the pkd- carrying evs to optimize biochemical procedures for their enrichment. results: our initial analysis revealed two populations of pkd- -carrying evs that differ in their densities: . - . versus . g/ml. we are currently characterizing these two distinct populations using transmission electron microscopy and refining our enrichment procedure for protein identification by mass spectrometry, sequencing of their rna cargoes and metabolome analysis. summary/conclusion: what function human pkd plays within the cilia and within the urinary evs is not well understood. identification of molecular mediators of c. elegans pkd- ev signalling will inform on the interactome of human pkd and its function in cilia versus evs. introduction: ectosomes play roles in many physiological and pathophysiological processes, and their precise is dependent on molecular cargo and parent cell type. a single cell can release distinct subpopulations of evs enriched with different molecular cargo, which adds complexity to elucidating cargo sorting and biogenesis mechanisms. in the nematode c. elegans, ectosomes bud from sensory neuron cilia and are released into the environment to modulate animal behaviour. methods: c. elegans is genetically tractable and optically transparent, allowing for live imaging of fluorescently tagged ev cargo. we express all tagged cargo at endogenous levels, adding physiological relevancy. results: we discovered that the calcium homoeostasis modulator ion channel clhm- localizes to cilia of ev-releasing neurons and observed gfp-tagged clhm- in ciliary evs. using super resolution microscopy, we imaged evs released from animals coexpressing tdtomato-tagged clhm- and gfp-tagged pkd- (another vesicle cargo) in the same neurons. while the two proteins colocalize in the cilia, clhm- ::tdtomato and pkd- ::gfp rarely colocalize in evs. this indicates that separate subpopulations of evs are being released from the same neurons. to determine how the clhm- subpopulation is formed, we are investigating candidate genes. anoh- , a homolog of the ca + scramblase tmem f, localizes to neuron cilia and induces phosphatidylserine exposure on the outer membrane leaflet. in anoh- mutants, the number of clhm- ::gfp evs released is significantly decreased but the number of pkd- ::gfp evs does not significantly change. in addition, i am using facs to isolate clhm- and pkd- containing evs and analysing the respective proteomes with lc-ms/ms. summary/conclusion: we are elucidating mechanisms that give rise to distinct subpopulations of ciliary evs in c. elegans and defining cargoes being enriched in these ev subpopulations to gain insight into ev cargo sorting and biogenesis mechanisms in ciliated neurons. ceramide accumulation induces exosome secretion through lysosomal protein laptm b kohei yuyama, hui sun and yasuyuki igarashi hokkaido university, sapporo, japan introduction: exosomes, a type of extracellular vesicles originated from multivesicular bodies (mvb), are important carriers of cellular molecules and have critical roles in intracellular communication in both health and disease. ceramides (cer) are implicated in biogenesis of exosome, however the molecular machinery that mediates exosome secretion remains obscure. lysosome-associated protein transmembrane- b (laptm b) is a lysosome/late endosome-resident transmembrane protein, which has been reported to bind cer. we demonstrate here that laptm b is involved in the exosome secretion, which are induced by exogenous cer treatment or lysosomal ceramidase inhibition in cultured neuronal cells. methods: neuroblastoma sh-sy y cells were treated with cer (porcine brain-derived cer or synthetic d : /c : ~c : cer) for h. exosomes were isolated from the culture supernatants by sequential centrifugation and their amounts were measured using ps capture exosome elisa kit. to analyse mvb transport, mvb and recycling endosomes are visualized with gfp-cd and rab immunostaining, respectively. results: we found that exogenous treatment of cer, especially those with c and c fatty acids, resulted in a marked increase in exosome secretion. in addition, lysosomal cer accumulation induced by acid ceramidase inhibition also accelerated exosome production. knockdown of laptm b significantly prevented the ceramide-dependent exosome release. in addition, we showed that these cer loading promoted colocalization of cd -positive mvb with rab -positive recycling endosomes, further demonstrated that laptm b knockdown cancelled the cer-dependent increase of the colocalization. summary/conclusion: these data suggest that lysosomal cer binds to laptm b and promote the transport of mvb to plasma membrane, resulting in an increase of exosome secretion in neuronal cells. chloroquine-mediated lysosomal inhibition alters composition and function of cancer-derived extracellular vesicles jing xu a , kevin yang a , shane colborne a , elham hosseini-beheshti b , gregg morin a , emma guns b and sharon m. gorski a a bc cancer, vancouver, canada; b the vancouver prostate centre, vancouver, canada introduction: small extracellular vesicles (sev) are signalling entities released by many types of eukaryotic cells. sev are of special interest in cancer due to their reported roles in modulating the cancer microenvironment and facilitating cancer cell invasion. macroautophagy (hereafter autophagy) is a catabolic process well-known for the recycling of cytosolic cargos through lysosome-mediated degradation. in this study, we profiled the changes in sev content and function under lysosome inhibition and investigated the involvement of autophagy machinery in sev content. methods: chloroquine (cq) was used to inhibit lysosomal degradation and autophagy turnover in triplenegative breast cancer (tnbc) cell lines. sev were collected via precipitation after pre-clearing and concentration of conditioned media. western blotting, nanosight and transmission electron microscopy were used to profile sev. quantitative mass spectrometry was used to characterize cq-induced changes in the sev proteome. antibody-conjugated magnetic beads were used in immunoprecipitation of sev. results: cq treatment did not substantially alter the physical properties of tnbc-derived sev. however, cq treatment altered the sev proteome and growth effects of sev on normal and endothelial recipient cells. cq treatment induced co-localization of mammalian atg proteins with endolysosomal markers in the cytoplasm, which coincided with an enrichment of atg s and their adaptor proteins in sev. cq-induced enrichment of atg s in sev required lipidation, and occured preferentially in one subset of sev. summary/conclusion: our study reveals changes in the content and function of cancer cell-derived sev in response to perturbation of intracellular trafficking pathways, demonstrates the flexibility and heterogeneity of sev composition, and has implications for cq efficacy in therapeutic settings. introduction: introduction: argonaute (ago ) is the essential component of the rna-induced silencing complex (risc) that binds mirnas and promotes mrna degradation. extracellular vesicle (ev)-carried mirnas have been shown to influence gene expression and functional phenotypes in recipient cells. many investigators have found ago in evs and it is postulated that ago is a major transporter of mirnas into small evs (sevs), such as exosomes. others have reported extracellular ago that is non-vesicular. we set out to evaluate the effect of growth factor signalling and serum contamination on the detection of ago in sevs. methods: methods: wildtype kras colorectal cancer cells, dks , were conditioned with different culture media (serum-free dmem, dmem supplemented with ev-depleted fbs, and opti-mem). evs were purified from conditioned media by cushion-density gradient ultracentrifugation. western blot analysis of dks total cell lysates, large evs and density gradient fractions was performed, probing for ago and ev marker proteins. the size and concentration of the evs were determined by particle metrix analysis. results: results: in all conditions, we found the highest abundance of sevs in fractions and , as assessed by western blot analysis. ago was detected in the same fractions as sevs in both the serum-free dmem and opti-mem conditions, although the levels of ago was higher in the serum-free dmem fractions compared to that of opti-mem. in contrast, ago was present in both vesicular and non-vesicular fractions in the dmem supplemented with ev-depleted fbs condition. no significant differences were observed in the size and number of evs collected in the three conditioning methods. summary/conclusion: summary/conclusion: the presence or absence of ago in evs has been controversial. multiple factors may affect the ability to detect vesicular ago , including serum and growth factors in the conditioned media that may provide sources of extravesicular ago and also regulate the trafficking of ago into vesicles. introduction: cancer-associated glycosphingolipids have been utilized as tumour markers and targets of cancer therapy. we have investigated roles of gangliosides in cancers, and clarified that cancerassociated gangliosides enhance malignant properties of cells by forming complexes with membrane molecules in lipid rafts. in this study, we analysed contents of gangliosides and membrane molecules on extracellular vesicles (ecvs) secreted from melanoma cell lines. methods: melanoma cell lines with various ganglioside patterns were used for isolation of ecvs. gangliosidemodified melanomas with genetic engineering were also used. genetic modification was done by cdnas of ganglioside synthase genes. ecvs were collected by ultra-centrifugation, or by tim -beads. contents in ecvs were analysed by immunoblotting or flow cytometry. roles of lipid rafts in the generation and secretion of ecvs were analysed by treating cells with mm methyl β-cyclodextrin. results: using melanoma cell lines, ecvs were isolated by ultra-centrifugation, and their sizes were analysed by nanosight. all samples showed uniform sizes between and nm. protein amounts in ecvs were measured, showing heterogeneous levels at ~ μg/ ml. then, gangliosides expressed on ecvs from these cell lines were analysed using tim beads and flow cytometry. gd and gd were detected on ecvs almost proportionally with expression levels of those gangliosides on the cell surface. then, immunoblotting was performed to analyse integrin levels in ecvs from transfectant cells expressing high levels of gd , showing increased levels of integrins in ecvs from gd + cells compared with those from gd -cell lines. integrin levels in cell lysates from these cells (gd + and gd cells) were almost equivalent. treatment of a gd -expressing melanoma cell line by mm methyl β-cyclodextrin resulted in marked reduction of secreted ecvs and amounts of tsg in them. summary/conclusion: ganglioside expression patterns on melanoma cells were well reflected in the expression of gangliosides on ecvs. these results as well as increased levels of integrins in ecvs from gd + cells suggest that gangliosides and lipid rafts are involved in the generation and secretion of ecvs. introduction: hypoxia, or low oxygen tension, is a common feature associated with tumour growth and is known to regulate tumour cell function, especially through rewiring of cell metabolism. however, how hypoxia influences tumour cell interactions with surrounding cells is not fully elucidated. we sought to evaluate how hypoxia alters metabolite and metabolism-associated mirna packaging in exosomes. methods: exosomes were isolated from t breast cancer cells cultured in normoxia ( % o ) and hypoxia ( % o ) via ultracentrifugation, optiprep gradients, and size exclusion chromatography. exosomes were further characterized by nanosight, qubit protein quantification, and flow cytometry analysis of exosome markers. metabolite and mirna profiling was performed on exosomes and exosome-producing cells in normoxia and hypoxia. results: secretion of exosomes was increased under hypoxic conditions. metabolite profiling revealed alterations in metabolites specific to exosomes derived from hypoxic cells. profiling of exosomal mirna showed packaging of metabolism-related mirna into exosomes derived from hypoxic cells. summary/conclusion: hypoxia alters the metabolite and mirna profiles of cancer cells, with selective packaging of these molecules into exosomes. we identified metabolites and mirna that are depleted and enriched in exosomes compared to cells. these studies identify hypoxia-associated shifts in exosome cargo, providing insight into exosome cargo packaging with potential implications for understanding how cancer cell-derived exosomes regulate recipient cell function. lysosomotropic agents prompts the release of extracellular vesicles carrying autophagy-associated markers: evidence of a general mechanism of secretion driven by lysosomal impairment introduction: drug-induced lysosomal storage disorders (lsds) are due to the transient intracellular accumulation, mostly of phospholipids, into multilamellar inclusion bodies within late endosomal/lysosomal compartment. they represent a major side-effect for many drugs of several pharmacological categories. most lsds inducers are cationic amphiphilic drug (cad), but the molecular mechanisms leading to accumulation of undigested substrates are unknown. extracellular vesicles (evs) have been implicated in cell waste disposal, but it is unclear whether they might be involved in extracellular release of undigested substrates. methods: to investigate this aspect, we developed hek cells stably expressing the fluorescent fusion proteins egfp-cd and mcherry-cd , separated evs by differential ultracentrifugation and quantified by evassociated fluorescence and nta particle count. results: evs released by these models upon treatment with drugs inducing the accumulation of phospholipids (amiodarone) or glycosaminoglycans (tilorone), showed the release of fluorescent medium/large evs ( k fraction) and small evs ( k fraction), whose size and distribution were similar to the same vesicles released by control cells, but enhanced the recovery of medium/large evs and to a lower extent of small evs, analysis of evs associated markers revealed a dosedependent increase of autophagy-associated markers in medium/large and small evs. similar results were obtained when autophagic flux was impaired by drugs raising lysosomal ph by different mechanisms, such as chloroquine and bafilomycin, but not when autophagic flux was stimulated by drugs such as curcumin or overexpression of the endosomal/lysosomal regulator tfeb. summary/conclusion: overall results show that impairment of autophagic flux, either by indigested substrates or higher lysosomal ph, is associated with an increased release evs enriched in autophagy markers, compatible with autophagomes and/or amphisomes, unravelling a connection with secretory autophagy. tomofumi yamamoto a , yusuke yamawaki b , yutaka hattori c and takahiro ochiya a a tokyo medical university, shinjuku-ku, japan; b national cancer center research institute, chuo-ku, japan; c keio university faculty of pharmacy, minato-ku, japan introduction: multiple myeloma (mm) is a haematological tumour. last decade, the prognosis of mm has improved by the development of therapeutic drugs; however, mm cells acquire drug resistance by longterm exposure of these therapeutic drugs. one of the possible explanations of drug resistance is that cells with drug resistance transmit information to other mm cells and their microenvironmental cells. although the elucidation of the mechanism of drug resistance in mm have been desired, it remains poorly understood. methods: in order to understand the mechanism of drug resistance in mm, lenalidomide resistant cell lines were established by long-term exposure of low concentration of lenalidomide. drug resistance was assessed by mts assay and caspase assay. the amount of ev was measured by exoscreen, which is ultra-sensitive detection method of evs by measuring surface protein of evs, such as, cd and cd (yoshioka et al., nat commun., ) . to identify the genes which involved in drug resistance, rna sequence among the drug-resistant cell lines and their parental cell lines was performed. results: firstly, characterization of these cells was confirmed. we found that all of the lenalidomide resistant cell lines secreted more evs than their parental cell lines. in addition to this, the size of ev derived from resistant cells are smaller than those of parental cells. next, we collected evs from resistant cells and parental cells by using ultracentrifugation, and added them to parental cells in the presence of lethal dose of lenalidomide. compared with ev derived from parental cell lines, the evs derived from lenalidomide resistant cell lines increased a number of living parental cells. these results suggested that the evs derived from lenalidomide resistant cells can affect the lenalidomide sensitive cells. as a result of rna sequence, several genes highly expressed in resistant cell line we found, which associated with lysosome pathway. among them, attenuating the sort and lamp genes could significantly reduce the ev secretion in mm cells, leading to enhance the lenalidomide sensitivity. summary/conclusion: our results showed that ev secretion via sort or lamp could induce the drug resistance in mm. study on biological stimulate mechanism of stem cell-derived exosome generation by nanoparticles introduction: mesenchymal stem cells (mscs) are pluripotent stromal cells known to release extracellular vesicles (evs) containing various growth factors and antioxidants that can positively affect surrounding cells. nanoscale msc-derived evs, such as exosomes, have been developed as bio-stable nano-type materials, but had low yield and were difficult to quantify. we hypothesized that the mechanism of nanoparticleenhanced exosome production would stimulate intracellular molecules. the aim of this study was to elucidate the molecular mechanisms of exosome generation by comparing the internalization of surface-modified positively charged nanoparticles and exosome generation from mscs. methods: mesenchymal stem cells (mscs) were cultured in mem-alpha with % fbs and × antibiotics. the positively charged nanoparticles were synthesized by poly-lactide-co-glicolide (plga) and polyethylenimine (pei) with cy . for tracking nanoparticles. all of the exosome image were identified using an electron microscope. additionally, it was confirmed the internalization of the nanoparticles by if. the primary antibodies used were anti-eea , anti-rab and anti-gm . in order to prove the development of exosomes, rt-pcr using autophagy-related mrna was performed. real-time rt-pcr was performed using the applied biosystems sequence detection system . lastly, mirna from msc-derived exosome analysed automatically in the affymetrix data extraction protocol using the provided affymetrix genechip® command console® software (agcc). all statistical testing and visualization of differentially expressed genes was conducted using r statistical language . . results: we determined that rab , located in the mvb and autolysosomal membrane, was increased upon exosome expression and was associated with autophagosome formation. these results suggested that nanoparticles migrated to lysosomes during treatment; however, intracellular exosome-forming factors were stimulated during endosomal maturation simultaneously. summary/conclusion: therefore, msc-derived exosome research using nanoparticles is useful for increasing exosome yield and the discovery of nanoparticleinduced genetic factors. theoretical description of formation of extracellular vesicles by budding of membrane introduction: understanding mechanisms of extracellular vesicles (evs) formation is of utmost importance for their effective use in science, medicine and technology. in particular, the discovery of universal mechanisms explaining the phenomena taking place in vesiculation appears to be crucial and highly warranted. mammalian erythrocytes and giant phospholipid vesicles have been largely used as model systems to study principles of membrane budding and vesiculation. the mechanisms conveniently studied in these simple systems are then generalized to other types of biological membranes. we present a theoretical description of membrane budding and compare the theoretically obtained shapes with the observed ones. methods: in accordance with the fluid crystal mosaic model, membrane is considered as composed of constituents (inclusions) subjected to the local curvature field created by surrounding constituents. constituents can attain different in-plane orientations in the membrane which correspond to different energies. the thermal motion oposes the complete orientational ordering. the single-constituent energy expresses a mismatch of the curvature of the membrane at the position of the constituent and the intrinsic principal curvatures of the constituent and inplane orientation of their principal axes. the free energy of the whole membrane is obtained by summing up (integration) the contributions of the constituents and using methods of statistical physics, and minimized by using numerical methods. results: to outline the principle of (outward and inward) budding, respective sequences of shapes corresponding to a formation of one (outward and inward) spherical bud were calculated by minimization of the free energy. also the corresponding shapes observed in evs (imaged by electron microscopy) and in erythrocytes and giant phospholipid vesicles (imaged by optical microscopy) are shown. it can be seen that theoretically calculated shapes and experimentally observed ones agree well over up to orders of magnitude (the order of the size of giant phospholipid vesicles is between and micro metres, in erythrocytes it is about micro metres and in evs it is about nanometres). summary/conclusion: budding of the membrane is an universal mechanism in formation of external and internal vesicles. introduction: the release of extracellular vesicles (evs) from cells is important for many cellular mechanisms both in normal physiology and in disease. arrdc (arrestin domain containing protein ) is an adaptor protein known to facilitate the ubiquitination of target substrates by nedd family ubiquitin ligases. it also traffics cargo to extracellular vesicles. previous studies show the involvement of arrdc in the trafficking of the divalent metal ion transporter dmt to evs in a ubiquitin-dependent manner, and we aimed to further understand this mechanism. methods: we performed mass spectrometry to identify ubiquitinated lysine residues in arrdc . we then generated arrdc wt and lysine mutant clones and expressed these in cells to determine the effect on ev biogenesis and protein trafficking. results: mass spectrometry data identified potential ubiquitinated lysine residues. out of these, lysine appeared to be the most important for arrdc function. arrdc k r mutation caused a decrease in the number of ev released by the cell compared to arrdc wt, and a reduction in trafficking of dmt to evs. furthermore, we also observed a decrease in dmt activity and an increase in its intracellular degradation in the presence of arrdc k r. k also appeared to be ubiquitinated with k polyubiquitin chains by the ubiquitin ligase smurf . summary/conclusion: our data suggests that k polyubiquitin chains are the signal for arrdc mediated ev biogenesis and protein trafficking, and loss of this signal causes cargo to be rerouted to intracellular degradation mechanisms. chair: tanina arab -department of molecular and comparative pathobiology, johns hopkins university school of medicine a d-printed model to represent the structure and nature of extracellular vesicles, for public engagement and education events. christian burton a , sara veiga a , jason webber a , kate milward a , muireann ni bhaoighill a , lauren evans a , andreia de almeida b , rachel j. errington a and aled clayton a a cardiff university, cardiff, uk; b cardiff university, research associate, uk introduction: explaining the field of extracellular vesicles to the lay public and young audiences can often be challenging. whilst diagrams and images of evs may be helpful, conveying clearly the shape and composition of an ev by these means is not always a success. whilst many members of the audience may be familiar with concepts of cells and related structures, others will find such discussions very abstract and challenging. in order to aid interactions with lay audiences we embarked on the design of a physical hand-held plastic model, representing a typical ev. incorporating flexibility in the design allowing the community to adapt it to showcase their own research. the second goal was to ensure manufacturability using widely available dprinting technologies. methods: the basic model design was conceived by dr c. burton, and iteratively developed using solidworks, , then exported for use in any cad environment (stl format). a model showing a halved ev hemisphere, with a visible lipid-bilayer was developed. attachable rings allow trans-membrane-molecules to be represented, current designs include mhc class-i, hspgs, integrins, tetraspanins and supported by handouts accompanying the models. intraluminal cargo is included via removeable "pegs", and examples representing rna or simple globular proteins, and a template has been created. results: the design is free and open source, and available to the community at: https://www.thingiverse. com/thing: . instructions for d printing are available from the uk extracellular vesicle society website; https://www.ukev.org.uk/public-engagementmaterials/. models have been produced using entrylevel d printers and trialled at engagement events with good early responses. summary/conclusion: the authors hope the community will use and develop this d-model design and that the approach provides an additional and helpful tool for educating audiences about the complexities and roles of evs in biology and disease. centrifugal filtration-sec is promising for extracellular vesicle isolation from d and d her + breast epithelial cell lines introduction: despite recent developments in breast cancer therapy, there is still need for a more targeted approach. extracellular vesicles (evs), endogenous nanovesicles released from human cells, are an attractive choice as nanodrug carriers due to their size, stability and their unique targeting specificity. the aim of this study was to determine if centrifugal filtration (cf) combined with size exclusion chromatography (cf-sec) would be useful for ev isolation from two epithelial breast cell lines d and d her +, representing the tissue of interest, and the amount of cell culture needed to get measurable ev concentrations. methods: cell culture media (without serum) from the immortalized breast epithelial cell lines d and d her + was concentrated with centrifugal filtration (cf) followed by isolation with size-exclusion chromatography (sec) using hiprep / sephacryl s- column run with Äkta start ( nm), min runs. each fraction ( - ml) was collected with fraction collector. dulbecco's particle free pbs was used as mobile phase. the resulting particles were analysed with nanoparticle tracking analysis (nta, nanosight ns , camera gain , static mode, capture time sec), western blotting (wb), microbca and transmission electron microscopy (tem, samples fixed with % formaldehyse and stained with % uranyl acetate, run at kv). results: although sec did not show any prevalent peaks from early eluting regions previously shown to contain extracellular vesicles, these fractions (f -f , - min) were collected from d her + cell culture medium. interestingly, both nta and tem suggest that f and f contained evs as the isolated particles measured and nm, respectively and tem revealed spherical particles - nm in diameter. wb was unable to detect the ev associated protein alix (but was present in the whole cell lysate). soluble proteins and protein aggregates eluted late in the sec chromatogram ( min), with protein analysis (microbca), tem and wb confirming their presence. summary/conclusion: cf-sec is a promising method for ev isolation for pharmaceutical applications, but further work is needed to optimize the isolation process using Äkta start for these cell lines. customer stories from the ev core of university of helsinki introduction: the ev core, world's first ev-dedicated technology platform established in , is a joint venture of two extracellular vesicle (ev) research laboratories at university of helsinki. as an academic research/service facility, the ev core provides infrastructure, state-of-the-art and emerging ev-technologies for research groups, hospitals, companies and authorities in the ev-field. the ev core provides ev isolation, purification and characterization services and offers contacts to downstream analyses in other core facilities based on optimized ev protocols. here, we present and discuss the customer experiences and prospects with the aim to further develop ev core services. methods: our most wanted services are nanoparticle tracking analysis, electron microscopy, ev isolation and rna isolation and consultation. currently, the key down-stream analysis methods are (mi)rna sequencing, metabolomics, flow cytometry and functional assays. results: we present the stories from our customers starting with their research questions and need for the ev expertise/consultation and equipment. next, we show how the projects advanced and what types of ev core -derived or other downstream services helped them to achieve their aims. in the end, we will acknowledge the customers experience and current status of their research. summary/conclusion: narratives of customer stories are an effective starting point for fruitful discussions about the current status and next developments in the young ev service field. recent isev workshops: open, reproducible and standardized ev research (ghent, ) and evs in immunology (buenos aires, ) introduction: since its founding in , isev has sought to further extracellular vesicle research in various ways including scientific meetings. these events encompass annual meetings as well as smaller, topically focused workshops, with the first isev workshop (on rna and evs) organized in new york city in october, . in december, , the workshop "open, reproducible, and standardized ev research" was held in ghent, belgium. in march, , the workshop, "evs in immunology" was held in buenos aires, argentina, with a preceding education day. methods: the international organizing committees of the and isev workshops prepared scientific programs around key themes of ev rigour and standardization (ghent, belgium, workshop) and evs in immunology (buenos aires, argentina, workshop). abstract and application submissions were invited. applications were reviewed and ranked by panels of ev experts for each event, and participants were invited. results: the and workshops assembled a total of more than individuals for talks and discussions around the themes of rigour and standardization and evs in immunology. the buenos aires workshop was preceded by an education day, coordinated by the isev executive committees for education and science and meetings. during these two workshops, poster presentations were permitted for the first time, affording additional presentation and interaction opportunities. the rigour and standardization workshop also featured real-time discussant polling to facilitate discussion. summary/conclusion: isev workshops such as those addressing rigour and standardization (ghent, ) and evs in immunology (buenos aires, ) continue to provide opportunities for focused discussion of small groups of experts on key topics in the field. often followed by published products, isev workshops help to lead and coordinate progress in ev science. for future isev workshops, educational activities may again expand the reach of each event, while poster sessions and app-driven real-time responses should be considered for enhanced interactions and participant canvassing. ev journal club: exchanging pizza for a worldwide audience during covid- kenneth w. witwer johns hopkins university school of medicine, baltimore, usa introduction: a monthly journal club focused on extracellular vesicle science was established at johns hopkins university in , featuring lunch and presentations by academic and industry participants. when covid- prevented in-person meetings beginning in march, , the journal club was converted to a virtual, weekly format on the popular online meeting app zoom. the journal club has persisted despite initial problems with online vandalism. most sessions are also made public on a youtube channel, https:// www.youtube.com/c/extracellularvesicleclub. methods: weekly ev club sessions are arranged by the host. most focus on a specific manuscript related to evs, but some weeks feature presentations of published or soon-to-be-published research by the presenting authors. sessions are advertised one week to several days in advance on social media platforms such as linkedin, twitter, and facebook, asking interested parties to sign up to join a mailing list via surveymonkey. the log-in information is then sent to the mailing list. upon clicking the link, participants are placed in a virtual waiting room for vetting by the host and volunteers. after admission, all parties but the host and presenter are muted to avoid distractions. questions and comments may be placed in a chat box. contributions are monitored and compiled by the host and volunteers to build a question-and-answer session at the end of the presentation. recorded sessions-with or without editing as needed-are placed on the youtube channel for additional access. results: despite initial problems with online vandalism known as "zoombombing," the journal club has continued weekly during the covid- shutdown in the host country (us). an audience of between and individuals is typical. participants typically ask more questions than can be answered in a one-hour time frame. the online format also allows for debate-style events and polling of the audience. summary/conclusion: this ev journal club is an example of how online tools can be used to facilitate international scientific interactions. further development of such formats could provide alternative approaches for isev activities in the science, education, and communication areas. the study aim is to assess whether the exposure to pm and pm , , chosen as paradigmatic environmental stressors, could modify the composition of nasal microbiota (nm) and extracellular vesicle (ev signalling network, showing a role in allergic ar exacerbation). methods: nm analysis were performed on v -v s rrna gene regions amplified from upper-airway tracts of ar cases and healthy individual controls to perform nm analyses. ev size, concentration and cellular origin for each subject were assessed by nanoparticle tracking analysis (nta) and flow-cytometry (fc). information on daily pm and pm , concentrations at the municipality of residence in the days preceding nasal sampling (i.e. day − to day − ) was assigned to each subject by arcgis software. multivariable and logistic analyses were applied on nm, nta and fc outcomes. results: when taxonomy composition was considered, in controls actinobacteria ( . %) was the most represented, followed by firmicutes ( . %) and proteobacteria ( . %) while in cases proteobacteria were . %, actinobacteria were . % and firmicutes were . %. cases showed a higher concentration of all the investigated ev types, derived from platelets (cd +), activated endothelium (cd e+), monocytes (cd +), eosinophils (cd +), neutrophils (cd +), mastocytes (cd c+), epithelial cells (epcam+), gram+ bacteria (lipoteichoic acid+), gram-bacteria (lps+). the effect was greatest in the case of mastocytes evs which were increased . fold in cases versus controls (p < . ). evs were modified by pm exposure at several time lags. in particular, a negative association between pm and eosinophil evs was observed (beta = − , ; pvalue = , ). as we clustered subjects according to their nm, we observed this variable was a strong effect modifier of the association between pm exposure and ev release. summary/conclusion: our findings start to provide an insight on the effect of air pollution on evs, taking into account the effect of nm, in patients with ar. further research is necessary to disentangle the mechanism exerted by inhaled pollutants in modulating evs and nm, and therefore ar exacerbation. funding: gsk investigator sponsered study aryl hydrocarbon receptor activation induces the expression of specific microrrnas in th cells that are release into extracellular vesicules and associated with arthritis introduction: in rheumatoid arthritis (ra), an autoimmune disorder characterized by a chronic sinovial inflammation, smoking is a major risk factor contributing to disease progression, and poor response to therapy. th cell is actively involved in worsening smooking-associates inflammation mediated by aryl hydrocarbon receptor (ahr), a cytoplasmic transcription factor involved in xenobiotic metabolism. both, ahr and th cells, has important implications during ra development. considering that cigarette smoke is a potent epigenetic modifier, we hypothesized that ahr activation, by cigarette components, would transcribe specific micrornas in th cells as a molecular mechanism to exacerbate inflammation in arthritis. methods: microrna expression was evaluated by largescale approach or real-time pcr. c /bl and ahr null mice were submitted to arthritis experimental models and exposed or not to cigarrete smoke (ethical committee approved / ). extracellular vesicles (evs) were isolated by ultracentrifugation, and characterized by western blot and nanosight. rankl-induced osteoclasts (ocs) differentiation in vitro was stained for trap. inhibition of mirnas were performed using anti-mirs transfection. results: we identified a specific group of mirnas induced in th cells after ahr activation. during arthritis progression, the micrornas are expressed and increases after exposure to cigarette smoke. in the absence of ahr their levels were drastically reduced. interestingly, we found that these micrornas are released by th cells into evs, and are able to promote osteoclastogenesis. ocs differentiation in vitro increases in the presence of th -derived evs, and this process is reduced in the absence of micrornas. summary/conclusion: microrna-mediated gene regulation plays crucial roles in the immune system functions, and their abnormal expression is highly correlated with the pathogenesis of ra. evs are known to function in cell-to-cell communication and are able to transmit their contents and cause changes in the target cell. our findings demonstrate a new molecular mechanism by which cigarette smoke could aggravate inflammation in arthritis; through the activation of ahr receptor in th cells, inducing the transcription of specific micrornas that are released into evs, and act as pro-inflammatory mediators. introduction: chagas disease (cd) is caused by the flagellated protozoan t. cruzi. trypomastigote forms are capable of releasing extracellular vesicles (evs) that contain the major surface molecules of the parasite. the parasite has a complex life cycle that leads to it a rapid adaptation in the environmental changes in the hosts. however, the effects of stress on on evs release are not completely understood. objetive: we evaluated the release of evs by trypomastigotes incubated under different stress conditions and the immunomodulatory role of these evs in pre-activated bone marrow-derived macrophages (bmdm). methods: nanoparticle tracking analysis (nta) and scanning electron microscopy (sem) showed an increase in evs releasing by trypomastigotes at °c under acidic conditions, evs released was affected and triggered amastigogenesis process. results: treatment with sodium azide (nan ) also caused changes in the release of evs regarding size and concentration. nitrosative stress caused by sodium nitrite (in culture medium mildly acidic, ph . ; in this condition nano releases nitric oxide) stimulated an increase in production of evs by t. cruzi. when the parasites were treated with nm s-nitrosoglutathione (snog), we observed a reduction in size and concentration of vesiculate material by trypomastigotes. at a higher snog concentration ( µm), the concentration of the vesiculate material increased. t. cruzi-derived evs exposed to stress conditions increased the expression of inos, arg , il- and il- genes in ifn-γ and lps pre-activated bmms. summary/conclusion: results suggest that the viability and/or integrity of the parasite are necessary for the evs releasing. in those in vitro conditions they triggered a proinflammatory response in host cells. this may be a strategy developed by the parasite to favour its establishment in the host. funding: fapesp, cnpq, capes and fapemig ppm-x / . immuno-toxicological evaluation of human mesenchymal stem cell- introduction: mesenchymal stem cells (mscs) have been widely used to the field of autoimmune diseases or tissue regeneration therapy. recently, many research groups have reported that mscs showed their ability via secreted paracrine mediators including extracellular vesicles (evs) rather than cell-to-cell contact. mscs mainly exist on bone marrow, peripheral blood, umbilical cord and adipose and can mostly secrete evs. it has emerged that evs alone are responsible for the therapeutic effect of mscs in plenty of animal diseases models. hence, msc-derived evs may be used as an alternative msc-based therapy in regenerative medicine. methods: as part of safety programme for human therapeutics, we performed immunotoxicological assessment of evs obtained from human mscs (hevs) in mice and human peripheral blood mononuclear cells (hpbmcs). firstly, mice were treated intravenously with a negative control, a positive control (lps; . mg/kg), or low-dose ( x e paticles/head) and high-dose ( x e paticles/ head) of hevs every other day for days and then analysed lymphocyte subsets from collected spleen by facs. next, we treated the evs on hpbmcs for days with low conc. ( x e particles/ml), high conc. ( x e particles/ml), pma/ionomycin as a cell activator or cpt ( μm) as an apoptotic inducer. annexin v/pi and csfe were analysed by facs. results: as a result, splenic nk cells and b cells were slightly increased about ~ % in hevs-treated mice, without biological significance, compared with a positive control (lps) as an immunogenicity inducer. and there were no effects on serum levels of inflammatory cytokines in mice. in addition, hevs had no cytotoxic effect on hpbmcs at both low and high conc. under the culture medium with evs-depleted fbs, the hevs appeared minimal anti-apoptotic effect on hpbmcs. for the cfse assay, the hevs showed slight proliferation on hpbmcs and pbmc activation induced by pma/ionomycin. summary/conclusion: in conclusion, the hevs have little immuno-toxicological effects in mice and hpbmcs. further detailed studies to elucidate immunological response of hevs for development of human therapeutics are needed. funding: this research was supported by a grant ( mfds ) from ministry of food and drug safety. investigation of immune response to mesenchymal stem cell-derived extracellular vesicles in the cancer setting introduction: mesenchymal stem cell-derived extracellular vesicles (msc-evs) are thought to be a fingerprint of the secreting cell and therefore may retain the cancer targeting and immune privilege of mscs. thus msc-evs hold immense potential as tumour-targeted therapeutics for breast cancer. the aim of this study was to determine whether msc-ev administration in tumour bearing immunocompetent animals would initiate an immune response. methods: evs were isolated from conditioned media of both human and murine bone marrow-derived mscs through sequential differential centrifugation, microfiltration and ultracentrifugation. evs were characterized by nanoparticle tracking analysis (nta), western blot and transmission electron microscopy (tem). x ( ) human or murine msc-evs were administered intravenously into t breast tumour bearing balb/c mice (n = ) and healthy controls (n = ). tumour tissue, draining lymph node and spleen were then harvested, dissociated and flow cytometry performed targeting markers associated with a range of immune cells including t-cells, macrophages and natural killer (nk) cells. results: evs were successfully isolated from murine and human mscs with the appropriate size of small evs (sevs: - nm) and morphology including a lipid bilayer observed by tem. evs expressed tetraspanins cd , cd , cd ; cytosolic protein tsg and were negative for calnexin. ev concentrations ranged from . x ( ) - . x ( )/ml. in order to study a range of immune cell populations two antibody panels were created using complimentary fluorescent dyes. the proportion of t-cells (cd +, cd +, cd +), neutrophils (gr- +, ly- c+), dendritic cells (cd c+), macrophages (cd b+, mhci+, mhcii+), nk cells (cd +) and b cells (cd +) remained stable in the tumour, draining lymph node and spleen of all tumour-bearing animals that received either human or murine msc-evs, with no significant change observed in any category. summary/conclusion: the data presented supports the hypothesis that msc-evs retain the immune privilege of the secretory cell, with human cell-derived evs illiciting no immune response in mice. this is encouraging and reinforces the potential for use of msc-evs in the therapeutic setting. introduction: mycobacterium avium (m. avium) is a slow growth rate non-tuberculous mycobacterium (ntm). m. avium infection is a severe global health problem. but the mechanisms of pathogenicity of m. avium are poorly understood. outer membrane vesicles (omvs) that traverse the cell wall and contain a varied bioactive components inculding dna, rna, protein and toxins. previous studies have suggested that these omvs are produced in vitro and during animal infection, but the role of omvs secretion during the interaction of m. avium with host cells remains unknown. methods: in this study, m. avium were grown in middlebrook h medium (m h ) supplemented with % (v/v) oadc enrichment and . % (v/v) glycero. m. avium omvs were isolated by ultracentrifugation method. characterization of omvs by transmission electron microscopy (tem) and nanoparticle tracking analysis (nta). the raw . murine macrophages were incubated with the m. avium omvs to analyse inflammatory response and production of nitric oxide (no) and reactive oxygen species (ros) of macrophage. results: in this study, we demonstrate by fluorescence microscopy that murine macrophages can phagocytosis omvs produced by m. avium. incubation of m. avium omvs with murine macrophages resulted in increased levels of extracellular tumour necrosis factor alpha (tnf-α), interleukin- β (il- β), terleukin- (il- ) and interleukin- (il- ). meanwhile omvs stimulated macrophages produce no and ros. introduction: hospital associated venous thromboembolism (ha-vte) in paediatric patients is the second most common contributor to harm in hospitalized children. platelet-endothelial interactions are integral to the formation of vte, especially in inflammatory conditions such as sepsis. small extracellular vesicles (sevs) have the ability to reprogramme target cell phenotypes via their microrna contents and are known to contribute to vte formation. we hypothesize that sepsis alters platelet-derived sev micrornas capable of net upregulation of vascular endothelial procoagulant and downregulation of anticoagulant pathways. methods: using a precipitation solution and size exclusion chromatography, we isolated sevs from platelet poor plasma of children admitted to the paediatric intensive care unit for sepsis and from healthy controls. we positively selected platelet-derived sevs using immunomagnetic isolation for cd b platelet antigen and confirmed selection using flow cytometry. microrna was profiled using affymetrix genechip mirna . array. results: microrna from sepsis patients (median age . years; iqr: . - and % female) with a median psofa score of (iqr: . - ) and from healthy controls (median age years; iqr: . - . and % female) was isolated and compared. in septic vs. healthy patients mirnas were differentially expressed (false discovery rate (fdr)< . ; fold change ≥| . |) affecting mrna pathways. in septic children, pathways affecting chemotaxis and cell movement of leukocytes were predicted to be activated with z-scores ≥ . summary/conclusion: we developed a method to successfully isolate platelet-derived sevs. sepsis alters the platelet-derived sev microrna profile in paediatric patients with sepsis. these micrornas are predicted to target chemotaxis and cell movement pathways, important contributors in the formation of ha-vte. further analysis into specifically targeted pathways should be conducted as a potential target for the prevention of ha-vte in sepsis. introduction: sjögren´s syndrome (ss) is a systemic autoimmune disease that mainly affects salivary and lacrimal glands. mechanisms of ss pathogenesis are poorly understood. it is thought that inflammation leads to destruction of exocrine glands, however the triggers of autoimmunity and the mechanisms by which inflammation drives immunopathology are not characterized. our work identifies t cell-exosomederived mir- - p as a pathogenic driver of immunopathology in ss. micrornas (mirnas) are endogenous small noncoding rna molecules that regulate the expression of target genes through translational repression of mrnas. through transcriptomic profiling studies our group had previously documented a significant upregulation of mir- - p in patient ss tissues and in serum exosomes. methods: structured search for target genes of mir- - p involved in salivary gland (sg) physiology was performed with mirdip . serca b, ryr and ac were selected for further validation and functional analysis. binding of the mirna was confirmed by luciferase reporter assays in hsg cell lines and human-derived primary epithelial cells. the mrna and protein levels of serca b, ryr and ac were determined by qpcr and western blot, respectively. to investigate the cell-specific distribution of mir- - p in relation to the expression levels of serca b, ryr , and ac , a double fluorescent in situ hybridization was performed. ca + signalling and camp levels were measured using fluorescent sensor. to isolate exosomes, the t cell medium and serum of ss-patients and healthy volunteers (hv) were collected. results: we show that mir- - p is over-expression in the sgs of ss-patients. next, we demonstrated that mir- - p is contained in exosomes in serum of sspatients significantly more than serum of hv. we also show that activated t cells secrete exosomes containing mir- - p which transfer into glandular cells and affecting intracellular ca + signalling, camp production and protein production by mir- - p targets (serca b, ryr and ac ). summary/conclusion: this study provides evidence for a functional role of the mir- - p in ss pathogenesis and promotes the concept that t cell-activation directly may impair epithelial cell function through secretion of mi-rna containing exosomes. treg-derived il -coated extracellular vesicles promote infectious tolerance (p ) subunits, yet the forms that il assumes and its role in peripheral tolerance, remain elusive. methods: we induce cba-specific, il -producing t regulatory (treg) cells in tregebi wt c bl/ reporter mice, and identify il producers by expression of ebi tdtom gene reporter, plus ebi and p proteins. results: curiously, both subunits of il were displayed on the surface of tolerogen-specific foxp + and foxp neg (itr ) t cells. furthermore, il producers, although rare, secrete ebi and p on extracellular vesicles (ev) targeting a -to -fold higher number of t and b lymphocytes, causing them to acquire surface il . this surface il is absent when ev/exosome production was inhibited, or if ebi is genetically deleted in treg cells. summary/conclusion: the unique ability of ev to coat bystander lymphocytes with il , promoting exhaustion in, and secondary suppression by, non-treg cells, identifies a novel mechanism of infectious tolerance. funding: nih grants r -ai - (to w.j.b.), r ca and p ca (to d.a.a.v.) and the university of wisconsin carbone cancer center support grant p ca . unique formulated dual targeting antigen specific and delivered mirna- gene regulating exosomes acting at the immune synapse to induce apc-derived secondary suppressive exosomes introduction: an exosome-apc circuit we uncovered may be applicable beyond skin immunity we study in mice. methods: high antigen dose tolerized cd + t cells make suppressive antigen-specific exosomes due to chosen surface antibody light chains that enable targeting antigen presenting cells (apc) antigen-specifically for delivery of also chosen inhibitory mirna- to mediate specific functional gene alterations. results: both antigen and gene specificity aspects are lent to naïve but activated exosomes by simple in vitro incubations alone. for mechanism, these primary exosomes bind antigen peptides in mhc on apc that in turn make secondary suppressive exosomes that act peptide/mhc-specifically on the effector t cells at the immune synapse. they transfer another mirna for strong prolonged inhibition of active delayed-type hypersensitivity (dth) for days even, when the primary mirna- -pos exosomes are administered orally at the height of the in vivo response, in a physiological dose. summary/conclusion: it is shown possible to induce therapeutic exosomes with ag targeting of choice due to placed ab on the surface and that also target specific gene functions of acceptor cells due to carriage of a selected mirna. this dual ag and gene-specific therapy has applications in treatment of cancer, autoimmunity and allergies. introduction: previously, our group characterized distinct populations of extracellular vesicle (ev) released from neutrophilic granulocytes: ev formed spontaneously (sev) and upon activation with opsonized particles (aev). the aev differs in protein cargo and its ability to inhibit bacterial growth. we described that mac- integrin (cr receptor) plays key role in the aev production and extracellular calcium supply is crucial in this signalization. in the present work, our aim was to investigate whether mac- activation or casignalling on their own are sufficient for the initiation of the aev biogeneis. methods: we isolated neutrophil derived evs from peripheral human blood and murine bone marrow by two-step centrifugation and filtration. we tested the effect of ca-ionophore and examined the ev production on c bi coated surface and in soluble form. we quantified the vesicles by flow cytometry and determined their protein content by bradford assay. we examined their antibacterial effect in parallel with optical density-based measurement and our flow cytometry based method. results: on c bi coated surface, we observed an increased ev production, and these evs possessed antibacterial capacity. however, in soluble condition, c bi did not induce further ev production, and these evs did not show any antibacterial property. we found that ca-ionophore initiated ev formation, but these ev did not show antibacterial effect. we observed ev production increase after ca-ionofore treatment both in the presence and in the absence of extracellular ca. the ca-ionophore slightly increased the opsonized particle induced ev production, but did not potentiate their antibacterial capacity. summary/conclusion: mac- activation is not just crucial, but sufficient in initiation of the aev biogenesis. clustering of this receptor is required. while the ca-signal is crucial, it is not sufficient in the generation of aevs. extracellular vesicles and their microrna cargo in retinal health and degeneration: mediators of homoeostasis, and immune modulation yvette s. m. wooff, adrian cioanca, riemke aggio-bruce, joshua chu-tan, ulrike schumann and riccardo natoli the australian national university, canberra, australia introduction: photoreceptor cell death and inflammation are known to occur progressively in retinal degenerative diseases such as age-related macular degeneration (amd). however, the molecular mechanisms regulating these biological processes are largely unknown. extracellular vesicles (ev) are essential mediators of cell-to-cell communication with emerging roles in the modulation of immune responses. evs, including exosomes, encapsulate and transfer microrna (mirna) to recipient cells and in this way can modulate the environment of recipient cells. dysregulation of evs however is correlated to a loss of cellular homoeostasis and increased inflammation. in this work we investigated the role of isolated retinal small-medium sized ev (s-mev) in the regulation of homoeostasis and immune modulation in both the healthy and degenerating retina. methods: isolated s-mev from healthy and degenerative (photo-oxidative damaged) mouse retinas were characterized using dynamic light scattering, transmission electron microscopy and western blot, and quantified using nanotracking analysis. small rna-seq was used to characterize the mirna cargo of retinal s-mev isolated from healthy and degenerating retinas. finally, the effect of exosome inhibition on s-mev-mediated immune modulation was investigated using systemic daily administration of exosome inhibitor gw and analysed by in situ hybridization of s-mev-abundant mirna. electroretinography and immunohistochemistry were performed to assess functional and morphological changes to the retina as a result of exosome depletion. results: our results demonstrated an inverse correlation between s-mev concentration and photoreceptor survival, with decreased s-mev numbers following retinal degeneration. small rna-seq revealed that s-mevs contained uniquely enriched mirnas, however no differential composition in s-mev mirna cargo following photo-oxidative damage was observed. exosome inhibition using gw exacerbated photoreceptor degeneration, with reduced retinal function and increased levels of inflammation and cell death seen following photo-oxidative damage. further, reduced translocation of the photoreceptor-derived s-mev was demonstrated following exosome-inhibition in photo-oxidative damaged mice. summary/conclusion: taken together, we propose that retinal s-mev and their mirna cargo play an essential role in maintaining retinal homoeostasis through immune-modulation, and have the potential to be targeted using gene therapy for retinal degenerative diseases. impacts of agricultural dust exposure on human lung-resident mesenchymal stromal/stem cells and their extracellular vesicles introduction: agricultural dust is considered a high-risk occupational hazard by the cdc, with impacts reaching throughout the communities surrounding these industries, leading to increased incidence of respiratory illness and disease among individuals within this occupation and these communities. lung-resident mesenchymal stromal/stem cells (lr-msc) have an important role in maintaining homoeostasis in the lung, and mediating pro-and anti-inflammatory effects, particularly during exposure to inhaled irritants, like agricultural dust. one way in which these lr-msc promote lung homoeostasis is through the release of extracellular vesicles (ev), with a variety of cargo that elicit changes among target cells. we hypothesize that exposure to agricultural dust modifies the quantity and cargo of ev released by lr-msc to promote lung tissue homoeostasis. methods: primary human lung-resident mesenchymal stromal cells were exposed to extracts of dusts collected from swine confinement facilities (de) for or hrs and the media from these exposures were collected and enriched for ev by opti-prep density gradient ultracentrifugation. the quantity of these ev were assessed by nanoparticle tracking analysis. additionally, cytokine and chemokine release by lr-msc were analysed by enzyme-linked immuno assays. results: as assessed at hr following treatment, deexposed lr-msc released pro-inflammatory cytokines, il- and il- , with il- release reaching statistical significance at . %, . %, and % de concentrations (p = . , < . , and < . respectively) and il- trending a similar dose response but only statistically significant at % de (p = < . ). de exposure of lr-msc also induced changes in the lr-msc-derived ev populations when compared to vehicle control, where lr-msc released significantly more ev in the and % iodixanol fractions (p = < . and . , respectively) at hr following de treatment. alternatively, there were significantly less ev in the and % density fractions in the media of deexposed lr-msc versus vehicle control. summary/conclusion: following exposure to agricultural dusts, lr-msc-derived ev populations more likely consist of exosomes and ectosomes, which play an important role in promoting lung tissue homoeostasis during exposure-related pulmonary inflammation. introduction: during analyses of single extracellular vesicles (evs) by flow cytometry (fcm), particles below the detection limit may exceed the trigger threshold, which is called swarm detection and generates false-positive counts. serial dilutions are recommended to find the minimal dilution for which swarm detection is absent. however, because particle concentrations in plasma vary, the optimal dilution differs > -fold between donors, but it is unfeasible to do serial dilutions for each clinical sample. therefore, our aims are to ( ) develop a faster method to avoid swarm detection, and ( ) increase the number of detected evs per second. methods: we measured serial dilutions of cd stained evs in platelet free plasma (pfp), with and without spiking of fitc beads, by fcm (apogee a -micro). we triggered either on side scatter or fluorescence. results: for scatter triggering with our fcm, swarm detection consistently occurred for plasma samples exceeding a (total particle) count rate of , - , events/s. the cd + evs concentration scaled linearly over . orders of magnitude of the dilution and most donors required > -fold dilution to avoid swarm detection, thereby reducing cd + ev counts. for fluorescence triggering, the cd + evs concentration scaled linearly over > orders of magnitude of the dilution. for all donors, swarm detection was absent after -fold dilution (relative to pure plasma). the count rates of cd + evs were - -fold higher compared to scatter triggering. the spiked fitc beads confirmed that the median signals remained constant. summary/conclusion: we have developed two clinically applicable ways to avoid swarm detection. for scatter triggering, the count rate provides direct feedback on the presence of swarm detection in plasma samples. for fluorescence triggering, swarm detection was absent for all plasma samples diluted ≥ -fold and compared to scatter triggering, count rates of cd + evs were - fold higher, thereby improving statistical significance. funding: edwin van der pol is supported by the netherlands organisation for scientific research -domain applied and engineering sciences (nwo-ttw), research programmes veni . benchmarking flow cytometric analysis of nanoparticles: a cross-platform study for single extracellular vesicle detection introduction: despite flow cytometry being widely used to analyse cells in suspension, most commercial instruments lack sensitivity when measuring nanoparticles (nps) and extracellular vesicles (evs). furthermore, the use of appropriate reference materials (rms) for calibration and quality control are essential to compare results acquired with different instruments. to work towards successful clinical applications for ev biomarker profiling, benchmarking studies including state-of-the-art flow cytometers are required. we here investigated the ability of three different flow cytometers to detect nps and evs. methods: the instrument sensitivity of light scattering detection was evaluated by using synthetic nps of different sizes and refractive indices. fluorescent calibration was investigated by using molecules of equivalent soluble fluorophores (mesf) beads. biological recombinant evs (revs) were used to validate the detection and quantification of fluorescent evs in a side-by-side cross-platform study using an n nanoflow analyser (nanofcm), an optimized bd influx and a cytoflex lx. results: we found that when light scatter based detection was used, the nanofcm detected the smallest non-fluorescent nps, the bd influx was able to provide reliable fsc information from the smallest detected nps and the cytoflex performance was greatly improved by the use of violet-ssc. biological revs showed that the nanofcm could clearly resolve fluorescent evs while the bd influx and cytoflex were unable to fully resolve revs from background, although fluorescence threshold improved detection. in addition, our findings revealed that different concentrations are required to ensure single ev detection in these platforms. summary/conclusion: we identified several strengths and limitations for each platform with respect to single ev analysis. furthermore, our results showed that proper calibration and rms are of utmost importance to ensure reliable interpretation of ev flow cytometric data. caution when using membrane dyes for sequential extracellular vesicle analysis diana pham, michael wong, desmond pink and john lewis nanostics, edmonton, canada introduction: confirmation that particles detected by microflow cytometry are actually extracellular vesicles (evs), or at least membranous in composition, can be achieved through a variety of methods. positively staining particles with a membrane dye strongly suggest that the particle contains a membrane; loss of stain (or detection) after detergent solubilization of the membrane-dyed particles provides even stronger evidence that the particles were evs. it is important to recognize that the labelling protocol provided by the membrane dye manufacturer may not be ideal for all types of evcontaining biological samples, such as blood, urine, semen etc.). removal of excess dye from stained evs is very difficult and can be impractical depending on the nature of the experiment. however, this means that the potential for excess dye to contaminate subsequent sampling is high. therefore, it is important to determine optimal working concentrations and labelling conditions when using membrane dyes for ev detection to understand properties that may impact your analyses. methods: to assess the utility of membrane dyes, titration curves were generated to determine the optimal working concentrations of membrane dyes for ev detection in conditioned media and human serum samples. once the optimal concentration was determined the potential of dye carry-over from sample to sample during microflow cytometry detection was evaluated by tracking dye positive (dye+) particles in phosphate buffered saline (pbs) blanks and matched, unlabelled, sample replicates. results: we found that optimal concentration of any membrane dye is dependent on sample type. even with the inclusion of system washes to prevent sample carryover, there was carryover of low amounts of dye+ particles into sequentially analysed pbs blanks. if unstained samples were analysed following a stained sample, excess dye (or at least dye+ events) appeared in the data. a sample concentration effect was also seen; samples of lower concentrations were more susceptible to dye carryover. summary/conclusion: when using membrane dyes to stain evs in biological samples, especially if an autosampler is employed to run a series of tests, it is critical to determine the optimal concentration of dye for each type of sample, as excess dye can carry over to the next sample in the queue. in addition, determining the necessary steps to clean any excess dye following each sample run will improve the accuracy of ev detection and analyses. funding: nanostics alberta innovates alberta cancer foundation correlation between size and protein expression of single exosomes by combined atomic force and fluorescence microscopy introduction: there are no universal markers of extracellular vesicles, but often they are identified by the presence of tetraspanins in their membrane. based on this, products have been developed to precipitate or quantify evs by acting upon cd , cd , and cd . however, evs also carry proteins from their parent cells, and capturing evs based their presence allows for a more complete understanding of vesicle heterogeneity from a single cell type, and for evs derived from specific tissues to be enriched from other biofluids in support of biomarker assessment. for example, evs derived from the brain could be captured from the general population of serum evs for better assessment of cargo associated with proteinopathy. the goal of this study was to identify specific antibodies to capture and label evs bearing the neural markers cd , snap , α-synuclein, tau, and ncam. methods: the targets were overexpressed in hek t cells through transient transfection of plasmids (origene). media was conditioned for - hours, and then centrifuged to remove cell debris. cell lysates and concentrated conditioned media (cm) were analysed by western blot. unpurified cm, or cm after performing size exclusion chromatography (sec, izon), were analysed in the exoview r system. diluted cm was incubated on custom antibody microarray chips overnight. then the chips were labelled with a cocktail of labelled antibodies, washed and imaged. vesicles were counted, sized, and phenotyped. next, commercially available pooled human csf was analysed in a similar fashion to determine their abundance in a relevant biofluid. results: multiple antibody clones were tested in different combinations for capture and labelling for the five different neuronal enriched proteins of interest, and optimal combinations were identified. some markers were identified on particles > nm in size that were negative for tetraspanins, while others colocalized with tetraspanins. through comparing permeabilized and intact evs with and without sec to remove non-vesicular proteins, we found that tau could be on the vesicle surface, within the vesicle, and free in solution. summary/conclusion: the exoview platform can be customized to enable the detection of proteins of interest and to determine whether they are on the ev surface, intravesicular, or non-ev associated. methods: forty non-smoking male and female subjects ( - y) at moderate risk for cvd were recruited for the study. evs from platelet-free plasma (pfp) were isolated using size exclusion chromatography (sec). the concentration and size distribution of evs were measured by nanoparticle tracking analysis (nta) and flow cytometry (fcm). three ev markers, including annexin v for the circulating phosphatidylserinepositive (ps+) evs, cd for platelet-derived evs and cd for endothelial-derived evs were used for phenotyping. in addition, coagulation and fibrinolysis were assessed using a thrombodynamics analyser (hemacore). platelet aggregation to determine platelet function was assessed by a high-throughput platelet function assay with a wide range of concentrations of agonists, including adenine diphosphate (adp), collagen-related peptides (crp-xl), epinephrine, thrombin receptor activating peptide (trap- ) and u . the association between thrombogenic risk markers for cvd and ev numbers was tested by pearson's correlation coefficient and linear regression model using the statistical program, spss. results: circulating ev concentration with threshold of nm, measured by nta, were positively associated with coagulation-related risk markers, including rate of clot growth (r = . ; p = . ) and clot size at min (r = . ; p = . ). ps+ evs derived from endothelial cells, determined by fcm, were negatively associated with lysis onset time (r = − . ; p = . ), whereas they were found positively correlated with lysis progression (r = . ; p = . ). both mean and mode size of cevs, detected by nta, were significantly correlated with u -induced platelet aggregation (r = − . ; p = . , r = − . ; p = . , respectively). summary/conclusion: in subjects at moderate risk for cvd, cev numbers were positively related to rate of clot growth and clot size and size of cevs was negatively related to platelet activity. higher numbers of endothelial cell-derived ps+ cevs were associated with lower rates of fibrinolysis. this suggests that cevs promote clot growth and reduce fibrinolysis, and may therefore be an indicator for greater risk of cvd. beyond stem cells: extracellular vesicles from human induced pluripotent stem cells (hipsc) and hipsc-cardiomyocytes as therapeutic approaches for heart failure introduction: heart failure is caused by a variety of underlying diseases, the most common being myocardial infarction. initially regarded as an alternative to pharmacological approaches, stem cell transplantation has failed to demonstrate clinically meaningful results. instead, it has become increasingly apparent that the therapeutic effects of transplanted cells are largely mediated by their secretome, while mounting evidence suggests extracellular vesicles (evs) play a major role in cardiac repair. within this framework, evs from human induced pluripotent stem cells (hipsc) and hipsc-derived cardiomyocytes (hipsc-cm), hold a tremendous potential to treat cardiovascular disease. we isolated evs from conditioned culture media at key stages of the hipsc-cm differentiation and maturation processes, i.e. from hipsc (hipsc-ev), cardiac progenitors (cpc-ev), immature (cmi-ev) and mature (cmm-ev) cardiomyocytes, with the aim of studying their potential role as therapeutics, and whether their effectiveness was influenced by the state of their parent cell. methods: hipsc were differentiated into cardiomyocytes in a d culture approach, using the protocols developed by our group. ev isolation was performed on an iodixanol density gradient, and the evs were characterized in terms of particle size and particle size distribution, presence of ev-specific markers, and imaging through transmission electron microscopy. functional studies were performed using human umbilical vein endothelial cells (huvecs) to evaluate evuptake, cell migration and angiogenesis. results: evs from all hipsc and cardiac derivatives presented a typical cup-shaped morphology and expressed cd and cd . ev yield varied along differentiation, with a minimum for cpc and a maximum for cmi. pkh -labelled evs were uptake by huvecs, and colocalized with calnexin, a protein from the endoplasmic reticulum. wound healing assays showed an increased cell migration in huvecs treated with cardiomyocyte-derived evs, in comparison with control evs isolated from foetal bovine serum. summary/conclusion: our findings suggest a different ev secretion profile along cm differentiation and maturation, with preliminary assays showing ev functionality. ongoing work aims at elucidating the possible differences in function and cargo amongst these types of evs. endothelial cells differentially load and secrete extracellular vesiclederived micrornas into apical and basolateral compartments this may play a role in microcalcification in calcific aortic valve disease (cavd), but this is poorly understood. annexin a is thought to be a marker of membrane-derived evs, but because it can be found on the cytoplasmic or extracellular side of the plasma membrane, its localization within or on the surface of evs is unclear. the goal of this study was to determine whether annexin a is found on the surface of evs in two cell lines relevant to cavd, and develop an assay that can be used to determine whether this changes under pathogenic conditions. methods: evs were isolated by differential ultracentrifugation from the conditioned medium (cm) of smooth muscle cells (smc) and valvular interstitial cells (vic). total protein in the cell lysates and ev pellets was analysed by western blot. evs from cells treated with control sirna or anxa -sirna were enumerated and phenotyped using the exoview r platform. evs with surface expression of cd , cd , cd , and annexin a were captured using a customized antibody microarray chip. then evs were labelled with fluorescent antibodies to assess ev number, size, and colocalization of ev proteins. the knockdown of annexin a allowed us to assess the specificity of the selected annexin a antibody. results: the ev fraction was positive for cd , and lacked markers of other vesicle types. western blot on the ev pellet and supernatant in ± edta indicated that there is annexin a both on the surface of and within the evs. using the antibody microarray chips, numerous annexin a + evs were captured on the annexin a spots from the control cm, and there was a marked decrease in capture and labelling from anxa -sirna treated cells. under both conditions, vesicles were also captured on tetraspanin probes, with the greatest number captured on cd , then cd and cd . there was a significant population of annexin a + evs that was negative for tetraspanins. summary/conclusion: annexin a is found on the surface of evs. the assay developed in collaboration with nanoview biosciences is well suited for assessing the number and phenotype of annexin a + evs derived from smc and vic cell lines, which could provide a useful method for understanding ev populations in cavd patient cell lines. funding: this work was supported by hl and hl . possibility of exosomal micrornas associated with chronic limb-threatening ischaemia, the end stage of atherosclerosis, as a promising biomarker introduction: chronic limb-threatening ischaemia (clti), the end stage of peripheral artery disease (pad), has poor prognosis and is attributed to lifestyle disease. with increasing of atherosclerotic disease all over the world, establishment of biomarker for should play a pivotal role for early detection and preventing aggravation of the disease. the aim of this study is to explore the possibility of liquid biopsy for atherosclerotic disease by analysis of clti-associated exosomal micrornas. methods: clti due to pad was diagnosed by anklebrachial blood pressure index, skin perfusion pressure (< mmhg) and angiography. ten preoperative clti patients and control patients without pad were analysed (all patients with diabetes and % of patients had end-stage renal failure [esrd] ). to identify biomarkers associated with clti, exosomes were extracted from patient's serum after ultracentrifugation and total rna including small rna was isolated from the exosomes. the expression profile of exosomal micrornas associated with clti were evaluated using a next generation sequencing. results: forty-three exosomal mirnas associated with clti were identified. intriguingly, these mirnas were clearly categorized with esrd, which was well known as end-stage of life-style disease: these were stratified into micrornas for esrd patients and micrornas for non-esrd patients. since esrd is the most important factor significantly related to patient's prognosis in clti, exosomal micrornas reflected patient's comorbidity onto the expression profile. summary/conclusion: a portion of the expression profile of exosomal micrornas associated with clti was identified. exosomal microrna could be a biomarker to stratify patient's condition along with their comorbidities and is very promising for individualized diagnosis in atherosclerotic diseases with risk diversity. postoperative plasma exosomal mir- and mir- a signature in patients with left ventricular reverse remodelling after surgical mitral valve repair underwent implantation of a prosthetic mitral ring. lv remodelling was assessed by cardiac magnetic resonance imaging and pexos were isolated by optimized ultracentrifugation before surgery (t ) and six months after surgery (t ). isolated pexos were quantified by nanoparticle tracking analysis and mir- , mir- , mir- a, and mir- a were measured by rt-qpcr. the same analysis was performed on healthy subjects with normal cardiac function (n = ). local ethical committee approved the study (emigrate study, approval n° ) and informed consent was obtained from all patients. results: pexos levels at t were lower (− %, p = . ) in patients with worst postoperative lv function, while they were higher at t (+ %, p = . ) in patients with reversed lv remodelling after surgery. at t , the increase in pexos levels was associated to decreased heart mass index (− %, p = . ) and higher levels of exosomal mir- (+ %, p = . ) and mir- a (+ %, p = . ) were detected in patients with improved lv function. summary/conclusion: higher postoperative levels of pexos delivering mir- and a depict lv reverse remodelling after surgical mitral valve repair. monitoring of exosomal micrornas cargo might predict postoperative outcome in patients with mr. expression of lipocalin- (lcn ) in circulating extracellular vesicles (evs) and femoral plaque-derived evs of peripheral arterial disease patients. introduction: clinically, the drug resistance situation of acinetobacter baumannii is becoming increasingly serious, and its drug resistance has become a difficult problem for nosocomial infection and clinical treatment. in view of the relatively slow development of antibacterial drugs, exploring the resistance mechanism of acinetobacter baumannii is of great significance to improve bacterial resistance and help clinical treatment. studies have shown that outer membrane vesicles (omvs) can transmit resistance genes to mediate the spread of drug resistance, and recent studies have confirmed that high expression of efflux pumps play an important role in the multidrug resistance of a. baumannii. in this study, we want to explore whether the outer membrane vesicles of acinetobacter baumannii can transfer the efflux pump related substances. methods: first, ultracentrifugation and density gradient centrifugation were used to extract the omvs of acinetobacter baumannii antimicrobial-sensitive strains (atcc ) and antimicrobial-resistant strains. then, nanoparticle tracking analysis (nta) technology was used to analyse the particle size and distribution range of omvs. transmission electron microscopy (tem) was used to identify their morphology and structure. bradford method was used to determine the protein concentration of omvs. next, the omvs of antimicrobial-resistant strains were incubated with the antimicrobial-sensitive strains and then the drug susceptibility test was done to determine whether omvs of antimicrobial-resistant strains could transmit antimicrobial-resistance information to the antimicrobial-sensitive strains. finally, pcr, qpcr and mass spectrometry were used to determine whether the efflux pump related genes were higher expression in omvs of antimicrobial-resistant strains than those in antimicrobial-sensitive strains. results: nanoparticle tracking analysis (nta) detected the concentration and size distribution of omvs of acinetobacter baumannii strains. it showed that the extracted omvs have a relatively uniform particle size and a size between - nm. tem showed that omvs had a typical vesicle structure. omvs coculture experiments showed that omvs of the antimicrobial-resistant strains can indeed pass resistance to the antimicrobial-sensitive strains. and the efflux pump related genes were higher expression in omvs of antimicrobial-resistant strains than those in antimicrobial-sensitive strains. summary/conclusion: omvs of the antimicrobialresistant strains can indeed pass resistance to the antimicrobial-sensitive strains. the cause of acquiring antimicrobial resistance in sensitive strains may be caused by resistant strains passing efflux pump-related genes or proteins to sensitive strains. characterization of melanocytic extracellular vesicles during ageing of the choroid kelly coutant a , léo piquet a , nathan schoonjans b , philippe gros-louis a , julie bérubé c , stéphanie proulx a , alain r. brisson d and solange landreville a a université laval, quebec city, canada; b université de lille, lille, france; c centre de recherche du chu de québec-université laval, quebec city, canada; d université de bordeaux, bordeaux, france introduction: the choroid is located at the backside of the light-sensitive retina and is highly vascularized. it contains pigmented melanocytes, and their melanin protects them against oxidative stress. since ageing reduces the number of melanosomes in melanocytes and generates a stiffer extracellular environment, our hypothesis is that surrounding choroidal cells and the retinal pigment epithelium (rpe) are subject to more oxidative stress-related damages. this study aimed to characterize evs released by human choroidal melanocytes in the context of intercellular cooperation during ocular ageing. methods: melanocytic evs were recovered from the conditioned culture medium of young/old melanocytes grown on hydrogels of varying stiffness ( . - kpa) by differential centrifugation. the concentration and size distribution of melanocytic evs were determined by high-sensitivity flow cytometry. cryo-transmission electron microscopy combined with receptor-specific gold labelling were used to reveal their morphology, size and phenotype. the relative abundance of surface markers was evaluated with the exo-check exosome antibody array. the uptake of fluorescent melanocytic evs by the rpe and choroidal endothelial cells was assessed by confocal microscopy. results: choroidal melanocytes released evs positive for annexin- and the tetraspanin cd . young melanocytes produced more annexin- positive evs and evs larger than nm compared to older donors. the stromal stiffness impacted the concentration and size of melanocytic evs. we confirmed the uptake of melanocytic evs by endothelial and rpe cells. summary/conclusion: evs from choroidal melanocytes are internalized by surrounding endothelial cells and rpe. age-related stressors modify the phenotype of melanocytic evs. the identification of melanocytic factors that can protect retina/choroid cells from oxidative stress-induced cell death could lead to more efficient therapy for patients suffering from dry agerelated macular degeneration. introduction: owing to their proposed biocompatibility and ability to cross biological barriers, evs represent an attractive therapeutic delivery platform. however, evs are eminently heterogeneous. a better understanding of ev heterogeneity and its origins will allow for improved design of ev-based therapeutics. ev heterogeneity is mainly studied by focusing on distinct ev subpopulations. other sources of heterogeneity, such as heterogeneity within ev secreting cells themselves, have been investigated in lesser detail. in this study, we assessed the phenotypic drift of cell derived evs to explore the origins of ev heterogeneity and its potential impact. methods: three independent samples of two mda-mb- breast cancer cell sub-clones were cultured for six weeks. evs were harvested weekly and analysed using the macsplex exosome flow cytometry kit. at two time points the proteome of evs was analysed by lc-ms/ms mass spectrometry with subsequent gene ontology and reactome pathway analysis. results: the expression of over proteins was deregulated in evs derived from the two different cell clones. many de-regulated proteins were associated with biological processes predicted to affect potential ev toxicity (platelet activation, neutrophil degranulation, blood coagulation) and ev biological activity (antigen presentation, inflammation, tgf-beta/ mtor/wnt signalling). more surprisingly, within only two weeks, over ev proteins, many associated with immune modulation, apoptosis, interleukins, cytokines and cell signalling pathways (including those affecting t-cell/b-cell receptors) were de-regulated between the two ev isolation time points. summary/conclusion: results suggest that temporal changes can be observed in the ev proteome (potentially by clonal drift, epigenetic changes or cellular genomic instability) over short time periods. these changes could cause significant differences in biological effects and delivery capabilities between evs harvested from the same cells at different time points and conditions. in vivo tracking and biodistribution analysis of mesenchymal stem cellderived extracellular vesicles in a radiation injury murine model introduction: recent studies indicated that extracellular vesicles (evs) play key roles in intercellular communication and have great potential for clinical application. understanding the biodistribution of evs is therefore essential. our previous works have shown the ability of mesenchymal stem cell (msc)derived evs to protect haematopoietic cells from radiation damage. in this study, we evaluated the biodistribution of msc-evs in a radiated mouse model. methods: human msc-evs were harvested by ultracentrifugation and labelled with did lipid dye. the reliability of the labelling evs was confirmed by sucrose gradient fractionation analysis. the distribution of evs in radiation-exposed mice after ev intravenous administration were evaluated by fluorescence molecular tomography and further confirmed by flow cytometry and confocal microscopy analysis. results: we observed that did labelled msc-evs appeared highest in liver and spleen, lower in bone marrow in tibias, femurs, and spine, and were undetectable in heart, kidney and lung. we found the significantly increased msc-ev accumulation in spleen and bone marrow post-radiation appeared with an increase of uptake of msc-ev by cd b+ and f / + cells, but not b + cells, compared to those organs from non-irradiated mice. however, there was a predominant ev accumulation in lung and less accumulation in spleen and liver; in mice infused with human lung fibroblast cell derived evs (lfc-evs) and there was no significant lfc-evs accumulation change in the spleen or liver after radiation. we further found that increasing levels of irradiation caused a selective increase in vesicle homing to marrow and spleen. this accumulation of msc-evs at the site of injured bone marrow could be detected as early as hour after msc-ev injection and was not significantly different between and hrs. post-msc-ev injection. summary/conclusion: this study indicated the specific accumulation of ms-evs at the site of injury of haematopoietic tissue in radiation injury mice. funding: this work was supported by the nih grants uh tr , uh tr - s , p gm , and t hl . linking fat to colorectal cancer: extracellular vesicle crosstalk sheffield hallam university, sheffield, uk introduction: colorectal cancer is the third most common cancer worldwide, and fourth leading cause of malignancy related mortality. understanding the mechanisms of its growth and metastasis is key to elucidating new therapeutic targets and developing treatments in the clinical setting. epidemiological evidence indicates an increased risk of cancer in obese patients, pointing to bidirectional communication between colon and adipose cells. extracellular vesicles (evs) are small membrane enclosed packages released by cells, capable of transporting bioactive cargo from donor to recipient cells and inducing phenotypic changes. adipocytes are a key component of the tumour microenvironment and interactions between adipose tissue and tumour cells may be important in the growth and metastasis of cancer. in this study, we investigate the effects of colorectal cancer evs on adipocytes in vitro, and potential induction of dedifferentiation to a more fibroblastic, pro-inflammatory phenotype. methods: evs were isolated from sw and ht human colorectal cancer cell lines by differential ultracentrifugation and mature adipocytes generated by differentiation of the sgbs human pre-adipocyte cell line. adipocytes were treated with evs and their lipid content measured by oil red o to determine loss of lipids. inflammatory cytokine profile was measured by elisa to assess any increase in pro-inflammatory behaviour, and expression of late adipogenesis markers were determined by western blot. results: ev treatment was shown to reduce lipid accumulation in adipocytes, with up to % reduction in lipids observed at the µg/ml dose. treatment was also shown to reduce the expression of late adipogenesis markers, and increase secreted levels of proinflammatory cytokines il- and il- by over fold and fold respectively. these results provide evidence for colorectal cancer derived ev involvement in the dedifferentiation observed in cancer associated adipocytes in vivo, displaying an altered phenotype, releasing lipid energy stores to fuel tumour growth and increasing pro-inflammatory signalling. summary/conclusion: studies have shown colorectal cancer evs may be involved in signalling which induces functional changes in cells within the tumour microenvironment. our work indicates that ev mediated dedifferentiation of resident adipocytes may potentially contribute to a microenvironment favouring cancer cell growth and metastasis. further work aims to elucidate the specific ev cargo which mediates these effects. introduction: ageing is a major risk factor for many human diseases. it is a complex process that progressively compromises most of the biological functions of the organisms, resulting in an increased susceptibility to disease and death. senescence is a cellular phenotype characterized by a stable cell cycle arrest. senescent cells are accumulated in the body during ageing. it contributes to develop age-related diseases and cancer. the alteration in intercellular communication with age has been demonstrated to be due to senescent cells developing a phenomenon denominated senescenceassociated secretory phenotype (sasp). exosomes are small extracellular vesicles (sev) ( - nm) of endocytic origin whereas microvesicles are formed by shedding of the plasma membrane. they contain nucleic acids, proteins and lipid that generally reflect the status of the parental cell and can influence the behaviour of neighbouring cells. methods: in this study, we demonstrated that the small extracellular vesicles (sev) contribute for transmitting paracrine senescence to proliferative cells firstly, we evaluated the presence of exosome-like particles in the sev from senescent cells by detection of exosome markers (alix, tsg and cd ), transmission electronic microscopy (tem) and nanoparticle tracking analysis (nta). to determine that sev from senescent cells are mediators of the paracrine senescence, we performed functional assays using cre-loxp reporter system and high-throughput results: besides, we confirmed at a single-cell level that the proliferative cells internalizing sev from senescent cells activate senescence process using the cre-reporter system. sev protein analysis from senescent cells by mass spectrometry (ms) and validation of top candidates using a functional sirna screen identify interferon induced transmembrane protein (ifitm ), a component of non-canonical interferon (ifn) pathway, as partially responsible for transmitting senescence to proliferative cells. summary/conclusion: in conclusion, we found that sev are regulators of paracrine senescence and ifitm contained in senescent sev has an important role in the intercellular communication mediated through sev during cellular senescence . bin wu a , lei guan a , ye xu a , likang chin a , ting li a , youhai chen a , gordon mills b , jinqi ren a , ravi radhakrishnan a , rebecca wells a and wei guo a a university of pennsylvania, philadelphia, usa; b oregon health & science university, portland, usa introduction: extracellular matrix (ecm) remodelling and stiffening are associated with solid tumour progression. stiff ecm promotes cell proliferation, epithelial-to-mesenchymal transition (emt), metastasis and chemoresistance. hepatocellular carcinoma (hcc) appears frequently in patients with liver cirrhosis or fibrosis while the mechanism remains unclear. exosomes have been determined to serve as messengers to mediate intercellular communication and influence the extracellular. tumour-derived exosomes have been shown to influence tumour progression, metastasis, drug resistance, angiogenesis and immune regulation. thus, determining whether exosomes provide a mechanism by which stiff matrix modulates tumour microenvironment for tumour progression opens a new way to understand cirrhosis and oncogenesis. here we identified the molecular mechanism of matrix stiffening induced exosome secretion and showed the different effect of exosomes induced by soft or stiff matrix on tumorigenesis. methods: huh cells were cultured on acrylamide gels with the stiffness was modulated to pa (soft) or k pa (stiff). the exosomes in conditioned media were collected and analysed by nanoparticle trafficking analysis (nta) and immunoblotting. protein expression level in cells was screened by reverse phase protein array (rppa). inhibitor or shrna were used to inhibit target proteins function. in vitro phosphorylation and gef assay were used to verify rabin phosphorylation and activation. exosomes from cells on soft or stiff matrix were injected into mice to study their effect on tumour growth. results: ( ) stiff matrix promoted exosomes secretion. ( ) akt was activated by stiff matrix and was required for exosome secretion. summary/conclusion: matrix stiffening promotes exosome secretion via akt-rabin -rab pathway, contributing to tumorigenesis. tridimensional fibroblast culture revealed a novel exososome-dependent extracellular matrix secretion mechanism vincent clément a , bastien paré b , cassandra goulet a , thiéry de serres-bérard a , stéphane bolduc a , françois berthod a and françois gros-louis a a université laval, québec, canada; b norgen biotek corp., thorold, canada introduction: the extracellular matrix (ecm) is constituted of a variety of proteins and polysaccharides that are secreted locally and assembled into a thick d meshwork to provide biophysical and biochemical support to the surrounding cells, and regulate numerous cellular functions such as adhesion, migration and proliferation. dysregulation of ecm components or aberrant ecm remodelling can lead to various pathologies, as well as to play important roles in wound healing. although ecm secretion pathways are still largely unknown, the current paradigm is that ecmassociated proteins are synthesized in the endoplasmic reticulum and transported via the endosomes to the golgi apparatus en route to the cell surface and released by exocytosis. methods: to study ecm secretion pathway, we used dimensional ( d) cultured fibroblasts. this culture method technique has been used widely to generate tissue-engineered self-assembled stromal tissues, free of exogenous materials, and rely on long-term supplementation of sodium ascorbate into the culture medium. non-cancerous fibroblasts, grown in conventional two-dimensional ( d) cellular cultures, are known to be a poor source of secreted exosomes when compared to cancerous fibroblasts. results: here, we provide evidence that non-cancerous dermal fibroblasts can secrete high amounts of exosomes, containing different ecm proteins, when cultivated in a d fashion. we also demonstrated that dermal fibroblast-derived exosomes had the capacity to travel from one cell to another, induce cellular migration and promote wound healing. summary/conclusion: altogether, these findings reveal a novel exosome-dependent ecm deposition mechanism and suggest that the use of d-fibroblast cellular culture may emerge as an innovative approach in precision medicine to better study the role of patient-derived exosomes and ecm proteins in the establishment of cellular microenvironment in health and disease. anthony yan-tang. wu a , charles lai, yun-chieh sung b , steven t. chou c , vanessa guo c , jasper c. chien c , john j. ko c , alan l. yang c , ju-chen chuang c , hsi-chien huang b , syuan wu c , meng-ru ho d , maria ericsson e , wan-wan lin f , koji ueda g , yunching chen h , chantal hoi yin cheung i and hsueh-fen juan j introduction: bionanoparticles including extracellular vesicles and exomeres (collectively termed evs), have been shown to play significant roles in diseases and therapeutic applications. however, their spatiotemporal dynamics in vivo have remained largely unresolved in detail due to the lack of a limited suitable method. methods: we developed a bioluminescence resonance energy transfer (bret)-based reporter, palmgret, to enable pan-bionanoparticle labelling ranging from exomeres (< nm) to small (< nm) and medium and large (> nm) evs and larger evs (> nm). results: palmgret emits robust, sustained signals and allows the visualization, tracking and quantification of bionanoparticles from whole-animal to nanoscopic resolutions under different imaging modalities, including bioluminescence, bret, and fluorescence. using palmgret, we show that evs released by lung metastatic hepatocellular carcinoma (hcc) exhibit lung tropism with varying distributions to other major organs in immunocompetent mice. ev proteomics identified hcc-ev lung tropic protein candidates associated with cancer progression, in which slco a and clic expression on non-tropic evs conferred lung-tropism, while cd gave spleen tropism. our results further demonstrate that redirected lung tropism decreases ev distribution to the liver, whereas the spleen tropism significantly reduces over time delivery to most major organs distribution including the liver and kidney. summary/conclusion: we established a multimodal and multi-resolution palmbret method to enable pan-bionanoparticle labelling and imaging and therefore quantification in live cells, whole animals, and preserved tissues. the method can resolve the intricate spatiotemporal dynamics of evs. palmgret revealed that evs derived from lung metastatic hcc are lung tropic, and the tropism can be conferred to non-lungtropic ev- t by decorating evs with identified hcc-ev membrane proteins. importantly, the enhanced ev delivery to tropic organs also significantly alters its distribution to other major organs. our findings suggest that the dynamics of ev biodistribution and targeted design should be investigated at the organ systems level in ev biology and therapeutic developments, respectively. tracking mesenchymal stem cell-derived extracellular vesicles (evs) in a in vivo cancer model introduction: small extracellular vesicles (sevs) are nanoparticles ( - mn) encircled by a phospholipid bilayer, derived from the endocytic pathway and released by all cells. sevs have an inherent role in cell communication and deliver cargo to target cells. mesenchymal stem cells (mscs) and have a natural ability to home to tumours and metastases while avoiding the host immune response. it is hypothesised that msc derived sevs (msc-sevs) also possess tumourhoming and immune-evading capacities therefore could provide a novel targeted delivery vehicle for treatment of cancer. it is imperative to elucidate msc-sevs migratory itinerary in vivo to support translation to the clinical setting. methods: this study aimed to image the interaction of labelled msc-sevs with cancer cells in real time in vivo. sevs were isolated from wildtype mscs and mscs with stably expressing red fluorescent protein (rfp) (via lentivirus) by the combined techniques of differential centrifugation, microfiltration and ultracentrifugation. isolated sevs were extensively characterised by transmission electron microscopy (tem), nanoparticle tracking analysis and western blot. nod scid gamma (nsg) mice with dorsal skinfold window chamber (dsfwc) were injected with either mda-mb- luciferase (luc) expressing cells or ht- -luc cells. bioluminescence imaging was performed to confirm tumour formation. a dose of x ^ msc-rfp-sevs was directly added to the window chamber and rfp expression detected using a microscope with rfp filter attachments. x ^ evs were incubated with the radionuclide, technetium- m tagged duramycin ( mtc-dur) for minutes at room temperature. excess radiolabel was removed using exosome spin column (invitrogen™). the mtc-dur-sevs were then added directly to the window chamber and charged particle imaging carried out. results: hours post-administration; the rfp signal was localised at the tumour site. radiolabelled sev signal could be detected minutes and hours after administration. msc-sevs were successfully detected at the tumour site following direct administration using two different tagging and imaging approaches. summary/conclusion: this promising preliminary data supports the potential of this approach for tracking msc-sev migration in vivo. future studies will investigate systemic tracking of msc-sev migration. vaughn garcia ; aejez sayeed ; rachel derita ; shiv ram krishn ; peter a. introduction: tumor-derived small extracellular vesicles (sevs) have emerged recently as mediators of tumorigenesis. however, the role of sevs in response to irradiation, a widely used therapy in prostate cancer, is not fully understood. methods: our study involved the tramp mouse model of prostate cancer. we used plasma sevs isolated using differential ultra-centrifugation and further isolated using iodixanol gradient fractionation. we also used nanoparticle tracking analysis (nta) to analyze sevs. mouse pelvises were irradiated using gy, for consecutive days. results: we first observed that upon pelvic irradiation of tramp mice, the levels of the signaling oncogene c-src are reduced in plasma-derived sevs, while the average size of sevs is increased from - nms to - nms. furthermore, we show that the sevs from irradiated cells lose the ability to stimulate anchorage independent growth and migration of recipient cancer cells. additionally, sevs from irradiated mice increase the amount of dna damage in recipient cancer cells. summary/conclusion: overall, our data show that irradiation of tramp mice (and prostate cancer cells) significantly reduces the pro-metastatic and pro-anchorage-independent growth potential of sevs when tested on human cells. changes to the composition and behavior of a cancer cell sev population via radiation therapy offers promise for future therapeutic approaches for prostate cancer. introduction: there are emerging physiological and pathological functions of extracellular vesicles (evs) in neurodegenerative diseases including alzheimer's disease (ad). brain derived-evs contain pathogenic proteins, such as tau, amyloid beta (aβ), which have been reported to contribute to cell-to-cell propagation in those diseases. investigation of the brain-derived ev cargo, therefore, is important to further understand the mechanisms of progression in neurodegenerative diseases. we developed the ev separation method from unfixed frozen mouse and human brain tissues and assessed the protein composition. methods: to establish the ev separation method, we separated evs from frozen mouse brain tissue using sucrose density gradient ultracentrifugation (sg-uc) or size exclusion chromatography to compare the results from the particle number, morphology and protein profiling by nta, tem and mass spectrometry. evs were then separated from cortical grey matter of ad (n = ) and control (n = ) by sg-uc. tau and aβ in the evs were measured by immunoassay. differentially expressed ev proteins were observed by quantitative proteomics employing machine learning. results: the separated evs were enriched in ev molecules and devoid of contaminant proteins by sg-uc, showing our method was successful. the levels of ps tau and aβ - were significantly increased in ad evs. annexin a (anxa ), neurosecretory protein vgf, neuronal membrane glycoprotein m -a (gpm a), and alpha-centractin (actz) were differentially expressed in ad evs. a combination of these proteins were confirmed to predict ad with the % accuracy by machine learning. summary/conclusion: these data suggest our method were suitable for the separation of brain-derived evs and ev anxa , vgf, gpm a and actz can be potential biomarkers for monitoring the progression of ad. edta stabilizes the concentrations of extracellular vesicles during blood collection introduction: to establish reliable biorepositories for research on extracellular vesicles (evs) as disease biomarkers, the release of evs during blood collection and handling must be avoided. currently, citrate is recommended as the anticoagulant for blood ev research, but citrate does not inhibit the release of evs from activated platelets. the release of platelet-derived evs excludes pneumatic tube transport and makes assays time dependent, thereby limiting clinical compatibility. therefore, we aim to stabilize the release of platelet ev concentrations. methods: blood samples were collected from healthy individuals and subjected to common circumstances known to induce platelet activation. blood was (i) incubated with or without thrombin receptor-activating peptide (trap; n = ), a potent platelet activator, (ii) send to the lab by a routine blood transport (pneumatic tube system; n = ), and (iii) stored at room temperature or at °c for hours (n = ). the concentrations of evs from platelets (cd +), activated platelets (p-selectin+), erythrocytes (cd a+), and leukocytes (cd +) were determined by flow cytometry (apogee a -micro). results: following activation by trap, concentrations of platelet-derived and activated platelet-derived evs increased . -fold and . -fold in citrate-anticoagulated blood, compared to . -fold and . -fold in edta-anticoagulated blood (edta vs citrate: p = . and p = . , respectively). preliminary data show that during pneumatic tube transport and routine sample handling, both platelet-and activated platelet-derived evs were more stable in edta compared to citrate. the concentrations of evs from erythrocytes and leukocytes were unaffected under all studied conditions. summary/conclusion: to conclude, edta stabilizes platelet ev concentrations during and after blood collection, which would facilitate pneumatic tube transport, enhance reliability and thereby improves the establishment of reliable biorepositories for ev research. introduction: cancer-cell secreted extracellular vesicles, called exosomes, are an emerging biomarker for cancer liquid biopsy. profiling of cancer-associated exosomes usually required lengthy, and multi-step procedures; therefore simple and easy-setup sensing methods are urgently needed for diagnosing cancer in a timely manner. chirality, the foundational property of all biomolecules, including exosomal proteins, can be utilized for exosome detection and differentiation using recent advances in chiral nanostructures. we found that microfluidic sensors can be successfully implemented for successful detection of cancer-associated exosomes taking advantage of unusually high circular dichroism (cd) of chiral gold nanoparticles (aunps). circular dichroism-based exosome (cdexo) detection utilizes chiroplasmonic enhancement of cd signatures of cancer-associated exosomes. we first synthesized donut-shaped aunps conjugated with l-cysteine and immobilized the aunps on a glass slide using a layer-by-layer assembly. the aunps on slide glass were surface functionalized by the standard biotin-avidin reaction after mua treatment. biotinylated annexin v marker, targeting phosphatidylserine (ps) expression on cancer-associated exosomes, was conjugated to the aunp surface. μl of exosome samples from cancer cells (a and h ) or normal cells (mrc ) were injected into the pdms microfluidic device and incubated for minutes. the cd signal before and after exosome exposure was monitored, compared, and systematically analysed as a rapid technique for the detection of exosomes with high sensitivity. results: we showed that the cdexo signals from cancer exosomes showed . folds absolute cd peak value change and . folds shift, respectively, compared to that of healthy exosomes. importantly, the cdexo sensing method takes less than mins in terms of total scanning time and requires minimal sample volumes. from the preclinical studies using blood samples from cancer patients and healthy donors, we found that cancer patients show stronger band shift and signal change comparing to that of healthy donors, implying our platform could be used for cancer diagnosis. summary/conclusion: this new versatile and sensitive method based on chiroplasmonic exosome detection paves the way to profiling disease-associated exosomes in a timely manner for minimal volumes of liquid biopsies. ev classification and fractionation strategy using surface charge labelling takanori ichiki a , hiroaki takehara a , hirofumi shiono b and hiromi kuramochi a a the university of tokyo, bunkyo, japan; b innovation center of nanomedicine, bunkyo, japan introduction: the development of new classification technology is required based on the evaluation of physicochemical properties of exosome surfaces and the diversity of constituent molecules. in this presentation, we present the electric charge activated exosome sorting platform comprising microfluidic device technology and electric charge labelling technique. methods: the single nanoparticle analysis platform, which has been developed by our research group, images rayleigh scattered light (elastically scattered light) obtained by irradiating nanoparticles with convergent laser light and provides information of individual particles by image processing. the method that utilizes electrokinetic phenomena, unlike the method using fluorescent labels, measures the properties of the particle surface without serious difficulty in principle even if the particle size is on the order of tens nanometres, and further enables to perform fractionation. since the number of particles usually handled in exosome research or its envisioned application is enormous, it is not realistic to take an approach such as a cell sorter in which particles are sequentially manipulated one by one following the measurement results of individual particles. results: particles receive attraction or repulsion by an external field according to the charge density on the surface, so there is no need to control the external force, and it is possible to design a device that can autonomously fractionate particles according to the difference in zeta potential. summary/conclusion: in conclusion, we have proposed and demonstrated the new concept of electric charge activated ev sorter. funding: this research was partially supported by the center of innovation program (coi stream) from the japan science and technology agency. high throughput exosome analysis by using reversible microfluidic electrochemical sensor system introduction: exosome is one of the important extracellular vesicles (evs) released from parental cells and it contains various types of molecular cargos from its original cell including proteins, messenger rna (mrna), and micro rna (mirnas) [ ] . the exosomes have recently emerged as biomarkers for early stage cancer detection because the number of exosomes originated from cancerous cells are significantly higher than those from normal cells [ ] . since many different types of exosomes exist in the whole blood, it is necessary to isolate and detect disease-specific exosomes. for this reason, the isolation and the detection of exosomes is an important research issue and has been studied by many groups. however, limitations such as low throughput and low recovery still make it difficult to use exosomes in diagnostics and therapeutics. methods: in this study, we developed an integrated microfluidic electrochemical biosensor to extract plasma from whole blood and subsequently detect cancer related exosomes in a continuous manner. this consists of two parts. the first part is a channel for extracting plasma containing exosomes from whole blood, and the second part is a channel combined with an electrochemical sensor for multiple detection of various exosomes in the extracted plasma. previously, a multi-orifice flow fractionation (moff) channel that consists of a series of expansion and contraction structures has been developed in our group. in this channel, the blood cells are moved to sides of channels by hydrodynamic forces and then are eliminated to outlets. at this time, the plasma is moved to the electrochemical sensor part, the exosomes in the plasma are captured to the electrodes immobilized with the specific antibodies and are quantified the amount of cancer-related exosomes. results: using this chip, blood cells were eliminated from the whole blood with over % of separation efficiency at µl/min flow rate and exosomes were collected continuously with high recovery (~ %). in order to quantify various types of exosomes, a labelfree electrochemical biosensor with electrochemical impedance spectroscopy (eis) was used for the continuous detection of exosomes. the limit of detection was x ^ exosomes/ml. summary/conclusion: the developed device is an integrated device capable of separating exosomes from whole blood with high purity and quantitating exosomes through the electrochemical sensor in a continuous manner. , , ) . the development of highthroughput techniques capable of simultaneously monitoring physical and biochemical properties of evs would significantly simplify and accelerate the characterization process. in this context, microfluidic technology is emerging as an attractive platform. here, we present a microfluidic device based on the combination of diffusion sizing and multi-wavelength fluorescence detection to simultaneously provide information on ev size, concentration and composition. methods: the diffusion of evs in the microfluidic channel provides information on their size distribution, and four different staining protocols with high signalto-noise ratios track different ev native molecules. evs are separated from unbound fluorophores directly during the microfluidic analysis, therefore avoiding the need for sample pretreatments and allowing to operate the device as a single-step immunoassay. results: the microfluidic device coupled with complementary staining techniques allows to individually detect and size particle populations with different ev components such as lipids, primary amines and the ev marker cd . we demonstrate that this approach can probe the abundance of ev-specific markers and impurities such as lipoproteins with high throughput and low sample consumption. summary/conclusion: we present a microfluidic technique capable of characterizing and quantifying evs at low costs, in a time-scale of minutes and requiring only up to µl of non-pretreated sample. this method is an important complementary tool to the current array of biophysical methods for ev characterization, in particular for high-throughput screening applications. funding: h -eu. . . -fet open programme via the grant agreement . immunomagnetic isolation of specific subpopulations of exosomes for liquid biopsy via nano-architected porous materials introduction: exosomes offer the potential to reveal significant biological information in many areas of clinical importance by virtue of their rna contents and protein surface markers. this abstract reports the fabrication of a device for high throughput targeted immunomagnetic capture of exosomes via the use of highly-ordered nano-architected porous metal lattice materials. methods: we have invented a fabrication technique to precisely make millions of nanoscale exosome sorting devices that can operate on unprocessed plasma. each nanoscale device can precisely sort targeted exosomes from background vesicles but is too slow for practical use individually. however, the operation of millions of these devices in parallel preserves the precision of nanoscale sorting while also enabling high throughput and robust use on raw plasma samples. the metal lattice within which these devices are contained is assembled via metal electroplating onto a selfassembled polystyrene bead lattice with face-centred cubic (fcc) symmetry with nanometre pores. the devices feature a conformally-coated layer of nickel-iron with gold passivation atop a base layer of nickel, resulting in a lattice of millions of nanoscale pores capable of magnetic sorting of exosomes tagged via surface-marker-based immunomagnetic labelling with magnetic nanoparticles. results: compared to our previous work on immunomagnetic exosome capture via commercial track-etched membranes (tempo), this device offers superior capture due to increased surface pore density (> x) and three-dimensional pore density (> x) alongside lower required sample volume due to decreased noncapturing volume in the device. finite-element analysis simulations show that strong magnetophoretic traps emerge at the pore boundaries in this structure between higher-permeability metals such as nickeliron permalloy and the lower-permeability sample fluid in the device. preliminary experimental data shows that this device can isolate iron nanoparticles in solution with > x enrichment from input and x capture efficacy versus tempo. summary/conclusion: current methods of exosome isolation such as ultracentrifugation and column chromatography all suffer from low throughput and limited yield. the application of inverse opal materials towards exosome capture offers the potential for isolation of specific exosome populations from very low clinical sample volumes or sparse biological signals. micropatterned growth surface topography affects extracellular vesicle production colin l. hisey a , james hearn b , yohanes nursalim a , vanessa chang a , cherie blenkiron a and lawrence w. chamley a a the university of auckland, auckland, new zealand; b university of auckland, grafton, new zealand introduction: extracellular vesicles are micro and nanoscale packages released by all cells and play an important role in cell-to-cell communication by shuttling biomolecules to nearby and distant cells. however, producing enough evs for many in vitro studies using conventional tissue culture techniques can be challenging, and despite the success of some bioreactors in increasing ev-production, it is still unknown how many independent culture conditions like growth surface topography can alter the production and content of evs. methods: standard mm petri dishes were patterned with µm tall polystyrene microtracks spaced by , and µm across a mm area using standard microfabrication techniques including photolithography, soft lithography and microtransfer printing. the micropatterns were characterized with sem and profilometry, then activated with oxygen plasma and uv sterilized. mdamb cells were seeded onto patterned and smooth (control) dishes and grown in serum-free media for the final hours of culture. evs were isolated using sequential ultracentrifugation of conditioned media and characterized using nta, tem and western blot. cell morphology was imaged using immunocytochemistry and single cell migration was characterized using time-lapse microscopy and manual single cell tracking in fiji. results: we demonstrate the simple and repeatable fabrication of microtracks across a large surface area in order to culture cells on topographically patterned growth surfaces. furthermore, we show that the µm spacing produced significantly more evs than other patterns as well as the highest cell aspect ratio and average single cell migration speed (p < . ). summary/conclusion: these findings have implications in both biomanufacturing of evs and potentially in enhancing the biomimicry of evs produced in vitro. however, further experimentation to assess the differences in cargo on patterned growth surface topographies compared to conventional methods is still required. funding: this project was funded by the maurice and phyllis paykel trust. using miscroscale thermophoresis and surface plasmon resonance to measure the interactions of extracellular vesicles mst is a quick method, easy to handle, has a low sample consumption, has no limitation on molecule size, and enables measurements in solution, either in various buffers or complex biological liquids. these properties make mst an interesting tool for research of extracellular vesicles (evs); therefore, our aim is to apply this method to evs. methods: evs were isolated from jurkat cell line by differential centrifugation. microscale thermophoresis (mst) and surface plasmon resonance (spr) were used to analyse the interaction between antibody and evs. results: we have demonstrated that interactions of evs with antibodies could be analysed by mst. however, the tiny glass capillaries for sample mounting represent a challenge due to adhesion of evs to their surface. we have tested commercial capillaries as well as prepared capillaries in house coated by liposomes or bovine serum albumin. the interactions between evs and antibodies were confirmed by surface plasmon resonance (spr), which is an established method for studying the interactions of evs. introduction: the isolation of extracellular vesicles (evs) from cell culture supernatants and complex body fluids, such as blood and urine, is of high importance for ev research as well as for future medical applications in diagnostics and therapy. nevertheless, it is still challenging to reach the desired recovery, purity and specificity due to many manual and time intensive sample preparation steps. conventional centrifugation for ev isolation or sample preparation prior to affinity-based separation methods can damage evs and cells, leading to misinterpretation of results or inactive evs. alternative field flow fractionation methods employing acoustic fields are highly promising, but so far limited to laboratory usage, based on a complex (moulding) fabrication and/or hardly reproducible. here, we present an innovative surface acoustic wave (saw)-based acoustofluidic device for gentle sorting of cells and particles. methods: our device consists of interdigital transducers patterned on a piezoelectric substrate generating saw propagating on the substrate surface. upon interaction of saw with our on-chip structured, fluid-loaden microchannels, an acoustic pressure field is developed across the fluid wherein particles are suspended. this pressure field can be employed to simply manipulate cells and particles based on their intrinsic properties, such as size, density and compressibility in continuous flow. the device is manufactured using precise and low-cost microtechnological methods and is suitable for reproducible mass fabrication. results: we demonstrated the separation of blood components, i.e. the sorting of erythrocytes and thrombocytes. furthermore, we could also show results on thrombocyte activation indicating a gentle separation without damaging these shear-sensitive cells, as well as first results on plasma separation from whole blood samples and nanoparticle sorting. summary/conclusion: our unique acoustofluidic sorting technology for complex suspensions has the potential to overcome the need for time-effective, cheap and gentle separation of evs. funding: this work was supported by efre infrapro project "champ: chip-based acoustofluidic medtech platform". nanophotonic platform for cancer-associated exosomal microrna detection introduction: exosomes have an important role in intercellular communication at physiological and pathological processes. their cargo includes micrornas (mirs), single-stranded non-coding rnas, involved in alterations on recipient cells, such as development of tumourous phenotype and metastasis. more particularly, mir- excels due to its association with several cancers. determining exosomal mirs as cancer indicators demands selective and accurate methods, which are not currently available or entail high costs. colorimetric photonic-based assays are a promising label-free alternative, which dismisses complex apparatus for signal reading since biorecognition is detected by colour change. moreover, the clinical and economic systems have also been demanding a decrease on the green footprint of biosensors, requirement fulfilled with naturally derived biomaterials. methods: herein, the biosensor is constructed on a biopolymer matrix to meet the requirements of an eco-friendly disposable device, and it is based on a photonic structure obtained by imprinting a nanopattern on the polymer surface. then, the surface is functionalized with the complementary oligonucleotide sequence of mir- as sensing probe. a labelfree detection is thus envisioned and the sensor performance is evaluated by changes in the optical properties when the target is present. results: the combination of biological materials conducted to a biosensor support with great flexibility and low water permeability, allowing easy surface functionalization. the self-reporting ability of the photonicbased sensor enables high intensity colours detected by naked eye. summary/conclusion: the alliance with the high selectivity of oligonucleotide hybridization is expected to offer great exosomal mir- recognition ability and an optimistic perspective for utilization in clinical setups. funding: the authors acknowledge the financial support from the european commission/h , through mindgap/fet-open/ga project. introduction: urinary extracellular vesicles (uevs) are important intercellular communicators. by systems biology integration, uevs prove to be relevant in genitourinary disease detection. however, it has recently been shown that labelled evs administered to the circulation can be detected in the urinary system, as well. thus, this pilot study aimed at phenotyping haematopoietic surface markers on uevs to create enough plausibility for future non-invasive biomarker studies of circulation and immune disorders that may translate into urine but are not yet timely recognized. methods: urine was obtained from healthy men signing a written informed consent (n = ). sampling was approved by the local ethics committee and in compliance with the declaration of helsinki. cell-free urine was obtained by serial centrifugation and ml, each, were utilized for the macsplex exosome kit, human (miltenyi biotec). the manufacturer's recommendations were followed to examine distinct uev surface markers of cd +/cd +/cd + vesicles in a multiplexed bead-based manner including respective controls. the accuri c (bd) was utilized for data acquisition. for further misev -compliant characterization, cd +/cd +/cd + uevs were isolated by immunoaffinity and analysed by fluorescence nanoparticle tracking (f-nta), transmission electron microscopy (tem) and western blotting (wb). urinary creatinine (ucrea) was determined to control for variances in urinary dilutions and used for data normalization. results: except cd , all other surface markers could be identified. the most abundant markers were cd and cd , which were detected in % of samples, followed by cd / ( %), cd ( %), cd and cd ( %, each). cd ( %), cd , cd ( %), cd e ( %) and cd showed similar relative median fluorescent intensities (rmfi), while cd yielded significantly higher (p = . ) and all other markers significantly lower rmfi (p < . ). tem and f-nta revealed cup-shaped vesicles ( ± nm) with . ± . e + particles/g ucrea. wb indicated uev isolates that were positive for alix, syntenin, tsg , cd , cd and cd without any uromodulin or calnexin contamination. summary/conclusion: our results imply that considerable quantities of circulatory evs are, indeed, filtered into urine and could serve as valuable non-invasive biomarkers for systemic dysfunctions. cardiovascular risk markers are strongly related to numbers of circulating extracellular vesicles ruihan zhou a , esra bozbas a , plinio ferreira b and parveen yaqoob a a university of reading, reading, uk; b imperial college london, london, uk introduction: extracellular vesicles (evs) are small plasma membrane-derived vesicles released from various cells, which potentially affect many physiological and pathophysiological processes, and are emerging as a potential novel biomarker in cardiovascular diseases (cvds). however, there is little information about the association of circulating ev levels with traditional cardiovascular risk markers and cvd risk score. methods: • subjects (n = ) aged - yrs with moderate risk of cvds were recruited and assessed for body mass index (bmi), blood pressure (bp) and plasma lipid profile (triacylglycerol, total cholesterol and high-density lipoprotein). • evs were isolated from platelet-free plasma by size exclusion chromatography and analysed by both nanoparticle tracking analysis (nta) and flow cytometry (fcm). nta was used to measure the concentration and size distribution of evs population, and evs were phenotyped by fcm via a colour panel, which included annexin v (for the majority of circulating evs), cd (for plateletderived evs) and cd (for endothelialderived evs). • the association between risk markers and ev numbers was examined by pearson's correlation coefficient and stepwise multivariate regression model. analysis of covariance (ancova) was performed after adjustment for various variables to determine the correlation between the quartile range of ev numbers and -yr cvd risk detected by qrisk . results: ev numbers, as determined by nta, were strongly associated with bmi (r = . , p < . ), blood pressure (systolic bp: r = . , p = . ; diastolic bp: r = . , p < . ) and plasma triacylglycerol levels (r = . , p < . ). plasma total cholesterol level was positively associated with platelet-derived evs, determined by fcm (r = . , p = . ). a multivariate regression model demonstrated that plasma triacylglycerol and diastolic bp independently predicted total ev numbers, with plasma triacylglycerol concentrations explaining . % of the variance for total ev numbers. an additional . % of the variance in total ev numbers was predicted by diastolic bp. ancova of the -yr cvd risk score in the quartile range of total ev numbers were positively and independently associated. summary/conclusion: bmi, blood pressure, plasma triacylglycerol and total cholesterol levels are strongly associated with ev numbers. plasma triacylglycerol and diastolic bp independently predict circulating ev numbers. elevated numbers of evs are independently associated with -yr cvd risk. introduction: extracellular vesicles from cardiospherederived cells (cdc-evs) are known to be anti-inflammatory in various disease models. to further dissect the mechanism, we examined the effects of cdc-evs on t lymphocytes. methods: naïve cd + t cells were isolated from secondary lymphoid organs of foxp -rfp reporter mice, using magnetic-activated and fluorescence-activated cell sorting. cells were subsequently polarized into effector subtypes (th , th , and th ), as well as regulatory t cells (tregs), and the effects of exposure to human-derived cdc-evs on proliferation and cytokine production were assessed. cdc-evs were isolated from serum-free, -day conditioned medium, using ultrafiltration by centrifugation. results: after polarization and culture for days, cdc-evs resulted in dose-dependent and cell-specific proliferative responses. effector t cells (th , th , th ) showed either no change in proliferation (th ) or decrease in proliferation (th , th ), compared to the vehicle control. in contrast, tregs proliferated much more than control (p < . ). next, we sought to characterize the changes in cytokine production by each effector t cell and tregs. compared to the vehicle control, exposure of polarized effector t cells to cdc-evs had little effect on the expression of characteristic cytokine genes, including ifnγ and tnfα (th ), il and il (th ), or il a and il f (th ). in contrast, exposure of tregs to cdc-evs resulted in~ -fold increase in expression of il , a key paracrine agent utilized by tregs in suppression of inflammation. this response was specific to cdc-evs insofar as it was not recapitulated with dermal fibroblast exosomes. concentrations of il- in the culture media of cdc-ev-conditioned tregs mirrored the increases in gene expression. summary/conclusion: cdc-evs potentiate tregs by increasing their proliferation and enhancing production of il- . this offers an attractive therapeutic approach to inflammatory diseases that relies on harnessing an endogenous mechanism of immunosuppression. funding: nih t hl prostanoids impair platelet reactivity, thrombus formation and platelet extracellular vesicle release in patients with pulmonary arterial hypertension aleksandra gąsecka a , marta banaszkiewicz b , rienk nieuwland c , edwin van der pol d , najat hajji e , hubert mutwil f , sylwester rogula a , wiktoria rutkowska a , szymon darocha g , grzegorz opolski a , krzysztof j. filipiak f , adam torbicki g and marcin kurzyna g introduction: prostanoids (epoprostenol, treprostinil and iloprost) induce vasodilation in advanced pulmonary arterial hypertension (pah) but also inhibit platelet activation, thereby increasing the risk of bleeding. therefore, the platelet function and extracellular vesicle (ev) concentrations were measured in pah patients treated with prostanoids and compared to patients with pah not receiving prostanoids. methods: venous blood was collected from patients treated with prostanoids (study group; n = , ± years, % female) and patients not treated with prostanoids (control group; n = , ± years, % female). platelet reactivity was analysed in whole blood by impedance aggregometry using arachidonic acid (aa; . mm), adenosine diphosphate (adp; . µm) and thrombin receptor-activating peptide (trap; µm) as agonists. in a subset of patients, concentrations of evs from platelets (cd + and cd p+; pevs), leukocytes (cd +, levs) and endothelial cells (cd +, eevs) were measured in plateletdepleted plasma by flow cytometry (a -micro). platelet-rich thrombus formation was measured using a whole blood perfusion system. results: compared to the control group, patients treated with prostanoids had lower platelet reactivity in response to aa and adp (p = . ) and lower concentrations of pevs and levs (p ≤ . ). furthermore, thrombus formation was delayed (p ≤ . ) and thrombus size was decreased (p = . ) on prostanoids. epoprostenol did not affect platelet reactivity in vitro, but decreased the concentrations of cd + pevs (p = . ). in contrast, treprostinil and iloprost decreased both platelet reactivity in response to aa and adp (p ≤ . ) and the concentrations of pevs (p ≤ . ). all prostanoids delayed thrombus formation and decreased thrombus size (p ≤ . ). summary/conclusion: patients with pah treated with prostanoids have increased risk of bleeding both due to impaired platelet aggregation, ev release and thrombus formation, compared to patients not treated with prostanoids. antiplatelet effect of prostanoids varies: whereas epoprostenol decreases the release of pevs, treprostinil and iloprost impair platelet aggregation. funding: ag is supported by the national science centre, research project preludium / /n/ nz / . evdp is supported by the netherlands organisation for scientific research -domain applied and engineering sciences (nwo-ttw), research programmes veni . nanoflow cytometry identifies an imbalance of epithelium-and neutrophil-derived extracellular vesicles in the airway environment of paediatric cystic fibrosis patients brian dobosh, vincent giacalone, milton brown, lucas silva, lokesh guglani and rabindra tirouvanziam emory university, atlanta, usa introduction: progressive lung disease is the leading cause of mortality in cystic fibrosis (cf), a chronic condition characterized by recruitment of polymorphonuclear neutrophils (pmns) into the airways. newly arrived pmns are exposed to extracellular vesicles (evs) from the airway epithelium and pmns recruited before them. in controlled experiments, these evs were necessary and sufficient to induce pathological changes including reduced bacterial killing and immunosuppressive activities towards macrophages and t-cells. however, children with cf do not always show a high pmn presence in their airways, which suggests that the balance between pmn recruitment and the activity of other cells is still in flux in early stage disease. methods: we utilized spectral nanoflow cytometry to profile the single ev content of the bronchoalveolar lavage fluid (balf) from cf children (< years of age). for nanoflow cytometry, evs were stained with di- -anepps, and with epcam, cd b and cd (to ascertain epithelial, pmn, and macrophage origins, respectively). violet side scatter and/or fluorescence threshold triggering were used for ev detection. the ratio of neutrophil-to epithelial-derived evs in cf balf correlated positively with the percentage of pmns that are present in the airways (p = . , spearman's rho = . ). this ratio also correlated with the pragma disease score, which quantifies airway damage by chest computed tomography (p = . , rho = . ). summary/conclusion: using a method to quantify evs from specific cell types in vivo, we demonstrated that the ratio of pmn-and epithelial cell-derived evs tracks with airway damage and neutrophil influx, suggesting a critical interplay between these cells in early cf disease. this ev-focused method can be applied to other diseases in which sampling cells is difficult. future experiments will use cf balf biobanks to strengthen data presented here. funding: cf foundation (tirouv a ), emory paediatrics flow core. the potential of crude extracellular vesicle micrornas for the diagnosis of community-acquired pneumonia and for the detection of pneumoniarelated sepsis as a severe secondary complication introduction: circulating cell-free micrornas (mirnas), often associated to extracellular vesicles (evs), are essential for cell-cell communication in the pathogenesis of infectious pulmonary disorders. as early pneumonia diagnosis is often clinically challenging, advances in disease detection could improve outcomes. we characterized crude ev mirnas as potential biomarkers for community-acquired pneumonia and sepsis as a severe secondary complication. methods: individuals were enrolled into our study, subdivided into a training (volunteer n = , pneumonia n = , sepsis n = ) and testing cohort (volunteer n = , pneumonia n = , sepsis n = ). after precipitating crude evs from sera (mircury exosome isolation kit-serum and plasma) and extracting total rna, small rna sequencing was performed. mirnas were selected as biomarker candidates by differential gene expression analysis (deseq ) and sparse partial-least-squares discriminant analysis (mixomics). technical and biological validation was performed by reverse transcription quantitative real-time pcr. group classification was predicted by partial-least-squares discriminant analysis. gene targets and causal networks were identified by ingenuity pathway analysis. results: differential gene expression analysis revealed significantly regulated mirnas in pneumonia compared to volunteers, and mirnas in pneumonia related to sepsis. based on sparse-partial least discriminant analysis, group separation was achieved by mirnas as discriminators with high sensitivity and specificity (area under the curve of the receiver operated curve: volunteer: . , pneumonia: . , sepsis: . ). mir- a- p (log fc = . , padj = . e- ) and mir- - p (log fc = . , padj = . e- ) differentiated between pneumonia and volunteers and mir- (log fc = − . , padj = . e- ) between pneumonia and sepsis. expression levels of mir- a- p and mir- were related to disease severity. mir- - p was higher expressed in pneumonia compared to volunteers and had equal expression in patient groups. prediction of group classification in the testing cohort was . %. signalling networks were constructed for "cellular and humoral immune response", "antimicrobial response" and "pathogen influenced signaling" involving the significantly regulated mirnas. summary/conclusion: crude ev mirnas are potentially novel biomarkers for community-acquired pneumonia and may help to identify patients at risk for progress to sepsis allowing early intervention and treatment. introduction: it remains unclear the specific mechanisms that lead to airways inflammation in asthma and the subsequent remodelling of the airways. exosomes, small extracellular vesicles, has become in an important mechanism of cell-to-cell communication and participate in diverse biological processes including inflammation. in this study, we hypothesize that exosomes and their mirna cargo play an important role in the proinflammatory status of the upper airway of asthma patients, especially in those patients with severe asthma. methods: in a pilot study, healthy subjects had induced sputum using standard methods. after several centrifugation steps, we were able to isolate exosomes from sputum supernatant by both precipitation and size exclusion cromatography (sec). exosome size was observed with transmission electron microscopy (tem) and the protein markers cd and cd were analysed by western blot (wb). then, total rnas were isolated from sputum exosomes and mirnas (mir- a-p, mir- - p, mir- a, mir- b- p, mir- - p, mir- - p, mir- - p, mir- - p, let- g- p), were evaluated by rt-qpcr. after the optimization of the methodology, healthy adults subjects and patients with persistent moderatesevere asthma, matched by age and sex were selected and induced sputum was collected. results: exosomes isolated with both methodologies (precipitation and sec) were observe under the tem with a correct range of size. furthermore, wb assay displayed a coherent protein profile for the exosome markers cd and cd . however, sec displayed low signal and the variability of between subjects was to higher. using the optimized method of precipitation, we observed that after normalization, mirna- a showed a significant increased (p = . ) in asthma patients compared to control. this mirna has been linked with an active proinflammatory status. summary/conclusion: our results confirm the presence of exosomes in induced sputum with promising applications in the field of asthma. the upregulation of exosomal mir- a, which is related with inflammation, suggest that exosomes could play a crucial role in the chronic inflammation of airway described in asthma patients. human nrf -active multipotent stromal cell exosomes reverse pathologic delay in the healing of cutaneous diabetic wounds introduction: multipotent stromal cells (mscs) have attracted much attention for their capacity to accelerate wound healing. exosomes, nanosized extracellular vesicles, may be key to translating msc therapy. we previously found that nuclear factor erythroid -related factor (nrf ) regulates msc promotion of diabetic tissue repair. here, we explore a novel role of nrf in exosome biogenesis and investigate whether exosome treatment recapitulates the effects mscs have on healing. methods: exosomes were harvested by differential ultracentrifugation of conditioned bone marrow derived msc media. for nrf -active exosomes, mscs were incubated with potent nrf activator, cddo-im. exosomes and mscs were vigorously characterized. full-thickness humanized-stented wounds were created on adult leprdb/db diabetic mice (db/db). exosomes were injected intradermally and circumferentially to the wound margin. results: mscs adopt an adherent fibroblast morphology, demonstrate robust osteogenic, chondrogenic, and adipogenic differentiation, express > % positive msc markers (cd , cd , cd , and cd ) and < % express negative markers (cd , cd , cd , cd , or hla-dr). immunoblotting of msc exosomes shows enrichment for positive exosomal markers cd , cd and tsg . nanoparticle tracking analysis (nta) shows a nanoparticle population with mean diameter of . ± . nm. transmission electron microscopy of exosomes reveals flattened cup-like spheres. nta demonstrates that nrf -active human mscs increase exosome secretion by %, compared to nrf -baseline mscs (p < . ). both nrf -baseline and nrf -active exosome treatment significantly reduced closure time to . and days respectively, compared to . days for vehicle-treated wounds (p < . ). this reduction eliminated the delay in closure time compared to wounds of c /b mice. nrf -active exosome treatment of db/db wounds reduced closure time by a further . days compared to untreated c /b wounds. at day , exosometreated db/db wounds have significant decreases in epithelial gap, expanded granulation tissue, and greater density of cd + vessels compared to vehicle-treated wounds. summary/conclusion: enhancing nrf function in mscs multiplies exosome yield. our results demonstrate exosome-based therapies hold tremendous promise and warrant further investigation for rapid translation. introduction: obesity increases prostate cancer aggressiveness and adipose tissue (at) is a rich source of extracellular vesicles (ev) that have been shown to contribute to pro-oncogenic effects in various malignancies. twist is a key mediator of tumour cell metastasis. the goal of this study was to determine molecular and phenotypic changes of prostate cancer cells in response to evs from obese human at and the role of different levels of endogenous twist . methods: ev were harvested from human at (atev) obtained from bariatric subjects or from at endothelial cells treated with proinflammatory cytokines (pic-ev) to mimic the obese at environment. evs were isolated by ultracentrifugation and characterized by electron microscopy, nta and protein markers. we determined the effect of atev and pic-ev on pc -ml prostate cancer cells proliferation and invasion. ev mirna cargo and transcriptome of pc -ml cells treated with atev or pic-ev were assessed using nanostring. to establish the contribution of twist to the ev-related phenotypic and molecular changes in recipient cells, we used pc -ml lines stably overexpressing or deficient in twist . results: atev from obese subjects and ev-pic from at endothelial cells both reduced invasion and increased proliferation in wild-type pc -ml cells. a molecular signature showing decreased expression of genes mediating invasion, adhesion and metabolism supported these functional effects. also atev and ev-pic shared a subset of mirna that target multiple mmps, inhibit glycolytic genes and target cell cycle inhibitory genes. pc -ml overexpressing twist showed an increase in both proliferation and invasiveness and this phenotype was supported by the transcriptomic analysis following ev treatment. summary/conclusion: ev produced by obese at or by at endothelial cells share a subset of mirna that in conjunction with increased twist expression contribute to tumorigenesis and metastasis of prostate cancer cells in vitro. introduction: exercise is associated with various health benefits, including the prevention and management of obesity and cardiometabolic risk factors. however, a strong heterogeneity in the adaptive response to exercise training exists. differential response to exercise training might be mediated by myokines (proteins, nucleic acids, metabolites) that can be released directly into the systemic circulation, or packaged within extracellular vesicles (evs). the objective of this study was to evaluate if changes in evs after acute aerobic exercise (ae) were associated with the responders phenotype following -week resistance exercise (re) training. methods: this is a secondary analysis of plasma samples from the exit trial (clinical trial # ). eleven sedentary obese youth ( . ± . years, bmi ≥ th percentile underwent an acute bout of ae ( % heart rate reserve, min). blood was collected before [time (at) − , min], during [at , , min], and after [ min (at ), min (at )] exercise. afterwards, youth participated in -week re programme, and were categorized into responders or non-responders (nr) based on changes in insulin sensitivity (above or below percentile). primary outcome: evs were isolated using size exclusion chromatography (izon®) at baseline (at ), immediately after ae (at ) and after recovery (at ). ev protein concentration, size, and zeta potential were analysed in a single-blind fashion. results: responders had larger evs (~ . nm) as opposed to nrs (~ . nm) at at (p < . ) and this pattern was maintained at at and at , though not significant (p = . ). nrs displayed differential ev size distribution (peaks at nm or nm), while ev distribution was highest at nm in responders. no difference in average zeta potential or total ev protein yield was observed between groups. an increase in ev yield with exercise time and recovery was observed in both groups. summary/conclusion: our preliminary data suggest that ev size is significantly increased after an acute bout of ae in obese youth responders. further research to delineate the role of evs as predictors of exercise adaptation is warranted. funding: funded by dream and research manitoba. using dual-fluorescent reporter mice to track tissue-specific extracellular vesicles andrea estrada, gabriella hehn, zackary valenti, christopher allen, nicole kruh-garcia and dan s. lark colorado state university, fort collins, usa introduction: extracellular vesicles (evs) from tissues like skeletal muscle (skm) and adipose tissue (at) have been implicated in human disease but are understudied. skm is likely a major player in ev biology as it accounts for~ % of total body mass. tools to define cellular ev origin are needed because tissues like skm are comprised of a variety of cell types. here, we describe our ongoing efforts using the dual fluorescent mg/mt mouse as a tool to analyse skm-myocyte derived evs. methods: wild-type (wt) and mg/mt mice were used for these studies. mg/mt mouse cells express membrane-tagged red (mt) or green (mg) fluorescent protein in the absence or presence of cre, respectively. we made skm myocyte mg expressing mice using a mouse expressing cre on the human skeletal actin promoter. blood was collected via cardiac puncture and platelet-free plasma was obtained via centrifugation. plasma evs were isolated using exoquick, exoquick-tc or size exclusion chromatography. skm and at were dissected into~ mm chunks, placed in serum-free dmem and incubated for hours. tissuederived evs were isolated using exoquick-tc. ev abundance was determined with a horiba viewsizer. individual evs were analysed with a cytek aurora spectral flow cytometer. settings were optimized using polystyrene beads and spectral unmixing was performed to allow detection of mg and mt. results: in wt mice, skm releases > times more evs than adipose tissue per unit of mass (p < . using paired student's t-test). since skm is also a major component of total body mass, these data further emphasize the importance of skm-derived evs. skmderived evs from wt mice were not fluorescent (< . % of events). evs from mg/mt mouse skm overwhelmingly expressed mg (> % of events) with negligible (< %) expression of mt. at-derived evs robustly expressed mt but lacked mg. summary/conclusion: these data provide "proof-ofprinciple" that mg and mt are readily incorporated into evs secreted ex vivo. surprisingly however, plasma evs from mg/mt mice expressed very little mg (~ %) or mt (~ %). this observation was confirmed with three separate isolation techniques. we are currently exploring possibilities to explain this finding, including: ) modification of evs post-secretion, ) clearance of fluorescent evs by the liver or ) that evs secreted from tissues remain predominantly in the interstitial space. funding: this work was supported by an innovative project award from the american heart association ( ipa ) to dsl. endothelial cd delivery of fa loaded extracellular vesicles is critical for thermogenesis. introduction: membrane cd facilitates tissue fatty acid (fa) uptake. we recently found that endothelial cell (ec) cd controls muscle and adipose tissue fa uptake, and influences the tissue's metabolic phenotype. the mechanism for cd -facilitated fa uptake is unknown. here we examined the role of ec cd in thermogenesis and in fa delivery to brown fat tissue. methods: adult male mice were housed individually, restricted from food during acute ( hr) cold exposure ( °c) with core temperature monitored every minutes. after hours, animals were sacrificed and samples collected for analysis. for cellular studies, human microvascular (lonza) or primary murine microvascular ec were used. for primary cells, crude cell pellets from lung homogenates were purified using mouse-cd magnetic beads (miltenyi). for microscopy studies, alkyne fa (cayman) was added to cells and to enable visualization of internalized fas, click chemistry (invitrogen) used to label alkyne-fa with alexa . for radioactive studies, primary lung ec were serum starved for hrs and incubated overnight with h-oleic acid bound to fa-free bsa ( : ratio). media was collected, clarified by centrifugation to remove microvesicles and debris. small extracellular vesicles (sevs) were isolated from clarified media using total exosome isolation reagent (invitrogen) and counted for radioactivity. results: basal core body temperatures are similar in mice lacking ec cd (eccd -/-) compared to controls (cd fl/fl). however, during cold exposure at °c , eccd -/-are unable to maintain body temperature (p < . ). plasma free fa are higher in cold exposed eccd -/-indicating fa clearance by brown fat is impaired. mitochondrial function and expression of thermogenic and mitochondrial genes in brown fat from eccd -/-and cd fl/fl mice were similar. these data suggested that endothelial delivery of fas is necessary for thermogenic maintenance of body temperature. to examine fa handling by ecs we used alkyne fas to visualize the process. we found that fas are transferred by microvascular ec through caveolae-mediated transcytosis involving src signalling and cav- phosphorylation. the internalized cav- and cd positive vesicles containing fas are released as sevs. to determine the dependence of cd on this process, we treated primary microvascular ec with radiolabeled fa and found that sevs secreted by cd -/-cells contain less labelled-fa (p = . ). summary/conclusion: endothelial delivery of fa is critical for thermogenesis. our working model for the mechanism of fa uptake by brown adipose tissue is the following: endothelial cells transfer the fa through caveolae-mediated transcytosis and secrete small extracellular vesicles (sevs) that help deliver fas to brown adipocytes. funding: this work is supported by nih grants dk and dk . introduction: diet-induced obesity modifies intestinal permeability leading to bacteria infiltration and to a decrease in the number of immune cells protecting mucosa. as orange consumption is beneficial for human health and used in preventive medicine, we determined whether orange juice-derived nanovesicles (onv) might be recommended as nutritional strategies for the treatment of intestinal complications associated with obesity. methods: onv isolated from fresh orange juices were characterized by lipidomic, metabolomic, microscopy, nta and for their stability during digestion. intestinal barrier (ib = caco- cells+ht- cells differentiated with oleic acid) were treated with onv and co-cultured with adipocytes to monitor ib fat absorption and release. obesity was induced in mice fed for weeks with a high-fat high-sucrose diet (hfhs mice vs standart chow diet mice). then half of the hfhs mice were gavaged with micrograms/day for weeks. results: onv did not modify high-fat high-sucrose diet-induced obesity and insulin resistance but reversed diet-induced gut modifications. six hours post-gavage, onv accumulated preferentially in jejunum involved in lipid absorption. in jejunum, and no other intestinal region, onv increased villi size, restored immune response and decreased barrier permeability in hfhsd mice. in addition, onv-treated mice had increased expressions of acat , angptl and dgat , but a decreased expression of fabp , fatp , mtp vs hfhsd animals, which indicated that fat absorption, tg synthesis and chylomicron release were strongly reduced. similarly to other plant-derived nanovesicles, these results were likely associated with onv lipid and metabolite compositions (strong enrichment in bioactive phospholipids: pe, pa, pc, pi and leucine) as onv did not resist to harsh digestive conditions in vitro and were poorly incorporated in enterocytes. as the effects of onv on the decrease in tg content and epithelial cell growth were also observed in vitro, gut microbiota unlikely participate to these effects. summary/conclusion: onv are important bioactive compounds of orange juice and for the first time we demonstrated that they can modulate lipid metabolism in the intestinal barrier associated with morphological changes. interestingly onv treatment targets mtp and angptl mrnas, therapeutic intestinal targets to reduce plasma lipids and for attenuating inflammation in gastrointestinal diseases. therefore, onv might be used to reduce the development of dyslipidemia-associated diseases and to restore intestinal functions in obese patients. funding: olga triballat institut; benjamin delessert institut, inrae institut. association, structure, and function of fibronectin in extracellular vesicles from hepatocytes xinlei li, ruju chen, sherri kemper and david brigstock nationwide children's hospital, columbus, usa introduction: we have shown that extracellular vesicles from normal hepatocytes have anti-fibrogenic activity and that they preferentially bind to hepatic stellate cells (hscs, the principal fibrosis-causing cell in the liver) and hepatocytes. in this study, our goal was to determine the molecular nature of the ev components involved in cell binding. fibronectin (fn ) is a key component of extracellular matrix, functioning in processes including cell adhesion, differentiation, and wound healing. two types of fn are present in vertebrates, of which the soluble plasma fn is derived principally from hepatocytes, while cell-associated fn is produced by numerous cell types. here we describe a novel function of plasma fn in facilitating binding of hepatocyte evs to target cells. methods: differential ultracentrifugation was used to collect evs released by parental mouse aml hepatocytes, fn ko aml cells in which fn was ablated using crispr-cas , primary human or mouse hepatocytes, or human hepg cells, or from human or mouse serum. evs were characterized by nanosight tracking analysis (nta), western blot, iodixanol gradient ultracentrifugation, and mass spectrometry. the binding efficiency of pkh -labelled evs from parental (ev-hep) or fn ko (ev-hepfn ko) aml cells was analysed in hepatocytes or hscs. swiss webster mice were injected with ccl for five weeks to induce liver fibrosis, with some mice also receiving i. p. administration of ev-hep or ev-hepfn ko over the last two weeks, followed by determination of hepatic fibrogenic genes by qrt-pcr. results: ev-hep or ev-hepfn ko were - nm in diameter and positive for common ev markers (cd , cd , flotillin- ). mass spectrometry showed that fn was the most abundant protein in ev-hep and comprised principally the plasma form. the abundant presence of ev fn was verified by western blot and co-immunoprecipitation with anti-cd or antiflotillin- . western blot showed that fn was also abundant in evs from primary human or mouse hepatocytes, hepg cells, and human or mouse serum. fn and ev-hep co-sedimented at a density of~ . g/ml. ev-hepfn ko yield and size-range were similar to those of ev-hep, suggesting that ev biogenesis is fn -independent. as compared to ev-hep, the binding of ev-hepfn ko to target cells was highly reduced whereas ev binding was independent of fn expression by the target cells themselves. both ev-hepfn ko and ev-hep were anti-fibrogenic in vivo but only ev-hep attenuated collagen ⍺ expression in mouse hscs in vitro. summary/conclusion: fn is abundantly associated with hepatocyte evs and facilitates ev binding to target hepatocytes or hscs. additional studies are needed to clarify the functional role of fn in mediating ev-hep anti-fibrogenic actions in vitro or in vivo. elevated glucose increases soluble and aggregated forms of human islet amyloid polypeptide in islet-derived extracellular vesicles -implications in type diabetes and islet transplantation introduction: type diabetes (t d) is characterized by reduced beta cell mass and function. islet amyloid, formed by aggregation of human islet amyloid polypeptide (hiapp), contributes to progressive beta cell loss in t d. amyloid also forms in human islets during pre-transplant culture and following transplantation in patients with type diabetes (t d) which is associated with graft failure. the cellular mechanisms underlying islet amyloid formation are still unclear. in this study, we examined the potential role of islet-derived extracellular vesicles (ev) in the clearance of soluble and aggregated (pro)iapp species from beta cells and amyloid formation. methods: human islets isolated from cadaveric pancreatic donors (n = donors) and wild-type or hiappexpressing (hiapp+) transgenic mouse islets (n = / group) were cultured in normal ( . mm) or elevated ( . mm) glucose to form amyloid. ev (exosomes) were isolated from culture medium using classical centrifugation and ultracentrifugation. purified ev were analysed by nanoparticle tracking analysis. western blot analysis and double immunogold transmission electron microscopy were performed to verify the presence of ev markers as well as (pro)hiapp species and oligomers (aggregates). results: human islets formed amyloid during culture with elevated glucose which was associated with progressive beta cell apoptosis. (pro)iapp species were detectable in ev released from human islets cultured in normal and elevated glucose. the latter markedly increased (pro)iapp content in islet-derived ev. interestingly, hiapp aggregates (oligomers) were present in the majority of ev released from human islets cultured in elevated glucose but were not detectable in islets cultured with normal glucose. similarly, ev released from hiapp+ mouse islets which formed amyloid during culture had higher (pro)iapp content compared to wild-type islet-derived ev. moreover, hiapp oligomers were present in ev derived from hiapp+ islets but not wt islets. summary/conclusion: in summary, our data show that (pro)iapp species are present in islet-derived ev and that elevated glucose increases (pro)hiapp and its aggregates in ev released from islets. islet-derived ev may play a key role in the process of amyloid formation in t d and human islet grafts. funding: university of manitoba research grants program (urgp). on. contraction, but not glycolysis, regulates the size of skeletal muscle evs secreted ex vivo. colorado state university, fort collins, usa introduction: skeletal muscle (skm) is a metabolically active tissue and accounts for~ % of total human body mass. acute exercise increases secretion of extracellular vesicles (evs), but the mechanisms responsible are unknown. muscle contraction increases the demand for atp which requires intercellular communication in order to adapt. we hypothesized that this "metabolic stress" during contraction increases skm ev secretion. methods: we tested our hypothesis using an ex vivo ev secretion assay. all studies were approved by the colorado state university institutional animal care and use committee. vastus medialis muscle (skm) from male c bl/ j mice (n = ) or female mt/mg mice (n = ) was cut into~ mg pieces and added to well plates (~ mg/well) filled with ml of serum-free dmem and placed in a cell culture incubator at c for hours. skm from male mice was treated with -deoxyglucose ( -dg) ( . nm - mm) to induce metabolic stress via inhibition of glycolysis. skm from female mice was treated with um of blebbistatin (bleb), a contraction inhibitor. after incubation, skm mass was measured and conditioned media was centrifuged ( , x g for min) to remove cell debris. evs were isolated using exoquick-tc. nta was performed on isolated evs using a horiba viewsizer . ev secretion was normalized to tissue mass and culture media volume then reported as ([particle]/ml/mg tissue). statistical comparisons for -dg experiments were made using a repeated measures -way anova. bleb experiments were analysed using a paired student's t-test. results: there was a trend towards greater ev abundance (p = . ) as a function of -dg treatment, but no effect on ev diameter (p = . ). bleb treatment did not alter ev abundance (p = . ), but significantly reduced ev mean diameter (p = . ; % decrease; dmso: . ± . vs. bleb: . ± . ). summary/conclusion: contrary to our hypothesis, inhibition of glycolysis with -dg did not stimulate skm ev secretion. however, bleb did appear to promote the release of small evs and/or inhibit secretion of larger evs. ongoing efforts are focused on testing other metabolic stressors and defining how blebbistatin promotes small ev secretion. funding: american heart association grant to dsl (ipa ). introduction: extracellular vesicles (evs, exosomes) are nanovesicles ( - nm) secreted from various types of cells. because of vesicular encapsulation of mirnas and enzymes, the evs play crucial roles in cell-to-cell communication by delivering these functional molecules to other cells [ ] . on the other hand, the evs are highly expected as next generation therapeutic tools due to pharmaceutical advantages such as controlled immunogenicity, effective usage of cell-tocell communication routes, artificial modification and encapsulation of functional molecules. however, cellular targeting and uptake efficacy of the evs are insufficient to be utilized as therapeutic tools [ , ] . in this study, we newly developed evs decorated with cellpenetrating sc or (sc ) peptides, which are derived from the c-terminal domain of the cationic antimicrobial protein, cap , because the peptides can be efficiently internalized by breast cancer cells. [ , ] . methods: all peptides were prepared by fmoc-solid phase synthesis. secreted evs from cd -gfp stably expressing hela cells were isolated by ultracentrifugation. cellular uptake of evs was analysed using a flow cytometer and a confocal laser microscope. encapsulation of saponin in the ev was conducted by electroporation. results: sc peptide is known as one of cell-penetrating peptides, and branched structure of sc peptides, (sc ) , further enhances the cellular uptake [ ] . in this research, we examined the effects of the peptide modification on cellular ev uptake, and modification of the sc or (sc ) peptides on ev membranes was conducted via stearyl moiety. as our results, increased macropinocytotic cellular uptake by modification of the peptides was successfully attained. especially, the modification of (sc ) peptides showed higher cellular uptake and macropinocytosis induction efficacy than that of sc peptides. in addition, anticancer protein, saporin toxin-encapsulated evs modified with the (sc ) peptides significantly enhanced their biological activity with dependency of glycosaminoglycan expression on targeted cells. summary/conclusion: the cell-penetrating (sc ) peptide-modified evs shows high abilities to be effectively internalized by cells and are applicable for intracellular delivery of therapeutic molecules. this study is expected to contribute to development of intracellular delivery techniques based on evs. [ introduction: rna therapeutics possess high potential which is yet to be realised, largely due to difficulties involved in delivery to the cytoplasm of target cells. extracellular vesicles (evs) possess numerous features that may help overcome this hurdle and have emerged as a promising rna delivery vehicle candidate. despite extensive research into the engineering of evs for rna delivery, little is known about how their intrinsic rna delivery efficiency compares to current synthetic rna delivery systems. using a novel crispr/cas based rna transfer fluorescent stoplight reporter system, we here compared the delivery efficiency of evs to state-of-the-art dlin-mc -dma lipid nanoparticles (lnps). methods: evs were isolated from mda-mb- cells expressing either a targeting or non-targeting control sgrna and applied to hek t stoplight+ reporter cells. lnps containing targeting sgrna were titrated onto hek t stoplight+ reporter cells to determine the minimum effective dose. lnp and ev particles were characterized using nanoparticle tracking analysis, dynamic light scattering and zeta potential analysis. sgrna copy number was determined using rt-qpcr. results: evs were ± nm in diameter as measured by dls and possessed a negative surface charge of − . ± . mv. rt-qpcr and nta analysis indicated that sgrna ev loading was low, with only in . e ± . e evs containing a single sgrna copy. nevertheless, evs containing targeting sgrna induced significant reporter activation while evs containing non-targeting sgrna did not. lnps were ± . nm in diameter and possessed a neutral charge. these particles also induced significant reporter activation when loaded with targeting sgrna. when delivered via evs, only between to sgrna copies per cell were required to induce statistically significant reporter activation. in contrast, the minimal effective sgrna dose when delivered by lnps was considerably higher at approximately e copies per cell. summary/conclusion: mda-mb- evs deliver rna in a highly efficient manner and are functional at sgrna concentrations several orders of magnitude lower than those required for lnp mediated delivery. this underlines the potential of evs as rna delivery vehicles and highlights the need to study the mechanisms by which evs achieve their efficiency in order for improved development of rna therapeutics. the role of circulating extracellular vesicles in patients with chronic chagas disease introduction: chagas disease is a neglected tropical disease (ntd) caused by the flagellated protozoan trypanosoma cruzi. it is a major public health problem in latin america, and it is now expanding over the globe through immigration of infected individuals. eukaryotic cells release extracellular vesicles (evs) that circulate in body fluids and have an important roles in intercellular communication, both in physiological and pathological conditions. objectives. our study proposes to characterize and to compare the circulating evs isolated from plasma of the chronic chagas disease (ccd) patients with healthy individuals (controls). methods: peripheral blood was collected from patients and controls in the presence of edta and evs enriched from plasma by differential ultracentrifugation. the obtained evs were characterized and quantified by nanoparticle tracking analysis (nta) and added to human thp- cells. after h, the cell supernatants were analysed by elisa for the presence of cytokines. results: lower amounts of evs were obtained from ccd patients in comparison with control individuals. however, the same amount of evs of ccd were more capable of inducing cytokines such as ifn-gamma and il- in relation to controls. summary/conclusion: although less evs are present in the blood of ccd, these evs induce high inflammatory reactions on macrophages suggesting a possible role of these evs in the establishment of chronic disease. funding: supported by fapesp, cnpq and capes. extracellular vesiclesa trojan horse for therapeutic agent delivery introduction: extracellular vesicles (evs) may prove to be one of the optimal payload carriers for therapeutic agents. while they travel through the extracellular space, the ev's lipid membrane layer shields their luminal cargo from deleterious external factors. when autologous evs are used to protect this therapeutic cargo, little immunogenic effects are expected compared to viral vectors and artificial structures, such as liposomes. their usage is potentially manifold, and they are ubiquitously present in all body tissues and fluids. the key is to develop a manageable ev loading agent for adoptive transfer therapies. methods: to exploit the unique properties of evs, highly positively charged proteins were used to load them with multiple biomolecules, such as a cas protein or dicer substrate dsrna as a functional payload and to improve their apparently inadequate natural ability to deposit cargo into the cytoplasm of recipient cells. results: highly positively charged proteins can associate with and/or diffuse through a phospholipid bilayer (thompson et al. ) . when these kinds of charged proteins are mixed with isolated evs in vitro, they are loaded into the evs. the positive charge of the protein has the advantage that it can associate with negatively charged agents, such as rna species, and aids the associated molecule to also incorporate into the ev. moreover, the positive charge of the protein helps with cargo delivery, and thus overcoming the bottleneck of the ev's cargo to escape the endosome post-uptake in a recipient cell. self-quenching fluorescent lipid dyes demonstrated that discharge of the highly positive ev cargo into the cytoplasm is concomitant with lipid mixing between the membrane of evs and the membrane of the recipient cell. when egfp-expressing microglia were exposed to evs loaded with a dicer substrate dsrna able to silence egfp via the positively charged protein, the uptake of dicer substrate dsrna was concomitant with a decrease in egfp expression in the microglia. a similar result was achieved when evs were loaded with cas protein conjugated to the highly positively charged protein. post-uptake of these cas -loaded evs, microglia expressing anti-egfp sgrna (single guide rna) lead to decreased egfp expression. summary/conclusion: our ev delivery technology has the capability of delivering multiple biomolecules, such as protein and rna cargo and demonstrates postuptake of the ev functionality of the ev delivered cargo in the recipient cell. hybrid extracellular vesiclesbiomimetic tool for drug delivery to repair endothelial cell dysfunction introduction: traditional drug delivery systems (dds) are usually based on liposomes, micelles or dendrimers. unfortunately, many dds cause side effects including organ toxicity and/or unexpected immune response. in living organisms, extracellular vesicles (evs) are responsible for delivering biologically active molecules to distant cells. in vitro loading of therapeutic compounds into evs is still not effective and needs developing new strategies. for these reasons we aimed to design hybrid extracellular vesicles (hevs) with high loading capacity for dds. methods: for hev synthesis, we used human endothelial derived evs. using freeze/thawing method we fused them with liposomes composed of cholesterol and one of the three lipids: dopc, sphingomyelin or phosphatidylserine. to confirm membrane fusion, we applied a spectroscopy ruler -fret (förster resonance energy transfer) and cryotem imaging technique. we characterized hevs using nta (for size distribution evaluation), dls (zeta potential) and western blot (for detection of evs markers). we evaluated loading efficiency using calcein as a model drug. additionally, we performed cytotoxicity tests. results: in the cryotem imaging, pure and homogenous hev population with a diameter of ± nm was detected. additionally, we observed changes in zeta potential and in size distribution after fusion. fret measurements showed increased fusion efficiency with the increasing number of freeze/thawing cycles and dependence on a lipid-to-protein ratio in evs. additionally, hev had higher loading efficiency than liposomes and sole evs and that their internalization by endothelial cells did not cause a cytotoxic effect. summary/conclusion: based on cryo-tem and fret, we confirmed that our fusion method of hybrid evs is effective and can be applied as a delivery platform for dds to endothelial cells. response to a range of stressors. the functional activity of these evs in recipient cells may, in part, be driven by changes to their biological cargoes. however, the molecular details of the underlying ev biogenesis and loading processes, and how this may vary in different conditions, is poorly understood. methods: we first studied the effect of oxidative stress on the functional activity of evs in recipient cells using cell viability and mitochondrial membrane potential assays in drosophila s r+ cells. we then carried out total rna sequencing of ev and cellular rna under three stress conditions and compared results to existing data in mouse cells. further to this we have used a bioinformatic pipeline to identify sequence motifs enriched in evs under stress. results: functional assays indicated changes to cell viability and mitochondrial membrane potential in recipient cells, which were donor cell-stress dependent. subsequent characterisation of rna showed an enrichment of ribosomal rna in evs relative to cells, but no significant changes to other biotypes. comparative analysis has also uncovered a set of genes enriched in evs under oxidative stress, and a further subset whose enrichment may be evolutionarily conserved in mouse. we also identified potential ev-loading motifs which may assist in rna loading specifically under stress. summary/conclusion: we have shown that evs derived from oxidatively stressed cells show dose-dependent differences in rna cargo and identified potential sequence motifs that may have a role in its loading. we are now validating the biological significance of these findings by combining different in vivo approaches in drosophila. this will enable us to gain insights into the basic mechanisms which govern ev loading in different contexts, and ultimately the molecular mechanisms underlying ev-mediated intercellular communication. ishai luz a , bibek bhatta a , kanaga sabapathy b and tomer cooks c a ben-gurion university of the negev, beer-sheva, israel; b national cancer centre, singapore, singapore, singapore; c ben-gurion university, beer sheva, israel introduction: mutations in tp are considered one of the most frequent genetic alterations in human cancer. besides the abrogation of the wild-type (wt) p -mediated tumour suppression, a distinct set of missense mutations was reported to endow mutant p proteins with novel activities termed gain-of-function (gof). even though mutations in tp are typically thought to arise in the tumour cells rather than in the stroma, the non-cell-autonomous effects of these mutants over the tumour microenvironment are poorly understood. in the presented studies, focusing on colon cancer as well as on lung cancer microbiome, we investigated intercellular interactions mediated by exosomes and outer membrane vesicles (omvs) in the context of cancers harbouring mutant p . methods: p results: in the colon, tumour cells harbouring mutp were found to exert a non-cell-autonomous effect over macrophages. when exposed to tumour cells harbouring mutp , monocytes became polarized towards a distinguished subset of macrophages characterized by tams-related markers. the mutant p affected tam were characterized as tnf-αlow/il- high, over expressing cd- and cd , with decreased phagocytic ability and increased invasion and matrix degradation potency. investigating the exosomal transfer from mutp tumour cells to macrophages, revealed a mutp -specific mirs signature led by mir- promoting the tam phenotype and creating an invasive front together with tumour cells. mir- was also found to be the top mutp -associated mir in a cohort of human colorectal resected tumours. separately, in two lung cancer cohorts, we identified a signature of microbiome members associated with p mutations. acidovorax temperans, a gram negative bacterium, was found to be abundant in tumours of patients with mutant p . we found a significant increase in tumour volume in animals inoculated with acidovorax temperans as compared to sham treated animals, and increased lung weight as a percent of total body weight. these preliminary data indicate that acidovorax temperans contributes to lung tumorigenesis in the presence of activated k-ras and mutant p . omvs shed by acidovorax temperans promoted inflammatory signalling in lung carcinoma cells and elevated cd expression on tumour cells and sirpα levels on macrophages. summary/conclusion: altogether, these findings are consistent with a microenvironmental role for specific "hot-spot" gof p mutants tightening the interaction between the tumour cell and the immune compartment in colon cancer. in both colon and lung cancer, mutant p facilitates cellular interactions within the tumour microenvironmets mediated by vesicles. funding: intramural funding from the national cancer institute, national institutes of health. lori zacharoff and mohamed el-naggar university of southern california, los angeles, usa introduction: the metal respiring bacterium s. oneidensis creates outer membrane extensions and outer membrane vesicles that are sculpted by the novel bar domain protein bdpa. these vesicles and extensions incorporate mutliheme cytochromes involved in extracellular electron transfer to metals and electrodes. however, the physiological relevance of incorporating these cytochromes into the higher order d architecture of a vesicle or extension is unknown. given that bar domains serve as a protein sorting mechanism in eukaryotes, we investigated the pathway crosstalk between bdpa and outer membrane multiheme cytochromes as means to understand the physiological significance of membrane architecture. methods: o this end, vesicle morphology and content was measured using dry weights, dynamic light scattering, fluorescence microscopy and comparative proteomics from wild type s. oneidensis and deletion strains. results: cells lacking bdpa make large amorphous vesicles that are dense with protein. in contrast, a strain lacking outer membrane cytochromes recruits less total protein into smaller vesicles. proteomics to show that both bdpa and multiheme cytochromes are involved in recruiting other proteins to outer membrane vesicles and have a reciprocal relationship. summary/conclusion: in the absence of bdpa, protein crowding has to become the main driving force of vesiculation and bdpa is essential for efficient incorporation of cytochromes. however, multiheme cytochromes are not only vesicular cargo, but are also important for shaping and loading vesicles. both of these situations make it clear that vesicles play a role in increasing the respiratory surface area of s. oneidensis cells. moving forward, we hope to be able to control bdpa and cytochrome levels for selective recruitment of technologically relevant payloads. introduction: fascioliasis caused by fasciola hepatica represents a major economic loss and clinical burden in cattle farming worldwide. extracellular vesicles (evs) contain pathogen-derived molecules that represent novel biomarkers of disease. in the present study, we have identified potential new biomarkers of f. hepatica infection in evs present in sera of infected cattle. methods: parasites and sera were obtained from local abattoirs (valencia, spain, and medellin, colombia, respectively). sera from infected and from healthy animals. parasites were cultured, and evs obtained by sizeexclusion chromatography (sec) and characterized by nta, tem and proteomic profiling. recombinant proteins from f. hepatica evs (enolase and fh . tegumentary protein) were produced, and coupled to magnetic beads. measurement of bovine igg antibodies was performed using luminex bead array technology. results: a total of proteins were identified associated with evs as shown by the presence of typical ev-markers (tsg , alix, cd ). two parasite proteins, enolase and the fh . tegumentary protein were produced as recombinant proteins and used for detection of cattle igg employing luminex bead array technology. interestingly, significant differences were found in the fluorescence values of both recombinant proteins allowing discrimination between sera from infected and non-infected cattle. the use of the fh . protein generated a highly significant difference between the two groups (p value = . ); as did enolase (p value was . ). summary/conclusion: this study demonstrates the usefulness of ev proteins as new biomarkers for early diagnosis of helminth infections using multiplex assays, a technology that may also be applied to other parasite ev molecules. life stage-specific glycosylation of schistosome-derived extracellular vesicles introduction: glycans play an essential role in pathogen-host interactions. larvae and adult worms from schistosoma mansoni release distinct subsets of glycoconjugates as excretory/secretory (es) products. extracellular vesicles (evs) are also among the es products. we recently found that schistosomuladerived evs are glycosylated and bind human dendritic cells via c-type lectin receptor (clr) dc-sign, leading to increased il- and il- release. here we investigated the glycosylation profile of evs released by s. mansoni adult worms, compared this to schistosomula evs, and addressed how this may affect parasite-host interactions via clrs. methods: evs from cultured s. mansoni parasites were obtained by ultracentrifugation and purified with iodixanol density gradients. isolated evs were analysed by nta and cryo em. n-glycan and lipid glycan content was determined by mass spectrometry. density gradient fractions with evs were loaded onto sds-page gels followed by western blot (wb) analysis using anti-glycan monoclonal antibodies (mabs). results: cryo em showed that adult worm evs lacked the long thin filaments that are characteristic for schistosomula evs. additionally, in contrast to schistosomula evs, glycolipids could not be detected in the adult worm evs. mass spectrometry analysis showed that the most abundant n-glycans in the adult worm evs contained galnacβ - glcnac (lacdinac, ldn) motifs, which correspond to previously published overall glycan profiles of this specific life stage. other differences in ev glycosylation between the two life stages were observed by wb using anti-glycan mabs: adult worm evs showed a paucimannosidic glycan motif whereas in the schistosomula evs galβ - (fucα - ) glcnac (lewis x) was detected in line with previous ms analysis. introduction: phloem plays a central role in plant function, as it is the responsible for the translocation of photoassimilates from source-to sink-organs, and a long-distance route for signals distribution. due to the sap high nutrient content, sieve elements are primary target for plant pathogens and pests. in this work we aimed to isolate and characterize extracellular vesicles (evs) from cucumis melo phloem sap, derived from plant either exposed or not to the melon aphid, aphis gossypii (hemiptera: aphididae). methods: phloem exudates from -week-old melon plants, either uninfested or infested with adults of a. gossypii (n = , replicates each), were collected by cutting the stem with a sterile razor blade between first and second expanded leaf from the top. evs were isolated by size exclusion chromatography, and analysed by nanoparticle tracking analysis (nta) and transmission electron microscopy. evs proteome was determined by quantitative mass spectrometry. results: evs from phloem sap were successfully isolated in every condition. no significant differences were detected among distinct samples, neither in particle concentration and size by nta, nor in protein concentration. most importantly, a total of different proteins were identified in phloem sap evs, including present in exosome databases (exocarta). on top of that, differentially expressed proteins were identified in evs derived from aphid infested or uninfested plants (p value < . ). summary/conclusion: understanding how plants trigger their defences against pests and pathogens is important to develop new control measures. the characterization of several proteins in evs from the phloem sap provide valuable information on long distance signalling in plants. moreover, as plants lack an immune system comparable to animals, the different protein content in phloem sap evs after exposure to aphids could indicate their important role in delivering inducible defences against invading pests and pathogens. extracellular vesicles from nematode species heligmosomoides bakeri and trichuris muris contain distinct small rnas that could enable niche specificity in the host introduction: gastrointestinal nematodes are extremely prevalent parasites that infect most animals and % of human population. their success as parasites is attributed to their ability to secrete diverse molecules that modulate the host immune system. extracellular vesicles (evs) are one of the immune modulatory compounds they release that directly modulate host cells. our goal is to understand how the small rna (srna) cargo underpins ev function, using a comparative analysis of ev cargo from diverse nematode species. methods: we first compared how different ev isolation methodologies (ultracentrifuge (uc), size fractionation, sucrose gradient floatation) effect the small rnas detected in h. bakeri evs using different library preparation kits (cleantag, truseq), with or without polyphosphatase treatment. we then compared this to small rna libraries from t. muris evs using comparable methods, uc ev purification, with or without polyphosphatase treatment and using the cleantag library preparation kit. results: evs from both species contained mirnas, however the mirna gene familes in h. bakeri and t. muris evs are distinct. the mirna content detected in ev samples collected by different purification protocols is robust. the largest difference in detected mirnas was found when comparing different library preparation kits. although both h. bakeri evs and t. muris evs were dominated by srnas derived from intergenic or repetitive elements in the parasite genomes, only in h. bakeri evs were these secondary sirnas. summary/conclusion: h. bakeri and t. muris evs contain distinct small rna cargos, which may underpin their ability to colonise different host niches, and/or modulate the host immune system differently. t. muris evs do not contain secondary sirnas, in contrast to h. bakeri, however they are dominated by srnas derived from intergenic or repetitive regions. comparative analysis of helminth evs could help pinpoint the srnas involved in cross-species communication. please provide any keywords if applicable.: nematode, cross-species communication, small rna introduction: extracellular vesicles (evs) are secreted from various cells including cancer cells and known to contain protein and small rnas including mirna isoforms (isomirs). therefore we also focused on isomirs including other small non-coding rnas for biomarker discovery. although liquid biopsies using small rnas are promising biomarkers for early detection of cancer, current approaches to detecting and analysing mirnas in the blood are still inadequate. artificial intelligence (ai) data analysis may provide better algorisms for diagnosing cancer. methods: small rnas were isolated from serum or purified evs using a mirneasy mini kit (qiagen) and quantified by using the ion s ™ next-generation sequencing system. (thermo fisher scientific). evs were purified using total exosome isolation reagent (invitrogen™). ai data analysis was performed using jmp® genomics and datarobot enterprise ai platform. results: three small rnas, isomir of mir- - p, mir- a- p, and trf-lys (ttt) were significantly upregulated in breast cancer patients compared with the healthy cancer-free individual. the combination algorithm using these three small rnas allows for a more accurate diagnosis of the area under the curve (auc) . . to test the possibilities that these small rnas are derived from cancer cells, we isolated evs from the serum and performed ngs analysis to profiled serum small rnas in evs. interestingly we found that two small rnas, mir- - p and mir- a- p, also high in breast cancer evs, indicating that these small rnas were expected to be derived from cancer cells. in oesophagus cancer, we also performed ngs analysis and identified twenty-four mir/isomirs candidates for diagnostic biomarkers. a multiple regression model selected mir- a- p and two isomirs (mir- - p and mir- - p) . the auc of the panel index was . . we also performed ai data analysis and discovered the novel algorisms that can diagnose breast and oesophagus cancer more accurately. summary/conclusion: we demonstrated combinations of circulating non-coding rnas containing evs potentially useful for the detection of early-stage breast and oesophagus cancers. in addition, the datarobot enterprise ai platform enables us to the more accurate diagnosis of cancers at the early stage. identification of novel ev-associated mirnas as toxic biomarkers in mouse introduction: recent findings reveal that extracellular vesicles (evs), secreted from cells, are circulating in the blood. evs are classified into exosomes ( - nm), microvesicles ( - , nm) and apoptotic bodies ( - , nm). evs contain mrnas, micrornas, and dnas and have the ability to transfer them from cell to cell. recently, especially in humans, the diagnostic accuracy of tumour cell type-specific evs as biomarkers is more than %. in addition, micrornas contained in the evs are being identified as specific biomarkers in blood for chemical-induced inflammation and organ damage. therefore, micrornas contained in the evs released into the blood from tissues and organs in response to adverse events such as chemical substances and medicine are expected to be useful as novel biomarkers for toxicity assessment. in this study, we aimed to identify target organs by comprehensive analysis of ev rnas in the blood of mice after chemical exposure to establish a highly sensitive "next generation type" toxicity test for chemical substances and medicine using ev rna in blood as a biomarker. methods: all animal studies were conducted in accordance with the helsinki declaration and the guidelines approved by the animal care committee of the national institute of health sciences. c bl/ j male mice ( weeks) were orally dosed with ccl (vehicle, , mg/kg). serum were separated from blood after , , and hours after ccl administration. the serum was centrifuged at , x g to remove cellular debris and subsequently ultracentrifuged , x g. the pellet is resuspended in pbs and ultracentrifuged , x g again. the comprehensive small rna-seq of collected evs were performed according to the manufacture's protocols. results: we succeeded in isolating more than novel small rnas, which could be used as novel highly sensitive biomarkers for hepatotoxicity due to carbon tetrachloride (ccl : mg/kg & mg/ kg). well known hepatotoxicity biomarkers, mirna- and mirna- were upregulated more than -fold in the administration of mg/kg ccl , but not responded in the administration of mg/kg ccl . summary/conclusion: these results suggest that mir- and mir- are mainly released from liver to blood directly only in the administration of mg/kg ccl , while novel more sensitive hepatotoxicity biomarkers which responded in the administration of both mg/kg and mg/kg ccl should be included in the ev. our novel biomarkers will accelerate a rapid evaluation of chemical substances and medicine in nonclinical safety evaluation. introduction: advancements in sequencing technologies have allowed analysis of the genomic landscape of cancer using circulating cell-free(cf) dna. however, cfdna does not originate only from tumour cells. we recently demonstrated that most of the dna circulating in plasma of cancer patients is associated with large evs (l-evs), and that l-ev-associated dna reflects genomic aberrations of the cells from which l-evs arise. since l-evs are specifically released by tumour cells, we explore their potential to report cancer-specific genomic alterations in patient plasma and compare it to cfdna. methods: differential ultracentrifugation, tunable resistive pulse sensing, qubit dsdna high sensitivity assay, capillary electrophoresis, whole exome sequencing ( - x), targeted sequencing (qiaseqtm), flow cytometry. results: we show here that l-evs in the size range of > micrometre are present exclusively in plasma obtained from cancer patients and absent in plasma from healthy donors. in agreement with this finding, double-stranded(ds) dna is detected only in l-ev fractions of patient plasma and not in those obtained from healthy donor plasma using the same protocol. we also demonstrate that the fragments of dsdna associated with circulating l-ev are larger in comparison with cfdna (> , bp versus~ bp). a large-scale analysis of l-ev dna obtained from plasma of patients with metastatic castration-resistant prostate cancer (mcrpc) as well as with non-small cell lung cancer (nsclc) demonstrates that dna associated with circulating l-evs reports cancer-specific genomic alterations in both types of cancer. we further investigate if l-evassociated dna is intra-or extravesicular and demonstrate that it is present in both forms. we finally compare the purity of the tumour signal in intravesicular l-ev dna, total l-ev dna, and cfdna obtained from patient plasma. summary/conclusion: our results demonstrate that circulating l-evs contain high quality, large molecular weight dna that contains cancer-specific genomic alterations, supporting the use of l-evs as a source of tumour-derived dna in plasma. introduction: epidermal growth factor receptor (egfr) mutation driven lung adenocarcinoma (ac) represents a unique subgroup that lends itself to treatment with oral egfr tyrosine kinase inhibitors. current methods that are used to detect these mutations (e.g. l r or the resistance mutation t m) involve invasive tumour biopsies or blood circulating tumour dna (ctdna) and cell free dna (cfdna). the sensitivity of blood ctdna and cfdna is limited by the frequency of genomic alterations in the egfr gene; additionally, ctdna does not reflect changes in the egfr protein, against which novel therapies are in development. there remains a need to develop bloodbased biomarkers that can circumvent these disadvantages and replace the more standard, invasive tumour biopsies. we propose the study of exosomes for treatment monitoring as well as to identify egfr resistance related genomic and proteomic changes. methods: we enrolled patients with metastatic lung ac: with egfr mutations and without (control). from the patients with egfr mutant lung ac, we processed blood samples through the patients' treatment course, using ultracentrifugation to isolate exosomes. we then used both droplet digital pcr (ddpcr) to test exosomal rna (exorna) for the mutation of interest and western blots to test protein resulting from exon deletion or l r mutations. results: from patients with egfr exon deletion mutations, we detected identical mutations in exorna from / samples. exorna based mutational load increased and mirrored clinical progression in patients. three patients whose cancer remained stable demonstrated a decrease in their exorna. one patient had blood drawn only at points and was therefore not plotted. exorna from patients with l r and t m mutations demonstrated the corresponding mutations; however, exorna did not mirror their disease course. we also demonstrated mutant egfr protein presence in exosomes from patients. finally, we tested cfdna for egfr mutations from four matched samples using ddpcr. we detected matched mutations in exosomes in all four, while cfdna mutations were only detected in / patients. summary/conclusion: in summary, we detected egfr mutations in / exosome samples isolated from metastatic lung ac. our results set the stage for optimization of exorna methods and inform future experiments relating to exosomal cargo in patients with egfr mutant lung ac. identification of plasma-derived, ev-based biomarkers for glioblastoma introduction: glioblastoma multiforme (gbm) is the most malignant and aggressive primary brain cancer in adults, with an incidence of . per , people. currently, diagnosis is only performed via histopathological investigation of a tissue sample from a gbm lesion, complemented with molecular diagnostics for identification of select biomarkers. mri is the standard of care for follow-up and monitoring of treatment response. therefore, development of a "liquid biopsy" to obtain disease-relevant information from patient's body fluids is highly desirable. methods: we present the results from a clinical study in which extracellular vesicle (ev)-derived mrnas and long non-coding rnas were profiled from the plasma of gbm patients and control individuals. we obtained plasma from patients at the time of initial diagnosis, and matched controls by sex and age. ev-associated rna was isolated from - ml plasma and rna-seq was performed using our proprietary pipeline. sequencing data was analysed for differential gene expression. results: we observed mrnas as differentially abundant between gbm and control samples, with mrnas enriched in gbm samples and mrnas enriched in control samples (p < . ). correlation based on differentially abundant mrnas separated gbm and control samples into two unique populations. eight differentially expressed mrnas were previously identified as part of the mesenchymal gbm subtype. these data, while preliminary, provide a potential basis for the further development of a noninvasive gbm gene panel test. summary/conclusion: we have identified a novel rna signature for gbm from plasma derived evs, which differs from previously identified biomarkers isolated from tissue. further work will refine this signature to enable detection, characterization, and patient monitoring for gbm with minimally invasive techniques. introduction: sjogren's syndrome (ss) is a systemic autoimmune disease in which inflammation progressively damages the moisture producing glands of the afflicted. million americans are estimated to be suffering from the disease, % of which are women with an average age of . overlapping symptoms with other health conditions and co-morbidities make ss particularly difficult to diagnose, with average time to diagnosis of years. saliva exosomal rna profiling has been primarily focused on small rnas and has been limited thus far due to the large contribution of sequencing reads from the oral microbiome. a noninvasive saliva exosomal rna (exorna) based test capable of diagnosis would be highly desirable. methods: we began by first developing a novel long rna-seq workflow to selectively enrich and profile human exosomal mrnas and long non-coding rnas (lncrnas) from saliva. we then profiled salivary exorna obtained from ss patients and healthy matched controls. finally, we performed differential gene expression analysis to obtain an exorna signature for ss. results: rna-seq data analysis demonstrated highly efficient enrichment of human transcriptome, with over % of reads mapping to the transcriptome. further rna biotype analysis showed over % of transcriptome reads mapped to protein coding genes and lncrnas. we detected over , mrnas and approx. lncrnas. differential expression analysis (dex) of ss vs. healthy control exorna identified upregulated genes, including mrnas and lncrnas (p < . ). genes were found to be downregulated in ss, including mrnas and lncrnas. gene ontology analysis of dex genes revealed enrichment of genes involved in various immune system related pathways. most importantly, principal component analysis (pca) resulted in clear separation of ss patients from healthy controls. summary/conclusion: our optimized rna-seq workflow enables saliva-based liquid biopsy for biomarker discovery. the gene signature identified in this ongoing study could potentially provide a non-invasive molecular means of diagnosing sjogren's syndrome. introduction: increasing embryo implantation rates has become one of the greatest challenges in assisted reproduction techniques. usually an endometrial biopsy is done to identify a receptive endometrium, which prevents embryo transfer in the same cycle, as it is detrimental for the implantation. the implantation is a complex process, which requires a synchrony between the development of the embryo and the endometrium, but also, an adequate embryo-endometrial cross talk. the presence of extracellular vesicles (evs) as mediators of this communication has been describe in the endometrial fluid. therefore, we hypothesize that the molecular analysis of the content of the evs and companion molecules from endometrial fluid could be a non-invasive method to recognize an implantative endometrium and consequently improve the implantation rates. methods: the objective is to define a simple, sensitive and reproducible non-invasive ev-based method that allow the quick identification of an implantative endometrium by means of mirna analysis. for the establishment of a robust methodology for analysing evs from endometrial fluid in clinical settings, where the sample is limited and no sophisticated equipment is available, five different methodologies were compared in triplicate. two of them consisted in the direct extraction of rna while in the other three, before the rna extraction an enrichment of evs was done. smallrnaseq was performed to determine the most efficient method. once the best method was selected, it was applied in a set of real samples with different implantation outcome. the content of mirnas (mainly associated with evs) of endometrial fluid samples from women in whom the implantation was successful (n = ) and unsuccessful (n = ) were analysed. results: our results show that the protocols with a previous enrichment step of evs obtained a higher mirna expression. the results obtained from the differential analysis of the set of samples with different implantation outcome are being analysed and it is expected that the results will be available by the time this communication is presented. summary/conclusion: this work demonstrates that it is possible to obtain and analyse evs and evs-associated mirnas from a small volume of endometrial fluid samples, which allows the use of ev-mirnas as a low-invasive biomarkers for the detection of an implantative endometrium. funding: jip is supported by a predoctoral grant from the basque government. small rna cargo of evs is affected by hormone treatment in prostate cancer introduction: small rnas are recently reported as a regulator for prostate cancer progression to castration-resistant disease. our previous work has shown that evs protein cargo is affected by male steroid hormone, dihydrotestosterone (dht). in this study, we assess the small rna cargo of evs in response to androgen manipulations. methods: androgen receptor-positive lncaps are grown in css medium to deplete the androgens. media were then replaced with vesicle-depleted css medium ± nm dihydrotestosterone (dht) ± µm enzalutamide (enz) for h. evs were isolated using sequential ultracentrifugation ( g for min, , g for min, , g for h), washed once in pbs. protein and rna were collected from both parent cells and conditioned medium to allow direct comparison between s-evs cargo and cells. small rna ngs libraries were prepared using the illumina's truseq small rna library prep kit and single-end sequenced at a read length of nucleotides (nt). fastq library files were processed using a custom-designed pipeline. adapters were removed using the cutadapt tool, trimmed reads were mapped with high stringency against ribosomal sequences using bowtie . snorna and trna fragments were identified using the flaimapper software. remaining reads were mapped against the human genome hg using bowtie . results: we found that the presence or absence of androgens does not significantly change the amount of total rna in small evs (s-evs). however, hormone stimulation altered the small rna content of s-evs, in parallel with our previous published data on ev protein cargo. dht increased the abundance of snorna in cells, while a reduction of snornas was observed in the s-evs fraction. interestingly, dht induced the formation of cell filopodia that are not inhibited by androgen inhibitor enzalutamide. pathway analysis indicates the p mediated regulation driven by mirnas found in s-evs upon exposure to dht. the expression profile of snorna and trna fragments in dht treated cells resembles results from clinical prostate cancer specimens. summary/conclusion: our findings show that androgen manipulation alters both s-ev derived protein and rna cargo. changes in the s-ev rna profile due to treatment with androgens are not identical to small rna profiles in parental cells, indicating a specific sorting mechanism of s-ev small rna upon androgen manipulation. further, dht induces the formation of cell filopodia irrespective of enzalutamide, suggesting cargo selection of s-evs. we conclude that small rna ev cargo can be utilised to as prostate cancer biomarkers in androgen targeted treatments. introduction: cancer immunotherapy, such as pd-l blockade, is a method to eliminate cancer cells. ectopic expression of pd-l , on the surface of tumour cells, has been associated with tumour persistence and as an important predictor of therapy response. a test that, specifically and accurately, detects pd-l is critically important in order to identify patients that would benefit from these treatments. emerging evidence has shown that extracellular vesicles (ev) can carry immune checkpoint molecules, such as pd-l , and whose expression have been correlated with tumour immunity response. with a multitude of commercially available antibodies identifying appropriate clones and associated assay is important in order to standardize the diagnostic modality used. methods: pd-l expressing cancer cell lines were used to generate evs. pd-l -myc vector was transfected to generate an overexpression system. exoview® sensors containing different anti-pd-l clones were generated. samples (cell derived and plasma) were incubated on chips to allow the antibody to bind the antigen on the ev. after incubation, chips were immunolabeled with fluorescently labelled antibodies against pdl- or ev associated markers. exoview r reader was used to enumerate the evs captured on the sensor surface and analyse the expression of pdl- on single vesicle through fluorescence imaging. immunoprecipitation and mass spectrometry (ip/ms) were employed as an orthogonal method to verify the specificity of the assay. results: to study the detection efficiency of the antibodies, engineered pd-l -myc evs were used. under these circumstances, all the tested antibodies were able to capture evs. when testing endogenous pd-l positive evs from different cancer cell lines, only . and clones consistently bound to evs. in addition, evs derived from plasma demonstrated to be positive for pd-l , however, only clone . was able to immobilize these evs. the results suggested that clone . could be a potential pd-l antibody to detect pd-l positive evs originating from various sources. to confirm these results, and assure the specificity of the antibody targeted ip/ms was employed. summary/conclusion: in combination with the exoview platform, anti-pd-l antibodies can be screened and potentially used to generate a non-invasive ev-specific assay that could detect this protein in patients. differences in extracellular vesicle protein cargo is dependent on head and neck squamous cell carcinoma cell of origin university of michigan, ann arbour, usa introduction: head and neck squamous cell carcinoma (hnscc) is the sixth most common, eighth most fatal cancer worldwide and includes cancers of the oropharynx, larynx, hypopharynx, and oral cavity. in , there were over , new cases and , deaths estimated in the usa alone. despite recent advances in treatment, including radiation, chemotherapy, surgery, concurrent chemoradiation, and immunotherapy, many tumours develop resistance and progress. patients develop metastases or tumours recur locally or regionally; the -year overall survival rate for hnscc is only - %. factors that contribute to poor survival for patients with hnscc include late stage diagnosis, lack of reliable markers for early stage detection, high level of biologic heterogeneity, and local recurrence and distant metastases after treatment. methods: this study used representative hpv-positive and hpv-negative hnscc cell lines, one hpvtransformed cell line. and two non-cancer oral keratinocyte cell lines. evs were isolated using differential ultracentrifugation and peg precipitation/ultracentrifugation. evs were characterized by tem, nta, and wes protein analysis for reported ev markers. ev and whole cell lysates were assessed by lc-ms/ms analysis using the tandem mass tag- plex kit. cluster analysis was performed on the fold-change peptide spectrum matches (psm) for the evs from the hnscc lines compared to the evs from the normal keratinocyte line (noksi). protein was measured using a capillarybased electrophoresis instrument. results: cd and annexinv were detected in all of the ev lysates tested, while calnexin was detected in all of the whole cell lysates and none of the ev samples tested. selected proteins stat , hla-a, tenascin, e-cadherin, β catenin, cytokeratin , epha , and cd , and hpv-related markers p , p , rb, cyclin d , and egfr were tested using the wes platform. evs from hpv-positive cell lines showed higher protein levels compared to evs from hpv-negative cell lines in stat , hla-a, and tenascin. only kert demonstrated lower protein levels in evs from hpvnegative cell lines. of the common hpv-associated hnscc markers: egfr, p , rb, cyclin d and p , only egfr was positive in any the evs tested. the remaining proteins queried, e-cadherin, β catenin, epha and cd showed varying protein levels in evs from both hpv positive and hpv-negative cell lines. summary/conclusion: our findings suggest that these proteins may be potential hnscc ev markers that may be ) selectively included in ev cargo for export from the cell as a strategy for metastasis, tumour cell survival, or modification of tumour microenvironment, or ) representative of originating cell composition, which may be developed for diagnostic or prognostic use in clinical liquid biopsy applications. validation of antibodies on western blot for extracellular vesicles from biological human samples and cancer cell conditioned media the brady urological institute, johns hopkins university school of medicine, baltimore, usa introduction: one of the major challenges in extracellular vesicles (evs) research is to prove the particles that are isolated are true evs, rather than other co-isolated contaminants, like lipoproteins. isev recommends using multiple assays to characterize evs. this study aims to validate the positive and negative protein markers for extracellular vesicles from plasma, urine and prostate cancer cell conditioned media (ccm). methods: membrane and cytosolic fractions of mcf cells served as positive and negative controls for all antibodies validated. evs were isolated from plasma of healthy volunteers, urine of healthy volunteers and ccm of pc- cells using differential ultracentrifugation. eight protein markers were assessed: positive markers cd , cd , cd , flotillin (flot ), alix and tumour susceptibility gene (tsg ), negative marker calnexin (canx), and contaminant markers apo-a for plasma and thp for urine. tetraspanins are small transmembrane proteins expressed in evs. flot is membrane protein that forms microdomains in the plasma. alix and tsg , an accessory protein of the endosomal sorting complex required for transport, are involved in the biogenesis of evs. they are positive markers for evs. canx is in the membrane of the endoplasmic reticulum. apolipoprotein-a (apo-a ) is the protein components of lipoproteins, therefore it is marker of contamination for plasma ev. tamm-horsfall protein (thp) is contamination marker for urine ev, because it is most abundant protein in human urine. results: all antibodies were validated in the correct positive and negative control, thus confirmed as usable and reliable antibodies for western blot. in plasma ev, cd , cd , cd and flot were positive and canx and apo-a were negative. in urine evs, cd , cd , flot- , alix and tsg were positive and canx and thp were negative. in ccm evs, cd , cd , flot , alix and tsg were positive and canx was negative. summary/conclusion: we confirmed a high degree of ev purity from sample types: urine, plasma, and ccm. of particular importance, we confirmed that evs isolated from biologic patient samples, plasma and urine, had low contamination. future work will use these methods to confirm purity of ev samples prior to addition analysis, such as examining ev cargo and biologic significance. proteomic study of mesenchymal stem cells derived exosomes modified using mir. introduction: the project we are working on is to modify the immunogenic profile of human cmms from the umbilical cord stroma through its stable transfection with anti-mir- - p, and therefore of the exosomes that these cells generate, for use in free-cell therapy to treat inflammatory process. methods: evs released from a primary culture of human umbilical cord mesenchymal stem cells and from primary culture of human umbilical cord mesenchymal stem cells mir -/-modified through stable lentiviral transfection were isolated by ultracentrifugation processes, characterized by transmission electron microscopy (tem) and measured by nanoparticles tracking analysis (nta). protein extraction from evs was made using ripa buffer and after checking protein integrity the total ev proteins. we performed a shotgun proteomic study using a tmt ( -plex) label of the total mir -/-exosomes protein comparing it with normal exosomes. after labelling the ltq-orbitrap platform of proteored was needed for fraction injections and data acquisition. proteome discoverer . (thermo) was used for protein processing and quantification. results: a total of . proteins were identified at least with a unique peptide and we have able to establish the proteomic profile of mir -/-exosomes against normal exosomes. we found out several protein modulated by mir and related to inflammation. summary/conclusion: we have able to establish the proteomic profile of mir -/-exosomes against normal exosomes focusing on proteins involving inflammation process. all those results seem indicate that exosomes could be modified, which could be used as an anti-inflammatory free-cell therapy. funding: proteored concept test project grant. a novel extracellular vesicle isolation method used to discover urine liver disease biomarkers introduction: hepatocellular carcinoma (hcc) is the th most common cancer worldwide and the rd most common cause of cancer death; additionally, its incidence is increasing. while outcomes for early hcc are superior to those for late stage disease, early detection of hcc remains a challenge. current guidelines have suboptimal sensitivity and specificity. in this pilot study, we hypothesize that urine extracellular vesicles (evs) may identify candidate biomarkers towards the development of an inexpensive, widely accessible screening assay for the early detection of hcc. methods: urine samples from healthy subjects, subjects with cirrhosis, and subjects with cirrhosis plus hcc were collected and processed using ymatrix columns to isolate ev-associated protein and mirna. protein was analysed using a tandem mass tag method on a thermo scientific orbitrap fusion mass spectrometer with comet/paws and edger processing. mirna was analysed using a targeted firefly microarray from abcam. differential expression and predictive modelling for the presence of hcc and cirrhosis was performed to identify candidate mirna and protein biomarkers. results: for mirna, samples were eligible for analysis after low expression filtering. we used pair-wise ratios of cancer-associated mirnas by gradient boosting of decision trees to develop a predictive model for hcc. our best model had a sensitivity and specificity of . and . respectively using mirnas to distinguish hcc from cirrhosis. all samples were eligible for protein analysis. based on differential expression and biologic relevance, we identified protein candidate biomarkers. interestingly, we found liver-selective proteins and known hcc/cirrhosis plasma/tissue markers, demonstrating proof-of-concept for the method. summary/conclusion: urine extracellular vesicles contain liver-selective proteins and known liver disease serum biomarkers as well as novel mirna and protein biomarkers that are significantly up-regulated in disease samples. the described candidate biomolecules may be easily accessible biomarkers with which to develop a sensitive and specific universal screening diagnostic for the early detection of cirrhosis and hcc. introduction: the peptidergic g-protein coupled receptors (gpcrs) are cell-signalling transmembrane proteins, which in their native form comprise of seven segments embedded in the cell membrane. this structural advancement is believed to be maintained in extracellular vesicles (evs). in autoimmune diseases, the presence of autoantibodies towards gpcrs is not uncommon, and to detect plasma autoantibodies, evs carrying gpcr will be used as template in a novel microarray screening tool. methods: purified evs from hek cells were printed on different types of surfaces; polymer coated glass slides and hydrophilic and hydrophobic plastic well plates. five different print buffers were tested in a multiplex assays. spots containing evs were stained with biotinylated antibodies (cd , cd , cd , adrβ , hsp , epcam and flotilin- ) followed by binding of cy -labelled streptavidin and visualized microarray scanner. results: the outcome of these experiments was promising, as some of the chosen printing buffers showed increased tendencies to bind evs. the ev presence was verified with a panel of markers known to be present on small evs. in addition, the ev content of the adrenergic beta- receptor (adrβ -receptor), which is a gpcr of interest in autoimmune diseases, was verified in some of the experimental setups. summary/conclusion: the approach of using evs as template in a screening tool possesses the potential to easily screen for autoimmune illness markers in diagnostic purposes. using the microarray technology allows the screening to be multivariate, specific and highly sensitive. circadian variation of extracellular vesicles secreted in urine: analysis of time point collection and normalization strategy. introduction: urinary extracellular vesicles (uevs) are an ideal source of biomarkers for kidney and urogenital diseases. despite the great deal of interest generated by uevs, little is known about its collection time and normalization approach. the majority of the studies on uevs focus on spot urine collection based on the assumption that it accurately reflects the renal function, although time point of collection is not standardized. therefore the practice to collect spot urine does not allow for calculating and standardizing accurately the uev excretion rate which may vary during the day. in addition, no research has been carried out yet to show the quantitative and qualitative difference of uevs between spot urine and h collections.the aim of this study is to compare uevs excreted in all single voids during a hour collection period and compare it with hour collection performed. methods: uevs were enriched by differential centrifugation and electron microscopy, western blot, nanoparticle tracking analysis, tuneable resistive pulse sensing and imaging flow cytometry were used to quantify uevs and associated markers variation during the hour. creatinine, urine osmolality and particle concentration were used to normalize the assessed analytes. results: electron microscopy showed a heterogeneous population of evs and western blot confirmed the presence of ev markers (tsg , alix and cd ). rna was extracted by a column-based method (mirna extraction kit qiagen) and cel- mirna was spiked in each sample. a multiparametric detection of nephron markers podocalyxin, aquaporin- and uevs pan tetraspanins (cd + dc + cd ) was performed utilizing imaging flow cytometry. whereas the uev composition did not change across the hours analysis, the quantity of uevs and related markers fluctuated during the day depending on the hydration and excretion rate.the results of a hour urine collection reflected the average results of all single voids over a hr period. creatinine and particle count normalization failed to normalize "outliers". summary/conclusion: this study represents the very first report which compares single void urine versus hour uev analysis. we concluded that the hour collection is the preferred choice for a robust and rigorous assessment of uevs and its associated markers. porcine body fluids differ in small extracellular vesicle counts: comparison of blood plasma, seminal plasma and cerebrospinal fluid as vesicle sources for proteomic analyses helena kupcova skalnikova a , jakub cervenka b , jaromir novak a , karolina turnovcova c , bozena levinska a , jana juhasova a , stefan juhas a and petr vodicka a a institute of animal physiology and genetics, czech academy of sciences, libechov, czech republic; b institute of animal physiology and genetics cas, v. v. i. libechov, libechov, czech republic; c analysis tools were used to identify in silico biological pathways and functions governed by detected mirnas. expression of putative targets of selected mirnas was tested using qpcr after in vitro delivery of uterine evs to ptr cells. results: careful characterisation confirmed that uterine lumen is enriched with a diverse population of evs caring mirnas. interestingly, out of detected mirnas showed difference in abundance between tested days of pregnancy and half of them was exclusively detected on d . identified mirnas were characterized as potent regulators of cellular development, growth, proliferation, and movement, in addition to their involvement in organismal and embryonic development. the expression of genes identified as a possible mirna targets was tested after evs delivery to ptr cells in vitro. both down-(e.g., ptger ) and up-regulated (e.g., lifr) genes were found (p < . ); involved in the same molecular and cellular functions enriched by detected mirnas. methods: evs were harvested from wild type and arrdc -/-epididymal cells using differential ultracentrifugation, then characterised using nanoparticle tracking analysis and transmission electron microscopy. sperm motility was measured using computer assisted sperm analysis and imagej. fertilisation capacity was measured using the following assays: capacitation-associated tyrosine phosphorylation, calcium ionophore induced acrosome reaction, zona pellucida binding assay and in vitro fertilization with time-lapse imaging of embryo development. immunohistochemistry was also used to visualise two pronuclei formation and blastocyst morphology. arrdc -/-sperm was supplemented with wild type evs in the above assays to assess whether they could restore function. results: sperm from arrdc -/-mice develop normally through the testis but fail to acquire adequate motility and fertilization capabilities through the epididymis, as evidenced by reduced motility, premature acrosome reaction, reduction in zona pellucida binding and production of two-cell embryos. we observed a significant reduction in ev production by arrdc -/-epididymal epithelial cells, and addition of wild type evs to arrdc -/-sperm dampens the acrosome reaction and restores zona pellucida binding. introduction: gestational diabetes (gdm) is among the most common pregnancy complications. despite treatment, up to % of pregnancies complicated by gdm result in infants being born large-for-gestational-age (lga). this not only causes problems at birth but predisposes offspring to developing cardio-metabolic disease in adulthood. there are no treatments for lga as the cause is unclear, although it is associated with altered placental vascular development. micrornas (mirnas) regulate placental development; they are produced within cells but can be released into the circulation inside evs, which in turn can be transported into target cells and tissues to influence cellular processes. we aimed to characterise circulating evs in pregnancies complicated by gdm-lga and determine if ev-derived mirnas have the potential to influence placental development. methods: maternal serum and plasma samples were collected from women with pregnancies complicated by gdm at - weeks gestation; placental tissue was collected at delivery and birth outcomes recorded. serum and plasma evs were isolated and characterised by electron microscopy (shape), nanoparticle tracking analysis (nta; size/concentration), and western blotting (ev-enriched proteins). mirna qpcr arrays were performed on evs. mirnas were quantified in placental tissue via qpcr. results: em and western blotting confirmed isolation of evs and nta revealed no significant difference in size/ concentration in gdm-lga pregnancies (n = ) compared to gdm-aga (n = ; p > . ). several ev mirnas were altered in maternal circulation in gdm-lga compared to gdm-aga (n = /group; >twofoldchange; p < . ), including four skeletal muscle-specific "myomirs": mir- - p, mir- a- p, mir- b, and mir- a- p (all increased). all four myomirs were present in placenta but only mir- - p was significantly altered in gdm-lga compared to gdm-aga (n = - /group; p < . ). summary/conclusion: ev-bound myomirs could have predictive value for aberrant foetal growth in cases of gdm. mir- - p regulates vascular development in other systems, so we propose that mir- - p contributes to lga by influencing placental vascular development, however further work is required to establish this. introduction: seminal plasma is particularly rich in extra cellular vesicles. myelinosomes are membranous organelles described throughout the seminiferous epithelium of the testis but never reported in semen. the aim of this study was to look for the presence of myelinosome vesicles in human seminal plasma. methods: because of the viscosity of seminal gel and its water-holding capacity, classical transmission electron microscopy does not seem to be an optimal technique to reveal the presence of myelinosomes in this fluid. cryo-electron microscopy is a technique that allows visualization of nanosized structures without prior fixation or addition of heavy metals for contrast. the sample is therefore visualized as close to its native state as possible. using standard myelinosome preparation from tm sertoli cells, we first analysed the appearance of "standard" native myelinosomes by cryo em and then compared it with the vesicles from human seminal plasma samples. results: we have specified by cry-em the morphological aspect of "standard" myelinosomes isolated from the culture media of tm sertoli cells. the vesicles with the same morphological appearance were revealed in human seminal plasma specimens. summary/conclusion: myelinosomes are membranous organelles found in the seminiferous epithelium of the testis and secreted by the somatic sertoli cells in the lumen of the seminiferous tubules.the preparations from human seminal plasma contains a population of large ev (average diameter nm) whose morphological appearance resemble those of myelinosomes. defining the specific biomarkers and functionalities of myelinosomes in human seminal plasma are the concerns to be addressed in our further research. introduction: more than one million patients worldwide suffer from tuberous sclerosis complex (tsc) and have mutations in either tsc or tsc genes. together, the tsc proteins regulate mtorc activity. all tsc patient post-mortem samples exhibit renal disease and % of patients with tsc experience a premature loss of renal function. mouse and human studies are incongruity with the second somatic hit mechanism of disease, because of the low percentage of cystic cells exhibiting loss of tsc expression. we posited that the loss of a tsc protein expression may alter extracellular vesicle (ev) biology and contribute to disease. methods: we used crispr/cas to disrupt the tsc gene in mouse inner medullary collecting duct (mimcd) cells, and isolated evs using gel filtration from the isogenic cell lines. we characterized the evs using tunable resistive pulse sensing (trps), dynamic light scattering (dls), transition electron microscopy (tem), and wester blot analysis. we further performed mass spectroscopy on the ev proteins. results: loss of the tsc gene in mimcd cells induced a greater than three-fold increase in ev production compared to the same cells having an intact tsc axis. electron microscopy confirmed the purity and spherical shape of evs. both trps and dls demonstrated that the isolated evs possessed a heterogenous size distribution. approximately % of the evs were in the - nm size range. western blot analysis using proteins isolated from the evs revealed the cellular proteins alix and tsg , the transmembrane proteins cd , cd and cd , and the primary cilia-related hedgehog signalling-related proteins arl b. proteomic analysis of evs identified a significant difference between the tsc -intact and tsc -deleted cells that correlated well with the increased production. summary/conclusion: evs may be involved in tissue homoeostasis and cause disease by overproduction and altered protein content. the evs released by renal cyst epithelia in tsc complex may serve as a tool to discover the mechanism of tsc cystogenesis and in developing potential therapeutic strategies. introduction: we have shown that evs derived from amniotic fluid stem cells (afsc) of mouse origin present therapeutic effect in an animal model of chronic kidney disease, alport syndrome (as). in light of clinical translation, we isolated afsc-evs of human origin, characterized their cargo and evaluated thier therapeutic effect in vivo. methods: human clonal afsc were derived from amniotic fluid collected after volunteer donors provided consent. evs were obtained from afsc and identity and purity were assessed by rna-seq and proteomics. potency of hafsc-evs was evaluated by performing in vivo studies. ev biodistribution was evaluated by mri and therapeutic effect by measuring renal function and mice life-span. bulk rna-seq was performed on glomeruli obtained from injected and non-injected mice to identify potential ev regulating targets. results: proteomic profiling identified intact proteins and rna-seq data identified , mirs in hafsc-evs. hafsc-ev "fingerprint" was assessed by performing go analysis on the most highly expressed proteins and mirs. the results identified pathways involved in tissue homoeostasis such as mtor pathway, tgfβ and vegf pathways. when injected in vivo into as mice, biodistribution studies showed that hafsc-evs localized in the kidney, corrected proteinuria. no side effects (including teratoma) were noted in the treated mice. rna-seq of glomeruli obtained from treated as mice showed similar gene expression patterns to wilt type mice, by cluster analysis. our data indicated that hevs highly modulated pathways involved in collagen and matrix deposition remodelling, in addition to downstream targets of vegf, fgf, tnf, angiotensin and preserved glomerular cells structure and function. summary/conclusion: our protocol for hevs derivation is reproducible and allows derivation of ev lots with the same identity (specific cargo of proteins and mirs) and potency (present therapeutic effect in as). hafsc-evs modulated signalling pathways that are central to maintaining glomerular homoeostasis and preserved glomeruli structure with improved kidney function. this suggests the possibility of using hafsc-evs as a new therapeutic option for treating renal failure in humans. introduction: recent studies have shown that stem cell-derived extracellular vesicles (msc-ev) therapy improves renal outcomes in models of acute ad chronic renal disease. however, to better investigate the molecular mechanisms of ev-induced regeneration, and to define new ev sources, devices that mimic d organ architecture and flow conditions are needed. the aim of our work is to evaluate the regenerative potential of naïve and engineered ev in a millifluidic in vitro d model of glomerular damage in continuous perfusion. methods: methods: we set a millifluidic in vitro d model of glomerular filtration, a three-layers structure composed by human podocytes and glomerular endothelial cells, and, in between, of a basement membrane of collagen type iv. the barrier thus formed is set up inside a bioreactor, in a closed milli-fluidic circuit in which fluid flows continuously at a certain flow rate. we reproduced different pathological conditions and tested the localization and effect of evs in a dynamic system. : results: we obtained a standardized protocol and an adequate configuration of the milli-fluidic circuit subject to continuous reperfusion. renal damage was induced by doxorubicin or by hypoxia-reperfusion injury. we evaluated uptake, cargo transfer and effect of naïve and mirna engineered msc-evs or of klotho engineered ineffective evs administered into the dynamic co-culture system. evs were able to pass through the system and to deliver to podocytes proregenerative factors, promoting survival and limiting permeability. introduction: worldwide, renal cell carcinoma (rcc) is th most common cancer in men and th most common in women. new biomarkers are needed to aid rcc-diagnosis, provide prognostic information, and to predict response to modern targeted therapies. extracellular vesicles (evs) are an emerging source of cancer biomarkers because all cells, including cancer cells, secrete evs into biofluids as blood and urine. however, benign cells contribute to ev populations isolated from blood and urine reducing the diseasespecificity. we have developed a protocol for ev isolation directly from human rcc tissue that can increase tumour-specificity of biomarkers. methods: we obtained technical and biological replicates from normal kidney tissue and clear cell rcc tissue. serum-free media was incubated with the specimens. a combination of differential centrifugation, filtration, and ultracentrifugation was used for ev isolation. evs were quantitated using two methods, allowing for comparison between nanosight ns and nanofcm. tem was used to determine presence of intact vesicles in the ev samples. presence of ev introduction: urothelial carcinoma (uc) is a malignant cancer that affects the urothelial cells, representing % of all bladder tumours. at diagnosis % of bladder cancers are non-muscle invasive tumours. importantly, upon transurethral resection of the bladder tumour, nearly - % of these patients will experience disease relapse and - % will progress to muscle invasive tumour, requiring thereby, a rigorous and expensive follow-up. currently, this is performed through the frequent use of highly invasive cystoscopy and the low sensitivity urine cytology. thus, innovative liquid biopsy-based biomarkers that circumvent these drawbacks are highly desirable for improved uc clinical management. here, we aim to implement a protocol for the isolation and characterization of extracellular vesicles (evs) from uc patients' urine samples. methods: a two-step protocol involving ultracentrifugation (uct) and by size-exclusion chromatography (sec) was optimized for urine samples. the isolated urine-derived evs from uc patients were then characterized according to their size, concentration (nta), morphology (tem), protein amount (lowry method), presence of ev-associated and disease-associated protein markers (western blot). results: isolated urinary evs from uc patients had a size ranging from nm to nm with characteristic ev morphology, express ev-associated markers as cd and hsp and were negative for cell debris markers. the recovery yield and purity of isolated evs following each isolation technique was characterized. upon uct, sec was required to deplete most of the ev-associated thp and albumin protein contaminants. some disease-associated protein markers were highly enriched in isolated urinary evs compared to crude urine. summary/conclusion: taken together, these results indicate that a two-step ev isolation protocol was properly implemented and validated in uc patients' urine samples. notably, several ev-associated disease biomarkers were detected in the urine of uc patients. this ev-based liquid biopsy might provide the means for real-time monitoring of residual disease and relapse in uc patients. introduction: glioblastoma multiforme (gbm) is a very aggressive type of brain tumour. different gbm molecular subtypes (proneural, mesenchymal and classical) often co-coexist within the same tumour, with the mesenchymal subtype driving the tumour progression. recently, our lab demonstrated that the cargo of extracellular vesicles (evs) could mirror the molecular background of the gbm cells from which they were derived. altogether, we believe that gbm cell-derived evs can be directly involved in the expansion of the mesenchymal signature in tumours, thus supporting gbm aggressiveness. methods: non-mesenchymal (t & u ) gbm cells were "primed" using evs derived from mesenchymallike (u & ln ) gbm cells. ev-primed gbm cells were then co-cultured with their non-primed counterparts to determine whether the mesenchymal signature can "spread" from cell to cell via evs. effect on cell proliferation, migration and invasion (in hyaluronic acid hydrogels) was assessed following ev treatment and co-culture. the expression of mesenchymal gbm markers was measured by western blotting. further mass spectrometry analysis of cell and ev content was undertaken to describe potential underlying mechanisms. results: co-culture with ev-primed gbm cells significantly increased proliferation and hydrogel invasiveness of non-mesenchymal cells. interestingly, the stimulating effect of co-culture was even stronger on the proliferation of ev-primed gbm cells. moreover, further proteomic analysis revealed that expression of mesenchymal gbm markers such as cd was increased in non-mesenchymal cells following coculture. summary/conclusion: our data suggest that evs from mesenchymal gbm cells can be uptaken by gbm cells from different subtypes, thus stimulating tumour progression. overall, we think the present study provides with new insights for the understanding of gbm recurrence and the development of potential therapeutic strategies. introduction: triple-negative breast cancer (tnbc) is the most aggressive form of breast cancer. previously we reported that the heterogenous population of evs released from tnbc cells promotes the growth and aggression of recipient cells. here we investigated if, by using compounds proposed to inhibit ev release i.e. calpeptin and y (to block those budding at cell membrane) and gw and manumycin a (to block evs from mvbs), we could reduce the associated transmission of aggressive phenotype. methods: evs were separated from medium conditioned by tnbc cell line hs ts(i) , using a discontinuous optiprep density gradient, after the cells were treatment for hrs with the compounds listed above. evs (pooled fractions - with a density range of . - . g/ml) were characterised by nta, bca, lipid assay, immunoblot, tem and flow cytometry. to investigate the functional effects of the evs released, proliferation and migration assays were performed on hs t and mda-mb- cells using the ev to cell ratios of × evs/ x cells, × evs/ x cells, × evs/ x cells to evaluate doseresponse. ev-track id ev (score of %). results: gw significantly (p = . ) decreased ev release from hs ts(i) cells. manumycin a and a combination of calpeptin and y (combo) decreased ev release, but significance was not reached. conversely, calpeptin and y actually increased ev release; but not significantly. of the reduced numbers of evs released following gw treatment, hla-dr+ evs were significantly (p = . ) enriched. none of the evs analysed significantly changed hs t or mda-mb- growth rates. however, evs from cells treated with calpeptin (p = . ), gw (p = . ), manumycin a (p = . ) and combo (p = . ) caused significant reduction in mda-mb- migration compared to the effects of evs from untreated cells. similarly, ev from cells treated with gw (p = . ), and combo (p = . ) caused significant reduction in hs t migration. summary/conclusion: while gw was the only compound that caused a significant decrease in quantities of ev released, the evs that continued to be released following treatment with gw or calpeptin and y significantly reduced migration of both recipient cell lines. funding: phd funding: tcd scholarship and carrick therapeutics ltd extracellular vesicles from highly metastatic lung cancer cells induce barrier impairment, permeability, and epithelial-to-mesenchymal plasticity in a -day mature bronchial epithelium purdue university, west lafayette, usa introduction: epithelial-to-mesenchymal (emt) transition plays an integral role in cancer metastasis, which is responsible for as much as % of cancer mortality. cancer exosomes induce emt in bronchial epithelial cells, however, the epithelial cells inhibit emt when allowed to form a mature epithelial barrier with apicalbasal polarity. it is not known if cancer-derived extracellular vesicles (evs) can induce emt and more importantly, barrier disruption in a mature epithelium. here, we show that evs from a highly metastatic lung cancer cell line (calu ) are) are not only sufficient to induce emt in non-tumorigenic bronchail epithelial cells (beas- b), but are also capable of disrupting a -day mature bronchial epithelial barrier by significantly reducing teer, inducing sixfold increase in permeability and complete loss of e-cadherin at cellcell tight junctions. methods: beas- b and calu evs were characterized using electron microscopy, nanosight and western blotting for exosome-specific features. for permeability studies, beas- b cells were cultured in transwell for days to establish an intact epitheliumconfirmed by measuring teer (trans-epithelial electrical resistance). intact beas- b monolayers were treated with calu evs at , and μg/ml for hrs, and barrier intactness and permeability were evaluated by measuring teer, apical-basolateral translocation of dextran beads and confocal imaging of tight junctions (e-cadherin). for emt experiments, beas- b cells treated with calu evs at and μg/ml were evaluated for ecadherin and vimentin levels by qrt-pcr and western blot after hrs. results: beas- b and calu evs were enriched in - nm size range, and cd and cd were enriched in the ev fraction in contrast to the cell lysate and vice versa for gp . calu evs significantly impaired day mature beas- b monolayer's barrier properties, which at the highest dose caused % reduction in teer from . ± . to . ± . Ω.cm (n = ). this was further confirmed by~sixfold increase in dextran beads' apical-basolateral translocation in min ( . ± ng/ml in control vs . ± ng/ml in treated) (n = ) and complete loss of e-cadherin expression at cell-cell tight junctions (n = ). at the transcript level, calu evs induced significant downregulation of e-cadherin by % and upregulation of vimentin (mesenchymal marker) twofold (n = ) in beas- b cells, indicating transition into mesenchymal phenotype. summary/conclusion: we demonstrated the involvement of evs derived from highly metastatic lung cancer cells in inducing emt in bronchial epithelial cells and epithelial barrier disruptionthe initial stage of the intravasation process. grp plays a crucial role in the extracellular vesicle-promoted radioresistance of irradiated head and neck cancer cells introduction: small evs released from irradiated head and neck squamous cell carcinoma (hnscc) cells increase resistance of recipient hnscc cells to radiation in vitro. we have identified the glucose-regulated protein (grp ), a chaperone protein of the hsp family which is involved in cellular stress responses and associated with worse survival in head and neck cancer patients, as an essential component of the ev-mediated radioresistance. methods: small evs were isolated from conditioned medium from irradiated and non-irradiated bhy hnscc cells by combined microfiltration ( . µm) and differential ultracentrifugation. grp surface expression was measured by proteomic analysis, immunoblotting and bead-facs. radiation resistance of bhy cells was determined by a clonogenic survival assay. results: increased grp was identified on the surface of evs from irradiated cells. the increase in ev grp correlated with increased grp expression at the donor cell surface. the grp content of recipient cells also increased upon transfer of evs from irradiated, but not non-irradiated cells, ultimately leading to enhanced cell survival. to check a potential role of elevated grp in radiation resistance we overexpressed grp . here the modest ( x) overexpression of grp was sufficient to confer an enhanced radioresistant phenotype to the bhy cells. a correlation between grp -dependent increase of radioresistance and activation of the akt pathway is yet to be determined. summary/conclusion: our results suggest a pivotal role for ev-transferred grp in modulating the radiation response of recipient hnscc cells. radiation directly increases the cellular and vesicular grp levels, and subsequent ev-mediated transfer leads to enhanced grp levels and radioresistance in recipient cells. this study provides new mechanistic insights into the effects of evs in radiation response and elucidates an interesting target protein and novel strategies for the improvement of radiotherapy. d modelling of ev release in progressing prostate cancer introduction: the modelling of cancer progression should be capable to translate acquired knowledge of cell behaviour to the real human body conditions. however, the extracellular vesicles (evs) isolated from d cell models are commonly exploited in research. taking into account the specificity of the prostate cancer (pc) environment, and a strong need of early diagnosis of castrate-resistance by prostate cancer (crpc) patients, we suggest in-depth profiling of different ev subtypes isolated from d culture as a new tool to model the progressing pc. methods: cells from hormone-resistant prostate carcinoma -rv line were cultured in d and d conditions, using d coseedistm. acd plasma controlled for haemolysis and remaining platelets was taken from patients with pc and crpc. the fractions of ev subtypes from cell culture and plasma were obtained by differential centrifugation (dc) followed by iodixanol density gradient purification. each of the fractions was measured by nanoparticle tracking analysis (nta), tunable resistive pulse sensing (trps) followed by elisa. for that, cd and cd were used as ev markers, apob and apoa for lipoprotein contaminants control, and cd , cd and psma as tissue-specific biomarkers for determination of fractions containing evs of different origin. ev-contained fractions were subjected to next generation sequencing (ngs). results: in d conditions, the -rv cells produce up to -times higher ev number than in d. size and density distribution of evs derived from d cultures but not of d resembled plasma evs. size distribution and biomarker expression among different ev subtypes allowed distinguishing between pc and cprcderived samples, indicating a potential to translate these results into clinics for early cprc detection. summary/conclusion: this work demonstrates a new approach to study the secretome of a progressing pc under d conditions. the profiles of ev subtypes produced by cancer cells growing in a d spatial architecture resemble the profiles of plasma evs and can serve a useful tool for the establishment of new biomarkers. introduction: renal cell carcinoma (rcc) is the most common primary renal neoplasm, with over , cases in the us alone each year. early detection of rcc leads to consistently better patient outcomes, and extracellular vesicles (evs) isolated from patient samples may prove to be a valuable clinical tool in the future. evs are abundant in blood and urine and show a large amount of heterogeneity but are difficult to analyse due to their small size and difficulty in isolation. here, we employ a multiparametric analysis of ev surface markers to identify a set of markers that may prove clinically relevant in future studies. methods: rcc cell lines vok , vok , and vok were cultured in flasks containing ml of ev-depleted media ( % fbs, centrifuged hr x , g). when cells reached~ % confluency, the conditioned media was collected and spun at , g for mins two times to deplete any remaining debris, leaving~ ml of media. this media was concentrated to a final volume of~ ml using a pall jumbosep kda mwco filter. this concentrate was purified from protein by using an izon qev- column, collecting ml fractions. protein content of each fraction was analysed using a absorbance while concentration and diameter distribution were determined through nanoparticle tracking analysis (nta). pooled samples made of the three most concentrated fractions were concentrated to a final volume of~ µl using the pall microsep kda filter and then used for analysis in the miltenyi macsplex exosome kit. flow cytometric data were generated by the cytoflex s and analysed using flowjo and mpapass software. these positive signals were verified through bead-only controls and titrations. results: the mpapass software allowed for heatmap generation, data reduction, clustering and visualization of expression patterns. of the detection antibodies used across capture beads, cd , cd , cd , beta- microglobulin, and cd were found to be prevalent in these rcc evs. these markers were found to be co-expressed particularly with cd , cd , and cd . summary/conclusion: the use of multiplex analysis allowed for detection of five distinctive surface markers found to be prevalent in evs collected from rcc cell lines. these results demonstrate the utility of multiplex analysis and mpapass software for identifying potential markers of interest and provide proteins that are worth exploring further. the next steps to this work will be developing custom multiplex arrays that tailor capture and detection of evs specifically for rcc pathology. low molecular weight protein tyrosine phosphatase (lmwptp) carried by colorectal cancer cells-derived extracellular vesicles as a player in tumour-educated human fibroblast university of campinas -unicamp, campinas, brazil introduction: extracellular vesicles (evs) are doublemembrane-bound nanovesicles released by cells playing a key role as mediators of intercellular communication. low molecular weight protein tyrosine phosphatase (lmwptp) is upregulated in several cancers type, including colorectal cancer (crc), and it has been correlated with aggressiveness, chemoresistance and poor prognostic. methods: the aim of this study was to determine whether crc cells release lmwptp-enriched-evs and influence tumour microenvironment-associated cells as a representative tumour education. crc cells, hct and ht , were cultured in serum-free medium for hours. conditioned medium was concentrated by ultrafiltration (mwco kda) and evs were isolated by total exosome isolation reagent (invitrogen). evs were characterized by nanoparticle tracking analysis (nta), transmission electron microscopy (tem) and western blotting (wb). lmwptp levels were analysed by wb and sandwich-elisa. to evaluate tumour education, hff- fibroblasts were used as recipient cells. the uptake of evs (pkh fluorescently labelled evs), proliferation (viability) and migration (wound healing assay) were analysed in a co-culture model of crc-derived evs and hff- . results: nta showed a higher concentration of evs released by ht . hct and ht evs displayed a mean diameter around nm and a cup-shaped morphology. isolated evs were positive for evs-markers cd and tsg and negative for gm a non-evs marker. ht lineage as well as derived-evs are lmwptp-enriched in comparison to hct cells and evs. upon incubation, fluorescently hct and ht derived evs were internalized into hff- cells in a perinuclear region. evs derived from both cells increased the viability and proliferation of hff- cells. intriguingly, evs derived from ht promoted cell migration. summary/conclusion: in conclusion, for the first time, we showed that lmwptp can be carried by evs derived from crc cells and lmwptp-enriched-evs can modulate biological aspects of hff- fibroblast. overall, our findings point lmwptp out as important player in tumour-educated fibroblast. exosomal mir- a inhibition by vincristine and prednisone in paediatric acute lymphoblastic leukaemia. introduction: vincristine and prednisone are standard agents in treatment of paediatric acute lymphocytic leukaemia (p-all). mechanistically, vincristine induces apoptosis by blocking microtubules formation, while prednisone binds to cytoplasmic receptors and inhibits dna synthesis, both of which lead to apoptosis. the effect of these agents on exosomal micro-rna expression and its functional regulation is not yet investigated. elevated levels of mir- a in circulating exosomes (nanoparticles) has been shown to lead to progression in several cancers, including all. we have previously shown that leukaemia-derived exosomes induce leukaemia cell proliferation via up-regulating of mir- a expression and silencing of exosomal mir- a reverses this exosomeinduced cell proliferating effect. the objective is to investigate the effect of vincristine and prednisone on exosomal mi-r a expression in all. methods: jm , sup-b , and nalm- leukaemic cell lines were treated in vitro with vincristine ( . to . µm) and prednisone ( . to . µm) in exo-free medium and apoptosis was measured by mts assay. total rna of exposed cell lines was isolated and cdna was prepared for mir- a analysis. expression of mir- a was analysed by q-pcr. exosomes from conditioned medium of exposed cell lines were isolated by ultracentrifugation method. purity and particle size of exosomes were confirmed by western blot and nanoparticle tracking analysis (nta) assay respectively. total exosomal rna was isolated from exosomes (exo-rna) by trizol method. synthesis of cdna was carried out with the miscript ii rt kit (qiagen). results: vincristine and prednisone promote apoptosis in leukaemia cell lines (jm and sup-b ) in a dosedependent manner. both cellular and exosomal mir- a expression was down-regulated by vincristine and prednisone exposure in all three leukaemia cell lines (jm , sup-b , and nalm- ). these observations demonstrate that cellular mir- a down regulation in the parental cells is stable and can be transferred to exosomes, confirming the concept that exosomes are the fingerprint of parent cells. summary/conclusion: our data suggest that the vincristine and prednisone anti-proliferative effect in p-all maybe induced by another yet unexplored pathway, that suppresses mir- a at a cellular and exosomal level in p-all, resulting in apoptosis. funding: this project is supported by the dimartino family foundation. secreted extracellular vesicles from renal cell carcinoma cells anatoliy samoylenko, artem zhyvolozhnyi, eslam abdelrady, naveed ahmad, genevieve bart and seppo vainio oulu university, oulu, finland introduction: clear cell renal cell carcinoma (ccrcc) represents the most common form of kidney cancer and is among the most lethal of all genitourinary cancers. despite surgery and medication therapy, most patients with metastatic ccrcc have a poor prognosis. intratumoural hypoxia is a key factor involved in renal cancer progression and it is known to promote secretion of evs by many types of tumour cells. methods: rcc-derived renca cells, embryonic kidney derived ub cells, and primary mouse hepatocytes were used in the study. evs were purified from cell culture media by gradient ultracentrifugation, sequential ultracentrifugation and exo-spin™ columns. before ev isolation cells were kept for h either under normoxia or hypoxia ( % oxygen). evs were analysed by transmission electron microscopy with negative staining and immunolabeling, by nanoparticle tracking analysis (nta) and western blotting. cells proliferation and viability were assayed by live cell imaging using incucyte zoom (essen bioscience), cell metabolic activity by seahorse xf analyser (agilent), rna expression by qpcr and ddpcr. proteins were identified by ultra-performance liquid chromatography-mass spectrometry (uplc-ms). rna libraries were made using nebnext small rna library prep kit, and sequenced on nextseq (illumina). results: we showed that hypoxia induced production of evs by rcc cells, and characterized differences in protein and rna content of evs generated by renca cells cultured under normoxic and hypoxic conditions. we also showed that rcc-produced vesicles modify key features of tumorigenesis (gene expression, metabolic activity, motility, and growth) of target cells. these data were obtained by using two target cell types: model mouse kidney cells and primary mouse hepatocytes, which represent typical site of rcc metastasis with an exceptionally poor prognosis. we proposed that a possible mechanism of ev action in rcc is related to changes in caveolin- function. we also tracked renca-derived evs in a chick embryo model and in a novel kidney organoid co-culture assay developed by our group (xu et al., ) . summary/conclusion: hypoxia may influence tumorigenic properties of rcc by changing rates of production and composition of evs. funding: the study was supported by finnish cancer foundation grants. exosomes synthesizing her mirna and engineered to adhere to her on tumour cells surface exhibit enhanced anti-tumour activity introduction: exosomes are small extracellular vesicles averaging - nm in diameter. they serve as a means of intercellular communication. typically they consist of structural proteins as well as selected proteins, mirnas, mrnas, and long noncoding rnas. thus in an earlier report this laboratory designed a mirna targeting a major herpes simplex virus regulatory protein. as predicted by the nucleotide packaging signal the mirnas were packed in exosomes and on exposure to infected cells significantly reduced virus yields. her (human epidermal growth factor receptor ) plays an important role in the neoplasia of some breast cancers. the protein is exhibited on the cell surface and is the target of therapeutic antibodies. methods: firstly, we report on the construction of a mirna targeting the synthesis of her both in cells constitutively expressing her and in cells transfected with a plasmid encoding her . secondly, we report that the mirna targeting the synthesis of her reduced the viability of her positive cancer cells both in cell culture and in implanted tumours. lastly, we enhanced the anti-tumour activity of the exosomes by binding to the exosome surface a ligand with affinity for the her on the surface of tumour cells. the -mir-her exosomes package with mirna designed to block her synthesis and deliver to cells. these exosomes kill cancer cells dependent on her for survival but have no effect on cells lacking her or which were engineered to have her but do not depend on it for survival. the -mir-xs-her exosomes carry in addition a peptide which enables the exosome to adhere her on the surface of the cancer cells. in consequence, these exosomes preferentially enter and kill cells exhibiting her on their surface. the exosomes with -mir-xs-her are significantly more effective in shrinking the size of her -positive tumours implanted in mice than the -mir-her exosomes. summary/conclusion: our studies indicate that exosomes carrying mirna against her have no effect on her negative cells it was nevertheless desirable to increase the uptake of exosomes carrying the her mirnas by her -positive tumour cells. to this end we modified the exosomes to exhibit on their surface a peptide that bound the exosomes to the her on the surface of cancer cells. in consequence, we significantly enhanced the uptake of exosomes carrying the mirnas directed against her by her positive cells. funding: these studies were supported by grants from shenzhen overseas high-calibre peacock foundation kqtd , shenzhen science and innovation commission project grants jcyj , jcyj to shenzhen international institute for biomedical research. systematic characterization of ovarian cancer-derived exosomes unveil mirnas interfering with cd + t cell activation introduction: cd + tumour-infiltrating lymphocytes (til) have been widely reported to correlate with cancer patient survival, including ovarian cancer. even with the presence of tils, immunotherapy has limited success in ovarian cancer. understanding the interaction between cd + til and tumour cells is thus important. our hypothesis is that tumour-derived exosomes are released and taken up by cd + til such that specific mirnas contained within modulate physiological processes that inhibit cd + t cell activation. we aim to identify mirnas carried in tumour-derived exosomes that inhibit cd + t cell activation in ovarian cancer. methods: we purified exosomes from nine ovarian cancer cell lines and stocked in high concentration. interferon-gamma (ifn-gamma expression screening was performed after days of co-incubation of tumour derived exosomes, cd + t cells, and activators in conditioned medium. cell counts and viability were tested by trypan blue staining at day and day . rna-seq for exosomes were generated to identify mirnas critical in differentiation effects on cd + t cell activations. microrna target matching uncovered target mrnas while enriched pathway analysis predicted potential signalling pathways involved. results: our ifn-gamma screening results indicated the exosomes exhibit different behaviours in interfering cd + t cell activation owing to different donors. exosomes derived from peo. and ovca cells have consistent polarized results in ifn-gamma expression. exosomes derived from peo. remained a low ifn-gamma expression and from ovca stayed at relatively high level. small rnas profiling analysis between the two cell lines identified mirnas (p < . ), and mirnas have been reported with validated targeting information, and out of have targets involved in immune signalling. mrna targets were uncovered by target matching. cmap search identified complex connections among mrnas with the top enriched pathways actively involved in cell cycle and immune related behaviours. summary/conclusion: our ifn-gamma screening identified crucial mirnas in ovarian cancer exosomes interfering cd + t cell activation. computational modelling on both experimental and public multiomics datasets predicted promising signalling pathways of tumour-immune crosstalk for functional validation. irradiation of breast cancer cells alters the quality of dna cargo in the exosomes that they produce sheila spada, paul zumbo, doron betel, tuo zhang, nils-petter rudqvist and sandra demaria weill cornell medicine, new york, usa introduction: irradiation of breast cancer cells with an immunogenic dose ( gyx ) leads to accumulation of cytosolic dna that is sensed by cgas leading to interferon type i (ifn-i) signalling via cgas/sting pathway [ ] [ ] [ ] . we previously showed that tumour-derived exosomes (tex) secreted by irradiated ( gyx ) (rt-tex) but not untreated (ut-tex) tsa carcinoma cells carry dna that stimulates the production of ifn-i in recipient dendritic cells (dc) via the cgas/ sting pathway [ ] . moreover, mice vaccination using rt-tex, but not ut-tex, elicited anti-tumour immune response inhibiting tumour growth [ ] . here, we hypothesized that the differential ability of rt-tex and ut-tex to activate ifn-i in recipient dcs is due to qualitative differences in dna cargo of rt-tex compare to ut-tex. methods: the length of dna purified from tex and from the cytosolic fraction of tsa cells was measured by agilent bioanalyzer. the dna cargo of tex was analysed by whole-genome sequencing (wgs) and whole-genome bisulphite sequencing. the percentage of methylation of total dna in tsa cells was quantified by -methyl cytosine dna elisa kit. results: dna fragments with size between and bp were enriched in rt-tex compared to ut-tex, as well as in the cytosolic fraction of irradiated compared to mock-treated tsa cells. wgs revealed that the entire genome was represented in tex dna cargo, regardless of rt. more than % of tex dna was of nuclear origin, but mitochondrial dna was increased in rt-tex. interestingly, we found that rt decreases the level of methylation in both exosomal and total dna in tsa cells compared to the controls. summary/conclusion: these data support the hypothesis that immunogenic rt alters some characteristics of the exosomal dna cargo, mirroring molecular changes occurring in parent irradiated breast cancer cells. the enrichment in dna fragments of - bp in rt-tex is intriguing considering that cgas is optimally activated by dna in this length range [ ] . we are currently investigating which features of the cargo dna that differ between ut-tex and rt-tex may explain the differential ability to induce ifn-i pathway activation in recipient dcs. the identification of a dna signature associated with the ability of tex to activate the cgas/sting pathway could provide a circulating biomarker of the rt-driven immunogenic tumour response. introduction: triple negative breast cancer (tnbc) is among the most difficult cancer subtypes to treat and continues to cause a high number of cancer-related deaths annually. extracellular vesicles (evs) transfer cell type-specific cargo and have important implications in disease initiation, therapy and outcome. upon treatment of cancer cells with low-dose chemotherapy, released evs are able to transfer phenotypic traits to other cancer cells. new treatment strategies for tnbc, like inhibitors of the er stress pathway (ire ) might impact on ev biogenesis, cargo delivery and response of cells in the cancer microenvironment. our aim is to identify immune modulatory alterations in breast cancer cells and cancer derived evs upon treatment with inhibitors of the er stress pathway. methods: human tnbc cell lines were treated with ire inhibitor mkc and cells were analysed for immune modulatory surface markers, like hla-i, b -h molecules and different integrins. mitochondrial and lysosomal activities were investigated by the use of a mito-and lysotracker and analysed by imagestream (isx) technology. extracellular vesicles were isolated from cell culture supernatants by sequential centrifugation, quantified by nanoparticle tracking (nta) and characterized by exosome bead array. single ev analysis of total cell free supernatants and of isolated evs was performed by isx and marker positive evs were quantified for absolute fluorescence signals and total amount by objectives/ml. ev uptake into t cells was investigated by the use of different ev labelling strategies. results: several immune relevant surface markers (hla-i and cd ) are downmodulated by ire inhibition across different cell lines. cell surface expressed cd and b -h show cell line specific downmodulation profiles upon ire inhibitor treatment. other immunomodulatory marker such as b -h and b -h , integrin cd , cell adhesion-promoting cd and stemness/metastasis marker (cd and ssea) are unaltered on ire treated breast cancer cells. cancer cell derived evs were tetraspanin positive (cd , cd , cd ), similar in number and showed differential expression of immune markers upon ire treatment. mitochondrial and lysosomal activities were unaltered under ire inhibition, whereas cell proliferation was diminished. no breast cancer-derived ev uptake of externally labelled evs into healthy t cells could be detected. summary/conclusion: ongoing analyses focus on the multicolour analysis of multiple markers on single evs by imaging flow cytometry and on the functional impact of cancer derived evs on t cells delivered by ev receptor binding. funding: dagmar quandt is supported by the sfi (cÚram research centre, /rc/ ), the european regional development fund and the dr. werner jackstädt-stiftung. chair: uta erdbrügger -university of virginia chair: larry harshyne -thomas jefferson university comparison of three isolation protocols to search extracellular vesicles signature in sickle cell disease patients introduction: sickle cell disease (scd) is an inherited disorder characterized by chronic haemolysis and continuous activation of different cell types. extracellular vesicles (evs) were described to be at increased levels in scd patient's plasma compared to healthy subjects and were associated with several clinical manifestations such as leg ulcers and stroke. scd patient's plasma has increased concentrations of haem, free-hb and other proteins and lipoproteins as chronic haemolysis consequence. here, we report the comparison of three mostly used isolation protocols to search ev signature in scd patient's plasma by flow cytometry. methods: blood samples were obtained from scd patients (n = ) following wisgrill et al., ( ) protocol. three different ev isolation protocols were used: differential centrifugation (dc), ultracentrifugation (uc) and size-exclusion chromatography (sec). lactadherin and calcein-am were used to detect phosphatidylserine (ps)+ vesicles and membrane integrity, respectively. platelet-derived evs (pevs), endothelialderived evs (eevs), leucocyte-derived evs (levs) and monocyte-derived evs (mevs) were quantified. silica beads were used to define evs gate and samples were acquired in the cytoflex cytometer platform. results: the quantification of pevs in uc, dc and sec samples was, respectively, x , , x and , x events/ml mean, eevs was , x , × and , x events/ml mean, levs was x , × and , x events/ml mean and mevs , x , , x and , x events/ml mean. uc samples demonstrated a higher concentration of evs, which could be more useful to functional studies than dc and sec, however, it took more time to separate than dc. dc was the fastest method to separate evs from plasma, being useful to study large patients cohorts, but showed the smallest overall number of evs. sec also demonstrated high capability to detect evs in plasma and the possibility of obtaining a purer sample, although it is the most expensive and time-consuming method among all tested. all evs populations were detected in the three protocols tested. summary/conclusion: in summary, all protocols tested were efficiently to detect evs in scd patient's plasma and the definition of the best protocol may vary based on the research aim and time and budget available. funding: fapesp / - . gabrielle lapping-carr, joanna gemel, yifan mao and eric beyer university of chicago, chicago, usa introduction: aberrant cell-cell interactions involving the endothelium are central to the pathophysiology of sickle cell disease (scd), including acute chest syndrome (acs), a deadly and unpredictable complication. we previously demonstrated that the plasma of scd patients contains increased circulating small extracellular vesicles (evs) compared to controls and that those vesicles can disrupt endothelial integrity in vitro by affecting adherens junctions and ve-cadherin. the current study was designed to examine the effects of those evs on other cellular junctions including tight (zonula occludens , zo- ) and gap junctions (con-nexin , cx ) and to test the hypothesis that the junctions would be more severely affected by evs isolated from patients during an episode of acs than by ones isolated from the same patient at baseline. methods: we identified subjects with scd in our biobank who had plasma isolated at baseline and at the beginning of an admission for acs. evs were isolated from platelet free plasma using established methodologies. to determine the effects on endothelium, cultures of human microvascular endothelial cells were treated with evs for h and studied by immunofluorescence, immunoblotting and rt-qpcr. gap junction-mediated intercellular communication was assessed following microinjection of lucifer yellow and neurobiotin. results: the distribution and abundance of zo- at the plasma membrane were minimally affected by scd evs. while baseline evs did not affect the distribution of cx , evs isolated during an episode of acs caused loss of cx from the plasma membrane. the integrated intensity of cx membrane staining was decreased bỹ % following treatment with acs evs. cx protein decreased on average by %, cx mrna levels by % and neurobiotin transfer by - % in cells treated with acs evs, compared to baseline evs. summary/conclusion: circulating evs in scd affect multiple components of endothelial junctions. gap junctions composed of cx are the most sensitive of the cellcell junctions, since their abundance and function are reduced by acs evs even when the endothelial monolayer appears intact. cx -mediated intercellular communication may be an early and sensitive event in the endothelial disturbance caused by evs in scd patients. funding: nih ul tr , comer hospital rbc race funds, ted mullin fund. the effects of platelet concentrate storage time on extracellular vesicle interactions associated with fibrin clot formation in-vitro jamie nash a , christine saunders b , amanda davies a and philip james a a cardiff metropolitan university, cardiff, uk; b welsh blood service, velindre university nhs trust, cardiff, uk introduction: platelet concentrates (pcs) have been utilised for decades to prevent bleeding in thrombocytopenic patients and to stop active bleeding. the storage of pcs however is a logistical challenge due to the limited day shelf life under standard conditions. during storage, platelets undergo a number of mechanical and biochemical changes contributing to the short shelf life of a pc. these changes are collectively known as the platelet storage lesion. platelet extracellular vesicles (pevs) are known to increase throughout pc storage, due to an increase in platelet activation. as pevs have previously been shown to be pro-coagulant and increase in annexin v binding over pc storage. the aim was to investigate the effect of pc storage time on extracellular vesicle interactions on fibrin clot formation. methods: pcs were sampled on alternate days up to days of storage and centrifuged to achieve acellular plasma. the plasma was subjected to ultracentrifugation ( , xg) to pellet evs. the size and concentration of evs was assessed using nanoparticle tracking analysis software, followed by a western blot to confirm evs were of platelet origin. the pevs were added at a fixed number to a control pooled plasma sample with added thrombin and tissue plasminogen activator. the time to clot and % lysis time were recorded by using the turbidometry of the plasma over time. results: evs isolated from the pc were confirmed to be of platelet origin by western blot using cd as a marker of platelet origin and cd as an ev marker. pevs caused a significant increase effect on the fibrin clot formation (p < . ) when compared to the control plasma. pevs also had a significant effect (p < . ) on the fibrinolysis time, extending the time taken to lyse the clot. characterization of mirna from serum derived exosomes in a mouse tibia fracture model of introduction: complex regional pain syndrome (crps) is a debilitating chronic disease that occurs after trauma to the periphery and is intimately associated with nerve injury. its presentation is often described as an injury that is disproportional to the inciting event and manifests neuropathic pain, systemic inflammation, and immune dysregulation. owing in part to our poor understanding of disease aetiology, current treatments for crps are insufficient and as a disease of exclusion there is a lack of quantitative diagnostic markers. exosomes are small extracellular vesicles (sevs) - nm in size which provide a means of cellular communication through their cargo molecules (protein, mirna, mrna, lipids) , and have demonstrated promise in uncovering mechanisms of disease manifestation and identifying potential diagnostic markers. we have shown previously that crps patients have differential expression of several mirnas in serum derived sevs as compared to healthy controls, but little is known on how this compares to the established mouse tibia fracture model of crps. methods: mice undergoing fracture were anesthetized and subjected to a unilateral tibia fracture followed by casting of the injured limb. after confirming the establishment of pain hypersensitivity, serum samples were collected from fracture model and control mice three weeks post-injury. sevs were isolated by differential centrifugation and characterized using nanoparticle tracking analysis, transmission electron microscopy and western blotting. rna-seq analysis is being performed to identify differentially expressed mirnas. results: nanoparticle tracking analysis showed no significant difference in the number or size of sevs present in the serum from the fracture model and control mice. rna-seq is ongoing and differential mirna expression in sevs from fracture model will be compared to control samples. comparative studies identifying mirnas that are common between crps patients and the rodent model will facilitate the development of correlational outcomes between preclinical and human studies. summary/conclusion: identification of similarities and differences between crps patients and animal models will aid in directing future studies at clinically relevant aspects of crps aetiology and identifying potential diagnostic markers for crps patients. extracellular vesicle-based liquid biopsy in acute myeloid leukaemia: a reliable source of residual disease biomarkers? introduction: acute myeloid leukaemia (aml) is an haematopoietic stem cell disorder with a poor -year survival rate. monitoring of measurable residual disease (mrd) in aml patients receiving chemotherapeutic treatment is useful to assess therapy response and predict relapse. indeed, many different leukaemia associated immunophenotypic protein markers (laips) are presently useful to detect mrd. nevertheless, their analysis currently requires invasive bone marrow aspirates, thus severely hindering real-time monitoring of the disease. therefore, alternative peripheral blood-based methods are highly desirable for an easy, real-time and costeffective monitoring of aml progression. this work aims was to assess the feasibility of a peripheral blood ev-based liquid biopsy method for aml disease monitoring, based on the detection of laips with a known negative impact on the prognosis of aml. methods: the profile of evs isolated from paired samples from aml patients' blood plasma collected at diagnosis, complete remission (and some at relapse) was compared and correlated with clinical data. for that, a size-exclusion chromatography (sec) method was optimized to isolate the circulating evs from the blood plasma. the evs of the paired aml patients' blood samples were then characterized according to their size (dls/nta), morphology (tem), proteinto-lipid ratio (lowry/sulpho phosphovanillin assay), surface charge (zeta-sizer) and protein cargo (western blot). results: sec allowed the isolation of size-resolved plasmaderived evs from the peripheral blood of aml patients. isolated evs had a size ranging from nm to nm with an intact morphology, expressing ev-associated markers such as hsp , cd , cd and cd . size-resolved evs also had a differential expression of mitofilin, actinin- , syntenin- and annexin-xi proteins. several laips were detected in the isolated evs and their relative abundance changed throughout the stage of the disease. summary/conclusion: our preliminary data shows that aml patients' circulating evs carry relevant immunophenotypic protein markers, which might predict aml clinical outcome. introduction: cell plasticity regulated by the balance between the epithelial-to-mesenchymal transition (emt) and met is critical in the metastatic cascade. extracellular vesicles (evs) may play an important role in this balance by shuttling molecular cargos into recipient cells. this study aims to evaluate the feasibility of profiling mrnas of parental prostate cancer (pca) cells with different phenotypes and their daughter evs using the nanostring low rna input ncounter assay. methods: pc -epi and pc -emt cell lines representing epithelial and mesenchymal phenotype, respectively, were generated from original pc cell line. the cell culture supernatant was first pre-cleared for any dead cells and debris by centrifugation at × g for min. without disturbing the pellet, the supernatant was then transferred to a fresh ultracentrifuge tube and centrifuged at , × g for min at °c. the remaining supernatant was then centrifuged to isolate the evs at , × g for min at °c. the evs pellet was further washed in × pbs followed by a second centrifugation at , × g for min at °c. the final evs pellet was resuspended in × pbs for subsequent characterization (transmission electron microscopy, nanoparticle tracking analysis and western blot) and ncounter assays. the total rna of cells and their daughter evs were assayed by the ncounter pancancer progression panel to determine expression of selected mrnas. the nanostring ncounter low rna input kit with the multiplex gene primer pool was used for the pre-amplification of mrna and overnight hybridization with the pancancer progression panel. each sample type was submitted to the assay in biological triplicate. results: when comparing all samples, eisen cluster analysis separated all the cells and all evs into two groups, regardless of their phenotypes. in subgroup analysis, the expression patterns between pc -epi and pc -emt cells were significantly different. clec b, kdr, crip , il ra , cc d b were significantly upregulated in pc -emt cells, while cxcl , epcam, esrp , tgfb , cdh , s a , ovol were significantly downregulated in pc -emt cells. the expression patterns between pc -epi and pc -emt evs were also significantly different. tbx , cav , col a , slc a , myc, itgb , timp , camk b, ptgds, p h , itgb , vim, stat were all significantly downregulated in pc -emt cell derived evs. summary/conclusion: the nanostring low rna input ncounter assay can provide reliable mrna expression profiling of evs. the mrna expression patterns are very different between cells and their daughter evs. both cells and evs with different phenotypes have different gene expressions. cancer cell-derived evs containing alphav beta integrin regulate cd , il- and il- levels in peripheral blood mononuclear cells introduction: extracellular vesicles (evs) mediate communication in the tumour microenvironment and play an important role in cancer progression. previously, we have shown the enrichment of alphav beta integrin in small extracellular vesicles (sevs) isolated by differential ultracentrifugation and iodixanol density gradient from pc prostate cancer cells. we have also shown in the past that alphav beta -positive sevs induce peripheral blood mononuclear cell (pbmc) polarization by increasing the expression of pro-tumorigenic m markers, such as cd and cd . finally, we have demonstrated that down-regulation of alphav beta integrin up-regulates the stat -interferon stimulated genes (isgs) pathway in cancer cells and in sevs released by them. methods: in order to investigate whether prostate cancer cell-derived vesicular stat has a causal effect in pbmc polarization, we down-regulated alphav beta and stat in prostate cancer cells derived sevs using sirna as well as crispr-cas strategies. the sevs isolated from these cells were used to analyse m polarization by measuring the levels of cd in pbmc. the results show that sevs lacking alphav beta inhibit cd levels in pbmc in a stat -independent manner. analysis of cytokines released by pbmc upon incubation with sevs lacking alphav beta , show that pbmc selectively up-regulate the levels of il- and il- , which are predominantly anti-tumorigenic cytokines. in contrast, sevs lacking alphav beta do not upregulate pro-angiogenic cytokines, such as vegf. summary/conclusion: these findings suggest that cancer cell-derived sevs containing alphav beta integrin promote a pro-tumorigenic pbmc phenotype in the tumour microenvironment by regulating cd , il- and il- levels. introduction: the recognition of donor-mhc molecules by recipient t cells triggers the immune response leading to rejection of allografts. our recent studies have documented the presence of high numbers of recipient apcs displaying donor-mhc molecules (cross-dressed) on their surface in the lymphoid organs of mice after skin, heart or pancreatic islet transplantation. in addition, we have reported that acquisition of allogeneic mhc molecules by host apcs (mhc crossdressing) is mediated by donor-derived extracellular vesicles (evs) trafficking through blood and lymphatic vessels (marino et al. science immunology, ) . in the present study, we investigated the ability of allogeneic evs and allo-mhc-cross-dressed cells to initiate a t cell alloresponse in vitro and in vivo. methods: evs were isolated (using differential centrifugation) from balb/c bone marrow derived dendritic cells (bmdcs). these evs were used to cross-dress b splenocytes in vitro. the transfer of donor mhc class i and ii on b cells was analysed by imaging flow cytometry. next, t cells from b mice were cultured in vitro with either allogeneic bmdc-derived balb/c evs or b spleen cells crossdressed with allogeneic balb/c mhc. alternatively, × balb/c or b bm derived evs or × balb/c bm cells were injected iv to b mice. in both cases, the t cell response was assessed by activation markers detection, infg production and cell proliferation. results: apcs cross-dressed with allogeneic mhc molecules can trigger a pro-inflammatory direct alloresponse by t cells in vitro and in vivo. on the other hand, allogeneic evs alone were only able to induce early t cell activation but not proliferation in vitro. furthermore, injection of mice with allogeneic evs alone could induce some but suboptimal alloresponse in vivo and only when administered with complete freund's adjuvant. summary/conclusion: blocking donor evs release and subsequent recipient apc cross-dressing may represent a promising target to selectively inhibit anti-donor t cell inflammatory responses thus achieving long-term allograft survival. funding: r dk . antifungal antibiotic activity of outer membrane vesicles from adherent lysobacter enzymogenes c against therapeutic and biocontrol targets. rutgers university, new brunswick, usa introduction: lysobacter enzymogenes is a predatory gram negative bacterial species being studied for biocontrol activity against fungi. planktonic l. enzymogenes c produces outer membrane vesicles (omv) harbouring small molecule antifungal antibiotics (meers et al. ) . we show here that the more biologically relevant surface-associated c exerts remote antifungal activity via omv as well. the results have important consequences regarding the natural mechanism of biocontrol of fungal pathogens by c as well as isolation and delivery of therapeutically relevant antifungal compounds. methods: omv were isolated from scraped adherent c culture on agar by similar methods to meers et al . omv were stained in some cases with fluorogenic syto dna stain for microscopic observation. fungal growth was monitored via turbidity readings in liquid culture or photomicrographs on agar. c was also grown on polycarbonate filter membranes with defined pore sizes to monitor growth of fungal cells on the opposite side. vesicles were also labelled with an amine-reactive probe alexa- and washed x by sedimentation. binding of labelled omv to fungal cells was observed by epifluorescence microscopy. results: syto -stained vesicles from surface-adherent c were similar to previously observed~ nm vesicles (meers et al., ) . the isolated vesicles inhibited growth of saccharomyces cerevisiae or candida albicans in liquid cultures at similar potency and were active against the filamentous species fusarium subglutinans grown on agar or maize leaves. c cultures grown on filters with nm pore size but not nm were able to inhibit the hyphal growth of f. subglutinans on the opposite side. similarly c on filters with a nm pore size were able to inhibit growth of c. albicans. observation of fluorescently-labelled c omv after interaction with c. albicans showed binding specifically to hyphae or pseudohyphae and for f. subglutinans to the growing hyphal tips. summary/conclusion: the omv of c specifically bind and inhibit the growth of fungal hyphae of various species without direct c cell contact. these data elucidate mechanisms of biocontrol and suggest strategies for production of therapeutic antifungal antibiotics. meers et al. elucidating the cellular uptake and tissue distribution mechanism of cell derived vesicles, a novel therapeutic carrier hui-chong lau a , jae young kim a , jin-hee park a , jun-sik yoon a , min jung kang a and seung wook oh b a mdimune inc, seoul, republic of korea; b mdimune inc, seattle, usa introduction: cell derived vesicles (cdvs) are emerging as a novel therapeutic carrier. one of the crucial factors in the development and therapeutic applications of cdvs is to understand the precise mechanism by which vesicles find and enter the target cells. in this study, we aim to investigate the uptake mechanism of cdvs produced from natural killer (nk) cells using a manufacturing process established at mdimune inc. both in vitro uptake assay and in vivo distribution analysis were performed to provide precise insights into how cdv exert its effect at the cellular level. methods: nk cells were mainly used to produce cdvs. breast cancer cells, bt , and human and rodent endothelial cells, with a varying degree of icam- expression, were used to determine the effect of lfa- expressed on the surface of nk-cdvs in cellular uptake using facs and confocal imaging analysis. next, various inhibitors for uptake pathways, such as phagocytosis, dynamin dependent endocytosis, and receptor mediated endocytosis, were used to understand the underlying mechanism of cellular uptake of cdvs. biodistribution profile of cdvs were characterized using both normal and tumour xenograft models by ivis imaging. results: using a recently established manufacturing process, we demonstrate that nk-cdvs can efficiently enter the target cells. this study also shows that the cellular uptake depends on the molecular interaction between icam- and lfa- . in vivo distribution profile of nk-cdvs are also assessed using various tumour models. furthermore, we present a cellular uptake mechanism involved in the entrance of cdvs into the target cells. summary/conclusion: this study demonstrates that the cdvs produced at the manufacturing scale can be easily taken up by cells via specific cellular pathways. this finding will facilitate the development of more efficient therapeutics for cancer and other debilitating diseases. myofibroblasts-derived microvesicles increase dermal fibroblasts collagen production through plgf- syrine arif, sebastien larochelle and véronique j. moulin chu de québec -université laval, loex, québec, canada introduction: a proper wound healing of the skin involves angiogenesis, extracellular matrix (ecm) remodelling and re-epithelialization. these three mechanisms require well-organized interactions between different cell populations. a key role in this context is played by myofibroblasts (wmyo), a cell population mainly differentiated from dermal fibroblasts. these cells contract wound edges and synthesize new ecm. we previously showed that myofibroblasts predominantly produces microvesicles (mvs) and can favour angiogenesis. however, proteomic analysis of mvs from our previous studies indicated some molecules that can potentially be implicated in ecm remodelling. in this study, we evaluated whether myofibroblasts-derived mvs could affect dermal fibroblasts who are highly responsible for ecm regulation. methods: mvs were isolated by differential centrifugation of medium collected from wmyo cells. number and size of mvs were characterized by transmission electron microscopy and nanosizer. multiplex assays of cytokines were evaluated in mvs samples, wmyo and mvs-depleted medium. to examine the interaction of mvs with fibroblasts, we evaluated the uptake of mvs isolated from wmyo transduced with a fluorescent protein. we then treated fibroblasts cultures with mvs or a selected cytokine for days and evaluated collagen production. lastly, we neutralized the selected cytokine in mvs samples before evaluating collagen production. results: plgf- was the cytokine detected in mvs samples in large amount ( . ± . pg/µg proteins in mvs). fibroblasts treated with mvs or plgf- significantly stimulated pro-collagen i level production with a fold change of . ± . and . ± . . moreover, the neutralization of plgf- present in mvs significantly inhibited the production of pro-collagen i by dermal fibroblasts. summary/conclusion: our results indicated that mvs influence fibroblasts pro-collagen production through plgf- signalling. funding: this work was supported by natural sciences and engineering research council of canada (nserc) (rgpin - ); les fonds de recherche du québec-santé (frqs) via the research centre funding grant; the quebec cell and tissue and gene therapy network-thécell (a thematic network supported by frqs). structural insights on fusion mechanisms of extracellular vesicles with model plasma membranes introduction: extracellular vesicles (evs) represent a potent intercellular communication system. while their functional biological properties are more and more investigated, the biophysical aspects of their interaction with recipient cells are often overlooked. small size ( to a few hundred nanometres in diameter) of evs and their heterogeneous origin still pose a great challenge for their isolation, quantification and biophysical/biochemical characterization. in particular the complex network of interactions between differently classified evs and recipient cells remains to be further revealed. here we deeply investigate the fusion mechanism between evs and a model plasma membrane system by an interplay of different structural/morphological techniques to get a molecular description of the interaction helping to clarify the role of different membrane compartments on the evs uptake mechanism. standardized protocols and good manufacturing practice conditions were employed to derive highly stable vesicles of defined size and reproducible molecular profiles from umbilical cord multipotent mesenchymal stem (stromal) cells. after a thorough biophysical and biochemical characterization of evs non-contact liquid imaging atomic force microscopy (afm) and, in parallel, neutron reflectometry (nr), as well as small angle neutron scattering (sans) experiments were performed on evs to determine their interaction with model plasma membranes in the form both of supported lipid bilayers and suspended unilamellar vesicles of variably complex composition. results: we observed that evs tend to fuse with the model membranes with a preferential interaction with the external layer of the fluid membrane. moreover we revealed a stronger interaction with the liquid ordered domains, strengthening the hypothesis of a critical role of lipid rafts in fusion mechanisms. summary/conclusion: our results on the analysis of the interaction of evs with artificial lipid membranes could provide insights on the internalization mechanisms of evs. the approach shown here can be further extended to convey incremental complexity, adding glycolipid and membrane proteins to the model lipid bilayers. this approach combined with data on the specific biological function of each ev subpopulation as retrieved by standard functional assays, will turn useful to select the crucial molecular aspects of evs internalization by cells. introduction: platelet-derived extracellular vesicles (pev) are the most abundant circulating extracellular vesicle (ev) and exhibit platelet-like properties, hence the original term "platelet dust". direct phenotyping of ev surface markers within biofluids is challenging often requiring time-intensive purification steps that can significantly alter resultant ev population characteristics. the exoview™ (nanoview biosciences) specifically captures ev sub-populations and was used to characterise the ev content of platelet free plasma (pfp) and a potential novel haemostatic agent designed for the treatment of severe trauma and haemorrhage, platelet enhanced plasma (pep). methods: freeze-thaw cycling of platelet rich plasma/ expired platelet concentrates was followed by centrifugation to remove platelet remnants and yeilded pep. pfp controls were prepared by double centrifugation ( g for minuntes followed by , g for minutes). rotational thromboelastometry (rotem) and calibrated automated thrombography (cat) were used to assess ev driven haemostasis and thrombin generation. a dilutional and hypothermic model of coagulopathy was designed to assess pep. ev capture arrays comprised of anti-cd , anti-cd , anti-cd and anti-cd were used (exoview™, nanoview biosciences). captured vesicles underewent interferometric imaging and were quantified, sized and further probed with fluorescent tetraspanin markers, annexin-v and intravesicular markers. results: pep is highly procoagulant, exhibits enhanced thrombin generation and can restore haemostasis in a dilutional model of coagulopatic whole blood. pep can be generated from expired platelet concentrates, potentially allowing for upscalable production. the predominant vesicle population were pev with a large cd /cd population that contained a smaller subpopulation of phosphatidyserine positive procoagulant vesicles. pfp as expected has a much lower number of pev and a cd positive ev population. summary/conclusion: pep is a unique resuscitation fluid containing high pev levels for the potential treatment of severe trauma and haemorrhage. exoview measurements can be performed in unpurified plasma and may be useful for measuring circulating ev in health and disease. funding: defence and security accelerator, dstl therapeutic effect of exosomes in mice model of autism daniel offen a , reut horev a , nisim perets a , ehud marom b , uri danon b and yona gefen b a tel aviv university, tel aviv, israel; b stem cell medicine ltd., jerusalem, israel introduction: during the recent decade, exosomes that derived from mesenchymal stem cells (msc-exo) have been spotlighted as a promising therapeutic target for various clinical indications, including neurological disorders. we have previously shown that intranasal administration of msc-exo, cross the bbb and significantly ameliorate autistic-like behavioural phenotype in btbr and shank animal models of autism, representing a potential therapeutic strategy to reduce symptoms of autism spectrum disorder (asd). our objective is to study the mechanism of action and the cellular pathways in which the msc-exo activate their target, we performed rna sequencing analysis of primary neurons isolated from shank mice treated with msc-exo. methods: primary neuronal cell cultures were prepared from newborn shank homozygotes mice model of autism. cultures were treated with msc-exo ( ^ particles/ul), isolated from human adipocytes, followed by rna sequencing. the alterations in gene expression between the treated and intact neurons were analysed for gene ontology and pathways and were also compared to proteomics analysis of the msc-exo in order to find regulatory proteins that may lead to these differences. results: bioinformatic analysis revealed several up-regulators proteins that might be responsible for the increase in anti-inflammatory and protective factors seen in the mice neurons treated with msc-exo. one of them is bdnf which is known as an essential growth factor responsible for neuroprotection and neurogenesis. importantly, no difference in the genetic expression of cancer-related genes was identified following msc-exo treatment indicating for their safety. summary/conclusion: our data suggest that adipocytederived msc-exo carry therapeutic potential in asd via alternation in gene-expression related mainly to immuno-modulation, reduce neuroinflammation and increase neuroprotection and neurogenesis. the beneficial effects of the exosomes treatment in mice models is being translated into a novel, easy to administer, a therapeutic strategy to reduce the symptoms of asd. introduction: autologous blood-derived products gain increasing focus in regenerative medicine, especially in orthopaedics and osteoarthritis therapy. this disease is characterised by cartilage degradation and inflammation among other symptoms, which are targeted by conventional therapies, but genuine cartilage regeneration is rarely achieved. citrate-anticoagulated platelet rich plasma (cprp) is often clinically applied to stimulate soft and hard tissue healing. recently, cell-free alternatives to cprp including hyperacute serum (hypact™ serum) have been developed. cprp and hypact™ serum contain specific profiles of growth factors, however, they also contain extracellular vesicles (evs) that harbour signal molecules including mirna. methods: evs were enriched by ultracentrifugation (uc) followed by size exclusion chromatography (sec) to obtain purified evs. particle size and concentration of each fraction was measured by nanoparticle tracking analysis (nta). fractions with the highest amount of particles were pooled and concentrated via uc, before mirna expression was assessed via screening with a panel of mirna-specific primer pairs by rt-qpcr. presence of evs was confirmed by cryoelectron microscopy. results: the ev concentration tended to be lower in hypact™ serum than in cprp as determined via nta. similarly, lower diversity of mirna species was found in hypact™ serum than cprp evs. around % of detected mirnas were found in both blood products, whereas only % of mirnas were shared between evs from cprp and hypact™ serum. while mirnas such as mir- were consistently depleted in evs compared to the corresponding blood product, others like mir- a were in enriched in hypact™ evs, but not cprp evs, indicating release of specific mirnas via evs in response to clotting. summary/conclusion: although the purification resulted in high loss of evs, we identified specific mirnas enriched in evs from cprp and hypact™ serum. their functional spectrum with respect to osteoarthritis therapy focuses on inhibition of inflammation, inhibition of tissue remodelling via matrix degrading enzymes as well as preventing senescence. this renders blood product derived evs as interesting candidates for in vitro and in vivo testing with respect to cartilage regeneration. funding: the work was jointly supported by the european fund for regional development (efre) and the fund for economy and tourism of lower austria, grant number wst -f- / - . protective role of shiitake mushroom-derived exosome-like nanoparticles in d-galactosamine and lipopolysaccharide-induced acute liver injury in mice baolong liu, xingyi chen and jiujiu yu university of nebraska lincoln, lincoln, usa introduction: fulminant hepatic failure (fhf) is a rare, life-threatening liver disease with poor prognosis. new therapeutic interventions are urgently needed to treat this disease. administration of d-galactosamine (galn) and a low dose of lipopolysaccharide (lps) triggers acute liver damage in mice, which simulates many clinical features of fhf in humans and therefore is widely used to investigate the molecular mechanisms and potential therapeutic interventions of fhf. recently, suppression of the nucleotide binding domain and leucine rich repeat related (nlr) family, pyrin domain containing (nlrp ) inflammasome was shown to alleviate the severity of lps/galn-induced liver injury in animal models. therefore, the goal of this study was to identify food-derived exosome-like nanoparticles (elns) with anti-nlrp inflammasome function to potentially control fhf. methods: seven commonly consumed mushrooms were used to extract elns, which were examined for anti-nlrp inflammasome activities in primary macrophages. results: it was found that these mushrooms contained elns composed of biomolecules including rnas, proteins, and lipids. among these mushroom-derived elns, only shiitake mushroom-derived elns (s-elns) strongly inhibited nlrp inflammasome activation by blocking the inflammasome assembly. this inhibitory effect was specific for the nlrp inflammasome because s-elns had no impact on activation of the absent in melanoma (aim ) inflammasome. s-elns also inhibited the secretion of interleukin (il)- and both protein and mrna levels of the il b gene in macrophages. remarkably, pre-treatment of s-elns protected mice from lps/galn-induced acute liver injury. summary/conclusion: therefore, s-elns, identified as potent inhibitors of the nlrp inflammasome, represent a new class of agents with the potential to combat fhf. approaches to assess clinically available exosomes' quality and safety introduction: recent adverse events resultant from an exosome product use in a nebraska clinic, highlight the importance of assuring product quality and safety standards. an often-overlooked safety risk is ancillary reagents remaining within a finished product. when processes to obtain exosomes utilize cow proteins such as fbs or bovine sera albumin, failure to adequately remove these can result in significant adverse allergic reactions. we evaluated different exosome products to test the hypothesis that purity of some products may not be consistent with actual product quality and safety profiles claimed. methods: three different exosome products (manufacturer a, b, and c) were prepared per their instructions for use. sample source identity was blinded from assaying scientists. an independent cro service was used to conduct the experiments to ensure unbiased assay execution and data collection. exosome suspensions were sampled undiluted for bovine protein content using commercially available bovine secretome protein arrays from ray biotech. a total of different proteins found in bovine serum were quantified. results: six of proteins were not detected in any sample. of array antibodies were found to cross react with human antigens. of the bovine proteins that were acceptable for analysis, manufacturers a, b, and c exosomes contained of proteins, of proteins, and of proteins, respectively. concentrations of individual bovine proteins ranged from . to , . ng/ml. summary/conclusion: these results indicate manufactures a and b are selling potentially dangerous products. the successful implementation of exosome products into the clinic requires equivalent demonstrations of safety and quality. this requires adopting strict quality standards and safety testing during their production. physicians must require safety data prior to clinical use. engineering pro-healing ev cargo using a closed-system bioreactor. introduction: chronic wounds, including diabetic ulcers and pressure ulcers, are difficult and expensive to treat. while tissue engineering approaches have largely failed as a viable treatment for chronic wounds, we hypothesize that stem cell-derived extracellular vesicles (evs) may provide several unique advantages. zenbio, inc has developed a methodology to generate commercial-scale stem cell-derived exosomes using a closedsystem hollow fibre bioreactor capable of continuous ev production. additionally, we have shown that by manipulating the cellular environment, we can improve the pro-healing capacity of the evs.this technology leverages the complex healing capabilities of stem cells without the obstacles of replicating cells. methods: we have demonstrated that a mild heat shock resulted in evs enriched for stress-response proteins and increased pro-healing activities in vitro. we extended this innovative approach to include stimulating adipose stem cells with combinations of heat shock and growth factors to generate differential extracellular vesicle packaging that enhances pro-healing activity. to monitor reproducibility across lots and batches, we rigorously characterized tuned evs for particle size and number as well as surface marker and cargo composition. results: our results using tuned evs showed efficacy using cellular models of inflammation, motility, vascularization, collagen production and metalloprotease activity. we utilized an established murine model of pressure ulcers to assess the in vivo efficacy of the tuned evs. these studies showed a single injection into the wound site activated a more rapid wound closure, increased collagen deposition and reduced dermal thickness compared to saline control. summary/conclusion: these data strongly support our hypothesis that evs may be selectively modified to improve their wound healing activity by modulating the culture or tissue microenvironment. future studies will use chronic wound models to determine optimal dosing and routes of administration. introduction: mesenchymal stem cell-derived extracellular vesicles (msc-evs) can reduce inflammation, promote healing and improve organ function thereby providing a potential "cell-free" therapy. prior to clinical translation, there is a critical need to synthesize existing preclinical evidence supporting their efficacy. this systematic review provides the most comprehensive evidence map of methods, safety and efficacy for msc-ev research to date. methods: medline and embase were systematically searched for in vivo interventional studies using msc-evs. two reviewers extracted data for: ) methodology, ) study design, ) intervention details and ) efficacy/ adverse events. results: after screening articles, studies met our eligibility criteria. msc-evs were used to treat a variety of diseases including renal ( %), neurological ( %) and cardiac ( %) conditions. benefits were described in % of studies across all organ systems and adverse effects were seen in only three studies; two showing tumour growth. however, several key methodological concerns were evident. based on size criteria for ev subtypes (exosomes/small evs~ - nm, microvesicles~ - nm) only % of studies used appropriate nomenclature. ultracentrifugation ( %) and isolation kits ( %) were the most common isolation methods despite marked differences in yield and purity. evs were inconsistently dosed by protein ( %), particle number ( %) or cell count ( %), hindering inter-study comparisons. two-thirds of studies used xenogeneic evs suggesting immunocompatibility. techniques to determine size, protein markers and morphology was highly heterogeneous, and only and studies met the characterization standards recommended in the misev and guidelines, respectively. finally, % of studies did not incorporate randomization which represents a high risk for bias and only a quarter performed biodistribution studies. summary/conclusion: this systematic review reveals extensive heterogeneity in methods and intervention details for animal studies of msc-evs. nonetheless, nearly all studies showed significant benefits in a wide range of distinct conditions. the knowledge gaps we identified highlight important opportunities for improving preclinical design and the need for more standardized approaches in this growing field of ev therapeutics. msc-exosomes as next generation therapeutics for atopic dermatitis exocobio inc, seoul, republic of korea introduction: atopic dermatitis (ad) is a systemic inflammatory disease with unknown cause. recent approval of a targeted therapy, dupilumab, opens new era of ad management. however, current therapeutic options for ad are only targeting inflammation, a component of ad vicious cycle including itching and barrier disruption. human mesenchymal stem cells (mscs) have been highlighted as a novel therapy for suppressing allergic progress of ad in clinical studies. unfortunately, phase iii clinical study of human umbilical cord blood mscs for ad was failed with unknown reason. previously, our group reported that exosomes derived from human adipose tissue-derived mscs (asc-exosomes) alleviated the pathological symptoms in a murine ad model with concomitant reduction of inflammation. methods: our group has further investigated the therapeutic effects of human asc-exosomes in an alternative murine ad model with skin barrier defects. large scale isolation of asc-exosomes was performed by tangential flow filtration and isolated asc-exosomes were characterized according to the recommendation by the isev. the protein and lipid cargo were also analysed. results: we found that asc-exosomes induced restoration of skin barrier by inducing de novo lipid synthesis and reduced the levels of multiple inflammatory cytokines. in addition, asc-exosomes suppressed the expression of itching-causing cytokines. transcriptomic analysis of ad skin lesions revealed that asc-exosomes reversed the abnormal expression of genes functioning in skin barrier function, lipid metabolism, and cell cycle. summary/conclusion: taken together, asc-exosomes could be a promising cell-free therapeutic option for the treatment of ad, which affecting inflammation, skin barrier function, and itching. cell derived vesicles: unravelling the science of novel vesicles with therapeutic promises introduction: cell derived vesicles (cdvs) are nanosized vesicles produced by serially extruding cells through small pores. a growing number of studies have implicated their therapeutic potentials, with superior yield compared to other extracellular vesicles (evs). however, two key objectives remain to be accomplished to demonstrate the utility of cdvs in clinical applications. first, a manufacturing process has to be developed to allow a large-scale production of cdvs. next, these novel vesicles need to be thoroughly characterized at multiple levels. methods: manufacturing-scale extruders were developed to allow extrusion of large volume of cell suspension in a single process. cdvs with approximately - nm in diameter were obtained by a serial extrusion. crude samples were then purified using the tangential flow filtration method to further remove cellular impurities. finally, physical and biochemical characteristics of purified cdvs were analysed using dls, nta, cryo-em, and facs analysis. additionally, cdvs were subject to multi-omics profiling to comprehend our understanding in molecular contents of cdvs. both mesenchymal stem cells (mscs) and natural killer (nk) cells were used for this study. results: in this study, we first demonstrate that the large-scale extruder efficiently produce cdvs with consistent quality at the scale that are compatible for clinical applications. surface marker and membrane composition analyses show that the cdvs are primarily formed using plasma membrane of source cells, with characteristic cellular markers enriched on the surface. comprehensive profiling of molecular components reveals the unique properties of cdvs as well as the underlying mechanism of formation of cdvs. summary/conclusion: recently, we have established a manufacturing process to enable clinical applications of cdvs. this study also highlights key molecular features of cdvs that can be harnessed to offer a powerful tool for regenerative and anticancer medicine. antifibrotic properties of extracellular vesicles derived from human induced pluripotent stem cells introduction: fibrosis is a pathological condition resulting from abnormal healing of various tissues. it is triggered by activation of fibroblasts and their subsequent transition to myofibroblast. in consequence, excessive deposition of extracellular matrix proteins leads to impaired organ function. to revert this process, we employed extracellular vesicles (evs) derived from human induced pluripotent stem cells (hipscs). as a model system, we used human cardiac fibroblasts (hcfs), since heart fibrosis constitutes a serious socioeconomic problem worldwide. methods: we isolated evs from conditioned media from three hipsc lines using ultrafiltration combined with size exclusion chromatography methods. next, we analysed the evs by nanosight, transmission electron microscopy, mass spectrometry and western blot methods. finally, we treated tgf-b-stimulated hcfs with hipsc-evs and evaluated expression of fibrosisrelated genes using real-time qpcr, western blot and fluorescence microscopy. results: we detected anti-fibrotic properties of hipsc-evs exerted on hcfs pre-stimulated with tgf-b. the evs significantly decreased the expression levels of acta , fn, tnc, snai , col a and reduced the number of myofibroblasts. the canonical profibrotic tgf-b-dependent smad / pathway was significantly attenuated in response to ev-treatment. summary/conclusion: in this study we demonstrated strong anti-fibrotic function of hipsc-evs. our findings can further be exploited for future medical applications to treat fibrotic diseases, such as heart fibrosis. funding: this work was supported by the project sonata : umo- / /d/nz / from the national science centre of poland to sbw. induced pluripotent stem cells-derived extracellular vesicles ameliorates d-galactosamine and lipopolysaccharide induced acute liver failure tianjin third central hospital affiliated to nankai university, tianjin, china (people's republic) introduction: liver failure is among the most causes of death in patients with liver disease. promoting liver regeneration will help patients with liver failure recover on their own. extracellular vesicles (evs) can released by induced pluripotent stem cells (ipscs) through paracrine effects and play a pivotal role in inter-cellular communication in the treatment of disease. in this study, we investigated whether the ipscs-evs have therapeutic effects on acute liver failure. methods: the ipscs-evs were isolated by ultracentrifugation and identified using nanoparticle tracking analysis, transmission electron microscopy and western blotting. the isolated ipscs-evs were administrated d-galactosamine-injured heprg cells in vitro and tail intravenously injected into d-galactosamine and lipopolysaccharide induced acute liver failure model mice in vivo, respectively. the anti-apoptosis role and potential mechanism were evaluated using flow cytometry and immunofluorescence staining. and alanine transaminase (alt) and aspartate transaminase (ast) in serum, h&e staining and tunel staining were explored the effect of ipscs-evs on liver injured and liver function. finally, high throughput sequencing of small rnas was performed to investigate mirna expression profiles in ipscs-evs and ipscs. results: the ipscs-evs that were all - nm, doublelayered and oval or round cellular vesicles and expressed the marker proteins cd , tsg and hsp . in vitro, the ipscs-evs treatment inhibited heprg apoptosis induced with d-galactosamine in a time-and dosedependent manner and promote the proliferation of hepatic stem cells. in vivo results showed that ipscs-evs significantly alleviated liver failure, improved liver function and prolonged the survival period. tunel assay showed that ipscs-evs suppress apoptosis of hepatocytes. moreover, mirna expression profiles analysis found that mir - a cluster and mir - cluster were enriched in ipscs-evs and ipscs. summary/conclusion: these findings indicated that ipscs-evs could ameliorate d-galactosamine and lipopolysaccharide induced acute liver failure to attenuate hepatocyte apoptosis, which will be benefit for therapy of liver disease in the future. msc-derived extracellular vesicles promote human cartilage regeneration by control of autophagy introduction: osteoarthritis (oa) is a rheumatic disease leading to chronic pain and disability with no effective treatment available. recently, allogeneic human mesenchymal stromal/stem cells (msc) entered clinical trials as a novel therapy for oa. increasing evidence suggests that therapeutic efficacy of msc depends on paracrine signalling. here we investigated the role of bone marrow msc-derived extracellular vesicles (bmmsc-evs), an important component of msc secretome, in cartilage repair. methods: to test the effect of bmmsc-evs on oa cartilage inflammation the tnf-alpha-stimulated human oa chondrocytes were treated with bmmsc-evs and inflammatory gene expression was measured by qrt-pcr after h. to access the impact of bmmsc-evs on cartilage regeneration the bmmsc-evs were added to the regeneration cultures of oa chondrocytes, which were analysed after weeks for glycosaminoglycan content by dmmb and qrt-pcr. paraffin sections of the regenerated tissue were stained for proteoglycans (safranin-o) and type ii collagen (immunostaining). results: we show that bmmsc-evs promote cartilage regeneration in vitro. treatment of oa chondrocytes with bmmsc-evs induces production of proteoglycans and type ii collagen and promotes proliferation of these cells. msc-evs also inhibit the adverse effects of inflammatory mediators on cartilage homoeostasis. our data show that bmmsc-evs downregulate tnfalpha induced expression of pro-inflammatory cox- , pro-inflammatory interleukins and collagenase activity in oa chondrocytes. the anti-inflammatory effect of bmmsc-evs involves the inhibition of nfκb signalling, activation of which is an important component of oa pathology. autophagy, a cellular homoeostatic mechanism for the removal of dysfunctional cellular organelles and macromolecules, is essential to maintaining chondrocytes survival and differentiation. the expression of autophagy regulators is reduced in osteoarthritic joints, which is also accompanied by increased chondrocyte apoptosis. our preliminary data indicate that bmmsc-evs carry mrna of natural autophagy inducers and promote autophagy in oa chondrocytes. therefore, we hypothesize that msc-evs exert their beneficial effects on cartilage regeneration by restoring the expression of autophagy regulators. summary/conclusion: in summary, our findings indicate that bmmsc-evs have ability to promote oa cartilage repair by reducing the inflammatory response and stimulation of oa chondrocytes to produce extracellular matrix, the essential processes for restoring and maintaining cartilage homoeostasis. thus, msc-evs hold great promise as a novel therapeutic for cartilage regeneration and osteoarthritis. large-scale preparations of small extracellular vesicles from conditioned media of mesenchymal stromal cells modulate therapeutic impacts on a newly established graft-versus-host-disease model in batch dependent manners introduction: extracellular vesicles (evs) harvested from supernatants of humane adult bone marrow-derived mesenchymal stem/stromal cells (mscs) can suppress acute inflammatory cues in a variety of different diseases, including graft-versus-host disease (gvhd) and ischaemic stroke. furthermore, they can promote regeneration of affected tissues. following a successful clinical treatment attempt of a steroid refractory gvhd patient, we intend to optimize msc-ev production strategies for further clinical applications. as we observed functional differences of independent msc-ev preparations in vitro, we aimed to adopt an in vivo gvhd model for the more advanced functional testing of different msc-ev preparations. methods: to this end we set up a bone marrow transplantation mouse model in which endogenous bone marrow was myeloablated by ionizing irradiation (iir). gvhd was induced by the transplantation of major histocompatibility mismatched allogeneic spleen-derived murine t cells. if not treated otherwise, myeloablated mice developed severe gvhd symptoms. results: the gvhd symptoms were effectively suppressed, when msc-ev preparations were applied at consecutive days, which exerted immune modulatory effects in a mixed-lymphocyte reaction assay. msc-ev preparations lacking in vitro immune modulating activities, however, hardly improved the symptoms of the gvhd mice. thus, our results demonstrate that not all msc-ev preparations harvested from adult bone marrow-derived mscs contain the same therapeutic potential. summary/conclusion: thus, successful transplantation of msc-evs into the clinics requires a platform allowing identification of msc-ev preparations with sufficient therapeutic, most probably immune modulating activities. funding: this research was funded by sevrit leitmarkt lifescience.nrw ls- - - g. introduction: malnutrition impacts approximately million children worldwide and is linked to % of global mortality in children below the age of five. severe acute malnutrition (sam) is associated with intestinal barrier breakdown and epithelial atrophy. extracellular vesicles including exosomes (evs; - nm) can travel to distant target cells through biofluids including milk. since milk-derived evs are known to induce intestinal stem cell proliferation, this study aimed to examine their potential efficacy in improving malnutrition-induced atrophy of intestinal mucosa and barrier dysfunction. methods: mice were fed either a control ( %) or a low protein ( %) diet for days to induce malnutrition. from day to , they received either bovine milk evs enriched using differential ultracentrifugation and sucrose gradient purification or control gavage and were sacrificed on day , hours after a fluorescein isothiocyanate (fitc) dose. tissue and blood were collected for histological and epithelial barrier function analyses. results: mice fed low protein diet developed intestinal villus atrophy and barrier dysfunction. despite continued low protein diet feeding, milk ev administration improved intestinal permeability, intestinal architecture and cellular proliferation. summary/conclusion: our results suggest that evs enriched from milk should be further explored as a valuable adjuvant therapy to standard clinical management of malnourished children with high risk of morbidity and mortality. funding: cb was generously awarded a catalyst grant from the centre for global child health at the hospital for sick children to support this work. the impact of spheroids culture on mesenchymal stem cells and ev production introduction: mesenchymal stem/stromal cells (mscs) are now widely believed as bio-factories releasing bioactive products responsible for their therapeutic effect, i.e. cytokines, chemokines, and extracellular vesicles (evs). mscs are highly sensitive to physical stimuli from their surrounding microenvironment and can change their characteristics in response to their environment. the application of d spheroids cell culture allows mscs to adapt to their cellular niche environment which, in turn, influences their paracrine signalling activity. we aim to determine how d and d culture microenvironments can modulate the ev production and investigate their anti-fibrotic activity. methods: for d culture, bone marrow-derived mscs were cultured on standard tissue culture plastic. for d culture, mscs were aggregated into spheroids using non-adherent -well plates and cultured with addition of . % methylcellulose. to collect conditioned media, both d and d mscs were cultured using serum free medium for days. evs were isolated by serial ultracentrifugation and were characterised on exoview platform which allows simultaneous detection of particle size and expression of cd /cd /cd . cell lysates were collected for mirna isolation and qrt-pcr was performed to analyse expression of candidate mirnas. to model the progress of lung fibrosis, human lung fibroblasts (hlfs) were cultured with tgf-β to induce fibroblast activation, subsequently exposed to d and d evs, and collagen production was measured. further, d and d msc-evs were added into human lung mscs isolated from healthy and ipf patients and cell proliferation was assessed using mts assay. results: d and d msc-evs have similar ev characteristics in terms of particle size and ev tetraspanin markers expression. exoview analysis showed expressions of cd /cd /cd and average particle diameters of < nm. on a cellular level, we identified a panel of anti/pro-fibrotic mirnas which are differentially expressed in d and d mscs. d and d msc-evs have similar anti-fibrotic activity shown by their ability to reduce collagen deposition in hlf cultures. both d and d msc-evs could promote cell proliferation on ipf lung mscs but no overall effect on healthy lung mscs. summary/conclusion: this concept of engineering the cellular microenvironment to promote ev production is as yet untouched and we foresee that in d cultures, we can culture mscs for longer timeframe and therefore maximising the overall ev production process. the outcome presents future potential for d culture of msc to increase the efficiency and feasibility of scalable ev production. outer membrane vesicles from photobacterium damselae subsp. piscicida: characterization and antigenic potential introduction: photobacterium damselae piscicida (phdp) is a gram-negative bacterium that causes a septicaemia in > fish species worldwide. it represents a major drawback for aquaculture, whose importance has been sharply growing as a food supplier. given the phdp massive mortality and widespread antibiotic resistance, an effective vaccine is highly needed. extracellular products (ecps) have an essential role in phdp virulence, containing important antigens. however, the ecps' identity remain undisclosed. in our efforts to dissect their composition, we found that they contain high amounts of outer membrane vesicles (omvs). these particles are potent weapons for bacteria and are being explored in the field of vaccinology, since omvs present antigens in native conformations and are strongly immunogenic, without requiring adjuvants. this potential associated to the urgent need for an anti-phdp vaccine prompted us to isolate and characterize the omvs shed by phdp. methods: in order to harvest high amounts of pure phdp omvs, a reproducible optimized protocol was developed: the bacteria-free supernatant from a phdp overnight culture is concentrated, dialysed and ultracentrifuged to collect the omvs. results: analysis of the obtained omvs preparations by transmission electronic microscopy and dynamic light scattering indicate that the main population of vesicles has sizes around - nm. proteomic analysis of the vesicles revealed the presence of the apoptogenic ab toxin aip that is known to play a major role in phdp virulence, a putative pore-forming toxin, a putative adhesin/invasin and several outer membrane proteins (omps), including a kda omp, predicted to be involved in iron acquisition, and other omps ( - kda), with an ompa-like structure that may act as adhesins. moreover, preliminary in vivo studies suggest that some of those proteins may have important roles for virulence, since injection of knock-out strains in sea bass induced a decreased mortality comparing to the wt strains. summary/conclusion: our findings suggest that omvs are a promising vaccine candidate and we are currently studying their biological activities and determining the antigenic potential of the identified proteins. introduction: whole body exposure to high doses of ionizing radiation (ir) can potentially be lethal if radiation injury is not diagnosed and treated expeditiously. when considering a non-invasive approach for the identification of biomarkers of ir exposure, we and others have studied molecules in plasma, serum, saliva, and urine. however, these matrices can potentially have significant background noise, obscuring potential biomarkers of biological importance. extracellular vesicles (evs) are fast becoming a platform for biomarker discovery in radiation research as well as in other pathologies. however, no groups have investigated the use of metabolomics to analyse evs derived from urine in the context of ir exposure. furthermore, the dominant protocols for ev isolation from urine require a large (up to ml) amount of starting volume, which may not be available for many studies. the aim of this study was to optimize ev isolation from rat urine and assess radiation-induced alterations in urine ev number and metabolic content. methods: as a proof of concept, we compared and optimized several ev isolation methods on small volumes of urine from male wag/rijcmcr rats exposed to gy or gy x-rays to the whole body except the hind leg. starting with either µl or µl of urine, we isolated evs using ultracentrifugation (uc) with filtration, size exclusion chromatography (sec), and a proprietary bead-based isolation method developed by a rd party provider. ev samples were characterized using nanoparticle tracking analysis. metabolomics profiles were measured using lc-qtof-ms. results: we found that sec resulted in the highest yield of evs from as little as µl of urine, while uc was the poorest performing. lc-qtof-ms analysis revealed that sec and uc had the most consistent identification of features, whereas the bead-based method contained artefacts likely as a result of the extraction method. we next used sec to isolate evs from a larger cohort of rats exposed to ir and analysed with ms. ev metabolic content will eb related to differences in survival and organ function between sham and irradiated groups. summary/conclusion: we conclude that sec is the preferred method for isolating evs from small volumes of urine for broad-based mass spectrometric analysis, and that the ev metabolome may be a sensitive and specific early indicator of radiation injury. introduction: there is growing evidence that contents (including rna and proteins) of exosomes may serve as biomarkers for early diagnosis and prognostic prediction of cancers. here we aim to identify potential protein markers for oesophageal cancer. methods: using our newly developed label-free exosome automated preparation system (leaps), exosomes were isolated from ml culture medium of various oesophageal cancer cells with different differentiation profiles and different sources of metastasis. exosomes from µl plasma of cancer patients at different clinical stages or with/without relapse and healthy controls were also prepared by leaps. matrix-assisted laser desorption ionization time-of-flight mass spectrometry (maldi-tof ms) was employed to directly analyse exosomes. protein identities of exosomal fingerprint peaks were tentatively assigned by correlation with top-down and bottom-up proteomics. results: start from ml culture medium or µl plasma, high-quality exosomes rapidly isolated by leaps are sufficient for maldi-tof mass spectrometry. it seemed that poorly differentiated cells showed more exosome release. maldi-tof ms fingerprints of exosomes in cells is cell line specific. ms profiles from poorly differentiated cells showed more peaks than that from highly differentiated cells. fingerprints also allowed classification of cancer cell lines through software mathematical analysis. we identified different numbers of significantly differentially expressed peaks in exosomes of various cancer cells. fingerprints of exosomes derived from the poorly differentiated cells showed more elevated peaks. top four peaks ( , m/z, , m/z, , m/z, , m/z) were commonly down-regulated in exosomes of most cancer cells. top four protein peaks ( , m/z, , m/z, , m/z, , m/z) that might be correlated to the differentiation profile of cancer cells were also identified. maldi-tof ms detection of exosomes in the plasma and clarifying identities of potential biomarker peaks will be done in the future. summary/conclusion: the combination of leaps and maldi-tof mass spectrometry provides a fast and high-throughput tool for exosomal marker discovery. potential biomarker identified in exosomes derived from oesophageal cancer cells or from plasma of cancer patients by this tool might be useful in cancer diagnosis and prognosis. fraction-based proteomic profiling of serum extracellular vesicles derived from cervical cancer patients introduction: current evidence indicates that extracellular vesicles (evs) can release from most of cell types and affect adjacent or distant cells by circulating in all bodily fluids. proteomic analysis of evs from clinical samples is complicated by the low abundance of ev proteins relative to highly abundant circulating proteins. size exclusion chromatography (sec) has been overcome as a method to deplete protein contaminants and enrich evs. methods: we collected serum of healthy women and cervical cancer patients with stage i-iii and then counted concentration and size distribution of the evs using nanoparticle tracking analysis (nta). differential ultracentrifugation combined with sec was used to isolate and purify evs from contaminant proteins. isolated evs were investigated their characteristic based on morphology using transmission electron microscope (tem) and on expression of cd , cd , cd protein markers using western blot analysis. fraction no. - of isolated evs in among sample groups were profiled by nano-liquid chromatography tandem mass spectrometry (nanolc-ms/ms) analysis. results: nta shows that the concentration of evs is increased in patients compared with healthy women. proteome profiles of evs isolated by sec were compared in each fraction. moreover, we detected molecular evidence for fraction-specific molecular pathways in connection with cancer progression and complied a set of protein signatures that closely reflect the associated clinical pathophysiology. summary/conclusion: these unique features in each fraction among sample groups would be the informative considering in order to select for further analysis as in vitro. introduction: recently, diagnostic biomarkers from exosomes by proteomic analysis have been reported, but it is required to optimize the isolation protocol to screen out more effective biomarkers. for serum-originated exosomes, it has been also reported to isolate them selectively, however, it is observed that a different method resulted in different protein profiles in -d gel electrophoresis. methods: we isolated exosomes by two discrete methods, using ultracentrifugation and magnetic separation. before ultracentrifugation and magnetic separation, precipitation using polymer materials was perforemd. the isolation of exosomes by these two methods followed by comparison of their size, total vesicle number, morphology, and protein markers. to identify protein biomarkers, proteomic analyis using -d gel electrophoresis was performed. results: both methods induced enrichment of exosome-specific proteins, but protein profiles in each exosome fraction was totally different. the protein profiles showd that the magnetic seperation following a polymer-based precipitation step was more efficient to screen out candidate biomarkers, which showed nearly protein profiles originated from exosomes. summary/conclusion: in our study, magnetic separation of exosomes from serum fraction was optimized for -d gel electrophoresis to observe identifiable biomarkers. an extracellular small rna-seq data processing pipeline optimized for high-performance computing chenghao zhu and angela zivkovic department of nutrition, uc davis, davis, usa introduction: a variety of rna species is found in extracellular biofluids such as blood, bile, and urine, carried by extracellular particles including extracellular vesicles (evs) and lipoproteins (e.g high density lipoproteins (hdls)). the extracellular rna (exrna) carried by evs and hdls is of great interest for two reasons: ) the exrna within different carriers could be diagnostic of the state of the tissues from which the particles originate, and ) exrna has been shown to affect gene expression in target cells. although the origin and functions of exrnas remain largely unknown, there is growing interest in exrna research for the development of diagnostics and new therapeutic targets. small rna sequencing is widely used to estimate the abundance of exrnas in biofluid samples. here we present a data processing pipeline for extracellular small rna sequencing. sequencing data are pre-processed through quality control, and then aligned to the endogenous genome to obtain the gene counts for various rna biotypes, including microrna, trna, rrna, piwi-interacting rna, long non-coding rna (lncrna) and protein coding rna. it also aligns sequencing reads to exogenous databases, including the ribosomal rna sequence database silva, and all sequenced bacteria genomes available on ensembl, to estimate the abundance of exogenous genes. results: we analysed a publicly available small rna-seq dataset of hdl from three systemic lupus erythematosus (sle) patients and three healthy controls using this pipeline. the mirna hsd-mir- , lncrna al . and ac . were elevated in sle patients compared to controls. exogenous rna reads mapped to bacteroidetes were also elevated in sle patients. summary/conclusion: our pipeline is able to process exrna sequencing data and estimate the abundance of major exrna species, as well as exogenous rna taxonomy. the pipeline is optimized for the job scheduler slurm, and can therefore utilize the full computational power of high-performance computers. the pipeline is publicly available on github (www.github. com/zhuchcn/excernapipeline). introduction: ibd is a chronic hyperinflammatory disorder that severely compromises the intestines. the aetiology of ibd is poorly understood. however, it has been associated with a dysregulation of the immune system and gut microbiota and with genetic and environmental factors. cumulative evidence indicates that evs play an essential role in modulating immune responses. recent research suggests that evs derived from dendritic cells, saliva and intestinal epithelial cells may be involved in the progression of ibd inflammation. however, little is known about the contribution of immune cells-derived evs with this pathology. the goal of this study is to shed light on the contribution of pbmc-derived evs on ibd pathogenesis. here we characterized and compared the composition of evs derived from pbmcs of ibd patients and healthy control. evs were isolated by differential centrifugations from the supernatant of pbmc activated with cd -cd beads for days in serum-free media. size and concentration were analysed using a nano sight instrument, while the presence of known evs markers (cd , cd , hsp ) was analysed by immunoblotting. whole evs proteome was performed by ms/ms and functional-enrichment analysis was done using funrich with uniprot database. results: proteomics analyses identified a total of proteins in the four groups. of those, ( . %) were present in both the ibd patients and control. this group of protein was composed of several ras-related proteins, eukaryotic initiation factors, granzyme, cd , tubulin, and serpins among others. patients' evs shared proteins in common such as proteasome subunit beta type- , t cell receptor beta, and the amine oxidase containing copper . interestingly, each patient sample had a unique group of proteins. among these are myeloperoxidase, neutrophil elastase, proteasome subunit alpha type- , and signalling lymphocytic activation molecule (slamf ). summary/conclusion: these preliminary studies show that the ev composition from pbmcs of ibd patients is specific and differs from a healthy control. this exclusive composition has the potential to be used as a biomarker for diagnostics and progression of the disease, and it could also provide new insights into our understanding of the cellular pathways involved in the pathogenesis of ibd. the studies were performed with corresponding irb approvals. proteomic analysis of exosomes isolated using precipitation and columnbased approaches introduction: exosomes are a subtype of small extracellular vesicles (evs) involved in various physiological and pathological processes with huge potential as biomarker resources or as therapeutic tools. although several exosome isolation approaches are available, complementary studies focusing on optimizing the methods for human bloodderived exosomes isolation and method-specific comparative exosomal proteomic profiles will be of clinical value. methods: blood-derived evs were isolated through precipitation-and column-based methods and characterized by transmission electron microscopy, nanoparticle tracking analysis and western blot analysis. serumderived exosomal proteomes were analysed by mass spectrometry (ms). the resulting proteomes were then overlapped with the proteomes obtained from exosome-related databases, to determine the % of similar content. in addition, bioinformatic analysis, including gene ontology (go) was carried out. results: both methodologies tested isolated particles with the expected morphology and size range, although the column-based method isolated a higher number of particles. about % of the exosomal proteins identified through ms overlapped with the proteomes extracted from the databases. go terms were similar for the proteomes isolated from the column-and precipitationbased methodologies. the top go terms identified for molecular function were ion binding, peptidase activity and enzyme regulator activity and for biological process were immune system process, transport and response to stress. further, partial least square analysis revealed a clear segregation of proteomes obtained by the distint methodologies and complementary statistical analysis revealed the proteins differently expressed. summary/conclusion: no major differences were found in the top biological processes and molecular function based on go analysis. nonetheless, the two approaches result in different evs yields and significant proteome differences were identified. characterization of distinct methods for blood-derived exosomes isolation can be useful in the context of evs potential in disease diagnostics/therapeutics. introduction: we and others are developing biomarkers for neurodegenerative diseases using neuronalenriched evs immunocaptured from a suspension of total plasma evs. here we assess how the isolation method for total evs affects the yield, purity and enrichment of neuronal evs. methods: for n = subjects, total evs were isolated by ev precipitation solution (exq), ev precipitation solution plus bipartite resin columns (exu) and size exclusion chromatography (qev) from . , . and . ml plasma, respectively. then, neuronal-enriched evs were immunoprecipitated using anti-l cam antibody. in total and l cam evs, we measured particle concentration by nanoparticle tracking analysis, protein concentration, and novel multiplex electrochemiluminescence immunoassays for tetraspanins cd , cd and cd on intact evs. results: for total evs, yield followed the order of exq > qev > exu, assessed by particle (p < . ) and protein concentrations (p < . ). l cam evs immunocaptured after exq showed -fold higher particle (p < . ) and fivefold higher protein (p < . ) concentrations compared to l cam evs after exu, and -fold higher particle (p < . ) and -fold higher protein (p < . ) concentration compared to l cam evs after qev. l cam evs after ev precipitation (exq) showed , and -fold higher cd , cd , and cd concentrations (p < . ) compared to l cam evs after exu, and , and -fold higher cd , cd , and cd concentrations (p < . ) compared to l cam evs after qev. l cam evs following different methods had equal purity assessed by ratios of particle/protein concentrations (p = ns), and tetraspanin/particle concentrations (p = ns). summary/conclusion: l cam ev immunocapture preceded by exq exceeded the yield of immunocapture preceded by exu or qev. recovered l cam evs showed equal purity by particle/protein and tetraspanin/particle metrics. neuronal enrichment results will be available by the time of isev. immunoprecipitation following exq, often considered impure, purifies final isolates as effectively as more onerous methods typically considered purer. balancing sensitivity, purity and scalability is essential for implementation of blood biomarkers in the clinical setting and may be achieved by combining techniques. funding: this research was supported in part by the intramural research program of the nih, national institute on aging. characterisation of breath exosomes: towards non-invasive diagnosis deanna ayupova a , renee goreham b and paul teesdale-spittle c a school of chemical and physcial sciences, victoria univeristy of wellington, wellington, new zealand; b university of newcastle, newcastle, australia; c school of biological sciences, victoria univeristy of wellington, wellington, new zealand introduction: breath-derived exosomes present new potential for non-invasive diagnosis of lung cancer. however, breath-derived exosomes have not been well characterized and methodology for their purification has not been optimised. in order to exploit their potential for diagnosis, it is first necessary to develop methods that reproducibly provide high quality pure exosomes from breath. in this study, we optimise methods for their isolation and characterise them in comparison to exosomes derived from cell culture models. methods: in order to characterize exosomes from exhaled breath condensate (ebc) it was first necessary to optimize methods for isolation of pure, intact, and high quality exosomes. to this end, isolation methods were optimised on cell-derived exosomes and then applied to ebc, yielding high quality exosomes from size exclusion chromatography (sec). ebc exosomes were compared with those from a and wi-cells using dls, tem, and cryo-sem. an immunoblotting-grid technique was used to validate the presence of exosome-specific markers cd and cd . protein content of exosomes were quantified and compared. results: sec-based isolation was more effective at isolation of pure and intact exosomes than ultracentrifugation, with the highest purity exosomes obtained in the middle fractions of the exosome-containing eluate. exosomes from ebc had a size range ( - nm), protein content ( - ug/ml) and molecular markers typical of cell-derived exosomes. summary/conclusion: breath-derived exosomes isolated through size exclusion chromatography are sufficiently pure for diagnostic purposes and are phenotypically similar to exosomes derived from other sources. we foresee their use in non-invasive diagnostics for lung cancer as an important future application. ligand-based exosome affinity purification (leap) is a rapid and reproducible method for the enrichment of functional evs introduction: platelet-derived extracellular vesicles (pevs) represent the next generation of therapeutic biologics as they enable a more refined and targeted approach when compared to crude blood derivatives currently used for treating diseases such as cancer, thrombocytopenia and chronic wounds. however, development of an ev-based therapeutic is hindered by the lack of a scalable, validated and reproducible purification process. in this study, pevs were isolated from activated platelet concentrates and purified using exopharm's ligand-based exosome affinity purification (leap) technology to produce a functionally active ev therapeutic. methods: platelet concentrates (n = ) were obtained from the australian red cross blood service and were activated by exopharm's proprietary process. activation was verified by measuring cd p using flow cytometry. the resulting platelet releasate ( ml) was subjected to leap purification to isolate pevs. for characterization, protein concentration was determined by a bicinchoninic acid assay, microfluidic resistive pulse sensing (mrps) was used to perform a particle count and transmission electron microscopy (tem) enabled visualization of ev morphology. key ev markers were detected using mass spectrometry (ms) and western blots. to confirm biological activity, human dermal fibroblasts were subjected to serum starvation for hours before treatment with pevs ( µg/ml). cell growth was recorded by the real-time xcelligence system and differences in proliferation were statistically analysed using a one-way anova. results: mrps and tem both revealed isolated pevs to be - nm in size. the final product was positive for platelet markers (cd , cd p) and key ev markers (tsg , alix, cd ). treatment with purified pevs significantly increased proliferation in serumstarved fibroblasts over hours. summary/conclusion: exopharm's leap technology is a rapid and reproducible purification process which produces pevs that adhere to misev guidelines and are functionally active. funding: all funding was through exopharm ltd (asx:ex ) a novel but simple method to obtain purified exosomes by one-step ultracentrifugation introduction: exosomes are extracellular vesicles (evs) that are derived from endosome membrane. they are usually - nm in diameter, actively secreted in most living cells. originally, exosomes were thought to act as cellular garbage disposals. recent studies showed that exosomes not only can serve as biomarkers for diagnosis, but also can be used as an ideal delivery vehicle for drugs in therapeutics. exosomes are natural carrier for mrna, mirna, sirna, protein, dna and peptide for long distance intercellular communication. isolation of exosomes is challenging due to their small size and heterogeneity. traditional differential ultracentrifugation method is still the gold standard for exosome purification. to further explore the potentials of exosomes being as the therapeutic delivery vehicle or diagnostic reagent, it is an essential step to purify them in high quality at high yield. methods: here, we report a novel method to obtain intact shape, high-quality and high purity exosomes with one-step ultracentrifugation by using "exojuice". results: data of nanoparticle tracking analysis (nta) and western blotting showed "exojuice" can yield exosomes with a simpler method to obtain higher purity exosomes in comparison to previous method of cushion ultracentrifugation using optiprep. summary/conclusion: our method can be used to purify exosomes from cell culture medium, serum, urine, saliva, and other biofluids. a straightforward device to extract apoplastic fluid from succulent fruits for higher purity of extracellular vesicles introduction: edible plants are emerging as a sustainable source for extracellular vesicle (ev)-based drug delivery vehicles. however, current isolation methods (e.g. grinding or squeezing) may cause destruction of plants' biostructures, and in turn leads to unwanted effects in downstream applications and complicates the study of nanovesiclescell. therefore, we designed a simple device that allows the extraction of apoplastic fluid (af) from succulent fruits, facilitating ev isolation as well as effective downstream applications. methods: an inner filter tube was designed to extract af with a determined membrane pore size. af was collected by low-speed centrifugation method and then filtered to eliminate the impurities from the cytoplasm and damaged cells. minced juice (mj) was homogenized by a blender and then centrifuged to remove large fragments. subsequently, the differential centrifugation method was employed to extract evs from af and mj. fourier-transform infrared spectroscopy (ftir), nanoparticle tracking analysis (nta), and transmission electron microscopy (tem) were performed to discriminate af, mj and their evs. results: the "spectroscopic" protein-to-lipid (p/l) ratio of af ( . ± . ) is significantly lower than that in mj ( . ± . ), showing the higher lipid contents in af, which may result from the loss of lipids in mj obtained from grinding or juicing methods. similarly, ftir showed the difference in p/l ratio between af and its evs ( . ± . and . ± . , respectively). nta showed the sharper peak and smaller vesicle size in the following order: mj ( . ± . nm), af ( ± . nm), af-derived evs collected at , × g and , × g ( . ± . nm and . ± . nm, respectively). furthermore, tem study indicated that the collected evs exhibited a typical lipid bilayer of extracellular nanovesicles. summary/conclusion: by using a reusable filter device, we successfully isolated af from succulent fruits, paving the way to collect plant evs without an interference of significant biodestruction or damaged cells, hence improving the purity of evs and facilitating downstream applications. moreover, this method is straightforward, reproductive, and can be potentially used in a large-scale production. method to simultaneously capture multiple classes of intact extracellular rna carriers including extracellular vesicles and lipoprotein particles introduction: extracellular particles including extracellular vesicles (evs), lipoproteins, and free proteins are carriers of extracellular rna (exrna), which has been shown to regulate cellular function. because these particles have different physiological origins, they have different rna signatures, so the first step to understanding the biology of exrna is to isolate individual particle fractions with high purity and efficiency. current methods for isolating evs are optimized for increased yields and purity of ev fractions but typically require multiple millilitres of starting plasma and do not capture the other exrna carrier particle types. methods that can capture evs from low starting plasma volumes and can also capture other exrna carriers simultaneously are needed for analysing samples from previously conducted large cohort studies, biorepositories, and in populations where sample volume is limiting. methods: we have developed a method adapted from lipoprotein isolation that requires only µl of starting plasma, and uses brief ultracentrifugation (uc) followed by fast protein liquid chromatography (fplc) to capture classes of purified exrna carriers including evs, ldl, hdl, lipidated albumin, proteins, and vldl/chylomicrons. we have validated successful capture of evs by microfluidic resistive pulse sensing (mrps, spectradyne), transmission electron microscopy (tem), and single particle interferometric reflectance imaging system (sp-iris; exoview) with optional fluorescence. results: we have observed . × particles per ml from a ml fplc fraction of evs measured from , events by mrps, confirming that evs are being captured by this method. there were also . × particles/ml and . × particles/ml in the two subsequent ml fractions that are known to contain lipoprotein particles, though these were measured from , events each. by tem we confirmed these observations that evs are eluting before lipoprotein particles with some evs eluting later in fractions containing lipoproteins. summary/conclusion: these results confirm the efficacy of the method in isolating multiple exrna carrier fractions simultaneously from a single ul plasma sample, making it amenable for the analysis of exrna in samples from large cohort studies, biorepositories, and vulnerable populations such as the elderly and young children. funding: nih/nia r ag ; nih ug ca - optimizing the isolation of placental mesenchymal stromal cell-derived extracellular vesicles in a d bioreactor system leora goldbloom-helzner a and aijun wang baaaaa a uc davis, davis, usa; b uc davis medical center, sacramento, usa introduction: extracellular vesicles (evs) derived from placental mesenchymal stromal cells (pmscs) have the potential to provide neuroprotection at sites of injury. however, a rate limiting step in ev research is the low yield, high technical time, and high cost of current isolation procedures. to address this inefficiency, we cultured pmscs in a unique bioreactor system to increase the absolute yield of evs per ml of media and per cell. future studies will determine if this system can improve pmsc ev yield without altering the demonstrated neuroprotective properties of pmsc-evs. methods: pmscs were cultured in the bioreactor for ten weeks. ev-conditioned media was collected weekly and evs were isolated through differential centrifugation. nanoparticle tracking analysis (nta) measured ev size and concentration. western blots tested for normal ev markers (cd , cd , and cd , calnexin(-)) and enzyme-linked immunosorbent assays (elisa) measured levels of characteristic growth factors in conditioned media including vascular endothelial growth factor (vegf), brain-derived neurotrophic factor (bdnf), and hepatocyte growth factor (hgf). results: evs remained consistent until week eight, after which a decrease in both ev size and concentration was seen. western blots revealed normal positive expressions of cd , cd , and cd and negative expressions of calnexin. levels of vegf, bndf, and hgf were comparable after weeks. cost analysis revealed an overall increase in ev yield for shorter labour time and lower material cost. summary/conclusion: this initial study uses a bioreactor system for a unique source of cells and has brought us closer to optimizing pmsc ev isolation protocols for increased yield, lower cost and time commitment, and maintained sample purity. preliminary data suggests the ev phenotype and cell secretome are consistent with those present in current culture settings. future experiments will assess the preserved neuroprotective properties of the pmsc evs. a novel method for isolating extracellular vesicles from cell culture media and plasma using polyethylenimine introduction: due to their ability to transport dna, rna, and protein cargoes between cells, extracellular vesicles (evs) are becoming popular for biomarker discovery as well as for therapeutic delivery. here we describe the development of a novel precipitation method for the isolation of evs from cell culture media and plasma that is based on polyethylenimine (pei), an inexpensive, water-soluble, and biocompatible cationic polymer. pei is a group of hydrophilic cationic polymers that are synthesized as either linear or branched forms of varying molecular masses ( , to , da) and are widely used in the biomedical field as a coating and transfection agent. methods: linear and branched pei of varying molecular weights (mw) were tested for their ability to precipitate evs from either conditioned culture media (ccm) or human plasma. isolated evs were characterized by western blotting and nanoparticle tracking analysis (nta). the small rna profile of evs isolated using pei from human plasma was analysed by ngs and ev-specific mirnas were confirmed by digital droplet pcr (ddpcr). mass spectrometry (ms) was used to analyse the proteome of pei-captured evs from plasma. hek cells producing gfp+ evs were used to optimize conditions for release of evs from both linear and branched pei by fluorescent spectrophotometry and flow cytometry measure-ments. results: linear and branched pei were both able to precipitate evs as determined by western blotting for ev protein markers; however, branched pei with mw > , da and linear pei with mw > , da were more efficient for ev precipitation than lower mw forms. despite its known ability to bind nucleic acids pei was unable to capture cell-free dna from plasma, although rna and in particular ev-associated mirnas such as mir- - p were recovered. ms revealed that pei enriches extracellular exosome proteins from plasma. evs captured from ccm by pei could be released from the complex using heparin or high salt conditions. summary/conclusion: pei has an unexpected preference for associating with evs compared to nucleic acids in complex biological samples and has a hitherto unrecognized application for ev precipitation. introduction: there is ongoing debate about which is the most appropriate method for isolation of evs, with most labs using some combination of differential ultracentrifugation (uc), size-exclusion chromatography (sec), and/or density gradient ultracentrifugation (dg). here we applied a surface-enhanced raman spectroscopy (sers) analysis platform to compare chemical composition of the isolate from each method against lipoprotein standards to assess the relative purity of the ev preps. methods: - ml of plasma was separated from whole blood collected from head and neck cancer patients. each sample was split into batches and evs were isolated by either uc, sec, or dg. following isolation, samples were incubated on commercial sers substrates and raman spectra were collected. lipoprotein standards were purchased and also measured for comparison. using principle component analysis (pca), spectra were analysed for chemical variability. results: sers analysis of sec, uc, and dg isolated evs were chemically distinguishable using simple pca. the chemical changes could in large part be attributed to fitting the differences in spectra to lipoprotein standards. we found that uc isolated populations clustered with the high-density lipoproteins (hdl), sec populations with the low-and very low-density lipoproteins (ldl, vldl), and dg populations were more variable, but mainly clustered together with the highdensity-lipoproteins (hdl). summary/conclusion: this set of experiments matches our expectation that various lipoprotein would contaminate ev preps according to their relative size and density distributions. no single isolation method could separate pure ev samples. this study also illustrates the utility of label-free sers analysis for rapid chemical characterization of evs. bioreactors: lessons to develop an extracellular vesicle factory vanessa chang, priscila dauros-singorenko, lawrence w. chamley, colin l. the university of auckland, auckland, new zealand introduction: high density mammalian cell culture systems (bioreactors) provide valuable advantage for large scale production of secreted products such as extracellular vesicles (ev). however, optimisation of design selection, handling and operational costs can be quite challenging. here we provide our experience with a celline bioreactor system. methods: cultures of adherent cell lines were established in celline ad bioreactors and propagated for up to weeks. media was changed twice weekly and cells shed into serum-free conditioned medium were counted and assessed for viability. nanoevs were isolated by sequential centrifugation ( g - , g - , g) and size exclusion chromatography (sec). nanoevs were characterised in their protein (bca) and particle (nanoparticle tracking analysis) amount, ev markers (western blotting) and morphology (transmission electron microscopy, tem). results: the viability of shed cells varied between cell lines and through time, suggesting a changing dynamic during reactor establishment and continuous growth phases, that was specific to each cell line. hdfa, bt and bt consistently shed mainly dead cells ( - %), as opposed to mcf and mda-mb- which predominantly shed live cells. sec fractionation of nanoevs identified a dominate ev-rich peak and significant quantities of smaller proteins, highlighting the need for further purification. nanoev yields from each - day culture averaged - × particles, representative of yields obtained from cells grown in to conventional t tissue culture flasks. ev markers and tem confirmed the protein profiles and morphology of evs obtained from bioreactors. summary/conclusion: high density bioreactor cultures offer a physiologically relevant, cost and space efficient approach to produce significant amounts of evs, providing sufficient material for numerous experimental uses. in our hands, with careful twice weekly management, they can be propagated for up to weeks without significant changes to the evs. introduction: extracellular vesicles (evs) have potential applications for clinical theranostics. ultracentri-fugation is most commonly adopted to the evs isolation, which is recommended as a gold standard method. however, ultracentrifugation is time-consuming and expensive equipment requirement, resulting in the coisolation of contaminants such as protein aggregates. therefore, our aim is to develop a rapid and efficient platform to isolate heterogonous evs based on the insertion of lipid molecules into the evs membrane to avoid co-isolation of non-membranous protein particles. methods: herein, a defected nanoscale functional metal organic framework (mof) was constructed as an efficient platform for evs isolation. typically, one single-stranded dna was designed and modified with a phosphate group at the ʹ-end and cholesterol at the ʹ-end to form a capture dna named phosphate−dna−cholesterol (pdc). the phosphate group forms a strong covalent bond with the designed defeated site of zr (iv) in mof uio- -nh and the cholesterol inserts into the phospholipid bilayer to capture evs without non-membranous particles contamination. the formed mof−phosphate−dna −cholesterol−evs (mof@pdc@evs) system was further treated with dnase i for dna hydrolysis to give high pure evs. results: a rapid and efficient isolation platform of evs based on a defected mof functionalized with phosphate-dna-cholesterol (mof@pdc) has been constructed successfully. compared with ultracentrifugation, mof@pdc platform promises to isolate size heterogeneous evs i) without non-membranous particles contamination, maintaining evs intact membrane structure, protein components, and biological functions; ii) with the ability to capture evs with % isolation efficiency; iii) makes evs isolation process simple and fast, which could be finished in minutes without requirement of the expensive equipment. summary/conclusion: in conclusion, this rapid and efficient platform is suitable for isolation evs from biological fluid for downstream protein analysis. this work opens a new perspective in mof-based separation researches and may shed light on further studies towards evs isolation. introduction: incorporation of pharmacologically active molecules on the surface or the lumen of extracellular vesicles (evs) is an important strategy for maximizing the therapeutic potential of evs. genetic engineering of producer cells by introducing dna through random or site-specific integration are promising strategies for creating engineered evs. longterm stability with consistent transgene expression in the ev producer cells and therapeutic potency of resulting engineered evs are crucial for biomanufacturing. we present a comprehensive study to investigate stability of transgene expression and potency of two potential therapeutic engineered evs derived from stably selected pools transfected by either random integration (ri) or site-specific integration (ssi). methods: producer cells were engineered to make evs displaying interleukin (il ) or interferon gamma (ifng) by ri or nuclease-mediated ssi into aavs locus. following puromycin (puro) selection, longterm cellular stability and transgene expression without selective pressure was investigated. evs were generated from stable cell pools at , , and months post-thaw and purified by density gradient ultracentrifugation. purified evs were biochemically characterized by nta, bca, western blot, and cholesterol quantitation. transgene expression and biological activity of evs displaying il and ifng were assessed by alphalisa and in vitro reporter assays. results: transfection by ssi resulted in faster recovery in puro selection compared to ri. all stable cell pools, regardless of integration method, resulted in comparable cell culture performance, ev yield, and lipid and protein content at all time points tested. the engineered evs also demonstrated long-term stability of il and ifng transgene expression and in vitro activity from both integration strategies. summary/conclusion: both methods for generating stable cell lines were comparable in terms of cell stability, transgene expression, ev titre and potency, with ssi having the advantage of speed, allowing for more rapid iteration cycle times. thus, both methods are suitable for the precision engineering of therapeutic evs. this work demonstrates feasibility to manufacture therapeutic engineered evs from stable cells from either integration strategy for clinical development. transport of outer membrane vesicles as a model therapeutic delivery system in pathogenic and commensal bacteria introduction: outer membrane vesicles (omvs) in gram-negative bacteria have been shown to be important carriers of biomolecules, including toxins and other virulence factors, peptidoglycan, and nucleic acids. it has been shown that omvs play an important role in the delivery of these biomolecules to host cells and bacterial cells. while many thorough studies have explored omv delivery to host cells, few studies have explored the mechanisms of delivery of omvs to bacterial cells. our goal was to study the delivery of omvs to other bacterial cells. specifically, we were studying the oral pathogen aggregatibacter actinomycetemcomitans (a.a.), a gram-negative organism associated with localized aggressive periodontitis, to study the process by which vesicles from this organism communicate with other bacterial cells. overall, we want to understand the roles specific surface components of omvs play in the transport of these omvs to other bacterial cells. methods: we studied omvs from two strains of a.a.: jp , a highly pathogenic strain, and , a natural commensal strain. af -labelled omvs were incubated with fresh bacterial cultures. association of the omvs with the bacterial cells was quantified using flow cytometry. to examine the role of surface-associated dna in this process, dna was digested with dnase, and the amount of surface-bound dna was quantified with the membrane impermeable dna stain, toto- . results: using flow cytometry, we observed jp omvs were delivered to , cells, and at a lesser amount to jp cells. alternatively, , omvs associated readily with jp cells, more than to , cells. this suggests that the delivery of omvs to bacterial cells may be a targeted delivery mechanism. furthermore, we hypothesized surface-associated dna may play a role in this interaction. we next digested the surface-associated dna on the omvs with dnase, and observed a decrease in association between the omvs and bacterial cells. this supports our hypothesis that dna on the surface of the omvs plays a role in association. current experiments are investigating this interaction in more detail. summary/conclusion: we have demonstrated that omvs are selectively delivered to bacterial cells, and surface-associated dna plays a role in this process. we propose to investigate this process to further understand omvs delivery to bacterial cells. funding: r de & r de . utilizing a gaucher's disease cell line for the evaluation of a novel exosome-based replacement therapy annie k. brown a , jiayi zhang b , brendan lawler b and biao lu b a santa clara university, san jose, usa; b santa clara university, santa clara, usa introduction: engineered nano-scale exosomes have great potential as new and targeted delivery vehicles for the treatment of gaucher's disease, the most common lysosomal storage disease. recently, we have reported the design, production, and isolation of exosomes loaded with lysosomal β-glucocerebrosidase (gba). people suffering from gaucher's disease do not have functional gba, which results in toxic build-up of undegraded substrates within the cell. methods: to evaluate the efficacy of this exosomebased therapy, a human gaucher's disease model is required. here, we have utilized near-haploid human cells (hap ) modified via crispr-cas to model gaucher's disease in vitro. these cells contain a bp insertion in the th exon of the gba gene, resulting in non-functional gba. pcr, enzyme activity assays, and flow cytometry have been employed to confirm the diseased genotype and phenotype. results: characterization of gba-knock out cells shows a total loss of gba enzyme activity. further characterization demonstrates a normal growth rate but an increased number of lysosomes, indicating a diseased phenotype. summary/conclusion: the utilization of a human gba-knock out cell line will enable the evaluation of the efficacy of our engineered exosomes. disease models will be an important resource for the evaluation of new biologic therapeutics, including exosomes. funding: we would like to acknowledge the santa clara university school of engineering for their support. thrxosomes: a novel exosomes based theranostic for lung cancer introduction: chemotherapy is the first-line of treatment for lung cancer. however, inefficient bio-distribution and reduced accumulation of drugs in the tumour results in treatment failure. therefore, improved drug delivery and diagnostic systems are warranted. herein, we propose a novel theranostic system "thrxosomes" where exosomes are loaded with super paramagnetic iron nanoparticles (spions) conjugated to an anticancer drug via a phresponsive linker for controlled release. we hypothesize that thrxosomes will exert profound anticancer tumour activity that can be concurrently be monitored by magnetic resonance imaging (mri). methods: thrxosomes were produced by combining normal human lung fibroblast (mrc ) cell-derived exosomes with spions conjugated to and anti-cancer drug (chemodrug or mirna) via a ph cleavable linker. the physical and biological properties of thrxosomes were determined using transmission electronic microscopy (tem), nanotracker-analysis (nta), inductively coupled plasma mass spectrometry (icpms), western blotting, cell viability, and mri. results: exosomes used in preparing thrxosomes were nm in size with a typical lipid bilayer structure, and were positive for cd , cd , flotillin and negative for annexin a confirming presence and purity of exosomes. charge analysis, tem, and icmps data showed successful loading of spion-drug conjugate. biological studies showed selective and enhanced drug release under acidic condition (ph . ) compared to drug release at ph . . cell uptake and viability studies demonstrated increased uptake and killing of thrxosome-treated human a lung cancer cells compared to mrc- cells. in vivo studies demonstrated accumulation and detection of spions by mri in in-situ tumours of a tumour-bearing mice. summary/conclusion: our study demonstrates thrxosomes will produce profound anticancer activity in lung cancer that is measurable by mri. exosome-modified nanoparticles as an alternative delivery system for small rnas in cancer therapy petro zhupanyn a , alexander ewe b , thomas büch c and achim aigner a a independent division for clinical pharmacology at rudolf-boehm-institute for pharmacology and toxicology, faculty of medicine, university of leipzig, germany, leipzig, germany; b dr., leipzig, germany; c rudolf-boehm-institute for pharmacology and toxicology, faculty of medicine, university of leipzig, germany, leipzig, germany introduction: gene knockdown by rna interference (rnai) is an alternative, non-invasive method for inhibiting proliferation or promoting apoptosis in tumour cells. this technique allows the specific targeting of key signalling proteins or mutated genes. most of the available transfection compounds suffer from rather profound cytotoxicity in vitro. the aim of our study was to establish a novel targeted small nucleic acid delivery system to the cells, with good cellular biocompatibility and applicability for in vivo studies. for this aim, we used native, cell own vesicles-exosomes. since exosomes are known to transport peptides and different rnas between cells and tissues, these unique, small extracellular vesicles (ev) may also be useful as transport vehicles for therapeutic sirna. methods: as detected by multiple cell surface protein expression analysis, exosomes carry specific surface expression markers, allowing the cellular uptake by the most of tissues. we established an ev purification protocol from tumour cell culture supernatants and a strategy for the efficient ev loading with our test sirnas or antimirs. here we used the combination of polyethylenimine (pei)-complexation of the rnas with ultrasound treatment for their loading into the evs. our ev-modified, ultrasound-treated nanoparticles were tested in vitro by measuring knockdown efficacies in luciferase reporter cell lines or by rt-qpcr gene expression analysis. results: more efficient cellular sirna uptake was observed upon ev-modification of our pei/rna nanoparticles, accompanied by efficient inhibition of gene expression. biological efficacies were retained also after storage for several days at room temperature. the monitoring of the ev-based particles by facs revealed a different time resolution of cellular uptake and nucleic acid release compared to the classically formulated peinanoparticles. in an in vivo therapy study in tumour xenograft-bearing mice, high biocompatibility, significant biological knock-down and tumour inhibition were observed after injection of anti-survivin sirnas formulated in our ecv-modified pei nanoparticles. summary/conclusion: our data demonstrate the usability of ecv-modified nanoparticles as efficient delivery system for small rnas in cancer therapy. microglial extracellular vesicles as therapeutic vector for neuroinflammation giulia marostica a , annamaria finardi b and roberto furlan a a san raffaele scientific institute, milan, italy; b san raffaele scientific institute, milan, italy introduction: microglia is considered an eligible target against the progressive multiple sclerosis (ms), but currently available therapies do not allow its efficient targeting. as many cell types, microglia communicate with the neighbouring cells through a complex system of extracellular vesicles (evs) exchange. recently my group described that microglia derived-evs, engineered to encapsulate il , are taken up by microglia itself, mediating a phenotype switch to a protective phenotype. in vivo studies suggest that these evs can ameliorate established neuroinflammation, thus making them a promising drug-delivery tool to target cns in ms. my project focuses on understanding the mechanism of action and the signalling pathway of evs delivery and to exploit this knowledge to specifically deliver different potential therapeutic molecules. for this purpose, we decided to characterize the evs through trps technology. methods: a murine microglia cell line (bv ) was engineered to stably overproduce endogenous il . this cell line was cultured in exosome-depleted rpmi and stimulated with pma( mg/ml) for min. evs isolation was carried out by collecting supernatant and subjecting it to consequential centrifugation of g, min, rt and g, min, °c. the resulting supernatant was filtered ( µm) and ultracentrifuged at , g for h at °c. the evs pellet was re-suspended in ice-cold pbs. the evs analysis with trps shows two populations of evs, one with a mean diameter of - nm and a broad zeta potential ranging from − mv to − mv, while the second population has a mean diameter of - nm and a zeta potential of − /- mv. this difference can be consistent with the different pathway formation of exosomes and microvesicles. we demonstrated in vivo the strong phenotypic change induced by our evs to resting microglia in a dose-and time-dependent effect. then, impairing the physiological procedure of the endosome acidification, the effect of our evs on recipient cells is higher. thus, suggesting an endocytic pathway for the internalization of the vesicles. we further demonstrate with gradient ultracentrifugation the capability of our formulation to vehicle endogenous il inside the vesicles. even if some protein is co-purified in the procedure, we know that the half-life of this cytokine is too short to elicit a strong in vivo response. consequently, we assume that the anti-inflammatory effect of our evs in vivo is a result of the il internalized in our formulation. summary/conclusion: these data help us understand more in detail the process of internalization and phenotype change mediated by these evs. our next goals are to discriminate between different internalization pathways and further validate the efficacy of our therapy on the eae mouse model. targeting il- rα on tumour-derived endothelial cells blunts metastatic spread of triple negative breast cancer via extracellular vesicle reprogramming introduction: the lack of an approved targeted therapy and the early onset of metastasis highlight the need for new treatments for triple-negative breast cancer (tnbc) patients. interleukin- acts as an autocrine factor for tumour-endothelial-cells (tec), and exerts pro-angiogenic paracrine action via extracellular vesicles (nevs). il- rα blockade on tec changes tec-ev (anti-il- r-evs) microrna cargo and promotes the regression of established tumour vessels. as tec are the doorway for "drug" entry into tumours, we have aimed to assess whether il- r blockade on tec impacts tumour progression via their unique ev cargo. methods: human tnbc samples, mda-mb- , mda-mb- and mcf cell lines were evaluated for the expression of il- rα. nevs and anti-il- r-evs were characterized by electron-microscopy, macsplex-exosome-kit and western blot. proliferation, migration, apoptosis and sphere formation were evaluated. scid mice were used for in vivo experiments. results: we noticed that, besides tec and inflammatory cells, tumour cells from . % of the human tnbc samples expressed il- rα. mda-mb- and mda-mb- , but not mda cells, expressed il- rα. in vitro, nevs provide survival and migratory signals, while anti-il- r-evs promoted apoptosis as well as reduced cell viability and migration of human tnbc cell lines. in vivo anti-il- r-ev treatment induced vessel regression in established tumours formed of mda-mb- cells and almost abolished the spread of liver and lung metastasis. moreover, decreased β-catenin and twist were found in tumours from animals treated with anti-il- r-evs. in addition, anti-il- r-evs reduced lung metastasis that was generated via the intravenous injection of mda-mb- cells. nevs that were depleted of mir- - p (antago-mir- - p-evs) were effective as anti-il- r-evs in down-regulating twist and reducing lung-vessel density and metastatic lesions in vivo. summary/conclusion: overall, these data provide the first evidence that il- rα is highly expressed in tnbc cells, tec and inflammatory cells, and that il- rα blockade on tec impacts tumour progression. introduction: high-grade serous ovarian cancer (hgsoc) is the deadliest gynaecologic cancer. its lethality is explained for late diagnosis at advanced stages and frequent recurrences despite achieving complete response with standard therapy. most of recurrences occurs at abdominal cavity with multiple metastasis. therefore, identifying key determinants of metastatic process remains as priority to find better therapies. current evidence assigns a central role of the exosomes in conditioning the metastatic niche in epithelial cancers. recently, we demonstrated that statins reduce metastasis in hgsoc in preclinical models. here, we decided to study the effects of statins on hgsoc-derived exosomes and its capability to condition the metastatic niche. methods: exosomes were isolated from heya ovarian cancer cell line and primary tissue cultures established from advanced-stage hgsocs (with signed informed consent and irb approval) by differential ultracentrifugation and quantified by nanoparticle tracking analysis (nta). enriched-cancer initiating cells (cic) spheroids were established from heya cells by using stem-selecting conditions. the paracrine effect of exosomes on cic migration/invasion was studied using either d migration or boyden chamber invasion assays. previous to exosome isolation, heya cells were treated with simvastatin ( um, h) or solvent and proteins involved in exosome biogenesis/uptake (alix, tsg ), its trafficking (rab a, rab a) and in conditioning the metastatic niche (emmprin) were measured by immunoblotting. results: exosomes isolated from heya cells or hgsocs enhance the metastatic potential of heya established spheroids in d migration or boyden chamber invasion assays. upon simvastatin treatment, we observed a significant reduction in migration/invasion induced by equivalent number of exosomes in heya -derived cics. under same treatment, we observed a significant decrease in protein levels of alix and tsg and an increase in the inactive forms of rab a and rab a in heya cells. we also observed a decrease in emmprin levels in heya -derived exosomes. summary/conclusion: here, we demonstrated a paracrine effect of hgsoc-derived exosomes that favour the metastasis process. in addition, we demonstrated that simvastatin reduces metastasis induced by cancerderived exosomes. such an effect is partially explained by changes in the expression of proteins involved in exosome biogenesis/uptake, its endocytic trafficking and in the content of proteins conditioning the metastatic niche. thus, simvastatin arises as potential therapeutic target to improve outcomes in this disease. funding: this research was supported by fondecyt granted to mauricio a. cuello label-free optical imaging and characterization of cancer-associated extracellular vesicles in tissues introduction: cancer-associated extracellular vesicles (evs) visualized in the tumour microenvironment have been identified as a potential biomarker for cancer-related tissue changes. analyses of evs have traditionally been performed in cells or isolated evs, with no temporal or spatial information that could be critically important for elucidating their roles in carcinogenesis. since the unperturbed distribution and organization of evs in the tumour microenvironment is associated with their cellular function and can potentially serve as a diagnostic and prognostic biomarker, there is a strong need for visualizing evs in freshly isolated tissue specimens. currently, only fluorescent labelling methods enable visualization and tracking of evs. we used a custom label-free multimodal multiphoton optical imaging system to detect and characterize evs and classify them using their optical signatures both in isolated tissues and in situ tumours. methods: label-free multimodal multiphoton imaging was used to provide simultaneous, co-registered structural and functional images of evs in untreated samples. heterogeneous populations of evs could be identified from their unique optical signatures. results: the intrinsic metabolic and structural properties of evs enabled reliable visualization and optical characterization of evs from cell cultures and in situ imaging of tumour-bearing rats. unique optical signatures were then used for identification of cancer-related evs in tissues from human breast cancer patients, and their density was found to be highly correlated with clinical diagnosis. in the current study, evs were isolated from urine of tumour-bearing dogs, and urine and serum from breast cancer patients. analysis of ev content showed higher concentration of nad(p)h in evs isolated from cancer subjects, than from healthy subjects, which reflects the reprogramming of cellular metabolism in carcinogenesis. summary/conclusion: these results suggest a potential label-free optical methodology to detect and characterize evs by their optical signatures, which can be utilized as possible diagnostic and prognostic biomarkers for cancer. funding: this research was conducted under protocols approved by the iacuc and irb at the university of illinois and carle foundation hospital, and supported by funding from nih. novel potential anticancer therapies based on interference with nuclear entry of cancer cell-derived extracellular vesicle components in recipient cells introduction: the intercellular communication mediated by extracellular vesicles (evs) in the tumour microenvironment plays an important role in tumour progression. experimental evidence indicates that evs derived from highly metastatic cells influence the behaviour of less aggressive cancer cells. we have previously described a novel intracellular pathway where a fraction of endocytosed ev-associated proteins and nucleic acids is transported into the nucleoplasm of the host cell via a subpopulation of late endosomes penetrating into nucleoplasmic reticulum (nr). here, we better characterize this pathway and report that it is required for the induction of an aggressive behaviour induced by evs released from highly metastatic sw colon cancer cells in isogenic primary cancer cells. methods: super resolution-structured illumination microscopy and magnetic-based co-immunoisolation studies were employed to identify the protein components of the nuclear pathway and to monitor the entry of ev-containing late endosomes into the nucleoplasmic reticulum. human sw carcinoma cells expressing er-gfp and rab -rfp were exposed to evs from sw cells and then live imaged. results: we have previously reported that the tripartite protein complex, containing vap-a, orp and rab orchestrates the localization of ev-carrying late endosomes into nr. we now report that silencing of orp or vap-a, but not its homologue vap-b, reverses the pro-metastatic changes induced by evs isolated from metastatic cells on their non-metastatic counterpart, including transition to an ameboid phenotype, cell rounding and blebbing. moreover, we found that certain nuclear pore complex proteins and importin-beta are co-immunoisolated with orp , vap-a and rab suggesting the formation of a large protein complex at the entry of nuclear pores. summary/conclusion: interfering with the mechanisms regulating this novel intracellular pathway may find therapeutic applications particularly in ev field and oncology. educated osteoblasts regulate breast cancer proliferation via small extracellular vesicles thomas jefferson university, philadelphia, usa introduction: breast cancer commonly traffics to bone, where breast cancer cells (bccs) can survive undetected for years before metastatic outgrowth. in bone, bccs interact with surrounding stromal cells, including osteoblasts (obs), to shape the metastatic niche. our lab discovered there are at least two subpopulations of obs in the bone-tumour niche, based on protein marker expression. one group, "educated osteoblasts" (eos) have engaged in crosstalk with bccs whereas another group, naïve obs, have not. we have novel evidence that eos regulate bcc proliferation. the purpose of this study was to determine if extracellular vesicles (evs) produced by eos play a role in regulating bcc proliferation. we hypothesized evs produced by eos would decrease bcc proliferation. methods: eo-derived small evs from culture media were isolated via ultracentrifugation and characterized evs for size, protein marker expression, and density floatation to validate the purity of ev samples. the functionality of eo-derived evs on bcc proliferation was examined using edu and checkpoint proteins p and p . bcc protection from chemotherapy induced cell death was also examined. results: we found that evs produced by eos, but not naïve obs, decreased both triple negative and erpositive bcc proliferation in a concentration dependent manner. furthermore, using an edu assay, we found that exposure to eo-derived evs induces bccs to undergo cell cycle arrest. interestingly, the cell cycle arrest was reversible and bcc proliferation was restored upon removal of eo-derived evs. in addition, exposure to eo-derived evs leads to increases in bcc expression of the g checkpoint proteins, p and p . we next wanted to investigate proliferative signalling pathways that may be deregulated in bccs following exposure to eo-derived evs. we found that eoderived evs reduce bcc levels of erk / . because our data indicate eo-derived evs induce sustained cell cycle arrest in bccs, we desired to know if eo-derived evs protected bccs from chemotherapy-induced cell death. we found that bccs exposed to eo-derived evs and the chemotherapy drug, doxorubicin, have decreased cell death compared to bccs exposed only to doxorubicin. summary/conclusion: altogether, our data suggest eos play a crucial role in bone-tumour microenvironment by regulating bcc proliferation. funding: supported by nih r ca and commonwealth of pennsylvania -department of health sap for kmb. phosphorylation of tyrosine in annexin a is essential for its association with exosomes and for imparting invasive and proliferative capacity to other cells priyanka prakash desai a , pankaj chaudhary b , xiangle sun b and jamboor vishwanatha a a unt health science center at fort worth, fort worth, usa; b unt health science center, fort worth, usa introduction: triple negative breast cancer (tnbc) accounts for %- % of all breast cancer cases. the lack of targeted-based therapies highlights the importance of studying tnbc. elevated levels of annexin a (anxa ), a ca+ -dependent phospholipid binding protein, has been correlated with worse overall survival in tnbc patients. our previous data implicate that exosomal anxa is involved in creating a pre-metastatic niche and facilitating metastasis in tnbc. moreover, n-terminal phosphorylation of tyrosine (tyr) in anxa has been implicated in regulating several anxa activities in cancer progression. here, we demonstrated that n-terminal phosphorylation of anxa at tyr is important for its association with exosomes which imparts invasive and proliferative phenotype to other cells. hence, dissecting the regulatory pathway will be critical for verifying the value of anxa as a therapeutic, diagnostic or prognostic marker in tnbc. methods: pn -egfp plasmids expressing the constitutive phosphomimetic (anxa -y e) and non-phosphomimetic mutant (anxa -y f) gene expressing mutation at tyr site were overexpressed in mda-mb- tnbc cells. mutant cells were experimentally validated for anxa specific functions like migration, invasion and proliferation. exosomes were isolated from the mutant phosphomimetic (exo-anxa -y e-gfp) and non-phosphomimetic (exo-anxa -y f-gfp) cells and were analysed for exosomal surface expression of anxa by immunoprecipitation and flowcytometry. cal- breast adenocarcinoma epithelial cells were treated with exo-anxa -y e-gfp and exo-anxa -y f-gfp to analyse the rate of invasion and proliferation by transwell invasion and proliferation assay, respectively. transfer of exosomal anxa in cal- was studied using immunofluorescence and its implications on signalling pathways were studied by western blot. results: mda-mb- phosphomimetic tnbc mutant cells showed increased migratory, invasive and proliferative capacity compared to non-phosphomimetic tnbc mutant cells. exo-anxa -y e-gfp had elevated surface anxa expression compared to exo-anxa -y f-gfp. cal- cells treated with exo-anxa -y e-gfp showed high migratory, invasive and proliferative characteristics, with a higher expression of p-anxa (tyr ), p-src(tyr ) and p-paxillin(tyr ) compared to exo-anxa -y f-gfp treated cells. summary/conclusion: n-terminal phosphorylation of tyr in anxa in mda-mb- tnbc cells (phosphomimetic mutant cells) is essential for its association with exosomes and for conferring increased invasive and proliferative capacity to other breast cancer cells. funding: the above study is funded by national institute of health ro ca and nimhd's u md to dr.j.k.vishwanatha. a novel method for epithelial-derived extracellular vesicle isolation and enrichment in patients with advanced prostate cancer arpit rao, helene barcelo and bharat thyagarajan university of minnesota, minneapolis, usa introduction: evaluation of changes in prostate cancer biology is difficult due to presence of lymph nodal or bony metastatic disease in a majority of patients. a number of liquid biopsy assays have shown clinical utility in prostate cancer, but are limited by low sensitivity (e.g. circulating tumour cells-based assays) or inability to perform transcriptome sequencing (cellfree dna-based assays). epithelial-derived extracellular vesicles (epi-ev)-based assays are uniquely positioned overcome both these limitations as evs are abundantly secreted into the blood and have rnacargo that mirrors the cell of origin. however, a reliable method to enrich for epi-evs is currently lacking. methods: plasma was isolated from the peripheral blood collected from patients with metastatic prostate cancer enrolled in an institutional biobanking study before initiation of systemic antineoplastic therapy. evs were isolated from µl of plasma using a commercially available qev size exclusion column (izon inc.). without subjecting the evs to any physical stressors such as centrifugation, cd magnetic beads were used to fractionate the evs into cd + (platelet derived) and cd -(non-platelet derived) fractions. multiparameter flow cytometry was used to evaluate evs that expressed cd and epcam and were negative for calnexin. nanotracking analysis (nta) was used to quantify both total ev and cd + and cd fractions in all patient samples. the average ± standard deviation of total evs obtained from the patients was . x ^ ± . x ^ evs/ml of plasma (coefficient of variation [cv] : %) while the average and standard deviation of cd -evs was . x ^ ± . x ^ (cv: %). the cd -ev fraction represented a variable amount of the total evs in prostate cancer patients ranging from . % to . %. multiparameter flow cytometry showed that over % of total evs were cd + and calnexin-, suggesting an endosomal origin for a vast majority of the evs in these plasma samples. however, the proportion of evs expressing epcam (marker of epi-evs) was higher among the cd -fraction ( % - %) as compared to the cd + fraction ( . % - %). summary/conclusion: our novel method was able to isolate and enrich the epi-ev from the plasma of advanced prostate cancer patients. correlation between clinical characteristics and ev quantity is being evaluated to identify the reason(s) for large variations in cd -ev fraction. future studies are planned to use our method in improving the sensitivity of ev-based assays and increase the rna yield to facilitate transcriptome sequencing. funding: this work was funded by grants from randy shaver community and research fund, minnetonka, mn. exosomes drive medulloblastoma metastasis in a mmp and emmprin dependent manner introduction: recurrent/metastatic medulloblastoma (mb) is a devastating disease with an abysmal prognosis of less than % -year survival. the secretion of extracellular vesicles (evs) has emerged as a pivotal mediator for communication in the tumour microenvironment during metastasis. the most investigated ev's are exosomes, nanovesicles secreted by all cell types and able to cross the blood-brain-barrier. matrix metalloproteinases (mmps) are enzymes secreted by tumour cells that can potentiate their dissemination by modification of the extracellular matrix. we hypothesise that exosomal mmp and its inducer emmprin could enhance metastasis of mb. methods: proliferation, invasion and migration assays were used to evaluate the phenotypic behaviour of primary cell lines pre-treated with metastatic tumour cell-derived exosomes. gelatin zymography and western blotting were performed to confirm mmp functional activity in cell lines and exosomes. nanoscale flow cytometry was used to measure surface exosomal emmprin levels. exosomal mmp and emmprin were modulated at the rna level. results: number of exosomes is directly related to the migratory behaviour of parental mb cell lines (p < . ). notably, functional exosomal mmp and emmprin levels also correlate with this. furthermore, exosomes from metastatic cell lines conferred enhanced migration and invasion on their matched isogenic primary (non-metastatic) cell line pair bỹ . -fold (p < . ). exosomes from metastatic cell lines also conferred increased migration on poorly migratory foetal neuronal stem cells. summary/conclusion: together this data suggests that exosomal mmp and emmprin may promote medulloblastoma metastasis and supports analysis of exosomal mmp and emmprin levels in patient cerebral spinal fluid samples. introduction: exosomes secreted from cancer cells harbour the potential to regulate intracellular signalling and promote metastasis. wherein, metastasis suppressor genes (msgs) play a pivotal role in regulating such signalling cascades. however, the regulation gets hampered due to low expression of msgs under metastatic conditions. nm -h , product of first identified metastasis suppressor gene nme , is significantly downregulated under metastatic conditions. nm -h serves as a regulator of small gtpases. several evidences have highlighted an involvement of small gtpases (such as rab , rab and rab ) in the biogenesis of exosomes. in addition, bacterial homolog of nm has been shown to interact with rab and rab . however, experimental evidence supporting a relationship between exosomes and nm -h is lacking. our current focus is to deduce the relationship between exosomes and msgs. methods: breast cancer cell lines were used to assess the effect of exosomes isolated from highly metastatic cells (mda-mb- cells) on lower/non metastatic cells (mcf- cells). nme was overexpressed in mda-mb- cells and subsequently used to isolate exosome fractions. equivalent amount of isolated exosome fractions from mda-mb- cells and mda-mb- /nme cells were utilized to access their effect on migration and difference in exosome markers. results: we observed an enrichment of nm -h in the exosomes isolated from mda-mb- cells upon overexpression of nme . proteinase k protection assay confirmed the packaging of nm -h inside the exosomes isolated from mda-mb- /nme cells and excluded the possibility of membrane association of nm -h . additionally, overexpression of nm -h led to a significant reduction in the ability of mda-mb- exosomes to stimulate movement of mcf- cells as confirmed by wound healing assays. our data also highlights a clear reduction in the protein levels of exosome markers such as cd , cd and alix in the exosome fraction isolated from mda-mb- /nme cells as compared to mda-mb- cells. interestingly, rab a, a protein involved in the endosome-lysosome fusion was also present in lower amount in the exosomes isolated from nm -h overexpressing cells. summary/conclusion: our data highlights an antimigratory effect of nm -h via exosomes. these findings support a regulatory role of nm -h in the packaging or release of exosomes in highly metastatic breast cancer cells, and further suggest that metastasis suppressor proteins may be involved in the regulation or packaging of exosomes. additional studies will be required to decipher the downstream signalling of nm -h which affects the biogenesis of exosomes as well as to assess the effect of nm -h overexpression on the content of exosomes. these insights could help us delineate the complex exosome biogenesis pathway and provide new potential drug targets for exosome regulation. introduction: exosomes (exs) are emerging as novel players in the beneficial effects induced by exercise on vascular diseases. our recent study has revealed that moderate exercise enhances the function of circulating endothelial progenitor cell-derived exosomes (cepc-exs) on protecting endothelial cells against hypoxia injury. in this study, we aimed to investigate whether exercise-regulated cepc-exs contribute to the beneficial effects of exercise on ischaemic stroke (is). methods: c bl/ mice performed moderate treadmill exercise ( m/min, -wks) before is induced by middle cerebral artery occlusion surgery. acute injury was evaluated at day by determining neurologic deficit, infarct volume, cell apoptosis in the penumbra and neurologic recovery was assessed by determining angiogenesis/neurogenesis, sensorimotor functions at day . the correlations of cepc-exs and their carried mir- with neurological parameters were analysed. the underlying mechanism of the effects of cepc-exs isolated from exercised mice was explored in a hypoxia neuron model. cellular mir- level, apoptosis, axon growth ability and gene expressions (cas- , bdnf and akt) were measured. results: ) exercised mice had a smaller infarct volume on day , which was associated with decreased cell apoptosis and cleaved cas- level, and a higher microvessel density than those in control; ) the elevated cepc-ex level positively correlated with tepc-exs in ischaemic brain of exercised mice on day . the upregulated mir- level positively correlated with the numbers of tepc-exs in ischaemic brain; ) the numbers of cepc-exs and their carried mir- level negatively correlated with the infarct volume, cell apoptosis and positively correlated with the microvessel density in the peri-infarct area on day ; ) exercised mice had decreased infarct volume, increased microvessel density, promoted angiogenesis/neurogenesis and improved sensorimotor functions on day , accompanying with upregulated levels of bdnf, p-trkb/trkb and p-akt/akt; ) cepc-exs of exercised mice protected neurons against hypoxia-induced apoptosis and compromised axon growth ability which were blocked by mir- and pi k inhibitors. summary/conclusion: our data suggest that the protective effects of moderate exercise intervention on the brain against mcao-induced ischaemic injury are ascribed to cepc-exs and their carried mir- . funding: this work was supported by american heart association ( post ) and nih ( r ns ). syndecan- regulates alveolar type epithelial cell senescence mediating through extracellular vesicles during lung fibroproliferation tanyalak parimon a , changfu yao a , adam aziz a , stephanie bora a , marilia zuttion zuttion a , dianhu jiang a , melanie koenigshoff b , cory hogaboam a , paul nobel a , barry stripp a and peter chen a a cedars-sinai medical center, los angeles, usa; b university of colorado, denver, usa introduction: alveolar type epithelial cell (at ) senescence is implicated in the pathogenesis of lung fibrosis, a progressive fatal condition. syndecan- , a heparan sulphate proteoglycan, is overexpressed by at cells of human idiopathic pulmonary fibrosis (ipf) and bleomycin-injured wt mice and the overexpression of syndecan- is profibrotic. moreover, syndecan- deficient (sdc -/-) mice had less lung fibrosis after bleomycin injury. we reported that extracellular vesicles (evs) in bronchoalveolar lavage (bal) of bleomycin-injured wt mice augmented lung fibrosis whereas the sdc -/--bal-evs attenuated the process. moreover, wt-bal-evs expressed lower level of anti-fibrotic mirnas (mir- b- p, − - p, − - p, and − - p) compared to the sdc -/-bal-evs. these mirnas targeted genes in the cellular senescence pathway indicating that syndecan- altered microrna profiles in the bal-evs to promote cellular senescence during lung fibrogenesis. we investigate how syndecan- regulates at senescence through evs. methods: bleomycin was intratracheally given into wt and sdc -/-mice. at day , lungs were processed for single-cell rna sequencing (scrnaseq) and western blot (wb). evs were isolated using ultrafiltration centrifugation method. human (a ) and mouse (mle- ) lung epithelial cell lines were used for in vitro experiments. results: scrnaseq analysis indicated while bleomycin stimulated an overexpression of cellular senescencespecific genes on at cells of wt mice, these genes were significantly downregulated on sdc -/-at cells. senescence proteins, p and p , were also less expressed in the lungs of sdc -/-than of the wt mice by wb. to determine the functional effects of evs in bal, a cells were treated with human ipf or control lung wash-evs and evaluated for beta-galactosidase activity. we found that ipf-evs markedly increased beta-galactosidase enzymatic activity. corroborating with these data, bleomycin-injured bal-wt-evs also significantly upregulated senescence marker, p , by wb on mle cells whereas sdc -/--bal-evs inhibited p expression. summary/conclusion: our data indicate that syndecan- regulates lung fibrosis through the senescence signalling pathway on at cells. furthermore, syndecan- controls at senescence mediating through extracellular vesicles in the bal. lastly, the most likely cargo molecules mediating this process are micrornas. immortalized cardiosphere-derived cell ev-associated pirna, imev-pi, protects against ischaemic injury in the heart alessandra ciullo, ahmed ibrahim, liang li, chang li, weixin liu and eduardo marbán smidt heart institute, cedars sinai medical center, los angeles, usa introduction: cardiosphere-derived cells (cdcs) are a population of heart-derived progenitors with demonstrated therapeutic efficacy in preclinical and clinical settings. cdcs function by secreting extracellular vesicles (evs), lipid-bilayer nanoparticles laden with bioactive molecules. recently our group developed a strategy for immortalizing cdcs (imcdc) that retains their therapeutic potential and enhances cdc function indirectly through their secreted evs. imcdc show a different rna content(mirna, mrna, rrna, trna and pirna) compared to primary cdc. in particular, we focus on piwi rnas (pirnas), small rnas bound by piwi proteins, important regulators of both the epigenome and transcriptome. we seek to explore the role of a pirna highly enriched in imcdc-evs (imev-pi). methods: evs are prepared by conditioning cells for hrs in serum-free basal media, in hypoxic culture. after hrs conditioned medium is cleared of cellular debris and evs isolated using ultrafiltration by centrifugation (ufc). fractions were analysed in terms of particle size, number, and concentration and pirna content. in vitro, bone marrow derived-macrophages (bmdm) were exposed to imcdc-ev, imev-pi and control and transcriptomic profile and potentially activated pathways were assessed. in vivo, - week-old wistar-kyoto female rats received ^ imcdc-ev, imev-pi, scramble or vehicle intracoronary minutes after ischaemiareperfusion(i/r). cardiac troponin i levels, scar size and monocytes were assessed at and hrs. results: by small-rna sequencing analysis we found that pirnas are enriched in both cdc-ev and imcdc-ev. imcdc show a different pirna composition compared to primary cdc. imev-pi was identified as one of the most highly-expressed non-coding rnas (the number of reads were x higher in imcdc-ev compared to cdc-ev). in vitro, imexo-pi-conditioned bmdm exhibit a different transcriptomic profile compared with control, with upregulation of pathways involved in the inflammatory response, cell death, and cell-to cell signalling. in vivo, imev-pi is cardioprotective, as shown by reduced scar size and lower cardiac troponin levels compared to vehicleand scramble-injected animals at hrs post i/r. imev-pi only minimally alters neutrophil counts profile in blood but it alters monocytes profile with a decreased number at hrs and an increase at hrs. summary/conclusion: we posit that imev-pi is a key determinant of imcdc-ev therapeutic efficacy. our results indicate that target cells may be macrophages/ monocytes, given that imev-pi exposure modifies their composition and mrna profile both in vitro and in vivo. introduction: extracellular vesicles (exosomes, evs) are cell membrane particles ( - nm) secreted by virtually cells. during intercellular communication in the body, secreted evs play crucial roles by carrying functional biomolecules (e.g., micrornas and enzymes) into other cells to affect cellular function, including disease progression and tissue regenerations. literature previously reported that the macropinocytosis pathway contributes greatly to the efficient cellular uptake of evs. the activation of growth factor receptors, such as epidermal growth factor receptor (egfr), induces macropinocytosis. in this study, we demonstrated the effects of evs on demal papilla and hair follicle regeneration. methods: identification of distinct nanoparticles and subsets of extracellular vesicles from umbilical cord blood stem cell by asymmetric flow field-flow fractionation. results: the effects of evs from umbilical cord blood stem cell on the propagation of demal papilla and hair follicle regeneration were observed. summary/conclusion: the enhancement of extracellular vesicles from umbilicalcord blood stem cell the propagation of demal papilla and hair follicle regeneration were observed and confirmed. mechanisms of host resistance to plasma membrane damage induced by pneumolysin attack introduction: bacterial pore-forming toxins (pfts) are major virulence factors produced by pathogens. pfts target host plasma membrane (pm) and create transmembrane pores, which allow uncontrolled flux of ions and small molecules across the pm disrupting cellular homoeostasis. to survive, cells display poorly understood repair mechanisms to recover the cell homoeostasis. several mechanisms were proposed to participate in cell recovery: exocytosis of cortical lysosomes; endocytosis of pfts pores; pm blebbing and shedding. methods: we used increasing concentrations of purified ply to intoxicate cells. pm permeability was assessed by flow cytometry using propidium iodide dye. cytoskeleton rearrangements were investigated by confocal immunofluorescence microscopy. extracellular vesicles released during pm repair were isolated by high-speed centrifugation and characterized by nanoparticle tracking analysis (nta), transmission electron microscopy (tem) and mass spectrometry/ liquid chromatography analysis. results: ply triggers a complete reorganization of the actomyosin cytoskeleton inducing the formation of cortical actomyosin bundles at sites of pm remodelling. these structures assemble upon loss of pm integrity and disassemble as pm recovers. we detected the release of microvesicles during the recovery of pm integrity. vesicle population is heterogeneous with sizes ranging from to nm, with the majority of them measuring - nm. vesicle proteomic analysis revealed that they contain ply, suggesting they participate in pore removal, proteins involved in vesicle trafficking, pm repair and exosome biogenesis. summary/conclusion: our data demonstrate that cells are able to recover from the damage induced by sublytic concentrations of ply. actomyosin cytoskeleton undergo massive changes with the assembly of cortical bundles possibly at sites of pm damage. we showed that cells produced extracellular vesicles during the process of repair. we are now focusing on understanding the biogenesis of those vesicles and its importance during the process of repair. introduction: despite of high expectations, mesenchymal stromal cell (msc)-based therapies still lack efficacy, partially due to loss of cell viability and function upon administration. msc-derived extracellular vesicles (msc-ev) emulate the regenerative potential of msc, shifting the field towards cell-free therapies. clinical applications require the establishment of a scalable and gmp-compliant processes for the production and isolation of msc-ev, combined with robust characterization platforms. methods: to develop a well-established process for the production of therapeutic msc-ev, we compared different msc sources (bone marrow, adipose tissue, umbilical cord matrix), culture media compositions (dmem supplemented with foetal bovine serum (thermo fisher scientific), dmem supplemented with human platelet lysate (aventacell biomedical) and stempro msc sfm xeno free medium (thermo fisher scientific)) and culture parameters (oxygen tension and shear stress) in two different culture platforms ( d static tissue culture flask vs d dynamic spinner vessels). subsequently, msc-ev were isolated by ultracentrifugation or a commercially available isolation kit and characterized according to isev guidelines. results: msc derived from different sources/donors were able to grow under normoxia and hypoxia in d t-flasks and d spinner vessel culture systems, while maintaining their immunophenotype and differentiation potential, according to the minimal criteria defined by the isct. the time point for pre-conditioning and collection of conditioned medium for msc-ev isolation was also optimized for both d and d culture systems. msc-ev were characterized according to misev guidelines, using techniques as nta, protein and lipid quantification, western blot, imaging and fourier-transform infrared spectroscopy (ftir). the results indicate that msc-ev derived from different sources/donors have similar size distribution, however, ev yields tend to be higher for the d culture system. of notice, several spectral regions were identified by ftir, enabling the detection of differences in the biomolecules present in msc-ev, msc-conditioned media and cells produced under different conditions. summary/conclusion: in summary, this study contributes to the establishment of a scalable process for msc-ev production. evaluation of three different isolation methods for small extracellular vesicles from human plasma in prostate cancer diagnosis introduction: extracellular vesicles (evs) have great potential in prostate cancer (pca) diagnosis and progression monitoring to complement the inaccurate prostate specific antigen (psa) screening and invasiveness of tissue biopsy. however, current methods cannot isolate pure evs and therefor evs characteristics remain largely unknown. in order to develop an accurate approach for ev isolation, we aimed to compare three emerging methods with different characteristics of small evs (sevs) from human pca plasma samples and to choose the best one for diagnostic and functional studies. methods: pca patients and age-matched healthy controls (hc) plasma (n = in each group) were used to isolate sevs with different isolation methods including commercial exoquick ultra kit, qev and qev size exclusion chromatography (sec). isolated sev were characterized by nanoparticle tracking analysis, immunoblotting, cyrogenic electron microscopy, flow cytometry (fc) and proteomics analysis. for fc characterizing surface marker expression, the sevs were further purified by cd and cd commercial immunoaffinity magnetic beads . lipoprotein was captured by streptavidin biotinylated apob magnetic beads to measuring the lipoprotein contamination. results: the sev size, morphology, surface protein and protein cargo with proteomics were analysed between the three isolation methods. sevs isolated from sec methods had a lower particle size, protein amount, protein/sev marker ratio and apob+/sev marker ratio than those from exoquick ultra method. in addition, sevs isolated from qev demonstrated a significantly higher sev content, more up-regulated and down-regulated pca proteins from proteomics but lower sev marker/protein ratio and a higher protein contamination than those from qev . furthermore, sev marker signal also showed a good correlation with particle numbers instead of protein content in all the methods. summary/conclusion: qev method demonstrated better performance in isolating relatively pure sevs from human plasma; qev has the better performance in isolating samples with higher sev content; exoquick ultra isolated samples with closely sev content to the qev but with the highest non-sev protein contaminations. people can choose higher sev content or higher sev purity according to the downstream analysis. moreover, sevs may also be used for treatment monitoring, as recent studies suggested that the expression levels of certain markers may change during therapy, reflecting tumour response. for cancer diagnostics and therapeutic purposes in clinical settings, it is important to have a device which allows multiplexed measurements, in order to scan a large number of markers simultaneously and compare the expression levels of different patients, or same patients at different treatment stages, in a time efficient manner. methods: herein, we propose a multiplexed platform for label-free detection and surface protein profiling of sevs. the technique is based on the electrokinetic phenomena of streaming current and zeta potential (\zeta*) and measures the\zeta* change upon sev binding on functionalized microcapillary surfaces. for the purpose, we used sevs derived from lung cancer cells. in its current form, the platform can measure up to channels simultaneously, however, it can be further expanded. results: having demonstrated that our electrokinetic sensor successfully detects sevs in a specific way, we tested its ability to measure the expression level of membrane proteins. the analysis showed that it could detect differences in the expressions of egfr on sevs, with a sensitivity of %. we then extended the platform for multiplexed analysis, by connecting and measuring four capillaries, functionalized with different capture probes, simultaneously. for the purpose, we targeted specific tumour markers, i.e. egfr, and exosomal tetraspanin family proteins, such as cd and cd . the results showed successful multiplexed ev detection. summary/conclusion: being the sensor suitable for multiplexed sev detection, we shall present our investigation on a set of pleural effusion samples collected from a cohort of lung-cancer patients with different genetic makeup. introduction: extracellular vesicles (evs) are released to biological fluids from different tissues and organs and they contain molecules proposed as biomarkers for multiple pathological conditions. however, most ev biomarkers have not been validated due to the lack of sensitive techniques compatible with high-throughput analysis required for routine screenings. using immunocapture techniques, combining antibodies against tetraspanins and candidate tumour-specific markers we have recently optimized several assays that greatly facilitate ev characterization. methods: we have improved flow cytometry and elisa assays, increasing substantially the sensitivity for ev detection. using dls, em and analytical ultracentrifugation, we have characterised the biophysical basis of this enhancement. the final methodology can be performed in any laboratory with access to conventional flow cytometry or elisa reader. results: using combinations of antibodies specific for the tetraspanins cd , cd and cd , it is possible to detect evs in minimal volumes of urine and plasma samples without previous enrichment. additionally antibodies against other less abundant markers, like the epithelial marker epcam, have been used to capture and identify evs directly in minimal volumes of urine or plasma with sensitivity higher than western blot analysis of isolated evs. furthermore, we demonstrate that additives altering the biophysical properties of an ev suspension, increased detection of tumour antigens in these immune-assays. summary/conclusion: the development of sensitive, high-throughput methods, easily translatable to clinical settings, as elisa and flow cytometry described here, opens a new avenue for the systematic identification of any surface marker on evs, even scarce proteins, using very small volumes of minimally processed biological samples. these methods will allow the validation of ev biomarkers in routine liquid biopsy tests. introduction: when ev subpopulations are enriched on antibody microarrays and probed for their surface proteins, the detection signal is biased towards abundant subpopulations as it is dependent on both the protein expression level and the number of evs captured. to address this challenge, we developed a novel normalization approach allowing: ) the estimation of a target signal independent of ev subpopulation size through dye-based ev quantification, and ) the assessment of subpopulation target enrichment relative to the population average by leveraging tim as an unbiased, lipid-based ev capture. here, we investigated the expression of cancer-associated proteins, particularly metastasis-associated integrins (itgs), in breast cancer evs with varying metastatic potential and organotropism. methods: the relative protein enrichment profiles for various ev subpopulations were established from evs of skbr (her +), t d and mcf- (er+pr+), bt and mda-mb- (triple negative) breast cancer cell lines, as well as five mda-mb- -derived cell lines of four different organotropisms (brain, bone, lung, liver) using our custom antibody microarrays with our normalization approach. results: as expected, her was broadly detected in her + skbr evs. interestingly, her -t d and mcf- evs also expressed her where it was highly enriched in its epcam+ subpopulations. itg α , β and β were only found in triple negative and organotropic evs with itg β and β differentially enriched based on the organotropism. the population average of mda-mb- and lung-tropic evs had high expression of itg β , where subpopulations of cd + evs showed positive enrichment while cd + and cd + evs showed negative enrichment. itg α , β and β were absent in the bone-tropic cd + ev subpopulation, a profile atypical in other organotropisms. lastly, egfr was negatively enriched in tetraspanin+ subpopulations in mda-mb- evs, but positively enriched in these subpopulations in organotropic evs, especially for brain-tropism. summary/conclusion: following normalization, we were able to quantify specific protein associations, uncovering a multitude of co-enrichment profiles that characterize specific metastatic and organotropic cell lines. notably, we found enrichment signatures that distinguish between different organotropisms derived from the same parental cancer line. introduction: the tissue microenvironment surrounding tumours is complex and the cross-talk between cancer and non-cancer cells is essential for tumour growth and progression. we have previously shown that heparan sulphate proteoglycans (hspgs), on the surface of prostate cancer evs, are required for delivery of tgfβ and initiation of a disease-supporting fibroblast phenotype. however, hspgs are known to bind numerous growth factors, so here we have explored the repertoire of such proteins tethered to evs by hspgs. methods: evs were isolated from du prostate cancer cell conditioned media by ultra-centrifugation onto a sucrose cushion. vesicular hspgs were modified either by removal of heparan sulphate (hs) glycosaminoglycan (gag) chains using the enzyme heparinase iii (hepiii), or attenuation of hspg core protein expression using shrnas to knockdown specific hspgs within the parent cell. differences in proteins present in control vs modified evs were identified by a sensitive protein array, based on proximity-ligation technology, and selected targets validated by elisa. functional delivery of growth factors by ev-associated hspgs to recipient fibroblasts is being explored using a variety of in vitro techniques. results: proteome analysis identified targets that bind to hs-gag chains, and also different proteins that showed altered expression following the loss of one or more hspgs from evs. using elisa, we have been able to quantify selected candidates on wild type vesicles, some of these are lost following hsdigestion. we were also able to validate proteins on hspg-deficient vesicles. gene ontology analysis suggests that ev hspg-mediated delivery of growth factors is important for control of processes such as angiogenesis, tumour invasion and immune regulation. functional validation of proteins identified is ongoing. summary/conclusion: here we demonstrate that hspgs play a key role in loading of evs with a complex assortment of growth factors, and therefore subsequent ev-mediated growth factor delivery. we anticipate that loss or damage of ev-associated hspgs will result in attenuation of ev induction of a tumour-supporting fibroblast phenotype. introduction: ovarian cancer (oc) is the fifth leading cause of cancer-related death in women, partly due to difficulty in early diagnosis. extracellular vesicles (evs) show promise for use in early diagnostics of oc. here, evs from cervical mucus (cm) of ovarian cancer patients were used for discovery of oc biomarkers for diagnostics. machine learning was used to mine ev mirna data to develop an oc biomarker panel (validation via the cancer genome atlas). examination of the mirna targets reveal that the panel is a sufficiently accurate predictor of oc. methods: evs from the cm of patients ( highgrade serous, low-grade, benign) were isolated for small rna-sequencing. the top differentially expressed mirnas were used in a random forest and "voom" (variance modelling at the observational level) model. unsupervised approaches were used and then vetted against patient symptomology data. a tcga ovarian cancer dataset (n = ) was used for validation. results: an oc biomarker panel of micrornas (voom: . % accuracy; random forest: % accuracy) was generated. the panel consists of members from the mir- family and the mir- family, among others. the mirna targets are associated with molecular functions and pathways specific in oc progression. summary/conclusion: our method has identified ev mirna biomarkers that may be crucial for early, noninvasive detection of oc. data science has been used to develop a feedback system integrating biochemical experiments, smaller datasets, and previously available data to identify and verify a biomarker panel for oc diagnostics. introduction: liver disease has become a significant cause of morbidity and mortality among hiv patients. alcohol exposure can further exacerbate liver damage by activating hepatic stellate cells (hscs), leading to hepatic fibrosis or cirrhosis, often seen at all levels of alcohol exposure among people with hiv. due to the potentiating effects of alcohol on hiv-induced hepatocytes (hep) damage, as well as the effect of ethanol in hsc-mediated extracellular remodelling, it is imperative to understand the interplay of hep and hscs. here, we focus on the exosomes released by hiv-and ethanol exposed hep and how these exosomes modulate the functional behaviour of hscs. methods: human hepatocyte huh . cyp e [hepatoma cells stably transfected with cyp e designated as rlw cells] were infected with hiv in the presence or absence of alcohol metabolite, acetaldehyde using the acetaldehyde-generating system (ags). the conditioned medium was collected from groups of cells: untreated, hiv-, ags-and hiv+ags. quantification of exosomes number and size were evaluated with zetaview or nanosight and further characterized for exosome markers following the guideline from minimal information for studies of evs (misev ). the human hepatic stellate lx- cell line was exposed to hepatocyte-derived exosomes and assessed for the activation using pro-inflammatory markers il- β, il- , tnfα, and fibrotic markers acta , and timp using quantitative pcr. we also analysed exosome mirna content in primary human hepatocytes (phh), which potentially regulates the function of recipient cells by "programming" their inflammation/fibrosis status. the network analysis for mrna and mirna were carried out using gene ontology consortium, and mirror . and david bioinformatics resources . . results: ags treatment further enhanced the release of hiv-induced exosome from hepatocytes. size distribution assessed by zeta view or nanosight revealed that approximately - % of particles distributed in the range of to nm, with a peak at~ nm. enriched expression of hiv protein p was observed in fractions f -f . western blotting of hepatocytederived exosome demonstrated positivity for exosome-enriched proteins alix, tsg and cd specifically in f -f fractions and negative for endoplasmic reticulum protein calnexin. the uptake of hepatocytederived exosomes by hscs was apparent as demonstrated by immunofluorescence. the internalization of hepatocyte-exosome induced activation of hscs as evidenced by increased expression of pro-inflammatory il- β, il- , tnfα markers in the latter cells. summary/conclusion: we conclude that ags treatment in hiv-infected hepatocytes potentiates the release of exosomes, which, following uptake by the hscs, leads to their activation. funding: this work is supported by nih- r aa - a . antimicrobial peptide ll- induces neutrophil-derived extracellular vesicles with antibacterial potential and protects murine sepsis yumi kumagai, taisuke murakami, kyoko kuwahara and isao nagaoka juntendo university, bunkyo-ku, japan introduction: extracellular vesicles (evs) released from immune cells or other host cells upon microbial infection modulate the immune response and thereby regulate the infection. sepsis is a life-threatening multiple organ dysfunction caused by systemic dysregulated inflammatory response to infection. nevertheless, numerous therapeutic trails concerning immune dysfunction have still been disappointing outcomes. we have previously shown that ll- , a human cathelicidin antimicrobial peptide, improves the survival of caecal ligation and puncture (clp) septic mice. here, we investigated the induction of ev release by ll- and functions of ll- -induced evs in murine sepsis. methods: evs were isolated from peritoneal exudates of clp mice and the supernatant of ll- -stimulated mouse bone marrow neutrophils by differential centrifugation or size exclusion chromatography. isolated evs were analysed by flow cytometry, western blotting, and nano particle analysis. neutrophil-derived evs were injected into clp mice to assess the protective function of evs against septic mice. the antibacterial activity of evs was evaluated by incubating with escherichia coli. results: in clp mice, ll- augmented the level of evs. evs from ll- -injected clp mice contained higher amounts of neutrophil-derived antibacterial proteins (lactoferrin and cramp, cathelicidin-related antimicrobial peptide) and exhibited higher antibacterial activity compared to evs from pbs-injected clp mice. furthermore, ll- stimulated mouse bone marrow neutrophils to release evs with antibacterial potential, and administration of the ll- -induced evs reduced the bacterial load and improved the survival of clp mice. summary/conclusion: ll- induces the release of antimicrobial evs from neutrophils in clp mice, thereby reducing the bacterial load and protecting mice from lethal septic condition. identification of mirna profiles of serum exosomes in active tuberculosis introduction: tuberculosis (tb) has exceeded hiv as the most lethal infectious disease globally for two consecutive years, mainly due to difficulties in achieving early and definitive diagnosis, and timely treatment. exosomes carrying rna, particularly mirna, have demonstrated their functional and diagnostic potential in diseases including tb. however, few published studies have explored whether exosomal mirnas could be used for diagnosis of tb. thus, more systematic and comprehensive study of exosomal mirnas with regard to their potential as non-invasive tb biomarkers is still urgently needed. methods: we searched the gene expression omnibus database for datasets published before december , and performed meta-analysis on available exosomal mirna profile data for healthy control (hc) and active tb clinical specimens . reprocessing next generation sequencing data under uniform parameters and utilizing state-of-the-art bioinformatics analysis. results: we identified many distinct up-regulated and down-regulated differentially expressed exosomal mirna across multiple studies, and further screened the top , which might provide a potential panel for differentiation of hc and tb. we classified all differentially expressed mirnas into six expression patterns and identified two persistently up-regulated mirna (hsa-mir- - p, and hsa-mir- - p) as potential markers during tb progression. moreover, the differential expressed exosomal genes that we screened from the datasets were consistent with the genes overlapped with predicted mrna targets of differentially expressed mirna. pathway and function analysis further demonstrated down-regulated signalling pathways/immune response and up-regulated metabolism and apoptosis/necrosis. introduction: trypanosoma cruzi is a protozoan parasite that causes chagas disease, a relevant source of morbidity in latin america, which has spread to many countries as result of immigration of the people from endemic areas. many studies have been showed that trypomastigote forms of t. cruzi release extracellular vesicles (ev) that increase parasite infection. objectives. here, we aim to test if previous immunization with evs in adjuvant can generate a protective immune response by decreasing the effects of evs in experimental chagas disease. methods: female balb/c mice were immunized by intra peritoneal (ip) administration with × or evs isolated from trypomastigotes forms, with aluminium hydroxide adjuvant (aloh). injections were administered intravenous in doses during days ( days interval). after immunization, mice were infected intra-peritoneally with trypomastigotes forms. parasitaemia was quantified by counting motile parasites in fresh blood sample drawn from lateral tail veins. mortality and weight were analysed during the infection. in control group, the mice were immunized with aioh. results: the immunization with evs with aloh decreased the blood parasitaemia and the animals survived, while all animals died in the group aloh alone. the animals immunized with evs had an increase of f / + cd b+ and cd /cd expression in cells isolated from the peritoneum. summary/conclusion: these results indicate that t. cruzi ev antigens can induce an immune response that controls the development and establishment of the experimental chagas disease. introduction: acinetobacter baumannii (ab) is a nosocomial pathogen, of major concern due to its multidrug resistance (mdr) and the recent appearance of hyper-virulent strains in the clinical setting. the world health organization included ab as a critical priority pathogen for the development of novel antibiotics. ab pathogenesis is associated with a multitude of potential virulence factors (vf) that remain poorly characterized. there is growing evidence that outer membrane vesicles (omv) are used as vehicles to transport bacterial proteins that contribute to set up the conditions for the infections. in the present work we studied the physiopathology of mdr ab. we focused on the contribution of non-characterized outer membrane proteins (omps) associated to omvs, with special focus on lipoproteins (lp). methods: we conducted a bioinformatic prediction using available datasets to construct a list of omv-associated omps putatively acting as vf in ab . seven genes were selected and the corresponding mutants were obtained from manoil lab collection. physiological analyses of the mutants were performed, and the involvement of the selected proteins in ab pathogenesis was evaluated by adherence, invasion, and cytotoxicity assays on human lung cells a . results: biochemical analysis indicated similar growth rates in rich media, as well as similar levels of omv production for all the mutants as compared to wt. also, no differences in susceptibility to chaotropic agents were observed, indicating no alteration of the om function as a general permeability barrier. all mutants similarly reduced a cell viability, but to a lesser extent than the wt. moreover, three of them exhibited less adhesion and invasion compared to the wt, and omv isolated from these mutants displayed variable levels of cytotoxicity. summary/conclusion: these results suggest roles for the mutant gene products in ab pathogenesis and contribute to the better understanding of ab virulence mechanisms, revealing novel possible targets for therapeutic development. funding: agencia nacional de promoción científica y tecnológica (anpcyt, pict - ) medicine, nanfang hospital, southern medical university, guangzhou, , china, guangzhou, china (people's republic); d zhujiang hospital, southern medical university, guangzhou, china, guangzhou, china (people's republic) introduction: talaromyces marneffei (t. marneffei) grows as a mycelial form in the environment but multiplies rapidly as a yeast form in the host and within macrophages. the yeast can cause disseminated and progressive infections or lethal talaromycosis. but the mechanisms of pathogenicity of t. marneffei are poorly understood. fungal extracellular vesicles (evs) have previously been shown to transmit a proinflammatory message to macrophages. however, the characteristics and effects of t. marneffei evs on the progress of infection have not yet been investigated. methods: in this study, evs of t. marneffei yeasts were isolated by ultracentrifugation method. evs were detected and confirmed by electron microscopy and nanoparticle tracking analysis (nta). the raw . murine macrophages were incubated with the t. marneffei vesicles to observe the changes of macrophage morphology and function, especially in inflammatory response. the proteins, dnas, rnas of t. marneffei vesicles were respectively removed with protease, dnase and rnase. all treated evs were used to incubate with murine macrophages observe the effect on macrophages in inflammatory response. results: we observed that evs secreted by t. marneffei have a typical spherical shape with a diameter of to nm. t. marneffei evs were internalized by raw . murine macrophages and promoted the production of no and proinflammatory cytokine by macrophages in a dose-dependent manner. t. marneffei evs stimulate macrophages to generate reactive oxygen species (ros). addition of t. marneffei evs to macrophages also promoted transcription of the m -polarization marker cd and diminish that of the m markers cd . incubation of t. marneffei vesicles with murine macrophages resulted in increased levels of extracellular interleukin- β(il- β), interleukin- (il- ) and interleukin- (il- ). the proinflammatory effect of vesicles was weakened when the proteins of the vesicles were destroyed. in contrast, no similar changes were observed in degraded dna and rna. summary/conclusion: our results indicate that the extracellular vesicles of t. marneffei can stimulate macrophage towards to m polarization phenotype and promote proinflammatory function. plasma-derived extracellular vesicles as potential biomarkers in chronic chagas disease patients introduction: chagas disease (cd), caused by the parasite trypanosoma cruzi (t. cruzi), is a neglected tropical disease affecting about million people worldwide. currently, one of the main clinical problems is the lack of effective biomarkers for therapeutic response and disease prognosis during chronic infections. in that context, extracellular vesicles (evs) are raising attention as novel, minimally invasive, and inexpensive method for diagnostic and screening of diseases, as well as a new source to identify new biomarkers. the main objective of this study is to use evs derived from biological fluids of chronic cd patients for identifying novel biomarkers, specifically in the context of therapeutic response and disease prognosis. methods: plasma, saliva and urine from a cohort of chronic cd patients are being collected before and at the end of benznidazole treatment. as negative controls, healthy donors have been also included. the purification and characterization of the evs was performed by size exclusion chromatography, followed by nanoparticle tracking analysis, bead-based flow cytometry assay and transmission electron microscopy. a proteomic analysis of the evs was also performed. results: proteins associated with evs secreted by infective t. cruzi have been previously identified in cell culture, but never in human samples. our results, based on the analysis of a single heart-transplanted patient with chronic cd, showed the presence of t. cruzi and human proteins specifically associated with plasma-derived evs. noticeably, several human and parasite proteins identified in evs obtained from plasma samples, were present or upregulated before chemotherapy and were absent or downregulated following treatment. currently, proteomics analyses are being performed with higher numbers of cd plasma samples. summary/conclusion: to the best of our knowledge, this is the first proteomic profiling of plasma-derived evs from a heart-transplanted patient with chronic cd. these results thus open the possibility of using evs from biological fluids as a tool for the identification of new biomarker candidates in chronic cd. these biomarkers are essential for assessing disease introduction: eukaryotic cells communicate with one another through multiple pathways. an established route of communication between eukaryotic cells is via the production of a range of different membrane bound signalling "packages", called extracellular vesicles (evs). evs are produced by all domains of life and carry proteins, nucleic acid (rna and dna), and other biological material, travelling between cells and around the body to deliver a range of chemical messages. bacteria can also produce evs that communicate with each other to coordinate population behaviour, as well as with eukaryotic cells to stimulate host defence or induce tolerance. here i investigate the poorly explored axis where evs are the vehicle for communication between eukaryotic cells and bacteria. methods: as a first step, i have isolated evs from tissue cultured eukaryotic cells grown in advanced rpmi media with minimal ev-depleted fbs. nanoevs were isolated from spent culture media using sequential centrifugation ( , × g, , × g) and concentration ( kda filter) before purifying using size exclusion chromatography columns. nanoev-rich fractions were pooled based on particle (nanoparticle tracking analysis) and protein quantity data. nanoevs were characterised by electron microscopy and expression of exosomal markers. eukaryotic nanoevs were then characterised in their effect upon the growth of escherichia coli as a model bacterium, also grown in tissue culture media to mimic relevant in vivo conditions. results: further experiments with increased dosages are required to determine the effect of human evs on bacteria. summary/conclusion: our work will investigate whether human evs communicate with the resident and pathogenic microbiota, while examining the mechanisms behind this communication. escherichia coli pathogenic bacteria commensal bacteria hydrogen sulphide (h s) derived extracellular vesicles: a potential protective role in response to respiratory syncytial virus (rsv) infection methods: evs were isolated from untreated (control evs) and gyy treated (gyy-evs) a cells, a human alveolar type ii-like epithelial cell line. evs were purified using a two-step enrichment procedure. evs were characterized using particle sizing (size and concentration) and western blot for the ev markers. electron microscopy and immunofluorescence staining were used to investigate presence of multivesicular bodies (mvbs), evs precursors, in both groups. recipient a cells were cultured for hours in the presence or absence of control-or gyy-evs, then infected with rsv for hours. viral titres by plaque assay were measured in recipient infected a cells. results: we confirmed the presence and purity of our evs. we found that gyy reduced the particles number of evs, but did not change ev size. a cells treated with gyy showed an accumulation of mvbs/lysosomes-like structures, as well as an increase in cd expression, a mvbs marker, compared to untreated cells. recipient a cells treated with gyy-evs showed lower viral replication than control ev-treated cells in response to rsv infection. we are currently investigating the potential mechanism for this observation and characterizing the rna cargo composition of gyy-evs. summary/conclusion: no vaccine or effective treatment is currently available for rsv. cellular pretreatment with gyy-evs reduced the rsv replication in airway epithelial recipient cells, suggesting that h s could exert its antiviral activity in the context of rsv infection potentially through modulation of ev composition. therefore, gyy-evs could represent a future novel pharmacological approach for ameliorating virus-induced lung disease. effects of extracellular vesicle-mediated transmission on reoviridae infection results: taken together, these data suggest that multiple particles of reovirus and rotavirus egress in large, virus-modulated evs, and that transmission in evs increases segment complementation compared to transmission as free particles. summary/conclusion: these discoveries may be broadly applicable to viruses that travel in evs and will contribute to general principles of virus transmission and diversification. continued studies will illuminate the specific cellular pathways reovirus and rotavirus utilize for successful egress. these pathways may prove to be critical targets for the improvement of vaccines and oncolytic therapy. multiparameter flow cytometry analysis of the human spleen and its interaction with plasma-derived evs from plasmodium vivax patients introduction: the spleen is a secondary lymph organ that filters blood and elicits immune responses against blood-borne pathogens, such as malaria parasites. extracellular vesicles (evs) are membrane-bound particles involved in intercellular communication. evs play several roles in malaria ranging from modulation of immune responses to induction of vascular alterations. here, we report the first integrated characterization of human spleen cells using multiparameter flow cytometry (mfc) describing subpopulations of splenic leukocytes and red blood cells (rbcs), and studied their interaction with plasma-derived evs from p. vivax patients (pvevs). methods: human spleens were obtained from organ transplantation donors. myeloid, lymphoid, erythroid and haematopoietic stem cells (hscs) were immunophenotyped by mfc. t cells, dendritic cells (dcs) and rbcs were enriched by density centrifugation and immunomagnetic isolation. pvevs and healthy donors evs (hevs) were purified by size-exclusion chromatography (sec) and characterized by bead-based flow cytometry. enriched evs were labelled with fluorescent lipophilic dyes and incubated with total splenocytes or enriched populations. evs-cells interaction was assessed by flow cytometry. results: human spleen immunophenotyping showed that cd + cells included b ( %), cd + t ( %), cd + t ( %), nk ( %) and nkt ( %) lymphocytes. myeloid cells comprised neutrophils ( %), monocytes ( %) and dcs ( . %). erythrocytes represented % whereas, unexpectedly, reticulocytes were . % of total cells. in addition, we also detected hscs, which accounted for . %. sec separated evs from the bulk of soluble plasma proteins as shown by the enrichment of cd , cd l and cd markers. interaction studies showed an increased proportion of t cells (cd + -fold and cd + -fold), monocytes ( . -fold) , b cells ( . -fold) and erythrocytes (threefold) interacting with pvevs as compared to hevs. summary/conclusion: the integrated cellular analysis of the human spleen and the methodology employed here allowed in vitro interaction studies of human spleen cells and evs. a larger proportion of monocytes, t and b lymphocytes as well as erythrocytes was found to interact with pvevs compared to hevs. future functional studies of these interactions can unveil pathophysiological processes involving the spleen in vivax malaria. neuroblastoma-secreted exosomes carrying mir- promote osteogenic differentiation of bone marrow mesenchymal stromal cells introduction: bone marrow (bm) is the major target organ for neuroblastoma (nb) metastasis and its involvement is associated with poor outcome. yet, the mechanism by which nb cells invade bm is largely unknown. tumour microenvironment represents a key element in tumour progression and mesenchymal stromal cells (mscs) have been recognized as a fundamental part of the associated tumour stroma. here, we explore the potential role of nb-derived exosomes in induction of a pro-osteogenic phenotype on bm-mscs. introduction: extracellular vesicles (evs) are nanosized particles delimited by a lipid bilayer which transfer functional molecular cargos from the cells of origin to target cells. this intercellular crosstalk controls both physiological and pathological conditions. given their presence in body fluids and their characteristics, these nanocarriers might be potentially used in diagnostics and/or therapy. breast cancer is the most frequently diagnosed malignancy and ranks as the leading cause of cancer mortality in women worldwide; the triple negative breast cancer, in particular, is the most aggressive subtype with a poor prognosis. since it is recognized that cell stiffness of cancer cells play a crucial role during the metastatic spreading, we set ourselves the goal of clarify the effects and the activity of small-evs (i.e. with a diameter below nm) in metastatic breast cancer, with a special attention on their correlation with the biomechanical properties of cells. methods: functional assays were performed on the non-invasive mcf breast cancer cell line, before and after the cellular uptake of small-evs originating from the invasive mda-mb- triple negative breast cancer cell line. the mechanical properties (cell stiffness, cytoskeleton organization and focal adhesions) of mcf cells were investigated before and after the vesicle uptake. results: the uptake of small-evs derived from mda-mb- significantly reduces the young's modulus values of mcf cell line making them more invasive. moreover actin and focal adhesion variations were observed in mcf cells before and after small-ev's uptake, suggesting a molecular rearrangement inside mcf cells upon uptake. summary/conclusion: our results evidence that small-evs play a key role in altering biomechanical properties of target cells and underline their relevance in cell-cell crosstalk. our approach is very promising to identify new molecular mechanisms through which evs perform their oncogenic function. stratification of angiogenic or non-angiogenic lesions in colorectal cancer liver metastases patients using extracellular vesicle mirna introduction: colorectal carcinoma (crc) is the second leading cause of cancer death in the western world. over % of the crc patients develop liver metastasis (lm) and % will die from metastatic disease. in the current clinical setting, liver resection provides the only possible cure, but only % of crclm patients are resectable. the combination of angiogenic inhibitors with chemotherapy is used to downsize crclm with the goal of converting unresectable patients to resectable ones. however, only - % of these patients can be successfully converted to a resectable state. we have no way of identifying those crclm patients that would respond/benefit to the addition of anti-angiogenic therapies (e.g. bevacizumab: bev)). proper stratification of patients into angiogenic inhibitor responders and non-responders will permit a proper assessment of the efficacy of angiogenic inhibitors. crclm forms distinct histopathological growth patterns (hgp): angiogenic (desmoplastic) and nonangiogenic (replacement) hgp. we demonstrated that crclm patients with predominant angiogenic lesions receiving bev plus chemotherapy have a more than double -year overall survival compared to patients with non-angiogenic lesions. therefore, nonangiogenic lesions do not respond to angiogenic inhibitors. our study focuses on stratifying angiogenic vs non-angiogenic lesions of crclm through extracellular vesicle mirnas. we are using two approaches in the selection of mirnas to target: . text mining of published ev mirna from crclm patients; and . differentially expressed mirnas present in tumour tissue from both lesion types, we have obtained by sequencing - patients. these two strategies will generate a list of mirnas that we will target using qpcr on plasma-derived ev mirna from the patients used in approach , where we have classified the lesions in the patients. preliminary data on patients will be presented. methods: ev isolation was performed using the gold standard centrifugation method. rnaseq and qpcr are used to generate the expression profile for angiogenic vs non-angiogenic type of crclm. results: the research is under progress. summary/conclusion: the research is under progress. the introduction: it is known that bone metastasis causes a reduction in the quality of life of cancer patients due to fractures and nerve compression. therefore, it is important to elucidate the mechanism of bone metastasis and develop new treatments. metastatic bone tumours occur at particularly high rates in cancers of the prostate, breast, and lung. in this study, we focused on extracellular vesicles (evs) in bone metastasis, and investigated that the role of evs derived from cancer cells in osteolysis. methods: the prostate, breast, and lung cancer cellderived evs were added to osteoclast precursors with rankls. the osteoclast differentiation was evaluated by tartrate-resistant acid phosphatase (trap) stain and by measuring the expression level of osteoclast markers using by qrt-pcr. a proteome analysis (lc-ms/ms) and sirna approaches were used to identify molecules which are responsible for promotion of osteoclast differentiation in the prostate cancer cellderived evs. to investigate whether the molecules are suitable for the detection of bone metastasis in serum evs, we isolated evs from serum of prostate cancer patients, and analysed the protein level of the molecules by western blot analysis. results: we found that the prostate cancer and lung cancer-derived evs significantly promoted the rankl-stimulated osteoclast differentiation. our analysis revealed that cub domain-containing protein (cdcp ), which is a membrane protein on the prostate cancer cell-derived evs, was responsible for promotion of osteoclast differentiation. moreover, cdcp was markedly detected in the evs-derived from serum of prostate cancer patients who had bone metastasis than that of normal subjects. we also found that cdcp exits on the breast and lung cancer cell-derived evs. summary/conclusion: we showed that the evsderived from bone metastatic tumours have a role in activation of osteoclastogenesis. moreover, we revealed that cdcp in the evs is responsible for promoting of osteoclast differentiation. these evs could be the novel diagnostic and therapeutic target for bone metastasis. increased expression of chemokine receptor cxcr in non-invasive colorectal cancer cells after incorporation of platelet-derived extracellular vesicles. introduction: blood platelets and platelet-derived extracellular vesicles (p-evs) play a crucial role in tumour growth and metastasis. p-evs, also referred to as platelet microparticles, are recognized as a carrier for proteins and nucleic acids that control cell-to-cell communication, mediate the formation of metastatic niches and affect tumour invasion and metastasis. among the other factors, p-evs contain the chemokine receptor cxcr , known as a co-receptor for hiv entry but also regarded as important in cancer development due to the importance of cxcr /cxcl signalling. overexpression of cxcr was reported in various, especially in invasive cancers, including colorectal cancer (crc). crc, the third most commonly diagnosed cancer, is usually diagnosed at the late stage and patient's death is mainly related to metastasis. increased levels of cxcr has been reported as a poor prognostic factor for survival of crc patients and its blocking has been suggested as therapeutic approach. the aim of this study was to analyse the effect of p-evs on the levels of cxcr in crc cells on various epithelial-to-mesenchymal transition stage. methods: we used crc cell lines ht and sw , which represent distant invasive potential and different phenotypes, epithelial and strongly mesenchymal, respectively. p-evs were isolated from outdated concentrates of human blood platelets after activation by thrombin in the presence of calcium ions, by subsequent centrifugation and ultracentrifugation. the p-evs were labelled using pkh fluorescent dye to visualize their uptake into cell lines by confocal microscopy. we also quantified the levels of cxcr in ht and sw by western blot analysis. the effect of p-evs uptake on the migration of crc cells was studied by "wound healing" method. results: we found that the levels of cxcr in crc lines used in the study were correlated with their emt stage. we show here that p-evs released by activated platelets were incorporated into both ht and sw cell lines. the expression of cxcr in ht was increased after the uptake of p-evs. additionally we observed that migration rate of ht cells with incorporated p-evs was elevated as compared to control cells. summary/conclusion: we posit that circulating p-evs can be incorporated into yet not invasive crc cells to significantly increase the level of cxcr receptors and that may lead to the their more invasive characteristics. introduction: for cancer therapy it is important to identify markers and key processes induced during cancer progression. one of them is epithelialmesenchymal transition (emt) which is associated with cell acquisition of invasiveness, stem cell characteristics and resistance to apoptosis and therapy. also the extracellular vesicles (evs) released from tumour cells, which can be taken up by cells constituting pre-metastatic niches, can alter cancer progression by promoting cells' reprogramming. our group has recently reported that snail transcription factor, a key factor of emt, when overexpressed in crc ht cells, drives their early emt and alters the expression of microrna (mirs). in the present study we analysed the mirs profile of evs released from those cells. methods: evs from three ht clones stably overexpressing snail and from control ht -pcdna were isolated by differential centrifugation and ultracentrifugation of conditioned media after h of culturing in serum-free medium. total rna was isolated and nextgeneration sequencing (ngs) analysis of the mirnas was performed followed by gene ontology ( introduction: prostate cancer (pca) is the most common malignant tumour in male urinary system and osteoblastic bone metastasis is the most observed metastasis in prostate cancer patients. it has been demonstrated that circulating micrornas contained in extracellular vesicles are potential early biomarkers and therapy targets for many diseases. however, the potential role of micrornas in prostate cancer bone metastasis, is not yet to be fully explored. methods: after isolation and purification evs using ultracentrifugation from conditioned media of bone metastatic co-opting prostate cancer cells and normal cells, total rna was extracted. subsequent to library preparation and small rna-seq, differential gene expression analysis was performed. data were filtered by mean mirna expression of ≥ reads, two fold up or down regulation between . − . and adjusted pvalue ≤ . . the uptake of pca-sevs was performed. three candidate mirnas (has-mir- c- p; has-mir- ; has-mir- - p) were internalized and osteoblast differentiation were detected by qpcr, histochemical staining and protein activity detection. results: total reads of mirnas in bone metastatic co-opting pca-evs exceeded significantly than that in normal evs (p < . ), indicating that mirnas delivered by pca cells play critical role in pca bone metastasis. pca-cm enhanced osteoblast differentiation and can be reversed by gw . the uptake of pca-evs by mc t -e was efficient. the high expression of the three candidate mirnas in pca-evs was verified by qpcr. all the three candidate mirnas promoted osteogenesis, verified by mrna expression of osteoblastic markers (alp, ocn, runx , osx), alp activity, alp staining and aliza red s staining. summary/conclusion: these findings suggest that mirna cargos in pca-evs play a pivotal role in the development of osteoblastic bone metastasis of pca, which can be potential early biomarkers and therapy targets for prostate cancer bone metastasis. funding: this work was supported by grants from the national natural science foundation of china ( ); xijing hospital science and technology foundation project (xjzt ptk ). introduction: retinoblastoma (rb) is the most common intraocular cancer of childhood. despite recent advances in conservative treatment have greatly improved the visual outcome, local tumour control remain difficult in presence of massive vitreous seeding. thus, the identification of new biomarkers is crucial to design more effective therapeutic approaches. traditional biopsy has long been considered unsafe in rb, due to the risk of extraocular spread. exosomes, nano-sized vesicles containing nucleic acids and proteins, represent an interesting alternative to detect tumour-associated biomarkers. the aim of this study was to determine the protein signature of exosomes derived from rb tumours (rbt) and vitreous seeding (rbvs) primary cell lines. methods: exosomes from rbt (hsjd-rbt , hsjd-rbt , hsjd-rbt , hsjd-rbt ) and rbvs (hsjd-rbvs , hsjd-rbvs , hsjd-rbvs ) cell lines were isolated by high speed ultracentrifugation. vesicles number and size were confirmed by nanosight and scanning electron microscopy. protein content was analysed by bicinchonic-acid assay and high resolution mass spectrometry. results: a total of proteins were identified. among these, and were expressed in exosomes rbt and one rbvs group respectively. gene enrichment analysis of exclusively and differentially expressed proteins and network analysis identified identified in rbvs exosomes upregulated proteins specifically related to invasion and metastasis such as proteins involved in extracellular matrix (ecm) remodelling and interaction, resistance to anoikis and metabolism/catabolism of glucose and aminoacids. summary/conclusion: in conclusion, in this study, we isolated exosomes from rb primary tumour and vitreous seeding cell lines and characterized their content with a proteomic approach. this is the first evidence describing a proteomic exosome signature specifically associated with vitreous seeding in rb. this characterization may represent a starting point for future analyses that allow defining exosomal markers as promising diagnostic and potential prognostic markers in rb as well as therapeutic targets. activation of hepatic stellate cells by extracellular vesicles released by uveal melanoma cells introduction: uveal melanoma (um) is the main intraocular tumour in adults, and is particularly resistant to treatments when disseminated to the liver. our hypothesis is that extracellular vesicles (evs) released by the primary tumour are priming the liver stroma for metastatic cell colonization by activating hepatic stellate cells (hstecs). this study aimed to characterize evs from um cells, and to determine their interactions with liver cells. methods: evs were isolated from cell lines derived from ocular tumours and liver metastases by differential centrifugation. their concentration/diameter range were determined by high-sensitivity flow cytometry. cryo-tem combined with receptor-specific gold labelling was used to reveal the morphology/size of melanomic evs. the presence of melanoma and ev markers was assessed by western blotting. the internalization of fluorescent melanomic evs in hstecs and their subsequent activation were assessed by confocal imaging using alpha-smooth muscle actin (alpha-sma) and phalloidin stainings. ev impact on invasion was measured with a tumour spheroid model embedded in extracellular matrix. melanomic evs were inoculated into the retro-orbital sinus of immunodeficient mice to study their selective organ distribution. results: melanomic evs were positive for annexin- , tetraspanins, as well as some melanoma markers. stellate cells with internalized melanomic evs expressed more alpha-sma, reflecting their activation. adding evs on tumour spheroids increased the invasion process. melanomic evs were localized into different murine organs, but mainly into the liver, as observed by in vivo fluorescent imaging. introduction: exosomes are being tested for their use as therapeutic agents in degenerative and chronic diseases. however, the optimal source of exosomes is currently under investigation. amniotic fluid (af) is a naturally-rich source of exosomes that is easily obtained for use in regenerative medicine. organicell flow™ is a minimally-manipulated, acellular product derived from human af and consist of over cytokines/chemokines as well as exosomes derived from the amniotic membrane and surrounding tissues. we characterized the exosome fraction of our product to elucidate the protein cargo of af exosomes and demonstrate the therapeutic potential as a novel regenerative therapy. methods: the exosome fraction of our product was analysed using nanosight nanoparticle imaging and macsplex exosome surface marker array analysis. exosomes were precipitated using size-exclusion filtration followed by ultracentrifugation from independent products (in triplicate) and subjected to protein lysis and preparation for mass spectrometry analysis using the easy nlc and q exactive instruments. tune (version . ) and xcalibur (version . ) was used to collect data while proteome discoverer (version . ) was used to analyse data. protein expression lists were created by merging the sample replicates together and commonly expressed proteins were determined using vinny . vin diagram analysis. webgestalt tool kit classification system was used to identify top protein function and pathway hits. results: organicell flow™ contain a mean concentration of . x ^ particles/ml (n = ) with a mean mode size of . nm (n = ). surface marker analysis confirms the presence of exosome associated proteins cd , cd , and cd in addition to a high expression of cd (n = ). the completed analysis revealed commonly detected proteins across products. the top molecular functions of identified proteins included protein-binding, ion-binding, and nucleic acid-binding with enzymes, transcription regulators, and transporter proteins representing the most abundant protein groups. pathway enrichment analysis revealed top hits for integrin, pdgf, and p pathways. a deeper dive into the enzyme category of the protein cargo further demonstrates the presence of proteins that promote dna repair such as dna polymerase (beta and lambda), telomerase reverse transcriptase, and brca . summary/conclusion: organicell flow™ characterization demonstrates the therapeutic potential of afderived exosomes. proteomic analysis revealed protein cargo that may regulate various growth factor and cellcycle associated pathways. furthermore, the presence of dna damage response proteins suggests a possible mechanism for induction of cellular repair. generation of car-t and γδt cell-derived exosomes for future cell free immunotherapies γδt cells are a subset of t cells with dual innate and adaptive qualities. this duality provides various advantages over their more studied and used counterpart, αβt cells. in the present study, we sought to compare the immunotherapeutic potential of car-t cell and γδt cell-derived exosomes as novel cell-free based alternatives. methods: cd -targeting car-t cells were obtained following the isolation, expansion and transduction of αβt cells using a lentiviral vector bearing the car construct. γδt cells were isolated and expanded from peripheral blood mononuclear cells (pbmcs) following innate or adaptive stimulation. exosomes from both cell sources were isolated after a -day culture in serum-free media using ultracentrifugation-based methods. exosomes were characterized by nanoparticle tracking analysis (determination of size) and western blot assays (detection of the appropriate surface markers). nalm- (b cell precursor leukaemia) cells were used as target cells for assessment of exosome cytotoxic/ killing function. car-t cell and γδt cell-derived exosomes were incubated at particles/target cell for -hours. total viable cell counts were assessed via imaging-based cytometry (nc- ) utilizing acridine orange and dapi staining. results: exosomes derived from γδt cells activated via innate mechanisms showed significant killing of nalm- as compared to exosomes from non-activated or adaptively activated γδt cells. in comparison, car-t cell-derived exosomes showed minor killing capabilities of the target cells. summary/conclusion: here, we report for the first time that exosomes derived from cd car-t cells and innately activated-γδt cells show/exert inhibitory action on nalm- cells. further studies are currently underway to identify the underlying mechanism(s) responsible. introduction: age-related cognitive dysfunction is associated with increased oxidative stress, low-level chronic neuroinflammation, and waned hippocampal neurogenesis in the brain. from this perspective, biologics capable of modulating oxidative stress and neuroinflammation, and stimulating neural stem cell activity in the brain might be useful as anti-ageing interventions. methods: we investigated the efficacy of intranasal administration of extracellular vesicles (evs) generated from cultures of rat subventricular zone neural stem cells (svz-nscs) in the middle-aged mice to alleviate cognitive and mood dysfunction, increased oxidative stress, neuroinflammation, and neurogenesis decline in old age. mice were treated intranasally with nsc-evs once weekly for three weeks ( billion per administration) starting from . months of age. a month later, the animals were examined for cognitive, memory, and mood function using multiple behavioural tests, and brain tissues were examined for oxidative stress, neuroinflammation, and neurogenesis. results: object-based tests revealed that aged animals receiving vehicle displayed cognitive impairments for discerning minor changes in the environment as well as for distinguishing similar but not identical experiences. these animals also exhibited spatial memory dysfunction and anhedonia. in contrast, aged animals receiving nsc-evs showed improved cognitive and mood function. biochemical analyses of brain tissues revealed that nsc-ev treatment normalized elevated concentrations of oxidative stress markers malondialdehyde and protein carbonyls and the proinflammatory cytokine interleukin- beta. moreover, nsc-ev treatment stimulated increased production of antiinflammatory protein interleukin- and the antioxidant superoxide dismutase. immunohistochemical analysis revealed modulation of neuroinflammation typified by reduced activity of reactive astrocytes and activated microglia and improved hippocampal neurogenesis. summary/conclusion: the results suggest that the intranasal administration of nsc-evs is a promising approach for maintaining better cognitive and mood function in ageing through modulation of oxidative stress, neuroinflammation, and neurogenesis. funding: supported by a grant from the national institute of neurological disorders and stroke ( r ns - to a.k.s.) chemically modified myocytes-derived evs for the treatment of cardiac fibrosis. marta prieto-vila a , asao muranaka a and takahiro ochiya b a tokyo medical university, tokyo, japan; b tokyo medical university, shinjuku-ku, japan introduction: myocardial fibrosis is a disorder that may occur after cardiac injure due to a malfunction of the cardiac remodelling. fibroblasts resident in myocardium are erroneously activated causing an excessive accumulation of extracellular matrix, which decreases cardiac function and eventually, leads to death. it is known that cardiomyocytes communicate with the surrounding cells such as fibroblast and endothelial cells by extracellular vesicles (evs). the loss of this communication is thought to play a central role in cardiac fibrosis. therefore, cardiomyocytes-derived evs may be a promising a cell-free system for the treatment of fibrosis inhibition. methods: a novel culture medium was stablished to improve the expansion of primary cardiac myocytes. this was tested using two commercially available primary myocytes cell lines. evs were collected by serial ultracentrifuges, and their effect on fibrosis was tested. for that, prior to any treatment, and to mimic fibrosis, primary cardiac fibroblast were activated overnight with tgfβ. results: by the use of a defined conjunct of chemicals, mature cardiomyocytes culture was highly improved to ensure a high collection of evs. terminal differentiation markers, as well as senesce apparition was delayed in comparison to predetermined culture medium. interestingly, those primary cells secreted a rather large amount of evs, which expressed common evs membrane marker. tgfβ-treated cardiac fibroblasts were co-cultured with myocytes showing a decrease of fibroblast activation markers both at mrna and protein levels. similar results were found when activated fibroblast were treated with evs. summary/conclusion: our findings indicate that the use of evs derived from chemically modified myocytes is a promising treatment for ischaemic myocardial fibrosis. however, further molecular experiments have to be done to identify the molecules within evs responsible for the inactivation of fibroblast. evaluation of osteoinductive and anti-inflammatory properties of spinederived exosomes renaud sicard a , tania del rivero b , jonathan messer c , shabnam namin c and timothy ganey c a vivex, biologics, inc., miami, usa; b vivex, biologics, inc., miami, usa; c vivex, biologics, inc., miami, usa introduction: over the last decades, mesenchymal stem cell-derived exosomes have been shown to play a crucial role in a myriad of cell function such as extracellular matrix synthesis, proliferation, differentiation or cell migration. biological sources of exosome (heterogeneous or homogeneous cell population, serum, urine etc.) have a direct influence on the content of their cargo and their therapeutic application and potential. in this study, we evaluated exosomes excreted from cadaveric spine-derived cells. we hypothesized that exosomes derived from a bone source such as the spine, will drive the osteogenic differentiation of progenitor cells. we also investigated their effects on inflammation in nucleus pulposus cells using an in-vitro assay. methods: after their isolation and characterization, exosomes derived from cadaveric human spines were assayed for osteoinductive properties. a c c myoblast cell line was treated with different concentrations of exosomes and expression of alkaline phosphatase was measured after days incubation. treatment with bmp- was used as positive control. anti-inflammatory properties were assessed by incubating tnf-treated nucleus pulposus cells with exosomes for days. qpcr analysis of mrna expression of inflammatory cytokines (il- , il -beta, il- ) metalloproteinases (mmp and adamts ), and apoptotic genes (bax, bcl ) was used to determine the effects of exosomes on inflammation. results: spine-derived exosomes positively expressed the exosome flow cytometry markers tested (cd , cd and cd ). the mean number of exosomes per microgram of protein was . ± . x indicating a relatively high purity. osteoinductive (oi) testing was performed using different concentrations of exosomes. the oi index of treatment of c c cells with bmp- , x , x , x , × or × exosomes alone was . , . , . , . , . and . respectively. anti-inflammatory properties of exosome are currently being assessed and will be presented at the time of the poster presentation. summary/conclusion: administering exosomes alone or in combination with an exogenous scaffold has the potential to repair injured tissue and to restore bone function. the clinical significance of this application is aimed to promote the patients' bone healing process and provide a cell-free therapeutic platform that is safe and effective. administration of human mesenchymal stem cell derived extracellular vesicles modulates the abnormal plasticity of newly born neurons and neuroinflammation in a rat model of status epilepticus maheedhar kodali a , daniel gitai b , dong ki kim a , mariam atobiloye a , bing shuai c , sahithi attaluri c , raghavendra upadhya c , leelavathi n madhu a , olagide w. castro a , darwin j. prockop a and ashok k. shetty c decline in the percentage of newly born neurons displaying basal dendrites. besides, ev treated animals displayed higher percentages of resting microglia (ramified microglia), reduced percentages of activated microglia (microglia expressing iba- and cd ), in comparison to animals receiving vehicle after se. interestingly, diminished abnormal plasticity of newly born neurons was accompanied by the preservation of interneurons positive for reelin; a protein believed to guide newly born neurons to their correct locations. summary/conclusion: the results suggest that even a low dose in administration of msc-derived evs after se can limit neurons loss, dampen the abnormal plasticity of newly born neurons, and modulate the activation of microglia. introduction: autism spectrum disorders (asd) are neurodevelopmental disorders characterized by three core symptoms that include social interaction deficits, cognitive inflexibility, and communication disorders. they have been steadily increasing in children over the past several years, with no effective treatment. two percent of all asd patients are suffering from a disorder caused by a mutation in the shank gene. shank is an important synaptic protein, disruption of this gene directly leads to cognitive and motor impairments. during the recent decade, exosomes that derived from mesenchymal stem cells (msc-exo) have been spotlighted as a promising therapeutic target for various clinical indications, including neurological disorders. here we test three different autistic mice models. btbr as a multifactorial mice model of autism and two different shank mutated mice. the first is a complete deletion of exon ( q . ) and the second is a specific insertion mutation of guanine to position in the gene (insg ) that leads to stop codon. methods: exosomes were isolated using differential centrifugation protocol and characterized using the misev guideline recommendations. each animal received an intranasal administration of ul containing exosomes/µl. for intravenous administration, the same number of exosomes, were used, injected in µl. results: all three animal models showed significant improvement in their autistic behavioural phenotypes following intranasal administration. the improvement seems to be dose-dependent and was better achieved via intranasal vs intravenous administration. biodistribution of msc-exo showed accumulation in the brain within hours, yet the reduction of the signal was observed in the kidneys, heart and lungs. summary/conclusion: our data suggest that exosomes derived from adipose msc, carry a therapeutic potential in asd, via non-invasive intranasal administration in three different mice models. these data further emphasize our potential therapeutic strategy to reduce symptoms of autism in clinical trials. funding: stem cell medicine ltd. israel. equine tendon injury treatment by evs: an in vitro study introduction: current treatment options for tendinopathies (chronic, painful tendon disorders), are not able to restore the functional properties of native tendons. hence, new treatment options are sought. the efficacy of mesenchymal stem cells (mscs) therapies, which combined with a rehabilitation programme including controlled exercise is the current gold standard in equine tendon treatment, has been shown to be largely due to the cells´paracrine activity. the aim of this study was therefore to evaluate the effect of bone marrow msc derived autologous and allogeneic conditioned medium (cm, full secretome) and their extracellular vesicles (evs) on "tendon healing" in vitro. methods: to compare the "therapeutic" effect of msc derived evs and cm, a standardized scratch assay (wound healing assay) was performed. cm from equine tenocytes, ev depleted medium and medium with or without fcs served as controls. tendons and bone marrow aspirates were obtained from three horses ( , and years) which were euthanized for reasons unrelated to this study. mscs were isolated by ficoll density gradient centrifugation and tenocytes were obtained by migration from tendon explants. for cm and ev production, cells were cultured in ev depleted medium. evs were harvested by a stepwise ultracentrifugation approach and characterized by nanoparticle tracking analysis (nta), western blot (cd , cd ) and transmission-electron microscopy (tem). results: western blot, nta and tem confirmed successful isolation of evs from equine mscs. the strongest positive effect on wound healing (fastest gap closure) was achieved by msc-cm (p < . ). the gap closure achieved with msc-evs was slower than with msc-cm (p < . ) but faster than with cm of tenocytes (p < . ). donor specific differences in wound healing capability were shown for both autologous and allogeneic application. summary/conclusion: treatment with msc-cm resulted in significantly faster wound healing of adult tenocytes in vitro than msc-evs or tenocyte-cm. mscs donor age shows a significant effect on gap closure following autologous but not allogeneic administration. ev-enriched secretome fraction from gmp-compatible, scalable, human ipsc-derived cardiac progenitors improve heart function in chronic heart failure mice introduction: we have shown that research-use-only grade (res) human ipsc-derived cardiac progenitors (cpcres) can produce a secretome whose small-evenriched fraction (svf) can treat chronic heart failure (chf) in mice. gmp-compatible, scalable processes for a cpc-derived svf suitable for human therapeutic use is needed. methods: ipsc-derived cpc were produced and cultured using gmp-compatible, scalable processes (cpctx). media without cells were "cultured" in parallel for "virgin media" controls (mv). cpcres were cultured as previously described. as a proof of concept, svfs were isolated from conditioned media by ultracentrifugation: cpctx-ev, cpcres-ev and mv. particle size distributions/concentrations (nanoparticle tracking analysis), protein levels (bsa), and the presence of cd- (elisa) were determined. in vitro activity was assessed by huvec scratch wound healing assay, and by rat and human cardiomyocyte (cm) survival assays. c bl/ mice in chf received echoguided myocardial injection of pbs vehicle control ( ul, n = ), cpctx-ev ( ul, n = ), or cpcres-ev ( ul, n = ). change in cardiac function was assessed by echocardiography. results: cpctx-ev particle sizes were polydisperse (mode~ nm) at a concentration of~ . e particles/ml (~ , particles/cell) and~ . mu cd /ug protein. cpctx-ev increased wound healing, human cm survival, and rat cm survival in vitro by . x, . x, and x, respectively over mv controls. in chf mice, significantly less cpctx-ev mice, and less cpcres-ev mice had severely progressive heart failure (left ventricular end systolic volume, lvesv, increased > %) than pbs control mice (pbs vs cpctx-ev, p < . ; pbs vs cpcres-ev, p < . ), and the average ejection fraction of the pbs group deteriorated . x more than the cpctx-ev group (− % vs − . %, respectively; ns). summary/conclusion: we have a process for cpc differentiation and production of conditioned media suitable for use in human clinical trials from which can be made an svf with the potential to treat chf, possibly through re-vascularization or preservation of cm viability. introduction: exosomes are nanoscale vesicles that mediate cell-to-cell communication via exchanging molecular cargo. mesenchymal stem cell (mscs) modification towards an osteogenic path can occur by uptake of exosomes from other cells. it is less clear whether vesicle placement in the absence of cells will facilitate site-specific delivery through acellular transfer of osteogenic activity. an electrospun fleece was combined with bone marrow-derived exosomes in the absence of cells to evaluate osteoinductive potential that might be thermo-stable and be used in a biologically neutral collagen carrier. comparisons were made of standard laboratory assay of osteoinductivity (oi), and in vivo expression in a mouse calvarial defect model. methods: electrospun type-i collagen was prepared with and without hydroxyapatite (ha) (spinplant gmbh, leipzig) as a foundation base for application of the bone marrow-derived exosomes. individual discs of the collagen enhanced scaffolds ( -mm) were prepared and placed in a mouse calvarial skull defect. animals were followed for and weeks. exosomes were isolated from qualified cadaveric human spines by differential ultracentrifugation. microscopic observation, quantitative assessment of oi with an alkaline phosphatase assay, and flow cytometry were used to evaluate the composition, the hybrid nature of the addition to the nano-collagen fibres. a fluorescent protein reporter transgenic mouse model expressing osteocalcin, type-i collagen, phex, and sp (osterix) was evaluated at and weeks to determine bone formation across the defect. results: alp activity on the scaffold with ha demonstrated an approximate tenfold increase to that of the collagen scaffold alone. while a dose-dependent effect, with higher doses of exosomes resulting in a greater amount of alkaline phosphatase expression, expression that exceeded that of the ng bmp- control. dose escalation from . , , and e resulted in similar increases in expression that was statistically greater with the combination of the fleece with the exosome component. bone formation in the mouse calvaium did not demonstrate gap closure at or at weeks, but did demonstrate enhanced osteoclastivity and robust bone remodelling at the margins of the defect. summary/conclusion: bone marrow-derived exosomes dried into an electrospun fibrillar collagen demonstrated in vitro osteoinductive potential that might provide site-specific placement that could enhance biologic potential. with the capacity for ambient temperature storage, the provision of site-specific placement becomes a technical consideration. placement of the human tissue derived exosomes in a transgenic mouse calvarial defect model did not demonstrate bridging bone across the defect. exosomes loaded with pten-interfering rna enables functional recovery in rats after complete spinal cord transection daniel offen a , nisim perets a , shaowei guo b , oshra betzer c , rachela popovtzer c and shulamit levenberg b a tel aviv university, tel aviv, israel; b technion, haifa, israel, haifa, israel; c bar ilan university, israel, ramt gan, israel introduction: complete spinal cord transection is a debilitating disease that usually leads to permanent functional impairments, with various complications and limited spontaneous recovery. the current investigation of molecular mechanisms controlling axon regeneration, (e.g., signalling networks and environmental cues), led to new strategies to enhance axonal regeneration. we have previously shown that intranasal administration of mesenchymal stem cells derived exosomes (msc-exo), cross the blood-brain barrier and significantly ameliorate motor and behavioural phenotype in several animal models of neurotrauma and neuropsychiatric disorders. methods: msc-exo were isolated from human bone marrow and were loaded with phosphatase and tensin homolog small interfering rna (pten-sirna). the exosomes were given intranasally to rats two hours after complete spinal cord transaction. eight weeks later we followed the motor function and histology and electrophysiology study was performed in order to reveal the connectivity and the biochemical changes in the treated rats. results: we demonstrate that intranasal (in) administrations of msc-derived exosomes could penetrate the blood-brain barrier, home selectively to spinal cord lesion via chemotaxis, and integrated in neurons within the lesion. furthermore, in rats with complete spinal cord transection, msc-exo loaded with pten-sirna silenced pten protein expression in the lesion and promoted robust axonal regeneration and angiogenesis, companied with decreased astrogliosis and microgliosis. moreover, the intranasal treatment partially restored electrophysiological and structural integrity, and most importantly, enabled the remarkable functional recovery and significant improvement in their movements. summary/conclusion: this rapid, non-invasive, approach, using cell-free nano-swimmers carrying molecules to target pathophysiological mechanisms suggest novel strategy for clinical translation to spinal cord injury and beyond. a novel umbilical cord derived wharton's jelly formulation for regenerative medicine applications introduction: musculoskeletal injuries have traditionally been treated with activity-modification, physical therapy, pharmacological agents and surgical procedures. these modalities have limitations, as well as potential side-effects. over the last decade, there has been an increased interest in the use of biologics for regenerative medicine applications (rma), including umbilical cord (uc) derived wharton's jelly (wj). despite this increase, there is insufficient literature assessing the amount of growth factors, cytokines, hyaluronic acid (ha) and extracellular vesicles (ev) including exosomes in these products. the purpose of this study was to develop a novel wj formulation and evaluate the presence of growth factors, cytokines, ha and ev including exosomes. methods: wj was isolated from human-uc obtained from consenting c-section donors and formulated into an injectable form. randomly selected samples from different batches were analysed for sterility testing and quantified for presence of growth factors, cytokines, ha and particles in ev size range. the results showed all samples passed the sterility test. growth factors including igfbp , , , and , tgfα, pdgf-aa were detected. expression of several immunomodulatory cytokines, rantes, il- r, il- , were also detected. expression of pro-inflammatory cytokines mcsfr, mip- a; anti-inflammatory cytokines tnf-ri, tnf-rii, il- ra; and homoeostatic cytokines timp- and timp- were observed. cytokines associated with wound-healing, icam- , g-csf, gdf- , and regenerative properties, gh were also expressed. high concentrations of ha were observed. particles in the ev size range ( - nm) were detected and were enclosed by the membrane, indicative of true ev. summary/conclusion: our results confirmed the presence of numerous growth factors, cytokines, ha and ev in the wj formulation. more studies are underway to confirm the presence of exosomes in detected ev using exosome-specific markers. we believe the presence of multiple factors within one wj formulation may play a role in reducing inflammation, pain and augment healing of musculoskeletal injuries. this offers a potential expanded use for rma. funding: this study was funded by biointegrate llc, new york, ny, usa. collagen sponge loaded with mesenchymal stem cell-derived small extracellular vesicles promote robust bone regeneration shang jiunn chuah a , chee weng yong a , jacob ren jie chew a , ruenn chai lai b , yi ann cheow a , raymond chung wen wong a , asher ah tong lim a , sai kiang lim c and wei seong toh d introduction: mesenchymal stem cell (msc) therapy has demonstrated effective bone regeneration in clinical studies. however, the therapeutic efficacy of mscs have been attributed to the secretion of extracellular vesicles (evs), particularly - nm small evs (sevs). here, we investigate the efficacy of msc-sevs loaded in collagen sponge in the regeneration of critical-sized calvarial defects in immunocompetent rats. methods: sevs were isolated from conditioned medium of human mscs and stored at − c. calvarial defects of -mm diameter were surgically created on thirty-two -week-old male sprague-dawley rats. these rats were then randomly assigned to groups (n = rats/group): defects treated with collagen sponge containing μg of sevs in μl saline (cs/sevs) and defects treated with control collagen sponge containing an equivalent volume of saline (cs/control). at and -week post-surgery, the calvarial bone samples was harvested for analyses by micro-computed tomography (micro-ct), histology, immunohistochemistry and histomorphometry. results: at -week post-surgery, micro-ct analysis showed little bone formation at the defect site in both cs/sevs and cs/control groups. no statistical differences were observed in micro-ct and histology scores in both groups. interestingly, cs/sevs group showed significantly higher osteocalcin (ocn)+ area of . ± . % than that of cs/control group ( . ± . %; p = . ). cd + microvessels at sizes ≤ µm and > µm in cs/sevs group ( . ± . and . ± . microvessels/hpf) were also significantly higher than that of cs/control ( . ± . and . ± . microvessels/hpf; p = . and p = . respectively). by weeks, cs/sevs group displayed enhanced new bone formation that completely bridged the calvaria defect. in contrast, rats in cs/control showed limited bone formation. consequently, cs/ sevs group displayed a micro-ct score of . ± . which was significantly better than that of cs/control group ( . ± . ; p = . ). cs/sevs group also exhibited >twofold increase in bone volume, and improved bone quality with higher trabecular thickness and number, and smaller separation (p < . ), compared to cs/control group. consistently, cs/sevs group displayed a significantly better histology score of . ± . than that of cs/control ( . ± . ; p = . ). moreover, cs/sevs group showed significantly higher ocn+ area of . ± . % than that of cs/control group ( . ± . %; p = . ). summary/conclusion: this study demonstrates that single-stage implantation of collagen sponge loaded with ready-to-use msc sevs can promote robust bone regeneration in a rat calvarial defect model. funding: national university of singapore, r , national medical research council singapore, r . immunomodulatory potential of extracellular vesicles derived from mesenchymal stromal cells introduction: extracellular vesicles (evs) derived from mesenchymal stem/stromal cells (mscs) are promising new agents in regenerative medicine and immunotherapy. considering that independent msc-ev preparations might differ in their therapeutic function, we have set up a functional assay allowing testing for the potential immunomodulatory properties of independent msc-ev preparations. methods: human peripheral blood-derived mononuclear cells (pbmcs) were pooled from up to different healthy donors warranting high allogeneic cross-reactivity, even following an optimized freezing and thawing procedure. after thawing, mixed pbmcs were cultured for days in the absence or presence of msc-evs. thereafter, cell morphologies were documented, supernatants were harvested for cytokines quantification and cells were phenotypically characterized by flow cytometry. by analysing the expression of a collection of different lineage and activation markers, we selected a panel of antigens apparently being regulated by msc-ev preparations considered to be therapeutically active. results: we observed that in the presence of active msc-ev preparations more cd + (monocytes) are recovered from the mlr assay than in corresponding control samples. focusing on t cells, we learned that active msc-ev preparations reduced the content of cd and cd t cells expressing activation markers like cd and cd . summary/conclusion: the mlr assay allows elaborated functional testing of immunomodulatory activities of given msc-ev preparations. currently, we are comparing the immune modulatory capabilities of evs derived from distinct sources and optimize the marker panel to distinguish discrete immune cell subtypes such as different cd cell types, i.e. th , th , th and tregs. extracellular vesicles in platelet-rich plasma: dependency on sample processing zala jan a , saba battelino b , darja božič c , matej hočevar d , ales iglič e , marko jeran c , manca pajnič a , ljubiša pađen a , domen vozel f and veronika kralj-iglič a introduction: platelet-rich plasma (prp) proved effective in regenerative medicine. numerous protocols for its preparation and application are available in the published literature. prp possesses important immune, haemostasis and regenerative factors, however, the mechanisms of their action are yet poorly understood. extracellular vesicles (evs) could be one of the important factors that would contribute to the beneficial effects of preparations. this study was performed as a part of a registered randomised controlled clinical trial (nr: nct ). prp was used to treat chronic middle ear inflammations. here we present the results of prp analyses from blood samples of volunteers with no record of disease. methods: plasma obtained from ml of blood was depleted of erythrocytes and enriched with other particles by repetitive centrifugation of samples. flow cytometry (fcm) was employed to monitor particle contents (cells and smaller particles) throughout the sample processing. the platelet gate was divided into two parts: intact platelets and smaller particles. identity and morphology of particles in the preparations were examined by scanning electron microscopy (sem). standard laboratory tests of blood were performed. results: sem images revealed the presence of heterogeneous population of particles in the preparation of prp, most of which were activated and partially fragmented platelets. the population of smaller particles measured with fcm, was identified as evs. the erythrocyte sedimentation rate was statistically significantly correlated to the volume of plasma obtained in the initial centrifugation step (r = , , p < , ) and to the concentration of evs (r = , ; p < , ). time from sample collection to the preparation of prp was negatively correlated with the concentration of platelets in prp and positively with the concentration of evs (r = , , p < , ). platelet concentration in preparation samples was found to depend on the concentration of platelets in the blood and parameters of sample processing connected with larger centrifugal and shear forces on the samples during centrifugation. these include: sample volume, the size and shape of the centrifuge tube and the distance of the sample from the rotor axis. summary/conclusion: evs are gradually forming upon activation and degradation of cells in the sample throughout the sample processing. optimal processing may importantly contribute to the healing properties of preparation. funding: authors acknowledge support from the european union's horizon research and innovation program under grant agreement no. (ves us project) and slovenian research agency (arrs, grants p - , p - , j - ). satellite cell-derived extracellular vesicles as a therapeutic for mitochondrial dysfunction in duchenne muscular dystrophy duchenne muscular dystrophy (dmd). sc-derived extracellular vesicles (sc-evs) may unlock the therapeutic potential of scs by overcoming these limitations. to investigate their therapeutic potential, we assessed the ability of sc-evs to reverse mitochondrial dysfunction, a key pathological feature of dmd, in oxidatively-damaged c c and primary dmd myotubes. methods: scs from c mice were isolated and cultured. evs were isolated from the supernatant of scs via polyethylene glycol precipitation and characterized using nanoparticle tracking analysis. the ability of sc-evs to deliver protein cargo to c c myotubes, and the localization of the cargo once delivered, were analysed using fluorescence microscopy. to examine sc-ev potential to restore the function of damaged mitochondria, c c myotubes were treated with µm h o for h followed by treatment with . x sc-evs for h. separately, cultured dystrophic myotubes were treated with . × evs every h for h. in both sets of experiments, maximal oxygen consumption rate (max ocr) was measured via seahorse xf cell mito stress test. where appropriate, a t-test was performed to test for statistical significance (p < . ). results: based on estimated cell number and ev quantification, each sc released approximately . × ± . x evs/day. evs delivered protein cargo into myotubes within h. fluorescent labelling of intracellular mitochondria showed co-localization of delivered protein and mitochondria. incubation of myotubes with h o resulted in a % decline in max ocr relative to untreated myotubes. subsequent treatment with sc-evs resulted in a % increase in max ocr. treatment of undamaged myotubes with sc-evs had no effect on max ocr. primary dmd myotubes treated with sc-evs showed a % increase in max ocr relative to untreated dmd myotubes. summary/conclusion: sc-evs rapidly deliver proteins into myotubes, much of which co-localizes with mitochondria, and reverses mitochondria dysfunction in oxidatively-damaged and dystrophic myotubes. introduction: flow cytometry has been used extensively for analysis of ev particles stained with fluorescent antibodies directed to the known cell surface markers. quantitation of the surface markers in terms of the number of molecules or the number of antibodies bound per specific marker has remained one of the largest challenges in the ev research field. changes in instrument setup as well as changes in fluorescent antibodies from different vendors, all impact the relative mfi values for the same ev sample. in this work we report a standardization method of quantitating extra-cellular vesicle surface markers with mesf liposomes. methods: liposomes labelled with fitc fluorescent dye were prepared with a bd proprietary technology. dynamic light scattering analysis was used for size determination of the liposomes. bd facsaria™ fusion system, modified with a small particle side scatter module (sp ssc), was used for analysis of the labelled liposomes by flow cytometry. results: we created a set of nm fitc-modified liposomes of various fluorescent intensities with a known number of fitc molecules incorporated in each liposome intensity. the mfi values of each liposome population (intensity) had a linear relationship to the amount of fitc used for labelling the liposome nanoparticles, suggesting that no self-quenching of fitc fluorescence had occurred. the number for the fitc fluorophores for each liposome intensity was expressed in the units of molecules of equivalents soluble fluorochrome (mesf). a plot of mesf vs. the fluorescent intensity of the liposomes (mfi values) obtained from flow cytometry analysis provided a calibration curve, from which the fluorescent intensity (mfi value) of a stained ev sample can be converted to the number of fluorophores bound (mesf value) to the surface of the ev particles. summary/conclusion: by this approach, the mfi values of stained ev particles are converted to standardized mesf values that are independent of instrument variation, resulting in further improvement of inter-laboratory standardization. furthermore, utilization of liposomes with similar size and refractive index to ev particles simplifies the data evaluation and improves the accuracy of ev surface marker quantitation by flow cytometry. currently, other fluorescent dyes are being explored to expand the utility of mesf liposomes with other fluorescent colours. measuring cholesterol as a high-throughput method for quantifying extracellular vesicles introduction: the extracellular vesicle (ev) field currently lacks a high-throughput method for accurately quantifying evs in solution. ev quantification has traditionally relied on nanoparticle tracking analysis (nta), which is time intensive and indiscriminately counts non-ev particles, such as membrane fragments and protein aggregates. we have rigorously assessed two commercially available methods for measuring cholesterol, a major lipid component of the ev lipid bilayer, and evaluated the utility of these assays to quantify evs in minimally processed samples. methods: the amplex® red cholesterol assay and cedex bio ht were used to quantify cholesterol in ev samples via enzymatic oxidation, with dynamic ranges of - , ng/ml and - µl/ml, respectively. samples throughout various stages of purification were analysed, from clarified cell culture medium to highly purified evs separated on an iodixanol gradient. we evaluated several pre-processing methods, to remove non-ev cholesterol content prior to analysis. results: the amplex® and cedex bio ht assays were found to perform comparably for quantifying cholesterol in purified evs (r = . ). importantly, cholesterol quantification on purified ev samples, ranging from e to e particles/ml, correlated well with nta measurements (r = . ). both µm filtration or an additional , rcf centrifugation step following clarification removed cholesterol associated with cellular debris or other non-ev sources, allowing for accurate quantification of conditioned medium samples or ultracentrifugation pellets (ucp) instead of needing to rigorously purify samples with an iodixanol density gradient. summary/conclusion: cholesterol quantitation can be used to accurately estimate ev concentration, allowing for rapid characterization of samples from clarified cell culture supernatant to highly purified evs. this highthroughput analytical capability may enable more comprehensive assessment of methods to boost ev yield through mass screening of cell culture conditions. optimization of nanoparticle tracking analysis of extracellular vesicles isolated from plasma and bronchopulmonary lavage fluid of patients with non-small cell lung cancer introduction: recent studies show that tumourderived extracellular vesicles (evs) greatly influence the tumour microenvironment and impact the therapy. in non-small cell lung cancer (nsclc), bronchopulmonary lavage fluid (balf) appears to be a good source of tumour-derived evs, providing more accurate information about the tumour microenvironment than evs from plasma. so far there is a lack of accurate and standardized methods for ev quantification. fluorescence nanoparticle tracking analysis (fl-nta) is an emerging method of ev-analysis, allowing discrimination of evs and exosomes from impurities. here we perform an optimization of the fl-nta method to compare evs from plasma and balf of nsclc patients and healthy controls (nc). methods: evs were isolated using homemade sizeexclusion chromatography (sec) columns (plasma) and ultrafiltration or differential ultracentrifugation (balf). nta was performed using zetaview pmx (particle metrix) after ev-staining with membrane dyes or fluorescence-labelled antibodies against typical ev-marker (cd , cd , cd ). results: nta scatter measurements showed a higher total particle concentration in plasma than in balf. however, membrane-specific staining showed a much greater purity of ev-preparations from balf, where nearly % of the particles detected in scatter mode showed positive membrane-staining. in contrast, only around - % of particles in the plasma ev-preparations were positive for the membrane dyes. fluorescence-staining for ev surface marker requires further optimization to obtain reproducible results. summary/conclusion: classical nta using only the scatter mode fails to discriminate between evs, lipoproteins and protein aggregates. for ev-analysis from complex biofluids like plasma, fla-nta and staining for specific ev marker is necessary to receive reliable data. balf seems to be a better source of tumourderived evs than plasma, since the obtained ev-preparations show a higher purity. improving conditions for fluorescence-staining and nta measurement of evs from plasma and balf of nsclc patients will provide an additional method for quantifying and phenotyping of evs. introduction: the exoviewer platform currently enables the user to capture extracellular vesicles (ev) by means of surface antigen-specific antibodies (e.g. targeting tetraspanins), making possible the enumeration of individual particles using single-particle interferometric reflectance imaging sensor (sp-iris, interferometric) imaging as well as fluorescence. currently, through interferometric imaging particles smaller than nm cannot be detected, while fluorescently stained ev smaller than nm can be well resolved. further, it is conceivable that small ev contain antigen numbers in the single digits, making antigen-specific immunostaining a challenge. to further characterize ev populations of different sizes and surface marker composition, it would be highly advantageous to target the vesicular nature of the detected particles linked to a fluorescence readout. methods: the goal of this project is to detect ev with a probe that is ubiquitously distributed across the surface (or lumen) of the vesicle. small ( - nm) ev present fairly distinctive lipid membrane features in the extracellular environment, turning the ev membrane into a "universal" marker, and as such may serve as an alternative marker that is complementary to canonical ev surface markers. results: here we present data on successfully staining ev with the membrane dye di- -anepps (di- ) and the luminal dye calcein-am. we demonstrate that ev from different sources can be efficiently stained with either dye, allowing the quantitative characterization of ev in an unbiased manner using exoviewer's fluorescence mode. while both dyes certainly have their own unique strengths, they exhibit the wanted linear correlation of ev staining versus concentration. further, both dyes are compatible with subsequent immunostaining applications, allowing the user to target specific surface or luminal markers (di- ). summary/conclusion: while a large-panel screening featuring other powerful dyes is continuously ongoing, the current data support the notion of providing the experimenter with a reference for total particle count and at the same time fully exploring the larger dynamic range of the fluorescence mode. moreover, the universal probe will enable the user to correlate intensity and particle size measurements, thereby significantly improving the exoviewer platform and its applications. membrane labelling is essential for the identification and quantification of extracellular vesicles via facs introduction: extracellular vesicle (ev) research is challenged by the lack of standard protocols to identify and distinguish between exosomes and ectosomes being released via exocytosis or plasma membrane shedding, respectively. analysis of small ev populations requires high-resolution technology and can be further improved using fluorescent labels such as carboxyfluorescein diacetate succinimidyl ester (cfse). at the inner leaflet of the plasma membrane, cfse is cleaved enzymatically resulting in covalent binding of the dye. in this study we optimized the conditions for membrane labelling of evs and their subsequent detection by flow cytometry to obtain a maximum yield of intact evs. methods: using sequential centrifugation, we separated ev subpopulations from supernatants of colo pancreas carcinoma cells based on size and mass. after , x g centrifugation, we reconstituted evs from the pellet. we used cfse for ev detection and analysed the expression of tetraspanins by facs to confirm the lipid bilayer structure. furthermore, we determined size distribution of evs by nanoparticle tracking analysis (nta) and electron microscopy. detecting evs as cfse+ events, we quantified our samples and investigated the impact of threshold adjustment on ev quantification. results: after high speed centrifugation of cell free supernatants, we identified cfse+ events as evs, which appeared as round structures under the microscope, and ranged from to nm in size. interestingly, tetraspanin markers cd and cd were detectable only on a subpopulation of purified evs, suggesting heterogeneity of our preparations. for sufficient labelling of evs, minimal temperature variations and short incubation times correlated with ev stability. of note, threshold adjustment significantly improved the sensitivity of the flow cytometer for the detection of labelled evs and hence, is central for data comparability. summary/conclusion: protocol standardization is of major importance for the use of evs as diagnostic markers in liquid biopsies. funding: this project has been supported in part by annelise-asmussen foundation, luebeck (grant ), leo pharma germany (grant ). surface plasmon field-enhanced fluorescence spectroscopy (spfs) system for quantitative and qualitative extracellular vesicles total evaluation without any sample pretreatment introduction: the function of extracellular vesicle (ev) is interested in the immunology and oncology fields as a key transmitter for cellular communication. however, the conventional ev evaluation methods are required complicated evs preconcentration from the sample, its leads ev analysis uncertainty. in this study, we applied the spfs highly sensitive automated system for quantitative and qualitative ev evaluation without any sample pre-concentration and preparation step. methods: spfs automated system and plastic disposable sensor had been developed by konica minolta corporation in house. anti-membrane protein (cd , cd , cd ) antibody was chemically bonded on hydrophilic polymer which was immobilized through the gold thin film on the spfs sensor. the concentration of standard ev materials was evaluated by the qnano system before using. ev detection without preconcentrating was achieved by sandwich immunoassay step in microchannel round-trip flow reaction (tat min) with the spfs system, and elisa was adapted as a conventional standard method. after spfs highly sensitive fluorescent measurements step, extracted and detected ev were effectively recovered by using the recovery buffer reaction. results: the ev sensitivity performance between spfs and elisa clearly showed a significant difference, and the lod of spfs ( . particles/μl) method was estimated times superior to the lod of conventional elisa ( , particles/μl). the spfs calibration curve showed a wide dynamic range at least over logs as an additional specificity. spfs method also showed fine results in the dilution linearity test with high reproducibility under the serum/plasma sample condition. the data for recovery test of ev expected us that highly accurate measurement can be guaranteed under the condition of dilution about times or less even in the whole blood sample. after the spfs measurement, extracted ev on the spfs sensor chip could be effectively recovered and could be analysed nucleic acid which contains micro rna. summary/conclusion: spfs system might have great potential for quantitative and qualitative ev evaluation. our strategy with spfs system for ev proteomic and genomic profiling will be possible for applying to ev quality control as well as a novel biomarker development. identification of a novel compound that inhibits small ev secretion and tumour progression by a sensitive elisa screening. yunfei ma a , takeshi yoshida a , duc tuan nguyen a , kazutaka matoba b , katsuhiko kida b , taito nishino b and rikinari hanayama c a kanazawa university, kanazawa, japan; b nissan chemical corporation, tokyo, japan; c wpi nano life science institute, kanazawa university, kanazawa, japan introduction: small evs from tumour cells are known to promote tumour progression, therefore, it is expected to develop drugs that regulate small ev secretion, which can be used in clinical applications. methods: to identify such regulators, we first developed a sensitive elisa system for the quantification of small ev secretion using a high-affinity ev binding protein tim . by using this elisa system, we screened for small compounds that promote or inhibit small ev secretion using a drug-repositioning compound library (about , compounds). results: as a result, we identified eight promoters and two inhibitors, including compound a, which significantly reduced small ev secretion from various cell types without affecting cell growth. we further investigated the effects of compound a on a mouse model of osteosarcoma and found that compound a suppressed tumour progression efficiently. summary/conclusion: these data suggest that compound a would be useful not only for the characterization of small ev function but also for the clinical therapy against tumour progression, by inhibiting small ev secretion. introduction: for many years it was believed that several proteins such as cd , cd and flotillin- were unique for exosomes, however recent studies have shown that several of these markers also can be present in other subpopulations of evs (kowal et al pnas ) . furthermore, few markers have been identified as uniquely present in microvesicles. the aim of this study was to in depth compare the proteome of microvesicles and exosomes. methods: mda-mb- -luc-d h , -d h ln and -bmd a were cultured in ev-depleted media. microvesicles ( , x g, min) and exosomes ( , x g . h) were isolated using a combination of differential ultracentrifugation and a density cushion (~ . g/ml). purity and yield of evs were determined by nanoparticle tracking analysis (nta), western blot, and electron microscopy (em). quantitative mass spectrometry (tmt-lc-ms/ms) was used to identify differently enriched proteins in microvesicles and exosomes (n = x cell lines). results: in total proteins were quantified, with being quantified in all samples. in total and proteins were significantly upregulated in exosomes and microvesicles, respectively. go terms associated with the proteins significantly upregulated in exosomes were "extracellular exosome" and "plasma membrane", while the microvesicle proteome was associated with "membrane" and mitochondrion". in exosomes tetraspanins, annexins, escrt and rab proteins were significantly upregulated. in contrast, proteins that were upregulated in microvesicles were involved in protein translocation into the mitochondrial membrane (timm and tomm proteins), in cytokinesis, and in micos complex. however, flotillin- was not differently expressed in the ev subtypes. summary/conclusion: this study identifies several proteins to be differently enriched in exosomes and microvesicles. several of the proteins suggest recently by kowal and colleagues, such as adam and mitofilin could be validated. additionally several novel proteins could be identified. identifying markers separating microvesicles and exosomes is of high importance for the ev field and future studies will have to validate them also in other cells to determine if they are generic. introduction: the cellular elements composing the lining of brain ventricles have drawn much attention from neuroscientists, especially the role of subependymal cells in neurogenesis, but the role of ependymal cells in brain function and disease is still neglected. our objective is to study the morphological aspects of rat brain ventricles and the ependymal cells as analysed by transmission and field emission scanning microscopy in normal or ischaemic rats. methods: for this purpose, male wistar rats were submitted to minutes of global brain ischaemia and divided into two groups: a) sham-operated animals and b) saline-treated ischaemic animals. all animals were allowed to survive for seven days. all procedures were approved by the ethics committee of the federal university of são paulo ( / ). transmission and scanning electron microscopic analysis of lateral brain ventricles were done in buffered , % glutaraldehyde/ %formaldehyde perfused brains. cerebrospinal fluid was collected for nta analysis. results: the morphological characterization of brain ventricle revealed a slight rarefaction of ciliary tufts of animals submitted to ischaemia when compared to normal animals. field emission electron microscopy revealed the secretion of vesicles by the ependymal cilia in the lateral ventricle. size and concentration of particles in the cerebrospinal fluid was confirmed by nta and transmission electron microscopy. summary/conclusion: our results are unprecedented and bring innovative potential regarding the role of extracellular vesicles in both the physiology and pathogenesis of the nervous system. these data may also contribute to the development of new technologies for diagnosis and therapy of chronic degenerative diseases. introduction: the function of mitochondria relies on precise and effective quality controls. neurons have high metabolic demands and employ multiple mechanisms to ensure functional mitochondria. we investigated mitochondrial vesiclesa less understood quality control mechanism for mitochondriaand assessed the effect of cellular stress. methods: we surveyed mitochondrial vesicles in rat and planaria brains with electron microscopy. we quantified these vesicles with serial-section electron microscopy (fib-sem). we also conducted confocal microscopy with airyscan analysis of cultured neurons expressing fluorescently tagged mitochondrial markers. results: electron microscopy showed the ultrastructure of various types of mitochondrial vesicles. serial-section electron microscopy revealed the d ultrastructure of mitochondrial vesicles and their prevalence in neurons. confocal microscopic analysis showed increased numbers of mitochondrial vesicles in neurons under mild stress. summary/conclusion: our findings provide direct structural evidence for mitochondrial vesicles in neurons and their abundance in response to neuronal stress. their detection in the extracellular compartment (evidence for which is expected to be presented by the time of isev) may allow for development of biomarkers for mitochondrial health, with relevance to numerous pathologic conditions. from endosomes, might be involved in the impairment of rna, specific feature of als disease. combining high-resolution flow cytometry and surface marker analysis using an automated platform to study extracellular vesicle in cerebrospinal fluid unity health toronto, toronto, canada introduction: there is growing enthusiasm that extracellular vesicles (evs) carry the potential for a variety of applications in medicine. as biomarkers, evs may aid clinicians in the evaluation of diagnoses, disease progression, or even response to therapy. however, proper characterization of the amount, size, and phenotype of evs in a given sample remains challenging due to their sub-micrometre size and heterogeneity. over the last years, technologies, including high-sensitivity flow cytometry and automated platforms that simultaneously assess ev amount, size, and phenotype, have matured, providing new opportunities to study evs for future clinical applications. using such technologies to analyse cerebrospinal fluid (csf), which is in direct contact with the brain and spinal cord, may yield valuable insights into neurological disease processes. while there is often uncertainty about the exact source of evs in a biological sample, cd has emerged as a surface marker that suggests a neuronal origin. methods: csf samples that had been stored at - degrees celsius for advanced biomarker studies were analysed using two distinct approaches. a becton, dickinson and company (bd) aria iii flow cytometer was converted into using violet side scatter (ssc) for improved detection of evs with instead of nm ssc. for the combined analysis of amount, size, and phenotype, samples were analysed with the nanoview bio r platform. phenotype analysis included probing for the classic tetraspanins associated with exosomes (cd , cd , cd ) and the neural cell adhesion molecule l (cd ). results: flow of csf samples showed similar vesicle counts in control vs. disease and an increase of counts in later disease stages when neurodegeneration is thought to be more prominent. all csf samples showed some binding to classic exosomal markers (cd , cd , cd ). the sample taken at the latest time point showed relatively high vesicle counts, overall larger vesicle size, and abundant cd binding. interestingly, the cd positive evs were not positive for any of the classic exosomal markers (cd , cd , and cd ). summary/conclusion: this data supports the notion that analysing the amount, size, and surface markers of evs in csf can reveal intriguing dynamics in such basic ev characteristics over time and suggests important differences between ev populations in different disease stages. while previous studies indicated that cd could identify an ev to be of neuronal origin, it remains to be determined whether such specific surface markers will emerge as clinically relevant tools to support the evaluation of people affected by neurological diseases. a distinct microrna signature in plasma derived small extracellular vesicles of different neurodegenerative diseases introduction: exploring identifying robust biomarkers is essential for early diagnosis of neurodegenerative diseases. blood stream transports large (levs) and small extracellular vesicles (sevs), which are extracellular vesicles of different sizes and biological functions that are transported in blood. aim of our study was to investigate mrna/mirna signatures in plasma derived levs and sevs of amyotrophic lateral sclerosis (als), alzheimer's disease (ad), parkinson's disease (pdpd), fronto-temporal dementia (ftd) and alzheimer's disease (ad) patients. methods: levs and sevs were isolated from plasma of patients and healthy volunteers (ctr) by ultracentrifugation and rna was extracted. whole transcriptome and mirna libraries were prepared with truseq stranded total rna kit and truseq small rna library kit (illumina). results: our data suggested that the rna cargo in levs and sevs varies among different diseases. mirna analysis in sevs provided the most informative disease specific signatures, while whole transcriptome analysis did not show any specific signature. als was characterized by a small but specific group of circulating mirnas. mirnas profiling revealed that pd and ftd can be subgrouped in two classes while ad appears to be a homogeneous disease population. furthermore, mirnas profiling show the presence of overlaps in the signatures between the analysed diseases. mirna profiling in levs is similar to that observed in sevs, although in levs the overall differences between diseases are less marked. summary/conclusion: in this study we have demonstrated that mirnas are the most interesting subpopulation of transcripts transported by plasma derived sevs since they discriminate a disease from the other and they can provide a signature for each neurodegenerative diseases. may be linked with apoe genotype, we investigated the possible effect of apoe genotype on brain-derived evs (bdevs) and their protein and rna molecular cargo. methods: cortical brain tissues of ad patients with different apoe genotypes [ε /ε (n = ), ε /ε ( ), ε /ε ( ), ε /ε ( )] and non-ad controls (n = ) were obtained. bdevs were separated by size exclusion chromatography plus ultracentrifugation (uc) and characterized per misev . proteins were analysed by mass spectrometry. after protein identification, data were normalized using the cyclicloess method and analysed by principal component analysis (pca). nested factorial design highlighted differentially expressed proteins. rna from bdevs was extracted by mirneasy mini kit. small rna libraries were constructed using the ion total rna-seq kit and sequenced on the ion torrent s ™ using ion™ chips. reads were aligned to human reference transcriptomes using bowtie. differential gene expression was quantified by edger and limma. results: among proteins dysregulated in ad bd-sevs, several have reported roles in ad, e.g., microtubule-associated protein tau and peroxiredoxin- . regarding apoe genotypes, proteins were differentially expressed between ε carriers (ε /ε and ε /ε ) with non ε carriers (ε /ε and ε /ε ). however, ev markers did not differ by apoe genotype. in contrast to protein cargo of bdevs, the overall small rna expression pattern was similar among ad patients with different apoe alleles and non-ad patients. only a few mirnas showed different abundance level between ε /ε and ε /ε groups, or between ad and non-ad groups. summary/conclusion: bdevs carry proteins and mirnas related to ad development and apoe genotypes. further verification of protein and rna expression in brain and plasma derived evs may reveal mechanisms of ev function in neuroinflammation and develop biomarkers for ad disease. funding: this project was funded by mh . efficient pathology spread by extracellular vesicles from human brain tissues in mouse brain and tissue cultured neurons: transmission and propagation to gabaergic neurons however, whether human brain-derived evs induce tau pathology has not yet been characterized in the mouse brain. here, we assess the mechanisms of disease spread after intrahippocampal injection of human brainderived evs into the aged mouse model. methods: ev-enriched fractions were isolated from unfixed frozen human brain samples from ad, prodromal ad (pad), control (ctrl) cases, and tau knockout (tko) mouse brains. isolated evs containing pg of human total tau were sterotaxically injected into the right outer molecular layer of the dentate gyrus of months-old c bl/ female mice. . months after the injection, hippocampal slices were prepared for whole-cell patch clamp recordings of ca pyramidal neurons were undertakent. hippocampi were analysed with immunohistochemistry using phosphorylated-tau (p-tau) epitopes including at . evs were examined for protein composition by protein mass-spectroscopy, the neuronal uptake in vitro, and structural analysis by the atomic force microscopy (afm). results: semiquantitative brain-wide immunohistochemistry of p-tau revealed that inoculation of ad or pad-evs induced tau propagation throughout the hippocampus, including the dentate gyrus, ca and ca subregions. at was localized primarily in gad + gabaergic neurons in pad and ad evs groups, accompanied with reduced amplitude of inhibitory postsynaptic currents and excitatory-inhibitory ratio in amplitube of postsynaptic currents in ca pyramidal neurons in pad evs. afm analysis showed higher density of tau oligomers in both ad and pad evs while only ad evs showed significantly higher neuronal uptake compared to ctrl evs. finally, proteomic analysis showed that ad evs are enriched in disease and glia-related molecules compared to ctrl evs, which may contribute to their enhanced neuronal uptake. summary/conclusion: intracranial injection of ad or pad evs induced p-tau accumulation primarily in gabaergic neurons throughout the hippocampus, resulted in higher uptake by neurons, and tau oligomer conformation, indicating of their pathogenic potency as seeding factors. gabaergic neuronal dysfunction in the hippocampal neuronal circuitry reported in early ad brains could be attributed to specific ev mediated tau propagation in this cell type, a phenomenon meriting further investigation and validation. funding: nih rf ag , nih r ag , nih r ag , cure alzheimer's fund, brightfocus foundation, curepsp, coins for alzheimer's research trust introduction: extracellular vesicles (evs) are released by cells of the central nervous system as a result of injury, including mild traumatic brain injury (mtbi). since mtbi may alter circulating levels of evs, this study aimed to investigate differences in circulating ev numbers between contact sport athletes with and without acute mtbi. methods: circulating evs containing cd (cd + ev), cd (cd + ev), and neural cell adhesion molecule (l cam+ev) were analysed in young, male athletes with or without mtbi ( - yo, n = per group). sodium citrate-treated blood samples were obtained from athletes with mtbi within -hours of injury and from control athletes free of mtbi for one year. athletes were best matched for age and history of prior mtbi. samples were double-centrifuged to obtain platelet-poor plasma and stored at − °c until analysed. quantification of evs was performed using a spectral flow cytometer. the study was approved by temple university's irb, and all athletes provided written informed consent. results: mann-whitney u tests showed that population percentages of small size ( - nm) cd + ev, cd + ev and l cam+evs were significantly higher in mtbi athletes (mean rank: . , . , . ) than controls (mean rank: . , . , . ) (u = . , p = . ; u = . , p > . ; u = . , p > . , respectively). population percentages of large size ( - nm) cd + ev, cd + ev and l cam+evs were also significantly higher in mtbi athletes (mean rank: . , . , . ) than controls (mean rank: . , . , . ) (u = . , p = . ; u = . , p > . ; u = . , p > . , respectively). there were no significant differences between percentages of evs associated with blood brain barrier function (cd + ev) or platelets (cd a+ev) among mtbi athletes or controls. introduction: parkinson's disease (pd) is characterized by clinical heterogeneity, different rates of progression and absence of definitive biomarkers. extracellular vesicles (evs) are easily isolated from plasma and play a central role in intercellular communication which is highly relevant for inflammatory processes implicated in protein misfolding-related neurodegenerative disorders. thus, we characterized distinctive plasmatic ev subpopulations of pd and atypical parkinsonisms (ap) patients, with the aim to identify candidate biomarkers among evs surface membraneproteins. methods: plasmatic evs were collected from pd, matched healthy controls (hc), ap with multiple system atrophy (msa) and ap with tauopathies (ap-tau). evs were quantified by nanoparticle tracking analysis. the expression of ev-surface markers, related to inflammatory and immune cells, were measured by macsplex and correlated to clinical scales. a diagnostic model based on ev markers expression was built via supervised machine learning algorithms and validated in an external cohort ( pd, hc, msa, ap-tau). the cantonal ethics committee approved the study protocol. all enrolled subjects gave written informed consent. results: pd showed the highest ev concentration compared to others groups. pd and msa displayed a greater pool of overexpressed immune markers compared to ap-tau. ev antigens correlate to cognitive impairment and disease gravity in pd and msa. the roc curve analysis of a compound ev marker showed optimal diagnostic performance for pd (auc . ; sensitivity . %, specificity . %) and msa (auc . ; sensi-tivity %,specificity . %)andgoodaccuracyforap-tau (auc . ; sensitivity . %, specificity . %). a diagnostic model based on ev markers expression, cor-rectlyclassified . %ofpatientswithreliablediagnostic performance after validation in an external cohort ( % of accuracy). summary/conclusion: this analysis of multiple immune surface markers of circulating evs in pd and ap well captured the clinical heterogeneity of pd and showed optimal diagnostic performance. furtherly it suggests a different immune dysregulation in pd and msa vs. ap-tau, to be confirmed by functional analysis in experimental models of disease. funding: supported by abreoc. separation and characterization of extracellular vesicles from human cerebrospinal fluid introduction: extracellular vesicles (ev) are released from cells to the surroundings and are found in human biofluids, where they constitute promising targets for novel biomarker identification. ev have been found in cerebrospinal fluid (csf) where they may provide with markers for neurological diseases. here, we aimed at purifying and characterizing ev from human csf. methods: csf was collected by lumbar puncture from patients with amyotrophic lateral sclerosis. patients gave written consent and studies were agreed by the local ethics committee. csf was fractionated by ultrafiltration (vivaspin, cut-off , ), and size-exclusion chromatography (sec; qevsingle izon science). eluted fractions were analysed by dynamic light scattering (dls) and electron microscopy. proteins were analysed by immunoblotting and nano-liquid chromatographytandem mass spectrometry. results: ev eluted in early fractions ( + ) after the sec void volume as evaluated by detection of cd and cd markers (immunoblotting) and annexin a (peptide mapping by nanolc-ms/ms). there, nanoparticles around nm were identified by dls. in agreement, electron microscopy showed ev with characteristic shape and sizes typically between and nm, with average diameter ± nm. cd was visualized by immunocytochemistry at the surface of ev around nm. on the other hand soluble proteins igg and albumin eluted in later fractions. curiously, galectin- binding protein (lgals bp or k) was also partially detected in early-eluting fractions as nanoparticles of irregular shapes and heterogeneous sizes typically between and nm; some of those nanoparticles had ring-like appearance. occasionally k also appeared on ev of variable dimensions. summary/conclusion: in conclusion, ev from the csf may be separated from soluble proteins and small molecules by a combination of ultrafiltration with sec fractionation. however, using this strategy a population of k-containing nanoparticles co-eluted with ev from the csf. further separation techniques need to be applied to separate ev from k nanoparticles to investigate their individual physiological relevance and biomarker potential. introduction: extracellular vesicles (ev) are released from cells to the surroundings and are found in human biofluids, where they constitute promising targets for novel biomarker identification. ev have been found in cerebrospinal fluid (csf) where they may provide with markers for neurological diseases. here, we aimed at purifying and characterizing ev from human csf. methods: csf was collected by lumbar puncture from patients with amyotrophic lateral sclerosis. patients gave written consent and studies were agreed by the local ethics committee. csf was fractionated by ultrafiltration (vivaspin, cut-off , ), and size-exclusion chromatography (sec; qevsingle izon science). eluted fractions were analysed by dynamic light scattering (dls) and electron microscopy. proteins were analysed by immunoblotting and nano-liquid chromatographytandem mass spectrometry. results: ev eluted in early fractions ( + ) after the sec void volume as evaluated by detection of cd and cd markers (immunoblotting) and annexin a (peptide mapping by nanolc-ms/ms). there, nanoparticles around nm were identified by dls. in agreement, electron microscopy showed ev with characteristic shape and sizes typically between and nm, with average diameter ± nm. cd was visualized by immunocytochemistry at the surface of ev around nm. on the other hand soluble proteins igg and albumin eluted in later fractions. curiously, galectin- binding protein (lgals bp or k) was also partially detected in early-eluting fractions as nanoparticles of irregular shapes and heterogeneous sizes typically between and nm; some of those nanoparticles had ring-like appearance. occasionally k also appeared on ev of variable dimensions. summary/conclusion: in conclusion, ev from the csf may be separated from soluble proteins and small molecules by a combination of ultrafiltration with sec fractionation. however, using this strategy a population of k-containing nanoparticles co-eluted with ev from the csf. further separation techniques need to be applied to separate ev from k nanoparticles to investigate their individual physiological relevance and biomarker potential. release of extracellular vesicles from platelets requires platelet-platelet interaction aleksandra gąsecka a , naomi c. buntsma b , sytske talsma c , krzysztof j. filipiak d , rienk nieuwland e and edwin van der pol f introduction: arterial thrombosis is a major and global cause of human death and disability, but a biomarker for early-diagnosis of thrombosis is absent. platelet activation and aggregation are the first steps of plateletrich thrombus formation, but their relative contribution to platelet extracellular vesicles (pevs) release is unknown. methods: to study the relation between pev release and platelet interaction (aggregation), citrate-anticoagulated whole blood (wb) from healthy donors was diluted , , , and -fold and activated by μm thrombin-receptor activating peptide (trap). in addition, undiluted wb and -fold diluted wb, which totally blocked pev release, were activated with various trap concentrations. concentrations of pevs (cd + and cd +, cd p + > nm) and activated platelets (cd +, cd p+ > nm) were measured by flow cytometry (apogee a -micro). platelet aggregation was assessed using impedance aggregometry. results: a -fold dilution of wb blocked both aggregation and the release of pevs. compared to baseline, activation of undiluted wb with trap increased the concentrations of cd + . -fold and cd +-cd p + pevs . -fold. the concentration of cd + (r = . ) and cd +-cd p+ (r = . ) pevs as well as platelet aggregation (r = . ) scaled inversely (reciprocal) with the dilution of wb. further, we found a linear correlation between the % of activated platelets and the concentration of cd + (r = . ) and cd +, cd p+ (r = . ) pevs in undiluted wb, which was absent in -fold diluted blood (r < . ). summary/conclusion: the absence of aggregation and pev release upon platelet activation in -fold diluted blood shows that aggregation directly depends on the distance between platelets, which is confirmed by the reciprocal relationship between pev release and blood dilution. because pevs are only released when platelet activation is followed by aggregation, pevs are a potential early biomarker of thrombosis. funding: ag is supported by the national science centre, research programme preludium / / n/nz / . evdp is supported by the netherlands organisation for scientific research -domain applied and engineering sciences (nwo-ttw), research programmes veni . age-dependent alteration in concentration and size distribution of extracellular vesicles in plasma of normotensive and hypertensive rats kosuke otani, muneyoshi okada and hideyuki yamawaki laboratory of veterinary pharmacology, school of veterinary medicine, kitasato university, towada, japan introduction: spontaneously hypertensive rats (shr) are the most widely used animal model of human essential hypertension. we previously reported that plasma small extracellular vesicles (sevs) in shr regulate systolic blood pressure, however, the mechanism has not been clarified. in the present study, we compared the concentration and size distribution of plasma evs (sevs and large evs) from young and aged normotensive wistar kyoto rats (wky) and shr. methods: heparin-anticoagulated plasma was collected from male wky and shr at ~ -(young) and -(aged) week-old. large evs were isolated from the plasma by centrifugation ( x g). sevs were isolated by ultracentrifugation ( , x g) following precipitation with polyethylene-glycol. the concentration and size distribution of sevs and large evs were measured by a tunable resistive pulse sensing analysis. results: there was no significant difference in the total concentration of plasma sevs between wky and shr or between young and aged rats. the mean diameter of plasma sevs from aged rats was larger than that from young rats in both wky and shr. also, the number of particles with a diameter of smaller than nm in plasma sevs from aged rats was lower than that from young rats. the concentration of plasma large evs from aged rats was higher than that from young rats in both wky and shr. there was no significant difference in the size distribution of plasma large evs between wky and shr or between young and aged rats. summary/conclusion: the present results for the first time demonstrate that the concentration of plasma large-sized evs is increased by ageing, while there is no difference in the concertation and size distribution of evs between wky and shr. further research is required to clarify the cause of age-dependent alternation in plasma ev size distribution and its physiological meaning. microrna profiling of circulating extracellular vesicles is involved with susceptibility to age-related diseases: relevance to cardiovascular signalling in ageing process ionara rodrigues siqueira a , laura cechinel b , rachael batabyal c and robert freishtat c a universidade federal do rio grande do sul (ufrgs), porto alegre, brazil; b universidade federal do rio grande do sul, porto alegre, brazil; c children's national hospital, washington, usa introduction: ageing represents a central risk factor for several diseases, such as cardiovascular diseases. our hypothesis is that extracellular vesicles (evs) can be potential mechanism of spreading molecules, such as micrornas, involved with susceptibility to chronic age-related diseases and geriatric syndromes. in this context, the role of micrornas in age-induced detrimental changes in the cardiovascular system has been suggested. although evs can protect micrornas from endogenous rnases and internalization of these vesicles into cells is involved with cell communication, delivering micrornas even to distant tissues, the relationships between evs micrornas profile and chronic age-related diseases has not been evaluated. our aim was to investigate the microrna profile of circulating evs during ageing process and their downstream signalling pathways. methods: the ethics committee (ceua -comissão de Ética no uso de animais -ufrgs; nr. , ) approved all animal procedures and experimental conditions. male wistar rats of -and -month-old were used, and plasma was obtained from the trunk blood. evs were isolated with exoquick following the manufacturer's instructions. microrna was isolated from evs and then amplified. microrna was labelled using the flashtag biotin hsr rna labelling kit and profiled on affymetrix genechip microrna . arrays. ingenuity pathway analysis (ipa) was used to identify pathways regulated by significantly altered micrornas. results: microarray analysis revealed micrornas. of these micrornas, were differentially expressed between aged and young-adult animals, micrornas were significantly upregulated and were downregulated in aged animals compared to young adult (p < . ; fold change of | . |). a conservative filter was applied on ipa and only experimentally validated and highly conserved predicted mrna targets for each microrna was used. ipa analysis showed that cardiac hypertrophic signalling is ranked as highly predicted targets for these differentially expressed micrornas (p < . ). moreover, ipa demonstrated that this canonical pathway is upregulated in aged animals when compared to young adult. in addition to cardiac hypertrophic signalling, other relevant cardiovascular canonical pathways, such as endothelin- signalling and intrinsic prothrombin activation pathway have predicted targets. summary/conclusion: our results showed for the first time that micrornas profile in circulating evs has a potential role to drive heart senescence and consequent cardiac diseases which represents the leading cause of death. introduction: introduction: the vascular endothelium and smooth muscle form adjacent cellular layers that comprise part of the vascular wall. here, we examined the extent to which extracellular vesicles (evs) vesicles participate in endothelial-vascular smooth muscle cell communication. methods: methods: evs were collected from rat aortic endothelial and smooth muscle cell serumfree media by ultracentrifugation. vesicle morphology, size and concentration were evaluated by transmission electron microscopy and nanoparticle tracking analysis. endothelial cell and vascular smooth muscle cell cultures were subjected to various concentrations of evs for various times. functional assays were performed. results: results: western blot as well as shot gun proteomic analyses revealed sets of proteins common to both endothelial-and smooth muscle-derived ev as well as proteins unique to each vascular cell type. functionally, endothelial-derived evs stimulated vascular cell adhesion molecule- (vcam- ) expression and enhanced leukocyte adhesion in vascular smooth muscle cells while smooth muscle evs did not elicit similar effects in endothelial cells. evs from endothelial cells also induced protein synthesis and senescenceassociated β galactosidase activity in vascular smooth muscle cells. proteomic analysis of vascular smooth muscle cells following exposure to endothelial cellderived evs revealed upregulation of several proteins including pro-inflammatory molecules, high-mobility group box (hmgb) and hmgb . pharmacological blockade of hmgb and hmgb and sirna depletion of hmgb in smooth muscle cells attenuated nfkb (p ) phosphorylation and nuclear translocation, vcam- expression and leukocyte adhesion induced by endothelial cell evs. summary/conclusion: conclusions: these data suggest that endothelial cell-derived evs can enhance signalling pathways that induce a pro inflammatory in vascular smooth muscle cells. introduction: graft patency is one of the major determinants of long-term outcome following coronary artery bypass graft surgery (cabg). biomarkers, if indicative of the underlying pathophysiological mechanisms, would suggest strategies to limit graft failure. many studies have generated compelling data on the sensitivity of mvs as biomarkers of cardiovascular disease progression and events. the mv usefulness in cabg has been tested only in a study that highlighted their importance in surgical haemostasis. no information is so far available on the association between the amount or pattern of circulating mvs and cabg outcome. we aimed to evaluate whether mv pre-operative signature could predict mid-term graft failure. methods: this was a nested case-control substudy of the coronary bypass grafting: factors related to late events and graft patency (cage) study that enrolled patients undergoing elective cabg. of these, underwent coronary computed tomography angiography months post-surgery showing % graft occlusion. flow cytometry mv analysis was performed in patients ( /group with occluded [cases] and patent [controls] grafts) on plasma samples collected the day before surgery and at follow-up. results: before surgery, cases had two-fold (p = . ) and four-fold (p = . ) more activated platelet-derived and tf+ mvs, respectively than controls. the mv thrombin generation capacity was also significantly greater (p < . ). this mv signature predicted graft occlusion (auc of . [ %ci: , - , ], p = . ). by using a mv-score ( - ), the or for re-occlusion for a score above was . ( % ci . - . , p < . ). summary/conclusion: the pre-operative signature of mvs is an independent predictor of mid-term graft occlusion in cabg patients and a cumulative mvscore stratifies patient's risk. since the mv signature mirrors platelet activation, patients with a high mvscore would benefit from a personalized antiplatelet therapy. exosomes from engineered immortalized human heart cells improve ventricular function and attenuate fibrosis in mice with arrhythmogenic cardiomyopathy yen-nien lin, lizbeth sanchez, rui zhang, thassio ricardo ribeiro mesquita, chang li, ahmed ibrahim, eduardo marbán and eugenio cingolani heart institude, cedars sinai medical center, los angeles, usa introduction: arrhythmogenic cardiomyopathy (ac) is characterized by progressive loss of cardiomyocytes and fibrofatty tissue replacement. currently, there is no effective treatment for this disease. exosomes (imexos) secreted by heart stromal cells, engineered to be immortal and overexpressing β-catenin, exert anti-inflammatory and anti-fibrotic effects and improve ventricular function in models of ischaemic injury (ibrahim et al., nature bme ). methods: to investigate the effectiveness of imexos in a murine model of ac, four-week old homozygous dsg knockout (dsgko) mice and wild type (wt, age-and strain-matched) mice were compared. dsgko mice were randomized to receive weekly imexos or vehicle via intravenous injection for weeks. neonatal rat ventricular myocyte (nrvm) proliferation and apoptotic assays were performed to explore potential effects of exosomes. results: biodistribution studies of dir-labelled imexos revealed some cardiac uptake, along with strong signals in spleen. at weeks, dsgko mice which had received intravenous imexos showed improved cardiac function (echocardiographic ejection fraction ± vs ± % in vehicle mice, p = . ), with an underlying attenuation in myocardial fibrosis by histology. electrophysiology test showed shorter qrs duration ( . ± . ms imexo vs . ± . ms vehicle, p = . ) and effective refractory period. programmed ventricular stimulation showed dsgko mice which had received imexos were remarkably less prone to ventricular tachycardia induction ( . ± % vs . ± % in vehicle, p = . ). in vitro study showed nrvm exposed to imexos for days exhibited higher brdu expression relative to vehicle group, and less annexin-v expression after oxidative stress induced by -minute illumination with nm uv. summary/conclusion: intravenous administration of imexos improved cardiac function, reduced cardiac fibrosis, and suppressed arrhythmogenesis in ac. our findings motivate clinical testing of imexos in ac, an orphan disease with great unmet medical need. funding: nih r hl (to em) cardiac-derived extracellular vesicles contribute to communication between heart and brain in chronic heart failure (chf) and target nrf / are signalling changhai tian a , lie gao b and irving zucker b a department of cellular and integrative physiology, university of nebraska medical center, omaha, usa; b department of cellular and integrative physiology, university of nebraska medical center, omaha, usa introduction: mirnas regulate the translation of proteins that are involved in redox homoeostasis in the heart and brain. intra-and/or inter-organ communication takes place by multiple mechanisms including extracellular vesicular (ev) transport. our previous studies suggested that cardiac derived mirna-enriched evs contribute to the dysregulation of nrf /antioxidant enzyme (are) signalling in the myocardium via intercellular cross-talk, and result in the decreased nrf /are signalling in the sympatho-regulatory areas of the brain in chf. however, it is unclear if cardiac derived evs circulate to the central nervous system evoking sympatho-excitation by disrupting central redox homoeostasis. methods: cardiac-specific membrane gfp+ mice were generated to track the brain distribution of cardiac evs in rats with chf (coronary ligation). the isolation and characterization of evs were carried out by differential ultracentrifugation, tem, nanosight, western blotting, and qrt-pcr. transfection, labelling, and microinjection of evs into the rostral ventrolateral medulla (rvlm) were performed. results: nrf protein was reduced in the rvlm of chf rats consistent with an upregulation of nrf -targeting mirnas. nrf -targeting mirnas were enriched in cardiac and circulating evs of chf rats. nrf -targeting and cardiac-specific mirnas were abundant in brain-derived evs. circulating evs were taken up by neurons in sympatho-regulatory areas of the brain. mirna-enriched evs from chf animals increased sympathetic tone which was prevented by a cocktail of nrf -targeting mirna inhibitors. summary/conclusion: myocardial infarction-induced mirna-enriched evs mediate the inter-organ crosstalk between heart and brain in the oxidative regulation of sympathetic outflow through targeting the nrf / are signalling pathway. these findings suggest that cardiac-derived ev mirnas targeting nrf /are signalling may act as an endocrine signalling mediator of chf that has potential as a novel therapeutic target. introduction: a fine-tuned communication between cardiac cells is vital to maintain myocardial integrity and contractility. not only an impairment of gap junction (gj)-mediated intercellular communication, but also defects in ev-mediated communication have been associated with ischaemic heart disease, a major causative factor of heart failure. we have previously shown that cx , the main ventricular gj protein, assembles into channels at the evs surface, mediating the release of vesicle content into target cells.the main objective of this work was to characterize the signals underlying protein sorting into extracellular vesicles (evs) in a human pathophysiological context, using connexin (cx ) as a model substrate. methods: animal models of ischaemia/reperfusion (i/ r) injury by ligation of the left anterior descending coronary artery, ex vivo and in vitro ischaemia models and human patients were used to investigate the secretion of ev-cx . results: release of cx was downregulated in circulating vesicles from i/r-injured mice and patients with st-segment elevation myocardial infarction, as well as in intracardiac and cardiomyocyte-derived evs. additionally, we show that ubiquitin signalled the release of cx in basal conditions but appeared to be dispensable during ischaemia. depletion of the autophagy adaptor p partially restored the secretion of cx , suggesting an interplay between ischaemiainduced cx degradation and secretion. summary/conclusion: overall, we demonstrated that ischaemia impairs the sorting of cx into evs, which may ultimately affect long-distance communication. through the identification of the underlying molecular mechanisms and players, these results pave the way towards the development of innovative diagnostic and therapeutic strategies for cardiovascular disorders. introduction: remote ischaemic conditioning is a cardioprotective intervention which protects the heart against ischaemia/reperfusion injury. transient activation of toll-like receptor (tlr ) and its downstream regulators (tnfα and il- ) have been implicated in cardioprotective interventions. extracellular vesicles (evs) play a role in cardioprotection through the activation of the tlrs. however, since isolation of evs in high amounts with suitable purity from blood is a challenge, our aim was to develop a cellular model system from which tlr-inducing, cardioprotective evs can be isolated in a reproducible manner. methods: ev release from hek cells was induced by calcium-ionophore a . evs were characterized, cytoprotection by evs against simulated ischaemia/ reperfusion injury and its mechanism were investigated in h c and ac cell lines. results: a induction of hek cell induced ev release and the isolates contained mostly large evs. evs decreased cytotoxicity and apoptosis due to h ischaemia followed by h reperfusion in h c and ac cells in a dose-dependent manner. evs activated tlr and its downstream signalling pathway in h c and ac cells as well as the expression of cytoprotective haem oxigenase (ho- ) in h c cells. summary/conclusion: a -induced evs exert cytoprotection in h c and ac cells by inducing tlr signalling and ho expression. therefore, evs released via calcium-ionophore treatment may serve as a basis of an efficient carpdioprotective therapy. introduction: biliary strictures may be benign or malignant. the major malignant causes of biliary stricture are a primary cholangiocarcinoma (cca) or pancreatic ductal adenocarcinoma (pdac). there is ongoing debate about adequate diagnostics in biliary strictures of unknown aetiology. micrornas (mirnas) are small non-coding rnas important in tumourigenesis. mirna have been found to be enriched in exosomes, small membrane-bound extracellular vesicles (ev) of endocytic origin, which is a novel pathway for intercellular signalling within the tumour microenvironment and have been implicated in loco-regional pre-metastatic niche formation. this project aims to investigate circulating-free and ev mirnas as biomarkers that can aid diagnosis in patients with a biliary stricture. we will ( ) isolate and characterise evs in plasma and bile from patients with benign and malignant biliary strictures (i.e. pancreaticobiliary cancers); and ( ) identify differentially expressed circulating-free and ev mirnas in plasma and bile suitable for detecting malignancy. methods: sample size (n = ) was calculated for a study power of % and α error of % for the ability of extracellular mirnas to discriminate benign from malignant biliary strictures. prospective matched plasma and bile samples will be collected from patients with benign (n = ) and malignant (n = ) biliary strictures undergoing endoscopic retrograde cholangiopancreatography (ercp). evs will be isolated from the biofluids by ultracentrifugation and/or size exclusion chromatography and then characterised (tem, nta and immunoblotting). circulating-free and ev-associated mirnas will be profiled using small rna sequencing. extracellular mirna "signatures" will then be validated by rt-qpcr, and diagnostic accuracy confirmed (sensitivity, specificity, auc). results: evs derived from patient samples have been characterised using nta, western blotting and tem. sec derived evs appear to be more well-defined than uc evs with marker positivity for cd , cd and cd . ongoing work will be focused on rna profiles of evs from both malignant and benign cohorts. summary/conclusion: there is currently no effective method to differentiate benign from malignant biliary strictures. novel plasma and bile circulating-free and ev-associated mirna biomarkers may improve the speed and accuracy of diagnosis, resulting in considerable patient benefits. furthermore, as little is known about the ev-associated function of these tumours, candidate ev-mirnas could be taken from "bedside to bench" and their function further investigated using in vivo, vitro and silico models. introduction: urine is a source of extracellular rna (exrna) biomarkers that can be obtained non-invasively throughout pregnancy. several studies have profiled extracellular mirnas in biofluids during pregnancy, but few have profiled extracellular mrnas (ex-mrnas) in urine. objective: to optimize methods for ex-mrna isolation and rna-seq library preparation from urine of healthy pregnant and non-pregnant females. methods: rna was isolated from pooled non-pregnant urine using kits based on ev precipitation (mircury exosome kit for csf/urine, seramir), ev affinity purification (exorneasy), and protein precipitation (mirneasy serum/plasma advanced). next, long (> nt) and short rnas were isolated from ev enriched urine of pregnant (n = ) and non-pregnant (n = ) individuals using the mircury kit followed by the mirneasy micro kit. rna-seq libraries were prepared using the smart-seq v ultra low input rna (oligo(dt) priming) and the smarter stranded total rna-seq kit v -pico input (random priming) methods (takara). preliminary data were obtained using the illumina miseq, and aligned using star v. . . .a. results: overall, rna isolation using mircury followed by the smart-seq v library preparation kit yielded the highest % of mapped reads: % in pooled non-pregnant, % in individual non-pregnant, and % in individual pregnant urine. for rna extracted using the mircury kit, the smart-seq v libraries had higher % of mapped mrna reads compared to pico libraries (p < . , t-test). in contrast for mirneasy advanced it was reversed ( % vs %). summary/conclusion: early results from low-depth sequencing show the highest mrna mapping rates for mircury followed by the smart-seq v kit. high-depth sequencing data are now being generated, which will enable us to perform detailed comparisons of different rna species from the rna profiles obtained using different library preparations and rna isolation methods from urine of pregnant and non-pregnant subjects. funding: this study was funded by nih k hd - , nih u hl , and a ucsd igm-illumina mini-grant. il- mutein-induced changes of exosomal mirna cargo in a humanized mouse model emily lurier, erik sampson, patrick halvey, mike cianci and katalin kis-toth pandion therapeutics, cambridge, usa introduction: regulatory t cells (tregs) are key contributors to immune homoeostasis. decreased number and/or function of these cells are frequent features of many autoimmune diseases linked to the development of tissue inflammation. while interleukin- (il- ) is essential for pan t cell proliferation and performance, low dose il- treatment has been shown to preferentially affect tregs and is being evaluated as an intervention in autoimmune diseases. pt is a novel il- mutein fc fusion molecule (il- m) designed to selectively engage with tregs. using a humanized nod-scid il rn-null (nsg) mouse model we have shown that pt expanded tregs without significant effects on other immune cells. we have also shown that tregs from pt -dosed humanized mice exhibit increased expression of foxp and cd , and demethylation of foxp and ctla- genes, suggesting enhanced function and stability. in the current study we investigated the mirna content of plasma exosomes isolated from pt -or vehicle-treated mice in order to identify treg specific mirnas from the il- m treated animals. methods: cd + haematopoietic stem cell humanized nsg mice were dosed once subcutaneously with pt or vehicle. plasma samples from mice were collected at day and exosome isolation was conducted using the exoquick method. small rna was extracted and quantified using the bioanalyzer small rna assay. an illumina nextseq instrument was used for library preparation and sequencing with bp single end reads at an approximate depth of - million reads per sample. raw sequences were mapped to human genome grch and analysed via a pipeline provided by the university of california santa cruz. results: rna within the exosomes from vehicle and il- m-treated groups was mostly comprised of mirna and trna. plasma was pooled from animals per treatment group and differential expression was determined using a twofold change cut-off. we found that pt treatment actively altered the mirna content of plasma exosomes, compared to exosomes from vehicle-treated mice. many of the differentially expressed mirnas are involved in immunoregulation. summary/conclusion: plasma exosomes from pt treated humanized mice encapsulated treatment-specific mirnas which can potentially be used as systemic biomarkers of treg expansion and function. identification of potential biomarkers in microglial specific exosomes isolated from prion-infected serum introduction: transmissible spongiform encephalopathies (tse) are neurodegenerative disorders caused by the misfolding of the cellular prion protein (prpc) to the beta-sheet rich abnormal prion protein (prpsc). prpsc aggregates in the brain and causes amyloid plaques, neuronal loss, spongiform degeneration and microglial activation. currently, definitive diagnosis of tse diseases is only confirmed post-mortem thus a diagnostic test in accessible body fluid is of interest. exosomes are a good resource for biomarker discovery since they cross the blood-brain barrier easily and contain protein, lipids and nucleic acids from the cells of origin. the goal of this study was to look at biomarkers from brain-originating exosomes (specifically microglia) isolated in the serum of prion-infected animals. methods: westerns and nanoparticle tracking analysis (nta) were used to look at the composition of microglial-specific exosomes. as proof of principle, exosomes were isolated from a microglial cell line (bv cells). a cd antibody was labelled with a fluorophore and binding to exosomes was visualized via nta. exosomes were isolated from serum of both prioninfected and mock-infected mice throughout disease course. a macrophage specific antibody (f / ) was bound to beads which were used to isolate exosomes which includes those of microglial origin. microrna was extracted from these exosomes and next-generation sequencing (ngs) was performed using the illumina platform. clc genomics workbench was used for bioinformatics analysis. results: microglial and macrophage proteins (tmem and iba ) were identified in exosomes isolated from bv cells and prion-infected mouse serum. macrophage exosomes were isolated via a novel antibody-bead based system. results of the ngs analysis of the microrna isolated from these exosomes indicated a series of mirna that could differentiate between control and infected samples as well as age-specific markers. summary/conclusion: to our knowledge, this is the first time microglial-specific exosomes have been isolated from prion-infected serum from early and end stage disease. the results of this analysis could facilitate the diagnosis of prion disease in easily-accessible biofluids pre-mortem. comparison of urinary extracellular vesicle isolation methods for transcriptomic biomarker research in diabetic kidney disease introduction: urinary extracellular vesicles (uevs) are emerging as a source for early biomarkers of kidney damage, holding the potential to replace the conventional invasive techniques including kidney biopsy. several methods are available for uev isolation. our aim was to compare different workflows and isolation by hydrostatic filtration dialysis (hfd), ultracentrifugation (uc) and a kit based isolation method for their subsequent use in mirna-seq and rna-seq for biomarker discovery in diabetic kidney disease. methods: type diabetic patients (t d) with macroalbuminuria and normoalbuminuric healthy controls were included in the study. sample collection and all experiments were performed in accordance with the declaration of helsinki. evs were isolated from - ml of h urine collections by uc, hfd, or a commercially available kit (purification based on spin column chromatography, urine exosome purification and rna isolation midi kit, norgen biotech, canada) each with different established urine clarification steps. quality control of the evs was performed with negative staining em, nta and western blotting. isolated rnas were profiled with bioanalyzer pico kit and subjected to mirna and mrna sequencing. for rna-seq, cdna library was prepared using smart-seq v ultra low input rna kit for sequencing (takara bio, japan). rna-seq was performed using hiseq (illumina). mirna-seq library was prepared using qiaseq mirna library kit (qiagen, germany). mirna-seq was performed on the illumina hiseq platform (illumina). results: our data showed that uev yield, morphology and size distribution were closely similar in hfd and uc preparations, while lower yields were obtained using the kit. by western blot, ev markers were detectable in samples isolated by hfd and uc but not readily in samples isolated with the kit. tamm-horsfall protein was detected in all the samples and albumin levels appeared higher in hfd and kit isolated samples relative to uc samples. the number of paired-end reads for rna-seq in hfd and uc samples (in both > m) were closely similar. instead, rna reads were lower than m for the kit samples. for mirna-seq, the number of reads as well as the molecular biotype distribution were similar for the three methods. by principal component analysis of the rna-seq data, we observed that hfd and uc grouped together showing similarities. however, for mirna-seq data such similarities were not obvious. this suggests that the three different workflows and isolation principles may enrich different mirna-rich uev preparation components. summary/conclusion: our transcriptomics data shows that hfd and uc are suitable methods to isolate uevs for mirna-seq and rna-seq. the kit based method appears better suited for mirna-seq. introduction: exosomes contain a variety of biomolecules including dna. knowledge of cfdna distribution and localization in bioliquid is important for understanding both biological function of cfdna and exosomes. some publications state that a large proportion of plasma cfdna is localized in exosomes. to quantify cfdna content in free vs. exosomal form in human plasma, urine, and saliva, we employed subx technology, which allows affinity capture dna via phosphates groups of the polynucleotide chain and exosomes via membrane surface phosphate moiety clusters. subx is a proprietary compound that can simultaneously bind to both cfdna and exosomes in bioliquids, thus allowing precipitation of the [subx-dna/ subx-exosomes] complexes without ultracentrifugation. methods: detection of subx-dna and exosomes binding was done by measurement of particle sizes using zetasizer nano zs and nanosight ns . the samples were processed with the subx exo-dna isolation kit following the standard protocols. dna, protein and lipid concentrations were measured by fluorescent assays using qubit fluorometer. results: subx efficiently and selectively captures and co-precipitates cfdna and exosomes directly from bioliquids. exosomes are easily extracted from the pellet in exosome reconstitution buffer (erb), followed by subsequent isolation of tightly bound cfdna from the subx pellet. erb does not extract dna form the [subx -dna] pellet and thus does not contaminate reconstituted exosomes with cfdna. thus, we separate two distinct types of extracellular materialintact exosomes and purified cfdna in a single protocol from the same sample. over % of dna in plasma and urine exist as a free circulating pool, while in saliva up to % is associated with exosomes. thus, cfdna distribution is probably bioliquid-specific and must be evaluated by methods that eliminate cfdna-outer exosomal membrane aggregation. summary/conclusion: subx technology is suitable for simultaneous isolation of both cfdna and exosomes from the same bioliquid sample. subx separates cfdna fragments non-specifically attached to the outer lipid layers of the exosome membrane from the true intra-exosomal cfdna. in contrast, salting-out peg technique is associated with aggregation of macromolecules and vesicles and thus leads to overestimation of exosome-associated polymers content, including cfdna. tracing extrachromosomal dna inheritance patterns in glioblastoma using crispr eunhee yi, amit gujar, hoon kim, albert cheng and roel verhaak jackson laboratory for genomic medicine, farmington, usa introduction: glioblastoma multiforme (gbm) is the most lethal brain tumour; it is characterized by poor response to standard post-resection radiation and cytotoxic therapy, resulting in a dismal prognosis with a five-year survival rate of %. recurrence after therapy for gbm is unavoidable. there are substantial differences among the cells of gbm tumours in the abundance and types of genetic material. this heterogeneity likely is the major cause of therapy failure, the development of treatment resistance, and ultimately recurrence. a recent study has suggested that the amount of a particular type of dnaextrachromosomal dna (ecdna)differs substantially among different gbm tumours, and differs within a given gbm tumour over time. despite the speculation that ecdna is a key factor of tumour heterogeneity, how ecdna is propagated and distributed amongand how it behaves withincancer cells is completely unknown. methods: to address this gap in knowledge, this study focused on developing a novel cytogenetic crisprbased tool that enables visualization and tracking ecdna behaviour in live gbm cells. results: we found breakpoint sequences resulting from genome rearrangements during ecdna formation by performing computational analysis from whole genome sequencing data. and each breakpoint was regarded as a unique target sequence for ecdnaspecific labelling. the uniqueness of each breakpoint was validated by breakpoint-pcr (bp-pcr). furthermore, the location and the amount of each breakpoint were observed by breakpoint-fish (bp-fish) analysis in gbm cells. summary/conclusion: this results will be strong evidence to make ecdna-specific crispr system in further research. tracing ecdna dynamics will provide new insight into the impact of ecdna on cancer evolution. introduction: small extracellular vesicles (sevs) are - nm vesicles that mediate intercellular communication by transferring rna and proteins to the recipient cells. these cargo molecules are selectively sorted into sevs and mirror the physiological state of the donor cells. given that sevs can cross the bloodbrain barrier and their composition can change in neurological disorders, there is an increasing interest in elucidating the molecular signatures of sevs in circulation as disease biomarkers. however, circulating sevs are derived from multiple cellular sources and determining their source is challenging. information on sev composition can be beneficial in predicting whether these sevs are released predominantly from central nervous system cells. we hypothesized that differentially expressed mirnas between neuronal sevs and astrocytic sevs could be used as cell-typespecific signatures. methods: small extracellular vesicles were isolated from cell culture media of postnatal mouse primary neurons and astrocytes using differential centrifugation and characterized using nanoparticle tracking analysis, transmission electron microscopy and western blotting. rna from neurons, astrocytes, and their respective sevs were used for transcriptome and small rna sequencing. results: we observed that only a subset of cellular mirnas was packaged into sevs; differential expression of specific mirnas between sevs and their corresponding cells suggest that cells employ special mechanisms to sort mirnas into sevs. these mechanisms could be celltype specific since neuronal sevs showed a different mirna profile compared to astrocytic sevs. exomotifs, the short sequence motifs that control the loading of rna into sevs, were present in differentially expressed mirnas. we also observed that five rnabinding proteins, which are associated with passive or active rna sorting into sevs, were differentially expressed between neuronal and astrocytic cells. summary/conclusion: mirna signatures of sevs from neurons and astrocytes could be beneficial in determining if these cell types contribute to the alterations of sev composition in circulation in neurological disorders. cell-type-specific selectivity in rna loading might be attributed to the differential expression of rna-binding proteins. introduction: analytes present in the extracellular fraction of bodily fluids (ex. blood, urine) have utility as a tool for uncovering the molecular landscape of tumours and hold great potential for discovery of individualized cancer medicine. urine, being noninvasive as a sample type, has an obvious advantage over blood when used for liquid biopsy purposes. however, potential for microbial proliferation and the labile nature of host cells and extracellular vesicles (evs) at the point of sample collection/transport to the lab drives the need for stabilization of urine samples. development of such sample stabilization opens up capability for the detection of various biomarkers present in the extracellular fraction to be used in liquid biopsy. this is of particular concern as studies around urinary analytes for cancer diagnosis, progression and therapeutic effect are rapidly expanding in cohort sizes. multi-site collections and at-clinic collections are increasingly prohibitive for large scale recruitment and also lead to variability in the time between collection and processing. methods: in this study, we have analysed two commercially available ev extraction kits and compared them with ultracentrifugation technique for size, concentration and specificity of the isolated evs from human urine samples with and without our proprietary preservation solution using nanoparticle tracking analysis and western blot analysis for exosomal membrane markers. ev rna contents in various urine fractions (first morning first void, random first void and midstream) were compared using rt-qpcr assay to provide better understanding of the collection techniques and fractionations that are ideal for ev research work. results: in our current work, we have bench-marked human urine collection and ev extraction in order to provide recommendations in standardization of sample acquisition and processing for urinary ev studies. we have utilized these standardization in order to develop a novel and efficient sample stabilization principle for preservation of evs and ev rna in urine samples during an ambient temperature hold. summary/conclusion: taken together, we have established a framework for evaluating technologies and techniques in the ev sample processing space, which can be utilized by other research groups. vn -isolated plasma extracellular vesicles improve tumour mutation detection by next-generation sequencing compared to cell-free dna and correlate with tissue biopsy of nsclc patients introduction: liquid biopsy is a minimally-invasive diagnostic method that detects circulating biomarkers and has the potential to improve access to molecular profiling for nsclc patients when tissue biopsy material is unavailable or insufficient. although isolation of cell-free dna (cfdna) from plasma is the standard liquid biopsy method for detecting dna mutations in cancer patients, the sensitivity can be highly variable. vn is a amino acid peptide with an affinity for heat shock proteins that are exposed on the surface of extracellular vesicles (evs); peptide-ev aggregates readily sediment using a benchtop centrifuge and therefore the vn peptide provides a rapid, clinically-amenable procedure for ev isolation. in this study, we determine whether isolation of evs from nsclc patient plasma improves the sensitivity of single nucleotide variants (snvs) detection compared to cfdna and correlate genetic changes observed by liquid biopsy with tumour ffpe tissue biopsy. methods: blood was collected from stage iii/iv nsclc patients with informed consent in either edta or cell-free dna bct® collection tubes and plasma was harvested within minutes. total nucleic acid (tna) was extracted from either vn -isolated evs from edta plasma or directly from plasma collected in edta or cell-free dna bct® tubes (cfdna). snvs were detected by next-generation sequencing (ngs) results: vn isolation of evs from plasma resulted in higher recovery of dna than cfdna isolation. the snvs detected in both ev-dna and cfdna correlated well with those reported in matched ffpe tumour tissue using ngs, including % specificity for egfr mutations. no improvement in snv detection was observed using cell-free dna bct® collection tubes compared to edta tubes. isolation of evs with the vn peptide prior to sequencing improved a number of ngs parameters including library yield, total reads, median read coverage and molecular coverage, resulting in improved sensitivity of snv detection. summary/conclusion: in summary, our research demonstrates that vn -based ev isolation is useful for molecular profiling of nsclc patients for whom tissue biopsy is not an option, thereby improving access to molecular profiling and targeted therapies. funding: atlantic canada opportunities agency novel markers for neuroendocrine prostate cancer divya bhagirath a , michael liston b , theresa akoto a and sharanjot saini a a augusta university, augusta, usa; b veteran affairs, san francisco, usa introduction: prostate cancer (pca) is fuelled by androgens and androgen receptor (ar) signalling. therefore, ablation of ar signalling by androgen deprivation therapy (adt) is the goal of first-line therapy that results in cancer regression initially. however, two to three years post-adt, the disease develops into castration-resistant prostate cancer (crpc). as a second-line of therapy, next generation of ar pathway inhibitors (api) such as enzalutamide (enz) are used that are effective initially followed by emergence of drug resistance. a subset of api-resistant tumours emerges to an ar independent state via undergoing a trans-differentiation to neuroendocrine lineage, a process referred to as neuroendocrine differentiation (ned). due to lack of ar signalling, these pca variants, referred to as neuroendocrine prostate cancer (nepc), are impervious to anti-androgen therapy and constitute an aggressive variant of advanced crpc with poor prognosis. currently, there is a lack of effective molecular biomarkers for predicting api therapy resistance and emergence of therapy-induced ned. methods: exosomes/evs were isolated from sera of a patient cohort with/without ned. the study was conducted in accordance with ethical guidelines of us common rule and was approved by the institutional committee on human research. written informed consent was obtained from all patients. following extensive characterization of evs by electron microscopy, nanosight tracking analyses and western blotting of exosomal markers, small rna sequencing was carried out on illumina hiseq platform to identify differentially expressed transcripts. machine learning algorithms were applied to clinical sequencing data to train a "mirna classifier". further, we probed the proteomic profile of exosomes isolated from nepc cellular model nci-h and enzalutamide resistant crpc cell lines by mass spectrometry. results: we identified that transition from crpc-adenocarcinomas to neuroendocrine states is associated with significant ev-mirna dysregulation, with a specific dysregulation in certain mirna families. with the application of machine learning algorithm, we identified an ev-based "molecular classifier" that can robustly stratify crpc-ne tumours from crpc-adenocarcinomas. proteomic analyses identified novel nepc-specific, glycosylated proteins that can be exploited for nepc diagnosis. summary/conclusion: our data suggest that ev mirna and protein profile can predict neuroendocrine differentiation in advanced castration-resistant prostate cancer patients. exosomal mrna in diagnosis strategy for hepatocellular carcinoma aleksandr abramov, alisa petkevich, vadim pospelov and pavel ogurtsov peoples' friendship university of russia (rudn university), moscow, russia introduction: exosomal cargo is informative source illustrating the genetic events happening in cells, what can be especially advantageous in case of cancer development for disease progression or treatment effectiveness monitoring. methods: plasma samples of hepatocellular carcinoma (hcc) patients, plasma samples of patients with liver cirrhosis - on the hepatitis c virus (hcv) background, healthy donors' plasma samples. exosomes were isolated with ultracentrifugation, western blot (cd , cd ) was performed. total mrna was isolated with exosomal rna isolation kit, norgen biotec corp. sequencing was carried out on a minion sequencer. housekeeping genes (gapdh, b m, actb, tuba a). detected mutations were confirmed by real-time pcr with specific highly sensitive lna probes. results: significant changes in expression levels were identified for genes in hcc and liver cirrhosis groups (increasing up to x compared to control samples and decreasing up to no detected expression). in out of patients with hcc mutant burden was significant increased compared to mutant burden in groups with cirrhotic samples. in out of patients with hcc increased expression for mrna line- was identified compared to cirrhotic patients. summary/conclusion: exosomal mrna expression levels may serve as a prognostic and diagnostic marker for patients with liver cirrhosis caused by hcv for hcc risk development. funding: research is supported with federal funds " - " circulating extracellular vesicle signatures in small cell lung cancer michela saviana a , giulia romano a , giovanni nigita b , robin toft a , patricia le a , kai wang c , mario acunzo a and patrick nana-sinkam a a virginia commonwealth university, richmond, usa; b the ohio state university, columbus, usa; c institute for systems biology, seattle, usa introduction: lung cancer is the leading cause of cancer deaths worldwide and classified primarily as either non-small cell lung cancer (nsclc) or small cell lung cancer (sclc). compared to nsclc, sclc has a faster growth rate, earlier widespread metastasis, and shorter overall survival. the early diagnosis of sclc and the development of novel therapeutics have proven challenging. thus, progression and recurrence rates remain high. non-invasive methods for cancer detection are increasingly being used to inform clinical decision making. extracellular vesicles (evs) have recently emerged as potential carriers of genetic contents such as micrornas (mirs) to induce reprogramming of components of the microenvironment in cancer initiation and progression. moreover, extracellular mirs expression profiles have been shown to have signatures related to tumour classification, diagnosis, and progression. methods: we selected a cohort of patients divided into groups: high-risk smokers, adenocarcinomas, squamous carcinomas, and sclc. we extracted total circulating ev and plasma rna from plasma ( patients in total) and rna from plasma in a separate group ( patients in total). utilizing both next-generation sequencing (ngs) and nanostring platforms, we analysed for global microrna (mirs) expression patterns. candidate mirs were then validated by qrt-pcr. results: we identified several deregulated mirs in both evs and plasma of sclc patients compared to the other groups. for evs, we validated mir- - p as a significant biomarker for the late stage of sclc compared to controls. in the case of plasma, we validated the upregulation of mir- in sclc compared to controls. summary/conclusion: our results indicate that a potential combination of plasma (mir- ) and ev-based (mir- - p) mirs be valuable biomarkers for sclc detection and serve as a basis for a non-invasive sclc classifier. funding: virginia commonwealth university, doim -nih/nci introduction: the isolation of evs from milk is technically challenging due to the complexity of milk. currently used separation procedures allow for the removal of milk fat globules and cells (by low speed centrifugation of fresh milk), removal (by acidification), or disruption (by addition of edta) of casein micelles. using these protocols the integrity, composition and targeting of bovine milk evs has been evaluated and has led to believe that milk evs might withstand these conditions. however, the effects on functionality of milk evs (i.e. immunomodulatory properties) after processing and isolation have not been studied. therefore, we have set up an in vitro culture system using a human t cell line that allows for the rapid screening of milk ev functionality. methods: fresh bovine milk was defatted and cells were removed after x , g centrifugation, followed by differential centrifugation at , g and , g. this milk was either subjected to acidification with hcl, or edta was added, or the milk supernatant remained untouched. top down optiprep density gradient separation followed by sec was used to further purify evs. these highly purified milk evs were added to human jurkat t cells, which were simultaneously stimulated using anti-cd and anti-cd antibodies. after h t cell activation was measured by il- cytokine production. results: precipitation or disruption of casein micelles allowed for the substantial removal of proteins during isolation compared to directly isolated evs, which aids in the purification of milk evs. in vitro analysis revealed that in the presence of directly isolated, or edta isolated milk evs, jurkat cells were suppressed in their activation as measured by il- production. remarkably, evs isolated from hcl-acidified milk were impaired in their suppressive capacity to inhibit il- production. summary/conclusion: although casein removal from bovine milk greatly improves purity of isolated milk evs, the detrimental effects on ev functionality should be considered. interestingly, evs exposed to acidic conditions lost their ability to modulate t cell activation, which is in contrast with the general believe that milk evs could withstand the gastro-intestinal tract. funding: this work is funded by the european union's horizon framework programme under the grant fetopen- evfoundry. optimising methods for separation and characterisation of extracellular vesicles from skim milk and infant milk formula introduction: infant milk formula (imf) is intended to impart nutrition to infants, similar to breast milk. however, although industrial imf production involves harsh treatment, potential consequences on extracellular vesicles (ev) in imf are not yet established. this study aimed to optimise methods for separating evs from imf and skim milk (sm) and to characterise the evs in accordance with misev . methods: sm and imf were either not treated (nt) or treated with acetic (aa) or hcl acid (isoelectric precipitation, ip), to remove caseins. samples were then subjected to differential ultracentrifugation (duc) or gradient ultracentrifugation using iodixanol solution (guc). for duc, ml samples were centrifuged at k g, k g, k g, k g and k g sequentially for min each and pellets re-suspended in ml pbs. preparation of agarose microspheres for high-efficient separation of extracellular vesicles cheng-tai chen, chien-an chen, carolyn yen and nien-tzu chou industrial technology research institute, chutung, taiwan (republic of china) introduction: size exclusion chromatography (sec) is becoming a widely used technique for separating of extracellular vesicles. various commercially available products were launched on the market, however, their separation efficiencies were not fully disclosed. herein, novel porous agarose microspheres with the tunable diameter and pore size were synthesized by emulsion reaction. the performance was evaluated and compared with commercial products. the modified sec column packing materials were shown to exhibit advantages for rapid, high-recovery and high-purity separation of extracellular vesicles from cell culture-conditioned medium and human plasma. methods: the homemade sec column was packed by gravity flow. μl of the sample was loaded and the pbs buffer was used as eluent. factions were collected and analysed by cd /cd sandwich elisa assay and by micro bca assay for determining respectively extracellular vesicles and total protein content. results: agarose microspheres were prepared by emulsification. the particle size can be controlled by the types and concentrations of surfactants. the product was collected by desired screen meshes and used as packing materials of the sec column. our results showed that the extracellular vesicles were clearly separated from proteins. more than . % of proteins were removed while the recovery of extracellular vesicles was close to %, which is much higher than % of the commercial product. the total separation time was less than min. summary/conclusion: we have established an approach for generating spherical agarose microspheres as packing materials of homemade sec columns, which are capable of separating extracellular vesicles from complex samples with high efficiency. further validations with additional samples are currently ongoing. immunomagnetic sequential ultrafiltration (isuf) platform for enrichment and purification of extracellular vesicles from large and small volumes of biofluid eduardo reategui, jingjing zhang, luong t. h. nguyen, richard hickey, nicole walters and andre f. palmer the ohio state university, columbus, usa introduction: evs derived from tumour cells have the potential to provide a much-needed source of non-invasive molecular biomarkers for liquid biopsies. however, compromises have to be made when using a particular technology/methodology for the isolation of evs. currently, there is a trade-off between sample volume and specificity in ev isolation technologies that limits quantitative molecular analysis of ev contents, ultimately impacting the utility of evs in cancer diagnostics. here, we present an approach called immunomagnetic sequential ultrafiltration (isuf). our platform combines ultrafiltration and immunoaffinity separation. using isuf, we demonstrate that small or large volumes of biofluid can be processed (~ µl or > ml) while concomitantly removing . % contaminating proteins. we also processed serum from breast cancer patients enabling the characterization of different tumour and immune biomarkers on the isolated evs. methods: human samples were collected under an approved irb. size distribution and concentration of evs were measured using a tunable resistive pulse sensing (trps) method. ev proteins and rnas were extracted and quantified using a bca protein assay and uv spectroscopy. isuf and other ev isolation methods were compared for ev concentration, protein, and rna quantity. results: ml of cell culture media (ccm), . ml serum, and ml urine samples were processed with the isuf platform and recovered in µl. for all cases, evs were enriched with recovery efficiency greater than %. the processing time for a ml sample was min with over % of purity. we compared ev concentration and purity isolate from . ml serum using isuf and other commercially available methods, isuf demonstrated superior performance on isolating evs at high concentrations and purities. analysis of total rna amounts in the isolated evs using different methods was corresponding to higher ev recovery efficiency of isuf. we also compared protein and rna levels of evs enriched with isuf present in urine and serum samples from the same donors (n = ), and we found that for the same number of evs, the ev rna concentration from both biofluids showed no significant difference. finally, we have processed serum samples from metastatic breast cancer patients and demonstrated that their isolated evs have expression levels of her , cd and mir biomarkers at significantly higher levels than healthy controls. summary/conclusion: the isuf platform can be scale-down or -up to work with small or large volumes of biofluids for the isolation of evs. using the isuf platform with clinical samples shows the potential of our platform to be used for cancer diagnosis or monitoring treatment response. funding: national institutes of health (nih) grants ug tr (e.r.); r hl , r hl , and r eb (afp). challenges in exosomes isolation from primary biological samples derived from multiple myeloma patients introduction: multiple myeloma (mm) remains incurable despite advances in its treatment and research progress on the crosstalk between mm and surrounding host cells. exosomes are important regulators of the cellular niche. their importance for diagnostic and therapeutic applications has been proven in many cancers. in this context we hypothesized that a better understanding of the molecular role and features of mm-derived exosomes would provide a basis for their use for both risk stratification and as predictive biomarkers of response to anti-mm drugs already in use in clinical settings, given the optimization and validity of their isolation/purification method. methods: exosomes were isolated from human mm cell lines (hmcls) supernatants and peripheral blood plasma (pbpl) isolated from healthy donors, mm and mgus (monoclonal gammopathy of undetermined significance) patients. both fresh and frozen samples were tested. we evaluated commercially-available kits, density-based separation and ultracentrifugation. results: higher purity and recovery, evaluated by western blotting, nanoparticle tracking analysis and electron microscopy, were observed for supernatant density-based purification and for pbpl resin-based isolation. exploring the function of mm-derived exosomes, we observed an increase in proliferation of the immortalized stromal cell (sc) line hs treated with exosomes when compared to untreated cells, and a higher increase in proliferation of scs treated with mm-exosomes when compared to exosomes derived from normal and mgus pbpl samples. summary/conclusion: the method of isolation represents a critical step in the study of exosomes as many factors can affect the purity, yield and downstream application. our data demonstrated that density and resin-based isolation methods provided functional mm-derived exosomes with proliferative effects on scs. altogether our findings may serve as a guide to choose exosome isolation methods for mm studies. further optimization steps, including albumin-depletion from plasma samples and use/type of serum in cell cultures, should be taken into consideration when planning proteomics and genomics as downstream applications. funding: australian government rtp and monash departmental scholarship. a rigorous method for exosome isolation from post-mortem eyes introduction: in order to determine and validate the tissue-specific content of extracellular vesicles (evs) in biofluids, robust ev isolation methods from tissues must be developed. however, to date very few rigorous methods to isolate or enrich for intact evs from tissues have been reported. we present a comprehensive exosome isolation method with a sufficient level of characterization to unequivocally demonstrate true ev identity from ex vivo eyes. methods: iodixanol (optiprep) buoyant density gradient ultracentrifugation (dguc), cushioned dguc (c-dguc), and our newly developed c-dguc immunocapture (c-dguc-ip) method were used to compare yield and enrichment of exosomes isolated from porcine eyes between to hours post-mortem. yield was assessed by nanoparticle tracking analysis (nta) and immunoblotting for exosomal markers along with total protein quantitation. enrichment was assessed by comparison of exosomal markers, ocular-specific markers and known contaminant markers, plus in-depth proteomic mass spectrometry analyses. results: high enrichment of posterior eyecup small evs (sev) were achieved by dguc and c-dguc, with c-dguc resulting in an eightfold increase in yield by nta and two to fivefold increases of exosomal protein markers such as syntenin- and cd by immuno-blotting compared to dguc. interestingly, in-depth proteomic analyses revealed that a majority of these sevs with densities of . - . g/ml isolated by dguc and c-dguc were likely of endoplasmic reticulum (er) and golgi origin, suggesting er-to-golgi transport vesicles resulting from post-mortem tissue cell rupture. in order to enrich further for sevs (including exosomes) we subjected sevs isolated by c-dguc to anti-cd immunocapture. the resulting sev proteome was enriched . -to -fold for bona fide sev and exosome markers compared to c-dguc. summary/conclusion: the c-dguc method provides an enhanced yield and purity of sevs and exosomes from ex vivo eye tissue. however, to avoid significant contamination with er and golgi-derived vesicles from postmortem eyes, a final ev-specific immunocapture step is required to achieve sufficient purity for subsequent analyses. our highly rigorous method paves the way for identification and validation of ocular-derived exosomes in blood and their potential use as eye disease biomarkers. characterization of the extraction of extracellular vesicles using a lab-ona-disc filtration system introduction: personalized treatment for cancer is a promising way to face the multiplicity of the disease, to increase the efficacy of drugs and to decrease their toxicity. as part of this strategy, liquid biopsy explores a new non-invasive approach to diagnose cancer, guide treatment and monitor its efficacy. extracellular vesicles (evs) are nanometric lipid bilayers micelles with high potential as biomarkers. they are involved in the transfer of information (proteins, rna and dna) between cells. evs include a broad spectrum of particle sizes, from the tens to thousands of nanometres. the isolation of evs from complex matrices is the first step of any protocol and is particularly important for the reproducibility and fidelity of the results presented, as it could bring bias in further analysis. in order to explore the heterogeneity of evs, a full characterization (physical and biological) of the extracted evs is needed. we evaluate and compare evs purification methods, including ultracentrifugation, sizeexclusion chromatography (sec) column and an emerging microfluidic technology: labspinner filtration labon-a-disc device isolating evs between two filters of and nm. methods: a cell supernatant was used as a model matrix. we compared three methods of extraction of evs: ultracentrifugation with two cycles of h at , g at degrees celsius (rotor type ti, beckman floor ultracentrifuge optima l k), qev size exclusion chromatography columns from izon (qevoriginal/ nm) and lab-on-a-disc filtration system (labspinner, exodisc c). evs characterization was conducted with nta (nanosightns ), trps (izon), nanodrop (spectrometernd ), tem (fei tecnai kv) and custom micro-immuno-assay. results: in this study, we characterize a filtration system made of two serial filters of nm and nm pores for isolation of evs. compared to ultracentrifugation and chromatography columns, yield of extraction is up to times higher and the size of the extracted particles is smaller. tem imaging was used for assessment of the quality of the extracted evs. however, albumin concentration measurement tends to show that the purity of the solution is decreased. the immuno-labelling analysis shows that the proteomic signature of the extracted evs differs according to the extraction methods. the new filtration technology seems to give us access to a broader range of evs compared to standard methods. summary/conclusion: in this study, we characterized purification methods including lab-on-a-disc filtration, and were able to demonstrate an increase of the concentration of evs by a factor of , a decrease of the size of the accessible extracted particles and access to new proteomic signatures. funding: we acknowledge the support of génome québec and action marie skłodowska-curie. effects of sample processing on isolation of extracellular vesicles from blood plasma by centrifugation darja božič a , matej hočevar b , veno kononenko c , marko jeran a , urška Štibler d , immacolata fiume e , manca pajnič f , ljubiša pađen f , ksenija kogej g , damjana drobne c , ales iglič h , gabriella pocsfalvi i , veronika kralj-iglič f and darja bozic j introduction: the isolation of extracellular vesicles (ev) from body fluids is still controversial and the poor understanding of vesicle stability and effects of sample processing is probably one of the core issues preventing the breakthrough of this field into applicative practices. methods: we performed an in-depth study of sample changes in blood, blood plasma and samples throughout the increasing speed of centrifugation, considering the number, size, contents and shape of particles in the isolates. flow cytometry, light scattering, mass spectrometry and scanning electron microscopy were employed to reveal the properties of material in the samples. results: the particles of size about - nm with characteristic topology of membrane vesicles without internal structure were observed by the scanning electron microscope only in ev isolates prepared from fresh blood sample. inspection of the tube surface in which the isolation took place suggests that those particles are likely formed from activated platelets tearing at the tube wall due to the centrifugal pull. the isolates prepared from frozen blood plasma prepared by centrifugation with different forces contained different amounts of particles with similar protein contents, predominated by highly abundant human plasma proteins, including albumins and immunoglobulins. some lipoprotein clearance and fibronectin precipitation were however observed through increased speed and time of centrifugation. summary/conclusion: the results of this study [ ] contribute to the understanding of stability and dynamics of membrane particles. the reported evidence provides the support for viewing ev isolates as a product, shaped by uniqueness of the starting samples and the thermal and mechanical stress applied upon processing. we believe this kind of insights strengthen our ability of reading the story of evs. introduction: apoptosis is a form of programmed cell death with diverse roles in the tumour microenvironment and emerging data show that, besides its role in tumour suppression, it can also promote oncogenic proliferation. highly aggressive tumours such as burkitt lymphoma (bl) show high levels of apoptosis, which has a diagnostic and prognostic value for classifying and staging the disease. we hypothesize that amongst other elements, extracellular vesicles (ev) are key mediators of apoptotic cell-derived tumour microenvironment signals. here, we report on ev released in vitro by apoptotic bl cells (apo-ev) in relation to their potential use as cancer biomarkers. methods: basic physical properties of apo-ev such as structure, size distribution, surface charge and membrane fluidity are discussed using cryo electron microscopy (em) and tomography, nanoparticle tracking analysis, dynamic light scattering and fluorescence anisotropy respectively. for phenotypic analysis we apply immunocapture and flow cytometry, immunogold labelling on transmission em, fluorescence microscopy and quantitative pcr. in addition, we study the interaction of apo-ev with blood components such as platelets, leucocytes and red cells, in order to understand their effects in the circulation and therefore their potential for analysis in blood samples. results: looking at the differences between apo-and non-apo-ev, apo-ev have larger diameter, while structurally are not different. however, we have identified distinct apo-ev markers such as active caspase and histones, or dna and small non-coding rna-y. there is also strong interaction of ev with platelets and leucocytes but not with red cells, indicating potential routes of transfer of ev cargo in the circulation. summary/conclusion: it is concluded that for the characterization of the heterogenous ev populations, combination of multiple techniques is often required, and also, understanding the strengths and limitations of each method is essential for choosing the appropriate set of analytical tools. finally, we consider that monitoring free circulating apo-ev or blood cells with which they have interacted is a promising approach to improve cancer diagnosis, prognosis and evaluation of therapeutic response. casting a small netrin: functional roles of a novel surface factor on stroma-derived extracellular vesicles in pancreatic cancer kristopher s. raghavan a , ralph francescone b , janusz franco-barraza b and edna cuckierman b a drexel university; fox chase cancer center, philadelphia, usa; b fox chase cancer center, philadelphia, usa introduction: pancreatic ductal adenocarcinoma (pdac) is a devastating disease driven and supported by changes in its microenvironment, or stroma. here we dissect the intercellular communication that exists between the primary stromal component, cancer-associated fibroblasts (cafs) and pdac. pdac communicates with its microenvironment, in part, through the exchange of specific types of extracellular vesicles (evs). specifically, we focus on the mechanism by which caf-secreted evs support pdac survival, with an additional goal to identify biomarkers suitable to generate a future "liquid biopsy" test for early pdac detection and prognosis. methods: evs are isolated from patient-derived pdac-associated fibroblasts via differential ultracentrifugation and validated by isev standards. human pdac cell lines used as recipient cells are treated with caf-evs to assess their role in supporting pdac survival. recombinant proteins, neutralizing peptides, and non-functional mutant proteins are used to block ev interaction with target cells. results: we observe sub-types of caf-evs containing unique surface receptors. one ev sub-population of interest contains a novel surface protein (nsp) expressed on the plasma membrane of pancreatic cafs, but not their healthy counterparts. further, pdac cells up-regulate nsp's lone binding partner, suggesting a role for these factors in pdac-selective ev uptake. functional assays designed to test pdac viability suggest these nsp(+)-evs protect pdac cells from programmed cell death as a result of physiological stress. this ev-mediated survival benefit can also be inhibited by blocking the interaction of nsp and its binding partner, suggesting the engagement of these two factors is necessary for cafs to support pdac via evs. pursuing our biomarker goal we confirm stromal nsp expression increases during early panin stages prior to tumour development, and we are currently seeking to validate nsp(+)-evs in blood of pdac patients. summary/conclusion: this research shines light on a novel mechanism of tumour-stroma communication that may be crucial for cancer progression during early disease stages and a potential target for disrupting the supportive role of the tumour microenvironment. additionally, we describe a sub-population of nsp (+)-evs that have the potential to serve as biomarkers for identifying pdac development. exosomes carry distinct mirnas that drive medulloblastoma progression introduction: extracellular vesicles (evs) represent an ideal source of functional biomarkers due to their role in intercellular communication and their ability to protect cargo, including rna, from degradation. the most investigated ev's are exosomes, nanovesicles secreted by all cell types and able to cross the bloodbrain-barrier. here we characterised the rna of exosomes isolated from medulloblastoma cell lines, with the aim of investigating exosomal rna cargo as potential functional biomarkers for medulloblastoma. methods: exosomes derived from a panel of matched (original tumour and metastasis) medulloblastoma cell lines were isolated and characterised by nanosight, electron microscopy, western blotting and nanoscale flow cytometry. exosomal mirna and mrna from our matched cell lines and foetal neuronal stem cells, which were used as a normal control, were analysed by rna-sequencing technology. results: based on hierarchical clustering, malignant derived exosomes were distinctly separated from normal control exosomes. mirna profiling revealed several established oncomirs identified in our malignant derived exosomes compared to control samples. using interaction pathway analysis, we identified that our malignant exosomes carry numerous mirnas implicated in migration, proliferation, cellular adhesion and tumour growth. several previously identified oncomirs were also identified to be present at higher levels in metastatic exosomes compared to primary and normal, including hsa-mir- - p and hsa-mir- a- p. summary/conclusion: this study shows that exosomes from mb cells carry a distinct mirna cargo which could enhance medulloblastoma progression. the use of circulating exosomes as markers of metastatic disease could be an innovative and powerful noninvasive tool. introduction: inflammatory changes in the bone marrow (bm) and suppression of haematopoietic stem and progenitor cell (hspc) function during acute myeloid leukaemia (aml) significantly contribute to patient morbidity and mortality. our laboratory has previously shown that aml-derived extracellular vesicle (ev-aml) trafficking confers a state of enforced quiescence and leads to lasting dna damage in hspcs. here we explore the underlying cause. specifically, we hypothesize that ev-aml incite inflammatory regulators as potential mediators of dna damage. methods: as a validated model of aml, we utilized the murine tib cell line as a source of ev-aml. ev-previous work has indicated that mirnas, notably mir- a and mir- , play a critical role in scc tumour development. evs are membrane-bound vesicles involved in cell-cell communication carrying actively sorted cargo, protected from degradation. the potential pathways these vesicular mirnas modulate and the implication they have on cancer biology is under active investigation. we have previously shown that the cadherin dsg , a stem cell marker, modulates ev release. dsg is upregulated in a number of cancers, including scc, and correlates with poor prognosis. here we aim to elucidate the impact of ev-associated mirnas in sccs by bioinformatic analysis. methods: scc cells stably expressing dsg were generated and evs isolated by sequential ultracentrifugation. total cellular and ev rna was isolated by mirneasy, analysed using rnaseq and identified by grch alignment. results were confirmed by qpcr. altered pathways based on targets were identified using mirnet and kegg pathway analysis. potential cancerassociated cytokine targets were confirmed by antibody array. results: rnaseq revealed cellular and ev mirnas that were differentially expressed in response to dsg with overlapping. the highest altered mirnas were validated by qpcr. kegg pathway analysis determined that these mirnas have the highest number of shared targets in cancer, cell cycle, and p signalling pathways. interestingly, mir- was upregulated while mir- a was dramatically downregulated in evs. targets of mir- a, icam- , il- , and il- , cytokines critical for cancer progression were upregulated. summary/conclusion: these results suggest that the mirna content of evs is tightly regulated. by altering the mirna profile, dsg contributes to the pathogenicity of these evs by increasing levels of cytokines important for cancer stem cell renewal and metastasis. in addition, these mirnas may serve as non-invasive diagnostic markers for sccs. funding: nih r cancer cells grown in d release distinct extracellular vesicles during tumour growth and invasion jens c. luoto, sara bengs, leila coelho rato, lea sistonen and eva henriksson Åbo akademi university, turku, finland introduction: cancer cells secrete extracellular vesicles (evs) that affect tumour progression. the characteristics of evs produced during tumour growth and invasion are however poorly understood. in this study, we identify the composition and characteristics of evs produced by noninvasive and invasive tumours and correlate these characteristics with the invasive status of the tumour. for that purpose, we established a protocol for isolating evs from extracellular matrix (ecm)-based three-dimensional ( d) cancer cell cultures. methods: human prostate cancer pc cells were grown in d cultures using ecm-based hydrogel, in standard d culture conditions and in bioreactor. evs were isolated from these cultures with differential and density gradient centrifugation. the isolated evs were characterized with nanoparticle tracking analyses, electron microscopy, immunoblotting and mass spectrometry (ms). results: our results demonstrate that d ecm-based hydrogel cell cultures secrete evs that can be isolated from both the conditioned media and the hydrogel. the invasive d cultivated pc organoids were found to secrete large amounts of evs compared to the non-invasive organoids. interestingly, our ms results revealed that non-invasive and invasive organoids secrete evs with partially distinct protein cargo. summary/conclusion: we have established a novel protocol for ev production in a d cell culture system utilizing ecm-based hydrogel, in which invasive tumour growth can be mimicked. our method allows the specific isolation and characterization of evs derived from different stages of d culture, such as non-invasive and invasive organoids. importantly, we found that tumour-derived evs change in composition during the tumour progression. taken together, our method can be used to define the distinct ev characteristics involved in cancer invasion. we previously showed extracellular vesicles (evs) to be causally involved in transmitting drug resistance. this study aimed to evaluate compounds proposed to reduce/block ev release. specifically, we selected calpeptin and y (proposed to inhibit evs budding from the cell membrane) and manumycin a and gw (proposed to inhibit evs deriving from mvbs). associated effects on -and consequences of-ev release were then investigated. methods: suitable compounds concentrations that were non-toxic to cells were first selected by performing cytotoxicity assay and flow cytometry (fc). conditioned medium (cm) was collected from docetaxel-resistant pc (pc rd) cells after h incubation in dfbs-medium with or without the compounds. evs were separated from tangential flow filtration concentrated cm using optiprep density gradient. . - . g/ml fractions were then pooled and washed. evs were characterised using nta, immunoblot, tem and lipid assay and fc. influences on growth and migration, of evs continuing to be released (at x evs/ x cells, x evs/ x cells), were evaluated on recipient du and rv cells. evtrack id ev , score % results: calpeptin and y , alone and in combination, did not significantly affect quantities of evs released. however, gw significantly (p < . ) increased quantities of released evs, of a larger size; very high protein to lipid ratio; and carrying grp compared to control evs (p < . ). this effect was reverted when gw was combined with manumycin a (p < . ). following all compounds treatments, x evs/ x cells inhibited rv proliferation (p < . ), while at x evs/ x cells only evs from manumycin a (p < . ) and y (p < . ) treated cells reduce rv proliferation. evs following gw treatment significantly (p < . ) inhibited du migration compared to bulk non-treated control and compared to the effect obtained using the entire pool of evs (p < . ). summary/conclusion: while none of the proposed inhibitors significantly reduced ev release, the resulting evs were less potent in transmitting aggressive behaviour, such as proliferation and migration, to receiving cell lines. patient-derived organoids represent a novel tool to study the effect of intra-tumoral heterogeneity on ev release in non-small cell lung cancer introduction: lung adenocarcinoma (luad) is the leading cause of cancer-related death with a low -year survival. although the importance of intra-tumoral cellular heterogeneity of solid tumors in the clinical outcome and treatment is emerging, proper models to study its effects on ev release and cargo in human tissues still lack. the d organoid technology maintains the cellular and genetic heterogeneity of in vivo tissues and has proved to be so far the best ex vivo model of human cancers. by using patient-derived and mouse organoids we set out i) to compare the ev release from normal and tumor tissues and ii) to follow changes in ev secretion when the relative ratio of tumor cell subpopulations is shifted. methods: we used mouse and luad patient-derived normal and tumor organoids. the medical research council of hungary approved our experiments with human samples and informed consent was obtained from patients. evs were detected by antibody-coated beads, nta and tem. intra-organoid heterogeneity was proved by immunostaining and rt-qpcr. results: we provide evidence that both mouse and human normal organoids contain all the bronchiolar cell types. interestingly, luad organoids selected for tp mutation contained not only ki + proliferating cells, but differentiated cell types as well. furthermore, all the lung organoid cultures produced evs and this was shifted to the smaller size range. interestingly however, when modifying the proportion of organoid cell types, we observed an increased ev release when more ki + proliferating cells were present both in normal and in luad samples. summary/conclusion: our data show that patientderived lung organoids represent a novel model to study the role of intra-tissue heterogeneity in ev functions in the humans, leading to improved diagnosis. funding: this work was funded by the national competitiveness and excellence program nvkp_ - (national research, development and innovation office, hungary) and by the national excellence program in higher education (ministry of human resources, hungary). exosome mediate heart-adipocyte communication after myocardial ischaemia/reperfusion and impairs adipocyte endocrine function yajing wang, lu gan, dina xie, wayne lau, theodore christopher, bernard lopez and xinliang ma thomas jefferson university, philadelphia, usa introduction: by incompletely understood mechanisms, mi patients sustain systemic metabolic disorder. adipocytes are an important cellular type regulating energy homoeostasis. the impact of mi upon adipocyte function remains unknown. exosomes (exo) are critical vehicles mediating organ-organ communication. however, whether and how exo may mediate post-mi cardiomyocyte/ adipocyte communication have not been previously investigated. methods: adult male mice were subjected to mi/r. serum exo were isolated hours after r and incubated with t l cells for hours. the effects of exo upon adipocyte function were determined. results: compared to control, mi/r exo significantly altered the expression of genes known to be important in adipocyte function. go analysis revealed that genes associated with endoplasmic reticulum (er) function and adipocyte endocrine function are the primary two pathways altered by mi/r exo. venn analysis identified mi-rnas as cardiac-enriched, adipocyte-poor, and er function-related mirnas. rt-qpcr confirmed the mir- a/ a/ - family members are the most markedly increased mi-rnas in mi/r exo. incubation of t l cells with mi-r a mimic significantly downregulated edem , dsba-l, and pparn, and upregulated perk and chop. conversely, mi-r a inhibitor significantly decreased the impact of mi/r exo upon er function genes. additional studies demonstrated edem and pparγ (two critical molecules maintaining er function and adipocyte endocrine function) to be direct targets of mi-r a. one of the most significant endocrine molecules of adipocyte origin, adiponectin is regulated by pparn at the transcriptional level and by dsba-l at the post-translational level. we next determined whether mi/r exo may affect adiponectin expression/ assembly. incubation of t l cells with mi/r exo significantly inhibited total and high molecular weight adiponectin expression, an effect blocked by mir a mimic. finally, in vivo administration of gw (exo biogenesis inhibitor) or mir a inhibitor attenuated adipocyte er dysfunction and restored plasma adiponectin level in mi/r animals. summary/conclusion: we demonstrate for the first time that mi/r causes significant adipocyte er and endocrine dysfunction by exo mediated cardiomyocyte/adipocyte communication via mir- a/ a/ - . funding: nih and american diabetes association pancreatic cancer cell extracellular vesicles drastically alter the behaviour of recipient normal pancreas cells charles p. hinzman a , yaoxiang li a , meth jayatilake a , jose trevino b , partha banerjee a and amrita cheema a a georgetown university medical center, washington, usa; b university of florida health science center, gainesville, usa introduction: pancreatic cancer (paca) is predicted to become the rd leading cause of cancer-related deaths by . patients diagnosed with pancreatic ductal adenocarcinoma (pdac) have a -year survival ratẽ %. detection of pre-neoplastic lesions can potentially improve survival. however, there is currently no screening test for early stage detection. importantly, paca tumours are % non-tumorigenic cells. a better understanding of early paca oncogenesis is needed. cancer cells shed extracellular vesicles (evs) that are internalized by neighbouring and distant cells to induce a myriad of cancer progression events. we hypothesize that in early paca oncogenesis, evs mediate a behavioural change in surrounding normal cells, leading to the formation of this unique stroma. the purpose of this study was to develop a model to examine the phenotypic changes undergone by normal human pancreas cells when they are exposed to paca cell evs. methods: evs were isolated using differential ultracentrifugation with filtration from established (panc- , sw- , capan- and miapaca- ) and patientderived xenograft (ppcl- and ppcl- ) paca cell lines. cells were grown using ev-depleted fbs. ev isolations were validated and quantified using transmission electron microscopy, quantitative elisa, immunoblot and nanoparticle tracking analysis. normal pancreas cells (htert-hpne and hpde-h c ) were co-cultured with cancer cell evs for - hours. metabolic activity was measured using a mito stress test on a seahorse xfe extracellular flux analyser. results: we discovered that normal cells undergo vast behavioural transformations, including significant morphological changes, increased proliferation and an uncharacteristic invasive capability, when co-cultured with paca cell evs. these responses were ev dose dependent. further, paca cell evs metabolically reprogrammed normal cells, causing a bioenergetic switch, from a quiescent, aerobic profile to a highly energetic and glycolytic profile. summary/conclusion: our results indicate that paca cell evs confer enormous transformational properties to normal human pancreas cells in vitro. we hypothesize that evs impart distinct transformational properties to normal cells in vivo and this influence could unveil novel mechanisms regulating cancer onset and progression. these signals may be detectable before progression of early-stage paca to pdac, leading to the development of assays for earlier diagnosis in patients. further studies are underway to identify the biochemical mediators of these changes. plasma extracellular vesicles-mirnas released by hypoxic cells are associated to pro-tumorigenic and immunosuppressive microenvironment in lung cancer introduction: extracellular vesicles (ev) containing specific subset of functional biomolecules, such as micrornas (mirnas) are released by all cell types. it has been widely demonstrated that ev-mirnas from cancer cells can manipulate the tumour microenvironment modulating the gene expression of recipient cells. we previously identified a three levels risk classifier (msc) based on plasma-mirnas associated with lung cancer development and prognosis. the aim of this study was to investigate the potential role of ev- mirnas as mediators of pro-tumorigenic features. methods: evs were isolated from plasma of heavysmoker individuals with high (mscpos) or low (mscneg) risk of lung cancer by differential centrifugation method. purity of evs isolated was confirmed by sizing using nta and tem analysis and expression of ev-enriched proteins. mirna levels were analysed by dpcr. in vitro and in vivo analyses were used to assess the biological effect of plasma evs on different recipient cells. results: levels of mirnas in evs correlated with determination of whole plasma ( % of correlation with msc classifier). mscpos-evs stimulated d and d proliferation of non-tumorigenic epithelial cells through c-myc transfer into recipient cells. furthermore, mscpos-evs increased the ability of huvec to form tubular structures compared to mscneg-evs. in vivo co-injection of mscpos-evstreated huvec with a lung cancer cells resulted in an increase of tumour growth compared to mscneg-evs-treated huvec. mir- modulation in evs with mirna mimics or in recipient cells using mirna inhibitors demonstrated that this mirna is implicated in the autocrine proangiogenic modulation of huvec phenotype. mscpos-evs induced m polarization of macrophages both in vitro and in vivo. we demonstrated using synthetic oligonucleotides that mir- is responsible for the immunosuppressive modulation of these cells. regarding the potential origin of evs in mscpos individuals, we observed that hypoxia stimulated the secretion of evs containing c-myc from fibroblasts, mir- -evs from endothelial cells and mir- -evs from granulocytes. summary/conclusion: these data show that plasma evs of high-risk individuals display pro-tumorigenic features, as documented by their ability to induce a pro-angiogenic and immunosuppressive microenvironment suggesting an involvement of evs in lung cancer development. exploration of ev-associated metastatic targets on melanoma cells introduction: cancer cells secrete evs that may harbour metastatic proteins. previous studies have demonstrated the decrease of cd tetraspanin expression in melanoma cells are correlated with enhancing its metastatic potential. however, other proteins, such as cd , cd , met and nrp which are overexpressed in melanoma cells, are also associated with the spread of cancer. in this study, we sought to investigate the expression of metastatic proteins in evs derived from murine melanoma b f lineage. methods: b f cells were cultured in dmem supplemented with % fbs and antibiotics. the cells supernatant were harvested each hours, filtered through . µm and ultracentrifuged at x g for min at ºc to pellet evs. next, size and concentration was determined using nanoparticle tracking analyses technique, and the morphology of evs were analysed by negative-staining transmission electron microscopy (tem). the ev's surface protein were characterized by flow cytometry and protein content was profiled by mass spectrometry. results: our flow cytometry results have shown the presence of tetraspanins markers cd , cd and cd on vesicle´s surface. in addition, our assay demonstrated a diminished expression of cd molecule in comparison to cd and cd . we have profiled melanoma-evs by mass spectrometry, identifying the presence of proteins that may be associated to metastasis, such as cd , cd , met and nrp . summary/conclusion: these preliminary results are consistent with the literature and suggest that melanoma-derived evs harbour proteins, which may contribute to promote tumour metastasis. in our next step, we plan to generate b f lineages harbouring shrna vectors, in order to knockdown gene expression of cd , cd , cd , met and nrp to investigate the metastatic potential in vitro, in comparison to parental cells. we also may use syngeneic mice models to explore metastatic potential of genetically modified b f -derived cells. introduction: overexpression of her occurs in % of breast cancers and confers aggressive behaviour and poorer prognosis. thankfully, a number of drugs such as neratinib have been developed to target her , potentially providing substantial benefit for many patients. nevertheless, it is estimated that up to % of patients with her -overexpressing tumours do not gain benefit, as a result of innate or acquired drug-resistance. this study aimed to investigate if nano-sized membrane-surrounded extracellular vesicles (evs) released from drug-sensitive and drug-resistant cells reflect the her status of their cells of origin and thus have potential as minimallyinvasive biomarkers. methods: evs were isolated from conditioned media (cm) of her -positive cell lines (hcc and skbr ) and their neratinib-resistant counterparts (hcc -nr and skbr -nr) that we developed in our laboratory. evs from cm of a triple-negative breast cancer (tnbc) cell line variant, hs ts(i) , were evaluated as negative control for her . in brief, cm was centrifuged at g, for min x to get rid of any cells. the supernatant was then centrifuged at , g for h at ºc to collect evs. the resulting evs were washed in pbs, centrifuged as before, and resuspended in μl pbs. ev quantities were estimated by nanoparticle tracking analysis (nts). ev lysates were characterised by immunoblots, for established positive and negative ev markers. particle concentration as well as size distribution of evs were measured using the zetaview (particle metrix, germany). surface proteins on single evs were analysed by highresolution flow cytometry (fc), using an amnis imagestreamx mark ii. all data was submitted to ev-track (ev-track id: ev ). results: neratinib-resistant cell line variants were found to have reduced her protein expression compared to their respective drug-sensitive counterparts. neratinib-resistant cell line variants released fewer evs, when normalised per number of secreting cells, compared to their-drug sensitive counterparts. furthermore, evs from drug-sensitive cells carried her , while those from drug-resistant cells lacked her (similar to the evs from the tnbc cells); reflecting the her status of their cells of origin. summary/conclusion: this study indicates that a reduction in her protein expression is a mechanism by which cancer cells manifest resistance to her -targeted drugs (i.e. by making fewer her receptors available on the cells surface to accommodate the drugs activity). furthermore, ev-carried her seems to reflect the her status of their cells of origin. this suggest that analysis of her on evs in the peripheral circulation may help predict response to her -targeted drugs. thus, analysis of evs in appropriate cohorts of patients' specimens is warranted. introduction: rab a, a small gtpase involved in exosome biogenesis by regulating mve docking at the plasma membrane, and its expression level highly correlated with ohsv replication ability in vitro. oncolytic viruses is a newly promising therapeutic agent for cancer treatment. however, more than % of tumours naturally showed highly resistant to oncolytic viruses for unknown reasons. uncovering the underlying mechanisms of resistance to ohsv can offer potential therapeutic targets to enhance ohsv activity to kill tumour cells. in addition, it will give new insights into the identification of therapeutic biomarkers, which can be used to predict patient response to ohsv in clinical. methods: deep-sequencing data showed that lower expression level of rab a is present in ohsv resistant tumour cells compared to that in sensitive tumour cells. then an ohsv resistant mc tumour cell line was established by repeated injections with ohsv in mc tumour-bear mouse model. lastly, it was verified that ohsv resistance is associated with a downregulation of rab a and overexpression of rab a can rescue ohsv replication. results: ) the lower expression level of rab a is shown in ohsv resistant tumour cells compared to that in sensitive tumour cells shows in deep-sequencing data. ) furthermore, we established an ohsv resistant mc tumour cell line by repeated injections with ohsv in mc tumour-bear mouse model. similarly, in ohsv resistant mc cell line, rab a expression was lower than parental mc cells. and the release of exosomes and virus was decreased in ohsv resistant mc cell line. these results were confirmed by rab a sirna knockdown. ) we verified that in ohsv naturally resistant human cancer cell lines, ohsv resistance is associated with a downregulation of rab a and overexpression of rab a can rescue ohsv replication. summary/conclusion: downregulation of rab a expression restricts the efficiency of ohsv replication and the spreading ability of the released progeny virus which also provide rab a as a potential target and biomarker for ohsv cancer therapy. funding: these studies were supported by grants from shenzhen overseas high-calibre peacock foundation kqtd , shenzhen science and innovation commission project grants jcyj , jcyj to shenzhen international institute for biomedical research. inactivation of emilin- by proteolysis and secretion in extracellular vesicles favours melanoma progression and metastasis introduction: studies have demonstrated that melanoma-derived extracellular vesicles (evs) home in distal organs and sentinel lymph nodes favouring metastasis. although lymph node metastases are themselves rarely life threatening, they could be considered as one of the first step of metastasis in many cancer types. therefore, defining the mechanisms involved in lymph node metastasis and pre-metastatic niche formation in lymph nodes could bring the clue to block the metastatic process from the beginning. methods: we have characterized secreted exosomes from a panel of mouse melanoma models representative of low metastatic potential (b -f ), high metastatic potential (b -f ) and lymph node metastasis (b -f r ). we analysed the rna expression in cells and protein content of exosomes derived from mouse melanoma lymph node metastatic models by rna sequencing and mass spectrometry respectively. we validated expression by western-blot, qpcr and immunofluorescence. to define the mechanism of emilin- secretion cells were treated with the ev secretion inhibitor (non-competitive inhibitor of sphingomyelinase), gw . cell viability and cell cycle assays with an overexpression model (b -f -emilin- ) were also performed. in vivo experiments based on subcutaneous and intrafootpad injection for studying the role of this protein during melanoma progression were performed to define the relevance of our findings in vivo. human paraffin samples were analysed for emilin- expression by immunohistochemistry. results: we found a signature of over-expressed genes and hyper-secreted proteins in exosomes related to lymph node metastasis in the b mouse melanoma model. among them, we found that emilin- , a protein with an important function in lymph node physiology, was hyper-secreted in exosomes. interestingly, we found that emilin- is degraded and secreted in exosomes as a mechanism favouring metastasis. further, we found that emilin- has a tumour suppressive-like role regulating negatively cell migration. importantly, our in vivo studies demonstrate that emilin- overexpression reduced primary tumour growth and metastasis in mouse melanoma models. analysis in human melanoma samples showed that cells expressing high levels of emilin- are reduced in metastatic lesions. summary/conclusion: overall, our analysis suggests that the inactivation of emilin- by proteolysis and secretion in exosomes reduce its intrinsic tumour suppressive activities in melanoma favouring tumour progression and metastasis. funding: this work was supported by grants from mineco (saf r), asociación española contra el cáncer, fundación de investigación oncológica fero and mineco-severo ochoa predoctoral program. introduction: the amplification of erbb gene and the consequent overexpression of the encoded protein her have an important role in breast cancer classification at diagnosis and subsequent treatment decision with the anti-her monoclonal antibody trastuzumab. fish and ihc have been used so far to detect erbb gene amplification and her protein overexpression respectively in tissue biopsies. in this context, a major goal for liquid biopsies is to take advantage of the information carried by circulating tumourderived materials (such as extracellular vesicles (evs) and cell free dna (cfdna)) to noninvasively detect erbb gene status in the blood. however, the isolation of diverse tumour-derived materials from a single aliquot of patients' plasma and the accurate detection of cancer biomarkers is still challenging. methods: by adopting a recently published nickelbased evs isolation (nbi) protocol that allows for recovery of cfdna after evs isolation, we generated a high-sensitivity molecular assay to accurately detect erbb amplification and consequent her overexpression on a limited volume of plasma ( . ml) collected from breast cancer patients (stage i, ii and iii) at diagnosis. results: ) we detected erbb amplifications by droplet digital pcr (ddpcr) on the cfdna isolated from the plasma of erbb positive patients; ) we confirmed her overexpression on a subset of patients by setting up an antibody-based affinity reaction designed to detect her protein on the surface of the isolated evs; ) we succeeded in the quantification of her transcripts enclosed within evs by performing ddpcr in samples of patients showing a range of circulating tumourderived material. the specificity and sensitivity of these novel methodological assays were tested on a cohort of healthy individuals (n = ) and on a cohort of her positive (n = ) or her negative breast cancer patients (tnbc; n = ). summary/conclusion: here we report a pilot study on a novel multimodal method for erbb detection from a minimal amount of plasma. this approach integrates information from cfdna with evs-derived rna and proteins analysis. this proof of concept may ultimately translate into relevant clinical applications for disease diagnosis as well as for therapy selection and monitoring of disease progression. introduction: the biological and medical importance of exosomes recognized over the last decade has given rise to a crucial need for the discrimination between true evs and co-purified nanoparticles, such as lipoproteins, protein aggregates or debris. additionally, it is imperative to develop methods to identify and characterize ev sub-populations. considering ev biology and the reliability of labelled biomolecules, we developed both exogenous and endogenous labelling protocols, taking advantage of different dye properties which can target a multitude of compartments. this approach reveals key aspects of ev structure and integrity. methods: nanosized evs/exosomes were purified by size exclusion chromatography (sec) from model cell lines and human plasma. diverse dyes were orthogonally evaluated through different single particle and bulk analysis technologies, such as high-resolution cytometry, nanoparticle tracking analysis and plate fluorimeter. concomitant profiling of specific ev subpopulations was optimized using antibodies (abs) against tetraspanins and cell type specific targets and assessed by single particle analysis and elisa. to assess specificity of labelling protocols we used specific controls such as recombinant rfp expressing vesicles and purified lipoproteins. results: our ev staining protocols allowed for high labelling efficiency and unprecedent ev discrimination, quantification and characterization by combining single particle analysis and bulk measurements in simple matrices (saline buffers) and in complex biofluids (i.e. plasma). different approaches have diverse and complementary advantages (costs, capacity, sensitivity, informative readout) for implementation in research and diagnostic development flows, directly feeding in-house r&d and qc pipeline. summary/conclusion: overall, fluorescent evs are versatile and valuable tracers that can be applied in the optimization of pre-analytical and purification protocols, selection of target biomarkers and diagnostic assay calibration and validation. funding: endevor (por-fse - ) region tuscany and exosomics r&d program. subpopulations of tissue-derived extracellular vesiclesmethodological evaluation for vesicle size measurement introduction: introduction: subpopulations of extracellular vesicle (evs) are commonly classified by their different size, however, the ev size cut off is still under discussion. the aim of the study was to evaluate size range of six ev subpopulations using three methods: electron microscopy (em), nanoparticle tracking analysis (nta) and exoview™. methods: methods: ev subpopulations were isolated from melanoma tissues by a centrifugation based protocol recently established in our lab. large and small evs (levs and sevs) were isolated with differential ultracentrifugation and these were further separated into low and high-density fractions (ld and hd). size of ev subpopulations was then evaluated by: em introduction: small-extracellular vesicles have an important role in cell metabolism and cell-to-cell communication. moreover, sevs when secreted from cells are capable to act as mediators of various neurological diseases. however, sevs show heterogeneity and this may impact their functions. therefore, to characterize sevs at a single-particle level is important to better detail the associated biological activity. in this scenario, we innovatively propose the structured illumination microscopy (sim) as a technique able to complement the non-optical methods (transmission electron microscopy, tem) to analyse single sevs and their markers. methods: human plasma sevs were separated from healthy cognitive control (ctrl), mild cognitive impairment (mci) and demented subjects. the sevscontaining pellet was resuspended in % paraformaldehyde or % glutaraldehyde solutions. for sim, sevsenriched preparations were washed with the blocking solution and incubated with the primary antibody (l cam). the secondary fluolabelled antibody was then added. between the steps with the blocking solution, the primary and secondary antibodies, two ultracentrifuge steps were performed. the image acquisition was done on a nikon sim system with a x oil immersion objective. sevs were imaged with a d-sim acquisition protocol. tem was performed on mesh formvar copper-coated grids. sevs-enriched preparations were incubated first with the blocking solution and then, immunogold-labelled for cd . samples were counterstained with uranyl acetate and observed under a jeol ex electron microscope. data were recorded with a morada digital camera system. participation to the present study was approved by relevant local ethics committee of mendrisio and lugano hospital and written informed consents were obtained from subjects. results: for sim methodology, only vesicles in the range from to nm were detected, as the final resolution achieved was nm. instead, for tem, sevs under nm were identified. none of these methods provided relevant information about possible difference in morphology of the ctrl-, mci or demented subjects-derived sevs. summary/conclusion: even if both methods identified cd or l cam-positive vesicles, sim resolution and the complexity of the protocol represent some disadvantages respect to tem, that may be the first choice screening technique for evs analysis to be then completed by sim for particular tasks. fabrication of nanopore structures via conformal metal-film deposition for ev sensing kwanjung kim a , seung-min han b , soo-hyun kim c and sung-wook nam d luminex corporation, seattle, usa introduction: extracellular vesicles (evs) are membrane-derived structures that include exosomes, microvesicles, and apoptotic bodies. these evsreleased under normal physiological conditions as well as in the pathogenesis of neurological, vascular, haematological, and autoimmune diseaseshave been shown to transfer biological molecules such as protein and rna between cells, potentially transmitting signals. to understand more about these signalling mechanisms, there is a need for detecting and quantifying evs with cargo protein and rna in a reproducible and reliable manner. however, this has been challenging due to the small size of evs (ranging from to nm in diameter), and the lack of specific staining reagents. methods: here, we utilize the amnis® cellstream® flow cytometer, which enables high-throughput flow cytometry with increased sensitivity for detecting small particles. we demonstrate that a charge-coupled device (ccd)-based, time-delay-integration image capturing system can be used to detect and quantify evs and their cargo labelled with exoglow™-protein or exoglow™-rna. results: in this study, we show flow cytometry data quantifying ev samples that have been labelled with cargo markers for proteins and rna. the ev cargo contents along with the appropriate control samples will be shown. summary/conclusion: the ccd based detection of the cellstream flow cytometer has the sensitivity to quantify evs and their cargo. single ev imaging reveals novel ev biomarkers and dna cargo siobhan m. king a , ricardo bastos a and andras miklosi b a oni, oxford, uk; b oni (oxford nanoimaging ltd), oxford, uk introduction: extracellular vesicles (evs) are cellderived membrane-bound particles that range in size from - nm and carry active molecules such as dna, rna and proteins. upon secretion, evs can execute many biological functions such as initiating intracellular communication or regulating immune responses. depending on their origin evs have different characteristics and cargo, making them attractive candidates for early diagnostic and therapeutic applications. however due to their small size and heterogeneity, direct visualization and characterization of the surface markers expressed remains a challenge since these vesicles are below the resolution limit of standard light microscopy. methods: here, we describe a method that provides size analysis of single-evs, which falls below the diffraction limit of light. this was done with purified evs, immunostained using fluorescently labelled primary antibodies raised against ev surface markers (cd , cd , cd ), specific cargo such as dna and probed for tissue specific cargo. characterization of the molecular content and structural properties of surfaceimmobilized evs was performed using single-molecule localisation microscopy (smlm) on the nanoimager platform. results: multicolour smlm was used to detect up to three ev biomarkers showing successful characterization of the molecular signature for different ev subpopulations. the distribution of novel components on urinary evs were visualized for the first time using this approach. in addition, smlm revealed the presence of dna on both the surface and also as a cargo inside evs isolated from tumour cell culture media, which was validated using complementary biochemical characterization. summary/conclusion: smlm is a powerful technique for single-ev analysis and characterization. visualization of single-urinary evs enabled accurate sizing and further insights into novel components expressed on the subpopulation's membrane surface. together, the data demonstrates that the quantitative abilities of smlm can significantly enhance our understanding of evs, as structure, phenotypes, and cargoes can now be successfully resolved. introduction: working skeletal muscle is a common site for injury due to unaccustomed exercise with or without underlying pathology. direct analysis of skm injury requires invasive tissue biopsies. circulating extracellular vesicles (evs) are abundant in blood and have been shown to be enriched in microrna; profiles of which may reflect the state of tissues. evs may therefore serve as a non-invasive indicator of muscle injury and regenerative processes in vivo. methods: two consecutive bouts of muscle-damaging exercise (plyometric jumping and downhill running) were performed by healthy male volunteers. serum creatine kinase (ck) and plasma evs were analysed at baseline, and hr post-exercise. perceived muscle pain (pmp) was assessed at and hr post-exercise. large evs were isolated using a g centrifugation step, and small evs were isolated using qev columns. ev-enriched isolates were visualized using tem, and size and numbers were quantified using nta. based on nta results the highest particle fractions ( - ) were pooled for rna analysis. qpcr was done on plasma, large evs and small evs. a group of muscle and immune cell-important mirs were analysed by means of normalization to an exogenous control. results: ck and pmp increased post-exercise, providing evidence for muscle damage. tem revealed an abundant and heterogeneous pool of evs. a concomitant abundance of evs was seen with nta (mean = . x particles/ml plasma). mean ev diameters were ± nm across all timepoints. no change in ev size nor number was seen over time, however, mir- decreased at hr when compared to hr in the small ev isolate only. plasma displayed an immediate increase in myomirs- and − at hr, which returned to baseline at hr. in contrast, myomirs- b and remained elevated over the hr period. myomir- b and , as well as immune-mirs, did not change in evs or plasma as a result of the intervention. summary/conclusion: the decrease in mir- in small evs at hr is consistent with previous data. no decrease in mir- in large evs suggests specific packaging and hence a specific response to the muscle damage in small evs. more changes occurred in plasma myomirs suggesting less specific passive leakage into circulation from damaged cell membranes. funding: south african national research foundation pulsed electromagnetic fields potentiate the paracrine function of mesenchymal stem cells for cartilage regeneration yingnan wu a , dinesh parate a , eng hin lee a , zheng yang a and alfredo franco-obregón b a national university of singpaore tissue engineering program, yll school of medicine., national university of singapore, singapore, singapore; b biolonic currents electromagnetic pulsing systems laboratory, biceps, national university of singapore, singapore, singapore introduction: the mesenchymal stem cell (msc) secretome, via the combined actions of its plethora of biologically active factors, is capable of orchestrating the regenerative responses of numerous tissues by both eliciting and amplifying biological responses within recipient cells. mscs are "environmentally-responsive" to local microenvironmental cues and biophysical perturbations, influencing their differentiation as well as secretion of bioactive factors. we have previously shown that exposures of mscs to pulsed electromagnetic fields (pemfs) enhanced msc chondrogenesis. here, we investigate the influence of pemf exposure over the paracrine activity of mscs and its significance to cartilage regeneration. also, the subsequent extracellular vesicles analysis and isolation are processed for the understanding of how the pemfs affect stem cell evs and consequent differentiation induction. methods: conditioned medium (cm) was generated from mscs subjected to either d or d culturing platforms, with or without pemf exposure. the paracrine effects of cm over chondrocytes and msc chondrogenesis, migration and proliferation, as well as the inflammatory status and induced apoptosis in chondrocytes and mscs was assessed. the cms which have significant effects during chondrogenesis will be analysed by protein and mirna studies. results: we show that the benefits of magnetic field stimulation over msc-derived chondrogenesis can be partly ascribed to its ability to modulate the msc secretome. mscs cultured on either d or d platforms displayed distinct magnetic sensitivities, whereby mscs grown in d or d platforms responded most favourably to pemf exposure at mt and mt amplitudes, respectively. ten minutes of pemf exposure was sufficient to substantially augment the chondrogenic potential of msc-derived cm generated from either platform. furthermore, pemf-induced cm was capable of enhancing the migration of chondrocytes and mscs as well as mitigating cellular inflammation and apoptosis. the cms protein results in the significant promotion chondrogenesis condition showed an increase in proliferation and anti-inflammatory cytokines. summary/conclusion: the findings reported here demonstrate that pemf-stimulation is capable of modulating the paracrine function of mscs for the enhancement and re-establishment of cartilage regeneration in states of cellular stress. the pemf-induced modulation of the msc-derived paracrine function for directed biological responses in recipient cells or tissues has broad clinical and practical ramifications with high translational value across numerous clinical application. effects of extracellular vesicles from blood derivatives on osteoarthritic chondrocytes within an inflammation model introduction: the degenerative disease osteoarthritis (oa) is one of the leading causes of disability especially of elderly people. besides various treatment options depending on the severity of the cartilage degradation, the application of blood derived products such as platelet rich plasma (prp) are getting more and more popular in clinical practice due to its high concentration of platelets and the perceived high growth factor levels. drawbacks of using prp include high donor variability, discrepancies among preparation protocols and the presence of cells (platelets, leukocytes) which can evoke cellular processes, especially inflammation, when injected into the diseased tissue. one possibility is to isolate only extracellular vesicles (evs) from blood derivatives to overcome these problems. in the current study the effects of evs isolated from blood derivatives on oa chondrocytes within an inflammation model was investigated. methods: cd positive primary monocytes were isolated from citrate anticoagulated whole blood by magnetic bead sorting. monocytes were differentiated into resting m macrophages and activated into m macrophages according to published protocols. elisa measurements verified successful differentiation and activation as il β and tnfα levels increased. as control, thp monocytes were used. patient-derived oa chondrocytes were grown in well plates and co-cultivated with activated m macrophages which were seeded into thincerts and added to the wells representing the inflammation model. furthermore, cells were treated for hours with media containing fcs, ev depleted fcs or evs isolated from prp or hypact serum. results: successful differentiation and activation of monocytes (thp and primary monocytes) into m macrophages was demonstrated by elevated levels of the inflammatory cytokines il β and tnfα. within the inflammation model (co-culture of oa chondrocytes with m macrophages), addition of evs isolated from prp or hypact serum resulted in decreased secretion levels of il β and tnfα compared to media supplemented with either fcs or ev depleted fcs. summary/conclusion: taken together, evs from blood derived products might be chondroprotective and anti-inflammatory mediators which protect cartilage from being degraded during oa. funding: the work was jointly supported by the european fund for regional development (efre) and the fund for economy and tourism of lower austria, grant number wst -f- / - . α, (oh) d regulates growth cartilage matrix vesicle micrornas niels asmussen, michael mcclure, zhao lin, zvi schwartz and barbara boyan virginia commonwealth university, richmond, usa introduction: matrix vesicles (mvs) are small ( - nm in diameter) lipid bound extracellular organelles isolated from calcifying tissues including the growth zone (gc) of growth plate cartilage. α, (oh) vitamin d ( α, ) is a regulator of gc chondrocytes and the mvs they produce. these mvs are key players in the mineralization process and are selectively enriched with enzymes and growth factors. we found that mvs are also selectively enriched with micrornas (mir), including mir- , mir- and mir- . the aim of this study was to determine the regulatory role of α, in the packaging of mirna in mvs by gc cells. methods: gc cells were isolated by enzymatic digestion from costochondral gc cartilage harvested from wk-old male sprague dawley rats (iacuc approved). confluent fourth passage gc cell cultures were treated with - m α, or vehicle for h. media were removed, cell monolayers digested with trypsin and cells and mvs isolated by differential ultracentrifugation. rna was precipitated from cells and mvs. small rnaseq data were trimmed, aligned and counted before undergoing differential expression analysis. experimental groups had an n = per variable. significant differences (p < . ) were determined using r v . . . results: α, treatment altered expression of mv mirs compared to control mvs, whereas cell mirnas were differentially expressed. . % of significantly up or down regulated mir found in mvs overlapped between α, and vehicle groups with the remaining being uniquely differentially expressed. α, increased mv mir- and decreased mir- - p two mirs known to regulate osteoblast proliferation ( increases, decreases). summary/conclusion: α, regulates gc chondrocyte and mv behaviour and this study demonstrates that it also impacts the mir packaging within mvs. mir discovered in mvs have been demonstrated to impact chondrocyte behaviour and the present study indicates that α, regulates the growth plate through mir delivered by mvs. introduction: increasing evidence has proposed extracellular vesicles (evs) as mediators of many of the therapeutic features of mesenchymal stromal cells (msc) that have been widely studied in clinical trials over the last years. these evs have been recognized as nanocarriers of important biological information, which play a central role in cell-to-cell communication. in this context, evs can be used as an alternative to a cell-based therapy, with reduced risks. the present work aimed to evaluate the impact of different culture conditions on the msc-derived evs molecular composition through fourier-transform infrared (ftir) spectroscopy. methods: evs derived from msc from different sources, expanded in two different culture media ((xenogeneic -free (xf) vs serum-containing medium (fbs)) were characterized by ftir spectroscopy, a highly sensitive, fast and high throughput technique. moreover, principal component analysis (pca) of preprocessed ftir spectra of purified evs was conducted, enabling the evaluation of the replica variance of the evs chemical fingerprint in a reduced dimensionality space. for that, different pre-processing methods were studied as baseline correction, standard normal variation and first and second derivative. results: evs secreted by mscs cultured with serumcontaining medium presented a more homogenous chemical fingerprint than evs obtained with xf medium. the regression vector of the pca enabled to identified relevant spectral bands that enabled the separation of samples in the score-plot of the previous analysis. ratios between these spectral bands were determined, since these attenuate artefacts due to cell quantity and baseline distortions underneath each band. statistically inference analysis of the ratios of spectral bands were conducted, by comparing the equality of the means of the populations using appropriate hypothesis tests and considering the significance level of %. it was possible to define ratios of spectral bands, that can be used as biomarkers, enabling the discrimination of evs chemical fingerprint in function of the culture medium used for msc expansion and the msc donor. summary/conclusion: this work is a step forward into understanding how different culture conditions affect msc-derived evs characteristics. funding: fundação para a ciência e tecnologia (ptdc/equ-equ/ / , uidb/ / ). performance qualification for microflow cytometers: understanding technical limitations to improve your research desmond pink a , michael wong a , diana pham a , renjith pillai a , leanne stifanyk b , sylvia koch b , rebecca hiebert a , oliver kenyon c and john lewis a a nanostics, edmonton, canada; b dynalife, edmonton, canada; c apogeeflow systems, hemel hempstead, uk introduction: as microflow cytometry and other techniques mature as validated modalities for analysing extracellular vesicles (ev), there has been a concerted effort to improve reproducibility . in order for this reproducibility to occur there has to be a critical understanding of advantages and limitations for each technology. for microflow cytometry, several instruments are available to analyse evs. each platform has different limitations as well as advantages over other platforms. to provide the optimal data for your specific research, it is critical to understand the limitations of your platform. to accurately define these limitations, a performance qualification (pq) of your instrument should be undertaken. methods: an apogee a platform was used in these experiments. experiments were designed with expected ranges and cut-offs for acceptance criteria.initial tests included autosampling of a well plate with either single or double aspiration, single sample reproducibility and linearity proportional to flow rate. other experiments designed to show machine performance included minimal time to achieve valid data, sample volume required for double aspiration, determination of coincidence; detection sensitivity using a spiked sample; flow rate stability for extended periods ( - minutes) . tests should also be performed to determine carryover at a range of sample concentrations. if present, the means to remove contaminating samples should be determined. any performance tests should be applicable to any instrument in the field. results: auto-sampling helped demonstrate consistent data; reproducibility of total events and biomarkers was - % c.v. detected bead concentrations were linear with flow rates between . and . ul/min. double well aspirations provided similar data with aspirations between - ul. valid data was achieved for a low abundant target (~ - events/ul) after only s, < %c.v. detection sensitivity was determined to be~ / , . carryover ranges were determined in the presence of nominal unstained serum. an optimal number of machine washes was determined. some membrane stains, such as cell mask and cfse require much more rigorous cleaning to remove stain carryover. summary/conclusion: to improve data reproducibility, performance qualification of any instrument is key. operational limitations help define optimal performance parameters of any technology. understanding the types of experiments to perform for your particular type of characterization technology depends on the requirements you set for your research. a good performance test should be applicable to any related instrument in the field. funding: funding provided by nanostics, the alberta cancer foundation, and alberta innovates. introduction: cancer cells release more evs than normal cells and evs secreted from tumour cells can promote tumour progression, survival, invasion and angiogenesis. the ev cargo may mirror the altered molecular state of the cell of origin. therefore, evs have potential for the development of non-invasive markers for early detection of cancers. evs and their cargo also have the potential to be multiplexed with other molecular markers or screening modalities (e.g., imaging) to develop integrated molecular-based computational tools for the early detection of cancer. one challenge with using evs as a biomarker is the lack of robust and reproducible methods for the isolation of a pure vesicular population. there is a lack of clear consensus for an optimal method of isolation of a pure ev population that is devoid of contamination with similar-sized vesicles of different origins. there is also a lack of standards to ensure rigour reproducibility. methods: the current funding opportunity announcement (foa), par - , is promoting research on the isolation and characterization of extracellular vesicles (evs) and their cargo for the discovery of biomarkers to predict cancer and cancer risk. results: the previous cycle of this foa, par - / , successfully funded r and r grants. these awards are focused on proteomics profiling of evs, effect of methodological and biological variability, asymmetric-flow field-flow technology, therapeutic monitoring, lss and sers lab on a chip optical spectroscopic, evs in obesity-driven hepatocellular carcinoma, nanoscale structure and bio-molecular heterogeneity, urinary ev dna, and ev markers in paediatric cancers. progress from these awardees have shown separation of two discernible exosome subpopulations and identified a distinct nanoparticle, the exomere (nature cell biology, ); and have shown that large-evs contain the entire genome of the cell of origin, including cancer-specific genomic alterations (journal of extracellular vesicles, ). protocols that critically evaluate and refine the existing methodologies to improve utilization of evs in clinical use have been shared (nature protocols, ). summary/conclusion: drs. sudhir srivastava and matthew young are the programme directors for the par which began accepting applications on january . this and other ev funding opportunities will be discussed. funding: this is a funding opportunity announcement offered by the national cancer institute. introduction: traumatic brain injury (tbi) is characterized by diverse primary mechanisms of injury that lead to the development of secondary pathological cascades that drive neurological deficit post-tbi. inability to separate patients based on the presence of these different endophenotypes represents a major challenge for diagnosis and treatment of tbi. extracellular vesicles including exosomes isolated from patient plasma have emerged as promising potential biomarkers for tbi due to their ability to cross the bbb into systemic circulation with molecular cargo intact for analysis. we have developed a novel microfluidic platform for rapid isolation of brain-derived evs providing a tool with which the biochemical state of neurons and glia can be directly assessed post-tbi. we used the ultra-sensitive, single molecule array (simoa) to quantify concentrations of protein biomarkers from the plasma and brain derived evs from mild tbi patients and controls. by combining multiple protein biomarkers, we could discriminate mtbi patients from controls in both the training and the blinded test set. building on this work, we are also characterizing single ev heterogeneity of neuron derived evs by developing novel droplet based digital assay for single ev quantification at ultra-low concentration. droplet based assay for single ev analysis would potentially be very informative for early disease diagnosis and therapy decision. methods: our microfluidic platform for ev isolation consists of tracked-etched membranes with millions of nanopores ( nm), coated with a magnetic film (nife) to precisely capture immunomagnetically labelled brain-specific evs from plasma. single molecule array (simoa) was used to quantify concentrations of the protein biomarkers (tau, uchl- , nfl, gfap, il , il , and tnf) in the plasma and brainderived exosomes of mild tbi (mtbi) patients and controls. to identify single ev, we applied droplet based enzyme-linked immunosorbent assay and encoded the fluorescent signal for single ev quantification within parallelized microfluidic platform. results: we report that concentrations of plasma and exosome gfap, nfl, and uchl were elevated in mtbi patients compared to controls (p < . ), and that each of these biomarkers are uncorrelated with one another. discrimination of mtbi patients from controls was most accurate when machine learning algorithms on the panel of biomarkers. specifically, combining plasma nfl, gfap, il and tnf-with tau from glur + evs showed % accuracy with % sensitivity and % specificity. summary/conclusion: this data suggests that neuronderived exosomes contain information that characterizes the injured and recovering brain. it also suggests that analysis of a panel of biomarkers from a combination of both blood and exosomal compartments could lead to more accurate diagnosis of mtbis. ps . = op . l cam is not associated with extracellular vesicles in cerebrospinal fluid or plasma maia norman, dmitry ter-ovanesyan, wendy trieu and david walt wyss institute, boston, usa introduction: neurons in living psychiatric and neurological patients are inaccessible for cell type specific analysis of rna and protein. our understanding of these diseases instead relies upon imperfect sources of biochemical information such as post-mortem brain tissue analysis and animal models. furthermore, there is a paucity of biochemical assays available to diagnose and manage brain diseases. extracellular vesicles (evs) present an opportunity to noninvasively sample the contents of neurons in cerebrospinal fluid (csf) and plasma. in order to isolate neuron-derived evs (ndevs), a cell type specific transmembrane protein is necessary for immunocapture. l cam, a protein abundant on the surface of neurons, has been used extensively in the literature for ndev isolation. however, l cam exists in humans in several isoforms without a transmembrane domain, and as such it can be secreted as a free protein. additionally, the ectodomain of l cam can be cleaved off of the cell surface in physiological processes. it remains to be demonstrated whether the l cam found in csf and plasma is ev associated, or if it is instead a spliced or cleaved isoform behaving as a free protein. methods: using single molecule arrays (simoa), a digital form of elisa, as well as western blotting, we quantify ev markers (cd , cd and cd ) as well as l cam and albumin. we use these assays to determine in which fractions of size exclusion chromatography (sec) and density gradient the l cam appears. we also immunocapture l cam from csf and plasma and perform western blots for the internal and external domains of l cam. results: simoa and western blot analysis of sec and density gradient fractions demonstrated that while the ev markers peaked all together, l cam eluted in the free protein fractions along with albumin in both csf and plasma. when immunoprecipitation was performed, western blotting revealed different isoforms of l cam in csf and plasma. summary/conclusion: our data utilize a multitude of distinct techniques that converge to demonstrate that l cam is not associated with evs in csf or plasma. furthermore, our data suggest that the isoforms present in csf and plasma are distinct, which indicates that the l cam in plasma is likely not coming from the brain. this data call into question the utility of l cam as a ndev marker and point to the need to find novel candidates for immunoprecipitation of ndevs. introduction: in parkinson's disease (pd), α-synuclein (α-syn) aggregates known as lewy bodies (lb) are present in both the central and peripheral nervous system. furthermore, data showing that α-syn can spread from pd patients to transplanted tissue has led to a new theory postulating that pathological forms of α-syn can drive disease by "infecting" healthy cells and corrupting normal proteins. the exact routes and mechanisms involved in such spreading are yet to be fully understood but it is known that α-syn can be secreted from cells and transported via extracellular vesicles (ev). ev derived from erythrocytes (eev) are of particular interest in this regard as they have been shown to contain α-syn. methods: we first optimized a protocol for the isolation of fluorescently labelled human eev. the capacity of these eev to cross the blood-brain barrier (bbb) was then evaluated in vitro using a boyden chamber composed of primary human brain endothelial cells. next, eev were added to a more complex and physiologically relevant d human bbb model including ipsc-derived brain microvascular endothelial cells. in both in vitro protocols, flow cytometry was performed on media collect from each compartment to determine the number of eev. immunofluorescence was performed to assess the localization of fluorophore tagged eev. we are also using an in vivo paradigm for the extraction and testing of eev spread and an in situ cerebral perfusion (isbp) model in wt mice to investigate if and how eev cross the bbb using confocal microscopy. results: in both in vitro models, flow cytometry analyses showed that fluorescently tagged eev added to the luminal side traversed the endothelial cell barrier. confocal analysis revealed that some eev could also be found within endothelial cells themselves. ongoing experiments are being conducted in our newly developed d bbb to further confirm these results. our preliminary in vivo experiments showed that fluorescently labelled beads, similar in size to eev, used in the isbp experiments are detectable in the brain parenchyma of injected wt mice using confocal microscopy. preliminary work also includes isbp injections of eev in -month-old wt mice, (n = /groups) derived from pd patients (at different stage of the disease) and a healthy individual as a control. summary/conclusion: our preliminary data suggests that eev can indeed move across the bbb in both in vitro and in vivo experimental setups. ongoing experiments will determine the dynamics and processes involved in this transport and whether eev can precipitate and/or exacerbate disease-related features. introduction: neuroblastoma accounts for % of childhood cancer mortality. amplification of the oncogene n-myc is a well-established poor prognostic marker for neuroblastoma. whilst n-myc amplification status strongly correlates with higher tumour aggression and resistance to treatment, the role of n-myc in the aggressiveness of the disease is poorly understood. exosomes are released by many cell types including cancer cells and are implicated as key mediators in cell-cell communication via the transfer of molecular cargo. hence, characterising the exosomal protein components from n-myc amplified and nonamplified neuroblastoma cells will improve our understanding on their role in the progression of neuroblastoma. methods: in this study, comparative proteomic analysis, nanoparticle tracking analysis, transmission electron microscopy, rnai-based knockdown, migration and cellular survivability assays were performed to understand the role of exosomes isolated from cells with varying n-myc amplification status. results: label-free quantitative proteomic profiling revealed proteins that are differentially abundant in exosomes released by the n-myc amplified and nonamplified neuroblastoma cells. gene ontology-based analysis highlighted the enrichment of proteins involved in cell communication and signal transduction in n-myc amplified exosomes. treatment of less aggressive sh-sy y cells with n-myc amplified sk-n-be cell-derived exosomes increased the migratory potential, colony forming abilities and conferred resistance to doxorubicin induced apoptosis. incubation of exosomes from n-myc knocked down sk-n-be cells abolished the transfer of resistance to doxorubicin induced apoptosis. summary/conclusion: these findings suggest that exosomes could play a pivotal role in n-myc-driven aggressive neuroblastoma and transfer of chemoresistance between cells. ps . = op . results: murine ctl evs were broadly divided into two populations that were eluted at low salt (l-s: . m- . m nacl) and high salt (h-s: . m- . m nacl) concentrations. l-s ctl evs were abundant in late endosome-related proteins, integrins, rabs, and effective mirnas, indicating exosome characteristics, and had biological activity for preventing tumour metastasis after depletion of tumoural mesenchymal cell populations by intratumoral administration (see seo et al., nat. commun. : , ) . contrary, h-s ctl evs were rich in dna, core histones, ribosomal proteins, cytoskeleton proteins, and housekeeping proteins, considering microvesicles and apoptotic bodies, and easily phagocytosed by a kupffer cell line (kup : kitani et al., results immunol. : - . ). in addition, there were noticeable differences between ls and h-s ctl evs in the negative zeta potential width and membrane glycan structure. summary/conclusion: thus, ion exchange can be an optimal mass fractionation method for discriminating bioactive exosomes from cargos for nucleic acids in evs. funding: cryotem was conducted in nara institute of science and technology (naist), supported by nanotechnology platform program (synthesis of molecules and materials: # ) of the ministry of education, culture, sports, science and technology (mext). this work was supported by grants from the japan agency for medical research and development (translational research network program (nagoya univ. seeds a )) and the japan science and technology agency (crest [jpmjcr h ]). clic is essential for breast cancer metastatic competence and predicts disease outcome introduction: metastatic breast cancer is a consequence of complex interactions between cancer cells and the host. clic , a member of a conserved gene family in the glutathione-s-transferase superfamily, mediates crosstalk between tumour and host in breast cancer. tcga and metabric data indicated that elevated clic expression was associated with breast cancers from young women, those with poor prognosis, and those with early stage metastatic disease. methods: since bulk tumour analysis does not distinguish between cancer and host stromal cells, we used genetic modifications of established syngeneic breast cancer mouse models to evaluate the contributions of clic in the host or tumour cells to develop metastases. results: experimentally, the essential clic host contributions for metastatic competence were related to circulating levels of pro-metastatic soluble factors, neoangiogenesis, tumour cell attachment to lung tissue, myofibroblast differentiation, and leukocyte migration. clic was detected as cargo in circulating extracellular vesicles (evs) from breast cancer patients. similarly, circulating evs from tumour-bearing mice have abundant clic in comparison to those from mice bearing tumours that lack clic . tumour cells released evs that induced myofibroblast conversion of wildtype but not clic ablated lung fibroblasts. summary/conclusion: these results illuminate clic expression as a prognostic marker for breast cancer patients, and experimentally, clic is a critical host factor for metastatic competence and potential target within host tissues for anti-metastatic therapy. funding: this work was supported by the intramural program of the national cancer institute under project zia bc . the application of flow cytometry in an ev-based liquid biopsy for the detection of cancer multidrug resistance in myeloma gabriele de rubis a , krishna sunkara a , sabna rajeev krishnan and mary bebawy b a laboratory of cancer cell biology and therapeutics, discipline of pharmacy, graduate school of health, the university of technology sydney, australia, sydney, australia; b the university of technology sydney, sydney, australia introduction: multiple myeloma (mm) is an incurable cancer of bone-marrow plasma cells. it is characterized by unpredictable and highly variable therapeutic response and poor survival, attributed to the development of multidrug resistance (mdr) to chemotherapy. presently, no clinical procedures allow for a continuous, minimally invasive monitoring of mdr. we identified unique extracellular vesicle (ev) populations in the blood of myeloma patients, which serve as biomarkers of disease evolution and mdr to combination chemotherapy. we describe approaches used to optimise the use of flow cytometry (fcm) for ev summary/conclusion: although further investigation is required, our results potentially promise an effective and inexpensive priming agent (i.e., ethanol) for the production of anti-inflammatory msc-evs. this, combined with the significant increase in yield via d dynamic culture, presents practical solutions to both ev manufacturing scalability and potency issues. donor source affects potency of mesenchymal stem cell-derived extracellular vesicles introduction: mesenchymal stem cell (msc) therapies have been heavily investigated for their utility in applications such as wound healing and regenerative medicine due to their angiogenic, immunomodulatory and anti-apoptotic effects. recently, msc-derived extracellular vesicles (evs) have been implicated as primary effectors in msc-based therapies via protein and nucleic acid cargo transfer to patient cells. msc evs represent a superior alternative to msc-based therapies, as they lack the ability to replicate and are much smaller in size, circumventing related safety concerns such as immunogenicity, teratoma formation and blood vessel occlusion. however, a key drawback with msc therapies in general is their variable therapeutic potency, which is dependent on donor source. as a cell derived therapeutic, this crucial limitation is hypothesized to exist in msc evs as well. here, we demonstrate the varying bioactivities of isolated msc evs from differing donors and tissue sources. methods: six separate msc lines were obtained from different donors, with three msc lines derived from donor adipose tissue, and the other three from the bone marrow of separate individuals. evs were isolated from each msc line at passage via differential centrifugation and ultrafiltration. these isolated msc evs were then characterized for size/concentration via nanoparticle tracking analysis, and ev markers (tsg , alix, cd ) via western blot. pro-vascularization capacities of msc evs were determined by a gap closure assay using human umbilical cord vein endothelial cells (huvecs). results: characterization of msc evs revealed similar sizes and ev marker expression across donor groups, frontiers in chemistry, submitted funding: this work was funded by the momentum programme (lp - ), by the national competitiveness and excellence program catalan institution for research and advanced studies (icrea) proteomic profiling of retinoblastoma-derived exosomes reveals potential biomarkers of vitreous seeding angel montero carcaboso g , andrea petretto b , franco locatelli a and angela di giannatale a a department of paediatric haematology/oncology and cell and gene therapy, irccs, ospedale pediatrico bambino gesù ps : separation and concentration a laboratory of clinical biophysics, faculty of health sciences ps : diverse ev biomarkers chair: pia siljander -faculty of biological and environmental sciences urinary evs were isolated using low vacuum filtration method followed by ultracentrifugation. raman spectra of urinary evs were recorded using a renishaw invia raman spectrometer. data analysis was performed using principal component analysis (pca) and hierarchical cluster analysis (hca). the size distribution and morphology of evs were analysed by transmission electron microscopy and nanoparticle tracking analysis methods. results: average raman spectra obtained for urinary evs from studied groups showed differences in intensities of specific bands in the region of - cm- . we found significant correlations between mean area under curve (auc) calculated for raman bands (phenylalanine, dna, proteins, lipids and amide i) and selected clinical parameters such as: egfr, serum creatinine, glucose, urine creatinine. chemometric methods showed spectral pattern responsible for separation between studied groups. nta measurements visualized evs with size of . ± . nm. summary/conclusion: our results showed that characteristic raman spectra of urinary evs are promising candidates for new, non-invasive biomarkers for dkd isolation of circulating extracellular vesicles and cfdna allows for erbb detection in a single aliquot of breast cancer patients plasma michela notarangelo a , mattia barbareschi b , antonella ferro c , orazio caffo c , vito d'agostino a and francesca demichelis d a department of cellular results: results: tissue-derived large and small evs showed difference in size (mean nm vs nm) when examined by em, whereas nta and exoview™ were unable to show a clear difference between the populations (nta: mean . nm vs nm nta can only detected vesicles above nm and exoview™ only measures vesicles between - nm. of the three different methods, em analysis of single vesicles visualized in a significant number of micrographs was the only one able to distinguish ev subpopulations by size. funding: funding: swedish research council knut och alice wallenberg foundation imaging of human plasma-derived small-extracellular vesicles using transmission electron microscopy and structured illumination microscopy mitovesicles: a new extracellular vesicle of mitochondrial origin altered in ageing and neurodegeneration alldred b , chris goulbourne b , hediye erdjument-bromage d , monika pawlik b , mitsuo saito e , mariko saito f , stephen d. ginsberg b an in vitro and in vivo perspective on the role of erythrocyte-derived extracellular vesicles in parkinson's disease pathology frédéric calon c , Éric boilard f and francesca cicchetti b a centre de recherche du chu de québec and faculté de médecine, département de psychiatrie & neurosciences département de microbiologie-infectiologie et d'immunologie evidences on microalgal extracellular vesicles: a morphological assessment antonella bongiovanni i , ales iglič j and veronika kralj-iglič j a laboratory of clinical biophysics, faculty of health sciences a faculty of dentistry, national university of singapore, singapore, singapore, singapore; b institute of medical biology, agency for science, technology and research, singapore, singapore, singapore; c exosome of cancer-associated fibroblast induce anti-cancer drug-resistance of nsclc so-young kim a and yeon-ju lee b a chonnam national university hwasun hospital biomedical research institute, gwangju, republic of korea; b chonnam national university hwasun hospital biomedical research institute, gwangju, republic of korea introduction: the understanding of interaction mechanisms between cancer cells and the tumour microenvironment (tme) is crucial for developing therapies that can arrest tumour progression and metastasis. cafs are the major constituent of the tme in many cancers. recent studies indicate that exosomes harbour the potential to regulate proliferation, survival and immune status in recipient cells. most of the current studies are focused on cancer cell secreted exosomes; and little is known about cafderived exosomes and their influence on cancer cells. methods: nsclc cell lines (pc gr) and mrc (normal fibroblast cells) were grown in culture with exosome-free fbs. cutured media was filtrated by tangential flow filtration systems. exosomes in supernatant were isolated with the exoquick-tc™ system. considering the important role of cell extrinsic factors on cell growth and survival, we assessed whether factors contained in the mpa exosome could affect proliferation and survival of recipient cancer cells. cells were then treated with μm osimertinib or pbs for days prior to cell quantification of live cells.to investigate mechanisms of resistance to osimertinib mediated by ma or mpa-exosome in nsclc cell lines, we test cell viability by crystal violet assay in trametinib or osimertinib treated after pretreated ma or mpa-exosome, pc gr during days. we will investigate how mpa-exsomes activate erk signalling pathway in pc gr cells to induce antitumor effects by western blot. results: mpa exosome increased proliferation of pc gr cells by more than % compared to control pbs. pc gr cells grown in mpa-exosome and subsequently treated with osimertinib showed a significant increase in cell survival compared to pc gr cells grown in ma-exosome. osimertinib is used to treat egfr-mutant non-small cell lung cancer (nsclc) with tyrosine kinase inhibitor resistance mediated by the egfr t m mutation.these data show that "mrc -pc gr-crosstalk factors" affect proliferation and adaptive drug resistance of cancer cells. mpa-exosome mediates erk signalling activation and attenuated after treatment of um osimertinib. summary/conclusion: cafs support cancer growth and invasion. co-cultured nsclc with mrc lung fibroblast increased cell viability and exosomal mir- through the tgf-ß pathway in treatment osimertinib. exosomal mir- up-regulation in cocultured nsclc with mrc- induced drug resistance to drug-induced apoptosis. thus, exosomal mir- expression in co-cultured nsclc with mrc may support drug tolerance persister cells. introduction: neural stem cell (nsc) therapy has shown promise for brain repair after injury or disease mostly through bystander effects. nevertheless, the translation of nscs derived from human induced pluripotent stem cells (hipscs) to the clinic remain constrained due to safety issues, which include immunogenic risks, tumorigenesis potential, and incomplete differentiation. a way to avoid these issues is by using extracellular vesicles (evs) generated from nscs, as nsc-evs likely have similar neuroprotective properties as nscs and are amenable for non-invasive administration as an autologous or allogeneic off-the-shelf product. however, this would require reliable purification and characterization processes, and testing of evs for composition and biological properties. methods: we generated evs from hipsc-derived nscs using a combination of ion-exchange chromatography (iex) and size-exclusion chromatography (sec) and investigated their composition through small rna sequencing and proteomics. we also performed in vitro and in vivo experiments to determine their biological and functional properties. results: iex and sec facilitated purification of hipsc-nsc evs nearly to homogeneity, which expressed ev markers such as cd , cd , cd , and alix with a mean size of nm. small rna sequencing revealed enrichment of mirnas related to different neuroprotective signalling pathways and diverse metabolic functions consistent with their role in cell-cell communication. the proteomic analysis identified > , proteins, including ev markers and many other proteins involved in central nervous system function and cellular processes. the evs also displayed antiinflammatory activity in an in vitro mouse macrophage assay. intranasal (in) administration of nsc-evs resulted in their rapid incorporation by neurons, microglia, and astrocytes in virtually all regions of the brain. functionally, in administration of nsc-evs reduced inflammatory activity in the brain in a model of status epilepticus, and increased hippocampal neurogenesis in the adult brain. summary/conclusion: biologically active evs with antiinflammatory and neurogenic properties could be purified and harvested from hipsc-nscs. such evs also contain many mirnas and proteins that are of interest for brain repair after injury or disease. funding: supported by a grant from the national institute of neurological disorders and stroke ( r ns - to a.k.s.) introduction: extracellular vesicles (evs) generated from human bone marrow-derived mesenchymal stem cells (hmscs) display anti-inflammatory and neuroprotective properties. our recent study has shown that intranasally (in) administered hmsc-evs incorporate into significant percentages of neurons and microglia in virtually all regions of the intact as well as the injured forebrain within hours (kodali et al., int j mol sci, ) . in this study, using a rat model, we investigated the efficacy of a low dose of hmsc-evs administered intranasally for alleviating the abnormal plasticity of newly born neurons and the activation of microglia after se. methods: approximately billion evs were dispensed bilaterally into both nostrils of young f rats that experienced two hours of kainate-induced se. animals were euthanized seven days after se, and brain tissue sections were processed for immunohistochemical staining of neun (a neuronal marker), dcx (a marker of newly born neurons), iba- (a microglial marker), and parvalbumin (pv) and reelin (markers of subclasses of interneurons). in addition, activated microglia were quantified using iba- and cd dual immunofluorescence. results: in administration of evs reduced the seinduced loss of pyramidal neurons in the hippocampal ca subfield. also, ev administration after se maintained higher levels of pv+ interneurons in the dentate gyrus. furthermore, ev treatment after se modulated abnormal neurogenesis, which was evidenced by a the role of small extracellular vesicles in chronic neuropathic pain zhucheng lin a , renee jean-toussaint b , yuzhen tian b , ahmet sacan a and seena ajit b a drexel university, philadelphia, usa; b drexel university college of medicine, philadelphia, usa introduction: chronic pain is the most prevalent, disabling, and expensive public health condition in the usa. exosomes are - nm extracellular vesicles that can transport rnas, proteins, and lipid mediators to recipient cells via circulation. exosomes can be beneficial or harmful depending on their source and contents. we hypothesized that the composition of small extracellular vesicles (sevs) can be altered following nerve injury and these alterations can provide insight into how the body responds to neuropathic pain. methods: to characterize changes following nerve injury, small extracellular vesicles (sevs) were purified by ultracentrifugation from mouse serum four weeks after spared nerve injury (sni) or sham surgery. mirna profiling and proteomics analysis using tandem mass spectrometry were performed to determine differential expression of mirnas and protein cargo respectively. for in vivo studies, sevs were administrated intrathecally into the mouse lumbar region. animals were evaluated for mechanical and thermal hypersensitivity over days after injection. results: our mirna profiling showed a distinct mirna signature in sni model compared to sham control. proteomics analysis detected gene products. of these, were unique to sni model. neuropathic pain can induce the activation of the complement cascade and we observed significant upregulation of complement component a (c a) in sevs from sni model. intercellular adhesion molecule (icam- ), required for the leukocyte recruitment, adhesion and homing of exosomes was also upregulated in sevs from sni model compared to sham control. administration of sevs from sni model increased paw withdraw threshold in naïve recipient mice and inflammatory pain model, indicating a protective role for sevs in attenuating chronic pain. summary/conclusion: our preliminary studies suggest a critical role for sevs cargo in regulating pain. additional studies are ongoing to determine the functional significance of alterations in sevs composition using mouse models of pain. introduction: amyotrophic lateral sclerosis (als) is a progressive adult-onset neurodegenerative disease caused by selective motor neurons (mns) death. the rapid disease progression strongly suggests that cell-tocell spreading of noxious factors could take place in als pathogenesis. extracellular vesicles could potentially spread the disease. in this study, we characterized large (levs) and small extracellular vesicles (sevs) isolated from plasma of sporadic als patients and healthy controls and determined their different composition in order to understand their neuroprotective or neurotoxic role in als pathogenesis. methods: levs and sevs were isolated from plasma of als patients and healthy volunteers by differential centrifugation and characterized by nanosight ns . cd , cd , cd , cd a and annexin v were used for flow cytometry. sod , tdp , fus protein level was investigated by western blot. for raman spectroscopy, evs were dried on top of a caf slide and raman spectra were acquired using a nm laser line. mirna libraries were prepared by truseq small rna library kit (illumina). results: the mean size both for levs and for sevs resulted increased in als patients compared to controls. levs derived from als patients were enriched in sod- , tdp- and fus proteins compared to ctrls. sevs showed a distinct spectral pattern from levs. in addition, levs of als patients were richer in lipids and had less intense bands relative to aromatic aminoacids compared to healthy controls. we also found a great presence of leukocyte derived levs (lmvs) in als patients compared to ad patients and healthy donors and significant correlation with the progression rate of the disease. on the other hand, mirna and rna whole transcriptome sequencing identified a specific signature of mirnas in plasma derived sevs of als patients compared to a group of healthy controls and three neurological groups of control. summary/conclusion: these data may suggest that levs derived from als patients, enriched in lipids and toxic proteins, might play a role in prion-like propagation and immunity of als disease, while sevs, deriving ps . introduction: dendritic spines are actin-rich structures at the postsynaptic sites of most excitatory synapses in the central nervous system. they are highly important structures for higher brain functions such as learning and memory. several live imaging studies have shown that long, thin, actinrich protrusions called dendritic filopodia are precursors of dendritic spines in hippocampal and cortical neurons. so far, many intracellular factors that regulate filopodia formation have been identified. however, extracellular mechanisms of filopodia formation are largely unknown. also, detailed molecular mechanisms by which astrocyte secreted factors regulate synaptogenesis are not well understood. small extracellular vesicles (sevs)/exosomes have potential to regulate filopodia, spine and synapse formation in autocrine or paracrine manner due to their unique cargo composition. here, we examine role of exosomes in filopodia, spine and synapse formation. methods: primary rat hippocampal and cortical neurons were transiently transfected with the multivesicular body (mvb) docking regulator gfp-rab b or with shrnas against the exosome secretion and biogenesis regulators rab b and hrs. transfected neurons were immunostained for synaptic proteins and analysed for filopodia at day in vitro (div) or spines at div . for rescue experiments, exosomes were isolated using differential ultracentrifugation method from conditioned media of div cortical neurons or primary astrocytes and characterized for their size, common protein markers and morphology. results: here, we find that mvb docking factor gfp-rab b localizes to both the tips and bases of actin-rich filopodia and spines in primary neurons. furthermore, genetic regulation of exosome secretion by overexpression or knockdown of rab b or hrs leads to respective increases or decreases in the number of filopodia, spines and synapses. the defects of exosome-inhibited neurons in filopodia density are rescued by add-back of neuronal exosomes. additionally, treatment of primary neurons with exosomes isolated from primary astrocyte cultures leads to enhanced spine and synapse formation. summary/conclusion: these results indicate that autocrine and paracrine communication via exosomes are a key part of the process of neuronal filopodia, spine and synapse formation. effects of apolipoprotein e genotype on protein and small rna profiles of brain tissue-derived extracellular vesicles of alzheimer's disease patients introduction: multiple sclerosis (ms) is the most frequent chronic inflammatory disease of the young adult central nervous system. nevertheless, the pathogenesis remains largely unknown. it is therefore relevant to better characterise in cerebrospinal fluid (csf), which irrigates the brain, novel bioactive compounds whose dysregulation could be involved in ms pathology. the concentration of extracellular vesicles (evs) has been already found affected in ms patient fluids but the content in bioactive molecules, particularly the micrornas (mirnas), remains barely investigated. the mirna are short oligonucleotides that are major posttranscriptional regulators and we previously showed the dysregulation of specific mirnas in csf of ms patients. evs can potentiate mirna effects by allowing remote action through the shuttling within biological fluids such as csf while providing a protection from circulating rnase. nevertheless, csf remains a challenging fluid to analyse due to limited access, low volume and presence of lipoproteins (other putative mirna carrier) that can be co-isolated with evs. methods: we performed a comparative analysis of ev isolation from csf by size-exclusion chromatography (sec), density-gradient ultracentrifugation, ultrafiltration or chemical precipitation (chemp) to determine the optimal technique(s) to enrich ev. results: sec applied on csf of control patients showed optimal ev purification with sufficient evs from . ml of csf for downstream ev characterization. furthermore, we were able to isolate mirnas from csf and determined their enrichment in evs by rnase-sensitivity treatments. finally, we have combined chemp and sec to enable a fast and largescale isolation of evs from > ml of csf, which successfully provided an increase in particles detected by nanoparticle tracking analysis. we are currently characterising the particles to confirm that they are purified evs, cleared from contaminants. summary/conclusion: this work opens perspective to analyse evs from ms patients and to determine whether mirnas participates in ms pathogenesis through their transit in evs. funding: fondation louvain, charcot foundation. differences in circulating number of extracellular vesicles between contact sport athletes with and without acute mtbi: a pilot study meghan rath a , jacqueline sayoc a , soo-young choi a , karlee burns b , aja corchado c , jane mcdevitt b , jingwie wu d , ryan tierney b , michael selzer e , xiaoxuan fan f and joon-young park a for bottom-up guc, increasing iodixanol gradients with . ml of samples were centrifuged at k g for h. fractions were then pooled based on densities ( . - . g/ml). bca and sds-page were used to analyse total protein; nanoparticle tracking (nta) and transmission electron microscopy (tem) for ev presence; and immunoblotting and imaging flow cytometry (ifcm) to evaluate ev specific markers. (ev-track id: ev ). results: immunoblotting showed absence of actinin from all samples, while cd and tsg were detected for all samples; apart from imf_ip. nt_samples were not analysed reliably by nta and ifcm, due to the high concentration of casein micelles present (~ ^ /ml in milk) that otherwise would be co-counted with evs. as expected, following ip, which most efficiently removed casein micelles, bca showed that samples had lowest total protein. this was confirmed by sds-page. thus, most effects were then focused on the ip casein-depleted samples. ifcm indicated that, post-guc, sm_ip evs had significantly (p < . ) more cd -positive particles/ml of milk vs all other guc and kduc samples. while there were no significant differences in sizes of ev separated from sm or imf, directly comparing the ip pre-treated samples, sm had significantly (p < . ) higher quantities of evs when compared to imf. additionally, tem indicated that evs separated from sm by guc were intact with limited background debris, whereas those separated from sm by duc and all imf evs were not. summary/conclusion: in conclusion, regardless of the method used, imf has fewer intact evs compared to sm. also, to obtain purest sm evs, ip followed by guc separation is optimal. introduction: extracellular vesicles (evs) exist as subpopulations with heterogeneous content. the surface heterogeneity of evs may reflect differences in functionality between ev subpopulations, as interactions with recipient cells may differ between ev subpopulations with different surface profiles. however, it is currently challenging to study functional differences between ev subpopulations due to the lack of suitable techniques to purify intact evs based on their surface signature. here, we showcase a novel capture-and-release platform to enrich intact ev subpopulations by their surface profile and compare their characteristics. methods: mda-mb- and skov- cell-derived evs were isolated using size exclusion chromatography. ev subpopulations were enriched based on surface markers cd , cd , cd or phosphatidylserine (ps) using a novel magnetic bead-based capture-and-release platform. obtained evs were characterized by transmission electron microscopy (tem), nanoparticle tracking analysis (nta) and western blotting. evs were fluorescently labelled using pkh and celltracker deep red (ctdr) and their uptake by recipient cells was examined using flow cytometry. results: western blot analysis showed that ev subpopulations enriched for the selected tetraspanins and ps were successfully isolated using a novel capture-andrelease platform. interestingly, evs isolated based on ps exposure (ps+) lacked most canonical ev markers. all ev subpopulations showed intact, cup-shaped morphology when analysed by tem, but contained less protein contaminants compared to the initial ev isolate. ps+ evs were slightly larger than other ev subpopulations when analysed by tem and nta. to test the capacity of ev subpopulations to interact with recipient cells, evs were labelled with pkh and ctdr prior to subpopulation fractionation. after fractionation, ps+ evs showed a significantly higher ctdr/pkh ratio than other ev subpopulations as determined by fluorescence spectroscopy, suggesting higher esterase activity of ps+ evs compared to other tested subpopulations. furthermore, mda-mb- derived evs isolated based on cd and cd expression were taken up more efficiently by hmec- and mda-mb- cells than evs isolated based on presence of cd or ps. summary/conclusion: using a novel technology to isolate ev subpopulations based on their surface profile, we here show that composition and cellular uptake efficiency differs between ev subpopulations. theoretically, this technology is applicable to any surface marker of interest, allowing its use to further establish ev surface-functionality relationships and enrich evs with desirable characteristics for therapeutic purposes. funding: this work was supported by a veni grant (no. ) of the dutch research council (nwo). aml were harvested from tib cells cultured in evfree medium using serial ultracentrifugation. hspc (ksl; lin-sca + ckit+) clonogenicity and inflammatory responses were assessed using colony-forming unit (cfu) assay and real-time polymerase-chain reaction, respectively. ifn-alpha receptor (ifnar ) expression and intracellular reactive oxygen species (ros) levels were assessed by flow cytometry. dna damage were assessed by quantifying nuclear γ-h ax using immunofluorescent microscopy. results: similar to evs derived from aml patients, tib ev-aml elicited double-stranded breaks in hspcs, and actively suppressed hspc clonogenicity. transcriptional profiling revealed that exposure to ev-aml induced the upregulation of several inflammatory mediators in hspcs, including isg , il- , ifnα, ch h. inflammatory signalling triggered by ev-aml did not depend on ifnα signalling as evident from suppression of clonogenicity in ifnar -null hspcs as well as the lack of evs-induced stat phosphorylation or ifnar downregulation. instead, we found increased levels of ros following ev-aml exposure. summary/conclusion: our findings support a model whereby ev-aml inflammatory signalling and oxidative stress lead to dna damage in hspcs. introduction: basic leucine zipper atf-like transcription factor (batf ) is implicated in inflammatory response and anti-tumour effects. although the tumour suppressive function of batf has been reported, its extracellular role in maintaining a non-supportive cancer microenvironment has not been explored. methods: in this study, we established gbm orthotopic and subcutaneous tumour models in nude and balb/c mice and flow cytometry analysis determined the batf inhibitory effects of mdscs recruiting. we used transwell assay to determine batf -positive evs (evs-batf ) inhibitory of the chemotaxis of myeloid-derived suppressor cells (mdscs) in vitro. in addition, exo-counter detection during the development of the gbm-batf model to demonstrate evs-batf crosstalk with distant tissues. amd blocking in tumour model confirms that evs-batf dominated by the sdf- a/cxcr signalling pathway. in addition, exo-counter detection of evs in pairs of gliomas in different stages proposes plasma-evsbatf (plevs-batf ) as a prognostic marker. results: we found that tumour-derived evs-batf regulate crosstalk between glioma cells and tumour microenvironment by inhibiting mdscs recruitment. evs-batf can be detected in plasma and bone marrow of glioma-bearing mice, this provides direct evidence that glioma-derived evs can communicate with distant site by crossing blood-brain barrier. besides, evs-batf injection significantly reduced sdf- α expression in the tumour tissues. after blocking sdf- α signalling by amd , the inhibitory effects of batf overexpression on mdscs recruitment were rescued. evs-batf inhibit mdscs recruiting and secreting mmp , mmp , and vegfa which promote gbm progression. strikingly, exo-counter detection of evs in pairs of gliomas in different stages reveals that the number of plevs-batf can distinguish stage iii-iv glioma from stage i-ii glioma and healthy donors. summary/conclusion: our results suggest that evs-batf may be an effective circulating biomarker associated with glioma progression. of note, we are the first to determine the regulatory role of evs-batf in regulating tumour microenvironment and propose plevs-batf as a prognostic marker predicting glioma progression and candidate target for gbm therapy. introduction: electrofluidics is an emerging technology of combining electronics and nanofluidics. one important device in electrofluidics is an ion transistor in which the ionic current through a nanopore is regulated by gate voltage bias. here, we suggest a fabrication method of nanopore by introducing focused ion beam (fib) and atomic layer deposition (ald) to sense extracellular vesicle (ev) via metal electrode structures. methods: we deposited nm-thick silicon-nitrite layers on both sides of silicon wafer by low-pressure chemical vapour deposition (lpcvd). we fabricated rectangular patterns by photolithography followed by reactive ion etching (rie) on the backside of the wafer. anisotropic silicon etching by koh was performed. the front side of the chip was patterned by photolithography followed by ti/au deposition for the fabrication of electrode structures. we drilled ~ nm pores in the si n membrane by fib. by the ald process, we deposited highly-conformal metal film, either platinum (pt) or ruthenium (ru) to shrink nanopores by a self limiting process. results: we expect that the ion current through the nanopore is efficiently controlled by the gate-surrounding structures. the nanopore ion transistor can be used to count the number of evs. summary/conclusion: we suggest a fabrication method of nanopore ion transistors by introducing focused ion beam (fib) and atomic layer deposition (ald). this device will be applicable for single ev sensing. introduction: extracellular vesicles (evs) are key players in cell-cell communication and increasing evidence has shown that evs function in cancer by promoting cancer cell motility and metastasis. analysing tumour-derived evs in biofluids is attractive because it would be a novel approach to a non-invasive liquid biopsy. unfortunately, evs are highly heterogeneous. they vary greatly in size, lipid composition, and cargo and are difficult to distinguish from other small particles in complex biofluids. we have developed a novel flow cytometry method to generate a distinct ev fingerprint to profile biological specimens. methods: evs from cell culture media (purified and unpurified) and biological fluids (plasma and urine) were detected by flow cytometry using features on individual evs produced by intrinsic (cd -phluorin) and extrinsic (lipophilic dye, di- -anepps, and antibodies) fluorescent labels. ev subpopulations were visualized with dimensional reduction (t-sne and umap) of - features that defined the vesicle size, shape, and fluorescent emission spectra associated with the fluorescent marker. unsupervised density based clustering (hdbscan) in conjunction with supervised machine learning (xgboost) was subsequently used to define subpopulations. we refer to this method as "ev fingerprinting". results: ev fingerprinting was successfully used to detect evs in complex biological specimens and trace their differential enrichment through conventional purification methods. evs were readily distinguished from protein complexes, lipoproteins and non-lipid particles. calibration with externally validated purified ev, as well as size, lipid, and fluorescence standards enabled ev fingerprinting as a rigorous and reproducible method for resolving heterogenous ev samples. ev fingerprinting applied to conditioned medium from tumour cells and biological fluids from cancer patients reveals unique ev profiles generated by cancer, further supporting the potential of ev fingerprinting as a liquid biopsy. summary/conclusion: our single-ev analysis approach characterizes whole ev populations in complex biological fluids without the need for purification, reducing time intensive purification protocols and subsequent sample loss, permitting efficient analysis of liquid biopsy samples. detection and quantification of extracellular vesicles with cargo protein and rna using the amnis® cellstream® flow cytometer introduction: the particle size distribution (psd) of extracellular vesicles (evs) is commonly measured by tunable resistive pulse sensing (trps) and nanoparticle tracking analysis (nta). both trps and nta have limitations that hamper the accurate measurement of the psd of evs, specifically in the size range from to nm. an alternative technique for measuring the psd of evs is micro-fluidic resistive pulse sensing (mrps). because a standard operating procedure (sop) for characterizing evs by mrps is absent, we aim to establish a reliable sop to ensure reproducible psd measurements of evs by mrps. methods: measurements (n = ) of red-blood cell, prostate cancer cell line supernatant, and human urine and plasma evs were acquired in × s acquisitions. two microfluidic cartridges were used to study a dynamic range of - nm. samples were diluted into phosphate buffered saline with different concentrations of tween or bsa. because the excess of particles affects the detection limit, serial dilutions were performed to find the optimal dilution for each sample. data were evaluated using data viewer software. results: the optimal dilution was determined for each sample by maximizing the particle rate and minimizing the measurement time while preserving a robust detection limit of or nm. moreover, we developed a procedure to optimize the peak filter settings of data viewer by fitting data to normal distributions and identifying threshold values for signal-to-noise ratio, symmetry, and transit time within % confidence. summary/conclusion: we recommend to use . % w/ v bsa in dpbs as sample diluent, because tween affects evs as confirmed by flow cytometry. by using orthogonal techniques and well-characterized biological test samples, we developed and validated a sop for ev detection by mrps, thereby making mrps a valuable tool for ev researchers. real-time measurements of extracellular vesicles binding kinetics achieved through interferometric imaging in a multiplexed microarray modality introduction: extracellular vesicles are very promising diagnostic biomarkers. as a matter of fact, the properties of these biological nanoparticles depend on the health conditions of each individual. however, experiments that involve evs phenotyping are time consuming, due to h-or overnight incubations. in order to get accurate results, maximizing binding efficiency is a necessity; that normally involves ensuring the saturation of the capture reaction, which can result in an unnecessarily long incubation time. with the ability of labelfree kinetic binding measurements using interferometric reflectance sensing in a microfluidic chamber, we perform an optimization of the incubation time in different flow conditions, while demonstrating a new way of multiplexing for real-time evs specific capture and detection.methods: all the real-time binding measurements were performed with the interferometric reflectance imaging sensor (iris). iris chips were first coated with an organic polymer (mcp- ), which provides an active surface for probe immobilization. then, antibodies against cd , cd , cd markers were spotted at different densities in a microarray modality. the chips were then encapsulated with a glass window to form a microfluidic chamber that allows for imaging the sensor surface. samples of hek-derived extracellular vesicles were flowed across the sensor surface in the iris system and real-time images were acquired. incubation was performed at different flow rates, and in static and stopflow modalities. results: in this work, we focus on the specific capture of evs under different flow conditions to achieve an optimization of the incubation time. indeed, through the acquisition of real time binding data, we are able to precisely monitor the equilibrium point of the capture reaction. in this configuration of iris, low magnification optics allow for simultaneous detection of binding on hundreds of capture ligand spots. therefore, surface probes (surface density and specificity) as well as assay conditions can be optimized. we report on the optimization of antibodies against cd , cd , and cd markers. since the sensor chips are identical to the single-particle detection assays developed by nanoview biosciences, the optimization of binding assays will directly impact the phenotyping of individual exosomes. summary/conclusion: our method proved to be very efficient in optimizing the most crucial aspects concerning evs captureflow conditions, incubation time, surface density and sample concentration. introduction: diabetes is a life treating diseases extending its impairing influence on more than billion of people around the world within upcoming years. the most harmful complication generating high treatment and social costs is diabetic nephropathy, which develops in about % of patients suffering diabetes. still we do not have an effective and direct prognostic biomarker to diagnose renal complications in the primary stage of renal disease. methods: extracellular vesicles were concentrated from diabetic patients' urine and washed to perform spectral analysis: fourier transform infrared spectroscopy (ftir), based on the molecular absorption of electromagnetic radiation in the infrared region of the spectrum in a range from cm- to cm- and raman spectroscopy (rs) as a technique based on inelastic scattering of monochromatic light. both techniques provide information on the chemical structure of compounds by identifying functional groups with high molecular specificity. results: average spectral signature obtained for evs from urine samples of patients in the different stage of kidney damage allowed distinguishing specific bands, representative for amide (i/ii), lipids, cholesterol and nucleic acids. spectral parameters correlated with a clinical stage and a commonly used indicator of renal function (creatinine) in diabetic patients. summary/conclusion: infrared and raman spectroscopy are promising tools to diagnose and monitor renal function in diabetes. introduction: several existing bioanalytical strategies for purifying and characterizing exosomes have allowed for fundamental progress to be made. mixtures of evs can be enriched for exosomes by techniques such as ultracentrifugation and size-exclusion chromatography. but, these processes require large amounts of material that are often difficult to obtain and many different types of particles have similar sizes and densities. it is likely that unique subfractions within enriched samples exist, particularly in complex biological matrices such as blood, urine or milk which remain difficult to characterize and isolate with existing analytical technologies. methods: bovine milk exosomes were isolated via differential ultracentrifugation and resolubilized in mm ammonium acetate. these data were recorded using charge detection mass spectrometry (cdms). in cdms, individual particles are reflected back and forth through an electrostatic ion trap where they pass through a sensitive charge detector. each time a trapped particle enters and exits the detector, its charge (z) and mass-to-charge (m/z) ratio is measured. mass distributions are generated by multiplying the m/z values by the charge measured for each ion and binning the resulting masses.results: the masses of particles in a bovine milk extracellular vesicle (ev) preparation enriched for exosomes were directly determined for the first time by cdms. particle masses and charges span a wide range from m~ to~ mda and z~ to~ e and are highly dependent upon the conditions used to extract and isolate the evs. in total, , particles were detected from eight cdms measurements. a simple two-dimensional gaussian model suggests that eight unique subpopulations of particles may be resolvable based on charge and mass. complementary em and proteomics analyses confirm that samples are enriched for exosomes. particles associated with the s , s , and s families that are centred at~ . ,~ . , and~ . mda, respectively, appear too small to be ascribed to exosomes. the remaining , ( %) particles detected by cdms are within the mass range expected for exosomes. while cdms measurements are at an early stage of development, this approach appears to provide a new physical basis for separating and characterizing ev particles. summary/conclusion: this work describes a novel biophysical approach for measuring and characterizing the masses and charges of the extremely heterogenous population of exosomes and other extracellular particles enriched in bovine milk. as new sample preparation methods, aimed at purifying specific types of exosomes from different cell lines, tissues, and other body fluids continue to evolve, rapid and sensitive cdms measurements of the physical properties of mass and charge may become an important means of assessing the efficacy of different protocols. funding: nih (r gm - ). bab is supported by indiana university quantitative chemical biology fellowship (t gm ). in situ detection of exosomal microrna- b by fusion with liposomeencapsulated nanomotor introduction: breast cancer is the most common cause of cancer-associated death in women and has raised global health concerns. early diagnosis and treatment are crucial to improve the prognosis and survival rate of breast cancer patients. liquid biopsy is expected to provide a strategy for early diagnosis of breast cancer. exosomes have been regarded as novel liquid biopsy biomarkers due to their stable cargo of rnas, lipids, and proteins from their origin cells. exosomal micro (mi)rnas have recently been recognized as promising indicators of cancer occurrence and progression. however, most of the reported exosomal mirna detection methods require the lysis or extraction process, which increases the possibility of sample loss. in situ detection strategies avoid interference from body fluid. in this study, we developed a gold nanomotor fluorescence platform based on liposome fusion for breast cancer exosomal mirna in situ detection. the exosomal mirna detection platform was constructed using a gold nanomotor (detector) and liposomes (carrier). the dnazyme amplification sequences which could be especially triggered by mirna- b were identified by sds-page before modified on gold nano-motor and the capacity of the nanomotor was assessed using synthetic target sequence, breast cancer cell mda-mb- , mirna- b-encapsulated anionic liposomes, and mirna- b-expressing exosomes. three kinds of liposomes were synthesized, characterized, and assessed for loading ability. membrane fusion effect was evaluated by confocal laser scanning microscopy (clsm) and nanoflow cytometry. the performance of this method to discriminate between breast cancer patients and healthy individuals was investigated. results: the chosen dnazyme amplification sequences transformed "locked" status to "cleavable" status on target addition, releasing a fluorescence signal. the modified gold nanomotor showed a ten times higher fluorescence signal in the presence of mirna- b than the background and no noticeable fluorescence changes from a single-base-mismatch sequence. moreover, among the three different liposomes, cationic liposomes exhibited great stability, high loading efficiency, and excellent membrane fusion effect. furthermore, the fluorescent experiments confirmed that cationic liposomes could load and transfer the nanomotors into exosomes for mirna- b detection. finally, we were able to distinguish breast cancer patients and healthy individuals by sensing exosomal mirna- b directly from plasma samples without exosome isolation. summary/conclusion: a separation-free and sensitive assay based on dnazyme amplification technique and membrane fusion effect was established for breast cancer-derived exosomal mirna- b detection, which could be a promising tool for the liquid biopsy of breast cancer. isolation of exosomes by membrane affinity column increases non-exosomal rna recovery in comparison to differential ultracentrifugation introduction: exosome-based liquid biopsy is a potential aid in the diagnosis and prognosis of cancer patients. however, in order to incorporate exosomes into clinical routine, there is a need to compare different isolation methods. here we analysed the impact, in exosomal rna yield, of two intermediate recovery/ intermediate specificity methods: differential ultracentrifugation (ucd) and a membrane-affinity column (mac) kit. although mac has a faster performance which is more suitable to the clinic, we found that ucd results in a higher recovery of exosomes and less contaminating non-exosomal rna.methods: exosomes were enriched by mac and ucd from identical volumes of human plasma ( , xg, min/ . ) m filtration/ , xg, h)(n = ) and lymphoma conditioned medium( xg, min/ xg, min/ xg, min/ , xg . h/ , xg, h) (n = ). all exosomes were characterized by nanoparticle tracking analysis (nta), immunoblotting of cd /cd /flotilin/alix and electron microscopy (tem). exosome pellets were pre-treated with proteinase k ( mg/ml/ °c/ min) and rnase a ( mg/ ml/ °c/ min) before phenol-chloroform/glycogen rna extraction. rna yield was measured by both fluorometer and bioanalyzer.results: isolation of exosomes by ucd, in both plasma and medium, resulted in a higher yield in comparison to mac. this was shown by an augmented intensity of marker bands in the ucd samples (p = . , n = ) as well as by an increased number of exosomes in tem.in contrast, mac final exosomal fraction (from both plasma and medium), resulted in a -fold and fold increase in rna, respectively, in comparison to ucd when measured by fluorometer. this was confirmed by bioanalyzer. introduction: there is a need for better techniques for characterizing ev populations. we developed a sensitive multiplexed electrochemiluminescence (ecl)based assay format to characterize evs in cell-conditioned medium (ccm) and human biofluids. here we use the format to analyse ev samples for the presence of ev surface proteins, and to identify changes in ev phenotype associated with different cell lines, purification methods and growth conditions. methods: multiplex plates were prepared on msd's u-plex® platform with antibodies for putative evsurface proteins. each well displayed an array of nine specific capture antibodies and a negative control antibody. evs from samples were captured on the arrays and then detected with a cocktail of anti-tetraspanin antibodies (cd , cd and cd ) conjugated to an ecl label. three distinct cell types were grown at two sites, msd and atcc. resulting ccm were each purified by four common methods: tangential flow filtration, peg-based precipitation, size-exclusion chromatography and centrifugal ultrafiltration. all samples were also assayed without purification.results: fifty-five of the surface markers were detected on intact evs from at least one evaluated cell type. datasets were analysed using correlation matrices, hierarchical clustering, and machine learning. for each cell type, when comparing unpurified ccm grown at different sites or evs prepared by different purification methods, we typically observed correlations above . , indicating that the purification methods did not introduce bias to ev phenotypes, and that the assay format can provide robust phenotypic information without any purification of evs. two unsupervised clustering analyseshierarchical clustering and t-distributed stochastic neighbour embeddingboth generated wellseparated clusters for each of the cell types, regardless of purification method or source. summary/conclusion: we developed multiplex ev surface marker assays and demonstrated their use for multimarker ev phenotyping. this flexible format enables rapid assay development for new ev subpopulations with or without sample purification. these results also demonstrate ev surface marker phenotyping via multiplex ecl assays may be used to distinguish ev populations from various cell types, and characterize bias introduced by purification. detection of misev recommended ev protein-markers using automated western blotting method for isolation of evs and a simple western blotting platform for automated protein separation and immunodetection of misev-recommended proteins.methods: total evs were isolated by affinity-membrane spin columns from pre-filtered . - ml plasma or - ml urine, respectively. intact vesicles were eluted and the ev-depleted biofluid fraction was collected from the flow-through. a small fraction ( μl) was analysed by a simple western blot workflow providing automated capillary electrophoresis-based protein separation and immunodetection, characterizing each fraction for presence or absence of misevrecommended proteins.results: a range of specific antibodies were identified and the ev fractions were shown to be enriched in evproteins, whereas contaminating non-ev proteins were significantly reduced. isolation of evs was necessary to allow detection of the low abundant ev protein markers, whereas non-ev proteins were readily detectable both in the neat biofluids and in the ev-depleted flowthrough. we characterized the effect of washing on the purity of ev isolates and defined the dynamic range of the workflow using titrations of input volume of both plasma and urine ev isolations. summary/conclusion: simple western blotting protocols were established for quality control of isolated evs in accordance with misev-guidelines. evs isolated using affinity-membrane spin columns were shown to be enriched in ev markers and depleted for non-ev proteins. al-pha beads: a library of extracellular vesicle-associated metalloproteinase biosensors (adams) and a disintegrin and metalloproteinase with thrombospondin motifs (adamtss) are highly promising cancer biomarker candidates that have complex roles in cancer pathogenesis and metastasis. importantly, within the context of lung cancer, the detection of adam proteolytic activity might be more informative than the level of adam protein.therefore, the development of low-cost metalloproteinase biosensors could serve as useful biomarker research tools. methods: to this end, we developed advanced proteolytic detector polyhydroxyalkanoates (al-pha) beadsa library of biodegradable, biopolymer-based protease biosensors. broadly, these biosensors utilise phac-reporter fusion proteins that are bound to microbially manufactured bioplastic beads. these phac-fusions also incorporate specific protease cleavage sites. in the presence of a specific protease, reporter proteins are cleaved off of the al-pha beadsresulting in a loss of bead fluorescence that can be measured using flow cytometry. these biosensors were assayed using either metalloproteinases, conditioned media or evs from in vitro cancer models.results: human metalloproteinase recognition motifs were identified in the literature and a total of different al-pha bead biosensors were designed. a control, tev-specific biosensor detected . introduction: brain extracellular vesicles (evs) are heterogenous and include previously described microvesicles and exosomes. herein we characterized a formerly unappreciated population of mitochondriaderived evs that we term "mitovesicles". mitochondrial dysfunction is a well-established hallmark of ageing and neurodegenerative disorders as down syndrome (ds). hence, we examined mitovesicle levels and cargo under these conditions to characterize in vivo mitovesicle biology and responsiveness to mitochondrial stressors. methods: employing a high-resolution density gradient, distinct and novel populations of evs were isolated from murine and human ds and diploid control postmortem brains or from cell media. morphometric ev features were analysed by nanoparticle tracking analysis and cryogenic electron microscopy, while ev constituents were characterized by western blotting, mass spectrometry, lipid profiling and mitochondrial rna qpcr.results: we identified a population of double-membrane, electron-dense brain evs containing multiple mitochondrial markers ("mitovesicles") that are highly distinct from microvesicles and exosomes. proteomic data show that mitovesicles contain a unique subset of mitochondrial proteins while lacking others, such as tom . mitovesicles have a lipid composition that is unlike that of previously described evs and is consistent with mitochondrial origin. functionally, the complex-iii inhibitor antimycin-a stimulated in vitro mitovesicle release into the cell media, suggesting an interrelationship between mitochondrial dysfunction and mitovesicle biology. in mouse brains, mitovesicle levels increased with age and were found to be higher in ds compared to diploid controls. mitochondrial rna and protein levels were also altered in ds compared to diploid controls. summary/conclusion: we describe a previously unidentified type of metabolically competent evs of mitochondrial origin that we designate mitovesicles. our data demonstrate that brain mitovesicle levels and cargo are tightly regulated in normal conditions and are modified during pathophysiological processes in which mitochondrial dysfunction occurs, suggesting that mitovesicles are a previously unrecognized player in mitochondria quality control and may have a role in the trans-cellular tissue response to oxidative stress. introduction: alzheimer's disease (ad) is a devastating neurodegenerative disease leading to progressive memory loss and ultimately death with limited therapeutic options. growing evidence supports the theory that toxic proteins, like tau and amyloid, may propagate from diseased cells by packaging toxic proteins into extracellular vesicles (evs) and releasing them to infect other cells. one enzyme involved in the isev abstract book biogenesis of evs is neutral sphingomyelinase (nsmase ), which catalyzes the hydrolysis of sphingomyelin to produce phosphorylcholine and ceramide. several groups have reported improved cognition and reduced tau propagation when nsmase is pharmacologically inhibited or genetically knocked down in ad mouse models. unfortunately, current nsmase inhibitors are not suitable for clinical development due to poor solubility and inadequate pharmacokinetic profiles.methods: our group carried out a high-throughput screening campaign followed by extensive medicinal chemistry efforts leading to the discovery of phenyl (r)-( -( -( , -dimethoxyphenyl)- , -dimethylimidazo [ , -b] pyridazin- -yl) pyrrolidin- -yl) carbamate (pddc), an orally active, nm potent inhibitor with excellent selectivity and brain penetration. we tested pddc's ability to inhibit exosome release in cultured primary glial cells as well as an in vivo model of acute ev release. we then treated xfad mice with mg/ kg of pddc daily for six months and monitored their behaviour in the fear conditioning assay.results: pddc dose dependently reduced ev release from cultured primary glial cells and significantly reduced plasma ev numbers in an in vivo model. following chronic treatment with pddc, xfad mice demonstrated significantly improved cognitive function in the fear conditioning assay. summary/conclusion: these promising findings are currently being expanded using mouse models of tau propagation. if successful, these data would support pddc as a novel compound for targeting the pathological spread of tau as a therapeutic for ad. profiling evs in the anterior cingulate cortex of individuals with major depressive disorder introduction: major depressive disorder (mdd) is one of the leading causes of disability worldwide, affecting % of the population. the environment has been thought to play a role in the disease development, resulting in biological changes mediated by epigenetic mechanisms. microrna's (mirna) are well known epigenetic regulators that are disrupted in the depressed brain, and they are packaged into extracellular vesicles (evs). evs have emerged as means of intercellular communication, a process that is also disrupted in mdd. they are thought to transfer mirna between cells, which can alter gene expression in recipient cells. therefore, we hypothesize that ev cargo is altered in mdd subjects compared to healthy controls (hc). the aim is to extract evs from human postmortem anterior cingulate cortex, a region previously associated with depression, and profile the mirna cargo and compare it between mdd subjects and hc. methods: post-mortem human brain tissue from the anterior cingulate cortex of mdd subjects and hc was mildly dissociated in the presence of collagenase type iii. residual tissue, cells, and large vesicles were eliminated, and evs were isolated using size exclusion chromatography. the quality was assessed by western blots and transmission electron microscopy (tem). rna was extracted and a small-rna library was constructed and sequenced using the illumina platform. differential expression analysis was then performed.results: western blots showed little to no endoplasmic reticulum (calnexin), golgi (bip), or mitochondrial (vdac) contamination, along with enrichment of the exosomal marker cd . tem images showed the typical cup-shaped morphology with sizes mostly between and nm. preliminary sequencing results revealed that mir- a- p, which is predicted to target glutamate receptors, is downregulated in evs from mdd subjects. summary/conclusion: high quality ev extractions can be obtained from post-mortem brain tissue using our method. this will be the first study to profile brainderived ev mirna in the context of depression. future studies will be needed to determine the effect of the different levels of mir- a- p. this could provide novel mechanistic insights into the pathophysiology of mdd and will serve as a starting point to examine the potential role of evs in mdd pathology. methods: we use ifc to characterize evs released by glioma using -ala, fluorescently labelled ev (cfda-se, cd ) and glioma specific (tenascin c and epidermal growth factor receptor viii, egfrviii) markers. furthermore, we characterized evs released by egfrviii positive glioma cells treated with dexamethasone, a steroid commonly used in glioma patients, to determine the effect of steroids on ev release. evs were quantified by ifc and results were confirmed by qpcr for the levels of egfrviii mrna. results: firstly, we optimized protocols to label glioma sevs using fluorescently labelled ev markers (cfda-se, cd ) and tumour specific markers (tenascin c and egfrviii). of the total evs (cfda-se), we demonstrate that % are tenascin c positive, . % are egfrviii positive and . % are -ala positive. there was only a minor overlap (< %) between the sub-populations. finally, we show that dexamethasone treated glioma cells release lower total evs ( . -fold), tumour specific evs ( . -fold; egfrviii), egfrviii mrna compared to mock treated cells. summary/conclusion: we demonstrate the potential of ifc to monitor sevs released by glioma cells exposed to different stimuli. this allows the characterization of ev sub-populations providing a working model to understand the dynamics of tumour evs at a single vesicle level. introduction: extracellular vesicles (ev) released by infective forms of trypanosoma cruzi, the agent of chagas' disease, modulate inflammatory response of macrophages through the activation of toll receptor (tlr ) via mitogen-activated protein kinase pathway. this induces the production of nitric oxide (no) and expression of the cytokines tnf-α, il- and il- , which could explain the inflammation observed in experimental chagas' disease, and eventually in the progression of human disease. evs released by the parasite are heterogeneous and it is unknown which factor, or factors present in the different vesicle populations act during the interaction with host cells.objectives. the goal of the present work was to characterize and isolate the different populations of evs released by t. cruzi and test their effects on macrophages. methods: ev released by trypomastigotes forms of t. cruzi (y strain) were purified by asymmetric flow field-flow fractionation (af ) and characterized by nanoparticles tracking analysis (nta). the different populations of evs were incubated with host human monocytes cells (thp- ) and cytokines production determined by elisa and qpcr. the different ev populations were also incubated with llcmk- epithelial cells and the infection by t. cruzi determined. results: we found two distinct populations of evs. a population with to nm (ev ) and another with to nm (ev ). ev induced more tnf-alpha, il- , ip- and ccl than ev . it was also more effective in promoting t. cruzi infection in epithelial cells. due to unknown reasons, making these systems insufficient for use in drug development and infectivity assays.noroviruses are known to attach to gram-negative enteric bacteria and this facilitates infection in vitro. however, the microbiome-norovirus-host communication link is missing. noroviruses infect immune cells present in lamina propria during acute infection, but bacteria themselves are large enough to cross the mucosal and the tight epithelial barrier which separates gut lumen from lamina propria. we hypothesized that binding of noroviruses to bacteria enhances extracellular vesicles (ev) production. because commensal bacterial evs by themselves do not have any detrimental effects on host cells, we believe using evs in in vitro culture will enhance norovirus infection, thus producing higher titre of viruses for vaccine and anti-viral drug development. methods: attachment assay: purified norovirus was incubated with enterobacter cloacae, lactobacillus acidophilus and bacteroides thethiotaomicron, and grown to produce evs. the attachment was confirmed via qpcr.isolation of evs: clarified media supernatants were subjected to ultracentrifugation at varying speeds and . um filtration. co-purification of norovirus with the evs was checked.ev quantification and characterization: ev total protein content was measured by microbca. the number of vesicles were quantified by nanoparticle tracking analysis. scanning and transmission electron microscopy was performed to check quality of ev preparation and determine if virus was attached to the vesicles. internal ev protein content was evaluated using ms-hplc. the evs were also check for infectivity via tcid assay. results: incubation of noroviruses with commensal bacteria resulted in significant increases in production of evs compared to uninfected controls. murine norovirus (mnv), used as a surrogate, was found to be associated with evs. em analysis determine association of viruses with the bacteria as well as the mvs, while also showing certain surface structural changes in virus attached bacteria compared to mock bacteria. the evs were found to cause infection in naive macrophages. summary/conclusion: changes in ev production and content by bacteria exposed to noroviruses will provide insight into its pathogenesis and possible solutions to the low viral output from hunov culture systems.ps . = op . kylie krohmaly a , claire hoptay b , andrea hahn a and robert freishtat a a children's national hospital, washington, usa; b childrens national hospital, washington, usa introduction: bacteria constitutively produce biologically active extracellular vesicles (evs), which contain rna, dna, and/or proteins. bacteria use these evs for communication with other bacteria and recent research suggests bacterial evs can also affect host cells. given these findings, it is necessary to examine the role of bacterial evs in human disease. current methods of bacterial ev isolation from human specimens cannot distinguish between bacterial species. however, there is utility in examining evs from specific species, as bacterial species and their evs may have unique contributions to human disease. our objective was to isolate circulating evs specifically from escherichia coli (eevs) and haemophilus influenzae (hevs), two known colonizers and pathogens in the gut and airway, respectively. methods: total evs were isolated from the blood of six healthy volunteers via precipitation and size exclusion chromatography. evs were then selected via a novel latex bead-based fluorescent antibody construct targeting species-specific outer membrane proteins. we used flow cytometry to evaluate the isolated evs. results: the constructs were saturated with eevs at an antibody concentration of . µg/ml of plasma, as geometric means ≥ . µg/ml were nearly equal. hevs were detected at µg/ml of plasma, but saturation is yet to be determined. eevs were imaged by a fei talos f x electron microscope and measured between - nm, and hevs were between - nm. both types of evs were spherical. summary/conclusion: using this novel technique, we were able to isolate, detect, and visualize eevs and hevs. this technique enables the study of specific bacterial evs. in the future, ev contents will be assayed. furthermore, this technique will be modified so that specific bacterial evs from body fluids can be used for downstream functional applications. this is the first time that bacterial evs from targeted bacterial species have been detected in blood from healthy humans. introduction: nasopharyngeal carcinoma (npc) is characterized by a large presence of regulatory t cells (tregs) and the production of tumour-derived exosomes with immunosuppressive properties. our team showed that npc-derived exosomes favour the suppressive activity and recruitment of human tregs via ccl chemokine, thus contributing to npc immune escape (mrizak et al., jnci, ) . more recently, our team has shown that npc-exosomes could induce tregs by altering the maturation of dendritic cells (dcs) and promoting tolerogenic dendritic cells (tdcs) (renaud et al., herpas congress ). our main objectives in this study are (i) to define and compare the metabolic status of mature dendritic cells (mdcs), control tdcs and tdcs generated in the presence of npc-exosomes (exocnptdc) and (ii) to evaluate the chemoattractive potential of npc-exosomes on exocnptdcs, and notably to investigate the involvement of ccl in this recruitment. methods: dcs are generated from human monocytes in the presence or absence of npc-exosomes. the maturation status of dcs was evaluated at a phenotypic level by studying the expression of maturation markers using flow cytometry and at a functional level by analysing cytokines secretion using elisa. this cytokine analyse has been performed in both conditions, on treated dcs and during co-culture assays of autologous cd t lymphocytes with treated dcs. in a second step, a mitochondrial metabolic and glycolytic study was performed using the seahorse technology (ocr and ecar measurement). finally, the chemoattractive potential of npc-derived exosomes on the different induced dcs was analysed (i) using boyden chamber chemoattraction assays or real-time videomicroscopy (chemotaxis µslide ibidi) and (ii) using rt-qpcr analysis of the receptor expression of ccl (ccr ).results: npc-exosomes alter dc maturation, which gives rise to tolerogenic dcs that favour the induction of tregs. in addition, the metabolic analysis of dcs seems to put foward a specific metabolic signature of the tdcs induced by npc-exosomes. and finally, chemoattraction assay suggests that npc-exosomes preferentially attract tdcs and exocnptdcs in a ccl dependant manner. summary/conclusion: taken together our results should allow us to characterize the major role of npc tumour exosomes on the maturation and the recruitment of dc and so identify them as anti-tumoural therapeutic targets. cytotoxic t lymphocyte ev that prevents tumour metastasis by collapse of tumoural mesenchymal stroma is classified into exosome, but not microvesicle or apoptotic body.naohiro seo a , junko nakamura a , tsuguhiro kaneda a , takanori ichiki b , asako shimoda c , kazunari akiyoshi c and hiroshi shiku a a mie university graduate school of medicine, mie, japan; b the university of tokyo, bunkyo, japan; c kyoto university, kyoto, japanintroduction: recently, instead of ultracentrifugation, development of new preparation protocol is demanded for research of reliable bioactivity and drug discovery of extracellular vesicles (evs). in this study, we propose a novel method for large scale preparation of highperformance extracellular vesicles focusing on membrane negative charge. methods: murine cytotoxic t lymphocyte (ctl) evs in supernatant were concentrated more than times at over % purity without leaking by kda mwco ultrafiltration, and subjected to ion exchange deae column chromatography after replacing with pbs. after ion exchange, evs were characterized by bca assay, nta assay, cryotem observation, proteome analysis, dna content measurement, mirna microarray analysis, zeta potential measurement, lectin array analysis, and target cell analysis.biomarker detection and analysis and detail strategies for cross-platform analytical validation. methods: we conducted a cross-platform analysis using two commercially available flow cytometers designed for ev detection. scatter resolution, enumeration accuracy and precision were determined across both platforms by analysing submicron silica beads (apogeemix, - nm) of known concentration.we detected large evs, as established by reference size beads, electron microscopy, expression of phosphatidylserine and the presence of integral membrane proteins of cell of origin. we analysed evs isolated from plasma by high-speed centrifugation ( , g) as well performing analysis by direct plasma labelling followed by validation by detergent lysis of vesicular constituents. a clinical operating range was defined which ensures linearity and avoids swarm detection. we observed comparable scatter resolution, enumeration accuracy (error ≤ %) and precision (cv ≤ %) across both platforms used. we defined two ev size gates: a "latex" gate ( to nm polystyrene latex beads), and a "silica" gate ( to nm silica beads) for evs at the lower end of our size range of interest. to improve detection sensitivity, we identified common contributors to signal noise and applied workflow strategies to minimize these. finally, we identified linear ranges which avoid swarm detection, and which ensures reproducible ev counts (cv < %) across both instruments. summary/conclusion: we present an optimised, standardised and cross-platform reproducible working protocol which supports the use of fcm in an ev-based liquid biopsy application. funding: the project is funded by spark oceania and uts innovation commercialisation seed fund scheme to mb. metabolomic profiling of serum and exosomes isolated from head and neck cancer patients after radiotherapy introduction: cancer radiotherapy (rt) induces the response of the whole body that could be detected at the blood level. searching for new molecular signatures which could correlate with treatment response in cancer patients is of particular importance. radiation-induced changes in proteome and transcriptome of serum have been widely described. however, metabolomic changes in serum, exosomes and other classes of small extracellular vesicles (ev) of cancer patients after rt have not been given as much attention. metabolomics of serum and ev of cancer patients could provide a valuable insight into the response of both tumour and whole organism to the treatment. the aim of the study was to compare serum and ev metabolomic profiles in head and neck cancer (hnc) patients before and after rt. methods: serum samples from hnc patients were taken before (a) and after (b) rt. healthy volunteers were used as a control group (c). ev were isolated from ml of serum using size-exclusion chromatography (sec). selected sec fractions were subjected to extraction of metabolites. a mixture of meoh/h o was used for extraction of metabolites from serum and ev samples. samples were analysed by gas chromatography-mass spectrometry (gc-ms).the study protocol adhered to the tenets of the declaration of helsinki and was approved by the bioethical committee of the maria skłodowska-curie national research institute of oncology, branch gliwice, poland (permit nr. do/dgp/ / / / / /g). results: an untargeted gc-ms-based approach allowed the detection of metabolites in serum samples and exosomal small molecules, of which joint. the identified compounds included amino acids, fatty acids, carboxylic acids, sugars, and others. there were metabolites which levels discriminated compared groups (a,b,c) of serum samples and compounds that discriminate the ev isolated from hnc serum before and after rt from hc. summary/conclusion: rt caused significant changes in levels of serum and ev metabolites witch are involved in amino acid metabolism, lipids metabolism, energy metabolism and oxidative stress response. capable of contributing to intercellular communication and metastasis. numerous studies have focused on elucidating their role in cancer progression. we recently showed that sevs isolated from pancreatic cancer cells can function as an initiator in malignant cell transformation. here, using a mass spectrometry (ms)-based proteomics approach, we analysed the differences in the protein cargo of sevs secreted from normal pancreatic and cancer cells to better understand their biological characteristics. methods: sevs were isolated from human pancreatic cancer cell lines (capan- , mia paca- , and panc- ) and normal pancreatic epithelial cells (hpde) using a combined ultrafiltration-ultracentrifugation method coupled with a sucrose density gradient purification. proteomic profiling of sevs was carried out using an lc-ms/ms method. protein identification from resulting ms/ms spectra was conducted using proteome database search software followed by gene ontology (go) enrichment and reactome pathway analysis.results: a total of , unique proteins were identified confidently across the combined samples. the proteins present in all four sev types ( , proteins) consist of general housekeeping proteins. proteins were uniquely found in all cancer sevs but not in the normal hpde sevs. this group contains an enrichment of proteins that function in the endosomal compartment of cells responsible for vesicle formation and secretion and suggest their important role in driving the increased production of sevs from cancer cells relative to normal cells. moreover, this group includes a set of proteins that have been implicated in malignant cell transformation, consistent with our previous work showing that each of the cancer sevs analysed here could initiate malignant transformation of nih/ t cells. conversely, there were proteins uniquely found in normal hpde sevs. this group includes a number of immune response proteins that are not found in any of the pancreatic cancer cell sevs. summary/conclusion: the differences in the proteomes of cancer and normal sevs may be indicative of their varying roles in cell transformation and helpful in delineating the types of evs that are being produced. in addition, these differences point towards their potential value as cancer biomarkers. proteomic profile of tumour-derived exosomes in plasma of melanoma patients introduction: in the past years, extracellular vesicles (evs) have attracted considerable interest due to their ability to provide valuable diagnostic information from liquid biopsies. the high abundance in all bodily fluids and their cargo stability confers evs the potential as a powerful tool to not only obtain novel biomarkers from inaccessible tissues, therapy response and monitoring, but also to reduce infection risks of conventional highly invasive biopsies. virtually all cells continuously release vesicles into the extracellular environment, diverse in size, content and features depending on the biogenesis, origin and function. this heterogeneity adds a layer of complexity when attempting to isolate and characterize tissue-specific vesicles. methods: hence, we aimed to use a immunomagnetic capture approach for prostate-derived evs from cell culture supernatants, with further investigation into human plasma and urine samples. analysis was performed by nanoparticle tracking analysis, western blotting and electron microscopy. additionally, an in-house spotted antibody microarray is in development. here, we intend to detect different ev sub-populations based on their surface markers. results: isolated immunocaptured ev populations based on the classical ev marker cd show an increased signal for the luminal protein tsg . ev populations targeting the tissue-specific marker prostate specific membrane antigen (psma), were found positive for tsg in a lower extent indicating a subpopulation of evs. the microarray uses less than µl of sample (concentrated cell culture supernatant, human plasma, urine) and leads to a faster characterization within h for ev surface marker as compared to western blot. summary/conclusion: immunomagnetic isolation might be a promising approach for liquid biopsy and thereby the microarray could be valuable to identify potential capture targets. the current design for different surface marker from samples simultaneously could be easily extended for sample size and surface profiling allowing for a more economical way to multiplex samples. paving the way for implementing a feasible and reliable technique for assessing urinary extracellular vesicles as biomarkers for bladder cancer in clinical practice introduction: extracellular vesicles (ev) in urine have been proposed as biomarkers for bladder cancer (bc). however, at present there are no standardized methods for ev isolation or urine sampling. our goal was to evaluate the ev isolation performance between different methods, the effect of the sampling time and the importance of urinary creatinine (ucr) normalization. methods: two urine samples of ml were collected from patients with non muscle-invasive bc: one from the first micturition and another from any time of the day. twenty ml were used for ucr measurement and ml were used for ev isolation by either precipitation with polyethylene glycol (peg), concentration by filtration (uf, centricon plus- , k, millipore), sepharose size exclusion column (sec), or combinations of these methods. additionally, the effect of protease inhibitors (pi) and dtt treatment after collection or during processing was analysed. size and number of particles were evaluated by nanosight and the presence of exosomal markers was evaluated by western blot. results: among the methods evaluated, uf + sec showed the best performance retrieving the highest number of particles in the range of - nm, and the highest protein expression of exosomal proteins. uf alone showed the highest concentration of ev, but with a tendency to isolate larger particles. particle concentration was positively correlated with ucr, reflecting the importance of ucr normalization before journal of extracellular vesicles comparing between patients. finally, no differences in the performance according to the time of collection, nor in the use of pi or dtt were observed. summary/conclusion: uf + sec gave the highest ev yield and was not affected by the time of urine collection. the use of pi and dtt can be avoided, and normalization to ucr should be considered when implementing this technique for assessing evs as biomarkers for bc in clinical practice. funding: pida . the introduction: human tumours, including pancreatic ductal adenocarcinoma (pdac), often harbour a subpopulation of cancer cells with extra centrosomes. we found, that these cells secrete an increased number of small extracellular vesicles (sevs), within the - nm size range. sevs play a role in cancer signalling and progression and are widely studied for their diagnostic potential. we aim to understand the role of sevs secreted by cells with extra centrosomes in shaping pdac-associated stroma, particularly fibrosis. methods: to study the sev mediated changes in the pdac microenvironment, we purified sevs through serial ultracentrifugation and size exclusion chromatography, characterised the content through silac-based proteomics, and assessed phenotypic changes in pancreatic stellate cells (pscs) and extracellular matrix (ecm) production through immunofluorescence staining. results: our data indicates, that the sevs secreted by cells with extra centrosomes are exosomes due to their endocytic origin, and we found, that they can activate pscs, key mediators of fibrosis in pdac. indeed, we observed an increased level of collagen i produced by pscs activated by sevs from cells with extra centrosomes as compared to cells without extra centrosomes. interestingly, we found, that psc activation through sevs is not mediated by tgf-β, assessed by the level of nuclear smad accumulation downstream of tgfβ activation, suggesting a novel mechanism of pscs activation. summary/conclusion: pdac cells with extra centrosomes contribute to a novel type of psc reprogramming, which could alter their ecm deposition and contribute to the extensive fibrosis observed in pdac. we are currently characterising the signalling pathways associated with sev mediated psc activation and how it impacts padc progression to better understand the role of centrosome amplification in the cancer-stromal crosstalk. exosomal carboxypeptidase e confers and cpe-shrna loaded exosomes inhibit growth and invasion of hepatocellular carcinoma cells. methods: exosomes were isolated from the culture media of high metastic hcc h cells and incubated with low metastatic hcc l cells. in other experiments, cpe-shna loaded exosomes from hek cells were incubated with hcc h cells. the recipient cells were analysed for proliferation using mtt assay, colony formation, and matrigel invasion. results: analysis of exosomes derived from hcc h cells revealed cpe-wt mrna and protein. exosomes released from hcc h cells were able to enhance proliferation and invasion of hcc l cells. when cpe expression was suppressed in the hcc h cells before exosome isolation, the exosomes had no effect on proliferation and invasion. these data demonstrate the ability of exosomes to confer growth and invasion in hcc cells and the role of exosomal cpe in driving the process.previously it was shown that down-regulation of cpe expression by shrna can reverse tumour growth and metastasis in an hcc mouse model. we therefore loaded cpe-shrna into exosomes by infecting hek (human embryonic kidney) cells with adenovirus carrying cpe-shrna-gfp. these modified isev abstract book exosomes were used to transfer cpe-shrna to hcc h cells, resulting in significant reduction in proliferation and colony-forming ability of these cells. cpe-shrna loaded exosomes were found to down-regulate the expression of cyclin d and c-myc, two genes with high relavance to tumour growth and metastasis. summary/conclusion: our results demonstrate the ability of exosomal cpe to enhance proliferation and invasion in low metastatic hcc cells and the potential to use shrna loaded exosomes to target cpe as a therapeutic strategy to treat liver cancer.funding: intramural program of the eunice kennedy shriver national institute of child health and human development, and national cancer institute, national institutes of health, bethesda, md. . stress hormones promote prostate cancer aggressiveness through modulation of mir- - p expression and exosome release north carolina central university, durham, usa introduction: despite proactive screening and steady declines in mortality, prostate cancer (pca) remains one of the most prevalent cancers among men. evidence suggests that chronic activation of stress signalling pathways can result in an altered mirnas transcriptome and affect exosomal content and release. here, we study the interaction between leptin and mir- - p expression, previously shown to be downregulated in pca patients. in addition, explored the effect of stress hormones cortisol and leptin on exosomal release and content from pca cells.methods: we utilized normal prostate cell line rwpe- , and pca cells pc , lncap and mda-pca- b. proliferation of cells treated with leptin in the presence or absence of mir- - p mimic or negative control was assessed by mtt, colony formation, wound healing, and expression of targets affected by mir- - p was assessed by western blotting. moreover, exosomes were isolated via differential centrifugation from pca cells treated with leptin or cortisol and exosome number was determined by nanotracking analysis. exosome content was determined by western blotting and proteomic analysis by mass spectrometry.results: we observed that leptin significantly decreased expression of mir- - p in rwpe- cells.co-treatment with mir- - p mimic and leptin abrogated these effects in a cell dependent manner. we also observed that co-treatment with leptin affected mir- - p target jag and other molecules involved in epithelial to mesenchymal transition. in parallel, we demonstrated that cortisol increases exosome secretion particularly in pc cell exosomes with a . -fold increase at nm cortisol compared to untreated. western blotting revealed the presence of gr in exosomes particularly at nm cortisol. summary/conclusion: understanding epigenetic regulation through mirnas and exosomes may be the key to understand stress hormone influence in pca progression. these findings suggest that stress hormones effectively affect mir- - p expression and exosomal release and signalling.introduction: extracellular vesicles (evs) are promising drug delivery vehicles for therapeutic microrna (mirna). for the loading of exogenous cargo, researchers broadly seek to either manipulate the evs directly or the cell that produce them. electroporation, sonication, and direct ev transfection are common methods that work by physical disruption or irreversible chemical addition, which may irreparably damage the molecules intended for therapy. on the other hand, transfection into the producer cells is a simple option that does not imperil ev integrity.methods: there are multiple factors that contribute to ev loading efficiency, including transfection reagent used, timing, and dosage. thus, we sought to establish a basic protocol and improve understanding of the underlying dynamics involved in a basic system consisting of hek t cells and mir- a- p mimic.results: in this work, we examined how different reagents lead to variable ev loading. then we looked at variable dosages, specifically the relationship between rna amount added to reagent, amount present in cell, and amount exported to evs. summary/conclusion: these results will help future studies produce evs with exogenously loaded small rna, and suggest future optimizations. funding: national institutes of health. r and t (host pathogen interactions at university of maryland). we report a single ev trapping method via aptamermediated assembly between au nanoparticle (aunp) and au superlattice template. we propose a chip-based ev trapping technique based on semiconductor processes. methods: we introduce aptamer coated au nanoparticle (aunp) and au superlattices as a template to capture evs. first, we fabricated poly(methyl methacrylate) (pmma) hole pattern on au-coated si substrates by using electron beam lithography (ebl). we designed nm-diameter hole patterns to capture one ev in each hole. to connect the aunp and the au superlattice template, we used an aptamer molecule as a linker strand. also, to capture individual evs, the aptamer molecule is designed to have a hairpin structure to specifically bind to cd , a protein marker of ev. we modified ʹ-terminal and ʹ-terminal of the cd aptamer with thiol group for the formation of self-assembly monolayer (sam) on both aunp and au superlattice surface. results: first, we coat the cd aptamer on the surface of aunp. afterwards, we load the aptamer-coated aunp into au superlattice template. ev solution is specifically bound to cd aptamer. after washing step, each ev is expected to locate within a single hole due to the size confinement of the hole. to separate the evs from the aptamer, we use restriction enzyme, bamhi, to recognize specific dna sequence and cleave them. summary/conclusion: in this report, we propose a aunp -linked au superlattice chip by aptamer molecules for trapping evs. we selected cd aptamer for specifically binding with cd in evs. in addition, we designed cd aptamer as a linker strand to connect introduction: a hallmark of platelet activation is the release of internal granules as extracellular vesicles/ microparticles. thrombolux is a dynamic-light-scattering-based (dls) instrument that was developed for use in clinical setting to check for platelet activation before transfusion. compared to traditional dls, the thrombolux requires no cleaning (single-use capillary) and requires very little sample ( µl). hence the thrombolux may be a useful instrument beyond platelet pack test in blood transfusion laboratory. we have evaluated its use as an in-process monitoring tool for industrial ev manufacturing, for both quantifying cells (input) and evs (output). methods: the thrombolux was used to test the activation status of expired platelet packs (donated by arcbs for research purpose). the readout was compared with platelet swirling test and flow cytometry data (surface marker). furthermore, the thrombolux was also tested for process development and ev manufacturing monitoring purposes at different stages of the process for its ability to rapidly obtain particle presence and size information on evs. time to result was also compared between different particle analysis methods. results: the thrombolux was a better predictor of platelet packs variability compared to the traditional platelet swirling method. however, we did not observe a strong correlation between the activation status and the flow cytometry-based activation marker data. the thrombolux was able to provide a useful estimation of particle presence and sizing of evs in-process.results are obtained rapidly, within minutes, with minimal sample prep. summary/conclusion: although we did not observe a significant direct correlation between flow cytometry activation data and the % microparticles (within a small sample size), the thrombolux has shown potential to become a useful tool for in-process monitoring for ev manufacturing and other ev research, in particular through its speed and ease of use. funding: all funding was through exopharm ltd (asx:ex ). secreted introduction: a major manufacturing challenge related to exosome bioprocessing is that of robust and scalable purification. as efforts to translate exosomes into clinics grows, the more important the design of quality systems which can reproducibly purify the product becomes. the current gold-standard, ultracentrifugation, was adopted from the viral vaccine industry, but remains imperfect in terms of scale up and manufacturing due to labour and time intensive process requirements. in order to follow the preferential adoption of more standard bioprocesses, as previously achieved by the viral vaccine industry, we show the development of two monolith chromatography steps which can be used to purify exosomes from a clinically relevant, allogeneic stem cell product (ctx e ). methods: t-flask expansion of ctx e cells was performed to yield batches of - l of conditioned medium. the medium was subsequently clarified by benchtop centrifugation, and concentrated into a crude concentrate by tangential flow filtration [tff], using a combination of . µm dead-end filtration prior to concentration in a kda hollow-fibre tff system. tff retentate was loaded onto ml hic or aex monoliths, for further purification. potency was assessed by a fibroblast wound healing assay in vitro. results: exosome presence was verified in the tff material by detection of cd and cd . exosomes recovered in this manner could achieve full wound closure in vitro over hours, when dosed at µg. further purification by monolith chromatography showed high levels of reduction of albumin, detected by western blot, as well as heightened ratios of particles to both total protein, and total dna. the results indicate that neither aex nor hic steps cause detrimental loss to product function, either alone or in combination with one another. introduction: custom-made platelet pellet lysate (ppl) and heat-treated ppl (hppl) exert strong neuroprotective effects of neurotoxin-exposed dopaminergic luhmes neuronal cell culture. this effect is significantly enhanced using hppl, which was also highly protective of th-expressing neurons in mice parkinson's disease (pd) model. introduction: there is a critical unmet medical need for new therapies to treat age-related diseases including cardiovascular diseases such as stroke. exosome derived from stem cells have shown intrinsic therapeutic potential in a variety of animal models of ischaemic diseases. we have identified scalable exosome production cell lines (purestem) as a source of angiogenic exosomes and are aiming to generate good manufacturing practice (gmp) grade therapeutic exosomes that can effectively mediate angiogenesis and tissue regeneration. we are developing exosome production and purification protocols that combine methods of tangential filtration flow (tff) and size exclusion chromatography (sec). the particle number and size were measured by both tunable resistive pulse sensing (trps) as well as nanoparticle tracking analysis (nta) for comparison. exosomes were characterized by detection of exosome surface markers and absence of cellular markers. purity was assessed by measuring particles per ug of total protein content. the angiogenic activity of purestem-exosomes was assessed using live-cell imaging to measure endothelial wound-healing and tube formation assays. we further investigated the molecular cargo of purestem-exosomes by screening mirnas targets, rna-seq analysis, and mass spectrometry analysis.results: the isolated purestem-exosomes using our developed protocols were highly purified, resulting purity in the range of e - e particles/ug. we selected angiogenic exosome-producing cell lines from our purestem library by screening for functional activity and characterizing their molecular cargo. we found that purestem progenitor-derived exosomes showed higher angiogenic potency than primary mesenchymal stem cell (msc)-derived exosomes. furthermore, angiogenic micrornas such as mir- were enriched in purestem-exosomes from certain producer cell lines. summary/conclusion: these data demonstrate the potential for using purestem lines as a highly scalable source of therapeutic exosomes. we were able to obtain highly pure exosomes that retain their angiogenic activity. we anticipate that purestem-exosomes will be a valuable resource for developing ev therapies for stroke and other ischaemic diseases. we have developed purification methodologies aimed at achieving a robust and scalable exosome production compatible with gmp for clinical grade purestem-exosomes. these developments have great potential as therapeutic agents for future preclinical in animal model of stroke and clinical trials. neuronal introduction: the hallmark of parkinson's disease (pd) is a-synuclein accumulation, predominantly in dopaminergic neurons, causing neurodegeneration. pd is also associated with insulin resistance, a condition characterized by phosphorylated insulin receptor substrate- (irs- ). besides motor symptoms, some pd patients develop mild cognitive impairment (pd-mci) or dementia (pd-d). given the importance for prognosis, there is an urgent need to develop biomarkers for distinguishing pd with normal cognition (pd-n) from pd-mci/d. neuronal-origin extracellular vesicles (nevs) contain cell signalling and pathogenic proteins (including a-synuclein), which may serve as biomarkers for alzheimer's disease, pd and other dementias.methods: from . ml of plasma from pd-n, pd-mci, and pd-d patients, we immunocaptured nevs using anti-l cam antibody. then, irs- pser and irs- ptyr and a-synuclein were measured in nevs using electrochemiluminescence immunoassays.results: a-synuclein was lower in pd-mci and pd-d compared to pd-n (p < . ) and significantly decreased with increasing motor symptom severity measured by mds-updrs iii score (p = . ). irs- pser was lower in pd-d than in pd-n. irs- ptyr significantly decreased with increasing mds-updrs iii score (p < . ). no biomarker was associated with disease duration. summary/conclusion: pd patients with cognitive impairment exhibited lower nev levels of a-synuclein than cognitively intact pd patients, whereas a-synuclein and irs- ptyr were inversely associated with pd motor symptom severity. additional biomarkers and measurements will be available by the time of isev. plasma nevs is a valuable tool for discovering biomarkers in pd and investigating aspects of disease progression. introduction: despite decades-long advancement in transplant medicine, there is a necessity for personalized approach regarding early kidney allograft injury recognition and immunosuppression therapy towards improved transplant outcomes. biopsy, a gold standard for assessment of kidney allograft injury, cannot be serially used for the diagnosis of subclinical injury due to it's invasiveness and possible sampling errors. instead, urine is easily obtainable and bearing extracellular vesicles (evs), potential carriers of pathological signals related to kidney injury. our aim was to set up a urinary ev (uev) isolation protocol that would allow consistent and reliable identification of their characteristics and cargo. methods: second morning urine sample ( ml) was collected from patients and processed within hours. oxalate precipitation, ph and dilution variability, uromodulin polymerization and high protein content were taken into account. isolated evs were defined by transmission electron microscopy (tem) and nanoparticle tracking analysis (nta). uev specific proteins and mirnas were analysed by western blot and qpcr, respectively. results: the optimal protocol relied on low speed urine centrifugation ( . x g, rt) for cell removal and storage at − °c prior to further analyses. after urine thawing at rt, added edta averted cryoprecipitate and uromodulin polymer formation, while concentrated pbs neutralized the ph. filtration through . µm pores was used for large particle removal, while centrifugal kda membrane units (amicon®, milipore) served for sample concentration followed by particle separation on sizeexclusion chromatography (sec; qevoriginal, izon q). protein vacant sec fractions (as rated at a ) were pooled and concentrated to a volume of µl. tem micrographs revealed high sample purity and cup-shaped morphology of uevs. as per nta results, the average mean size of evs was , nm with concentration range of × particles/ml of starting urine. uevs were positive for the tested marker proteins hsc , flotillin, tubulin, gadph and cd . qpcr verified mirna presence in uevs, with ct for mir let- i at . summary/conclusion: we successfully isolated pure uevs. the set up protocol will be used to assess uevs as non-invasive biomarkers of allograft injury in kidney transplant recipients. astrocyte-derived extracellular vesicles regulate dendritic spine formation and neuronal network connectivity introduction: recent advancements in the biology of extracellular vesicles have begun to implicate glial released microvesicles as mediators of glia to neuron communication, suggesting that alterations in the release and/or composition of astrocyte microvesicles could impact neuronal function. methods: astrocytes were allowed to constitutively release extracellular vesicles (adev-cr), or stimulated with atp (adev-atp). adevs were isolated by ultracentrifugation followed by proteomic analysis. we developed a normative whole transcriptome database using primary neurons exposed to adev-cr, and identified changes in neuronal gene expression produced by exposure of neurons to adev-atp. we identified a number of pathways associated with the biological response of synapse, spine and neurite outgrowth that were regulated by adev-atp. the molecular cargo of adev-atp responsible for regulating synaptic functions in neurons were characterized by biochemical, molecular, and functional assays. results: adev-atp enhanced the maturation of dendritic spines and produced functional enhancements in neuronal activity and network connectivity. the mechanism for this effect involved the delivery of integrin- and epha that were enriched in adev-atp. integrin- facilitated binding of adevs to the neuronal surface, and epha -receptor signalled through ephrin to the tyrosine kinase erbb / that regulated the phosphorylation and activation of trkb without increasing expression of the natural ligands bdnf or ntf . this direct activation of trkb increased the expression of the synaptic scaffolding proteins disc , arc, and cplx to promote the maturation of dendritic spines. this increase in mature dendritic spines was associated with increased neuronal activity and network connectivity demonstrating a functional strengthening of synapses. summary/conclusion: these data identify a molecular mechanism whereby modifications in adev protein cargo produced by the stimulation of astrocytes with atp regulates synaptic maturation through activation of trkb in a manner independent of growth factors. stephanie kronstadt and steven m. jay university of maryland, college park, college park, usa introduction: mesenchymal stem cell extracellular vesicles (msc-evs) have been shown to have an immunosuppressive effect in both autoimmune and inflammatory disorders. despite this, clinical translation of ev therapies is hindered by potentially low potency in vivo and the lack of a scalable biomanufacturing process. cell culture parameters are critical in modulating both yield and bioactivity of evs. thus, we hypothesized that the combination of chemical priming and d dynamic culture would enhance the yield and potency of immunosuppressive msc-evs. methods: bone marrow-derived mscs cultured in flasks were chemically primed using ethanol or curcumin. mscs were also cultured using a d-printed scaffold-perfusion bioreactor using a flow rate of ml/min. anti-inflammatory effects were assessed following application of msc-evs to lipopolysaccharide (lps)-stimulated murine macrophages. subsequent inhibition of the production of the pro-inflammatory cytokine il- , quantified using an elisa, was used to characterize evs as anti-inflammatory. in addition, both chemical priming and the bioreactor will be simultaneously utilized to potentially uncover any synergistic effects on ev immunomodulation abilities. nanoparticle tracking analysis (nta) was used to assess ev size and concentration while protein mass was measured via a bca assay. results: preliminary data suggests that priming mscs with µm ethanol for hours prior to ev collection results in a strong inhibition of il- production in stimulated murine macrophages. nta revealed that msc-ev yield increased by about two orders of magnitude in the bioreactor ( . e ± . e ) when compared with flasks ( . e ± . e ). protein measurements also indicated that ev production in the bioreactor (~ µg) was much greater compared with production in the flasks (~ µg). additionally, average protein content per ev was reduced in the bioreactor when compared with flask evs. regardless of tissue source. furthermore, comparison of adipose tissue-derived (ad) msc evs from three donors indicates varying pro-vascularization bioactivity between those donors evaluated in vitro via gap closure assay. similar results were observed for the bone marrow-derived (bm) msc ev donor groups. summary/conclusion: this work highlights the need for screening of donor derived-mscs before use for therapeutic ev production. additionally, standardized criteria for msc donor selection are needed before isolated msc evs can be used as a large-scale, repeatable therapeutic treatment. analysis of extracellular vesicle populations from malaria-infected erythrocytes by field-flow fractionation reveal distinct sub-sets alicia rojas a , paula abou-karam a , anna rivkin a , yael fridmann-sirkis b , yifat ofir-birin c and neta regev-rudzi c a department of biochemical sciences, weizmann institute of sciences, rehovot, israel, rehovot, israel; b wis, rehovot, israel; c weizmann institute of science, rehovot, israel introduction: malaria is one the most devastating infectious disease in the world and plasmodium falciparum (pf) represents the deadliest species. this parasite invades human red blood cells (rbcs) and releases extracellular vesicles (evs) carrying dna, rna and protein cargo components which are involved in the pathogenesis of the disease. recently, it has been shown in mammalian systems that evs are subdivided into different subpopulations, each with a distinct biological function. however, it is still unknown whether pfinfected rbcs (pf-evs) release different ev subpopulations with distinct cargo. methods: we isolated evs from pf-infected and uninfected rbcs, pf-evs or ui-evs, respectively, using differential centrifugation. the ev pellet was subjected to field flow fractionation (fff). the different subpopulations were collected, concentrated with size-exclusion filters and evaluated by nanoparticle tracking analysis. additionally, the presence of ev markers (sr and hsp ) were examined by western blot analysis. results: the fff analysis showed four particle subpopulations derived from the pf-evs and five in the ui-evs. the first three subpopulations were similar in their detection signals in both samples, but the fourth subpopulation was consistently higher in ui-evs than in pf-evs. moreover, hsp was detected in subpopulations and of both pf-evs and ui-evs, whereas sr only in subpopulation . isev abstract book summary/conclusion: pf-ev and ui-ev have similar separation profiles and proteins markers in their subpopulations, consistent with the fact that both samples are derived from host rbcs. additional data regarding the dna and rna cargo, as well as microscopic observations of the pf-ev and ui-ev subpopulations is necessary. this will clarify how malaria parasites sort their components into evs and which fractions are associated to immune evasion and pathogenesis. we have established a small size laboratory production of the microalgae culture in order to harvest the extracellular vesicles (evs) for pharmaceutical and medical uses. in this work we report on globular particles in the isolates from media of microalgae of two types, that we recognize as evs. we observed changes in their production at different temperatures and conditions. methods: samples were fixed by various combinations of aldehyde fixatives and/or osmium tetroxide. they were dehydrated in a graded series of ethanol, hexamethyldisilazane, and air dried. they were au/pd coated for inspection with scanning electron microscopes (sem) crossbeam fib-sem gemini ii (zeiss, germany) and jsm- f field emission scanning electron microscope (jeol ltd., tokyo, japan). results: microalgae were incubated overnight at °c and °c in growth medium and in growth medium supplemented with detergent. the samples obtained from the microalgae culture contained particles that we recognized as extracellular vesicles, however, these particles do not correspond to characteristic shapes of membrane enclosed entities without internal structure. increased temperature and/or presence of surfactant (triton x- and sodium dodecyl sulphate) stimulated formation of evs of different shapes and sizes. the isolates of these samples were rich with evs. in the presence of surfactant, the cell-walls detached from the cell and collapsed upon dehydration. this was documented by sem. summary/conclusion: focused ion beam technique revealed complex internal structure of the algae. it seems from the shapes of the observed structures that the particles deposited on the surface of the microalgae do not derive from budding of the membrane surface, but are instead shed by the cells from the cell interior upon the rupture of the cell wall. key: cord- -kt gt t authors: nan title: poster session abstracts date: - - journal: pediatr pulmonol doi: . /ppul. sha: doc_id: cord_uid: kt gt t nan causing mutation is ∆f , making studies of nbd essential in understanding cftr function. crystal structures have been solved of wild-type (wt) and mutant cftr nbd , including variants containing ∆f (lewis et al. embo j. ; lewis et al. j. biol. chem. ; thibodeau et al. nat. struct. mol. biol. ) . cftr nbd contains a regulatory insertion (ri) and a regulatory extension (re) that contain pka phosphorylation sites. although the nbd core is very similar in the various structures, the ri and re in some structures of human wt nbd differ by °rotations, indicating that they are mobile. we have used nmr spectroscopy to study wt and ∆f nbd in the phosphorylated (phospho) and non-phosphorylated (non-phospho) states, and bound to different regions of cftr. despite their similar structures, differences are observed in the nmr spectra of wt and ∆f nbd . these changes, possibly due to altered dynamics or interactions of the ri and re with the nbd core, will be discussed. the inherent stability and function of nbd could be affected by these changes, accounting for part of the ∆f defect. different interactions between wt and ∆f nbd with the icls could also affect channel function. to identify residues comprising the icls, we generated a homology model of cftr based on the crystal structure of sav in which nbd and nbd form a productive dimer (dawson and locher. nature. ) . our data on a peptide corresponding to the first intracellular loop (icl ) indicate that binding to nbd requires phosphorylation of the ri. our homology model shows that the icl binding site is buried when the ri is bound to the nbd core. nmr data comparing phospho and non-phospho nbd indicates that phosphorylation of the ri and re disrupts their interactions with the nbd core, likely exposing the site for icl binding. the structure of the molybdate transporter modb c , with the nbds in an open conformation, shows icl binding is cognate nbd at a different interface (hollenstein et al. nature. ) . icl in modb c interacts with the nbd near y , the analogous residue to f in cftr. we will show binding data of icl with wt and ∆f nbd in different states and also present resonance assignments of nbd to map the icl interaction sites. since the sav and modb c structures were solved in different states, the different icl interactions may represent structural changes during the reaction cycle. the ∆f mutation may thus affect intramolecular associations in certain conformations, which would account for another part of the ∆f defect. probing differences in interactions of wt and ∆f nbd is critical for understanding the molecular basis of normal cftr function and of the altered cftr function in cystic fibrosis. shsps target ∆f cftr for erad via the sumo pathway ahner, a. ; brodsky, j.l. ; frizzell, r.a. . cell biology and physiology, university of pittsburgh, pittsburgh, pa, usa; . biological sciences, university of pittsburgh, pittsburgh, pa, usa the most frequent disease-causing mutation in cystic fibrosis (cf) is a deletion of phenylalanine at position (df ) in the first nucleotide binding domain of cftr. essentially all of the mutant protein, as well as % of wild type (wt) cftr, is retained in the er and targeted for erassociated degradation (erad) by the s proteasome. key mediators of cftr folding and degradation are molecular chaperones, which help protein substrates fold, but can target them for degradation if folding efficiency is compromised. to identify factors modulating erad of cftr we performed a microarray analysis, screening for transcription profile differences between yeast expressing cftr and control strains, and identified enhanced transcript levels for the small heat shock protein hsp in yeast expressing cftr. we then demonstrated that cftr degradation was blocked in strains lacking the genes encoding the two yeast shsps, hsp and hsp ( ) . to examine whether shsps regulate cftr biogenesis in mammalian cells, we asked which of the human shsp homologues are endogenously co-expressed with cftr using rt-pcr and western blot analysis. we detected shsps, including alphaa-crystallin and hsp , in calu- and primary hbe cells. co-expression of alphaacrystallin or hsp together with wt or df -cftr in hek cells selectively decreased the steady state levels of df -cftr. pulse-chase experiments showed that the rate of df -cftr degradation was enhanced when either shsp was over-expressed, but wt cftr biogenesis was unchanged. co-immuno-precipitation experiments in hek cells revealed that alphaa-crystallin and hsp interacted preferentially with df -cftr. thus far, our results suggest that shsps selectively increase df -cftr's accessibility to proteasome-mediated degradation pathways. previously, hsp was reported to interact with ubc , the sumo (small ubiquitin-like modifier) conjugating enzyme ( ) . sumo is covalently linked to its substrates, often at sumoylation consensus sites; similarly to ubiquitin, this occurs through a series of thiol transfer reactions. we confirmed this interaction by co-immuno-precipitation and found that ubc as well as the sumo specific protease, senp , are expressed in airway epithelial cells. over-expression of ubc decreased, while over-expression of senp increased df -cftr protein levels. pulse-chase experiments indicated that these enzymes regulate selectively the degradation of df -cftr, similarly to the shsps. preliminary studies in vitro and in vivo indicate that cftr, and particularly purified nbd , is sumoylated and that hsp facilitates this process. mutational analysis of the sumoylation consensus sites within cftr suggests a cycle of sumo modification that facilitates wt cftr biosynthesis but targets df -cftr for degradation, probably because the balance between sumo addition and removal is impaired by an excessive interaction of shsps with the mutant protein. [supported by grants from the nih and the cystic young, a. physiology and biophysics, chicago medical school, north chicago, il, usa efficient retrieval of wtcftr from the cell surface is mediated by components of the clathrin mediated endocytic pathway; however many aspects of cftr endocytosis remain to be elucidated. we have previously shown that endocytosis of wtcftr is dependent upon recognition of a tyrosinebased motif in the carboxyl tail of cftr by the ap endocytic adaptor complex. in contrast to wtcftr, less is known about the mechanisms whereby mutant cftr undergoes internalization. according to one model, df cftr undergoes accelerated endocytosis compared to wtcftr, whereas others have argued that endocytic rates of df cftr are comparable to those of wild-type cftr. to further define the function of proteins involved in the initial steps of wt and mutant cftr endocytosis, we investigated the role of the gtpase dynamin. dynamin has been proposed to play a key role in endocytosis by facilitating the scission of nascent clathrin coated vesicles from the cell surface. until recently, very few tools were available to modulate clathrin mediated events. we took advantage of dynasore, a newly discovered small molecule, non-competitive inhibitor of the gtpase dynamin. dynasore acts as a potent, rapid and reversible inhibitor of endocytic pathways known to depend upon dynamin, by blocking coated vesicle formation within seconds of dynasore addition. endocytosis of wtcftr was inhibited rapidly and efficiently upon addition of dynasore. furthermore, within minutes of dynasore washout, endocytosis of wtcftr resumed completely. also upon dynamin washout, normal trafficking and recycling of wtcftr was also observed. temperature correction of df causes expression of cftr at the cell surface, however such 'rescued' df cftr is not stabilized upon returning cells to c. application of dynasore prevented such rapid loss of mutant cftr from the cell surface. since maintenance of mutant cftr at the cell surface is critical for effective therapeutic intervention in patients with cf, our results provide insight into additional potential targets for pharmacological manipulation for the treatment of cf. richardson, j.m.; thibodeau, p.h.; watson, j.; thomas, p.j. physiology, ut southwestern medical center, dallas, tx, usa protein misfolding is the basis for a multitude of human diseases; however, the mechanisms underlying misfolding are not well understood. most cases of cystic fibrosis are associated with mutations-including the most common, deletion of phenylalanine (∆f ) in nbd -that interfere with the folding of the cystic fibrosis transmembrane conductance regulator (cftr). the resulting loss of functional cftr causes the disease. thus, elucidating how ∆f , affects the folding of cftr is critical to understanding the pathology of the disease. cftr is composed of five domains: two integral membrane transmembrane domains (tmds), a regulatory domain (r), and two nucleotide binding domains (nbds). ∆f occurs in the n-terminal nbd . both the murine wildtype and ∆f nbd can be expressed in bacteria and purified to near homogeneity. while the soluble expression yield of ∆f is lower than the wild type under identical conditions, ∆f achieves a native conformation similar to wild type as monitored by a variety of hydrodynamic and spectroscopic characteristics such as analytical size exclusion chromatography, circular dichroism, and fluorescence. recently, a non-native, but folded, species has been detected under mildly denaturing conditions. far-uv cd reveals a time and temperature dependent conversion from a mixed alpha/beta native conformation to a less helical non-native conformation, while fluorescence measurements reveal a parallel blue shift in the peak emission intensity from to nm. this non-native species is in a more open conformation as determined by both limited proteolysis and the change in retention time on analytical size exclusion chromatography. the conversion to this state is inhibited by the native state ligand (atp) and by the presence of the second site suppressors (g e, r m, and r k). notably, the ∆f nbd protein populates the non-native state under milder conditions than the wild type nbd . these studies reveal the properties of the native state and its conversion to a partially folded state that is affected by the ∆f mutation. this data highlight the proximal affect of the disease causing mutation and provide a means of assessing strategies designed to correct the defect. this work is supported by the cystic fibrosis foundation through a postdoctoral training grant and by the nih we have previously demonstrated that the stability of cftr is negatively modulated by the cftr interacting protein, cal. cal competes with nherf in binding to the pdz motif at the c-terminus of cftr and directs cftr trafficking to the lysosome. recently, silencing of cal or overexpression of nherf has been shown to restore chloride transport activity in ∆f -cftr cell lines. to identify the molecular machineries involved in cal dependent degradation of cftr, we investigated the role of the cal interacting snare protein syntaxin (stx ). stx protein expression and function were manipulated by sirna silencing, over-expression, and a dominant-negative mutant. we also performed a functional study of chloride transport activity in cystic fibrosis bronchial epithelial cell line cfbe o-stably expressing either wt-cftr or ∆f -cftr. the endogenous stx and cal protein expressions were knocked down by sirna techniques in multiple human cell lines including hek , hela cell stably expressing ∆f -cftr and cfbe o-stably expressing either wt-cftr or ∆f -cftr. in hek cells, knockdown of either stx or cal augments the expression of transfected gfp-tagged cftr (gfp-cftr), which is consistent with the observation that overexpression of either stx or cal enhances the degradation of cftr. stx interaction with cal was confirmed by a co-immunoprecipitation assay. because of this interaction, we hypothesize that stx negatively regulates cftr protein stability by interacting with cal which in turn binds to the c-ter-minal pdz motif of cftr. consistent with his hypothesis, stx knockdown has no effect on protein levels of a cftr mutant lacking the pdz motif (gfp-cftr∆trl). more importantly, cal knockdown eliminates the inhibitory effect of stx overexpression on gfp-cftr. a cell surface biotinylation assay and confocal microscopy showed significant increases in plasma membrane expression of cftr in stx knockdown cells without gross changes in the expression pattern. furthermore, consistent with its trans-golgi localization, stx knockdown has no effect on gfp-∆f -cftr expression. however, ∆f -cftr increases significantly in stx knockdown, temperature-rescued cells. to assess the effect of stx in bronchial epithelial cells, stx was knocked down in cfbe o-stably expressing wt-cftr. consistent with the observations in hek and hela cells, the expression of untagged wt-cftr is also augmented. knockdown of stx increases cftr expression in a does-dependent manner, reciprocal to the expression level of stx , without changing the level of expression of cal or gapdh. furthermore, stx knockdown lead to a does-dependent increase in cftr mediated short-circuit current that is stimulated by forskolin and inhibited by cftr chloride channel inhibitor cftrinh- . more importantly, in temperaturerescued cfbe o-stably expressing ∆f -cftr, stx knockdown tripled the cftr mediated short-circuit chloride current. these results not only delineate the effect of stx on post-golgi trafficking of cftr and its dependence on cal-mediated pdz-based interaction, but also points to it as a potential therapeutic target in conjunction with the er-quality control based rescue of ∆f -cftr. supported by the cystic fibrosis foundation and the national institutes of health. seavilleklein, g. ; evagelidis, a. ; amer, n. ; chappe, f. ; hanrahan, j. ; chappe, v. . physiology & biophysics, dalhousie university, halifax, ns, canada; . physiology, mcgill university, montreal, qc, canada cftr channel activity is regulated by phosphorylation and de-phosphorylation of the r-domain. direct pkc phosphorylation at specific sites in the r domain and nbd is essential and modulates cftr activation by pka. however, the molecular mechanism by which phosphorylation of the r domain induces channel activity remains unknown. we have reported biochemical evidence for the role of phosphorylation in domain-domain association using a deltar-split construct encoding the front (tmd -nbd ) and back (nbd -tmd ) halves of cftr as separate polypeptides ( ) . in agreement with the inhibitory role of the r domain, iodide effluxes and patch-clamp experiments confirmed that deltar-split channels were functional and constitutively active without camp stimulation. co-transfecting a plasmid that directs expression of the r domain restored the pka-dependence of re-assembled channels. co-immunoprecipitations of the halves of the deltar-split with a gst-r domain fusion protein in vitro or with the cotransfected r domain in bhk cells revealed that pka phosphorylation enhances the r domain binding to the rest of the channel. we have now investigated the effect of pkc phosphorylation on the r domain binding. our data demonstrate that pkc phosphorylation also induces a strong binding of gst-r domain in vitro or co-transfected r domain in vivo when bhk cells are stimulated with pma. interestingly, the combination of pkc+pka phosphorylation induced a stronger binding than pka alone. mutation of all consensus pkc sites on the r domain to alanine ( ca) eliminated specific pkc phosphorylation but did not alter pka phosphorylation in gst-rd. western blotting and confocal microscopy experiments confirmed that expression levels of all domains of the split- ca/rd are unchanged compared to split-rd and co-localize at the plasma membrane of bhk cells. basal activity of the split- ca/rd was similar to that of the split-rd in iodide efflux experiments. however, activation by camp+ibmx was delayed and significantly reduced ( . % of split-rd and . % of wt-cftr activation). these results confirm the functional re-assembly of the ca-rd with the front and back halves of the channel and the inhibitory role of the r domain. in co-immunoprecipitation experiments, the same level of interaction was observed between the ca/rd and rd with the split halves suggesting that basal, inhibitory interaction of the r domain is independent of pkc phosphorylation. however, ca/rd binding with the split channel was not increased by pkc or pka stimulation. we conclude that pkc phosphorylation is essential for pka-dependent binding of the r domain to the rest of the channel, and that the combined effect of these kinases further strengthens the r domain interaction. these structural changes are consistent with the essential role of pkc sites for cftr activation by pka as previously reported ( ) . supported by cihr, nshrf, dmrf & ccff. g.seavilleklein was supported by nserc. ( ) the in vivo folding mechanism of multidomain membrane proteins, including the cystic fibrosis transmembrane conductance regulator (cftr), is poorly understood. cftr, a member of the abc transporter superfamily, contains two symmetrical halves, each consisting of a membrane spanning domain (msd and msd ) and a cytosolic nucleotide binding domains (nbd and nbd ), connected by the regulatory (r) domain. to address whether cftr follows the archetypical domain-wise folding of multidomain soluble polypeptides and/or the cooperative domain folding mechanism, first we determined domain combinations that are necessary and sufficient to escape the endoplasmic reticulum (er) quality control. one-, twoor three-domain assemblies failed to be processed efficiently in cells, measured by biochemical and morphological assays. the smallest folding unit that escaped the er quality control was composed of a four-domain assembly, containing msd , nbd , r or msd as linear or split polypeptide. as a second approach we showed that cf-causing point mutations in the msd , nbd or msd provoked the er retention of the full-length cftr with severe conformational defect in the nbd , measured by limited proteolysis and immunoblotting, using domain spacific antibodies. the posttranslational folding kinetics of the four-domain folding unit was comparable to that of cftr, suggesting that cooperative domain assembly is essential for the channel biogenesis. this mechanism provides an explanation for the processing defect caused by numerous cf mutations and outlines a possible paradigm for cftr folding in living cells. cftr in post-golgi compartments. our results, jointly, uncover the role of n-glycans as a critical structural determinant of cftr conformational maturation/stability in a chaperone-independent manner. karamyshev, a.l. ; patrick, a.e. ; johnson, a.e. ; thomas, p.j. . ut southwestern medical center at dallas, dallas, tx, usa; . texas a&m university, college station, tx, usa cystic fibrosis (cf) is caused by mutations in cftr (cystic fibrosis transmembrane conductance regulator). cftr, a member of the abc transporter family, is a multispanning membrane protein critical to chloride and water movement in secretory epithelia. over one thousand mutations, in all parts of the cftr sequence, have been identified, many of which disrupt the function of the protein to cause the disease. some of the disease-associated mutations in the n-terminal portion of cftr were predicted to alter cotranslational interactions with factors involved in the membrane integration of cftr required for its proper function. to test this hypothesis, a photoreactive probe was incorporated into different positions of the n-terminal portion of cftr by introducing amber stop codons and translating these messages in vitro in the presence of the amber suppressor trna n ε -( -azido- nitrobenzoyl)-lys-trna amb . ribosome-associated nascent chains of a specific length containing the modified amino acid at a defined position were produced using truncated mrnas. some of these cftr nascent chains photocrosslinked with proteins of approximately and kda molecular mass. crosslinking to the ~ kda protein was observed only after the tm span had emerged from the ribosomal tunnel. moreover, truncation analysis demonstrated that tm is necessary for this interaction. in evaluating a range of nascent chains, the extents of photocrosslinking to the ~ kda and kda proteins often varied inversely. immunochemical and knockdown/depletion studies indicate the ~ kda protein is srp , a subunit of the signal recognition particle involved in targeting ribosome nascent chain complexes to the translocon in the endoplasmic reticulum membrane. highlighting the functional relevance of this interaction, in cells with reduced srp levels, nonglycosylated forms of cftr accumulate and total cftr levels decrease. notably, some cf-causing mutations in tm also reduce crosslinking to srp. these data reveal that mutational inhibition of nascent cftr association with srp likely contributes to or mediates some types of cf. aleksandrov, l.; aleksandrov, a.a.; riordan, j.r. the initial reversible steps of atp binding to the first (nbd ) and second (nbd ) nucleotide binding sites of cftr are divalent cation independent and dependent, respectively (aleksandrov et al, j biol chem , - , ) . subsequent to the initial binding, either mg or mn drive rapid hydrolysis at the second site while promoting trapping of the nucleotide at the first site. this occlusion at the first site of functional wildtype cftr is somewhat similar to that which occurs when the catalytic glutamates in both of the hydrolytic sites of p-glycoprotein are mutated. this occluded state has been proposed to be the result of dimerization of the two nbds and represent a transient intermediate formed during atp hydrolysis (tombline and senior, j bioenerg biomembr , - , ) . to test the possible relevance of this interpretation to cftr, we have now characterized the process by which nbd occludes [ p]- n atp and [ p]- n adp. we have now found that only n atp and not n adp can be bound initially at nbd in the absence of mg. consistent with this mg requirement for the ndp binding, n adp or adp is able to compete for the n atp binding site only when the divalent ion is present. despite the lack of a requirement for mg for atp binding, retention of the nucleotide triphosphate at the binding site at °c was dependent on the cation. however, at reduced temperature ( °c), n atp remains locked in the binding pocket with virtually no reduction over a hour period even in the absence of mg. these apparently paradoxical temperature effects hint that different molecular events can contribute to the occlusion of nucleotide at nbd . that the occlusion process occurs exclusively at nbd and does not depend on its interaction with nbd , was shown by experiments in which identical behavior was exhibited by ∆nbd constructs of cftr. thus, nucleotide trapping by wild-type cftr, unlike that by mutant p-glycoprotein, is accomplished by events at a single domain (nbd ), rather than by dimerization of the two nbds. (supported by the nih and cff). cftr consists of five domains or regions, two regions that span the cell membrane (the tmds), two cytoplasmic nucleotide-binding domains (the nbds), and a regulatory (r) domain. the r domain is unique to cftr: without phosphorylation of this domain, the cftr chloride channel does not open. the tmds and the nbds of cftr are similar to those found in many atp-binding cassette proteins, the majority of which are transporters. the domains are fused in a single polypeptide chain, linked in the sequence: tmd , nbd , r domain, tmd , nbd . the first nbd is the site of the location of the mutation that is responsible for the development of cystic fibrosis in the majority of human patients (loss of phenylalanine ). although there is a structure for nbd , we are still lacking a structure for the entire cftr protein. we have employed electron microscopy to provide low-to medium-resolution structures for the complete cftr protein which was expressed in bhk cells and purified by affinity chromatography. using single particle analysis combined with site-specific labelling using . nm diameter ni-nta nanogold, we have identified the locations of nbd and the region around residue of the r-domain within the low resolution d structure. by a process of elimination, this has also allowed the identification of nbd and the region occupied by the two tmds. these low resolution studies have now enabled us to interpret medium resolution d structural data obtained for cftr. progress in obtaining two-dimensional arrays (crystals a single molecule thick) should allow higher resolution structural data to be obtained for the entire cftr protein -sufficient for the identification of the transmembrane alpha helices. these data should allow a much clearer picture of the transmembrane channel formed by cftr and will inform molecular modelling approaches aimed at the development of novel drugs for the treatment of cystic fibrosis. we explored the gating process by introducing cysteines along the entire lengths of various transmembrane domains and extra-cellular loops, and studying their ability to chemically modified. we study the changes in their reactivity in closed and open channel states to characterize the changes in transmembrane domain conformations during gating. using membrane impermeant mts-reagents, we have characterized the conformational changes in tm -ecl , ecl -tm , ecl -tm , and ecl -tm of cftr. our results suggest that atp binding induces modest changes in the transmembrane domains and extracellular loops. channel opening involves a rearrangement of the tm helices leading to decreased hydration of residues near the extracellular end. this conformational change is either a part of, or allosterically coupled to the gating mechanism. physiological role of nherf in the regulation of cftr raghuram, v.; yang, y.; yaemsiri, s. laboratory of kidney and electrolyte metabolism, nhlbi/nih, bethesda, md, usa in the apical membrane of epithelial cells, cftr seems to exist within a multiprotein complex, in which its activity is regulated by interactions with other proteins. the c-terminus of cftr contains a pdz-binding motif, which binds to several pdz domain-containing proteins, including members of na+/h+ exchanger regulatory factor (nherf) family, nherf /ebp , nherf /e karp, cap /nherf , nherf , and cal/pist/gopc. pdz domain proteins act as scaffolds to assemble signaling proteins to form signaling complexes. these signaling complexes are central to achieve specificity and efficiency of signal transduction. nherf and nherf have a c-terminal erm domain, which interacts with cytoskeleton-associated erm proteins. this interaction may anchor cftr to the actin-cytoskeleton and restrict its movement within the membrane. in addition, several functional roles have been proposed for cftr-pdz domain interactions, including as an adapter to promote efficient pka phosphorylation of cftr, apical targeting and recycling of cftr, regulation of single-channel activity, organization of β-adrenergic, and lysophosphatidic acid- receptor signaling complex, and trafficking of romk channels. our current understandings of the role of cftr-pdz interactions are mostly obtained from heterologous expression studies. very few studies have addressed the role of pdz domain proteins in native epithelial cells or in organ physiology. we have used rna interference techniques to produce a knockout or knockdown (kd) calu- cell lines using a lenti-viral vector for stable suppression. using four different shrnas, we have generated four stable nherf -kd cell lines with varying levels of nherf suppression. in these cells, nherf expression ranged from - % of calu- nt cells (control cells). in nherf -kd cells, steady-state cftr protein level was greatly reduced, whereas its apical localization was unaffected. we also found structural defects in the apical microvilli. in polarized nherf -kd epithelia, membrane-permeable camp analogs were unable to stimulate cftr mediated short-circuit currents. however, forskolin induced response was nearly normal despite reduced cftr expression. examinations of βadrenergic-, and adenosine-receptor signaling pathways suggest that nherf plays a unique role in organizing the adenosine receptor signaling pathway. abnormal retention of the df cftr mutated protein in lung epithelial cells underlies the pathology in a large proportion of individuals with cystic fibrosis. a drug discovery alliance between cfft and biofocus dpi was initiated with the aim to identify novel genes which upon shrna-mediated knockdown were able to efficiently restore df cftr activity. a high-throughput assay based on an yfp halide reporter was developed in a human cystic fibrosis bronchial epithelial cell line (cfbe o-), whereby the rate of halide influx directly correlated to df cftr activity. biofocus dpi's proprietary adenoviral shrna libraries totalling , viruses were screened in this functional assay and yielded hits. these hits were repropogated to generate new adenoviral stocks and rescreened at three concentrations on the yfp halide reporter assay. genes were eliminated due to potential cytotoxic effects, and hit calling was performed on the remaining hits. a total of duplicate hits were confirmed ( %). of the confirmed hits had more than one shrnas which targeted the respective gene. confirmed hits were stronger in restoring df cftr function than the most potent positive control: syntaxin- . in order to elucidate which of these hits induce halide influx via direct rescue and activation of df cftr, the yfp halide reporter assay was performed in the presence or absence of the cftrinh . these cftr dependent hits will move forward into subsequent validation experiments whereby hits will be categorised into potentiators and correctors. the final goal of this program is to prioritize targets for entry into drug discovery. restoring expression and/or function of mutated cftr provides a means to treat cystic fibrosis. biofocus dpi -a galapagos company -in collaboration with the cystic fibrosis foundation therapeutics (cfft) has established a drug target discovery project that will enable the selection of proteins that rescue mutated cftr. by screening biofocus dpi's proprietary adenoviral shrna libraries in a high-throughput primary functional assay for cftr activity in human cells, we have identified and confirmed hits out of , that entered the primary screen. to further validate these proteins as potential drug targets, we have set up a series of experiments and assays focused on the rescue of cftr expression and its maturation by knock-down of specific targets. ) in order to establish whether the knock-down of a specific target was able to promote df -cftr plasma membrane localization, we performed immunofluorescence stainings of df -cftr in a patient-derived human cell line carrying the df -cftr mutation (cfbe o-) grown in air-liquid interface culture. a transduction protocol to deliver shrna adenovirus to this polarized airway cell model system has been optimized and preliminary results showed increased levels of expression of df -cftr at the plasma membrane level upon knock-down of syntaxin- , our positive control. experiments performed at οc also demonstrated redistribution of df -cftr to the apical plasma membrane at low temperature. we will show the results of all the shrna hits in this assay. ) changes in the post-translational maturation levels of df -cftr upon knock-down of specific proteins were evaluated by determining the accumulation of band b and c by using a western blot approach. in this way, we can select targets able to promote post-translational maturation of df -cftr and/or its escape from er in the cfbe o-cell line. accumulation of band b and c indicates maturation of cftr and will be considered as a beneficial effect. preliminary results show increased accumulation of band b upon knock-down of specific targets and overexpression of wild-type cftr induces band c accumulation. experiments performed at οc also demonstrated increased band b expression. we will show the effect of all the shrna hits in this assay on band b and c expression. we believe that the results obtained by the combination of those two approaches will guide us in obtaining very strong drug targets for entry into a drug discovery phase. because gene therapy as a corrective measure for cf is possible in the foreseeable future, it is important to understand whether the global effects of gene introduction will have significant detrimental impact on cell function. to address this question, global gene mrna expression profiles were created with the affymetrix u a & b chip sets. wild type cftr (n= ), ∆f (n= ) and g d (n= ) were transfected in separate ib cell cultures using lipofectamine ™. control expression profiles were generated from untreated ib cells; one from this experimental set and five from previous control experiments. differential gene expression was determined by anova using a % false discovery rate threshold. genespring was used for data analysis and visualization. genego was used to determine which functional categories were over represented in the list and further assisted in interpretation of the results. rt-pcr was used to confirm differential expression of selected genes and examine the response in more detail. all transfected constructs produced very similar gene expression profiles quite distinct from the untransfected cells. probe sets representing uniquely named genes were significantly different between transfected and untransfected cells. cftr mrna expression in all forms was increased an average of -fold. transfection of ib - cells resulted in upregulation of a variety of genes involved in several basic processes: inflammation, protein folding and cytoskeleton framework regulation. genego canonical pathways with a significantly enriched number of genes from the data set involved glucocorticoid signaling and regulation, cytoskeleton remodeling and adhesion plus a variety of other signaling pathways including il , chemokines, g-protein and mapk cascades. cell processes overrepresented in the list were predominately associated with protein folding, inflammatory responses and cytoskeleton regulation. geneontology categories over represented in the list were regulation of signal transduction, receptor mediated endocytosis and protein folding. the proinflammatory interleukins il and il were upregulated . fold and fold respectively. the genes most upregulated, samd ( -fold) and oasl ( fold), are not well annotated. as controls, rt-pcr analysis of cells treated with either lipofectamine ™ alone or any form of cftr dna alone did not show upregulation of il , il , samd or oasl. in cells transfected with lipofectamine containing dna, upregulation of il (n= ), oasl (n= ) and samd (n= ) was confirmed by rt-pcr. oasl is known to interact with methyl cpg-binding protein , a dna transcription repressor acting at methylated promoter regions. thus it is possible that the global effects noted here leading to dramatic upregulation of two novel genes oasl and smad could be previously unrecognized consequences of gene therapies. cystic fibrosis (cf) is a disease which is caused by mutations of the cf gene, which codes for the cftr chloride channel. the ∆f -cftr mutation results in a mutant protein that undergoes transcription but fails to localize to the apical membrane, where it normally functions as a camp-regulated clchannel. however, clchannel activity can be detected at the plasma membrane when ∆f -cftr is overexpressed, synthesized at a reduced temperature, or in the presence of chemical chaperones. thus, strategies that facilitate the translocation of ∆f -cftr to the plasma membrane by increasing cftr transcription may be beneficial for the treat-ment of cf. previous studies performed using fluorescence halide efflux measurements and short-circuit current voltage clamp have shown that treatment with pparγ (peroxisome proliferator activated receptor gamma) agonists, such as pioglitazone and fll (fmoc-l-leucine), resulted in an increased biosynthesis and trafficking of ∆f -cftr to the cell surface. this effect was at least partially due to increased ∆f -cftr expression. further studies were designed to investigate the effect of pparγ agonists on cftr promoter activity. two firefly luciferase-conjugated cftr promoter plasmids, containing either . kb or . kb fragments, were transfected together with a control plasmid containing a renilla luciferase in human alveolar basal epithelial (a ) cells. the ratio of the firefly to renilla luminescence was used to measure cftr promoter activity. pparγ agonists increased cftr expression acting both on . kb and . kb promoter fragments. these results were confirmed in quantitative rt-pcr studies. transcription factor binding site analysis using current online programs and matrices showed three putative ppre (peroxisome proliferator response element) consensus sequences in the cftr promoter. the interaction of these sites with pparγ was shown using emsa (electrophoretic mobility shift assay). interestingly, none of these consensus sequences appear in the . kb construct, which still maintained a significant pparγ activation response. therefore, we hypothesized that a novel pparγ agonist effector site(s) is involved in transcription activation of cftr. to characterize this novel site(s), a series of deletion constructs to the promoter region were created, using mutation-introduced restriction sites in the original promoter construct. the new constructs ranged from . kb to base pairs upstream of the transcription start site. in preliminary studies we found that at least one previously uncharacterized pparγ effector site was present in the . kb promoter fragment that regulated cftr expression. additional experiments were performed using emsa to determine the proteins and cofactors which controlled the optimal conditions for cftr expression. the results of this study show that pparγ agonists, currently prescribed to treat hyperglycemia and improve peripheral insulin resistance, may also have additional benefits for the treatment of cf. our studies also show that pioglitazone and other pparγ agonists, by increasing cftr expression, are potent activators of clflux in epithelial cells expressing ∆f -cftr. this work was supported by grant from the canadian cystic fibrosis foundation decreased by µm gsno. the effect of gsno on hop expression was post-transcriptional. in cfbe o-cells transfected with sirna duplex specific for hop ( hr), hop expression was modestly inhibited; however, gsno dramatically augmented the inhibition. we next showed that knockdown of endogenous hop by sirna increased the steady-state levels of immature and mature forms of cftr. though overexpression of hop did not significantly affect cftr expression (immature and mature forms increased . ± . and . ± . fold respectively), in the presence of µm gsno, expression of both immature and mature cftr forms significantly increased in the hop overexpressing cells ( . ± . and . ± . fold respectively). next, we treated cfbe o-cells with a pulse of gsno ( µm; hr); isolated cytosol, er and golgi fractions; immunoprecipitated cftr from these fractions; and studied whether hop, co-immunoprecipitated with cftr, was s-nitrosylated (using a biotin substitution technique followed by streptavadin isolation). we found that sno-modified hop coimmunoprecipitated with ∆f cftr in the cytosol and er at baseline; and that gsno decreased the association between sno-modified hop and ∆f cftr. we speculate that the effect of gsno to decrease hop-cftr association may ) result from increased hop s-nitrosylation; and ) be permissive for ∆f cftr maturation. these data may be of relevance to the development of no donor-based therapies for cf. supported by cf foundation and the nih. mouse models for cystic fibrosis (cf) provide new possibilities to study disease pathogenesis, to correlate genotype and phenotype, and to test novel cf therapies. we use the cftr tm eu mice homozygous for the f del mutation in a congenic fvb background as a tool in pre-clinical testing of therapeutic strategies aimed at the correction of the f del folding and trafficking defect. these mice show characteristic cf ion transport abnormalities such as reduced camp-and cgmp activated intestinal chloride secretion and an increase of the amiloride-sensitive sodium absorption in nasal epithelium. we developed an ex vivo assay for the rescue studies of the f del trafficking defect in murine intestine by proteasome inhibitors based on periodical measurements of camp-activated transepithlial chloride currents in the ussing chamber. muscle-stripped ileum was incubated at c in william's e glutamax medium supplemented with insulin ( µg/ml) and dexamethasone ( µg/ml), in the presence of vehicle or proteasome inhibitor. a partial gain of chloride secretory function was achieved by incubating the tissue for - h at c in the presence of the proteasome inhibitors (pi) epoxomicin ( µm), mg ( µm), nip-(leu) -vinyl and adalys(bio)ahx l vs (both µm) and bortezomib ( nm). exposure of the tissue for h at c to alln ( µm) revealed a functional recovery up to wt levels. immunohistochemical and western blot analysis of all pitreated tissues showed the appearance of cftr positive crypt cells and mature band c cftr protein. rescue by alln remained detectable following inhibition of the er to golgi transport by brefeldin a. our ex vivo experiments demonstrate that proteasome inhibitors can enhance the amount of mature, functionally active murine f cftr mutant protein in intact tissue by preventing proteolytic degradation and increasing its expression at the cell surface. in vivo treatment of homozygous f del mice with bortezomib at a dose of . mg/kg/day for days resulted in a high morbidity. administration of a lower dose ( . mg/kg/day ) was well tolerated and no mortality or significant weight loss could be observed. as outcome parameters for the in vivo treatment with pis we used a salivary secretion assay and bioelectric measurements of intestinal and nasal epithelium in the ussing chamber. there was no difference in isoproterenol-induced salivary gland secretion or and in cftr-mediated intestinal chloride currents between control -and bortezomib-treated animals. however we found a reduction ( . fold) of the amiloride-sensitive short circuit current (isc) in the nasal epithelium of bortezomib-treated mice, indicating a moderate correction of the enacmediated sodium hyper absorption, that was not paralleled by a gain of the forskolin-mediated transepithelial chloride secretion. current in vivo experiments focus on the effects of pis upon short term ( h- h) rather than chronic treatment, to allow a direct comparison with the ex vivo treatment data. supported by grants from the cf foundation, bethesda, the sophia foundation and the dutch stomach-liver-intestine foundation. rotterdam, netherlands; . physiology and biophysics, university of alabama birmingham, birmingham, al, usa; . physiology, school of medical sciences, university of bristol, bristol, united kingdom recent progress in the development of small molecule correctors and potentiators capable of restoring cftr function have increased the need for pre-clinical test models including cultured airway epithelial cells from human cf patients as well as cf mouse models. the validity of cf mice as a surrogate for human cf patients depends on the assumption that human and mouse cftr, despite their limited sequence homology ( %) and differences in open probability (p o ) and channel subconductance state, behave similarly in their response to pharmacological correctors and potentiators. to verify this assumption, we used the iodide efflux technique to compare the cftr chloride channel activity in (i) cho cells stably transfected with mouse f del-cftr, (ii) nih- t cells stably expressing human f del-cftr, and (iii) their wt-counterparts. forskolin ( µm)-stimulated iodide efflux was very low in both mouse and human f del cftr cells ( . respectively % of wt values) and was stimulated to a similar extent ( . and . fold) by the cftr potentiator genistein ( µm). preincubation for h at c, or at c in the presence of the vertex corrector cfcor- ( µm) caused a - fold increase in forskolin-activated i-efflux in both cell types that was further enhanced by . - fold upon addition of genistein. functional correction was paralleled by the appearance of mature, complex-glycosylated mouse or human cftr on western blots. surprisingly, two other, structurally unrelated compounds known to correct the gating defect in hcftr, i.e. the vertex potentiator cfpot- ( µm) and the uncharged nppb analogue nppb-am ( µm; cf. wang w et al. ; j biol chem : ) mimicked the effect of genistein on hf del-cftr but failed to potentiate mf del-cftr in the t cells. both cfpot- and nppb-am likewise failed to potentiate forskolin-stimulated, cftr-mediated transepithelial chloride currents ex vivo in muscle-stripped ileal mucosa from homozygous f del-cftr tm eu mice (congenic fvb) mounted in ussing chambers under conditions in which genistein ( µm) enhanced the current response by - fold. in contrast, preincubation of the tissue in william's e glutamax medium for h at c, or for h at c in the presence of cfcor- ( µm) enhanced the forskolin+genisteininduced transepithelial clsecretion up to - % of the response in cftr+/+ littermates, and was associated with the appearance of band c on western blots. these results indicate that mouse and human f del-cftr ( ) respond to low temperature incubation or to a small molecule corrector with a similar gain in surface expression and function, supporting the use of cf mouse models for in vivo tests of cftr correctors; ( ) respond differently to various classes of cftr potentiators, in line with their pronounced differences in gating behaviour, and emphasizing the specificity of cftr-potentiator interaction. further studies focussing on human/mouse cftr chimera and aimed to identify the domain(s) accounting for the differential response to the cftr potentiators are in progress. cell apoptosis is a highly regulated process which is central to the maintenance of pulmonary vascular homeostasis. recent investigations implicated cftr in the regulation of intracellular sphingolipid levels. our laboratory demonstrated that an imbalance in endothelial sphingolipid levels triggers apoptotic cell death. we therefore hypothesized that a dysregulated cftr function in human lung endothelial cells inhibits programmed cell death in response to stress. methods: patch clamp analysis was used to verify the expression of cftr on human lung endothelial cells and the effect of specific inhibitors. we inhibited cftr with dpc, cftr inh - , and nppb or specific sirna followed by treatment with prototypical stressors that induce endothelial cell apoptosis, staurosporine, h o , or tnf-α. apoptosis was quantified by caspase- activity or annexin/ propidium iodide staining. lipid extraction was performed by a modified bligh and dyer method and intracellular sphingolipids were measured by tandem mass spectrometry. results: patch clamp analysis confirmed functional cftr expression on endothelial cells and a complete inhibitory effect of dpc and nppb but no effect of dids. as expected, treatment with staurosporine, a general protein kinase inhibitor and h o , as an oxidative stress stimulus resulted in significant endothelial cell apoptosis. similarly, tnf-α treatment ( ng/ml) in combination with cycloheximide caused activation of caspase- associated with a significant upregulation of the pro-apoptotic sphingolipid ceramide. knock-down with specific cftr sirna was validated with western blot and whole cell patch clamp analysis. dpc, nppb, and cftr inh - consistently and significantly inhibited apoptosis in human lung and primary mouse lung endothelial cells. caspase- activity was decreased by - % (% decrease in caspase units/µg/min) in hlmvec treated with h o after pretreatment with dpc ( %), cftr inh - ( %), and nppb ( %). however, we observed that dids, a non-specific chloride channel inhibitor also inhibited staurosporine induced caspase- activation ( %), suggesting the possible involvement of other chloride channels. in conclusion, our data suggest a role of cftr in modulating endothelial cell apoptosis in response to various stresses. the absence of functional cftr impaired stress-induced apoptosis, a process which may be mediated via alterations in sphingolipid trafficking. this abnormal cftr function in cf leading to aberrant cellular responses to stress may perpetuate an activated, pro-inflammatory phenotype of the lung vasculature. in turn, aberrant vascular activation may have a significant impact to the abnormal airway remodeling and persistence of a chronic inflammatory state characteristic of cf. supported by: cystic fibrosis foundation clinical fellowship training grant hegedus, t. , ; serohijos, a.w. , ; dokholyan, n.v. ; he, l. , ; riordan, j.r. , . department of biochemistry and biophysics, unc at chapel hill, chapel hill, nc, usa; . cystic fibrosis treatment and research center, unc at chapel hill, chapel hill, nc, usa; . department of physics and astronomy, unc at chapel hill, chapel hill, nc, usa cftr (cystic fibrosis transmembrane conductance regulator) is a camp dependent chloride channel. the phosphorylation of its amino acid r domain by protein kinase a (pka) is obligatory for channel gating under normal conditions. r domain contains multiple pka phosphorylation sites, which participate in activation, but no specific site is essential for channel function. in spite of numerous studies of the role of r domain in cftr regulation, the mechanism is largely unknown. one reason for this is the lack of information on r domain structure and its interactions with other parts of cftr. it has been shown that r domain lacks well-defined secondary structural elements and is intrinsically disordered. in this study, we used computational methods to explore the structure and function of the disordered r domain. our results show a specific disorder-order pattern in r domain, which is conserved among species even though the primary structure is not conserved. this result implies a role of the disorder-order pattern in r domain function and provides a basis of a novel working model. to gain some insight into the three dimensional structure of r domain, we per-formed ab initio folding of r-domain using discrete molecular dynamics with an all-atom force field. as disordered domains can not be characterized with a single structure, an ensemble was generated by selection of structures with the lowest energy from a large number of folding simulations. these structures were clustered to determine the dominant r domain conformations. they reveal features of secondary and tertiary structure, the positions of phosphorylation sites in d, and residue contacts within the r region that might be important for structure and function. to computationally confirm the mechanism proposed in our working model, an ensemble of phosphorylated r domain structures was also produced and characterized. supported by nih (r dk ), cff (dokhol i ), and novartis ( ). physiology system that utilises the planar patch clamp technique (finkel et al ) . this format utilises perforated patch clamp recording of wells in parallel, permitting the generation of up to data points per day. we have employed this system to develop assays to detect modulators of mutant cftr. human cftr-g d and df , over expressed in bhk and cho cells respectively, were cultured to approximately % confluence before harvesting. df cells were temperature corrected at °c for hours prior to experimentation to facilitate channel trafficking to the plasma membrane. cells were placed on the quattro system and whole cell chloride currents measured using asymmetric chloride selective solutions such that the chloride reversal potential was significantly different from that of leak currents ( mv). currents were assessed using a voltage ramp from - to + mv applied prior and min subsequent to compound addition. to validate the assays we used the prototypical cftr potentiator, genistein, which was added simultaneously to cells under study with µm forskolin. after assessing the effects of genistein, the specific cftr inhibitor glyh , was added at µm to confirm the currents were mediated via activation of cftr. the ec values determined for genistein were . ± . µm (mean ± s.e.m, n= ) for df and . ± . µm (mean ± s.e.m, n= ) for g d. these values agree with literature values (bulteau-pignoux et al ) . two alternative potentiators, phenylglycine- (pg- ) and vrt- were also tested in the presence of µm forskolin and were active against both df and g d. the ec values for pg- were . ± . µm (mean ± s.e.m, n= ) for df and . ± . µm (mean ± s.e.m, n= ) for g d. the ec values for vrt- were . ± . µm (mean ± s.d, n= ) for df and . µm (mean, n= ) for g d. these values agree with published data from ion transport studies (pedemonte et al ; van goor et al ) . in summary the ionworks quattro can be used to identify compounds which modulate the activity of cftr. this assay has a number of advantages over other formats in that it is direct, sensitive and employs voltage control of the cells under study. these assays can be used for primary screening of small to medium compound sets or as a secondary screen to triage the output from higher throughput indirect formats such as membrane potential. references it is very well agreed that ∆f cftr is recognized as a misfolded protein by the er quality control and targeted for degradation by the proteasome. during the course of experiments for aav gene therapy for cf we created an aav / vector expressing a truncated cftr insert (∆ ) driven by a chicken beta actin promoter (raav-cb-∆ -cftr). ∆ -cftr contains the normal coding sequence of wt cftr except that it is missing the first amino acids. we observed that cos cells transiently transfected with raav-cb-∆ cftr express cftr at significantly lower levels as compared to cells similarly transfected with a wt-cftr construct. to examine why, cells were transfected with raav-cb-∆ -cftr and treated for hours with µm of proteasome inhibitor, mg . in the cells treated with mg , ∆ -cftr protein expression was significantly increased. because lysosomal inhibitors did not have a significant effect, the data suggest that a large frac-tion of ∆ -cftr protein is degraded in the proteasome. to evaluate the rate of degradation, cells were treated with cycloheximide. time course experiments showed that ∆ is more rapidly degraded than ∆f -cftr. importantly, in cos cells cotransfected with ∆f and ∆ -cftr, there was a significant increase in the mature c-band of ∆f cftr compared to cells transfected with ∆f alone. this suggests that ∆ -cftr enhanced the maturation of ∆f from b to c bands. enhanced maturation of cftr from b band to c band also occurred when ∆ -cftr was transfected into hela cells stably expressing ∆ cftr (hela ∆ ) and cfbe o-cells stably expressing ∆ (cfbe o-∆ ). a number of proteins participate in the er to determine the fate of cftr. ∆f -cftr is known to associate with retrograde translocation protein complex on the er membrane containing vcp (valosin-containing protein) and derlin . vcp is also found in aggresomes containing ∆f -cftr in association with cytosolic histone deacetylase, hdac . we hypothesized that since ∆ is more efficiently degraded as compared to ∆f -cftr it may have higher affinity with vcp protein complex. we verified that ∆ -cftr interacts more readily with vcp and also has a higher degree of association with hdac as compared to ∆f -cftr. our data suggest that ∆ is more readily degraded by the proteasome as compared to ∆f -cftr and lacks biosynthetic arrest in the er. ∆ -cftr binds more readily to vcp and associates avidly with hdac , two outcomes that are expected to enhance proteasomal degradation. by engaging key quality control proteins, ∆ -cftr allows more ∆f -cftr to mature. our data suggest that truncated forms of cftr (∆ ) may be useful for cf gene therapy by affecting the maturation of endogenous cftr. ∆f , the most common disease causing cftr mutation, has impaired conformational maturation during its biosynthesis, and thus is unable to exit the endoplasmic reticulum (er). a well-defined di-acidic er exit code has been identified within the first nucleotide binding domain (nbd ) of wildtype cftr (jcb : - , ) , the disruption of which prevents cftr from efficiently exiting the er. this is consistent with the role of the diacidic code in the copii-mediated cargo concentration process. however, it is not known whether other types of er-to-golgi trafficking signals play a role in cftr trafficking in the early secretory pathway. even less is known about the trafficking signal(s) that are responsible for the rescue of ∆f cftr by a number of means. to achieve a better understanding of the trafficking signals dictating the export of cftr from the er, we constructed a series of cftr mutants to probe the potential functional targeting signals within both wild-type and ∆f cftr. it has been shown that nbd is not required for the export of wild-type cftr from the er. we further found that an internal deletion mutant lacking membrane spanning domain (msd ) and nbd -r but retaining the n-terminal cytoplasmic region, msd and nbd does not have sufficient signal to allow er export, suggesting that the signal(s) that are responsible for cftr export largely reside within the nbd -r. unlike ∆f cftr, the di-acidic code mutants are not temperature-sensitive. dissecting the di-acidic code revealed that both acidic residues contribute to er export, and that simultaneous substitution of both leads to a much decreased export of cftr. we found that the temperature-dependent export of ∆f relies on the presence of the di-acidic code within the nbd , suggesting that this code has a functional role in the temperature-dependent rescue of ∆f cftr. in our effort to further examine the role of the di-acidic code in the rescue of ∆f cftr by second site mutations, we found that ∆f cftr rescue by the simultaneous disruption of two of the arginine-framed tripeptides (afts) is also dependent upon the di-acidic code. surprisingly, we found that while nbd is not necessary for the export of wild-type cftr, it is required for the aft-mediated rescue of ∆f cftr, suggesting a role for cytoplasmic inter-domain interactions in the aft-mediated rescue of ∆f cftr. a detailed biochemical analysis of these mutants provides valuable insights into the roles of different cftr "trafficking" motifs in the er-to-golgi transport of cftr, the specific conformational defects of ∆f cftr and the potential molecular mechanism underlying the rescue of ∆f cftr. supported by cff. sonawane, n.d. ; zhao, d. ; galietta, l. ; zegarra-moran, o. ; verkman, a. . medicine, university of california san francisco, san francisco, ca, usa; . genetica molecolare, istituto gianina gaslini, genova, italy cftr inhibitors are predicted to prevent intestinal fluid secretion in enterotoxin-mediated secretory diarrheas such as cholera. cftr inhibitors that are not absorbed across the intestinal wall are attractive for diarrhea therapy because they may provide safe oral therapy. we previously discovered low affinity glycine hydrazide (glyh, ic ~ µm) cftr inhibitors that block cftr at its external pore (muanprasat et al., j. gen. physiol. ; : - ) . in order to increase inhibitor potency and prevent washout during severe secretory diarrhea, we synthesized a series of glycocalyx interactive cftr inhibitors containing a malonic acid hydrazide (malh) cftr pore blocking moiety linked to a lectin (sonawane et al., gastroenterology ; : - . lectin conjugation improved cftr inhibitory potency by ~ -fold (ic to nm). high-affinity cftr inhibition was abolished by malh-lectin heat denaturation, protease digestion, or competition by mannose or unconjugated lectin. fluorescently labeled malh-lectin remained membrane-bound for > hours after washout, whereas washout occurred in a few minutes without the lectin. malh-lectins blocked cholera toxin-induced intestinal fluid secretion in closed intestinal loops in mice with ec - pmol, and greatly reduced mortality in a suckling mouse model of cholera. we recently synthesized mono-and divalent cftr inhibitors consisting of malh coupled via a disulfonic stilbene linker to flexible mono and bifunctional polyethyleneglycols (pegs) of molecular size . , . , , , , , , and kda, with calculated solution lengths of - nm, with the larger size pegs potentially spanning cftr dimers or inducing their formation. ic for inhibition of cftr chloride current was - µm for monovalent malh-pegs, but substantially lower and size-dependent for divalent malh-pegs, decreasing from . µm to nm with increasing peg size. the mechanisms responsible for the improved and size-dependent potency of divalent malh-pegs were studied by whole cell, single-channel patch-clamp and by functional analysis of multivalent malh-conjugated dextrans and asymmetric divalent pegs. whole-cell experiments revealed reversible voltage-dependent block of cftr currents, with outward (positive) currents being more strongly blocked. in outside-out patch-clamp experiments, inhibitors caused a reduction of the mean open time. for malh-peg kda-malh ( µm) , the mean open time decreased from ± to ± ms. the effect on currentvoltage relationship and channel kinetics are consistent with a mechanism involving occlusion of the cftr pore from the extracellular side. luminally added divalent malh-pegs blocked by > % cholera toxin-induced fluid secretion in mouse intestinal loops with ic s of < pmol/loop, and greatly reduced mortality in a suckling mouse model of cholera. nonabsorbable, multivalent cftr inhibitor-macromolecule conjugates may be useful as anti-secretory agents in the treatment of enterotoxin-mediated diarrheas. supported by cff and nih. activation of the cystic fibrosis transmembrane conductance regulator (cftr) clchannel is primarily controlled by pka-dependent phosphory-lation of the r domain. once the r domain is phosphorylated, atp binding and enzymatic activity at two nucleotide-binding domains (nbds) open and close the cftr clchannel. we hypothesized that r domain phosphorylation regulates cftr activity by modulating atp interactions within two nbds. to test this hypothesis, we studied the activity of wild-type cftr and five variants with deletions of portions of the r domain between residues and . to alter atp-dependent channel gating, we tested three cftr stimulators, pyrophosphate (pp i , mm), 'deoxy-atp ( 'datp, mm) and caatp ( mm) . consistent with previous studies, pp i , 'datp and caatp all increased current of wild-type cftr about two-fold. each stimulator produced a similar increase in the r domain variants. the similar effects of stimulators on the channel activities of wild-type cftr and r domain variants suggest that the r domain does not have a major role in regulating atp-dependent channel gating. the r domain deletions did not alter the single-channel current amplitude of the r domain variants. however, r domain variants missing the sequence between residues and markedly reduced the open state probability, suggesting that this region is required for normal gating. these data suggest that the r domain does not control cftr activity by modulating atp interactions with nbd binding sites. instead, we speculate that the c-terminal part of r domain might participate in the channel-gating machinery downstream of atp regulation. supported by the cystic fibrosis foundation and howard hughes medical institute. haggie, p.m.; verkman, a. medicine and physiology, cvri, u.c.s.f., san francisco, ca, usa it was recently reported that phagolysosomes of alveolar macrophages from cf mice acidify in a cftr-dependent manner and that defective phagolysosomal acidification impairs bactericidal activity (di et al., nat. cell biol. , : - ) . these findings suggested a unifying hypothesis for cf disease progression: defective phagolysosomal acidification in cf macrophages permits the initiation and promotes progression of bacterial infection in the lungs. to reassess the central finding of that study we measured phagolysosomal ph using a fluorescent ph indicator containing oregon green® (pka ~ . ) and tetramethylrhodamine covalently bound to zymosan. phagolysosomal ph was insensitive to cftr inhibition ( µm cftrinh- ) in j macrophages (ph . ± . vs. . ± . ), alveolar macrophages from mouse lung (ph . ± . vs. . ± . ), and alveolar macrophages from human lung (ph . ± . vs. . ± . ). phagolysosomal ph in alveolar macrophages from ∆f cf mice was not significantly different from that in alveolar macrophages from wild-type mice (ph . ± . vs. . ± . ). to account for their finding of defective phagolysosomal acidification in alveolar macrophages, di et al. reported that lysosomal acidification was cftr-dependent and that fusion of lysosomes to phagosomes (at ~ min after phagocytic uptake) was responsible for phagosomal acidification. we measured lysosomal acidification in j macrophages using a dextran-conjugate containing oregon green® and tetramethylrhodamine and found that acidification was not impaired by cftr inhibition (ph . ± . vs. . ± . ) . we also measured the kinetics of phagosomal acidification using a zymosan-conjugate containing -(and- )-carboxyfluorescein (pka ~ . ) and tetramethylrhodamine. phagosomal acidification in j macrophages and murine alveolar macrophages began within min of phagocytosis and reached steady-state by - min, in agreement with prior data in murine peritoneal and bone marrow-derived macrophages. acidification kinetics in j macrophages was not altered by cftr inhibition, nor was acidification kinetics different in wild-type vs. cf alveolar macrophages. our findings do not support the conclusion that phagolysosomal acidification in alveolar macrophages is cftr-dependent, nor that it is impaired in cf. the mechanism of phagosomal acidification proposed by di et al. is not in accord with our data or precedents in the literature. because phagolysosomal acidification is central to the proposed mechanism linking defective cftr chloride channel function with cf lung disease, our results do not support the involvement of cftr in defective macrophage function in the pathogenesis of cf lung disease. supported by cff and nih. pared to that of wild-type cftr. the hsp family of molecular chaperones play important roles in the protein quality control process within the er. hsp atpase activity is regulated by multiple co-chaperones such as hsp , bag- and hspbp . hsp , a high-molecular-weight member of the hsp family was recently shown to display nucleotide exchange activity for hsp s in vitro. furthermore, hsp was identified as a component of a cftr-associated multiple protein complex using a global proteomic approach (cell : - , ) . in an attempt to explore the role of hsp in cftr conformational maturation in the er, we over-expressed the co-chaperone in hek cells and quantitatively analyzed its effect on the maturation and degradation of cftr. consistent with its role as a nucleotide exchange factor for hsp s, over-expression of hsp inhibits the er export of wild-type cftr and promotes its degradation. however, in striking contrast, over-expressing hsp stabilizes ∆f cftr and promotes its er export at reduced temperature. this effect is less pronounced at physiological temperature. the apparently opposite effects of hsp on wild-type and ∆f cftr maturation and quality control, suggests distinct conformational maturation pathways for the two cftr molecules, and reveals a specific role for hsp in regulating ∆f cftr refolding. such conclusion is reinforced by rnai experiments and further supported by quantitative co-immunoprecipitation. rnai-mediated down-regulation of hsp expression destabilizes ∆f cftr and reduces its export at reduced temperature, and hsp shows more extensive association with the er form of ∆f cftr than that of wild-type cftr. further analysis revealed that hsp not only modulates the chaperone activities of hsp s but also alters their steady state levels within the cell, creating secondary effects on cftr maturation and quality control. further studies are necessary to achieve a better understanding of the machinery, pathway and mechanism of ∆f cftr conformational maturation at reduced as well as physiological temperatures, and this in turn will provide critical insights and key factors that are of potential value to the rescue of the trafficking defect of ∆f cftr. farinha, c.m. , ; pissarra, l.s. ; amaral, m.d. , . department of chemistry and biochemistry, faculty of sciences, university of lisboa, lisboa, portugal; . ctr hum genet, nat inst health, lisboa, portugal the most frequent mutation in the cystic fibrosis (cf) gene, f del, causes retention of its protein product, f del-cf transmembrane conductance regulator (cftr) in the endoplasmic reticulum (er) as a core-glycosylated intermediate that is rapidly degraded. therefore, most f del-cftr fails to traffic to the plasma membrane, where wild-type (wt) cftr normally functions as a chloride (cl-) channel. retention, however, is not due to lack of function, since the mutant still retains some function if it reaches the membrane. instead, it results from misfolding which is recognized by the er quality control (erqc) in which many intervenients, including molecular chaperones, participate. identification of molecular partners involved in the disposal of f del-cftr to the proteasome is therefore crutial. it was recently shown that casein kinase ii (ck ), a pleiotropic constitutively active protein kinase involved in several processes, such as neoplasia, cell survival and viral infections, binds wt-cftr near the f residue, phosphorylating its first nucleotide binding domain (nbd ) at s [ ] . interestingly, deletion of f abrogates this interaction, which is the first described f del-dependent protein-protein interaction. our aim here was to identify whether ck interaction affects the early steps of cftr biogenesis, turnover and processing. to this end, novel bhk cells were produced which stably express wt-or f del-cftr in which the consensus residue s was substituted by either a neutral (alanine -s a) or an acidic residue (aspartate -s d). initial biochemical analyses of these cell lines revealed that: ) cftr proteins bearing d or a at position are processed; ) despite producing equivalent levels of cftr transcripts, s a expressing lines consistently show lower levels of cftr protein. metabolic labelling and pulse-chase experiments followed by cftr immunoprecipitation were also performed in these lines. after quantification of bands b (immature form) and c (mature form) of cftr, these preliminary results show that substitution of s (by a or d) does not affect the turnover or processing of either wt-or f del-cftr. the effect of ck inhibition on the turnover and processing of cftr was also studied, by incubating cells with µm of the ck inhibitor tetrabromobenzotriazole (tbb) for min. pulse-chase experiments under these conditions show that: ) the steady-state levels of both wt-and f del-cftr are reduced; ) the turnover of wt-cftr (but not of f del-cftr) is increased; and ) processing of wt-cftr is decreased. such data are consistent with a putative stabilizing role for ck upon wt-cftr [ ] . however, in our preliminary results this effect does not appear to be dependent on residue (nor on the putative charge added by the kinase on this residue), thus suggesting that this effect may be indirect. further studies are underway to identify the mechanism by which ck affects the turnover and processing of cftr. . treharne et al ( ) pissarra, l.s. ; xu, z. ; farinha, c.m. , ; sheppard, d.n. ; amaral, m.d. , . department of chemistry and biochemistry, faculty of sciences, university of lisboa, lisboa, portugal; . dep physiology, univ bristol, school med sciences, bristol, united kingdom; . ctr hum genet, nat inst health, lisboa, portugal g e and rk (the simultaneous mutation of four arginine-framed tripeptides (afts): r k, r k, r k and r k) are second site mutations that rescue the processing and function of f del-cftr [ , ] . these revertant mutations rescue f del-cftr from retention within the endoplasmic reticulum by distinct mechanisms: g e likely alters the conformation of the first nucleotide-binding domain (nbd ), whereas rk plausibly allows f del-cftr to escape er retention/retrieval mediated by afts [ ] . both g e and one of the afts (r k) lie close to the residue g , where a common cf-causing mutation g d occurs, generating a correctly localized clchannel with a severe gating defect. our aim here was to assess whether the revertants g e and rk influence the folding, processing and gating behaviour of the g d mutation, by employing biochemical and functional approaches. to test this idea, we introduced the g d mutation into cftr cdnas containing either g e or rk in the pnut vector and stably expressed these constructs in bhk cells. preliminary results from biochemical studies indicate that the mature fully-glycosylated form of cftr protein (band c) of both g e-g d-cftr and rk-g d-cftr were present at similar levels to those of g d-cftr. analysis of cftr-mediated iodide efflux from these cells revealed that g e is unable to rescue the functional defect of g d. however, rk generated an efflux of iodide larger than that elicited by cells expressing g d, albeit smaller than that of cells expressing wild-type (wt) cftr. consistent with these data neither rk nor g e rescued the defective channel gating of g d-cftr. however, both revertants caused a small increase in g d-cftr activity by attenuating the prolonged interburst interval of g d. we conclude that f del and g d disrupt cftr channel gating by distinct mechanisms. altogether, our data suggest that at least when in cis with the g d mutation, the afts (together or individually) might have a direct effect on cftr channel gating. this raises the possibility that rk, in addition to its well-described effect on trafficking, may act on cftr structure and/or folding, as previously suggested for r k [ ] . work supported by grant pocti/sau/mmo/ / and pluriannual funding of cigmh (fct, portugal) and the uk cf trust. l. pissarra was a recipient of bd/ / doctoral fellowship (fct, portugal) . [ ] carvalho ac, gansheroff lj, teem jl ( ) j biol chem , - . [ ] chang xb, cui l, hou yx, jensen tj, aleksandrov aa, mengos a, riordan jr ( ) mol cell , [ ] [ ] [ ] [ ] [ ] [ ] [ ] roxo-rosa m, xu z, schmidt a, neto m, cai z, soares cm, sheppard dn, amaral md ( ) proc natl acad sci usa , - . [ ] teem jl, carson mr, welsh mj ( ) improves ∆f -cftr intracellular trafficking in cf epithelial cells, although the mechanism by which this occurs is not clear. to identify gene products with atlered abundance in response to pba, we performed a differential display rt-pcr screen on rna isolated from ib - cf epithelial cells treated with pba for - hours. we isolated a cdna encoding stip- , a putative human stat- (signal transducer and activator of transcription- ) interacting protein and confirmed that pba causes transiently increased stip- mrna and protein abundance after hours of exposure. stip- was originally described as a scaffold protein that is required for ligand-dependent activation of stat- . stip- is also identical to elongator protein (elp- ), a subunit of the multicomponent elongator complex that stimulates rna polymerase ii activity. interestingly, recent data suggests that elongator may also regulate polarized secretion (rahl, et al. ( ) , mol. cell, : - ). we therefore hypothesized that stip- /elp- would regulate ∆f -cftr intracellular trafficking. in ib - cells, stip- /elp- associates with ∆f -cftr in coimmunoprecipitation experiments after pba treatment. in ib - cells overexpressing stip- /elp- , immunofluorescence experiments suggested that ∆f -cftr trafficked to the plasma membrane even in the absence of pba treatment. stip- /elp co-localized with markers of the golgi ( kda golgi protein) and exocytic vesicles (snap- ) when overexpressed in these cells. in contrast, overexpression of a deletion mutant of stip- /elp- lacking the wd domain, or stat- binding region, blocks the improvement of ∆f -cftr trafficking in response to pba in immunofluorescence and surface biotinylation experiments. this stip- /elp- deletion mutant did not co-localize with golgi or exocytic vesicle markers. furthermore, immunofluorescence experiments also suggested that pba causes colocalization of stip- /elp- and elp- , another member of the elongator complex that is often defective in familial dysautonomia. these data are consistent with stip- /elp- regulating ∆f -cftr intracellular trafficking in response to pba. this regulation may occur in the context of pba stimulating assembly of elongator, which in turn may promote trafficking of exocytic vesicles carrying ∆f -cftr to the plasma membrane. supported by grants from niddk. fold [ , ] . however, functional studies demonstrate significant differences in the gating behaviour of hcftr and mcftr [ ] . a powerful approach to investigate cftr structure and function is to examine interspecies differences and identify regions of conservation and divergence. to understand the structural basis for the functional differences between hcftr and mcftr, we generated hmcftr chimeras containing mcftr domains on an hcftr backbone. for this purpose, we replaced all or part of nbd , nbd or the rdomain of hcftr with the equivalent regions of mcftr and investigated their biochemical and functional properties. the in vivo folding of these hmcftr chimeric proteins was indirectly evaluated from their maturation status, after their stable expression in novel bhk cell lines. like wt-hcftr, most chimeric proteins were processed within the cell. however, two chimeras failed to mature: clone b (mnbd , amino acid (aa) residues - ) and clone c (mnbd , aa - ). we compared the murine sequence of these two chimeras with that of hcftr to determine the respective physico-chemical distances (pcds) of their aa changes. changes with higher pcd values were selected and in vitro mutagenesis performed to introduce these aa alterations into hcftr cdna. for clone b the selected changes were: e q, e q (pcd = ); s t (pcd = ); k q (pcd = ), i t (pcd = ) and k e (pcd = ). for clone c the changes were: p t (pcd = ), k q (pcd = ), y n (pcd = ), s k (pcd = ), c y (pcd = ), d g (pcd = ) and e d (pcd = ). biochemical analyses of these mutants stably expressed in bhk cells revealed that for clone b, e q, s t, k q and i t were processed, whereas k e was not. for clone c, p t, k q, y n, d g and e d were processed. iodide efflux showed that s t, k q, i t, e d and d g were functional, but not k e. we consider that with additional functional analyses, our approach will identify critical residues responsible for conformational changes and hence, functional differences between hcftr and mcftr. work supported by the bbsrc and grant pocti/mgi/ / (fct, portugal) and pluriannual funding of cigmh (fct, portugal) . ac dapaula is a recipient of phd fellowship sfrh/ bd/ / (fct, portugal we recently reported that the upr decreases endogenous wild-type (wt) cftr expression. as a follow up of these studies, we investigated the role of the folding deficient, ∆f cftr on upr induction and identified a mechanism by which the upr decreases cftr expression. for these studies, we developed a cell line expressing recombinant ∆f cftr on the endogenous wt background (calu- ∆f) and established individual clones with different ratios of endogenous (wt) to recombinant (∆f cftr) expression. two clones which express : (calu- ∆fc ) and : (calu- ∆fc ) ratios of wt to ∆f cftr and cfpac- cells expressing endogenous ∆f cftr were tested as models. the upr was constitutively active in calu- ∆fc cells only. in calu- ∆fc induction of ∆f cftr expression resulted in upr activation, indicating that high expression of ∆f cftr is required for upr induction. furthermore, following pharmacological induction of the upr, endogenous cftr mrna decreased to undetectable amounts both in calu- ∆f and cfpac- cells. the decrease in cftr mrna levels was not the result of shortened mrna half-life. in contrast, using a human cftr promoter driven reporter (pcftr-pluc), we demonstrate suppression of the cftr promoter when the upr is activated. considering that correction of ∆f cftr is the main therapeutic approach for cf, it is important that ∆f cftr expressed at low levels, such as in vivo, does not activate the upr. however, our results also reveal that ∆f cftr correctors have to be tested for upr activation, since transcriptional inhibition in this setting may contribute to inefficient rescue in native cells or in vivo. genistein, a naturally occurring tryrosine kinase inhibitor, at low concentrations ( µm) stimulates wt -cftr-mediated chloride secretion in a variety of cell and tissue types by an adenosine ', '-cyclic monophosphate (camp)-independent pathway. genistein has been shown to potentiate the channel function of ∆f -cftr and g d-cftr channels by prolonging channel open time (hwanget al., am j physiol cell physiol : c -c , ; illeck et al. am j physiol cell physiol : c -c , ) patch clamp studies have provided indirect evidence that genistein may bind directly to cftr and potentiate channel open probability by modifying the function of the nucleotide binding domains (weinreich et al., pflügers arch : - , ) . the goal of the present study was to determine whether genistein activated purified and reconstituted cftr protein as an initial step toward defining its molecular mechanism of action. the effect of genistein on purified and reconstituted wt-cftr was first studied following its reconstitution into planar lipid bilayers. in the presence of µm atp, phosphorylated cftr exhibited a low open probability (po= . ). the addition of µm genistein, caused a significant increase to the open probability of cftr (po= . ). using an electrogenic flux assay on a population of purified and phosphorylated cftr molecules, we determined that µm genistein caused a potentiation ( % increase) of chloride channel activity in the presence of µm atp. these results suggest that this macroscopic assay of purified cftr function is sufficiently sensitive to detect the effect of this potentiator. currently, the consequences of genistein on the channel activity and atpase activity of purified reconstituted ∆f -cftr and g d-cftr are being assessed. preliminary results suggest that in the presence of low atp concentrations ( - µm), µm genistein inhibits the atpase activity of ∆f -cftr, suggesting that it interacts directly with the nucleotide binding domains to alter their function. previous studies indicated an acute coordination between the activities of cystic fibrosis transmembrane conductance regulator (cftr) chloride channel and the amiloride sensitive epithelial sodium channel (enac) so that both these channels are either activated or deactivated in a synchronous fashion in the human sweat duct (reddy et.al, ) . however, the mechanisms responsible for such cooperativity between these ion channels are unknown. previous studies indicated that: cytoplasmic ph controls cftr activity through effects on phosphorylation (reddy et.al. ) . at ph . , cftr chloride conductance was reduced to ± ms/cm , but incresed to ± ms/cm at ph . (n=number of ducts= ). enac channel activity is also a function of cytosolic ph in heterologous expression systems (chalfant et.al, ) . the objective of this study was to test the hypothesis that the cytosolic ph may mediate the cooperative effects that occur between cftr and enac. we used basolaterally a-toxin permeabilized apical membrane preparations of native human sweat duct which expresses enac and cftr robustly as an experimental system. we showed that while luminal ph had no effect, changes in cytosolic ph acutely affected enac activity. that is, acidic ph inhibited, while basic ph activated enac activity. alkalinizing cytosolic ph from . to . increased enac conductance (genac) by ± . ms/cm (n= ). ph regulation of enac activity appears to be independent of cftr and endogenous kinase activities because basic ph stimulated enac: a.) after deactivating cftr by removing camp and atp in normal ducts, b.) in the absence of cftr in cf ducts, and c.) after blocking endogenous kinase activity with the non-specific kinase inhibitor, staurosporine. na + /h + exchanger (nhe) may mediate changes in cytosolic ph as a function of intracellular na + activity. nhe mrna is expressed in the sweat duct and cytosolic ph responds to changes in na + gradients across the basolateral membrane. conceivably, when transport conditions are favorable and intracellular na + is low, alkaline ph would allow enac and cftr to cooperatively admit na + and clthrough the apical membrane. when conditions are unfavorable and intracellular na + is excessive, acid ph would limit na + and clentry to protect the cell from disruptive changes in cell volume. thus, changes in cytosolic ph may play a crucial role in coordinating the activities of enac and cftr during transepithelial salt transport. acknowledgements: kirk taylor and sucharitha madireddi for expert technical assistance. funded by nih-ro de , nih-ro hl and the nancy olmsted trust. the cystic fibrosis transmembrane conductance regulator (cftr) is an anion channel that is normally expressed in the plasma membrane but is mislocalized in cf. to monitor trafficking we inserted super ecliptic phluorin, a ph-sensitive variant of green fluorescent protein (gfp) (miesenbock g, de angelis da, rothman je ( ) nature , - ) into the fourth extracellular loop of cftr. this construct, which we call cftr-phluor, has weak fluorescence intracellularly that increases ~ -fold when it is inserted into the plasma membrane and the phlour becomes exposed to more alkaline extracellular ph . thus cftr-phluor should be less fluorescent than cftr with the normal gfp fused to the n or c termini when situated in intracellular vesicles. despite the insertion of an entire gfp in the middle of the channel protein, cftr-phluor trafficked to the membrane and patch clamp studies revealed little change in unitary conductance or open probability compared to wild-type cftr. cftr-phluor enabled visualization of vesicle insertion events at the plasma membrane by total internal reflection fluorescence (tirf) microscopy. faintly fluorescent vesicles containing cftr were observed under the plasma membrane and eventually fusing with it to generate a burst of fluorescence. phluorin inserted at the same position in ∆f -cftr, the predominant mutant in cf, was retained in the er where the phluor remained partially quenched. when the folding defect was corrected by culturing cells at °c or treating them with known correctors, ∆f -phluor trafficking was partially restored and total cell fluorescence increased. this enabled a quantitative analysis of ∆f -cftr correction based on the increase in fluorescence in individual cells by flow cytometry. cftr-phluor may be most useful for microscope-based high-content screening studies of cftr and ∆f -cftr trafficking. support: the breathe program, canadian cf foundation, canadian institutes of health research and cf foundation therapeutics (usa). tukaye, d.n.; guggino, w.b. physiology, johns hopkins university, baltimore, md, usa type iii secretion system (t ss) toxins of p. aeruginosa are important virulence factors in p. aeruginosa infections. one of the t ss toxins, exos, has been shown to facilitate uptake and invasion of p. aeruginosa at airway surface epithelium. in mouse models of pseudomonas pneumonia, infection with p. aeruginosa exos+ strains caused increased levels of fluid in lungs as determined at autopsy in contrast to infection with p. aeruginosa exosstrains. the exact molecular mechanism underlying these observations is not known. exos is a bifunctional protein with gtpase activating protein (gap) activity at the n terminal domain and adp ribosyl transferase (adprt) activity at the c terminal domain. we found that exos-gap activity increases total cellular levels of mature (c band) wild type (wt) cftr in cfbe o-cells as measured by western blot analysis. this increase was mediated by decreased delivery of wt cftr for lysosomal degradation. in contrast, exos-gap failed to increase total levels of ∆ cftr (b band). interestingly, exos-gap increased total levels of ∆ cftr, bands c and b, following rescue with -phenylbutyrate. this indicates that targets of exos-gap exist in cftr trafficking beyond the er degradation pathway. exos-gap also brought about a corresponding increase in surface levels of mature wt cftr as measured by surface biotinylation. in conclusion, we have shown that p. aeruginosa t ss exos-gap, upregulates total and surface levels of mature wt cftr by modulating cftr trafficking beyond er, most likely by decreasing lysosomal degradation. the decrease in lysosomal degradation could be occurring in part by inactivation of small molecular weight g-proteins involved in delivery of wt cftr to lysosomes. increases in total and hence surface levels of cftr could in part explain increased amounts of fluids seen in p. aeruginosa pneumonia mouse model treated with exos+ strains. the post-maturational trafficking and localization of cftr is regulated by a wide variety of proteins. among the most prominent are several pdz domain proteins that bind to the c-terminal residues of cftr. in particular, two of these proteins, nherf and cal, have been shown to mediate opposing effects on the apical membrane levels of the disease-associated ∆f -cftr mutant, by controlling the balance between endocytic recycling and lysosomal degradation. in particular, whereas overexpression of nherf rescues ∆f -cftr at the cell surface, overexpression of cal has the opposite effect on cftr. cal-mediated downregulation of cftr requires a functional pdz binding site. we have now shown that suppression of endogenous cal cooperates with temperature rescue to stabilize functional ∆f -cftr channels at the apical membrane in polarized bronchial epithelial cells. this work supports the hypothesis that the cal pdz binding site is a therapeutic target for treatment of cystic fibrosis. a more direct test of this hypothesis will require competitive inhibitors that can efficiently block the cal:cftr interaction. in addition, given the large-number of protein-protein interactions formed by both the cal pdz domain and the cftr c-terminus, these inhibitors should ideally exhibit bidirectional selectivity, neither disrupting the favorable nherf :cftr interaction, nor strongly displacing other membrane proteins that interact with cal. using a fluorescence polarization assay and peptide-array technology, we have now identified a high-affinity, cal-selective peptide inhibitor. compared to cftr, this sequence binds cal more tightly, but nherf more weakly. furthermore, we have shown that the cal:cftr interaction is the weakest among the known cal-binding proteins, and that cftr should thus be susceptible to selective displacement from the cal binding site. finally, we have designed cell-permeable peptides that allow us to test the hypotheses (a) that such compounds will specifically disrupt the cal:cftr interaction and (b) that such targeted stereochemical inhibition will stabilize the functional cell-surface expression of ∆f -cftr in airway epithelial cells. the pathways for the endoplasmic reticulum associated protein degradation (erad) of misfolded proteins in the mammalian cells are incompletely understood. we investigated the role of molecular chaperones in erad for the cystic fibrosis transmembrane conductance regulator (cftr) and its common folding mutant, ∆f cftr. we found that hsp and its cochaperone hdj- interacted significantly with ∆f cftr and wt-cftr in airway epithelial cells. hsp interacted with the immature form of cftr while hdj- associated with both immature form and ubiquitylated cftr. structure-function studies showed that hdj- recognized ubiquitylated cftr via its zn-binding domain. immunoprecipitation and gst-fusion protein pulldown experiments revealed that hdj- interacted with ubiquitin. in steady-state, over-expression of hdj- elicited increasing both immature and mature forms of wt-cftr, but it resulted in decreasing immature form of ∆f cftr. pulse-chase studies showed that co-expression of hdj- promoted ∆f cftr degradation, reducing its half-life from to min. in contrast, hdj- expression did not significantly affect wt-cftr biogenesis. these data are consistent with our finding that hdj- shows a selective physical interaction with ∆f cftr, which translates into a functional discrimination between the mutant and its wt counterpart. the hpd motif within the j-domain of hdj- , which is necessary for the co-chaperone to bind hsp and stimulate its atpase activity, was required for hdj- mediated ∆f cftr degradation. mutation of the hpd motif retarded the degradation of ∆f cftr and increased the steady-state levels of ∆f cftr -fold. a role for hsp in hdj- mediated ∆f cftr degradation was tested in which hsp knockdown increased ∆f cftr expression - fold. hjd- regulates ∆f cftr maturation and knockdown of endogenous hdj- promoted ∆f cftr and its camp-dependent anion conductance in airway epithelial cells. in contrast, we were unable to detect maturation of ∆f cftr in hsp knockdown experiments, indicating that the maturation of ∆f cftr mediated by reduced hdj- expression may be independent of hsp function. taken together, our data demonstrate that hdj- is a molecular sensor that can detect differences in the folding related to the ∆f cftr maturation. these data also highlight a novel pathway for hdj- linked, ubiquitin-dependent degradation of ∆f cftr. reduction of hdj- expression or its association with ∆f cftr promotes maturation of this mutant and therefore represents a potential therapeutic approach for cystic fibrosis. [supported by nih and cf foundation] how the loss of cftr function results in cholesterol accumulation within the cell is currently unknown. cftr activation is driven by the camp and we propose that cf cells respond to the loss of cftr activity by increasing the camp pathway in order to increase cftr expression. to test this hypothesis, epithelial cells were treated with the phosphodiesterase- (pde ) and pde inhibitors milrinone and rolipram, respectively. inhibition of pde function with rolipram in wildtype cells leads to peri-nuclear free cholesterol accumulation identical to what is observed in cf epithelial cells as viewed by filipin staining, strongly implicating a camp-dependent pathway in the regulation of cholesterol trafficking. the pde -selective inhibitor milrinone had no effect on cholesterol trafficking suggesting specificity for the pde pathway. our preliminary data support this hypothesis in that both cf-model hteo-pcepr cells and mouse nasal epithelium (mne) from cftr -/-mice exhibit reduced protein expression of the campspecific phosphodiesterases pde compared to wt controls. the proposed consequence of a chronic amplification in camp signaling is altered cholesterol transport regulation. to address this potential role of pde in cholesterol trafficking, free cholesterol was visualized using filipin staining in wild type cells that were treated with rolipram, a pde -specific inhibitor. conversely, treatment of cf-model pcepr cells with the pka inhibitor rp-camps significantly improves cholesterol processing, further pointing to the involvement of the camp pathway. we propose that the camp pathway influences cholesterol processing through the regulation of β-arrestin- (βarr ) according to the premise that chronic increase in the camp pathway would initiate an elevation in βarr expression. βarr is an important regulator of adrenergic receptor recycling and organelle trafficking. because βarr is pivotal in regulating endocytotic recycling pathways, it could also impact cholesterol processing. this predicted increase in βarr protein expression is observed in both cf-model pcepr cells and cftr -/-mne compared to respective controls. over expression of βarr in wt epithelial cells leads to cf-like peri-nuclear cholesterol accumulation further implicating a role for βarr in the development of this phenotype. altered cholesterol trafficking in cftr would lead one to expect different βarr localization throughout the cell. further understanding of the implications of altered camp signaling and its relationship to aberrant cholesterol accumu-rationale: we recently reported that vcp (valosin containing protein) physically interacts with gp /amfr (autocrine motility factor receptor) to couple retrograde translocation of ∆f -cftr to proteasomal degradation (vij et al. jbc ) . recent studies have revealed an alternative system to the proteasome for degradation of polyubiquitinated misfolded proteins termed the aggresome. histone deacetylase- (hdac ) is a unique cytoplasmic deacetylase capable of interacting with ubiquitin and mediating the accumulation of ∆f -cftr in aggresome bodies. hypothesis: vcp competes with hdac to dissociate ∆f -cftr perinuclear aggregates. methods: ib - (∆f /w x) cf bronchial epithelial cells were transiently transfected with ∆f -cftr and vcp-gfp constructs and treated with a hdac inhibitor (tubacin), proteasome inhibitor (ps- or mg- ), lysosome inhibitor (baflomycin a ) or nil-tubacin (control) for , or hrs. the effect of these inhibitors on ∆f -cftr and vcp:∆f -cftr interactions were quantified by immunoprecipitating these protein complexes followed by immunoblotting with vcp, hdac or clathrin antibody. ∆f -cftr levels were measured by metabolic labeling using tran- s-label ( µci/ml) for a min pulse and hrs chase. the subsequent effects of these inhibitors on vcp localization was detected by fluorescence microscopy of gfp moiety. results: the co-immunoprecipitation experiments showed that hdac inhibition by µm tubacin ( hrs) promotes vcp:∆f -cftr and prevents hdac :∆f -cftr interaction. the proteasome inhibition (mg µm) resulted in maximal hdac :∆f -cftr at hrs with minimal at hrs while vcp:∆f -cftr increased overtime ( to hrs). adding tubacin ( µm; hrs) reversed the proteasomal inhibitor effects. in pulse-chase experiments, mg- increased the accumulation of cftr bform and poly-ubiquitinated-∆f -cftr as compared to the untreated control. tubacin suppressed the levels of mg- induced poly-ubiquinated-∆f -cftr as well as ∆f -cftr b-form. the vcp localization by microscopy showed the accumulation of vcp-gfp in perinuclear aggregates in the presence of proteasome inhibitor (ps- µm). the µm tubacin blocked these ps- induced perinuclear aggregates. we observed that vcp is associated with endocytic protein-complexes containing clathrin in the presence of ps- indicating cytosolic re-localization of vcp. we further confirmed the cytosolic re-localization of vcp-gfp in the presence of lysosome inhibitor (bafilomycin a µm) by fluorescent microscopy. conclusion: we found that a small molecule inhibitor of aggresome function prevents hdac :∆f -cftr interaction, promotes vcp:∆f -cftr interaction, and suppresses the accumulation of poly-ubiquinated-∆f -cftr. wang, y. ; jiang, y. ; zhu, n. ; feng, x. ; yang, h. , ; ma, t. , . membrane channel research laboratory, northeast normal university, changchun, china; . biopharmaceutical center, liaoning normal university, dalian, china deletion of the codon encoding the phenylalanine residue at position (∆f ) in the cystic fibrosis transmembrane conductance regulator (cftr) is the most common mutation causing cystic fibrosis (cf). the ∆f mutation results in a cftr protein with impaired folding, trafficking and gating in human and rodents. the consequences of ∆f -cftr mutation in other species have not been studied. the purpose of the present study was to characterize the ∆f mutant of porcine cftr in cell culture model. cdna sequence encoding full-length porcine cftr (pcftr) was cloned from the lung by rt-pcr using primers designed according to porcine genomic sequences in two bac clones (genbank accession no. ac and ac ) containing all exons of the porcine cftr. pcftr encodes a -amino-acid protein that shows . % identity to human cftr. the phenylalanine residue at position is conserved in pcftr. functional analysis of pcftr stably expressed in fisher rat thyroid (frt) epithelial cells by short-circuit current assays indicated a campactivated cl-channel similar to human cftr except that the sensitivity of pcftr to the specific cftr inhibitor cftrinh- is -fold lower than that of human cftr. a ∆f mutation of porcine cftr was generated by site-directed mutagenesis. surprisingly, ∆f -pcftr expressed as a func-tional chloride channel activated by forskolin in stably transfected frt cells. western blot analysis of cos and bhk cells transiently transfected with cmyc-tagged ∆f -pcftr indicated a protein pattern identical to that of wildtype pcftr. immunofluorescence analysis of the transiently transfected cells demonstrated similar plasma membrane expression pattern of ∆f -pcftr and wildtype pcftr. these results clearly demonstrated unimpaired cellular processing of ∆f -pcftr. however, the sensitivity of ∆f -pcftr to forskolin activation (ic ~ . µm) was dramatically lower than that of wildtype pcftr (ic ~ . µm), indicating a defect of channel gating by pka phosphorylation. the present study provided the first evidence that the ∆f mutation in mammalian cftr does not result in impaired cellular processing that is closely associated with cf phenotype. we have optimized airway epithelial cell spheroid cultures for functional measurements of cftr chloride conductance in order to screen smallmolecule activators and inhibitors. spheroids, which are sealed spheroidal monolayers of airway surface epithelial cells with diameter - micron, were generated within to days after nasal brushing and suspension culture. yields of up to spheroids were obtained from nasal brushing of a non-cf or cf subject. approximately , spheroids could be prepared from one million freshly isolated human bronchial epithelial cells. we compared several electrophysiological and fluorescence methods to assay cftr function in spheroids. whole-cell current was measured in single spheroids following pipette immobilization and micropipette puncture of the solutionexposed apical cell membrane. large, forskolin-stimulated whole-cell chloride currents were measured in non-cf spheroids. as an independent approach, transepithelial potential difference was measured by micropipette insertion into the spheroid lumen. for fluorescence detection, cell cytoplasm was stained with the halide indicators mqae or lzq. fluorescently stained spheroids were immobilized on a coated coverglass or in a custom microfluidic chamber. halide flux across the apical surface was measured in response to chloride-nitrate or iodide-chloride solution exchange, in the absence and presence of camp activators. the fluorescence assay is suitable for medium-throughput screening of ∆f correctors and potentiators, with definitive verification of cftr chloride channel function by whole-cell current analysis. an airway spheroid cultures provide a powerful approach to prioritize the efficacy of ∆f correctors because of their rapid generation without prolonged culture, and their suitability for quantitative analysis of cftr function. supported by cff and nih. background and aims: cftr has been shown to be expressed and functional in ventricular cardiomyocytes. however, to date, no physiological role for cftr in the heart has been established. the aim of this study was to determine if cftr plays a role in the regulation of cardiomyocyte contraction rate. methods: cardiomyocyte contraction rate was assessed using the neonatal mouse beating assay. ventricular myocytes were isolated from neonatal mice and cultured for - days until they formed a functional in sitium. contraction rates were captured by video imaging and analyzed by metamorph in the presence and absence of beta-adrenergic receptor stimulation by isoproteronol ( µm). assessment of cftr activity was determined by pharmacological inhibition by glibenclamide ( µm), dpc ( µm), and cftr inh- ( and µm). the role of k atp channels was assessed by -hd ( µm). results: baseline contraction rate of vehicletreated (dmso or h o) cardiomyocytes was unchanged. addition of isoproteronol caused a significant increase in contraction rate, which was sustained for at least minutes. in contrast, application of glibenclamide to beating ventricular myocytes significantly, but transiently, inhibited cardiomyocyte contraction rate, with recovery occurring within minutes. glibenclamide did not affect the initial increase in contraction rate caused by isoproteronol stimulation, but inhibited sustained increases in isoproteronol-stimulated contraction rate. to determine if this was an effect of k atp channel inhibition we examined the effect of -hd on contraction rate. in contrast to glibenclamide, -hd had no inhibitory affect on basal or isoproteronol-stimulated contraction rate, indicating glibenclamide's inhibitory effect was due to its inhibition of cftr. to further verify this, we examined the effect of two other cftr inhibitors, dpc and cftr inh- on ventricular cardiomyocyte contraction rate. both dpc and cftr inh- elicited similar responses as glibenclamide, with a quick, but transient inhibition of baseline contraction rate and inhibition of sustained isoproteronol-stimulated increases in cardiomyocyte beating. summary and conclusions: using neonatal murine ventricular myocyte cultures inhibition of cftr leads to a significant, but transient inhibition of baseline ventricular cardiomyocyte contraction rate. additionally, while inhibition of cftr did not affect initial increases in beta-adrenergic receptor-stimulated contraction rate, cftr inhibition did prevent sustained increases in contraction rate. the data suggest that cftr plays a role in modulating ventricular cardiomyocyte contraction rate during resting and adrenergic-stimulated conditions. drevillon, l. , ; tanguy, g. , ; hinzpeter, a. , ; arous, n. , ; goossens, m. , ; fanen, p. , . génétique, inserm u. , créteil, france; . faculté de médecine, université paris , créteil, france cystic fibrosis is mainly caused by mutations that interfere with the biosynthetic folding of the cftr protein. the aim of this study was to find cellular proteins capable of interacting with cftr and modifying its processing or its trafficking. we have used a genetic screen in yeast to identify such proteins and identified commd that preferentially interacted with the third cytoplasmic loop of cftr. commd is a regulator of copper transporter atp b in hepatocyte and sodium epithelial channel enac, but its exact biochemical function and physiological relevance remain elusive. here, we report that first, commd interacted with wild-type and f del-cftr in epithelial cells and second, commd subcellular distribution was not only both nuclear and cytoplasmic, but also in cytoplasmic vesicular compartments. we wondered if commd was involved in the processing and/or the trafficking of cftr protein in epithelial cells. we demonstrated that commd was not involved in cftr processing but that cell surface expression of wild-type cftr at the plasma membrane was cut by half when commd expression was % reduced by rna interference. we drew the conclusion that commd is a major component of cftr trafficking and/or recycling at the plasma membrane, precise characterization of commd in these vesicular compartments is under process to decipher its function in cftr trafficking. commd has been labelled as the prototype of a newly described protein family that plays a role in inhibiting nf-kappab signalling. moreover, subcellular distribution of commd was different in cf and non cf cells, nuclear form of commd being increased in f del cells compared to wild-type cells. thus, this opens up a new area of investigation about commd nuclear function. finally, these data indicate that commd is involved in multiple cellular processes of outstanding interest in cf pathophysiology. understanding how its modulation modifies transepithelial transport and inflammation in cf versus non cf cells should give new therapeutic clues to reduce exaggerated inflammation and improve fluid secretion in cf patients. supported ; fanen, p. ; galietta, l.j. . lab. of molecular genetics, istituto giannina gaslini, genova, italy; . inserm u. , creteil, france class iii cystic fibrosis mutations cause reduced cltransport by impairing the process of cftr channel opening. typical class iii mutations include g d, g d, and f del, all of them affecting nucleotide binding domains. it has been reported that mutations residing in other regions of the cftr protein may also reduce channel activity. we investigated the extent of loss of function caused by such mutations, in particular by missense mutations localized in cftr intracellular loops, and the sensitivity to pharmacological stimulation with cftr potentiators. to determine cftr activity, plasmids carrying the coding sequence for wild type or mutant cftr were co-transfected in cos- cells together with the halide-sensitive yellow fluorescent protein yfp-h q/i l. the rate of cftr-dependent anion transport was measured after short-term stimulation with forskolin alone or in combination with potentiators (felodipine, phenylglycine pg- , or sulfonamide sf- ). processing of cftr protein was assessed by examining glycosylation status of cftr by immunoprecipitation experiments. when stimulated with forskolin alone, cftr mutants showed the following anion transport compared to the wild type protein: i t ( %), i v ( %), q k ( %), e k ( %), g r ( %), d h ( %). interestingly, all mutants having a reduced anion transport responded to stimulation with potentiators pg- , sf- , and felodipine ( µm) with an increase in anion transport that approached the level of normal cftr protein. the correction of defective cftr activity by potentiators was confirmed in frt cells expressing e k and d h mutants using short-circuit current measurements. our results indicate that some missense cf mutations, like e k and g r, really cause a severe reduction in cftr activity. others, like d h, cause only a partial loss of function which may explain their association with a milder form of the disease. conversely, some other amino acid changes (with the extreme example of i v that seems to cause a gain of function) do not impair cftr activity and therefore may represent polymorphisms and not cf-causing mutations. interestingly, all mutants having a mild or more severe anion transport defect were sensitive to potentiators. this finding indicates that cftr potentiators have a wide efficacy on many class iii mutants and therefore may represent a promising therapeutic strategy for a significant number of cf patients. supported by cfft and telethon-italy. cftr, a cl-channel membrane protein, is a member of the atp-binding cassette (abc) super-family. mutations in cftr result in a misfolded protein which fails to mature, leading to impaired flow of cl-ions across the membrane of the epithelial cells lining the airway, and to cf. the most common mutation leading to cf is df . currently available structural information relevant to cftr modeling includes low resolution structures of cftr ( Å) and p-gp ( Å) and high resolution structures of other abc transporters as well as nucleotide binding domains (nbd) . of particular relevance is the newly published structure of the bacterial multidrug abc transporter, sav , whose topology can serve as a starting point for cftr modeling. as a first step towards the development of cf therapeutics, epix has undertaken the modeling of the d structure of cftr (excluding the r domain) in its conducting (i.e., open-channel, nbd-dimer) state, using our proprietary membrane-protein modeling technology (predicttm) in combination with other modeling tools. to date, we have modeled the cytosolic part of the receptor, namely the nbds:icls construct and work is ongoing on modeling the transmembrane (msd :msd ) region. the now available partial model has been scanned for putative binding sites for df -cftr correctors/potentiators. several potential sites have been located, and epix has initiated its in silico screening technology in order to identify potential drug candidates. this work is supported by the cfft. the deletion of a phenylalanine residue at position (f del) in the first nucleotide-binding domain (nbd ) of cftr is the principal cause of cystic fibrosis (cf). the altered interaction of f del cftr with endoplasmic reticulum (er) quality control proteins, primarily chaperones, promotes its proteasomal degradation. however, it is believed that crucial cftr-interacting proteins (cips) remain unknown [ ] . moreover, there is little information currently available on the strength of cip-cftr interactions. our goals here are two-fold: ) to quantify cftr-cip interactions; and ) to isolate and characterise unidentified cips. to address our first goal, we used surface plasmon resonance (spr; biacore tm ) to quantify real-time binding of hsc (a well-known cip) to bacterially expressed wt-and f del-nbd . hsc /hsp was covalently immobilised onto the surface of carboxymethyl dextran (cm ) sensor chips ( µm) and the real-time binding of purified nbd and control proteins quantified. in control experiments, anti-hsc antibody b ( nm; against residues - ) bound specifically to immobilised hsc with high affinity (k d , . ± . nm; n = ) whereas bovine serum albumin ( µm) did not interact (n = ). given the difficulties associated with expression of hnbd carrying the f del mutation [ ] it was not used in our spr analyses. instead, we used purified murine (m) nbd to quantify the impact of f del on the strength of the hsc -nbd binding. in the presence of low concentrations of atp (≤ um), the affinity of nbd binding to immobilised hsc was strengthened when f was deleted (wt, k d app , . ± . µm; f del, k d app , . ± . µm; n = ; p < . ). interestingly, raising the concentration of mgatp weakened the binding of nbd ( . µm) to hsc (wt, k i , µm atp; n = ). furthermore, deletion of f dramatically increased the concentration of mgatp required to destabilise the nbd -hsc interaction (f del, k i , µm atp; n = ). in summary, we used spr to demonstrate that: (i) nbd of cftr binds specifically to hsc , and (ii) the f del mutation enhances the association of nbd with hsc . we are presently investigating the effect of co-chaperones and small molecules on the interaction of hsc/hsp with wt-and f del-nbd . to identify novel cips, purified nbd was immobilised onto metalaffinity resin and used to capture cips from epithelial cell lysates. by this approach, cips were captured from human respiratory cell (calu- ) lysates and analysed by d-electrophoresis. relevant protein spots so far identified by mass spectrometry include: ) raichu x (thr/ser kinase); ) profilin isoform b; ) annexin a ; ) ifapsoriasin (intermediate filament-associated ca + regulatory protein); ) mgc (member of the er reticulon family). current analyses are underway to determine their functional roles in cftr trafficking and function. [ ] amaral ( billet, a. ; melin, p. ; norez, c. ; bilan, f. ; vandebrouck, c. ; mettey, y. ; becq, f. . physiologie, cnrs umr , poitiers, france; . gmas umr , poitiers, france; . faculté medecine et pharmacie, poitiers, france in order to gain a better insight into the structure-activity relationship (sar) of cftr protein, we have functionally characterized a cystic fibrosis mutation: the cftr-g d, identified in patients with oligospermia (vankeerberghen et al., ) . we examined cftr chloride channel activity by patch-clamp analysis, in whole cell configuration, using cos- cells transiently transfected with gfp-cftr-wt or with the mutant gfp-cftr-g d. cftr channels were activated by µm forskolin (fsk), and, after stable activation µm cftrinh- was added. as gfp-cftr-wt, g d chloride channels elicit a time and voltage independent current in presence of µm fsk. gfp-cftr-g d current density at + mv ( . ± . pa/pf) is . fold less than gfp-cftr-wt current density at + mv ( . ± . pa/pf). g d mediated currents are blocked by µm of the specific cftr-inhibitor, cftrinh- (ma et al., ) . as expected, gfp-cftr-wt channels are stimulated by µm of the potent activator -butyl- -hydroxy- -chlorobenzo[c]quinolizinium chloride (mpb- , derand et al., ) with a current density at + mv of . ± . pa/pf. interestingly, no activation of g d channels is recorded in presence of mpb- : difference between currents recorded in basal condition or in presence of mpb- is not significant. using iodide efflux experiments, we showed that cftr-g d mutant does not respond to several benzo[c]quinolizinium derivatives. however, the mutant channel can be activated by forskolin, ibmx and genistein. thus, the mechanisms of activation by xanthine and isoflavone are not affected by the mutation. these results show that: ( ) cftr-g d channels are functional: activation by camp pathway, inhibition by cftrinh- . ( ) camp chloride currents elicited by cftr-g d protein are weaker than cftr-wt. ( ) cftr-g d channels are refractory to benzo[c]quinolizinium activation. the replacement of the glycine by a negative charged amino acid in position affects cftr chloride channel activity and prevents activation by benzo [c] quinolizinium. this amino acid g , localized in the interface of nbd and r domain between two β sheets (callebaut et al., ) seems to have a crucial role in pharmacological activation of cftr by mpbs. further experiments will be performed to evaluate the role of the charge and of the nature of the amino acid at position in cftr protein. supported by vaincre la mucoviscidose and cnrs. cysteine string protein (csp) is a j-domain containing protein that regulates the extent of wild type cftr maturation. over-expression of csp blocks the formation of mature, glycosylated (band c) cftr and increases the level of immature (band b) cftr, whereas knockdown of csp elicits a marked increase in cftr maturation (zhang et al., j. biol. chem. : , ) . thus, csp appears to block the exit of cftr from the er. when the er exit of cftr is blocked by a different mechanism, i.e. the expression of mutant sar -gtp (sar h g interferes with the formation of copii vesicles), the steady-state level of immature cftr is two-fold greater than that resulting from csp over-expression. this finding suggests that csp, in addition to blocking er exit of cftr, may facilitate the degradation of the immature protein. in agreement with this concept, csp expression increased cftr ubiquitylation. a mutation within the conserved hpd motif of the j-domain (h q) disrupts the ability of csp to interact with and stimulate the atpase activity of hsp . csp h q does not block cftr maturation (above ref); in addition, the level of immature cftr was ~ % higher when h q csp was over-expressed than the level observed with mutant sar . this finding suggests that the pro-degradative effect of csp on immature cftr requires hsp binding/activation. chip (carboxy terminus of hsp -interacting protein) is an e ubiquitin protein ligase that can target wt and ∆f cftr for proteasome-mediated degradation (meacham et al., nature cell biol. : , ) . we sought to determine weather chip was involved in the csp-mediated degradation of cftr. indeed, co-immunoprecipitation (co-ip) experiments showed that chip associated with csp. also, csp h q, which has reduced interaction with hsp , showed a similar level of chip interaction by co-ip, suggesting that their association did not depend on hsp as a linker between the two proteins. a csp mutant that is truncated after the cysteine string domain, which is its site of membrane attachment, blocked cftr maturation as did wt csp; however, this mutant did not decrease cftr band b levels like wt csp did. in addition, the csp mutant missing its cterminus did not associate with chip. these findings further implicate chip in the csp-mediated degradation of immature cftr, and they suggest that the c-terminus of csp is a chip binding domain. when a dominant-negative mutant of chip, which lacks the u-box needed for chip-mediated ubiquitylation (chip∆ub), was co-expressed with cftr, csp was no longer able to facilitate the degradation of cftr in the er. these data show that it is possible to separate the effect of csp on cftr maturation from its ability to mediate the degradation of immature cftr. thus, csp blocks the exit of cftr from the er, and this requires a j-domain mediated activation of hsp but not the csp c-terminus. the action of csp on cftr degradation requires both a functional j-domain and the csp c-terminus, and this may require a trimeric complex involving csp, hsp and chip. [ the cystic fibrosis transmembrane conductance regulator (cftr) represents the main clchannel in the apical membrane of epithelial cells for camp-dependent clsecretion. mutations of this channel causes cystic fibrosis disease ; thus discovery of pharmacological activators of cftr is crucial to design future medicament for protein therapy. recently, we reported on the synthesis and screening of a small library of -phenylpyrrolo [ , b] pyrazines (named rp derivatives) evaluated as activators of wild-type cftr, g d-cftr and f del-cftr clchannels (noël et al, ) . this preliminary structure-activity relationship study identified -hydroxyphenyl and -n-butyl as determinants required for activation of cftr (rp- and rp- ). here we studied structure-function relationship of more than compounds prepared by chemical synthesis, and the subsequent activation of cftr channels. within the -phenylpyrrolo [ , -b] pyrazines family, we observed by iodide efflux technique that rp- bearing a hydroxyphenyl substituant is more potent (ec = nm) than rp- having -hydroxyphenyl substituant (ec = nm). by whole-cell patch clamp recording analysis, we confirmed that nanomolar concentrations of rp- activate linear chloride current in cho cells stably transfected with human wild-type cftr. this current was inhibited by µm of cftr inh - . we also found significant stimulation of short circuit current (i sc ) by rp- (ec = nm) on colon of cftr +/+ but not of cftr -/mice mounted in ussing chamber. stimulation of i sc by rp derivatives was inhibited by glibenclamide. the structural analogue , was less potent (ec = nm). as for rp- compound, we found that the -nbutyl chain is crucial for rp- and rp- activity, and that the -hydroxyphenyl compounds without -n-butyl chain are unactive on cftr. in this study, we showed that the presence of an hydroxyl group at position , or of the pyrrolopyrazine cycle determined the highest activity on cftr. the most potent compound is the -n-butyl- -( -hydroxyphenyl) hpyrrolo [ , b] pyrazine . the potency of these agents indicates that compounds in this class may be of therapeutic benefit in cftr-related diseases, including cystic fibrosis. the most common mutation causing cystic fibrosis (cf) is the deletion of f in the cftr gene, which results in inefficient trafficking of the cftr protein from the endoplasmic reticulum to the plasma membrane. the surface expression of the cftr is usually quantified biochemically with western blot or functionally with electrophysiological assays. however, these methods are labor intensive. we explore using fluorescence-based techniques for monitoring cftr surface expression. wild-type cftr was tagged at the amino terminus with green fluorescent polypeptides (gfp-cftr). the gfp-cftr was either transiently or stably expressed in chinese hamster ovary (cho) cells. the cells were then treated with a red fluorophore that specifically labels plasma membranes. the fluorescent images were acquired with an olympus laser scanning confocal microscope. two image channels were taken simultaneously. the green channel was excited with a nm laser, and signals within - nm were collected. the red channel was excited with a nm laser, and signals within - nm were collected. for a single focal plane, each image was composed of * pixels. threshold values were given to both green and red channels. pixels with an intensity value above the threshold were marked as green/red pixels. the green pixels in the first channel represent the signal of gfp-cftr, and the red pixels in the second channel represent the location of the plasma membrane. thus, pixels that are both green in the first channel and red in the second channel are the cftr surface expression, and denoted as yellow pixels. the percentage of successfully trafficked cftr is thus quantitatively estimated by the ratio of yellow to green pixels in the single plane. alternatively, a stack of images from the same cell at different depth was collected and analyzed. the total amount and surface portion of cftr expression are then determined by summing the correspondent pixels. the percentage of cftr surface expression was then calculated and will be verified both biochemically and functionally. the approach is modified to study deltaf cftr trafficking. we anticipate that the method can be used to screen cftr correctors from a small or intermediate sized chemical database. supported by cfft and nih. the most common mutation of the cystic fibrosis transmembrane conductance regulator (cftr) gene (deletion of phe- (∆f ) in the first nucleotide binding domain (nbd )) causes retention of the ∆f protein in the endoplasmic reticulum (er). ∆f mutation prevents conformational maturation of cftr protein without altering profoundly the local structure of nbd , but possibly by disrupting the interaction between nbd and nbd . however, the individual role of nbd and nbd in biogenesis, folding, maturation and membrane stability of a full length cftr is still under debate. using splicing by overlap extension method (soe by pcr), we generated six recombinant cftr proteins with inverted or suppressed nbds. the native boundary domains of nbd and nbd were respected for all mutants (riordan j.r. et al. ) and their c-terminal tails preserved. the constructs were transiently or stably expressed in cos- and bhk cells respectively. immmunodetection and immunolocalization assays confirmed that deletion of nbd domain (n -cftr) did not alter the trafficking or the plasma membrane expression of cftr. both processes were abolished in the presence of ∆f mutation. however, using whole cell patch-clamp configuration, we did not detect any camp-stimulated clcurrent. pulse-chase experiments established that the turnover of n -cftr is - fold faster (t / ~ h) than its wild type counterpart (t / ~ h). when we replaced nbd with nbd ( n -cftr), we could detect a faint plasma membrane expression associated with a weak cl-channel activation. more surprisingly, the t / of n -cftr coreglycosylated form was around h. when nbd was deleted (n -cftr) or substituted with nbd domain ( n -cftr), the expression and functional pattern were similar to cftr ∆f . furthermore, we showed that the n -cftr folding was not attenuated as demonstrated by protease susceptibility and sdsresistant thermoaggregation tendency, supporting the notion that n -cftr membrane stability requires nbd -nbd interaction. altogether, our results suggest that nbd domain is essential to form a foldable cftr that satisfies er quality control (erqc) but correct inter-domain assembly is mandatory for its stability at plasma membrane. the results are also in agreement with previous suggestion that ∆f mutation alters the interactions between nbds and transmembrane domains of cftr. supported . inserm u , inserm, paris, france; . inserm u , inserm, paris, france; . inserm u , inserm, paris, france; . inserm u , inserm, paris, france; . inserm u , inserm, paris, france; . inserm u , inserm, paris, france; . inserm , inserm, paris, france; . inserm u , inserm, paris, france the physiological role of cftr in renal epithelium is not known. in the proximal tubule, cftr co-localizes with the sodium-phosphate cotransporter npt a, a protein involved in phosphate (pi) reabsorption. the aim of our work is to determine if there is a functional interaction between cftr and npt a. to this end, the arnm coding for cftr and the arn coding for npt a (bearing a myc tag in c-ter) were injected in xenopus laevis oocytes. electrophysiological or biochemical experiments were performed days after arns injection. currents induced by mm pi (ipi) were measured in voltage-clamped (- mv) oocytes that expressed npt a alone, or in oocytes co-expressing cftr + npt a. for immunoprecipitation experiments, - cftrab and myc ab were used. results ipi was significantly reduced in oocytes expressing cftr + npt a compared to that measured in oocytes expressing npt a alone. using a two-bath electrodes configuration during voltage-clamping did not change this observation. this result suggests that when expressed in oocytes, cftr is in functional interaction with npt a. kinetic analysis (ipi as a function of pi concentration) of npt a showed a reduction in vmax but not in phosphate km. this suggests that npt a membrane expression and/or activity is altered when cftr is co-expressed. co-immunoprecipitation experiments performed on protein lysate derived from oocytes co-expressing cftr and npt a suggested an interaction between the transporters. using a pka activating cocktail as a pkc activator induced cftr-mediated currents in cftr+npt a expressing oocytes. in these oocytes, ipi is decreased by pkc activation (as expected from npt a regulation) but is increased shortly after exposure to the pka activating cocktail. the increase of ipi observed under this condition is at variance to that expected from classical npt a regulation. in oocytes expressing npt a alone, both pka activation and pkc activation reduced ipi. these observations suggest that in oocytes, cftr expression modifies npt a regulation. our study suggests that, after expression in xenopus laevis oocytes, cftr interacts functionally with npt a, and modifies its regulation. an interaction is also suggested by the co-immunoprecipitation of cftr and npt a. these results cannot discriminate between a direct or an indirect (via a third protein) interaction. to determine if an intracellular chloride concentration change may participate to the functional interaction, we are now measuring in cftr+npt a expressing oocytes intracellular chloride activity (using chloride-sensitive microelectrodes) during the use of the pka activating cocktail, and measuring ipi when pka is stimulated in the presence of a specific cftr inhibitor. cystic fibrosis (cf), the most commonly inherited lethal pulmonary disorder in caucasians, is caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (cftr). to date, more than mutations were identified in cftr and were associated with clinical disease. a phenylalanine deletion at position accounts for % of cf genotypes in caucasian populations, and determines cftr misfolding and degradation by proteasome. consequently, limited cftr abundance leads to multiorgan disease, affecting the lung, pancreas, gut, liver, sweat glands and the reproductive organs. the past several years have witnessed an explosion of information regarding the identification and elucidation of molecules and pathways that are regulated by cftr or that regulate cftr activity. genomics and proteomics technologies now offer the opportunity to examine global alterations in the mrna and protein expression patterns of cf cells and tissues to elucidate the pathways linking defective cftr to clinical disease. here we describe systems biology methods that integrate heterogeneous datasets, including protein-protein interaction networks, gene expression and mass-spectrometry profiles, and mutation and genetic variation information, in order to identify the regulatory circuits and active subnetworks that are responsible for the progression and the severity of cf disease. the study will focus on correlations that define the key events in the cftr folding and trafficking pathways that lead to pathogenesis and dysfunctions, and could provide insights and targets for intervention and therapy. the number of cftr cl-channels in the plasma membrane, and thus the transepithelial cl-secretion is controlled, in part, by the endocytic trafficking of cftr. rab gtpases regulate the endocytic trafficking by acting as molecular switches that cycle between the gdp-bound (i.e. inactive) and the gtp-bound (i.e. active) state, associate with target membranes, and recruit downstream effectors. rab facilitates cftr endocytosis and rab a facilitates the recycling of internalized cftr to the plasma membrane. furthermore, rab and rab facilitate cftr trafficking to the lysosome. the mechanism of sorting internalized cftr for either recycling to the plasma membrane or degradation in the lysosome is not well understood. recent evidence demonstrates that protein sorting occurs in rab specific sorting endosomes. presence of cftr in rab compartment has been previously demonstrated in human airway epithelial cells by electron microscopy. yet, the role of rab in cftr trafficking has not been determined in these cells. rab did not control cftr trafficking in fibroblasts (bhk cells). however, it negatively regulated the plasma membrane expression of cftr in the colorectal cells . the goal of this study was to elucidate the role of rab in the endocytic trafficking of cftr in polarized human airway epithelial cells (calu- cells and cfbe o-cells stably expressing wt-cftr or deltaf -cftr). endogenous rab coimmunoprecipitated with cftr in calu- cell and cfbe o-cells. to determine if rab plays a role in cftr trafficking, cfbe o-cells were transfected with either the flag-tagged, dominant negative (dn) rab mutant deficient in gtp binding (gdp-locked; flag-rab -n i) or with a non-specific cdna control (gfp). if rab sorts internalized cftr to the recycling pathway, the dn rab should inhibit recycling, and, thus, should decrease the plasma membrane expression of cftr. if, on the other hand, rab sorts internalized cftr for degradation the dn rab should inhibit cftr degradation, increase cftr recycling and thus, should increase the expression of cftr in the plasma membrane. the dn rab increased cftr expression in the plasma membrane as determined by cell surface biotinylation. these data are consistent with the role of rab in sorting internalized cftr for degradation. similar to overexpression of the dn rab , silencing endogenous rab with double stranded small interfering rna specific for the human rab sequence increased the plasma membrane expression of cftr and increased the cftr mediated cl-secretion across polarized cfbe o-cells in ussing chamber experiments. the plasma membrane half-life of deltaf -cftr is decreased compared to wt-cftr. however, after silencing rab , the plasma membrane half-life of deltaf -cftr was similar to that of wt-cftr, as determined by cftr disappearance from the plasma membrane following incubation with cycloheximide. our data are consistent with the role of rab in sorting the internalized cftr for degradation and are consistent with the changing paradigm for the role of rab in protein trafficking (supported by the nih grant p rr and the cff swiate qo). the conformational changes underlying activation of phosphorylated cftr by atp remain unclear. in the present study we assessed the utility of labeling endogenous cysteine(s) in cftr using an environmentally-sensitive fluorescent probe alexa- in monitoring such changes in structure. these studies were performed using purified and functionally reconstituted wild-type cftr. we labeled pka-phosphorylated cftr with µm alexa- maleimide prior to reconstitution in phospholipids liposomes. changes in fluorescence intensity were monitored in a suspension of proteoliposomes following addition of and mm mg-atp or mm mg-amp-pnp in order to evaluate nucleotide dependent changes in conformation. in order to identify the labeled cysteines, the protein was subjected trypsin-mediated proteolysis and analysis by mass spectrometry. we have shown that cftr is labeled using alexa- maleimide. a significant increase in fluorescence emission occurred in alexa- labeled cftr of ± (au) relative to empty liposomes one minute after the addition of activating nucleotide, mm mg-atp (n = ). the non-hydrolyzable analogue, mm mg-amp-pnp, failed to cause an increase in fluorescence of alexa- -labeled cftr. however, the higher concentration of mm mg-amp-pnp evoked an increase in fluorescence similar in magnitude to that of mm mg-atp suggesting that the fluorescence response reflects structural changes relating to atp binding to cftr. analysis by mass spectrometry identified the labeled residue as c . this cysteine resides at the junction between nbd and the "r" domain in a region which has been suggested to be highly dynamic on the basis of multiple crystal structures. our studies suggest that this flexible region around c of cftr undergoes conformational change upon mg-atp binding such that it moves the alexa fluorophore into a relatively more polar environment. this finding presents a first case where conformational changes in the cftr nbds induced by atp binding could lead to changes in the conformation of the r domain. further, these studies suggest that fluorescence methods can be used to probe dynamic conformational changes in purified reconstituted cftr. acknowledgements cystic fibrosis is caused by mutations in the apical chloride channel cftr with % of patients carrying at least one allele of the ∆f mutation. this mutant form of cftr is characterized by a trafficking defect in which it fails to exit the endoplasmic reticulum (er). we have previously reported that ∆f cftr is found in complex with hsp and its co-chaperones in cell extracts suggesting that either this mutant form of the protein accumulates in on-pathway folding intermediates which result in its accumulation in the er or that these intermediates include inhibitory factors leading to retention of ∆f cftr in this compartment. we have examined the role of one of these hsp co-chaperones, fk binding protein (fkbp ), in the life cycle of cftr. fkbp is a unique member of the immunophilin family of peptidyl-prolyl isomerases (ppiases) which mediate the cis/trans interconversion of peptidyl-prolyl bonds, which is thought to represent a rate limiting step in protein folding. in order to establish a functional role for fkbp in cftr stability and trafficking, we modulated the expression level of this protein in human bronchial epithelial cells endogenously expressing either wild type or ∆f cftr. a knockdown of fkbp by sirna resulted in a destabilization of both wild type and ∆f cftr as well as a concomitant loss in channel activity of wild type and temperature corrected ∆f cftr. conversely, over expression of fkbp resulted in a stabilization of the er glycoform of ∆f cftr in both mammalian and yeast systems. these data suggest that fkbp is an essential component of the hsp -mediated folding machinery for both wild type and ∆f cftr, which supports the hypothesis that ∆f cftr accumulates in the er in an on-pathway folding intermediate. dmh cftr folding involves a series of sequential, although potentially overlapping steps that involve i) formation, orientation and integration of (tms), ii) helical packing, iii) folding of cytosolic and extracytosolic domains, and iv) establishing proper domain-domain contacts. this process begins cotranslationally and is facilitated by the sec er translocation machinery and a diverse group of lumenal and cytosolic chaperones. the most common inherited cftr mutation, deltaf , disrupts one or more early steps along this folding pathway. a major obstacle in understanding how deltaf causes cf is that the native folding environment, comprised of lipids, cytosolic and er proteins, is not amenable to traditional biochemical and biophysical approaches. to overcome this problem we are developing a flu-orescence based analytical approach that enables one to directly access nascent ribosome-attached folding intermediates at virtually any stage of synthesis. the strategy requires incorporation of fluorescent probes into the substrate protein, which provide a highly selective means to monitor structural features at sub-nanomolar concentrations in highly complex biological mixtures. polypeptides are synthesized in vitro from truncated mrna templates to generate uniform cohorts of ribosome-bound nascent chains. progressive stages of folding can then be evaluated using mrna templates of different length. as proof-of-principle we have shown that fully synthesized gfp variants can be readily trapped in an unfolded state and complete their folding only after the entire nascent peptide has exited the large ribosomal subunit. this approach enabled us to measure real-time folding kinetics of extended peptide domains following synchronous ribosome release and compare fluorophore maturation of four fluorescent protein variants with different spectral properties cfp, gfp, venus and mcherry. to extend this approach to cftr and other general protein substrates, we are employing fluorescence resonance energy transfer (fret) using "donor" and "acceptor" probes incorporated simultaneously into the substrate protein. an in frame n-terminal fusion to cfp provides the "donor" fluorophore, while the "acceptor" fluorophore is incorporated at an engineered stop codon using a synthetic modified aminoacyl-suppressor trna. we are currently developing a 'molecular ruler' to correlate the efficiency of energy transfer with changes in the relative distance between probes in ribosome-bound and ribosome-released cftr folding intermediates. fret-based experiments are underway to evaluate folding of both n-terminal ( - aa) and nbd ( - aa) domains. these studies will provide new insight into the mechanism of cotranslational cftr domain folding and provide a potential means to identify agents that correct the deltaf folding defect. relationships between hypoxia, ado release, and prostenoid production in ariway cells are not defined. in the present studies, we examined mechanisms governing hypoxia-induced production of prostenoids in cfbe o-and calu- monolayers. using elisa-based detection of prostenoid and leukotriene production, both calu- and cfbe omonolayers treated with ado ( µm) or arachidonic acid (aa, µm, the parent molecule of prostenoids and leukotrienes) for hrs led to increases in pge levels ( - ± - pg/mg of total proteins vs controls ± pg/mg in calu- cells, - ± - pg/mg vs controls ± pg/mg in cfbe o-cells, p< . in both airway cell types compared with control). in contrast, ltb and ltc release were not stimulated by both maneuvers. we next used sirna techniques to knockdown a b adenosine receptor expression, and demonstrated durable knockdown of a b ar expression to~ % of scrambled sirna-treated controls following lipid-based transfection with sirna directed against the a b ar in cfbe o-cells. following sirna knockdown of a b ar expression, cells were exposed to hypoxic conditions (mucosal volume = ml in -well plate), normoxic conditions (mucosal volume = . ml, fio = . ), and ado ( µm). hypoxic stress led to high levels of mucosal pge production ( ± . pg/mg vs controls ± . pg/mg, p< . ) that was sensitive to a b ar knockdown ( ± . pg/mg, p< . ). exogenous ado also stimulated pge production, but this effect was small relative to hypoxia. we next examined basolateral effects of ado and aa on transmucosal clsecretion in cfbe o-and calu- cells grown as monolayers on permeable supports and conducted ussing chamber analysis under voltage clamp conditions. addition of ado and aa to the basolateral membrane ( µm) activated transmucosal clsecretion [ . ± . µa/cm (ado) and . ± . µa/cm (aa) for calu- cells; and . ± . µa/cm (ado) and . ± . µa/cm (aa) for cfbe o-cells expressing wtcftr] that was sensitive to basolateral blockade with barium cl -( mm). isc by either agonist was stimulat-ed in the presence and absence of apical glybenclamide ( µm), adenosine deaminase (ada - u/ml) and hexokinase (hexo - u/ml), confirming that the effects were specific for the basolateral membrane and could be accomplished with or without cftr activity. these results demonstrate that hypoxia is sufficient to stimulate pge production through a b ars, and that both ado and aa activate basolateral k + channels to promote transepithelial clsecretion. the findings further support a model in which ado (and prostenoids) regulate cltransport through effects on both the apical and the basolateral membrane, and elucidate molecules that couple the two membranes as part of the normal clsecretory response. hypoxia would be predicted to increase local prostenoid production through ado release and stimulation of a b ars, and further highlight a unifying model by which local hypoxia promotes inflammation in airway epithelia. supported by the nih and cff. low levels of tissue oxygen -observed in lung diseases such as cystic fibrosis -initiate a signaling cascade resulting in altered transcription of genes possessing a hypoxia response element (hre). we recently used gene chip mrna array, quantitative rt-pcr, protein analysis, and functional (short circuit current) assays to show that cftr expression is strongly repressed by hypoxia in vitro and in vivo. among several thousand human mrnas suppressed by low ambient oxygen, cftr was among the most strongly inhibited (by - fold) in a cell model system. however, cftr and the vast majority of other repressed genes lack a traditional hre. because hypoxia inducible factor (hif) acts primarily as a transcriptional activator, the mechanisms underlying hypoxia mediated suppression of mammalian genes are not well understood. we therefore tested the hypothesis that micrornas play a central role during transcriptional regulation of genes such as cftr. micrornas are evolutionarily conserved, short noncoding rna sequences believed important for repression of gene expression. some reports have estimated that at least % of protein-coding genes are regulated by micrornas. thousands of micrornas have been predicted in the human genome by available algorithms (e.g. palgrade, mirscan, and promir) and several hundred experimentally validated. the cftr gene contains predicted target sites for micrornas in the 'utr, including hsa-mir- (nucleotides - of the cftr 'utr), hsa-mir- (nt - ) and hsa-mir- (nt - ). however, no study to date has examined the effect of micrornas on cftr message levels, or the influence of micrornas during the cftr response to environmental perturbations such as hypoxia. we therefore performed a global, genome-wide analyses of both mrnas and micrornas expressed during hypoxia (using air/liquid or liquid/liquid interface culture systems) in ht (colonic epithelial) cells that robustly express cftr. we found that approximately % of micrornas were significantly altered by hypoxia, as judged by mirna profiling (ambion mirvana™ bioarray v , asuragen microrna expression profiling service). comparison of these hypoxia-related micrornas with expression profiles of their predicted targets indicated a much lower level of correlation than has been previously hypothesized by others. we show that none of the micrornas predicted to regulate cftr are altered in ht (in response to hypoxia). these findings are therefore consistent with the notion that highly expressed genes in a particular tissue type are not co-expressed with their predicted regulatory micrornas. our data also indicate that micrornas do not mediate the hypoxic repression of cftr via the ' utr. supported by nih and cff. . physiology, mcgill university, montreal, qc, canada; . biochemistry, mcgill university, montreal, qc, canada the ∆f mutation impairs cftr maturation and trafficking to the plasma membrane and results in a partially functional chloride channel that is retained in the endoplasmic reticulum and degraded. using our highthroughput trafficking assay, we previously showed that the quinoline km , a structural analogue of sildenafil, corrects the ∆f -cftr processing defect and leads to a significant increase in cftr surface expression after h treatment with µm and after h treatment with nm according to detection of mature cftr in western-blots. additional studies have now been carried out with other preparations to assess the functionality of ∆f -cftr following rescue by km treatment. different time and concentration exposures were tested on the cftr function using iodide efflux assays with both bhk and human bronchial epithelial cells over-expressing ∆f -cftr. exposure to µm km for h partially restored iodide efflux responses to µm forskolin + µm genistein in both cell line. after h incubation, rescue of the mutant protein in bhk cells still required µm, whereas nm was sufficient in the human cell line cfbe. iodide efflux assays were performed at different times following km wash out to functionally assess the stability of the rescued mutant protein. camp-stimulated iodide efflux was still detectable h after removing km , but responsiveness was lost after h in both cell types. electrophysiological studies were performed to examine the channel activity of ∆f -cftr rescued by km treatment. incubation with µm km for h increased camp-responsive ∆f -cftr current density in hek whole-cell patches ~ -fold. the conductance was time-and voltage-independent, anion selective, and strongly inhibited by glibenclamide. incubating polarized cfbe monolayers with µm km increased the forskolin + genistein-stimulated short-circuit current after h, and this response was inhibited by cftrinh ( µm). permeabilization of the basolateral membrane with nystatin confirmed the apical location of the forskolin + genistein-stimulation. ussing chamber experiments also revealed trafficking correction in ileum that had been excised from cftrtm eu mice homozygous for ∆f . when the ileum was pre-incubated with µm km for - h, the forskolin + genistein-stimulation of short-circuit current was significantly increased when compared to untreated intestine. these results confirm partial correction of ∆f -cftr by km in unpolarized cells and in human polarized epithelial cell monolayers and ex-vivo intestine isolated from ∆f -cftr mice. this work was supported by the breathe program funded by the ccff and cihr, cihr, cfft, and génome québec. primary sequence and overall structure of human cftr overlaps significantly with murine, rabbit and other homologues, a feature that has limited generation of conformation specific reagents to address cftr folding. because regions of ∆f cftr exposed extracellularly may exhibit aberrant topology (jbc : - ), probes that identify cftr from the extracellular surface could be useful to ) verify plasma membrane localization of ∆f cftr, ) discriminate wild-type from mutant protein, and ) provide a means of determining whether small molecule "correctors" of the ∆f processing lead to a native (wildtype) cftr configuration. our laboratories are investigating phage-display techniques suitable for detecting properly folded extracellular domains of cftr. two large phage libraries encoding or amino acid sequences (approximately phage per library) were panned against extracellular regions of cftr in hela cells. following incubation in blocking solution containing pbs and % bsa at °c, unbound fraction was incubated with recombinant helas expressing wild-type or temperature corrected ∆f cftr (protein expression verified biochemically and functionally). bound bacteriophage was dislodged after multiple washings by exposure to an acidic elution buffer and amplified to increase copy number (five rounds of antigen selection for each library, eur j biochem : - ). supernatants from microtiter wells expressing phage were analyzed for binding to parental (no cftr), wt or ∆f cftr expressing hela cells. using this method we obtained enrichment of phage at the surface of cells expressing wt or ∆f cftr. an example using a -amino acid sequence ( phage) with specificity for ∆f cftr (representative of four repeat experiments) is shown in a binding assay with hela cells/condition. reagents such as these can be further optimized by constructing second generation libraries using core recognition sequences (derived from the initial screen) and surrounding the core epitopes with random amino acid diversity. these experiments are intended to identify peptides with high binding affinity (nm binding constants) that recognize native cftr epitopes exposed extracellularly. because there is evidence that low temperature corrected ∆f cftr in the plasma membrane is misfolded, these probes may ultimately distinguish wild-type cftr from the surface targeted ∆f mutant and be useful as drug discovery reagents. supported by cff and nih. bridges, r.; nagubadi, s.; thakerar, a.; jia, y.; bradbury, n.a. physiology and biophysics, rosalind franklin university of medicine and science, north chicago, il, usa the goal of this project was to compare several reported correctors of ∆f -cftr biosynthesis for their efficacy on chloride channel activity under a standard set of experimental conditions. fisher rat thyroid (frt) cells were transfected with ∆f -cftr and a stable cell line selected with g . cells were grown on transwell filters and studied under short circuit current (i sc ) conditions in ussing chambers. i sc measurements were performed at °c. the basolateral membrane was permeabilized with α toxin and chloride currents were measured with a serosal to mucosal chloride gradient. atp ( mm) and gtp ( µm) were added to the serosal bath. cftr channel activity was stimulated with camp ( µm), ibmx ( µm) and genistein ( µm). cells were treated with correctors or vehicle (dmso) at and hours before the i sc measurements. a subgroup of cells were temperature corrected at °c for hours for comparison. because only modest currents were observed even in the temperature corrected cells ( - µa/cm ) cells were treated for hours with mm sodium butyrate and nm dexamethasone to enhance the observed currents. under these conditions temperature corrected cells displayed stimulated chloride currents of ~ µa/cm that were - -fold greater than cells maintained at °c. correctors c (n- ' ]bithiazolyl- '-yl]-benzamide) and c ( -( h-benzomidazol- -ylsulfanylmethyl)- -( -methoxy- -methyl-quinazolin- -ylamino)pyrimidin- -ol) caused a modest rescue of ∆f -cftr chloride currents compared to cells maintained at °c (c , . -fold; c , . -fold) but far below the effect of temperature correction. rescue by c was concentration dependent with a significant observable effect at nm and a maximum effect at µm. rescue by c was observed at only µm, lower concentrations had no effect and higher concentrations caused an inhibition of the stimulated chloride currents. compounds c -piperazin- -yl]-ethyl}- -piperidin- -yl-quinazoline) ( to µm), c ( -cyclohexyloxy- -{ - [ -( - methoxy-benzensulfonyl)-piperazin- yl]-ethyl}-quinazoline) ( to µm) and phenylbutyrate ( mm) did not significantly increase the stimulated chloride currents. all four compounds c -c caused significant decreases in the transepithelial resistances at the higher concentrations ( - µm) suggesting some degree of cytotoxicity. our results indicate only c and c demonstrate efficacy in frt cells and that these effects are modest compared to temperature correction. supported by the cystic fibrosis foundation. cftr contains two membrane-spanning domains (msd), two nucleotide-binding domains (nbd), and a regulatory domain that require proper assembly for chloride channel activity. the most prevalent disease causing mutation, cftr∆f , arises from deletion of phenylalanine in the nbd domain. the cftr∆f mutant is translated and inserted into the endoplasmic reticulum (er) membrane, but is unable to attain its native state and accumulates in a kinetically trapped conformation that is degraded by the ubiquitin proteasome system ( ) . the nature of the misfolded cftr∆f intermediate that is selected for premature degradation is not clear and is a topic of great interest ( ) . recent work in the cyr lab indicate that defects in cftr and cftr∆f folding are sensed by two different e ubiquitin ligase complexes that are located in the er membrane and cytosol ( ) . the membrane-associated derlin- /rma e complex appears to recognize early folding defects of cftr that may involve assembly of msd into a complex with the amino-terminal regions of cftr. in contrast, the cytosolic hsc /chip e complex appears to sense folding defects that occur after synthesis of the nbd domain. misfolded cftrs recognized at either checkpoint are retained and degraded via the proteasomal pathway. the presence of dual protein quality control (qc) checkpoints suggests a mechanism by which the folding status of cftrs membrane and cytosolic domains are sequentially monitored prior to its escape from the er. currently little is known about how these erqc complexes sense misfolded substrates. small-molecule chemical correctors have recently been identified which rescue the folding defect of cftr∆f in model cell culture systems. in the presence of these chemical correctors a subset of cftr∆f is able to localize to the cell surface and retain channel function. however, how the chemical correctors rescue cftr∆f folding is not clear. we have examined the extent to which the molecules correct folding defects that are recognized by the derlin- /rma or hsc /chip qc checkpoints. in addition, because it is unknown at what point of cftr biogenesis that the chemical correctors act to facilitate folding, we have determined how the chemical correctors affect the folding of different biogenic intermediates. our results indicate that the correctors act in a post-translational manner to influence assembly of cftr sub-domains. interactions with members of the qc pathway have also been used to monitor the folding and assembly process of cftr in the presence of chemical correctors. ultimately the results from these studies will enhance our knowledge of how chemical correctors permit passage of the cftr mutants past different erqc checkpoints. supported by -cystic fibrosis foundation combination therapy has proven successful in treating a wide variety of human diseases, including cancer, infectious disease, and diabetes. historically, combination treatments have been discovered through trial and error using drugs with proven disease modifying effects. we have developed a combination high-throughput screening (chts™) technology to systematically and efficiently find synergistic combinations that target multiple pathways underlying disease biology, and that may be developed into new therapeutics. we are applying chts technology to discover and develop combination therapeutics for the treatment of cystic fibrosis. we have optimized two high throughput screening assays for identifying correctors and potentiators of the cftr (cystic fibrosis transmembrane protein regulator) ∆f mutation. one assay uses the flipr membrane potential dye (fmp) and the other uses genetically modified yellow fluorescent protein yfp h q/i l to measure changes in halide flux through ∆f cftr. both assays were optimized and validated on the flipr tetra using the panel of cf correctors, potentiators and inhibitors provided by the cf foundation. using our automated chts, we systematically evaluate pairwise combinations of a library of ~ , approved drugs and other bioactive molecules to identify novel disease-relevant biological interactions. candidate single agents and combinations are tested in secondary assays and prioritized for testing in animal efficacy models. combination therapies can act on multiple pathways in a coordinated way. chts may prove to be a useful tool for identifying combination therapeutic candidates with novel multi-target mechanisms and a way to enhance co-therapy regimens for the treatment of cystic fibrosis. supported the highly variable clinical phenotypes of cf airway disease suggest that a number of genetic factors, other than cftr, play a role in its pathophysiology. some of these modifier genes are expected to play a role in the endoplasmic reticulum quality control (erqc), since the major defect caused by f del is misfolding, retention and degradation of the mutant protein by this cellular surveillance system. the nematode caenorhabditis elegans is an excellent multicellular genetic model, which has been successfully used in studies of human diseases. its ~ , -genes genome has been fully sequenced, it has short life-span ( - weeks) and life cycle (~ . days) and it is easily cultured and amenable for gene disruption, both by knockout or rnai. our goal is to generate a c. elegans model for the cftr folding defect, so as to take advantage of this model for the identification of genes involved in cftr folding and/or degradation. to this end, and because no cftr orthologue has been described in c. elegans, we constructed two previously described human p-gp-/cftr chimeras (p-gp/wt-cftr and p-gp/f del-cftr) [ , ] to be used as an in vivo folding substrate in this organism. the two chimeric cdnas and intact human p-gp cdna were cloned into the c. elegans ubiquitous expression vector ps and injected into mutant nematode strains for multidrug resistance genes [ ] . the effect on the nematodes phenotype was evaluated by an assay of heavy metal sensitivity ( . mm arsenite), as described [ ] . our quantitative results show that the p-gp/wt-cftr chimera increases the resistance to arsenite when injected into the pgp- /pgp- c. elegans double mutant, whereas p-gp/f del-cftr causes no effect. these preliminary results indicate it is possible to generate two distinct nematode phenotypes caused by each of the transgenic chimeras (p-gp/wt-cftr and pgp/f del-cftr). they also suggest that the same folding defect impairing f del-cftr function in cf may be responsible for the loss of heavy metal resistance function by the p-gp/f del-cftr chimera. these chimeras are currently under test in pgp- /pgp- double and in pgp- /pgp- /mrp- triple c. elegans mutants. analysis of these strains by rt-pcr showed that the mrnas from the two p-gp/cftr chimeras have low expression levels. this led us to produce more robust constructs, including: ) to test chimera expression under the c. elegans endogenous pgp- or intestinal-specific (potent) promoters; and ) to develop p-gp chimeras with a full cftr-nbd . these c. elegans models will be used in genome-wide rnai screens to identify genes involved in the erqc of the p-gp/cftr chimeras. work channel gating of cftr is regulated by atp binding and hydrolysis at the nbds and by pka phosphorylation at multiple sites, primarily within the intrinsically disordered regulatory (r) region. phosphorylation at multiple sites is required for full activation of channel function, but no one specific phosphorylation site is required. the phospho-regulation of r region interactions with nbd likely contributes to the regulation of cftr channel function by modulating r region interactions with nbd at the nbd /nbd dimerization interface, consistent with nbd crystal structures that include the regulatory extension (re) comprising the first residues of the r region ( ). this regulation likely occurs via a dynamic complex where multiple segments of nonphosphorylated r region with αhelical propensity transiently engage nbd and are released. pka phosphorylation disrupts r region α-helical propensity and decreases binding of each interacting segment ( ) . regulation of the ∆f cftr by pka is altered, with the rate of channel activation by pka -fold slower than for wild-type ( ). we hypothesize that the dynamic interactions between the r region and nbd are affected by the ∆f mutation. to characterize the differences in r region binding between wild-type and ∆f nbd at residue-level resolution, we use nuclear magnetic resonance (nmr) techniques. experiments are performed using either r region or nbd samples labeled with nmr-active nuclei and monitoring their structural changes upon the addition of unlabeled binding partner, allowing us to confirm r region binding to nbd at the nbd /nbd dimerization interface and to identify other potential interaction surfaces on both the r region and nbd . together, these experiments will allow us to characterize the dynamic associations between the r region and nbd in order to better understand the molecular basis for the pathogenesis associated with ∆f cftr. funded by the canadian institutes for health research, the canadian cystic fibrosis foundation, and the us cystic fibrosis foundation . lewis, h.a., et al, embo j. , - ( ) . . baker, j.m.r., hudson, r.p., kanelis, v., choy, w-y, thibodeau, p.h., thomas, p.j., and forman-kay, j.d. submitted. the ∆f -cftr mutation, the most common gene mutation in cystic fibrosis (cf), results in diminished plasma membrane expression of cftr, leading to loss of functional cftr and altered mucociliary clearance. this impairment promotes chronic infection of cf patients by the opportunistic pathogen, pseudomonas aeruginosa. we previously reported that a secreted protein from p. aeruginosa (cftr inhibitory factor (cif)) reduces the wt-cftr and ∆f -cftr-mediated chloride secretion and the plasma membrane expression of cftr by decreasing endocytic recycling of cftr. the aim of the current study was to investigate the mechanism by which cif is secreted by p. aeruginosa and delivered to human airway epithelial cells (cfbe o-). in this study, we show that cif is packaged in outer membrane vesicles (omv) for secretion from p. aeruginosa. interestingly, cif secretion via omv was increased when the bacteria were exposed to airway epithelial cells. changes in cif protein level or bacterial growth did not account for the increase in cif secretion. purification and application of p. aeruginosa omv to airway epithelial cells caused a decrease in plasma membrane expression of cftr. the decreased plasma membrane cftr expression was followed by increased ubiquitination and lysosomal degradation of cftr. cif delivery to the airway epithelial cells via omv showed a more robust decrease in plasma membrane cftr expression, in comparison with cif delivery as purified protein. to investigate the mechanisms whereby cif enters epithelial cells, optiprep gradient fractionation experiments were conducted in polarized airway epithelial cells. in airway epithelial cells treated with p. aeruginosa omv, cif was associated with airway cell lipid rafts. pharmacological inhibition of lipid raft formation with filipin iii blocked the decrease in cftr plasma membrane expression upon treatment with omv. these studies demonstrate that cif is released by p. aeruginosa via omv and enters the airway epithelial cells through lipid rafts. upon entry into the epithelial cell, cif increases the ubiquitination and degradation of cftr and redirects cftr trafficking from the recycling pathway to the lysosomal-degradative pathway. these data suggest that chronic infection of p. aeruginosa in the cf lung may reduce the efficacy of therapeutics developed to increase plasma membrane expression of the mutant ∆f -cftr (supported by the nih ro hl - and t -dk - ). . physiology, dartmouth medical school, hanover, nh, usa; . microbiology and immunology, dartmouth medical school, hanover, nh, usa we have identified and cloned a secreted protein from pseudomonas aeruginosa (p. aeruginosa pa and clinical isolates), designated cftr inhibitory factor (cif). cif reduces the apical membrane expression of cftr and inhibits the cftr-mediated chloride ion secretion by human airway epithelial cells expressing wt-cftr and df -cftr. cif does not have general effects on protein trafficking, as the localization and expression of gp , na + -k + -atpase, and the transferrin receptor were not affected. cftr is a member of the atp-binding cassette (abc) transporter superfamily. abc proteins are atp-dependent transporters involved in exporting a wide variety of cytotoxic agents across the plasma membrane. p-glycoprotein (pgp; abcb , mdr gene product), also an abc transporter, is one of the major drug-efflux pumps expressed in normal tissues, as well as in many human cancers. over-expression of pgp results in reduced intracellular drug concentration and reduced cytotoxicity, conferring multi-drug resistance (mdr) to cancer cells. the aim of the current study was to examine whether cif also affects pgp expression in the plasma membrane, which could be exploited as a potential therapeutic strategy for restoring the sensitivity to pgp-transported drugs in cancer chemotherapy. cif significantly reduced the apical expression of pgp in mdck cells stably transfected with gfp-tagged pgp (mdck-gfp-mdr cells) and in caco- and calu- cells, which express endogenous pgp in the apical plasma membrane. the drug sensitivity of mdck-gfp-mdr cells to doxorubicin, a pgp substrate, was evaluated in the absence and presence of cif by measuring the ec . cif reduced the cytotoxicity of doxorubicin by -fold. neither cif nor vehicle alone had a cytotoxic effect. the drug sensitivity of the parental cell line, mdck-c , that expresses -fold less pgp than the transfected mdck cells, to doxorubicin was also increased by -fold. by contrast, cif had no effect on the ec values of cisplatin (a substrate of mrp ) and etoposide (a substrate of mrp ), suggesting that the alteration of the sensitivity to doxorubicin was due to decreased apical expression of pgp, but not other abc transporters. these results suggest that although cif may be an obstacle to therapeutic attempts to restore the apical expression of cftr in the cf lung, it could be useful for the development of a novel class of inhibitors of pgp aimed at increasing the sensitivity of tumors to chemotherapeutic drugs (supported by the nih ( ro hl )). hamai, h. ; keyserman, f. ; worgall, t.s. , . pathology, columbia university, new york, ny, usa; . pediatrics, columbia university, new york, ny, usa cystic fibrosis (cf) is characterized by an inflammatory state and susceptibility to chronic lung infections. a common clinical finding is dyslipidemia characterized by altered plasma free fatty acid patterns, low plasma hdl levels and increased phospholipase a (pla ) activity. it is not clear how cftr mutations relate to lipid abnormalities. it was shown that defective cftr is associated with decreased uptake of sphingolipids (sl). sl homeostasis is tightly regulated and metabolites are relevant to lipid metabolism and cf. sl synthesis (sls) correlates with activity of srebp, a key transcription factor of lipid metabolism; ceramide- -phosphate, a sl metabolite, is a regulator of pla that is highly activated in cf. we investigated the hypothesis that sls is increased in cf. experiments were carried out in airway epithelial cell lines that express defective cftr (c / ib ), no cftr ( hbe sense / antisense) and overexpress cftr (a cells infected with adcftr or adcontrol). sls was assessed using radioactive tracers h-serine (de-novo sls) and h-sphinganine (recycling pathways). ceramide and sphingomyelin mass were determined enzymatically. neutral and alkaline sphingomyelinase activity was assessed using h-sphingomyelin as substrate. srebp mediated gene transcription was assessed using sre-promoter assays. cholesterol synthesis was evaluated by incorporation of h-acetic and h-mevalonic acid. cholesterol mass was determined by gas-chromatography. abca- expression was determined by western blot analysis. pla activity was measured using fluorescent phospholipid substrates. sls was post-transcriptionally increased in cells expressing defective or no cftr through the de-novo pathway ( % ± % for ib ; %± % for hbe-antisense; p< . ) and the recycling pathways ( % ± % or ib ; % ± % for hbe-antisense; p< . ). ceramide mass was increased by % (± %) in hbe antisense, % ± % in ib cells. sphingomyelin mass was increased -fold in ib , up to -fold in hbe antisense cells. neutral and alkaline sphingomyelinase activity was increased up to -fold in both cell lines. sre-mediated gene expression was increased up to -fold in ib and by % in hbe-antisense cells. overexpression of cftr in a decreased sls and srebp activity up to %. free cholesterol synthesis was increased by % in ib and by % in hbe antisense. expression of the cholesterol efflux receptor abca- was decreased in ib and hbe antisense compared to controls. sl mass, sre-mediated gene transcription and pla activity were decreased following incubation with inhibitors of sls in ib and hbe antisense cells. conclusion: defective cftr or lack of cftr expression correlates with ) increased sls synthesis and sl mass; ) increased sremediated gene transcription and free cholesterol synthesis; ) decreased expression of the abca- cholesterol efflux receptor. inhibition of sls decreases sre-mediated gene transcription and pla activity. dysfunctional sls is a newly recognized pathway associated with defective cftr and a possible therapeutic target in cf. the ability to make accurate, reproducible measures of mucociliary and cough clearance (mcc/cc) in cf patients is critical to assessment of new therapies designed to improve mcc/cc function, and thus decrease pulmonary infections and decline in lung function. for example, using mcc/cc methods developed in our laboratory, we showed that -week treatment with aerosolized hypertonic ( %) saline (hs) led to a sustained increase in mcc and improved lung function in cf patients (donaldson et al, n eng j med ; : ) . the mcc/cc technique involves patient inhalation of radiolabeled particles followed by gamma camera scanning to determine the rate of particle movement from the lungs. while a few cf centers have shown the capability to measure mcc/cc, the techniques across these centers are not standardized, making comparison of results difficult. it is vital to develop standard, simplified techniques for these measures to make larger cf patient populations available for such studies. to standardize a radiolabeled particle inhalation technique that can be easily used by other cf centers, we have compared three protocols in healthy and cf subjects. protocol # was identical to that used in recent studies, and incorporates a devilbiss jet nebulizer to generate µm mmad (gsd = . ), aqueous droplets containing suspended tc m-sulfur colloid particles. tidal volume and inhaled/exhaled flow rates were guided by a volume signal displayed on an oscilloscope. protocol # was further standardized and simplified to allow use at other centers. this protocol utilized the same nebulizer, but triggered by a commercial dosimeter (spira electro , finland) during inhalation as the subject breathed in time to a metronome ( breaths/min) at flow rate of . l/sec. subjects controlled inspiratory flow rate by feedback from a digital flow readout on the dosimeter. repeat measurements of mcc/cc were made in healthy subjects and mild cf patients to compare mcc/cc variables following radioaerosol inhalation using protocols # and # . there were no differences in particle deposition (i.e. central to peripheral ratio, or "c/p"), or clearance of particles through minutes and hours in either subject group. these data suggest that the new methodology (protocol # ) will be useful and easily employed by other cf centers to test effectiveness of new therapies for cf. the third protocol (# ) was then tested in five of the healthy subjects. this protocol used a nebulizer that generated very large droplets ( . um mmad, gsd = . ) and subjects inhaled at very slow flow rates (~ ml/sec). it was designed to provide greater bronchial airway deposition and thus an "improved signal" for mcc/cc evaluation. indeed, this methodology produced a larger average c/p and significantly greater particle clearance from the lung compared to the other standardized methodology (protocol # ) (c/p = . vs. . ; min clearance = % vs. %; hr clearance = % vs. %, respectively). interestingly, the greater fraction of clearance between min and hr may also reflect heightened ability to assess clearance from smaller airways, which could be particularly important in cf. protocol # will be tested further in cf patients. supported by cf foundation. previous clinical trials in cystic fibrosis (cf) indicate that anti-inflammatory therapy probably will not result in improvement in lung function, but will slow the rate of decline. this imposes constraints on study design for new anti-inflammatory agents, requiring that they use many patients over long periods. it is highly desirable to design a strategy for evaluation of antiinflammatory agents that will allow for the selection of only the most promising agents for phase iii trials. sputum induction (si) samples lower respiratory tract secretions and permits measurement of inflammatory markers. the purpose of this study was to assess the measurement of inflammatory markers in induced sputum as one such strategy by using ibuprofen as the test agent because it has demonstrated clinical benefit in cf. if changes in inflammatory markers are detectable in ibuprofen treated patients, then si might serve as a quick, noninvasive method by which to select the most promising anti-inflammatory agents for further study. methods: in this twoarm (ibuprofen and no treatment), open-label, parallel group study, cf patients >/= years of age with mild to moderate lung disease were screened at sites. si was performed on days , , and . patients in the ibuprofen group received drug on days - . subjects met eligibility criteria. were randomized to ibuprofen and to no treatment. subjects ( ibuprofen and no treatment) completed the study through day and comprise the intent-to-treat efficacy population. a maximum of subjects ( ibuprofen and no treatment) and a minimum of subjects ( ibuprofen and no treatment) across all markers had acceptable inflammatory marker values at both days and . within group and between group comparisons were made. results: with respect to the within group comparisons (before and after ibuprofen), most inflammatory markers slightly decreased in the ibuprofen group and increased in the no treatment group. for ibuprofen patients, mean changes were most apparent for il- (- . log pg/ml, p= . ) and % neutrophils (pmns) (- %, p= . ). with respect to the between group comparisons (ibuprofen vs. no treatment), differences were strongest for il- ( . log pg/ml, p= . ), percent pmns ( %, p= . ), and absolute pmn count ( . cells/ml, p= . ). fourteen days after discontinuing ibuprofen, inflammatory mediators tended to increase. there was no difference in aes between the two groups, and si was generally safe. conclusions: . overall, si is well-tolerated in patients >/= years, . a -week treatment period may not be long enough to study anti-inflammatory drugs, . with respect to changes in inflammatory markers associated with anti-inflammatory therapy, the effect size is small and the variance of the effect is small in clinically stable patients. . measuring inflammatory markers in induced sputum has potential as a screening tool for studying anti-inflammatory drugs in cf, . further studies of si in cf are warranted. supported by cystic fibrosis foundation therapeutics, inc. in preparation for our gene therapy clinical trial programme we are currently assessing a number of sputum biomarkers including viscosity and elasticity, total solids and dna content and hr sputum weight. we tracked and correlated these biomarkers in cf patients ( years and over) during a course of iv antibiotics (ab) by collecting samples on several occasions [visit (v) : at the start of ab treatment, v : at the end of ab treatment (generally after weeks) and interim periods]. to ensure adequate reproducibility of the results viscosity/elasticity measurements were carried out in triplicate using a csl rheometer which required a comparatively large volume ( mls) of spontaneously expectorated sputum. because of this requirement paired samples could only be obtained from approximately % of the patients. there was no change in viscosity/elasticity (n= ), solid or dna (n= ) content when comparing samples at the beginning and end of iv ab. in contrast, hr sputum weight was significantly (p< . ) lower at the end of the ab treatment (visit : . ± . g, visit : . ± . g, n= ). there was a strong and significant correlation between solid content and viscosity/elasticity (r= . , p< . ) and a modest correlation between hr sputum weight and % predicted fev (r=- . , p< . ) and hr sputum weight and patient scored symptom severity (r= . , p< . ). surprisingly, sputum dna content did not correlate with viscosity/elasticity, despite being generally thought of as a contributor to viscosity, which may in part be related to the assay not being able to discriminate between free and cell-enclosed genomic dna.in summary, after a course of iv antibiotics which lead to significant subjective and objective (fev ) improvement the overall quantity of expectorated sputum was significantly reduced but, based on analysis available to date, none of the other parameters (viscosity, elasticity, solid and dna content) changed significantly. considering the difficulties we encountered in collecting sufficient sputum during this period of an exacerbation, sputum viscosity/elasticity measurements may not be feasible parameters to measure in a gene therapy trial to which stable patients re likely to be recruited. this study was funded by the cf trust. chronic neutrophilic inflammation is a feature of cystic fibrotic (cf)related inflammation and serves as a significant contributor to morbidity and mortality associated with the condition. recently, our group has described a novel collagen-derived neutrophil chemoattractant, proline-glycine-proline (pgp), in a murine model of lipopolysaccharide-induced inflammation. the purpose of this study was to explore both the presence of this peptide in sputum from cf patients and the determination of a proteolytic system involved in pgp generation. we show that lower airway secretions from cf subjects have -fold increase in pgp levels compared to normal controls and that cf sputum (p< . ) is able to generate pgp from intact type i and ii collagen -fold above that seen from normal control sputum ex vivo (p< . ). we further demonstrate that production of pgp is dependent on two specific matrix metalloproteases (mmps), mmp- and mmp- . finally, we show that the generation of pgp can be significantly inhibited ( - % inhibition) by the use of either mmp- or mmp- specific inhibitors (p< . versus no inhibitor); a nonspecific mmp inhibitor (doxycycline) also demonstrates significant attenuation of pgp production. the determination of the requirements of these proteases in pgp generation allows for the identification of logical targets for disease-modifying therapeutics in cf. . cfrc, uab, birmingham, al, usa; . cfrc, university of north carolina, chapel hill, nc, usa cystic fibrosis (cf) lung disease is characterized by chronic neutrophilic inflammation. the discrete collagen breakdown product prolineglycine-proline (pgp) is a potent neutrophil chemoattractant thought to be generated by a specific proteolytic cascade present during cf pulmonary exacerbation (nat med ( ): , ) . high-mobility group box (hmgb ) is a potent inflammatory mediator found in sepsis, rheumatoid arthritis, and other inflammatory diseases characterized by neutrophilic inflammation. the role of pgp and hmgb as pmn attractants in cf is unknown. in this study we utilized specimens derived both from humans and scnn b-transgenic mice (βenac mice) in which overexpression of the βenac subunit in the airways leads to sodium hyperabsorption and airway surface liquid depletion, mimicking the cf muco-obstructive phenotype. pgp was quantified using tandem ms. neutrophil chemotaxis was measured in vitro following preincuabtion of sputum or balf with anti-hmgb neutralizing antibody. murine balf was assayed for hmgb by western blot and elisa. human sputum was similarly evaluated after normalizing for total protein concentration. in vivo hmgb activity was measured after intratracheal instillation in wt mice. βenac mice had % higher cell counts and a greater percentage of pmns ( . % vs. . %) in bal samples than wild-type (wt) littermates (p< . , n= /genotype). pgp was elevated in balf of scnn b animals ( . vs. below limits of detection in wt controls, p< . , n= ), increased in serum from cf subjects ( vs. pg/ml normal controls, p< . , n= ), and detected at high levels in cf sputum ( , pg/ml). balf screened for hmgb by western blot revealed elevated levels in βenac mice (p< . by densitometry, n= /genotype); elisa confirmed % greater hmgb concentration ( . vs. . ng/ml, p< . ). hmgb was also detected by western blot in human cf sputum ( of ) and at higher levels than secretions from normal subjects (p< . by densitometry; n= cf, control). purified hmgb induced dose-dependent pmn chemotaxis in vitro (peak - ng/ml, p< . , n= ) and cf sputum caused potent chemotaxis ( -fold over control, p< . , n= ) that was inhibited by preincubation with hmgb antibody (p< . ). balf from βenac mice (but not wt controls) was also chemotactic ( -fold greater than media control, p< . , n= ) and inhibited by anti-hmgb antibody (p< . ). exogenous hmgb ( ng) administered intratracheally to lps resistant mice (c hej, toll- receptor mutant) and balb/c mice caused pmn influx in balf at hours (c hej: . x pmns after hmgb vs. . x after vehicle, p< . ; balb/c: . x vs. . x , p< . ), and generation of pgp ( . vs. . pg/ml in c hej, p< . , n= ) . in summary, pgp and hmgb are elevated in balf of βenac mice that present with cf-like lung pathology; in cf airway secretions, hmgb is present in vivo at concentrations that induce neutrophil chemotaxis in vitro and pgp production in vivo, and is inhibited by blocking antibody. the role of pgp and hmgb in cf deserve further evaluation as they may be potential therapeutic targets of dysregulated inflammation. esther, c.r. ; jasin, h.m. ; collins, l.p. ; boysen, g. ; boucher, r.c. . pediatric pulmonology, university of north carolina at chapel hill, chapel hill, nc, usa; . cf research center, univeristy of north carolina at chapel hill, chapel hill, nc, usa; . center for environmental health and susecptibility, univeristy of north carolina at chapel hill, chapel hill, nc, usa biomarkers of airway inflammation are needed in cystic fibrosis (cf) to aid clinical management and assess the efficacy of novel therapies. we describe a simple and non-invasive method to measure airway purines, signaling molecules previously shown to be biomarkers of neutrophilic airway inflammation. airway secretions were obtained using exhaled breath condensate (ebc) collection, a technique that was easily performed in the outpatient setting on children as young as three years. the purines adenosine and amp in ebc were measured using ultra-sensitive liquid chromatography/tandem mass spectrometry (lc/ms/ms). in addition, we used lc/ms/ms to simultaneously measure urea as a dilution marker to control for the known dilutional variability of airway secretions within ebc. detection of adenonosine, amp, and urea was optimized using positive mode ms with selected reaction monitoring, and stable isotope dilution was utilized to improve quantification. detection was linear with concentration over four orders of magnitude, with coefficients of variation < %. the method was sensitive, with limits of detection for adenosine and amp (~ . nm) and urea (~ . µm), below expected ebc concentrations. applying the methods to ebc demonstrated that detection of purines and urea was reliable, with intraclass correlation coefficients greater than . between duplicate measures (n= ). we also measured purines and urea in ebc samples collected prospectively from children with cf (n= ) and healthy controls (n= ) during regular clinic visits. all samples were lyophilized and reconstituted to increase concentration -fold. adenosine, amp, and urea were detected in all samples. neither adenosine nor amp levels differed between groups, but urea was significantly lower in cf ( . ± . µm) versus healthy control ( . ± . µm, p= . ), suggesting that airway secretions were more dilute in cf samples. when dilutional variability was controlled using purine to urea ratios, the amp to urea ratio was found to be elevated in subjects with cf ( . ± . ) compared to healthy controls ( . ± . , p= . ), consistent with previous findings. the amp to adenosine ratio was also elevated in cf ( . ± . ) compared to control ( . ± . ), although the difference did not reach statistical significance in this small sample set (p= . ). experiments are ongoing to increase sample size and assess ebc purine levels before and after treatment of a cf exacerbation. these results demonstrate that lc/ms/ms analysis of ebc provides a non-invasive method to measure purine biomarkers of cf airway inflammation. given the flexibility of ms, this methodology could also prove applicable for study of other small molecule biomarkers. importantly, we also show that dilution of airway secretions in ebc may be altered in cf, and lc/ms/ms can be used to control for this variability through measurement of urea as a dilution marker. introduction: mucociliary clearance and antimicrobial peptide activity may be affected by airway surface liquid (asl) ph, which is regulated by epithelial ion transport. asl ph is abnormal in disease states in which il- a is elevated, including cystic fibrosis (cf), and the il- a receptor is expressed at the basolateral surface of bronchial epithelium. therefore, we investigated the effects of il- a on vectorial ion transport in well-differentiated hbe cells. methods: hbe cells were grown with an apical air interface and incubated with il- a at or ng/ml in the basolateral medium for hours prior to being studied with standard short-circuit current (isc) techniques. results: il- a treated cells had a minimal increase in resting and amiloride-sensitive isc compared to control cells, and had a dosedependent, statistically significant increase in forskolin-stimulated isc. at a dose of ng/ml, il- a doubled forskolin-stimulated isc ( . ± . µa/cm for control vs. . µa/cm for il- a). the increased isc was not bumetanide-sensitive, but was sensitive to acetazolamide and dnds, and was not present in hco --free solutions, suggesting it was due to hco secretion. to investigate whether this hco secretion was cl --dependent, we studied hbe cells in cl --free solutions, and there was no difference between untreated and il- a treated hbe cells. to test the hypothesis that il- a promoted cl -/hco exchange, we performed experiments in hbe cells mounted in cl --free solutions, and after addition of amiloride and forskolin, mm nacl was added to the mucosal bath and mm nagluconate was added to the serosal bath. under these conditions, one would predict a decrease in isc due to mucosal to serosal clmovement down its electrochemical gradient. in untreated hbe cells, we saw a decrease in isc followed by a recovery. in il- a treated cells there was a sustained increase in isc, suggesting that movement of cldown its electrochemical gradient resulted in exchange of clfor an anion at the apical membrane. in the absence of hco -, both untreated and il- a treated cells responded to claddition to the mucosal bath with decreases in isc. to assess the cftr dependence of the il- a-induced isc, we stimulated primary cf hbe cells treated with il- a. in cf cells, there was a minimal increase in amiloride-sensitive isc ( . ± . µa/cm for control vs. ± . µa/cm for il- a ) and in forskolin-stimulated isc ( . ± . µa/cm for control vs. . ± . µa/cm for il- a ). conclusion: the proinflammatory cytokine il- a induces cl -/hco exchange in hbe cells. notably, the observed cl -/hco exchange appears to be cftr-independent. however, cftr activation is required for maximum cl -/hco exchange as demonstrated by the smaller isc generated in cf cells. these data suggest that the cytokine milieu of the airway epithelium can alter ion transport and potentially asl physiology. furthermore, they suggest that cftr may interact with or regulate novel proteins in the presence of inflammation. supported by cff and nih periciliary fluid balance is maintained by the coordination of sodium and chloride (cl-) channels in the apical membranes of the respiratory epithelia. in the absence of the cystic fibrosis transmembrane conductance regulator (cftr), cl-secretion is diminished and sodium reabsorption becomes exaggerated. activation of non-cftr-dependent cl-channels can provide an alternate pathway for cl-secretion in the airways and gastrointestinal tract. the ph and voltage-dependent type- cl-channel (clc- ) is expressed in airway epithelial cell luminal membranes. we hypothesize that topically applied clc- agonists may restore cl-secretion in cystic fibrosis (cf) murine airways. using in vivo nasal potential difference measurements, we quantified clc- -mediated cl-transport in both cf and wild-type mice during nasal perfusion with lubiprostone (a prostone compound and specific clc- agonist; sucampo, pharmaceuticals, inc., bethesda md) or vehicle control. wild-type (c bl , n = ) and cf knock-out (cfko; n= (jax stock# )(jackson laboratories, bar harbor, me) mice were sedated and intubated to protect the lower airways from aspiration of perfusate. nasal and subcutaneous bridges were connected to fluid-filled silver chloride electrodes. baseline (in ringer's), amiloride-inhibited, low cl-(cl-free, gluconate-substituted ringer's with amiloride), and low cl-plus lubiprostone (with increasing concentrations of lubiprostone or vehicle) mice were perfused and potential differences were measured with a high impedance voltmeter (world precision instruments, sarasota fl) and continuously recorded. a clear dose-response relationship was detected in both wild-type and cf mice. at µm lubiprostone, wild-type mice showed hyperpolarization of - . ± . mv and cf mice responded with - . ± . mv hyperpolarization. a paired t-test of low cl-perfusion and lubiprostone perfusions revealed significant (p< . ) differences in both genotypes. five clc- cfko mice were similarly tested and showed no response to lubiprostone (+ . ± / mv). cftr inhibitor- ( µmol) (calbiochem, san diego ca) added to the low cl-perfusion in additional wild-type mice eliminated the low cl-response but did not abolish the lubiprostone response, confirming that clc- is present and independent of cftr regulation. we conclude that direct application of a clc- agonist in the cf murine upper airways restores near normal levels of cl-secretion. cftr is an apical membrane chloride channel whose activity is required to maintain airway surface liquid (asl) volume and efficient mucociliary clearance. cftr is activated by camp dependent stimulation of protein kinase a. however, studies have suggested that the actin cytoskeleton may also be required for regulation of cftr, by acting as a structural element in second messenger compartmentalization ( ) . to determine whether an intact cytoskeleton is required for asl volume homeostasis, adenosine (ado), atp, or isoproterenol (iso) (all at µm) were added apically to human bronchial epithelial cultures (hbec's) before or after the cytoskeleton had been disrupted by cytochalasin d exposure ( µm for h). in control cultures ado, atp and iso all resulted in a significant increase in asl height (∆asl) within min (∆asl; ado, . ± . µm; atp, . ± . µm; iso, . ± . µm). however, in cytochalasin d exposed cultures ado was without effect while atp and iso were still able to evoke an increase in height (∆asl; ado, - . ± . µm; atp, . ± . µm; iso, . ± . µm). however, we showed that a b receptor function was not compromised by cytoskeleton disruption as camp levels were seen to increase both after addition of adenosine and adenosine in the presence of cytochalasin d. thus, the effect of cytochalasin d on asl height appeared specific for adenosine-mediated cl-secretion. to further test the effect of cytochalasin d on the relationship between adenosine and cftr, we designed a fluorescent resonance energy transfer (fret) pair of cfp-cftr (labeled at the n-terminus) and yfp-a b receptor (labeled at the c-terminus). addition of adenosine ( µm) increased fret by . times (% efficiency; control, . ± . %, ado, . ± . %) compared to the vehicle control (n= ). cytochalasin d exposure ( µm for h) completely inhibited the increase in fret that was observed after adenosine addition (p< . ). in contrast, cytochalasin d had no effect on fret between cftr and the β adrenoceptor. to determine whether cytochalasin d treatment ( µm for h) had any direct effect on cftr protein levels and localization we used a cell line stably expressed with an exotope-tagged cftr (hb cells) ( ) . cftr protein levels were found to be significantly reduced as compared to nontreated controls by both western blot analysis and chemiluminescence detection (n= ). cells were also stained with an antibody against the external cftr exotope and imaged by confocal microscopy and a significant decrease in staining at the plasma membrane was observed after cytochalasin d treatment (n= ). we conclude that cytochalasin d exposure decreases cftr protein levels and specifically inhibits adenosine-mediated regulation of cftr activity in the airways, leading to disruption of asl homeostasis and inhibition of mucociliary clearance. numerous studies have reported evidence that cftr is expressed in the apical membrane of serous cells in the airway submucosal glands. thus, it has been reasoned that ( ) cftr plays some role in normal anion and fluid secretion by airway glands and ( ) loss of this channel's function in cystic fibrosis (cf) airways plays some role in the etiology of cf lung disease. a recent study by joo et al. (jbc : - , ) indicates that cf airway glands lose the ability to secrete fluid when treated with forskolin or vip but maintain the ability to secrete when treated with muscarinic agonists. this result suggests that vip and muscarinic agonists mediate secretion by cftr-dependent and cftr-independent pathways, respectively. the present study was undertaken to explore the dynamics of fluid secretion by individual submucosal glands to determine if further differences in their responses to these two agonists could be distinguished. pig tracheas were obtained from a local slaughterhouse. after removal of the cartilage, tissues were mounted in a warm ( °c) observation chamber that permitted exposure of the submucosa to krebs solution. the mucosal surfaces of the airways were dried with a stream of warm dry air and then layered with mineral oil. liquid secreted from the gland duct openings formed spherical aqueous droplets within the oil layer, and their volumes were estimated from spatial dimensions taken from sequential digital images. liquid secretion rates were estimated for individual glands present within a . mm region of interest. rates were determined for two experimental protocols. for one group of tissues, secretion rates were determined for a control period, followed by a period of exposure to µm vip, and then a period of exposure to both vip and µm acetylcholine (ach). in the second group, the order of vip and ach treatment was reversed. these agonist concentrations were expected to produce near maximum rates of secretion. when the agonists were applied first, secretion rates were significantly (p< . ) increased from the control period by both ach ( . ± . nl/min to . ± . nl/min) and vip ( . ± . nl/min to . ± . nl/min). the secretion rates for individual glands were highly variable with both agonists (ach: . - . nl/min; vip: . - . nl/min). when ach application followed vip treatment, the mean secretion rate was significantly increased further to . ± . nl/min; however, when vip application followed ach treatment, the secretion rate was only marginally and insignificantly increased to . ± . nl/min. we conclude that both ach and vip are efficacious secretagogues for porcine airway glands and that the rate of secretion from individual glands to either agonist is very heterogenous. because vip has no effect following stimulation by ach, we speculate that ach induces secretion by activating all transporters relevant to vip-induced secretion including cftr. however, ach must also activate channels and/or transporters not induced by vip since its effects are additive to vip. this most likely includes activation of a non-cftr anion channel. ( serous cells are thought to serve as the principal anion and fluidsecreting cells of airway submucosal glands. mucous cells primarily secrete gel-forming mucins. these respective physiological roles for these two cell types imply that the mucous cells, which secrete the thick mucus gel, should lie downstream of the serous cells so that their watery secretions can flush the mucins out of the ducts and onto the airway surface. early studies indicated that such arrangements existed within glands but reports of cell distributions have been descriptive rather than quantitative. because of resurgent interest in serous cell function, we revisited this issue to formally document the distribution of these cell types within the secretory tubules of the submucosal glands as well as at the surfaces of the glands, where serous cells are most accessible for study. a piece of porcine lung, containing a mm diameter bronchus, was treated with formalin fixative, and consecutive µm sections were taken. the apices of virtually all gland epithelial cells, except the cells lining the collecting and ciliated ducts, stain positive for pas indicating that these cells are either serous or mucous cells. however, pas staining does not allow distinctions to be made between these two cell types. consequently, all slide sections were stained with hematoxylin and eosin. with these stains, mucous cells were identified as cells containing lucent granules in their apices whereas serous cells were considered to be cells where the apical cytoplasm stained a uniform dark reddish color. first, the distribution of serous and mucous cells in the secretory tubules were determined. the acini of individual tubules were located in the slide sections, and cells were identified and counted in consecutive sections that approached the collecting ducts until the tubules were no longer distinguishable as discrete tubes. secretory tubules from individual glands were studied. in the acini, serous cells accounted for . ± . % of total cells whereas mucous cells accounted for . ± . % of total cells (remaining cells were not distinguishable as either cell type). there was a significant (p<. ) negative correlation of serous cell numbers with distance from the acini so that at µm from the acinus serous cells accounted for only . ± . % of total cells. there was a coincident significant positive correlation with mucous cell numbers so that at µm from the acini mucous cells accounted for . ± . % of total cells. next, we examined the cell distribution at the adventitial and mucosal surfaces and at the lateral margins of the glands. serous cells accounted for . %, . %, and . % of the total cells at these respective locales. mucous cells represented . %, . %, and . % of total cells in these same respective regions. we conclude that serous cells are by far the dominant cell type in secretory tubules of porcine submucosal glands. mucous cells are rare in acini but increase in frequency with distance from the acini. the outer surfaces of the glands are dominated by serous cells as well. at the lateral margin of the glands, serous cells outnumber mucous cells by -to- . (supported by nih hl ) . many of the membrane transporters that participate in this process have been identified; however, the identity of the class of kchannels that maintains resting membrane potential and/or cell membrane polarization during the secretion process remains poorly characterized. liquid secretion by porcine submucosal glands is insensitive to numerous k + channel blockers including ba + , tetraethylammonium, apamin, charybdotoxin, iberiotoxin, clotrimazole, penitrem a, -aminopyridine, and quinidine. recently, a new class of k + channels, the "tandem-pore" or k p channels, have been described. potassium channels of this class are insensitive to many traditional k + channel inhibitors but are blocked by local anesthetics, such as lidocaine and bupivacaine. additionally, many k p channels are sensitive to changes in extracellular ph. in the present study, we considered whether k p channels might participate in the liquid secretion response to acetylcholine (ach). intrapulmonary bronchi, excised intact from the lungs of pigs, were cleared of accessible luminal liquid, cannulated, and treated with µm ach to induce secretion. paired airways were pretreated for h with mm bupivacaine. bupivacaine blocked . ± . % of the liquid secretion response to ach. the ic for the bupivacaine inhibition was approximately µm. the inhibitory effect of bupivacaine was not due to nonspecific toxicity since tissues exposed to bupivacaine for h and then washed with fresh inhibitor-free buffer recovered to % the control achinduced secretory rate. the bupivacaine effect was not due to inhibition of voltage-gated na + channels in intrinsic neurons since µm tetrodotoxin did not inhibit ach-induced secretion. no significant effect was seen in the secretion rate when the extracellular solution ph was lowered from . ± . (normal hco --buffered krebs gassed with % co ) to . ± . by gassing with % co gas (balance o ). while we recognize the relative nonspecificity of this agent, the inhibitory effects of bupivacaine on liquid secretion by porcine bronchi are consistent with k p channel participation in this secretory response. the failure to demonstrate inhibition with acidification of extracellular solution could signify a k p channel subtype that is insensitive to low extracellular ph. ( . novartis, horsham, united kingdom; . rosalind franklin university, chicago, il, usa; . mount sinai medical center, miami, fl, usa; . genomics institute of the novartis foundation, la jolla, ca, usa enac activity in the human airway epithelium has been reported to be partially-sensitive to the broad spectrum trypsin-like protease inhibitors aprotinin and placental bikunin (bridges et al., am j physiol :l - ; donaldson et al., jbc : - ) . a low molecular weight inhibitor of the airway channel activating protease (cap) would represent a potential therapeutic approach to attenuating enac function in cystic fibrosis (cf) lung disease. the aims of the current study were to: ( ) further characterise the in vitro efficacy of cap inhibitors in human bronchial epithelial cells (hbes), ( ) to evaluate the relevance of a cap mechanism to the regulation of enac function in vivo, and ( ) to establish whether the inhibition of the airway cap can modulate mucociliary clearance in vivo. primary cultures of hbes (normal and cf), cultured under air-liquid interface conditions, demonstrated an amiloride-sensitive short circuit current (isc), that was sensitive to aprotinin but that was insensitive to inhibition by sbti, α -anti-trypsin and α -pdx. the amiloride sensitive isc was also attenuated by the low molecular weight cap inhibitor nvp-qau (hbc ) with an ic value of approximately nm. nvp-qau caused a time dependent inhibition of the isc with a t ⁄ of approximately min and this effect could be reversed by the addition of excess trypsin. in vivo, aprotinin attenuated the guinea pig tracheal potential difference (tpd) from - . ± . mv to - . ± . mv (n= - ) with an ed value of pmoles/kg, measured at hours following intra-tracheal instillation. the tpd was unaffected by either sbti or α -anti-trypsin at doses up to pmoles/kg. the combination dosing of an enac blocker with aprotinin did not attenuate the tpd beyond the effect observed with either agent alone, consistent with aprotinin attenuating enac function in this model system. intra-tracheal instillation of nvp-qau attenuated the tpd from - . ± . mv to - . mv with an ed value of µg/kg (n= - ). in the sheep, the administration of nvp-qau into the airways by aerosolisation of a mg/ml solution resulted in a - fold enhancement of the rate of clearance of mtc sulfur colloid from the lungs compared with the vehicle control. these studies indicate that the cap mechanism of enac regulation can translate through in vitro human assays, into in vivo models. cap inhibitors can attenuate tpd in the guinea pig in vivo and enhance the rate of mucociliary clearance in the sheep, thereby representing an approach to the therapeutic regulation of enac function in cf lung disease. cotton, c. pediatrics, physiology and biophysics, case western reserve university, cleveland, oh, usa cystic fibrosis is caused by mutations in the gene that encodes cftr, a camp-activated apical membrane anion channel. loss of cftr-mediated anion conductance is a primary defect in cf but secondary defects such a sodium hyperabsorption are also implicated in cf pathophysiology. it is generally accepted that mucociliary clearance is compromised in cf airways due to reduced fluid secretion and/or increased fluid absorption. the recent generation of a transgenic mouse model with enac overexpression that exhibits cf-like lung disease highlights the importance of sodium hyperabsorption. therapies designed to reduce mucus production and viscosity, increase lumenal water content, stimulate fluid secretion, and inhibit fluid absorption are under development for treatment of cf airways. although controversial, several recent studies have demonstrated that serca pump inhibitors partially correct the trafficking defect associated with delta f mutant cftr. the goal of this work is to determine if serca pump inhibitors affect enac-mediated sodium absorption. a renal collecting duct epithelial cell line and primary cultures of well-differentiated human tracheal epithelial cells (air/liquid interface culture) were used for these studies. amiloride-sensitive short circuit current was determined as a measure of enac-mediated sodium absorption. quantitative rt-pcr was used to evaluate expression levels for each of the subunits of enac (alpha, beta, and gamma). epithelial monolayers were treated for - hours with serca pump inhibitors (thapsigargin, - nm; dbhq, - um; and curcumin, - um). treatment with serca pump inhibitors decreased amiloride-sensitive short circuit current by - %. in contrast, campstimulated short circuit current was not reduced by treatment with serca pump inhibitors. the steady-state levels of alpha, beta, and gamma enac were reduced by - % in renal epithelial monolayers treated with thapsigargin whereas beta and gamma enac were reduced by - % with no significant change in alpha enac in airway epithelial cells. the results of these studies demonstrate that sustained depletion of endoplasmic reticulum calcium stores and/or elevation of intracellular calcium by inhibition of serca pump activity reduces enac mrna expression and enac-mediated sodium absorption. thus, inhibition of serca pump activity in airway epithelial cells of cf patients that carry the delta f mutation may provide dual benefit by promoting delivery of mutant cftr to the membrane . pediatrics, national jewish medical and research center, denver, co, usa; . integrated department of immunology, national jewish medical and research center, denver, co, usa; . department of medicine, cystic fibrosis/pulmonary research and treatment center, the university of north carolina at chapel hill, chapel hill, nc, usa increased cytosolic calcium ([ca + ] i ) initiated by the release of stored ca + from the endoplasmic reticulum (er) is a key signal to elicit a wide range of essential cellular responses, including secretory functions of the respiratory epithelium that are modified in cf. serca pumps are responsible for (re)filling the er ca + stores, and serca blockers such as thapsigargin are very potent and commonly used pharmacological triggers of ca + -signals. modulation of the activity of sercas can profoundly affect ca + homeostasis. although defective calcium homeostasis is a characteristic of several pulmonary diseases including cf, the role of serca is unknown. lung tissue samples (bronchus and distal lungs) from normal (n= ) and cf subjects (n= ) were evaluated by immunohistochemistry. serca expression was decreased in the bronchial and bronchiolar epithelia of cf. non-cf and cf bronchial epithelial cell line pairs including calu- and jme/cf , c- and ib - , hbe o-( hbe), cfbe o-(cf o-) and cfbe o-(cf o-) were also probed. given certain limitations of such cells, several consistent findings still emerged. a % and % decrease in serca expression was observed in cf o-and cf ocells as compared to hbe cells. immunocytochemical studies in these cells confirmed that the serca was localized in the er and that the decreased serca expression was not associated with decreased er content. reduced serca expression and activity ( . ± . vs . ± . and . ± . pmol/min/mg protein in hbe vs. cf o-and cf ocells respectively) was observed in the purified er membranes from cf cell lines. northern blot analysis revealed a parallel reduced mrna expression as well. decreased serca was accompanied by increase in the low affinity isoform serca ( . ± . vs. . ± . and . ± . arbitrary serca intensity/b-actin in respectively) . we have also evaluated a limited number of primary airway epithelial cells isolated from lung samples of normal and cf subjects for serca expression. serca expression in polarized tracheobronchial epithelial cell lysates from cf subjects was decreased by about % as compared to those from normal subjects ( . ± . vs. . ± . arbitrary serca intensity/b-actin units in cf and normal respectively, n= ). expression of serca could also be suppressed by inhibiting cftr with cftr inh in normal human bronchial epithelial cells ( various studies indicate that the airway surface fluid possess an antibacterial system based on the combined action of lactoperoxidase, h o , and scn -(thiocyanate). the enzyme lactoperoxidase (secreted by submucosal glands) utilizes h o and scnto generate oscn -(hypothiocyanite) a molecule with antimicrobial activity. h o is produced by dual oxidases expressed on the apical membrane of airway epithelial cells whereas scnis transported across the epithelium through anion transporters and channels. in particular, scntransport seems to occur through cftr and other ca +dependent clchannels. therefore, reduced scntransport in the airways of cystic fibrosis patients may contribute to impaired antimicrobial activity. in a recent study, we have found that cytokines, in particular il- , cause a strong increase in the ability of cultured bronchial epithelia to transport scnfrom the basolateral to the apical membrane. this effect is mediated by upregulation of ca + -dependent clchannels and of the slc a (pendrin) anion transporter. these findings suggest that under proinflammatory conditions the activity of the scn -/h o /lactoperoxidase system is potentiated. we evaluated the antimicrobial activity of airway surface by seeding bacteria on the apical membrane of human bronchial epithelia grown with an air liquid interface on a porous membrane. an inoculum corresponding to , cfu of s. aureus was added to the cells in µl of saline solution with and without lactoperoxidase ( . µg/ml). experiments were performed in the presence or absence of scn -( µm) in the basolateral solution. bacteria were recovered after four hours and plated on agar plates for colony counting. our preliminary results show that simple addition of lactoperoxidase to the apical surface decreases bacterial survival. in addition, bacterial killing is strongly enhanced by prestimulation of cells for hours with il- ( ng/ml). the effect of lactoperoxidase is dependent on h o as it is prevented by addition of catalase and is absent in cell-free experiments when h o is omitted from the reaction mixture. surprisingly, the presence of scnin the basolateral compartment did not increase bacterial killing and, in some cases, appeared to generate a protective effect. our results suggest that lactoperoxidase, in the presence of h o , is an effective antimicrobial molecule. the contribution of scnand the mechanism of the potentiation caused by cytokines is less clear and requires further investigation. a possibility is that, in the absence of scn -, lactoperoxidase generates an oxidant molecule that is more toxic to bacteria than oscn -. elucidation of this mechanism and comparison between cf and non-cf epithelia is in progress to assess the role of cftr and of other anion channels. supported by cfft and telethon-italy. cf patients become infected with pseudomonas aeruginosa, which release flagellin into the airway surface liquid to activate toll-like receptor and proinflammatory signaling. flagellin has been shown to inhibit na absorption by airway epithelia. we tested flagellin on cl secretion and proinflammatory signaling (nf-κb activation) by calu cells, a cftrexpressing, serous-like airway gland cell line. calu cells were grown on filters and either mounted in ussing chambers (clamped to zero mv) in the presence of a serosa-to-mucosa gradient of [cl] for measurements of transepithelial cl secretion (i cl ) or treated with an adenovirus expressing nf-κb-controlled luciferase to assay nf-κb activation. flagellin ( - g/ml) on either the apical or basolateral surface of cells increased apparent anion secretion that began in - mins and increased over - mins by - µa/cm . this i cl was blocked by glibenclamide and glyh , indicating that it resulted at least in part from activation of cftr. flagellin also stimulated fluid secretion by intact human tracheal glands. flagellin activated nf-κb (luciferase assays) in both calu- cells and in the cf cell line cf , while i cl was stimulated in calu but not in cf cells. flagellin-stimulated i cl in calu- cells was blocked % by sb (a p mapk blocker) and by a similar amount by wortmannin (pi kinase blocker). interleukin β and the tlr -agonist pam cys also activated i cl . the effect of flagellin was not due to increases in cytosolic [ca + ] (ca i ) because flagellin did not alter ca i . in contrast to the slow effects of flagellin, pam cys and il β, atp rapidly increased i cl (within secs, up to µa/cm ) followed by slower decrease to steady i cl = - µa/cm that had a similar time signature as the increases in ca i . forskolin (to increase cytosolic [cyclic amp]) increased i cl within mins to a steady value = - µa/cm . flagellin had small or no effects on i cl following maximal stimulation with either atp or forskolin. atp and forskolin on their own had no effect on nf-κb. flagellin increased phosphorylation of p mapk and of akt (down-stream kinase phosphorylated by pi k) with a time course similar to the increase in i cl . flagellin-activated nf-κb was reduced by roughly % by either wortmannin or sb . these results indicated that tlr agonists and inflammatory cytokines stimulate both nf-κb and proinflammatory processes and also cftr-dependent cl and fluid secretion by airway gland cells. these responses are mediated in part by activation of both p mapk and pi kinase. flagellin-tlr -activated increases in cftr-mediated cl secretion and reduction in enac-mediated na absorption will increase fluid secretion into the airways, which may facilitate bacterial removal by the mucociliary escalator and thereby reduce the proinflammatory stimulus. in cf it is expected that bacteria will activate tlr signaling to trigger innate immune responses, but cl and water secretion and the resulting "bacterial flush," will be missing, leading to sustained inflammation. ( , ) . treatment with hs improved several measures of lung function including mucociliary clearance. in one study amiloride was seen to have a negative impact on the benefit of hs ( ) . the long duration of action of hs and the paradoxical effect of amiloride are surprising and require further investigation. aims: our goal was to study the effect of exposure to a hypertonic challenge (hc) on amiloride sensitive na+ transport (ina) in primary cultures of human bronchial epithelial (hbe) cells from non-cf and cf patients. methods: hbe cells were grown at an air liquid interface and studied under short circuit current conditions. cells were challenged with hs by exchange of the apical or basolateral baths. in some experiments the timedependent effect of a small ( µl) isosmotic or hs volume at the airway surface was tested on ina and apical osmolality. results: application of hypertonic nacl and mannitol solutions from either the mucosal or serosal sides of the epithelium inhibited ina. the degree of inhibition was a saturable function of the imposed hypertonicity with an ic of (mannitol) and (nacl) mosmol/kg h o and a maximal inhibition of %. the inclusion of amiloride ( - µm) did not affect the hc-induced reduction in ina. the inhibition in ina by hc was rapid in onset and accompanied by a fall in the transepithelial resistance and the total transepithelial capacitance as expected for cell shrinkage. the inhibition in ina was only partially reversible (~ %) after returning to isotonic solutions for min. exposure of the airway surface to hypertonicity ( mosmol/kg h o) inhibited ina by - % at min of exposure. as osmotically-driven water moved from the serosal to the mucosal side, the apical volume increased concomitant with a decrease in its osmolality and a recovery of ina. the osmolality declined exponentially and reached the isosmotic value after ~ hrs. the recovery of ina lagged behind the recovery of apical osmolality by , and % at . , and hrs of incubation, respectively. after hrs of airway hc exposure, the osmolality and ina recovered completely. our results demonstrate that hc causes a rapid, dramatic and prolonged decrease in na+ absorption and that continuous presence of amiloride had no effect on the inhibition of ina or the recovery of apical osmolality. hc also causes the epithelium to shrink. no appreciable differences were observed between cf and non-cf hbe cells. conclusions: we propose cell shrinkage together with the continued influx of na+ increases intracellular na+ ([na]i) and lead to an inhibition of na+ transport. elevated [na] i is a known inhibitor of epithelial na+ channels ( ). the sustained nature of the inhibition in na+ transport to a hc may help explain the longer than expected duration of action of hs in the clinical trials ( , ) . explanation of the paradoxical effects of amiloride observed in a clinical trial ( ) requires further investigation. references: . n engl j med : - , ; . n engl j med : - , ; . physiol rev : - , reduced airway surface liquid (asl) volume resulting from enac mediated na+ hyperabsorption and cftr-mediated hyposecretion plays a critical role in cf lung disease pathogenesis. however, the mechanisms involved in enac/cftr regulation are poorly understood. using human bronchial epithelial cultures (hbecs) under thick film conditions, i.e. in the ussing chamber with the asl washed away, adenosine (ado) directly stimulates cl-secretion. however, in a cl-free environment, ado has no effect on the amiloride-sensitive current, which is mediated by enac. mean change in i sc following µm ado to the apical chamber was . µa/cm from baseline compared to vehicle alone (- . µa/cm ;ns;n= ) in hbecs. mean delta amiloride ( µm) was - . and - . µa/cm respectively (ns; n= each). in contrast, under thin film conditions we have previously shown that ado stimulates sustained asl secretion via activation of cftr in the presence of the protease-inhibitor aprotinin, whereas trypsin pre-treatment abolished this secretion, likely by activating enac and abolishing the electrical driving force for cl-secretion . further, ado-mediated asl secretion is significantly increased if hbecs (thin film conditions) are left with intact asl for h, an action that is abolished by acutely washing the apical surface . thus, we hypothesised that a soluble enac inhibitor was secreted into the asl under thin film conditions, which could accumulate sufficiently to inhibit enac and provide the necessary driving force for cftr-mediated cl-secretion after ado-addition. to search for such an inhibitor, we incubated asl with trypsin-coated beads and identified bound proteins by mass spectrometry (albumin-coated beads were used as a control to exclude non-specific binding). the major identified protein was plunc, a protein that is secreted into the asl both in vivo and in vitro which has no current known function. secretion of plunc into hbec asl was confirmed by western blot. to test whether plunc could alter asl volume homeostasis, we made an anti-plunc shrna retroviral construct which we used to infect hbecs. unlike the control anti-luciferase shrna infected hbecs, anti-plunc shrna-infected hbecs exhibited > % knockdown of plunc and a failure to regulate asl volume (ctrl, . ± . µm; ± . µm; n= ) . when co-expressed with enac in oocytes, plunc inhibited enac currents by % (n= ). cftr function in contrast was unaffected by plunc expression (n= ). thus,it is likely that ado stimulates cftr mediated cl-secretion but is not involved in enac regulation. however, experiments were performed in a very simplified environment i.e. static cultures with little mucosal atp or mucus and these findings will need to be expanded and further evaluated in native tissues. we conclude that plunc, rather than ado may be the soluble mediator in the asl which regulates enac function by reducing its activity. . tarran, r. et al., j. gen. physiol., . ( ) airway surface liquid (asl) absorption is mediated by epithelial na + channels (enac), which establish an osmotic gradient that drives fluid absorption. we and others have recently reported that a protease / anti-protease balance regulates enac in normal human bronchial epithelial cells (hbe) and provides a mechanism for auto-regulation of asl volume. in cf, this balance is disturbed, leading to constitutive proteolytic activation of enac and the pathological na + hyper-absorption characteristic of the disease. to determine if channel activating protease expression is regulated by changes in asl volume and is altered in cf, we examined prostasin expression in control and cf hbe under basal and asl volume expansion conditions using western blotting. prostasin migrates as kda and kda bands in apically biotinylated proteins, apical secretions, and whole cell lysates of primary hbe. following apical aprotinin exposure, only the kda prostasin molecular species was present in the biotinylated proteins, suggesting that the proteolytic conversion of prostasin zymogen to active enzyme occurs on the cell surface. following asl volume expansion, cell surface prostasin expression increased by > % (p= . , n= tissue donors, > cultures from each). as our recent studies indicated that increased proteolytic activation of enac occurs in cf airway epithelia, we next compared prostasin expression in cf and control hbe cells. prostasin expression in the apical biotinylate of cf hbe was . fold greater than in control hbe (p= . , n= tissue donors). furthermore, the ratio of the kda (active enzyme) to kda (zymogen) prostasin molecular species was . fold greater in cf (p= . ), indicating that increased activation of prostasin occurs in cf airway epithelium. we next determined whether increased prostasin activation in cf may reflect the deficiency of a protease inhibitor. serpin e , also known as protease nexin- (pn- ), forms an ~ kda complex with prostasin and permanently inactivates its protease activity. while no significant difference in pn- expression was observed between control and cf hbe, pn- was found to contribute to prostasin regulation by (i) forming an inactive prostasin complex, (ii) inhibiting the amiloride-sensitive short-circuit current across hbe, and (iii) preventing conversion of prostasin zymogen to active enzyme. these findings demonstrate that cellular mechanisms coordinate prostasin expression and activity with asl volume. accordingly, at times when the asl volume is high, prostasin expression increases, and this presumably augments na + and asl absorption to absorb the excess luminal fluid. these regulatory mechanisms govern the apical membrane expression and processing of zymogen to active enzyme. because prostasin is incapable of autocatalysis, these findings support the existence of a proteolytic cascade that controls enac activity during asl volume homeostasis, perhaps reflecting the recently reported matriptase-prostasin cascade. furthermore, the increased expression of prostasin in cf suggests that abnormal regulation of prostasin contributes to na + hyper-absorption in cf airways. supported by the nih, cff, and ala. we report that annexin (anx )-s a forms a functional camp/pka/calcineurin (can)-dependent complex with cftr. cell stimulation with forskolin/ibmx significantly increases the amount of anx -s a that reciprocally co-immunoprecipitates with cell surface cftr and can a. pre-inhibition of the cells with pka or can inhibitors attenuates the interaction. furthermore, we find that the acetylated peptide (stvheilcklsleg, ac - ), but not the non-acetylated equivalent n - , corresponding to the s a binding site on anx , disrupts the anx -s a /cftr complex. analysis of dids and cftr inh -sensitive currents, taken as indication of the outwardly rectifying cl-channels (orcc) and cftr-mediated currents, respectively, showed that ac - , but not n - , inhibits both the camp/pka-dependent orcc and cftr activities. can inhibitors (cypermethrin, cyclosporin a) discriminated between orcc/cftr by inhibiting the cftr inh , but not the dids, sensitive currents, by more than %. furthermore, peptide ac - inhibited acetylcholine-induced short-circuit current measured across a sheet of intact intestinal biopsy. our data suggests that the anx -s a /cftr complex is important for cftr function across epithelia. the content and water (h o) mobility in airway epithelium is tighly coupled to airway function. in cystic fibrosis (cf), h o epithelial permeability is reduced in airways. the demand of noninvasively imaging techniques with high spatial resolution potential is rising because such imaging tools would expedite anatomical and functional phenotyping in the genetically altered mice. magnetic resonance microscopy (mrm) is a noninvasive, inherently three-dimensional ( d) imaging technique capable of visualizing anatomical structures in the mouse and allows for interpretation of complex spatial relationships between substructures and h o. in this study, we explore different mr contrast parameters and signal-to-noise ratios at a µm pixel size to characterize microstructure and h o mobility in ex vivo trachea of cf transmembrane conductance regulator (cftr)-deficient (cftr knockout, cftr tm unc ) mice and their aged-matched wt littermates. this study is performed using a bruker mrm system at . tesla. we demonstrate for the first time the ability of d-mrm to map the h o content and mobility in trachea epithelium. from the d-mrm videoimages, differential h o content was visualized in different levels of trachea in wt and cf mice. t mrm images depicting the h o rotational mobility which is related to environmental viscosity of trachea epithelium will be also shown. finally, this d-mrm imaging method is a valuable method for measuring h o content and permeability in airways and can serve for assessing the effects of drugs on h o mobility in cf airways. supported by grants from inserm, upmc-paris , cnrs, and the french cystic fibrosis association (vlm). cystic fibrosis (cf) is a lethal inherited disorder caused by mutations in a single gene encoding the cystic fibrosis transmembrane conductance regulator (cftr) protein resulting in progressive lung oxidative damage. in this study, we evaluated the role of cftr in the control of ubiquitin-proteasome activity (ups system) and the nf-κb / iκb-αsignaling after lung oxidative stress. we exposed cftr deficient (cftr-/-) and wild type mice for h to hyperoxia-mediated oxidative stress. cftr deficient mice exhibited significantly higher lung proteasomal activity than cftr+/+ animals after oxidative stress. this was accompagnied by a strong reduction of lung caspase- activity and an absence of degradation of nf-κb inhibitor iκb-α. in vitro, human cftr-deficient lung cells also exhibited higher proteasomal activity and a lack of increased nf-κbdependent transcriptional activity compared to cftr-sufficient lung cells after oxidative stress. furthermore, inhibition of the proteasomal activity by mg in cftr-deficient lung cells restored the nf-κb/iκb-α signaling to that of cftr-sufficient lung cells. inhibition of caspase- by z-dqmd in cftr-sufficient lung cells mimicked the response profile of increased proteasomal degradation and lowered nf-κb transcriptional activity of cftr-deficient lung cells when exposed to oxidative stress. all together, these results suggest that cftr is a crucial molecule in regulating proteasomal degradation and nf-κb activity in lung epithelium under oxidative stress. staphylococcus aureus, one of the major pathogen involved in airway infections, releases in the airway lumen virulence factors that may impair the airway epithelial functionality. to date, the effect of s. aureus virulence factors on the loss of electrolyte homeostasis of the airway epithelium has not been investigated. we have previously shown that the combination of a corticosteroid and a long-acting beta agonist attenuated the airway epithelial cell inflammatory response induced by s. aureus virulence factors. the aim of the present work was to investigate the effect of s. aureus virulence factors on the airway epithelial tightness and on the chloride efflux of airway epithelial cells incubated or not with salmeterol hydroxynaphthoate × - m and fluticasone propionate × - m (sm/fp). the airway epithelial tightness was assessed by immunocytochemistry, western blotting and transepithelial resistance measurement. the chloride efflux was evaluated by dynamic imaging using the -methoxy-n-( -sulfopropyl) quinolinium (spq) fluorescent probe. s. aureus (strain - ) virulence factors were obtained by growing bacteria at × cfu/ml for h at °c. the bacteria supernatant containing s. aureus soluble virulence factors was then centrifuged, filtered and diluted at % in cell culture medium (a concentration that did not alter cell viability). human airway epithelial cells (mm ) were incubated with % s. aureus virulence factors for h and then co-incubated or not with sm/fp for h. we observed that the incubation with % s. aureus virulence factors alone did not significantly alter the epithelial integrity assessed by the expression of tight junction proteins and transepithelial resistance measurement. however, when the cells were incubated with sm/fp alone or with % s. aureus virulence factors plus sm/fp, the expression of tight junction proteins was significantly increased (p< . ) as compared to cells incubated with s. aureus virulence factors. interestingly, the chloride efflux in airway epithelial cells was significantly decreased in a time-dependent way by s. aureus virulence factors ( fold decrease after a h incubation). the co-incubation of airway epithelial cells with s. aureus virulence factors and sm/fp prevented the s. aureus virulence factorsdependent chloride efflux decrease. our results demonstrate that bacterial virulence factors induce the loss of the electrolyte homeostasis and suggest that the treatment by the combination of a corticosteroid and a long-acting beta agonist may preserve the airway epithelial functionality. supported by association vaincre la mucoviscidose and glaxo-smithkline nilsson, h.e. ; dragomir, a. ; ahlander, a. ; johannesson, m. ; roomans, g.m. . medical cell biology, uppsala university, uppsala, sweden; . women's and children's health, uppsala university, uppsala, sweden inhalation of hyperosmotic solutions (salt, mannitol) has been used in the treatment of patients with cystic fibrosis or asthma, but the mechanism behind the effect of hyperosmotic solutions on mucociliary clearance (mcc) is unclear. one explanation has been suggested, namely that hypertonic solutions open tight junctions, which may lead to increased water transport followed by an increased airway surface liquid (asl) volume. furthermore, the role in cftr-mediated hco -conductance in regulating asl ph has led us to investigate if ph changes may have an effect on the tightness of tight junctions. the effect of osmolarity was investigated on the hbe o-cell line by the addition of nacl, nabr, licl, mannitol or xylitol ( - mosm). the effect of ph was investigated on the hbe o-, calu- and t cell lines as well as the cystic fibrosis cell line cfbe o-. transepithelial resistance was measured as indicator of the tightness of the cultures. cell-cell contacts and morphology were investigated by immuno-fluorescence and by transmission electron microscopy, with lanthanum nitrate added to the luminal side of the epithelium to investigate tight junction permeability. the electrolyte solutions caused a significant decrease in transepithelial resistance from mosm on, when the hyperosmolar exposure was gradually increased from to mosm, whereas the non-electrolyte solutions caused a decrease in transepithelial resistance from mosm on. immunofluorescence micrographs showed weaker staining for the proteins zo- , claudin- in treated samples compared to the control. the ultrastructure revealed an increased number of open tight junctions as well as a disturbed morphology with increasing osmolarity, and with electrolyte solutions opening a larger proportion of tight junctions than non-electrolyte solutions. it was noted that during the exposure of the cultured cells to the hyperosmolar solutions, the ph of the medium increased from . to . . hbe o-cells exposed to both a rise in ph and hyperosmotic stress showed an overall lower teer and a significantly reduced ability to recover from stress compared to cultures at a ph hold constant at . . without exposure to hyperosmolar solutions, a rise in ph caused a significant decrease in transepithelial resistance in hbe o-cells, calu- and t cell lines but not in the cfbe o-cell lines, where the reaction was significantly less and delayed. in conclusion, hyperosmolar solutions caused a reversible opening of the tj in hbe o-cell cultures with electrolytes having stronger effects than non-electrolytes, where one of the effects on mcc may be due to increased water transport across the leaky paracellular space. an increase in ph caused a significant decrease in teer in the healthy cell lines compared to the cfbe o-cell line. we speculated that an impaired alkalinisation of the apical fluid due to a defective cftr also will cause the tight junctions to react different to other external stimuli, such as osmolarity, compared to healthy cells, which is also indicated by preliminary experiments on the effect of nacl on teer in cfbe o-cultures. extracellular nucleotides regulate surfactant secretion in alveoli and mucociliary clearance in airway epithelia, but the mechanism(s) of their release and their regulatory pathways remain incompletely understood. previously, we showed that hypotonic swelling of a epithelial cells induces ca + -dependent secretion of several adenosine and uridine nucleotides, implicating regulated exocytosis. in this study, we examined sources of intracellular ca + ([ca + ] i ) elevation evoked by acute % hypotonic stress and the role of autocrine purinergic signaling in ca + -dependent atp release. we found that atp release does not directly involve ca + influx from extracellular spaces, but depends entirely on ca + mobilization from intracellular stores. the [ca + ] i response consisted of slowly-rising elevation representing mobilization from thapsigargin (tg)-insensitive stores and a superimposed rapid spike due to ca + release from tg-sensitive endoplasmic reticulum (er) stores. the latter could be abolished by hydrolysis of extracellular tri-and di-nucleotides with apyrase; blocking p y /p y receptors of a cells with suramin; blocking udp receptors (p y ) by ppads; emptying tg-sensitive stores downstream with µm tg or mm caffeine in ca + -free extracellular solution; or blocking the ca + -release inositol , , -triphosphate (ip ) receptor channel of the er with µm aminoethyl diphenylborinate. these results demonstrate that the rapid [ca + ] i spike results from the autocrine stimulation of ip )/ca + -coupled p y /p y receptors, which accounts for ~ % of total ca + -dependent atp release evoked by hypotonic shock. our study reveals a novel paradigm in which atp release is amplified by the synergistic autocrine/paracrine action of co-released uridine and adenosine nucleotides. we suggest that a similar mechanism of purinergic signal propagation operates in other cell types. (this study was supported in part by the canadian institutes of health research and the canadian cystic fibrosis foundation (ccff). s.t. was the recipient of a ccff studentship). the maintenance of a thin liquid layer on the airways and alveoli surfaces is essential for normal lung physiology.yet, the mechanism sensing the height of the layer remains obscure. in this study, we examined atp secretion from human lung a epithelial cell monolayers mounted in a closed, flowthrough chamber ( . mm height) and found that passage of an air bubble over the monolayer caused transient (< . min) but significant atp release ( to , pmoles/ cells). air bubble-induced atp release was reduced by~ % from cells loaded with the intracellular ca + chelator bapta-am, and was completely abolished in n-methylmalemide-treated ( mm) cells, suggesting the involvement of ca + -dependent exocytosis. fura- intracellular ca + ([ca + ] i ) imaging experiments revealed transient [ca + ] i elevation during the passage of an air bubble over the cell monolayer, but the [ca + ] i response did not involve non-specific ca + influx from the extracellular space, e.g. due to cell damage, since similar responses were observed in ca + -free extracellular solution. ethidium bromide staining did not disclose any cell damage in these experiments. confocal fluorescence microscopy study showed reversible cell deformation (flattening) of % to % in height in the monolayer part in contact with the air bubble and confirmed that cell integrity was entirely preserved. these experiments demonstrate that in close proximity to the air-liquid interface (i.e. between the air bubble and the wet cell surface), surface tension forces are transmitted directly on cells, causing their mechanical deformation, elevation of [ca + ] i and subsequent atp release. we propose that a similar mechanism may operate in vivo in the airways, where surface tension forces acting directly on exposed epithelial cell surfaces may provide a fail-proof mechanism to protect proper airway surface liquid volume via mechanosensitive, ca + -dependent atp release and purinergic modulation of fluid secretion. this mechanism may be defective in cystic fibrosis due to excessive mucus layer covering the airways. introduction : cystic fibrosis is a genetic disease that reflects the consequences of mutations in the cystic fibrosis transmembrane conductance reg-ulator (cftr) gene, affecting anionic transport in epithelia. slc family members are anionic transporters involved in cl-and hco -absorption or secretion in epithelia. in addition, the activation of some slc family members by cftr has been demonstrated (nature cell biology, , : - ) . slc a is a poorly characterized member of this family, being the solely expressed in lung. putative interaction domains with cftr are also present in the slc a protein. in this study, we have investigated the transport properties of slc a (human) to determine the functional and pharmacological characteristics of this transporter. methods : to this aim, electrophysiological studies (two-electrode voltage clamp or current clamp methods and intracellular ph measurements using ph sensitive microelectrode) were performed in xenopus laevis oocytes expressing slc a proteins. complementary ( cl) transport studies were also undertaken. results : the protein expression results in the appearance of an anionic current showing a linear current/voltage relationship. cl influxes experiments confirmed the induced cl-permeability following slc a protein expression. the sequences of conductivity, cl->i-> no -≥ gluconate > so -and selectivity (px/pcl), i-> cl-= no -> gluconate > so -are found. the hco -conductance mediated by the slc a protein expression is low. using co /hco -containing ringer solutions, no intracellular phi changes were detectable in conditions (low chloride in external medium) favoring the cl-/hco -exchange whereas phi changes (alkalinization) were observed with the expression of the ae exchanger. however, phi changes could be detected in conditions largely favoring the driving force for hco -entry. dids and ns inhibited slc a associated currents. the specific cftr inhibitor (cftrinh ) or glybenclamide had little effect. elevation of intracellular camp (a cftr activator) was also ineffective while maneuvers increasing intracellular calcium blocked the slc a associated currents. conclusions : our study demonstrates that slc a presents an electrogenic anion conductance (characteristics of an anionic channel) when expressed in xenopus oocytes (however a transporter functioning as a ncl-/hco -exchanger can not be excluded). this function (channel) has also been described for another member of this family, slc a (j biol. chem, , : - ) . the physiological role of slc a , present in the bronchial cells of airway epithelia and its potential interaction with cftr has to be precised in situ or in oocytes and in mammal expression systems. medicine, women's & children's hospital, nth adelaide, sa, australia; . cf research & treatment center, university of nth carolina at chapel hill, chapel hill, nc, usa; [ ] [ ] [ ] [ ] [ ] [ ] jasri, hyogo, japan; . physics and synchrotron science, monash university, clayton, vic, australia; . paediatrics, university of adelaide, adelaide, sa, australia non-invasive imaging of lung (e.g. hrct, mri, pet) is a valuable modality for detection and monitoring of the effects of cf in human airways, but resolution is limited; e.g. airway hrct detects airways no smaller than ~ . mm dia. detection of wall or lumen changes in smaller airways, or the detection and monitoring of induced airway disease in live rodent models, demands significantly greater resolution coupled with rapid image capture. we report substantially increased airway resolution is achievable using synchrotron phase-contrast x-ray and can produce three-dimensional reconstructions encompassing the smallest mouse lung airways. nembutal-anaesthetised c bl/ mice (~ gm) were imaged at the spring- synchrotron in hyogo, japan. -d phase-contrast airway images were obtained on ccd detectors ( . µm pixels) with a cm phase-contrast propagation distance at kev; nose and trachea were imaged at or sec intervals ( - ms exposures) over mins. mice killed with nembutal overdose were imaged in axially aligned segments to obtain dimensional ct data voume of approximately x x mm with x x um voxels of nose, trachea, and lung (hammamatsu ccd detector). volume renderings were produced using volview or osirix software. live, -d nasal or tracheal imaging revealed airway-surface activity in some live mice consistent with dynamic mucociliary clearance activity. individual ct slices revealed the fine detail in the mouse lung, with dynamic ("fly through") sequences of lung ct slices permitting visual identification and tracking of lung tree branching from trachea (~ mm dia) into small airways (approx um dia). adjustment of x-ray contrast, opacity, and range in these volume reconstructions permitted selective display of the mouse lung conducting-airway tree and airway surfaces. selected regions of the lung could be examined statically, or in rotation through different orientations in high-resolution -d. synchrotron phase contrast x-ray provides a new option for non-invasive imaging of intact mouse lung, at a resolution that permits examination of small conducting airways in mice. combined with volume-reconstruction software these ultract images can provide a powerful option for understanding the structure-function relationships produced by airway disease. ultra-ct -d studies are underway to compare lung airways at high resoltuion in normal and transgenic mice having altered airway pathophysiology. the epithelial sodium channel, enac, has a vital role in the function of the pulmonary epithelia and significantly contributes to the pathophysiology of the cf airway. thus, strategies to repair mutant cftr dysfunction must also consider the influence of such repair on enac functional expression. the ∆f trafficking repair agent -phenylbutyrate modulates the expression of the kda molecular chaperones hsc and hsp in cf epithelial cells. we therefore assessed the role of these chaperones in the regulation of enac trafficking. in xenopus oocytes, we previously observed that overexpression of hsc inhibits murine enac (menac) functional and surface expression. in contrast, hsp can either enhance or inhibit menac functional and surface expression depending on the extent of overexpression [goldfarb et al. ( ), proc. natl. acad. sci. : - ]. here, we tested the hypothesis that these differential effects of hsc and hsp on menac expression also occur in epithelial cells. mdck cell lines with stable expression of α-ha, β-v , γ-myc-menac and tetracyclineinducible expression of either hsc , atpase-deficient hsc , hsp or atpase-deficient hsp containing myc/his epitope-tags were selected. all epitopes were fused to the respective c-termini. these cells were grown as high-resistance (> Ω/cm ) monolayers on permeable supports. doxycycline-induced overexpression of hsc decreased amiloride-sensitive i sc and the whole cell content of α-haand β-v -menac. in contrast, lower amounts of tetracycline-induced hsp overexpression increased amiloridesensitive i sc and whole cell α-haand β-v -menac expression; these effects were absent at higher levels of hsp overexpression. these data are consistent with our previous data in xenopus oocytes. interestingly, the atpase-deficient chaperones had the opposite effect on menac functional expression. modest doxycycline-induced expression of atpase-deficient hsc increased amiloride-sensitive i sc in these cells, while modest overexpression of atpase-deficient hsp decreased amiloride-sensitive i sc . these effects may result from "dominant negative" interference with hsc and hsp function, respectively. these data are consistent with hsc and hsp having different effects on menac functional expression in epithelial cells, and that these effects are dependent upon the atpase activity of the respective chaperones. supported by grants from niddk. we have studied survival, airway epithelia bioelectrics and lung pathology of mice over-expressing various combinations of the epithelial na + channel (enac) subunits (α, β and γ, genes scnn a, scnn b and scnn g). we generated double-transgenic mice and crossed them with single-transgenic mice to obtain litters comprising all possible genotypes. survival analysis revealed that overexpression of βenac in combination with either αenac or γenac significantly reduced survival in comparison to wild-type (wt) littermates. strikingly, at days of age, all genotypes were represented according to the expected mendelian proportion, except for the triple transgenic αβγenac, which was significantly under represented. in utero studies are ongoing to understand if over-expression of αβγ affects the fetuses or the newborn pups during the first hours post-partum. due to the high mortality of mice over-expressing βenac subunit combinations, we studied tracheas from pups at days of age. as previously reported, mice overexpressing β, but not the α or γ enac subunits, exhibit airways hyperabsorption of na + and lung pathology (mall et all ) . the βenac tracheas exhibited na + absorption [as measured by the change in short circuit current (isc) in response to amiloride in the ussing chamber] that was ~ . fold greater (∆isc . ± . µa×cm - , n= , means±sem) than wt ( . ± . µa×cm - , n= ). over-expressing αβ or αγ enac subunits resulted in rates of na + absorption that did not differ significantly from those of βenac or γenac subunit alone, respectively. however over-expressing the β and γ enac subunits together resulted in an amiloride sensitive isc that was ~ . fold greater (∆isc . ± . µa×cm - , n= ) than wt. we also studied the tracheal bioelectrics of the few day-old αβγenac pups available. tracheas from the αbγ pups exhibited na + absorption that was ~ . fold greater (∆isc ± . µa×cm - , n= ) than wt. due to the small sample size, we cannot determine if this response is significantly different from the response of the βγenac pups. analysis of lung pathology in day-old pups revealed that all the combinations that exhibited increased airway na + absorption and decreased survival in comparison to wt littermates, e.g. αβ, βγ and αβγ, also presented with alveolar space enlargement, maybe due to postobstructive air trapping, and airway epithelia necrotic degeneration associated with ab-pas negative bronchial obstruction. from these data, it appears that the rate of airway na + absorption negatively correlates with the survival of the pups. thus, it is likely that the mucus and airway surface liquid is more dehydrated as a function of the rates of na + absorption (aβγ > βγ > αβ, β > wt) which produces gradual failure to survive due to asphyxia secondary to airway obstruction. supported by nih scor p hl choi, j. , ; joo, n. ; wu, j. ; krouse, m. ; wine, j.j. . cystic fibrosis research laboratory, , stanford, ca, usa; . otorhinolaryngology, yonsei university, seoul, south korea submucosal glands, which produce most airway mucus, are mainly controlled by parasympathetic pathways that stimulate glands via airway intrinsic neurons distributed along the airway walls. the intrinsic neurons can be stimulated via axon reflexes from receptors in the mucosa. in previous experiments, it was shown that capsaicin applied to the mucosa stimulated gland secretion in the upper tracheas of wt but not cftr -/-mice (ianowski et al., j physiol, , , ) . it seemed important to assess a possible role of capsaicin-sensitive pathways in pigs and humans. we obtained pig tracheas following acute experiments carried out for other reasons, and human airways following lung transplants from donor tracheas and from the excised lungs of the transplant recipients. secretion from individual submucosal glands was quantified optically by time-lapse digital imaging of the growth of spherical bubbles of mucus in an oil layer in humans, commercial chili oil ( µm dispersed in µm mineral oil) stimulated mucus secretion from submucosal glands from both donors (n = glands from subjects, . ± . nl/min/gland) and disease control subjects (n = glands from subjects, . ± . nl/min/gland). however, there were no responses to chili oil in submucosal glands from cf subjects (n = glands from subjects, . ± . nl/min/gland). in pigs, we determined concentrationresponse relations for gland secretion in response to purified capsaicin (sigma). the threshold was ~ µm and the ec was . ± . µm. the response to µm capsaicin was partially blocked by high dose ttx (> µm). as in humans, the capsaicin response required cftr, because it was blocked ~ % by cftrinh- , (n = glands, from pigs, p < . ). cftrinh- is presently the most specific inhibitor of cftr that can be used with glands, where we do not have access to the apical membrane. in ferrets, intrinsic airway neurons express acetylcholine, vip and sp, often colocalized within the same neurons (dey et al., am j respir cell mol biol, , , ) . in pigs, all three of these transmitter systems appear to be involved in gland secretion to capsaicin, because the response was partially blocked by the nk- receptor blocker l (oxalate salt, µm), the vip receptor inhibitor l k ( µm), and the muscarinic receptor blocker atropine ( µm); each of these blocked to % of the response (n = to glands from to pigs). indeed, most of the local pathways are probably damaged, which may account for the relatively small responses to strong stimulation. relatively few experiments have been carried out in intact airways with uninterrupted innervation and circulation, but in those experiments the glands were highly responsive to modest mucosal stimuli (reviewed in wine, auto neurosci, , , ) . hence, we hypothesize that ( ) reflex stimulation of glands plays an important role in lung innate defenses; ( ) defects in these responses is an important reason that cf airways are prone to infection, and ( ) local reflexes may assume greater importance for maintaining the mucosal defenses of transplanted lungs. supported by niddk ro - (jjw), cff and cfri. most airway mucus arises from submucosal glands, which express cftr in their ciliated ducts and in serous cells. the airway glands secrete to agonists that either increase intracellular ca + (e.g. acetylcholine) or camp (e.g. vip).substance p(sp) also stimulates airway gland liquid secretion in pig (trout l et al., am j physiol lung cell mol physiol. , ,:l ) , suggesting that sensory afferents or local neurons that express sp play a role in stimulating submucosal glands. we used pharmacological methods to dissect the secretory mechanism of sp-induced gland secretion in comparison with responses to carbachol. tracheas were obtained from pigs after acute surgeries carried out for other reasons. the ventral mucosa with underlying glands was dissected from the cartilage, pinned mucosal side up at the gas/bath interface of a physiological chamber, and covered with oil. secretions from individual glands could be visualized as spherical bubbles in the oil, and secretion rates determined by optical monitoring of bubble diameters. concentration-response relations for gland secretion were determined for sp ( - bubbles at each concentration from pigs). the threshold was ~ nm for sp and the ec was . ± . µm. the maximum secretion rate to sp was . ± . nl/min.gland, which is only ~ / of the maximal secretion rate to carbachol ( . ± . nl/min.gland). the inhibition profile for responses to sp ( µm) was quite different from those to carbachol ( nm, a value chose to mimic the secretion rate to µm sp). gland secretory responses to µm sp ( . ± . nl/min.gland, glands from pigs) were strongly inhibited by µm cftrinh- ( . ± . nl/min.gland, glands from pigs, p< . ), and by µm clotrimazole ( . ± . nl/min.gland, glands from pigs), whereas µm niflumic acid did not inhibit ( . ± . nl/min.gland, glands from pigs). in contrast, secretory response to nm carbachol ( . ± . nl/min.gland, glands from pigs) were only weakly inhibited by cftrinh- ( . ± . nl/min.gland, glands from pigs, p< . ), and by clotrimazole ( . ± . nl/min.gland, glands from pigs, p> . ), but niflumic acid, which was ineffective with sp, inhibited the carbachol response ( . ± . nl/min.gland, glands from pigs, p< . ). gland secretion to sp depends at least partially on intracellular ca + release, because the response was partially inhibited by µm bapta-am ( . ± . nl/min.gland, glands from pigs, p< . ). there was no additional effect of sp on top of µm carbachol, but a subthreshold level of sp ( nm) showed synergic response ( . ± . nl/min.gland, glands from pigs) with subthreshold level of vip ( nm). we are presently testing how sp activates cftr-dependent mucus secretion. in initial experiments to determine if pkc was being recruited, however the pkc inhibitor (gf , sigma) did not suppress sp-induced mucus secretion. in contrast, the nkcc inhibitor bumetanide ( µm) markedly suppressed secretion to sp ( . ± . nl/min.gland, glands from pigs, p< . ). supported by niddk ro - (jjw), cff and cfri. mucus obstruction of airways is considered the most vicious agent of morbidity and mortality in cystic fibrosis (cf). to provide optimal defense of the small airways the volume/thickness of airway surface fluid must be controlled according to physiological demand. to date we have no clear understandings of how these fluids are maintained in a steady state between two pathological extremes (too little or too much) in native small airways. earlier studies have attempted to measure the ion transport properties of small airways of sheep (al-bazzaz et. al. ) and pigs (ballard et. al. ), but the complicated branching structure of small airways may have compromised electrical signals. to avoid dissection trauma wang et. al. ( ) examined the electrophysiological properties of undissected intact small airways from pigs in vitro by microperfusing bronchioles (diam. - mm) embedded in the lung parenchyma; however, these studies were limited to luminal manipulations and transbronchiolar electrical potentials only. to more rigorously define small airway properties, we designed a special micro ussing chamber ( area ≈ mm ). we excised and opened small airways (diam. - mm) of pig lung that we mounted as a flat sheet over a pvc supporting filter ( - µm holes). both sides of the tissue could then be bathed with different solutions at °c. the luminal side was isolated by pressing a pipette (≈ mm diam., tip fire polished) on to the apical surface of the tissue until it sealed electrically. constant current pulses ( . - . µa) were passed across the tissue. transepithelial potential (tep) and resistance in bilateral mm nacl ringers were, - . ± . mv and . ± . Ω.cm (mean ± se) respectively. when mm naglu ringers replaced the luminal solution, tep increased significantly to - . ± . mv (n= , p< . ) and resistance increased to . ± . Ω.cm (n= , p< . . adding forkskolin (fsk, µm) plus ibmx ( . mm) hyperpolarized the tep to - . ± . mv (n= , p< . ), but decreased the resistance to . ± . Ω.cm (n= , p< . ). luminal amiloride ( µm) depolarized the tep to - . ± . mv (n= , p< . ) but significantly increased resistance to . ± . Ω.cm (n= , p< . ). these results show that this system supports measurements of ion transport properties of airways smaller than mm diameter where cf lung pathology is thought to originate. these airway epithelia express a very high constitutively active clconductance and are apparently capable of secretory as well as absorptive functions. amiloride sensitive na + conductance (≈ ms/cm ) was not significantly affected by fsk added to activate secretion. the observation that stimulation with fsk did not significantly suppress na + conductance (≈ ms/cm ) (p= . ) may suggest that separate groups of reabsorptive and secretory cells comprise small airway epithelia. mucins are class of proteins uniquely characterized by large glycosylated domains. these proteins likely play a key role in cystic fibrosis (cf) lung disease as major constituents of mucus. in airways of both humans and mice, the gel-forming secreted mucins are represented by muc ac and muc b, and the transmembrane mucins are represented primarily by muc , muc , and muc . we have been interested in the role of the transmembrane mucins in airways and have previously demonstrated that lack of muc in a knock-out mouse model (provided by s. gendler) shows increased susceptibility to adenoviral-mediated gene transfer (stonebraker, j. virology, ) . to expand upon these findings, mice deficient for muc were developed using embryonic stem cell technology by deleting exon of the mouse muc gene, which contains the atg start codon and signal peptide. rna analysis reveals loss of exon in homozygous mutant mice and immunohistochemistry using a vntr antibody indicated a loss of full length muc protein. muc homozygous mice are viable and do not develop any spontaneous, readily detectable disease phenotype. although the muc deficient mice do have reduced fertility consisting of both reduced litter size and number, all major tissue systems where the muc protein is easily detected, including the eye, respiratory tract, intestine, and reproductive tracts, appear normal in year old mice after histological analyses. in contrast to the results we reported in the muc mice, the muc deficient mice do not show an increase in adenoviral mediated gene transfer, suggesting that muc is not involved in the barrier aspect of the airway glycocalxy, at least for adenoviral infection. interestingly, quantitative real-time pcr analysis revealed up-regulation of muc protein in the trachea of the muc deficient mice, suggesting that muc may be able to compensate for loss of muc . the expression of muc in human ciliated cells as revealed by recent antibody studies prompted us to evaluate the potential role of muc in ciliary function. ciliary beat frequency (cbf) was monitored in a shear chamber using microscopy image analysis. cbf under baseline conditions for this study (low shear stress) from muc deficient trachea (c bl/ j congenic n ) was significantly lower than that for littermate controls ( . vs. . , sem ± . , p = . ). application of shear resulted in increased cbf in both groups, although the wt response to shear was smaller in magnitude. thus, muc may have a role in supporting basal cbf and may make cbf less vulnerable to stimulation by shear, perhaps by reducing the inherent friction of ciliary motion. this finding underscores the usefulness of this model for dissecting out the normal roles that the transmembrane mucins play in airway biology. supported by nih (hl ) and cff (r -cr ). enac (joo et al. jbc , and jbc , ) . in a search for a model to test the hypothesis we found that ussing chamber short-circuit current(i sc ) measurements with freshly obtained airway mucosa provided useful information about enac activity in response to various drug treatments. in these studies we observed an as yet unidentified pathway that regulates surface enac activity. airway mucosa from sheep tracheas displays a large, amiloride/benzamil-sensitive i sc prior to stimulation. when stimulated with basolateral carbachol, the enac i sc was almost abolished, as indicated by a sustained decrease ( . ± . %, n= ) in i sc after an initial increase and left a small effect to subsequent amiloride or benzamil. in contrast, when carbachol was given after benzamil, it again produced a peak response followed by a sustained increase (~ %, n= ) in the i sc . enac inhibition by carbachol was dose dependent with a minimal sensitivity between . ~ µm and was slowly reversible. apical µm atp also inhibited enac i sc by . ± . % (n= ) and carbachol on top of atp further reduced the enac i sc by . ± . % (n= ). however, u/ml of apyrase pretreatment, in attempt to breakdown atp, failed to inhibit carbachol-induced enac inhibition, indicating that atp release is not on obligatory component of carbachol inhibition of enac. carbachol inhibition of enac could result from a direct action of carbachol on the surface epithelia, by activation of airway intrinsic neurons, or by activation of the glands, which were intact in these preparations and are strongly stimulated by carbachol. rabbit tracheas, which lack airway submucosal glands, displayed large enac currents and these were not inhibited by cholinergic stimulation (n= ). carbachol-induced enac inhibition, like gland stimulation but unlike stimulation of neurons, was mediated by muscarinic receptors since it was eliminated by atropine but not by the nicotinic receptor antagonist, hexomethonium bromide (n= ). however, inhibition of gland secretion with hepes/bumetanide did not eliminate the ability of carbachol to inhibit enac. the effects of carbachol on surface na + transport in our study are consistent with previous observations in microperfused sheep bronchiole preparations (al-bazzaz, ajp-lung , ). although we do not yet know the mechanism, it would be physiologically efficient if parasympathetic, cholinergic activation of airway gland secretion was accompanied by enac inhibition, which would minimize the fluid absorption by surface epithelia and increase clearance. it will be important to determine if cholinergic inhibition of enac is present in human tissues, and if so, if it is defective in cf. supported by cff and nih. introduction: lung disease accounts for more than % of the current morbidity and mortality associated with cf. the majority of research aiming to elucidate the reason(s) of severe lung disease has focused on airway surface liquid (asl) layer homeostasis or composition. the cftr gene encodes a camp-regulated clchannel located on the apical side of the epithelium and is instrumental in maintaining a hydrated asl layer and promoting effective mucociliary clearance (mcc). in patients with class i or class ii cftr genetic mutations, normal cftr clchannel activity in the epithelium is impaired resulting in na + hyperabsorption and consequently in the generation of a thick and static overlaying asl layer creating an environment that favors progressively worsening bacterial infections. in this study we investigated the potential contributory role of altered respiratory cilia function to inefficient mcc in cf airways allowing for bacterial infestations. our ex vivo model system was the nasal airways of the cftr gene knockout (cftr tm unc ) mouse in which key aspects of the phenotype of human cf lung disease, such as dehydrated asl layer and goblet hyperplasia, are observed. methods and results: ciliary beat frequency (cbf) was measured using a dual temperature controlled perfusion chamber, differential interference contrast microscopy and high speed digital video analysis system. we evaluated the cbf of nasal septa explants from homozygote cf (affected) and non-affected littermate wild type (wt) and heterozygote mice congenic on the c bl/ background. the basal cbf of homozygote cf mice ( ± hz, n= ) was significantly lower than that of wt mice ( ± hz, n= ) and also heterozygote mice ( ± hz, n= ) (p< . , anova, snk test). to determine whether the dehydrated asl layer was still present on the surface of the cf mouse nasal airway epithelium and as such impeding normal cilial beating, we fixed nasal septa (n= ) of cf and wt mice immediately following isolation using the pfc/oso fixation method to preserve the fragile asl layer and compared the height of the asl layer to nasal septa (n= ) from cf and wt mice fixed with pfc/oso immediately following cbf analysis. electron microscopy analysis revealed that although the asl layer was present at the time of isolation, there was no asl layer following the perfusion of solutions necessary for the cbf measurements. this finding demonstrated that the activity of cilia was not impaired in the cf nasal airway epithelium due to the presence of a dehydrated asl layer. cbf frequency was also significantly reduced in three different codes of mouse ciliated nasal airway epithelial cultures derived from affected cf mice compared to those derived from non-affected wt mice. conclusion: we show that the absence of the cftr gene product in the cf knockout mouse nasal airway epithelium is associated with significantly decreased basal cbf compared to the non-affected wt and heterozygote mice. this observation potentially provides an additional mechanism as to why lung disease persists in human cf lungs. ml and mba contributed equally. clinical studies have linked increased sputum and peripheral blood neutrophil mpo activity with increased airflow obstruction in cystic fibrosis (cf) patients of the same age, gender, airway bacterial flora, and cftr genotype. variations in the tgf-β gene associated with increased tgf-β production have been linked to worse airflow obstruction in cf patients of similar age, gender and cftr genotype. we hypothesized that in the presence of mpo, tgf-β production would increase in airway epithelial cells. we obtained normal human airway epithelial cells (hae) and cultured them in trans-wells. tgf-β mrna was measured by pcr. tgf-β protein was detected by immunofluorescence staining and immunoprecipitation. we found increased tgf-β mrna after hae were exposed to mpo (at activities found in cf sputum) normalized to mrna of constitutive beta actin. under identical conditions, tgf-β protein expression was increased in hae. we conclude that neutrophil mpo present on the apical surface of cultured hae induces transcription and protein synthesis of tgf-β . these results suggest that mpo induction of airway epithelial tgf-β is one molecular system linking the chronic neutrophilic endobronchitis seen in cf and subsequent airway wall fibrosis. are more oxidatively stressed than normal. as oxidative stress (os) is a potent activator of nf-κb, these data supported our hypothesis that cftr dysfunction causes alterations in the expression and/or modification of redox related proteins thereby enhancing activation of nf-κb. delineation of this interaction will produce good therapeutic targets. in this report we examine the role of os in cf inflammation. methods: to address our hypothesis, we used both in vitro and in vivo models of cf. our airway epithelial cell models are the hteo-pcep and pcep-r cell pair; and the hbeo-sense (s) and antisense (as) cell pair. additionally, we examined whole lungs from - week old b . s -cftr tm mrc mice (r h mutants back-crossed into the c bl j background). proteins were prepared from homogenates of whole samples, run on -d gels, and their densities compared. the gel spots of differentially expressed proteins were excised, subjected to in-gel trypsin digestion, and identified by lc ms/ms. for biochemical analysis, we tested for the levels of h o , glutathione, and lipid peroxidation. we correlated these data with measurements of nf-κb activity (elisa and cytokine production), and the activity of nrf- , an antioxidant (ao) response pathway transcription factor. we studied cultured cells in the presence and absence of inflammatory stimulation (tnf-α/il- β), and compared non-stimulated cf mouse lungs to lungs from normal litter mates. results: our proteomic analysis revealed that in the absence of inflammatory stimulation, a paradoxical decrease in ao proteins exists in cf cells by or more fold compared to matched pairs. this is despite a significant increase in intracellular os, which we confirmed by different biochemical measures. activity of nrf- was decreased by ~ % in cf cells vs. normal. the data predict an increase in h o , which is a potent activator of nf-κb, and we confirmed both a significant elevation in h o (up - fold in cf) and a related significant increase in nf-κb activity, as assessed by elisa for nuclear p , and il- and il- production. when we analyzed whole lungs from cf mice for antioxidant protein levels, we similarly observed decreases vs. normal littermates. when cells were stimulated, differences in protein expression and oxidative stress between cf and normal were further enhanced, corresponding to an excessive activation of nf-κb and increased production of il- and il- . treatment with the antioxidants nac or selenium decreased the activation of nf-κb to normal levels. members of the clc family of proteins form either voltage-gated chloride channels or cl -/h + exchangers; nine subtypes are found in mammals. among these, clc- is one of the most ubiquitous variants, which is expressed in several of the types of epithelial cells that also express the cystic fibrosis transmembrane conductance regulator (cftr). it has been postulated that clc- may be a suitable alternative chloride pathway in these cells; however the contribution of clc- to chloride transport in these cells is poorly understood. this is partly due to the lack of appropriate pharmacological tools that are capable of specifically inhibiting clc- . we describe here the isolation, from venom of the scorpion leiurus quinquestriatus hebraeus, of the first peptide inhibitor of any clc channel. this toxin, which we have named gatx , specifically inhibits the clc- channel. gatx bears primary sequence identical to that of a toxin thought to serve as a k + channel inhibitor, although no molecular target for this toxin was previously known. a homology model of gatx reveals that it adopts a similar fold to other scorpion toxins. gatx was prepared via solid-phase chemical synthesis, and the synthetic toxin inhibits clc- with higher affinity than any other inhibitor of clc- or any other chloride channel. the k d for gatx mediated inhibition of clc- in multichannel patches is only pm at v m = - mv, and pm at - mv. single channel experiments show that gatx does not alter single channel amplitude, but may alter channel gating. these experiments provide the basis for developing gatx into a useful tool that can be used to define the role of clc- in airway and intestinal epithelial cells. furthermore, the high affinity and specificity of inhibition suggest that gatx interacts intimately with the channel, and thus may the exploitation of alternative chloride channels to bypass the defect in cftr-mediated chloride secretion is an established therapeutic strategy in cf. in search of such potential therapeutic targets, we assessed camp/pkadependent chloride secretion across excised nasal epithelium of cftr null mice and wildtype controls, by measuring short-circuit currents (isc) in an ussing chamber set-up. all experiments were performed in the presence of amiloride to block the contribution of epithelial sodium channels to the isc. in cftr null mice of mixed background (c bl/ ; ), the camp agonist forskolin activated a bumetanide-sensitive rise in isc that was comparable in magnitude to the cftr-mediated isc response of littermate controls ( ± vs. ± µa/cm , respectively). the forskolin-induced isc in these null mice was inhibited by the general chloride channel inhibitor dpc, but appeared insensitive to the cftr inhibitors cftrinh- and glibenclamide, the orcc and calcium-activated chloride channel inhibitor dids, and the clc- inhibitors cadmium and pre-activated omeprazol. moreover, the isc response to forskolin greatly exceeded the response to the calciumlinked agonists ionomycin and carbachol. intriguingly, in nasal epithelium of cftr null mice with a different genetic background (fvb), no such forskolin-inducible secretory pathway was evident (cftr-/-: ± vs. cftr+/+: ± µa/cm ). when, in nasal epithelium of cftr null mice (c bl/ ; ), the basolateral membrane electrical resistance was negated by nystatin treatment, and in the presence of a transepithelial chloride gradient, forskolin failed to elicit a rise in isc, suggesting that camp/pka signaling targets a basolaterally located ion transport system. indeed, in intact epithelium, addition of chromanol b, a selective inhibitor of camp/pka-activated potassium channels, to the serosal bath, blocked the forskolin-induced rise in isc. we tentatively conclude that the hyperpolarization of nasal epithelial cells, which ensues from camp/pka-induced potassium efflux across the basolateral membrane, drives chloride exit across the apical membrane via a constitutively active anion conductive pathway that is absent from fvb cftr null mice. rt-pcr and immunostaining demonstrated that, among the candidate anion channels that fit the above pharmacological profile, clc-ka, but not clc-kb, was expressed in nasal epithelium of both cftr null mice and controls (c bl/ ; ), together with the accessory protein barttin. importantly, immunostaining suggested a strong increase in clc-k protein abundance in the cftr null mice, as compared to wildtype animals. on the basis of these findings, we postulate that clc-ka channels may serve as cftr bypass channels in nasal epithelium of cftr null mice that, albeit indirectly, are responsive to camp/pka signaling. future studies aim to characterize clc-ka and barttin in the airways from cf patients and controls, and to identify the post-transcriptional mechanism resulting in the up-regulation of clc-ka protein in cftr null mice. in the disease cystic fibrosis (cf), the most common mutation f del results in endoplasmic reticulum retention of misfolded cf gene proteins (cftr). the endoplasmic reticulum (er) f del-cftr protein retention is dependent upon chaperone proteins, many of which require ca + for optimal activity. an increase in cytosolic free ca + concentration is dependent on the activity of ion channels in the membrane of er and on extracellular ca + influx. this influx, named capacitative ca + entry (cce), induced by calcium store depletion, represents the major ca + influx mechanism in cells (putney et al., ) . during the cce, ca + influx can be mediated by one or several of the isoforms of transient receptor potential canonical channels (trpc) (putney et al., ) . aims & methods: in the present work, we studied the cce in human cf tracheal gland serous cfkm cells compared to non cf human tracheal mm cells. for this, we characterized the molecular identity of trpc channels responsible of cce by rt pcr technique and we compared global ca + influx in cf and non cf cells by using fluo -am probes. moreover, we measured the single channel activity of plasma membrane trpc channels by cellattached patch-clamp configuration in cf and non cf cells. we also studied the cce in cf cells after correction of the abnormal f del-cftr trafficking by miglustat ( h at µm of n-butyldeoxynojirimycin, nb-dnj) (norez et al., ) and low temperature ( h at °c) (denning et al., ) . results: we detected by rt-pcr technique the presence of trpc and trpc in both cell lines and we observed that cce was increased -fold in cf-km cells compare to non-cf mm cells. following stimulation by histamine, we recorded elementary ca + currents with similar amplitude ( . pa at - mv) and conductance ( ps) whatever the cells tested (cf-km or mm cells). however, we observed a -fold increase of the npo (number of channels in each patch × open probability of single channel) in cf cells compare to non-cf cells. in f del-cftr corrected-cf cells, the ca + influx stimulated by the ca + stores depletion was reduced to a similar level to non cf mm cells. the permanent contact of the airway epithelium with the external milieu induces frequent injuries caused by inhaled pathogens and particles. regeneration of the wounded airway epithelium needs to respect its prior structure in order to restore its defence functions. the underlying molecular mechanisms associated with the regeneration of the human airway epithelium are poorly described. the aim of our study was to determine, using a global expression approach, the transcriptional profile of human airway cells during the epithelial regeneration process. human airway epithelial cells collected from nasal polyps were seeded on type iv collagen-coated porous membranes, cultured up to confluence in liquid-liquid(ll) conditions, then put at the air-liquid interface (ali) allowing epithelial mucociliary differentiation. rna were extracted from epithelial cells at % confluence in ll conditions (epithelial cell proliferation and migration stage, step i), after days in ali conditions (beginning of epithelial cell differentiation, step ii), when first ciliated cells appeared (step iii) and when cultures were completely differentiated (step iv). our results demonstrated that between step i and step ii, genes were significantly modulated (ratio r<- or r> , p< . ), among them were activated and were repressed. between step ii and step iii, transcripts were up-regulated (tubulin, dynein, stath, microtubule associated proteins…) and only one gene (fatty acid desaturase- ) was repressed. finally, between step iii and step iv, except stath which was~ -fold down-regulated, we observed only few genes differentially and slightly modulated. all along the regeneration process, between step i and step iv, genes were significantly modulated with % of activated and % of repressed genes, among them a family of genes encoding proteins involved in the ciliary differentiation (foxj , tektin, dynein, tubulin, …), extracellular matrix proteins (collagen, laminin, …) and inflammatory-related genes like cytokines, growth factors and their receptors (ccl , vegfc, csf , il , il ra …). by elucidating the specific transcriptome involved in each step of the human airway epithelium regeneration, our study could allow to better understand the different cellular and molecular mechanisms, as well as the chronological events involved in these processes, and will further be applied to the study of the regeneration process in cystic fibrosis airway epithelium. supported by french association vaincre la mucoviscidose. electrolytes may be transported either through cells, the transcellular pathway, or between cells, the paracellular pathway. most research has focused on the channels and transporters of the transcellular pathway, while much less is known about paracellular transport. paracellular transport is regulated by tight junctions. claudins, the main component of tight junctions, are necessary and sufficient for tight junction formation and are important in establishing ion selectivity. the goal of this study was to further investigate the role of tight junctions in human airway epithelia. real-time pcr identified claudins , , , , , and in primary human airway epithelia. immunocytochemistry localized claudins , , , and to tight junctions. to measure the relative ion selectivity of tight junctions, we studied well-differentiated human airway epithelia in ussing chambers. to minimize transcellular transport, we used cf epithelia, which lack cftr and blocked enac with amiloride. we found the pna/pcl is . ± . , suggesting tight juctions are more permeable to cations than anions. the relative cation selectivity was na+ > k+ > cs+ > tma+ > mg + and the relative anion selectivity was cl-> br-≥ i-> no -> gluconate-. we compared these in vitro results to in vivo studies in people with cf and measured nasal voltage in the presence of amiloride. we found the pna/pcl is . ± . . tight junctions were slightly more selective for cations than anions in vitro and in vivo. to determine the effect of specific claudins on the selectivity of the paracellular pathway, primary human airway epithelia were infected with adenovirus expressing claudin or gfp. epithelia overexpressing claudin had a higher transepithelial resistance (ter) and the pna/pcl was . . however, infection efficiency was only %. this lead us to investigate if transformed human airway epithelial cells lines may be a better model to study permeability. cufi cells also express claudins , , , , , and . furthermore, like the primary human airway, cufi cells are more selective for cations; pna/pcl = . ± . . future studies will investigate the effect of claudin expression on the paracellular permeability of these cells. the results will help identify the properties of a pathway critical to epithelial transport and barrier functions. altered adenosine (ado) signaling is associated with chronic lung disease and increased asl ado levels are thought to drive inflammation by stimulation of a b receptors (a b-r) in airway epithelia, which can activate nfκb. when measured by lavage, asl ado is ≤ nm. due to its relatively low affinity to ado, a b-r are thought to be inactive under normal conditions (ec is ~ . µm), and only stimulated during chronic lung disease. however, we have previously demonstrated that the ado-receptor (ado-r) antagonist -spt inhibits asl volume regulation, and that a b-r is in close proximity to cftr. thus, we hypothesized that the asl ado concentration close to the apical membrane is higher than measured by asl lavage in bulk solution. we tested this hypothesis by looking for ado-r expression and function in well-differentiated human bronchial epithelia cultures (hbecs) and in vivo. by pcr analysis, we found that a b-r was the only ado-r expressed in hbecs. we then measured asl height over h ± ado-r antagonists by xz confocal microcopy when asl height was preset to ~ µm at t= . at µm, caffeine is a non-specific ado-r antagonist, which inhibited asl volume regulation ( h asl height was . µm vs. . µm for controls, n= ). similar inhibition was observed with alloxazine, which is relatively specific for a b-r (n= ). in contrast, dpcpx, zm & mrs , which inhibit a , a a & a -r respectively had no effect on asl volume regulation. asl height regulation was also ablated by the addition of adenosine deaminase (ada) to the asl (n = ). we next attempted to recapitulate the changes in asl height seen under control conditions, i.e. increase from to µm over the initial h followed by a plateau (the remainder of the h) by adding the non-hydrolyzable ado analogue neca to the asl in the presence of ada. nm neca was insufficient to regulate asl volume and height remained at µm over h. in contrast, addition of µm neca to the asl resulted asl volume regulation that was indistinguishable from control conditions (n= ), suggesting that near-membrane asl is much higher than has been measured by lavage. to confirm that a b-r stimulates cftr chloride secretion, we measured transepithelial potential differences of hbecs mounted in ussing chambers. a dose-dependent increase in cl-secretion was observed by ado addition with an ec of . +/- . µm, similar to the concentration of neca required to regulate asl height. to demonstrate which ado-r are expressed in human airways in vivo, we used laser capture to obtain ciliated cells from frozen tissue sections of excised donor bronchi. relative expression of a b-r by qpcr was found to be . +/- . fold higher than the a , a a-r, or a receptors (n= ). to confirm that ado-r activate cftr in vivo, we measured nasal potential differences. we found that ado activated chloride secretion at similar levels to isoproterenol. the effect of ado was inhibited by pre-exposure to µm caffeine (ado, . +/- . mv; ado + caffeine, . +/- . mv). we conclude that a b-r has an important role in asl homeostasis through regulation of cftr and it is active in human airway epithelia under normal physiological conditions. through osmotic forces, hypertonic saline (hs) may increase the volume of cf airway surface liquid, restore mucus clearance, and improve lung function. the na + channel antagonist amiloride is predicted to increase the duration of action of hs. however, patients taking amiloride with hs showed less clinical benefits than patients taking placebo with hs. in vitro data suggested that amiloride inhibits transepithelial water flux (pf) in cystic fibrosis airway epithelia. we have extended this study by looking at the effects of amiloride on aquaporins in a heterologous expression system (mdck cells) and have used immunofluorescence to determine which aquaporins are expressed in human airways in vivo and in vitro. inhibition of enac by aprotinin pretreatment had no effect on hs-induced pf (n= ), suggesting that amiloride did not exert its actions on pf by inhibiting enac. however, since hs-induced pf was also inhibited with hgcl , we speculated that amiloride directly blocked an aquaporin. to test this hypothesis, we measured the rate of cell shrinkage of wild-type mdck cells, and those stably-expressing aqp or , or expressing aqp (after h exposure to a mildly hypertonic media; mosm). following mucosal exposure to a mosm hypertonic ringer solution, mdck cultures grown to confluency on permeable filters shrunk by % after s (n = ), as measured by xz confocal microscopy following staining with calcein-am. in contrast mdck/aqp or mdck/aqp cultures shrunk by % over the same period and mdck/aqp cultures shrunk by % (all n= ). a min pretreatment with µm amiloride abolished hypertonic ringer-induced cell shrinkage in mdck/aqp cultures ( % at s; n= ) but had no affect on cell shrinkage in aqp or aqp expressing cultures. amiloride is fluorescent and can be imaged in epithelia by xz confocal scanning under uv excitation. amiloride was not internalized immediately (< s) before hypertonic ringer addition and did not block aqp -mediated cell shrinkage ( % at s; n = ). however, a min pretreatment with amiloride caused it be internalized and at this time aqp -induced cell shrinkage was blocked, suggesting that amiloride blocks aqp intracellularly. to confirm that aqp is present in airway epithelia, we performed immunostaining with an aqp antibody and found that aqp is highly expressed at the apical membrane of ciliated cells in human bronchial tissue and well-differentiated bronchial cultures. we conclude that amiloride blocks aqp , which is abundantly expressed in the superficial airway epithelia. supported we have previously observed that cftr, annexin (a ) and cytosolic phospholipase a (cpla ) are partially recruited in detergent insoluble microdomains (dim) upon proinflammatory stimulation. this is prevented by specific inhibition of cftr. a participates in the regulation of inflammation by inhibiting cpla activity. we have also shown that a expression is decreased in cftr-/-mouse tissues. we postulate that cftr, a and cpla participate in the regulation of inflammation by their dynamic interaction within dim. in this study we aimed to (i) demonstrate and characterize this interaction, and (ii) identify other potential partners of this complex in dim. by coimmunoprecipitation, we examined the interaction between cftr, a and cpla in human pulmonary epithelial cells calu . to search for additional partners we developed a new proteomic approach based on double sds-page (dsds-page), which is compatible with membrane protein analysis. calu cells and human epithelial bronchial cfbe cells (expressing either wt or f delcftr) were subjected or not to proinflammatory conditions (tnfα). dim were isolated by density gradient and analyzed by dsds-page. cftr immunoprecipitated with a and cpla in calu cells. dsds-page analyses showed significant differential expression of cytokeratin , actin and protein disulfide isomerase (pdi, involved in protein folding) in dim between control and proinflammatory conditions in calu cells, and between wt and f del cfbe cells, as determined by mass spectrometry. in conclusion, cftr, a and cpla interact in basal conditions. proinflammatory treatment causes differential expression of cytoskeletal proteins and pdi depending on the expression of normal and mutated cftr. this work suggests that cftr could participate in the regulation of inflammation within a complex with a and cpla . cytoskeletal proteins and pdi could be involved in the dynamics of this complex. this could be related to the abnormal inflammatory response characteristic of cf. we acute and chronic inflammation, common features of cf airways disease, have been linked to higher atp release and changes in atp metabolic patterns in vivo, e.g., in induced sputum and exhaled breath condensate from cf patients and bronchoalveolar lavage fluid from inflamed lobes (esther, ped pulm sppl , ) . we, therefore, investigated the contribution of airway epithelia per se to the raised atp concentrations on cf airway surface during inflammation. when exposed to supernatant of mucopurulent material (smm) from cf airways for h, or infected with respiratory syncytial virus (rsv) h prior to the study, basal atp release from well-differentiated primary human bronchial epithelial (hbe) cultures did not differ from that of control cultures. however, basal atp release was increased weeks after rsv infection when inflammation had waned (as indexed by il- secretion). the increase in basal atp release correlated with increased basal udp-glucose and mucin release, and goblet cell metaplasia. these data indicate that the increased basal atp release following rsv infection reflected increased goblet cell secretion. a different pattern was observed for stimulated atp release. hbes exposed to smm for h, or infected with rsv h prior, released - times more atp following hypotonic challenge than control cultures (okada, ped pulm sppl, ) . the increase in peak atp concentrations correlated with the degree of inflammation as indexed by il- secretion and the increase in intracellular calcium (ca + i ) mobilization in response to hypotonic challenge, but not with the goblet cell number. ca + i mobilization in response to hypotonic challenge in control hbes was almost completely due to extracellular ca + i influx which was inhibited by antagonists of a transient receptor potential v (trpv ) ca + channel. in contrast, an additional component of atp-induced ca + i release from endoplasmic reticulum (er) was observed in smm-treated hbes. the increase in hypotonicity-induced atp release in smm-treated hbes compared to control hbes was almost completely sensitive to chelation of ca + i by bapta ( µm; h), or inhibition of exocytosis by brefeldin a ( µm; . h), monensin ( µm; h) or n-ethylmaleimide ( mm; min), whereas these reagents were ineffective against hypotonicity-induced atp release from control hbes. these data suggest a positive feedback loop between the increased atp release linked to ca + i -dependent exocytosis and the increase in atp-induced ca + i mobolization linked to increased er ca + i stores (ribeiro, jbc , ) in inflamed hbes. in conclusion, we propose that basal and hypotonicity-induced atp release from hbes are regulated in distinct mechanisms during inflammation. basal atp release reflects continuous exocytosis processes independent of ca + i mobilization and differs as a function of cell type (ciliated vs goblet cell), whereas acute inflammation upregulates hypotonicity-induced atp release via increased ca + i mobilization and ca + i -dependent exocytosis. we conclude that airway epithelia exhibit multiple mechanisms of increasing atp release into the lumen in response to inflammation. supported by cff grant okada i to sfo and grants from nih. the main biological role of vitamin d is in control of bone and mineral metabolism, but it has also been discovered to have many immune system functions. vitamin d, a fat soluble vitamin, is poorly absorbed in cf leading to low serum levels. we sought to investigate the involvement of vitamin d in the control of inflammation in a cf model. methods cf tracheal epithelial cells (cfte) were pretreated with vitamin d ( , [oh] d ) or control and exposed to p aeruginosa lipopolysaccharide (lps) for hrs before collecting the cell supernatants. cytokine levels in the supernatants were assessed by cytokine array and subsequent elisas specific for il- , il- and mcp- . vitamin d was measured in the plasma of children with cystic fibrosis aged months to years. neutrophil numbers, neutrophil elastase (ne) activity and il- concentrations were measured in the bronchoalveolar lavage (bal) of these children. children with cystic fibrosis were deemed to be non-colonised, intermittently colonised or chronically colonised with p aeruginosa on the basis of the leeds criteria. pretreatment with vitamin d significantly reduced secretion of il- , il- and mcp- from cftes after challenge with p aeruginosa lps in a dose dependant manner. plasma vitamin d levels were highest in the noncolonised group and lowest in the chronically colonised group with intermediate levels in the intermittently colonised group. bal neutrophil counts, ne activity and il- concentration showed a reciprocal pattern. conclusions vitamin d acts on cf lung epithelial cells to protect against excessive cytokine production in response to lps. levels of vitamin d are inversely related to both p aeruginosa colonisation status and bal markers of inflammation. low levels of vitamin d may contribute to advancing lung disease in children with cystic fibrosis. more aggressive correction of vitamin d deficiency may have a role in protecting against excessive inflammatory response to infection. sleeping in a mist tent has been used as a treatment for cystic fibrosis (cf) patients, in order to hydrate the viscous mucus and make it easier to be removed by coughing (matthews et al., ) . however, the efficiency of the method has been questioned, and its use was largely discontinued. with a new method to measure the ion content of human nasal fluid the effect of sleeping in a moisture tent on the ion content of nasal airway surface liquid (asl) in cf-patients and healthy controls was determined. the cf-patients and healthy controls spent a night ( h) in a mist tent, and samples of the nasal asl were taken before the experiment, after the period in the tent, and then at each hour during h after the persons had left the tent. samples of nasal fluid were collected on sephadex g- beads that had been mounted on strips of filter paper, which were then put into the nasal cavity of the cf patients or controls for - min. the strips were removed, and the beads were isolated, dried, and analyzed by x-ray microanalysis (vanthanouvong et al., ) . the concentration of na, cl, and k in the nasal fluid of cf patients was , , and mm, respectively, before the patient entered the tent, significantly higher than the levels in the nasal fluid of the controls (na mm, cl mm, and k mm). during the period in the tent, the ion content decreased, to levels of na mm, cl mm, and k mm (cfpatients) and na mm, cl mm, and k mm (controls) immediately after leaving the tent. after leaving the mist tent the ion levels in the nasal fluid increased, reaching after h values of na mm, cl mm, and k mm (cf-patients) and na mm, cl mm and k mm (controls), which was for both groups higher than before entering the tent. no major changes in the ion content of the asl occurred after h. the salt content of the asl may be relevant in cf, since the asl is known to contain anti-bacterial proteins, defensins, which are sensitive to the salt concentration (bals et al., ) . hence, the higher salt concentra-tions in the asl of cf-patients may have negative consequences for the anti-bacterial defense system in the lungs, and conversely, the decrease in ion concentrations, caused by spending time in a mist tent, may have positive effects. (we have previously shown that none of our patients was chronically colonized with pseudomonas aeruginosa while regularly sleeping in mist tents.) however, with currently used procedures, the effect of sleeping in a mist tent on the ion content of the nasal asl is short-lived. bals background: antibody microarrays for clinical applications have been anticipated for more than a decade but the technology has only recently become sufficiently mature for reproducible, robust detection of low abundance proteins. the advantage of this new technology is the high throughput, parallel detection of identified, known, low abundance proteins. antibody microarray results are usually given in terms of ratios between two samples (e.g., "experiment" and "control"). however, given the printing and calibration issues inherent with antibody microarrays, this ratio'ing approach precludes statistically valid inter-array comparisons of the individual protein concentrations. we have therefore developed and implemented a different calibration strategy using a benchmark mixture that is applied to every array as an internal standard, thus controlling for any differences in antibody activities as well as printing imperfections. the amount of protein bound to each spot is determined relative to the respective protein in the internal standard. since all samples in the study are compared to the same internal standard, this semi-quantitative approach permits the comparison of multiple samples. all data for the same antibody on multiple arrays can then be used to calculate the average and standard deviation for a given population (such as patients or control groups) in a parallel fashion. methods: cf lung epithelial cultured cells ib - and the cftrrepaired ib - /s were biosynthetically labeled with a hour pulse of [s]methionine, and then chased with cold methionine for , or hours. we then isolated total protein from each sample, and labeled the proteins with cy . labeled proteins were applied to clontech® antibody arrays ( antibodies printed in duplicates) according to the manufacturer's protocols. the internal standard, consisting of a mixture of cell cultures and tissue extracts known to contain the cognate protein for each antibody on the array, was labeled with cy . the data were then used to compare the cf and the corrected cells in terms of amount, biosynthesis rate and degradation half-life (t / ) for each one of the features on the array. data analysis: for each spot on the array the ratio of cy /cy (after background subtraction) was calculated, normalized and stored for further analysis. for "pulse-chase" experiments the ratio of [s] counts to cy at each time point gives a measure of the fraction of newly synthesized (radiolabeled) to total protein (dye labeled) bound to each given antibody on the array. the addition of the cy labeled internal standard enables the calibration of these totals, allowing the comparison all the spots for any given antibody on multiple arrays. furthermore, the internal standard calibration makes it possible to compare not only t / values but also to calculate the relative degradation rates in different cell lines even when the amounts of that protein are different in the "control" and "experiment" cells. the new approach quantitatively identifies and subjects to analysis the effect of the cftr mutation on protein biosynthesis and degradation of the cellular signaling proteome. supported by nih no -hv- (hbp) and ro - (hbp) in cf patients, defective apical chloride secretion, due to lack of functional cftr, leads to dehydration of airway surface liquid (asl) and mucus plugging. this defect is likely ameliorated, in part, by an alternative route of chloride conductance, the calcium activated chloride channel (cacc) . female gender appears to be associated with more rapid decline of lung function and earlier death in cf. this suggests that gender differences in hormonal environments might influence asl height regulation, thus modifying the pulmonary phenotype. we hypothesized that changes in estrogen concentrations might alter asl hydration by affecting cacc. consistent with this, we have shown that estrogen attenuates increases in intracellular calcium and asl height that follow purinergic receptor activation in primary cultures of human bronchial epithelial cells (hbecs, see accompanying abstract by hengrui sun et al). here, we report correlations between estrogen levels and nasal ion transport in vivo and further probe hormonal effects on ion transport and asl height regulation in vitro. in vivo studies: in healthy, spontaneously menstruating females, nasal potential difference (npd) was measured (both nostrils) on two occasions during a single monthly cycle. based on a history of the most recent menstrual cycles, we measured npd on a day when estrogen levels were predicted to be low (generally - days following onset of menses) and subsequently on the day when the pre-ovulatory estrogen surge was predicted. the recording electrode was positioned at the location where maximal stable basal pd was detected and changes in pd recorded continuously during sequential perfusion with ] ringer's solution, ] ringer's solution containing amiloride (to inhibit sodium absorption via enac), ] modified (low chloride) ringer's containing amiloride and finally ] a similar solution also containing utp (to activate cacc). as predicted, blood estrogen levels were higher on the "pre-ovulatory" day ( . ± . vs . ± . nm, p< . ). utp-stimulated change in pd was significantly lower on the study day when estrogen levels were elevated ( . ± . vs . ± . mvs, p< . ). further, the amiloride-sensitive component of basal pd was higher on this day ( . ± . vs . ± . mvs, p< . ). the change in pd in response to perfusion with a low chloride solution was not different. in vitro studies: expression of estrogen receptor (er) α (but not β) was confirmed in non-cf and cf hbecs by real time pcr. erα was detected in hbecs from males and female donors. in cf and non-cf hbecs mounted in modified ussing chambers, exposure to nm e was associated with a significant (~ %) reduction in utp-stimulated short circuit current. e inhibited the atp-stimulated increase in asl height in male and female hbecs and this asl regulatory phenotype was not reproduced by testosterone or progesterone. in conclusion, estrogen inhibits utp-activated chloride secretion in vivo and in vitro suggesting that elevated pre-ovulatory estrogen levels may further impair mucociliary clearance in females with cf. all data mean±sem. supported by cff leroy matthews award [ ] . data demonstrating an upregulation of caccs when cftr is absent or defective [ , ] have triggered further studies of these channels and prompted further elucidation of the functional (and possibly also physical) interactions between these two channels. although searched for long, the molecular identity of caccs is still under debate. the role of bestrophin family proteins as putative candidates for caccs is discussed controversially. bestrophin (best- ) has been proposed to form ca + activated clchannels in epithelial cells. moreover, caccs have been shown to support cell proliferation, namely in the development of cancer [ ] . here, our goal is two-fold: ) to investigate the correlation between expression levels of best- and epithelial cell proliferation; and ) to pursue a search for interacting protein partners of best- to better understand the role and function of bestrophin(s) in epithelial cells. to study the correlation between best- expression and cell proliferation, we analyzed two populations of the t colonic carcinoma cell line with very different proliferation rates: the original (t -slow) and the spontaneously transformed t cells (t -fast). we observed that t -slow cells have a small proliferation rate and express low amounts of best- , while t -fast cells express high levels of this protein. best- is spontaneously active in t -fast cells. best- -rnai inhibited ca + activated clconductance and cell proliferation, therefore establishing a novel role of bestrophins in cell proliferation, which may be of relevance for the regeneration of the epithelia in cf and also for alternative therapies for cf. for the second objective, two putative cytoplasmic domains of best , polyhistidine -tagged (phis) n-term (aa - , best -n) and c-term (aa - , best -c), were cloned into the pet-sumo bacterial expression vector. these domains were then immobilized onto metal-affinity resin to capture interacting proteins from human t whole cell and sub-cellular lysates. protein-containing fractions recovered from best -n/c-coated and blank resins were subjected to d-gel and protein identification. it is expected that functional characterization of the interaction between the captured proteins and best- will give new insights into the biological role, plausibly of relevance to cf. work supported by dfg-sfb -a grant (germany) and pluriannual funding of cigmh (feder-eu and fct, portugal extracellular nucleotides acting on epithelial cell surface purinergic receptors regulate the mucociliary clearance process that removes noxious materials from the lung. however, how epithelial cells release nucleotides into the airways remains largely unknown. cftr has been suggested to mediate atp release in airway epithelia, but conclusive demonstration remains elusive. calu- is a human airway derived cell line often described as a model of airway gland serous cells. at least % of cells in a monolayer expressed high levels of cftr protein and activity at the apical membrane. atp, adenosine, and udp-glucose, the naturally occurring agonists for the p y , a b, and p y receptors, respectively, were quantified in airway surface liquid with nanomolar sensitivity using radiolabeling and fluorescence hplc-based techniques, and real-time atp measurements. atp (and udp-glucose) was found to be released constitutively and selectively onto the mucosal surface of calu- cells. however, the specific cftr inhibitor ( µm, - min) failed to decrease atp concentration in the mucosal bath of resting calu- cells. forskolin ( µm, min) stimulated cftr channel activity, but did not affect basal levels of atp (or udpglucose) release. in contrast, elevation of intracellular calcium using ionomycin ( - µm, - min) significantly increased atp (and udp-glucose) release into the mucosal (but not serosal) bath of calu- cells. calciummediated nucleotide release enhancement was not abolished by cftr inhibitor . maneuvers like pre-incubation in nominally free-calcium solution, bapta ( µm, min), or cytochalasin d ( µm, min) to disrupt actin cytoskeleton, which inhibited regulated exocytosis in calu- cells, also reduced calcium-mediated increase in nucleotide release. calu- monolayers presented a fraction of cells (< %) exhibiting negligible cftr immunostaining signal, but displaying airway goblet cell mucin granules. remarkably, stimulation of atp and udp-sugar release with ionomycin resulted in concomitant enhanced exocytotic secretion of muc ac. these results suggest that cftr is not involved in regulation of atp release in airway epithelial cells. more likely, nucleotides and nucleotide-sugars residing in the biosynthetic pathway involved in modification of apically targeted glycoconjugates (e.g. mucins) are released to the extracellular environment via calcium-triggered exocytosis. supported although the regulation of na+ transport is relatively well described in normal and cf airway epithelial cells, the impact of inflammation and infection, two major components of cf lung disease, on na+ transport has not been studied extensively. significant amounts of evidence suggest that bacterial by-products may influence na+ transport in airway epithelium. furthermore, the secretion of exoenzyme s by pseudomonas aeruginosa is enhanced during an exacerbation of lung disease in cystic fibrosis. in the past few years, our laboratory has shown that exposition of lung epithelial cells to p. aeruginosa lps leads to a rapid decrease in activity and expression of enac channel. the aim of our study was to evaluate the mechanism involved in the modulation of enac expression and activity by lps. primary culture of lung epithelial cells grown on filters were treated with lps ( µg/ml) for different time ( min, , , h). at each time point, the total and amiloride-sensitive short circuit current (isc) was determined in ussing chamber. in parallel, the cell proteins were collected to investigate by western blot the signalling pathways involved in this response to lps. a hours lps treatment leads to a significant decrease in alpha and gamma enac mrna to % and % of control respectively. similarly, the amiloridesensitive current was decreased by % of the current in untreated cells after a min exposure to lps. by western blot, we demonstrated the strong implication of the pi k/akt pathway in response to lps in the regulation enac mrna and the potential implication of tyrosine/kinase receptors signalling pathway through mtor in the regulation of enac channel activity. in conclusion, we demonstrate that inflammatory molecules can modulate the expression and function of the na+ transport mechanism in lung epithelial cells and could contribute to the modulation of airway surface fluid in the chronic infectious process associated with cf. serous acinar cells are thought to be crucial for secretion of airway surface liquid by submucosal glands in large airways, but the molecular mechanisms of serous cell ion and fluid transport are not well understood. to address this, we isolated murine nasal serous acinar cells and studied them using simultaneous dic and quantitative fluorescence microscopy to track changes in cell volume and intracellular concentrations of ions involved in fluid secretion ([cl -] i ; spq) and its regulation ([ca + ] i ; fura- ). previous results indicated that serous acinar cell volume changes were indicative of solute efflux (shrinking) and solute influx (swelling), reflecting the secretory state of the cell. acinar cells stimulated with µm carbachol (cch) exhibited a rapid increase in [ca + ] i and subsequent ~ % cell shrinkage due to efflux of ~ % of cell clcontent and parallel loss of k + and osmotically obliged h o. because murine serous cells are sites of apical cftr expression in the airway, we tested the requirement for cftr in cch/ca +activated shrinkage/clefflux. acinar cells isolated from either cftr tm unc-/mice or wt cells pre-treated with cftr inh exhibited identical rates and magnitudes of ca + -induced cell shrinkage and clloss compared to wt control cells. resting [cl -] i was also identical in wt and cftr tm unc-/serous acinar cells ( ± and ± mm, respectively), suggesting similar levels of resting clconductance. thus, murine serous acinar cells can secrete kcl/h o by ca + -dependent mechanisms in the absence of functional cftr, in agreement with studies of intact glands suggesting cch-induced glandular fluid secretion is cftr-independent. wt serous acinar cells were stimulated with camp-agonists, including µm forskolin, µm isoproterenol, µm adenosine, and µm vip. however, no camp-induced shrinkage was observed, even in the presence of a cocktail of inhibitors to block solute uptake. to determine if cftr activation could enhance cch/ca + -induced cell shrinkage at a sub-optimal [cch], serous acinar cells were pre-treated with forskolin followed by stimulation with µm cch in the continued presence of forskolin. however, similar rates and magnitudes of cell shrinkage were observed in forskolin-treated and control cells. these results suggest that the magnitude of the cftr clconductance in murine serous acinar cells is much smaller than the ca + -activated clconductance, below the resolution of these optical assays. more sensitive electrophysiological approaches are being used to determine the molecular identity of the non-cftr clconductance(s) and the role of cftr in murine serous acinar cell ion transport. females with cf exhibit more rapid decline in fev than males, which reduces their life span. normal airway epithelia regulate airway surface liquid (asl) volume and mucus transport by secretion through both cftr and the ca +-activated cl-channel (cacc), and inhibition of either of these pathways in isolation does not abolish mucus transport. however, a loss of both pathways is predicted to result in mucus plugging which contributes to lung disease. despite a loss of cftr, cacc is functional in cf airways due to mechanical stimulation of atp release and activation of the p y pathway which serves to raise intracellular ca +. we hypothesized that e may inhibit cacc, leaving females with cf more prone to lung disease than males due to a greater depletion of asl volume. using confocal microscopy, we found that acute pre-exposure with e inhibited atp induced asl secretion in normal and cf human bronchial epithelial cultures (hbecs) from both male and female donors. the ic for e -inhibition of atp-induced asl secretion was . nm. to better understand how estrogen receptors (ers) interact with the p y pathway, we transiently transfected er-α or er-β linked to an orange fluorescent protein in an er null bhk cell line. with the co-transfection of ha tagged p y -r, we observed that e addition did not induce internalization of p y -r, neither did it alter the internalization induced by atp. we then measured total cell inositol phosphates (ip) in cf airway cells +/-e pretreatment. cell ip levels were not different between groups (n= ), suggesting that the effect of e is downstream of ip production. the investigation of the changes of the intracellular ca + (ca +i) by recording fura- emission ratio over time in er-α or er-β expressed cells provided clues. after nm e exposure, the fura- emission ratio in untransfected cells (n= ) or er-β transfected cells (n= ) increased by . following um atp addition. this increase was significantly inhibited by~ % in er-α transfected cells (n= ), providing good evidence that er-α is respon-sible for inhibiting the p y pathway. we next measured ca +i in normal hbecs. while acute nm e addition had no affect on basal ca +i levels, which were around nm, this maneuver reduced the ca +i response to nm following atp addition (both n= ), suggesting that e addition altered ca + signaling in response to atp. thapsigargin induces ca +i release from the endoplasmic reticulum, but the magnitude of ca +i release after thapsigargin addition was not altered by e pretreatment, indicating that e works further up stream to affect the p y signaling pathway. together, these results suggest that e directly interacts with the p y signaling pathway through er-α by altering ca +i. we propose that this interaction leaves females more vulnerable to infections due to a reduced asl volume during periods of high e , such as occur prior to ovulation. the nasal potential difference (or npd) is a sensitive, real time bioelectric assay of cftr-dependent and independent ion transport that is a commonly used study endpoint in early clinical stages of drug development. technical limitations of the assay must be controlled to facilitate its use in multicenter trials and limited intra-site variability. two forms of equipment are commonly employed to evaluate nasal potential difference in humans: silver-silver chloride electrodes (agcl) with saline bridges or calomel electrodes with agar bridges; however, these techniques have never been directly compared. we repeatedly evaluated (within days) the basal potential difference of five normal subjects using the two available techniques, then compared variance and repeatability of the measures. basal pd was measured during perfusion of ringer's solution, and measured at . , . , . , . , and . cm within the inferior meatus, as indicated in the cf-tdn standard operating procedures. mean basal pd were within . mv using the two techniques, with very similar coefficients of variation (cvar; . vs. . in agcl vs. calomel, respectively). maximum basal pds were also similar between the two methodologies (mean difference . mv; cvar . and . , respectively). the correlation between first and second measures of mean pd was highly reproducible, and similar between the two techniques (r= . vs. . in agcl vs. calomel, respectively); however, when each individual basal pd measure was compared, correlation between first and second pd using the agcl device appeared superior (r= . vs. . respectively; n= measures per device). comparison of values obtained in cf subjects are currently in progress and will also be reported. next, we examined diagnostic npd tracings in cf subjects for whether the effect of amiloride might influence values obtained in the contralateral nare. an effect of cross-contamination by amiloride or other agent (gene therapy vectors or other nasal administered compounds, for example) could influence accurate measure of npd values. when basal pds were performed in both nostrils prior to amiloride exposure in either nostril, mean basal pd between nares remained stable (< mv, p=ns for change). however, if the basal pd in one nostril was measured after amiloride exposure in the contralateral side, mean pd depolarized by mv (from - to - mv; p< . ), indicating the presence of a 'cross-nostril' amiloride effect that might potentially affect interpretation of this endpoint if not performed in a sequence designed to avoid amiloride exposure. in summary, we show that calomel and agcl electrodes perform similarly in repeated measures from the same normal subjects, and that improper npd sequence can adversely affect basal pd measures. other potential impediments to the pd measurement (e.g. solution temperature, tonicity, ionic content, and sources of electrical offset) will also be discussed. these findings are relevant to the optimizing the use of the npd assay in clinical trials. supported by nih, cff, and cfft. the gene defective in patients with cystic fibrosis (cf) is the cystic fibrosis transmembrane conductance regulator (cftr), a camp-activated chloride channel in the apical membrane of epithelia, but alteration of expression and function of many genes undoubtedly contributes to the pleiotropic manifestations of cf. a hallmark of cf in airway epithelia is the hyperabsorption of sodium through the epithelial sodium channel (enac) in the apical membrane followed by extrusion through the na,k-atpase in the basolateral membrane. this results in airway surface liquid (asl) dehydration, mucus buildup and bacterial infection characteristically observed in the lungs of cf patients. previous studies have shown that the na,k-atpase is increased in cf airway epithelia, consistent with increased sodium transport along the enac/na,k-atpase axis. we have previously demonstrated that fxyd , a member of a small family of proteins known to regulate the na,k-atpase, is increased in s x cf mouse lung and nasal epithelia and in primary human tracheal epithelial cells (hte) after treatment with an inhibitor of cftr ( ). recent evidence has indicated that enac activation occurs through modification of the serine protease-protease inhibitor balance in asl. thus, we hypothesized that enac-mediated increases in sodium absorption may be partially responsible for the increase in fxyd expression observed in cf airway epithelia. we now report that increasing asl volume on hte cells upregulated fxyd expression (p< . ) after hours, an effect that was further increased after trypsin addition and abrogated by the serine-protease inhibitor aprotinin. treatment of hte cells with nystatin also increased fxyd expression measured by quantitative rt-pcr (p< . ). based on these results and the observation that mice overexpressing enac beta-subunit exhibit cystic fibrosis-like lung pathophysiology, we examined fxyd expression in the lungs of mice overexpressing the scnn b-transgene. contrary to our expectations, fxyd lung expression was decreased as assessed by quantitative rt-pcr and immunoblot analysis (p< . ), indicating fxyd may be alternatively regulated in epithelia under conditions of acute versus chronic sodium hyperabsorption. therefore, we measured fxyd expression in an epithelial cell culture model using an inducible, tri-cistronic vector capable of expressing enac alpha, beta and gamma subunits. we determined that fxyd expression is decreased after chronic enac induction. we conclude that fxyd expression is coordinately regulated based on acute versus chronic enac-mediated sodium absorption and suggest that fxyd is increased in cystic fibrosis airway epithelia as a result of an imbalance between increased inflammatory signaling and altered ion transport. picher, m.; van heusden, c. cystic fibrosis center, university of north carolina, chapel hill, nc, usa airway infection by the respiratory syncytial virus (rsv) is considered an important cause of pulmonary exacerbation and hospitalization in cystic fibrosis (cf) children. this disease is characterized by functional mutations in the cf transmembrane regulator (cftr). the inability of cftr -/-mice to clear rsv is associated with exaggerated inflammatory responses. a relationship was recently established between airway inflammation and chronically-elevated airway adenosine. this signaling molecule regulates key aspects of lung defenses, including bacterial clearance and inflammatory cells. high adenosine levels measured in bronchoalveolar lavage fluid of asthmatic or cf patients cause airway inflammation, remodeling and fibrosis in animal models. the objective of this study was to investigate the longterm effects of rsv infection on adenosine regulation and inflammatory responses in human airway epithelia. methods. polarized primary cultures of bronchial epithelial cells from healthy subjects and cf patients were exposed to vehicle, uv-inactivated or active green fluorescent protein (gfp)-rsv. the cultures were monitored days for infection (fluorescence microscopy), adenosine levels (fluorescence chromatography), the release of cytokines regulating adenosine tnfα) and enzyme activities (ecto '-nucleotidase [ecto '-nt: amp Ç adenosine] and adenosine deaminase [ada : adenosine Ç inosine]). results. (a) both normal and cf cultures exhibited detectable rsv infection over days, with peak density on day - . (b) we demonstrate that rsv enhances the release of all five cytokines from normal cultures over days. in contrast, cf cultures responded to rsv by a transient decrease in il- β, il- and tnfα (peak on day ), but a sustained decrease in il- , and a sustained increase in il- , secretion. (c) on normal epithelia, rsv transiently stimulated ecto '-nt (peak on day ) but caused a sustained increase in ada activity, resulting in chronically-low adenosine levels. in cf, rsv raised adenosine levels by the combined effects of a sustained increase in ecto '-nt activity and the lack ada response. (d) chronically-elevated adenosine generated by ada inhibition [erythro -( -hydroxy- -nonyl)adenine] stimulated the secretion of all cytokines, except il- . conclusion. these data support a complex relationship between adenosine and cytokine regulation in human airway epithelia. the il- -adenosine amplification pathways identified in rsv-infected cf epithelia are in agreement with animal models and their key role in chronic airway inflammation/remodeling. on the other hand, the data also suggest that ada activity is influenced by il- , known to stimulate the expression of a major protein anchoring soluble ada to epithelial surfaces: cd . altogether, this study suggests that an rsv infection in cf children may accelerate the progression of the disease by setting the stage for airway adenosine-cytokine amplification pathways. supported by the cystic fibrosis foundation (picher g ). cystic fibrosis (cf) airway submucosal glands are defective in mucus secretion rate (jayaraman, pnas, , , ; joo, j biol chem, , , ; joo, pediatr. pulmonol., , suppl. , ; wine, proc ats, , , ; thiagarajah, faseb j, , , ; song, ajp cell, ; salinas, faseb j, , , ) . one hypothesis is that loss of cftr results in reduced secretion of electrolytes leading to underhydration of mucins, thereby altering the mucus properties. it has been generally believed that the cf mucus in response to cholinergic agonists is thicker and more viscous. however, comparisons of pure gland mucus are difficult because of variability in gland secretion rates and a decline in mucus solids with repeated stimulations (wu, pediatr. pulmonol. , suppl. , ) . in the present study, we directly compared the solid content of airway submucosal gland mucus between control and cf individuals. human donor tracheas (hn) or disease control bronchi (dc) were dissected and mounted as previously described but without mineral oil. tissue was kept warmed and humidified in a chamber with two layers of saturated degree c / % co /o water vapor. the mucosal surface was blotted dry and coated with a thin layer of dimethylpolysiloxane to prevent re-absorption by the surface epithelium. the tissue surface was covered with a cover slip, leaving minimal air space between it and the surface to further reduce evaporation. a µl droplet of % nacl was placed at the side of every tissue as a standard to assess evaporation and measurement error. any basal secretions were removed prior to stimulation with µm carbachol. mucus secretion from individual glands was pooled using forceps and then drawn into a constant bore microcapillary (drummond) that had been alcohol-cleaned, dried, and weighed. stimulation time was allowed to vary so that at least µl of mucus was collected. after mucus collection, both the standard and the bath solution were refreshed. mucus filled capillaries were weighed with a mettler microbalance before and after overnight degree c oven drying to obtain nonvolatile solid content. the average solids measured in six repeated measures of % salt solutions was ± . % with no trend over time. results for hn (n= ) and dc (n= ) did not differ and were pooled as 'control'. the initial control secretion yielded . ± . % solids (mean ± sem, n= subjects) and required ± min to produce adequate mucus. the initial cf secretion gave . ± . % solids (n= ) in ± min. the second control secretion yielded . ± . % solids (n= ) and required ± min, whereas the second cf secretion gave . ± . % solids (n= ) and required ± min. in summary, cf gland mucus solids content was % higher than control following the first stimulation (p = . ). after the second stimulation, cf solids content was % higher than the control (p = . ). solids contents of controls observed in our experiment are much higher than was found for mucus collected from airway lumens after hr stimulation (c. %, trout, am j physiol, , , l ) . supported by niddk ro - (jjw) and cff. in patients with cystic fibrosis (cf) as well as in some animal models of cf lung disease, higher levels of prostaglandin e (pge ) have been detected compared to control subjects. it is not clear whether this excess of pge contributes to the airway pathology of cf, or whether it may serve a protective function against airway constriction. inhaled pge has been tried in asthma and may be of benefit as a bronchodilator. interestingly, pge has shown benefit in aspirin-sensitive asthmatics, a population that shares some features with the cf airway phenotype including nasal polyposis. the mechanism of airway relaxation to pge is mediated primarily by ep receptors signaling through camp. in cf, beta-adrenergic bronchodilators are commonly used, but are often only partially effective at reversing bronchospasm. beta-agonists and pge both signal through g-proteins coupled to camp and pka signaling, thus pge may offer another treatment when beta-agonists are ineffective at reversing bronchospasm in cf. in mouse tracheas, the beta-agonist isoproterenol (iso) will partially relax a pre-contracted airway, but ± % of contractile force remains (n= ). in contrast to the limited relaxation with iso, constricted mouse airways demonstrate significantly greater relaxation to pge , with only ± % of force remaining (n= , p< . compared to iso). this difference in relaxation responses suggests different intracellular signals are activated by each agonist despite both signaling through pka. to investigate phosphorylation events in response to iso or pge , we developed a new method for perfusing isolated mouse lungs. using this technique, tissues were perfused with buffered solution containing p for radiolabeling. lungs were then stimulated with either iso or pge , and phosphoproteins were compared from each condition. radiolabeling was effective under all three conditions. targets were chosen from each group (unstimulated, iso, or pge treated) that showed phosphorylation or dephosphorylation, and proteins were identified by mass spectrometry. over differentially phosphorylated proteins were identified, and two of these (rho gdi and inositol-requiring protein ) were selected as candidates to be tested in the context of relaxation to pge compared to iso. by identifying signaling targets involved in airway relaxation to pge , it may be possible to activate beneficial airway relaxation responses without the limitations seen in iso relaxation and without activating pge responses other than relaxation that may link pge to the airway pathology seen in cf. the parental ∆f /∆f cf bronchial epithelial cell line (cfbe o-) was compared to clones of cfbe o-complemented with . kb wild type (wt) or . kb ∆f cftr cdna in terms of their ion transport characteristics. the transepithelial resistance (rt) (~ (cm ) observed in the parental cfbe o-was maintained in the complemented cells. two clones complemented with wt cftr cdna (c - . wt and c - . wt) demonstrated rt ~ Ωcm , while another clone (c - . ∆f) complemented with the ∆f cftr cdna showed an rt of ~ Ωcm . two stable clones expressing wt cftr were selected based on electrophysiological characteristics and stability of cftr expression. forskolin-stimulated cl secretion in clone c - . wt was on average . ± . µa/cm and . ± . µa/cm in clone c - . wt. furthermore small camp-stimulated transepithelial current of . ± . µa/cm was observed in clone c - . ∆f indicating that ∆f cftr can traffic to the plasma membrane if present at high enough levels at physiological temperatures. the role that cftr plays in ca-activated cl conductance was investigated following treatment of the cells with atp in parental cfbe o-and wtcftr . -cfbe o-monolayers. in symmetrical cl-containing solutions, mucosal application of atp ( µm) elicited a small, transient cl secretory response in the parental cfbe ocells ( . ± . µa/cm ), while the magnitude of the atp-stimulated cl current was markedly enhanced in cftr-corrected cfbe o-cells with peak currents at . ± . µa/cm . recordings done in presence of a serosal-tomucosal cl gradient to increase the driving force for cl secretion showed a transient atp-dependent activation of cl currents in parental cfbe ocells with peak cl currents of . ± . µa/cm (n= ). cftr corrected cfbe o-cells showed a sustained activation of cl currents of similar magnitude ( . ± . µa/cm ; n= ). atp-stimulated cl currents were effectively blocked by the cftr inhibitor glyh ( µm). histochemical analysis showed prominent epithelial characteristics in all clones. these observations indicate that i) the apical driving force is limiting for atp-stimulated cl secretion and that ii) cftr participates in the cl secretory response to atp. furthermore, the findings suggest that these novel clonal isolates of the cfbe o-bronchial epithelial cell line can be useful for the investigation of cf therapies. this work was supported by grants from the cff, cfri, nih, elizabeth nash foundation, and pacfi. the lactoperoxidase (lpo) system functions in normal airways to prevent infection and may be compromised in cystic fibrosis (cf) due to defective transport of its substrate thiocyanate. a computational model of normal airway surface liquid suggests that hydrogen peroxide levels regulate lpo system activity and that the lower thiocyanate in cf airways may result in elevated levels of hydrogen peroxide that is likely produced by duox the major nadph oxidase in airway epithelial cells. to investigate the regulation of duox by bacterial products, passage normal human airway epithelial cells were grown and redifferentiated at the air liquid interface and treated for , or h with either pseudomonas aeruginosa flagellin or lps, applied apically. treated and mock treated cultures were analyzed for levels of duox and lpo mrna and protein, and for hydrogen peroxide production. quantitative pcr was performed using taqman kits, protein was measured by western blots, and hydrogen peroxide was quantified using amplex red-hrp coupled fluorescence. the data showed the duox and lpo mrna but not duox mrna, were significantly upregulated at all measured time points following apical treatment of the cultures with purified flagellin compared to mock treated. at h duox was increased . +/- . fold and lpo was increased . +/- . fold (means +/-s.e.m., n = lungs, - cultures of each lung). muc ac mrna was also upregulated by flagellin treatment ( . +/- . fold, n = lungs, - cultures of each lung). apical treatment with lps had no effect on any tested mrna and induced minimal il- secretion in these cultures. western blots using anti-duox antibodies (courtesy of f. miot, brussels, belgium) showed corresponding increases in duox protein following flagellin treatments. hydrogen peroxide production by flagellin treated cultures was increased in proportion to increases in duox mrna. application of flagellin alone to the apical surface of previously untreated cultures did not increase hydrogen peroxide production. thus, pseudomonas flagellin up-regulates duox and results in higher activity in normal human airway epithelial cells perhaps serving to increase innate host defense activity of the lpo system. chloride ion in phagolysosomes is an essential substrate for neutrophils to produce hypochlorous acid (hocl), an important bactericidal agent. we have previously shown that cftr was present in the phagolysosomal membrane of neutrophils and the cftr defect led to deficient chlorination of ingested pseudomonas aeruginosa (pa) (painter et al, biochemistry, : - , ) . these results suggested that chloride accessibility to the phagolysosomal lumen might be impaired in cf neutrophils thereby affecting neutrophil-mediated bacterial killing. to understand the role of chloride ion in the process, we first assessed the effect of extracellular chloride concentration on pa killing by normal neutrophils under iso-osmotic conditions. surprisingly, bacterial killing was strongly dependent on extracellular chloride concentration. the initial rate of killing of pa was~ fold less in a chloride-free medium than a physiological chloride medium. killing efficiency increased as the chloride concentration was raised in a dosedependent fashion and plateaued at~ mm chloride concentration. to determine what percent of the chloride-dependent killing was oxidantdependent, we applied the cftr inhibitor glyh- , the myeloperoxidase (mpo) inhibitor aminobenzoic acid hydrazide (abah) and the nadph-oxidase inhibitor diphenylene iodium chloride (dpi), respectively to bacterial killing assays. the results showed that the killing of pa could be divided into three discernible components: ( ) a component accounting for about - % of the total killing rate that was inhibited by glyh- , abah or dpi; ( ) a second component amounting to~ - % that was chloride-dependent and partially sensitive to glyh- and; ( ) a third accounting for~ % of the total killing activity that was not sensitive to oxidant inhibitors, or extracellular chloride concentration. dpi and abah had no effect on killing of pa in the absence of extracellular chloride suggesting that most of the oxidant-mediated killing activity toward pa was due to hocl. none of the inhibitors had any significant effect on phagocytosis. finally, the rate of pa killing by cf neutrophils was found to be significantly lower ( - %) than that seen for normal neutrophils and was comparable to the effect of glyh- on killing of pa by normal neutrophils. the data suggest that cftr plays a significant role in killing of pa by normal neutrophils and, when defective as in cf, may compromise the ability of neutrophils to efficiently kill pa. this research was supported by a cff grant to gw. %. the cf group demonstrated a significantly higher frequency of pseudomonas respiratory infections than the non-cf group. interestingly, no significant differences were detected in any infections from other systems including blood, sinuses, skin, wounds, oral cavity, bowel, eyes, peritoneal cavity, and urinary tract. moreover, the cf lung-transplant patients had significantly less time free from pseudomonas infections in the transplanted lungs. conclusion normal lungs transplanted into cf patients had significantly higher susceptibility to pseudomonas infections than those transplanted into non-cf patients, which strongly suggests that defective host defense mechanism(s) beyond the respiratory system contribute to cf lung pathogenesis. pyocyanin (n-methyl- -hydroxyphenazine, pcn) is produced by pseudomonas aeruginosa and is found in pulmonary secretions of infected cf patients at concentrations up to µm. pcn is a redox-active compound that generates superoxide and hydrogen peroxide in the presence of cellular reductants. since cftr was reported to be modulated by h o , we hypothesized that pcn, through its ability to redox cycle and to produce h o , would affect cftr-mediated cl transport. we therefore tested pcn and h o for effects on cltransport (transepithelial short-circuit currents in ussing chambers measured with serosal-to-mucosal clgradient) and cytosolic redox potential (imaging microscopy on cells transfected with a redox sensitive gfp, rogfp ) using cftr-corrected cf bronchial epithelial cells (wtcftr . . under resting conditions, acute exposure of the apical epithelial surface to µm pcn elicited a small clsecretory response ( . ± . µa/cm , n= ) that was completely blocked by the cftr inhibitor glyh ( µm). in contrast, in presence of the camp-elevating agonist forskolin, pcn caused a time-dependent decline of forskolin-stimulated cftr clcurrents by %. exposure to h o ( µm) elicited a more pronounced clsecretory response than pcn in resting cells and h o -stimulated clcurrents peaked at . ± . µa/cm (n= ). h o was less effective than pcn at inhibiting forskolin-stimulated cftr clcurrents and currents declined by %. both pcn and h o oxidized the cytosolic redox potential from a resting value of - mv by similar amounts ( ± mv for pcn vs. ± mv for h o ), but rates of oxidation were faster for h o ( . ± . mv/min) than for pcn ( . ± . mv/min). inhibition of camp-stimulated cftr clcurrents by oxidation was a linear function of the cytosolic redox potential, but pcn ( µa×cm - ×mv - ) was more potent than h o ( . µa×cm - ×mv - ). cf bronchial epithelial cells homozygous for ∆f -cftr did not show a clsecretory response to pcn or h o . these data suggest that cftr-corrected cf bronchial epithelial cells respond acutely to oxidative stress by turning on cftr activity probably as part of the innate host defense response and this mechanism is absent in cf airways. furthermore, prolonged exposure to pcn and h o is detrimental for the proper function of the camp-regulated cftr clconductance. we conclude that oxidative stress in p. aeruginosa-infected airways is a key factor that needs to be considered for successfully correcting cftr function in cf airways. supported by nih (nccam p at ), cff (fischer g ) the lung is continually exposed to environmental agents and pathogens. the lung epithelial lining fluid (elf) is first to encounter these agents and gsh is a major antioxidant found in this apical fluid. the cystic fibrosis transmembrane conductance regulator protein (cftr) is the only known gsh transporter that maintains lung elf gsh levels. cystic fibrosis (cf) patient have low levels of gsh in their elf and have copious and viscous mucus. hypertonic saline is used in cf patient to help clear mucous and improve lung function however the mechanism(s) by which it does so are poorly understood. the purpose of these studies was to examine whether hypertonic ( . %) saline can modulate apical gsh levels and protect lung epithelial cells against the oxidant, t-butyl hydroperoxide (t-booh). we used human lung epithelial cell lines sufficient (c ) and deficient (ib ) in cftr. the cftr deficient ib cells have % lower basal levels of apical gsh as compare to cftr sufficient c cells. cells were exposed separately and in combination to % saline, and t-booh ( um) for hours. the cftr deficient ib cells were much more sensitive to t-booh-mediated oxidative injury as measured by lactate dehydrogenase (ldh) release. hypertonic saline exposure was associated with an increase in apical gsh levels in both cftr sufficient and deficient cells and decreased t-boohmediated oxidative injury in both cells lines. hypertonic saline increased gsh in the apical compartment, which appeared to be largely cftr mediated. we examined this issue in mice given a clinically used dosing regimen of % hypertonic saline nebulized twice daily and examined changes in elf gsh levels hours after last treatment. mice receiving the hypertonic saline treatment had significantly higher gsh levels in their elf than untreated controls. this data suggests that cftr and gsh adaptive responses play an important role in lung's reaction to oxidants. we propose that factors, which interfere with the lung's capability to mount and maintain an adequate adaptive apical gsh response, may compromise and sensitize the lung to oxidative injury. (supported in part from nih grant hl ). the absence of functional cftr in airway epithelia of cf patients leads to abnormal airway surface liquid, which favors chronic bacterial infection. this chronic infection in cf lungs is associated with an exaggerated inflammatory response characterized by abnormal cytokines secretion and massive lung neutrophils infiltration. we have previous recapitulated the abnormal inflammatory response in cftr-/-(cf) mice by repeated administration of pseudomonas aeruginosa lipopolysaccharide (lps) (ped. pulmonology, supp. , ) . we demonstrated that the bronchoalveolar lavage (bal) fluid of cf and heterozygous (het, cftr+/-) littermate control mice when compared to the bal from wt mice shows ) higher numbers of neutrophils and ) higher concentrations of cytokines (il- alpha, il- , kc, mcp- , gm-csf, m-csf) as assessed using multiplex analysis of selected cytokines. of note, airway epithelial cells from cf patients are known to have elevated secretion of some of these cytokines. however to date, the contribution of immune cells to these abnormal cytokine levels has not been well characterized. this study aims to investigate the contribution of cf macrophages in this altered cytokine profile/response. we examined cytokine secretion from two different macrophage populations: alveolar macrophages (am) obtained from the bal fluid and bone marrow (bm)-derived macrophages differentiated in presence of m-csf. both macrophage populations were cultured in the presence or absence of lps, and the supernatants were tested for the concentration of the cytokines that we had shown to be abnormal in vivo. these macrophage populations were examined from cf, het and wt mice. in absence of lps exposure, bm-derived macrophages from cf mice have lower levels of il- and kc compared to wt and het mice. in contrast, am of cf mice have higher levels of il- and kc than am of wt mice. this difference may be due to am's prior exposure to antigens in vivo whereas the bm-derived macrophages are a truly naïve population. interestingly, after lps stimulation, il- a, il- , mcp- , kc, g-csf and gm-csf are higher in cf macrophages compare to wt and het mice suggesting that cf affected macrophages display an altered inflammatory response when compared to wt and het. the abnormal production of these cytokines was also detected at the transcriptional level using quantitative rt-pcr analysis. in conclusion, our data support the hypothesis that cftr-null macrophages may have an impaired cytokine profile at baseline as well as after stimulation. furthermore, cftr deficient macrophages may contribute directly to the abnormal inflammatory response in cf. (supported by cff, nih, niddk) cystic fibrosis lung disease is associated with an excess of free neutrophil elastase (ne) in the airway arising from persistent neutrophil inflammation. dx- is a potent small protein inhibitor of neutrophil elastase that could be a useful therapy for diseases involving neutrophilic inflammation such as cystic fibrosis and copd. dx- effectively inhibits ne activity in cf sputum sol (ic = pm). however the physical barriers posed by cf whole sputum may prevent dx- accessing and inhibiting ne ensconced within the mucus. the mucolytic dnase acts by cleaving extracellular dna in mucus thereby reducing viscosity. however dna binds and inactivates ne and therefore dnase treatment of mucus results in an acute increase in active ne. the lipid portion of pulmonary surfactant acts as a lubricant and our group has previously shown that bovine lung extract surfactant (bles) increases the fluidity of whole sputum. ( aim we aimed to show that, by reducing the surface tension of sputum, bles would enhance the anti-elastase effect of dx- in whole sputum. methods whole sputum ( . g per well in a -well plate) was pre-incubated with µl saline as control, µl dnase ( µg/ml) or µl bovine lung extract surfactant (bles, µg/ml) for minutes at °c followed by addition of µl dx- ( µm or µm) for h at °c. the contents of each well were aspirated, added to ml nacl ( . %) and centrifuged at , g for min. the resulting sputum sol was assayed for ne activity using the colorimetric substrate n-meosuc-aapv-pna. results ne activity in whole sputum was significantly inhibited by µm dx- (p= . ) and µm dx- (p= . ). µm dx- inhibited significantly more ne when sputum was pre-treated with bles (p= . ) treatment of sputum with dnase caused a significant increase in ne activity compared to untreated sputum (p= . ) whereas treatment of sputum with bles did not have this effect. conclusion dx- effectively inhibits elastase in cf sol. pre-treatment of cf whole sputum with bles enhanced the anti-elastase effect of dx- and treatment of sputum with bles alone did not cause an increase in ne activity in contrast to treatment with dnase. dx- inhibitory capacity in whole sputum could be improved by co-treatment with surfactant which increases fluidity of sputum and could allow greater access of drugs to sputum components. bovine lung extract surfactant, bles™ was a kind gift from bles biochemicals inc., on, canada. dx- was a kind gift from dyax corp, ma who also funded part of this research. table : results are summarised as neutrophil elastase activity µg/ml and show the mean and sem of n= experiments. cystic fibrosis lung disease is associated with an excess of free neutrophil elastase (ne) in the airway arising from persistent neutrophil inflammation. dx- is a potent small protein inhibitor of neutrophil elastase that could be a useful therapy for diseases involving neutrophilic inflammation such as cystic fibrosis and copd. dx- effectively inhibits ne activity in cf sputum sol (ic = pm). however the physical barriers posed by cf whole sputum may prevent dx- accessing and inhibiting ne ensconced within the mucus. the mucolytic dnase acts by cleaving extracellular dna in mucus thereby reducing viscosity. however dna binds and inactivates ne and therefore dnase treatment of mucus results in an acute increase in active ne. pulmonary surfactant acts as a lubricant and our group has previously shown that bovine lung extract surfactant (bles™) increases the fluidity of whole sputum. ( we aimed to show that, by reducing the surface tension of sputum, bles™ would enhance the anti-elastase effect of dx- in whole sputum. methods whole sputum ( . g per well in a -well plate) was pre-incubated with µl saline as control, µl dnase ( µg/ml) or µl bovine lung extract surfactant (bles™, µg/ml) for minutes at °c followed by addition of µl dx- ( µm or µm) for h at °c. the contents of each well were aspirated, added to ml nacl ( . %) and centrifuged at , g for min. the resulting sputum sol was assayed for ne activity using the colorimetric substrate n-meosuc-aapv-pna. results ne activity in whole sputum was significantly inhibited by µm dx- (p= . ) and µm dx- (p= . ). µm dx- inhibited significantly more ne when sputum was pre-treated with bles™ (p= . ) treatment of sputum with dnase caused a significant increase in ne activity compared to untreated sputum (p= . ) whereas treatment of sputum with bles did not have this effect. conclusion dx- effectively inhibits elastase in cf sol. pre-treatment of cf whole sputum with bles™ enhanced the anti-elastase effect of dx- and treatment of sputum with bles alone did not cause an increase in ne activity in contrast to treatment with dnase. dx- inhibitory capacity in whole sputum could be improved by co-treatment with surfactant which increases fluidity of sputum and could allow greater access of drugs to sputum components. we would like to thank prof. f. possmayer, on, canada and bles biochemicals inc., on, canada for the bovine lung extract surfactant, bles™. dx- was a kind gift from dyax corp, ma who also funded part of this research. table results are summarised as neutrophil elastase activity µg/ml and show the mean and sem of n= experiments. cf patients infected with p. aeruginosa (pa) have increased levels of proinflammatory cytokines, such as tnf-α, il- β, il- , and il- ; but have low levels of the anti-inflammatory cytokine, il- in their airways. the proinflammatory mediators in the cf airway recruit a large number of polymorphonuclear neukocytes (pmns), which release high levels of neutrophil elastase and human neutrophil peptides and damage the lungs. the intense neutrophil-dominated inflammatory response associated with chronic pa infection results in progressive airway obstruction, the cause of death of over three-quarters of cf patients. recent studies highlight the significance of sphingolipid metabolism in cell growth, differentiation, membrane traffic, apoptosis, senescence, and immune regulation. acid sphingomyelinase (asm) plays a critical role in sphingolipid metabolism by hydrolyzing sphingomyelin to ceramide. ceramide self-associates to form ceramide-enriched membrane platforms(also called lipid rafts). these platforms have been shown to be involved in the internalization of pa; induction of apoptosis and immune regulation. our in vitro and in vivo studies of asm confirm that there is a loss of induction of asm after pa infection in both cf bronchial epithelial cells and cftr knock out mice as compared with controls. in vitro, we examined the asm activity in human bronchial epithelial cells with normal cftr expression (s cells) and with mutant cftr expression (ib cells ) after pa infection at , , , , , and min. asm activity was up-regulated in s cells following pa infection after min and kept increasing until min, while asm induction was lost in ib cells. asm protein and ceramide levels also increased in s cells following pa infection compared with ib cells. in our in vivo studies, cftr knock out mice and control c bl/ mice were both administered various doses of pa orally ( x cfu to x cfu) . six hours after pa infection, lung homogenates from the c bl/ mice indicated increasing asm protein and ceramide production, which corresponded to the dose of pa given. in contrast, asm protein and total ceramide production remained at baseline levels in the lung homogenates of cf mice regardless of the dose of pa given. rna interference (sirna) assays were performed by transfecting a bp pre-designed sirna specific for human asm in s cells. these cells were then infected with pa for hrs to see if they had similar il- level compared with infected ib cells. we demonstrated a significantly elevated il- expression in s cells tranfected with asm sirna compare with s cells transfected with a scramble sirna (p< . ). our results suggest that the loss of asm induction, caused by mutant cftr, is involved in pa clearance and contributes to the exaggerated proinflammatory cytokine response in cf airway. we are conducting research on defining the roles of asm and the sphingolipids in pa infection; cell signaling; as well as cytokine induction, we are also looking at the therapeutic potential of aav.asm vectors in pa-infected cf bronchial epithelial cells and cftr knock out mice. work supported by nhlbi introduction: new therapies are critically needed to treat cystic fibrosis (cf) lung disease, which is characterized by chronic infection and inflammation. novel therapies to target airway inflammation are likely to improve the clinical outcome in cf. as yet there is no consensus regarding the molecular pathway(s) mediating inflammation in the cf lung. objective: to establish whether toll-like receptor (tlr) signalling is the key molecular pathway responsible for the inflammation seen in cf lung disease and to determine whether inhibitors of tlr signalling can reduce this damaging inflammatory response. methods and results: to quantify the airway inflammatory response, we used established cf and cf-corrected lung epithelial cells (ib - and c ). since artefacts may alter the inflammatory phenotype of cell lines, we validated all findings with fresh peripheral blood mononuclear cells (pbmcs) from cf patients (n= ) and healthy controls. we measured the inflammatory response (il , il , tnfα) of these cells following stimulation with: (i) cf pathogens (p. aeurginosa, b. multivorans, b. cenocepacia); (ii) purified tlr ligands. both cf respiratory epithelial cells and cf pbmcs had a greatly increased inflammatory response compared to matched controls when stimulated with whole bacteria and purified tlr ligands (p< . ). interestingly, the cf airway cells responded most vigorously to the tlr ligand, flagellin (p< . ). to investigate tlr as a potential anti-inflammatory target, we found that tlr mrna (p< . ) and protein (p< . ) were expressed at a higher level in cf compared to cf-corrected lung epithelial cells. finally, to determine the contribution of tlr activation to cf lung inflammation we exposed the airway cells to wild-type (pak & pao ) and isogenic flagellin-deficient strains of p. aeruginosa (pak∆flic, pao ∆flic, pao ∆flge & pao ∆flim) . strikingly, the absence of the tlr -flagellin interaction significantly reduced the proinflammatory response of cf respiratory epithelial cells to p. aeruginosa (p< . ). we show that tlr-mediated innate immune responsiveness is increased in both cf respiratory epithelial cells and fresh blood cells from cf patients. moreover, inhibition of the tlr -flagellin interaction markedly reduced proinflammatory response of cf respiratory epithelial cells following exposure to predominant cf pathogen p. aeruginosa. these data suggest that tlr activation may represent a novel anti-inflammatory target for improving cf lung disease. supported by the canadian cf foundation. cf airway cells (ib - ) were stimulated with wild-type (pak) and flagellin-deficient p. aeruginosa (pak∆flic). introduction: genetic polymorphisms for tgf-β have recently been identified as a significant modifier of cf lung disease severity. however, at present little is known about protein measurements of tgf-β in bronchoalveolar lavage fluid (balf) or serum obtained from pediatric cf patients during an acute respiratory exacerbation. hypothesis: tgf-β in cf balf and serum is increased in association with markers of cf lung disease severity, and is decreased after completion of iv antibiotic (iv abx) therapy. study design and methods: in a descriptive, prospective study of children with cf presenting for hospital admission to treat a pulmonary exacerbation, balf specimens were obtained from patients undergoing clinically-indicated flexible bronchoscopy on admission. balf was immediately analyzed for total and differential cell counts with the cell-free balf supernatant stored at - °c. serum specimens were obtained at the initiation and conclusion of iv abx and similarly stored at - °c. measurement of total tgf-β utilized a commercial elisa kit. t-test statistical analysis assumed significance at p<. . results: balf was obtained from cf patients with a mean age of . ± . years (range - years). three balf cultures were pseudomonas aeruginosa positive (psa+). balf tgf-β levels ranged from - pg/ml (mean ± . pg/ml) with the highest tgf-β measured in a patient with abpa and culture positive for m. abscessus. in balf, higher neutrophil counts (greater than the median, . x pmn/ml) were associated with increased balf tgf-β ( ± . pg/ml vs. ± . pg/ml, p<. ). serum tgf-β was collected in cf patients with a mean age of . ± . years (range - years), four of whom were psa+. serum tgf-β ranged from . - . ng/ml (mean . ± . ng/ml). in both balf and serum, neither patient age nor psa culture status was associated with increased tgf-β at admission. however, previous hospitalization in the last months was significantly associated with an elevated admission tgf-β in both balf ( ± . pg/ml vs. ± . pg/ml, p<. ) and serum ( . ± . ng/ml vs. . ± . ng/ml, p<. ). after completing the clinically-indicated course of iv abx, / psa-cf patients had a reduction in serum tgf-β (mean . ± . ng/ml to . ± . ng/ml, p=ns). in contrast, / psa+ cf patients had an increase in serum tgf-β (mean . ± . ng/ml to . ± . ng/ml, p=ns) after completing iv abx therapy. conclusions: tgf-β is measurable in balf and serum from pediatric cf patients and varies in association with clinical parameters. these preliminary results support our hypothesis that increased tgf-β may be associated with more severe lung disease as evidenced by ) association of increased tgf-β in balf and serum obtained from cf patients with previous hospitalization in the last months, and ) association of increased tgf-β in balf with increased cellular inflammation. the direction of change in serum tgf-β after iv abx therapy may be influenced by the presence or absence of pseudomonas infection. acknowledgments: supported by cff (harris ao). paula murphy and cassidy henegar provided technical support. background: cystic fibrosis is one of the leading genetic causes of end stage lung disease for which the treatment is lung transplantation. however, mortality due to chronic rejection known as obliterative bronchiolitis (ob) remains high due to small airway obliteration. ob is associated with increased levels of matrix metalloproteinase- (mmp- ), an interstitial collagenase, expressed by polymorphonuclear cells (pmns). pmns are among the first inflammatory cells to be detected in ob but very little is known about the role of pmn mmp- or the mechanisms used by pmns to migrate through extracellular collagen matrices. objective: to identify the molecular mechanism for neutrophil recruitment into the airway. we hypothesize that mmp- promotes accumulation of pmns in ob lesions by promoting their migration through the extracellular matrix protein barriers by its pericellular collagenase activity. method: the heterotopic airway transplant model was used to study the role of neutrophil mmp- in promoting pmn migration through extracellular matrices. mhc-disparate balb/c tracheas were subcutaneously transplanted into c bl/ wild-type (wt) or mmp- deficient (mmp- -/-) mice. to further dissect the mechanism of pmn migration in vitro their migration through collagen gels was studied. collagen gels were made in transwells using rat-tail collagen providing a highly cross-linked thick collagen barrier. bone marrow derived pmns from wt or mmp -/-mice were fluorescently labeled and stimulated to induce the surface expression of mmp- . pmns were placed on thick collagen gels in traswells. the transwells had polycarbonate membranes at the bottom of the gels. the pore size of the membranes did not allow pmns to pass through into the lower chambers that had flmp a chemo attractant. at hours and hours the gels were fixed and the polycarbonate membrane at the bottom of the collagen gel was gently peeled off. pmns on the membrane were counted under a fluorescent microscope. results: significantly decreased migration of pmns into the airway lumen was noted in the mmp- deficient mice (p< . ) weeks post-transplantation compared to wt mice in the heterotopic airway transplant model. in addition there was more than a three-fold increase in the pmns outside the lumen in the extracellular collagen matix (p< . ). wt-pmns were able to penetrate collagen barriers in greater numbers both at and hours compared to mmp -/-pmns in vitro (p < . for hours and . for hours). in addition, mmp inhibitor gm -treated wt cells demonstrated decreased migration through collagen gels ( hours p< . and hours p< . ). conclusions: mmp- -/-mice has significantly less pmns in the airway lumen due to decreased migration through collagen matrices providing protection against ob. this has great significance for conditions such as cystic fibrosis, lung transplant rejection (obliterative bronchiolitis) and other inflammatory diseases mediated by pmns. mmp- blockade can potentially be a novel therapeutic target to decrease neutrophil efflux into the airways and thus reduce airway inflammation. background: our laboratory has demonstrated alterations in cf-related regulation of cholesterol processing. hydroxycholesterols (oxysterols) are oxygenated derivatives of cholesterol formed from autoxidation of cholesterol and from de novo synthesis. oxysterols are known to be key regulators of cholesterol metabolism and cellular signaling. the role of oxysterols in the cellular pathways involved in cholesterol processing in cystic fibrosis has not been elucidated. based on our previous findings of increased de novo cholesterol synthesis in cf tissues, we hypothesize that oxysterol production in cf would be positively impacted. increased oxysterol production may lead to abnormalities in cellular signaling associated with oxidative stress and may influence the inflammatory response in cf. the goals of this research are to investigate the effect of oxysterols on inflammation in cf and to determine if there is a difference in oxysterol production in cf compared to wild type controls. results: the goal of these studies is to determine if cf cells respond uniquely to oxysterols compared to control cells. initial studies demonstrate that cf-model /hteo-pcepr cells failed to respond to -oh cholesterol ( -oh) with regards to nfκb activation. control /hteo-pcep cells, however, exhibited a significant -fold increase in nfκb activity upon -oh stimulation ( . -fold +/- . increase from il- , tnf alpha alone). however, il- promoter activation in response to -oh was not significantly altered in either cf or wt cells ( . -fold +/- . (cf) increase from il- , tnf alpha alone, . -fold +/- . (wt) increase from il- , tnf alpha alone). in order to verify il- promoter results, il- protein production was measured directly in response to pseudomonas aeruginosa. il- protein level in response to -oh was increased in cf cells ( . -fold) and decreased in wt cells ( . -fold), although not significantly in either group. oxysterol content and de novo synthesis are currently being examined in vivo in cf and wild type mouse models. mice lacking cftr on a c bl/ background and wild type controls were obtained from our animal core and the amount of oxysterol present in these mouse models is currently being studied. conclusion: these findings suggest that endogenous oxysterol production or other components of oxidative stress are pre-stimulating nfκb activation via the oxysterol-sensitive pathway. interestingly, although oxysterols robustly activate nfκb in wt cells, oxysterol-dependent pathways are not involved in il- production. these data suggest that il- is not a primary cause for increased oxysterol mediated inflammation. elucidating the role of oxysterols in cf will be an important contribution to our understanding of the inflammatory pathways in cf and will contribute to the growing body of evidence for aberrant cholesterol regulation in cf. this work is supported by grants from the cff and the nhlbi. cystic fibrosis (cf) is a common genetic disorder caused by a mutation in the cftr, a chloride transporter of the abc transporter family. innate immunity, the first line in host defense against infectious agents, is abnormal in cf patients. cf patients are commonly colonized with bacterial pathogens, which is the major clinical problem of this disease. despite a marked difference in the clinical outcome amongst cf patients infected with pseudomonas aeruginosa and burkholderia cepacia, the mechanisms under-lying these differences remain unclear. understanding the innate immune response, specifically the role of cftr, in p. aeruginosa and b. cepacia infections will lead to new insights into mechanisms that limit these infections. our objective is to develop a cf model in the simple organism, the worm, c.elegans, and to test the role of the cftr orthologue, mrp- , in host defense. using this model, we have compared the survival of c. elegans between virulent p.aeruginosa (pa ) and b.cepacia (bc). our experiments have demonstrated that, when compared to growth on their natural food source, e.coli op , worms fed on pa and bc die prematurely. specifically, worms survive up to hours on op , whereas pa fed worms die with in hours. death on pa also requires active infection, as heat inactivation of these bacteria destroys their killing capacity. two bc strains have been tested, a virulent strain ( ) and an avirulent strain ( ). worms infected with bc show an intermediate phenotype, surviving approximately hours. our future work will define whether synergy occurs during coinfection with p.aeruginosa and b.cepacia, as would be suggested by clinical data. in addition, we will define the transcriptional changes induced in c.elegans fed on pa and bc, and use these data to identify common and pathogen specific responses to these infections. finally, mrp- knock-down worms will be generated using rna interference, and the role of the cftr orthologue in infection will be tested. in conclusion, c.elegans offers a genetically tractable model organism in which the innate immune response to pseudomonas aeruginosa and burkholderia cepacia can be studied. in addition, this simple model system will allow us to define the role of the cftr orthologue in host defense. supported serine proteases released from neutrophils are considered central to the pathogenesis of cf lung disease, and have been obvious therapeutic targets. although intracellular serine proteases are important in host defense and bacterial killing, extracellular enzymes may contribute to bacterial persistence and promote airway inflammation. neutrophil elastase (ne) digests key opsonins present in the lung and has been shown to disrupt phagocytosis, allowing the bacteria to persist in the face of established pulmonary inflammation. in addition we have found that other proteases, like cathepsin g (cg), an abundant serine protease found in human and murine neutrophils, may also have other, critical roles in development of cf lung disease. indeed, using murine models of endobronchial inflammation, cg inhibits airway defenses and interferes with the host's ability to clear pseudomonas aeruginosa from the lung with effects quite distinct from ne. to test the hypothesis that differences in bacterial killing are due to defects in innate defenses created by excess, unopposed cg at the apical surface of the airway epithelium, we have examined profiles of proteins secreted into the airway lumen by epithelial cells, which are necessary for p. aeruginosa clearance and susceptible to cg proteolysis. analysis of lavage fluid utilizing -d page indicated a total of spots which were increased in pooled lavage fluid from infected cg-deficient mice as compared to wild-type littermates. tandem mass spectrometry analysis identified several novel proteins that were cleaved by cg, including inter-alpha inhibitor protein, selenium-binding protein, sec -like protein, beta- -glycoprotein, and annexin a . another protein candidate, serum amyloid p component (sap), is a novel opsonin and may be involved in airway defenses. immunoblot analy-sis demonstrates that cg cleaves sap. these results suggest that cg, like ne, plays an important role in the pathogenesis of lung diseases. we have previously demonstrated in ∆f -cf mice that in vivo treatment with azithromycin (azm) attenuates cellular infiltration in baseline and lipopolysaccharide (lps)-induced inflammation, and inhibits proinflammatory cytokine release in induced inflammatory condition (legssyer et al, ) . this study aimed at investigating macrophages isolated from wild type (wt) or ∆f -cf mice (van doorninck et al, ) and the effect of azm on these cells. purified peritoneal and alveolar macrophages were stimulated with lps (p. aeruginosa, . µg/ml) plus ifnγ ( . µg/ml), with or without azm ( µg/ml). macrophage inflammatory status was investigated by assessing different pro-and anti-inflammatory mediators at mrna level h after stimulation. pro and anti-inflammatory markers seemed to be reduced in naive alveolar cf macrophages. by contrast, a trend to overexpression of these markers was seen in naive peritoneal cf cells. pro-inflammatory status, as assessed by il- β, was induced in stimulated alveolar and peritoneal cf macrophages and in naive peritoneal cf cells. moreover, nos- was found to be overexpressed in stimulated alveolar cf macrophages while il- was downregulated. azm significantly reduced il- β expression in stimulated alveolar macrophages. in conclusion, peritoneal and alveolar macrophages exhibit distinct phenotype. a pro-inflammatory status was more prominent in stimulated cf alveolar and peritoneal cells. interestingly, azm modulates il- β overexpression only in cf stimulated alveolar macrophages, supporting the antiinflammatory activity of this macrolide and identifying, at least partly, alveolar macrophages as possible target cells for its effects. supported it has been recently reported by perez and coll. that the inhibition of function of w/t cftr produces an inflammatory profile that resembles that observed in cf patients [am j physiol lung cell mol physiol, ] , whereas we demonstrated that correction of f del cftr function with mpb- down modulates the pseudomonas aeruginosa (p.aeruginosa) dependent expression of the pro-inflammatory mediators il- and icam- in cf bronchial cells [am. j. resp. cell. mol biology, ] . since both evidence support a direct link between cftr function and inflammatory response in respiratory epithelial cells, we extended our investigation to other f del cftr correctors, such as miglustat [norez et al, febs letters, ] , which is an approved drug for gaucher disease, in comparison with an isomer without any correcting effect, namely nb-dgj. human bronchial ib - (cftr f del/w x), cufi- (cftr f del/f del) and nuli- (cftr w/t) cells were exposed to the laboratory strain of p.aeruginosa (pao ) or tnf-alpha or il- beta and the transcription of icam- and il- were quantitated by real time rt pcr. cftr function was assayed by single-cell fluorescence imaging, using the potential-sensitive probe disbac [renier et al, hum. gene ther. ]. analysis of binding of nf-kb and ap- transcription factors to labelled dna target was performed with electrophoretic mobility shift assay (emsa) [borgatti et al, j. biol. chem. ]. miglustat significantly reduced the expression of il- by % in ib - and by % in cufi- cells and of icam- by % in ib - and by % in cufi- cells, upon infection by pao . in parallel, correction of f del cftr function was observed after miglustat treatment in both ib - and cufi- cells. miglustat had no major effects on overall binding activity of transcription factors nf-kb and ap- , activated by pao in ib - cells. the inflammatory response induced by tnf-alpha or il- beta was also significantly reduced in ib - and cufi- cells treated with miglustat. nb-dgj, which is not a corrector of function of f del cftr, down modulated the induction of il- and icam- in ib - and cufi- cells. in addition, both miglustat and nb-dgj reduced the inflammatory response to pao in non cf nuli- cells. in conclusion, miglustat and nb-dgj have an anti-inflammatory effect in bronchial cells independently of the correction of f del cftr, by interfering with the pro-inflammatory signaling downstream the receptors for pathogens and pro-inflammatory cytokines. since miglustat is already approved for the treatment of gaucher disease and other glycosphingolipidoses, it may be an interesting candidate to ameliorate lung inflammation in cf patients. background: the primary cause of mortality and morbidity in cystic fibrosis (cf) is lung disease, which is characterized as a chronic cycle of infection and inflammation dominated by a neutrophilic infiltrate and increased levels of circulating mediators such as il- . a wide variation in the rate of progression of lung disease is observed in cf. hypothesis: we postulate the presence of a brisk systemic inflammatory response in cf patients and that this systemic response reflects the magnitude and progression of lung disease. methods: lung function and clinical data were obtained from cf patients treated and followed in the adult cf clinic, st paul's hospital. forced expiratory volume at s (fev ) and forced vital capacity (fvc) values dating back to were used in regression equations to group patients into quartiles based on their decline in predicted fev %. rate of decline in lung function was compared with clinical characteristics and various systemic inflammatory biomarkers, including wbc, band cell count, crp, il- β, il- , il- , mcp- , and gm-csf results: the quartiles exemplified significantly different rates of decline in fev /fvc% values. subjects with a rapid decline in lung function were younger and were colonized/infected more commonly with methicillinresistant staphylococcus aureus (mrsa) (p< . ). in addition, altered calcium, magnesium, and albumin levels were found in subjects with increased rates of decline in lung function (p< . ). preliminary results show that patients with cf have significantly elevated levels of wbc ( . e /l), crp ( . e ng/ml), and il- ( . pg/ml) in serum when compared to control subjects (p< . ). there were trends in relationships between levels of il- , il- and mcp- and rates of decline in lung function, however none reached statistical significance. conclusion: there is a variable rate in the decline in lung function among cf patients and one factor may be a heterogeneous systemic inflammatory response. circulating pro-inflammatory mediators may not only impact progression of lung disease, but could also be novel biomarkers to monitor and assess disease severity in cystic fibrosis. nutritional status in addition effects the rate of decline in lung function perhaps by modifying the inflammatory response. this research is supported by the bc lung association. overexpression of the epithelial na + channel β subunit (protein = βenac, gene = scnn b) in transgenic mice results in cf-like lung pathology, characterized by neonatal mortality, mucus obstruction and airway inflammation (mall et al., nat. med. ) . breeding βenac mice into gene-deleted mice enables quick and efficient determination of the specific pathways relevant to the development of lung pathology. tnfα is a pleiotropic pro-inflammatory cytokine released by many different cell types, including t cells, macrophages, granulocytes, and epithelial cells. tnfα levels are significantly elevated in bronchoalveolar lavage (bal) from βenac mice in comparison to wild-type (wt) littermates. we crossbred βenac mice (heterozygous, inbred line c - ) with tnfα knockout mice, generating four types of mice: wt, tnfα +/-; wt, tnfα -/-; βenac, tnfα +/-; and βenac, tnfα -/-. all mice had comparable, high survival, ranging between - %. the lack of tnfα did not prevent neutrophil and eosinophil infiltration and did not significantly modify the lung pathology characteristic of βenac mice, namely mucus plugs, mucous secretory cell hyperplasia, airway inflammation and emphysema. the absence of tnfα in bal from βenac, tnfα -/-mice, in comparison to βenac, tnfα +/mice, was confirmed by luminex cytokine assay. the levels of kc, a potent neutrophil chemoattractant, were significantly elevated in bal fluid from βenac mice, regardless of the presence or absence of tnfα. in wt mice, no granulocytic infiltrate or morphologic changes were observed and tnfα and kc were undetectable. similarly, the pathologic changes in βenac mice were not mitigated by crossbreeding to knockout mice for tnfα receptor (tnfαr ), the major mediator of biologic responses classically attributed to tnfα. these results suggest that tnfα per se does not have a critical pro-inflammatory role in the development of inflammation in the βenac mouse model. the production of alternative cytokines may compensate for the loss of tnfα bioactivity in the tnfα and tnfαr -/-mice. since tnfα has been suggested to play a role in the regulation of enac, ussing chamber studies are underway to test whether the bioelectric features of tracheobronchial epithelia from βenac mice are altered by the absence of tnfα in vivo. these data suggest that a tnfα-independent signaling cascade links airway surface dehydration to airway inflammation in the βenac mouse model. innovative pharmacological approaches to control the excessive neutrophil infiltrates into the bronchial lumen of cf patients are thought to be beneficial to reduce the extensive airway tissue damage. the activation of expression of proinflammatory genes by p.aeruginosa with bronchial epithelial cells is a central mechanism to be targeted with novel therapies. medicinal plants from the socalled traditional asian medicine are attracting a growing interest because of their potential safety, already tested in large scale applications in human diseases. however, due to the presence of different active principles in each plant extract, whose multifunctional effects may even result contradictory, understanding the effect of each component is mandatory to pursue selective and reproducible applications. a panel of medicinal plant extracts have been firstly screened for their capacity to interfere in the binding of nuclear transcription factor proteins (tf) with dna consensus sequences identified in the promoters of the pro-inflammatory genes, thus for their potential inhibitory action on gene expression. extracts from several medicinal plants have been screened for their ability to interfere with the tf nf-kb, ap- and creb, which are induced by p.aeruginosa, and some of them, such as emblica officinalis (eo), aegle marmelos (am), polyalthia longifolia (pl), have been shown to inhibit tf/dna interactions, opening the possibility of potential applications to downregulate expression of pro-inflammatory genes. extracts from eo, am, pl were tested in ib - cf bronchial cells exposed to the p.aeruginosa laboratory strain pao . eo, am and pl strongly inhibited the pao -dependent transcription of il- in ib - cells. pyrogallol, one active principle of eo, was tested in ib - cells, where it inhibited the transcription of the neutrophil chemokines il- , gro-alpha and gamma, of the intercellular adhesion molecule icam- and the pro-inflammatory cytokine interleukin , similarly to the whole eo extract, whereas a second active principle from eo, namely hydroxy-isoquinoline, had no effect. similar results were obtained with eo and pyrogallol in the monocyte-derived macrophage cell line thp- exposed to pao . in conclusion, extracts from plants of the traditional medicine can inhibit expression of pro-inflammatory genes and screening active principles purified from medicinal plants could result useful to identify safe and innovative pharmaceutical molecules to control lung inflammation in the lung of cf patients. supported by italian cystic fibrosis research foundation and by fondazione cariverona -bando -malattie rare e della povertà. massive infiltrates of neutrophils in the mucosal wall and lumen of the conductive airways of cf patients contribute to the progressive lung func-tion decline by releasing different proteases responsible for the progressive airway tissue damage. bacterial products and pathogens themselves within the mucopurulent material of the airway surface fluid induce the activation of transcription factors such as nf-kappab, ap- , sp , nf-il , nf-at, elk- , creb resulting in expression of chemo/cytokine genes driving the recruitment of leukocytes inside the bronchial lumen. to find new treatment options focused on the reduction of neutrophil chemotaxis, without abolishing the immune response against pathogens, we are exploring the transcription factor (tf) "decoy" strategy, in which oligodeoxynucleotides (odn) mimicking the consensus sequences for the tfs proteins identified in the promoters of different chemo/cytokines are delivered inside the cell in order to interfere with gene transcription. cf bronchial epithelial cells ib - have been exposed to the (i)p.aeruginosa(/i) strain pao . a clear pao -dependent activation of tfs such as nf-κb, sp , ap- , nf-at, nf-il was confirmed by electrophoretic mobility shift assay (emsa). in parallel, transcription of genes involved in innate immune response has been quantified by real-time rt pcr. transcription factor "decoy" odns directed against the consensus sequences identified in the promoters of different genes have been designed and validated by testing their interference in tf protein/dna binding assays. transfection of ib - cells with hiv- ltr and igk chain nf-κb odn "decoys" complexed with lipofectamine, performed hrs before challenge with pao , was shown previously to inhibit strongly pao -dependent transcription of il- but not gro, il- β, il- and icam- . therefore other tf "decoy" odns have been also tested: a) odn for nf-κb from il- promoter inhibited il- , gro-gamma and il- by %, b) odn for sp from hiv- genome inhibited il- by %, c) odn for ap- from il- promoter inhibited both il- and gro-gamma by %. a tf "decoy" molecule designed as peptide-dna chimera mimicking the consensus sequence of hiv ltr nf-κb strongly and selectively inhibited il- transcription. in conclusion, transcription of chemo/cytokines induced by (i)p.aeruginosa(/i) in cf bronchial epithelial cells (i)in vitro(/i) can be inhibited with different efficiency and selectivity by tf "decoy" molecules. these results provide useful hints for a gene-targeted anti-inflammatory approach and add further information on the regulation of expression of pro-inflammatory genes induced by (i)p.aeruginosa(/i) in bronchial epithelial cells. supported by italian cystic fibrosis research foundation and by fondazione cariverona -bando -malattie rare e della povertà. background: cf is characterized by hypersecretion of the pro-inflammatory cytokine il- from airway epithelial cells. however the mechanism by which il- gene expression is dysregulated in cf is not known. the expression of cytokine and chemokine genes is known to be regulated at multiple mechanistic steps including transcription, mrna decay, translation, and various post-translational steps. sequence-specific mrna degradation is now recognized to be an important site controlling the expression of several chemokine mrnas. this selective behavior is conferred by cisacting elements in the mrna composed of au-rich sequences (ares). the regulatory function of ares is thought to be mediated via rna-binding proteins that specifically recognize the are motifs. the steps required for mrna decay is comprised of deadenylation, decapping and body decay. deadenylation or removal of the poly (a) tail by poly(a)-specific ribonuclease (parn) appears to be the first and perhaps the rate-limiting step. this is accompanied by decapping or enzymatic removal of the ' methylate guanosine cap. subsequently, exonuclease activities in the ' to ' or ' to ' direction predominate to degrade the remaining mrna. hypothesis: we have hypothesized that defects in transcription as well as modulation of the post-transcriptional stability of il- mrna might contribute to hyper-production of il- protein in the cystic fibrosis. methods: il- mrna stability was assessed in cystic fibrosis ib - lung epithelial cells and in aav-cftr-repaired ib - /s cells, by measuring residual il- mrna levels at various intervals of time after addition of actinomycin-d to the culture. protein s extracts, prepared from the cf cell line as well as the cftr-corrected cell line, were analyzed by western blot. the expression levels of the various factors known to participate in are-mediated mrna decay, including ttp (an are-binding protein), parn and the exosome were compared in the two sets of s extracts. results: we find that the levels of il- mrna in the cf cells are greater than the levels found in the cftr-repaired cells. in addition, we found that the rate of decay of il- mrna in cf cells was significantly less than that in the repaired cell line. the levels of ttp, parn and exosome proteins were significantly reduced in the cf cells compared to the cftrrepaired controls. however, expression of ttp in ib - cells causes significant destabilization of il- mrna. conclusion: we conclude that the high levels of il- protein expression in cf lung epithelial cells can be partly due to enhanced stability of the il- mrna. consistently, the actual levels of il- mrna are higher in the cf cells compared to controls. understanding the mechanism by which ttp promotes enhanced destabilization of il- mrna in cf cells may be important for developing novel therapeutic targets to alleviate the pulmonary pathophysiology of this disease manifested by hyper-secretion of il- protein. understanding the mechanism of airway remodeling could lead to the identification of novel therapeutic targets for the prevention of irreversible lung damage in cf. we have investigated transcriptional responses to epithelial injury in a mouse model of cf that was developed in our laboratory (f del, cftr tm eur fvb backcross f ). to induce transient epithelial lung injury, homozygous normal and mutant age matched littermates were treated in parallel with naphthalene ( mg/kg ip) or carrier (corn oil) as control (n= x ). naphthalene causes an almost complete selective ablation of clara cells overnight, followed by migration of resistant ciliated cells and rapid proliferation of undifferentiated progenitor cells. two and seven days after treatment, lungs were collected for histological and transcriptome analysis. based on a previous affymetrix microarray analysis (n= ), a quantitative pcr array was designed containing genes differentially expressed after naphthalene injury. as expected, naphthalene injury results in a transient increase of cell proliferation markers (mki , cdc , cdc a etc), and a strong ( %) reduction of clara cell markers (cc /scgb a ; claudin /cldn ; cyp f ). in addition, we have now identified twenty-five genes that are increased (p< . ) two-to fifty-fold after naphthalene injury in both normal and mutant mice. this includes known and novel markers of epithelial tissue injury such as timp , retlna, mmp , serpin n, and lipocalin. in particular, a group of egf receptor agonists is strongly induced: amphiregulin (areg, fold), epiregulin (ereg fold) and heparin binding egf ( fold). amphiregulin mrna can be detected by in situ hybridization only in airway epithelial cells after injury. these egfr agonists are involved in stimulating epithelial repair, but can also activate cells in the underlying mesenchyma. indeed, we observe a substantial increase of major extracellular matrix mrna's two days after injury (co a : -fold, col a : -fold, elastin: -fold). whereas expression levels were substantially reduced in normal animals seven days after injury, in cf mutant animals two to three fold higher levels were observed for these three genes at day seven (p< . ). conclusions. we have designed and validated a gene array that can be used to study distal lung injury and repair. egfr agonists produced by epithelial cells are the most prominent growth factors after injury, involved in both epithelial repair and extracellular matrix production by mesenchymal cells. ecm production in cf mouse lung is sustained compared to normal mice, suggesting an inherent tendency towards fibrosis in cf lungs. our data suggest that the mesenchymal egf receptor and regulation of its agonists are important future targets of novel therapeutic strategies. supported by eec th fw projects eurocarecf, improved precision the mucosa of the proximal airways defends itself and the lower airways from inhaled irritants, allergens, and microbial and viral infections by several mechanisms. sensory nerves monitor the luminal microenvironment and trigger reflexes in the central nervous system (cns) that alter breathing, induce cough and stimulate mucus secretion when challenged with noxious stimuli. sensory nerves also release the tachykinins substance p (sp), neurokinin a and calcitonin gene-related peptide through axon reflexes in neighboring tissues, and these locally released tachykinins stimulate mucus secretion by binding to neurokinin receptors on submucosal glands. recently we reported that local fluid secretory responses to noxious stimuli are dependent on the clchannel cftr in mouse airway submucosal glands and are defective in glands from cftr knockout mice (ianowski et al., j physiol, , , ) . we have now tested the effects of sp directly and examined the possible role of cftr in mediating these responses using tracheas from congenic wild-type and cftr knockout mice (cftr m unc /cftr m unc ) that had been bred onto c bl/ j or balb/c genetic backgrounds. we compared single gland secretion rates using optical methods as described previously. the cftr genotype of each mouse was assessed by using pcr to amplify genomic dna from tail clippings obtained at age days. after the cftr knockout mice were weaned, intestinal obstruction was minimized by supplementing their water with peglite. capsaicinoids (chili pepper oil) increased fluid secretion from glands of wild-type mice from ± pl/min (n= tracheas, glands) to ± pl/min (n= tracheas, glands, anova p< . , tukey-kramer multiple comparison test p< . ). this response was abolished by exposing the basolateral surface of the tracheas to l- , ( µmol/l), a known sp (nk- ) receptor antagonist ( ± pl/min, not different from control, tukey-kramer multiple comparison test p> . ). secretion was stimulated from ± pl/min to ± pl/min (n= tracheas, glands, student's t-test p< . ) by the direct application of sp, and this response was strongly inhibited by pre-incubation with the cftr inhibitor cftrinh (saturating concentration, nominal µmol/l; n= tracheas, glands). finally, submucosal glands from cftr knockout mice failed to secrete when exposed to sp ( µmol/l) whereas wild-type littermates were responsive. these results indicate that sp mediates local responses to capsaicinoids through a cftr-dependent mechanism. loss of this local regulation in cf may contribute to the susceptibility of cf airways. support: canadian cf foundation, canadian institutes of health research, nih (dk ), and the cff (usa). chronically infected/inflamed cf human bronchial epithelia (hbe), or normal hbe exposed to supernatant from mucopurulent material (smm) from human cf airways, exhibit expansion of the endoplasmic reticulum (er) ca + stores and amplification of ca + -mediated inflammatory responses. we have shown that infection/inflammation of hbe triggers an unfolded protein response (upr) coupled to mrna splicing of x-box binding protein- (xbp- ). spliced xbp- (xbp- s) is a transcription factor that promotes er expansion to augment the protein folding capacity during increased protein synthesis. because we have shown that hbe inflammation couples to increases in protein synthesis, we hypothesized that hbe infection/inflammation-induced er ca + store expansion is mediated by xbp- s. to test this hypothesis, we constructed four retrovirally-tranduced stable hbe o-cell lines containing empty vector (ev), xbp- unspliced (xbp- u), xbp- s, or dominant negative xbp- (dn-xbp- ). cells were grown to confluence under an air-liquid interface and er ca + store expansion and il- secretion studied in the absence or following hr mucosal pbs or smm exposure. consistent with our hypothesis, expression of xbp- s in the absence of stimulation induced er expansion and increased mucosal utp-sensitive er ca + stores ( to confirm that these effects were mediated by upr activation, cells were transfected with a upr response element luciferase reporter plasmid, and luciferase activity was measured in the absence or presence of the upr inducer tunicamycin (tm). cells expressing xbp- s exhibited higher baseline luciferase activity than cells expressing ev or xbp- u, whereas luciferase activity was decreased in the dn-xbp- expressing cells. furthermore, while tm increased luciferase activity in cultures expressing ev or xbp- u, its effect was blocked in dn-xbp- expressing cells. these findings suggest that xbp- s is the major trigger of er ca + store expansion, which mediates the amplified ca + -dependent inflammatory response in infected/inflamed airway epithelia. although it remains to be established whether upr-dependent xbp- s is a beneficial or a maladaptive response in infected/inflamed airways, therapies aimed at manipulating this upr pathway may be beneficial for patients with chronic inflammatory lung diseases. luminal exposure of well-differentiated normal human bronchial epithelia (hbe) to supernatant from mucopurulent material (smm) from human cf airways increases the secretion of inflammatory mediators and triggers an unfolded protein response (upr) mediated by the mrna splicing of the x-box binding protein- (xbp- ). spliced xbp- (xbp- s) is a transcription factor that expands the endoplasmic reticulum (er) and protein secretory pathway during er stress induced by accumulation of unfolded proteins due to increases in protein synthesis. our previous studies in primary cultures of hbe and hbe o-cells stably expressing empty vector, inactive unspliced xbp- (xbp- u), xbp- s or a dominant negative xbp- (dn-xbp- ) suggest that induction of xbp- s by smm exposure promotes er ca + store expansion, which mediates a ca + -dependent hyperinflammatory response. the present studies were designed to determine if pseudomonas aeruginosa pak strain (p.a.) would reproduce the effects of smm in both in vitro and, importantly, in vivo models. for in vitro studies, hbe were exposed to broth (tsb) or to a % p.a. extract for hr, and inflammation, xbp- s, and er ca + store expansion investigated by il- secretion, southern blots and calreticulin expression, respectively. similar to smm, p.a. induced a . fold increase in il- secretion coupled to a fold increase in xbp- s and larger er ca + stores as compared with tsbexposed hbe. these data demonstrate the link between p.a. infection, upr activation and xbp- s in vitro. we next tested the relevance of these findings in vivo. wild-type mice airways were challenged with pbs or cfu of p.a., and airway epithelial er density was assessed by calreticulin staining hr later. in comparison with pbs challenges, p.a.-challenged mice exhibited airway epithelial er expansion, which correlated with the degree of airway inflammation, based on the presence of inflammatory cells. to test whether this p.a.-induced er expansion was linked to xbp- s, we utilized "er stress activated indicator" (erai) mice expressing a fusion protein consisting of xbp- u and the fluorescent protein venus. in erai mice, upr activation-dependent xbp- splicing leads to venus expression; hence, venus fluorescence is an index of xbp- s. consistent with our hypothesis that p.a. infection-triggered inflammation would induce upr-dependent xbp- s, venus fluorescence, after hr challenge with cfu of p.a., was increased in inflamed as compared with non-inflamed airways. these findings suggest that ) airway epithelia respond to bacterial infection-induced inflammation by up-regulating the er ca + stores and ) activation of the xbp- s pathway by bacterial infection may be relevant to airway inflammatory responses in vivo. funded by the cff. we have shown that luminal exposure of well-differentiated primary cultures of normal human bronchial epithelia (hbe) to supernatant from mucopurulent material (smm) from human cf airways increases total cellular protein synthesis, which reflects the increased secretion of inflammatory factors induced by the infectious and inflammatory process. in the present studies, we first investigated whether these hbe responses to smm were linked with an increased metabolic rate by measuring lactate accumulation into the serosal media. smm increased lactate production, and this effect was maximal within hrs ( . + . vs. . + . , and . + . vs. . + . mmol/l in and hr pbs vs. smm, respectively; n= - ), suggesting that the increase in protein synthesis couples to a hyper-metabolic state in infected/inflamed hbe. in agreement with this notion, or hr smm, as compared with pbs exposure, induced the expression of genes associated with amino acid transport and metabolism (n= ). in addition, or hr smm exposure up-regulated genes involved in oxidative stress (n= ). we hypothesized that these hbe responses were linked to an unfolded protein response (upr) mediated by activation of the pkr-like er kinase/pancreatic eif α kinase (perk)-induced activating transcription factor (atf ), since this pathway has been shown to confer protection against amino acid loss and oxidative stress in other cells. we first tested whether smm induced activation of perk/atf in hbe by performing western blot analyzes of the components of this pathway. twenty-four hr smm exposure induced perk activation, as indexed by phosphorylation of perk, in comparison with pbs-exposed hbe. on the other hand, total perk protein levels were unchanged in smm-treated hbe. phosphorylation of eif α, the downstream effector of perk, and increased atf protein levels, which depend on the phosphorylated status of eif α, provided additional evidence that the perk/atf pathway was activated by smm. these data are consistent with the hypothesis that induction of atf is triggered by upr activation resulting from increased synthesis of inflammatory factors. we next utilized rna microarrays to test whether atf target genes were induced by smm. six or hr smm exposure induced atf target genes (e.g., ero , an oxido-reductase that provides protection against the accumulation of endogenous peroxides during er stress; stanniocalcin , whose expression is associated with anti-apoptotic functions; and heme oxygenase ). these findings suggest that ) activation of the upr-dependent atf pathway is a compensatory component of the airway epithelial adaptive response to luminal infection/inflammation, and ) activation of the atf pathway protects against inflammation-induced amino acid loss and oxidative stress by up-regulating genes involved in amino acid transport/metabolism and oxidative stress responses. unraveling the functions of atf should help determine if therapies targeted to manipulate pathway activity would be likely to improve lung function in patients with cf or other chronic inflammatory airway diseases. funded by the cff. miller, t.j.; perez, a.; qian, y.; davis, p. pediatrics, case western reserve university, cleveland, oh, usa fxyd is a cell surface protein originally identified in a screen for molecular markers of tumorigenesis. increased fxyd expression was found in tumors from stomach, thyroid, colon, pancreatic, breast and lung cancers and correlated with down-regulation of e-cadherin and poor patient prognosis. recent studies have shown that overexpression of fxyd promotes cell motility, decreases cell-cell attachment and increases tumor metastasis. fxyd , also known as dysadherin, is a member of small family of proteins known to regulate the na,k-atpase. we now report that fxyd is upregulated in cystic fibrosis (cf) airway epithelia and modulates wound healing. we show by immunohistochemistry and immunoblot analyses that fxyd is increased in the lungs of s x cf mice, and demonstrate an almost -fold increase in fxyd expression in the nasal epithelia of cf mice compared to wild-type littermates (p< . ). furthermore, we show that fxyd is upregulated in nasal scrapings from human cf patients compared to controls (p< . ). immunofluorescence data show that flag-tagged fxyd co-localizes with the na,k-atpase in epithelial cells, suggesting that fxyd , similar to other members of the fxyd family, may regulate na,k-atpase function. it has previously been shown that expression and localization of the na,k-atpase is required for efficient polarization and suppression of cell motility in epithelial cells. the recurrent remodeling of pulmonary epithelium as a result of bacterial infection in cf requires that airway epithelial cells polarize and migrate to wound sites in order to maintain lung integrity. thus we hypothesized that fxyd may be involved in wound healing after infection. laser-capture microdissection and microarray analysis of murine lung epithelia after hours treatment with p. aeruginosa indicated a significant, -fold increase in expression of fxyd that was confirmed by immunoblot analysis. others have shown that fxyd may mediate expression of mcp- , a critical determinant in monocyte recruitment, through activation of the nf-kb pathway. treatment of human tracheal epithelial (hte) cells with a cftr inhibitor ( ) confirmed that loss of cftr function correlated with increased fxyd expression by quantitative rt-pcr (p< . ), an effect that was abrogated with treatment of pdtc, an inhibitor of nf-kb (p< . ). we speculated that fxyd -induced increases in cell motility may be due in part to phosphorylation at serine . in a murine airway epithelial cell wound healing model, serine to alanine (s a) mutations at serine inhibited wound healing compared to wild type fxyd overexpression, whereas aspartic acid (s d) mutations increased wound healing (p< . ). immunoblot and immunofluorescence analyses of these mutants suggest phosphorylation at ser regulates membrane localization. we conclude that fxyd is increased in cystic fibrosis epithelia due to increased inflammatory mediators and suggest that fxyd may modulate airway epithelia wound healing after infection with p. aeruginosa through phosphorylation at ser . the inflammatory response to bacterial infection in the cf airway is exaggerated compared to normal, leading to the accumulation of millions of necrotic neutrophils. extracellular neutrophil elastase (ne) activity in the cf airway not only compromises innate defences by cleaving opsonins and reducing ciliary activity, but amplifies the inflammatory response by stimulating expression of the neutrophil chemoattractant il- and increases mucus production, in addition to degrading the tissue matrix leading to fatal bronchiectasis. ne therefore represents an important target for the development of new therapies. however, this strategy requires consideration of the normal physiological function of this enzyme, since previous studies in knock-out mice indicated an essential role for ne in cell migration, bacterial phagocytosis and killing. our approach was to use the intracellular neutrophil elastase inhibitor gw a to 'knock-out' ne activity in neutrophils in normal human blood and test the function of isolated cells in chemotaxis, bacterial phagocytosis and killing assays. whole normal human blood was incubated with gw a, or pbs control, for h at °c. neutrophils were isolated by sedimentation of red blood cells (rbc) on dextran , purification on lymphoprep and hypotonic lysis to remove contaminating rbc. chemotaxis towards il- was measured using a modified micro-boyden chamber. phagocytosis was assayed by the depletion of staphylococcus aureus (sa), pseudomonas aeruginosa (pa) and e. coli (ec) in supernatants following culture in a : ratio with pmn for h at °c. supernatants were diluted and remaining organisms were plated on agar and grown overnight to count viable colonies. bacterial killing was assayed by incubating bacteria and cells in a : ratio for minutes to allow phagocytosis, washing off remaining organisms, and incubating for h at °c. neutrophils were lysed with water and lysates plated on agar to test for bacterial growth overnight. results; gw a inhibited intracellular ne dose-dependently and at µm . ± . % ne activity remained (n= ). there was no significant effect of µm gw a on neutrophil chemotaxis, bacterial phagocytosis or killing of any organism compared to pbs-treated controls. phagocytosis data is shown in the table. thus, in the absence of ne activity, human neutrophils remain wellequipped with other defence molecules including myeloperoxidase and defensins to successfully maintain the role of the neutrophil in innate immunity. however, mouse neutrophils which lack defensins require ne activity for optimal intracellular bacterial killing, and mice are not a perfect model for studies of human infection. the development of novel inhibitors of ne to treat lung disease in cf therefore remains an important goal. supported by the cf trust of great britain. background: sphingolipid signalling may differ between individuals with cf and healthy controls. the response to bacterial inflammation is different, and uptake and inactivation of sphingosine- -phosphate, an intracel-lular pro-inflammatory mediator, is reduced in cf cells. it may therefore continue to act on g-protein coupled receptors in the plasma membrane. (boujaoude et al, j biol chem ) . furthermore, ceramide originating from basolateral sphingomyelin hinders augmentation of cftr-mediated anion conductance across the apical membrane, resulting in reduction of transepithelial airway anion secretion (ito et al, bbrc ) . aim of study: to determine if there is a difference in the levels of alkaline, neutral or acid sphingomyelinase (smase), or in the levels of neutral or acid ceramidase, in the intestinal or bronchial mucosa and some other tissues, between wildtype, homozygous (+/+) and heterozygous (+/-) delta-f cftr mice. methods: enzyme activities (duan and nilsson meth enzymol ) were determined in intestine (and content) divided into four regions, liver, lungs, kidney and spleen from deltaf -cftr mice (+/+) and controls (wildtype, +/-). results: there was an increased amount of neutral ceramidase in spleens from deltaf -cftr mice (+/+) in comparison to control mice (p= . ). no other significant differences were seen. conclusion: delta-f mutation did not influence the levels of alkaline smase and neutral ceramidase acting as ectoenzymes, or the levels of intracellular smases and ceramidases, which may all generate bioactive sphingolipid metabolites in intestine and lungs. the implications of the increased level of neutral ceramidase in spleen are not known. in children with cystic fibrosis (cf) there is a clear correlation between the development of chronic p aeruginosa infection and acceleration in the decline of lung function. when chronically present, p aeruginosa takes on a mucoid phenotype and is impossible to eradicate. prior to this, when colonisation is intermittent, it is possible to eradicate it with aggressive antibiotic regimes. we sought to examine the degree of inflammation and innate defence status in the lungs of children with cystic fibrosis in various stages of colonisation by looking at a range of proteases, innate defence proteins and markers of inflammation in broncho alveolar lavage (bal). children with cf were allocated to one of three groups in relation to p aeruginosa infection; chronically colonised, intermittently colonised and non-colonised, on the basis of the leeds criteria. bal was collected as per ers guidelines as part of each patient's routine clinical care. bal was collected from control patients undergoing elective non-pulmonary surgical procedures. differential cell counts in bal were performed manually. secretory leukocyte protease inhibitor (slpi), elafin, alpha- antitrypsin (a at) and lactoferrin concentrations were measured by elisa. neutrophil elastase activity and cathepsin activity were assayed by colorometric activity assays. fifty two patients were included in the study ranging in age from months to years ( chronic, intermittent, non colonised and controls). neutrophil counts, neutrophil elastase activity and cathepsin activity were markedly increased in children chronically colonised with p aeruginosa compared to those in the intermittent and non colonised groups. in contrast, levels of the antiproteases slpi and a at and the antimicrobial peptides elafin and lactoferrin were highest in the control group and decreased as colonisation progressed, with levels in the chronically colonised group markedly lower than those with intermittent colonisation. this study demonstrates that in children with chronic p aeruginosa colonisation, there is a marked decrease in antiproteases and antimicrobial factors and a marked increase in protease activity and neutrophil influx in comparison with those who are non-colonised or intermittently colonised. these findings underline the importance of careful microbiological surveillance and early aggressive treatment of p aeruginosa infection in children with cf in order to avoid chronic colonisation. background : abnormal bronchial angiogenesis is responsible for hemoptysis in cystic fibrosis (cf). expression of vegf-a in airway epithelium induces bronchial angiogenesis in animal models. we have recently found that vegf-a and egf receptors (egfr) are increased in the airway epithelium of subjects with advanced cf lung disease. aims: to examine the effects of pa bacterial products and egfr inhibition on vegf synthesis in airway epithelium. methods: culture of non cf (nci-h ) and cf (cfte o-) human airway epithelial cell lines. stimulation with pa lipopolysaccharide (lps). assessment of vegf mrna and protein by rt-pcr and elisa. use of chemical inhibitors, blocking antibodies and sirna. results: pa lps increased vegf gene expression and protein production time-and dose-dependently in both cells lines. using chemical inhibitors, we show that egfr and erk / activation are required for lpsinduced vegf production. using blocking antibodies to egfr and its ligands, we show that tgf-alpha-dependent egfr activation mediates pa lps-induced vegf gene and protein synthesis. using pharmacological inhibitors (an ros scavenger and an nadph oxidase inhibitor) and using small interfering rna of dual oxidase (duox) and tnf-alpha converting enzyme (tace),we show that lps-induced vegf upregulation is dependent on duox -mediated ros release and tace activation. thus, pa products induce vegf synthesis in airway epithelium via a duox -ros-tace-tgf-alpha-egfr-erk / cascade. conclusions: these results describe a novel pathway by which bacterial products induce angiogenic signaling in cf and non cf airway epithelium. background: unlike bronchoalveolar lavage (bal), the airway mucosa has been under-investigated in cystic fibrosis (cf), despite the fact that irreversible airway wall changes (bronchiectasis) are a feature of endstage disease. cf is characterized by a neutrophil-dominated inflammation in bal, but little is known about the pattern of inflammation in the airway mucosa, especially in children with relatively early stage disease. we aimed to assess whether the pattern of inflammation seen in cf bal was also found in the airway mucosa in cf children. methods: to date, endobronchial biopsies and bal from children ( - years) with cf and control children ( - years) without lower respiratory disease have been assessed. bal cell differential was assessed on may-grünwald-stained cytospins. endobronchial biopsies were stained for neutrophils (neutrophil elastase, ne), t-(cd ) and b-(cd ) lymphocytes, eosinophils (eg ), and macrophages (cd ). area profile counts of immunopositive cells in subepithelial tissue were performed by investigators blinded to disease group. results: all cell types were increased in cf bal compared to controls. cf bal was characterized by an abundance of neutrophils ( x /ml vs. x /ml in controls, p< . ) with moderate numbers of lymphocytes ( x /ml vs. x /ml in controls, p< . ). in contrast, cf subepithelial tissue was characterized by a lymphocytic infiltrate ( cells/mm vs. cells/mm , p< . ) with only very few neutrophils ( cells/mm vs. cell/mm , p< . ). the lymphocytic infiltrate in cf consisted mainly of t lymphocytes ( %). eosinophil counts in subepithelial tissue did not differ between cf and controls. for all cell types, there was no correlation between counts in bal and counts in subepithelial tissue. conclusions: in contrast to the neutrophil-dominated inflammation in the airway lumen, cf is characterized by a lymphocytic inflammation in the airway mucosa. the lymphocytic infiltrate consists mainly of t lymphocytes, the pathophysiological function of which may be important and is being investigated in future work. support: ers long-term fellowship and swiss national foundation grant to nr the epithelium serves as a barrier to the penetration of foreign antigens, particles, and infectious agents across the airway. the integrity of this barrier is dependent, in part, upon the apical junctional complex consisting of the tight junction (tj) and the adherens junction. alterations in tj permeability have been linked to the pathogenesis of inflammatory bowel disease and this increased intestinal permeability may actually precede the onset of chronic inflammation. in cystic fibrosis (cf), the airway lumen is filled with high concentrations of inflammatory cells, bacteria, and inflammatory mediators. since tj barrier function can be significantly reduced by inflammatory mediators, we hypothesized that measures that enhance airway tj barrier function will decrease airway responses to the continuous presence of inflammatory mediators in the lumen. to test this hypothesis, we examined the relationship between lung inflammation and epithelial permeability in vivo using a lipopolysaccharide (lps) model of lung inflammation. pseudomonas aeruginosa lps was instilled intratracheally into the lungs of c bl/ mice which were then euthanized at , and hrs. lung inflammation was assessed by total cell counts using a hemacytometer and differential counts by wrights staining of cytospin preparations of the bronchoalveolar lavage fluid (balf). lps increased total cell counts and neutrophil concentrations that peaked at hours after lps administration, compared to saline controls. measurements of the proinflammatory murine cytokine kc in balf, by a cytokine antibody bead technique, showed an increase in murine kc at hr following lps administration, which correlated with the substantial increase in neutrophil concentration. changes in lung permeability with inflammation were assessed by elisa measurements of the levels of serum protein murine albumin in balf. correlating with changes in cellular inflammation and murine kc levels, albumin concentration peaked at hr after lps administration. this increase was subsequently resolved, consistent with the restoration of barrier function. an examination of frozen sections of lung from lps-treated animals showed a redistribution of the tight junction protein zo- consistent with the disruption of barrier function. since the p map kinase signaling pathway has been implicated in lps-induced airway inflammation, and an inhibitor of this kinase, sb has been shown to reduce this inflammation, the effect of this inhibitor on barrier function is being investigated. in initial studies, sb appears to reduce total and neutrophil cell counts by % in vivo. the effect of sb on murine albumin concentrations in balf and on tj protein localization is currently being evaluated. a reduction in these parameters will be used as indices of improved barrier function with sb . these studies will determine whether a reduction in lung inflammation correlates with a restoration of barrier function. the degree of protection provided by the p map kinase inhibitor sb could have important implications for inflammatory lung diseases such as cf. background: chronic pulmonary inflammation in cf is characterized by a robust neutrophil response associated with airway damage and failure to eliminate the pathogen, p. aeruginosa (pa). pa is a highly adaptable opportunist which quickly develops resistance to antimicrobials. thus, the development of specific immunotherapy targeting the neutrophil recruitment without ablating the host's immune response to infection or promoting pathogen resistance would be ideal. our group has identified il- as a prime target for the development of immunotherapy due to the central role that the il- /il- proinflammatory axis plays in neutrophil recruitment. however il- does not mediate the early neutrophil recruitment seen in response to infection. hypothesis: il- , acting synergistically with il- , is critical to early neutrophil recruitment during pulmonary pa infection. the primary effector cells are the il- -producing antigen presenting cells: alveolar macrophages (ams) and myeloid dendritic cells (dcs). methods: wt and il- -deficient mice were infected with pa at x cfu/ ul by intratracheal (it) inoculation for hours. bal inflammatory cell counts, and cytokines and chemokines were measured. am and dc cultures were infected for hours in vitro and supernatant cytokines/chemokines were measured by luminex and elisa; il- levels were measured by taqman. these studies were designed to elucidate the role of il- in the early neutrophil peak and define am-and dc-mediated cytokine/chemokine production. recombinant murine il- , il- , and il- + il- were instilled via it into wt and il- -deficient mice. bal inflammatory cell counts and cytokines/chemokines were measured at hours. these studies were designed to elucidate the role of il- and il- in the early neutrophil peak and define am-and dc-mediated cytokine and chemokine production. results: at hours post-infection, il- deficient mice had significantly lower percent neutrophils (p< . ) and lower mip α, kc, and il- (p< . ) in the bal. il- was undetectable. there was no significant difference in bacterial load that could account for these cytokine/chemokine differences. infected wt ams elaborated significantly more mip a, gm-csf, mcp- , il- , g-csf, ip- , kc and il- than the il- -deficient ams (p< . ) and the response was inoculum-dependent (p< . ). dcs elaborated no il- and exhibited il- -dependent differences in mip- α and mcp- production (p< . ). in vivo studies of il- and il- effect demonstrated a synergistic increase in bal neutrophil recruitment (p< . ) and cytokine and chemokine induction (p< . ). conclusion: the first wave of neutrophil recruitment seen during pa infection is il- -dependent and il- -independent. ams and dcs are critical to il- and il- indcution of this neutrophil recruitment. these studies identify il- as a key mediator of neutrophil recruitment in the early stages of infection as well as the proximate mediator in the il- /il- pro-inflammatory axis and suggest il- as a potential target for anti-inflammatory therapy in the treatment of pa pulmonary infection. supported by the cystic fibrosis foundation, american lung association and the nih the airways are under constant assault from air-borne pathogenic material. despite the intake of up to , bacteria per hour, the airways are sterile below the larynx in healthy individuals. the task of maintaining this sterility falls to the airway surface liquid layer (asl), the protective twophased system consisting of the viscoelastic mucus layer and the periciliary layer (pcl) through which cilia beat, sweeping the mucus layer away from the lungs. the mucus layer, which is responsible for trapping pathogenic material, is comprised of mucins (high molecular weight glycoproteins), cellular debris, dna, neurtifils, and more than other proteins. this chemically heterogeneous mixture forms a viscoelastic gel that is thick enough to trap pathogens of various sizes and surface chemistries, while not sticking to the underling cellular / cilia layer, allowing the transport of trapped pathogenic materials away from the lungs. the performance of this trapping / transportation system is defined by the rheological properties of the mucus layer and the force imparted on the mucus. therefore, understanding how mucins and other chemical components of the mucus layer interact with each other to form a successful mucus gel (i.e. one that is cleared from the airways) is crucial to understanding airway defense. here we present the results of physical and chemical composition studies of sputum samples collected from patients with chronic obstructive pulmonary disease (copd) and cystic fibrosis (cf). the rheological properties of each sample was assayed using parallel plate rheolometry, probing the materials non-linear viscoelastic properties such as viscosity, elasticity, and yield stress. the physical properties of the sample are then correlated to the sample's chemical properties such as percents solids (divided between salts, proteins, and mucins), as well as the relative concentrations of the key airway mucins muc b and muc ac. our results indicate that the physical properties of sputum are not well predicted from the total amount of biosolids in a given sputum sample, but by the relative concentrations of muc b and muc ac and the interactions of these molecules with themselves and the other proteins present. further, we establish that the heterogeneous physical properties within a given sputum sample correlate to differences in the muc b and muc ac concentrations. the gene modifier study (gms) was established as an effort to identify potential genetic modifiers of cystic fibrosis pulmonary disease and survival. during the course of this study over delta f homozygous cf patients classified as having mild lung disease, severe lung disease, or increased survival, have submitted both clinical data and blood samples for single nucleotide polymorphism (snp) analysis. the original snp analyses have shown a significant association between variants in the endothelin receptor a (ednra) gene, and cf survival, most markedly in female cf patients. sixteen additional snps within and around the ednra gene have now been genotyped, and have implicated the ' and ' untranslated regions of the gene as having the most significant association with pulmonary disease in females (p< . ), suggesting quantitative differences as a possible mechanism for the association with pulmonary phenotype. we are in the process of saturating the ' and ' regions of ednra with an additional snps to further delineate the genetic association. ednra binds endothelin- (et- ) in airway smooth muscle cells, causing increased cell proliferation, smooth muscle contraction, and stimulation of inflammatory molecules. because each of these effects is known to be deleterious to the cf lung, we hypothesize that the ednra variants found more commonly in "severe" cf females ("severe" alleles) are marking increased ednra expression compared to alleles found more commonly in "mild" cf females ("mild" allelels). because the genetic association was strongest in cf females, we used the matinspector software to analyze kb of ednra promoter sequence and found several putative binding sites for both estrogen and progesterone. we then used a brdu assay to measure cell proliferation after stimulation with both estrogen and progesterone. these experiments showed that the asm cells with the "severe" ' ednra genotype proliferated at levels approximately twice that of the asm cells with the "mild" ' ednra genotype following stimulation with either estrogen or progesterone. using a single base extension protocol and quantitative pcr on human airway smooth muscle cells, we were also able to compare ednra expression from the "severe" allele, and the "mild" allele. these comparisons of ednra expression demonstrate that expression levels appear to be approximately % higher from alleles found more frequently in the "severe" cf females. in addition, preliminary data suggest that stimulating the asm cells with estrogen increases ednra expression by approximately fold, and like the cell proliferation experiments, these increases are most pronounced in cell lines with the "severe" genotypes. these data suggest that the "severe" genotypes are marking alleles with increased expression, perhaps due to estrogen binding, that leads to increased et- functional effects that over time are deleterious to the cf lung. cf patients do present with variable spectra of lung disease, of which infections are most life-threatening. β-defensins have an antimicrobial activity against a broad spectrum of microorganisms and are chemotactic agents for cells of the adaptive immune system, and therefore assist in combating these infections. β-defensins - are part of a repeat region. this repeat region is polymorphic between individuals and therefore the dosage of these defensin genes/proteins varies. we developed a real time pcr assay to quantify the number of βdefensin repeats in this region. appropriate controls are needed for an accurate quantitative assay. therefore we made concatemeric constructs with copy of defb and a particular number of defb copies, which ranged from to copies. using these controls as standards, the number of defensin repeats could be accurately determined in dna samples. we then tested f del homozygous cf patients from belgian ( patients), czech ( patients) and south-italian ( patients) origin. the diploid number of repeats varied between and . for each patient group, a higher number of repeats was found in the group of patients with milder disease (fev > %) compared to the group of patients with more severe disease (fev < %) (student t test, p-values of . , . and . respectively). moreover, in our cohort of belgian cf patients, cf patients of years or older have a significant higher number of repeats than the cf patient group below (p-value . ). to evaluate this at the functional level, we cultured nasal epithelial cells from individuals with a low number of repeats (i.e. or repeats) and individuals with a high number of repeats (i.e. repeats). the cells where grown in air liquid interface cultures. after differentiation, the cells were stimulated with ng tnfα. in cells with a high number of repeats, defb expression, as measured by the extent of transcription, was strongly upregulated by tnfα. in cells with a low number of repeats, defb was not upregulated (p-value = . ). we also tested the antimicrobial activity of epithelial cells. we challenged epithelial cells from individuals ( repeats) with a laboratory strain (pa ) of pseudomonas aeruginosa and a clinical isolate ( - cfu), either in combination with tnfα or without tnfα. after h, surviving bacteria were counted by a plating out method. cells that were stimulated with tnfα h prior to the bacterial challenges were more bactericidal. the clinical strain was more vulnerable to the surface liquid than the laboratory strain. in epithelial cells from individuals having a low number of repeats, these effects were very variable from individual to individual. in summary, the β-defensin region is a modulator of cystic fibrosis lung disease. the pro-inflammatory response in cultured epithelium cells strongly correlates with the number of β-defensin repeats. cells with a higher number of repeats respond to tnfα treatment, which in turn results in a better antimicrobial activity of the surface liquid. rationale: studies of affected twins and siblings demonstrate that modifier genes are major contributors to variation in cystic fibrosis (cf) lung disease severity. we performed genome wide linkage analysis to identify regions likely to contain modifier genes affecting severity of cf lung disease. methods: individuals with cf from families were analyzed. pulmonary function data were collected from patient chart review and were supplemented with data from the us cystic fibrosis foundation patient registry. to minimize environmental variation, only data obtained while subjects were living with an affected twin or sibling were analyzed. the pulmonary phenotypes were defined using the best cf-specific percentile for fev (kulich, et al) within the last year of available pft data as a crosssectional measure (maxfev cf%) and using two longitudinal measures: the lifetime average cf-specific % for fev (avgfev cf%) and the estimated percent-predicted fev at age (estfev %pred@ yrs, schluchter, et al). longitudinal measures were derived from a minimum of years of pft data. short tandem repeat markers (strs) were typed in all affected individuals and their parents (marshfield genotyping center: markers or decode genotyping center: markers). two-point and multipoint linkage analyses were performed using sequential oligogenic linkage analysis routines (solar). results: patients represented the spectrum of lung disease severity, with maxfev cf%'s ranging from to , mean . ± . and avgfev cf% ranging from . to . , mean . ± . . the maxfev cf% was predictive of avgfev cf% (r= . , p< . ) for the individuals for whom both measures were available. the two longitudinal measures were also highly correlated (r= . , p< . ). linkage was found at chromosome for all three phenotype definitions. peak multipoint lod scores on chromosome occurred at cm for maxfev cf% and avgfev cf% (lod . and lod . , respectively) and at cm for estfev %pred@ yrs (lod . ). single point lod scores on chromosome peaked at marker aaat ( . for maxfev cf%, . for avgfev cf%, and . for est-fev %pred@ yrs). the region of linkage encompasses approximately megabases near the telomere of chromosome q. conclusions: chromosome appears to contain one or more genetic modifiers of cf lung disease severity. supported by the nhlbi, cff and genome canada through the ogi. cystic fibrosis-related diabetes (cfrd) is the most common extrapulmonary complication of cf and is an increasingly important contributor to morbidity and mortality as cf patients live longer. while pancreatic fibrosis and loss of exocrine and endocrine tissue are common in cf, - % of cf adults develop defects in insulin secretion and accumulation of islet amyloid polypeptide, features typical of type diabetes (t dm) in the general population. to test whether modifier genes play a role in cfrd, we compared concordance rates for cfrd in pairs of monozygous (mz) twins, sets of dizygous (dz) twins, and sets of or more siblings ( individuals with cf). criteria for defining cfrd included physician diagnosis, treatment with insulin/oral agent, and episodes of glucose ≥ mg/dl. mz twins were highly concordant for cfrd ( of pairs, %). the young age of dz twin recruits precluded analysis of this group in isolation ( of pairs were concordant). twelve of ( %) sibling pairs were concordant for cfrd. with heritability defined as: h = *(mz concordance -dz concordance), and including siblings as a proxy for dz twins, heritability is estimated as ~ . . the same results were obtained considering only same-sex dz twins and siblings, correcting for differences in age and duration of clinical follow-up, or restricting analysis to ∆f homozygotes. these data support a significant role for one or more modifier genes in development of cfrd. we then tested whether cfrd correlated with a strong family history of adult-onset diabetes (at least first-degree or second-degree relatives on the same side of the family). of those reporting family history of diabetes, of had cfrd, compared to of with no family history (or= . [ . - . ]; p= . ). this correlation persisted after adjusting for age, sex, and pancreatic insufficiency (or= . ; p= . ). thus, family history of diabetes correlated with increased risk of cfrd. we then tested whether variants in tcf l , a transcription factor in the wnt signaling pathway, that have been reproducibly associated with t dm in the general population were associated with cfrd in our study subjects. genotyping of four single nucleotide polymorphisms associated with t dm (rs , rs , rs , rs , here termed snpa-d) and transmission disequilibrium testing (tdt) of parent-parent-child trios revealed significant overtransmission for snpb ( : , p= . ) and snpc ( : , p= . ), and possible overtransmission for snpa ( : , p= . ) and snpd ( : , p= . ). in every case, the tcf l allele overtransmitted to patients with cfrd is the same allele that confers increased risk for t dm. furthermore, individuals with cfrd who were homozygous for risk alleles were diagnosed at a significantly earlier age (average . vs. . ; p= . ). these data support a key role for modifier genes in development of cf-related diabetes, and demonstrate that cfrd and type diabetes may share disease mechanisms such as alterations in wnt signaling. supported by nih dk , hd , dk and hl , and cf foundation grant cuttin p . cystic fibrosis (cf) phenotypes and survival are highly variable among df homozygous patients, pointing to the existence of modifier genes and/or environmental factors that contribute to this disease. studies to identify genetic modifiers of cf are being carried out using dna from homozygous df cf patients. important clinical features, such as severity of lung disease, liver disease and meconium ileus (mi) status, are well defined. tgfβ has been previously identified as a modifier of cf lung disease (drumm et al., nejm, ( ) : [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ) , but it does not explain all of the genetic heterogeneity in this population. current evidence suggests that mucus is involved in the progression of cf, making the muc genes prime candidates as modifiers of lung disease and/or other phenotypes. methods: our approach to evaluate muc genes utilizes both variable number tandem repeat (vntr) polymorphisms and single nucleotide polymorphisms (snps). vntr polymorphisms in the muc and muc ac genes (n= and n= patients, respectively) were detected by southern blotting under conditions fully optimized to maximize the allele size resolution. to minimize gel to gel variation, a genomic dna mixture with the most common muc and muc ac alleles was used as internal markers. accuracy and reproducibility were evaluated by duplicating the southern on the critical dna samples for muc and muc ac genes (n= and n= patients, respectively). fifty snps in muc ac, muc b, muc , muc and muc genes are being tested using illumina technology in cf patients. results: preliminary analysis suggests there are significant differences between cf patients with "severe" and "mild" lung disease for both muc and muc ac allele distribution, which is mainly driven by the male population; exhaustive statistical data analysis still is underway. the vntr data also suggest significant association between the larger muc allele size and cf patients with mi. the ongoing vntr analysis will be complemented by muc gene snps being genotyped. conclusions: initial results indicate that we can reproducibly characterize muc and muc ac vntr alleles. additional characterization of muc and muc ac vntr alleles, coupled to snp data, will allow us to better define the significance of muc gene variations as modifiers of different cf phenotypes. supported by cff perezv g (jpv), cff knowle a (mk), cff r -cr (wko), nih rr , r hl , and cff drumm a . reporting for the gene modifier study group (mrk). we have previously reported that βenac transgenic mice, which overexpress the beta subunit of the amiloride sensitive sodium channel (scnn b) specifically in the airways, share common features with cf, including increased enac activity, reduced airway surface liquid, mucus accumulation and obstruction, inflammation, and death. interestingly, analysis of this model also suggested the existence of potential genetic modifiers of phenotype severity, and we speculated that identification of these modifiers would provide novel insights into disease phenotype. to establish a set of reagents that could be used to uncover genetic modifiers, we have bred βenac transgenic mice from two independent founder lines ( and , b :c background) onto several strains of inbred mice, including c bl/ n, c h/hen, balb/cj, fvb/j, and /svj. these studies revealed dramatic phenotypic differences as measured by survival ( to %) among strains and between lines. all lines thus far tested show ~ - fold increases in amiloride sensitive short-circuit current as measured by ussing chambers in the trachea. complete phenotypic characterization of c bl/ n line at backcross generation reveals high survival ( ± % in comparison to ± % of the mixed b :c background), yet the mice maintain the pulmonary features of the originally reported mice, including increased mucus plugging, mucous cell hyperplasia, neutrophilic and eosinophilic inflammation peaking at early timepoints ( days - weeks). lymphocytic nodules, which are not commonly seen in - weeks-old animals with mixed strain background, are a common feature in the c bl/ n congenic line. emphysema and early airway epithelial cell necrosis, two phenotypes initially not strongly associated with transgene expression, are also observed. line on balb/cj and c h/hen backgrounds has reduced survival compared to the c bl/ n background, and generation backcross lines are now being evaluated in these two strains for other phenotypic characteristics. analysis of backcross data from line , which has low survival on all genetic backgrounds tested to date, including c bl/ n, suggested that the transgene may have integrated onto a c h locus with a dominant negative effect on survival. genetic analysis using genome-wide snp genotyping revealed a region of chromosome linked to the transgene in line (lod score > . ). further analysis of this region is underway. in summary, backcrossing onto different genetic backgrounds is revealing genetic modifiers for phenotypes in the βenac overexpressing model. characterization of these phenotypic and genetic differences should provide clues about the mechanisms relevant to disease development. furthermore, inbred lines with variable phenotype will likely be an important reagent for the cf community as the utility of this model is evaluated in future studies. supported by nih (scor p hl ) and cff (mall go, oneal go). introduction: new york state screens newborns with immunoreactive trypsinogen levels within the top % of all infants, for common cf gene mutations. infants found to be heterozygote carriers are referred to a cf center to determine sweat chloride concentration. the proposed abnormal sweat chloride value for this group of newborns is ≥ mmol/l, ≥ standard deviation (sd) above the mean (farrell, ) . this retrospective study reports on the mean sweat chloride value + sd in cf heterozygote newborns who have been referred for evaluation to suny upstate medical university cystic fibrosis center, and regarding the genotype of these infants with abnormal sweat chloride levels. method: from october, to december, , infants were referred for positive cf screening, and of these ( %) were identified as heterozygote carriers by the screening program. at our center, these patients underwent pilocarpine iontophoresis, followed by collection of sweat (≥ µl) in macroduct ® coils. sweat testing was performed successfully in of the ( %) patients. the patients' age (mean ± sd) at the time of sweat testing was . ± . days. for infants with an initial sweat chloride level ≥ mmol/l, the sweat test was repeated within a week and a complete gene sequencing was requested (quest laboratory, ca). results: the sweat chloride level (mean ± sd) in newborns who were heterozygous for a cf mutation (excluding those who were found to have an additional mutation or deletion) was . ± . mmol/l (n = ). the mean + sd was mmol/l, which defined our minimal value for an abnormal test. eleven infants had sweat chloride values of - mmol/l (table) ; of them ( %) were subsequently diagnosed with cf by complete gene sequencing. four of the remaining patients ( %) had ∆f mutation coupled to the t variant on the opposite chromosome (sweat chloride levels, - mmol/l). conclusions: the reference range for sweat chloride in cf heterozygote infants appears to be significantly lower for some centers than previously reported. thus, each cf center should consider evaluating the cutoff values for the test at their site. moreover, the t polymorphism may account for sweat chloride elevations in heterozygote infants. method: we reviewed the charts of all patients with cf who were referred as a result of the new york state newborn screening program to the suny upstate medical university cf center from october, through april, . we included the patients who were identified by the state as heterozygote carriers of one of common cf gene mutations, and whose second mutation was identified only after complete cf gene sequencing. results: seven of the patients ( %) met inclusion criteria. six of the patients ( %) were compound heterozygous for a novel or a rare cf gene mutation. one patient was compound heterozygous for a large deletion in the cf gene. the genotype and clinical status of the patients are shown in table. all patients have been pancreatic sufficient to date. conclusions:the clinical effect of compound heterozygosity as a result of novel or rare cf gene mutations appears to be mild in early childhood. prior to newborn screening (with the exception of the patient with cftr deletion), these patients may not have been diagnosed with cf in the first few years of life. these patients may have subclinical airway inflammation and thus benefit from early treatment. patients with cf manifest symptoms in the pancreas, respiratory tract, male reproductive tract and sweat gland due to mutations in cftr. patients with non-classic cf have disease in a subset of these organ systems. most non-classic cf patients have two disease-causing mutations in cftr and at least one mutation permits residual cftr function. a subset of non-classic cf patients have only one cf-causing mutation after screening for a panel of common cf-causing mutations or following mutation scanning of the coding region of cftr. these patients present a diagnostic dilemma and a challenge for genetic counseling. we evaluated cf patients with only one cf-causing mutation identified after a screen of cftr mutations ( patients) or scanning of the coding region of cftr ( patients). nine of these patients, including one set of siblings, have non-classic cf with borderline or elevated sweat [cl -] plus lung disease (table) . one patient is pancreatic insufficient and has classic cf. many of these patients have features which are consistent with cftr dysfunction including a cf-like nasal potential difference (npd),p. aeruginosa infection, or congenital bilateral absence of the vas deferens (cbavd), suggesting that they have a second cftr mutation. mutations that are not detected by screening methods include insertions or deletions, mutations outside of the cftr coding region that affect rna splicing or expression, or mutations in the coding region of cftr that were missed by screening methods. to exclude the third possiblity, dna sequencing of the exons and flanking introns of cftr was performed. a second mutation was identified in the coding region of cftr in of the patients; had screening for known cftr mutations (genzyme), while the remaining had comprehensive scanning of the coding region of cftr by modified tgge (ambry). each of the mutations identified by sequencing has been previously described in patients with cf and is predicted to cause cftr dysfunction. these results reaffirm that patients with one cftr mutation who have biochemical and clinical features of cf are likely to have a second mutation in the coding region of cftr. thus, we suggest sequencing cftr in patients with only one mutation after mutation screening before employing other more complex genotyping methods (insertion/deletion or rna analysis). diagnostic criteria for confirming cf in symptomatic individuals includes two positive sweat tests or two known disease-causing cftr mutations. accurate sweat testing is performed at accredited cf centers, while cftr testing is available through national and specialty genetic labs. most labs offer analysis of a basic panel of cftr mutations as recommended by the american college of medical genetics (acmg), while specialty laboratories may offer an expanded panel or full sequencing/scanning. we report on one center's use of these methodologies to confirm the diagnosis of cf presenting in adulthood. the charts of patients diagnosed with cf at 〉 years of age were reviewed. were sweat tested. all were genotyped. results: twelve ( %) pts had two positive sweat tests (> mmol/l). an additional five ( %) had at least one borderline result ( - mmol/l). two pts had negative results ( - mmol/l). one refused sweat testing because dna analysis through the acmg panel had confirmed the diagnosis prior to initial consultation. genotyping results for the pts are summarized in the table below. rare mutations identifiable only through gene sequencing accounted for / alleles ( %) in our population. importantly, among this group, only one pt had a negative sweat test, a suggestive mmol/l. two pts with positive sweat tests and clinical symptoms failed to reveal any cf mutations after sequencing. we continue to follow pts without genotypic confirmation, based on their clinical presentation and sweat chloride levels, and have recommended additional evaluation, including nasal potential difference studies in our series of adult-diagnosed patients, sweat test results were positive, borderline, or suggestive in all cases tested. sweat testing costs $ -$ and results are ready in a day. genotyping costs $ -$ and takes several weeks. we acknowledge that circumstances may arise where reliable sweat testing is not conveniently available; but in our series, genotyping with the acmg panel would have diagnosed % of pts; using an expanded panel would have diagnosed %. genotyping is an important tool for genetic counseling, determination of eligibility for research studies, furthering knowledge of cftr dysfunction and cf pathophysiology, and for confirming a cf diagnosis after borderline or suggestive sweat test results. based on our findings and the dramatic difference in cost, we conclude that sweat testing should remain the first approach in the diagnostic workup of adult patients with a clinical presentation suggestive of cf. in colorado, infants with cf (non-meconium ileus) have been diagnosed with cf by a two tiered immunoreactive trypsinogen (irt/irt) based newborn screening approach. the irt/irt algorithm has been recently adopted by other screening programs with two mandatory screening tests. while most infants in colorado have been successfully identified, the program has had a missed case rate of approximately %. the more common approach to cf newborn screening is the irt/dna method in which the blood spot of infants with an initial elevated irt is tested for the most common cf mutations. the initial irt cutoff is lower in the irt/dna programs than in the irt/irt programs, resulting in a lower missed case rate. the considerable number of carriers identified through the irt/dna approach puts a significant burden on the genetic counseling community, as carriers are identified at a rate of / - / of positive irts we propose an irt/irt/dna newborn screening algorithm that will maximize sensitivity and specificity while minimizing the number of identified carriers. using new database technologies in the newborn screening lab we will be able to identify those infants with an elevated first irt (> ng/ml, approximately th percentile). all infants with an irt > ng/ml will have a repeat irt on their second state mandated blood-spot. if the second screen is also elevated (> ng/ml), the blood spot will be tested using a panel of mutations, including mutations specific to the hispanic community. infants with one or two cftr mutations will have a sweat test to confirm the diagnosis, or rule out cf. we compared the projected statistics of our current method irt/irt to the new irt/irt/dna method, and to irt/dna is presented in the table, based on , births per year in colorado. four infants ( . %) identified under the current irt/irt protocol would not have been identified by the mutation panel proposed in the new algorithm, out of genotyped, non-meconium ileus infants. three of these missed cases are hispanic. two would be identified using an extreme irt cutoff of the . th percentile ( ng/ml). the projected missed case rate would be < . % ( . - . %, % ci), using the irt/irt/dna algorithm, with carriers identified, maximizing both sensitivity and specificity. this algorithm may provide a better alternative to the irt/irt screening methods in states with two mandatory screening tests, and has advantages over both the irt/irt and irt/dna methods. newborn screening for cystic fibrosis (cf) is rapidly expanding and has been implemented in at least states. although most newborn screening assays are done using biochemical testing, many laboratories screening for cf include both biochemical and molecular testing of multiple alleles in the cystic fibrosis transmembrane conductance regulator gene. in response to the growing need for proficiency testing (pt) materials for molecular testing, the centers for disease control's newborn screening quality assurance program (nsqap) in collaboration with the university of wisconsin school of medicine and public health, the johns hopkins hospital, and case western reserve university, created a repository of dried-blood spot specimens with known mutations in the cftr gene to be used in a pt program. twenty milliliters of blood was collected voluntarily from adult donors with cf and sent to the nsqap laboratory. each specimen was adjusted to a hematocrit of % before being spotted onto whatman paper ( µl per spot), dried, and stored at - °c with desiccant. proficiency testing (pt) panels consisted of to blind-coded specimens from adult donors. the panels were sent quarterly to laboratories worldwide that test specimens for cf using molecular methods. laboratories were asked to report the genotype, method used, and the presumptive clinical assessment of each specimen. twenty-two laboratories participated during both quarters and , . the laboratories used different methods ranging from in-house assays to commercially available kits. most reporting laboratories tested the following alleles -∆f , g x, and g d. another twelve alleles were detected by most participants. nine more alleles were common among commercially available kits. laboratories were evaluated based on the clinical assessments. mutations that were not detected by a particular method were not evaluated. overall, the laboratories performed well. data compiled from both quarters demonstrated that there was incorrect clinical assessment and amplification failures. developing a pt program for dna-based testing is complicated by the number of methods and different alleles each laboratory chooses to test. though molecular testing for cf may be complex, pt monitors the laboratory's ability to test multiple alleles, including uncommon alleles, the limitations of various assays, and the different algorithms used for screening. the repository will also allow storage and access to rare specimens that may be useful for future research but are not readily available. who did could not reproduce. genetic counseling focused on a patient's parents, who were counseled about their recurrence risk at the time of the child's diagnosis. today, cf is a disease of adulthood. in , > % of cf patients in the us were > ; by it will be > %. together with advances in assisted reproductive technology (art), reproduction and recurrence risk are now important issues for adolescent and young adult cf patients. methods: a item questionnaire was developed from the results of prior semi-structured interviews with cf patients age - years. knowledge based questions (medical issues, inheritance, and reproductive options/risks) as well as communication patterns (preferred resources for learning about cf and preferred people with whom to talk about reproductive issues) were addressed. recruited from the uab cf clinic population, patients age - (mean ), male ( %), female ( %), completed the questionnaire. results: regarding autosomal recessive inheritance of cf, only % knew that two carriers have a % chance of having a child with cf, and % knew that two carriers have a % chance of having a child who is a carrier. however, % knew that two carriers could have a child who did not have cf, and % knew that two carriers could have a child who did not carry cf. on their own reproductive risks, % knew that a cf patient had a % chance of having a child with cf if their partner was not a carrier, but only % knew that all their children would be carriers even if their partner was not a carrier. in the scenario of a cf patient with a cf carrier partner, % knew that a child had a % chance of having cf, and % knew that a child had a % chance of being a cf carrier. most patients knew about their reproductive potential, as % responded that cf patients are able to have children. however, when asked about whether the chance for having children was different for males and females with cf, % answered that it was more difficult for males, % that it was more difficult for females, and % answered "not sure." while % reported that they knew that there were options for male cf patients who wanted to have children, only % knew of art. conclusions: despite widespread availability, the lack of knowledge of adolescents and young adults with cf about the genetics of their disease continues. furthermore, these patients are unaware of both modern technologies that could enable them to have biological children and the risk of those children having cf. this study illustrates the changing needs of patient education as medical knowledge progresses. cf patients would benefit from further genetic knowledge and counseling to enable them to make informed decisions about reproduction as they mature into adulthood. center at the university of minnesota is one of three sites in the state providing confirmatory testing and follow up services for newborns identified by screening. while cf nbs identifies children with cf, most of the infants with positive screening results are carriers. our goal is to provide genetic counseling to every cf nbs patient seen at our center, and we believe that a protocol incorporating genetic counseling in the initial care plan for infants both with and without cf is imperative. the literature has shown that families who obtain genetic counseling through the cf nbs process recall genetic information more easily and accurately and are more likely to have testing to determine parental carrier status. for parents of a child that is determined to be a cf carrier, it is especially important to find the optimal method and timing of genetic counseling as many of these families are from several hours away and therefore less likely to return to clinic. to provide genetic information and emotional support for the families of infants screening positive on cf nbs, our center has a genetic counselor who serves as the cf nbs coordinator and clinical contact for the family. this allows many opportunities to speak with the genetic counselor and ask questions, as well as learn about their child's diagnosis or carrier status and the subsequent carrier testing recommendations for the infant's parents and families. to assess the success and impact of the minnesota cystic fibrosis center's nbs follow-up program and the incorporation of genetic counseling, an anonymous questionnaire was developed for parents of infants who were seen at our center due to a positive cf nbs result. questionnaires were mailed to parents of all infants seen at our center for a sweat test and genetic counseling due to a positive cystic fibrosis newborn screening result. as of the last mailing, this totals families. two questionnaires were returned due to incorrect address and questionnaires were returned answered, indicating a response rate of % ( / ). responses overwhelmingly indicated that parents were satisfied with our center's algorithm for the cf nbs follow-up program and found the information and support provided through genetic counseling to be a useful and recommended portion of the program. as cf nbs continues, it is critical that we learn about the patient's experience with genetic counseling and the nbs program, as well as identify areas needing improvement. genetic counseling is vital to the comprehensive success of our center's program, and we will report on the responses gathered from the families identified through cf nbs this first year, as well as discuss the lessons learned from setting up such projects on a state-wide basis. background & aims: aberrant splicing and nonsense mediated decay (nmd) lead to dysfunctional mrnas by skipping exons and to a reduced number of functional mrna respectively. both mechanisms have a strong quantitative aspect and may determine whether a cf patient develops a classic or atypical disease phenotype. in order to approach these highly important questions we wanted to establish a new quantitative real-time pcr based assay which allows allele specific quantification on cdna level. using this assay we like to determine the exact proportions of the f del and non-f del cftr mrna in cf patients compound heterozygous for the f del mutation (for example in cf patients carrying the f del and a nonsense mutation such as the r x). material & methods: materials: we used genomic dna (gdna) and total rna (extracted from white blood cells and nasal epithelial cells respectively) from cf patients with compound heterozygosity for the f del mutation, homozygosity for f del mutation and from healthy individuals (controls). methods: the lasq (ligation dependent allele specific quantification) assay comprises reactions: . reverse transcription of cftr mrna into cdna using gene specific primers (all rna specific). . overnight hybridization ( - h) of the cftr cdna with either the f del specific or the wt specific oligo probe pair provided by jan schouten (mrc holland). . ligation of the hybridized oligo pairs using the ligase enzyme from mrc holland. . quantitative real-time pcr of the allele specific ligation products on the lightcycler (roche). results: in order to establish the lasq assay we first validated it using gdna instead of cdna as template. mixing experiments were performed to verify the accuracy of the assay. in brief, gdna (c= mg/l) of a f del homozygous and a f del compound heterozygous cf patient were mixed in such a manner that . , . amplification products of the f del and the wt allele (both bp long) were analyzed by gel electrophoresis (page) and direct sequencing (abi ) to control specificity. our results using gdna and cdna showed that there occurs unspecific hybridization/ ligation for both probe pairs. the proportion of unspecific amplification products varies between . and . and increases the lower the initial number of templates is. however, the specificity of this assay can be significantly improved by increasing the hybridization temperature and/or decreasing the ligation time. conclusion: although some minor limitations concerning allele specificity the lasq assay has been proven to be an accurate, reliable and reproducible method for allele specific quantification and may be applied for several important questions in cystic fibrosis such as the exact determination of the amount of nmd of cftr mrna containing a premature termination codon (ptc) or the allele specific determination of aberrant splicing of cftr mrna . background & aims: as clinical presentation varies significantly among cf patients with the same genotype, e.g. in the f del homozygous, it is evident that factors in addition to the cftr genotype such as modifier genes, are involved in determining disease severity. however, only one of several previously postulated modifier genes, the tgfβ gene, could recently be confirmed in a large association study. hence the identification of new modifier genes is a very important task in order to find new explanations for the heterogeneity of pulmonary disease in cf patients. we decided to search for new potential modifier genes applying a quantitative proteomic approach comparing the proteomes of a wild type ( hbe o-) and a f del homozygous bronchial epithelial cell line (cfbe o-). the main goal of this study is the identification of up or down regulated proteins in the cfbe cell line which may act as modifiers of cf disease. material & methods: materials: we used two bronchial epithelial cell lines, e.g. a wild type ( hbe o-) and a f del homozygous (cfbe o-) cell line which we obtained from dr. gruenert (california, usa). additionally, we also used nasal cells from f del homozygous cf patients obtained either from nasal brushings or nasal polyps. methods: proteome analysis was performed by making d-gels using high sensitive staining protocols (ruthenium and deep purple). quantitative analysis was accomplished applying the powerful dige (difference in gel electrophoresis) method. for each dige experiment we made gels whereby each cell line was twice labelled with cy and cy (= technical replicates). finally, identification of the protein spots was done by the use of a maldi-tof mass spectrometer. results: in a first step we established the proteomes of the two bronchial epithelial cell lines. we were able to optimize protein extraction and d gel electrophoresis in such a manner that the proteomes of the two cell-lines looked very similarly and the assignment of spots could easily be done. protein spots from both cell lines were analyzed using our mass spectrometry (maldi-tof ms) and allowed the identification of more than different proteins so far. in a next step we quantitatively compared the proteome of the two cell lines using the d-dige method leading to the identification of proteins which are down regulated and proteins which are up regulated at least twofold in the cfbe cell line. out of the aforementioned differently expressed proteins could already be identified because they were among the previously determined proteins. while glutathione s-transferase p and protein s -a (s calcium binding protein) were down regulated ( . and . -fold respectively), superoxide dismutase was up regulated ( . -fold) in the cfbe cell line. conclusion: comparative quantitative proteomics using the dige method is a promising tool in search of potential new modifier genes which may unravel one of the key problems in cf: the large heterogeneity of pulmonary disease in f del homozygous patients. background: cystic fibrosis is one of the most common autosomal recessive disorders among caucasians, and manifests a wide range of disease severity. although this range of disease expression can be attributed, in part, to specific mutations within the cftr gene, much of this variability has not been adequately explained. from the gene modifier study (gms-a multicenter study of , cf patients), ten genes were tested as potential modifiers of cf, and the tgfβ codon cc genotype was associated with lung disease severity. objective: to test whether the adverse codon cc genotype is associated with higher circulating levels of tgfβ , compared to the tt genotype in cf patients and healthy controls. if true, then a link will be established between genotype, disease severity, and circulating levels of tgfβ , and have implications for novel treatment of cf patients. methods: the study includes clinically stable cf patients and healthy control subjects equally distributed between the cc and tt genotypes. the cf patients enrolled are age and older, of both genders, and all ethnicities that fulfilled the standard diagnostic criteria for cf, using the genetic information from the gms. healthy controls are age and older, caucasian males and females, obtained from the environmental polymorphism registry (epr), a dna registry of , self-reported normal volunteers. we genotyped blood samples from the epr to define healthy control subjects for each of the cc and tt cohorts. subjects have a blood draw of ml. blood is divided into tubes and each tube is used to measure a different parameter: cbc with differential; tgfβ levels in platelet poor plasma by quantikine human tgfβ elisa kit; tgfβ levels in the buffy coatwhich includes platelets; and tgfβ mrna levels in lymphocytes from the buffy coat, using real-time pcr roche light cycler. cbc with differential is performed to quantitate lymphocytes and platelets in order to reference tgfβ protein and rna levels to the number of circulating blood cells. analyses will include graphical comparisons between the two groups, chi square analysis, student's two-sample t test, and one way analysis of variance (anova). results: pilot studies show that elisa tgfβ measurements are reproducible and mrna levels can be quantified. we have currently enrolled cc genotype and tt genotype of the cf subjects and cc genotype and tt genotype of the healthy controls. blood samples have been collected and processed. conclusion: genetic variants that predispose to more severe cf disease are potential targets for new therapies. we are testing the hypothesis that the adverse (codon ) cc genotype is likely to reflect increased transcription and/ or tgfβ protein synthesis/ secretion. if true, "anti-tgfβ " therapies could provide a novel therapy in cf. *reporting for the gene modifier study group. supported by cff knowle a , cff drumm a , gcrc rr , nih r hl . aim: centralized periodic evaluations of data from the screening laboratories and cf centers by afdphe (french association for screening and prevention of infant handicaps), were analysed to optimize the efficiency of the program. methods: the strategy combined d irt assay/dna analysis (kit elucigen cf- arms) /d -fail-safe irt. revised irt-cut off levels were decided in order to maintain the percentage of positive screens around a . % target. a questionnaire yearly sent to the cf centers collected the cf false negative cases. results: from to december, , cf cases were detected through nbs ( screened infants). the i period (p) (n= ); d -irt: µg/l and d -irt: µg/l showed a) . % infant above the d cut-off, generating an excess of costly dna tests b) % had a d -irt above the cut-off leading to a very high number of st (n= ) with an extremely low rate of cf (n= ). by increasing slightly both d /d irt cutoff levels ( µg/l, µg/l) during the ii p (n= ) a) the number of positive screens decreased to . % b) . % of infants with elevated d -irt had to be referred for st (n= ) with cf diagnosis. since the risk to had true cf remained very low among infants with no detected mutations, during the iii p (n= ), d -irt concerned only the ones with d -irt> µg/l and the percentage of infants requiring a st was reduced to . % (n= , cf). the incidence of cf detected during these p did not vary significantly ( / - / - / ). another point of concern was the false negative cohort; with a follow-up period over months, cf were detected on clinical symptoms ( . %) at a mean age of months. only were directly related to the modifications of the strategy. conclusion: centralisation of data made possible changes in the flow charts of the screening strategy to limit the number of false positive cases without significant alteration of the global performance of the program. there were three infants of mixed aa/caucasian origin diagnosed with cf, all had ∆ /genotype on screening. mutation data on the additional aa cf patients followed at the cf centers in new york was collected. six patients have not been genotyped. there were patients who were homozygous for ∆ , patients had only one mutation identified and were not found to have any cf mutations. ∆ ( %) and + g>a( %) were the most common mutations. there are five cf patients (including infants diagnosed by nbs) with mixed racial origin who are not included in this analysis. mutations results: the median (range) mbl plasma level was . ( . - . ) µg/ml in cf patients, compared to . ( . - . ) µg/ml in controls. mbl genotype frequencies were similar in patients and controls. lung function level was not correlated with mbl genotype or plasma level. the frequency of colonization with pseudomonas aeruginosa was % in mbldeficient (xa/o and o/o genotype) children with cf and % in mbl-sufficient (a/a and ya/o genotypes) children (p= . ). we found a trend of a decreased age of first onset of colonization with staphylococcus aureus, haemophilus influenza and pseudomonas aeruginosa in mbl-deficient cf patients (p= . , p= . , and p= . , respectively). conclusions: mbl-deficiency was associated with an increased frequency of pseudomonas aeruginosa colonization in children. mbl deficiency was not associated with lung function deterioration. in a larger cohort we hope to confirm that mbl deficiency influences age at onset of bacterial colonization in cf patients. acknowledgments although there is some evidence that cftr gene mutations may be associated with respiratory diseases, little is known about the relationship between cftr gene mutations and idiopathic bronchiectasis (ngiam et al. ) . we have recently showed that a rare allele (- g>a) in the minimal cftr promoter, previously reported in patients with idiopathic bronchiectasis ( using supershift assays, we demonstrated that these transcription factors bind cftr promoter in vitro. a functional analysis, by using co-transfection assays with expression vectors of each transcription factor in pulmonary epithelial cells, showed that nrf , irf significantly decrease cftr expression, whereas irf and sp increase it. in an attempt to further elucidate the mechanisms involving these factors in the cftr transcriptional regulation, for instance, to determine whether these factors could interact together in order to regulate cftr transcription, we started several experiments such as co-immnoprecipitation, multiple co-transfection and rnai. taken together, these data evidence that the variant - a in cftr promoter should be considered as an important risk factor in bronchiectasis pathogenesis. furthermore, we have identified a novel regulatory complex on the minimal cftr promoter, which will enlighten the understanding of the transcriptional regulation of the cftr gene. a better knowledge of cftr cis-and trans-acting elements will allow to consider new approaches to modulate and/or control more specifically cftr expression. this work is supported in part by the association vaincre la mucoviscidose. (nat genet, ) . a recent study reports strong genetic influences for mi (blackman et al., gastroenterology, ) , but it did not replicate the modifier locus on chr. q . inconsistency of results may reflect the variability of data reporting and different classifications of mi (i.e. surgically or medically treated). we tested the accuracy of reporting of mi on case report forms (crfs), as compared to primary source documents. methods: the crf for the gms has a checklist for past medical history. for example, we requested "yes" or "no" for mi (an obstruction of the terminal ileum at birth), but did not require source documents or identification of the type of treatment. to evaluate crf reporting of mi, we requested source documents for patients with reported mi, including a surgical or medical treatment report. if a written report from the time of birth was unavailable, a clinic note, detailing mi at birth, treatment, and evidence of a surgical, abdominal scar (if applicable), was required. verbal confirmation by the patient and evidence of a surgical scar was also accepted, if no written documentation was available. results: on crfs, of patients ( %) were initially reported to have mi. to date, source documentation has been obtained for patients, and of those ( %) have been confirmed to have mi ( % by written report, % by verbal report). of the with confirmed mi, ( %) had surgery, and ( %) had medical treatment. there were false reports of mi ( . %). mi could not be confirmed or refuted for patients ( . %) because of insufficient information (n= ) or confounding circumstances (n= ). to date, documentation for patients who were reported to have no mi has been obtained, and no false negatives have been found. additional documentation for mi from other sites is expected. conclusion: at least %, and perhaps as many as %, of the reports of mi on crfs were inaccurate. gene modifier studies must include rigorous documentation of mi to ensure an accurate correlation between phenotype and genotype. studies should also characterize different classifications (i.e. surgical vs. medical treatment) to assist the detection of gene modifiers of mi. reporting for the gene modifier study group (mrk); supported by cff knowle a , cff drumm a , nih r dk , nih r hl , and gcrc rr . any mutation that disrupts or diminishes the efficiency of the splicing process will have an impact on disease manifestation. in the cystic fibrosis (cf) transmembrane conductance regulator (cftr) gene more than , mutations were identified, most of them being disease-causing [ ] . among these,~ % are classified as missense and about % are classified as splicing mutations, given that they disrupt the consensus splice sites [ ] . however, the splicing mutation concept is evolving, as it nowadays also includes mutations other than just those within or close to the consensus splice sites, rendering prediction of mutation consequences a hard task. nevertheless, it is still very important for the clinical settings to determine the functional effect of gene mutations. our aim here was to study the effect of i v, a rare cftr missense mutation in nbd (exon ), directly in native tissues of a cf patient bearing f del in the other allele, so as to gain insight on how it influence the disease outcome. to look for a possible effect at the rna level, total rna was extracted from native nasal cells and colonic tissue, and cdna producd using random primers. rt-pcr amplification was performed in the region spanning exons - , being one of the primers fluorescently labelled. the products were analysed as described before [ ] . the i v( a>g) mutation which creates both a novel acceptor and a novel donor, was found here to cause alternative spliced cftr transcripts lacking the last nucleotides of exon , thus showing that only the novel donor is used in vivo. moreover, our data show that no normal (only alternatively spliced) transcripts result from this allele. we have also analysed the i v mutation at the protein level by producing a stable bhk cell line expressing the i v-cftr, after generating the respective mutant cdna construct by site-direct mutagenesis. protein expression and function was determined by immunoblot and iodide efflux assay, respectively. results show that i v-cftr protein is processed and functional. however, given the above-described absence of normally spliced mrna coding for this cftr variant, we have to conclude that this protein is not produced in vivo. indeed, since only the mrna coding for cftr lacking the last six aminoacids of exon was detected, we are currently characterizing in vitro the proprerties of this truncated protein. altogether, our data clearly demonstrate that the functional effect of this mutation is not due to the amino acid change but to abnormal splicing. in conclusion, characterization of the consequences of mutations in native affected tissues is important, not just because this provides unexpected information about the mechanisms underlying the basic defect but also for disease diagnosis and prognosis. (darrah et al, nacfc ) . the purpose of this study was to examine the influence of common genetic variation in the endothelin pathway on the cf phenotype. methods: patients were recruited from the cf clinics in dublin, belfast and seattle. serial clinical data were abstracted from medical charts and local clinical databases. studentized residuals of maximum fev , after adjusting for age, gender and height, was the phenotype of interest. twentyone maximally informative tagsnps were identified in the edn , edn , ednra and ednrb genes using the niehs environmental genome project and were genotyped using the illumina beadarray system. genotype and haplotype analysis were carried out using helixtree genetic analysis software. results: clinical and genetic data were available on cf patients ( from seattle and from dublin/belfast). in the combined cohorts, tagsnps in the ednra gene were significantly associated with differences in cf lung disease severity (p= . ). this was observed in both the irish cohort (p= . ) and the seattle cohort (p= . ). the effects were independent of gender and cftr genotype. four common haplotypes (haplotype frequency> %) were identified in the ednra gene. there was significant association between ednra haplotypes and cf lung disease severity (table ). there was no association between genetic variants in ednrb, edn and edn and cf lung disease severity. conclusions: the ednra gene is a genetic modifier of the cf phenotype. our findings were seen in two independent cohorts and verify existing associations found in other populations. the endothelin pathway may be a novel therapeutic target for the treatment of cf lung disease. background: cystic fibrosis (cf) is a recessive "monogenetic" disorder, but there is heterogeneity of lung disease severity and survival reflecting environment and non-cftr genetic modifiers. the unc/cwru multisite gene modifier study (gms) identified patients who were "severe" or "mild" as teenagers or young adults and pertinent cross-sectional data was collected; however, we did not collect all pertinent information about early (age < years) clinical features. objective: we sought to retrospectively evaluate the early clinical features of "severe" and "young mild" patients enrolled in the gms. methods: we obtained all cff registry data available on patients. there were "severe" (worst th percentile of birth cohort, age range - ) and "mild" (best th percentile, age range - ). initial analyses focused on cross-sectional plots of patient age versus multiple clinical features, including age at diagnosis, hospitalizations, cdc height and weight percentiles, presence/absence of ps. aeruginosa from respiratory cultures, and fev (% pred). results: there were . and . years of cff registry data per patient for "severes" and "milds," respectively. preliminary results indicate "severe" patients were diagnosed earlier in life than "mild" patients (mean: . vs. . years, p= . ). this difference is greater after omitting patients who had meconium ileus (diagnosis: . vs . years, "severes" and "milds" respectively, p= . ). between and years of age, "severe" patients were hospitalized more frequently than "mild" patients and, from age onwards, the disparity in frequency of hospitalization increased. "severes" and "milds" had similar cdc height percentiles (~ th percentile) until age , at which point percentiles increased more for "mild" versus "severe" patients. by age , "severe" patients already had lower cdc weight percentiles than "milds" ( nd versus st percentile) and this disparity increased throughout adolescence. from age to , "severe" patients had a - fold higher prevalence of ps. aeruginosa than "mild" patients. as early as years of age, fev (% pred) was ~ points lower in "severes" as compared to "milds." however, some "severe" patients had normal lung function at ages - and overlapped with mild patients; thus phenotyping of lung severity for young patients is not optimal. all of these results were similar for males and females. summary: retrospective analysis of the cff registry data indicates that patients classified later in life as being "severe" experience a worse course of disease from early in life. understanding the early clinical course may prove helpful in defining surrogate phenotypes for modifier studies, and help define appropriate therapeutic targets. cystic fibrosis is mainly caused by small molecular defects of the cftr gene; despite the genotype is defined in the majority of patients, a number of cf cases still remain uncharacterised. the cf mutation database lists more than large rearrangements that may escape detection using pcrbase techniques. the innogenetics assay, the dhplc and sequencing screening showed a mutation detection rate of . % in our population. we report here the results of mlpa screening for ctfr gene rearrangements, performed on the unidentified alleles of our cf patients. our sample population consists of unrelated italian cf patients (for a total of alleles), followed at cf centres of lombardia region. mlpa analysis was performed in patients who still had one or two unidentified alleles after extensive analysis of cftr gene. all patients studied had classical clinical cf symptoms. subjects presented with persistent or recurrent respiratory symptoms, failure to thrive, salt loss syndrome and gastro-intestinal findings. we characterized different deletions and a new duplication (dup promoter-ex. ). thus, . % ( / ) tested alleles had a large gene rearrangement. the deletion of exons - ( / ) was the most frequent in our cohort. all patients had positive sweat chloride values (above meq/l), except the patient carrying duplication who has borderline sweat chloride value. out of patients, ( %) had fecal elastase levels consistent with a preserved pancreatic function: of these patients, had mild mutation, had severe, and had unknown mutation. six patients present liver involvement. the results of the present study could indicate that compound heterozygosity for large rearrangements in cftr gene, is strongly associated with severe pancreatic disease as mutations in classes i, ii, and/or iii. l f is a missense substitution which changes from leucine to phenylalanine at position , resulting from a g/c transition at position in exon a of the cftr gene. it has been described in patients with disseminated bronchiectasis, recurrent idiopathic pancreatitis, sarcoidosis, newborns with hypertripsinemia,. recently derichs et al. reported one healthy -year-old girl homozygous for l f; therefore the pathogenic role of the variant is still unclear. in this study we present subjects compound heterozygotes with l f. no other mutations have been identified after molecular analysis performed using sequencing analysis of the whole coding region of cftr gene and mlpa technique in order to search for gene rearrangements.none of them presented ivs t allele. three of them had a severe mutation in trans (r x, aa>g, and n k), while the other three had a mild mutation on the complementary allele (d h, r q, and r w). individuals with a severe mutation in trans presented a remarkably different clinical picture compared to those with a mild mutation in trans. the formers were diagnosed at a mean age of . years, had an average sweat chloride of . meq/l; besides, all of them have respiratory symptoms, staphilococcus aureus in sputum cultures (one had pseudomonas aeruginosa as well), and in two the chest x-rays was abnormal. the three l f/ps mutation individuals were diagnosed at a mean age of . years, had an average sweat chloride of . meq/l; besides none of them had respiratory symptoms, abnormal chest-xrays, or positive sputum cultures, but two had a history of pancreatitis. these data seem to suggest that l f cannot in any case be considered a neutral polymorphism. the variability of its clinical expression seems to be influenced by the mutation in trans. further studies are needed in order to support our results. expression of the cystic fibrosis transmembrane conductance regulator (cftr) is tightly regulated both spatially and temporally, yet the molecular mechanism of this regulation is not well understood. because no tissue-specific regulatory elements were recognized in the basal cftr promoter, the crucial cis-regulatory elements are likely to be located elsewhere within the cftr locus. a number of the non-coding regions of the cftr gene were found by our laboratory to contain dnase i hypersensitive sites (dhs), suggesting the presence of regulatory elements at these sites. studies presented here investigate the role of an intron dhs (dhs ) in the tissue-specific regulation of cftr gene transcription. the elucidation of molecular mechanisms underlying the temporal and spatial expression of cftr may aid in developing more specific targeted therapies for cystic fibrosis (cf). footprinting analysis of cftr intron revealed a protected region within the core of dhs at + kb. in silico analysis of this sequence uncovered a number of transcription factors binding motifs, including a consensus binding sequence for hepatocyte nuclear factor (hnf ). we have previously identified a role for this factor in the regulation of cftr expression. results of electrophoretic mobility shift assays (emsa) demonstrate that hnf α specifically binds to the motif in cftr intron in vitro. in addition, chromatin immunoprecipitation (chip) analysis of cells expressing both cftr and hnf α factor shows that this factor binds to the intron site in vivo. when cloned as an enhancer, the dhs element was found to augment minimal cftr promoter activity in a luciferase reporter based assay. this increase in luciferase activity was abolished when two nucleotides within the core hnf binding site were mutated, suggesting a functional role for hnf α in cftr gene transcription. further experiments are underway to determine whether additional transcription factors can be recruited to the core of the intron dhs regulatory element and can interact with hnf and the cftr basal promoter to modulate tissue-specific cftr gene expression. the promoter of the cftr (cystic fibrosis transmembrane conductance regulator) gene is not solely responsible for its complex pattern of expression. to identify potential regulatory elements for cftr we previously mapped dnase i hypersensitive sites (dhs) across kb spanning the locus. in addition to intronic dhs, a number of sites were observed that flank the cftr gene. we hypothesized that these may include insulator elements that establish the chromatin expression domain, within which the cftr gene is regulated. insulators are elements that shield against the effects of regulatory elements from adjacent genes, and they may also block silencing of integrated transgenes. using a well-established insulator assay, in which dna regions of interest are assayed for their ability to interfere with communication between a chicken β-globin enhancer and a neomycin resistance gene, we demonstrated that two dhs, at - . kb from the cftr translational start site, and at + . kb from the ' end of the gene, exhibit enhancer blocking activity comparable to known insulator elements. electrophoretic mobility shift assays (emsa), demonstrated in vitro binding of the well-characterized insulator protein ctcf (ccctc-binding factor) at the - . kb dhs. this was confirmed in vivo in both cftr-expressing and non-expressing cell types using chromatin immunoprecipitation (chip). in contrast, although the + . kb dhs did not bind ctcf, we obtained in vitro evidence for the interaction of other factors that may be involved in insulator activity. furthermore, chip analysis of histone modifications across the cftr locus revealed striking differences between the - . kb and + . kb dhs, again suggesting mechanistic differences between these elements. the characterization of insulator elements flanking the cftr locus may be of direct practical relevance in the design of vectors for effective cf gene therapy. one of the problems encountered in gene therapy protocols is the relatively rapid loss of expression from the cftr cdna once it is introduced into mammalian cells. incorporation of the - . kb and + . aminoglycosides, particularly tobramycin, are primary antibiotics used to treat the airway infections in cystic fibrosis patients. lifetime, systemic exposure to these antibiotics is significant and can be associated with significant nephro-and ototoxicity. these toxicities have the potential to significantly reduce the quality of life in the aging adult cf population. we previously reported an incidence of aminoglycoside ototoxicity of greater than % with sensitive audiometric studies (i.e. pure tone audiometry and distortion product otoacoustic emissions). these studies indicated considerable variability in ototoxicity in patients with similar systemic aminoglycoside exposure. the literature suggests that genetic variability in mitochondrial dna can partially explain these differences. single nucleotide polymorphisms (snps) in the mitochondrial srrna gene are associated with aminoglycoside ototoxicity. we genotyped unselected adult cf patients for four of the mitochondrial s-rrna polymorphisms associated with aminoglycoside ototoxicity by direct sequencing of dna harvested from peripheral blood. four patients exhibited polymorphisms in this gene. two patients possessed the a g transition, while another patient each revealed a polymorphism at a g and c t transitions. the patient with the a g polymorphism died prior to audiometric testing. both patients with the a g had audiometric studies consistent with moderate to severe ototoxicity. while the patient with the c t had mild aminoglycoside ototoxicity. all four patients had severe airway obstruction and at least one full course of parenteral tobramycin at mg/kg/d. there was no history of toxic serum levels aminoglycosides during this therapy. both patients with the a g polymorphism had a family history consistent with the expected maternal inheritance pattern associated with the mitochondrial s-rrna gene. we found no evidence of the delt polymorphism in this population. these studies indicate a higher than expected frequency ( %) of mitochondrial s rrna polymorphisms associated with aminoglycoside ototoxicity. these studies demonstrate that genetic screen-ing provides valuable susceptibility information and may change clinical decision-making. support: rising hope foundation/national cf foundation. cystic fibrosis is a genetic autosomal recessive disease that is caused by deleterious mutations in the cftr gene. in its most severe form, cf results in abnormal sweat electrolytes, sino-pulmonary disease, male infertility, and pancreatic exocrine insufficiency. in fact, cf is the most frequent cause of pancreatic insufficiency in humans. carriers of cf (i.e., heterozygotes) do not express any of these classic symptoms. recent studies indicate that significant numbers of non-cf patients diagnosed with congenital bilateral absence of the vas deferens (cbavd) or idiopathic chronic pancreatitis (icp) are compound heterozygotes and carry two cftr mutations of varying severity. in dogs, especially german shepherds and rough-coated collies, exocrine pancreatic insufficiency (epi) has been observed with symptoms similar to those seen in human patients. epi in dogs is most often caused by pancreatic acinar atrophy (paa), a degenerative disease of the exocrine pancreas that has been shown to be inherited as an autosomal recessive trait. the locus responsible for canine paa has not been identified to date. we considered the possibility that cftr mutations might be responsible for this disease. a dog that had been diagnosed with paa and one that was a known carrier of paa were screened for cftr mutations. our samples also included dogs diagnosed with idiopathic pancreatitis as well as healthy dogs. we have established protocols for detecting putative mutations in the canine cftr gene using temporal temperature gradient gel electrophoresis (ttge). in the dog with paa and in the carrier of paa, our screening methodology identified mutations in of the amplicons that span the exons of the cftr gene. dna sequencing revealed silent mutations in exons , and . in exons (l ) and (l ), single nucleotide polymorphisms converted ctt codons into ctc codons, both leucine codons. in exon (p ) another single nucleotide polymorphism converted a ccc proline codon into a cct proline codon. the other identified mutations (amplicons , and , , , , , and ) were found in intronic sequences and have no predicted effect on cftr expression or function. based on these findings, we conclude that cftr mutations are not responsible for paa and the epi that it causes in dogs. hypothesis: specific polymorphisms in genes of the innate immunity may modify the severity and progression of cf pulmonary disease and influence pseudomonas aeruginosa colonisation. methods: single nucleotide polymorphisms (snps) analysis of multiple genes contributing to the innate immunity ( (mbl ), masp (mbl associated serine protease) Ç , fcn (ficolin) Ç , lbp (lipopolysaccharide -binding protein), cd , tlr (toll-like receptor Ç ) ) was performed in cf patients (age - years). spirometric data and bacterial colonisation status with pseudomonas aeruginosa were collected retrospectively. a decline in fev of % over a years period and a percent predicted fev value of % were used to discriminate mild from more severe affected adult cf patients (≥ years). the frequency of single and combined snps in well-defined subgroups of the cf population was compared. results: in adult cf patients with a mean fev lower than %, the combinations of snps of cd (promoter) with tlr (promoter), fcn with tlr (exon ), and masp with tlr (promoter) were more common than in adult cf patients with mean fev > % (odds ratio (or) , ( % confidential interval (ci) : , - , ) , or , ( % ci: , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ) and or , ( % ci: , - , ) respectively). the frequency of combination snps of masp with tlr and masp with tlr was significantly higher in cf adults with more than % decline in fev compared to cf adults with less than % decline in fev (or , ( % ci: , - , ) and or , ( % ci: , - , ) respectively). however, the single snps of masp , tlr , tlr were not more frequently found. in pseudomonas aeruginosa colonised cf patients, an increased frequency of combined snps of tlr (exon ) with tlr (exon ), lbp (exon ) with tlr (exon ) and mbl with tlr (exon ) (or , ( % ci: , - , ), or , ( % ci: , - , ), or , ( % ci: , - , ) respectively) was found. in contrast, for single snp tlr (exon ) no statistically significant differences were found between the groups (or : , ( % ci: , - , )). in general, the odds ratios for the combined snps were higher than for the single snps and remained significant when the different groups were subdivided according to cftr genotype. conclusion: certain combinations of snps of genes of the innate immunity are more frequently found in cf patients with a lower fev , a stronger decline in pulmonary function and pseudomonas aeruginosa colonisation. however, for the single snps this trend was less pronounced or absent. congenital bilateral absence of the vas deferens (cbavd) is a rare condition associated with mutations in cftr. cbavd lacks extra-genital organ system abnormalities; it has been viewed as a mild (variant) form of cf. the disease serves as an excellent model for studying the clinical and experimental ramifications of marginal cftr activity, and the functional thresholds necessary to confer a cf-related phenotype. recently, drumm et al. (nejm, ) found that the codon cc genotype of tgfβ- is associated with more severe pulmonary disease, particularly in individuals homozygous for ∆f cftr. tgfβ is also known to play a critical role during normal development and differentiation of the human vas deferens (han-nema et al., horm res, ) . because % of cbavd subjects carry at least one defective cftr allele (but millions of males worldwide are heterozygous for cftr mutations without developing cbavd), the present study was designed to test whether tgfβ polymorphism might serve as a genetic modifier underlying isolated vas deferens loss in the setting of cbavd. we sequenced the ' end of the tgfβ gene in eighty cbavd individuals possessing at least one cftr mutation, and compared genotype frequency with results previously published from demographically similar controls. when cc frequency in cbavd subjects was compared to pooled estimates from earlier trials, no significant differences were seen using a non-dominant model. compared to frequencies reported by arkwright et al. and garrote et al., a greater number of homozygous cc subjects ( . % vs. . %, p< . ) was observed. our preliminary results therefore indicate that if tgfβ- codon polymorphism is associated with cbavd penetrance, it does not represent the major modifier of disease phenotype. in order to evaluate this possibility further, we plan analysis of a larger (matched) control set of cbavd and non-cbavd individuals, studies of tgfβ- activity in vas deferens epithelial cells with and without cftr, and experiments to investigate the effects of tgfβ on cftr biogenesis and function. supported by nih and cff. newborn screening (nbs) for cf has been successful in identifying cf infants, particularly those who have positive sweat tests immediately following the positive screening result. tracking by personnel at the central nbs program facilitates resolution of diagnostic status because cf nbs presents a challenge to cf center (cfc) systems and primary care providers when apparently asymptomatic infants have inadequate (quantity not sufficient (qns)), borderline or indeterminate sweat tests. short-term follow up is more problematic when the screening algorithm requests earlier sweat tests and defines a lower cutoff for indeterminate sweat test results than is used for older infants, children and adults. the massachusetts (ma) cf nbs workgroup chose a low cutoff for indeterminate results of meq/l up to months of age, reverting to the accepted meq/l cutoff thereafter. follow up by the central nbs program identified infants whose diagnostic status remained unresolved more than weeks after a qns or borderline sweat test and eliminated the need for individual cfcs to follow infants tested at more than one cfc. the program sent informational letters and a reminder recommendation to finalize the diagnosis to primary care providers of infants with unresolved diagnoses. of the cf infants identified by ma cf nbs, had an initial sweat result that was indeterminate and another had an initial result that was qns. the central screening program has the capacity to track infant and child outcomes across cfcs; for example, we have been tracking growth outcomes of all cf infants identified by ma cf nbs, despite of the cf infants having moved from one cfc to another during their first years of life. tracking and outcomes data will be presented. when more than one cfc operates within a screening program, centralized tracking ensures capture of quality data. despite several educational campaigns cf is often not diagnosed in early infancy in poland (the mean age of diagnosis months, median is months). after several years of discussions,in september a neonatal screening for cf supported by the ministery of health was started as the third obligatory neonatal screening in poland. more than , neonates were screened in the first seven months using the existing screening infrastructure and spare blood spots after pku and congenital hypothyreosis screening. the protocol was two stage irt/dna (covering the most common mutations in the polish population). the final diagnosis was based on sweat tests (conventional pilocarpine-iontophoresis and conductometric nanoduct) as well as clinical examination. values of chloride in sweat > mmol/l and nacl > mmol/l were considered diagnostic for cf. children were confirmed as cf. although delta f is the most common mutation in the polish population, only two infants were delta f homozygotes, six were delta f heterozygotes and the remaining had different mutations ( children had + kbc>). half of the diagnosed group was still pancreatic sufficient. the children had confirmed cf diagnosis at the mean age of weeks. only children were free from any radiological changes in lungs. the most common pathogen in this group was staphylococcus aureus. at the first visit information for parents about cf was given along with physiotherapy and dietary education. anthropometric measurements, airways bacteriology and elastase - in stool were performed. psychologist consultations were available. neonatal screening for cf enabled earlier diagnosis and the introduction of complex therapy in comparision with symptomatic screening. objectives: the clinical course of cystic fibrosis (cf) varies widely among patients carrying the same cftr gene mutations supporting that additional genetic modifiers could affect the cf phenotype. as inflammation is a central contributor to the pathogenesis of cf lung disease, genes involved in the inflammatory response are potential modifier candidate genes. methods: we examined the influence of polymorphisms in genes involved in the inflammatory response (tnf, il- β, il- receptor antagonist (rn), il- , il- , il- and tgf-β ), on disease progression in a group of caucasian children with cf. the genotypes were tested for an association with changes in lung function tests, pseudomonas aeruginosa colonization and nutritional status by multivariable analysis. results: we found a significant association between tgf-β + t/c variants and decline in lung function measured as the forced expiratory volume in one second (fev ) and the forced vital capacity (fvc) (p= . and . respectively). il- gene polymorphisms (- a/t, + g/t and + t/c) were associated with the occurrence of pseudomonas aeruginosa colonization (p< . ). conclusions: this study suggest that il- variants may influence pseudomonas aeruginosa colonization in cf, however this need further confirmation. moreover, we provide additional support to the results of other trials and strongly suggest an association between tgf-β variants and lung disease phenotype in cf. if such a role of tgf-β is confirmed by functional studies, it may have important clinical impact for the identification of patients at risk to develop more severe respiratory manifestations and for the development of new therapeutic strategies aimed at adequately balancing tgf-β production and action introduction: cystic fibrosis (cf) is the most common monogenic disease in caucasian population, its frequency is about one in live born. the estimated frequency in hispanic population is about one in , with differences in mutation frequency especially for low frequent alleles. heterogeneity in pulmonary manifestations cannot be explained by the cftr mutation genotype. recent studies are focused in the study of modifier genes. these are genes different from the primary cftr gene that could influence the fq phenotype, acting trough mechanisms like inflammation, repair and remodeling, protease-anti protease balance, innate immune response, etc. this work shows some advances in cf diagnosis and modifier genes analysis in mexican population. methods: we tested the effectiveness of innolipa cftr kit (innogenetics) in clinically diagnosed cf patients. thirty six frequent mutations in cftr gene were analyzed by pcr and reverse hybridization with allele specific oligonucleotides probes in dna samples extracted from blood or oral swabs. for frequency analysis of modifier genes we tested about seventy dna samples from a dna bank of patients previously tested positive for cftr mutation. the polymorphism analysis of modifier genes was made by pcr-rflp. for alpha antitrypsin (aat), z and s alleles were analyzed according stanford and cols. ( ) . for tumor necrosis factor alpha (tnfα) - polymorphism in the promoter region was analyzed according chen and cols. ( ) . results: mutation was detected in in of patients ( % of effectiveness). df allele frequency was % (caucasian - %) followed by + g-a, s n, g x, g e and delt ( %). % of the mutant alleles remain undetected. preliminary results in modifier genes show low frequency for aat mutant alleles ( . % for aat s and z alelle respectively) and . % for tnf- . conclusions: df alelle frequency was %, and we found the low fequency alleles s n and + g-a. frequency of modifier genes mutant alleles in cf patients are low. this work is in progress of selection and recruitment of patients and controls, as well as standardization of the other genes modifiers, along with some other biomarkers that will allow to understand the physiopathology of cf and to define a possible genetic risk profile of severe pulmonary disease. over putative cystic fibrosis (cf)-causing mutations have been reported to the cf mutation database and almost half (~ ) are rare missense mutations that are predicted to substitute a single amino acid. to determine if any of these rare missense mutations cause cf by altering localiza-tion of cftr, we examined mutations in the cytosolic loops of cftr since localization signals have been identified in cytosolic loops of other ion channels. amino acids alignments comparing human cftr sequence to that of species revealed that cytosolic loop (cl ) was the most conserved loop by identity. furthermore, cl had the most naturally observed mutations: of the amino acids were found to be mutated in cf patients and sites had multiple missense mutations at the same amino acid. in addition, different substitutions of the same cl amino acid have been associated with different disease consequences, such as the missense mutations in the arginine at codon . patients with the r p mutation have pancreatic insufficient (pi)-cf, those with r w mutation have pancreatic sufficient (ps)-cf or cbavd, while patients with r q primarily had pi-cf. to determine whether the r mutations cause disease by affecting cftr localization, we used the flp-in system to create stable polarized mdck-gfp-cftr cells to produce isogenic clones with cftr expressed from the same integration site. confocal microscopy studies of stable mdck-frt-gfp-cftr-r mutant cell lines revealed that cftr-r p was cytoplasmic, cftr-r w was apical and cytoplasmic, and cftr-r q was apical. to confirm these observations, quantitative biotinylation studies were performed. biotinylation assays of mdck cells stably expressing cftr-r p confirmed that this mutant was absent from the apical surface, cftr-r w had a low level of fully glycosylated protein at the apical membrane, while cftr-r q had fully glycosylated protein at the apical membrane comparable to wild-type cftr. thus, cftr-r p and cftr-r w displayed properties in polarized cells that were distinctly different from wild-type cftr consistent with their associated cf phenotypes. however, the profile of cftr-r q (apical localization and cl-channel function (seibert et al. , mickle et al. ) appears inconsistent with a pi-cf phenotype. indeed, re-analysis of cftr genes bearing r q revealed that of carried a second mutation in cis (s x). in nine of these patients in whom cftr genotype and pancreatic status is known, presence of the nonsense mutation s x is associated with the pi-cf phenotype. since nonsense mutations are known to cause severe gene dysfunction by promoting decay of their rna transcript, we concluded that the s x mutation rather than r q is responsible for the pi-cf phenotype. the finding of s x explained the otherwise enigmatic functional studies of r q and clinical observations in patients bearing this mutation. this study also demonstrates that stable expression of cftr mutants in polarized mdck cells using the flp-in system provides a useful screen for evaluating the disease potential of rare missense mutations. the clinical course of cf is characterized by recurrent pulmonary infections and chronic inflammation. in cf patients, up-regulation of toll-like receptor- (tlr ) gene in airway epithelial cells is believed to enhance proinflammatory responses towards bacterial tlr ligands. we have recently shown that decreased methylation (demethylation) of the tlr promoter is responsible for cf-related up-regulation of tlr in bronchial epithelial cells (shuto. t. et al., faseb j, ) . however, the molecular mechanisms responsible for dna demethylation-dependent tlr gene upregulation in cf cells remain unknown. here, we identified minimum region of the tlr promoter critical for its expression at - /+ , which contains one putative binding site for sp . sp inhibitor mithramycin a treatment reduced tlr promoter activity and its expression in cf bronchial epithelial cells (cfbe o-), suggesting the importance of this sp site for the tlr gene regulation. moreover, bisulfite sequence analysis revealed the hypomethylation of adjacent this sp binding motif within tlr promoter in cfbe o-cells, implying the cf-specific demethylation of adjacent sp binding motif. although mithramycin a rarely affected basal expression of tlr gene in hbe o-cells (non-cf bronchial epithelial cells), mithramycin a inhibited tlr expression in -azacytidine (dna-demethylating agent)-treated hbe o-cells. taken together, our results suggest that sp is crucial for dna demethylation-induced gene upregulation of tlr in cf bronchial epithelial cells. mucus hypersecretion due to goblet cell metaplasia is a critical feature of cf lung disease, affecting both mucociliary clearance and drug delivery. various studies in cf lungs and cf primary cultures have shown increased expression of muc ac, the main gel-forming mucin produced by goblet cells; however, little is known about the role of muc ac in the progression of lung disease. to determine whether overexpression of muc ac alone is sufficient to induce lung pathology, we generated a mouse model overexpressing muc ac. the entire muc ac cdna (minus % of the predicted vntr sequence, final size ~ kb) was cloned and tagged with an internal gfp epitope at a site close to the vntr to preserve the integrity of mucin domains. gfp-muc ac was linked to the rat ccsp promoter to drive specific airway expression, primarily in clara cells since these cells are capable of secreting mucins. blastocyst injections were used to generate transgenic founder lines in c bl/ background. although muc ac-gfp mrnas were expressed in transgenic mice, lung histology did not show significant increases in inflammation, ab/pas-positive cells, or luminal secretion compared to wild-type littermates. gfp fluorescence from freshly excised lung was weak but immunohistochemistry showed that the distribution of gfp correlated with the expected expression pattern, with the majority of positive cells localized to the airways and few positive cells in the alveolar space. at higher magnification, gfp-positive granules were observed in dome-shaped cells that were also positive with cc antibody, suggesting proper muc ac-gfp packaging in clara cells. bronchoalveolar lavage samples separated by agarose gel western blots and probed with gfp antibody confirmed that each line secreted muc ac-gfp, with a migration pattern comparable to a mucin. to further characterize the secreted transgenic mucin, a pool of lavage samples was separated into supernatant and pellet. the pellet was resolubilized in m guanidine. both elements were passed through a s size-exclusion column. analysis by light scattering and refractometry to determine molecular weight and concentration revealed that the solubilized pellet contained an enormous but compact complex ( x g/mol and nm radius of gyration). gfp-rich fractions were pas-positive, indicating proper glycosylation of the transgenic muc ac-gfp. cscl density gradient studies revealed that the gfp-rich fractions had a high molecular weight but did not appear to be a prominent part of the megacomplex retrieved in lavages, unlike other tested mucins. in conclusion, we showed that the overexpressed muc ac-gfp was a high-molecular-weight protein that was glycosylated, packaged in clara cells and formed multimers. lack of discernable phenotype in the transgenic lungs suggests that mucin overproduction alone is not sufficient to trigger goblet cell metaplasia and mucus accumulation, supporting the protective role of mucins. currently we are crossing the muc ac-gfp with the scnn b mouse model to study muc ac-gfp under dehydrated conditions and we are conducting allergen-challenges in the transgenic mice. results of such studies will provide novel insights into the role of secreted muc ac in development of lung disease in these models. supported by grants from the cf foundation. the major characteristic in cystic fibrosis (cf) is acquirement of chronic lung infections with p. aeruginosa. once established the chronic infection can only be suppressed, not eliminated. the cf patients harbour one single strain of p. aeruginosa which only rarely is replaced by other strains. during the chronic lung infection there is a year long mutual impact of the bactaria on the host, and of the host response and antibiotics on the bacteria, resulting in a continous adaptation of the bactaria in the lungs of the cf patients with selection for different clones. in contrast, mouse models of the lung infection in cf is limited to two weeks! therefore, we introduced a new strategy infecting different groups of balb/c mice with pulsed field gel electrophoresis-(pfge) identical clones isolated from one cf patient during -years of p. aeruginosa lung infection. ) infecting the mice with non-mucoid isolate showed that the early isolates were significantly better in establishing an infection with reduced clearance of the bacteria from the lungs (p< . ), increased dissimination of the macroscopic pathology (p< . ) and a more acute type and a higher degree of inflammation (p< . ) as compared to mice infected with a late isolate. in addition, the pulmonary cytokine response was comparable with observations in cf where gm-csf and il- correlated to a milder disease, whereas g-csf, mip- (il- analog), and il- b correlated to severe disease. ) infecting the mice with the mucoid pfge-identical isolates from the same cf patient revealed a signicantly higher mortality in mice infected with the late isolates (p< . ). in addition, the pulmonary g-csf and mip- response increased in the groups of mice that were infected with the late isolates as compared to the early isolate (p< . ). moreover, the g-csf and mip- increased during the first three days of infection with the late mucoid isolates (p< . ), indicating an impairment of the host response in controlling the lung infection. ) when tobramycin treatment was initiated h after infection of mice with a mucoid isolate, the number of bacteria were cleared or reduced to a significantly lower level (p< . ), and a significant decrease of both g-csf and mip- at day one, two and three was observed (p< . ). in contrast, when treatment was initiated after h the number of bacteria did not change significantly, and the reduction in inflammatory cytokine response became significant for both late isolates only at day three of treatment (p< . ). moreover, cytokine levels were significantly lower in the h as compared to the h group (p< . ) confirming the importance of early and aggressive antibiotic treatment in cf. non-mucoid isolates were reduced during the first three days of infection independent of treatment. in conclusion, we have implemented a clinical important cf-time perspective (years) in experimental p. aeruginosa lung infection, useful for patho-physiological and treatment studies. cystic fibrosis (cf) is a complex disease that affects multiple organs and results in a wide range of phenotypes in humans. mouse models of cf display many of the phenotypes observed in human patients and provide necessary tools to dissect this multifaceted disease. however, due to the involvement of cftr in various organ systems, the ability to discern the contribution of each organ system to each phenotype of cf is unknown. for example, we have found that mouse growth is dramatically reduced compared to wildtype, and mechanisms ranging from neuroendocrine disruption, intestinal malabsorption and cacchexia have been proposed. we have found that cf mice consume more calories per gram body weight than non-cf mice and intestinal absorption of dietary lipids is similar between the two. cf animals absorb % and non-cf % (p > . ). to identify the source of the growth deficit, it would be advantageous to restrict expression of cftr in specific tissues to determine each tissue's contribution to the phenotype. to accomplish this, we have created a conditional, null murine cftr allele. a conditional null allele is one in which the gene of interest is induced to be non-functional in either a specific tissue or cell type, or at a specific developmental time. examination of cftr absence on a tissue-by-tissue basis will greatly facilitate our understanding of the disease. the conditional cftr allele was created by "floxing" exon of cftr, known to be critical for function, with flanking loxp sites that allow for the deletion of exon when bacterial recombinase cre is present. to date, mice stably transmitting the floxed allele have been created. these founder mice have been crossed with mice expressing cre from the promoter of the villin gene, which restricts transcription of cre to intestinal epithelium. first generation animals show appropriate recombination and deletion of exon in the gut. however, these first generation animals are heterozygous for the floxed allele. we are currently backcrossing these animals with the founders to generate animals with homozygous deletion of cftr in the intestinal epithelium. similar crosses are planned to target other tissues. this new animal model of cf will be available to the cf research community to further our understanding of cf pathophysiology. this work was supported by a grant from the cf foundation increased enac-mediated na+ absorption is a hallmark of cystic fibrosis (cf) airways. we demonstrated that mimicking na+ hyperabsorption by overexpression of βenac in mouse airways results in airway surface liquid (asl) depletion and reduced mucus clearance causing a spontaneous cflike lung disease with high pulmonary mortality, and airway mucus obstruction, mucous cell metaplasia and chronic airway inflammation in adult survivors (mall et al., nature med. : , ) . the aim of this study was to identify the initiating lesions and investigate the natural history of lung disease caused by asl depletion in βenac overexpressing (βenac-tg) mice. to achieve this goal, we performed longitudinal studies on lung morphology, airway mucus obstruction, bronchoalveolar lavage inflammatory cell counts, expression levels of mucins (muc ac, muc b, muc ) and proinflammatory cytokines (tnfα, kc, ifnγ, il- , eotaxin- ), lung volume, lung mechanics, and airway na+ transport in neonatal to adult βenac-tg mice and wild-type littermate controls. we show that airway mucus obstruction in βenac-tg mice originated in the trachea in the absence of mucous cell metaplasia in the first days of life, and was associated with hypoxic degeneration and necrosis of airway epithelium, and death. in surviving βenac-tg mice, mucus obstruction extended into the lungs and was accompanied by secondary mucous cell metaplasia, increased expression of muc ac, muc b and muc , and airway inflammation with transient increases in macrophages and tnfα, in eosinophils and il- , and persistent increases in neutrophils and kc in the lung. βenac-tg mice also developed emphysema with increased lung volumes, distal airspace enlargement, and increased lung compliance. several disease surrogate markers waned in parallel with βenac transcript levels and airway na+ absorption in adult βenac-tg mice indicating that the level of enac overexpression was critical in determining disease severity. in summary, our results provide several novel insights into the in vivo pathogenesis of lung disease caused by asl depletion. we show that (i) asl depletion is sufficient to initiate severe airway mucus obstruction in the absence of mucous cell metaplasia and/or mucus hypersecretion; and (ii) airway na+ hyperabsorption and mucus plugging-induced hypoxia is associated with airway epithelial necrosis constituting a mechanism that initiates airway inflammation in the absence of infection. further, our results point to a novel link between airway na+ hyperabsorption, eosinophilic inflammation, and emphysema. the high frequency of allergic airway inflammation and emphysema in cf patients warrants further studies of the mechanisms that link asl depletion with these associated pathologies. the balance of airway surface liquid (asl) at a height compatible with efficient clearance of mucus is a process regulated by two main pathways: the secretion of clvia cftr, and absorption of na + via the epithelial na + channel (enac). to study this interplay, an immortal cell line capable of growing at an air liquid interface and having properties of human bronchiolar epithelium would be desirable for its simplicity in preparation and ease of molecular manipulation. an immortal human bronchial adenocarcinoma cell line (hbac) was infected with a lcfsn retrovirus carrying either cftr cdna (hbac cftr ) or the blank vector (hbac mock ). - day monolayers, grown on transwell clear membranes, were mounted in ussing chambers and studied under open-circuit conditions. to study regulation of clsecretion, the equivalent short circuit current was calculated in the presence of amiloride (apical, µm). in hbac mock cells, forskolin ( µm, basolateral) had no effect upon i sc . in contrast, hbac cftr cells demonstrated a robust increase in i sc ; a rapid increase to a peak (∆~ µa cm ), followed by relaxation to a stable and sustained plateau. genistein ( µm) also stimulated i sc in hbac cftr (∆i sc µa cm ) but not hbac mock cells. amiloride resistant basal current was consistently higher, by - µa cm , in hbac cftr versus hbac mock cells, presumably reflecting low basal cftr activity. we next examined the ability of endogenous receptors present in hbacs to regulate transfected cftr. stimulation of i sc in response to apical adenosine ( µm) in hbac cftr was qualitatively and quantatively similar to the forskolin response. while the above experiments provided evidence to suggest that hbac cftr cells possess the bioelectric capability of sustaining anion transport via nucleoside-stimulated cftr activity, it does not guarantee a resultant vectorial flow of fluid. in the final series of experiments, we examined whether adenosine was indeed able to stimulate secretion across the hbac-cftr monolayer by assaying asl height. monolayers were loaded apically with fitc-labeled dextran ( µl), and the majority of dye was then aspirated. monolayers were then allowed to equilibrate over a hour period, and asl measured by xz scanning on a leica confocal microscope. ~ µm adenosine and ~ µm benzamil was then added apically in pfc and asl height measured after and minutes. in both hbac mock and hbac-cftr cells, the basal asl height was ~ µm. over minutes asl height of hbac mock cells did not change in response to adenosine. in contrast, adenosine stimulated hbac cftr cells demonstrated a doubling of asl height (to µm) over the same period. this response is consistent with the prolonged increase in i sc observed upon stimulation with adenosine. in summary, the hbac model which displays only absorptive currents endogenously, can be transformed into a cell line in which cftr-mediated secretion can be stimulated and sustained by endogenous receptor systems present in the apical membrane. this new model will allow us to study cftr-mediated fluid transport in a defined manipulatable system. despite the identification of the cftr channel more than years ago, many questions remain about its regulation and in vivo function. in order to better understand how cftr functions in vivo, numerous studies have searched for and identified potential interacting partners but to date, there is little evidence linking any of the known cftr interactors to its activity. during a forward genetic screen in zebrafish, aiming to identify genes regulating endodermal organ development, we identified a mutant, which we named baobab (bao), after the african tree that accumulates water during the wet season, which exhibits a dramatically enlarged fluid-filled gut tube. most interestingly, we have shown that various cftr inhibitors reduced the appearance of enlarged guts in bao mutant embryos, suggesting that the fluid accumulation phenotype resulted from an increase in cftr activity. to test whether increased cftr activity resulted from changes in cftr protein levels or localization we raised antibodies against zebrafish cftr. western-blot and immunolocalization studies revealed that the channel was expressed and localized as in wild type in bao mutant embryos indicating that the increased cftr function most likely resulted from the activation of the channel. these data, together with the recessive character of the mutation, suggest that the bao protein is a negative regulator of cftr activity. we have positionally cloned the bao locus and isolated the affected gene. bao encodes a cytosolic protein that has not been previously shown to regulate cftr. in agreement with the pharmacological data pointing to a gut-specific function of bao, in situ hybridization studies showed that bao is higly expressed in gut. we are currently investigating how the bao protein regulates cftr function. in addition, we are also investigating the function of cftr during zebrafish development. these studies lay the groundwork to take advantage of the zebrafish system to further investigate cftr biology. we establish a genetic model system for studying the regulation of the cftr channel that will contribute to the understanding of the pathophysiology of cystic fibrosis and several intestinal secretory conditions. acknowledegements: this work was supported by an embo long-term postdoctoral fellowship to mb and by nih grants to d.y.r.s.. we thank a. verkman for providing cftr inhibitors and k. brand for technical assistance. the trachea of the adult cf mouse (cftr tm unc or cftr tm kth ) expresses little cftr and exhibits neither a defect in camp-mediated clsecretion nor hyperabsorption of na + , both signature abnormalities in human cf airways. we generated a mouse exhibiting hyperabsorption of na + in the airways by over-expressing the β subunit of the epithelial na + channel (βenac, gene scnn b). βenac mice exhibit airway pathology (decreased mucociliary transport, mucus plugging, and airway inflammation) similar to human cf airways. to determine whether inactivation of cftr in βenac mice results in a more severe phenotype, we generated a cf/βenac mouse by crossing the ∆f (c bl/ n) mouse (cf mouse) with the βenac mouse (c bl/ n). survival analysis showed a drastic reduction in survival for cf/βenac mice in comparison to cf mice or βenac mice. we studied the airway bioelectrics of day-old pups of each genotype. the basal short circuit current (i sc ) was ~ fold greater in wild-type (wt) ( ± µa . cm - ; mean ± sem, n= ) vs. cf ( . µa . cm - , n= ) neonatal tracheas. as previously reported, the basal i sc in βenac preps was significantly elevated ( . ± µa . cm - , n= ) vs. wt tracheas. however, the basal i sc in the cf/βenac tracheas ( . ± µa . cm - , n= ) was intermediate between the cf and the βenac tracheas. the amiloride-sensitive i sc was elevated similarly in the tracheas of the βenac and cf/βenac pups and was ~ x greater than that exhibited by the wt or cf tracheas. the post-amiloride i sc was strikingly reduced in both cf and cf/βenac tracheas compared to the wt and βenac tracheas. we suggest that the post-amiloride i sc reflects constitutive cftr-mediated clsecretion, which may help hydrate airways in the neonate. unlike the tracheas of adult cf mice, neonatal cf tracheas exhibited the classic defect in camp-mediated clsecretion, with the forskolin response in the cf and cf/βenac tracheas being reduced ~ fold compared to the response in the wt or βenac preps. the response to utp (ca ++ -mediated clsecretion) was similar in the four genotypes. absence of cftr-mediated clsecretion does not appear to cause airway pathology in neonatal cf mice, but when this defect is coupled with na + hyperabsorption, it results in a significant increase in neonatal mortality. due to lack of cftr-mediated clsecretion, cf/βenac mice appear to experience greater mucus adhesion, producing death due to asphyxiation. in addition, the tracheas of cf mice (especially congenic c bl/ n) often appear constricted or collapsed due to a cartilaginous defect resulting in increased compliance. this anatomical defect does not appear to be detrimental to the cf mouse. however, in cf/βenac pups, that accumulate thick, sticky mucus in their tracheas, this tracheal constriction may contribute to their asphyxiation. these results demonstrate the importance of both abnormal na + absorption and decreased clsecretion in the pathogenesis of cf lung disease. supported by nih scor p hl . recently, we reported that species-specific differences in the airway biology of raav transduction exist between mice and human (am j respir cell mol biol, , : - ) . these differences significantly interfere with the use of mice as surrogate models for human lung gene therapy with certain raav serotypes. to survey which species might be an optimal animal model for raav preclinical testing, we utilized differentiated airway epithelia grown at an air-liquid interface (ali) from five species (human, non-human primate [nhp] , pig, ferret, and mouse) to test the efficiency of transduction with three serotypes of raav (type , , and ). our results demonstrated significant species-specific differences in raav transduction biology in airway epithelia. the serotype preferences of raav transduction from the apical membrane in human, ferret, and pig epithelia was raav >raav ≈raav , different from that seen in mouse epithelia (raav >>raav >>raav ). surprisingly, the profile of raav transduction with these three serotypes in nhp rhesus monkey airway epithelia (raav ≈raav >>raav ) was less similar to human than pig and ferret. additionally, raav lacked a bias in polarity of transduction in human airway epithelia, however, in nhp epithelia, raav transduced the basolateral membrane -fold more effectively than the apical membrane. furthermore, differences in the properties of raav receptors between these species was also seen. using neuraminidase treatment of the apical surface of ali cultures, we tested whether n-linked sialic acids were potential receptors for raav binding and/or transduction. these studies demonstrated removal of n-linked sialic acids inhibited raav transduction of human, nhp, and mouse airway epithelia, but not ferret and pig. however, raav binding in this setting did not correlate with changes in transduction; neuraminidase treatment dramatically reduced raav binding to nhp epithelia, but enhanced binding to human epithelia (~ fold). these findings suggest viral binding to airway epithelial cells may not be a major barrier for raav transduction. evidence that proteasome-modulating agents enhancing raav transduction - logs for all serotypes and species tested, suggest that the ubiquitin/proteasome pathway represents a common intracellular block in raav transduction of airway epithelia. these findings support the notion that viral interactions at the surface of airway epithelial cells (with co-receptors and receptors) influence intracellular processing aav virions that can alter the efficiency of productive transduction. enac surface density in cortical collecting duct (ccd) epithelial cells is established by camp/pka regulated insertion and retrieval of the channel at the apical membrane, together with the recycling of internalized enac back to the apical surface (butterworth et al. ( ) journal of general physiology : - ). ubiquitination by the nedd - ligase elicits endocytosis of surface enac, which may lead to its degradation. however, enac recycling to the apical surface requires ubiquitin removal by a deubiquitinating enzyme (dub). using a chemical probe approach, we identified ubiquitin carboxy-terminal hydrolase (uch-l ) as the predominant dub in vesicular endocytic and recycling compartments in mouse ccd epithelia. specific inhibition of uch-l ( µm of , , , -tetrachloroindan- , -dione) in filter-cultured ccd cells resulted in a rapid decline in enac currents, which was accelerated by forskolin stimulation, suggesting that uch is responsible for recycling enac to the apical surface. knockdown of uch-l significantly reduced both basal and camp-stimulated enac currents (from . ± . µa/cm in control sirna treated to . ± . µa/cm in uch-l knockdown cells). to determine whether the same dub was responsible for the regulation of enac in human bronchial epithelial (hbe) airway cells, differentiated cultures derived from cf and non-cf lung tissue were treated with the uch inhibitor. to address the possibility that protease cleaved enac may be regulated differently from unprocessed channels, differentiated hbe cultures were either maintained at air/liquid interface or were submerged under liquid/liquid conditions to alter the cleavage state of enac, as we have previously described (myerburg et al. ( ) journal of biological chemistry : - ). in all cases, no significant difference in enac-mediated na+ transport was observed between control and uch-l inhibited conditions. these data are in sharp contrast with the mechanisms of enac regulation observed in the ccd model system, and they support our previous findings that enac turnover and mode of regulation in hbe are distinct from the recycling mechanisms defined in the ccd epithelia (butterworth et al. primary human tracheobronchial epithelial (htbe) cells cultured at an air-liquid interface (ali) regulate ion transport, mucin secretion, and cilia beating to recapitulate directional mucus clearance, a key innate defense mechanism that fails in cystic fibrosis (cf). thus, ali htbe cell cultures are crucial for studying cf pathogenesis and for developing/validating novel therapies. different research centers use alternative protocols that enable reproducible creation of ali htbe cell cultures. however, limited primary htbe cell availability has driven the creation of cognate cell lines, accomplished mainly by the introduction of viral oncogenes. viral oncogenes disrupt normal cell physiology causing cellular hyperplasia, abundant apoptosis, genomic instability and, sometimes, poor polarization/differentiation, rendering the cells less useful than primary cells for assessing cftr function. furthermore, genetically unstable cell lines will continuously acquire changes independent of cftr genotype. bmi- is a polycomb group protein that controls cell cycle and cell identity via epigenetic regulation of chromatin, maintaining stem cells by suppressing expression of p , an inhibitor of cyclin dependent kinases. bmi- expression has been used to create cell lines that recapitulate normal cell structure and function better than viral oncogene-immortalized cell lines. using hiv lentiviral vectors, we introduced bmi- and the catalytic subunit of telomerase to enhance the growth of different ∆f homozygous cf and non-cf primary human airway epithelial cell preparations. all new cell lines grew for at least population doublings, while their normal counterparts senesced prior to a maximum of doublings. at passage - , the new cell lines had a diploid karyotype compared to grossly abnormal chromosomes in cells immortalized by viral oncogenes. ussing chamber analysis of ali cultures at passage (p) - revealed variable transepithelial resistances among the cell lines ranging from -> Ω*cm , but short circuit current (isc) responses stimulated by forskolin and inhibited by cftr were true to the cell's genotype and comparable to early passage primary cells, except for one non-cf cell line with relatively low cftr isc's at p . amiloride sensitive and utp-stimulated isc's were present but were more variable in magnitude in comparison to the currents observed in low passage primary cells. ali cultures exhibited a pseudostratified morphology with prominent apical membrane polarization, few apoptotic bodies, numerous mucous secretory cells and occasional ciliated cells. all cf and non-cf cell lines in culture produced similar levels of il- at baseline and cf and non-cf cells equally increased il- secretion in response to il- β, tnfα and the tolllike receptor agonist, pam cys. these novel cell lines will help fill gaps currently hindering cf research and therapeutic development. supported by cystic fibrosis foundation grant randel g . we recently considered that domestic dogs could represent a large natural reservoir to search for cftr mutations and, ultimately, to identify and breed animals with cf traits. the canine genome has been completely sequenced, and the canine cftr gene, found on chromosome , consists of exons spread over kb. an animal model that faithfully expresses human cf lung pathology is critical for the development of effective treatments for this most lethal aspect of cf disease. while transgenic mouse models of cf replicate numerous aspects of cf disease, they unfortunately do not develop the characteristic lung pathology including mucus plugging of airways, chronic bacterial infections, and bronchiectasis. the reason for this is not completely understood but it is likely related to significant differences in lung morphology between these species. to date, no non-murine genetic model of cf has been developed, and naturally-occurring, cf-causing mutations have not been identified in any non-human animal species. temporal temperature gradient gel electrophoresis (ttge), developed as a screening tool to identify sequence variations and mutations in the human cftr gene, was used to screen dogs for cftr mutations. dna was purified from whole blood received from veterinary clinics treating dogs for a variety of ailments. we have performed ttge analysis on dna from dogs, which includes dogs with pancreatitis and dogs with bronchiec-tasis, and have found hundreds of potential mutations amongst the thousands of amplicons tested. ttge can detect single base pair changes within a fragment of dna. as the temperature increases, the migrating dna becomes partially melted, creating single stranded regions (internal bubbles and branched ends) that decrease its mobility through the gel. the dna fragments have distinct melting profiles that are sequence dependent; therefore, changes in sequence of even a point mutation can alter the profile and result in a change in the rate of migration through the gel that can distinguish wild type from even a homozygous mutant. thus far exonic mutations have been sequenced but none have caused changes to cftr at the amino acid level (i.e., the mutations have all been silent). we conclude that ttge is a sensitive and effective method for screening large canine populations for cftr mutations. ( introduction: the search for "correctors" of defective protein processing of ∆f cftr has led to the development of high throughput screening assays to find compounds that promote the surface activity of the mutant protein. by necessity, primary screening assays typically use cell lines expressing recombinant ∆f cftr. however, "hits" identified in primary screening require scrutiny for false positives whose activity is either dependent on the ∆f cftr expression system or insufficiently robust to correct processing in the complex physiological setting of a native epithelium. to this end, we have developed a secondary functional assay that measures the shortcircuit current (isc) responses in organ cultures of gallbladder mucosa from ∆f cftr mice (cftr tm kth ). previous studies have shown that murine ∆f cftr recapitulates the protein processing defect including the capability to increase processing when mucosa is incubated at reduced temperature ( °c) (j. clin. invest. : invest. : , . gallbladder mucosa was chosen for this assay because the murine gallbladder can be removed aseptically for organ culture, the epithelium is essentially a monolayer, and the mucosal wall is transparent which facilitates morphological assessment. methods: using either wild-type (wt), ∆f cftr (df) mutant or cftr knockout (cftr ko) mice, excised gallbladders were opened longitudinally, rinsed and positioned on loose nylon mesh. the preparation was mounted between two halves of a polycarbonate culture cup with . mm aperture and placed in submersion culture for - days at either °c or °c using supplemented ham's f medium mixed : with nih t fibroblast-conditioned dmem containing % calf serum. after incubation, the culture cups were placed in custom designed ussing chambers for measurement of isc responses to a cocktail of µm forskolin and µm ibmx (camp isc) at °c. contribution of calcium-activated clchannels to the camp isc was eliminated by addition of the distilbene dids ( µm) to the luminal bath. results: the mean ∆camp isc responses (in µa/cm ) after two days incubation were as follows: wt at °c = . ± . (n = ); df at °c = . ± . (n = ); df at °c = . ± . (n = ); and cftr ko at °c = . ± . (n = ). in a pairwise comparison, the wt ( °c) was significantly greater than the df ( °c) and cftr ko ( °c) but not significantly different from df ( °c). conclusions: incubation of ∆f cftr mutant murine gallbladder mucosa at °c for two days in organ culture restored approximately % of the cftr-dependent isc responses to camp stimulation. the temperature-dependent effect is consistent with correction of murine ∆f cftr processing, indicating a physiological screening assay that is useful for preclinical testing of candidate drugs targeted at correcting the protein processing defect of ∆f cftr. supported by cfft and nih. the lack of a large animal model that replicates the lung disease found in humans with cystic fibrosis is a major impediment to understanding disease pathogenesis and the development of effective therapies. although cystic fibrosis is a multi-system disease, airway infection and inflammation currently cause most of the morbidity and mortality in patients. several homozygote null and missense cftr mutations have been developed in mice, but they do not exhibit the lung phenotype observed in humans. in contrast to mice, the structure and physiology of porcine lung and airways closely resemble those of humans. therefore, the goal of this project is to develop a swine model for cystic fibrosis by combining gene targeting and nuclear transfer to produce a pig with a cftr null genotype. we employed homologous recombination to disrupt exon of pig cftr with a neomycin resistance cassette. we generated primary male fetal fibroblasts that were positive for cftr-targeting at one allele as detected by multiple pcr screens. genomic southern blot analysis confirmed proper gene targeting and revealed most clones were free of random integration events. these cells were used for somatic cell nuclear transfer that resulted in the birth of ten cftr null heterozygote piglets. subsequent breeding of these male pigs to wild-type females has yielded at least female cftr null heterozygotes. breeding male and female heterozygotes will provide cftrnull homozygotes. our recent published and preliminary studies demonstrate that mouse slc a , a newly discovered member of slc family of anion exchangers, is expressed on the apical membrane of tracheal epithelial cells and gastric surface epithelial cells and in the tubulovesicle membranes of gastric parietal cells. the functional properties of mouse slc a were examined in xenopus oocytes and in stably transfected cultured cells. in xenopus oocytes injected with the slc a crna and studied with the voltage and/or ph sensitive microelectrodes, the rank order for anion selectivity at + mv was cl->i-> no ->gluconate > sulfate, and that of the selectivity (px/pcl) was i-> cl-> no -= gluconate > sulfate. inhibitor profile studies demonstrated that nppb at µm inhibited the current at min by % (p< . compared to control period, n= ). the effect of nppb was partly reversible. dids at µm had no significant effect on the slc a -mediated current (p> . compared to control period, n= ). niflumic acid at µm inhibited the current at min by ~ % (p< . compared to control period, n= ) which was partly reversible. cftr inh at µm inhibited the current at min by ~ % (p< . compared to control period, n= ) which was reversible. clamping currents in a mm k-gluconate medium and using mm k-cl or k-bicarbonate revealed very low bicarbonate conductance for slc a compared to that of cl-. no significant phi changes were observed in oocytes when favoring cl-exit by substituting perfusate cl-with gluconate in the presence of co /hco -. conversely we observed significant alkalinization in ae -expressing oocytes in the same experimental solutions. interestingly, in hek cells stably transfected with the mouse slc a , a significant intracellular alkalinization was observed upon switching to a chloride-free perfusate. the alkalization in slc a -expressing cells was inhibited by ~ % in the presence of µm dids. baseline intracellular ph was significantly increased in slc a expressing hek cells vs. non transfected cells. however, baseline phi was comparable in control and slc a -expressing cells in the presence of µm eipa, indicating the activation of endogenous nhe by slc a . in conclusion, slc a displays characteristics of an anionic channel in xenopus oocytes, with low bicarbonate permeability. in mammalian expression systems, slc a can also function in cl-/hco -exchange mode with moderate sensitivity to inhibition by dids. we propose that slc a plays an important role in anion (chloride) secretion and/or ph regulation in tracheal and gastric epithelial cells. no cell lines exist at present that robustly express endogenous wt and ∆f cftr on the same genetic background. zinc finger nucleases (zfns) allow the introduction of specific genetic changes at loci within the human genome (nature : [ ] [ ] [ ] [ ] [ ] [ ] ) . we have been using this technology in an attempt to generate human cells carrying the major cf-causing genotype -∆f -at the endogenous cftr locus. such lines could be expected to provide better understanding of the ∆f maturational processing defect, as well as new model systems for cell based assays of ∆f repair. we performed a bioinformatic analysis of cftr exon , and designed multiple distinct zfns against the ∆f dna sequence. all of these nucleases were assembled, cloned into mammalian expression vectors, and assayed for dna binding affinity and specificity. the zfns were then assayed for endogenous cftr locus editing in calu- and ht- cells using a novel, massively parallel methodology applied for the detection of genome editing events. the effort demonstrated that specific zfn pairs successfully bind to, and cleave, within exon of the endogenous cftr locus in both cell types. we will describe experiments that yielded optimized zfns for the cftr locus, and a robust viral platform to transiently deliver genome editing reagents to the human pulmonary cell line, calu- . supported by cff. the localization of slc anion transporters, as well as their function vary among members. some (slc a , slc a , slc a , slc a ) are widespread, implying specific physiological function in diverse tissues. others are expressed only in a single epithelia, e.g., slc a (prestin) in the inner ear. we have previously found that slc a is a highly electrogenic cl --hco exchanger with a large anion conductance. to determine the possible physiologic roles of slc a , we developed a chicken antibody (designed against sulfate transporter and anti-sigma domain, stas, of slc a ) to localize the protein. to test the antibody specificity for functional protein, we inject xenopus oocytes with crna for slc a , slc a or slc a and documented appropriate ph and membrane potential changes. only oocytes functionally expressing slc a were stained using our primary slc a antibody. further, specific staining was eliminated when either pre-immune serum or blocking peptide was used. slc a was only found in epithelia. in the respiratory system, slc a was in the apical membrane of nasal epithelial cells and tracheal submucosal glands. membranes of calu- cells, derived from human tracheal submucosal glands, stained indicating that our antibody also recognizes the human slc a protein. slc a is found in the proximal parts of the gastrointestinal tract (esophagus, stomach), with lesser amounts in the small intestine and colon. however, slc a is not present in liver. slc a is present in ducts of secretory epithelia such as salivary glands and pancreas. in the urinary tract, slc a is localized to the renal proximal tubule apical membrane but not in urinary bladder. in the reproductive system, we found slc a in epithelia of the uterus and seminal vesicles but not the vas deference or the prostate. finally, slc a is found on the eye surface, i.e., corneal epithelial cells. together, these results and the varied slc a physiology suggest that several epithelial function models will need to be re-evaluated to incorporate an electrogenic cl --hco exchanger and/or another apical anion channel. (support: romero- g , sindic- f ). a tgfβ- polymorphism modifies severity of cystic fibrosis lung disease in the setting of ∆f cftr homozygosity (drumm et al., nejm, ) . the mechanism(s) underlying this observation, and whether elevated or repressed tgfβ are responsible, are not known. in addition, the pathogenic role of tgfβ in other cf organs and in murine models of the disease has not been well studied. because the tgfβ codon cc genotype is associated with worsened cf pulmonary prognosis, and since high levels of tgfβ are also known to suppress cftr mrna expression and apical cftr protein expression in colonic epithelia (howe et al., exp cell res, ) , we developed a mouse model to test tgfβ/cftr interactions that may directly influence cf phenotype. we bred ∆f heterozygous mice (cftr tm kth , ∆f cftr) against the tgfβ receptor-deficient murine strain (mtrii ). mtrii mice express a dominant-negative mutant form of the tgfβ type ii receptor which lacks the intracellular (kinase) receptor domain. the receptor binds tgfβ, but fails to transmit a tgfβ dependent signal. the tgfβ receptor transgene was inserted downstream of a metallothionein-derived promoter and expressed in numerous murine tissues following induction with mm znso in drinking water. an antibody raised against active tgfβ (lc- - - , gift of dr. kathy flanders, national cancer institute) was used to establish tgfβ in murine tissues including lung and intestinal tract of the mtrii strain. rt-pcr confirmed high level transgene expression similar to gapdh internal control. nasal potential difference measurements in cftr +/+ mice expressing the tgfβ transgene indicated intact camp dependent cltransport following induction of the dominant-negative receptor. homozygous cf animals deficient for tgfβ signaling have recently been obtained and exhibit the expected litter sizes and distribution of the ∆f allele. studies of cf homozygous mice following induction with znso are in progress, and will be used to determine whether a failure to signal through tgfβ in vivo ) influences the severity of cf intestinal disease, ) alters airway or intestinal bioelectric findings characteristic of cf, or ) changes survival, histopathology or other features of the disease. these experiments are intended to provide an in vivo means of investigating tgfβ signaling and resultant cf modifier effects in vivo. supported by nih and cff. objective: to determine if the abpa phenotype observed in cftr mice was preventable by corrective gene replacement, we delivered aav cftr to the lung. in addition to further assess whether cftr-/-lymphocytes alone where responsible for the phenotype, we performed adoptive transfers of cftr-/-lymphocytes into cftr+/+ mice and proceeded to expose the mice to the abpa model. design/methods: cftr-/-mice underwent it administration of raav cftr or raav gfp. all mice were then sensitized with ug of crude af antigen via intraperitoneal route. non-injected mice were sensitized without prior virus exposure. challenges were performed on all mice, using an af solution delivered by aerosol inhalation in a closed chamber on consecutive days. serum ige levels were then obtained, as well as bronchoalveolar lavage fluid for differential cell counts, histologic and cytokine analysis. adoptive transfer experiments were done using spleens cells isolated from af sensitized delaf -/-and f +/+ mice and transfer into c /bl _rag-/-mice. after weeks post transfer c /bl _rag-/-mice with f -/-and f +/+ lymphocytes were then challenged with af. results: following challenge with af, raav -cftr mice had significantly lower total serum ige levels ( , ) as compared to raav -gfp controls ( , ) (p= . ). analysis of af-specific ige also revealed a two-fold reduction in the mice receiving the corrective gene therapy. curiously, gene transfer to the lung lowered the secretion of inflammatory cytokines from activated splenocytes of raav -cftr when compared to raav -gfp controls. these results suggest that correction of immune cells in the lung that traffic to the spleen may be partially responsible for the attenuation of the phenotype. in addition the transfer experiments showed that the hyper-ige syndrome can be partially transferred into b rag-/-by f -/-splenocytes. conclusions: correction of cftr deficiency by aav lung transfer attenuates the inflammatory phenotype associated with abpa. epithelial cells lining the vas deferens express cyclooxygenase (cox) when exposed to testosterone in vivo, although no functional link to ion transport has been established. we hypothesized that testosterone modulates cftr-mediated anion secretion across vas deferens epithelia that is stimulated via cox-dependent pathways. vas deferens epithelial cells were isolated from human and porcine tissues and cultured as monolayers on permeable supports until assayed in modified ussing chambers. rna was isolated concurrently for semi-quantitative gene expression analysis. bradykinin (bk) caused an increase in anion secretion (measured as short circuit current) and this effect was inhibited by indomethacin, which indicates cox dependency. testosterone treated monolayers exhibited a bkinduced response that is % greater than paired vehicle treated monolayers. qrt-pcr revealed that cox- mrna is times more abundant than cox- mrna in cultured vas deferens epithelial cells. the effects of bk on ion transport are inhibited by the b receptor antagonist hoe while the b receptor agonist des-arg -bk produced no effect. prostaglandin e and selective agonists of the ep ( -oh-pge ) and ep (butaprost) receptors produced concentration dependent increases in short circuit current whereas sulprostone, an agonist of ep and ep receptors, had no effect. taken together, these results suggest that testosterone plays a key permissive role for bk-stimulated anion secretion by inducing cox- expression. bradykinin acts at the b receptor to increase cox- activity and the synthesis of prostaglandins that ultimately bind to ep receptors to promote cftr-dependent hco -and cl-secretion that would be expected to modify the environment for sperm. the results further suggest that developmentally associated increases in testosterone levels during gestation and puberty will produce physiological changes in vas deferens luminal environment that are likely compromised in cystic fibrosis patients. supported by cystic fibrosis foundation schult po and ksu cobre nih rr- . background: recent studies in cystic fibrosis (cf) suggest a prominent t h profile; a facet that may interfere with normal bacterial clearance. this phenotype appears to increase secondary to infection with aspergillus fumigatus or pseudomonas aeruginosa and also, may interfere with macrophage activation. these same characteristics are present in cftr-deficient mice (cftr s x -/-fabp-hcftr (+/+), hereafter designated cf mice). as a study of pathogenesis of cf related diabetes (cfrd), we previously observed increased il- and splenocyte proliferation in cf mice subject to streptozotocin-induced hyperglycemia. herein, we hypothesized that differences in glucose concentration would increase t h profiles of splenocytes in vitro. methods: cf mice and c bl/ j mice (b mice; n= /group) were sacrificed at wk of age. splenocytes were collected and cultured at the following glucose concentrations: , , and mm. each mouse and glucose concentration was stimulated with cona, lps and cd /cd . supernatants were collected at hr and measured by luminex analysis. statistical analysis was performed by a one-way anova. results: stimulation by cona produced increased levels of il- , il- , tnf-a, gm-csf and il- in splenocytes of cf mice (p< . for all). stimulation by lps produced decreased concentrations of il- in cf mice(p< . ). stimulation of cf mice by cd /cd produced decreased concentrations of il- (p< . ). interestingly, no difference was observed in cytokine elaboration as a function of glucose concentration. data are presented in table as fold increase of cytokine concentration for cf mice compared to b mice. discussion: profound differences in cytokine production were measured in stimulated splenocyte responses of cf in comparison to b mice. the profiles did support the notion for a prominent t h response, with the potential to interference of normal macrophage activation. in addition, differences in cytokine production profiles appear related to the nature of adaptive and innate stimuli provided (cona vs. lps). the absence of influence by glucose concentration in vitro suggests that local/immediate concentration of glucose is not relevant to prolonged expression of cytokine responses to stimuli. further studies on the impact of chronic hyperglycemia and its relation to immune response are necessary. an improved understanding of these mechanisms holds hope for a better overall outcome in patients with cf and cfrd. studies from different laboratories to characterize ∆f cftr clchannel activity have yielded disparate results, likely due to variations in technique, cell type, or method of analysis. in the present study, we compared ∆f cftr activation in two well characterized polarizing cell models. fisher rat thyroid (frt) cells have been used in high-throughput drug screening to identify ∆f cftr active agents, whereas cfbe ocells have been transduced to provide robust ∆f cftr expression in an airway epithelial background. ∆f cftr biogenesis following low temperature correction was compared, indicating qualitatively similar levels of cftr transcripts and band b and c cftr. in paired studies in ussing chambers, distinct differences in isc were seen following exposure to cftr activating stimuli. in frt cell monolayers grown at °c, forskolin ( µm) or genistein ( µm) were strong stimuli of isc, producing mean isc of . +/- . (forskolin) and . +/- . (genistein) µa/cm . regardless of exposure sequence, both agents induced equivalent changes in maximal current ( % of . µa/cm by forskolin when administered prior to genistein; % of . µa/cm by forskolin in converse order). growth at °c for hours increased currents approximately . fold, but relative contributions of the two stimuli were similar to cells grown at °c ( % of total current stimulated by forskolin). in cfbe o -∆f cells grown at °c, forskolin-dependant currents were minimal and genistein-stimulated currents small ( . +/- . µa/cm , compared to zero current in parental, nontransduced cfbe ocells, p< . ). in temperature-corrected ∆f cfbe ocells, forskolin was a poor stimulus of cl-transport relative to genistein, contributing % to the total current produced by both agents (p< . ). genistein-stimulated currents did not require pre-treatment with forskolin to produce maximal effects ( . µa/cm with genistein alone vs. . µa/cm after forskolin and genistein), and forskolin did not cause further isc when added after genistein ( µa/cm ). levels of camp did not account for these differences, since forskolin markedly increased camp in both models. to determine whether excessive phosphodiesterase (pde) or phosphatase (pp) activity limited ∆f cftr activation by forskolin in the cfbe o -∆f cells, monolayers were pretreated with the nonspecific pde inhibitor papaverine ( µm) or the pp inhibitor endothall ( µm), each sufficient to activate isc in cfbe oexpressing wtcftr (papverine: . vs. . µa/cm with vehicle, p< . ; endothal: . vs. . ua/cm , p= . ). neither agent restored isc in ∆f cfbe oby forskolin ( . mv difference, p=ns). these results indicate that activity of ∆f cftr in polarizing monolayers is exquisitely dependant on the model chosen for study. substantial differences in ∆f cftr behavior in two commonly used cell models, and suggest that the failure of camp to activate ∆f cftr in cfbe ocells is due to cell-specific factors that may ) influence ∆f cftr folding and tertiary structure, or ) favor interactions of ∆f cftr with inhibitory binding partners that limit activation by camp. adverse immune responses to viral vectors or vector-encoded proteins may hinder the practical application of gene therapy. to date, little effort has been devoted to determining the immunological consequences of topically delivered lentiviral vectors to respiratory epithelia. previously, we demonstrated that an integrating feline immunodeficiency virus-based lentiviral vector pseudotyped with a baculoviral envelope glycoprotein (gp -fiv) readily transduced differentiated airway epithelia in vitro when applied to the apical surface. furthermore, using a luciferase reporter gene and bioluminescence imaging, we observed in vivo gene transfer to murine nasal epithelia following a single application of gp -fiv. longitudinal bioluminescence analysis documented persistent expression in nasal epithelia for greater than months without significant decline. by histological analysis, surface epithelial cells were transduced following a single nasal application of gp -fiv expressing beta-galactosidase. importantly, we observed that consecutive doses of gp -fiv delivered over consecutive days greatly increased the number of beta-galactosidase positive epithelial cells. using quantitative bioluminescent imaging, we observed that repeated doses given over consecutive days resulted in a linear additive increase in gene expression. in addition, we performed studies investigating gene transfer efficacy and immune responses following lentiviral vector readministration with greater intervening time periods. in control mice, we observed that giving nasally instilled doses ( weeks apart) of an e /e deleted ad vector resulted in significantly attenuated expression after the second dose. the attenuated expression correlated to the production of neutralizing antibodies. in contrast, using the same delivery protocol, doses of gp -fiv resulted in gene transfer without loss of expression of the forth dose. no significant production of neutralizing antibodies was observed following the lentiviral delivery protocol. we further report the additive increase in reporter gene expression following doses of lentiviral vector delivered over consecutive weeks ( dose/week), as well as the lack of production of systemic and local neutralizing antibodies. gp -fiv efficiently transduces and persistently expresses a transgene in respiratory epithelia and has the potential for repeat administration without eliciting adverse immune responses. these data have important implications for the application of lentiviral gene therapy to pulmonary diseases. uniform delivery of gene therapy vectors to all lung lobes is important for cf gene therapy. however, this important objective has not been achieved in large animals. to address this obstacle, we have developed an approach to specifically deliver vector into all lung lobes. in this strategy, an intracorporeal nebulizing catheter (aero-probe) is inserted into a bronchoscope to permit visual targeted aerosolization of vector specifically into each lung lobe. using this approach, ml of . % lpc (to transiently open tight junctions) containing x vp of hdad-k lacz was sequentially aerosolized into each of the six major lung lobes of a baboon. aerosolization was triggered by the ventilator upon inspiration at . ml/pulse at a rate of to pulses/min. halfway through the procedure a very slight, transient and fluctuating decrease in oxygen saturation of no more than % was noted that did not warrant supplemental oxygen. the entire procedure was otherwise uneventful and well tolerated with no clinical manifestations of toxicity. no changes in chest x-rays taken at h, h and at necropsy h post-vector were observed compared to baseline. x-gal staining of the lungs at h revealed extensive transduction of the epithelium in the large and small airways in all six major targeted lung lobes. x-gal histochemistry revealed substantial transduction that was exclusively restricted to the airway epithelial cells and submucosal glands, the target cells for cf gene therapy. assessment of the proximal major bronchi from each lung lobe revealed that~ % of the airway epithelial cells were transduced in the left upper lobe (lul), ≥ % were transduced in the lml, lll, rul, rml and rll, and~ % were transduced in the accessory lobe. approximately % of the airway epithelial cells in the tracheal sections examined were transduced. we also investigated the duration of pulmonary transgene expression. in these studies, we aerosolized a hdad expressing the baboon α-fetoprotein from the k promoter into the lungs of baboons. by measuring serum bafp levels, we found that pulmonary transgene expression from transduced airway epithelial cells can be detected for at least days post-vector. these results demonstrate for the first time that exceedingly high levels of transduction of the airway epithelial cells and submucosal glands throughout all lung lobes in a large animal can be achieved with negligible toxicity resulting in long term trangene expression and should pave the way towards successful clinical cf gene therapy. inefficient gene transfer after repeat administration of most viral vectors has limited their suitability for cf gene therapy. however, lentiviral vectors are reported to be less immunogenic. this property combined with their capacity to integrate into the genome of transduced cells and, therefore, allowing gene expression for the life-time of the cell (approximately months for airway epithelial cells and theoretically unlimited for stem cells) justify further investigation of these vectors for cf gene therapy. to ensure high transduction efficiency in the lung without the need for any pre-conditioning strategies we pseudotyped a simian immunodeficiency virus (sivagm)-based vector with the sendai virus f and hn proteins (f/hn-siv-gfp) and transduced mouse nasal epithelium ( x e tu/mouse). at this titre transduction efficiency along the nasal septum was up to % when measured and days after transduction (n= /time-point). the vast majority (approx. %) of gfp positive cells across all time-points were respiratory epithelial cells followed by neuronal and sustenticular cells (approx. %) in the olfactory epithelium. interestingly, we observed two types of duration patterns of gfp expression in mouse nose. in some mice the number of gfp positive cells decreased over time (day : ± cells/section, n= , day : ± cells/section, n= ). however, in other mice strong gfp expression persisted for up to days (day : ± cells/section, n= ), significantly longer than the proposed life-span of airway epithelial cells. in order to support the idea of f/hn-siv integration into nasal respiratory stem/progenitor cells we artificially induced cell division after siv vector transduction by damaging the nasal tissue with the detergent (polidocanol), which strips of the epithelium within a few hours, while retaining the basal cell layer. the epithelium completely regenerates over the course of a few days. seven and days after the vector transduction ( x e tu/mouse, n= ) the nasal tissue was perfused with % polidocanol ( µl/mouse) and gene expression was analyzed weeks after the last detergent treatment. gfp expression was detectable in various cell types of the regenerating epithelium including ciliated and basal cells. importantly, gfp-expressing cells were more clustered after polidocanol treatment, possibly indicating origination from a common progenitor. these data suggest that, f/hn-siv integration may have occurred into differentiated airway epithelial cells as well as into stem/progenitor cells involved in the regeneration of the airway epithelium. introduction: hypersecretion of mucus is a major cause of airway obstruction in cystic fibrosis, asthma and chronic bronchitis. though mucus is a complex, non-homogeneous mixture of secretions, one group of its constituents, the mucous glycoproteins or mucins, contributes greatly to the obstruction associated with airway diseases. currently, there are a limited number of therapeutic agents available for controlling mucin overproduction and hypersecretion. the objective of this study is to develop a sensitive, cost-effective technique for reliably quantifying total mucin concentration in a robust mucin-secreting cell line for use in high throughput screening of small molecule compounds with potential to inhibit mucin secretion. methods: an enzyme-linked lectin assay (ella) was adapted using a lectin from helix pomatia (hpa), which binds specifically to n-acetylgalactosamine residues. bovine sub-maxillary mucin was used for test development and later, as a mucin standard. the test sample was "sandwiched" between hpa pre-coated onto -well microtiter plates and biotinylated hpa lectin. the plate was developed with a fluorescent hrp substrate, and measured at nm using a standard fluorescence plate reader. using the ella, a number of carcinoma (hm , a , ls , nci-h ) and immortalized lung epithelial ( hbe, cfsme) cell lines were tested to determine their responsiveness to various secretagogues. preliminary experiments indicated that the nci-h cells, derived from a human mucoepidermoid lung carcinoma, were most suitable for adaptation to high throughput studies. nci-h cells were seeded into -plates at a density sufficient to produce confluence in approximately hours. then, individual wells were incubated for hour in either control or secretagogue-containing media. supernatants were collected and mucin concentration of individual wells was determined using the ella. results: optimization of the ella provided a sensitive calibration curve from ng/ml to . mg/ml. mucin secretory ratios (sr) were calculated by comparing mucin release from test and control wells. significant (p ≤ . ) secretory ratios were obtained following incubation with utp at µm ( . ± . ), vip at nm ( . ± . ), carbachol at . mm ( . ± . ), interleukin- β at ng/ml ( . ± . ) and heparin-binding egf-like growth factor at ng/ml ( . ± . ); all data expressed as sr ± sem, n = . histamine at µm did not result in significant mucin secretion. conclusion: a relatively simple and economical assay has been developed for high throughput screening of small molecules for detecting their effects on mucin secretion. cystic fibrosis (cf) is the most common lethal genetic disease in north america, where it occurs with a frequency of one in ~ live births. it is caused by mutations in the cystic fibrosis transmembrane conductance regulator (cftr) gene, the most common being the deletion of phenylalanine (delf ). the delf mutation causes a folding defect that inhibits maturation and trafficking to the plasma membrane, however the mutant still functions as a chloride ion channel when delivered to the plasma membrane, and partial rescue ( - %) may be sufficient to alleviate disease symptoms. thus cftr is an attractive target for the development of pharmaceutical therapies. we recently described a novel, cell-based assay for protein trafficking which directly monitors expression of delf cftr on the surface of baby hamster kidney (bhk) cells and is suitable for high throughput screening of small molecule libraries. in this study we have extended the approach to natural products by studying extracts derived from marine sponges that were collected from the coasts of indonesia and papua new guinea. sponges are a particularly rich source of novel bioactives and secondary metabolites. iterative use of our high throughput assay and chemical deconvolution of active fractions yielded four alkaloids jmcg , jmcg , jmcg and jmcg . these compounds caused a detectable increase in delf cftr trafficking to the plasma membrane within h, and after exposure to pm for h all four increased surface delf cftr expression - % and enhanced camp-stimulated halide conductance. we tested a series of analogues of the alkaloids and found three others jmcg , jmcg and jmcg that also caused trafficking correction and functional ion channels in the plasma membrane. these results were confirmed in functional studies of the human bronchial epithelial cell-line cfbe stably overexpressing delf cftr. these compounds increased surface delf cftr by - % within h when treated with nm. these results identify a promising group of delf cftr trafficking correctors that are now being actively pursued as potential pharmacotherapeutics for cystic fibrosis along with other hits from compound library screening campaigns. work supported as part of the breathe program jointly funded by the ccff and cihr, and grants from cystic fibrosis foundation therapeutics, (cfft). kim chiaw, p. , ; bear, c.e. background/rationale: biosynthetic misprocessing of the major cf mutant ∆f -cftr has been proposed to result from the disruption in domain-domain interactions (du et al. nature struct and mol biol, ) that may lead to the exposure of di-arginine (rxr) retention/retrieval motifs. simultaneous site directed mutagenesis of all four rxr motifs to rxk or kxr motifs overcame retention and promoted cell surface expression of ∆f -cftr (chang et al. mol cell, ) . we hypothesize that it is possible to compete inhibitory interactions with rxr motifs exposed in ∆f -cftr by delivering synthetically derived cftr peptides bearing one or a combination of rxr motifs (cf-rxr), each conjugated to cell penetrating peptides (cpp) to permit intracellular delivery. results: confocal immunofluorescence microscopy confirms that alexa- -tagged cpp-cf-rxr peptides can be taken up rapidly (within min) by bhk cells stably expressing recombinant human ∆f -cftr. fluorophore-conjugated peptides appear to be concentrated in vesicular compartments, consistent with the current hypothesis that cpp-peptides enter cells into endosomal vesicles via macropinocytosis from which efflux into the cytosol occurs (wadia et al. nature med, ) . we determined that treatment of cell cultures with cpp-cf-rxr peptide at a final concentration [ µm] for minutes is effective in inducing cell surface functional expression of human ∆f -cftr in bhk cells as observed by a . -fold increase in cyclic amp-mediated iodide efflux over control ∆f -cftr (n= ; p < . ). this rescue is specific to the cf-rxr peptide as controls (cpp alone or peptides bearing a kxk motif in place of rxr) show no functional rescue of ∆f -cftr (n= ; p > . ). preliminary cyclic ampmediated iodide efflux results also indicate that ∆f -cftr functional rescue by cpp-cf-rxr peptide is as effective as temperature ( o c) rescue. although we hypothesize that the peptide acts to facilitate trafficking of the major mutant to the cell surface, immunoblotting studies fail to show a clear conversion of the er-form (band b) to the fully processed, band c form of the protein. currently, we are developing more sensitive assays of cell surface expression as well as evaluating the possible efficacy of this peptide in potentiating function of ∆f -cftr. discussion: future efforts include investigating the relative efficacy of peptides corresponding to the four different rxr motifs in rescuing ∆f -cftr trafficking. the promising candidate described above is currently being evaluated with respect to functional rescue of the major mutant in primary human bronchial epithelial cells obtained from patients homozygous for ∆f -cftr (joseph zabner and phil karp, university of iowa) and in cftr-∆f mice. these studies may provide the basis for future pre-clinical studies of peptide based therapies of cystic fibrosis (supported by ccff (breathe program grant) and cihr -(strategic training program in the structural biology of membrane proteins linked to disease)). several promising methods of promoting ∆f cftr escape from er degradation increase cftr chloride channel activity at the cell surface. however, increasing data from a number of laboratories indicate that in both epithelial and non-epithelial cell lines expressing ∆f cftr, the rescued chloride channel is unstable at the cell surface compared to wild-type cftr. small molecule correctors have therefore become a widely used approach to enhance ∆f cftr function at the cell surface. in most cases, the exact mechanism of these promising compounds has not been established. in the present study, we use stably transfected, polarized cfbe o-cells to elucidate the effect of two of these compounds, cfcor- and corr- a, on the surface stability of wild type and low temperature rescued ∆f cftr. specifically, we cultured cells expressing ∆f cftr at c for hours to establish a large pool of surface-expressed ∆f cftr; we then monitored cftr internalization rates and half-life with and without small molecule treatment using surface biotinylation-based assays, performed as described previously (jurkuvenaite et al. j. biol. chem. : , ) . as a control, internalization and half-life of transferrin receptor was also followed. our results indicate that both cfcor- and corr- a slow ∆f cftr endocytosis from ~ %/ . min to ~ %/ . min, and this is similar to wild type cftr internalization rates. neither wild type cftr nor transferrin receptor internalization rates were affected by corrector treatment, suggesting that the effects of the correctors were specific for ∆f cftr. in the surface half-life studies, both correctors doubled ∆f cftr halflife from to hours, but they did not correct the half-life to wild type levels (~ h). again, the correctors had no effect on either wild type cftr or transferrin receptor surface half-lives. our results suggest that small molecule correctors may not only rescue cftr from erad during biogenesis, but can contribute to the stabilization of ∆f cftr at the cell surface, and this effect seems to be ∆f cftr-specific. the ∆f mutation in cftr causes defects in chloride channel gating and cellular processing. cell cultures models suggest the need for both a potentiator and corrector to restore ∆f -cftr channel function and processing, respectively. previously, we identified several classes of potentiators, including 'phenylglycines', which probably bind to cftr at the cell surface and restore normal channel gating (pedemonte et al., mol. pharm. ; : - . we also identified several classes of correctors, including 'bisaminomethylbithiazoles', which may bind to ∆f -cftr in the er and partially restore its folding and plasma membrane targeting (pedemonte, et al., j. clin. invest. ; : - . these compounds were identified from screening a diverse collection of , small molecules; the most active potentiator was phenylglycine pg { -[( - h-indol- -ylacetyl)-methyl-amino]-n-( -isopropylphenyl)- -phenyl-acetamide} and the most potent corrector was the bisaminomethylbithiazole corr- a {n- ( -[ -chloro- -methoxyphenylamino]- '-methyl- , '-bithiazol- 'yl) benzamide}. here we synthesized and characterized a hybrid molecule (corr- a-linker-pg ) containing both corrector and potentiator fragments. based on sar data of potentiators and correctors, a hybrid molecule was designed that incorporated an enzymatically hydrolysable linker to generate and deliver the potentiator and corrector-linker fragments. using synthetic organic chemistry we synthesized a hybrid molecule containing a pg -oh moiety and a corr- a-linker-cooh moiety, linked with an ethylene glycol spacer through an ester bond. the potentiator and corrector fragments (after cleavage) had low micromolar potency or better. however, the intact hybrid molecule was inactive, likely because of its large size ( daltons) and hence poor cell penetration. as proof-of-principle evidence that the hybrid molecule can be hydrolyzed to the active fragments, treatment with carbonic anhydrase or rodent stool specimens gave the appropriate potentiator and corrector-linker fragments identified by lcms. smallmolecule corrector-potentiator hybrids have potential utility as single drug therapy for cf caused by the ∆f mutation. supported by cff and nih. cftr is a chloride channel gated by atp binding and hydrolysis at its two nucleotide binding domains (nbd and nbd ). the two nbds may dimerize in a head-to-tail configuration, as observed in other abc transporters, forming two atp binding pockets (abp and abp ), with the atp molecules buried at the dimer interface. abp , formed by the walker a and b motifs of nbd and the signature sequence of nbd , was proposed as the site critical for the atp-dependent opening of the cftr channel, while abp (consisting of the walker a and b motifs of nbd and the signature sequence of nbd ) may contribute to the stabilization of the open channel conformation. g d, the third most common cf-associated mutation, is characterized by a very low open probability, and patients carrying this mutation present a severe phenotype. this mutation is located in the signature sequence of nbd (and thus in abp ). we have recently characterized this mutant channel and found that its low activity is atp-independent. this behavior corroborates with the idea that the occupancy of abp by atp is crucial for catalyzing the opening of the channel. interestingly, we now found that a high affinity atp analog, n -( -phenylethyl)-atp (p-atp), increases g d currents primarily by increasing the open time of the channel. this effect of p-atp is reduced when p-atp was applied together with atp, suggesting a competition between atp and p-atp for a common binding site. introducing the mutation y g (located at abp ), which reduces by more than -fold the atp-binding affinity in wild-type channels, does not alter the effect of p-atp in the g d/y g mutant. in contrast, when we introduce the mutation w g (located at abp ) in the g d background, the effect of p-atp is reduced remarkably, suggesting that abp is the binding site where p-atp binds to increase the activity of g d channels. these new results further confirm the idea that nucleotide binding at abp could stabilize the open channel conformation. since the competition experiments show that atp and p-atp are readily exchanged in the binding site, we conclude that the atp molecule is not occluded in abp , at least under the g d background. our observation that p-atp enhances the g d activity by binding at abp implicates that abp can potentially be a target for drugs to bind and increase the channel activity. supported by cff grant to s. bompadre and nih grants to t.-c. hwang (hl r , dk r ) and s. bompadre (dk k ). van goor, f.; hadida, s.; grootenhuis, p. vertex pharmaceuticals, san diego, ca, usa several cf-causing mutations, including ∆f , g d, and r h, cause gating defects in cftr that decrease the open probability (po) of the channel, resulting in decreased cl-secretion across epithelia of multiple organs, including the lung. a therapeutic strategy for the treatment of cf is to develop pharmacological potentiators that increase po leading to increased fluid transport in affected tissues. vx- was identified through cell-based screening and chemistry optimization as a potent potentiator of cftr, including ∆f -, g d-, and r h-cftr. in single-channel recordings of cftr expressed in fisher rat thyroid cells, vx- increased the po of ∆f -cftr from . ± . to . ± . . the po of g d increased from . ± . - . ± . . these levels are similar to the po of wild-type cftr, indicating that vx- restores normal gating at the single channel level. to assess the potency and efficacy of vx- in native airway cells, cl-secretion was monitored in ussing chamber studies using human bronchial epithelia (hbe) cultured from the bronchi of cf and non-cf donors. in the absence of the potentiator vx- , cftr-mediated clsecretion in ∆f /∆f or g d/∆f -hbe reached a maximum amplitude of . ± . and . ± . ua/cm , respectively. this is ~ % of that observed in wild-type-hbe ( ± ua/cm ) and consistent with the low level of residual cftr activity reported in some individuals with severe cf disease. in ∆f /∆f -hbe, vx- caused a -fold increase (peak response; . ± . ua/cm ) in cftr mediated cl-secretion with an ec of ± nm, whereas in g d/∆f -hbe, cl-secretion increased fold (peak response; ± ua/cm ) with an ec of ± nm. the improved efficacy observed in g d/∆f -hbe is consistent with a higher cell surface density of g d-cftr compared to ∆f -cftr, which has defective trafficking to the cell membrane. vx- was found to be highly selective for cftr and to have good oral bioavailability in multiple species with a half-life of - hours. the biological and pharmacokinetic data support the clinical development of vx- for the treatment of cystic fibrosis. we would like to acknowledge the cfft for their support and dr. joseph pilewski for providing cf hbe. the pari eflow ® rapid nebulizer is a member of the "soft mist" inhaler class for the administration of aerosolized antibiotics that eliminates the need for a compressor. objectives: to compare the pharmacokinetics and safety of tobramycin inhalation solution (tobi ® ) delivered via the pari eflow ® rapid electronic nebulizer vs the pari lc ® plus jet nebulizer with compressor. methods: we compared tobramycin levels in sputum and serum, incidence of bronchospasm and frequency of adverse events (ae) of mg tobi, administered twice-daily for weeks from these two delivery systems in a randomized, crossover study in cystic fibrosis patients. blood and sputum samples were collected over a dosing interval after the first and last dose on each device (days and ). tobramycin serum and sputum area under the concentration-time curves (auc) were derived and the therapeutic ratio (mean sputum auc / mean serum auc) calculated. pulmonary function tests were performed before and min after nebulization on days and , and ae were recorded. results: the total nebulization time was reduced by half for the pari eflow rapid vs pari lc plus ( . ± . min vs . ± . min [p< . ] on day , and . ± . vs . ± . [p< . ] on day , respectively). tobramycin predose serum levels > µg/ml or cmax > µg/ml, predefined as a potential for increased risk of systemic toxicity, were not exceeded in any patient during use of either nebulizer. as tabulated below, mean systemic exposure to tobramycin from the pari eflow rapid on day was slightly lower, by %, whereas mean sputum exposure was higher, by %, compared with the pari lc plus. consequently, the therapeutic ratio was nearly -fold higher for the pari eflow rapid due mainly to higher sputum concentrations. adverse events occurred in patients using the pari lc plus nebulizer (primarily headache and abdominal pain) and in patients using the pari eflow rapid nebulizer (primarily headache, cough, dyspnea). on day , no clinically relevant bronchospasm (defined as ≥ % decrease in fev ) was observed for either treatment. the mean percentage change in fev at mins from predose on day was - . % for the lc plus and - . % for the eflow rapid. conclusions: tobi delivered via the pari eflow rapid electronic nebulizer required shorter delivery times with similar safety and a higher therapeutic ratio (sputum/serum exposure) compared with the pari lc plus jet nebulizer. sponsored by novartis pharma ag which acknowledges support from pari gmbh introduction alpha- antitrypsin (aat) is a protein protecting lung tissues by inhibition of neutrophil elastase. the function of neutrophil elastase is to digest bacteria and other foreign objects in the lungs. in a person deficient of aat, the neutrophil elastase acts uncontrolled, destroying healthy tissue. the result of the damage is emphysema, which progresses if not treated. patients show a specific impaired breathing pattern of short inhalation followed by prolonged exhalation. current therapy consists of weekly i.v. infusion of aat of about - mg/kg body weight. by this method only about % of the administered dose is estimated to reach the lungs. since aat can currently be derived only from human blood serum by an expensive purification process, the worldwide supplies are limited and do not allow to treat all patients. an improvement of the delivery efficiency is highly desirable. inhaled treatment would offer a targeted therapy by delivering aat directly to the lungs and allow for a better use of the limited drug. the eflow ® , a novel electronic nebulizer, is more efficient than conventional nebulizers. it operates by means of a perforated vibrating membrane, applying no or only little stress to the nebulized substances. hence it is well suited for the pulmonary delivery of proteins. it has been shown previously, that a % aat solution can be nebulized by eflow ® without a loss of biological activity. we investigated two eflow ® configurations comparing the in-vitro delivered dose for different breathing patterns. method a highly purified % aat preparation (kamada ltd, rehovot, israel) was aerosolized with the eflow ® electronic nebulizer. initial studies were conducted using the l configuration of eflow ® . delivery performance is compared to a specially designed xl configuration, with increased aerosol chamber volume. aerosol delivery efficiency was determined by breath simulation using an emphysema breathing pattern (tidal volume ml, breaths/min, inh:exh ratio = : . ) generated by a pari compas™ breath simulator. as reference, a standardized regular breathing pattern (tidal volume ml, breaths/min, inh:exh = : ) was also investigated. the respirable fraction (drug in droplets < and . µm) was determined using the andersen cascade impactor at . l/min. samples were analysed for aat activity by an elastase inhibition assay. when the eflow ® l configuration was investigated for the standardized breathing pattern, a delivered dose (dd) of ± % of the loaded dose ( mg) was found. this value was reduced to ± % when the emphysema pattern was applied. with the new eflow ® xl configuration an improved dd of ± % was obtained. furthermore, when the emphysema pattern was applied, the dd did not drop strongly, but remained at values above %. with % of aerosol droplets in the respirable size range (< µm) and % < . µm, a high % of aat can be expected to reach both, the central and peripheral lungs of patients. conclusion inhaled aat has the potential to significantly improve the efficiency of aat replacement therapy. a customized eflow ® electronic nebulizer produced delivered doses > % even when breathing patterns of patients with impaired breathing capabilities were mimicked. the formulation is optimized for rapid aerosol administration through use of higher drug concentrations and is taste-masked. this study was conducted to evaluate the aerosol performance of novel higher dosed levofloxacin inhalation solutions in a customized eflow ® electronic nebulizer. methods two concentrations of mp- ( mg/ml and mg/ml of levofloxacin) were nebulized at fill volumes of and ml using the eflow ® l nebulizer configuration. the in-vitro delivered dose (dd) was determined by breath simulation using a standardized breathing pattern (tidal volume ml, breaths/min, inh:exh ratio = : ) generated by a pari compas™ breath simulator. the geometric droplet size distribution was determined by laser diffraction (ld). the aerodynamic droplet size distribution was determined using the andersen cascade impactor (aci). the invitro respirable dose (rd) was calculated by multiplying the respirable fraction (rf, %droplets < µm) derived from the cascade impaction experiment and the dd derived from breath simulation. nebulization time was determined by the electronic shut-off of the eflow ® control unit. all tests were conducted for three devices in duplicate, each (n= ). the eflow ® electronic nebulizer delivered mp- at both concentrations and fill volumes with equal efficiency, obtaining in-vitro dds of around ± %. the average nebulization times were between and minutes for the ml fill volume and between and minutes for the ml fill volume and were also independent of the concentration in the investigated range. determination of the mass median diameter (mmd) by ld resulted in values between . and . µm. values of the mass median aerodynamic diameter (mmad) obtained by cascade impaction were similar (between . and . µm). the average respirable fraction was in the range between % and % for both experimental methods. conclusion the eflow ® electronic nebulizer can be customized to deliver high doses of mp- . the short nebulization times achieved are patient friendly and should support high patient compliance. mp- , with the potential for once daily administration, provides significant advantages over currently avail-able aerosol antibiotics. clinical evaluation of mp- delivered by the eflow ® nebulizer in cf patients is in progress. lavange, l. ; engels, j. ; accurso, f.j. . university of north carolina, chapel hill, nc, usa; . inspire pharmaceuticals, inc., durham, nc, usa; . university of colorado, denver, co, usa introduction: percent change from baseline in a continuous outcome variable, such as lung function, is a useful descriptive measure in therapeutic clinical trials. while often favored by clinicians as the primary efficacy measure, the use of percent change as the basis for statistical comparisons raises a number of issues. defined as *(post-pre)/pre, where pre and post represent baseline and follow-up values, respectively, percent change is a ratio of random variables and as such, does not follow a normal distribution. analyzing percent change with standard methods can result in inefficiences and may be inappropriate without a suitable transformation (e.g., logarithmic). furthermore, the treatment effect estimated on the transformed scale is difficult to interpret. the purpose of this abstract is to describe an alternative method for analyzing percent change and illustrate its utility in cf clinical trials. methods: probability plots are generated to compare the distributions of percent change in fev from baseline to study endpoint among treatment groups. the plots are similar to kaplan-meier curves, with percent change on the horizontal axis in lieu of survival time. the percentage of patients on the vertical axis depicts the cumulative percentage of patients who had a percent change at least as great as the corresponding value on the horizontal axis. a log-rank test statistic provides an overall test for any difference in distributions among treatment groups. the methods are illustrated using data from a -day, phase , multicenter, randomized, double-blind, placebo-controlled clinical trial of denufosol in cf patients with a screening fev ≥ % predicted. results: a probability plot of denufosol (active doses combined, n= ) vs. placebo (n= ) is shown below. more denufosol patients ( %) experienced improvement compared to placebo ( %). the log rank test, adjusted for study site, showed that the overall distributions of percent change in fev between denufosol and placebo were significantly different (p-value= . ). conclusions: the proposed methods provide a convenient means for assessing differences in percent change and may be useful in cf clinical trials. comparisons of both a descriptive and inferential nature can be made with minimal assumptions, thereby avoiding the pitfalls in analyzing percent change with standard techniques. the methods are easy to implement with existing software packages (e.g., sas). trial supported by inspire pharmaceuticals, inc. and cf foundation therapeutics, inc. chen, x. , ; davis, p. compacted dna nanoparticles formulated with pegylated polylysine and plasmid dna transfect airway cells in vivo at high efficiency, so they have great potential for gene therapy. the mechanism by which dna nanoparticles enter the cell and get expressed is not well understood. we previously showed that rhodamine labeled dna nanoparticles enter primary human tracheal epithelial (hte) cells within min, and accumulate in the nucleus in hr, where the transgene can be expressed. dna nanoparticles do not enter via clathrin-mediated endocytosis (cme), which leads to the degradation of internalized material in lysosomes. further characterization of the kinetics and trafficking pathway may allow us to improve the formulation of the nanoparticles and facilitate its gene delivery. as they do in hte cells, rhodamine labeled dna nanoparticles enter hela and hbeo-, an immortalized human bronchial epithelial cell line, within min, and by - hr accumulate in the nucleus, especially the nucleolus, where they colocalize with nucleolin. expression of the gfp reporter gene in the nanoparticles was observed at hr, which confirms the functionality of the labeled dna nanoparticles. cellular entry is energy dependent, as little or no intracellular rhodamine was observed at °c by hr. we also applied rhodamine labeled nanoparticles and biotin-conjugated transferrin, a marker for cme, simultaneously. little colocalization was observed during a min to hr time course, so the nanoparticles do not follow this pathway. we then found a direct and strong relationship of cell surface nucleolin and the ability of the cells to take up and express dna nanoparticles. nucleolin directly binds to dna nanoparticles with kd= . nm. manipulations of cell surface nucleolin in hela cells affect transfection of dna nanoparticles with a positive correlation. we therefore costained nucleolin in both hela and hbeo-cells treated with the rhodamine labeled dna nanoparticle. the rhodamine fluorescence colocalizes with nucleolin both in the cytoplasm at early time points and in the nucleus/nucleolus at hr and hr, which further supports that nucleolin might be associated with the nanoparticles during their trafficking. to confirm this association, we performed uptake experiments with ms- , a mouse monoclonal antibody against nucleolin, alone or with rhodamine labeled nanoparticles over a min to hr time course. the ms- antibody enters hela cells at similar kinetics to the nanoparticles. at min, we observed substantial amount of cytoplasmic staining, while nuclear staining tends to increase by time until hr. it has little or no colocalization with transferrin in the cytoplasm, which suggests that this antibody also enters the cell via a pathway other than cme. in contrast, it showed extensive colocalization with dna nanoparticles in both cytoplasm and nucleus at all time points, which suggests that cell surface nucleolin and dna nanoparticles are associated or at least in close vicinity during their trafficking inside the cell. therefore, we suggest that dna nanoparticles enter the cell by binding to cell surface nucleolin and enter the cell via the same pathway as nucleolin antibody. chen, x. , ; davis, p. dna nanoparticles are non-viral gene delivery vectors in clinical trial for treating genetic disorders including cystic fibrosis. we previously discovered that cell surface nucleolin serves as a receptor for the dna nanoparticles, and is important for their gene delivery efficiency. as nucleolin has no signal sequence or membrane-spanning domain, it is not clear how nucleolin are expressed on the outer surface of plasma membrane or what signals increase its surface delivery. we initially observed that the transfection efficiency of dna nanoparticles in hela cell decreases by . % following hr serum-free medium treatment, which reduces cell surface nucleolin by . % as determined by cell surface biotinylation and streptavidin bead pulldown. since serum depletion inhibits cell proliferation and affects cell cycle progression, we examined the level of cell surface nucleolin at different stages of cell cycle. hela cells were arrested at s phase by high concentration of thymidine, and allowed to progress synchronously through cell cycle after removing this block. surprisingly, we observed substantial increase of cell surface nucleolin at the onset of m phase, about hours after release of thymidine block. it has been reported that nucleolin is phosphorylated by a cell cycle dependent kinase (cdk) cdc , and has eight consecutive cdk phosphorylation sites. therefore it is appealing to speculate that phosphorylation by cdc might increase the targeting of nucleolin to the cell surface. we then determined the potential export signal in nucleolin by serial deletions. deletion of c-terminal aa of nucleolin, including the c-terminal glycine/arginine rich (gar) domain and the four rna recognition motifs (rrm) does not affect its arrival at the cell surface. the n-terminal aa of nucleolin is sufficient to target a gfp protein to the cell surface. in contrast, when we further delete the cdk phosphorylation sites from aa to , cell surface expression of nucleolin is significantly diminished. furthermore, nucleolin lacking the n-terminal aa is not present on the cell surface. therefore, phosphorylation on the cdk sites may serve as a signal to enhance the cell surface expression of nucleolin. there has been recent interest in dry powder inhaled mannitol as a therapeutic agent in patients with cystic fibrosis (cf). it is has been shown to increase mucociliary clearance (mcc) by airway rehydration. whilst there has been one short term clinical trial of mannitol in adult subjects [ ] , to date no studies have been conducted looking at its potential as a therapy in children with cf. it could be argued that children may have the potential to benefit most from a therapeutic agent that acts relatively proximally in the cf pathogenic pathway. the aim of this study was to determine acute tolerability of inhaled mannitol in children with cf. we recruited children (aged to years) with cf. inclusion criteria were either rhdnase treatment or an fev > % and < % of mean predicted normal value. bronchial provocation challenges with incrementally increasing doses ( , , , , , , mg) of dry powder mannitol were carried out. subjects were pre-treated with bronchodilator minutes prior to the challenge ( mcg of salbutamol or mg of terbutaline). fev was measured following each dose increase up to a maximum cumulative dose of mg. oxygen saturation monitoring was carried out throughout. a positive challenge was defined by a drop in fev of > % from baseline. these subjects received bronchodilator treatment and had spirometry repeated at minute intervals until fev returned to within % of baseline. those children with a negative challenge had spirometry repeated minutes post-completion of the challenge results mean baseline fev was % predicted ( - , sd . ). / subjects ( %) had a positive challenge. mean pd (dose of mannitol required to cause a % reduction in fev ) was mg ( - , sd ). the mean time to complete the challenge was minutes ( - , sd . ) for negative challenges and minutes ( - , sd . ) to pd for those subjects with a positive challenge. we found no association between a positive challenge and age, sex, weight, height, baseline fev , pseudomonas aeruginosa colonization, bronchodilator reversibility, previous aspergillus fumigatus sen-sitization, atopy or corticosteroid use. there was a non-significant trend for lower fef - (means . versus . ; p= . ) and higher prevalence of aspergillus fumigatus cultured in sputum at baseline ( / versus / children; p= . ) amongst those children who went on to have a positive challenge. there was no significant drop in oxygen saturation in either group. although cough was common during the challenge, other adverse events were infrequent. we find that % of children with cf could not tolerate inhaled mannitol as compared with % of adult subjects as reported in the previous study [ ] . we could not identify factors predictive of a positive mannitol challenge in these patients. the most common cause of cystic fibrosis (cf) is the deletion of phenylalanine (∆f ) in the cf transmembrane conductance regulator (cftr) chloride channel [ ] . a major problem with ∆f cftr is that the protein is defective in folding so that little mature protein is delivered to the cell surface [ ] . expression of ∆f cftr in the presence of small molecules known as correctors or pharmacological chaperones can increase the level of mature protein [ ] [ ] [ ] [ ] . unfortunately, the efficiency of correctorinduced maturation of ∆f cftr is low and other approaches are needed to increase the therapeutic value of correctors. we postulated that expression of ∆f cftr in the presence of multiple correctors that bound to different sites in the protein may have an additive effect on maturation. in support of this mechanism, we found that expression of p-glycoprotein processing mutants (cftr's sister protein) in the presence of two compounds that bind to different sites (rhodamine b and hoechst ) had an additive effect on maturation. therefore we tested whether expression of ∆f cftr in the presence of combinations of three different classes of corrector molecules would increase its maturation efficiency. it was found that the combination of the quinazoline vrt- together with the thiazole corr- b or bisaminomethylbithiazole corr- a doubled the steady-state maturation efficiency of ∆f cftr (about % of total cftr was mature protein) compared to expression in the presence of a single compound. the additive effect of the correctors on ∆f cftr maturation suggests that they may directly interact at different sites of the protein. the use of multiple correctors has the potential to increase the therapeutic value of pharmacological chaperones. pharmacol. , - . the cftr protein is expressed at the surface of the airway epithelium, where it plays a critical role in maintaining airway hydration by secretion of chloride. for gene therapy of cf lung disease to be successful, it is critical that the endogenous transgene be expressed in the correct cell type and at levels sufficient to restore normal function. the expression of high levels of cftr has resulted in cftr mediated chloride secretion in a wide range of experimental systems. however, the cell type or types that need to be corrected and the level of cftr expression required to ameliorate the pulmonary manifestations of cf are not yet clear. because cftr has been localized to the ciliated cells of human airway epithelial cells, we have been investigating the use of ciliated cell-specific promoters to improve the efficiency and safety of gene therapy for cf and other airway diseases. in previous studies a fragment of the human foxj promoter was shown to target transgene expression specifically to ciliated cells. however, expression of cftr from this promoter in transgenic cf mice did not significantly improve the cf phenotype, as measured by the nasal potential difference (pd) technique (ostrowski et al, ) . this may be due to the inability of this promoter to correct the olfactory epithelium, which comprises approximately % of the mouse nasal cavity. in cf mice, the olfactory epithelium exhibits sodium hyperabsorption and an absence of cftr mediated chloride secretion, similar to the respiratory epithelium (grubb et al, ) . in this work, we used a fragment of the ciliated cell-specific promoter foxj to drive cftr expression specifically in human ciliated cells. replication deficient adenovirus expressing either egfp or cftr from the foxj promoter were used to transduce well-differentiated cultures of human cf cells following treatment with c to disrupt tight-junctions. cultures were studied hours after treatment, and those treated with ad-foxj /egfp showed strong expression of egfp that was dependant on ciliated cell differentiation. rna analysis demonstrated strong expression of cftr from the foxj promoter, and western blotting demonstrated levels of protein that were much greater than the level in normal airway cells (> -fold). immuno-localization of cftr with specific antibodies resulted in a strong signal at the apical membrane of ciliated cells. cultures treated with ad-foxj /cftr demonstrated a significant increase in forskolin-stimulated short circuit current (isc; . +/- . µa/cm , mean +/-sem, n= ), that was approximately -fold greater than the response in ad-foxj /egfp treated cultures ( . +/- . µa/cm , n= ). the increase in isc was blocked by inh , an inhibitor of cftr mediated secretion. under these conditions, ad-foxj /cftr restored approximately % of the forskolin response of normal human airway cells ( . +/- . µa/cm , n= ). because patients expressing low levels of normal cftr mrna ( - %) have mild disease symptoms, these studies demonstrate that the incorporation of the ciliated cell-specific foxj promoter into gene therapy vectors may be useful for treatment of cf. supported by nhlbi ro hl and the cff. there is a compelling need for safe and effective ai therapy for cf lung disease. low-dose methotrexate (mtx) has been used to treat inflammatory diseases and its use has been reported in cf. encouraging results previously described regarding mtx in cf prompted this study. the study objective was to determine if mtx safely reduces inflammation in the airways of cf patients. methods: this was a single-center, open label study of mtx in stable cf patients with mild to moderate lung disease. baseline levels of neutrophils, free elastase, pro-inflammatory cytokines, and bacteria were determined from an induced sputum specimen. subjects received mg/m /week of oral mtx (single dose). after weeks of treatment, a second induced sputum specimen was obtained for the same inflammatory indices. within subject comparisons (end of treatment vs baseline) were performed for the following primary endpoints: total white cell and neutrophil counts, percent neutrophils, and concentrations of free elastase, il- , il- , tnf-α, and il- β. sputum quantitative microbiology was obtained at baseline and end of therapy. routine laboratories and spirometry were performed monthly. pharmacokinetic testing was performed on the final day of treatment. results: thirteen subjects were screened with started on mtx. six completed the protocol and withdrew early secondary to adverse events (gastrointestinal [gi] complaints and pulmonary exacerbation). five of subjects completing the protocol had declines in pulmonary function: mean change in fvc was - . l (range - . to - . ); mean change in fev was - . l (range - . to - . ); change in fef - was . l (range - . to . ). mean change in weight was - . pounds (range - to . ). mean change in free elastase was . µg/ml (range - . to . ; sd . ). mean change in il- was pg/ml (range - to ; sd ). similar results were obtained for the other cytokines. analysis of cytology specimens is pending. esr and crp did not show significant changes. two subjects completing the protocol had significant gi side effects, including requiring admission for severe abdominal discomfort. quantitative microbiology specimens revealed an increase in colony counts in subjects, decreases in , and mixed results in the th. other safety laboratories were not remarkable. pharmacokinetic studies are pending. complete statistical analysis and safety assessment are also underway. conclusions: the small sample size of this study precludes definitive conclusions regarding the safety or efficacy of mtx. however, the data suggest that ) induced sputum inflammatory indices can be used to assess an ai in clinically stable patients, and ) mtx therapy may not be beneficial, and may be difficult to tolerate as along-term ai therapy for cf lung disease. additional analyses of cell counts and microbiology from this study are ongoing, and may provide additional insight into the effect of mtx in the cf lung. sponsored by the cf foundation , a cellular component of many gram negative bacteria (e.g. pseudomonas aeruginosa), is a common airway stimulus. dampening the inflammatory response in cf reduces the progressive decline in lung function, so anti-inflammatory agents have become both a cornerstone of cf clinical care and a focus of therapeutics research development. reactive oxygen species [ros] can play a role in proinflammatory signaling, including the activation of nuclear factor κb [nfκb] . research has demonstrated increased oxidative stress in cf epithelial cells, potentially highlighting one of the mechanisms responsible for the exuberant inflammation observed. we have previously shown that cddo, a novel anti-inflammatory agent, significantly limits nfκb activation in cftr deficient cell culture models at nanomolar concentrations. using cf mice, we have also shown that intra-tracheal cddo limits neutrophil accumulation and the concentrations of proinflammatory cytokines and chemokines in bronchoalveolar lavage [bal] fluid in response to lps. the synthetic triterpenoids have been shown to activate the nrf transcription factor, thereby inducing several genes involved in redox balance. we now present data demonstrating that cddo may inhibit inflammation in models of cf pulmonary disease by reducing the oxidative stress within cells. methods: b . s -cftr tm mrc cf mice received µl of µm cddo in pbs with %dmso or vehicle (control) daily. drug was administered intratracheally with an atomizer (penncentury) for three days before stimulation. in separate experiments, all mice received µg lps or free pseudomonas aeruginosa intratracheally. mice were sacrificed by co asphyxiation and cardiac puncture hours after stimulation and bal, serum and lung tissue were obtained. the lungs were incubated in lysis buffer containing protease inhibitors and immediately frozen. upon thawing, the tissue was homogenized and whole lung protein extracted. one mg of lung protein from each experimental animal was separated by two dimensional gel electrophoresis. gels were then imaged and analyzed with pdquest to identify protein spots that were differentially expressed in drugtreated mice compared with controls. spots of interest were excised from the gels, trypsin digested and analyzed by liquid chromatography/tandem mass spectroscopy. results: over proteins with greater than -fold differences in expression were identified. we categorized proteins appearing in repeated analyses and identified several affecting intracellular redox regulation; including glutathione s-transferase mu and , peroxiredoxin and , enolase (alpha non-neuron), contrapsin, alpha- antiproteinase and cu/zn superoxide dismutase. these proteins were expressed at significantly greater concentration in the lungs of mice treated with cddo rather than vehicle. conclusion: cddo exhibits anti-inflammatory effects in mouse models of cf pulmonary disease and one potential mechanism for this effect may be the upregulation of reducing proteins to combat oxidative stress. methods: lpc ( ul of . %, . % or . % (w/v) in pbs) was administered (trans-orally via a cannula) to trachea of c l/b mice. in sheep ( of completed) we targeted delivery of . ml of . % lpc dissolved in pbs to the main bronchus of the right lung at branch in a month old sheep (bronchoscope, via tracheostomy). one hour later ul (mice) or . ml (sheep) of a lv-lacz vector ( . x tu/ml) was administered in the same manner. one week post-exposure lungs were inflation-fixed in situ, removed, stained using x-gal, sectioned, and counterstained with saf-o or h/e. results: no lacz gene expression (blue cells) was found in lungs of control (pbs-treated) mice or in the left (untreated side) of sheep lung. blue cells were found in scattered punctuate groups, or in lines of stained cells in mice and sheep. mice given the highest lpc dose ( . %) had extensive gene transfer in larynx, trachea, carina, and in large, middle and small airways of most lobes of the lung, reaching % airway perimeter cell transduction in one animal. with decreasing lpc dose the distribution and number of blues cells was reduced, but transduced epithelial cells including nonciliated columnar cells, ciliated cells, and basal cells were seen in all cases. in sheep, blue cells appeared in the right main bronchus between branch and branch , and in the branch airway, and with highest expression found near branch and . cross-sections revealed that primarily ciliated columnar cells and basal cells (and no goblet cells or macrophages) were transduced. conclusions: lv gene transfer into mouse or sheep lung can be enhanced by pretreatment with lpc. in mice this early data suggests an lpc dose-dependency. for sheep, we await further results to confirm the encouraging finding in this first study; however, it does confirm that gene transfer into the airways of large animals with a lung size similar to humans can also be achieved using lpc pre-treatment and a vsv-g pseudotyped lentivirus. the transduction of both ciliated and basal lung epithelial cells in-vivo in both models is an encouraging funding for the development and understanding of our continuing efforts to produce life-long airway gene transfer suitable for a cf airway gene therapy. supported by: nh&mrc, usa cff, sa channel crf, philanthropic donations. increased airway na+ absorption is a characteristic abnormality in cystic fibrosis (cf), and is thought to play a key role in the pathogenesis of cf lung disease. we have previously demonstrated that mimicking na+ hyperabsorption by overexpression of βenac in mouse airways results in airway surface liquid (asl) volume depletion and reduced mucus clearance causing a spontaneous cf-like lung disease with high pulmonary mortality, and airway mucus plugging, mucous cell metaplasia and chronic airway inflammation in surviving βenac-transgenic (βenac-tg) mice (mall et al., nature med. : , ). in the present study, we used βenac-tg mice to test if inhibition of increased airway na+ absorption by the classic enac blocker amiloride has therapeutic effects on cf-like lung disease in vivo. specifically, we determined the effects of 'early' and 'late' enac blocker intervention on mortality, airway mucus obstruction and inflammation by starting intrapulmonary amiloride therapy in βenac-tg mice either at birth, i.e. prior to the onset of lung disease, or after mucus obstruction and inflammation had established. to achieve this goal, newborn, day, or week old βenac-tg mice and wild-type littermate controls were treated by intranasal instillation of amiloride ( mmol/l; µl/g body weight, tid) or vehicle (h o) alone for a period of days. initial deposition studies showed that this treatment protocol resulted in pulmonary amiloride concentrations sufficient for inhibition of enac. during amiloride therapy, growth and survival were monitored, and any loss in body volume due to diuretic side effects was substituted by subcutaneous injection of isotonic saline (nacl . %). after the day treatment period, mice were euthanized, bronchoalveolar lavage (bal) was performed to determine inflammatory cell counts, and lungs were processed for histology and morphometry to quantitate airway mucus obstruction and mucous cell metaplasia. we show that amiloride significantly reduced pulmonary mortality by~ % (p < . ), when therapy was started in newborn βenac-tg mice. further, early amiloride treatment significantly reduced airway mucus content (p < . ), mucous cell numbers (p < . ), and bal eosinophils (p < . ) compared to vehicle treated βenac-tg mice. in contrast, amiloride therapy had no benefits on airway mucus obstruction, mucous cell metaplasia or inflammation, if treatment was started in day, or week old βenac-tg mice with established lung disease. taken together, our results are consistent with previous human studies where amiloride lacked therapeutic benefits in cf patients with established lung disease, and demonstrate for the first time that early inhibition of na+ hyperabsorption is an effective therapy for cf-like lung disease in vivo. these results warrant an evaluation of more potent and longer acting amiloride derivatives in chronic lung disease in mice, and a clinical evaluation of preventive enac blocker therapy in human trials. supported by cff (mall g ) and ec (mext- - objective: hypertonic saline aerosol delivered intranasally is currently being studied to enhance mucociliary clearance. there is evidence of patients' reluctance to use concentrations above % due to potential discomfort. our study was performed to determine the short term tolerance of . % and % hypertonic saline versus normal saline delivered intranasally via a nebulizer/compressor system (pari sinustartm, pari respiratory equipment, midlothian, va). methods: using the sinustartm nasal aerosol delivery system, we administered concentrations of saline solution, ( . %, . %, %) to healthy, adult volunteers for minutes each. a washout period of minutes between treatments allowed volunteers to wipe their nose and cleanse their mouth with water. a question self-administered questionnaire was completed following each treatment using a point scale ( =high tolerability; =low tolerability). burning sensation, cold, cough, throat irritation, runny nose, and overall comfort were measured. the order of treatments was randomized and volunteers were blinded to the concentration. data: for all measurements . % and % were equally well tolerated compared to normal saline with burning, cough, and throat irritation measuring . or below. no variable measured more than . . out of volunteers stated they would continue this treatment on a regular basis. conclusions: our study indicates that during the time of treatment nasal aerosol delivery of hypertonic saline up to % is well tolerated in healthy adults. there appears to be no issue of discomfort associated with hypertonic saline that would prevent nasal aerosol treatment compliance. our overall research goal is to discover new drugs for clinical treatment of cystic fibrosis (cf) that will correct the biochemical defect in the predominant cf mutation, the ∆f form of the cystic fibrosis transmembrane conductance regulator (cftr) protein, which accounts for over % of all cf cases. this mutation leads to a misfolded protein which is rapidly degraded as well as changes in its function and half-life at the cell surface. however, it has been proposed that only a fraction of the normal surface expression level is needed to provide a clinically significant impact on the disease. thus, from a pharmacological standpoint, strategies that can partially but effectively correct these defects would be expected to have a clear clinical benefit to these cf patients, and these strategies may also be applicable to other cftr mutations. we previously demonstrated that several members of a particular class of related drugs, the anthracyclines, anthraquinones, and anthracenediones, significantly increased cftr cell surface expression and function in cell culture. in particular, we demonstrated that a non-cytotoxic concentration of doxorubicin (dox), a model anthracycline drug, significantly increased total cellular and membrane-associated cftr protein levels and cftr-associated chloride currents in human colon cancer t cells, and also caused a two-fold increase in ∆f cftr-associated chloride current in a canine mdck-c -derived cell line that expresses a stably transfected copy of human ∆f cftr. our previous studies also demonstrated that dox is able to impart structural integrity to ∆f cftr, increasing its half-life and decreasing its proteolytic sensitivity, which is indicative of efficient folding. additionally, two other structurally related drugs, i.e., the anthracycline, daunorubicin, and the anthraquinone, mitoxantrone, were shown to have similar effects on ∆f cftr expression. in the current work, we have extended these effects to two other related aza-anthracenedione molecules, bbr (pixantrone) which is a cancer chemotherapy drug with lower non-target toxicity than dox and which is in phase iii clinical trials, and a structurally related analog, bbr , which is essentially non-cytotoxic with a -fold lower cytotoxic potency than bbr . both compounds increased cftr-associated chloride currents to a similar extent as dox in cfbe human airway epithelial cells expressing ∆f cftr, as measured by ussing chamber experiments. the ability of the non-cytotoxic analog bbr to do so is particularly important, since it indicates that correction of the ∆f cftr defect is not directly tied to the toxicity of this class of compounds per se but rather is a result of other structural features. thus, it is likely that this and/or other non-toxic analogs in this chemical class can be developed that have potential as clinically useful agents for treatment of cf in patients. supported by a cystic fibrosis foundation (cff) grant to jwh and rm, and by the cff-supported dartmouth cf program. cystic fibrosis (cf) is caused by mutations in cystic fibrosis transmembrane conductance regulator (cftr). cftr is not only an epithelial chloride channel, but it also regulates other transporter and ion channels, including epithelial sodium channels (enac) and aquaporin water channels. in patients with cf, absent or dysfunctional cftr results in abnormal electrolyte and fluid content on the epithelial surface. treatments intended to normalize ion transport in cf airways through non-cftr dependent pathways represent attractive approaches to alleviate the underlying pathologic defect. calcium (ca +)-activated cl-channels have been proposed as rescue channel for the cyclic amp-dependent cftr cl-channels, offering a target for cf pharmacotherapy. increases in cytosolic ca + concentration activate epithelial cl-channels, but inhibit epithelial na+ channels, which is also beneficial in correcting the hyperabsorption of na+ in cf. herein we developed a high throughput screening (hts) assay for screening compound libraries with the intention of finding compounds which can increase intracellular ca +. in the pilot primary screening, we screened a mssp library which contains known bioactive compounds and natural products. compounds were classified as the real hits after the secondary screening validation with the hit percentage . %. we have found that the ec of these compounds are less than um in dose-response studies and none of those compounds were toxic to the cells. we have further discovered that those compounds stimulate cl-secretion through activating ca + dependent cl-channels in cf and non-cf human airway epithelial cells in short-circuit current experiments. in summary, our data suggest that modulation of intracellular ca + is a target for cf therapy. the compounds identified by our study will provide possible therapeutic leads in the treatment of cf. non-viral gene delivery particles with various synthetic polymer coatings have been developed for cystic fibrosis (cf). intracellular trafficking and in vivo tissue distribution of these particles must be carefully monitored to guide rational design of efficient delivery system. fluorescent semiconductor quantum dots (qds) allow sensitive, long-term and multi-target imaging in cellular environment, and so are a promising tool in tracing gene delivery materials in vitro and in vivo. however, application has been limited by the lack of efficient and reproducible techniques of qd bioconjugation. here, we describe a method of labeling dna nanoparticles with tunable qds that may enable us to study gene delivery in vitro and in vivo. highly fluorescent zns-coated cdse qds were first synthesized and encapsulated in amine-containing phospholipid micelles. the non-viral vector system we used is based on a polymer backbone consisting of lysines with a cysteine on the n-terminus (ck ) which is conjugated to polyethylene glycol (peg). this polymer vector condenses dna plasmids into stable nanoparticles that efficiently transfect airway epithelial cells in vivo. to introduce the aminecontaining qds, we utilized a hetero-bifunctional peg with an nhs ester at one end, which was first reacted with the amine groups of the qds and a maleimide group at the other end which was subsequently reacted with the thiol group in ck . excess reagents were removed from the final conjugates qd-peg-ck by filtration. conjugation was first confirmed by . % agarose gel electrophoresis. free qds were positively charged and migrated toward the cathode. pegylated qds migrated significantly slower than free qds, and qd-peg-ck conjugates migrated at still a different rate. to further demonstrate conjugation, we biotin-labeled the ck and assessed the binding of qd-peg-ck -biotin to avidin coated agarose beads by monitoring the yield of fluorescent beads. qds, pegylated qds, and physical mixture of qds and ck -biotin were also incubated with the agarose beads as controls. fluorescent beads were detected only when qd-peg-ck -biotin was present. to directly evaluate the ability of qd-peg-ck to bind double stranded dna, we also immobilized double stranded dna fragments to agarose beads. qd-peg-ck , but not unconjugated qd bound to the immobilized dna fragments as determined by the yield of fluorescent beads. qd-peg-ck was then used to compact luciferase reporter plasmids. electron microscopy images of the compacted dna showed that qds were integrated into some dna nanoparticles. we transfected hela cells with qd-labeled and unlabeled dna nanoparticles. luciferase assay and toxicity assay indicated that qd-labeled dna nanoparticles transfected hela cells, though with less reporter gene expression than unlabeled nanoparticles and more toxicity than uncompacted qd. in conclusion, we have developed a method to conjugate qd to dna nanoparticles. improvement of the quality and biocompatibility of the qd may be required for further in vitro and in vivo studies. this system is also versatile. other less toxic optical makers can be readily tested. this study was supported by dk and the cff. in cystic fibrosis (cf) the epithelial sodium channel (enac) hyperactivity plays a role in the pathogenesis of chronic lung disease. the missing enac regulation by the cf transmembrane conductance regulator (cftr) causes increased absorption of sodium ions and fluid across airway epithelia leading to the depletion of the perciliary liquid layer and to the consequent inhibition of mucus clearance. we developed a hiv-based lentiviral (lv) vector containing a sirna cassette to efficiently knockdown the expression and activity of enac in human respiratory cells. background: sildenafil has been implicated in the relocation of cystic fibrosis transmembrane conductance regulator (cftr) protein. the effect was observed in vitro and in the presence of doses roughly times larger than those commonly used for treating erectile dysfunction. aim: to evaluate in vivo therapeutic efficiency of clinical doses of sildenafil and vardenafil, two approved type v phosphodiesterase inhibitors, for correcting chloride transport defect in ∆f mice. methods: we measured transepithelial potential difference in vivo across the nasal mucosa as a readout for sodium and chloride conductance. the effect of a single intraperitoneal injection of sildenafil ( . mg/kg) or vardenafil ( . mg/kg) was investigated in df /df and normal homozygous mice. results: in df /df mice, chloride conductance, evaluated by perfusing the nasal mucosa with a chloride-free solution in the presence of amiloride and with forskolin, was corrected h after sildenafil administration. a more prolonged effect, persisting at least for h, was observed with vardenafil. the forskolin response was increased after sildenafil and vardenafil in both normal and df mutant animals. no effect on sodium conductance was detected in any group of animals. conclusion: our results provide preclinical evidence of effectiveness of both drugs for correcting chloride transport defects in the cf. acknowledgments: this work was supported by grants from the french cf association, vaincre la mucoviscidose and by an educational grant from pfizer belgium. there is a strong interest in developing small molecules able to correct the phenotypic effects of cystic fibrosis (cf) mutations. many mutations (e.g. ∆f ) impair the function of cftr protein, by altering the protein targeting to the plasma membrane and/or by causing an abnormally low open channel probability. drug-like organic compounds may restore the correct membrane localization ("correctors") or increase channel activity ("potentiators") of mutant cftr. in the last years, various research teams have identified molecules with activity as correctors (corr- a, vrt- , miglustat, curcumin) or as potentiators (tetrahydrobenzothiophenes, phenylglycines, sulfonamides, vrt- , , -dihydropyridines). however, these compounds have been tested using different assays and the results are sometimes controversial, with marked differences in declared efficacy and potency of compounds. we have tested a panel of correctors and potentiators to directly compare their effects under the same experimental conditions. for this purpose, we have used the functional assay based on the halide-sensitive eyfp-h q/i l to measure ∆f -cftr activity in frt and a cells. the assay for potentiators consisted in stimulation with the test compound ( . - µm) plus forskolin (on cells previously incubated at °c for hours). the assay for correctors consisted in hours incubation of ∆f cells with test compounds and then determination of cftr activity in the presence of forskolin plus genistein ( µm). our results indicate that all potentiators are active in our assay with a comparable maximal effect but with values of potency that vary significantly among compounds. the potency order measured in frt cells was: dhp- = pg- (ka~ nm) > sf- (ka = nm) > dhp- = dhp- (ka~ nm) > act- b (ka = nm) > felodipine (ka = nm) > vrt- (ka = . µm) > genistein (ka = . µm). a similar order of potency was found also in a cells expressing ∆f . the activity of correctors showed a more marked dependence on cell line. while the potency was comparable between frt and a cells, the maximal effects showed clear differences. in frt cells, corr- a, corr- b and corr- c generated a maximal effect that was . - -fold higher than that obtained by incubating the cells at low temperature. conversely, vrt- and vrt- were less effective (maximal correction equivalent to - % of low temperature rescue). in a cells, all compounds were instead less effective than low temperature, with vrt- being the molecule eliciting the highest activity ( - % of low temperature). our results indicate the , -dihydropyridine dhp- and the phenylglycine pg- among the most potent activators of the mutant cftr channel. the similar behavior of potentiators in two different cell lines is consistent with the assumption that all potentiators act with a similar mechanism, by interacting with the cftr protein itself. in contrast, the cell line dependence of correctors suggests that they act with indirect mechanisms, possibly by interacting with proteins involved in cftr biosynthesis and trafficking. supported by cfft and telethon-italy. we also thank cfft and rfums for providing chemical compounds. lung damage in cystic fibrosis (cf) patients is determined by mucus accumulation, pseudomonas aeruginosa infection and chronic inflammation. extracellular gsh is a scavenger of free radicals produced by neutrophils in inflamed tissues. glutathione transferases (gst) are a superfamily of dimeric proteins which conjugate glutathione to a wide range of substrates including oxidants and are involved the synthesis of leukotriens. clinical beneficial effects have been reported in cf patients following treatment with the macrolide azythromicin (azm); anti-inflammatory properties have been proposed as possible mechanism. the aim of this study is to investigate the regulation of the gstt and gstm activity and expression by azm. reductions of about % and % on gst enzymatic activity were detected in ib - and cfsmeo-cells respectively. gsts mrna expression in cf airway epithelial cell lines was analysed by quantitative pcr (qpcr). the level of gstt and gstm basal expression in cf cells ib - was significantly higher than in isogenic non-cf cells c . we found statistically significant decreases of gstt and gstm mrna of about % and % respectively in ib - cells after treatment with azm for hours, restoring the levels observed in c cells. in cfsmeo-cells after exposure to azm we observed % and % reductions in gstt and gstm mrna respectively. the macrolide jm, known to lack clinical anti-inflammatory properties, had no significant effects on gstt and gstm mrna expression in all cell lines. furthermore, azm did not alter the mrna expression levels of gstp , a glutathione-s-transferase not differentially expressed in cf and isogenic non-cf cells. decreased expression of % and % of gstt protein has been detected by immunoblotting in ib - and cfsmeo-cells, respectively, following treatment with azm. in the same conditions we found a drastic reduction of protein level of gstm in both cf cell lines. finally, gsts activity and the expression of gstt and gstm proteins in cf cells, were reduced approximately to the same level detected after treatment with interleukin (il- ), an anti-inflammatory cytokine, shedding light on a possible correlation between gsts inhibition and antiinflammatory properties of azm. the effects of azm described in this study suggest that downregulation of gstt and gstm expression may result in increased availability of intracellular gsh making cf cells less susceptible to oxidative stress induced by chronic inflammation. inhibition of gstt and gstm might provide a therapeutic approach for limiting the effects of inflammation critical for lung damage in cf patients. this study is supported by italian cf research foundation; comitato di vicenza-associazione veneta per la lotta contro la fibrosi cistica; azienda ospedaliera verona, italy. tradtrantip, l.; padmawar, p.; yangthara, b.; verkman, a. activation of cftr chloride conductance by gpcrs involves camp elevation and pka-mediated cftr phosphorylation. we developed a 'pathway screen' in which cftr-mediated iodide influx is used as a read-out of gpcr activation. the cell-based fluorescence assay utilizes multiply transfected epithelial cells expressing wildtype cftr, yfp-h q/i l and a specified gpcr. we recently used this assay to identify a new class of nanomolar-affinity, -aryl- -benzoyl- hydroxy- -( -arylethyl)- h-pyrrol- -one vasopressin- receptor antagonists (yangthara et al., mol. pharm. , in press ). additional screening of , diverse small molecules yielded novel chemical classes of inhibitors of cftr activation. the potential molecular targets of pathway inhibitors include the gpcr, gs or gi proteins, adenylyl cyclase, phosphodiesterase, pka and cftr. a series of target identification studies was done to classify the new pathway inhibitors, which involved the use of agonists acting at different sites in the activation pathway, and specific site-of-action assays. the pathway screen yielded new small-molecule cftr inhibitors that are unrelated to thiazolidinone and glycine hydrazide inhibitors. one interesting class of pathway inhibitors, thiophenecarboxylates, represent the first small-molecule phosphodiesterase activators, which strongly reduce camp and cgmp concentration in many cell types. the best thiophenecarboxylate greatly reduced intestinal fluid secretion in closed loop mouse models of cholera and travelers' diarrhea, and slowed cyst growth in a model of polycystic kidney disease. other pathway inhibitors, which are potential effectors of g-proteins and pka, are under evaluation. the gpcr-linked cftr pathway screen developed here is useful for high-throughput parallel identification of small-molecule inhibitors of multiple targets in the cftr activation pathway. potential uses of the inhibitors identified here include therapy of secretory diarrheas, polycystic kidney disease, and cyclic nucleotide-dependent tumor growth, as well as pharmacological creation of cf-phenotype in ex vivo human tissues and animal models. supported by cff and nih. inhibitors of intestinal caccs are predicted to have anti-secretory effects in certain diarrheas, and activators of airway and intestinal caccs are of potential use in cystic fibrosis therapy (activation of 'alternative' chloride channels). the purpose of this study was to identify small-molecule cacc inhibitors and activators that target caccs directly, rather than ubiquitous upstream processes such as calcium signaling. we screened a collection of , chemically diverse small molecules using a novel high-throughput screening assay involving lentiviral introduction of a yfp-based halide sensor in cacc-natively expressing human epithelial cells. cacc inhibitors were identified from iodide influx following cacc simulation by carbachol/atp. we identified five classes of cacc inhibitors with micromolar potency, including tetrahydro-cyclopentaquinolines, and -aryl- -(trifluoromethyl)-pyrazoles each of which was unrelated to known transport modulators. two classes of compounds inhibited calcium-activated halide flux following stimulation by multiple types of agonists, including thapsigargin and calcium ionophores, and by patch-clamp analysis appear to target cacc directly. structure-activity analysis of analogs of 'hits' yielded compounds with improved potency, which have been resynthesized and characterized for use in assays of antidiarrheal effiacy in rodent models of viral and drug-induced secretory diarrheas. screening for caccselective activators that act in a sustained manner (non-transiently) was accomplished using a similar cell-based fluorescence assay, but instead testing for increased halide influx. several classes of putative cacc activators with micromolar-potency were identified in screening of , small molecules, whose mechanism-of-action and specificity are under investigation. small-molecule modulators of cacc function that target caccs directly have potential clinical applications, and may be useful in defining the physiological roles and molecular identity of caccs. supported by cff and nih. introduction: inhaled hypertonic saline 〈hs〉improves lung function and decreases pulmonary exacerbations in older children and adults with cf. initiating therapeutic interventions in the youngest patients, particularly those that target the underlying cf defect of airway surface liquid volume depletion, has potential to preserve lung function and improve prognosis. subbarao et al performed baseline, post-albuterol and post-hs lung function testing in infants using the raised volume rapid thoracoabdominal compression technique 〈rvrtc〉and demonstrated no significant drop in lung function 〈 pediatric pulmonology, 〉. however, performing three sets of rvrtc maneuvers under the same sedation could prove difficult. before conducting a therapeutic trial of hs in this population, a simplified protocol must be possible at multiple centers. we sought to evaluate a simplified approach as well as to analyze changes in lung function and clinical findings after acute administration of hs. methods: in this ongoing study, clinically stable children with cf between the ages of months and years inhale . mg of albuterol prior to sedation with chloral hydrate. rvrtc and plethysmography are then performed before and after inhalation of ml of ‰ hs. fvc, forced expiratory volume in . seconds 〈fev . 〉, fef - , frc, rv/tlc ratio, respiratory rate, oxygen saturation, and chest exam findings are recorded. predefined stopping criteria include a % drop in fev . or in oxygen saturation to below ‰. results: six subjects 〈mean age . ± . years〉 have completed the protocol with ‰ hs. comparison of post-albuterol lung function to that obtained minutes after ‰ hs revealed no changes in mean fvc 〈 vs ml; p= . 〉, mean fev . 〈 vs ml; p= . 〉, mean fef - 〈 vs ml/sec; p= . 〉, frc 〈measured after each inhaled therapy in of subjects; vs ml; p= . 〉, or rv/tlc 〈measured after each inhaled therapy in of subjects; . vs . ; p= . 〉. respiratory rate, oxygen saturation and chest exam were unchanged. conclusions: results from this study demonstrate that a two-step protocol may used to evaluate the safety of hs. based on these findings, acute administration of ‰ hs is safe in children ages months to years with cf. despite the known improvement in mucociliary clearance, preliminary findings demonstrate a lack of an immediate response in lung volume measures. given the demonstrated benefits in older children and adults, a multicenter therapeutic trial of hs is warranted. supported by the cystic fibrosis foundation. methods and results: we have characterized aav serotypes - in addition to twenty novel vectors isolated from human or macaque tissues to transduce the murine airway epithelium in vivo. vectors [ e+ genome copies (gc)/mouse] expressing α- -antitrypsin (aat) and βgalactosidase (β-gal) were co-instilled into the mouse lung or nose. transgene expression levels were monitored by assaying aat concentration in serum as well as the number and cell-types positive for (β-gal) expression in lung and nasal airways. of all vectors tested aav and aav were the two most efficient vectors in conducting airways. when these aav vectors ( e+ gc) were subsequently evaluated on human ciliated airway epithelial cultures (haec), in contrast to our findings in mouse airways, aav failed to transduce haec, whereas aav resulted in~ % of the haec expressing transgene. since aav was the most efficient vector in mouse and human airway epithelium we performed structure-function analyses of the aav vector capsid and found two atypical capsid residues that were unique in otherwise conserved positions (f , k ). to generate a potentially fitter vector, residue f was mutated to its conserved state. residue k was found to confer lung tropism and was left unchanged. the resulting vector, aav . , transduced mouse lung and nasal airway with greater efficiency than all aav vectors tested. the increased transduction efficiency of this vector was also observed (~ %) in haec derived from six different human subjects. to continue our preclinical studies in a more relevant model, aav / . expressing egfp was tested in ciliated cultures derived from macaque airways and showed - % of cells expressing egfp one week after inoculation with e+ gc. confocal microscopy revealed that aav . targeted a significant number of ciliated cells: the airway cell-types that likely require cftr expression in cf patients. aav . expressing rhesus α-fetoprotein (rhafp) was then inoculated in the nasal airways of a rhesus macaque and transduction evaluated by monitoring concentration of rhafp in the nasal lavage fluid. we have found that rhafp expression remained high (~ ng/mg) and stable for at least days. conclusion: the enhanced transduction efficiency of aav / . vector in human and macaque airway cultures and its ability to stably transduce the nasal airway of a rhesus macaque in vivo demonstrates that aav / . is a good candidate gene transfer agent for the efficient expression of cftr in human cf airway epithelium. submitted for presentation at the american society of gene therapy. supported by gsk, cff, cfpo , p , mtcc. pharmacological correction of ∆f cftr cellular processing is a potential therapeutic strategy for cystic fibrosis. recent high-throughput screening has identified synthetic small molecules, such as bisaminomethylbithiazoles (corr- ), which partially restore chloride permeability in ∆f mutant cells. the purpose of this study was to examine the utility of natural compounds (chinese medicinal herbs) as ∆f correctors. a herbal compound fraction library was constructed from herbs most frequently used in traditional chinese medicine (tcm) that are believed to contain therapeutic compounds for a broad spectrum of human diseases including lung disease. for construction of the tcm fraction library, crude herbal extracts were first prepared by % ethanol extraction on soxhlet reflux apparatus followed by automated fractionation by preparative hplc. eighty fractions were collected from each of the herbs. each fraction contained to (average . ) individual compounds as determined by analytical hplc. collected fractions were dried and milligram of the material was dissolved in µl dmso to generate mg/ml solutions in -well plates. each -well plate contained fractions from one herb. high-throughout screening was done using the frt cell-based fluorescence assay developed previously (j. clin. invest. : - , . of , fractions screened, active fractions from herbs were identified, with positive fractions verified in secondary screening. the positive fraction did not increase halide transport in control non-transfected cells, and halide transport in ∆f -corrected cells was fully abolished by cftrinh- . we have fractionated some of the most active fractions by preparative hplc to identify which compound(s) conferred activity. for example, in one fraction there were single compounds, of which conferred corrector activity with ic s < µm and efficacy comparable to that of low temperature rescue. these results demonstrate the feasibility of ∆f -cftr corrector discovery from natural compounds. further fractionation, characterization and structure determination are in progress. the unexpectedly high 'hit'rate for the natural compounds suggests their further exploration in cf therapy. cystic fibrosis (cf) is the most common genetic disease affecting the caucasian population, with an incidence of approximately one in three thousand births. cf transpires as a result from a mutation in the cystic fibrosis transmembrane conductance regulator protein (cftr), which regulates ion transport across epithelial membranes. subsequently, patients afflicted with cf have an abnormally excessive incidence of chronic lung infection, with organisms such as pseudomonas aeruginosa. because cystic fibrosis is characterized by chronic bacterial infections, excessive neutrophil recruitment to the lungs, and a coinciding increase in pro-inflammatory cytokine production and nuclear factor-kappa b (nf-κb) activation, we hypothesized that exogenous addition of the nf-κb inhibitor iκbα might ameliorate this phenotype. we cloned the human iκbα gene as well as a mutated iκbα gene into plasmids with chicken-beta actin hybrid promoters. we then tested the new plasmids, paav .cb-hiκbα and paav .cb-hiκbαm, in vitro in the presence and absence of pseudomonas aeruginosa infection in the ib - and s cell lines. both plasmids produce iκbα at high levels as shown by enzyme linked immunosorbant assays (elisas). we also show that paav .cb-hiκbα transfected ib - cells, after infection with pseudomonas aeruginosa, express significantly reduced levels of interleukin (il)- β ( fold, p< . ), il- ( fold, p< . ), and tnf ( fold, p= . ) as well as nf-κb activation ( fold, p< . ) compared to p. aeruginosa-infected ib - controls as determined by human cytokine and nf-κb phosphoprotein bio-plex assays; cytokine expression and nf-κb activation levels in infected paav .cb-hiκbα transfected ib - cells were between levels found in infected ib - and s- cells, excluding il- levels which were below s- levels of expression. flavonoids are among the most potent cftr modulators known. equol [ hydroxy- -( '-hydroxyphenyl)-chroman] is a product of intestinal metabolism of dietary isoflavones such as daidzein, and has been of recent interest in studies of cancer, cardiovascular risk, and neurologic disease. equol is metabolically stable, and % circulates in the free (non-protein bound) form, which is considerably greater than the proportion of free daidzein ( %). structural differences such as modification of ring c (e.g. saturation at c- / , and absence of carbonyl group at c- ) and an absent hydroxyl at position c- distinguish equol from compounds previously reported to modulate cftr activity. in prior work by our center, we showed that equol activates wt and ∆f cftr in membrane patches excised from bhk cells and in cfbe o-monolayers studied in ussing chambers. activation by equol occurred only after r-domain phosphorylation in wt and ∆f cftr constructs, but was independent of rdomain phosphorylation in ∆r cftr, indicating activity may be related to dimerization of the nbds or other domain-domain interaction. because a molecule that alters nbd interactions might have effects on the aberrant processing of ∆f cftr, we screened equol and other flavonoids by preincubating compounds with cfbe o-cells expressing ∆f cftr for hours, and then tested for rescue of cftr dependent cl-channel activity after exchange of media solution. we found that preincubation with equol ( - µm) induced rescue of short-circuit current compared to vehicle treated cells ( vs. µa/cm , p< . , n= ). we then evaluated hour preincubation with equol ( um) for biochemical evidence of cftr processing correction in ∆f cfbe o-cells grown in polarizing conditions. immunoprecipitation and in vitro phosphorylation demonstrated minimal formation of band c compared to vehicle treated cells, but adaptation of a more sensitive avidin label/biotinylation assay specific for surface localized cftr revealed clear evidence that equol preincubation led to cftr at the plasma membrane. next, we examined equol in hela cells stably transduced with ∆f cftr. preincubation of equol led to dose-dependent increases in halide transport measured by fluorescence-based halide efflux (spq) after stimulation by genistein ( . and . fluorescence slope (∆%/sec) with equol and µm respectively, compared to . ∆%/sec in cells pretreated with vehicle alone; p< . , n= - cells/condition). immunohistochemical staining of ∆f hela cells for cftr with - c-terminus antibody showed rescue of surface localized protein with equol ( µm) preincubation for hours, while vehicle treated cells showed only perinuclear staining. in summary, we show functional, biochemical, and immunohistochemical evidence that the naturally occurring flavonoid equol corrects the ∆f processing defect in two model systems. a naturally occurring agent that both activates and corrects ∆f cftr deserves further exploration as a potential cf therapeutic, and may lead to new insights regarding domain-domain interactions that influence the activation and biogenesis of the mutant channel. supported by nih and cff. mote ∆f cftr maturation has been identified. although several small molecule agents have been described that overcome ∆f cftr processing defects in specific cellular models, few studies have directly compared the activity of temperature corrected and chemically corrected ∆f cftr in polarized cell systems. in the present study, we examined chemical and temperature corrected activity of ∆f cftr. correctors included all members of the cfft modulator library (c , c , c , and c ; rosalind franklin university, chicago, il). in a screen using ∆f cfbe omonolayers, maximal corrector activity across the two model systems exhibited a rank order of c >> c > c = c , using forskolin ( µm) and genistein ( µm) stimulated isc as a sensitive test for ∆f cftr activity at the plasma membrane. no change in isc was observed in matched control (parental) cells lacking ∆f cftr expression. based on these results, we further defined the activity of c in ∆f cfbe oand frt model systems, including dose/response and time dependence for peak isc rescue. in cfbe o-cells, exposure to µm c for hrs produced maximal reproducible correction of ∆f cftr processing, with loss of activity following prolonged or high concentration exposure; in frt cells, peak effects were seen at hours. ∆f cftr activity following small molecule treatment qualitatively mirrored temperature correction ( °c growth for - hrs) in both cell types. maximal currents produced by stimulation with forskolin ( um) and genistein ( µm) in ∆f frt monolayers following c pretreatment were % of that produced by temperature correction (p< . ). forskolin was responsible for % of maximal current in frt ∆f cells following chemical correction and % of maximal currents following temperature correction. in ∆f cfbe o-cells, maximal currents following chemical correction were % of that produced by temperature correction (p< . ). forskolin was responsible for . % of maximal currents in cfbe o -∆f cells following chemical correction and . % of maximal currents following temperature correction. these results illustrate dose and time response with a small molecule corrector in two polarizing epithelial model systems, and provide reassurance that observations based upon ∆f cftr following low temperature incubation are relevant to functional analysis after chemical correction. similarity in activation properties between chemical and low temperature correction suggest it is unlikely that the two maneuvers result in ∆f cftr with significantly different structural properties. the studies also indicate fundamental differences in ∆f cftr behavior in frt compared to cfbe ocells, and emphasize the importance of identifying an agent that can restore camp dependent regulation to bronchial epithelial cell types. supported by the nih, cff and cfft. participants: n= patients ( randomised to azm and to placebo) who had successfully completed a course of intravenous antipseudomonal antibiotics immediately before the trial (mean age: . years; mean fev : % of predicted). measurements and results: after treatment (mean dose of . mg/kg body weight once a week) pulmonary function declined in both groups compared to baseline (i.e. after cessation of iv antibiotics). the azithromycin group had signifcantly better results regarding the mean changes in serum crp (azm: + . mg/l, placebo: + . mg/l, p= . ), lipopolysaccharide binding protein in serum, lbp (azm: + . µg/ml, placebo: + . µg/ml, p= . ), serum interleukin- (azm: - . pg/ml, placebo: + . pg/ml, p= . ) and alginate in sputum (azm: + µg/ml, placebo: + µg/ml, p= . ). quality of life (german version of the cfq) showed significantly better results after azm in adolescents and adults. azithromycin was well tolerated with no increase in treatment-related adverse events. conclusion: once-weekly azithromycin ameliorated inflammatory reactions and improved quality of life. a decline of pulmonary function after cessation of intravenous antibiotics could not be prevented, however. this study has been sponsored pfizer gmbh, germany this open-label, multicenter study was conducted in the usa and australia to evaluate the clinical responsiveness of a patient-reported outcome measure, the cfq-r respiratory scale, by determining the minimal clinically important difference (mcid) following a -day course of tobramycin inhalation solution (tis). cf patients (n= [≥ to < yrs, n= ; ≥ yrs, n= ]) with pseudomonas aeruginosa and clinical symptoms predictive of a pulmonary exacerbation (increased cough, increased sputum /chest congestion, decreased exercise tolerance or decreased appetite) were enrolled. three efficacy measures were included: )change in forced expiratory volume in second (fev ) from baseline (day ) to end of treatment (day ) or end of study (day ); )one question about change in respiratory function (days and ; global rating of change questionnaire, respiratory domain, grcq rd; =no change; =maximal improvement or worsening);and )change in cfq r-respiratory scale (day , ). average change from baseline fev (mean [standard deviation, sd]) was . % ( . ) at day and . % ( . ) at day . based on grcq-rd at day , patients ( %) reported no change in respiratory symptoms (score < . ), ( %) a minimal change (≥ . to < . ), ( %) a moderate change (≥ . to < . ), and ( %) a large change (≥ . ). mean (sd) change from baseline cfq-r respiratory was . ( . ) at day and . ( . ) at day . at day , change in cfq-r was moderately correlated with change in fev and with grcq-rd; the correlation was stronger for patients with baseline fev < % of predicted fev values (see table) . mean change from baseline on the cfq-r respiratory scale was . at day for patients with grcq-rd scores indicating minimal change in symptoms (≥ . to < . ; n= ); this provided an estimate of the mcid for the cfq-r respiratory scale for those in exacerbation. this mcid value was consistent with estimates from distribution-based methods( ⁄ sd and standard error of measurement). the cfq-r was responsive to changes in pulmonary symptoms in patients in mild exacerbation following tis treatment; the mcid in this population was . points. responses on the cfq-r-respiratory scale were moderately correlated with changes in fev . funded by gilead sciences, inc. this was an open-label, multicenter study conducted in the usa. we determined the minimal clinically important difference (mcid) for the cfq-r, respiratory scale following a -day course of tobramycin inhalation solution (tis) in patients with cf and chronic pa infection (n= , children [< yrs]). patients had received ≥ courses of tis (mean = . ) within the previous year, however their respiratory symptoms were stable at study entry, with forced expiratory volume in second (fev ) between % to % of predicted values. efficacy measures included: ) percent change in fev (l) from baseline (day ) to treatment end (day ); ) a single question about change in respiratory function (day ; global rating of change questionnaire; respiratory domain, grcq-rd; =no change, =maximal improvement or worsening); ) change in cfq-r-respiratory scale (day to ); and ) change in log pa colony-forming unit (cfu) density in sputum (day to ). the mcid for cfq-r was estimated using three methods: ) change in cfq-r (day to ) for the patient subset with minimal change in respiratory function, as determined by the grcq-rd at day ; ) cfq-r standard error of measurement (sem) from a validation sample), and ) ⁄ standard deviation (sd) of the cfq-r respiratory scale scores. at day , change from baseline fev (mean [sd]) was . % ( . ), change from baseline cfq-r was - . ( . ), and change in log pa cfus was - . ( . ) . based on the grcq-rd at day , patients ( %) reported no change in respiratory symptoms (score < . ), ( %) a minimal change (≥ . to < . ), ( %) a moderate change (≥ . to < . ), and ( %) a large change (≥ . ). pearson r-values for the correlation of change in the cfq-r-respiratory scale and change in fev , log cfus and grcq-rd were . , . , and . , respectively. estimates of the mcid for cfq-r-rd ranged from . to . for adults/adolescent patients (table) . in patients with cf who had no immediate need for antipseudomonal therapy at study entry, the cfq-r-respiratory scale appeared responsive to changes in patient disease perception following days of tis treatment; the mcid for cfq-r was approximately points for the adolescent/adult patient population. funded by gilead sciences, inc. ex vivo chloride secretion measurements (intestinal current measurement, icm) in cf patients have been established over the past years to study the cftr-basic defect in more functional detail. modified micro-ussingchambers are used to registrate the transepithelial short-circuit current (isc) in freshly obtained human rectal suction biopsies as a measure of ion transport after stimulation with secretagogues. hereby, the cftr clchannel, its amount of residual function in cf and alternative cl-channels can be investigated by a standardised protocol.in the course of the development of cftr pharmacotherapeutics as well as agents activating alternative cl-channels, icm may function as an useful outcome parameter in preclinical and clinical trials. it is easy to perform repeatedly at all patients ages and comprises the safety advantages of an ex vivo method which is relevant especially for early study phases. aim of this study was to describe reference values and quantify the intraindividual variability of different icm parameters. methods: a total of n= rectal biopsies from n= individuals; with pancreatic insufficient (pi)-cf (n= ; mean age . years), pancreatic sufficient (ps)-cf (n= ; . years), excluded cf by icm diagnostics (n= ; . years) and healthy control (n= ; . years) were included into analysis. for calculation of intraindividual variability, - biopsies/patient were compared with respect to basal tissue resistance (rt basal), basal open circuit potential difference (pd basal), basal short circuit current (isc basal) and the isc responses to stimulation with carbachol ( - mol/l, serosal), -bromocyclic monophosphate (camp) ( - mol/l, mucosal+serosal) + forskoline ( - mol/l, serosal) and histamine ( x - mol/l, serosal).results:we determined icm reference values for the groups of pi-cf (isc basal . ± . µa/cm , ∆isc carbachol . ± . µa/cm ,∆isc camp/forskoline . ± . µa/cm ,∆isc histamine - . ± . µa/cm );ps-cf (isc basal . ± . µa/cm ,∆isc carbachol . ± . µa/cm ,∆isc camp/forskoline . ± . µa/cm ,∆isc histamine . ± . µa/cm ),and healthy control (isc basal . ± . µa/cm , ∆isc carbachol . ± . µa/cm , ∆isc camp/forskoline . ± . µa/cm , ∆isc histamine . ± . µa/cm ). for the total cohort, mean coefficients of variation were: rt basal %, pd basal %, isc basal %, ∆isc carbachol %, ∆isc camp/forskoline %, ∆isc histamine %. conclusion:this first comprehensive analysis of the intraindividual variability of icm basal tissue and cl-secretion parameters provides the basis for the method as an useful outcome measure for future clinical trials aiming to rescue the cftr basic defect. possible effects of pharmacological therapeutics in cf relevant human epithelia have to be adequately interpreted with respect to subject variability and laboratories reference data. ex vivo cl-secretion measurements have the potential of being an essential step in the evaluation process of cftr-correcting/potentiating agents on their way from laboratory screening to the application in human cf tissue without any risk of toxicity. center, placebo-controlled, double-blinded pilot study we assessed safety and tolerability of . mg/d moli versus placebo (normal saline) administered by inhalation (pari lc plus) once daily for days. patients included were ≥ years of age in phase i and ≥ years of age in phase ii, with a fev > % predicted and stable lung disease. overall, subjects received moli and placebo. exclusion criteria included abpa, b. cepacia infection and severe liver disease. the study involved clinic visits over a period of weeks to assess adverse events, spirometry, pulse oximetry and quality of life. a total of adverse events (aes) were observed in subjects ( ae in subjects receiving moli ), with only (productive cough) in the moli group being of severe intensity. the most frequent aes related to the study medication were (productive) cough ( x) and dry throat/throat irritation ( x), and most of these resolved within hour after inhalation. in the moli group no significant ae, defined as a decline of fev ≥ % from baseline accompanied by symptoms, a decrease in oxygen saturation to < % or a fall of % from baseline requiring therapeutic intervention, or a change in safety parameters judged to be clinically significant was observed. this trial was not primarily designed to show efficacy; however, the median change in fev from day to day was - % in the placebo group, and + % in the moli group, and this difference was significant (wilcoxon test, p= . ). similarly, there was a significant difference between the median change in fef - % from day to day in the placebo group as compared to the moli group (- % vs. + %, p= . ). no significant changes were observed for the other study days or for fvc and pulse oximetry. moli was well tolerated in this trial, with the observed aes generally being mild and of short duration. these encouraging explorative results are currently being further evaluated in explorative and confirmatory trials. introduction: results from published data elucidate that microbes found in the upper respiratory tract are similar or the same as those found in the lower airways of cf patients. inhaled, aerosolized drug delivery to the lower respiratory tract is an established treatment route. however, drug delivery systems capable of depositing drug to the paranasal cavities are not yet established and require evidence of deposition and efficacy. pari developed the vibrent™ paranasal drug delivery system to enable the aerosol and drug to penetrate into the nose and sinuses. objectives: this study was conducted to demonstrate that the pari vibrent™ pulsating drug delivery system is capable of ventilating the human paranasal sinuses of healthy volunteers. methods: mkr-gas was continuously ventilated through the nasal tract of three healthy non-smokers in front of a single-head gamma camera (diacam, siemens, germany), using the pari vibent™ pulsating drug delivery system. the nebulizer was coupled to the right nostril and a flow resistor to an output tube was inserted into the left nostril. during ventilation with the krgas (about sec) the subject closed their soft palate to transmit the pulsation and to prevent penetration into the lower respiratory tract. the gas supply of the vibrent™ was directly taken from the mkr-gas generator output channel. kr-gas ventilation imaging was performed with and without pulsation. serial images were recorded with anterior and lateral views. additionally, mri (magnetic resonance imaging) lateral slices of the subjects' head were recorded. the gamma camera images were superimposed to the mri images by adjusting the spatial resolution. with no pulsation from the vibrent™ no ventilation of sinus cavity was visualized by gamma camera images and radioactivity was detected in the nose only. when pulsation was added the maxillary sinuses can be visualized in the gamma camera images of all volunteers. conclusions: without pulsation no ventilation was observed. gas penetration to the paranasal sinuses can be demonstrated using the pulsating action of the pari vibrent™, potentially enabling drug delivery via aerosols. this confirms results of in vitro studies using a cast model. mkr-gas ventilation of the nasal cavities during sec breath holding in front of a planar gamma camera head (anterior) using the pari vibrent without (w/o, left image) and with (w, right image) the pulsation system. the delivery and the exhaust tubing of the kr-gas are shown together with the outline of the head, obtained from mri pictures. introduction: pediatric patients with cf were previously studied in clinical trial studies of denufosol, a novel selective p y agonist that enhances ciliary beat frequency and activates chloride secretion to hydrate the airways in the lung. pediatric patients are often discouraged from participation in clinical trials until later stages of drug development. aims: in order to evaluate the safety experience with denufosol in this population, we have retrospectively examined integrated data for pediatric cf patients with mild to moderate pulmonary disease that participated in three phase studies. demographic and baseline characteristics in addition to safety and tolerability results for cf patients aged - years old are reported. methods: three phase , multicenter, randomized, double-blind, placebo-controlled, parallel group studies were conducted. patients were randomized to receive either denufosol ( , or mg) or placebo (normal saline) tid for days by inhalation. only study included denufosol mg, while all studies included denufosol mg and mg. the fev predicted normal required to be eligible was > % (study ); %- %, inclusive (study ); > % (study ). all three studies included a one-week pre-randomization period during which reproducibility of fev (l) ± % was required in order to be randomized to double-blind study medication. patients were allowed to use bronchodilators, dornase alfa and corticosteroids in studies , and . patients were allowed to use oral antibiotics including macrolides and inhaled tobramycin solution in studies and . results: a total of cf patients - years old were randomized and dosed in three -day studies. eighty-one received denufosol (active doses combined) and received placebo. demographics were similar for all treatment groups -denufosol pediatric patients had a mean (sd) age of . (± . ) years old compared to placebo pediatric patients who had a mean (sd) age of . (± . ) years. denufosoltreated patients were % male and placebo-treated patients were % male. the mean (sd) percent predicted fev at baseline was similar between treatment groups [ . % (± . ) and . % (± . ) for denufosol and placebo, respectively]. the overall incidence of treatment emergent adverse events (ae) was similar between treatment groups ( % denufosol, % placebo). the most common ae was cough, reported by % and % of patients that received denufosol and placebo, respectively. seven percent of denufosol and % of placebo patients prematurely discontinued from the study due to aes. there were no differences in compliance with administration of study drug ( % in patients given denufosol and % in patients given placebo). conclusion: doses of denufosol up to mg given tid for days were well tolerated in pediatric cf patients - years old. these data demonstrate that inclusion of patients - years old is feasible regardless of administration of denufosol or placebo. a longer term phase study of denufosol in cf patients > years old in north america is currently ongoing. acknowledgements: this research was funded by inspire and the cystic fibrosis foundation. objective: pulmonary delivery of anti-infectives provides the potential to attain pk-pd indices exceeding those which can be achieved with systemic dosing. mp- is a novel formulation of levofloxacin that enables delivery of high concentrations over a short period, and provides taste masking. the objective of this study was to: i) determine the safety of aerosol doses of mp- and, ii) determine pharmacokinetics of levofloxacin in serum, urine, and sputum following aerosol doses of mp- using the pari eflow nebulizer in normal healthy volunteers (nhv) and patients with cystic fibrosis (cf). methods: nhv and patients with stable cf were enrolled in a single within-subject ascending dose study of dose levels (loaded doses of , , and mg) of inhalational mp- (levofloxacin solution for inhalation) or placebo. study participants were monitored for safety and changes in pulmonary function. serum, urine, and sputum (cf patients only) samples were collected at various times following the dose and assayed for levofloxacin concentration using hplc. in addition, each participant received a single iv dose of levofloxacin to determine the systemic bioavailability following aerosol mp- . noncompartmental and compartmental methods were used to determine serum, sputum and urinary pharmacokinetic parameters. pharmacokinetic deconvolution methods were used to determine the amount of the dose remaining in the lung over time. results: this study is ongoing. dosing in nhvs ( active mp- , placebo) has been completed. there were no serious adverse effects, and no significant changes in pulmonary function were noted. preliminary pharmacokinetic data from nhvs show a proportional increase in serum levofloxacin concentrations with increasing aerosol mp- dose; serum aucs were . , . , . , and . mg-h/l for the iv dose, low, mid, and high aerosol doses, respectively. deconvolution of the serum levofloxacin concentrations from aerosol mp- and iv dosing shows absorption of drug from lung over time. conclusion: preliminary results show that mp- is well tolerated following aerosol administration in normal volunteers. serum concentration data following aerosol dosing suggests that absorption of levofloxacin into the systemic circulation is a major route of elimination from the lung. studies in cf patients are in progress and will be presented. methods: a mg/kg aerosol dose of mp- was given using a microspray aerosol generation device. when placed just above the tracheal bifurcation, and activated, it delivers a bolus aerosol dose to the lung. a mg/kg intravenous (iv) dose of lvx was given as a slow bolus into the lateral tail vein. plasma lvx was determined in all rats up to sacrifice at , , , or hours, at which time rats were humanely euthanized and bronchialalveolar lavage (bal) performed. fluids were analyzed for lvx concentration using an hplc/ms method. results: the plasma lvx versus time profiles of both iv and aerosol doses were best described by a two compartment model. the plasma auc after an iv dose of levofloxacin and an aerosol dose mp- were similar ( . mg᭹hr/l vs. . mg᭹hr/l, respectively) suggesting near % bioavailability from the lung. after an aerosol dose, the mean residence time (mrt) was prolonged when compared to the intravenous dose ( . vs. . hours). this delay in absorption was associated with an increase in bal lvx auc - h in bal ( . mg᭹hr/l vs. . mg᭹hr/l for iv vs. aerosol dosing, respectively). conclusion: these data show that levofloxacin is highly available to plasma following a single microspray aerosol dose of mp- . the aerosol dose does produce a slightly longer mean residence time in plasma, suggesting delayed, but complete absorption from the lung; this delay in absorption was associated with increase in bal aucs. these data suggest that high concentrations of lvx can be attained in lung fluids following an aerosol dose of mp- . auc: area under the time-concentration curve; cl: clearance; cmax: maximum concentration; f: bioavailability; mrt: mean residence time. methods: animals were assigned to one of five exposure groups for all studies. for the -day toxicology studies, animals were exposed for up to four hours daily for twenty-eight consecutive days, followed by a -day recovery period. respiratory function and cardiovascular safety were conducted on the first and last day of the -day study in dogs. rats were exposed to , , or mg/kg/day, and dogs were exposed to , , or mg/kg/day. at the conclusion of the study, necropsy was performed and all respiratory tissues were harvested, weighed, and underwent gross and microscopic examination. a separate dog respiratory safety study was conducted in which dogs were exposed to , , or mg/kg. results: four week repeat dose inhalational exposure of mp- at target doses up to mg/kg/day in dogs and mg/kg/day in rats was not associated with any test article-related changes in any respiratory tissues. aerosol administration of mp- at doses of up to mg/kg in dogs was found to have no acute effects on minute volume, tidal volume, respiratory rate, or ecg. conclusion: local effects due to the inhalational administration of levofloxacin formulated as mp- were not observed in either rats or dogs. these data suggest that the risk of respiratory toxicity from nebulized doses of mp- is low. methods: bacteria were grown overnight in mueller-hinton broth (mhb) at °c and then sub-cultured into fresh mhb and allowed to reach log phase ( hours). female swiss mice were infected under anesthesia by intratracheal instillation of . ml of a x cfu/ml bacterial suspension. ip doses were selected to provide lvx exposures (as auc) comparable to that obtained with systemic dosing regimens. for the p. aeruginosa infection, treatment was initiated by either the ip or aerosol route hours post-infection. mice were euthanized and hours after the start of treatment, lungs harvested, and bacterial counts in lung determined. results: lvx plasma pharmacokinetic profiles were nearly identical following intraperitoneal or aerosol dosing. the geometric mean log cfu/lung pair (sd) for the p. aeruginosa are shown in the table below: in both the k. pneumoniae and p. aeruginosa lung infection studies, aerosol administration was more effective than systemic administration; for k. pneumoniae, the extent of bacterial killing at hrs was ca. . log cfu greater with aerosol mp- than with systemic lvx. conclusion: aerosol administration of mp- produces a greater extent of bacterial killing than systemic dosing of lvx in mouse models of pneumonia due to k. pneumoniae and p. aeruginosa. the majority of morbidity and mortality in cystic fibrosis patients is caused by chronic and persistent lung infections especially with pseudomonas aeruginosa. since galactosyl ceramide had been previously shown to be involved in pseudomonas internalization, ceramide levels in the plasma of cf patients were assessed and compared to healthy volunteers using hplc followed by mass spectrometry. the results demonstrated that cf patients displayed significantly lower levels of several ceramide species. also, cftr-knockout mice displayed diminished ceramide levels in cf related organs (lung, pancreas, and ileum) and plasma compared to wildtype controls. treatment with a semi-synthetic retinoid (fenretinide), which was previously reported to induce ceramide in neuroblastoma cell lines, was able to increase ceramide concentrations in cf related organs in cftr-knockout mice to the levels of wildtype mice. treatment also dramatically improved the ability of cftr-knockout mice to control pseudomonas infection. following infection with pseudomonas-impregnated agar beads, fenretinide treated cftr-knockout mice were able to clear bacterial infection as efficiently as wildtype mice. overall, these findings not only documented a novel deficiency of ceramide in cf patients but also demonstrated a pharmacological means to correct this defect in cftr-knockout mice. our data provides a strong rationale for clinical intervention that may benefit cystic fibrosis patients suffering from cf lung disease. recent reports show that adult bone marrow-derived stem cells can localize to and acquire phenotypic and functional markers of lung epithelium. these findings raise the novel possibility of stem cell therapy for multitude of lung diseases. however, only small numbers of adult marrowderived stem cells localize to lung and it is not clear whether these cells will be clinically useful. we investigated whether mesenchymal stem cells (mscs) obtained from human cord blood might have increased potential to participate in structural lung remodeling. cord blood was obtained from normal deliveries at the university of vermont. mononuclear cells were isolated and plastic adherent cells were expanded and characterized as mscs according to international society for stem cells research (isscr) criteria. following systemic ( x cells/mouse by tail vein) administration to sublethally irradiated ( . gy) immunodeficient (nod/scid) mice, lungs harvested day, weeks, or months later demonstrated small numbers of human b -microglobulin positive cells in the airway epithelium at all time points. small number of cells was found also stain positive for pancytokeratin (pan-ck) and rarely, we identified cells of b -microglobulin+/pan-ck+ in the airway epithelium of these mice after weeks. we are currently characterizing the phenotype of these cells with ccsp and cftr but these data suggest that cb-mscs may be a potential alternative source of stem cells for use in lung remodeling. high-throughput screening (hts) and other drug development programs have identified cftr activators and potentiators that require secondary evaluation and mechanistic confirmation. we recently evaluated the cfft modulator library (rosalind franklin university, chicago, il) and found some, but not all, cftr potentiators induced potent phosphorylation of the regulatory-domain (r-d), conventionally viewed as the first step in cftr activation. to assess whether this observation has functional significance regarding ion channel activation by these agents, we evaluated cftr potentiators in cfbe o-and fisher rat thyroid (frt) cells stabily transduced with ∆f cftr. cells were studied in modified ussing chambers under control conditions and after correction of ∆f cftr misprocessing using either low temperature ( οc x hrs) or pre-incubation with the chemical corrector c . total currents were determined following potentiator (at reported ec ), forskolin ( µm), and genistein ( µm) stimulation. in temperature corrected ∆f cfbe ocells, potentiator (p , cfpot- ), an agent that does not induce r-d phosphyorylation, caused modest activation when acutely administered ( µm) ( . vs . µa/cm in vehicle treated cells, p < . ). importantly, this agent potentiated forskolin mediated short-circuit current (isc: . vs . µa/cm , p < . ). forskolin accounted for % (vs. % with vehicle) of total current, demonstrating potent rescue of camp dependent cftr activity, an effect not previously reported in this cell type (p< . ). in contrast, two cftr potentiators that induce r-d phosphorylation, p (uccf- , a benzoflavone intermediate, µm) and p (uccf- , an isoxazole, µm) induced modest direct activation of cftr ( . and . µa/cm respectively, p= . and p< . ), but no evidence of forskolin potentiation (p : . µa/cm , % of total stimulated current p=ns; p : . µa/cm , %, p=ns). in ∆f frt cells grown at low temperate, p elicited strong potentiation of forskolin mediated currents ( . vs. . µa/cm with vehicle, p < . ), and modest direct cftr activation ( . vs. . µa/cm , p < . ). p and p were limited to minimal (not significant) activation and no evidence of forskolin potentiation. in cfbe oand frt cells pretreated with c (to chemically correct ∆f cftr processing), p again potentiated forskolin mediated current ( . vs. . µa/cm with vehicle, p< . in the frt model) but did not directly activate i sc . findings with p and p were otherwise as seen with low temperature corrected cells. in summary, in ∆f cftr polarized epithelia, p both activates and potentiates cftr activity (with potentiation being the predominant effect, a unique observation in cfbe ocells), while p and p confer activation of cftr without forskolin potentiation. given that p and p induce r-d phosphorylation while p does not, our findings suggest that agents that do not confer phosphorylation of the r-d may be better suited to rescue the endogenous camp mediated component of i sc . screening of potential therapeutic agents for effects on r-d phosphorylation may help predict utility at restoring ∆f chloride channel activity. supported by nih and cff. activation of cftr is conventionally viewed as a two step process: pka-regulated phosphorylation of multiple sites within the regulatory domain (r-domain), followed by atp dependent gating mediated by binding sites at the interface of the two nucleotide binding domains (nbds). cftr 'potentiators', small organic molecules that overcome mutant cftr gating defects at the cell surface, have been proposed as therapies for cf. although a number of these agents are advancing to the clinical testing phase, their mechanism(s) of action are not well understood. as a step towards better characterizing cftr potentiators available through the cfft modulator library or other resources, we are developing standardized biochemical and functional assays to evaluate the r-domain during cftr activation in living cells. we have previously described a gel-shift method by which phosphorylation of isolated r-domain (residues - ) can be monitored. using this method, we have confirmed that one potentiator, p (cfpot- ), does not induce phosphorylation of the r-domain ( % of forskolin response, n= , p = ns). unexpectedly, two potentiators, p (uccf- , a benzoflavone intermediate) and p (uccf- , an isoxazole), robustly confer phosphorylation of the r-domain (p : % of forskolin response, n= , p = . , p : % of forskolin response, n= , p = . ). we found the phosphorylation conferred by either p or p could be inhibited with the pka inhibitor h ( µm). maximal stimulation of phosphorylation occurs within minutes, indicating time dependence similar to forskolin. importantly, neither p nor p increased total cellular camp, a finding confirmed by a number of other laboratories. the results may therefore implicate compartmental inhibition of cftr-associated phosphotases (eg. pp a) or phosphodiestereases (eg. pde ) as an underlying mechanism by which isoxazole or certain flavone-derivative compounds stimulate cftr. to further test this hypothesis, p , p and p are being examined for effects on ∆r-cftr chloride channel activity. these studies provide a means by which novel cftr potentiators can be biochemically categorized based on r-domain phosphorylation, a measure of the first step of cftr activation. compounds working through distinct mechanisms may have particular relevance to certain cftr mutations, and could provide synergy in the clinical setting. supported by nih and cff. mucociliary clearance (mcc) is an innate defense mechanism that protects the lungs from bacteria and viruses. mcc requires maintenance of a thin layer of airway surface liquid (asl) to eliminate inhaled particles. the asl volume is tightly regulated by a balance of ion and water transport across the airway epithelia. in cf, the loss of cftr clsecretion coupled with unregulated na + absorption via enac results in asl volume depletion and impaired mcc. although significant progress has been made in the identification of the basic disease mechanism in cf, therapeutic approaches that address abnormal cftr biogenesis are not currently available. the extracellular nucleotides atp and utp are important mediators of asl volume and mcc. in the airways, secreted atp acts on the g-protein coupled p y receptors to fine-tune mcc via the regulation of apical ion transport, ciliary beating, and mucin secretion. we have previously demonstrated that atp controls asl volume by inhibiting absorption through enac and increasing secretion through apical membrane chloride channels. p y receptor agonists are good candidates to treat cf. however, the rapid hydrolysis of atp and utp on the airway surface of cf patients limits the effectiveness of this approach. consequently, inspire pharmaceuticals has developed di-nucleotide molecules which retain the ability to activate p y receptors, but are more resistant to hydrolysis by ectonucleotidases. denufosol, inspire's lead compound for cf treatment, potently activates p y receptors to stimulate cl -/water secretion, ciliary beating, and mucin release in epithelial tissues. however, the stability and potency of denufosol has not been determined using human bronchial epithelial (hbe) cells. in primary cultures of hbes, denufosol was significantly more stable than utp. under thin-film conditions, the initial hydrolysis rate of a therapeutic concentration of denufosol ( mm) was . nmol × min - × cm - with a half-life of approximately hour. by comparison, denufosol is more than times more stable than utp on the mucosal surface of hbes. importantly, the increased stability of denufosol translated into an increase in efficacy for this compound in vitro. equipotent concentrations of denufosol and utp significantly increased asl height. however, denufosol produced maximum asl height increases that were both greater ( . % versus . % maximum increase over basal asl for denufosol and utp, respectively) and longer-lasting ( . % versus . % increase over basal asl following a minute application of denufosol and utp, respectively) compared to utp. furthermore, the asl height increases are specific for denufosol as the addition of apyrase blunts the response to utp, but not denufosol. our data demonstrate that denufosol is more stable than utp on the mucosal surface of human airway epithelia, which results in larger and more sustained increases in asl volume. recent phase ii clinical trials show that administration of denufosol over days was well tolerated and associated with improved lung function in mild cf patients. taken together, these data suggest that denufosol is a promising candidate molecule for cf therapeutics. mucociliary clearance (mcc) is the primary airway host defense against chronic exposure to infectious and noxious agents. mcc is dependent upon ciliary beating and the volume and composition of the airway surface liquid (asl). asl volume is regulated via isotonic fluid transport which is dominated by na + absorption in the superficial epithelium. in cf, unregulated na + absorption through enac drives asl volume depletion and a subsequent decline in mcc. the accumulation of mucus in the airways of cf patients supports persistent and life-threatening bacterial infection. increasing airway surface hydration represents a promising therapeutic approach for treating cf. in vivo, aerosolized osmotic agents such as hypertonic saline (hs) improve mcc and lung function in cf patients. however, the benefits derived from hs treatments are transient, as nacl is relatively rapidly absorbed by airway epithelia. previously, donaldson et al. (nejm ) tested the hypothesis that blocking na + absorption with the enac inhibitor amiloride would increase the efficacy and extend the benefit of hs. surprisingly, amiloride blunted the response to hs, which resulted from a previously unrecognized property of amiloride to inhibit aquaporin-mediated water transport. while potent inhibitors of enac would likely be of great benefit to the cf treatment milieu, the compounds then available were not adequate for this purpose. parion sciences has developed novel -substituted pyrazinoylguanidine compounds that selectively inhibit enac and are > -fold more potent than amiloride. in the present study, we evaluated the ability of two compounds, - and , to increase asl volume under thin film conditions. in primary cultures of human bronchial epithelial cells (hbes), - alone produced a small, but significant increase in asl height. in cf cultures exposed to phasic motion that simulates the shear stress generated by normal tidal breathing, - alone increased asl heights by . %. additionally, we tested the effects of - pre-addition in combination with % hs. as expected, hs alone produced a rapid and substantial increase in asl height ( . % by minutes) which declined to % bỹ hour post-treatment. pre-treatment of cultures with - prior to hs was both more potent ( . % by minutes) and longer-lasting ( % initial response at ~ hours) than hs alone. on normal hbes cultures, the compound alone increased the basal asl height by . % following a hour exposure. similar to - , in combination with hs likewise extended the longevity of the hs effect (t / ~ h versus ~ h for + hs and hs, respectively). by blocking na + absorption, the parion compounds alone increased asl volume in both normal and cf hbes. strikingly, when used in combination with hs, the parion compounds enhance and sustain the increase in asl volume associated with hs alone. our data demonstrate that the parion compounds alone are sufficient to increase basal asl volume. furthermore, these data provide a proof-of-concept that combinational therapies utilizing osmotic agents and compounds that regulate ion transport will provide therapeutic benefits to cf patients. methods: a total of patients (with fev ≥ % of predicted) received mg of arikace by inhalation for days. drug was administered using the pari lc star nebulizer. laboratory parameters, adverse events and pulmonary function tests were collected for all study subjects in order to determine safety and tolerability of arikace™. sputum samples were collected to determine changes in bacterial density, and amikacin pharmacokinetic parameters were assessed at selected time points in urine, serum and sputum specimens. change in fev , fev % predicted, fef - % and fvc relative to baseline and change in log cfu on days and were assessed. results: on day , and , the observed change for fef - % was . (p < . ), . (p = . ) and . l/sec (p = . ), respectively. on day and , the observed change for fev was . (p = . ) and . l (p = . ), respectively, and was . (p < . ) and . l/sec (p = . ) for fev % predicted. significant relationships (p ≤ . ) between log cfu and serum auc:mic ratio, and between changes in log cfu and fev , fev % predicted and fvc were identified. treatment was safe and well tolerated with the most frequent adverse events being dyspnea and headache of mild to moderate severity. conclusion: inhaled arikace™ mg/ml was well tolerated and in select patients improved pulmonary function. together these clinically relevant changes from baseline likely represent drug effect and warrant further development of liposomal amikacin for inhalation in patients with cf. allergic bronchopulmonary aspergillosis (abpa) is a disease caused by hypersensitivity to aspergillus fumigatus (af). the prevalence of abpa is approximately %- % of patients with cf and can contribute to worsening of their pulmonary disease. the treatment of abpa consists of high-dose oral corticosteroids for many months and may include also antifungal antibiotic such as itraconazole. the clinical effectiveness of corticosteroids is usually shown by an improvement in clinical symptoms and radiological parameters, as well as a reduction in total ige. the prolonged period of systemic steroids may cause significant side effects such as cushingoid appearance, hypertension, glucose intolerance and more. over the past years, we have used pulse high-dose methylprednisolone in patients with cf. method:all patients were diagnosed as suffering from abpa by the standard criteria as the may have mucoid impaction or central bronchiectasis on chest radiography, an elevated ige (> iu/ml), the presence of specific ige anti af and an elevated eosinophil count. the patient may have a positive skin prick test to af allergen. they were treated once a month with iv pulse high-dose methylprednisolone ( mg/kg/a day) for days once a month for several months until the total ige decreased to normal values. three patients were treated also with itraconazole. in patients itraconazole was discontinues due to side effects. results: we treated patients age - years old ( m/ f). four patient are pancreatic insufficient and pancreatic sufficient. ige was +/- ( - ) at the time of diagnosis and decrease to +/- ( - ). fev increased from +/- to +/- . the patients also gained weight and improved their chest x-ray finding. side effect were minor and mainly during the treatment days and resolved - days after each treatment and included malaise during the infusions, glucose intolerance on infusion days in one case, and hypertension in another case. conclusion: iv pulse steroid treatment should be considered in abpa as it was found effective with fewer side effects that should be expected from prolonged oral corticosteroid treatment. lung pathology in individuals with cystic fibrosis (cf) is linked to sodium hyper-absorption. the defective regulation of the epithelial sodium channel enac is thought to be a major contributor to reduced airway surface liquid (asl) volume and impaired mucociliary clearance of the airways. thus, strategies designed to inhibit enac function may result in clinical benefit. rna interference (rnai), mediated by short, double-stranded rna molecules, can be used to target complementary rna sequences for degradation via the rna induced silencing complex (risc). we investigated the possibility of using rnai to reduce expression of enac in the mouse lung, as proof of principle for a strategy to reduce sodium hyperabsorption in the cf lung. potent sirna molecules capable of efficient knockdown of enac alpha, beta and gamma subunits were identified in an optimised cell culture system utilising mouse kidney m cells ( , cells per -well; pmol sirna complexed with lipofectamine ; hour harvest) in which enac expression was quantified using real-time rt-pcr. approximately % knockdown of each enac subunit was observed with the most potent sirna molecules in this system. subsequently, sirna molecules were delivered to the lungs of female balb/c mice via hydrodynamic tail vein injection ( µg naked sirna, n= - ). after hours, lungs were harvested, rna extracted and enac mrna measured using real-time taqman pcr. whereas, enac subunit mrna levels in mice treated with a negative control sirna were not different from untreated mice (p < . ), delivery of enac alpha and beta-specific sirna molecules resulted in a reduction to . ± . % or . ± . % of the expression levels observed in untreated mice, respectively (p < . , mann-whitney u). interestingly, despite efficient knockdown in cell culture, in vivo treatment with the most potent enac gamma-specific sirna molecule led to no reduction in enac gamma expression compared with untreated controls (p > . ). these data show proof of principle that enac expression can be reduced in the lung using sirna. further work is needed to assess the functional consequences of inhibiting enac in the lung. gene therapy for cystic fibrosis lung disease will likely require longterm transgene expression; however, in the lung, many gene transfer agents have resulted in only transient gene expression. previously we have shown that the choice of enhancer/promoter elements has a strong influence on the duration of reporter gene expression following delivery of non-viral vectors to the mouse lung (gill et al., , gene ther, , . however, it is also possible that other elements of plasmid vector design play an important role. using a clinically relevant mouse lung aerosol model, we have studied the effect of varying the plasmid cpg-dinucleotide content on the duration of expression from plasmids that had an identical enhancer/promoter sequence. firstly we constructed a cpg-free plasmid vector containing a synthetic cpg-free luciferase gene under the transcriptional control of the human cmv enhancer/ef α promoter. secondly, we constructed a similar vector containing an identical enhancer and promoter but with a small number of cpg motifs, in the luciferase gene ( cpgs) and in the backbone sequence ( cpgs). we then investigated the level and duration of transgene expression following aerosol delivery of these plasmids complexed with the genzyme lipid gl to the lungs of balb/c mice ( . mg/ml pdna, mm gl , ml, pari lc+ nebuliser). total lung extracts were assayed for luciferase activity at days , , , & (n= per time-point). the cpgcontaining plasmid initially directed approximately -fold higher levels of reporter gene expression than the cpg-free plasmid (p< . both days and post-delivery). however, while expression from the cpg-containing plasmid subsequently fell slowly to background levels, expression from the cpg-free plasmid increased -fold to day , and remained at the peak levels observed with the cpg-containing plasmid until the end of the experiment at day (cpg-free plasmid -fold, -fold and -fold higher expression at days , and post-delivery respectively; p< . for each). these data suggest that the ability of a promoter/enhancer sequence to direct long-term expression in the mouse lung is dependent by the sequence of the plasmid backbone. one explanation for this observation is that a specific, but as yet unidentified, sequence exists in the cpg containing backbone that triggers transcriptional silencing. alternatively, the well-described host inflammatory response to cpg-containing dna, may be involved and the cpg content of the vector could be responsible for these differences. we are currently evaluating the potency and duration of effect of cftr expressing, cpgfree, plasmids in late-stage pre-clinical studies prior to the initiation of a further round of clinical studies of non-viral gene transfer in cf patients. background: novel approaches to cf therapy by improving/restoring cftr chloride channel function using gene therapeutic tools or small molecules are currently undergoing phase i and phase ii clinical trials. there remains a great need for accurate and practical methods of measuring cftr function in vivo to act as primary outcome measures of efficacy. we propose that an in vivo assay measuring cftr function in sweat glands offers several potential advantages over the nasal potential difference test. objective: to develop a reliable test of sweat gland function that is capable of measuring the range of cftr function in vivo. methods: we are performing repeated measures of sweat gland function in healthy controls (n= ), obligate heterozygotes (n= ), pancreatic sufficient (cfps, n= ) and insufficient cf patients (cfpi, n= ). sweat secretion is stimulated by iontophoresis ( µa/cm ) with % pilocarpine. to elucidate the most sensitive and discriminatory parameter of sweat gland function within this study group we are performing simultaneous measurements of: a) the transglandular potential difference of a stimulated skin area using an ecg electrode (espd) and b) single sweat gland potential differences in single sweat glands (spd). using a wescor® collector cup we also measure: c) sweat secretion rate and (d) sweat chloride concentration (sweat cl-). results: we report interim results of our ongoing study (table) . sweat rate was not different between the groups, but a gender difference was observed (mean±sd; male: . ± . µl/min/cm ; female: . ± . µl/min/cm , p< . ) as previously described. our preliminary data show that espd as well as sweat cl-allow good discrimination among healthy controls, cfps and cfpi (p< . and p< . respectively). spd show similar trends to espd but with greater overlap (differences not significant). conclusion: these encouraging preliminary results, particularly the espd method and sweat cl-following stimulation with a lower iontophoretic current, justify further efforts to complete enrolment of subjects and to further refine technical challenges such as minimizing the effect liquid junction potentials across the ecg electrode. results will also be compared to the classical sweat test using higher iontophoresis current and npd measurements in these patients. this study is supported by the ccff and genome canada. ino- , a prodrug, has been demonstrated to inhibit nasal potential difference (pd)in human cf nasal airway epithelia and cf mice and this effect is more potent with repeated dosing. as such it is being developed as potential therapeutic for cystic fibrosis. ino- cell entry is facilitated by its hydrophobic propionoxy(methyl)ester protecting groups which can be hydrolyzed by intracellular carboxyesterases after the prodrug enters the cell. once the protecting groups are removed, the drug (ino- ) with the ether-linked octyl group is expected to be more slowly metabolized. however, the precise kinetics of the uptake of ino- , its conversion to its active metabolite, and methods. confluent cultures of hela or t cells were incubated for varying periods of time ranging from min to hrs with medium containing [ h] ino- ( x cpm/ml) and µm ino- cold carrier to ascertain the rate of uptake. in pulse chase experiments after cultures were incubated for hours, they were washed and incubated with media without radiolabel for varying periods of time. after the indicated times, the media was removed and the cells were washed, harvested, extracted and the aqueous and organic extracts subjected to sax and reversed phase hplc respectively. results ino- , a prodrug, has been demonstrated to inhibit nasal pd in human cf nasal airway epithelia and cf mice and this effect is more potent with repeated dosing. ino- cell entry is facilitated by its hydrophobic propionoxy(methyl)ester protecting groups which can be hydrolyzed by intracellular carboxyesterases after the prodrug enters the cell. once the protecting groups are removed, the drug (ino- ) with the ether-linked octyl group is expected to be more slowly metabolized. however, it is not known whether the mucus present in cf airways could impede its access to airway epithelia or whether ino- would cross the epithelial layer to enter the bloodstream. here, in order to address these issues we measured its uptake into human tracheal epithelial sheets in the presence and absence of mucus and compared its transepithelial permeation to mannitol. we studied the uptake and transepithelial fluxes of [ h]ino- in primary cultures of human tracheal epithelium with transepithelial resistance of from to ohms.cm . the mucosal surface of some tissues was rinsed three times with pbs to remove mucus for comparison with control cells which were not rinsed. elisa for human airway mucins performed on the rinsings confirmed that three rinses sufficed to remove the mucosal mucous blanket. the development of long-term, safe and efficacious gene therapies for lung disease will require efficient gene delivery to stem cells which produce the differentiated progeny of affected epithelial tissues. recombinant lentiviral vectors are being considered for gene therapy applications for the lung because they can efficiently transduce a wide variety of stem and progenitor cell types. however, it is of concern that strong constitutive viral promoters have produced oncogenesis in x-scid patient recipients of retroviral vectors in part due to the disregulation of proto-oncogenes by the strong viral promoter/enhancer located near the integration site. we propose that regulation of transgene expression to the tissue or cell type of interest will enhance the safety of gene therapy for respiratory disease. in this study we have investigated the potential for respiratory specific transgene expression from integrated lentiproviruses in cell lines in vitro and in mice in vivo. the lentiviral vectors constructed for this study contained regulatory elements predicted to produce lung specific transgene expression: the surfactant protein c promoter (spc) for alveolar epithelial type ii cell (aecii) expression, the clara cell kd protein (cc ) for clara cell expression in the airway, and the jaagskiete sheep retrovirus (jsrv) promoter for expression in both cell types. transgene expression from the spc and cc vectors was restricted to aecii and clara cell lines respectively, while expression from the jsrv vector was observed in multiple respiratory and non-respiratory cell types. following intra-tracheal delivery of lentivector supernatant to mice, transgene expression was observed in aecii from the spc lentivector (n= mice), and in clara cells from the cc promoted lentivector (n= mice). transgene expression was not detected in non-respiratory tissues following intravenous delivery of cc and spc lentiviral vectors to neonatal murine recipients (n= and n= respectively). in summary, incorporation of genomic regulatory elements from the spc and cc genes resulted in respiratory specific transgene expression in vitro and in vivo. these vectors will provide a useful tool for the study of lung biology and the development of gene therapies for lung disorders such as cf. cystic fibrosis (cf) is associated with chronic pulmonary inflammation and progressive lung dysfunction, associated with the formation of oxidants derived from neutrophil myeloperoxidase (mpo). indeed, respiratory tract levels of mpo are extensive, and have been found to correlate with decreases in respiratory parameters or disease severity in cf. mpo-derived oxidants thus may play a role in perpetuating the progressive lung dysfunction associated with cf. based upon these premises, we reasoned that mpo could represent a novel therapeutic target for the treatment of inflammatory diseases such as cf. herein we report that substituted urea and thiourea compounds are potent inhibitors of mpo. utilizing tetramethylbenzidine (tmb) as a classical heme peroxidase substrate, we screened a large number of structurally varied urea and thiourea derivatives. alkyl, cycloalkyl, and aromatic ureas and thioureas all proved to be effective mpo inhibitors, however, with a high degree of variability in potency. of those compounds examined, substituted phenylthioureas proved to be the most potent mpo inhibitors. various ortho-, meta-, and para-substituted phenylthioureas (ptus) were then assessed for inhibitor potency. the most potent inhibitor of mpo was -chlorophenylthiourea (ic = nm, ki = nm). the potency of ortho-ptus as mpo inhibitors showed a linear relationship with ortho sigma substituent constants (reflecting resonance, steric and inductive effects). moreover, cyclic voltametry experiments with ortho-substituted ptu compounds revealed a linear relationship between inhibitor potency and thiourea oxidation potential. this correlation is indicative of a substituent effect on the relative ease of sulfur oxidation within the thiourea moiety. this finding suggests that the redox-active sulfur plays a crucial role in mpo inhibition, perhaps via a direct interaction with the heme of mpo. thioureas proved to also inhibit the chlorinating activity of mpo, whereas sustituted ureas were not effective in this regard. both ureas and thioureas were also effective inhibitors of dityrosine formation by mpo. these findings will help facilitate the rational design and optimization of mpo inhibitors with even greater potency, which could serve as novel therapeutic agents for the treatment of cf. gene therapy is the transfer of a normal copy of the cftr gene and should, in theory, provide a long-term cure for cf. the development of prenatal screening programs for cystic fibrosis has provided an opportunity to identify cf patients in utero, and may provide an opportunity to treat the disease before birth, prior to the onset of disease symptoms. the long-term goal of our study is to investigate the therapeutic potential of delivering the normal cftr gene to epithelial stem cells in fetal cf mice by in utero administration of recombinant lentiviral vectors carrying the normal cftr gene. this would provide us with the unique opportunity to "correct" the chloride channel defect in the epithelial stem cells, which give rise to the epithelial cells in the tissues affected by cf at a very early stage. the aim of the current study is to optimize the in utero gene delivery of recombinant lentiviral vectors, carrying an egfp reporter gene to epithelial stem cells in fetal mice. mice have been used for these studies, as mouse strains with a variety of cf mutations are available. in all studies, the uterus was exteriorized following a mid-line laparotomy, and the injections were made directly into the amniotic fluid of individual fetuses. in the first studies, the survival of pregnant females and pups following saline injection at e . (n= ) and e . (n= ) was evaluated. all five females survived the procedure and an average of % ( / ) of the pups present at the time of injection were born alive. this experiment demonstrated that the surgical procedures were reasonably safe. in the next experiments, we injected . ul lentiviral supernatant (~ . x iu/pup) into the amniotic fluid of each pup and evaluated the effect of the gestational age pups on transduction efficiency. the pregnant mice were allowed to carry the fetuses to term. at p , p and p , - pups of each litter were sacrificed and skin, trachea, lung, stomach and intestines were harvested for analysis of gene transfer and egfp transgene expression. all but one pup ( / ) from the e . time point aborted prior to parturition. from the e . and e . injections, / and / pups were born naturally, respectively. at e . and e . , proviral sequences were observed in the skin, trachea and stomach, although the level of transduction varied among individual fetuses and tissues. immunofluorescence analysis on the skin of one mouse with a high proviral copy number demonstrated that some of the egfp positive cells were also positive for vimentin or f / , markers of mesenchymal cells and macrophages, respectively but negative for pancytokeratin. our preliminary studies suggest that in utero gene therapy with lentiviral gene delivery may be safe, however ongoing studies will provide more in depth analysis on safety and feasibility of delivering the normal cftr cdna to epithelial stem cells in the tissues affected by cf in utero. future studies will investigate the therapeutic potential of delivering a lentiviral vector containing the cftr gene to fetal pups in a murine model of cf. supported background & objectives: cystic fibrosis is characterized by progressive loss of lung function resulting from chronic bacterial infection and concomitant airway inflammation. while a number of bacteria are known pathogens in cf, mounting evidence suggests that the lungs of cf patients harbor numerous bacterial species, and that interactions within this ecosystem may affect outcomes. however, the composition of this community is not well characterized. we therefore initiated work to profile the bacterial diversity of the lungs of cf patients with end-stage disease by s rrna gene sequencing. methods: genomic dna was purified from snap-frozen explanted lung tissue of randomly selected cf patients undergoing transplantation in toronto between and . bacterial s rrna genes were amplified from each sample using broad-spectrum pcr primers, and pcr products used to generate s rdna libraries for each patient. s rdna profiles for each patient were generated by automated dna sequencing of up to randomly selected clones per library, and tentative species assignments made by comparison to known s rrna gene sequences using phylogenetically-based analyses. results: sequence analysis of s rdna sequences from this preliminary set of patients (avg. = sequences/patient; range = - ) demonstrates a range of complexity of the microflora in cf lungs, with some patients showing a single dominant organism and others showing multiple co-existing species. in total, over species have been identified to date in this set of patients. these include species identified previously by clinical microbiology, including pseudomonas aeruginosa (detected in / patients), and burkholderia species (detected in / patients known clinically to be infected). numerous organisms not previously reported in the lung were also identified, including several from the taxonomic order burkholderiales. interestingly, while organisms commonly associated with cf were detected in patients transplanted throughout the year, a cluster of four plant-associated species demonstrated striking seasonal variation in rates of detection. conclusions: the lungs of cf lung transplant patients harbor a diverse bacterial community. sequencing of s rrna genes is a powerful method to profile this diversity. correlation of these profiles with clinical information may help identify new patterns of infection and important interactions between species in this community. introduction and aims: pulmonary infection, primarily with pseudomonas aeruginosa, is the leading cause of morbidity and mortality in cystic fibrosis (cf) patients. we have demonstrated that anaerobic bacteria, belonging primarily to the genera prevotella, veillonella, propionibacterium and actinomyces, can be cultured from the sputum of cf patients. the aim of this study was to determine the antibiotic susceptibility of these anaerobic isolates grown planktonically and as biofilms. methods: the planktonic susceptibility of anaerobic isolates to antibiotics used in the treatment of cf (meropenem, piperacillin/tazobactam) as well as to antibiotics traditionally used to treat anaerobic infection (clindamycin, metronidazole and ampicillin) was examined using e-test® strips. selected anaerobic isolates were grown as biofilms for or hours in well trays prior to challenge with antibiotic. bacterial biofilm formation was determined using crystal violet. results: the planktonic susceptibility of anaerobic isolates ( prevotella, veillonella, propionibacterium, actinomyces and additional isolates) comprising different genera was determined. although all of the anaerobic isolates examined were sensitive to meropenem when grown planktonically, high levels of resistance to the anti-anaerobic drugs, metronidazole and clindamycin were observed. examining susceptibility by genera, the veillonella were the most resistant to piperacillin/tazobactam and the propionibacterium the most resistant to metronidazole. furthermore, several isolates, including of the prevotella isolates examined, were found to be resistant to multiple antibiotics. the susceptibility of anaerobic biofilms to antibiotics was found to be isolate and antibiotic dependent. generally antibiotic treatment of hour old biofilms was often able to eradicate biomass and prevent biofilm formation; however, antibiotic treatment after hours of biofilm formation with up to x the mic of an antibiotic often failed to eradicate the biofilm. conclusion: these results suggest that alternative antibiotic treatment regimes may be necessary to treat cf pulmonary infection if anaerobes are present. these results also suggest, based on in vitro susceptibility, that meropenem and not metronidazole or clindamycin would be the most effective antibiotic in treating any anaerobes present in cf pulmonary infection. early eradication protocols for the first appearance of pseudomonas aeruginosa (psa) have become standard practice in many cystic fibrosis (cf) clinics. the details of such protocols often vary significantly between clinics, making it important to compare efficacy from one approach to another. however, comparison of reported results is currently very difficult. this is not only because of variations in completeness of reporting, but especially because calculation of average time to regrowth cannot by definition include patients who remain free of psa regrowth at the time the "average" is calculated (nor those in whom a re-appearance of psa is found to be due to a different strain). such results would be better expressed using "time to event" (tte) statistics (analogous to "life tables") and different protocols can be better compared using this methodology. we report for the first time, results from our psa early aggressive eradication protocol for the period - , expressed using tte statistical analysis. children are seen in clinic on average times yearly for full assessment, and at each clinic visit sputum or pharyngeal cultures obtained by the physiotherapist. standard treatment for "first growth" pseudomonas aeruginosa consists of weeks of intravenous antibiotics (piperacillin mg/kg/day plus tobramycin mg/kg/day) followed by weeks of oral ciprofloxacin ( - mg/kg/day) and inhaled colymycin ( mg bid) for months. a total of treatment courses have been completed for the period - with a % clearance rate, defined as or more consecutive negative cultures for pseudomonas aeruginosa over months. since the initiation of this protocol, the percentage of patients colonized with psa in our cf pediatric clinic has declined from % in , down to % in . subsequent regrowth of psa occurred in / of patients. treatment courses may then be repeated a second, third, or fourth time. to determine whether these repeat psa isolates are newly acquired or identical to the previous isolate, rapd typing of these isolates is routinely assessed. a total of isolates have been rapd tested and have identified the same strain as the previous isolate in ( %) and a different strain in ( %)treatment courses. the average time to regrowth for those who had a recurrence of psa following this treatment protocol was months, but this does not include those patients who have had no further growth of psa for periods up to years. to reflect the results for all patients, including those who have not as yet had further regrowth of psa, we have constructed tte curves. these measures can be used to compare results using our protocol with results from other approaches to clearing pseudomonas aeruginosa in cf. retsch-bogart, g.z. inhaled antibiotics have been part of the therapeutic armamentarium for patients with cf for decades. we studied the effect of inhaled aztreonam on respiratory symptoms in cf in a phase , double blind, placebo (pl) controlled trial of azli, a novel formulation of aztreonam, which enrolled patients with cf from centers in the us, canada, australia, and new zealand. all patients have completed study participation. inclusion criteria included age ≥ years, pa in sputum or throat swab, fev ≥ % to ≤ % predicted, and no use of anti-pseudomonal antibiotics in the previous days. following a day screening period, patients were treated for days with either azli mg or pl. study drug was administered tid using the pari eflow® electronic nebulizer after pre-treatment with bronchodilator. concomitant standard cf therapies were allowed, with the exception of anti-pseudomonal antibiotics, azithromycin and hypertonic saline. the primary endpoint was change from baseline to day in scores from the cystic fibrosis questionnaire-revised (cfq-r) respiratory domain, a validated patient-reported measure of respiratory symptoms. other efficacy measures included change in pulmonary function (fev , fvc, and fef - ), number of hospital days and number of courses of iv, oral or inhaled anti-pseudomonal antibiotics. microbiological endpoints included change in pa bacterial density in sputum, change in susceptibility of pa to aztreonam, and emergence or disappearance of other pathogens. safety evaluations included adverse events, airway reactivity (defined as acute decrease in fev ≥ % at minutes after dosing) and clinical chemistry and hematology. complete efficacy and safety results from this trial will be presented. salam, a.p.; orchard, c.; wee, a.; hodson, m. introduction: oral ciprofloxacin is often used for pseudomonas aeruginosa (psa) infections in patients with cystic fibrosis (cf) not requiring intravenous antibiotics. oral chloramphenicol is commonly used at the royal brompton hospital (rbh) as an alternative to ciprofloxacin, but less so in other cf centres due to concerns about aplastic anaemia and uncertainty regarding effectiveness. this study aimed to address three questions: ) what proportion of psa is sensitive to ciprofloxacin and/or chloramphenicol? ) were there any adverse haematological effects with the use of oral chloramphenicol at rbh? ) how effective is chloramphenicol in comparison to ciprofloxacin? study design: we carried out a retrospective review from the rbh cf database ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) together with analysis of patients' notes. methods: ) psa sensitivities to ciprofloxacin and chloramphenicol were recorded from sputum samples taken in late . ) all blood dyscrasias in patients who had received chloramphenicol from to were reviewed. ) the database was searched for patients who had received a course of either oral chloramphenicol or ciprofloxacin from to . patients from each group were randomly selected. fev , fvc and oxygen saturations pre and post treatment completion were analysed, as were markers of symptom improvement. results: ) sputum samples from patients grew psa. some were sensitive to only one antibiotic, and there were some cross-sensitivities, but in total . % were sensitive to chloramphenicol and . % to ciprofloxacin. ) patients received oral chloramphenicol and oral ciprofloxacin from - . multiple courses were often given, but usually no more frequent than monthly with chloramphenicol. over years, cases of blood dyscrasia were recorded, none due to chloramphenicol, and all recovered. ) in the ciprofloxacin group: the mean oxygen saturations improved by . % (p . ), the mean fev improved by . % (p . ), and the mean fvc by . % (p . ). in the chloramphenicol group: the mean oxygen saturations improved by . % (p . ). ), the mean fev improved by % (p . ) and the mean fvc improved by . % (p . ). % of patients in the chloramphenicol group and % of patients in the ciprofloxacin group reported symptom improvement. a significant proportion of psa is sensitive to chloramphenicol and in over years there have been no documented cases of related aplastic anaemia. clinical improvements with choramphenicol comparable to ciprofloxacin were also recorded. our data shows chloramphenicol is safe and may be the oral antibiotic of choice in ciprofloxacin allergy or psa resistance to ciprofloxacin. a randomised prospective study is needed to further evaluate the efficacy of chloramphenicol in comparison to ciprofloxacin. pseudomonas aeruginosa lung infections are one of the main factors causing disease in cf patients. at present the generally recommended treatment is repeated courses of antibiotics. this has resulted in fewer infections and a markedly improved prognosis for the patients. but it has also severe drawbacks such as antibiotic resistance, disturbed normal flora and allergenicity. antibiotic resistance is an emerging problem world wide and alternatives are urgently needed. specific chicken antibodies, anti-pseudomonas igy, have potential both as an alternative and a complement to antibiotics (pediatr pulmonol ; : ) . anti-pseudomonas igy prevents p. aeruginosa from adhesion to epithelial cells and has affinity for flagellin in vitro. humans do not produce anti-igy antibodies and oral administration of igy is generally regarded as safe. the risk that bacteria should develop resistance to igy is extremely low. in total, cf patients have received antipseudomonas igy for up to years. since november, the drug is given to a group of cf patients (at present patients) on individual permissions granted by the swedish mpa and reimbursed by the swedish government. in our first study about the effect of igy there were . positive cultures/ months, compared to . / months in a control group. up to december , , nearly five years later, the effect is maintained with . / months for the whole study period. we are at present compiling the data from the control group during the same period. only two siblings in the igy-treated group have been chronically colonized (with an identical p. aeruginosa strain), which still is non-mucoid after . and . years. during the whole treatment period there have been no cultures positive for mucoid p. aeruginosa or b. cepacia and other pathogens (s. maltophilia, a. xyloxidans, atypical mycobacteria and a. fumigatus) have only appeared sporadically -possibly due to the relatively low use of antibiotics. all patients have preserved pulmonary functions and nutritional status. there have been no adverse events. thus treatment with anti-pseudomonas igy has diminished the number of positive cultures and delayed the onset of chronic infection. the need for antibiotics is reduced as well as their adverse side effects. in conclusion, igy is an important complement to antibiotics for prevention of p. aeruginosa infections in cf. introduction and aims: we have previously shown by culture that the lungs of clinically stable cystic fibrosis (cf) patients are not only colonised by commonly recognised aerobic bacteria, such as pseudomonas aeruginosa, but also by a range of potentially pathogenic anaerobic species. the aim of this study was to determine whether anaerobes are also present in the sputum of cf patients with an acute exacerbation of pulmonary infection. methods: sputum samples were collected and processed using strict anaerobic bacteriological techniques, prior to commencing and at the end of antibiotic therapy, from adult cf patients admitted for treatment of an acute exacerbation of pulmonary infection. bacteria within the samples were detected by plating on selective agars, quantified by total viable count and identified by pcr and sequencing of s ribosomal rna genes. as a control induced sputum samples were collected, using hypertonic saline, from healthy volunteers who did not have cf and processed using similar methods. results: anaerobes from a range of species including prevotella, veillonella, propionibacterium, actinomyces and gemella were detected in high numbers (up to x cfu/g of sputum) from all patients prior to commencing antibiotic therapy with the predominant primary pathogens (p. aeruginosa or b. cepacia complex) detected in similar numbers. anaerobic bacteria were also detected in sputum samples at the end of antibiotic treatment and for / ( %) patients they were present in lower numbers than detected before antibiotic treatment. moreover, in / ( %) patients there was greater than a one log reduction in the total viable count of anaerobes. similar data for detection of the predominant aerobic pathogens was also collected for of these patients. p. aeruginosa or b. cepacia complex was present in lower numbers following antibiotic treatment in / ( %) patients and in / ( %) patients there was more than a one log reduction in the total viable count. anaerobes were not detected in the induced sputum samples from volunteers and in the remaining samples they were present in much smaller numbers, ranging from to cfu/g of induced sputum, than detected in cf sputum. the most commonly isolated bacteria from the induced sputum samples were the actinomyces and streptococcus, with prevotella cultured from only samples. no veillonella and propionibacterium species were isolated from the induced sputum samples and combined with the low frequency of isolation of prevotella species, these results strongly suggests that the prevotella, veillonella and propionibacterium species recovered from cf sputum samples are not oral contaminants. conclusion: these results indicate that anaerobes are present in large numbers within the cf lung during an acute exacerbation of pulmonary infection. their presence could be of important clinical relevance to cf patients as they may contribute significantly to the inflammatory process. airway infection and inflammation cause the majority of morbidity and mortality in cystic fibrosis (cf). the microbiology of cf is complex. not only are the routinely identified pathogenic bacteria present, increasingly a number of other bacteria are associated with pulmonary disease. in addition, the normal microbiota likely contribute to airway disease in cf via intracellular signaling, but remains largely unstudied. the application of culture independent methods for bacterial identification in cf airway secretions provides for the first time the ability to characterize the bacterial communities in the cf airway comprehensively and efficiently. here we present the results of a two-year study of cf airway samples to describe the bacterial communities present by ribosomal rna sequences. we have examined airway specimens from subjects ( cf, controls). these results provide a unique perspective on cf airway microbiology. bacterial communities identified in sputum were more complex than communities identified in bronchoalveolar lavage fluid (balf). however, pulmonary status determined the level of complexity, with higher complexity observed during stable pulmonary function. we identified a collection of anaerobic bacteria that were present at high levels during pulmonary exacerbation in a subset of cf subjects. detection rates ranged from - % of cf subjects examined depending on the specific organism (table ). these organisms of interest are suspected to be involved in pulmonary exacerbation especially when cultures for standard cf pathogens are negative (~ % in our center). preliminary data will be presented from quantitative real-time pcr experiments to track these organisms. we also will present the application of new meth-ods for community comparisons. this analysis provides the ability to look at how clinical information from individuals with cf corresponds to the microbial communities that occur in the airway. overall, the results point to a much richer bacterial community associated with the cf airway than previously thought. the results also point to new opportunities to understand the differences within the cf population, and thus potentially improve individual patient outcomes. cystic fibrosis (cf) patients are characterized by persistent microbial colonization of the airways and recurrent pulmonary infection. however, due to the domination of bacterial communities present in cf sputum by a small number of species (typically pseudomonas aeruginosa), culture-independent approaches to characterize the depth of bacterial diversity in cf sputum have yielded limited information. here we describe application of a novel microarray, the s rrna phylochip to comprehensively describe the bacterial diversity present in temporal sputum samples of cf patients who experienced periods of exacerbation (defined as hospitalization) and remission. in addition, these samples permitted assessment of bacterial community dynamism during periods of antimicrobial administration in these patients. phylochip analysis identified bacterial taxa present across these patient samples, including a large number of known human pathogens. antimicrobial administration caused a profound decrease in bacterial diversity in all patients examined. despite this, a sizeable fraction of each community remained stable or proliferated during the period of antimicrobial administration, suggesting many of the bacterial taxa present were resistant to the antimicrobials administered. taxa exhibiting this behavior included p. aeruginosa, arcobacter cryaerophilus, streptococcus constellatus and burkholderia mallei amongst other pathogens. these results suggest that complex polymicrobial communities exist in cf sputum that exhibit significant antimicrobial driven dynamism and consist of multiple antimicrobial resistant pathogens. future studies will focus on whole community gene expression to determine which virulence systems are activated in response to bacterial community perturbations and contribute to pathogenic processes in cf airways. the lungs of chronically infected cystic fibrosis (cf) patients have been intensively studied. it is well documented that pseudomonas aeruginosa is the predominant pathogen in cf (høiby , baurnfeind et al. , koch , and that p. aeruginosa accumulates in heaps (aggregates), detected in cf sputum (høiby ) and intraluminal in the lung (baltimore et al. ) . the bacterial density is highest in the bronchi (potts et al ) and these p. aeruginosa microcolonies are embedded in a matrix (lam et al. ) . the morbidity of the infection is due to an extensive and ongoing pmn response which apparently does not eradicate the bacteria, instead leading to slow degradation of the lung. in addition, even highest deliverable doses of antibiotics fail to clear the bacteria completely, though survival has significantly increased the last years due to aggressive anti p. aeruginosa therapy (koch and høiby ) the present study was performed to investigate the significance of the aggressive therapy for the distribution of p. aeruginosa and how p. aeruginosa is organized and persists in the cf lung. the material used was: autopsies from dead short term colonized cf patients (n= ) obtained before today's aggressive antibiotic treatment , and explanted lungs from long term p. aeruginosa infected cf patients (n= ) (i.v. antibiotics every rd month since + inhalation of colistin daily since . histological sections of the lungs were investigated using he stain, gram stain, alcian blue, antibodies against alginate and p. aeruginosa specific pna-fish combined with bacuni pna-fish and with dapi as counter stains. due to the high specificity of the pna-fish probes employed in this study, our results provide strong evidence for that before the aggressive antibiotic therapy, p. aeruginosa infected and destroyed the cf lung due to fast spreading into the respiratory zone. today antibiotics suppress but can not eradicate the bacteria from the conductive zone, whereas the remaining respiratory zone is protected from massive biofilm infection for prolonged time. the conductive zone serves as a reservoir; here the bacteria are organized in microcolonies embedded in puss, a trait which is independent on the time course of the infection and amounts of antibiotics. these microcolonies consisted solely of p. aeruginosa. in addition, we found no bacteria adhering to the epithelial tissue. the pus consisted mainly of leukocytes, surrounding the microcolonies. a smear of dna and dead leukocytes were detected just around the microcolonies, possible due to the quorum sensing dependent rhamnolipid killing recently described by us (jensen et al. ) . we conclude that p. aeruginosa persists in the cf lung due to its ability to create microcolonies i.e. biofilms. within these biofilms the bacteria are protected against antibiotics and the host defence. respiratory viral infections, including influenza (flu) and respiratory syncytial virus (rsv) contribute to the morbidity of children with cf. additional human respiratory viruses have been identified in children such as human metapneumovirus (hmpv) and coronavirus (cov), but these have not been examined in cf patients. in our laboratory, pcr assays have been developed to identify both traditional and newer respiratory viruses in healthy and immunocompromised children. these assays are usually performed on nasal swabs or nasal wash samples. however, oropharyngeal (op) and sputum samples are routinely obtained in cf for bacterial culture. the goals of this trial are to determine whether these available samples can be used for viral pcr studies and to examine the epidemiology of respiratory viruses in cf. for this single center observational trial, cf patients, ages . to . yrs (mean . yrs), have been recruited. subjects provide op and nasopharyngeal (np) samples at quarterly clinic visits and with acute pulmonary exacerbations requiring hospitalization; sputum is also collected if patients can expectorate. np and op samples are pooled when both are available. viruses assayed are flu a and b, rsv, parainfluenza (piv) , , and , hmpv, cov, adenovirus (adv), and rhinovirus (rhv). subjects are being followed for two respiratory viral seasons. we report here the methodology of viral pcr detection in cf sputum, as well as the results of viral detection during the first respiratory viral season in the seattle community. the mean length of time subjects were followed was . yrs (median: . yrs, range: . - . yrs). overall, samples ( % of the samples analyzed) were positive. twenty-four subjects ( . %) had at least one viral infection detected and ( . %) had two viruses at different visits. both sputum and pooled op/np samples were available at visits and were concordant in % of cases. among the discordant cases, it was more common for sputum to be positive and op/np to be negative ( . %) than the converse ( . %). the most frequently identified viruses were rhv ( isolations); piv and cov were the next most prevalent with and isolations, respectively. in conclusion, sputum viral pcr shows promise for the diagnosis of both traditional and novel viral infections in cf. as part of ongoing clinical trials in cf, non-fermenting gram negative bacilli (nfgnr) identified at site laboratories are often sent to the tdn core microbiology laboratory for confirmation of identification. among nfgnr from cf patients, p. aeruginosa are the most frequently isolated and also are acknowledged to be the easiest to identify. however, in a survey of "p. aeruginosa" isolates sent to the laboratory in the past year, pcr identified ( %) as alternate species. these included: non-aeruginosa pseudomonads, stenotrophomonas maltophilia, achromobacter xylosoxidans, ralstonia spp., alcaligenes spp., brevidimonas spp., and bordetella bronchiseptica. identification of these nfgnr can be difficult; however, p. aeruginosa is an important isolate for the clinical microbiology laboratories in all cf centers to be able to identify. many methods are available including conventional biochemical testing and rapid identification kits. in the tdn core laboratory we previously demonstrated the importance of combining phenotypic identification (morphology plus a short set of biochemical tests) and genotypic identification ( s rdna sequencing of v and/or pcr using three primer sets) and developed an algorithm for testing. in this study, we examined all of the cf cultures performed in the clinical microbiology laboratory at chrmc over the past two years to determine the number of isolates for which it was necessary to perform genotypic identification in nfgnr. among this collection of nfgnr from clinical cultures sent to the microbiology laboratory from the seattle children's hospital cf center, pcr identified isolates as burkholderia cepacia complex. next most commonly identified were a. xylosoxidans ( ), p. aeruginosa ( ) and non-aeruginosa pseudomonas spp. ( ). three isolates remained unidentified, even with pcr sequencing of the v determinant. the clinical microbiology laboratory at chrmc has served as the core microbiology laboratory for tdn clinical trials for years and thousands of cf samples are processed annually. the result is that there is increased experience with cf isolates among the laboratory personnel. however, even with experienced technologists, a significant number of nfgnr required genotypic identification in the past two years. thus, it is important for clinical microbiology laboratories with less experience in the identification of cf nfgnr to utilize additional molecular techniques or send problematic isolates to a reference laboratory. based on these results we propose an algorithm for the most efficient means of identifying nfgnr from cf samples using a combination of tests including phenotypic (colony morphology, biochemical testing) and genotypic identification (pcr, sequencing). background: the epidemiology of s. aureus has been changing in both cf and non-cf patients. in non-cf patients, methicillin-resistant s. aureus (mrsa) has traditionally caused infections in patients with risk factors such as hospitalization, surgery, and anterior nares colonization. recently, community-acquired (ca-) mrsa infections have increased among patients without known risks. in cf, colonization of the anterior nares with methicillin-susceptible s. aureus (mssa) is associated with transmission between patients and household members. the prevalence of respiratory tract colonization/ infection with mrsa has increased in cf, but little is known about risk factors for mrsa, including possible transmission among families, and if strains are ca-or healthcare-acquired. methods: this is a case-control, multicenter study to investigate potential risk factors for mrsa among children with cf - years of age. subjects are enrolled from cf centers in the new york metropolitan area that have a mrsa prevalence ranging from % to %. cases are children with a positive respiratory tract culture for mrsa within the past years. controls ( age and center matched per case) are negative for mrsa. the aims of the study are: [ ] to determine the point prevalence of anterior nares colonization with s. aureus in children with cf and their household members; [ ] to assess potential risk factors for mrsa by administering a survey to the parents of subjects inquiring about factors such as crowding, pets, and participation in contact sports and by reviewing the medical record for antibiotic use, hospitalization, and surgery; [ ] to determine potential transmission of s. aureus within families and centers by assessing the molecular epidemiology of strains using pulsed field gel electrophoresis and meca type of mrsa isolates. results: to date, cf subjects ( cases, controls), mean age . years, and household members ( to per subject) have been enrolled. preliminary data demonstrate positive anterior nares cultures among cases, controls, and household members to be / ( %), / ( %), and / ( %), respectively. of the s. aureus isolates, were mssa and were mrsa ( cases, household members of cases, and household members of controls). during the past months, household members have been hospitalized and one has stayed overnight in a nursing home; had staphylococcal infections and had skin infections. data collection and molecular analysis are ongoing. conclusions: we expect this study to provide insight into risk factors for respiratory tract colonization/ infection with mrsa, the role of ca-mrsa in cf, and potential strategies to prevent mrsa. aims: . revise infection control guidelines for the institution . establish outpatient infection control policies consistent with those used for hospital inpatients . cohort young and recently diagnosed cf patients without pseudomonas or mrsa to clinic days separate from colonized patients . establish a protocol for eradication of mrsa in newly colonized patients. methods: working with representatives from hospital epidemiology, the cf center, inpatient units, outpatient clinics, respiratory therapy, and cf families, a uniform infection control policy for cf patients in all settings (inpatient, outpatient, home care) was drafted and revised. arrangements were made to cohort newly diagnosed patients and children < years free of pseudomonas and mrsa to separate clinic days from colonized patients. review of the mrsa positive patients in our pediatric population, for whom data was available, showed median age of acquisition of years (range mo to yrs). for patients the first culture obtained at diagnosis or transfer from another center was mrsa positive ( mo, yr, yr). an eradication protocol for newly acquired mrsa has been developed involving treatment with oral and topical antibiotics and phisohex or clorox washes for days, plus changing disposable respiratory care equipment at the beginning and end of the treatment period. surveillance cultures of nose, groin, and airway or sputum are done after completion of the treatment protocol and re-treatment initiated if still positive for mrsa. conclusion: using a three-tiered approach of infection control guidelines, cohorting, and mrsa eradication protocol we expect to decrease our overall rate of mrsa and protect our newly diagnosed and young cf patients from acquisition. objective: studies have shown that respiratory pathogens can be transmitted within cf centers. in a recent cross-sectional study at cf centers, we assessed the rate of bacterial shedding by cf patients during office visits and noted that % of patients carried respiratory flora on their hands. the current study was performed to assess the effectiveness of alcohol-based rubs on hand carriage of respiratory pathogens by patients during the course of office visits. methods: four bacterial organisms were chosen for study: pseudomonas aeruginosa (pa), staphylococcus aureus (sa) [including methicillin-resistant (mrsa) strains], stenotrophomonas maltophilia (sm) and burkholderia cepacia complex (bcc). at the beginning of clinic visits, hands of study patients were cultured using the "glove-juice" technique (gjt) [ ] . hand hygiene was then performed using an alcohol-based rub. at the end of the visit, hands were again sampled using the gjt. samples were sent to the microbiology laboratory at dartmouth-hitchcock medical center for culture and identification of study organisms. hand and respiratory tract isolates were compared using pulsed field gel electrophoresis (pfge). recovery experiments were also performed to assess the sensitivity of the gjt. results: samples were collected from encounters ( adult; pediatric). median encounter time was min (range . - . min). recovery experiments determined that the level of detection for each study organism using the gjt was ~ colony forming units. confirmation of matches between hand and respiratory isolates was determined by pfge. the number of patients with study organisms recovered from the respiratory tract was: pa, sa, mrsa, sm, and bcc. the overall hand contamination rate with a matched respiratory tract isolate at either culturing time was % ( % ci, . - . %). before use of the alcohol rub, hand contamination was . % ( % ci, . - . %). in those patients with hand contamination by respiratory pathogens prior to alcohol rub, . % ( % ci, - %) had negative cultures at the end of the clinic visit. however, there was strong evidence of hand contamination during the encounter, despite hand hygiene, as the overall rate at the end of visits was . % ( % ci, . - . %). there was a trend toward increased hand contamination in those suffering from pulmonary exacerbations versus clinically stable patients ( . % [ % ci, . - %] vs. . % [ % ci, . - %], p= . ). conclusions: contamination of patient hands by respiratory tract pathogens is observed in the outpatient setting in approximately % of patients. a single application of an alcohol-based hand rub, while safe and inexpensive, has limited antiseptic effectiveness during the full course of a typical cf clinic visit. repeated use of alcohol-based hand rubs during outpatient encounters would likely be more effective. these data support recent recommendations in the cf foundation consensus guidelines for infection control [ ] (nthi) is the most common initial bacterium infecting the airways in cystic fibrosis (cf). nthi infection usually precedes pseudomonas aeruginosa infection in many chronic lung diseases, including cf, emphysema, diffuse panbronchiolitis, and immotile cilia syndrome. it is hypothesized that nthi acts as a gateway organism paving the way for subsequent infection with p. aeruginosa, however, the mechanism by which this occurs remains poorly understood. we used a novel co-culture model of persistent nthi biofilm infection on airway epithelial cultures to study epithelial immune responses following nthi biofilm infections. objective: we hypothesized that prolonged exposure to nthi biofilms causes airway epithelia to become tolerant to inflammatory stimuli and show subsequent decreased innate immune responses. methods: nthi were inoculated onto the apical surface of calu air-liquid interface epithelia and grown in co-culture. rna from sets of paired airway epithelial cultures at , , and hours both with and without nthi inoculation was isolated and hybridized on custom affymetrix chips. data were normalized with rma and data biases were removed using the mixed model anova method. differentially expressed gene analyses were performed using anova, and pathways analyses were performed using gene set enrichment analysis (gsea). to test the hypothesis that prolonged nthi infection induces tolerance, epithelial cultures were pre-treated with either days of nthi or days of pbs before stimulation with pbs, il β ( ng/ml) or th cfus of live p. aeruginosa before subsequent measurement of epithelial responses. results: both rma and gsea analyses of microarray data showed many innate immune and pro-inflammatory responses at early timepoints that largely returned to baseline after days of co-culture, despite an increasing apical nthi biofilm infection. stimulation with il β or p. aeruginosa after days of nthi infection resulted in significantly decreased il production compared to uninfected controls. nthi treated cultures subsequently infected with p. aeruginosa also displayed increased tight junction integrity and resistance to cell culture death. conclusions: persistent nthi infections on airway epithelia resulted in increased tolerance to bacteria and inflammatory stimuli and increased resistance to bacterial toxicity. airway epithelial tolerance may contribute to a failure to clear subsequent bacterial infections. these phenomena would help to explain how early infections with nthi may promote chronic bacterial infections and p. aeruginosa acquisition in cf and other nthi related airway diseases. the colonization of cf airways by p. aeruginosa leads to intractable and persistent lung infections that resist permanent eradication by antibiotics. the lack of efficacy of current therapies is believed to be due to the formation of p. aeruginosa antibiotic resistant biofilms in the cf airways. however, little is known about biofilm formation on human airway epithelial cells. thus, we designed a continuous flow, live cell imaging system to grow p. aeruginosa biofilms on glass or on the apical surface of polarized human cells. cfbe ohuman airway epithelial cells homozygous for the ∆f mutation (cfbe), and cfbe o-cells complemented with wt-cftr (cfbe+wt-cftr) were grown as polarized monolayers at an air-liquid interface and gfp-labeled p. aeruginosa were applied to the apical surface of the cells. after hours biofilms did not form on glass but large bacterial macrocolonies developed on cfbe cells. surprisingly, biofilms also developed on cfbe+wt-cftr cells: however, p. aeruginosa biomass on cfbe+wt-cftr cells was significantly reduced compared to cfbe cells ( . ± . µm /µm vs . ± . µm /µm ). the minimal bactericidal concentration for tobramycin was > , µg/ml for p. aeruginosa grown on cfbe and cfbe+wt-cftr cells. these results explain why tobramycin given clinically, which is~ , µg/ml in bronchoalveolar fluid collected from cf patients, fails to eradicate the persistent infection by p. aeruginosa. p. aeruginosa also formed biofilms on human bronchial epithelial cells, which produce mucus. indeed, biomass was similar on cfbe+wt-cftr cells, which do not produce mucus, and human bronchial epithelial cells. to determine if human airway epithelial cells secrete factors that facilitate biofilm formation, conditioned medium was collected from cfbe and cfbe+wt-cftr cells and applied to p. aeruginosa grown on glass. under these conditions, biofilm formation was dramatically facilitated, with a biomass of . ± . µm /µm when p. aeruginosa was grown in conditioned medium collected from cfbe+wt-cftr cells and . ± . µm /µm when grown in conditioned medium collected from cfbe cells. the secreted factor(s) were greater than kda, as assessed by size exclusion. our results suggest that human airway epithelial cells secrete factor(s) that facilitate biofilm formation by p. aeruginosa, in the presence and absence of mucus, and that secretions from cf cells are more effective in promoting biofilms than secretions from non-cf cells. the ability of non-cf cells to facilitate biofilm formation appears to contradict the observation that non-cf lungs do not harbor p. aeruginosa. we propose that the innate immune response in the non-cf lung effectively eliminates p. aeruginosa from the lung, thus, biofilms do not form even thought the airway epithelial cells secrete factors that have the potential to facilitate biofilm formation. however, in cf when mucociliary clearance is compromised, p. aeruginosa accumulates in the lung and secretions by cf airway epithelial cells greatly enhances the formation of drug resistant biofilms (supported by the nih (ro ai , ro -hl- , p -rr , t -dk- ), and the cff (stanto ro, anders fo). ma, l. ; parsek, m.r. ; wozniak, d.j. . microbiology and immunology, wake forest university health science, winstonsalem, nc, usa; . university of washington, seattle, nc, usa bacteria in natural, industrial and clinical settings live in surface-associated communities termed biofilms, which are the source of persistent infection and resistance to antimicrobial treatment. biofilm development is initiated by the attachment of planktonic cells to a surface, followed by formation of microcolnies, and finally disperses swimming cells from microcolnies to occupy a new surface. to maintain the community structure, bacteria in a biofilm are usually enmeshed in an extracellular polymeric matrix, which consists of nucleic acids, proteins, and polysaccharides. exopolysaccharides (eps) have been known as a component of the biofilm matrix for years. however, little is know about how the eps matrix forms and develops and whether an eps can form a matrix independently. in the present report, we use lectins that specifically detect the mannose or galactose structure in pseudomonas aeruginosa psl eps. this technique allows us to visualize psl eps on the bacteria cell surface and in the biofilm matrix. our results indicate that psl eps is likely anchored on the cell surface in a helical pattern and clearly forms a matrix, which holds bacteria in the biofilm and on the surface. in a flat multiple-layer biofilm, psl eps is equally distributed in the entire biofilm. however, microcolonies reveal peripheral staining of psl eps with minimal staining of matrix in the center of the microcolonies. instead, this region has swimming cells indicating a biofilm development stage prior to dispersion. more strikingly, this area also has concentrated dead cells and/or extracellular dna, which fills up the viod spaces in the lower center of microcolonies at the stage prior to dispersion. these data provides a plausible mechanism for how p. aeruginosa sacrifices a portion of cells to make void spaces and free another portion of cells for future dispersion. in addition, our data also show that psl eps matrix and dna matrix are not overlapping, which suggests that these two components of the matrix work coordinately to encase bacteria in the biofilm. finally we show that the psl eps matrix is present in biofilms formed by mucoid p. aeruginosa, and formation of the psl eps matrix is independent of the production of alginate and pel, two eps that also contribute to p. aeruginosa biofilm formation. overall, our data indicates that psl eps functions as a primary scaffold, holding biofilm cells together in the matrix. moreover, our data along with published literature suggest a possible model for how p. aeruginosa biofilms persist in cystic fibrosis patients. . clinical microbiology, rigshospitalet, copenhagen, denmark; . biocentrum, technical university of denmark, kgs. lyngby, denmark; . department of paediatrics, copenhagen cystic fibrosis centre, rigshospitalet, copenhagen, denmark pseudomonas aeruginosa predominates chronic lung infections in patients with cystic fibrosis (cf). a hallmark of chronic p. aeruginosa lung infections in cf patients is the presence of mucoid p. aeruginosa biofilms surrounded by numerous polymorphonuclear leukocytes (pmns) in the lower airways. how p. aeruginosa escapes the bactericidal pmns is not fully understood. several virulence factors of p. aeruginosa is controlled by quorum sensing (qs); a density dependent mode of inter-bacterial communication based on signal transmitter molecules. active qs is present during chronic p. aeruginosa lung infections in cf patients and we have previously demonstrated a qs-regulated tolerance of biofilm bacteria to the antimicrobial properties of the pmns. the precise qs-regulated effect on the pmns is, however, unknown. in the present study, further elaboration using flow cytometry and microscopy revealed that qs-competent p. aeruginosa (pao and ∆rhli lasi complemented with c -hsl and -oxo-c -hsl) induces rapid necrosis of the pmns. this mechanism was also observed in in vitro biofilms and in mouse lungs infected with p. aeruginosa embedded in alginate. by using hplc fractionation the toxicity could be ascribed to a single substance. using lc-ms and d nmr this substance was identified as rhamnolipid b. in accordance, concentrations of rhamnolipid exceeding µg/ml were found in the toxic supernatants from wild-type p. aeruginosa (pao ) whereas non-toxic supernatants from qs-deficient knock-out mutants (∆rhlr lasr and ∆rhli lasi) and from ∆rhla mutants contained low amounts of rhamnolipids approaching µg/ml. our results demonstrate the potential of the qs system to facilitate infections with p. aeruginosa by utilizing rhamnolipid to disable a major first line of defense of the host -the pmns. furthermore, our study emphasizes the inhibition of qs as a target for the treatment of infections with p. aeruginosa. carlsson, m. , ; pettersson, a. ; andersson, c. ; wieslander, j. ; eriksson, l. ; segelmark, m. ; hellmark, t. . dept of microbiology, immunology, and glycobiology, university of lund, lund, sweden; . department of nephrology, lund university, lund, sweden; . heart lung division, the cystic fibrosis center, lund university hospital, lund, sweden; . wieslab analys ab, lund, sweden the clinical consequence of chronic pseudomonas (p) aeruginosa colonization in cystic fibrosis (cf) varies between individuals for unknown reasons. auto-antibodies against bactericidal/ permeability increasing protein (bpi-anca) are associated with poor prognosis in cf. we hypothesize that there is a correlation between the presence of bpi-anca, the biological properties of the colonizing bacteria and the clinical conditions of the hosts. we have compared isolates of p aeruginosa from two groups of cf patients: one with positive serum levels of bpi-anca and deteriorating lung disease, and one with negative bpi-anca levels and stable clinical conditions. epithelial cells (a ) and isolated polymorphonuclear granulocytes (pmns) were stimulated with the clinical isolates and cell death was analyzed with flow cytometry. interleukin- (il- ) released into the supernatant was measured by elisa. we found that the anca associated strains in most cases showed a pyocyanin negative phenotype. these strains also induced less inflammatory response than the non-anca associated strains as shown by the number of necrotic cells and il- release, yet elevated compared to control. we conclude that colonization with strains of p aeruginosa that induce a weak inflammatory response is associated with unfavourable outcome in cf. we speculate that inadequate control of pathogen proliferation through an insufficient inflammatory response results in a slowly increasing number of bacteria and accumulation of dying pmns in the airways, contributing to the progressive lung disease seen in many cf patients. . pulmonary critical care, northwestern university, chicago, il, usa; . microbiology/immunology, northwestern university, chicago, il, usa; . pulmonary medicine, children's memorial hospital, chicago, il, usa; . pediatrics, children's memorial hospital, chicago, il, usa purpose: most cystic fibrosis (cf) patients are infected with pseudomonas aeruginosa (pa) and have progressive loss of lung function. among individual patients, however, there are marked differences in rates of lung function deterioration. although the reasons for this variability are not completely understood, it is likely that microbiological factors play a role. type iii secretion in pseudomonas aeruginosa (pa) isolates from non-cf patients have been associated with poorer outcome. the aim of this study was to determine whether there is an association between type iii secretion properties and deterioration in lung function in cf. methods: we prospectively enrolled cf children and adults over years. demographics, clinical characteristics, spirometry and a respiratory culture were obtained at the first visit. the age at time of first postive pa culture was also determined. subsequently, spirometry and respiratory cultures were obtained and clinical characteristics recorded every months until december . from each sputum culture individual isolates were selected for evaluation and type iii secretion was evaluated for each isolate by western blot analysis. the children were subcohorted into those newly infected ( st positive pa culture) or those chronically infected (minimum sputum pa (+) > year duration). results: there were patients evaluated, of which were adults and were children. ninety percent of our population cohort was caucasian and % were female. overall % of the patients were on tobi and % on macrolides chronically. the mean age at time of a first pa positive culture was years for the chronically infected subjects(adults and children) and years for the st time infected subcohort. a total of sputum samples were collected for the current study. the prevalence of type iii secretion properties for the overall population was . %. foe adults, of ( . %) pa isolates were type iii secretion positive compared to of isolates ( . %) in chronically infected children and of isolates( . %) from newly infected children. at study entry the mean fev % predicted was +/- for the whole cohort. for the adult subcohort the fev % predicted was % +/- %, for chronically infected children it was % +/- % and newly infected children is was %+/- %. the average annual rate of fev % change was - . % for adults, - . % for chronically infected children and + . for newly infected children. analysis on the relationship between type iii secretion pa isolates and fev % decline is ongoing and will be presented at nacf. conclusion: prevalence of type iii secretion in pa isolates from cf patients decreases with age. there is a slower decline in fev % in chronically pa infected adults compared to chronically pa infected children. the relationship of type iii secretion to fev % change and pulmonary exacerbations will be presented at nacf. a limited number of bacterial species -in example pseudomonas aeruginosa, staphylococcus aureus, burkholderia cepacia complex, stenotrophomonas maltophilia -is typically found in the cf lung. these facultative anaerobic bacteria exhibit the capability to switch from aerobic to anaerobic metabolism, which enables them to survive in the hypoxic environment in the cf lung [ ] . absence of oxygen would also favour the growth of fastidious anaerobes and, indeed, sequencing of s rrna resulted in the detection of strict anaerobic species in cf sputum [ ] . thus, we identified and quantified strict anaerobes in cf sputum using conventional anaerobic microbiological methods, examined the recovery rate of identical species in sputum samples from the same patients, and determined the antibiotic susceptibilities. sputum samples were collected from cf children and cf adults (mean age . ± . yrs). samples were incubated aerobically on columbia agar, supplemented with % sheep blood, and anaerobically on brain heart infusion agar and schaedler agar supplemented with % mutton blood, for up to days. bacteria were identified (rapid ana ii identification system, remel, lenexa, ks) and cfu's were determined by dilution plate counting. fastidious anaerobes ( strains) were submitted to e-test ® susceptibility testing (ab biodisk, solna, sweden) using the anaerobically active antibiotics ceftazidime, clindamycin, meropenem, metronidazole, and piperacillin/tazobactam. in . % of the patients, the following strict anaerobes were detected with a mean of . x cfu/ml (range . x to . x cfu/ml): peptostreptococcus spp., clostridium spp., actinomyces spp., prevotella spp., wolinella spp., propionibacterium spp., streptococcus spp., lactobacillus spp., gemella spp., bacteroides spp. and eubacterium spp., whereas p. aeruginosa, s. aureus and b. cepacia revealed . x cfu/ml (range . x to . x cfu/ml). in sputum samples contaminated with p. aeruginosa together with strict anaerobes . x ± . x cfu/ml p. aeruginosa were counted, whereas in samples with p. aeruginosa without strict anaerobes only . x ± . x cfu/ml were found (p= . ). for s. aureus, no significant difference was observed. identical strict anaerobic species were detected in out of patients with two or more repeated sputum samples ( %). e-test® sensitivity testing for strict anaerobes yielded high sensitivity for meropenem (only . % resistant strains), piperacillin/tazobactam ( . %), and clindamycin ( . %), but not ceftazidime ( . %) or metronidazole ( . %). high numbers of strict anaerobes are present in the majority of cf patients. possibly, strict anaerobes promote growth of p. aeruginosa but not s. aureus. the high persistence of identical anaerobic strains reflects chronic lung infection and may be caused by their increased resistence against standard antibiotics such as ceftazidime. [ in sputum in the cf lungs bacteria such as pseudomonas aeruginosa have to metabolize anaerobically [ ] . for anaerobic energy generation, p. aeruginosa can use nitrate and arginine, but also pyruvate which is produced from glucose via anaerobic glycolysis [ ] and can be metabolized to lactate and vice versa. in order to investigate if p. aeruginosa may benefit from externally produced lactate, we measured the concentration of lactate in p. aeruginosa, staphylococcus aureus, burkholderia cenocepacia and polymorphonuclear neutrophils in vitro and in cf sputum. in sputum samples of cf patients and in neutrophils ( x /ml) from healthy donors lactate concentrations were determined. in addition, p. aeruginosa (starting with x cfu/ml in tryptone soy broth), s. aureus ( x cfu/ml), and b. cenocepacia ( x cfu/ml)were grown aerobically ( through hrs) and anaerobically ( through days). l-lactate was measured spectrophotometrically (detection limit: . mmol/l), and total lactate gaschromatographically. aerobic and anaerobic gene expression of p. aeruginosa strain pao was determined using affymetrix® microarrays. lactate concentrations in cf sputum amounted to . ± . mmol/l (range . to . mmol/l). concentrations were similar in sputum samples colonized with p. aeruginosa, s. aureus ( . ± . vs. . ± . mmol/l, p= . ) and b. cenocepacia ( . mmol/l). neutrophils produced . mmol/l. in all samples exclusively l-lactate was found. during in vitro experiments, p. aeruginosa did not generate any lactate at all, neither aerobically nor anaerobically. in contrast, anaerobically grown s. aureus produced up to . mmol/l lactate, and b. cenocepacia up to . mmol/l. a p. aeruginosa suspension [ x cfu/ml] spiked with mmol/l l-lactate did not change its concentration, indicating that p. aeruginosa does not metabolize lactate. similar results were obtained in our gene chip experiments: after three days of anaerobic growth, the genes encoding for the lactate dehydrogenases were downregulated (pa ldha - . fold, pa llda - . fold) or unchanged (pa lldd . fold). in contrast, the genes encoding for pyruvate decomposition to acetyl coa (pa and pa , both encoding for pyruvate dehygrogenase e components) were upregulated by and fold, respectively. we could demonstrate that p. aeruginosa does not benefit from externally produced lactate. we confirmed the important role of pyruvate metabolism for anaerobic p. aeruginosa energy generation. whether lactate production of neutrophils, s. aureus or b. cenocepacia contributes to cf lung pathophysiology still remains to be investigated. references: [ ] macleod, d. ; barker, l. ; gurgel, j. ; kenney, t. ; burns, j. ; baker, w. . gilead sciences, inc., seattle, wa, usa; . university of washington, seattle, wa, usa antibiotic resistance may severely limit therapeutic options in individuals with cystic fibrosis (cf) or bronchiectasis. because of frequent antibiotic treatment courses, resistance continues to emerge, even to newer agents. treatment with multiple antibiotics in a single aerosol formulation may be a promising approach to slow development of resistance. fosfomycin is a phosphonic acid antibiotic that is bactericidal against both gram positive and gram negative organisms. fosfomycin inhibits the first committed step in the synthesis of peptidoglycan, suggesting cross resistance to other cell wall acting antibiotics will not occur. the aminoglycoside tobramycin is one of the most commonly used antimicrobials in cf, with potent activity against gram negative bacteria and the majority of staphylococcus aureus isolates. a : (wt/wt) fixed combination of fosfomycin:tobramycin (gs- / ) was used to determine the in vitro susceptibilities of a panel of respiratory pathogens: cf pseudomonas aeruginosa ( ), s. aureus ( ), haemophilus influenzae ( ), stenotrophomonas maltophilia ( ) and burkholderia cepacia complex ( ), including minimal inhibitory concentration (mic) and time-kill experiments in the absence and presence of % porcine mucin. synergy was evaluated using the checkerboard method, and spontaneous resistance mutation frequencies were determined in antibiotic-containing agar ( x, x and x mic). in vivo drug efficacy was examined using a rat agar bead pneumonia model of either p. aeruginosa or s. aureus. all experiments compared gs- / to fosfomycin and tobramycin as single agents. gs- / had a lower mic than tobramycin for the s. aureus strains, % of which were methicillin resistant (mrsa). for p. aeruginosa, gs- / had a lower mic and mic than fosfomycin alone, but tobramycin was more active than either. for h. influenzae and s. maltophilia, gs- / , fosfomycin and tobramycin had similar mic and mic . b. cepacia complex were resistant to all three drugs. results in the presence of mucin were similar. time-kill studies showed a more rapid and prolonged killing of s. aureus and p. aeruginosa by gs- / compared with either agent alone at the same drug concentrations. gs- / was bactericidal and exhibited concentration-dependent killing. synergy studies showed no antagonism between fosfomycin and tobramycin, and the majority of p. aeruginosa and all of the s. aureus tested demonstrated indifference for the combination. at x mic concentrations the mutation frequency of gs- / was at least - logs lower than tobramycin and - logs lower than fosfomycin alone for s. aureus. for p. aeruginosa the mutation frequency of gs- / was - logs lower than fosfomycin and - logs lower than tobramycin. in the rat pneumonia model, gs- / and tobramycin alone demonstrated bactericidal killing of p. aeruginosa; both were more active than fosfomycin alone. in vivo killing of s. aureus by gs- / was also demonstrated. gs- / appears to have advantages over single agents for the treatment of both gram positive and gram negative bacterial lung infections in cf and bronchiectasis. britton, l.j. antibiotic resistance is becoming a major problem in the treatment of pulmonary exacerbations in cystic fibrosis (cf). organ-isms that are resistant to multiple antibiotics infect the airways of an estimated - % of adults with cf. as a result of the growing resistance in cf patients, many centers have been performing synergy testing of sputum cultures in addition to the conventional culture and sensitivity testing. the purpose of our investigation was to determine if antibiotic synergy studies and the use of synergistic combinations of antibiotics improve therapeutic outcomes in cystic fibrosis patients with acute pulmonary exacerbations. methods: this study is a retrospective chart review of cf patients who had antibiotic synergy testing performed while hospitalized for respiratory tract infections. eligibility criteria included cf patients hospitalized with respiratory tract infections from to . laboratory data from each hospital admission was reviewed for synergy among those antibiotics commonly used against pseudomonas aeruginosa. a review of medical charts ascertained each patient's pulmonary function (determined by fev before and after antibiotic therapy), weight z-score, organism cultured from sputum sample, time to next hospital admission, and the antibiotic(s) actually prescribed. primary endpoints were determined to be the change in fev and the time to next admission. results: four hundred seventy-five hospital admissions were analyzed. a total of cystic fibrosis patients, age birth to years, were included in the study. patients receiving antibiotic synergy experienced a significant decrease in mean time to next admission ( days with synergy vs. days without synergy, p= . ). no significant difference was found in the change in fev before or after antibiotic therapy, with an increase of . % with synergy vs. . % without synergy (p= . ). patients infected with non-mucoid p. aeruginosa experienced days to the next hospital admission, while patients infected with mucoid p. aeruginosa experienced days to the next hospital admission (p = . ). no statistically significant difference was observed between the synergy and non-synergy groups in regards to nutritional status and lung function prior to antibiotic therapy. speculations: antibiotic resistance in cystic fibrosis patients increases the morbidity and mortality caused by this disorder with each exacerbation the patient experiences. synergistic antibiotic therapies should improve patient outcomes through more efficient bacterial eradication and increased time to next hospital admission; however, we were unable to substantiate this assumption based on the results from this study. synergistic antibiotics did not show an improvement in the therapeutic outcomes of days to next admission or change in fev . background: expectorated sputum (es) technique is currently the most frequently used method for routine assessment of lower airway infection in patients with cystic fibrosis (cf). induced sputum (is) using hypertonic saline (hs) has been successfully used in cf patients unable to produce sputum spontaneously, but only limited data are available comparing the diagnostic yield of expectorated versus induced sputum in cf patients. while ultrasonic nebuliser have been used in the majority of studies, new high output jet nebulisers may offer a suitable alternative technique to induce sputum expectoration. aim: to assess the feasibility of sputum induction using a pari e-flow nebulizer and to compare the diagnostic yield of is and es in children with cf . methods: this is a preliminary report of an ongoing study in routine clinical care in sputum producing children with cf. es is being obtained before sputum induction. subsequently, sputum induction is performed using stepwise inhalation of nebulized ml of %, % and % hypertonic saline with an e-flow nebulizer (pari, starnberg, germany). lung function is assessed by portable spirometry before the procedure and after inhalation of each saline concentration. results: so far cf patients ( females) with a mean age of . years (range to years) and fev between - % predicted (mean %) have been included in the study. all subjects provided es samples and all produced sputum after induction. discrepancies in cultures between es and is samples were seen in cases ( . %). in cases additional cf pathogens were found in is samples, whereas in cases es yielded additional organisms compared to is. the number of distinct pathogens was similar in the remaining patient, but different bacteria were found with the techniques. in cases p. aeruginosa was detected only with one of the techniques ( es versus is). the spectrum of side effects was similar to previous reports using other nebulizer systems, with throat irritation being the most common adverse event of the is technique. all patients who had symptoms during the procedure became asymptomatic at the time of discharge from the clinic - minutes after the procedure. patients did not finish all cycles of sputum induction procedure due to symptoms of shortness of breath and/or drop in fev > % from baseline (in patients); all reversed after salbutamol inhalation. vomiting occurred in one patient. patients refused to complete the procedure due to unpleasant taste and/or throat irritation. conclusion: these preliminary results show discrepancies between expectorated sputum and induced sputum cultures in a significant proportion of cf patients. this may be explained by the previously described regional differences in lower airway infection in cf airways rather than by a higher diagnostic yield of one of the techniques. the results also demonstrate that the e-flow system is an efficient and safe device for sputum induction in sputum producing children with cf. seidler, m.j.; salvenmoser, s.; müller, f.c. dept. pediatrics iii, university heidelberg, pediatric pulmonology, cystic fibrosis centre & infectious diseases, heidelberg, germany background: the preferred growth form of bacteria is a biofilm. s. aureus, h. influenzae, and p. aeruginosa can produce an extracellular matrix (ecm) with implications in cystic fibrosis (cf) lung disease. the biofilm can protect against host defenses and antimicrobials. a. fumigatus is a frequent colonizer of the cf respiratory tract and can cause allergic bronchopulmonary aspergillosis (abpa). while antifungals in vitro are active against a. fumigatus, in vivo antifungal therapy is often complicated or resistance is observable. the aim of this study was to investigate the ability of a. fumigatus to form a biofilm-like matrix in vitro on polystyrene (ps), human bronchial epithelia cells ( hbe) and human bronchial epithelia cells with f del/f del (cfbe o-). methods: a. fumigatus atcc # was incubated in rpmi at different ph and concentrations of fbs. temperature, production time and different flow conditions were varied on ps, hbe and cfbe o-. dry weight measurement and antifungal drug susceptibility testing was performed. scanning electron microscopy (sem) and confocal scanning laser microscopy (cslm) images were analyzed. results: the thickest biofilm was produced on ps with rpmi (+ % fbs, ph= . ) at °c for h slightly rocking. biofilm dry weight on ps was . mg after h and . mg after h. the dry weight of produced biofilm exceeded . mg on hbe and . mg on cfbe o-cells after h of biofilm production. there was no significant difference in dry weight increase between the cell lines and ps. planktonic a. fumigatus was susceptible to itraconazole ( . µg/ml), voriconazole ( . µg/ml) and amphotericin b ( µg/ml). aspergillus in biofilm was resistant against all drugs (> µg/ml). the sem pictures displayed a network of hyphal structures and matrix at h. characteristic flow channels were observed at h. cslm images displayed conidia and hyphal structures embedded in matrix formations. a-alexafluor dyed polysaccharides of the cell wall and of the ecm in the biofilm. three dimensional constructs of the cslm pictures displayed biofilm on hbe and proofed viability of the cells after h co-incubation. differences in biofilm production between hbe and cfbe o-were not significant. conclusions: a. fumigatus is able to form a biofilm structure in vitro on ps, hbe and cfbe o-. a biofilm-like matrix produced by a. fumigatus was evidenced by dry weight measurement, sem, cslm and antifungal drug resist-ance in comparison to planktonic cells. potential clinical implications of a. fumigatus biofilm formation in vivo require further attention and investigations. etherington, c. ; peckham, d. ; conway, s. ; hall, m. ; denton, m. . seacroft hospital, regional adult cf unit, leeds, united kingdom; . microbiology department, leeds teaching hospitals, leeds, united kingdom susceptibility testing results are not predictive of clinical response to antibiotic therapy in chronic pseudomonas aeruginosa infections in cystic fibrosis. we assessed the impact of reducing the number of routine susceptibility tests performed on clinical outcome in these cases. in june we introduced a protocol of limiting susceptibility tests to p. aeruginosa isolates obtained from respiratory samples taken at the commencement of antibiotic therapy, when there was evidence of clinical failure, or routinely if not tested in the previous three months. at all other times, isolates were identified and reported as normal but p. aeruginosa isolates were not subjected to susceptibility tests. between st june and th november p. aeruginosa, was isolated on at least one occasion from patients attending our adult cf unit. in this six month period we reduced the number of susceptibility tests by % (from a projected , tests on samples to an actual tests on samples). this resulted in projected savings of $ , in consumables and hours (costed at $ , ) of laboratory staff time per annum, a total saving of $ , (£ , ) per annum. we assessed the response to intravenous antibiotic treatment between the study period in and the same period in . no significant differences in median change of fev , fvc, crp, white cell count, weight, or duration of intravenous antibiotics were observed. for cf units sending regular, routine sputum samples, a reduction in the number of susceptibility tests performed in cases of chronic p. aeruginosa can be carried out without impacting on clinical outcomes. we report our preliminary results for a total of strains obtained from patients ( patients harbored more than strains). these patients were classified in groups of respiratory insufficiency according to their fev : severe in patients (fev < %), moderate in patients (fev : - %) and mild in patients (fev > %). the clonal distribution was analyzed for the different strains of sa ( to ) isolated from patients sputum. these strains were analyzed for their antibiotic susceptibility and typed by pulsed-field electrophoresis gel (pfge) after smai digestion of chromosomal dna. thirty seven patients ( %) were colonized with mrsa. nine patients were both colonized with mrsa and mssa. among mrsa strains, / ( %) were also resistant to more than three other antibiotic family. strains harboring minor differences in the banding pattern (> % similarity as assessed by the dice coefficient) were considered clonal. our results show that % of the patients were colonized with a single persistent strain during the year of follow-up. consecutive isolates with different pfge profiles were obtained from only / patients ( %). pfge analysis revealed that mrsa isolated from patients were grouped in clusters. these results revealed a possible clonal relationship between mrsa isolated from different patients with cf. we did not find any difference in the distribution of sa strains in our cf patients among the groups of respiratory insufficiency. the study is ongoing in our adult cf population and in necker pediatric cf centre in paris. giusti, r. ; furfaro, s. . pediatrics, long island college hospital, brooklyn, ny, usa; . research, lumina fund, new york, ny, usa palivizumab(synagis) is a humanized monoclonal antibody to rsv. the redbook acknowledges that some patients with cystic fibrosis may be at increased risk of rsv infection but that there is insufficient data to determine the effectiveness of this therapy. the objective of this study was to assess: )practice patterns of cf physicians in the us and canada )the severity of rsv disease in cf infants during the past rsv season. )if there is a standard of care concerning the use of synagis for cf infants. methods a questionnaire was developed using a web-based commercial vendor. an embedded web link was distributed via an e-mail sent to all us and canadian pediatric cf center directors. respondents clicked on a link to respond to the survey and results were automatically tabulated in real-time. completed responses were received from center directors ( us and canadian) for a response rate of % in us and % in canada. most responders ( %) have prescribed synagis for infants with cf in the first rsv season, however only % routinely prescribed synagis for all infants with cf. only % expressed having had difficulty in obtaining insurance approval for this medication. many physicians indicated that synagis was frequently prescribed by the general pediatrician and that infants living at long distances from a cf center may be hospitalized at local hospitals. these issues may affect the accuracy of the data and result in an underestimate of actual synagis prescription and hospitalization rates. there were infants diagnosed with cf in the past year and of these infants were reported as symptomatic. there were cf infants ( %) reported as having a documented rsv infection and ( %) of these infants had received synagis. there were infants ( %) with documented rsv infection who responded to outpatient management.there were no deaths but patients were hospitalized and of these had received synagis. of the infants that required admission to an icu had received synagis. of the patients who were noted as having persistent chest x-ray changes had received treatment with synagis. there were infants with persistent wheezing and of these infants had been treated with synagis. conclusions despite the limitations of a retrospective survey, this data demonstrates that rsv can cause significant and prolonged pulmonary disease and is a significant precipitating factor resulting in hospitalization of cf infants. the data also notes that many cf infants infected during the past rsv season have a mild illness and respond to outpatient management. a surprising finding of this survey is that infants at cf care centers where synagis was prescribed for all cf infants continued to have significant rsv related hospitalizations, persistent wheezing or prolonged chest x-ray abnormalities. there are different opinions among cf physicians concerning the routine use of synagis and currently the data suggests that there is not a standard of care concerning the prophylaxis of cf infants with synagis. this results of this survey should encourage physicians to prospectively study the efficacy of synagis prophylaxis in preventing hospitalization, persistent wheezing and chest x-ray abnormalities in cf infants. milani, a. ; cisbani, g. ; macchi, r. ; vidal-aroca, f. ; bertoni, g. . biomolecular sciences and biotechnology, university of milan, milano, italy; . basilea pharmaceutica ltd., basel, switzerland with ever increasing frequency, we now observe several examples of bacteria being resistant to every clinically available drug. therefore this urgently calls for the development of novel and improved antibiotics that may escape the extant mechanisms of bacterial resistance. one recent and promising development of the genome-wide search for target functions for antibiotics led to the identification of essential genes of pathogens by interfering antisense rnas. the first step is the construction of shotgun antisense libraries (sals) as follows. genomic dna is extracted from the bacterial strain of interest, fragmented by shearing into short pieces of dna, blunt-ended and cloned in an expression vector under the control of a regulatable promoter. the library is then reintroduced into the cognate bacterial strain and screened by replica plating colonies both in the presence and the absence of an inducer of the vector promoter. by this method, insert sequencing of clones showing conditional growth phenotypes is expected to lead to the identification of essential genes that can be silenced via antisense rna activity. we adopted this technology in order to generate a panel of essential functions of the cystic fibrosis-related opportunistic pathogen pseudomonas aeruginosa. so far, we tuned the protocol for sal generation in p. aeruginosa and identified a number of sequences conferring different levels of growth inhibition we are now characterizing these putative antisense rnas in order to define the minimal sequence able to cause the toxic effect and, on the other hand, to understand the cellular role of their targets. pseudomonas aeruginosa (pa) is an extremely versatile microbe with a vast array of pathogenic and metabolic mechanisms that allow it to form persistent infections in select patient populations, especially patients with cystic fibrosis. an ineffective immune response is considered partially to blame for failure of pa eradication. we have discovered that some strains of pa express peptidylarginine deiminase (padi) activity. padi is an enzyme that post-translationally modifies peptidylarginine to peptidylcitrulline with ammonia as a byproduct. our lab has shown that human padi can modulate the immune system through downregulation of tlr and ikk-gamma signaling. characterization of the pa padi will offer insights into a completely novel method of immune modulation by pseudomonas aeruginosa. using a widely published colorimetric assay for padi activity we have found the specific activity of crude pa cell lysates is very low. however this is similar to the only other known prokaryotic padi described in porphyromonas gingivalis. a protein homology search with the porphyromonas padi has revealed the likely genetic locus of pseudomonas padi in a . kb operon that appears in the genome of the pathogenic pseudomonas isolate pa . this operon appears to contain two candidate genes for padi activity based on conserved motif searches (padi and padi ). both have been cloned into expression vectors, partially purified by affinity tag technology and tested in the colorimetric padi assay. curiously padi autocitrullinates itself while padi has not shown activity. reaction conditions and substrate specificities for pa-padi are not like either the human or porphyromonas padi. furthermore analysis of up to clinically diverse strains has demonstrated % carry the gene for padi . this work is only the second description of padi activity in any prokaryote. pa padi could clearly have a dramatic impact on the local inflammatory milieu if it can access the same targets as human padi. the description of this activity in pseudomonas will advance our knowledge of the human-pathogen relationship and give insight into new therapies. which functions by translocating toxins into the cytoplasm of host cells. these toxins cause disease by damaging the surrounding host tissue, promoting dissemination of the organism and paralyzing the phagocytic mechanism of macrophages. pcrv is a factor required for the translocation of the toxins. the bases of these studies were to evaluate pcrv as a protective antigen in "p. aeruginosa" pneumonia and cldc as a vaccine adjuvant. methods: mice were vaccinated , , or times with the pcrv antigen combined with either aluminum hydroxide (alum) or cldc by various routes of administration. efficacy of vaccination was evaluated by challenge with "p. aeruginosa" and evaluation of survival and/or measurement of various parameters associated with lung injury. results: increase in median survival time was highly significant when cldc/pcrv was compared with cldc or pcrv alone. following subcutaneous administrations cldc/pcrv showed an increase in median survival time ( hours versus hours) and overall survival benefit following intraperitoneal administrations ( % versus %). mice with anti-pcrv antibody levels above µg/ml were significantly protected. conclusion: the investigators establish the efficacy of cldc/pcrv vaccines via several parenteral routes of administration compared to no treatment as well as cldc and pcrv-only controls. differences were demonstrated between performance of cldc/pcrv and alum/pcrv in measures of lung injury, median survival, and overall survival. the results correlated with antibody levels and histological examination of the lung tissues. importantly, these studies indicate that protection can be achieved against "p. aeruginosa" infection by targeting an antigen associated with the type iii secretion system. background: there has been a recent increase in the number of reported cases of acute renal failure (arf) in cystic fibrosis (cf). our group have undertaken a national survey, which measured the incidence risk of arf in cf patients at between . and . cases / , cf patients / year. we have now conducted a case control study to determine which factors which are associated with an increased risk of arf. methods: in our initial survey we confirmed cases of arf, in cf patients from uk cf centres, presenting between & . using the uk cf database, we identified sex and age (within months) matched controls. informed consent was sought from the control patients, or their parents, for access to the case notes and clinical data were extracted. analysis of risk factors was by conditional logistic regression, using stata (version ) and by fisher's exact test. results: there were cases of arf ( male, median age y, range m- y) and controls ( male, y, m- y). in the group of patients with arf, / had received an aminoglycoside at the time of their episode of arf or in the preceding week, compared with only of the controls for the same time period (p< . ). the median number of days of aminoglycoside in the year prior to the index case developing acute renal failure was ( - ) for cases and ( - ) for controls. conditional logistic regression showed that the odds ratio for arf per each day of aminoglycoside was . ( % ci . to . , p= . it is well-known that pa senses the environment and changes its phenotype. for instance, it produces greater amounts of the extracellular polysaccharide alginate in the cf lung, characterized by a microaerobic environment. little is known about the changes in protein secretion induced by oxygen limitation in pao , the proto-typical pa laboratory strain. no data are available on this regard about pa clinical strains. our work was aimed to study the differential regulation of proteins secreted by pa strains grown in microaerobic or aerobic conditions. a pa clinical isolate and pao were grown overnight in aerobiosis and in microaerophilic conditions. the supernatants were collected and proteomic analysis was carried over by two-dimensional capillary chromatography -tandem mass spectrometry (mauri et al., faseb j ) to evaluate the differential protein expression. in the pa clinical isolate, we identified proteins down-regulated and proteins upregulated in aerobic conditions in comparison with microaerophilic culture while in pao proteins were down-regulated and were up-regulated. proteins were down-regulated and up-regulated both in the clinical and in the laboratory strain. these proteins can mediate different biological functions since they are enzymes, heat shock proteins, chaperones, proteins involved in adaptation, motility and in the transport of small molecules. among all these proteins, as the alkaline metalloproteinase is associated with tissue invasion not only by causing rupture of epithelial tight-junctions but also by degrading several chemokines, we decided to validate its up-regulation observed in aerobic conditions by zymography. we found that the proteolytic activity of the supernatants of pa grown in aerobic conditions was higher than in microaerophilic culture both for the laboratory and clinical strain, indicating the functional relevance of data obtained by proteomic analysis. the identification of proteins differentially regulated in aerobiosis and oxygen limitated conditions in pa laboratory and clinical strains might be helpful for the knowledge of the mechanisms of colonization and lung damage due to pa in cf patients. for instance, the validation of the upregulation of the alkaline metalloproteinase in aerobic condition may shed light on these mechanisms of cf lung disease. further studies are in progress to evaluate the function of other bacterial exoproducts regulated in this model and to extend the analysis to other clinical strains. supported by the italian cystic fibrosis research foundation (ffc-grant# / ), comitato di vicenza dell'associazione veneta per la lotta contro la fibrosi cistica and azienda ospedaliera di verona, italy. cystic fibrosis (cf) sufferers are subject to repeated lung infections most commonly with the bacterium pseudomonas aeruginosa. in spite of antibiotic treatment p. aeruginosa tends to become established giving rise to persistent chronic infection. in the lung environment, the bacterium grows as a highly structured biofilm consisting of a complex community of cells embedded within a self-secreted polysaccharide matrix. investigations of p. aeruginosa biofilm growth using model strains have elucidated mechanisms which appear to govern the biofilm life-cycle. our research aims to test whether observation of these mechanisms in planktonic culture can be related to the efficiency of biofilm formation. biofilm initiation has been linked to cell motility and in particular to the presence of flagella and pili, which are thought to be important for cell attachment and the formation of microcolonies. on testing a large, genetically diverse group of clinical p. aeruginosa isolates retrieved from the lungs of cf patients we found no definitive correlation between the degree of motility of an isolate in planktonic culture and its ability to form a biofilm in vitro. the development of biofilm architecture has been demonstrated in model systems to be coordinated by the production and secretion of n-acylhomoserine lactone (ahl) quorum sensing molecules. however among the clinical isolates tested we observed no obvious correlation between the amount of ahls produced in planktonic culture and the extent of biofilm formation. overall we have found observation of phenotypic characteristics in planktonic culture to be poor predictors of efficient biofilm formation. a proteomics approach was adopted to provide further insight into the physiology of biofilm growth of p. aeruginosa isolates by comparison with planktonic growth of the same isolates. two genetically unrelated clinical isolates, demonstrated as being capable of efficient in vitro biofilm formation, were selected from our culture collection on the basis of their diverse phenotypic characteristics when cultured planktonically. one displays both twitching and swimming motilities, is mucoid and expresses ahls, while the other is non-motile, non-mucoid and no ahls have been detected in planktonic culture. we have developed a simple flow-through bioreactor to provide sufficient quantities of biofilm for proteomic analysis. gel-based and gel-free techniques were employed to study protein expression patterns for both biofilm and planktonic cultures of each isolate by mass spectrometry. proteins specific for each growth phase could be detected and may prove suitable biomarkers for monitoring the physiological status of biofilm forming strains when a larger bank of clinical isolates are examined. liposomal amikacin (arikace tm ) is a liposome-encapsulated form of amikacin that is formulated to treat chronic p. aeruginosa infections in cystic fibrosis patients. these liposomes carry a zwitterionic surface charge and are composed of lipids found naturally within the lung. a key aspect of the activity of the formulation is the ability to penetrate to the sites of pseudomonas biofilm-like growth in the lung. experiments were designed to investigate the penetration of liposomes into p. aeruginosa biofilms and in vitro activity. methods and results: model liposomes of the same size and lipid composition as liposomal amikacin (arikace tm ) were prepared with membrane-associated or encapsulated fluorescent labels, a hydrophobic carbocyanine dye and calcein, respectively. a mucoid strain of pseudomonas aeruginosa (pa ) was used to establish biofilms in rectangular optical grade glass flow cells. biofilms were observed after four days of growth by confocal laser scanning microscopy using a focal plane set to view within the biofilm cluster or outside as a control. time dependent accumulation of fluorescent liposomes within the biofilms was measured by the spatial distribution of fluorescence intensity in regions within or outside of the biofilm. images indicated significant penetration of liposomes into the interior of biofilms under these conditions. the rate of penetration was considerably slower than typical rates for small molecules, consistent with the size of the liposomes. liposome concentrations were higher near the periphery than the interior. however, even the interior concentration was at least as high as the concentration of liposomes in the fluid outside of the biofilm, suggesting some binding or trapping of the liposomes within the biofilm. penetration of liposomes was observed under flow or static conditions. in a "washout" experiment, where medium is passed through the biofilms previously treated with liposomes, a significant portion of the liposomes remained associated with the biofilms for an extended period of time. the penetration of liposomes was reflected in the observation of killing of bacteria in colonies in the interior of agar beads. exposure of these cultures to liposomal amikacin resulted in a large reduction of viable bacteria throughout the beads as monitored by a fluorescent dna content assay. similar colony forming unit reductions in animal models (to be shown in other poster presentations) suggest that these principles also operate in vivo. liposomes similar to liposomal amikacin (arikace tm ) readily penetrate into biofilms of pseudomonas aeruginosa and may even have enhanced binding to biofilms. this binding along with localized release can explain the substantial efficacy observed in animal models. coates, a.l. adherence to recommended therapy in cf has always been a challenge, in part, due to the time demands of the daily therapy. while twice daily inhaled tobramycin for those infected with pseudomonas aeruginosa (pa) has become an accepted standard of care, as much as minutes a day may be consumed inhaling mg in ml of tobramycin (tobi ® ) from the pari lc plus ® breath enhanced jet nebulizer. the purpose of this study was to determine if equivalent levels of pulmonary deposition could be achieved in a much shorter time period using . ml of a more concentrated ( mg/ml) tobramycin solution delivered by a perforated vibrating membrane nebulizer (eflow ® membrane configuration l) both, developed by pari pharma; germany. methods with a goal to study children and adults, to date, the subjects are children years and older and adult males, all with an fev > % predicted, with stable cf. all were receiving inhaled tobramycin for positive sputum cultures of pa. following pretreatment with albuterol, they inhaled both preparations on two occasions with m tc-dtpa added to the tobramycin in the nuclear medicine facility. in vitro preliminary work demonstrated that the radiolabel tracked with both formulations of tobramycin. deposition was measured by a gamma camera taking both tissue attenuation and mucociliary clearance during nebulization into account (pediatric pulmonol suppl : a ; ) . in order to have a continuous rate of deposition, the pari lc plus ® was run for a timed minutes and then both the total deposition and time of nebulization "scaled up" from in vitro testing when the nebulizer was run to dryness. this was done by multiplying the deposition by the total output when run to dryness divided by the total output in minutes. (blood samples were taken for quantification of tobramycin in the serum but not yet analysed). the rate of output per minute was calculated from the minute run and the total time was total output from in vitro testing divided by the rate of output. the eflow ® pro-vides a continuous output and stops automatically at dryness. quality assurance was the agreement between total radioactivity pre nebulization (in the nebulizer) and post which included the subject, the nebulizer, the connectors and the expiratory filter. the pari lc plus ® delivered . ± . mg in . ± . minutes compared to . ± . mg in . ± . minutes for the eflow ® . only the time of delivery was significantly different with p< . (paired t-test). tolerability of the treatment was comparable for both inhalation regimes, but the shorter treatment was preferred by all patients. these results demonstrate the possibility of delivering equivalent levels of tobramycin in much shorter periods of time into the lungs of cf patients when using eflow ® , a very efficient electronic nebulizer. this time saving may improve adherence to recommended therapy. (pediatrics ; : ) . in order to properly interpret op cultures from nbs infants, especially those with non-classic cf mutations, we need to know the op flora of non-cf infants. we obtained op cultures from healthy infants under yr of age. op specimens were plated on standard cf culture media. exclusion criteria included a first degree relative with cf, respiratory illness at the time of culture, or positive newborn screen for cf. data on cigarette smoke exposure, animal exposure, and exposure to hot tubs/swimming pools was collected. samples have been collected to date. in healthy, non-cf infants, the most common finding is non-specific mixed gram negative and positive growth. however, infants have grown pa (ages months, months, and months). many infants have grown multiple organisms. the following bacteria have been found in (n) number of infants: s. aureus ( ), e. coli ( ), e. cloacae ( ) , h. flu ( ), klebsiella ( ), pseudomonas aeruginosa ( ), h. parainflu ( ), unidentified non-lactose fermenting ( ), other ( ). data on infants including correlation to environmental factors will be presented. conclusion: non-cf infants commonly have s. aureus and many gram negative organisms including pa in their oropharynx. these results may have some bearing on interpetting colonization and clearance of pa in infants identified through nbs and in epic study participants. # ) ). identification of smg by sputum cultivation represents a significant challenge because the organisms are phenotypically diverse, grow poorly on routine culture media, and are very difficult to discriminate from other members of the oropharyngeal flora. we have developed a solid media for the selective isolation of smg from sputum. the value of the media is highlighted by the identification of smg as the quantitatively dominant organism in sputum samples of three cf patients admitted to hospital for an acute pulmonary exacerbation. in all three, the smg species failed to be identified on routine or selective media currently described for the culture of cf-specific sputum pathogens. antibiotic treatment directed against the smg correlated with clinical resolution of acute symptoms as well the reduction of smg on daily serial sputum cultures during hospitalization. this novel selective media makes use of antibiotics (colistin, sulfadiazine and oxolinic acid) inhibitory to the growth of principal cf pathogens and much of the usual oropharyngeal flora. smg agar utilizes a colorimetric indicator to uniquely identify smg colonies. the sensitivity and specificity of the selective media has been evaluated by molecular methods using terminal restriction fragment length polymorphism analysis. smg organisms do not respond well to anti-pseudomonal therapy, therefore proper detection and culture-directed antibiotic therapy is paramount. we believe that smg represent significant respiratory pathogens in cf, and because of the inability to effectively culture and identify smg they have largely gone unrecognized. pseudomonas aeruginosa releases substantial amounts of the blue antibiotic pigment pyocyanin. in presence of a reductant (such as nadph), pyocyanin redox-cycles and generates superoxide and h o . in infected cf airways, pyocyanin concentrations can reach high micromolar levels and contribute to oxidative stress of the airways. the structural basis of the pyocyanin molecule that underlies the redox-cycling with reductants of the airways is not clear. we therefore investigated i) the ability of physiologically or pharmacologically relevant reductants of the airways to support the redox-cycle activity of pyocyanin, and ii) the molecular features of pyocyanin that support redox cycling. dose-and time-dependent h o production by pyocyanin was measured by amplex red oxidation in presence of horseradish peroxidase. rates of h o production by µm pyocyanin in presence of µm reductant were: nadph ( pmole/min) > l-ascorbate ( pmole/min) > reduced glutathione ( pmole/min) > α-tocopherol ( pmole/min). in contrast, lipoic acid, genistein, or resveratrol did not significantly support pyocyanin-mediated h o production. in absence of a reductant, pyocyanin showed no measurable formation of h o . to identify the structural characteristics of the pyocyanin molecule that allow for its redox-cycling activity we synthesized a number of new pyocyanins containing electron-donating or electron-withdrawing substituents. functional assays were performed in presence of l-ascorbate as reductant. molecular substituents that donated electrons to the positively charged core of pyocyanin, either by hyperconjugative or resonance effects, reduced the h o output of the corresponding pyocyanin. in contrast, a closely related analog ( -hydroxyphenazine- , dioxide) showed significantly increased activity ( . x compared to pyocyanin) suggesting that the electron-withdrawing effect of the n-oxide functionality led to an increase in the redox-cycle activity. these data indicate that the functional characteristics of pyocyanin as a redox-cycling compound are governed by its positively charged core. in the airways, pyocyanin is predicted to utilize several reductants that are present in the airway surface liquid or intracellularly, thus contributing to cf airway disease. because pyocyanin utilizes a variety of reductants, it appears prudent to test whether inhaled small-molecular cf therapeutics support pyocyanin function. supported by nih (hl- , p at ), cfri, philip morris usa inc and philip morris international, and cff (fischer g ). taccetti, g.; braggion, c.; ravenni, n.; zavataro, l.; neri, a.; festini, f.; campana, s. meyer hospital, university of florence, cf center of tuscany, florence, italy for practical purposes, after early eradication treatment, at least three consecutive negative respiratory cultures over a -month period would indicate that the organism has been eradicated (cf trust guidelines). this recommendation is based on opinion/clinical experience of respected authorities in the absence of directly applicable studies of good quality. aims: using molecular biological techniques, we evaluated whether this -month interval is really trustworthy for distinguishing between regrowth of the same strain, suppressed but not eradicated by treatment, and new pseudomonas aeruginosa (pa) colonization. patients and methods: cystic fibrosis (cf) patients were treated with oral ciprofloxacin and nebulized colistin at detection of pa. all pa colonization episodes were recorded in an appropriate database. molecular study of each bacterial isolate from each colonization episode was performed with the rapd-pcr. results: between between - of patients in follow-up in our center had repeated pa colonization. the patients' mean age at first pa colonization was ± . months. a total of episodes was observed (mean of . episodes per patient, median of , range - ). molecular typing on strains indicated that ( . %) were a different genotype from later colonization episodes while ( . %) isolates had the same genotype as those of the preceding episode. the same genotype as preceding colonization was observed in ( . %) of isolates from patients in which a successive colonization was verified in less than months, and was verified in ( . %) of strains in patients having a successive colonization in over months (or= . ). colonization was due to a genotypically diverse strain in % of cases where colonization occurred within months of eradication. during the observation period ( %) of patients acquired chronic pa infection. conclusion: re-colonization by pa following eradication therapy is mostly ( %) caused by strains with a different genotype, suggesting acquisition from an external source. a short pa-free period is mainly due to transient suppression of pa growth, and true eradication followed by acquisition of a new pa genotype occurred in most cases only after a pa-free period longer than months. those patients with a pa-free interval of less than months had a six-times higher chance that the pa was not eradicated compared to those with a germ-free interval of over months. this evidence demonstrates that the definition of successful eradication should be reconsidered, taking into account additional parameters such as molecular analysis. cystic fibrosis (cf) patients appear to have an increased risk of urolithiasis. while a number of possible explanations for this have been proposed and investigated, no definitive mechanism has yet been demonstrated. as cf patients frequently get respiratory tract infections they regularly receive ciprofloxacin treatment, often given in doses well in excess of conventional prescription regimes. there are occasional reports of ciprofloxacin crystals urine and stones in the urinary tract. here we investigate the hypothesis that ciprofloxacin excreted in urine might act as a promoter of crystallisation of calcium or magnesium salts and thereby increase the risk of kidney stone disease. the effect of ciprofloxacin was tested in artificial urine (au). in vitro crystallisation was tested using a well plate turbidity method, to identify a metastable limit of oxalate concentration (ml) and a growth and nucleation parameter, the turbidity rate index (tri). the nucleation ph of urine was examined by tritrating oxalate free au through a ph range of . to . and monitoring the solution/suspension turbidity. in au at ph . , ciprofloxacin, at , , or mg/l, had no detectable effect on initiation of calcium oxalate crystallisation (ml) or its progress (tri) (n= for each concentration). when au with mm ca, mm mg and mm po was titrated there were two distinct nucleation points; a slow event starting at about ph . and a much faster event at about ph . . omitting ca or mg confirmed that the first event was due to calcium phosphate precipitation and the second to struvite. including ciprofloxacin at , or mg/l did not alter these nucleation ph values, but the magnitude of the turbidity rise showed that the ciprofloxacin co-precipitated with the struvite. ciprofloxacin at mg/l and without ca or mg began to precipitate at ph . and could be held in solution until ph . when ca and mg were included. even at high concentrations, ciprofloxacin does not influence calcium oxalate crystallisation. nor does it promote calcium phosphate or struvite precipitation; on the contrary, while the calcium and magnesium remain in solution, they help to prevent precipitation of the ciprofloxacin itself. urinary ciprofloxacin does not appear to act as a stone or crystal promoter. pseudomonas aeruginosa is a significant cause of mortality in cystic fibrosis (cf) sufferers. cf patients were thought to acquire p. aeruginosa from the environment; however genotyping over recent years has revealed clonal strains in sputa from cf patients in the uk, australia, and canada that are transmitted person to person or from a common source. one clonal strain, australian epidemic strain- (aes- ), (formerly melbourne epidemic strain, m or pi) currently infects up to % of patients in five cf clinics on the eastern seaboard of australia. most cf clonal strains have been associated with increased virulence not fully explained by greater antibiotic resistance. both genotypic and phenotypic differences have been postulated as important in enabling transmission of clonal strains. in order to compare the expression profile of aes- to the type strain p. aeruginosa pao , the cf research group at the university of sydney compared the genome expression data of four clonal aes- isolates and pao , when grown as planktonic and as -hr biofilm cultures. in aes- , a set of significantly differentially expressed genes (all downregulated) were identified, including the quorum sensing genes lasa, lasb and rhll. in contrast, both upregulated and downregulated genes were differentially expressed in pao biofilm compared to pao planktonic culture. expression data was validated using quantitative real-time pcr. to compare biofilm growth at the phenotypic level, the four clonal strains and pao were grown as -hr biofilms in a double-blind study, and the size of ten randomly selected biofilms per isolate, stained with syto® green fluorescent stain was measured. at hr, the biofilms formed by aes- isolates were significantly larger (ca. -fold) than pao (average size: ± µm vs ± µm )(p< . ). the average thickness of three biofilms per isolate, measured by confocal microscopy, showed aes- biofilms to be approximately . -fold thicker than those formed by pao ( . ± . µm vs . ± . µm). the general gene downregulation observed in aes- biofilms suggests an adaptation to the cf host, while a larger biofilm would provide for more effective bacterial dispersal. thus the transmissibility of aes- may be linked to enhanced biofilm formation upon colonisation. background -objective: whilst influence induced by bacterial colonization in cystic fibrosis (cf) is established, risk induced by fungal colonization is less defined. prevalence of other species than aspergillus sp. or candida sp., and factors associated with fungal presence are also poorly documented. our preliminary study aimed to determine which fungal species were present in sputum collected from adult cf patients, and which factors were associated with fungal presence. methods: in a monocenter, transversal prospective study, cf adult patients were included to determine fungal presence in sputum using semi selective growing media. clinical parameters (shwachman score, respiratory function, nutritional status, gastro-oesophageal reflux, pancreatic insufficiency and diabetes); therapeutics used (including oral or intravenous antibiotics, systemic or inhaled corticosteroids or bronchodilatators, antifungal treatments); microbiological data of bacterial colonization and environmental parameters (potted plants or domestic animals presence) were determined for each patient. correlation between fungal, clinical, environmental, therapeutic or microbiological data was evaluated by mann-whitney non parametric u test. results: patients ( %) presented with fungal presence in sputum. % presented with yeasts species, % with moulds. aspergillus fumigatus and candida albicans were the predominant species in moulds and yeasts respectively, but less common mould species such as exophiala dermatidis or paecilomyces variotii were also recovered. factors associated with fungal presence were pancreatic insufficiency (p= , ); malnutrition (p= , ), bacterial colonization and inhaled corticosteroids. candida albicans was correlated with more severe shwachman score (p= , ), bacterial colonization (p= , ), notably with pseudomonas aeruginosa (p= , ) , and intravenous antibiotics use (p= , ). moulds species were significantly associated with inhaled corticosteroids (p= , ). antifungal use was associated with frequent resistance to azoles treatments ( resistant isolates out of patients treated). conclusion: fungal presence in cf appears frequent. some species could have been previously overlooked due to diagnosis difficulties. the effect of corticosteroids on moulds species, already found in other pathologies, appears important in cf. influence of fungal presence on cf course needs prospective studies, in order to establish if patients could benefit from antifungal treatments or preventive measures. dren on a ventilator without a previous diagnosis of cystic fibrosis is unknown. the aim of our study was to investigate the prevalence of these microorganisms in routine sputum cultures in young children on a ventilator in a pediatric intensive care unit (picu). methods: from all ventilated children aged - years admitted from - , sputum culture results obtained from tracheal aspirates within the first week of admission were retrospectively analysed. three patient subgroups were identified: respiratory failure due to pulmonary disease (group ), ventilation after elective surgical procedures (group ) and other ventilated children (group ). children with a previous intensive care admission or cf were excluded. the cf database was checked ( ) to identify any children with a new diagnosis of cf that were included in the study. results: . % of all ventilated children had a positive sputum culture with one of the "cf-bacteria" s. aureus, h. influenzae and p. aeruginosa these were found mainly in group (see table) . / of these children were admitted for treatment of respiratory syncytial virus (rsv) bronchiolitis. a sweat test was performed in children, all admitted with pulmonary disease and because of co-existing clinical signs or symptoms. one sweat test was positive and subsequently cf was diagnosed in this child. no other children out of the study group have since then been diagnosed with cf. as the population in the northern part of the netherlands is very stable, it is unlikely a diagnosis of cf has been missed. conclusion: s. aureus and h. influenzae are cultured frequently in ventilated children without cf, especially when ventilated because of pulmonary disease. the opportunistic pathogen p. aeruginosa causes both acute and chronic human infections. the balance between systemic infection and mortality or chronic persistence and morbidity depends on complex relationships in which the immunological status and genetic potential of the host but also the bacterial biodiversity are determinant factors. p. aeruginosa extensive genetic adaptation and microevolution have been repeatedly observed in chronic infections of cf patients in contrast to what it is documented in acute infections. whether these p. aeruginosa clonal variants differ in their pathogenic potential is not yet known. a total of clinical p. aeruginosa isolates from six cf patients which carried unique clonal lineage from the onset of colonization over - years were selected (bragonzi et al, ; montanari et al, ) . five p. aeruginosa environmental strains, which represent the source of acquisition for cf patients, and two laboratory strains pao and pa were also used as references. multiple genotypic analysis of sequential p. aeruginosa isolates which included pfge, atchip and multilocus snps showed intraclonal diversity with genome rearrangements, variations in pathogenic islands and acquisition of prothoadaptive mutations in the muc genes. the largest divergence was observed between the completely sequenced reference strains pao and pa , the latter represented in our panel by three cf isolates. p. aeruginosa virulence has been assessed by monitoring its capacity to induce bacteremia and to establish chronic infection in a murine model (bragonzi et al, ) . overall, p. aeruginosa environmental strains increased five times the risk of bacteremia when compared to clinical strains (test of proportion: p< . ). the high risk of mortality was also evidenced for p. aeruginosa cf strains isolated at the onset of the infection when compared with those isolated after years of colonization (p= . ) indicating that environmental strains or newly acquired strains were similar in their virulence. p. aeruginosa clinical strains isolated after years of colonization increased chronic persistence and reduced the risk of bacteremia (p= . ) when compared to strains isolated at the onset of the infection. the strain pa was found to be lethal in contrast to p. aeruginosa clonal strains of clinical origin that established chronic persistent infection in the murine lung. furthermore, our data showed that adaptive traits commonly associated with the chronic p. aeruginosa infections in cf patients, such as transition to the mucoid phenotype, did not confer a selective advantage to bacterial cells in colonizing the murine lung (p= . ). these results suggest that p. aeruginosa pathogenicity is independent of the strain's genotype but rather genetic adaptation to the cf airways plays an important role in the development of persistence and in the resistance to host defences. supported by telethon and italian cf research foundation. beringer, p. the combination of antipseudomonal beta-lactam and aminoglycoside antibiotics is frequently prescribed during acute pulmonary exacerbations. since the introduction of the beta-lactam compounds there has been numerous reports citing altered pharmacokinetics of several compounds (but not all) within the beta-lactam class. recently it has been suggested that substrate specificity for the renal transporter pgp may explain the variability in renal drug clearance observed in patients with cf. pgp is structurally related to cftr and evidence suggests it serves a complementary role in modulating alternative chloride channel function. in a cftr knockout model mdr expression was reported to be increased nearly four-fold. in an effort to elucidate the potential contribution of pgp to the renal clearance of drugs in patients with cf, we conducted a controlled clinical trial comparing the pharmacokinetics of fexofenadine (fx) in patients with cf and age matched healthy volunteers (hv). fexofenadine is not significantly metabolized and is a relatively specific substrate for the membrane transporters pgp and oatp. probenecid (pb) is a selective inhibitor of oatp and was used pharmacologically to block the activity of this transporter in-vivo. our hypothesis is that if pgp is upregulated within the renal tubules of patients with cf we would expect to see an enhanced renal clearance of fexofenadine in these patients when compared with control subjects. methods: (n= cf, hv) subjects underwent this prospective controlled study which fx was received alone or in combination with probenecid (pb). iothalamate was given each day to measure glomerular filtration rate (gfr). blood and urine samples were obtained at specified times over hours each study day for determination of fexofenadine and iothalamate concentrations. fexofenadine and iothalamate concentrations were assayed using liquid chromatography-tandem mass spectrometry and hplc-uv respectively. pharmacokinetic analysis was performed using a -compartment cumulative urinary excretion model with the adapt ii software. differences between groups were determined using a paired t-test or mann whitney-u. results: patients were included, cf patients were slightly older and had lower body mass index when compared to hv, (mean±sd, ± . vs. . ± years p= . , . ± . vs. . ± . p= . respectively) but did not differ in gfr ( ± . vs. ± ml/min- . m p= . respectively). no significant differences were found between cf and hv in clr of fx alone and fx in combination with pb clr ( . ± vs. . ± . ml/min- . m p= . and . ± vs . ± . ml/min- . m p= . respectively). conclusion: the results of this study indicate the enhanced clearance of certain antibiotics previously reported in patients with cf do not appear to be due to upregulation of renal tubular pgp or increased gfr. maceachran, d.p. department of microbiology and immunology, dartmouth medical school, hanover, nh, usa p. aeruginosa is the leading cause of morbidity and mortality in cystic fibrosis (cf) patients. shortly after birth cf patients are colonized by p. aeruginosa, which leads to a lifelong chronic infection. the mechanisms behind p. aeruginosa colonization of the cf lung are still poorly understood, however several secreted toxins have been associated with this phenomenon. cf is characterized by a loss of mucocilliary clearance, a key component of innate immunity in the lung, and an increase in sputum viscosity as a result of mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (cftr). we have previously shown that the novel p. aeruginosa secreted protein cif is capable of reducing apical membrane expression of cftr. we have begun to characterize cif expression and have identified a key regulator of cif transcription. mutations affecting the divergently transcribed putative tetr family repressor encoded by the pa gene result in a significant increase in cif expression. furthermore, we have demonstrated that the pa gene product is capable of binding the promoter region located immediately upstream of the cif-containing operon. this work has been supported by the nih t -dk - . ryall, b. ; davies, j.c. , ; wilson, r. ; shoemark, a. ; williams, h.d. . division of biology, imperial college london, london, united kingdom; . department of gene therapy, imperial college london, london, united kingdom; . host defence unit, royal brompton hospital, london, united kingdom; . paediatric respiratory medicine, royal brompton hospital, london, united kingdom pseudomonas aeruginosa is the most important respiratory pathogen in patients with cystic fibrosis (cf) and non-cf bronchiectasis. the factors which enable p. aeruginosa to establish chronic, debilitating respiratory infections are unclear. however p. aeruginosa is one of a limited number of bacteria that is able to synthesise hydrogen cyanide, a potent inhibitor of cellular respiration. this study examined whether hydrogen cyanide is produced by p. aeruginosa in cf and non-cf bronchiectasis airway infection. cyanide concentration was measured in fresh sputum from cf and non-cf bronchiectasis patients with and without p. aeruginosa lung infection using a cyanide ion sensing electrode. cyanide was detected in sputum from / cf and non-cf bronchiectasis patients with current p. aeruginosa infection, whereas it was not detected in any of the patients without this organism (p< . ). maximum lev-els were µm (mean ±se: ± . µm), which compares with levels of above µm which would be considered toxic in blood. concurrent lung function data were available on all p. aeruginosainfected cf patients; the group with measurable sputum cyanide (n= ) was not different from those without (n= ) on the basis of age, gender or p. aeruginosa phenotype (mucoid/ non-mucoid). however, those with detectable cyanide had significantly poorer lung function than those without (fev % predicted: . ± . % versus . ± . %, p< . ; fvc% predicted: . ± . % versus . ± . , p< . ). we have shown that cyanide is detectable at clinically significant levels in sputum from cf and non-cf bronchiectasis patients infected with p. aeruginosa and is associated with significantly impaired lung function. we have recently reported a dose regimen for once-daily tobramycin dosing in patients with cystic fibrosis which was based on a retrospective study (lam w et al., j antimicrob chemother ) . we present here an interim analysis of an ongoing prospective study to evaluate the proposed dosing in paediatric patients with cystic fibrosis. as part of this protocol pharmacokinetic parameters were determined in cystic fibrosis (cf) patients receiving intravenous antibiotic therapy for a pulmonary exacerbation. serum creatinine, audiometric testing, respiratory cultures and fev were assessed at baseline and after a week treatment period. serum tobramycin concentrations were interpreted by clinical pharmacists performing therapeutic drug monitoring (tdm) to optimize dosing. serum tobramycin levels were drawn after the first dose, repeated with each dose adjustment and weekly thereafter. the proposed sample size of this study is patients; an interim analysis was performed to identify trends for pharmacokinetic and safety outcomes from patients admitted from november through april . seven ( . %) patients achieved the target c max of - mg/l, ( . %) patients remained under the target range and patient was above the target range with the st dose. with nd, rd, and th tdm performed for either routine weekly levels or dose adjustment levels, , , and patients met the target c max respectively. the percentage of patients who achieved target c max increased to . % with the rd set of tdm levels and . % with the th set of tdm levels (p-value< . ). there were no significant changes in k e , t / and v d /kg for each patient from st to nd set of tdm levels over time. compared to the previous retrospective study the mean v d /kg was significantly higher ( . l/kg versus . l/kg, p= . ) and the mean t / was significantly shorter ( . hrs versus . hrs; p< . ). none of the patients' serum creatinine increased ≥ % from baseline. baseline audiometric tests were within normal limits in ( . %) patients. p. aeruginosa was isolated from respiratory specimens of patients on initial culture. of the patients with follow-up cultures, patients grew p. aeruginosa sensitive to tobramycin, demonstrating no decrease in sensitivity. our preliminary results suggest the pharmacokinetic data differ from the expected profile suggested by the retrospective study. all study patients treated according to the guidelines were clinically well after their course of therapy and did not experience clinically significant toxicity from once-daily tobramycin dosing. the once-daily tobramycin dosing will continue to be evaluated until the proposed sample size is achieved for final analysis. anderson, g.g.; o'toole, g.a. dartmouth medical school, hanover, nh, usa pseudomonas aeruginosa chronically colonizes the lungs of individuals with cystic fibrosis (cf). evidence suggests that this infection progresses through distinct phases, wherein initial colonizing strains undergo a switch to mucoidy that corresponds to formation of bacterial microcolonies in the airways and life-long persistence. currently, the environmental signals and mechanisms regulating conversion from the acute, highly virulent form to a less virulent biofilm state remain obscure. to investigate these issues, we developed a tissue culture model system for growth of p. aeruginosa biofilms on cf-derived human airway epithelial cells in vitro. biofilms grown on these cells appear similar to those found in cf airways as well as to abiotic biofilms. eventually, epithelial cells were killed by the p. aeruginosa biofilms. however, we discovered that addition of the antibiotic tobramycin preserved the monolayer integrity for at least hours. bacteria were not completely eliminated by this treatment, suggesting that tobramycin influenced the virulence of the biofilm bacteria. microarray analysis of cf cell grown biofilms treated with tobramycin revealed marked alterations in transcriptional profiles compared to untreated controls. two tobramycin-induced genes were identified from this screen which, when deleted, exerted altered virulence phenotypes on airway epithelial cells in culture. quantification of cytotoxicity of these mutants revealed either increased or decreased ability to kill the epithelial cells. complementation with full-length copies of these genes restored wild-type function. these studies suggest a complex relationship between acute and chronic forms of p. aeruginosa, and that antibiotic treatment may influence transitions between these modes. it is this interplay that may determine the virulence status of the microorganism upon initial infection as well as during cf exacerbations. neville, m.; sardaryan, g.; ejaz, s.; scotto, a.w.; gupta, r. transave, inc., monmouth junction, nj, usa purpose: during the course of treatment, patients with cystic fibrosis and chronic pseudomonas aeruginosa infections in their lungs may be treated with nebulized tobramycin (tobi®). reduction in treatment times or dosing frequency may improve compliance and outcome. nebulized arikace™ may offer advantages to tobramycin by producing sustained lung levels of drug, reducing dosing frequency and increasing antimicrobial efficacy. herein are presented biodistribution data and efficacy data in a chronic rat model of p aeruginosa comparing arikace™, soluble amikacin and tobramycin. method: female rats (charles river) received (~ mg / kg) of arikace™, soluble amikacin and tobramycin by inhalation. test articles were aerosolized in a pari lc star nebulizer at psi using devilbiss compressors (sunrise medical) for min. - rats were euthanized at time and hr post inhalation by co asphyxiation. blood was collected by a cardiac puncture. serum was collected from coagulated blood by centrifugation and stored at oc until analysis for drug content. in addition lungs, kidneys, intestinal contents, urine and brains were harvested. tissues and biological samples were homogenized in saline and antibiotic concentrations determined by tdx analyzer (abbott). chronic lung infections were produced in rats by intratracheal instillation of agar beads containing p. aeruginosa. three days post instillation, rats (n= /group) were treated with aerosolized saline or once daily with arikace™, or twice daily with tobramycin (total daily dose = mg /kg) for days. rats were euthanized days post final dose and the pseudomonas cfu counts per lung were determined. results: the biodistribution and pharmacokinetics of aerosolized arikace™ was significantly different from that of amikacin and tobramycin. the primary difference was the amount of antibiotic retained in the lungs. rats that inhaled soluble amikacin cleared approximately % of the amikacin from the lungs after hr with concomitant % increase in drug levels in the urine. rats that inhaled arikace™ cleared only % of the deposited amikacin from the lungs after hrs. this initial clearance of drug most likely reflected the amikacin released during nebulization. extended clearance of arikace™ from the lungs was measured by recovery of amikacin in the urine at later time points. in a separate longer-term study comparing arikace™ and tobramycin, auc ( - hr)for arikace™ in the lung was ug*hr/g while only ug*hr/ g for tobramycin which demonstrates a -fold increase in retention of antibiotic in the lung with arikace™. eradication of p. aeruginosa from the lungs of rats with arikace™ was equal to tobramycin (given equivalent daily doses). however, arikace™ reduced the number of cfu in the lungs with a single daily exposure to the same degree as inhalation of tobramycin twice a day. conclusions: inhalation of arikace™ achieved higher concentrations and increased retention time of the antibiotic in the lungs of rats when compared to inhaled amikacin or tobramycin. it is expected that these properties of arikace™ will reduce dosing frequency and improve antimicrobial efficacy in patients. nielsen, x.c. ; johansen, u.r. ; nørregaard, l. ; vandamme, p. ; høiby, n. . clinical microbiology, rigshospitalet, copenhagen, denmark; . laboratory for microbiology, university of ghent, ghent, belgium background: the burhkolderia cepacia complex (bcc) is a diverse group of bacteria with at least genomovars (gv) or species. accurate species identification is necessary for better understanding of the epidemiology and pathogenesis of bcc. species identification based on phenotypic characters is difficult when it is based on few if any differences. crossed immunoelectrophoresis (cie) has been performed routinely to identify all burkholderia and other difficult isolates in our department (høiby et al.: "taxonomic application of crossed immunoelectrophoresis." int. j. syst. bact. ( ): - .) . in this study, we compared the results obtained by cie with those obtained by the standard molecular biological method using reca gene pcr and rflp to identify bcc at the species level (golden standard). methods: between and , burkholdreia isolates were recovered from patients in the two danish cystic fibrosis (cf) centres. . cie method: immunological cross-reactivity between antigens from the reference strains and the strains isolated from our cf patients were compared by employing cie and rabbit standard-antibodies (purified igg) raised against water-soluble antigens from reference strains of the bcc. species identification was made based on matching coefficient (mc). . reca gene-based identification: bcc specific primers were used to amplify reca gene. species-specific pcr is then performed using b. multivorans specific primers to identify the b. multivorans gv ii. rflp patterns were generated for those non-multivorans bcc by digesting the bcc-specific pcr products with haeiii. the patterns were then compared with the patterns generated from reference strains in the database. pulsed field gel electrophoresis (pfge) genotyping was performed for all strains to detect patient-topatient transmission. results: / isolates were bcc-specific pcr positive. species-specific pcr identified isolates as b. multivorans (gv ii). the other three isolates were identified as b. cenocepacia (gv iii) based on rflp patterns. two isolates with negative bcc-specific pcr reactions were identified as b. gladioli by cie (confirmed by reference laboratory in belgium). results from cie showed that strains from bcc were immunologically closely related with only a few non-cross-reactive antigens. compared with the standard reca gene-based method, cie method resulted in correct identifications in isolates ( . %), misidentifications in isolates ( . %) and uncertain identifications in isolates ( . %). genotyping by pfge showed unique patterns for all isolates except for those from two sisters. this suggested that patient-to-patient transmission of bcc among the danish cf patients has remained very rare. conclusions: reca gene-based pcr and rflp is a quick and reliable method for identification of bcc at the species level. cie proved useful in initial classification of bcc strains, but additional information is obtained by the reca gene-based species identification and pfge method. background: in childhood, the cystic fibrosis airway is characterized by persistent inflammation with high quantities of neutrophils. in this setting, recurrent exposure to low numbers of pseudomonas aeruginosa occurs routinely. chronic infection by p. aeruginosa is associated with a significant increase in morbidity and mortality in cf, and is believed to occur by formation of a biofilm in the cf airway. previously, we have shown a significant neutrophil dependent enhancement of p. aeruginosa biofilm density that is greatest for low initial concentrations of p. aeruginosa. biofilm enhancement is facilitated via neutrophil-derived dna and f-actin, which form a framework that p. aeruginosa can exploit for growth. dna and factin polymerize via positively charged molecules such as histones. p. aeruginosa then associates with these polymers, which results in increased early biofilm density, displaying an increase of over fold at hours compared to p. aeruginosa in the absence of neutrophils. hypothesis: compounds that disperse dna and/or f-actin can disrupt neutrophil-enhanced biofilms. methods: poly(aspartic acid), a negatively charged amino acid chain with the capacity to disrupt f-actin, was examined singly and in combination with dnase and/or antibiotics (ciprofloxacin and tobramycin). the nunc-tsp system was used to allow for high throughput assessment of the density of biofilm formation in the presence of neutrophils and the effect of various agents in disrupting the biofilm. the biofilm assay is independent of cellular settling, as the reactor is vertically suspended in the culture. p. aeruginosa strains tested were pao , and two isogenic cf isolates recovered from an initial infection (early), and following established infection ~ years later (late). biofilms were formed by incubation of p. aeruginosa with human neutrophils for to hours in rpmi with % heat inactivated platelet poor plasma. results: antibiotics were incapable of disrupting biofilms. poly(asp) significantly disrupted neutrophil-induced biofilms. in the presence of neutrophil products this action is sensitive to proteolytic degradation, but can be protected by the presence of protease inhibitors. dnase also disrupts a biofilm formed in the presence of human neutrophils. the combination of poly(asp) + dnase resulted in an increase in biofilm disruption for a hour biofilm. biofilms allowed to form for hours were more resistant to disruption by dnase and poly(asp) as single agents, but the combination of both demonstrated a synergistic effect. similar disruption of biofilms were observed when pao was compared to early and late cf strains. conclusions: pseudomonas aeruginosa biofilms formed in the presence of neutrophils in vitro can be disrupted by agents that disperse the f-actin and dna framework. support: cff, max and yetta karasik foundation cystic fibrosis (cf) patients may suffer increased morbidity and mortality through colonisation, allergy and invasive infection from fungi. the black yeast, exophiala dermatitidis (synonym wangiella dermatitidis) has been found with increasing frequency in sputum specimens of cf patients, with reported isolation rates ranging from between . and . %. at present, no species-specific diagnostic pcr exists to aid with the clinical laboratory detection and identification of this organism. a novel species-specific pcr-based assay was developed for the detection of e. dermatitidis, based on employment of rdna operons and interspacer (its) regions between these rdna operons. two novel primers, (designated exdf & exdr) were designed in silico with the aid of computer-aided alignment software and with the alignment of multiple species of exophiala, as well as with other commonly described yeasts and filamentous fungi within cf sputum, including candida, aspergillus and scedosporium. pcr employing exdf (forward primer [ -,mer], '-ccg cct att cag gtc c- ') and exdr (reverse primer [ -mer], '-tct ctc cca ctc ccg c- '), was employed and optimized on extracted genomic dna from a well characterized culture of e. dermatitidis, as well as with high quality genomic dna template from a further unrelated fungi, including candida albicans, c. dubliniensis, c. parapsilosis, c. glabrata, scedosporium apiospermum, penicillium sp., aspergillus fumigatus, aspergillus versicolor, pichia guilliermondii, rhodotorula sp., trichosporon sp., aureobasidium pullulans, fusarium sp., mucor hiemalis, bionectria ochroleuca, gibberella pulicaris. results demonstrated that only dna from e. dermatitidis gave an amplification product of the expected size, whilst none of the other fungi were amplifiable. subsequent direct employment of this primer pair detected this yeast in the sputum of / ( %) adult cf patients, employing a nested pcr approach with panfungal outer flanking primers of the its -its region. these two patients were the only patients who were previously shown to have a cultural history of e. dermititidis from their sputum. e. dermatitidis is a slow-growing fungus, which usually takes up to two weeks to culture in the microbiology laboratory and therefore is slow to detect conventionally, with the risk of bacterial overgrowth from common co-habiting pan-and multiresistant bacterial pathogens from sputum, namely pseudomonas aeruginosa and burkholderia cepacia complex organisms, hence this species-specific pcr assay may help detect this organism from cf sputum more specifically and rapidly. overall, employment of this novel assay may help in the understanding of the occurrence, aetiology and epidemiology of exophiala dermatitidis, as an emerging fungal agent in patients with cf. cystic fibrosis patients are particularly susceptible to infection by strains of pseudomonas aeruginosa and burkholderia cepacia complex. since , the pseudomonas genome database has been a resource for peer-reviewed, continually-updated annotations for the pseudomonas aeruginosa pao reference strain's genome and, more recently, comparative analyses to several closely-related pseudomonas species. in order to facilitate better cross-strain and cross-species genome comparisons, we have developed or are incorporating methods to improve the identification of orthologs (genes diverged due to speciation) and identify genes undergoing unusual selection. our analysis of several completely sequenced pseudomonas genomes has revealed that approximately % of ortholog predictions by the classic "reciprocal best blast hit method" are likely incorrect. we have also performed an analysis of unusually large intergenic regions in p. aeruginosa pao that appear to be essential (according to saturation transposon mutagenesis) or involved in virulence (according to signature tagged mutagenesis). we identified at least new putative protein-coding regions and non-coding rnas that were not previously annotated and may play critical roles in microbial pathogenesis or viability. we are also focusing our attention on facilitating better cross-strain comparisons between recently sequenced burkholderia cepacia complex genomes through development of a new online burkholderia genome database. in addition to improved ortholog prediction, this new database will provide access to a very flexible boolean search feature that allows researchers to search and compare annotations within or between the genomes of burkholderia strains, returning annotations from multiple genomes suitable for simultaneous viewing and downstream analyses. we have also included new, very accurate protein subcellular localization predictions for the deduced proteome from each of these genomes -predictions that can aid the identification of new cell surface or secreted therapeutic targets or vaccine candidates. further comparative analyses with other newly-sequenced, related strains should provide insight into strain-specific features that may be exploited to better understand virulence and antimicrobial resistance exhibited by cf-relevant pathogens. funding provided by cystic fibrosis foundation, therapeutics (usa). popova, a.p.; verma, r.; zanni, r.l.; sembrano, e. pediatrics, monmouth medical center, long brnach, nj, usa background: cystic fibrosis (cf) is a chronic, progressive, genetic disease leading to poor lung function, chronic bronchial infection with certain bacteria and recurrent pulmonary exacerbations. despite evidence of inflammation within the airway, it is unclear which circulating inflammatory biomarker best reflects the airway inflammation and lung function in cf. objective: to determine the utility of c-reactive protein (crp) value in evaluating patients with cystic fibrosis pulmonary exacerbation. design: a retrospective chart review was carried out on patients followed at the cystic fibrosis center at the children's hospital. information about serum crp values, pulmonary function test results(pft), presence of pulmonary exacerbation as documented in the chart, sputum culture results, current medications, as well as demographic data, genetic compositions and weight percentiles between january and march was collected from the charts and was analyzed. results: of patients whose charts were reviewed, were excluded because they underwent lung transplant, were excluded because they had other mild infection, not diagnosed as pulmonary exacerbation. for patients crp was not measured and they were also excluded from the analysis. total of patients were included in the study. for patients information about crp was obtained on more than one occasion. the patients were divided into groups based on the presence of pulmonary exacerbation and elevation of crp values -in the group with pulmonary exacerbation, patients had elevated crp and had normal crp and in the nonexacerbation group had elevated crp and had normal. fisher's exact test (two -tail) was applied (p= . ) and confirmed statistically significant elevation of crp in patients with pulmonary exacerbation of cystic fibrosis. the means and standard deviations of the crp values for the patients without pulmonary exacerbation and the patients with pulmonary exacerbation were calculated and analyzed using independent group t-test (two tail). mean crp value for the exacerbation group was . with standard deviation of . and mean crp value for the non-exacerbation group of . with a standard deviation of . . statistically significant difference between the two means was noted (p= . ). conclusion: serum crp values in patients with cf pulmonary exacerbation were significantly higher than in patients without pulmonary exacerbation. serum crp may be a useful marker of pulmonary exacerbation in children. in the absence of pulmonary exacerbation, cf is not associated with elevated crp values in children. the findings of this study could aid in identifying patients with pulmonary exacerbation earlier and initiate appropriate therapy sooner. further studies are needed to determine whether crp values are associated with the severity of the underlying disease, to what extent the pft are affected, the age of the patient, the type of bacterial pathogen, and genetic factors. background pulmonary infections with nontuberculous mycobacteria (ntm) are commonly associated with structural lung diseases or airways clearance disorders such as cystic fibrosis and primary ciliary dyskinesia. we follow approximately individuals with ntm pulmonary disease who share common clinical features but have no identifiable systemic immune defect or clearly defined predisposing conditions. the pulmonary disease and clinical presentation suggest these individuals may have disorders of ion transport or ciliary function. while some cftr mutations and ciliary abnormalities are difficult to detect through standard genetic and ultrastructural analyses, in vivo physiologic tests of ion transport and measures of ciliary function may allow us to identify ion transport abnormalities in subjects with no identified cftr abnormalities, determine the functional significance of novel cftr mutations, and distinguish disorders of ciliary function from other airways clearance disorders. methods we evaluated the medical and family histories, chest and sinus ct's, sputum cultures, cftr genotypes, and sweat chloride levels of individuals with nontuberculous mycobacterial pulmonary disease and individuals with chronic airways disease and bronchiectasis. we systematically tested the pulmonary function (pft), nasal nitric oxide production (nno), and nasal potential difference (npd) of each subject. nasal mucosal scrape biopsies were performed and sent to unc-chapel hill for assessment of ciliary ultrastructure as part of the genetic diseases of mucociliary clearance consortium. pulmonary function, nno, and npd were measured on healthy controls. results all subjects had sino-pulmonary symptoms consistent with those associated with cf and pcd. subjects had low nno ( . ± . nl/min) consistent with levels reported in subjects with pcd (< nl/min, noone ). one subject with variant cf (∆f /r h) displayed low basal pd (- . mv), elevated na+ absorption ( . mv), and no cftrmediated cl-conductance (- . mv). subjects with cftr mutations, subject with isolated elevated sweat chloride ( mmol/l), and subject with isolated borderline sweat chloride ( mmol/l) displayed normal basal potential difference (- . ± . mv) and na+ absorption ( . ± . mv) but decreased cftr-mediated cl-conductance (- . ± . mv) relative to controls (- . ± . mv). conclusions ntm patients heterozygous for cftr-mutations or with elevated sweat chloride (and no identified mutations with coding region sequencing of cftr) may have detectable subtle differences in cftr clconductance, while measurement of nno may identify disease associated with ciliary abnormalities. these findings suggest subtle abnormalities in airways clearance may predispose to airway infection with environmental organisms such as ntm. support: intramural funds, national institute of allergy and infectious diseases muhlebach, m. ; goodrich, j. ; sutton-shields, t.n. ; wedd, j.p. ; henegar, c. ; miller, m.b. ; gilligan, p.h. . dept. of pediatrics, unc chapel hill, chapel hill, nc, usa; . university of north carolina hospitals, chapel hill, nc, usa; . school of public health, university of north carolina hospitals, chapel hill, nc, usa; . dept. pathology and laboratory medicine, university of north carolina hospitals, chapel hill, nc, usa introduction: proportion of methicillin resistant (mrsa) vs. susceptible sa infections increased from % in to % in in us hospitals. similarly, the prevalence of mrsa in the community has risen in recent years. these community acquired (ca) mrsa isolates are genotypically different, have different antibiotic susceptibility, and often carry the pvl virulence gene. this virulence gene is associated with more invasive, mostly skin infections in healthy individuals. reports on impact of mrsa in cf show conflicting data. , . the aim of this study was to determine the prevalence of ca-mrsa strains at our pediatric and adult cf center and to assess the impact of these infections on clinical outcome. methods: all mrsa isolates recovered from routine clinical cultures, were prospectively collected and analyzed for months ( / - / ). using molecular assays to determine the pvl status and to identify the sccmec (staphylococcal cassette chromosome) type, the organisms were classified as ca or ha. classification as ca mrsa was based on presence of pvl and sccmec iv type. clinical outcomes included lung function measurements in patients reliably performing spirometry and nutritional parameters (bmi%). patients who had undergone lung transplant were excluded as their lung functions vary depending on transplant outcome. results: of the patients identified in this month period, % had ha and % ca mrsa. neither current age (mean . yrs. in ha vs. . years in ca) nor age at acquisition of mrsa ( . vs. years) was significantly different. sixty-eight % of all mrsa infected patients had chronic p. aeruginosa infection, ( % in the ha and % in ca, relative risk . of having ha in presence of chronic p. aeruginosa infection). crosssectional comparison of patients infected with ca vs. ha acquired strains showed no difference in nutritional status (bmi vs. %). lung function (fev and fef - ) was not different in ca vs. ha patients, neither when including all mrsa infected patients or those who had no concomitant infection with other organisms (n= ). we will present further comparisons to age and gender matched children with ossa infection in the final report. conclusion: comparison to ossa in matched patients will assess clinical outcomes of infections with mrsa. initial data collected at our center did not show a significant difference in outcomes between patients infected with ca vs. ha mrsa. a multicenter study would most efficiently assess the impact on clinical outcomes and determine regional differences of mrsa patterns (ca vs. ha) in different cf centers. ref : the stringent response is a global regulatory mechanism used by bacteria to adapt to nutrient limitation and other environmental stresses, and is mediated by the signaling molecules (p)ppgpp (phosphorylated guanosine nucleotides). increases in (p)ppgpp repress metabolic processes and growth, and regulate genes involved in long-term stress and starvation survival in several species ((i)e. coli, m. tuberculosis(i)), bacterial multicellular behavior ((i)myxococcus xanthus(i)), and pathogenesis ((i)p. aeruginosa(i)). while many studies have investigated bacterial functions (such as attachment and motility) involved in biofilm development, less is known about the physiological adaptation occurring in biofilm growth. when cells grow to high densities entrapped in a polymer matrix, they must adapt to heterogeneous micro-environments where gradients in nutrients, oxygen and metabolic waste exist. nutrient may thus become depleted as they are consumed by overlying cells, and some biofilm subpopulations are likely starved. we hypothesized that the stringent response mediates adaptation to nutrient starvation in (i)p. aeruginosa(i) and is important for biofilm formation. we tested a (i)rela(i) deletion mutant unable to synthesize (p)ppgpp in response to amino acid starvation, and its isogenic wildtype parent pao . we measured the starvation survival of planktonic bacteria in phosphate buffered saline and observed that the (i)rela(i) has a to fold greater loss in viability compared to wild type after h. we also tested biofilm formation in a static biofilm assay using polystyrene peg lids and estimated biomass accumulation by crystal violet staining. in this system, the (i)rela(i) accumulates times less biomass than wild type. in a flow-cell reactor system, the wildtype strain forms complex mushroom-like biofilm structures, but the (i)rela(i) mutant forms flat, thin biofilms. we determined cell attachment to polystyrene by crystal violet staining, and to glass surfaces by direct visualization under microscopy. the attachment to both surfaces is similar in the (i)rela(i) and wildtype strains, and the strains have identical planktonic growth rates. our findings suggest that the stringent response is involved in starvation survival, a physiological condition relevant to growth in biofilm. furthermore, (i)rela(i) inactivation impairs biofilm formation, suggesting that the stringent response may be an important stress adaptation in biofilm. belfast, united kingdom; . northern ireland public health laboratory, belfast city hospital, belfast, united kingdom; . northern ireland regional adult cf unit, belfast city hospital, belfast, united kingdom; . department of respiratory medicine, queen's university, belfast, united kingdom pseudomonas aeruginosa (pa) is a clinically significant bacterial pathogen responsible for increased morbidity and mortality in patients with cystic fibrosis. small non-coding micro(mi)rna species (generally ~ nts or less) in prokaryotes are involved in numerous cellular processes as is the case with eukaryotes. as in eukaryotes, these mirnas act by base pairing with target mrnas imposing translational and stability changes of mrnas and thus culminating in the control and regulation of target mrnas, crucial for bacterial stress responses and virulence to changing environments surrounding the infected zone or host cell (e.g. human airway epithelial cells). we recruited a commercial kit (biodynamics dynaexpress mirna cloning kit av, tokyo, japan) and isolated mirnas from a variety of pseudomonas pathogens from plant, soil / agricultural environment (pseudomonas syringae -ps), type strain (pseudomonas aeruginosa -pa) and a collection of clinical isolates of pseudomonas aeruginosa (cfpa) from adult cf patients, who were chronically infected with p. aeruginosa. the results on the mirna clones obtained exhibited a wide variation in the occurrence of mirnas in this bacterium. the putative mirnas obtained, cfpa in particular, yielded small non-coding rnas of sizes from as little as nts to well over nts. an initial search using the bioinformatic tool srnapredict (www.waldorlab/tufts.edu) has not as yet, fully revealed the genomic annotations for these novel srna sequences in the inter-generic regions of the pathogenic pseudomonad group of bacteria. we present our data on these unknown cellular mirnas cloned from cfpa isolates from cf patients and discuss their significance as novel regulators to bacterial stress responses and in the context of the bacterium's involvement as the predominant pulmonary pathogen, associated with cystic fibrosis. airway function is diminished in infants with cf diagnosed clinically but whether this is true for those identified by newborn screening remains unclear. we investigated whether lung function is diminished in infants with cf diagnosed by newborn screening and if this occurs in association with pulmonary infection. methods:lung function was measured in asymptomatic infants with cf following sedation with chloral hydrate ( - mg/kg) using the raised volume rapid thoracic compression technique at an inflation pressure of cmh . we measured forced vital capacity (fvc), forced expired volume after . seconds (fev . ) and forced expired flow at % expired fvc (fef ) which were then expressed as z scores. broncho-alveolar lavage (bal) was performed under general anesthesia within forty-eight hours following infant lung function testing. three separate aliquots of ml/kg sterile saline were instilled into the right middle lobe and a further aliquot in the left lingula and the bal fluid analyzed for bacterial, fungal and viral pathogens using routine culture techniques. results: bal was successful in all infants studied aged . to . yrs (median . yrs) and lung function in infants. median (range) z scores for fvc, fev . and fef were - . (- . to . ), - . (- . to . ) and - . (- . to . ) respectively. these decrements in lung function were all significant (p< . ). bal cultures were positive in ( %) infants of whom ( %) had evidence of infection with s. aureus and ( %) with p. aeruginosa. in infants there was no growth in bal or growth only of mixed oral flora. viruses were not identified in any infants. there was no association between any of the lung function parameters and pulmonary infection detected by bal. lung function was successful in a subgroup of of infants who were less than . yrs (median age . yrs) when tested. diminished airway function was identified in this younger subgroup even though only one infant had evidence of pulmonary infection with respiratory organisms in bal. in this younger subgroup median fev . z score was - . which was significantly below that predicted ( % ci: - . ,- . and p= . ). conclusions: airway function is diminished in infants with cf diagnosed by newborn screening and occurs irrespective of infection identified by bal. lung function is abnormal in very young infants before pulmonary symptoms develop and when no evidence of pulmonary infection can be detected in bal. inhaled high-dose tobramycin appears to transiently clear p. aeruginosa (pa) from airway secretions in young children with cf, though inflammation may not be markedly reduced (am j resp crit care med ; : ) . because inhaled antibiotics may not reach some areas of infection, we hypothesized that intravenous (i.v.) antibiotics may be more effective than inhaled high-dose tobramycin for reducing lower airways inflammation in cf children with early pa infection. study design: we initiated a single-center, randomized, prospective study comparing the effects of two antibiotic treatment regimens on bronchoalveolar lavage fluid (balf) inflammatory markers. clinically stable cf children with a recent isolate of pa from surveillance cultures are randomized to receive weeks of tobi® (group ) or weeks of i.v. ceftazidime and tobramycin (group ). balf is obtained just before treatment, and repeated - weeks after completion of treatment. the primary study outcome is change in balf % neutrophils (pmn). multiple secondary outcomes are assessed including quantitative bacterial cultures and cytokines in balf. this report summarizes interim analysis at the halfway point of the study. results: to date, subjects have enrolled. five subjects ( in group , in group ) dropped out after the initial bronchoscopy due to a decision by the primary cf physician to use other treatments based on balf culture results ( ) , or because of the development of a new respiratory illness before the second bronchoscopy ( ). a total of adverse events ( significant, unrelated to protocol) were reported in subjects, none resulting in a change of protocol request by the irb or data safety monitor. of the subjects completing the protocol to date, were in group , and were in group . the groups were well matched in terms of age, pa in initial balf, and initial balf %pmn. a majority of subjects in each group were children with their first isolation of pa on surveillance cultures. there was a tendency for group , but not group , to have mild reductions in balf %pmn (- ± %) after treatment. interim efficacy analysis did not suggest a significant difference between the treatment groups at this stage. similar changes were seen in group for pmn/ml (- ± x /ml) and several cytokines including gm-csf (- ± pg/ml) and mcp- (- ± pg/ml). two group subjects were unable to have stable i.v. access established and were treated with oral ciprofloxacin and inhaled tobi® for weeks as alternative systemic antibiotics, per protocol. dropping these subjects from group data did not change data trends. conclusions: the protocol appears to have adequate safety. parents of children with first-time pa infection may be more likely to consent to treatment randomization, than those with repeated pa infection. interim data analysis suggests that continuation of the study toward the original sample estimate is appropriate. acknowledgements: supported by cff(noah a ) and nih (gcrc r ). we thank tracy callahan rn, lupe haynes rrt, justin hubbard, paula murphy, cassidy henegar, nancy lee rn, benjamin butler rn, and sheree berckmans rn for logistical and technical support. methods: our local cf database was searched for patients born - . patients were included in the analysis if they were followed in our cf center before the age of . patients were separated into two birth cohorts, born - (cohort ) and - (cohort ). yearly peak fev % predicted from ages - were obtained using knudson reference equations. yearly peak bmi% and weight-for-age% at each age were also obtained. type tests of fixed effects and repeated measures analyses were used to assess mean peak bmi%, mean peak weight-for-age%, mean peak fev % predicted at first pft, mean peak fev % predicted from ages - , and mean rate of change (slope analysis) in peak fev % predicted from ages - . chi-square test for association was used to analyze differences in demographic parameters. wilcoxon rank-sum test was used to assess differences in age at diagnosis. wilcoxon non-parametric test was used to analyze differences in mean clinic visits per year. chi-square testing with a two sided alpha of p < . was used to determine significance. results: cf patients were included in the analysis. there were no significant differences between birth cohorts and for age of diagnosis (p= . ), sex (p= . ), race (p= . ), genotype (p= . ), pancreatic insufficiency (p= . ), or the mean number of clinic visits per year from ages of - (p= . ). there was a significant difference in the mean number of clinic visits per year from ages of - (p=. ; see table ) . birth cohort had a significant effect on the rate of decline in lung function from ages - . birth cohort and age had significant effects on the mean weight-for-age%, mean fev % predicted at first pft, and mean fev % predicted from ages - . sex had a trend for significant effects on lung function parameters. adjusted for age and sex, there were significant differences between birth cohorts and in mean bmi% and mean weight-for-age% at ages and , mean fev % predicted at first pft, mean fev % predicted from ages - , and in the mean rate of decline in fev % predicted (see table ). conclusions: improvements in lung function and nutritional outcomes in our cf center are associated with an increase in the mean number of clinical visits per year for cf patients ages - . the frequency of clinical follow-up is a marker of cf care that may have positive impacts on cf outcomes. background: current management of cf airway disease includes the use of key pharmacological therapies designed to combat the chronic cycle of obstruction, infection, and inflammation. randomized controlled trials have demonstrated that maintenance therapy with azithromycin (azm), dornase alfa (dnase), and tobramycin for inhalation (tob) each improves pulmonary function and reduces the frequency of exacerbations in patients with p. aeruginosa. the purpose of this study was to determine if cf patients in an uncontrolled environment (clinical setting), experience a reduced rate of pulmonary function decline when receiving azm + dnase + tob compared to those not on all three therapies. methods: adult cf patients were selected based on positive sputum cultures for p. aeruginosa, pulmonary function test results (fev % predicted) for the past year, and receiving one of the key pharmacological therapies. in addition data was collected on cfrd, depression, medication adherence, airway clearance, and nutritional status. patients were stratified according to baseline pulmonary function: mild (fev ), moderate (fev of - %), and severe (fev < %). patients were then sub-grouped as to whether they were receiving all or ≤ of the key therapies. the annual rate of decline in % predicted fev was determined using linear regression on non-exacerbation data and was categorized as stable (< . % decline), intermediate ( . - . %) , or rapidly declining (> . %). time to intermediate or rapid decline in pulmonary function was determined using cox proportional hazard modeling. results: patients ( males/ females, median age years) were included in this study; with mild, with moderate, and with severe pulmonary disease. overall, the mild and moderate groups experienced median declines of . % and . %, while the severe group experienced a . % improvement in fev % predicted respectively. the use of regimens containing azm therapy significantly delayed time to intermediate or rapid decline in lung function in the mild group (median days vs. . days, hr= . , p= . ). similarly, regimens containing dnase + azm + tob significantly delayed time to intermediate or rapid decline in lung function in the mild group (median days vs. days, hr= . , p= . ). no significant differences were seen in the moderate or severe group for patients receiving all or individual therapies. con-clusion: administration of pharmacological regimens containing dnase + azm + tob, or azm + tob or azm + dnase significantly delay progression of pulmonary disease in cf patients with mild lung disease. sawhney, v. ; arthur, b. ; seltzer, r. ; kraynack, n.c. . internal medicine, akron general medical center, akron, oh, usa; . pediatrics, akron children's hospital, akron, oh, usa; . biostatistics, neoucom, rootstown, oh, usa; . pulmonology, akron children's hospital, akron, oh, usa early and efficient diagnosis of pulmonary exacerbations (pex) in patients with cystic fibrosis (cf) is crucial in the management of cf. in oct , we implemented a pulmonary exacerbation scoring (pes) system to uniformly and consistently identify pex in cf patients as part of a quality improvement project at the cf center at akron children's hospital. we have previously reported improved pulmonary function outcomes in pediatric patients at our center after implementation of the pes. we now describe the impact of the pes on clinical practice in the management of pex and pulmonary function in our adult cf patients. the pes was developed by our multidisciplinary cf team after an extensive literature review and was implemented in oct . the pes has been previously described and consists of clinical questions divided into three domains: systemic and pulmonary symptoms and signs, and objective measurements. these elements are scored individually and a cumulative pes of ≥ (range - ) is considered a pex and treatment with antibiotics is recommended. the course and choice of antibiotic regimen is left to the physician's discretion. we examined median percent predicted fev decline from quarterly percent predicted fev measurements obtained from port cf. we calculated this for all the patients seen in our adult cf clinic (age> ) from oct till sep during which the pes was in use. we compared this rate of decline to median percent predicted fev decline for the same cohort of patients over the preceding two years (oct till sep , during which the pes was not in use. the percent utilization of the pes was calculated individually for all adult cf care providers. the influence of individual components of pes in decision to treat was also studied. we found no significant difference between the rates of decline in percent predicted fev during the pre-pes and post-pes periods (mean = -. , sd = . vs mean = -. , sd = . , p= . ) as measured by paired samples t-test. logistic regression analysis was done to evaluate the relative importance of each pes component in predicting decision to treat a pex. we found that changes in fev (or= . ), cough (or= . ), sputum (or= . ), and chest examination (or= . ) are most likely to affect the decision to treat where as dyspnea (or= . ) has the little effect on decision to treat. we found a high degree of correlation between adult provider use of the pes and an independent chart reviewer (correlation coefficient . - . ; p< . ). in contrast to cf patients ages - years, we found no significant effect of use of the pes on pulmonary function in our adult population, despite the high frequency of use by our adult providers. it is unclear why this is the case given the robust effect of the pes on our pediatric population. this study suggests that a simplified score that includes fev , cough, sputum, and chest exam may be useful in identifying a pex in adult cf patients. special thanks to tasha capozzi for her help with chart review. this project was supported by the akron children's hospital foundation. objective chronic pulmonary infection with pseudomonas aeruginosa is the main cause for morbidity and mortality in cystic fibrosis (cf) patients. tobramycin and colistin, widely used inhaled antibiotics in cf patients, were found to affect elastase activity in vitro. we showed recently that cxcr on neutrophils mediates bacterial killing, but is cleaved in cf airways by elastase. therefore, we examined the effects of inhalation with tobramycin and colistin on cxcr expression and antibacterial capacities by airway neutrophils in cf patients in vivo. methods cxcr expression was quantified by flow cytometry on neutrophils in peripheral blood and induced sputum of cf patients without inhalation therapy, cf patients with tobramycin and cf patients with colistin inhalation therapy. the longitudinal effect of a weeks tobramycin or colistin inhalation period on cxcr expression, free elastase levels and bacterial killing by airway neutrophils was assessed. results inhalation with tobramycin increased, while inhalation with colistin decreased cxcr expression on neutrophils in induced sputum of cf patients. neutrophils isolated from sputum of cf patients after tobramycin therapy had higher, whereas patients after colistin therapy had lower bacterial killing capacity and α-defensin release as compared to neutrophils before antibiotic inhalation therapy. conclusions tobramycin and colistin differentially modulate the pulmonary host defense in cf patients in vivo via cxcr on neutrophils. these findings may have clinical implications when considering antibiotic treatment in cf patients. sharp, j. ; sheehan, d. ; ren, c.l. . pediatrics, women & children's hospital, buffalo, ny, usa; . pediatrics, university of rochester, rochester, ny, usa background: airway infection and inflammation occur early in infants with cf. the raised volume/rapid thoracoabdominal (rv/rtc) technique is more sensitive than older infant pulmonary function test (ipft) methods in detecting airflow obstruction in these patients. however, there are limited data regarding lung function measured by rv/rtc in infants with cf nbs. we hypothesized that abnormalities of lung function measured by rv/rtc are present in cf nbs infants. to test this hypothesis, we reviewed the ipft data from cf nbs infants followed at our cf centers. methods: this study was a retrospective chart review. we identified infants with cf nbs followed at our cf centers who had had at least ipft performed in the first year of life. ipfts were performed using the collins ipl. forced expiratory flows were measured using rv/rtc as described by feher et al (j appl physiol : , and fractional lung volumes were measured by whole body plethysmography (bp) as described by castile, et al (ped pulm : , ) . data from the cf infants were compared to normal data reported by jones, et al (ajrccm : , ) for rv/rtc and castile, et al for bp. results: we reviewed data from infants. their mean age was . m (range= - ). pancreatic insufficiency was present in ( %) infants, and ( . %) had been treated for pseudomonas infection. their pft findings are summarized in the table. fvc and fev . were not significantly different from the normal predicted values. however, the mean fef and fef - z scores were significantly below the mean of normal infants (p= . and . respectively). gas trapping was present, as evidenced by significantly increased frc, rv, and rv/tlc (p< . for all measures). six infants ( %) had an fef z score of < - . , and ( %) had an frc > % predicted. conclusions: infants with cf nbs have abnormal lung function early in life. our results suggest that pfts in cf nbs infants can be useful in detecting early changes in lung function. we speculate that early therapeutic interventions may improve lung function in this group of patients. table. pft findings in infants with cf nbs. forced expiratory flows are reported as z scores, while fractional lung volumes are reported as % predicted. objective: clinicians use pfts to assess effectiveness of therapy in hospitalized cf patients. however, the variability of multiple pft efforts has not been described in this group of patients. previous study of within day variation of pfts has been reported in healthy adults and children (enright, , and nickerson, ) and clinically stable outpatients with cf (nickerson, , and cooper, ) . nickerson and cooper found coefficient of variations (cv) for fvc of . - %, fev of . - . %, and fef - of . - . % for adults and children with cf. no one has studied the within day variation of pfts in cf patients during pulmonary exacerbations. our objective was to determine the within day variation of pfts during hospitalizations for cf pulmonary exacerbations. we hypothesized that within day variation would improve during treatment. methods: we retrospectively reviewed pft data for all patients admitted to childrens' hospital and regional medical center for a cf pulmonary exacerbation in . patients performed pfts under the direction of experienced pulmonary function technicians. the test was complete when the patient performed efforts that met ats/ers criteria for acceptability. if the patient was unable to produce efforts because of coughing, efforts were accepted if the fvc and fev were within % or ml of each other. for analysis, we included only those pfts: ( ) with at least - flow-volume curves that fulfilled ats/ers criteria, and ( ) were performed within days of hospital admission or discharge. for patients with multiple admissions, we used pft data for their first admission only. for patients with admission and discharge pfts available, we compared the coefficient of variation (cv) for pfts (fvc, fev , ). there were at least days between admission and discharge pfts. the wilcoxon rank-order test was used to compare the cv for admission and discharge pfts. data: sixty-one patients were admitted times in . excluding repeat hospital admissions, patients had individual pft trials available at the time of admission and patients at the time of discharge. seven admission and discharge pfts included only trials. three patients at admission, and patients at discharge, were unable to produce pft curves that met ats/ers criteria, despite previously having done so when well. nine patients had paired admission and discharge pfts from the same hospitalization. the cv for fvc at discharge was significantly better than at admission, p = . (see table) . conclusions: within day variation of pfts during hospitalizations for cf pulmonary exacerbations is comparable to published outpatient data. cv for fvc improves significantly during exacerbation. changes in fvc > %, fev > %, and fef - > % can be considered a significant spirometric response to therapy in hospitalized cf patients. background: lung clearance index (lci), a measure of ventilation heterogeneity, can be calculated from multiple breath washouts of inert gas. in cross sectional studies it is a more sensitive measure of early airway dysfunction in cf than spirometry alone. in normal subjects, the technique is reproducible, with a narrow normal range of . - . . we hypothesized that it may be helpful in detecting change longitudinally, and may have value in assessing novel therapeutic interventions such as the upcoming gene therapy by the uk cfgt consortium. methods: patients aged years and over, presenting with an exacerbation requiring intravenous antibiotics, were recruited. lci was assessed in triplicate by multiple breath washout of . % sulphur hexafluoride (sf ), using a novel gas analyser (innocor, denmark) and custom-built software within the first hours of starting treatment. the test was repeated at the end of parenteral antibiotic therapy and again - weeks later when clinically stable. patients were recruited at three sites and assessed using standardised equipment and a data analysis protocol. results: paired lci measurements were available in patients, mean age (range - ) years, male. in a further patients, technically acceptable washouts could not be obtained at one or other time point. mean (sd) lci improved from . ( . ) to . ( . ), p< . . in / patients there was an improvement in lci of > % after antibiotics. patients showed a > % deterioration in lci. mean fev was significantly greater after treatment ( . v . l/s, p< . , n= ). there was a weak correlation between percent change in fev and lci (r = . , p< . ). patients also had lci measured at the follow up. mean (sd) lci at this visit was . ( . ), p< . vs visit , no significant difference vs visit . conclusions: lci can be used to demonstrate changes in the lung after antibiotic therapy. this is the first study in cf to demonstrate a lon-gitudinal improvement after intervention in a marker of ventilation heterogeneity, and offers different but complimentary information to spirometry. the higher sensitivity of lci may make possible measurement of improvement in mild patients after novel therapies such as gene therapy. this work was funded by the cystic fibrosis trust. aim: inhaled hypertonic saline positively affects sputum production in patients with cystic fibrosis (cf) by improving mucociliary clearance. this study was conducted to compare the aerosol delivery performance of various nebulizers upon aerosolization of hypertonic saline (hyper-sal™) . % and %, available as preservative-free, sterile ml solution in single dose, patient friendly blow-fill seal vials. method: ml hypertonic saline (hyper-sal™) % was nebulized by three jet nebulizers the pari lc plus®, lc star® and hudson rci micro mist as well as the eflow® for hypertonic saline (hs) generating aerosols via a perforated vibrating membrane. hs . % was characterized in the eflow®hs. droplet size distribution patterns were assessed by laser diffraction at a flow rate of l/min and aerosol delivery performance by breath simulation tests mimicking a sinusoidal breathing pattern. the delivered dose (dd) was determined by gravimetric analysis of the inhalation filters. inhalation and exhalation filters were changed after min and the drug delivery rate (ddr) was calculated as nacl found on inhalation filters per min. respirable drug delivery rate (rddr; ddr x respirable fraction < µm) indicates how much nacl can reach the lungs per minute. results: aerosol characteristics of the different nebulizers after aerosolization of ml hs % are summarized in the table. data for hs . % nebulized by the eflow®hs were as follows: dd= . %, ddr= . %, nebulization time = . min and comparable to data for hs %. the lower recoveries for the jet nebulizers are probably associated with increased evaporation and longer nebulization times. conclusion: about - % of the charged sodium chloride from ml hs was delivered and % - % of these droplets were in a respirable size range < µm resulting in vitro respirable doses of about % - % of the label claim. ddr and rddr are about -to -fold higher for eflow®hs indicating a much higher delivery efficiency compared to conventional jet nebulizers. further studies will be needed to demonstrate that about ml hs may be sufficient to deliver a comparable dose to the lungs using an eflow®hs compared with jet nebulizers. this bears the potential to further reduce nebulization time and increase acceptance by patients. objectives: individuals with cf frequently have complicated and time consuming treatment regimens consisting of inhaled antibiotics, mucolytics, and airway clearance. patients and clinicians may have difficulty determining which specific therapies are effective for any individual patient or whether a particular treatment is causing adverse effects. this study assessed the utility of home spirometry for providing an objective measure of pulmonary status in between scheduled clinic visits. methods: consecutive adults with cf were recruited at a single academic medical center and were asked to measure fev twice daily for six months using the piko- meter (nspire health™, llc). subjects agreed to participate and completed the study. subjects were given diaries to record changes in respiratory symptoms and changes in medications during the study period. subjects were evaluated in clinic at least every three months and fev values obtained in the pulmonary function lab were compared to home fev from the same day using linear regression and bland-altman analyses. graphical displays of home fev measures were visually inspected for changing trends and these were compared with medication changes. results: the mean fev measured at home was . ± . l. the mean fev in the pulmonary lab was . ± . l. the home fev was significantly correlated with the clinic value from the same day, r = . , p< . . fev measures fell within the limits of agreement (± sd) by bland-altman plot. in of subjects ( . %) there was at least one sustained change in fev that correlated with either stopping or starting a particular treatment. the figure shows two significant changes in fev in one patient related to medication changes. there is an increase in fev at the initiation of aerosolized % saline and a fall in fev that corresponded to inhaled tobramycin use. the improvement on hypertonic saline increased this patient's enthusiasm for this treatment. conclusions: home fev monitoring is feasible and appears to provide accurate measurement of pulmonary function. it can provide clinicians with objective data to tailor treatments without necessitating a clinic visit. feedback from home monitoring may improve medication adherence and may allow treatments to be individually tailored based on patient responses to the medications. . respiratory unit, royal hospital for sick children, glasgow, united kingdom; . diagnostic imaging, nhs greater glasgow & clyde, glasgow, united kingdom; . medical statistics, royal hospital for sick children, glasgow, united kingdom; . medical physics department, gartnavel hospital, glasgow, united kingdom background: there have been calls for caution regarding the suggested use of regular chest computed tomography (ct) scans for monitoring disease progression in cf . consideration of the associations of higher ionizing radiation exposure must be made before regular chest cts are performed on the wider cf population. aim: we tested the hypothesis that higher ionizing radiation exposure from chest radiographs (cxr) and chest cts would be associated with markers of severity including gender, genotype (class i/ii or iii-v), height sds, weight sds, bmi, fev sds, length of hospital admission (days), scottish index of multiple deprivation (simd) scores and pa/bc microbiological infection (chronic or intermittent colonisation with pseudomonas aeruginosa and/or burkholderia complex). methods: effective doses of ionizing radiation (msv) from cxrs and chest cts (including hr scans) were determined for all cf children at our centre between st january and th july ( weeks). the most recent anthropometric and lung function data to july were used. simd scores report a data zone that encompasses ~ individuals and comprises indicators in six domains (current income, employment, health, education, housing and access). results: cf patients ( male: female) underwent radiation exposure procedures (mean of . procedures per patient per year). the average effective doses were calculated for of the procedures ( . %) including cxrs and chest cts. lung function data were available on patients ( %). univariate analysis revealed that higher ionizing radiation exposure was significantly associated with fev sds (p< . ), length of stay (p< . ) and pa/bc infection (p< . ). the regression line indicates that for each drop of two fev sds, each child receives an extra msv in radiation exposure from cxrs and chest cts alone. similarly, each child receives an extra msv in radiation exposure from cxrs and chest cts for every days of hospital admission. multivariate analysis with backward selection demonstrated that length of hospital admission (p< . ) and pa/bc infection (p= . ) were associated with higher ionizing radiation exposure. discussion: in cf, higher ionizing radiation exposure from cxrs and chest cts in childhood are associated with poor lung function, pa/bc infection and length of hospital admission but not other indicators of severity such as gender, severe genotype, anthropometric measures or socio-economic status. using effective doses of ionizing radiation as an outcome marker in cf is an alternative means of assessing the impact of the disease and its management on the child. background: exhaled volatile compounds have attracted attention as potential non-invasive biomarkers of chronic lung disease. early sustained airway inflammation and bacterial infection are characteristic features of pulmonary involvement in cystic fibrosis (cf). the cellular response is dominated by neutrophils, which have been shown to generate oxygen-and halogen-derived radicals as part of their host defense armamentarium. aims: to gather pilot data on the pulmonary output or uptake of selected organic trace gases in cf patients and healthy controls; and to explore the impact of pulmonary exacerbations and antibiotic treatment. methods: samples of ambient air and exhaled breath were collected from children, adolescents and young adults with cf and from healthy controls. the patients' age ranged from - years and their fev between - % predicted, were pseudomonas positive. twelve patients were studied at the start of i.v. antibiotic treatment for a pulmonary exacerbation; repeated sampling after the course was performed in subjects. all samples were analyzed on a customized gas chromatography system with flame ionization and electron capture detectors. after determining the mixing ratios of a variety of volatile organic compounds, alveolar gradients were calculated from the corresponding exhaled and ambient values. results: compared to healthy control subjects, cf patients showed a significantly lower respiratory output of methanol, ethanol, acetaldehyde and dimethylsulfide but a higher net release of benzene, methylchloride, trichloromethane and trichloroethene. these differences were most pronounced in patients with acute exacerbations. a relevant scatter and overlap between groups was noted for ethane, pentane, acetone, isoprene, toluene, and tetrachloromethane. after antibiotic treatment, the output of benzene, methylchloride and trichloromethane decreased by - %. conclusion: although important pathways remain incompletely understood, the local biochemical milieu in cf lungs may be informative and accessible through non-invasive breath testing. in this context, the diagnostic potential of exhaled organic traces gases (especially oxygenated, halogenated and aromatic compounds) should be further explored. supported by: german research foundation (dfg ba / - ). toy, e. ; weiner, j. ; sacco, p. ; duh, m.s. . health economics, novartis pharmaceuticals, east hanover, nj, usa; . analysis group, inc., boston, ma, usa objective: to review up-to-date economic outcomes data in patients with cystic fibrosis (cf), and in particular, costs related to respiratory infection by pseudomonas aeruginosa (pa), which is the leading cause of morbidity and mortality in cf patients. methods: a systematic search of the medline database from - was conducted to identify major and review articles that contained the terms "cystic fibrosis" and "cost" and which were published in english language journals. selected conference abstracts were searched, and additional articles were identified through bibliographies of retrieved articles. recent articles that contained economic data on antibiotic and mucolytic therapies were selected for in-depth review. cost estimates were converted to us dollars for comparability. results: approximately articles fulfilled the initial search criteria, and were selected for in-depth review. articles were divided into four categories: economic impact of inhaled tobramycin ( articles), the effect of home-vs. hos-pital-based antibiotic therapies for pulmonary exacerbations ( articles), economic impact of recombinant human deoxyribonuclease (rhdnase) ( articles), and cost-of-illness for cf ( articles). inhaled tobramycin (actual or recommended dose of mg/ ml) has been associated with reductions in health care costs; these cost savings offset between % and % of the cost of this therapy. antibiotic therapy for pulmonary exacerbations generally resulted in lower health care costs when administered in a home setting rather than a hospital setting. use of rhdnase led to reduction in health care costs, with higher reductions observed in patients with higher levels of use; the cost savings offset between % and % of the drug cost. cost-of-illness studies have been conducted in different countries; the economic estimates varied widely across studies due to differences in treatment patterns, health systems, methodologies, and patient populations. most cost-of-illness studies were retrospective observational studies from the perspective of a hospital or third-party payer, and the cost of cf ranged from $ , to $ , per patient per year. the largest cost categories included hospitalizations, out-patient visits, rhdnase and antibiotics, while disease severity and presence of pa infection were common determinants of cost. cost-of-illness studies have underestimated societal costs because they have rarely considered patient time costs and none have considered indirect costs (e.g., burden on lost productivity or informal caregivers). conclusions: studies show that inhaled tobramycin and rhdnase can partially offset medical costs; home-based iv antibiotic therapy is likely to reduce costs; and direct costs associated with cf can be high but vary widely across countries and analytical methodologies. areas for future research include direct comparisons of inhaled antibiotic therapies, examination of the relationship between treatment adherence and economic outcomes, and estimation of societal cost-of illness. this study was sponsored by novartis pharmaceuticals corporation, east hanover, nj. background: sputum interleukin (il ) and myeloperoxidase (mpo) are advocated as markers for infective exacerbations in cf. we have previously shown that the neutrophil protein, calprotectin, is a sputum biomarker of disease activity in cross sectional and longitudinal studies. in the present study we evaluate longitudinal measurement of sputum and serum calprotectin during infective exacerbation. methods: sputum and venous blood samples were taken at the beginning (visit ) and end of a course of iv antibiotics (visit ), and at time of clinical stability following the infective episode (visit ). processed sputum supernatant was analysed by elisa for calprotectin, il and mpo. serum calprotectin was assayed by elisa. results: paired sputum samples samples were available for visits and and paired samples for visits and . all data are presented as mean(sem). sputum calprotectin decreased from ( ) to ( ) µg/ml, p< . , from visit to . there was a non-significant increase to ( ) µg/ml at visit . sputum il did not change from visit to [ . ( . ) ( . ) ]. serum calprotectin decreased from ( . ) to . ( . ) ng/ml, p< . (n= paired samples) from visit to . at visit there was an increase to ( ) ng/ml, p< . (n= ). discussion: calprotectin appears a valuable marker both in sputum and serum for tracking changes in lung inflammation during an exacerbation of cf and may be more sensitive to change than il or mpo. on stopping antibiotic therapy sputum and serum calprotectin increase implying that this marker may be sensitive to small sub-clinical changes in lung inflammation. these data suggest that sputum and serum calprotectin might be employed to assess responses to drug therapies in cf such as gene therapy. this research was funded by the cf trust uk. chronic lung disorders are usually associated with a hypoxia driven increase in red cell mass. however, patients with cystic fibrosis often have normal or decreased haemoglobin levels. the present prospective observational study in cystic fibrosis (cf) patients was performed to determine which factors were involved in alterations in the hematopoetic response to corresponding arterial oxygen pressure. sixty adult patients (age - ) with stable cf were included. they all had vitamin a, d, e and k but no vitamin b supplementation. patients were on oral fe + ( mg/day). resting arterial blood gases, lung function, complete blood counts, parameters of iron status, crp, sputum microbiology and serum erythropoietin were measured at recruitment and after and months. patients had varying degrees of pulmonary functional impairment and % were hypoxemic (arterial oxygen pressure < mm hg). low-grade systemic inflammation (crp > . mg/dl) was present in % of the patients, who all had bacterial colonization. none of the patient had erythrocytosis and patients had anemia . there was no significant difference in iron status between patients with or without chronic iron supplementation and erythropoietin levels were normal. during the months observation period no significant changes occurred. the patients exhibited an impaired erythropoietic response to hypoxemia with normal or low hematocrit in spite of chronic lung disease. linear multivariate regression analysis (table) revealed crp and iron levels but neither iron substitution, nor erythropoietin levels nor lung function parameters as independent determinant of haemoglobin levels. cf may be associated with anemia of variable severity as expression of the chronic inflammation present in these patients. the therapeutic consequences are to treat the underlying inflammation rather than to supplement iron. variables included in initial model: background improvement in the treatment and survival of females with cf has led to many leading a normal adult lifestyle, and some can now expect to raise a family. however, pregnancy can have a profound impact on wellbeing, and caring for a young child can affect compliance with cf treatment. to look at this further, we surveyed the experience of pregnancy and motherhood in cf women attending our large adult cf centre ( patients). methods we reviewed the records of cf women who chose to remain pregnant ( babies [ male] from pregnancies), looking at changes in lung function, bmi, respiratory exacerbations during pregnancy, clinic attendance, and oral and iv antibiotic therapy (home and inpatient care) during pregnancy and also in the first year post delivery. we devised a questionnaire to examine how patients coped with pregnancy and the demands of motherhood, focussing on changes in compliance with therapy, worries regarding the effect of cf treatments on the baby, and support given by the cf team. results at conception mean fev was % predicted (range to ) and mean bmi (range to ).there were no miscarriages or neonatal mortalities and all children remain well, but the sickest mother died at years. mean fev following pregnancy was % [range to ] (p= . ), with mother losing % by one year post delivery. at one year, bmi was unchanged (mean , range to ). rates of respiratory exacerbations and iv antibiotics were similar pre and during pregnancy, although during the first year post delivery mothers avoided hospital based treatment preferring home care. six mothers' clinic attendances increased during and post pregnancy ( attended outpatients times in the first year post delivery). nine mothers worried about the effects of cf medication on their unborn child: the remaining mother was in her nd pregnancy. all managed their health differently during pregnancy: whilst increased compliance, worked harder to stay healthy, and increased clinic attendances; avoided clinic, postponed treatment, avoided any medication in the first trimester, and reduced oral antibiotic use to prevent harm to their unborn child. following delivery, mothers spent less time on their own health due to motherhood demands, and missed treatments during the first year: missed all except iv antibiotics. seven mothers ceased chest clearance and nebuliser therapy, and whilst missed oral therapies only avoided iv therapy. one mother who returned to work early felt this had contributed to her post natal depression. nine mothers indicated that cf team support was adequate, but the remaining mother felt that she should have been offered more frequent home visits post delivery due to lack of family support. conclusions the experience of pregnancy in our centre has positive outcomes for most mothers. however, compliance with treatment was affected and a number of mothers lost significant pulmonary function due to pregnancy. our study has shown that the demands of motherhood especially in the first year challenge the management of their own health. we offer a proactive service to women considering pregnancy, with early participation of the multidisciplinary team, and have increased home visit frequency to during the first year post-delivery. geller, d.e.; kesser, k.c. research, nemours children's clinic, orlando, fl, usa background: alpha- antiprotease (α- ap) is being developed as an inhaled drug in cf to inactivate excess elastase in the lungs. it is estimated that tens of milligrams of α- ap need to be deposited in the lungs to be effective. delivering a high dose with conventional jet nebulizers may be too time-consuming. we speculated that slowing and prolonging inspiration could optimize α- ap delivery. the i-neb® adaptive aerosol delivery (aad® system, respironics) is a portable, electronic, battery-powered, mesh nebulizer that uses the aad algorithms to deliver drug in the first portion of inspiration. interchangeable mouthpieces allow the i-neb to be used in a conventional tidal breathing mode (tbm) or in the target inhalation mode (tim) that guides the patient to inhale very slowly. the low residual volume after nebulization reduces drug waste and optimizes efficiency. we compared delivery efficiency of α- ap in vitro with the i-neb in the tbm vs. tim modes. methods: we studied new i-neb devices using tbm (tidal volume [vt] . l, respiratory rate [rr]= , i-time= seconds), tim- (vt . l, rr= , i-time= sec), and tim- (vt . l, rr= , i-time= sec) using a breath simulator. nebulizers were filled with . ml α- ap ( mg/ml). nebulized drug was captured on a filter and measured via spectrophotometer (inhaled dose), and particle size was measured with an insitec laser system. results: median particle sizes for the modes were similar ( . - . µm) . the dose emitted from the i-neb was very high, and similar between modes ( - % of nominal dose). predicted lung doses for these experiments were calculated from a previous scintigraphy study (nikander ) that showed % of the emitted dose deposited with tbm and % with tim. predicted lung dose of α- ap (as % of nominal) and delivery times were tbm: . % in . min, tim- : . % in . min, and tim- : . % in . min. discussion: the i-neb has a very high predicted lung dose with both tbm and tim. however, tim reduces the time of administration to as little as / that of tidal breathing. if larger predicted lung dose is necessary to see a clinical effect, either the initial drug volume or drug concentration may be increased. we conclude that slow, deep, controlled inspirations using i-neb is a very efficient method to deliver α- ap, and is faster than tidal breathing. we also speculate that tim has the additional potential advantage of better distribution of drug in the lungs because of slower flow rate and particle velocity. objective: early inflammation was observed in infants with cf regardless the occurrence of bacterial infection. lung function (lf) is a tool to assess bronchial inflammation consequences. few data are available on the pronostic value of impaired lf in infancy. methods: in infants, airflow obstruction (t ptef/te , curve shapes, tidal volume, functional residual capacity (frc, nitrogen washout technique) were assessed with the sensor medics and interpreted with stocks reference values. at y, fev and pulmonary flows (spiro , nova medical sc, zapletal reference values) and frc he , were measured. symptomatic children were defined when cough, wheeze or rattle were recurrent. all symptomatic infants have been treated with inhaled steroids as any infantile wheezer. results: screened infants ( ∆f /∆f , ∆f /other, other/other) were investigated at mean age mo and years. ( %) had respiratory symptoms (rs); the mean tidal volume was normal ( . ml/kg), ( %) had a marked obstructive curve shape, ( %) a decreased t ptef/te , and ( %) hyperinflation. then, ( %) had normal lf, ( %) ao, ( %) hyperinflation and ( %) both. at age , % had rs, and lf with mild alteration as mean flows ≥ %, and frc he = . %. accurate analyses showed that ( %) had normal lf, ( %) ao and ( %) small airway diseases (only distal ao and/or hyperinflation). frc in infancy and at age were correlated (r= . ; p= . ). conversely, no correlation between ao parameters in infancy and at age was found. the lf alteration was independent of the genotype, bacterial colonization and recurrent symptoms at both ages. conclusion: ao in infancy is frequent as found in half of the screened cf infants. the lack of correlation with the ao observed at age suggested that ao in infants might be due to a non specific inflammation (i.e viral), transient and then relevant from a treatment. hyperinflation, although mild, is conversely an early marker of the cf disease as persistent between infancy and age . we describe the efficacy of a novel system using an electronic real-time distant home monitoring of symptoms and spirometry in cf patients with the aim of early detection of p exs patients and methods: this is a month prospective observational study. all patients were provided with a set consisted of a mobile phone-palm pc (the x daii, o -uk) and with a spirometer (vitalograph) with an attachment to the pc. the pc contained a purpose-built software on which cf symptoms and spirometric values were recorded once daily. these were then sent on a real time to a computer server where the data were plotted on a time-magnitude graph. the site was accessed by a password-permitted cf physicians. patients were asked to score four symptoms (cough, sputum, breathlessness and fatigue) once a day and to record at least three attempts of spirometry. these were accepted by the software only if they met the ats criteria. preset criteria for p exs were stipulated. when the recordings met the criteria for p exs patients were invited to come to the cf clinic for review and were managed by either watchful waiting, oral or iv antibiotics. a prodrome phase is deemed to be present when a patient had an increase in one or more symptom for one week prior to the p ex. twenty one evaluable patients were included in the study. in ( %) there were an increase in one or more than one recorded symptoms throughout the study. a total of p exs median . per patient were detected within the months study period. in ( %) p exs, symptom increase and/or decline in fev met the preset criteria for p ex and in cases patients presented despite that symptom changes were not sufficient to meet the pre-set definition of p ex. prodromal phase prior to p exs were seen in ( %) patients. adherence and technical issues were the main two problems throughout the study. distant daily monitoring of patients symptom is easy and sensitive. it can be used to monitor symptoms and early detect p exs. the vast majority of our patients have an increase in their symptoms even when they perceive their disease to be stable. a prodormal phase preceded p ex is seen in the majority of patients. the pre-set criteria for defining p ex may have been too stringent. the research was funded by o telecommunication, uk the monitoring system used in the study. a combination of a mobile phone-palm computer (x daii, o uk) and a spirometer (vitalograph uk) .. the aim of this research was to examine whether systemic inflammation is present in adult patients with cf during their stable status. patients and methods: blood samples were obtained from a total of cf patients female, mean age , range( - ) males mean . range - and from control subjects, female, mean age . , range( - ) males,mean age . yrs, range( - ) all cf patients were included when they were at least weeks away from any pulmonary exacerbations. all control subjects were not known to have any pulmonary or systemic disease. venous blood samples were taken and spun at rpm.separated sera was aliquted and frozen at - degrees c. they were analysed in batches for cytokines il- b,il- ,il- , il- , ilp ,and tnf-alpa using a cba human inflammatory kit(bd bioscience). whitney-u test was used to compare values of serum cytokines between stable cf patients and control subjects.. serum il- and il- were raised in cf patients compared to control subjects. the mean value for il- was . pg/ml (sd . ) for patients and . pg/ml (sd . ) for controls, p< . . similarly, the mean value of serum il was . pg/ml (sd . ) in cf patients compared to . pg/ml (sd . ) in the control group, p< . (figure below). there was no difference between cf patients and control with regards to serum il- b, il- and il- p . conclusion: serum il- and il- were raised in cf patients compared to an agematched control subjects. inflammatory markers were raised in the sera of cf patients even during disease stability. even during disease stability, the lungs remain an inflamed organ which could explain the decline in lung function tests and the countinued daily systemic symptoms experienced by patients with cf. serum il and il in cf patients with stable disease compared to the control group (please see abstract body) we have shown that even during disease stability, an increase in inflammatory markers are present in the sera of cf patients compared to an agematched control group. this longitudinal study examines changes in serum inflammatory markers during p exs. we investigated cf patients female, mean age , range - years. their mean baseline fev was . % of predicted (range . - %). a baseline venous blood sample was withdrawn and sera were stored at - degree c from patients during disease stability. out of those there experienced p exs. blood samples were obtained at the beginning and at the end of the p exs.the following inflammatory markers were analysed: white blood counts, c -reactive protein (crp), il- , il- , il- , il- b, il- p and tnf-α. patients recorded their symptoms and their fev on a daily basis on an electronic diary and the data were beamed daily to a research centre where data were analysed. p ex was defined as ). an increase in two symptoms for successive days, or ). an increase in symptoms and % reduced fev from baseline over successive days or ). % decline in fev over successive days or ).when a patient felt that he/she was going through a p ex. paired t tests were used to analyse changes in inflammatory markers. there was no change in the number of circulating neutrophils, lymphocytes or eosinophiles at the start compared with the end of p ex. there was an increase in mean crp values at the start of p ex and reduction at the end of p ex, but this did not attain statistical significance. mean (sd) of serum il increased from . pg/ml ( . ) at baseline to . pg/ml ( . ) at the start of p ex (p= . ) and returned to base line . pg/ml ( . ) at the end of p exs (p= . compared to values at the start of p ex). (figure ) there were no changes in the values of serum il- , il- , il- b, il p or tnf-α at the start and at the end of p exs. serum il increased at the start of p ex and returned at the baseline at the end of p ex. crp was the closest inflammatory marker to follow that pattern. none of the other inflammatory markers changed with the diagnosis of p ex's. this and other studies call for the price of analysis of il- to be reduced to become available in routine clinical practice. background. abpa can be a therapeutic challenge in children with cf. aim was to describe the tolerability of voriconazole and nebulised liposomal amphotericin in the treatment of abpa in children with cf. methods. we performed a retrospective case note review of voriconazole and nebulised liposomal amphotericin use, in the treatment of abpa in children with cf, at our centre. results. children diagnosed with abpa according to the current criteria, failed to improve despite previous treatment with itraconazole and steroids, and received voriconazole as a second line agent. the dose of voriconazole was as recommended by the manufacturer's summary of product characteristics and the duration of treatment was two weeks. significant side effects noted in children. observed side effects included visual hallucinations (n= ), photopsia (n= ), sleep disturbances (n= ), severe headache (n= ), influenza like symptoms (n= ), gastrointestinal side effects (n= ) and elevation of alkaline phosphatase (n= ). of these children, one child tolerated voriconazole when concomitant treatment with omeprazole was withdrawn. a second course of voriconazole, given at a lower dose and without the loading dose was tolerated by two children. voriconazole blood levels were not measured in any patient. we used nebulised liposomal amphotericin in of these children. one child received amphotericin as a second line agent when voriconazole had to be stopped because of significant side effects. children had active abpa despite treatment with first line (itraconazole and steroids) and second line (voriconazole) agents and received amphotericin as a third line agent. amphotericin was used at a starting dose of mg nebulised twice daily, with weekly increments as tolerated, up to a maximum dose of mg twice daily for four weeks. the starting dose and all subsequent increments in dose were supervised. all children received salbutamol immediately before nebulised amphotericin. the standard formulation available for intravenous infusion was made up into single doses for nebulisation by the pharmacy. this was stored at c and diluted with water for injection for use with the nebuliser. the only side effect, experienced by all children was a dry irritant cough at the start of nebulisation that settled soon without need for discontinuation of the medication. improvement in clinical symptoms, lung function and total serum ige was noted in all children. the improvement in fev and fvc, expressed as mean (range) of percentage predicted were and respectively. the mean (range) fall in serum total ige and aspergillus specific ige were ku/l ( - ) and mg/l ( - ) respectively. conclusion. voriconazole use was associated with significant side effects. avoidance of drugs that may potentially result in interaction is important. omitting a loading dose and using a lower maintenance dose may help selected patients tolerate voriconazole. nebulised liposomal amphotericin was tolerated by our children with cf and abpa who did not tolerate or responded poorly to voriconazole. recent studies have shown that high resolution computed tomography (hrct) is a more accurate method of evaluating early lung disease in cystic fibrosis (cf) than pulmonary function tests (pfts) or chest x-rays. since young children can not perform the breathing maneuvers required for high quality hrct, lung volume control is necessary. positive pressure facemask ventilation using chloral hydrate allows full inspiration and end exhalation images, but requires special expertise. controlled ventilation can also be produced with general anesthesia (ga). ga has not been reported in children undergoing hrct for cf research. the advantages of ga over sedation include rapid onset, faster recovery and reliability of scan completion. the undesirable effects of sedation are unpredictability and the higher failure rates. additionally, failed sedation contributed significantly to parental dissatisfaction. (malviya, et al., ) . the primary outcome of our study was to examine the safety of using ga for hrct scans and the quality of images obtained. a secondary outcome was parental satisfaction. methods: participants were recruited from a larger nih/cffti funded multi-site randomized clinical trial focusing on behavior and nutrition treatment to improve growth in preschoolers with cf. hrct in subjects to years old (mean age ± months) was performed with ga. parents accompanied the child during induction and left after the child was asleep prior to the intravenous (iv) being placed. after induction of anesthesia, a proseal®, specialized laryngeal mask airway (lma), was placed which allowed ventilation with higher pressures and the ability to suction the stomach. volumetric thin section ct scans were obtained. ga using inhalation (sevoflurane) and/or iv (propofol) agents was used and out of families who were eligible ( % recruitment rate) participated in the sub-study. results: the quality of the hrct scans in all subjects were optimal. there were no anesthetic complications upon emergence and none of the patients experienced any side effects after follow-up phone calls at hours. parents also expressed that the clinical interpretation of the hrct scan provided beneficial information about their child's current lung disease status. the mean time from completion of scan to discharge was . ± minutes. conclusions: the use of ga in radiology departments is becoming more common in pediatric centers. our initial findings of this study demonstrate that ga can be safely administered for children with cf. in addition to very good quality scans being obtained, parental satisfaction was also noted. the use of the proseal® lma for hrct may improve the image quality and aid in the diagnosis of early lung disease in preschoolers with cf. supported by nih grant r dk and a cf foundation clinical research grant. whey protein is rich in sulfhydryl (sh) groups and is recognized for its ability to increase glutathione and reduce oxidative stress. hyperbaric pressure treatment of whey protein increases its digestibility, promotes the release of novel peptides for absorption, increases intracellular glutathione in healthy subjects (zavorsky et al, int j vit nutr res, ) , and reduces in vitro production of il- (vilela et al, inter immnuopharmacol, ) . we present here the initial results of a pilot study of supplementation with pressurized whey in cf patients. children and adults with cf were supplemented with hyperbaric whey protein for month ( gm/day in patients less than years of age, gm/day in older patients). anthropometric measures, pulmonary function, serum c-reactive protein (crp) and plasma lipid peroxides were measured before and after supplementation. the results of the first patients to have completed the study are reported, with further data to be presented. in / subjects weight increased and in / subjects lean body mass increased. there were no significant changes in fev or fev/fvc ratio. in / patients the crp fell ( / by more than %) and in / the crp increased. in the remaining two patients, the crp was undetectable at baseline and did not change at follow-up. plasma lipid peroxides decreased in the three patients in whom it could be measured ( / to undetectable levels). these preliminary results suggest that supplementation with pressurized whey protein can reduce systemic inflammation in cf patients and has the potential to beneficially modulate the inflammatory response in cf patients. supported by the breathe initiative of the canadian cf foundation with pseudomonas aeruginosa. soon after its introduction, the mesh technology based pari eflow® nebulizer gained preference over jet nebulization in the cf community because of its short nebulization time, portability and noiseless operation. concerns were raised about the durability of the device due to expected vulnerability of the mesh. the aim of this study was to compare the particle size distributions in the aerosol, delivered doses and output rates from the eflow and the pari lc plus®(lc+)with paritur-boboy compressor before and after use in daily practice. lc+ nebulizers in use by the adult patients taking part in the study were taken in for testing and new eflows, cleaning instructions and a disinfection device were provided. after months use ( cycles of x doses tsi: mg/ ml), the eflows were collected by the local cf center. particle size distributions were measured at a constant flow rate of l/min with a laser diffraction apparatus. because of uncertainty regarding the cleaning procedure after last use, additional e-flows were collected from the same patients after another period of months use. conclusions: new eflows produce slightly larger droplets in a narrower size range than new lc+ nebulizers. after use in daily practice, droplet size distribution changes for both devices. output rate (delivered dose per minute) decreases for both devices. for used eflows delivered dose is lower than for used lc+ nebulizers, because the eflow is programmed to switch off after min, even when nebulization is still incomplete ( out of tested eflows). this may result in a considerable reduction of delivered dose. however, when a mesh is replaced, performance is the same as that of a new device, suggesting that the changes in performance originate in the vibrating mesh. for daily practice these results indicate: ( ) the performance of both nebulizers changes during use but the changes are smaller for eflow than for lc+ ( ) changes in droplet size distribution are relatively small for both devices;in contrast with output rate and delivered dose ( ) proper functioning of eflow can be improved by timely replacement of the mesh, ultimately when eflow switches off after minutes. without replacement, the delivered dose is unpredictable. mean values for size distribution, delivered dose, nebulization time and output rate (x is volume median diameter) backgrounds: lung surfactant protein a (sp-a) belongs to the family of collagen containing, c-type lectins, called collectins. sp-a plays a critical role in the innate immune defense of the lung. sp-a binds calcium dependent to carbohydrate structures on pathogens via its carbohydrate-binding domain (crd). cystic fibrosis (cf) lung disease is characterized by chronic inflammatory and proteolytic processes in the airways, leading to destruction of the lungs and early death. aims: we hypothesized that sp-a might have a reduced function in cf. this might result from sp-a degradation by neutrophil proteases, an altered macromolecular organization of sp-a, or inhibition of crd-binding by airway components. methods: we studied bronchoalveolar lavage fluid (balf) from clinically stable patients with cf and from children suffering from bronchitis. balf samples were purified by carbohydrate-binding assay on fucose attached to sepharose resin with and without calcium. in the absence of calcium sp-a cannot bind via its crd. each fraction was analysed for sp-a and igg by immunoblotting. macromolecular organization of sp-a was determined by gel chromatography. results: only % of total sp-a of cf balf bound to the fucose column, whereas % bound from the bronchitis balf. the structural organization of the sp-a oligomers did not differ between the non-binding and the binding fractions of both groups. of the cf balf had sp-a at kd that did not bind to fucose. these balfs had a high percentage of neutrophils (> %). in vitro human leucocyte elastase decreased sp-a to less than % of its initial levels and sp-a fragments at kd were detected. conclusions: neutrophil proteases degrade sp-a in cf lungs. the carbohydrate dependent binding of sp-a is diminished due to a reduced function of the crd. these alterations contribute to the reduced ability in cf to remove specific pathogens from the lungs. chiron, r. , ; murris-espin, m. , ; crampette, l. , ; varrin, m. ; didier, a. , ; wallaert, b. , ; chanez, p. , ; leroy, s. , . crcm, chu, montpellier, france; . crcm, chu, toulouse, france; . orl, chu, montpellier, france; . iurc, chu, montpellier, france; . crcm, chu, lille, france; montpellier, france in cystic fibrosis (cf) patients, upper airways involvement is frequent but its impact and relationships on the course and severity of the disease has been rarely investigated. objective: to relate upper airways involvement with lower airways parameters and quality of life in cf patients. methods: clinical and radiological prospective study of subjects with cf at french cf centres. clinical rhino-sinusitis (rs) was defined by the presence of more than episodes of nasal symptoms during the last year, current nasal obstruction or rhinorrhea. nasal investigations included: anterior rhinoscopy, sinus ct scan analysis and rs quality of life questionnaire. lower airways were assessed using a thoracic ct scan (bhalla score), spirometry and sputum culture and a cf quality of life questionnaire (cfq +). results: ( f/ m) patients aged . ( - ) years old were enrolled. endoscopy demonstrated polyposis in % and nasal hyper secretions in . % of the patients. spirometry, bhalla score, sputum colonization did not discriminate patients with (n= ) or without rs (n= ), and regardless the presence of polyps. cfq + was significantly lower in patients with of rs (p= . ). conclusions: rs involvement altered quality of life in cf patients, although, they do not have a more severe pulmonary disease. a specific attention and treatment to rs should be paid in cf patients. the impact on different outcomes deserves to be examined in longitudinal studies. as part of the uk cystic fibrosis gene therapy consortium "tracking study", ebc was measured (ecoscreen, jaeger) in adult and paediatric patients presenting with an infective exacerbation requiring iv antibiotics. exacerbation was defined by pre-determined criteria. ebc was repeated at the end of parenteral antibiotic therapy and again - weeks later. patients were recruited at three sites and assessed using standardised equipment. samples were obtained according to ers guidelines. exhaled breath ph was assayed (shindengen ph meter, camlab, uk) immediately after collection without a de-aeration step. results: paired ebc measurements at the start and end of antibiotic therapy were available in patients. mean age was yrs (range - ). data from patients were excluded because at least one ph measurement was > , suggesting salivary contamination. for the remaining patients mean ebc (sd) ph increased from . ( . ) to . ( . ), p< . . of these patients, also had measurements available at a third visit, - weeks after completion of antibiotics. were excluded because of salivary contamination. no difference in mean(sd) ebc ph was found ( . ( . ) vs. . ( . )) conclusions: ebc ph increases after treatment with antibiotics, and may offer a non-invasive means of assessing airways inflammation in chest exacerbations. further work is required to follow the longitudinal change in ebc ph and other inflammatory markers in clinically stable patients, as ebc may be a useful tool in assessing response to novel treatments, such as gene therapy. this work was funded by the cystic fibrosis trust. background: children with cystic fibrosis (cf) undergo multiple procedures exposing them to ionizing radiation in hospital. some specialist centres have advocated regular chest computed tomography (ct) as a means of better quantifying lung pathology. the clinical benefit of this approach is unproven and previous calculations of risk have failed to account for other ionizing radiation exposure procedures . aim: to quantify the exposure of ionizing radiation in the pediatric cf population of a tertiary level children's hospital. to determine the potential difference in exposure of introducing biannual chest ct scans. methods: effective doses of ionizing radiation from chest (cxr) and abdominal (axr) radiographs, fluoroscopy and cts (sinuses and chest including hr scans) were determined for cf children at our centre between st january and th july ( weeks). the maximal effect was tested i.e. introduction of biannual chest ct scans from age years abolishing the requirement for cxrs and the assumption was made that axrs, sinus cts and fluoroscopy rates were unchanged (median exposure values calculated for age groups -< year, -< year, -< year, -< year, -< year, > year) . results: cf patients (n= , gender ratio : ) underwent a mean of . ionizing radiation exposure procedures per patient over the study period (total of procedures). the average effective ionizing radiation doses (msv) were calculated for ( . %) procedures. each cf child received a mean (median) effective radiation dose of . ( . ) msv/year with a range of . - . msv/year. the reason for the skewed distribution was identified by analysing the proportions of the procedures to the total effective dose in this population: fluoroscopy . %, chest ct . %, sinus ct . %, cxrs . %, axrs . % and hrct . %. it was calculated that these patients undergo . cxrs per patient per year. we calculated that an average patient diagnosed by newborn screening could expect to receive . msv from ionizing radiation procedures between birth and age . biannual ct chest scans in addition to median exposures for each age group from axrs, sinus cts and fluoroscopy would potentially result in . msv by age , an fold increase. discussion: there is a in lifetime risk of developing a solid or haematological cancer with each exposure to msv of ionizing radiation . although fluoroscopy forms the most significant contribution to a pediatric cf patient's ionizing radiation exposure in our centre, introducing biannual chest cts would markedly increase exposure. as cf survival improves, cf physicians must be cognisant of the potential health cost of survival. references : previous studies have used generic measures of hrqol, which are not sensitive to important changes that may occur as a result of treatment. this study assesses the impact of iv antibiotic treatment for a pulmonary exacerbation on both generic and disease-specific hrqol of children and adolescents with cf. method: participants included patients (m age = . years) from the cincinnati children's hospital and university of florida cf centers. fiftyfour percent of the sample were male. patients completed two hrqol measures, the pedsql™ (generic) and cfq-r child or teen/adult version (cf-specific), which have excellent reliability and validity. these measures were completed within hours of hospital admission (pre) and hours of discontinuation of iv antibiotics (post). note that approximately % of patients went home on iv antibiotics and % remained in the hospital for approximately weeks. results: significant improvements were found in pulmonary functioning (fev % predicted) pre to post iv antibiotic treatment (paired t ( ) = - . , p < . ), with an average of % increase in fev % predicted. paired t-tests were conducted to examine changes in hrqol scores, using holm's procedure to correct for multiple comparisons. significant improvements were found on the cfq-r respiratory (paired t ( ) = - . , p < . ) and weight scales (paired t ( ) = - . , p < . ). trends were also noted for improved emotional functioning (paired t ( ) = - . , p = . ) and vitality (paired t ( ) = - . , p = . ), and worse treatment burden (paired t ( ) = . , p = . ). on the pedsql, only emotional functioning improved significantly (paired t ( ) = - . , p < . ). conclusions: results of this study confirm the effectiveness of iv antibiotics for the treatment of pulmonary exacerbations, with significant improvements found for both pulmonary functioning and hrqol. these results also highlighted the importance of using a disease-specific hrqol instrument, which proved to be more sensitive than the generic measure. on the cfq-r, significant improvements were observed in respiratory symptoms and weight, aspects of functioning that are not assessed on generic measures. fukushima, l.k.; hsu, e.; woo, m.s. division of pediatric pulmonology, childrens hospital los angeles, los angeles, ca, usa do hispanic and caucasian cf patients share the same frequency of pulmonary exacerbations? increased frequency of pulmonary exacerbations is linked to increased risk for cf morbidity and mortality. we have previously reported that pediatric hispanic cf patients have greater mortality than our caucasian cf patients who attend the same accredited cf center. we performed a retrospective review of cf pulmonary exacerbations (defined as physician prescription of intravenous or oral antibiotic therapy) in our cf patients who routinely attended the cf clinic during the period of january , through december , . data collected included demographics, number of intravenous antibiotic courses and number of oral antibiotic courses. unpaired student's t-test was used to compare age of the groups and chi-square was used to compare use of intravenous and oral antibiotics between hispanic and caucasian cf patients as well as between males and females. during the study period, there was a total of cf patients ( males: females; mean age . ± . years). of the patients, ( %) were classified as hispanic ( males: females; mean age . ± . years) and ( %)were classified as caucasian ( males: females; mean age . ± . years; p= . , ns). hispanic cf patients ( patients had episodes; males: females) had significantly greater number of pulmonary exacerbations (received treatment with intravenous and/or oral antibiotics) than caucasian cf patients ( patients had episodes; p = . ). hispanic cf patients who required intravenous antibiotic therapy were significantly younger ( . ± . years vs . ± . years; p< . ) than the caucasian cf patients who were treated with intravenous antibiotics. there was no significant difference in age between hispanic and caucasian cf patients who received oral antibiotic courses ( . ± . years vs . ± . years; p= . , ns). gender did not have a significant impact on pulmonary exacerbation risk in our population (hispanic iv use by gender p= . ; hispanic oral use by gender p= . ; caucasian iv use by gender p= . ; caucasian oral use by gender p= . ). we conclude that hispanic cf patients had increased incidence of pulmonary exacerbations than caucasian cf patients who attend the same cf clinic. hispanic patients who were treated with intravenous antibiotics were younger than the caucasian cf patients. we speculate that hispanic ethnicity has a major impact on the incidence of pulmonary exacerbations. other factors, such as modifier genes, environmental exposures, or inflammatory responses, may play a role in the increased risk of pulmonary exacerbations in the hispanic cf population. introduction: one of the main goals of the multidisciplinary team of the cf association of minas gerais, brazil is to find strategies to preserve lung tissue from the bacterial exacerbation in cystic fibrosis patients. objective: to investigate clues on onset of bacterial exacerbation. to our knowledge, no studies were found regarding the identification of the early warning signs of bacterial exacerbation with cf patients. we wanted to know whether cf patients present early warning signs before onset of fever and/or breathing difficulties. methods: telephone interviews were carried out with the cf patients registered in the association, by a physiotherapist academic who did not know the patients and just ask two questions. the first question was about the use of antibiotics during the year of and the second was related to early signs before the onset of fever. results: from the patients, only patients replied. patients who were using antibiotics therapy during and had fever: / patients who were using antibiotics and did not have fever: / patients who had fever and did not use antibiotics: / patients who did not have fever and did not use antibiotics: / the results revealed the following warning signs and also that some patients had more than one sign. tiredness, quietness, slowness ( / ); increased cough ( / ); lack of appetite ( / ); sticky sputum ( / ); sleepiness ( / ); headache ( / ); increased excitement, nervousness ( / ); crybaby ( / ); dry mouth ( / ); clammy ( / ); difficult to walk ( / ); gastric upset ( / ); itchy throat ( / ); abdominal pain ( / ) ; itchy eyes ( / ); breathing difficulties ( / ); tremulous ( / ); paleness ( / ); burning ear ( / ); chest pain ( / ); hissing ( / ) ; and syncope( / ). conclusions: it is helpful to know these early warning sings. we assume that the earlier the diagnosis of the exacerbation is made, the easier it is to resolve the problem and the less destruction of the lung tissues. reference: lung line, national jewish center for immunology and respiratory medicine . background: cystic fibrosis patients are experiencing increasing survival and are being treated with more chronic therapies. how these changes are reflected in day to day clinical symptoms has not been evaluated. objective: we examined data from the epidemiologic study of cystic fibrosis, a large longitudinal observational study, to characterize the change in respiratory symptoms over time. for each year between and data from patients with at least one visit recorded as occurring while clinically stable were included. data from all visits, including sick as well as stable, were used for each year in which at least one clinically stable visit was recorded. patients were separated into age groups less than , to , to , and or older. note that in data collection changed, potentially confounding the results. results: the average number of patients per year was . the percent of patients with no reported cough progressively increased over time (table) . results were similar for pateints with no reported sputum production at clinically stable visits. when sick visits were added there were similar results. the frequency of cough reported at greater than percent of visits progressively decreased over time (table) . again the results for sputum production were similar. these findings were seen in all age groups although older patients are more likely to experience these symptoms. conclusions: these data suggest that cf patients are experiencing fewer respiratory symptoms of cough and sputum production over the last decade. this improvement has occurred in patients with either intermittent or chronic symptoms. how clinical care has impacted on these symptoms has yet to be determined. also, more people are being diagnosed with cf at an earlier age or with milder disease, potentially resulting in the entire cf population having fewer symptoms. lung disease in children with cf begins in early childhood, with intermittent and then chronic infection, associated with an exaggerated inflammatory response. the extent to which inflammation contributes to bronchiectasis has not yet been identified. the aim of this study was examine relationships between inflammation and early lung damage methods: twenty eight children with cf were assessed at diagnosis and annually thereafter with a bronchoalveolar lavage (bal). bal fluid was used to assess micro-organisms and inflammation, including total and differential cell counts. a three slice hrct technique (inspiration and expiration) was performed under general anesthesia at age - y, immediately preceding the annual bal. hrct scans were scored by an independent and experienced radiologist using a modification of the brody score. results. twelve children ( %) had evidence of bronchiectasis on hrct. inflammation present in bal at months of age predicted bronchiectasis at - y. total cell count at mo, predominantly neutrophils, was higher in those who developed bronchiectasis compared to those who did not ( . vs . x cells/ml bal; p= . ). the total number of infections detected in bal was not related to bronchectasis. however, children with bronchiectasis had a higher incidence of pseudomonas aeruginosa within the first years of life ( % vs. %). conclusions: bronchiectasis is evident within the first five years of life and appears to be associated with an exaggerated inflammatory response and the early acquisition of pseudomonas aeruginosa. supported by: cystic fibrosis foundation (usa), national health and medical research council (australia). fortunately the survival of patients with cystic fibrosis has improved remarkably over the last few decades. many patients can life quite "normal". therefore family planning becomes more important. several reports docu-mented the good maternal and fetal outcome in pregnant women with cystic fibrosis. besides the data from the french cf registry no longterm data about the impact of paternity of male patients with cystic fibrosis are available until now. in a retrospective analysis we gathered data about all our male patients who became father while being followed-up in our cystic fibrosis center for adults. from male patients, patients became father between and . patient no. became father twice (second time father of twins); patients no. became also father of twins .we looked at fev % year prior to childbirth, perinatal and and years after the childbirth. for the fev % one year prior to birth as well as for fev % and years after birth, we calculated the difference to the fev % at time of birth. between and , male adult patients with cystic fibrosis became fathers of a total of children, all by the assistance of reproductive techniques. their fev % are shown in table . although the male patients are not directly influenced by a pregenancy (as the pregnant women are by growth of the uterus), there seems to be more than ususal differences in this lung function parameter. there were also more fluctuations in fev per year. the biggest differences in fev % were seen year after birth of their children. further investigations in a larger group (in cooperation with other centers) are planned; especially with a focus on the frequency of infections and quality of life compared to male patients without children. intravenous (i.v) aminoglycosides are widely used in cystic fibrosis (cf) patients as treatment for pulmonary exacerbations. in an effort to reduce undesiderable effects of these antibiotics it is recommended to measure drug serum levels. recent experience suggests that tobramycin (to) may be administered as a single daily dose with equal efficacy as multiple doses but less risk of nephrotoxicity. aim: we evaluated the efficacy of once-daily i.v to treatment to improve pulmonary function expressed as forced expiratory volume in one second (fev ) in cf patients with pulmonary exacerbations compared to the same group of patients who were previously treated with to in multiple daily i.v doses, as historic control. we evaluated baseline and peak drug serum levels of to administered once daily compared to multiple dose levels. methods: all patients were treated with i.v to in combination with a beta-lactam antibiotic for two weeks. respiratory exacerbation was defined according to cff criteria. all cf patients were colonized by non-fermentative gram-negative bacteria. serum concentrations of to were measured (fluorescent polarisation immunoassays) before the fifth infusion (basal) and minutes after the fifth infusion (peak). reference to blood levels were considered to be ≤ mg/l (basal) and < mg/l (peak) for multiple daily doses, and ≤ mg/l (basal) and - mg/l (peak) for single daily dose. treatment efficacy was evaluated on the basis of improved clinical condition and increased fev values compared to the beginning of therapy. results: patients with respiratory exacerbation were evaluated, with a mean (± ds) age of . years (± . ; median . , range . - . ) . out of patients were chronically colonized by pseudomonas aeruginosa. the total mean (± sd) daily dose of to administered in multiple doses was (± . ) mg whereas the mean single dose was . (± . ) mg. of those patients given multiple doses of to, only one patient ( . %) had basal to values outside the range and patients ( . %) had peak values outside the range. of those patients given a single daily dose, no patients had basal to values outside the range whereas out of ( %) had peak values of less than mg/l and patients out of ( %) had peaks between and mg/l. the mean (± sd) increase in fev was . % (± . ) in patients treated with a single dose and % (± . ) in those treated with multiple doses. the mean (± sd) period between fev measurements before and after exacerbation was . days (± . , median , range - ) in case of monodose therapy, and . (± . , median , range - ) in case of therapy with multiple doses. conclusions: both single and multiple-dose i.v to therapy caused a comparable fev increase and were effective in resolving the respiratory exacerbation. although clinical improvement was observed, the mean i.v to dose of mg/day in patients treated with once-daily i.v to determined peak values between and mg/l in % of subjects. intravenously administered aminoglycosides are efficacious for the treatment of pulmonary infections of cystic fibrosis (cf) patients, with the principal adverse effects of these molecules being acoustic nerve damage and nephropathy. damage to the eighth cranial nerve varies from . to % in cf patients treated with multiple daily doses of aminoglycosides. recent experience suggests that tobramycin can be administered as a single daily dose, resulting in less nephrotoxicity but equal efficacy. patients and methods: we evaluated the prevalence of oto-and nephrotoxicity due to aminoglycosides administered to cf patients followed at the tuscan regional center where we are currently following patients. cochlear damage was evaluated using tonal audiometry (amplifon amplaid s). renal damage was evaluated by measuring patients' creatinine clearance. results: ( %) patients ( males and females) between the ages of and (mean age . years, median , sd ) were tested with audiometry. all these patients had been repeatedly treated with multiple daily doses of intravenous aminoglycosides. ( . %) ( males and females) out of patients between and years (mean age . , median . , sd . ) had their creatinine clearance measured. we found that patients ( . %) had an abnormal audiometry, typically attributable to aminoglycoside ototoxicity (high frequency deficit). two patients required a hearing aid. one patient with normal cochlear function had labyrinth deficit. one out of patients tested had pathological creatinine clearance values. conclusions: our data indicate that ( . %) of our patients had damage to the eighth cranial nerve due to repeated multiple daily doses of aminoglycosides. we recommend that cf patients under aminoglycoside therapy be routinely given audiometric and creatinine clearance exams in an attempt to reduce the incidence of undesirable side effects of these drugs. davies, j. , ; voase, n. , ; dewar, m. , ; mullard, k. , ; gammie, f. , ; saunders, c. , ; horsley, a. , ; gray, r. , ; macleod, k. , ; somerton, l. , ; higgins, t. , ; donovan, j. , ; cornish, n. , ; hansell, d. , ; aziz, z. , ; ashby, d. ; geddes, d. , ; greening, a. , ; cunningham, s. , ; innes, a. , ; alton, e. , . gene therapy, imperial college, london, united kingdom; . western general hospital, edinburgh, united kingdom; . royal hospital for sick children, edinburgh, united kingdom; . royal brompton hospital, london, united kingdom; . queen mary, university of london, london, united kingdom; . uk cf gene therapy consortium, edinburgh, london, oxford, united kingdom the improving clinical status of patients with cf and the slow rate of decline of conventional outcome measures such as fev necessitate the development of clinically-relevant surrogate outcome assays for use in clinical trials. in our forthcoming clinical trial of cftr gene therapy, the uk cf gene therapy consortium will use both established, clinically-available assays and more novel measures designed specifically for this purpose. both groups of assays are being subjected to rigorous testing prior to use to establish their utility as disease biomarkers. in this study, we examined their performance in the context of an infective exacerbation treated with iv antibiotics. this abstract will report the response of established, clinically-available assays; available data from novel assays will be reported separately. children ( years and above) and adults with cf were recruited from three centres at the time of a clinician-defined infective exacerbation requiring iv antibiotic treatment. exclusion criteria included fev < % predicted and requirement for supplemental oxygen. a panel of assays was performed at the start and end of treatment, which was most commonly days. data are presented as mean (sd) or median (range) and differences compared with either parametric or non-parametric paired analysis as appropriate. clinical assays included spirometry, sputum microbiology, sputum cell count and differential, serum inflammatory markers (crp, white blood cell (wbc) count,) and chest ct. patients also completed a symptom score sheet. to date, patients (mean age . [ . ] years, male) have paired data available from the start and end of the course of ivs. at baseline, were infected with p. aeruginosa, with b. cepacia complex organisms and with s. aureus ( mrsa). significant changes from baseline were observed in fev ( . [ . ] the clinical response to any novel intervention, for example, cftr gene therapy is difficult to predict. prior testing of experimental assays in a study such as this provides data on the variability of the measurements within the disease population and the degree of change observed with an intervention known to lead to clinical benefit. this should aid the design of rational, powered clinical trials. funded by the cf trust influenza can worsen cf and lead to serious complications and mortality. influenza vaccine is safe and effective in cf patients. in france, annual influenza vaccination is recommended for all cf patients aged over months and for healthcare workers in regular and prolonged contact with high-risk patients. objective: to estimate the influenza vaccination coverage rate for - season in cf patients and healthcare workers in cf care centres of the great south region of france. methods: a multicentric observational study between february and september was conducted. healthcare workers were contacted by telephone and were questioned about their - influenza vaccination status, and the reason for not getting vaccinated. for patients aged over months whose vaccination booklet was available, the physician filled in a written questionnaire. results: a total of professionals were interviewed. the survey included . % of doctors, . % of nurses, . % of dieticians, . % of physiotherapists, . % of social workers and . % of psychologists. the overall influenza vc rate was . % and varied considerably according to profession: . % of doctors, . % of physiotherapists, . % of dieticians, % of psychologists, . % of nurses and . % of social workers. the overall influenza vc rate in cf patients was . % ( . % in children (age < years old) versus . % in adults; p< . ). receiving a voucher for free vaccination from the national insurance increased the influenza vc rate. for healthcare workers, the main reason for non vaccination was that flu was considered as a benign disease (useless vaccine); for patients, it was a lack of time. conclusion methods: adult patients with cf were studied. patients completed a standardised questionnaire. the questionnaire identified which changes and duration of symptoms would cause them to contact the cf team and/or change treatment. results: all patients would contact the cf team for haemoptysis but % would wait for more than hours and % would wait for more than hours before doing so. up to % of patients would wait a week or more before contacting the cf team because of sputum purulence. % of patients would not contact the cf team if they developed new chest pain. conclusion: there appears to be a disconnect between recognizing a change in symptoms and the length of time before acting on the change. this has major implications for the delivery of cf care. supported by cystic fibrosis association of ireland. aim: to determine the efficacy of standardized desensitization protocols in treating antibiotic allergies in adults with cystic fibrosis (cf). background: dependence on high dose and multiple combination intravenous (iv) antibiotics to treat pulmonary exacerbations in adult cf patients has resulted in an increased frequency of allergic reactions when compared with the general population. ( , ) strategies to address these allergies are essential to maintain effective antibiotic treatment in this population. antibiotic desensitization is the process by which one induces immune tolerance by incremental exposure to the antibiotic, which may induce stabilization of mast cells. the mechanism remains undetermined but may be mediated via ige or memory t-cells. prior to the study period, standardized antibiotic desensitization protocols were developed by an allergist/immunologist based on published reports and were approved through the hospital's pharmacy and therapeutics committee. methods: the toronto cf database was accessed to generate a list of hospital admissions for iv antibiotics during the -year study period ( ) ( ) ( ) ( ) ( ) ( ) . patients who underwent desensitization were identified and each of their case notes underwent retrospective analysis. data were collected with respect to age, organism requiring antibiotic therapy, antibiotic allergy requiring desensitization, reactions during desensitization, reactions post desensitization, treatments required to treat these reactions, and the completeness of antibiotic therapy post desensitization. statistical analyses were performed using graph pad prism®. results: during the study period the cf unit dispensed and initiated approximately courses of iv antibiotics. a total of desensitizations were performed in patients ( female). although some patients were desensitized to only one drug or to the same drug more than once, there were patient-drug combinations with a median of (+/= . ) unique combinations per patient. of the desensitizations protocols initiated, patients developed documented reaction to the antibiotic during desensitization however only desensitization protocol was not completed due to an adverse event. there were no episodes of anaphylaxis either during or after desensitization. a total of patients developed documented reactions during the subsequent antibiotic course - required termination of the antibiotic and completed the course of iv antibiotics with management of allergic type reactions with antihistamines. conclusions: standardized desensitization protocols were initiated in patients during the study period. the success rate for desensitizations in these patients was % however when the data is analyzed for true allergic phenomenon, the success rate rises to %. this demonstrates the efficacy of these standardized protocols in the treatment of ige-mediated allergic reactions to antibiotics in the adult cf patient. references: . burrows, j., toon, m., bell, s., ( ) . antibiotic desensitization in adults with cystic fibrosis. respirology, : - . . ramesh, s. ( ) . antibiotic hypersensitivity in patients with cf. clin rev allerg immun, : - . surgery for symptomatic disease. there is a long-held suspicion that poorly-controlled chronic sinusitis negatively impacts the respiratory status of cf patients, but whether sinus surgery improves the clinical course of cf lung disease is unclear. aim: assess the impact of functional endoscopic sinus surgery (fess) on respiratory exacerbation rate and lung function in adult cf patients methods: this is a single-center, retrospective study based on chart review. during the study period, - , adult patients underwent separate fess procedures. this abstract contains results so far from the analysis of patients that underwent a total of surgeries. primary outcomes were -year change pre to post fess in pulmonary function parameters and pulmonary exacerbation rate. results: at the time of surgery, the mean fev was % of predicted (ppd), the mean fvc . ppd, and the median age was . years (range . - . ). . % of patients were female. the median number of iv antibiotic courses for respiratory exacerbations in the months prior to fess was (range - ) compared to (range - ) in the year after surgery (p = . ). further,there were fewer days on intravenous (iv) antibiotics for respiratory exacerbations in the months prior to fess than in the month post-operative period (median vs. days), but this difference was not statistically significant (p = . ). the use of oral antibiotics for respiratory flares was comparable in the months before and after fess (median vs. days, respectively, p = . ). the median number of hospital admissions and median number of days hospitalized for respiratory exacerbations did not differ in the months pre vs. post-fess (p = . and p = . , respectively). the mean fev and fvc in the months pre and post surgery did not differ significantly ( . vs. . ppd, p = . , and . vs. . ppd, p = . ). the linear rate of change in fev and fvc did not differ significantly in the months prior to and following fess (-. l/month vs. +. l/month, p = . and -. l/month vs. +. l/month, p = . , respectively). there were no significant differences (p > . ) for any of the above comparisons when the analysis was limited to months before and after fess. there were no significant differences (p > . ) in antibiotic use and hospitalizations when the analysis was limited to the surgeries in which patients were experiencing both respiratory and sinus symptoms. this latter group had lower fev and fvc ppd in the month post-fess period ( . vs. . ppd, p = . , and . vs. . ppd, p = . , respectively). however, the rate of decline in fev and fvc in the months before and after surgery did not differ significantly ( p = . and p = . , respectively). conclusion: fess did not significantly affect the rate of respiratory exacerbations or rate of decline in pulmonary function in adults with cystic fibrosis. cumulative decline in lung function as measured by fev in cystic fibrosis (cf) pulmonary exacerbation is a major cause of cf-related morbidity and mortality. high-dose extended interval aminoglycoside (hdei ag) use may be more effective in improving lung function than traditional multiple daily dosing in cf-related pulmonary exacerbation. intermittent dosing of beta-lactam antibiotics are often used with hdei ag in patients with cf pulmonary exacerbation, which does not optimize betalactam pharmacodynamics. the primary purpose of this study is to determine the effect of hdei ag plus continuous infusion beta-lactam (cibl) on patients' return to best baseline fev from the previous months. this study was conducted at university of kentucky healthcare. this was a concurrent observational trial with patients serving as their own historic controls via review of the medical record. all pediatric and adult patients with cf respiratory disease hospitalized with an acute pulmonary exacerbation between november , and may , , with pseudomonas aeruginosa in their respiratory culture and were followed for at least one year were included. excluded were patients with known hearing disability or renal insufficiency or an inability to reliably perform spirometry (ie.less than six years old). patients served as their own historic controls via conventional treatment (control) versus hdei ag plus cibl (treatment). the primary endpoint was to determine the percent of patient courses that return to best baseline fev in the last twelve months after treatment with hdei ag plus cibl. secondary endpoints included determination of patient courses that returned to mean baseline fev in the last twelve months after study treatments, and description of beta-lactam dosing characteristics. thirteen patients were included in the pilot analysis which totaled patient courses (mean . courses per patient). average patient age was ± . years with a mean fev of %; . % were female. after hdei ag plus cibl no patients showed a return to their best baseline fev in the previous months with the exception of one patient whose value was unchanged. average overall percent change in best baseline fev group was - . ± . at follow up. % of patient courses exhibited a return to their mean baseline fev . average overall percent change in mean fev baseline was . ± . at follow up. five of the ( . %) study patients who had serum beta-lactam concentrations obtained were able to achieve at least four times the mic of the target organism. more data will be collected on subsequent courses and follow up lung function. our preliminary data suggest that our patients lost % of their best fev with an acute exacerbation and returned to % above mean baseline after treatment with hdei ag and cibl therapy. this evidence suggests that this might be an alternative to intermittent beta-lactam therapy. follow up data on remaining patients may provide sufficient evidence to validate this hypothesis. respiratory exacerbation in cystic fibrosis patients is characterized by increased sputum that may become more purulent. the detection of the exacerbation is based mainly on clinical subjective parameters. fev measurements are accepted as gold standard to be used for the life-time of the patient but do not adapt fast enough after resolution of an exacerbation. we compared parameters that are affected by the uneven distribution of ventilation in the lung due to increase sputum. methods: a zen body plethysmograph was used to measure tlc, fev and dlco (co/ch mixture). tlc was also calculated by the volume of ch , the angle of the slope of phase iii was calculated in cf patients at the beginning of the respiratory exacerbation and after weeks of antibiotic treatment. patients were - years old ( m/ f). results: fev changed by . + . % at the end of the antibiotic treatment, the tlc(pleth) however was stable and changed by only . + . %. the rv/tlc decreased by . + . % and the tlc(gas) was increased by + . %. the difference between tlc (pleth) and tlc (gas) before and after the antibiotic treatment decreased from . + . % to . + . %. conclusion: the changes in the parameters affected by the unevenness of distribution of ventilation (m/p reflecting the increased sputum in the airway) are more pronounced than fev and may be used as a more sensitive parameter for assessing the course of respiratory exacerbation. fibreoptic bronchoscopy was performed in children aged - years: with cf, with non-cf bronchiectasis (bx), with asthma, and control children without lower respiratory disease. endobronchial biopsies were taken and stained with haematoxylin and eosin. asm content, myocyte number and size were quantified using stereology. results: the volume fraction of asm in subepithelial tissue (median; iqr) was . ( . - . ) in controls. by comparison, it was . ( . - . , p< . ) in cf, . ( . - . , p< . ) in bx, and . ( . - . , p< . ) in asthma. myocyte number (cells per mm of reticular basement membrane) was ( - ) in controls, ( - , ns) in cf, ( - , ns) in bx, and ( - , p< . ) in asthma. myocyte size (um , mean; sd) was ( ) in controls, ( , p< . ) in cf, ( , p< . ) in bx, and ( , p< . ) in asthma. in cf, the volume fraction of asm in subepithelial tissue was related to myocyte number (r= . , p= . ), but not to myocyte size. conclusions: asm remodeling occurs in cf children. this, however, is not disease-specific and is also found in non-cf bronchiectasis and in asthma. both myocyte hypertrophy and hyperplasia contribute to increase in asm in cf. support: ers long-term fellowship and swiss national foundation grant to nr purpose: ct scans are increasingly being ordered on a routine basis in patients with cystic fibrosis. while prior studies have investigated their clinical utility in children, there is limited and conflicting information on their utility in adults. the purpose of this study was to determine the relationship between clinical disease severity as assessed by spirometry and ct findings as well as initial ct score ability to predict change in lung function in a nonselected group of adult cf patients. methods: a retrospective review of cf patients (mean age . +/- . , male %) who had undergone a routine chest ct was performed. the ct scans were scored using the brody method by a blinded reader with the degree of bronchiectasis, mucous plugging, peribronchial thickening, parenchymal disease, and air trapping assessed individually and a composite score generated. the ct metrics were first compared with spirometric test that were temporally related to the date of the ct scan, and then with future spirometric tests. results: for the cohort the patients had a wide range of severity of disease with the average fev % . +/- . , and the average fvc% was . +/- . . no significant correlation was found between initial fev % and total ct score ( . p value . ), or initial fvc% and total ct score (- . p value . ). for ct metrics and future spirometry, the average time from ct to pft's was days +/- with median of days. predictors for a significant decline in pft's were a decreasing bmi and male sex (p-values . and . respectively). further, for patients with a low fev % (< % at baseline), the fvc% change over time was associated with total ct score, male gender, and bmi (p-values . , . , and . respectively) conclusions: in adult cf patients, ct scan findings did not correlate with lung function. however in longitudinal analysis, bmi and gender had an effect on fvc change. for patients with low lung function, total ct score did have an association with change in fvc along with bmi and gender. pulmonary exacerbations (pes) are episodes of worsening of respiratory symptoms that commonly occur in patients with cystic fibrosis (cf). in clinical trials of new therapies in cf, pulmonary exacerbation is widely used as a primary or secondary endpoint. despite its importance, no single clearly agreed upon definition exists. some are based on patient reported symptoms while others require hospitalization or antibiotic prescription. use of different definitions makes it difficult to compare the results across studies and also to plan future studies. moreover, the rate of pes (proportion of patients with at least one exacerbation over a given time-period) is affected by baseline patient characteristics such as age, fev and medication use at the start of the clinical trial; factors which also contribute to the difficulty in comparing trials. using data from the cf therapeutics development network (tdn) data bank, we examined several pe definitions and baseline characteristics for their effect on pe rates. we find that pulmonary exacerbation rates vary substantially depending on both the pe definition and the baseline characteristics. for example, in one study, the proportion of subjects hospitalized and/or prescribed antibiotics during the month treatment period was %, while the proportion who met a symptoms based criteria for pe during the same period was %. we also examined the implications of varying control group exacerbation rates on sample size requirements for clinical trials and find that the control group pe rate has a large impact. for illustration, we assume a two group study with two-sided significance level . and % power. if the control group rate is %, then approximately subjects per group are required to detect a relative rate reduction of % (corresponding to a treatment group rate of %). however, if the control group rate is %, then only approximately subjects per group are required to detect the same relative rate reduction of % (corresponding to a treatment group rate of %). thus, a pe definition that selects for a greater pe rate in the control group will require fewer study subjects to detect the same relative rate reduction given that all other variables remain equal. sample size requirements can be further reduced by analyzing the number of events or the time to first event instead of the proportion of subjects who have at least one pe during a given time period. supported by cystic fibrosis foundation therapeutics, inc. (ram-sey y ), the national center for research resources (ncrr mo -rr ) and csl behring. briglia, h.; hilliard, j.; chmiel, j.f.; krenicky, j.; konstan, m.w. pediatrics, case western reserve university, cleveland, oh, usa cystic fibrosis (cf) is characterized by a vicious cycle of obstruction, infection and inflammation. the inflammatory response, which is profound and excessive relative to the burden of bacteria, is characterized by a massive neutrophil (pmn) influx. cf patients often experience intermittent declines in lung function associated with increases in both bacterial burden and pmn influx. these pulmonary exacerbations require treatment with antibiotics. there are substantive issues in the identification of pulmonary exacerbations and the assessment of therapies. a marker which indicates the inflammatory state of the lung would be useful to identify infective/inflammatory exacerbations, as opposed to worsening due to pulmonary vascular disease, or simply upper airway infection (cold), and might guide therapy for intensity and duration. g-csf and gro-α are cytokines produced in the lung by a variety of cells, including macrophages and epithelial cells. g-csf is involved in the proliferation, differentiation and activation of pmn precursors, while gro-α has known chemotactic and activating effects on pmns. transfer of these cytokines through the basolateral membrane into the bloodstream results in increased pmn production and activation in the bone marrow. the plasma concentrations of g-csf and gro-α might serve as potential biomarkers for a pulmonary exacerbation. in this pilot study we measured g-csf and gro-α in the plasma of healthy volunteers (n= ), clinically stable cf patients (n= ) and cf patients experiencing a pulmonary exacerbation, both before (n= ) and following (n= ) - days of antibiotic (abx) therapy. cytokines were measured using commercially available elisa kits (r&d systems, minneapolis, mn) . t-tests were performed on natural log transformed cytokine data (sigmastat v . , systat software inc., san jose, ca). ᭹ the lack of a significant difference between g-csf and gro-α plasma levels in healthy and clinically stable cf patients suggests that these cytokines are not indicators of chronic inflammation. ᭹ both cytokines are increased during pulmonary exacerbations in patients with cf and decrease to baseline values following antibiotic therapy, suggesting that these cytokines might serve as potential biomarkers for cf pulmonary exacerbations. ᭹ although not statistically significant, there was a trend towards an inverse correlation between both cytokines and fev during pulmonary exacerbations in this small study (data not shown). ᭹ further studies are warranted to increase our understanding of g-csf and gro-α as potential biomarkers of pulmonary exacerbation in cystic fibrosis. this work was supported in part by the cystic fibrosis foundation through an institutional research development grant. pletcher, s.d. ; koff, j.l. ; kleinhenz, m. . otolaryngology, university of california, san francisco, san francisco, ca, usa; . medicine, university of california, san francisco, san francisco, ca, usa introduction: while sinus disease is common in adult patients with cystic fibrosis (cf), the severity of sinus symptoms and relationship of sinus disease to other manifestations of cf are relatively unknown. objectives: to evaluate the severity of sinus symptoms in adult patients with cf and correlate these findings with other cf manifestations. methods: twenty-six consecutive adult patients with cystic fibrosis were surveyed during routine clinic visits for sinus specific and overall quality of life outcomes using both the sino-nasal outcome test (snot- ) and the cystic fibrosis questionaire (cfqr). analysis of snot- and cfqr scores were compared to %fev and sputum culture growth of pseudomonas for all patients. results: snot- scores ranged from . to . with a mean of . on a - point scale. for the purpose of data analysis, the patients were divided into two patient cohorts: group with minimal sinus symptoms (snot- < . ) and group with mild to moderate sinus symptoms (snot- > . ). the mean %fev for group was significantly higher than the mean %fev for group ( . % vs. . % respectively, p< . ). patients in group were more likely to have sputum culture growth of pseudomonas although this trend did not reach statistical significance ( % vs %, p= . ). cfqr scores differed significantly among the two groups with group patients reporting more disability in the physical, role, vitality, body, eating, health, respiratory, weight, and digestion domains but not in the emotion, social, and treatment domains. conclusions: this cohort of adult cf patients show minimal to moderate symptoms of sinonasal disability. those patients with mild to moderate sinonasal symptoms have decreased pulmonary function and decreased overall quality of life compared to cf patients with minimal sinus symptoms. , . internal medicine, university of iowa hospital and clinics, iowa city, ia, usa; . translational lung imaging program, university of iowa hospitals and clinics, iowa city, ia, usa; . pulmonary and critical care medicine, mount sinai medical center, miami beach, fl, usa mucociliary transport is an important component of airway host defense against inhaled particles and microbial pathogens. mucociliary clearance is hypothesized to be defective in cystic fibrosis allowing for airway colonization and infection. current methods for measuring mucociliary transport and clearance lack the ability to measure mucociliary transport in specific proximal and distal airway segments. using a -slice high resolution ct scanner (somatom , siemens), we are developing a method to track radiopaque particle movement in the swine airway. swine were induced with ketamine/acepromazine, intubated, and then anesthesia maintained with propofol infusions. animals spontaneously breathed humidified air with the endotracheal tube cuff deflated. radiopaque teflon/bismuth trioxide disks ( mm diameter, . mm thick) were instilled via a catheter into the airways. serial ct images ( . mm slice thickness and slice increment of . mm) were obtained every minutes for a total of minutes. airway tree segmentation was performed using pulmonary workstation . (vida diagnostics, iowa city, ia). this program allows for fully automated airway tree segmentation. baseline mucociliary transport rates ( -dimensional) were . ± . mm/min. following aerosol delivery of human sputum leukocyte elastase ( mu), mucociliary transport was markedly diminished ( . ± . mm/min) at minutes. thus far, we are able to measure mucociliary transport down to the rd generation airways. future studies include more distal deposition and measurement of particle movement. in summary, this novel method measures mucociliary transport in defined airways and will allow us to examine the effect of altered airway epithelial function on mucociliary transport. lung transplant. purpose: to design a ct scoring system to quantify structural abnormalities on ct scans from cf patients with severe advanced lung disease (sald) and to investigate the correlation between ct scores and survival. materials and methods: ct scans of cf patients screened for lung transplant between and were collected from transplant centres. all scans were scored with the brody ii scoring system. to design a new scoring system sensitive for the specific changes in sald, a panel of experts reviewed a random set of ct scans on eligible items to be used in a pilot analysis. the resulting sald scoring system consisted of four items: bulla/cysts; areas with consolidation/mucous; areas with hypo perfusion/air trapping and hyper/normal perfusion. in each ct slice ( mm spacing) and for each of the items the surface area of corresponding lung tissue involved was estimated on a - % scale. total surface area for the items and each slice added up to %. the final sald score was a mean volume estimate of abnormal and normal lung tissue. results: pilot analysis of a set of ct scans showed a a spectrum of abnormalities, ranging from predominantly bronchiectasis to predominantly mosaic perfusion. a moderate correlation was found between the sald components inflammation and hypo perfusion/ air trapping and brody score (p= . , r= . and p= . , r= . ). the inter-observer variability of both scoring systems was comparable. (ri ranging from . to . ) analysis of the complete cohort with the brody ii score showed a significant correlation with survival with a hazard ratio of . ( % ci of . to . ) for dying on the waiting list for every ten point increase in brody ii score. (p= . ). the brody component scores for bronchiectasis, airway wall thickening and parenchyma showed a similar correlation with hazard ratios ( % ci's) of respectively . ( . - . ), . ( . - . ) and . ( . - . ) for every ten point increase in score. (p= . , . and . ). the preliminary results with the brody score suggest that it is useful to include ct information in prediction models. it is likely that the predictive value of the ct can for survival can be further improved using the dedicated sald-ct score. scoring of the remaining scans according to the sald system is ongoing and will be completed shortly. snell, g. . physiotherapy, the alfred, melbourne, vic, australia; . allergy, immunology and respiratory medicine, the alfred, melbourne, vic, australia; . epidemiology and preventative medicine, monash university, melbourne, vic, australia; . radiology, the alfred, melbourne, vic, australia background despite the widespread use of airway clearance (ac) techniques to clear excessive secretions and improve lung function, little is known about their efficacy following lung transplant (lt). this study aimed to compare the effects of two ac strategies (proactive vs reactive) on a range of clinical outcomes following lt. methods a prospective randomized trial design with stratification for suppurative pre-lt disease was used. consecutive patients at month post lt were eligible for inclusion. subjects were excluded if medically unstable, ventilator dependent or had a contraindication to performing positive expiratory pressure (pep) therapy. patients performed ac using pep either twice daily (proactive strategy) or only in the presence chest infection (reactive strategy). lung function (fev and fvc), chest radiography (brasfield score), exercise capacity ( minute walk) and bronchoscopic airway characteristics (anastomotic healing, patency and secretions) were assessed at , and months post lt. patient adherence and satisfaction were measured. results of consecutive patients, ( in each group) were recruited and completed the study. both groups improved lung function (fev ± % to ± % p< . ; fvc ± % to ± % p< . ), brasfield scores ( . ± . to . ± . p< . ) and minute walk ( ± m to ± m p< . ) over the study period. no significant differences between groups for any outcome were found. the vast majority had fully healed, % patent anastomoses without secretions at months. there were no significant differences between airway characteristics and incidence of chest infection. adherence to both strategies was high ( % proactive, % reactive). conclusion in the absence of significant differences in outcomes, it is recommended that ac only be performed in the presence of chest infection based on greater treatment cost and treatment time required by a proactive strategy. further studies in those with reduced anastomotic patency and in those with recurrent chest infections later post-transplant are warranted. supported by an alfred trusts grant. munro, p.e. ; button, b. , ; bailey, m. ; holland, a. , ; snell, g. . physiotherapy, the alfred, prahran, vic, australia; . allergy, immunology and respiratory medicine, the alfred, melbourne, vic, australia; . epidemiology and preventative medicine, monash university, melbourne, vic, australia; . school of physiotherapy, la trobe university, melbourne, vic, australia background the optimal duration and structure of pulmonary rehabilitation (pr) for lung transplant (lt) recipients is not known. this study aimed to describe changes in functional outcomes in lt recipients who participated in a post lt pr program at the alfred, melbourne, australia. methods prospective, repeated measures design. functional exercise capacity (six minute walk test- mwt), lung function (fev , fvc) and quality of life (sf ) were assessed at , and months following lt. following discharge, all subjects attended a hour supervised outpatient exercise training class days per week until weeks post lt and education sessions. patients with post-operative complications were excluded. data were analysed using descriptive statistics and anova with repeated measures. results consecutive lt recipients from sept to mar were assessed for inclusion. ( % male) subjects, mean age ± yrs were recruited and completed the study. % had undergone bilateral lt. % had cystic fibrosis, % chronic obstructive pulmonary disease. significant improvements were demonstrated in mwt ( ± m to ± m, p< . ), fev ( ± % to ± %, p< . ), fvc ( ± % to ± %, p< . ) and all quality of life domains, p< . . the greatest changes in mwt and lung function were seen between and months. conclusion functional exercise capacity, lung function and quality of life improved significantly over the first months in lt recipients who participated in pr at our institution. these data will allow benchmarking with other centres and program structures. supported by an alfred trusts grant. does gender play a role in perception of cf quality of life domains before and after lung transplantation? the cf quality of life (cfq-r) for patients м years of age is a disease-specific instrument. previous studies using this instrument have reported that cf females have higher scores in weight and body perception than cf males. to determine the impact of lung transplantation on gender-based perception of quality of life, we reviewed the cfq-r on all cf patients м years of age and who underwent successful lung transplantation at our center. cfq-r was administered - months prior to transplant surgery and then months after lung transplantation while the patients were in the outpatient clinic setting. domains measured: physical, role, vitality, emotion, social, body image, eating, treatment burden, health status, weight, respiratory symptoms, and digestion. the cfq-r responses were computer-scored and domain scores were then generated (score - , =better). during the ⁄ year study period, cf patients underwent successful lung transplantation ( males: females; mean age . ± . years). prior to lung transplantation, female cf patients had lower physical domain scores but higher scores in health status perceptions than the male cf lung transplant candidates (see graph). all patients had marked improvement in all domains months after lung transplantation (see graph). after transplant surgery, cf males had higher scores than cf females in emotion and in eating domains. scores for body image domain were similar for both genders before and -months after lung transplantation. we conclude that although there were differences in physical and health domain perceptions prior to lung transplantation, both male and female pediatric cf patients had marked improvement in all domains after lung transplantation. however, female cf lung transplant recipients had lower emotion and eating domain scores after transplantation. we speculate that lower scores in these domains may reflect increased post-traumatic stress and depression in female lung transplant recipients. further study on cf quality of life in pediatric lung transplant recipients is on-going. background cf liver disease (cfld) with severe cirrhosis develops in - % of patients, usually within the first decades of life. most patients with cfld suffer from complications of portal hypertension but hepatocellular function usually remains intact for many years, even decades. patients with cfld referred for lung transplantation (tx) may be offered lung tx, lung-liver tx or be excluded from lung tx. however, variceal hemorrhage due to portal hypertension can be managed by banding, sclerotherapy or shunt procedures without liver tx. the aim of this study was to examine outcome of cfld patients who underwent lung tx and the subsequent progression of cfld, particularly with the use of potentially hepatotoxic immunosuppressive drugs. we conducted a retrospective cohort study to compare cf patients undergoing lung tx with and without cfld. b. cepacia-negative cf patients undergoing lung tx at toronto general hospital from - were eligible for inclusion. liver cirrhosis was defined by histology, esophageal varices on endoscopy or imaging evidence of splenomegaly. each patient with cfld was matched for age and year of tx with cf patients without cfld. demographic data, survival and liver function tests (lfts)(ast, alt, alp, inr, albumin, protein at week , month , , , , , , , , and post tx) were obtained by chart review. results of the groups were compared using unpaired t-test (parametric data) or mann-whitney u test (non-parametric data). results b. cepacia-negative cf patients underwent lung tx over this period, ( . %) having cfld with cirrhosis. data from the cfld patients vs. matched cf patients without cfld were analyzed. no significant difference was found between the groups in pre-tx age, gender, bmi, pi status, cfrd status, fev % predicted, -minute walk distance or lfts. in the cfld group albumin was lower week post-tx ( . + . g/l vs. . + . g/l, p< . ), alp was higher month post lung tx ( . vs. . , p< . ) and alt was higher months post-tx ( . + . u/l vs. . + . u/l, p< . ). there was no significant difference in the other blood tests. azathioprine was changed to mycophenolate mofetil in cfld patients due to liver test abnormalities. cfld patient developed hepatic encephalopathy and ascites years post lung tx and is being assessed for liver tx. no difference was seen in the number of episodes of acute rejection or post-tx survival ( . % vs. . % alive at years). discussion cfld patients undergoing lung tx did not have a worse prognosis than patients without cfld. transient elevation of lfts were seen in the immediate post-tx period but settled either spontaneously or after discontinuation of azathioprine. we conclude that selected patients with cirrhosis due to cfld may be safely offered lung tx without concomitant liver tx. infectious mononucleosis, commonly called mononucleosis, or "mono," is an illness caused by the epstein-barr virus. mononucleosis has been nicknamed the "kissing disease" because the epstein-barr virus commonly is transmitted in saliva during kissing. however, sneezes and coughs also can transmit the virus occasionally. the epstein-barr virus (ebv) permanently infects more than % of the people on earth, but it causes symptomatic mononucleosis only in a small number. in developed nations, such as the united states, mononucleosis most often occurs between the ages of and . unfortunately ebv can not only infect, but can also transform, b cells after transplant and may lead to lymphoma with a resulting ~ % mortality rate. the source of ebv is presumed to be passenger lymphocytes in the donor tissue and those at greatest risk are patients who are ebv naive before transplant. since most cf patients with endstage lung disease in the developed world are now being referred for transplant given the success of this intervention, we reviewed our entire lung transplant database to determine the nature of ebv infection and the likelihood of developing lymphoma following lung transplantation. of first-time lung transplants, ( %) had cf. ebv serology was not available in ( cf, others) before transplant. of ( %) cf patients were ebv seronegative before transplant as compared to of ( %) patients with other end stage lung diseases (composed mostly of copd and ipf) in the control population (chi square p < . ). the cf ebv seronegative cohort was (sd ) years old on average, an age when most seroconversion has already occurred in our society. of the cf ebv seronegatives, survived beyond months and were thus at risk for post transplant lymphoma. of these , developed lymphoma (incidence = %) in comparison to of the ebv seronegative controls (incidence = %, p = ns). all lymphomas were stage iv at presentation and the majority ( of ) arose in the first post transplant year. the cf ebv seronegatives who developed lymphoma did not differ from those who did not with regard to levels of immunosuppression (cyclosporine, methylprednisolone or prednisone) or doses of antiviral therapy (i.e., ganciclovir which is active against both cmv and ebv). in addition, the cessation of anti-lymphocyte antibody induction therapy in the summer of , somewhat surprisingly, had no impact on the development of lymphoma. two cases of lymphoma developed in cf ebv seropositives (incidence = . %) and no cases of lymphoma developed in the control ebv seropositives (p = ns). thus ebv seronegativity raises the lymphoma risk fold in cf patients (p < . ). two of the ( %) cases of lymphoma resulted in death (one cf and one other) and one cf patient probably died of complications related to chemotherapy even though he was tumor free at that time. lymphoma outcome was not stage or clonality dependent. in conclusion, cf lung transplant recipients are at an increased risk for lymphoma largely due to their lack of exposure to ebv before transplant. kissing more before transplant may lower the risk of lymphoma afterward. university hospital uzb, brussels, belgium; . chest medicine, erasme university hospital, brussels, belgium; . dermatology, erasme university hospital, brussels, belgium; . gynaecology, erasme university hospital, brussels, belgium; . gastroenterology, erasme university hospital, brussels, belgium; . pathology, erasme university hospital, brussels, belgium hpv infection is an underestimated phenomenon in organ transplantation (tx) recipients, being unable during immunosuppression (is) to mount an adequate anti-viral immune response and risking genital/anal warts (condylomata acuminata) as well as pre-cancerous/cancerous lesions. we retrospectively assessed the incidence/treatment of genital/anal hpv-associated lesions in cystic fibrosis (cf) lung tx recipients. the files of all tx patients transplanted in the ulb center between and / ( men/ women, median age ± . , range - yrs) were reviewed. maintenance is consisted in chronic triple therapy combining a calcineurin inhibitor, a cell cycle inhibitor and corticosteroids. median survival was . ± . months (range - ). ten of sexually active patients ( %) who survived ≥ months post-tx developed hpv-associated genital/anal lesions: men ( - yrs) and women ( - yrs). genital/anal hpv-pcr proven condylomata were diagnosed between - months in the men and between - months post-tx in the women. all underwent local treatment (cryotherapy ± laser ± topical imiquimod), / patients underwent multiple treatments under general anesthesia. recurrence of the condylomata was high. one male patient presented high-grade anal dysplasia and women moderate to high-grade cervical dysplasia; underwent conization and one complete hysterectomy after conizations. one of the females presented both condylomata and a high grade cervical dysplasia. these retrospective data in a cf population indicate that ) hpv infection may cause significant morbidity in young subjects with chronic disease needing organ tx. it may even compromise life expectancy after tx. ) data on the effectiveness of treatment strategies -including topical immunomodulators -have to be collected ) the potentially protective effects of the now available hpv vaccination in females and males? with chronic disease at risk for future organ tx, when administrated before sexual activity and before tx, should be rapidly evaluated in a multi-center effort. ballmann, m. ; pfister, e. ; schlueter, k. ; becker, t. ; melter, m. ; junge, s. . pediatric pulmonology and neonatology, medical school, hannover, germany; . pediatric nephrology and hepatology, medical school, hannover, germany; . visceral and transplant surgury, medical school, hannover, germany liver disease is an important comortality and comorbidity in cf patients even in the pediatric age group. liver transplantation (ltx) is an accepted option in end stage liver disease. nevertheless the clinical outcome in cf patients is still under discussion. here we reported the clinical outcome months after isolated liver tx in pediatric cf-patients.since n= ltx were performed in children (male n= , female n= ; age (mean±sd) , ± years) . immunosuppressive therapy was done with ciclosporin ± basilixumab and steroid. we followed pulmonary function, cn-score, nutritional status (bmi z-score) and body com-position, airway cultures and igg.results:all were still alive months after ltx. before ltx all patients had a mild to moderate pulmonary disease: fvc(%pred) , ± ( , %- ), fev (%pred) , ± , ( - ), mef (%pred) , ± , ( - ) , cn-score ± , points. nutritional status: bmi(mean) , ± , % (range: , %- , %, z-score: , ± , ), upper arm circumference(cm)(mean±sd) , ± , , triceps skin fat fold(cm) , ± , . body composition: , ± , % of bodyweight were fat. one year after ltx all patients were still in a stable pulmonary situation: fvc(%pred) , ± (range: , %- %), fev (%pred) , ± , (range: %- %), mef (%pred) , ± , (range: , %- %), cn-score , ± , points. there weren't any significant changes in the airway microbiology under immunosuppressive therapy (steroid, ciclosporin±basiliximab), the serum igg levels declined significant from , ± , g/l to , ± , /l(p< . ). the growth over the year was , ± cm and the increase of weight come to , ± , kg, while the bmi didn't increase in this first year after transplantation. certainly we found an increase of the body fat mass (to , ± , % of body weight), of the upper arm circumference (to ± , cm) und of the triceps skin fat fold (to , ± , cm) .conclusion:liver transplantation is an effective therapy even in children with cf related liver disease and stabilise pulmonary function and improves nutritional status in patients with cf and mild or moderate pulmonary involvement before ltx. (neglia jp et al., n engl j med ; : - ) , and colon cancer has been described in young adults with cf (chaun h et al., can j gastroenterol ; : - ) . three of lung transplant (ltx) recipients from our center developed colon cancer after successful bilateral lung transplant for end-stage lung disease. data from these individuals are given in table below . an additional recipients with cystic fibrosis were screened via colonoscopy. colonic polyps were detected in and included lesions up to cm in diameter. five years after a colonoscopy that had shown a mm polyp, one patient underwent a nd colonoscopy that revealed multiple polyps, including a cm diameter sessile polyp in the sigmoid colon. colonic malignancies appear to arise from mucosal polyps, and screening via colonoscopy can detect and remove premalignant lesions. lung transplant recipients have an increased risk of gi malignancies and tumor surveillance is impaired by post-transplant immunosuppression, colonoscopic screening should be considered for transplant recipients with cystic fibrosis. additionally, colonic malignancy should be considered as a potential cause of unexplained gastrointestinal symptoms such as constipation. nossent, g. ; kastelijn, e. ; teding van berkhout, f. ; zanen, p. ; van den bosch, j. ; van de graaf, e. . respiratory dis, university medical centre, utrecht, netherlands; . respiratory diseases, st. antonius hospital, nieuwegein, netherlands objective: in the netherlands lung allocation is based on waiting time. to prioritize patients with a poor prognosis and to reduce waiting list mortality the high urgency (hu) status was introduced in . to be considered for hu status there has to be a limited life expectancy on clinical judgement and all three transplantation centers have to audit this decision. the aim of this study is to analyze the potential effect of las on patients with cystic fibrosis (cf) who became hu or died on the waiting list. it would be expected that patients who became hu had a higher las. methods: from till in all cf patients placed on the hu waiting list, the median las ( th, th) was calculated at the moment of listing and at the moment the patients became hu. also for the cf patients who died on the waiting list the mean las (sd) was calculated at the moment of listing. results: from the patients on the waiting list patients had cf. of these patient were placed on hu list and died on the waiting list. in the cf patients that became hu the median las on the moment of listing was . ( . ; . ) . at the moment they were placed on the hu waiting list the median las was . ( . ; . )(p= . ) . the median las of the patients who died on the waitinglist was . ( . ) . this las did not differ significantly from the las of listing of the patients that became hu (p= . ). conclusion: the las does not detect the deterioration in patients with cf as diagnosed by clinical judgement. this may be due to the exclusion of specific prognostic factors of cf in the calculation of the las. besides that, the las does not differentiate cf patients on the waiting list with a high chance of mortality. introduction: lutx recipients have one of the highest rates of ia in solid organ transplantation causing high morbidity and mortality rates. nowadays antifungal prophylaxis is extensively used but clinical trials are rare. methods: all patients who received lutx in our hospital were included in this retrospective study. aspergillus airway colonization was defined as aspergillus cultured twice from airway specimens in the absence of ia or tracheobronchitis (tb). definition of ia and tb was according to literature. after the death of cf-patients due to ia, a prophylaxis protocol was introduced containing avoidance of use of the cell saver during the operation in pretx colonized patients to avoid hematological fungal spreading and targeted prophylaxis with itraconazole or voriconazole in all pre-and posttx colonized patients during months and inhaled amphotericin during hospital admission. results: a total of lutx were performed (in patients) ( % cf patients) from - - until - - . patients ( . %) were colonized with aspergillus pretx and patients posttx. only patients were colonized before and after tx. before introduction of the protocol patients ( / = . %) ( % cf patients) developed an aspergillus infection: ( . %) patients developed a. fumigatus tb and ( . %) patient suffered from ia: cerebral a. fumigatus infection (proven ia). two patients, both cf patients, ( / = %) died due toaspergillus infection (cerebral and tb).after introduction of the protocol only one a. fumigatus tb occurred ( / = . %) but no invasive fungal infection. conclusions: the rate of fungal infections in our lutx patients was comparable with data from literature. since introduction of azole prophylaxis protocol and avoidance of use of cellsaver no ia occurred. fischer, a. ; müllinger, b. ; arendt, t. ; bernhardt, t. ; hannah, k. ; scheuch, g. . activaero gmbh, gemuenden, germany; . biological products, bayer healthcare, leverkusen, germany; . activaero gmbh, gauting, germany; . talecris biotherapeutics, research triangle park, nc, usa background patient compliance during a study is an important factor in view of assessing the clinical effect of a treatment. this is especially true when patients administer the drug at home. usually, patient records, counting of returned doses or mechanical counters are used to track compliance, which may be biased by the study subjects. this report is about an aerosol study in cf which used a device for controlled breathing (akita® inhalation system, activaero, germany). the device works with a patient-individual smart card that records every single breath during a treatment including a date/time stamp in an encrypted manner. each patient's inhalation protocol can be displayed by loading the smart card into the proprietary "compliance manager" software. methods for this report, the data set of a recent controlled study (griese et al., ) was analyzed. cf-patients were instructed to inhale with the akita inhalation system at home for days. after the study, the smart cards were returned to activaero, and the inhalation records on the chip were analysed. we analysed the compliance of patients who participated at least days ( out of patients, others deemed to be drop-outs . most of the patients showed a dsc which is lower than the tdc, indicating that they had missing treatment days, which were compensated by additional inhalations on other days. we found patients with a dsc more than % lower than their tdc (max difference: . %). conclusion this aerosol study with home treatment demonstrated a high compliance rate for tdc and dsc. this information is more reliable for compliance than study participation in days alone. for future studies it is recommended to define in the protocol not only a minimum of study participation in days but also a minimum in tdc and dsc. in addition the compliance thresholds may be defined with regard to the drug's pharmacokinetic profile. in general, the inhalation record of the akita smart card provides an unbiased view of the inhalations treatments during a study especially when the subjects perform the inhalations at home. compliance calculations as shown above may be performed and linked to other outcomes of a study for validation. compliance data like these may also be used routinely by the treating physician in order to guide and supervise his patients. introduction: adaptive aerosol delivery (the i-neb ® aad ® system) has been developed to deliver precise, reproducible doses of aerosol into inhalation. i-neb utilizes a medication chamber, which incorporates a metering chamber to deliver a preset (metered) dose of aerosol. inhalation of hypertonic ( %) saline has been shown to aid mucociliary clearance in patients with cystic fibrosis. [ ] we performed an in vitro test to determine the emitted, delivered and residual doses of hypertonic saline from the i-neb aad system with . ml metering chamber, and a conventional jet nebulizer (lc plus ® ) with a ml fill. the results were then entered into a model to predict the likely lung dose from the in vitro tests, in order to determine the equivalent i-neb dose to the ml conventional jet nebulizer fill. methods:an i-neb device configured to operate at power level (of ) was fitted with a metering chamber designed to deliver a preset dose of . ml. the i-neb was weighed, loaded with ml of % saline and run on the cen simulated breathing pattern. this process was repeated using an lc plus nebulizer loaded with ml of % saline. aerosol was collected on a filter placed between a harvard pump and the device. emitted dose and residual dose were determined by gravimetric output, and delivered dose was determined by ion analysis. all tests were conducted in triplicate. results: as shown in table , the emitted dose approximated the delivered dose for i-neb, whereas for lc plus the emitted dose was approximately twice the delivered dose, due to the wastage of aerosol upon simulated exhalation. as i-neb has a delivered dose of mg, and i-neb has been shown to deposit . % of the dose in the lung, [ ] this would result in a lung dose of mg. the pari lc plus has a higher delivered dose, but the overall lung deposition is only % of the fill volume, i.e. mg. [ ] conclusion: the results of this test suggest that two treatments would be required in order to deliver an equivalent lung dose of hypertonic saline from an i-neb device fitted with a . ml metering chamber, compared with a ml fill in an lc plus jet nebulizer. references introduction: inhaled colistimethate sodium can be used for the eradication of pseudomonas aeruginosa in patients with cystic fibrosis. the tonicity of colistimethate sodium inhalation solution has been linked to the occurrence of bronchospasm in some patients. it has been suggested that this bronchospasm can be avoided by using an isotonic solution of colistimethate sodium. [ ] this can be complicated when using conventional jet nebulizers, since evaporation during nebulization tends to change the tonicity of the solution remaining in the nebulizer. however, because very little evaporation occurs within the i-neb medication chamber during nebulization, the tonicity of the loaded solution at the beginning and end of nebulization remains almost constant. aim: we investigated the effects of various saline concentrations upon the tonicity of two concentrations of colistimethate sodium solution ( . miu/ml and miu/ml). methods: diluent containing sodium chloride concentrations of: % (water for injection), . %, . %, . %, and . % (normal saline) were used to reconstitute vials of colistimethate sodium (promixin ® , profile pharma, uk). each vial was reconstituted with either or ml of diluent to make up miu/ml or . miu/ml colistimethate sodium solution, respectively. the tonicity of each diluent concentration/colistimethate sodium concentration combination was made up and tested in triplicate using an advanced micro osmometer (advanced instruments, norwood, usa). the results were plotted on a graph along with a line of best fit, to determine the diluent concentration that gave a mean tonicity closest to that of an isotonic solution. results: tonicity increased linearly with increasing concentrations of saline solution for both miu/ml and . miu/ml concentrations. the tonicity of the miu/ml solution was approximately mosm higher than the . miu/ml solution across the range of saline concentrations tested. the best fit line passed through the isotonic solution point ( mosm) closest to the . % saline concentration point. conclusions: the diluent that produces the closest approximation of isotonicity for the mean of . miu/ml and miu/ml colistimethate sodium concentrations was . % saline. references . transave, inc., monmouth junction, nj, usa; . activaero, gmbh, gauting, germany arikace™ is an inhalation formulation of a liposomal amikacin suspension that is designed to treat chronic pseudomonas aeruginosa infections in cystic fibrosis patients. liposome encapsulation of amikacin reduces nonspecific binding of this cationic aminoglycoside drug to the negatively charged mucus and biofilm surfaces and allows penetration and delivery of packets of highly concentrated drug to the otherwise protected bacteria. as nebulized and delivered to the lungs, arikace™ comprises % liposomal amikacin and % 'free' amikacin that is not entrapped by liposomes; free drug is produced by liposome leakage during nebulization. this profile provides an initial high peak concentration of amikacin followed by a sustained level as drug leaks from the liposomes. nebulized arikace™ with this profile was evaluated previously in human clinical studies using the lc star® nebulizer. although the . µm liposomes of arikace™ are efficiently loaded with drug, the expected effective dose in humans will require relatively large volumes of drug solution to be administered. to minimize treatment time and improve patient convenience we sought to find a nebulizer that would efficiently deposit drug and produce aerosol at a high output rate, while still producing the same level of free drug during nebulization. methods: nebulization of liposomal amikacin was compared using several nebulizers, including jet nebulizers: lc star® and lc sprint® (both from pari), and electronic mesh nebulizers: microair® ne u v (omron), aeronebgo® (nektar), and eflow® (pari). additionally, meshes of different pore sizes were examined for the microair® and eflow® devices. output rates were measured by gravimetric differences. droplet size distribution was assessed by a laser light scattering method and by cascade impaction; mass median aerodynamic diameters (mmad), geometric standard deviation (gsd), and fine particle fraction (fpf) (i.e., % droplet mass < µm). amikacin association with the liposomes was measured using a centrifugation-filtration assay. results: in terms of output rate, the order of performance was eflow® (~ . g/min) > lc sprint® > lc star® > aeronebgo® > microair®. while the aeronebgo® and microair® mesh nebulizers performed well with saline, their performance declined dramatically when nebulizing the liposomal amikacin solution, and, in fact, the microair device clogged. in additional studies with the eflow® where different meshes were compared, the l mesh was selected as it provided ideal aerosol properties (mmad = . µm, gsd = . , fpf > %) with a ratio of entrapped to free amikacin of approximately %/ %. importantly, from in vitro breath simulation studies it is estimated that about twice of the nominal drug dose is expected to deposit in the lungs compared to the lc star®. conclusions: for nebulization of liposomal amikacin, the eflow® configured with a l mesh provided optimum aerosol characteristics with a high output rate and ideal size for distribution throughout the lung. the eflow® reduces dose administration time not only because of its faster output rate but also because its higher deposition factor greatly reduces the amount of drug needed. zeman, k.l.; bennett, w.d. cemalb, university of north carolina, chapel hill, nc, usa many inhaled, therapeutic drugs have their site of action in the conducting airways, but may deposit elsewhere in the respiratory tract, resulting in unwanted side effects and waste. the quantity and location of particle deposition in the respiratory tract depends on both the particles' aerodynamic size and breathing pattern. in subjects with normal lung function, we evaluated three methodologies for their ability to deliver radiolabeled particles ( mtc-labeled sulfur colloid) to the conducting airways: . inhalation of µm mmad particles (devilbiss jet nebulizer) using a resting tidal volume and flow (mean . l and . lps); . inhalation from the same nebulizer using a very large inspiratory capacity volume and higher flow rate (mean . l and . lps); and . inhalation of larger . µm mmad (heart jet nebulizer) particles with a large tidal volume and very low flow rate (mean . l and . lps). gamma scintigraphy gave an estimate of % conducting airway deposition (%cad) by multiplying the percent of activity in the lungs immediately post-deposition relative to the total deposition (i.e. lung+mouth +esophagus+stomach) times the percent of activity cleared from the lungs over hours. %cad was %, %, and % for the three methods, respectively, significantly greater for the large particle, very slow inhalation technique when compared to the other two methods. the amount deposited in the mouth and larynx was %, and %. in addition, we evaluated protocols and above in a preliminary group of cf patients. for protocol , mean inhaled volume and rate was . l and . lps. mean inhaled volume and flow were . l and . lps for protocol . the %cad for the two protocols and were % and %, respectively. the amount deposited in the mouth and larynx was % and %. higher therapeutic value of a medication delivered to the airways where the primary defect is associated with cf disease, and with fewer losses to the extrathoracic airways, can be obtained by using a "very large particle/slow inhaled flow" routine when compared to normal or higher flow breathing associated with typical nebulizers. supported by cf foundation. positive effects of physical conditioning in cystic fibrosis (cf) have been reported for quality of life (qol) in addition to effectis on fitness and lung functions. the objective of this study was to identify the effects of different modes of training on qol in cf. cf-patients (age - yr., fev > %pred.) were randomized into groups: germany: unsupervised homebased training, hours per week, free choice of training mode and means (ut, n= ) and controls (con , n= ); switzerland: aerobic training (at, n= ) or weight training (wt, n= ) in a fitness center, x min per week, or controls (con , n= ). subjects in the training groups were asked to train for months. at study entry and after and months, qol was assessed by questionnaire and maximal physical working capacity (pwc, %pred) was determined during cycle ergometry (godfrey protocol). changes from baseline values were calculated and anova for repeated measures was used to test for differences among groups. weight training resulted in a significant decrease in qol (generic dimension) at and months compared with all other training modes including controls. there was no significant difference between at or ut and controls in qol during the program. however, there was a positive association between the change in pwc and the change in qol (r= . , p< . ) in the entire sample. in conclusion, physical training may not always result in an increase in ql in cf. possibly, the decrease in qol in the wt group may have resulted from tiredness induced by the training. thus, a different outcome might have been found with less intense training. positive effects may be achieved by improving aerobic fitness. supported introduction: exercise and exercise training programs are felt to be important to improve aerobic exercise capacity, muscle strength, lung function, and quality of life in cf-patients. recent studies in children and adults with cf have demonstrated an increase of motor performance after exercise training. less is known about trainability of motor performance in younger children with cf. the aim of this study was to assess the effects of a training program on motor performance in preschool children with cf. methods: preschool children with cf ( female and male patients, mean age . yrs; range - yrs, fev %pred. , ± . rage from . - . ) participated. at baseline (t ) and at the end of the training program (t ) the "motorik test (mot)" was used to assess motor performance and to evaluate training effects. components of motor performance tested with the mot are balance, agility and flexibility, strength, coordination, fine motor skills, reactivity, accuracy of movement). lung function in patients with cf was measured by spirometric techniques. the duration of training was - weeks ( times/week, minutes per session) with different kinds of sport activities. results: at t , strength and balance had increased significantly (p< . ). no other components of the mot increased, but the increases in strength and balance were enough to cause a significant (p< . ) increase overall motor performance, as indicated by an increase of . % in mq. the mq is the sum of all test-items of the mot. no correlation was found between lung function and motor performance. discussion: to our knowledge this was the first study that examined the effects of an exercise training program on motor performance in preschool children with cf. main findings of this study were that a training program of - week of duration improved some aspects of motor performance. the improvements may be explained by different kinds of sports activities during training when compared with activities before training. classification of motor performance of the tested children was normal (mq = . ± . ). this could be the reason that only some aspects of motor performance increased. parents of the children were asked about habitual physical activity at home. all children were physically active and some of the children participated in organized sports. a recent study showed that with an increase in age lower motor performance in children and adolescent with cf was seen compared to healthy children. we speculate that time spent in physical activity in preschool children with cf is comparable to healthy children. conclusion: an exercise program offered to preschool-children with cf may lead to an improvement in motor performance. pre-school children with cf have normal motor performance. to prevent a decline of motor performance, exercise programs should be available for preschool children. gruber, w. ; braumann, k.m. ; orenstein, d.m. ; huels, g. . dept. for sports and exercise, clinic sattelduene for children and adolescent, nebel/amrum, germany; . institute for sport and exercise medicine, university of hamburg, hamburg, germany; . school of medicine, university of pittsburgh, pittsburgh, pa, usa; . dept. of medicine, clinic sattelduene for children and adolescent, nebel/amrum, germany introduction: a decline in physical activity with age is one of the main problems in patients with cf. as a consequence, lower exercise capacity and a more rapid decline in lung function may be seen in those patients who are physical inactive. furthermore, motor performance is lower in children and adolescents with cf compared to healthy children of the same age. little attention has been paid to younger children with cf and motor performance. the aim of the study was to compare motor performance in preschool-children with cf with healthy children of the same age. methods: children with cf (mean age . yrs; range - yrs) and healthy children (mean age . yrs; range - yrs) participated. the "motorik test" (mot) was used to assess motor performance. the mot consisted of test-items which evaluated seven different components of motor performance (balance, agility and flexibility, strength, coordination, fine motor skills, reactivity, accuracy of movement). lung function in patients with cf was measured by spirometric techniques results: results of the mot showed no difference either for single testitems or for parameter "motorische quotient (mq)" (p> . ). this parameter classified motor performance of all aspects tested with the mot. classification of children with cf and of healthy children was normal. mean value for parameter mq (cf: . ± . vs. healthy . ± . ) was slight higher in patients with cf than in healthy children (p> . ). differences between healthy and cf for different components of motor performance were not significant (p< . ) discussion: the mot is a reliable and valid test method in germany to determine motor performance in younger children. to our knowledge this was the first study to examine motor performance in preschool-children with cf. furthermore, we compared patients with cf and healthy children. we observed no differences between groups for motor performance. in older children with cf, lower motor performance has previously been found compared to healthy children of the same age. we do not have any information about the level of habitual activity in tested healthy children. we therefore speculate that time spent in physical activity in cf is similar to that of healthy children at this age. a recent study showed a decline in physical activity in cf when age and problems during physical activity increased. we think that motor performance is normal in children with cf up to an age of yrs. when age increased, time spent in physical activity decreases (treatment, school) and therefore motor performance decreases. conclusion: motor performance is normal in preschool-children with cf compared to healthy children. to prevent a decline or to maintain motor performance it is recommended to motivate cf-children from early infancy to participate in sports activities or other physical activities to maintain or to increase their physical fitness. . respiratory medicine, the hospital for sick children, toronto, on, canada; . respirology, st. michael's hospital, toronto, on, canada; . respiratory medicine, montreal children's hospital, montreal, qc, canada purpose: the habitual activity estimation scale (haes) questionnaire has been shown to be a feasible tool to measure physical activity however the reliability has yet to be determined in cf populations. we therefore assessed the reliability and validity of the haes questionnaire in paediatric and adult cystic fibrosis (cf) populations. methods: fourteen ( male, female) patients aged . + . yrs with cystic fibrosis from the cf clinics at the hospital for sick children and st. michael's hospital participated in this study. participants were clinically stable at the time of the study (fev > % predicted) and participating in their 'habitual' physical activity. to assess test-retest reliability, patients completed each of the haes (schneiderman-walker et al., j pediatr. ; ( ): - .), and a validated -day activity diary (bratteby et al., eur j clin nutr. ; ( ) : - ), and wore an actigraphtm tri-axial accelerometer for two consecutive weeks. validity was assessed by comparing the activity results of each of the three instruments over a single week time period. results: intra-class correlation coefficient estimates of reliability for the haes, diary and accelerometer were . (p< . ), . (p< . ), . (p< . ), respectively. validity was estimated by comparing the results from each instrument using a one-way anova with block design. there were no overall differences among the participants' activity results as estimated by the haes, diary and accelerometer. furthermore, there were no significant differences between activity measures among the instruments when broken into morning, afternoon, or evening periods, or between measures from weekday or weekend days. while there were no significant differences among the instruments when recording 'very active' intensity levels, they did detect differences during the inactive / somewhat inactive (p = . ) and somewhat active (p = . ) intensity levels. conclusion: the findings of this study suggest that the haes questionnaire is a reliable and valid instrument that can be used to assess activity levels in patients with cystic fibrosis. in agreement with others, our patients found it easiest to recall and record accurately those periods in which they are participating in vigorous activity. acknowledgement: this research was supported by the canadian cystic fibrosis foundation. . respiratory medicine, hospital for sick children, toronto, on, canada; . respiratory medicine, montreal children's hospital, montreal, qc, canada; . respirology, st. michael's hospital, toronto, on, canada objective: previous work showed a relationship between habitual physical activity (hpa) and decline of lung function (fev ) in cf females (sneiderman-walker, j peds : , ) . this study only included a small number of patients and the follow-up period was limited to months. the purpose of this study was to evaluate daily activity levels and decline in fev in a larger cohort over a longer period of time. methods: a total of (n= female) patients with cf (age - yrs) were studied over a six year period at the hospital for sick children, st. michael's hospital and montreal children's hospital. at each quarterly cf clinic visit, patients performed pulmonary function tests to determine fev and completed the habitual activity estimation scale (haes). weekday total activity (wdta), expressed as hours/day (h/day), was calculated from the sum of 'very active' (wdva) and 'somewhat active' (wdsa) categories as previously described. a repeated measures linear regression was performed to evaluate the relationship of fev and hpa over time. results: at baseline, mean fev was % predicted (range - % pred). mean baseline wdta was . h/d (wdva = . h/d and wdsa = . h/d) similar to that previously reported. subjects were divided into high and low activity groups based on the group median wdta. those in the low activity group had a significantly (p= . ) steeper rate of decline (- . % predicted per year) compared to those in the high activity group (- . % predicted per year) . unlike in the in the previous study this difference was observed in males as well as females. conclusions: six year follow-up of this patient cohort has shown that higher activity levels are clearly associated with a slower rate of decline of fev . we are currently evaluating whether strategies aimed at increasing physical activity will have an impact on lung function decline in cf patients. background: knowledge of exercise principles, benefits, and techniques has not been assessed in children with cystic fibrosis (cf) or their parents. regular exercise is a recommended cf treatment regimen that is often not followed, but one which might improve cardiovascular fitness, lung function, sputum clearance, and health-related quality of life. further, aerobic fitness, measured as peak oxygen uptake (vo ), is positively related to survival in cf. construction of a reliable and valid measure of exercise knowledge is necessary to assess level of knowledge and study its relationship to exercise behavior. the aim of this study was to develop and test a measure of exercise knowledge for pediatric patients with cf and their parents. methods: we constructed a -item exercise knowledge test (ekt) and then examined its psychometric properties. a comprehensive review of the exercise literature guided the formulation of objectives and development of items. to establish content validity, a panel of experts (cf physician, two exercise physiologists, and a clinical cf nurse) evaluated the objectives and test items for relevance, clarity, and accuracy. after feedback from the panel, we revised the ekt through an iterative process. in consultation with a reading level expert, we wrote the ekt items so that the test could be administered to children and adults. a measurement and evaluation expert also reviewed the test, and additional revisions were made. during routine clinic visits, the ekt was administered to subjects with cf and parents. the cf subjects included boys and girls, age to years, fev range: - % predicted. the parents had an average age of years and most (n= ) were female. results: the ekt includes items, true-false and multiple choice. it is self-administered, yields a single summed score, and has a flesch-kincaid rd grade reading level. the means were . + . ( % correct) and . + . ( % correct) for the cf subjects and parents, respectively; % of the parents scored between and . internal consistency reliability estimates were . for the children and . for the parents, reflecting both the multidimensionality of the material and the lack of variability in the parent scores. item difficulty ranged from . to . for the cf subjects and from . to . for the parents. in both groups, the discrimination values were positive for all items with difficulty values less than . . conclusions: we were able to develop a measure of exercise knowledge with an acceptable reading level for children with cf and their parents. this sample of cf subjects and parents showed substantial exercise knowledge. the ekt can be a useful tool to identify gaps in exercise knowledge in cf patients and their parents, develop educational programs to increase knowledge, and examine how knowledge relates to exercise behavior in children with cf. supported by cystic fibrosis foundation therapeutics, inc. purpose: although physical activity is routinely recommended for adolescents with cystic fibrosis, past research suggests that many patients do not regularly participate in physical activity. there may be a variety of reasons, both related to disease and otherwise, for this inconsistent participation. in this study, qualitative methods were used to describe factors that facilitated or served as barriers to physical activity in adolescents with cf and compared these factors to those in a healthy peer group. methods: ten subjects with cf and ten subjects without cf aged - volunteered for this study. each subject participated in two, open-ended semi-structured interviews with - weeks between each interview. subjects were questioned about their current and past physical activity as well as the benefits and barriers to participation. the adolescents with cf were also asked about their perceptions of physical activity on their own health and other adolescents with cf. interviews were transcribed and coded by three investigator groups. analysis was conducted using the grounded theory approach at two points in the study, following each interview and again at the completion of all interviews. at the first point, individual interviews were coded using line-by-line coding, and similar information was grouped into categories. in the second interview, categories were confirmed and subjects were also asked to elaborate or clarify points from the first interview. interviews were continued until themes reached a saturation point. upon completion of all interviews, data for each group were organized by question. data categories appearing throughout the interviews became major themes describing facilitators and barriers to exercise. the major themes were identified by the three researcher groups and then through collaborative group analysis. results: the central theme for both groups was perceived importance of physical activity for health benefits. for the cf group, facilitators and barriers were both psychological and physical. physical benefits were categorized as either relating to their general health (such as feeling energized) or as benefiting their disease (such as improving breathing). in the non-cf group, facilitators included social factors in addition to general health-related and psychological benefits. in the non-cf group, barriers were categorized as either internal or external, with the internal barriers being similar to the cf group (physical and psychological), however, the non-cf participants also indicated external barriers (such as time) not articulated by the cf group. conclusions: in general, we found very similar facilitators and barriers to physical activity in adolescents with cf and those without. however, the adolescents with cf strongly indicated that physical activity would have positive effects on their physical health. further work is needed to determine ways to accentuate facilitators and remove barriers to improve physical activity levels in adolescents with cf. this project was supported by a research grant from the cardiovascular & pulmonary section, apta. lloyd, e. ; dodd, m. . paediatric cystic fibrosis service, central manchester and manchester children's university hospital, manchester, united kingdom; . adult cystic fibrosis centre, wythenshawe hospital, university hospital of south manchester nhs foundation trust, manchester, united kingdom background: training in the skills of communication and self care are considered important components of transitional care . physiotherapists work to involve families and young people to encourage this independence. the cf manager training programme (cfmtp) is a recognised tool for measuring age appropriate tasks in cf care. aim: the aim of the study was to survey the current levels of autonomy in all patients with cystic fibrosis in the north west of england from birth to years to provide a baseline measure for encouraging autonomy in preparation for transition. method: three areas of the cfmtp were selected for the survey: physiotherapy (p), inhalation therapy ( it) and communication (c) for the age groups: - . , . - , - , - , - , - , - , - , - ,and + years. physiotherapists from all the north west cf clinics were invited to assess all the patients under their care. data collected were age, gender and whether the patients skills fell "below", "same" or "above" the age appropriate self management tasks as described in the cfmtp. 'not applicable' was indicated where the skills did not apply. results: / clinics responded. out of a total of patients in the nw were assessed, three patients were excluded. population by clinic ranged from - . population by age category: ( - . years) ( . - years) ( - years) ( - years) ( - years) ( - years) ( - years) ( - years) ( - years and ( + years). / ( %) were male. for p (n= ), it (n= ), c (n= ), %, %, % = below, %, %, % = same, %, %, % = above respectively. there were no gender differences. conclusions: overall scores revealed that / rd of patients were gaining age appropriate independence in all areas. there was the least autonomy with it. it was rare for a child to be setting up and cleaning equipment regularly at - years, but this continued throughout the adolescent years. overall % were 'below' for p and it; this was highest after the age of years. autonomy with physiotherapy appeared slow to develop. some categories in cfmtp were different from our practice for e.g. patients are not invited to be seen alone in our clinics until years v - years and this may account for the lower scores in communication below this age. this study has highlighted that some of our patients are not fully independent in the time up to transfer. we need to encourage independence from an early age with all aspects of care and integrate the process into our routine clinical practice. refs mcdonagh and viner bmj ; : - , . parents' guide to the cf manager training programme. children's hospital of eastern ontario planner, s. ; morrison, l. . physiotherapy, gartnavel general hospital, glasgow, united kingdom; cystic fibrosis (cf) causes a deterioration in lung function and exercise tolerance. assessing exercise capacity is therefore essential in order to monitor changes in disease severity. the -minute walk test ( mwt) and minute step test ( mst) are validated and widely used outcome measures for identifying exercise capacity in patients with cf. exercise testing is part of annual review of all patients in the west of scotland. previous studies identified that the mst was a sensitive measure of exercise tolerance, but did not successfully challenge patients with mild or moderate lung disease as defined by fev(sub) (/sub)> % (morrison ) . further analysis of the mst, where step height was increased to inches did not show any difference in sao(sub) (/sub). decline or rate of perceived exertion (rpe) in patients with mild disease. the chester step test (cst) was developed to assess aerobic fitness which is used in the uk cardiac population for exercise prescription (sykes ) . it is a minute progressive, sub-maximal test with a variable step height ( - inches) , making it suitable for those with a wide range of exercise capacities. in addition, cst results extrapolate to a mets value (oxygen utilisation: met = rate of oxygen utilisation at rest) as an outcome measure which can facilitate exercise prescription based on established values for physical activities. we aim to determine whether the cst is a more sensitive measure of exercise capacity than the mwt or mst when considering decrease in sao(sub) (/sub) and rpe. patients ( aged - mean fev(sub) (/sub) . l) with mild-moderate cf performed the cst and mwt and results were correlated with fev(sub) (/sub). decrease in sao(sub) (/sub), increase in hr and rpe scores were also compared between the cst, mwt and recent mst data. patients were exercised to a symptom limited maximum which differed from the cardiac cst where patients are exercised to % of maximal hr. the cst appears to be more challenging as only patients completed it, all with an fev(sub) (/sub)of > % predicted, whereas in the mst and the mwt all patients completed the test. mets levels for the cst were weakly correlated with fev(sub) (/sub)(r= . ) and the mwt (r= . ) . this could be explained by the narrow range of mets data available or the size of the sample population. there were significantly greater rises in hr (p= . , p= . ), decrease in sao(sub) (/sub) (p= . , p= . ) and rpe (p= . , p= . ) , between the cst, mst and mwt respectively, suggesting that the cst is a more sensitive measure of cardiorespiratory effort. conclusions the cst appears to be a more challenging exercise test for those with mild to moderate disease and therefore may be a better reflection of exercise capabilities. cst results are useful for prescribing exercise and monitoring programmes appropriately for this patient group. further study could determine whether the cst is also suitable for those with severe disease. lee, a.l. ; button, b. ; holdsworth, m. ; holland, a. . physiotherapy, the alfred, melbourne, vic, australia; . latrobe university, melbourne, vic, australia musculoskeletal pain is prevalent in cystic fibrosis (cf). while the chest and back are frequently reported regions of acute or chronic pain, it is unclear if pain in these areas is related to disease severity or respiratory symptoms. manual therapy and postural advice has been shown to alleviate pain, but the effects of a combined approach of musculoskeletal physiotherapy and massage has not been studied. the aim of this study was to identify the primary regions of musculoskeletal pain, the relationship between pain severity and respiratory symptoms and to examine the effect of musculoskeletal physiotherapy techniques and soft tissue therapy, including remedial massage on pain and ease of breathing (eob) in adults with cf. one hundred and twenty-nine adults with cf ( with acute exacerbation, post lung transplant) aged ± years (mean ±sd) with fev(sub) (/sub) of ± % participated in this study. following assessment of primary pain regions, each subject underwent a single treatment session including spinal joint / intercostal mobilisation and soft tissue therapy. pain and eob on a visual analogue scale were measured before and after treatment. changes were compared using paired-samples t-tests. pain was frequently reported in the thoracic spine region ( % of subjects), followed by the shoulder region ( %), cervical spine region ( %) and chest wall region ( %). equivalent ratings of pain and eob prior to treatment were reported for all regions of pain. eob rating prior to treatment were worse in those with low bmi (r=- . , p= . ) and low fev(sub) (/sub) (r=- . , p= . ). a single treatment session was associated with reduction in pain ( %ci . to . , p< . ) and improvement in eob ( %ci . to . , p< . ), irrespective of clinical status. improvement in pain was equivalent for all primary pain regions. however, greater improvement in eob was evident in subjects with shoulder pain compared to other regions ( %ci - . to - . , p= . ). a combination of musculoskeletal physiotherapy techniques and soft tissue therapy reduces musculoskeletal pain and improves eob in adults with stable cf, during an acute exacerbation and post lung transplantation. service cystic fibrosis is characterized by an excessive production of airway secretions responsible for bronchial obstruction and recurrent infections of the respiratory tract. the transport by ciliary activity and cough may be dependent on the unsteady rheological properties of the respiratory mucus such as thixotropy and shear-thinning properties which correspond to a decrease of viscosity with time or flow rate, respectively. we have previously shown that respiratory mucus with high shear-thinning and thixotropic properties is better transported by the cough mechanism (zahm et al, eur. respir. j., ) . to promote mucus clearance and to finally expectorate the mucus by a cough manoeuvre, an innovative device, the airhelp, has been developed and aimed to improve chest physiotherapy effectiveness. during a prolonged expiratory manoeuvre, the airhelp delivers negative pressure variations (maximal amplitude: - kpa) at an oscillatory frequency of hz. the aim of the present work was to test the effect of the airhelp device on the in vitro clearance of mucus by airflow. a plexiglass tube of mm in diameter and mm in length has been connected by one part on the airhelp and by the other part on a l thank mimicking the lung compliance. a µl drop of mucus simulant at different concentrations (viscogum at . % or . % with actigum at . % to . %) or of respiratory mucus, was deposited within the plastic tube and the distance travelled by the sample under a sec-airflow was measured. twelve respiratory mucus samples collected from cf patients were analyzed and the viscosity of the samples was measured before and after the airhelp experiment. we observed a significant (p < . ) and negative relationship (r = - . ) between the viscosity of mucus simulants and oscillatory airflow transport: the lower the viscosity, the higher the mucus transport by oscillatory airflow. using cf respiratory mucus samples, we observed that without the oscillatory airflow, no transport occurred, whereas when the samples were submitted to the oscillatory airflow, a significant increase in mucus transportability was measured. in addition, the viscosity of the cf respiratory mucus samples was significantly decreased after oscillatory airflow treatment ( ± pa.s to ± pa.s, p < . ). these results demonstrate that the mucus transport efficiency by an oscillatory airflow is dependent on mucus viscosity and that the improvement in mucus transport is related to the thixotropic and shear thinning properties of the airway mucus. we conclude that the airhelp device may improve chest physiotherapy effectiveness. potter, e. ; nufer, j. ; cullina, j. , ; quinton, h. ; jain, m. , ; mccolley, s.a. , . northwestern university feinberg school of medicine, chicago, il, usa; . children's memorial hospital, chicago, il, usa; lebanon, nh, usa; . northwestern memorial hospital, chicago, il, usa socioeconomic status (ses) significantly impacts health outcomes in cystic fibrosis (cf). the cystic fibrosis foundation (cff) is currently using median income estimated by zip code from the us census of the population for ses risk adjustment of data reported publicly from the cff patient registry. median income estimated by zip code is an "ecologic" variable that may misclassify patient and family income. in order to more fully assess the impact of ses on cf health outcomes, the cff began to ask centers to collect data on household income directly from patients and families. these data were captured for . % of patients and families in and . % in in the national registry. we report a method of collecting ses data from children and adults implemented at the children's memorial hospital and northwestern university cystic fibrosis center, and compare directly collected income to income based on zip code. methods: a short survey with the questions on household composition, income, and education level from the cff patient registry was developed. a letter from the center director and adult program director was mailed to patients and families informing them of the survey's purpose and confidentiality measures, i.e. responses would be seen only by the individual entering the data and identified by cff id number. the survey was distributed to cf patients and families in clinic by clerical staff. completed surveys were placed in an envelope and sealed by the patient or family. both directly collected income and income from zip code was classified into groups (<$ k, - k, - k, - k, - k, - k, - k, - k, and > k) . percent concordance and spearman's rank correlation were calculated. data were used for patients who provided data in ; otherwise, data were used. each patient's data was used only once in the analysis. results: unique patients were identified, pediatric and adult patients. eighteen percent of pediatric and % of adult patients preferred not to answer income questions or did not complete the survey. the mean income by zip code was $ k for patients who did not report income data and $ k for the entire group. for the responses, reported income data was moderately correlated with income by zip code (rho= . , p< . ). concordance between the categories was %. the correlation was stronger for pediatric patients (rho= . , p< . ) than for adults (rho= . , p= . ). re-classifying income as "low", "medium" and "high" (<$ k, $ - k, >$ ) led to a concordance of %. conclusion: survey methods that protect patient and family confidentiality result in good return rates of full ses data from patients and families. zip code derived median income shows moderate concordance with reported income. comparing the associations of the forms of income data with important cf outcomes needs to be done in a larger data set. we acknowledge the cystic fibrosis foundation, gerald o'connor, kathryn sabadosa, and participating patients and families. the patient registry provided a unique opportunity to assess changes in the epidemiology of respiratory pathogens in cf. while the incidence of p. aeruginosa remained stable, the prevalence decreased, suggestive of the impact of antibiotic eradication strategies. in contrast, the increasing incidence and prevalence of s. aureus and mrsa may reflect improved microbiological surveillance and laboratory techniques. the trends noted for b. cepacia complex may reflect successful implementation of infection control strategies. future studies are needed to better define the association between these observed trends and improving care for cf patients. vanderwyden, a. , ; drumm, m.l. , ; schluchter, m. . pediatrics, case western reserve university, cleveland, oh, usa; . epidemiology and biostatistics, case western reserve university, cleveland, oh, usa; . genetics, case western reserve university, cleveland, oh, usa in the genetic modifier study (gms) of lung disease of cystic fibrosis (cf), patients, homozygous for the ∆f mutation, were classified as having either severe or mild lung disease, as defined by the lowest or highest quartile of forced expiratory volume in one second (fev ) and a linear model was fit from these patients' longitudinal data (drumm , schluchter . while these models successfully dichotomized patients for the association study, we assessed whether a single slope model could be improved. here we have begun to compare models with one, two and three slopes, each representing different age ranges, for their use in this type of association study. as an example, interleukin- (il- ), a neutrophil chemoattractant, associates with the severe lung inflammation seen in cf patients. we have observed the tt genotype of the il- snp rs , correlated with severe pulmonary function in the gms sample of patients, relative to the aa and at genotypes of this snp. in a replicate study of patients, with cftr genotypes associated with exocrine insuffiency, longitudinal linear mixed models were used to further characterize the decline in lung function over time as associated with the il- snp, rs . three linear regression models of fev % predicted of patients were fit over age and compared with likelihood ratio tests. the three models differed with respect to the number of nodes. the first model had a single slope while the second and the third were piecewise linear models, with nodes at age , and ages and , respectively. age was chosen as the first node, as by this age most cf patients have reached puberty, an event believed to coincide with changes in pulmonary function. the second node, age , was arbitrarily chosen as a point in the third decade, as survivor effect becomes a significant issue during this period. when compared to the single slope and two slope models, the three slope model with nodes at age and age better explained the pulmonary function of this study (p< . , - loglikelihood chi-squared tests). when the explanatory variable, il- rs genotype, was added, the single slope model and the three slope model were again compared. the three slope with nodes at age and age again resulted in a better fit of fev % predicted over age as correlated with rs genotype (p< . , - loglikelihood chi-squared test). estimates of fev % predicted at ages , , , and for each rs genotype (aa, at/ta, or tt) were determined using the three slope model (p< . for all estimates). other explanatory variables, like gender and survival (p< . ), were added to further elucidate the relation between il- genotype and lung disease phenotype. we propose to use this piecewise three slope model with nodes at age and age to look at the pulmonary function of patients in the cf foundation registry data to better explain the decline in fev % predicted over time seen in cf patients. (supported by hl- and grants from the cf foundation) stephenson, a.l. purpose: the purpose of this study is to determine the annual incidence of hospitalizations for individuals with cf living in ontario and to examine predictors of hospitalization, specifically gender. methods: this is a retrospective cohort study from to , using newly linked clinical and administrative databases. the canadian cf foundation patient data registry (cpdr), contains detailed clinical information on all cf patients receiving care at one of accredited cf centres across canada. the institute for clinical evaluative sciences (ices), holds linked administrative databases containing information on all publicly reimbursed health care services delivered in ontario. canadian institute for health information (cihi) discharge abstracts database (dad), ontario health insurance plan (ohip), and homecare databases were used for this study. the cpdr was probabilistically linked (automatch software) to the ices administrative database using name, gender and date of birth. all individuals were then linked to the cihi dad, ohip, and homecare databases using unique numeric identifiers. with these databases, longitudinal records for each individual were created. cihi-dad, ohip, and homecare claims data were used to calculate the number of hospitalizations per year for pulmonary infections. males and females were analyzed separately to elucidate any gender disparities. other predictor variables include age, geographical location, number of clinic visits, socioeconomic status (income quintile) as well as clinical variables such as pulmonary function tests, nutritional status, diabetes mellitus, sputum bacteriology and pancreatic status. results: the cpdr contained data on individuals with cf followed within canada between - . of those individuals, could be found within the ices administrative databases using probabilistic linkage. of those individuals matched to the administrative databases, ( . % female) had hospitalizations. a total of , ohip claims were made during this -year period which represents any encounter between an individual with cf and a physician in ontario. multivariable analysis of hospitalization predictors is in progress. zhang, z.; lai, h. uw-madison, madison, wi, usa the cff practice guidelines recommend adjusting for genetic potential when evaluating height status in children with cf. however, calculation of adjusted height percentile is not recommended due to the complexity of method involved in such calculation. instead, a simple method to estimate a target height based on mid parental height was recommended. however, this target height includes a -cm confidence interval, which spans most of the channels on the growth chart. therefore, a child's unadjusted height may be considerably below the target height, yet remains above the lower bound of the target height range. in addition, there is a paucity of data on the associations between adjusted height to lung disease outcomes to justify whether adjustment for genetic potential is necessary. the objective of this study is to compare two methods of incorporating genetic potential in identifying short stature, namely, the cff's target method and the method developed by himes et al (pediatr ; : - ) , which applies positive adjustments to children with short parents and negative adjustments to those with tall parents. data from - cff registry were utilized. a total of children born between - who had complete height data from age - years and their parental height data were analyzed. short stature was defined by unadjusted height < th percentile, unadjusted height below the lower bound of cff's target height, or himes' adjusted height < th percentile. our results showed that adjusted height percentiles are consistently lower than unadjusted height percentiles, with an average difference of . z-score unit between ages - years and . z-score unit between ages - . proportionately more children were classified as short stature based on himes method compared to unadjusted height ( % vs. %, p = . ). in contrast, proportionately fewer children were below the lower bound of cff's target height ( %) compared to unadjusted height < th percentile ( %), p < . . among children with average parents, unadjusted and adjusted height percentiles were similar. however, among children with short parents (< th percentile), the percentage children with of short stature decreased from % with unadjusted height to % with himes' adjusted height and % with cff's target height method. among children with tall parents (> th percentile), the percentage of short stature increased from % with unadjusted height to % with himes method and % with cff's target height method. taken together, these results demonstrate that, without incorporating genetic height potential, - times more cf children who have short parents would be classified as short stature, while times fewer cf children who have tall parents would be classified as short stature. further analyses show that, compared to unadjusted height percentile, himes adjusted height percentile is more sensitive to, and has stronger association with, percent predicted fev- . in conclusion, our findings provide evidence that genetic potential should be incorporated in evaluating short statue in cf children, particularly for children with short or tall parents. the cff's target height method and himes method differ significantly in identifying short stature in cf children with short parents. cohort study of individuals in the cff patient registry from - . all individuals years of age or older and diagnosed before age were included. the new, persistent mrsa infection cohort was defined by having at least two cultures negative for mrsa over a two-year lead-in period followed by at least two mrsa positive cultures. individuals with only one mrsa culture (transient infection) were excluded. we developed multiple linear regression models using generalized estimating equations to assess the effect of mrsa on fev %predicted (%fev ) and rate of change of %fev . results: during the study period , individuals cultured mrsa. of these, , ( %) cultured mrsa only once (transient infection) and were excluded from subsequent analysis. , individuals met the criteria for new and persistent mrsa infection. , met the criteria for never having mrsa resulting in a total study cohort of , . participants were followed for a median of years. the median time to first isolation of mrsa in the persistent mrsa cohort was . years and the mean number of positive mrsa cultures per patient was . . a comparison of baseline characteristics between the persistent mrsa and never mrsa cohorts revealed the mrsa cohort to be younger ( . vs . years p< . ), equivalent in lung function (%fev . % vs . % p=ns), and with higher likelihood of methicillin sensitive staph aureus colonization ( % vs. . % p< . ). . years into the study period (the median time of acquisition for new mrsa), the mrsa cohort had a lower %fev ( . % vs . % p< . ), and averaged twice as many hospital admissions per year ( . vs . p< . ) and home iv courses per year ( . vs . p< . ). in an unadjusted analysis, the presence of mrsa was associated with a mean %fev that was . % lower than the mrsa cohort ( %ci . %- . %). however, after adjusting for sex, age, pancreatic status, p. aeruginosa, and b. cenocepacia, the presence of mrsa was associated with a mean %fev . % lower ( %ci . %- . %). most importantly, an analysis investigating the interaction between mrsa and time, after adjusting for confounders, indicated no statistically nor clinically meaningful difference in the mean rate of lung function decline between the two groups. conclusions: approximately one-third of patients who culture mrsa do so only once and do not subsequently develop persistent mrsa infection. mrsa infection occurs in a group with more severe structural lung disease as measured by fev , and frequent hospitalizations and iv antibiotics are strongly associated with mrsa acquisition. after adjusting for confounders, mrsa infection is not statistically significantly associated with a more rapid decline in %fev over time. simmonds, n.j. , ; macneill, s. ; cullinan, p. , ; hodson, m.e. , . department of cystic fibrosis, royal brompton hospital, london, united kingdom; . national heart and lung institute, imperial college, london, united kingdom introduction: disentangling the influence different factors have on long-term survival in cf is complex. there are a myriad of influences (including environmental, healthcare-related, psychological and socioeconomic) which may be important, as there is generally a poor genotype-phenotype correlation. the aim of this case-control study was to identify these factors. the cf database at the royal brompton hospital was used to identify long-term survivors. they were classified as cases and were patients who had reached years of age (without transplantation). each case was agematched with at least one control who died of cf (or a cf-related condition) or was transplanted before reaching years of age. late diagnosis patients were excluded. conditional logistic regression models were used to identify potential influences on survival. results: cases and controls were analysed, producing matched pairs. % of the patients were pancreatic insufficient. of the many factors investigated, those resulting in increased probabilities of survival included: increased body mass index (or= . , % ci . - . ), fev (or per % increase= . , ) at transfer to the adult clinic (after adjusting for age and sex) and the use of oral antibiotics before attending the adult clinic (or= . , . - . ) after adjusting for age at first attendance. factors associated with a reduced probability of survival included: cf diagnosis < years old (or= . , . - . ); initial presentation of chest symptoms or malabsorption (or= . , . - . and or= . , . - . , respectively); referral from a paediatric clinic in a deprived area (or= . , . - . ); pneumothorax before transfer to the adult clinic (or= . , . - . ) after adjusting for age at first attendance; and s. aureus or p. aeruginosa colonisation before years of age (or= . , . - . and . , . - . , respectively). factors not influencing survival included: sex, h. influenzae colonisation before years of age, development of diabetes before years of age, major haemoptysis before transfer to the adult clinic, parents' occupation, number of siblings (cf and non-cf) and school achievements. in a very carefully matched study we failed to identify major non-clinical determinants of long-term survival in early diagnosis cf. findings suggest that the majority of significant factors were directly related to optimal physical parameters, the use of oral antibiotics, avoiding bacterial colonisation and a low pneumothorax rate. adler, a. , ; bilton, d. ; haworth, c. ; gunn, e. ; shine, b. . addenbrooke's cambridge university hospitals, cambridge, united kingdom; . papworth hospital, papworth everard, united kingdom; . oxford centre for diabetes and endocrinology, oxford, united kingdom background and aims: although the prevalence of diabetes in patients with cf (cfrd) is high, few prospective studies exist, and even for genet-ic factors, cross-sectional studies may bias the magnitude of associations. this study sought to identify risk factors for cfrd from a predominantly white cohort of individuals with cf in the united kingdom. methods: , individuals aged - were identified from the uk cf database, a registry of patients coordinated by the uk cystic fibrosis trust with data from over specialists centres from england, scotland and wales, countries which provide medical care free of charge. of the patients, , had no diabetes at baseline ( - ) , and also had at least one follow-up annual visit. diabetes was defined as either a physician diagnosis of diabetes, a blood glucose of > . mmol/l two hours following an oral glucose tolerance test, or treatment with insulin or oral hypoglycemic drugs. follow-up was calculated as time from baseline to the first detection of new diabetes or censoring. risk factors included clinical, genetic (functional classes i,ii,iii vs iv,v), and anthropometric characteristics measured at baseline. survival analyses were limited to patients with complete data. cox proportional hazards modeling was used to estimate the magnitude of the association between potential risk factors and incident diabetes. one-way interactions with sex were tested. results: the median age of entry into the cohort was years and bmi was . kg/m . % were male, and % were white. patients developed diabetes during , person-years of follow-up (mean . years); the incidence was . % per year. patients who developed diabetes were more likely in univariate analyses to be older, have a high body mass index (bmi), bacterial pulmonary infections, liver and pulmonary function abnormalities, poor oxygen saturation, a history of organ transplantation, supplemental feeds, and take pancreatic enzymes or bile acids. in addition, patients with class iv,v genotypes were less likely to develop diabetes relative to patients with other genotypes, as were patients screened for cf at birth. not associated with incident diabetes was age at diagnosis of cf and ethnicity. in multivariate analyses (n= , ) age (hazard ratio . per year, % ci . - . ), female sex ( . ( % ci . - . ), % predicted forced expiratory volume in second ( . , . - . per % decrement), dose of pancreatic enzyme replacement ( . , . - . ), above vs below median dosage, use of bile acid supplements ( . , . - . ) , and genotype class i,ii, iii ( . , . - . ) relative to class iv/v. these associations were not confounded by bmi or history of transplantation. there were no significant interactions between sex and other risk factors. conclusions: the study confirms the high incidence of cfrd, and shows that patients with class iv/v genotypes are less likely to develop diabetes independent of other cf-related complications. females are disproportionately at risk for diabetes which is not explained by a higher prevalence of known risk factors nor by effect modification. cystic fibrosis children are at increased risk for hearing disorders due aminoglycoside exposure, mucopurulent upper respiratory tract infections, and increased inflammatory responses. pure tone audiometry is the standard instrument used to determine hearing loss. however, it can be expensive and time-consuming to use audiometry for routine hearing screening. hearing questionnaires have been used as a screening tool to assess possible hearing loss in otherwise healthy populations. what are the results of hearing questionnaires administered to pediatric cf patients? does cf patient age or ethnicity increase the likelihood of hearing loss as determined by a hearing questionnaire? a hearing questionnaire (english and spanish versions) was administered to pediatric cf patients over a week period at our accredited cf center. data collected included demographic information. inclusion criteria: age м years and in baseline health status. possible hearing loss was defined as at least "yes" responses (> ) to the quesions administered to the patients. fisher's exact test was used to perform the analyses. a total of cf patients were seen in the cf clinic during the study period. cf patients completed the hearing questionnaire. mean age was . ± . years (median . years). there were males and females; were classified as hispanic and were classified as caucasian. patients were older than . years (the median age) and patients were less than . years. patients who were older than age . years had a score of > and patients < age . years had scores > (p= . , ns). thus, of cf patients ( %) were classified as having possible hearing loss as per their questionnaire scores. of hispanic cf patients had hearing scores > and of caucasian patients had hearing score > (p= . , ns) . we found that a significant proportion ( %) of our cf children may require formal hearing evaluation. we speculate that hearing questionnaires may be useful to screen for hearing loss in the cf patient population. hispanic cf patients have increased morbidity and mortality risks compared to caucasian cf patients who attend our accredited cystic fibrosis center in southern california. is the increased morbidity/mortality risk due to decreased access to outpatient cf care? we performed a retrospective review of all patient visits to our cf clinic between january , through december , . data collected included demographic information (age, ethnicity, gender) , and the number of clinic visits during the study period. data was analyzed by unpaired student's t-test as well as by chi-square. during the study period, a total of cf patients ( males: females; mean age . ± . years; range months to years) were routinely followed in our cf clinic. these patients had a total of outpatient visits to the cf clinic ( . clinic visits/patient over the year study period). there were hispanic cf patients ( males: females; mean age . ± . years) and caucasian cf patients ( males: females; mean age . ± . years; p= . , ns). there was no difference in gender distribution between the group (p= . , ns). hispanic patients had a greater number of cf clinic visits ( . visits/patient) as compared to caucasian patients ( . vists/patient; p= . ) during the same period. we conclude that the increased morbidity and mortality of hispanic cf patients in california cannot be attributed to barriers to outpatient care. in fact, hispanic cf patients were seen more frequently in cf clinic compared to caucasian cf patients who attend the same clinic. we speculate that hispanic cf patients are seen more frequently in cf clinic due to increased severity of cf disease manifestations. duguépéroux, i. ; scotet, v. ; audrézet, m. , ; blayau, m. ; parent, p. ; journel, h. ; boisseau, p. ; férec, c. , . inserm u , brest, france; . dep. of genetics, brest, france; . dep. of genetics, rennes, france; . unit of medical genetics, brest, france; . unit of medical genetics, vannes, france; . dep. of genetics, nantes, france aim: the aim of this study was to describe -year experience of prenatal diagnosis (pd) for cf in brittany (western france) and to assess its impact on incidence. method: we registered, by the genetic laboratories of our region, all the pds performed in women living in brittany over the period - . we described the number of pds made for each reason (way by which the one-in-four risk was identified: previous affected child, family testing, echogenic bowel, etc). we reported the proportion of cf-affected fetuses and of consecutive terminations, and assessed the incidence modification due to pd. results: over the -year period, a total of pds were performed in couples living in brittany. most of them were done in couples already having cf child(ren) (n= , . %). extended testing in families -a practice largely proposed in brittany -led to the identification of new onein-four risk couples among the relatives of cf patients who opted for pd times over the study period ( . %). the other pds were made in couples without previous history of cf. the one-in-four risk was mainly identified following the detection of an echogenic bowel during pregnancy ultrasound examination ( pds were done directly following the positive ultrasound ( . %), whereas others were done for subsequent pregnancies ( . %)). the other pds were consecutive to the detection of an heterozygote through newborn screening (n= , . %) or for an other reasons (n= , . %). over the study period, the number of pds per couple varied between one and five, the mean being . . overall, a total of cf fetuses were identified, among whom were terminated ( . %). the inclusion in the incidence calculation of these pregnancy terminations led that to an incidence modification of . % over the study period. conclusion: this study shows that pd for cf is commonly used in brittany and highlights the impact of family testing and of routine ultrasound examination of pregnancies in that region. supported context: hispanics with cystic fibrosis (cf) in the united states experience an % increased annual risk of death from cf compared to non-hispanic patients. when adjusted for socioeconomic status this disparity persists. studies characterizing this at-risk population do not exist. objective: to characterize the center-reported us hispanic population with cystic fibrosis through a cystic fibrosis foundation (cff) patient registry analysis and to elucidate factors present within this hispanic population that may affect outcomes. design: retrospective cross sectional study of the cff patient registry. patients: all , patients in the cff patient registry were included in the analysis. there were no exclusion criteria. the hispanic population is defined as those who were entered by their respective care center as hispanic ethnicity regardless of race. main study measures: prior to analysis, study measures were selected to demographically and genetically characterize the hispanic cf population. genotype, state of residence, mean age and mean age of diagnosis were obtained in the entire population. maternal education (for patients < years of age) and insurance status data were captured as indicators of socioeconomic status. complication rates, fev percent predicted (under years of age) and absolute fev (over years old of age) were obtained as measures of clinical status. t-tests and logistic regression analyses were used to compare measures between hispanic and non-hispanic groups. odds ratios (or) and % confidence intervals are reported. results: center-reported hispanic patients with cf were identified. over % of hispanic patients resided in california, florida, texas or new york. the most common genotype was delta f homozygous, accounting for % of the population. mean age was . years +/- . years for hispanic patients and . years +/- . years for non-hispanic patients. hispanic patients were diagnosed at an earlier age ( . years vs. . years, p= . ). they were more likely to have mothers with education at less than a high school level (or . ( . , . ) ) and have government insurance (or . ( . , . ) ). complication patterns differed between the two groups; non-hispanics were more likely to have complications reported that were related to depression (or . ( . , . ) ), bone health (or . ( . , . ) ) and cf related diabetes (or . ( . , . )) compared to hispanics. hispanics however, were more likely to have complications reported from cf liver disease (or . ( . , . ) ). pulmonary function, as measured by fev percent predicted in children (p< . ) and absolute fev for adults (p= . ) were lower for hispanic patients. conclusions: differences exist between hispanics and non-hispanics with cf with respect to maternal education, insurance status, complications rates and pulmonary function. the lower reported rates of depression, bone complications and cf related diabetes may represent ascertainment bias. further study is needed into the etiology of health outcome disparities in this population, and to design interventions for this high-risk population. mastella, g.; baldo, e.; forneris, m.; furnari, m.; lucidi, v.; manunza, d.; marinelli, i.; messore, b.; neri, a.; raia, v.; salvatore, d.; buzzetti, r.; cairo group, italian cf research foundation, verona, italy with our work we reviewed the international scientific literature coming from cf registries worldwide. our aims were: to verify what has been produced in scientific literature thanks to the material derived from fc registries and to see which clinical problems have been tackled and which clinical questions were answered correctly and exhaustively. a search in medline and embase produced articles (our strategy was "cystic fibrosis"[mesh] and ("registry"[mesh]) or registr$) updated to the / / ). out of these articles have been selected by two independent assessors on the basis of their pertinence (primary studies that used data taken from a formally established registry, at least national, to test some hypothesis of research). each article has been examined with the help of a pre-defined form that in particular evaluated skills of different registry data, selected clinical characteristics of the study populations and statistical methods. more than one half of cases data came from usa-cff foundation patient registry, the remaining coming from canadian, uk, french, italian, german, and other european registries. the main focuses of the articles were included in the following topics: ) incidence/prevalence of the disease and survival ( studies); ) neonatal screening, growth and nutrition ( ); ) genotype/phenotype correlation/ different ethnical groups and twin/brothers ( ); ) complications and outcomes (abpa, diabetes, nasal polyposis, pregnancy and paternity, etc) ( ); ) microbiology ( ). our assessment scores were good/excellent for / studies for the relevance of the problem addressed; / for the usefulness of the outcomes and / for the usefulness of the implications for research from the italian registry. thirty-two articles gave a significant contribution to the analysis of the studied problem, but of them left a partial uncertainty, while left complete uncertainty. cf registries are a very important source of data to provide a powerful tool for clinical and research questions. however, further comparable studies have to be planned to assess registry data as a platform for improvement in clinical practice. vandenbranden, s.l. ; mcmullen, a. ; konstan, m. ; morgan, w. ; wagener, j. ; schechter, m. ; watts, k. , ; mccolley, s. , . children's memorial medical center, chicago, il, usa; . emory university, atlanta, ga, usa; . university of arizona, tuscon, az, usa; . case western reserve university, cleveland, oh, usa; . northwestern university feinberg school of medicine, chicago, il, usa; . university of colorado, denver, co, usa; . university of rochester, rochester, ny, usa background: although life expectancy in cf has dramatically improved over the last years, most of the improvement is due to improved survival during early childhood. adolescence and young adulthood continue to be a time of worsening pulmonary disease and mortality. study objective: data from a large longitudinal observational study, the epidemiologic study of cystic fibrosis (escf), was examined to characterize lung function decline in subjects with cf during the periods preceding and after the age of . design: escf data from individuals with cf collected during the period of - were analyzed. inclusion criteria required individuals to have a minimum of encounters in each of the . year time periods before and after the age of . the cohort was strati-fied by disease severity based on the best fev at the th year (+/- months). one hundred thirty seven subjects had severe lung disease (fev < % predicted), had moderate-severe lung disease (fev -< % predicted), had mild lung disease (fev -< % predicted), and had very mild disease (fev > % predicted) the study compared the annualized rate of fev decline during the adolescent period, defined as ages - . years, and in the young adult period defined as . - years. results: in the entire cohort, the annualized rate of decline was less in the young adult period than in the adolescent period (p< . ). the most dramatic improvement in slope of decline was seen in the severe group, and significant improvements were also noted in the moderate group (p< . ). the mild group demonstrated insignificant change; the very mild group was the only group that demonstrated a more negative annualized rate of the decline in young adulthood than in adolescence (p= . ). conclusions: these data suggest that, overall, young adults have a slower rate of decline in fev in the period after age . this "stabilizing" of lung function is most prominent in those individuals with moderate-to-severe lung disease. only those with very mild lung disease have a slightly more rapid rate of decline between the ages of and . overall there is less variation in the rate of fev decline across disease strata after the th birthday. further analysis is underway to better understand the factors leading to these findings. . department of paediatrics, university of florence, cystic fibrosis centre, florence, italy; . italian cystic fibrosis microbiology group, florence, italy; . institute of medical microbiology and hygiene, tübingen, germany mrsa is now a worldwide public health problem, due to the increasing rate of infection in several settings as well as in cf patients. mrsa was first recognized as being acquired from hospitalized patients (ha-mrsa), but the onset of mrsa infection outside the hospital setting, in communityassociated strains (ca-mrsa), has recently been described with increasing frequency. ha-mrsa are known to be responsible for infections in hospitalized patients, although highly virulent ca-mrsa are increasingly reported worldwide as the cause of severe outbreaks, replacing ha-mrsa strains in nosocomial infections. little evidence is available about the characterization (sccmec type) and epidemiology of community-acquired mrsa (ca-mrsa) or hospital-acquired mrsa (ha-mrsa), transmissibility, antibiotic susceptibility and virulence in the cf population. the present multicenter study investigated the susceptibility pattern, epidemiology, and sccmec type of mrsa strains isolated from nine italian cf centers. the susceptibility pattern was determined by the disk diffusion method and pulsed field gel electrophoresis (pfge) was performed for epidemiological purposes. the sccmec type in order to identify hospital-and community-acquired mrsa strains (ha-mrsa and ca-mrsa respectively) was determined with molecular methods, and the presence of gene encoding panton-valentine leukocidin (pvl) was tested. during the study period, mrsa strains were isolated from out of ( . %) patients attending these cf centers. of the sccmec type representing ha-mrsa, our data showed an high prevalence of sccmec i ( . %) while the prevalence of sccmec ii, (reported as the most represented among ha-mrsa) was only . %. furthermore a high prevalence ( . %) was found of sccmec iv (suggestive of ca-mrsa strains). epidemiological analysis showed that ( . %) out of analyzed mrsa strains belong to the same pfge clone shared among six centers, belonging to the sccmec type iv, suggestive of ca-mrsa isolates. twenty-four ( . %) out of these strains were sccmec type iv, indicative of ca-mrsa, as suggested by the good susceptibility rate to trimethoprim-sulfamethoxazole and rifampin. surprisingly all the isolates belonging to this epidemic clone were negative for the pvl genes. these results show that ca-mrsa is now spreading in the cf population, documenting the first epidemic of ca-mrsa in cf patients who are considered at risk for ha-mrsa acquisition due to frequent hospitalizations. these data show a peculiar picture of mrsa epidemiology indicating that further studies are required to clarify the role of ca-mrsa in global epidemiology, their pathogenicity and clinical impact on cf patients. we thank the fondazione per la ricerca sulla fibrosi cistica-onlus for its grant (ffc# ) . there are concerns regarding an increased risk of cancer in patients with cystic fibrosis (cf). this study aimed to review the occurrence of malignant disease in a large population of adult patients with cf. the case notes of patients attending the leeds adult cf unit were reviewed. demographics and data regarding a diagnosis of malignant disease were recorded. a total of eight patients ( male, female) were diagnosed with a malignant condition over the year period. the median (range) age of diagnosis of a malignant condition was . years ( - years). five patients were diagnosed with malignancy post-transplantation. within this group of patients there were two cases of lymphoproliferative disease, one case of basal cell carcinoma, one case of liver cancer and one case of small cell lung cancer related to the donor lung. the median (range) time to diagnosis post-transplantation was two years ( - years) with a median (range) age of years ( - years). three patients were diagnosed with a malignant condition without solid organ transplantation. this group consisted of one case of pancreatic carcinoma, one case of oesophageal carcinoma and one case of bowel carcinoma. the median age of diagnosis in this group of patients was years ( - years) . prior to transplantation we found all malignant diagnoses were related to the gastrointestinal tract. post-transplantation we found a wider variety of malignant diagnoses, with lymphoproliferative disease being most common. awareness of the increased risk of malignant disease is important and appropriate investigations should be undertaken particularly in patients with atypical symptoms. background: patient outcomes can be improved by implementing evidence-based guidelines for initiating therapies. in our cf center, practitioners have traditionally prescribed medications without a centralized database to screen patients for evidence-based criteria. goals: the primary goal of this project was to increase cf practitioner, family and patient awareness of evidence-based medication guidelines to facilitate discussions regarding therapies. the secondary goal of this quality improvement (qi) project was to determine if cf practitioner prescribing patterns would be altered by increasing awareness. methods: the cf steering committee at cincinnati children's hospital medical center (cchmc) identified oral azithromycin, inhaled tobramycin, and dornase alpha as target medications for this qi project. an evidence-based medicine committee was formed, including a parent advocate, to direct the project. a consensus was reached among cf practitioners for when to initiate therapy discussions. dornase alpha was recommended for cf patients ages six and older. inhaled tobramycin was recommended for cf patients ages six and older with chronic pseudomonas (defined as positive cultures in past months). oral azithromycin was recommended for cf patients ages six and older, with chronic pseudomonas, and a weight of kilograms or more. the cf database was screened to determine each patient's medication eligibilities. eligibilities were reviewed weekly at our cf chart conference. medication specific family and patient handouts were developed, reviewed by the parent advocate, and given to eligible families and patients on arrival to clinic. data was collected to document the number of discussions facilitated at clinic visits, the decisions regarding medications, and the number of new treatment prescriptions that were facilitated. after seven weeks of the initiative, statistical analysis was conducted using mcnemar's test for differences. a chi-square test with a two sided alpha of p < . was used to determine significance. results: of the cf patients in our center were eligible for pulmonary medications using our evidence-based criteria. there were individual prescribing opportunities in these patients (taking each medication for each patient as a unique prescribing opportunity). at the start of the project, . % ( / ) of the prescribing opportunities were fulfilled. % ( / ) of patients were prescribed all of their recommended medications. during the first seven weeks of the project, cf practitioners discussed initiating new pulmonary medications with cf families and patients. the proportion of prescribing opportunities fulfilled increased from . % ( / ) to . % ( / ) (p value < . ; % ci . %- . %). the proportion of patients prescribed all of their recommended medications increased from % ( / ) to . % ( / ) (p value < . ; % ci . %- . %). conclusions: increasing awareness of evidence-based pulmonary medication recommendations with cf practitioners, families and patients facilitates therapy discussions, increases the proportion of therapies prescribed and increases the proportion of patients on all recommended therapies. purpose: our purpose was to optimize lung function (fev ) by consistently and aggressively diagnosing and treating pulmonary exacerbations (pex) in all cf patients at our center. it was important to work on this because our families wanted standardized care and we felt our patients' fev 's were suboptimal. methods: our core llc team included two parents of cf patients and one adult patient. we met weekly, received coaching every other week, and learned and applied quality improvement techniques. physicians began monthly meetings, and monthly meetings of the entire cf team continued. the fab met every other month. we identified our specific aims, and approached them systematically. the providers, starting with criteria used by several authors, agreed upon the definition of pex and created a pulmonary exacerbation score sheet (pexss). we selected a score of as the threshold for defining a pex. after using the pexss for three months, all providers reviewed the individual pexsss and validated our original choice of . run charts were produced and reviewed periodically to display each provider's percent of pexsss used. when the use of the pexss was not standardized after a year, it was reprinted on bright yellow paper to make it stand out. to educate the families and patients about the early symptoms of pex, a refrigerator magnet was designed and distributed that included all pex criteria. results: we standardized the use of our pexss by all providers on all patients seen in cf clinic with ongoing measurement of its use and regular feedback. many patients and families gained greater knowledge about what a pex was, and the importance of early recognition and treatment. the cf nurses note that ) many patients call with milder pex symptoms than they did before, ) providers treat pex earlier, ) many patients call with their pex score as well as their symptoms. we hospitalized more patients for pex after this project began, with an average of patients/year in the years prior, and in . the median fev % predicted of our - conclusions: participating in the llc changed the culture and functioning of our cf team. limitations to our study include: ) fev change may not have been due solely to this project, ) the groups who's fev 's were averaged over the various years are different. we will continue to optimize and standardize ) prevention of pex, ) inpatient care, ) care of pex in cf providers' private offices, and ) follow up of patients with pex. patient adherence to airway clearance techniques (act) in the treatment of cystic fibrosis is known to be sub-optimal resulting in deterioration of lung function. the global aim of our project was to improve median fev . the specific aim was to improve adherence to act. we believed that these goals could be acheived by implementing a program which included re-education of airway clearance techniques (react) for our cystic fibrosis patients. methods: the initial phase of this project began with the administration of an anonymous survey to patients/families. this survey was designed to assess act practices, knowledge of the rationale for performing act and barriers that prevent patients/families from adhereing to act. the next step included an in-clinic questionnaire and patient/family demonstration of current aerosol therapy and act. based on individual results patients were identified as: adherent with correct technique, adherent with incorrect technique or non-adherent. we defined adherent as correct performance of act one to two times daily as prescribed. then all patients entered the react program which included a standardized tutorial on proper act and correction of techniques if necessary. in the non-adherent group barriers to performing act were discussed and problem solving techniques were used to overcome barriers. the patients in the non-adherent group were re-evaluated in clinic monthly while the adherent patients returned on an every two month schedule. results: analysis of our initial survey revealed that while the majority of patients/families reported performing act daily, the duration of treatments was less then medically prescribed. many patients/families reported barriers which decreased adherence to therapy. to date % ( / ) of our patients have completed the in-clinic questionnaire and entered the react program. the results of these evaluations and patient demonstrations revealed: % ( / ) were adherent with correct technique, % ( / ) were adherent with incorrect technique and % ( / ) were non-adherent. in the six months since the implementation of the react program, patients/families are self-reporting an increased adherence to act and the median fev for our center has increased form . % ( cff registry data, ages - years) to . % (cff registry, first quarter, , ages - years). conclusion: while adherence to act in our center was poor, we were able to improve adherence and ultimately improve the median fev by implementing a program of re-education for act (react). we believe re-assessment of adherence to therapy and reviewing correct technique is necessary at every clinic visit. continuous quality improvement (cqi) techniques have recently been employed in medicine to improve quality of care. although there is evidence that cqi improves efficiency in hospitals, little is known about effects of cqi on improving patient outcomes. we have previously reported creation of a unique nutritional risk pathway (nrp) to care for cystic fibrosis (cf) patients who are at nutritional risk. the goal of this study is to assess perceived impact and objective clinical efficiency of our nrp. patient data was recorded from all children attending the cf clinic over an -month observation period (between sep and mar ). bmi percentiles were assessed and nutritional risk zones -green: acceptable (bmi > %ile), yellow: at risk (bmi - %ile) or red: at urgent risk (bmi < %ile) -assigned at each visit. the visit intervals were every to weeks based on severity of nutritional risk. patients that remained in the red zone for an average of to months with no improvement were considered for gastrostomy tube (gt) placement. the cf dietitian updated the data weekly and e-mailed quarterly to cf physicians for review of their patients' status. patient/family perceptions and understanding of the nrp was assessed by questionnaire. of the children in the study, ( %) were identified to be at nutritional risk and were enrolled into the nrp: in red and in yellow zones; ( %) male, ( %) female; ( %) age < yrs, ( %) age ≥ yrs. over the -month observation period, ( %) showed improvement ( moved from red to yellow, from red to green and from yellow to green zones). thirteen ( %) females improved, whereas ( %) males improved. in the under yrs age group, ( %) improved compared to ( %) in the ≥ yrs age group. as a result of participation in the nrp, ( %) children had gt placed: six of the showed improvement with four moving from the red to green zone. from the survey responders, % stated they understood the meaning of the cf nrp. all responders were extremely to somewhat motivated to focus on their child's nutrition when asked to return for growth monitoring or follow-up visits. having specific goals for weight gain, calorie requirements and bmi were considered extremely helpful by % of families. when gt placement for their child was recommended, % felt they lacked all the necessary information to make a decision. our new cf nutrition education booklet was not received by % of families. some of the physicians who received the quarterly nutritional patient data provided positive feedback and found this information beneficial for patient care. nutritional cqi in our cf center utilizing a specific nrp and monitoring of nutritional risk status resulted in improved patient outcomes. overall perceptions of our nrp were positive from both patients and cf physicians. these results suggest that this cqi strategy improved clinical outcomes in our patient population. patients with cf require frequent healthcare visits to optimize growth, initiate early interventions and delay progression of lung disease. the cff recommends evaluation at a cf center at least quarterly. as part of a llciv quality improvement (qi) initiative, our cf team set a goal to increase the percent of cf patients attending clinic or more times a year to % or greater by developing strategies to monitor and improve adherence rates. methods: we reviewed cf patients who attended clinic times or less in to determine patterns of non-adherence in regards to age, gender, distance from center, season, physician or insurer. a patient survey was used to assess clinic attendance barriers from a patient/family perspective. a brainstorming session identified other barriers. a fishbone diagram was drafted into barrier categories: external, internal, communication, and patient/family perceptions. after reviewing all barriers, we concluded the most effective improvements could be made to internal clinic barriers. clinic processes were then assessed including making of appointments, reminder letters/calls, a time cycle analysis, and number of cf appointments available per patient per physician. our family advisory board participated by focusing on the importance of or more clinic visits in their newsletter and by assignment of a member to the llciv team. we discovered a lack of a standardized process to follow-up (fu) patients who "did not keep appointments" (dnka). a spreadsheet was created to monitor physician dnka rates. a primary fu strategy was devised to identify patients who dnka through daily auto-emails to the cf office assistant and utilization of a rescheduling decision tree for reappointment of these patients by our appointment center. two secondary fu strategies were developed. the first involves the clinical coordinator printing a monthly report off port cf entitled "patients due a visit" then emailing this list to the cf team to contact and reappoint. the second involves the cf social worker tracking patients seen per quarter on a spreadsheet and emailing the spreadsheet quarterly to the cf team as well as posting on our datawall. results: in , and , %, %, and % of our patients attended clinic or more times, respectively. in in , and , %, %, and % of our patients were at nutritional risk, respectively. in and , mean fev % predicted was . % and . %, respectively. conclusion: the number of cf patients attending clinic quarterly at our center increased by implementing processes to track dnka patients and reappoint them to clinic promptly. however, some of the early improvement in clinic attendance was partially due to an increased awareness of our qi efforts by our clinic staff, cf team and patients/families. we speculate that improvements in nutritional status and lung function are at least partially related to more frequent clinic attendance by our patients. continued monitoring of the number of dnka patients and patients who attend clinic quarterly is required to sustain these gains in improved patient outcomes. data when we first measured patient/family satisfaction in , parents and children were given questionnaires to measure their satisfaction with services provided at the clinic. at that time, responses in our survey were very positive. with regards to our new model we received very positive feedback, encouraging us to continue with the model. parents and children liked the elimination of duplication in questions and they also like having the whole team involved in the meeting and problem-solving. final results for our second satisfaction questionnaire will not be available until june of this year as the questionnaires are being given at clinics over a month period this spring. the children complete a questionnaire with the help of their parents if needed and the parents also complete a survey. results to date are very positive for both the parents and the children. of the children who have completed the survey to date, all stated they would like to continue to meet with the group as a whole as opposed to meeting individually with team members. of the parents surveyed to date, all are either very satisfied or satisfied with the group format. conclusion: we have not yet completed the survey but results to date clearly show that both the patients and their families are happy with the round table model. results will be compiled early this summer and would be ready for the poster and/or presentation in the fall. the cff clinical care guidelines recommend routine clinic visits for assessments, interventions, monitoring, education and counseling every months or more often as indicated. adherence to these guidelines is important in ensuring better clinical outcomes. in reviewing our cf center data from the cff registry report, . % of pediatric patients ( - yrs.) and . % of adults ( yrs. and older) adhered to the guidelines. national goals were not provided then; instead data was given for the top ten cf centers ( . % and . % for pediatric and adult patients respectively) as well as the national rates ( . % and . % for pediatric and adult patients respectively). our cf care center acknowledged the need to improve adherence to these guidelines and at the beginning of the year , embarked on a quality improvement project to address this issue. following the pdsa (plan-do-study-act) steps, we agreed on the objective of increasing the percentage of pediatric and adult patients adhering to the guidelines by at least % and at the same time, to exceed the national average in year or by the end of the year . the physicians and social worker obtained feedback from patients and/or families during a clinic visit regarding barriers in complying with scheduled clinic visits. suggestions were elicited to overcome these barriers. the team members then identified barriers in meeting the guidelines and implemented measures to improve compliance with clinical care guidelines. among these measures were sharing the information about clinical care guidelines and importance of adherence through our cf newsletter and at every clinic visit, reminder calls to patients or caregivers at least days prior to the visit, appointments for the next visit given prior to discharge from the clinic, and same day calls to patients who failed to keep their appointment for rescheduling. our social worker identified patients with difficulties or problems in consistently keeping appointments and helped address these problems. progress made in this endeavor was shared with parents and caregivers during the "parents' night" held in the fall of . indeed, with consistent implementation of these measures, we have seen improvement in the percentage of patients adhering to guidelines. in our cf care center data, . % of pediatric patients and . % of adult patients adhered to recommended clinical care guidelines, approximating and in fact for the adults, exceeding, our target of % increase in the percentage ( % and % increase respectively for pediatric and adult patients). these data were better than the national average for ( . % and . % for pediatric and adult patients respectively) but for pediatric patients, still % below the national goal of %. adults approximated the national goal of %. we are committed to continually engage in the process of quality improvement in further improving adherence to clinical care guidelines and to hold on to the gains. weight z-scores and height z-scores were monitored prospectively for all infants, children, and adolescents at a pediatric outpatient cf clinic during an ongoing, multidisciplinary quality improvement (qi) initiative. all cf team members participated in this qi project which emphasized the importance of good nutrition and adequate growth for optimal lung health. specific strategies for improvement included education to families at each visit with a written nutrition action plan, increased emphasis on liquid supplements and enteral feeds as needed, and a consist message from all cf clinic staff in encouraging attention to nutrition. the study objective was to determine if multidisciplinary qi interventions to improve nutritional status had any effect on weight z-scores and/or height z-scores over a -year period. the null hypothesis was that z-scores for weight and height would either not improve or worsen. inclusion criterion for this study was attendance at cf oupatient clinic during the first months of the qi initiative (time ) with a subsequent clinic visit years later (time ). patients not attending clinic at both time and time were excluded. a cohort of patients ( male, female) met the inclusion criterion. the mean time to time interval was . ± . years (range . to . years) with mean patient age at time of . ± . years (range . to . years). pancreatic enzyme replacement therapy was used for ( . %) of the cohort. paired sample t-tests indicated significant improvements for weight z-score (p <. , t ( ) = . ) and height z-score (p <. , t ( ) = . ) after the -year intervention. at time , children ( . %) were underweight or less than th percentile while children ( . %) were normal-weight, at or more than th percentile for age. mean differences in both weight and height z-scores in the initially underweight patients were greater than those for the normal-weight patients over the year interval. no significant differences were found for the initially normal weight-for-age children between time and time for weight z-score ( p = . ) or height z-score (p =. ). children who were not underweight at time grew normally, without acceleration over the yr intervention. therefore, we conclude that a longterm multidisciplinary qi project to enhance nutritional status resulted in significantly improved weight z-scores and height z-scores for those individuals who were initially underweight for age. the most common cause of morbidity and mortality for patients with cystic fibrosis (cf) is pulmonary exacerbations. these exacerbations lead to hospital admissions and treatment with intravenous (iv) antibiotics. all cf patients at our facility are admitted to a teaching service which includes house officers and students. standardized order forms are used to improve accuracy and timeliness of medications and therapies. the efficiency of this process was brought into question in / , when several parents complained about a delay in the receipt of first dose iv antibiotics after hospital admission. the objectives of this study were to determine what were the actual delivery times of the iv antibiotics to the patients (both retrospectively and prospectively), and areas where improvements could be made. our predetermined target time was < hours. two analyses were performed in order to answer the objectives. the first analysis was done in a retrospective fashion. this analysis included cf patients admitted from / / - / / . a total of patients were included. total median time from admission to receipt of first dose of iv antibiotics was analyzed. a second collection of blinded prospective data was performed between / / - / / . only the pulmonologist, pharmacist collecting data, and pharmacy clinical manager were aware of the data collection. a total of patients were included in the prospective project. each part of the process model were recorded and analyzed so that areas of improvement could be realized. the median time from hospital admission to first dose of antibiotics was hours (range - hours) (retrospective cohort). the prospective cohort median time from hospital admission to first dose of antibiotics was hours (range min- hrs). the median time from admission to order entry by pharmacy: hrs, min (range min- hrs, min). the median time from the medication order being written by medical practitioner to order entry by pharmacy: minutes (range min- hrs, min). the median turn-around time by the hospital pharmacy of order entry until reaching the nursing unit: min (range min- min). the median time required by the nursing staff to administer the antibiotic: hrs, minutes (range min- hrs). this project was initiated in response to parental concerns. from the results of the retrospective and prospective analyses, it was determined that a delay is occurring in the receipt of iv antibiotics, which translates to hours in the hospital without therapy. this is both inconvenient and uncomfortable to the family, and wasteful of medical resources. the findings of this project were presented to the pharmacy and therapeutic committee of the hospital, to the nursing directors, and to the parents of cystic fibrosis patients. the delay is greater than expected before this project was initiated. as a result of these findings a "fast track" medication order set was developed to expedite initiation of inpatient therapy. four key areas included in this order set are sputum, iv access, diet, and iv antibiotics. we speculate that time to first drug delivery will decrease and patient satisfaction with improve. the pediatric intermountain cystic fibrosis center (imcfc) provides multidisciplinary cystic fibrosis (cf) care to pediatric patients from utah and parts of idaho, wyoming, and nevada. historically the imcfc has embraced early intervention. as new therapies have been added to the cf armamentarium it has become necessary to have a method of keeping track of which patients are candidates for various therapies and also which medications have been prescribed, utilized or even should be discontinued. in order to address this quality issue, the imcfc decided to develop and implement the use of a medication tracker (mt). the imcfc decided to focus on key therapies which were felt to be at risk for variability in prescriptive practice. the five therapies included were: pulmozyme<®>, tobi<™>, hypertonic saline(hs), azithromycin and asthma medications (inhaled corticosteroids and/or leukotriene receptor antagonists). ibuprofen was intially included and later removed from the mt. prior to implementing the mt, the imcfc met to review literature, medication trackers from other centers, and develop consensus among team members. six versions of the mt have been developed between / / - / / . each mt has been tested in a pdsa cycle and revised. since initating the mt, patients have been seen at least once. there have been mt encounters. the mt is used only after the first visit to the imcfc and reflects changes to established cf care. there have been a total of changes in prescribed therapies in different patients( % of patients seen). a minority of patients were receiving therapies that did not meet our pre-determined consensus for prescription. the most common therapies in this category were tobi™ and azithromycin. primary reasons cited for prescribing were: chronic suppressive antibiotic use, pseudomonas positive and < years old, and significant changes on chest imaging. reasons patients were not prescribed clinically indicated therapies were: patient refusal, participation in clinical trials, cost, and lack of compliance with other therapies. changes in asthma therapy included the discontinuation of medication in encounters and the initiation of therapy in . the use of a specific cf patient mt allows consistent practice in selection of therapies. it can be used by all team members and helps to identify discrepancies in therapy. assuring all patients receive information and are considered for new therapies is increasingly important as our treatment options expand. the mt also highlights the need to consider discontinuing therapies. we speculate the mt will become an increasingly valuable tool over time and improve patient care. rationale: recent reports document increasing prevalence of antimicrobial resistance across a range of pathogens in patients with cf. the increase in multidrug resistance of these organisms complicates the therapeutic management of these patients. objective: to describe patterns of antibiotic use among physicians treating cf patients. methods: for the original purpose of evaluating site characteristics that act as effect modifiers on the relative efficacy or cost of novel therapies, we administered a survey to acquire information on practice patterns and characteristics of physicians participating in a clinical study of an investigational drug for patients with cystic fibrosis. chi-square and spearman rank test were used to evaluate associations between physician characteristics and patterns of reported antibiotic use. results: fifty-nine physicians, representing study sites, completed the survey. ninety percent reported a medical specialty of pulmonology and % reported practicing at a teaching hospital. on average, respondents reported that about half of their practice consisted of patients with mild cf (defined by fev ≥ % of predicted normal.). forty-two percent of respondents reported prescribing oral antibiotics and % of respondents reported prescribing inhaled antibiotics for maintenance therapy for at least % of their mild cf patients. eight-five percent and % of respondents reported prescribing iv antibiotics for pulmonary exacerbation and pulmonary 'clean out', respectively, with about a third of these cases being prescribed for outpatient iv use. physicians that prescribed iv antibiotics for pulmonary clean out were more likely to pre-scribe oral (p= . ) and inhaled antibiotics (p=. ) for maintenance therapy than physicians that did not prescribe iv antibiotics for pulmonary clean out. conclusions: antibiotic use for maintenance therapy and iv antibiotic use for pulmonary exacerbation and pulmonary clean out acted as 'complements' rather than 'substitutes' as there was a group of physicians who reported less use of antibiotics. our findings warrant further research as well as additional exploratory analyses with a larger and broader sample to examine associations between physician characteristics and patterns of antibiotic use. understanding the relationship between physician characteristics and antibiotic use has the potential to influence the approach to management of cf patients in an environment of increasing antimicrobial resistance. introduction: hypertonic saline (hs) has been shown to increase mucus clearance and improve lung function in cf, and is now increasingly being used as part of routine therapy. one of the goals of our cf center quality improvement (qi) initiative is to increase use of hs among all eligible patients. objectives: to assess adherence to treatment with hs, parental opinions on treatment, perception of side effects, and the effect of initiating hs on the use of other nebulized medications in pediatric cf patients started on hs therapy during . methods: patients under years of age were identified as initiating hs therapy during . a telephone survey of their parents using a -item multiple-option questionnaire was administered in april as part of our cf center qi initiative. results: parents ( %) completed the survey: ( %) patients were still using hs. of those still reporting use of hs, ( %) were using it twice a day; ( %) once a day; and less often than daily. among the patients who discontinued hs, ( %) stopped following recommendations from their pulmonologist; ( %) stopped due to side effects; ( %) felt hs took too long to administer; and ( %) felt hs solution was too difficult to mix. the most common side effect reported was cough in patients ( %); ( %) reported no side effects. the majority of patients ( %) spent - minutes per day on hs therapy. thirteen ( %) patients discontinued another nebulized medication when they initiated hs; ( %) of those stopped pulmozyme. reasons for stopping pulmozyme included: time to nebulize both medications was too long ( %), perception that hs was working better for their symptoms ( %); and doctor's instructions ( %). overall, ( %) parents felt hs improved their child's symptoms; ( %) saw no difference; and ( %) believed hs made symptoms worse. there was a significant difference in the proportion of patients that reported improvement of their symptoms in the group using hs twice a day ( / , %) when compared to those using it once a day ( / , %) (p= . ). discussion: the majority of patients continued to report the use of hs therapy several months after our initial intervention, with the most parents reporting improvement in their child's symptoms. physician recommendation was the most common reason cited for discontinuation of hs therapy. further study of physician opinions on hs therapy, particularly the timing of initiation and discontinuation, is warranted. one-half of patients reported using hs only once a day; however those reporting twice daily use were more likely to report subjective improvement of symptoms. since treatment complexity in cf increasing, determining the effectiveness of once daily hs therapy, either alone, in place of, or in combination with other therapies such as pulmozyme is essential. long-term follow up of these hs-initiated patients will focus on effects on exacerbations, pulmonary function, and hospitalizations. the uab/children's hospital (chs) cf center participated in learning and leadership collaborative ii. as part of the qi journey, the chs cf qi team thoroughly evaluated the current system of care in an effort to identify areas of needed improvement. one area assessed was clinic attendance. it was discovered that % of patients failed to attend clinic appointments every week. according to cff care guidelines, patients with cf should be seen a minimum of times each year at the cf center. the cff has also found that those centers that see their patients more frequently have improved outcomes for fev and nutritional status. aim to decrease the number of missed appointments at the chs cf clinic. the chs cf center administered a qi survey to identify the reasons patients and families were missing appointments. this survey was a item questionnaire that evaluated: reasons for missed appointments, if the families rescheduled missed appointments, difficulties with rescheduling, the need for reminder calls, distance traveled to the cf center, and insurance status. results completed surveys were collected. % of the families reported medicaid as their insurance provider. % of families live more than miles away from the cf center. % of the families confirmed that they had missed an appointment in cf clinic over the last months. the reasons for the missed appointments were widely variable; however, . % of those who missed appointments reported that they did not have access to transportation to get to the appointment. overall, families did not report difficulties in rescheduling appointments. % of those that missed appointments identified medicaid as their insurance which can be correlated with lower income families and . % of those that missed lived greater than miles away from the cf center. % of families reported that a reminder call would be helpful for them. interventions after evaluation of the survey data, the center quickly initiated reminder calls for cf clinics. a student employed by the cf center made these telephone calls to the patients scheduled for clinic every week. the majority of the time the student caller left a message for the family. with the implementation of this simple intervention, missed appointments have decreased from % to % which is a % improvement rate. reminder calls are now a routine part of the cf center's system of care. other ongoing interventions include increased involvement of primary care doctors for patients lost to follow up for more than months and a system for quickly rescheduling patients. next steps in addition to reminder calls, the cf team will now determine where patients who routinely miss appointments live. if there are pockets of patients in common areas of the state, the team will attempt to maximize services for those patients such as linking them with community transportation resources, community health advocates, or providing satellite clinics. this will be accomplished through geomapping of the alabama cf population based on zip code. we designed a scoring system to uniformly identify patients experiencing a pulmonary exacerbation. the elements of this pul-monary exacerbation score (pes) have been previously described. at the time this project began the median fev % predicted of the cf patients at our center - years old was below the national average ( cf registry data). our hypothesis was that greater uniformity of identification of pulmonary exacerbations, would improve pulmonary function in the pediatric age group at our center. this abstract describes continued improvement in the pulmonary function of this age group at our center now that the pes has become standardized in our clinical practice. methods: beginning in october , a pes was calculated for all patients presenting to our center age - years of age. any patient with a pes of > was defined as having a pulmonary exacerbation, and treatment with antibiotics was recommended. the patient's cf physician dictated the ultimate decision for treatment and specific course of treatment. median fev of all patients age - years was calculated quarterly to determine the effect of the pes use on pulmonary function. in october the pes was incorporated into our cf clinic medical record and its use was standardized. the use of the pes, the adherence to the recommendation for treatment, and quarterly fev of the population has continued to be monitored. results: from / to / (the duration of the original qi project), patient visits occurred, with a pes calculated on (utilization of . %). patients were treated for a pulmonary exacerbation of the patients with a pes of > (adherence of . %). . % of exacerbations were treated with oral and/or inhaled antibiotics, and . % were treated with intravenous antibiotics. since standardization of the pes in / , visits have occured. use of the score has decreased slightly ( . % utilization), however adherence to the score has increased ( . % of pes > have been treated). use of oral vs. iv antibiotics has been similar ( . % oral vs. . % iv). the median fev of our patient population has improved from an average of . % predicted during the months before implementing the pes to an average of . % predicted during the ensuing months of the original qi project (a . % improvement). since standardization of the pes in / , the average median fev of the population has continued to increase to . % predicted (an . % increase from the baseline period). this improvement in median fev is reflected in our cf registry data. summary: implementation of a pulmonary exacerbation score, designed to encourage uniform identification of pulmonary exacerbations in patients with cf, is associated with an improvement in median fev in children - years of age. standardization of the pes has resulted in continued improvement in pulmonary function of this population at our cf center. this work has been supported in part by the cff and akron children's hospital. our cf family council parent and patient advisory group has been involved in the development and implementation of this project. utilizing data from the cff patient registry, we determined that our center has had nutritional outcomes consistently below the national average for pediatric and near the average for our adult patients. as a result, we developed the global aim to improve and maintain optimal growth and nutrition in all patients at our cf center. our specific aims included: )to record growth parameters at all outpatient visits; )to identify patients at nutrition risk, categorizing them based on anthropometric data; ) to develop and implement a specific nutrition plan based on nutritional category; and ) to educate staff, patients,and families on the benefits of normal growth and nutrition. we surveyed our patients to determine their overall attitudes toward nutrition and nutritional care received at our center. we discovered that many patients and families did not feel that nutrition was addressed at every cf visit nor that they had a nutritional plan at each encounter. this led to the development of an intake form/home-going sheet for our patients to complete that included data elements necessary for assessment of nutrition and that clearly outlined a nutrition action plan. a standardized nutritional assessment score (nas) was devised that incorporated weight, height/length, bmi and weight for length percentiles as well as weight loss, failure to gain or maintain, and failure to remain in the expected growth channel. the nas was developed after examining the medical literature, reviewing work done by other qi teams, and acquiring input from our pediatric gastroenterologist and endocrinologist. we then developed nutritional algorithms to address nutritional intake, malabsorption, evaluation for cfrd, and short stature. these four algorithms were used to develop a nutrition action plan for each individual. beginning in march , a nutritional category (optimal/acceptable, concerning, at risk or failure) was assigned to each patient at each visit based on their nas. an individualized nutrition action plan was developed with each patient. at the time of project implementation, patients age - at our center had a median bmi %ile of . % with . % of our patients with a bmi < th%ile. our patients > years had a median bmi of . with . % having a bmi< th%ile. after one month of implementation of the nas, % of our patients had been categorized with % of our patients having individual nutrition action plan developed. to track our progress, we will monitor median bmi, % of patients with bmi < %ile, and % of our patients in each nutritional category quarterly using portcf. our goal is to have all our patients assessed and a nutrition action plan implemented by the end of . we have developed a nutritional assessment score and nutrition treatment algorithms for pediatric and adult patients with cf as part of a qi initiative to improve nutritional status of patients at our center. the project has been supported, in part, by akron children's hospital. we are indebted to lori lundquist, a parent of two of our cf patients, who has been an integral part of our nutriton qi team. in germany there are about , cf patients who are usually treated in special centres by a team of trained and experienced health care professionals. the result of this structured approach to patient care, including frequent clinical assessments, monitoring, and aggressive interventions is an improved mean survival and a higher quality of life of pediatric and adult cf patients. however, a recent survey showed that german reimbursement schemes cover only about % of resources used within these centres. objective to assess and evaluate direct costs of outpatient treatment of cf patients in germany. a micro-costing approach was used to record resource use data directly within representative cf centres. results of the evaluation may be used to initiate and support discussions between health care providers and insurances about adequate reimbursement schemes in germany. outpatient care was evaluated in seven different centres for pediatric and adult cf patients. patient reported outcomes, clinical patient data, resource utilisation, and physician related time consumption for treatment was recorded for every patient during one representative month in . cost data for staff, materials, medical equipment, rooms, etc. were assessed within the respective centres. data for cf patients were collected. about half of them were children and adolescents, and half of them were adults. a comparison of the resource uses to the actual remuneration of these services indicates that only about % of the costs are covered by the reimbursement system. correlation analysis identified significant cost drivers like the age of the patient, comorbidities like pancreatic insufficiency and hepatobiliary complications, lung function (% fev ), and the presence of certain pathogens in the lungs. as the actual reimbursement covers only about % of the resource usage costs in germany, the existence of the cf centres and sufficient treatment for cf patients is uncertain in the future. in order to ensure the existence of the centres it would be necessary to agree to a cost-covering reimbursement at minimum. this may be based on a lump-sum payment that could be differentiated for pediatric and adult centres or according to comorbidities or clinical parameters. the data calculated in this study could be used to trigger and support discussions between health care providers and insurances about a new cost-covering reimbursement system for the out-patient treatment of cf patients in germany. the impact of the new us cystic fibrosis foundation (cff) nutrition practice guidelines, i.e., discontinuing the use of percentage of ideal body weight (%ibw) to define "nutritional failure" and setting body mass index (bmi) below the th percentile to define "nutritional risk", on evaluating nutritional care practices is unclear. methods: data from children reported to the cff patient registry were analyzed to compare the rates of malnutrition among cf centers. results: nationally, eliminating %ibw < % as a criterion for underweight resulted in a . % reduction (from . % to . %) in "nutritional failure" rate. misclassification of "nutritional failure" by %ibw < % ranged . - . % among centers and was greater for centers having a larger proportion of tall patients. one-third of centers switched to a different tertile ranking, after correcting for misclassification by %ibw. use of bmi < th percentile led to the classification of . % of patients as "nutritional risk". more than half of the centers had different tertile rankings on "nutritional risk" compared to "nutritional failure". a total of . % ( - . % among centers) of patients who had height < th percentile but bmi ≥ percentile were not considered at nutritional risk. the cystic fibrosis questionnaire-revised (cfqr) is a disease specific quality of life measure that is currently undergoing clinical validation and may prove to be a useful adjunct to provider assessments. we proposed the routine use of the cfqr in a busy adult cystic fibrosis (cf) clinic would assist in identifying patients in need of limited clinic resources. this performance improvement project focused on new and infrequently-seen patients (i.e. fewer than three outpatient clinic visits/year) to determine the frequency of patients with domain scores that fall more than one standard deviation below our clinic mean. methods: the cfqr is routinely self-administered to all adult cf patients in clinic at least once every year. these assessments occur after vital signs are obtained and prior to any other clinical assessments. patients were deemed to be at baseline if there were no changes made to their pharmacologic or chest physiotherapy regimens. the mean and standard deviation of each domain of baseline cfqr assessments were determined. the frequency of rarely seen and new patients with domain scores less than one standard deviation below the mean was determined. results ( ). of these baseline assessments, forty five were obtained from patients who were rarely seen or new to the clinic. . % (n= ) of these patients had one or more domain scores falling more than one standard deviation below the mean. . % (n= ) of these patients had two or more domain scores below these cutoffs. % (n= ) of the rarely evaluated patients had domain composite scores (i.e. the sum of the individual domains) that fell below the standard deviation cutoff. the most common outlying domains in these infrequently seen or new patients were body ( ), weight ( ), physical ( ), health perceptions ( ) and the composite domain ( ). conclusions: the cfqr is easy to administer and score during routine adult cf clinical visits. as many as % of our infrequently evaluated patients had multiple scores that were more than one standard deviation below our clinic mean. we conclude that the cfqr is a useful adjunctive measure to provider assessments and can help focus limited personnel resources. aminoglycoside antibiotics are known to be toxic to the inner ear. however, they continue to have a leading role in the treatment of certain infections and in the treatment of pulmonary exacerbations in patients having cystic fibrosis (cf). while there is ample evidence in the literature of ototoxicity, and there are established protocols for monitoring the effects of toxic agents on hearing, less is known about the prevalence of vestibular toxicity among patients who are exposed, and there are no commonly accepted protocols for monitoring vestibular system function. oscillopsia and unsteadiness when standing and walking are the two most commonly reported and disturbing symptoms associated with bilateral loss of vestibular system function. oscillopsia is the perception that viewed stationary objects or surroundings move coincident with head movement. when it is severe, oscillopsia can prevent an individual from having clear vision with even the slightest head movement. the unsteadiness that accompanies vestibular loss can range from mild to disabling. because of the referral of a number of patients to the vestibular testing center (vtc) at the university of michigan in whom severe bilateral vestibular loss was evident secondary to apparent aminoglycoside toxicity and our concern about quality of life issues, we initiated a quality improvement clinical protocol with our cf patients. vestibular system function and hearing are evaluated at the time of admission for pulmonary exacerbations and coincident with the three month follow-up visit with the pulmonologist. our goal is to understand the incidence of ototoxicity in this patient population, and to determine how best to measure incremental changes over time. the ultimate desired outcome is to investigate the efficacy of protective agents for limiting the toxic impact of these drugs on hearing and vestibular function. to date, we have completed vestibular testing on patients with cf ranging in age from - years, some of whom were referred prior to the initiation of this monitoring program. of the sample, individuals have completely normal vestibular test results, and of the had no prior aminoglycoside exposure. of the remaining patients, have bilateral vestibular loss ranging from mild to severe, two have evidence of a unilateral vestibular loss, and each of the others has non-lateralizing evidence of vestibular involvement. we have also documented change in the three individuals we have seen for repeat testing. specifically, repeat testing has shown incremental decline in vestibular function with repeated exposure to aminoglycosides. interestingly, only individuals in the group have documented hearing loss, and although most report that they do not and have never had any problems with dizziness or balance, many patients have evidence of oscillopsia or abnormal postural control test results. it is clear from our limited data that it is important to monitor vestibular system function whenever potentially toxic agents are used. while monitoring hearing is also warranted, our data suggest that a monitoring program that includes only hearing is insufficient. background: the correlation between bmi, lung function, and longterm outcomes in cystic fibrosis patients has been well documented. based on this data the cf foundation (cff) has established adult patient care guidelines for bmi of or greater for women and or greater for men. in our cf center implementation of nutrition therapies in cf adult patients has been difficult due to lack of acceptance of proposed interventions. due to the importance of optimal nutrition to attain the cff recommendation for bmi, university of cincinnati adult cf center implemented a weight management quality improvement protocol to improve the nutritional status of patients below the cff recommended bmi. our aim is to improve to the nutritional outcomes in our center through patients' acceptance of our nutrition interventions. strategies: as part of the weight management protocol we developed a nutrition action plan, nutrition algorithm, and weight management education record and learning log. the action plan took a systematic approach to weight management, classifying patients into different risk categories based on bmi. patients were categorized as mild nutritional insufficiency (bmi - ), moderate nutritional insufficiency , and severe nutritional insufficiency . each category required a specific action to address weight loss or inability to gain weight. areas of focus for the action plan include; intake assessment through computerized dietary analysis, assessment of pancreatic function, pancreatic enzyme replacement therapy (pert) regimen assessment, hour stool collection for malabsorption, assessment of glycemic control, assessment for gerd, and enteral/parental feeding. the dietitian completed a chart review and nutrition assessment with the patients and classified them into one of the three nutritional risk categories. eleven patients with a bmi below were initially identified. nine ( %) of the eleven patients with a bmi of or less have been approached with the protocol and algorithm. patients completed a three day food journal and oral intake was assessed via computerized dietary analysis. oral enteral supplements were recommended - times per day. g-tube placement was discussed and an educational material on tube feeding was distributed. the protocol and algorithm guided identification and assessment of contributing factors to weight loss. results: of eighty patients seen in the adult center since the beginning of october, thirty-seven of the patients ( %) have a bmi below and eleven patients ( %) have a bmi below . out of the nine patients approached with the protocol % (n= ) completed and returned the three day food journal. of the patients that completed their food journal % (n= ) agreed to a peg placement, one of the patients is still contemplating peg placement while sorting through some psychosocial issues, and the other patient has had a differential diagnosis that might negate the necessity for a peg placement. conclusion: we developed a systematic approach to weight management within our adult cf program. this systematic approach to weight management, which includes patient involvement and specific education, has improved the overall acceptance of nutrition interventions in our adult population. the inpatient care of adults with cystic fibrosis can be a challenging given complicated medical regimens, specialized respiratory care orders, and unique dietary requirements. a large proportion of these patients are admitted to academic centers with rotating house staff that may have never treated an adult with cystic fibrosis. to improve the quality of care of our adult patients we have implemented a standardized electronic order set, a recurring educational program, and a "pocket card" to help the house staff. the adult cystic fibrosis program at vanderbilt cares for over patients, with - inpatient admissions per month. vanderbilt's inpatient order system is completely electronic. we have devised an order set based on our agreed standard of care for inpatients with pneumonia. the order set guides the house staff through most aspects of inpatient care including culturing, antibiotic choice and dosing, dietary consultation, nutritional recommendations, and respiratory care orders. this order set not only educates the house staff on our standard of care but also provides direct access to each order they may wish to enter. we have reiterated this educational process with a pocket sized handout that guides them through the typical issues in adults with cf as well as provide phone numbers to members of the cf team. these resources are presented in short talk given at the beginning of each month to incoming house staff. a preliminary data analysis suggests that over % of all adults with cf are admitted using some component of this order set. an informal survey of the house staff also concludes that the pocket card and lectures have been helpful. all three aspects of this quality improvement have been implemented since december of . we plan to formally test the impact of this intervention when a meaningful number of house staff have been through the service. * background: malnutrition is a common complication of cystic fibrosis (cf). the median bmi percentile in cf patients between the ages of and years is . % but ranges between various centers from . to . percent. previous research has shown a trend toward improvement of pulmonary disease and a significant improvement in weight, height, and bmi z-scores after nutritional supplementation via a gastrostomy tube ; furthermore, fev is positively correlated with bmi percentile. higher weight at three years of age predicts better pulmonary function at six years of age. the effect of gastrostomy tube feedings on pulmonary exacerbation frequency has not been well defined. hypothesis: the incidence of pulmonary exacerbations in children will decrease after the initiation of supplemental gastrostomy feedings. methods: a retrospective chart review of all pediatric patients seen at the cmh cf center, and who received gastrostomy tubes between and was performed. data was evaluated in six month time intervals for two years prior and two years after gastrostomy tube placement. we assessed weight, height and bmi percentiles, fev percent predicted, number of courses of exacerbation therapy (intravenous and oral) and microbiologic data. results: we identified patients, females and males, with ages ranging from months to years of age ( . years mean) at time of gastrostomy tube insertion. all patients had increases in their weight-forage percentile (mean of all patients of . percentile pre to . percentile post, p< . ), height-for-age percentile ( . percentile pre to . percentile post, p< . ), and bmi percentiles ( . percentile pre to . percentile post, p< . ). the mean fev percent predicted declined overall from . % to . % ( . percent predicted per year) over four years in patients. there was a trend of exacerbation reduction after gastrostomy tube placement from to total courses (p= . ). this decline in exacerbations after gastrostomy tube placement was most dramatic in those patients growing pseudomonas aeruginosa without other organisms, with a decline from total courses to total courses in patients (p= . ). discussion: weight, height and bmi percentile improved after initiation of gastrostomy tube feedings. lung function (fev % predicted) decreased at the rate predicted in spite of gastrostomy feedings. there is a trend toward decrease in exacerbation frequency after improved nutrition with gastrostomy tube feedings, especially in patients infected with pseudomonas aeruginosa. limitations of this study include the small sample size, relatively short follow-up time, and the retrospective nature of the study which did not allow assessment of adherence. conclusion: improved nutrition through gastrostomy tube placement may decrease the frequency of pulmonary exacerbations. evidence of an association of improved pulmonary function with improved nutritional status has motivated aggressive nutritional intervention. percutaneous endoscopic gastrostomies (pegs) are employed increasingly in cystic fibrosis to optimize nutritional status. we reviewed our center data over the past years to assess changes in our peg practice and effects on nutritional and pulmonary outcomes. we hypothesized that our change in practice to earlier peg intervention improved patient's nutritional and pulmonary outcomes. we conducted a case controlled retrospective study of pediatric cf patients spanning the past years divided into two periods: p (peg placed before ) and p (peg placed or later) compared with their respective age/sex matched controls. we compared peg vs. control cf patients with respect to age, nutritional and pulmonary status. we compared weight, height and bmi directly preceding peg placement and at approximately one-year post peg. we compared available data for best yearly fev and fvc before peg and at , and -years post-peg. we then compared data between the two time periods. results: peg patients in p (n= ; mean age . + . yrs.) had lower bmi ( z = - . v. - . , p< . ) pre-peg placement compared to controls. by year post peg, the bmi normalized and equaled controls (z=- . v. - . , p= . ). peg patients' fev %ile prior to placement was lower than controls ( v. , p= . ) as was their fvc %ile ( v. , p= . ) and remained lower at year post-peg. following peg placement, absolute bmi increased ( . kg/m , p= . ), as did bmi z-score ( . , p= . ). we did not see significant changes in pulmonary function tests (pft). peg patients in p , (n= ; mean age . + . yrs.) also had lower bmi (z= - . v. - . , p= . ) pre-peg than controls. at year post-peg, the peg patients still had a significantly lower bmi (z = - . v. - . , p= . ). fev was lower in peg patients compared to controls in pre-, year, and -year post-peg data (p< . ). variability in pfts and small sample size limited our analysis. pre-peg weight, height, bmi, fev and fvc were lower in p patients compared to the younger p patients. at -year post-peg, similar bmi zscores (- . , p= . ) was obtained between the two periods. variability in pfts among the patients within each period prevented valid comparisons, although p tended to have better pft values. conclusions: peg placement was effective at improving nutritional status in patients with cystic fibrosis. placement in younger patients with better pre-peg nutritional and pulmonary status allowed more complete nutritional recovery. we speculate that improved height z-scores with apparently stable fev is consistent with increased pulmonary growth. demonstration of an attenuated rate of pft decline to correlate with this earlier intervention will require a larger population and longer observation period. a multi-center longitudinal outcome study would help determine optimal timing and criteria for peg intervention. . pediatric gastroenterology and hepatology, university medical center groningen, groningen, netherlands; . biochemistery, erasmus medical center, rotterdam, netherlands background: ursodeoxycholic acid (udca) treatment is frequently applied for cystic fibrosis-related liver disease (cfld). it has been hypothesized that udca is beneficial through its choleretic activity, i.e. its capacity to induce bile flow. the hepatic expression of the cftr protein is restricted to the cholangiocytes lining the bile ducts. cholangiocytic cftr is involved in the generation of ductular bile flow. it is not known to what extent the choleretic activity of bile salt treatment depends on expression of competent cftr protein, and whether or not udca differs in this respect from other bile salts. we evaluated the role of cftr in the acute and chronic choleretic effect of bile salt treatment, through comparative studies in cftr-null, homozygous f del, and corresponding wild type control (wt) mice. methods: bile flow was determined after gallbladder cannulation. bile flow during the first minutes after acute interruption of the enterohepatic circulation was regarded as basal bile flow. after min, taurocholic acid (tca) or tauroursodeoxycholic acid (tudca) were iv administered in stepwise increasing dosages, up to nmol/min/ g bw, to cftr-null mice and wt littermates fed a standard chow diet. other cftr-null, homozygous f del mice and their respective wt littermates were fed either the standard chow, or the same diet supplemented with cholic acid (ca, . wt%) for weeks. finally, cftr-null mice and wt littermates were fed the standard chow or the same diet supplemented with ursodeoxycholic acid (udca, . wt%) for weeks. : upon a regular chow diet, the basal bile flow was similar in cftr-null and homozygous f del mice, compared to their respective wt littermates (cftr-null, . ± . vs. . ± . ; and, f del(∆/∆), . ± . vs. . ± . µl/min. g bw, resp.; ns). iv administration of tca or tudca to cftr-null mice and wt littermates increased bile flow to similar extents. dietary ca treatment increased basal bile flow significantly less in cftr-null and homozygous f del mice than in their respective wt littermates (cftr-null, . ± . vs. . ± . , p< . ; and, f del(∆/∆), . ± . vs. . ± . µl/min. g bw, p= . ; resp.). interestingly, dietary udca treatment increased basal bile flow more profoundly than ca treatment, and to similar levels in cftr-null mice and wt littermates (+~ %, . ± . vs. . ± . µl/min/ g bw, resp.; ns). conclusion: upon chronic treatment, udca is highly choleretic in mice, independently of the presence of functional cftr. the independence of functional cftr is udca specific, since the choleretic activity of chronic ca treatment is diminished in cftr-null and homozygous f del mice. we speculate that this specific, cftr-independent choleretic effect of chronic udca treatment could be therapeutically important for cystic fibrosis. most cystic fibrosis (cf) patients have mild to moderate focal portal tract changes (ductal obstruction and cholangitis) and hepatosteatosis. etiology of severe liver disease (ld)with cirrhosis and portal hypertension which occurs in - % of cf patients is unknown. the c bl/ j congenic cf mice develop progressive cf-like ld. as a useful therapeutic model, we have shown that dietary addition of docosahexaenoic acid (dha) significantly ameliorates inflammation but has little effect on ductal obstruction (am j physiol gastrointest liver physiol : g -g , ) . we hypothesized that treatment with udca,a hydrophilic bile acid will ameliorate portal tract ductal obstruction and have an additive effect with dha in preventing ld. methods: thirty-day old wild-type and cf littermates are fed peptamen and either olive oil or dha, with and without . % udca ( groups, mice/group). mice are killed following days of treatment and bile salts, serum liver enzymes and serum and tissue lipids analyzed. h&e stained liver tissues are coded and assessed blindly by an expert hepatopathologist (jp) for evidence of hepatosteatosis, inflammation, bile duct obstruction, fibrosis and other signs of liver pathology. an arbitrary scale of to is used, with representing no pathology and being the most severe. we present interim statistical analysis performed for pathology results only for untreated mice (wt, n= ; cf, n= ); treated mice with dha, (wt, n= ; cf, n= ), treated mice with udca (wt, n= ; cf, n= ) and treated mice with udca and dha (wt, n= ; cf, n= ). results: mean pathology scores from groups of mice are pooled to examine the effects of genotype, treatment and diet (table) . at present,the bile,lipid and liver biochemical test data are insufficient to perform statistical analysis. udca-treated mice have significantly increased bile duct obstructed scores compared to the untreated mice which subanalyzed (not shown) reveals increased bile duct obstruction in the wild-type littermates and no effect in cf mice. hepatosteatosis and inflammatory scores in the untreated mice (fed olive oil) are increased compared to the mice treated with dha. summary: we anticipate completing and performing final analysis within six months. however, these preliminary data confirm that dha reduces inflammation in c bl/ j mice livers and suggest that udca therapy has an adverse effect in wt mice and is not effective in ameliorating liver disease in cf mice. udca with dha therapy does not appear to have an additive effect in ameliorating cfld in this mouse model. supported by axcan pharma inc. background the cf mouse intestine has an innate response associated with small intestinal bacterial overgrowth (sibo). a previous affymetrix genechip analysis of the cf mouse intestine showed altered expression of several eicosanoid metabolic genes. eicosanoids are biologically active arachidonic acid metabolites with a variety of pro-and anti-inflammatory actions as well as effects on mucus production, electrolyte transport, and gastrointestinal motility, all of which are of potential importance to the cf phenotype of the intestine. methods wild type (wt) and cf mouse (cftr tm unc ) littermates congenic on the c bl/ j background were fed an elemental liquid diet (peptamen). intestinal rna was isolated for quantitative rt-pcr of expression levels of genes involved in eicosanoid metabolism to confirm and extend the genechip analysis. various eicosanoids were measured in small intestinal lavage fluid using specific enzyme immunoassays. results we confirmed microarray results by qrt-pcr that the following genes were differentially expressed in the cf small intestine relative to wt: cyp a (cytochrome p a ; -hete synthesis; % of wt); ltb dh (leukotriene b dehydrogenase; metabolizes prostaglandins and leukotrienes; % of wt); cyp c (cytochrome p c ; -hete synthesis; % of wt); ltc s (leukotriene c synthase; ltc synthesis; % of wt); hpgd (hydroxyprostaglandin dehydrogenase ; metabolizes prostaglandins; % of wt); and pla g (group phospholipase a ; potentiates arachidonic acid generation; % of wt). non-significant or less than -fold changes in gene expression were measured for: cyclooxygenase (cox) genes, pge synthase, pgi synthase, and -and -lipoxygenases. as measured by their stable metabolic products, pge and pgf α were significantly increased in the cf mouse intestine ( % and % of wt, respectively). the following eicosanoids were significantly decreased in the cf mice: -hete ( % of wt), -hete ( % of wt) and -hete ( % of wt). there were not significant changes in the levels of pgi , pgd , ltb , cys-lts, or lxa . the changed levels of eicosanoids are generally consistent with the differential gene expression. conclusions there are significant changes in expression of eicosanoid metabolic genes and certain eicosanoids in the cf mouse small intestine. the increase in pla g and decrease in cyp expression levels are expected to make more arachidonic acid available for eicosanoids except hetes. decreases in degradative genes (hpgd, ltb dh) will prolong availability of prostaglandins. altered levels of eicosanoids are expected to play important roles in the pathophysiology of the cf intestine. elevated pge may contribute to increased mucus secretion which is characteristic of cf. pgf α and pge regulate intestinal motility and small intestinal transit is slower in cf patients and in cf mice. the hetes are mostly known for their effects on vascular tone and electrolyte transport in the kidney but are less-well characterized in the intestine. their dramatic decrease in the cf intestine may affect blood flow, electrolyte transport, intestinal motility, or other gi functions. the specific effects of the altered eicosanoid metabolism in cf remain to be explored. supported by cff grant delisl g . for the scandinavian study consortium. background: progressive pulmonary disease, correlating with igg levels, is a major cause of morbidity and mortality in cf patients. recently, immunosuppressive effects of vitamin d and a potential role of vitamin d deficiency in the pathophysiology of autoimmune diseases have been described. we investigated whether vitamin d deficiency could contribute to the chronic proinflammatory state characterizing cf and the higher occurrence of immune-hyperreactivity-related conditions in cf patients. methods: multiple linear regression analysis, taking the confounding factor of age into account, was used to evaluate the relationship between cross-sectionally determined vitamin d variables and: lung function parameters, inflammatory parameters and immune hyperreactivity markers in patients followed at the cf centers in sweden, norway and denmark. vitamin d intake was based on a -day precoded dietary food record, calculated in the national food databases. findings: in the population of all patients included in the study (n= ), blood vitamin d positively correlated with fev (p< , ) as well as with fvc (p< , ). negative correlation was found between blood vitamin d and igg (p< , ), supplemented vitamin d per kg bw and igg (p< , ), total vitamin d intake per kg bw and igg (p< , ). additionally, lower blood vitamin d levels were related to both presence of pathological ogtt (p< , ) and occurrence of cfrdm (p< , ). correspondingly, food vitamin d (p< , ), food vitamin d per kg bw (p< , ), supplemented vitamin d (p< , ), supplemented vitamin d per kg bw (p< , ), total vitamin d intake (p< , ) and total vitamin d intake per kg bw (p< , ) all correlated negatively with hba c values. most of the studied vitamin d variables were lower in patients with endomysial antibodies positivity (n= ) and higher in patients with abpa (n= ) than in the rest of cf population studied (ns). interestingly, in children (n= ) blood vitamin d correlated negatively with transglutaminase levels (p< , ) and there was also a significant negative correlation between hba c and vitamin d intake. surprisingly, in adult cf patients (n= ) iga positively correlated with total vitamin d intake (p< . ) and total vitamin d intake per kg bw (p< . ). in the population of stockholm cf-center patients (n= ), where it was possible to allow for the influence of both age and compliance, negative correlation between blood vitamin d and igg (p< . ) was found. after allowing for age, compliance showed significant correlation only with igg (p< . ) and esr (p< . ), and with none of the rest of the studied variables. conclusions: vitamin d deficiency might contribute to the continuous shift towards inflammation as well as to the higher prevalence of dm and celiac disease in cf patients. new recommendations for vitamin d supplementation and monitoring of blood vitamin d levels are needed, in order to improve immunological balance and decrease the need for anti-inflammatory therapy in cf patients. supported by swedish heart lung foundation, stiftelsen frimurare-barnhuset i stockholm, norwegian and swedish cystic fibrosis associations, and by an unrestricted grant from solvay pharma. von drygalski, a.; biller, j.a. medical college of wisconsin, milwaukee, wi, usa anemia is associated with increased morbidity and mortality in other chronic diseases, but little is known about anemia in cf. recognition and correction of anemia in cf might lead to better outcomes. chronic inflammation, iron deficiency and malabsorption may be important mechanisms contributing to the anemia in cf patients. we hypothesized that anemia might be correlated with poor lung function. methods: clinical charts and portcf.org visit logs of patients of all ages ( m to yrs) with cf were reviewed for as many years as charts permitted (range - yrs). the following data were extracted: cbc, iron studies, pulmonary function tests (pfts), vitamins a, d, and e levels, creatinine, pertinent medical history, and current medications. anemia was defined by age-and gender-specific world health organization (who) criteria. patients were considered anemic if low hemoglobin(hb) was present on two separate occasions at least two months apart, or average annual hb met who criteria. pfts in patients (all > yrs) included forced expiratory volume (fev ) and forced vital capacity (fvc) as percent predicted of normal and were considered a reflection of patient performance. the most representative annual pft from at least annual tests was chosen for analysis since acute infection adversely influences daily performance. results: of patients were anemic (prevalence %). prevalence of anemia increased with age from . % in patients < age to % in patients > age and was significantly higher in patients with vitamin deficiencies. % vitamin-deficient patients vs. . % non-vitamin deficient patients were anemic (p= . ). mean hb was . mg/dl (range . - . ). roughly two thirds of patients had moderate and severe anemia (hb < mg/dl) with no gender difference in regard to prevalence or severity identified. complete iron studies were available in / patients. were identified as iron deficiency anemia (ida), as anemia of chronic illness(aci), and as combined; others had renal failure. in patients, iron studies were incomplete, but renal failure, hemoptysis, hematochezia and solid organ transplants were contributing factors in half. pfts obtained in patients ≥ yrs were compared in anemic and non-anemic patients. mean fev and fvc at all ages were statistically significantly poorer in the anemic patients (p < . ). complete iron studies were also available in non-anemic patients with impaired lung function. / patients had ferritins < ng/ml suggesting relative iron deficiency. conclusions: the prevalence of anemia in cf patients is high and increases with age. in patients for whom iron studies were available, ida was the prominent underlying mechanism, followed by aci or a combination of both. in addition, a group of non-anemic patients with poor lung function had relative iron deficiency. the strikingly significant association between anemia and poor pulmonary function as well as the higher prevalence of anemia in vitamin deficient patients uncover anemia as significant co-morbidity in cf. identification of iron deficiency in anemic as well as in non-anemic patients with poor lung function could be important since iron supplementation might allow hb levels to increase as an appropriate response to hypoxia. oral administration of dha to a cf mouse model corrected the lipid imbalance and reversed certain pathological manifestations in tissues affected by cf. the aim of this study was to investigate the metabolism of la through the n- pathway, and to determine the effect of dha supplementation on la metabolism in cultured cells with a cf phenotype. methods: sense (wt) and antisense (cf) cftr hbe cells were cultured in mem containing % horse serum. cells were supplemented with various concentrations of la (ranging from to µm) and dha ( , , and µm) for one week. cellular lipids were extracted from confluent monolayers, and fatty acids were methylated and analyzed by gc-ms. metabolism through the n- pathway was studied by incubating the cells with . µm [ c] la for hours, and quantitating its downstream metabolites by hplc. results: the levels of la and dha were significantly decreased in cf cells compared to wt cells (la in wt: . ± . , cf: . ± . %, p< . ; dha in wt: . ± . , cf: . ± . %, p< . , mean ± sem for each). la supplementation resulted in an increase in la and aa levels in both wt and cf cells. at most la concentrations tested, la levels were significantly lower and aa levels were significantly higher in cf cells, indicating an increased production of aa from la in cf cells. the study of [ c] la metabolites showed an increased production of multiple downstream fatty acids including : n- ( . fold), aa ( . fold), and : n- ( . fold) in cf cells compared to wt cells, further substantiating an increased metabolism through the n- pathway. supplementing hbe cells with dha for week resulted in a significant increase in la levels and a corresponding decrease in aa levels in wt and cf cells. when supplemental dha was added in combination with la, dha inhibited the la-derived increase in aa levels in cf cells and normalized them to the wt values. the formation of [ c] la metabolites was inhibited after -hour supplementation with dha. this inhibitory effect of dha was greatest on aa production, and it was more prominent in cf than in wt cells (wt: . fold, cf: . fold decrease in aa production). conclusions: decreased la levels in cf are at least in part related to increased la metabolism through the n- pathway. the fact that dha supplementation decreased the flux from la to fatty acid metabolites in the n- pathway, especially to aa, may partially explain dha's mechanism of therapeutic benefit. these data would also suggest that dha combined with la in the diet may normalize fatty acid metabolism in cf patients. background: decreased levels of linoleic acid (la) and docosahexaenoic acid (dha) have been found in the blood and tissues of cf patients. studies on cultured cf cells and cftr-/-mice have indicated that decreased la levels in cf might be related to its increased metabolism through the n- pathway. other potential mechanisms of the observed decreased la and dha levels in cf could be related to altered cellular uptake of these two essential fatty acids. the goal of this study was to investigate the uptake of la and dha into cultured cf cells, and their distribution among the major lipid classes. methods: sense (wt) and antisense (cf) cftr hbe cells were cultured in mem containing % horse serum. the uptake of la and dha into the cells was studied by incubating the cells with . µm [ c] la or [ c] dha for hour and hours, followed by measurement of radioactivity in the supernatant and cell lysate. lipid fractions from the cells (total phospholipids, triglycerides, cholesteryl esters, and free fatty acids) were separated by thin layer chromatography (tlc), and the fatty acid composition was determined by gc-ms after methylation. incorporation of la and dha into cellular lipid fractions was determined by incubating the cells with . µm [ c] la or . µm [ c] dha for hour and hours, followed by tlc separation of lipids and measurement of radioactivity associated with the various fractions. (all data are mean±sem). results: the levels of la and dha were significantly decreased in cf cells compared to wt cells (la in wt: . ± . , cf: . ± . %, p< . ; dha in wt: . ± . , cf: . ± . %, p< . ). fatty acid composition analysis of lipid fractions indicated that la levels were significantly decreased in the phospholipid and triglyceride fractions of cf cells ( . % and . % of the wt values, respectively), and dha levels were significantly decreased in the phospholipid fraction of cf cells ( . % of the wt values). the uptake of la was significantly higher into cf cells compared to wt cells at hour (wt: . ± . , cf: . ± . dpm/µg protein, p< . ) and hours (wt: . ± . , cf: . ± . dpm/µg protein, p< . ). the uptake of dha was also significantly higher in cf cells compared to wt cells at hour (wt: . ± . , cf: . ± . dpm/µg protein, p< . ) and hours (wt: . ± . , cf: . ± . dpm/µg protein, p< . ). incorporation of la and dha was increased in the phospholipid, cholesteryl ester, and free fatty acid fractions of cf cells at hours. dha, but not la, incorporation was significantly increased in the triglyceride fraction of cf cells. conclusions: low la and dha levels in cf cells are associated with a decrease in cellular lipid fractions, the largest of which is in total phospholipids. there is an increased uptake of la and dha into cf cells, and into most lipid fractions of these cells. the decreased levels of la and dha in cf cells, despite their increased cellular uptake and esterification in lipid classes, indicate that there is increased mobilization of these fatty acids in cultured cf cells. perez, a.; issler, a.; davis, p.b. pediatrics, cwru, cleveland, oh, usa even though lung disease continues to be the primary cause of death in cystic fibrosis (cf), the considerable increase in life expectancy of the cf patient over the last decades, has made management of the diverse gastrointestinal and nutritional complications in the adult cf increasingly important. malnutrition and fatty acid deficiency has been a hallmark of cf and its impact in long-term lung health has been established. data obtained by us using affymetrix mg-u av arrays from ileum and liver of pristine -week-old male mice bearing the y x cf mutation and their normal littermates, indicates that there is a profound disturbance in genes associated with lipid metabolism, many of which are regulated by the peroxisome proliferator-activated receptor-gamma (pparγ), a ligand-regulated transcription factor that regulates lipid metabolism and possess anti-inflammatory properties. we hypothesize that lack of cftr function results in altered expression of pparγ regulated genes in the gut and liver, leading to the fatty acid metabolism abnormalities observed in cf. pparγ mrna and protein levels were measured in ileum, colon, liver, and fat of week old female mice bearing the cf mutation s x and its wt littermates (c bl/ j background), and fabpcftr mice (gut corrected, mixed background) after the administration of ethanol or pioglitazone (a pparγ agonist, mg/kg) by gavage every h for days, sacrificed h after last dose. highest pparγ mrna levels were found in fat, followed by colon, liver, and lowest in ileum. at basal state, mrna levels were higher only in fabp ileum vs. wt and fabp colon vs. cf. after treatment, higher expression was seen in cf and fabp ileums and cf liver vs. controls and wt treated mice. however, there were striking differences at the protein level, especially in the colon. at basal state, pparγ was localized, mainly at the surface of the epithelia in wt, in crypts in cf, and mainly in crypts but extending towards the surface in fabp mice. upon treatment, pparγ location in wt colon moved almost exclusively to the crypts, and in the cf and fabp mice, although expression remained mainly in crypts, pparγ could now be seen at the surface. changes in pparγ protein were also seen in ileum and especially liver, between wt and cf, at basal and upon treatment, but changes were subtle and will require more sensitive methods to further examine them. we also examined mrna and protein levels for several genes involved in fat processing that could be related directly or indirectly to pparγ. mrna and protein levels for aqp and cubilin, and protein levels for megalin, apoa- , and apob- were reduced in the ileum of cf mice compared to wt. our data indicates a pparγ dysfunction in the gastrointestinal system of cf mice, which could be responsible for the gastrointestinal manifestations observed in cf. understanding the association between pparγ, cftr, and fat processing may help us to develop new therapies directed towards improving the nutritional status of cf patients that, in the long run, will influence their lung health. cf is characterized by low linoleic acid (la) and docosahexaenoic acid (dha) levels. although this could be due to a primary abnormality in fatty acid biosynthesis, this could also be explained by alterations in phosphatidylcholine (pc) formation. pc are formed by two pathways. the most utilized is the de novo conversion of choline to pc via the cdp-choline pathway, which preferentially forms pc containing saturated and monounsaturated fatty acids. in the other pathway phosphatidylethanolamine (pe) is converted to pc through three methylation steps utilizing the enzyme phosphatidylethanolamine n-methyltransferase (pemt). this pathway preferentially forms pc containing polyunsaturated fatty acids. thus decreased pemt activity would be predicted to result in decreased dha and la levels. this is supported by results seen in pemt knockout mice (watkins et al, j nutrition, ) showing decreased dha and la levels, and variable to increased levels of arachidonic acid (aa). furthermore, innis et al have shown that cf patients have decreased choline levels making them more dependent on the pemt pathway. we hypothesized that alterations in fatty acid levels in cf may be due at least in part to decreased pemt activity. methods: cftr -/mice (cf) and wt littermates were fed with either a diet containing choline (control) or, in order to mimic choline status in humans, an otherwise matched choline deficient (cd) liquid diet for days. choline levels were measured using a kit. livers were perfused and microsomes prepared. to measure pemt activity, microsomes were incubated with radioactive s-adenosylmethionine in the presence of dimethyl pe. the pc product was extracted and radioactivity measured. fatty acids from liver and pancreas were analyzed by gc/ms. results: there was no difference in choline levels in serum or liver comparing wt and cf mice. there was no difference in pemt activity in livers from cf mice compared to wt on control diet. cf mice on cd diet had decreased pemt activity in the liver compared to wt mice on cd diet (wt: ± ; cf: ± cpm/mg protein/min; p< . conclusion: induction of choline deficiency in cf mice to mimic the situation in cf patients, leads to decreased pemt activity. this resulted in low la levels compared to wt mice in both the liver and pancreas and indicates that the fatty acid abnormality in cf is at least in part due to defective pc metabolism through the pemt pathway. ( ). urolithiasis appears to be more common in adult cystic fibrosis (cf) patients than in the general population. a variety of urinary findings have been observed which could account for this, including low volumes, raised oxalate and low citrate, but not all the reports are consistent. most cf patients have pancreatic insufficiency and a plausible explanation for an elevated oxalate excretion in these patients is malabsorbed fat in the intestinal tract sequestering calcium and permitting more oxalate to be absorbed from their food. if this were the mechanism for the elevated stone risk in cf patients then we would expect to observe differences in stone incidence and oxalate excretion between cf patients with exocrine pancreatic sufficiency (ps) and pancreatic insufficiency (pi). methods diagnosis of cf is by clinical and genetic criteria and ps or pi status is based on faecal elastase measurement. patients and healthy volunteers (hv) provided h urines for analysis of volume, ph, ca, mg, na, k, po , oxalate, citrate, creatinine, urea. supersaturations were calculated using equil . there is a history of renal stone disease in out of cf patients attending our unit. patients have pi and include of the stone formers. the remaining stone former is amongst the ps patients. no significant differences in urine chemistry or derived supersaturations were found between the cf patients without a history of stones that were ps (n= ) or pi (n= ). nor were any such differences found when stone forming cf patients were included (ps, n= ; pi, n= ) or when the healthy volunteers were included in the ps group (ps, n= ; pi, n= ). amongst the cf patients, only citrate and creatinine excretions were significantly different between stone formers (n= ) and non stone formers (n= ) (medians (mmol/ h), citrate, . vs. . , p= . ; creatinine, . vs. . , p= . ) . the difference in creatinine excretion can be accounted for by the difference in body mass of these two groups (median (kg) . vs. . , p= . ) compared to the hv, the cf patients with a history of stones had significantly lower citrate excretion and higher calcium oxalate supersaturation (medians, citrate (mmol/ h), . vs. . , p= . ; . vs. . , p= . ) conclusions there is no evidence to suggest that the prevalence of stones is different in the ps and pi groups nor could we detect any difference in excretion of oxalate or any other urine variable related to pancreatic sufficiency. comparing those with and without stones, the only consistent finding was a lower citrate excretion by stone formers. between the hv and stone formers this was also associated with a significantly reduced calcium oxalate supersaturation. our evidence does not support the hypothesis that fat malabsorption by the majority of cf patients contributes to an elevated risk of renal stone disease. we undertook assessment of the use of a hypopharyngeal sensor for detection of laryngopharyngeal reflux in infants and children. gastric reflux in the airway, or supraesophageal reflux, commonly takes a gaseous form that cannot easily be measured using conventional technology. the miniaturized ph sensor at the tip of the dx-ph probe is the only sensor able to measure ph in the airway. fifteen infants and children referred to the pediatric pulmonary center for assessment of chronic cough, hoarse voice, or uncontrolled asthma unresponsive to usual medical intervention underwent - hour to placement of the dx-ph probe in supplementation of radiologic assessment of gastresophageal reflux. the probe was placed transnasally and visualzed in the hypopharynx with the red light at the tip. drops in ph < were correlated with food and symptom diaries. positive results were found in / patients, leading to surgical intervention in three patients. one patient with cystic fibrosis age months with failure to thrive underwent study for symptoms of cough. upper gi demonstrated no gross gastroesophageal reflux or anatomic obstruction.gastric emptying scan was mildly prolonged. outpatient supraesophageal ph probe demonstrated drops to ph< associated with respiratory symptoms. patient underwent nissen and g tube placement with resolution at six week follow-up.the patient returned at age six months with onset cough and underwent repeat study. there were no drops in ph and the cough responded to oral steroid burst. the device was tolerated in / patients with early removal in one young adult with endstage neuromuscular disease and in an infant with "colic" due to a hysterical parental reaction.as a result of our experience we have incorporated the use of supraesophageal ph monitoring into our practice, with particular attention to infants with persistent asthma and children with histories suggestive of gastric asthma. we have now incorporated it into our vivometrics lifeshirt, an ambulatory physiologic monitoring device which will allow us to examine supraesophageal ph and its relationship to sleep apnea, cough, tachycardia, tachypnea, and hypoxemia. aim: monitoring and adjusting dose requirements of pancreatic enzyme replacement therapy (pert) are an integral part of the dietetic assessment of patients with cf. we investigated characteristics of enzyme use in our adult clinic population and determine the extent to which inappropriate enzyme use contributed to poor nutritional and clinical state. method: information was collected using an annonymous self-administered questionnaire developed to measure patient practice, knowledge and beliefs on pert. exclusion criteria included pancreatic sufficiency, < units lipase/kg/d, and fev < %. results: out of potential clinic population of patients completed the questionnaire ( - y, % male, fev - %). % of participants reported to never miss enzymes with meals; this was considerably lower for snacks ( %). those patients who omit enzymes with meals also missed enzymes with snacks (r = %, p< . ). a more appropriate use of pert was observed in patients with lower as opposed to higher bmi. despite intensive dietetic input % of patients missed pert with foods containing fat and % took pert inappropriately with food and drink that did not contain fat. the results identified potentially better practices for measuring pert behaviour and knowledge: ) taking pert with all meals, ) taking pert with majority of snacks ) swallowing capsules intact rather than splitting them open, ) carrying enzymes around with them and ) the ability to titrate pert in accordance to the fat content of food. patients were scored on the basis of the above criteria to differentiate between prudent enzyme users and those who are compromising therapy. in conjunction with their nutritional status score (bmi and gastrointestinal symptoms) risk for intervention can be assessed. discussion:underweight patients have more optimal enzyme use, suggesting greater dietetic involvement in these patients. schall et al also found this to be the case in children. the findings emphasised the need for targeted and effective input in patients where problems are less obvious. the questionnaire has been a useful research tool, and has been adapted as a screening tool for dietitians to gain a subjective perspective of patient's enzyme management and identify patients who need support. the combination of patient's pert usage and their nutritional status could help capture and identify risk objectively and quickly and allows resources to be allocated most effectively. schall . ), nutritional failure, measured as bmi< , (p> . ), or lung transplant status (p> . ) between patients with and without colonic wall redundancy. there was no significant difference in cftr function, per sweat chloride values, between groups (p> . ). conclusion: we report a previously undescribed colonic wall abnormality seen on ct in patients with cystic fibrosis: proximal colonic wall redundancy was found in % of adults with cystic fibrosis, but not in children, appeared to be chronic, and was associated with non-∆f cftr gene mutations, particularly the g x mutation. recognition of the ct appearances will prevent erroneous diagnosis of acute colonic disease in this patient population, and stimulate investigation of a biologic basis for this finding as a recognizable feature of adult cystic fibrosis. intro: infants and children with cystic fibrosis, both pancreatic sufficient and insufficient, are at risk for developing hyponatremia due to an excess loss of salt though skin during sweating. these children should receive daily supplemental sodium. children without cf require - meq/kg of sodium daily; and one could argue children with cf need more. a historical recommendation is / tsp table salt daily for infants; this contains to meq of sodium based on the density of the table salt. parents may measure the salt inaccurately, and it may be difficult logistically to distribute the salt throughout the day. if supplementation is given as sodium chloride solutions distributed through pharmacies, as suggested in a recent consensus conference, co-pays or full payments may be required. our aim is to provide a simple, inexpensive, and more accessible method for accurately giving sodium supplementation. do pre-packaged salt packets contain a precise amount of sodium and thus could be recommended to fulfill the daily requirement of sodium in patients with cf? method: pre-packaged table salt packets were collected with permission from four national fast food restaurants and one commercial salt producer. contents (sodium chloride) of ten packets from each source were weighed individually. measurements were made using a scientific scale with the ability to measure up to / , of a gram. weights of sodium chloride from individual packets were converted to meq sodium. means, ranges and standard deviations were calculated for the data using microsoft office excel ™ program. see table conclusions: there is some variability in the content of pre-packaged salt packets, both between and within brands, though some brands have less variance than others. measurements showed that packages contained mean values of approximately to meq of sodium. there is a range in the sodium dose recommendations, and spot urine sodium can be used to determine if patients are sodium depleted. pre-packaged salt packets lack the precision of pharmacological dosing but are inexpensive, practical, and supply a reasonably predictable dose of sodium. background: ten to forty percent of people with cf have vitamin d deficiency secondary to pancreatic insufficiency. obtaining optimal vitamin d levels ( -ohd) has been a challenge. for vitamin d deficient individuals, current supplementation strategies ( , iu weekly for months) have been mostly unsuccessful. objectives: to identify individuals at risk for vitamin d deficiency and to evaluate the efficacy of a week repletion course of high dose ergocalciferol in children and young adults with cf. methods: as part of a quality improvement initiative, a prospective cohort study was performed from january to april . phase i included querying our cf practice database to identify patients who were due for annual routine blood tests or who had recently documented levels of -ohd less than ng/ml. in phase ii, , iu of daily ergocalciferol was prescribed for a day period as part of routine inpatient or outpatient care. during phase iii, a post treatment -ohd level was obtained. baseline subject characteristics were obtained at entry and included age, gender, pubertal status, pancreatic status, and fev %. post -ohd levels were classified as sub therapeutic (< ng/ml), therapeutic ( - ), high therapeutic ( - ), or potentially toxic (> ). a paired t test was performed to evaluate pre and post intervention differences. the impact of age, gender, and lung function on the response to vitamin d supplementation were also analyzed. all values are expressed as mean ± standard deviation. results: eighteen individuals with cf participated in the study. the mean age was years with a range of to years. . % were male, and % were pancreatic insufficient requiring pancreatic enzyme replacement. fev % was . ± . %. all participants had -ohd levels less than ng/dl pre-treatment. -ohd levels increased from . ± . to . ± . (p< . ). of the participants ( . %) became therapeutic over the two week interval. an increase of . ± . (ng/ml) or % was seen in the two week period. no correlation was seen on the extent of increase in -ohd and baseline lung function. pre and peri pubertal individuals had a greater increase in -ohd levels than post pubertal individuals ( . ± . vs . ± . , p< . ) although the magnitude of change did not reach significance. the degree of response also appeared related to age (r=- . ). no impact of gender was seen. seven individuals achieved normal therapeutic values while were in the high therapeutic range. no participants had toxic levels. conclusions: the results of this study demonstrate that high dosing of ergocalciferol over a day period is an effective strategy in achieving therapeutic levels of vitamin d. it is unclear whether a pubertal effect on the degree of response exists or if this response is merely age related. further research is needed to evaluate whether this strategy is able to maintain therapeutic levels after completion of the intervention. additional studies that monitor compliance and evaluate responders and non-responders are needed. foundation consensus committee established guidelines for optimizing bone health in individuals with cf, including screening for vitamin d insufficiency and a treatment protocol for achieving and maintaining normal serum vitamin d concentrations. however, since the guidelines were published, there has been additional research assessing various vitamin d treatment regimens and their effect on serum vitamin d concentrations. in addition, vitamin d assay variability has recently been well documented. the objective of this study was to evaluate variability of vitamin d surveillance methods as well as treatment protocols used to treat low vitamin d concentrations at cf centers. methods: a survey was created via survey monkey, and was sent to the cf nutrition listserv, which includes over cf healthcare providers. spss version . was used for statistical analysis. results: fifty-nine listserv members responded to the survey. the majority ( %) of respondents measured oh vitamin d concentrations, and % considered patients to be vitamin d deficient when serum concentrations were below ng/ml. however, % were monitoring only , oh vitamin d or a combination of both concentrations. there was considerable variability in vitamin d supplementation protocols for treating low oh vitamin d concentrations. only % were following the consensus guidelines of , units of ergocalciferol once/week for ages through adults. one third indicated that they followed consensus guidelines for patients less than age ; however nearly half of the remaining two thirds were not sure what is prescribed or have not observed low vitamin d concentrations in this age group. fifty-two percent of respondents were aware of vitamin d assay variability. despite this knowledge, the majority were unaware of the type of vitamin d assay used to measure serum vitamin d concentrations at their institution. conclusions: there is considerable variability in the measurement and treatment of vitamin d concentrations across cf centers despite guidelines provided via a cff consensus report. the consensus guidelines for measuring vitamin d concentrations and treatment of vitamin d insufficiency should be revisited based on recent studies and expert opinion. children with cystic fibrosis (cf) who suffer from sub-optimal growth are offered overnight enteral feeding via gastrostomy tube. this study aimed to assess the nutritional impact of overnight feeding over a year period using a retrospective study design. data, including height, weight, fev , pancreatic function were collated for all patients with cf who had a gastrostomy tube placed in the past years. data were collected at baseline and then every months for years post-gastrostomy insertion. data for height, weight and body mass index (bmi) were converted into z scores using centre for disease control reference ranges and the lms method ( , ) . a total of patients ( . % of the clinic population) were identified as having a gastrostomy tube. two were no longer receiving feeds and there were incomplete data for subjects. on placement of the tubes, there were substantial deficits in nutritional status with mean z scores for height - . (sd . ), weight - . (sd . ) and bmi - . (sd . ), with compromised lung function (mean fev %, range - ). using a repeated measures anova with fev as a co-variate, there were no significant differences in weight, height or bmi from baseline to years (at years post placement: mean z scores for height - . (sd . ), weight - . (sd . ) and bmi - . (sd . ). fev remained stable over time. these results indicate that gastrostomy feeding can potentially halt the decline in nutritional status that is a feature of cf. however, patient expectations that over night enteral feeding will lead to an increase in nutritional status need to be sensitively managed. background : cystic fibrosis (cf) is the most common life threatening autosomal recessive disorder in caucasians.following a landmark paper by corrie et al a high fat,high calorie diet has promoted a normal growth pattern.improved nutritional status together with prevention and early treatment of respiratory infections has contributed to improved survival. objective: the aim of this study was to assess the relationship between lean body mass and disease severity in children with cf. methods: the nutritional status and body composition of children with cf was measured using a harpenden stadiometer, calibrated electronic scales and a ge lunar prodigy densitometer. the following indices were calculated; body mass index (bmi), fat mass (expressed as logarithm % total body fat (ln%tbf) and fat free mass (ffm). body composition data were expressed as z-scores using dutch reference values. correlations were performed between indices of nutrition (bmi, height, ffm, tbf) z scores and markers of disease severity (percent predicted fev and shwachman kulczycki (sk) scores).the nutritional status of these children was also compared with healthy controls. statistical analyses were performed using microsoft excel . results: there was a weak but significant correlation between sk scores and height z score and ffm (r = . , p< . & r = . , p= < . respectively). there was also a weak but significant correlation between % predicted fev and bmi and ffm (r= . , p < . , r= . , p< . ). height and weight z scores were significantly lower in children with cf (- . and - . respectively) than in control subjects ( . and . respectively) with p < . in both groups. conclusion: this study demonstrates that there is an association between indices of nutrition and disease severity in children with cystic fibrosis. muscle mass (ffm), assessed by dual energy x-ray absorptiometry (dxa) correlates with lung function and with sk scores. the strongest correlation between markers of disease severity (sk scores and % predicted fev ) was with ffm. ffm may be expected to be associated with respiratory function tests.however,sk scores are a more general assessment of well being and less obviously directly related to muscle mass. ffm and growth may reflect long term nutrition and health in contrast to bmi and ln%tbf which may reflect acute deterioration or short term nutritional intervention. therefore ffm may prove to be a useful tool to assess quality of nutrition and may be predictive of respiratory and general decline. body composition assessment has an important role in chronic conditions such as cystic fibrosis, in order to identify nutritional depletion and evaluate nutritional interventions. it is important that reliable and accurate methods are used. the usefulness of non-invasive methods, such as skinfold thickness measurements (sft) to measure change in body composition has not been evaluated in cf. this study aimed to compare changes in fat-free mass (ffm) and fat mass measured using sft and dual-energy x-ray absorptiometry (dxa) in adults with cf. methods: adults with cf ( % male, % pancreatic insufficient, mean age at baseline . (sd . ) years, mean fev at baseline . (sd . ) % predicted) were studied. they underwent body composition assessment using dxa and sft, at baseline and a mean of . (sd . ) years later. durnin and womersley equations were used to estimate ffm and fat mass from sft. estimates of change in ffm and fat mass obtained using sft were compared with dxa using paired t-tests, univariate analysis and bland and altman analysis. results: at baseline, mean ffm was . (sd . ) kg; mean fat mass was . (sd . ) kg and mean bmi was . (sd . ) kg/m . mean ffm, fat mass and bmi were not significantly different at follow-up, indicating no change in nutritional status for the population overall. individual change in ffm ranged from - . to + . kg, while change in fat mass ranged from - . to + . kg. the table shows mean change (∆) in ffm and fat mass by each method, correlations (r ) between sft and dxa, and the % limits of agreements (loa) between sft and dxa for change in ffm and fat mass. mean change in ffm and fat mass estimated using sft did not differ significantly from dxa. mean bias between the methods was small, and overall correlations between sft and dxa were strong for changes in both ffm, suggesting good agreement for the overall population. however the % loa between the two methods were wide for change in both ffm and fat mass. this suggests that sft will not accurately predict changes in body composition in all cf patients. conclusion: sft measurements do not accurately estimate change in ffm or fat mass over time in individual adult cf patients compared with dxa. body composition changes measured using sft tended to more closely reflect changes detected by dxa in male patients compared with females. caution should be exercised when interpreting the results of serial measurements of body composition using sft in individuals with cf. analysed were significantly correlated with age-cml or srage levels (age, gender, bmi, presence of cf-related diabetes mellitus (cfrd) or hba c level). conclusions: serum levels of ages are elevated in adults with cf. elevation is not restricted to those with cfrd. the levels of srage reflect ability to respond to this pathway and are associated with poorer lung function. modification of the diet in cf may reduce this mediator and may have the potential to modify lung and renal injury. mouse models of cf generated by expression of improperly processed cftr (∆f ) or lacking cftr expression (ko) demonstrate selective induction of sult e in the liver (falany et al., biochem. j. : , ) . sult e is responsible for the sulfation and inactivation of β-estradiol (e ) at physiological concentrations. the increase in sult e expression is specific to the hepatocyte, whereas cftr is selectively expressed in cholangiocytes. the induction of sult e activity is associated with changes in levels of e regulated proteins in cftr (-/-) liver (li and falany, j cystic fibrosis : , ) . increased sult e activity is correlated with low body weight and decreased igf- message levels in livers of cftr(-/-). the mechanism for the induction of sult e in hepatocytes by cholanigiocytes lacking cftr expression, and the mechanism for the inhibition of igf- expression by increasing sult e expression was investigated in human cells. human mmnk- cholangiocytes in which cftr expression was inhibited by short interfering rna (sirna) induced sult e expression in human hepg hepatocytes when cocultured in a permeable membrane separated system. sult a expression was not altered in this model. the data suggests that loss of cftr function in cholangiocytes is capable of selectively modulating sult e expression in hepatocytes. to investigate the ability of increased sult e activity to modulate expression of igf- , hepg cells were stably transformed with sult e /pcdna to activity levels intermediate to the levels observed in cftr(-/-) mice. the increased sult e activity was associated with decreased igf- message expression in the hepg cells. since a major pathway for igf- regulation involves growth hormone (gh) stimulation of stat b phosphorylation, the effects of e and increased sult e activity on gh stimulated stat b phosphorylation were examined. stat b activation was identified using immunoblot analysis with a rabbit anti-tyrosine phosphorylated-stat b antibody. treatment of control hepg with nm e prior to the addition of gh increases stat b phosphorylation. in hepg cells expressing increased sult e activity, the stimulation of stat b phosphorylation by nm e was significantly decreased. e had an apparent rapid action on stat b phosphorylation that is not attenuated by the estrogen receptor antagonist, ici , . e was effective at increasing stat b phosphorylation when applied to hepg cells - min before gh. increased stat b phosphorylation was observed in control hepg cells at nm e although -fold higher e concentrations were required in the sult e -hepg cells consistent with the increased e sulfation. no differences were observed in total stat b in any of the studies. this study demonstrates that human cholangiocytes with low levels of functional cftr are capable of inducing sult e expression in human hepatocytes and the increase in e sulfation inhibits the gh stimulation of igf- expression via a decrease in stat b phosphorylation. supported by grants from the cystic fibrosis research, inc. and nih (gm ). williams, j.e. ; benden, c. ; jaffe, a. ; suri, r. ; wells, j.c. ; fewtrell, m.s. . mrc childhood nutrition unit, institute of child health, london, united kingdom; . portex respiratory medicine unit, great ormond street hospital, london, united kingdom background: patients with cystic fibrosis (cf) are at high risk of malnutrition. to our knowledge, no study so far has employed a reference fourcomponent model(sup) (/sup)( cm) to assess body composition (bc) in cf children, which allows accurate evaluation of both fat mass (fm) and the components of fat-free mass (ffm; mineral, protein, water) and most studies have been cross sectional. methods: cf subjects, aged - yrs, were compared with age-and sex-matched healthy controls for assessment of bc using a reference cm. comparison between groups was performed using paired t-tests and general linear models to adjust for age, height and pubertal status. in addition fm index (fmi; fm/height ) and ffmi (ffm/height ) standard deviation scores (sds) were calculated using measurements performed in a reference population of healthy children aged to yrs. repeat measurements were made in children with cf ( boys) after yrs and the change in fmi sds and ffmi sds investigated. results: for the initial measurement at baseline, boys with cf (n= ) were significantly shorter (mean (se) height sds: - . ( . , p< . ) compared to uk reference data; girls with cf were lighter, bmi sds (- . ( . ), p< . ). at baseline, comparison of cf children with pair-matched controls indicated that there was no difference in the boys but cf girls had less fm (cf minus control) (- . ( . )kg, p< . ) and mineral mass (- . ( . )kg, p< . ) after adjustment for age, height and puberty. fmi sds adjusted for age and puberty was also significantly lower in girls with cf (- . ( . ), p< . ) whereas ffmi sds was not. for comparison with the large reference population there was no difference in the boys at either time point for fmi sds and ffmi sds (paired t-test). for girls however, there was a significant difference at both baseline fmi sds (- . ( . ), p< . ), ffmi sds (- . ( . ), p< . ) and yrs fmi sds (- . ( . ), p< . ) and ffmi sds (- . ( . ), p< . ). paired t-test analysis between baseline and yr followup measurements indicated that fmi sds and ffmi sds were not significantly different at each point in time in either boys and girls. conclusion: at baseline, the bc of cf boys appears to be within normal range, but the cf girls already have lower fm and mineral than their healthy pair matched controls, even when adjustment for size and puberty is made. follow-up measures in cf children indicated that these descriptions of nutritional status of cf boys and girls remained consistent over the following years, with no evidence of any further deterioration in girls. however, nutritional surveillance is important in both sexes in cf at around puberty to address potential deterioration and this study suggests that the cm is a useful tool for this purpose. fuller background: although lung function (lf) in children with cystic fibrosis (cf) has been reported to be related to their nutritional status, particularly body fat, the latter has usually been assumed from anthropometric measurements and not measured using an appropriate reference method. habal, h. ; al-turkmani, m. ; freedman, s. ; laposata, m. . pathology, massachusetts general hospital and harvard medical school, boston, ma, usa; . medicine, bidmc and harvard medical school, boston, ma, usa background: increased dietary intake of fatty acids is recommended for cf patients. it is unknown if this results in increased incorporation of all fatty acids-saturated, monounsaturated, and polyunsaturated-into cells with a cf phenotype to a greater extent than into non-cf cells. docosahexaenoic acid (dha) supplementation of cftr-/-mice has been shown to reverse the pathologic cf phenotype. aim: to investigate whether the uptake of multiple predominant fatty acids and the release of membrane-bound arachidonic acid (aa) are altered in cultured epithelial cells with a cf phenotype; in addition, to determine whether fatty acid uptake is selectively inhibited by dha supplementation. methods: sense (wt) and antisense (cf) cftr hbe cells were cultured in mem containing % horse serum. aa release from membrane phospholipids (pls) was studied by incubating the cells with µm [ c] aa for min, and then with serum free medium containing . % fatty acid free human serum albumin in the presence or absence of µm adenosine for min, followed by measurement of radioactivity in the supernatant. the uptake of fatty acids into the cells was studied by incubating the cells for different time points with . µm [ c] linoleic acid (la), [ c] aa, [ c] dha, [ c] palmitic acid or [ c] oleic acid, in the presence and absence of µm or µm dha, followed by measurement of radioactivity in the cell lysate. results: basal and adenosine-stimulated aa release from membrane pls was significantly higher in cf cells compared to wt cells (basal wt: . ± . , cf: . ± . dpm/ cells, p< . ; stimulated wt: . ± . , cf: . ± . dpm/ cells, p< . ; mean±sem for each). the uptake of la, aa, dha, palmitic acid and oleic acid were significantly higher into cf cells compared to wt cells. dha significantly decreased the elevated uptake of these fatty acids into cf cells, but not into wt cells. data are shown in the table below. conclusion: in this cell culture model, these data demonstrate that aa uptake and release are increased in cf cells, which may suggest elevated mobilization of this fatty acid by cf cells. our data also show that cftr dysfunction leads to increased uptake of all tested fatty acids into the cells. downregulation of this increased uptake may be one mechanism by which dha exerts its therapeutic benefits. low bmi in cf correlates directly with worsening lung function and is an independent risk factor for mortality. the causes for this decreased bmi are incompletely understood but could be related to levels of the orexigenic peptide ghrelin, and to levels of leptin, an anorexigen. studies assessing plasma levels of leptin in cf have shown contradictory results, while the data on ghrelin in cf are scarce. we assessed both leptin and ghrelin plasma levels in mild (fev > % predicted), moderate (fev % to % predicted), and severe (fev < % predicted) adult cf patients. we compared these levels in cf subjects to age matched controls with normal bmi (range . to . kg/m ). we conclude that leptin levels are decreased, while ghrelin levels are increased only in cf patients with severe disease and low bmi. plasma levels of ghrelin and leptin did not differ from normals in those with mild and moderate disease. gastroesophageal reflux (ger) is common in cf before and after lung transplant (ltx). laparascopic fundoplication (lapf) is used when patients fail on conservative medical therapy. aims: to review longer term clinical outcomes of lapf in patients with cf in terms of lung function, body mass index (bmi), reflux symptoms, patient satisfaction with surgery and complications. method: retrospective review of patient records and patient questionnaire to gather relevant information. results:thirty-two patients with cf underwent lap.f recently; transplanted (ltxgroup), female (mean age [ - ]) years, and not transplanted (cf group), female ( ). the median time between ltx & lapf was ( ; ) days, range - . endoscopy was undertaken in patients prior to surgery and all underwent hr ph monitoring. complete (nissen) lapf was undertaken in % patients and a partial (toupet) in the other %. total median time in the operating theatre was ( - ) minutes. the average length of stay in hospital (in days) in the ltxgroup: pre-op. was . ; post-op. was . days; and in the cf group was: . and . respectively. lung function (fev and fvc in percent predicted) and bmi were measured at minus(-) , plus (+) , + and + days from lapf. patient satisfaction with lapf was a mean of % in the ltx group and % in the cf group and improvement in reflux symptoms of % in both groups. nocturnal cough reduced markedly in the cf group and there was an overall improvement in quality of life in both groups. complications consisted of port site hernias, failed lapf with re-operation months later; esophageal dilatations and patient with lower esophageal sphincter hypotonia. there were deaths on day , and post fundoplication. one was in a non-transplanted patient with recurrent episodes of hemoptysis. she succumbed to a catastrophic hemoptysis episode not related to lapf. the other were in transplanted patients. conclusions: in this relatively small series of patients there was a small but significant drop in fev in the ltx group days after lapf, but not in the cf group who remained stable throughout the review period. there was a significant drop in bmi in both groups after lapf, but the cf group returned to pre lapf values at + days. patient satisfaction with the procedure was high. fundoplication is an expensibe complicated procedure not without risk. a long term prospective study is warranted. historically this phenomenon has been attributed to intestinal malabsorption and cachexia as a consequence of chronic lung infection. however, cf mice exhibit similar growth and weight deficits but are not afflicted with malabsorption or lung infection, suggesting other mechanisms are participating as well. we hypothesize that small size is in part a result of increased β-oxidation in adipose stores. to test this we utilized a mouse model to study lipid metabolism. our study includes female cf mice with a null mutation in the cftr gene. these mice are congenic on the c bl/ j background as well as age matched with their controls (wt) to - days. liver, adipose and skeletal muscle (calf) were dissected from wt and cf mice in a fed state. tissues were immediately flash frozen in liquid nitrogen and used for rna and protein lysate harvest. quantitative pcr (qpcr) was performed using primers for the lipogenic isoform of acetyl-coa carboxylase α (acc α) in the liver and adipocytes, whereas the non-lipogenic isoform acc β was assessed in the skeletal muscle. primers for acyl coa oxidase (aox), carnitine palmitoyltransferase (cpt ) and long acyl-coa dehydrogenase (lcad) were used as gene markers for β-oxidation steps in the peroxisome, carnitine shuttle and mitochondrion, respectively. western blots were performed to detect acc (active) and phospho-acc (inactive) forms. the results show that there is a decrease in the total amount of acc protein in the fat as well as the skeletal muscle of the cf mice and the ratio of acc:phospho-acc is substantially lower in the cf tissues. phosphorylation and/or decreased expression of acc results in decreased expression of malonyl coa, leading to a cascade of events that increase β-oxidation. in addition, increased expression of aox and lcad, two downstream β-oxidation genes, is also observed. hyperactivity of the β-oxidation pathway could account for the decreased adipose stores seen in cf mice compared to the wt. such patterns of expression are reminiscent of starvation. caloric intake and intestinal absorption of dietary lipid were measured and cf animals consumed approximately % the calories of wt animals (per gram body weight) and lipid absorption was not significantly different ( % for cf, % for wt), indicating the cf animals have sufficient access to nutrition. we will continue to investigate fatty acid synthesis and degradation pathways during both the fed and fasted states to delineate the mechanism. this research was supported by grant hl . effect of gender on dha levels in p and rbc with lower values for male cf patients. acknowledgments: this work was supported by the belgian cf association (ablm), the french cf association (vlm) and by a grant from frs (ucl). data are expressed as means ± sd. * and indicate a significant difference with controls and cf ps patients respectively, p< . . background: deficit of antioxidant systems and increased oxidative stress have been demonstrated in cf patients with respect to healthy controls. the origin of this imbalance is known to be multifactorial. objective: the aim of our study was to assess changes in antioxidant systems and oxidative stress parameters in cf pediatric patients in presence or absence of pancreatic insufficiency material and methods: we recruited patients ( with pancreatic insufficiency pi and with pancreatic sufficiency ps) attending the cystic fibrosis centre of turin ( ) ( ) . during the annual routine control visit were performed antropometric measurements, pulmonary function tests (fev ), and were collected fasting blood samples and last days food record. vitamin e, a, c, selenium, glutathione (gsh, gshpx, gssg), dehydroascorbic acid, pcr were assessed. data were analysed using the two sample t-test with equal variances. results: there are not statistically significant differences between two groups for sex, age and fev . / of pi patients and / of ps patients took oral vitamin supplementation. serum levels of alpha-tocopherol and beta-carotene resulted within the local reference range either in pi or ps patients showing no significative differences. the mean serum levels were respectively suboptimal and optimal for vitamin e and a (biesalsky ' ). pi and ps patients did not show significant intergroup differences of water soluble and enzymatic antioxidants levels as well. serum levels of vitamin c, selenium, erythrocyte reduced glutathione (gsh) and glutathione peroxidase activity (gshpx) resulted within the local reference range. plasma vitamin c concentrations resulted slightly inferior to the optimal values (biesalsky ' ) in both groups. oxidative stress parameters (erythrocyte glutathione disulfide-gssg, dehydroascorbic acid) and inflammatory parameters (pcr) did not differ significantly, presenting values within the local reference range, in both groups. nutritional data revealed significantly reduced zscore for weight (p< . ) and height (p< . ) in pi patients vs ps patients. our data did not evidence significant differences in total energy (kcal/kg) and macronutrients (% of total kcal) intake in both pi and ps patients. dietary intake of antioxidant vitamins resulted slightly superior the rda (larn- ) presenting no significant intergroup differences. conclusions: this study shows that pancreatic insufficiency do not influence oxidant/antioxidant balance in cf patients at least in paediatric age. until innovative supplementation guidelines will be proposed for application, nutritional education and monitoring and correct dietary habits are useful to optimize antioxidant status in cf patients. there are more than cf mutations that are responsible for the spectrum of disease severity in cystic fibrosis (cf). the most common, f del mutation, is associated with pancreatic insufficiency (pi), and a classic phenotype. . yet even within a group of f del homozygotes, there is a small minority of patients ( . %) who are not taking pancreatic supplements, and are therefore presumably pancreatic sufficient (ps) (data from * cff registry). this observation prompted us to look more closely at enzyme use in cf patients with non-f del homozygous genotype. we reviewed enzyme use in cf patients who were fully genotyped and whose enzyme use was documented in the * cf registry. heterozygotes with one or two non-f del mutations were included in this analysis. using the assumption that the milder mutation is dominant, if the patient was ps and heterozygous with one f del mutation, the second mutation was classified as ps; if the person was pi, the second mutation was classified as pi. we also reviewed the pancreatic status and genotype of patients participating in the wisconsin newborn screening study. in this randomized control trial (rct), pancreatic status was determined by hour fecal fat analysis. enzyme use was extracted from the database for the year . there were patients in the cff registry with a total of different mutations. twenty-one of these mutations were associated with pi. this finding correlated well with the per cent of patients who were on enzyme supplements (range of per cent patients on enzymes by mutation - %). however when we looked at the six mutations associated with ps, the distribution of patients on enzymes vs. not, was much closer. see table below. possible reasons for enzyme supplement use by patients with ps mutations include a) treatment for recurrent pancreatitis, b)the fact that this is a cross-sectional analysis and patient may be on enzymes temporarily, c) patient may truly be pi, d) overuse of enzymes due to unreliable test to measure pancreatic status in patient, e) patients who are on enzymes are older, and have changed from ps to pi phenotype. this analysis suggests a need for better assessment of pancreatic status in patients with cf. * this analysis was originally done by request for the ecfs conference at garda on cftr genetics, and focused on the suggested " main mutations", and will be updated with the cff registry data. (supported by nih grants dk , dk , mo rr , mo rr ) number of patients in rct with each mutation: , , , , , respectively. dutta, a. ; woo, k. ; fitz, j.g. ; feranchak, a.p. . department of pediatrics, ut southwestern medical center/children's medical center, dallas, tx, usa; . medicine, ut southwestern, dallas, tx, usa extracellular atp is an important signaling molecule contributing to bile formation by the liver through binding biliary epithelial cell (cholangiocyte) membrane p receptors and stimulating cl-efflux. importantly, atp appears to work through cl-channels unrelated to cftr, suggesting it may be a potential way to modulate bile flow in cf liver disease. however, the signaling pathways linking receptor binding to channel activation in cholangiocytes are unknown. consequently, the aim of the present study was to identify the pathways responsible for atp-stimulated increases in [ca +]i and membrane clpermeability in a human biliary epithelial cell model. studies were performed in mz-cha- human biliary cells; [ca +]i was measured by fura- and membrane cl-currents by patch-clamp techniques. results: exposure of cells to atp ( µm) resulted in a rapid increase in [ca +]i to . ± . nm (n= ) which was abolished by prior depletion of intracellular ca + by thapsigargin ( . ± . nm; n= ), but unaffected by removal of extracellular ca + (egta, . ± . nm; n= ). in parallel studies, atp ( µm) increased current density from - . ± pa/pf to - . ± . pa/pf (n= ) . currents reversed at mv (ecl-), were outwardly rectifying, and were inhibited by the cl-channel inhibitor nppb (- . ± . pa/pf, n= ), but not the cftr inhibitor cftrinh . atp failed to activate currents after depletion of intracellular ca + stores by bapta-am (- . ± . pa/pf, n= ). the p y receptor antagonist suramin inhibited atp-stimulated increases in [ca +]i ( . ± . nm; n= ) and cl-channel activity (- . ± . pa/pf; n= ). however, the p x receptor antagonist brilliant blue g did not affect the magnitude of atp-stimulated cl-currents (- . ± . pa/pf, n= ). together these findings suggest that atp activates cl-currents primarily through p y, but not p x, receptors in human biliary cells. since p y are g-proteincoupled receptors and modulate [ca +]i by phospholipase c (plc) generation of inositol , , -trisphosphate (ip ), a series of experiments were designed to directly assess the effects of ip receptor inhibition or stimulation on atpstimulated cl-currents. first, the plc inhibitor, u blocked atp-stimulated cl-currents ( . ± . pa/pf; n= ). second, the specific ip receptor blocker -apb, inhibited both the atp-stimulated increase in [ca +]i ( . ± . nm, n= ) as well as membrane cl-currents (- . ± . pa/pf, n= ). lastly, intracellular dialysis with purified ip during whole-cell patch clamp activated cl-currents with identical properties to those activated by atp (- . ± . pa/pf, n= ). together these studies demonstrate that extracellular atp stimulates ca +-activated cl-channels in cholangiocytes primarily through p y receptor binding and plc/ip -dependent release of intracellular ca + stores. thus, the p y-ip receptor signaling complex represents a potential target to increase cholangiocyte secretion, and hence augment bile flow, in the treatment of the cholestatic liver disease associated with cf. supported by grants from the nih niddk and the cff to a. feranchak. background: supplemental pep therapy is used to treat cf patients with epi to assist with digestion, food absorption. this open-label, multipledose, single-treatment, multicenter study evaluated efficacy and safety of eur- for malabsorption treatment in young patients with cf and epi. eur- is a novel, zero-overfill, highly-stable formulation of porcinederived pancreatic enzymes, and was specifically developed for use in very young children allowing the product to be sprinkled on food. methods: eligible patients were < years with cf and epi, in acceptable nutritional status, needing peps and clinically stable. patients consumed a standard cf recommended diet, could not take drugs affecting gastric ph or motility. the trial involved a -day dose stabilization period and a -day treatment period. patients switched from their baseline enzyme treatment without washout. the optimal dose of eur- , determined during dose stabilization, was used during treatment. primary endpoint compared malabsorption of fat after eur- vs. previous treatment. clinical symptoms were also evaluated, as was physicians' and parents' judgment on the control of malabsorption signs and symptoms. safety measures included aes, physical exams, vital signs and changes in clinical laboratory findings, including cholesterol levels and fat-soluble vitamins. results: nineteen patients ( male) enrolled and completed all study phases. mean age was . years (range - ). percentage of responders (patients without steatorrhea (< % fecal fat content) and without signs and symptoms of malabsorption after and weeks of treatment) was . at baseline, . (p= . vs. base) after stabilization and . (p = . vs. base) at study end. frequency and oily stools showed a significant decrease vs. baseline at study end. the mean number of stools per day was . stools at screening and . stools during treatment (p< . ). the mean proportion of stool samples with visible oil or grease at screening was . % and . % during treatment (p= . ). a statistically significant improvement was seen in the incidence of moderate bloating. physicians characterized % of patients as "improved" in the control of epi signs and symptoms vs. previous therapies and % as "unchanged" at the end of the treatment phase, while parent assessments were % "improved" and % "unchanged." vitamin k absorption (measured through pivka) improved with eur- treatment over prior pep treatment measured at baseline. eur- was safe and well tolerated, with minimal aes. no aes led to discontinuation of study drug interruption. no incidences of uric acid toxicity or fibrosing colonopathy following eur- treatment. no trends were seen in lab parameters, physical examination, or vital signs. conclusion: eur- is effective, safe, well-tolerated in the treatment of epi in young cf patients. treatment controlled malabsorption and clinical symptoms of epi in young patients with cf. safety and efficacy of eur- in this trial is consistent with the pivotal phase iii trial. there is an association between the presence of cf and alterations in fatty acid metabolism, consisting of a decrease in linoleate ( : n- ). more recently, a deficiency of docosahexaenoate ( : n- , dha) has arisen as a prominent fatty acid abnormality in cf. an established breeding colony of exon cftr knockout (cftr-/-, cf, n= ) and wild type (wt, n= ) mice was used for this study. all mice were weaned at days of age and then raised on peptamen and water until days of age, and then continued for days with ml/day of peptamen to homogenize the nutritional status. after this period, some mice (n= per group) were subjected to dha treatment ( mg/day) for one week. after sacrifice, pancreatic tissue was collected, homogenized and extracted. lipid classes were separated by sequential elution in aminopropyl columns. the eluted fractions were transmethylated and injected into a hewlett-packard a gas chromatograph with a flame ionization detector for quantitative analysis. the vast majority of fatty acids were mainly distributed in phospholipids, except those with -carbons. wt and cf mice were studied a gender gap in the survival of cystic fibrosis (cf) is well-documented. however, little is known whether there is a gender différence in pubertal growth pattern in cf children. delayed and attenuated pubertal growth are commonly observed in adolescents with cf. however, characterizing height velocity (hv) pattern is very challenging because of errors associated with calculating hv, e.g. interpolation and extrapolation between heights measured in irregular intervals, as well as difficulties in determining the location and the magnitude of peak height velocity (phv). in this study, we applied a new statistical method, namely, the semi-parametric shape-invariant model, to characterize the hv pattern of cf children. this method is based on the assumption that all individuals' growth curves have a common shape. therefore, this method attempts to shift and stretch (squeeze) individual's growth data until they can be modeled by the common shape curve. the semi-parametric shape-invariant model has the advantage of fitting measured heights directly, thereby eliminating the errors associated with calculating hv. the study population included cf children who had height measurements between ages to years documented in the - cff registry. because fitting each individual height curve is computing-intensive, boys and girls were randomly chosen for this preliminary analysis. our results showed that girls exhibited a typical growth deceleration during late preadolescence, followed by accelerated hv and a single phv during adolescence (figure) . unexpectedly, cf boys exhibited a notable hv peak during late preadolescence in addition to the typical adolescent phv (figure) . overall, the mean magnitude and age of phv was . cm at . years for boys and . cm at . years for girls. when compared to phv of non-cf children based on tanner's reference (pediatr. , : ) , cf boys showed a greater delay in the age of phv while cf girls showed a greater attenuation in the magnitude of phv. further analyses are being performed to characterize the hv patterns of all children to confirm these results. (supported by nih-dk ). anelli, m.; foresti, r.; peloso, l.; ortenzi, g. medical affairs, eurand, pessano con bo, italy background: given their inherent instability, the dose units of all pancreatic enzyme preparations (peps) have always been "overfilled" to compensate for enzyme degradation over time and assure at least % of the label lipase content at the end of their shelf life. this overfill issue was first examined by whitehead who reported overfills ranging from to % after analyzing several commercially available peps (whitehead am. pharm weekbl sci. ) . the usp presently allows for pep overfills up to %. thus, the actual active lipase content of a pep capsule labelled at , iu might vary from , to , units according to its "freshness." as a result, patients taking peps receive a product with variable potency, which can lead to efficacy issues, increased pill burden, unnecessary drug interactions, and product switching. the fda has also noted the potential safety risk posed by overfilling and, in a recently published guidance, it imposed a zero overfill requirement for all peps to be marketed in the usa after april . methods: over a period of two years, we ran a series of tests similar to those conducted by whitehead to evaluate overfill on commercial peps currently available in the u.s. and europe. results: no finished product was formulated to % of the labelclaimed lipase enzyme activity. overfill on commercially available peps ranged from % to %, with a median value of %. the peps analyzed were to months old on average; therefore, the actual amount of overfill is likely underestimated. conclusion: our findings confirm those reported earlier by whitehead and demonstrate that none of the currently available peps complies with the zero overfill requirement of the fda guidance. these findings highlight a potential cause of suboptimal therapy with currently available peps. references: -whitehead am. study to compare the enzyme activity, acid resistance and dissolution characteristics of currently available pancreatic enzyme preparations. pharm weekbl sci. feb ; ( ): - . -food and drug administration. guidance for industry: exocrine pancreatic insufficiency drug products -submitting ndas. april . heubi, j. ; boas, s.r. ; blake, k. ; nasr, s.z. ; woo, m.s. ; graff, g.r. ; hardy, k.a. ; amaro-galvez, r. ; latino, m. ; lee, c. . children 's hospital medical center, cincinnati, oh, usa; . chicago cf care, glenview, il, usa; . nemours, jacksonville, fl, usa; . michigan u, ann arbor, mi, usa; . children's h, los angeles, ca, usa; . penn state h, hershey, pa, usa; . children's h, oakland, ca, usa; . texas u, tyler, tx, usa; . eurand, milan, italy; . eurand, vandalia, oh, usa background: supplemental pep therapy helps prevent maldigestion and malabsorption in cf patients. eur- is a novel, zero-overfill, highly-stable formulation of porcine-derived pancreatic enzymes developed to treat epi in cf patients. this phase iii, randomized, doubleblind, placebo-controlled, two-treatment, crossover, multicenter trial evaluated the efficacy and safety of eur- to treat epi in cf patients. methods: patients with confirmed cf and epi, age ≥ years, good nutritional status and weight ≤ kg were enrolled. no drugs affecting gastric ph or motility were allowed. after open-label dose titration, patients were randomized to receive eur- or placebo over a week-long period. following another open-label normalization, all patients were crossed over to the alternative treatment arm. primary endpoint was change in coefficient of fat absorption (cfa) following oral administration of eur- vs. placebo. secondary endpoints were change in coefficient of nitrogen absorption (cna), cholesterol, fat soluble vitamins, body weight, bmi and epi symptoms following oral administration of eur- vs. placebo. safety endpoints included aes, clinical laboratory parameters, physical exams and vital signs. results: thirty-four patients were enrolled ( female); were evaluated. mean age was . years (range - ). eur- treatment was associated with statistically significant (p< . ) increases in both cfa and cna vs. placebo. eur- treatment was associated with decreases in pivka ii (an indicator of vitamin k status). epi symptoms consistently improved during eur- treatment, including a statistically significant reduction in stool frequency and fewer soft and watery stools. treatment with eur- improved the signs and symptoms of malabsorption even in the small subset of patients with cfa values greater than % under placebo treatment. there were no notable changes in serum lipids, vitamin a and e values, mean weight or bmi in patients who received eur- . eur- was safe and well-tolerated. there were no unexpected or significant differences in the frequency or type of aes between eur- and placebo, and no trends were seen in lab parameters or vital signs. no patient discontinued from the study due to an ae. two serious aes were considered unrelated to the study drug and both resolved. conclusion: in this randomized, double blind, placebo-controlled study, eur- was safe, well tolerated and effective in the treatment of cf patients with epi, with clinically and statistically significant improvements in cfa, cna, and epi signs and symptoms in the absence of any concomitant treatment affecting gastrointestinal motility and ph. the therapeutic benefit of eur- was seen even in patients with very high cfa values (> %). the relative infrequency of cystic fibrosis related diabetes (cfrd), until recently, means little is known about hypoglycaemia ('hypos') associated with its treatment. insulin is frequently taken by those who have cfrd, for whom the risk of hypoglycaemia may be high due to dramatic changes in insulin sensitivity that can occur with pulmonary exacerbations and a decreased ability to secrete glucagon. a number of studies involving people with type diabetes mellitus (t dm) have reported on their experiences of hypoglycaemia, but whether results from such investigations apply to cfrd is unclear. an exploratory investigation was conducted to compare the type, frequency and severity of hypoglycaemic symptoms experienced by patients who had cfrd with patients who had t dm. method a cross sectional study was conducted, involving adults ( - years) with t dm or cfrd, recruited from two hospitals in england. a questionnaire, sent to patients with t dm and with cfrd, included investigator-developed items about knowledge and frequency of hypoglycaemia and the edinburgh hypoglycaemia scale (ehs), a standardised measure that assesses autonomic (e.g. sweating, hunger) and neuroglycopenic (e.g. confusion, poor co-ordination) symptoms associated with hypoglycaemia. questionnaire data were entered into spss for analysis. comparisons were conducted using t-tests, mann whitney tests or chi square tests as appropriate. results questionnaires were returned by patients with t dm and with cfrd. five of those with t dm were over years of age and, therefore, excluded from analysis. almost all participants had experienced a 'hypo' ( %). comparisons between the two groups are reported in table . conclusion patients with cfrd reported fewer and tended to have less severe episodes of hypoglycaemia compared to those with t dm. fewer neuroglycopenic symptoms in the former could be related to their shorter duration of diabetes or to how cfrd was managed; patients with cfrd were given shorter acting insulin, making them less prone to 'hypos' during the night, and had a high sugar diet to ensure weight maintenance. differences between the groups could also be related to specific characteristics of these two forms of diabetes. table : data from questionnaire insulin resistance. the purpose of this study was to investigate the relationship between pem and the risk of developing glucose intolerance and/or cfrd and the effect on lung function in children ≤ years of age whose history suggests pem. patients whose diagnosis of cf was suggested by failure to thrive(ftt) and whose growth parameters remain < % for weight and height for age were identified as having incurred pem. a retrospective analysis of our cf registry database was performed. cf patients ≤ years old were identified. of patients( . %) had cfrd. ( . %) of these plotted < % for weight and height (pem)and ( . %) were diagnosed with cfrd. further review of the patients showed whose diagnosis of cf was suggested by ftt and ( . %) of those had cfrd. statistical analysis did not show a clinical significance, but a strong relationship is implied. pulmonary function, fev %, was significantly lower in the pem/cfrd patients (mean fev . % ± . %) than the pem only group, (mean fev . % ± . %)(p= . ). this review provides further support that patients who have pem are at increased risk of developing cfrd. a significant risk was not established; however a strong association is suggested. assessment of a larger cohort of patients may show a stronger relationship. findings from this study would suggest that further research into the physiological relationship between pem and cfrd should be initiated. cfrd screening should be considered at a younger age than the current cff recommended age of > years. diagnosing cfrd/glucose intolerance early may contribute to improve growth parameters, preserve lung function, and improve survival. based on this review, our center is initiating cfrd screening for all patients ≥ years, and considers screening for all pem patients ≥ years. background: patients with cf are thought to have a rapid postprandial rise in plasma glucose which may be followed by a delayed and prolonged insulin response and hypoglycemia. we sought to estimate the prevalance, cumulative one year incidence, and factors associated with hypoglycemia during oral glucose tolerance testing in non-diabetic adults with cf. we also compared the prevalence of hypoglycemia to a geographically matched cohort of young adults without cf. methods: we performed a prospective study of individuals aged over years with cf followed in clinic providing population-based, specialized care in cambridgeshire, england. we excluded patients with previously-diagnosed diabetes or taking anti-diabetic medications. we evaluated individuals who underwent g hour oral glucose tolerance testing (ogtt) in . of these, individuals had neither new diabetes nor hypoglycemia in and were retested using ogtt in . we testing the association between biochemical and anthropometric factors measured in with the prevalence of hypoglycemia in , and incident hypoglycemia in . biochemical hypoglycemia was defined as a blood glucose between < . mmol/l ( mg/dl), but greater or equal to . mmol/l ( mg/dl) and severe hypoglycemia as a value lower than . mmol/l on either the fasting or post glucose challenge value. for comparison, we used data from the cambridgeshire-based young ely study of individuals age - years who underwent g ogtt testing in - . results: in , in patients with cf, the prevalence of fasting hypoglycemia was . %. no patients had fasting severe hypoglycemia. following glucose challenge, . % and . % of patients had glucose values consistent with hypoglycemia and severe hypoglycemia respectively. there were no diferrences in age, hepatic, pulmonary or renal function, but patients with hypoglycemia at two hours were five times more like to be male (odds ratio . , % ci . - . ) and more likely to have a higher bmi. the prevalence of a hours value of < . mmol/l in patients with cf at . % ( / ) was not different than in controls . % ( / ). of patients who did not have hypoglycemic readings in , at one year, patient developed fasting hypoglycemia and individuals developed post-challenge hypoglycemic for a cumulative incidence . %. of these . % ( ) had values in the range of severe hypoglycemia. there were no differences between age, sex, bmi, liver, renal, or pulmonary function in those who did and did not develop hypoglycemia. conclusions: hypoglycemia appears common in patients with cf but is also common in a healthy population. male sex and bmi were associated with post load hypoglycemia in patients with cf in cross-sectional analyses only. further research needs to assess whether these low blood glucose values are associated with symptoms. it is well recognised that cystic fibrosis related diabetes (cfrd) is a poor prognostic indicator in cf, and therefore early recognition is imperative to allow effective treatment in an attempt to prevent the associated decline in pulmonary function and nutritional status. although the oral glucose tolerance test (ogtt) is the accepted method of detecting diabetes mellitus in non cf individuals, glucose intolerance and lack of insulin can be variable in cf patients such that the reliability of ogtt in the diagnosis of cfrd has been questioned. under these circumstances, other methods of monitoring glucose intolerance, such as serial post prandial glucose monitoring (sgm), may be more appropriate. to look at this further, we prospectively compared ogtt with sgm in a group of adult cf patients. fourteen patients with no previous history of cfrd ( ], df homozygous, male) admitted for acute pulmonary exacerbations were evaluated. all were exocrine pancreatic insufficient, and was on long term oral steroids. eleven patients received mg/day oral prednisolone in addition to iv antibiotic therapy for the duration of their admission. ogtt was performed in the standard fashion (ingestion of . g glucose/kg body weight [maximum g] within minutes, after an overnight fast). sgm was performed hours post prandially and before bedtime over the period of the admission. the local ethics committee approved the study and informed consent was obtained from all patients. results sgm revealed elevated postprandial blood glucose values in all patients. there were episodes (mean per patient [ to ]) with glucose > . mmol/l (frank diabetes, who criteria) and episodes (mean per patient [ to ] ) with glucose . to . mmol/l (impaired glucose tolerance, who criteria). however, ogtt revealed impaired glucose tolerance in only one patient ( hour glucose value . mmol/l). in the remaining patients, ogtt revealed normal hour glucose values (mean . mmol/l [range . to . ]). fasting plasma glucose values were also normal (mean . mmol/l [ . to . ]) in all patients. we have shown a high prevalence of hyperglycaemia in our adult patients admitted for pulmonary exacerbations that was only detected using serial glucose monitoring. this study suggests that sgm may be more sensitive at detecting early cfrd than the ogtt. further work needs to be carried out to look at the best methods of diagnosing cfrd, to facilitate early treatment and thereby improve the prognosis. the diagnosis of cystic fibrosis related diabetes (cfrd) is associated with a decline in pulmonary function and nutritional state and is a poor prognostic indicator: the risk of its development increases with age. furthermore, the development of cfrd is due to a gradual loss of insulin production, and the disease may therefore be preceded by a period of glucose intolerance. despite this, the incidence of hyperglycaemia in cf patients is unknown. to study this further, we monitored the blood glucose profiles of adult cf patients admitted for the treatment of a pulmonary exacerbation. we looked at consecutive cf patient admissions for iv antibiotic therapy to our large adult unit. of these, had established cfrd and were excluded. the remaining patients (mean age years [range to ], male) formed the study population. none were on long term oral steroids, but received mg/day prednisolone as part of their routine exacerbation treatment. all patients underwent blood glucose measurement hours after each meal and before bedtime as part of a standard assessment protocol in our clinic. thirty patients ( %, / on steroids) demonstrated post prandial hyperglycaemia: exhibited values between . and . mmol/l (impaired glucose tolerance, who criteria) and > . mmol/l (frank diabetes, who criteria). there were no significant differences in age, genotype, fev , fvc, bmi, or duration of hospital stay between the two groups. of the hyperglycaemic patients, ( %) had abnormal glucose levels detected before bed time, significantly more than hours post breakfast ( %, p< . ), post lunch ( %, p< . ), but not post dinner ( %, p= . ). this study has shown a high prevalence of hyperglycaemia in adult cf patients treated for a pulmonary exacerbation who were not otherwise known to suffer from cfrd. nearly all our patients demonstrated glucose intolerance later in the day, when the recommended screening test for cfrd (the fasting oral glucose tolerance challenge) cannot be used. post prandial glucose monitoring of such patients during times of stress may therefore be advisable, allowing the introduction of insulin treatment at an earlier stage in the disease process. whittaker, l.a.; tilluckdharry, l.; christian, r. medicine, university of vermont, burlington, vt, usa objective: bone disease is a recognized complication of cystic fibrosis (cf). there are currently no guidelines for bone mineral density (bmd) testing or tools for fracture risk assessment in cf adults. we piloted a survey designed to identify cf-specific risk factors for low bone density and fracture. methods: a question survey was completed by patients prior to measurement of bmd at the hip, spine and radius by densitometry (dxa). the survey was comprised of questions on calcium and vitamin d intake, exercise, family history, medications, reproductive health, ultraviolet exposure, cf related diabetes (cfrd), weight loss, severity of cf lung disease and quality of life. z scores (bmd adjusted for age and gender) were used so that male and female data could be analyzed collectively. patient perceived health (pph) was rated by four questions and totaled for the pph score, which ranged from - with the highest score indicating the best health. functional status (fs) was rated by three questions that reflect the degree to which cf interferes with school, work or play. the total fs score ranged from - , with the highest score representing the least interference with life. questions on pph and fs were derived from a validated clinical assessment tool, the cf questionnaire (cfq) ( ) . results: all cf adult patients (n= , age and older) were offered participation in the study. dxa of total hip and lumbar spine was performed on patients ( %); ( %) of these patients also completed the survey. % of patients had low bone density on dxa (z score <- ). % of male cf adults had low bone density vs % of females. fracture was reported by % of patients who completed the survey ( % female and % male). lower pph score was associated with lower z score at the hip and spine. lower fs score was associated with lower z score at the hip and spine. low body mass index was associated with low z scores at both hip and spine. patients with bmi below kg/m were most likely to have osteopenia. conclusions: osteopenia and history of fracture were common in our adult cf population regardless of gender. bmi, pph and fs predicted bone density. risk stratification by a cf-specific bone health survey may guide bone density screening strategies for cf adults. prospective studies in cf adults are needed to determine whether a cf-specific survey can predict fracture risk as well as bone density. references : as life expectancy in cystic fibrosis (cf) increase, osteoporosis has become more prevalent. vitamin d status is one of many factors that contribute to optimal bone health. previous data has shown, the majority of cf patients are deficient in vitamin d despite being prescribed daily cf specific vitamin therapy. previous research has also shown that the current recommendations for vitamin d supplementation and repletion provided by the cystic fibrosis foundation bone health consensus ( ) are not sufficient to achieve optimal vitamin d levels in the majority of patients. moreover, the recommendations fail to sustain levels in the optimal range for bone health. purpose: we demonstrate a successful supplementation algorithm for individualized high dose vitamin d with continued high dose maintenance therapy which achieves and sustains appropriate levels. methods: all patients followed at the pediatric center are prescribed routine cf vitamin supplementation. vitamin d levels are obtained as part of routine clinical care via mass spectroscopy. if serum vitamin d level is < ng/dl, vitamin d therapy with ergocalciferol is initiated one time per week. patients < years of age receive iu/ dose and > years of age receive iu/dose. at the next routine quarterly visit, vitamin d levels are re-assessed via mass spectroscopy to determine efficacy of once weekly supplementation. if levels are > ng/dl, patients continue high dose supplementation time a week as maintenance therapy. however, if levels remain < ng/dl, the ergocalciferol is increased by time a week in month increments until adequate levels are obtained. once adequate serum vitamin d levels are obtained, patients remain on the level of supplementation needed to achieve levels > ng/dl as maintenance therapy. all patients are instructed to take vitamin d and their cf vitamins with enzymes and food to optimize absorption. results: after implementation of the algorithm, . % of patients, with varying levels of supplementation, had vitamin d levels > ng/dl (mean= . ng/dl). of the patients that achieved goal vitamin d levels, % were able to achieve and sustain adequate vitamin d levels without additional vitamin d supplementation while on standard cf vitamins (mean vitamin d level = . ng/dl). the remaining patients required chronic maintenance vitamin d: % required vitamin d supplementation once per week with a resulting mean vitamin d level of . ng/dl, . % required maintenance vitamin d supplementation twice per week with a resulting mean vitamin d level of . ng/dl, and, . % required vitamin d supplementation or more times per week with a resulting mean vitamin d level of . ng/dl. another % of patients take vitamin d supplements other than ergocalciferol and were not included in this study. conclusions: individualized vitamin d repletion and maintenance therapy as outlined in our algorithm is a successful mechanism to obtain optimal vitamin d levels in patients with cf. inhaled tobramycin ( %). sixty-two percent reported using at least different types of oral medications on that day including pancreatic enzymes ( %), oral antibiotics ( %), anti-reflux medication ( %), azithromycin ( %), anti-histamine ( %), and a decongestant ( %). twenty percent reported taking at least one medication for pain. forty-nine percent reported performing airway clearance at least twice during the day with % using a vest, % performing standard chest physiotherapy, and % using a flutter or acapella device. there was no difference in the reported number of medications or the reported time needed to complete therapies based on respondent age. although respondents with severe lung disease (fev < % predicted) reported a higher median number of therapies ( vs. , p= . ), the reported time for completing therapies was not associated with fev (p= . ). conclusion: daily treatment burden and complexity in all adults with cf is high, and is only marginally increased in those with worse pulmonary function. given this already high load of daily therapies, efforts to assess the treatment burden of new cf therapies are warranted. the impact of this high treatment burden on overall health related quality of life for adults with cf needs to be assessed. objective: the cystic fibrosis foundation (cff) has a network of over accredited care centers throughout the united states. data is collected from these care centers to help better understand the disease and describe changes in survival, standards of care, and health outcomes. when the cff was founded in few children survived to school age. now the predicted survival extends beyond the mid 's (cff, ) . with this change has come the need for age appropriate care in many areas including reproduction. the adult cystic fibrosis center at morristown memorial hospital presently follows adults. of those, women( %) with cf have had children affected with cystic fibrosis. each woman was diagnosed after her child. the cf data registry does not have any means of identifying if a patient has an affected child. as the average age of survival of cf patients has increased, so has the likelihood of cf patients having cf offspring. what is the estimated incidence of cf patients having cf affected children attending accredited cf centers in the united states? method: questionnaires were sent to care centers and satellite centers via standard mail or facsimile based on information from the cff directory. one month after the last questionnaire was mailed, the cf foundation sent out a reminder email to the nurse coordinators. questions asked were as follows: ) name of center ) do you presently care for any patients with cf who have given birth to, or fathered a cf affected child? ) how many patients at your center have had cf affected children? ) was the parent diagnosed before or after pregnancy was confirmed? ) if parent's diagnosis came after conception, was this after child was diagnosed? ) was the parent's diagnosis a result of prenatal testing? results: a total of questionnaires were mailed or faxed. this netted a result of responses. after email reminder, an additional questionnaires were returned for a total of responses, an % response rate. name of center was checked to remove duplicate information. the number of parents with cystic fibrosis who have had children with cf numbered . of those, ( %) were diagnosed before pregnancy; ( %) were diagnosed after pregnancy. of those diagnosed after pregnancy, the majority ( %) were diagnosed as a result of their child's diagnosis. only ( %) were diagnosed as a result of prenatal testing. conclusion: based on the responses from accredited cf care centers, patients with cf have children also affected with this disorder. prior to conceiving their child, % knew that they had cf. having cf did not appear to be a deterrent to delivering cf offspring. this study did not look at the age of the parent or child; they may have conceived their child before genetic screening was common for spouses. as the median survival of this population increases, the incidence of cf adults with cf children may also increase. the cff may choose to add a question to the cf data registry, identifying cf patients with cf offspring. the potential physical and psychosocial impact can then be tracked. anderson, a.; popli, k.; stewart, j.; heslop, k.; gascoigne, a.; bourke, s. adult cystic fibrosis centre and fertility centre, royal victoria infirmary, newcastle upon tyne, united kingdom as the outcome of patients with cf improves fertility issues become increasingly important. almost all men with cf are infertile because of azoospermia due to congenital absence of the vas deferens. these men can attain genetic paternity by sperm aspiration from the epididymis or testis with intracytoplasmic sperm injection (icsi), but few men with cf seek fertility treatment to assess their knowledge and attitude towards fertility issues a questionnaire was sent to men with cf: ( %) responded; ( %) declined to give information and ( %) replied in full. overall % thought that nearly all men with cf were infertile, % did not know how many were affected, and % could describe the exact nature of infertility. only % knew that treatment for infertility was available: % of these thought it was rarely successful and % were unsure of success rates. although % had thought of having children at some stage, only % had sought investigation of infertility. two men had children, one by icsi and one by donor insemination. overall % feared that the child could have cf and % worried that their health would affect their ability to function as a parent. in terms of receiving advice % thought that this should be given by a fertility specialist, % by a cf physician and % by a primary care physician. with regard to fertility treatment % thought that decisions should be made by the patients and their partners, % by the patient alone, and % by a doctor. the reluctance of men with cf to seek fertility treatment appears to be due to many factors including lack of knowledge of available treatments, fears about the child inheriting cf, and concerns that their health would adversely affect their role as a parent. in addiition to information provided by the cf team advice from a specialist in fertility treatment may help to improve the provision of reproductive counselling to these patients. nash, e.f. , ; coonar, a.s. ; stephenson, a.l. , ; delgado, d.h. ; singer, l.g. , ; tullis, e. , ; chaparro-mutis, c. studies have shown varying degrees of right ventricular (rv) dysfunction in adult cf patients with severe lung disease. left ventricular (lv) dysfunction is much less common with conflicting reports as to its prevalence. pulmonary hypertension (ph) is commonly seen, the severity correlating with fev . one group reported that ph was more prevalent in b. cepaciacolonized patients, albeit in a small sample. the aim of this study was to determine the true prevalence of ph and cardiac dysfunction in cf patients with severe lung disease. we carried out a retrospective cohort study of all adult cf patients referred to the lung transplant program at toronto general hospital from - . adult patients referred with non-cf bronchiectasis were included for comparison. demographic data, lung function, -minute walk, micro-biology, doppler echocardiography and resting and stress radionuclide ventriculography (muga) results were analyzed by students t-test (parametric) or mann whitney u test (non-parametric). results adult cf patients ( % non-b. cepacia, % b. cepacia) and non-cf bronchiectasis patients were included. data from these groups are summarized in the table. we found higher minute walk and bmi values and a higher percentage of males in the b. cepacia compared to non-b. cepacia group. ph was present in % of the cf patients in whom pasp could be obtained. b. cepacia patients had lower first pass rvef compared to non-b. cepacia patients. % of the b. cepacia patients had an abnormal lvef response to exercise (defined as < % increase on exercise) compared to % of the non-b. cepacia patients. we found a high prevalence of ph as well as rv and lv dysfunction in cf patients with severe lung disease. b. cepacia-colonized patients had worse rv function compared to non-b. cepacia-colonized patients on first pass radionuclide ventriculography. *non-cf bronchiectasis group older than cf groups (p< . ) b. cepacia cf group higher than non-b. cepacia cf group (p< . ) b. cepacia cf group lower than non-b. cepacia cf group (p<. ) perobelli, s. ; dorazio, c. ; assael, b. ; tamanini, a. ; castellani, c. . cystic fibrosis center, verona, italy; . molecular biology laboratory, verona, italy cf neonatal screeening (nbs) can identify neonates with elevated irt, normal or borderline sweat chloride concentrations, and one cf mutation plus an additional cftr sequence variant, or, alternatively, two cftr sequence variants. the combination of hypertrypsinogenemia and two cftr gene defects is consistent with the presence of a cftr-related disorder (cftr-rd). although these neonates are usually healthy at diagnosis, the long-term phenotypical consequences may be highly variable. such variability makes impossible to predict the clinical outcome, and provide satisfactory genetic counselling for the family. the diagnosis of such a vague condition could potentially cause considerable family stress. this study aimed at assessing how parents of infants diagnosed with a cftr-rd through nbs perceive their children's health. a questionnaire for parents was designed, consisting of three sections. the first section asked about information given to the family in the neonatal period, and about emotional reactions to the cftr-rd communication of diagnosis. the second section investigated the present perception of the child's health, and the parents level of anxiety/concern. the third section focused on the impact of the diagnosis on family planning, on relationships inside the couple and with the child, and on emotional distress; this section was concerned also with the use of carrier testing in relatives. parents were also asked to fill in a standardized form on child behaviour (achenbach child behaviour check-list). the questionnaire was distributed to two populations, the parents of children diagnosed with cftr-rd through nbs (group a), and the parents of children diagnosed with cf through nbs as the life expectancy of children with cf increases new issues arise that have not been dealt with in the past. in the past,issues related to the support and care of a young adult with cf were rarely raised because only small numbers of children with cf lived into adulthood. however, with an increasing young adult population, young adults without family support have surfaced in significant numbers and their needs should be identified and understood by the cf center care team. these young adults face the same range of issues others with cf face related to health insurance, government benefits, housing, education and employment. however, they do not have a family to help them wiht support, health insurance benefits and care needs. method in october , the cf legal information hotline ("hotline") began to provide free and confidential information to people with cf, family members and healthcare providers in the u.s. the hotline is staffed by attorneys, beth sufian and james passamano(bs and jp) and is accessed by dialing a toll free number or by e-mail. information about the hotline is available at cf care centers, in cf publications and on the web. information regarding the content of each call is recorded on an intake form and includes caller name, address, phone number, age of person with cf, cf center attended, and information related to the caller's question, attempts made to access legal information prior to calling the hotline, relation of access to information to access to care. calls and e-mails are tracked by seven major categories which are divided into subcategories. if a caller has more than one question, the call is tracked using the caller's primary issue. results from november to april the cf legal information hotline has received calls from young adults with cf who do not have family support and have issues related to obtaining health insur-ance, government benefits, educational oppornties or employment opportunities. as well as issues related to lack of support for care. the hotline has also received calls from cf care centers team members with questions related to the needs of young adults without family support. over an eight year period the cf hotline received _______calls. the categories for the calls were: social security benefits ( , ) , health insurance ( , ) , educational ( ), employment ( ), long term disability benefits ( ), family law ( ) and miscellaneous legal issues ( ). discussion people with cf face obstacles to care if they are unaware of their legal rights especially in the areas of health insurance and government benefits. healthcare providers are busy addressing the medical needs of patients and are often not well equipped to provide detailed information about the legal rights of their patients. the hotline provides important information to both patients and healthcare providers that can optimize both patient care and outcomes. access to legal information can result in individuals obtaining health insurance, medicaid, medicare or other state coverage that in turn results in better access to treatment. legal information can also lead to important modifications in the educational or employment environment for the individual with cf. these modifications can be critical in allowing those individuals access to important care and treatments. providing information that can help individuals obtain social security or long term disability benefits can result in a monthly benefit and health insurance that then allows the individual to devote more time to their healthcare. many people with cf and healthcare providers lack information about the legal rights of individuals with cf. understanding the legal rights of people with cf can reduce obstacles to obtaining care, optimize education and employment, and increase access to retirement benefits. further work needs to be done to identify ways to make individuals with cf and healthcare providers aware of the hotline. the cf hotline provides a unique service to cf patients and healthcare providers by providing information regarding the legal rights of people with cf. identification of ways to assist young adults without family support should be identified and cf center xcare teams shoudl be given the tools to allow them to help their patients access government assistance and other programs that can help make sure that the young adults have acces to care and support they need to live with cf. the dvd gives healthcare staff, patients and families a rare glimpse of ten couples with cystic fibrosis (cf) interacting at a cf care center-sponsored 'couples support group'. they discuss the impact this forum has in their daily lives for gainingo information about how cf affects their lives as individuals, and as couples, and how the 'non-cf partner' learns from others. o skills at networking and personal coping strategies. o reflections on 'family life and cf', and o observations on ways cf care centers can join them in greater health partnership. lastly, this dvd provides a 'starter kit' for ways to establish such a group. one of the realities of a life-threatening illness can be that of social isolation, and of separation from others who share the same disease. having the opportunity of participate in a support group can significantly reduce that sense of isolation and give the participants an ability to share experiences and give support to others with this disease. cystic fibrosis as an illness has growing ranks of 'survivors' who live longer, fuller lives, thanks to the advances made on multiple-research fronts. historically, adults with cystic fibrosis have not had much occasion to share person-to-person experiences within the structure of their cf care centers. justifiably, with the potential for cross-infection, the cystic fibrosis foundation has urged caution for the cf population in having face-to-face interactions. in , our cf care center recognized this need and sponsoredwith guidance from staff facilitators-an on-going adult support group. prior to the formation of the group pre-and on-site group ic protocols were developed. at the pre-group stage, the physician initially certifies that the patient is free from aggressive pathogens. each time the patient rsvp's permission to attend a group session, the physician must 'recertify' this status. on-site protocols emphasize a number of ic precautions to minimize crossinfection. the center recognized that by developing such a support group, 'patient and family centered care' could be enhanced. a goal in the group's formation was to create 'mutually beneficial partnerships among patients, families, and healthcare providers'. group structure/successes to date: the group is limited to participants. two cf care center staff act as facilitators. this is a support, not a therapy group. compliance with therapies is reported to have increased as one outcome of this forum. formal topics are purposefully not predetermined; the group's success is predicated on the personal responsibility of each participant to bring topics/issues from their daily life experiences. the group has gained identity and trust and has evolved over three years into two separate groups: an individual cf group and a couples cf group. ownership of these groups by those who attend is apparent. both the care center and the couples support group are forums for learning 'how to live with cf'. future goals will call for using identified quality of life tools to measure psychosocial and emotional growth in individuals through their participation over time in the group. sawicki, g. ; asher, d. ; dill, e. ; sellers, d. ; robinson, w. . children's hospital, boston, ma, usa; . education development center, newton, ma, usa background: advance care planning (acp) is an important tool to promote alignment of the patient's care with his or her needs, goals and values, and is particularly useful if the patient's ability to make decision becomes compromised. the purpose of acp is to develop a treatment plan promoting a high quality of life for those near the end of life. such planning is especially appropriate in persons with life limiting disease. minimal research exists on the advance care planning process of adults with cf.<p>methods: in the fifth survey wave of the project on adult care in cystic fibrosis (pac-cf), an ongoing longitudinal panel study of cf adults from cf centers in the us, participants were asked to report on their opinions and experiences with advance care planning. the survey asked about opinions on advance care planning documents, communication about advance care planning with family and clinicians and whether a directive had been completed. clinical data were obtained from the participating centers. preliminary bivariate analyses were conducted to examine factors associated with completing an advance care directive.<p>results: / ( %) surveys were completed. the mean age was ± years, % were female, and the mean fev (% predicted) was ± . % reported that they had spoken to someone about the care they would want if they became too ill to make their own decisions in the future, % had thought about whom they would want as their healthcare proxy, and % reported having specific wishes about the types of medical treatment they want or would not want if they became too ill to make decisions for themselves in the future. however, only % reported completing a healthcare proxy form, living will or any other type of written instructions concerning advance care directives. % of respondents reported feeling comfortable talking to their clinician about acp. % of subjects said that their cf clinicians have asked them about acp, and only % reported that they have discussed acp with their cf clinician. the following characteristics were significantly related (p<. ) to completion of an advance care directive: female gender ( % vs. %), age (mean years vs. years), having a college degree ( % vs. %), being married currently or in the past ( % vs. %), having children ( % vs. %), having diabetes ( % vs. %), having specific wishes about treatment decisions ( % vs. %), and reporting that a clinician had discussed acp with them ( % vs. %).<p>discussion: though the majority of cf adults report thinking about, communicating with family and deciding on their advance care wishes, only a minority report completing any legal documentation supporting their decision. additionally, very few cf adults report being asked about acp by their clinicians and even fewer report discussing acp with them. formulating specific wishes and discussing acp with a clinician are strongly associated with completing an advance care directive, suggesting that if clinicians were more active in talking to their patients about acp, patients are much more likely to complete advance care directives. efforts to improve clinician communication with cf adults around the issues of advance care planning are needed. objectives: caregiver involvement in children's cf treatments fosters better adherence and improved health outcomes. several studies have suggested that parents of children with cf report elevated symptoms of depression (quittner et al., ) , however, there is no data on the effects of caregiver depression on adherence. adherence to treatment is difficult to measure in patients with cf, and prior research has relied primarily on self-report, which is likely to be inflated. the purpose of this study was to examine the effects of caregiver depression on adherence to enzymes using both parent-reported and electronically monitored enzyme adherence over a three-month period. in addition, we examined the relationship between depression, rates of enzyme adherence and short-term changes in weight. methods: as part of a larger intervention study at cf centers, children with cf ages to and their parents were recruited. parents completed a standardized measure of depression (cesd) at enrollment and were provided with mems caps that recorded the date and time of each bottle opening. three months later, the electronic data from the pill caps was downloaded and parents completed a structured interview reporting their child's adherence to all components of the cf treatment regimen. only the section addressing enzyme adherence was included in this analysis. standard health outcomes, such as weight and height, were also assessed at enrollment and months later. results: preliminary analyses indicated that caregivers reported elevated levels of depression, with % scoring in the clinical range. rates of adherence to enzymes were poor ( % at home and % at school). caregiver depression was negatively associated with adherence, with depressed caregivers demonstrating lower rates of adherence ( percentage points). adherence to enzymes was associated with changes in weight, with a % adherence translating into percentile points of weight gain. final analyses will also include parent-reported adherence to enzymes. conclusions: a substantial number of parents scored in the clinical range on a depression screening measure. in addition, depression was associated with poorer adherence. rates of adherence to enzymes were surprisingly low, both at home and at school. furthermore, poor adherence was associated with a decrease in weight three months later. caregiver depression appears to be under-diagnosed and these results suggest that screening and intervention may be warranted. adherence to enzymes should also be targeted in clinical interventions. funding was provided by nih grant #hl of non-compliance with medical treatment and greater behavioral and emotional distress. adherence is particularly problematic during adolescence, however, few family-based interventions have been developed to target adherence behaviors in this population. the goal of this study was to compare the effects of behavioral-family systems therapy (bfst), an empirically-supported treatment, to both family education (fe) and standard care (sc). methods: as part of a larger intervention study, adolescents with cf ages to and their parents completed the conflict behavior questionnaire (cbq) and the parent-adolescent relationship questionnaire (parq) developed by robin and foster ( ) . adolescents completed these measures for each parent and both parents, if available, completed them with respect to their teen. the problem-solving, communication, and beliefs subscales from the parq were administered. participants were then randomly assigned to one of three treatment arms: bfst, fe or sc. families in the bfst group received ten -minute sessions over a month period, including family problem-solving, communication skills training, and cognitive restructuring. families assigned to the fe group received ten minute psychoeducational sessions over months aimed at increasing knowledge about cf and its management. those in the sc group received their usual care at the cf center. the cbq and parq were completed pre, post and , and months following the intervention. results: adolescents reported that the bfst intervention, relative to the other two, improved their communication with their primary caregivers (p < . ) and caregivers improved their communication with their adolescents (p < . ). in addition, caregivers reported improved problem-solving with their adolescents (p < . ). on the cbq, bfst reduced both adolescent and primary caregiver conflict and fe reduced caregiver conflict. no significant improvements were found for those in sc. conclusions: preliminary findings suggest that bfst is effective in improving communication skills and reducing conflict in adolescents with cf and their caregivers. future analyses will evaluate the effectiveness of bfst and fe on adherence and other family functioning measures over the course of the study. funding was provided by nih (hl # ) background: cases of diabetes among people with cystic fibrosis (cf) have increased as life expectancy for these patients improves, yet the impact of this additional illness on daily functioning is under-researched. to explore this issue, a study was conducted comparing the views of patients with either cystic fibrosis related diabetes (cfrd) or type diabetes mellitus (t dm) about being diagnosed and living with diabetes. methods: qualitative research was used because the study was concerned with understanding diabetes from patients' perspectives. purposive sampling was employed to achieve maximum variation in terms of duration and type of diabetes experienced by participants. data were collected via semi-structured interviews, all of which were taped and transcribed verbatim. recruitment continued until no new ideas or insights emerged from additional participants. a framework approach to analysis was adopted. this involved coding and summarising interview data into charts to explore and develop main themes. initial analysis was undertaken by two researchers (st and cd) and amended after comments from the remaining authors. results: interviews, carried out with cfrd and similarly aged t dm patients, lasted for an average of minutes (range - minutes). the following themes were derived from the data collected: evolving vs fracturing identity; diabetes in context; self-management motivators. for patients with cfrd, diagnosis represented a progression in their health status, which called on them to adapt existing treatment regimens to accommodate this additional condition. in contrast, interviewees with t dm had to re-evaluate their previous sense of self as 'healthy' and adjust to manag-ing a long-term complaint. these individuals were more likely to talk about diabetes in relation to a range of competing commitments (e.g. work or family related) and to describe feeling psychologically low due to diabetes compared to patients with cfrd, the latter depicting demands from their primary illness (cf) as a major obstacle to caring for diabetes. participants with cfrd recalled feeling lucky when told they would not face strict dietary restraints, which they associated with other forms of diabetes, and seemed less concerned about diabetic complications than those with t dm. for interviewees with t dm, a desire to reduce future health risks motivated their self-management efforts, whilst those with cfrd were driven by the negative effect poor control of diabetes had on their chest and weight. conclusions: both sets of interviewees found diabetes time consuming and, on occasions, frustrating to accommodate into daily life. findings from the study act as a reminder that patients manage their condition in the real world, against a plethora of other demands on their time and energy. in the narratives of participants with cfrd, these demands were often related to the existence of their primary illness. ing the course of development. the purpose of this study is to examine rates of medication adherence across a wide age span using an objective measure of adherence and to compare adherence to health outcomes. methods: patients with cf age years or older who are prescribed azithromycin, colistin, hypertonic saline, pulmozyme, and/or tobi for at least one year are eligible. recruitment is ongoing. the previous year's medication refill records were requested from participant-identified pharmacies. a medication possession ratio (mpr) was calculated for each drug and was defined as the sum of all days of medication supply received during the months divided by the number of days the medication was prescribed. values were truncated to % and averaged across all medications to obtain a composite score. medical records were abstracted to identify the prescribed drug regimen and health outcomes (e.g., lung function, exacerbations, bmi) over the same time period. results: thus far, patients have joined the study ( % participation rate), resulting in pharmacy records requested ( unique pharmacies). of the first participants with complete pharmacy data, the mean age was . years (sd= . ; range= - ) and % were female. the table presents mpr data for each drug and composite score overall and stratified by child, adolescent, and adult participants. the complexity of the drug regimen and age were significantly correlated with the composite score (rho= -. and rho= -. respectively; p<. ). future analyses will compare the composite score and each drug's mpr with health outcomes. con-clusions: participants had suboptimal medication adherence similar to that reported in the literature. as mpr provides the maximal possible level of adherence, actual adherence may be even lower. poor adherence spanned the age groups and was associated with regimen complexity. children had the highest adherence, but also the least complicated drug regimen. these results suggest that obtaining pharmacy records is a viable means to objectively assessing medication adherence. moreover, results suggest that interventions targeting medication adherence may be a strategy for improving health outcomes, particularly for adolescents and adults. * fewer than participants within this cell (cf) whose exchange capacities for oxygen and carbon dioxide are already diminished. lack of rem and delta sleep have significant implications for the daytime functioning of pediatric patients with cf, potentially leading to impaired memory and attention which would reduce their ability to complete typical activities, such as school work and disease-specific tasks related to self care and adherence. based on these clinic findings, the authors are beginning a prospective study on the effects of sleep on neuropsychological functioning, well-being and compliance in pediatric patients with cf. future research should examine the relationship between decreased slow wave, rem sleep and psychosocial outcomes, including attention, memory, and health-related quality of life. there are diagnostic challenges related to non-classic cf (knowles and durie, ) particularly patients presenting in adolescence or adulthood with obstructive azoospermia, chronic sinopulmonary disease and chronic or acute, recurrent pancreatitis. practitioners are often unable to provide clarity to patients about diagnosis, disease course, and prognosis. potential harms and benefits of relaying a clear or unclear diagnosis may be psychological rather than physical and have long-term implications for mental and social well-being. the aim of the current study is to assess the psychological impact of diagnostic information for adults presenting with a cf phenotype, be it confirmatory (cf or most likely not cf) or unclear. method: we administered self report measures pertaining to psychological state, cognitive appraisal and uncertainty in a welldefined cohort of adult patients. presenting symptoms of patients include chronic sinopulmonary disease, obstructive azoospermia, pancreatitis, and those presenting with more than more cf phenotype. patients completed the self-report measures on two occasions: at the time of diagnostic testing and months after being counseled and notified of the diagnostic results. at the time of the initial assessment, the subjects were unaware of the test results and had not been seen by a cf physician. results: we provide interim observations on patients (sinopulmonary (n= ), obstructive azoospermia (n= ), pancreatitis (n= ), and multiple phenotypes (n= ) who completed the initial assessment. at the time of diagnostic testing, the level of depression, hostility, anxiety and interpersonal sensitivity were elevated in each group (t score > ); male patients reported significantly greater depression, anxiety and interpersonal sensitivity than female patients. compared to published normative data of patients with mixed chronic illnesses, patients presenting with sinopulmonary or pancreatic disorders reported significantly more uncertainty(p < . ). to date, of these patients have completed the measures months after receiving the diagnostic information. preliminary review of results shows that the level of depression, hostility, anxiety and interpersonal sensitivity remained elevated while the degree of uncertainty decreased. among the patients who completed assessment at months, patients were told that it was unlikely that they had cf, were told that they had mild cf, while were given an unclear diagnosis. those who were told that they did not have cf reported more uncertainty than those who were told they had cf (p < . ). detailed analyses related to outcome diagnosis will be conducted at the time of study completion (october ) . when this study is complete, the information will assist in our understanding of the psychological impact of a genetic diagnosis at an older age, identify issues confronting those individuals and help to establish appropriate paradigms for delivering complex information about genetic diseases. supported by grants from niddk [scor] and the canadian cf foundation. ( ). in accordance with these guidelines, we report our experience in using our nutrition action plan (nap) in patients with suboptimal nutrition. objective: to determine if implementing our nap would improve the bmi of these patients. methods: our nap is a written contract between the patient, caregiver and cf team. for clarity of nutritional goals, the nap is based on the colors of a traffic light. the red zone indicates a bmi less than the th percentile and poor nutritional status. the yellow zone indicates a bmi between the th and th percentile and fair nutritional status. the green zone indicates a bmi greater or equal to the th percentile and desired nutritional status. within each color zone, specific recommendations to improve nutritional status are listed. the ultimate goal for each patient is reaching the green zone. the caregiver, patient and nutritionist review the current nutritional status and a mutually accepted weight gain goal is established. the goal is recorded on the nap which is then signed by the nutritionist and patient. this contract establishes a commitment to achieve the goal by the next clinic visit. a reward system is implemented in conjunction with the nap. for patients reaching their goal, a previously agreed upon prize is given. patients achieving some weight gain but falling short of their goal receive a gift such as candy or small toys that serve as an incentive for continued weight gain. patients chosen for inclusion into this study demonstrated a bmi less than the th percentile; those on growth hormone, appetite stimulants or chronic systemic steroids were excluded. results: twenty-one patients, male and female, with a mean age of . years (range to years) were included in this study. fifteen patients demonstrated an improvement in their bmi with use of the nap: improved by - %; improved by - %; improved by - %; improved by - %; improved by - %. six participants had a negative change in bmi which we attribute to family upheaval, missed appointments with the cf team, exacerbations of disease and non-adherence. conclusion: we intervened in patients with a bmi less than the th percentile using a nap and a reward system. the results of this pilot study suggest that our nap and reward system may be of benefit in achieving positive gains in bmi. reference: ( ) previously it was rare for women with severe cf to undertake pregnancy and doctors tended to advise against pregnancy if the forced expiratory volume in one second (fev ) was less than %. the attitudes of doctors and patients are changing and many women living their lives with cf nowadays chose to undertake pregnancy despite the severity of their health problems. over the last years females (age range - years) have attended the newcastle adult cf centre. there have been a total of pregnancies involving women with live births ( ectopic). a further women had termination of unwanted pregnancy. mean age at the time of pregnancy was years. we compared an age-matched cohort of never pregnant women to see if there were differences in key variables making successful pregnancy more likely. the mean fev was % (range - %) and % (range - %) of predicted in the pregnant and non-pregnant group respectively (p= . ); rates of chronic pseudomonas aeuriginosa infection were ( %) and ( %) respectively (p= . ). cases of burkholderia cenocepacia were identified in the non pregnant group. there were no cases in the pregnant group (p= . ); ( %) had diabetes with patients having cf related diabetes prior to pregnancy and patients developing gestational diabetes; this compared with ( %) of non pregnant women (p= . ); there was impaired nutritional status within both groups with a mean bmi of . pre pregnancy (range - ) and . in the non pregnant group (range - ) (p= . ); ( %) and ( %) patients had had previous gastrostomy feeding (p= . ). pancreatic insufficiency was present in ( %) and ( %) patients in respective groups (p= . ). no child had cf but one child had complex congenital heart disease. there were no maternal deaths but patients subsequently underwent lung transplantation and patients died leaving young children (ages and years). patients in the non-pregnant group have also died. conclusion the profile of women with cf undertaking pregnancy is now similar to the overall cf population. the woman's decision to undertake pregnancy is often not directly related to the severity of her disease. amongst those who successfully completed pregnancy maternal and fetal outcomes are generally favorable. objectives: health-related quality of life (hrqol) instruments provide important information on disease progression and are increasingly used as patient-reported outcomes (pros) for behavioral and pharmacological trials (goss & quittner, in press) . recent advances in medical care have improved the long-term stability of pulmonary functioning, which has made it more difficult to detect small improvements in fev % predicted. new outcome measures are needed; the cystic fibrosis questionnaire-revised (cfq-r; quittner et al., ) , a disease-specific hrqol measure, has shown promise in this regard. the purpose of this study was to examine the longitudinal relationships between fev % predicted, weight percentiles, and cfq-r scores for adolescents with cf. methods: as part of a larger study evaluating adherence interventions, adolescents with cf, ages to , and their parents were enrolled at six centers. parents reported on their adolescents using the cfq-parent version, and adolescents completed the cfq-teen/adult version as a selfreport. measures were completed pre and post treatment and at , , and month follow-up visits. spirometry and weight data were collected at these same time points. results: empirical bayes estimates were used to examine change over time. preliminary results indicated that adolescents' scores on the cfq-r respiratory scale were significantly and positively correlated with changes in pulmonary functioning. parents' scores on the cfq-r vitality scale were also correlated with changes in pulmonary functioning, with higher cfq-r vitality scores associated with better lung function. in addition, parents' scores on the cfq-r weight scale were significantly and positively correlated with changes in adolescents' weight. conclusions: these results support the validity and sensitivity of the cfq-r to changes in both pulmonary functioning and weight for adolescents with cf. additional analyses will be conducted to determine the relative stability of hrqol scores over time and to examine other cfq-r scales that may reflect positive changes in adolescent functioning (e.g., reduced treatment burden background: development of cf specific patient symptom tools as outcome measures is a critical step toward the development of potential new therapies for cf. we have developed a novel instrument to assess respiratory symptoms in cf patients. methods: we conducted in-depth qualitative interviews using the day reconstruction method and cognitive debriefing interviews ( adults, youth, and parents) at two cf programs, the university of washington and children's hospital and regional medical center. interviews were conducted until no new symptoms were mentioned by interviewees (i.e., data saturation). the interviews were audio-recorded, transcribed, coded and analyzed for themes. results: six pulmonary symptoms were identified in the interviews: cough, sputum production, wheeze, chest tightness, difficulty breathing/shortness of breath and fever. in addition, the most commonly cited activity and emotional impacts identified in the interviews were also included on the questionnaire. these emotions included: frustration, sadness/depression, irritability, worry, difficulty sleeping, and activities included time spent sitting or lying down, reduction of usual activities, and missing school or work. in all, eight symptom items were selected for inclusion on the self-administered questionnaire (see table) . as a result of the cognitive debriefing interviews, we changed initial language and adjusted the response options on some of the items in order to create greater distinctions between them. no important issues were felt to be omitted by patients who were interviewed. conclusions: using qualitative inductive methodology, we have created a novel patient reported outcome measure for cf. further study will be required to assess validation of the instrument. or more signs and symptoms occurred for more than two days, antibiotics were ordered with physician input and phone follow-up in days. if symptoms occurred less than two days, chest clearance was increased with phone follow-up in hours. the initial algorithm (ia) was piloted by nurses on patient calls on the emr and reviewed for adherence. subsequently, the cf phone note was revised, expanding the symptoms to include gi issues, last visit date and last hospitalization. the revised algorithm (ra) was used for a one month trial period with the entire nursing staff. a total of patient calls relating to pulmonary exacerbation were again reviewed for adherence to the algorithm. results: the review of phone notes following the ia found ( %) of the phone notes to be adherent to the assessment and treatment plan on the algorithm. several exceptions included calls received for gi issues, medications, complaints of general pediatric symptoms, and lab results. the phone follow-up within - hrs was also difficult to accomplish secondary to nursing responsibilities and difficulty locating the patient. the review of calls following the ra found ( %) notes were adherent to the algorithm. discussion: the revised algorithm and cf phone note for pulmonary exacerbation improved consistency in nursing assessment of signs and symptoms, evaluation of the current plan of care, and triggered appropriate interventions based on symptoms and length of illness. this improvement was particularly pronounced among the part-time rns who found it easier to obtain a more accurate history of patient's illnesses without asking for assistance from other cf team members. since the algorithm and cf phone note are newly implemented, tracking for consistency with the cf phone note will continue, revisions will be made as needed, and phone notes for conditions other than pulmonary exacerbation may be developed. methods: in , helen devos children's hospital integrated research coordinators (rc) from the spectrum health research department into the cf care team. this model incorporates two key aspects: . a shared resource of experienced, centrally educated and trained research coordinators (rc) with three rc's having a primary cf focus . incorporation of rc's into helen devos cf care centers' multidisciplinary team for weekly contact with patients. using the cf registry consent as a starting point of research discussion with the patients and families, the rc's present new and upcoming opportunities and answer questions about research. with attendance at local and national cf meetings, rc's have expanded their cf knowledge, networked with other rc's and explored new research trials. our site also integrated education about various aspects of research into both the clinical staff and cf family education meetings. a review of the research database was completed to assess our cf center's research program. results: over the years reviewed, participation in clinical research trials has seen significant growth and patient participation has exceeded patients enrolled into clinical studies. conclusion: utilizing a team of dedicated rc's from the research department has proven to be effective in advancing our cf center's clinical research program. discussion: to meet the growing demand of patients needed to participate in clinical trials, cf centers will need to provide research education to both the patients and families and the clinical team, improve on skills needed to identify and enroll our patients into clinical trials and include the rc's into the multi-disciplinary cf patient care team. centralized research represents an excellent model for cf centers that want to excel in clinical research. integration of rc's into the cf care team, along with education initiatives for both clinical staff and patients and families has led to successful participation and enrollment in clinical trials. further studies need to be done to better identify barriers that patients and families have with participation in research. the goal of "cystic fibrosis transition care" is to ensure that youth with cystic fibrosis (cf) are adequately prepared to participate in the management of their health condition into adulthood and as they graduate to the adult health care system. in canada, it is unknown if, or to what extent, cf transition care is practiced in individual clinics. we therefore surveyed all canadian pediatric cf clinic coordinators (n= )using a brief standardized questionnaire; surveys ( %) were completed and returned for analysis. findings: all responding clinics transfer their pediatric patients to a distinct adult cf clinic, however almost half of these share one or more team members between the two clinics. transition care is recognized and practiced by % of responding clinics: % follow a formal transition program, and % follow an "informal program". "informal" practices vary widely from one clinic to another. formal programs were created by individual clinics, and share the common properties of being based on current research, having set goals, and utilizing a tool to document the process. the average age of transfer to the adult clinic is years. most clinics have "rare exceptions only" to delaying the age of transfer, the most commonly cited reason being "very ill/palliative patient". a small number of clinics also cited "intellectually challenged patient" and "reluctant patient" as reasons to delay transfer. only % of pediatric clinics hold a formal "transition" or "graduation" clinic, which allows adult team members to be introduced to youth before the actual transfer of care takes place. conclusions: although sharing team members between pediatric and adult clinics may ease the change from pediatric to adult care, it can also create barriers to the transition process. therefore, it is encouraging to learn that the majority of canadian pediatric cf clinics view adult care as distinct from pediatric care, and that there is a consistent age of transfer across the country. transition preparedness is also recognized as an important component of care, as evidenced by the number of clinics following a formal or informal transition process. it remains undetermined, however, whether in general there is adequate preparation for adult care. with only % of clinics following a formal transition process (and % not following any type of transition program), further assessment seems warranted and may reveal inadequacies requiring remediation. future direction: to strengthen cf transition care in canada, a "patient readiness to transition" questionnaire has been developed and will be administered to canadian cf patients transferring to adult cf clinics in . analysis of this questionnaire will provide insight into how well canadian cf clinics are preparing their youth for transfer to adult life, and may provide directions for further improvement. nearly all females with cf now survive into adulthood, and advice regarding pregnancy and contraception is becoming increasingly important as part of their sexual health education. we set out to identify the level of knowledge of these issues in patients attending our large adult cf unit. using a structured questionnaire, we surveyed consecutive cf females (age range to years) attending routine cf clinics. we asked about knowledge and usage of contraception, and issues relating to pregnancy and its possible effect in cf, ensuring the opportunity for a one to one discussion with the individual on completion of the questionnaire. only ( %) claimed to have had previous advice relating to contraception and ( %) pregnancy education. of these, ( %) stated that this was from adult cf nurse specialists, ( %) from adult cf physicians and the remainder from other sources (general medical practitioners, written literature, and the internet). only patients ( %) recalled that they had been given information in the paediatric sector before transition. the majority of all advice was given verbally. of the ( %) who were using some form of contraception ( [ %] condoms, [ %] combined pill, [ %] depot injection, [ %] a coil, and [ %] the mini pill); ( %) had experienced problems, and in some cases patients had been misinformed about the reliability of their contraceptive choice. as regards pregnancy, ( %) had undergone previous termination and ( %) already had children. thirty six ( %) had considered becoming pregnant but ( %) said they would discuss this with a member of the cf team beforehand: ( %) were aware this might impact on their health. of these, ( %) believed a reduction in lung capacity to be the biggest possible problem. other problems identified were alteration of medication, tiredness, and weight loss or gain. overall, ( %) felt they did not have enough information regarding pregnancy in cf and ( %) that insufficient contraceptive advice was given, expressing a wish for further knowledge. conclusion this survey has shown that a significant number of adult females with cf require further education about contraception and pregnancy. in some cases the advice they had already been given may not have been appropriate for patients with cf. furthermore, few patients appeared to have had effective counselling in the paediatric sector, despite the risk of pregnancy. we are working with patients and paediatric colleagues to improve the education in this important area for our women with cf. the state of minnesota added cystic fibrosis to the newborn screening panel on march , . one-year follow-up to date reveals infants have screened positive, of the screen positive, have been diagnosed with cystic fibrosis, infants are pending sweat chloride tests and evaluation. of this population infants are being followed by the minnesota cf center. the university has a comprehensive, interdisciplinary team available to provide care to infants, children and adults with cystic fibrosis. in response to newborn screening for cystic fibrosis, the center has implemented a prophylactic care program to include clinical care through early intervention and education within the first two weeks of life. benefits of collaborative care have been previously identified in the literature, showing improved quality of care with increased patient satisfaction, lower mortality, and improved outcomes. infants who are identified by newborn screening and receive earlier treatment have the potential for improved physical health and development. the inclusion of comprehensive genetic consultation as an integral component of cf education, allows for informed reproductive decisions. the newborn screening intervention plan is supervised by a nurse practitioner and consists of initial evaluation, initial therapy implementation, and a teaching/consultation schedule, which covers a minimum of four appointments scheduled over weeks. follow-up visits are scheduled within hours of referral and then at one week, days and weeks post referral. the schedule is adjusted based on infant acuity and family needs. initial evaluation consists of the confirmatory sweat chloride testing and/or genetic mutation analysis, serum laboratories, chest x-ray, stool for fecal elastase, and nasopharyngeal culture. general, supportive teaching with evidence from the literature is provided. preliminary results to date demonstrate that our infants have had no pulmonary exacerbations, no pulmonary hospitalizations with pulmonary functions obtained in over half of the infants. nasopharyngeal cultures obtained at the time of ascertainment demonstrate newborn screening patients have positive cultures for staphylococcus aureus, haemophilus influenzae, escherichia coli, streptococcus pneumoniae, klebsiella pneumoniae, acinetobacter junii and alcaligenes faecalis. one infant was culture positive for pseudomonas aeruginosa at the first out-patient clinic visit. this demonstrates colonization of organisms prior to clinic exposure. through minnesota's early intervention and education plan, the families will be more adherent to prescribed care, which will result in improved cf infant outcomes as monitored by number of exacerbations, pulmonary functions and potential complications. a detailed outline of medical intervention, education, and outcomes will be presented. cf-pediatric centre we developed a malabsorption blood test (mbt) using odd-chain length fatty acids, pentadecanoic acid (pa) and heptadecanoic acid (ha), to assess fat absorption in subjects with cf. pa is a free fatty acid, while ha requires hydrolysis of triheptadecanoic acid (tha) by pancreatic enzymes for absorption. objective: to determine the mbt reproducibility in healthy subjects and subjects with cf. methods: subjects with cf ingested a liquid test meal including fats (pa and tha) on occasions at least days apart. for each subject, a standard dose of pert ( , lipase units) or the subject's usual dose (if higher) was given with each test meal. serum was analyzed for pa and ha levels at baseline and then hourly for hours. a non-compartmental pharmacokinetic analysis was performed using pa and ha concentration-time data from subjects with cf. c max was directly observed from the individual subject profiles, and auc was calculated using the linear trapezoid rule. summary statistics (mean±s.d.) were calculated for all parameters. reproducibility of the mbt was also assessed in healthy adult controls tested on occasions. between subject (bsv, %) and within subject variability (wsv, %) was calculated for pa, ha and the ha/pa ratio. results: bsv (%) and wsv (%) of pa and ha absorption are presented in the table for subjects with cf (age . ± . y, females) and healthy adult subjects (age . ± . y, females). as expected, the mbt had greater reproducibility in healthy controls than in subjects with cf. in healthy controls, the reproducibility for pa and ha absorption was comparable; there is less wsv than bsv, and the ha/pa ratio has the best reproducibility. in subjects with cf, pa and the ha/pa ratio show less wsv than ha. conclusion: wider variability in fat absorption in subjects with cf reflects the complex interactions of the biliary, pancreatic and intestinal factors background: the international, multi-center gene modifier study of cystic fibrosis liver disease (cfld) initially enrolled patients with severe liver disease (i.e. cirrhosis and portal hypertension). for the initial population despite severe liver disease with portal hypertension, many of these patients had normal liver biochemical function tests and/or inr ( - %), depending on the test. the mean age of enrollment into the study was years, with the mean age of cfld diagnosis of years. candidate gene testing revealed an increased prevalence of the alpha- antitrypsin z allele in cfld patients, particularly in females, as compared to cf patients (> yrs.) without cfld, and an association with tgfβ variants (- and codon ) in males smoothing reference centile curves: the lms method and penalized likelihood hepb) and inactivated hepatitis a (hepa) vaccines in cystic fibrosis (cf) patients chronic liver disease (cld) remains the second mortality cause in cf. we evaluated the immunogenicity of hepa-and hepb-vaccines in cf-patients because it is described lower in patients with cld. patients and methods: blood samples of cf-patients aged . - . y (mean . y) were tested twice (time interval y) by chemiluminescent microparticle immunoassay to asses the presence of hbs-ab and ha-igg, and their vaccination status was recorded. seronegative patients for hbs-ab and/or ha-igg were vaccinated in between tests with engerix®, havrix® or twinrix®. results: / ( . %) patients tested positive for hbs-ab. / cld ( steatosis and cirrhosis). / tested positive at first but negative at present data are incomplete for hbs-ab and ha-igg for and patients. conclusions: . immunogenicity of hepa-and hepb-vaccines is comparable in cf-patients and healthy subjects. . cf-patients are at risk for cld and seroconversion must be checked after vaccination. . vaccination records are important as numbers of antibodies may decline beneath the detection limit in time methods: lf was assessed in children with cf, aged - yrs, using forced expiratory volume (fev(sub) (/sub)% predicted). body composition was measured using a reference four-component model(sup) (/sup) ( cm) allowing accurate evaluation of both fat mass (fm) and the components of fat-free mass dual-energy xray absorptiometry (dxa; lunar prodigy, g.e. medical systems) was used to measure fat, lean (non mineral) and bone mass and skin-fold thicknesses (bicep, tricep, sub-scapular) were measured. strength of relationship was assessed using pearson's correlation coefficient (r) and significant body composition components were fitted into a regression model. difference in fev(sub) (/sub) between the sexes was assessed with an independent t test. results: boys with cf did not differ from the reference population conclusion: our results confirm that body fat, but not ffm or bone mass, are related to the severity of impaired lf in children with cf. it is likely that the significant association between fev(sub) (/sub)and fat in girls but not boys reflects the poor body composition of the girls with cf. longitudinal follow-up of these children should indicate whether this sex difference persists after puberty. given the fact that prognosis is worse in girls the sex difference we've identified merits more attention. fuller nj et al. four-component model for the assessment of body composition in humans: comparison with alternative methods and evaluation of the density and hydration of fat-free mass conclusions: these results show that pancreatic status plays an important role in relation with pufa status in cf patients and particularly in (the initial fatty acids in the n- and n- pathways) in neutral lipids, nonesterified fatty acids, and phospholipids this may, in some way, account for the fact that : n- is more readily metabolized, and therefore depleted, in cf animals than in wt mice. conclusion: dha supplementation increased the localization does an integrated clinical and nutritional approach prevent this case control study examines the impact of clinical approach on pre and post-cfrd clinical course. patients with cfrd (mean age . + . yrs) matched to cf controls ( . + . yrs) for age, sex and pseudomonal status had parameters of clinical status and nutritional intervention recorded annually from six years pre diagnosis of cfrd to two years post. weight and body mass index (bmi) were lower at all time points to diagnosis of cfrd (ns) but were stable as a % of control values intravenous antibiotic treatment intensified, peaking at one year post diagnosis . days/yr (cfrd) v . (cf) ns but there was no difference in nebulised antibiotic use. an aggressive clinical approach prevents nutritional decline and delays respiratory decline until the year preceding diagnosis of cfrd data show mean (sd) or % , ; battezzati with advancing age insulin secretory defects and insulin resistance cause glucose intolerance and diabetes in an increasing proportion of cystic fibrosis (cf) patients. prediction of diabetes development in cf is made difficult by unique features: many patients are normoglycemic or even hypoglycemic after overnight fast, and there are repeated changes of glucose tolerance status from normal to diabetes and vice-versa, for many years and for unclear reasons. aim of the study was to detect predictive factors of definitive cf related diabetes (cfrd) development in patients undergoing oral glucose tolerance test (ogtt) evaluations routinarily. methods. starting from , all patients followed at the cf center in milan aged > years and without established cfrd undergo ogtt yearly. among those who received their first ogtt between and , had developed definitive diabetes by µu/ml, and . ± . vs . ± . ng/ml). glucose (p= . ) and insulin aucs (p= . ) were the most important predictive variables, respectively directly and inversely related to cfrd development (pseudo r for the model: . , p< . ). glycated hemoglobin and baseline glucose concentrations were directly related with outcome at univariate analysis whereas c-peptide concentrations were inversely related. in contrast, no relationship emerged between insulin-sensitivity indexes and outcome. anthropometric (weight, height z scores, bmi) and pulmonary function indexes were also unrelated. conclusions. insulin secretory defects are an important determinant of subsequent cfrd developement response rate was / in group a and / in group b. group a included males and females, mean age was months, sd . ; group b included males and females, mean age months, sd . . followgroup b parents do ("poor-very poor" health % vs %, p< . ), but over time health perception in parents of children with cf improves, and the gap vanishes ("poor-very poor" health in last year % in group b). post-diagnosis anxiety, depressive symptoms, sleep disturbances, mood changes, and nervousness are less frequently reported in group a ( % vs % p< . ), and the diagnosis was considered to affect the parent-child relationship more in group b than in a (p< . ). the two groups did not differ in the assessment scores of internalizing, externalizing and total behavioural/emotional problems. however, group b performed worse than the general control population average (mean . sd . vs mean sd . , p< . ). thirty-five% of parents in group a and % in group b changed their family planning projects following the diagnosis physical health and anxiety symptoms: does monitoring mediate the relation? results: physical health, monitoring, and anxiety symptoms were related. children who perceived themselves to be relatively healthy were less likely to have an information processing style characterized by high levels of monitoring (r = -. , p < . ), and they reported fewer trait anxiety symptoms (r = -. , p < . ). monitoring, as hypothesized, was associated with increased trait anxiety (r = . , p = . ). low bmi, a potential indicator of poor nutritional status and physical health among youth with cf, also was related to higher monitoring (r = -. , p = . ) and trait anxiety (r = -. , p = . ). tests of mediation indicate that monitoring partially mediated the relation between poor health and trait anxiety for both child report of physical health (goodman test: z = . , p = . ) and for bmi (goodman test: z = . , p = . ), but not when parental report or fev were used to assess youth's physical health, or when parental report of youth's internalizing symptoms was used as the outcome. conclusion: youth who tend to generally scan for and are attentive to changes in their environment, which may include their internal physical environment, were found to report more anxiety. moreover, this monitoring style appears to partially mediate the relation between physical health and anxiety among youth with cf. if monitoring is found to be a risk factor leading to an increase in future anxiety symptoms among youth with cf data was analysed using a thematic analysis: "framework" results: young people say that befriending is fun, offers opportunities for new experiences, a confidant outside the family, and gives them a boost psychologically. carers see befriending as confidence building for their children, providing time out for themselves, and helping with the big questions. befrienders see their role as mentoring, broadening young people's horizons and providing a safe place physically and emotionally. challenges include: forming and ending relationships, having multiple befrienders, ongoing support and training for befrienders, maintaining boundaries, sibling rivalry, and cost. conclusions: befriending is a new innovation in cf, and has the potential to make a difference to young people's lives. careful planning at the outset, ongoing support for befrienders and regular evaluation are essential factors in ensuring its success good practice in befriending services for people with learning disabilities qualitative research practice sage london ଙ in addition, there was high use of non-cf vitamins (n= ) and protein shakes (n= ) which were not included as a cam therapies. when prayer was excluded (which was used by % of respondents), . % of the patients are still using some form of cam. the most commonly used cam therapies in cf were relaxation therapies (n= ), massage therapy (n= ), chiropractor care (n= ), herb/plant product therapies (n= ), homeopathy (n= ), and yoga (n= ). an analysis of the relaxation therapies revealed that deep breathing exercises (n= ) were the most common and frequently combined with other relaxation techniques: meditation (n= ), progressive relaxation (n= ) and guided imagery (n= ). the most common herb/plant products used were: echinacea (n= ), garlic (n= ), selenium (n= ), ragweed or chamomile (n= ) and ginseng (n= ). the use of possible cf specific cam therapies were fish oils/omega fatty acids (n= ), glutathione (n= ), docosahexaenoic acid (n= ) and curcumin (n= ). in conclusion, cam is widely used by the cf population with "prayer for health" being the most common modality, however, many patients are utilizing multiple cam therapies (data not presented) within the framework of a weighted satisfaction model of quality of life, we investigated the importance ratings of adolescent and adult patients regarding disease-specific aspects of living with cf. method: outpatients (aged - years, m= . , sd= . ; fev - %, m= . , sd= . ) repeatedly filled in the cf-specific module of the questions on life satisfaction (flz-cf) to measure satisfaction with nine cf-specific aspects of life in relation to the subjective importance of each life domain. a ranking list of the most important life domains across the study group was determined, and intra-individual changes of the importance scores were analysed. associations of importance ratings with changes of pulmonary functioning (fev %) were examined. results: the most important aspects (% of "very important" or "extremely important" answers) are sleep ( . %), integration of therapy into daily routine ( . %), breathing ( . %), gastrointestinal functioning ( . %), and eating ( . %), less important is understanding by others ( %) , ; durieu, i. , . département d'information médicale, hospices civils de lyon dr. von hauner children`s hospital helios hospital, e.v. behring, berlin, germany supported by the cystic fibrosis foundation leroy matthews physician scientist award, the national heart, lung, and blood institute cystic fibrosis foundation therapeutics inc. symptoms patient-reported respiratory symptoms in cystic fibrosis: initial validation children's hospital and regional medical center the first author is supported by a second year clinical fellowship from razvi, s. ; quittell, l.m. ; sewall, a. ; marshall, b.c. ; saiman, l. . pediatric pulmonary medicine, columbia university, new york, ny, usa; . cystic fibrosis foundation, bethesda, md, usa significance: significant improvements have been made in diagnostic and therapeutic strategies for cf patients in recent decades. we hypothesized that these changes could potentially impact cf respiratory microbiology. thus, we examined longitudinal trends in the annual incidence and prevalence of cf respiratory pathogens from to .methods: cf foundation patient registry data, estimated to include % of the cf population in the u.s., was utilized in this analysis. patients were included if results from at least one respiratory culture were in the registry from january to december . patients were excluded after organ transplantation. to avoid misclassification of incident cases, a retrospective review of registry data from to was performed to establish the culture status of included patients prior to . thus, incident cases were subjects with first detection of a given pathogen in a given year. prevalent cases were defined as subjects with at least one positive respiratory culture for a given pathogen in a given year. all submitted culture results were included.results: the number of cf patients submitting registry data increased from , in to , in . the proportion of subjects meeting inclusion criteria remained relatively constant ( % to % per year). the median age of the study cohort increased from . years in to . years in . during the study period, the incidence of hemophilus influenzae remained stable ( . % to . %) as did the prevalence ( . % to . %). the incidence of pseudomonas aeruginosa ranged from . % in to a peak of . % in . the prevalence of p. aeruginosa declined from . % in to . % in . there is a trend for both an increasing incidence and prevalence of staphylococcus aureus; the incidence increased from . % in to . % in and the prevalence increased from . % in to . % in . the age specific prevalence of s. aureus remained highest in children aged - years. the incidence of methicillin-resistant s. aureus (mrsa) increased from . % in to . % in , with a parallel increase in prevalence from . % to . %. the highest prevalence of mrsa was noted in subjects years of age and older. the incidence of burkholderia cepacia complex decreased from . % in to . % in , while the prevalence remained relatively stable (range . % to . %). both the incidence and prevalence of stenotrophomonas maltophilia increased (incidence: . % to . %, prevalence: . % to . %).this retrospective study aims at assessing the respiratory function and the cystic fibrosis (cf) co-morbid conditions according to the patients' nutritional status.method: data were collected from the french observatory (onm) (n= ) (vlm, ined). the last year data for weight (w), height (h), fev , fvc were extracted from the database (exclusion criteria: transplan-tation); were also analysed the genotype, pancreatic enzymes consumption, pseudomonas aeruginosa (pa) colonization, cirrhosis, diabetes, pulmonary transplantation and mortality (sas . ). we used international age-adjusted standards for bmi z-score for children (c) and bmi for adults (over years) (a) (cole tj bmj ) to define underw (uw), normal w (nw), overw (ow) and obesity (ob) .results: data are available for patients in the c cohort and for patients in the a cohort ( %). nutritional status subgroups prevalence (%) is: . , . , . and . in c and . , . , . and . in a for respectively uw, nw, ow and ob. mean age in a is significantly increasing with bmi (p<. ). frequency of del/ del in c and a is lower in ow/ob (p<. ) as well as the use of pancreatic enzymes (p<. ). ow/ob patients have the best fev and fvc values whatever the gender and the age, with significantly less pa colonization in a (p<. ). we could identify a positive correlation between the pulmonary function and bmi. the cf co-morbid conditions demonstrated a lower prevalence of diabetes ( / ) and cirrhosis ( / ) in ow/ob. none of the ow/ob c or a are on a transplant waiting list versus . % in uw/nw (n= / ) and none died versus % in uw/nw (n= / ).conclusion: the observed prevalence of ow and ob in cf is respectively . and % whereas the french obepi study collected and %. our results suggest that increased bmi is associated with better fev , fvc, lower prevalence of pa, cirrhosis and diabetes. the potential risks of chronically high bmi have not been studied in this population yet, but justify further investigations in this longer life expectancy cohort. materials and methods: we retrospectively identified abdominal ct scans of consecutive patients ( females, males, mean age of years) with cystic fibrosis and a control group of consecutive patients ( females, males, mean age of years) scanned as potential renal donors. three readers reviewed all scans and recorded the presence and location of colonic wall redundancy, and the wall thickness of the ascending, transverse, and descending colon. clinical information on cystic fibrosis patients, including cftr gene mutations, was queried from our cystic fibrosis patient registry database. additionally, medical records of all cystic fibrosis patients were reviewed to determine the indication for abdominal ct. results: colonic wall redundancy was seen exclusively in patients with cystic fibrosis, and was noted in of patients each for reviewers , , and (p< . ). colonic wall redundancy was seen in of adult patients with cystic fibrosis ( %), but was not seen in any children age or younger (p< . ). excellent agreement was found for the ct identification of colonic wall redundancy among readers (kappa= . , p< . ). cystic fibrosis patients with colonic wall redundancy had significantly thicker ascending colonic walls (mean . mm) vs. those without wall redundancy (mean . mm) or controls (mean . mm), (p= . ). three patients with colonic wall redundancy had follow-up ct, and all showed temporal stability (mean of months). among adult cystic fibrosis patients, cftr gene mutations were available for of patients with and of without colonic wall redundancy. while the common ∆f mutation was the predominant mutant allele among patients with normal colons at ct (only of patients, or %, had an identified mutation other than ∆f on either allele), a higher prevalence of less common non-∆f mutations was seen patients with colonic wall redundancy ( of patients, or %, p< . ). the g x mutation was seen exclusively in patients with colonic wall redundancy ( of patients, or %, p= . ). there was no significant difference in the proportion of patients with abdominal pain (> . ), pancreatic insufficiency (p> . ), diabetes mellitus (p> . ), history of meconium ileus background: airway inflammation in cf is associated with marked remodelling and bronchiectasis. pro-inflammatory mediators such as advanced glycation end-products (ages) and the soluble receptor for age (srage) may perpetuate this response in the lung and in other organs such as the kidney . the total body burden of ages reflects exogenous sources from the diet, endogenous production by the body, tissue degradation and renal clearance, which may be reduced in renal impairment. the accumulation of endogenous age is accelerated in conditions of high oxidative stress and inflammation, and ages have been implicated in the pathogenesis of diabetic nephropathy. the burden and significance of ages in cf has not been determined, but if elevated, dietary modification of ages may represent a novel anti-inflammatory approach for cf. the aim of this study was to determine serum levels of age and srage, and identify clinical correlates of age and srage levels.methods: adults with cf (n= , males, % pancreatic insufficient, mean age . ± . years (range to years), mean fev %predicted . ± . %predicted (range - )) and healthy adults (n= , male, mean age . ± . years (range - years)) were studied. cf participants provided - serum samples each over a six month period, while healthy controls provided a single sample. serum was analysed for levels of advanced glycation end-product (age-cml) and soluble receptor for age (srage) (elisa). for each cf participant, levels in the multiple samples were averaged, and the median for the study sample reported. clinical data, including bmi, presence of cf-related diabetes mellitus (cfrd) and serum hba c level were collected from the medical record. mann-whitney tests were used to compare cf and control levels, while spearman rank correlations were used to identify clinical correlates of age and srage.results: prevalence of cfrd was . %. mean bmi was . ± . kg/m and mean hba c level was . ± . %. median (iqr) levels for age-cml were ( , ) background: osteopenia is diagnosed in cystic fibrosis (cf) using dual-energy x-ray absorptiometry (dxa). areal bmd from dxa is subject to error when bones are smaller in volume than reference standards (t-scores). normalization of bone size by use of zscores in cf is controversial and not widely utilized, thus comparing to larger bone areas. reports in cftr-deficient mice (cf mice) reveal osteopenia when measured by dxa. we hypothesized that use of pqct, which eliminates bias of size, would more accurately analyze bmd.methods: femurs were collected at necropsy from cf mice and c bl/ j (b ) mice at wks, and cf mice and b mice at wks (all female) for pqct. time points were chosen to coincide with pre-pubertal and adult ages for comparison to human disease. total mineral, trabecular and cortical densities were measured. student's t-test was used to detect significant differences (p< . ).results: femur measurements from both cf mice and b mice are listed as mean ± sd in the table (below). length was greater in b mice compared to cf mice at and wks. total area at the metaphysis and diaphysis were greater in b mice at and wks. data are consistent with larger bones in b mice. total mineral density , trabecular density and cortical density , however, were greater in cf mice compared to b ( & wks, wks, wks).discussion: our study demonstrates greater bmd in cf mice when volumetric data are analyzed and size differences are accounted for by use of pqct. these findings persisted at adult age, suggesting a normal deposition of bone. in clinical management, dxa imaging is readily available compared to pqct, and can be used as a predictor of bmd and fracture risk. correct reference ranges must be utilized to minimize erroneous values secondary to size. z-scores allow correction for differences in bone area and size, even in adults. more studies into understanding bone mineral deficit and tools of measurement are needed in cf. meanwhile, bone measurements by radiographic imaging must be taken in context of overall health, pubertal progression, and size of individuals. gainesville, fl, usa; . nemours children's clinic, orlando, fl, usa; . suny upstate medical university, syracuse, ny, usa; . case western reserve university, cleveland, oh, usa; . genentech, san fancisco, ca, usa background: greater growth rates, bmi and fat-free mass are associated with improved lung function in individuals with cf (pedreira, et al. pediatr pulmonol. ) . current treatments for weight gain have focused on nutritional supplements and appetite stimulation, and not underlying issues of catabolism or chronic disease. we hypothesized that anabolic effects of gh would not only improve weight and height, but lbm as well.methods: sixty-seven prepubertal children with cf and height less than or equal to the th percentile were randomized to daily gh (n= ) or observation (n= ) for a period of year, followed by an off-treatment observation period of months. children randomized to gh received somatropin injections daily at . mg/kg/wk. height and weight were measured every three months. height was evaluated as height standard deviation score (sds) to control for differences in age and sex. lbm was measured by dexa scan at time zero, at and months, and at the end of the -month observation period. in addition, for lbm, change from baseline was calculated for subjects for whom the same dexa equipment was used at both time points. the preliminary results of the first months of the study are presented here.results: data for subjects in the observation arm and subjects in the gh arm, who completed months, are available. as shown in the table below (listed as mean ± sd), gain in height, weight and lbm were significantly greater in the gh treated group than in the observation group over the month period.discussion: gh improves growth in prepubertal children with cf as measured by height sds. in addition, gh significantly improves weight gain and this gain is, in part a result of the significantly greater increase in lbm in the gh-treated group than in the observation group. the relationship between the improvement in lbm and other outcomes in cf deserves further exploration. the national cystic fibrosis(cf) registry database notes cystic fibrosis related diabetes mellitus(cfrd) as a complication in . % of cf patients at all ages. cfrd is associated with poorer nutrition and increased pulmonary morbidity. the cause of cfrd is not completely known but insulin resistance may be associated with fibrotic damage to pancreatic islet cells due to chronic inflammation. other studies suggest protein energy malnutrition(pem) in early life leads to impairment of insulin secretion and cystic fibrosis-related diabetes (cfrd) accounts for increased morbidity and mortality in patients with cf and occurs in approximately % of patients by the age of years. cfrd net (network for epidemiology and trials) is a consortium of four large uk cf centres caring for adult patients with cf. the aim of this group is to undertake research into the diagnosis, investigation and management of cfrd. in this abstract, we estimate the prevalence of diabetes in a screened population of adults with cf. methods annual review data were collected on attending patients with cf aged and above during . g ogtts were performed after fasting for at least hours on patients without known diabetes. each ogtt was categorised as either normal ( hour glucose . - . mmol/l), impaired ( hour glucose ≥ . mmol/l and < . mmol/l) or diabetic ( hour glucose ≥ . mmol/l). all patients with a diabetic ogtt were followed up with serial bm monitoring to determine whether or not they had cfrd. fasting plasma glucose (fpg) was considered to be elevated ≥ . mmol/l, isolated impaired fasting glucose (igf) was defined as a value between . mmol/l and . mmol/l and hypoglycaemia was defined as a blood glucose < . mmol/l. in addition, we obtained information on age, sex and prescription of anti-diabetic medications subsequent to ogtt. in three of four centres, hba c was routinely performed on all patients. of patients (median age years, % male), ( %) had established diabetes and were therefore excluded from further screening. of the remaining patients, ( %) underwent formal ogtt testing. in this latter group ( %) had a diabetic ogtt, ( %) an impaired ogtt and ( %) had evidence of reactive hypoglycaemia at hours. of the patients with a diabetic ogtt, ( %) only had an abnormal hour value, ( %) only had an elevated fpg and (< %) had both. all patients with diabetic ogtts underwent blood glucose monitoring and ( %) went on to treatment with hypoglycaemic agents in the calendar year. none of the patients with an isolated elevated fpg had diabetes.hba cs were available in of newly diagnosed patients with diabetes (in the centres which performed hba c). in this group median hba c was . %. this study confirms the high prevalence of diabetes among screened patients and the growing burden of diabetes management in adult cystic fibrosis clinics. it further highlights the importance of performing screening for diabetes in this population; the majority of patients were identified on the basis of abnormal hour values. despite this only half of all suitable patients underwent ogtts in the four centres committed to screening. these patients merit further study, as previous work suggests that the decline in clinical status occurs several years before diabetes becomes apparent. cystic fibrosis related diabetes (cfrd) occurs in up to % of patients with cystic fibrosis (cf), the incidence rising with increasing age. the likelihood of developing long-term complications secondary to diabetes increases with poor glycaemic control and duration of diabetes. as survival has improved for people with cf those who develop cfrd may live with diabetes for several years. aim: to establish the frequency of diabetic complications in patients with cystic fibrosis related diabetes.method : patients with cfrd attending the adult cf service at the royal brompton hospital between april -march were screened for diabetic complications and cardiovascular risk factors. a total of patients (male/female: / ) including post transplant patients were screened. mean age was . years ( - ), mean hba c . % ( . - . % ) and average duration of diabetes was . years (< - years).as immunosuppressive therapy can also cause many of the complications associated with diabetes the results are presented separately for nontransplant and post transplant patients (table ) . patients in total had retinopathy: background retinopathy, proliferative retinopathy undergoing laser therapy and maculopathy. the average duration of diabetes in those with background retinopathy was . years with only patient having diabetes for > years. the patient with proliferative retinopathy (non-transplant) had been diabetic for years. a raised creatinine level was identified in all transplanted patients with microalbuminuria but none of the non-transplanted patients. patient (nontransplant) had macroalbuminuria. post transplant patients were on treatment for hypertension and a further patients ( post transplant and nontransplant) had elevated blood pressure (> / ) at screening requiring follow-up. none of the patients had evidence of cardiovascular disease or stroke.conclusion: macrovascular complications were not seen. microvascular complications occurred but were less common than the reported incidence in type and type diabetes. this may reflect the relatively short duration of diabetes (mean . years) of patients in this study. a further study comparing cfrd patients with a non-cf diabetic control group of similar duration of diabetes is warranted. cystic fibrosis (cf) is a disease that leads to serious disturbances in nutritional status and bone calcification. comparison of two methods for assessment of bone mineralization: dexa and hand radiograms in diagnosing osteopenia or osteoporosis. study was performed in a group of cf patients ( f, m), aged - yrs. nutritional status was assessed using bmi, cole's index and bmc. radiograms of non-dominant hand were assessed according to normalized optical density comparing to aluminum standard. bone density was also assessed using dexa. for statistical analysis, backward stepwise binary logistic regression (wald's test) was used. analysis of data revealed that using bmc, cole's index and hand radiograms markers we can diagnose bone mass disturbances (z score < sd) with precision up to . % comparing to dexa. sensitivity and specificity of this method was respectively . % and . %. false negative results were obtained in patients and false positive were also in patients. hand radiograms method could be an alternative for dexa in screening of bone density disturbances in cf patients. the study was partly supported by grant of ministry of science and higher education no p e .in a very recent publication( ) a high rate of fasting ( %) and reactive hypoglycaemia ( %) was described in a group of n= cf patients older than years who received an oral glucose tolerance test. reactive hypoglycaemia was related to the minute glucose concentration in the ogt test. we were surprised by the high percentage of asymptomatic hypoglycaemic situations in cf patients. as a part of a prospective intervention study in cf patients with cfrd, we screened more than patients years or older with cf for cfrd. in this multi-centres population ogt was performed in the morning after overnight fasting according to who standards. using the same definition for fasting hypoglycaemia (glucose < mg/dl; . mmol/l) and reactive hypoglycaemia (glucose < mg/dl; . mmol/l at minute during ogt test)as in the other study we calculated the percentage of cf patients with fasting or reactive hypoglycaemia in our cohort. results: ogt were done in patients with cf. age(mean±sd) , ± , years; bmi z-score - . ± , ;height z-score - , ± , and weight z-score - , ± , . ogt was categorised according to ada criteria. normal ogt (n= all ; fasting hypoglycaemia (fh) n= ( , %), reactive hypoglycaemia (rh)n= ( , %),ifg (n= all, fh n= , rh n= ( , %), igt(n= ,fh n= ( , %), rh n= ),fgt (n= all, fh n= ,rh n= ) and diabetes mellitus (n= all, fh n= ( , %)). fh was observed in ( , %) and rh in ( , %) out of ogt tests. there were no difference related to age, bmi z-score, height z-score and weight z-score comparing those patients with fh or rh to those without fh or rh in all categories of ogt tests.in this large multi-centres cohort of cf patients we were neither able to confirm the high percentage of fasting nor of reactive asymptomatic hypoglycaemia which was reported recently on a small group of cf patients. nutritional status measured by bmi z-scores, height and weight z-scores were unaffected by hypoglycaemia in our cohort as noticed also in the small group. as the ogt tests in the other study were done in a single centre it might by that a centre specific situation influenced the frequency of hypoglycaemia in that small group. we conclude that large numbers of investigations might be needed to come up with firm conclusions related to frequency of specific aspects of glucose disturbance in cf patients.fh does not exclude diabetes mellitus or igt. as survival from cf improves, surveillance to identify and treat complications associated with longevity is an important component of management. renal disease has been reported in adults with cf. risk factors may include cf-related diabetes mellitus (cfrd) and use of nephrotoxic medications. diabetic nephropathy has been observed in the absence of cfrd , highlighting the need for screening for renal impairment. this study aimed to measure renal function in a sample of adults with cf, and to compare estimated glomerular filtration rate (egfr) and urinary creatinine clearance (urcrcl) as markers of renal function.methods: adults with cf aged years or over (n= , % male, % pancreatic insufficient, mean age . ± . years) underwent screening for renal impairment. of these patients, ( males) had cfrd, and ( males) had no history or clinical features of cfrd. hour urine collections were analysed for urcrcl, with renal impairment being defined as < ml/min. serum creatinine level was used to calculate egfr (mdrd formula), which was classified renal function as normal or mildly impaired (> ml/min); moderately impaired ( - ml/min) or severely impaired (< ml/min). sensitivity of egfr for identification of renal function was calculated using urinary creatinine clearance as the reference method. unpaired t-tests were used to compared cfrd with non-cfrd patients.results: mean urcrcl was ± ml/min, and eight patients ( . %) had renal impairment (< ml/min). of these, only had egfr suggestive of renal impairment. the sensitivity of egfr for identifying renal impairment in this sample was %. a further patients had moderately impaired egfr, but normal urcrcl. five of the patients with impaired urcrcl had cfrd. age, gender, cfrd, fev %predicted and hba c level did not correlate with urcrcl. the table compares cfrd patients with non-cfrd patients. hba c level was higher in cfrd patients. there were no other significant differences in clinical or renal function parameters.conclusion: renal impairment, is common in the adult cf population and is not confined to patients with cfrd. screening using egfr is poorly predictive of impaired urcrcl in this population. these results suggest that surveillance to monitor renal function is indicated in the adult cf population, including in patients without cfrd, and that determination of urcrcl should be included in the screening process. cystic fibrosis (cf) patients suffer from pancreatic insufficiency resulting in malabsorption of fat soluble vitamins including vitamin d. many fac-tors contribute to low bone mass and fractures in patients with cf; however, chronic vitamin d deficiency plays a major role. the prevalence of vitamin d deficiency has been reported as high as % in some specialized care centers. in addition, reports of occult vertebral fractures have been reported as high as %. we sought to determine the prevalence of vitamin d deficiency (defined as -hydroxyvitamin d ( (oh)d) < ng/ml) and of vertebral fractures at our cf center. we obtained irb approval to review the records of all patients seen at our cf center during [ ] [ ] . we collected information related to bone health including (oh)d, bone mineral density and lateral spine or chest x-rays to examine for the presence of a vertebral fracture. we reviewed the records of subjects who were seen at our center during the study period. the mean age of the subjects was ± years. subjects had a mean bmi of . ± and an fev % predicted of . ± %. the percentage of subjects having an annual (oh)d level checked was only % and % in and , respectively. the mean (oh)d was . ± and . ± ng/ml in and . the prevalence of vitamin deficiency was % and % in and . about one quarter of the subjects had bone mineral density testing with half of the tests showing osteopenia or osteoporosis. twenty-seven percent of subjects had vertebral abnormalites detected on lateral chest x-ray. we sought to determine whether any factors were associated with vitamin d deficiency. we found that taking a multivitamin did not significantly protect against vitamin d deficiency. however, not taking a supplement containing vitamin d other than a multivitamin was associated with a % risk of vitamin d deficiency (p= . ). subjects having (oh)d levels determined in the winter or spring was associated with a % risk of vitamin d deficiency (p= . ). in summary, we found annual testing for vitamin d status was inadequate in our cf center and that when (oh)d level was determined, over % of subjects were vitamin d deficient. nearly one quarter of our adult patients already had evidence of vertebral compression fracture seen in lateral chest x-ray. we urge greater screening for vitamin d deficiency in the cf population. effective protocols to prevent and/or treat vitamin d deficiency are urgently needed in the cf population. improved vitamin d status in cf patients is one factor that may reduce the high prevalence of vetebral fractures. support for this study was provided by proctor and gamble pharmaceuticals background: more cf patients are surviving into adulthood, in part due to the increasing use of new therapies and more aggressive management of chronic respiratory and gi disease. as a result, the recommended daily treatment regimens for most cf adults are both complex and time consuming. objective: to assess the self-reported daily treatment burden of cf therapies in a cohort of adults with cf. methods: in the sixth survey wave of the project on adult care in cf (pac-cf), an ongoing longitudinal panel study of adults with cf from us cf centers, respondents were asked to report the type of medications, inhaled therapies, and airway clearance therapies they used during the day prior to completing the survey, as well as estimate the time generally required to complete each type of therapy.results: of the respondents (response rate %), the median number of therapies reported was (iqr - ) and the median reported amount of time usually spent on treatments was minutes per day (iqr - minutes). forty-eight percent reported using or more different inhaled therapies or using inhaled therapies or more times during that one day. the most commonly reported inhaled therapies were a bronchodilator ( %), pulmozyme ( %), an inhaled steroid ( %), hypertonic saline ( %), and objective: caring for a child with cf is stressful and often leads to adverse effects for both children and caregivers. support from family and friends can help reduce the adverse effects of chronic stress, however, there is little research documenting types of support provision as well as sources of support. moreover, social support may be related to important health outcomes. this study described the types of support provided to caregivers of children with cf and examined relationships between support and health outcomes.method: as part of a larger intervention study to improve adherence to medical regimens, children with cf ages to and their caregivers were enrolled across cf centers in florida: university of florida, nemours children's clinic, and all children's hospital. caregivers completed the norbeck social support questionnaire (nssq; norbeck, ) in addition to other measures at baseline and follow up. this questionnaire provides information about the number and type of people providing support, as well as its type and quality.results: caregivers received the majority of support from their families, spouses and friends and characterized these relationships as longer in duration, with more frequent provision of support. other individuals who provided support included the cf team, religious leaders, counselors or psychologists, and work associates. overall, caregivers rated family members and spouses as providing the greatest amount (i.e., "quite a bit") of emotional support. spouses received the highest ratings for tangible support; they were perceived as providing "quite a bit" of support in comparison to families (i.e., moderate) and friends (i.e., a little). in terms of size of the support network, caregivers in this study had similar networks to those of a normative sample of healthy adults. in addition, there was no difference in amount of emotional or tangible support received by these caregivers. preliminary baseline results indicated that social support was positively related to both adherence, children's growth (i.e., height and weight percentile), and family income.discussion: caregivers of young children with cf reported similar support networks as other healthy adults. however, there was variability in the amount of support provided by source, with spouses providing the greatest amount. social support appeared to be a protective factor, and lack of support was related to negative health consequences for their children. more attention should be focused on the potentially beneficial effects of social support.funding was provided by nih grant # befriending is reported to be valued by those who have been befriended, offering opportunities for social activities and new experiences and can impact positively on self confidence and self esteem . whilst government policy supports befriending, few schemes collect evidence to demonstrate the effectiveness of these services . young people with cf may suffer social isolation due to chronic illness and treatment demands and a befriend-ing service was offered to those considered socially vulnerable in the edinburgh area.this project aims to evaluate the effectiveness of this befriending service on young people with cf and their carers. methods liverpool, united kingdom; . child mental health, university of liverpool, liverpool, united kingdom; . child health, university of liverpool, liverpool, united kingdom; . psychology, university of miami, miami, fl, usa; . psychology, birkbeck college, university of london, london, united kingdom; . mathematics and statistics, university of lancaster, lancaster, united kingdom; . psychiatry, university of manchester, manchester, united kingdom introduction: existing treatments for cystic fibrosis (cf) are time-consuming and labour intensive and biomedical advances are likely to lead to further novel interventions. there is concern amongst clinicians that a high care burden associated with these interventions may compromise the wellbeing of caregivers, reduce adherence to the treatment protocol and increase the likelihood of inadvertent errors in the delivery of interventions. at present, there is no measure of 'treatment burden' and therefore no way to assess the impact of new and increasingly more complex interventions conducted by lay caregivers at home. furthermore, trials are hampered by the lack of measurable patient-reported outcomes beyond a broad quality of life assessment. to address this gap we have developed a measure of treatment burden for cf using qualitative methods (focus groups, action research, in-depth interviews) with participating parents and a working group of cf team professional staff. this measure addresses the time, effort, meaning and ease of management for caregivers of children with cf up to years and post the first year following a diagnosis. methods: we report here the pilot phase of the validation of this instrument, which involved (i) cognitive interviewing with n= caregivers; and, (ii) n= caregivers completing the clcf instrument together with a quality of life measure at time points. this yielded data for stability, reliability and coherence of the hypothesized constructs. results: the clcf takes minutes to complete. face validity and acceptability are established. for the first pilot sample the age of the child ranged from . - . years. treatments took, on average minutes per day to complete and an average of minutes of this was devoted to administering enzymes particularly for carers of infants. a prominent concern amongst these parents was their child's height and weight and their efforts were directed at optimising growth. for the child however, management of nebulised medications and physiotherapy were more frequently flagged as a concern. these data suggest this is a reliable, coherent and useful, although complex tool. conclusion: the caregiver challenge for cf cannot be understood simply in terms of time and effort involved in delivery of treatments. it also involves contextualising interventions for their meaning at multiple levels of explanation for the individual concerned. structural equation modelling would be an appropriate way to proceed with the main validation when such complex relationships occur between latent variables and would serve as a confirmatory factor analysis for the hypothesised relationships. sponsored by: royal liverpool children's nhs trust and university of liverpool. funded by: national institute for health research (nihr) research for patient benefit programme.introduction: the french cf practice recommendations, published at the end of in parallel with the creation of cf reference centres, advise that each patient should be seen at least every months at a cystic fibrosis reference centre.objective: to investigate the impact of these recommendations on the effectiveness of the follow-up of patients at the four reference centres in the rhône-alpes region.methods: all patients with cystic fibrosis attending one of the four cf centres between and were retrospectively included. the total number of visits was recorded for each patient and each year of the study period. to determine the evolution slope for each patient followed for at least years, a negative binomial regression of number of visits versus time was carried out (confirmed with a repeated model). to estimate the impact of the recommendations, the analysis was restricted to patients with at least two visits before and after and a second model was adjusted with a new intercept in to estimate the change in slope as of this point.results: a total of patients were included in the cohort. the average number of visits per patient rose from . in to . in (p< . ). the proportion of patients with at least visits per year increased from % to %. the negative binomial regression for the patients having had at least years follow-up confirmed this trend with an average slope of + . (sd= . ). a total of patients were evaluable for the change in slope. no significant change in trend was observed in : only % of patients had a higher rate of growth (this change was significant for only subjects). at the last follow-up visit, patients with increasing rates of number of visits were older ( yrs vs. yrs, p< . ), had lower %fev ( vs. , p= . ), had a similar average number of visits before ( . vs. . , p= . ), and a similar weight-for-age z-score (- . vs. - . , p= . ) .conclusion: the number of visits per patient is regularly increasing. since the publication of the practice recommendations in , the growth has tended to slow down: clinicians were already convinced by the need for closer follow-up and had begun to increase the rate of visits. methods: patients > years who presented for routine healthcare voluntarily completed four surveys: ) the revised eating attitudes test (eat), validated for cf patients by abbott and colleagues, where higher scores reflect worse attitudes towards eating; ) the rosenberg self-esteem scale (rse), where higher scores reflect better self-esteem; ) the body image scale (bis), developed for cf patients by wenninger and colleagues, where higher scores reflect better body image; ) the cystic fibrosis questionnaire (cfq), developed by quittner and colleagues, where higher scores reflect better hrqol. also, fev %, body mass index (bmi), and pancreatic sufficiency or insufficiency (based on the need for pancreatic enzymes) was recorded. regression analyses controlling for age, gender and bmi were used to examine the associations between the surveys. results: this study included patients with % males and a mean age of years. the eat was negatively associated with the rse (p= . , adjusted r = . ), bis (p= . , adjusted r = . ) and cfq (p= . , adjusted r = . ). the rse was positively associated with the cfq (p= . , adjusted r = . ). the bis was positively associated with the rse (p= . , adjusted r = . ) and cfq (p= . , adjusted r = . ). neither bmi nor pancreatic function was associated with the surveys (p=ns). fev % was positively associated with the cfq (p= . , adjusted r = . ) but was not associated with the other surveys.conclusions: more negative attitudes towards eating predict worse selfesteem and body image, while more positive body image predicts better self-esteem. also, attitudes towards eating, self-esteem and body image are significant predictors of hrqol with body image being the most important predictor.clinical importance: hrqol is an important clinical outcome measure in cf. clinicians need to be sensitive to attitudes towards eating, self-esteem and body image in cf patients, because they are important predictors of hrqol. riekert, k.a. ; mogayzel, p.j. ; bilderback, a. ; hale, w. ; boyle, m.p. . medicine, johns hopkins university, baltimore, md, usa; . pediatrics, johns hopkins university, baltimore, md, usa background: existing research suggests that self-reported adherence to all aspects of the regimen is likely suboptimal and objective measures suggest even poorer adherence. there is little empirical data on how much adherence is necessary to achieve desired health outcomes. moreover, there is limited data on how adherence changes dur-objective: cf may be associated with pain attributed to several etiologies. this study evaluates the prevalence of pain symptoms in adult cf patients and the influence on patients` lives.methods: patients of adult cf centers in germany completed a validated, self report questionnaire during a routine clinic visit assessing characteristics of chronic pain (prevalence, duration, location, quality and intensity of pain symptoms). furthermore, the impact of pain on different aspects of life was explored using a numeric scale from to with being the worst. every-day-life was divided into categories: duties at home, recreation, social activities, occupation, sexual life, autonomy, vital activities and cf-therapy. the average intensity of pain within the last weeks was correlated to fev , bmi and age.results: patients ( male) completed the questionnaire. the age was - years (mean . ± . ). patients ( %) aged - years (mean . ± . )reported pain within the last months. bmi was . - . kg/m (mean . ± . ), fev - % (mean . ± . ). if asked for pain within the last weeks ( %) reported painful episodes lasting from - days with a mean of . days (± . ). most patients described pain occurring at more than one site with the head being the most localized site, followed by chest and abdomen. concerning the quality of pain . % characterized their pain episodes as attacks whereas . % reported them as continious, . % had continious pain combined with pain attacks. patients desribed the intensity of pain as . (± . ) on a scale from to with being the most severe pain. the category of life negatively influenced most of all by pain episodes was recreation, followed by occupation and duties at home. female patients were more limited in their acitivites by pain symptoms than male patients with the highest difference being reported in sexual life. there was no correlation of the average intensity of pain within the last weeks to fev , bmi or age.conclusion: the prelevance of pain in cf is often underestimated. painful episodes can be the cause of worsening the quality of life for adult cf patients. assessment of pain should be routinely performed as part of care in cf centers. objective. sleep has been examined in a number of pediatric conditions, with impaired sleep resulting in worse neurobehavioral functioning (beebe, ) . recent research has shown that slow wave sleep is critical for memory (marshall, helgadottir, molle, & born, ) . to the authors' knowledge, no studies have examined slow wave and rapid-eye-movement (rem) sleep in pediatric patients with cystic fibrosis. methods. retrospective medical record review revealed two patients who underwent clinically indicated polysomnography (psg) who reported feeling tired and lacking energy. patients were females with cystic fibrosis, ages and years. results. psg revealed an increase in stage sleep for both patients, resulting in increased risk and observed hypopneic episodes. patient -a experienced . % in stage , . % in stage , and % in stages and combined. this patient also had no rem sleep. patient -b experienced % in stage , . % in stage , and % in delta wave sleep, with no rem sleep. respiratory disturbance index (rdi) for patients a and b were calculated at . and . , respectively. respiratory effort related arousal (rera) contributed to rdi by . and . points in patients a and b, respectively. in addition, endtidal co for patient a and b were maxed at mm hg and mmhg, respectively. their epworth sleepiness scales were scored at and for patients a and b, respectively. conclusions. structure of sleep was abnormal in both patients with decreased slow wave (delta) sleep in patient b and lack of delta sleep in patient a. neither patient experienced rem sleep. compensatory increased stage (nrem) sleep could cause respiratory related abnormalities, such as hypopnea and apnea in patients with cystic fibrosis background: understanding and advancing the application of tools to measure patients' symptoms is critical to advancing our evaluation of potential new therapies used to treat cf. we performed initial evaluation of the measurement properties of a cf-specific respiratory symptom daily questionnaire. methods: we planned to enroll cf subjects, stratified by age, at three cf programs, the university of washington medical center and children's hospital and regional medical center in seattle and mary bridge children's hospital in tacoma in a prospective assessment of a novel cf specific respiratory questionnaire. cf subjects years and older were eligible for enrollment. patients (parents for children under the age of ) completed a daily symptom questionnaire during two -day periods of clinical stability and one -day period when patients were ill for a total of days. the ill state began when patients/parents sought medical attention for respiratory symptoms. the questionnaires were completed using a secure web-based application or via paper for those patients without access to the internet. two health related quality of life measures (generic and cf specific) were completed by the subjects at the end of each -day period to assess the relationship between the novel questionnaire and health related quality of life. patients also used pedometers during the well and ill states to assess relationship between symptoms and activity level.results: cf subjects have been enrolled to date. at the time of this interim analysis, patients had completed questionnaires. of this total, only individual questionnaire entries have been missed in patients (< % of possible questionnaires). interim review of cross-sectional data suggests clear differences in symptom reporting between the ill and well periods. examples include % ( / ) noted difficulty breathing and % ( / ) noted tightness in the chest in the preceding hours during the initial well period compared to % ( / ) noting difficulty breathing and % ( / ) noting tightness in the chest during the ill period.conclusions: interim evaluation of this novel instrument demonstrates feasibility of deploying this instrument via the internet with an extremely high completion rate. using a cross-sectional analysis, the instrument can discriminate between the ill and well state. further data will be presented regarding within patient variability and discrimination of the instrument.supported by the cystic fibrosis foundation leroy matthews physician scientist award, the national heart, lung, and blood institute (hl - ), national institute of health (rr- - ), cystic fibrosis foundation therapeutics inc. background: improved communication in today's health care delivery system is a critically important aspect of patient care. nurses are in an effective position to improve communication due to their close interaction with patients. as a result, they are able to identify potential problems, communicate them to the healthcare team and improve patient care and satisfaction.hypothesis: improving communication between the patient/family and the health care team will improve patient/family satisfaction.method: as a member hospital partnering with the institute for healthcare improvement's (ihi) initiative, transforming care at the bedside (tcab), the children's hospital of philadelphia (chop) introduced the use of daily patient goal sheets (dpgs) as a vehicle to improve communication between members of the healthcare team, patients and families and the dpgs was refined to meet the needs of an inpatient medical unit to which cf patients are admitted. using rapid plan-do-study-act (pdsa) cycles, a multidisciplinary team engaged in small tests of change in different stages. initially, patient goals and discharge criteria generated in daily rounds were posted in each room on easel paper. then large "whiteboards" were placed in each patient room for documenting patient goals, discharge criteria, names of the care team members and questions from patients and families. as a final step in the process, we shifted the health care team rounds to the patient's bedside and implemented bedside shift to shift nurse report and safety checks. patient satisfaction was continuously monitored over this process using the press ganey survey.results: over an eight month period, the press ganey survey results for the unit showed an average increase of %. we attribute this increase to better information sharing among patient, family and hospital staff regarding the plan of care for the patient's hospitalization. the health care team also had an improved dialog with the patients/families. conclusion: patient/family and healthcare team communication and satisfaction were improved using an organized and stepwise pdsa process to develop daily patient goal sheets on a medical unit. background: the uab/chs pediatric cf center provides care for approximately patients. the pulmonary division has full-time rns and part-time rns who rotate phone triage for all calls (including cf) of patients seen by the faculty physicians. historically, patient calls received during office hours are taken by the receptionist and put into the emr, then returned by the rn on call. the rn contacts one of the staff md's, gives report of the patient's condition,treatment orders are received, and the patient notified. there are several issues with this phone triage system, which include inconsistencies in rn and md response as well as differences in documentation. global aim: to improve the consistency among rns in phone triage of sick cf patients.method: an algorithm for pulmonary exacerbation phone triage was developed by the cf center director and lead rns to improve consistency in symptom assessment and treatment plan. a specific list of systemic and pulmonary signs and symptoms was formed including length of illness. if three objective: patients with cf often require intravenous antibiotics for treatment of pulmonary exacerbations. patients often receive peripherally inserted central catheter (picc) lines or totally implantable venous access devices (tivads) for venous access. few studies have examined complications of tivad implantation and little published data exists concerning picc line complications in cf patients. this study sought to assess the complication rates of both tivad and picc lines as well as to identify possible risk factors for developing complications.methods: this retrospective study included patients from cf centers in northern new england. data was obtained from each patient's local medical record, port cf, and patient interviews. demographic data was recorded for all cf patients between / / - / / . for each tivad or picc line, the following information was recorded: type of line placed, history of prior line placement during the study period, patient age, history of use for blood draws, method of line flushing, and status of the line at the end of the study (if still in use). complications were defined as catheter occlusion, vascular thrombosis/stenosis, infection, or other local inflammatory reactions.results: data was collected for pediatric and adult cf patients during the defined study period. seventy-three tivads and picc lines were placed during the study period in patients. the tivad and picc line complication rates were % ( / ) and . % ( / ), respectively. in pediatric patients, % ( of ) tivads and . % ( / ) picc lines had a complication. of the tivad complications, three were systemic infections and three were catheter occlusions. of the picc line complications, there were two venous thromboses, one line occlusion, two systemic infections, and nine minor incidents of localized phlebitis. in adults, complications were recorded in % ( / ) of tivads and . % ( / ) of picc lines. of the tivad complications, there were five systemic infections, seven catheter occlusions, and four venous thromboses. of the picc line complications, there were three venous thromboses, three line occlusions, and four minor incidents of localized phlebitis. all adult patients who developed a deep venous thrombosis(dvt) associated with tivad implantation were homozygous for the deltaf mutation. the presence of diabetes or burkholderia cepacia complex(bcc) lung infection was associated with dvt with odds ratios of . ( % ci . - . ) and . ( %ci . - . ), respectively. conclusions: complications of picc lines were uncommon and usually minor. the rate of tivad complication observed was more common over the lifetime of the catheter and was similar to previously published reports. we identified potential risk factors for the development of dvt associated with tivads and picc lines, specifically cf related diabetes, bcc infection, and homozygous deltaf genotype. the mechanism by which these factors are associated with catheter complications is unclear and warrants further investigation. key: cord- -uj fe y authors: nan title: scientific abstracts date: - - journal: reprod sci doi: . / sha: doc_id: cord_uid: uj fe y nan by reduced placental oxygenation, hypoxia-induced oxidative stress is a predominant mechanism. we investigated the effect of hypoxic pregnancy, with and without antioxidant treatment, on placental and maternal circulatory indices of oxidative stress in rats. methods: on pregnancy day , wistar rats were randomised into: normoxia ( % o litters), hypoxia ( % o litters) and hypoxia + vit c ( % o + mg. ml - vit c in water, litters). on day , dams were anaesthetised, maternal blood taken, pups measured and weighed, placentae weighed and frozen. only placentae from two male pups from any one litter were investigated. blood was processed for ascorbate, urate, l-cysteine and glutathione (gsh) measurement. placental protein was analysed for heat shock protein (hsp ; western). results: hypoxia + vit c did not affect maternal food or water intake. vit c elevated maternal ascorbate by % of baseline; a similar increment to human trials (poston et al. ) . hypoxia elevated placental hsp and maternal plasma urate and l-cysteine, but decreased gsh. vit c in hypoxic pregnancies prevented all stimulated effects, but the reduction in gsh persisted. hypoxic pups had a reduced ponderal index and elevated head diameter: body weight ratio; effects also prevented by vit c. fetal oxygen uptake in normal and gdm pregnancies. emanuela taricco, tatjana radaelli, veronica cozzi, gabriele rossi, danila puglia, giorgio pardi, irene cetin. department of obstetrics gynecology "l. mangiagalli", irccs policlinico, mangiagalli e regina elena, milan, italy. background. diabetes in pregnancy has been associated with alterations of fetal growth probably due to increased nutrient availability and placental transport. fetal hypoxia and acidemia have been reported in pregestational diabetic pregnancies with poor glicemic control but this is still uncertain in well controlled patients. the role of placental function and the relationship between maternal and fetal circulation is crucial for efficient exchanges of oxygen and nutrients. since umbilical blood flow can be obtained by us in utero, we studied normal and gdm pregnancies in order to evaluate fetal oxygen uptake. methods. normal (n) and gdm pregnancies were studied at term, at the time of elective caesarean section. umbilical vein volume flow (qumb) was measured by us before caesarean section and blood samples from umbilical vein (uv) and artery (ua) were obtained. blood gases and acid-base balance were evaluated. results. average fetal weights were similar in both groups (n= ± ; gdm= ± g) while placental weights were significantly different (n= ± ; gdm= ± g). n and gdm pregnancies showed similar values of qumb and qumb/kg of fetal weight. (qumb: . ± . in n and . ± . ml/min in gdm; qumb/kg: . ± . in n and . ± . ml/min/kg in gdm). in fetuses from gdm pregnancies a significant reduction in o sat, o cont and po and a significant increased lactate conc was found in both uv and ua compared to n (table ) . o umb uptake (n= . ± . ; gdm= . ± . mmo/l/min) and o umb uptake/kg (n= . ± . ; gdm= . ± . mmo/l/ min/kg) were significantly lower in gdm compared to n fetuses. conclusions. our data indicate that fetuses from gdm pregnancies show a significant reduction in oxygen supply despite a normal blood flow/kg of fetal weight. these data may suggest that a good maternal metabolic control is not sufficient to ensure normal placental oxygen supply and/or utilization by the gdm fetus. background: synthetic glucocorticoids (sgc) are given to mothers at risk of preterm delivery to promote fetal lung maturation. evidence is emerging indicating long-term effects of such treatment on endocrine function and behavior in offspring. however, virtually nothing is known concerning potential transgenerational influences on growth, endocrine function and behaviour. we hypothesize that repeated treatment of grandmothers (f ) with sgc will alter hypothalamic-pituitary-adrenal (hpa) function in f offspring with no manipulation of the f pregnancy. methods: pregnant guinea pigs (f ) were subcutaneously injected with betamethasone (beta; mg/kg) or vehicle (veh) on gestational days / , / / . adult f female offspring from each group were mated with control males. hpa function was assessed in adult f offspring by non-invasive measurement of salivary cortisol concentrations: ) under basal conditions, ) during and following exposure to psychological stress (high frequency strobe light) or psychological/physical stress (forced swim) and, ) following dexamethasone suppression. results: there was no effect of beta (f ) on bodyweight from birth to adulthood in f offspring. basal salivary cortisol in the betaf females was lower than in the vehf group in the morning but not the afternoon; there were no differences in male f offspring. in contrast, both male and female betaf failed to mount adrenocortical responses to psychological stress compared to vehf offspring that mounted robust responses (p< . ). swim stress induced robust adrenocortical responses in all groups (p< . ), however, the response was consistently lower in betaf offspring. beta exposure also led to a significant difference (p< . ) in the cortisol response to dexamethasone suppression in female (f ) offspring, with a similar trend in males. conclusion: prenatal exposure to sgc (f ) causes transgenerational programming of adrenocortical function in adult f offspring. grand maternal exposure to beta results reduced basal adrenocortical activity in betaf female offspring, and caused stress hypo-responsiveness in both males and females. dexamethasone suppression tests indicate altered central glucocorticoid feedback. these findings have important ramifications for the management of human preterm labor. support: canadian institutes of health research. in the uterine and umbilical vasculatures, we hypothesized that their remodeling would also be blunted in pregnant enos -/-mice, leading to an elevated vascular resistance and decreased blood flow to the placenta contributing to fetal growth restriction. methods: utero-and umbilical-placental blood velocity waveforms and umbilical arterial diameters were measured using mhz ultrasound biomicroscopy in control (c bl/ j) and enos -/-mice at . days of pregnancy (n= mothers). spiral artery and fetal capillary morphologies, and uterine arterial diameters were evaluated from vascular corrosion casts. tissues were collected for hydroxyprobe- and actin immunohistochemistry to identify hypoxic and smooth muscle regions. we calculated resistance index ((s-d)/s) from systolic (s) and diastolic (d) velocities, and blood flow from mean velocity and vessel area. results: calculated uterine blood flow normalized to the weight of the uterus and its contents was % lower (p< . ) in pregnant enos -/-mothers due to large reductions in uterine artery diameter (- %, p< . ) and mean velocity (- %, p< . ). uterine arterial resistance index was % higher (p< . ), and the spiral arteries were less coiled and contained more smooth muscle actin in enos -/-mice than controls. more intense hypoxic immunoreactivity was detected in the spongiotrophoblast and trophoblast giant cell layers of the junctional zone of enos -/-placentas, whereas fainter staining was only detected in the spongiotrophoblast cell layer in controls. in the umbilical circulation, flow normalized to fetal weight was not significantly changed although the resistance index was slightly elevated ( %, p< . ) and capillary lobule length was reduced by (- %, p< . ). fetal organs showed increased hypoxic immunoreactivity suggesting reduced organ oxygen delivery in enos -/-fetuses. conclusions: results suggest that enos plays an important role in uterine and spiral artery remodeling and in augmenting utero-placental blood flow during pregnancy. it also appears to enhance oxygen delivery to the placenta and fetus. enos may contribute to normal fetal growth by these mechanisms. integrin methods: hpmcs isolated from term placentas were assessed for their phenotype markers, mutilineage capacity, and the expression of integrin molecules. the hpmcs were induced to endothelial cell differentiation in the presence of endothelial cell growth medium with % of fcs and ng/ml vegf for to days. the angiogenesis ability of these cells was demonstrated by using an in vitro angiogenesis kit and in vivo chick chorioallantoic membrane assay from ten-day-old embryos. blocking antibodies specific to integrin , associated with gdm. this study was adequately powered to detect association in the caucasian and asian group. the absence of association suggests that gdm and t dm may have more divergent molecular pathophysiology than previously suspected. background: uterine endometrium has a unique cycle of physiological angiogenesis. in mice, endothelial progenitor cells (epc) contribute to endometrial angiogenesis being incorporated after oestradiol administration. objective: to determine whether circulating © epc number and function vary through the menstrual cycle in response to changes in circulating sex steroid concentrations. methods: ten healthy, nulliparous, pre-menopausal, non-smoking women (mean age years) with regular menses ( - days) were studied. venous blood was collected during menstrual, follicular, periovulatory and luteal phases of a single cycle (days - , - , - , - ) . cepcs, serum oestradiol (e) and progesterone (p) were measured at each phase. cepcs were quantified by phenotype using flow cytometry (leukocytes co-expressing cd , kdr and cd ) and function by the epc-colony forming unit (cfu) assay. epc-cfus were stained for endothelial markers including uptake of acetylated low-density lipoprotein, binding of lectin (ulex europaeus) and endoglin. results: luteal p was > nmol/l in all women. cepc numbers increased in the menstrual and follicular phases being -fold higher in the follicular compared to periovulatory phase (p< . ). there was no significant variation in cepc function over the menstrual cycle. there was no correlation between serum e or p levels and cepc number or function. conclusion: cepcs number but not function (epc-cfu assay) vary during the menstrual cycle with numbers increasing during the menstrual and follicular phase and falling in the periovulatory and luteal phase of the menstrual cycle. this may represent mobilisation of cepcs from the bone marrow and subsequent incorporation into the endometrium. neither function nor cepc number correlate with serum p or e. our previous studies demonstrated that tissue factor (tf), which binds factor vii to act as a potent pro-coagulant and angiogenic factor, is over-expressed in endometriotic lesions. thus, we determined whether tf could serve as a target for the elimination of pre-established ectopic human endometrial growth in a mouse model. icon is composed of a mutated factor vii (fvii) domain targeting tf and an igg fc (fvii/igg fc) effector domain that activates antibody-dependent immune responses against tf bearing endothelial cells. methods: athymic, ovariectomized and estrogen-treated mice received intraperitoneal (i.p.) injections of . mg of proliferative phase human endometrial tissue derived from normal (disease-free) women. twelve days after inoculation to establish lesions, icon protein ( ug) was delivered i.p. once a week for weeks. after sacrifice, animals were subject to gross inspection. residual endometriotic tissue was formalin fixed, paraffin embedded and immunostained for von willebrand's factor (vwf) and tf. results: compared to control mice, treatment with ug of icon abolished all lesions in of mice and reduced both size ( . to . mm) and number of lesions ( . to per diseased mouse) in the remaining mice. moreover, residual lesions from icon treated mice were atrophic and displayed significant reductions in vessel areas of % +/- % (p= . , mean +/-sem, n= ) as determined by vwf immunostaining. no hemorrhagic or thrombotic sequelae were observed in icon treated mice. conclusions: unlike other treatments that target developing angiogenesis, icon can target both developing and established human endometriotic lesions in athymic mice. the gross and microscopic vessel analysis suggests that icon directly or indirectly destroys endometriotic vessels. thus, icon presents a novel, non-toxic therapy for endometriosis. compared to the miscarriage and top groups. ihc for crisp demonstrated increased secretion of crisp in the glandular epithelium and expression in the leucocytes of the tubal uterine decidua. conclusions: there are differences in decidual gene expression in tubal compared to iu pregnancies. we believe that potential biomarkers of tubal pregnancy can be discovered by focusing on secreted proteins associated with uterine decidualization. one of these proteins, crisp , is significantly increased in decidua of tubal ectopic pregnancies and we are currently investigating its expression pattern in sera from women with tubal compared to iu pregnancies. progesterone and hoxa regulate gaba-a pi receptor expression, membrane translocation and activation. homayoun sadeghi, hugh s taylor. obstetrics, gynecology and reproductive sciences, yale school of medicine, new haven, ct, usa. objective the expression of the gaba-a pi receptor has been previously described in the human endometrium in both luminal epithelium and stroma. it is upregulated during stromal decidualization in the rat and in the implantation window of human endometrium. the gaba receptor is modulated by progesterone metabolites, with the resultant opening of the receptor ion channels which allow the water flux necessary for trophoblast attachment. here we identified regulators of pi subunit receptor gene expression and activity. the well-differentiated human endometrial adenocarcinoma cell line (ishikawa) and human endometrial stromal cells (hesc) were transfected with hoxa , treated with progesterone, or treated with vehicle or empty plasmid controls. gaba-a pi receptor mrna upregulation was evaluated by real time rt-pcr. protein expression was evaluated using immunohistochemistry. results gaba-a pi receptor mrna expression was increased with either progesterone treatment ( %, p= . ) or hoxa transfection ( %, p= . ). coadministration of progesterone along with increased hoxa transfection had no additive effect on the expression of gaba-a pi receptor mrna (p= . ). gaba-a pi receptor protein expression was similarly increased by each treatment. either hoxa or progesterone independently caused translocation of the gaba receptor from the cytoplasm to the cell membrane in ishikawa cells. conclusion gaba-a pi receptor expression is increased in the human luminal epithelium and stroma in the window of implantation. activation of the pi subunit leads to opening of ion channels, likely allowing flux of water into the epithelial cells and out of the uterine lumen. progesterone and hoxa each increase both pi subunit receptor expression and membrane translocation. the lack of additive effect suggests progesterone induced pi subunit receptor expression is likely mediated indirectly through progesterone's regulation of hoxa expression. finally, after receptor expression and translocation, progesterone mediated gaba receptor ion channel activation mediates water resorption necessary for implantation. cancer. suzy davies, donghai dai, kimberly k leslie. obstetrics and gynecology, university of new mexico, albuquerque, nm, usa. objectives: endometrial cancer is the most frequent gynecologic cancer in women. it affects an estimated , women in the us every year, and long term outcomes for patients with advanced stage or recurrent disease are poor. targeted molecular therapy against the vascular endothelial growth factor (vegf) and its receptors constitute a new therapeutic option for patients that is now under study by the gynecologic oncology group in a phase trial, gog e (now in second stage accrual). the goal of our work was to assess the potential effectiveness of vegf/vegfr blockade in preclinical endometrial cancer models. methods: these studies employed two agents, bevacizumab (avastin, a vegfa blocking antibody) and vandetanib (zactima, a tyrosine kinase inhibitor of egfr and vegfr ). ic experiments were performed on endometrial cancer cells in culture using four established cell line models. xenografted athymic mice were also employed to test the ability of compounds to inhibit tumor growth in vivo. tumors were isolated from controls and treated animals, mrna was extracted, and affymetrix gene expression arrays were performed to determine the genes consistently modulated by treatment. results: compared to vandetanib with an ic of . m, bevacizumab showed little activity on cell proliferation in vitro, and cell numbers were not reduced by % using concentrations up to . m. however, bevacizumab demonstrated robust activity in the athymic mouse model, resulting in a significant decrease in tumor formation and growth compared to vehicle treated animals when dosed bi-weekly in a concentration of . mg/mouse ip. tumors from this model demonstrated that eighteen genes were consistently up or down regulated in the presence of bevacizumab. among the regulated transcripts was microrna , which was significantly down-regulated. microrna is an anti-apoptotic factor, and its inhibition by bevacizumab predicts for increased expression of the tumor suppressor pten, decreased cell proliferation, and a reduced capacity for metastasis. conclusions: these studies confirm that the vegf pathway is a good target for new therapies against endometrial cancer. blocking this pathway not only inhibits angiogenesis, but also results in changes in gene expression that enhance apoptosis and reduce cellular proliferation and tumor invasion. we collected fibroid and adjacent normal tissues following hysterectomy with patient consent and institutional irbs. to date, data points for each gene from arrays have been collected from patients. the fluorescence readings were log-transformed and normalized with the robust quartile normalization method. quality control of the normalized data was performed to remove arrays that deviated from twice the inter-quartile range calculated from the array signals. results: the custom microarray profiling identified genes that were differentially expressed between normal and fibroid tissues (p < . ; fdr< . ). among them, genes encode receptor tks, genes encode tk ligands, and genes encode cell cycle and apoptosis proteins. clustering analysis of the mean log ratios of these genes has led to the division of the patients into two major groups. thirty four ( %) belong to a group characterized by the significant downregulation of the cyr (ccn ) gene in fibroid tissues. the cyr -down group can be further divided into two sub-groups based on the expression of another tk ligand, efn a. other receptor differentially expressed tk did not segregate with the three defined sub-groups. finally, there was no sub-group segregation based on age of the patient, menstrual phase, or weight of the fibroid. conclusion: our results have demonstrated that tyrosine kinases and their ligands are uniquely differentially expressed between normal and fibroid tissues. unique tyrosine kinase ligands in our population were cyr and efn a, and these markers created three molecular classification groups based on their differential expression. these results support the hypothesis that tyrosine kinase ligands are involved in fibroid growth, and may offer targets for a strategy to avoid hysterectomy. microvascular perfusion sonographic imaging to detect early stage ovarian cancer. joanie mayer hope, arthur c fleischer, brian day, stephanie v blank, bhavana pothuri, robert wallach, john p curtin, david a fishman. gynecologic oncology, new york university school of medicine, new york, ny, usa; radiology, vanderbilt university medical center, nashville, tn, usa. objective: epithelial ovarian cancer (eoc) is the th leading cause of death in us women due to the inability to detect early stage disease. recent sonographic developments involving harmonics, pulse inversion, and the use of contrast agents justify the hope that depiction of aberrant tumor microvascularity associated with early disease can occur. this study utilizes these new techniques to assess the unique microvascularity associated with early stage eoc. methods: we used pulse inversion harmonic microvascular imaging (mvi) technology to depict differences between benign and malignant ovarian lesions. contrast enhanced harmonic transvaginal (tv) sonography was performed using the philips iu scanner after intravenous injection of g of definity (bristol-myer-squibb). split screen real-time images were acquired displaying conventional sonographic views adjacent to harmonic, low mechanical index images. morphologic features (thickened wall, papillary excrescence, calcifications) and aberrant vascularity were noted. q-lab quantification of wash-in, peak enhancement, and wash-out times as well as area-under-thecurve (corresponding to microvessel perfusion) were compared using student's t-tests. results: to date, contrast enhancement patterns of ovaries have been analyzed, benign and malignant. of the malignancies ( fallopian tube, ovarian: stage i, stage iii), / women were correctly identified using conventional tv imaging and others were identified using mvi / . all benign lesions were correctly identified by mvi / while tv detected normals ( false negatives). the lesions detected as malignant by mvi were -stage i fallopian tube and -stage i eoc. contrast enhancement kinetics of malignant lesions demonstrated similar wash-in ( . ± . vs . ± . , p = . ), greater peak enhancement ( . ± . vs . ± . , p < . ), longer wash-out ( . ± . vs . ± . (p < . ), and greater perfusion ( . ± . vs . ± , p < . ) when compared to benign lesions. conclusion: contrast enhancement patterns are significantly different in benign vs. malignant ovarian masses. this technique has clear potential in differentiating benign from malignant lesions and for detecting occult stage i disease. identification and characterization of mir- a as regulator of ikk expression and its function in ovarian cancer cells. rui chen, ayesha b alvero, thomas rutherford, gil mor. department of obstetrics and gynecology, yale university school of medicine, new haven, ct, usa. introduction: the proinflammatory environment associated with tumor growth and chemoresistance is produced by both immune cells and cancer cells. the nf-b pathway plays a critical role mediating the capacity of cancer cells to produce pro-inflammatory cytokines. recently, we described a group of epithelial ovarian cancer (eoc) cells characterized by ikk expression as the main factor promoting nf-b activation and cytokine production. in this study we evaluated the regulation of ikk in eoc cells. we describe the identification and characterization of mir- a as a regulator of ikk expression, thus indirectly of nf-b activity and function. materials and methods: human eoc cell lines were established from malignant ovarian cancer ascites. protein expression were determined by western blotting. ikk mrna was measured by rt-pcr. cytokines were profiled by the luminex system. ikk transfection was done with roche fugene transfection reagent. mirna microarray was done with invitrogen ncode multi-species mirna microarray kit. mir- a qrt-pcr was performed with invitrogen ncode sybr greener mirna qrt-pcr analysis kit. mirna transfection was done with ambion siport neofx agent. the ikk '-utr luciferase reporter plasmid was established based on ambion pmir-report mirna expression reporter vector. results: ikk expression was associated with nf-b cyclic activity and the ability of type i eoc cells (but not type ii) to produce inflammatory cytokines. transfection of ikk into type ii eoc cells reversed their phenotype. mirna microarray identified mirnas differentially expressed in type i versus type ii cells, one of which, mir- a, had putative binding sites in the '-utr of ikk mrna. mir- a introduction into type i cells inhibited ikk expression, and direct inhibition through ikk 's '-utr was confirmed by luciferase assay. conclusion: we describe for the first time the identification of ikk as a potential key switch between chemo-resistant and chemo-sensitive phenotypes, by regulating nf-b activity in eoc cells. furthermore, we identified mir- a as a direct regulator of ikk expression. ikk expression may represent an adaptational stage in tumor progression allowing cancer cells to create their own inflammatory environment. these findings may provide novel molecular targets and potential markers for individual therapy selection. regulation of cd in the rat uterus by nitric oxide and the involvement of p kinase pathway. uma yallampalli, rebakah elkins, pawel goluszko, chandra yallampalli. obstetrics gynecology, university of texas medical branch, galveston, tx, usa. objective. cd is expressed in many cell types including uterine cells and has been shown to play an important role in protecting against compliment attack. we have previously shown in endometrial cell lines that cd levels were down regulated by nitric oxide (no) . in this study we extend our previous observations to determine if no down regulates cd in rat uterine tissues and assess its mechanisms of action. methods. non pregnant rats ( g; b wt) were bilaterally ovariectomized under ketamine anesthesia. groups of ovariectomized rats were implanted with alzet mini pumps to deliver nitro-l-arginine melthylester (l-name) at or /mg/rat/day in saline or saline alone. uteri were obtained from these rats at or hours after infusion. in another set of experiments uteri were obtained from ovariectomized rats and cut in to small pieces and incubated in vitro with either l-name ( mm), diethylenetriamine-no mm) or worthmanin ( . m) in mem without phenol red for hours. uterine tissues were homogenized in trizol and mrna levels for cd were measured using rt-pcr and expressed as a ratio to s. results. results show that in vivo treatment with l-name to ovariectomized rats caused elevations in uterine cd mrna levels in a time and dose dependent manner with maximal responses seen with mg l-name and at hours. in vitro studies show that deta-no suppressed cd mrna levels in the rat uterus. both l-name and pi kinase inhibitor, worthmanin caused increases in cd levels in these tissues and the effects of worthmanin are reversed by deta-no. these results suggest that cd levels in the rat uterus are down regulated by no and are upregulated when no synthesis is inhibited by l-name. further, pi kinase appears to be involved in cd regulation in the rat uterus and no donor appears to modulate this response. lung. lakeitha r foster, daniel b hardy, carole r mendelson. dept of pediatrics; depts of biochemistry and ob/gyn, ut southwestern medical center, dallas, tx, usa. during % of human pregnancy, the maternal uterus is maintained in a state of almost complete quiescence by elevated circulating levels of progesterone (p ). we previously observed that p acting through the progesterone receptor (pr) serves an anti-inflammatory role and inhibits uterine contractility by antagonizing nuclear factor b activation and cyclooxygenase- (cox- ) expression (hardy et al., ) . our previous findings also suggest that the fetus provides an important signal for the initiation of labor near term through augmented secretion of the major lung surfactant protein, sp-a, into amniotic fluid (condon et al., ) . secreted sp-a, in turn, activates fetal macrophages which migrate to the maternal uterus where they release cytokines and promote an inflammatory response, leading to labor. in the present study, we tested the hypothesis that maternal p also maintains uterine quiescence by inhibiting sp-a production by the fetal lung. age matched icr mice were injected s.c. once daily either with sesame oil (control) or p ( mg/ml) from to days post-coitum (dpc). as expected, treatment with p delayed parturition by - h. this also was associated with a decrease in uterine cox- mrna expression, as compared to the vehicle-injected controls. interestingly, maternal p treatment caused a marked decrease in the levels of immunoreactive sp-a protein secreted by the fetal lungs into in amniotic fluid at dpc. furthermore, sp-a protein and mrna levels were reduced in the fetal lungs of p -injected mothers, as compared to controls. this was associated with an inhibitory effect of p treatment on cox- protein and mrna levels in the fetal lungs. these findings were of interest, since cox- expression is markedly upregulated during differentiation of human fetal lung (hfl) explants in culture (hardy et al., ) and endogenous and exogenous prostaglandins increase sp-a expression in hfl (acarregui et al., ) . collectively, these findings suggest that maternal p treatment prevents increased uterine contractility, in part, by inhibiting inflammatory response pathways within the fetal lung. this, in turn, blocks the developmental induction of sp-a expression and its secretion into amniotic fluid. in this manner, maternal p inhibits an important fetal signal leading to labor. supported by nih p hd ; nih r hl . recent evidence suggests that leukocytes infiltrate uterine tissues at the time of parturition implicating inflammation as a key mechanism of human labor. ccl- is a pro-inflammatory cytokine that may contribute to the development of inflammatory reaction in the myometrium. previously we showed upregulation of rat ccl- gene expression in myometrium during term and ru -induced preterm labor. also ccl- was elevated specifically in the gravid horn of unilaterally pregnant rats suggesting that mechanical strain imposed by the growing fetus controls its expression in the myometrium. the objective of this study was to investigate the role of mechanical stretch as a possible regulator of myometrial leukocyte infiltration and ccl- as a mediator of this stretch response. we also studied the effect of progesterone (p ) on the myometrial secretion of ccl- . methods. we used primary culture of rat myometrium smooth muscle cells (smcs) to study in vitro ccl- gene and protein induction by static mechanical stretch. ccl- gene expression analysis was performed by real-time rt-pcr and immunoreactive (ir) protein content was measured by elisa assay. we used primary rat monocytes to access whether stretch-induced ccl- production by myometrial smcs resulted in enhanced monocyte chemotactic activity. results. myometrial cells were stretched for - hours and the supernatants collected. analysis of media conditioned by primary myometrial smcs revealed that static mechanical stretch ( % elongation for hours) caused a significant accumulation in ir ccl- which was repressed by pretreatment with p ( um). the rise in ccl- protein levels was preceded by a transient increase on ccl- mrna. the migration of primary rat monocytes in response to conditioned medium from stretched myometrial smcs was much greater than that of conditioned medium from control non-stretched cells. co-incubation with a neutralizing antibody to ccl- significantly reduced the chemotaxis of monocytes in response to the stretch-conditioned medium. conclusion: uterine smcs play an active role in uterine inflammation by producing chemokines and promoting the chemotaxis of immune cells into the myometrium. the blockade of this effect by p offers a potential explanation for the therapeutic actions of this hormone in the prevention of preterm birth. membranes during human labor. nardhy gomez-lopez, , guadalupe estrada-gutierrez, lourdes vadillo-perez, felipe vadillo-ortega. direction of research, instituto nacional de perinatologia, mexico city, df, mexico; escuela nacional de ciencias biologicas, ipn, mexico city, df, mexico. introduction. leukocytes arriving to the choriodecidua (chd) during labor are capable to secrete cytokines and matrix metalloproteinases that may play a role in the fetal membranes (fm) extracellular matrix degradation. objective. the aim of this work was to identify changes in the leukocyte subpopulations in the chd and fm during human labor. methods: fm were obtained from two groups of women: ) term without labor (n= ) and ) term with spontaneous labor (n= ). chd cells were isolated and analyzed by flow cytometry. explants of fm were embedded in paraffin and analyzed by confocal microscopy. in both techniques, cd , cd , cd , cd and cd subpopulations of leukocytes were identified. intracellular mmp- was also identified in these cells. results: major changes in leukocytes subpopulations during labor involved a higher amount of cd +, cd+ and cd + cells, both in the chd and inside the fm. mmp- was associated to cd + cells. cd + exhibited a more widespread localization in the fm and cd + cells were localized in the contact with the trophoblast layer during labor. conclusions: leukocyte populations changes both in the chd and fm during labor and are characterized by arrival and infiltration of specific subpopulation of lymphocytes and monocytes. nk cells are enriched in mmp- , which may be related to a role in extracellular matrix degradation leading to the rupture of fetal membranes. women with a history of a pregnancy complicated by preeclampsia or intrauterine growth restriction (iugr) have an increased risk of future cardiovascular disease. excessive weight, particularly abdominal fat mass, is associated with cardiovascular morbidity and mortality. objectives: the aim of this study was to investigate differences in body composition and fat distribution between women with a history of preeclampsia or iugr and uncomplicated pregnancies. methods: from a genetically isolated population in the southwest of the netherlands, non-pregnant women with a history of preeclampsia (n= ), iugr (n= ) and uncomplicated pregnancies (n= ) were recruited at a mean follow up time of . years after pregnancy. body composition and fat distribution were assessed by dual energy-x-ray absorptiometry (dxa) and anthropometric measurements. results: women with a history of preeclampsia compared to controls had higher mean total-, fat-and lean mass (p < . ) as well as higher mean indices of body mass, fat mass and lean mass (p< . ). no significant differences were found for these variables between women with a history of iugr and controls. women with a history of preeclampsia had higher waist circumferences and waistto-hip ratios (p< . ) as well as excess of android fat mass and increased android-to-fat ratios (p< . ). women after pregnancies complicated by iugr had higher waist-to-hip ratios (p < . ). after controlling for body mass index, both women with a history of preeclampsia or iugr had higher waist circumferences (p < . ) and waist-to-hip ratios (p < . ) as well as smaller hip circumferences (p < . ). conclusion: despite differences in body mass index, both women with a history of preeclampsia and women after pregnancies complicated by iugr have a metabolically adverse fat distribution, marked by an excess of fat deposition in the abdominal region relatively to the hip region. these findings may explain, at least partly, their increased cardiovascular risk. women with a history of preeclampsia. marc ea spaanderman, timo h ekhart, robert aardenburg, louis lh peeters. obstetrics and gynecology, radboud university medical center, nijmegen, netherlands; obstetrics and gynecology, university medical center maastricht, maastricht, netherlands. background: a history of preeclampsia is associated with persistent short-term alterations in circulatory function and remote cardiovascular disease. in this study we tested the hypothesis at least years after preeclamptic pregnancy renal and central hemodynamic function is impaired as compared to women with uncomplicated pregnancy. methods: in formerly preeclamptic women (pe) and healthy parous controls (control) who were normotensive at weeks post-partum follow up, we assessed at least years after delivery blood pressure (mmhg), cardiac output (co, doppler ultrasonography, l/min) and effective renal plasma flow (erpf, pah clearance, ml/min/ . m ) after which we calculated total peripheral vascular resistance (. dyne.s/cm ) and renal vascular resistance (. dyne.s/cm ). data were analyzed parametrically (p< . ). results: age and bmi were comparable between groups. blood pressure was higher in formerly pe. moreover, % of formerly pe women and % of control were hypertensive (p< . ). although cardiac out was comparable between groups, total peripheral and renal vascular resistance were about % higher and erpf % lower in formerly pe women as compared to control. conclusion: at least years after gestational hypertensive disease, women who were normotensive at direct follow up have impaired renal and central hemodynamic function and developed more often chronic hypertension. long-term follow up may also be warranted in apparently healthy formerly pe women who are normotensive at post-partum follow up. circulatory function in formerly preeclamptic women and healthy parous controls co erpf tpvr rvr formerly pe . ± . ± * ± * ± * control . ± . ± ± ± * = p< . pregnancy increases blood-brain barrier permeability coefficient (l p ) to lucifer yellow: role of estrogen. marchien j wiegman, , marilyn j cipolla. neurology, ob/gyn and pharmacology, university of vermont, burlington, vt, usa; ob/gyn, university medical center groningen, groningen, netherlands. background: eclampsia is similar to posterior reversible encephalopathy syndrome in which an acute rise in blood pressure causes breakthrough of autoregulation, blood-brain barrier (bbb) disruption, and cerebral edema formation. we previously showed that late-pregnant (lp) animals developed cerebral edema during breakthrough, a response that was absent in nonpregnant (np) animals. in the current study we hypothesized that pregnancy predisposes the brain to edema during acute hypertension by enhanced bbb permeability. we further hypothesized that the underlying effect of pregnancy on the bbb permeability is due to elevated estrogen levels. methods: permeability coefficients (l p ) to lucifer yellow (ly), a polar compound that does not pass through tight junctions, were compared in posterior cerebral arteries (pca) from groups of sprague dawley rats: np (n= ), lp (d ; n= ), ovariectomized and implanted with -estradiol ( . mg, -day release) and estriol ( . mg, -day release) pellets for days (ovx+e; n= ), and ovariectomized and implanted with placebo pellets for days (ovx; n= ). pcas were isolated, pressurized in an arteriograph, and perfused with . mg/ml ly in saline. concentration changes of ly outside the vessel wall were determined at pressures from - mmhg. the slope of the pressure vs. permeability curve is the rate of flux, or l p for ly. results: l p for ly was significantly increased in pcas from lp and ovx animals vs. np (p< . ; figure ). estrogen was protective of the bbb only in ovariectomized animals, decreasing l p % in ovx+e vs. ovx. however, pregnancy did not afford protection and had a l p that was % greater than np. conclusions: pregnancy significantly increases bbb permeability to ly, an effect that may predispose the brain to edema formation during acute hypertension. these data also show that estrogen modulates l p in ovariectomized animals differentially than pregnancy, suggesting that the increased bbb permeability in pregnancy is caused by a mechanism other than elevated estrogen levels. in both pre-and early pregnancy, the sympathoinhibitory response to volume expansion is blunted in formerly preeclamptic women with low plasma volume. ineke krabbendam, marc ea spaanderman, dorette a courtar, robert aardenburg, ben j janssen, fred k lotgering, louis lh peeters. obstetrics and gynecology, radboud university nijmegen medical centre, nijmegen, netherlands; obstetrics and gynecology, university hospital maastricht, maastricht, netherlands; pharmacology and toxicology, university of maastricht, maastricht, netherlands. background: the circulation of formerly preeclamptic women with a low plasma volume (lpv) is characterized by sympathetic dominance. these women respond to a new pregnancy with an aberrant rise in atrial natriuretic peptide (anp) and a times higher chance to develop recurrent gestational hypertensive disease compared to their counterparts with normal plasma volume (npv). anp has sympathicomimetic capacity. we postulate that the sympathetic overdrive in lpv-women is associated with a reduced venous capacitance. to this end, we compared the response to volume expansion (ve) in women with lpv and npv, both before and in pregnancy. method: in non-pregnant normotensive formerly preeclamptic women, we measured pv (hsa i indicator dilution method) at least months post partum. we intravenously infused ml of iso-oncotic fluid over minutes. during the infusion, we recorded changes in heart rate (hr, bpm), blood pressure (bp, mmhg), cardiac output (co, l/min), sympathetic activity (lfsys, mmhg , low frequency component of spontaneous fluctuations in systolic bp, portapress) and anp (nmol/l). eight women became pregnant within year and were evaluated at weeks gestation. changes in circulatory and autonomic function between and within groups were analyzed non-parametrically (p< . ). results: before pregnancy, ve leads to comparable changes in hr, bp and co in women with lpv ( / ) and npv ( / ). in npv, lfsys decreased %, but only % in lpv (p< . ). anp remained unaltered in npv, but increased in lpv. in the pregnant group, women had lpv and had npv. in both groups, pregnancy did not alter the response to ve. conclusion: irrespective of pregnancy, the sympathoinhibitory response to ve is diminished in lpv. these data suggest that in these women ve leads to venous overfill, giving rise to anp-release and consequently sympathetic activation, flattening the normal baroreceptor-mediated sympathoinhibitory response. we speculate that this mechanism contribute to circulatory maladaptation to pregnancy, sympathetic dominance and subsequent gestational hypertensive disease. in both pe and ht, serum sflt- was increased, and plgf reduced at all gestations (p< . ). seng levels were also increased in pe. after weeks (but not before) antihypertensive treatment was associated with a significant fall in serum sflt- and seng, in pe only. the concentrations of both sflt- and seng were significantly higher in the placentas of women with pe, but not ht, compared with controls (p= . ). only sflt- was significantly reduced in the placenta in women who received antihypertensive therapy. conclusion in pe, antihypertensive therapy after weeks' gestation is associated with a significant fall in serum sflt- and seng, and in placental sflt- . these findings raise the possibility that these drugs may have an effect on the pathophysiology of pe other than their known antihypertensive action. the synergistic effect of soluble vegf receptor pre-treatment and small doses of tnf-on endothelial cells. tereza cindrova-davies, debbie a sanders, olivera spasic-boskovic, graham j burton, d stephen charnock-jones. dept of pdn, university of cambridge, united kingdom; dept of obstetrics and gynaecology, university of cambridge, united kingdom. introduction: preeclampsia is marked by an enhanced endothelial inflammatory response manifested by maternal endothelial activation. soluble fms-like tyrosine kinase- (sflt- , svegf-r ), a naturally occurring circulating antagonist of vegf-a and plgf, is one of the secreted factors implicated in the pathogenesis of preeclampsia. in women who develop preeclampsia, sflt rises sharply, preceding the onset of the clinical disease. the aim of this study was to examine the effect of a combined treatment with recombinant sflt- and tnf-on the activation of human umbilical cord endothelial cells (huvec) by examining leukocyte adhesion, and the expression of icam, vcam, endothelin and vwf. methods: huvec were seeded, grown overnight and pre-treated with recombinant sflt ( - ng/ml) in a basic % fbs-dmem medium for hr. on day , low doses of tnf-( . ng/ml) were applied to pre-treated cells for hr. antagonism of the vegf-a action was mimicked at the protein level by pre-incubating huvec with anti-flt antibody ( - g/ml), anti-kdr antibody ( g/ml), anti-vegf antibody ( g/ml), or vegf receptor inhibitor su ( m). at the rna level, the effect of sflt was mimicked by sirna transfection of huvec with siflt or sikdr. at the end of each experiment cells were either harvested for western blotting, fixed in % pfa for immunofluorescence or incubated with labelled hl leukocyte cells, followed by fluorescent detection of adhesion. results: pre-incubation of huvec with sflt and subsequent treatment with low doses of tnf-increased the adhesion of hl leukocytes and increased icam- , vcam- , endothelin and vwf, compared to tnf-treatment alone. similar results were obtained when cells were pre-treated with su , anti-flt, anti-kdr or anti-vegf. transfection knock-down of flt or kdr gene also significantly increased leukocyte adhesion when small doses of tnfwere added. conclusions: pre-incubation with recombinant sflt, anti-flt, anti-kdr, anti-vegf, su or knocking-down flt or kdr transcripts all antagonised the autocrine actions of vegf and/or plgf. this predisposes huvecs to be more sensitive to the effect of tnf-. our study shows that sflt and tnfcombine to induce an enhanced synergistic effect, activating endothelial cells. maternal obesity is associated with increased production of inflammatory cytokines and risk of poor perinatal outcome. inflammatory cytokines can stimulate production of reactive oxygen (ros) and nitrogen (rns) species, which can covalently modify protein function. placental oxidative and nitrative stress are increased in pathological pregnancies and associated with altered placental function. objectives: determine the effect of increasing body mass index (bmi) on placental nitrative stress, measured by the expression and localization of nitrated (nitrotyrosine) and oxidized proteins. methods: placental tissue was collected at term ( - wks) from lean kg/m ), overweight ( - . kg/m ) and obese ( - kg/m ) patients (n= or /group). tissue was sectioned for immunostaining with nitrotyrosine antibody. protein samples were either dot blotted onto nitrocellulose membrane, probed with nitrotyrosine ab, and nitrated proteins detected using ecl, or derivatized using , -dinitrophenylhydrazine (dnph) (oxyblot® kit), separated on sds-page, probed with anti-dnph ab ( : ) and oxidized proteins detected using ecl. protein band intensity was measured by densitometry. oxidized proteins were selected for maldi mass spectrometry analysis. results: nitrotyrosine residues were immunolocalized primarily in the fetal capillary endothelial cells and the villous stroma, but were almost absent in the syncytiotrophoblast. by dot blot, nitrotyrosine expression differed across the three groups (p< . , anova) with expression in tissue from obese women being significantly increased compared to lean (p< . ) and overweight (p< . , tukey test). several oxidized proteins were detected with significantly greater expression seen in the lean versus overweight groups (p< . , mann-whitney u). one oxidized protein was identified by maldi-ms with four peptide matches and . % coverage as -beta hydroxysteroid dehydrogenase ( bhsd). discussion: with increasing bmi, an increase in nitrative stress appears to occur in parallel with a decrease in oxidative stress. oxidative stress is apparently reduced as ros are consumed by the interaction with rns to give nitrative stress. bhsd is involved in the biosynthesis of steroid hormones and glucocorticoids. its activity and hence steroid metabolism in the placenta may be regulated by oxidation with implications for fetal development. background teenagers are susceptible to delivering small-for-gestationalage (sga) infants. previous studies implicate continued maternal growth as a causal factor . growing adolescent sheep have reduced fetal birthweight due to impaired placental development and nutrient transfer . we hypothesized that placental function is impaired in human teenage pregnancy if there is maternal growth. methods placentas were collected from teenagers ( - years) and adults. activity of the amino acid transporter, system a, was quantified by the sodium-dependent uptake of c-methylaminoisobutyric acid into placental fragments. teenagers were defined as growing (> mm increase in kneeheight per days) or non-growing. system a activity was analysed in relation to individualised birthweight centile and maternal growth. results placental system a activity was significantly lower in teenage compared to adult pregnancies (p< . ). this was unrelated to birthweight; teenagers who delivered infants appropriate-for-gestational age (aga) had significantly lower placental system a activity compared to aga infants delivered to adults (p< . ). growing teenagers did not deliver lower birthweight infants than non-growing teenagers (median birthweight g and g respectively). furthermore, system a activity in placentas from growing teenagers was significantly elevated compared to that in non-growing teenagers (p< . ), and was similar to placental activity in adults. conclusions system a activity was reduced in placentas from teenagers compared to adults. this suggests that inherently lower placental function predisposes teenagers to sga, but that other factors (e.g. adequate nutrition) can compensate in those teenagers delivering aga infants. in contrast to our hypothesis, placental system a was elevated in growing teenagers and mimicked that of adults. this may be related to a hormonal-milieu in growing teenagers that is conducive to fetal growth, in part through stimulating placental transport. homocysteine inhibition of system a amino acid transport activity in human placenta. eleni tsitsiou, susan l greenwood, colin p sibley, stephen w d'souza, jocelyn d glazier. maternal and fetal health research group, st. mary's hospital, university of manchester, manchester, united kingdom. background: elevated plasma levels of the amino acid homocysteine (hcy) during pregnancy are associated with vascular-related complications and adverse neonatal outcomes including a reduced birthweight. fetal and maternal plasma hcy concentrations are positively correlated suggesting placental transport of hcy may be an important determinant of fetal plasma hcy. the mechanisms involved in placental hcy transport are uncharacterised. evidence that the system a amino acid transporter, which transports neutral amino acids in a na + -dependent manner, is important in promoting fetal growth and that a reduced system a activity is associated with intrauterine growth restriction (iugr), led to our hypothesis that system a provides one mechanism for placental hcy transport. this hypothesis was tested by measuring the ability of hcy to inhibit system a activity in isolated microvillous plasma membrane (mvm) vesicles and placental fragments. materials and methods: mvm vesicles and placental fragments were isolated from placentas of normal pregnancies at term. system a activity was measured at initial rate ( s and min respectively) as na + -dependent cmethylaminoisobutyric acid (meaib) uptake into mvm vesicles ( . mm) or fragments ( . mm) in the absence (control) or presence of l-hcy and dl-hcy or model substrates. results: mm l-hcy (custom-synthesised) and dl-hcy (commercial source) significantly (p< . ) inhibited na + -dependent c-meaib uptake into mvm vesicles compared to control; comparable in magnitude to other model substrates (meaib, l-ala, l-ser, l-met; n= , kruskal-wallis with dunn's multiple comparison test). l-hcy, l-met and meaib ( . - mm) caused a dose-dependent inhibition of na + -dependent c-meaib uptake into mvm with ec values (in mm; mean ± se) of . ± . , . ± . , and . ± . respectively, n= ). na + -dependent c-meaib uptake into fragments was reduced substantially in the presence of mm l-hcy or dl-hcy causing a ± and ± % reduction (mean ± sd, n= - ) of control respectively. conclusion: these observations suggest that hcy is a relatively high affinity substrate for system a in the human placenta. we speculate that inhibition of placental amino acid uptake by hcy could impact on fetal growth and development. supported by the mrc and action research endowment fund. glucose regulates placental mtor activity and glucose deprivation down-regulates placental system l activity in an mtor-dependent manner. sara roos, theresa l powell, thomas jansson. department of physiology, institute of neuroscience and physiology, gothenburg, sweden; department of obstetrics and gynecology, university of cincinnati, cincinnati, oh, usa. placental amino acid transporters are down-regulated in intrauterine growth restriction (iugr). we have previously shown that mammalian target of rapamycin (mtor) regulates placental system l transporter activity and that placental mtor activity is decreased in iugr. however, the upstream regulators of placental mtor are unknown. in iugr, fetal hypoglycemia and reduced maternal glucose levels are common and the placenta may therefore be exposed to low glucose levels. hypothesis: we hypothesized that glucose availability regulates placental amino acid transporter activity mediated by changes in mtor signaling. methods: cytotrophoblast cells were isolated and cultured until syncytialization at hours. cells were cultured for an additional hours in culture media containing . mm, . mm, or mm glucose (control), which corresponds to standard culture media. at hrs, the activity of the mtor signaling pathway was assessed by measuring the protein expression of s k phosphorylated at thr- , the primary site of mtor phosphorylation. in another set of cells, system l activity was assessed by measuring the bchinhibitable uptake of h-leucine. results: as compared to control, phospho-thr- -s k expression was reduced by % in cells incubated in . mm results: fgr pregnancies were delivered earlier ( ± . v ± . w; p< . ), weighed less ( . ± . v . ± . kg; p< . ) and had higher s/d ratios ( . ± . v . ± . ; p< . ). eaa concentrations were similar between groups, but there were differences (non-parametric testing; p< . ) in transport rates between groups with his crossing considerably faster and ile crossing slower in fgr . the table shows fetal vein/maternal (fv/m) ratios standardized to the leu fv/m ratio for all eaa for both groups. amino acids fell into groups for fgr pregnancies: ) his (ratio . ), ) leu, phe, met (ratios ), and ) val, thr, ile, trp, lys with intermediate ratios ( . ns conclusion: this is the st study to compare the relative rates of in vivo placental transport for all eaa between normal and fgr human pregnancies showing striking differences in the transport rates of two eaa. in the absence of eaa concentration differences between groups, the higher his transport rate in fgr suggests higher utilization by the placenta. vasculature of women who received exogenous estrogen compared to those who received placebo. the purpose of this investigation was to identify the gene expression in response to the estrogen, equilin, as the major component of conjugated equine estrogen and genistein, a phytoestrogen in human coronary artery endothelial cells. human coronary artery endothelial cells from a -year old female were used. cells were treated with estradiol [e ], equilin [eq] ( . nm) or genistein [gen] ( micromolar) for hours. focused oligomicroarrays for cardiovascular disease and endothelial cell function were used to study gene expression. rt-pcr was used to confirm the transcription of genes analyzed in oligoarrays. vascular adhesion molecules and genes involved in the inflammatory response were most affected with the three estrogen sources. significant reduction of integrin alphae was seen with all three . , . and . for e , eq and gen respectively. similarly nfkb was also reduced . , . and . fold compared to controls. significant reduction of ccl was demonstrated with eq ( . fold) and gen ( . fold). tnfreceptor a and b were only significantly decreased by gen. vcam- which is regulated by proatherogenic factors and upregulated mainly at atherosclerosis -prone sites, was significantly reduced ( . -fold) with genistein, and a slight reduction was seen with e and no change was observed with eq. our data support the clinical findings that estrogen has favorable effects on the genes involved in atherosclerotic disease. while e has been shown to be slightly more effective than equilin in the modulation of gene expression, genistein was significantly more effective in the favorable expression of genes involved in particularly the inflammatory response. olga n lekontseva, sandra t davidge. physiology and obstetrics/gynecology, university of alberta, edmonton, ab, canada. introduction: the prevalence of cardiovascular disease in women dramatically rises in the postmenopausal period. although deficiency of estrogen has been implicated in the pathophysiology of systemic vascular dysfunction, the effects of estrogen on vasculature are complex and not completely understood. we have previously shown that estrogen exerts a beneficial effect on the aging vascular system by reducing circulating levels of the inflammatory cytokine tnf . tnf is a known regulator of matrix metalloproteinases (mmps), proteolytic enzymes that may modulate vascular tone through cleavage of vasoactive peptides such as big endothelin- (et- ). the role of estrogen in this pathway is unknown. we tested the hypothesis that in aging/estrogen deficiency, tnf -induced mmp activity mediates greater vasoconstriction, in part, through the et- pathway. we further hypothesized that estrogen replacement reduces vascular sensitivity to the constriction by preventing mmp activation. methods: aged ( month old) female sprague dawley rats were ovariectomized and treated with either placebo [ovx] , -estradiol [ovx+e ], or the tnf inhibitor etanercept [ovx+etan] . after four weeks, resistance mesenteric arteries were isolated and studied on the pressure arteriograph. concentrationresponse to exogenous big in the absence or presence of mmp inhibitor (gm , μm) was assessed in the vessels. results: treatment of "menopausal" [ovx] rats with either estrogen or the tnf inhibitor reduced sensitivity of arteries to big et- (ec = . ± . μm in ovx versus . ± . μm in ovx+e or . ± . μm in ovx+etan groups; p< . ). although mmp inhibition attenuated maximal constriction in all of the arteries, there was a significantly greater (p< . ) role of mmps in big et- -induced vasoconstriction in the ovx+e group (reduction in max constriction= . ± . %) compared to ovx ( . ± . %) and ovx+etan groups ( . ± . %). conclusions: both estrogen and tnf inhibition reduced big et- vasoconstriction. however, contrary to our hypothesis, tnf is not contributing to mmp modulation of et- vasoconstriction. interestingly, our study demonstrates a novel role for estrogen to increase mmp contribution to big et- vasoactivity with the net effect being less vasoconstrictive. understanding this unique pathway of regulation by estrogen in the aged vasculature will allow for development of new therapeutic options for women. mo, usa. introduction: the etiology of pelvic organ prolapse is multifactorial, with both inherited and acquired components. the molecular mechanisms of prolapse have not been established yet. we have previously shown that lysyl oxidase (lox) expression is suppressed in uterosacral ligaments of women with pelvic organ prolapse. it has also been shown that lox is a tumor suppressor gene inactivated by methylation in human gastric cancers. hypothesis: the aim of this study was to analyze the dna sequence of the promoter region of the lysyl oxidase gene in tissues from women with pelvic organ prolapse and identify whether methylation is present. our hypothesis is that the promoters of the lox gene in women with pelvic organ prolapse have significantly more methylation sites than women without prolapse. materials and methods: genomic dna was isolated from the uterosacral ligaments of eight women with pelvic organ prolapse and women without prolapse (controls). genomic dna samples were treated with ez dna methylation kit (zemo research, orange, ca). the lox gene promoter region of - to + was amplified by pcr and then cloned into pcr . -topo (invitrogen, carlsbad, ca) and transformed into an e. coli dh a strain. amplified plasmid dna samples containing the lox gene promoter region from each woman were sequenced and methylated cpg islands were identified by sequence comparison. results: a total of methylated cpg sites were found in the patient group with pelvic organ prolapse while only methylated cpg site was found in the non-prolapse control group. conclusion: these findings suggest that methylation in the promoter region suppresses lox gene expression in women with pelvic organ prolapse. background: reports from case-control and cohort studies have suggested an inverse association between lactation and breast cancer risk, but findings have been inconsistent. methods: we conducted a prospective observational cohort study of , parous women participating in the nurses' health study ii from to . our primary outcome was incident premenopausal breast cancer. results: during the study period, cases of premenopausal breast cancer were diagnosed during , person-years of follow-up. women who had ever breastfed had a % lower incidence of premenopausal breast cancer ( % confidence interval [ci] - %) compared with women who never breastfed, adjusting for parity, age at first birth, year of first birth, height, body mass index (bmi), bmi at age , family history, personal history of benign breast disease, participant birth weight, preterm birth, age at menarche, oral contraceptive use, physical activity, and alcohol consumption. no trend was observed with duration of lactation (p= . ). the association between ever-breastfeeding and premenopausal breast cancer was modified by use of medication to suppress lactation (p= . ); in analyses restricted to women who had never used suppressive medication, ever-breastfeeding was associated with a % ( %ci - %) reduction in incident disease. the association between lactation and premenopausal breast cancer was further modified by family history of breast cancer (p= . ). among women who had never used suppressive medication and reported a family history, those who had breastfed had a % lower covariate-adjusted risk of premenopausal breast cancer ( % ci - %) than women who had never breastfed. among women without a family history, ever-breastfeeding was not associated with breast cancer incidence (hazard ratio . , % ci . - . ). among women who had ever breastfed, we observed no association between breast cancer risk and duration of lactation amenorrhea or exclusive breastfeeding. conclusion: in a large, prospective cohort study, ever-breastfeeding was inversely associated with risk for premenopausal breast cancer. at the durations observed in our cohort, we observed no trend with duration of breastfeeding or lactation amenorrhea. the inverse association with ever-breastfeeding was stronger among women with a family history of breast cancer. regulatory and nkt cells at the maternal-fetal interface. thomas f mcelrath, rachael a clark. brigham women's hospital, boston, ma, usa; harvard skin disease research center, brigham women's hospital, boston, ma, usa. introduction: pregnancy represents an immunologically challenging event requiring maternal tolerance of the fetal semi-allograft. an increase in decidual cd + cd + t cells has been documented but it is unclear if these represent foxp + t cells (tregs) . the possibility also exists that other cd + lineages with potential regulatory function exist within the decidua parientalis. we examined if cd + cd + cells are true foxp + tregs and evaluated the frequency of other t cell subsets with possible regulatory potential. methods: we extracted t cells from term deciduas of planned cesarean deliveries. cells were stained with directly conjugated monoclonal antibodies and were analyzed on a six color flow cytometer. results: we found that only a subset (median %) of cd + t cells were true foxp + regulatory t cells. from donors, foxp + tregs accounted for a median of . % of all cd + cells. these cells were memory cd ro + t cells lacking ccr , and l-selectin but expressing cd , ctla- , gitr, and hla-dr. additionally we found that a median % of cd + t cells expressed the nkt marker cd . these cells were a mixture of cd and cd -cd -t cells, with variable numbers of cd t cells, suggesting they do not represent merely recently activated t cells. there was enrichment for t cells with nkt markers after culture on hela cells expressing cd d, suggesting that these cell represent true nkt cells. nkt cells were of the non-classical type, with a diverse t cell repertoire ( . % iv jq) and also expressed cd , hla class ii, cd ro but were ccr and l-selectin low. comments: we find that only a minority of cd -expressing t cell in the decidua are true foxp + tregs. because much of the work on treg in pregnancy has used cd as a treg marker, this suggests additional studies are needed to confirm the role of these cells in pregnancy. we find that a novel population of non-classical nkt cells exists in the human decidua. nkt cells can either promote or antagonize tolerance, depending on the immunologic context. the large number of these cells in the decidua suggests they may play a role equal or exceeding that of regulatory t cells. prolapse. marsha k guess, kathleen a connell, richard bercik, lloyd g cantley. obstetrics, gynecology reproductive sciences, yale university school of medicine, new haven, ct, usa; internal medicine/neprhology, yale university school of medicine, new haven, ct, usa. objectives: thy- is a cell surface glycoprotien expressed in human fibroblasts, neurons, hematopoetic stems cells and endothelial cells. thy- expression affects fibroblast proliferation and migration, cell-cell, as well as, cell-matrix interactions. moreover, thy- expression has been shown to play a critical role in fibroblast dedifferentiation into myofibroblasts, as well as in extracellular matrix (ecm) production and fibrosis. women with pelvic organ prolapse (pop) have alterations in vaginal ecm protein expression and metabolism, as well as decreases in smooth muscle fractional content. in the current study, we evaluated thy- as well as the smooth muscle markers alpha-smooth muscle actin (asma) and desmin expression in women with pelvic organ prolapse compared to women with normal pelvic support (controls). methods: anterior apical vaginal wall specimens from women with pop and controls were collected at the time of hysterectomy. messenger rna and protein expression of thy- , asma and desmin were evaluated using semiquantitative rt-pcr, real-time pcr and western blot analysis. gapdh and beta actin were used as internal controls. results: rt-pcr demonstrated the presence of thy- , asma and desmin in vaginal tissue from women with pop and controls. further, thy- mrna expression was downregulated % in women with pop compared to controls (p = . ). a parallel decrease in thy- protein was seen in women with pop compared to controls (p= . ). although a % and a % decease were seen in asma and desmin mrna, these differences were not statistically significant. similarly, no differences were seen in asma and desmin protein expression. conclusion: we demonstrate that there is significantly less thy- expression in vaginal tissue from women with pop compared to controls. differential expression of thy- in prolapsed vaginal tissues suggests that thy- may have a functional role in mediating ecm metabolism in the female genitourinary tract. hur expression is altered in ectopic endometrium. s karipcin, t altun, ua kayisli, e seli. ob gyn, yale u., new haven, ct, usa. introduction: cytokines and growth factors contribute to cyclic turnover of the normal endomterium and to the pathogenesis of endometriosis. cytokine and growth factor messenger rnas (mrnas) undergo rapid turnover that is primarily mediated by au-rich elements (are) that consist of multiple stretches of adenylate and uridylate residues located in the ' untranslated region ( '-utr) of their mrnas. hur is a ubiquitously expressed rna-binding protein that stabilizes are containing mrnas and prolongs their expression. we hypothesized that hur may play a role in the regulation of cytokine expression during normal menstrual cycle and in endometriosis. methods: tissue sections obtained from normal (n= ) and ectopic (n= ) endometrium were immunostained for hur. staining intensity was evaluated by hscore and grouped according to menstrual cycle phase. statistical analysis was done with one-way anova. cultured stromal cells isolated from normal endometrium were treated with vehicle, estradiol (e ; - m), or progesterone (p, - m) for and h, and hur expression was determined using western analysis and normalized to ß-actin. results: hur immunostaining was nuclear in endometrial cells. hur immunoreactivity was significantly lower in the early proliferative and late secretory phases ( . ± . and . ± . , respectively), compared to the mid-late proliferative ( . ± . ) and early-mid secretory phases ( . ± . )(p< . ). moreover, hur expression was significantly lower in ectopic endometrial cells when compared to normal endometrium in midlate proliferative and early and mid-secretory phases (p< . ). progesterone suppressed hur levels significantly in cultured endometrial stromal cells at both and h compared to control (p< . ) while estrogen did not cause a significant change. discussion: decreased hur levels in the late secretory and early proliferative phases are likely to contribute to degradation of cytokines and result in lower cytokine levels observed mid-cycle. late secretory decrease in hur levels may be mediated by progesterone as suggested by in vitro findings. in ectopic endometrium, persistent low expression of hur compared to normal endometrium most probably results from elevated cytokine levels associated with endometriosis. the effect of lower hur expression in ectopic endometrium on other are-containing transcripts, and on the pathogenesis of endometriosis remains to be elucidated. tissue. hong zhao, joy innes, scott reierstad, mehmet bertan yilmaz, serdar e bulun. department of obstetrics and gynecology, northwestern university, chicago, il, usa. background: aromatase is the key enzyme for estrogen biosythesis, and is encoded by the cyp a gene. thus far, only three unique untranslated first exons associated with distinct promoters in the mouse cyp a gene were described (brain-, ovary and testis-specific exon i). however, it remains unknown whether aromatase is expressed in other mouse tissues via previously unknown tissue-specific promoters activating new exon is. methods: real-time pcr was used to examine the aromatase expression levels in various c bl mouse tissues. '-rapid amplification of cdna ends ( '-race) was used to determine the transcriptional start sites of cyp a transcripts. promoter activity was measured using serial deletion mutants of dna fused to the luciferase reporter gene. results: real-time pcr results showed that aromatase was expressed in male gonadal fat and the expression level is lower than that in testis. the adipose tissue-specific untranslated exon i of cyp a transcript was isolated using '-race and this novel gonadal fat-specific exon i of cyp a mrna did not show sequence similarities to previously reported ones. this new adiposespecific exon i was mapped to kb upstream of the translation start site in the coding exon ii. the genomic region upstream of the adipose-specific exon i was cloned into luciferase plasmids. transfection of murine t -l cells with these plasmids showed that promoter activity was conferred by the sequence located at - to - bp upstream of the transcriptional start site. dexamethasone significantly induced activity of the adipose-specific promoter region. conclusion: taken together, our results suggest that a novel cyp a transcript is regulated by a tissue-specific promoter in male murine gonadal fat. these sacrificed; reproductive and selected other organs were removed, weighed and evaluated histologically by an observer blinded to treatment. results: the table below shows that tcc augmented effects of tp on weights of accessory sex organs. histological assessment revealed that tcc induced greater glandular distention with more secretions compared to the effects of tp alone; furthermore, mitotic figures were seen only in prostates from rats exposed to tp+tcc. conclusions: tcc is a newly identified endocrine disruptor with unique and novel actions resulting in potentiation of androgen effects on sex organs. these observations underscore possible environmental risks related to exposure to tcc. background: blastocyst implantation is dependent on the differentiation of human endometrial stromal cells (hesc) into decidual cells. transforming growth factor family members have well defined roles in cell differentiation and proliferation. activin a, a tgf superfamily member, enhances hesc decidualization and localizes to decidualized cells in human endometrium. other tgf superfamily members, including bmp , bmp , bmp , gdf , gdf (myostatin), gdf and nodal, may also be present in decidual cells and therefore may also play a role during this important process. this study aimed to determine whether activin is the major family member driving decidualization or whether other family members contribute to the process. methods: broad ranging activin inhibitors (activin-m a and sb ) that effect receptor-ligand interactions of other tgf superfamily members were used in hesc decidualization. protein localization was examined in secretory phase endometrium and first trimester decidua by immunohistochemistry and mrna expression was examined in an ex vivo model. the secretion of candidate proteins was measured during hesc decidualization and certain recombinant proteins added during decidualization to examine their effect. results: m a ( nm) and sb ( m) significantly reduced decidualization ( % and % respectively) demonstrating that activin and possibly other tgf family members are involved in decidualization in vitro. bmp , gdf and tgf protein were detected in decidual cells of mid-late secretory endometrium and first trimester decidua whilst all ligands except nodal, were expressed by hesc. both bmp and tgf secretion increased during hesc decidualization and administration of both these proteins significantly enhanced decidualization in vitro. conclusions: these data support a role for activin a, bmp and tgf in hesc decidualization. this is important as the elucidation of factors involved during decidualization will aid in better understanding implantation and fertility. abnormal chromatin remodeling in diabetic murine oocytes. laura lawrence, ann ratchford, cybill esguerra, qiang wang, kelle moley. ob/ gyn, washington university, st. louis, mo, usa. background: diabetic women experience increased miscarriages and adverse pregnancy outcomes. previous studies suggest adverse diabetic outcomes may occur earlier than the preimplantation period, particularly during oogenesis. we hypothesize that diabetes affects chromatin remodeling and chromosomal condensation in murine oocytes. methods: mii oocytes from diabetic and control mice were fixed with % pfa, permeabilized with . % triton x- for min, and immunostained against a-tubulin and the nucleus for hr at rt. images were obtained with laser confocal scanning microscope. protein expression levels of chromatin with dimethyl h k modifications were measured in nondiabetic and diabetic denuded murine oocytes at hours post pmsg ( hour) and at six hours post hcg ( hour) via western immunoblots. mature nondiabetic denuded oocytes were fixed in %pfa and permeabilized with . % triton x- . they were stained via immunohistochemistry against histone protein h at lysine (h k me ) and heterochromatin. results: immunohistochemistry reveals that diabetic mii oocytes have aberrant spindle formations and metaphase chromosome alignment. approximately / ( %) diabetic oocytes examined had abnormal spindles and metaphase alignments compared with only about / normal oocytes ( . %). western blot demonstrated times higher expression of dimethylated chromatin in diabetic oocytes at time compared with nondiabetic oocytes. at time hours, diabetic oocytes had significantly fewer h k modifications than the controls. when staining mature murine nondiabetic oocytes for dimethylation of h k me by immunohistochemistry, we demonstrated h k me expression in a condensed heterochromatin ring surrounding the nucleolus, consistent with transcriptional silencing. conclusions: diabetic mii oocytes have a significant increase in abnormal spindle formation and metaphase chromosome alignment. they also have increased dimethylation compared with normal oocytes at a time point when they should be transcriptionally active, storing maternal mrna in preparation for the silencing period. in addition, after hcg injection to trigger maturation and gene silencing, the diabetic oocytes had decreased dimethylated chromatin changes. our findings suggest that diabetic oocytes may be exiting the transcriptionally active period prematurely and may ultimately experience decreased, partial, and incomplete gene silencing. and xenopus epab genes and proteins were performed. expression of human epab and pabpc mrna was tested in ten different somatic tissues, testes, and ovaries by rt-pcr. amplification with actin primers provided a positive control and allowed semi-quantitative analysis. epab and pabpc expression in human prophase i (pi) and metaphase ii (mii) oocytes, -cell embryos and blastocysts was evaluated using quantitative real time pcr. results: human epab is a aa protein with % identity and % similarity to mouse epab and contains rna recognition motifs and a pabp domain. human epab mrna is detected in ovaries and to a lesser extent in testes and several somatic tissues including kidney, liver, and muscle. similar to its mouse orthologue, human epab mrna is expressed in pi and mii oocytes, but not in -cell embryos or blastocysts. pabpc mrna is ubiquitously present in all tissues as well as -cell, and blastocyst stage embryos. however, its levels are significantly lower than that of epab in oocytes. conclusions: in this study we report the identification of human epab. similar to that observed in xenopus and mouse, human epab is the predominant poly(a) binding protein in oocytes and it is replaced by pabpc following zga, which occurs at -to -cell stage in human. our findings suggest that the unique translational regulatory pathways that control gene expression during oogenesis and early embryo development may be common between model organisms and humans. objective: c-jun nh -terminal kinase (jnk), a member of mitogen-activated protein (map) kinase family, is involved in cell proliferation, differentiation, and survival. fsh is required for granulosa cell proliferation and antral follicle growth but its mechanism of action in preantral stages is not well defined. we previously showed that pharmacological inhibition of jnk pathway halts invitro growth of murine preantral follicles in serum free media supplemented with fsh ( miu/ml). sigcs (spontaneously immortalized rat granulosa cell line) have characteristics similar to preantral granulosa cells and hence they were used in this study to determine whether the jnk pathway plays a key role in preantral granulosa cell proliferation. our specific aims were to determine whether: a) fsh activates jnk pathway in granulosa cells; b) the inhibition of jnk pathway blocks cell cycle progression. material and methods: sigcs were treated with miu/ml recombinant fsh in serum free media two days after serum starvation. activation of jnk pathway was analyzed with if and wb using phospho-jnk and phospho-cjun expression, respectively. fsh receptor expression (fshr) was analyzed with if. the inhibition of jnk pathway on cell cycle progression was analyzed by facs using a jnk inhibitor sp . results: fshr protein was expressed in sigc indicating that they can respond to fsh (fig a) . likewise by if, phospho-jnk expression was significantly increased in sigc hour post fsh exposure (fig- a) . similarly on wb, phospho-cjun expression increased as early as min after fsh exposure and peaked at hrs. cjun phosphorylation was abolished hr after treatment with sp ( mm) (fig b) . facs analysis showed that the inhibition of jnk by sp resulted in cell cycle arrest at g /m transition in a dose dependent fashion (fig c) . conclusion: these results strongly suggest that the proliferative effect of fsh on immature granulosa cells is mediated through the activation of jnk pathway. this is the first experimental observation implicating jnk signaling in granulosa cell cycle control. (nichd - ). the induction of {alpha}-hydroxylase (cyp ) expression in granulosa cells. satin s patel, victor e beshay, william e rainey, bruce r carr. reproductive endocrinology and infertility, university of texas at southwestern medical center at dallas, dallas, tx, usa; physiology, medical college of georgia, augusta, ga, usa. according to the traditional two-cell two-gonadotropin hypothesis of the ovary, androgen production arises exclusively from theca cells. the granulosa cells, in turn, utilize androstenedione, which is aromatized eventually to estradiol. studies involving immunohistochemical analysis of normal ovaries have shown that granulosa cells express significantly higher levels of the activator protein- (ap- ) transcription factor, cfos compared to theca cells, where cfos expression is virtually absent. we hypothesize that cfos functions to inhibit the expression of cyp in granulosa cells, thereby suppressing androgen production. hence, the inhibition of cfos activity might result in cyp expression in the granulosa cell. our objective was to define the role of cfos, in the regulation of cyp expression in granulosa cells. transformed human luteinized granulosa (hgl ) cells were utilized for all experiments. hgl cells were cultured in monolayer for h. cells were treated for h with and without pd (pd), a mapkk inhibitor, which also blocks cfos expression. rna was isolated and real time rt-pcr was performed for cyp . cfos rna interference experiments were carried out using rnaifect, cfos smartpool sirna and scrambled sirna for h. rna was isolated and rt-pcr was also performed for cyp . immunochistochemical studies were performed on normal ovaries, staining for cfos and cyp . treatment of hgl cells with the mapkk inhibitor pd for h, resulted in a -fold increase in cyp mrna expression compared to basal conditions. in cfos gene silenced cells, cyp mrna expression also increased by -fold compared to control sirna conditions. immunohistochemical staining for cfos and cyp showed significant staining of cfos in the granulosa cell layer, but absent staining for cyp . conversely, the theca cell layer did not stain for cfos, but staining was evident for cyp . these results suggest that the ap- transcription factor, cfos, may play a role in the inhibition of cyp expression in granulosa cells. this may provide an explanation for the lack of cyp expression in granulosa cells. the g /m stages of the granulosa cell cycle. as clip- has been identified as an mtor substrate, we hypothesized that its function at the mitotic spindle would be positively regulated by mtor during the late g and m phases of the cell cycle in granulosa cells. during periods of stress (e.g., mtor inhibition), mtor would fail to phosphorylate clip- , leading to spindle checkpoint failure and follicle undergrowth. objectives: the expression of clip- and mtor were evaluated. computational analysis of potential clip- phosphorylation sites and comparison with residues on known mtor targets were performed. clip- threonine and serine were chosen and evaluated as bona fide mtor phosphorylation sites. a preliminary assessment of the effects of mtor inhibition upon clip- function was performed. methods: for protein expression analyses, western blots, immunostaining of tissues and primary granulosa cells in culture were performed. computational analysis of potential clip- phosphorylation sites was followed by in vitro assessment of mtor kinase activity upon clip- and a peptide substrate. results: clip- was expressed in the ovarian stroma, blood vessels (including the endothelial cells of both arteries and veins), granulosa cells, and in the oocytes of primordial and growing follicles. overlapping expression was found between clip- , mtor, and the mtor cofactors raptor and rictor in granulosa cells. this expression was conserved between the mouse and the human. evaluation of clip- phosphorylation supported thr as a bona fide mtor target. conclusions: clip- was supported as an mtor substrate protein during granulosa cell mitosis. the mechanism of mtor action during granulosa cell growth and survival is likely to include the phosphorylation of clip- and subsequent positive regulation of mitotic spindle function. the effect of a selective oxytocin antagonist (barusiban) in threatened preterm labour: a randomised, double-blind, placebo-controlled trial. steven thornton, thomas m goodwin, gorm greisen, morten hedegaard, joan-carles arce. warwick medical school, university of warwick, coventry, united kingdom; maternal-fetal medicine, university of southern california, los angeles, usa; dept of neonatology, rigshospitalet, copenhagen, denmark; dept of obstetrics, rigshospitalet, copenhagen, denmark; clinical research development, ferring pharmaceuticals, copenhagen, denmark. objective: a mixed oxytocin/vasopressin v a antagonist, atosiban, has been shown to reduce uterine contractions in placebo-controlled clinical trials and is useful in the management of preterm labour. the objective of this study was to determine the effect of a selective oxytocin antagonist, barusiban, in delaying delivery and reducing uterine contractions in women with threatened preterm labour at a late gestational age and relatively high risk of delivery. methods: this was a randomised, double-blind, placebo-controlled multicentre study in countries. a total of women between + and + weeks gestation, and with uterine contractions of sec duration during min, cervical length mm, and cervical dilatation > and < cm were randomised to receive a single intravenous bolus dose of either barusiban . mg, mg, mg, mg or placebo. rescue tocolytics were prohibited. the primary end-point was percentage of women who did not deliver within h. uterine contractions were monitored by cardiotocography. obstetrical and neonatal outcomes were determined. results: there were no significant differences between the placebo and any barusiban group in percentage of women who did not deliver within h ( % in the placebo group and % to % in the barusiban groups). there was no dose-effect relationship nor an effect at or h. none of the barusiban groups were associated with a significant reduction in number of uterine contractions compared to placebo at any time point up to h post-dosing. postpartum blood loss and time to established lactation were not significantly increased with barusiban. barusiban was well tolerated and was not associated with safety concerns for the women, fetus or neonates. conclusion: a single intravenous bolus of a selective oxytocin antagonist, barusiban (dose range . - mg), did not delay delivery or reduce uterine contractions compared to placebo in women with preterm labour at late gestational age and with short cervical length. the results contrast those of the mixed oxytocin/vasopressin v a antagonist, atosiban. prolonged delivery intervals in triplet gestations. tracy a manuck, heather l mertz, leah passmore, david c merrill. obstetrics and gynecology, wake forest university health sciences, winston-salem, nc, usa. objective: delayed interval delivery is one management strategy for previable preterm labor affecting multiple gestations. prior reports of asynchronous deliveries have examined twins and higher-order multiples as a group. this study was conducted to analyze the unique situation of asynchronous triplet deliveries. study design: cases of asynchronous triplet deliveries resulting in an ongoing twin gestation were ascertained through medline. data were abstracted and combined with two similar previously unpublished cases. patients were grouped by management with and without rescue cerclage. variables compared included use of tocolytics, antibiotic administration, gestational age at delivery of each fetus, interdelivery interval, delivery mode, birthweights, and short and long term outcomes. chi-square or t-test analyses were used where appropriate. results: fifty-one cases of asynchronous triplet deliveries met inclusion criteria and were analyzed. twenty-three patients ( . %) underwent placement of a rescue cerclage following delivery of the first infant. these patients delivered the first fetus at a significantly earlier gestational age as compared to those patients without a cerclage ( . +/- . weeks vs. . +/- . weeks, p= . ). patients with a rescue cerclage had a significantly longer prolongation of the remaining twin gestation ( . +/- . days vs. . +/- . days, p= . ). no significant differences in use of tocolytics or antibiotics, gestational age at delivery of triplets "b" and "c," mode of delivery, short term outcome (alive at hours), or long term outcome (alive at discharge) were noted, despite delivery of triplet "a" at a significantly younger gestational age. conclusion: rescue cerclage, particularly when placed following previable delivery of a first triplet, may significantly prolong the delivery interval for the remaining twin gestation. mean pregnancy prolongation (days). objective the aim of our study was to evaluate the role of amnioinfusion in pregnancies complicated by pprom. we studied singleton pregnancies with pprom at < weeks gestation. all patients were managed conservatively with bed-rest, prophilactic antibiotics, tocolytics and steroids. only patients without vaginal bleeding and/or contractions were included: patients showed an amniotic fluid pocket (afp) persistently cm and did not undergo amnioinfusion (group b) whereas had a maximum afp < cm and were offered amnioinfusion to restore an adequate amount of amniotic fluid (group a). in patients of group a amnioinfusion was successful (afp cm for hours following the procedure: group a ) whilst in it was unsuccessful (afp< for hours: group a ) and repeated. results were analized with the student t test for unpaired samples and with the when appropriate. p values < . were considered significant. results the group where amnioinfusion was not successful (group a ) showed the worst outcome (see table) . there were intrauterine deaths, all in this group. pulmonary hypoplasia was present in / ( . %) newborns (both survived and deceased) newborns, / in group a . no maternal complications were recorded. conclusions our data confirm that a conservative-active management with amnioinfusion can be considered a reasonable option in women with pprom. in our series it was effective in preventing both neonatal death and pulmonary hypoplasia. group a (infusion successful) background. synthetic progestogens are effective in reducing the risk of spontaneous preterm birth in high risk singleton, but not multiple, pregnancy. we hypothesized that myometrial stretch may inhibit the response of human myometrium to progestogens. methods. myometrial strips obtained with written consent at the time of term planned cesarean section were studied using a modification of the method of young and zhang (jsgi ; : - ) . strips were maintained in individual tubes in tissue culture media in an incubator for a period of three days. the effect of prolonged stretch was assessed by comparing strips connected to a . g weight with those connected to a . g weight. the effect of prolonged exposure to progestogen was studied by adding medroxyprogesterone acetate (mpa, nm or nm). following the day incubation, myometrial strips were transferred to an organ bath containing kreb's solution. all were placed under g tension and responses obtained to mm potassium then oxytocin. contractility was expressed as the ratio of the maximum response to potassium or oxytocin to the wet weight of the tissue (units = g.tension per gram), summarized as the mean (sem) and compared using student's paired t test. prolonged stretch increased the maximum response to both potassium ( . g weight = . [ . ]; . g weight = . [ . ], n= , p= . ) and oxytocin ( . g weight = . [ . ]; . g weight = . [ . ], n= , p= . ). in strips with a . g weight, incubation in mpa for three days reduced the maximum response to potassium (vehicle = . [ . ]; mpa = . [ . ], n= , p= . ) and there was a trend towards a reduced maximum response to oxytocin (vehicle = . [ . ]; mpa = . [ . ], n= , p= . ). in strips with a . g weight, incubation in mpa for three days had no effect on either the maximum response to potassium . prolonged stretch increases human myometrial contractility in vitro. . prolonged exposure to a progestogen inhibits the contractility of human myometrium but this effect is blocked by prolonged stretch. these properties of human myometrium may explain the failure of oh progesterone caproate to reduce the incidence of spontaneous preterm birth in multiple gestations. sangeeta jain, william l maner, janet l brandon, gary dv hankins, robert e garfield. obstetrics and gynecology, the university of texas medical branch, galveston, tx, usa. objective: to determine if transabdominal uterine electromyography (emg) correlates with parturition factors such as measurement-to-delivery time (mtdt), cervical dilation (cd), cervical effacement (ce), and station (s) for preterm labor patients with and without tocolysis. materials and methods: pregnant preterm labor women were included. uterine electromyography (emg) was measured for minutes. cd, ce, and s were assessed at or near the time of uterine emg measurement. the power density spectrum peak frequency (pdspf) was calculated on emg. patients were grouped (g : tocolysis, n= ; g : no tocolysis, n= ). pearson-product-moment test was used for correlation. significant differences were sought between groups using student-t test. p< . significant. results: there was a significantly higher uterine emg activity (pdspf: . ± . vs. . ± . ), but no difference in cd ( . ± . vs. . ± . ), for patients delivering within days of emg recording compared to those who delivered later, regardless of tocolysis. there was no apparent difference in uterine emg in tocolytic vs. non-tocolytic patients, regardless of mtdt (table ) . conclusions: uterine emg activity is significantly greater in patients in preterm labor who delivered within days of measurement, making it a viable alternative diagnostic parameter for assessing the state of parturition. tocolytics may not affect uterine emg, but this should be further verified with larger studies. supported by grant nih r -hd . objective: calcium sensitizers are a novel class of drugs with unique molecular and phsiological actions. levosimendan, the best characterized of these compounds and is used in the treatment of acute and chronic heart failure. levosimendan can exert an inotropic effect via sensitization of myofilaments to calcium. it also exerts a relaxant effect on vascular smooth muscle through the opening of atp-dependent potassium channels and has been shown to be a potent inhibitor of human uterine contractions in vitro. for these reasons we investigated the effects of levosimendan on uterine contractions, both spontaneous and agonist induced, in the presence of glibenclamide, a k-atp channel blocker. method: biopsies of human myometrium were obtained at elective caesarean section (n= ). dissected myometrial strips suspended under isometric conditions, undergoing spontaneous and oxytocin-induced contractions, were exposed to glibenclmide ( mmol) followed by cumulative additions of levosimendan in the concentration range of nmol/l to mmol/l. control experiments were performed simultaneously. results: levosimendan exerted an inhibitory effect on spontaneous and oxytocin induced contractions in human m yometrium in vitro, in comparison to control experiments. the effect of levosimendan was significantly antagonized by glibenclamide with the mean maximal inhibition seen due to levosimendan greatly reduced (n= , p< . ). conclusion: the calcium sensitizer levosimendan exerted a potent relaxant effect on human uterine contractility in vitro. this action was antagonized by glibanclamide and this study demonstrates that the effect of levosimendan on uterine smooth muscle is mediated at least in part through the k-atp channel. introduction: the determination of the beginning and ending points for "bursts" of electrical activity occurring during uterine contractions is sometimes difficult. if bursts cannot be discerned, the preferred burst-by-burst analysis cannot be performed. one solution to is to analyze any given electrical recording in its entirety. but this approach has often lead to meaningless results when traditional analytic methods are applied. chaos analysis, using lyapunov exponents, may provide an answer. materials and methods: term patients were included in the analysis: were in labor (group ), while were non-labor (group ). minute recordings were analyzed using "lyapunov exponent." for each pair of subsequent trajectories in phase space, only the most positive lyapunov exponent was calculated. the mean largest exponent was found by averaging over all of the trajectories in the recording. the lyapunov exponent is given in units of bits per data sample. thus a value of + means that the separation of nearby orbits doubles on the average in the time interval between data samples. the mean largest exponent was found for each patient recording. these values were compared using t-test (p < . considered significant). results: the mean and sd of the lyapunov exponent for all the patients was . ± . . moreover, the lyapunov exponent calculated for each patient was positive. comparing lyapunov exponents of the two groups showed a statistically low value (low chaos) for the laboring group, compared to the non-laboring group (figure) . conclusions: there is a chaotic component associated with uterine emg traces, since small but non-negative lyapunov exponents were found in all the traces observed. the lyapunov exponent indicated significantly lower chaotic behavior in the whole emg traces of patients who were in labor than found in those who were not in labor, implying that this measure could be a good diagnostic parameter for labor, possibly eliminating the need for tedious burst-by-burst analysis. supported by grant nih r -hd . comparing uterine emg to tocodynamometer for monitoring contractions. robert e garfield, lynette b mackay, sangeeta jain, william l maner. obstetrics and gynecology, the university of texas medical branch, galveston, tx, usa. objective: to determine whether uterine electromyography (emg) plots contractions similarly to tocodynamometer (toco). study design: pregnant term labor patients were recorded using both uterine emg and toco simultaneously. uterine emg signals were sampled at hz and band-pass filtered in the . to . hz range. root-mean-square (rms) function was calculated from the uterine emg signals in order to produce a "toco-like" trace from the original emg trace. emg-generated rms contraction plots were then compared to toco contraction plots using the following criteria: contractions were assigned a marker value of " ." in-between contraction periods were assigned a " ." from these marker values, contraction rates were established. correlation was found between the contraction rates of rms and toco. temporal overlap of contractions plotted by the two methods was used to find overall percent agreement (opa), positive percent agreement (ppa), and negative percent agreement (npa). these parameters were corrected for within-patient variation using a bootstrap method. results: uterine rms contraction plots were seen to correspond with toco contraction plots (fig. ) . corrected opa, ppa, and npa were high at . %, . %, and . %, respectively. there was a large, statistically significant correlation between uterine emg and toco contraction frequency (fig. ) . conclusions: the similarity between toco and uterine emg contraction plots (specifically, using rms to convert) will allow emg to be used interchangeably with toco in the clinic. supported by grant nih r -hd . introduction -this is a secondary analysis of women participating in a center randomized placebo controlled trial (rct) evaluating the impact of -ohpc in preterm birth (ptb) prevention among women with twins. objective -to evaluate the relationship between plasma -ohpc concentrations and gestational age (ga) at delivery in women with twins receiving weekly injections of -ohpc. methods -women with twins were randomized between - weeks to receive weekly im injections of either -ohpc ( mg) or placebo until weeks. after a minimum of five consecutive injections had been administered to assure steady state concentrations a plasma sample was collected between - weeks. the sample drawn just prior to the next scheduled injection was analyzed for -ohpc by hplc-ms in a blinded manner. the lower limit of quantification of -ohpc was ng/ml. we conducted univariate analyses to assess the association of -ohpc concentration and ga at delivery. we also conducted a proportional hazards model to evaluate the time from sample draw to spontaneous delivery (censoring indicated preterm deliveries), and a logistic regression to evaluate ptb< weeks; in both analyses we adjusted for bmi, race and ga at sample draw. results - women assigned to -ohpc were included; all received all of their scheduled injections. the concentration of -ohpc was significantly higher in women delivering < weeks compared with those women delivering > weeks (p= . , table) . concentration of -ohpc was significantly correlated with ga at delivery (r = - . , p= . ). each unit increase of -ohpc was associated with a % increased odds of delivering < weeks (odds ratio . , % ci, . - . , p= . ) and a % increase in hazard of spontaneous delivery (hazard ratio . , % ci, . - . , p= . ) after adjusting for confounders. gestational age at delivery mean (sd) -ng/ml < weeks (n= ) . ( . ) > weeks (n= ) . ( . ) conclusion plasma -ohpc concentrations after weekly injections were inversely related to ga at delivery in women with twins. since -ohpc induces its own metabolism it is possible that higher concentrations during initial treatment are associated with lower plasma concentrations and reduced efficacy in later pregnancy . clearly more studies are needed. objective: in many non-human species, maternal circulating progesterone levels fall prior to delivery, leading to the theory that in humans progesterone withdrawal occurs on a local and/or functional level. our objective was to characterize maternal and fetal progesterone in human preterm and term labor. methods: women between . and . weeks' gestation (cases) or term controls ( - weeks) with either labor with intact membranes or premature rupture of the membranes prior to labor (prom) were enrolled in a prospective case-control study. progesterone was measured by immulite assay in maternal serum collected upon enrollment and again within minutes after delivery and in umbilical cord serum obtained at delivery. maternal progesterone treatment was not used in any subjects. results: cases and controls were studied (see table for comparisons). controls p value ga at enrollment, weeks . ± . . ± . < . interval to delivery, days (median, range) ( - ) ( - . ) < . maternal progesterone at enrollment, ng/ml ± ± < . maternal progesterone after delivery, ng/ml ± ± . cord progesterone, ng/ml ± ± . among cases, fetal but not maternal progesterone was significantly lower in preterm labor with intact membranes ( ± ng/ml, n = ), as compared to prom ( ± , n = ), p< . . this difference increased further when cases of clinical chorioamnionitis were excluded. conclusions: serum progesterone in laboring patients prior to delivery is higher at term than in the preterm period, which may be attributable to increased placental mass in late pregnancy. this disparity disappears shortly after delivery of the fetus and placenta. fetal progesterone levels are several-fold higher than peripartum maternal levels. preterm labor with intact membranes is associated with diminished fetal progesterone, a phenomenon unrelated to clinical infection. these findings suggest the possibility of fetally regulated progesterone withdrawal as a mechanism underlying preterm labor with intact membranes. [snps] and ptb. however, many of these studies are inconclusive and non reproducible. the challenge of identifying robust associations between genetic variation and either susceptibility or protection from ptb is enormous. a systematic review of literature followed by metaanalysis was performed to understand true associations between snps and ptb. methods: for systematic review, articles were chosen based on medline and embase searches ( ( -april and relevant articles were chosen based on stringent inclusion criteria. primarily, studies reporting genetic associations between snps in maternal dna in singleton pregnancies and spontaneous ptb were included. other criteria included, but not limited to, provided genetic data in a complete enough format so that it could be evaluated in meta-analysis and defined the clinical outcome clearly. meta-analysis was performed wherever > replication data sets were available results: a total of abstracts were reviewed and were selected for full text review. data were extracted from articles. over associations were reported between snps on various candidate genes and ptb; however only had replication dataset. meta-analysis documented significant association between pon a g (odds ratio [or]= . ( %ci . - . ), pon (rs# )(or= . ; ci- . - . ), tnfrsf - a/g (fas) (or= . ; ci- . - . ) and ptb. two snps pon s c (or= . ; ci- . - . ) and ifn gamma (rs ; or- . ; ci- . - . ) documented protective effect. conclusions: systematic review concludes significant heterogeneities leading to lack of reproducible data in genetic association studies of ptb. heterogeneities are contributed predominantly by lack of adequate power, poor phenotype selection, and population admixture. the functional relevance of the risk and protective alleles needs to be verified. jignesh parvadia, mounira habli, jeff livingstone, william polzin, foong lim, timothy crombleholme. pediatric and thoracic surgery, cincinnati children's hospital medical center, cincinnati, oh, usa; obstetric and gynecology, university of cincinnati, cincinnati, oh, usa; obstetric and gynecology, good samaritan hospital, cincinnati, oh, usa. objective little is known about the response of ttts to treatment either by amnioreduction or selective fetospopic laser in triplet pregnancy, particularly the survival of the bystander fetuses. in order to define the response of triplet pregnancies to treatment for ttts we reviewed our experience with higher order multifetal gestations complicated by ttts. study design retrospective chart review of patients diagnosed with in high order gestation from - was performed. results among cases of ttts / ( . %) patients with high order gestations were identified (n= ) with a mean ga at diagnosis of . ± . weeks. pregnancies ( . %) were dichorionic triamniotic and ( . %) were monochorionic triamniotic. cincinnati modification of quinterro staging was utilized to characterize recipient cardiomyopathy as mild (stage iiia, n= ), moderate (stage iiib, n= ) and severe (stage iiic or iv, n= ) categories. / ( . %) were treated with amnioreduction alone (ar), / ( . %) with selective fetoscopic laser photocoagulation (sflp) alone, / ( . %) with ar followed by sflp and / ( . %) with ar followed by intrafetal radio frequency ablation (rfa). / ( . %) patient had a cervical cerclage. / ( . %) patients were treated but remain undelivered. mean ga at delivery was . ± . weeks. overall survival was / ( . %) with bystander survival was / ( %), donor survival / ( . %), recipient survival was / ( . %). conclusion triplet pregnancies treated for ttts have % survival rate for bystander fetuses and have . % survival rates for donor and recipients comparable to twins treated for ttts. ga at diagnosis . ± . weeks cincinnati modification of quinterro (i), (ii), (iii), (iiia), (iiib), (iv) ga at delivery . ± . weeks live birth -donor / ( . %)* # -recipient / ( . %)*# -bystander / ( %) # birth weight -donor to assess the effect of breast feeding (bf) on perinatal outcome in relation to maternal antenatal methadone dose. study design a retrospective chart review study of methadone dependent mother and infant pairs. patients were categorized into groups based on maternal dose at time of delivery: group : dose mg, group : dose - mg, group : dose > mg. the finnegan's scoring system was used to monitor neonatal abstinence syndrome(nas). treatment for nas was initiated if there were scores of . neonatal outcome data included:% nicu admission, % of babies discharged(d/c) at time of maternal d/c, % nas, % treated for nas and total hospital stay. data were analyzed by t-test and fisher's exact test. maternal characteristics between the groups were similar. regardless of maternal methadone dose, bf infants have shorter hospital stays and higher rates of d/c at time of maternal d/c, lower incidence of nas and fewer nicu admission (table) . in all three groups, breast feeding did not impact the severity of nas as reflected in finnegan's score(fs). (table) conclusion regardless of maternal methadone dose, breast feeding improves perinatal objective: to evaluate the effects of preventive collagen plugging of the fetoscopic access port at the time of balloon removal on pregnancy outcome in fetoscopic endoluminal tracheal occlusion pregnancies. study design: fifty-one pregnancies involving fetuses with severe congenital diaphragmatic hernia (cdh) were studied. all patients underwent feto between - weeks gestational age (ga) and fetoscopic balloon removal around weeks ga. at the time of balloon removal, a purified dried collagen plug was inserted through the fetoscopic access port in consecutive pregnancies but not in the first pregnancies considered as controls. all patients underwent post-plugging ultrasound and magnetic resonance imaging studies to evaluate for membrane dehiscence, amniotic fluid volume and fetal well being. ga at delivery, incidence of premature rupture of the membranes (pprom), bleeding at port retrieval and adverse fetal effects were compared in both groups. results: mean (sd) ga at feto [ . ( . ) vs. . ( . ) weeks; p= ns] and balloon removal [ . ( . ) vs. . ( . ) weeks; p= ns] was similar in the treatment and in the control group. incidence of pprom following the second fetoscopy was / in the study group compared to / in the control group (p< . ). mean (sd) ga at delivery was . ( . ) weeks in the study group, compared to . ( . ) in the control group (p= . ). bleeding from the trocar insertion site occurred in cases in both groups, but clinically significant bleeding occurred only in one of the controls. membrane dehiscence was noted in patient in the treatment group compared to in the control group (p=ns). conclusion: preventive collagen plugging of the fetal membrane defect created by the fetoscopic access resulted in a significant reduction in pprom rates and a trend towards increased ga at birth without adverse fetal effects in feto pregnancies. wider application of this technique should be considered, but needs evaluation in larger, randomized trials. hydrops fetalis is an uncommon fetal condition characterised by the abnormal accumulation of fluid in two or more body cavities, traditionally associated with a poor prognosis. the relative rarity of this presentation has meant that published case series have consisted of small numbers. a retrospective review of case notes of all cases managed at kemh between and was performed. in western australia, kemh is the only tertiary maternity hospital incorporating a maternal-fetal medicine unit. cases were obtained from the mfm database. in the period to there was a total of pregnancies affected by hydrops (incidence . per births). the average maternal age was years. in cases a fetal abnormality had occurred in a previous pregnancy. the median gestational age at diagnosis was weeks (range - weeks). in just over half ( %) of cases, the diagnosis was confirmed prior to delivery. a post-mortem was performed on all but of the babies not born alive. edema was present in at least cavities in over half of cases (n= ). chromosomal anomalies included trisomy , trisomy and turners syndrome. in all cases of infection, parvovirus b was implicated. cardiac arrythmias included svt and atrial flutter. cases classified as other included alpha thalassemia and syndromic disorders. in cases an interruption of pregnancy was performed at a mean gestational age of weeks. of those who did not elect to terminate the pregnancy, there were fetal deaths in utero, live borns with neonatal death. for the live borns, the median gestational at delivery was . weeks (range to . weeks). the causes of hydrops in live birth cases included cardiac arrythmia (n= ), infection (n= ), chromosomal abnormality (n= ), unknown (n= ) and other (n= (dmv) have been noted to play a role in the development of hemorrhagic and periventricular leukomalacic lesions in premature babies. since deep vein drainage system is relatively more prominent in the developmental brain than adult brain, we investigated if dmv anomalies could be associated with clastic lesions in-utero. methods two senior neuroradiologists reviewed fetal brain exam performed between and , seeking for unequivocal anomalies in dmv, such as periventricular venular engorgement. all mr scanning is performed at . tesla, using surface abdominal coils and single-shot fast spin-echo t -weighted - mm thick sections, with . - . mm in planar spatial resolution. we found cases with dmv anomalies (tab. ). most of the dmv engorgement is located at frontal lobes level. from this limited preliminary series it appears that dmv involvement plays a role in the development of periventricular leukomalacia and periventricular hemorrhagic necrosis. the observation that these lesions are mostly located at frontal level may suggest that some of the term neonates carrying sequelae of atypically located leukomalacia (i.e. deep frontal lobe) might have developed these lesions in-utero. it is of interest to notice that most of our cases were related to heart failure. therefore, central venous hypertension affecting immature deep cerebral venous system has to be taken into account. in our center, patients with an estimated risk for chromosomal abnormalities at term greater that in after st trimester combined screening test (ft) are offered non-directive genetic counseling. the aim of this study was to evaluate the responses of women younger than attending this genetic counseling session. material and methods: data from patients referred for a positive ft from september , to july , was retrieved from our database. information concerning women younger than years of age at the estimated date of delivery was extracted and tabulated. results: during the study period women had genetic counseling for positive ft. thirteen patients were excluded from further analysis ( had incomplete clinical documentation and had spontaneous miscarriages prior to weeks gestation). four hundred and twenty-five patients were older than and were younger than at the estimated date of delivery. table depicts summary statistics for studied variables in this younger group of women. conclusions: overall this data suggests that approximately % of this younger group of women opted for chorionic villous sampling (cvs), % for amniocentesis and more than % declined prenatal genetic testing. moreover, this data also suggests that: ) these women opted for cvs when the ft risk (mean = in ) almost doubled the cvs procedure related risk quoted at % and, ) when the ft risk is between in and in almost half ( out of ) declined not only the st trimester cvs but also the nd trimester amniocentesis. we believe that understanding our patient population is important to optimize both the efficiency and efficacy of the alternative prenatal screening programs. acceptance for prenatal genetic testing after a positive first trimester combined screening test in women of less than to determine the optimal diagnostic test using prenatal ultrasonography and mri for predicting pulmonary hypoplasia in fetuses with congenital diaphragmatic hernia. methods: relevant papers were identified by searching medline ( medline ( - , embase ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) and the cochrane library ( issue ). in addition, the specialist literature on the topic and reference lists were hand searched for relevant articles. studies were selected if they examined diagnostic tests for the prenatal prediction of pulmonary hypoplasia in fetuses with congenital diaphragmatic hernia. the primary outcome measure was perinatal survival. study selection, quality assessment and data abstraction were performed independently and in duplicate by separate observers. results: of a total number of articles (published studies), there were eighteen studies that fulfilled the entry criteria. six examined entirely unique heterogeneous parameters. of the remainder, all examined the ultrasound measurement of lung to head ratios (lhr) at a 'cut-off of greater than or less than the thresholds of . , . , . , . . in addition, the presence of liver in the fetal thorax was included (if present) as a co-variable (liver "up"). a lhr > . compared to < . provided the strongest association with perinatal survival (peto or . , % ci . - . ). the finding of "liver up"in the fetal thorax had a negative association with survival (peto or . , % ci . - . ). only three studies provided data for lhr in conjunction with the presence of liver in the fetal thorax (peto or survival for lhr > . compared to < . was or . , % ci . - . ). data was also available for liver up and lhr > . which had a peto or of . ( % ci . - . ). discussion: the data supports the view that lhr may be a useful prognostic indicator of perinatal survival in fetuses with congenital diaphragmatic hernia. however, heterogeneity still exists regarding the timing of ultrasound measurement and the use of mri. the majority of studies have small sample sizes. objective: to estimate, in fetal anaemia, the diagnostic value of fetal ultrasonography and doppler blood flow in the evaluation of fetal anaemia methods: literature from to was identified using medline and embase, the cochrane library and relevant specialist register of the cochrane collaboration, and by checking reference lists of known primary studies and review articles. studies were selected if the accuracy of the fetal ultrasound parameters or doppler studies of blood flow in the fetal vessels was estimated compared with a reference standard. diagnostic tests evaluated were ultrasound measurement of the fetal spleen and liver length and doppler studies from the umbilical vein and middle cerebral artery. data from the selected studies were abstracted as x tables comparing the diagnostic test result with the reference standard. results were pooled where appropriate. diagnostic accuracy was expressed as sensitivity and specificity. twenty-nine primary studies were identified containing suitable data. twentyone of these gave data on middle cerebral artery doppler peak systolic velocity (mca-psv) and could be pooled in the meta-analysis giving a sensitivity of . ( . - . ) and a specificity of . ( . - . ) for cases in detecting severe anaemia. four studies gave data on spleen perimeter and it was possible to pool three of these giving a sensitivity of . ( . - . ) and a specificity of . ( . - . ) for cases. three studies had data for liver length measurements and two were pooled. the sensitivity was . ( . - . ) and the specificity was . ( . - . ) but only cases were used in the analysis. there were three studies on umbilical vein maximum velocity doppler studies and all were suitable for meta-analysis giving a sensitivity of . ( . - . ) and a specificity of . ( . - . ) with cases analysed. two studies gave data on middle cerebral artery time-averaged mean velocity score giving cases. the sensitivity was . ( . - . ) and specificity was . ( . - . ). discussion: middle cerebral artery peak systolic velocity dopplers remain the gold standard for non-invasive screening of fetal anaemia. middle cerebral artery time-averaged mean velocity scores require further investigation. other tests perform poorly when diagnostic accuracy is assessed. cochrane review of treatment in twin to twin transfusion syndrome. twin-twin transfusion syndrome is a condition affecting monochorionic twin pregnancies associated with a high risk of perinatal mortality and morbidity. the objective of this review was to evaluate the impact (maternal,fetal and pediatric)of treatment modalities in twin-twin transfusion syndrome. register and cochrane controlled trials register. we also searched conference proceedings and made personal contact with experts active in the area of the review. randomised and quasi-randomised studies of amnioreduction versus laser coagulation, septostomy versus laser coagulation or septostomy versus amnioreduction. eligibility was assessed by one reviewer. study authors were contacted for additional information. two studies were included. this review shows that laser coagulation of anastomotic vessels results in less fetal (rr . ; % ci . , . ) and neonatal deaths (rr . ; % ci . , . ) than amnioreduction ( figure ). there is no difference in perinatal outcome between amnioreduction and septostomy. more babies in the laser arm are alive without neurological abnormality at six months of age (developmental delay at < years rr . , % ci . , . )( figure ). conclusions: endoscopic laser coagulation of anastomotic vessels should be considered in the treatment of all stages of twin twin transfusion syndrome to improve perinatal outcome. further research on the effect of treatment on milder forms of twin twin transfusion syndrome (quintero stage and ) are required. (produced in collaration with the cochrane collaboration, uk). (cvs) concluded that although the risks of pregnancy loss are relatively low, lack of adequate controls tends to underestimate the true added risk of prenatal invasive procedures (obstet gynecol. ; ( ): - ). the west midlands is a large region within the uk containing approximately % of the total uk population. the congenital anomaly register for this region is able to monitor pregnancy outcomes with accurate deminator data. results: there were first trimester cvs performed, by ten operators, in the west midlands (uk) in . this equates to . procedures per births. significantly higher rates were noted in areas of high socioeconomic status. the median number of procedures performed per operator was (range - ). all operators were performing other invasive tests such as amniocentesis or cordocentesis,etc. the most common indication for cvs was: i) fetal anomaly on dating scan ( . %) ii) abnormal (> in ) risk on combined first trimester screening ( . %) iii) molecular genetic diagnosis ( . %) and iv) maternal request ( . %). using a combination of first trimester scanning and cvs, % had abnormal karyotype/structural anomaly.the corrected loss rate (background and procedure-related) following cvs in normally formed, singleton pregnancies was . % ( thci . - . %) up to days postnatal (perinatal loss) and . % ( thci . - . %) with days of procedure. the proportion of cvs in which an adequate sample was not obtained was . % ( th ci . - . %). conclusions: this epidemiological study using accurate demoninator data provide interesting statistics relating to the uptake and prenatal risks of first trimester cvs. with the increasing prevalence of obesity in the last two decades, we have seen a tremendous increase in bariatric procedures in reproductive aged women. malnourishment and vitamin deficiency are common complications after gastric bypass which may impact on fetal development. we present the case of a yo who underwent a duodenal switch procedure in . she was discovered to be pregnant during evaluation for persistent malnutrition in . multiple prenatal ultrasounds were performed; the first at weeks gestation was unremarkable. the week sono revealed a male fetus with shortened femurs and humeri bilaterally, nasal bridge hypoplasia, macroglossia, poorly defined hands, and possible clubbed feet. amniocentesis revealed a normal karyotype. d ultrasound redemonstrated the abnormal facial findings. a fetal echocardiogram was normal. the lagging long bone measurements continued to worsen, ultimately with femurs weeks behind. the fetal thoracic circumference was two standard deviations below the mean, giving rise to concern for pulmonary sequelae. growth restriction was noted at the wk sonogram. delivery by cesarean section was at / weeks secondary to nonreasuring fetal status. the birthweight was gm; apgars were , , and . postnatal radiographs confirmed antenatal ultrasonographic findings and demonstrated evidence of epiphyseal stippling. the infant remained intubated until weeks of life, after which it died secondary to respiratory complications associated with pulmonary hypoplasia. gene mapping studies have not found any point mutations on the recessive gene as an etiology of this disorder. rhizomelic chondrodysplasia punctata refers to a heterogeneous group of bone dysplasias with a familiar radiographic phenotype involving punctate calcifications and epiphyseal stippling. the etiology of this may be secondary to chromosomal abnormalities, mendelian gene disorders, or teratogens, notably warfarin. this case may be explained by vitamin k deficiency of the embryo due to maternal malabsorption after bariatric surgery. the maternal vit k level was <. ng/dl at time of delivery(normal > . ). the teratogenic effects of vitamin k deficiency in this instance highlight the need for strict counseling and screening for vitamin deficiency in those women undergoing bariatric surgery since previous obesity-related anovulation is reversed as patients lose weight, resulting in unexpected pregnancies and potentially preventable fetal abnormalities. term preeclampsia: any risk for the neonate? sindhu k srinivas, jamie bastek, christina m andrela, emmanuelle pare, michal a elovitz. obstetrics and gynecology; crrwh, university of pennsylvania, philadelphia, pa, usa. introduction: preeclampsia continues to be a major contributor to maternal morbidity and mortality worldwide. preeclampsia at term is not associated with the same risk of neonatal morbidty and mortality as preterm preeclampsia . however, neonatal outcomes in term women with preeclampsia have not been adequately studied. we sought to compare short term neonatal outcomes in term infants born to women with and without preeclampsia. methods: this study was part of a large case control study. women with preeclampsia (n= ) and term controls (n= ) were prospectively identified. infants with congenital anomalies were excluded. hospital length of stay (los), admission or transfer to the nicu, and use of mechanical ventilation or cpap within first week of life were assessed. associations between neonatal outcomes and preeclampsia were evaluated using chi-square analysis and wilcoxon rank sum test. significant confounders were controlled for using multivariable logistic regression. results: discharge day of life was significantly different between neonates born to women with preeclampsia (median = ; mean = . ) versus those born to women with uncomplicated term deliveries (median = ; mean = . , p< . ). this difference persisted even when neonates with iugr and those born to diabetic mothers were excluded (p< . ). term infants born to women with preeclampsia have a higher odds of being admitted or transferred to the nicu (aor= . [ . - . ], p= . ) after controlling for iugr, delivery mode, race, and gestational age at delivery. these infants also have a higher odds of mechanical ventilation (aor= [ . - . ], p= . ) and cpap use (aor= . [ . - . ], p= . ) after controlling for the same confounders. there was no difference in ivh or nec between the two groups. conclusion: neonates born to women with preeclampsia have differences in short-term morbidity when compared to neonates born to women without preeclampsia, despite being born at term. whether this increase in neonatal morbidity is attributable to medications used in preeclampsia, such as magnesium sulfate, is unclear. these findings may have implications for patient counseling as well as hospital resource allocation. further investigation correlating these findings with long-term morbidity is warranted. could confined placental mosaicism account for adverse perinatal outcomes in ivf pregnancies? benoit c jacod, gh schuring-blom, kd lichtenbelt, jse laven, d van opstal, mjc eijkemans, nick s macklon. reproductive medicine gynaecology, university medical centre, utrecht, netherlands; clinical genetics, university medical centre, utrecht, netherlands; obstetrics gynaecology, erasmus medical centre, rotterdam, netherlands; clinical genetics, erasmus medical centre, rotterdam, netherlands. background: ivf singletons have poorer perinatal outcomes than singletons from spontaneous conceptions. this may be due to the influence of ovarian stimulation on the chromosomal constitution of the embryos which could be translated into localized chromosomal anomalies in the placenta. aim: to compare the incidence of confined placental mosaicism (cpm) in ivf/icsi pregnancies and spontaneous conceptions. methods: multicentre retrospective analysis of karyotype results obtained by chorionic villus sampling (cvs) performed because of advanced maternal age ( years at weeks of gestation) in the netherlands between and . results: from a total of pregnancies, cvs results were analysed: in the ivf/icsi group and in the control group. the mean age of women in both groups was . years (mean difference - . , % ci - . - . ). foetal karyotype was missing in cases of possible cpm, all in the control group. when taking into account missing data, the incidence of cpm was lower in the ivf-icsi group than in the control group, . % vs. . % (odds ratio . , % ci . - . ) whereas the incidence of foetal chromosomal anomalies was increased . % vs. . % (odds ratio . , % ci . - . ) although both differences are not significant. conclusions: the incidence of confined placental mosaicism is not increased in ivf/icsi pregnancies compared to spontaneous conceptions. cpm probably does not account for the adverse perinatal outcomes following ivf/icsi. fetal rh d, cc and ee genotyping using fetal dna from maternal blood is not impaired by the presence of maternal alloimmunization. chad a grotegut, stacey l jeronis, john p gaughan, enrique hernandez, ossie geifman-holtzman. obstetrics and gynecology, duke university, durham, nc, usa; obstetrics, gynecology and reproductive sciences, temple university, philadelphia, pa, usa; biostatistics, temple university, philadelphia, pa, usa. this study was conducted to assess the impact of maternal alloimmunization on the accuracy of fetal rh d, cc and ee genotyping from maternal blood. we performed a literature search of english-written articles describing fetal rh d, cc and ee determination from maternal blood (am j obstet gynecol ; : - ) . using this database, we determined the accuracy of rh d, cc and ee genotyping in the presence of maternal alloimmunization. for each subgroup, % confidence intervals for a proportion were calculated and compared between groups. we found english-written publications reporting non-invasive rhd genotyping and reporting non-invasive rh ce genotyping from maternal blood. fourteen ( . %) of the rh d articles and three ( %) of the rh ce articles provided accuracy results in the setting of alloimmunization. the accuracy results are reported as follows: . % of the samples were determined correctly in the presence of alloimmunization.* this accuracy was significantly lower than the accuracy reported for all rh cc samples. when only studies utilizing free fetal dna for rh cc genotyping were used (vs fetal cells), fetal cc genotype was determined correctly in / ( %, % ci . , ), which was similar to the overall success rate for rh cc determination. overall, there were no differences in the success of rh d, cc or ee determination in the setting of alloimmunization compared to the overall accuracy seen when free fetal dna was used. the presence of maternal alloimmunization does not reduce the accuracy of fetal rh d, cc or ee non-invasive genotyping from maternal blood utilizing free fetal dna. further research into structure and rearrangements of the rh d, cc and ee genes may further improve diagnostic accuracy of free fetal dna from maternal blood. fetal dna, most likely of trophoblast origin, is present in both the plasma of pregnant women and provides a potential basis for non-invasive fetal diagnostic tests. however, fetal dna in maternal plasma is highly contaminated with maternal dna, and this contamination is the main technical challenge in trying to accomplish non-invasive detection of fetal chromosome abnormalities. existing methods for the selective amplification of fetal dna have generally relied on specific sequence differences between the mother and fetus. as an alternative, we have developed a method for selective amplification of fetal dna that makes use of observation that trophoblast dna is globally hypomethylated in comparison with dna from other sources. in this method, a dna mixture is first digested with a methylation sensitive restriction enzyme and then amplified by linker-mediated pcr. after an initial amplification, a second isothermal rolling-circle amplification is performed. this procedure results in the differential amplification of short, relatively hypomethylated fragments. after amplification, the resulting "representations" are comparatively hybridized to a microarray consisting of oligonucleotides that correspond to restriction fragments generated by the initial digest. copy number differences are then detected through statistical analysis of array addresses that show significant amplification. to test the feasibility of this method for detecting aneuploidy, we have prepared mixtures of peripheral blood dna and first trimester trophoblast dna from either normal or from samples with trisomy and trisomy . we present data showing that aneuploidy can be detected even when % of the starting dna sample was derived from a euploid source and only % was from an aneuploid trophoblast sample. two different approaches to data analysis are presented. one relies on prior analyses of trophoblast methylation and the second is independent of any prior knowledge or analysis. both methods provide similar ability to detect aneuploidy. future work will focus on testing whether this approach can be used for non-invasive prenatal diagnosis. chromatin immunoprecipitation and emsa analysis of nf-b and c/ ebp synergism on the otr promoter. shirin khanjani, yun s lee, vasso terzidou, mark r johnson, phillip r bennett. institute of reproductive developmental biology, imperial college, london, united kingdom. we have shown, the transient transfections of the transcription factors nf-bp and c/ebp leads to a synergistic increase in otr promoter activity in human myocytes. this effect is mediated through a bp region of the promoter between - to - from tss. we now report that this sequence binds both nf-bp and c/ebp in vitro and chip studies show binding of both transcription factors to be increased by il- in vivo. materials and methods: emsa studies were performed using a bp oligonocleotide sequence (- to - ) , containing the bp region responsible for the synergistic activation of the otr promoter and nuclear extracts from primary human myocytes treated with il- ng/ml for hours. for chip analysis, dna protein complexes were crosslinked and antibodies recognizing p , c/ebp and h (positive control) and igg (negative control) were used for immunoprecipitation. primers were designed to amplify the region - to - , which includes the bp response sequence. to further confirm the interaction between nf-bp with c/ebp a xnf-b consensus/luc reporter construct was cotransfected with an expression vector for either nf-bp or c/ebp . results: specific nf-bp and c/ebp binding was seen in the emsa study. preincubation with antibodies to nf-bp and c/ebp led, not to supershift, but to elimination of dna binding for both nf-kb p and c/ebp . chip analysis confirmed increased in vivo binding of nf-b p and c/ebp to this region of the otr promoter following stimulation with il- . transfection of the nf-b/luc reporter construct with an expression vector for c/ebp caused a significant reduction in the basal promoter activity suggesting that c/ebp is binding to nf-kb. this interaction was further confirmed using a tf-tf interaction array. conclusion: these data support the role of nf-b and c/ebp in regulation of otr. the bp region contains a c/ebp but not a nf-b dna binding site suggesting that c/ebp primarily binds to this part of the otr promoter but also interacts with nf-b. the emsa data shows that the bp region binds both nf-b and c/ebp . the loss of the supershift observed in previous emsa studies suggests that the antibodies inhibit the interaction between c/ebp and nf-b, therefore inhibiting dna binding. chip analysis supports the concept that il- leads to binding of nf-b and c/ebp to the bp region. regulation of pro-labour genes by c/ebp, nf-b and ap- in human uterine myocytes. suren r sooranna, shirin khanjani, yun s lee, phillip r bennett, mark r johnson. imperial college parturition research group, imperial college, london, united kingdom; irdb, imperial college, london. introduction: the transcription factors c/ebp (lap), nf-b (p ) and ap- (c-fos and c-jun) are implicated in inflammatory processes such as parturition. the promoter regions of the pro-labour genes il- , pghs- and otr contain putative transcription factor-binding sites for these transcription factors. our aim was to determine the effect of transfecting these transcription factors either alone or in combination into uterine myocytes and to determine their effects on the expression of pro-labour genes including il- , pghs- , otr, connexin- and fp. methods: primary cultures of human uterine myocytes (n= ) were grown from myometrial samples obtained at the time of elective lscs. cells were cultured in well plates to % confluence at which point expression constructs for c/ ebp (lap), nf-b(p ), ap- (c-fos and c-jun) were transfected either alone or in different combinations. the empty expression vector psg was used as a filler construct. cells were cultured for and h after which culture medium was collected for elisa and cells frozen at - °c prior to rna extraction. copy numbers of il- , pghs- , otr, fp, connexin- and gapdh were measured by qpcr using a rotor-genetm (corbett research). results: h post transfection with c/ebp (lap), nf-b (p ), c-fos and c-jun alone or in combination showed no significant changes in pghs - , otr and connexin- expression. over expression of p alone or together with either c-fos or c-jun increased fp expression by , and % respectively (p= . ). nf-bp consistently increased il- expression either alone (by %; p= . ) or in combination with lap, c-fos or c-jun (by , and % respectively; n= ; p= , ). rel a, lap, c-fos and c-jun together also increased il- expression (by %; p= . ) and a small but significant increase was seen with a combination c-fos and c-jun (by %; p= . ). the changes observed in il- expression were reflected in the medium il- concentration at h post transfection. in the presence of rela and c-fos il- increased from . ± . to . ± . pg/ml of culture medium (mean ± sem; n= ). conclusions: nf-b (p ) consistently increased fp and il- expression in human myometrium. the data suggest that pghs- activation has greater dependence upon other transcription factor(s) in addition to p . true identity of myometrial pr-c: fact or fiction? yun s lee, suren r soorana, mark r johnson, jan brosens, phillip r bennett. imperial college parturition research group, hammersmith campus, london, united kingdom. progesterone is thought to be central to maintenance of pregnancy. multiple progesterone receptor (pr) isoforms underlie complex and diverse biological action of progestins. previously two human pr isoforms have been identified: pr-b and pr-a. pr-a is n-terminally truncated form of pr-b (initiation site methionine ). in some cells pr-a inhibits pr-b. it has been proposed that increased expression of pr-a in myometrium underlies a 'functional progesterone withdrawal'. the breast cancer cell t d contains a kda progestin-specific binding protein that is not found in pr negative cells. it was proposed that there is a downstream methionine (met ) which serves as a translation initiation site for the generation of a pr isoform of kda. based on such findings condon et al (mol endo ) have focused on the possible role of kda pr-c in human parturition. they found that expression of "pr-c" using pr antibody (sc- santa cruz, sc) is increased in upper segment myometrium with labour and that overexpression of this protein inhibits pr-b function. we cloned the same human pr cdna into psg expression vector. in vivo translation produced a protein of only kda. furthermore overexpression of pr-c did not significantly decrease pr-b activity in human myocytes. we examined other downstream initiation sites, which may produce a kda protein. we constructed potential pr isoforms in psg vector with initiation sites at met and . in vivo translation produced proteins of approximately and kda respectively. to determine the effect of pr isoforms on pr-b function, myocytes were co-transfected with pr-a, pr-c , pr-c and pr-c with constant amounts of pr-b. the progesterone dependent pre/luc was used as reporter. unlike pr-c both pr-c and pr-c significantly inhibited ligand dependent pr-b mediated transcriptional activity. we found in western analysis that the antibodies pgr- (nc) and the sc- (sc) detected both endogenous and overexpressed pr-a and pr-b but none of the pr-c isoforms. the sc- antibody detected only pr-a and pr-b very poorly. our data suggests that the sc- antibody would not detect any pr-c protein and that none of the commercially available antibodies in the uk do so. great care needs to be taken when over-expressing pr isoforms to ensure that proteins are of the expected size. if pr-c does exist in vivo then the kda but not the kda isoform might inhibit pr-b function. tnf receptor antibody (tnf ri ab), nf-b inhibitor (nf-b activation inhibitor) and erk inhibitor (u ) (p< . ), but not by tnf receptor antibody (tnf rii ab), p mapk inhibitor (sb ) and jnk inhibitor (sp ). by western blot analysis, we found that the protein level of tnf receptor associate factor (traf ) was higher than that of tnf receptor associate factor (traf ) (traf >traf ) in untreated ct cells. however, after tnf treatment for h to h, traf protein level was increased, but traf protein level was reduced (traf >traf ). the increase of traf and decrease of traf were blocked by tnf ri ab, but not by tnf rii ab. we also found that tnf rapidly (within - min) and significantly increased phosphorylation of ikk , erk / and jnk / / and the phosphorylation of these protein kinases by tnf was reduced significantly by tnf ri ab, but not by tnf rii ab. moreover, we found that the changes of increased traf and decreased traf in ct cells (traf >traf ) resulted in a dramatic deficiency in phosphorylation of the above protein kinases induced by tnf compared with the normal ct cells (traf >traf ). nuclear localization of nf-b p in tnf treated cells was increased compared to untreated controls. conclusion: we have demonstrated for the first time that tnf stimulates mmp- production in ct cells through tnf ri-trafs-ikk /erk-nf-b signaling pathways, but not through the jnk/p -ap- pathway. these studies reveal steps within this pathway as possible therapeutic targets to inhibit mmp- expression potentially attenuating tnf -induced degradation of extracellular matrix and pre-term rupture of the fetal membranes. objective: women with preterm birth are at elevated risk of cardiovascular disease, but mechanisms that might relate these conditions are not understood. we hypothesized that women with spontaneous preterm vs. term births may have early gestation evidence of activation of the fibrinolytic cascade, as measured by the thrombin-antithrombin iii (tat) complex. we also tested if this relation may be associated with inflammation. methods: tat was measured in plasma collected < weeks gestation (mean . weeks, sd . ) among women without chronic medical conditions, preeclampsia or growth restriction who delivered singleton liveborn infants. inflammation was assessed by c-reactive protein (crp) measured in serum from the same samples. women with spontaneous preterm birth (sptb) < weeks (n= ) and -< weeks (n= ) were compared to women with term births >= weeks (n= ). high tat was defined as > . ng/ml (highest quartile among women with term births) and high crp was defined as >= ug/ml. multinomial logistic regression was utilized to relate elevated tat and inflammation to risk of sptb subtypes. results: women with sptb were more likely to have tat concentrations in the highest quartile compared to women with term births (< weeks, . %; -< weeks, . %; >= weeks . %, p< . ). women with high tat concentrations had a . -fold ( % ci: . - . ) increased risk for sptb < weeks, after adjustment for body mass index, race, age and gestational age at sampling. there was no relation between high tat and sptb -< weeks (or . , % ci . - . ). additional adjustment for elevated crp (>= ug/ ml) did not effect the estimates associated with tat, and elevated crp was independently related to risk for both sptb subtypes (< weeks, or . [ . - . ]; -< weeks, or . [ . - . ]). thus, women with high tat and elevated crp appeared to be at particularly elevated risk of sptb < weeks (or . , % ci . - . ). conclusions: the thrombin-antithrombin iii complex was elevated early in gestation among women with sptb < weeks, perhaps secondary to microvascular injury. this effect was independent of inflammation, suggesting that the elevated fibrinolytic cascade may function independently from inflammation among women with sptb. plasma cortico-releasing hormone and cortisol concentrations and psychological stress among pregnant women. katherine p himes, hyagriv n simhan. obstetrics and gynecology, university of pittsburgh medical center, magee womens hospital, pittsburgh, pa, usa. objective: many studies have found an association between psychological stress and preterm birth. we sought to determine if women with greater psychological stress during pregnancy had higher concentrations of plasma cortico-releasing hormone (crh) or cortisol. study design: this is a secondary analysis of a multicenter case-control study, nested within an observational cohort. of , participants, plasma crh and cortisol concentrations at and weeks gestation were available in controls who delivered after weeks and cases who delivered before weeks. the abbreviated scale for the assessment of psychosocial status in pregnancy (asaps) was available for all women. concentrations of crh and cortisol were compared between women above and below the lowest quartile score on the asaps among cases and controls. the same analysis was done for the portion of the scale related to psychological stress. concentrations of crh and cortisol and psychological stress were also compared between black and non-black cases and controls. univariate analysis was performed with kruskal wallis or chi-square. results: there was no difference in crh or cortisol concentrations at or weeks among women above or below the lowest quartile on the asaps (controls:p= . - . cases:p= . - . ). greater psychological stress was not associated with higher concentrations of crh or cortisol at or weeks(controls:p= . - . cases:p= . - . ). crh concentrations were not different between blacks and non-blacks. among both cases and controls, cortisol concentrations at and weeks were lower in black women than non-black women (controls:p< . cases:p< . ). the median cortisol concentration among control black women was . g/ml at weeks compared to . g/ml among non-black women and . g/ml compared to . g/ml at weeks. black women reported less psychological stress than non-black women (p= . ) conclusion: we found no relationship between psychological stress and plasma crh or cortisol. furthermore, while stress is hypothesized to play a role in the racial disparity of preterm birth, black women reported less psychological stress and had lower cortisol concentrations than non-black women. improved assessments of psychological stress and additional biomarkers involved in the stress response may broaden our understanding of how stress contributes to preterm birth. expression, tissular traffic and activation of mmp- in human fetal membranes during labor. rodrigo vega-sanchez, arturo flores, marisol castillo, nardhy gomez, felipe vadillo-ortega. direction of research, instituto nacional de perinatologia, mexico city, df, mexico. introduction. rupture of the fetal membranes (fm) during human labor occurs as a consequence of extracellular matrix degradation. this process is controlled by increased secretion and activity of matrix metalloproteinases, particularly mmp- .several evidences suggest that mmp- is mainly produced by infiltrating choriodecidual leukocytes that could arrive from placental circulation. characterization of the synthesis, transport and activation of mmp- within the fm is critical to understand the process of membrane rupture during human labor. objectives. expression and secretion of mmp- in placental leukocytes, trafficking of the enzyme through the fm and one possible mechanism for its activation were analyzed. methods. leukocytes were isolated from placental blood of women after active labor. maternal leukocytes were used as controls. cells were cultured for h. relative expression of mmp- by rt-pcr and enzyme secretion by elisa and zymography were followed at different times. to analyze the traffic of mmp- through the fm, fluorescein-conjugated prommp- was added to the choriodecidual side of the fm in an in vitro system that allows the separation of amnion and chorion. labeled mmp- was localized at distinct times by confocal microscopy. the protease responsible for the activation of mmp- was identified using neutralizing antibodies and specific inhibitors. results. no difference in the relative expression of mmp- in leukocytes throughout the culture was found. however, secretion of the enzyme significantly increased since h (p< . ). experiments using labeled mmp- , repeatedly showed that after h in culture, the enzyme was mainly localized within the amniotic epithelium. specific inhibition of mmp- significantly decreased the activation of pro-mmp- . conclusions. our results demonstrate that the increased secretion of mmp- by placental leukocytes is not associated to increased gene expression, suggesting that homing of a specific leukocyte subpopulation to the choriodecidua is occurring during labor. mmp- can be trafficked from the choriodecidua to the amnion, suggesting a transmembranal pathway that may regulate the tissular localization of the enzyme to the area of the fm with high content of connective tissue. once secreted by the placental leukocytes, activation of mmp- depends mainly on mmp- , which seems to be derived from the same leukocytes. objective: preterm labour is a major problem in terms of perinatal morbidity and mortality. the histone-deacetylase inhibitor (hdaci), trichostatin a (tsa) has been shown to have an inhibitory effect on myometrial contractility. the aim of this study was to evaluate the effect of the hdaci's suberic bishydroxymate (sbha) and valproic acid (vpa) on human uterine contractions and hence their potential role as tocolytic agents. methods: biopsies of human myometrium were obtained at elective caesarean section (n= ). dissected myometrial strips suspended under isometric conditions, undergoing spontaneous and oxytocin-induced contractions, were exposed to cumulative additions of sbha in the concentration range of nmol/l to mmol/l and vpa ( nmol/l- mmol/l). control experiments were run simultaneously. results: sbha and vpa exerted a potent and cumulative inhibitory effect on spontaneous and oxytocin-induced contractions, compared to control strips. the mean maximal inhibition (mmi) values for sbha were . % for spontaneous contractions (n= ; p< . ), and . % for oxytocin-induced contractions (n= ; p< . ). the mmi values for vpa were . % for spontaneous contractions (n= ; p< . ), and . % for oxytocin-induced contractions (n= ; p< . ). conclusion: these results raise the possibility that hdaci's may have tocolytic potential, in addition to their current clinical indications. the inhibitory effect observed may be linked to the ability of hdac inhibitors to induce the expression of genes involved in the maintenance of myometrial quiescence via epigenetic mechanisms but may potentially also involve non-epigenetic pathways. progestin suppresses thrombin-enhanced interleukin- expression in term decidual cells: implications for abruption-induced preterm delivery. edward kuczynski, lynn f buchwalder, frederick schatz, charles j lockwood. obstetrics/gynecology reprod. sciences, yale university school of medicine, new haven, ct, usa. background and objective: decidual hemorrhage (abruption) promotes binding of factor vii to decidual cell (dc)-expressed tissue factor to generate thrombin. thrombin in turn induces several biological effects leading to preterm delivery via activation of cell surface protease-activated receptors (pars). interleukin- (il- ) is a pleiotropic proinflammatory cytokine induced by pars. this study assessed the separate and interactive effects of thrombin and medroxyprogesterone acetate (mpa) on il- expression in term dcs. methods: term decidua from stripped fetal membranes were isolated and the dcs were purified on a percoll gradient, grown to confluence and passaged until leukocyte-free. confluent dcs were primed in - m estradiol (e ) of e + - m mpa for days, then incubated in a defined medium (dm) with corresponding steroids ± thrombin. after hours, il- levels in conditioned dm were measured by elisa and western blotting. in parallel -hour incubations, il- mrna levels were assessed by quantitative rt-pcr and normalized to -actin mrna. results: secreted il- levels were similar in cultures maintained in e alone ( . ± . ) and e + mpa ( . ± . pg/ml/ug protein; mean ± sem; n = ). the addition of thrombin ( . u/ml) enhanced secreted il- levels by . ± . fold (p< . ) in incubations with e and by . ± . -fold (p< . ) in incubations with e + mpa. the inhibitory effect of mpa was statistically significant (p< . ). in confluent dcs incubated with e + mpa, exogenous thrombin ( . - . u/ml) elicited a concentration-dependent increase in secreted il- levels. hirudin acted as a pure thrombin antagonist, exerting no agonist effects alone, but counteracting thrombin-enhanced il- secretion. western blotting confirmed the elisa results. quantitative rt-pcr confirmed that il- m-rna levels corresponded to protein changes. conclusion: thrombin enhances il- mrna and protein expression in term dcs and progestin blunts these effects. since thrombin-generating abruption is closely associated with preterm delivery, anti-inflammatory effects induced by progestin inhibition of dc-derived il- may contribute to the protection against ptd, and may explain the reported protective effects of administration of -oh-progesterone in recent clinical trials. introduction: catechol-o-methyltransferase (comt) catalyzes the methylation of the phenolic hydroxyl groups in a variety of catechols. during estrogen metabolism, this enzyme converts the catechol estrogen, -hydroxyestrogen ( ohe ), to -ethoxyestrogen ( meohe ). the comt substrate, -ohe , can exhibit an anti-estrogenic effect in multiple biologic assays while the methoxyestrogen ( -meoe ) can exhibit an estrogenic effect. the biologic activities of these estrogen metabolites ( ohe meoe) depend upon their concentrations and tissue type. since comt activity ultimately controls levels of these metabolites, it appears to be a key factor in regulating the cellular estrogenic milieu. we have recently reported that amnion layers of human fetal membranes from laboring women exhibited folds higher comt mrna expression when compared to non-laboring women (wentz et. al. sgi ) . objective: to investigate the impact of comt inhibition on prostaglandin e (pge ) production by human amniotic membrane explants. study design: explants consisting of -cm circular sections of the amnion layer (obtained from term pregnant women who underwent elective repeat cesarean section) were prepared and placed in tissue culture explants media at ºc. after a -hour incubation, explants were treated with the selective comt inhibitor ro - , at and m concentrations. the incubation media was harvested after and -hour intervals. the levels of prostaglandin e (pge ) in the media were measured by sensitive elisa and were normalized against total protein concentration. results: ro - comt inhibitor induced major reductions in pge production in media collected from amnion explants of human fetal membranes. in the group treated with m of ro - for hours there was %± % reduction of pge after hours compared to untreated control (p< . ). in the amnion explants treated with m of ro - , there was %± % after hours and %± % after hours of treatment compared to untreated control (p< . ). conclusions: this finding indicates that comt activity in the amnion layer of human fetal membranes affects pge production. by facilitating a pro-estrogenic milieu in human fetal membranes in late gestation, increased comt activity may indirectly increases production of factors associated with labor such as pge . hypothesis: an extensive remodeling of the human cervical connective tissue takes place throughout pregnancy with a decrease in the total concentration of collagen and proteoglycans due to an altered higher metabolic turnover. we hypothesize that the profound changes in proteoglycan production in the human pregnant cervix can be seen in corresponding cervical fibroblasts as well. we also hypothesize that proteoglycan production in cervical fibroblasts from preterm partal women are simmilar to the production in fibroblasts from partal women. method: cervical biopsies were obtained from non-pregnant women, women during elective cesarean section, woman after spontaneous parturition and after a preterm vaginal delivery. by explant technique fibroblasts were cultured from the biopsies. produced proteoglycans were metabolically labeled with s during hours and then purified by ion-exchange chromatography and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. results: the total proteoglycan production decreases with approximately % in partal and preterm partal cell cultures. the reduction of proteoglycans in preterm partal and partal cell cultures is significant compared to non pregnant fibroblast cultures. the distribution of biglycan and perlecan are similar in partal and preterm partal cells. biglycan are significantly reduced by around % and perlecan are significantly increased by around % compared to non pregnant cultures. preterm partal cervical fibroblasts secreates significantly more heparan sulfate proteoglycans compared to non pregnant cultures. the changes in total proteoglycan production in the human pregnant cervix can be seen in corresponding cervical fibroblasts as well. both partal and preterm partal cell cultures differ in their proteoglycan production compared to their non pregnant counterpart, suggesting a role for proteoglycans in cervical ripening. the role of the oxytocin/oxytocin receptor system is not well defined in human amnion. previous studies in rabbit amnion have demonstrated an up-regulation of oxytocin receptors in the end of pregnancy and have shown that there is a large increase in the ability of ot to stimulate pge production. we and others have previously shown a role for nf-b in otr regulation. in whole genome array analysis of human amnion we found that otr was the gene with the second highest increase associated with activation of nf-b (the highest being cox- ). the present work was directed towards further understanding of otr expression and function in human amnion at term. we have shown that pge release by pre-labour primary human amnion cells is significantly increased after oxytocin treatment for hrs (ot: - m; n= ; triplicate samples; fold increase p< . ). the expression of otr in labour (+) and labour (-) primary amnion cells was measured with real time rt-pcr. we found a significantly higher level of expression in the labour (+) cells (n= ; duplicate samples; p< . ). western blot analysis confirmed the upregulation of otr in labouring amnion. treatment with il- b resulted in a significant upregulation of otr which peaked with a fold induction after hours (p< . ). il- caused a fold increase in otr mrna levels in labour (-) cells, bringing the expression level up to that found in labour (+) cells. our findings provide further evidence for a role of otr in human amnion. expression of otr in human amnion is significantly increased after the onset of labour. term non laboured amnion can be stimulated with il- to increase otr expression to levels of laboured amnion. one of the functions of otr in amnion cells is stimulation of prostaglandin synthesis. expression of antioxidant defence proteins in human myometrium before and after the onset of labour. vasso terzidou, mandeep s kandola, shirin khanjani, jan j brosens, phillip r bennett. parturition reserach group, irdb, imperial college, london, united kingdom. background oxidative stress is a result of an imbalance between the production of reactive oxygen species (ros) and the antioxidant defence mechanisms present in biological systems. parturition and infection-induced preterm labour resemble inflammatory processes that are linked to the production of ros including super-oxide (o -), hydrogen peroxide (h o ) and peroxynitrite. low concentrations of ros can act as second messengers in the regulation of several cellular functions. in an attempt to maintain redox homeostasis cells are equipped with machineries to both produce and scavenge ros. enzymatic scavengers include superoxide dismutase (sod), glutathione peroxidise and catalase. sod is the first enzymatic step in the defence system against oxidative stress. nuclear factor-kappa b (nf-b), a transcription factor family classically associated with inflammation, is activated in response to infection and proinflammatory cytokines, such as those prevalent during labour. labour is associated with an increase in nf-b activity in human myometrium and in fetal membranes. nf-b is known to regulate a range of genes associated with the onset of labour. in a study using affymetrix whole genome arrays analysis nf-b overexpression was associated with increased expression of sod- ( . fold). to determine changes in ros scavenging potential upon onset of labour, lower segment myometrial biopsies were taken at term before and after the onset of labour (n= ; each group). cdna was extracted from the tissues and real time rt-pcr was performed for sod- , serum/glucocorticoidinduced protein kinase- (sgk- ) and the dna repair enzyme gadd a. we found that labour onset is associated with a two fold increase in the levels of expression of each. oxidative damage at the fetomaternal interphase has been extensively studied in the placenta as part of investigations for first trimester pregnancy losses, iugr and pre-eclampsia. the role of ros is less well defined in human decidua and the underlying myometrium. our results suggest a role for oxidative stress and redox homeostasis in the maintenance of myometrial quiescene during pregnancy and onset of labour. plasma anandamide levels increase during labour induction and appear to delay labour progression. vijianitha nallendran, anthony h taylor, patricia mw lam, stephen c bell, david j taylor, justin c konje. cancer studies and molecular medicine, university of leicester, leicester, united kingdom. background: the evidence for a role of the endocannabinoid, anandamide (aea) in labour is conflicting. we previously showed elevated aea plasma levels in labouring women whilst another group showed that activation of functional receptors for anandamide actually inhibited uterotonin-induced contractions through an adenylate cyclase pathway . the aim of this study was to explore further the relationship between labour and plasma aea levels. methods: plasma aea levels in women undergoing induction of labour for various indications at term, were measured by a sensitive isotope dilution method using hplc-ms/ms. each volunteer had an assessment of her cervix prior to the start of the induction when the first sample for aea was collected. once active labour was established (cervix cm dilated; contractions every - minutes), a second sample was collected. results: seventeen ( %) of the subjects were multigravida. the inductions were for postdates(n= ); decreased fetal movements(n= ), fetal growth restriction(n= ), symphysis pubis dysfunction(n= ), diabetes mellitus(n= ), pre-eclampsia(n= ), fetal cardiac complication(n= ), spontaneous rupture of newborn offspring with persistent pulmonary hypertension, despite enhanced fetal membranes(n= ). plasma aea levels increased significantly once labour was established (figure) and demonstrated in ( %) of the cases. the median (interquartile range) plasma aea level increased from . nm ( . - . ) at induction to . nm ( . - . ); *p= . ; mann-whitney u-test) at active labour. there was a positive correlation between plasma anandamide levels taken before induction and the time taken for the women to enter into active labour (r = . , p= . pearson correlation). plasma aea levels were higher in labouring women compared to non-labouring women after the induction of labour confirming our previous observations and suggesting a direct role for this endocannabinoid in labour. further studies are required to elucidate this role. nuclear factor-kappa b (nf-b) is a transcription factor family classically associated with inflammation, activated in response to infection and proinflammatory cytokines, such as those prevalent during labour. as a cytokineinducible transcription factor it plays a key role in the expression of a variety of genes involved in inflammatory responses and cell survival. nf-b dna binding and transcriptional activity plays an important role in labour associated gene expression in myometrium. in this study we examined the range of genes regulated by nf-b in myometrium using transient transfections and wholegenome array analysis. myometrial cells were extracted from myometrial biopsies taken at the time of elective caesarean sections at term. transient transfections of primary myocytes were performed using expression vectors for nf-b p .the amount of dna was constant and psg was used as a control construct. cdna was made from myocytes transfected with either nf-b or psg . affymetrix genechip u microarray was performed (n= , each group). we found that genes were significantly differentially expressed between control and overexpressed nf-b samples. twenty eight of these genes were upregulated with nf-b and were down regulated. several chemokines and cytokines were identified in the upregulated group. interleukin demonstrated the highest, fold, induction in the upregulated group, followed by tumor necrosis factor, a-induced protein (tnfaip ), chemokine ligand (ccl ), chemokine ligand (ccl ), pentaxin related gene (ptx ), interleukin (il ) and superoxide dismutase (sod- ). nine genes were present in the nf-b group that were absent in the control group. these included chemokine ligand (ccl ), chemokine ligand (ccl ), chemokine ligand (cxcl ) and il . ingenuity pathway analysis demonstrated that immune response, inflammatory, cell growth and proliferation and cell death were the main pathways involved. standardisation experiments have been performed, and the microarray results were confirmed with real time rt-pcr in several candidate genes. our results provide further support in the role of nf-b in human labour and suggest its direct link in upregulation of inflammatory genes, cytokines and chemokines, consistent with the imflammatory nature of the biochemistry of labour. inflammation is widely accepted to be a key feature of human labor. secretory leukocyte protease inhibitor (slpi), an innate immune molecule, has been shown to be an antimicrobial and anti-inflammatory protein. the aims of this study were to verify its expression and localization in human myometrium methods: specimens were obtained at time of cesarean delivery with or without labor. expression and localization of slpi was detected using immunohistochemistry. slpi expression pattern relative to nf-b p subunit was compared between not in labor and in labor subjects, between different tissue sections as well as in in vitro model systems including myometrial explants, uterine smooth muscle cells (usmc) and ishikawa endometrial adenocarcinoma cells. slpi was predominantly localized to the nuclei of myocytes. the observed nuclear immunoreactivity of myocytes was increased during the labor relative to not in labor, paralleled with p nuclear translocation. the nuclear pattern of slpi is specific to myometrium since slpi immunostaining was present exclusively in the cytoplasm of all other tissues examined, including amnion, chorion, decidua and endometrium. slpi staining was also positive in macrophages, indicated by co-localization of slpi with cd positive cells. treatment with il- or tnf-induced nuclear translocation of p in myometrium explants and usmc, but not in ishikawa cells. in human myometrium, slpi is predominantly localized in the nuclei of myocytes and in macrophages. the nuclear expression pattern of slpi is myometrium-specific and increased following the onset of labor and correlated with nf-b activation. further understanding of its physiological significance may suggest new strategies aimed at preventing preterm birth. application of a new proteomic technology on amniotic fluid in prom. sara consonni, niccolo bosso, marianna andreani, agnese pizzardi, fulvio magni, anna locatelli. obstetrics and gynaecology, university of milano-bicocca, monza, italy; experimental medicine, university of milano-bicocca, monza, italy. objective: mass spectrometry (ms) is the obligatory tool for proteomics studies. biological samples must be purified before ms analysis due to the matrix complexity. a recent approach combines active surface prepurification with maldi-tof (matrix assisted laser desorption) analysis. clinprot technology provides the prepurification of the sample through the use of magnetic beads (mb) with activated surface. this technique can be carried out by robot in an automated way on a large number of samples. a unique example of the use of mb before maldi-tof analysis to determine proteomic profiles of amniotic fluid (af) is reported for rapid detection of fetal aneuploidies (wang ) . the objective of our study was to verify the applicability of clinprot prepurification before maldi-tof analysis on amniotic fluid collected noninvasively in premature rupture of membranes (prom). methods: we sampled af from vaginal posterior fornix of women with preterm prom (group , n= ) and term prom (group , n= ). samples were prepared with mb and analyzed with ms maldi-tof in order to generate proteomic profiles. results: it was possible to generate average proteomic profiles in the two study groups and the observed profiles were different. samples of af non invasively collected in prom can be analyzed by ms maldi-tof after preparation with mb. this technique allows to retain part of the eluted sample for characterization of protein peaks of interest. due to the less laborious characteristics of this method in comparison with techniques based on bidimensional electrophoresis its application can be useful in clinical proteomics. progestins accentuate the maternal but not fetal inflammatory response of women with intra-amniotic inflammation. sonya abdel-razeq, irina a buhimschi, michael cackovic, guoyang luo, antonette dulay, victor rosenberg, mert bahtiyar, errol norwitz, edmund funai, catalin buhimschi. ob./gyn. reprod.sci., yale university, new haven, ct, usa. introduction: data from animal research suggests that progestins have a marked pro-inflammatory capacity. recent studies support the administration of -hydroxyprogesterone caproate in women at risk for preterm birth. we sought to determine the impact of progestins during gestation on the extent of maternal and fetal inflammatory responses in pregnant women with intraamniotic inflammation. methods: amniotic fluid, placenta and cord blood were obtained from women who delivered preterm (median[range], ga: [ - ] wks). an amniocentesis was done to rule out infection. women exposed to progestins (n= ) within one week prior to amniocentesis were matched to controls (crl) by age, parity, history of preterm birth ga, membrane status and interval to delivery. proteomic profiling of amniotic fluid [mass restricted (mr) score] identified the presence or absence of intra-amniotic inflammation. an mr score of or confirmed intra-amniotic inflammation. amniotic fluid and umbilical cord interleukin- (il- ) levels were measured by elisa. histological chorioamnionitis was graded based on recognized criteria. results: overall, women and their fetuses exposed to progestin did not exhibit an increased inflammatory response (table) . however, sub-analysis restricted to women with mr or (n= ) showed that in the context of intraamniotic inflammation, progestins were associated with significantly elevated amniotic fluid il- levels compared to unexposed women (progestins (n= ): vs. crl (n= ): [ . - ] ng/ml, p= . ). these relationships were maintained after correction for steroid and antibiotic exposure. such significance was not found for amniotic fluid glucose, ldh, wbc count or cord blood il- . conclusion: our results suggest that progestins may amplify the maternal, but not the fetal inflammatory response of women with intraamniotic inflammation. objective: recently progestins have been shown to reduce the incidence of recurrent preterm labor in women. progestins have long been known to inhibit preterm labor in some species including rats and are also known to delay term labor. nitric oxide (no) donors, including ng, inhibit uterine contractility and have been used as tocolytics. the aim of this study was to examine the inhibitory effects of r on preterm labor in rats induced with a progesterone antagonist (onapristone, zk) with and without ng. materials and methods: charles river s-d timed pregnant rats (n= /group) were treated with zk ( mg/rat, s.c.) alone or vehicle (controls) on day of gestation. other groups of rats were treated with zk in combination with various doses of r ( . , or mg/rat s.c) with and without ng ( mg s.c. pellet) from days to of gestation. all rats were sacrificed on day of gestation and the number of fetuses and implantation sites were counted to determine the preterm delivery rate. one way anova was used for statistical analysis. p< . was considered significantly different. results: rats treated with zk alone delivered all their fetuses prematurely compared with controls (p< . ) treated with vehicle only (ca. % fetuses delivered). ng treatment alone did not affect the delivery rate (p> . ) compared to controls. similarly zk + ng did not reduce the preterm delivery rate compared to zk alone (p> . ). however r dose dependently reduced (p< . at all doses) the number of fetuses delivered prematurely in response to zk and the premature delivery rate was further reduced when treatment included the combination of r plus ng (p< . ). conclusions: zk effectively induces premature delivery. premature delivery produced by zk can be effectively reduced with a r , a progestin known to bind with high affinity to nuclear progesterone receptors. ng by itself, at the dosage used, does not reduce the prematurity rate caused by zk. however, r and ng act synergistically to reduce the preterm delivery rate. this study indicates that combinations of a progestin with an no donor may be an effective treatment for preterm labor and delivery. monkeys. pl grigsby, jp rasanen, , dw sadowsky, m bertolino, m carbonatto, e gillio tos, s canali, j lacy, a chollet, mj novy. reprod sci, oregon primate res ctr, beaverton, or; ob/gyn, oregon health sci univ, or, usa; rbm, merck serono, italy; merck serono, switzerland. objective: to investigate the pharmacokinetics (pk) and pharmacodynamics (pd) of as , a novel orally active non-peptide oxytocin (ot) antagonist in rhesus monkeys. study design: dose finding and pk/pd studies were done in chronically instrumented non-pregnant (n= ) and pregnant ( - dga, term d; n= ) monkeys. maternal and amniotic fluid compartments were serially sampled during dosing and washout. treatment phases included: ot infusion control, ot infusion + as , and ot rescue therapy. ot infusions ( - mu/kg/hr, iv) were given to determine the lowest dose required to produce stable, sub-maximal uterine contractions in each animal. as was administered p.o. at , , mg/kg and the effects on inhibition of uterine activity (hca, mmhg.sec/hr) were compared. to verify antagonist reversibility, objectives: infection such as chorioamnionitis is thought to be the cause of premature rupture of the membrane and induce uterine contractions leading to preterm delivery. however, the mechanism for enhancement of uterine contractility is not well understood. we have reported that non-selective cation channels (nsccs) regulate pacemaker potentials to generate rhythmical contractions and should be targets of magnesium ions used for tocolysis. the purpose of this study is to investigate the changes of the expression of nsccs during normal pregnancy and the effect of inflammation in preterm. methods; atp receptors (p x) and transient receptor potential canonical (trpc) channels were examined as uterine nsccs. the mrna was extracted from the rat myometrium of non-pregnant and pregnant rats at days , , and . the expression of each subtype of p x and trpc channels was measured by real time rt-pcr (abi ) with taqman probes (abi). as an inflammatory model lipopolysaccharide (lps; . mg/kg) was injected into intraperitoneal cavity at day and the tissue was sampled after six hours. results; p x and p x were determined to be dominant subtypes of p x channel. the expression of p x was increased by % at day , compared with day and that of p x was enhanced by %. on the other hands, trpc and trpc were detected dominantly. the expression of trpc was increased three times in the late stages of gestation. however, trpc was suppressed by %. in the lps treated rat myometrium cox- mrna expression was measured to be fold higher than that of the control rat, showing inflammatory effects in the myometrium. in this model the expressions of p x , p x and trpc were enhanced by . , . and . times, but trpc was not changed. the mrna expression of p x , p x and trpc channels in rat myometrium was increased in the late stages of pregnancy. these channels are suggested to be concerned with onset of labor. in the inflammatory model the expression of these channels was accelerated dramatically and these values were much higher than those in normal pregnancy. this finding supposed inflammation may enhance some types of nsccs to accelerate uterine contractility and induce preterm delivery. anandamide ( to determine the mechanism of rho protein activation during oxytocin and carbachol induced contraction, freshly prepared myometrial strips in krebs henseleit buffer were treated with oxytocin ( nm) and carbachol ( μm) under isometric and tension free conditions. control strips were exposed to buffer only. treated myometrial strips were solubilised and separated into membrane and cytosolic extracts and equal aliquots were immunoblotted with rhoa and rhob antibodies. rhoa translocated to the membrane after oxytocin and carbachol stimulation under both isometric and tension free conditions (p< . ). there were no significant changes in rhob membrane to cytosol ratios relative to control. agonist induced contraction in human myometrium is associated with rhoa but not rhob membrane translocation. during pregnancy components of the intracellular camp signalling pathway show increased gene expression resulting in the maintenance of myometrial quiescence until term where a substantial decrease in expression of these genes is observed. protein kinase a regulatory subunit riia (rii ) is upregulated at both the mrna and protein levels in the human myometrium during pregnancy. this particular subunit is membrane-bound and by directing phosphorylation to myometrial cytoskeletal proteins may affect contractile machinery thus playing a role in maintaining uterine relaxation. acetylation of histones promotes a favourable chromatin environment for transcriptional activity of many genes. this process is largely inhibited by histone deacetylases (hdacs), whereby its activity leads to transcriptional repression. hdacs can be recruited to the promoter region of a gene by other transcription factors such as sp proteins. since the rii promoter contains three sp - consensus binding sequences in its proximal part, we investigated whether this gene is a target for transcriptional regulation by hdacs. using dna precipitation assays we found that sp , and as well as hdac and form complexes with biotin-labelled fragments relating to sp - elements in the promoter region of rii . additionally, treatment of myometrial primary cell cultures with hdac inhibitor trichostatin a (tsa), or with the methyltransferase inhibitor -azac resulted in increased mrna and proteins levels. further studies with full and truncated luciferase constructs of the promoter region of the rii gene in transiently transfected myometrial cells confirmed that all three sp - elements are involved in the transcriptional regulation of the gene. this process involves hdacs, as h treatment with tsa significantly increased the luciferase signal. changes in the binding of sp proteins and hdacs to the promoter after tsa and azac treatment were investigated employing chromatin immunoprecipitation assays. alterations in the methylation status of the promoter after treatment were examined by bisulfite modification and dna sequencing. together, this study highlights the importance of chromatin modifications in the maintenance of uterine quiescence during pregnancy as well as identifying a potential mechanistic target for drugs that may reduce the incidence of preterm labour. objective: progesterone maintains pregnancy by promoting myometrial quiescence. typically progesterone effects are thought to be mediated through the classic genomic pathway. there is evidence, however, that progesterone also acts via a non-genomic pathway by interacting with specific membrane progesterone receptors (mprs) and in particular progesterone receptor membrane component - (pgrmc- ). the role of non-genomic progesterone actions in human pregnancy and parturition is not clearly understood. the goal of this study was to measure the extent of mpr expression in biopsy specimens of human myometrium obtained at cesarean delivery, and to determine whether expression changes with advancing gestation or the onset of labor. study design: lower uterine segment myometrial biopsies were obtained at the time of delivery from consenting women who were at term and not in labor (n= ), preterm and not in labor (n= ) and term and in labor (n= ). protein extracts were prepared and subjected to polyacrylamide gel electrophoresis and immunoblot analyses for pgrmc- and gapdh. abundance of pgrmc- protein relative to gapdh was determined by digital densitometry. we also performed immunohistochemistry (ihc) to determine the cellular localization of pgrmc- in the human pregnancy myometrium. results: pgrmc- protein was identified in each biopsy specimen. there was a -fold increase in pgrmc- protein in term compared with preterm biopsies (relative to gapdh p< . ). the relative level of pgrmc- protein was not different between biopsy specimens from laboring and non-laboring women at term (compared to gapdh, p= . ). pgrmc- immunoreactivity was localized to granular cytoplasmic staining. conclusion: this is the first description of the presence of pgrmc- protein in the human myometrium during pregnancy. its presence suggests that progesterone may influence contractility non-genomically via these receptors. the functional significance of the gestational age associated two fold increase in pgrmc- is unclear. changes in expression of this receptor during pregnancy may be important for the hormonal control of parturition and can be the focus of future studies. (ruddock et. al., abstract smfm, ) . the aim was to examine the mechanism of p inhibition. methods: uterine tissues from women (n= ) at term with cesarean section, were suspended in organ chambers and exposed to various agents or solvents. contractility was registered, stored, analyzed and compared before and after addition of agents or kcl. tissues were treated with p alone ( - to - m) or p bound to bovine serum albumin (bsa/p , - to - m p ), a progestin with low affinity to mpr (r , - - - m), or a non-sex steroid (cholesterol, - to - m). other tissues were pretreated with selective inhibitors of adenylate cyclase (sq , - m), guanylate cylase (odq, - m), phosphodiesterase (rolipram, - m), nitric oxide (no) synthases (l-name, - m) or a nuclear p receptor antagonist (mifepristone, mif, - m), followed by p . data were analyzed by anova for statistical differences (p< . ). results: p rapidly (< hour), effectively ( %) and dose-dependently inhibits spontaneous and kclinduced contractility (ed of < - m incubation of strips with zd- , at concentrations up to m for one hour prior to the experiment, led to no significant reduction in the total work done. however, incubation of strips with l- with a concentration of nm for one hour prior to the experiment, led to a statistically significant reduction in the total work done after one and a half hours. furthermore, when increasing concentrations of pge ( - to - ) were added to l- ( nm) pre-treated strips, total work done per contraction was comparable to that of non-treated controls. similar effects were not observed in zd- ( nm) pre-treated strips. since the reduction in contractility caused by l- was greater than that caused by zd- , it is likely that the stimulating effect of pge acts predominantly via ep receptors. taken together with our previous data showing an increase in ep at labour, this data shows that targetting an ep receptor may be a useful strategy in managaing pre-term labour. recently a truncated kda isoform of the er-has been described in human endothelial and testicular cells. we describe the presence of this kda erisoform in pregnant and immortalized non-pregnant human myometrial cells. methods: myometrial tissue obtained from non-laboring pregnant women undergoing cesarean section at term (n= ) was dissected prior to being finely minced. a portion of the tissue was placed in pbs and sonicated, while the remainder of the tissue was dissociated with collagenase prior to filtration and placement on culture plates. cells were then cultured in mem w/ % fetal bovine serum (fbs) until confluence. cultured cells were then dissociated with . % typsin/edta, subsequently re-plated and grown to confluence. immunohistochemistry directed toward smooth muscle protein was used to verify myometrial phenotype.) protein was extracted and quantified. western blot was performed with mouse monoclonal anti-er-receptor antibody (santa cruz biotechnology) to the f- domain of the human er-receptor. an immortalized non-pregnant human myometrial cell line provided by ann word, htert, was cultured and isolated as described above. results: htert cells expressed both the truncated ( kda) and the full length ( kda) er-isoforms. fresh and cultured myometrial cells from non-labored term pregnant patients also expressed both isoforms of er-. subsequent subcultures of myometrial tissue continued to express the kda erisoform, yet the expression of the kda isoform was lost. a representative blot is below. conclusions: we demonstrate, for the first time, the presence of a kda erisoform in cultured and non-cultured pregnant and cultured nonpregnant human myometrial tissue. discovery of this isoform of er-in myometrial tissue could provide insight into the molecular mechanisms involved in parturition. the action of prostaglandin f (pgf ), a potent uterotonic stimulant that is associated with labour at term and preterm, is mediated by its receptor, ptgfr. myometrial ptgfr mrna levels fall during pregnancy and this likely plays a role in uterine quiescence. however, the mechanisms by which this occurs are poorly understood. we previously reported that pgf downregulates fp mrna expression in cultured human myometrial ultr cells in a protein kinase c (pkc) dependent manner. in addition to the downregulation of mrna levels, receptor desensitization may also represent another mechanism of decreasing ptgfr activity because ligand binding to g protein coupled receptors often results in receptor internalization. we therefore hypothesized that pgf treatment of ultr cells also results in pkc dependent ptgfr internalization. methods: near confluent cultured human myometrial ultr cells were treated +/- - m or - m pgf for , , or hr. cells were fixed with formaldehyde and visualized for localization of ptgfr by immunofluorescence. to examine the potential involvement of pkc in the process, ultr cells were treated +/- - m pgf and +/- m myristoylated pkc inhibitor ( - ) and examined for ptgfr cellular localization as described above. results: pgf treatment resulted in a dose dependent decrease in ptgfr membrane signal at , , and h. this decrease was dependent on pkc as cotreatment with the myristoylated pkc inhibitor ( - ) prevented the pgf induced decrease in membrane ptgfr at h treatment. there was no visible effect of the pkc inhibitor on ptgfr membrane signal on its own. conclusion: we conclude that pgf decreases membrane levels of ptgfr protein in human myometrial ultr cells in a pkc dependent manner. these results suggest that pkc may be required for both the pgf induced internalization and desensitization of ptgfr protein and downregulation of ptgfr mrna in human myometrial ultr cells. therefore pkc may play a crucial role in downregulating ptgfr expression and activity and maintaining uterine quiescence during pregnancy. rhoa is a small gtpase that acts as a molecular switch to control a variety of signalling pathways in smooth muscle, including contractility. it is thought that increases in rhoa-gtp levels facilitates phosphorylation of target proteins such as cpi- , promoting contractility at pre-term and term labour in humans. however, in situations of acute or chronic hypoxia in the uterus, it is important that myometrial contractility and subsequent labour is not facilitated prematurely. given the importance of rhoa to a cell's response to hypoxia in other cell types, it was hypothesised that rhoa plays a central role in the mechanism controlling smooth muscle contraction in the uterus too. following acute hypoxia ( . % o ) for one to six hours, rhoa mrna, total protein and activation (rhoa-gtp) levels were analysed, using semi-quantitative pcrs and western blot, and compared to normoxic non-pregnant human uterine smooth muscle control cells. next, we investigated whether reduced oxygen conditions affected oxytocin induced activation of rhoa, following a two hour treatment of nm oxytocin. firstly, our results demonstrate that the rhoa itself is significantly activated under low oxygen conditions, resulting in phosphorylation of myosin phosphatase, myosin light chain and cofilin, three proteins known to be central in contraction and actin filament organisation. secondly, hypoxia significantly reduced the coupling of oxytocin to rhoa activation under the conditions examined. we observed a significantly reduced level of rhoa expression and activation which correlated with an increase in the level of another rhogtpase protein, rhoe. we propose that rhoa inactivation occurs through a rhoe-mediated mechanism, suggesting a balance in the activity of these two antagonistic rhogtpases in oxytocin-induced hypoxic human uterine smooth muscle cells. these results provide a possible explanation for the reduced coupling of oxytocin as a stimulant of myometrial contractions during slowly progressing labours. oxytocin-induced release of calcium ions (ca + ) from sarcoplasmic reticulum (sr) and sensitisation of contractile proteins to ca + have been suggested to mediate the oxytocin-induced potentiation of myometrial contractions. objective: we investigated the effects of oxytocin in the presence of nifedipine, a known inhibitor of the l-type calcium channel (ltcc). method: samples of myometrium were obtained from women undergoing term caesarean section with the approval of the local ethics committee. a standard organ bath system (ad instruments, uk) was employed to analyse contractile activity. stable spontaneous contractions were recorded for - minutes before addition of nifedipine. results: in agreement with our previous findings, application of oxytocin to spontaneously active strips produced a two-component effect: a transient tetanus-like contraction, followed by prolonged augmentation of phasic contractions. nifedipine ( um) rapidly abolishes spontaneous contractions, subsequent addition of nm oxytocin produced an initial, transient rise in force, approxiamately % compared to oxytocin alone, followed by high frequency oscillations in > % of strips. calcium-free solutions were used to confirm that oscillations were due to ca + entry. disabling the sr store using thapsigargin ( um) had no effect on oscillations, confirming the sr not to be involved. the t-type calcium channel blocker, mibefradil ( um ) showed no inhibition of oscillations. an ip receptor and store-operated calcium channel inhibitor, -aminoethyldiphenylborate ( -apb) um also had no effect on oxytocin-induced oscillations. the store-operated calcium channel inhibitor skf- ( um) showed partial inhibition of oscillations. conclusions: based on these results, we propose that the most likely mechanism of ca + entry producing oxytocin-induced oscillations in the presence of nifedipine is the transient receptor channel-c (trpc) channel, known to be present in the human myometrium. further work needs to be completed to clarify this further. identification of stim and orai in human myometrium. a new paradigm in store-operated calcium signaling. evonne c chin-smith, mark r johnson, rachel m tribe. division of reproduction and endocrinology, king's college london, london, united kingdom; department of maternal fetal medicine, imperial college school of medicine, chelsea and wesminster hospital, london, united kingdom. background: recent reports have suggested that two novel proteins, stim and orai, are involved in the regulation of store-operated calcium entry. we have previously reported that members of the trpc family, putative basal and store operated calcium entry channels, are present in human myometrium and regulated by labour associated stimuli il- beta and mechanical stretch. although stim and orai isoforms have been reported in other smooth muscle cell types, there are no published reports of stim and orai expression in human myometrium. the aim of this study was to identify mrna expression of stim , stim , orai and orai in human myometrium. methods: human myometrial biopsies were obtained from women undergoing elective caesarean section at term (prior to labour) with informed written consent and institutional ethics committee approval. whole myometrial tissue was either snap frozen and stored at - o c or used for cell culture. rna was extracted from whole tissue (n= ), primary cultured myometrial cells (n= ) and passaged (p ) myometrial cells (n= ) and stim , stim , orai and orai mrna expression was assesed by quantitative real-time pcr. results: all four genes were expressed in whole myometrial tissue and cells. stim and stim mrna expression in cultured myometrial smooth muscle cells (primary and passaged) was significantly reduced compared to myometrial tissue expression (p< . ). however, there was no significant difference in either orai or orai expression in whole tissue versus cultured myometrial smooth muscle cells. conclusion: to our knowledge this is the first report of stim / and orai / mrna expression in human myometrium. these genes may contribute to the regulation of calcium signalling in human myometrium, but the functional significance of their expression remains to be determined. funded by the bbsrc. obstetrics and gynecology, national university of ireland, galway, galway, ireland. objective: the ability of uterine smooth muscle cells to stimulate collagen contraction has been well established as an in vitro model of myometrial contractility. devost and zingg ( ) reported that the contractility of human myometrial cell lines layered onto collagen matrices was increased by oxytocin while another group described the stimulation of human uterine smooth muscle cell contractility, cultured within collagen lattices, with endothelin- (dallot et al., ) . our study investigated the response of human primary uterine smooth muscle cells cultured within collagen lattices, to various compounds, including the non-specific depolarizing agent potassium chloride (kcl), the inflammatory cytokine tnf , the rock- inhibitor y- , oxytocin, and oxytocin plus its clinically used antagonist, atosiban. methods: human primary uterine smooth muscle cells (utsmc) (lonza) were maintained in dmem high glucose media. cells ( , per well) passage - , were embedded in . mg/ml collagen, in . ml aliquots, in dmem-f media on well plates (invitrogen) (dallot et al., ) . effects on contraction were studied by monitoring changes in gel area (alpha innotech imager, image j software (nih)). statistical significance was determined by the student t test. results: the utsmcs displayed basal contraction of the collagen gels while the non-contractile cell line hek cells, did not. kcl ( nm) stimulated an % increase in contractility (n= , p= . ) while nm tnf resulted in an . % increase (n= , p= . ), in comparison to unstimulated cells embedded in gel. the rock inhibitor y- ( m) inhibited contractility, with a . % decrease in collagen gel contractility (n= , p= . ). a % increase (n= , p= . ) in utsmc embedded collagen contractility was observed with nm oxytocin, which was antagonized by atosiban ( m) (n= ). conclusion: this study highlights the importance of the development and optimisation of a reproducible human in vitro myometrial contractility model, to evaluate the effect of various known labor-associated, and also unknown compounds. this should aid in our understanding of the many complex biochemical pathways involved in myometrial contractility at labor, and ultimately contribute to the prevention of preterm labor. changes in intra-mural myometrial blood flow during spontaneous labour using d power doppler angiography (pda). nw jones, , nj raine-fenning, h mousa, mj taggart, k jayaprakasan, gj bugg. nottingham university hospitals nhs trust, united kingdom; nottingham university, united kingdom; university of newcastle upon tyne, united kingdom. methods d pda was used to measure the percentage (%) change in the vascularization index (vi), the flow index (fi) and the vascularization flow index (vfi) in a volume of myometrium at the uterine fundus of nulliparous women at term, in the first stage of uncomplicated spontaneous labour. d data sets were obtained during a single cycle of uterine relaxation (r ), contraction and subsequent relaxation (r ). measurements were made independantly by two authors (nwj and gjb) using vocal® (ge kretz) and the mean value was used for analysis. the results from each woman are presented as a % change of r . data is presented as medians [interquartile range (iqr)] and analysed using non-parametric tests. the median volume (cm ) of interest for r was . cm ( . - . cm ), for the contraction was . (fig. ) . the % change for all three indices between r and r was not significantly different. however, there was a significant difference between the contraction and r (p< . ). the mean intra-class correlation coefficient and % confidence interval (ci) of vi, fi and vfi for the authors (nwj and gjb) were . ( . - . ), . ( . - . ) and . ( . - . ), indicative of good inter-observer reliability. conclusion d pda is a useful and reliable tool in the assessment of changes in intramural myometrial blood flow, which was found to reduce significantly during a contraction but increase again during the following uterine relaxation. . raine-fenning nj, et al. the inter-observer reliability of d pda acquisition within the female pelvis. ultrasound obstet gynecol. ; : - . the role of pgf on myometrial contractility; studied with a selective fp antagonist. shankari arulkumaran, andre chollet, phillip r bennet. imperial college parturition research group, institute of reproductive and developmental biology, hammersmith hospital campus, london, united kingdom; merck serono, geneva, switzerland. objectives: human myometrial strips established in culture will usually begin contracting after one hour. we have previously shown that stretch upregulates prostaglandin synthesis and have therefore hypothesized that spontaneous contractions occur because of stretch-related prostaglandin synthesis. methods: experiments were performed using x mm human pre-labour, lower uterine segment myometrial strips in a dmt myograph ms in oxygenated kreb's solution, with adi powerlab software. spontaneous contractions required stretch force and initial experiments determined that the maximum number of strips attaining spontaneous contractions was greatest at a force of - g. a novel antagonist to pgf (fpa) was studied in this model. the fpa has a ki of nm for fp and is - fold selective for fp compared with other prostanoid receptors. results: addition of pge and pgf after the commencement of spontaneous contractions caused a statistically significant increase in the total work done by the strips. the effect of pge was being greater than that of pgf . pre-incubation of the baths with a novel and selective fp antagonist (fpa) with concentrations up to m ( - ) did not affect the total work done by spontaneous contractions compared to non-treated controls. however, increasing concentrations of the fpa ( - to - ) decreased the total work done by -fold on strips treated with pgf beforehand in comparison the pgf -treated strips alone. conclusion: these data suggest that stretch and synthesis of prostaglandins is essential for spontaneous contractility in human myometrial strips. since fpa is able to block pgf induced but not spontaneous contractions, it is likely that pgf does not play a role in spontaneous myometrial contractility in vitro, although it may do so in vivo. because other factors may combine to increase contractility, the combination of an fp antagonist with other inhibitors may therefore, be a more effective strategy in reducing pre-term deliveries. objectives: brain natriuretic peptide (bnp) is synthesized in fetal membranes and inhibits oxytocin-induced contraction of preterm human myometrium. we hypothesized that bnp may be a paracrine mediator of human myometrial quiescence. we showed bnp content is higher in membranes from preterm pregnancies absent labor and significantly decreased with idiopathic preterm labor. while bnp activates natriuretic peptides receptors a (npr-a), b (npr-b), and its clearance receptor (npr-c), we have shown bnp does not inhibit myometrial contraction via npr-a or npr-b. herein, we test in part the hypothesis that bnp inhibits myometrial contractions by activating npr-c by quantitating npr-c in human myometrium at different gestations and labor status. methods: myometrial samples were obtained at the time of cesarean section after informed consent from groups of patients: preterm not in labor (pt-nl), preterm in labor (pt-l), term not in labor (t-nl) and term in labor (t-l). myometrial samples were obtained from women - weeks' gestation, and term between and weeks. mrna for npr-a, b and c were semiquantitated by realtime pcr (normalized by s mrna), and npr-c protein by western blot. results: natriuretic peptide receptors a, b and c mrnas were identified in all groups. while there were no differences in mrna levels among groups, npr-c protein was increased in samples from term laboring women. conclusion: since bnp inhibits the contraction of human preterm but not term myometrium independent of npr-a and npr-b, we have previously speculated that bnp activates another "unknown" receptor. herein we find that myometrial npr-c protein but not mrna increases during human labor at term. the increase in npr-c at term could compete with the unknown quiescent receptor to functionally reduce the availability of bnp and thus permit or promote myometrial activation/contraction. (supported by a grant from chilean government fondecyt ). objective: bnp is synthesized within human chorion and amnion and inhibits oxytocin-induced contraction of human myometrium. bnp activates guanylate cyclase (gc) natriuretic peptides receptors a (npr-a) and b (npr-b). bnp also stimulates the clearance receptor (npr-c) whose action is not mediated by gc. the intracellular pathway of bnp/npr-c inhibition is not known. we determined that bnp does not inhibit myometrial contraction via npr-a or -b. we hypothesized that bnp inhibits myometrium by npr-c activation. we test aspects of our hypothesis by determining whether bnp/npr-c pathway inhibits myometrial contractions via myometrial nos pathway. methods: bnp and canp (specific npr-c agonist) were added to primary human myometrial cell cultures and enos/inos activity/expression determined. cell cultures (n= ) were prepared from myometrial samples of nonlaboring, term pregnant women. after confluence ( d), the cells were incubated ( h) with nm bnp and/or canp. nos activity was measured ( l-citruline assay) and transcription/translation semi quantitated (realtime pcr and western blotting). results: bnp had no effect on either nos expression or activity. canp significantly reduced inos but not enos mrna level. canp did not alter the protein level of nos but significantly reduced nos activity. co-incubation with bnp and canp reduced both mrna levels (p< . ) and significantly decreased enos, but not inos, protein without change in overall nos activity. within the female pelvis. ultrasound obstet gynecol. ; : - . conclusion: the activation of npr-c by canp reduces nos activity and expression. the same effect was not observed by bnp. the difference may be explained because bnp (but not canp) activates all natriuretic peptide receptors, and they may have opposite actions on nos pathway. we conclude unlikely bnp inhibits human myometrial contraction by npr-c activation via the nos pathway. ( introduction: ultr is a retroviral immortalized human uterine smooth muscle cell line which we use as a model for uterine hypertrophy studies. however, this cell line has limited usage due to a reduced rate of cell division and eventually replicative senescence in culture. one of the mechanisms of human somatic cellular senescence is un-compensated shortening of telomeres, the specialized dna structures located at the ends of eukaryotic chromosomes. introduction of human telomere reverse transcriptase (htert) has been shown to induce telomerase activity and telomere elongation, and extend life-span of normal human cells. objective: to recover ultr cell division without altering the phenotypic characteristics of smooth muscle cells by introducing htert, therefore improving ultr cell line. methods: ultr cells were transfected with a modified htert expression vector at a relatively early stage (passage ) in the presence of selective antibiotic. ultr cell growth rate, with (ultr-ht) or without transfection, was determined by plating cells in multiple plates at a fixed density and counting cell numbers after days. single cell clones of stably transfected cells were further isolated by plating in well plates. expression of htert, smooth muscle specific genes (smc-sgs), and target genes was identified by rt-pcr of total rna extracted from ultr and ultr-ht cells. results: rt-pcr demonstrated successful introduction of htert into ultr cells. the growth rate of ultr-ht cells was increased and cell morphology was improved (free of the typical aneupoid appearance). at passage , ultr-ht cells grew . fold (p< . ) and . fold (p< . ) faster than ultr cells at passages and , respectively. currently single cell clones have been selected. ultr-ht cells express a set of smc-sgs, including -actin, caldesmon, calponin, myosin heavy chain, sm , and smoothelin, confirming that the smooth muscle phenotype is preserved. a panel of genes involved in angiotensin ii signalling, including angiotensin ii receptors (at / ), nox family (nox , , and duox ), nox-associated genes (p phox, p phox, p phox, and rac / ), was also preserved. conclusion: this is the first report of rescuing uterine smooth muscle cell replicative senescence via activation of telomerase. we have established a human uterine smooth muscle cell line which will provide an improved in vitro model for studying human myometrium. at term, myometrium is characterized by spontaneous contractions which vary in the amplitude, frequency and duration depending on specie and hormonal status. repetitive depolarization of plasma membrane followed by transient elevation in [ca + ] i is thought to underlie the contractions. however, the detailed pathways which regulate the spontaneous contractility remain unclear. objective: this study was designed to elucidate the effect of protein kinase c (pkc) on the spontaneous contractions and [ca + ] i transients in the myometria from term pregnant mice and women. methods: the human samples were obtained from women undergoing cesarean section with the approval from the institutional review board committee while mice myometria were dissected from the days pregnant mice sacrificed according to the animal study protocol. the isometric force and [ca + ] i were measured simultaneously in fura- loaded myometrial strips using spectrofluorometer equipped with force transducer. results: the pkc activator phorbol , -dibutyrate (pdbu) applied at - m first stimulated the amplitude of spontaneous contractions in mice and human myometria followed by their inhibition. under the pdbu treatment the frequency of the contractions was first increased and then decreased in both species. at the same time, pdbu didn't initially increase the amplitude of [ca + ] i transients but attenuated it over time. however, the frequency of the transients was first increased and later decreased upon the pdbu exposure. in addition, pkc activation with pdbu resulted in the elevation of the uterine basal tone without corresponding changes in the basal level of [ca + ] i in human myometrium. in mice myometrium, on the contrary, pkc activation didn't result in an increase of the muscle tone and the basal level of [ca + ] i . conclusions: we propose that pkc causes bi-phasic effect on uterine spontaneous contraction in mice and human first to potentiate the amplitudes and the frequencies and later decreases them. the amplitude of [ca + ] i was not initially potentiated by pdbu suggestive of the dissociation of the contractile and [ca + ] i response. the stimulatory effect of pdbu on the basal muscle tone in human myometrium and the absence of the effect in mouse suggest different mode of pkc action on uterine contraction in human and mice. introduction: mechanical stretch of uterine myocytes is detected through integrins on the cell surface which form part of the focal adhesion complex, this signals through mapk, ultimately leading to the up-regulation of various pro-labour genes including pghs- and il- . on integrin activation fak is recruited to the focal adhesion complex and activated by autophosphorylation at tyr- , creating a src binding site. this promotes further fak phosphorylation at tyr- , and , enhancing fak catalytic activity. however fak can also act as a scaffold protein and its exact role in the expression of pro-labour genes is uncertain. in this study we used inhibitors for the kinase activity of fak and mek / in order to test the affect of fak kinase activity on expression on pghs- mrna. methods: primary human myometrial cell cultures were grown from myometrial biopsies taken from women undergoing elective caesarean section. cells were plated onto -well flexible bottom plates coated in type i collagen. cells were subjected to % static stretch for up to minutes. cells were also incubated with either the rho kinase inhibitor y or the mek / inhibitor u prior to being stretched. western blots were performed using antibodies to fak phospho- , fak phospho- and erk / phospho- / , -actin was used as a loading control. rna was also extracted and levels of pghs- measured using q-pcr. results: stretch increased levels of phosphorylation at both tyr- and tyr- with the greatest increases occurring at and minutes. however the increase at tyr- appeared to be greater than at tyr- . incubation with the rho kinase inhibitor reduced phosphorylation of fak- , however this did not affect phosphorylation of erk / or stretch induced up regulation of pghs- mrna. in contrast incubation with the mek / inhibitor reduced erk / phosphorylation and expression of pghs- mrna, whilst also reducing fak phosphorylation (n= ; p= . ). conclusions: fak has previously been shown to be important in activation of the stretch induced mapk cascade and pghs- expression. however these data suggest that while stretch causes fak phosphorylation fak kinase activity is not essential to pghs- expression. this suggests fak may be acting as a protein scaffold. . our aim was to examine the effects of both natural progesterone and hp on spontaneous myometrial contractions. myometrial biopsies were taken with informed consent and ethics approval from non-labouring women at elective caesarean section weeks gestation. strips of myometrium mm long , mm wide were cut and suspended under a resting tension of mn in organ baths of krebs gassed with % o / % co within hours of collection. progesterone or hp were added in a cumulative manner at -minute intervals. changes in amplitude were recorded. results were compared using anova. following equilibration for hours, myometrial strips contracted in a rhythmic manner (amplitude . ± . mn, n= pairs). progesterone ( nm- m) produced a concentration dependent inhibitory effect on myometrial contractions (fig ), which was greater than that of vehicle (p< . ). maximum inhibition measured . ± . % and . ± . % for progesterone and vehicle, respectively. hp exerted an inhibitory effect, this was not significantly different from the vehicle (p> . ). progesterone exerts an inhibitory effect on myometrial contractility in vitro. this is apparent within minutes suggesting a nongenomic action. our data are in agreement with some reports in the literature but conflict with others[ ]. this acute inhibition of myometrial contractility may contribute to the mechanism by which progesterone prevents preterm birth. in contrast, we were unable to demonstrate an inhibitory effect of hp on contractility despite its demonstrated ability to reduce the incidence of preterm delivery [ ] . our data suggest that natural progesterone may be a more effective tocolytic agent in the acute setting than hp. obstetrics and gynecology, ramathibodi hospital, mahidol university, bangkok, thailand; pathology, ramathibodi hospital, mahidol university, bangkok, thailand; obstetrics and gynecology, samuel lunenfeld research institute, toronto, canada. objective: chemokines has been shown to play an important role in regulating uterine function. recent evidence demonstrates that monocyte chemotactic protein (mcp) level in amniotic fluid increases during spontaneous labor. the aim of this study was to examine the expression of mcp in human myometrium. methods: myometrial biopsies were taken from nonpregnant women undergoing hysterectomy and term pregnant women undergoing cesarean section followed written consent and local ethics committee approval. elective cesarean section was performed before the onset of labor while emegency section was done after the onset of labor. immunolocalization (n = each) was performed on paraffin sections by avidin biotin complex (abc) technique using monoclonal antibody specific to human mcp . reverse transcriptionpolymerase chain reaction (n = each) using gene specific primer against mcp and mcp receptor was performed to identify mcp messenger(m) rna in human myometrium . results: immunohistochemical findings demonstrated mcp in human myometrial cells from nonpregnant, term pregnant women before and after the onset of labor. mcp was labelled on plasma membrane and cytoplasm of myocytes from these three groups of women. similarly, mcp and mcp receptor mrna were found in nonpregnant and term pregnant women before and after the onset of labor. in this prospective study a group of fetuses was consecutively enrolled. one ultrasound examination was performed to each patient within days from delivery. we considered fetal macrosomia a birthweight g and big babies a birthweight g. cut-off points for identifying the best value of fetal abdominal circumference for fetal macrosomia prediction were chosen by receiving operator characteristics' curve (roc) analysis. using the best cut-off indicated by roc analysis, specificity and sensitivity were calculated. mean gestational age at delivery was + weeks ( + sd). neonates weighted less than g, had a weight range between and g and weighted more than g. the fetal ac measurement was the selected criterium to evaluate the risk of fetal macrosomia. to identify macrosomic fetuses the roc curve analysis identified a cut off of ac > mm which allowed to select fetuses. analysing this population we found true negative cases, false negatives, false positives and true positives, with a sensitivity of . % and a specificity of %. to detect big babies the roc curve analysis identified a cut off of ac > mm (n. fetuses), reaching a sensitivity of % and a specificity of %, without false negative cases and with false positives ( true positives, true negatives). the cases that weighted between and g, were included in the cases considered false positives by this cut-off. conclusions. these results clearly indicated that ultrasounds alone can not be used to manage a pregnancy suspected for fetal macrosomia or big babies. in fact, to avoid shoulder dystocia and all other complications strictly related to macrosomic fetuses, clinicians should perform a large number of useless elective cesarean sections. . pathway analysis of differentially expressed genes (pa; z-score . ) showed up-regulation of axon guidance, folate biosynthesis, nitrogen metabolism and down-regulation of steroid biosynthesis, insulin signaling, oxidative phosphorylation, tgf-beta signaling, and ubiquinone biosynthesis pathways in cm vs cf. comparison of mnr vs c in f showed genes up-and down-regulated (n= , ; p< . ). pa showed up-regulation steroid biosynthesis, fatty acid metabolism, glycolysis/gluconeogenesis, phosphatidylinositol signaling, ketone body metabolism, and ubiquinone biosynthesis pathways and down-regulation of bile acid biosynthesis, cell adhesion molecules, dna polymerase, notch signaling and ti diabetes mellitus pathways in mnr vs c. comparison of mnr vs c in m showed genes up-and down-regulated (n= , ; p< . ). pa showed up-regulation of jak-stat signaling, autophagy, renin-angiotensin system (ras), and ubiquinone biosynthesis pathways and down-regulation of apoptosis, basal transcription, folate biosynthesis, nitrogen metabolism, protein export, and snare interaction pathways in mnr vs c. only the ubiquinone biosynthesis pathway up-regulation of mnr vs. c was common to both m and f. conclusions: ta and pa demonstrate sex-specific transcriptome expression in fetal kidneys at . g. in addition, these results show sexspecificity in response to mnr. we have seen similar effects of mnr on gene expression for autophagy, apoptosis, ras, ubiquinone and cell adhesion pathways at . g, suggesting these pathways may contribute to persistent affects of mnr. finally, we postulate that stress in utero may contribute to sex differences in risk of hypertension in adult life. leptin and neuropeptied y protein expression paradoxally increased in gestationall food restricted dams. louiza belkacemi, chun-hung chen, andrea jelks, michael g ross, mina desai. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: placental insufficiency is associated with marked increase in placental leptin production. this results in a rise in maternal leptin levels that serves as an early index of placental dysfunction. further increased placental leptin and suppressed neuropeptide y (npy) are associated with preeclampsia. leptin, an anorexigenic hormone and npy, an orexigenic peptide regulate food intake. importantly, leptin also serves as a placental and fetal growth factor. we have shown that maternal food restriction (mfr) results in intrauterine growth ... sf ()to:!)) df(,..)l) ...._ bf(jtolo) ... -m bf(,..lj) =- ( . ) <(u.)) ( . ) )( . )"" ' ~. ) !( . )' d.'cbom< ( ) f( ))""" s( l) background teenagers are more likely to deliver small-for-gestational age (sga) infants than adults, even after adjustment for socioeconomic factors , . previous studies of mostly black and hispanic subjects in the usa have suggested that maternal growth may contribute to reduced infant birthweight, due to preferential nutrient partitioning to the mother . the impact of maternal growth on birthweight and nutrient partitioning in pregnant teenagers in the uk has not been examined. methods skeletal growth (change in knee-height from st to rd trimester), weight gain and skinfold thicknesses were measured in pregnant teenagers (n= , % non-white) in london and manchester. key mediators of nutrient partitioning and metabolism: insulin-like growth factor(igf)- , igf binding protein(bp)- and leptin, were measured in maternal plasma ( weeks gestation). results maternal growth (defined as increase in knee-height > mm/ days) was detected in % of pregnant teenagers. this growth was not associated with sga birth; in fact these mothers were more likely to deliver large-forgestational age (lga) infants (p< . ). maternal weight gain and fat accrual at peripheral and central sites were greater in growers (p< . ). these parameters correlated positively with maternal igf- and leptin but negatively with the igf inhibitor, igfbp- (p< . for all). subjects delivering sga infants gained significantly less weight (p< . ) and had lower igf- levels (p< . ) than those delivering non-sga infants. conclusion maternal growth in teenage pregnancy was not associated with reduced birthweight. indeed the increased weight gain and fat accrual observed in growing teenagers may protect against sga birth and promote fetal growth. igf- and leptin promote fetal growth, primarily through effects on maternal metabolism and nutrient partitioning to the fetoplacental unit. these data suggest that higher maternal igf- and leptin in growing teenagers may provide an anabolic drive for both maternal and fetal growth. background. umbilical oxygen uptake (o umb uptake) has been estimated in human pregnancies only in acute experiments at the time of caesarean section. the recently developed possibility to measure umbilical blood flow by ultrasound in utero, prompted us to study normal and iugr pregnancies in order to evaluate fetal oxygen uptake utilizing the fick principle, i.e. uptake equals umbilical blood flow times (a-v) differences. methods. thirty-six iugr pregnancies were studied at the time of elective caesarean section and compared to twenty-one controls (c) (gestational age: c= . ± . and iugr= . ± . wks). an ultrasound examination was performed within hours from the caesarean section in all the recruited patients. umbilical vein absolute volume flow (qumb) was measured as the result between umbilical vein area and the time-averaged peak velocity * . . blood samples from umbilical vein (uv) and artery (ua) were obtained after the delivery and blood gases and acid-base balance were evaluated. umbilical oxygen uptake was calculated as o umb uptake = qumb*(uv-ua) o content. results. as expected, average fetal and placental weights were significantly different in the studied groups ( ± and ± g in iugr vs ± and ± g in n) . iugr pregnancies showed a significant reduction in qumb ( . ± . vs . ± . ml/min; p< . ) but no differences in the qumb/kg of fetal weight. iugr fetuses showed a significant reduction in o sat, o cont and po in both uv and ua compared to n. (uv-ua)o content ( . ± . vs . ± . mmol/l; p< . ) and o umb uptake/kg ( . ± . vs . ± . mmol/min/kg; p< . ) were significantly reduced in iugr. conclusions. we here report an evaluation of fetal oxygen uptake that proved surprisingly similar to the values reported in chronically catheterized animals. however, iugr fetuses showed a significant reduction in both blood and oxygen supply: this latter was reduced more that % on a per kg basis. iugr fetuses therefore utilize less oxygen than normally grown fetuses. circulating levels of vitamin d and il- in pregnancies with iugr. calvin j hobel, chander p arora, adegoke adeniji, priya arora, susan e jackman, olga miadel, baldjyan lilit. ob-gyn, cedars-sinai medical center, burns ans allen research institute, los angeles, ca, usa; university of california los angeles, los angeles, ca, usa. background: any condition resulting in under exposure to sunlight, including the use of sun block or poor nutrition may result in insufficiency ( . - nmol/l) or even deficiency of vitamin d (< . nmol/l).vitamin d regulates placental development and function. vitamin d deficiency has been linked to increased risk of serious chronic and inflammatory diseases. objective: to determine if the circulating levels of vitamin d and interleukin - (il- ) in maternal plasma correlates to pregnancies resulting in fetus with intrauterine growth restriction (iugr). hypothesis: the metabolism of vitamin d initiates the biochemical cascade of events leading to the expression of il- and the inflammatory response in iugr births. study design: in a behavior in pregnancy study, plasma samples at all three time points were analyzed in a cohort of women for (oh)d using elisa. the samples were also analyzed for il- at three stages of pregnancy: t ( - weeks), t ( - weeks) and t ( - weeks). iugr was defined as birth weight below the tenth percentile for gestational age. none of the iugr cases had spontaneous preterm birth. results: iugr was diagnosed in of women with available samples from a behavior in pregnancy study (bips) . out of these subjects, were selected as matched case controls. circulating levels of vitamin d ( (oh)d) were significantly lower in iugr cases at each visit (p<. ). the levels indicated deficient ( . ± . nmol/l) vitamin d in iugr group at t but sufficient vitamin d levels ( . ± . nmol/l) in controls. subsequent visits also showed lower levels in the iugr cases compared to the control group (t : ± . nmol/l vs ± . nmol /l; t : ± . nmol/l vs ± . nmol /l). at all three time intervals, significantly (p<. ) higher levels of il- were associated with the iugr cases ( pg/ml, pg/ml and pg/ml respectively) as compared to the controls ( pg/ml, pg/ml and pg/ml respectively). conclusions: vitamin d deficiency or even insufficiency may be an unrecognized cause of iugr. it is possible that a primary non-infectious inflammatory process is activated by vitamin d deficiency. combined assessment of vitamin d deficiency and il- expression during different stages of pregnancy may facilitate the resognition of the risk of developing iugr. response to global % maternal nutrient restriction (mnr). nathan drever, thomas j mcdonald, peter w nathanielsz, cun li. obstetrics and gynecology, university of texas health science center at san antonio, san antonio, tx, usa. background: igf-ii is a major growth factor in the developing pancreas and studies in the human fetus demonstrate that the peptide localizes to b cells of the islets (j endocrinology : ). rodent studies show decreased fetal pancreatic growth and igf-ii and ins abundance and increased apoptosis with mnr (j endocrinology : ). we previously demonstrated a fall in most components of the placental and fetal baboon liver igf systems with mnr. here we have evaluated fetal pancreatic igf-ii and ins changes in response to mnr. methods: pregnant baboons were fed ad lib (ctr, n= ) or % of wt adjusted ctr diet (mnr, n= ) from . gestation (g) and fetuses were recovered at c-section under general anesthesia at . (n= ; ctr and mnr) and . g (n= ; ctr and mnr). igf-ii and ins expression were determined by immunohistochemistry (ihc) and quantified by image analysis for fraction (area immunostained/area of the field x %) and density. data are expressed as mean + sem; ctr data are expressed] first; comparison made with two tailed t-test. results: at . g there was no difference in igf-ii or insulin fraction or density between groups. at . g, igf-ii fraction ( . ± . vs . ± . ,p< . ) and density ( . x ± . x vs . x ± . x , p< . ) and insulin fraction ( . ± . vs . ± . , p= . ) were reduced. conclusion: moderate mnr decreases abundance of fetal pancreatic igf-ii and ins at . g. in the fetal baboon and supports the extant evidence for impaired pancreatic development with mnr seen in rodents. maintenance of liver growth in the hypoxic growth restricted fetal sheep: a role for intrahepatic glut ? sheridan gentili, janna l morrison, i caroline mcmillen. sansom institute, unisa, adelaide, south australia, australia. objective: we have previously demonstrated that there is a differential tissue response to chronic placental and fetal growth restriction. growth of fetal tissues such as the brain and the adrenal are consistently spared in the face of chronic substrate restriction, whilst we have demonstrated that the growth of the fetal liver may be either maintained or reduced. it is unclear whether the growth response of the fetal liver to hypoxia and hypoglycemia are determined by intrahepatic metabolic adaptations. hypothesis: we hypothesize that there will be a differential profile of hepatic expression of glut , hsd , the gluconeogenic and glycolytic enzymes pepck and g pdh and the transcription factors pgc and ppar in animals in which liver growth is maintained or reduced. methods: carunclectomy was performed in non-pregnant ewes to induce placental restriction (pr). vascular catheters were inserted in pr and control (c) fetuses at - d and arterial blood samples were collected for blood gas analysis. mean gestation po < mmhg was defined as hypoxic (h; normoxia, n). post mortem was performed at - d. hepatic mrna expression of glut , hsd , pepck, g pdh, pgc and ppar was determined using qrt-pcr. results: four experimental groups were defined by fetal po and liver growth (c-n, pr-n, pr-h and pr-h-reduced liver growth). fetal weight correlated with mean gestational po (r = . , y= . x+ . , p< . ). liver weight was significantly lower in a cohort of pr-h fetuses (c, . ± . ; pr-n . ± . ; pr-h . ± . ; pr-h-rlg . ± . g:kg; p< . ). glut expression was highest in those pr-h fetuses in which liver growth was maintained, whilst the expression of hsd , pgc , ppar and pepck was highest in the pr-h fetuses in which liver growth was reduced (p< . ). conclusions: in the pr-h fetuses in which liver growth was reduced, the increase in hsd expression may be associated with an increase in hepatic exposure to cortisol and an associated increase in pepck, pgc and ppar . interestingly the compensatory increase in hepatic glut expression did not occur in fetuses in which liver growth was reduced. predicting the trajectory of fetal growth. racine n edwards-silva, jeffrey gornbein, calvin j hobel. obstetrics gynecology, los angeles, ca, usa; biomathematics, david geffen school of medicine at university of california, los angeles, ca, usa; obstetrics gynecology, david geffen school of medicine at university of california, los angeles, ca, usa. objective: to evaluate twelve potential predictors of fetal growth trajectory defined as the rate of fetal weight change over time. study design: a longitudinal prospective study of singleton fetal growth trajectory. the twelve potential predictors considered were: fetal gender, gestational age, parity, race, bmi, age, weight at st clinical exam, cumulative weight gain at each exam, smoking, and alcohol use. estimated fetal weight was computed using the hadlock formula and sonographic fetal biometric parameters at - weeks, - weeks, and - weeks. the rate of change in fetal weight was defined as x (fetal wt at exam j -fetal wt at exam i )(j > i)/ (gestational age at exam j -gestational age at exam i ). bivariate statistical analysis included the non-parametric spearman rank correlation and wilcoxon rank sum test. all factors were assessed multivariately using multiple linear regression. results: there were multi-ethnic women included in the study, after were excluded. they underwent a total of , exams. fetal gender (p= . ), maternal weight at st exam (p= . ), and cumulative maternal weight gain at exam (p < . ) were significant predictors of fetal growth trajectory. in this model, male fetuses had an average rate of fetal weight change of . grams per days higher than females. the rate of fetal weight change increased by an average of . grams per days for each lb increase in maternal weight at the st exam. the fetal growth rate increased . grams per days for each lb of cumulative weight gain at the rd exam. conclusions: in this study, maternal weight at the st exam and cumulative maternal weight gain were the significant determinants of fetal growth trajectory. adequate initial maternal weight and cumulative gestational weight gains probably ensure sufficient nourishment for normal placental growth, uteroplacental blood flow, and fetal nutrient uptake. this supports the emphasis on periconceptual nutritional counseling for all pregnant women. the implication of this study is that similar to fetal programming of adult diseases, there is nutritional programming of fetal growth trajectory. high risk patients. other predictors include the known risk factors of age < and > , single, tobacco/alcohol use, and african american race. interestingly, hispanic and native american patients have a lower lbw rate compared to other groups. introduction: catecholamines released by the sympathetic nervous system and adrenal medulla act via b-ars to regulate glucose and insulin function in liver, pancreas, adipose and muscle tissue. b-ar knock out mice show increased fat mass and glucose intolerance (asenslo et al.,diabetes, ) . mnr animal models have increased sympathetic activity. we, therefore, evaluated effects of mnr on baboon fetal liver b -ar. methods: baboons were fed as ad lib controls (ctr) or % of wt adjusted ctr diet (mnr) from . gestation(g) with fetuses retrieved at c-section under general anesthesia at . or . g. protein expression determined by immunohistochemistry for b -ar in the central liver lobule was quantified by image anaysis and expressed as fraction = area immuno-stained/area of the field x %. all data are expressed as mean + sem with ctr data presented first. liver glycogen expression was determined by the periodic accid schiiff (pas) method. comparisions were made with student's t-test with alpha level set at . . results: fetal body and liver wts were not changed by mnr at either age. pas stained liver glycogen at . g = . ± . vs. . ± . %, p< . . b -ar fraction was lower following mnr at . g and at . g (p< . ; fig ) conclusions: mnr decreased b -ar over % at . g and % at . g. decreased fetal liver b -ar in mnr alters glucose metabolisam and may result in reduced lipolysis predisposing to fatty liver. infection with noncytopathic bovine viral diarrhea virus (ncpbvdv) during early bovine pregnancy (< d gestation) results in fetal immunotolerance, persistent infection (pi) and intrauterine growth restriction (iugr). in contrast, infection after the development of adaptive immune competence (> d gestation or postnatal) results in a transient infection (ti). we have previously reported an iugr in pi fetuses presenting as decreased body weight and ponderal index. a growth defect can be the result of many factors, including placental insufficiency and nutrient restriction, however it was hypothesized that the iugr seen in bvdv pi fetuses may be an immunopathological effect caused by the persisting virus. our two part experimental design examined the relationship between bvdv and its pi host. in experiment (exp) , blood cell mrna was collected from pi steers (n= ; confirmed by virus isolation), or uninfected control steers (n= ) and used to identify differentially expressed genes using microarray (affymetrix) and qtrt-pcr approaches. in exp , bvdv naïve pregnant heifers (n= per group) were not infected (control) or infected with ncpbvdv on d. or d. of pregnancy creating pi and ti fetuses, respectively. fetuses were collected by c-section on d. ; infection was confirmed by elisa and qtrt-pcr. histology of placental tissue revealed no placentitis or pathology, and glucose and lactate levels in fetal serum were normal. microarray analysis revealed genes that were differentially regulated in pi vs. controls (p< . , > . fold). qtrt-pcr of steer and fetal blood revealed a significant upregulation of activators and products of the antiviral type-i interferon (ifn-i) pathway. as ifn-i can act as a growthsuppressive cytokine, a long-term upregulation may contribute to the iugr seen in persistent bvdv infection and in other viral infections observed during pregnancy. nricg - from the csrees. patients with a short cervix are at an increased risk for spontaneous preterm delivery. therefore, it is possible that women with a short cervix during pregnancy are also at risk to deliver an sga neonate. this study was conducted to address this question. study design: patients > weeks of gestation were prospectively enrolled into an observational study ( / to / ). transvaginal sonographic examinations were performed every weeks until delivery. the shortest cervical length between - weeks was used for analysis. sga was defined as less than the tenth percentile of birth weight. results: asymptomatic patients were studied. . % of patients delivered an sga neonate ( / ). the median cervical length was mm ( to mm); patients had a cervical length < mm. of these, % ( / ) delivered an sga neonate. similarly, % ( / ) patients with a cervical length < mm had an sga neonate. no relationship was found between a short cervix and sga (short cervix was defined as either < mm and < mm). the frequency of sga was not significantly different between women with a short cervix and those with a long cervix [ % ( / ) vs. % ( / ); p= . ]. newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced background folate is an essential micronutrient for cellular growth. recommendations on periconceptional folic acid use are mainly focussed on prevention of neural tube defects, despite growing evidence that folic acid use may have positive effects on birth weight. objective to examine associations between folic acid use, intrauterine fetal growth and birth weight. design the study was embedded in the generation r study in rotterdam, the netherlands, a population-based prospective cohort study from early pregnancy onwards. methods information on folic acid use was obtained by questionnaires and categorized into three groups: ) preconception start of folic acid use; ) start of folic acid use in first ten weeks of gestation; ) no folic acid use at all. fetal growth measurements included head circumference, abdominal circumference and femur length measured in mid-and late pregnancy, i.e., gestational age - and > weeks, respectively, and birth weight. fetal weight in mid-and late pregnancy was estimated using the haddock method. results data from , pregnant women were available. overall, folic acid use was positively associated with fetal growth. preconceptional folic acid use resulted in an increased growth of grams ( %ci . - . , p< . ) per week from late pregnancy to birth, compared to no folic acid use. similarly, start of folic acid use in the first ten weeks of gestation resulted in an increased growth of grams ( %ci . - . , p= . ) per week from late pregnancy to birth. both preconceptional folic acid use and folic acid use started in the first ten weeks of gestation resulted in higher birth weights of grams ( %ci - ) and grams ( %ci - ), respectively, compared to no folic acid use. a tendency was found for an increased risk of birth weight less than grams when folic acid was not started preconceptionally (or . , %ci . - . ). conclusion periconceptional folic acid use is significantly associated with increased fetal growth resulting in a higher birth weight. ductus venosus isovolumetric relaxation in severely premature growth-restricted fetuses. jason l picconi, katherine drennan, farhan hanif, michael kruger, giancarlo mari. obstetrics and gynecology, wayne state university/dmc, detroit, mi, usa. objective: ductus venosus (dv) doppler waveforms are characterized by two periods in which blood velocity decreases. the first represents the isovolumetric relaxation (ir) at the end of ventricular systole and the second represents atrial contraction at the end of ventricular diastole (a). ductus venosus reversed flow (dvrf) occurring at the time of the a-wave is considered a risk factor for intrauterine fetal demise (iufd). we have previously reported that absent or reversed a-wave flow can be present for weeks before iufd occurs or delivery is performed for non-reassuring fetal testing. the guiding hypothesis for this study is that decreased flow at the time of ir in combination with absent or reversed a-wave flow allows a more accurate prediction of fetal outcome than a-wave absent or reversed flow alone. material and methods: ductus venosus doppler was serially studied in severely premature iugr fetuses (estimated fetal weight < th percentile and umbilical artery pulsatility index > th percentile) from diagnosis until demise or delivery. ductus venosus waveforms were assessed quantitatively for peak systolic velocity (psv), isovolumetric relaxation velocity (irv), and end diastolic velocity (edv). the psv/irv + edv were compared to fetal and neonatal outcome. a kruskal-wallis one way anova, mann whitney u post-hoc test, and a roc, were used for statistical analysis. a p < . was considered statistically significant. results: all fetuses were delivered at < weeks. six cases resulted in iufd, five cases resulted in neonatal demise (nd), and six cases resulted in neonatal survival (ns) at the time of discharge from the hospital. the psv/irv+edv correlated better than a-wave reversal of flow with perinatal outcome. a psv/irv+edv score less than - . resulted in iufd, whereas a score greater than - . resulted in live birth. live births segregated based on estimated gestational age, where those fetuses at less than weeks resulted in nd and those fetuses at or greater than weeks resulted in ns. all results were statistically significant. conclusions: the isovolumetric relaxation velocity is a novel doppler parameter in the assessment of severely premature iugr fetuses. these data indicate that assessment of irv should be considered part of the evaluation of severely iugr fetuses. background: fetal growth restriction has been linked to an increased incidence of chronic hypertension, which may be the result of extracellular matrix changes (ecm) within the vascular tree. in previous studies, ecm changes were observed in the umbilical arteries of preterm growth restricted infants. objective: to determine if there are alterations in collagen subtypes within the umbilical cords from growth restricted fetal rat pups after a period of maternal nutrient restriction. methods: timed pregnant sprague-dawley rats were fed either a % food restricted diet (mfr; n= ) or were fed ad libidum (control; n= ) from d until d of gestation. litter size, fetal weights and placental weights were then noted and umbilical cords from randomly selected pups in each litter were snap frozen. gene expression for collagens i, iii, xiv and decorin was evaluated by real-time rt-pcr with normalization to the gadph housekeeping gene. data were analyzed from fitting general linear regression models with estimation based on the quasi-likelihood estimation (generalized estimating equations) to account for clustering of responses within litters. data are shown as cycles to amplification (ct), which is inversely proportional to mrna levels. results: no difference in median litter size was detected. however, fetal and placental weights in the mfr group were significantly less than those from control dams. no significant differences in umbilical cord gene expression for collagens i, iii, xiv or decorin were detected between mfr and control pups. conclusions: maternal food restriction does not result in any detectable alterations in collagen or decorin expression within the intact umbilical cord, despite causing significant reductions in fetal growth. control (n= ) p-value litter size (median, range) ( , ) ( , ) . fetal weight (mean ± sd) g . ± . . ± . < . placental weight (mean ± sd) g . ± . . ± . . collagen i (mean ± sem) ct . ± . . we have previously demonstrated that the restriction of placental and fetal growth results in fetal hypoxia and fetal brain sparing suggesting a redistribution of cardiac output. furthermore, placentally restricted (pr) hypoxic fetuses are more dependent on their sympathetic nervous system for the maintenance of blood pressure during late gestation. nerve growth factor (ngf) plays a significant role in sympathetic innervation. hypothesis: we hypothesize that the expression of ngf will be higher in the aorta and femoral artery of the pr hypoxic compared to the control fetus. method: carunclectomy was performed in non-pregnant ewes to induce pr. vascular catheters were inserted in pr and control (c) fetuses at - d and arterial blood samples were collected for blood gas analysis. all pr fetuses introduction elevated sflt- levels have been shown to be a feature of pre-eclampsia and is considered to play a significant role in the pathogenesis of the condition. the role of angiogenic factors in fetal growth restriction has not been as well established. this study evaluated the levels of circulating vascular endothelial growth factor (vegf) and its soluble receptor sflt- , in normal pregnancies and isolated placental vascular disease without evidence of pre-eclampsia. method maternal peripheral venous samples were collected antenatally from two groups of pregnant women between - weeks of gestation. group a: uncomplicated normal pregnancies (n = ) and group b: pregnancies complicated by isolated placental vascular disease( n = ) as defined by birth weight less than th centile for gestation and umbilical artery doppler s:d ratio above the th centile for gestation, with no evidence of maternal preeclampsia or pre-existing hypertension. plasma vegf and sflt- levels were measured using standard eliza techniques. comparison between groups were performed by using one way analysis of variance. the maternal plasma sflt- levels (figure ) in group a: normal pregnancies increased with gestation (p < . ). the sflt- levels in group b: isolated placental vascular disease were significantly higher throughout all gestations (p = . ) and did not show a significant variation with gestation ( p = . ). the plasma vegf levels were below the detectable levels of the assay in all samples except normal pregnancies. the significantly increased maternal plasma sflt- levels in established isolated placental vascular disease without evidence of pre-eclampsia suggest a disease of placental origin. the dysregulation of angiogenic factors may be part of a repair and regeneration process in the placenta. objectives: production of -reduced neurosteroids is critical for reducing fetal vulnerability to stressors in pregnancy and inhibition of -reductases ( r) increase acute hypoxia-induced apoptosis in the fetal brain (yawno et al neurosci : . we have developed a model of placental insufficiency that results in fetal growth restriction (gr) in the guinea pig (palliser et al repro sci # ). the aim of the present study was to determine the effects of suppression of -reduced steroid synthesis on apoptosis in vulnerable regions of the fetal brain and on neurosteroid synthetic enzymes in pregnancies compromised by chronic placental insufficiency. methods: placental insufficiency was induced in guinea pig dams by surgical ablation of uterine artery branches at mid gestation (term d). sham operated or gr dams received finasteride (a r inhibitor; mg/kg/day) during late gestation ( d until term). activated caspase- , a marker of apoptotic cell death, was measured by immunohistochemistry and steroidogenic enzymes, r and cytochrome p side chain cleavage (p scc) were measured by real time pcr and western blotting in fetuses ( d) and neonates h after birth. results: placental insufficiency significantly reduced fetal body and organ weight by % whilst sparing brain weight. the number of activated caspase- positive cells was significantly increased in the fetal hippocampus of gr fetuses and further increased in the cortex of gr fetuses receiving finasteride. the neonatal brain also exhibited changes in caspase- activation following gr and finasteride treatment. the fetal adrenal and the placenta responded to the compromise with increased expression of p scc mrna and r protein, respectively. conclusion: the combination of placental insufficiency and suppressed neurosteroid system leads to markedly increased apoptotic cell death in the fetal brain which continues to affect the neonatal brain. the fetus responds to these conditions by increasing steroid synthetic enzyme expression in the placenta and adrenal glands suggestive of a possible neuroprotective feedback process. to study the effect of smoking by mothers on the fetal and neonatal brain using non-invasive magnetoencephalography technique (meg). materials and methods: using fetal magnetoencephalography, cortical auditory evoked responses (aer) were measured from fetuses ranging from to weeks gestational age for a total of recordings. measurements were taken from mothers with a history of smoking (sm) and from mothers with no smoking history (ns). after delivery, five sm and five ns newborns had meg aer measurements twice for a total of recordings. aer was quantified by cross-correlation analysis and its significance was assessed by boot-strap technique. results: aers were detectable in out of fetal recordings in the ns group and of in the sm group. the neonatal response rate was % for each group. the latencies were divided into three components: c ( - ms), c ( - ms) and c ( - ms). in both fetuses and neonates as well, there was a statistically significant difference (p< . ) between the two groups in the c -component and the sm group showed faster aer compared to the lr group. conclusion: meg technique provides a non-invasive approach to study the effects of smoking on developing fetal and neonatal brain. the observed decrease in the latency of fetuses and neonates of the smoking mothers could indicate a hypersensitive cortical response to auditory tone. adenosine (ado) modulates metabolism in adult mammals through multiple mechanisms that involve ado a and a a receptors. objective: this study was designed to test the hypothesis that ado a and a a receptors participate in fetal metabolic homeostasis. methods: experiments were performed in chronically catheterized fetal sheep (> . term). intravascular infusion for h of dpcpx (a receptor antagonist) or zm (zm, a a receptor antagonist) was performed alone or in concert with ado administration. the highly selective ado receptor antagonists were also infused in fetuses in which hypoxia was induced for h by having the ewe breathe a hypoxic gas mixture (fio = . ). data were analyzed by two-way repeated measures of anova. results: blockade of ado a receptors (n= ) increased significantly (p < . ) fetal concentrations of glucose [control (c): . ± . (se)]; experiment (e): . ± . mg/dl] and lactate (c: . ± . ; e: . ± mg/dl) without significantly altering insulin levels and arterial blood gases or ph. antagonism of ado a a receptors (n= ) did not affect plasma levels of glucose, lactate, or insulin. intravenous infusion of ado (n= ), which did not alter pao or paco , increased concentrations of glucose (c: . ± . ; e: . ± . mg/dl) and lactate (c: . ± . ; e: . ± . ). zm (n= ), but not dpcpx (n= ), abolished ado-induced rise in glucose and lactate concentrations. isocapnic hypoxia (pao torr), which increases ( - fold) fetal plasma ado levels to those similar to ado infusion, decreased arterial ph (c: . ± . ; e: . ± . ), and increased fetal levels of glucose (c: . ± . ; e: . ± . mg/dl) and lactate (c: . ± . ; e: . ± . mg/dl) without altering changing insulin concentrations. these effects of hypoxia were not altered by dpcpx or zm. conclusions: ) a receptors modulate plasma levels of glucose and lactate in normoxic fetuses; ) a a receptors mediate the adoinduced rise in plasma glucose and lactate; and ) a and a a receptors are not significant modulators of hypoxia-induced changes in plasma levels of glucose and lactate. supported by usphs hd- . objective: to investigate the dynamic changes of the relationship between leptin, adiponectin and resistin in maternal and fetal circulation during pregnancy and in the early post-natal period. materials and methods: thirty pregnant women with uncomplicated singleton pregnancy delivered at term. maternal and fetal/neonatal venous blood samples were obtained at delivery and at hours from birth. leptin, adiponectin and resistin were measured by specific elisa assays. neonatal anthropometric measurements, glucose metabolism and lipid profile, blood pressure information were obtained. statistical analysis was performed by anova followed by student t test or duncan's test whenever appropriate. correlations were calculated by using the pearson coefficient. results: adipokines concentration at birth and at h from birth was showed in table. multivariate regression analysis showed that fetal leptin levels were positively associated with female gender and adiponectin levels, but not with anthropometric characteristics. fetal leptin and adiponectin levels were not correlated with maternal concentration, whereas fetal and maternal resistin levels were. fetal and maternal resistin concentration was positively associated with gestational age and birth weight and fetal resistin levels correlated negatively with lipid profile. after birth leptin concentration in maternal and neonatal circulation decreased dramatically and the correlation between leptin and adiponectin levels was lost. a positive correlation between resistin and leptin concentration in neonatal circulation was found at h from birth. conclusions: in contrast to adult life a positive correlation between leptin and adiponectin is present in fetal life. placental secretion of leptin, but not of adiponectin and resistin, contributes significantly to maternal and fetal circulating levels. significant changes in the relationship among adipokines occurred immediately after birth and may affect growth and development in early post-natal period. adipokines concentration in maternal and fetal circulation ) antagonism has been shown to normalize placental perfusion and fetal growth in several rat models of fetal growth restriction. however, direct administration of et a antagonists to newborn rats within hours of delivery has been consistently associated with neonatal demise due to failure of the ductus arteriosus to close, raising concerns about the safety of their use late in pregnancy. perinatal exposure to et a antagonists (maternal administration in late gestation) and its impact on rat pup survival and oxygen saturation has not been investigated. objective: to determine the impact of a maternally administered et a antagonist on oxygen saturation in newborn and -day-old rat pups. methods: timed pregnant sprague-dawley rats were treated with fr ( mg/kg/day; et a antagonist) or . % nahco vehicle, by subcutaneous osmotic pump connected to an intravenous catheter, from gestational day (term= days) through parturition. all five pregnant rats in each group delivered spontaneously and nursed their pups through postpartum day . oxygen saturation of each rat pup was measured by pulse oximeter on postpartum days and . results are presented as means ± se. newborn -day neonate vehicle . ± . . ± . eta antagonist . ± . . ± . there were no statistically significant differences between the treatment groups. maternal administration of an et a antagonist from gestational day through parturition, at a dose sufficient to ameliorate fetal growth restriction, has no adverse impact on oxygen saturation in neonatal rat pups. helen l torrance, jan b derks, martijn a oudijk, avnesh s thakor, tereza cindrova-davies, frank van bel, gerard ha visser, graham j burton, dino a giussani. perinatal center, university medical center utrecht, netherlands; department of physiology, development neuroscience, university of cambridge, united kingdom. introduction: the management of perinatal asphyxia remains a major concerns in obstetrics. umbilical cord compressions (ucc) induce fetal asphyxia and ischaemia-reperfusion (i/r). i/r increases reactive oxygen species, for instance via activation of the xanthine oxidase (xo) pathway, which may promote oxidative stress in the fetal circulation. while treatment with allopurinol of asphyxic human neonates reduced free radicals and improved cardiovascular status, treatment started postnatally was deemed too late to prevent oxidative damage (benders et al. arch dis child : , ) . consequently, in complicated pregnancy, recommendations to treat the fetus via the mother, rather than the neonate, with allopurinol are being entertained today. we investigated the effects of maternal allopurinol treatment on indices of oxidative stress in the fetal heart following repeated ucc in sheep. methods: at . of gestation, surgically instrumented sheep fetuses were submitted to i/r ( x min repeated ucc) under maternal allopurinol (n= ) or saline vehicle (n= ) infusion. fetal hearts were collected h after i/r and snap frozen for measurement of (anti)oxidant proteins by western blot. hearts from uninstrumented fetal sheep at . gestation served as controls. statistical comparisons were made using one-way anova. results: i/r episodes led to increased expression of cox- , enos, hsp and decreased expression of sod and gluthatione peroxidase (gpx) in the fetal heart, findings consistent with cardiac oxidative stress. maternal treatment with allopurinol ameliorated these effects (fig. objectives: hypoxic-ischemic fetal brain injury (hie) is a major cause of neonatal death and morbidity. evidence suggests that the brain cell injury associated with chronic hpx (in contrast to an acute ischemic reperfusion injury) is a complex process reflecting a series of adaptive intracellular events that are duration and gestational age dependent. we applied advanced proteomic tools as a next step toward ascertaining a more complete understanding of the impact of hpx on the fetal brain proteome. methods: time-mated guinea pigs were housed in a chamber beginning on day for d, breathing either room air (nmx), or . % or % o ( % o hpx or . % o hpx). on day (term), the fetal brains were removed and preserved for study. total protein was extracted, and the proteome first characterized by d gel electrophoresis. the density of the resulting protein spots were acquired and analyzed using the gs densitometer and pdquest software. identified spots of interest were trypsin digested and subject to maldi mass spectrometry using the proteomics analyzer mass spectrometer for peptide mapping or sequencing. the hpx-induced protein spots were identified based on a minimum of a x change from nmx. results: hpx had a clear effect on the fetal brain proteome. superoxide dismutase (sod), heat shock protein (hsp ), actin (actg ), interleukin- (il- ), and glutamine synthetase (gs) were each up regulated, while cofilin- , brain-type creatine kinase (bb-ck), and - gtp binding protein were each down regulated. all protein changes were proportional to the hpx ( % o hpx vs nmx, % o hpx vs . % o hpx, p< . ). conclusions: this initial application of proteomic techniques confirms that fetal brain damage secondary to chronic hpx (in contrast to acute hpx) is a complex processes characterized by fetal adaptations mediated by multiple protein activations and inactivations. while sod, il- , and gs have each been previously investigated, the potential roles of actg , bb-ck, beta- gtp binding protein, and cofilin- in the fetal response to hpx and the associated brain damage are unclear and represent strong candidates for future investigation. acknowlegement: this study was supported by grants from the phs (r hl - , cpw) and cdc grant (u dp - , cpw). intermittent umbilical cord occlusion occurs in % of human pregnancies.given that elastogenesis within the vascular wall is in part mediated by hemodynamic conditions during development, the blood pressure response to acute hypoxic insults such as cord occlusion may alter arterial composition. elastin content of a central and peripheral artery and blood pressure responses in fetal sheep exposed to varying degrees of cord occlusion were determined. methods: over a day period, near term fetal sheep received total umbilical cord occlusion (uco) lasting min/ hour (mild group; n= ), min/hour (moderate group; n= ), min/hour (severe group; n = ) or no occlusion (control group; n= ). fetal arterial blood samples were drawn min prior to and at the end of cord occlusions. mean arterial pressure (map) was monitored continuously. the carotid and superior mesenteric arteries were excised and a colorimetric assay (biocolor) performed for determination of elastin content. results are presented as mean ± sem. results:umbilical cord occlusions produced decreases in fetal arterial oxygen pressure and oxygen saturation that were progressively more pronounced across mild, moderate and severe uco groups (p < . ). lactate concentration rose during occlusions in the moderate and severe groups, but not the mild group (p < . ).elastin content of the superior mesenteric artery did not differ between the experimental groups and the control group. elastin content of the carotid artery and map response elastin content (μg/mg tissue) max ∆ in map duration of rise in map (min) control . ± . . ± . -mild . ± . . ± . ** . ± . moderate . ± . . ± . ** † . ± . † severe . ± . * . ± . ** † † . ± . † † * p < . ; ** p < . : experimental groups compared to control † p < . ; † † p < . : compared to mild group the max in map was positively correlated with elastin content (r = . , p < . ). conclusions:the transient rise in blood pressure and preferential blood flow to the brain that occur in response to acute hypoxemia during severe intermittent umbilical cord occlusion induce an increase in elastin synthesis in the carotid artery.this may give rise to adaptive programming of postnatal central arterial compliance. electrocortical activity during repetitive umbilical cord occlusions (uco) with worsening acidemia in the ovine fetus near term. martin g frasch, roy mansano, michael g ross, robert gagnon, bryan s richardson. obgyn, chri, univ of western ontario, london, canada; obgyn, harbor-ucla med ctr, torrance, ca. objective: uterine contractions during labour can restrict umbilical blood flow compromising fetal oxygenation and leading to adverse neonatal outcome including newborn encephalopathy/subsequent cerebral palsy. while electronic fetal heart rate (fhr) monitoring is widely used for assessment during labour, abnormal fhr patterns as used clinically have a poor positive predictive value for concerning/significant acidosis at birth. there is limited study of fetal electrocortical activity (ecog) in animal models with induced hypoxia/ acidemia as might be seen during labour. thus, we aimed to induce repetitive ucos in fetal sheep leading to worsening acidemia to determine the predictive value of ecog activity for fetal compromise. methods: near-term fetal sheep (n= ) underwent chronic preparation with artery catheters, ecg/ecog electrodes and placement of an inflatable umbilical cord occluder. following a baseline recording period, fetuses underwent a series of mild ( min every min), moderate ( min every min) and severe ( min every min) ucos with each series lasting h or until fetal arterial ph decreased to < . . fetal arterial blood samples for blood gases and ph were taken at selected time points during the baseline, ucos, and recovery periods. arterial blood pressure (abp) and fhr were continuously monitored and spectral edge frequency (sef) was calculated from ecog. correlation of sef with fhr change was calculated for time intervals using sef maxima and fhr minima during uco series. data are presented as means±sem. results: repetitive ucos led to development of a marked acidosis (ph . ± . to . ± . , p< . ). ± min prior to fetal ph drop < . , the sef of ecog began to increase abruptly during each fhr deceleration from ± hz up to ± hz (p< . ) and was correlated to both fhr change and a decrease in fetal abp during each fhr deceleration at this time (p< . ). conclusion: our findings suggest that in the animal model studied ( ) fetal ecog activity is impaired with progressive acidemia accompanied by fhr decelerations and pathological abp decreases; ( ) there is a consistent temporal relationship between the occurrence of ecog alterations and the subsequent critical drop of ph < . . these findings could contribute to improvement in the clinical ability to predict fetal compromise during labour using ecog/fhr monitoring. background: the ductus arteriosus (da) plays a pivotal role in fetal development and circulation. during gestation, patency of the fetal da is maintained by nitric oxide (no) and prostaglandins (pg). no and pgs are downstream effectors of estrogen (e ) and progesterone (p ). however, the roles of e and p in da regulation have not been studied. objective: we hypothesized that: e and p have opposite effects in the da; that rising e and falling p levels help trigger da closure via specific hormone receptors; and that no and/or pgs mediate da responses to e and p . methods: expression of er , er , pra, prb, and the putative membrane receptors mpr-, -, -, pgrmc- , - , and serbp- was examined by rt-pcr and qpcr on days , , (term), and p . the effects of e and p ( - - - m) on the d fetal da were examined in a cannulated microvessel myography system. e and p effects were also studied in the presence of receptor antagonists (ici , ru ) and no and pg inhibitors (l-name, indomethacin). results: er and pr receptors were expressed at low levels; pgrmc- expression was stronger than other membrane prs, and increased with advancing gestation. under fetal o conditions, e induced rapid, concentrationdependent dilation at - - - m (n= ); p induced progressive vasodilation at all doses (n= ). e and p -induced dilation occurred within - seconds of exposure. while e -dilated das did not respond to ici, ici-pretreated das failed to dilate to the same dose of e (n= ) suggesting antagonism of e effects. the vasodilatory effects of e were partially inhibited by pretreatment with l-name. p -dilated das did not respond to ru , whereas ru pretreated das showed a small, significant response to p (n= ), suggesting partial antagonism or signaling via alternative, ru -independent pathways. pre-treatment with indocin did not block the vasodilatory effects of p . conclusions: contrary to our expectation, both e and p have dilating effects on the fetal da, via no, pgs and non-no, non-pg pathways. expression studies and the rapid response of ex vivo das are consistent with non-genomic actions via membrane receptors. hormone shifts in parturition may have longterm effects on da preparation for postnatal closure, but strategies to maintain fetal da patency or treat newborns with pda will require better understanding of this process. background. for the fetus, arterial blood gases are critical in the regulation of cerebral blood flow (cbf) and cerebral oxygenation. however, the relation of cbf, cortical tissue po (tpo ), and electrocorticographic (ecog) activity to arterial o tension (pao ) are not well defined. in an effort to elucidate these interrelations, we tested the null hypothesis that in the near-term fetus, acute hypoxic-associated cerebral oxygenation and related variables are not closely associated with ecog state. methods. by use of a laser doppler flowmeter with a fluorescent tissue o probe, and with fluorescent-labeled microspheres, and with ecog electrodes, in near-term fetal sheep (n = ) we measured laser doppler cbf (ld-cbf), tpo , ecog (root mean square (rms) voltage with high voltage low frequency, hvlf versus low voltage high frequency, lvhf) and spectral edge frequency- % (sef ) in response to min moderate isocapnic hypoxia. results. ld-cbf, cerebral o delivery, tpo , and several other variables correlated highly with ecog state. in the normoxic control fetus, in association with a shift from hvlf to lvhf ecog activity, tpo decreased briefly to ± from a control value of ± torr; however, as ld-cbf increased ± %, and sef increased to ± from ± %, tpo returned to near normal value. with acute hypoxia (pao = ± torr) when in the lvhf state ld-cbf increased only ± %, as opposed to a ± % increase when in hvlf ecog state. with this degree of hypoxia, tpo decreased to ± torr, sef remained at ± %, and cerebral metabolic rate for o (cmro ) decreased ± % (p< . ). conclusions. for the near-term fetus, normoxia with changes in ecog state was associated with brief periods of decrease in tpo , which were restored quickly by increased ld-cbf. in contrast, acute hypoxia was associated with a significant depression of cortical tpo , cmro , and ecog state, with increased ld-cbf failing to restore cortical tpo . thus, we reject the null hypothesis that in such fetuses, hypoxia demonstrates no compromise in cerebral oxygenation. (supported by usphs hd- ). releasing hormone and arginine vasopressin in the ovine fetus. charles a ducsay, kanchan m kaushal, malgorzata mlynarczyk, kimberly hyatt, dean a myers. ctr. for perinatal biol., loma linda univ., loma linda, ca; ob/gyn, univ. oklahoma hlth. sci. ctr., oklahoma city, ok. background: long term hypoxia (lth) profoundly affects the hypothalamicpituitary-adrenal axis of the fetal sheep. we previously showed that lth causes augmented corticotrope function. the present study was designed to test the hypothesis that lth enhances sensitivity to the acth secretagogues; cortiocotrophic releasing hormone (crh) and arginine vasopressin (avp) resulting in increased anterior pituitary corticotrope secretion of acth. methods: pregnant ewes were maintained at high altitude ( , m) from day to - of gestation (dg), when they were returned to the lab and a maternal tracheal catheter was implanted. maternal po was maintained at a level comparable to that observed at altitude ( mmhg) by nitrogen infusion. on dg, lth (n = ) and age-matched, normoxic control (n = ) fetuses were implantated with vascular catheters. each fetus received a min infusion of either saline vehicle, ng/kg of ovine crh or ng/kg of avp (estimated body weight/min) in a randomized order over consecutive days ( - dg). blood samples were collected at min (baseline prior to infusion), , , and min following the start of the infusion and analyzed for acth, as well as the acth precursors pro-opiomelanocortin and the major processing intermediate kda proacth. results: vehicle had no effect on any of the measured parameters. with crh infusion, acth (pg/ml) increased in both groups over the course of the study. however, peak concentrations (at min) were significantly higher in the lth group compared to control ( ± vs. ± , respectively; p< . ). acth precursor secretion (pm) was greater in lth fetuses compared to controls during the experiment (p< . ). in response to avp, peak acth concentrations were also higher in the lth fetuses compared to control ( ± vs. ± respectively; p< . ), however peak levels were reached at between and min after start of infusion with levels in both groups returning to pre-infusion values. a similar pattern was observed with precursor levels ( . ± . vs. . ± . , p< . , lth vs. control). conclusions: lth significantly increases pituitary sensitivity to both crh and avp. this enhanced sensitivity may be mechanism of our previously observed enhanced corticotrope function. (supported my nih grants hd and hd ). martin g frasch, roy mansano, michael g ross, robert gagnon, bryan s richardson. ob/gyn, chri, university of western ontario, london, canada; ob/gyn, harbor-ucla med. ctr., torrance, ca. objective: repetitive uco leading to worsening fetal acidosis with fetal heart rate (fhr) decelerations (fhr dec ) are accompanied by pathological decreases of fetal arterial blood pressure (bp). we hypothesized this bp change may be caused by the bjr, a vagally mediated reflex with bradycardia and hypotension to reduce cardiac workload, via stimulation of cardiac chemoreceptors during systemic acidemia. methods: ten near-term fetal sheep ( ± dga) underwent chronic preparation with brachial artery catheters and placement of an inflatable umbilical cord occluder. after a control period, fetuses underwent a series of mild ( min every min), moderate ( min every min) and severe ( min every min) uco each lasting h or until ph decreased to < . . fetal arterial blood samples were taken at selected time points during the control and uco periods. bp and fhr were continuously monitored. individual fhr nadir during each fhr dec and accompanying bp change ( bp = [bp at the time of a fhr nadir] -[bp at baseline preceding a uco series]) were determined during all uco. data are presented as means±sem. results: control period ph ( . ± . ), fhr ( ± bpm) and bp ( ± mmhg) were within the physiological range. average depth of fhr dec for all uco was ± bpm and increased with higher lactate concentrations (r = . , p < . ) and lower ph (r = . , p = . ). bp during all uco demonstrated an initial hypertensive response to fhr dec , which decreased with lower ph (r = . , p < . ). bp increased ± mmhg (p < . ) during mild uco (ph to . ± . ), ± mmhg during moderate uco (ph to . ± . , p < . ) and ± mmhg during severe uco (ph to . ± . , p < . ). conclusion: these results suggest that bjr, a short-acting, ph dependent depressor reflex, blunts the physiologic hypertensive response to cord occlusion insults leading to worsening acidemia as might be seen in the human fetus during labour with repetitive variable fhr dec . the failure to increase bp may prevent optimal blood flow distribution responses necessary for preservation of vital organs. role of nos and pde in placental dysfunction following fetal bypass. mitali basu, r scott baker, christopher t lam, kenneth e clark, pirooz eghtesady. cardiothoracic surgery, cincinnati children's hospital, cincinnati, oh, usa; ob/gyn, university of cincinnati, cincinnati, oh, usa. introduction rising placental vascular resistance following fetal cardiopulmonary bypass (bypass) remains the achilles' heel of fetal cardiac surgery. we have previously shown in real-time that nitric oxide (no) production rises during bypass and falls post-bypass, while cgmp levels rise throughout. using immunohistochemical and western analysis of placenta, we examined the involvement no pathway components in this placental vascular pathophysiology. ovine fetuses at - days gestation were placed on bypass for minutes and followed post-bypass for hours. placental samples were collected immediately prior to bypass and at and min post-bypass (n= ) and compared to a group of similarly instrumented controls, (n= ). placental enos, inos and pde protein expression was measured using standard methods and relative expression normalized to beta-actin. statistical analysis utilized students t-test, and anova for trend and group-wise analysis, (significance at p= . ). pre-bypass protein expression did not differ between groups. pde protein levels and phosphorylated pde- expression were both elevated min post bypass, and reduced min post bypass compared to sham, (p= . ). enos levels in the bypass group increased linearly from pre-bypass to min postbypass (p< . ), and were also elevated compared with shams (p< . ), while shams had declined significantly by min post bypass, (p< . ). similarly, phosphorylated enos expression in the bypass group increased linearly from pre-bypass to min post-bypass, (p= . ), while shams trended towards decline by min post-bypass. simultaneously, placental inos expression remained stable within groups, but was lower in the bypass group at and min post-bypass, (p< . ). the preceding data correlated with observed immunohistological changes in the same placental cotyledons. fetal bypass leads to significant increases in placental protein levels of pde , phosphorylated pde- , enos and ostensibly phosphorylated enos. increased pde expression may be a response to increased no and the generated cgmp. this data suggests a compensatory upregulation of pde and enos that eventually fails, leading to increasing placental vascular resistance and subsequent lethal placental dysfunction. angiotensin receptors (rat-ii) in neuronal nuclei of the brainstem have been implicated in integration of baroreceptor responses. in near term fetal sheep, we have previously shown that days of mild chronic hypoxemia increases heart rate (hr) and blood pressure (bp). the aim of the present study was to investigate the effects of chronic mild hypoxemia on the expression of rat-ii in the medulla oblongata and on baroreflex control of the circulation. methods: at days of gestational age, pregnant sheep were submitted to days of hypoxemia ( % of fetal arterial po ; at day fetus ± vs ± . ; mother ± vs ± mmhg). hr and arterial bp were continuously recorded from mother and fetus in control (n= ) and hypoxemic (n= ) animals. baroreflex sensitivity (brs) as well as hr and diastolic bp variability were analyzed (nevrokard software). brainstem was collected and rat-ii expression in the nucleus tractus solitarius (nts) and dorsal motor nucleus of the vagus (dmnx) was measured by autoradiography using i-sarthran labeling. data are shown as mean±sem and were analyzed by t-test. results: hypoxemia induced a greater total binding of i-sarthran in nts and dmnx resulting from an increased expression of at receptors. in the time-domain analysis of brs hypoxemia induces lower baroreflex sensitivity in the fetus (brs in ms/mmhg; . ± . vs. . ± . , p< . ). in the frequency domain, hypoxemia changes the relative power of low frequencies (lf: . - . hz) and high frequencies (hf: . - . hz) of rri and dbp with an increased value of the lf/hf ratio (rri lf/hf . ± . vs ± . , p< . ; dbp lf/hf . ± . vs . ± . , p< . ). also the alpha index for baroreflex sensitivity is decreased in hypoxemic animals (alpha lf . ± vs . ± . , p< . ; alpha hf . ± . vs . ± . , p< . ). no differences were observed in maternal variables. conclusions: our results suggest a link between a prenatal insult, alterations in cns receptors and functional alterations of baroreflex responses. a higher sympathetic outflow, suggested by a greater lf/hf ratio, and impaired reflex gain, both possibly mediated by increases in nts rat , may have potential long-term consequences for the development of hypertension. hd ;hd ;hl . objective : we evaluated velocity profiles, relative wall distension rate (rwdr), and wall shear rate (wsr) of fetal descending aorta (fda) in uncomplicated singleton gestations. study design: ninety seven uncomplicated singleton fetuses were studied throughout gestation using multigate spectral doppler analysis (msda) working with gasp software. this consists of a personal computer (pc) add-on board including a single high-speed digital signal processor. the analysis of echo-signals backscattered from range cells located along the axis of the interrogating ultrasound (us) beam. post-processing was accomplished using gasp software. statistical analysis consisted of spearman correlation and chi-square test. results: velocity profiles, wall distension, wall shear rate were obtained from fetal descending aorta throughout gestation establishing gestational age specific norms. wdr[%] is highly correlated with gestational age in appropriate growth fetuses ( . ± . , rs= . p< . ) with linear regression with standardized coefficient of . (p< . ). in contrast, wsr ( ± ) is unchanged during the first , second and third trimesters (p= . ).conclusion: we speculate that the relative wdr changes observed during gestation, in normally grown fetuses, may be secondary to adaptive vascular and autonomic responses and the evolving composition of the vessel wall, particularly with respect to elastin. conversely, the mean wsr for the study group was independent and constant throughout the gestation. these findings suggest that there is an increase in the diameter of the fetal aorta, which provides adaptation to the progressive flow demands, while preserving other key hemodynamic parameters. in this study, we have established normative values of rwdr and wsr, which are new rheological parameters that may be useful in distinguishing normal and pathologic hemodynamic states. objective: placental dysfunction is a key barrier to successful fetal cardiopulmonary bypass (cpb) for repair of congenital heart defects in utero. endothelial cells regulate vascular tone during fetal cpb through interactions of vasodilation by nitric oxide (no) and endothelin- (et- )-mediated vasoconstriction. the objective was to determine the time during fetal cpb when endothelial cell-mediated changes occur. methods: human umbilical vein endothelial cells (huvec) were cultured in media containing % serum collected from ovine fetuses (n= ) that underwent min of cpb, then were maintained for min. serum was collected before cpb, from pump prime before initiation of cpb, min on cpb, or and min after fetal cpb. control cells were cultured in normal fetal serum. cells were harvested and hr after addition of fetal serum. no production was measured in real time with an electrochemical detection system (inno-t, harvard apparatus). et- was measured in the culture media by elisa. results: no production by huvec after and hr was stimulated above control levels by fetal serum collected during and up to min after fetal cpb (p< . ). serum collected from fetuses that were surgically instrumented, but not yet subjected to cpb, decreased no levels below controls (p<. ). stimulation of et- after and hr of huvec culture peaked with serum collected at min after fetal cpb (p<. compared with control), but was elevated above control levels at each collection time point (p<. , table ). conclusions: fetal cpb releases serum proteins that elevate endothelial cell no and et- production during and for at least min after cpb. although the specific regulatory proteins remain to be identified, the no and et- pathways share circulating mediators and participate in a feedback loop to modulate vascular tone. to test the hypothesis that hypoxia-induced upregulation of no is linked to cardiac inos expression, a selective inos inhibitor, l-nil (l-n -( -iminoethyl)-lysine), was administered in vivo to fetal guinea pigs and no levels measured in fetal hearts. methods: pregnant guinea pigs were exposed to either normal room air (normoxia; %o ) or . %o (hypoxia) in a hypoxic chamber for days prior to term (term= d). l-nil was administered to pregnant normoxic and hypoxic guinea pigs via their drinking water at a dose of - mg/kg/d for days. at d gestation, pregnant sows were anesthetized and near-term fetuses removed via hysterotomy. the fetal hearts were excised, weighed, and normalized to their respective fetal body weights. left cardiac ventricles were obtained and frozen in liquid n and stored at - o c until ready for analysis. the effect of total no product (no and no -, nox) of left ventricles of fetuses exposed to normoxia (n= ), hypoxia (n= ) and hypoxia plus l-nil (n= ) was quantified by a commercial fluorometric no assay kit. results: intrauterine hypoxia significantly reduced fetal body weight by % and increased placenta/fetal body wt by % as expected for hypoxic stress. hypoxia induced a slight increase in heart/fetal body wt by %. fetal cardiac nox levels (pmoles/mg) were increased by hypoxia ( . + . ) by . fold compared to normoxic controls ( . + . ). l-nil significantly decreased (p< . ) nox levels in hypoxic hearts by % ( . + . vs . + . ; hypoxic vs hypoxic+l-nil, respectively). conclusion: l-nil inhibits inos-derived no generation in the hypoxic fetal guinea pig heart. since previous study in our lab showed a significant increase in inos expression but a decrease in enos and no change in nnos expression, we hypothesize that hypoxia upregulates cardiac no generation via the inos pathway. further study is needed to identify the important role of cardiac inos in the adaptive response of fetal hearts to chronic intrauterine hypoxia. objective: fetal asphyxia-mediated metabolic acidosis results in a ph decrease and an increase of lactate and base deficit in blood (bd blood ) and extracellular fluid (bd ecf ). it is not clear whether bd blood and bd ecf are similarly altered with worsening fetal acidosis. in the present study we sought to study the dynamic relations of bd blood and bd ecf to lactate in the ovine fetus subjected to repetitive uco with worsening acidemia. methods: ten near-term fetal sheep ( ± dga) underwent chronic preparation with brachial artery catheters and placement of an inflatable umbilical cord occluder. following a baseline recording period, fetuses underwent a series of mild ( min every min), moderate ( min every min) and severe ( min every min) uco with each series lasting h or until fetal arterial ph decreased to < . . fetal arterial blood was sampled at baseline, immediately before and during the first mild, moderate and severe uco and at min intervals during the moderate and severe uco. bd gap (bd blood -bd ecf ) was studied in relation to lactate. presented as means±sem. results: lactate correlated to bd blood (r= . , p< . ). bd ecf correlated strongly with bd blood (r= , p< . ). bd ecf increased by . ± . mmol/l more than bd blood from baseline to ph nadir, with bd gap therefore decreasing with increasing lactic acidemia ( fig. ) . lactate, bd blood , bd ecf and bd gap correlated to ph (r= . , r= . , r= . and r= , respectively, all p< . ) and ph could therefore be predicted with any of the four acid-base parameters. conclusion: the increases of bd blood and bd ecf and the decrease of bd gap with increasing lactic acidemia suggest a relatively more rapid accumulation of [h+] in ecf during uco of increasing severity. this may be because the metabolic build-up of acidosis occurs primarily in the tissues and the endothelial permeability for [h+] is impeded during increasing acidosis with atp depletion thus decreasing [h+] movement from ecf to plasma. previous studies have shown that intraperitoneal transplantation of human umbilical cord blood (hucb)-derived mononuclear cells led to the specific 'homing' of these cells to a hypoxic-ischemic brain lesion in perinatal rats. motor deficits resulting from the lesion were alleviated upon transplantation. thus, the presence of hucb cells at the lesion site seems to be a major prerequisite for their potential beneficial effect. however, the mechanisms of cell 'homing' are still unclear. in this study, we focused on elucidating mechanisms underlying the specific migration of hucb-derived mononuclear cells to the brain lesion. one possibility to induce cell 'homing' are chemotactic signals present at the lesion site. the cxc chemokine stromal derived factor- (sdf- ), which was previously shown to be a potent chemoattractant for directed migration of other stem and progenitor cells, is a putative candidate in our lesion paradigm. therefore we investigated the spatial and temporal expression of sdf- in brain hemispheres with or without hypoxic-ischemic lesion. sdf- expression was substantially increased at the lesion site during the investigated period of fourteen days after the insult. furthermore, hla-positive hucb cells were mainly detected in sdf- expressing brain regions and we were able to show that these cells express the sdf- receptor cxcr on their surface. the functional implication of sdf- in directing hucb cell migration was determined by application of neutralizing sdf- antibodies in vivo, resulting in a reduced number of hucb-derived mononuclear cells at the lesion site. with these functional effects, together with the observed timing and location of its expression, the involvement of the chemokine sdf- in hucb cell 'homing' seems conceivable. novel pathways in inflammation-induced fetal brain injury. michal a elovitz, jinghua chai. obstetrics and gynecology; crrwh, university of pennsylvania, philadelphia, pa, usa. intro: while survival of extremely preterm infants continues to improve, the number of children with cognitive impairment and/or cerebral palsy is increasing. if preterm birth (ptb) cannot be prevented, then strategies to identify and treat fetal brain injury in the setting of a ptb must be investigated. these studies were performed to explore novel pathways involved in fetal brain injury in the setting of inflammation-induced ptb. methods: cd- mice on e receive intrauterine injection of lipopolysaccharide (lps) or saline. hours after injection, fetal brains from the left upper horn were harvested. fetal brains were removed from each dam with dams per treatment group. separate rna samples were prepared and used for microarray (ma) analysis. all protocols were conducted as described in the affymetrix genechip expression analysis technical manual in the ma core facility using the moe av chip. data analysis was performed using significance analysis for microarray (sam). the brain samples from the same dam were considered as dependent samples. pathway analysis and functional annotation clustering tools were used with david. validations studies using qpcr with fetal brain samples (n= - per group) were performed. results: while there was significant differences in gene expression between lps and saline exposed fetal brains, variability existed even between pups from the same dam. genes were significantly differentially expressed between lps and saline brains (p< . ). with a p value of < . , genes were differentially expressed. pathway analysis revealed significant involvement of ) the cadeherin, cadherin-like, cell fraction and calcium binding (enrichment score . ) and ) ribosomal processing, rna metabolism, pyrimidine metabolism (enrichment score . ). specifically, genes involved in neurogenesis, synaptic function, and neuronal and glial metabolism were most differentially regulated. qpcr confirmed observed fold changes in / genes analyzed. inflammatory pathways were not differentially regulated. conclusions: current theories regarding fetal brain injury in ptb focus on activation of inflammatory processes as essential events. this data suggests that long-term neurological injury in a ptb may be secondary to altered neuronal function, metabolism and/or communication. disruptions in these pathways should be explored as key mechanisms to adverse neurological outcomes in preterm neonates and should be targets for future investigations. regulation of microglial activation in the developing murine brain. mariya hristova, virginia zbarsky, daniel cuthill, adam wallace, donald peebles, gennadij raivich. institute for women's health, university college london, london, united kingdom. introduction: the aim of this study was to assess the process of microglial differentiation in developing white matter, an area of the brain that is particularly vulnerable to damage pre-myelination (approx weeks gestation). microglia form a distinctive non-neuronal component. although related to peripheral macrophages they undergo highly specific processes of regional maturation and differentiation inside the brain with a slimming of the cell body, development of very elaborate crenulated arborised branches and downregulation of most macrophage activation markers. this process is relatively rapid in most grey matter brain regions, but is retarded in and around the subcortical white matter (swm) giving rise to the phagocytic fountains of microglia (fom). methods:we examined the process of deactivation and morphological differentiation in the cortex and swm of mice - days after birth (p -p ) using confocal microscopy for monoclonal antibodies against alpha and beta integrin subunits and the costimulatory factor b . , colocalised with standard microglial marker iba . results: strikingly, only the fom macrophages, but not cortical microglia, strongly expressed typical activation markers alpha- , alpha- , alpha-m, alpha-x, beta- and b . . the data for alpha-x are shown as an example in the figure; cortical microglia are shown in the grey bars and swm in black. fom activation was maximal at p , decreased linearly over p and p and disappeared at p . this process followed the presence of ingested phagocytic material but correlated only moderately with ramification, demonstrated by non-ramified but inactive p cortical microglia and formation of stubby processes in p fom. conclusion: these data describe strong and selective biochemical activation of fom phagocytes in p -p swm, roughly equivalent to early rd trimester human foetal development. this presence of highly active phagocytes in the neighbourhood of vulnerable wm could play an important role in the genesis of axonal damage in the foetus and premature neonate. , pp. - ) . mbp is expressed in mature oligodendrocytes (jakovcevski and zecevic, glia ) . although myelination in the fetal sheep brain, a model extensively used to evaluate fetal development, has been described using conventional staining techniques (barlow, j comp neurol ) the use of specific mbp staining allows a more precise determination of the onset of myelination (antonow-schlorke et al., reprod sci . , , a) . aim: to use mbp expression to determine the trajectory of development of different white matter tracts of fetal sheep brain. methods: the ontogenetic profile of mbp expression was estimated in healthy fetuses at . (n= ), . (n= ), . (n= ), . (n= ), . (n= ), . (n= ) and . (n= ) of gestation (term days). after brain fixation and embedding in wax, sections at the level of the optic chiasm were stained with a monoclonal anti-mbp antibody using the abc-technique. immunohistochemical distribution of mbp was morphometrically assessed in the dorsal internal capsule and cortical white matter tracts, i.e. the lateral centrum semiovale, superficial white matter and median corpus callosum using an image analysis system (scion image . , nih, usa). results: mbp expression of various fetal white matter structures started at different time points; initially in the internal capsule at . of gestation followed by the cortical white matter structures at . of gestation. cortical myelination advanced from the cortical deep white matter via superficial white matter to the corpus callosum reflecting different rates of progression. conclusions: the onset of mbp expression estimated here explicitly antedates previous observations in sheep (barlow, ) the way the embryonic heart functions before cardiac morphogenesis is completed is still subject of many studies. to gain more insight into the early cardiac structure-function relationship, doppler blood flow velocity waveforms at four different locations in the embryonic chicken heart during cardiovascular development were assessed. we collected waveforms using high frequency ultrasound biomicroscopy with a -mhz transducer at hh stages , and which are comparable to humans at to weeks of gestation. waveforms were obtained at the inflow tract, the primitive left ventricle, the primitive right ventricle, and at the outflow tract in ten different embryos per stage. by exploring the time relation between the waveforms, cardiac cycle events were outlined. our results demonstrate that stage and location dependent, intracardiac blood flow velocity waveforms can be obtained in the chicken embryo which reflect stage dependent pumping mechanisms. the blood flow profiles assessed at the four locations in the embryonic heart demonstrated a developmental related increase in velocity. in the primitive ventricle the passive filling wave decreased, whereas the active filling wave increased resulting in a decreased p to a ratio in the course of time. high frequency derived cardiac blood flow velocity characteristics support previous findings that the embryonic heart functions like a suction pump at early development and transforms towards another pumping mechanism at later developmental stages. these findings are of importance for the interpretation of human first trimester cardiac velocimetry studies. figure a . changes in the perimeter of the thymus with gestational age by fetal gender are depicted in figure b . background. for the fetus, the roles of arterial blood gases are recognized to be critical in the regulation of cerebral blood flow (cbf) and cerebral metabolic rate for oxygen (cmro ), cortical tissue po (tpo ), and electrocorticographic (ecog) activity. in addition, metabolites such as adenosine (ado) are important in this regard. nonetheless, the relation of adenosine and its metabolites to these indices of cerebral oxygenation are not well defined. in an effort to elucidate these interrelations, we tested the null hypothesis that acute hypoxic-associated cerebral oxygenation and related variables are not closely associated with adenosine. the most common cause of perinatal brain injury is chronic hypoxia/ischemia. the mechanisms are complex and poorly understood. applying proteomics tools, we identified a set of hypoxia-related proteins in the fetal guinea pig (gp) brain (companion abstract). one protein, cofilin- , which regulates the rapid cycling of actin assembly and disassembly, was dramatically altered by chronic brain hpx. herein, we test the hypothesis that confilin- modifications during hpx contributes to brain damage via apoptosis and is an example of an adaptive response that becomes maladaptive. methods: time-mated gps were housed in a chamber from d to d (term) with either room air (nmx) or . % or % o . fetal brains were removed and sections prepared for double staining specific to bax and dephosporylated cofilin- . total protein was isolated and equal amounts loaded on a sds gel, separated by electrophoresis, and transfered to pvdf membranes probed with primary antibodies to dephosphorylated and phosphorylated cofilin- , bax and bcl-xl, and incubated with second antibody. protein signals were detected, quantified by densitometry, and normalized to -actin. total rna was isolated, reverse-transcribed, and quantified by real-time pcr using sybr green i labeling. expression was calculated by the ct method using s for control. melt analysis was performed to confirm specificity of the amplification, and efficiency determined by the slope of the standard curve. the mitogen activated protein kinase (mapk) signalling pathway is upregulated in perinatal hypoxic-ischaemic brain. the role of the extracellular signal-regulated kinase (erk) cascade, a pivotal component of mapk signalling, remains unclear with reported actions ranging from mitogenic and trophic effects to a neuronal death-promoting role. the aim of this study was to assess the role of neuronal erk activity using selective neuronal inhibition in a perinatal model of hypoxic-ischemic brain injury. methods: we explored the effects of selective neuronal inhibition of erk activation using transgenic mice expressing dominant-negative (dn) mek , the upstream activator of erk / , which was under the control of the pan-neuronal talpha alpha-tubulin promoter. p-erk immunoreactivity was quantified following a hypoxic inschemic (hi) insult in p c bl/ mice, caused by unilateral occlusion of the carotid artery, followed by hypoxia with % oxygen for mins at °c. the volume of surviving brain on the affected hemisphere was expressed as a % relative to the control side. results: expression of the mek dn was associated with a reduction in erk / activation following hypoxia ischaemia, as assessed by p-erk immunoreativity. compared with the wild type, littermate controls, mek dn animals exhibited significantly decreased volume of forebrain damage brain following unilateral, min hi insult (*p< . , **p< . , anova). similar protective effects of mek dn were observed in cerebral cortex, hippocampus, thalamus and striatum when compared to wild type littermate controls and correlated with a significant reduction in microglial activation in all brain areas. conclusion: overall, these results suggest that neuronal mek and its downstream signals have an important death-inducing role in this model of perinatal brain injury and could serve as potential targets for therapeutic intervention. oup, ) . however, whether melatonin protects placento-fetal development during hypoxic or undernourished pregnancy is unknown. we investigated in rats the effects on placental and fetal growth of maternal treatment with melatonin in hypoxic and undernourished pregnancy. methods: on pregnancy day , wistar rats were divided: control ( % o ) + melatonin ( g.ml - drinking water), hypoxia ( % o ) + melatonin, and undernutrition ( % reduction in food intake) + melatonin (n= per group). on day , dams were anaesthetised, the pups, placentae and fetal brain were weighed. results: relative to controls (fetal weight: . ± . g; brain/body weight ratio: . ± . mg/g; n= ), both hypoxic ( . ± . g; . ± . mg/g, n= ) and undernourished pregnancy ( . ± . g; . ± . mg/g, n= ) promoted asymmetric iugr (p< . ), without affecting placental weight. melatonin treatment had no effect on iugr, but it decreased placental weight in normoxic ( . + . , n= ), hypoxic ( . ± . , n= ) and undernourished ( . ± . , n= ) pregnancies relative to untreated controls ( . ± . , n= ; p< . ). when fetal body weight was expressed relative to placental weight, a measure of placental efficiency, melatonin prevented the fall in the ratio in undernourished, but not hypoxic pregnancies (fig. objective: midkine (mk) is a -kda heparin-binding growth factor with various functions including cell proliferation, migration, differentiation and angiogenesis. mk expression is strictly regulated in temporal sequence; it is highly expressed during midgestation. we recently studied mk expression in the midgestation human fetus, and showed that the highest mk expression were observed in the adrenal gland, brain, lung and kidney. in the present study, we investigated the profile of mk in human amniotic fluid (af) and amnion (am). methods: amniotic fluid and amniotic membranes were collected at diagnostic amniocentesis, preterm no labor, and term no labor. mk protein levels were analysed by western blot. expression of transcripts encoding mk and putative mk receptors were examined by rt-pcr and real-time quantitative rt-pcr (qpcr). results: ) western blot analysis demonstrated abundant mk protein in the human am; mk levels were higher at midgestation than at term ( -fold: wks vs. wks). ) tissue transglutaminase known to polymerize mk was abundant in af. ) qpcr revealed that mk mrna was not expressed in am whereas it was highly expressed in the fetal adrenal, kidney and lung (positive controls). ) among the receptors implicated in mk signaling, lowdensity lipoprotein receptor-related protein and syndecan- were expressed in am while protein-tyrosine phosphatase, anaplastic lymphoma kinase, and syndecan- were not. conclusions: abundant mk protein in the midgestation af is likely to be derived from the fetus. mk in af may play a role in feto-amniotic communications and/or development of fetal organs exposed to af. short term hfix expression. we hypothesized that long term hfix expression could be achieved in fetal sheep using an aav vector, without stimulating an immune response to the transgenic hfix protein. we injected aav hfix vector ( - x p/kg) into the peritoneal cavity of fetal sheep under ultrasound guidance in early (n = ) or late (n = ) gestation. fetal blood was retrieved by ultrasound guided sampling from the intra-hepatic umbilical vein. fetal and lamb blood was tested for hfix expression using elisa, antibody responses using functional assays, and for liver damage up to a year after birth. vector spread was detected in maternal and fetal tissues by quantitative pcr analysis. results: the highest level hfix was detected days after late ip injection ( % and % normal human levels). early gestation ip injection gave . % and . % at and days after injection. hfix levels dropped rapidly correlating with the increase in size of the fetal liver and lamb. however, hfix was detectable at a low levels ( . %) year after birth in early and months after birth in late gestation injected lambs. up to year after birth, liver function tests and bile acid levels were normal, showing no evidence of liver pathology. no functional antibodies to the hfix protein or aav vector were detectable. high vector levels were detected in the fetal liver, and other peritoneal organs; no vector was present in fetal gonadal tissue. conclusion: hfix expression is detectable up to year after delivery of aav vector to the fetal sheep using a clinically applicable method. this is the first study to show long term hfix expression after fetal gene therapy in a large animal. further work will include testing for immune tolerance to exogenous hfix protein in these animals. objective: placental estrogens play a pivotal role in the endocrine control of pregnancy and may be involved in the key changes occuring during parturition. it has been established that crh interacting with crh receptor has a positive effect on estrogen production throughout pregnancy. urocortin (ucn ), a novel peptide of the crh family binding exclusively crh receptor , is expressed by human placenta and the aim of the present study was to evaluate the influence of ucn on estrogen biosynthesis in cultured trophoblast cells. methods: cultured term placental cells were treated with various concentrations of ucn in the presence of the estrogens precursors dehydroepiandrosterone sulphate (dhea-s), androstenedione or testosterone. estradiol secretion was measured in the culture medium using a specific elisa, p arom mrna and protein expression were evaluated by real time pcr and western blot analysis respectively. results: the addition of ucn , in presence of dheas significantly increased e levels at , and hours treatment, while in presence of androstenedione and testosterone an increase in e secretion was detected only at , and hours (at different ucn doses). both p arom mrna and protein were up-regulated in presence of each estrogen precursor and the addition of ucn caused a synergistic increase. anti-sauvagine (a crh type receptor antagonist) resulted in a significantly attenuated ucn effects on e secretion and on p arom mrna and protein expression. conclusions: the present study supports a possible role of ucn on placental e biosynthesis: e secretion and p arom transcript and protein expression were significantly increased after ucn treatment. in conclusion, the crf family may play a major role on placental steroidogenesis, stimulating dheas secretion in fetal adrenals by crh and controlling placental estrogen biosynthesis through ucn . a possible influence on the mechanisms of parturition may be hypothesized. increased expression of kiss- gene in preterm placenta. letizia galleri, michela torricelli, chiara voltolini, fernando m reis, felice petraglia. department of pediatrics, obstetrics and reproductive medicine, university of siena, siena, italy. introduction placenta expresses a large number of peptides involved in delivery. neuropeptides, cytokines, are expressed and secreted by human placenta at the time of preterm delivery. human placenta is a major source of kiss- , a -amino-acid peptide encoded by a putative metastasis suppressor gene. kiss- acts on its placental g protein-coupled receptors (kiss- r); this peptide stimulates release of oxytocin in rats, the most potent known uterine stimulant, suggesting a possible role of kiss- in the mechanisms of labor. aim of study the aim of this study was to evaluate placental expression of kiss- at preterm labor. material and methods placental tissue and plasma samples (both maternal and fetal) were collected at term in the absence of labor (tnl), at term spontaneous vaginal delivery (tl), and at preterm labor (ptl). changes in placental mrna expression were determined by real-time quantitative rt-pcr analysis. kiss- protein levels were measured by specific immunoenzymatic assay (elisa). results placental kiss- mrna expression was significantly higher (p< . ) in ptl than in tnl and in tl. however, maternal and fetal plasma kiss- levels did not differ among tnl, tl, and ptl samples. conclusion the present study showed that placental kiss- mrna expression is increased in preterm delivery. further studies are needed to better understand the role of kiss- on cascade leading term and preterm labor. placental angiogenesis is essential for placental development, which occurs under a hypoxic environment ( - % o ) during normal pregnancy. it has been reported that in transformed human dermal microvascular endothelial cells, hypoxia activates erk / , which stabilizes hypoxia-inducible transcription factor- (hif- ). however, signaling mechanisms governing placental angiogenesis under hypoxia is largely unknown. herein, we tested whether hypoxia affected fgf -and vegf-stimulated cell proliferation partly via activating erk / and stabilizing hif- protein levels in human placental artery endothelial (hpae) and transformed human placental microvascular endothelial (hpme) cells. methods: cells cultured under normoxia ( % o ) or hypoxia ( % o ) for days were treated with fgf and vegf. after days, the number of cells was determined. activation of erk / and levels of hif- protein were determined by western analysis. results: under normoxia, fgf and vegf stimulated (p < . ) cell proliferation and induced ( min, p < . ) erk / phosphorylation in hpae and hpme cells. hypoxia promoted (p < . ) fgf -and vegf-stimulated cell proliferation in hpae cells, whereas hypoxia blocked (p < . ) such actions in hpme cells. hypoxia enhanced fgf -, but not vegf-induced erk / phosphorylation in hpae cells. in contrast, hypoxia promoted (p < . ) vegf-, but not fgf -induced erk / phosphorylation in hpme cells. hypoxia increased (p < . ) hif- levels in the hpae cells: first detected at . hr and maintained up to hr. hpme cells had high basal levels of hif- ( folds; p < . ) compared with hpae cells. hypoxia did not alter hif- levels up to hr and decreased (p < . ) hif- levels at hr in hpme cells, conclusions: in hpae cells, hypoxia enhances fgf -and vegf-stimulated cell proliferation possibly partially via promoting activation of different protein kinases. this stimulatory effect is associated with increased hif- protein levels. however, inhibition by hypoxia on fgf -and vegf-stimulated hpme cell proliferation is associated with relatively high hif- levels in the first hr and decreased hif- levels at hr. thus, these data suggest that different signaling mechanisms are involved in hypoxia-modulated growth factor-induced placental angiogenesis in which an increase in hif- levels plays a critical role. objective: corticotrophin releasing factor (crf) is a well established neurohormone involved in the initiation of the stress response. we recently documented that rat placenta is analogous to human placenta in the expression of crf-mrna and protein, and that placental crf release may contribute to in utero meconium passage. time-of-day variation in crf expression is a highly recognized phenomenon at the brain cellular sites of crf synthesis. we sought to determine whether crf expression in rat placenta is subject to time-of-day variation. method: time-dated pregnant rats were obtained on day of gestation (term= days), housed in under h- h light-dark conditions (lights on ). lab chow and water were continuously available. on day , pregnant rats were quickly anesthetized by exposure to isoflurane, abdomen opened and fetuses and placenta exteriorized either at - hr (zeitgeiber time, zt ) or - hr (zt ). individual placentas were processed either for rna extraction or immunohistochemical investigation. the levels of crf-mrna were assessed by pcr using rat specific pcr primers. pcr bands were subsequently cloned and sequenced. bouin's solution fixed paraffin sections of placenta were subjected to crf immunohistochemistry with antibodies specific to rat species and intensity of immunostaining was analyzed using image pro-plus software and expressed in arbitrary units (au). results: one specific pcr band of bp size was consistently identifiable in placenta harvested at zt but not at zt . nucleotide sequence of the pcr band confirmed its identity as crf-mrna. intensity of crf-immunostaining was significantly greater at zt in giant trophoblast (gt) cells (gt-crf: zt = . ± . au, zt = . ± . au, p= . ) but not in spongiotrophoblast cells (stc) (stc-crf: zt = . ± . au, zt = . ± . au, p=ns) or labyrinth cells (lbc) (lbc-crf: zt = . ± . , zt = . ± . au, p=ns) . conclusion: similar to adult brain, rat placenta expresses crf mrna and protein. time-of-day variation of crf expression originally seen in central nervous system neurons is also identifiable in giant trophoblasts cells at zt . these findings suggest that stress-mediated placental crf release, and potentially fetal meconium passage, may be dependent upon time-of-day. objectve: transplacental water flow is essential for the provision and maintenance of fetal body water and amniotic fluid. water flow across membranes is regulated by aquaporin (aqp) water channels in many tissues. thus aqp , expressed in the placenta, is a candidate to regulate maternal to fetal fluid exchange. maternal beta-mimetics have been hypothesized to augment fetal growth by increasing the availability of nutrients and perhaps water. as camp acts as a second messenger to increase expression of selected aqps in other tissues, we sought to determine if betamimetics acting through camp could modulate placental aqp , and potentially influence placental water transfer. methods: trophoblastic cell cultures were established in first trimester-derived extravillous htr- /svneo cells and term placenta-like trophoblast carcinoma cell line jeg- . cultures were treated with sp-camp, a membrane-permeable and phosphodiesterase resistant camp, and forskolin, an adenylate cyclase stimulator, in doses of . , and m for hrs (aqp mrna expression) and hrs (aqp protein expression). for time course experiments, cells were incubated with μm of either sp-camp or forskolin for , and hrs at °c, % co . after cell harvest, mrna and protein expression were assayed using real time pcr and western blotting. results: sp-camp and forskolin increased aqp mrna expression in both cell lines after hrs (p< . ) in a dose-dependent manner. protein expression paralleled the increase seen in the mrna. m of sp-camp and forskolin stimulated aqp mrna expression after hrs in htr- /svneo cells and after hrs in jeg- cells (p< . ). m of either sp-camp or forskolin stimulated aqp protein expression in both cell lines after hrs (p< . ) and the expression remained high at hrs. conclusion: aqp gene expression in trophoblast cells is up-regulated by camp agonists. these results suggest that maternal beta-adrenergic agonists or antagonists may modulate maternal-fetal water flux via modulation of aqp water channels. rat placenta expresses corticotrophin releasing factor-binding protein and mrna. jayaraman lakshmanan, thomas r magee, bindu cherian, sharon k sugano, hanalise huff, michael g ross. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: corticotrophin releasing factor-binding protein (crf-bp), a kda secreted glycoprotein, was originally isolated from human plasma. it binds crf and urocortin i with an equal or greater affinity than does the crf receptor. crf-bp expression has been reported in target tissues such as brain and placenta. based on its ability to inhibit crf actions in vitro, it is speculated to function as a "gate keeper" for crf-initiated stress responses. we recently documented expression of crf and urocortin- in rat placenta. in the present study we sought to establish the expression of crf-bp in rat placenta by immunohistochemical and pcr-analyses. methods: placental tissues (n= ) collected from sprague-dawley pregnant rats on day of gestation (term= ) were either fixed in % paraformaldehyde solution (ph . ) and paraffin embedded or processed for total rna extraction. paraffin sections of five micron thickness were subjected to immunohistochemical analysis with goat polyclonal antibodies to human crf-bp precursor (sc- santa cruz biotechnology, ca) by avidinbiotin-peroxidase complex technique. the structure of cloned human crf-bp precursor exhibit significant amino acid sequence homology among all species studied. immunoreactivities on the sections were quantified using image pro . software and staining intensity (od/area) expressed as arbitrary units (au). all values are expressed as mean±sem. for pcr, cdna was synthesized and pcr amplified with a one step rt-pcr kit. fragments were gel purified, clone into plasmid vector and dna sequenced. results: the crf-bp antibody elicited strong positive staining in decidua, giant trophoblasts, spongiotrophoblasts and labyrinth cells. the results of image analyses revealed (au): decidua: . ± . , giant trophoblast cells: . ± . , spongiotrophoblasts: . ± . , fetal membranes: . ± . . pcr analyses identified a single bp band, consistent with crf-bp. conclusion: our study establishes for the first time that rat placenta is analogous to humans in that both express crf-bp mrna and protein. immunohistochemical findings reveal that crf-bp protein expression occurs at multiple sites within the placenta. expression of per clock protein in rat placenta: an internal timer for placental functions. jayaraman lakshmanan, reuben lakshmanan, sharon k sugano, michael g ross. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: corticotrophin releasing factor (crf) expression time-of-day variation occurs in giant trophoblast cells on the maternal side of the rat placenta. this temporal change in crf expression pattern is very similar to that of central nervous system neurons, suggesting that placental cells may use a similar mechanism to read time of the day. the self-sustaining rhythm generating capacity of the suprachiasmatic nuclei is believed to be derived from cell-autonomous, transcriptional feed-back loops dependent on a number of canonical clock genes. in the present study we sought to determine whether rat placenta expresses period gene (per ), one of the clock/cycle related genes. methods: time-dated pregnant sprague-dawley rats were received on day of gestation and housed in a controlled environment ( - h lights on) with free assess to food and water. placentas collected on days and of gestation (term= days) between and . am were fixed in % paraformaldehyde and paraffin embedded. for each gestational ages a total of placentas from different pregnant rats were used. paraffin sections ( sections per placenta) were subjected to immunohistochemical analysis with antibody to per ( : , santa cruz biotechnology, ca) by abc technique and , 'diaminobenzidine as a chromagen. sections were examined under microscope, imunostaining quantified by image pro . software and expressed in arbitrary units (au). data are presented as mean ± sem. statistical significance was analyzed by anova with a p value < . as significant. conclusion: the present results indicate that rat placental cells, devoid of any neural innervations, express per clock protein, similar to central nervous system neurons. based on the differences in the relative intensities between trophoblasts and labyrinth cells on day of gestation, we conclude that fetalmaternal interactions in per regulation disappear at term (or at the time of birth.). our findings imply that per may function as internal physiological modulator in placenta. to determine the mechanism(s) underlying placental time-of-day crf variation, we examined the effect of maternal administration of betamethasone (a synthetic glucocorticoid) on nuclear gc receptor expression in placental cells. method: time-dated pregnant rats (n= ) on day of gestation were given a single subcutaneous injection (at : am) of betamethasone ( μg/kg body weight) while control pregnant rats (n= ) received saline. dams were exposed to isoflurane anesthesia at : am, and placentas harvested, fixed in bouin's solution and paraffin embedded. sections were subjected to immunohistochemical analysis with gc-receptor antibody (sc- , santa cruz biotechnology), with immunoreactive material identified by abc technique using , ' diaminobenzidine as a chromagen. percentages of gc-nuclear receptor (gc-nr) positive cells and their intensities (od/area) were quantified using image pro . plus software. all values are expressed as mean ± sem. statistical analysis was by anova with p< . considered significant. results: in placentas of saline exposed pregnant rats, isolated labyrinth (lb) cells (< %) were positive to gc-nr. no positive staining for gc-nrs was seen either in giant trophoblasts (gt) or in spongiotrophoblasts (st). betamethasone administration was associated with a significant and marked increase in gc-nr staining in all three placental cell types (p< . ). among the cells, gt ( ± %) demonstrated a greater percentage of cells expressing gc-nr than did st ( ± %) or (lb= ± %) (p< . ), though there was similar immunoreactive intensities (gt: . ± . , st: . ± . , lb: ± . au; p=ns) conclusion: our findings indicate that all placental cells respond to gc with upregulation of gc-nr with an enhanced response among gt cells. these results suggest that endogenous or exogenous maternal stress-induced gc exposure may influence signaling responses within both maternal and fetal placental compartments. ovine placenta at very-preterm gestation expresses corticotrophin releasing factor (crf), crf-binding protein (crf-bp) and glucocorticoid receptors (gr). jayaraman lakshmanan, john d richard, guo l liu, sharon k sugano, michael g ross. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: in humans, the placenta is the major source of maternal and fetal plasma crf. based on its critical functions, crf is considered as a "placental clock" of parturition. we recently reported that ovine fetal as well as maternal plasma contain measurable amounts of crf at near-term but not at very preterm gestation. we interpret the absence of crf in plasma at very preterm gestation is either due to lack of placental crf expression and/or placental crf release. here, we examined the expression status of crf, crf-bp (a known specific binding protein for crf and a known regulator of crf functions in human placenta) and gr (a known positive regulator of crf expression in human placenta) in ovine placenta collected at very-preterm gestation. method: placenta harvested from time-dated pregnant ewes on ± days gestation were fixed in bouins's solution and processed for paraffin embedding. paraffin sections were subjected to immunohistochemical analysis with polyclonal antibodies specific to ovine crf( : - : , phoenix pharmaceuticals, ca,) crf binding protein ( : - : , sc ) and gr ( : - : , sc: , santacruz biotechnology, ca). immunoreactive material on the sections were identified as brown staining by abc technique using diaminobenzidine as chormagen. immunoreactive material was quantified by image analysis using image pro . plus software and the immuoreactive intensity (od/area) expressed as arbitrary units (au). results: strong positive staining for crf ( . ± . au), crf-bp ( . ± . au) and nuclear-gr ( . ± . au) were noticed in syncytiotrophoblast cells in all placental sections obtained from pregnant ewes at days gestation. control sections exhibited no positive staining. conclusion: our findings indicated that ovine placenta at very-preterm gestation expresses crf, crf-bp and gr. these results suggest that absence of measurable crf in maternal and fetal plasma at very-preterm gestation is not due to lack of placental crf expression but rather due to the absence of regulated crf secretory mechanisms. rat placenta expresses urocortin i protein and mrna. thomas r magee, michael g ross, john d richard, sharon k sugano, jayaraman lakshmanan. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: urocortin i (ucn- ), a amino acid neuropeptide belongs to corticotrophin releasing factor (crf) stress hormone family. in humans, the placenta and several gestational tissues have been reported to express ucn- protein and ucn- mrna. a number of published studies indicate that ucn- is similar to crf in expression pattern and biological functions. we recently reported that rat placenta is a site of crf protein and crf mrna expression. in the present study we sought to determine whether rat placenta expresses ucn- protein and ucn- mrna. methods: placenta (n= ) collected from pregnant rats at day gestation were either fixed in bouin's solution and processed for paraffin embedding or placed in rna later preservative and frozen for rna extraction. for immunohistochemical localization, five micron thickness paraffin sections were cut and immunostained with rabbit polyclonal antibodies to ucn- ( : to : , sigma) by standard abc technique. control sections were incubated with omission of ucn- antibody. immunoreactive materials on the sections were identified using , ' daminobenzidine as chromagen. immunostaining intensity (od/area) was quantified by image-pro plus software and expressed in arbitrary units (au). for pcr, cdna was synthesized and pcr amplified with a one step rt-pcr kit. fragments were gel purified, cloned into a plasmid vector and dna sequenced. all values are given as mean ± sem. results: ucn- polyclonal antibody elicited strong immunostaining in placental trophoblast cells with variable intensity. the pattern of immunostaining is as follows: giant trophoblast cells: . ± . au, spongiotrophoblast cells: . ± . au and labyrinth cells: . ± . au. the relative intensity in labyrinth cells was significantly lower than the other two cell types (p< . ). pcr analyses revealed the presence of a single band of bp, consistent with ucn- mrna. conclusion: similar to human placenta, rat placenta expresses ucn- protein and ucn- mrna, with most expression localized to the maternal side trophoblast cells. these results support our hypothesis that rat placenta can be used as a model to understand the role of this peptide in feto-maternal stress. the role of the nuclear hormone receptors fxr, pxr and car in placental bile acid homeostasis in normal and pathological pregnancy. victoria geenes, peter dixon, selina raguz, jenny chambers, kishore bhakoo, catherine williamson. imperial college, london, united kingdom. background: obstetric cholestasis (oc) is a pregnancy specific liver disorder characterised by raised maternal serum bile acid levels and associated with adverse fetal outcome. the aetiology of oc is complex and not fully understood, but the fetal complications are likely to result from an accumulation of bile acids in the fetal circulation. bile acids are the toxic end products of hepatic cholesterol metabolism and are synthesised from weeks gestation. in common with other waste products, accumulation in the fetal compartment is prevented by excretion across the placenta into the maternal compartment. however, studies of maternal and cord serum from normal and oc pregnancies have suggested that bidirectional transfer of bile acids is possible. hepatic bile acid transport and metabolism is regulated by members of the nuclear hormone receptor family, namely fxr, pxr and car, but the mechanisms for regulating placental transfer are unknown. objectives: this study used placenta from normal and cholestatic pregnancies to investigate the expression of genes involved in hepatic bile acid homeostasis. methods: villous trophoblast samples from oc and normal pregnancies (np), and human livers were collected and preserved in rnalater. explant cultures were prepared from a further np placentas and cultured for days at % oxygen. on day they were treated with chenodeoxycholic acid, lithocholic acid or vehicle for hours prior to fixing in rnalater. total rna was extracted using trizol, and reverse transcribed to cdna. quantitative real-time pcr was performed using sybr green. target gene mrna abundance was calculated from a standard curve and normalised to l . results: the expression of fxr, pxr and car, the nuclear hormone receptors responsible for hepatic bile acid homeostasis and several fxr target genes (shp, mdr and bsep) was found to be very low in np, and unaffected by the presence of maternal cholestasis. furthermore, the expression of these genes could not be induced by bile acid treatment in an in vitro model. here we have shown that the nuclear hormone receptors (fxr, pxr and car) involved in hepatic bile acid homeostasis are expressed at very low levels in normal and pathological pregnancy and are thus likely to be of limited importance in placental bile acid transfer. a placental phenotype for obstetric cholestasis. victoria geenes, jenny chambers, jo wyatt-ashmead, kishore bhakoo, catherine williamson. imperial college, london, united kingdom; hammersmith hospital, london, united kingdom. background: obstetric cholestasis (oc) is a pregnancy specific liver disorder that affects . % of pregnant women in the uk. biochemically, oc is characterised by liver dysfunction with elevated maternal serum bile acids (sba), and clinically by an increased risk of fetal complications including fetal distress, meconium staining of the amniotic fluid, preterm labour and sudden intrauterine death. the aetiology of oc is complex and not fully understood, but the fetal complications are likely to result from the toxic effects of bile acids, which can cause vasoconstriction in the placenta and fetal cardiac dysrrhythmias. furthermore, in a rodent model of oc, bile acids cause oxidative stress in the placenta and a reduction in litter size. these changes are absent in animals treated with ursodeoxycholic acid (udca), a drug used to treat oc. however human data are lacking. objectives: this study used whole placenta and explant cultures to investigate the effects of bile acids on human placental architecture and to establish whether udca protects the placenta against bile acid induced damage. methods: villous trophoblast samples were collected from oc and normal pregnancies (np) and fixed in formalin. explant cultures were prepared from a further np placentas, and cultured for days at % oxygen. on day a subset were treated with udca overnight, and on day the explants were treated with taurocholic acid, taurochenodeoxycholic acid or vehicle for hours prior to fixing. m sections were stained with haematoxyllin and eosin. slides were reviewed by a perinatal pathologist and syncytial knots (sk) counted. results: oc tissues showed marked alterations in morphology, including congestion of the terminal villi, and loosening of the stroma and fibrotic change of the membranes of the stem villi. there were significantly more sk the oc samples (mann-whitney u test p= . ). furthermore, sk were increased in explants treated with bile acids, but not those treated with udca prior to the addition of bile acids. conclusions: here we describe several morphological abnormalities of the placenta associated with maternal cholestasis. these changes were confirmed using a placental explant model, which also showed that udca protects the placenta against bile acid induced damage. in summary, this study indicates that udca is likely to protect the fetus in oc. the placenta has complex metabolic and endocrine activities and is essential for the growth and survival of the fetus in utero. ultrasound is the most sensitive and less invasive method to evaluate placental size, morphology and function. the three-dimensional approach allows to calculate placental volume in the i and ii trimester of pregnancy. pregnancies from assisted reproduction technologies (art) show an increased rate of pathologies potentially related to placental insufficiency such as intrauterine growth restriction and preterm delivery. aim. to determine placental volume in art pregnancies in a cross sectional study in the first trimester of gestation. design. using three-dimensional ultrasound machine (ge ) placental volume measurement from art pregnancies (n= ; - wks) were compared with data from normal controls (n= ) matched for gestational age. data were analysed with software stata . results. mean placental volume was . ml (sd± . ) in art pregnancies and . ml (sd± . ) in normal controls. mean difference resulted in . ml (- - . ) and was statistically different (p= . ). multiple linear regression analysis showed a statistically significant interaction (p= . ) between gestational age and case status, i.e. differences in placental volume increase significantly with advancing gestational age between cases and normal controls. mean gestational age at birth was not essentially different between the two subsets ( . weeks ± . ) however a statistically significantly lower mean fetal birthweight was found in art pregnancies: g (sd± ) vs .(sd± . ) (p= . ). conclusions. placental volume is slightly decreased in art pregnancies. measurements of placenta volume by d ultrasound may play a role in identifying the degree of placental growth early in gestation in a risk population. in vitro effects of adenovirus-mediated gene transfer of insulin like growth factor- (ad-igf- ) in trophoblast cells. mounira habli, datis alae, foong yen lim, jignesh parvadia, timothy crombleholme. obstetrics and gynecology; pediatric surgery, university of cincinnati/children's hospital, usa. objective:recently, ad-igf- corrected both fetal and placental weights in rat model of fetal growth restriction (fgr) . to elucidate the mechanism of action of ad-igf- ;we examined the effect of ad-igf- on trophoblast proliferation, differentiation and invasion. methods: rcho- trophoblast cell line derived from rat choricarcinoma was used. ad-igf- and galactosidase transgene (ad-lacz) were given at moi of : and : in all the experiments.transduction efficiency was assessed hr after infection with ad-lacz by galactosidase enzyme activity. the invasiveness of rcho- was measured by using bdmatrigel invasion chamber kit. rcho- proliferation cell density was assessed by crystal violet assay. morphologic analysis of rcho- differentiation in response to treatment (differentiated cells are multinucleated giant cells) was assessed byimunocytochemistry staining using placental lactogen- antibodies(specific hormone produced by giant cells). results: % efficiency of gene transfer of ad-lacz to rcho- cells was observed at both moi's.there was no significant difference in proliferation between treated control group regardless of the dose at and hrs. ad-igf- induced morphologic differentiation of trophoblast cells compared to control (fig ) at hrs.ad-igf- induced higher rate invasion in a dose dependent fashion as compared to control(ad-lacz) group( % at moi vs . % in control p< . )(fig ). conclusion: ad-igf- gene transfer induces differentiation and invasiveness of trophoblast cells. these may be the mechansim of correction of fgr in rat model of placental insufficiency. placental gene transfer may be an effective treatment strategy for placental insufficiency. types of human placenta. martina dieber-rotheneder, ursula hiden, gernot desoye, mila cervar-zivkovic. department of obstetrics and gynaecology, medical university graz, graz, austria. background and hypothesis: endothelin- is a polypeptide with a wide range of functions. in the placenta it acts as a potent regulator of vasotonus on endothelial and smooth muscle cells, whereas on the trophoblast it regulates cell proliferation, invasion and apoptosis. endothelin- action is differently signaled through two endothelin receptor (etr) subtypes, etr-a and etr-b. several alternative splice variants of etr were identified. here we hypothesize that the etr-a splicing varies with gestational age and is different in trophoblast vs endothelial cells of the human term placenta. methods: mrna of placental tissue from first trimester (pregnancy terminations, missed abortions) and term of gestation, first trimester and term trophoblasts, arterial and venous placental endothelial cells as well as cell lines representing first trimester trophoblast (ach p) and term placental endothelial cells (hpec) were analyzed for full length etr-a and known as well as unknown splice variants by sqrt-pcr using primers spanning the exons - . the predominant dna bands in agarose gel were excised and sequenced. results: all tissues and cells expressed full length etr-a. in all tissues an additional -deletion variant was identified which was not found in the isolated cells. trophoblasts expressed another yet unidentified splice variant. the endothelial cells expressed a -deletion variant and a novel splice variant, which was identified as partial -partial deletion. this novel splice variant was found regardless of the arterial or venous origin of the cells as well as in the cell line (sv -transformed). in tissues und trophoblast no difference in splicing was found between first trimester and term. conclusion: gestational age does not alter splicing of endothelin receptor-a. splicing is different between trophoblasts and endothelial cells. the endothelial cells contain a novel splice variant. the differential splicing may allow maternal and fetal endothelin- to induce different effects on the two major cell types of the placenta. (supported by grant , jubilee fund, austrian national bank, vienna). adrenomedullin production and secretion by human trophoblast cells are regulated by glucocorticoids and hypoxia. francesca ciardo, katia pacioni, emanuela marinoni, giovanna corona, massimo moscarini, alfredo patella, romolo di iorio. department of gynecology, perinatology and child health, university "la sapienza", rome, italy; department of obstetetrics and gynecology, university of ferrara, ferrara, italy. objectives: adrenomedullin (am) is produced by intrauterine tissues and is involved in the regulation of implantation, placental hemodynamics and endocrine function. circulating am is increased in pregnancy complications such as preeclampsia and intrauterine growth retardation. we investigated whether am output by human trophoblast cells is regulated by hypoxia and/or glucocorticoids. study design: trophoblast cells obtained by human placentas at term (n= ) were cultured in presence or absence of hypoxia ( % o ) and treated with or without betamethasone at the dose of - , - , - m. media and cells were collected at h and at , and h from syncytiotrophoblast cultures. am was measured in cultured media by specific ria kit. protein expression in trophoblast cells was evaluated with immunohistochemistry and western blot. results: hypoxia stimulated am output and protein expression by cytotrophoblast and syncytiotrophoblast cells. betamethasone induced an increase in am production and secretion in a time-and dose-dependent manner (figure). effects of hypoxia were partially reversed by betamethasone in a dose and time-dependent fashion. conclusions: am production in trophoblast cells is up-regulated by hypoxia and glucocorticoids, independently. increased am levels in pregnancy complications characterized by placental insufficiency might derived by an increase in am placental secretion stimulated directly by hypoxia or indirectly by an increase in fetal cortisol levels. preeclampsia complicates - % of pregnancies, and while associated with significant maternal and fetal morbidity and mortality, the mechanisms responsible for preeclampsia are not completely understood. recent advances suggest there are imbalances of pro-and antiangiogenic factors, present from the time of implantation affecting vascular responsiveness of the fetal placental unit and maternal vasculature. in this study, we investigate vasomotor responsiveness of placental arteries from normal and preeclamptic (pet) pregnancies using an in vitro muscle bath contractility assay. transverse rings of placental arteries were cut and equilibrated in the muscle bath containing a bicarbonate buffer aerated with % o / % co at °c. viability was demonstrated by contraction to mm kcl prior to all experiments. cumulative logarithmic dose dependent responses to four different contractile agents: pgf , serotonin, carbochol, and norepinephrine were compared. placental arteries precontracted with pgf at half maximal concentration were relaxed with three vasorelaxants: sodium nitroprusside (snp), forskolin (fsk) and lactate (lct). agonist studies revealed contraction to both pgf and serotonin but not to carbochol or norepinephrine. there were no statistically significant differences between the responses of normal and pet arteries. both normal and pet arteries demonstrated heightened responsiveness to sodium nitroprusside compared with forskolin and lactate. this study reveals that the placental vessels are more sensitive to sodium nitroprusside, that mediates relaxation through nitric oxide -cyclic gmp signal transduction pathway, than forskolin that induces relaxation through the cyclic amp pathway. cancer complicates approximately one in , pregnancies. depending on the type of cancer and trimester, patients can terminate the pregnancy or choose treatment such as chemotherapy. since there is limited knowledge on the safety of chemotherapy on fetal tissues in utero, clinicians cannot efficiently analyze the risks of particular anticancer agents when deciding on a course of therapy. since carboplatin is among the most common anticancer agents used for treatment of cancer during pregnancy the primary objective of this study was to evaluate if carboplatin will readily cross to placental barrier and determine total potential platinum fetal exposure. this project utilizes an ex vivo placenta perfusion model to determine the concentration of carboplatin that crosses the human placental barrier. placentas are obtained within minutes of spontaneous delivery and re-perfused. two carboplatin concentrations were selected of ng/ml and ng/ml to represent clinically relevant maternal plasma concentrations. antipyrine was used as the internal control. serial samples were collected every minutes for total minutes in open-open model then experiment repeated with closed circuit model. antipyrine concentrations were determined by hplc methods. platinum concentrations in media samples were determined with a validated atomic absorption assay. a cell culture-based approach will be used to determine whether cell cycle or apoptotic proteins are altered (p , bax, bcl- , aif, and pro-caspase- ) using western blotting as a detection method. a total of two placentas have been completed at the low carboplatin concentration and one at the high concentration. the mean carboplatin clearance was . +/- . ml/min and . +/- . ml/min at the low and high carboplatin concentrations, respectively. an estimated % increase in the transport fraction was observed in the high concentration experiments compared to the low concentrations model. fetal carboplatin exposure ranged from to ng/ml. the placental perfusion experiments conducted at carboplatin and ng/ml indicate that carboplatin crosses the placenta through simple diffusion. the toxicology assays are ongoing to determine potential effect on fetal tissues. preterm delivery represents one of the predominant causes of perinatal mortality and morbidity, and is one of the most unpredictable gestational disturbances. urocortin is a peptide expressed by trophoblast and gestational tissues (amnion and chorion), whose maternal levels correlate with gestational length, since they are increased at preterm delivery and decreased in post-term pregnancy, when compared to term pregnancy. the addiction of urocortin enhances contractility of human myometrial strips, suggesting a possible role on uterine contractility. high maternal urocortin levels in threatened preterm delivery correlates with the timing of delivery, suggesting a possible role in the predictivity pf preterm delivery. in the present study urocortin concentrations in amniotic fluid collected at mid gestation for amniocentesis were correlated with gestational age at delivery. a case-control study with amniotic fluid obtained from healthy women undergoing amniocentesis for genetic indications was performed; urocortin concentrations were measured by a specific elisa. amniotic fluid urocortin concentrations resulted significantly lower (p= . ) in women delivering preterm (n= ; ucn= . ± . pg/ml) (m ± se) than in those delivering at term (n= ; ucn= . ± . pg/ml). in conclusion, the present preliminary data showed that amniotic fluid urocortin concentrations at mid gestation may represent predictive marker of preterm delivery. human placentas throughout pregnancy. annunziata mastrogiacomo, elisabetta federico, francesca caprio, maria teresa schettino, gabriele coppola, antonio de luca, luigi cobellis. department of obstetrics and gynecology, second university of naples, naples, italy; department of medicine and public health, section of anatomy, second university of naples, nales, italy. hypothesis apoptosis is intimately involved in placental homeostasis, growth and remodeling and the apoptotic rates increase progressively during normal pregnancy as part of normal placental development. moreover, apoptosis increases in pregnancies complicated by some pathologies such as preeclampsia, fetal growth restriction, diabetes. in the present study, we describe differences in the expression of pro-apoptotic protein bax, in first trimester voluntary termination of pregnancy, first trimester abortion (reserved abortion), caesarean birth, spontaneous birth, preeclampsia and diabetes. material and methods human placental samples were obtained with informed consent from patients undergoing surgery such as first trimester voluntary termination of pregnancy (n= ), first trimester abortion (miscarriage) (n= ), delivery section (n= ), spontaneous birth (n= ), preeclampsia (n= ) and diabetes (n= ). the gestation period ranged from to weeks. the specimens were immediately fixed in formalin for immunohistochemistry. we first observed a strong increase of bax expression in the cytotrophoblast, stroma, endothelial cells and decidua of placentas of the first trimester abortion compared to the low/moderate bax immunopositivity in all the placental compartments during the first trimester voluntary termination of pregnancy. secondly, we showed a more intense immunopositivity for bax in the third trimester spontaneous birth respect to the third trimester caesarean birth. thirdly, we observed an increase of bax expression in preeclamptic placentas compared to the normal full-term placentas. on the contrary, we observed a moderate bax expression in diabetic placentas only slightly decreased compared to the normal full-term placentas. our results seem to suggest that deregulation of apoptotic turnover may lead to placental dysfunction and pathologies. objectives: maternal endothelial activation in preeclampsia (pe) is attributed to the release of unknown factors from a hypoperfused placenta. the application of proteomic technologies such as mass spectrometry promises the reward of identifying and characterising these factors. using a placental explantconditioned culture media model system we have developed a proteomics workflow to obtain relative quantification of proteins released into placental explant culture media. methods: term villous explants were cultured in serum-free conditions and exposed to differing oxygen concentrations ( % %) to mimic physiological and non-physiological intervillous o tensions. the d media was concentrated, immunodepleted to remove fibrinogen (using beckman igy- columns) and proteins labelled using an itraq (applied biosystems) kit following the manufacturer's protocol. labelled peptides were fractionated by strong cation exchange and then analysed using lc-maldi on a maldi-tof/tof (applied biosystems) mass spectrometer. data was analysed using protein pilot . (applied biosystems). results: when matched pooled (n= ) media samples were subjected to our proteomics workflow over proteins were identified with a calculated false positive identification rate of < %. of these proteins a total of display a statistically significant difference in protein levels between the % % o samples (p=< . ). from a list of proteins of interest we selected proteins for validation using elisa/western blotting to measure their relative abundance in both pooled and individual explant media samples. interleukin is an example of a low-abundance differentially expressed protein identified in our proteomic workflow and subsequently validated by elisa; il- (pg/ml) in % o : . , range . to . ; % o : . , range . to . ; values are median with interquartile range. *** p< . , mann-whitney test (n= ) conclusions: we demonstrate a successful reduction to practice of a relative quantitative proteomics strategy applied to placental explant-conditioned culture media. having optimised and validated this proteomic workflow we will apply the same methods to compare proteins released by normal and preeclamptic placentas. a proteomics screen of human placental microvillous syncytiotrophoblast (stb) revealed the expression of dysferlin (dysf), a membrane repair protein associated with certain muscular dystrophies (vandré et al., ) . a second ferlin protein, myoferlin (myof), was also discovered which has not yet been characterized in the placenta. in human c c myoblasts, dysf and myof are reciprocally expressed as a function of differentiation into multinucleate syncytial myotubes, with dysf predominating in differentiated cells. we hypothesized that dysf and myof would show a similar reciprocal expression pattern in human trophoblasts as a function of syncytialization. methods term placentas from uncomplicated pregnancies were obtained with informed consent (n= ) and either fixed for immunofluorescence (if) labeling or flash frozen for subsequent immunoblotting (ib). human trophoblastic (bewo, jar, and jeg- ) and other cell lines (mrc- , hl- , huvec) were cultured in the absence or presence of μm forskolin or solvent control for - days. dysf and myof expression was examined by rt-pcr, ib, and if. results ib validated proteomics data showing myof expression in term placenta. by if, myof labeling was predominant in apical and basal stb plasma membranes. myof was expressed in trophoblastic cell lines (bewo, jar, and jeg- ), cultured endothelium (huvec), and a fibroblast cell line (mrc- ), but was not detected in a leukemic cell line (hl- ). dysf was constitutively expressed in jar, but minimally expressed in unstimulated bewo. following forskolin-induced syncytialization, we observed a time-dependent increase in dysf expression in bewo concomitant with increasing syncytin- levels. by if, dysf expression was restricted to bewo cells in syncytial structures. in contrast, myof expression was robust in mononuclear bewo cells and not down-regulated over the course of days of differentiation. conclusions two ferlin-family genes are expressed in trophoblasts. fusion-competent bewo cells behave similarly to cultured myoblasts and ctbs with regard to dysf expression, which is restricted to syncytializing cells. myof, in contrast, is expressed constitutively in bewo and other models. while both proteins are likely to function in stb plasma membrane repair (e.g., following syncytial sprouting), the relative contributions of each to this process awaits clarification. features of chronic placental insufficiency are significantly more common in small for gestational age placentas from infants with intrauterine growth retardation. emily king, violetta kolesnikova, carri tillotson, jean-marie guise, terry morgan. pathology; center for biostatistics; obstetrics and gynecology, ohsu, portland, or, usa. background: there is a strong association between intrauterine growth restriction (iugr) and small for gestational age (sga) placentas (heinonen et al. placenta ( ), : - ) . the cause is likely multifactorial, but we hypothesize that chronic uteroplacental insufficiency may play a role. our objective was to test whether sga placentas from human neonates with iugr show significantly more pathologic features of placental insufficiency compared to controls. design: we performed a retrospective review of consecutive singleton placentas submitted to ohsu pathology ( - ), excluding elective terminations and spontaneous abortions before weeks gestation. clinical records were reviewed and pregnancy outcomes recorded, including: ) neonatal sex; ) weight (iugr calculated by routine methods), ) trimmed weight of the placenta (sga calculated by routine methods), ) gross placental infarctions, ) gestational age at delivery, and ) maternal features (e.g. race, gravida). controls were defined as cases within the series without sga or iugr (e.g. submitted for meconium, infection, etc). histologic sections were scored by two pathologists while blinded to clinical diagnoses as positive or negative for features of placental insufficiency, including accelerated villous maturation (avm), chorangiosis, and microscopic infarctions. significant associations were tested by analysis and logistic regression for multiple variables. results: similar to prior reports, we observed a strong association between iugr and sga placentas ( . ; p < . ). this relationship was independent of maternal race, fetal sex, and parity, although it was more common in primigravidas. avm and placental infarctions were significantly more frequent in sga placentas with superimposed iugr. controls ( introduction: messenger rna expression peripheral blood cells (pbc) has been recently used as biomarkers of environmental exposures (ionizing radiation or tobacco), physiological conditions (stress) and diseases (hypertension, neurological disorders). pbc evaluation is a useful diagnostic tool in an era of individualized medicine. since there is an urgent need for non-invasive methods for determination of fetal (f) and placental (p) function, this study was designed to evaluate the genes differently and commonly expressed in p tissue and leukocytes in maternal (m) and f circulation.material and methods. p (n= ), f (n= ) and m blood (n= ) were obtained during cesarean section in pregnant baboons at term. total rna from a buffy coat pellet was isolated using a modified procedure of the qiagen rneasy mini kit (qiagen). anti-sense rna (arna) was synthesized and purified using the illumina rna amplification kit (ambion, usa). hybridization of arna to illumina sentrix human whole genome (wg- )beadchips and subsequent washing, blocking and detection was performed using illumina's beadchip ´ protocol. samples were scanned on the illumina beadarrayer gx reader using illumina beadscan image data acquisition software (ver. . . . ). differential gene expression data analysis was performed using illumina beadstudio software (ver. . . . ). results. the detection level of gene transcripts using illumina methodology with control human rna was - at p< . . gene transcripts were detected in f blood and in maternal blood. transcripts were uniquely expressed in fetal blood. transcripts were found in m, but not in f leukocytes. the number of gene transcripts expressed in p tissue was and of these genes were not expressed in m leukocytes. conclusion. despite white blood cells trafficking through the p barrier there is a set of unique genes expressed only in p or in the m or f circulation. the application of these genes as the biomarkers of p barrier function still need to be evaluated. objective: to evaluate if a relationship exists between duration of placental exposure to meconium in vivo and histologic evidence of severity and extent of meconium uptake by macrophages. study design: from a cohort of / ( %) consecutive singleton liveborn infants delivered at term with thick meconium-stained fluid, ( %) had placental histologic examination performed, and in / the timing of meconium appearance after membrane rupture was documented. placental histologic examination quantitatively evaluated the intensity of meconium uptake by resident macrophages based on the number of macrophages/field, and the extent of uptake based on histologic location, graded in a score to . results: mean interval between meconium appearance and delivery was . ± . min (range - ). after exclusion of cases in which severe placental inflammation interfered with analysis, meconium uptake by macrophages was documented in / cases at the level of amniochorionic membranes, in / cases at the placenta, and in / cases at the umbilical cord. there was no correlation between the interval meconium appearance-to-delivery in relation to presence of meconium in the membranes (p= . ), in the placenta (p= . ), in the cord (p= . ), or score of severity of meconium uptake (p= . ). the results did not change after correcting for gestational age, oligohydramnios, presence of placental acute inflammatory or vascular lesions. conclusion: there is no relationship between duration of placental exposure to meconium and the extent and intensity of its uptake by macrophages in cases with exposure up to . hours. transfer of bisphenol a across the human placenta. biju balakrishnan, , kimiora henare, , eric thorstensen, , murray d mitchell. , the liggins institute, university of auckland; national research centre for growth and development, auckland, new zealand. introduction: there are growing concerns over the effects of developmental exposure to the xenoestrogen bisphenol a (bpa). animal studies have shown that bpa is transferred through the placenta and can cause deleterious effects to the fetus. the presence of bpa in feto-placental tissues in humans has also been reported. however, a detailed study of the time-course of bpa transfer across the human placenta has not been performed. the aim of this research was to study the transfer of bpa in ex-vivo perfused human placental tissues. methods: a dual recirculating single cotyledon perfusion was used to monitor the placental transfer of bpa. bpa ( ng/ml), antipyrine (ap) ( μg/ml), and fitc dextran (fitc-dx) ( . μg/ml) were added to the maternal perfusate, and perfusion was continued for hours. perfusate samples were collected from both reservoirs at timed intervals and analysed. bpa, ap, and fitc-dx were determined by hplc with fluorescent detection, hplc with uv detection, and spectrofluorometry respectively. the viability and metabolic activity of the placentae were assessed by measuring -hcg (elisa), glucose utilization, and lactate production (autoanalyzer). fetal pressure, ph, flow rates and fluid shifts were monitored continuously. results: the biochemical validation parameters (glucose consumption, lactate production and -hcg secretion) indicated that the placental tissue was metabolically active and viable throughout the -hour perfusion period. the physical parameters observed (fetal pressure < mmhg, ph range . - . ) were in concordance with other published works for placental perfusion. membrane integrity was confirmed by fluid shifts from either circuit of < ml/hr, and by < % materno-fetal transfer of fitc-dx. an observed ap transfer of - % further validated our model. bpa first appeared in the fetal compartment within minutes of perfusion and reached a peak of about - % of maternal concentration within hours of perfusion. this figure is likely to be an underestimate since it does not include conjugated bpas. conclusion: this first study of bpa transfer in ex-vivo perfused human placental tissue shows that our model can serve as a useful tool to study the transfer kinetics and metabolism of bpa in human term placentae. we found that bpa rapidly crosses the human placenta at environmentally-relevant doses, with potentially harmful effects on the human fetus. angiogenic growth factor secretion by uterine natural killer cells in co-culture with extravillous trophoblast. gendie e lash, katsu naruse, , barbara a innes, stephen c robson, judith n bulmer. institue of cellular medicine, newcastle university, newcastle upon tyne, tyne and wear, united kingdom; obstetrics and gynaecology, nara medical university, nara, japan. objectives. uterine natural killer (unk) and extravillous trophoblast (evt) cells have been proposed to play roles in remodeling of uterine spiral arteries through secretion of various angiogenic growth factors. we have previously demonstrated that unk cells are a significant source of angiogenic growth factors within the decidua, many of which alter with gestational age. however whether the secretion of these proteins is altered by interactions of unk cells with evt is unclear. hypothesis. unk cell angiogenic growth factor secretion is regulated by evt in early pregnant decidua. methods. placental and decidual samples were collected from women undergoing termination of pregnancy with written informed consent ( - and - weeks gestation, n= each group). . × cd + unk cells were positively selected from decidua and co-cultured with evt ( . × ) or cytotrophoblast (ctb; . × ) purified from the same placenta for h in direct or indirect contact (n= each group). angiogenin, ang , pdgf-bb, fgf-basic, timp , icam and vegf-a were measured by fast quant ® angiogenesis multiplex assay system, and vegf-c, ang and plgf by elisa. results of unk co-culture with evt or ctb (negative control) at each gestational age were analyzed with wilcoxon rank test. in addition, the effect of direct and indirect co-culture of unk cells with evt at each gestational age was compared with mann whitney u test. results. at - weeks gestation unk secretion of ang (p= . ), icam (p= . ) and plgf (p= . ) was increased in the presence of evt compared with ctb. no differences were observed at - weeks gestation. in addition, at - weeks gestation unk secretion of ang (p= . ), vegf-c (p= . ) and ang (p= . ) was increased in direct co-culture with evt compared with indirect co-culture. at - weeks gestation unk secretion of timp- (p= . ) was reduced in direct co-culture with evt compared with indirect co-culture. conclusions. unk cell secretion of several key angiogenic growth factors was altered by direct culture with evt. these data suggest that a membrane bound molecule (such as hla-g) mediates this modulation of unk cell activity. abnormalities in placentation and impaired placental circulation can lead to fetal growth restriction. -d ultrasound can be used to evaluate this through the quantification of the power doppler signal that may be expressed as a percentage of colour voxels within a user-defined volume (vi: vascularisation index). we aimed to test the hypothesis that increased fetoplacental blood flow correlates with an increased vi, using the in vitro dual perfusion model of the human placental lobule. three term lobules were dually perfused through both circulations with earles bicarbonate buffer (ebb), and supplemented on the fetal-side only with adult erythrocytes, prepared to a % haematocrit. following initial equilibration perfusion at normal flow values, fetal-side flow was varied between and ml/min, whilst maternal-side flow was held at ml/min. images were obtained with a 'voluson i' ultrasound machine and a neonatal transducer (pulse repetition frequency = . hz, wall motion filter = low , and gain = . ). three -d datasets were acquired at each flow rate from each placental lobule and these were measured in triplicate using vocal. a sphere was centred on a visibly perfused cotyledon along the chorionic-decidual axis, with a diameter corresponding to placental thickness. linear regression analysis was used to assess the relationship between the total fetal-side flow and mean vi. the mean vi showed a high degree of correlation with total fetal-side flow for each lobule ( figure ) suggesting increased vascular perfusion and the inclusion of perfused vessels that cross the detection threshold with increased flow. this data provides qualifying information for translation to a clinical application, where early gestational fetoplacental blood flow will be assessed to predict the onset of fetal growth restriction. anthony n imudia, brian a kilburn, anelia petkova, samuel s edwin, roberto romero, d randall armant. objective survival of first trimester cytotrophoblast cells depends on their upregulation of heparin-binding egf-like growth factor (hbegf), which is downregulated in placental tissues diagnosed with preeclampsia. we have examined the expression and cytoprotective activity of hbegf in term villous explants subjected to hypoxic stress in vitro. non-pathological placentas were collected by cesarean section at term (n= ). chorionic villous explants were prepared and cultured at either % or % o and treated with the hbegf antagonist crm or recombinant hbegf. paraffin sections were assayed for trophoblast cell death by the tunel assay, proliferation by immunohistochemical labeling of nuclear ki and hbegf expression by semi-quantitative immunohistochemistry. data were compared using anova and the student-newman-keuls posthoc test. results crm ( mg/ml) increased trophoblast cell death after culturing villous explants h at % o (p< . ), but only slightly affected proliferative capacity. culture at % o increased trophoblast cell death % above explants incubated at % o (p< . ). trophoblast cell proliferation decreased after h in explants cultured at either % or % o (p< . ). exogenous hbegf ( nm) prevented the elevation of cell death during hypoxia (p< . ) and maintained nuclear ki expression at %, but not %, o . contrary to first trimester trophoblast, hbegf was not upregulated by hypoxia in term trophoblast. the failure of term trophoblast to elevate hbegf expression in response to hypoxia could contribute to their decreased survival at low o compared to early gestation. endogenous hbegf signaling appears to facilitate survival of term trophoblast during villous explants culture. exogenous hbegf supplementation prevented cell death due to hypoxia and maintained trophoblast proliferation rates under in vitro conditions. therefore, hbegf, which is downregulated in preeclampsia, could have significant impact on trophoblast survival during late gestation. supported by the intramural research program of nichd. conspicuous meconium-laden macrophages in the chorionic plate are an independent predictor of clinically significant fetal distress. violetta kolesnikova, emily king, carrie tillotson, jean-marie guise, terry morgan. pathology; center for biostatistics; obstetrics and gynecology, ohsu, portland, or, usa. background: meconium is a common indication for placental examination, present in approximately % of placentas routinely submitted to pathologists (beebe et al. obstet gynecol ; : - ) . prolonged meconium exposure leads to accumulation of meconium-laden macrophages in the chorionic plate (estimated to require at least hours). our objective was to test whether prolonged meconium exposure is associated with clinically significant fetal distress. design: retrospective review of consecutive singleton placentas submitted to ohsu pathology ( - ) was performed, excluding abortions before weeks gestation, and placentas without representative sections of the chorionic plate. clinical records were reviewed and pregnancy outcomes recorded, including: ) evidence of fetal distress prompting c-section (non-reassuring fetal heart rate), ) gestational age, ) maternal diagnosis, ) apgar scores, and ) neonatal length of hospital stay. routine histologic sections were independently scored by two pathologists while blinded to clinical diagnoses and outcomes. cases were scored as positive or negative for: ) diffusely conspicuous meconium-laden macrophages in the chorionic plate (at least / hpf), ) chorioamnionitis, and ) features of placental insufficiency. significant associations were tested by the mann-whitney u test for paired comparisons, x analysis, and logistic regression for multiple variables. results: conspicuous meconium staining of the chorionic plate was common ( / , %) and was significantly more frequent in c-sections performed for fetal distress ( / cases, %) (x . ; p-value < . ). logistic regression modeling showed that this association was independent of chorioamnionitis and features of placental insufficiency. there was no association between meconium and neonatal outcome, including no difference in apgar scores or length of hospital stay. conclusions: our data support the hypothesis that meconium-laden macrophages in the chorionic plate are associated with fetal distress prompting c-section. whether meconium is the cause or consequence of this distress is uncertain. however, given the acute time frame between clinical diagnosis and c-section, we suspect prolonged meconium exposure may be a significant cause of non-reassuring fetal heart rate changes. apoptotic index in normal and intrauterine growth-restricted rat placentas. elissa scotland, tri nguyen, s chiang, radmila runic. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: intra-uterine stress caused by maternal food-restriction may have adverse fetal and placental effects. we sought to assess the effect of maternal food restriction during pregnancy on placental apoptosis. methods: rat placentas were analyzed from control dams fed ad libitum and dams % food-restricted from day of gestation. placentas were harvested at embryonic days and (n= ). placentas were fixed in % pfa and two methods of analysis were utilized to determine the proportion of apoptotic to non-apoptotic cells within maternal food restricted and control rat placentas: immunohistochemistry (ih) using fas-ligand and in situ tunel (terminal deoxynucleotidyl transferase biotin-dutp nick end labeling). tunel: after rehydration, slides were subjected to tdt enzyme. dab was used to visualize brown apoptotic nuclei. dna-se treated slide was used as a positive control. ih: primary rabbit polyclonal fas-ligand antibody was used at : dilution, after which secondary antibody linked to peroxidase and dab staining was performed. results: food-restricted placentas demonstrated . % relative apoptotic index when compared to . % in the control group. the iugr iod (integrated optical density) per unit area in the placental membrane was higher than in the control group. fasl demonstrates an iod of . + . in food restricted placentas as compared to . + . per μm surface area in controls. apoptosis was seen in the amnion as well as trophoblast (syncytialtrophoblasts and some cytotrophoblasts). conclusion: the increased apoptotic index in maternal food restricted placentas suggests that the accompanying iugr may be a result of both maternal/fetal nutrient restriction and increased fetal stress. this study found that maternal food restriction during pregnancy affects placental apoptosis. there is more apoptosis in the iugr placental bed at e than at e , with the reverse being true for the placental membrane. more research is required to statistically validate the data. the suggested next step is to evaluate fas and fas-l and to address possible mechanisms of placental apoptosis. cord. jayaraman lakshmanan, avish arora, lilit baldjyan, sharon k sugano, olga miadel, adegoke adeniji, michael g ross, calvin j hobel. ob-gyn, harbor-ucla medical center, torrance, ca, usa; ob-gyn, cedars-sinai medical center, los angeles, ca, usa. background: crf-bp is a kda protein, that specifically binds corticotrophin releasing factor (crf). the structure of cloned crf cdnas in all species examined predicts that the precursor is larger than the kda in size and contains one n-glycosylation site. marked reductions in plasma crf-bp levels seen in pregnant women prior to both preterm and term delivery led to the notion that crf-bp is a "gatekeeper" of crf responses. recently we identified crf-bp expression in term human umbilical cord (uc) by immunohistochemical analyses. objective: to characterized the expression of crf-bp by immunohistochemical and biochemical analyses in human uc. methods: freshly obtained human preterm ( to < weeks, n= ) umbilical cord ( pieces of mm thickness taken at - cm intervals close to placenta) were fixed in bouin's solution and paraffin embedded. also, pieces of ucs were dissected, arteries and vein separated, weighed and frozen at - c. for immunohistochemical localization, uc sections were subjected to immunostaining with polyclonal antibodies to human crf-bp precursors. for western blot analyses, whole uc as well as isolated arteries and vein were homogenized in a buffer containing detergents and protease inhibitors. the homogenate supernatant proteins were subjected to western analyses by standard protocol. immunoreactive protein bands were identified by chemiluminescent reagent. results: both goat and rabbit crf-bp polyclonal antibodies elicited weak to moderate positive staining in uc epithelial layers, vascular musculature and barely endothelial cells. they identified a strong kda band in whole uc as well as in isolated artery and venous preparations. in addition minor immunoreactive protein bands of , , kda in size were noticed in all three preparations. conclusion: we conclude that preterm uc expresses crf-bp. we postulate that the kda major band is either glycosylated crf-bp precursor or glycosylated kda mature protein. the low molecular weight immunoreactive proteins likely represent proteolytically processed, glycosylated crf-bp precursor or a proetolytically processed mature da crf-bp. uc obtained at delivery could be useful as a tool to understand the critical functions of crf-bp in feto-placental unit. objective: adenosine, known to be released from inflammatory sites and tisse ischemia, has many important biologic roles. four specific adenosine receptors have been cloned to date, termed a , a a, a b, and a . recently our study has shown that increased a receptor in the trophoblast of preeclamptic pregnancy was noted and non-vascular and trophoblast-mediated a receptor may play an important role in the pathogenesis of preeclampsia. there are evidences of impaired trophoblast invasion related to matrix metalloproteinase (mmp) in preeclampsia and the relationship between adenosine receptor and mmp in other fields. the objective of this study is to evaluate the effect of mmp expression by adenosine a receptor in preeclamptic villous explants at different oxygen conditions. methods: placental villous explants from normal (n= ) and preeclamptic (n= ) pregnancies were cultured at high ( %) and low ( %) oxygen levels for days. explants were analyzed for mmp- /- and timp- /- by rt-pcr and western blot. preeclamptic villous explants in hypoxic culture condition were treated with a receptor agonist, cl-ib-meca and a receptor antagonist, mre. mmp- / - expression was determined in a time-and dose-dependent manner by rt-pcr, western blot. also mmp- /- activity was evaluated by zymogram assay. results: there were significantly increased a receptor intensity and reduced mmp- /- and timp- /- expression at low oxygen level in normal and preeclamptic villous explants. interestingly, in preeclamptic villous explants, after high oxygen culture mmp- /- and timp- /- expression were recovered to almost same level compared to those in normal villous explants. treatment of preeclamptic villous explants with cl-ib-meca in low oxygen level resulted in a time-and dose-dependent enhanced expression of mmp- /- . this cl-ib-meca-induced expression of mmp- /- was inhibited by pretreatment with mre. conclusion: to our knowledge, this study is the first to evaluate modulation of mmp secretion by adenosine a receptor in preeclamptic villous explant. our results provide evidence for the existence of functional adenosine a receptors in the trophoblast and suggest that adenosine a receptor will be investigated as a therapeutic target in preeclampsia. murray d mitchell, timothy a sato, anderson wang, jeffrey a keelan, anna p ponnampalam, michelle glass. liggins institute; department of pharmacology and clinical pharmacology, university of auckland, auckland, new zealand. introduction: it is well established that prostaglandins play critical roles in multiple aspects of pregnancy and that the fetal membranes are an important site of intrauterine prostaglandin production. endocannabinoids have been implicated in the maintenance of pregnancy and parturition in women and are a source of arachidonic acid which is a substrate for the production of prostaglandins. the aim of the present study was to determine the effects of endocannabinoids on the production of prostaglandins in extraplacental membranes -amnion, chorion and decidua. methods: explants of term amnion and choriodecidua were established and treated with endogenous endocannabinoids -arachidonoyl glycerol ( ag) and anandamide (aea) and with the synthetic cannabinoid cp , , to determine the ability of these substances to modulate prostaglandin e (pge ) production. pge was measured by radioimmunoassay. the explants were also treated with cp , in the presence of either sr a (a selective antagonist of the cannabinoid receptor cb ) or ns (a cox- inhibitor), to determine whether any observed stimulation of pge production was mediated through cox- activity and/or the cb receptor. cox- , cox- , cpla and pgdh protein levels were measured by western blotting. results: all three cannabinoids caused a significant increase in pge production in amnion but not in choriodecidua. however, separated fetal (chorion) explants responded to cannabinoid treatment in a similar manner to amnion, whereas maternal (decidual) explants did not. the enhanced pge production caused by cp , was abrogated by co-treatment with either sr a or ns , illustrating that the cannabinoid action on prostaglandin production in fetal membranes is mediated by cb agonism and cox- . preliminary data from western blotting show that cannabinoid treatment results in the up-regulation of cox- expression. however, there was no change in cox- expression and no evidence either for up-regulation of cpla or for down-regulation of pgdh expression. conclusion: this study demonstrates a potential role for endocannabinoids in the modulation of prostaglandin production in late human pregnancy, with potentially important implications for the timing and progression of term and preterm labour and membrane rupture. inactivation of vegf receptor- , but not vegfr- or vegfr- , during the peri-implantation period prevents normal pregnancy development in the rodent through disruption of uterine angiogenesis. nataki c douglas, hongyan tang, raul gomez, bronislaw pytowski, daniel j hicklin, jan kitajewski, mark v sauer, ralf c zimmermann. division of reproductive endocrinology and infertility, columbia university, new york, ny, usa; imclone systems, inc., new york. objective: vegf is involved in the regulation of uterine angiogenesis and implantation in both rodents and non-human primates. vegfr- , r- , and r- are expressed in the uterine decidua and are involved in the regulation of vessel formation in many systems. to determine if these receptors have a functional role in the regulation of post-implantation angiogenesis and pregnancy development, we examined the effects of blocking vegfr- , r- , and r- function. design: prospective animal laboratory material and methods: to avoid effects of vegf receptor neutralization on ovarian function, we utilized a progesterone replaced, ovariectomized mouse model. vegf receptor blocking antibodies were administered on ed . , prior to embryonic expression of these receptors. embryonic development was evaluated on ed . , blood vessel density and apoptosis on ed . , and cellular proliferation on ed . (n= per time point in each group). anova with bonferroni correction was used to compare sample means. results: see tables. conclusions: neutralization of vegfr- and vegfr- , but not vegfr- resulted in a significant reduction in cellular proliferation and decidual angiogenesis. vegfr- mediates decidual angiogenesis, but is not required for normal pregnancy development. in contrast, an intact vegf/vegfr- pathway is required for the decidual angiogenesis that mediates early pregnancy development. effect of vegfr neutralization on ed . control anti r- anti r- anti r- no. of implantation sites . +/- . +/- . . +/- . . +/- . hoxa encodes a transcription factor required for endometrial receptivity and embryo implantation. our objective was to identify and to characterize those molecular markers regulated by hoxa in multiple cellular model-systems. using microarray technologies, we identified putative hoxa target genes involved in early implantation. liposome-mediated transfection delivering either empty vector or the same plasmid constitutively expressing hoxa was introduced into newly impregnated mice during laparotomy or layered onto cultured human endometrial stromal-cells (hescs). rna products from these in vivo and in vitro transfections were used to identify targets and to validate the microarray screen employing semi-quantitative real-time pcr (qrt-pcr). we identified statistically-significant genes regulated by hoxa overexpression of which genes were down-regulated greater than -fold when compared to controls. cellular ontogenies of differentially-expressed genes include: cell adhesion molecules, signal transduction factors as well as metabolic regulators. furthermore, we identified the -phosphoglycerate dehydrogenase gene, (pgdh) whose products are regulated by hoxa during implantation in both murine model systems in and cell culture. this genes codes for an enzyme critical to de novo l-serine biosynthesis via a phosphorylation-dependent pathway. microarray analysis demonstrated a fold expression decrease when hoxa is overexpressed. this diminution in pgdh expression was noted in the validation experiments using qrt-pcr and corroborated in hesc cells where the mrna levels decreased to % when compared to controls. the repression of pgdh during the implantation window may represent a conservation of activity as secretory-phase protein synthesis may be suppressed in order to promote cellular differentiation and resultant implantation. these regulatory relationships identified in mouse implantation likely function to enhance uterine receptivity and may have a role in human implantation. objective: hoxa is expressed in endometrium, where it is regulated by sex steroids and is necessary for endometrial receptivity and implantation. hoxa is also expressed in leukocytes. here we hypothesized that hoxa would be regulated by sex steroids in both cell types. we further hypothesized a correlation between expression of hoxa in peripheral blood cell (pbcs) and endometrium in both mice and in humans. methods: real-time pcr was used to determine differential expression of hoxa mrna in u cells, a monomyelocytic cell line, in response to increasing concentrations of estradiol. to determine if hoxa is expressed in leukocytes in vivo, peripheral leukocyte hoxa mrna expression was measured over sequential estrus cycles in mature cd nulliparous mice and correlated to vaginal smear-cytology. additionally, peripheral leukocytes were isolated either from pregnant or normally-cycling women to assess hoxa mrna expression. results: there was a direct, dose-responsive correlation between exposure to increasing estradiol and hoxa mrna expression levels in u cells. in a murine model, we demonstrated that hoxa mrna expression-levels varies throughout the estrus cycle with a marked increase in expression following vaginal plug detection. the nadir of hoxa expression is prior to proestrus and increased up to -fold during the receptive phase. this increased expression continues throughout gestation. the heightened expression in murine leukocytederived hoxa mrna also is demonstrated across species. our preliminary data suggests that the greatest fold-increase of expression occurs during the window of implantation of the secretory phase in normal, cycling women. this level was sustained in pregnancy. there appears to be a trend with the highest levels of expression associated with viable gestations. women with attenuated expression profiles had non-viable gestations. conclusions: hoxa expression is regulated by sex steroids in both leukocytes and endometrium. the temporal pattern of peripheral hoxa transcript expression demonstrated in mice and humans mimics the differential rna expression documented within the uterus. leukocyte hoxa expression during the reproductive cycle in mice and humans is a marker of endometrial receptivity. peripheral leukocyte expression of hoxa mrna may correlate with implantation success. carolien m boomsma, annemieke kavelaars, marinus jc eijkemans, gijs teklenburg, bart cjm fauser, cobi heijnen, nick s macklon. reproductive medicine and gynaecology and laboratory of psychoneuroimmunology, university medical center, utrecht, netherlands. the purpose of this study is to assess the association between the intra-uterine cytokine expression profile at the time of embryo transfer and successful embryo implantation following ivf/ icsi treatment. materials and methods women undergoing ivf/ icsi underwent endometrial secretion aspiration prior to embryo transfer. known soluble mediators of implantation were measured using a multiplex immunoassay, namely il ß, il , il , il , il , il , il , il , tnf , vegf, ifn , eotaxin, mcp- , ip- , dkk- and hbegf. mif was determined using an elisa. the total protein concentration was measured for normalization purposes. data were log transformed to obtain normal distribution. multivariable logistic regression analysis with a backward elimination procedure (p< . ) was used, potential confounders (age, blood contamination, embryo quality) were included in a forward stepwise model. ten mediators of the analysed were detectable in - % of the samples. il was detectable in % of samples, dkk- %, il- %, il- %, il- % and hbegf was detectable in % of samples. ifn-was not detectable in any of the samples. multivariable logistic regression showed only logmif concentrations to have a significant correlation with achieving clinical pregnancy (p = . ). higher mif concentrations were correlated with a higher chance of conceiving. endometrial secretion analysis represents a novel means of assessing the intra-uterine milieu encountered by the embryo and offers new perspectives in the study of endometrial receptivity. in this large prospective study assessing an array of cytokines, mif was found to be significantly correlated with pregnancy. mif, macrophage migration inhibitory factor, is a cytokine with numerous proinflammatory, immunomodulatory, angiogenic and tissue remodelling properties. mif induces the synthesis and secretion of matrix metalloproteinases by endometrial cells, which may contribute to embryo invasion. its expression is particularly increased during the secretory phase, suggesting a role in reproductive processes. analysis of aspirated endometrial secretions offers a direct clinical test of endometrial receptivity which can be applied during treatment cycles without disrupting implantation. endometrial receptivity and secretory differentiation require progesterone (p). it has been hypothesized that low p levels result in delayed endometrial differentiation and infertility due to reduced receptivity. objective: test the effects of low luteal p on histologic and molecular markers of differentiation and function. methods: normal cycling women (n= ) were treated with daily leuprolide ( . mg/d) beginning in the midluteal phase and continuing through the protocol. after menses, subjects received transdermal estradiol (e, . mg/d) for days. after day of e, subjects also received daily i.m. injections of p, randomized to mg/d (sub-physiological) or mg/d (physiological). endometrial biopsy was performed after days of combined e and p treatment. additional untreated women had biopsies performed days after spontaneous lh surge. endometrial histologic dating was performed by two individuals according to the criteria of noyes et al. mrna levels were assessed using real-time rt-pcr. results: mean(s.d.) of peak and trough p serum concentrations in the mg/d p group were . ( . ) and . ( . ) ng/dl, respectively, while those in the mg/d group were . ( . ) and . ( . ) ng/dl. there were no differences between treatment groups for histologic dating; the mean(s.d.) histologic date was . ( . ), . ( . ), and . ( . ) for the mg, mg, and spontaneous cycle groups, respectively. there also were no differences among the three groups in mrna levels of ten functional markers (er , pr, integrin subunit, osteopontin, cyr , egr- , fkb , c-fos, and cd ), although variability of gene expression was greater in those who received mg/d p than in those who received mg/d p. there was also no correlation between serum progesterone level and gene expression or histologic date. conclusions: sub-physiological levels of progesterone, in the range seen in ovulatory women, do not induce detectable changes in expression of marker genes or histological dating, although low p levels were associated with greater variability of gene expression. these data suggest that abnormalities in endometrial histologic development and function likely result from intrinsic abnormalities rather than from low levels of p secretion. (supported by unc nova carta fund and nih u hd- ). endometrial ip attracts trophectoderm through cxcr interaction. simcha yagel, caryn greenfield, hen sela, jacob hanna, irit manaster, orna singer, ronit haimov-kochman, shira natanson-yaron, diana prus, benjamin reubinoff, debra s goldman-wohl, ofer mandelboim. obstetrics and gynecology, hadassah-hebrew university medical centers, jerusalem, israel; lautenberg center for immunology, jerusalem, israel; human embryonic stem cell research center, jerusalem, israel. introduction: implantation is initiated in part by attraction of the blastocyst to the endometrium lining the uterus. we hypothesized that this process is partly accomplished by chemokines expressed by the endometrium interacting with chemokine receptors on the blastocyst, suggesting that they play an important role in implantation. materials and methods: chemokine receptors were characterized in jeg cells, placental villi, primary trophoblast cell culture, trophectoderm cells derived from human es cells, blastocyst trophectoderm, and st trimester placental tissue sections. expression of chemokines was tested in decidua, endometrium, and ishikawa and hec- cell lines. immunohistochemistry, intracellular staining, elisa, facs analysis, and rt-pcr were employed to characterize chemokine receptor and ligand expression. functional testing was performed using transwell migration assays and in a nude mouse model using a matrigel gel plug cell attraction assay followed by facs analysis. results: trophoblasts demonstrated expression of various chemokine receptors, most prominently cxcr and cxcr . immunohistochemistry of trophoblast from placental villi plated on matrigel expressed cxcr and cxcr as well as hla-g. noteworthy is that trophectoderm cells derived from hes cells treated with bmp- and jeg cells, and blastocyst trophectoderm expressed principally cxcr . elisa and immunohistochemistry showed that decidua and endometrium expressed chemokines ip and il . migration assays demonstrated that ip significantly attracted various trophoblast and trophectoderm cells in vitro, and in the mouse model in vivo. taken together these results demonstrate the interaction between trophoblasts and endometrial cells is mediated by cxcr and cxcr , and il and ip . these interactions are important in the attraction of trophoblasts at the feto-maternal interface. however, ip -cxcr is the most relevant to early implantation as only cxcr is expressed consistently by trophectoderm. the endometrial proteome: changes from proliferative to secretory phase. lois a salamonsen, jenny i-c chen, xian mak, peter j stanton, david m robertson, andrew n stephens. prince henry's institute of medical research, melbourne, vic, australia. global gene analyses have demonstrated major changes across the menstrual cycle, but which of these are reflected in the proteome is not known. this study aimed to globally assess proteins differentially expressed in the endometrium between the proliferative and. secretory phases. d page analysis with dige minimal dye labelling was conducted across the pi range - on endometrial tissue from either the mid-proliferative or midsecretory phases (n= /group). profiles were assessed using samespots software. differentially expressed proteins were identified using maldi-tof ms and the interrelationship of proteins examined using ingenuity software. a total of spots were detected: were differentially expressed (p < . ) with spots having an overall false discovery rate < % (q < . ). hierarchical clustering analysis revealed that these proteins lay within three main branches in the protein dendrogram. one cluster had proteins upregulated in the proliferative phase and two contained proteins up-regulated in the secretory phase. the unique protein profiles were also revealed using principle component analysis (pca): proteins clustered into two main groups, according to cycle phase. pca thus indicated similar unique protein signatures as suggested by hierarchical clustering. thirty one of the differentially expressed proteins were identified using maldi-tof ms. these proteins could be grouped into seven categories, which included structural ( ), transport ( ), regulatory ( ), membrane ( ), enzyme ( ), motor ( ) and others ( ). proteins involved in matrix assembly and those needed for subsequent establishment of secretory endometrium were up-regulated in proliferative endometrium. proteins important for cellular organisation and communication as well as products responding to environmental stress and the immune system were highly up-regulated during the secretory phase. biological pathways were constructed based on the proteins identified. the top network for secretory endometrium clustered around tgf-b. others related to inhibition of cell death / cell viability and leukocyte extravasation. these studies provide a global approach to the cyclic changes of the endometrium and highlight the complex dynamics of protein expression in human endometrium. hormonal regulation of prokineticins in the human fallopian tube: potential regulators of embryo transport. aw horne, hn jabbour, p lourenco, s wright, s battersby, arw williams, hod critchley. reproductive and developmental sciences, university of edinburgh, edinburgh, united kingdom; mrc human reproductive sciences unit, queen's medical research institute, edinburgh, united kingdom. background: understanding the factors regulating embryo transport in the fallopian tube (ft) has important clinical implications. embryo retention in the tube due to ft dysfunction is thought to lead to tubal pregnancy, a considerable cause of morbidity and occasional mortality. transport of the embryo through the ft is, for the greater part, accomplished by smooth muscle contraction. a group of multi-functional proteins and their receptors, called prokineticins, have been shown to affect smooth muscle function in other tissues, such as the intestine. the expression pattern of prokineticins, and their receptors, was examined in normal human ft obtained throughout the menstrual cycle, and the effect of the sex steroids on prokineticin expression was examined in an in-vitro model of the ft. methods: ft biopsies (n= ) and sera (for measurement of oestradiol and progesterone for endocrine staging) were collected from women undergoing gynaecological procedures for benign conditions. using a combination of quantitative taqman rt-pcr and immunohistochemistry, the mrna and protein expression pattern of prokineticins, and their receptors, were examined in the ft throughout the menstrual cycle. tubal explant culture was established using surgical tissue from the biopsies and exposed to varying concentrations, and time courses, of oestrogen and progesterone. results: prokineticin (pk ) and prokineticin receptor (pkr ) mrna are up-regulated in the progesterone-dominant mid-secretory phase. pk and pkr protein are expressed in the epithelium, smooth muscle and around the blood vessels of the ft. stimulation of tubal explant cultures with a physiological concentration of progesterone showed an up-regulation of pk and pkr . conclusions: prokineticins show temporal variation in expression in human ft and appear to be regulated by progesterone. their role in embryo transport needs to be investigated to further understanding of pregnancy complications, such as tubal pregnancy. translating mouse to human: a dynamic model of xenografted human endometrium. alex j polotsky, liyin zhu, nanette santoro, jeffrey w pollard. albert einstein college of medicine, bronx, ny, usa. in the mouse endometrium, the hormonal environment controls cellular proliferation and cell cycle activity. estradiol (e ) inhibits glycogen synthase kinase beta (gsk- ), resulting in nuclear accumulation of cyclin d and progression of the cell cycle, as well as dna replication licensing. in utero administration of the gsk- inhibitor, licl, results in epithelial cell proliferation in the absence of e (zhu, pollard. pnas. ; : ) . in this study, we derived a functional model of xenografted human endometrium to perform mechanistic studies of human endometrial proliferation. methods: human endometrial samples were obtained from volunteers aged - . immuno-compromised mice were transplanted with disaggregated/ recombined human epithelial glands and stroma under the kidney capsule. after weeks of out-growth, mice were ovariectomized, and replaced with e or licl. xenografts were harvested and processed for immunohistochemistry (ihc) and glandular labeling index (li). t test was used to compare group means. results: - % of engraftments were successful, resulting in a vascularized endometrium with characteristic architecture (a, b). hoechst staining confirmed that xenografts were made up of human cells ©. e (e) induced significantly greater proliferation compared to control (d) as assessed by ihc for ki , with li of . ± . and . ± . , respectively, p = . ). minichromosome maintenance- , a protein involved in dna replication licensing, was more sensitive for e -treated cells synthesizing dna than was ki staining (i). estrogen (g) and progesterone receptors (h) were expressed in xenotransplant tissue, the latter being up-regulated by e . licl (f) induced proliferation similar to e and greater than control (li = . ± . , p= . for e vs. licl). conclusion: xenografted human endometrium provides a dynamic model of endometrial proliferation that is well suited for translational studies. administration of licl in the absence of e induced glandular proliferation, supporting the notion that similar mechanisms are operative in human proliferation as in the mouse. gnrh analogs have been extensively used in assisted reproduction. although the main effects of gnrh analogs are via gnrh receptors on the pituitary gonadotrope, gnrh and gnrh receptors have been identified in many reproductive tissues including human endometrium, suggesting their potential action at endometrial level. in the present study, we examined the potential regulatory action of gnrha, la on sex steroid mediated gene expression in human endometrium. human endometrial surface epithelial (hes) cells and isolated endometrial stromal (esc) cells were used as in vitro models. all experiments lasted for h. the cells were treated with estradiol (e , nm, h), progesterone (p , nm, h), gnrha (la μm, h), e ( h) followed by p (last h) and gnrha plus e plus p where, gnrha added either first, second or last in order at h intervals. total rna was extracted, reversed-transcribed and subjected to real-time pcr simultaneously, measuring the expressions of il- , il- , il- , il- , il- , il- , il- , il- p , il- p , il- , ifn-and tnf . both hes and esc expressed majority of these cytokines with the exception of low to undetectable levels of il- , il- , il- and ifn-. treatment with e and p , either alone, or in combination significantly upregulated the expression of many of these cytokines at varying extend as compared to controls. however, gnrha either alone or in combination with e and p significantly diminished e , p or e plus p induced mrna expression of cytokines. the suppressive effects of gnrha on some of the cytokines varied significantly by the order which gnrha was introduced into the culture medium. we conclude that gnrha by acting directly on the endometrial cells effectively suppresses the mrna expression of several key proinflammatory cytokines upregulated by ovarian steroids. our results imply that gnrha therapy during assisted reproduction may modify endometrial receptivity via downregulation of proinflammatory cytokines induced by estradiol and progesterone. supported by nih grant hd . introduction: endometrial angiogenesis is characterized by a rapid increase during the early proliferative phase that peaks midcycle, followed by a gradual decrease in the secretory phase, while menses involves generalized endometrial inflammation, necrosis, and vascular thrombosis. vascular endothelial growth factor (vegf), whose expression is regulated by sex steroids, is a mediator of endometrial angiogenesis. recently, myoferlin, a kd transmembrane protein, was identified in endothelial cells where it mediates vegf-dependent endothelial cell proliferation, migration, and nitric oxide synthesis by affecting vegf receptor- function and stability. myoferlin also appears to play a role in vesicle trafficking and membrane repair. objective: to characterize myoferlin protein expression in endometrium. methods: western analysis of cultured human endometrial stromal cells (hesc) and human endometrial endothelial cells (heec) treated with physiologic concentrations of estradiol and progesterone was performed using a polyclonal rabbit antibody against myoferlin. subsequently, immunohistochemistry (ihc) was performed on human endometrium samples obtained from various stages of the menstrual cycle. results: myoferlin protein was expressed in hescs and heecs, but expression was not affected by sex steroids. ihc staining for myoferlin was specific, intense, and localized to the apical membrane of glandular epithelial cells and endothelial cells, with less intense staining in the stroma. h-score quantification showed that in endometrial endothelial cells, myoferlin protein expression was highest during the early proliferative and early secretory phases, while glandular and stromal myoferlin expression peaked during the late proliferative/early secretory phase. conclusion: myoferlin expression in human endometrium correlates with periods of greatest endometrial angiogenesis, and expression is not limited to endothelial cells, but also includes glandular and stromal cells. given the involvement of myoferlin in vegf signaling as well as membrane repair, understanding its role in human endometrium may further elucidate an understanding of endometrial development both under physiologic and pathologic conditions. the human embryo is the primary regulator of embryo-endometrial molecular cross talk during early implantation. gijs teklenburg, , cobi heijnen, esther baart, karima amarouchi, carolien boomsma, janet carver, helen mardon, annemieke kavelaars, nick macklon. reproductive medicine and gynaecology, and laboratory of psychoneuroimmunology, university medical center, utrecht, netherlands; nuffield department of obstetrics and gynaecology, university of oxford, women's centre, john radcliffe hospital, oxford, united kingdom. introduction: uterine receptivity and implantation are controlled by locally acting trophic factors and cytokines. in humans, the regulation of the embryo-endometrial dialogue beyond the early blastocyst stage is poorly understood. we hypothesized that the interaction between a healthy conceptus and the receptive endometrium is associated with a distinct local regulation of cytokine production favouring implantation. methods: human embryos, cryopreserved at day after fertilization and donated for research, were thawed and cultured under standard conditions until day five. forty-two embryos from donors developed to the blastocyst stage. following removal of the zona pellucida, they were placed in individual coculture on a confluent monolayer of endometrial stromal cells. on day , the developmental potential of each embryo was assessed as early arrested, late arrested or developing. culture supernatants were analysed for concentrations of il- , il- , il- , il- , il- , il- , il- , il- , tnf-, mcp- , ip- , eotaxin and hb-egf using a multiplex immunoassay. day supernatants from culture systems in which no embryo had been placed were also analysed as controls. results: out of ( %) embryos continued to develop and were able to attach and invade into the stromal cell compartment of the co-culture environment. twelve late arrested embryos showed signs of degradation on day and the totally disintegrated embryos were assigned to the early arrested group. supernatants from both early and late arrested embryo cultures contained significantly lower levels of a number of cytokines and growth factors in comparison to developing embryo cultures. moreover, the levels of these mediators in co-cultures were significantly lower than those in non-embryo control stromal cell culture supernatants. conclusion: these data suggest a pivotal role of the embryo in embryoendometrial cross talk. whether reduced mediator expression in co-cultures reflects a selective down regulation of stromal cell cytokine and growth factor production is now being investigated. epithelial cell dynamics during human implantation. hiroshi uchida, tetsuo maruyama, toru arase, masanori ono, takashi kajitani, maki kagami, hideyuku oda, sayaka nishikawa, yasunori yoshimura. department of obstetrics and gynecology, keio university school of medicine, shinjuku, tokyo, japan. epithelio-mesenchymal transition (emt) is thought to play a role in functional differentiation of endometrial epithelial cells during human implantation. molecular mechanism of epithelial sheet remodeling caused by embryo invasion remains elusive. to address this, we investigated cellular dynamics of n-cadherin and vimentin, the two representative major markers of emt, during implantation. in in vitro implantation assay using a human endometrial epithelial cell line, ishikawa, and a human choriocarcinoma cell line, jar (uchida et al., hum reprod ), we pre-treated human ishikawa cells with or without ovarian steroid hormones ( -estradiol + progesterone; ep), fa- (n-cadherin blocking ab), or suberoylanilide hydroxamic acid (saha), one of histone deacetylase inhibitors, which has a potential to improve in vitro implantation (ibid). implantation or treatment with or without ep or saha enhanced the expression of n-cadherin and vimentin but down-regulated ecadherin. furthermore, treatment with ep or saha accelerated ishikawa cell motility and increased the number and spreading area of jar spheroids. in vitro implantation assay, the most prominent staining intensity of n-cadherin was observed just around the adhered spheroid from which its intensity decreased away. functional blockade of n-cadherin by fa- resulted in the complete suppression of ishikawa cell motility, the unique distribution of n-cadherin around jar spheroids, and the spreading area of jar spheroids, while it did not affect the number of the adhered spheroids. human implantation consists of the multiple steps, including apposition, adhesion and penetration. thus, these results collectively indicate that emt may take place after the apposition and that n-cadherin may be required for the remodeling and emt of the epithelial sheet during embryo invasion. n-cadherin may enhance the recruitment of spheroid-neighboring cells, suggesting its role in the covering-up of the invading embryo through acceleration of epithelial cell motility. endocannabinoid regulation in human endometrium. jessica g scotchie, marc a fritz, steven l young. obstetrics and gynecology, university of north carolina, chapel hill, nc, usa. background: research in mouse models demonstrates two cyclically regulated endocannabinoids produced in murine endometrium, anandamide and -arachidonoyl glycerol ( -ag); both play critical roles in murine embryonic implantation. no studies of endocannabinoids in human endometrium have been performed. objectives: determine menstrual cycle expression and localization of synthetic and degradative enzymes for anandamide and ag in human endometrium. methods: human endometrium was collected from volunteers across the menstrual cycle (n= ). quantitative rt-pcr was performed analyzing the expression of: n-acylphosphatidylethanolamine (nape) and fatty acid amide hydrolase (faah), the synthetic and degradative enzymes for anandamide, respectively; sn- -diacylglycerol lipase-a (dagla), and b (daglb), the synthesis enzymes for ag; and monoacylglycerol lipase (magl) and cyclooxygenase- (cox ), the degradative enzymes for ag. the constitutive gene ppia was used for comparison. immunohistochemical localization of nape protein was performed using nape-pld polyclonal antibody (cayman chemical, # ). anova and student's t-test analysis performed on samples grouped by proliferative (pro), early, mid-, and late secretory (es, ms, ls) phases. results: mrna expression of all enzymes responsible for synthesis and degradation of anandamide and ag was detected throughout the cycle. no significant cyclic change in nape, faah, or daglb gene expression was seen. a decrease in dagla gene expression in the ms and ls phases compared to the pro and es phases (p= . ) was seen. magl gene expression was higher in the secretory phase than the pro phase (p= . ). cox gene expression was detected at low levels in the pro, es and ms phases, with marked increase in the ls phase (p< . for all comparisons). protein localization of nape showed a cytoplasmic epithelial location, with increased staining on pro and es samples compared to ms and ls samples. immunolocalization of remaining proteins is ongoing. conclusion: this is the first report documenting the presence of endocannabinoid synthetic and degradative enzymes in human endometrium. genes controlling anandamide expression do not fluctuate significantly across the cycle. however, ag's degradative enzymes increase in the secretory phase, suggesting that lower ag levels may be advantageous for embryo implantation. our findings suggest that human endometrial endocannabinoid regulation differs from murine regulation. interleukin- beta (il- ) regulates il- signaling in decidua-implication in the pathophysiology of preeclampsia (pe). sj huang, cf yen, cp chen, f schatz, cj lockwood. obstetrics, gynecology and reproductive sciences, yale university, new haven, ct, usa; ob/gyn, chang gung memorial hospital, tao-yuan, taiwan; ob/gyn, mackay memorial hospital, taipei, taiwan. objective: previously, we found much higher cytoplasmic immunoreactive il- levels in the preeclamptic decidual cells than in adjacent interstitial trophoblasts. such decidual cell-derived il- contributes to the systemic endothelial cell dysfunction that elicits the proteinuria and hypertension of the maternal syndrome. il- promotes the transition from innate to adaptive immunity. moreover, by skewing monocyte differentiation from a dendritic to a macrophage phenotype, decidual il- may promote the macrophage excess observed in the preeclamptic decidua. macrophages impair trophoblast decidual invasion to foster incomplete spiral artery remodeling that elicits placental ischemia and hypoxia. the current study: ) localized il- mrna levels in preeclamptic versus normal decidual sections; ) evaluated mechanisms regulating il- synthesis by targeting intracellular signaling pathways with specific inhibitors; ) identified potential il- targets by immunolocalizing the il- receptor (il- r) to specific cell types in placental bed biopsies. methods: in situ hybridization localized il- mrna in normal versus preeclamptic decidua. il- r was immunolocalized in placental bed biopsies. leukocyte-free first trimester decidual cells were incubated with e and mpa ± il- ( ng/ml) ± an inhibitor of p mapk (sb ) or protein kinase c (calphostin c) or nf b (activation inhibitor iii) for hrs. an elisa measured secreted il- levels. results: il- mrna was present primarily in decidual cells with increased il- mrna levels observed in pe. preferential expression of the il- r was observed on decidual cells in placental bed biopsies. compared with basal il- levels ( . ± . pg/ml/ g cell protein) by decidual cells, il- enhanced il- output by decidual cells ( . ± . pg/ml/ g cell protein). only the p mapk inhibitor significantly reduced this output to . ± . pg/ml/ g cell protein (n= , p< . ). our results indicate that inflammatory cytokine enhances il- synthesis in decidual cells of the preeclamptic decidua by a mechanism involving p mapk. such il- is likely to act as an autocrine/paracrine effector via decidual cell-expressed il- r to contribute to the macrophage excess observed in the preeclamptic decidua. heparanase is up-regulated by estrogen and during the secretory phase of the human endometrium. ronit haimov-kochman, shira natanson-yaron, caryn greenfield, achinoam lev-sagie, lichtenstein michal, haya lorberboum-galsky, israel vlodavsky, simcha yagel, arye hurwitz. ob/ gyn, jerusalem, israel; cellular biochemistry human genetics, hadassah hebrew university medical centers, jerusalem, israel; cancer and vascular biology research center, technion school of medicine, haifa, israel. introduction: heparanase is an endoglycosidase that cleaves heparan sulfate (hs) proteoglycan of the extracellular matrix. the full-length proheparanase is activated by cleavage into an active isoenzyme, resulting in the release of hsbound cell-differentiation factors, such as hb-egf. the cycling endometrium involves remarkable steroid hormone-induced tissue remodeling. in vivo, increasing exposure to unopposed estrogen may lead to endometrial malignant transformation. aim: to investigate heparanase expression and regulation in the cycling endometrium. materials and methods: heparanase mrna levels were measured by quatitative rt-pcr in naturally menstruating women and in hec a, estrogen receptor (er)-negative and ishikawa, er-positive endometrial carcinoma cell lines exposed to increasing doses of estradiol. heparanase isoenzymes were localized by immunohistochemistry using specific anitbodies in murine endometrium and human normal, hyperplastic and malignant endometrium. results: heparanase mrna level increased fold in secretory phase (d ) compared to proliferative phase (d ) endometrium. heparanase transcript levels increased fold during hr culture in er positive adenocarcinoma cell line exposed to increasing doses of estradiol, but not in hec a, er negative cell line. both heparanase isoforms were localized to murine glandular endometrium. human glandular endometrium at both proliferative and secretory phases was immunoreactive with the active isoform of heparanase. proheparanase was detected in basal membrane of endometrial glands and endometrial stroma during secretory phase. along with malignant transformation of the endometrium the presence of proheparanase increased dramatically from none in stroma of normal and hyperplastic endometrium to abundance in malignant tumors. conclusions: heparanase gene expression is higher during the window of implantation and up-regulated with estrogen in endometrial cells via er in vitro and vivo. heparanase is differentially localized in the secretory phase of the endometrium compared to the proliferative phase, suggesting a role for this molecule during the window of implantation in man. interleukin- (il- ) system mrna and protein expression in the human fallopian tube with ectopic implantation. hong-yuan huang, , tien-hung huang, chin-jung li, chyi-long lee, , hsin-shih wang, , yung-kuei soong. , obstetrics and gynecology, chang gung memorial hospital, kwei-shan, tao-yuan, taiwan; obstetrics and gynecology, chang gung university and school of medicine, kwei-shan, tao-yuan, taiwan. objective: ectopic pregnancy, an abnormal implantation of a fertilized ovum outside the uterine cavity, has been increasing in number at a staggering pace of all pregnancies. il- system is one of the major cytokines involved in human endometrium during embryo implantation and might perform a defensive role against maternal immune response. very little information is available regarding the expression and synthesis of cytokines in the pathogenesis of fallopian tube with ectopic gestation. the purpose of this study is to investigate il- system expression in human fallopian tubes with ectopic pregnancy. methods: paired segments of human fallopian tubes with ectopic implantation site and side portion close to ectopic gestation (n= ) were collected from women undergoing laparoscopic salpingectomy after informed consent and irb approval. segments of fallopian tubes from women undergoing tubal ligation (n= ) were used as control groups. total extracted rna was reverse transcribed and amplified by pcr using specific primers for gapdh ( bp), il- ( bp), il- bp ( bp) and il- r ( bp). quantitative il- and il- bp mrna expression in human fallopian tube was determined by real-time pcr. to determine the presence of il- system proteins, tissues were fixed and processed for immunohistochemical study. data analysis was done with anova and pearson's correlation. results: il- and il- bp as well as il- r mrna were all expressed in tubal ectopic implantation and normal tubes. according to real-time pcr with c t value quantification and -ct method, a significantly higher il- expression in tubal ectopic implantation and lower ratio of il- antagonist to agonist in portion close to ectopic implantation is demonstrated in comparison to normal tubes (p< . ). immunoreactive il- system at the protein levels was also present in human fallopian tubes with ectopic implantation and normal tubes. conclusions: these results suggest that fallopian tube il- system expression may play a crucial role during the process of early embryonic implantation. the expression and ratio of antagonist to agonist in fallopian tubes may indicate an earlier "dialogue " in human fallopian tubal gestation prior to uterine implantation. replacement. marcia c ferreira, ines kd cavallo, fernando m reis. gynecology, ufmg, belo horizonte, mg, brazil. activin a is a growth factor expressed in the endometrium, where it modulates tissue remodelling and enhances decidualization. the effects of activin a are counteracted by two binding proteins, namely follistatin and follistatin-like (fstl ). while the endometrial expression of activin a increases during the secretory phase of menstrual cycle, the effects of ovarian steroids on these proteins and their mrnas has not been assessed yet in postmenopausal women or in ovariectomized animals. we have evaluated the effects of estrogen alone or estrogen plus progestin on the endometrial expression of activin beta-a subunit, follistatin and fstl in ovariectomized rats. adult female wistar rats (n= ) were ovariectomized and received one week later a single dose of estradiol benzoate ( . mg/kg body weight, i.m. injection), either alone (n= ) or associated with depot medroxyprogesterone acetate ( . mg/kg body weight, i.m. injection, n= ), or oil vehicle (control group, n= ). one week after the hormone or placebo treatment, the animals were sacrificed and their uteri were removed and processed by immunohistochemistry and real-time pcr. data were normalized to the expression of ribosomal phosphoprotein p (rpp ) and analyzed with the delta-delta ct method, anova and newman-keuls test. activin beta-a subunit mrna levels increased significantly in the uteri of rats treated with estradiol alone ( . fold increase over controls, p< . ) and to the same extent in rats receiving estradiol plus medroxyprogesterone ( . fold increase over controls, p< . ). this was accompanied by increase of beta-a subunit immunostaining in estradiol and estroprogestin-treated rats, which was noted only in the surface endometrial epithelium. follistatin mrna expression, conversely, showed a significant decrease in the groups treated with estrogen alone ( . fold compared to controls, p< . ) and estrogen plus progestin ( . fold compared to controls, p< . ), while follistatin immunostaining in the glandular epithelium was weaker in estradiol and estroprotestin-treated rats compared to controls. fstl expression was similar in the groups. in conclusion, the expression of activin beta-a subunit increases and that of follistatin decreases following estrogen replacement in the endometrium of ovariectomized rats, and these effects are not further altered by the addition of progestin. endometrial nk cells are a unique inert nk subset until pregnancy. simcha yagel, irit manaster, jacob hanna, ronit haimov-kochman, miri godin, yuval bdolach, caryn greenfield, shira natanson-yaron, arye hurwitz, debra s goldman-wohl, ofer mandelboim. obstetrics and gynecology, hadassah-hebrew university medical center, jerusalem, israel; lautenberg center for tumor immunology, hadassah-hebrew university medical center, jerusalem, israel. introduction: we recently demonstrated that nk (natural killer) cells play a critical role in trophoblast migration and angiogenesis at the fetal maternal interface. nk cells populate the endometrium at the secretory phase of the menstrual cycle, the time of anticipated blastocyst implantation. peripheral blood (pb) nk cells and decidual nk (dnk) cells express a variety of activating receptors, including nkp , nkp and nkp , collectively known as natural cytotoxicity receptors (ncrs), and nkg d which regulate nk cell killing and growth factor production. to compare endometrial nk cell (enk) activating receptor expression and function to pbnk and dnk cells and endometrial ligand expression, with a focus on their roles in blastocyst implantation. patients and methods: subjects were ivf patients undergoing natural menstrual cycles. endometrium samples were collected on treatment days and . a lymphocyte profile of the endometrial cells and pb was performed. facs analysis was performed on isolated endometrial nk cells, pbnk cells and dnk cells for cd , cd , nkp , nkp , nkp and nkg d. ncr ligand expression was characterized on adherent endometrial cells using ncr-ig fusion proteins and nkgd -ig and nkg d specific ligands as well as control ccmi-ig. redirected killing assays and cytokine secretion assays of ifn , vegf, plgf, and il- with and without il- were performed. results: endometrial lymphocytes of day and in these women are mostly cd bright cd -nk cells, with a significant amount of t cells, similar to pbnk cells and in marked contrast to dnk cells. unlike pbnk and dnk cells, enk receptors do not express nkp , nkp . nkp and nkg d are the only activating enk receptor expressed. like decidual cells, adherent stromal endometrial cells expressed the ligands for nkp , nkp and nkg d, suggesting that these nk cells have potential for activation. finally enk cells could not kill or secrete cytokines. conclusions: these findings of a unique activating receptor profile on endometrial nk cells, unlike that of dnk and pbnk, suggest that enk cells are a special local population of nk cells that change dramatically and are activated at the onset of pregnancy. variation in platelet activation throughout the menstrual cycle. fiona c denison, amy o robb, imogen b smith, nicholas l mills, hilary od critchley, david e newby. centre for reproductive biology; centre for cardiovascular sciences, the university of edinburgh, united kingdom. background: platelet-monocyte aggregation (pma) is a sensitive and novel measure of platelet activation with important proinflammatory consequences including release of cytokines and chemokines. previous studies using less sensitive techniques suggest that platelet activation alters during the menstrual cycle in response to circulating concentrations of sex steroids. the effect of sex steroids on circulating (c) pmas during a single menstrual cycle is not known. objective: to determine whether cpmas, platelet surface (ps) p-selectin and plasma (p) p-selectin vary through the menstrual cycle in response to changes in circulating sex steroid concentrations. methods: healthy, nulliparous, pre-menopausal, non-smoking women (mean age years), with regular menses ( - days) were studied. subjects gave written informed consent and the study had ethical approval. serial venous blood samples were taken at menstrual, follicular, periovulatory and luteal phases of a single cycle (days - , - , - and - ). cpmas (monocytes positive for the platelet marker cd a) were measured by flow-cytometry. psp-selectin expression was calculated on cd a positive cells. isotype-matched controls were used. serum oestradiol (e) and progesterone (p), plasma and pp-selectin were measured by elisas. data were analysed by one-way anova with repeated measures and bonferroni's post-tests for multiple comparisons. results: luteal phase p was > nmol/l in all women. numbers of cpmas and expression of psp-selectin were both significantly higher during menstrual compared with periovulatory phase of the menstrual cycle ( . ± . vs. . ± . %, p= . and . ± . vs . ± . %, p< . , respectively). there was no significant difference in pp-selectin concentration during the menstrual cycle (p= . ). there was no correlation between levels of serum e or p and numbers of cpmas, expression of psp-selectin or pp-selectin concentration. conclusions: numbers of cpmas and expression of pspselectin are maximal at menstruation with neither numbers of cpmas nor expression of psp-selectin correlating with serum e or p levels. this study suggests that activated platelets may potentially contribute to the inflammatory response at menstruation by releasing inflammatory mediators. by angiogenic factors and peri-cellular proteases in decidual secretory endometrium (dse), decidua parietalis (dp), and basalis (db) of miscarriage patients and matched controls. comparison of these parameters between the two groups enabled hypothesizing about their correlation with the occurrence of miscarriages. methods: decidua was obtained during st trimester termination of pregnancy (control group) and vacuum aspiration of missed abortions (case group). vascularization was studied by cd -immunohistochemistry. the expression of vascular endothelial growth factor-a, placental growth factor, flt- , kdr, angiopoietin- , angiopoietin- , tie- and the membrane-type matrix metalloproteinases mt -, mt -, mt -and mt -mmp were determined at mrna and antigen level and cd -positive unk cells, cd -positive macrophages, proliferation (ki ) and apoptosis (activated caspase- ) were evaluated by immunohistochemistry in consecutive serial sections. results: the decidual vascularization pattern showed differences between cases and controls: i.e. fewer vessels with larger circumference in cases, and this correlated with the differential expression of various angiogenic factors and proteases at mrna and antigen level. moreover, the endothelial protein expression of flt , kdr, mt -and mt -mmp was increased at the implantation site of cases. ki and active caspase- showed similar levels in the two groups and also the immune cells, both unk cells and macrophages, showed no differences at the implantation site between both groups. conclusion: differences between cases and controls appeared not to be based on altered proliferation, apoptosis, and/or inflammation. the differences in vascularization pattern and in the expression of angiogenic factors and proteases between both study groups suggest a correlation between decidual vascularization and the occurrence of miscarriages. respond to adrenomedullin. yaun-lin dong, hong y wen, janice endsley, alison hogg, hui-qun wang, manubai nagamani, chandra yallampalli. background: natural killer (nk) cells are the predominant lymphocytes present in human implantation site. decidual nk cells express perforin, an essential molecule required for lysis. formation of the placenta involves cooperation between maternal nk cells and fetal trophoblast cells that remodels the blood supply; however, the interaction between trophoblasts and decidual nk cells is largely unknown. adrenomedullin (adm) has been implicated in regulating early placental function and fetal growth. objective: to determine the role of multifunctional peptide adm in the decidual nk cells and fetal trophoblast cells interactions. methods: decidual and placental tissues were obtained from normal firsttrimester pregnancies terminated for social reasons. ethical approval to use these tissues was obtained from the irb of university of texas medical branch. cell preparations containing all decidual mononuclear cells were isolated by collagenase enzymatic disaggregation. cd decidual nk cells were purified by magnetic bead isolation. results: ) immunohistochemical analysis showed that adm is expressed primarily in decidual cells and trophoblast cells at the human implantation site; ) confocal imaging analysis demonstrated that decidual nk cells, which were identified by anti-cd staining, express adm receptor components crlr/ramp /ramp and their mrna expressions were futher confirmed by rt-pcr; ) k target cell killing assay indicates that adm inhibits cytokine il- /il- -induced decidual nk cell cytotoxicity; and ) immunofluorescent labeling and flow cytometric analysis revealed that adm suppresses perforin expression by decidual nk cells. conclusion: trophoblast-derived adm inhibits decidual nk cell cytotoxicity via suppressing perforin expression, thus, our results provide evidence for a new paradigm of embryonic-maternal communication involving a adm mediated interaction between decidual nk cells and fetal trophoblasts. leandro g oliveira, gendie e lash, judith n bulmer, barbara a innes, roger f searle, stephen c robson. institute of cellular medicine, newcastle university, newcastle upon tyne, tyne and wear, united kingdom. background: we have previously demonstrated that co-culture with extravillous trophoblast cells (evt) (expressing hla-g) alters cytokine secretion by uterine natural killer (unk) cells, particularly at - weeks gestation. we have also reported that unk cells can stimulate evt invasion, but only at - weeks gestation (not at - weeks gestation). in addition, unk cell cytokine profiles alter with increasing gestational age. other reports have suggested that evt or hla-g expressing cells may alter the expression of cytokines and angiogenic growth factors by unk cells. hypothesis: hla-g expressing cells alters unk secretion of cytokines. methods: cd + unk cells were isolated from early pregnancy decidua ( - and - weeks gestation, n= each group) using enzyme digestion and positive immunomagnetic bead separation. the human b lymphoblastoid . transfected with either hla-g ( g) or a mock cdna ( cdna) were obtained as a kind gift from mr r apps (university of cambridge, uk). isolated unk cells were cultured in the presence or absence of the two cell lines in either direct or indirect contact (n= each group and each gestational age) for hours. cell supernatants were analysed for cytokines using a fastquant® th /th multiplex protein assay (il- , il- , il- , il- , il- , tnf-, il- , il- , ifn-) or by standard elisa (tgf- ). the effect of direct co-culture of unk cells with g compared with co-culture with cdna at each gestational age was tested using mann whitney u test. the effect of co-culture of unk cells with g in both direct and indirect contact was also tested using mann whitney u test. results: there was no difference in the level of cytokines secreted by the g or cdna cells. cytokine secretion by unk cells was not altered after direct co-culture with either g or cdna cells at either gestational age. in addition, direct or indirect co-culture of g or cdna with unk did not alter cytokine secretion at either gestional age. conclusions: hla-g does not alter the secretion of cytokines by unk cells from either - or - weeks gestation. other evt or decidua derived factors (including hla-e) may be responsible for the alteration in secretion of cytokines by unks with increasing gestational age. introduction: natural killer (nk) lymphocytes are central to innate immunity and contribute to tissue homeostasis by eliminating altered cells. their nkg d receptor pathway plays a fundamental role in target elimination through binding nkg d ligands on the cell surface. reduction in the nkg d ligand, ulbp , expression is associated with immune resistance in neoplastic processes. we have previously shown that fibroblasts from adhesion tissue (at) are characterized by increased extracellular matrix molecules and inflammatory cytokines compared with normal peritoneal (np) fibroblasts. objective: to determine if there is a difference in nk lymphocyte-mediated elimination between np and at fibroblasts and to investigate potential role of nkg d pathway in this process. material and methods: expression of nkg d ligands; ulbp , ulbp , mica, and micb was evaluated by flow cytometry and western blot in primary cell cultures of fibroblasts from np and at, established from two patients. peripheral blood nk lymphocytes (cd +cd -) from three healthy volunteers were isolated using macs system with purity greater than % and kept in interleukin overnight. fibroblast elimination with and without ulbp blocking was investigated following -hour co-incubation with allogeneic nk lymphocytes using our established flow cytometric cell mediated cytotoxicity assay. paired t test was used in statistical analysis. results: the flow cytometry studies showed that nkg d ligands (ulbp , ulbp , mica and micb) were lower in at compared to np fibroblasts, reaching a statistical significance in ulbp expression (p = . ). western blot analysis also revealed a lower ulbp protein level in at than np fibroblasts. furthermore, nk lymphocyte-mediated elimination was % lower in at in comparison with np fibroblasts. blocking ulbp expression resulted in decreased nk lymphocyte-mediated np fibroblast elimination by %, supporting the role of nkg d receptor pathway in the process. conclusions: our results demonstrate that nkg d pathway is operational in at fibroblast resistance to immune elimination, and extends our prior observations of the potential role of immunological mechanisms in the pathogenesis of adhesion development. objective: galectin- is an anti-inflammatory lectin that has pleiotropic regulatory functions at the crossroad of innate and adaptive immunity. human galectin- is expressed in the placenta and immune privileged sites and it has been implicated in establishing immune tolerance. the aim of this study was to examine the evolution and placental expression of the lgals gene in primates. methods: seven primate nucleotide sequences were generated, aligned to vertebrate orthologs from all classes and subjected to phylogenetic analysis. deduced amino acid sequences were analyzed for functionally important substitutions. placental galectin- expression was studied by immunohistochemistry and western blot. results: ) the lgals gene had high sequence identity among all investigated species. ) phylogenetic analysis revealed that intense purifying selection had been acting on the lgals gene in placental mammals (dn/ds= . ); ) residues responsible for sugar binding or molecule stabilizing were highly conserved in primates. ) immunostaining showed a uniformly abundant and ubiquitous galectin- expression pattern in human, old and new world monkey and prosimian placentas, regardless the type of placentation. the lgals gene has conserved sequence and placental expression pattern in primates that may suggest its important function in maternal-fetal immune interactions. these results support the view that immune interactions at the maternal-fetal interface evolved in concert with invasive placentation and that these interactions have been maintained regardless of the degree of placental invasion in primates and other mammals. expression of interleukin- in human endometrium throughout the menstrual cycle and early pregnancy. yesim h uz, , william murk, umit a kayisli, aydin arici. department of obstetrics, gynecology and reproductive sciences, yale university school of medicine, new haven, ct, usa; department of histology and embryology, trakya university school of medicine, edirne, turkey. background: interleukin- (il- ) is a recently discovered heterodimeric cytokine, comprised by a novel p subunit and a p subunit shared by il- . it has biological activities that are similar to but distinct from il- , and is known to be involved in th /th cell class switching and the regulation of cytokines such as ifn-gamma, il- , tnf-alpha, and il- . early pregnancy is associated with alterations in the maternal immune response, such as changes in cytokine expression, and leukocyte recruitment and subtype switching. we hypothesized that expression of il- in the human endometrium is menstrual cycle-and pregnancy-dependent. materials and methods: endometrial samples from women (n= ) undergoing surgery for benign gynecologic conditions, and decidual tissues from women (n= ) with clinically normal pregnancies terminated voluntarily in the first trimester, were obtained after receiving informed consent. endometrial samples were grouped according to menstrual phase. paraffin sections were stained with il- p antibodies and evaluated semi-quantitatively with hscore. statistical analysis of the data was done using anova, with p< . considered significant. results: il- immunoreactivity was predominantly located in the cytoplasm of both endometrial stromal (esc) and glandular (egc) cells. escs showed mild il- immunoreactivity without significant changes in intensity throughout the menstrual cycle. on the other hand, first trimester decidual cells showed significantly stronger il- staining compared to escs from non-pregnant endometrium (p< . ). il- immunoreactivity in egcs was high in the late proliferative phase, as compared to other cycle phases and first trimester tissues (p< . ). moreover, egcs from the early secretory phase (p< . ) and first trimester tissues (p< . ) showed higher il- immunoreactivity compared to the early proliferative and late secretory phases. conclusions: this is the first study describing il- expression in the human endometrium and decidua. these results suggest that il- has a cycledependent expression in endometrial cells and may be involved in regulating cytokine expression and immune cell modulation during the menstrual cycle and early pregnancy. gercel-taylor, douglas d taylor. obstetrics, gynecology, and women's health, university of louisville, louisville, ky, usa. objective: estrogen appears appear to be a critical regulator of the immune system. since hypoestrogenism is present in the postmenopausal woman, our objective was to determine whether t cell activation and function, defined as il- production and signaling molecule expressions at the transcriptional and translational levels, were affected by a low estrogen environment. design: prospective study in a university research laboratory. materials and methods: jurkat . t cells, initially grown in estrogen free media, were incubated in pm (representing postmenopausal levels) or pm (premenopausal levels) of estradiol (e ) for hours. cells were either resting or activated with a phorbol ester, -phorbol -myristate -acetate (pma), and ionomycin. enzyme-linked immunosorbent spot assay (elispot) was performed to analyze production of il- . expression of signaling protein components, cd and jak, were determined by western immunoblotting. real time-polymerase chain reaction was performed to quantify cd , jak , and jak gene expression. a p value of < . was considered significant. results: jurkat cells exposed to pm e and activated exhibited significantly diminished numbers of il- producing colonies compared to t cells exposed to pm ( . ± . vs. . ± . colonies, p< . ). analysis of cellular cd and jak protein demonstrated that jurkat cells incubated in pm e expressed a . -fold decrease in cd and . -fold decrease in jak compared with cells incubated in pm e (p< . ). these diminished protein levels appeared to be the consequence of suppressed transcription, as the mrna levels of cd , jak and jak were significantly decreased in jurkat cells incubated in low levels of estrogen ( . , . , and . fold, respectively, compared to pm). conclusions: jurkat cells exposed to low postmenopausal estrogen levels produce significantly less il- following activation, which was associated with a significant decrease in signaling proteins. the diminished levels of signaling proteins appear to result from decreased cd , jak and jak gene expression in the presence of low estrogen. these findings support the observation of decreased cellular immune response in postmenopausal women and may provide a basis for the increased risk of infections and cancer proliferation associated with aging. support: dept. of ob/gyn research seed fund. the expression pattern of novel cytokines (il- and ) in human fetal membranes. judith eckardt, , stephen j fortunato, holger maul, ramkumar menon. the perinatal research center, nashville, tn, usa; womens' hospital, university of heidelberg, heidelberg, baden-wuerttemberg, germany. objective: interleukin (il) and are novel cytokines produced by various immunological cells in response to microbial antigens. the functions of these cytokines in reproductive system is unknown. this study examines the expression pattern of il- and il- in human fetal membranes from preterm and term births and in in vitro in normal term membranes in response to bacterial endotoxin (lipopolysaccharide-lps). methods: fetal membranes collected (n= ) from cesareans at term (normal, not in labor) were placed in an organ explant system for hours and were stimulated with lps for an additional hrs. fetal membranes were also collected (n= ) either at preterm or term after vaginal deliveries. in a case -control study (preterm birth vs. normal term deliveries) amniotic fluids (af) (n= ) were collected to document the role of il- and il- in ptb. tissue expressions of il- and il- were studied by rt-pcr using specific primers. elisa documented culture media and af cytokine concentrations. statistical analysis was performed using non-parametric mann-whitney u test. results: both il- and il - expressions were seen in fetal membranes in culture (in vitro) regardless of stimulation. in vivo in membranes from preterm and term deliveries and membranes at term not in labor also documented the expression of these cytokines. culture media analysis documented higher concentration of il- after lps stimulation (lps- . vs. . pg/ml; p= . ) whereas no difference was noticed in il- concentration (lps- . control- . pg/ml; p= . ) between the two groups. af analysis, regardless of the status, did not document detectable concentrations of either of the cytokines (lower limit . pg/ml for both). conclusion: this is the first study to document the expression of two novel cytokines in laboring and non laboring human fetal membranes and also in membranes from preterm deliveries. il- production was stimulated by lps whereas il- was not affected. these cytokines are not physiological components of af and their role in fetal membranes is unclear. higher il- concentration in response to lps but lack of its presence in term or preterm af is suggestive of an autocrine immune response during pregnancy in response to a microbial antigen. evidence for a selective migration of fetus specific cd + cd bright regulatory t cells from the peripheral blood to the decidua in human pregnancy. tamara tilburg, , dave l roelen, barbara j van der mast, godelieve m de groot, carin kleijburg, sicco a scherjon, frans hj claas. department of obstetrics, leiden university medical centre, leiden, netherlands; department of immunohematologie and bloodbank, leiden university medical centre, leiden, netherlands. during pregnancy the maternal immune system has to tolerate the persistence of fetal alloantigens. many mechanisms contribute to the prevention of a destructive immune response mediated by maternal alloreactive lymphocytes directed against the allogeneic fetus. murine studies suggest that cd + cd + t cells provide mechanisms of specific immune tolerance to fetal alloantigens during pregnancy. previous studies by our group demonstrate that a significantly higher percentage of activated t cells and cd + cd bright t cells are present in decidual tissue in comparison with maternal peripheral blood in human pregnancy ( ) . in this study we examined the phenotypic and functional properties of cd + cd bright t cells derived from maternal peripheral blood and decidual tissue. depletion of cd + cd bright t cells from maternal peripheral blood demonstrates regulation to a rd party umbilical cord blood cells comparable to non-pregnant controls, whereas the suppression capacity to umbilical cord blood cells of her own child is absent. furthermore, maternal peripheral blood shows a reduced percentage of cd + cd bright foxp + and cd + cd bright hla-dr + cells compared to peripheral blood of non-pregnant controls. in contrast, decidual lymphocyte isolates contain high percentages of cd + cd bright t cells with a regulatory phenotype that are able to down regulate fetus-specific and non-specific immune responses. these data suggest a preferential recruitment of fetus-specific regulatory t cells from maternal peripheral blood to the fetal-maternal interface where they may contribute to the local regulation of fetus specific responses. ( ) tilburgs t, roelen dl, van der mast bj, van schip jj, kleijburg c, de groot-swings gm, kanhai hh, claas fh, scherjon sa. differential distribution of cd (+) cd (bright) and cd (+) cd (-) t-cells in decidua and maternal blood during human pregnancy. placenta. apr; suppl a:s - . alicia del toro-arreola, lourdes nunez-atahualpa, juan velazquez-rodriguez, laura gonzalez-lopez, jorge i gamez-nava, adrian daneri-navarro. there is evidence that the maternal immune system is influence by changes in the hormonal levels during the menstrual cycle (mc). so far, the information related to the levels of t, treg, nk cells and receptors of activation and inhibitors is scarce. aim: to analyze the populations of t, treg, nk cells and their receptors of peripheral blood of healthy women and their correlation with hormones during mc. material and methods: we studied to women not using hormonal contraceptives in the day th of the follicular phase and st of the luteal phase of the mc. pbmc subsets and their receptors were determined by flow cytometry and hormone levels by chemiluminescence method. we found that the progesterone and prolactin were positive correlated (rho= . , p< . and rho= . , p< . , respectively) with cd /nkg in t cells and negative correlated (rho= - . , p< . and rho= - . , p< . , respectively) with nk cells. meanwhile cortisol was positive correlated (rho= . , p< . ) with the receptor nkg d expressed in nk cells. the results observed in this study in the luteal phase of mc on the expression of cd /nkg inhibitor receptor and nkg d activator receptor were related to a particular hormone (progesterone, prolactin and cortisol) might contribute to understanding the physiological role of the neuroendocrine axis on the expression of some receptors of the immune system in order to keep the homeostasis milieu of the mc. objective: sp-d, a key component of the innate immune system, is detected in amniotic fluid (af) and believed to originate in the fetal lung. however, sp-d is produced by other cells and therefore extra-pulmonary sources must be considered. the objective of this study was to determine the maternal and fetal plasma and af concentrations of sp-d to gain insight into the behavior of this natural antimicrobial peptide in pregnancy. moreover, we studied sp-d expression in maternal and fetal peripheral leukocytes. methods: maternal and fetal plasma and af samples were obtained from patients in the following groups: ) term not in labor (tnl; n= ); ) term in labor (til; n= ); ) preterm labor without histologic chorioamnionitis (ptl; n= ) and ) preterm labor with histologic chorioamnionitis (ptl-hc; n= ). sp-d concentration was measured by elisa. sp-d mrna expression in maternal and fetal leukocytes was evaluated by real-time qrt-pcr. flow cytometry and confocal microscopy were used to study the localization of sp-d in leukocytes. results: ) the af sp-d concentration increased as a function of gestational age (mean, til: , . ng/ml vs. ptl: , . ng/ml; p< . ); ) in contrast, the maternal and fetal plasma sp-d concentrations decreased with advancing gestational age (mean, til: . ng/ml vs. ptl: . ng/ml; p< . , and til: . ng/ml vs. ptl: . ng/ml; p< . , respectively); ) the maternal plasma sp-d concentration was lower than that of fetal plasma (mean: . ng/ml vs. . ng/ml; p< . ); ) however, sp-d mrna expression in maternal leukocytes was . fold higher than that of fetal leukocytes (p< . ); ) neutrophils (both maternal and fetal) expressed sp-d as demonstrated by flow cytometry and confocal microscopy. conclusion: ) the concentrations of sp-d in the maternal and fetal circulation decreased with gestational age while the af concentration increased; ) the expression of sp-d mrna is higher in maternal leukocytes than in fetal leukocytes; ) we report for the first time that maternal and fetal neutrophils are a source of sp-d and propose that this molecule plays a role in host defense against infection and in the modulation of the maternal and fetal immune response. introduction intrauterine insemination (iui) is a fertility technique that allows for couples to have intercourse after the procedure is performed. it has been postulated that intercourse after iui may increase the pregnancy rate by either endometrial stimulation or because it may represent a second spermatic flow in the periovulatory period. in the present study we evaluate the effect of intercourse on the pregnancy rate of patients undergoing iui. material and methods: from to patients were enrolled in the study. every couple undergoing iui was instructed at the moment of insemination to decide whether to have or not intercourse on the same day of the procedure. all couples were abstinent three to seven days before iui. the information regarding intercourse was recorded the day after treatment. ovulatory, insulin resistant, cervical, male, tubal and endometrial factors as well as parity and time of infertility were compared between the two groups. all these factors were analyzed based on number of follicles that ovulated in each group. our principal outcome was to determine the pregnancy rate. intercourse was practiced by . % of the couples. the global pregnancy rate was . %. the pregnancy rate for the couples who had intercourse was . % and . % for those who did not have intercourse (p< . ). even though age, parity, time of infertility and stimulation protocols were similarly distributed in both groups, the proportion of tubal and endometrial factors were higher among those who had intercourse (p< . ). when subjects with tubal and endometrial factors were excluded, the pregnancy rate between both groups (n= ) was similar ( . % vs . % for positive and negative intercourse, respectively). the average number of ovulatory follicles was . + . for the group that had intercourse and . + . for those who did not. according to our results, intercourse after iui does not improve pregnancy rate after this procedure is performed. furthermore our study indicates that iui does not interfere with sexual intimacy since almost % of the couples decided to have intercourse on the same day of the procedure. . we sought to investigate the effects of gravidity on mmc and fmc in healthy, parous women. methods: mc was assayed in dna extracted from peripheral blood mononuclear cells (pbmc). hla-genotyping was first conducted and mc quantified employing a q-pcr assay targeting a non-shared maternal-or fetalspecific hla polymorphism. gravidity was dichotomized as a history of one pregnancy compared with two or more, and the prevalence of mc was analyzed using logistic regression. possible confounders were included as appropriate, including subject age and time since last pregnancy. adjustment for possible correlation between values was also made when there were repeated measures for the same subject. results: for the mmc analysis, there were subjects with observations. for the fmc analysis, there were subjects with observations. table provides a summary of mmc and fmc by gravidity. mmc was significantly decreased with higher gravidity. fmc was not affected by gravidity. gravida gravida or more adjusted* or ( %ci) mmc / ( %) / ( %) . ( . - . ) fmc / ( %) / ( %) . ( . - . ) *adjusted for possible correlation between values within a subject, subject age, and time from last pregnancy, as appropriate. increasing gravidity is significantly associated with a decreased prevalence of mmc. despite additional sources of fmc, there does not appear to be an increase in fmc prevalence with increasing gravidity. the biology of mc is incompletely understood, and the nature of mmc and fmc are likely to be different given that acquisition of the former, but not the latter, occurs within a nascent immune system. these data raise interesting questions when considered as interactions of acquired grafts within a host, including whether emergence and persistence of one dominant source of mc may be most advantageous for an individual. anti-igd antibody treatment as a novel immunosuppressive agent for autoimmune diseases and its effects on th /th gene expression. tommie g nguyen, eileen d gallery, , jonathan m morris. elevated t-helper cell type- (th ) and type- (th ) cytokine expression have a role in autoimmune diseases, allograft rejection and pregnancy-related complications. thus, molecules that can shift the immunity away from th /th responses toward a th response represent a novel therapeutic treatment for these conditions. we have previously demonstrated that pregnancy is associated with a suppression of t-bet in peripheral t cells. in this study, we examined a novel effect of anti-igd antibody on t-bet expression, th /th gene expression in human peripheral blood mononuclear cells (pbmc) and its therapeutic effects in an animal model of collagen-induced arthritis. methods: human pbmc were isolated and then cultured in the presence of anti-igd antibody at various time points followed by stimulation with pma/ionomycin (p/i) for hrs. gene expression was examined by rt-pcr, western blotting and elisa. for in vivo study, arthritis-prone dba/j mice were induced to undergo joint inflammation by intradermal injections with bovine type-ii collagen. these mice were then given daily doses of mg/kg of intravenous injection with anti-igd antibodies as preventive or therapeutic treatments (n = per group). results: treatment with anti-igd antibodies significantly suppressed p/iinduced expression of t-bet (a master regulator of th immunity), tnf-(a classical pro-inflammatory th cytokine), and il- (a critical proinflammatory th cytokine) in human pbmc. this suppression is highly specific to these genes because anti-igd antibodies have no effects on the expression of ifn-g and il- (two classical th cytokines). in vivo experiment showed that anti-igd antibody treatment markedly reduced clinical severity of joint inflammation when comparing the clinical score of control mice group ( . ± . , mean ± s.e.m), preventive group ( . ± . ) and therapeutic group ( . ± . ) . conclusions: our study has demonstrated that suppression of t-bet by anti-igd antibodies, similar to the changes seen in human pregnancy is a novel in vivo anti-inflammatory effect. given the essential role of t-bet, tnf-and il- in the pathogenesis of human autoimmune diseases, anti-igd antibodies may represent a novel immunosuppressive treatment that needs further studies and evaluation. objective: women with circulating anti-phospholipid antibodies (apl) are at risk for recurrent miscarriage, preeclampsia and preterm labor. apl antibodies directly target the placenta by binding to phospholipids or phospholipid-binding proteins expressed on the surface of viable trophoblasts. the objective of this study was to determine the effects of apl antibodies on first trimester trophoblast cells. methods: two mouse igg anti-human beta -glycoprotein i monoclonal antibodies (mabs), designated id and iic , were used in these studies. the first trimester trophoblast cell line, htr , was incubated with either medium, a mouse igg control, id or iic ( - g/ml), in the presence or absence of unfractionated heparin ( ng/ml). trophoblast cell death and apoptosis was determined using a viability assay, hoechst staining and a caspase activity assay. cytokine production was evaluated by multiplex analysis. results: following a hour incubation, significant trophoblast cell death was induced by iic ( . ± . %) and id ( . ± . %) at the high dose of g/ml, when compared to the medium and mouse igg controls (p< . ). hoechst staining showed that id -and iic -induced trophoblast cell death was a result of apoptosis. moreover, id and iic induced a significant increase in caspase- , - and - activity (p< . ). treatment of trophoblasts with heparin significantly inhibited the effects of iic and id on cell death by . ± . % and . ± . % %, respectively (p< . ). following a hour incubation at lower concentrations ( g/ml), treatment of trophoblast cells with id or iic resulted in a significant upregulation of il- , mcp- , gro production (p< . ), and a significant reduction in il- secretion (p< . ). conclusion: this study demonstrates that at low levels apl antibodies can modulate trophoblast cytokine production, while at higher levels, the same antibodies induce trophoblast apoptosis in a caspase-dependent manner. these findings shed new light on the mechanisms by which apl antibodies may impact placental survival and function. antigenic targets for the diagnosis of premature ovarian failure. hc bohler, c gercel-taylor, lt ku, st nakajima, dd taylor. obstetrics, gynecology, women's health, university of louisville, louisville, ky, usa. objective: premature ovarian failure (pof) is a premature depletion of ovarian follicles before the age of , affecting approximately % of women < years. the involvement of autoimmune mechanisms in pof has been suggested and similar mechanisms have been postulated for other ovarian pathologies, including idiopathic infertility, polycystic ovary syndrome (pcos), or endometriosis. while the association of autoantibodies has been demonstrated for these ovarian pathologies, variation in specificity and frequency of false positivity have limited the diagnostic use of autoantibodies. the objective of this study was to develop an antigen array to differentiate antibody recognition patterns of pof from other infertility pathologies. design: prospective study in a university research laboratory. materials and methods: patients diagnosed with infertility were included in this pilot study: endometriosis (n= ), pcos (n= ) and pof (n= ). autoantibodies were assayed by dot immunoblotting using an antigen array derived from endometrial and ovarian cells. for the cellular antigen preparations, solubilized total proteins were separated by reverse phase-hplcquid chromatography and the individual proteins were blotted onto nitrocellulose membranes and reactivity visualized by peroxidase-labeled antihuman igg. results: patients with pof, endometriosis, and pcos all exhibited autoantibodies reactive with these cellular proteins. while some antigenic reactivities were shared by all infertility patients, the pattern of antigen recognition was distinct for patients with pof. patients with pof all recognized a common antigenic proteins (row , antigens a,b,d-g). conclusions: alterations in autoreactivity are observed in patients with the diagnosis of infertility; however, distinct patterns of autoantibody recognition can be demonstrated for patients with different pathologies. while this study needs to be expanded to reliably establish the specificity, sensitivity and positive and negative predictive values, patients with pof clearly exhibit a shared recognition pattern that may be useful a diagnostic marker. cicek gercel-taylor. ob/gyn, university of louisville, louisville, ky, usa. objective: americans' consumption of nutraceuticals is one of the most rapidly expanding health markets, growing at a rate of % annually. multiple nutraceuticals containing phytoestrogens have been marketed as "immune boosters" despite suboptimal evidence-based medicine to support such statements. as immunomodulatory therapies should affect downstream cytokine expression, the relative effects of estradiol and genistein in regulating expression of cd -and jak were tested. these markers were chosen since they are central to t cell signaling. cd -is a critical transducer of tcr activation and regulates t cell proliferation and cytokine production. jak upregulation is a specific marker for hematopoietic cell stimulation. methods: to test the immunomodulatory effects of phytoestrogens and estrogen, jurkat . (t cell leukemia) cells were grown in estrogen-free, phenol red-free media for hours. these cells were then exposed for hours to pm, pm (postmenopausal), or pm (premenopausual) of estradiol in the presence of increasing concentrations of genistein ( , . , . , . , , and m). cells were then solubilized and cellular protein quantitated. protein concentrations were standardized and western blots for each set of culturing conditions were run in triplicate. cd -and jak expression were quantitated following visualization with chemiluminescence by digital pixel quantification. results: our findings show that in the absence of estradiol and at postmenopausal levels of estradiol, genistein induced a dose dependent increase in cd -reaching a maximum of fold. although cultivation of t cells in pm of estradiol significantly increased the levels of cd -and jak relative to hypoestrogenic conditions, the genistein mediated dose response was not observed. conclusions: these in vitro results indicate that genistein can at least partially reverse suppression of signaling molecules observed in postmenopausal estrogen environments. clinically, this suggests that phytoestrogens may have greater immunomodulatory properties for postmenopausal females than those that are premenopausal. maternal serum il- as a biomarker of acute immunologic rejection of pregnancy. joaquin santolaya-forgas, juan deleon-luis, isabel galan. obstetrics and gynecology, brigham and women's hospital, boston, ma, usa; amarillo women's health research institute, texas tech university health science center, amarillo, tx, usa. objective: markers of acute rejection of pregnancy are very scarce. in this study we aimed at determining if rapid changes in maternal serum concetration of a variety of biomarkers could be used for this purpose. we used an established baboon model for in utero stem cell therapy to introduce at - days from conception and via ultrasound-guided celocentesis, human hematopoietic stem cells with different proportions of natural killer t-cells (nk). maternal blood was collected before and hours after celocentesis for quantification of hormones and il- using solid phase, enzyme labeled, chemiluminescent sequential immunometric assays. pearson correlation analysis was used for determination of significant changes from baseline (p< . ). results: all animals survived their pregnancies. seven animals receiving < % concentration of nk delivered at term ( days gestation) while animal receiving more than % concentration of nk had dead fetuses on ultrasound evaluation hours after celocentesis. table depicts mean maternal serum concentration of the biomarkers investigated (all n.s.). figure shows mean il- changes from baseline in continuing (n.s.) and rejected pregnancies (p< . ). conclusions: we have described a model in which in utero graft vs host disease can be studied. these preliminary results suggest that of all the biomarkers investigated, il- might be the most sensitive for detection of an acute rejection of pregnancy. biomarkers of acute immunologic rejection of pregnancy biomarker unit pre-celocentesis ( ) the activity of cytotoxic cd + t cells during pregnancy protects the mother and fetus from infection. however, pregnancy's effect on the proliferation and apoptosis of cd + t cells has not been clearly defined. objective: to determine if normal pregnancy changes the number of proliferating or apoptotic splenic cd +t cells. methods: female c bl/ mice were used unmated (um) or underwent timed mating. one day prior to harvest, mice were i.p. injected with bromodeoxyuridine (brdu), which is incorporated into replicating dna. harvested spleens were homogenized, enumerated, and stained for cell surface expression of cd and t cell receptor beta chain (tcr ). apoptotic cells were detected by treatment with terminal transferase and fitc-dutp (tunel). the numbers of cd +tcr + cells that were brdu+ or tunel+ were calculated from the percentage of positive cells obtained by flow cytometry and the absolute number of cells counted. for each experiment, the ratio of the number of positive cells in pregnant to um mice was compared by anova with dunnett's post-test. results: at day of pregnancy (n= ), the number of brdu positive cd + t cells was two fold higher than that found in um (n= , p< . ). the number of proliferating cd + t cells continued to be non-significantly elevated at day ( . x, n= ), day ( . x, n= ), and day of pregnancy ( . x, n= ) compared to um. by day of pregnancy (n= ) the number of proliferating cd + t cells returned to the um level, however by this time the total number of splenic cd + t cells was . fold higher than um (n= p< . ). on gestational day , the number of proliferating cd + t cells declined further ( . x, n= ), and the number of splenic cd +t cells returned to the um level (n= , p> . ). compared to um mice, there was no significant difference in the number of cd + t cells undergoing apoptosis at any gestational day examined ( . - . x, p> . ). in normal murine pregnancy, the number of cd + t cells is increased in late gestation, and then returns to baseline at the end of pregnancy. this is due to an early increase then gradual decline in cd + t cell proliferation, accompanied by a steady rate of apoptosis. this argues that the maternal immune system undergoes dynamic homeostatic changes, and is not globally suppressed. supported by nihro hd - niht ai . arturo cerbulo-vazquez, cun li, , gene hubbard, natalia e schlabritz-loutsevitch, , peter w nathanielsz. objectives: early thymocyte (t) maturation occurs in the cortex while later stages occur in the medulla. thymic epithelial cells (tec) synthesize gc and t express gr. tec may influence t cell maturation by regulating apoptosisinduced gc-gr interactions. igf- (also synthesized by tec) may support thymocyte prolifetarion. human fetuses of mothers in premature labor are exposed to gc. gc administered to pregnant baboons at . , . , and . gestation (g) alters fetal lymphocyte populations at . (g) (j repro immunol, , : ) . we determined if fetal gc exposure alters thymic ) structure; ) gr and igf-i protein. methods: pregnant baboons received saline (control ctr; n= ) or betamethasone i.m. ( μg/kg daily for two days at . , . and . g; gc group; n= ), c-sectioned at at . g under general anesthesia and thymic gr and igf-i evaluated by immunohistochemistry. results: gr localized to medulla and a few cortical cells. igf-i localized to cortex with little medullary expression. medullary necrosis was greater in ctr than gc fetuses. t gr was located in cytoplasm. no gross differences were observed between ctr or gc fetuses for either igf-i or gr. conclusions: a) early thymocyte maturation may be supported by igf- , b) later differentiation involves gr, and c) after exposure to gc doses equivalent to human therapy, no gross effects were detected on gr or igf, but d) natural thymic necrosis was inhibited. lindsay s christensen, peyman bizargity, elizabeth a bonney. ob/gyn, university of vermont, burlington, vt, usa. background: the exact mechanism by which bacterial products trigger preterm delivery and the immune cellular circuits involved remain unclear. our recent data in normal c bl/ (b ) or recombinase deficient c bl/ mice (rag-ko) indicates that t and b cells are not critical for lps-induced preterm delivery and stresses the importance of related innate mechanisms. rag-ko mice are more susceptible to lps, suggesting that t or b cells may control the innate response. macrophages are vital to innate immunity and produce proinflammatory cytokines that can activate prostaglandin synthesis and myometrial contraction. we questioned whether differences in susceptibility between the strains are due to differences in the uterine macrophage response to lps and thus examined macrophages at the maternal-fetal interface early after injection. objective: to compare uterine macrophages levels at and hours after lps injection in pregnant b and rag-ko mice. methods: b and rag-ko mice were mated and on gestation day , females were injected intraperitoneally with μg lps in l saline (pbs) or l pbs alone. euthanasia and uterine harvest occurred or hours after injection. frozen uterine sections were stained with the macrophage marker f / or an isotype-matched control followed by an alexafluor -conjugated secondary and a nuclear stain (dapi). sections were visualized by fluorescence microscopy. for each mouse, f / + dapi+ and total dapi + cells were counted in areas of representative section and the percentage of f / + dapi+ cells was calculated. the mean percentage for at least representative areas per experimental group was analyzed by anova. results: two hours post-injection, macrophages levels were similar in b and rag-ko mice injected with pbs (b , n= , . ± . ; rag-ko, n= , . ± . % + per area). lps injection increased macrophages (p< . ) in both strains (b , n= , . ± . ; rag-ko, n= , . ± . ,); no difference was evident between strains. the percentage of f / + cells was similar hours post-injection (b , n= , . ± . v. rag-ko, n= , . ± . ), and not elevated relative to the hour time point. objective. decay-accelerating factor (cd ), is expressed in the plasma membranes and protects mammalian cells against the lytic action of serum complement. phosphoinositide -kinases (pi ks) are involved in the regulation of cell functions by synthesizing a second messenger molecule ptdins ( , , ) p . akt, a serine-threonine kinase acts downstream of pi k and regulates cell survival, growth and proliferation. the pi k-akt activity is controlled by tumor suppressor gene pten. in this study we assessed whether the pi k-akt activity affects the expression of cd in human endometrial and cervical cells. methods. endometrial and cervical cell lines which differ in the constitutive pi k activity were used in this study. ishikawa and rl - endometrial cell lines harbor pten mutation and have high levels of phosphorylated akt (p-akt). hec- -a and kle endometrial cell lines and hela cells express wild-type pten and have minimal or no demonstrable levels of p-akt. the expression of cd was evaluated by rt-pcr, immunoblotting and flow cytometry. the pi k activity was assessed by immunoblotting with anti-p-akt antibodies. the effect of inhibition of pi k-akt pathway on cd expression was evaluated in cells treated with wortmannin ( nm), ly ( mm), or with akt inhibitor sh ( mm). results. immunoblotting densitometry and measurements of mean fluorescence intensities showed that the level of cd expression correlates with the status of pi k-akt pathway. the cd expression was lowest in hec- -a, ishikawa and rl - cells which constitutively express p-akt. higher cd expression was found in hela cells and kle cells which express wild-type pten product and has no detectable phospho-akt. mean fluorescence intensities were . -fold higher for kle cells and -fold higher for hela cells compared to hec- -a cells. treatment of cells with akt inhibitor led to . - . -fold increase in cd expression. the . - . -fold increase following treatment with pi k inhibitor wortmannin was found in ishikawa cells, rl - and kle cells. conclusions. human endometrial cell lines with elevated pi k-akt activity express lower level of cd compared to cell lines with intact pten gene function. these findings may indicate that structural alteration at the dna level and resultant overexpression of pi k-akt pathway are involved in the downregulation of cd . endometrial and cervical cells. pawel goluszko, chandra yallampalli. obstetrics gynecology, university of texas medical branch, galveston, tx, usa. objective. cell shape is determined by the cytoskeleton, which provides the mechanical support and is involved in cellular signaling. apoptotic cells undergo major morphological changes such as rounding and contraction, a process regulated by caspases, the cysteine proteases responsible for events controlling the cell disassembly. the motifs in certain cytokeratins make them substrates for caspase degradation. anti-apoptotic serine/threonine kinase akt provides a survival signal by phosphorylating downstream effector molecules including caspase- . while studying the akt distribution in human endometrial cell line we found that akt shows filamentous pattern of staining resembling cytoskeleton organization. in this study we evaluated whether akt staining correlates with the microfilaments (mf), microtubules (mt) or intermediate filaments. endometrial ishikawa, rl - , hec- -a, kle and cervical hela cell lines were used in the study. incubation with cytochalasin d, ( ug/ml) or nocodazole ( mg/ml) and labeling with bodipy fl phallacidin, anti -tubulin or anti-akt antibody were used to assess whether cytoskeleton disruptors affect akt distribution and mf and mt organization. for colocalization, cells were stained with anti-cytokeratin mouse antibody followed by anti-mouse alexa conjugate, and then stained with anti-akt rabbit antibody and anti-rabbit alexa conjugate. the scans collected with laser scanning confocal microscope from channels filtered for alexa and alexa were combined digitally and evaluated with imaris colocalization analysis software. filamentous pattern of akt staining was most pronounced in ishikawa and less obvious in hec- -a cells. treatment with cytochalasin d or nocodazole resulted in disruption of mf and mt but had no effect on cytokeratin organization and akt distribution. double staining with anti-cytokeratin- and anti-akt antibody showed overlapping staining for cytokeratin and akt. analysis of digitally acquired images showed highest correlation for colocalized channels in rl - cells ( . ) followed by ishikawa ( . ) and hela cells ( . ). lowest correlation was found for kle ( . ) and hec- -a cells ( . ) conclusions. this study indicates a strong colocalization pattern of serine/ threonine kinase akt with cytokeratins, and suggests a mechanism by which cytokeratins might be protected against cleavage by caspase- and caspase- in the early apoptotic stages. background: cd + cd + t regulatory cells (t-reg), express foxp ,suppress antigen-specific immune responses and are important for allograft tolerance. during pregnancy the mother tolerates an allograft expressing paternal antigens (the fetus), requiring substantial changes in immune regulation over a programmed period of time. the presence of t-reg cells (cd + cd highfoxp +) was assessed in the peripheral venous blood of non-pregnant, pregnant and seven postnatal healthy women by flow cytometry. human decidua was obtained by surgical termination of pregnancy in the first (n= ), second (n= ) and third (n= ) trimester of human pregnancy. paraffin sections were immunostained for foxp and cd . foxp +cells were quantified in x fields and results compared between first, second and third trimester samples and according to the presence of extravillous trophoblast. results: fluorometric studies of blood samples indicate an increase % of circulating cd + cd highfoxp t-cells in pregnant ( . % [range . - . %]) vs. non-pregnant controls ( . % [range . - . %]; p< . ). a progression from st, nd and rd trimesters indicated the % of cd + cd highfoxp t-cells was . %, . % and . %, respectively. low numbers of foxp + cells were detected in all decidua samples and their distribution mirrored that of cd + cells. in st trimester samples, foxp + cells were often detected in lymphoid aggregates adjacent to endometrial glands. increased numbers of foxp + cells were detected in st ( . ± . ) compared with nd trimester decidua ( . ± . ; p< . ) but there was no difference between st and rd trimester ( . ± . ), nor between nd and rd trimester decidua. in st trimester decidua, numbers of foxp + cells were increased in areas without extravillous trophoblast. conclusion: normal human pregnancy is associated with an increase in the number of circulating cd + cd highfoxp t-cells. the presence of foxp + cells in early gestation human decidua may be important in the initiation of materno-fetal tolerance at an autocrine level. (supported by mrc). aims: -defensins are small cationic peptides with antibiotic and antimicotic activity. hyaluronan and its degradation products have been described as endogenous ligands for tlr and tlr , whose involvement in -defensin expression has been reported in different epithelial tissues and cell lines. we aim to investigate weather low molecular weight hyaluronic acid induces -defensin release by keratinocytes, via tlr and tlr . methods: the induction of -defensin production following in vitro treatment of human keratinocytes with a low molecular weight hyaluronic acid solution was evaluated by pcr-analysis and elisa techniques. studies on the involvement of tlr and tlr in -defensin production have been performed using specific blocking antibodies. results: pcr and elisa revealed an intense -defensin production following hyaluronic acid treatment in human keratinocytes. the -defensin production induced by hyaluronan was abolished following block of tlr and tlr by specific antibodies, demonstrating the involvement of these receptors. the same hyaluronic acid treatment did not induce activation of inflammatory genes, such as il- , tnf-, il- and il- . conclusion: our data show that hyaluronic acid is an efficient inducer ofdefensin production in keratinocytes, via tlr and tlr . this observation might be important to open new perspectives in the development of possible topical products containing hyaluronic acid, to improve the release ofdefensins by keratinocytes, ameliorating the self-defence of the skin in case of skin infections. therefore, one of the possible applications for this kind of topical products might be the treatment of infective vulvitis, one of the most distressing gynaecological diseases for adult women. pregnancy outcome? kiera von besser, serena wu, mary d stephenson. , obstetrics and gynecology, university of chicago, chicago, il, usa; obstetrics and gynaecology, university of british columbia, vancouver, bc, canada. objective: to investigate whether gender of an ongoing pregnancy impacts the probability of a successful outcome, and, to ascertain whether the gender of prior live birth(s) impacts subsequent pregnancy outcome, among women with rm/aps. materials and method: cohort-control study. rm subjects were evaluated by mds between - (stephenson, . couples who met aps criteria (wilson et al, ) , restricted to rm only, were followed prospectively. cohort data was compared to live birth data from the vital statistics agency of british columbia from - . secondary sex ratios (ssrs) among successful pregnancy outcomes were calculated by dividing the number of male live births by female. sex ratios were calculated for all pregnancies weeks, regardless if they ended in success or demise. pearsons test with yates continuity correction was applied. results: subjects were identified. subjects had prior live births of known gender ( male/ female), giving a ssr of . . there were also prior fetal demises wks of known gender ( / ) giving a sex ratio for all prior pregnancies at wks of . . subjects delivered subsequent live births of known gender ( / ), giving a ssr of . . there were also subsequent fetal demises ( / ) giving a sex ratio for all subsequent pregnancies of . . subjects delivered both prior and subsequent live births. the ssr was . ( / ) among their prior and . ( / ) among their subsequent live births. including fetal demises, subjects had ongoing prior and subsequent pregnancies. the sex ratio was . among their prior pregnancies and . among their subsequent. as the control, a ssr of . ( , / , ) was calculated from vital statistics data. when the prior and subsequent ssrs of the cohort were compared to each other, as well as to the control, there were no statistically significant differences. conclusions: our findings, from the largest study of its kind to date, suggest that, in patients with rm/aps, the gender of an ongoing pregnancy does not significantly affect the probability of a successful outcome, to any greater degree than it does in the general population. also, the gender of a prior ongoing pregnancy does not significantly impact the likelihood of developing rm/aps. oocyte maturation. jk friend, fb bezirci, e seli. ob gyn, yale u., new haven, ct, usa. introduction: oocyte maturation is associated with repression of transcription. during oocyte maturation, fertilization, and early embryo development until the onset of zygotic gene expression, proteins are synthesized from maternallyderived mrnas. the regulation of protein expression from these maternal mrnas is post-transcriptional, and occurs mainly via poly(a)-tail elongation. the embryonic poly(a)-binding protein (epab) is the predominant poly(a)binding protein before the activation of the zygotic genome, and plays a critical role in the activation of certain maternal mrnas, those bound by cpeb and probably pumilio. we are characterizing additional functions of epab during the process of oocyte maturation. methods and results: our model system is the xenopus laevis oocyte where oocyte maturation is induced by the addition of progesterone. our preliminary findings indicate that epab is phosphorylated, and that levels of phosphorylated epab increase upon progesterone-induced oocyte maturation. moreover, glycerol gradient centrifugation revealed that nonphosphorylated and phosphorylated epab are contained in distinct complexes that change mobility upon oocyte maturation. furthermore, oligo-dt selection for poly(a)-containing mrnas strongly suggests that these mrnas are bound exclusively by phosphorylated epab. using affinity purification, we have determined that nonphosphorylated epab exists primarily in a large protein complex prior to oocyte maturation that is later disassembled after the addition of progesterone. conclusions: based on these preliminary findings, we conclude that prior to oocyte maturation, the bulk of epab is nonphosphorylated and is found in a protein-rich complex separate from poly(a)-containing mrnas. upon oocyte maturation (when certain maternally-derived mrnas are activated for translation), the majority of epab becomes phosphorylated, and this phosphorylated form of epab is likely bound to translationally-active mrnas. we are currently investigating what kinase phosphorylates epab and whether this phosphorylation plays a role in translational up-regulation of epab-bound mrnas. introduction: reactive oxygen species (ros) play important roles in all aspects of cellular fate. nadph oxidase isoforms, a family of genetically preserved enzyme complexes, have been shown to be the main sources of ros in various cell types. however, the role of nadph oxidase isoforms in human myometrium proliferation and differentiation has not been defined. in the myometrium, different smooth muscle phenotypes maybe associated with specific physiologic functions. we have shown that angiotensin ii (angii) stimulates hypertrophy but not cell proliferation in ultr cells, an in vitro model of human myometrium. ultr cells at greater than passages display a replicative senescent phenotype. by introducing human telomerase reverse transcriptase (htert) gene, we have obtained a stable cell line (ultr-ht) which has a significantly increased division rate and distinct cellular morphology than the original ultr cells. objective: to determine the relationship of expression of nadph oxidase to ultr cellular fate. methods: early and late passages (p - ) of ultr and ultr-ht (p - ) cells were grown on either plastic or collagen iv (cn )-coated surfaces. ultr-ht cells were further stimulated with angii ( . um) for hrs. expression of nadph oxidase core (nox - and duox / ) and associated subunits (p phox, p phox, noxo , p phox, noxa , p phox, and rac / ), and angii receptors at / was identified by rt-pcr from cellular total rna. fluorescent immunohistochemistry (ihc) was employed to determine protein expression and localization. results: the mrna level of house keeping gene -actin was unchanged by any cellular manipulation. the senescent phenotype of ultr cells was accompanied by an apparent down-regulation of nox , p phox, and noxa genes, and an up-regulation of at / . overexpression of htert did not reverse nox , p phox and noxa expression while cell division rate was increased. however, there was a down-regulation of nox , at / and rac . plating ultr-ht cells on cn induced nox / down-regulation and up-regulation of duox / , with no apparent change of at / . however, exposure to cn re-directed cellular response to angii such that only nox was induced by angii stimulation. conclusion: expression of nadph oxidase isoforms nox , , , and duox / are correlated with ultr cell differentiation and cell fate control. data also suggests that ang ii-induced myometrial hypertrophy involves nox mediated ros generation. fluids from reproductive women -the influence of aging. eriko y fujii, masahiro nakayama. women's health, national center for child health and development, tokyo, japan; aska reproductive clinic, nara, japan. [introduction] receptor for advanced glycation end products (rage) is a multiligand type glycoprotein, and is characterized based on its ability to bind ages, adducts formed by non-enzymatic glycation and oxidation of protein and lipids. this process occurs during normal course of aging. ages/rage interaction regulates various physiological function, such as inflammation, angiogenesis through vegf inducement. a soluble form of rage (srage) works as a decoy in the body and inhibits intracellular signaling. [objectives] the balance of these factors may contribute to reproductive dysfunction by aging. we aimed to measure the ages, srage and vegf concentrations in plasma and follicular fluids from reproductive women, and examined the differences of those factors between young group and old group. [material and methods] patients' plasma and follicular fluid were collected with consent based on regulations of the ethical committee, and we measured ages (pentosidine, cml), srage and vegf in duplicate using commercially available elisa kits (fushimi co, r d and cyelex). concentrations were calculated from each standard curve, and compared between the young group under years old, and the old group over y.o. data were evaluated for the difference in two groups by student's t test , and the significance was determined by p< . . [results] ) srage in plasma, ± pg/ml (mean±sd), n= in the young group was significantly higher than ± pg/ml, n= in the old group. there was no significant difference in plasma vegf. ) vegf in follicular fluid was ± pg /mg protein, n= in the young, and ± pg /mg protein, n= in the old was increased significantly. ) we could not see statistical difference of pentosidine nor cml concentrations between two groups in plasma and follicular fluid samples. [conclusions] it has been reported that higher concentration of vegf in follicular fluid may relate to worse pregnancy rate in art. there was a significant decrease of plasma srage in older group in our result, and because of this decrease of 'decoy', focal ages-rage-vegf signaling might be activated in older women. our results showed the possibility that ages/rage and vegf regulation may contribute to the reproductive dysfunction by aging. and leiomyosarcoma cells. qun pan, xiaoping luo, nasser chegini. ob/ gyn, university of florida, gainesville, fl, usa. as a part of a novel endogenous rna silencing machinery, a noncoding short rna strand referred to as "microrna" (mirna) has been identified to regulate the stability of the target gene expression through mrna degradation and repression. we have identified the expression of many of these mirnas in leiomyoma, myometrium, their isolated smooth muscle cells (lsmc and msmc), transformed lsmc (t-lsmc) and sklm (leiomyosarcoma cell line), including mir- which is predicted to target the expression of many genes, including tgf-b and tgf-b type ii receptor (tgf-brii). however, the biological significance of these mirnas in various cellular processes remains to be established. as such in the present study we examined the expression, regulation and function of mir- in lsmc, msmc, t-lsmc and sklm. we found that mir- is expressed and regulated by b-estridiol and medroxyprogesterone acetate ( - m) in these cells (p< . ). we further assessed the regulatory function of gain of and loss of function of mir- on the expression of tgf-brii. transfection of lsmc, msmc, t-lsmc and sklm with pre-mir-and anti-mir- oligonueclotides resulted in a significant increase and/or inhibition of mir- expression in these cells, respectively as determined by realtime pcr (p< . ). over-expression of mir- resulted in a significant reduction, while transfection with antimir- increased the expression of tgfbrii mrna and protein in these cells as compared to controls (p< . ). we concluded that mir- is expressed in leiomyoma and myometrial cells, its expression is regulated by the ovarian steroids and it functions by targeting the expression of tgfbrii and possibly other genes with key regulatory action on cell growth, angiogenesis, transcription regulation, ecm turnover and apoptosis that results in leiomyomas growth and regression. (supported by nih grant hd ). objective: placenta and a number of gestational tissues are well recognized to express corticotrophin releasing factor (crf), urocortin (ucn ), ucn , ucn and crf -r and crf-r receptor subtypes together with crfbinding proteins locally. ucn and ucn are implicated in the reversal of stress responses initiated by crf. in the present investigation, we evaluated functions of crf and ucns by quantifying their contents in venous smooth muscle layers using image pro . in human umbilical cords collected at preterm and term gestation methods: umbilical cord specimens ( - mm thickness, pieces per umbilical cord) collected at preterm and term (n= each) at delivery were fixed in bouin's solution and paraffin embedded. sections were subjected to immunohistochemical analyses with polyclonal antibodies to crf ( : ), ucn ( : ), ucn ( : ) and ucn ( : ) (peninsula laboratory, pa and sigma aldridge, ms) by standard abc technique. immunoreactive materials on the sections were identified using , '-diaminobenzidine as a chromagen. immunostaining intensities (od/area) on uc-sections were quantified by image pro . software and expressed as arbitrary units (au). all values were expressed as mean ± sem. differences between groups were evaluated by anova, followed by the post-hoc tukey test for multiple comparison. results: antibodies to crf and ucns elicited positive immunostaining of variable intensity in venous smooth muscle layers in uc-sections of preterm and term gestations. immunostaining intensity (au) of venous smooth muscle layers at preterm (pt) and term (t) are as follows: crf-pt= . ± . vs crf-t= . ± . (p< . ); ucn -pt= . ± . vs ucn -t= . ± . (p< . ); ucn -pt= . ± . vs ucn -t= . ± . (p=ns); ucn -pt= . ± . vs . ± . (p< . ). conclusion: crf content in venous smooth muscle layer markedly increased at term, while ucn and ucn contents significantly decreased and no significant change occurred in ucn content. based on the opposing changes in crf vs ucn and ucn immunostaining, we speculate that crf, but not ucn or ucn , is the major key player of vasodilation and function locally at the level of venous smooth muscle cells at term. ucn and ucn may perform cytoprotective functions at preterm. physiol. ; : - , am j physiol. ; : - ) . these studies showed that when ad libitum (al) feeding was resumed in cr females, fertility was sustained well beyond the age at which al-fed females became infertile. however, much of what is known on the effects of cr on fertility derives from models in which cr was initiated at weaning. further, there is large variation in how the experiments were conducted. objective: herein we tested if adult-onset cr could delay age-related infertility in females. methods: cr ( %, nia protocol as described in j geront. a:b -b ) was initiated in c bl/ female mice at months and continued until . months of age, at which time al feeding was resumed. matings were initiated at months of age. for mating during cr, a male mouse was housed overnight in a cage with a female and removed the next morning, so that the female mice could be fed their dietary food ration. al-fed and cr females followed the same mating regimen. the number of offspring born and that survived to weaning (day ) were recorded. results: fertility was lost in of al-fed females by months of age and continued to decline through . months of age. age-matched females maintained on cr during the same period exhibited poor fertility, with a total of pregnancies achieved out of females. although cr females showed poor fertility while on cr, their fertility improved dramatically after the reinitiation of al feeding at . months of age. while only out of , al-fed females achieved a pregnancy between . - . months of age, out of age-matched cr-then-al-fed females achieved a total of pregnancies in this -month time. notably, % of the pups born to cr-then-al-fed females between . - . months of age survived and developed to weaning without complications. conclusions: adult-onset cr allows maintenance of female fertility into advanced maternal age after the reinitiation of al feeding. how long fertility can be maintained and the minimum time needed for the beneficial effects of cr to be realized remain to be investigated. nonetheless, these observations suggest that there may be ways to safely extend fertility in females at ages when reproductive function is suboptimal (nih r -ag ). bile acids in human ovarian follicular fluid. laura p smith, , kaila deiorio-haggar, jason reindollar, alan s penzias, , anny usheva-simidjiyska. ob/gyn reproductive endocrinology infertility, bidmc, boston, ma, usa; boston ivf, boston, ma, usa; endocrinology, bidmc, boston, ma, usa. introduction: bile acids are known to serve important functions in the hepatobiliary and gastrointestinal systems. the presence of bile acids in the human ovary and relation with fertility potential have never been previously evaluated. methods: human follicular fluid (ff) from large follicles and small follicles was obtained at vaginal oocyte retrieval. human serum was obtained before and hours after human chorionic gonadotropin (h-cg). follicular fluid and serum samples were analyzed for total bile acids by spectrophotometry. bile acid concentrations were correlated with age, number of retrieved and mature oocytes, number of fertilized oocytes, and pregnancy. results: bile acid concentrations were analyzed and compared to the normal human serum bile acid concentration which ranges from to micromoles/l. bile acids are present in follicular fluid with a mean concentration of . micromoles/l in large follicles and . micromoles/l in small follicles (p= . ). pre and post h-cg serum bile acid concentrations differed significantly ( . micromoles/l vs. . micromoles/l, p= . ). there was a trend toward higher bile acid concentration in large follicles of young patients < years old compared to older patients years old ( . ± . vs. . ± . , p= . ). there was also a trend toward higher pre h-cg serum bile acid concentrations in older patients ( . ± . vs. . ± . , p= . ). there was no correlation between serum and follicular fluid bile acid concentrations and number of oocytes retrieved or number of mature oocytes, but there did appear to be a positive correlation between pre h-cg serum bile acid concentration and number of fertilized oocytes (spearman's correlation coefficient . , p= . ). conclusions: this is the first demonstration of the presence of bile acids within human ovarian follicular fluid. there may be a relationship between bile acid concentration and fertility potential. the precise function of bile acids in human ovarian follicular fluid is under investigation. objective to investigate the impact of ovarian hyperstimulation treatment on the biomarkers of the homocysteine pathway in blood and follicular fluid, and their association with the follicle diameter as a measure of follicular maturation. methods in women undergoing ivf/icsi treatment blood samples were collected on cycle day and the day of hcg administration. during oocyte retrieval in each woman the diameter of two follicles was measured and the corresponding follicular fluids were collected. in blood and follicular fluid total homocysteine (thcy), folate, cobalamin and pyridoxal' 'phosphate (plp) concentrations were determined. women with a serum folate . nmol/l were classified as folic acid supplemented. results ovarian hyperstimulation significantly decreased thcy and cobalamin blood levels (both p . ). the blood and follicular fluid concentrations of thcy, folate, cobalamin and plp were significantly correlated (all p . ). in the total group, a two-fold increase of thcy in follicular fluid resulted in a . mm decrease of the follicular diameter (p . ). in non-supplemented women this decrease was . mm (p . ). in supplemented women a twofold increase of follicular fluid folate resulted in a . mm decrease of the follicular diameter (p . ). conclusions ovarian hyperstimulation reduces thcy blood levels independent of folic acid supplementation. however, high follicular fluid thcy and folic acid supplementation may have detrimental effects on the maturation of the follicle. ), post-fixed for rain in % w/v osmium tetroxide in the same buffer, quickly dehydrated in a series of ethanol solutions, and embedded in epon. thin sections were stained with uranyl acetate followed by lead citrate and were observed at electron microscope. results: ten blastocysts from each group were collected and analyzed. compared to the in vivo embryos, blastocysts generated in vitro exhibited significant differences. the surface of their trophectoderm (te) layer had a reduced number of microvilli, the number of the apoptotic cells in the inner cell mass (icm) was higher and the presence of non functional mitochondria was elevated. in this study we have, for the first time, compared the ultrastructure of the in vivo and in vitro conceived blastocysts. taken together, these results suggest that both, the higher rate of apoptosis and the morphological alterations in the mitochondrial structure in ivf embryos, are associated with stress during in vitro embryo culture. therefore, these parameters can be used, in the future, as markers for the assessment of the embryo wellbeing in the ivf settings. deteriorating oocyte quality is a critical hurdle in the management of infertility, especially one associated with advancing age.here, we explore a newly discovered role of nitric oxide (no) in the sustenance of oocyte quality. methods: sibling oocytes from superovulated mice were subjected to intracytoplasmic sperm injection (icsi) with cauda-epididymal spermatozoa following exposure to either the no donor, s-nitroso n-acetyl penicillamine (snap, . m/min); a soluble guanylyl cyclase inhibitor, h-[ , , ] oxadiazolo [ , -a] quinoxalin- -one (odq, m) or an no synthase inhibitor, n w -nitro-l-arginine methyl ester (l-name, mm). their sibling oocytes were subjected to icsi either before (young) or after culture for the corresponding period (old). outcomes of fertilization, cleavage and development to the morula and blastocyst stages were compared. some embryos from each subgroup were also subjected to tunel assay for apoptosis. results: oocytes deteriorated in their ability to undergo normal fertilization and development to morulae/blastocysts after aging in culture, as compared to their sibling cohorts subjected to icsi immediately after ovulation (p< . ). this deterioration was prevented after oocyte exposure to snap. while, exposure to l-name or odq resulted in a significant compromise in fertilization and development to the morulae/blastocysts (p< . ) with detection of apoptosis, which was also noted in embryos derived from aged oocytes but not in those from young or snap exposed oocytes. conclusions: no is essential to sustain oocyte fertilizability and developmental ability, and to prevent blastomere apoptosis. objective to investigate the effects of the levels of the biomarkers of the homocysteine pathway on ivf outcome. methods from women undergoing an ivf or icsi procedures, two blood samples and two mono follicular fluid samples were collected for determination of folate, cobalamin, and total homocysteine (thcy). total protein was determined in follicular fluid to adjust the biomarker concentrations for follicular maturation. primary endpoint of the study was oocyte quality measured by fertilization and embryo quality (range - ; with being best quality). secondary endpoint was the occurrence of pregnancy. results % of the included women used a folic acid supplement (serum folate . nmol/l). in non-supplemented women higher cobalamin levels in follicular fluid correlated with a better embryo quality (estimate - . ; p . ) and higher thcy levels (median . mol/l, range . - . ) correlated with a worse embryo quality (estimate . ; p . ). in supplemented women higher follicular fluid thcy (median . mol/l, range . - . ) correlated with better embryo quality (estimate - . ; p . ). the follicular fluid folate level of oocytes that did not fertilize was . -fold higher than in the fluids of a fertilized oocyte ( % ci . - . ; p . ). a two-fold increase of follicular folate corresponded with a . higher chance to achieve pregnancy ( % ci . - . ). conclusions cobalamin levels in follicular fluid are correlated with embryo quality. folic acid supplementation modifies the thcy and folate levels in follicular fluid and thereby affects oocyte quality. the level of folate in follicular fluid is important in the fertilization process. we recently reported that increasing relative oocyte immaturity is associated with worsening outcome, and that cycles with many immature oocytes are more common in younger women. (moore et al, asrm annual meeting, ) to further investigate this trend, we conducted a case/control analysis of patients with repeated cycles of high-level oocyte immaturity (hloi). methods: oocyte maturity data was collected on all icsi cycles starting in . we defined a cycle with hloi as having > % immature oocytes (> sd's above the median). a case was defined as a patient with hloi on more than one cycle. control subjects were age-matched and defined as having </= % ( sd above median) immature oocytes on all cycles. results: from subjects, we identified cases of recurrent hloi (comprising icsi cycles) and control subjects. at baseline, cases had more oocytes retrieved per cycle than controls ( . vs. . , p<. ). all other baseline characteristics were similar. all case subjects were younger than . outcomes are shown in table . discussion: clinical pregnancy and live births were reduced in the case group, but more than expected based on % immaturity. during cycles, there were no live births among the case group. patients with recurrent hloi represent a previously undescribed group of young patients with a very poor prognosis during icsi treatment. this study may suggest that the problem underlying the immaturity may relate to overproduction of oocytes during stimulation, a factor which may be modifiable. however, the additional trend (though not significant) that even the mature oocytes from the case patients tend to have poor outcomes suggests the possibility of a maturation defect and warrants further consideration. data on response to gonadotropins, fert rates, and implantation rates will be presented. amongst those, were performed for an initial diagnosis of premature ovarian aging (poa), for the diagnosis of premature ovarian failure, and for repeated pregnancy loss. in women under age , b-fsh levels over miu/ml and up to < miu/ml denoted a diagnosis of poa, while levels of miu/ml were considered diagnostic for pof. all patients signed consent permitting review of medical records for clinical research purposes. results: day fsh levels ranged from . to . miu/ml (mean . ± . miu/ml). amh levels ranged from < . to . ng/ml (mean . ± . ) and allele repeats ranged from to , (mean for allele- , . ± . ; allele- , . ± . ). mixed model analysis of variance for all patients, adjusted for age, race, and allele- revealed a statistically significant correlation between b-fsh levels and number of repeats in allele- ( figure ; p < . ). we did not observe a significant linear relationship between serum amh levels and cgg repeat counts, however cgg repeat counts above were significantly associated with serum amh levels less than ng/ml (chi square = . ). conclusions: triple repeats above , a level currently considered well within normal, are associated with evidence of premature decline in ovarian function. the evaluation of triple repeats in frm gene offers a first objective assessment of ovarian function and should be predictive of autologous reproductive life span in most women. objective: the levels of mis in the serum and in the ovary have been demonstrated to decrease in response to increasing doses of cisplatin. we hypothesize that ovarian stimulation also will reveal dose related decreases in ovarian and serum mis levels after cisplatin exposure, further demonstrating the follicular damage from chemotherapy. methods: adult female sprague-dawley rats were treated with saline, . mg/kg cisplatin or . mg/kg cisplatin as two weekly injections. five days following the last cisplatin injection, the rats were injected with pregnant mare serum gonadotropin (pmsg) and euthanized hours following the pmsg injection. serum was collected and both ovaries were obtained for protein analysis. serum and ovarian levels of mis were determined using an elisa kit. western blot analysis, immunohistochemical analysis, and tunel analysis were also performed on the ovarian proteins. results: stimulated mis levels declined in a linear fashion in response to increasing cisplatin doses (p< . ). ovarian protein levels measured by elisa and western blot both indicated that stimulated ovarian mis levels also decrease in a linear fashion (p= . and p< . respectively.) immunohistochemical analysis revealed that while the total number of follicles remains the same, both the total number of mis positive follicles, and the percentage of mis positive follicles declines in a linear fashion (p= . for both). tunel analysis reveals that there is no change in the incidence of apoptosis in the stimulated ovaries from any treatment group. conclusions: the administration of pmsg after treatment with cisplatin causes a dose dependent decrease in the levels of mis in both the serum and in the ovary. serum mis levels following ovarian stimulation may be useful as a serum biomarker in this animal model to assess ovarian damage following the administration of cisplatin. correlations of micro-rnas a, b, - pp, and - p with inflammatory and steroidogenic factors in human granulosa/cumulus cells. tannaz toloubeydokhti, orhan bukulmez, kenneth c drury, r stan williams, nasser chegini. obstetrics and gynecology, university of florida college of medicine, gaineville, fl, usa. as part of the novel endogenous rna silencing machinery, noncoding short rna strands, referred to as micrornas (mirnas), have been identified to regulate the stability of the target gene expression through degradation and repression. the expression of many of these mirnas has been identified in human tissues however, their biological significance in various cellular processes remains to be elucidated. in this cross-sectional study of human granulosa cells, we aimed to study the potential correlations between five selected mirnas, mir- a, mir- b, mir- - p, mir- and mir- - p, and mrna expressions of some of their predicted target genes namely, cyclooxygenase (cox)- , il- , steroidogenic acute regulatory protein (star), aromatase and estrogen receptor (er) . granulosa/cumulus cells were obtained from women (age range - ; mean age . ± . ) undergoing oocyte retrieval for assisted reproduction. total rna was extracted from these cells and reversed-transcribed and real-time pcr was performed to measure steady-state levels of mirnas and mrna transcripts. we observed that the expression of mir- a, mir- - p, mir- and mir- - p displayed a significant and positive correlation with the expression of cox- . moreover, mir- b significantly correlated with star mrna expression, but not with other genes tested (p< . ). with advancing age, the expression of mir- - p and cox- was significantly decreased, with cox- expression displaying a positive correlation with aromatase, star and er mrna expressions (p< . ). none of the factors tested showed any significant correlations with peak estradiol levels and number of oocytes retrieved. receiver operating characteristic curve analysis revealed that female age cut-off value of . y best predicted whether mir- - p was below its mean arbitrary value. in conclusion, given the importance of mirnas' regulatory function in gene expression, our results suggest that mir- b may play an important role in gene expression related to granulosa cell luteinization, while variation in mir- - p expression with female age may suggest its potential role as a predictor of oocyte quality and granulosa cell function. given the importance of mirnas in gene regulation, the role of mirnas in ovarian physiology and aging requires further investigation. support: nih grant . introduction: a symbiotic relationship between ovarian granulosa cells (gc) and the enclosed developing oocyte is critical to reproductive efficiency. genetic modulations in gc's can lead to reproductive insufficiency, highlighting the critical role of gc's in reproductive competence. defects in the oocyte-gc repertoire in women with dor include lower e levels, luteal deficiency, poor fertilization rates, higher incidence of aneuploidy, lower pregnancy and higher miscarriage rates. utilizing global gene expression analyses in cumulus gc's, we attempt to enhance our understanding of biological mechanisms that may contribute to poor reproductive capacity in young women with dor. methods: infertile women (< years old) undergoing ivf were prospectively enrolled into two groups based on ovarian reserve (normal reserve, fsh< dor, fsh > miu/ml). at egg retrieval, cumulus gc's were isolated, rna extracted transcribed into cdna. microarray targets were generated cdna hybridized to affymetrix human genome u plus . genechips. microarray data were analyzed (array assist) and normalized (robust multichip analysis). a difference in gene expression of > fold was considered biologically relevant. results: of the , genes identified to be differentially expressed in young women with dor compared to normal reserve, genes demonstrated consistency of expression across five different normalization schema; were down regulated and up-regulated. expression of gremlin, a member of the dan family of genes known for its highly regulated expression pattern during folliculogenesis, was noted to be down-regulated -fold over two probe sets (- . ) in women with dor versus normal reserve; this down-regulation was confirmed by real-time pcr (- . ) . conclusions: this is the first demonstration linking differential expression of gremlin with etiology of infertility in women. gremlin is a downstream effector of oocyte-derived gdf- which facilitates cumulus cell expansion, a critical event in reproductive physiology. our finding of a significant downregulation of gremlin expression in cumulus gc's associated with dor may partly explain the physiology of poor reproductive performance. nih k cd . nih k rr . ferring pharmaceuticals. the objective of the present study was to investigate the effects of the gnrh antagonist ca on expression of mis and aromatase (cyp ) using luteinized human granulosa cells obtained during in-vitro fertilization cycles and an immortalized human granulosa cell line (hgl ). the granulosa cells were cultured +/-dibutyryl camp ( mm) and incubated +/-the gnrh antagonist, ca, for - h. rna was isolated, reversed-transcribed and real-time pcr was performed to measure mrna transcripts for ovary-specific cypiia and mis. we observed that camp markedly induced aromatase mrna in both the primary and immortalized human granulosa cells. interestingly, camp treatment of these cells also caused an upregulation of mis mrna. objectives: bisphenol a (bpa), a known endocrine disruptor, is a chemical used as a plasticizer in the manufacture of polycarbonate plastics and epoxy resins and is present in multitude products, including the interior coating of food cans, milk containers, and baby formula bottles. bpa can leach into foods during heat processing and is known to exert a variety of endocrine-like effects on different cell types acting as an estrogen because it contains two hydroxyl groups in its diphenyl structure. in this study we focused on the effects of bpa on aromatase expression and estradiol production in the human granulosa kgn cell line. we also evaluated its effects on several transcription factors crucial in cyp expression. materials and methods: kgn cells were cultured in f- dmem and were starved for h before experiments. subsequently they were treated for h with vehicle (control), fsh ( ng/ml), and/or bpa ( , , , , um) . messenger rna expression was quantified by real time pcr and estradiol secrection was measured in supernatants by elisa. results: fsh induced a -fold increase in aromatase expression. bpa induced a dose-dependent decrease in cyp production, with the greatest effect at um (p< . ), resulting in +/- % (mean+/-sem) inhibition, compared to aromatase expression induced by fsh alone. bpa also reduced levels of estradiol secretion in a dose-dependent manner, with the greatest inhibition at um (p< . ) resulting in +/- % decrease. we also evaluated expression of transcription factors known to be involved in regulating the activity of the ovary-specific aromatase proximal pii promoter. interestingly factors known for induction of aromatase such us steroidogenic factor- and gata- , mimic the pattern of cyp expression after bpa treatment, whereas, other receptors previously reported to act as aromatase inhibitor, such as ppar gamma were up-regulated by the addition of bpa. moreover, expression of creb remained virtually intact, suggesting that most likely mechanisms governing endocrine disruption by bpa are highly selective. conclusions: bpa inhibited fsh stimulated aromatase expression and downstream estradiol secretion. we propose that constant exposure to this chemical may result in endocrine disruption which may contribute to reduced fertility and early ovarian senescence. oocyte maturation occurs during folliculogenesis as a result of complex cell-tocell communications between the granulosa cells and the oocyte. maintaining the granulosa cells' spherical structure and network of gap junctions surrounding the oocyte is critical. this project tests the ability of a novel substrate-free threedimensional culture system to form viable granulosa cell spheroids. methods: after irb approval, freshly obtained follicular fluid from in vitro fertilization was obtained and granulosa cells were purified by percol gradient. nonadhesive agarose hydrogels, containing cylindrical round bottom recesses m in diameter, were cast from micro-molds designed using computer-assisted rapid prototyping. granulosa cells seeded at a density of , cells per gel were incubated for up to days. cellular viability was assessed with live:dead assay. results: after three days in culture, granulosa cells formed spheroids of densely packed cells that were difficult to disperse with multiple pipettings. the cells remained viable for at least days. conclusions: granulosa cells can be cultured in a novel substrate-free threedimensional culture system. the cells form tightly adherent spheroids that remain viable for extended culture. the cohesiveness of the cells suggests the formation of gap junctions. this is under investigation with immunohistochemistry and electron microscopy. these experiments suggest that a substrate-free threedimensional hydrogel culture system may be ideal to reassemble follicular structure important for future in vitro evidence testing and oocyte maturation. known to function as responders to pathogens, inflammation, and tissue injury. previous studies in our laboratory demonstrated that saa was produced in mouse granulosa and production was regulated by cytokines. ovulation has long been considered an inflammatory reaction and patients with chronic inflammatory conditions often experience infertility. the present study was undertaken to explore the role of saa in human ovarian function. methods: ovarian granulosa and luteal cells were obtained from surgically removed specimens and mural and cumulus granulosa-luteal cells were obtained from ivf aspirates. rna was extracted from fresh or cultured cells. some cells were treated in vitro with tnf or other cytokines for h. expression of saa was assessed by quantitative rt-pcr. in addition, serum levels of saa were determined using a commercial elisa in women undergoing ovulation induction (oi) and art. saa levels were measured at baseline, during oi, on the day of hcg administration and at the time of the pregnancy test. results: saa mrna was expressed in theca, granulosa, and granulosa-luteal cells. in granulosa-luteal cells both saa and saa mrnas were expressed at higher levels in cumulus compared to mural granulosa. expression of saa and saa in theca was increased following treatment with tnf in vitro. serum levels indicated that patients with ovulatory dysfunction had increased levels of saa at the time of hcg injection while patients without ovulatory dysfunction had lower saa levels as compared to the baseline level. in addition, patients undergoing oi who achieved pregnancy exhibited increased levels of saa at the time of the pregnancy test compared to baseline levels, whereas patients who did not become pregnant had lower post-cycle levels of saa. interestingly, saa levels did not change in art patients that became pregnant without undergoing oi (donor egg or frozen embryo transfer) conclusions: human ovarian cells express saa mrnas which can be altered in vitro. serum levels of saa may correlate with the responsiveness of the ovary to gonadotropins as evidenced by altered levels in women with ovulatory dysfunction, and by an increase in pregnant patients following ovarian stimulation. yeh, beom su kim, felicia hercules, jennifer peresie, armando arroyo. gynecology-obstetrics, university at buffalo, buffalo, ny, usa. objective: cisplatin is a common chemotherapeutic agent given to women for treatment for a wide variety of cancers. we hypothesize that one mechanism by which cisplatin may cause damage to ovarian structures is by decreasing the amount of anti-oxidant activity in the ovary. we examined super-oxide dismutase (sod ), a critical anti-oxidant enzyme that has been shown to be affected by cisplatin in other tissues, in the ovaries of cisplatin treated animals. methods: adult female sprague dawley rats were injected with saline, cisplatin . mg/kg or cisplatin . mg/kg as weekly doses. five days following the last injection, the rats were euthanized and both ovaries were excised. one ovary was processed for immunohistochemistry and the other was processed for protein analysis using western blot techniques for sod . the anti-sod antibody was purchased from santa cruz. the immunohistochemical sections were scored using a semiquantitative h scoring method. results: immunohistochemistry analysis of the expression pattern of sod following cisplatin administration revealed that there was a significant linear decrease in a dose response pattern in the expression of sod in antral follicles and in corpora lutea (p< . for both). no change was found in the h score of sod in other ovarian structures. western blot analysis of sod in the ovaries following increasing doses of cisplatin revealed no changes in the overall protein levels of sod in the ovary. conclusions: this is the first report that administration of cisplatin causes changes in the expression pattern of sod in antral follicles and in copora lutea. cisplatin decreases the amount of sod available in these structures, possibly leading to increased oxidative stress and free radical damage, thereby leading to ovarian damage found after cisplatin administration. is there evidence for aromatase activity in the stroma of postmenopausal ovaries? mf landay, rh fogle, rb allen, s patel, fz stanczyk, rj paulson. ob/gyn, usc, keck school of medicine, los angeles, ca, usa. background: following menopause, the ovaries continue to secrete androgens and estrogens. we have recently confirmed the production of androstenedione, testosterone and estradiol (e ) up to ten years after menopause by measuring gradients from ovarian venous effluent and peripheral blood. anti-müllerian hormone (amh) and inhibin b have been shown to be markers of follicular activity. peripheral levels of these hormones have previously been found to be undetectable in menopause, suggesting the absence of follicular activity in the postmenopausal ovary. objective: to investigate if the postmenopausal ovary continues to demonstrate evidence of follicular activity as the source of steroid production. design: observational study materials and methods: eight subjects aged ± . yr (range - ) were enrolled. postmenopausal status was confirmed by preoperative fsh levels of more than u/l and/or amenorrhea greater than months. serum was collected from the ovarian veins during total abdominal hysterectomy and bilateral oophorectomy. peripheral blood was also collected pre-operatively, intraoperatively and postoperatively. all samples were analyzed for amh and inhibin b using elisas with sensitivities of . ng/ml and pg/ml, respectively. androgen and estrogen levels in these samples have previously been reported, and documented a gradient between ovarian venous effluent and peripheral serum in all cases. results: ) six patients had no detectable follicular activity by amh and inhibin b levels. ) one patient demonstrated detectable inhibin b levels with an -fold gradient between ovarian venous effluent ( pg/ml) and peripheral blood ( pg/ml), however no amh was detected. ) in one patient, aged and months postmenopause, both amh and inhibin b were detected. peripheral inhibin b levels were high at pg/ml. amh was detectable at levels of . ng/ml. conclusions: ) in the majority of patients, continued e and androgen production in the ovary occurs in the absence of follicular activity as detected by amh and inhibin b production. ) some patients have evidence of follicular function up to ten years after menopause. ) since e is produced in post-menopausal ovaries in the absence of follicular activity, these data provide evidence for aromatase activity in the stroma of post-menopausal ovaries. to elucidate the process by which prostaglandin f (pgf ) mediates luteal regression, this study examined the role that the transcription factor yin yang (yy ) and histone deacetylase (hdac ) play in altering luteal cholesterol uptake by the scavenger receptor class b type i (sr-bi), intracellular cholesterol transport by steroidogenic acute regulatory protein (star), and cholesterol processing by p side chain cleavage enzyme (p scc) expression. rat luteal cells ( days post-ovulation) were treated with pgf ( hr) and trichostatin a (tsa; hr), a potent hdac inhibitor. protein expression was measured post-treatment by western blot and cholesterol was localized via filipin staining. star and sr-bi promoter activation was also assessed in hek t cells treated with yy , myy , a deletion mutant lacking an essential region required for transcriptional repression, and tsa. pgf caused a significant -fold decline in star (p< . ), and smaller declines in sr-bi and p scc which occurred concomitantly with an increase in yy ( -fold, p< . ) and intracellular lipid staining ( % increase). in contrast, nm tsa treatment caused a dose dependent increase in sr-bi, star, and p scc protein levels, . -fold (p< . ), . -fold (p< . ), . -fold (p< . ), respectively, and a . -fold decline (p< . ) in intracellular lipid levels. tsa prevented the pgf -induced decline in sr-bi, star, and p scc expression. promoter analysis demonstrated that wildtype yy , but not myy , repressed sr-bi and star activation while the addition of tsa overcame the repressive effects. this study shows the critical role that yy plays in pgf induced luteal regression by recruiting a histone deacetylase to block three essential processes in steroid production. in luteal cells yy -mediated global repressive action prevents cholesterol metabolism by inhibiting cholesterol uptake, intracellular transport, and processing thus leading to functional and structural luteal demise. supported by nih hd and nih hl . regulation of intermedin expression in cycling rat ovary. madhu chauhan, rebekah elkins, chandra yallampalli. obstetrics gynecology, university of texas medical branch, galveston, tx, usa. background: intermedin (imd) is a ct/cgrp family peptide involved in a variety of physiological functions, including vasodilatation and fetoplacental growth. this peptide is expressed in a variety of tissues such as stomach, placenta, uterus, pituitary and ovary suggesting its different functions including in reproduction. imd gene has an estrogen response element and we have shown that the plasma concentration of immuno-reactive imd is elevated in rats with pregnancy. however, expression of imd in the ovary and its regulation during estrous cycle is not known. we hypothesize that imd is expressed in the ovary and its expression is hormonally regulated throughout the estrous cycle in rat. objective: ) to assess expression of imd mrna and its receptors components calcitonin receptor like receptor (crlr), and receptor activity modifying proteins, ramp and ramp mrna in rat ovary; on diestrus, proestrus and estrus stages of rat estrus cycle and ; ) to demonstrate immunohistolocalization of imd, crlr, ramp , ramp and ramp in rat ovary. methods: groups of sprague-dawley non-pregnant and pregnant rats on day of gestation were used in this study. non-pregnant rats were sacrificed on diestrus, proestrus and estrus stage. ovaries were collected and total rna was extracted using trizol method. rna was treated with dnase before performing the reverse transcriptase polymerase chain reaction (rt-pcr). immunohistolocalization of imd, ramp , ramp and ramp proteins were assessed in tissue sections of ovaries from pregnant rats sacrificed on day . results: ) imd mrna is regulated in rat estrus cycle and its expression is significantly downregulated in estrus stage compared to the diestrus and proestrus; )expression of imd receptor crlr is highest in the diestrus stage, followed by a decline in proestrus which further declined during estrus; ) expression ramp mrna is higher in proestrus compared to diestrus and estrus (p< . ) but there is no significant change in the ramp expression during the estrus cycle and ; ) imd, ramp , ramp and ramp are immunolocalized in rat ovary in granulose cells of all follicles and granulosa cells of the corpus luteum. conclusion: imd mrna and protein are expressed in rat ovary suggesting a possible role for imd in ovarian function. defining and defying deterioration in oocyte quality with advancing chronological age: role of nitric oxide. pt goud, ap goud, mp diamond, b gonik, hm abu-soud. div reprod endocrinology, dept ob/gyn, wayne state university, detroit, mi, usa; div maternal and fetal medicine, sinai grace hospital, wayne state university, detroit, mi, usa. nitric oxide (no) is a ubiquitous free radical essential for oocyte maturation, function and sustenance of oocyte quality post-ovulation. the current study investigates the role of no insufficiency in the causation of oocyte quality deterioration with advancing chronological age. methods: in set , oocytes were retrieved from the following superovulated b d f mice: (a) young breeders (yb, n= ); (b) retired breeders (rb, n= ), and © old animals (oa, n= ), aged - , - , and - weeks respectively; and studied for zona pellucida dissolution time (zpdt), spindle ( -tubulin fluorescence immunocytochemistry, sigma) and chromosome morphology (dapi, vector), ooplasmic microtubule (mt) dynamics (omd) in response to taxol [ ], and cortical granule (cg) status (rhodamine conjugated lectin, vector). in set (n= ), oocytes from retired breeders were studied after exposure in vitro to an no-donor, s-nitroso acetyl penicillamine (snap in m- , . m no/min , n= , h, °c, % co ), while their sibling control oocytes were cultured for corresponding period under identical conditions without snap. [ ]. zpdt, spindle and chromosome morphology, omd and cg status were assessed with a confocal microscope and compared between the subgroups using anova, chi square and/or fisher's exacts test and appropriate post-hoc tests. results: in set , a significantly fewer oocytes were retrieved per animal (mean) in rb ( ) and oa ( ) compared to yb ( . , p< . ). advancing age was also associated with a significant increase in zpdt, omd and cg loss in rb compared to yb (p< . ). furthermore, significantly fewer oocytes from rb than yb had normal spindle and chromosome morphology (p< . ). oocytes from oa had significant spindle and chromosome disarray, a near total cg loss and significantly harder zp (p< . ). these oocytes also exhibited diminished omd in response to taxol although, metaphases were exquisitely sensitive to disruption with taxol. in set , exposure to snap in oocytes from rb resulted in a significantly lowerzpdt, omd and cg loss, and significantly higher incidence of normal spindles ( the gamma-aminobutyric acid (gaba), its biosnthetic enzyme gad and gaba-a receptors have been found in the oviduct and ovary. objective: this study examined the expression of gaba-a receptor subunit genes and gad and whether the gaba-a receptor could alter cytosolic ca + in gc. methods: for qrt-pcr, both human cumulus and mural gc were obtained at the time of oocyte retrieval for ivf and cultured separately. total rna was isolated separately from each gc type and from human brain ( positive control). the total rna from patients was pooled for each gc type and all rna was treated with dnase i. two-step qrt -pcr was performed using gene-specific lux™ primers for all gaba-a receptor subunits, gad and gad plus gapdh. single and specific qrt-pcr products were verified by melting curve analysis, gel electrophoresis, and dna sequencing. for ca + study, gc were cultured on coverslips. gc were loaded with fura- -am and changes in ca + concentration of gc were studied using a dynamic digital ca + imaging system. gaba-a agonist, muscimol, was used to study any dose-dependent effects on gc. gaba-a antagonist, m bicuculline, were perfused min. prior to and during application of muscimol. the responses were represented as changes in the / nm fluorescence ratio over time. m atp was used as positive control. results: the qrt-pcr results indicated that all gaba-a receptor subunits were expressed to various degrees in both types of gc, with the expressed highest in both cell types. gaba-a receptor subunits showing the next highest expression in both cell types were , and . gad isoenzyme was more abundantly expressed in cumulus and mural gc than gad . ca + imaging showed that muscimol, had the ability to increase ca ++ in gc, about % gc (n= ) cells responded to muscimol. muscimol increased intracellular ca + in a dose-dependent manner. the muscimol responsive cells was reduced by bicuculline, from % to % (n= , p< . ). conclusion: qrt-pcr indicates that gaba-a receptor subunit gene and gad expression occurs in both cumulus and mural gc. the ability of bicuculline to inhibit the ca + response to muscimol suggests the activation of gaba-a receptor. the current study confirms the presence of functional gaba-a in gc for the first time, and suggests that gaba may exert trophic effects in the ovary via gaba-a receptor. the present study test the hypothesis that administration of l-nitro-l-arginine methyl ester (l-name), a nos inhibitor, prior to hypoxia prevents the hypoxiainduced activation of p mapk, erk and jnk in the cerebral cortex of the guinea pig fetus during gestation. to test this hypothesis guinea pig fetuses at and days gestation were assigned to normoxic (nx, n= ), hypoxic (hx, n= ) and hypoxic pretreated with nos inhibitor (hx+l-name, mg/kg i.p., n= ) groups. hypoxia in the fetuses was induced by exposing the pregnant guinea pigs at both gestational ages to an fio of . for min. cerebral tissue hypoxia was documented biochemically by determining the tissue levels of atp and phosphocreatine (pcr). neuronal nuclei were isolated, purified and proteins separated by sds-page, and then probed with specific phosporylated erk, jnk and p antibodies. in the days gestation group: expression of p-p was . ± . (nx), . ± . (hx) . ± . (hx+l-name). p-erk expression was . ± . (nx), . ± . (hx), . ± . (hx+l-name). p-jnk expression was . ± . (nx), . ± . (hx), . ± . (hx+l-name). in the days gestation group: expression of p-p was . ± . (nx), . ± . (hx), . ± . (hx+l-name). p-erk expression was . ± . (nx), . ± . (hx), . ± . (hx+l-name). p-jnk expression was . ± . (nx), . ± . (hx) . ± . (hx+l-name). the data show that administration of l-name prior to hypoxia decreased the expression of phosporylated p , erk and jnk at both gestation ages however expression of phosporylation was higher at term as compared to preterm. since a nos inhibitor prevented the hypoxia-induced phosphorylation of p , erk and jnk in both gestational ages, we conclude that the hypoxia-induced activation of p , erk and jnk in the cerebral cortical nuclei of preterm and term guinea pig fetus is no-mediated. we speculate that no-mediated modification of cysteine residue leading to inhibition of map kinase phosphatases results in increased activation of p , erk and jnk in the guinea pig fetus. (nih-hd , nih-hd and st. christophers foundation for children). the the endocannabinoid, anandamide (aea), is involved in the hormone-cytokine network that regulates implantation and early pregnancy maintenance with levels at the endometrial level considered a major checkpoint . high levels ( nm) in culture are associated with embryo death while plasma levels (> nm) at weeks in women undergoing ivf-et are associated with failed pregnancy . what is uncertain is whether systemic aea levels after spontaneous conception in women presenting with threatened miscarriage are predictive of pregnancy outcome. our aim was therefore to measure plasma aea levels in women presenting with threatened miscarriage and to relate these to outcomes. methods: plasma aea levels were measured using a sensitive and previously validated hplc-ms method at - weeks gestation in women (nonsmokers, bmi < kg/m ) presenting with threatened miscarriage and in whom a viable pregnancy was confirmed by ultrasound scan. results: nine of the women subsequently had a miscarriage. the plasma aea levels in those women who had live births was . -fold lower than that in those who subsequently miscarried ( . ± . nm versus . ± . nm, mean ± sem; respectively; p< . unpaired student's t-test). the roc analysis revealed an area under the curve of . ± . with a sensitivity of % ( %ci of . % to . %) and a specificity of . % ( % ci of . % to . %). using an aea level of . nm as the optimal cut-off point, a single plasma aea measurement provided a sensitivity of % and a specificity of % with a negative predictive value of % and a positive predictive value of % for subsequent miscarriage. conclusion: these findings suggest a possible predictive role for plasma aea in women presenting with threatened miscarriage. the data also indicate that systemic aea levels may reflect local endometrial levels and therefore the local hormonal milieu in the early stages of normal pregnancies and in those complicated by threatened miscarriage. introduction: follicle stimulating hormone (fsh) mediates cyclic follicle growth and development and is widely used for controlled ovarian stimulation. the ovarian response of different women to fsh is variable, ranging from poor response to ovarian hyperstimulation and has been partly attributed to two common variants of the fsh receptor (fshr). we have previously identified four abnormal fshr splicing products ( exon deletions and intron insertion) in women with low and high response to fsh. two of the splice variants, deletion of exon and deletion of exon , showed a correlation with low and high response to fsh, respectively. in the current study, we evaluated the functional competence of the mutant fshrs in vitro. methods: we established stable hek cell lines expressing wild-type (wt) and splice-variant (del) fshr under the control of the inducible tet on/off system. the cells were transfected with a camp-responsive luciferase reporter plasmid and stimulated with fsh. results: the subcellular distribution of all splicing variants was the same as the controls and the protein localized mainly on the cell surface. all four splicing variants showed markedly decreased camp activation compared to controls when stimulated with fsh. however, all variants were able to form functional heterodimers with the wt receptor when co-expressed. interestingly, the heterodimer containing the form of fshr lacking exon , found in patients with decreased response to fsh, resulted in lower intracellular camp compared with the wild-type homodimer. conclusion: our findings suggest that fshr variants can contribute to abnormal response to stimulation in certain women undergoing ivf treatment. the variants require the presence of wild-type receptor in order to initiate signaling in response to fsh. further analysis of this signaling cascade in granulosa cells is underway to estimate the final production of estrogen from these heterodimeric receptors. objective: to compare the proteome of human endometrium in the prereceptive versus the receptive phases of the menstrual cycle. m m: endometrial biopsies were collected and days after urinary lh surge in the same menstrual cycle from three fertile women (n= ). proteins were extracted using sample grinding kit (ge healthcare) and interfering substances removed using -d clean-up kit (ge healthcare). labelling was performed with cydye dige fluors (ge healthcare) and proteins were separated using difference gel electrophoresis (dige). for the isoelectric focusing, cm ipg-strips in the nonlinear range of ph - were used. the second dimension was carried out using sds-page. differentially expressed proteins were detected by image analysis using decyder v . and the statistical module eda (ge healthcare). the spots of interest were subject to protein identification based on in-gel digestion, maldi-tof/tof mass spectrometry and database searching. western blot analysis were performed in the same biopsies in order to validate some candidate proteins. results: table displays the differentially protein abundance between days lh+ and lh+ (> -fold change). of these proteins, were increased at lh+ in comparison with lh+ , whereas only proteins were decreased. stathmin was found more than -fold decrease in lh+ compared to lh+ in the three patients studied in the validation studies performed by western blot. conclusions: this study shows that the human endometrium has a differential protein repertoire during the window of implantation compared to the prereceptive phase. the role of these proteins in the molecular events directed to embryo implantation is under research. differential protein abundance in human endometrium ( the extracellular signal-regulated kinases and (erk / ) are a mitogen-activated protein kinase (mapk) subfamily that act as key links in eukaryotic intracellular signaling transduction. activated by phosphorylation in response to specific stimuli, erk / is known to play a role in the regulation of cellular proliferation and survival. the human myometrium is a tissue known to undergo cycle-dependent proliferative and apoptotic changes in response to sex steroids. we hypothesized that erk / activity is involved in mediating menstrual cycle-dependent changes in the myometrium. materials and methods: immunostaining for phospho-erk and total-erk was performed on myometrial tissues obtained from normal women (n= ) at different phases of the menstrual cycle. staining intensities were evaluated by hscore. myometrial smooth muscle cells were isolated and cultured from normal women and treated with vehicle, estrogen ( - m), and progesterone ( - m) for and minutes, and then subjected to western blot analysis for p-and t-erk. statistical analysis was performed using one-way anova. results: tissue staining revealed that p-erk was mostly nuclear in all tissues, while t-erk was cytoplasmic and nuclear. p-erk staining was significantly stronger in the secretory phase and strongest at the early secretory phase, compared to other phases (p< . ). t-erk staining intensity was moderatehigh without variation across the menstrual cycle. in cultured myometrial cells, progesterone significantly increased p-erk levels within and minutes (p< . ) when compared to control. our results suggest that erk activity in the human myometrium is regulated in a menstrual cycle-dependent manner. the increased phosphorylation in the secretory phase suggests the involvement of progesterone in erk activation, as supported by our in vitro results. this increased erk activity may play a role in regulating myometrial proliferation. measures of insulin resistance (ir) including: fasting serum insulin, gir and homa. measures of plasma levels of endothelin- (et- ), soluble intercellular adhesion molecule- (sicam- ), soluble vascular cell adhesion molecule- (svcam- ) and high sensitivity c-reactive proteins (hscrp). results: women with pcos exhibited significantly higher levels of et- (p < . ), sicamp- (p < . ), svcam- (p < . ) and hscrp (p < . ) as compared with age-matched controls, respectively. positive correlations were evident between et- and fai (r = . ; p < . ) but et- negatively correlated with shbg (r = - . ; p < . ). svcam- positively correlated with total t (r = . ; . ), hscrp correlated with: bmi (r = . ; p < . ), and homa (r = - . ; p < . ), respectively. conclusions: women with pcos exhibited abnormal levels of endothelial and inflammatory markers, which appear to be inter-related to hyperandrogenaemia. ( ), and a higher expression of fatty acid amide hydrolase (faah) in peripheral lymphocytes post-ovulation ( ), suggest an involvement of the endocannabinoid system in menstrual cycle control. our aims were to investigate changes in plasma aea levels and in endometrial faah expression throughout the menstrual cycle. methods: plasma aea levels were measured using a hplc ms/ms method from women, median age yrs (range - ) and bmi kg/m (range - ), with regular menstrual cycles, with no medical problem and not on any medication for the preceding months. the menstrual cycle was divided to early follicular d - (n= ), late follicular d - (n= ), ovulatory d - (n= ), early luteal d - (n- ) and late luteal phases d - (n= ). uterine biopsies were taken from hysterectomy specimens taken for benign conditions and subjected to immunohistochemistry for faah with polyclonal antibodies. results: aea levels were significantly higher around ovulation in comparison to the pre-ovulatory or post-ovulatory phases as shown in the figure. faah expression in the endometrial stroma was unchanged throughout the follicular phase but increased during the mid to late luteal phase reaching a maximum in the late luteal phase. a high aea level in the early follicular phase and during ovulation suggests a role for aea in ovulation. the lower levels of aea in the luteal phase, essential for successful implantation, may be regulated by increased faah expression at the uterine level. the modulation of plasma aea levels during the menstrual cycle strongly suggests that it is regulated by gonadal steroid hormones. ( , , , , , , and , as well as at pregnancy. binding activities of igfbp and their protein levels (igfbp , , , , , ) were assessed by western ligand blot (wlb) and western blot (wb), respectively. the glyscosylation and phosphorylation status of igfbps were examined by deglycosylation treatment. our results showed that both glycosylated and unglycosylated igfbp elevate in fetal and newborn rat and graduately decline to a lower level at day and kept lower constant level since then. interestingly, biding activity of glycosylated igfbp was not detected by wlb assay. the igfbp- cleaved products were observed after rat day age day , which associated with a decrease in full length of igfbp- , suggesting endoproteolytic processing may involved in decreasing igfbp content. igfbp- , with heterogeneous glycosylation, were appeared after age day and disappeared during pregnancy, recurrence again postpartum. glycosylation of igfbp has no effect on its binding activity. unglycosylated and glycosylated igfbp were constant in life time. physiologically constant igfbp were detected by wb in protein, but not by wlb in binging activity. conclusion: highly elevated circulation igfbp suggest physiological role in new born and early postnatal development. its binding activity are well regulated by its posttranslation modification, such as glycosylation of igfbp in inactivating binding activity and possible involvement of active endoproteolytic processing in maintaining binding active igfbp- at low background: cigarette smoking affects hormone biosynthesis, storage, release, binding, transport and clearance, resulting in changes in circulating hormone levels. we used metabolomics to analyze the effects of cigarette smoking and second hand smoke (shs) on changes in hormone levels in women of childbearing age. methods: this is a three arm study; women aged - years who are active smokers, exposed to shs (passive smokers), or non-exposed were recruited from the washington d.c. area. all women completed a detailed staffadministered questionnaire probing their medical history, occupational, lifestyle factors and diet. blood and urine samples were collected at the follicular phase. hormone profiles were determined using metabolomics for serum thyroxine and triiodothyronine and steroid hormones, as well as for cotinine using isotopedilution tandem mass spectrometry (lc/ms/ms-api ). in addition, all samples were analyzed for serum lh, fsh, tsh and creatinine. results: the relationships of cigarette smoking, age, relative weight, and dietary intake to serum thyroxine, triiodothyronine, estradiol (e ), estrone (e ), progesterone (p), -hydroxyprogesterone ( ohp),dehydroepiandrosterone (dhea), dehydroepiandrosterone sulfate (dheas), androstenedione, cortisol, -deoxycortisol, corticosterone, testosterone, aldosterone and vitamin d were analyzed using lc/ms/ms. mean tsh in non-exposed, shs-exposed and smokers: . , . , and . miu/ml respectively. similarly, mean t . , . ( % increase) (p= . ), . μg/dl (- %) in active smokers; (active vs. exposed p= . ). shs increased dhea levels ( % higher, p = . ), dheas ( % higher, p = . ), cortisol ( % lower, p = . ), aldosterone ( % lower, p = . ) and androstenedione ( % higher, p = . ). these data suggest that active smoking and shs can have a profound effect on serum t , adrenal steroid and sex hormone concentrations in women of childbearing age. objective: to determine the prevalence and predictors of the metabolic syndrome (mbs) among in saudi women with polycystic ovary syndrome (pcos) in comparison to women without pcos and to assess the role of androgens and insulin resistance (ir) in mbs development. design: a prospective case control study. setting: tertiary referral university hospital. subjects: six hundreds saudi women living in the jeddah area were classified as follows: with pcos and age-matched women without pcos. interventions: blood samples were collected from all women with or without pcos between : - : , after an overnight fast. main outcome measures: measures of abdominal obesity, blood pressure and that of serum levels of lh, fsh, tsh, ft , -ohp, -a, dheas, total t, free t, shbg, insulin, hdl-c, triglycerides and plasma levels of glucose. measures ir including: fasting serum insulin, gir and homa. results: age-adjusted prevalence of mbs was higher in women with pcos ( . %, % ci: . - . %) as compared with women without pcos ( . %, % ci: . - . %) (p< . ). in the same age group, the risk of mbs in women with pcos was greater than that for women without pcos (p< . ). markers of ir were significantly abnormal in women with both pcos and mbs in comparison to those without mbs (p< . ). the most common abnormal components of mbs in women with both pcos and mbs (after adjustment for age) were: decreased hdl-c ( . ± . %); increased triglycerides ( . ± . %); and increased bmi ( . ± . %), respectively. the prevalence of mbs from lowest to highest tertile of free t level was . , . and . %, respectively; in women with both pcos and mbs. in women with pcos, % exhibited all components of mbs; . % had components, and . % had components. conclusions: women with pcos exhibited significantly higher prevalence of mbs ( . -fold) as compared with age-matched control without pcos. ir is a possible common pathogenetic factor for both mbs and the pcos. it is suggested that more intensive screening and/or therapy monitoring of mbs among women with pcos should be part of the therapeutic modalities of the condition. case of sisters with complete androgen insensitivity syndrome and discordant mullerian remnants. jennifer l nichols, jennifer j gell, eric j bieber. reproductive endocrinology and infertility, geisinger medical center, danville, pa, usa. complete androgen insensitivity is an x-linked recessive disorder resulting in the abnormal expression of the androgen receptor. affected individuals are most commonly phenotypically female but genotypically male. the prevalence of this disorder is in , live male births. we present a case of complete androgen insensitivity in members of the same family with differing residual mullerian tissue. sister a presented at age for evaluation of primary amenorrhea. a karyotype revealed ,xy. an mri of the pelvis showed a hypoplastic uterus but no ovaries. this patient underwent laparoscopic bilateral gonadectomy and hemihysterectomy. on examination under anesthesia, she was noted to have a normal vagina with no cervix noted. at laparoscopic evaluation, she was noted to have bilateral elongated gonads and what appeared to be a remnant of uterine tissue. pathology revealed portions of immature testicles and fragments of smooth muscle in what grossly appeared to be the uterine remnant. the patient's total testosterone following surgery was noted to be elevated at . ng/ml. other laboratory evaluation showed fsh . miu/ml, lh . miu/ml, free testosterone . pg/ml, and estradiol . pg/ml. approximately two years later sister b presented at age for evaluation of primary amenorrhea. no uterus or ovaries were visualized on pelvic ultrasound. again a karyotype revealed ,xy. laboratory evaluation demonstrated fsh . miu/ml, lh . miu/ml, estradiol . pg/ml, total testosterone . ng/ml, and free testosterone . pg/ml. she underwent a laparoscopic bilateral gonadectomy. no uterus, cervix or pelvic masses were identified on exam under anesthesia. at laparoscopy, both gonads were visualized and removed without difficulty but no uterus was visualized. pathology reported two testicular and epididymal-like structures. this case demonstrates the presentation and laparoscopic results of complete androgen insensitivity syndrome discovered in two siblings. although both girls are genotypically male, they differ in the presence of vestigial mullerian tissue. the case shows with complete androgen insensitivity, as an x-linked defect, one should consider apparent sisters of affected individuals, as well as offspring of unaffected individuals with a family member diagnosed. background: anandamide (n-arachidonoylethanolamine, aea) is an endocannabinoid that binds to and activates the cannabinoid receptors, cb and cb and may have important roles in the regulation of human reproduction. the traditional lipid extraction methods used for aea are cumbersome, slow and of low efficiency. the aim of this study was to determine whether the use of a solid phase (spe) method of aea extraction from human plasma would offer any advantages over the traditional liquid phase (lpe) method. methods: pooled human plasma was obtained from the local blood transfusion unit and aliquots stored at - °c prior to extraction. an internal standard of . pmol of deuterated aea (aea-d ) was added to each plasma sample and aea extracted from aliquots on each occasion over three days using the lpe and spe methods. spe was performed with waters oasis hlb cc/ mg cartridges on a waters vacuum manifold. cartridges were activated with methanol and water, the samples applied and washed with % methanol at ml/min. aea was eluted with ml acetonitrile and the eluents dried under a stream of nitrogen before reconstitution in ml acetonitrile. aea levels were measured using uplc-ms/ms against authentic standards. results: these are shown in the table. conclusion: the spe method was -fold more efficient at extracting aea compared to the traditional lpe method. the intra-day and inter-day assay variability were similar for both techniques, although the spe method was quicker, cheaper and required less plasma to generate data similar to that from the traditional lpe method, suggesting that the spe method may be more efficient than the lpe method, and thus making it more suitable for routine analysis of multiple plasma aea samples. background: the precise role of the endocannabinoid, anandamide (narachidonoylethanolamine, aea) in reproduction has been hampered by difficulties in its accurate measurement. aea levels have previously been measured by tlc, gc-ms and hplc-ms but these are laborious. our aim was to improve the analysis of aea using uplc and tandem ms/ms with a standard isotope-dilution method , . methods: authentic non-labelled and deuterium-labelled aea (aea-d ) diluted in acetonitrile were maintained at °c before analysis and separation by uplc on a c ( . x mm) column maintained at °c using a gradient of % a, . min: % a, . min: % a, . min: % a, . min: % a with a flow rate of . ml/min. the mobile phases were a ( mm ammonium acetate, . % formic acid) and b (acetonitrile, . % formic acid). the analytes were quantified using multiple-reaction monitoring in positive ion mode with a quattro premier mass spectrometer. entry, collision and exit energies were - , and - ev, respectively. calibration curves were performed in triplicate with . pmol aea-d as the internal standard. transitions employed were . > . and . > . for aea and aea-d , respectively. results: calibration curves ( . to fmol aea on column; n= ) were linear, producing a mean (±sd) gradient of y = . ± . x, crossing the y-axis very close to the origin ( . ± . units). variability was limited, with an r = . . measurements were precise, fmol aea produced a cv of only . %, and the retention time was consistent at . ± . min (n= ). the limit of quantification (signal/noise> ) was . fmol on column and the limit of detection (lod) was . fmol on column (signal/noise= ). accuracy for . fmol, . fmol and fmol aea was . ± . %, . ± . % and . ± . %, respectively. conclusions: the method described is an improvement over other lc-ms/ms methods , with a lower lod [ . fmol v. fmol or fmol ] and shorter run time [ min v. min or . min ]. thus, an improvement in terms of speed, increased sensitivity and better reproducibility will allow for a more accurate assessment of aea concentrations in a number of biological samples. objectives: the gata family of transcription factors consists of six zinc-finger proteins with a critical role in tissue-specific and developmentally-regulated gene expression. gata factors exert their effects alone and through interactions with cofactors, such as friend of gata (fog), as well as with nuclear hormone receptors, including steroidogenic factor- (sf- ), a well-described activator of gonadotropin gene expression. the objective of these studies was to define the role of gata family members in the gonadotrope. methods: ) total rna was extracted from the gonadotrope cell line, l t , and analyzed by standard rt-pcr analysis. ) the cv- fibroblast cell line was transiently transfected by the calcium phosphate precipitation method with a rat - /+ lh promoterreporter vector as well as cmv-driven expression vectors for gata- , gata- , dngata- , fog- and/or sf- . results: gonadotrope l t cells were found to express transcripts encoding gata- , gata- , and gata- as well as fog- and fog- . gata- and gata- stimulated lh gene promoter activity by approximately -fold (p< . ) and synergistically increased the sf- response ( -fold versus -fold for sf- alone; p< . ). the gata-mediated increase in lh gene expression was nearly eliminated with co-transfection of fog- . similarly, co-transfection with a gata- dominant negative construct blunted wild-type gata- effects in a dose-dependent fashion. conclusions: these data demonstrate expression of both gata and the functionally related fog proteins in a well-characterized gonadotrope cell line. furthermore, they demonstrate a functional role for these factors in regulation of gonadotrope function, specifically expression of the lh gene. low-dose dexamethasone (dex) therapy early in pregnancy is used in fetuses with suspected risk of congenital adrenal hyperplasia. several adverse neurological events in prenatally treated children have been reported and the fetal hypothalamic-pituitary-adrenal (hpa) axis may be involved. aim: to investigate the immediate and long-term effects of early maternal dex administration on fetal growth and pituitary-adrenal activity in sheep. method: pregnant ewes carrying singleton fetuses (total n= ) were randomized to control ( ml saline/ewe) or dex treatment ( . mg/kg ewe weight) consisting of four intramuscular injections at -hourly intervals over hours on - days of gestation (dg). at , , , and dg fetal weights were recorded. i -ria, qrt-pcr and in-situ hybridisation were used to measure fetal plasma cortisol and acth levels and to analyse adrenal and pituitary mrna expression. results: dex-exposed fetuses were lighter than control animals at dg*, but not at other times; in general fetal organ weights were similar between treatment groups. fetal plasma acth was unaffected by dex and did not differ between genders. similarly, pomc and pc- mrna in pars distalis were unaltered after dex. however, fetal plasma cortisol was reduced after dex in both male and females at dg*, was similar at and dg, then elevated at dg*. plasma cortisol in female fetuses in control and after dex was significantly higher than in males. the increases in cortisol after dex at dg* were associated with increased fetal adrenal expression of p c and bhsd mrna in females, reduced expression of mc r in males, but no difference in star mrna. conclusion: we conclude that in sheep, early dex programs the developmental trajectory of the fetal hpa with increased activation directly of the adrenal, but not pars distalis function. in females this effect may be attributed to increased fetal adrenal steroidogenic activity. the effect of dex in increasing cortisol in males, albeit at a significantly lower level than in females, appears to be independent of the enzymes that we have measured.*p< . . synaptophysin and gonadotropin-releasing hormone (gnrh) are colocalized in rat hypothalamus. armando arroyo, beom su kim, amanda biehl, blenna cl bett, , john yeh. gynecology-obstetrics, university of buffalo, buffalo, ny, usa; physiology and biophysics, university at buffalo, buffalo, ny, usa. the cellular and molecular mechanisms that control gonadotropin-releasing hormone (gnrh) release are not completely understood. gnrh is stored in synaptic vesicles and released by exocytosis at gnrh nerve terminals. there are currently nine families of synaptic vesicle proteins that are involved in neurotransmitter release by exocytosis. synaptophysin is one of the most common synaptic vesicle proteins present in synaptic vesicles in neurons. the hypothesis of this study is that synaptophysin is expressed in gnrh neurons. we obtained sections from the hypothalamus of female sprague dawley rats. double-label fluorescence immunohistochemistry was performed on free-floating sections. sections were incubated with a mixture of mouse monoclonal antibody against gnrh ( : -chemicon international) and with a rabbit polyclonal antibody against synaptophysin ( : -santa cruz biotechnology) for h. after incubation the sections were washed and incubated with a mixture of alexa conjugated goat antimouse and alexa conjugated goat antirabbit ( : ; molecular probes) for h. slices were visualized with confocal microscopy (zeiss lsm- ). fifteen out of a total of fifteen gnrh cell bodies in the medial preoptic area and most gnrh neuron terminals in the median eminence showed intense immunostaining for synaptophysin. this is the first study to demonstrate that synaptophysin is expressed in rat gnrh neuron terminals. this suggests that synaptophysin is present in gnrh neuron vesicles. thus, gnrh release by exocytosis may be mediated by synaptic vesicle proteins. objective: our aim was to identify the effects of early gestation gc exposure on fetal and postnatal hpa axis development and function in postnatal life. method: pregnant ewes carrying singleton fetuses were randomized to control ( ml saline/ewe) or dex groups ( . mg/kg), consisting of four at h intervals on days - of pregnancy. at months postnatal age, catheters were implanted; a bolus injection of crh ( . mg/body weight) and avp ( . mg/body weight) were administered and arterial blood samples were taken at - , - , , , , , , , and min. levels of hepatic cbg mrna were determined by qpcr, expressed relative to s rrna. plasma cortisol and cbg levels were measured by radioimmunoassay. results: both total and free cortisol levels in the dex females (n= ) were lower than in dex males (n= ) from to min (p . ) and lower than in control females (n= ) at minutes (p . ), also in the dex-m group were higher than in control males (n= ) at min (p . ). plasma cbg levels and cbg mrna expression were not altered by dexamethasone exposure or sex. conclusions: these findings suggest that prenatal glucocorticoid exposure alters the development of the hpa axis differentially according to the sex of the exposed fetus. to determine maternal injections of synthetic glucocorticoids in early gestation can alter expression of hippocampal corticosteroid receptors at months postnatal age. method: pregnant ewes carrying singleton fetuses were randomized to control ( ml saline/ewe) or dexamethasone treatment ( . mg/kg) consisting of four injections at h intervals on days - . hippocampal was collected from the offspring at months postnatal age. levels of mrna of gr and mr were determined using qpcr and levels relatived to s rrna. results: dexamethasone-treated male animals (n= ) had significantly higher levels of mr gene expression than both control males (n= ; p= . ) and females (n= ; p= . ). gr gene expression levels were higher in treated vs. control males (p= . ), but in females levels in treated and control animals were similar. total body, brain and hippocampus weights were similar. conclusions: maternal dexamethasone administration in early pregnancy resulted in gender-dependent changes in hippocampal gene expression when measured in the offspring seven months after birth. objective: diminished ovarian reserve (dor) affects many younger women. we analyzed superovulation with intrauterine insemination (so) cycles in vitro fertilization (ivf) cycles of women with dor to determine if women < with dor differ from their older counterparts. method: irb approved retrospective review of so ivf cycles from / - / with follicle stimulating hormone (fsh) levels based on age < or . results: so cycles were performed in women. no differences were noted in clinical pregnancy. women < were more likely to have inseminates with a lower mean tms/ins, median tms/ins was . among pregnancy cycles compared to for unsuccessful cycles. for ivf, mean total gonadotropin dosage was significantly lower in women < , mean number of follicles mm peak e were significantly higher in women < . so ivf measures are in tables , respectively. conclusions: similar clinical pregnancy outcomes were seen despite age. in ivf, women < required less gonadotropins and generated more follicles but with no significant difference in number of mature oocytes or clinical pregnancies. of note, pregnancy rates for so in women < with dor are substantially lower than expected in our clinical practice. as ivf yielded a substantially higher chance of pregnancy, consideration should be made to expedite progression to ivf. to assess the effects of intraovarian injection of ad-fshr on the reproductive system of fshr (-/-) mice. methods: about l containing x pfu of ad-hfshr were injected directly into each ovary of treated group and same amount of ad-lacz were injected into the ovaries of control animals. vaginal smears were collected and body weight was measured daily. four weeks after the injection animals were sacrificed and all organs were weighted and evaluated by h e. fsh, e and p measured before and after treatment. results: ad-hfshrtreated mice showed obvious estrogenic changes in vaginal smear while in control animals vaginal smear remained at diestrus stage. significant increase in total body weight and estrogen dependent organs weight (uterus, ovary, vagina) was observed in treated animals compare to control group (p< . ). no significant weight changes were observed in other organs. h e evaluation of the ovaries showed significant increases in both the total number of follicles and the collective diameter of the follicles in treated animals compare to controls. on average follicles/ovary were observed in ad-hfshr-treated group of which follicles were at the antral stage while only follicles observed in ad-lacz control group, with zero follicles at antral stage. a . to folds increase in e and about % decrease of fsh observed in treated animals compared to control mice. there was no significant change in serum progesterone level between treated and control groups. conclusion: intraovarian injection of an adenovirus expressing human fshr gene is able to restore folliculogenesis and resume estrogen hormone production in female forko mice. objective: the sentinel issue surrounding multiple gestations following ivf is the inability to precisely estimate the reproductive potential of individual embryos with the currently used embryo grading systems based on embryo cleavage rate and morphology. recently, metabolomic profiling of spent culture media using raman and near-infrared spectroscopy have been reported to predict reproductive potential of embryos. in this study we applied proton nuclear magnetic resonance (¹h nmr) spectroscopy to analyze metabolomic profile of embryo culture media and to identify components of the media that correlate with reproductive potential. methods: eighteen spent media samples from embryos that failed to implant, and samples from embryos that resulted in pregnancy and delivery were individually collected after embryo transfer on day , and evaluated using ¹h nmr. the spectra obtained were analyzed using a selective genetic algorithm (ga) to determine regions predictive of pregnancy outcome as determined by logistic regression. to avoid random correlations, a leave-one out crossvalidation was used. sensitivity and specificity of predicting pregnancy (described as implantation and delivery) were calculated. results: using the ga, two areas in the ¹h nmr spectral region were identified as most discriminatory between the two groups. viability indices calculated by ¹h nmr using these regions were significantly higher in culture media of embryos with proven reproductive potential ( . ± . ) compared to those that failed to implant ( . ± . ) (p< . ). compiled outcomes from the leave-one-out cross-validation of the logistic regression using the ¹h nmr measurements resulted in a sensitivity of % and a specificity of . %. quantification by integration showed significantly decreased glutamate levels (p= . ) and a trend toward an increase in pyruvate levels (p= . ) in culture media of embryos that did not cause pregnancy. conclusion: metabolomic profile of spent embryo culture media using ¹h nmr correlates with the reproductive potential of embryos. the lower glutamate levels detected in culture media of embryos that failed to implant could potentially be due to the toxicity associated with increased embryonic glutamate uptake. additional studies using complementary approaches are needed to further delineate molecular components associated with reproductive potential. objective: beta-carotene and other carotenoids are known antioxidants previously identified in human follicular fluid (ff). in addition to their inherent antioxidant properties, carotenoids have been identified as precursors of the antioxidant retinol in bovine ff. high retinol levels in bovine ff are associated with non-atretic follicles. this would suggest a possible role for retinol and its carotenoid precursors in follicular heath and the general oxidative state of the follicle. we sought to measure carotenoids and retinol in the ff of women undergoing ivf and correlate these levels with normal fertilization as a marker of follicular/oocyte health. design: prospective cohort study materials and methods: ff from a single - mm follicle was obtained from women (age - ) undergoing ivf and intracytoplasmic sperm injection (icsi). serum was also obtained at the time of oocyte retrieval. retinol, vitamin e ( , , and tocopherol) and carotenoids ( -carotene,cryptoxanthin, lycopene and lutein/zeaxanthin) were measured using hplc. we correlated ff carotenoid and retinol levels with oocyte fertilization status following icsi. results: as previously reported, retinol, vitamin e and carotenoids were all identified in the ff. each fat-soluble vitamin level was significantly lower in ff compared to serum (p< . for all analytes) and the levels were strongly correlated (r > . , p< . for all analytes). mean levels of ff -carotene were significantly higher in those follicles that resulted in a fertilized oocyte ( . +/- . g/ml vs. . +/- . g/ml, p= . ). other carotenoids, retinol, and vitamin e levels did not correlate with fertilization outcomes. conclusions: our finding of a strong association between ff -carotene concentration and subsequent normal fertilization of the oocytes suggests an important antioxidant role for -carotene in the health of the human ovarian follicle/oocyte. the lack of correlation with other carotenoids, retinol and vitamin e suggests that the antioxidant properties of -carotene act by preventing singlet oxygen and scavenging the peroxyl radical and may directly influence oocyte competence. mature oocytes obtained during egg retrieval have been shown to undergo spindle depolymerization as a result of cooling. for this reason, ivf programs strive to maintain constant temperature during follicle aspiration. we sought to elucidate if there was a difference in temperature of fluid aspirated into a hand held (hh) or heating block (hb) encased collection tube. the experiment was performed in an ambient temperature of °c. thermistors, pinhead sized sensors with an accuracy of %, were used to monitor temperature differences between control fluid (cf) (sterile water ºc) and aspirated fluid. data was recorded using an -channel data acquisition system from dataq instruments. one thermistor was placed into the cf, the other was placed into an empty collection tube set in the heating block or held in a gloved hand. a cm, gauge, single lumen needle connected to cm of tubing was used to aspirate into the empty collection tube. temperature was recorded x/second, each experiment was repeated times. baseline empty collection tube temperature was significantly cooler in the hh vs the hb group ( . ± . °c vs . ±. °c, p=. ). two seconds after aspiration, the lowest aspirate temperature was observed, (hh . ±. °c, hb . ±. °c, p>. ) no difference between groups. in both groups, temperature quickly increased as aspiration progressed (hh . ±. °c, hb . ±. °c, p>. ). the hb group took an average of . min to return to baseline ( °c), the hh group never returned to baseline. substantial cooling of aspirated fluid occurs during oocyte retrieval, with a mean temperature decrease of . ±. °c corresponding to . °c. considering this dramatic decrease, the difference between temperatures in the hh vs. the hb group is negligible. current aspiration systems poorly regulate temperature, thus the choice of aspirating into a test tube warmed by hand or by heating block is inconsequential. clinical management of twin gestations with recurrent preterm labor symptoms. lesley de la torre, luis roca, niki b istwan, debbie j rhea, gary j stanziano, victor hugo gonzalez-quintero. obstetrics and gynecology, university of miami medical center, miami, fl, usa; clinical research, matria healthcare, marietta, ga, usa. objective to examine pregnancy outcomes in women with twin pregnancies receiving nifedipine tocolysis (nt) who experienced recurrent preterm labor symptoms (rptlsx). study design twin pregnancies enrolled for outpatient preterm labor (ptl) surveillance services prescribed nt following an initial episode of ptl were identified from a database (n= ). eligible for inclusion were patients later hospitalized with acute rptlsx (n= ). included were those < weeks' gestational age (ga), with intact membranes, remaining undelivered for > hours after rptlsx . pregnancy outcomes of women resuming nt (rnt group, n= ) following hospitalization were compared to those having an alteration in treatment (alttx group, n= ) to continuous subcutaneous terbutaline. per normality assumptions, either independent student´s t or mann-whitney u test statistics were used for continuous variables; pearson´s chi-square for categoric. all p-values presented as two-sided, significant at < . . results overall, ( . %) of twin pregnancies prescribed nt experienced rptlsx; ( . %) were not eligible for continued tocolysis. pregnancy outcomes are presented in table. conclusion rptlsx are common. in twin pregnancies receiving nt, alteration of tocolytic treatment following rptlsx had a positive impact on pregnancy prolongation and neonatal outcomes. routine cervical dilatation during elective cesarean section -is it really necessary? arie koifman, avi harlev, eyal sheiner, fernanda press, arnon wiznitzer. obstetrics and gynecology, soroka university medical center, ben-gurion university of the negev, beer-sheva, israel. objective: the purpose of this study was to examine the necessity of routine cervical dilatation during elective cesarean section. material and methods a retrospective cohort study was performed, including all cases of elective cesarean sections performed at a tertiary medical center during . stratified analysis, using the mantel-haenszel technique, was done to control for confounders. results out of a total of elective cesarean deliveries (cd), underwent routine cervical dilatation. the overall rate of febrile morbidity was . %. no significant differences in postpartum febrile morbidity were noted between the groups ( . and . %; p= . ). in addition, hospitalization duration did not differ between the groups ( . ± . and . ± . days p= . ). about % of all elective operations were repeated cd. there was no difference in febrile morbidity between the groups even in that subgroup of the elective cd. likewise, there was no difference in anemia rate between the two groups (hemoglobin . ± . mg and . ± . mg p= . ). controlling for a previous vaginal delivery, using the mantel-haenszel technique, no significant association was noted between cervical dilatation and fever (weighted or= . ; % ci . - . ; p= . ). nevertheless, in a subgroup of patients following a previous vaginal delivery, cervical dilatation was significantly associated with post-operative fever (or= . the majority of women with an unfavorable cervix undergoing iol at term deliver vaginally, even with a prolonged first stage of labor. this is important information to discuss with women prior to iol when establishing labor expectations. providers should consider the ongoing success of labor induction when contemplating a diagnosis of "failed induction". objective: this study was performed to investigate and compare changes of the lipid peroxide levels and the protein carbonyls formation in the maternal venous plasma of preterm premature rupture of membrane (pprom) during antibiotics administration. materials and methods: thirty-six pregnant women with pprom between and weeks of gestation were chosen for this study. eighteen patients (group ) were treated with amoxicillin and erythromycin for day period while the other patients (group ) were treated with rd generation cephalosporin and erythromycin for the same period. samples of maternal blood were obtained from the two groups at before the antibiotics administration, day , and day after the antibiotics administration. lipid peroxide levels were measured by thiobarbituric acid reaction and protein carbonyl contents were determined by the , -dinitrophenylhydrazine method. results: . the lipid peroxide levels and protein carbonyls formation in the maternal venous plasma of pprom was significantly higher than that of normal pregnancy ( . ± . vs . ± . nmol/mg protein, p< . ), ( . ± . vs . ± . nmol/mg protein, p< . ). . there were no significant differences in the lipid peroxide levels and protein carbonyls formation of the maternal venous plasma with pprom mixed and incubated by amoxicillin, cefodizime, and erythromycin (in vitro). . there were no significant differences in the lipid peroxide levels and protein carbonyls formation of the venous plasma of group among before the antibiotics administration, day , and day after the antibiotics administration. . the protein carbonyls formation in the venous plasma of group was significantly decreased at day and day after the antibiotics administration than that of before the antibiotics administration ( . ± . , . ± . , . ± . nmol/mg protein, p< . ). conclusion: in the maternal venous plasma of pprom, the lipid peroxide levels and protein carbonyls formation were increased. the results suggest that reactive oxygen species formation by inflammatory reaction is suppressed by combined treatment of rd generation cephalosporin and erythromycin. objective: the aim of the present prospective cohort study was to evaluate the correlation between blood flow pulsatility index in fetal middle cerebral artery (mca) and the onset of spontaneous labor. study design: doppler evaluation of fetal mca were performed between and weeks gestation in consecutive pregnant women with a singleton pregnancy and known gestational age. the study population was divided according to the mca pi (< . or >/= . mom) and, using survival analysis and cox regression, the two subgroups obtained were compared. in these analyses, the event was delivery (following spontaneous labor) and the time variable was time elapsed since doppler exam. results: the median time elapsed between doppler evaluation and spontaneous labor was significantly shorter in the women with mca pi lower than . mom ( . days, interquartile range - ) in comparison with the women with mca pi higher or equal than . mom ( . days, interquartile - . days (p< . , mann-whitney test). after correction for birth weight and umbilical artery pi, survival analysis and cox regression confirmed that mca pi was independently associated with the number of days elapsed from doppler to spontaneous labor and delivery (p< . , exp(b) . , ci % . - . ). conclusions: the present data show that, at term of pregnancy, fetal cerebral resistance reduction anticipates the onset of spontaneous labor. novel calcium sensitizers and human uterine contractility. mark p hehir, audrey t moynihan, terry j smith, john j morrison. department of obstetrics and gynaecology, national university of ireland, galway, ireland. objective: the factors regulating contractility of uterine smooth muscle are central to the occurrence of preterm labour and delivery. calcium sensitizers are a novel class of drugs with unique molecular actions. levosimendan, the best characterized of these compounds, is used in the treatment of acute and chronic heart failure and is a compound which exerts a number of effects on smooth muscle. it can exert an inotropic effect via sensitization of myofilaments to calcium and also exerts a relaxant effect by opening atp-dependent potassium channels. for these reasons we hypothesized that levosimendan may have an effect on myometrial contractility and investigated its action on both spontaneous and agonist induced contractions. method: biopsies of human myometrium were obtained at elective caesarean section (n= ). dissected myometrial strips suspended under isometric conditions, undergoing spontaneous and oxytocin-induced contractions, were exposed to cumulative additions of levosimendan in the concentration range of nmol/l to mmol/l. two sets of control experiments were performed simultaneously as follows: . strips exposed to either physiological salt solution (pss) only, for spontaneous contractions, or . nmol/l oxytocin; . strips exposed to pss/oxytocin and vehicle for levosimendan. results: levosimendan exerted an inhibitory effect on spontaneous and agonist induced contractions, compared to control strips. the mean maximal inhibition values were as follows: . ± . % for spontaneous contractions (n= ; p< . ) and . ± . % for oxytocin-induced contractions (n= ; p< . ). no significant difference was found between control and control for both spontaneous and oxytocin induced contractions. conclusion: the calcium sensitizer levosimendan exerted a potent relaxant effect on uterine contractility in vitro. this action was seen in both spontaneous and agonist induced contractions. the results from this study raise the possibility of calcium sensitizers holding potential tocolytic properties in vivo and further studies are required to investigate the potential benefits of this novel class of drugs. to determine to what degree extensive patient movement affects uterine emg signals and toco signals. study design: pregnant term labor patients were recorded using both uterine emg and toco simultaneously. baseline recordings were obtained when patients were still, and these were used as control records. test recordings for both devices were obtained from all patients by asking the patients to perform movements. area under the rectified-voltage amplitude curve of the uterine emg signals and area under the curve for the toco signals were found. mean %-increase was calculated for the uterine emg and toco devices (each device %-increase values were averaged over all patients). mann-whitney rank-sum test was used to look for any statistical differences in %-increase in area for uterine emg vs. toco methods (p < . considered significant). results: there was a large increase in activity (artifact) on both devices' signals during patient movement (figure ) . both devices' traces eventually returned to baseline after the patient movement stopped. toco movement artifact was significantly higher than emg movement artifact ( table ) . conclusions: both uterine emg and toco signals experience artifact when patients move the uterine emg electrodes and toco pressure transducer, respectively. toco seems to be more adversely affected by such movements. uterine emg may be a preferred method for monitoring contractions of laboring patients in the clinic. supported by grant nih r -hd . results: % of obstetricians completed the questionnaire. none would counsel patients against labour unless there were contraindications. the majority would recommend labour for all indications for previous caesarean section investigated although in all instances, personal preferences were lower (p< . ); after a failed instrumental delivery, % obstetricians would recommend labour but only % would choose that option for themselves (p< . ). overall, female obstetricians would contemplate and recommend labour more readily than male obstetricians. labour augmentation and induction were recommended to patients more frequently ( % and % respectively) than chosen for personal care ( % and %). reluctance for labour augmentation and induction was greatest among younger consultants. conclusion: consultants have responded to consumerism and aim to meet the requests of their patients. they more readily recommend labour than they would choose for personal care, and a majority would recommend labour induction when necessary. informed patient choice is paramount rather than attaining a target figure for women attempting to labour, and the views of those requesting delivery by caesarean section should be respected. (table) . cd increased in nullipara singleton, cephalic, term pregnancy in spontaneous labor, mostly due to dystocia; nullipara singleton, cephalic term pregnancy with induced labor, mostly due to non-reassuring fetal status and dystocia; singleton, cephalic term pregnancy with previous cd, mostly due to elective cd; singleton cephalic preterm. no changes in neonatal outcome were observed. conclusion: clinical audit was useful to keep under control cd rate in our institution in comparison with italian reality, but it was not sufficient to maintain stable cd rate suggesting the need of other, multifaceted strategies. elective delivery at weeks' gestation is a common obstetric intervention. the purpose of this study is to estimate the maternal mortality rates (mmr) associated with this acog -approved intervention. there are no reliable us data describing mmr by mode of delivery. therefore, we base our estimates on british data indicating a procedure related mmr of . / , , . / , and . / , for vaginal, elective cesarean section (c/s) and emergency-unplanned c/s deliveries, respectively. a decision tree model was constructed assuming that all eligible patients (approx. , , /annually in the u.s.) would be delivered at weeks by either an elective c/s or an induction of labor. we further assumed that % of inductions would result in a vaginal delivery. our estimates show that the annual, delivery-related maternal mortality associated with an elective delivery of all patients at weeks would be and for elective c/s and induction of labor, respectively. because vaginal delivery results in the lowest mmr, we performed a oneway sensitivity analysis to identify the impact of changing the success rate of induction on the estimated mmr. the results indicate that once the success rate exceeds %, this intervention would be associated with a lower mmr as compared to elective c/s. our estimates indicate that although the overall mmr associated with elective delivery at weeks is relatively low (approximately . / , deliveries), it is certainly not negligible. in addition, the mmr is highly dependent on the likelihood of a successful vaginal delivery following induction of labor. when the success rate for induction of labor falls below %, an elective c/s appears to be the safer delivery method. background: the first obstetric visit is an opportunity to provide the pregnant patient information regarding substances that can cause potential harm to the pregnancy. little is known about how obstetric care providers handle these topics. objective: to examine patient-provider communication about substance use during the first prenatal visit. methods: we audiotaped first prenatal visits and qualitatively analyzed those tapes in which patients disclosed substance use. we invited patient participants to return for a semi structured interview during which they reviewed their audiotaped conversations and described their reactions to the providers' communication styles. results: providers ( residents, midwives, nurse practitioners) and patients participated. providers asked about smoking, alcohol and drug use in all ( %) visits. patients reported being smokers, reported alcohol use, and reported drug use. provider responses to smoking disclosures included brief discussions of smoking effects on pregnancy, encouragement to quit/cut down, and referral to smoking cessation programs. provider responses to alcohol or drug disclosures included only general statements regarding effects on pregnancy (e.g., "we find that this is bad for babies.") and referral to ultrasound/genetics for reassurance. few alcohol or drug discussions assessed whether the patient had intentions or concerns regarding continued use during the pregnancy. few discussions addressed strategies for behavioral change. none included assessment for motivations, readiness, or barriers to change. in follow up interviews, patient participants said they expected to be asked about substance use but advised providers to ask non-judgmentally. those who used alcohol/drugs wanted more information on potential effects of these substances on the pregnancy/fetus and appreciated the reassurance from referrals to ultrasound/genetics. discussion: counseling for risky behaviors in the first obstetric visits contained only limited discussion of the effects of the risky behaviors and primarily focused on referral-which may be a proxy for avoiding a difficult and time consuming conversations. background. there are conflicting results regarding the association of maternal psychiatric disorders or psychological distress due to stressful life events with pre-term birth and low birth weight. aims. to investigate the association between maternal psychiatric disorders and/or stressful life events and intrauterine growth abnormality, low birth weight or preterm birth. method. three mutually exclusive and homogeneous groups of pregnant women ( with actual psychiatric disorder, with stressful life events, and healthy comparisons) underwent serial fetal ultrasound examinations and uterine and umbilical artery doppler velocimetry at (+ ), (+ ) and (+ ) weeks of gestational age. subjects were recruited from all consecutive women attending two antenatal clinics. the presence of any maternal medical illness, drug treatments, fetal chromosomal and/or structural malformations, were exclusion criteria. all women recruited underwent a structured interview at - weeks for the psychiatric diagnosis (mini international neuropsychiatric interview -mini) (sheehan et al, ) ; moreover, the -item hamilton rating scale for depression and the hamilton rating scale for anxiety were included in the assessment. the person who obtained obstetrical clinical data was blind to the results of psychological evaluations. results. the three groups were comparable for: age, parity, socioeconomic status, smoking, alcohol consumption, body mass index. gestational age at birth was not different in the three groups. infants of women with psychiatric disorders had significantly lower birthweight ( + g) and higher percentage of birth weight below the th centile for gestational age ( %) than infants of healthy mothers ( + g and %, respectively). there was also a trend towards lower mean birth weight ( + g) and higher incidence of birth weight below the th centile ( %) in the stressful life event group. there was no significant difference among groups in the percentage of abnormal uterine or umbilical doppler results. conclusions. maternal psychiatric disorders are associated with a lower birth-weight, but the effect is unlikely to be due to abnormal utero-placental or feto-placental vascularisation. objective: the purpose of this study was to evaluate antenatal ultrasound as a tool for the detection of intrauterine growth restriction (iugr) and small for gestational age (sga) infants among subjects with elevated human chorionic gonadotropin (hcg) levels on second trimester serum screening. although iugr has been linked to elevated hcg levels, the optimal screening regimen for antenatal sonographic surveillance has not been previously established. methods: a retrospective cohort study was performed at saddleback memorial medical center where serial ultrasounds from weeks-delivery are generally recommended for patients with hcg levels > . mom. all pregnancies were dated by second trimester ultrasound ± last menstrual period. subjects with an hcg > . mom, who had at least one antenatal ultrasound evaluation for iugr, were identified from an electronic ultrasound database used for clinical report generation. ultrasound data were then linked to an obstetrical birth outcomes database for relevant demographic/delivery information using unique identifiers. iugr was defined as a sonographic estimated fetal weight (efw) < %ile for the estimated gestational age (ega). sga was defined as an actual birthweight < %ile for the ega at the time of delivery. results: from - , there were subjects with elevated hcg levels who underwent antenatal ultrasound surveillance for iugr and who had known delivery information. a total of ultrasound examinations were performed. the median number of examinations per subject was with a range from - examinations per subject. the incidence of iugr and of sga were . % (n= / ) and . % (n= / ), respectively. no fetus with iugr demonstrated absent or reverse end diastolic umbilical artery doppler flow. antenatal ultrasound examinations only identified . % (n= / ) of sga infants. however, the sensitivity for the detection of sga was % when an efw cut-off of % was used. conclusions: although the majority of sga infants did not demonstrate growth restriction on antenatal ultrasound, a sonographic efw > %ile appears to be a safe cut-off to rule out fetuses at risk for sga. neonatal dry lung syndrome after pprom: reason to change intrauterine referral practice in the netherlands? ellen s hoogakker, christiaan v hulzebos, gerda g zeeman. obstetrics and gynecology, university medical center groningen, groningen, netherlands; pediatrics, division of neonatology, university medical center groningen, groningen, netherlands. background: neonatal dry lung syndrome (dls) is a distinct clinical entity following pprom, mimicking pulmonary hypoplasia but with dramatic respiratory improvement during the first - h . its pathogenesis implies complete collapse of small airways to a degree that capillary forces impede distension by ordinary ventilatory pressures. in the netherlands, women with pprom remain admitted at a level iii nicu perinatal center until they reach wks. when they are referred back to their community hospital, unless they live in the neighborhood of the tertiary center. we question this referral pattern because we still see severe respiratory problems occur > wks. we sought to determine the prevalence of such morbidity, in particular dls, in women with pprom who deliver > wks. methods: retrospective descriptive study of neonatal outcome data of singleton pregnancies complicated by pprom between - wks, who deliver > wks (latency > h) , during the -yr period of - at a single academic center. data were extracted from medical records and electronic department databases. results: pprom pregnancies were identified. all but received at least full course of steroids. ( %) delivered > wks. newborns born at the community hospital needed emergency transportation to a level iii nicu for respiratory morbidity. neonatal outcome data (means ± sd) are listed in the table. there were no cases of late onset sepsis, nec or perinatal mortality. conclusions: respiratory morbidity still occurs after pprom with delivery > wks. further investigation of pregnancy-related characteristics, such as the presence of anhydramnios and the latency period, with regards to dls is needed. modification of current referral practice depends upon complete data derived from all dutch level iii perinatal care centers (n = ). tertiary care center (n = ) to determine if an association exists between macrosomia (birthweight > g) and perinatal outcomes in women with and without gestational diabetes mellitus (gdm). study design: this is a retrospective cohort study of , singleton pregnancies. the study cohort was stratified by the diagnosis of gdm, with the presence or absence of macrosomia as the dependent variable. perinatal outcomes examined included neonatal hyperbilirubinemia, hypoglycemia, respiratory distress syndrome (rds), shoulder dystocia and erb's palsy. chisquare tests were performed as well as multivariable analyses controlling for confounders, using p< . to indicate statistical significance. results: in women diagnosed with gdm, macrosomia is associated with a higher frequency of hypoglycemia, respiratory distress syndrome, shoulder dystocia and erb's palsy. though the prevalence of these outcomes is relatively decreased in patients without gdm, they are still more prevalent in macrosomic patients. macrosomia is associated with a higher prevalence of adverse perinatal outcomes in women with and without gdm. therefore, it is important to evaluate neonates with birthweights greater than grams for hypoglycemia and unrecognized erb's palsy. conclusion a significant proportion of our obstetrical patients are obese and many do not perceive themselves to be obese. while our finding of an inverse correlation between level of education and bmi may be confounded by socioeconomic status, our results suggest that in order to address the problems of obesity in our population an important first step will be improving the education of our reproductive age women regarding normal weight gain and nutrition in pregnancy. this may have a significant impact on improving pregnancy outcomes of today's obstetrical population. objective: the aim of the study was to evaluate the impact of a "flat" oral gct upon the outcome of pregnancy. the gct was considered flat when the difference between the basal value and the after load value was %. methods: we prospectively analyzed the outcomes of pregnancies who delivered at our department. inclusion criteria were singleton pregnancy; bmi < kg/m , absence of major risk factors for diabetes and of pregestational diabetes. g -hour oral gct was performed at - weeks of gestation. women were subdivided into groups according to the result of the gct: group = negative (load glucose > % and < mg/dl) women ( . %); group =flat (load glucose % than basal and < mg/dl) women ( . %); group =positive gct/negative ogtt, women ( . %). data are mean ± sd. differences were calculated with the student t test for unpaired samples and test. regression analysis were performed by the least squared method. p-values were considered significant at p< . . results: the characteristics and obstetric outcomes in the three groups are presented in the table . in all patients there was a significant linear relationship between the load and basal glucose values (load value= . + . basal value; r= . ; p< . ) and between birthweight and load values (birthweight= . + . load value; r= . ; p< . ). the relationships were significant also in group and separately but not in group . in this preliminary study we found no major outcome differences in women with flat gct compared to women with normal and gct positive/ ogtt negative results. it seems important, however, to futher investigate the meaning of a flat curve in a bigger population and/or by means of metabolic studies with the use of stable isotopes. results characteristic of the study population and obstetric outcomes are presented in table . the probability to be primiparous and to deliver babies < ° centile decreased significantly with bmi whereas the risk of cesarean section, of post partum hemorrhage at vaginal delivery and to deliver babies > ° centile increased significantly. also the risk to develop preeclampsia and gestational diabetes was increased, although not significantly, with bmi. compared to lean and normal, obese were more likely to be hypertensive and diabetic before pregnancy (p< . ) and to start pregnancy without any medical and obstetrical risk but obesity ( . % vs . and . %; p< . ). african women exhibited the highest bmi and the highest rate of obesity (table ). conclusions we confirm that obese women have an increased risk of prepregnancy and pregnancy complications: less than % have an uncomplicated pregnancy. at delivery there is an increased risk of cesarean section and post partum haemorrhage. objective: overweight and obese women often give birth to larger babies, which is associated with traumatic birth injuries and an increased risk to develop obesity, diabetes and hypertension in childhood and later in life. the mechanisms underlying fetal overgrowth in obese pregnant women are largely unknown. the aim of this study was to establish a mouse model of obesity/high fat diet in pregnancy. we hypothesized that a moderately high fat diet prior to and during pregnancy would result in increased maternal adiposity, fetal overgrowth and a metabolic profile similar to that of obese newborn offspring with persistent pulmonary hypertension, despite enhanced pregnant women. method: c bl/ j female mice were fed control (c, % of energy from fat) or isocaloric high fat (hf, % of energy from fat) diets ad libitum for weeks prior to mating and during gestation. at gestational day . maternal blood samples were obtained to measure adiponectin, leptin and cytokine levels and maternal fat pads were isolated and weighed. in a sub set of animals measurements of transplacental transport of neutral amino acids and glucose were performed in vivo under ketamine anesthesia using h-meaib, and c-glucose. results: no significant differences were observed in maternal pre-pregnancy bodyweight, total caloric intake, weight of the dam at e . or litter size between treatment groups. fetal weight was increased in the hf group by % (p< . ). maternal adiponectin levels were significantly (p< . , n= ) decreased (hf ± , c ± g/ml) and leptin levels increased by % in animals fed a high fat diet, but this difference did not reach statistical significance (n= ). adiposity (fat pad weight) was increased by % (p< . , n= in the dams fed high fat diet, however no difference was observed in maternal il- levels and neither group had measurable levels of tnf-. maternal red blood cell lipid profiles were altered in high fat animals with an increase in stearic and linoleic acids but decreased oleic acid levels. preliminary data showed that placental uptake and transfer to the fetus of glucose and meaib were increased by at least % in dams fed high fat diet. conclusion: this murine high fat diet model has several features consistent with human obesity in pregnancy and the maternal metabolic environment is similar to that seen in the human. the increase in placental uptake and transfer of nutrients constitute a key mechanism underlying fetal overgrowth in overweight/obesity in pregnancy. objective: epidemiological studies have demonstrated a positive relationship between maternal overnutrition and the development of the metabolic syndrome in the offspring. we previously reported that lambs of well fed ewes have increased plasma glucose levels in early life, this may be a consequence of altered hepatic glucose production. increased hsd expression is associated with an increase in intracellular cortisol, promotion of hepatic insulin resistance and a consequential increase in gluconeogenic activity. hypothesis: we hypothesised that maternal overnutrition in late gestation in the sheep results in increased hepatic expression of hsd in the lamb before and after birth. methods: ewes were provided with either % (control,c) or % (well fed, wf) of maintenance energy requirements from d gestation until delivery. post-mortem was performed on either ± d gestation (c= ,wf= ) or and postnatal day (c= ,wf= ). plasma glucose and leptin concentrations in the fetuses and lambs were determined. the relative hepatic mrna expression of hsd and reference gene rpp were determined by qrt-pcr. results: relative liver weight was significantly higher in lambs of wf ewes compared to c at d (p< . ). the expression of hsd mrna was significantly higher in the postnatal compared to the fetal lamb (p< . ) independent of maternal nutritional treatment. however, there was no effect of maternal overnutrition on the hepatic expression of hsd mrna before or after birth. there was no effect of prenatal nutrition on fetal or postnatal plasma cortisol concentrations. the expression of hsd in the liver of lambs of wf, but not c ewes, was inversely related to plasma glucose concentrations in the first hrs after birth (r =- . , p= . ). we have therefore demonstrated that exposure to prenatal overnutrition results in an inverse relationship between hsd mrna expression in the liver at d and plasma glucose concentrations on the first day of life. this suggests that exposure to high glucose levels before and immediately after birth results in a reduced expression of hepatic hsd . we would expect a reduction, rather than promotion of intra-hepatic cortisol production, and therefore it is unlikely to explain the hyperglycemia present in lambs of wf ewes in early life. ninety-seven ( %) of mothers were classified as obese (bmi> ) based on their first pregnancy weight. glycemic control at weeks was superior in the non-obese group (table ) . there were no differences in glycemic control during the last week of pregnancy. obesity was significantly associated with increased maternal weight gain during pregnancy. mean birth weights, ponderal indices and rates of macrosomia were significantly higher in infants born to obese women when compared to non-obese (table ) . primary cesarean deliveries rates were comparable. the rate of neonatal hypoglycemia, hyperbilirubinemia, phototherapy and neonatal icu admissions did not differ between obese and non-obese diabetic women (table ) . although not statistically significant, there was a trend towards an increased rate of birth injuries in the obese group. a similar comparison between obese and non-obese women treated with medication (glyburide or insulin) demonstrated a higher mean birth weight ( g+ vs. g+ , p=. ) and higher rate of macrosomia ( vs. %, p= . ). there were no differences in glycemic control, cesarean delivery rates and other neonatal outcomes between the obese and non-obese treated with medication. conclusion: obese women with type ii and gdm give birth to larger infants than their non-obese counterparts and have a higher incidence of fetal macrosomia. there was a trend towards increased rate of birth injuries in the obese group. despite these differences other maternal and neonatal outcomes were similar which may be a reflection of glycemic control. introduction: tlrs are key components of the innate immune system which recognize conserved sequences on the surface of pathogens and trigger effector cell functions. the placenta, and more specifically the trophoblast, may play an important role in the response to infection. previously, we described the expression of tlr- by human trophoblast and their ability to respond to polyinosinic-polycytidylic acid (polyi:c), a synthetic double strand rna which mimics viral rna. in the present study we evaluate the effect of polyi:c in mouse pregnancy and characterize the local and systemic response. material and methods: human first trimester trophoblast cell line, htr , was treated with polyi:c. c b/ wild type and tlr- knock out mice were injected intraperitoneally with polyi:c at . gestation day. cytokine and chemokine level were determined in supernatant and lysates using the bio-rad multiplex assay and analysis was done using the bio-plex is. results: polyi:c induced cytokine (il , il and il ) and chemokine (il , rantes, gro , mcp and mip ) secretion and production by human cultured trophoblast in a time ( - h) and a dose ( - g/ml) dependent manner. injection of polyi:c to c b/ wild type mice induced preterm delivery within h at a dose of mg/kg body weight. no effect was observed in tlr- knock out mice. a robust systemic (spleen and serum) and local (placenta and amniotic fluid) inflammatory response was observed and h following polyi:c treatment. trophoblast cell cultures from tlr- ko mice confirmed that the response to polyi:c is tlr- dependent. conclusion: we demonstrate that viral infection may trigger an immune response leading to preterm labor. furthermore we show that the trophoblast is able to recognize and respond to viruses through the expression of tlr- . our findings provide a novel mechanism of pathogenesis of preterm labor associated with tlr- mediated inflammatory response. introduction: adverse neurological outcome is a major cause of neonatal morbidity after a preterm birth (ptb). a growing body of evidence demonstrates the involvement of inflammatory pathways in ptb and implicates these pathways as a causative factor in fetal brain injury. however, activations of cytokine pathways in normal labor (whether iatrogenic preterm or at term) has been observed. what remains understudied is the effect of labor on the preterm fetal brain and whether an inflammatory stimulus is essential for fetal brain injury. methods: mouse models were utilized: ( ) a model of intrauterine inflammation (lps into uterine horn) (n= ); controls received intrauterine saline (n= ) and ( ) autism is a neurodevelopmental disorder with a strong genetic component and several known environmental risk factors, such as infection. in addition, its onset of etiology is likely to occur during prenatal development. we propose that subjecting fetal sheep via amniocentesis to the bacterial endotoxin lipopolysaccharide (lps) injected to the amniotic fluid at gestational day (gd) will result in morphological alterations in the offsprings' cerebellum resembling alterations found in the cerebellum of patients with autism. using high precision design-based stereology, we investigated mean total-and layer-specific volume and mean total granule and purkinje cell (pc) number in the cerebellum of lps infected animals and controls. the results of the present study showed preserved volumes of the total cerebellum as well as of the molecular layer, outer and inner granular cell layers and white matter. interestingly, compared to controls, the lps infected brains showed a statistically significant increase (+ . %) in the mean total number of granule cells, whereas the pcs did not show any difference between the groups. these seemingly paradoxical results might be explained by ( ) the so-called time of origin of these neurons, i.e. the pcs develop prenatally whereas the granule cells develop postnatally or ( ) the direct correlation between pcs and granule cell number in the cerebellum. these results might contribute, as an animal model, to our understanding of the biological basis for interindividual differences in morphological alterations found in the brains of patients with autism. the previous studies have shown that there is a higher incidence of spontaneous preterm birth and poorer neonatal outcome in pregnancies with a male fetus. in vitro studies also report a sex dependent pattern in different placental enzymatic systems. we have shown previously that lactobacillus rhamnosus gr- supernatant is able to antagonize the actions of lps on cytokines and ptgs in placental trophoblasts. we hypothesize that fetal sex will influence the production of cytokines and prostaglandin regulating enzymes in lps and lactobacilli treated placental trophoblast cells. methods: term placentae were collected from women undergoing elective caesarean section. placental trophoblasts were isolated using established primary culture protocols. cells were pretreated with lactobacilli supernatant and subsequently treated with lps. pgdh and ptgs expression levels were measured by western blot analysis and tnf-, and il- concentrations measured by elisa. results: lps stimulation caused a marked increase in production of tnf-( . pg/ml to . pg/ml, n= , p< . ), an effect that was greater in placentae of the male fetuses ( . pg/ml, n= ) compared to female fetuses ( . pg/ml, n= ). lactobacilli supernatant abolished this response in both sexes. lps-activated trophoblasts from placentae of the male fetuses showed an increase in il- production (n= , p< . ) and ptgs expression (n= , p< . ). however, there was no response to lps in placentae of the female fetuses. lactobacilli supernatant up-regulated pgdh (n= ) by %, and this effect was greater in placentae of the female fetuses (n= ). conclusion: we conclude that human placentae from pregnancies carrying male fetuses are more responsive to lps by producing more pro-and anti-inflammatory cytokines, as well as ptgs . conversely, placentae of the female fetuses upregulate pgdh with lactobacilli treatment. these findings may explain the underlying mechanism for the higher incidence of preterm birth and adverse pregnancy outcomes seen with male fetuses in the clinical setting. , ) . this study investigates whether iai is associated also with altered expression of neuropilin- . methods: (i) immunohistochemistry (ihc) was performed on tissue sections of term decidua with or without clinical / histologic evidence of iai (n= for each). neuropilin- expression was scored by an investigator blinded to the identity of the samples. (ii) cultured term dscs were retrieved from elective cesarean (n= ), purified, and depleted of leukocytes. after treatment with - m estradiol (e ), - m medroxyprogesterone acetate (mpa), both, or vehicle for days, dscs were stimulated with il- b ( - ng/ml), tnfa ( ng/ ml), or thrombin ( . iu/ml) for h. since no elisa exists for neuropilin- , protein expression was determined by immunocytochemistry (icc). (iii) total rna was extracted and the effect of il- b on neuropilin- mrna expression measured by real-time rt-qpcr and corrected for b-actin mrna. results: neuropilin- expression in term decidua was increased in tissues with iai vs controls (p< . ), and localized primarily to dscs. using icc, an increase in neuropilin- was noted after stimulation with il- b and tnfa, but not thrombin. il- b increased neuropilin- mrna expression in dscs by . ± . -fold (from . ± . to . ± . neuropilin- mrna/b-actin mrna; p= . ). conclusions: iai is associated with increased expression of the vegf receptor, neuropilin- , in term decidual tissues. il- b and tnfa (but not thrombin) stimulated neuropilin- expression in term dscs, and this effect appears to be mediated at the level of gene transcription. since aberrant vegf function alters vascular permeability, these data provide a mechanism by which iai can promote 'decidual activation' and preterm labor. inflammation, university of glasgow, glasgow, united kingdom. objective: asthma is associated with inappropriate activation of airway smooth muscle, chemokine expression and accumulation of mast cells which drive smooth muscle reactivity. labour is similarly associated with smooth muscle activation and expression of cxcr and cxcr ligands. the role of mast cells in human parturition is unknown; however, mast cell products can stimulate myometrial contractions and preterm labour in animal models. we have quantified mast cells in association with human labour and determined whether they express cxcr and cxcr . methods: lower segment myometrial and cervical biopsies were taken at term caesarean section from women not in labour (nil) (myometrium n= ; cervix n= ) and in labour (il) (myometrium n= ; cervix n= ). mast cells were localised in myometrial and cervical sections by icc with a primary antibody against c-kit. the number of cell transects in randomly selected high-powered fields ( x) was quantified blindly by two observers for each specimen, with median density and interquartile range (iqr) calculated. backto-back icc was performed to determine whether c-kit co-localised with the chemokine receptors cxcr and cxcr . results: mast cells were in close association with myometrial smooth muscle in non-labouring lower segment myometrium. labour was associated with a significant influx and increase in mast cells numbers (nil median . , iqr . - . ; il median . , iqr . - . , p= . ). in contrast no significant increase in mast cells was observed in cervical tissue in association with labour (nil median . , iqr . - . ; il median . iqr . - . , p= . ). analysis of chemokine receptor expression demonstrated co-localisation of cxcr to c-kit positive cells present within the myometrium. conclusions: human labour at term is associated with an increase in mast cells within the myometrium, with close approximation to smooth muscle bundles. these mast cells express the chemokine receptor cxcr , the ligands of which we have previously shown to be up-regulated in labouring myometrium. mast cells are not accumulated in cervix in association with labour suggesting a less critical role in cervical ripening. further analysis of the role of mast cells in modulating myometrial smooth muscle physiology is warranted. progestins and the glucocorticoid receptor in human myometrial and amnion-derived wish cells. alison j tyson-capper, stephen c robson. reproductive sciences, newcastle university, newcastle upon tyne, united kingdom. background and objectives: progesterone (p) can reduce the risk of preterm birth in some high risk women. in this context there is accumulating evidence that this, at least in part, may be due to anti-inflammatory and immunoregulatory properties of p. target tissue responsiveness to p is considered to be determined by the progesterone receptors (pr) and nuclear co-factors that directly interact with pr. pr and glucocorticoid receptors (gr) share several structural and functional characteristics, including similarities in dna sequence recognition by binding to the same hormone response elements. pr and gr interact with similar chaperones in the absence of ligand and with a similar group of co-activators in the presence of hormones; both can display comparable anti-inflammatory activities under specific physiological conditions. in this study we aimed to investigate whether the anti-inflammatory properties of progestins may be mediated by pr and gr signalling. methods: primary cultures of non-pregnant and term pregnant human myometrial (passage - ) and wish cells were serum starved for hrs and treated with -hydroxyprogesterone ( -hp), progesterone (p), dexamethasone (dex) and immunofluorescent staining and immunoblotting analyses performed. in some experiments cells were pre-treated with ru (a pr/gr antagonist) or org (a pure pr antagonist). results: in the absence of hormone gr appeared to be predominantly cytoplasmic, whereas, upon treatment with -hp and p ( and m) and dex ( nm) gr was abundant within the nuclei of myometrial cells. immunoblotting analyses demonstrated that levels of gr progressively increased within the nuclear fractions of both pregnant myometrial and wish cells in response to increasing concentrations of p ( nm to m), and decreased sequentially within cytoplasmic fractions. in the presence of org gr protein levels remained constant within the cytoplasm. there also appeared to be a slight increase in gr expression, though not statistically significant (p> . ) within both cells types in response to p. conclusion: in this study we show that gr is activated by -hp and p and translocates to the nuclei of human myometrial and amnion-derived cells. in addition, levels of gr increase in response to p. whether p and -hp act as agonists or antagonists for gr in the regulation of hormone response genes associated with the onset of term and preterm labour remains to be elucidated. objective: using a mouse model of inflammation-induced preterm birth (ptb), we have demonstrated dramatic cytokine elevations in the uterus and placenta with concomitant, though less dramatic, cytokine elevations in the fetal liver and brain, associated with neuronal injury. because precise mechanisms of fetal injury in ptb remain unclear, we sought to examine inflammatory cell trafficking, and target organ damage by histopathologic assessment of the placenta, fetus, and fetal brain. study design: hours after intrauterine infusion of saline or lps into the right lower uterine horn of cd- mice, the left upper horn, with the gestational sacs(gs) in situ, was removed en bloc(n= per group) each with - gs with fetuses/treatment group. specimens were fixed, bisected and processed for histology and ihc. inflammatory and hematopoietic cells were quantified using pas, gata- (erythroid precursors), cd , and bm (macrophage-mp) within the placenta, liver, extremity mesenchyme, brain and leptomeninges. the presence of hemorrhage, necrosis, and apoptosis (h ax stain) was assessed. erythopoietin (epo) levels were measured in brain and liver by elisa. results: more neutrophils were present in maternal decidual vessels in lps compared to saline (p= . ). in lps-exposed, fetal mp were increased in the placenta (p= . ), fetal extremity mesenchyme (p= . ), fetal liver (p= . ) and leptomeninges (p= . ) but not in the brain or spinal cord compared to saline. no necrosis, hemorrhage or increased apoptosis was noted in the fetal brains. % of lps-exposed fetuses and % of saline-exposed had liver hemorrhages (p< . ). increases in nucleated erythrocytes and erythroid precursors were found in fetal vasculature of the placenta in lps-exposed (p= . ). epo levels were not elevated in either group. conclusion: intrauterine lps infusion induces acute inflammation predominantly in the maternal circulation of the placenta. in the fetus, there is widespread mp activation, liver hemorrhage and increased erythroid precursors seen in the fetal circulation of the placenta. although histologic evidence of cns damage was not evident, the increased mps present in the leptomeninges may play an important role in inflammatory-mediated cns damage. non-toll-like innate immune proteins: do they change during pregnancy? juan m gonzalez, hua xu, ella ofori, michal a elovitz. obgyn; crrwh, university of pennsylvania, philadelphia, pa, usa. introduction: trem- (trigger receptors expressed on myeloid cells) is an important regulator of innate immunity. the natural ligand for trem has not been identified. activation of trem- in the presence of toll-like receptors results in substantial amplification of the host inflammatory response (klesney-tait et al nature immunology ). since inflammatory pathways are implicated in adverse pregnancy outcomes, this novel mediator of inflammation may play a critical role in preterm birth (ptb). therefore, we sought to determine trem- expression in the uterus, cervix, and placenta across gestation and to determine if trem- levels are altered by intrauterine inflammation. methods: in cd- mice, trem- was investigated in non-pregnant (np) and throughout gestation e , e (n= - per group). uterine, cervical, and placental tissues were harvested. using an established mouse model of inflammation-induced ptb, uterine tissue was collected hours after intrauterine infusion of saline (n= ) or lipopolysaccharide (lps) (n= ). for a non-pregnant model, using cd- mice, lps (n= ) or saline (n= ) was injected into the uterine horn following same procedures as with pregnant mice. uteri were harvested hrs later. quantikine ® mouse trem- immunoassay was utilized for these studies. statistical analysis was performed using oneway anova followed by pair-wise comparison if statistical significance was reached (p< . ) results: trem levels are significantly different between np and pregnant uterine tissues (p= . ). e and e trem expression is significantly increased . and . -fold compared to np (p= . and . respectively). trem- levels in the placenta and cervix were not significantly different between e and e . trem levels increased about -fold in the uterus after intrauterine infusion of saline or lps compared to e controls. in nonpregnant, trem levels were significantly different (p= . ) with a -fold increase in trem expression in uteri exposed to lps or saline compared to controls. conclusions: non-toll-like innate immune proteins are differentially regulated during pregnancy compared to the non-pregnant state. the role of trem- in inflammation-induced ptb requires further study. research is warranted to determine if uterine up-regulation of trem in gestation is associated with an increased likelihood of responding to pathogens or severe as a protective mechanism. reduced plasma levels of vitamin d in caucasian women at term are associated with increased rate of infection. chander p arora, adegoke adeniji, susan e jackman, babak forooghi, isaac mostadim, phillip yadegari, calvin j hobel. og-gyn, cedars-siani medical center, los angeles, ca, usa; university of california los angeles, los angeles, ca, usa. background: vitamin d plays an important role in human pregnancy by acting as a regulator of immunity at the fetal-maternal interface. inflammatory changes associated with pro-inflammatory cytokines were reduced by vitamin d while anti-inflammatory cytokines were increased in t lymphocytes. vitamin d status has been defined as deficiency (< . nmol/l), insufficiency ( . to nmol/l) and sufficiency (> nmol/l) . objective: to assess the involvement of vitamin d in the occurrence of maternal infection during pregnacy in women with term deliveries. hypothesis: vitamin d metabolism could affect the rate of infection during pregnancy. study design plasma levels of (oh)d were determined by elisa and the rate of infection was recorded in a behavior in pregnancy study. in this study, ethnically diverse women were evaluated at - weeks (t ), - weeks (t ) and - weeks (t ). maternal infections were documented at each stage as well as at baseline visit with history of infection in current pregnancy. of these subjects who delivered at term two groups ( with no infection during pregnancy, with infection or history of infection) were matched further for non-smoking status, non-diabetics, ethnicity and maternal age. plasma from these were assayed for (oh)d and analyzed for the rate of maternal infection using fisher´s exact test or chi-square test. results although the women delivered at term, the levels of (oh)d in caucasians were significantly lower in the subjects with infection than the ones without (p<. ). women with vitamin d insufficiecy in the first trimester were more likely to develop infection during pregnancy ( . nmol/l ± . at t , . nmol/l ± . at t and . nmol/l ± . at t ; all p<. ) but not subjects with sufficient vitamin d at t ( . nmol/l ± . at t , . nmol/l ± . at t and . nmol/l ± . at t ; all p<. ). conclusion the results reveal a positive association between (oh) d concentrations and greater risk of infection. vitamin d deficiency or even insufficiency may, therefore be involved in the pathogenesis of maternal infection during pregnancy. it is probable that vitamin d deficiency or even insufficiency could modulate the maternal susceptibility to infection during pregnancy by a proinflammatory mechanism. in vitro and in vivo observational data suggest that infection leads to caspase activation and apoptosis in the placenta and membranes, however currently there are no data on the role of apoptosis in the pathogenesis of infection associated preterm delivery. here we used group b streptococcus (gbs) as a model pathogen and examined the role of caspase dependent and independent apoptosis in preterm delivery. methods: we injected ( . x ) heat killed group b streptococcus organisms (hk-gbs) intraperitoneally (i.p.) in . day pregnant c b/l mice. the mice were euthanized at hr (n= ) and hr (n= ), the placentas and membranes were removed and assessed for apoptosis by tunel staining. caspase activation and expression were determined by immuno-histochemistry (ih) and western blotting. the effect of apoptosis on preterm delivery was assessed by i.p. treating the pregnant mice with pbs (n= ), dmso (n= ) or pancaspase inhibitor z-vad-fmk (n= ) prior to hk-gbs and observing the animal for delivery for hrs. results: there was a time dependent, gbs-induced increase in apoptosis by tunel assay and caspase activation in the placenta and membranes. in addition hk-gbs-induced the expression of caspase and caspase independent m-calpain in the placenta. z-vad-fmk ( mg/kg), at the maximum concentration that did not induce maternal illness, did not prevent hk-gbsinduced preterm delivery. conclusions: caspase dependent and independent mechanisms are activated in the placenta upon exposure to gbs. systemic adminstration of a pan-caspase inhibitor did not impact upon the occurance or timing of bacterially induced preterm delivery. further studies are needed to assess the role of apoptosis in the pathogenesis of infection associated preterm delivery. early and ( . %) had a late sptb. the mean + sd gestational age at blood draw was + weeks. the median level of bb was higher in women with early as compared with late sptb or term births (p= . for trend). women with bb in the top quartile were . times more likely to have an early sptb as compared with women who had lower levels of bb ( % ci . to , p = . ). there was no association between bb and late sptb (rr= . , % ci = . to ). the adjusted or of an elevated bb for early sptb was . ( % ci = . to , p= . ). when the analysis was restricted to the women with sptb the rr of an elevated bb for early sptb was . ( % ci . to . , p = . ). conclusions: a significant relationship was found between an elevated bb in early pregnancy and early sptb suggesting inflammatory events in early pregnancy, perhaps infection-related, are part of the pathogenic mechanisms. objective: genital tract infection and/or inflammation appears to contribute to the majority of ptds preceding weeks of gestation. ptd in humans has been associated with colonization and/or infection with a variety of different organisms including gram positive and negative bacteria, mycoplasma, ureaplasma, trichomonads, malaria parasites and viruses. the innate immune response to these pathogens is produced by a family of pattern-recognition cell membrane receptors known as the toll-like receptors (tlrs). these studies sought to characterize the tlr isoforms expressed in the preterm mouse uterus, and their modulation during lipopolysaccharide (lps)-induced ptd. methods: using sterile surgical technique, day- pregnant cd- mice underwent intrauterine injection of g lps. subsequently, the mice were euthanized at , , , , and hours after lps injection. uterine tissue was harvested and placed in rnalater; subsequently total rna was isolated using the trizol reagent and genomic dna was removed using turbo dnafree. cdna was made using iscript cdna synthesis kit. pcr primers were designed using published mouse tlr gene sequences. real-time quantitative rt-pcr was performed using the power sybr green master mix and an abi prism multicycler. to confirm tlr amplicon sizes, the rt-pcr products were visualized on a % agarose/tbe gel stained with gelred. results: these studies have confirmed mrna expression of all of the reported mouse tlr isoforms. these tlr amplicons range in size from to bp as expected; amplicon sequences are pending. quantitative rt-pcr studies performed using uterine tissues from five mice at each time point demonstrated that at hours after lps injection, tlr increased -fold and tlr increased -fold (both p< . ). in contrast, the expression of tlr (the ligand for lps) remained stable during the hours after lps; the expression of tlr , , , , , and also remained stable. tlr expression trended upward and tlr trended downward, although neither was statistically significant. conclusions: these studies have confirmed expression of all tlrs within the preterm pregnant mouse uterus, along with characterization of their modulation during lps-induced ptd. these observations are important because they contribute to our understanding of the immunologic signaling events leading to ptd. (funded by nih hd ). udp-glucose and its receptor p y as a new innate immune system in the female reproductive tract. toru arase, tetsuo maruyama, hiroshi uchida, takashi kajitani, masanori ono, maki kagami, hironori asada, yasunori yoshimura. obstetrics and gynecology, keio university, shinjukuku, tokyo, japan. objective: innate immune system involving toll-like receptors has recently emerged in the female reproductive tract (frt). we hypothesize that there may exist new other mucosal immunity in frt. recently, it has been reported that p y , a g protein-coupled p y receptor for udp-glucose (udp-g), is involved in the lung epithelial immune system. the aim of this study is to investigate whether udp-g and p y have a potential as the defense immune system in frt, in particular endometrium. materials and methods: we obtained human endometrial tissues from consenting reproductive-aged patients. the spatiotemporal expression of p y in human and mouse endometrial tissues was analyzed using rt-pcr and ihc. isolated human endometrial cells and a human endometrial epithelial cell line, ishikawa, were cultured, treated with udp-g, and subjected to rt-pcr analysis for il- mrna expression. we also measured the il- secretion using elisa. small interfering rna was used to knock down p y expression. the chemotactic activity of udp-g on neutrophils was tested using transwell assay with ishikawa cells. lastly, mouse uterine tissues were incubated with udp-g and subjected to rt-pcr analysis for mrna expressions of kc and mip- , the il- homologues in mice. results: p y was exclusively expressed in the glandular and luminal epithelium both in human and mouse uteri. treatment with udp-g induced the secretion of il- in ishikawa and human endometrial glandular cells, but not stromal cells, in a dose-and a time-dependent manner. p y knockdown abrogated udp-g-induced il- production. treatment with udp-g also significantly increased the chemotaxis of neutrophils, which was attenuated by co-addition of anti-human il- neutralizing antibody. in the mouse uterus stimulation of udp-g significantly up-regulated the expressions of kc and mip- mrna. conclusions: udp-g is an endogenous molecule and released into the extracellular environment in a lytic manner after cell damage. taken together, our results collectively substantiate a model in which udp-g released from endometrial cells damaged by infection stimulates il- production via p y in endometrial glandular epithelium, which, in turn, recruits neutrophils thereby preventing the progression of infection. thus, udp-g and its receptor p y may be involved in the trans-species mucosal immune system in frt. (jsgi ; , (suppl) , abst # ) but in utero effects on fetal lung remain to be established. we have examined the relationship between duration of iai and subsequent azi treatment on the severity of fetal lung histopathology. we hypothesized that early treatment would prevent the development of advanced lesions, while late treatment may reduce the severity of lung damage. study design. thirteen chronically instrumented rhesus monkeys received intraamniotic inoculation of u. parvum (serovar ; - x cfu) at ± . days gest. age (dga, mean ± sem, term= d). six of these animals received maternal i.v. azi ( . mg/kg q h or q h for d) either alone (n= ) or in combination (n= ) with dexamethasone (dex; mg/kg/d i.v. for d) and indomethacin (indo; mg/d p.o for d). tissues were obtained at cesarean section for histopathologic assessment. leukocytic infiltration of aveolar spaces and septal walls, type ii pneumocyte hyperplasia and peribronchiolar lymphocytic aggregates were scored as absent ( ), minimal ( ), moderate ( ) and severe ( ). results. inoculation-to-delivery interval was - d for combined treatment groups and was similar to long duration infection without treatment ( - d). treatment effects were tabulated as mean scores and compared as follows: control (n= ), score ; short duration infection ( - d; n= ), score ; long duration infection ( - d; n= ), score ; short duration infection + treatment (n= ), score ; long duration infection + treatment (n= ), score . conclusions. histopathologic findings of fetal pneumonia progressively worsen with duration of u. parvum iai. early maternal azi treatment prevents development of advanced lung lesions, while later treatment may reduce the severity of fetal lung damage. our results suggest that in utero treatment of iai may lower the risk of neonatal bronchopulmonary dysplasia. support: nih hd , rr . brain associated with adverse neurodevelopmental outcome. lps triggers proinflammatory responses through toll-like receptor- (tlr ). mitogen activated protein kinases including c-jun-n-terminal kinase (jnk) have been reported to be implicated in tlr signalling pathways and play important role in both onset of labor and brain injury. in the present study, we used a mouse model of intrauterine infection-associated preterm labor to determine whether the administration of specific inhibitor of jnk signaling, d-jnk inhibitory peptide (d-jnki) can (i) inhibit jnk activity in vivo, (ii) delay lps-induced preterm delivery, and (iii) improve neonatal outcome in the presence of intrauterine inflammation. intrauterine administration of tlr- specific lps to cd pregnant mice at day of pregnancy caused preterm delivery after to h with % pup mortality. in vitro kinase assay demonstrated the activation of jnk in response to lps in the maternal uterus and fetal brain. furthermore, the brain specific jnk was found to be the major jnk isoform activated by lps in the fetal brain. co-administration of d-jnki with lps to pregnant mice delayed significantly (p< . ) lps-induced preterm delivery and reduced pup mortality up to %. this was associated with inhibition of jnk activity in both maternal uterus and fetal brain. in addition, we have found that treatment with lps significantly up-regulated cox- , cxcl (il- equivalent) and ccl in myometrium and this is significantly suppressed after co-administration of d-jnki. we conclude that specific inhibition of jnk signaling may have a potential of controlling preterm labor and preventing fetal brain damage as a result of infection/inflammation. the prostaglandin -deoxy- , -prostaglandin j delays lps-induced preterm delivery and reduces mortality in the mouse. intrauterine infection is a common trigger for preterm birth, and is also a risk factor for the development of neurodevelopmental abnormalities in the neonate. bacterial lipopolysaccharide (lps) binds to toll-like receptor- (tlr ) to activate pro-inflammatory signaling pathways. the transcription factor nuclear factor kappa b (nf-kb) is a key player in the orchestration of the inflammatory response and has a central role in parturition. we have previously shown that exposure to the anti-inflammatory cyclopentenone prostaglandin -deoxy- , -prostaglandin j ( d-pgj ) inhibits il- b-induced nf-kb activity and cox- expression in human myometrial and amnion epithelial cells in vitro. in the present study, we used a mouse model of intrauterine infection-associated preterm labor to determine whether the administration of d-pgj can (i) inhibit nf-kb in vivo, (ii) delay lps-induced preterm delivery, and (iii) improve neonatal outcome in the presence of intrauterine inflammation. intrauterine administration of tlr specific lps to cd pregnant mice at day of pregnancy caused preterm delivery after to h with % pup mortality. co-administration of d-pgj with lps to pregnant mice delayed significantly (p< . ) lps-induced preterm delivery and conferred protection from lps-induced fetal mortality up to %. we have looked at the expression profile of several labor associated genes in myometrium hours after lps administration. (otr, connexin and , cox- , cox- , cxcl (il- equivalent) and ccl ). we have found that treatment with lps significantly up-regulated cox- , cxcl and ccl and this is significantly suppressed after with co-administration of d-pgj . western analysis for ser -phosphorylated p and ikkb in-vitro kinase assay has demonstrated that lps induced activation of nf-kb at both h and h. co-administration of d-pgj was associated with inhibition of nf-kb activation in both the maternal uterus and the fetal brain. conclusion d-pgj may have potential as a therapeutic agent in the management of preterm labor and, by targeting the player nf-kb, may have the added advantage of preventing detrimental effects to the fetus that may result from infection/inflammation. synergistic macrophage response to co-activation of tlr- and tlr- : mechanisms and implications for bacterial/viral co-infection. vladimir ilievski, emmet hirsch. , department of ob/gyn, evanston northwestern healthcare, evanston, il; department of ob/gyn, feinberg school of medicine, northwestern university, chicago, il. background: toll-like receptors (tlrs) recognize structural components of pathogens and initiate host defenses. tlr- responds to gram-positive organisms and peptidoglycan (pgn), a gram-positive cell wall constituent. tlr- is activated by viral infection in response to double-stranded rna. polyinosinic-cytidylic acid (poly(i:c)) is a tlr- ligand. we have shown that pgn and poly(i:c) have a synergistic effect on the expression of downstream genes for both tlr- and tlr- . here we identify mechanisms underlying this synergy. methods: mouse peritoneal macrophages or a mouse macrophage cell line (raw . ) were treated in tissue culture with either pgn ( g/ml), poly(i: c) ( g/ml) or both pgn and poly(i:c) either simultaneously or sequentially for - hours. total rna was extracted and duplex rt-pcr was performed for inducible nitric oxide synthase (inos), interleukin (il- ), tumor necrosis factor (tnf), the chemokine rantes and tlr- , normalized to the housekeeping gene gapdh. results: compared to stimulation with either pgn or poly(i:c) alone, costimulation of raw . cells with both pgn and poly(i:c) resulted in synergistic expression of inos, il- , tnf and rantes (p< . for all) at and hours . sequential stimulation with either pgn or poly(i:c) for h followed by incubation for an additional h with the alternate ligand also induced synergistic expression of the same rnas, albeit at lower levels than were elicited by simultaneous stimulation. in contrast, incubation with either pgn or poly(i:c) for h followed by medium for h induced minimal to no gene expression. both pgn and poly(i:c) induced tlr- mrna after h but not h. tlr- mrna was not detectable by rt-pcr. in primary peritoneal macrophages, similar synergy due to pgn and poly(i:c) was seen. conclusions: simultaneous or sequential exposure to pgn and poly(i:c) exerts a synergistic effect on the expression of inflammatory mediators in macrophages. interestingly, either one of these two tlr pathways can prime cells for activation of the other pathway. a possible mechanism for this effect may be induction of tlr- by either tlr- or tlr- activation. these observations have implications for bacterial/viral co-infection. this is a secondary analysis of a prospective cohort study. after irb approval, daily blood samples were obtained from pprom subjects and analyzed for il- by elisa. paired maternal serum il- levels from subjects were divided into groups: il- levels obtained - hours prior to completion of antibiotics and those obtained - hours after completion of antibiotics. the wilcoxon signed rank test was used to perform the data comparison on the analyze-it statistical software program. statistical significance was defined as p< . . of the pprom subjects, the maternal age was . yrs; gestational age at admission was . weeks; latency was . days; gestational age at delivery was weeks; and infant birth weight was grams. median maternal serum il- levels obtained off antibiotics were significantly higher when compared to those on antibiotics ( . vs. . pg/ml, p< . ). the results of this investigation suggest that maternal serum il- levels rise after discontinuation of antibiotics. the optimal duration of antibiotics administration in the setting of pprom is unknown. this data suggests a role for continuation of antibiotics in women with pprom in order to prolong the latency period and potentially decrease neonatal morbidity. to identify clinical and pathologic factors associated with fetal inflammatory responses in the placenta from term parturients. methods: retrospective cohort study of consecutive term parturients with submitted placentas in . placentas with histologic chorioamnionitis were divided into two cohorts: group -maternal inflammatory responses only, and group -maternal and fetal inflammatory responses. maternal and fetal inflammatory responses in the placenta were classified according to guidelines established by the amniotic fluid infection nosology committee of the perinatal section of the society of pediatric pathologists. selected demographic, intrapartum, newborn and placental characteristics were analyzed with t-tests and chi-square tests as appropriate. multiple logistic regression was used to identify independent predictors of fetal inflammatory responses in the placenta. results: of consecutively submitted placentas, had histologic chorioamnionitis: with maternal inflammatory responses only (group ) and with both maternal and fetal inflammatory responses (group ). fetal inflammatory responses observed in group were associated with higher stages of maternal inflammatory responses (p<. ). % of fetal inflammatory responses were stage i (acute chorionic vasculitis or phlebitis).group patients were more likely to have had amnioinfusion ( % v %, p=. ) and less likely induction of labor ( % v %, p=. ). group was more likely to have had intrapartum fever ( % v %, p=. ) and maternal tachycardia ( % v %, p=. ). newborns from group were more likely to have been observed for sepsis ( % v %, p <. ) and have an apgar score at minutes ( % v %, p=. ). a logistic regression model showed that stage ii or greater maternal inflammatory responses (or . ) and amnioinfusion (or . ) were independent predictors of fetal inflammatory responses. conclusion: higher stages of maternal inflammatory responses in the placenta and amnioinfusion were independent predictors of fetal inflammatory responses in term parturients with histologic chorioamnionitis. objective: previous studies have shown that sulfasalazaine (sasp) inhibits lipopolysaccharide (lps)-induced nf-b activation and decreases lpsstimulated interleukin- (il- ) production by cultured explants of placenta, amnion and choriodecidua with no effect on cell viability. bacteria-induced il- production in the cervix is a potential mechanism for premature cervical ripening that may lead to preterm birth. it is unclear, however, whether sasp can inhibit il- production by endocervical cells. therefore, we performed a series of in vitro studies to determine if sasp inhibits il- production by endocervical epithelial cells stimulated with bacterial pathogens associated with preterm birth. methods: human endocervical epithelial cells were incubated with - . mm sasp overnight and then stimulated with ccu/ml ureaplasma parvum, cfu/ml escherichia coli, or x cfu/ml gardnerella vaginalis for another overnight incubation in -well cultures. conditioned medium was then harvested and production of il- was quantified by elisa. viability of the cells was ascertained at the end of the experiment with the mtt-assay. each treatment was applied in quadruplicate wells and experiments were repeated times using different batches of cells for each pathogen. results were evaluated using the general linear models procedure of sas for a randomized block design. results: sasp had no detectible effect on il- production by endocervical cells not treated with bacteria. at the highest concentration tested ( . mm), sasp significantly inhibited il- production by cultures stimulated with e. coli (p< . ), u. parvum (p< . ), and g. vaginalis (p< . ). viability of the cells, however, was significantly reduced by sasp at . and . mm in the both the presence and absence of bacteria for all experiments. conclusion: although high concentrations of sasp inhibit il- production by cultures of endocervical cells stimulated with pathogens associated with preterm birth, this effect may be due to toxicity of the antibiotic on the cells. the effect of -hydroxyprogesterone caproate on lipopolysaccharide stimulated peripheral blood mononuclear cells from pregnant women. richard h lee, aimin li, frank z stanczyk, jinwen i lin, t murphy goodwin. obstetrics and gynecology, university of southern california, los angeles, ca, usa. introduction: -hydroxyprogesterone caproate ( -ohpc) reduces the rate of recurrent preterm birth in high-risk women. however, there are lines of evidence to suggest that -ohpc alters inflammatory response in the setting of gram-negative infection. in a mouse model, -ohpc decreased the rate of preterm birth but was associated with significantly increased maternal morbidity when mice were exposed to lipopolysaccharide (lps). in non-pregnant women, -ohpc pre-treatment of whole blood exposed to lps significantly increased the production of tnf-. objective: to determine if -ohpc has an effect on the production of proinflammatory cytokines from peripheral blood mononuclear cells (pbmc) in pregnant women. methods: pbmc were isolated from fresh whole blood samples of pregnant women between and weeks. pregnant women had no prior history of preterm birth. cells were treated with -ohpc ( m) and escherichia coli lipopolysaccharide (lps, g/ml) alone or in combination. after and hours of culture, supernatants were collected and tested for tnf-and il- levels by enzyme-linked immunosorbent assay (elisa). statistical analysis was performed using non-parametric tests. p < . was considered significant. results: pbmc exposed to lps significantly increased tnf-and il- secretion compared to untreated controls (p= . and p= . ). tnf-concentrations were not significantly different between lps and lps+ -ohpc treated cells at both and hours (p= . and p= . ). il- production was significantly increased after -hour treatment with lps+ -ohpc compared to lps alone ( , [ , - , ] background. recent clinical trials indicate that progestins reduce the incidence of preterm birth in some high risk pregnancies. it has been proposed that progesterone promotes uterine quiescence, in part, via its antiinflammatory properties with inhibition of pro-inflammatory gene expression. it is intriguing that progestins are clinically effective given the considerably increased background circulating levels of the hormone during pregnancy. we hypothesised that non-classical progesterone signalling pathways contribute towards mediation of the anti inflammatory effects of progestins. to the endotoxin lipopolysaccharide (lps) by activation of inflammatory pathways. myometrial cell cultures were treated with lps ( g/ml)+/progestin ( nm). two progestin compounds were investigated. natural progesterone (p) is known to have a strong affinity with progesterone receptor (pr) analogues in contrast to -hydroxyprogesterone ( -hp) which, in the absence of esterification with caproate or acetate, has been reported to have no progestogenic activity at pr. the effect of p and -hp on the activation of two inflammatory genes known to be associated with labour (cycloxygenase [cox- ], toll-like receptor [tlr- ]) was evaluated. cox- and tlr- were detected at the protein and mrna levels using sds-page and rt-pcr. results. lps-induced expression of cox- and tlr- proteins were significantly inhibited by both p (p< . and p< . , respectively) and -hp (p< . and p< . , respectively). furthermore, preliminary results indicate that co-incubation with the anti-progesterone mifepristone, fail to abrogate the anti inflammatory effect associated with progestin treatment. conclusion. non-classical progesterone signalling pathways have a role in mediating the anti-inflammatory properties of progestins. further elucidation of the molecular actions of progestins may allow novel approaches to ameliorate the inflammatory response associated with preterm labour. detection and transcriptional expression of tlr- , tlr- and tlr- at the maternal-fetal interface. jacqueline p tilak, karen zakharian, alexandra tungol, gabriel o schubiner, david m svinarich. patient care research, providence hospital, southfield, mi, usa. preterm labor and delivery remains a leading cause of neonatal morbidity and mortality and bacterial infection is considered to be the most common etiology. toll-like receptors (tlr's) and the associated components of the innate immune system may represent a first line of defense against these pathogens. tlr's belong to a family of pattern-recognition receptors that bind to highly conserved pathogen-associated molecular patterns (pamps) including lipopolysaccharide (lps), lipotechoic acid (lta) and cpg dna, and are a key component of the innate immune system. this study was undertaken to characterize the transcriptional expression and regulation of tlr- , tlr- and tlr- within gynecologic and gestational tissues. human first trimester trophoblasts, endometrial mesoderm, ectocervix, choriocarcinoma and neonatal epithelium, were cultured in media alone or in the presence of either lps ( mg/ml) or lta ( mg/ml) for , , , , and hours. total rna was isolated and semi-quantitative rt-pcr was performed using intron-spanning amplimers to tlr- , tlr- , tlr- and gapdh. following gel electrophoresis, the integrated optical densities were determined for the corresponding pcr products and normalized with respect to gapdh levels. inducible transcription of tlr- with lta was observed in choriocarcinoma cells ( -fold, h), and endometrial mesoderm cells ( -fold, h). tlr- induction with lps was observed in ectocervical cells ( -fold, h), choriocarcinoma cells ( -fold, h) and endometrial mesoderm cells ( -fold, h). tlr- induction with lps was observed in choriocarcinoma cells ( fold, h) and neonatal epithelial cells ( -fold, h). all cell lines showed at least constitutive levels of transcription for tlr- , tlr- and tlr- . these data demonstrate that tlr- , tlr- and tlr- are transcriptionally expressed either constitutively or inducibly in both gynecologic (endometrial mesoderm, ectocervix) and gestational (chorion, trophoblast), tissues. translation of these receptors suggests that the innate immune system may function at the maternal-fetal interface to help protect the fetus against infection and prevent or diminish the likelihood of prematurity. objective: further to our development of a sheep model of intrauterine ureaplasma spp infection, we aimed to examine the capability of ureaplasma colonization in the amniotic fluid to infect the fetus and alter lung and brain development. methods: at days of gestation (d, term= d) ewes bearing single fetuses were given a single ultrasound-guided intra-amniotic injection of (a) x colony forming units (cfu) of u parvum (serovar , n= ; serovar , n= ), (b) x cfu of u parvum (serovar , n= ; serovar , n= ) or (c) media control (n= ). at d, fetuses were delivered by cesarean section. amniotic fluid and umbilical arterial blood samples were collected. fetal body weight was recorded, fetal cerebrospinal fluid (csf) collected and a descending pressurevolume curve constructed after inflation of the lungs to cmh o. fetal membranes were immersion fixed and the fetal brain was perfusion fixed with % paraformaldehyde. the fetal brain and membranes were blocked, paraffin embedded, stained and viewed under the light microscope. results: chronic intra-amniotic colonisation with u parvum serovar or (ureaplasmas) did not result in fetal abortion or death. amniotic fluid ureaplasma titers at delivery were not dose-dependent. chronic ureaplasma exposure did not affect fetal body or brain weights, or result in a fetal systemic inflammatory response. umbilical arterial blood gases at delivery were similar between ureaplasma-and media-exposed fetuses. chronic intra-amniotic exposure to ureaplasmas resulted in higher inflammatory cell scores in the fetal membranes compared to media controls (p< . ). lung compliance was increased in ureaplasma-exposed fetuses compared to controls (p< . ). there were no gross anatomical changes observed in the white or grey matter in the cerebral hemispheres of fetuses that had been exposed to ureaplasmas; even in animals (n= ) that had csf ureaplasma colonisation. conclusion: chronic ureaplasma colonisation enhances fetal lung compliance without gross anatomical changes in the fetal brain. background: an important source of vitamin d is its synthesis by skin from uv solar radiations. the skin pigment melanin absorbs uv photons thus reducing the vitamin d synthesis by more than % making african americans at high risk for vitamin d deficiency. low maternal vitamin d status during pregnancy has been linked to molecular pathways for adverse outcomes. objective: the nf b transcription factor regulates genes involved in inflammation and immune processes, and is proposed to play a major role in the successful outcome of pregnancy and labor. prior immunohistochemical (ihc) and biochemical studies have been conflicting regarding nf b activation in human intrauterine tissues. the aim of this study was to quantify nuclear localization of p , the major tranactivating nf b subunit, in full-thickness fetal membranes (fm) and myometrium using ihc. methods: a retrospective analysis of paired fm, decidua, and myometrial samples was conducted using tissues collected from women in cohorts: preterm no labor (pnl, n= ), preterm labor (ptl, n= ), spontaneous term labor (stl, n= ), and term no labor (tnl, n= ). subjects were delivered between gestational ages - weeks (preterm) and - weeks (term) by cesarean. paraffin sections were stained with polyclonal (rabbit) anti-human p and microscopically examined. myometrial samples were categorized in a blinded fashion to groups of staining: no nuclear p (-), rare nuclear p (+), and > % nuclear p (++). a p nuclear labeling index (nli; % nuclear p /total cells) was calculated for each histological layer in full-thickness fm specimens using a blinded targeted randomization scheme consisting of non-overlapping low-magnification fields. results: nuclear p labeling was rare in amnion and inconsistently present in chorion. in decidua, p nuclear labeling was observed in all cases; however, decidual nli did not vary significantly across cohorts [pnl- % ( - %); ptl- % ( - %); tnl- % ( - %); stl- % ( - %); all values are median (iqr)]. in myometrium, ++ p nuclear labeling was significantly associated with labor, but present only in a portion of cases (ptl- %; stl- %). despite a trend, decidual nli was not significantly correlated with myometrial nuclear p labeling: myometrial specimens classified as -, +, and ++ corresponded with decidual nli of % ( - %) [median (iqr)], % ( - %), and % ( - %), respectively. conclusions: in a comprehensive ihc analysis, significant nuclear p immunolabeling was observed in myometrial cells following labor. nuclear p labeling in decidua was present in all cases, and did not vary significantly among the cohorts. the inflammatory cytokines interleukin- and tnf-increase g-csf expression in term decidual cells. objectives: chorioamnionitis (cam) elicits premature rupture of the membranes and pre-term delivery. previously, we found that the decidua from cam patients contains much higher neutrophil numbers than control decidua, but macrophage numbers are similar in both groups. granulocyte colony-stimulating factor (g-csf) enhances granulocyte colony formation chemoattraction and activation. the amniotic fluid during cam contains elevated tnf-and il- levels. to account for the marked influx of neutrophils infiltration in cam-complicated decidua, tnf-and il-effects were assessed on g-csf expression in term decidual cell (dc) monolayers. methods: confluent leukocyte-free term dcs from normal term deliveries were primed with - m estradiol (e ) + - m medroxyprogesterone acetate (mpa) for days, then switched to serum-free defined medium (dm) with steroids ± tnf-or il- . after h, aliquots of conditioned dm supernatants, cell lysates and extracted rna from h parallel incubations were frozen. secreted g-csf was measured by elisa in conditioned dm and normalized to cell protein and mrna was assessed by quantitative real time rt-pcr and normalized to -actin mrna. results: in cultured first trimester dcs, basal g-csf levels in conditioned dm were . ± . pg/ml/ug cell protein [mean ± sem, n= ] and did not differ from e +mpa basal levels. the addition of ng/ml of tnf-or il- significantly elevated g-csf output to . ± . and ± respectively (p< . ), representing more than a -fold and , -fold change; respectively. western blotting confirmed the elisa results. quantitative rt-pcr demonstrated corresponding changes in g-csf mrna levels as found for the elisa measurements. concentration-dependent effects for both tnfand il- from . to . ng/ml were observed. conclusions: when extrapolated to the placental milieu of cam, the marked increase in g-csf elicited in term dcs by tnf-and il- may provide a mechanism to account for the selective increase in decidual neutrophils versus macrophages. background. the immunological mechanisms of the female reproductive tract are unclear. toll-like receptors (tlrs), innate immune receptors that combat microbial infections and establish adaptive immunity, may play a role. infection in pregnancy has been associated with preterm birth and tlrs may modulate the host response to such infections. we hypothesised that the distribution of tlr and tlr in cervical epithelial cells may differ with pregnancy, and that oestrogen may contribute to the modulation of these receptors. aims and objectives. . to compare tlr and tlr protein expression, using immunocytochemistry, in the cervical epithelium of pre-menopausal non-pregnant women with pregnant women at term. methods. fresh non-pregnant (n= ) and pregnant (n= ) human cervical cells were obtained by smear and tlr and tlr expression investigated by immunocytochemistry. cervical epithelial cells from nonpregnant women obtained by smears were then coincubated with varying concentrations of estradiol, and tlr and tlr protein expression quantified by immunocytochemistry. results. using pixel-based image analysis software, we demonstrated a reduction in tlr expression in late pregnant compared with non-pregnant cervical epithelium (p= . ), whilst tlr did not appear to differ. there was an apparent up-regulation of tlr protein expression in response to beta-oestradiol (n= ) objective: to evaluate in vitro antimicrobial activity of cranberry-exposed urine against common urinary pathogens. subjects were pregnant women enrolled in a clinical trial evaluating the effect of daily cranberry juice cocktail or matching placebo on asymptomatic bacteriuria and other urinary tract infections. methods: -hour urine samples from pregnant women who were randomized to cranberry or placebo in three treatment arms: a. cranberry two times daily with meals (c, c; n= ), b: cranberry in the am, then placebo at dinner (c, p; n= ), c: placebo two times daily with meals (p, p; n= ). we identified non-pregnant, reproductive-aged controls, randomized them to the same treatment groups and collected -hour urine specimens from them. the ph of all urine specimens was adjusted to ph= and filtered. aliquots of each were independently inoculated with overnight culture of - cell/ml each of single strains of e. coli with fimbriae type i and type ii, k. pneumoniae, and c. albicans. after hours of incubation for e. coli and k. pneumoniae, and hours for c. albicans cfu/ml of each specimen were enumerated by subculture with quantitative plate counts in duplicate. results: there were no differences for any of the antimicrobial activities between pregnant and non-pregnant groups, or based on treatment allocation. we were able to perform antimicrobial assays on the urine of women exposed to cranberry juice or placebo, but unable to demonstrate differences based on treatment allocation or pregnancy. this may be due to beta-error. further studies are planned. supported by r dk - ; clinical trials registration nct . ...,.. e. coli . x .:!: . x . x .:!: . x group a (c, c) group b ( c, p) . x + . x . x + . x . . . group c (p, p) . x o"::!>s x o" . x ~ .ox: o• k. ~neumoniae group a ( c, c) . x + . x . x + . x . . . group b (c, p) . x + . x . x + . x group c (p, pl . x ~ . x . x ~ . x c. al bicans group a (c, c) . x '+ . x . x + . x group b (c, p) . x o•+ . x . x + . x . . . group c (p, p) . x ~ . x . x ~ . x • objective: to measure compliance, we sought to evaluate the use of a bioassay for cranberry in the urine of women enrolled in a clinical trial designed to determine the effect of daily dosing of cranberry juice cocktail or matching placebo on the incidence of asymptomatic bacteriuria (asb) and other urinary tract infections (uti) during pregnancy. methods: we collected -hour urine specimens from pregnant women who were randomized to ingest cranberry or placebo in three treatment arms: a: cranberry two times daily with meals (n= ), b: cranberry in the am, then placebo at dinner (n= ), c. placebo two times daily with meals (n= ). we identified non-pregnant, reproductive-aged controls, randomized them to the same treatment regimens (group a: n= , group b: n= , group c: n= ), and collected -hour urine specimens from them. bacterial anti-adhesion effects of the cranberry-exposed urine were evaluated utilizing a human red blood cell hemagglutination assay specific for uropathogenic p-fimbriated e. coli. activity was quantified as , , and %. results: when combining all subjects and dosing regimens, the sensitivity of the assay was %, range % in the pregnant group assigned single daily dose of cranberry to % in the non-pregnant group assigned to multiple daily doses. the specificity ranged from % to %. conclusions: these data indicate that the bioassay can be applied to the pregnant patient population, although the sensitivity of the assay is variable. higher daily dosing appears to confer greater sensitivity. further studies are needed to evaluate the utility of this bioassay for compliance. supported by r dk - . clinical trials registration nct . objective: increasing evidence suggests that inflammation plays a crucial role in initiation of labour both at term and preterm. we have previously shown upregulation of pro-inflammatory cytokines in myometrium, cervix and membranes at term labour. we have also shown that myometrium and cervix is invaded by leukocytes at the time of labour, and these leukocytes express pro-inflammatory cytokines. in this study, we aimed to test the hypothesis that pro-inflammatory cytokines and toll-like receptor and (tlr and ) mrna expression are up-regulated in circulating leukocytes during term and preterm labour. methods: peripheral blood samples were taken from pregnant women: - weeks gestation (w) preterm not in labour (ptnl) n= ; - w preterm in labour (ptl), n= ; - w term not in labour (tnl) n= ; and - w term in labour (tl) n= . leukocytes were isolated using dextran sedimentation. total rna was isolated and reverse transcribed using high capacity cdna archive kit, and mrna expression determined by real-time pcr (abi, taqman). student's t-test was used to compare outcomes between groups. results: messenger rna expression of il- , icam- , mcp- , tlr and tlr was significantly increased in tl compared to gestation matched tnl. the expression levels of il- b, il- and tlr- were significantly greater in ptl compared with gestation matched ptnl. (table i) . conclusions: we show up-regulation of pro-inflammatory cytokine production in circulating leukocytes in both term and preterm labour. thus, the pathophysiology of labour seems to involve the upregulation of proinflammatory cytokine production in peripheral blood leukocytes, followed by their invasion into the myometrium and cervix. this work further contributes to our understanding of the pathophysiology of parturition, and suggests that peripheral blood leukocytes may be potential targets for therapeutic agents aimed at modifying the time course of parturition. introduction. the mechanisms of innate immunity and tolerance in the female reproductive tract (frt) are ill-understood but involve the pattern recognition toll-like receptors (tlrs). we have previously demonstrated high expression levels of tlr gene and protein in fresh human cervical epithelium. aims. in order to gain insight into the immunological role of cervical epithelial cells (cecs), we sought to determine cec responses to the following tlr- agonists: macrophage-activating lipopeptide (malp- ), pam csk , and peptidoglycan. methods. cecs were isolated by smears and compared between pregnant ( rd trimester) and nonpregnant women. following cell preparation, flow cytometry was performed using a direct staining procedure with mouse anti-human tlr primary antibody and its igg , isotype control, to determine tlr- protein expression. a further nonpregnant smear samples were each prepared, and seeded at a concentration of , cervical epithelial cells/ml into -well cell plates. cells were incubated at °c in % co overnight with the tlr agonists malp- and pam csk (at concentrations of and ng/ml), peptidoglycan( ng/ml), il- ( ng/ml, positive control) and rpmi + -glutamine media only (vehicle). following centrifugation, all resulting supernatants were stored at - °c until the concentration of il- was determined by elisa and an array of cytokines by bead assays. results. both pregnant and nonpregnant cecs expressed tlr (specific mean fluorescence . vs . respectively) but did not differ (p = . ). unstimulated cells incubated with buffer alone demonstrated high il- levels ( pg/ml), which did not differ following stimulation with malp- ( pg/ml), pam csk ( pg/ml) or peptidoglycan ( pg/ml). results of an array of pro-and anti-inflammatory cytokines following incubation of cells stimulated with tlr agonists are pending. conclusion. high basal il- levels suggest constitutive expression by cecs but the physiological relevance of this intriguing observation is unclear. that cec stimulation with tlr- agonists did not lead to further release of il- may epitomise the resistance of these cells to antigenic challenge by the vaginal commensal flora. cecs may play a pivotal role in modulating the immunological tolerance necessary to minimise inappropriate inflammation in the cervix. there are several epidemiological and clinical studies that omega- fatty acids and related fish oils can decrease the premature labor and birth. however, the scientific mechanisms are not well elucidated. this study was carried out to investigate the effects of omega- fatty acids on producing pge and il- , in human umbilical vascular endothelial cell(huvec) media with artificial inflammation as an infection-induced premature labor tissue model. also, if there are some significant effects with omega- fatty acids, we want to investigate the possible mechanisms. materials and methods; human umbilical vascular vascular cell primary culture was done. in the adequate cell confluence in each cell dish, pretreatment with various concentrations of epa, dha and troglitazone (another ppar-ligand) and incubation were done for hours in °c, co incubator. after that, g/ml conc. of lipopolysaccharide(lps) was treated to the each cell dishes and incubated for hours. the cell media were collected, and pge and il- concentration were checked by elisa. the each cell lysates were collected and western blot anaysis for cyclooxygenase(cox)- were done. on the other hand, nuclear factor kappa b (nf b) luciferase vector was transfected and then did the same pretreatment with epa, dha and troglitazone and lps treatment to each cell dishes. after that, nf b luciferase activity was checked with luminometer. statistical analysis was done by student t-test. results: epa, dha and troglitazone decreased the pge (p< . ) and il- (p< . ) significantly in elisa. cox- expression revealed the significant reduction with pretreatment of epa, dha and troglitazone in higher concentration ( , m) in the western blotting (p< . ). nf b luciferase activity were also significantly decresed with pretreatment of epa, dha and troglitazone in higher concentration (p< . ). conclusion; this study offers some scientific mechanisms, by which omega- fatty acids (epa, dha) and troglitazone may be one kind of the preventive medicine for infection-induced preterm labor and delivery. also, these effects may come from the common mechanism of epa, dha and troglitazone, ppar-ligands. the next study would be how the cox- , nf b and pparare related. ( mg/ml). cytokine expression was measured in medium using the bio-plex suspension array system (bio-rad). mean expression was compared between the two groups using t test. results: -aminopurine significantly inhibited lps stimulated cytokine production by human myometrium. in contrast, there were no significant differences in expression after mpa treatment, although a trend towards inhibition of proinflammatory cytokine and an increase in il- production was noted. introduction: intrauterine infection is now recognised as a major antecedent of white matter injury (wmi) in the preterm infant brain, which can manifest later as cerebral palsy or as varying degrees of cognitive dysfunction. wmi in these infants is characterized by damage to immature oligodendrocytes, and has been linked both to microglial activation and to elevated levels of tnfa, il- b and il- . we have developed a fetal sheep model for diffuse and focal wmi, based on repeated administration of e. coli lipopolysaccharide (lps) as the stimulus for an inflammatory response. methods: surgery to implant fetal vascular catheters was performed on pregnant ewes carrying single fetuses at d - of gestation. fetuses received repeated iv injections of lps ( ng/kg estimated fetal weight/day) between d and d . plasma levels of pro-inflammatory cytokines were determined in fetal arterial blood samples taken between d and d . at d ewes and fetuses were euthanized and fetal brain tissue samples collected for histological analysis. results: five days after final administration of lps to fetuses we observed a pattern of wmi similar to that seen clinically, ranging from focal to diffuse injury within the periventricular region, impairment of white matter (cnpase; marker for immature/mature oligodendrocytes) tracts, and thinning of the corpus callosum, characterised by oligodendrocyte disruption and microglial proliferation in the surrounding and sub-cortical white matter. we also found a progressive rise in fetal plasma tnf levels between days and (day two of treatment to third day following final dose of lps). background: plasma fatty acid (fa) levels are associated with altered host inflammatory responses, blood pressure regulation, and insulin resistance, characteristic features of pregnancy-induced hypertension (pih). most studies compare the n- and n- polyunsaturated fatty acids (pufas). in addition, recent data demonstrate that saturated and trans-fas promote inflammation. based on the immunomodulatory activity of various fas, we explored their effects on placenta inflammatory responses ex vivo. methods: placenta cells were isolated from fresh human (anonymous), term placentas and treated with/without lipopolysaccharide (lps) with various fas, including saturated fas, trans-fas, and n- and n- pufas (at physiological concentrations). after an overnight treatment, tnf-alpha (tnf) production was determined. data were analyzed by analysis of variance (anova) followed by the dunnett's test. results: oleic acid ( : n- ), a cis-monosaturated fa common in the mediterranean diet, did not induce significant placenta tnf production (fig. a) . by contrast, elaidic acid ( : n- ), a trans-monosaturated fa, induced tnf production by -fold compared to vehicle (fig. a) . likewise, palmitic acid ( : ), a saturated fa, induced placenta tnf production by -fold (fig. a) . to mimic inflammation, placenta cells were treated with lps ex vivo in the presence/absence of fas. docosahexanoic acid ( : n- , dha) reduced placenta tnf production by up to % following stimulation (fig. b) . similarly, treatment of placenta cells with linoleic acid ( : n- , la) or arachidonic acid (n : n- , aa) suppressed tnf production by up to % and %, respectively (fig. b) . conclusions: both saturated fas and trans-fas, which positively correlate with hypertension, induce placental tnf production. the n- fa precursors to prostaglandins, aa and linoleic acid, reduce placental tnf production following stimulation. likewise, dha, a product of n- fa metabolism commonly consumed in fish oil and associated with improved blood pressure, suppresses tnf production by stimulated placenta cells. vegf and flk mediated glomerulogenesis in offspring exposed to maternal hypernatremia. roy z mansano, mina desai, ahmed abdel-hakeem, thomas r magee, tazmia q henry, cynthia nast, john s torday, michael g ross. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: growth restricted human and animal offspring, resulting from maternal nutrient restriction or uteroplacental insufficiency, consistently demonstrate reduced glomerular number and a predisposition to adult systemic hypertension and renal compromise. in contrast, maternal water restriction (wr) produces newborns with a unique programmed phenotype of increased glomerular number. glomerulogenesis is dependent, in part, on appropriately timed and quantified vascular development. as vegf and its receptor flk have crucial stimulatory effects on renal vasculogenesis, we hypothesized that maternal wr-induced morphologic renal changes are secondary to vegfmediated vasculogenic effects. methods: from day to term gestation, pregnant rats received either ad libitum food and water (control, n= ), or wr to produce an increment of meq/l in plasma sodium (n= ). following delivery, all dams received ad libitum food and water. at d after birth, offspring kidneys were extracted for mrna. vegf and its receptor, flk , mrna levels were determined using real-time rt-pcr (presented as fold difference normalized to s). at d after birth, offspring glomerular number were determined in formalin fixed m sections using histomorphometric analysis. values are means±se. results: wr offspring (day ) had higher glomerular number than control (wr ± , control ± per mm , p< . ). flk mrna expression was increased in wr offspring kidneys (wr . ± . , control . ± . , p< . ) as compared to controls. in contrast, vegf mrna expression in wr offspring kidney was comparable to control (wr . ± . , control . ± . , p= . ). conclusion:prenatally wr offspring demonstrate significantly increased glomerular number. as vegf recruits angioblasts to the developing glomerulus via its receptor flk , increased receptors (flk ) with the same level of the ligand (vegf) suggest that enhanced vasculogenesis may represent a putative mechanism for increased nephrogenesis in wr offspring. modulation of newborn vasculogenesis via vegf and flk expression may represent a therapeutic option for growth restricted offspring with decreased glomerular number. objective: maternal food restriction (mfr) during gestation (embryonic day to birth) reduces rat kidney glomerular number by % at weeks of age. undernutrition during human gestation leads to similar impaired nephrogenesis and increased hypertension in adults. renal development may be delineated into stages including ureteric bud branching, mesenchymal to epithelial transformation and glomerulogenesis. previously, we detected dysregulation of genes controlling nephrogenesis at gestational ages, e -e , suggesting that mfr has significant effects on ureteric bud branching. in the present study we evaluated the effect of this dysregulation on early nephron development in e fetal kidney explants from dams with mfr. methods: e fetal kidneys were collected from % mfr pregnant rats and ad libitum control rats (n= per group and time point), incubated in dmem/f k medium for , , , , and days, and fixed with % paraformaldehyde. kidneys were stained with fluorescein-labeled dolichos biflorus agglutinin (dba), images captured and terminal ureteric buds quantitated. all values are presented as mean ± sem. differences were considered significant at p< . . results: mfr ex-vivo kidneys at day demonstrated an initial moderate reduction in terminal branches versus control (c) samples (mfr: ± vs c: ± terminal branch ends per kidney). both mfr and control (c) kidneys demonstrated significant (p< . ) increases in branching in explant culture to maximum values at days (mfr: ± vs c: ± ). with increasing culture days, the percent reduction in mfr branching increased with terminal branch point decreases of % at day , % at day , % at day , % at day , and % at day (p< . , mfr vs c at day ). conclusion: kidney explant cultures from mfr treated fetuses display basal and culture-based decreased branching compared to controls. this decrease in terminal ureteric buds, in combination with our previous findings of dysregulation of branching-associated kidney transcription and growth factors, suggests mfr induces early (e ) dysregulation of branching as a major mechanism of the associated nephropenia. these results indicate that early programming events in kidney development induce nephropenia and renal disease in adults. intrauterine growth restriction (iugr) has important implications for the neonate not only at the time of birth, but also as an adult. in humans and animals with iugr, the elevated risk of developing hypertension is thought to involve an upregulation of the renin-angiotensin system (ras). however, the link between the intrauterine insult and enhanced ras is not known. a novel mechanism leading to hypertension has emerged recently involving two orphan g-protein coupled receptors (gcr and gcr ) and their endogenous ligands, succinate and -ketoglutarate. infusion of succinate into adult mice induces hypertension, an effect which was eliminated in gcr knockout mice or by pretreatment with ace inhibitors. interestingly, succinate levels have been shown to increase in the circulation under conditions of oxidative stress, one of the hypothesized mediators of developmental programming of hypertension. thus, we hypothesized that gcr and gcr are upregulated in a rat model of iugr and may contribute to in utero programming of hypertension. timedpregnant rats were fed either control (c, % casein) or low protein (lp, % casein) diet throughout gestation. kidneys were collected on embryonic day (e ), post-natal day (d ) or (d ), n . real-time pcr was used to compare gcr , gcr and renin expression. data were standardized to a housekeeping gene (s ) and are expressed as fold increase/ decrease compared to control. a student's t-test was used to determine significance between groups (p< . ). offspring from lp dams were significantly smaller at birth and did not display catch-up growth. in kidneys from lp fetuses, both gcr and gcr were significantly elevated compared with controls ( . fold and . fold respectively; p= . ), whereas renin was . fold lower. on post-natal d , there was a trend towards increased expression of both gcr ( . fold; p= . ) and renin ( . fold; p= . ) in offspring from lp dams. at d , there were no significant differences between groups. in conclusion, these preliminary data suggest that in iugr offspring from lp rats, the gcr / pathway may be enhanced, indicating a novel mechanism for the programming of hypertension. objective: in addition to excess adipose tissue, obesity is accompanied by increased fat storage in organs such as the liver. peroxisome proliferatoractivated receptors (ppars) are ligand-activated transcription factors involved in the regulation of lipid metabolism, and lipid-associated inflammatory response. obesity represents a state of chronic low-level inflammation, with ppar and ppar implicated in this process. we have previously shown that nutrient restriction in pregnancy results in intrauterine growth restricted (iugr) newborns which develop adult obesity with elevated c-reactive protein (crp) levels. as crp is derived from the liver, we hypothesized that iugr-induced obesity inhibition of hepatic ppar and ppar is associated with an increased inflammatory response. methods: control dams received ad libitum food, whereas study dams were % food-restricted from pregnancy day to to produce iugr newborns. all pups were nursed by control dams and weaned at weeks to ad libitum feed. at day and months of age, male offspring were analyzed for hepatic ppar , ppar and crp mrna (real time rt-pcr) and protein (western blot) expression. data was normalized to -actin and presented as fold change for protein levels. at months, hepatic triglyceride content was determined enzymatically. results: at d of age, iugr pups showed significant downregulation of ppar ( . -fold) and ppar ( . -fold) expression, though crp expression was significantly upregulated ( -fold). findings persisted at months of age, with continued downregulation of ppar and ppar ( . -fold) and upregulation of crp expression ( -fold). furthermore, iugr adults had significantly increased hepatic triglyceride content ( ± vs. ± mg/g liver, p< . ). conclusions: reduced expression of hepatic ppar and ppar in iugr offspring may contribute to elevated hepatic crp levels and triglyceride content. thus, developmental hepatic dysregulation leads to programmed obesity-induced inflammation in iugr offspring. offspring. mina desai, ederlen casillas, guang han, darran n tosh, , michael g ross. dept. of ob/gyn, labiomed at harbor-ucla med. ctr., torrance, ca, usa; dept. of physiology, univ. of adelaide, australia. objective: leptin and insulin mediate central anorexigenic signaling responses via different receptor molecules: leptin binds to the obrb receptor activating jak-stat and pi k pathways, whereas insulin activates pi k pathway by binding to its receptor (ir) and substrate (irs ). maternal food restriction in pregnancy results in iugr newborns that develop hyperphagia and adult obesity. the iugr newborns have significantly decreased plasma leptin levels with increased hypothalamic expression of basal obrb, stat and decreased expression of ir and irs , suggesting altered anorexigenic pathways. we studied the response of hypothalamic leptin/insulin signal molecules to peripheral leptin in iugr newborns. methods: control dams received ad libitum food, whereas study dams were % food-restricted from pregnancy day to . day old male offspring were given saline or leptin ( g/g, i.p). hypothalamus was dissected at , and minutes and protein expression of total stat , phosphorylated stat (p-stat ), inhibitor of leptin signal (socs ), ir, irs , total akt and phosphorylated akt was determined by western blot (normalized to -actin). data is compared between leptin and saline treatments in iugr and controls. results: in response to peripheral leptin, iugr newborns showed marked dysfunction in stimulated hypothalamic leptin and insulin signaling responses. ( ) jak-stat : leptin-treated controls show progressively increased pstat ( -fold) with initial suppression of socs ( . -fold) as compared to salinetreated controls. conversely in leptin-treated iugr, the pattern is reversed such that there is sustained decline in pstat expression ( . -fold) with failure to downregulate socs . ( ) pi k pathway: leptin-treated controls showed a significant reduction in irs ( . -fold) and pakt ( . -fold) as compared to saline-treated controls. however, leptin-treated iugr newborns exhibited a paradoxical increase expression of irs ( -fold) and pakt ( -fold). conclusion:the iugr offspring demonstrate persistent upregulation of leptin receptor, a reduced phosphorylated stat (p-stat ) response in conjunction with an enhanced socs- response. the persistent increase in insulin responses indicates a dysfunction in dynamic signaling, leading to altered anorexigenic response and development of programmed obesity. we have previously demonstrated that maternal food restriction (mfr) in rats induces a marked increase in the expression of vegf protein in the aorta and mesenteric arterioles, accompanied by an increase in tgf-and collagen in both vessel types in adult rat offspring (am j physiol, ) . the aim of this study was to determine if this in vivo finding could be reproduced in an in vitro preparation. methods: two types of preparations were used in this study. we isolated endothelial cells from week old male control rat aortas. these cells were used after the third passage. staining with von willerbrand factor demonstrated that these cells were pure endothelial cells. the second type of preparation was aortic explants from week old male control rats. endothelial cells and aortic explants were transfected with a vegf adenovirus ( - viral infective particles) or a -galactosidase-adenovirus as a control. after hours of culture protein was isolated from cells and explants for western blot analysis using rat specific antibodies. culture media was assayed for vegf by elisa. results: transfection of vegf adenovirus induced a dose-dependant increase in the expression of vegf protein in primary endothelial cells and aortic explants. the transfection of vegf into the endothelial cells showed a bell shaped curve, and was accompanied by an increase in media levels of vegf protein. maximal secretion of vegf was found with viral infective particles. vegf adenovirus transfection induced a dose-dependant increase in c-reactive protein (crp) (inflammatory marker), and tgf-protein in both aortic explants and primary endothelial cells. these results indicate that upregulation of vegf in blood vessels induces inflammation and tgf-expression which in turn can induce collagen synthesis. thus the increased collagen expression and reduced compliance previously reported by us in vessels of mfr offspring can be explained by the over-expression of vegf which we reported. therapeutic intervention aimed at prevention of the increase in vascular vegf expression in mfr offspring could potentially prevent programmed hypertension. maternal regulation of high fat nourishment during lactation period reduce a hypertension of male offspring. hidenori takahashi, toshiaki okawa, keiya fujimori, akira sato. obgyn, fukushima med.univ., fukushima, fukushima, japan. objective: exposure to undernutrition or high fat nourishment during fetal life has been proposed as an underlying cause of adult hypertension, but the effect of maternal feeding regulation during lactation period on blood pressure of offspring is unclear. our objective was to investigate the effects of either high-fat diet (hfd) during gestation to lactation period or restrictive fed a hfd during lactation period on blood pressure in male rat offspring. we use types pregnant wistar rats as fed with normal nutrition (group a), with a high fat diet (hfd) during gestation to lactation period (group b) and with hfd nutritionally restricted by feeding with % of the normal lactationmatched dietary intake from the day of delivery to the end of lactation period (group c). the male offspring was measured blood pressure at , and weeks by using indirect tail-cuff method. statically analysis was performed using oneway annova. results: body weight was significantly reduced in c offspring compared to a and b in male offspring at day after delivery (p< . ). at weeks old, the body weight of c offspring was no difference to catch up compared to a and b offspring. systolic and diastolic blood pressures were significantly elevated at all , and weeks in offspring of b > c >a. (p< . , vs. a) conclusions: under high-fat nutrition during gestation to lactation period induced hypertension in male rat offspring. maternal high fat environment make a hypertensive offspring, but regulation of fat feeding during lactation period may reduce adulthood hypertension. background: uterine artery (uta) doppler velocimetry has been validated in populations of heterogeneous parity in the second trimester for prediction of obstetric outcomes requiring preterm delivery to include: fetal growth restriction, fetal demise, hypertensive disorders of pregnancy, abruption, and indicated preterm delivery. understanding that parity may affect uta doppler indices in subsequent pregnancies, we sought to validate these predictive values in the first trimester in a homogeneous population of multiparous women. study design: multiparous women undergoing first trimester screening of singleton pregnancies were enrolled and followed prospectively until delivery (n= ). these women were divided into controls, ri < . (n= ) and cases, ri . (n= ), based on prior studies. demographic, clinical, and sonographic data (including uta indices and assessment of notching) were obtained. statistical analysis included student's t test and chi square. results: cases were not significantly different from controls in terms of maternal age, ethnicity, bmi, or medical history. uta doppler indices were significantly different between the two cohorts in terms of the presence of unilateral or bilateral notching ( % vs, % p< . and % vs. %, p< . , respectively). in contrast to that observed in patients of heterogenous parity previously, ri . was not associated with adverse pregnancy outcomes despite an average ri of . , significantly above this threshold. conclusions: in this multiparous cohort ri was not predictive of adverse obstetrical outcome, in contrast to that observed in cohorts including nulliparous patient. parity may affect uta vasculature and obscure the ability of doppler velocimetry to predict adverse obstetric outcome in multiparous women. presence of uta doppler notching in the first trimester remained a robust predictor of adverse obstetric outcomes in multiparous patients. objective: epidemiological studies have shown that offspring exposed to preeclampsia during fetal development are more susceptible to airway disease later in life. we have shown previously that gender, but not sflt- over-expression during pregnancy determines higher reactivity in the offspring airways at months of age. the objective of this study was to examine the effect of preeclampsia on the trachea from female and male offspring in our model of sflt- induced preeclampsia at year of age and compare responses between the two age groups. methods: cd- mice at day of gestation were injected via the tail vein with adenovirus carrying sflt (adsflt , pfu/ l) or mfc (admfc, pfu/ l). mice were allowed to deliver. tracheas were isolated from female and male offspring at months and year of age, and rings were mounted in organ chambers for isometric tension recording. responses to potassium chloride (kcl, mm), the mast cell degranulating agent compound / ( / , g/ml), and concentration-responses curve to acetylcholine ( - - - m) were obtained. results: there was no significant difference in responses to acetylcholine, kcl, or compound / between year old offspring born to the sflt and mfc groups. when comparing offspring within the same pregnancy exposure groups, responses to acetylcholine in adsflt -treated group were significantly higher in year old females than males. comparison between age groups by pregnancy exposure revealed that in the mfc group, year old male offspring had higher responses to compound / and acetylcholine than months old males. responses to kcl were significantly higher in months old males than year olds independent of maternal treatment during pregnancy. in females, the only difference between age groups was observed in the mfc group, where months old offspring demonstrated significantly higher responses to acetylcholine compared to year old offspring. conclusions: our findings did not show that airways of year old offspring born to mice with a preeclampsia-like syndrome induced by sflt- over-expression have airway hyperreactivity. however, sex and age differences in airway responses dependent on maternal exposure during pregnancy were observed, and needs to be explored further to elucidate underlying mechanisms. objective: maternal food restriction (fr) results in iugr newborns that when normally nursed exhibit rapid catch-up growth and adult obesity. continued fr during nursing delays catch-up growth and prevents adult obesity. igf- , which modulates growth and is secreted by the liver, may contribute to these morbidities. igf- is epigenetically regulated involving two promoters, alternative exon splicing and multiple transcription termination sites. we determined if hepatic igf- mrna levels correlate with obesity, and whether these changes are due to programmed epigenetic modification. methods: control pregnant rats received ad libitum food from gestation day to and lactation, whereas study dams were % fr. fr pups were nursed by either control (fr/adlib) or fr dams (fr/fr) and weaned to ad libitum feed. at day and months, male livers were analyzed for igf- mrna variant levels (real time rt-pcr). chromatin immunoprecipitation (chip) was performed using the antibody for h k trimethyl, and associated levels of each igf- species were measured by pcr. results: at months, obese fr/adlib males showed increased mrna levels of igf- a, igf- b, igf- exon , and igf- exon as compared to controls ( ± , ± , ± , and ± %). comparing fr/adlib month to newborn offspring, h /k was increased at igf -promoter , promoter , exon , utr# and utr# ( ± , ± , ± , ± , and ± %), though there was no differences between control month and newborns. in contrast, month fr/fr males had comparable mrna levels to the controls except for igf- b (% of control: ± ). further, fr/fr month h /k was only different from newborns at utr# (% of newborn: ± ). conclusion: iugr newborns with rapid catch-up growth and adult obesity have increased postnatal hepatic igf- mrna levels, likely a result of igf- histone and chromatin structure modifications to h k trimethylation. conversely, iugr with delayed catch-up growth and absence of adult obesity have levels similar to that of controls. thus, modulation of the rate of iugr newborn catch-up growth may protect against igf- epigenetic modifications. introduction: igf-ii is synthesized as a pro-hormone (proigf-ii; -amino acid peptide) which is then processed into its active forms: "big" igf-ii ( - ) and mature igf-ii ( - ). these active forms are essential for placental and fetal development and have also been shown to persist into postnatal life. since maternal smoking is known to adversely affect feto-placental growth and postnatal development, we postulated that these effects might be mediated through nicotine-induced alterations in igf-ii processing. methods: in the present study, nulliparous female wistar rats ( - g) were given nicotine ( mg/kg/day) or saline for days prior to mating, during pregnancy, and throughout lactation. at gestational day , and , dams were euthanized and we collected serum (fetal and maternal), amniotic fluid and recorded fetal body weight. a subset of dams were allowed to deliver at term. following parturition, serum samples from the offspring were collected at birth (pnd ) and weaning (pnd ). body weight was recorded weekly from birth to weaning. pro, "big" and mature igf-ii levels were determined by western blot analysis. results: maternal nicotine exposure during pregnancy resulted in a significant reduction in fetal body weight by gestational day . however, there was no effect of nicotine on fetal serum or amniotic fluid igf-ii levels at any gestational age examined. in maternal serum, mature igf-ii in control animals decreased with advancing gestational age such that igf-ii levels were lowest at gestational day . nicotine administration prevented this decline, which resulted in significantly higher mature igf-ii levels in nicotine-exposed mothers at gestational day . in postnatal life, nicotine exposed offspring had significantly lower levels of "big" igf-ii expression at weaning (pnd ). conclusions: these data demonstrate that nicotine can alter the amount of the active forms of igf-ii in the mother and the newborn. dysregulation of maternal igf-ii occurs concomitantly with suboptimal fetal growth. results from this study suggest a mechanism by which maternal smoking causes impaired fetal growth and adverse postnatal health outcomes. objective: maternal food restriction in pregnancy results in iugr newborns which develop adult metabolic syndrome. programming of both increased appetite-mediated hyperphagia and enhanced adipogenesis contribute to the development of obesity. transcription factors, peroxisome-proliferatoractivated-receptor (ppar ), ccaat/enhancer binding-protein (c/ebp ), and sterol regulatory element binding-protein (srebp c) regulate adipogenesis and lipogenesis. although iugr offspring exhibit acute upregulation of the adipogenesis signaling cascade prior to the development of obesity, we determined if this increased adipogenic potential was an intrinsic cellular response, and thus maintained in cell culture. we further examined the responses to adipocyte stimulators (ppar activator-ligand rosiglitazone) and inhibitors (ppar repressor-ligand badge). methods: control dams received ad libitum food, whereas study dams were % food-restricted from pregnancy day to term. adipocytes from day old iugr and controls were isolated and cell proliferation rate was determined (mtt). primary adipocyte cell cultures were established and following % confluence, iugr and control adipocytes were treated to two doses ( and m) of either rosiglitazone or badge for h. mrna and protein was extracted for expression of ppar , c/ebp , srebp c. data was normalized to -actin and compared to the respective untreated cells. results: iugr adipocytes had significantly increased protein expression of ppar ( . -fold) and c/ebp ( -fold) as compared to control adipocytes, though srebp c levels were unchanged. mrna levels showed similar changes in iugr newborns. importantly, iugr adipocytes exhibited increased cell proliferation ( % of control, p< . ) and showed greater response to rosiglitazone ( . -fold), though similar response to badge, as the control adipocytes conclusion: iugr primary adipocytes cell culture exhibit basal phenotypic characteristic of programmed upregulation of adipogenic transcription factors which promote adipose cell proliferation. the enhanced response to the adipogenic stimulant is further evidence of the predisposition to obesity. in contrast, the normal suppressive response to the inhibitor suggests that iugr adipocytes may respond to pharmacologic approaches to prevent obesity during this period. objective: maternal nutrient restriction results in intrauterine growth restricted (iugr) newborns which develop programmed obesity despite a normal post-weaning diet. the epidemic of obesity has been attributed in part to programmed "thrifty phenotype" and exposure to "western" diets. hepatic igf- is epigenetically regulated involving two promoters, alternative exon splicing, and multiple transcription termination sites. iugr offspring with normal post-weaning diet have increased postnatal hepatic igf- mrna levels, likely a result of igf- histone and chromatin structure modifications to h k trimethylation. we hypothesized that iugr newborns that develop programmed obesity would demonstrate discernable hepatic igf- changes which are distinct from diet-induced obesity. we determined igf- hepatic mrna levels and epigenetic characteristics in programmed (iugr) and dietinduced (dio) offspring. methods:: control pregnant rats received ad libitum food whereas study dams were % maternal food restricted from day to . all pups were nursed on ad libitum fed dams. controls were weaned to high-fat (fat, %) diet whereas iugr were weaned to normal ad libitum diet (fat, %) to produce diet-induced (dio) and programmed obese groups, respectively. at months, male hepatic igf- were analyzed for igf- mrna variant levels (real time rt-pcr). chromatin immunoprecipitation (chip) was performed using the antibody for h k dimethyl and h k trimethyl, and associated levels of each igf- species were measured by pcr. result: relative to dio control males, iugr had increased mrna of igf- a, exon and exon ( ± , ± , ± %). chip with h k dimethyl showed increased igf- exon ( ± %) and with h k trimethyl, increased igf- promoter and promoter ( ± , ± %) as compared to dio controls. conclusion: adult obese iugr males exposed to normal postweaning diet have increased hepatic igf- a mrna and h k dimethylation and trimethylation of igf- than dio controls. changes in igf- in adulthood from a prenatal insult thus suggest that igf- is programmed during the fetal period and may be associated with programmed adult obesity. rebekah elkins, pandu gangula, chandra yallampalli. obstetrics gynecology, university of texas medical branch, galveston, tx, usa; internal medicine, university of texas medical branch, galveston, tx, usa. objectives: we previously reported that the offspring of rats fed % protein during gestation develop hypertension at two to four months and that the hypertension is exacerbated in males. this study is to evaluate: ) changes in estrogen receptor (er) angiotensin ii subtype receptor (at -r) and endothelial nitric oxide synthase (enos) in the mesenteric artery and aorta of offspring and assess if these changes, if any, are gender specific. methods: pregnant sprague dawley rats were fed either % protein (ctrl) or % protein (lpd) from day of gestation. the offspring were evaluated for hypertension by means of systolic blood pressure measurements. at four months for the males and nine months for the females, mesenteric artery and aorta were collected in rnalater. expression of estrogen receptor a (er-a) and b (er-b), at -r, and enos were analyzed by western immunoblotting and rt-pcr and expressed relative to b-actin or s. results: mesenteric artery shows no differences between ctrl and lpd female offspring in at -r, enos, er-a or er-b. similarly mesenteric artery shows no diet exposure related changes in at -r, er-a or er-b in male offspring. however, enos expression was lower in mesenteric artery of lpd male offspring. on the other hand, in the aorta both er-a and er-b levels are lower in lpd female offspring while there were no changes in at -r or enos. no changes in at -r, er-a or er-b were observed in male offspring aorta of ctrl and lpd rats. conclusion: the in utero exposure to lpd results in adult hypertension in both male and female offspring. some mechanisms for hypertension include the decrease in er-a and er-b but not at -r or enos in females, and the decrease in enos but not at -r or er in males indicating gender related differences. offspring. hidenori takahashi, toshiaki okawa, keiya fujimori, akira sato. obgyn, fukushima med. univ., fukushima, fukushima, japan. our objective was to investigate the effects of either severe undernutrition during late gestation or lactation period on blood pressure and the development of vascular function in male rat offspring. we use normal pregnant wistar rats (group a), nutritionally restricted by feeding with % of the normal gestation-matched dietary intake from day of gestation to delivery (group b) and % restricted after delivery to the end of lactation period (group c). the offspring was measured blood pressure at and weeks by using indirect tail-cuff method. rings of thoracic aorta with intact endothelium from the male offspring of a and b at weeks, were equilibrated at g passive tension in organ chambers filled with krebs-henseleit solution continuously bubbled with %co in air ( °, ph . ) for isometric tension recording. concentration-response relationships to norepinephrine (ne) and angiotensinii(atii) were obtained in the absence or presence of n(omega)-nitro-l-arginine methyl ester (l-name) or a selective atii type- receptor blocker (valsartan). responses to cumulative concentrations of sodium nitroprusside (snp) and to - m oxyhemoglobin (hb, nitric oxide scavenger) were also determined. contractions were expressed as a percent of the reference contraction induced by potassium chloride ( mm). statically analysis was performed using one-way annova. results: body weight was significantly reduced in b offspring compared to a and c in male offspring at day (p< . ). at weeks the body weight of offspring of b was no difference to catch up compared to a and c offspring. systolic and diastolic blood pressures were significantly elevated at both and weeks in offspring of b > c >a. ne concentration-dependently stimulated tension of aortic rings from in a and b offspring, which was not significantly (n= ). maximal contractions to ne were significantly stimulated by l-name in a (p< . ), but not b offspring. valsartan significantly inhibited aortic contractions by ne in r (p< . ), but not a offspring. there was no significant difference on responses of aortic rings by atii, snp and hb in a and b offspring. conclusions: severe under nutrition during not only late gestation but also lactation period induced hypertension in male rat offspring in adulthood. fetal origin of adult hypertension might be vascular endothelial dysfunction. fujimori, akira sato. obgyn, fukushima med. univ., fukushima, fukushima, japan. objective: exposure to undernutrition during fetal life has been proposed as an underlying cause of adult hypertension, but the effect of either high fat nourishment or undernutrition during lactation period on blood pressure is unclear. our objective was to investigate the most effective maternal nourishment and feeding period for offspring induced adulthood hypertension in using high-fat diet (hfd). study design: we use types pregnant wistar rats as fed with normal nutrition (group a), nutritionally restricted by feeding with % of the normal gestation-matched dietary intake from day of gestation to delivery (group b), % restricted after delivery to the end of lactation period (group c), with a high fat diet (hfd) during gestation to lactation period (group d) and with hfd nutritionally % restricted from the day of delivery to the end of lactation period (group e). the offspring was measured body weight (bw) and measured blood pressure at , and weeks by using indirect tail-cuff method. statically analysis was performed using one-way annova. results: bw was significantly reduced in b offspring compared to another (a, c, d, e) male offspring at day (p< . ). at day after delivery, bw was significantly reduced in c, e offspring compared to a, d in male offspring (p< . ). at weeks old, bw of all type offspring was no difference. systolic and diastolic blood pressures were significantly elevated at weeks in offspring of d> b > e > c >a. (p< . , vs. a). at weeks, hypertensive offspring as b>d>e>c>a (p< . , vs. a). at weeks, d> e > b > c >a (p< . , vs. a). conclusions: maternal high fat environment make a hypertensive offspring, but regulation of fat feeding during lactation period may reduce adulthood hypertension. in case with normal food, restrictive feeding during late gestation is more effective than lactation period for inducing hypertensive male offspring. regulation of maternal feeding not only during late gestation but also lactation period may control adulthood hypertension. the strongest epigenetical factor of maternal nutrition is high fat feeding during pregnancy to lactation period for f blood pressure, respectively. or residence at high altitude, impacts fetal growth and development. in a preliminary study, we observed a significant decrease in birth weight, subsequent compensatory postnatal growth, and an increase in relative right ventricular (rv) weight at postnatal (pn) day in female offspring of rats exposed to hypoxia ( , ft; . % po ) from days thru of gestation (dga). thus, our objective was to further elucidate the impact of prenatal hypoxia on fetal growth and postnatal development. methods: pregnant dams (hx, n= ) were hypoxic from - dga with additional control dams either fed ad libitum (al, n= ), pair-fed with the hx dams throughout gestation (pg, n= ), or only pair-fed during the window of hypoxia (ph, n= ). female offspring from hx, pg, and ph dams were cross-fostered onto additional al dams (n= /litter) by h after birth. results: at birth, there was no difference in litter size; however, body weight (bw) of the hx, pg, and ph pups was lower (p< . ) than that of al pups, and hx pups were lighter (p< . ) than ph pups. weight of hx offspring remained lower (p< . ) than al pups until the termination of the study at pn , while the pg and ph pups reached weights comparable to the al offspring by pn . relative to bw, heart weight and left ventricular/septal (lvs) weight was not different among groups; however, right ventricular weight (rv/bw) was greater (p< . ) in the hx offspring at pn as was rv/lvs (p< . ). cardiac function was evaluated by echocardiography at pn . rv wall thickness was % greater (p< . ) in hx pups as compared to al pups, confirming the significantly higher relative rv weight observed at necropsy. pep, pep/at, and pep/et were %, %, and % higher respectively in the hx offspring relative to the al offspring. lv end diastolic and end systolic diameters were smaller (p< . ) in hx and ph offspring relative to the al group. myosin heavy chain (mhc) and mrna concentrations in the rv were evaluated by qrt-pcr, and the mhc / mrna ratio was greater (p< . ) in the hx pups. conclusion: prenatal hypoxia from - dga impacted both fetal and postnatal growth, altered postnatal heart development and function, with the primary impact being on the rv. supported by nih hd . reproductive sciences; pharmacology experimental therapeutics, university of maryland, baltimore, md, usa. background: exposure to nicotine (nic) is a significant risk to normal fetal development. fetal nic, which readily crosses the placenta, can be acquired from pregnant mothers by smoking or nicotine replacement therapy. the impact of nic on fetal organs may be mediated directly and/or via intrauterine hypoxia (hpx) via constriction of the uterine circulation. in adult hearts, both nic and hypoxia stimulate gene expression of matrix metalloproteinases (mmp), although the study of nic and hypoxia on gene expression in fetal organs remains incomplete. because mmps are involved in the regulation of extracellular matrix turnover and cardiac remodeling, we tested the hypothesis that prenatal nic and intrauterine hpx upregulate protein expression and activity of mmp in the fetal guinea pig heart. methods: pregnant guinea pigs were placed in either normoxia (nmx) or hpx ( . %o in chamber) for d prior to term ( d). in two separate groups, nic was also added to the drinking water ( mg/kg/d) for d at a dose that generates fetal nic levels ( ng/ml cotinine) equivalent to a moderate smoker. anesthetized near-term fetuses ( d) were excised and weighed. left ventricles of hearts were obtained and frozen at - c for storage. mmp protein levels and enzymatic activity were measured by western analysis and gel zymography, respectively. results: nic alone (nmx+nic) decreased (p< . ) fetal body wt by %, increased (p< . ) the relative fetal brain wt (brain wt/fetal body wt ratio) by . % and had no effect on relative placental or fetal heart wts. hpx alone decreased (p< . ) fetal body wt by . %, increased the relative fetal brain wt by % but, in contrast to nic alone, increased relative placental wt by . %. both mmp protein levels (mmp /a-actin density values) and activity (clear band density) were increased (p< . ) by nic alone (by . and . fold, respectively) and hpx alone (by . and . fold, respectively). in addition, both protein and activity levels of hpx hearts were further increased by nic (by . and . fold) compared to hpx alone. conclusion: prenatal nic upregulates mmp expression in nmx fetal hearts and is potentiated by hpx. this suggests that under conditions of intrauterine stress cardiac remodeling by mmp activation may be an important mechanism by which nic and hpx affect fetal heart function. objective: in addition to peripheral hypoglycemic effects, insulin induces central anorexigenic responses via stimulation of the phosphoinositide- kinase (pi k) pathway and cellular growth by mitogen activated protein kinase (mapk) pathway. the pi k signaling cascade is activated by insulin binding to its receptor (ir), recruiting ir substrate (irs- ), and phosphorylating pi k. activated pi k in turn causes phosphorylation of protein kinase b/akt which subsequently modulates hypothalamic anorexigenic responses. in contrast, the mapk (erk /erk ) signaling pathway likely involves irs- . further, insulin signaling is inhibited by the lipid phosphatase pten. we have previously shown that maternal food restriction (mfr) during pregnancy results in iugr newborns that develop hyperphagia, obesity and insulin resistance as adults. we sought to determine if altered hypothalamic basal insulin signaling expression of pi k and mapk pathways contribute to reduced satiety responses and thus enhanced growth in iugr newborns methods: pregnant control dams received ad libitum (n= ) food, whereas study dams were % mfr (n= ) from pregnancy day to to produce iugr newborns. at day , hypothalamic region was dissected and analyzed for mrna levels (real time rt-pcr) of insulin signaling components via pi k (ir, irs , pi k and akt) and mapk (irs , erk , erk ) pathways, and pten. data is presented as fold difference normalized to beta- -microglobulin. results: at d of age, iugr pups exhibited downregulation of the entire pi k pathway with significantly decreased (p< . ) mrna levels of ir ( . -fold), irs- ( . -fold), pi k ( . -fold) and akt ( . -fold). further, iugr pups showed similar decreased mrna expression of erk ( . -fold) and erk ( . -fold). however pten expression was similar to the controls. conclusion: reduced insulin-mediated pi k signaling likely contributes to the suppressed anorexigenic responses and development of obesity in iugr offspring. reduction of central mapk signaling suggests a potential maldevelopment of additional neuronal pathways in iugr offspring. objectives: in the rat, uteroplacental insufficiency restricts fetal growth and impairs mammary development further compromising postnatal growth. both male and female growth-restricted offspring have a reduced nephron endowment but only males develop hypertension with glomerular hypertrophy, which can be reversed by improving the lactational environment. this study used cross-fostering to assess the influence of the prenatal and postnatal environments on renal development and nephrogenesis. methods: bilateral uterine vessel ligation (restricted, r) or sham surgery (control, c) was performed on day of gestation in wky rats. control and restricted pups were cross-fostered onto c or r mothers on postnatal day (pn ). post mortem was carried out on pn (c and r) and pn (c-on-c, c-on-r, r-on-c, r-on-r). results: body and kidney weights were decreased in r and r-on-r pups on pn and pn (p< . ). there was some evidence of accelerated pup growth for r-on-c relative to r-on-r on pn . male, but not female, relative bmp mrna expression on pn was higher in r than c (p< . ) while gdnf, tgf and at receptor were not different. on pn , wnt (but not at r, vegf-a) mrna expression (males only) was relatively higher in r-on-r (p< . ) when compared to c-on-c (p< . ). this and the histological analyses suggests an up-regulation of nephrogenic activity with more immature nephrons (males and females) in r-on-r (p< . ) when compared to c-on-c, while r-on-c remained intermediate. intrauterine growth-restricted pups were born lighter and with smaller kidneys. this was partially rescued by improving lactational nutrition (r-on-c) at pn . higher bmp mrna expression indicates impaired branching morphogenesis in pn r male, but not female kidneys, suggesting the timing and/or molecular mechanisms underlying the nephron deficit may be sex specific. at pn there was evidence of extended and increased nephrogenic activity in r-on-r, however, this was unable to restore the later nephron deficit. improved lactation for r-on-c, which prevented the adult nephron deficit and hypertension, increased and extended nephrogenesis to a lesser degree than r-on-r suggesting that the restoration of nephron endowment was likely to have occurred prior to pn . amy m tetrault, sarah b lieber, marya shanabrough, tamas l horvath, hugh s taylor. obstetrics, gynecology and reproductive sciences, yale university school of medicine, new haven, ct, usa. objective: classically recognized for its role in energy balance, body weight and appetite, ghrelin has also been implicated in reproduction. ghrelin (-/-) mice are infertile while administration of ghrelin to wt mice results in decreased litter size and constrained embryonic growth. here we investigate the effect of maternal ghrelin deficiency on in utero developmental programming of the female reproductive tract. hox genes determine developmental identity of the paramesonephric duct. we determined that hoxa is regulated by ghrelin in vitro and that in utero ghrelin deficency alters f hoxa gene expression and reproductive success. methods: wild-type females mice parented by ghrelin +/-b d f (ghrellin deficient) mice were analyzed for litter size, oocyte, and corpus luteum number. rna was extracted from the uterus of mice exposed to ghrelin deficiency in utero. ishikawa cells were treated with ghrelin with/without receptor (ghsr) antagonist, or saline and rna extracted. in both hoxa expression was analysed by real time rt-pcr normalized to -actin and also determined by ihc. experiments were repeated in triplicate and mrna expression compared by student's t-test. results: wild-type female offspring of ghrelin deficient dams had smaller litter sizes than controls (n= , . ± . pups; n= , . ± . pups, respectively; p< . ). no differences were seen in oocyte or corpus luteum number suggesting a uterine defect. hoxa mrna and protein expression were decreased in the uterus of the f females. ghsr was expressed in uterine endometrium. treatment of ishikawa cells with nm to nm ghrelin resulted in a to % increase (p< . ) in hoxa expression. treatment with ghrelin and ghsr antagonist resulted in similar increases in hoxa expression indicating a non-receptor mediated mechanism. conclusion: ghrelin contributes to reproductive tract developmental programming; in utero ghrelin deficiency compromises reproduction in female offspring. the developmental effects of ghrelin were mediated by alteration in hox gene expression and not through the classic ghsr receptor. obesity and decreased ghrelin may lead to defects in developmental programming of the reproductive tract. these findings demonstrate the importance of nutrition, energy utilization and appropriate ghrelin levels on normal uterine development. we have previously studied the deleterious effects of lack of the endothelial nitric oxid synthase (nos ) in mouse dams and their offspring. our laboratory demonstrated that adaptive responses in subsequent pregnancies may offset the harmful effects of the genetic deficiency of nos . in this study we aimed to determine hepatic and renal histopathologic damage in nos deficient pregnant mice comparing animals carrying their first versus their second pregnancy. study design: gravid nos +/+wt and nos -/-ko mice during their first (p ) or second (p ) pregnancy were sacrificed at day of gestation. livers and kidneys were stained and analyzed for the presence and extent of histopathologic lesions. results: nos +/+wt dams displayed a low incidence of significant renal or hepatic lesions in either the first or the second pregnancy. in nos -/-ko mice the incidence of liver necrosis and inflammation during the first pregnancy was % and %, respectively. in nos -/-ko dams sacrificed during the second gestation the incidence rates for the same lesions were % and %, respectively (p< . ). this correlation persisted when we analyzed the relative severity of hepatic lesions between p and p animals. although a similar trend was observed, the difference between p and p animals with regards to kidney lesions did not reach the level of statistical significance in our study. conclusions: a second pregnancy in this animal model of hypertension was associated with a significantly improved hepatic histopathology compared with the first pregnancy. this observation is consistent with our previous studies showing a decrease in systemic vascular resistance in p versus p nos -/-ko mice. the beneficial effects of a prior pregnancy may partially underlie the phenomenon of a decreased risk of preeclampsia in multiparous versus nulliparous women. further studies are required to delineate the counterregulatory mechanisms leading to improved cardiovascular function in subsequent pregnancies in these genetically modified animals. maternal hypomethylation is associated with congenital heart defects in down syndrome. lmjw van driel, , r de jonge, wa helbing, bd van zelst, j lindemans, eap steegers, rpm steegers-theunissen. , , , obstetrics gynecology; pediatrics; clinical chemistry; epidemiolog y biostatistics; clinical genetics; erasmus university mc, rotterdam, netherlands. background: maternal age and hyperhomocysteinemia are risk factors for having a child with down syndrome (ds) and congenital heart defects (chds), respectively. evidence is rising that ageing is associated with a state of hypomethylation. objectives: to investigate whether the risk of a child with ds and chd is associated with maternal hypomethylation. methods: we conducted a case-control triad study at months after the index-pregnancy. case-children (n= ) were included if they had ds and chd. children (n= ) without a major congenital malformation served as controls. the concentrations of s-adenosyl methionine (sam), s-adenosyl homocysteine (sah), sam/sah ratio, and homocysteine in maternal blood were measured as biomarkers for methylation. the data were analyzed using the mann-whitney u test and a logistic regression model. results: maternal age was included in the model as potential confounder. the levels and the crude and adjusted or( %ci) of the biomarkers are shown in table . an increase of the sam/sah ratio with unit decreases the risk of a child with ds and chd with percent. moreover, every increase of mol/l of homocysteine . fold increases this risk. conclusions: maternal hypomethylation is significantly associated with an increased risk of having a child with ds and chd. since, the effects are confounded by maternal age, hypomethylation can be considered as feature of ageing. the developmental origins hypothesis postulates that during critical ontogenetic periods, transient environmental stimuli perturb developmental pathways and induce permanent changes in gene expression, metabolism, and chronic disease susceptibility. one likely mechanism is via early nutritional influences on epigenetic gene modification consisting of the presence of a methyl group on the carbon of a cytosine residue. this modification is responsible for an important form of gene regulation in eukaryotes. in the present study, we have tested the hypothesis that maternal low-protein diet altered epigenetic regulation of specific gene of the offspring. c bl/ female mice were mated and on the day the plug was detected, these females were then randomly allocated to be fed isocaloric diets consisting % protein or % protein. at delivery, offspring were killed and the livers were removed immediately, frozen in liquid nitrogen and stored at - c. genomic sequencing after bisulfite modification is used to study site-specific dna methylation. dna methylation status of oct- and sphk- gene upstream regions in the mouse liver was analyzed. hepatic oct- or sphk- promoter methylation was not significantly different between both groups. however, dna methylation pattern of the genomic dna is specific in low-protein diet group. aberrant oct- and sphk- gene expression may cause perturbations in cell differentiation. we suggest that the epigenetic mechanism consisting of dna methylation underlies the fetal programming theory. primary human cytomegalovirus (hcmv) infection during pregnancy can have devastating consequences for both the mother and fetus. hcmv infection has been implicated in the development of pre-eclampsia and intrauterine growth retardation (iugr), as well as congenital cmv syndrome in newborns exposed in utero. previously, we have shown that hcmv infection of placental cytotrophoblasts inhibits their normal invasion, proliferation, and migration. however, the mechanisms occurring during early establishment of placental infection are largely unknown. we assessed the impact of hcmv infection on cytotrophoblasts by performing immuno-based assays for various cytokines and cellular growth factors. we detected significant cytokine dysregulation at both and hours after in vitro hcmv infection of cytotrophoblast cells. soluble cytokines involved in recruitment of monocytes and macrophages (gro-a, mcp- ) were downregulated at both and hours after infection. sdf- , which is chemotactic for lymphocytes during early inflammation, was also decreased. these results suggest that recruitment of cells involved in the anti-viral immune response is being interrupted early in the course of infection by hcmv. additionally, a large decrease in the amount of soluble hgf was seen. hgf normally induces migration of cytotrophoblasts along the invasive pathway, and downregulation of this factor could severely affect these processes. finally, we saw increased amounts of soluble icam- , contrasted by decreased amounts of vcam- , indicating dysregulation of adhesion molecules that are necessary for successful placental invasion to occur. all together, these data indicate significant alterations in cytokine profiles as early as hours after hcmv infection, which could provide important clues to the pathogenesis of hcmv in placental invasion and inflammation. additional studies will further elucidate this dysregulation, and determine whether these effects are due to alterations in pre-existing cellular factors or if transcriptional alterations are involved. method: studies were performed in pregnant ewes (n= in each group) with twin fetuses at - days (very preterm) and - days (near-term). neither mother nor fetuses were instrumented prior to the time of blood collection. maternal blood was collected from the jugular vein before sedation. anesthesia was then induced with isofluorane, the abdomen was opened, fetuses exteriorized and blood collected from umbilical cords. blood samples were transferred to plastic tubes containing ethylene-diamino tetraacetic acid and reduced glutathione, plasma separated and stored at - c. commercial radioimmunoassay kits for ovine-crf (phoenix pharmaceuticals, b : . ± . pg/ml) and cortisol (diagnostic laboratory, b : . ± . ng/ml) were used according to the manufacturer's instructions. results: in very-preterm gestations, maternal and fetal plasma crf levels were undetectable. maternal, but not fetal, plasma cortisol levels were measurable ( ± ng/ml). in near-term gestations, both cortisol and crf were measurable in maternal (crf: ± pg/ml; cortisol: ± ng/m) and fetal plasma (crf: ± pg/ml; cortisol: ± ng/ml). plasma crf levels were higher in nearterm fetuses than in their maternal ewes (p < . ). conclusion: ovine plasma crf levels are measurable in maternal and fetal plasma in near-term but not very-preterm gestations. the absence of crf in preterm plasma, perhaps due to reduced placental expression and/or placental crf release, may contribute to the rarity of in utero meconium passage in preterm gestations. interleukin - objectives: interleukin- (il- ) is a pro-inflammatory cytokine produced in adipose cells. recent studies suggest il- may be a marker of maternal obesity and in utero fetal programming. our hypotheses were ) il- correlates with maternal obesity and ) il- mediates the effect of maternal obesity on infant birth weight. methods: the parity, inflammation, and diabetes (pid) study is a longitudinal study of adipokine levels in a diverse sample of pregnant women. we present a cross-sectional analysis of first trimester il- levels from non-diabetic women who underwent a live birth. the independent variable was il- (pg/ml), measured with monoclonal antibody elisa assays. the dependent variable was infant birth weight (gms). maternal bmi categories were: normal/underweight (< kg/m ); overweight ( - kg/m ); and obese (> kg/m ). data on demographic and clinical factors, nutrition and physical activity were collected at baseline. average il- levels were compared across bmi categories using anova. the association of il- levels with infant birth weight was estimated using multiple linear regression, adjusting for covariates. results: average il- levels were significantly higher in obese women ( . ± . ) compared to overweight ( . ± . ) and normal/underweight women ( . ± . ) [p< . ]. after adjustment, il- levels was positively correlated with pre-pregnancy bmi [regression coefficient (rc) . ; % ci: ( . , . )]. as shown in the table below, elevated levels of il- were statistically significantly associated with a . gm higher infant birth weight after full adjustment. each unit increase in pre-pregnancy bmi was associated with a gm higher infant birth weight. table . association of il- with infant birth weight characteristic regression coefficient % confidence interval interleukin- . . , . pre-pregnancy bmi . , gestational weight gain - - , bmi = body mass index; coefficients adjusted for demographics, clinical factors, pre-pregnancy bmi, gestational weight gain, non-fasting first trimester glucose levels, gestational age, nutritional intake and physical activity conclusion: il- and pre-pregnancy bmi were associated with infant birth weight after adjustment for covariates. our findings suggest that the effect of maternal obesity on infant birth weight may be mediated through il- or an alternative independent pathway. we have demonstrated that -tetrahydrocannabinol (thc), in physiologically relevant concentrations, inhibits the growth and tight transcriptional control of the bewo trophoblast cell line . the mechanism involved the decreased expression of the transcriptional regulator histone deacetylase (hdac ). in these experiments we sought to answer the question, 'does anandamide (aea) work in the same manner as thc?' methods: the first trimester human trophoblast cell, bewo were plated at or x cells /well to -well and -well plates for growth and rna experiments, respectively. after growing to - % confluence, cultures were treated with varying concentrations of aea up to a maximum of m for hr. cell numbers were determined using the xtt apoptosis/proliferation assay. total cellular rna was prepared and the relative levels of hdac and gapdh determined by end-point rt-pcr. aea exhibited an inhibitory effect on the bewo cell cultures, but only at concentrations in excess of m where confluency was significantly reduced from % at m to - % at m and m (*p< . ; one-way anova with tukey's hsd test; n= ). cultures treated with m and m aea did not exhibit increased cell death or failure to attach to the substratum, as evidenced by the lack of increase in the shedding of cells into the spent medium. bewo cells treated with aea showed a dose-dependent decrease in hdac mrna expression with a significant effect at . m (*p< . ; oneway anova with tukey's hsd test; n= ). at this dose, the effect of aea had reached an effective maximum decrease in hdac mrna levels ( %) because hdac mrna levels were not decreased further by either m aea ( %) or m aea ( %). the alteration of hdac gene expression by aea in bewo cells with its associated decrease in cell number suggest that the trophoblast cell may be an important target for circulating endocannabinoids during the st trimester of pregnancy. the data indicates that although exocannabinoids and endocannabinoids both inhibit bewo cell growth, they do so using different transcriptional mechanisms. further understanding of the mechanism(s) by which aea alters placental physiology may lead to new strategies for the prevention of pregnancy complications such as st trimester miscarriages. ( ) taylor background: anandamide (aea) exerts its effects by acting on two cannabinoid receptors, cb and cb with the main regulator for aea levels being the metabolising enzyme, fatty acid amide hydrolase (faah). aea, cb , cb and faah constitute the endocannabinoid system and previous studies have shown that faah and cb are expressed in term human placenta( ) suggesting that the endocannabinoid system might be present earlier in gestation. this study aimed to document changes in faah, cb and cb expression in the placenta during the first trimester of pregnancy. methods: first trimester samples ( to weeks gestation) were fixed in % neutral-buffered formalin for days before embedding into paraffin wax or frozen in liquid nitrogen for rna analysis. for immunohistochemistry, faah polyclonal antibodies were used at an optimal dilution of : in pbs, cb at : and cb at : . transcripts were measured using q-pcr with gene-specific primers. results: immunoreactive faah, cb and cb were detected in all samples. faah immunoreactivity in the syncytiotrophoblast increased between the th and th gestational week and by week faah was barely detectable within large parts of the placenta. simultaneously, faah immunoreactivity increased in the mesenchymal core of the developing villi. immunoreactive cb and cb localised to the syncytiotrophoblast, cytotrophoblast and mesenchymal core with cb immunoreactivity showing diminished intensity after week , although this did not reach significance at the transcript level. cb immunoreactivity was absent from fetal blood cells and infiltrating maternal plasma cells, whereas cb and cb immunoreactivity was detected in endothelial cells but not in the vascular smooth muscle cells of blood vessels. the intensity of cb immunoreactivity in the syncytiotrophoblast differed from that of cb and faah in that it remained constant throughout. conclusion: the data suggest that placental faah and cb levels do not alter significantly during the first trimester, but alter their cellular distribution from the syncytiotrophoblast to the mesenchymal core. the significant loss of cb expression from the syncytiotrophoblast after the th week of gestation, a point of critical alteration in the developing placenta, suggests that its retention may be detrimental to normal placental development. reference: park, b. et al., ( ) hankins, chandra yallampalli. obstetrics gynecology, university of texas medical branch, galveston, tx, usa. background: numerous angiogenic proteins synthesized in the placenta are thought to be involved in placental vascularization and development; however, the molecular mechanism modeling the angiogenic process in early pregnancy remains elusive. calatonin gene-related peptide (cgrp) is a multifunctional peptide expressed at the human implantation site; but its influence on in vitro angiogenesis by human micro vascular endothelial cells is not known. objective: the present study was designed to determine the influence of cgrp on angiogenesis by human dermal microvascular endothelial cell (hdmvec) in vitro. methods: hdmvecs (vec technologies) were cultured in mcdb- complete solution containing cgrp ( - m), cgrp plus its antagonist cgrp - ( - m), in well plates with x cells per well. the existence of cgrp receptor components calcitonin receptor-like receptor (crlr) and receptor activity modifying protein (ramp ) was determined using immunofluorescent staining. cell proliferation was examined using methylthiazoltetrazolium (mtt) assay. the pro-angiogenic bioactivity of cgrp was evaluated using cell migration and capillary like tube formation on the matrigel. results: ) immunofluorescent staining showed that cgrp receptor components crlr and ramp are abundantly expressed by hdmvecs. replacement of the primary antibodies with preimmune serum resulted in a negative staining; ) cgrp dose-dependently ( - to - m) stimulated hdmvec proliferation, and this effect was totally blocked by cgrp antagonist, cgrp - ; ) quantitative analysis for cell migration revealed that cgrp increases hdmvec migration in a dose and time-dependant manner; and ) cgrp promotes hdmvec capillary like tube formation, and the length of capillary tube induced by cgrp ( - m) was significantly increased over that of the untreated controls. this increase was observed at hours of treatment and further increase was noted at hours of culture. conclusion: cgrp induces in vitro angiogenesis by promoting microvascular endothelial cell proliferation, migration and capillary like tube formation. therefore, trophoblast derived cgrp at the implantation site may play a role in placental angiogenesis and fetal growth. over-expression of socs- gene promotes il- production by jeg- trophoblast cells. qin dong, , ruping fan, yang gu, david f lewis, yuping wang. biochemistry, harbin medical university, harbin, heilongjiang, china; obstetrics and gynecology, lsuhsc-shreveport, shreveport, la, usa. objective: suppressor of cytokine signaling- (socs- ) plays an important role in negative regulation of inflammatory response in biological cells. evidence has shown anti-inflammatory cytokine il- expression was significantly reduced in trophoblasts of preeclamptic (pe) placentas. we sought to determine if over-expression of socs- in placental trophoblasts could promote il- production. methods: full-length socs- open reading frame (socs- cdna) was generated by rt-pcr from total rna samples isolated from human leukocytes and cloned into a pzsgreen -n vector, which encodes a green fluorescent protein zsgreen . successful socs- cloning was confirmed by sequencing. for transfection, jeg- cells were placed into well/cluster plates at a concentration of . x /well. the tranfection was carried out with . g of socs- /zsgreen plasmid (psocs- /zsgreen ) for hours when cell reached - % confluence. siport lipid were used. jeg- cells transfected with zsgreen plasmid (pzsgreen ) only was used as control. after approximately hours of transfection, cells were treated with il- at and ng/ml. medium was then collected and measured for il- by elisa. il- production was calculated as the percentage of increase by psocs- /zsgreen transfected cells compared to the cells transfected with pzsgreen only. result: il- production was increased by psocs- /zsgreen transfected jeg- cells compared to the cells transfected with pzsgreen only when stimulated by il- , control: . % increase; ng/ml il- : . % increase and ng/ml il- : % increase, p< . , respectively. data are means from three independent experiments. conclusion: over-expression of socs- gene could promote il- production by placental trophoblast cells. reduced sil- r release and increased ratio of sgp /sil- r production by placental tissues from women with preeclampsia. shuang zhao, ruping fan, jingxia sun, yang gu, david f lewis, yuping wang. obstetrics and gynecology, lsuhsc-shreveport, shreveport, la, usa; obstetrics and gynecology, first hospital, harbin medical university, harbin, heilongjiang, china. objective: placental tissue/trophoblasts release more inflammatory cytokines (il- , il- and tnf ) in preeclampsia (pe) than in normal pregnancies. however, the reason for increased inflammatory cytokines released by pe placentas is not clear. soluble il- receptor (sil- r) and membrane receptor il- /gp complex play an important role in the negative regulation of cytokine signaling in suppressor of cytokine signaling (socs) pathway. in contrast, soluble gp (sgp ) is an antagonist for il- /il- r trans-signaling. this study was undertaken to determine sil- r and sgp production by villous tissues from normal and pe placentas. methods: placentas delivered by normal and pe pregnant women were used in this study. placental explants were incubated with dmem for h. the culture medium was collected. placental villous tissue productions of sil- r and sgp were measured by elisa. all samples were assayed in duplicate. data are expressed as mean ± se and analyzed by mann whitney test. a p level < . was considered statistically different. results: placental tissues from pe produced significantly less sil- r than tissues from normal pregnancies, . ± . vs. . ± . pg/mg of wet tissue, p< . . soluble gp production was relatively compatible between pe and normal placental tissues: . ± . vs. . ± . pg/mg of wet tissue. the ratio of sgp /sil- r release was significantly higher in pe than in normal placentas, . ± . vs. . ± . , p< . . conclusion: reduced sil- r production and/or increased ratio of sgp /sil- r production by pe placentas suggest less cytokine inhibitory activity in pe placentas, which may contribute to the increased toxic cytokine production in placentas from pe. using chemiluminescence immunoassays. peter s uzelac, jing dai, frank z stanczyk, daniel r mishell, jr. obstetrics and gynecology, university of louisville, louisville, ky, usa; obstetrics and gynecology, university of southern california, los angeles, ca, usa. objective: characterizing human chorionic gonadotropin (hcg) levels throughout normal pregnancy is critical to its use as a bio-marker for abnormal gestations. there is a paucity of data describing hcg trends during pregnancy and most of the relevant studies use older, less specific assays. our first objective was to characterize hcg levels throughout normal gestation using two different contemporary chemiluminescence immunoassays. our second objective was to compare hcg patterns in healthy gestations with pregnancies affected by diabetes, a common obstetrical complication. methods: a single blood sample was collected from healthy pregnant women and diabetic pregnancies. gestational age was confirmed by ultrasound. serum hcg levels were quantified by chemiluminescence immunoassays using the acs- and immulite systems. data was grouped in -week intervals until weeks of gestation and -week intervals thereafter, with to samples in each interval for healthy women and to samples in each interval for diabetic women. paired t-test and wilcoxon rank sum test were used for statistical analysis. results: using the acs- system, mean hcg levels (miu/ml) for healthy pregnant women were , at - weeks, , at - weeks, , at - weeks, , at - weeks, , at - weeks, and , at - weeks. mean hcg levels obtained from the immulite system were similar. for diabetic pregnancies, mean hcg levels (miu/ml) were , , , , , , , , , and , , respectively. between - weeks of gestation, the hcg levels were significantly lower in diabetic pregnancies (p= . ) compared to healthy controls. ) compared to healthy pregnancies, diabetic gestations have significantly lower peak hcg levels. the cause of this difference is an area deserving further investigation. background: mouse and human placentae share several cellular and molecular features, including a haemochorial interface, allowing the murine labyrinth to be compared with the human fetal placenta. there is an autonomous embryonic ras from at least the time of implantation. ace converts angiotensin i to the active angiotensin ii (angii). angii's actions as a pro-inflammatory agent, in promoting cell migration, angiogenesis and cellular growth and apoptosis, strongly suggest a rôle in placentation. hypothesis: there would be counterregulation of the placental ras if the effect of ace were removed. this study investigated for the first time whether the knockout of somatic ace affected the expression and localisation of various components of the ras in the placentae of wild-type (wt/wt), heterozygous (wt/ ) and ace knockout ( / ) mice. methods: immunohistochemistry (dako envision plus) was used to localise and semiquantify ang type receptor (at r), ang type receptor (at r), ace, and ace- in the three genotypes (n= /group). placental sections were blinded to genotype; a score range of - was used. the test was used (spss version . ) to analyse the difference in staining score by genotype. results: immunoreactivity of all antigens increased in the placental labyrinth of / mice compared to wt/wt and wt/ mice (at r p< . ; at r p<< . ; ace p<< . ; ace- p<< . ). at r and ace- displayed increased staining in the fetal vascular endothelium of / mice (at r p<< . ; ace- p= . ), and in the cells lining the maternal central artery (at r p<< . ; ace- p<< . ). ace- expression was very high in cytotrophoblast lining the maternal blood space in all genotypes. no gross structural differences were seen. comment: the antibody used did not differentiate between ace and the membrane-bound "testicular"-ace (tace). immunohistochemically-identified ace expression was upregulated in / placentae despite the loss of somatic ace, suggesting that t-ace can be expressed placentally. we believe this is the first demonstration of such expression. ace and t-ace are catalyticallysimilar in converting angi to angii. furthermore, angii acting via the at rs is vasodilatory and ace- catalyses production of the vasodilatory ang ( - ); placental blood flow was presumably well-maintained and pregnancy outcome is normal in / mice. it is generally accepted that prostaglandin production plays a crucial role in both term and preterm parturition, and recently the administration of alpha hydroxyprogesterone acetate has been shown useful in preventing preterm labor. what is not clear is the biochemical pathway of prostaglandin production during labor and what, if any, effect alpha hydroxyprogesterone acetate has on this pathway. in order to address this question, we used immunohistochemical staining techniques to evaluate the effect of treating human placental amnion and chorion decidua with alpha hydroxyprogesterone acetate. membranes from unlabored patients were obtained at cesarean section and immediately seperated into control and drug treated specimens. controls were from the same placenta and were subjected to all experimental procedures except for the addition of alpha hydroxyprogesterone acetate to the culture media. specimens were compared at zero, six, and twenty four hour intervals. at the appropriate time, each specimen was formalin fixed and then paraffin blocked. tissue sections were then mounted on slides which were immunohistochemically stained using appropriate primary and secondary antibodies and standard techniques. the slides were then analyzed via light microscopy for changes in staining of three enzymes involved in prostaglandin production--cyclooxygenase (cox- ), cyclooxygenase (cox- ), and -hydroxy prostaglandin dehydrogenase (pgdh). compared to control, the slides treated with alpha hydroxyprogesterone acetate had differing amounts of enzyme expression. cox- was relatively unchanged and pgdh was only slightly increased, but cox- was noticeably decreased in the treated slides. these results were time dependent. this data suggests that alpha hydroxyprogesterone acetate decreases prostaglandin production in fetal membranes primarily by downregulation of the cyclooxygenase enzyme. objectives: in the human placenta, proliferation, differentiation and fusion of cytotrophoblasts (ct) are essential events in the formation of the multinucleated syncytiotrophoblast, however the regulation of these processes is poorly understood. using an explant model of human first trimester placenta we have established that both igf-i and -ii enhance ct proliferation, differentiation and survival mediated via igf r signalling. we have also shown that non-specific inhibition of protein tyrosine phosphatases inhibits igf-mediated signalling in trophoblast; therefore, we have now used sirna-mediated knockdown to investigate the role of the tyrosine phosphatase shp- in this pathway. methods: amaxa nucleofector technology was used to deliver shp- or scrambled sirna ( nm) to bewo cells or first trimester villous tissue fragments. knockdown was confirmed by q-pcr and western blotting. transfected cells and tissue were maintained in culture for hours, then treated with igf-i or igf-ii ( nm) for a further hours before immunohistochemical (ihc) analysis for cell proliferation (ki , brdu) or apoptosis (m ). results: sirna-mediated knockdown of shp- in bewo cells ( % reduction on western blot) demonstrated that igf-induced proliferation was reduced from . ± . % to . ± . % (p< . , n= ). ihc analysis of tissue demonstrated that shp- is localised to ct. following knockdown ( % decrease by q-pcr), igf-i-and igf-ii-induced ct proliferation was decreased by . ± . % and . ± . % respectively (p< . , n= ). furthermore, the ability of igf-i-and igf-ii to prevent ct apoptosis (m staining) was reduced by . ± . % and . ± . % respectively (p< . , n= ) after shp- knockdown. conclusions: igf stimulation of cytotrophoblast proliferation is mediated by shp- . exogenous igf rescues cytotrophoblast from apoptosis, and this pathway is also shp- -dependent. villous endothelial cells. emily j su, zhi-hong lin, ping yin, scott reierstad, joy innes, serdar e bulun. obstetrics and gynecology, northwestern university feinberg school of medicine, chicago, il, usa. background: within the human vascular system, estrogens have been shown to enhance vasodilatation in both normal and abnormal endothelium. estrogenic function occurs by activation of one or both of two estrogen receptors, estrogen receptor-alpha (esr ) and estrogen receptor-beta (esr ). these estrogen receptors are expressed in a wide variety of tissues. within the vasculature, estrogen receptors regulate the expression of multiple vasodilator and vasoconstrictor proteins. specifically, esr has been shown to be critical in maintaining normal vascular physiology in a murine model, where esr knock-out mice demonstrate significant systolic and diastolic hypertension. we hypothesize that within placental endothelium, estrogen plays an important role in maintaining normal vascular function that is critical for normal fetal growth and development. methods: term placentas from uncomplicated pregnancies were obtained, and the decidua was removed. an iv cannula was inserted into the umbilical vein, which was perfused with a collagenase/dispase solution. the perfusate was collected and subjected to further purification. these cells were cultured in complete medium, and after the initial passage of these cells, purity was confirmed via immunofluorescence and flow cytometric studies. estrogen receptor expression was determined in these cells via western blotting. additionally, these endothelial cells underwent treatment with varying doses of estradiol ( - m to - m), and quantitative real-time pcr was performed thereafter for mrna levels of various genes important in prostanoid production. results: western blotting demonstrated that esr is the only estrogen receptor expressed within villous placental endothelial cells. estradiol induced cyclooxygenase- (cox- ) mrna levels -to -fold, as quantified by real-time pcr (p< . ). conversely, there was no effect of estradiol on cyclooxgenase- (cox- ). conclusion: these results suggest that estradiol and esr are important in mediating the balance of prostanoid production that is essential in maintaining placental vascular health. future studies will further delineate estrogenic effects on prostanoid production within placental endothelial cells in health and disease. supported by the smfm/aaogf scholarship award and the nih grant u -hd . previously we established that proteins secreted by the decidua promote the differentiation of extravillous trophoblasts (evt) from a proliferative phenotype (characterized by cx , her- and alpha integrin protein expression) to an invasive phenotype (characterized by her- and alpha protein expression). the ability of decidua-conditioned media (dcm) to induce trophoblast differentiation was inhibited in the presence of the her- receptor antagonist, ag . furthermore, dcm-induced jar cell migration was also attenuated in the presence of ag . thus, the purpose of this study is to define the role of her signaling in evt differentiation and invasion. methods: evt differentiation was assessed in placental villous explant outgrowths and jar cells using antibodies against markers of the proliferative and invasive phenotypes. trophoblast migration was assessed using jar cells in transwell migration assays. results: treatment of placental villous explants with egf, a her- ligand, resulted in the downregulation of her- and an upregulation of her- expression, as well as an induction of alpha integrin expression. pre-treatment of placental villous explants with ag blocked this effect. in the jar cell line, egf treatment mimicked the differentiation-promoting effects of dcm by downregulating her- and upregulating her- expression, effects that were both blocked when jar cells were pre-incubated with ag . in contrast to dcm however, egf stimulation did not induce jar migration. stimulation of jar cells with hb-egf, a her- /her- heterodimer ligand, induced jar migration in a dose-dependent manner. analysis of dcm using antibody arrays confirmed the presence of many members of the egf family including hb-egf. immunohistochemical assessments of placental villous explants verified the expression of her- in evt outgrowths and in jar cells; her- expression was not affected by stimulation with either egf or hb-egf. conclusions: her signaling is an important and necessary component of the invasive evt differentiation cascade. our data supports a role for her- signaling in the induction of the invasive evt phenotype. chandrasekhar thota, chandra yallampalli. obstetrics gynecology, university of texas medical branch, galveston, tx, usa. background: parathyroid hormone related peptide (pthrp) is expressed in trophoblast cells and may play a role in placental growth and function. studies conducted in pregnant rats using pthrp antagonist showed decreases in fetoplacental growth during mid gestation. objective: to assess the effects of pthrp silencing on the expression of growth factors in immortalized first trimester trophoblast cells (htr- /svneo cells). methods: htr- /svneo cells cultured at º c and %co in rpmi- medium supplemented with % fbs were transiently transfected with nm of three different sirna sequences of pthrp, si , auaccuaacucaggaaacuuu; s i , g a g c u g u g u c u g a a c a u c a u u ; a n d s i , caagauuuacggcgacgauuu. for control, a scrambled sirna sequence was used. at % confluency, cells from each well were split and transfected again in triplicates with respective sirna sequences. total rna was isolated using trizol reagent h after transfection, and protein was extracted using lysis buffer h after transfection. the isolated rna and protein were subjected to reverse transcription and polymerase chain reaction (rt-pcr) and western analysis, respectively, using primers and antibodies specific for pthrp, plgf, vegf and lif. the results are expressed relative to either s for changes in mrna expression or -actin for changes in protein expression. results: rt-pcr of total rna obtained from htr cells subjected to double transfection with sirnas for pthrp showed a significant decrease in pthrp expression. expression of growth factors plgf, vegf and lif showed decreases with all the three sirna sequences used compared to the scrambled sequence. western analysis of cell lysates obtained from htr cells subjected to transfection with sirnas for pthrp showed a significant decrease in protein expression of pthrp and vegf. however the protein expression for plgf, and lif decreased in cells transfected with only si sequence of pthrp. conclusions: our studies showed that transient transfection of htr cells with sirna for pthrp caused decreases in both mrna and protein expression of pthrp. our results further suggests that decrease in pthrp peptide in transfected cells resulted in a decrease in vegf, plgf and lif suggesting that pthrp may play role in regulating trophoblast cell functions. objective. maternal obesity poses an increased risk to the fetus during pregnancy, and has long term consequences for the progeny. we tested the hypothesis that maternal caloric excess effects growth-related gene expression changes in the murine placenta. methods. female c bl/ mice were fed a hypercaloric diet ( % fat, % sugar) or standard chow for six weeks prior to mating and throughout pregnancy. near-term (day gestation) the dams ( controls, overfed) were sacrificed. following placental rna extraction, we used the affymetrix mouse a_ . array to measure gene expression changes. we performed pathway analysis on regulated genes. results. maternal overfeeding was associated with a two-fold increase in body fat mass. probe sets, corresponding to genes showed differential expression (p < . ); twenty-seven of which were up-regulated, and ten down-regulated, as compared to the placenta of control fed dams. of note, several genes related to obesity, diabetes, dna methylation, and the tgf beta pathway were differentially expressed. conclusions. diet-induced obesity in mice was associated with altered placental gene expression, including genes involved in tgf beta signaling and dna methylation pathways. these findings may have important implications for placental growth and epigenetic regulation. (supported by usphs hd- , earnest eu framework , tommy's the baby charity, uk). objective. maternal dietary protein restriction has been shown to have deleterious effects on placental development, and has long-term consequences for the progeny. to comprehend more completely stress responses to maternal protein restriction, we measured gene expression changes in the mouse placenta. methods. pregnant fvb/nj mice were fed an isocaloric diet containing % less protein than normal chow ( % vs. % protein content) from embryonic day . (e . ) to e . . following placental rna extraction, we used the affymetrix mouse a_ . array to measure gene expression changes. we performed pathway analysis on the regulated genes, and used both qrt-pcr and immunohistochemistry to verify the results. results. the weights of the e . pups were decreased % (p< . ). probe sets, corresponding to genes, were regulated by protein restriction (p< . ); ninety-one being up-regulated and down-regulated. of particular note, several genes related to the p pathway were up-regulated. along with p itself, positive regulators of p (zmiz , jmy, hipk ) and genes activated by p (inpp d, cebpa) were induced. for selected genes we confirmed these results using qrt-pcr and immunohistochemistry. conclusions. by microarray analysis, we have described the genetic response to maternal protein deprivation in the mouse placenta. we observed that pups were growth restricted, and genes related to the p pathway were regulated. we propose a model through which intrauterine growth restriction is triggered, in part, by activation of the p pathway. (supported by usphs hd- and the sgi medical student grant to cpg). purpose: human placental villous tissue cultures have been underused in the study of placental drug disposition. thus we assessed the utility of this model by studying the effect of time in culture on the viability and integrity of the tissue and the, expression and function of proteins involved in the formation and efflux of -chloro- , -dinitrobenzene (cdnb) conjugate , -dinitrophenyl-s-glutathione (dnp-sg) as a model system for phase ii metabolism and cellular efflux. methods: placental tissue samples were obtained within minutes of cesarean deliveries following normal pregnancies in three patients. villous tissue was cultured in m medium to hr. at , , , , , and hr post culture, villous tissue was preincubated without or with atpase inhibitor sodium orthovanadate, exposed to μm cdnb, rinsed and incubated in buffer at °c to determine formation and efflux of dnp-sg, which was assayed by hplc. changes in expression of gstp - , abc transporter isoforms b , c and g (abcb , abcc , and abcg , resp.) were assessed by immunoblotting. lactate dehydrogenase (ldh) release, methyl tetrazolium thiazolyl blue (mtt) incorporation, and total tissue glutathione content were monitored up to hr. villous tissue morphology was assessed by immunohistochemistry. results: villous tissue structure and protein expression of glutathione-stransferase isoform p - (gstp - ) and abcg remained unchanged over hr in culture. expression of abcb and abcc , and total tissue glutathione decreased with culture time. ldh release was unchanged up to hr and increased at hr, while mtt incorporation remained constant to hr and decreased at and hr suggesting a decline in tissue integrity and viability at hr. however, dnp-sg formation, dnp-sg buffer/tissue ratio, and the extent of inhibition of dnp-sg efflux by sodium orthovanadate remained unchanged through hr. sodium orthovanadate decreased the dnp-sg buffer/tissue ratio by . ± . % (p< . ), consistent with inhibition of apical abc transporters. conclusions: these results support the use of this model to study the coordinated function of metabolizing enzyme gstp - and apical abc transporters in the formation and efflux of the model substrate dnp-sg. the model may be useful to study metabolism and transport of other compounds. syncytiotrophoblast. shauna f williams, ewa fik-rymarkiewicz, stacy zamudio, nicholas p illsley. obstetrics, gynecology and women's health, umd-new jersey medical school, newark, nj, usa. introduction: multiple inputs influence placental protein synthesis. nutritional, endocrine and metabolic factors have been implicated but its regulation has not been investigated. one of the factors shown to be associated with the inhibition of protein synthesis is hypoxia. the goal of this study was to determine the effects of hypoxia on a marker of placental protein synthesis. eukaryotic initiation factor (eif ) is a subunit of eif which is required for initiation of translation however when phosphorylated, eif is unable to participate in the assembly of the initiation complex. hypoxia has been shown previously to cause increased phosphorylation of eif . we hypothesized that hypoxia would increase the levels of phosphorylated eif in term syncytiotrophoblast, thus inhibiting protein synthesis. methods: primary syncytiotrophoblast cultured from term cytotrophoblast were incubated for hr in atmospheres of , , or % o or in the presence of the hypoxia-mimetic, dimethyloxalylglycine (dmog, . - . mm) in % o . cell extracts were analyzed by western blotting to determine the degree of eif phosphorylation. results: incubation in , , or % o did not increase eif phosphorylation relative to the % o control (n= , separate placental preparations). incubation in dmog concentrations up to . mm did not affect eif phosphorylation however incubation in . mm dmog increased eif phosphorylation by ± % (p < . , n= ). conclusions: contrary to our expectations, inhibition of protein synthesis via the eif regulatory pathway was not apparent except when induced by the highest concentration of dmog, consistent with severe hypoxia. thus while eif phosphorylation does occur, we did not observe changes at dissolved oxygen levels of %. these data suggest that a reduction in syncytial protein synthesis via the eif pathway takes place only under severe hypoxic stress. (supported by nih hd ). the increasing prevalence of overweight and obese women of childbearing age is a growing public health concern. the impact of maternal obesity on placental aa transport, which is essential for normal fetal development, remains poorly defined. there are three sub-types of the placental na-dependent system a transporter, snat , and which mediate neutral aa transport. snat is ubiquitously expressed in mammalian tissues and is likely responsible for the majority of placental system a activity. objective: to examine the impact of maternal obesity and over-nutrition on the fetal: maternal (f:m) aa ratio and placental protein abundance for snat . methods: nonpregnant ewes were randomly assigned to a control (c, % of nrc recommendations) or obesogenic (ob, % of nrc) diet from - to days of gestation (dg). under isofluorane anesthesia, maternal and fetal blood samples were collected for aa analysis by hplc from five twin bearing ewes in each dietary group. after euthanasia, placental cotyledonary (cot) tissue was separated from caruncular tissue, frozen in liquid nitrogen and stored at - c for western blot analysis. results: fetuses from ob ewes were % heavier (p< . ) than those from c ewes at dg ( ± vs. ± g). blood concentrations of asn, thr, cit, arg, tau, tyr, trp, val, phe, leu and orn were higher (p< . ), or tended to be higher (met and lys, p< . ) in ob than c ewes. in contrast, f:m ratios, for asn, ser, gln, his, gly, thr, cit, arg, b-ala, tau, ala, tyr, trp, met, val, phe, leu, orn and lys were reduced (p< . ) in ob compared to c ewes. snat content in cot tissue was reduced in ob when compared to c ewes ( . ± . vs. . ± . arbitrary units; p< . ). conclusions: maternal obesity in pregnancy reduced expression of placental snat protein and efficiency of placental aa transport in ewes, providing a mechanism whereby fetuses may mitigate excessive delivery of aa under conditions of maternal obesity and over-nutrition. decreased aa transport to the fetus may play a role in altered cellular structure and function. nih inbre p rr . early gestation utero-placental hemodynamics in an ovine model of fetal growth restriction. lucia dohnal, james s barry, henry l galan, randall b wilkening, russell v anthony. perinatal research center, university of colorado health sciences center, aurora, co. objective: fetal growth restricted (fgr) pregnancies, during late gestation, exhibit altered placental hemodynamics, and reduced capacity for o and nutrient transfer. it was our objective to examine utero-placental hemodynamics and o uptake during early gestation in an ovine model of fgr. methods: singleton-bearing ewes were instrumented with uterine artery flow probes, uterine venous and femoral artery catheters before being placed into a highambient temperature (fgr; n= ) or normothermic (con; n= ) environment at days of gestation (dga). maternal arterial and venous blood, uterine artery flow, heart rate, arterial pressure and respiration rate was collected until dga, at which time umbilical venous blood, fetal weight, placental weight and tissue were harvested. data reported here were analyzed by students t-test. results: maternal respiration rate ( . ± . vs . ± . breaths/min) and arterial po ( . ± . vs . ± . mmhg) were increased (p . ), whereas maternal heart rate ( . ± . vs . ± . beats/min), blood pressure ( . ± . vs . ± . mmhg) and arterial pco ( . ± . vs . ± . mmhg) were reduced (p . ) in fgr pregnancies. at dga, fetal weight was not different (p . ), but placental (total placentome) weight ( . ± . vs . ± . g) was reduced (p . ) in fgr pregnancies. while uterine artery (pregnant horn) flow ( . ± . vs . ± . ml/min) tended (p= . ) to be reduced in fgr pregnancies, relative uterine artery flow ( . ± . vs . ± . ml/min/g fetus; . ± . vs . ± . ml/min/ g placenta) was not different (p . ). uterine o uptake (mmol/min), relative uterine o uptake (ml/min/g fetus or ml/min/ g placenta) and uterine o extraction (%) were not different (p . ) between fgr and con pregnancies. at dga, umbilical vein po (mmhg), o content (mm) and o capacity (mm) were also not different between fgr and con pregnancies. conclusions: reduction in absolute uterine artery flow (ml/min) did not impact utero-placental o uptake or transfer to the umbilical vein, and may have resulted from reductions in maternal cardiac output. relative uterine artery flow was not reduced, suggesting that uterine blood flow and delivery of o to the conceptus does not mediate the ongoing placental growth restriction initiated during early gestation. supported by nih hd . barton c staat, anna maria marconi, cinzia paolini, alex cheung, henry l galan, frederick c battaglia. obstetrics gynecology and pediatrics, univ of colorado at denver health sciences center, aurora, co, usa; dept of obstetrics and gynecology, san paolo institute of biomedical sciences, university of milano, milano, italy. objective: to determine relative contributions of transplacental flux vs fetal production for myo-inositol and mannose in normal term pregnancies using stable isotopic methodolgy. background: myo-inositol and mannose are important in biologic functions. an external supply of mannose may be required for glycoprotein synthesis. low maternal myo-inositol is associated with spina bifida. mannose concentrations are known to be higher in the mother than the fetus. in contrast, myo-inositol concentrations are higher in the fetus than the mother. what remains unknown is whether fetal levels of these polyols are a result of direct maternal transport or from conversion of glucose. design: four term uncomplicated pregnancies undergoing an elective ceasaran section were infused with c labeled isotopes of glucose, myo-inositol and mannose over hours prior to delivery. maternal samples were obtained prior to infusate being administered, and at hour (h), . h and h. fetal concentrations were measured from umbilical artery and vein plasma. the concentrations of labeled and unlabeled glucose, mannose and myo-inositol were measured using high pressure anion exchange chromatogrpahy permitting detection of polyols and sugars at concentrations in the μm range. the feto-maternal molar percent enrichment (mpe) ratio was calculated for each glucose, mannose, and myo-inositol as the ratio between fetal plasma enrichment and the maternal plasma enrichment at steady state. steady state was calculated as the mean of the three maternal samples taken during infusion. results: the feto-maternal mpe ratios of mannose ( . ± . , p= . ) and glucose ( . ± . , p= . ) were not significantly different from . , consistent with transplacental supply. the feto-maternal ratio for myo-inositol ( . ± . , p= . ) indicates little transplacental flux ( % of fetal inositol derived from maternal plasma). conclusion: in normal term pregnancies, fetal mannose and glucose concentrations are dependent upon maternal transplacental supply. in contrast, fetal myo-inositol concentration is not dependent upon transplacental supply, but fetal demands are met by placental conversion, likely from glucose. christian wadsack, manuela augsten, christian guelly, ursula hiden, ingrid lang, manfred moertl, uwe lang, gernot desoye. clinic ob/gyn; center of med res; inst cell biol, histol embryol, med univ graz, austria. background placenta and fetus need lipids for growth and synthesis functions. part of the lipids is supplied from maternal sources by transplacental transfer. recently, we identified in human placenta the high density lipoprotein (hdl) receptor scavenger receptor class b type i (sr-bi). among other functions it mediates hdl-induced ser -phosphorylation of endothelial nitric oxide synthase (enos) resulting in enos activation. this mechanism allows hdl to contribute to regulation of vasotonus in arteries. we hypothesized that term placental endothelial cells (ec) express sr-bi at levels different between arteries and veins. methods sr-bi was localized by ihc and quantified by qrt-pcr in rna isolated from arterial and venous vessels. arterial (eca) and venous (ecv) placental ec were rigorously characterized. sr-bi levels were measured by qrt-pcr and immunoblotting. hdl binding and uptake was measured in eca and ecv with i-labelled hdl. hdl from human donors was used to stimulate ser enos phosphorylation. epigenetic regulation was studied by methylationspecific pcrs for cpg-rich promoter regions of sr-bi. pdzk a key adaptor for sr-bi mediated enos activation was measured by sqrt-pcr. in situ analyses (ihc, qrt-pcr) showed more sr-bi in arteries than in the vein. the differential expression persisted in vitro in isolated eca and ecv even after passages and culture under same conditions suggesting epigenetic mechanisms regulating sr-bi. however, no methylation was found in eca or ecv. sr-bi was functional since hdl cell association was -fold higher in eca than in ecv ( ± . vs ± . ng hdl/mg prot). hdl did not induce ser enos phosporylation in eca or ecv, which was stimulated by ionomycin about -fold in both cell types. pdzk was undetectable in eca and ecv, whereas it was expressed in placental tissue. conclusion more sr-bi is expressed in ec from arteries than from veins in situ and in vitro. this is not the result of different methylation of sr-bi promoter and, hence, unlikely an epigenetic phenomenon. mechanism of differential expression and its functional consequences for vasotonus regulation is yet unknown. the lack of pdzk may account for the failure of hdl to activate enos. (grants , , oenb). cells. juan a arroyo, brad ziebell, mi-hye park, henry l galan. obstetrics and gynecology, university of colorado andgealth sciences center, aurora, co, usa. introduction: mtor is a protein that regulates cell growth in response to nutrients and growth factors. downstream effectors of the mtor pathway include the p and the ebp proteins. activation by phosphorylation of these proteins increases protein synthesis. given that various signaling pathways are regulated by hypoxia in human trophoblast and that mtor is expressed in human trophoblast, our objective was to determine the effects of hypoxia in the activation of mtor, p and ebp in cultured human trophoblast. study designs: trophoblast cell were isolated from term uncomplicated placentas using a trypsin, dnase and disapase solution. cytokeratin immunocytochemistry confirmed trophoblast cells culture purity. trophoblast cells were treated with hypoxia ( % o ) or normoxia ( % o ) for and hours. western blot for p-mtor, mtor, p-p , p , p- ebp , and ebp were done for each time studied. results: trophoblast cells demonstrated: ) positive staining for cytokeratin, ) non significant differences for mtor at either ( . -fold; p= . ) or hours ( . -fold, p= . ), ) no differences in p protein at ( . fold; p= . ) or hours ( . -fold, p= . . ), ) no differences for ebp at either or hour. conclusion: we conclude that the mtor pathway is not regulated under hypoxic conditions in cultured trophoblast, which suggests that hypoxia does not affect protein synthesis in cultured human trophoblast. however, this may not reflect what happens in vivo in iugr. (supported by nih grant r hl - a ). increased expression of phospho-mtor, phospho-p , phospho-akt and phospho-erk in an ovine model of fetal growth restriction. juan a arroyo, brad ziebell, henry l galan. obstetrics and gynecology, university of colorado and health sciences center, aurora, co, usa. objective: both phosphorylated (p) mtor and p are known to be involved in protein synthesis and are regulated by physiological conditions such as fetal growth restriction (fgr). in a hyperthermic (ht) ovine model of fgr we hypothesize that mtor, p , ebp , erk and akt will be phosphorylated (activated) in the placentae of age (dga) animals. study design: ewes were exposed to ht conditions for days to induce iugr and were placed in ambient conditions. at necropsy ( dga), placentomes were separated into the maternal (caruncle) and fetal (cotyledon) components and frozen for western blot analysis with antibodies against (p) mtor, mtor, (p) p , p , (p) ebp , ebp , (p) erk, erk, (p)akt and akt. results: compared to control animals, fgr animals had smaller fetuses ( ± g v. ± g; p= . ) and smaller placentae ( ± g v. ± g; p= . ) at dga. fgr cotyledon showed an increase in p-mtor ( . -fold; p= . ), p-p ( . -fold; p< . ), p-erk ( . -fold; p< . ) and p-akt ( . -fold; p< . ). in contrast, caruncle (maternal) did not show any changes for the mtor pathway. conclusion: in fgr ovine pregnancies, the fetal placental tissues (cotyledons) showed upregulation of the mtor pathway for protein synthesis via phosphorylation of the p but not ebp while this was not seen in the maternal (caruncle) tissues. in addition neither the cotyledon or caruncle tissues at mid-gestation ( dga) showed changes in these endpoints, which is prior to the exponential fetal growth that starts at mid-gestation. (supported by nih grant r hl - a ). hyperuricemia has long been recognized as a common clinical finding in preeclamptic (pe) women. to date, elevated uric acid concentrations in these women have been considered a marker of disease severity. however preeclamptic pregnancies with hyperuricemia, are associated with an increased frequency of preterm birth and fetal growth restriction. over the past decade several pathogenic roles for uric acid have become evident, raising the possibility of a role(s) for uric acid in the altered vascular and placental functions associated with pe. objective: examine the effects of syncytial uric acid uptake on system a amino acid transport across the human placenta using a primary placental villous explant model. methods: placental villous explants from placentae of healthy, term pregnancies were incubated for hours with uric acid ( . mg/dl), corresponding to concentrations of uric acid observed in pe women. these experiments were conducted in the presence or absence of probenecid ( m), a uric acid cellular uptake inhibitor. system a amino acid transport was subsequently assayed using a radiochemical assay in which na+-dependant uptake of radio-labeled system a substrate, [ c] methyl-amino-isobutyric acid, was measured over minutes. data were analyzed using a paired student's t-test and presented as mean ± sem. results: uric acid attenuated system a amino acid placental transport by % (± . %, p< . ). this inhibitory effect of uric acid on system a activity was prevented by probenecid. conclusions: uric acid reduces placental amino acid transport at concentrations observed in pe women. this inhibitory effect of uric acid is dependant upon syncytial uptake of uric acid, being inhibited by the uric acid transporter inhibitor probenecid. these results may be relevant to the increased frequency of fetal growth restriction observed in hyperuricemic pe. additionally the results of this study, indicating a detrimental effect of hyperuricemia on placental function, also suggest a role for uric acid in the pathophysiology of pe. hyperuricemia, a well-documented clinical finding in preeclamptic women, is associated with pre-term birth and intrauterine growth restriction. uric acid is higher in women destined to develop preeclampsia as early as weeks of gestation at a time when cytotrophoblast are invading decidua and myometrium and remodeling uterine spiral arterioles. we propose that elevated concentrations of uric acid may have detrimental effects on placental development in part through inhibition of trophoblast invasion through the decidua. objective: examine the effects of increasing concentrations of uric acid on trophoblast invasion through a reconstituted extracellular matrix. methods: using the in-vitro matrigel invasion assay, the effects of increasing concentrations of dissolved uric acid ( . mg/dl, . mg/dl and . mg/dl) on the ability of immortalized first trimester extravillous trophoblast cells (htr -svneo) to invade through a reconstituted extracellular membrane were assessed. the concentrations of uric acid used were comparable to those measured in healthy pregnant women and preeclamptic women with an increase in uric acid of two or four standard deviations above normal. cells that successfully invaded through the matrigel membrane within hours were fixed with methanol, stained with hematoxylin and counted. data were analyzed using a one-way analysis of variance with fisher's post-hoc analysis. results: uric acid attenuated trophoblast invasion in a dose-dependent fashion (p< . ), with decreases of % (± . %), % (± . %) and % (± . %) respectively compared to untreated controls. conclusions: exogenous uric acid, at physiological and pathological concentrations, is capable of attenuating trophoblast invasion through a reconstituted extracellular membrane in a dose dependent fashion. these results suggest uric acid is a potential contributor to the pathophysiology of altered placental perfusion in preeclamptic pregnancies. previous studies have shown that transforming growth factor (tgf)-is a key inhibitory factors in the invasion of early trophoblast cells, suggesting therefore that overcoming tgf-beta signaling may be necessary for successful implantation. smad ubiquitin regulatory factor (smurf ), a hect type e ubiquitin ligase, is a key regulator of tgf-signaling pathway, targeting tgf-receptors and various smads for proteasome-mediated degradation. in this context, smurf has been shown to play important roles in embryonic development, cell senescence and tumor formation. as a key regulator of tgfbeta signaling, we wished to determine whether smurf has a physiological role during embryo implantation, especially in trophoblast invasion. we have examined the spatio-temporal expression of smurf in human placental villi during pregnancy. we have also investigated the possible function of smurf in trophoblast cell migration and invasion in a model system involving a human extravillous trophoblast cell line, htr /svneo. our results showed that expression of smurf in placental villi was the highest during the first trimester and the expression decreased in the nd trimester. expression of smurf was lowest in placental villi at parturition. overexpression of smurf in htr /svneo cells reduced tgf beta type i receptor levels and attenuated the inhibitory effect of tgf-on cell migration and invasion. conversely, rnai-mediated down-regulation of smurf resulted in significant increase of tgf-type i receptor protein levels. in contrast, the levels of smad , another potential target of smurf , was unchanged. in conclusion, the present study suggests that smurf participates in trophoblast cell migration and invasion by down-regulating the expression of tgf-type i receptor. our previous data demonstrated the extensive expression of mmp- in various kinds of trophoblast cells in human placenta at the early pregnancy. however, the modulation of the enzyme in trophoblasts is largely unclear. in the present study, the effects of the two types of gonadotropin releasing hormone (gnrh) on mmp- expression were examined in an immortalized human cytotrophoblast cell line, b tert- that has been established in this lab. real-time quantitative pcr and western blot analysis revealed that both types of gnrh (gnrh i and gnrh ii) could increase mmp- mrna and protein levels in b tert- cells in time-dependent manners. in particular, regulatory effect of gnrh i on mmp- expression was concentration-independent, whereas that of gnrh ii was dose-dependent. moreover, both gnrh i and gnrh ii could evidently activate jnk kinase, and sp , an inhibitor of a jnk kinase, reversed the up-regulation of mmp- induced by either gnrh i or gnrh ii. on the other hand, it is not likely that erk / pathway participates in the signaling of gnrh i or gnrh ii. collectively, our observations suggest that gnrh i and gnrh ii elicit their modulation effects in human trophoblastic cells through jnk pathway leading to up-regulation of mmp- . during the first trimester of pregnancy, the oxygen tension of the developing trophoblast cells is less than %. however, the majority of studies on primary trophoblast cell development have been performed at % oxygen. primary third-trimester trophoblast cells are believed to be nonproliferative syncytiotrophoblast cells. we have previously demonstrated that low oxygen tension dramatically affects the differentiation pathway of these cells. we now hypothesize that cell culture in low oxygen tension will improve cell growth and restore proliferation. methods: primary trophoblast cells were purified from third-trimester placenta by enzymatic dispersion and cd- negative selection and cultured at %, % or . % oxygen tension for up to days. the number of cells in culture was assessed by cell counting and by measuring genomic dna. live:dead and mtt assays were used to determine viability. proliferation was assayed with brdu and immunohistochemistry for proliferating cell nuclear antigen. to assess cellular activity, radioactivity of protein precipitated from cells cultured in the presence of tritiated leucine was measured. results: there were no obvious morphologic changes in the cells cultured in different oxygen tensions. the amount of cell loss was directly proportional to oxygen tension: at % oxygen % of the cells remain in culture; at . % oxygen tension % of the cells remained. the cells at . % oxygen tension were proliferating and had a five-fold increased metabolic activity. conclusions: it was previously believed that third-trimester trophoblast cells are non-proliferative. we have demonstrated that low oxygen tension increases the survival of primary third-trimester trophoblast cells. this may reflect the change in the differentiation pathway of these cells. however, the cells also begin to proliferate and increase their metabolic activity. trophoblast cells in vivo form a three dimensional structure which promotes critical complex cell-to-cell interactions that cannot be studied with traditional monolayer cell culture. we developed a substrate-free three-dimensional trophoblast culture system capable of studying cellular interactions without a confounding artificial matrix. methods: nonadhesive agarose hydrogels containing cylindrical recesses m in diameter were cast from molds designed using computer-assisted prototyping. tcl trophoblast cells were seeded into the gels ( , cells per) for up to days. viability and cellular stress were assessed and the threedimensional structures of the spheroids were analyzed. results: tcl trophoblast cells formed uniform spheroids within three days of seeding. the spheroids remained intact after being removed from the mold. when placed in traditional cell culture dishes the cells adhered to the plate within one hour and rapidly proliferated into a monolayer. repetitive reseeding allowed easy transition between monolayer and spheroid without affecting cellular morphology. serial sectioning on days , and revealed central vacuolization forming a trophoblast vesicle with an outer rim . m (+/- m) thick. this rim size remained constant for at least days. live:dead assay demonstrated that the outer cells remained viable and staining against proliferating cell nuclear antigen demonstrated that the cells were proliferating. the inner cells undergo apoptosis as demonstrated by caspase- staining. there is an abundance of vegf staining in the cells remaining in the on the inside of the sphere suggesting a gradient of nutrient or oxidative stress. the formation of a vesicle has been confirmed with confocal imaging. em imaging revealed the structure of the rim. conclusions: trophoblast cells cultured in a novel substrate-free three dimensional system form trophoblast vesicles within days of seeding. these vesicles remain viable after long-term culture and can be repeatedly reformed with repetitive seeding. this new cell culture technique allows us to better study placental cell-cell interactions with the potential of forming microtissues. the transcription factor glial cell missing- (gcm ) mediates cell cycle arrest and differentiation of human trophoblast progenitors into villous syncytiotrophoblast and invasive extravillous cytotrophoblast (evt). micro-array analysis of total rna extracted from cultured bewo cells, in which gcm mrna and protein were repressed using sirna, identified tissue inhibitor of metalloproteinase- (timp- ) in the highest ( -fold) upregulated group of genes. confirmatory rtpcr demonstrated a -fold mrna induction. in placental villi, gcm acts as a transcription factor promoting expression of the fusogenic protein syncytin that mediates syncytial fusion into the overlying syncytiotrophoblast. by contrast, syncytial fusion is uncommon in evt. rather these cells invade several millimeters into the distal myometrium where they transform spiral arterioles. to investigate the role of timp and gcm in the trophoblast we assessed its mrna by qrt-pcr and protein by western blot in cellular extracts from both bewo cells grown under standard cultivation conditions (synchronized by prior thymidine exposure) and floating cultured first trimester villous explants cultured in % oxygen with prior exposure to either gcm sirna or anti-sense oligo-nucleotides to gcm . gcm inhibition in the bewo system was associated with a - % increase in timp- protein expression and alteration of cell proliferation and differentiation in both models. we are presently utilizing the explant model of evt invasion (explant tips cultured on matrigel in % oxygen) to test the hypothesis that gcm mediates metallo-proteinase expression and evt invasion via timp- . presently we conclude that gcm -mediated evt differentiation involves more than an arrest of mitosis and may include promotion of invasion via repression of timp- . funding: cihr. scott h purcell, jeremy d cantlon, virginia d winn, russell v anthony. , colorado state university, fort collins, co; university of colorado health sciences center, aurora, co. background: periattachment factor (pf) is a nuclear protein first described in the bovine conceptus. our research in sheep has shown pf mrna concentration peaks when the conceptus is undergoing elongation and initial apposition to the endometrium, and that pf is a nuclear protein localized to the trophoblast. in silico analysis identified a human homolog, hprr . objective: the objective of this experiment was to determine if pf was expressed in the human placenta, and to develop short-hairpin (sh) rnas for hpf to begin investigating its function. materials and methods: immunohistochemistry was performed on paraffin embedded first and second trimester human placental samples. placental sections were immuno-stained using rabbit polyclonal antiovine pf or anti-human cytokeratin- . cytotrophoblasts from first trimester pregnancies (n= ) were subjected to an in vitro invasion assay and rna was harvested following , , and h. quantitative rt-pcr was performed on these samples with intron-spanning primers for hpf, and normalized on hs mrna concentrations. based on the human pf sequence, four putative shrna constructs were generated and cloned into a lentiviral expression vector. bewo human choriocarcinoma cells were treated with one of four shrna contructs or an empty vector for h and then rna was harvested from cells for analysis by quantitative rt-pcr. results: periattachment factor was present in the nuclei of both first and second trimester cytotrophoblasts. hpf mrna concentration increased as invasion occurred from , , to h in all samples; while hypoxia decreased expression at h of invasion compared to h under normoxic conditions. the four lentiviral vectors expressing shrna against hpf resulted in hpf mrna concentrations at , , and % of hpf mrna concentration with the control vector. conclusion: the presence of pf in the human placenta and the increase in pf mrna during cytotrophoblast invasion may indicate this gene plays a role during implantation. we have developed shrnas against pf that result in greater than % mrna knockdown and will be using these to begin to elucidate the function of pf in the human placenta, specifically during the invasion process. recently we demonstrated that infusion of imd antagonist (imd - ) in rat caused distorted labyrinth indicative of a deficient vasculature in placenta. we hypothesize that imd has a role in migration of first trimester trophoblast cell (htr- /svneo) via regulating human leukocyte antigen (hla-g) and stimulating mek / / erk / phosphorylation. objectives: ) to asses the effect of imd on migrating capacity of htr- sv/neo cells using scratch assay in presence or absence of mek and ras/raf inhibitor, u and manumycin a, respectively ; ) to assess the effect of imd peptide on phosphorylation of mek / and erk / in first trimester htr cells ) to analyze the effects of imd on the expression of human leukocyte antigen, hla-g, a critical factor involved in the invasion and vascular remodeling of spiral uterine arteries and subsequent pregnancy in human. methods: htr- sv/neo cells were used to assess the effect of imd ( - m) on the expression of hla-g mrna and phosphorylation of erk / and mek / protein by reverse transcriptase polymeration chain reaction (rt-pcr) and western blot analysis respectively. scratch wound assay was used to determine the migration capacity of htr cells. total rna was isolated from cells using trizol reagent and processed for rt-pcr and results are expressed relative to s mrna. trichloroacetic acid was used for the extraction of total protein for western blot analyses. results: our data demonstrates that, ) imd enhances the migrating capacity of htr cells (compared to the untreated cells) and these effects are inhibited by mek and ras/raf inhibitors, u and manumycin a, respectively; ) imd ( - m ) stimulates phosphorylation of erk / and mek / proteins in htr cells, ) imd increases the expression of hla-g mrna in htr cells. conclusion: imd promotes migration of first trimester htr cells through mek/ erk signaling pathway and modulates the expression of immunoregulatory molecule, hla-g in these cells. chymotrypsin-like protease promotes the placenta tissue release of sflt- . yang gu, shuang zhao, david f lewis, yuping wang. obstetrics and gynecology, lsuhsc-shreveport, shreveport, la, usa. objective: the placenta is a major source of soluble vegf receptor- (sflt- ) in the maternal circulation during pregnancy. increased placental release of sflt- is believed to play an important role in the pathophysiology and pathogenesis in pe. however, the mechanism of increased placental sflt- release in pe is unknown. we recently reported increased chymotrypsin-like protease (clp) activity and expression in placental trophoblasts from pe. in this study, we tested if proteolytic effects of chymotrypsin may play a role in promoting sflt- release by placental trophoblasts. methods: placentas delivered by normal pregnant women (n = ) were used. we tested if chymotrypsin could promote sflt- release by placental tissue, in which villous explants were cultured with dmem containing chymotrypsin at . , . , and . g/ml for hours. the culture medium was then collected for measuring sflt- . we then determined the specificity of chymotrypsin induced sflt- release. villous tissues were cultured with or without chymotrypsin inhibitor (ci) in culture and then the medium was collected and measured for sflt- . soluble flt- was measured by elisa. all samples were assayed in duplicate. data are presented as mean ± se and analyzed by anova. a p level < . is considered as statistically different. results: ) sflt- concentrations in the medium were increased when chymotrypsin was present in culture and the increased sflt- release induced by chymotrypsin was in a concentration-dependent manner: control: . ± . ; . g/ml: . ± . ; . g/ml: . ± . ; and . g/ml: . ± . (p< . ) pg/mg tissue/hour. ) ci could attenuate sflt- release. this inhibitory effect was also revealed in a concentration-dependent manner: control: . ± . ; ci . g/ml: . ± . ; and ci . g/ml: . ± . (p< . ) pg/mg tissue/hour. conclusions: increased placental sflt- release stimulated by chymotrypsin and decreased placental sflt- release inhibited by chymotrypsin inhibitor suggest that the proteolytic effect of clp may play a role in sflt- generation. therefore, increased clp activity in placental trophoblasts may contribute to the increased placental sflt- production in pe. (supported nih grants hl and hd ). the change of autophagy-related proteins, lc and beclin- , by tnfa stimulation in cultured primary trophoblasts. soo-young oh, kyung hee kim, suk-joo choi, jong-hwa kim, cheong-rae roh. department of obstetrics and gynecology, samsung medical center, sungkyunkwan university school of medicine, seoul, korea. objective: our previous work have demonstrated that the expression of lc , but not beclin- , was increased in placentas from pregnancies complicated by severe preeclampsia (sgi abstract # ) . to understand the regulatory mechanism of these autophagy-related proteins in trophoblast cells, we investigated the changes in these proteins in response to cytokine or hypoxic stimulation in cultured primary trophoblast. material and methods: primary human cytotrophoblasts obtained from normal term placenta were cultured with stimulation of tnf-a or cocl for a given time and the changes of beclin- and lc were assessed using immunoblot analysis. paired t test was used for statistic analysis. results: tnf-a stimulation induced a significant increase of the expression of lc -ii in cultured primary trophoblasts while decreasing the expression of beclin- (p< . for each). however, cocl stimulation did not induce a significant change of both lc -ii and beclin- . conclusions: our data suggests that tnf-a stimulation in cultured primary trophoblasts is associated with increased autophagic activity. background: thyroid hormones play vital roles in the development of the fetal brain. mutations in mct , recently recognised as a specific thyroid hormone transporter, define a novel syndrome of severe x-linked psychomotor retardation accompanied by elevated serum t . we previously reported that mct expression in n-tera- (nt ) cells (a human embryonal cell line with characteristics of cns precursors), as well as mct -null jeg- choriocarcinoma cells, resulted in markedly reduced cell proliferation. further, the s x mct mutation, as reported in males affected by severe psychomotor impairment, resulted in a similar repression of proliferation to wild type, whereas the l p mutant failed to influence cell turnover compared with control. methods: we now examine the effect of "knocking down" mct via sirna and evaluate the effects of cell proliferation (mtt and [ h]-thymidine assays) and tri-iodothyronine (t ) uptake. results: repression of endogenous mct expression in nt cells by % caused a significant increase in proliferation compared to matched-dose nonspecific sirna treatment, independent of t concentration ( . %, . % and . % induction at , and nm t , n= , p< . ). we also sought to examine the role of mct in t uptake. in jeg- cells, wild type mct induced a . -fold increase in the uptake of i-labelled t . by contrast, mutants s x and l p failed to significantly augment t uptake, though r h caused a mild but significant . fold induction in uptake, hence retaining approximately % of wt activity. in parallel experiments, co-transfection of mu-crystallin, a t binding protein, resulted in a similar increase in t uptake compared with control ( . -fold; n= ; p< . ), implying that mct plays only a minor role in thyroid hormone efflux in jeg- cells. mutants s x, l p and r h showed analogous responses to those in the absence of mu-crystallin. conclusion: these results further extend the evidence of a potential role for mct in the modulation of cell proliferation, independent of t transport. to determine predictors of failure for labor induction in women with preeclampsia. we conducted a retrospective cohort study to examine cesarean delivery rates in all the preeclamptic women at a single institution undergoing labor induction between - with a singleton pregnancy >= weeks gestational age (ga). bivariate analyses informed the creation of multivariable logistic models to predict the risk of cesarean delivery using multiple predictors (maternal age, race/ethnicity, unfavorable cervix, gestational diabetes, diabetes, and gestational age). analyses were stratified by parity. our study population included , preeclamptic women undergoing labor induction. in the bivariate analyses, the risk of cesarean delivery ranged from as low as . % (p= . ) among multiparous women - weeks ga to as high as . % (p< . ) among nulliparous women with diabetes. a total of , women had adequate data to be included in the multivariable analyses. odds ratios of the predictors are presented in the objective: preeclampsia and cardiovascular disease share many risk factors, and women with preeclampsia are at increased risk of cardiovascular mortality later in life. we investigated whether risk factors associated with cardiovascular disease and preeclampsia remain elevated months postpartum. methods: we measured plasma sflt , endoglin, plgf, cellular fibronectin (cfn), uric acid, homocysteine, and asymmetric dimethylarginine (adma) in women with uncomplicated normotensive pregnancies compared to women with preeclampsia in samples collected at pre-delivery and again several months postpartum (average . ± . months). data are mean±sd or median (interquartile range). statistical analysis was by wilcoxin rank-sum or students unpaired t-tests with statistical significance accepted at p< . . results: the mean concentration of sflt , endoglin, plgf, homocysteine, adma, cfn, and uric acid were all significantly different in samples collected pre-delivery in subjects with preeclampsia compared to controls (table). adma, cfn and uric acid remained significantly higher postpartum in subjects with previous preeclampsia compared to postpartum controls (table) . conclusions: biological markers associated with altered vascular function or cardiovascular risk are elevated in women with preeclampsia, and some remain significantly higher in postpartum preeclamptic women. these data suggest that vascular dysfunction persists in women with previous preeclampsia, and may contribute to the increased risk of future cardiovascular disease. funded in part by national institutes of health nih- mo -rr and nih- po -hd . (ajp, ) . as leptin is a known potent angiogenic factor we hypothesized that leptin deficiency and/or resistance to leptin-induced vegf expression might be a mechanism for reduced angiogenesis in mfr offspring. methods: pregnant sprague-dawley rats had % mfr from day of gestation until delivery. mfr and control offspring were sacrificed on day of life (p ). some tissues were used to determine the expression of leptin by western blot analysis. for culture experiments, thoracic aortas were dissected, cut into - mm explants and incubated with leptin ( - ng/ml) in dmem ( % fbs). after hours of culture, rna was extracted from the tissues and subjected to real time rt-pcr using specific rat primers for vegf, vegfr and r , and ob-ra, stat and socs ( s mrna as control). culture media was analyzed for vegf protein by elisa. results: expression of leptin mrna and protein in p mfr aortas was significantly reduced. in culture, leptin significantly increased expression of vegf, vegfr and vegfr mrna in explants of aortas obtained from the control but not mfr tissues. as expected, control but not mfr aortic explants secreted significantly more vegf in vitro. to determine the mechanism for resistance to leptin-induced vegf in mfr offspring, we assessed expression of leptin receptor (ob-ra) in explants treated with leptin. leptin was found to induce the expression of ob-ra in aortas from both dietary groups. this upregulation of leptin receptor was accompanied by significant upregulation of stat and socs mrna in the control tissues. in contrast, in mfr explants only the ng/ml concentration of leptin induced an increase in stat mrna, and the magnitude of socs mrna increase by both concentrations of leptin was significantly less in the mfr explants. conclusion: these results indicate that reduced angiogenesis in mfr vessels is in part due to reduced leptin expression and ability of leptin to stimulate vegf expression. although in vitro leptin induced the expression of its receptor in both groups, it was only in the mfr group in which leptin up-regulated vegf and its receptors. our results suggest that this defect in leptin receptor function in mfr vessels is due, in part, to defects in jak/stat signaling. objective: women with a history of early-onset preeclampsia are at increased risk of developing major cardiovascular disease (cvd) related events, that have a detrimental effect on their long-term health and life expectancy. in this follow-up study, we measured established risk factors predictive of first cvd events after early-onset preeclampsia. study design: over a -year interval, primiparous women with a history of early-onset preeclampsia (delivery < weeks gestation) were included and tested for major cardiovascular risk factors at least six months after delivery, in addition to a population-based control group of healthy non-pregnant women. women with chronic hypertension were excluded. results: mean age was . years for cases compared to . years for controls (p<. ). after adjustment for age, we observed significantly increased mean values for weight (p=. ), body-mass index (p<. ), systolic blood pressure (p<. ), diastolic blood pressure (p<. ), total cholesterol (p=. ), ldl cholesterol (p<. ), triglycerides (p=. ), fasting blood glucose (p<. ), and lower hdl cholesterol (p<. ) in women with previous early-onset preeclampsia. no difference was found for height, smoking, diabetes, and ethnicity. estimated -year risk of first cvd events by framingham risk scores remained < % for all women (low-risk). nonetheless, at mean (sd) . ( . ) years after early-onset preeclampsia, % of women met the criteria for metabolic syndrome, % of women exhibited >= , % of women >= and % of women >= major cvd risk factors. conclusion: the majority of women with a history of early-onset preeclampsia exhibit at least one modifiable risk factor for future cvd. although most of these women are classified as low-risk according to the current aha guidelines, this is mainly due to their young age masking other, mostly modifiable, major risk factors. our data thus support life-style intervention programs aimed at primary prevention of cvd in women with a history of early-onset preeclampsia. it has recently been shown that antihypertensive drugs can stimulate cytokine release in normal and hypertensive pregnancy. there is evidence that these cytokines alter the secretion of inhibin a. inhibin a and activin a levels are increased in pre-eclampsia (pe), but it is not known if antihypertensive therapy can affect their secretion. patients and methods we recruited women with hypertensive disorders in pregnancy ( pe and non-proteinuric hypertension [ht]) and matched normotensive controls. inhibin a and activin a levels, before and - hours after initiating antihypertensives, were measured in serum and urine, using an elisa. the same markers were measured using validated assays in placentas delivered at cesarean section at similar gestational age ( pe, ht and controls). analysis the three study groups were compared using anova multiple comparisons with bonferroni post hoc testing. the data were normally distributed after logarithmic transformation. marker levels before and after antihypertensive therapy were compared using paired t-test. we compared placental concentrations between the group which received antihypertensive therapy and the group which did not, using an independent t-test. data were analysed using spss®. in pe, both serum and urine levels of inibin a and activin a were increased at all gestations (p< . ). activin a (but not inhibin a) level was also increased at all gestations in ht (p< . ). after weeks' gestation (but not before), antihypertensive treatment was associated with a significant fall in both inhibin a and activin a serum levels, and urinary inhibin a, in both pe and ht. the placental concentration of inhibin a, but not activin a, was significantly higher in women with pe compared with controls (p< . ). there was no significant difference in either marker between controls and women with ht. the fall in serum levels of inhibin a and activin a following antihypertensive treatment after weeks' gestation may indicate that these drugs have an effect on the pathophysiology of pe other than their known antihypertensive action. pre-eclampsia (pe) is a placental disease of unknown etiology. anti-angiogenic factors, such as soluble fms-like tyrosine kinase (sflt- ) and soluble endoglin (seng), and pro-angiogenic factors, such as vascular endothelial growth factor (vegf) and placental growth factor (plgf), are believed to play an important role in its pathophysiology. maternal plasma concentrations of these markers are altered in pe, even weeks before the clinical manifestations. the aim of this study was to compare the concentration of these markers in placental extracts of normotensive pregnant women, and women with pe and non-proteinuric hypertension (ht). patients and methods placental samples were collected at cesarean section from women with pe (n = ), ht (n = ) and normotensive pregnancies of similar gestational age (n = ). these samples were stored at - ºc. the frozen tissue samples were homogenised and these four markers measured by specific, validated enzymelinked immunosorbent assays. analysis the three study groups were compared using anova multiple comparisons with bonferroni post hoc testing. the data were normally distributed after logarithmic transformation. data were analysed using spss®. the concentrations of both sflt- and seng were significantly higher in the placentas of women with pe, but not ht, compared with controls (p= . ). there was no significant difference in plgf concentration between controls and women with pe or ht. placental vegf concentrations in both pe and ht were higher than in controls (p< . ); there was no significant difference between the levels in pe and ht (p= . ). the fact that placental concentrations of sflt- and seng mirrored the known rise in serum levels in pe suggests that the placenta is the main source of these circulating factors. although sflt- was significantly raised in pe, plgf was not reduced. this suggests that the lower levels of free plgf found in the serum of women with pe are not the result of impaired placental production or secretion, but are due to increased binding by (the increased levels of) sflt- in the serum. objective. the purpose of this study was to evaluate whether systematic screening with uterine artery doppler (utad) and serum biochemical markers of oxidative stress, endothelial dysfunction and vasculogenesis performed during the first trimester predict efficiently pre-eclampsia (pet), specifically early-onset pet, in an unselected chilean population. methods. this nested case-control study involved asymptomatic pregnancies scanned at + - + week of gestation. the subjects for biochemical testing were women who were delivered due to pet (n= ) and normotensive controls (n= ) that were enrolled during the first trimester scan. mean pulsatility index (pi) of the utad was calculated. blood samples were obtained and stored at - o c until biochemical analysis of oxidative stress, endothelial dysfunction and vasculogenesis were performed. normally distributed data were analysed by the unpaired t test, and non-normally distributed data by the mann-whitney rank sum test. chi-square tests were used for the comparison of categorical variables. a probability level of p< . was considered significant. multiple logistic regressions were used to develop a combined predictive index. results. there was % and % significantly increased of the mean pi utad in women who later developed pet or early-onset pet compared to control pregnancies during the first trimester scan. although oxidative stress and endothelial dysfunction biochemical markers were not different between all pet pregnancies and control groups, plasma levels of sflt ( . ± . vs. . ± . pg/ml, p< . ) and placenta growth factor ( . ± . vs. . ± . pg/ml, p< . ) were significantly higher in women who subsequently developed early-onset pet compared to controls. multivariate logistic regression showed that a combination between abnormal utad and both biochemical markers of abnormal vasculogenesis were the best predictor test for early-onset pet, being its detection rate % with % false positive rate. conclusion. this study has shown early and selective changes in markers of impaired placentation and angiogenic state in women who later developed earlyonset pet, without alteration in oxidative stress and endothelial dysfunction. supported by fondecyt . currently it is unknown whether maternal inflammatory changes are specific to pregnancy or reflect an innate susceptibility to inflammation. c-reactive protein (crp) and interleukin- (il- ) are markers of the acute-phase inflammatory response and predictive of future cardiovascular events. we compared crp and il- levels after influenza vaccination, as an in vivo model for lowgrade inflammation, in non-pregnant women with a history of early-onset preeclampsia and controls with only uneventful pregnancies. methods: forty-four women with a history of early-onset preeclampsia (delivery < weeks' gestation) and twenty-nine controls with at least one uneventful pregnancy received an influenza vaccination. we then compared plasma levels of crp and il- at baseline, . days and . days after vaccination. results: median baseline crp and il- levels of women with a history of early-onset preeclampsia were comparable to controls ( . versus . mg/l; p= . and . versus . pg/l; p= . , respectively). however, high crp and il- responses to vaccination were more common in cases (ors for response > th, > th, > th, > th and > th percentile based on the distribution of control values of . , . , . , . and for crp [p for trend . ] and of . , . , . , . and . for il- [p for trend . ], respectively). the relationship between high il- responses and early-onset preeclampsia persisted after adjustment for body-mass index (p for trend . ). conclusion: women with a history of early-onset preeclampsia more frequently exhibit an innate pro-inflammatory phenotype not specific to pregnancy. background altered maternal inflammatory responses play a role in the development of preeclampsia and the hemolysis, elevated liver enzymes and low platelets (hellp) syndrome. we examined whether allelic variants of the innate immune receptors toll-like receptor (tlr ) and nucleotide-binding oligomerization domain (nod ), that impair the inflammatory response to endotoxin, are related to preeclampsia and hellp syndrome. we determined five common mutations in tlr (d g and t i) and nod (r w, g r and l fs) in primiparous women with a history of early-onset preeclampsia, of whom women developed hellp syndrome and in women with a history of only uneventful pregnancies as controls. in addition, we assessed plasma levels of pro-inflammatory biomarkers c-rp, il- , sicam- , fibrinogen and von willebrand factor in a subset of women included at least six months after delivery. after adjustment for maternal age and chronic hypertension, attenuating allelic variants of tlr were more common in women with a history of early-onset preeclampsia than in controls (or . [ % ci . - . ]). highest frequencies for tlr variants were observed in women who developed hellp syndrome (adjusted or . [ % ci . - . ]). in addition, high levels of il- and fibrinogen were associated with a history of early-onset preeclampsia. combined positivity for any of the tlr and nod allelic variants and high levels of il- was . -fold more common in women with a history of early-onset preeclampsia ( % ci . - . ) compared to controls. we observed an association of common tlr and nod gene variants, and pro-inflammatory phenotype with a history of early-onset preeclampsia and hellp syndrome, that suggests involvement of the maternal innate immune system in severe hypertensive disorders of pregnancy. thus, we sought changes in pbef in the serum of patients with mild and severe pe, compared with matched controls. immunodistribution of pbef in fetal membranes and placentas from similar patients was also studied. methods: serum samples ( ) were collected with clinical data including; gestational age, medications, ethnicity and recognized complications. patients in labor or infection were excluded. the standard bp and proteinuria criteria was utilized to classify cases for pe grouping; no pe (n= ), mild pe (n= bp; / - / , proteinuria; trace to + or - mg/ hr urine) and severe pe (n= bp; > / , proteinuria; > + or > g/ hr, or other associated symptoms). pbef concentration was determined by eia (phoenix pharmaceuticals) in accordance with the manufacturers instructions. fetal membranes and placentas of additional patients, no pe (n= ) and with pe (n= ) were fixed and embedded in paraffin. sections ( um) were immunostained with pbef antibody / (pheonix) and treated with abc reagent (vector labs) followed by dab ( . % mg/ml), washed, counterstained, mounted and viewed under brightfield microscopy. results: the concentrations of pbef in serum were between - ng/ml and were significantly higher in those patients with mild pe (p= . ) and further significantly elevated in those with severe pe (p= . ) compared with the matched controls. pbef was detected by immunocytochemistry in the placental syncytiotrophoblast and in the amnion and choriodecidua of the fetal membranes. conclusions: pbef was elevated in the serum of patients according to the degree of pe severity and may be derived from the placenta and/or fetal membranes. (supported by nih #u rr - to the pacific research center for early human development, university of hawaii). background: platelet-monocyte aggregation (pma) is a novel sensitive measure of platelet activation and indicates a proinflammatory state (cytokine release). less sensitive techniques demonstrate platelet activation during pregnancy and pre-eclampsia (pe) but platelet activation has not been assessed by pma.objective: longitudinal study of pma in normal pregnancy and pe. methods: healthy, non-smoking primigravida with an uncomplicated pregnancy and primigravida women with pe were studied. pe was defined by standard definitions. informed consent was obtained and the study had ethical approval. serial venous blood collected at , , , wks in controls, at time of diagnosis in pe cases and wks post-natal (pn) in all. pma, platelet surface p-selectin (psp-sel) and monocyte cd expression (mcd ) were analysed by flow-cytometry and plasma (p) p-sel by elisa. results: groups were matched for mean age and bmi. in controls, pmas, psp-sel and mcd expression and pp-sel increased with gestation and decreased post-natally (table ) . for pe analysis, data was divided into pre-term (sampled at mean wks), and term (mean wks). pp-sel was lower in pre-term pe than control (normal pregnancy wks; p= . ) there was no significant difference in other measures between pe and control ( objective: much effort has been put into the evaluation of novel markers to identify pregnant women at risk for the development of pre-eclampsia. soluble endoglin (seng) and soluble fms-like tyrosine kinase (sflt ), two antiangiogenic agents, appear to be involved in the pathogenesis of pre-eclampsia. despite several studies describing higher midtrimester serum concentrations of these markers in women with subsequent pre-eclampsia, information on first trimester serum levels is scarce. the aim of this study was to assess seng and sflt as first trimester serum markers for the prediction of pre-eclampsia. methods: sera were obtained between + and + weeks of gestation from women who later developed late-onset pre-eclampsia and from controls matched for gestational age, maternal age, maternal pre-pregnancy weight, and storage. using commercially available microplate enzyme immunoanalytical methods, seng and sflt were determined and the results analyzed using non-parametric statistical tests. results: the serum concentration of seng was found to be increased in women with subsequent pre-eclampsia when compared to controls (mean ± sd, . ± . ng/ml versus . ± . ng/ml, p = . , unpaired mann-whitney test). similarly, the serum levels of sflt were higher in women later developing late-onset pre-eclampsia ( ± ng/ml) when compared to controls ( ± ng/ml, p = . ). sensitivities and specificities for predicting pre-eclampsia were % and % for seng and % and % for sflt- , respectively. the combination of the two markers by multiplication yielded a sensitivity of % and a specificity of %. conclusion: seng and sflt , showing increased first trimester serum levels in women with subsequent pre-eclampsia, might both fulfill the characteristics of first trimester markers to predict pre-eclampsia. the combination of the two, however, did not improve the sensitivity nor the specificity compared to their individual determinations. moderate sensitivities and specifities, however, limit the clinical use of these molecules as single markers. pregnancy is associated with increased monocyte/platelet aggregate formation in whole blood. beth a bouchard, adrienne schonberg, gary j badger, ira m bernstein. biochemistry; obstetrics and gynecology; medical biostatistics, univ of vt, burlington, vt, usa. background preeclampsia is associated with increased rates of platelet clearance, changes in platelet function and platelet activation. the goal of the current study was to examine basal levels of platelet activation through pregnancy beginning prior to conception, and to examine the association of platelet activation with the development of hypertensive complications during pregnancy. methods two indices of platelet activation, platelet cd expression (%cd ) and monocyte/platelet aggregate (%mp) formation, were measured in whole blood by flow cytometry using specific, fluorescentlylabeled monoclonal antibodies in healthy, nonsmoking women during the follicular phase of their menstrual cycle (pp, cycle day . + . ). all women subsequently conceived singleton pregnancies and were re-examined in early (ep, - wks) and late pregnancy (lp, - wks). five of these women were diagnosed with hypertensive complications ( hypertension, preeclampsia) at term although hypertension was not observed at any study time point. data are expressed as mean±sem. p< . was accepted for significance. results subjects were . + . years old with a bmi of . + . kg/m at the time of prepregnancy studies. a significant increase in the %mp formed over time of pregnancy was observed (p= . ). there was little change in the %mp formed between pp and ep (pp, . ± . % and ep, . ± . %, p= . ). however, the %mp increased significantly in lp ( . ± . %) as compared to pp (p= . ) and ep (p= . ). this increase occurred independent of the development of hypertensive complications (p= . ) and independent of pp platelet activation status. although statistically significant increases in cd expression were not observed, the change in cd expression over pregnancy correlated with the change in %mp over pregnancy (r= . , p= . ) and cd expression correlated with %mp in lp (r= . , p= . ). conclusion these combined observations suggest that pregnancy is associated with increases in levels of unstimulated platelet activation and that these increases occur in the presence or absence of subsequent hypertensive complications. furthermore, we observed a correlation between the changes in two distinct platelet activation events, %mp and cd expression, during pregnancy. supported by nih hl . backgound sildenafil citrate (sc) has been proposed as a therapy to improve uterine perfusion in pregnancies complicated by iugr or preeclampsia. we sought to determine the effects of sc on uterine vascular resistance, uterine blood flow and cardiac output in young healthy nulliparous women. methods eleven young healthy nulligravid women were studied during the luteal phase (cycle day + ) of the menstrual cycle. women were randomized in a double-blind fashion to receive placebo (pl), or sc at a dose of or mg. uterine artery vascular resistance, uterine artery volumetric flow, brachial artery volumetric flow and cardiac output were measured at baseline and at and hours post dosing employing color doppler ultrasound. comparisons were made by anova between those randomized to pl (n= ) versus sc (n= ). p< . was accepted for significance. data are expressed as mean + s.e.m. results there were no significant differences in subject age, cycle day, body mass index, uterine blood flow, brachial blood flow or cardiac output at baseline comparing the two groups. there was a tendency towards increased uterine blood flow in subjects randomized to receive sc ( % increase) compared to pl (no change), changes in uterine blood flow, brachial blood flow and cardiac output are outlined in the objective reduced maternal plasma volume in the third trimester has been associated with both fetal growth restriction and preeclampisa. we sought to determine the degree to which third trimester plasma volume is dependent on plasma volume prior to pregnancy. methods sixteen young ( . + . years) healthy nulligravid women had their plasma volume measured during the follicular phase (cycle day . + . ) of the menstrual cycle and subsequently conceived. subjects were predominantly caucasian ( / ) with a mean prepregnancy bmi of . + . kg/m . plasma volume was re-estimated at - weeks gestation. all patients were placed on sodium and total calorie balanced diets for days prior to each plasma volume determination. plasma volume was determined employing evans blue dilution with multiple post injection sampling time points. data are expressed as mean + s.d. results baseline prepregnant plasma volume was , + ml or + ml/unit bmi. third trimester plasma volume was , + ml representing a % increase (p< . ). the range of plasma volume expansion was - % dependent upon prepregnant plasma volume. plasma volume in the third trimester of pregnancy was strongly correlated to prepregnant plasma volume r= . (p= . ). plasma volume expansion was consistent across the range of prepregnancy plasma volume. conclusions pre-pregnancy plasma volume contributes approximately % of the variance in third trimester plasma volume. the observed increase is plasma volume is independent of prepregnancy volume resulting in a greater percentage increase for those starting at the lower end of the plasma volume range. as third trimester plasma volume is strongly associated with pregnancy outcome the correlation of prepregnancy plasma volume to third trimester plasma volume suggests that prepregnancy status contributes to these adverse reproductive events.this work was supported by nih hl . background: hydatidiform mole is a rare disorder of pregnancy and may predispose the mother to severe morbidity. molar pregnancies are known to be associated with high risk for the development of early onset preeclampsia. in recent years, the expression of sflt- (soluble vegfr- ) was found to be increased in preeclampsia, and contributes to the pathogenesis of the maternal systemic disease. the objective of the present study was to examine the expression of sflt- in placentae from molar pregnancies. methods: placental samples from unique cases of twin pregnancies with complete molar pregnancy in one sac and developing fetus in the other sac were prospectively collected (n= ). the first set delivered at weeks due to excessive bleeding. the second set delivered at weeks due to severe iugr and elevated blood pressure. mrna level of sflt- was measured by quantitative real-time pcr using specific taqman primers and probe. protein expression of sflt- in placental tissue lysates were measured by western blot analysis using a polyclonal antibody against flt- . immunohistochemistry of paraffin embedded samples was performed using specific antibody for sftl- . results: mrna level of sflt- was increased by . fold in the molar placentae compared to matched controls. the placentae of the developing fetuses which were growth restricted exhibited . fold increase compared to controls. sflt- protein expression in the molar placentae was increased by . fold compared to controls, while the co-twin placentae exhibited a . fold increase compared to controls. immunohistochemistry revealed strong positive immunoreactivity for sflt- in the trophoblast layer of both molar pregnancies and iugr co-twin relative to controls. conclusion: our data suggest that sflt- expression is increased in placentae from molar pregnancies and thus may explain the increased risk for developing early onset preeclampsia. the expression of sflt- in the growth restricted twin placenta is also increased compared to controls and support our previous observation (supported by cihr and owh/igh). (n= ); ) neonates of patients with pe (n= ); and ) sga neonates (n= ). cord blood was collected immediately after delivery and pz plasma concentrations were measured by elisa. pz deficiency was defined as a cord plasma concentration < th percentile of the normal pregnancy group. non-parametric statistics were used for analysis. results: ) cord plasma pz concentration differed significantly among the study groups (kruskal wallis, p< . ); ) neonates of patients with pe and sga neonates had a significantly lower median cord plasma pz concentration than those delivered after normal pregnancy (pe: median . g/ml, range . - . , p< . ; sga: median . g/ml, range . - . , p= . ; normal pregnancy: median . g/ml, range . - . ); ) there were no differences in the rate of pz deficiency among the groups; and ) there was no relationship between placental histologic findings and median cord plasma pz concentrations between and among the sga and pe groups. conclusions: ) at the time of delivery, the median cord plasma pz concentration was lower in sga neonates and those born to women with pe than in neonates born to normal pregnancies; ) there was no difference in the rate of pz deficiency among the study groups, suggesting that the lower median pz cord blood concentrations in pe and sga groups may result from activation of the coagulation cascade rather than an inherited pz deficiency. objective: periconceptional multivitamin (mv) use may be related preeclampsia risk. we examined the relation between timing and frequency of periconceptional multivitamin use and the risk of preeclampsia. methods: women in the danish national birth cohort who delivered singleton liveborn infants (n= , ) reported upon enrollment at . weeks (sd . ) the number of weeks of regular multivitamin use during a week periconceptional period (lmp- to lmp+ ). preeclampsia cases were identified using icd- codes (n= , . %). logistic regression was used to estimate the effect of frequency (number of weeks of use) and timing of use to lmp+ ] and post-conception [lmp+ to lmp+ ]). results were stratified a priori by overweight status. results: overall, , women ( %) reported mv use in the periconceptional period. after adjustment for bmi, smoking, parity and chronic hypertension, infrequent mv use (< weeks of use) had no relation to risk (or . ; % ci . , . ) but regular use (>= weeks) was associated with modestly reduced risk ( . ( . , . ). similarly, when mv use was modeled as a continuous variable, each additional week of use was related to reduced risk for preeclampsia (or . , % ci . - . ). this potential dose effect of periconceptional mv use appeared to be limited to normal weight women (bmi < kg/m², or . ; % ci . - . ), with no apparent effect among overweight women (bmi kg/m², or . ; % ci . - . ). a total of , women reported regular mv use in both the preconception and post-conception periods, and , women reported regular use only in the post-conception period. among normal weight women, regular use in the preconception period had no effect on preeclampsia risk (or . , % ci . - . ). in contrast, use in the post conception period was associated with reduced risk for preeclampsia (or . , % ci . - . ). conclusions: regular periconceptional mv use was associated with a modestly reduced risk for preeclampsia among normal weight women. if causal, mv use immediately after conception appeared to be the critical exposure window. background: pre-eclampsia (pe), a disorder of pregnancy characterized by maternal inflammation, results in immune, cardiovascular and metabolic dysfunction. in non-pregnant persons, inflammatory disorders are treated with and prevented by pharmaceuticals and lifestyle methods such as physical activity (pa). while most pharmaceuticals are contraindicated for pregnant women, pa during pregnancy has been found safe, healthy and beneficial for both mother and baby. clinical evidence has found pa can beneficially affect pregnancy outcome, decrease excessive inflammation and decrease the risk of pe. epidemiological studies indicate that pa may be useful in preventing pe. unfortunately, previous studies have quantified pa based on recall of postpartum women and have not controlled for differences in women's interpretations of amount, type or intensity of pa. however, investigating pa utilizing a laboratory-based exercise intervention to control these variables inflicts difficulties translating the intervention into a community-based program that attracts and retains pregnant women in order to enhance public health. method: a retrospective study was performed to determine the rates of pe among the , women who gave birth at yale-new haven hospital (ynhh) during and , and a person subset of this group who performed prenatal pa in a community-based program that is evidence-based and standardized, thereby controlling for type and intensity of pa. additionally, the program is established in the community, has been offered to the public for years and is internationally known. results: during during - , the pe rate for the general population at ynhh was . %. for the pa group, the rate was . %. two women in the pa group were diagnosed with pe in the last month of pregnancy and delivered normal infants at term. no pe was observed in this group (pa group) during the second or early third trimesters nor was there any prematurity in this group. significance: these findings support the hypothesis that adequate physical activity provided in a standardized community-based group setting may provide a non-pharmacological approach for preventing pe. growth restriction. margreet plaisier, esther streefland, pieter koolwijk, frans m helmerhorst, jan jaap hm erwich. department of gynecology and reproductive medicine, leiden university medical center, leiden, netherlands; department of obstetrics and gynecology, university medical center groningen, groningen, netherlands; department of physiology, vu univerity medical center, amsterdam, netherlands. objective: disturbances in decidual and placental vascular development may play a role in the pathogenesis of pregnancy complications, like pre-eclampsia (pe) or fetal growth restriction (fgr). whether the regulation of decidual vascular adaptation to implantation is altered in these illnesses, is not elucidated yet. the present study focused on the role of first-trimester angiogenic factors in the pathogenesis of pe and or fgr. methods: first-trimester decidua samples were obtained during routine chorionic villous sampling. the expression of vascular endothelial growth factor (vegf-a), placental growth factor (plgf), flt- , kdr, angiopoietin- (ang- ), angiopoietin- (ang- ) and tie- mrna was determined by rt-pcr. the expression of the angiogenic factors was related to the pregnancy outcome, i.e. uncomplicated, pe or fgr. results: the first-trimester decidual tissues expressed all angiogenic factors. mrna levels of vegf-a, plgf, kdr, ang- , ang- and tie- appeared increased in fgr cases compared to matched controls. in addition, plgf, ang- and tie- mrna appeared increased in pe cases compared to matched controls. the differential expression of angiogenic factors was more pronounced in cases with fgr than pe. the large inter-individual variation disallowed a significant outcome. conclusions: various angiogenic factors showed differential mrna expression in st trimester decidua of patients developing pe or fgr in later pregnancy compared to their matched controls. the first-trimester decidual samples provided a unique opportunity to obtain information regarding the onset of pe and fgr. early st trimester changes in angiogenic factor expression may well occur as a compensatory mechanism. in turn, this may set the stage for increased non-branching angiogenesis and altered decidual and placental vascular adaptation, which may be part of the pathogenesis of pe and/or fgr. complications. beth a bouchard, adrienne schonberg, gary j badger, ira m bernstein. biochemistry; obstetrics and gynecology; medical biostatistics, univ of vt, burlington, vt, usa. background preeclampsia is characterized by endothelial dysfunction. the goal of the current study was to prospectively measure plasma levels of the soluble endothelial cell adhesion molecules, sicam- and svcam- , beginning prior to pregnancy and determine if subjects destined to develop hypertension complicating pregnancy had differences in the concentrations of these molecules. methods serum levels of sicam- and svcam- were measured in healthy, nonsmoking women (cycle day . + . , prepregnancy) by elisa. all women subsequently conceived singleton pregnancies and were re-examined in early (ep, - weeks) and late pregnancy (lp, - weeks). five of these women developed hypertensive complications ( gestational hypertension, pre-eclampsia) near term. all subjects were normotensive at all study time points. data are expressed as mean ± sem. p< . was accepted as significant. results subjects were . + . years old with a mean bmi of . + . kg/m at the time of prepregnancy studies. significant differences in sicam- levels as a function of pregnancy were observed (p= . ) and are outlined in table p= . differences were dependent upon the stage of pregnancy in those women who were not diagnosed with hypertensive complications with a decrease in sicam- levels in ep (p= . ) followed by an increase in sicam- levels in lp (p= . ). in women with hypertension in pregnancy, these differences in sicam- levels were not evident (p= . ). there were no differences in sicam- levels comparing women with or women without hypertensive complications prior to pregnancy (p= . ). in contrast to sicam- , we observed no significant differences in svcam- levels over pregnancy or between those with and without hypertension. conclusions these combined observations suggest that levels of the soluble adhesion molecule sicam- change significantly over time in normal pregnancies. subjects destined to develop hypertension did not demonstrate the early pregnancy reduction in sicam- . supported by nih hl . yuval bdolah, uriel elchalal, shira natanson-yaron, hadas caspi, tali bdolah-abram, angelika bord, caryn greenfield, debra goldman-wohl, ariel milwidsky, franklin h epstein, s ananth karumanchi, simcha yagel, drorith hochner-celnikier. ob/ gyn, jerusalem, israel; ob/gyn medicine, beth israel deaconess medical center, harvard medical school, boston, ma, usa. objectives: nulliparity is a risk factor for preeclampsia (pe) with a reported incidence of up to - times higher than multiparous pregnancies. soluble fms-like tyrosine kinase- (sflt ), a circulating anti-angiogenic molecule of placental origin plays a pivotal role in pe by antagonizing placental growth factor (plgf). increased sflt and sflt /plgf have been shown to antedate clinical signs in pe. we therefore hypothesized, that the higher risk of pe in nulliparous pregnancies is associated with high sflt (or sflt /plgf). methods: maternal serum samples from nulliparous (n= ) and multiparous (n= ) term singleton pregnancies without pe, at the time of admission to delivery room, were used. serum samples were analyzed for levels of sflt and plgf by elisa. statistical analysis was performed applying t-test and the kruskall-wallis test and using spss software. results: for nulliparous and multiparous pregnancies, the mean serum sflt levels were , ± and , ± , (p= . ), the mean serum plgf levels were ± and ± (p= . ), and the mean ratios of sflt- /plgf were ± and ± (p= . ), accordingly. in a subgroup of multigravidous nulliparous pregnancies, sflt levels were , ± . correcting for maternal age did not alter the results. moreover, results did not differ between multiparous pregnancies with a - years interpregnancy interval compared with a - years interval. conclusions: in nulliparous pregnancies, circulating sflt levels and sflt / plgf ratios are significantly higher than in multiparous pregnancies. these findings suggest that the increased risk of pe in nulliparous pregnancies may involve anti-angiogenic imbalance. nulliparity may be more substantial than primigravity, as a risk factor for pe, suggesting that first semester abortions in primigravidas may not protect from pe in a subsequent term pregnancy. nevertheless, even - years intervals from the previous gestation do not increase the risk for pe. different normograms of angiogenesis should be used, when assessing the risk for pe in multiparous versus nulliparous pregnancies. objective: we determined whether maternal serum levels of angiogenic proteins namely soluble fms like tyrosine kinase (sflt- ), soluble endoglin (seng), and placental growth factor (plgf) -measured during the first trimester are associated with the subsequent development of placental abruption. methods: we performed a prospective, nested case-control study of women enrolled in the massachusetts general hospital obstetric maternal study (moms). first trimester serum samples from placental abruption cases and normal pregnancies were measured for angiogenic factors. cases and controls were matched by body mass index and age. placenta abruption was diagnosed by standard clinical findings and pathological examination of the placenta. women with confirmed preeclampsia or chronic hypertension were excluded. results: compared to controls, cases had more pregnancies, delivered infants at an earlier gestational age and with lower birth weight. first trimester levels of seng were significantly increased in cases compared to controls: . ± . ng/ml vs. . ± . ng/ml, p < . . there were no significant difference in serum levels of plgf, . ± . ng/ml versus . ± . ng/ml, p=ns, although sflt levels were lower in cases: . ± . ng/ml vs. . ± . ng/ml, p=ns. in logistic regression analysis adjusted for age, race, smoking, number of pregnancies, gestational age at delivery, gestational age of blood sampling, and blood pressure at first prenatal, seng levels remained independently associated with subsequent risk (odds ratio . , % ci . - . ) of placental abruption. examining this relationship by tertiles of seng, in the unadjusted model, women in the second (or . , % ci . - . ) and third (or . , % ci . - . ) tertiles were at increased risk of developing placental abruption compared with women in the lowest tertile. after adjusting for known risk factors of placental abruption, women in the second (or . , % ci . - . ) and third (or . , % ci . . ) tertiles remained at increased risk for placental abruption. conclusion: increased first trimester maternal serum levels of seng are associated with increased risk of subsequent placental abruption. ob/gyn, univ of vermont. shear stress is the most potent physiologic stimulus for elevating endothelial no production for flow-mediated vasodilatation. we measured in vivo shear stress during the proliferative and secretory phases and at and weeks of gestation hypothesizing that uterine blood flow (ubf) elevations in turn increase shear stress in early and late gestation. methods: during proliferative, secretory phases, and at and - weeks of pregnancy ua blood velocity and internal radius were measured bilaterally using color doppler ultrasound. blood viscosity was measured at shear rates in excess of /sec. results: compared to the proliferative phase viscosity was decreased at weeks and more so at weeks gestation (p< . ). . ± . . ± . nsd . ± . *** . ± . *** ua velocity (cm/sec) . ± . . ± . *** . ± . *** . ± . *** ubf (ml/min) . ± . ± *** . ± . *** . ± . *** shear stress (dynes/cm ) . ± . . ± . *** . ± . *** . ± . *** internal ua radius was not altered by the menstrual cycle, and was greater at weeks (+ %) and weeks (+ %). compared to proliferative, secretory phase showed significant rises in unilateral ubf and velocity that rose progressively during gestation. in contrast, shear stress increased in secretory ( %) and did not rise further in early pregnancy but by weeks shear stresses was further elevated %. conclusions: equivalent rises in shear stress during the secretory phase and weeks gestation demonstrate increases in radius and profound remodeling of uas that reflect the physiologic process of "normalization of shear". by late gestation, continued but modest rises in radius illustrate that further increases in shear stress occur almost solely due to rises in ubf via falls in down stream impedance. continued rises in shear stress into late gestation provide progressive stimuli for no production by ua endothelium. nih hl , hd , hl background: hypertension during pregnancy is associated with altered uterine vascular reactivity and blood flow, although its effects on arterial myogenic behavior have not been explored. the purpose of this study was to evaluate the effects of hypertension and no inhibition on myogenic tone in pregnancy, as the ability of a vessel to constrict and dilate in response to pressure plays a key role in regulating blood flow to the uterus. methods: three groups of sprague dawley late pregnant (day ) rats were used: control (n= ), hypertensive ( . g/l l-name in the drinking water, n= ), and treated with l-name and hydralazine (also in the drinking water, . g/l, n= ) to prevent the blood pressure increase, yet maintain no inhibition. resistance-sized radial arteries (< m) were mounted in a pressure arteriograph and equilibrated at mmhg (in pss containing l-nna and indomethacin) to induce a myogenic response. vessels were then subjected to pressure steps from to mmhg. tone (%) was calculated by comparing the vessel diameter at each pressure with the passive diameter at the same pressure (determined by incubation with . mm papaverine and m diltiazem). results: myogenic tone developed in controls ( ± % maximal), and was maintained over a broad range of transmural pressures . arteries from the l-name group did not develop tone at any pressure. co-treatment with hydralazine reinstated tone ( ± % maximal) over the same range of pressures as in the control group. the reduction in average placental weights in the l-name group ( vs. mg, p< . ) was restored by hydralazine ( mg, p< . vs. l-name). average fetal weights were also reduced in the l-name group ( . vs. . g, p< . ), but only partially restored by hydralazine co-treatment ( . g, p< . vs. control and l-name groups). conclusions: surprisingly, radial uterine arteries from the l-name group did not develop tone over any range of pressures, despite the fact that matched arteries from late-pregnant controls developed significant myogenic tone. this abolishment of tone was reversed by hydralazine, which also had beneficial effects on fetal and placental growth. these results implicate hypertension rather than no inhibition as the key factor in the suppression of myogenicitiy and dysregulation of uterine blood flow. charles r rosenfeld, timothy roy. division of neonatal-perinatal medicine, ut southwestern medical center at dallas, dallas, tx, usa. background: upbf b rises -fold in ovine pregnancy; but the mechanisms responsible for the rise and maintenance are unclear. we (jsgi ) reported that uterine vascular smooth muscle bk ca k + channels contribute to uterine vasodilation and upbf b maintenance; but up-stream activators are unclear. uterine vascular prostacyclin synthesis increases in pregnancy, but cyclooxygenase inhibition does not alter upbf b (ajp ) . vascular nitric oxide synthase (nos) also increases; but acute inhibition with l-name decreases upbf b only % (jci ) . it is unclear if l-name doses were insufficient or if prolonged nos inhibition has a greater effect. objective: to determine if local nos inhibition with l-name dose-dependently decreases upbf b and if prolonged inhibition exerts a greater effect on upbf b . methods: pregnant ewes were studied at - d gestation age (ga). had doseresponse studies with uterine artery l-name infusions to achieve . - . mg/ml over min. had h arterial l-name infusions to achieve and maintain levels of . mg/ml at (n= ), (n= ) and d (n= ) ga, while measuring arterial pressure (map), heart rate (hr) and upbf b before, during ( , , , , , , and h) and after ( , h) infusions. uterine arterial and venous cgmp were measured. data were analyzed by anova. results: acute nos inhibition decreased upbf b - %, but was not doserelated. h arterial l-name infusions decreased upbf b by - h at each ga (p . , anova) and values returned to baseline by h postinfusion. sensitivity did not differ between ga (p= . , anova), upbf b falling - % at each ga. contralateral upbf b was unaffected at all ga. map rose and hr fell during infusions at and d ga, p . ; but were unaffected at d ga. venous-arterial cgmp concentration differences were seen at d and absent at h of l-name infusion, p= . . conclusions: uterine vascular nos increases in ovine pregnancy, but its inhibition decreases upbf b %, suggesting study doses were insufficient to fully inhibit vascular nos activity. alternatively, nos contributes to the maintenance of upbf b , but other mediators, not yet identified, are more important in activating bk ca and regulating upbf b . notably, l-name reached the systemic circulation, and although further diluted, map rose - %, suggesting the systemic vasculature may be more sensitive to nos inhibition than upbf b . charles r rosenfeld, xiao-tie liu. division of neonatal-perinatal medicine, university of texas southwestern medical school, dallas, tx, usa. background: uteroplacental blood flow (upbf) rises -fold in ovine pregnancy, reflecting increases during pre-implantation, placentation and finally, in the last third of gestation. we reported that bk ca density in uterine vascular smooth muscle (uvsm) increases in ovine pregnancy (sgi ) and inhibition with tetraethylammonium ( . mm) dose-dependently decreases basal upbf % in the last third of pregnancy (jsgi ) . it is unclear how bk ca subunit expression changes and activity is regulated in pregnancy; since uterine vascular nitric oxide is increased, signaling via cgmp-dependent protein kinase g (pkg) is one potential pathway. objective: to determine simultaneous changes in uvsm bk ca density and regulatory -subunit expression and the cgmp signaling pathway for activation in ovine pregnancy. methods: endothelium-denuded segments of nd generation uterine arteries obtained from nonpregnant (n= ), pregnant (n= , - d gestation; term d) and postpartum (n= , - d) sheep were used to measure expression of bk ca -and regulatory -subunits and signaling proteins in uvsm by immunoblot analysis and immunohistochemistry. results: uvsm bk ca density, reflected as a change in the kda -subunit, rose % with placentation (p< . ) and was unchanged thereafter. expression of the kda regulatory -subunit paralleled the rise in bk ca density during placentation, increasing % (p< . ), but increased another -fold and exponentially in the last third of pregnancy (p< . , r = . , n= ). changes in subunit immunostaining in uvsm paralleled increases in protein. although uvsm soluble guanylyl cyclase was unchanged in pregnancy (p= . , r= . , n= ), pkg expression rose -fold (p= . , r= . , n= ) and gradually returned to nonpregnant levels by d postpartum (p= . , r= . , n= ). uvsm cgmp is being measured. conclusions: these are the st data suggesting that increases in ovine upbf during placentation involve vascular growth and bk camediated vasodilation. the rise in upbf in the last third of pregnancy reflects bk ca -mediated vasodilation due to enhanced channel activation via increases in uvsm pkg, bk ca phosphorylation and changes in subunit stoichiometry due to an exponential rise in the regulatory -subunit. it is unclear what initiates and directs these changes in bk ca expression and sensitivity. relaxin (rlx) is a hormone traditionally associated with changes in the female reproductive tract during pregnacy. recent evidence suggests that rlx may play a pivotal role in regulating cardiovascular function during gestation. analogous to pregnancy, administration of recombinant human relaxin (rhrlx) to nonpregnant female rats reduces systemic vascular resistance, as well as increases global arterial compliance. additionally, we demonstrated that, concurrent with rlx´s influence on overall cardiovascular function, small renal arteries (sra) from rhrlx-treated mice and rats are characterized by reduced passive stiffness and increased arterial wall area whereas external iliac arteries (eia) are not. we hypothesized that rlx regulates arterial passive mechanical properties by altering the cellular and biochemical composition of the arterial wall. nonpregnant female mice were administered rhrlx for days after which sra and eia were isolated. we measured arterial collagen, elastin, and total protein using the sircol collagen assay, the fastin elastin assay, and the pierce bca protein assay, respectively. additionally, we quantified arterial smooth muscle cell (smc) density using immunofluorescent techniques. sra isolated from rhrlx-treated mice were characterized by a significant reduction in collagen to total protein ratio ( . ± . vs . ± . μg collagen/μg protein; mean±sem; p< . ) as well as a significant increase in smc density ( . ± . vs . ± . cells/ μm ; p< . ) compared to control mice with no significant change in elastin content ( ± vs ± μg elastin/mg dry weight). in contrast, there were no significant changes in collagen to total protein ratio ( . ± . vs . ± . μg collagen/μg protein), smc density ( . ± . vs . ± . cells/ μm ) or elastin content ( ± vs ± μg elastin/mg dry weight) in eia from the rhrlx-treated mice compared to control mice. of note, comparable results were observed for rlx knock-out (rlx -/-) and wild-type mice with rlx -/mice exhibiting increased arterial collagen and decreased smc density. we conclude that the rlx-induced decrease in passive stiffness of sra that we previously reported is, at least in part, due to rlx-induced alterations in arterial wall cellular and biochemical composition. further, our findings suggest that these vessel wall remodeling effects of rlx are artery-type specific. relaxin is a peptide hormone that emanates from the corpus luteum of the ovary and circulates during pregnancy. this hormone plays an important role in renal vasodilation and hyperfiltration, two fundamental maternal adaptations in pregnancy. chronic administration of recombinant human relaxin (rhrlx) to both virgin rats and mice inhibits myogenic reactivity and increases compliance of small renal arteries, thus further mimicking pregnancy. we hypothesize that these arterial responses to rhrlx are mediated by the lgr , and not the lgr receptor. both lgr and lgr receptor-deficient, and wild-type virgin mice were investigated. the mice were chronically infused with rhrlx or vehicle (veh) for days. small renal arteries were isolated and mounted in a pressure arteriograph and myogenic reactivity was assessed (% change in diameter over baseline in response to a mmhg step increase in intraluminal pressure). small renal arteries from rhrlx-infused lgr wild-type mice showed inhibited myogenic reactivity with a . ± . % increase in diameter whereas the arteries from rhrlx-infused lgr knock-out mice exhibited robust myogenic reactivity with only a . ± . % change in diameter (p= . ). in contrast, myogenic reactivity of small renal arteries was inhibited in both the rhrlx-infused lgr knock-out and wild-type mice. the veh-treated lgr and lgr mice, regardless of genotype, exhibited robust myogenic reactivity. arterial compliance was also assessed for each genotype/treatment group. rhrlx infusion increased arterial compliance of small renal arteries from lgr wild-type, but not from lgr knock-out mice (p= . ). in contrast, the arteries from rhrlx-infused lgr wild-type and knock-out mice showed increased compliance relative to veh-infused animals. conclusion: relaxin-induced inhibition of myogenic reacitivity and increase in compliance of small renal arteries is mediated by the lgr , and not the lgr receptor. smoking is associated with adverse pregnancy outcomes including fetal growth restriction. pathologic effects of smoking on maternal vasculature is a potential mechanism leading to fetal growth restriction. the objective of this study was to determine whether cigarette smoke exposure during pregnancy affects the functional properties of uterine and peripheral arteries using a gravid murine model. study design: pregnant and virgin c bl/cj mice were exposed to whole body side stream smoke using an inhalational chamber for hours/day. smoke exposure was increased from day of gestation until late pregnancy (day - ) with mean total suspended particle levels of mg/m , representative of moderate to heavy smoking in humans. control animals were exposed to ambient room air. late pregnant and virgin mice were sacrificed and uterine, mesenteric, and renal arteries were isolated and studied in a pressure arteriograph system (n= - in each group). plasma cotinine was measured by elisa. means were compared using t-test or analysis of variance. results: fetal weights were significantly reduced in mice exposed to smoke compared to control fetuses ( . ± . g vs . ± . g, p= . ), while litter sizes were not different. cotinine levels in smoking mice were significantly elevated compared to control mice ( . ± . vs . ± . ng/ml for virgin mice and . ± vs . ± . ng/ml for pregnant mice). there was no significant difference in phenylephrine responses between groups. endothelial mediated relaxation responses to methacholine were significantly impaired in both the uterine and mesenteric vasculature of pregnant mice exposed to cigarette smoke during gestation. no difference in endothelial-mediated relaxation was seen in isolated renal arteries in pregnant mice exposed to cigarette smoke, however relaxation was significantly reduced in renal arteries from smokeexposed virgin mice. conclusions: passive cigarette smoke exposure is associated with impaired vascular relaxation of uterine and mesenteric arteries in a gravid murine model. functional vascular perturbations during pregnancy, specifically reduced uterine blood flow and impaired peripheral vasodilation, may be a mechanism by which smoking results in fetal growth restriction. human chorionic gonadotropin (hcg) is essential during early human gestation for "rescue" of the corpus luteum. however, its potential contribution to the widespread maternal vasodilation of pregnancy that occurs at this stage of pregnancy remains uncertain. our objective was to use the renal circulation of conscious rats as an experimental model in which to test the vasodilatory potential of hcg. in addition, we investigated both myogenic reactivity and relaxation responses in small renal and mesenteric arteries isolated from rats, as well as in small human subcutaneous arteries using a pressure arteriograph. we chronically instrumented rats for measurement of renal function. five were ovariectomized, and sham-ovariectomized. after days of surgical recovery, baseline glomerular filtration (gfr) and effective renal plasma flow (erpf) were measured on two separate days and the values averaged. then, an osmotic minipump containing hcg ( iu/min) was implanted s.c. and renal function was again assessed and days thereafter. gfr and erpf significantly increased and calculated effective renal vascular resistance decreased from baseline in the intact (p< . vs. baseline), but not ovariectomized (p=ns) rats on both days and of hcg administration. in the intact, but not ovariectomized rats, plasma osmolality declined and progesterone increased (both p< . vs baseline). plasma hcg concentrations were , miu/ml and comparable in both groups of rats. incubation of small renal arteries from rats with recombinant human relaxin (rhrlx, - ng/ml), but not hcg ( , - , miu/ml) in vitro inhibited myogenic reactivity and relaxed phenylephrine (pe)-constricted arteries. in contrast, both rhrlx and hcg inhibited myogenic reactivity and relaxed pe-constricted small mesenteric arteries from rats and small human subcutaneously arteries. in conclusion, consistent with our earlier work showing a virtually exclusive role for relaxin in mediating the renal circulatory changes of pregnancy, hcg is likely to play little or no role. in contrast, hcg and relaxin are both likely to contribute to the vasodilation of other organ circulations during pregnancy. pierre-andre scott, , michele brochu, jean st-louis. , research centre, chu ste-justine, montréal, qc, canada; pharmacology, université de montréal, montréal, qc, canada. uterine vasculature undergoes major structural and functional changes during pregnancy. estrogens have been shown to induce increased uterine blood flow in this circulation. we have reported that estradiol ( -e ) induced direct vasorelaxant response on smooth muscle of the uterine arteries that, for it major part, is not mediated through tissue nitric oxide. to investigate the cellular effectors mediating this vasorelaxant effect of -e , we set-up uterine arteries of non-pregnant rats in wire myograph systems for microvessels. -e and -e were equipotent in relaxing phe ( μmol/l)-preconstricted uterine arteries, although the later produced significant smaller relaxations. genistein, a phytoestrogen presumed to inhibit phosphatases, also produced uterine artery relaxation with significant lower potency. to try interfere with the vasorelaxant effect of -e , tissues were preincubated with different substances. cycloheximide (protein biosysthesis inhibitor), ici , (estrogen receptor modulator), and kt (pka inhibitor) did not significantly influenced the response to -e . rp- -pcpt-cgmps (pkg inhibitor) slightly displaced the concentration-relaxation curve to -e to the right. inhibitors of potassium channels, penitrem a (bkca) and glybenclamide (katp), showed opposite effects for -e concentration-response curve; the former producing a right shift and the latter a small not significant left shift. these data indicated that the direct acute effect of -e in uterine artery is the result of complex interactions within smooth muscle cells, involving potassium channels, and protein kinases and phosphatases. the renin-angiotensin-aldosterone system is paradoxically activated during pregnancy, since blood pressure decreased. earlier data showed that the high levels of aldosterone present during pregnancy may be involved in cardiovascular adaptation to pregnancy. in order to delineate this effect of aldosterone on vascular tone, potassium canrenoate, an antagonist of mineralocorticoid receptors (mr), was administered ( mg/kg/day) to pregnant rats from the day to of gestation. rats were sacrificed at day , and of gestation together with untreated and day pregnant rats. the thoracic aorta was quickly removed and set up in tissue baths as ring ( - mm) preparation. as observed previously, canrenoate enhanced responsiveness to phe for all time of treatment tested, but only at day ( days treatment) for kcl responsiveness. aortic contractile responses to tea (tetraethylammonium) gradually decreased during pregnancy to almost disappear at the end of gestation. in the present results, canrenoate treatment made the response statistically different by day of pregnancy. finally, the activity of na/k-atpase was measured by the relaxant effect of kcl added to physiological solution without potassium. the activity of the pump was decreased when approaching parturition compare to day of pregnancy. canrenoate treatment abolished this effect. the present data show that vascular changes that occurred during pregnancy are markedly modified by treatment of rats with an antagonist of mineralocorticoid receptors, suggesting that aldosterone may be involved in vascular adaptation to pregnancy. background impaired endothelium-dependent vasodilatation has previously been demonstrated in small myometrial arteries from women with gestational diabetes. this impairment may play a role in mediating the complications observed in diabetic pregnancies. it is not known which mechanisms of endothelium-dependent vasodilatation are affected in myometrial arteries by gestational diabetes. aim to investigate mechanisms of endothelium-dependent vasodilatation in uterine arteries using a mouse model of pregnancy complicated by diabetes. methods diabetes was induced in female c bl /j mice (streptozotocin; mg/kg) prior to mating. mice were culled at day of pregnancy (term) and uterine arteries dissected, mounted on a wire myograph, normalised to mmhg and equilibrated ( °c; %co /air). arteries were constricted with phenylephrine ( m) and a concentration-response curve to the endotheliumdependent vasodilator acetylcholine (ach; . nm- m) constructed in the presence and absence of a nitric oxide synthase inhibitor (l-nna; m), a cyclooxygenase inhibitor (indomethacin; m) or a combination of the two to determine the contribution of nitric oxide (no), prostacyclin and endothelium derived hyperpolarising factor (edhf) to vasodilatation. results sensitivity to ach was comparable between diabetic and vehicle treated mice (ec . ± . nm vs . ± . nm). the contribution of individual endothelium-dependent vasodilators was significantly altered in arteries from diabetic mice. at m ach, edhf-mediated relaxation was significantly reduced (p= . , one-way anova) compared with controls ( . ± . vs . ± ). in comparison, no-mediated relaxation was significantly increased (p= . , one-way anova) compared with controls ( . ± . vs . ± . %). conclusions endothelium-dependent relaxation was not reduced in uterine arteries of diabetic mice compared with controls. however, there was a profound change in the contribution of endothelium-dependent vasodilators in arteries of diabetic mice. this may alter compensatory capacity as disease progresses. supported by mfn training grant (cihr). raf- serine/threonine protein kinase has been extensively studied as the upstream kinase linking ras activation to the mek/erk module. mek/erk has been shown to play a role in the modulation of vascular contraction. however, the role of raf kinase in vascular contraction and its possible involvement in alteration of maternal vascular function during pregnancy is not known. objectives: to determine ( ) if raf kinase contributes to phenylephrine (pe)induced contractile response, ( ) the role of raf kinase inhibitor (gw ) in regulating vascular tone during pregnancy, and ( ) mechanism by which gw produces vasodilatation in rat mesenteric arteries. methods and results: conscious non pregnant (np) and pregnant (p) sprague dawley rats received increasing doses of pe in the absence or presence of gw . pe induced a dose-dependent increase in map in both np and p rats but responses were substantially depressed in pregnancy. gw shifted the pe-induced dose response to the right in both np and p rats. gw itself did not affect basal blood pressure. isometric tension studies in mesenteric arteries showed that gw did not change the kcl-evoked contraction but significantly inhibited the contraction to pe in both np and p arteries. interestingly, at a given concentration, gw produced greater inhibition of pe-induced contractile response in p than in np arteries. also, in p arteries the inhibitory effects of gw were greater as the pregnancy progressed from day to day . raf kinase expression and activity was significantly decreased in arteries of p compared to np rats. in mesenteric vascular smooth muscle cells (vsmcs), pe stimulated activation of raf kinase as indicated by phosphorylation of its immediate target, mek / in a time-and dose-dependent manner. measurement of [ca + ] i with fura- showed that gw -mediated inhibition of pe-induced contraction was not associated with decrease in [ca + ] i . vsmcs treated with pe exhibited higher levels of the contractile proteins, p-mypt and p-mlc which was inhibited by gw . conclusion: to our knowledge, for the first time we show that raf kinase plays an important role in the regulation of vascular contractility. decreased expression and/or activity of raf kinase during pregnancy may explain in part, for decreased systemic vascular reactivity during late gestation. the mechanism(s) that contribute to reduced vascular sensitivity to phenyleperine (pe) during pregnancy is not well understood. pe, in addition to activating the classical contractile pathways, also stimulates growth factor pathways that results in activation of ras/mitogen-activated protein kinases. it is not clear whether these pathways play a role in the modulation of vascular contraction. hence, the present study was designed to determine ) if ras is involved in mediating the pressor response to pe in non pregnant (np) and pregnant (p) rats, ) if so, the mechanism by which ras protein contributes to pe-induced vascular contraction, and ) any differential expression and/or activity of ras during pregnancy that might contribute to altered vascular response. methods: ) mean arterial pressure (map) was measured in conscious np and p rats. ) isometric tension was measured in mesenteric arteries of np and p rats. ) expression of contractile proteins, p-mlc and p-mypt were studied in cultured vascular smooth muscle cells (vsmcs). ) expression and activity of ras in mesenteric arteries of np and p rats were measured by western blot analysis. results: ( ) intravenous administration of pe resulted in a dose-dependent increase in map but responses were substantially depressed in pregnancy. inhibiting ras activation with manumycin, decreased pe-induced increase in map in both np and p rats. manumycin by itself had no effects on basal map. ( ) isometric contraction studies in myograph with mesenteric arteries showed that manumycin shifted pe-induced dose response curve to the right with a decrease in e max in np and p rats. higher concentration of manumycin was required to inhibit pe-induced contraction in np ( μm) compared to p ( - μm) rats. ( ) pretreatment with manumycin inhibited pe-induced increase in the extent of phosphorylation of both mlc and mypt in mvsmcs. ( ) compared to p rats, mesenteric arteries of np rats revealed increased basal and pe-induced expression and activity of ras. reduced expression of this contractile ras pathway might contribute to decreased vascular reactivity during pregnancy. conclusion: ras protein plays a role in regulation of vascular contraction. the decreased amount and activity of vascular ras may be a novel mechanism that explains the reduced vascular resistance during pregnancy. overweight affects the relaxin-induced response of mesenteric arteries. joris van drongelen, arianne van koppen, marc ea spaanderman, paul abm smits, frederik k lotgering. obstetrics and gynecology, radboud university nijmegen medical centre, nijmegen, netherlands; pharmacology and toxicology, radboud university nijmegen medical centre, nijmegen, netherlands. objective: overweight affects pregnancy-induced vascular adaptation and predisposes to gestational hypertensive disease. relaxin plays a key role in normal gestational vascular adaptation by increasing endotheliumdependent vasodilation and compliance, and reducing myogenic reactivity. we hypothesized that overweight blunts the vascular response to relaxin. the vascular responses to flow and pressure of mesenteric arteries after pre-treatment to human recombinant relaxin (rlx) or placebo (plac) were examined in overweight (ow) and normal weight (nw) rats in a pressure-perfusion myograph. overweight was established by a high-fat diet. the endothelium-dependent vasodilatation was measured in response to flow: e (flow inducing % dilatation) and e max (maximal dilative effect). active contractile (myogenic reactivity) and passive dilative (compliance) vascular responses to pressure were determined. all vascular responses were calculated as the proportional change in diameter to % precontraction with u . results: in nw rats rlx decreased e and increased e max to flow and attenuated myogenic reactivity without affecting vascular compliance. in ow plac-treated rats, compared to nw rats, an upregulated vasodilative state was present (decreased e and increased e max , and lower myogenic reactivity). in ow rats rlx increased e to flow and unaffected e max . rlx increased myogenic reactivity mildly in absence of changes in vascular compliance. values are presented as mean±sem; * p< . between nw/ow, # p< . within nw/ow. whereas rlx stimulates endothelium-dependent vasodilation to flow and myogenic reactivity in nw rats, ow overturns the flow-induced response and decreases myogenic reactivity to a lesser extent than present in nw rats. we speculate that these overweight-induced adverse effects of rlx prelude to vascular maladaptation and gestational hypertensive disease. compared to the luteal (lut) phase, uterine blood flow is increased in vivo during the follicular (fol) phase and more so during pregnancy (preg). both of these are physiologic states of high estrogen and shear stress. endothelial cells express enos and produce greater amounts of nitric oxide (no) in response to elevations of shear stress. phosphorylation of enos is a signaling marker of activation. we have recently validated lut, fol and preg uaec culture models for evaluating enos phosphorylation responses to shear stress and vascular mediators. we hypothesized that uaecs derived from fol and preg sheep will show greater enos phosphorylation than lut phase uaecs, and with more robust responses in the presence of estrogen (e ). methods: uaecs were cultured until % confluence, and then subjected to (static control), or dynes/cm for hrs in the absence or presence of e ( nm). western analysis was used to compare optical densities of ser -penos (penos) normalized to total-enos (mean + sem). results: in lut uaec penos was equally increased two fold by and dynes/cm (from . + . to . + . and . + . , respectively). compared to lut uaecs, the static control fol uaecs appeared to have higher penos levels ( . + . ) and this was further increased . - . fold with dynes/cm ( . + . ) and dynes/cm ( . + . ). as seen with fol uaecs, preg uaec static penos ( . + . ) appeared higher than lut uaec, but unexpectedly, neither nor dynes/cm significantly raised these levels of penos ( . + . and . + . ). regardless of shear stress level, e replacement in the culture media only increased the penos levels in the nonpregnant, but not the pregnant derived uaecs. conclusions: increasing amounts of shear stress have a corresponding increase in the ratio of enos that is phosphorylated at serine in lut and fol uaecs. pregnant uaecs do not appear to increase the constitutive ratio of serine phosphorylation of enos at either or dynes/cm . in contrast to our hypothesis, chronic treatment with e for hrs did not augment the ratio of enos phosphorylation in pregnancy. nih hl , hd , hl . cells. honghai zhang, wu xiang liao, dong-bao chen. reproductive medicine, university of california san diego, la jolla, ca, usa. s-nitrosylation (sno) is a rapid, reversible, and nitric oxide (no) dependent post-translational protein modification critical for signal transduction. estrogen stimulates endothelial cell (ec) no production but yet to be determined is if this leads to increased formation of sno-proteins in ec. hypothesis: we hypothesize that estrogen stimulates the formation of sno via a receptor and endogenous no dependent pathway in huvec or ovine uterine artery ec. methods: cells on glass coverslips were treated with mm of no donor gsno or dea nonoate, estradiol- ( nm) in the presence of l-name ( mm) for min. the cells were fixed with methanol. in situ sno-proteins were detected by a rapid biotin derivatization method by blocking thiols by methylmethanethiosulfonate (mmts), followed by ascorbate reduction and labeling with texas red-labeled ethylmethanethiosulfonate (mtsea) and examined by fluorescence microscopy. cells ( x /group) were lysed in hen buffer, and then similarly prepared but labeled with biotin-mtsea. a portion of the samples were separated on sds-page and blotted with anti-biotin antibody for total sno-protein patterns. the biotin-labeled sno-proteins were captured by avidin, followed by immunoblotting with specific antibodies. results: basal sno-protein labeling was apparent in both ec types. exogenous no donated by gsno or dea nonoate significantly increased red fluorescence labeling of sno-proteins in the cytosol of both ec types. treatment with estradiol- also increased total sno-protein labeling in both ec types. pretreatment with l-name significantly reduced sno-protein labeling. sds-page and immunoblotting with anti-biotin antibody analyses identified multiple bands of sno-proteins. in avidin captured biotinylated samples, multiple proteins were indentified. conclusion: exogenous no donated by gsno or dea nonoate and endogenous no upon estrogen stimulation increased s-nitrosylated proteins in ec (supported by nih ro hl and ). background and objective: the renin-angiotensin system (ras) functions both systemically and locally as a primary regulator of blood pressure and fluid volume and is therefore involved in the etiopathogenesis of essential hypertension as well as preeclampsia, a pregnancy-induced hypertensive disorder in pregnant women. while much progress has been made in the development of strategies to block the systemic ras and thus treat essential hypertension, relatively less advance has been achieved in the development of effective local ras inhibitors to treat preeclampsia, primarily due to the fact the conventional systemic ras inhibitors generate potential teratogenic risks to pregnant women during critical stages of their pregnancies. ginger has been widely and effectively used in pregnant women to treat pregnancyinduced nausea and vomiting with minimal adverse effects. the present study was designed to investigate whether ginger could down-regulate renin secretion by human decidual cells (the major source of renin in the tissue-based uteroplacental ras), as an initial step toward a long-term goal to develop potential safe and effective ras inhibitors for use in obstetric patients. methods: full-term normal human placentas were obtained within one hour of vaginal deliveries or cesarean sections. decidual cells were isolated from the decidua parietalis. after an initial culture for days in a serum-containing medium, the decidual cells were treated with ginger juice at various doses in a serum-free medium for hours. the culture supernatants were then harvested and subject to western blot analyses of renin protein contents. the ginger juice used was freshly prepared from the ginger roots purchased from a local grocery store and different batches of juice preparations were standardized based on their optical density values at a wavelength of μm on a spectrophotometer. results: a dominant band of renin at approximately kd was detected in all samples. when compared with the control (i.e. %), ginger juice decreased the renin protein contents in the culture supernatants in a dose-dependent manner. no significant difference in the cell morphology was observed between the control and treatment groups. conclusion: ginger root juice down-regulates renin secretion in human decidua, showing a great potential of a novel ras inhibitor, particularly, in obstetric patients. noninvasive quantification of the autonomic nervous system throughout pregnancy. dietmar schlembach, karoline pickel, daniela ulrich, philipp klaritsch, isa alkan, uwe lang, manfred g moertl. obstetrics and gynecology, medical university of graz, graz, styria, austria; cnsystems medizintechnik ag, graz, styria, austria. introduction: the analysis of heart rate variability (hrv), blood pressure variability (bpv), and baroreflex sensitivity (brs) has become a powerful tool for the assessment of autonomic control. although hrv analysis was initially developed for risk stratification in cardiology, the field of clinical application has broadened in recent years. the aim of our study was to investigate the adaptation of autonomic control during pregnancy based on analysis of heart rate variability and baroreceptor sensitivity. methods: patients with uncomplicated pregnancy were measured with the taskforce® monitor i made by cnsystems. all measurements were performed in supine position under standardized resting conditions. autonomic parameters were recorded at rest and within the "deep breathing method" as a "cardiovascular challenge test". results: throughout pregnancy a shift to higher sympathetic activity respectively a lower parasympathetic activity was observed. , and . ± . [> wks]) (figure ). during the deep breathing maneuver we could show an increase of the sympathetic / parasympathetic ratio (figure ). the noninvasive determination of autonomic parameters throughout pregnancy is possible. these results can be used as basic parameters for classifying and assessing autonomic changes in pathological conditions in pregnancy such as hypertensive disorders. how far the characterization and challenge of these autonomic functions can be utilized for diagnosis and prediction of preeclampsia has to be shown in future studies. hemodynamic parameters measured noninvasively throughout pregnancy. daniela ulrich, manfred g moertl, karoline pickel, philipp klaritsch, isa alkan, uwe lang, dietmar schlembach. obstetrics and gynecology, medical university of graz, graz, styria, austria; cnsystems medizintechnik ag, graz, styria, austria. background: hemodynamic changes throughout pregnancy have been measured predominantly by invasive techniques. the discussion on the valence und the low acceptance of these invasive procedures by pregnant women demands a noninvasive method for evaluating and distinguishing cardiovascular adaptative mechanisms throughout normal and especially complicated pregnancy. method: healthy patients with uncomplicated pregnancy were measured with the taskforce® monitor i (cnsystems, austria) at different time points throughout pregnancy. cardiovascular parameters were recorded in supine and left lateral position under standardized conditions. results: throughout pregnancy an increase of global parameters such as heart rate (hr), blood pressure (bp), total peripheral resistance (tpr) and total peripheral resistance index (tpri) were observed. stroke volume (sv), stroke index (si), cardiac output (co), contractility index (ci), acceleration index (aci), and left ventricular ejection time (lvet) decreased throughout pregnancy (table). discussion: the noninvasive determination of cardiovascular parameters throughout pregnancy is possible and the results of this pilot study can serve as basic parameters for classifying and assessing cardiovascular changes in pathological conditions in pregnancy such as hypertensive disorders. assigning pregnant hypertensive women into hyper-and hypodynamic groups may aid in planning individual therapeutic strategies. objective: both gnrh i and gnrh ii are expressed in humans. gnrh i is produced by the hypothalamus under the regulation of gonadal steroids and stimulates pituitary gonadotropins. during pregnancy gnrh i is also produced by the placenta and affects hcg. gnrh ii, the ancient isoform, is produced by numerous human tissues including immune and reproductive tissues and circulates in blood in quantifiable levels. we have demonstrated that gnrh ii analogs directly affect fertilization and uterine function, and propose that it acts via immune regulation. during pregnancy it is known to regulate numerous hormone productions, yet the levels of gnrh ii has not been reported. in these studies we have determined the circulating levels of gnrh ii throughout pregnancy and in early pregnancy loss. study design: thirty-three women having normal pregnancies were followed prospectively. plasma samples were drawn at , , , , , , weeks gestation and during labor. plasma was also collected from patients have spontaneous abortions (n= ). circulating gnrh ii and crh and gnrh i were measured by specific radioimmunoassays. results: circulating gnrh ii increased from non-pregnant levels ( +/- pg/ ml, mean+/-sem) to +/- pg/ml by -week lmp. gnrh ii concentrations continued to increase through weeks gestation over the -week levels, although a significant increase was only attained by weeks. gnrh ii continued to increase in late term, attaining levels of +/- pg/ml which was significantly higher than that of early gestation. during labor and delivery gnrh ii in maternal plasma was further increased to +/- pg/ml, i.e., . times that of -week gestation ( +/- pg/ml). circulating gnrh ii did not parallel gnrh i in early pregnancy but did in late gestation. gnrh ii did correlate with crh in early pregnancy but not in late gestation. patients having spontaneous abortion had increased circulating gnrh ii at -weeks lmp ( +/- pg/ml) as compared to normal pregnancies ( +/- pg/ml). conclusion: gnrh ii increased throughout pregnancy attaining highest concentrations during labor and delivery. patient with early pregnancy loss had increased gnrh ii expression. the function of gnrh ii or factors affecting this increased gnrh ii in normal or abnormal human pregnancy should be investigated. introduction: plasma levels of the endocannabinoid, anandamide (aea) decrease during the luteal phase of the menstrual cycle and early pregnancy and increase during parturition. high plasma aea levels at weeks in women undergoing ivf-et was associated with a failure to achieve an ongoing pregnancy . what is uncertain is what happens to aea levels when the pregnancy has already failed. we aimed to quantify aea levels in women presenting in early pregnancy with a diagnosed non-viable pregnancy and to compare them to those of a viable pregnancy. methods: plasma aea was measured by a sensitive isotope dilution hplc-ms/ms method from women in early pregnancy ( - weeks) of whom had a viable pregnancy and had a non-viable pregnancy. serum hcg and progesterone were measured in blood samples by standard elisa methods. results: the ages and bmi of both groups were similar ( ± . versus ± . years; mean ± sd; p= . ; student's unpaired t-test and ± . versus ± . kg/m ; p= . ) respectively. plasma aea levels in women with non-viable pregnancies were significantly higher than those in women with viable pregnancies ( . ± . versus . ± . nm; p= . ). there was no correlation found between aea levels and hcg or progesterone levels p= . and r= . ; p= . , respectively) , despite progesterone being significantly lower in the non-viable group ( . ± . versus . ± . ng/ml; p= . ). conclusion: higher plasma aea levels were associated with early pregnancy failure, and this association appeared to be independent of serum hcg or progesterone concentrations. these data suggest that plasma aea levels are linked with pregnancy failure through a mechanism that does not involve hcg or progesterone production. the precise involvement of anandamide in early pregnancy needs to be investigated further. n- ) and arachidonic (aa, : n- ) acids, the major brain long-chain polyunsaturated fatty acids (lc-pufa) are generated by elongation-desaturation of dietary essential fatty acids (efa). the maternal liver is principally responsible for efa elongation-desaturation. objetive. we determined effects of protein restriction in pregnancy on expression of d, d and elongases and (elov and ) in the maternal liver rat. methods. pregnant rats were fed control ( % casein; c) or restricted ( % casein; r) isocaloric diets. at day gestation maternal blood and livers were collected. serum triglycerides, cholesterol and glucose were determinate with the synchron cx autoanalyser and leptin and insulin by ria. liver fat determination was performed by the soxhlet method. liver ara and dha were calculated by gas chromatography based upon retention times from methyl ester standards. liver d, d, elov and mrna were measured by rt-pcr and northern blot. data are m ± sem; analysis by t-test. results. liver weights did not differ in c and r. total liver fat ( ± vs ± mg) serum leptin ( ± . vs ± . ng/ml) and insulin ( . ± . vs . ± . ng/ml) differed in c and r respectively (p< . ). serum triglycerides, cholesterol, and glucose did not differ. desaturase and elongase mrna and % of maternal liver aa and dha were lower in r (fig ) . conclusion. low liver fat content and desaturase and elongase mrna in r indicate impaired lc-pufa synthesis which may adversely impact fetal development, especially the brain. objective: to test the hypothesis that obstetrical dic results from an excessive leak of fetal material into the maternal circulation. a secondary objective was to assess maternal morbidity. methods: all cesarean hysterectomy cases for hemorrhage at our hospital from to were included. intravascular presence of fetal material was determined by two pathologists, blinded to each other and any clinical information. the percentage of those with any fetal material in the maternal circulation was calculated for each diagnosis for hemorrhage. for a given diagnosis, the percentage of intravascular fetal material in those with the given diagnosis was compared to those without that diagnosis using fisher's exact test. most patients had multiple hemorrhage diagnoses. a two sample t-test was used to evaluate the difference in mean blood loss between those with and without intravascular fetal material. results: primary outcome* % of patients received blood products and % received clotting agents. the average blood loss was cc. there were no maternal deaths and injuries to adjacent organs, all to the bladder. conclusions: there was no association between the presence of intravascular fetal material and any specific hemorrhage diagnosis or amount of blood loss. although the power to detect a relationship was low, the excessive leakage of fetal material as a specific and exclusive mechanism of obstetrical dic, as postulated, seems unlikely. while % of the population was transfused, there were few intraoperative complications and no maternal deaths. hypertension, tel-aviv university, sheba medical center, ramat-gan, israel. objective: the joint national committee on high blood pressure (jnc ) determined blood pressure of - / - in adults to be prehypertension that requires health promoting life style modifications to prevent cardiovascular disease. similarly, we hypothesize that gestational prehypertension in pregnancy is associated with increased risk of pregnancy complications and its management would improve pregnancy outcome. methods: prospective and retrospective recruitment resulted in a study group consisting of patients who were diagnosed in the first and early second trimester (< weeks) with blood pressure of - / , and a control group consisting of patients with blood pressure < / at similar gestational age. comparisons between the two groups were accomplished for demographic characteristics and outcome measures using the chi-square test statistic and two-sample t-tests for categorical and continuous variables, respectively. results: all outcome measures analyzed resulted in poorer results being associated with the study group compared with control as follows: % versus % of the study and control group pregnancies, respectively, experienced preeclampsia (p= . ); and % versus % of the study and control group, respectively, were associated with gestational hypertension (p= . ). % of the control group deliveries were associated with admission to the nicu, compared to only % in the control group (p= . ); % versus % of the study and control group deliveries, respectively, were preterm (p= . ); twenty-nine percent of the study group were cesarean deliveries, compared to % in the control group (p= . ). on admission mean sbp and dbp was and mmhg, respectively, in the study group, compared to and mmhg in the control group (p= . for sbp and p= . for dbp). conclusions: "gestational pre-hypertension" may be a real pregnancy condition that when is recognized, physician attention, patient close monitoring during prenatal care and delivery and early intervention in patients at risk, may all promote improved pregnancy outcome. larger scale study is in progress to evaluate and confirm these findings in the larger pregnant population. are contractions at - weeks gestation less painful than those at full term? kristy a ruis, karin blakemore, abimbola aina-mumuney, valerie jones. gynecology and obstetrics, johns hopkins hospital, baltimore, md, usa. objective to determine if gestational age plays a role in severity of pain associated with uterine contractions. study design self-reported pain from women in labor, on a scale of - , is assessed and recorded in an obstetrical database by nurses at regular intervals at two hospitals within our medical system and was retrospectively reviewed from - . pain at various dilatations ( - cm) of women who were laboring and then delivered at - weeks' gestation was compared to women in labor at term. maternal demographics of age, race, education level, bmi, presence of support person, request for analgesia at any point in labor, and epidural placement were abstracted from maternal records. categorical data were analyzed using chi square or fisher exact test; continuous variables using student t-test. results laboring patients between - weeks gestation and at term were identified. term and preterm patients did not differ with respect to maternal demographics. pain reported by preterm patients was significantly less at - cm dilatation ( . vs. . ‚ p= . ) and significantly greater at - cm ( . vs. . ‚ p= . ). pain reported at - cm and - cm was not statistically different between the groups. of note, fewer of the preterm patients received an epidural despite the request for analgesia. conclusion preterm laboring patients between - weeks gestation may perceive less pain at - cm dilatation compared to term laboring patients; however, their pain perception at advanced dilatation was comparable to those at term despite the fact that they were less likely to have an epidural in place at the time of pain evaluation. in light of these findings, the widely accepted etiology of labor pain as the result of cervical dilatation may need to be re-examined. also, factoring in the patient's discomfort level into the evaluation for onset of early active labor may not be valid in the preterm patient. background and objective: asthma is a risk factor for adverse pregnancy outcome. this has been attributed to the effect of allergy in pregnancy. mast cell mediators can induce uterine contractility. the objective of the study was to test the hypothesis that pregnant women with asthma have different uterine electrical signals from those generated by normal pregnant women. materials and methods: uterine electromyography (emg) recordings from gestational age matched pregnant women at term were analyzed. the cases consisted of patients with asthma and the controls, those women without asthma. emg was recorded for approximately minutes from surface electrodes placed upon the maternal abdomen, with the electrical signals filtered in the uterine-specific range of . to . hz to remove noise components. the recordings were analyzed in their entirety first by power spectrum analysis and then by lyapunov analysis. the power-spectrum-largest-peak frequency and the largest lyapunov exponent were calculated for each patient, and then the mean and standard deviation of these parameters was compared using student's t-test. results: patients with asthma had significant lower power spectrum frequency and higher lyapunov exponent than women without asthma. see ) the power spectrum frequency and the lyapunov exponent used to analyze uterine emg signals were different in women with and without asthma; ) the results suggest that uterine electrical activity is altered in women affected by asthma; ) these findings may explain the observed increased frequency of preterm birth in women with asthma. further studies are required to dissect the mechanisms. fetal microchimeric cells trafficked into the maternal circulation persist in blood and tissues for years after pregnancy. increasing data suggest that microchimerism occurs after every pregnancy, but the biological role of fetal microchimerism or the cell types involved is unclear. while persistent fetal cells were initially implicated in autoimmune disease, animal studies suggest these fetal cells play a broader role in response to tissue injury. methods: appendix specimens were acquired from women undergoing appendicectomy during pregnancy. detailed reproductive histories were obtained. fluorescence in situ hybridisation (fish) with two different probes allowed investigation of the presence of male presumed-fetal cells and nested pcr amplification of sry gene confirmed male dna in the appendix. immunostaining was used to determine the fetal cell phenotype. results: male cells were identified in appendix tissues from women with known male pregnancies (n= ) and also from a woman with no sons and a previous miscarriage of undetermined gender (n= ). no male cells were observed in the control (n= ), a woman with daughters. male cells of presumed fetal origin were evenly distributed in the muscle, mucosa and submucosa layers of the appendix. morphology and co-localisation analysis suggested the identified male cells had differentiated primarily into muscle cells or lymphocytes. combined immunostaining and y-fish demonstrated male desmin+ muscle cells and cd + and cd + lymphocytes. finally, the presence of male dna in the appendix specimens was confirmed by nested pcr. male cells of presumed fetal origin were identified in the appendix of pregnant women. microchimerism frequency varied according to the reproductive history and the degree of inflammation. microchimerism rates were higher in the appendix from women with current male pregnancies than in those with previous male pregnancies and were higher in those with histological acute inflammation, when compared to milder cases. male microchimeric cells identified were of haematopoietic and mesenchymal origin. this study suggests that fetal cells are present in sites of tissue injury and may participate in tissue repair during pregnancy. collagen i and collagen-binding integrins mrna expression in rat cervix during gestation. huiling ji, tanya l dailey, vit long, edward ks chien. obstetrics and gynecology, maternal fetal medicine, women and infants' hospital of rhode island, providence, ri, usa. objective cervical remodeling is associated with cell proliferation and extracellular matrix (ecm) remodeling. integrins are a family of multifunctional cell adhesion receptors which mediate ecm-cell interactions including binding collagen. of the integrin (i) heterodimers, , , and are primary receptors for collagen. the predominant cervical collagen is collagen type . the purpose of this study is to investigate collagen and integrin expression in the pregnant rat cervix. the cervix was harvested from timed pregnant sprague-dawley rats. non pregnant (np)and timed pregnant day , , , , and animals were euthanized using a protocol approved by the iacuc. four animals were sacrificed on each day of gestation. quantitative rt-pcr was used to evaluate mrna expression and normalized to -actin. a standard curve was generated from a single sample. data was analyzed using anova and multiple comparisons testing. collagen i mrna expression increased through mid-gestation but decreased to np levels on day . a similar pattern was seen with the integrin beta subunit. the pattern of integrin alpha subunit expression was different for each subunit during pregnancy. the changes in collagen, integrin alpha , , and integrin beta were found to be statistically significant. (figure) . the pattern of collagen binding integrin alpha subunit expression during the rat gestation appears to vary independently of each other. the expression of the subunit paralleled col expression. collagen binding integrins may play independent roles in signaling and biomechanics during gestation. funding: women infants' hospital research fund; phs nih-ncrr p rr p rr . background. the effects of chemotherapy on human fetal development are poorly studied, mainly due to the lack of adequate animal models in which placentation and embryological development resembles humans and pharmacokinetic studies, prenatal sonography as well as histological studies can be performed. aim of the study. to test the baboon as model for studying pharmacokinetics and fetal effects of chemotherapy administered during pregnancy. methods. experiments were performed in pregnant baboons at a mean gestational age of (+/- ) days. detailed ultrasound examination (biometry, doppler, screening) was performed days before, at and day after the drug administration. the administration of different schemes of chemotherapy and the fetal samplings occurred under endotracheal anaesthesia. maternal blood samplings, as well as percutaneous ultrasound guided fetal blood ( ml) and amniotic fluid ( ml) samplings were performed at least once immediately after drug administration. in case of fetal demise, the fetus underwent detailed macro-and microscopic examination. in the other animals mother and fetus were euthanized h after the experiments, with collection of blood, amniotic fluid and tissues for further analysis. results. all mothers survived the experiments, one mother developed paralitic ileus. none of the fetuses died during the acute phase of the experiment but / ( %) fetuses died within h following the experiment. none of the animals showed significant clinical or histological signs of infection or anaemia. a total of ultrasound examinations were performed in fetuses, allowing the creation of sonographic growth charts in this model. at the time of drug administration the mean maternal weight was . kg (range . - . ) and the estimated fetal weight was gr (range - ). sixteen out of cordocenteses ( %) and all amniocenteses ( %) were successful in obtaining the required samples for analysis. conclusion. the pregnant baboon can be used as a model for studies on pharmacokinetics and fetal effects of chemotherapy administered during gestation. prenatal ultrasound is comparable to the human situation, and amniocentesis and cordocentesis can be performed with a high success rate and short term survival. therapy to prevent preterm birth is limited". moreover, -hydroxyprogesterone ( ohp) caproate is a category d drug according to the fda (evidence of fetal harm). p is produced in the adrenal glands, gonads and brain. after weeks gestation p is secreted from the placenta independently to the mother and the fetus. p is the precursor of aldosterone and after conversion to ohp, of cortisol and androstenedione. baboons have similar reproductive system and development to humans and are used to study mechanisms of labor and prevention of preterm labor. currently, there are no normative data on the concentration of p, ohp, cortisol ©, estradiol (e ) or inhibin (i) throughout their pregnancy. therefore, a doseresponse or the teratogenic effects of p or ohp caproate cannot be studied in a controlled manner in this animal model. the aim of this study was to generate reference values for these hormones during baboon gestation and early postpartum life. material and methods: hormonal quantitative measurements were performed (immulite analyzer) results: the mean concentrations of these hormones are depicted in figure (maternal serum, from conception to term days) and figure (newborn serum) conclusion: this study provides reference values for hormones quantified during the baboon gestation and early postpartum life. this may be useful for future research concerning the effects of p and ohp administration during pregnancy. ( )non-pregnant women eligible for ivf treatment and ( )pregnant women receiving prenatal care. the pre- / samples were drawn in the year prior to / / ; the post- / samples were drawn within months after. the pre- / and post- / normal pregnant samples were drawn at + weeks' gestation. the non-pregnant samples were from ivf candidates with serum e levels< newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced pg/ml and fsh serum levels< . miu/ml. the samples were assayed for acth and cortisol by a commercially available immulite™ system. hsp and hsp were measured by commercially available elisa. these results were compared in the patient populations before and after / . t-tests were used for analysis. statistical significance was set at p< . . results: in the pregnant subjects, there were no statistical differences in the mean levels of acth, cortisol, and hsp pre-and post- / (table ) . only serum hsp levels were significantly increased in the pregnant women post- / .* mean serum acth, cortisol, hsp , and hsp levels were all significantly different in non-pregnant subjects pre-and post- / . conclusions: except for hsp , serum markers for stress were unchanged in pregnant subjects after the stress of / / in comparison to non-pregnant women whose serum hsp , hsp , cortisol, and acth levels were significantly different. the lack of change in serum markers of stress in pregnant women suggests that the hormonal milieu of pregnancy may buffer against acute environmental stressors. objective: in human pregnancy, maternal body composition provides an indicator of maternal nutritional status and metabolic capacity. previously, we reported that ß hsd- activity was significantly reduced in term placentas from thin women and women with a smaller mid-upper arm circumference before conception. this suggests that these fetuses may have been exposed to inappropriate levels of maternal cortisol, which is a mediator of gestation length. in this study, we hypothesize that the duration of gestation will be shorter in women who tend to be thin and have a lower mid-upper arm circumference prior to conception. methods: within the longitudinal, population-based southampton women's survey (sws), analyses were performed on women whose estimated date of conception was set using an algorithm that combined menstrual and early ultrasound scan data and who had a spontaneous onset of labour and delivered after weeks of gestation. linear regression was used to examine the relationships between maternal body composition and the duration of gestation. results: within the women surveyed, lower maternal body mass index, mid-upper arm circumference and arm muscle area before conception were all associated with shorter duration of gestation at delivery (r= . , p= . ; r= . , p= . ; and r= . , p= . , respectively). a lower subscapular skinfold thickness, sum of skinfolds and height were also associated with shorter duration of gestation (r= . , p= . ; r= . , p= . and r= . , p= . , respectively). mother's age, own birthweight and ratio of subscapular/triceps skinfold thickness were not related to the duration of gestation. conclusions: in this study, we found that thinner women with a lower midupper arm circumference and arm muscle area tended to have a shorter duration of gestation. our findings of shorter gestation length in thinner women are in keeping with our previous observation of lower ß hsd- activity in term placentas from thinner women. we conclude that metabolic capacity prior to conception could influence duration of gestation through mechanisms that include alteration in placental metabolism and fetal cortisol exposure. fecal incontinence (fi) is a debilitating condition affecting - % of the us. our prior studies found that % of women report new onset fi after childbirth. the goal of our study was to examine the impact of fi on postpartum quality of life (qol). women reporting fi on a statewide survey who agreed to participate in a -year study of qol were included in the analysis. women were considered to have fi based upon the nih definition of fi. the quality of life survey was based upon the uebersax incontinence impact questionnaire and was administered every months for years. qol in women with fi was examined using chi square, and impact of severe fi (stool incontinence) was determined by multivariate logistic regression. results women with fi were surveyed and of those, ( %) returned at least survey during the study period, completed all survey questions, and were included in the final analysis. among women with fi, % felt frustrated due to fi, % reported fi impacted their emotional health, . % reported fi impacted child-caring abilities, and . % reported a negative impact on social activities. qol was similar across survey periods. one out of three women with fi reported severe symptoms (incontinence of stool). women with severe symptoms were - times more likely to report negative impacts on qol compared to milder (e.g. flatus) fi after adjusting for age, parity and urinary incontinence. . % felt their stool was stored elsewhere before bowel movements, . % reported using digital defecation and more than half ( %) reported symptoms of urinary leakage. despite the substantial impact on postpartum quality of life, few women sought medical help with only % of women at months, . % at year, and . % at years ever reporting their symptoms to a medical provider. postpartum women report that fecal incontinence has a substantial negative impact on their qol after delivery including their emotional health and ability to care for their newborn. despite this profound impact, few women will discuss fi with medical providers. these data suggests there may be a benefit for providers to inquire about fi at postpartum visits. objective: the objective of this study was to determine what characteristics contribute to racial/ethnic differences in breastfeeding rates. study design: a retrospective cohort study of all women who delivered a viable infant (n= , ) was conducted. the primary outcome was breastfeeding upon discharge of the hospital. we first examined the association between race/ ethnicity and breastfeeding. next, we conducted stratified analyses examining a variety of predictors of breastfeeding within the racial/ethnic groups including maternal demographics, obstetric interventions, and perinatal complications. results: we found that both asians (or . , % ci . - . ) and blacks ( . , % ci . - . ) had statistically significant lower rates of breastfeeding, while latinas (or . , % ci . - . ) showed a trend towards higher rates of breastfeeding. in latinas, we found that rd and th degree lacerations were significantly associated with lower rates of breastfeeding, but were not predictive of breastfeeding in the other racial/ethnic groups. epidural use was predictive of lower rates of breastfeeding in caucasians and blacks, but was not predictive in latinos and asians. some of the other predictors which differed between the racial/ethnic groups were obesity and induction of labor (table ) . conclusion: race/ethnicity is significantly associated with breastfeeding, with blacks and asians having the lowest rates of breastfeeding at discharge. a variety of other factors are associated with breastfeeding and interestingly, their effect appears to differ between the racial/ethnic groups. future studies might elucidate the sociocultural and biomedical reasons that explain the differences. these results help focus our efforts during the peripartum period to advocate for mothers who have factors that might impede breast feeding. epidemiology, erasmus medical centre, rotterdam, netherlands; pediatrics, erasmus medical centre, rotterdam, netherlands; public health, erasmus medical centre, rotterdam, netherlands; clinical genetics, erasmus medical centre, rotterdam, netherlands. background recommendations on folic acid use to prevent neural tube defects are launched in several countries. however, the adequate use of folic acid supplements during the periconception period seems to be low. objective to assess the prevalence of adequate folic acid use, defined as the preconception start of supplements, and to identify its determinants in a multi-ethnic population. design the study was embedded in the generation r study in rotterdam, the netherlands, a population-based prospective cohort study from early pregnancy onwards. methods from all women in the cohort who delivered between april and january information on folic acid use and potential determinants was obtained by questionnaires and physical examination. logistic regression models were used to identify determinants of periconception folic acid use. results. data from , pregnant women were available. of all women % adequately used folic acid supplements. the most important risk factors for inadequate use were unplanned pregnancy (or . , p< . ), nonwestern ethnicity (or . , ci . - . , p < . ) and a low educational level (or . , ci . - . , p< . ). other risk factors were single marital status, smoking, multiparity (all p< . ) and alcohol use (p< . ). prior spontaneous abortion was associated with increased adequate folic acid use (p< . ). conclusion adequate periconceptional folic acid use is low. improved preconception care and public health education programs are necessary to improve the uptake of folic acid. although methamphetamine (ma) use has been front and center of the united states drug control policy since , little attention has been given to ma use in pregnancy, despite the fact that pregnant admissions to drug treatment for ma have been rising sharply. to determine trends in the prevalence of ma treatment admissions during pregnancy, we undertook a secondary analysis of the treatment episode data set (teds), an administrative data set that captures at least % of all known treatment admissions in the us. in particular, we investigated risk factors for ma use and how these characteristics have changed over time. demographic, geographic and substance use data were collected for the , pregnant admissions captured between and . logistic regression models were constructed by year. confounding was assessed via backwards elimination with a change-in-estimate criteria of . considered substantial. trend results were reported as adjusted proportions and represented graphically. overall ma prevalence, reported as the primary drug of use upon admission, rose from . % in to . % in . although white women had times the odds of using ma in (adjusted or ( %ci) . [ . , . ]), this had dropped to . [ . , . ] by , mostly due to an increase in latina ma use in pregnancy. pregnant women admitted for ma had fewer prior treatment admissions, were more likely to be unemployed, and less likely to use alcohol or marijuana. we found great geographic variability in use. ma was more common in the pacific region and least common in new england. there was little change in regional variation over the study time period. less is known about the perinatal effects of ma compared with other substances, yet it is the primary drug of choice for pregnant women admitted into treatment. as pregnant women occupy a unique place in drug treatment, analysis of national trend data is essential to guide both policy and research. background: epidemiologists have grouped the multiple disorders that lead to extremely preterm delivery in a variety of ways. we sought to identify characteristics that would support the combining or dividing of the disorders that lead to preterm delivery. methods: we enrolled , women who delivered a live born singleton infant between and completed weeks gestation at tertiary centers in the united states. each delivery was classified according to the complication that prompted presentation: preterm labor, preterm premature rupture of newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced fetal membranes (pprom), preeclampsia, placental abruption, cervical incompetence, and fetal indication/intrauterine growth restriction (iugr). we compared these entities on the frequency of characteristics identified by standardized interview, chart review, histological examination of the placenta, and culture of placenta parenchyma. results: the percents of women who presented with each antenatal complication were: preterm labor ( ), pprom ( ), preeclampsia ( ), placental abruption ( ), cervical insufficiency ( ), and fetal indication/iugr ( ). after considering antecedents and correlates of the processes leading to preterm delivery, we observed two overarching epidemiologic patterns. the first pattern, characterized by recovery of organisms from the placenta and by histologic chorioamnionitis, tended to be associated with preterm labor, pprom, placental abruption, and cervical insufficiency. the second pattern, characterized by a paucity of organisms and inflammation and the presence of histologic features of dysfunctional placentation, tended to be associated with preeclampsia and fetal indications/iugr. conclusions: disorders leading to preterm delivery can be categorized broadly into two groups: those associated with intrauterine inflammation and those with aberrations of placentation. predictors of compliance with tuberculosis screening in pregnancy. nadav schwartz, sarah wagner, sean keeler, may tam, julian mierlak, , aaron caughey. obstetrics and gynecology, nyu school of medicine, new york, ny; obstetrics and gynecology, ucsf, san francisco, ca; obstetrics and gynecology, gouverneur health care services, new york, ny. objective: poor compliance with tuberculosis screening and treatment is a major obstacle to the containment of this disease. we sought to identify predictors of ppd and cxr compliance during pregnancy. methods: a retrospective cohort study at a single institution which serves a largely immigrant population in nyc, from november , through june , . data on maternal age, ethnicity, country of origin, level of education, ppd and cxr status were collected. results: of the , pregnancies, asian and hispanic race/ethnicities accounted for . % and . % of the population, respectively, with . % being us-born. the mean age was . years and the overall ppd+ rate was . %. there was % non-compliance with ppd testing, and % of ppd+ patients were non-compliant with their chest x-rays. asian women were more likely to be ppd-compliant than hispanic or caucasian women. ppd+ asian women were also more likely to be compliant with cxr. us-born women were significantly less likely to be compliant with their ppd or with their cxr. women > years old were less likely to be compliant with their cxr, while women with an elementary school education or less were more likely to be compliant. (see table for odds ratios and %ci) conclusions: age, education, immigrant status and other cultural and ethnic factors appear to play a role in compliance with tuberculosis screening. further elucidation of these effects may help clinicians target at-risk subpopulations and improve overall compliance, working towards better control of this disease. background: in the us, differential outcomes in cervical cancer among medically underserved women are linked to multiple barriers impacting loss to follow-up and failure or delay in diagnostic resolution. prior studies have found risk factors for acquisition of hpv and for inability to obtain a pap smear. no study has explored health literacy and physician communication as a key factor. methods: this research represents the formative phase of a randomized controlled trial of african american (aa) and hispanic women aimed to improve communication and abnormal pap smear follow-up in chicago. semi-structured interviews and focus groups were conducted face to face in spanish or english. each interview lasted minutes, and each focus group lasted - minutes. we recruited patients ( hispanic, aa) and providers from a purposive sample representative of two large clinics that serve low-income women. results: all interviews were transcribed by two investigators. each interview was coded by two investigators separately and then a third investigator reviewed the transcripts and coding to achieve triangulation. codes for these themes were developed and the responses were tabulated using the coding scheme. atlas ti was utilized to analyze all qualitative data. from these data, we uncovered provider-patient challenges in cancer communication including: the providers' trade off between medical accuracy and/or literacy when communicating with their patients. for the patients, the word cancer was important to hear since they wanted the truth and needed to hear this word in order to encourage them to respond more quickly. however, many providers believed that the word cancer was too "scary" and to "extreme" of a word that may communicate too much exaggerated information. both the hispanic and aa patients did not seem to differ in their responses. conclusion: results exemplify not only the importance of health literacy and patient provider communication, but demonstrate the wealth of information gained from qualitative research-a method of data collection seldom utilized in ob/gyn investigations. funding: women's reproductive health research award: hd - ; r ca (spring). population. kristine beckers, isabelle guelinckx, greet vansant, roland devlieger. obstetrics and gynaecology, university hospital gasthuisberg, leuven, belgium; clinical nutrition, katholic university leuven, leuven, belgium. objective: to generate reference charts for weight gain during pregnancy for the different bmi-categories (underweight, normal weight, overweight, obesity), based on recent data in a homogeneous caucasian population. methods: in a retrospective study at the department of obstetrics of the leuven university hospital (belgium), weight gain and pre-pregnancy bmi were determined in belgian pregnant women with accurately dateable, uncomplicated singleton pregnancies. centile curves for the different bmicategories were constructed using of the linear mixed model, one set of charts based on the absolute weight gain, another set based on the relative (expressed as percentage) weight gain. the effect of parity on weight gain was examined. results: overall mean weight gain was . ± . kg ( . ± . lbs). mean weight gain was . ± . kg ( . ± . lbs) in the underweight population, . ± . kg ( . ± . lbs) in the population with normal weight, . ± . kg ( . ± . lbs) in the overweight population and . ± . kg ( . ± . lbs) in the obese population. weight gain (pattern and amount) of the underweight and normal weight patients differed significantly of the overweight and obese patients. parity had a statistical, but no clinical significant influence on amount and evolution of weight gain. conclusion: by using strict inclusion criteria, bmi-category-specific reference charts were generated representing the optimal gestational weight gain, rather than the mean weight gain. this enables the weight charts to be used as a clinical tool during the counselling of pregnant women. further studies are required to assess the effectiveness of this clinical tool. female fshr(-/-) mice are sterile, because of a block in folliculogenesis at the primary follicle stage, show decrease in e and elevated fsh. this animal model is an appropriate model for studying hypergonadotropic ovarian dysgenesis and infertility, caused by c t mutation in fshr gene. objective: to investigate the effects of bmt on serum hormonal levels, follicular maturation and fertility of fshr(-/-) mice. methods: fshr (-/-) mice at - weeks of age were randomized into treated versus control groups.bm from syngenic female donor was injected into the tails of recipients. control group received vehicle alone. vaginal smears were collected, body weight was measured daily. sample animals were sacrificed at , , , , and weeks post bmt. all organs were weighted and examined by h e. fsh, e , and p were measured before and after treatment. for donor cell tracking, dna was extracted from various organs. specific primer sets were designed for normal and mutant hfshr gene. pcr amplification was done and pcr products were analyzed in % agarose gel. results: total body weight significantly increased in treated animals(p< . ). significant increase in both the total number of follicles, and the collective diameter of the follicles in treated animals observed(p< . and p< . ). six out of treated animals showed estrogenic changes in daily vaginal smear. e level increased . - . times and fsh level dropped to % in treated animals. normal (donor allele) fshr gene was amplifiable in out of recipient mice, and was detected only in the ovaries and uterus but not in any other tested organs.control group did not show any changes in vaginal smear, hormonal level, and normal fshr allele. conclusion: intravenously injected syngeneic bone marrow cells were able to home to the ovaries of female fshr (-/-) mice and restore folliculogenesis and resume steroid hormone production. potential mechanisms for these observations will be discussed. conclusion: neural stem cells (nscs) give rise to progenitors, which expand by rapid proliferation until cell cycle arrest followed by differentiation along one of cns cell lineages: neurons, astrocytes and oligodendrocytes. increased differentiation of neuronal cells with ins and even more with leptin suggests that iugr-associated reduction of these growth factors may contribute to impaired hypothalamic pathway development. objective: to develop a technology of modulating hucb in order to achieve neuronal cells for future potential replacement of damaged neuronal tissue. design: we developed a two-dimensional ( d) tissue culture technology for isolation and differentiation of collagen-adherent hucb neural progenitors (hucbnps). we further used the extracellular matrix protein collagen, organized in a three-dimensional ( d) gel, supplemented with neuronal conditioning medium and nerve growth factor (ngf), to facilitate ex-vivo long term neuronal differentiation of hucbnps. we developed a stable green fluorescence protein (gfp)-pc cell model, to be used as a positive control for monitoring neuronal outgrowth for proper evaluation of the hucbnps growth in the d scaffold, mimicking a neural tissue organization. results: our experimental data indicate that d collagen environment is neuronal biocompatible, supporting attachment, long-term survival, proliferation and differentiation of both hucbnps and gfp-pc cells. conclusions: improvement of the d technology with cultures of hucbnps that is in progress in our laboratory might be the first step in validating the concept for the feasibility of generating a neuronal d tissue construct for future potential treatment of neurodegenerative disorders. piane, justin tsai, gnanaratnam giritharan, emin maltepe, paolo rinaudo. reproductive sciences, university of california san francisco, san francisco, ca, usa. objective: ivf embryos are often used to generate stem cells. however, it is unknown if stem cell lines originated from in vitro generated embryos are different from stem cell originated from in vivo embryos. trophoblastic cells, due to their external position within the embryo, are most susceptible to environmental factors encountered in vitro. furthermore, our previous data showed that trophoblast transport functions may be impaired after culture in vitro and that the number of trophoblastic cells is reduced in the embryo after ivf. in this study, we assess the differentiation characteristics of ts cell lines obtained from in vivo and in vitro fertilized embryos. methods: oocytes were isolated from superovulated c bl/ j female mice and in vitro fertilized with sperm from male c bl/ j mice. the resulting late-cavitating blastocysts were harvested. in vivo controls were obtained by flushing the blastocysts from the uteri of superovulated pregnant mice days post hcg. ts cells from the two groups were allowed to develop and maintained in vitro in the presence of fgf and heparin, using a feeder layer of human placental fibroblasts. ts cells were then allowed to differentiate without fgf and heparin, fixed on day , stained with -tubulin and zo- antibodies and then observed under fluorescence microscopy. three ts cell lines per group were analyzed and their differentiative capacity was evaluated using morphological criteria. results: in vivo and ivf derived ts exhibit a similar differentiation pattern. in particular, the number and timing of trophoblast giant cells and spongiotrophoblasts derivation is similar in the two groups. there are no obvious abnormalities in the immunologic staining morphology of the different cell lines at different time points. conclusion: there are no apparent morphological alterations in ts cells lines derived from ivf embryos as compared to in vivo embryos. this finding in an animal model increases our confidence in the reliability of human stem cells derived from ivf. as a further investigation, markers of trophoblast giant cells, spongiotrophoblast, syncytiotrophoblast and chorionic trophoblast cells will be examined and compared in the two different groups by northern blot hybridization. genomewide high density snp-cgh reveals several new deletion copy number variants on the x chromosome in pof patients. erik ah knauff, cisca wijmenga, ruben van 't slot, lude franke, bart cjm fauser. reproduction gynecology, university medical centre, utrecht, netherlands; complex genetics group, university medical centre, utrecht, netherlands. introduction: around % of women have a post-menopausal hormonal profile before age , referred to as premature ovarian failure (pof). pof could act as a genetic model for accelerated follicle loss and may render useful information about the polygenetic background of major individual differences in menopausal age. macrodeletions on the x chromosome are associated with pof but karyotyping has a maximal resolution of mb. when using genotyping whole genome arrays it is now possible to detect submicroscopic deletions and duplications (copy number variants (cnv)) up to kb effective resolution. we have screened for microdeletions on the x chromosome in a well-phenotyped cohort of pof patients. methods: our study included caucasian, ,xx patients with spontaneous secondary amenorrhea before age , fsh > iu/l and absence of (low) x/ xx mosaicism and fmr premutations. dna analysis was performed using illumina k cnv arrays containing , probes. , probes were located on the x chromosome ( probe for every kb), of which , probes were specifically designed to detect known cnvs. ten samples with call rates < % were removed from the analysis and one sample gave inconsistent x probe intensities. we screened for deletions ( kb) using an algorithm detecting at least five consecutive probes with intensities (logr > - . ) below the mean probe intensity. we identified a total of x chromosomal microdeletions, divided in loci and varying between - kb in size. of the samples showed at least one deletion on the x chromosome. % of the identified deletions had already been recorded in the database of genomic variants. in the newly identified newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced loci, we found coding regions containing genes. eight of these are established or potential pof candidate genes, including five that are clustered on the terminal xq critical pof region. conclusions: we observed abundant variation in the cnv regions, a proof-of-principle for this specially designed cnv-chip. snp comparative genomic hybridization revealed possible new deletions specific to pof on the x chromosome. data on duplications, validation and whole genome cnv analysis, compared to a control cohort, will become available soon and will be presented at the sgi annual scientific meeting. unexplained intrauterine fetal death (iufd) is associated with long qt syndrome. irene cetin, patrizio antonazzo, sabrina cozzolino, stefania calabrese, lia crotti, francesca ferrari, roberto insolia, fabio facchinetti, peter j schwartz. dept. of obst/gynecol, univ. of milano, milano, italy; molec cardiol lab, policlinico san matteo, pavia, italy; mother-infant dept., univ. of modena/reggio emilia, modena, italy. introduction: in developed countries, nearly in every pregnancies ends in late fetal loss. many iufds can be attributed to maternal disorders, fetal pathology, placental pathology and fewer to complications of labor and delivery. however, - % of cases remain unexplained. we recently demonstrated that % of sudden infant death syndrome (sids) cases carry functionally relevant genetic variants in long qt syndrome (lqts) genes. aim of the study was to analyze whether lqts genes are associated with iufd. materials and methods: patients with iufd were enrolled in two years, as part of an italian multicentre study. iufd was defined as fetal death at weeks or more of gestation according to the definition of late fetal death of the who. out of cases were classified as "unexplained stillbirths" according to the wigglesworth and aberdeen classifications. at birth placental and/cord samples were collected and dna extracted. the main lqts genes kcnq , kcnh , scn a, kcne and kcne were screened through dhplc and sequence analysis. any amino-acid substitution identified in the samples was checked for in a control population of caucasian women with uneventful pregnancies. preliminary data on the first cases (gestational age of death: - weeks) are reported. results: a total of missense mutations were identified in of stillbirths ( %), two on scn a and one on kcnh . the two mutations on scn a (v l; p a) were observed in two iufd occurred at term; they had been previously associated to sids and shown to increase the late sodium current. the mutation on kcnh is a novel genetic variant absent in reference alleles, never described in any control populations; this mutations was present in a case of iufd diagnosed at weeks of gestation. we are currently performing the electrophysiological cellular studies to define its functional effect. conclusions: these preliminary data indicate that a potentially significant number of currently unexplained iufd might be caused by ion channel diseases such as lqts. the potential prevention of sids or iufd recurrence and the identification of other affected family members could have important implications for the affected families. alternative splicing of epab is regulated by exonic splicing enhancers. e seli, a yaba, o guzeloglu-kayisli, md lalioti. ob gyn, yale u., new haven, ct, usa. introduction: alternative splicing is an important mechanism by which the genome gives rise to the observed diversity of proteins. embryonic poly(a) binding protein (epab), expressed exclusively in oocytes and early embryos, mediates translation of maternal mrnas. we identified an alternatively spliced form of epab lacking exon (cex del), and investigated its regulation as a model for alternative splicing in early development. specifically, we evaluated: imprinting (expression from maternal or paternal allele only); rna editing (post-transcriptional single nucleotide substitution); and exonic splicing enhancers (eses, exonic sites that bind splicing proteins). methods: a single nucleotide polymorphism (snp) detected in exon (c a/g) served as a marker for the parental origin of the spliced form. snp genotyping was performed by pcr amplification of exon followed by restriction enzyme digestion. to evaluate imprinting, we characterized heterozygous mice (a/g) that inherited the snp from either the mother or the father. to test for rna-editing and exonic enhancer contribution we tested mice homozygous for the exon snp (a/a or g/g). efficiency of alternative splicing in different genetic backgrounds was tested using real-time pcr normalized to actin expression. results: in mice heterozygous (a/g) for the exon snp, cex del was only expressed from the (a) allele. however, this was independent of the parental origin of the allele, ruling out imprinting. in mice homozygous (g/g) for the exon snp, the cex del variant also contained (g). therefore, rna editing did not occur. further sequence analysis led to the identification of an additional snp in exon (c g/c) that co-segregated with the exon snp. presence of c g led to the formation of an ese that binds splicing regulatory protein srp , leading to efficient exclusion of exon . real time pcr revealed a five-fold increase in the expression of the cex del alternative splicing variant in animals carrying the enhancer (homozygous g/g) for the exon snp compared to those that did not (homozygous c/c) at the same locus (p < . ). conclusions: in this study, we found that eses mediate the alternative splicing of oocyte-specific transcripts. our findings suggest that single nucleotide polymorphisms may alter the ratio between alternative splicing variants of oocyte-specific proteins. the role that these subtle differences play in determining individual reproductive outcome remains to be identified. epigenetics and chromosomal abnormalities in human oocytes. ilse van den berg, , joop se laven, robert jan galjaard, j hikke van doorninck. , obstetrics gynaecology; clinical genetics, erasmus medical center, rotterdam, netherlands. humans have a low fertility rate compared to other mammalian species. moreover their fertility declines with increasing age. both phenomena are largely due to an increasing number of chromosomal abnormalities in human oocytes during life. identifying factors that cause aneuploidy in oocytes may offer possibilities to diminish these abnormalities in vitro or in vivo. chromosomal segregation errors can result from aberrant recombination but little is known about epigenetic factors that may cause aneuploidy. epigenetic modifications such as histone acetylation and subsequent de-acetylation in oocytes are necessary for a correct progress through meiosis. if disturbed it may lead to aberrant segregation of chromosomes or chromatids due to decreased kinetochore function and imperfect spindle figures resulting in aneuploidy. mice oocyte data have shown a correlation of abnormal histone de-acetylation and aneuploidy and a correlation between age, remaining histone acetylation and aneuploidy (akiyama et al., ) . our research focused on human oocytes and investigated whether a similar relationship between histone acetylation, age and aneuploidy is present. human oocytes were surplus from standard ivf/icsi treatments (ic+). human oocytes showed immunostaining for histone , lysine acetylation (h k ) at the germinal vesicle stage and complete deacetylation at the mii stage in % of the oocytes, while % keep high levels of h k acetylation. treatment of germinal vesicle oocytes with a histon deacetylase inhibitor (tsa) during in vitro maturation until mii stage resulted in high levels of acetylation. remaining acetylation in tsa treated oocytes was correlated significantly with abnormal spindle figures (tubulin staining), a hallmark of developing aneuploidy. similarly, % of oocytes with naturally remaining h k acetylation showed abnormal spindle figures. in contrast % of the normal de-acetylated oocytes showed normal spindles (p= . ). this suggests that defective de-acetylation of h k in human oocytes leads to abnormal spindle figures and subsequent aneuploidy. advanced maternal age is associated with a reduction in de-acetylation during in vitro maturation and an increase in spindle abnormalities. these results may stimulate the development of assays for histone modifications as biomarkers to follow oocyte quality in in vitro maturation studies or in optimizing general ivf treatments. objective: racial disparity in preterm birth (ptb) is unexplained and genetic risk factors are suspected as a major cause. this large scale candidate gene study examines differences association of single nucleotide polymorphisms [snps] in caucasians (c) and african americans (aa) to help elucidate racial disparity in preterm birth (ptb) methods: in this case (preterm birth < weeks) control study (term birth > weeks) maternal and fetal dna from ( cases and controls) c and ( ptb and term) aa were collected. a high-throughput candidate gene association study was performed examining snps in genes of selected from hypothesized ptb pathways. single locus association analyses were performed separately on maternal and fetal samples. results: snps in genes associated with ptb (p< . ) were common between races with both maternal and fetal dna analyses. however, snps in genes in c and in aa in both maternal and fetal dna differed in its association with ptb. in c maternal dna, the single strongest association between ptb and snp was in plasminogen activator tissue (plat) gene (c- t; rs ) at both allelic (p = . x - ) and genotypic (p= . x - ) level with an odds ratio (or) of . ]. the single strongest effect in c fetal dna was observed in a snp in the interleukin- receptor antagonist gene (a g; rs ) for both allele (p= . ) and genotype (p= . x - ), or . ). in aa, the strongest associations were in interluekin- (c t-rs , allele p= . x - , genotype p= . x - ) in maternal dna with an or= . [ci . - . ] and in fetal dna interleukin- receptor b (a g; rs , allele p= . x - , genotype p= . x - ) with an or . ]. conclusion: large scale high throughput analyses of snps in candidate genes of ptb pathway reveals significant differences between races at both maternal and fetal levels. we found that the strongest single locus associations differed in the two races in both maternal and fetal dna samples. these findings support the hypothesis that underlying genetic predispositions may differ between these populations, perhaps partly explaining racial disparity in preterm birth. candidate gene association study indicates differential etiologies in gdm and in t dm. johann urschitz, tarik sultan, kenneth ward. obgyn, jabsom, university of hawaii, honolulu, hi, usa. objective: recently several genome-wide association studies have associated multiple single nucleotide polymorphisms (snps) in multiple genes with increased risk of type diabetes. as risk factors are similar between t dm and gestational diabetes (gdm), we investigated a possible association of those snps with gdm. study design: blood was collected from caucasian ( cases, controls) women who met coustan-carpenter criteria for gdm and non diabetic controls. dna was extracted and a candidate gene association study was performed (taqman, abi). results: chi contingency tests were used to analyze genotype and allele frequencies in controls and gdm affected pregnancies (see table below). none of the snps showed a significant association after bonferroni correction. conclusion: several polymorphisms which displayed highly significant associations with type diabetes are not associated with gestational diabetes. these results suggest that gdm is not a simple unmasking of a t dm predisposition by the metabolic demands of pregnancy; rather it appears that different biological mechanisms are responsible for the respective diseases. introduction: histone acetylation/deacetylation plays an important role in the regulation of gene expression. histone deacetylase inhibitors (hdaci), in general, maintain gene expression, although in some cases they cause repression of specific genes. in this context we have previously shown that the hdaci tsa suppresses activation of the pro-contractile gene cox- in human myometrial and amnion-derived cells [reproductive sciences, vol , no (supplement, # )]. we have also shown that expression profiles for hdacs ( - , ) differ significantly within upper and lower regions of the myometrium during pregnancy and in labour. objectives: since changes in both acetylation and phosphoryation status can influence the activity of individual hdacs we aimed to define whether there are any changes in the levels of acetylation and phosphoryation of myometrial hdac , and during pregnancy and labour. methods: protein homogenates prepared from non-pregnant, term and labouring myometrium were treated +/-shrimp alkaline phosphatase and subjected to sds-page and western immunoblotting (wb) using antibodies to hdac , and . the acetylation status of the hdacs was assessed by a co-immunoprecipitation assay using anti-hdac and anti-acetylated lysine antibodies. results: distinct patterns of acetylation were observed for the individual hdacs; hdac and appeared to be more acetylated in non-pregnant and labouring lower tissues when compared to pregnant myometrium. in contrast, hdac appeared to be slightly more acetylated in lower uterine samples from pregnant and labouring myometrium. in experiments to evaluate changes in phosphorylation status we observed that ) myometrial hdac appeared to less phosphorylated in both upper and lower labouring samples when compared to non-pregnant and pregnant samples; ) hdac appeared to be more phosphorylated in labouring samples than non-pregnant and pregnant myometrium; ) no changes in phosphorylation levels were observed for hdac using this test system. conclusions: the difference in hdac acetylation and phosphorylation levels in the human myometrium may indicate differential regulation of the activity of the hdacs within the distinct myometrial regions, perhaps leading to the alteration of their epigenetic effect on genes related to myometrial quiescence and contraction and the subsequent onset of both term and preterm labour. objective: native hawaiians and pacific islanders experience a higher perinatal morbidity secondary to the increased incidence and earlier onset of heart failure. this increased incidence is likely multi-factorial and includes co-morbidities and genetic factors. mutations in the human adrenergic receptor gene may play a role in the determination of heart function. mutations in the b -adrenergic receptor (adrb ) located on chromosome have been identified as a cause progressive cardiomyocyte loss leading to a dilated cardiomyopathy. the minor allele frequencies for most of these polymorphisms have been determined for caucasian, japanese, african-american and chinese as part of the hapmap project. the frequencies have not been determined in the pacific islander groups. this study helped determine the hawaiian allele frequency and determine the allele frequency in the presence of cardiac or other vascular co-morbidities such as hypertension, preeclampsia, and gestational diabetes. study design: real time pcr technology (taqman genotyping assays, applied biosystems) was used to screen maternal dna (n= ) from the phenotyping sample core of the pacific center for early human development (prcehd) for the rs single nucleotide polymorphisms (snps). genotype frequencies were also determined in % complete ethnic controls as determined by a four grandparent descent. results: chi square was used to analyze allele and genotype frequencies differences between controls and affected pregnancies. the results are summarized in the following tables. conclusion: the rs snp was not significantly associated with hypertension, preeclampsia or gdm in our overall hawaiian population. the genotype frequency in the % pacific islander group was significantly different from the caucasians (p= . ) and asians (p= . ) in our overall hawaiian population. angiotensin-converting enzyme and -adducin polymorphisms in preeclamptic mothers and fetuses. c mando, p antonazzo, s tabano, f colleoni, a martinelli, s calabrese, c benedetto, l marozio, f facchinetti, m miozzo, i cetin. inst. obstetrics and gynecology, fondaz. irccs policlinico mangiagalli regina elena, univ. milan, milan, italy; dept. biology and genetics, univ. milan, italy; dept.obstetrics and gynecology, univ. torino, italy; univ. modena and reggio emilia, modena, italy. background the genes encoding angiotensin-converting enzyme (ace) and -adducin (add ) share the potential of influencing blood pressure. previous studies demonstrated in humans the association of hypertension with the combined effect of both ace insertion/deletion (i/d) polymorphism, which leads to a different activity of the enzyme, and add g w non-sense single nucleotide polymorphism (snp). ace i-i genotype has been associated with low serum ace activity. controversial studies concerning the association of ace polymorphism with preeclampsia (pe) were reported and the possible combined effect of both ace i/d and add g w polymorphisms yet remains to be investigated. population association study we genotyped polymorphisms (ace i/d and add g w) in women: with pe ( / with severe pe) and controls. moreover, we investigated a subset of their fetuses: from severe pe, from mild pe and controls, in order to identify specific maternal and/or fetal genotypes conferring a higher risk to develop pe. we both evaluated the single and the combined effects of ace and add genotypes on mother-fetus couples and singularly on mothers and fetuses. ace i/d genotype was analyzed using a pcr method; an allelic discrimination approach was performed to detect the add snp. in mother-fetus genotype couples, neither ace nor add polymorphisms are associated with pe, nor are the combination of their genotypes separately in mothers and in fetuses. nevertheless in mothers with mild pe ace i-i genotype is significantly less frequent ( % vs %; p< . ) and ace i-d is significantly more frequent ( % vs %; p< . ) compared to controls. ace i allele frequency is not significantly different in mild pe compared to controls ( % vs %). in women with mild pe the i allele seems to move from i-i to i-d genotype. it leads to the increase in mild pe women of those genotypes with a higher ace activity conferring a higher susceptibility to develop pe. this association with mild pe and not with severe pe could be due to the contribution in the latter of many other genetic and environmental factors. introduction: maternal mrnas stored in the oocyte are critical for early development as transcription ceases upon oocyte maturation, and gene expression until zygotic genome activation (zga) is mediated by translation of maternal transcripts. cytoplasmic polyadenylation element binding protein (cpeb) plays a central role in this process by stabilizing maternal mrnas and regulating their timely activation. this function has been well characterized in xenpous, and a similar role in mammals is suspected as the cpeb knockout mouse displays infertility as a result of arrested oogenesis. in order to investigate if translational regulation of maternal transcripts by cpeb is maintained in mammals, we characterized the spatial and temporal expression of cpeb- in the mouse and human. methods: ten different somatic tissues, testes, and ovaries were tested by rt-pcr for the expression of cpeb mrna in mouse and human. cpeb mrna expression in mouse was also tested in prophase i (pi) and metaphase ii (mii) oocytes, -cell, -cell, -cell, -cell embryos and blastocysts. in human, pi and mii oocytes, -cell embryos and blastocysts were evaluated. sequencing of the pcr products was performed to confirm specific amplification of cpeb. amplification with actin primers provided a positive control and allowed semi-quantitative analysis. results and discussion: highest level of cpeb mrna expression was detected in ovaries and testis of both mouse and human. in addition, differential expression of cpeb in somatic tissues was also observed. among these, prominent expression was present in brain, where a role for cpeb in facilitating gene expression through cytoplasmic polyadenylation has been proposed. these data would suggest that translational control of mrna by cpeb may be a mechanism utilized by multiple somatic tissues to regulate gene expression. in the mouse, cpeb was expressed in pi and mii oocytes and -cell and -cell embryos, and became undetectable in -cell or more advanced embryos. human cpeb was expressed in pi and mii oocytes, but not in -cell embryos or blastocysts. zga occurs at late -cell stage and -to- -cell stage, in mouse and human, respectively. the tightly controlled temporal expression of cpeb prior to zga in both mouse and human is consistent with a conserved role for cpeb in the regulation of maternal mrna expression during early development in mammals. background embryonic stem cells (esc) express specific transcription factors that are indicative of their ability to maintain pluripotency. these factors are activated during preimplantation embryo development. the interplay between the transcription factors pou f (oct / ) and caudal-homeobox domain (cdx ) is thought to regulate inner cell mass (icm) and trophectoderm (te) differentiation; however, the mechanism of cell fate determination in mammalian embryos is poorly understood. genetic ablation of oct / in mice prevents icm development, while cdx knockout embryos fail to implant. esc deficient in nanog, a second key regulator of pluripotency, fail to maintain pluripotency and undergo differentiation. expression of nanog in primate embryos has not been investigated, therefore the localization of oct / , nanog and cdx was determined in rhesus macaque blastocysts. methods oocytes were collected from rhesus macaque females, fertilized and cultured in vitro to the blastocyst stage. embryos were collected hours post insemination, then fixed prior to incubation with primary antibodies directed against oct / , nanog and cdx . following incubation with cy conjugated secondary antibodies, nuclei were counterstained with dapi and embryos visualized using an olympus bx fluorescence microscope. results nanog protein was restricted to the icm of monkey blastocysts. unlike the mouse embryo, oct / protein was detected in both the icm and te, based on two different antibodies. the expression of cdx was localized specifically to the te. conclusions the ubiquitous pattern of oct / expression is consistent with observations in human, cow and pig embryos. significantly, lack of restricted oct / protein, and icm localization of nanog in primate blastocysts, suggests that nanog more specifically determines cell fate in primate embryos. these results contrast markedly with current mechanistic hypotheses, although other factors may lie upstream of nanog. importantly, this difference may underlie observations that regulatory mechanisms in esc differ between mice and primates. further investigations will focus on determining the onset of marker expression, and the upstream regulators of nanog activation. supported by nih grants r hd r rr , and the intramural research program of nichd. in vitro-conceived mice tend to be smaller at birth and throughout their life. luisa delle piane, annemarie donjacour, francesca di sebastiano, gnanaratnam giritharan, paolo rinaudo. obstetrics, gynecology and reproductive sciences, university of california san francisco, san francisco, ca, usa. objective: epidemiological evidence indicates that ivf is associated with an increased incidence of low birth weight. this phenomenon has not been studied in a mouse model; in addition, it is unknown if the resulting offspring show different growth pattern later in life. we therefore created an ivf mouse model to follow growth patterns till adulthood. methods: we generated experimental groups of b mice: one cohort of in vivo generated mice (in vivo group), one cohort of in vitro generated animals (ivf group) transferred to cd foster mothers and one cohort of animals fertilized in vivo and transferred to cd foster mothers (flushed blastocysts group, fb). the fb group was generated because our preliminary results showed that embryo transfer to b foster mothers was not successful. all pups were delivered at term, measured and weighed at birth and then weekly up to weeks. parametric tests (anova with bonferroni correction) were used as appropriate. a mixed model was used to compare growth curves. results: average litter size was . (in vivo), . (fb) and . (ivf). birth weight of male mice both ivf ( . mg) and fb ( . mg) was larger than male in vivo mice ( . mg) (p< . ). ivf mice tended to be smaller than fb mice in both sexes (p= . ). the bmi (body mass index) was not different among all the groups. male fb growth curves were different from in vivo mice (p< . ) and more importantly from ivf growth curves (p= . ) (fig. ) . conclusion: the method of conception and the maternal environment play a significant role in determining birth weight in this mouse model, emphasizing that the fb group is the best control for the effects of ivf in this model. these preliminary results confirmed for the first time an ivf effect on growth and interestingly the ivf males did not show a catch-up growth. the finding of smaller ivf mice when compared to fb is both noteworthy, because it confirms human data, and worrisome, because lower birth weight is associated with long term sequelae according to the barker hypothesis. the effect of betamethasone exposure in mid-gestation on renal sodium excretion in male sheep. lijun tang, luke carrey, nancy valego, philip deibel, james perrott, jorge figueroa, mark chappell, james c rose. obestetrics and gynecology, wake forest university, medical school, nc, usa; hypertension center, wake forest university, medical school, nc, usa. objective: whereas prenatal exposure of ovine fetuses to clinically relevant doses of glucocorticoids during the time of peak nephrogenesis results in a reduction in nephron number in adulthood, there is little information about its effect on sodium excretion. in the present study, we evaluated the effect of exposure to betamethasone on renal sodium excretion in adult male sheep. methods: we studied nineteen conscious adult rams at . years of age which were exposed to either vehicle or betamethasone at - days gestation. we implanted vascular and bladder catheters and then allowed the animals a - days recovery period prior to study. inulin and para-aminohippuric acid (pah) clearances were performed for estimating glomerular filtration rate (gfr) and renal plasma flow (rpf) respectively. following determination under basal conditions, an acute hypertonic sodium load was administrated intravenously by a continuous infusion of nacl ( . meq/kg/min at . ml/min) for minutes. urine was continuously collected for determination of na + excretion. results: basal gfr was decreased in steroid exposed adults ( . ± . ml/min/kg) compared with the vehicle animals ( . ± . ml/min/kg) (p= . ). rpf was similar in the vehicle and steroid exposed group ( . ± . ml/min/kg vs . ± . ml/min/kg). at basal conditions, na + excretion (u na v) was similar in vehicle and steroid exposed group ( . ± . μmol/h vs . ± . μmol/h). this similarity was also present after normalization by body weight ( . ± . μmol/h/kg vs . ± . μmol/h/kg). the vehicle group excreted . ± . % of the dose of na during the experiment while the betamethasone exposed group excreted only . ± . % (p< . ). gfr and prpf did not change during the experiments. these results suggest that prenatal exposure to glucocorticoids affects renal function in adult male sheep which results in a decreased basal gfr and an attenuated ability to excrete on acute sodium load. the elevated blood pressure previously observed associated with this prenatal steroid treatment may be related to the alteration in renal function. the study is supported by nih grants hd , hl and hd . characterisation of human fetal cardiac stem cells. marah alfakir, nicholas dawe, annette meeson, stephen c robson. school of surgical reproductive sciences, newcastle university, newcastle upon tyne, united kingdom. objectives: for many patients with severe cardiac disease treatment is limited to surgical intervention and/or heart transplantation. the heart has a limited regenerative capacity but it is insufficient to repair extensive damage. cellular therapies might provide an alternative treatment. we have identified stem cells (sp cells) in the adult mouse and human heart and have shown that the mouse cardiac sp cells can differentiate along a cardiac lineage. sp cells can be identified using facs combined with hoechst dye efflux. this ability to efflux hoechst dye is due to expression of abcg (an abc transporter). we have hypothesized that the fetal heart would contain more cardiac stem cells, relative to the adult, and the aim of this project was to determine the spatial and temporal expression of known stem cells markers in the embryonic/fetal heart. methods: human fetal hearts were collected aged between - wk. we used rt-pcr, using primers for several stem cell (abcg , cd , cd ) and cardiac specific (mlc- v, mhc) markers, and ihc on frozen and paraffin sections, using a panel of antibodies (abcg , cd , cd , islet ). cdna was generated from the following regions of the heart at different developmental stages: right and left atrium, right and left ventricle, and outflow tract. results: abcg mrna was highly expressed in all regions of the fetal heart between - wk. expression was down regulated at - wk especially in the la and lv. a similar pattern of expression was observed for cd . cd showed low/moderate expression in all heart regions examined; however, this expression was absent in the lv at - wk. mrna and protein analysis showed similar results. whilst protein expression of abcg was robust at wk (approximately - %), it declined with increasing gestational age. cd was also strongly expressed at weeks of age. there was no co-localisation between abcg and islet . conclusion: abcg and cd are both expressed between - wk of age. based on this analysis, further studies are underway to isolate and characterise both the abcg and cd expressing fetal cardiac cells which might be a potential source of cardiac stem/progenitor cells. xanthine oxidase plays a significant role in the fetal cardiovascular defence to hypoxia. ja hansell, a kane, e herrera, da giussani. physiology, cambridge university, united kingdom. prenatal hypoxia remains a major concern in obstetrics. the fetal defence to hypoxia includes redistribution of the cardiac output, away from peripheral and towards essential ciculations, such as those perfusing the brain (cohn et al . ajog : , ) . the physiology underlying this response is well characterised and involves chemoreflex and endocrine responses (giussani et al. fet mat med rev : , ) . more recently, local factors such as nitric oxide (no) and reactive oxygen species (ros), and the interaction between them, have been shown to play a role. antioxidants, such as vitamin c, scavenge hypoxia-induced ros, maintain no high and thereby depress peripheral constriction (thakor et al., sgi ) . in this study, we address the source of hypoxia-induced ros production and investigated the effects on the in vivo fetal femoral constrictor response to hypoxia of maternal treatment with the xanthine oxidase inhibitor allopurinol in sheep. methods: under anaesthesia, sheep at . gestation, were instrumented with maternal and fetal catheters and a fetal femoral transonic probe. five days later, all animals were subjected to h normoxia, . h hypoxia (mat fio to reduce fetal pao to ca. mmhg) and h recovery, either following maternal i.v. treatment with vehicle or allopurinol in low ( mg.kg - over min) or high ( mg.kg - over min ) doses. treatments finished min prior to hypoxia. the low allopurinol dose was adopted from human studies (benders et al. arch dis child : , ) . the timing of the hypoxic challenge coincided with peak concentrations in fetal sheep plasma of oxypurinol (active metabolite). we evaluated the effect of bpa exposure on uterine gene expression in a nonhuman primate model. method: a total of nine vervet monkeys (cercopithecus aethiops sabaeus) were ovariectomized. three monkeys were treated with bpa via alzet pumps ( microgram/kg/day), two with estradiol benzoate and three received combined treatment with bpa and estradiol. the remaining monkey was untreated and served as a control. following days of treatment the monkeys were hysterectomized and immunohistochemistry performed to examine endometrial gene expression. we evaluated hoxa , proliferating cell nuclear antigen (pcna), and caspase iii gene expression as markers of differentiation, proliferation, and apoptosis respectively. differential expression was evaluated by h-score. results: exposure to a combination of estradiol and bpa resulted in increased expression of hoxa and pcna compared to control (p< . ). lower, but increased, levels of hoxa and pcna gene expression were seen in the specimens exposed solely to estradiol (p< . ). those specimens exposed to bpa alone demonstrated a small induction in hoxa expression (p< . ) with no change in caspase iii or pcna expression when compared to the control. conclusion: bpa induced the expression of hoxa in the uteri of a non-human primate. exposure in the presence of endogenous estrogen further augmented estrogenic stimulation confirming that bpa is a xenoestrogen. bpa's effect of developmental gene expression may alter uterine differentiation. bpa acts as an endocrine disruptor by altering estrogen regulation on a gene required for fertility. contractility and meconium passage. jayaraman lakshmanan, john d richard, guo l liu, sharon k sugano, ahmet karadag, michael g ross. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa; dept. of pediatrics, harbor-ucla med. ctr., torrance, ca, usa. objective: endogenous glucocorticoids (gcs) increase with advancing fetal age suggesting that gcs function as a hormonal modulator of organ maturation. although fetal colonic motility and meconium passage primarily occur near term, the role of gc in colonic maturation is poorly understood. we sought to examine gc-nuclear receptor (gc-nr) expression in ovine fetal distal colon smooth muscle from very-preterm to term gestation to define the cascade of gc initiated biochemical changes as a prerequisite for maturation-associated meconium passage. methods: ovine fetal distal colonic segments removed at very-preterm (vpt: - d gestation), preterm (pt: - d), near term (nt: - d) and term (t: - d) (n= fetuses in each group) were fixed in bouin's solution and paraffin embedded. sections were subjected to immunohistochmical analysis with gc-receptor antibody ( : , sc- , santacruz biotechnology, ca) using standard abc regimen. immunoreactive material identified by , 'diaminobenzidine as chromogen. the percentage of gc-nr staining in smooth muscle-enteric unit was analyzed by counting dark brown immunostained nuclei at different fields at x magnification. all values are expressed as mean ± sem. results: the gc-receptor antibody immunostained nuclei both in smooth muscle and enteric units and the observed pattern (expressed as percentage of total nuclei) are as follows: nuclear gc expression varies with gestational age with maximal expression occurring near-term in smooth muscle and enteric neurons, in a synchronized manner. a near total disappearance of gc-nr expression occurs at term. these results suggest that peak gastrointestinal gc-nr mediated effects may occur prior to term with secondary signaling effects resulting in distal colonic motility maturation and meconium passage. the rna-binding protein hud co-localizes with choline acetyl mechanisms of in utero meconium passage. jayaraman lakshmanan, john d richard, guo l liu, sharon k sugano, octavio balbuena, michael g ross. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: we have previously speculated that crf, acting through its receptor crf-r , increases gastrointestinal (gi) motility and potentiates in utero meconium passage. patients with paraneoplastic syndrome with auto-antibodies to hud, a neuronal rna binding protein, develop severe gi dysmotility, indicating a role for hud in gi motility. hud binds mrna for actylcholinesterase implying a role in the cholinergic neurotransmitter system. based on these known functions, we hypothesized that hud may regulate post-transcriptional activity in fetal colonic cholinergic neurons expressing crf-r . method: bouin's solution-fixed paraffin-embedded sections of distal colonic segments were prepared from very preterm (vpt: - days), preterm (pt: - days), near term (nt: - days) and term ( - days) ovine fetuses (n= at each age). sections were immunostained with anti-human neuronal protein huc/hud antibodies ( : to : ) with abc reagents and examined at x. neurons positive for hud staining were quantified. double immunofluorescence and laser confocal analyses evaluated co-expression of hud in neurons expressing peripheral choline acetyl transferase (pchat) (a marker for peripheral cholinergic neurons) and crf-r receptor. results: hud immunostaining was seen in either entire cytoplasm (c) or cytoplasm and nuclear regions (c+n) in both submucosal and myenteric neurons. a greater percentage of submucosal (vpt: ± , pt: ± , nt: ± , and t: ± %) than myenteric neurons (vpt: ± , pt: ± , nt: ± , t: ± %) exhibited positive staining at all ages. the percentages of neurons with c+n staining in submucosal neurons were significantly lower at very-preterm gestation. confocal studies co-localized the hud staining with pchat and crf-r receptor immunoreactivity both in submucosal and myenteric neurons. conclusion: in the fetal enteric nervous system hud may function both as a rna-binding protein and as a nuclear cytoplasmic shuttling protein. colocalizaion studies suggest that both pchat-mrna and crf-r mrna species are target transcripts for hud in myenteric neurons. we speculate upregulation of hud contributes to in utero meconium passage. meconium passage during mild stressors. jayaraman lakshmanan, guo l liu, john d richard, sharon k sugano, raina khan, octavio balbuena, kimberly chap, virender rehan, michael g ross. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa; dept. of pediatrics, harbor-ucla med. ctr., torrance, ca, usa. objective: mr exhibit a greater affinity to glucocorticoids (gc) than do glucocorticoid receptors (gr). thus low circulating gc levels may preferentially bind mr during mild-stress periods, while the high gc levels may bind gc-receptors. mr antagonism increases gc-mediated responses, suggesting that mrs have a regulatory impact on gc-mediated stress responses. we recently documented that fetal in utero meconium passage is a neurovisceral stress response. to test our hypothesis that mrs play a primary role in mediating suppressive gc effects, we examined the expression patterns of mr in ovine fetal distal colon. methods: bouin's solution fixed paraffin sections of distal colonic segments collected from ovine fetuses (n= for each gestational ages) at very preterm (vpt: - days gestation), preterm (pt: - days), near term (nt: - days) and term (t: - days) were subjected to immunohistochemistry with polyclonal antibodies to mr. digital photos ( field per colonic ring, rings each gestational age) taken at x were used to count the number of intensely stained neurons, referred to as "mr-capped neurons". differences over time were determined with anova. results: mr antibody elicited a punctate staining pattern in all layers of ovine fetal distal colon. significant immunoreactive intensity was observed in submucosal and myenteric ganglia at all ages: submucosal ganglia: vpt= . ± . , pt= . ± . , nt= . ± . , t= . ± . ; a subpopulation of enteric neurons exhibited dense staining ("mr-capped"). advancing gestation was associated with a significant decreased in the percentage of mrcapped myenteric (vpt = ± , pt = ± , nt = ± , t = ± %; p< . ), though not submucosal ganglia neurons. results indicate a significant decrease in the percentage of mr capped myentric neurons with advancing gestation. in view of the potential inhibitory effect of mr on gc-mediated stress responses, these results suggest that the decrease in mr expression may contribute to near term and term in utero meconium passage. the ambiguity of myometrial progesterone receptor expression in pregnancy and labour. alison j tyson-capper, elizabeth a shiells, stephen c robson. surgical reproductive sciences, institute of cellular medicine, newcastle university, newcastle upon tyne, united kingdom. background and aims: in contrast to many other species, human parturition is not due to a reduction in circulating levels of progesterone (p) but appears to be related to changes in expression, ratios or signalling events of p receptors (pr-a and pr-b). the literature on pr expression in human myometrium in pregnancy and labour remains conflicting. we hypothesise this is due, at least in part, to differing specificities of the various pr antibodies employed. we carried out a comprehensive analysis of pr expression using ten 'pr-specific' antibodies that recognise different amino acid epitopes within the pr proteins. two phosphorylation-specific antibodies for pr were also included to define whether phosphorylation status of myometrial pr changes in pregnancy and in labour. methods: western immunoblotting and semi quantitative rt-pcr were undertaken using protein lysates and rna prepared from myometrial tissue from: -first (n= ) and second (n= ) trimesters, paired upper and lower segment myometrium from preterm ( - wks, n = ), term not in labour (n = ), term spontaneous labour (n = ) and non pregnant (n = ). results: using the same set of myometrial lysates for each antibody, we found that the specificity of individual pr antibodies, in particular those raised against internal and c-terminal epitopes of the pr proteins, varied considerably resulting in different patterns of expression. there also appeared to be temporal and spatial differences in levels of myometrial pr proteins ( - kda/ - kda) in pregnancy and in labour with use of the phosphorylation-specific pr antibodies, however, it remains to be resolved which pr isoforms these may be. in contrast, data using four antibodies all of which react with the amino terminal domain of pr consistently indicated that expression of pr, in particular pr-b, decreased significantly at term (p < . ) and in labour (p < . ) when compared to non-pregnant levels. a decrease in levels of pr-b protein was also observed when comparing preterm levels to non-pregnant levels. rt-pcr using primers specific to pr-b consistently indicated that levels of pr-b mrna decreased at term and in labour. conclusion: interpretation of gestation-related changes in myometrial pr expression and phosphorylation status must take into account the pr antibodies used. further studies are now underway to validate the specificity of the pr antibodies. objective to test the hypothesis that expression of fshr in mural gl cells from ivf follicles varies with the infertility diagnosis and correlates with the outcome of ovulation induction (oi). materials and methods women undergoing ivf were classified as: . "no ovarian factor", (tubal or male factor and egg donors, nof;n= ); . endometriosis (endo; n= ); . poor responders (pr; n= ); . polycystic ovary syndrome (pcos; n= ). ovulation induction was carried out using a long or microflare or antagonist protocol based on clinical parameters and gonadotrophin doses were selected based on ovarian reserve (day fsh and e and basal antral follicle count) and adjusted to the individual response. after ultrasound guided egg retrieval, mural gl cells were isolated from pooled follicular fluids from each patient using a percoll gradient and anti-cd immunobeads to eliminate wbcs, viability was assessed by trypan blue. fshr was measured by rt-pcr as relative expression compared to beta actin. statistical analysis was performed with the spss using pearson´s correlation, one way anova and student´s t-test. results fshr expression in nof ( ± . ) was statistically significantly higher than in pr ( ± . ) and lower than in pcos ( ± . ). both endo ( ± . ) and pr levels of fshr were statistically significantly lower than in pcos. analysis of all cycles showed that fshr expression correlates positively with the number of total (r= , ; p< , ) and mii (r= , ; p< , ) oocytes and negatively with the units of fsh (r=- , ; p< , ) and lh (r=- , ; p< , ) administered for oi. separate analysis of nof showed a positive correlation with the number of total and mii oocytes retrieved and with estradiol levels on day of hcg but not with the total dose of gonadotropins received during oi. fshr expression correlates negatively with day fsh levels in all patients except in pcos (r=- , ; p< , ). conclusions these results suggest that expression of fshr in the hyperstimulated ovarian follicle is "average" in normoresponders, high in high responders (pcos) and low in poor responders (pr and endo). knowledge of the level of expression of fshr might be useful to individualize gonadotrophin doses and oi protocols thus improving pregnancy rates. comparison of intracellular atp levels between non-vitrified and surviving post-vitrification/thawed human oocytes. somjate manipalviratn, zhi-bin tong, eric widra, , alan h decherney. reproductive biology and medicine branch, nichd/nih, bethesda, md, usa; department of obstetrics and gynecology, georgetown university hospital, washington, dc, usa; shady grove reproductive science center, washington, dc, usa. objective: to compare intracellular atp level in non-vitrified and surviving post-vitrification/thawed human oocytes using an atp bioanalysis assay. design: prospective match-controlled laboratory study material and methods: all oocytes were obtained from women undergoing controlled ovarian stimulation for ivf/icsi. oocytes were discarded due to nuclear immaturity at the time of planned icsi. all immature oocytes (gv and mi) were incubated overnight. oocytes from patients with or more discarded eggs which matured to mii stage were used in this study. oocytes from each women were randomly divided into groups. in group , the oocytes were placed in l of ultrapure water and kept at - o c for further atp analysis. in group , the oocytes were vitrified using % ethylene glycol, % dimethyl sulphoxide and . m sucrose as cryoprotectant. these oocytes were kept in liquid nitrogen for - days before thawing with a rapid thawing method. oocyte survival was determined by morphological assessment after minutes of incubation then oocytes were placed in l of ultrapure water and kept at - o c for further atp analysis. intracellular atp level was determined using luciferin-luciferase bioluminescent assay. result: ninety-one discarded human oocytes were obtained from women. oocytes were vitrified/thawed before measuring for atp content. the other oocytes were measured for their atp content without undergoing vitrification/thawing process. oocyte survival rate after vitrification/thawing is . % ( / ). mean oocyte atp levels in non-vitrified oocytes is significantly higher than surviving post-vitrification/thawed oocytes (table ). coefficient of variation of luciferin-luciferase bioluminescent assay is less than %. conclusion: oocyte atp level in surviving post-vitrification/thawed human oocytes is significantly lower than non-vitrified oocytes. objective to study the expression of genes involved in cell proliferation and steroidogenesis in the human follicle (kl , kl , fshr, papp, p ) and its relationship with ivf outcome. materials and methods patients with different infertility diagnosis underwent ovulation induction with either a long or microflare or antagonist protocol based on clinical parameters; the dose of gonadotrophin used for ovulation induction was selected based on the ovarian reserve (day fsh and e and basal antral follicle count) and adjusted to the individual patient response. ultrasound guided egg retrieval was performed hours after administration of . iu of hcg. mural granulosa-lutein cells (gl cells) were isolated from pooled follicular fluids (ff) from each patient using a percoll gradient and anti-cd immunbeads to eliminate wbcs, viability was assessed by trypan blue. the genes under study were measured by rt-pcr as relative expression compared to actin. statistical analysis was performed with the spss statistical software using pearson´s correlation and mann-whitney u. results kl expression correlates positively with kl levels (r= , ; p< , ) and with genes implicated in granulosa cell function such as fshr (r= , ; p< , ), papp (r= , ; p< , ) and p (r= , ; p< , ). the number of mii oocytes obtained is positively correlated with both fshr (r= , ; p< , ) and papp (r= , ; p< , ) expression, but not with the other genes. kl expression in women who became pregnant during the ivf cycle studied was higher ( , ± ) than in non pregnant women ( , ± , ).(n= , mann-whitney u p< , ). conclusions patients who become pregnant have increased expression of kl , which is associated with optimal granulosa cell proliferation and maturation. this is in accordance with the presence of lower apoptosis in granulosa cells in the same patients. syndrome during assisted reproduction treatment. k jayaprakasan, r jayaprakasan, h al-hasie, js clewes, bk campbell, ir johnson, nj raine-fenning. school of human development, university of nottingham, nottingham, united kingdom. objective: to test the hypothesis that an increased pre-treatment ovarian blood flow is associated with the development of ovarian hyperstimulation syndrome (ohss) and to evaluate ovarian vascularity as a predictor of ohss during invitro fertilization (ivf). methods: subjects undergoing first cycle of ivf had d transvaginal ultrasound in the early follicular phase of the menstrual cycle preceding ivf. of them developed ovarian hyper-response, defined as retrieval of oocytes and ohss. subjects had normal ovarian response with retrieval of to oocytes in the absence of ohss. antral follicle count (afc), ovarian volume (ov), and ovarian vascularity (vascularisation index, vi; flow index, fi and vascularisation flow index, vfi) were measured and an unpaired t-test was used to compare these parameters between the ohss and the control groups. multiple logistic regression analysis was used to assess the predictive value of these variables against age, bmi and basal fsh for the development of ohss. results: the ovarian vi ( . ± . vs. . ± . ), fi ( . ± . vs. . ± . ) and vfi ( . ± . vs. . ± . ) were similar in both the groups. afc and ov were significantly higher (p< . ) in the ohss group ( . ± . and . ± . cm respectively) than in the control group ( . ± . and . ± . cm respectively). afc was the only significant (p= . ) predictor of ohss on multiple regression analysis (table ) . conclusion: women developing ohss during ivf do not demonstrate an increased pre-treatment ovarian blood flow as measured by d ultrasound but do have a significantly higher afc, which is the only significant predictor of ohss. compared to other species, little is known about the steroid biosynthetic capacity and gene expression profiles of human cumulus cells. objective: to examine the ability of human cumulus cells in primary and long-term culture to synthesize steroids and respond to gonadotropin or camp-dependent stimulation. methods: human cumulus cells were isolated from cumulusoocyte complexes during assisted reproductive technology (art) and placed in primary culture or propagated to third passage. at subconfluence, cells were transferred into serum-free conditions in the presence of vehicle, forskolin, fsh, or hcg. at h, media was collected and progesterone (p ) and estradiol (e ) levels were determined by eia. aromatase activity was measured by the tritiated water assay. aromatase (cyp ) and cholesterol side-chain cleavage (cyp a ) mrna abundance was determined by quantitative real-time pcr (qrt-pcr). transcriptional regulation of the cyp and cyp a promoters was investigated by transient transfection of cumulus cells with promoter luciferase constructs. results: substantial amounts of p and e were synthesized by cumulus cells in culture, which were further elevated by forskolin, hcg, or fsh treatment. the presence of cyp and cyp a mrna in cumulus cells was confirmed by qrt-pcr, and the relative mrna abundance of both transcripts was induced by forskolin, hcg, or fsh treatment. changes in cyp and cyp a gene expression in response to camp and gonadotropin stimulation were associated with increased transcriptional regulation of both the cyp and cyp a gene promoters. our studies demonstrate that human cumulus cells are sites of significant p and e biosynthesis and respond to camp-and gonadotropin-stimulation in vitro. the ability to examine human cumulus cells in primary and long-term culture provides a unique model system to investigate cumulus cell function, and the paracrine role of cumulus cells in oocyte development, maturation, and subsequent fertilization. background: there is increasing evidence suggesting that events occurring during fetal development may result in increased predisposition to adult conditions, such as diabetes. in vitro fertilization (ivf) is a new environmental stressor and the long-term effects of these manipulations are currently unknown. there is some evidence that lower number of insulin-secreting cells at birth signals predisposition to diabetes later in life. in this study, we compare areas of pancreatic insulin-secreting cells in newborn mice conceived in vivo versus in vitro. methods: oocytes were collected from super ovulated cf- mice and fertilized in vitro with cauda epididymal sperm from b d f /j mice. fertilized eggs were cultured in whitten medium under % co in humidified air at °c for h. blastocysts were transferred to the uteri of pseudo-pregnant recipients. control mice were allowed to conceive in vivo. the newborn animals were sacrificed within hours of birth. pancreases were sectioned, immunostained with anti-insulin, and total areas and stained areas were compared using t-test with two-tailed distribution. p value of < . was considered significant. results: there were total of newborn animals: females ( ivf, controls) and males ( ivf, controls). percentage of total pancreatic area occupied by insulin-secreting cells was lower in female ivf ( . mm , . %) animals compared to female controls ( . mm , . %) and higher in male ivf ( . mm , . %) animals compared to controls ( . mm , . %). the average of males and females was slightly lower for ivf ( . mm , . %) than controls ( . mm , . %). none of these differences were statistically significant. there was a trend in newborn female mice towards lower amount of insulin-secreting cells in the ivf offspring, suggesting possible predisposition to diabetes later in life. this effect was not observed in newborn males. while our study failed to demonstrate consistent and significant differences in the areas of insulin-secreting cells between newborn mice conceived in vitro and in vivo, the number of animals in the study may have been too small to show significant results. larger studies are needed to further investigate this question. peter s uzelac, phyllis risch, kassi shelton, steven t nakajima. obstetrics and gynecology, university of louisville, louisville, ky, usa. objective: several fertility preservation methods currently available may not be viable options to certain patients due to their high cost and limited number of centers offering these services. for women undergoing bilateral oophorectomy, in vitro maturation (ivm) of oocytes retrieved from unstimulated whole ovary specimens may represent a more practical solution. here we describe out initial experience in offering ivm as a fertility-preserving measure to two patients undergoing total abdominal hysterectomy and bilateral salpingo-oophoectomy (tah-bso). case one was a year old nulligravid with chronic pelvic inflammatory disease unresponsive to medical management. case two was a year old nulligravid with stage ia endometrial carcinoma. surgery was planned for the mid-follicular phase in order to collect prophase i oocytes before the onset of late-follicular phase atresia. upon surgical removal, suction aspiration of all identifiable follicles was performed. ovaries were then serially sectioned and all tissue was examined for the presence of prophase i oocytes. results: total number of prophase i oocytes retrieved was and , maturation rate after hours was % ( / ) and % ( / ), fertilization rate after hours was % ( / ) and % ( / ), maturation rate after hours was % ( / ) and % ( / ) and fertilization rates after hours % ( / ) and % ( / ), for case and case respectively. ivm of oocytes retrieved from unstimulated whole ovary specimens may be a simple and inexpensive approach to fertility preservation in women undergoing bilateral oophorectomy. by requiring minimal adjustments to in vitro fertilization protocols, this treatment could easily be implemented in centers which currently have no fertility preservation program. patient age at time of retrieval may be predictive of developmental potential. continued patient enrollment and follow-up studies on the developmental potential of embryos derived from this technique are necessary to fully evaluate its potential for a role in fertility preservation. the routine use of progesterone as luteal phase support in women with a diagnosis of polycystic ovary syndrome (pcos) undergoing ovulation induction cycles with oral agents has not been fully elucidated. we hypothesize that women with pcos utilizing either clomiphene citrate or letrozole, an aromatase inhibitor, should administer intravaginal progesterone in the luteal phase to increase pregnancy rates. design: retrospective chart review. materials and methods: cycle data from women with pcos undergoing ovulation induction with clomiphene citrate or letrozole at the university of cincinnati medical center from to were evaluated. diagnosis of pcos was based on rotterdam criteria and all other infertility diagnoses were excluded. clinical pregnancy rates (presence of fetal cardiac activity on ultrasound at - weeks gestation) in women who received intravaginal micronized progesterone ( mg bid) following ovulation induction (with and without intrauterine insemination) were compared to those who did not receive progesterone. results: no significant differences were noted in demographic parameters, including patient age or bmi. a total of cycles were evaluated in women treated with clomiphene citrate. clinical pregnancies were documented in . % ( / ) of cycles in the progesterone group compared to . % ( / ) of the non-progesterone group (p < . ). forty-three cycles were evaluated in patients treated with letrozole. clinical pregnancies were documented in . % ( / ) of cycles in the progesterone group compared to none ( / ) in the non-progesterone group (p < . ). conclusions: patients with pcos who used letrozole for ovulation induction had superior clinical pregnancy rates when using intravaginal micronized progesterone compared to women who did not receive luteal phase support. there was a trend toward increased clinical pregnancy rates in pcos patients utilizing luteal support following clomiphene citrate for ovulation induction. therefore, in women with pcos undergoing ovulation induction with oral agents, luteal supplementation with progesterone should be strongly considered, especially in those using letrozole. were measured and an unpaired t-test was used to compare these parameters between poor and normal responders. multiple logistic regression analysis was used to assess the predictive value of these variables against age and basal fsh for poor ovarian response. results: the ovarian vi ( . ± . vs. . ± . ), fi ( . ± . vs. . ± . ) and vfi ( . ± . vs. . ± . ) were similar in both poor and normal responders. afc and ov were significantly lower (p< . ) in poor responders ( ± . and . ± . cm respectively) than in normal responders ( . ± . and . ± . cm respectively). afc was the best (p< . ) predictor of poor ovarian response on multiple regression analysis ( objective: gay male couples increasingly seek parenthood through in vitro fertilization using an oocyte donor and a gestational carrier, but no studies describe this unique experience. the purpose of this study was to determine medical and psychosocial issues unique to gay men using art. design: qualitative analysis of semi-structured interviews with gay male couples seeking parenthood through art. materials and methods: sixteen gay males (eight couples) entering an art program were assessed through the use of a semi-structured interview. characteristics evaluated included age, relationship status, duration of their relationship, psychological health and stability, how the decision was made concerning who would donate the sperm, the decision to use an anonymous or known oocyte donor, and whether their gestational carrier was someone previously known to them or not. results: the average age of the men in this study was years. all eight couples were in a committed relationship and had been together for an average of . years. five of the couples ( %) had been joined in a civil union which is legal in the state of connecticut. all subjects were psychologically stable and in good health. six couples ( %) were very clear about which partner would inseminate the oocytes. of those couples, two felt that the older partner should donate; two felt that the partner who cared more about a genetic connection to the child should donate; and two felt that the partner with "better genes" should donate. the remaining two couples chose to inseminate equal numbers of oocytes in order to transfer an embryo from each partner. all couples chose an anonymous oocyte donor, two couples chose relatives as their gestational carriers, while the others chose carriers recruited through an agency. conclusions: participants in this study were determined to become parents through assisted reproduction. they had given thoughtful consideration to the medical and psychosocial issues unique to this process including which partner would be the genetic parent, and why. clomiphene superovulation. rb allen, az steiner, rh fogle, mj kalan, rj paulson. ob/gyn, usc keck school of medicine, la, ca, usa; ob/gyn, unc, chapel hill, nc, usa. intro: micro-dose hcg has been demonstrated to increase ovulation and pregnancy rates following clomiphene administration in women resistant to clomiphene. since micro-dose hcg stimulates growth in follicles expressing the lh receptor, we hypothesized that its use following clomiphene in women with unexplained infertility would increase the number of follicles that ovulate and subsequently, pregnancy rates. m m: irb approval was obtained for this prospective pilot study of women with unexplained infertility. on day # a baseline ultrasound was performed and serum fsh and e levels were measured. subjects were given mg of clomiphene daily from days - and then returned for serial ultrasounds on day # . when at least follicles mm were present, iu of hcg im daily was initiated. cycles were monitored by ultrasound every days until a +lh surge occurred. all subjects had previously undergone a cycle using clomiphene alone for superovulation followed by iui and these cycles were used for comparison. results: subjects, aged . ± . yrs (mean±sem) and bmi . ± . kg/m with . ± . yrs of infertility were enrolled. the mean fsh and e levels were . ± . miu/ml and . ± . pg/ml, respectively. there was an average of . ± . days from starting clomiphene to the attainment of follicles mm. out of the subjects had follicles mm by day # . a mean of . ± . days of treatment were required until ovulation occurred. there were twice as many follicles in the micro-dose hcg cycles, although due to the small sample size this did not reach statistical significance ( . ± . follicles, measuring . ± . mm in the micro-dose hcg cycles, compared to . ± . (p= . ), measuring . ± . mm (p= . ) in the control cycles). there was no difference in the endometrial thickness at the time of ovulation between the micro-dose hcg cycles and the control cycles: . ± . mm vs. . ± . mm (p= . ), respectively. all subjects had at least ovulatory sized follicles in the study cycles. % of subjects had an iui performed, however there were no pregnancies resulting from this study. conclusions: ) the addition of micro-dose hcg to clomiphene may allow more follicles to reach an ovulatory size, which should theoretically increase pregnancy rates ) this pilot study demonstrates that adding micro-dose hcg may increase the effectiveness of clomiphene when given in a superovulatory protocol. jessica salas, donald maier, john nulsen, claudio benadiva, lawrence engmann. obstetrics gynecology, university of connecticut, farmington, ct, usa. objective: to evaluate the antepartum, intrapartum, and neonatal complications in patients undergoing ivf using a gnrh-antagonist protocol where a gnrhagonist was used to induce final oocyte maturation. materials and methods: a retrospective review of data from high responders undergoing their st or nd ivf cycle using a gnrh-antagonist protocol who were triggered with leuprolide acetate (study group) or hcg (control) and achieved at least a singleton pregnancy reaching the third trimester. patients younger than years old were included. both groups received luteal phase support with intramuscular progesterone. the study group received estrogen patch supplementation. outcomes measured were antenatal and intrapartum complications, order of gestation, gestational age at delivery, birth weight, neonatal adverse outcomes, and congenital anomalies. pearson chi square, fisher's exact test, or independent t-test were used as appropriate. results: the baseline characteristics were different (table ) . maternal antenatal and intrapartum complications were similar in both groups ( . % vs . %, p= . ). there were more singletons in the study group ( . % vs . %, p< . ). a subgroup analysis of gestational age at delivery, birth weight, neonatal complications and congenital anomalies in singletons showed no difference (table ) . conclusions: this is the first study reporting the maternal and perinatal outcomes after gnrh-agonist trigger. differences in response to ovarian stimulation may dictate use of gnrh-agonist triggering for prevention of ohss, which may explain the differences in baseline characteristics. maternal and neonatal complications remain unaffected. table lupron (n= ) objective: moderate to severe ovarian hyperstimulation syndrome (ohss) occurs in - % of assisted technology cycles and carries the potential for severe complications. traditional management consists of hospitalization with intravenous fluids (ivf), bedrest, and close monitoring. early and aggressive paracentesis is an alternative method of treatment for ohss in an outpatient setting, but the economic consequences of the two treatment regimens have not been compared. design: cost-effectiveness analysis materials and methods: two scenarios, outpatient management with transvaginal paracentesis and conservative therapy with hospitalization, were compared. potential initial outcomes were analyzed for the conservative group to include hospitalization either in a ward hospital bed or the intensive care unit (icu) for an average of seven days. costs included ivf administration, ultrasound, and daily bloodwork. initial outcomes for the outpatient management group included no further therapy beyond the initial transvaginal paracentesis, bloodwork, ivfs, and ultrasound versus admission for an average of three days to a ward bed with similar management. the probability of the negative outcome for the conservative group was set at % (icu admission) and % for the outpatient group (regular hospitalization). results: the cost of conservative therapy including first tier complications ranged from a low of $ , to a high of $ , . the cost of outpatient management with aggressive paracentesis and its first tier complications ranged from a low of $ to a high of $ , . this resulted in an estimated cost burden of $ , to $ , for conservative management with hospitalization. the main improvement factor was that patients in the outpatient management group had a much lower likelihood for prolonged hospitalization than the conservatively managed group. conclusions: aggressive outpatient treatment of moderate to severe ohss with early paracentesis appears to be a cost-effective strategy. induction. zeynep alpay, michael p diamond, michael l kruger, elizabeth e puscheck. obstetrics and gynecology, division of reproductive endocrinology and infertility, hutzel hospital, wayne state university, detroit, mi, usa. introduction: aromatase inhibitors (ai) are a new method of ovulation induction and is proposed to replace clomiphene citrate (cc) due to their reported advantages. their superior effect is thought to be due to up-regulation of the estrogen receptors and increase in the sensitivity of the endometrium, resulting in better proliferation despite low estrogen levels in the circulation. objective: to compare the effect of different ovulation induction methods, including ai, cc and gonadotropins (gt) on the endometrium in infertility patients. methods: we reviewed ovulation induction cycles performed in our institution in the last one-year period retrospectively. there were gt, cc and ai cycles. age, gravida, day (d et) and midcycle (mcet) endometrial thickness, endometrial pattern (ep) on day of hcg injection, endometrial growth during the induction cycle (d-et), which was calculated by the difference between mcet and d et, and the pregnancy rates (pr) were compared. the ep was categorized as type a (homogenous and hyperechogenic), type b (intermediate isoechogenic pattern and a poorly defined central echogenic line), and type c (multilayered triple-line). chi-square, anova, t test and pearson correlation were used for statistical analysis. results: in all cycles, ep was closely related to the d-et (p < . ). this correlation appeared to be more pronounced when observed ep was compared with d-et (r = . ; p < . ) rather than with mcet (r = . ; p = . ). there was a reverse relationship between the age of the patients and ep (r = - . ; p < . ). a negative correlation was also found between the gravida and ep (r = - . ; p < . ). pregnancy rate was significantly correlated with ep (r = . ; p < . ). pregnancy rate was % in type a ep, . % in b, % in c when all cycles were analyzed. aromatase inhibitors and gt treatments each resulted in % type c pattern in contrast to % with cc (p = . for ai vs. cc; p = . for gt vs. cc). the d-et was greater in gt cycles compared with ai or cc (p < . ). conclusion: these results show that ep has a strong correlation with pr in ovulation induction cycles. additionally, ai and gt treatments have similar effects on ep. both ep and the growth of the endometrium during the induction (d-et) are good prognostic variables for the successful pregnancy initiation. ovarian response in patients undergoing ovarian stimulation after myomectomy. hyacinth browne, , desiree mccarthy-keith, , barbara stegmann, , alicia armstrong. , reproductive biology and medicine branch, nichd, nih, bethesda, md, usa; reproductive endocrinology and infertility, walter reed army medical center, washington, dc, usa. objective: the impact of myomectomy on ovarian function has not been wellstudied. other surgical treatments of fibroids, such as hysterectomy and uterine artery embolization, have shown an increase of fsh into the peri-menopausal range. the objective of this study is to examine ovarian response in infertile women undergoing ovarian stimulation after abdominal myomectomy. design: retrospective analysis. materials and methods: a retrospective analysis of all infertile women with known fibroids who had a failed art cycle, from january to , followed by an abdominal myomectomy and a subsequent art cycle was performed. women served as their own controls. ovarian function pre and post-myomectomy was assessed by age, day and fsh levels, days of stimulation, total gonadotropins used, peak estradiol level, number of oocytes retrieved, embryos obtained, and high-grade embryos, and pregnancy outcome. quantitative results are presented as mean + sd. results: four women had a failed art cycle and underwent an abdominal myomectomy prior to a subsequent art cycle. the mean age was and pre-and post-myomectomy, respectively. all subjects had uterine factor infertility. two of these women also had tubal factor infertility, and one had endometriosis and male factor infertility. we found no difference in ovarian response pre and post-myomectomy. pre-myomectomy post-myomectomy age (years) + . + . day fsh (u/l) . + . . + . day fsh (u/l) . + . . + . objective: anti-mullerian hormone ( amh) is produced by developing primordial follicles. amh levels are stable through the menstrual cycle and therefore can be drawn randomly. the objective of this study was to assess the association of anti-mullerian hormone ( amh) and oocytes obtained at ivf retrieval. design: cross-sectional cohort study. materials and methods: the study cohort is comprised of thirty women in cycles of in vitro fertiization (ivf). serum levels antimullerian hormone (amh) were drawn before starting an ovulation induction cycle for ivf. standard ovulation induction was performed with leuprolide flare and miu/ml per day of follicle stimulating hormone. we performed linear regression of log convert d oocyte number against the log converted amh level. serum amh was measured using an enzymatically amplified two-site immunoassay dsl- - active mis/amh elisa. statistical analysis was performed using spss version . . continuous values are presented as mean std error. results: the log number of oocytes retrieved was directly related to the log value of amh (p = . ). conclusions: our data demonstrate an association between serum amh and oocytes produced in response to ovulation induction. using amh levels in addition to fsh levels in evaluating for ovarian reserve and pregnancy outcome may help improve predictions of reproductive performance. support: foundation for reproductive medicine. context: prediction of outcome after in vitro fertilization (ivf) can be difficult due multiple factors. human chorionic gonadotropin (hcg) levels correlate with pregnancy outcome, but data that can be used to easily counsel patients on their possible outcome is lacking. objective: to investigate the use of hcg levels along with other significant factors to predict the likelihood of an ivf pregnancy progressing to the point of detection of cardiac activity by ultrasound design: retrospective data analysis of ivf cycles performed from january to july resulting in pregnancies. multiple logistic regression analysis modeling was performed to determine the factors most predictive of an ongoing early pregnancy and to assess possible confounding variables. setting: an academic fertility center. patients: patients undergoing in vitro fertilization using autologous fresh embryos. intervention: none main outcome measure: pregnancy continuation to the documentation of cardiac activity by ultrasound. results: maternal age, day (post-oocyte aspiration) hcg level, and day hcg levels were significant in predicting pregnancy outcome. day and day hcg levels were highly correlated and can be considered proxies for each other. the most accurate predictive model used only a single day hcg level and maternal age. the type of fertilization method used, the cycle number for that patient, and the number of embryos transferred were not found to be significantly different. ongoing pregnancy rates were directly proportional to day hcg level, and inversely proportional to maternal age. the incidence of multiple pregnancies also increased proportionally to the initial hcg level. % of ongoing pregnancies had hcg level > miu/ml. conclusions: a single day post-oocyte aspiration hcg level and maternal age are the most predictive of an ongoing ivf pregnancy. there was no difference in outcome between fertilization methods or number of embryos transferred. there is no benefit to obtaining serial hcg levels after the initial one. erk activation with u ( m)) reduced fsh stimulated cyclin d mrna expression by %. fsh has also been shown to stimulate mtor, a regulator of growth and proliferation of many cell types. inhibiting mtor activation with nm rapamycin for min significantly reduced fsh-mediated cyclin d mrna expression. dht exposure for hours also inhibited fsh stimulated mtor signaling, as shown by a % reduction in the phosphorylation of its down stream target p s kinase. furthermore, pretreatment with dht resulted in significantly reduced fsh-mediated tsc- phosphorylation, which is an upstream regulator of mtor pathway. further studies revealed that fsh mediated tsc- phosphorylation and mtor signaling is regulated by erk, but independent of akt. based on these results we conclude that elevated reduced metabolites of androgens inhibit fsh-stimulated pka-erk pathway resulting in the inhibition of multiple mitogenic signaling pathways leading to defective follicle maturation culminating in anovulation. thus, rescuing the erk activation might serve as a potential therapeutic target to restore normal granulosa cell proliferation in hyperandrogenic states. (supported by nih grant hd- ). searching pcos-genes: results of a genome screen for pcos in a dutch founder population. olivier valkenburg, annemarie g mulders, aida bertoli-avella, ben a oostra, joop se laven. department of gynecology and obstetrics, erasmusmc, rotterdam, netherlands; department of internal medicine, erasmusmc, rotterdam, netherlands. context: although the etiology of pcos is not yet fully understood, evidence has accumulated for a complex genetic background i.e. a combination of multiple genetic and environmental factors. to reduce genetic heterogeneity, this study was conducted in a genetically isolated population in the netherlands . objective: in order to identify genomic loci that are associated with pcos, a whole genome screen using highly polymorphic microsatellite markers was performed in a founder population. subjects and methods: pcos patients ( rotterdam criteria) were identified in the founder population (rucphen, the netherlands) of ± . inhabitants. patients underwent a standardized screening procedure that included clinical, ultrasound and endocrine evaluation. all patients plus unaffected first-degree family members were genotyped using polymorphic markers on (microsatellites) from the abi prism ® linkage mapping set md- (average spacing cm). association-analysis was performed with the transmission disequilibrium test (tdt, genehunter software). linkage analysis was performed in clusters of or more closely related patients (simwalk ). results: there was an average number of . alleles per polymorphic marker. the tdt identified two non-adjacent markers on chrome (d s and d s ) that showed significant association with pcos (p , ). other markers that showed significant association were positioned on chromosomes (d s ), (d s ), (d s ) , (d s ), (d s ) and seventeen (d s ) (all p values . ). linkage analysis in sub-pedigrees revealed no significant linkage for these, or other, loci with pcos. moreover the results of a prior report of linkage of a marker on chromosome (d s ) could not be confirmed. conclusions: the lack of consistent results suggests the absence of a consistent genetic background in this select group of pcos patients. this supports the hypothesis of a complex genetic background for pcos that allows relatively small contributions of multiple risk-genes to be involved in the pathogenesis of this syndrome. in this founder-population the genetic heterogeneity was not sufficiently reduced to find risk-loci in a genome wide screen using highly polymorphic markers with an average spacing of cm. objectives: to objectively quantify uterine and endometrial blood flow in women with polycystic ovarian syndrome (pcos) and to examine if this was different in women with different phenotypic expressions of the disease. methods: transvaginal d and d ultrasound was performed in women with pcos, as defined by the rotterdam criteria, and sub-group analysis conducted based on the subjects' body mass index (bmi), ovulation status, and hirsutism score. anova was used to compare the mean values between the groups. results: pcos women with clinical hyperandrogenaemia had significantly lower endometrial and subendometrial blood flow than their anovulatory normoandrogenic counterparts (table ). there were no differences between lean and obese women or between anovulatory and ovulatory women with pcos. the pulsed wave doppler parameters were similar in all three phenotypic groups. conclusions: hirsute women with pcos have impaired endometrial perfusion compared to their normoandrogenic counterparts which is only evident with d ultrasound and not conventional pulsed wave doppler. nusayba a bagegni, jill blaine, anuja dokras. obstetrics gynecology, university of iowa hospitals clinics, iowa city, ia, usa; obstetrics gynecology, university of pennsylvania, philadelphia, pa, usa. background: there is conflicting evidence on the association between pcos and early and late obstetric complications. it is unclear if the reported risks are independent of bmi, preexisting hypertension and diabetes. we examined the risk of early and late obstetrical complications in a large group of women with pcos compared to controls. methods: we reviewed pregnancy records of women with pcos (rotterdam criteria, n= ) and controls (tubal infertility, n= ) after in vitro fertilization at university of iowa from - . the wilcoxon rank sum test and fisher's exact test were used to evaluate differences between variables and logistic regression analysis was used to determine the independent risk of pcos. results: subject demographics and medical history are shown in table. the first trimester miscarriage rate was % in women with pcos and % in controls. after logistic regression analysis pcos was not associated with miscarriage (p= . ). the prevalence of gestational dm (gdm) was similar in both groups % pcos vs % controls. pcos was not associated with gdm after adjusting for age and bmi (p= . ). however, bmi was significantly associated with gdm after adjusting for age and pcos (p= . ). risk of both pre-eclampsia and pih was % in pcos and % in controls, but not statistically significant after adjusting for age, bmi and twin gestation. preexisting htn showed a significant association with preeclampsia (p< . ). there was no significant difference in preterm delivery, cesarean section, twin gestation, intrauterine fetal death and intrauterine growth restriction in the groups. conclusion: despite adequate power, our study did not detect an increased risk of miscarriage in women with pcos. obesity was a significant contributor to late obstetric complications, namely gdm. these findings may warrant aggressive counseling of women with pcos on the potential benefits of weight loss prior to pregnancy. objective: to assess the prevalence of polycystic appearing ovaries (pcao) as defined by the rotterdam criteria; and to determine whether metabolic parameters differ between regularly cycling women with pcao and those with normal ovaries. studies have demonstrated a population frequency of pcao of - %, with - % among women with regular cycles. most were performed in a young reproductive age population; none examined the impact of age. all were performed prior to adoption of the rotterdam criteria. recent studies have shown an increased prevalence of metabolic syndrome among women diagnosed with polycystic ovarian syndrome (pcos). however studies focused on women in their s found no increase in bmi or fasting insulin with pcao but regular menses. participants: women aged - with regular cycles (every - days) enrolled in the ova study, a population-based study of ovarian aging. each subject underwent a transvaginal ultrasound for assessment of ovarian volume and antral follicle count (afc). outcomes collected included waist measurements and hdl, ldl, total cholesterol, triglycerides, fasting glucose and insulin. pcao were determined by the rotterdam criteria (afc of on one ovary or ovarian volume > cc). student's t-test and chi-square tests were used to assess differences between those with and without pcao for continuous and categorical variables, respectively. the prevalence of pcao decreased with increasing age (table ) . of the women with pcao, % met the afc criterion only, while % met the volume criterion only, and % met both. women with and without pcao did not differ in waist measurements, or in fasting lipids, insulin, or glucose. the rotterdam criteria, while less subjective than those described by adams in , have led to an increased prevalence of pcao among women with regular menses, over / of women in their s. women with pcao do not differ from those with normal ovaries in metabolic parameters associated with pcos. consideration should be given to adopting an age-adjusted criterion for afc, or a combination of afc and ovarian volume, for diagnosing pcos. background: pcos, especially accompanied by obesity, has been reported to be associated with a characteristic dyslipidemia comprising elevated triglycerides (tgs) and depressed hdl, especially the hdl- fraction. this is an atherogenic profile; in fact, studies suggest low hdl- may correlate most strongly with cardiovascular disease risk. weight loss is a mainstay of treatment and improves all manifestations of the disease, but the optimal diet to recommend remains undetermined. preliminary studies show a high protein diet may improve total hdl levels and insulin responses. however, the effect of weight loss and dietary composition on hdl- levels in pcos has not been investigated. objective: to evaluate the fasting lipid profile in newly diagnosed obese pcos patients and to determine the effects of a high-protein diet with or without metformin on weight loss, hdl- and other lipoproteins, and menstrual cyclicity. methods: in this pilot retrospective observational study, the fasting lipid profile of obese women newly diagnosed with pcos was determined. they were then placed on a high protein ( - g/day), low carbohydrate ( - g/day) diet with or without metformin ( and %, respectively) and followed monthly for an average of months (range - ). results: at diagnosis, % had low hdl and % had low hdl- ; only % had elevated tgs. on the diet, the patients demonstrated an average weight loss of . lbs ( . to . , p< . ) and decreased bmi of . kg/m ( . to . , p< . ). hdl levels increased significantly ( % increase from . to . , p< . ), especially the hdl- fraction ( % increase from . to . , p< . ). triglycerides decreased as well ( . to . , p< . ). ldl decreased but did not reach statistical significance. resumed menstrual cycles, were started on oral contraceptives, and had a hysterectomy. pregnancies occurred. no difference was seen with metformin use. conclusion: a majority demonstrated decreased hdl and hdl- at diagnosis. the high protein diet resulted in significant weight loss and improvement in hdl- levels, as well as improvements in total hdl, tgs and menstrual cyclicity. metformin produced no added benefit. prospective trials based on these data will help determine the optimal diet to reduce the significant short-and long-term morbidity in the large population of women with pcos. resistin is an adipokine that has been associated with obesity and insulin resistance in animal models. studies on the role of resistin on insulin resistance in humans have been controversial. recently resistin has been shown to exert atherosclerotic effects and elevated resistin levels have been observed in women with coronary heart disease (chd). women with polycystic ovary syndrome (pcos) are at high risk for chd. our present study investigates potential association of resistin and markers of inflammation, c-reactive protein (crp) and insulin resistance in women with pcos. methods: thirty two women with pcos participated in the study. all were hirsute and had irregular cycles. nineteen women with normal ovulatory cycles who matched the pcos patients in bmi served as controls. fasting glucose, insulin, resistin and crp levels were measured in all women. after a high carbohydrate diet for days, a standard oral glucose tolerance test (ogtt) was performed. blood samples were collected for glucose and insulin before and , and hrs after g oral glucose. the area under the curve (auc) for insulin and glucose was calculated. women with overt diabetes were excluded from the study. results: fasting insulin levels ( . + . μu [±se] /ml) and insulin response to oral glucose (auc) ( . + . μu/ml) were higher (p < . ) in women with pcos compared to controls. all were insulin resistant with homa-ir value > . mol x μu / l . resistin levels in women with pcos ( . ± . ng/ml) was significantly (p < . ) higher compared to control women ( . ± . ng/ml). crp levels in women with pcos ( . ± ng/ml) was also significantly (p < . ) higher than the controls ( . ± ng/ml). there was a significant positive correlation between resistin and crp levels (r = . , p < . ). there was no correlation between resistin levels and fasting insulin levels or insulin auc. there was also no correlation between resistin levels and fasting glucose or glucose auc. conclusions: our results indicate that ( )women with pcos and insulin resistance have increased resistin levels, ( ) there is a strong association between resistin and crp ( ) elevated resistin and crp levels may predict women who are at increased risk for chd ( ) ) it is unlikely that resistin plays a major role on insulin resistance in women with pcos. vuk p jovanovic, enrico carmina, prati vardhana, michel ferin, rogerio a lobo. department of obstetrics and gynecology, columbia university, new york, ny, usa; department of internal medicine, university of palermo, palermo, italy. current diagnostic criteria for pcos includes both ovulatory (ov) and anovulatory or "classic" (c) phenotypes. in an effort to further characterize differences and /or similarities between these phenotypes, we studied hyperandrogenic women with pcos (age . ± . , bmi . ± . ) and age matched controls (age . ± . , bmi . ± . ). women with pcos were divided into weight matched (ov) n= and (c) n= groups. fat and weight distribution were assessed by dexa (total fat, r fat, trunk fat) as well as fasting levels of lh, e , mis/amh, kisspeptin and testosterone, the adipocytokines (leptin, adiponectin, visfatin and retinol-binding-protein- rbp ) and serum glucose, insulin and crp. the hyperandrogenic pcos groups had characteristically altered hormone profiles compared to matched controls. although total fat mass was comparable, women with c-pcos had a significantly larger waist circumference ( . . vs. cm, p< . ) trunk fat, r fat and %trunk and %r fat compared to ov-pcos (p< . ). leptin, rbp and visfatin did not significantly differ among the pcos subgroups although adiponectin was lower in the c-pcos group (p< . ). quicki was significantly lower in c-pcos ( . ± . vs. . ± . , p< . ) and insulin was higher ( . ± . vs. . ± . , p< . ). serum lh was also higher ( . ± . vs. . ± . , p< . ) but kisspeptin, testosterone and estradiol were similar. mis/amh ( . ± . vs. . ± . ng/ml, not significant) and crp ( . ± . vs. . ± . , p< . ) were higher in c-pcos. significant correlations (p< . ) were noted among kisspeptin/rbp (r . ), mis/testosterone (r . ), insulin/ %trunk fat (r . , p< , ), total fat/leptin (r . , p< . ), %trunk fat/adiponectin (r - . ). in conclusion, women with c-pcos when compared to similarly hyperandrogenic women with ov-pcos with similar bmi, have increased abdominal fat, and appear to have differences in serum lh, crp and increased insulin resistance. the latter, as well as subtle differences in the adipocytokines may explain these differences in anthropometric findings. problems of normal oogenesis and folliculogenesis but also disturbed oogenesis and folliculogenesis in polycystic ovaries are not fully understood. oocyte specific genes play an essential role in oogenesis and folliculogenesis. there are suggestions about possible role of some oocyte specific genes in etiopathophysiology of pcos. zona pellucida gene (zp ) is recently identified gene, which belongs to zona pellucida genes such as zp , zp and zp . the role of zp , contrary to above-mentioned other zp genes is not well described. the aim of this study was to analyze zp coding sequence and expression in patients with polycystic ovary syndrome. material included blood received from patients (mean age , +/- , years; mean bmi , +/- , kg/m ) with polycystic ovary syndrome. all patients with pcos were diagnosed with the use of eshre/asrm criteria from . dna was isolated from blood cells( after separation of blood cells from serum) using a dna isolation kit (qiagen ). genomic dna was used for in vitro amplification by pcr with a specific set of primers complementary to the coding sequence of the zp gene. products from each pcr reaction were examined by sscp method. samples with changes detected by sscp in comparison to control probes were cloned into plasmid vector and then automatically sequenced from a total of patient samples with pcos, we identified nucleotide changes in the zp coding seguence : silent nucleotide changes in exons , , , , and nucleotide change in the exon ( position , t>g). the mutation in exon ( t>g ) results in substitution of cystein for glycin of amino acid in position of zp protein. in summary, our data demonstrate that zp nucleotide changes account for % of patients with pcos. relationship between serum mullerian inhibiting substance levels and insulin in women with polycystic ovary syndrome. rebecca a chilvers, shilla chakrabarthy, summer james, xin ma, manubai nagamani. ob/gyn, university of texas medical branch, galveston, tx, usa. müllerian-inhibiting substance (mis) is a member of the transforming growth factor-superfamily of growth factors. it is expressed exclusively in granulosa cells and is believed to play a role in the regulation of follicle selection and maturation. women with pcos have anovulation and most of them have hyperinsulinemia. the purpose of our study is to investigate possible association between insulin and mis in the dysregulation of folliculogenesis in pcos. methods: twenty one women with pcos who had anovulatory cycles and hyperandrogenism were recruited for the study. sixteen women with ovulatory cycles and matched the pcos patients in age and bmi served as controls. an oral glucose tolerance test (ogtt) was performed in all women. blood samples were obtained at fasting and , and hours after glucose ingestion for measurement of glucose and insulin. to investigate the effect of hyperinsulinemia on mis secretion, mis levels were measured in ten patients during the ogtt. fasting mis, testosterone, dheas, fsh, and lh levels were measured in all patients. results: fasting insulin levels ( . + . μu/ml) vs . + μ u/ml [+ se]) (p < . ) and area under the curve (auc) of insulin ( . + . vs . + . μu/ml) (p < . ) were significantly increased in women with pcos compared to control women, while the glucose levels were normal indicating insulin resistance. mis levels were significantly (p < . ) increased in women with pcos ( . + . ng/ml) compared to controls ( . + . ng/ml). there was no correlation between age and mis levels in pcos patients while there was a highly significant negative correlation between the age and mis levels in the control women ( r = - . , p< . ). there was significant negative correlation between mis and fasting insulin levels (r = - . , p < . ) and insulin auc during the ogtt (r = - . , p < . ). there were no changes in mis levels during ogtt. conclusions: results of our study indicate that in women with pcos, ( ) there is an increase in secretion of mis, ( ) higher insulin levels are associated with lower mis levels, ( ) acute increase in insulin levels has no effect on mis levels, ( ) lower mis levels in women with severe hyperinsulinemia could be due to associated increased follicular atresia and a decrease in the ovarian reserve. further studies are needed to investigate the role of insulin on mis secretion. hiroyuki asakura, noriko tanaka, kyoko nishio. ohgimachi ladies' clinic, osaka, japan. objective: elevation of serum anti-müllerian hormone (amh) levels among polycystic ovary syndrome (pcos) patients has been reported. however, the regulatory factors of amh in pcos remain unknown. we examined correlations between amh values and various indices of insulin resistance in pcos women. methods: infertile women compatible with rotterdam criteria for pcos were recruited under informed consent. the subjects underwent g oral glucose tolerance test ( ggtt, sampling at , , minutes), and -step elisa for amh (sensitivity . pmol/l, inter intra-assay c.v. : . %, . %, respectively). p< . was considered as statistical significance. results: average amh level of the subjects (age: . + . years, bmi: . + . kg/m , mean+s.d.) was . + . pmol/l, which was higher than normo-ovulatory women ( . + . pmol/l, n= ). amh levels had significant positive correlation with total ovarian volume by ultrasound ( . + . cc), but not with bmi, waist-hip ratio ( . + . ), and levels of serum lh ( . + . iu/l), total testosterone ( . + . ng/dl). although fasting glucose levels ( . + . mg/dl) were positively correlated with total ovarian volume (r= . ), amh levels had no significant correlation with fasting insulin ( . + . iu/l), fasting glucose/insulin ration ( . + . ), homa-ir ( . + . ), and the sum of insulin levels ( . + . iu/l)during ggtt. conclusions: increased serum amh level of pcos and its positive correlation with total ovarian volume implies that determination of serum amh level would aid in confirming diagnosis of the ovulatory disorder. absence of relationship between amh and various clinical indices of insulin resistance suggests alternative regulatory factors of amh gene expression in the follicular compartment. reproductive tissues. amisra a nikrodhanond, keeley l mui, helen h kim. ob/gyn, university of chicago, chicago, il, usa. background: although primarily a neuroendocrine hormone, gonadotropinreleasing hormone (gnrh) is also produced in reproductive tissues, where it acts locally. the study of gnrh regulation in these tissues is limited by low levels of expression. objective: to elucidate mechanisms that regulate gnrh expression in male tissues, our objective was to identify regions of the gnrh gene that target expression in vivo. methods: transgenic mice were generated with different fragments of the mouse gnrh gene promoter (- /+ and - /+ bps), fused to the luciferase reporter gene. in these mice, luciferase activity (detected as light) reflects gnrh promoter activity. using bioluminescent imaging, gnrh promoter activity was assayed in live gnrh-luc mice and in their reproductive tissues ex vivo. to confirm imaging results, luciferase activity was also measured as relative light units (rlu) in tissue homogenates. for each dna construct, male mice were examined. with whole-body imaging, bioluminescence was detected in the genital region of the gnrh-luc transgenic mice (fig.a) . in mice that incorporated the - luc transgene, examination of reproductive tissues ex vivo revealed bioluminescence in the prostate and penis, but not in the seminal vesicles, epididymis, or testes (fig.b) . in contrast, in the - luc mice, bioluminescence was detected in the testes, but not in other tissues (fig.c) . examination of luciferase activity in tissue homogenates from - luc mice confirmed gnrh promoter activity in the prostate ( ± rlu) and penis ( ± rlu) and absence in the testes ( ± rlu). in the - luc mice, however, luciferase activity was detected only in the testes ( ± rlu) and not in the prostate ( ± rlu) or penis ( ± rlu). conclusions: our studies demonstrate that gnrh is present in male reproductive tissues, but may be differentially regulated in the testes vs. prostate/penis. promoter elements, contained within the proximal - bp of the mouse gnrh gene, are sufficient to mediate testicular gnrh expression while - bp of the gene promoter are necessary to direct expression to the prostate and penis. characterization of mouse ringo/speedy homologues. z walton, s uckac, o guzeloglu-kayisli, md lalioti, d sakkas, e seli. ob gyn, yale u., new haven, ct, usa. introduction: ringo/speedy (ringo/spy) is a recently discovered cyclindependent kinase (cdk) activator that functions similar to cyclins in controlling the cell cycle. ringo/spy plays a crucial role during meiotic maturation in xenopus by inducing meiotic g /m progression and germinal vesicle breakdown (gvbd). more recently, padmanabhan and richter demonstrated that ringo/spy mrna is repressed in the xenopus oocyte cytoplasm by pumilio . upon meiotic reactivation, pumilio loses its interactions with ringo/spy permitting its translation. ringo/spy is then expressed, leading to activation of cpeb by phosphorylation, which in turn elicits polyadenylation and translation activation of the mrna for a critical oocyte maturation factor, the mos kinase. in this study, we investigated the expression of ringo/spy in mouse. methods: ten different somatic tissues, testes, and ovaries were tested by reverse transcription-polymerase chain reaction (rt-pcr) for the expression of ringo/spy homologues and their alternative splicing variants. ringo/spy mrna expression was also tested in prophase i (pi) and metaphase ii (mii) oocytes, -cell, -cell, -cell, -cell embryos and blastocysts. amplification with actin primers provided a positive control and allowed semi-quantitative analysis. results: we analyzed the two previously identified homologues of ringo/spy (a and b). the two alternative splicing variants of ringo/spy a (a and a ) were separately studied for their expression profile. we also identified an additional alternative splicing variant of ringo/spy b and evaluated similarly. ringo/spy a ( and ) were expressed in testes, ovaries, and certain somatic tissues including brain, and spleen. ringo/spy b (both variants) were only expressed in testis. ringo/spy a or b were not present in mouse oocytes or early embryos. conclusions: our findings indicate that the previopusly described ringo/spy homologues a and b are not expressed in mouse oocytes or early embryos suggesting that either an alternative cdk-activator or a yet to be identified homologue of ringo/spy may be mediating meiotic g /m progression in mouse. testis specific expression of ringo/spy b, and previously described role of ringo/spy in meiosis suggests a role for this protein in male gametogenesis in mouse. treated with glyburide. jennifer aguayo, gladys a ramos, alethea hanley, carri r warshak, thomas r moore. reproductive medicine, university of california, san diego, san diego, ca, usa. objective: to determine the risk factors that may predict inadequate response to first-line glyburide monotherapy in pregnant women with type diabetes mellitus (dm) or gdm and to assess if non-responders are at increased risk of adverse pregnancy and neonatal outcomes. study design: this was a retrospective cohort of women diagnosed with type ii or gdm initially treated with glyburide at a single institution from - . maternal characteristics, adequate glycemic control defined as more than % of fasting glucose < mg/dl and one-hour post-prandials < mg/dl, and neonatal outcomes were assessed. non-responders were defined as failure to achieve adequate glycemic control on maximum daily doses of glyburide or intolerance due to side effects, necessitating switch to insulin therapy. statistical methods included bivariate analyses. results: of the women initially treated with glyburide, ( %) failed to achieve adequate glycemic control or did not tolerate glyburide. reasons for glyburide non-response were maximum dose ( mg/d) reached ( %), maternal hypoglycemia ( %) and other side effects ( %). there were no statistically significant differences between non-responders and responders with respect to family history of diabetes ( % vs. %, p= . ), prior history of gdm ( % vs. %, p= . ) and macrosomia (> g) ( % vs. %, p= . ) or hour glucose challenge test ( + vs. + mg/dl, p= . ). non-responders had a higher rate of obesity (bmi> ) ( % vs. %, p= . ) and earlier gestational age at initiation of therapy ( + . vs. + . wks, p= . ). there were no differences between the groups in the mean -week fasting ( + vs. + mg/dl, p= . ) and post-prandial glucose values ( + vs. + mg/dl, p= . ). no significant differences were observed in incidence of pre-eclampsia, primary cesarean delivery or birth weight. neonatal outcomes including ponderal index, neonatal hypoglycemia, nicu admission, and birth injuries also did not differ between the groups. conclusion: women who are obese and who require earlier initiation of glyburide therapy are at increased risk of non-response to glyburide monotherapy. however, despite non-response, these women can be managed with subsequent insulin therapy to achieve similar glycemic control and pregnancy and neonatal outcomes. a different diagnostic strategy using the gram, -hour glucose tolerance test for the diagnosis of gestational diabetes mellitus. yvonne w cheng, ingrid block-kurbisch, jennifer lydell, aaron b caughey. obstetrics, gynecology and reproductive sciences, university of california, san francisco, san francisco, ca, usa. objective: to examine whether a simplified strategy using the gm, -hour glucose tolerance test (gtt) may be useful for the diagnosis of gestational diabetes mellitus (gdm). methods: this is a retrospective cohort study of women with singleton pregnancy who received the -gm, -hour glucose challenging test (gct) for initial screening and the gm, -hour gtt as confirmatory tests for the diagnosis of gdm between and . various combinations of the gm, -hour gtt results were examined and compared to the diagnostic criteria for gdm established by the carpenter and coustan criteria using the receiver-operator characteristic (roc) curves. perinatal outcomes of women who would have had a false-positive or false-negative test results were compared to those who did not have gdm using the carpenter and coustan criteria. potential confounding factors were controlled for using multivariable regression models. results: , women had gm, -hour gtt results available for analysis during the study period. using gdm diagnosed by the carpenter and coustan criteria as reference, various diagnostic strategies was compared using roc curves (figure ) . summation of the -hour and -hour gtt results with a diagnostic threshold of mg/dl yielded the most optimal balance between sensitivity ( . %) and specificity ( . %). when compared to women without gdm, women who were diagnosed with gdm by c c criteria but not by summation of -hour and -hour gtts had higher odds of operative vaginal delivery (aor= . , % confidence interval [ci] . - . ) and neonatal birthweight > gm (aor= . , % ci . - . ). conclusion: using only the summation of only the -hour and -hour gtt results of the gm -hour gtt offers an alternative test strategy which may be more convenient and less costly for the diagnosis of gdm. however, women who would have gdm by the carpenter and coustan criteria but not by the summation method have higher odds of having an operative vaginal delivery and neonatal birthweight > gm. outcome of induction of labor indicated for term prom among women with or without a uterine scar. yifat ochshorn, avital skornick rapaport, adi reches, joseph b lessing, ariel many. obstetrics gynecology, lis maternity hospital, tel aviv sourasky medical center, tel aviv, israel. objective: we aimed at comparing the outcome of induced term deliveries presenting with prom with or without a previous uterine scar. methods: the computerized files of women delivered following induction of labor due to term prom, were reviewed. perinatal outcome parameters such as the mode of delivery, indication for cesarean section, rate of low ' apgar scores and nicu admissions were compared between women with and without a previous cesarean. results: during the study period women delivered in our institution following prom and induction of labor , of them had a previous uterine scar. parturients of both groups (with and without a scar) were similar with regard to age, gestational age at delivery and parity. cesarean section rate was higher for the previous scar group ( % vs %, p< . ). the most common indication for cesarean was arrest of dilatation and/or descent among women with previous scar accounting for % and % (p< . ) for women with and with no uterine scar, respectively. no differences were noted in neonatal outcome parameters such as rate of low ' apgar scores and nicu admission rate between the two groups. conclusion: induction of labor due to prom culminates in higher cesarean rate in women with one previous scar compared with parturients with no scar. perinatal outcome is similar between the two groups. acid base balance and immediate perinatal outcome of vertex compared with breech presentation in elective cesarean section. avital skornick rapaport, yifat ochshorn, joseph b lessing, yuval yaron, michael kupferminc, ariel many. obstetrics gynecology, lis maternity hospital, tel aviv sourasky medical center, tel aviv, israel. backround: apgar scores, umbilical blood ph and bicarbonate are generally lower and pco levels are higher in vaginally delivered breech neonates compared to cephalic deliveries. although cesarean delivery improved apgar scores there is a debate wether it improved the acid base status. this retroprospective study compared umbilical cord blood acid-base values and perinatal outcome of elective cesarean breech-delivery with those of elective cephalic cesarean delivery and to determine whether a different metabolic status and perinatal outcome should be expected in neonates in breech presentation. study design: the study group included singleton pregnancies delivered by elective cesarean section at term between january and march . computerized files of singleton breech presentation elective cesarean sections were compared to those of singleton vertex neonates delivered by elective cesarean section. demographic data included: maternal age, pregnancy week at delivery and parity. perinatal outcome measures checked were: birth weight, apgar scores at 'and ', umbilical cord venous and arterial ph and base excess. results: during the period between january and march there were singleton elective cesarean sections, of them were breech and vertex. the mean age, gravida and parity were significantly different between groups ( . vs. . , . vs. . . and . vs. . respectively, p< . ). the birth weight was significantly different - . gram for the vertex and . gram in the breech deliveries (p< . ) there were no differences in either apgar scores or umbilical ph between the breech and vertex neonates delivered by cesarean section at term ( - + w). venous and arterial po and pco levels were significantly different, though the differences were very small and we doubt if these differences have any clinical importance. conclusion: although vaginal breech deliveries are associated with increased risk of asphyxia during delivery, elective cesarean breech deliveries are not at increased risk for lower ph levels or apgar scores. the clinical significance of bleeding during the second trimester of pregnancy. arie koifman, amalia levi, yaron zaulan, avi harlev, eyal sheiner. obstetrics gynecology, soroka university medical center, ben gurion university of the negev, beer-sheva, israel; epidemiology and health services evaluation, faculty of health sciences, ben gurion university of the negev, beer-sheva, israel. objective: this study aimed at investigating clinical importance and pregnancy outcome in women suffering from bleeding during the second half of their pregnancies. methods: a population based study including all deliveries which took place in the soroka university medical center between the years - were examined. comparison was performed between patients with and without second trimester bleeding pregnancies terminated before weeks, multiple gestations and women lacking prenatal care were excluded . stratified analysis, using the mantel-haenszel technique, and a multiple logistic regression model were performed . results: during the study period, , singleton deliveries occurred in our institute. of these, ( . %) were complicated with bleeding upon admission during the second half of pregnancy. the cases were attributed to placental abruption ( . %; n= ) and placenta previa ( . %; n= ). independent risk factors associated with bleeding, were oligohydramnios, polyhydramnions, (odds ratio [or]= . ; % confidence interval [ci] . - . ; p= and . ; . - . ;p< . respectively ), suspected intra uterine growth restriction (iugr, . ; . - . ; p<. ), gestational age, previos abortions and maternal age. these patients subsequently were more likely to deliver by cesarean section (cs, . % vs. . %, or= . ; %ci . - . ; p< . ). perinatal mortality among patients admitted due to second half bleeding was significantly higher as compared to patients without bleeding (p<. ). conclusion: bleeding upon admission during the second half of pregnancy is an independent risk factor for perinatal mortality. careful surveillance, including fetal monitoring, is suggested in these cases in order to reduce the adverse perinatal outcome. crude and adjusted odds ratios for perinatal mortality among patients with vaginal bleeding. characteristics or % ci p crude or for perinatal mortality . . - . < . or adjusted for: < . iugr . . - . < . oligohydramnios . . - . < . premature rupture of membranes . . - . < . the predictors included neonatal pi, bmi, or birthweight while the outcomes included hypoglycemia, shoulder dystocia, acidemia and hyperbilirubenemia. roc curve analyses were utilized to evaluate the relationship between sensitivity and specificity for each of the predictors and outcomes, with an area under the curve (auc) significantly greater than . indicating a screening test that is better than chance. results: neonatal pi, bmi and birthweight are poor predictors of nicu admission, acidemia and hypoglycemia with all auc values not statistically significantly different than chance alone. they are a reasonable predictor of shoulder dystocia, with birthweight functioning the best (p< . ). in general, neonatal anthropometric measurements do not appear to be good predictors of short term neonatal outcomes. although they are a reasonable predictor of shoulder dystocia, they are not useful clinically for this outcome since they cannot be calculated until after birth. in future studies, new models of neonatal anthropometrics should be created to better predict adverse neonatal outcomes. neonatal anthropometric measurements and their relationship to perinatal outcomes expressed as auc and % confidence intervals history of at least one spontaneous preterm delivery were offered protocolbased prenatal care including first-or second trimester bacterial vaginosis screening, repeated ultrasound cervical length assessment, and supportive care by specialized nurses. women with a positive test for bacterial vaginosis were treated with oral metronidazol therapy, and women with a cervical length below . cm (before weeks of gestation) underwent a vaginal cerclage. progesterone administration was not provided. data on obstetrical and medical history, pregnancy, delivery and neonatal outcome were prospectively collected and evaluated. results: in total, pregnant women were prospectively followedup. eighty-seven women had experienced a preterm delivery in their last pregnancy, of whom had delivered before weeks. three women were diagnosed previously with a bicornual or septal uterus. twelve women received metronidazol therapy and women underwent a vaginal cerclage. eighty-four women ( . %) delivered at weeks or beyond. one-hundred and three women ( . %) delivered no earlier than weeks, and only one woman delivered at weeks. two women gave birth to a twin (at and weeks, respectively). two neonatal deaths due to pulmonary hypoplasia were recorded. conclusion: women at high risk of preterm delivery who were offered protocol-based prenatal care not including progesterone therapy had a better pregnancy outcome than would be expected from previous studies. our findings may question the reported beneficial effects of prenatal progesterone administration. premature rupture of membranes. tracy a manuck, alexandra g eller, m sean esplin, robert m silver. obstetrics gynecology, university of utah health sciences, salt lake city, ut, usa. objective: historically, outcomes following expectant management of midtrimester preterm premature rupture of membranes (pprom) have been uniformly poor. thus, many patients elected pregnancy termination. however, outcomes may be improved with recent advances in neonatal medicine. our purpose was to assess outcomes in expectantly managed early pprom in an era of improved maternal and neonatal care. study design: this is a retrospective cohort of patients from tertiary healthcare systems from - experiencing pprom . weeks gestation. patients electing immediate termination of pregnancy, carrying fetus(es) with lethal anomalies, or delivering within hours of pprom were excluded. survival without major morbidity was the primary outcome. data were analyzed using student t-test and chi-square as appropriate. results: a total of women carrying fetuses ( singleton, twin, triplet, quadruplet) met inclusion criteria. only the fetus in the "ruptured sac" was studied. pprom occurred at a mean of . (+/- . , range . - ) weeks. the average latency period was . (+/- . , range . - ) days, with a mean delivery gestational age of . (+/- . , range . - ) weeks. this communication provides the first report of fundal uterine necrosis following placement of multiple uterine compression sutures. only two previous published cases report occurrence of uterine necrosis following application of a uterine compression suture --both identified as the b-lynch technique --and neither of these cases report necrosis confined to the fundus. in our case, uterine atony was refractory to pharmacologic therapy and manual compression of the uterus appeared to decrease the amount of blood loss. therefore, we applied a traditional b-lynch which effectively contracted the majority of the uterus, while the fundus remained atonic. a second horizontal, square suture was then placed between the cornua and carried over the fundus (see figure one: red reflects b-lynch suture; blue represents second fundal, square stitch). the patient subsequently experienced significant, persistent fevers despite antibiotic therapy. a repeat laparotomy was performed, at which time we found uterine necrosis confined to the uterine fundus (see figure : photograph taken at the time of hysterectomy). the placenta showed no histologic evidence of chorioamniotis --hence, the patient's main risk factor for fundal necrosis was the compression sutures. physicians should be aware of the risk of uterine necrosis, especially after placement of multiple compression sutures and, more specifically, after placement of a compression suture confined to the uterine fundus. background: migraines are far more common in women than in men. the incidence of migraines increases in girls after puberty, reaching an incidence of % in women who experience migraine at least once a year around middle age. migraine has been postulated as one of the major risk factors for stroke during pregnancy and the puerperium.triptans are a class of serotonin receptor agonists used in the treatment of migraine headaches. triptans administered in combination with other drugs have been known to precipitate serotonin syndrome, a rare but potentially life-threatening condition clinically manifested by a triad consisting of mental-status changes, autonomic hyperactivity, and neuromuscular abnormalities. objective: to determine whether triptan monotherapy is associated with serotonin syndrome methods: using data mining techniques we analyzed the entire food and drug administration's (fda) adverse event reporting system (aers) database. results: after excluding reports of concomitant serotonergic medication or other potentially confounding medication, eleven reports remained of serotonin syndrome associated with triptan use without concomitant serotonergic medication. the mean age for these eleven cases was . years; nine cases occurred in females and two occurred in males. there were no apparent instances of overdose among these eleven cases. conclusions: serotonin syndrome is a rare but serious occurrence with the use of triptan monotherapy. such cases were seen with eletriptan, rizatriptan, sumatriptan, and zolmitriptan. because of the spontaneous nature of voluntary reporting to aers, the actual number of occurrences of serotonin syndrome in patients using triptans is probably higher and cannot be assessed from aers data. users of triptan in combination with an ssri or snri should be warned of this rare but serious adverse effect. during periods of drug initiation, dose escalation, or the addition of another serotonergic agent, patients should be particularly vigilant for symptoms of concern and seek urgent medical attention if any occur. the offending agents should be withdrawn and the patients closely monitored and treated with supportive measures as required. rising maternal mortality in the midst of modern technology. oormila p kovilam, jane khoury, padmini c sekar, ralph c buncher. obstetrics gynecology, university of cincinnati, cincinnati, oh, usa; center for epidemiology and biostatistics, cincinnati children's hospital medical center, cincinnati, oh, usa; environmental health, university of cincinnatic, cincinnati, oh, usa. introduction: maternal mortality rate (mmr) is an index of overall wellbeing of the community and safe motherhood should be given utmost priority in obstetric care. as per , who estimates % of maternal mortality occurs in developed countries . this rate has established without much decline in recent years. the mmr for ohio for to was reported by cdc to be . per , live births with % confidence interval . to . . objective: our objective was to audit the trend in maternal mortality in the state of ohio for the last years. methods: information from the death certificates completed by attending physicians, medical examiners, coroners, funeral directors filed with ohio state registration offices were analyzed. we only used women who were residents of the state of ohio at the time of death, and cause of death was coded as a "complication of pregnancy, childbirth and the puerperium", icd codes to and icd codes o to o . five year maternal mortality pattern and long term trend was assessed. results: the number of maternal deaths recorded as due to pregnancy complications varied over the years from a high of in to a low of in and . in general the five-year mortality rate was decreasing over time until the last four years to , when we may be seeing an upward trend compared to to (p= . ). the rates and associated % confidence intervals (ci) are shown in the table below. years mmr % ci ci - ci . . - . ci - . . - . - . . - . - . . - . - . . - . the rate of maternal mortality is on the rise after a period of downward trend. we should develop a multidisciplinary approach to analyze and target the root causes of maternal death. introduction. women with thrombophilia are at higher risk of vte during pregnancy due to the acquired hypercoagulable state. it is likely that not only maternal veins but also placental vessels are more prone to the development of thrombosis. our objective was to compare maternal and fetal placental circulation disorders in women with intra-uterine fetal death (iufd) and thrombophilia and women without thrombophilia. methods. in a dutch multi-centre study on iufd, during the period - we studied singleton deaths > weeks of gestation for which the diagnosis of iufd was determined before labour. factor v leiden and prothrombin g a were tested at induction of labour. panel classification of cause according to the tulip classification was performed by assessors after individual investigation of structured patient information. we studied the cause of death group "maternal and fetal placental circulation disorders": placenta bed pathology with abruption or infarction as origin of mechanism and placental parenchyma pathology with fetal thrombotic vasculopathy and massive perivillous fibrin deposition as origin of mechanism. results. of the women tested for factor v leiden, ( . %) were carriers. of the deaths caused by "maternal and fetal placental circulation disorders" ( . %) mothers were carriers of factor v leiden. of the deaths with another cause of death ( . %) mothers were carriers of factor v leiden (p= . ). of the women tested for prothrombin g a, ( . %) were carriers. of the deaths caused by "maternal and fetal placental circulation disorders" ( . %) mothers were carriers of prothrombin g a mutation. of the deaths with another cause of death ( . %) mothers were carrier of prothrombin g a mutation (p= . ). in women with iufd and factor v leiden or prothrombin g a mutation "maternal and fetal placental circulation disorders" did not seem to cause iufd more often than in non-carriers. , university medical center groningen, groningen, netherlands; pathology, university medical center groningen, groningen, netherlands; trial coordinating center, dept of epidemiology, university medical center groningen, groningen, netherlands; hematology, university medical center groningen, groningen, netherlands. introduction. growing evidence suggests that women with thrombophilic defects may be at higher risk of fetal loss. although some paternal components to the predisposition of preeclampsia have been demonstrated it is not known whether paternal components contribute to intra-uterine fetal death (iufd). our objective was to investigate the relation between paternal thrombophilic defects and iufd. methods. in a dutch multi-centre study on iufd, from - we studied singleton deaths > weeks of gestation for which the diagnosis of iufd was determined before labour. we tested male partners of women with iufd for antithrombin (at), protein c, protein s type i and iii, factor v leiden, prothrombin g a (factor ii) and factor viii: ag. standard tests were performed in one laboratory. normal ranges were determined in healthy male blood donors. we compared prevalence of thrombophilic defects to reference values from the literature. . of the men tested for factor v leiden, ( . %) were carrier versus ( . %) non-carriers. prevalence of factor v leiden in the normal population is % (p= . ). men were tested for prothrombin g a, ( . %) were carriers and ( . %) non-carriers. all were heterozygous. prevalence of prothrombin g a mutation in the normal population is % (p= . ). decreased levels of antithrombin, protein c, protein s type i and iii and increased levels of factor viii: ag were observed significantly more often in male partners of women with iufd compared to the normal population. conclusion. in our iufd group the prevalence of male factor v leiden carriers was comparable to the normal population, prevalence of prothrombin g a mutation was lower. the difference in other factors imply an as yet unexplained association of male thrombophilia with fetal death in their partner. objective fetal macrosomia, the most important complication of gdm, increases the risk of shoulder dystocia, erb's palsy and intrauterine hypoxia. we compared frequencies of fetal macrosomia and erb's palsy in two gdm cohorts with different severity of glycemic disturbance and in healthy controls. methods we studied consecutive gdm women with singleton childbirth and or abnormal values in -h ogtt with g of glucose. abnormal plasma glucose values of the ogtt were: fasting . , -h . and -h . mmol/l. insulin treatment was started when values were . preprandially or . mmol/l postprandially in the -h glucose profile done within days of diagnosis. if the same woman had more than childbirth during the study period, only the last pregnancy was included. the control group consisted of women from a nearby town with singleton childbirth in the same hospital. women with diabetes were excluded from controls. macrosomia was defined as birth weight (bw) > . sd above the mean of a standard population. erb's palsy was diagnosed by pediatric surgeons. results gdm women were older, more obese, had more childbirths and more often a previous child with bw > g than controls. insulin was started in gdm women ( . %). c/s rate was . % in controls, . % in diettreated and . % in insulin-treated gdm women. macrosomia rate was . % in controls, . % in diet-treated and . % in insulin-treated gdm women (p< . compared with controls or diet-treated gdm women). the frequency of macrosomia did not differ between the diet-treated gdm and control women. erb's palsy occurred in . % of controls, in . % of diet-treated and in . % of insulin-treated gdm women. the frequency of erb's palsy was significantly higher in both gdm groups than in controls (p= . ). by regression analysis, previous child with bw > g (p< . ), previous c/s (p= . ), mother's age (p= . ), bmi (p= . ), insulin treatment (p= . ) and fasting plasma glucose of the -h profile (p= . ) were significant independent predictors of fetal macrosomia. conclusions the -h glucose profile done shortly after the diagnosis of gdm clearly distinguishes between low-risk (diet-treated) and high-risk (insulintreated) gdm pregnancies for fetal macrosomia. unfavourable fetal body composition of diet-treated gdm women is the likely explanation for the high rate of erb's palsy in this group. . '-:·:tpartq,-, '-:·:tpartq,-, '-:·:tpartq,-, % p= . ). first trimester uta doppler indices were similar in the two cohorts in terms of the resistance and pulsatility indices (ri . vs. . , p= . ; pi . vs. . , p= . ) . however, bilateral notching was much more common in the cohort of prior adverse outcomes ( % vs. % p= . ) as well as in patients destined to have a subsequent poor outcome (p= . ). conclusions: not surprisingly, prior poor obstetric outcome was strongly associated with recurrent adverse obstetric outcome. uta notching was robustly associated with prior poor obstetric history as well as a recurrent poor outcome. this clinical history had no discernable influence on ri or pi. reassurance may not be offered based on first trimester uta ri and pi. anti-retroviral therapy is associated with increased blood loss and uterine atony for patients undergoing primary cesarean section. carey eppes, alice cootauco, melissa russo, jessica bienstock. gynecology and obstetrics, johns hopkins university school of medicine, baltimore, md, usa. objective: anti-retroviral therapy has been associated with gastrointestinal smooth muscle dysfunction. it has also been hypothesized that myopathies can occur secondary to anti-retroviral therapy due to mitchondrial alterations. in our experience, we have noticed an increased incidence of uterine atony in our hiv patients on anti-retroviral therapy. we sought to examine the incidence of postpartum hemorrhage and uterine atony in patients currently on anti-retroviral therapy. study design: a retrospective case-controlled study was conducted on all hiv positive pregnant women on anti-retroviral therapy undergoing a primary low segment transverse cesarean section from through . these patients were obtained from an irb approved database containing hiv positive patients within our institution. patients' medical records were abstracted for demographic data, use of uterotonics, preoperative and postoperative hematocrits, and incidence of blood transfusions. controls were matched for age, parity, gestational age and surgical indication. data was analyzed using the t-test, fischer's exact test and chi square. results: there were no differences in demographics, incidence of chorioamnionitis or magnesium sulfate use between groups. patients on anti-retroviral therapy had a statistically greater decrease in hematocrit and estimated blood loss compared to controls. they also had an increased need for uterotonics and blood transfusions. conclusion: anti-retroviral therapy may impact uterine smooth muscle contractility in pregnancy, as evident by the increased incidence of uterine atony and change in hematocrit. additional research is needed to elucidate the mechanism. clinicians should be aware of the potential for uterine atony and excessive blood loss in patients on anti-retroviral therapy. background: adolescent pregnancy is frequently associated with adverse outcomes, especially small-for-gestational age (sga) deliveries. some studies have implicated maternal nutritional status in these poor outcomes. methods: in a prospective longitudinal study, ethnically-diverse, pregnant adolescents were studied from booking to parturition, with collection of anthropometric and nutritional variables. blood samples ( - weeks' gestation; n= subjects) were assayed for a spectrum of nutritional biomarkers. logistic regression was used to determine significant associations between studied variables and pregnancy outcomes. results: median age at recruitment was . years (iqr: . - . ). outcome data was available for subjects. ( . %) had uncomplicated pregnancies. median birthweight was , g (iqr: , - , g) and median birthweight centile was . % (iqr: . - . %). ( . %) infants were born sga and ( . %) were preterm. spontaneous vaginal deliveries occurred in . % of cases. there were ( . %) cases of pre-eclampsia and ( . %) admissions to neonatal care. . % of subjects reported smoking at booking. iron deficiency anaemia was prevalent in . % of subjects by - weeks and was strongly associated with higher infant birthweight centile (p< . ). . % had serum -hydroxy vitamin d concentrations < ng/ml, although this was not associated with any outcomes. low folate status, as indicated by low red cell folate (p= . ), low serum folate (p= . ) and high serum homocysteine (p= . ) concentrations, was associated with higher rates of sga birth. conclusion: adolescent pregnancy in inner-city populations is associated with a high risk of sga and preterm birth, increasing the likelihood of health problems in later life and perpetuating social disparities in health. the association between anaemia and higher birthweight is unexplained. impaired maternal folate status may contribute to impaired fetal growth. we suggest that sga birth in this population could be reduced by the use of antenatal supplements or the mandatory fortification of flour with folic acid. amount of cigarette use pre-pregnancy and during pregnancy were self-reported and subgrouped into nonsmokers, quitting during pregnancy, and continued smoking throughout pregnancy. categorical outcomes were compared using chi-square test. multivariable logistic regression analyses were used to control for potential confounders (continued smoking compared to quitting during pregnancy). results: women who quit smoking during pregnancy had higher rates of pregnancy-associated hypertension and cesarean delivery but lower rates of preterm delivery and neonatal birthweight < gm compared to non-smokers. compared to women who quit smoking during pregnancy, those who continue to smoke have lower odds of pregnancy-associated hypertension and cesarean delivery, but higher odds of preterm delivery, neonatal birthweight < gm, and apgar score < at minutes (see table) conclusion: quitting cigarette smoking during pregnancy appears to reduce undesirable neonatal outcomes, though an increase in pregnancy associated hypertension. these findings should be emphasized to women who are smoking during pregnancy. to achieve effective tamponade the balloon was inflated initially up to - ml with incremental increases of volume by ml until bleeding stops. to measure intrauterine balloon pressures we used a standard pressure transducer (edwards lifesciences) connected to the balloon port and to a pressure monitor (ge dash ). the pressure transducer was mounted on the bedrail at uterine level, primed with sterile saline and then connected to the balloon port of the catheter. the system was zeroed to atmospheric pressure, the stopcock of the intrauterine balloon port was opened and the pressure was recorded (p : the total pressure on the fluid). to verify the accuracy of measurements we used another balloon catheter (reference), inflated with the same amount of saline, connected to the pressure transducer and zeroed to atmospheric pressure. the reference balloon measured p (the intrinsic balloon pressure caused by distension at that volume). the system was then zeroed to the reference balloon. the pressure transducer was then reconnected with the intrauterine balloon and the actual intrauterine pressure was recorded (p ). we calculated the correlation coefficient between p and systolic, diastolic and mean blood pressure using a linear regression (statsdirect statistical software there is a growing body of evidence to suggest that peripartum assessment of fetal or neonatal lactate levels are as good as or better than standard blood gas analysis in the prediction of neonatal outcome. in this study we have evaluated the ability of umbilical cord blood gases and lactate levels in the prediction of neonatal hypoxic-ischaemic encephalopathy (hie). department of neonatal paediatrics, king edward memorial hospital, perth, western australia, australia; women and infants research foundation, king edward memorial hospital, perth, western australia, australia. objective: current evidence suggests that umbilical cord ph at delivery provides the most sensitive reflection of birth asphyxia. paired umbilical artery/vein blood gases have been routinely collected at king edward memorial hospital over the last years. the objective of this study was to determine: local reference ranges; accuracy of sampling; and the rates of metabolic acidosis. of the , births ( ) ( ) ( ) ( ) , accurate paired results were available on % of births. over the study period there was a progressive improvements in accuracy rates of paired sampling (p< . ). the median ( . th , . th centile) values for cord arterial blood gases were: ph . ( . , . ); po . mmhg ( . , . ); pco . mmhg ( . , . ); base excess - . (- . , . ); and lactate . mmol/l ( . , . ). there was a progressive improvement in all blood gas measures over the years of this study (all p< . ). moreover, there were significant reductions in all measures of metabolic acidosis (see table) . the progressive improvement in the measures of metabolic acidosis remained significant after multivariate analysis including obstetric, fetal, and demographic factors associated with metabolic acidosis. the introduction of universal umbilical cord blood gas analysis to all births is associated with significant improvements in all markers of metabolic acidosis. together with other compelling evidence in the literature, these data support the routine use of cord arterial and venous gases at all births; however, improved accuracy rates on paired sampling requires an ongoing education program umbilical artery indicators of acidosis, percentage of total ph < . ph < th centile* objective: intrapartum pcn prophylaxis aims to prevent early-onset gbs sepsis by interrupting vertical transmission from colonized mothers to their newborns. however, despite its wide clinical use, systematic pharmacokinetic evidence in support of the current pcn dosage regimen is lacking. current cdc guidelines recommend intensified surveillance and testing of infants exposed to < h of prophylaxis. our goal was to examine the relationship between maternal time of exposure to pcn and fetal serum pcn levels among maternal-fetal dyads exposed to short durations of pcn prophylaxis (< h) compared to those exposed to longer durations. study design: ninety-eight laboring gbs positive women were administered million units (mu) of intravenous pcn to be followed by . mu every hours until delivery (cdc ) . subjects with renal disease, multiple gestation, and preterm delivery (< wks) were excluded. umbilical cord blood samples were collected at delivery and pcn levels measured by high-performance liquid chromatography. intra and inter-assay coefficients of variation were < %. results: the pcn concentrations (mean±sd) by duration of prophylaxis were: < h, . ± . g/ml (n= ); - h, . ± . g/ml (n= ); - h, . ± . g/ ml (n= ); - h, . ± . g/ml (n= ); - h, . ± . g/ml (n= );> h . ± . g/ml (n= ); and for those without a second dose after h, . ± . g/ml (n= ). fetuses exposed to short duration (< h) had higher levels of pcn than those exposed to > h (p= . ). in multivariable linear regression analysis, fetal pcn levels were determined by total duration of exposure, time since last dose, dosage, and number of doses, but not maternal bmi. pcn levels in cord serum increased linearly until hour; thereafter, they decreased rapidly, but all groups were significantly above the minimum inhibitory concentration (mic) for gbs ( . g/ml)(p< . ). furthermore, every sample individually remained - fold above the mic. conclusion: in this study, even short durations of prophylaxis achieved levels above the mic, suggesting a benefit to prophylaxis even in precipitous labors. the data also suggests that the current cdc designation of infants exposed to < h of pcn prophylaxis as particularly at risk for gbs sepsis may be inaccurate from a pharmacokinetic standpoint. objective: -oh aa is a metabolite of tryptophan with pro-oxidant and proapoptotic properties and has been shown to increase in umbilical cord blood in pregnancies with intra-uterine infection. since labour-related events may also activate inflammatory pathways, we sought to determine the placental release of -oh aa into the umbilical circulation in labouring vs non-labouring patients at term. methods: twenty-six patients were studied (term labour n= , and term elective cesarean section n= ) with blood sampling from a clamped segment of umbilical cord after delivery of the fetus and from the cord at its insertion into the placenta after delivery of the placenta, with subsequent measurement of blood gases/ph and -oh aa (isocratic hplc using fluorometric detection with assay sensitivity at pmol). results: -oh aa measurements from respective umbilical and placental cord vessels were all variably higher in the labouring group vs the elective cesarean group patients (table ) . for labouring group patients, the -oh aa levels from the umbilical vein were significantly higher than those from the umbilical artery, indicating net release from the placenta into the fetal circulation. placental vein levels were also significantly higher than those from the umbilical vein, indicating continued placental release of -oh aa into the cord blood after delivery of the fetus. conclusion: labour at term is associated with changes in the placental metabolism of tryptophan resulting in the increased release of -oh aa into the fetal circulation with the potential for pro-oxidative and apoptotic effects in many tissues, including the brain. obstetrics, gynecology, and reproductive sciences, new brunswick, nj, usa; department of obstetrics and gynecology, mineola, ny, usa. objective: ischemic placental disease (preeclampsia, small for gestational age, sga and placental abruption) is a major contributor to pregnancy-related morbidity. although the placenta is considered a fetal organ, it is accepted that ischemic placental disease (ipd) can present clinically with either fetal or maternal manifestations. we hypothesized that the pattern of diagnosis (maternal versus fetal) varies by gestational age and would provide insights in to origins of indicated and spontaneous preterm birth. methods: this was a retrospective cohort study utilizing the maternallylinked reproductive history data for missouri residents , restricted to singleton live births. women who experienced spontaneous onset of labor and subsequently delivered preterm were classified as spontaneous preterm birth. medically indicated preterm birth included women who delivered preterm through a labor induction or (prelabor) cesarean delivery. ipd was classified as maternal (preeclampsia only), fetal (sga only) or both (preeclampsia with sga or abruption, and all conditions). results: among term births with ipd, . % presented as maternal disease only, . % as fetal disease, and the remainder ( . %) as both. among spontaneous preterm births with ipd, a greater proportion were of fetal presentation ( bethesda, md, usa; dept ob/gyn, wayne state univ, detroit, mi, usa; dept pathology, wayne state univ, detroit, mi, usa; dept ob/gyn, soroka univ medical center, israel. objective: hemoglobin (hg) and its catabolic products have been observed in cases of amniotic fluid (af) discoloration, which is a risk factor for intraamniotic infection/inflammation (iai). the study aimed to determine the association between af fetal hg concentration and gestational age, term and preterm labor and iai. study design: this cross-sectional study included: ) mid-trimester (n= ); ) term not in labor (tnl) (n= ); ) term in labor (tin) (n= ); ) preterm labor (ptl) who delivered at term (n= ); ) ptl without iai (n= ); ) ptl with iai (n= ); ) preterm prelabor rupture of membranes (pprom) with (n= ) and without iai (n= ). af fetal hg concentrations were determined by elisa. results: ) fetal hg was detected in . % of all af and . % of mid-trimester samples; ) women at tnl had a higher median af fetal hg concentration than patients at mid-trimester ( . ng/ml, iqr - vs . ng/ml, iqr . - . , p= . ); ) no differences were found in median af fetal hg concentration among patients with and without labor at term (til: . ng/ml, iqr - . ; p= . ); ) median af fetal hg concentration was not significantly different among the ptl subgroups [ptl with iai: . ng/ml, ptl who delivered preterm: . ng/ml, ptl without iai who delivered at term: . ng/ml, p= . (kruskal wallis) ]; ) in pprom, there were no differences among patients with and without iai ( . ng/ml, respectively; p= . ) ; ) median af fetal hg concentrations were significantly higher in ptl or pprom, with or without iai than in pregnant women at term, with and without labor (p< . for all comparisons). conclusions: ) immunoreactive af fetal hg increases with gestational age; ) among women with ptl or pprom, the median af fetal hg concentration is not associated with iai; ) the median af fetal hg concentration is higher in pregnancies complicated with ptl or pprom than in term pregnancies. the impact of latency time to delivery after preterm premature rupture of membranes (pprom) on neonatal outcome. dan nayot, deborah penava, barbra de vrijer, orlando da silva, bryan s richardson. obstetrics and gynaecology; pediatrics, university of western ontario, london, on, canada. objective: there continues to be controversy as to the management of pprom with conservative management to advance gestational age (ga) versus aggressive management with early induction to avoid chorioamnionitis. we have therefore used the perinatal and neonatal databases of a large regional patient population to determine the association of pregnancy variables with latency time to delivery after pprom and the impact of latency duration on adverse neonatal outcomes. methods: the perinatal/neonatal database of st. joseph's health care, london, ontario was used to obtain demographic and neonatal outcome information for all patients with pprom > and < weeks gestation, singleton and no major anomalies, delivering between january , and december , . patients were grouped according to ga at pprom stratified for latency time < hrs vs > hrs with incidences for those pregnancy related variables and neonatal outcomes available from the database then compared with the use of logistic regression analysis. results: there were patients who met the inclusion criteria of whom %, %, and % had pprom at - wks , at - wks, or at - wks, respectively, and with these pprom groupings showing a stepwise decrease in the percentage of patients with latency to delivery > hrs, at %, %, and %, respectively. pregnancy related variables and neonatal outcomes for these patient groupings are as shown in table . conclusion: despite a to fold increase in the incidence of chorioamnionitis with latency to delivery > hrs, a policy of conservative management to advance ga after pprom will result in decreased severe infant morbidity until weeks, and moderate infant morbidity until weeks. hospital. outcomes studied were prolongation of latency period with intact membranes and prom using magnesium sulfate (mgso ), nifedipine, terbutaline and tocolytic agents. secondary outcomes examined were maternal complications. statistical analysis performed were t-test and . comparisons were expressed as odds ratios ( % ci). results: in a -year period, twin gestations were admitted in ptl and administered tocolytic agent(s) with mean gestational age of . weeks. when tocolytic agents were used, maternal complications were: ( . %) with intact membranes and ( . %) with prom had pulmonary edema (or= . , ci . - . ); ( . %) with intact membranes and ( . %) with prom had chorioamnionitis (or= . , ci . - . ); ( . %) with intact membranes and ( . %) with prom had postpartum hemorrhage (or= . , ci . - . ). conclusion: there is no difference in prolongation of latency period achieved in twin gestations in ptl with intact membranes or prom using tocolytic agents. similarly, there is no difference between the groups with latency period hrs. there is an increased likelihood of pulmonary edema and chorioamnionitis with use of tocolytic agents. however, results are not significant due to type ii error. mean latency period and latency ≥ hrs using single or multiple tocolytic agent ( introduction: high sensitivity crp (hscrp) is a serum marker of inflammation and has proven clinical utlility in predicting cardiovascular disease (cvd). considering the hypothesized association between preeclampsia (pre) and inflammation and cvd, it is plausible that hscrp may have utility in predicting pre. prior to widespread utilization of this marker, the affect of labor on levels of crp needs to be clarified. we assessed the association between labor and hscrp levels in term deliveries and between elevated hscrp and adverse perinatal outcomes. a secondary analysis comparing hscrp in women with preeclampsia (pre) to those without was performed. methods: women presenting for term delivery or pre were prospectively identified as part of a case-control study. clinical data and serum were collected for all subjects. a standard immunoturbidimetric assay was used to measure hscrp levels. women presenting for induction of labor or planned cesarean delivery (non-labor) were compared to women presenting in labor. a secondary analysis comparing non labor women with and without pre was performed. serum was collected prior to labor induction in the non-labor group. nonparametric comparisons were made using wilcoxon rank sum tests. mvlr was used to evaluate dichotomous outcomes and control for confounders. results: women were included (non-labor group (n= ), labor group (n= ), non-labor pre (n= )). the median and mean hscrp levels were and and and in the non-labor and labor groups respectively (p= . ). elevated levels of hscrp in these term deliveries were not associated with chorioamnionitis (p= . ), maternal postpartum complications (endometritis, hemorrhage, transfusion) (p= . ), mode of delivery (p= . ), or admission to the nicu ( . ). levels of hscrp were significantly greater in the non-labor pre group compared to non labor without pre (mean . vs. . , median vs. , p< . ). conclusion: use of hscrp as a biomarker may improve clinical prediction of obstetrical complications such as pre. levels of hscrp are affected by labor and this should be taken into account when studying the utility of this biomarker. further, crp levels are elevated in women with pre even after excluding patients in labor. further investigations to determine if crp elevation in term labor is associated with adverse outcomes may be warranted. the role of hscrp as a valid and discriminating biomarker in pre should be assessed. review of the electronic labor record facilitated collection of maternal demographic, intrapartum, and newborn data. data analyzed with t-tests, chi-square tests, and calculated odds ratios with % cis as appropriate. a forward stepwise logistic regression analysis was used to identify predictors of histologic chorioamnionitis. results: of submitted placentas, had histologic chorioamnionitis (cases) and did not (controls). the groups were similar with respect to age, race, gbs status, and mode of delivery. gestational age, birthweight, duration of labor and ruptured membranes, and number of vaginal exams were greater in the cases (p . ). the cases were more likely to have had epidural anesthesia (or . ), internal monitoring (or . ), fever (or . ), maternal tachycardia (or . ) and fetal tachycardia (or . ) and less likely to have had induction of labor (or . ). newborns in the histologic chorioamnionitis group were more likely to have been observed for sepsis (or . severe sepsis defined as sepsis associated with acute respiratory distress syndrome (ards) or cardiovascular dysfunction(cvd) or with or more other organ dysfunction.all patients were resuscitated with fluids and treated with broad spectrum antibiotics and supportive care as needed. outcome data were: etiology, management, maternal complications, duration of icu stay and perinatal survival. results patients were young (mean age= . ± . years) with a mean gestational age at delivery . ± . weeks. etiologies were pyelonephritis(n= ), septic abortion(n= ), endomyometritis (n= ), chorioamnionitis (n= ), ruptured appendix(n= ), pneumonia( n= ) and one unknown. eighteen ( %) were diagnosed during antepartum and ( %) postpartum period.there were ( %) maternal deaths and high rate of major morbidities (table) . among the antepartum patients,there were, abortions, iufd, neonatal death for a perinatal survival rate of only %. conclusion pregnancies complicated with severe sepsis/septic shock are associated with substantial maternal and perinatal morbidities. the low maternal mortality rate in our study as compared to previous reports is attributed to early diagnosis and aggressive management of maternal complications. fetal loss rate, however continues to be high when septic shock develops antepartum. and tnfa levels are elevated. we developed a dynamic computer (in silico) model of pregnancy (uterine myometrial environment -infection/inflammation and endocrine crosstalk). mathematical differential equations were used to describe the interactions between molecules. infection was represented as increased levels of activated, nuclear transcription factor nf-kb. in the model ru inhibited both the glucocorticoid receptor and progesterone receptors. simulations were run adding different concentrations of ru ( . um= dose used in patients, . um, . um) at different time points during infection (before, at the time of or after nf-kb activation). ru degradation kinetics was also included. the effect of ru on nf-kb induced il- and tnfa levels was assessed. results: infection induced nf-kb activation led to increased il- and tnfa levels. there was a subsequent increase in cortisol that led to dampening of nf-kb activation, il- and tnfa levels. in the presence of ru , il- and tnfa levels continued to rise. the effect of ru on nf-kb induced il- and tnfa was dose dependent and was more prominent in slow metabolizers who had ru in the system for a longer time. addition of ru after the onset of nf-kb activation led to increased il- and tnfa levels above those observed without ru . the addition of misoprostol (prostaglandin e analogue), at the concentrations used together with ru for medical abortion, did not add to the effect of ru on nf-kb induced il- or tnf. conclusions: ru has dose dependent effects on infection induced immune activation and may contribute to the pathogenesis of c sordelii induced sepsis syndrome. the increased susceptibility of neonates to infection remains a major clinical problem. we previously found that after intraperitoneal (ip) listeria monocytogenes infection, neonatal mice have an ld that is orders of magnitude lower than adults. we also found that the inflammatory response of neonatal mouse macrophages, but not neutrophils, in the peritoneal fluid was deficient, and that this correlated with low levels of macrophage chemokines mcp- and rantes. given that the liver and spleen are important organs in listeria infection, we sought to characterize the innate immune response in these organs. a sublethal dose of listeria was injected intraperitoneally into balb/c - week old adult mice and - day old neonatal mice. liver and spleen was collected at , , and hours, sectioned serially for staining with hematoxylin-eosin and primary antibodies (rabbit anti-listeria monocytogenes igg; mhc class ii rat anti-mouse igg, a marker for activated macrophages; f / rat antimouse igg, a marker for macrophages) and secondary antibodies (cy- goat anti-rabbit igg; af goat anti-rat igg) were applied. negative controls used only secondary antibody. real time pcr was used to compare the levels of the chemokines mcp- and rantes in adult and neonatal liver. after ip infection, both adults and neonates showed similar influx of neutrophils to the sites of infection within the liver and spleen. at hours post-infection with listeria, adult liver and spleen showed increased staining for f / and class ii, which increased further and became confluent surrounding microabscesses at and hours. in contrast, the listeria infected neonatal mouse showed some increase in f / around microabscesses but no apparent increase in staining for mhc class ii. the neonates showed greater staining for listeria at each time point. mcp- and rantes levels were higher in infected neonatal liver compared to adults. in the neonatal mouse, the innate immune response in the liver and spleen was characterized by a deficiency of activated macrophages. this deficiency was not correlated with hepatic expression of macrophage chemokines. is there a seasonal pattern in the incidence of post-cesarean endometritis? tamula m patterson, alan tn tita, william w andrews. obstetrics and gynecology, the university of alabama at birmingham, birmingham, al, usa. objective: several theories, including one suggesting a peak in july coincident with resident turnover, postulate seasonality in post-cesarean infections. we assessed whether there is seasonal variation in endometritis. a retrospective cohort study of post-cesarean endometritis at our university-based institution using our obstetric computerized database to compare annual variation in monthly incidence patterns from to . prior to establishing an average aggregate seasonal pattern for all years, years were assessed separately for a recurrent pattern of peaks and nadirs in incidence. peak incidence (or nadir) was defined as any monthly incidence that differed from the mean incidence for the year by over %. results: a total of . % ( , ) of , deliveries from to were by cesarean. annual cesarean rates increased by an absolute rate of over %; while, post-cesarean endometritis rates decreased from % to %. monthly incidence of post-cesarean endometritis did not reveal a consistent recurrent pattern of peaks (figure ) or nadirs. the month of july accounted for only out of a total of peaks for all years. the adjacent months of june and august accounted for only each. the month with the highest number of peaks was april with only . these findings contraindicated the establishment of an average aggregate monthly pattern for all years. the incidence of post-cesarean endometritis did not follow a seasonal pattern. chi-square analyses were used to compare associations between race (black (bl) vs non-black (nbl)) and dichotomous characteristics. student´s t-test was used to compare continuous variables. results , patients were evaluated ( cases and controls). % and % of cases and % and % of the controls were bl and nbl respectively. the baseline prevalence of chtn in bl and nbl controls was . % and . % (p= . ). when comparing bl and nbl cases, bl women had a higher mean systolic blood pressure and screening bmi. bl women also had a trend toward being discharged on post partum blood pressure medicine when compared to nbl women. there was no difference in chtn, diabetes, severity of disease, iugr, or delivery < wks between the two groups (table) . to determine the leading causes of death in a case series of stillborn infants examined in a large hospital autopsy service, and to describe the most common post-mortem observations. study design: one hundred and sixty one stillborn infants were examined. gross pathology observations were recorded at autopsy and during the placental exam, and tissue sections were collected and examined microscopically. immediate and underlying cause of death (cod) were recorded, along with contributory cod, concomitant/significant cod, and incidental findings. statistical analysis was conducted using the software spss v. . . results: the immediate anatomic cod could be determined in . % of all infants examined. in over % of these cases, cod was attributable to placental or umbilical cord findings affecting the maternal-fetal blood supply. the most prevalent among these findings were placental lesions (maternal floor infarction, placental abruption, fetal thrombotic vasculopathy), umbilical cord lesions (entanglement, true knot, compression, excessive length/twisting), and infectious/inflammatory processes (chorioamnionitis, chronic villitis objective: advanced maternal age (ama) is associated with increased risk of intrauterine fetal demise (iufd). antenatal testing (at) is widely used in clinical practice to prevent iufd due to uteroplacental insufficiency and has been suggested to reduce the risk of iufd in ama women. we sought to assess the impact of at on obstetrical interventions and compliance with new practice recommendations. methods: retrospective cohort of ama women ( and older at their due date) who delivered at or after weeks. non-exposed women delivered from july to december (when at for ama was not routinely recommended); exposed women delivered from july to december (after at for ama was introduced at our institution). subjects were identified through the perinatal database; records were abstracted for demographics, medical history, and labor/delivery variables. outcomes included rates of at and induction of labor (iol) and mode of delivery. associations between at for ama and outcomes were tested using t test and chi square. assuming a baseline rate of iol of %, we had % power to detect an increase to % after the introduction of at. results: women met the inclusion criteria: delivered before the introduction of at (non-exposed=before at) and delivered after the introduction of at (exposed=after at). baseline clinical characteristics were similar in both groups. as anticipated, at was more common in the after at group than in the before at group ( % vs %; p< . ). women were not eligible for or declined trial of labor, thus not "at risk" for iol and not included in all analyses. ob intervention rates were increased after at compared to before at (table ) . the corrected iufd rates were similar in both groups ( / vs / ). conclusion: at an academic center, compliance with new practice recommendations was excellent. introducing at testing for a new indication seemed to increase iol and cs rates. these findings should be considered when assessing the risks:benefits ratio of antenatal testing. . we stratified indications for cesarean into the following: failed induction (cervix < cm dilated), arrest of dilation, arrest of descent, failed operative delivery, fetal intolerance of labor (fil), and "other" reasons (e.g., pre-eclampsia, abruption, chorioamnionitis, malpresentation). both fil and "other" reasons were more likely to occur among the medically indicated group (rr . , . physicians in solo practice had higher rates of elective inductions (p< . ), but there was no association between cesarean and practice type. conclusions: inductions accounted for . % of cesareans. these results suggest an increased risk for cs for patients undergoing medically indicated inductions at our institution. there was no association between cesarean and type of practice, whether solo or group, suggesting institutional clinical policies may be more important than practice type in determining delivery outcome after induction. further research is needed to understand how age, race/ethnicity, or other unmeasured patient factors may impact these findings. given rising rates of both cesarean delivery and inductions, this information may be pertinent to women considering elective induction prior to weeks. objective: fetal demise can lead to a consumptive coagulophathy ("fetal death syndrome") traditionally attributed to the release of "tissue thromboplastin", now known as "tissue factor" (tf). tf is the most potent activator of coagulation. despite the appeal and acceptance of this proposed pathophysiology, there is no evidence supporting this view. this study was undertaken to determine if fetal death prior to development of fetal death syndrome is associated with changes in maternal plasma concentration of cd l (a marker of platelet activation), tf and its soluble inhibitor (tfpi). methods: a cross-sectional study included the following groups: ) women with normal pregnancy (n= ) and ) patients with fetal demise without disseminated intravascular coagulation (n= ). plasma concentrations of scd l, tf and tfpi were measured by elisa. standard coagulation tests were performed. non-parametric statistics were used for analysis. results: ) patients with fetal demise had a higher median maternal plasma scd l concentration than women with normal pregnancy (median . pg/ml, range - vs. median . pg/ml, range . - . , p< . ); ) there was no significant difference between the groups in the median maternal plasma tf concentration and ) in contrast, the median maternal plasma tfpi concentration was significantly lower in patients with fetal demise than in women with normal pregnancy (median . ng/ml, range . - . vs. median . ng/ml, range . - . , p< . ). conclusions: ) a change in the plasma concentration of tf was not demonstrated; ) a change in the ratio of tf/tf inhibitor pathway may predispose to thrombin generation and activation of the coagulation cascade; ) however, maternal platelet activation is present in patients with a fetal demise without fetal death syndrome and ) the role of tf in fetal death syndrome remains to be proven. lipoic acid inhibits matrix metalloproteinase production, activity and prostaglandin e secretion by cultured amnion epithelial and mesenchymal cells. r moore, j novak, d kumar, j moore. case western reserve university, cleveland, oh, usa. introduction: cytokines, free radicals, matrix metalloproteinases (mmp) and prostaglandins (pg) have been implicated in processes of fetal membrane rupture and labor. dietary anti-oxidant supplementation has been suggested as a possible therapy for high risk patients, however, clincal evidence supporting the efficacy of agents such as vitamin c or n-acetylcysteine remains controversial. in fact, we have previously shown that vitamin c increases matrix metalloproteinase (mmp) activity in isolated fetal membrane fragments and fails to inhibit tumor necrosis factor (tnf) induced fetal membrane weakening in vitro. in this study, we examine the effect of the naturally occurring anti-oxidant, -lipoic acid, on tnf induced mmp activity/protein and pge secretion in isolated amnion epithelial and mesenchymal cells. methods: amnion epithelial and mesenchymal cells were pre-treated with increasing doses of -lipoic acid ( - mm/ h), then with increasing doses of tnf ( - ng/ml/ h). medium and cells were analyzed by gelatin zymography/western blotting for mmp /mmp activities/protein. pge output was determined by immunoassay. results: tnf induced a dose dependent increase in mmp production, secretion and activity in amnion epithelial cells. tnf ( ng/ml) induced an fold increase in cellular active mmp production and fold increase in secreted mmp enzyme activity by amnion epithelial cells. these increases were reduced - % following h pre-treatment with . - . mm -lipoic acid. mmp protein/activity and pge secretion by amnion epithelial cells were barely detectable and unaffected by tnf and/or -lipoic acid treatment. in striking contrast, mesenchymal cells exhibited little basal or tnf induced mmp protein/activity. mmp protein/activity in mesenchymal cells were unaffected by either tnf and/or -lipoic acid. however, tnf treated mesemchymal cells exhibited a dose dependent increase in pge production ( fold increase/ ng/ml tnf/ h) that was inhibited by %- % following . objective: nearly fifty years after the discovery of microphthalmia-associated transcription factor (mitf), its gene was identified as a specialized transcription factor that dictates cell-specific differentiation. unique mitf isoforms are generated from alternative promoter usage. an isoform of mitf (mitf-cx) is down-regulated in cervical stromal cells of the ripened cervix. further, mitf-cx inhibits il- gene expression and thereby suppresses signaling of the final pathway in cervical ripening. since mitf binds to canonical eboxes (canntg) in promoter regions of target genes, we sought to determine if mitf regulated its own promoter through ebox motifs. methods: the kb genomic dna sequence upstream of the mitf-cx transcription start site was cloned into pgl luciferase reporter vectors which were co-transfected with wild type or mutmitf-cx (impaired dna binding) into cervical stromal cells or hek cells. at h, promoter activity was determined and normalized for transfection efficiency. results: gel-shift assays conducted with oligonucleotides corresponding to eboxes in the mitf-cx promoter revealed two strong binding sites (eboxes - to - and - to - ). specific binding was established using oligonucleotides with or without mutated ebox, antibody supershift experiments, competition with cold probe, and absence of binding to mutmitf-cx. binding specificities were confirmed in nuclear extracts from cells that overexpressed mitf-cx, but not control or mutmitf-cx. reporter gene studies indicated that mitf-cx, but not mitf-m, increased mitf-cx promoter activity -to -fold. whereas mutations in ebox or resulted in significant decreases in mitf-stimulated promoter activity, mitfinduced increases in promoter activity were abolished by mutations in both eboxes. conclusions: collectively these experiments indicate that mitf-cx is a vital regulator of its own promoter activity and acts in a positive feedforward loop through two specific binding sites in its promoter. moreover, isoform-specific amino acids are important to mediate mitf-induced mitf-cx promoter activity. decreasing mitf protein or mutating its promoter would interrupt this loop resulting in rapid reduction of mitf synthesis. since mitf-cx suppresses il- gene expression in cervical stromal cells, we suggest that preservation of mitf-cx-induced mitf gene expression is an important mechanism to maintain the "brake" on cervical ripening and ensure cervical competency during pregnancy. the role of cd in cervical remodeling. denisse sanchez, brenda timmons, mala mahendroo. obstetrics and gynecology, ut southwestern medical center, dallas, tx, usa. objective: prior to the onset of parturition, the uterine cervix undergoes a remodeling process from a closed, rigid structure, to one that is soft and dilatable. many changes occur, including increases in hyaluronan (ha), a glycosaminoglycan that facilitates loosening of the collagen matrix. in the postpartum period, the concentration of ha is reduced to that of the nonpregnant state. cd , a transmembrane glycoprotein expressed in hematopoietic and epithelial cells, is a receptor for ha. cd expression by immune cells is important in extravasation of leukocytes into tissue. cd may also be required for ha catabolism through the action of hyaluronidases and . in the cervix, cd is expressed in the endo-cervical epithelia as well as in immune cells localized in the stromal matrix. to study the importance of cd in cervical remodeling, mice with a null mutation for cd (cd -/-) were evaluated during pregnancy, parturition and postpartum. methods: changes in ha amount and size distribution were assessed using ha molecular weight gels and fluorophore assisted carbohydrate electrophoresis. to identify defects in cervical remodeling in the cd -/mice, differences in expression for genes regulated in the cervix were studied by quantitative real time pcr . to determine whether cd plays a role in the recruitment of immune cells during cervical ripening and postpartum repair, immunohistochemistry with antibodies against leukocytes was done. in the postpartum period, there is a higher ratio of high molecular weight ha relative to low molecular weight ha in the cd -/mice. this suggests that postpartum breakdown of ha in cd -/cervices is delayed. as compared to wt cervix, there was a significant increase in hyaluronidase (hyal- ) mrna in the postpartum period. the distribution and relative numbers of immune cells in the cd -/cervix was similar to wt. conclusion: these studies provide evidence that cd may play a role in remodeling of the postpartum cervix back to the nonpregnant state. our current data suggests that the catabolism of ha after birth is delayed in the mutant mice and upregulation of hyal may compensate to allow ha removal. furthermore, the activity of hyal- may be dependent on cd . little difference in the recruitment of immune cells between cd and wt animals suggest that cd expression is not required for this process. these experiments provide a greater understanding for the role of cd and ha in cervical remodeling. in background: during in vitro experiments we have shown that separation of amnion from choriodecidua occurs as an integral part of the process of fetal membrane (fm) rupture. although spontaneous amnion and choriodecidual separation is seen in fm after both svd and elective c/s deliveries, its etiology is uncertain. biochemical degradation at the amnion-choriodecidua interface may be a key contributing factor. our previous biomechanical studies have demonstrated that separated fm require less physical work to rupture than intact membranes. the purpose of this study was to determine whether fm separation was associated with clinical differences in the birth process. hypothesis: during term, normal labor, spontaneous separation of fm is associated with differences in the clinical parameters of labor and delivery. study design: fm from consecutive term deliveries were cut off the placental disk. separated areas of fm were cut from the intact areas. both were weighed and their weight ratios determined. maternal medical, pregnancy, and delivery data were collected and analyzed. results: term fm had the following characteristics: maternal age ± . yr, gravida . ± . , gestation ± . wks, elec. cs . %, duration of rom ± min, duration of contractions ± min, african american %. % of the fm had < % separation; % had more than % separation. srom fm with > % separation (vs. < %) had significantly shorter duration of rom (p= . ) and admission to birth (p= . ) times. srom fm with > % separation (vs. < %) had even shorter rom (p= . ), duration of contractions (p= . ) and admission to birth (p= . ). the > % group (vs. < %) was further along, gestationally (p= . ). srom fm (vs. arom) had shorter admission to birth (p= . ), but longer rom to birth (p< . ) times. absence of epidural (p= . ), srom mode of rupture (p= . ), svd (vs. elec. c/s) (p= . ), and the presence of meconium (p= . ), were all associated with increased fm separation. conclusion: spontaneous separation of fetal membranes is nearly universal and is associated with increased gestation, spontaneous rupture of membranes, shorter duration of contractions, and svd. speculation: we speculate that programmed biochemical changes initiate fm separation which then facilitates rom and childbirth. whole genome array and si-rna investigation of the function of nfkb in human amnion epithelial cells. sheri e lim, shirin khanjani, yun s lee, tg teoh, , philip r bennett. institute of reproductive and developmental biology, imperial college, london, united kingdom; maternal fetal medicine, st. mary's hospital, london, united kingdom. introduction: labour is associated with activation of nfkappab in the amnion. nfkappab increases prostaglandin synthesis through the upregulation of cyclooxygenase- (cox- ), which is essential to the labour process. cox- mrna expression increases with gestation in the amnion. primary amnion epithelial cells cultivated from tissue collected prior to the onset of labour display a spectrum of nfkappab activation, similar to the spectrum of cox- expression presumably relating to the nearness of labour. our aim was to investigate the full range of genes under nfkappab control in amnion epithelial cells by using whole genome arrays. methods: amnion from women undergoing elective caesarean section was collected and primary cell cultures established. total rna and protein were extracted from each culture. nuclear localization of nfkappab is required for its activation. western analysis of nuclear p was therefore performed to identify the samples displaying the lowest and highest nfkappab activity. the corresponding rna samples displaying the three lowest and three highest nuclear p protein concentrations were used for whole genome analysis using affymetrix u arrays. results and conclusions: we identified significantly regulated genes. the gene with the highest fold change was cox- (x . ) followed by oxytocin receptor (x . ), ch orf (x . ), integrina (x . ), and connective tissue growth factor (x . ). other significant genes included interleukin- (il- ) (x . ). pathway analysis revealed the majority of other nfkappab associated genes were involved in cell signaling, turnover and proliferation. we used real-time pcr (rtq-pcr) to validate cox- and il- expression. to prove that cox- is directly regulated by nfkappab, primary amnion epithelial cells were then transiently transfected with nfkappab p sigenome smart pool and sicontrol non-targeting sirna pool. western blot analysis confirmed knockdown of nfkappab p associated with inhibition of cox- demonstrating that nfkappab is essential for cox- expression. objective: premature birth is a major public problem accounting for over , deaths and , surviving infants with life-long morbidity yearly. in order to develop a rational basis for treatment and prevention of premature fetal membrane (fm) failure, we first need to understand the sub-failure fm structural and mechanical behavior at near full term. methods: we utilized planar biaxial mechanical testing, which approximates the physiologic loading state, for mechanical evaluation of the fm, and a structural constitutive model approach was used to offer insight into the structure-strength of the fm by integrating information on tissue composition and structure. small angle light scattering (sals) was used to nondestructively quantify the collagen fiber architecture of both intact and separated fm layers. results: in the stress free state, the gross collagen fiber architecture of the fm and separated layers were not homogenously align but exhibited small regions of fiber alignment. the amnion layer displayed the greatest alignment. the model fit the equi-biaxial strain data well (r = . ) and indicated that fm collagen fibers were rapidly recruited and straightened well below failure stress levels. collagen fibers were gradually recruited followed by a drastic increase in fiber recruitment. conclusion: this study provided the first data on the effective collagen fiber stiffness in the intact fm under physiologic biaxial loading, which was related to quantitative collagen fiber architectural measures. modeling results indicated that the collagen fibers became fully loaded and straighten well below physiological loading levels. failure did not occur during physiological loading, indicating that fibers do not begin to fail until all collagen fibers are fully straightened and bearing load. this result suggested modest structural reserve in the fm collagen architecture, and may be an important aspect of its failure properties. previously, we demonstrated that a physically "weak zone" exists overlying the cervix in the fm, evident of collagen remodeling and cellular apoptosis. we are currently extending the present study to include the "weak zone" tissues, allowing us to elucidate the micro-mechanical mechanisms that facilitate failure in this newly identified fm zone. supported by nih . the extracellular matrix of the cervix undergoes extensive remodeling during parturition. hyaluronan (ha) is a major constituent of the extracellular matrix of the term pregnant cervix. the onset of labor is preceded by an increase in ha and after delivery, the concentration of cervical ha gradually decreases to that of the non-pregnant state. these dramatic changes suggest that ha plays an important role during parturition. hyaluronan synthase (has ) is one of three known ha synthases and the most abundant isoform in the pregnant cervix. transcripts for has are regulated by two alternative promoters, one upstream of the first coding exon (proximal promoter) and another upstream on an untranslated exon (distal promoter). the focus of the current study is to further our understanding of the transcriptional regulation of has during cervical ripening. methods: rna blotting was carried out using transcript specific probes corresponding to the distal and proximal promoter of the mouse has gene. the regulation of has was evaluated in a cervical epithelial cancer cell line (caski cells) which we have previously shown to express endogenous has . regulation of has expression by epidermal growth factor was assessed in the caski cells by western blotting and quantitative real time pcr assessment of transcripts. results: has mrnas in the nonpregnant (np) and pregnant cervix are transcribed from the distal promoter upstream of exon . two transcripts of approximately . kb and . kb were detected that arise from use of polyadenylation sequences. as compared to np, the expression of has is increased , , and fold on gestation days , and shortly postpartum respectively. has mrna expression is increased upon treatment of caski cells with epidermal growth factor ( ng/ml) and is suppressed in cells treated with ag ( m), an inhibitor of egf receptor phosphorylation. maximal stimulation was observed at and hours of treatment. conclusion: has is the major ha synthase expressed during cervical ripening and the majority of transcripts are driven by the distal promoter in the has gene. in vitro studies using caski cells suggest has is regulated in part by the egf signaling pathway resulting in a several fold increase in has expression. these results provide an understanding of has gene regulation at the time of cervical ripening which will ultimately enhance our understanding of the molecular mechanisms important to cervical ripening. chorion and decidua cells. chad a grotegut, bernard j canzoneri, liping feng, phil heine, amy p murtha. obstetrics and gynecology, duke university, durham, nc, usa. objective: preterm premature rupture of the fetal membranes accounts for approximately % of all preterm deliveries. cigarette smoking independently carries a fourfold increase risk for pprom. our laboratory has previously demonstrated that the chorion layer undergoes apoptosis in women with pprom. this study was conducted to determine if extract of cigarette smoke causes cell death in specific cells of the fetal membrane. fetal membranes were collected at the time of elective cesarean section from women without labor and at term. the chorion and decidua layers were separated and purified on a gradient spin column and then plated near confluence. cigarette smoke extract (cse) was collected in cell media and used to treat chorion and decidua cells in culture at concentrations ranging from % to % in -well plates. cell viability was determined at , and hours following treatment with a non-radioactive cell viability assay. data were analyzed using paired t test (analyse-it, leeds, uk). chorion and decidua cells underwent cell death when exposed to cse in a dose dependent fashion. increasing concentrations of cse resulted in increased cell death at hours in both cell types (figure ). at hours, chorion exhibited greater percent cell death compared to decidua at concentrations of , and % cse (p= . , . , and . , respectively). for any given concentration of cse, the degree of cell death increased with increasing length of exposure ( , and hours) for each cell type. chorion cells routinely exhibited greater percentage of cell death following treatment with cse at concentrations ranging from - % compared to decidua cells. human chorion and decidua cells in primary cell culture exhibit a dose-response and time dependent cell death in the presence of cse. human chorion cells show greater sensitivity to cell death when compared to decidua cells. further studies are needed to determine the mechanisms through which these cell types undergo cell death and the implications for the differential sensitivity to cigarette smoke. yoon ha kim, tae-bok song, cheol hong kim, jong woon kim, moon kyoung cho, sung yeul yang, bong whan ahn. obstetrics gynecology, chonnam national university medical school, gwangju, korea; biochemistry, chonnam national university medical school, gwangju, korea. objective: to investigate the lipid peroxide levels and protein carbonyls levels in the amniotic fluid of pregnant women with preterm premature rupture of membranes (pprom). the lipid peroxide levels in the amniotic fluid of normal pregnancy (n= ) and pregnant women with pprom (n= ) were newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced measured by thiobarbituric acid reaction. the protein carbonyl contents in the amniotic fluid of normal pregnancy (n= ) and pregnant women with pprom (n= ) were determined by the , -dinitrophenylhydrazine method. after amniotic fluid of them were mixed and incubated up to hours with . ml of mm moxalactam, cefodizime, amoxacillin, erythromycin, the lipid peroxide levels and protein carbonyl contents in them were measured. results: . the lipid peroxide levels in the amniotic fluid of pregnant women with pprom was significantly higher than that of normal pregnancy ( . ± . vs. . ± . nmol/mg protein, p< . ). . the protein carbonyl levels in the amniotic fluid of pregnant women with pprom was significantly higher than that of normal pregnancy ( . ± . vs. . ± . nmol/mg protein p< . ). . the lipid peroxide levels and protein carbonyls formation by moxalactam in the amniotic fluid of pregnant women with pprom was significantly higher than basal level ( . ± . vs. . ± . nmol/mg protein, . ± . vs. . ± . nmol/mg protein, p< . ). . the lipid peroxide levels and protein carbonyls formation by cefodizime in the amniotic fluid of pregnant women with pprom was significantly lower than basal level ( . ± . vs. . ± . nmol/mg protein, . ± . vs. . ± . nmol/mg protein, p< . ). . there were no significant differences in the levels of lipid peroxide and protein carbonyls by amoxacillin and erythromycin in the amniotic fluid of pregnant women with pprom between antibiotics-induced and basal levels. background: chorioamnionitis (cam) is a major antecedent of preterm delivery (ptd) associated with elevated amniotic fluid tnf and il . we hypothesized that these cytokines enhance the term decidual cell (dc) expression of the matrix metalloproteinases (mmp) and , which can then promote ptd by degrading the extracellular matrix of the decidua, fetal membranes, and cervix. methods: immunostaining for mmp- , mmp- , and vimentin (a dc marker) was performed on cam-complicated (n= ) and gestational age-matched control decidua (n= ), and staining intensities were evaluated by hscore. confluent, leukocyte-free term dcs were primed with - m estradiol (e ) or e + - m medroxyprogesterone acetate (mpa), and then switched to a defined medium with e +/-mpa with or without ng/ml of il or tnf . secreted mmp- and mmp- levels were measured by elisa (n= ), and quantitative rt-pcr assessed mmp- and mmp- mrna levels (n= ). results: tissue staining revealed that mmp- and mmp- levels in cam-complicated decidua (hscore mean±sem: ± and ± , respectively) were significantly higher than in control decidua ( ± , and ± respectively; p< . ). in cultured term dcs incubated with e , tnf and il significantly increased secreted levels of mmp- compared to e alone (pg/ml/ g protein: . ± . and . ± . , respectively, vs. . ± . ; p< . ). in parallel incubations with e +mpa, basal mmp- output was lowered by %, and tnf -and il -elicited mmp levels were blunted by % and %, respectively. rt-pcr confirmed that tnf and il increased mmp mrna levels (p< . ), although mrna levels in e +mpa incubations were not different from those of e alone. mmp levels in all treatments were similar. conclusions: mmp- and mmp- are elevated in cam decidua compared to controls. our in vitro results suggest that mmp- expression is enhanced by the high levels of il and tnf associated with cam, and that mpa may be able to blunt this effect. we have previously found a similar regulatory mechanism of mmp- and mmp- and their over-expression in cam-complicated tissues. synergy among these mmps may represent a potent pathogenic mechanism of cam that can be targeted through the therapeutic use of progestins in preventing cam-induced ptd. domerudee preechapornprasert, patama promsonthi, wasun chantratita, mana rochanawutanon, patcharee karnsombut, chutatip srichunrusami, stephen j lye, boonsri chanrachakul. obstetrics and gynecology, ramathibodi hospital, mahidol university, bangkok, thailand; pathology, ramathibodi hospital, mahidol university, bangkok, thailand; obstetrics and gynecology, samuel lunenfeld research institute, toronto, canada. objective: cervical ripening is an inflammatory process involving chemokines, cytokines and various mediators. recent study has shown that the level of monocyte chemotactic protein (mcp) , a chemokine, increases in amniotic fluid during spontaneous labor. the aim of this study was to examine the localization and expression of mcp in human cervix before pregnancy, during pregnancy and after the onset of labor. methods: this study was approved by the local ethics committee and written informed consent was obtained from each participant. cervical biopsies were taken from groups of women; nonpregnant women, first trimester pregnant women, term pregnant women with and without labor. tissue samples were fixed in % formal saline for paraffin section. immunohistochemistry (n = each) was performed by avidin biotin complex (abc) technique using monoclonal antibody specific to human mcp . the mcp messenger(m) rna was identified by reverse transcription-polymerase chain reaction using gene specific primer against mcp and mcp receptor (n = each). results: immunohistochemistry demonstrated mcp in cervical tissues from all four groups of women. mcp was localized on plasma membrane and cytoplasm of both squamous epithelial and columnar cell lining of endocervical gland. mcp and mcp receptor mrna were identified in nonpregnant, first trimester and term with and without labor human cervix. conclusion: mcp and mcp receptor were located in cervical tissues of nonpregnant and pregnant women at different gestation both before and after the onset of labor.ongoing studies are investigating the role of this chemokine during pregnancy and labor. whether preterm cervical ripening is just an aberrant regulation in timing or whether divergent mechanisms and pathways are involved in preterm versus term cervical ripening remains to be elucidated. methods: cervical tissue was collected from groups of cd- mice. group : mouse model of ptb that utilizes intrauterine infusion of lipopolysaccharide (lps). group : e dams representing preterm controls. group : e . - dams selected from a timed pregnant batch where half of the dams had delivered representing term cervical ripening. n= dams/treatment group. separate rna samples were used for microarray analysis (ma). significance analysis for ma and partek software was used for biostatistical analysis. pathway analysis was performed using david. quantitative pcr was performed to confirm the most differentially regulated genes. results: using a cut-off of -fold change with p value of < . , genes in the cervix were differently regulated between the groups. principal component analysis revealed three distinct groups (see graph). functional annotation clustering demonstrated the following pathways: ) in preterm cervical ripening (e lps vs e ): immune and inflammation response, defense response ) in term cervical ripening compared to preterm controls: negative regulation of cellular process and biological process, ecm, cell-cell communication. qprc confirmed the highly significant differences found in ma (see table) . conclusions: the molecular mechanisms and pathways governing preterm and term cervical ripening are distinctly different. elucidating these unique pathways can lead to improved therapeutics for prevention of ptb as well as for postdate pregnancies. cervical ripening at term involves activation of apoptotic enzymes. maria kb sennstrom, valentina ciani, gunvor e ekman. obstetrics and gynecology, women and child health, karolinska institute, stockholm, sweden; obstetric and gynecology, university of siena, siena, italy. aim: to investigate if the human cervical ripening at term involves programmed cell death. during the final cervical ripening the extracellular matrix dominated cervix undergoes an extensive remodelling of the tissue. inflammatory mediators such as the cytokines increase in ripening cervical tissue at term. programmed cell death, apoptosis, has been suggested as important in this process. apoptosis can be induced by inflammatory mediators such as cytokines. we also looked upon the distribution of inflammatory cells in cervical tissue. materials and methods: cervical biopsies from pregnant women at term and post partum women with fully ripened cervix were studied. biopsies from non-pregnant women served as controls. immunohistochemical analysis of the apoptotic enzyme caspase- and the inflammatory cell marker cd was performed on paraffin embedded sections of cervical tissue. double staining was performed. mann-whitney u-test was used for statistical analysis. results: there was a significantly higher frequency of caspase- staining in the post partal sections from ripened cervical tissue compared to tissue from term pregnant (p= . ) with unripe cervix and from non-pregnant patients (p= . ). the inflammatory cells staining for cd increased in post partal and term pregnant sections compared to non-pregnant (p= . ). there was a higher frequency of caspase- positive cells in the post partal tissue than of cd positive cells (p= . ). the localization of cd- positive staining was highest in the epithelia and basal lamina while caspase- staining was most pronounced in stromal tissue and around vessels. conclusion: our data show apoptotic activity in stromal tissue in fully ripened human cervix at term of pregnancy, suggesting that apoptotic mechanisms are involved in the extracellular matrix remodelling at term. the apoptotic activity is not co-localized with inflammatory cells suggesting non-infectous inflammation with apoptosis as important for cervical ripening at term. objective: cervical biomechanical responses are important for accommodating the increased stress induced by an enlarging uterus. a mechanical testing system was modified to evaluate the stress relaxation response in the pregnant cervix. methods: tissue harvested from the non pregnant and timed pregnant (days , , , , and ) sprague-dawley rats underwent tensile testing using an instron material testing system. the testing regimen consisted of tissue extension to near maximal strain over seconds followed by a period of constant strain for minutes, then return to rest over seconds. this cycle was repeated additional times with a minute rest period between cycles. strain and force measurements were recorded at second intervals. - animals were used for each time point. in addition, stress and strain at the yield point was also determined for each gestational time point. results: the pregnant and non pregnant samples exhibit marked differences in response in both stress and strain. the timed pregnant tissue demonstrated progressively increased compliance and lower nominal stress compared to non pregnant tissue. peak nominal stress declined with each successive cycle. this was demonstrated in the peak nominal stress and strain values at the yield point. conclusion: the cervix becomes more distensible (compliant) but less resistant to force with increasing gestational age. ...,.. e. coli . x .:!: . x . x .:!: . x group a (c, c) group b ( c, p) . x + . x . x + . x . . . group c (p, p) . x o"::!>s x o" . x ~ .ox: o• k. ~neumoniae group a ( c, c) . x + . x . x + . x . . . group b (c, p) . x + . x . x + . x group c (p, pl . x ~ . x . x ~ . x c. al bicans group a (c, c) . x '+ . x . x + . x group b (c, p) . x o•+ . x . x + . x . . . group c (p, p) . x ~ . x . x ~ . x • introduction: a growing body of evidence supports that inflammatory processes are implicated in spontaneous preterm birth (ptb). using an inflammatory and non-inflammatory mouse model of ptb, we sought to determine if activation of these inflammatory pathways are essential for ptb and/or cervical ripening to occur. methods: timed pregnant cd- mice were used in these two models of ptb: ) a model of intrauterine inflammation where lipopolysaccharide (lps) is injected into the uterine horn (n= ); controls for this model received intrauterine saline (n= ) and ) a non-infectious model of ptb using ru sq ( ugrams/dam) (n= ); controls for this model received no intervention (n= ) were used for these studies. for both models, hours later uterine and cervical tissues were harvested. the tissues were processed for protein and rna studies. elisas were performed to assess il- and tnf-alpha in the uterine tissue. mrna expression of ifn , il- , il- , il- , tnf-alpha were assessed in cervical tissue from both models by quantitative pcr. results: in uterine tissues, both il- and tnf were significantly elevated in the lps-induced ptb when compared to control, saline, and non-infectiousinduced preterm birth (p< . ) (figure ). in cervical tissue, an increase in il- , il- beta and tnf mrnawas observed in both models, while ifn-gamma and il- were only increased in the lps model. (figure ). conclusions: up-regulation of pro-inflammatory cytokines in the uterus do not appear to be essential for ptb. cytokine expression in the cervix is greater in an inflammatory model of ptb but is also present in a non-infectious model. these studies suggest that targeting a cytokine response in the cervix may hold the most promise in prevention of ptb. the initiation of labor at term and preterm is associated with an inflammatory response, with increased interleukins in amniotic fluid (af) and infiltration of the myometrium by neutrophils and macrophages (m ). whereas, in preterm labor, intra-amniotic infection may provide the stimulus for increased af interleukins and inflammatory cell migration, the stimulus for these events at term has remained uncertain. in studies using pregnant mice, we observed that the m that invade the maternal uterus near term arise from the fetus. furthermore, we obtained compelling evidence that surfactant protein-a (sp-a), a developmentally regulated c-type lectin secreted by the fetal lung into af near term, activates af m , which migrate to the uterus where they promote an inflammatory response culminating in labor. we propose that interactions of m surface receptors with sp-a, at term, or bacterial lipopolysaccharide at preterm, initiate changes in m phenotypic properties, resulting in the enhanced expression of genes that promote their migration to the uterus. the objectives of the present study were to analyze the numbers and phenotypic properties of mouse af m during late gestation and to identify their putative tissue source(s) of origin. to assess changes in the number of m in af during late gestation, af cells were isolated and stained for the m marker f / . the density of adherent f / + cells greatly increased in equivalent volumes of af between . and . days postcoitum (dpc) ( . dpc = term). interestingly, the f / + cells at . dpc were highly similar in morphology to those present in . dpc fetal lung, but distinctly different from those in fetal liver, suggesting their pulmonary origin. the af m were foam cell-like, suggesting the presence of lipid inclusions, a property shared by adult alveolar m . to further analyze gestational changes in the m population(s) in mouse af, we used flow cytometric analysis. in our initial studies, cells isolated from af were stained for f / and for cd , a pan-leukocyte marker. we observed that af from . and . dpc mice contained two sub-populations of cd + f / + cells. studies are in progress to analyze these af m populations for expression of cell surface antigens indicative of their maturity, activation state and chemotactic properties in association with the developmental induction of sp-a synthesis and secretion by the fetal lung. april bleich, patrick keller, r ann word. ob-gyn, ut southwestern, dallas, tx, usa. the role of prs, proinflammatory cytokines, cell adhesion molecules, cox- , and toll-like receptors in mediating cervical ripening prior to labor is not clear. the objective of this study was to quantify pr isoforms and determine the relative expression of certain inflammation-related genes in cervical stroma from nonpregnant and pregnant women in early gestation (eg), term before and after cervical ripening, and during labor. methods: standard curves of pr-b, -a, pra+b and qpcr were used to quantify total and pr-b in cervical stroma from nonpregnant (proliferative, n = ; progestin treatment, n = ) and pregnant women undergoing hysterectomy (eg, n = ; term before ripening, n = ; after ripening, n = ; in labor, n = ). cervical status was determined by modified bishop scoring. results: total pr expression was maximal in nonpregnant women in the proliferative phase ( . ± . pg/ug cdna) and decreased % by progestins ( . ± . pg/ug cdna). this level was maintained in stroma from pregnant women before labor ( . ± . , eg.; . ± . before ripening; . ± . after ripening pg/ug cdna). in contrast, total pr was decreased significantly in the dilated cervix ( . ± . pg/ug, p < . ). interestingly, whereas pr-b was ± % that of total pr in the nonpregnant cervix, pr-b mrna levels were ± % to ± % in cervical tissues from all pregnant women and did not vary with labor status. using immunoblot analysis and pr-specific antibodies (pgr ), pr-b immunoreactivity was % that of total pr in all samples from pregnant women, and both pr-a and -b were downregulated significantly in the dilated cervix (from ± to ± units/atub). decreased expression of pr in the dilated cervix was accompanied by significant increases in il- , mcp- , tlr- , cd l, cox- , and s a mrna (all p < . , anova) but not cd b or tlr- . pgdh mrna was decreased significantly in the dilated cervix. with the exception of cox- , expression of these genes was similar before labor regardless of cervical ripening. conclusions: both pr and pr-b are decreased proportionately in the dilated cervix, but not during cervical ripening. further, a number of inflammatory gene products are increased dramatically in the cervix during labor, but not before. taken together, the results suggest that cervical ripening is distinct from cervical dilation and involves upregulation of cox- but not il- , mcp- , or toll-like receptors. ifn-y il- il- , il- tnf- "p value < . lps/saline ru /controls . "" . " . " . * " " . * . . " association between interleukin- (il- ) and il- to examine association of amniotic fluid interleukin (il- ) concentration with il- and its receptor il -r haplotypes in term and preterm caucasians (c) and african americans (aa) samples. methods: in this study case (preterm birth -ptb [< weeks]) and control (term [> weeks]) amniotic fluid (af) il- concentrations were analyzed for association with haplotypes of the il- and il r genes in aa and c separately. in il- , eight, and in il- r, single nucleotide polymorphisms (snps) were examined. aa and c maternal and fetal genotypes were assessed (aa: maternal:cases- controls- fetal: cases- fetal controls-; c: maternal cases- , controls- , fetal:cases- ; controls= ). haplotype associations were performed by using a sliding window with outcome il- concentration. analyses were performed separately on maternal and fetal dna. results: the strongest haplotype associations were observed in il- r rather than in il- . in c fetal dna il- r haplotypes defined by markers - bp from the transcription start site associated most strongly with af il- concentrations (global p= . x - ) and in aa maternal il- r haplotype markers at - - - (global p= . x - associated with il- concentrations. in the c fetal cases the - haplotype with the highest concentration was a-g (log(cytokine) = . pg/ml). in aa maternal samples the highest concentration was observed for haplotype t-t-g-c at - - - .this was seen in both cases and controls at (case log (cytokine) = . ; control log(cytokine) = . pg/ml). significant associations from haplotype analyses converged on three regions of the il- r in both races. no strong differences were observed between the haplotypes of cases with and without microbial invasion of the amniotic cavity (miac), with the exception of aa fetal samples that showed two overlapping haplotypes in il- that associated in cases with miac but not in cases without miac (- and - ; - and - )(both with p < x - ) conclusion: differences in the af il- concentration in ptb do not result from single snp effect on il- but are a result of complex relationships between il- and il- r haplotypes. these associations exhibit racial disparity. the role of tnf-in parturition. helen alexander, amanda tattersall, mark tattersall, suren sooranna, peta grigsby, leslie myatt, mark johnson. obstetrics gynaecology, imperial college, london, united kingdom; obstetrics gynaecology, university of cincinnati college of medcine, cincinnati, oh, usa. introduction: tumour necrosis factor-alpha (tnf-) is thought to play a role in inflammation-induced preterm labour since the decidua produces tnfin response to bacterial products and amniotic fluid tnf-concentrations are increased in the presence of intra-amniotic infection. the aims of this study were to (i) investigate the expression of myometrial tnf-and its receptors in relation to the onset of preterm and term labour; (ii) to identify which intracellular pathways are activated by tnf-; and to investigate the effect of tnf-alone and in combination with il- or il- on gene expression in uterine myocytes. methods: biopsies of human myometrium were taken at caesarean section from women before and after the onset of preterm and term labour and analysed for tnf-and its receptor mrna expression. a further samples were obtained before the onset of labour (n= ) from which myocytes were isolated and cultured in -well plates. when cells were - % confluent either tnf-at a concentration of ng/ml was added to the cells for , , , and min and the cells analysed by western blotting or tnf-at concentrations of , . and ng/ml was added either alone or in combination with similar concentrations of il- or il- to cells for hours and mrna was extracted and converted to cdna to determine il- and gapdh gene expression by qpcr. results: there was no change in tnf-mrna expression in relation to the onset of labour, but the expression of tnfr and tnfr mrna levels were significantly increased with gestation and further increased with the onset of labour. incubation of uterine myocytes with tnf-( ng/ml) activated all three mapk substypes: erk, jnk, and p , activation peaked between - minutes. preliminary data suggest that tnf-induces il- mrna expression but exposure to il- or il- itself did not enhance this response. conclusions: although there is no significant increase in tnf-concentration from baseline during labour we have shown tnf receptor mrna levels do increase with labour at term. exposure of isolated uterine myocytes to tnfcauses activation of all mapk subtypes and an increase in il- mrna expression. this enhanced mapk-dependent il- expression at term may be mediated via increased myometrial sensitivity to tnf-through increased tnf receptor expression. remodeling. brenda c timmons, anna-marie fairhurst, mala s mahendroo. obstetrics and gynecology, ut southwestern medical center, dallas, tx, usa; immunology, ut southwestern medical center, dallas, tx, usa. objective: the molecular mechanisms involved in cervical ripening are not well understood. immunohistochemical studies from our lab report a recruitment of inflammatory cells to the cervical stroma one day before birth in the mouse using a neutrophil/monocyte marker (neutrophil / ). in this study, we sought to identify and quantitate inflammatory cells migrating into the mouse cervix and to determine if this recruitment was affected by changes in progesterone levels. peripheral blood was also evaluated to see if changes in the cervix was paralleled in blood. methods: flow cytometric analysis was performed using cervical cells and peripheral blood obtained before and during cervical ripening along with - h postpartum. dispersion of cervical cells was optimized. these cells were stained with a panel of fluorescent conjugated antibodies directed against leukocyte antigens and analyzed on an lsrii flow cytometer. cells were also sorted and stained to visualize cell morphologies. to determine the effect of progesterone on the migration of leukocytes, gestation d mice were treated for h with a progesterone receptor (pr) antagonist prior to tissue collection. results: neutrophils do not appear to increase in the cervix until after birth. monocyte (mo) numbers do increase during cervical ripening (late day , d . ) and remain high through postpartum (pp). macrophages (mØ) are present prior to cervical ripening and steady state levels are maintained during labor and pp. pr antagonist treatment on d resulted in a premature increase in mo but not neutrophils or mØ. in contrast to the cervix, mo and neutrophil numbers do not significantly increase in the peripheral blood until pp. results from lymphocyte studies suggest a low level of b and t cells in the cervix. in the peripheral blood, b cells remain consistent through parturition and the t cells decrease by d . and continue to decrease pp. conclusion: tissue mo are increased in the cervix during ripening. this recruitment is dependant on loss of pr function. in contrast, neutrophils are increased in the pp cervix while mØ numbers appear constant. timing of changes in mo and neutrophil numbers in the peripheral blood differed from that observed in the cervix suggesting quantitation of these cell types in blood is not reflective of what is occurring in the cervix during ripening, dilation and pp repair. novel interactions between nf-b and other labour-associated transcription factors identified by a tf-tf array. shirin khanjani, yun s lee, suren r sooranna, mark r johnson, phillip r bennett. irdb, imperial college, london, united kingdom. introduction: external stimuli lead to changes in cellular gene expression through activation of inducible transcription factors. nf-b is a ubiquitous transcription factor classically associated with inflammation, which is activated in response to infection and proinflammatory cytokines such as those prevalent during the labour. the tf-tf interaction array uses a novel technology for detecting interactions between transcription factors based on binding of tfs to their own consensus dna binding sequence. materials and methods: primary myometrial cells were grown until - % confluent and stimulated with ng/ml il- prior to nuclear protein extraction. the nuclear extracts were incubated with the provided set of biotin-labeled, double-stranded oligonucleotide probes, which represent a known library of cis-elements. during the incubation step, these tf probes bind to their specific tfs in the nuclear extract. next, immunoprecipitation was performed using an antibody against nf-bp , which pulled out nf-bp and any tfs bound to it, bound to corresponding cis-elements. normal igg was used in a parallel experiment to represent a negative control. free cis-elements and nonspecific binding proteins were washed away and the cis-elements were finally eluted and hybridized to the array membrane, which is spotted with different tf consensus sequences. results: table shows the different tfs interacting with nf-bp based on the degree of binding stimulated by il- compared to no-il- control. conclusion: these data show that il- stimulation causes nf-b to bind to a wide variety of other treansciption factors. of particular interest in the area of parturition is binding to the other pro-inlammatory tfs such as c/ebp and ap- , which are known to regulate labour-associated genes in synergy with nf-b. the lack of association between nf-b and pr without il- stimulation supports the concept that with the onset of labour inflammation leads to functional progesterone withdrawal, rather than pr acting to inhibit inflammation. table high ap- , c/ebp, cbf, creb , c-myb, e f- , ets, ets- /pea ,fast- , gas/isre, hse, mef- , mef- , myc-max, nf- , nfatc, nf-e , nf-e , pax- , objective: pre-partum cervical ripening involves remodeling of collagen structure and inflammatory immune cell activity (jsgi : , ; reprod biol endo : , ) . although parturition is associated with systemic or local progesterone withdrawal (ajog : , ) , effects of a decline in progesterone on cervical ripening is not known. the present study tested the hypothesis that progesterone withdrawal promotes collagen degradation, innervation, and immune cell trafficking in the cervix of nonpregnant mice. methods: adult virgin female c bl mice received capsules (sc) with oil vehicle (v) or estradiol (e) and progesterone (p) to simulate concentrations in pregnancy (hum reprod : , ) . after days, mice in the v and e+p groups were euthanized. the p capsule was removed from some mice on day (e-p) and groups killed on days and . cervix sections were stained for collagen, nerve fibers, macrophages, or neutrophils (n= /group/day; sections/ cervix; biol reprod : , ; jsgi : , ) . stained macrophages and neutrophils were counted (image pro-plus , media cybernetics). results: e+p treatment for days promoted hypertrophy of the cervix compared to v controls, i.e., collagen content and structure diminished, cell nuclei density declined, and nerve fibers increased. removal of p did not affect these endpoints. for immune cells, e+p for , , or days decreased immune cell numbers. by contrast, p removal increased macrophages and neutrophils in the cervix on days and (p< . , e-p vs e+p groups, respectively). the census of resident immune cells in e-p groups at and h after p removal equaled that in the v group. conclusions: mimicking gonadal steroid concentrations in circulation during pregnancy promotes hypertrophy and suppresses immigration of immune cells in the cervix. in this non-pregnant murine model for parturition, progesterone withdrawal recruits immune cells, but fails to promote further remodeling or hyperplasia of nerve fibers in the cervix. the findings raise the possibility that ripening of the cervix requires not only recruitment, but also activation of immune cells. whether proinflammatory activities by specific immune cells affect nerve fiber hypertrophy or neural activity, as part of the mechanism for ripening of the cervix, remains to be determined. we have previously identified and characterized a truncated pr (pr-m), that localizes to the mitochondrion by multiple experimental techniques, including confocal imaging of a recombinant gfp fusion protein, western blot analysis after cellular fractionation of nuclear pr negative t d-y breast cancer cells and western blot analysis of purified human heart mitochondrial proteins. initial studies with nuclear pr negative mcf- a breast epithelial cells shown to express pr-m demonstrated an increase in mitochondrial membrane potential (mmp) with progesterone/progestin treatment. these studies led to the hypothesis that progesterone modulates cellular respiration via the mitochondrial receptor, pr-m. objectives: the present studies sought to further localize pr-m to the outer, inner or matrix portion of the mitochondrion and to correlate the increase in mmp with total cellular atp production. additionally, the potency of progesterone and synthetic progestins on the change in mmp was evaluated. methods: the location of pr-m was determined by western blot analysis after fractionation of human heart mitochondria with digitonin treatment and differential centrifugation. mmp was determined in mcf- a breast epithelial cells and a rhabdomyosarcoma cells by the change in fluorescent emission of jc- . total atp was determined by a bioluminescent assay. results: western blot analysis after mitochondrial fractionation showed pr-m localization exclusively in the outer membrane. a dose-dependent increase in mmp was seen in cell lines with - min treatment with progesterone and r which were inhibited by a specific pr antagonist, rti- - b, and not affected by the translational inhibitor, cycloheximide. similar changes in mmp were seen with the same concentration of progesterone, mpa and r . progesterone/progestin treatment for min led to an increase in total cellular atp without a change in cell number. conclusions: progesterone/progestin treatment results in an increase in cellular respiration in cells expressing an outer mitochondrial membrane pr and known to lack nuclear pr expression. this may represent a mechanism whereby progesterone enhances cellular energy production to meet the demands of pregnancy. introduction pge is a major product of the fetal membranes, decidua and myometrium and plays an important role in cervical ripening and myometrial contractions. there are four pge receptors, ep- and ep- mediate contractions whilst ep- and ep- mediate quiescence. ep- contains multiple consensus sequences for transcription factors known to be of importance in labour, in particular nfkappab. we therefore performed experiments to determine the effect of activation and inhibition of nfkappab upon ep- expression. myocytes plated in well plates were treated with il- ( ng/ml), to activate nfkappab. myometrial cells were also transiently transfected with nfkappab p sigenome smart pool and sicontrol non-targeting sirna to knock down nfkappab p . rna was extracted for amplifying ep- using quantitative rt-pcr with amplification of l as a control to normalise data. il- caused an increase in expression of ep- . knock down of nfkappab using si-rna resulted in a further increase in ep- . this data suggests that although il- stimulates expression of ep- it does so through an nfkappab independent mechanism. the upregulation of ep- with sirna knock down of p suggests that activation of nfkappab would inhibit ep- expression consistent with the concept that activation of nfkappab at term causes the myometrium to adopt a more contractile phenotype. introduction: a role for the pro-inflammatory cytokine il- is suggested in preterm and term birth, independent of the presence of infection. we previously showed that mice with a null mutation in the il- gene (ko) delivered one day later than wild type (wt) mice due at least in part to altered timing of uterine expression of prostaglandin (pg) f receptor, ptgfr, mrna. we also observed differences in mrna expression of other uterine activation proteins (uaps), pg h synthase (pghs)- , oxytocin receptor (otr) and connexin- (cx- ), suggesting multiple physiological effects for il- in term delivery. objective: to examine the effect of il- deficiency on the peri-partal uterine mrna expression of the pge receptors (ep) , and ; the post-partum changes of all uaps; and the relationship of these to serum progesterone (p ) concentrations. methods: gestational length was observed in pregnant c bl/ wt (n= ) and ko (n= ) mice. uap mrna levels were measured by real time rt-pcr in wt and ko dams sacrificed from d through delivery and up to h postdelivery. serum p was determined by ria. data were analyzed by one-way and two-way anova using the holm-sidak test to differentiate treatment effects at p< . . results: birth was delayed in the il- ko mice ( . ± . d vs. . ± . d), and this affected the timing of peri-partal changes for all uaps similarly. both ep and ep (relaxatory receptors) were elevated at d in ko dams, but returned to low levels before delivery and were elevated at delivery (ep ) or afterwards (ep ). ep levels did not change. otr increased several hours before delivery in all dams. cx- increased at delivery, then fell, while pghs- increased at delivery and remained elevated afterwards. ptgfr mrna increased - -fold at delivery and a further - -fold after delivery, suggesting loss of pgf permitted enhanced ptgfr expression. p serum concentrations fell pre-partum in both groups. conclusions: il- ko alters the expression pattern of several pregnancy and parturition-related genes and may delay the pre-partum p fall, suggesting a potential ovarian effect. a uterine role for il- in regulating the timing of normal term parturition cannot be ruled out. objective: recent studies have highlighted the prevalence of vitamin d deficiency in pregnant women, particularly in those from ethnic groups with darker skin who require higher levels of uv light to make parental vitamin d. as the active form of vitamin d, , -dihydroxyvitamin d ( , (oh) d ) is a potent immunomodulator, we postulated that vitamin d deficiency may lead to dysregulated placental immunity. both trophoblast and decidua express the enzyme -hydroxylase (cyp b ) which catalyzes synthesis of , (oh) d from the inactive pro-hormone -hydroxyvitamin d ( ohd ). in view of the role of trophoblast as a barrier site protecting the fetus against infection, we investigated the impact of cyp b , ohd and , (oh) d on innate immune responses in trophoblastic cells. methods: a trophoblast cell line was used to assess the effect of vitamin d on innate immune responses. the cells were treated for hrs with various concentrations of , (oh) d ( - nm) and antimicrobial cathelicidin expression was assessed by real time pcr. to assess whether expression of cyp b was affected by pathogenic stimuli, a cells were treated with ligands for toll-like receptor (tlr) - , cyp b expression (real time pcr) and ohd utilization were assessed. results: a trophoblast cell line; which shows temperature-sensitive differentiation, revealed expression of cyp b with higher levels of enzyme activity under conditions of syncytiotrophoblast development. cells treated for hrs with , (oh) d showed dose-dependent induction of the antimicrobial defensin cathelicidin expression ( . - fold induction), whilst cells treated pro-hormone ohd ( nm) showed . -fold induction of cathelicidin expression. activation of tlr (poly i:c) and tlr (lipopolysaccharide) enhanced the expression of cyp b ( -and . -fold) and increased the sensitivity to ohd as a consequence. conclusion: these data show that autocrine synthesis of , (oh) d from ohd can stimulate trophoblast immune responses in a similar fashion to macrophages. as ohd is the major circulating form of vitamin d, we hypothesize that trophoblast innate immunity may be significantly compromised under conditions of vitamin d deficiency. introduction: secretory leukocyte protease inhibitor (slpi) is a potent -kda protein inhibitor of neutrophil elastase and it is a mediator of mucosal immunity and an inhibitor of nfkb regulated inflammatory responses. however, its source, function and regulation within the uterus during pregnancy and at parturition are not well defined. it has previously been shown to be present in fetal membranes and cervical mucus and in amniotic fluid, where its levels is increased from second trimester to term and with a further increase at parturition. slpi has also been shown to be responsive to progesterone in human epithelial cells. our aim was to determine the effects of il- on slpi gene expression in human myometrium. methods: primary human uterine myocytes were isolated from non labouring myometrium and cultured in well plates and when cells were - % confluent they were serum starved overnight and incubated with m methyl- -hydroxy-progesterone acetate for h and with or without ng/ml il- for a further hours. at the end of incubations rna was extracted and converted to cdna. paired upper and lower segment myometrial tissue was collected at caesarean section either before or after the onset of term or pre-term labour and frozen for extraction of rna (n= for ptnl, ptl, tnl and l). copy numbers of slpi, gapdh and beta-actin were measured by qpcr. results: h incubation of uterine myocytes with ng/ml il- caused a marked increase in slpi by % (n= ; p< . ). incubation of uterine myocytes with m progesterone for h also increased slpi by % (n= ; p< . ). incubation with il- for h in the presence of h with progesterone increased slpi: gapdh mrna ratio from . ± . to . ± . (mean ± sem; p< . ). slpi expression was similar in the upper and lower segment myometrium in preterm patients. in term myometrium the slpi: beta-actin mrna ratio was increased by -and -fold in term labour versus term non labour samples in the lower and upper segment respectively (mean ± sem; p< . ). these data show slpi is present in human myometrium and that it is increased by il- . progesterone also increases its expression. its expression is increased in term labour where its anti-bacterial, anti-fungal and anti-viral properties could allow it to act as an endogenous block to infections. objective: pre-b cell colony-enhancing factor (pbef) downstream mapk signaling and transcription factor activation. introduction: pbef is expressed in all layers of the human fetal membranes and the myometrium and is upregulated by labor, infection, nf-kb, ap- and stretching. pbef has the ability to protect a variety of cells from apoptosis, however it also appears to act as an insulin mimetic via the insulin receptor (fukuhara et al. science : ; - ) . its levels are elevated in obese patients, those with type diabetes and in gestational diabetes. because pbef has poorly understood biological activities an investigation of mapk signaling and transcription factor activation induced by pbef has been undertaken in order to gain insights into its mechanisms of action. methods: primary aec were isolated from fetal membranes and treated with rhpbef ( ng/ml) for h, lysed and the resultant proteins labeled with cy or cy (amersham). there were used on a protein microarray containing constituents of the mapk signaling pathways (sigma). isolated aec were transfected with luciferase constructs for nf-kb, ap- , cre, hse and gre response elements using the exgen reagent. the cells were treated with rhpbef ( . , . , , ng/ml) hrs, lysed and ul of sample was analyzed for luciferase activity with dual-luciferase reporter assay system (promega). co-transfection with gfp and sv luciferase constructs to control for transfection efficiency and cell number (respectively) was also performed for each experiment. results: pbef significantly upregulated mapk signaling components including functioning as part of the erk pathways and belonging to p signaling. it also activated nf-kb and camp response elements. however, it significantly down regulated signaling molecules belonging to the jnk pathway that resulted in decreased c-jun phosphorylation. conclusions: pbef signaling in aec is consistent with its anti-apoptotic ability and its upregulation of the pro-inflammatory cytokines. although some mapk components responsive to pbef could be associated with insulin receptor signaling, some may not. therefore, it appears likely that pbef interacts with another potentially unique, but currently unidentified receptor. was the first effector to be characterised, but camp is now known to have other effectors, including camp receptor protein, cyclic nucleotide-gated channels and camp-guanine nucleotide exchange factor/exchange protein directly activated by camp (camp-gef/epac). two epac's have been identified and they consist of functional domains: camp-binding domains, a dep domain, a ras exchange motif and a gef domain. our aim was to determine the presence of epac's in human myometrium and to study the effect of camp responses on prolabour genes such as il- , pghs- , otr and fp. methods: primary human uterine myocytes were isolated from non labouring myometrium and cultured in well plates until - % confluent. cells were serum starved overnight and incubated with μm methyl progesterone, . mm sodium -bromo-camp, . mm forskolin, μm kt , ng/ml brefeldin a and . mm -pmeopt- 'o-me-camp either alone or in combination for h. rna was extracted and converted to cdna. paired upper and lower segment myometrial tissue was collected at caesarean section either before or after the onset of term or pre-term labour and frozen for extraction of rna (n= for ptnl, ptl, tnl and l). copy numbers of epac , epac , il- , pghs- , otr, fp, gapdh and -actin were measured by qpcr. results: epac and epac were present in the upper and lower segment of myometrium with epac levels being some -fold higher than those of epac . there was no change with the onset of labour at or before term. treatment with il- for h significantly increased epac (n= ; p< . ) but had no effect on epac . when conditions mimicked pregnancy, (the presence of forskolin and progesterone), brefeldin a, an epac antagonist, increased basal pghs- and fp mrna expression (n= ; p< . ), but had no effect on il- and tended to reduce otr. the il- -induced increase in pghs- and il- , but not fp, was greater with the epac antagonist. conclusions: these data show epac's are present in human myometrium and that camp acts via epacs to reduce pghs- and fp expression during pregnancy. background: inflammatory events have been implicated in the process of labour. glucocorticoids mediate strong anti-inflammatory effects through binding to the glucocorticoid receptor (gr), which on activation translocates to the nucleus and either increases or decreases the expression of responsive genes thereby suppressing inflammation. objective: to characterise the expression profile for gr protein and mrna in human myometrium during fetal maturation and parturition. methods: western immunoblotting (wb) was employed to characterise gr protein expression in first (n= ) and second trimester myometrium (n= ), and in paired upper and lower segment pregnant (non-labouring, p, n= ) and labouring (l, n= ) myometrium; as compared to non-pregnant (np, n= ) control samples. immunofluorescence staining with confocal microscopy and rt-pcr were also undertaken. results: detection of gr protein by wb revealed two bands at - kda, representing the alternatively spliced isoforms, gr-and gr-. densitometric analysis showed that gr levels decreased significantly during pregnancy and remained at very low levels at term and in labour when compared with np samples (p< . ); this decrease was seen for gr-and gr-. no significant temporal variations were observed in gr protein levels at term or during labour. gr protein was localised by immunofluorescence staining to the nuclei and cytoplasm of cells. less intense staining was apparent in p compared to np tissues; consistent with wb data. rt-pcr showed a consistent predominance of gr-to gr-mrna in all the tissues used. the observed decrease in protein expression was also mirrored at the mrna level: gr-mrna levels appeared to gradually decrease throughout gestation (p< . ). differences between the upper and lower myometrial regions were only observed in the labouring samples, where gr-mrna levels were significantly decreased in the lower segment (p< . ). nested pcr was additionally used to amplify gr-, but no consistent pattern of mrna expression was obtained. conclusions: these data are the first to characterise gr expression in human myometrium. spatial and temporal variations have been found with expression evolving through the three trimesters of pregnancy and in labour. further studies are now underway to evaluate whether gr contributes to the sequence of inflammatory events implicated in triggering term and preterm labour. these studies sought to determine the mechanism by which pas modulate the immune response. methods: ) an in vitro co-culture model mixed with human cervical epithelial hela cells and pma-induced human macrophage u cells at epithelial/macrophage cell ratio of : was employed. ) the co-culture was pre-treated with medroxyprogesterone acetate (mpa), progesterone (prog) and dexamethasone (dex) at concentrations of , , and nm for hrs followed by day stimulation of g/ml lipopolysaccharide (lps). these experiments were repeated using hela cells transfected with sirna for glucocorticoid receptor (gr). the production of cytokines il- , il- and il- , il- and tnf were determined using elisas. ) the co-culture was pre-treated with nm of each pas for hours prior to lps stimulation at g/ml for . , . , , , and hours. the phosphorylation of p mapk and gr and the expression of mapk phosphatase (mkp ) were determined by western-blotting. results: il- , il- and il- , il- and tnf were elevated by . fold, . fold, fold, . fold and . fold in the co-culture model in response to lps(p< . for all). pretreatment of mpa and dex, but not prog, inhibited the lps-induced cytokine production. the anti-inflammatory effect of mpa occurs in a dose-dependent manner. in the absence of gr, the inhibitory effect of mpa and dex on il- , but not on il- , was lost. mpa and dex, but not prog, induced the phosphorylation of gr and expression of mkp . p mapk was activated by lps for up to hrs and pretreatment of mpa and dex attenuated this response. the ability of mpa and dex to suppress the inflammatory response in cervical tissues appears to be mediated through a gr-dependent pathway, specifically though p mapk. our studies suggest that prog is not a significant immunomodulator in these tissues. background: progesterone (p ) has been shown to play a critical role in maintaining pregnancy and preventing preterm birth. these effects are likely related to its immunomodulatory properties. we investigated whether camp plays a role in the immunosuppressive action of progesterone on fetal mononuclear cells. methods: umbilical cord blood mononuclear cells were isolated using density gradient centrifugation. to establish a p effect and optimize concentrations, cells were pretreated with p ( - m- - m), dexamethasone ( um) or vehicle for hour prior to overnight lps ( ng/ml) stimulation. supernatants were assayed for tnf-using elisa. ldh assays confirmed the absence of a cytotoxic effect. next, cells were pre-incubated with forskolin (adenylate cyclase activator) or db-camp (camp agonist) prior to lps to determine the effects of camp on tnf production. finally, cells were pretreated with rp-camp (camp antagonist) prior to p incubation and lps stimulation to determine whether the p effect was reversed by a camp antagonist. results: p significantly inhibited lps-induced tnf production in a dose dependent manner, with maximum suppression observed at - m. both forskolin and db-camp suppressed lps-induced tnf production in a doserelated manner (fig ) . finally, a dose-dependent partial reversal of tnf production was observed when cells were pretreated with rp-camp. (fig ) conclusions: progesterone suppresses the inflammatory response in fetal mononuclear cells as measured by tnf expression. this effect is mimicked by adenylate cyclase activators and camp agonists and partially reversed by camp antagonists. this implicates the camp pathway in mediating the immunomodulatory actions of p . objectives estrogen improves endothelial function after vascular injury via largely unknown mechanisms. endothelial progenitor cells (epcs) are known to be implicated in various vascular events requiring endothelialization. we hypothesized that estrogen and progesterone could influence the function and the change of epcs in menstrual cycle. material and methods peripheral blood mononuclear cells (pbmcs) were isolated from peripheral blood of healthy young women in each menstrual period by density gradient centrifugation with ficoll separating solution. pbmcs were seeded in endothelial basal medium with or without estrogen or progesterone. on the th day in culture, nonadherent cells were removed. on the th day in culture, adherent cells were incubated with di-ldl, fixed with paraformaldehyde, and stained with fluorescein isothiocyanate-labeled lectin. the number of ldl-and lectin-positive cells was measured as epcs using flowcytometry. the expression of estrogen and progesterone receptor mrna in epcs were measured by real time pcr in menstrual and luteal period. the number of epcs was significantly increased in the menstrual and luteal period compared with the follicular phase. estrogen and progesterone significantly increased the number of adherent epcs dose dependently in menstrual period, but not in luteal period. the expression of estrogen-alpha receptor in menstrual period was higher than luteal period. also, estrogen-beta receptor in luteal period was strongly expressed compared with menstrual period. these results suggest the expression of estrogen receptor and progesterone receptor play important roles to regulate epcs' proliferation during menstrual cycle. objective: elevated levels of fetal plasma avp are associated with the development of oligohydramnios, in part a result of avp-mediated reduction in fetal urine production. as avp urinary concentration effects are mediated via upregulation of renal tubular aqp water channels, we propose that avp modulates placental aqp channels, influencing bidirectional maternal-fetal water flow. we sought to study the effect of avp on trophoblast aqp gene expression using the first trimester-derived extravillous htr- /svneo cells and the term placenta-like trophoblast carcinoma cells jeg- . methods: cultures of both cell lines were treated with a physiological concentration of avp ( . nm) to determine aqp mrna and protein expression. negative controls consisted of cells incubated in medium supplemented with % fbs without avp. to determine whether avp regulation of aqp occurs by a camp signaling pathway, jeg- cells were preincubated with μm -(tetrahydro- '-furyl) adenine (sq ), a cell-permeable camp inhibitor, before being treated with avp ( . nm). cells were incubated for hrs for aqp mrna expression and for hrs for protein extraction at °c. after harvest, real time pcr and western blotting analysis were used to detect the aqp mrna and protein expression levels, respectively. results: avp increased aqp mrna expression in both cell lines by . and . fold after hrs. aqp protein expression paralleled the increase seen in the mrna (p< . ). pretreatment of jeg- cells with sq inhibitor completely blocked the stimulatory effect of avp. conclusion: aqp gene expression is up-regulated by avp in first trimester and term trophoblast cells, with a higher induction in the later. avp activation of aqp gene expression occurs via a camp mediated pathway, as the adenyl cyclase inhibitor blocked avp effects on aqp gene expression. these results suggest that increased fetal plasma avp may contribute to oligohydramnios by an increase in aqp-mediated fetal to maternal water flow. objective: changes in expression, ratio and activity of progesterone receptors within human myometrium have been proposed as potential contributory mechanisms for the functional progesterone withdrawal effect which precedes labour. progesterone receptor c (pr-c) is an n terminally truncated isoform of the full length progesterone receptor; pr-c has been shown to be abundant in fetal membranes, placenta and upper segment of laboring myometrium ( , ). the function and regulation of pr-c is at present unclear. in this study we aimed to investigate the regulation of pr-c in cultured human myometrial and amnion-derived wish cells. methods: myometrial primary cell cultures were prepared from non pregnant and term pregnant uteri. both myometrial and wish cell cultures were treated with natural progesterone ( , nm, mm, mm, mm) for , and hrs. western immunoblotting was employed using nuclear and cytoplasmic extracts prepared from treated cells to observe the effect of progesterone on a) expression and b) subcellular localisation of pr-c within both cell types. immunofluorescent staining and confocal microscopy were also employed. experiments were also undertaken whereby nuclear and cytoplasmic extracts were subjected to shrimp alkaline phosphatase treatment to evaluate the phosphorylation status of pr-c in response to hormone. results: data indicates that a kda cytoplasmic protein, representing pr-c is abundant in myometrial and amnion cell cultures. we show that expression of pr-c increases in both cytoplasmic and nuclear fractions within both cell types in response to progesterone. evidence also suggests that pr-c is phosphorylated in a progesterone-independent manner. concurrent treatment with progesterone and the pr-antagonists ru and organon in amnion-derived cells still led to an increased abundance of pr-c but seemed to alter the phosphorylation status of pr-c. conclusion: pr-c expression and subcellular localisation within myometrial and wish cells alters in response to progesterone. speculation is generated about potential role of pr-c in reproductive tissues and its contribution to functional progesterone withdrawal. . taylor smooth muscle responses have been studied, data on regional blood flow (bf)distribution are scarce. we have studied the effect of a single dex course on relative bf distribution within the brain, skeletal muscle, heart, omental fat, placenta and adrenal in fetal sheep at . g, the equivalent of weeks. methods: fetal sheep received amniotic, jugular, carotid and femoral artery and vein catheters at days g (dg). experiments started at dg in dex ( mg) and saline treated controls (ctr), each receiving injections h apart. fetal blood pressure was recorded continuously (windaq pro+). microspheres ( . x total; sterispheres, biopal) were injected at : am into fetal jugular and femoral veins before maternal i.m. injection (t ) and h after the rd injection (t ) in both groups. tissues were collected hours after the th injection for assessment of bf (biopal). results: fetal bp increased above baseline after dex ( . + . mmhg; p= . ) with no change in ctr (- . + . mmhg). regional bf distribution is presented as a percent of the total counts per g tissue (table ) and after normalization within each animal to placentomal flow, shown previously to be unchanged by dex, in table . no significant differences in flow were found. discussion: although dex altered bp, regional distribution of bf among key organs was unaffected. whether gc does not affect regional bf, or the rise in bp is the result of a non-specific, system-wide vasoconstriction, or the impact of gc on bf is similar in all organs, will be subject of further investigation that will include determination of regional vascular resistance and absolute flow. stimulator while crf-r is an inhibitor of gi motility at times of stress. we recently hypothesized that stress-induced in utero meconium passage in fetuses is analogous to stress-induced defecation in adult rats and likely mediated by crf pathway. in support, we demonstrated hypoxia-induced meconium passage in conjunction with marked increases in fetal plasma crf levels. as the mouse is a common experimental model, with known genome, we sought to determine the presence of crf-r and crf-r receptors in mouse fetuses. methods: time-dated cd- pregnant mouse were anaesthetized and fetuses (n= ) were collected at e (term = e ). whole gi tracts were dissected, fixed in bouin's solution, paraffin embedded and sectioned at five micron thickness. sections were immunostained with rabbit polyclonal antibodies to crf-r and crf-r by abc technique with vector abc reagents. immunoreactivity was identified as brown staining using ', diaminobenzamidine as chromagen. slides were counter stained with mayer's hematoxylin and examined under the microscope. immunoreactive signals in the smooth muscle layers of gi tracts were quantified by image analysis using image pro . plus software. results: immunostaining for both crf-r and crf-r was seen in the smooth muscle layers of all lumens examined ( lumens per section). the immunostaining intensity expressed as intensity over density (iod) in arbitrary units for crf-r (maximal iod: . ± . au and minimal iod: . ± . au) and for crf-r (maximal iod: . ± . au and minimal iod: . ± . au) greatly varied between lumens. the mouse fetal gi tract expressed both crf-r and crf-r receptors, suggesting that the mouse is an appropriate model for fetal-stress induced neurovisceral motor responses. the non-uniform distribution of crf receptors in the fetal gi tract suggests the existence of regional differences in the expression patterns of crf-receptors of both types. we speculate that mouse devoid of crf and crf-r or r receptors will be useful to confirm the participation of crf pathway in stress-induced in utero meconium passage. antenatal exposure to glucocorticoids (gc) is associated with a reduction in nephron number and hypertension in adult life. one of the mechanisms for the development of hypertension is thought to be the long term effect of single nephron hyperfiltration. however, in most studies there is only a - % reduction in nephron number with no change in gfr. thus, although a reduction in nephron number may be a contributing factor it is unlikely the only variable. the aim of the present study was to evaluate nephron mass, renal function and blood pressure in a cohort of adult sheep exposed antenatally to betamethasone. methods: pregnant sheep were treated with two im doses of betamethasone (bm, . mg/kg) or vehicle (ctr) -hs apart at days gestational age and allowed to deliver at term. at . yr of age in female (f) and male (m) offspring, glomerular filtration rate (gfr) was measured as inulin clearance and effective renal plasma flow (erpf) as pah clearance. blood pressure was measured though an indwelling catheter in the femoral artery over a -hour period. nephron number was measured by the acid maceration technique. data mean±sem were analyzed by anova and/or two sample t test. results: antenatal bm was associated with an elevation in arterial blood pressure and a reduction in nephron number in both f and m offspring. in contrast, a reduction in gfr and erpf was present only in males. plasma and urinary electrolytes as well as urinary protein excretion were not different from ctr. conclusion: our data show that prenatal exposure to a single course of gc at . gestation has long-term effects on blood pressure regulation. interestingly, while the decrease in nephron number was of similar magnitude in m and f, evidence of alterations in renal function was present only in males. these suggest that the decrease in renal function observed in males is not solely a consequence of the decrease in nephron number. furthermore, it is possible that different mechanisms are responsible for the elevation in arterial blood pressure observed in m and f. hl ; hd p hd . clr l>t-x to evaluate the effects on blood pressure, urine output, sodium excretion and gfr of the intrarenal infusion of angiotensin - in the male sheep with or without prenatal exposure to b. we studied male sheep which had received either b (n= ) or vehicle (n= ) at - days gestation and were born at term. catheters were placed in the femoral artery, femoral vein, left renal artery and the bladder. the right kidney was removed. after days recovery the sheep received an infusion of ang - ( ng/kg/min) with or without its antagonist, [d-ala ]-ang-( - )(d-ala, ng/kg/min), into the renal artery for hours. blood pressure, gfr, urine output and sodium excretion were measured before and after the infusion. two-way analysis of variance (anova) was used to test mean values between the groups. with or without d-ala, map and urine output didn't change significantly during ang - infusion. however, the infusion of ang - resulted in a decrease (change from baseline) (p= . ) in na + excretion of . ± . meq/kg/ h in the vehicle animals and . ± . meq/kg/ h in the b animals. there was no significant change in gfr during ang - infusion. gfr was lower in the b group during the combined ang - and d-ala infusion ( . ± . ml/min vs . ± in vehicle group, p= . ). intrarenal infusion of ang - at ng/kg/min is followed by a significant decrease in sodium excretion but no significant change in urine output and gfr. the decrease tended to be greater in the vehicle animals and was not markedly attenuated by the antagonist d-ala. this suggests the effect of ang - may be mediated by a receptor other than the mas receptor for ang - and may be of lesser magnitude in animals exposed to b before birth. and norepinephrine ( . - . g/kg/min) and to an l-name ( mg/kg) bolus were studied. vascular reactivity was analyzed by curve fitting of either the blood pressure or hr responses to obtain the maximal response. data are expressed as mean±sem of change from baseline. statistical significance was established using t test. results: bm-exposed animals displayed a higher blood pressure response to at-ii (atii max ± vs ± % of baseline, p< . , figure left panel). no differences in blood pressure or heart rate responses to norepinephrine infusion were detected in bm when compared to control animals (figure right panel) . maximal response to l-name was also elevated in bm-exposed animals but did not reach statistical significance with the current sample size. conclusion: we have shown that antenatal gc treatment significantly increases blood pressure in sheep. here we show an enhanced in vivo response to at-ii. this response may contribute to the increased blood pressure observed. the greater response to l-name most likely represent an increased no production as a compensatory mechanism to the higher blood pressure observed. hl . women at risk of delivering preterm are frequently treated with glucocorticoids to facilitate fetal lung maturation. animal studies have revealed that prenatal exposure to glucocorticoids may alter aspects of hypothalamic-pituitary adrenal functioning in later life. in this study we examined the effects of clinically relevant prenatal betamethasone treatment on the responsiveness of pituitary cells (in terms of adrenocorticotropin, acth, secretion) isolated from adult sheep. time-dated pregnant sheep were injected with betamethasone ( . mg/kg) or vehicle on days and of gestation. thereafter pregnancy was allowed to continue unimpeded and offspring were born. pituitaries were isolated from adult sheep aged between and months and cells were dispersed and plated at a density × cells per well in well plates. after h, cells were stimulated with normal medium, nm arginine vasopressin (avp), nm avp, nm corticotropin releasing factor (crf) or nm crf for h. medium was collected and analyzed for acth using a commercially available kit. acth secretion (given as % increase from untreated cells) was similar between the control and betamethasone groups following nm avp ( . ± . vs. . ± . ) and nm crf ( . ± . vs. . ± . ). at higher stimulatory concentrations of both avp and crf, acth secretion remained similar in control cells ( . ± . and . ± . respectively), but was significantly decreased in betamethasone cells ( . ± . and . ± . respectively). these findings suggest that prenatal exposure to clinically relevant doses of betamethasone can alter pituitary responsiveness to both avp and crf in adulthood. this research was supported by nih grants hd and hd . lcc is supported by an rjr-leon golberg fellowship in pharmacology and toxicology. objective: newborn meconium (mec) passage normally occurs within the first - h after birth. in contrast, more than half a million infants born annually in the us pass mec in utero. neither the physiological mechanism(s) nor causes for in utero mec passage are well understood, though both fetal maturation and stress are associated. we recently reported in utero mec passage and marked increases in plasma crf levels in term fetal rats exposed to maternal hypoxic stress. we hypothesize that stress-induced in utero mec passage is mediated by the crf pathway in a manner analogous to stress-induced defecation in adult rats. in the present investigation we examined placental crf content prior to and following maternal hypoxia, to determine the source of increased plasma crf. methods: time-dated pregnant rats (n= ) were exposed to a paradigm of graded, stepwise hypoxia on day (term= ) (pediatr. res. : - , ) . control pregnant rats were exposed to % oxygen for a similar duration as experimental animals. at the end of the study, placentas were harvested, fixed in % paraformaldehyde and paraffin embedded. sections (n= per placenta) were subjected to immunohistochemical analyses with rat/human crf antibody (peninsula laboratory) by abc technique. immunoreactivities on placental sections were quantified using the image pro . software and intensity expressed as arbitrary unit (au). all values are mean± sem. results: in control maternal rats exposed to normoxia, positive staining for crf was seen in decidua ( . ± . au) and in all three major placental cells types (giant trophoblast cells: . ± . au, spongiotrophoblast cells: . ± . au and labyrinth cells: . ± . au.) in contrast, in animals exposed to hypoxia, there was a total absence of crf staining in the placental cells, with only positive crf staining seen only in the decidua ( . ± . au). conclusion: the total absence of crf staining in both the basal and labyrinth zones in placentas of pregnant rats subjected to hypoxic stress suggests that placental cells rapidly release crf into the maternal and fetal circulation in response to hypoxic-stress. our findings support our hypothesis that the peripheral crf pathway mediates in utero mec passage. offspring. angela g massmann, jie zhang, jorge p figueroa. department of obstetrics and gynecology, wake forest university school of medicine, winston-salem, nc, usa. objective: in rats and sheep, exposure to glucocorticoids (gc) in the perinatal period induces hypertension in adult life. recent evidence suggests that endothelin b (etb) receptor plays an important role in the regulation of sodium balance and blood pressure. renal medulla is an important site of expression and action of etb receptor. furthermore, in kidney it is the medulla (km) where the highest concentrations of immunoreactive et- and etb receptor are found. the aim of this study was to measure eta and etb expression in adult sheep kidney exposed antenatally to gc. methods: pregnant sheep were treated with two im doses of betamethasone (bm, . mg/kg) or vehicle (v) hours apart at days of gestational age and allowed to deliver at term. at . yr of age, male and female offspring exposed to either vehicle or bm were euthanized and the kidneys harvested. kidney medulla (km) was obtained by sharp dissection. eta and etb protein and mrna expression were evaluated by western blot and rnaase protection assay. data are expressed as mean±sem and were analyzed by two way anova. results: a significant decrease in etb (f= . , p= . ) but not eta protein was observed in km of bm exposed male and female adult sheep. at the mrna level, bm exposure also affected etb, but not, eta expression. however, bm treated animals had higher mrna levels than control sheep (f= . ; p= . ). conclusion: our data show that prenatal exposure to a single course of gc at . gestation has long-term effects. the activation of etb receptor by et- inhibits sodium transport function in the collecting duct and the selective gene deletion of the etb in medulla results in hypertension.therefore, our finding of a significant reduction in etb protein suggests that there may be an impairment in the kidney's ability to regulate sodium reabsorption. the functional relevance of the alterations in etb protein expression as well as the discrepancies in mrna regulation need to be established. hl ; hd p hd . in the placenta, -hydroxysteroid dehydrogenase type ( hsd ) regulates the transfer of cortisol. maternal glucocorticoid (gc) administration has been shown to affect the ovine fetal growth trajectory and regulate hsd expression in mid and late gestation. however, little is know about the effects of gc administration in early gestation. we hypothesized that maternally administered low-dose dexamethasone (dex) in early gestation would affect hsd expression, which will subsequently alter fetal birth weight. pregnant ewes were randomized and received intramuscular injections consisting of either saline ( ml saline/ewe) or dex ( . mg/kg) given hours apart starting on days of gestation (dg). the animals were sacrificed at , , , and dg and fetal weights were recorded and placental tissue was collected. qrt-pcr was used to measure hsd placental mrna expression. statistical analysis was done by anova with student's t-test posthoc. overall, there was no significant effect of dex treatment on placental hsd mrna expression across gestation. however at dg, there was a significant increase in hsd expression after dex (p= . ). there was an increase in placental hsd mrna progressively from dg (mean = . ) to dg (mean = . ), independent of treatment. overall, a positive correlation between hsd and fetal weight (r = . ) was apparent at dg and at dg (p= . ) in both control (p= . ) and dex (p= . ). significant positive correlations between placental hsd and fetal weight (r = . ) were found in females (p= . ) and a similar trend was found in male fetuses (p= . ). we conclude that in the sheep placenta, hsd expression increases throughout gestation and may respond, at least transiently in early gestation, to elevations in maternal gc. the positive correlation between hsd and fetal weight is consistent with the thesis that the fetal growth trajectory may be influenced by maternal cortisol levels and that placental hsd is an important mediator of these effects. antenatal gc exposure induces hypertension in the young adult rat (neuroendocrinol; ; : ) and increases femoral vascular resistance in the lamb (jphysiol; ; : ) . unpublished own examinations in preparation of this study have shown age dependency of the vascular reactivity. vascular contractility of the middle cerebral artery (mca) decreased in response to k + and noradrenaline (na) between mo and , y of age (p< . ). vascular relaxation to acetylcholine (ach) but not to pge decreased during the same time (p< . ). vascular contractility of the renal artery (ra) increased in response to k + (p< . ). aims: to examine if antenatal gc alter vascular reactivity in the aged individual when cardiovascular diseases are predominant. we studied the ra because its vascular tone is involved in modulation of arterial pressure control (amjphysiol; ; :r ) and the mca because depressive disorders that are associated with dysregulation of the hpa axis (jcem; ; : ) increase stroke mortality for unknown reasons (amjpsychiatry; ; : ) . to be clinically relevant, we administered dexamethasone at the dose used to enhance lung maturation in babies threaten premature labor. methods: pregnant dams received saline (n= ) or μg/kg dexamethasone (n= ) i.p. at day e / equivalent to x mg dexamethasone administered to a kg pregnant woman. at . years of age, vascular response of the ra and mca to endothelium-dependent and independent mediators was measured using wire myography. vessels were inspected histologically for intact endothelium. results: basal vasoconstrictory and dilatory responses to all mediators were less pronounced in the mca than in the ra probably reflecting autoregulatory properties of cerebral vessels (p< . ). after prenatal dexamethasone exposure, contractility but not sensitivity to k + and na was enhanced in the mca and even more pronounced in the ra (p< . ). relaxation to ach and pge was similarly diminished in both vessels after precontraction with na (p< . ). conclusions: prenatal dexamethasone exposure at the dose used clinically increases renal and cerebral vascular tone in the aged rat by endotheliumdependent and independent mechanisms. this effect may be a potential mechanism of fetal programming of cardiovascular diseases in later life. betamethasone (bmz) therapy during pregnancy to improve fetal lung maturity may result in long term side effects, including hypertension, during adulthood. the kidney is an important organ which regulates systemic blood pressure via the local renin angiotensin system (ras). the major enzymes of the intrarenal ras include angiotensin converting enzyme (ace), angiotensin converting enzyme (ace ), and neprilysin. the hypothesis of this study is that prenatal bmz exposure will result in alterations of the enzymes regulating the intrarenal ras. specifically, sheep that are treated with bmz in utero will have higher amounts of ace activity in kidney cortex compared to control animals. pregnant sheep of known mating date were either treated with vehicle (n= ) or with two doses of bmz (n= ), . mg/kg, hours apart at days of gestation. renal cortex from the male offspring was harvested at - months of age. cortical membranes were then solubilized and incubated at °c with either i-ang i or i-ang ii in the presence or absence of lisinopril to inhibit ace activity, mln to inhibit ace activity, or sch to inhibit neprilysin activity. the metabolic products were separated by reversephase high performance liquid chromatography (rp-hplc). the rate of enzyme activity was quantified by calculating the area under the curve for each product and converted to fmol of product per mg protein per minute of incubation. statistical analysis was performed using student's t-test. p< . was considered significant. results: bmz treatment resulted in a significant increase in ace activity compared to control animals (p= . ). ace and neprilysin levels were not significantly different between treatment groups. when expressed as a ratio of ace/ace activity, bmz treated animals exhibited a . fold greater proportion of ace activity versus control animals (p= . ). this study demonstrates that bmz exposure during fetal life results in a significant increase in renal ace activity during adult life. this increase in enzyme activity would be expected to be associated with increased levels of ang ii in the kidney and na + retention, possibly underlying the development of hypertension. hd , hd . we recently hypothesized that stress-induced in utero meconium passage in term fetuses utilizes peripheral and/or central crf pathways in a manner analogous to stress-induced defecation in adult rats. as participation of central crf pathway requires the crf circuitry system in brain-gut axis, we exploited immunohistochemical techniques to address whether term fetal colon is wired by crf-nerve fibers. methods: fetal distal colon segments (n= ) were dissected from term ovine fetuses ( - d gestation). in addition, lumbosacral spinal cord with spinal roots and dorsal root ganglia (drg) (n= ) were dissected from ovine fetuses, and vagal nerve trunk with nodose ganglia (n= ) were dissected from mouse fetuses. paraffin sections fixed either in bouin's solution or zamboni's were subjected to immunohistochemistry with rabbit polyclonal antibodies specific to ovine-crf (ocrf). immunoreactivity on the sections was identified by standard abc technique, immunostaining quantified by image-pro plus software and expressed (intensity over area) as arbitrary units (au). results: ocrf antibody elicited strong positive staining on the serosal surface (port of entry for extrinsic nerve fibers) in distal colonic segments objective: we hypothesize that stress-induced in utero meconium passage is mediated by the crf pathway. ucn-i, similar to crf, is a potent contractility inhibitor of preterm ovine fetal distal colon, which has abundant crf-r receptors. both ucn- and crf thus may inhibit colonic motility and prevent preterm in utero meconium passage via crf-r receptors. however, ucn-i is reported to stimulate contractility of adult rat colonic smooth muscle strips more strongly than crf. we sought to study the pattern of ucn-i innervation in distal colon of ovine fetuses to delineate whether ucn- functions as a facilitator or inhibitor of colonic propulsive motility. methods: bouin's solution fixed, paraffin sections of very preterm (vpt: - days gestation), preterm (pt: - days), near term (nt: - ) and term (t: - days) ovine fetal distal colon rings were subjected to immunohistochemistry with polyclonal antibodies to human ucn- ( : sigma) by abc system. intensity of ucn- immunostaining in smooth muscle layers and myenteric neurons were quantified by image-pro plus software and expressed (intensity/area) in arbitrary units (au). differences over time were assessed with anova. in all groups very preterm was significantly greater than term (p< . ). conclusion: ucn- immunoreactivity in all three muscle layers, as well as myenteric and submucosal neurons, is highest at very preterm as compared to more mature gestations. these results suggest that the reduction of ucn- inhibitory function may facilitate stress-induced meconium passage at term. to evaluate the effect on blood pressure (bp), urinary output (uop), sodium excretion (nae) and gfr of the intrarenal infusion of ang ii in the male sheep after prenatal exposure to b. methods: we studied male sheep which had received either b (n= ) or vehicle (n= ) at - days gestation. catheters were placed in the femoral artery and vein, left renal artery and in the bladder. a right side nephrectomy was performed. after days of recovery, the sheep were infused with ang ii ( ng/kg/min) with or without candesartan ( ng/kg/day) or pd (pd g loading dose and ng/kg/min infusion) into the renal artery over hours. bp, gfr, uop and nae were measured before and after the infusion. two-way analysis of variance was used to test mean values between the groups. results: bp and uop did not change with ang ii infusion with or without candesartan. the infusion of pd did not affect uop but did increase bp in the b sheep (p= . ). ang ii decreased the nae in both groups(p= . ). candesartan increased nae among controls(controls . and b . meq/kg/h, p= . ). pd infusion decreased nae among controls but this was not significant. baseline gfr was higher among vehicle compared to b animals (p= . ). during ang ii infusion there was a decrease in gfr among control compared to b sheep (- . vs. - . ml/min, p= . ). this difference was not seen during candesartan or pd infusion. conclusion: ang ii infusion led to a decrease in nae in both groups and gfr among controls but not b animals. infusion of ang ii with candesartan increased nae while pd decreased nae among controls but not b animals. this suggests that prenatal steroid exposure can alter at receptor mediated response in renal function by decreasing the effect of ang ii on renal sodium excretion. the differences in gfr suggest that this may be due to an effect on tubular sodium reabsorption rather than glomerular perfusion. antalarmin antagonism of acth-induced cortisol secretion by ovine adrenal cells probably not at acth receptor. nancy k valego, james c rose. center of research for ob/gyn, wake forest u. school of medicine, winston-salem, nc, usa. in addition to regulating the hpa axis, crh and its type receptor (crhr- ) occur peripherally and in the female reproductive system. late in human pregnancy, a surge in placental crh probably stimulates adrenal secretion of cortisol and dheas requisite to parturition. we previously reported that, in dispersed fetal or adult ovine adrenal cortical cells, hour incubation with the specific crh-r antagonist, antalarmin (ant), significantly reduced both cyclicamp and cortisol responses to acth, suggesting an interaction with acth-r (like crh-r , a g-protein-coupled membrane receptor). however, forskolin (fsk; direct stimulant of adenylyl cyclase)-stimulated cortisol secretion was also attenuated by hour co-incubation with ant. our objective was to clarify the effect of ant on binding of acth to its receptor after a short ( . hours) treatment. method: acth binding: the adrenals from adult sheep were obtained at necropsy and the cortex cells dispersed and plated @ cells/well. after hours in culture, wells were rinsed x and refilled with serum-free dmem/ f containing vehicle (dmso) with or without ant. after . hours, wells were washed very gently and the binding assay (adapted from rainey et al; j biol chem, : , ) completed. triplicate vehicle or ant wells were treated with i -tyrosine human acth ( - ) with or without - m acth (for non-specific binding). after hour, wells were chilled, washed, and the cells lysed and counted on a gamma counter. fsk treatment: cells were treated as above except that fsk ( - m) was added to the wells. after . hours, medium was removed and stored @ - ºc for camp eia and cortisol ria. results (mean±se) of . hour incubation on acth binding and fskinduced secretion. vehicle antalarmin acth binding (net cpm) n= ± ± cortisol (ng/ml/ . hr) n= ± ± * (p=. ) camp (pmol/ml/ . hr) n= . ± . . ± . * (p=. ) * indicates significant difference from vehicle alone. conclusion: the crh-r antagonist, antalarmin, attenuates acthstimulated cortisol secretion but not by preventing acth receptor binding. however, its effect is early in the secretory process and is associated with a reduced camp response. objective glucocorticoids are often administered to pregnant women to prevent neonatal respiratory distress syndrome. prenatal steroid treatment increases blood pressure in adult sheep. exposure to excess corticosteroids before birth is hypothesized to be a key mechanism underlying the fetal origins of adult disease hypothesis and effects on the renin-angiotension system (ras) may modulate the steroid-induced increases in blood pressure. we therefore sought to determine if renin processing and secretion were altered in adult female sheep exposed antenatally to betamethasone ( ) and to compare them with data from males studied previously. methods pregnant sheep were randomized to receive doses of . mg/kg of or vehicle, at and days of gestation; the offspring were studied at and months of age. in female offspring, active renin concentration (arc) and total renin concentration in plasma were measured by ria of angiotensin i generated by incubation with excess substrate. prorenin concentration (prc) is the difference between total and active renin. nine or control exposed female animals born at term ( - days of gestation) were brought from the farm at - months of age, and had vascular and bladder catheters placed. five days after surgery, a sodium load of hypertonic nacl ( . meq/kg/min at . ml/min) was given for min. blood samples were obtained. data are expressed as mean sem and were analyzed by t test. the prc was significantly higher in the females than in the males ( . ± . vs . ± . p< . ) but there was no effect of prenatal steroid treatment. arc was similar in both genders. however, arc was a significantly greater percent of the total plasma renin concentration in the males ( . ± . vs . ± . p< . ) during the na infusion experiment, the exposed females had lower arc than did the control females ( . ± . vs . ± . p< . ). conclusion the data suggest that prenatal exposure to didn't alter the processing and secretion of renin in adult female sheep. prenatal steroid treatment does not appear to alter the effect of gender on plasma renin levels in adult sheep.it seems unlikely that the elevated blood pressure seen in adult ewes after prenatal exposure is the result of increased secretion or processing of renin. supported by hd and hd . background: mrap is a recently discovered protein with alpha and beta isoforms, a common amino terminal region and divergent c-terminal sequences generated by alternative splicing. in human and mouse mrap plays an essential role in the generation of a functional, g-protein coupled acth receptor (melanocortin- receptor; mc r) but has not been described for ruminants. we previously reported that lth fetal sheep exhibited elevated basal acth - yet decreased expression of key steroidogenic enzymes and the mc r in the adrenal cortex while basal cortisol levels were not different from control. we hypothesized that mrap could play a key role in mediating the effect of lth as well as developmental regulation of fetal adrenal cortisol synthesis in fetal sheep. the goals of the present study were to determine the ontogenic pattern of mrap expression in the sheep fetus and to determine if lth alters mrap expression. methods: we searched the bovine genomic sequence database (www.ncbi.nlm. nih.gov/genome/guide/cow/) and found a sequence with > % homology to the human mrap n-terminal residues, with partial homology to the beta isoform in the carboxyl region ( %). using primers based on the bovine sequence, we confirmed the presence of mrap in the ovine fetal adrenal cortex. adrenal cortical tissue was collected from sheep fetuses from - (n= ) and near term ( - ; n= ) days of gestation (dg) as well as from lth (n= ) fetuses (exposed to high altitude hypoxia from to - dg) and age-matched normoxic controls (n= ). cyclophilin was used as a housekeeping mrna. data are expressed in fg mrna/ ng total rna. results: the expression of mrap, based on quantitative rt-pcr, was low between - dg ( . ± . ) and increased (p< . ) approx. -fold near term ( - dg; . ± . ). levels of mrna for mrap were highly correlated to cyp mrna in individual samples. mrap expression in control ( . ± . ) and lth ( . ± . ) did not differ between groups. gender differences in hypertension are well described and there is growing evidence that the regulation of the renin angiotensin system (ras) is influenced by sex hormones. the major enzymes of the ras include angiotensin converting enzyme (ace), angiotensin converting enzyme (ace ), and neprilysin. the peptide products of these enzymes have opposing actions. angiotensin ii (ang ii), the product of ace, is a potent vasoconstrictor. on the other hand, the peptide products of ace and neprilysin, ang ( - ) and ang ( - ), exhibit vasodilatory properties. thus, modifications in the relative proportions of these enzymes and their peptide products can result in alterations of systemic blood pressure. the purpose of this study was to describe the gender differences in angiotensin converting enzyme (ace), angiotensin converting enzyme (ace ), and neprilysin (nep) enzyme activities in adult sheep kidney cortex. renal cortex from male (n= ) and female (n= ) sheep were harvested at - months of age. the tissue membranes were solubilized and incubated at °c with either i-ang i or i-ang ii in the presence or absence of lisinopril to inhibit ace activity, mln to inhibit ace activity, or sch to inhibit neprilysin activity. the metabolic products were then separated by reverse-phase high performance liquid chromatography (rp-hplc). the rate of enzyme activity was then quantified by calculating the area under the curve for each product and converted to fmol of product per mg protein per minute of incubation. statistical analysis was performed using student's t-test. p< . was considered significant. results ace activity was over times greater ( ± . vs. . ± . fmol/mg/min, p< . ), ace activity was . times greater ( ± . vs. . ± . fmol/mg/min, p= . ) and nep activity was nearly times greater ( . ± . vs. . ± . fmol/mg/min, p= . ) in female kidney cortex. in adult sheep, key enzymes of the intrarenal ras have signficantly greater activity in female kidney cortex. these findings suggest that there are fundamental, gender specific, differences in the regulation of the enzymes of the intrarenal ras. the physiologic significance of these findings remain to be elucidated. hd , hd . in rats and sheep exposure to glucocorticoids (gc) in the perinatal period is associated with a reduction in nephron number and hypertension in adult life. furthermore, antenatal exposure to gc alters glucose tolerance in animals and in people. the aim of the present study was to determine ) if insulin resistance is a contributing factor for the development of hypertension in adult sheep exposed antenatally to gc and ) if diet-induced obesity has a more pronounced effect in sheep exposed antenatally to gc. methods: pregnant sheep were treated with two im doses of betamethasone (bm, . mg/kg) or vehicle (ctr) -hours apart at days gestational age and allowed to deliver at term. at mo of age, female sheep were randomly allocated to be fed at either % of recommended nutritional allowance or ad libitum for three months. sheep were chronically instrumented under general anesthesia to place intravascular catheters. insulin sensitivity was evaluated both by iv glucose tolerance test (ivgtt) and euglycemic clamp (hec)techniques. for the ivgtt a . g/kg glucose bolus was used and for the hec mu/kg human insulin was used. data mean±sem were analyzed by anova and/or two sample t test. results: ad lib fed sheep gain > % of the original weight. antenatal bm was associated with an elevation in basal and ivgtt plasma insulin values. as shown on the figure, diet-induced obesity significantly increased insulin plasma levels during ivgtt in bm-exposed adult female sheep (f= . ;p< . ). insulin sensitivity derived from hec was significantly decreased by obesity in bm-exposed adult female sheep. conclusion: our data show that prenatal exposure to a single course of gc at . gestation has long-term effects on glucose metabolism regulation. bm-exposed sheep exhibit alterations in glucose tolerance, hyperinsulinemia and insulin resistance. these abnormalities are exagertated by diet-induced obesity. hl . syngergistic induction of -hydroxysteroid deyhdrogenase type expression by cortisol and interleukin- in human fetal lung fibroblasts. z yang, p zhu, cm guo, l myatt, k sun. school of life sciences, fudan university, shanghai, china; obstetrics and gynecology, university of cincinnati, cincinnati, oh, usa. objectives: glucocorticoids acting via glucocorticoid receptor (gr), serve as crucial hormones in fetal lung maturation. glucocorticoids and proinflammatory cytokines to induce b-hydroxysteroid dehydrogenase type ( b-hsd ) which converts inactive cortisone to active cortisol, but their effect on b-hsd expression has not been addressed in human fetal lung. we examined the interactions and mechanism of cortisol and interleukin- b (il- b) effect on b-hsd in human fetal lung fibroblasts (hfl- cells). methods: the expression of b-hsd in hfl- was examined with immunocytochemistry and pcr. b-hsd , prostaglandin h synthase- (pghs- ) and cytosolic phospholipase a a (cpla ) mrna levels in cultured human fetal lung fibroblasts treated with cortisol and il- b were measured with real time pcr. the roles of gr and c/ebps in the effect of cortisol and il- b were studied using a gr antagonist (ru ) and transfection of plasmid carrying c/ebp-specific dominant-negative gene (cmv -a/cebp). results: hfl- cells expressed a high level of b-hsd . both cortisol ( - - - m) and il- b ( . - ng/ml) induced b-hsd mrna expression in a concentration-dependent manner, an effect blocked by the mrna transcription inhibitor , -dichlorobenzimidazole riboside ( μm). ru ( - m) blocked the induction of b-hsd by cortisol. induction of b-hsd mrna expression by cortisol ( - m) was synergistically increased by co-treatment with il- b ( . - ng/ml) in a concentration-dependent manner. in contrast, the induction of cpla and pghs- expression by il- b was inhibited by cortisol, suggesting a different mechanism of interaction. transfection of the cells with c/ebp-specific dominant-negative plasmid attenuated induction of b-hsd mrna expression by either cortisol or il- b. these data suggest induction of b-hsd expression by cortisol is a gr dependent process involving c/ebps, which also mediate induction of b-hsd expression by il- b. conclusions: cortisol and il- b synergistically induce b-hsd expression in human fetal lung fibroblasts. this would lead to greater local cortisol production, perhaps providing either a self-attenuating mechanism for control of inflammation or a mechanism for enhancing fetal lung maturation when the fetus is exposed to cytokines e.g. with infection-induced preterm labor. do alterations in placental -hydroxysteroid dehydrogenase ( hsd) activities explain differences in fetal hypothalamic pituitary adrenal function following periconceptional undernutrition or twinning in sheep? kl connor, pl van zijl, cw rumball, , al jaquiery, , je harding, , mh oliver, , fh bloomfield, , jrg challis. medicine, university of toronto. periconceptional undernutrition (pcun) leads to activation of fetal hypothalamic pituitary adrenal (hpa) function, whereas twinning results in delayed fetal hpa activation. we hypothesized that these differences in fetal hpa activity were the result of altered patterns of expression of placental of hsd isozymes and hence of the maternal glucocorticoid (gc) effect on the fetus. we developed a mass spectrometric assay for the measurement of hsd- and - activities and validated this method against a widely used thin layer chromatography method. sheep were randomly assigned to ad libitum (n control) concentrates throughout gestation or were undernourished (un) from days before until days after mating to reduce maternal body weight by %, with ad libitum feeding thereafter. placentomes were collected on days , , , and of gestation (term, d) and hsd- and - activities were determined by measuring the rate of interconversion between cortisone to cortisol. with un, there was a trend towards lower hsd- activity at d (p= . ), a significant reduction at d (p< . ), but no difference at d or d . hsd- activity was not different between n and un animals at any time. there was no effect of twinning on hsd- or - at d . however, with twins both hsd- (p= . ) and - (p< . ) activities increased at d . hsd- activity was reduced in twins (p= . ) at d but was higher than any singleton pregnancies at d (p= . ). hsd- was not different between singletons and twins at either d or d . overall, hsd- was lower in placentae of male compared to female fetuses in late gestation; hsd- was higher in male than female fetuses. there was no interaction with un in either sex. we conclude that pcun and twinning result in alterations of placental hsd activities in sheep. modifications in these enzymes during critical periods of fetal development may affect transplacental transfer or placental generation of gcs reaching the fetus potentially influencing the timing of activation of the fetal hpa axis, fetal maturation and later life development. methods: pregnant sprague-dawley rats had % mfr from day of gestation until delivery. control animals had ad libitum food. offspring were sacrificed on day of life (p ) and at months (n= - per group). adrenals were dissected and snap frozen in liquid nitrogen for later extraction of rna. real time rt-pcr using specific rat primers was used to quantify mrna levels ( s as control). we evaluated the expression of -beta hydroxylase (cyp b ), aldosterone synthase (cyp b ), acth receptor (mcr ), p side chain cleavage enzyme (cyp a ), star protein, -hydroxysteroid dehydrogenase type (hsd ) and type (hsd ), glucocorticoid receptor (gr), and mineralocorticoid receptor (nr c ). fold changes in mrna expression in controls and mfr offspring was compared by student's t-test. results: there was a marked downregulation in expression of cyp b (p=. ), cyp b (p=. ), hsd (p=. ), p (p=. ), acth receptor (p=. ), star (p=. ) and nr c (p=. ) mrna in p mfr offspring, with no changes in hsd and gr. gender specific differences were found in the adult mfr offspring. in the male mfr offspring the expression of hsd and gr were significantly upregulated with a trend towards an increase in acth receptor (p=. ) whereas in the female mfr the expression of acth receptor (p=. ) was increased and nr c (p=. ) and cyp b were decreased (p=. ). in combined data for adult male and female offspring the expression of acth receptor was significantly (p=. ) increased. conclusion: these results indicate that mfr has a suppressive effect on steroidogenic enzymes of the newborn offspring regardless of gender. this may be an adaptive mechanism in the fetus/newborn to offset the high circulating maternal glucocorticoids in response to undernutrition. in adult male mfr offspring, increased hsd indicates an increase in gc synthesis whereas in the females there were no changes in gc synthesizing enzymes with a suppression of mc synthesizing pathway. the common finding of an increased acth receptor expression in both genders would suggest an increased sensitivity of adult mfr offspring adrenals to the effects of stress. objective: during gestation the fetal adrenal undergoes phases of active growth ( - d), quiescence ( - d) followed by reactivation (> d) before birth. insulin like growth factors play an important role in stimulating adrenal growth throughout late gestation. interestingly the prepartum activation of the adrenal is delayed in the female compared with the male fetus, but the mechanisms underlying this delay are unknown. hypothesis: we hypothesize that there are gender specific differences in the gestational profile of adrenal igf mrna expression between male and female fetuses. methods: a total of twin fetuses were used in this study. post mortem was performed at either - d, - d or - d gestation. adrenal mrna expression of igf , igf , igf r, igf r and cyp was determined by qrt-pcr. results: fetal weight was not different between males and females, and increased (p< . ) with increasing gestational age. the relative adrenal weight was lower however after d when compared to the earlier gestation age group (p< . ). adrenal igf mrna expression was lower (p< . ) at - d when compared to the earlier gestation age groups. interestingly, igf r expression was highest at - d (p< . ). there was an interaction between the effects of age and gender on adrenal igf and igf r mrna expression such that the expression was higher in males compared to females at - d (p< . ), but was not different to either the earlier or later gestational ages. there was no effect of either gender or gestational age on adrenal cyp mrna expression. conclusions: it has been speculated that a delay in prepartum activation of the adrenal in female fetuses may be due to gender specific differences in the intra-adrenal bioavailability of igf . in this study we have demonstrated there is an increase in both igf and igf r mrna expression in males compared to female fetuses. this may be evidence that adrenal igf expression plays a role in the earlier activation of the adrenal gland in male fetuses. we have previously shown that in response to a secondary stressor, in vivo cortisol secretion is elevated in long term hypoxic (lth) ovine fetus despite lower acth receptor mrna expression, and no differences in plasma adrenocorticotropic hormone (acth) levels when compared to normoxic controls. the present study was designed to determine the potential mechanism(s) of this enhanced cortisol secretion. specifically we tested the hypothesis that post receptor signaling events including camp production and expression of steroidogenic acute regulatory protein (star) are enhanced following lth. methods: for the lth group, pregnant sheep were maintained at high altitude ( , m) from day to near term. on days - (term = days), fetal adrenal glands were collected from lth (n= ) and age-matched, normoxic signal transduction pathways that control embryogenesis, cell differentiation, cell proliferation and cell death. the roles of extracellular signal-regulated kinase / (erk / ) and p map kinase in the differentiation and invasion of human trophoblasts have been studied. however, the in vivo expression and activation of erk / and p at the placental bed has not been elucidated. the study group consisted of placental bed biopsy tissues obtained from the pregnancies without preeclampsia (n= ) and with preeclampsia (n= ) between and weeks of gestation. we evaluated the expressions and phosphorylations of erk / and p map kinase in the invasive trophoblasts in the placental bed tissues using immunohistochemistry. results: p and phospho-p map kinase were not detected in invasive trophoblasts in cases or controls. erk / and phospho-erk / were positive in invasive trophoblasts albeit with variable staining. phosphorylation of erk / was significantly less frequent in invasive trophoblasts in placental bed biopsies from women with preeclampsia compared with normotensive controls. conclusion: these findings suggest that preeclampsia is associated with decreased activation of erk / in invasive trophoblasts in vivo. objective: placentae from preeclamptic pregnancies are characterized by an excess of immature hyperproliferative trophoblast cells, however the molecular mechanisms regulating cell cycle progression in this pathology are unclear. our aim was to examine the expression of g phase cell cycle regulators in normal and preeclamptic placentae and to establish whether the hyperproliferative state of trophoblast cells in preeclampsia may result from a developmental delay. methods: human placental samples were collected from normal pregnancies throughout gestation (n= ) and from severe early onset preeclamptic (n= ), and age-matched control placentae (n= ). protein expression of cyclin e , cyclin d , cyclin d and cell cycle inhibitors, p , p , p , and p was assessed by western blot analysis. spatial and temporal localization of cyclins e , d , d , p and p was determined by fluorescence immunohistochemistry. expression of cyclin e and cyclin d mrna was evaluated by qpcr analysis. results: immunohistochemical analysis showed cyclin e and cyclin d to be localized to cytotrophoblast (ct) cells of the chorionic villi; additionally cyclin e was also expressed in the extravillous trophoblast cells of the anchoring columns. in contrast, cyclin d expression was predominantly restricted to the villous stroma. cell cycle inhibitor p was expressed in both ct and syncytiotrophoblast (st) cells whereas p was restricted to the st. during normal placentation, levels of both cyclin e and cyclin d were high in the first trimester and decreased with advancing gestation. the expression of cyclin d , p and p showed an inverse correlation to cyclins e and d whereby their expression increased towards term. levels of p and p remained constant throughout pregnancy. preeclamptic placentae showed a significant increase in both cyclin e and p and a decrease cyclin d and p expression, as compared to age-matched control tissues. interestingly, preeclampsia was associated with an increased number of cyclin e positive progenitor ct cells in the floating villi and an increased expression of p in the endothelial cells lining the villous vessels. conclusion: preeclampsia displays an altered expression of g phase cell cycle regulatory molecules portraying an expression profile that closely resembles that of first trimester placentae. (supported by cihr, owh/igh). we have also demonstrated elevated, hif- -mediated placental and circulating sflt- at high altitude. we sought to correlate circulating levels of plgf, free vegf and sflt- with maternal and fetal oxygen tensions. we hypothesized that circulating sflt- and free vegf would be increased at m, and that plgf, known to be up-regulated by higher oxygen tension, would be decreased. methods: we collected both serum and plasma samples, the latter treated with inhibitors of platelet activation. maternal and umbilical samples were obtained from and healthy mother-infant pairs living at m and m respectively. plgf, free vegf and sflt- were measured in both serum and plasma using commercially available elisa kits (r d). data were log-transformed and analyzed by unpaired student's t test or anova as appropriate. results: plgf did not differ between altitudes. free vegf ( ± pg/ml vs. ± pg/ml, p<. ) and sflt- ( . ± . vs. . ± . ng/ml) were increased at m. however concentrations of all the angiogenic growth factors were reduced when platelet activation was prevented (- ± % plgf; - ± % free vegf; - ± % sflt- , p<. ). cord blood free vegf was -fold greater than in the mothers, but inhibition of peripheral cell activation abolished detectable levels in % of the babies. similar results were obtained for sflt- . thus, while free and total maternal circulating vegf and sflt- are elevated at high altitude, these increases are due to activation of peripheral blood cells. moreover, free vegf does not exist in detectable amounts in human pregnancy. there was no relationship between variation in the angiogenic growth factors and maternal or fetal oxygen tensions. the objective of this study is to identify candidate genes responsible for the variance between severe preeclampsia (spe) and hellp syndrome (hs). placental biopsies from spe (n= ) and hs (n= ) were collected. diagnosis of spe was confirmed by blood pressure and protein criteria. hs was diagnosed in preeclamptic patients who developed characteristic laboratory abnormalities. placental tissues were embedded in oct for sectioning, h e staining and rna isolation (invitrogen, carlsbad). gene expression data was obtained by hybridizing fluorescently labeled reverse transcription products to spotted cdna microarrays. the microarrays were produced by the laboratory of microarray technology at the van andel institute (grand rapids, mi) using a custom microarrayer. microarrays were scanned using an agilent g b scanner. images were analyzed using genepix . (axon). limmagui was used to generate lists of discriminating genes for these data, while david provided functional annotations. there were differentially expressed genes between spe and hs (p<. ). among these candidate genes, were up-regulated and were downregulated. the most up-regulated genes are ( )-deoxyribonucleotidase (dnt- ), superoxide dismutase , hydroxy-delta- -steroid dehydrogenase, caspase , and general transcription factor iih. further analysis of functional groups revealed the most enriched gene categories are related to cellular energy (mitochondria), cell cycle regulation, and protein metabolism. the underlying role of the placenta in the development of hs is currently unknown. this study shows that there is a relatively mdest number of genes that are differentially expressed between hs versus spe placentals. thes data provide the molecular context for placental changes seen in this variant of preeclampsia and provides an opportunity to study the role of the effected genes in disease variance. ( ), severe preeclampsia ( ), hellp syndrome ( ) and eclampsia ( ) and control group ( patients) uncomplicated pregnancies. total rna was isolated and reversely transcribed to c-dna. the mrna level of tert was detected with a probe-specific real-time quantitative pcr assay using ß-actin as the reference gene. crossing point (cp) values were obtained during the pcr amplification and the relative expression level of htert equals to (cp htert -cp ß-actin) . statistical analysis was performed using the student's t test. immunohistochemistry (ihc) staining was employed to localize htert protein on placenta tissue sections using abc method, incubation with rabbit anti-htert antibody followed by application of a goat anti-rabbit antibody with results evaluation using microscope. objective tgf s are involved in the regulation of trophoblast differentiation and invasion, and we have previously reported that tgf- is over expressed in pre-eclamptic placentae. tgf s signal via a receptor complex composed of type ii (t r-ii) and type i (t r-i) receptor. to date, seven type i receptors, designated as activin receptor-like kinase (alk - ) have been identified. our aim was to investigate the expression pattern of alk and alk receptors, known to exert tgf signaling, in preeclamptic and iugr placentae. methods human placental tissue throughout gestation was used in order to determine the development profile of the receptors. in addition, placental tissue from preeclamptic and iugr pregnancies and from age matched controls was collected. all iugr pregnancies were characterized by absence of end diastolic velocity in the umbilical artery and had no evidence of preeclampsia. expression of alk and alk mrna was measured by real-time pcr analysis, and protein by western blot analysis using alk and alk antibodies. immunoblot analysis demonstrated a unique developmental profile whereby alk expression increased with advancing gestation. in preeclamptic placentae alk expression was significantly increased compared to preterm and term controls, whereas alk expression was significantly decreased. preeclamptic placentae also exhibited decreased phosphorylation of smad , a tgf signaling molecule, which is activated by alk and increased phosphorylation of smad , which is triggered by alk . the expression of alk and alk in iugr placentae differed from that of preeclampsia as both alk and alk mrna levels were significantly increased in iugr compared to preterm and term controls. however, only alk expression was significantly increased in iugr placentae at the protein level, while no differences in alk protein levels were noted between iugr and controls. conclusions imbalance between alk and alk signaling pathways might play a role in the pathogenesis of preeclampsia and iugr. as tgf signaling via either alk or alk has been found to differentially regulate vasculogenesis, changes in these signaling pathways may contribute to the altered vasculogenesis found in these pregnancy-related disorders. (supported by cihr and owh/igh). ( ), severe preeclampsia ( ), hellp syndrome ( ) and eclampsia ( ) and patients, the control group who had uncomplicated pregnancies. total protein was isolated from placental tissue. gadd a and its downstream signal proteins--phospo-p , phospho-mkk /mkk were assessed by western blot. immunohistochemistry (ihc) staining was employed to localize the expression of gadd a and sflt- proteins in placenta tissue sections using abc method. huvec cells were cultured to - % confluence and were divided into groups: control, stress induction (sorbitol, . m, h), p inhibition (sb- , ug/ml, ug/ml and ug/ml, hour) + sorbitol ( . m, h). total protein was isolated from cells and the supernatant of huvec was collected. western blot was processed to detect the induction of gadd a and phospho-p . supernatant sflt- was measured with an eia kit and the results were read at nm wavelength. results: gadd a protein was elevated in the preeclamptic placentas with its downstream proteins (mkk and p ) activation compared with control. overexpression of gadd a and sflt- in preeclamptic placentas was observed with ihc staining. in huvec cells, gadd a was induced by sorbitol, triggering the activation of the downstream p- pathway and the accumulation of sflt- in the supernatant. the up-regulation of sflt- secretion by inducing gadd a was depleted when treated with p- inhibitor. conclusions: our study reveals that gadd a and its down stream p- pathway were activated in preeclamptic placentas and this stress inducible signal pathway regulates the secretion of sflt- , which is a key player in preeclampsia. it provides novel evidence that links placental stress to sflt- secretion via the gadd . trophoblast adipose triglyceride lipase (atgl) expression is upregulated in preeclampsia. beth a plunkett, jennifer a doll, emily j su, serdar e bulun, mona cornwell, susan e crawford. obstetrics and gynecology, northwestern university, chicago, il, usa; pathology, northwestern university, chicago, il, usa. objective: preeclampsia is characterized by placental endothelial cell dysfunction and elevated maternal triglyceridemia (tg). tg traverse the placenta in a process of uptake followed by lipolysis with subsequent release of fatty acids to the fetus. although three placental lipases (hormone sensitive lipase, endothelial lipase and lipoprotein lipase) have been identified, they do not account for all lipolytic activity. here, we introduce a new lipase, adipose triglyceride lipase (atgl), which is responsible for the hydrolysis of triglycerides to diglycerides in adipocytes and was recently identified as a receptor for endothelial cell modulator pigment epithelium-derived factor. the purpose of this study is to determine if expression of atgl, a potential modulator of both lipid metabolism and vasculature, is upregulated in preeclampsia. methods: immunohistochemical studies were performed on placental tissues from normal pregnancies (n= ) and those complicated by severe preeclampsia (n= ) with anti-atgl antibodies. the degree of positivity in the trophoblasts and endothelial cell was scored ( =none, =spotty, light, =consistent, dark). to determine if atgl is a product of placental endothelial cells (plec), microvascular cells were isolated from normal placental tissue. purity of the sample was confirmed using flowcytometry (> / positivity for factor antigen). atgl was detected immunohistochemically and via western blot using anti-atgl antibodies. results: mean atgl expression in preeclamptic trophoblast was significantly higher than normal placentas ( . + standard deviation versus . + . , p = . ). endothelial expression was not significantly different in preeclamptic ( . + . ) versus normal placentas ( . + . , p> . ). atgl stained intensely and demonstrated a beaded pattern in the endothelial cells, suggestive of a lipid droplet pattern. immunohistochemistry of plec and western blot analysis of cell lysates revealed strong immunopositivity for atgl, although at a smaller size than anticipated. conclusion: these findings demonstrate that a novel lipase, atgl, is produced by trophoblasts and is upregulated in severe preeclampsia. plec express high levels of atgl, suggesting that atgl could prove to be important in the vascular dysfunction and lipid abnormalities characteristic of preeclampsia. increased endothelial chymotrypsin-like protease (chymase) expression is responsible for endothelial activation in preeclampsia. yuping wang, yang gu, yanping zhang, david f lewis. obstetrics and gynecology, lsuhsc-shreveport, shreveport, la, usa. objective: endothelial (ec) activation is an important component of inflammatory phenotypic changes in preeclampsia (pe). our previous study showed enhanced chymotrypsin-like protease (clp)/chymase expression in the maternal vessel endothelium in women with pe. in this study, specific effect of placental-derived clp on ec activation was examined. methods: human uterine microvascular endothelial cells (utmvecs) were used. placental conditioned medium (cm) was prepared by culturing villous explants from normal and pe placentas. confluent utmvecs were treated with placental cm with or without depletion of chymotrypsin. ec adhesion molecule expressions for icam, vcam, p-selectin and e-selectin were determined by a colorimetric assay at od nm. depletion of chymotrypsin from cm was performed by immunoprecipitation. to further determine if activation of endogenous clp/chymase in ecs is responsible for up-regulation of p-selectin and e-selectin expression, chymase sirna was applied to ec culture before the cells were treated with normal or pe cm and then ec adhesion molecule expressions were examined. data was expressed as mean ± se and analyzed by anova. a p level < . was set for statistically different. results: ) expressions of vcam, p-selectin and e-selectin, but not icam, were significantly increased in pe-cm treated utmvecs compared to those of normal-cm treated cells and untreated controls, p< . ; ) there was no difference for adhesion molecule expression in utmvecs between normal-cm treated with untreated controls; ) utmvecs transfected with chymase sirna significantly reduced p-selectin and e-selectin expressions when exposed to pe-cm, p< . . conclusion: placental-derived clp/chymase is responsible for activating ecs and inducing ec adhesion molecule expression. activation of ec chymase may be directly related to the inflammatory phenotypic changes that occur in ecs in pe. (supported nih grants hd and hl ). corin is a transmembrane serine protease that is important in processing natriuretic peptides (nps) and maintaining normal blood pressure. genetically modified mice without corin function develop a syndrome during pregnancy similar to preeclampsia. corin is present in the pregnant uterus and a deficiency in the enzyme may lead to hypertension and preeclampsia. we tested the hypothesis that corin expression was increased in human myometrium from women with preeclampsia. methods: myometrium was obtained from groups of women at the time of primary cesarean section: (i) preterm no labor with preeclampsia (n= , ptspe, . weeks); (ii) preterm labor (n= , ptl, . weeks); (iv) term no labor (n= , tnl, . weeks); (v) term labor (n= , tl, . weeks). microarray gene profiling was performed using affymetrix human genome u plus . arrays. women who were not in active labor at the time of delivery (tnl) served as the control group. conventional and real time pcr was performed to verify corin expression in the arrays was directionally accurate. corin protein expression was examined by western blot. results: compared to tnl control, corin levels decreased nearly -fold in myometrium from women in term labor (tl). there was a small increase in corin gene expression of preeclamptic women (ptspe) and little-to-no change in myometrium from women in preterm labor (ptl). these results were confirmed by pcr analysis. conclusion: blood volume surges during pregnancy, increasing the potential for hypertension and preeclampsia in the mother. the corin-np control system could contribute to this condition. however, there are no reports on corin message or protein levels in women with preeclampsia. our preliminary results suggest that preeclampsia is not a consequence of a corin deficiency (decreased corin preeclampsia). acknowlegement: supported by grants from the phs (r hl - , cpw) and cdc grant (u dp - , cpw). the expression of gp at implantation site: implication for the pathogenesis of preeclampsia. objective: gp is a common shard signal transducing subunit of the il- cytokine family which is critical for implantation, and viewed as marker of endometrial blastocyst receptivity. preeclampsia (pe) is highly related to the restricted trophoblast invasion, which leads to impaired spiral artery remodeling. our hypothesis was that the poor placentation of pe is associated with the altered expression of gp at the implantation site. methods: human decidua from patients with pe and uncomplicated term deliveries (n = , respectively) were immunostained for gp . the intensity and distribution of immunostaining on decidual cells and extravillous traphoblasts were evaluated with hscore. statistical analysis of the data was performed using student's t-test and kruskal-wallis one way anova on ranks followed by post hoc test. results: immunostaining of gp was significantly higher in decidual cells of patients with pe compared with normal specimens, with hscore (median, [interquartile range]) value [ . - . ] and , respectively (p< . ). in pe specimen, the hscore of decidual cells was also significantly higher than that of trophoblast ( [ - ]; p< . ). there were no difference in the comparison of hscore of trophoblasts between the pe and normal specimens, and in normal specimens between decidal cells and trophoblasts. conclusions: the increase of gp expression in the decidual cells of preeclamptic placenta may be implied to the pathogenesis of poor placentation in preeclampsia. we have previously demonstrated that decidual cells are the major source of renin at the human uteroplacental interface, but little is known regarding the human decidual renin expression in hypoxic conditions. therefore, the present study was undertaken to determine the effect of hypoxia on renin secretion by human decidual cells in vitro. methods: full-term normal human placentas were obtained within one hour of vaginal deliveries or cesarean sections. decidual cells were isolated from the decidua parietalis. after an initial culture for days in a serum-containing medium, the decidual cells were exposed to normoxia or hypoxia ( % or % oxygen) in a serum-free medium for hours. the culture supernatants were then harvested and subject to western blot analyses of renin protein contents. a dominant band of renin at approximately kd was detected in all samples. when compared with the cells cultured in the normoxic condition, the cells cultured in both hypoxic conditions (i.e. % and % oxygen) had significantly lower renin protein contents in their culture supernatants. conclusion: our data for the first time show that hypoxia down-regulates renin secretion in human decidua, suggesting a link between the uteroplacental ras and oxygen tension during human gestation. the effect of nucleated fetal red blood cells derived from preeclamptic patients on endothelial progenitor cell proliferation. keiichi matsubara, emiko abe, yuko matsubara, shinji hyodo, masaharu ito. obstetrics and gynecology, ehime university school of medicine, toon, ehime, japan. objectives inadequate uteroplacental circulation results in placental ischemia and the development of preeclampsia (pe). endothelial progenitor cells (epcs) are thought to be a key player in the fetal angiogenesis. vascular endothelial growth factor (vegf), which is up regulated in pe, is involved in epcs proliferation. recently, it was reported that fetal nucleated red blood cells (nrbcs) have the capability to generate vegf. we hypothesized that nrbcs could influence epcs proliferation in the placenta and may be involved in the pathogenesis of pe. material and methods mononuclear cells (mncs) were isolated from the umbilical venous blood of normal pregnant women and preeclamptic patients by density gradient centrifugation. mncs were incubated with anti-cd antibody conjugated with microbeads. nrbcs were collected using a mini macs separator. nrbcs were incubated for hours with or without angiotensin ii (ang ii) and erythropoietin (epo). vegf and placental growth factor (plgf) concentrations in the supernatant were measured using elisa. also, pbmcs without nrbcs were seeded in endothelial basal medium with or without nrbcs using a boyden chamber. these samples were incubated for days with or without ang ii and epo. the adherent cells were incubated with di-ldl, fixed with paraformaldehyde, and stained with fluorescein isothiocyanate-labeled lectin. di-ldl and lectin positive cells was considered to represent epcs and the number was measured using flowcytometry. the number of nrbcs derived from umbilical venous blood was significantly increased in pe. both ang ii and epo significantly increased vegf concentration in the supernatant of nrbcs derived from normal pregnant women. however, ang ii and epo did not influence the nrbcs' vegf production in pe patients. plgf was not detectable in the supernatant. the number of epcs in the umbilical venous blood was significantly decreased in pe and the number was not changed by nrbcs. on the other hand, the number of epcs was significantly decreased in the culture with nrbcs. epo significantly increased the number of epcs in pe at the lower concentration of epo compared with normal pregnant women. conclusions it appears that fetal nrbcs may inhibit fetal epcs proliferation in pe. since epo reduced the inhibitory reaction of nrbcs without vegf production epo may affect epcs proliferation independently of nrbcs. relationship between the hypoxia-inducible factor- (hif- ) and the receptor svegf-r /sflt- : implication for phatophysiology of preeclampsia. julio e valdivia-silva, , juan c gonzalez-altamirano, keisy lopez- the trophoblast invasion is critical for the establishment of the uteroplacental circulation. at early phases of this process local oxygen pressure in the placenta is lower, that pathologically in preeclampsia remain constant. because of this, is important to understand the response of placental cells against these stimuli. in the present work, we use primary cultures of trophoblast cells, fibroblasts of the villous, and human umbilical endothelial cells, isolated of preterm and term placentas (with and without preeclampsia), to explore the effect of the oxygen pressure in the expression and synthesis of vegf, svegfr- /sflt- , hif- and- . our results show that the low pressure of oxygen resulted in a significant increase of the mrna and the protein of the receptor svegf-r selectively in the cts. the vegf's expression and synthesis was raised in three cellular types, but the free protein (not bounded to svegf-r ) of the cts was diminished. on the other hand, the expression of the arnm of hif- or - in cells was comparable in all the types of placentas, nevertheless, the protein hif- was more increased in the cts of preeclamptic placentas. to evaluate the relation of hif- and the increase of the receptor svegfr- , we used sirna-hif . in response to the inhibition, the expression of the receptor svegf-r diminished dramatically. the blockade of hif- did not alter vegf's expression. our data are the first that propose that the protein of the factor of transcription hif- is one of the molecules involved in the selective expression of the receptor svegf-r in trophoblast cells during hypoxia. figure: reduction of the expression to soluble receptor flt- /svegfr- after transfection with sirna-hif in trophoblast cells. sirna= interference rna, lu=luciferase. luc-sirna was used as control. effects of estradiol on synthesis, secretion, and activation of von willebrand factor in endometrial endothelial cell. shumei zhao, chainarong choksuchat, michael s scholfield, todd d deutch, thomas d kimble, david f archer. obstetrics and gynecology, eastern virginia medical school, norfolk, va, usa. objectives: heavy menstrual bleeding (hmb) is a serious clinical condition affecting % of women. due to inefficient and ineffective medical interventions, women with hmb often elect endometrial ablation or hysterectomy to eliminate hmb symptoms. morbidity and loss of fertility linked to these surgical treatments support the search for more effective medical remedies. von willebrand factor (vwf), a principle initiator of blood clotting produced by endothelial cells. women with von willebrand disease (vwd), have a high incidence of hmb indicating poor clotting. these findings suggest vwf heavily impacts the amount of blood loss during menstruation. estrogen stops/reduces hmb and has been used to treat hmb in women with vwd, although the mechanism(s) is unknown. this proposal addresses the hypothesis that estradiol (e ) increases the synthesis and activation of vwf, promoting clotting. materials and methods: immortalized human endometrial endothelia cells (heecs) were used for the studies. to determine if e increases synthesis of vwf, we treated heecs with e at . μm, . μm and . μm for hours. vwf protein and mrna levels were determined by western blotting and real time pcr, respectively. to establish if e can convert vwf from an inactive to an active conformation, the release of activated vwf by cells into culture medium will be assessed by elisa. decreased adamts (a disintegrin and metalloproteinase with thrombospondin type motif) activity results in increased amounts of active vwf. rrelease of activated adamts will be determined by fret. to ascertain if e regulated vwf secretion is by genomic pathway, heecs will be exposed to the estrogen receptor antagonist ici , . elisa and fret will assess the release of active vwf and adamts , respectively. results: western blotting demonstrated e increases vwf mrna and protein levels in a dose-dependent manner in heecs.conclusion: the project will determine if e acts at the heecs to increase synthesis, secretion and activation of vwf. preliminary results show e increases intracellular vwf protein and mrna levels in heecs, supporting our hypothesis that e increases synthesis of vwf in heecs in vitro. if e increases activated vwf, it could reduce/stop hmb by increasing activated vwf, providing justification for a clinical trial of e to treat hmb. over-expression of vegf-d does not induce lymphangiogenesis in the mouse endometrium. peter a rogers, jacqui f donoghue, marc g achen, steven a stacker, jane e girling. obstetrics gynaecology, monash university, melbourne, victoria, australia; ludwig institute for cancer research, melbourne, victoria, australia. background: the human endometrial functionalis has reduced lymphatics compared to the basalis and myometrium . this study examines the distribution of lymphatics in mouse uterus and investigates if over-expression of the lymphangiogenic growth factor vascular endothelial growth factor-d (vegf-d) stimulates growth of new endometrial lymphatic vessels. methods: the distribution of uterine lymphatics was examined in c bl/ jxcba mice collected during the oestrus cycle, early pregnancy and following oestrogen and progesterone treatment. human ebna cells with/without stable transfection of vegf-d were injected into the uterine horn of nod/scid mice. uteri were collected after weeks. serial sections were immunostained with lyve- and/or vegfr (lymphatic endothelial cell markers), cd (blood endothelial cell marker), mab (human vegf-d), mab (human mitochondria) and pcna (proliferative cell nuclear antigen). results: lymphatic vessel profiles were mostly found in the connective tissue between the longitudinal and circular muscle layers of the myometrium. they were rare in the endometrium and only observed in % of the sections. when present in endometrium, lymphatic vessel profiles were usually situated adjacent to the endometrial/myometrial border. ebna tumours formed inside and outside the uterine horn of both the control (n= of ) and vegf-d group (n= of ). localization of ebna cells within the mouse uterus was confirmed by anti-human mitochondrial expression. vegf-d immunostaining confirmed that transfected ebna cells expressed vegf-d in vivo. over-expression of vegf-d did not stimulate endometrial lymphangiogenesis, although there was an increase in vessel diameter of lymphatics in the myometrium adjacent to tumours. initial analysis shows no significant effect of vegf-d ebna cells on endometrial blood vascular density or endothelial cell proliferation. conclusions: minimal lymphatics are present in the mouse endometrium, as is the case for lymphatic vessels in the human endometrial functionalis. the lack of endometrial lymphangiogenesis in response to vegf-d suggests the presence of an inhibitory factor limiting lymphatic growth in this tissue. in early pregnancy, decidual-derived vegf mediates angiogenesis and is required for implantation and placentation. the role of decidual vegf in later pregnancy is poorly understood. decidual hemorrhage (placental abruption) generates excess thrombin and is a major risk factor for pprom and preterm birth. this study compares immunohistochemical (ihc) localization of vegf in decidual tissue sections from term pregnancies complicated by abruption and gestational age-matched controls, and investigates the effect of thrombin on vegf expression by cultured human term decidual stromal cells (dscs). study design: ihc was performed on serial sections of term placental tissues (no labor) with (n= ) and without (n= ) abruption. purified term dscs were passaged until > % free of cd + cells by facs. confluent dscs were primed with - m estradiol (e ), - m medroxyprogesterone acetate (mpa), both, or vehicle for days. after h incubation in defined medium with corresponding steroids ± thrombin ( . - . iu/ml), conditioned supernatants were analyzed for vegf by elisa. extracted total rna was used to assess vegf mrna levels by quantitative rt-pcr using established primers. results: vegf expression was localized by ihc primarily to dscs in placental tissue sections, and was increased in tissues from placental abruption vs controls. in term dscs, thrombin increased vegf secretion in a dosedependent fashion irrespective of the hormonal milieu (eg, . -fold stimulation by . iu/ml thrombin from . ± . to . ± . pg/ml per mcg protein for e +mpa; p= . ). this effect was abrogated by the thrombin inactivator, hirudin. vegf mrna were similarly increased by thrombin with or without steroid hormones (eg, . -fold for e +mpa; p< . ). conclusions: placental abruption is associated with increased vegf expression in term decidual tissues in vivo with thrombin enhancing vegf mrna and protein expression in term dscs in vitro. excess thrombinmediated vegf expression in term decidua aberrantly increases endothelial cell permeability to further generate thrombin by continuous exposure of tissue factor-expressing decidual cells to circulating factor vii. thrombin-enhanced matrix metalloproteinase expression in term dscs would degrade decidual and fetal membrane extracellular matrix to induce pprom and preterm birth. basal directional release of angiotensin ii by endothelial cells stimulated by chymotrypsin-like protease (clp)/chymase. yuping wang, david f lewis, yang gu. obstetrics and gynecology, lsuhsc-shreveport, shreveport, la, usa. objective: chymotrypsin-like protease (clp)/chymase is a serine protease which plays a major role in angiotensin ii (ang ii) generation in the human heart. our previous study showed a higher clp activity in the maternal plasma in women with pe than in normal pregnancies. we also found enhanced chymase expression in the maternal vessel endothelium in women with pe. in this study, we determined if clp could promote endothelial cell (ec) generation of ang ii. methods: we specifically examined basal directional release of ang ii by cultured ecs. ecs were grown on cell culture insert ( well/plate, micron pore size). when ecs reached confluence, chymotrypsin (chy) at concentrations of . , . , . , . , and . g/ml were added to the upper chamber of the cell insert. after hours of culture, medium in the lower chamber was collected. medium concentrations of ang ii were measured by enzymelinked immunoassay (eia). all samples were measured in duplicate. data are expressed as mean ± se and analyzed by anova. a p level < . was considered statistically different. results: chymotrypsin produced a concentration-dependent increase in basal directional release of ang ii by cultured ecs, control: . ± . g/ml; chy . : . ± . g/ml; chy . : . ± . g/ml; chy . : . ± . g/ml; chy . : . ± . g/ml (p< . ); chy . : . ± . g/ml (p< . ), respectively. data are means from independent experiments. conclusion: apical exposure of ecs with chymotrypsin-like protease could promote basal directional release of ang ii. our result implicates that in pe, elevated clp levels in the maternal circulation are very likely to affect ec generation of ang ii. basal directional released ang ii may bind to its receptor on underlying vascular smooth muscle cells and contribute to the increased vasoconstriction in pe. (supported nih grants hd and hl ). vegf stimulates angiogenesis and vasodilation critical for dramatic rises in materno-feto interface blood flows directly linked to fetal growth/survival. extracellular signal-regulated kinase (erk / ) pathway mediates partially vegf-induced angiogenic and vasodilatory responses in placental endothelial cells (ec). it is, however, unknown how this vegf-induced signaling is organized in placental ec. objectives: ovine fetoplacental artery ec (ofpaec) and its transformed counterpart, sv -of to test whether: ) vegf-activated erk / signaling is compartmentalized in the caveolae and disruption of caveolae interferes vegf-induced erk / activation and; ) caveolin- , the structure protein of caveolae, regulates vegf-stimulated erk / phosphorylation. methods: ofpaec or sv -of cells were cultured in mcdb- / % fbs/antibiotics. serum-starved subconfluent ( %) cells were treated with rhvegf ( to ng/ml) for various times. caveolae were disrupted by -cyclodextrin ( -cd, mm, min) or caveolin- scaffolding domain (cav-sd, m, hr). sv -of cells were used for fractionation of caveolae membranes by discontinuous sucrose gradient ( %/ %/ %) ultracentrifugation. activation of erk / signaling pathway were analyzed by western-blotting with specific antibodies. results: in total cell extracts, vegf stimulates erk / phosphorylation in a time-and dose-dependent manner. erk / phosphorylation maximized by vegf ( ng/ml) at - min, which was abrogated by -cd or cav-sd. all the molecules for compromising the erk / signaling module, plc , pkc , src, ras, raf- , mek / and erk / , were detectable in purified caveolae membranes positive for various markers including caveolin- , enos, flotillin- , and -adaptin. in caveolae, vegf dramatically increased phosphorylated erk / without altering total erk / in a time-dependent manner similar to that in total cell extracts, which also maximized at - min. pretreatment with -cd or cav-sd blocked vegf stimulation of erk / phosphorylation in caveolae. conclusion: vegf activates the erk / signaling pathway in caveolae and caveolae integrity is essential for vegf-activated erk / signaling pathway. we conclude that caveolae/caveolin- serves as a platform for compartmentalizing the vegf-induced erk / signaling pathway in placental ec (hl and hl ). hypoxia upregulates gcm in human placenta. david mccaig, fiona lyall. institute of medical genetics, university of glasgow, glasgow, united kingdom. introduction: studies in transgenic mice have shown that a variety of genes regulate the differentiation of trophoblast cells. these genes include gcm . gcm is also expressed in the human placenta. placental gcm- protein has been reported to be reduced in pre-eclampsia, in view of the close link between hypoxia, hypoxia-reoxygneation, pre-eclampsia, placental development and the reported reduction in gcm we hypothesised that gcm expression would be affected by hypoxia. aim: the aim of this study was to determine the effects of hypoxia on gcm expression in the human placenta. two model systems were used; ( ) free floating villous explants and ( ) cultured primary cytotrophoblast and syncytiotrophoblast cells as described previously*. methods: explants or cell cultures were exposed to either hypoxia or hypoxia followed by re-oxygenation. western blot analysis was used to assess gcm protein levels. bands on the gels were quantified using scanning densitometry. statistical differences (n= experiments for both models used) were calulated by anova and turkey's post-hoc test. results: gcm protein was detectable at a low level in villous explants maintained for h in % o . a striking increase in gcm protein was observed when villous explants were incubated for h in % o (p< . ). incubation of villous explants for h in % o followed by re-oxygenation for h in % o resulted in a marked decline in gcm protein (p< . ). expression of gcm was also analysed in primary cytotrophoblast and syncytiotrophoblast cultured in % o or reduced oxygen ( % o ) conditions. gcm protein was not detected in any of the experimental conditions used. discussion: the present study has shown that acute hypoxia increases gcm- protein in villous explants. the experiments with purified trophoblast do not support a role for hypoxia increasing gcm- in these cells under the experimental conditions used. the present findings are in keeping with the complex effects of oxygen depending on the conditions used. the observed hypoxic effects on gcm warrant further investigation. the effect of acute alcohol exposure on histone -lys modification in the mid-gestation embryonic lung. xiangyuan wang, debra wolgemuth, , , laxmi baxi. ob/gyn; genetics development; human nutrition, columbia university medical center, new york, ny, usa. objective maternal alcohol abuse during pregnancy produces an array of birth defects comprising fetal alcohol syndrome. lung development depends on a balance between cell proliferation and apoptosis. we have previously shown that the acute alcohol exposure in the mid-gestation embryo can delay lung development and induce apoptosis. acute exposure to ethanol of selected tissues in mouse embryos has been reported by others to initiate apoptosis within hours after exposure and result in histone modifications. specifically, histone acetylation and deacetylation are involved in transcriptional activation and repression, respectively, but can also involve apoptosis. in the present study, we have investigated the effect of alcohol on acetylation of histone at lysine (ach lys ) in the mid-gestation embryonic lung. pregnant c bl/ j mice at day . of gestation (e . ) were injected intraperitoneally with doses of % ethanol ( . g/kg ), hr apart (alcoholexposed: ae) or with ringers solution (controls: c). ae and five c fetuses were retrieved and hr later and the lungs were fixed and processed for morphological evaluation and staining with rabbit polyclonal anti-ach ly antibody. the entire lung tissue field was evaluated for the levels of ach lys staining and scored as (-) to (++++). three areas were selected randomly from each sample and the total number of cells and staining positive cells were counted in the bronchial epithelium and in the mesenchyme. twelve hr after alcohol exposure at e . , the morphology of ae embryonic lungs was normal. however, high levels of ach lys were detected in % of the bronchial epithelial cells and % of the mesenchymal cells. the expression level in both lineages decreased hr after alcohol exposure. in the controls, the expression of ach lys was virtually undetectable in both the bronchial epithelium and the mesenchymal cells. our previous study showed that ae e . lungs significantly increased apoptotic cell in both bronchial epithelium and mesenchyme hours after alcohol treatment. we now observe that elevated expression of ach lys in the embryonic lung preceded the observation of apoptosis, suggesting that alteration in the acetylation of h could be one of the molecular mechanisms involved in the induction of apoptosis following acute alcohol exposure. objective: our purpose was to evaluate the effect of vitamin c and e supplementation on lipid peroxide levels, total antioxidant ability, and antioxidant levels in the umbilical venous plasma. materials and methods: women at risk for preeclampsia (nullipara, previous preeclampsia, chronic hypertension) were recruited at to weeksgestation and randomly assigned to receive either mg of vitamin c and iu of vitamin e (study group, n= ) or placebo (control group, n= ) daily until delivery. umbilical venous blood were collected after full term delivery. lipid peroxide levels, oxygen-radical absorbance capacity (orac) values, antioxidant levels were measured by each method (thiobarbituric acid reaction, cao's method, and high performance liquid chromatography). results: . the lipid peroxide levels in the umbilical venous plasma of study group were significantly lower than that of control group ( . ± . vs. . ± . nmol/mg protein, p< . ). . the orac values in the umbilical venous plasma of study group were significantly higher than that of control group ( . ± . vs. . ± . u/ml, p< . ). . the -tocopherol levels in the umbilical venous plasma of study group were significantly higher than that of control group ( . ± . vs. . ± . nmol/ml, p< . ). . there were no significant differences in the ascorbic acid, uric acid, -carotene, retinol, and -tocopherol levels in the umbilical venous plasma between control and study group. conclusion: this suggested that maternal vitamin c and e supplementation may affect the oxidant-antioxidants balance of the utero-placenta unit and fetus. placental tnf-related apoptosis-inducing ligand (trail) in normal pregnancy and pre-eclampsia. xilian bai, jenny e myers, philip n baker, john d aplin, ian p crocker. maternal and fetal health research group, the university of manchester. introduction: enhanced placental trophoblast apoptosis is well known to occur in pre-eclampsia. however, the potential role of membrane associated or soluble trail, an apoptosis-inducing ligand, and its death receptor, dr , is not known. we tested the hypotheses that the trail/trail-receptor system is compromised in pre-eclampsia and that soluble trail is a circulating factor which triggers the vascular complications of pre-eclampsia. method: this study was conducted on placental samples and plasma (edta) from women with uncomplicated pregnancies (n= ) and with pre-eclampsia (n= ) at - weeks gestation. protein expression levels and tissue localisation of trail and dr were defined in villous tissue by western blotting and immunohistochemistry. soluble trail (strail) was measured in maternal plasma from both groups using a commercial elisa (diaclone). results: placental villous trail and dr protein was unaltered in preeclampsia compared to normal pregnancy. whilst there was differential distribution of trail and dr within the component cells, this also was unaffected in pre-eclampsia. within the villi, trail was mainly confined to the cytoplasm and perinuclear regions of cytotrophoblast, syncytiotrophoblast, stromal cells and the fetal capillary endothelium. conversely, dr was restricted to trophoblast only, distributed evenly between cytoplasm, plasma membranes and nucleus. strail was present in plasma from non-pregnant women of childbearing age [ . ( . - . ) , median (interquartile range), pg/ml, n= ]. however, there was no significant increase either in pregnancy [ . ( . - . ) pg/ml, n= ] or pre-eclampsia [ . ( . - . ) pg/ ml, n= ] (kruskal-wallis test, p> . ). conclusions: these results suggest that placental villous trail is not adversely regulated in pre-eclampsia. the absence of dr in stromal and endothelial cells may increase resistance to apoptotic stimuli in cells of the villous core. the co-localisation of trail and dr in trophoblast suggests a role in autocrine regulation of cell turnover in this cell type. introduction: preeclampsia is associated with apoptosis of the syncytial layer of placenta and the release of particulate and soluble factors which are deleterious to maternal endothelial function. the goal of the current study was to determine whether laser capture microdissection (lcmd) and western blotting could be used to assess levels of syncytial fas ligand (fasl), a key protein in the apoptotic cascade. methods: frozen sections of term placenta delivered from uncomplicated pregnancies at term were used for study (n= ). following staining with mayer's hematoxylin (n= ), lcmd (leica instruments) of an intact terminal villus was carried out using a focused laser pulse directed to the area of interest using a microscope. in the first round of microdissection the placental villus core consisting of fibroblast, macrophages, fetal vessels, and connective tissue, were removed. in the second round of lcmd, the syncytial layer of that same villus was removed and collected in lysis buffer containing detergent and protease inhibitors, and the number of nuclei per sample was recorded. this procedure was repeated until syncytial tissue from - villi were collected. electrophoretic separation of lysate proteins was then carried out, and western blotting and immunodetection using an anti-fasl antibody was performed. results: we observed that fasl was detected in microdissected syncytial specimens at a molecular weight of approximately kda ( figure) , consistent with our previous reports. it is of note, that western blotting of samples containing approximately (lane ), (lane ), (lane ), and (lane ) nuclei revealed that fasl could be reliably detected in specimens containing as few as nuclei. conclusions: since fasl is a cytokine of low to moderate abundance in placenta, this suggests that lcmd coupled with western blotting will be a valuable methodology to elucidate pathways of syncytial apoptosis and pathophysiology in pregnancies complicated by preeclampsia. supported in part by nih grant hd . the placenta of preeclamptic pregnancies shows oxidative and nitrative stress. we have shown low protein abundance but paradoxically high activity of inducible nitric oxide synthase (inos) in the placenta with severe preeclampsia compared with normal pregnancies. protein nitration and phosphorylation are post-translational modifications possibly involved in nos activity. objectives: examine inos localization and tyrosine phosphorylation in the preeclamptic placenta and the effect of a peroxynitrite generator (sin- ) on inos expression in primary human placental microvascular endothelial cell (hpmec) cultures. methods: placental lysates from normal (n= ), mild (n= ) and severe (n= ) preeclamptic pregnancies were western blotted for inos and what are the roles played by peroxynitrite in preeclamptic women? yoshikatsu suzuki, tamao yamamoto, yoshimasa watanabe, takeo itoh, hidetaka izumi. obstetrics and gynecology, nagoya city university, nagoya, japan; pharmacology, nagoya city university, nagoya, japan; obstetrics and gynecology, izumi women's hospital, fukuoka, japan. aim preeclampsia is characterized by hypertension plus proteinuria. it is hypothesized that the endothelial cell function might be activated by placental faculty in early pregnancy and the activation might cause the vascular disease in preeclampsia. peroxynitrite, which is produced by combination with superoxide (o -) and nitric oxide (no), is strong oxidant as well as o -.we investigated whether or not the localization of peroxynitrite in both placenta and resistance artery might play important roles of developing preeclampsia. omental arteries or placentas were obtained from severe preeclamptic and term-normotensive pregnant women at cesarean section. they were stained by ant-nitrotyrosine (nt, a marker of peroxynitrite) antibody. furthermore, the concentration of nt was measured in omental arteries. in formed consent was obtained from all patients in written. preeclampsia was diagnosed according to the criteria of japan society of obstetrics and gynecology. the localization of nt was seen in placentas obtained from sever preeclamptic women ( / ), although it was not seen from normotensive pregnant women ( / ). the localization was also seen in placentas from of severe preeclamptic women with intrauterine fetal restriction. in omental arteries from both groups, the localization of nt was seen to same degree, and the concentration of nt was similar ( . ± . for sever preeclampsia and . ± . for normotensive pregnant women). from these results, it was suggested that an increase in o production, a decrease in no as well as peroxynitrite production in the placentas might cause the vascular dysfunction and subsequently damage uteroplacental blood circulation in preeclampsia. however, the role played by peroxynitrite in resistance artery might be different and more complicated. background: preeclampsia (pe) is associated with shallow cytotrophoblast (ct) invasion of the decidua, leading to impaired vascular transformation and poor uteroplacental perfusion. ct invasion requires the selective proteolysis of the peri-decidual cell (dc) extracellular matrix. we hypothesized that il and tnf , cytokines that have been linked to pe, may induce aberrant expression of the matrix metalloproteinases (mmp) and in dcs, thereby preferentially degrading the decidual ecm and interfering with sequential ct invasion. methods: immunostaining for mmp- , mmp- , and vimentin (a dc marker) was performed on decidua from normal (n= ) and preeclamptic (n= ) women, and staining intensities were evaluated by hscore. confluent, leukocyte-free first trimester dcs were primed with - m estradiol (e ) or e + - m medroxyprogesterone acetate (mpa), and then switched to a defined medium with e +/-mpa with or without ng/ml of il or tnf . secreted mmp- and mmp- levels were measured by elisa (n= ) and confirmed by western blotting. quantitative rt-pcr assessed mmp- and mmp- mrna levels (n= ). results: tissue staining revealed that mmp- and mmp- levels in preeclamptic decidua (hscore mean±sem: ± and ± , respectively) were significantly higher than in normal decidua ( ± and ± , respectively; p< . ). in cultured first trimester dcs incubated with e , tnf increased secreted mmp- and mmp- levels by ± and ± -fold, respectively, while il increased them by ± and ± fold, respectively (p< . ). in parallel incubations with e +mpa, basal mmp- and mmp- output were lowered by approximately % and %, respectively, while tnf -and il elicited mmp- and mmp- levels were blunted by - %. western blotting confirmed the elisa results, and mrna levels corresponded to changes in mmp- and mmp- secreted protein levels. conclusions: over-expression of mmp- and mmp- in decidual cells may promote pe by disrupting decidual ecm and impairing normal ct invasion. the high levels of il and tnf associated with pe may be contributing to this over-expression. our in vitro observations that mpa blunts tnf -and il -elicited mmp expression suggests that exogenous progestin may offer a novel therapeutic approach in preventing pe. to produce -methoxyestradiol ( -me), a compound with diverse biological activities including inhibition of hif- , a transcription factor that mediates cellular response to hypoxia. circulating levels of -me and placental comt activity are significantly reduced in preeclampsia, raising the possibility that reduced production of -me contributes to the pathophysiology of preeclampsia by altering placental response to hypoxia. genetic variation in the comt gene is linked to comt activity and has been associated with intrauterine fetal growth restriction. we determined if a snp in exon of the comt gene (rs ), which does not change amino acid sequence ( leu ), but reduces comt mrna translation, was associated with preeclampsia. we analyzed comt genotypes in paired dna samples extracted from maternal and cord blood from normal pregnancies and pregnancies complicated by preeclampsia by allele discrimination. the study population was predominantly (> %) african-american. the frequency of the minor rs "g" allele, which is associated with low comt activity, was similar in maternal cases and controls (cases: %; controls: %, p= . ), but was significantly greater in fetal dna from pregnancies complicated by preeclampsia compared to control pregnancies (g allele frequency cases: %; control: %; p< . ). likewise, fetal carriage of the rs "g" allele conferred a significantly greater risk of preeclampsia (odds ratio: . ; % c.i.: . , . , p< . ). there was also a significant discordance between paired maternal and fetal rs genotypes with significantly greater discordance for the "g" allele in fetuses hosted in preeclamptic pregnancies (odds ratio: . ; % c.i.: . , . ). we conclude that genetic variation in the comt gene is associated with risk of preeclampsia, possibly through a mechanism involving reduced production of -me. supported by nih p md . smoking is associated with elevated adma in preeclampsia. michael p frank, robert w powers. , magee-womens research institute, pittsburgh, pa, usa; obstetrics gynecology, university of pittsburgh, pittsburgh, pa, usa. objective: cigarette smoke exposure paradoxically reduces the risk of preeclampsia. asymmetric dimethylarginine (adma) is an endogenous competitive inhibitor of nitric oxide synthase (nos), an independent risk factor for cardiovascular mortality, adma is elevated in women who develop preeclampsia, and adma has been reported to be both higher and lower in smokers. the objective of this study was to investigate the concentration of adma in pregnant smokers and nonsmokers with and without preeclampsia. study design: case-control study of women with uncomplicated pregnancy (controls), and women with preeclampsia matched for gestational age at sample collection. adma was measured by hplc. cigarette smoke exposure was determined by questionnaire and confirmed by plasma cotinine. data are mean±sd. analysis was by two factor anova with fishers post-hoc testing, significance accepted at p< . results: as previously reported, maternal plasma adma concentrations were higher in women with preeclampsia compared to controls (p< . ). in addition, the concentration of adma was significantly higher in preeclampsia smokers compared to controls and preeclampsia nonsmokers (p< . ). in contrast, there was no difference in adma concentration between control smokers and nonsmokers. conclusion: these data may suggest a differential effect of cigarette smoke exposure on circulating adma concentrations between women who do and do not develop preeclampsia. previous data has suggested that cigarette smoking is associated with lower adma in low risk elderly patients, and higher adma in high-risk subjects with diabetes. therefore, the data in preeclamptic and non-preeclamptic subjects may be consistent with these studies, however, the underlying biological explanation for this differential effect has yet to be determined. objective: eclampsia is similar to hypertensive encephalopathy in which an acute elevation in blood pressure causes autoregulatory breakthrough, hyperperfusion and edema formation. we previously reported that the pressure of breakthrough was similar between nonpregnant (np) and late-pregnant (lp) rats, but only lp animals developed edema. this study tested the hypothesis that lp animals have decreased in cerebrovascular resistance (cvr) and hyperperfusion in response to breakthrough vs. np. we further hypothesized that acute hypertension would cause greater blood-brain barrier (bbb) permeability in lp rats due to elevated hydrostatic pressure. methods: in vivo models of bbb permeability and cerebral blood flow (cbf) were used in np (n= ) and lp (d - ; n= ) rats that were either normotensive or hypertensive (np-htn, lp-htn) by infusion of phenylephrine to raise mean arterial pressure. permeability was determined in anterior and posterior brain regions by calculating the flux of kd dextran into the brain tissue, measured by a fluorescent spectrophotometer after flushing the vasculature with saline. cbf and cvr were measured by infusion of m fluorescent microspheres and determined based on the flow rate and fluorescence intensity of a reference sample for each animal. animals were ventilated to maintain blood gases within normal ranges (po > mmhg, pco = - mmhg). results: although the pressure change was similar between np and lp ( and mm hg), lp animals responded to acute hypertension with hyperperfusion. cbf increased from ± to ± ml/ g/min in np ( %) and ± to ± ml/ g/min in lp ( %; p< . vs. np). hyperperfusion in lp animals was associate with decreased cvr vs. np ( . ± . vs. . ± . mm hg/(ml/ g/min); p< . ). bbb permeability was significantly increased in lp animals at breakthrough vs. np in both anterior and posterior brain regions. the flux of dextran in anterior and posterior brain regions for np vs. lp animals was: ± vs. ± for anterior (p< . ) and ± vs. ± for posterior (p< . ). conclusions: these data demonstrate that pregnancy decreases cvr and causes hyperperfusion of the brain during acute hypertension. because increases in cvr is a protective function in the brain, impairment of these mechanisms during pregnancy may predispose the brain to edema when blood pressure is elevated, as in eclampsia. introduction: this study used the in vitro dually perfused human placental lobule to test the hypothesese that placental release of vegf and the fetoplacental vasodilatory response to exogenous vegf- are altered by tissue oxygenations that mimic healthy and preeclamptic pregancies. methods: lobules were dually perfused for six hours under one of two oxygenation conditions, representing 'normoxia' and 'hypoxia' (n = each): delivering maternal side inflow perfusate at oxygen concentrations of . % and %, respectively, distributed via cannulae; and fetal side inflow perfusate oxygen concentration of - % in both systems. venous perfusates were sampled and assayed, appropriate to side of release, for erythropoietin (epo), macrophage inflammatory protein- alpha (mip- alpha) (reference oxygen sensitive hormones), free vegf, svegfr- and plgf. in separate perfusions, fetoplacental vasodilation in response to pm vegf was investigated, following preconstriction of the fetoplacental vasculature to steady state fetal-side inflow hydrostatic pressure (fihp) with the thromboxane mimetic, u , (n = each). results: maternal-side mip- alpha release was higher in the 'hypoxic' than the 'normoxic' system ( ± and ± pg/ml, respectively, at hours, mean ± se; -way anova: p< . ). maternal-side epo and fetal-side soluble free vegf and svegfr- release were not different between groups. fetal-side release of plgf was higher in the 'hypoxic' group than the 'normoxic' group ( . ± . and . ± . pm, respectively, at hours, mean ± se; -way anova: p < . ). there was no difference in the vasodilatory response to vegf- in the fetoplacental vasculature between the groups ( . ± . and . ± . % change in fihp, mean ± se). discussion: differences in mip- alpha and plgf release provide evidence for metabolic separation of the adapted systems, caused by a changed oxygen environment. our failure to observe differences in epo, vegf and svegfr- release may be explained by longer lag-times for up regulation of their gene expression. vegf associated endothelial signalling appears to be unaffected by 'hypoxia' in the placenta over the time course studied here. background: there is a placental renin-angiotensin system (ras) from very early pregnancy. ang iv mediates various effects by binding to its specific receptor, the at r, the active site of which is an insulin-regulated aminopeptidase (irap). there is at r expression in both endothelial and smooth muscle cells. ang iv at low concentrations is vasodilatory, increasing blood flow via the at rs; it also stimulates nf-kappa beta and modulates glut- . to date at r expression has not been investigated in the placenta. we propose that at r plays a part in the placental vascular development necessary for successful pregnancy, and that reduced at r expression may be associated with inadequate vascular adaptation contributing to pre-eclampsia (pe). aim: to identify and locate at r expression in both np and pe placentae. methods: the study had hospital ethical approval; written informed consent was obtained from all women. placental samples were obtained from np and pe at delivery (gestational ages: and weeks respectively). samples were taken from areas, near cord, middle and outer edge of the placenta. paraffin embedded sections were immunostained for irap reactivity using a rabbit polyclonal antibody (gift from professor david james, garvan institute, australia). immunoreactivity of trophoblast and uterine cell populations was assessed using a semi-quantitative grading system. grade = no positive labelling, = - %, = - %, = - % and = - % of cells positively labelled. median (max, min) are shown. results: ) at r immunostaining was prominent in the syncytiotrophoblast and hofbauer cells of all placental villi examined, with no differences in expression between sampling sites. ) at r positivity was reduced in near cord pe samples ( . ( . , . )) compared to np ( . ( . , . ) p= . ). conclusion: we have shown for the first time, dense at rs in syncytiotrophoblast and hofbauer cells in np placentae, and their down-regulation in pe. reduced ang iv/at r binding may contribute to increased placental vasoconstriction resulting in increased ischemia/reperfusion. this in turn may stimulate xanthine oxidase, which is itself stimulated by angii, leading to increased superoxide production. further work is needed to clearly define the role of this newly identified component of the renin-angiotensin system in normal pregnancy and pe. pre-eclampsia is associated with lower percentages of regulatory t cells in maternal blood. jr prins, hm boelens, jj hm erwich, j heimweg, s van der heide, ajm van oosterhout, aej dubois, jg aarnoudse. obstetrics, university medical center groningen, groningen, netherlands; laboratory of allergology and pulmonary disease, university medical center groningen; pediatric allergology, beatrix children's clinic, university medical center groningen. pre-eclampsia is a serious disease of human pregnancy and immunological mechanisms play a role in its pathophysiology. normal pregnancy is associated with an increase in regulatory t (treg) cells and with a predominant th immune profile. treg cells are a subpopulation of cd + lymphocytes and are specifically characterized by the lineage specific transcription factor foxp . treg cells seem to induce immunological changes that have a protective role in maintaining normal pregnancy. we hypothesised that percentages of treg cells are decreased in pregnancies complicated by pre-eclampsia. methods in total, women with pregnancies complicated by pre-eclampsia and healthy pregnant controls were enrolled. to obtain control umbilical cord blood as well, control group i consisted of eighteen healthy pregnant women at term. in addition, since women with pre-eclampsia delivered preterm, control group ii (peripheral blood only) consisted of women during normal pregnancy with a gestational age matched for the preterm pre-eclamptic group. treg cells were measured from whole blood using four-color flow-cytometry. women with a pregnancy complicated by pre-eclampsia had a significantly lower percentage of cd + foxp + treg cells ( . vs . %; p< . ). in the pre-term group the pregnancies complicated by pre-eclampsia showed a significantly lower percentage of cd + foxp + cells in the peripheral blood as compared to the healthy pregnant controls. at term this percentage was also lower but not significantly so. between pre-term and term pregnancies both complicated by pre-eclampsia no significant difference was found in the percentage of cd + foxp + treg cells. no difference was found in umbilical cord blood ( . vs . %). conclusions our data suggest that pre-eclampsia is associated with a diminished percentage of treg cells in peripheral blood. we conclude that a deficiency of regulatory t cells may play a role in the pathophysiology of pre-eclampsia. background: preeclampsia and eclampsia are significant causes of maternal and fetal death. however, the pathophysiology of these conditions is unclear. we and others have reported that inhibition of endogenous nitric oxide (no) synthesis produces symptoms similar to preeclampsia in pregnant rats. several studies demonstrate that fetoplacental weights are altered in pregnancies of spontaneous hypertensive (shr) rats. in addition, impaired synthesis of tetrahydrobiopterin (bh ), a major co-factor for endothelial nitric oxide synthase (enos) activity and enhanced expression of enos has been observed in the pathogenesis of hypertension. in the current study, we examined whether supplementation of bh and sepiapterin (sep, a precursor for bh biosynthesis in the salvage pathway) reduces increased blood pressure and improves fetoplacental weights in shr pregnant rats. methods: groups ( - ) of shr pregnant rats were either treated with bh , sep ( mg/k.g body weight/day/rat, oral tablets) or vehicle (normal diet) beginning from day of pregnancy until day of gestation. animals were sacrificed on day of gestation and fetoplacental weights were recorded immediately. western blot analysis was performed to determine vascular enos expression (enos/gapdh). results: significant (p< . ) elevations in blood pressure (bp, mmhg) were observed in shr (shr, ± . vs. wky, ± . mmhg) compared to wistar-kyoto (wky) group. supplementation of either bh or sep ( ± . ), significantly (p< . ) reduced elevated bp beginning from day of pregnancy. fetal but not placental weights were significantly (p< . ) reduced in shr ( . ± . grams) compared to wky ( . ± . ) rats. bh ( . ± . ) treatment partially (p< . ) increased fetal weights compared to the shr group. vascular enos expression is significantly (p< . ) elevated in shr ( . ± . ) compared to wky rats ( . ± . ). further, treatment with bh ( . ± . ) but not sep ( . ± . ) significantly (p< . ) reduced elevated enos protein expression in shr rats. conclusions: bh may be beneficial treatment of preeclampsia to reduce blood pressure and improve fetal perfusion to increase fetal weights. genetic risk factor for severe preeclampsia: significance of endothelial nitric oxide synthase gene t- ->c and missense glu asp variants. toshihiro yoshimura, michihiro yoshimura, masafumi nakayama. obstetrics and gynecology, kumamoto university school of medicine, kumamoto, japan; cardiovascular medicine, jikei university school of medicine, tokyo, japan; cardiovascular medicine, kumamoto university school of medicine, kumamoto, japan. introduction: we recently identified two endothelial nitric oxide synthase (enos) gene polymorphisms, a glu asp missense variant in exon and a t- -->c variant in the '-franking region, which are associated with coronary spasm and myocardial infarction in japanese population. and we also identified a missense glu asp variant is associated with severe preeclampsia and placental abruption. our objective was to analyze the association between the t- -->c and severe preeclampsia. materials and methods: the study participants included patients with histories of severe preeclampsia. this is a preliminary study, therefore, the comparisons were made with the general normal population. results: the analyses revealed that the frequency of the missense glu asp variant (n= / , %) was significantly higher than the general population (n= / , %), as we previously published. however, the frequency of the t- -->c variant (n= / , %) was not different from the general population (n= / , %). interestingly, only one patient had both t- -->c and missense glu asp variants, and she developed placental abruption. conclusion: although our sample size is small, it is very unlikely that the t- -->c variant is associated with severe preeclampsia. the t- -->c variant may not be a genetic susceptibility factor to severe preeclampsia. the t- -->c may have some reproductive significance in combination with missense glu asp variant, however huge number of patients would be needed to analyze such rare ( . %) combination of the variance. the association between the development of preeclampsia and methylenetetrahydrofolate reductase, angiotensinogen, vascular endothelial growth factor single nucleotide polymorphism genotype combinations. hyun soo park, jong kwan jun, chan-wook park, joong shin park, bo hyun yoon, hee chul syn. obstetrics and gynecology, dongguk university international hospital, goyang-si, gyeonggi-do, republic of korea; obstetrics and gynecology, seoul national university college of medicine, seoul, republic of korea. objective this study was conducted to investigate if there exists any genotype combination of multiple single nucleotide polymorphism (snp)s which is frequently found in preeclampsia patients. study design one hundred sixty two preeclampsia patients and normotensive pregnant women were included in this study between jan and jul . diagnosis of preeclampsia and assignment of severity were made according to the criteria by national high blood pressure education working group and american college of obstetricians and gynecologists. the patients were reclassified as early ( weeks or before) and late-onset ( weeks or beyond) disease. genotypes were measured with pcr-rflp for methylenetetrahydrofolate reductase (mthfr) c t, angiotensinogen (agt) m t, vascular endothelial growth factor (vegf) c t with the dna extracted from maternal blood. case-control study for each snp was done and the frequencies of genotype combination were compared. anova, t-test, chi-square test, fisher's exact test and logistic regression analysis were used for statistical analysis. a p value of < . was considered statistically significant. results genotypes of mthfr polymorphism showed significant difference between late onset preeclampsia and control (cc+ct/tt, or: . , p< . ) but agt and vegf polymorphism did not show statistical difference between any case-control combination. only out of possible genotype combinations were found and there was no statistical difference in the frequencies of genotype combination between case and control group. conclusion mthfr polymorphism might be associated with the development of preeclampsia, but there was no combination of mthfr, agt and vegf polymorphisms which is associated with the development of preeclampsia. diffuse staining and vascular smooth muscle (vsm) staining. resistance-sized vessels ( - μm) were evaluated. results: for mpo, the intensity of staining (fig) and the % vessels with neutrophil, diffuse and vsm staining was significantly greater for obese than for overweight or normal weight patients: % diffuse staining ( . ± . vs. . ± . vs. . ± . , p< . ); % vsm staining ( . ± . vs. . ± . vs. . ± . , p< . ). for mmp , obese and overweight patients had a greater (p< . ) % vessels with neutrophil, diffuse and vsm staining than normal weight patients: % diffuse staining ( . ± . vs. . ± . vs. . ± . ); % vsm staining ( . ± . vs. . ± . vs. . ± . ). conclusions: neutrophils infiltrate the systemic vasculature of obese women and release mpo and mmp . speculation: obesity may put women at risk for pe because their vasculature may already be dysfunctional due to neutrophil infiltration and release of mpo and mmp . hl . collagen is an important protein that maintains the structural integrity of tissues. disruption of vascular smooth muscle collagen could result in vascular dysfunction in women with preeclampsia. recently, neutrophil infiltration of the systemic vasculature was demonstrated in preeclamptic women. neutrophils produce inflammatory mediators, such as reactive oxygen species (ros) and tnf-. we hypothesized that neutrophils, ros and tnf-would alter expression of collagen regulating genes. methods: primary cultures of human vascular smooth muscle cells (vsmc) were seeded into t- flasks ( , cells/flask) and grown for days to % confluence. the cells were treated for hours with medium control, ros (hx, . mm + xo . u/ml), tnf-( ng/ml); and neutrophils ( , ) activated with arachidonic acid, μm, ( : ratio of neutrophils to vsmc). rna was extracted from cell homogenates and analyzed for gene expression with an rt profiler pcr array system for human extracellular matrix genes (superarray). to determine the fold-change of gene expression, the results were first normalized to a housekeeping gene and then ct was calculated across two rt-pcr arrays where group was the control and group was the experimental treatment. results: table . conclusions: neutrophils, ros and tnf increased mmp expression. interestingly, genes involved in collagen synthesis (col a ) or inhibition of mmp- activity (timp ) were either not affected or down-regulated. these data suggest that neutrophil infiltration in preeclamptic women could cause vascular dysfunction by creating an imbalance between collagen synthesis and collagen breakdown favoring breakdown. hl , fogarty d tw , p md . objective: hypoxia increases membrane attack complex (mac) binding to cultured human trophoblasts, and mac enhances apoptosis in trophoblasts exposed to low compared to normal fio . trophoblast microparticles and cellular fragments released into the maternal circulation in vivo may contribute to the systemic pathophysiology of preeclampsia. we tested the hypothesis that hypoxia induced mac deposition on cultured human trophoblasts yields microparticles and fragments coated with mac. study design: primary cytotrophoblasts from term human placentas (n= ) were cultured h in % and % oxygen in dmem with % human serum with active mac or heat inactivated serum (control). media were centrifuged to obtain pellets of microparticles and cell debris which were immunostained for mac or exposed - h to confluent, phorbol myristate acetate differentiated u macrophages. the percentage of macrophages that ingested trophoblast debris was quantified by counting the number of macrophages with immunofluorescence for trophoblast cytokeratin filament staining, as assessed by confocal microscopy. results: cultures exposed to normal human serum, but not heat inactivated control serum, showed mac immunofluorescence on microparticles and fragments in medium, with the highest level of mac in cultures exposed to extreme hypoxia. the maximal percentage of macrophages that ingested the trophoblast debris coated with mac from cultures with % oxygen was . %, not different from the . % from cultures exposed a % fio and control serum. conclusion: trophoblasts exposed to hypoxia and active complement release microparticles and cellular fragments coated with mac into the extracellular environment. mac coating does not influence phagocytic removal of the debris by macrophages suggesting that placental derived, membrane bound mac could circulate to yield systemic affects on maternal endothelium. supported by nih hd and hd . is there a role for fatty acids in the pathogenesis of pre-eclampsia? nicola j robinson, laura j minchell, jenny e myers, philip n baker, carl a hubel, ian p crocker. maternal and fetal health research group, the university of manchester; obstetrics, gynecology and reproductive sciences, university of pittsburgh. objectives: women with pre-eclampsia (pe) display altered lipid metabolism as characterized by elevated circulating triglycerides and non-esterified fatty acids (nefa) and these changes are evident before the disease is clinically apparent. we have tested the hypothesis that the increased circulating levels of nefa contribute to endothelial dysfunction in pe. methods: human umbilical vein endothelial cells were incubated for h with pooled plasma ( %) from normal or pe pregnancies, or with palmitic, oleic and linoleic acid in culture media at the concentrations and molar ratios to albumin identified in normal ( , , m, ratio . ) and pe pregnancies ( , , m, ratio . ) , . lipid droplet accumulation was determined using an oil red o absorbance assay. endothelial metabolism was measured using the mtt test and mitochondrial membrane potential determined by jc- assay as a marker of early apoptosis. results: plasma from pe pregnancies increased endothelial cell lipid droplet accumulation compared to normal plasma (p< . , wilcoxon signed ranks, n= ). this change was replicated following exposure to nefa at the combined concentrations found in pe compared to normal pregnant controls (p< . , n= ). plasma from women with pe caused a significant decrease in mitochondrial dehydrogenase activity (mtt test; p< . , n= ) and a reduction in jc- fluorescence (p< . , n= ), compared to normal plasma, suggestive of mitochondrial membrane depolarization and increased cellular apoptosis. again these effects were replicated using nefa in culture medium at the levels found in pe compared to normal pregnancies (mtt test: p< . , n= ; jc- assay: p< . , n= ). conclusions: in endothelial cell cultures, plasma from women with pe caused increased lipid droplet accumulation, decreased cellular metabolism and increased apoptosis. these changes to cellular function were mirrored using nefa in culture medium at the concentrations and molar ratios to albumin previously reported in pe. these findings provide evidence that the changes in endothelial cell function induced by plasma from women with pe may be due to the increased nefa circulating levels and that increased palmitic, oleic and linoleic acid, in combination, could play a role in pathogenesis of pe. lorentzen ( ) bjog; endresen ( ) ajog. background: skewing of the maternal endothelial phenotype in pre-eclampsia (pe) is attributed to the release of unknown factors from a hypoperfused placenta. we hypothesise that factors secreted from pe placental tissue will impair endothelial cell function. we have tested the effect of soluble factors by menopausal status, there was a significant increase in bmi in pre-but not in postmenopausal women. in postmenopausal women there was insufficient power to note a statistically significant change in bmi. results are summarized with the follow-up for each group represented by "n" in the graph below. premenopausal patients were divided into hysterectomy with oophorectomy (pbso) versus hysterectomy alone (ph). the ph group showed an increase in bmi that plateaus at time . in the pbso group the bmi continued to increase over time. subgroup analysis comparing ph to pbso demonstrates initial weight loss in pbso but a significant increase in bmi from baseline at time compared to ph. conclusions: hysterectomy appears to be associated with an increase in bmi over time. subgroup analysis suggests that, in premenopausal women, oophorectomy is more strongly associated with continuing weight gain than hysterectomy alone. purpose: obesity is implicated as a key risk factor in chronic disease, but no studies have associated central obesity to the presence of chronic abdominal and/or pelvic pain. we set out to identify the prevalence of chronic abdominal/ pelvic pain in an underserved, primarily latina population by a cross-sectional study in the olive view-ucla outpatient gynecology clinic. we sought to identify an association between the presence and severity of abdominal/pelvic pain and central obesity. methods: nonpregnant women presenting to the gynecology clinic were prospectively evaluated and grouped according to the presence of abdominal/ pelvic pain ('none-mild' or 'moderate-severe' pain). body mass index (bmi) and abdominal circumference (ac) were measured. patients with 'moderate-severe' pain completed standardized questionnaires for pelvic pain and global health scores. results: / ( %) of patients has 'none-mild' pain, and ( %) had 'moderate-severe' pain. pain prevalence was not significantly associated with bmi (mean: 'none-mild' . + . kg/m ; 'moderate-severe' . + . kg/m , p= . ), nor was pain severity (p= . ). pain prevalence was significantly associated with ac (mean: 'none-mild' . + . inches; 'moderate-severe' . + . , p= . ). a borderline positive association exists between ac and pain severity (p= . ). conclusions: we demonstrate an association between both the presence and severity of chronic abdominal/pelvic pain and central obesity, independent of bmi. ac appears to be a more relevant factor than other traditional measures of habitus in patients with this chronic malady. to improve preventative care in women's health management, further evaluation of the role of central obesity in the pathogenesis of chronic pain is necessary. aims: vulvitis is one of the most frequently diagnosed gynaecological infections. we aim to assess the efficacy against infective vulvitis of a new topical medical device containing low molecular weight hyaluronic acid (lmw-ha). the ability of this molecule to stimulate -defensin release in keratinocytes has been recently shown. methods: we report preliminary data regarding women suffering from infective vulvitis, as assessed by a gyneacologist: patients were randomly selected to receive sphg ( patients), a cream containing low-molecular weigh hyaluronic acid, or vehicle ( patients). patients were asked to apply the cream to the vulva twice-daily for days. at the end of treatment, evaluation of efficacy, tolerability and acceptability of the cream was assessed by a specific questionnaire. results: preliminary results show that patients receiving sphg report a significative improvement of vulvitis symptoms, in terms of itch, redness of the skin and burning, in comparison to vehicle. sphg also showed a good tolerability, cosmetic acceptability and symptomatic relief perceived by patients. conclusion: sphg seems to be efficacious in ameliorating the symptoms of infective vulvitis. this activity may be probably related to the lmw-ha presents in this formulation: in fact, low molecular weight hyaluronic acid has been recently shown to induce -defensin production by human keratinocytes. since this peptide exerts antimicrobial and antimicotic activity, the improvement of symptoms assessed in patients receiving sphg might be linked to a reduced infective charge, attributable to the activity of -defensin . background: allogeneic hematopoietic stem cell transplant (hsct) is a treatment used for many malignant and nonmalignant diseases of the bone marrow and immune system. hsct may be complicated by chronic graft-versus-hostdisease (cgvhd) in to % from matched unrelated donors. genital cgvhd complicates about % of hsct and may uncommonly result in labial fusion. case: year old woman with a history of ewing's sarcoma and acute myelogenous leukemia, had received chemotherapy and total body irradiation (tbi) followed by a matched unrelated donor hsct. menarche occurred at years of age after normal pubertal development. she menstruated regularly until cancer diagnosis. premature ovarian failure resulted after chemotherapy and tbi and oral contraceptive pills were used for hormone replacement. after transplant, she developed chronic gvhd involving the skin, eyes, mouth and joints, and concomitantly complained of vulvar pruritus. she was presumed to have a yeast infection which was treated with fluconazole without a pelvic exam. she was evaluated by a gynecologist when she was unable to insert a tampon. pelvic exam revealed dense labia minora adhesions from the clitoris to urethral meatus and posteriorly leaving a cm opening at the urethra. pelvic mri revealed a normal uterus and ovaries. after weeks of topical estrogen cream, the adhesions remained dense and were lysed under general anesthesia. vaginal examination revealed pale, minimally rugated, normal mucosa. cervical cytology was normal. post-operatively she used daily topical estrogen and hydrocortisone creams. at months after surgery, her urinary stream was stronger. on pelvic exam, the labial opening was cm, but a small posterior forchette adhesion elicited severe pain. after using dilators coated with topical steroids and estrogen, she was able to insert a tampon. conclusion: genital gvhd should be considered in women with genital tract complaints after hsct. labial fusion secondary to chronic gvhd may be treated successfully with surgery and medical therapy. support: rbmb/nichd/nih. dana r ambler, mazen e abdallah, rahi victory, michael p diamond, elizabeth e puscheck, jay m berman. obstetrics and gynecology, wayne state university/detroit medical center, detroit, mi, usa. objective: to determine which factors are predictive of a ruptured ectopic pregnancy, and whether endometrial stripe thickness can be used as an alternative to such criteria in the diagnosis of an ectopic pregnancy. design: retrospectively collected ectopic pregnancy database. setting: detroit medical center, detroit, michigan. patients or participants: women with a diagnosis of an ectopic pregnancy were studied, with surgically confirmed tubal rupture cases. interventions: abstracted data included hcg(iu), gestational age (days), presenting symptoms of pain and/or bleeding, hemoglobin (hgb), hematocrit (hct), historical risk factors, ultrasound-determined ectopic size, endometrial stripe thickness (mm), amount of cul de sac fluid (cds), tubal rupture at time of surgery, and estimated blood loss (ebl)(ml). covariates included demographics. results were significant when p< . . results: chi square analysis revealed that there is a relationship between endometrial stripe thickness and hcg levels. logistic regression models demonstrated that endometrial stripe thickness was not predictive of ectopic rupture, (or . , p= . ) . however, logistic regression, both forced and forward stepwise analysis, demonstrated that hcg (or= . , p< . ), a large volume of cul-de-sac fluid (or= . , p< . ), and increasing pain (or= . , p= . ) were associated with increased risks of rupture. gestational age, and ectopic related risk factors were also not predictive of rupture. conclusions: endometrial stripe thickness is not a useful predictor for the diagnosis of a ruptured ectopic pregnancy. serum hcg measurement, cds fluid volume and the presence of pain are much stronger diagnostic indicators of ectopic pregnancies. clinical profile of migraineurs in a university hospital gynecology department in japan. [objectives] the changes in hormonal milieu associated with menarche, pregnancy, menopause, and post-menopause are frequently accompanied by changes in the patterns and frequency of migraine. little is known on the relationship of women's issues of migraine though the balance between estrogen and progesterone is critical in the elimination of migraine. our aim was to investigate the relations among the prevalence of migraine, the reproductive stage, and gynecologic diseases. [materials and methods] female patients (average age: . years old) who consulted a physician and agreed to answer about the questionnaires during september, -june, . migraineurs were diagnosed with the migraine screener by the japanese headache medical treatment promotion committee in . and the patients answered questionnaires that screened about menopausal disorder and abnormal menstruation at once. they were conducted a survey in the form of a questionnaire and sometimes were taken blood samples. [results] in patients had migraine ( . % average age: . years old). prevalence were . % in one's twenties, higher than another ages. most patients with migraine was complicated by menstruation disorders (premenstrual syndrome %, dysmenorrhea . %), sterility ( . %), and severe menopausal disorder ( %). when trh and the lh-rh test were examined for the sterility patient, migraineurs had higher prolactin basal level and lh level after lord but lower fsh level before and after lord than nonmigraineurs. on the other hand, the prevalence of migraine for postmenopausal women and women who had gynecology cancer treatment was low, and there was no relation between migraine and pregnancy history. [conclusions] this study provides migraine headache is influenced by reproductive stage and that women with migraine are frequently complicated by menstruation disorder. it is thought that migraine account for abnormality of hormone milieu including abnormality of the hypothalamus-pituitary system. objective: this study evaluated the efficacy of doses of estradiol (e ) gel . % (divigel ® ), a novel formulation of e consisting of mg e per g transdermal gel, to reduce frequency and severity of vasomotor symptoms and signs of vulvar and vaginal atrophy (vva) associated with menopause. design: postmenopausal women were evaluated in a -week study comparing placebo to e gel . % at doses of . g/day, . g/day, and . g/day with estimated nominal daily deliveries of . mg, . mg, and . mg of e respectively. endpoints included mean change from baseline in daily frequency and severity of moderate to severe hot flashes (msvs). vaginal ph and % superficial cells were collected at baseline and end of study. results: e gel . % showed statistically significant improvements from placebo gel as early as week (table) that were maintained throughout treatment. signs of vva (vaginal ph and % of superficial cells) showed statistically significant improvements from baseline with all doses of e gel . % compared to placebo. conclusion: e gel . % significantly decreased the frequency and severity of msvs at all doses evaluated in this trial. e gel . % offers multiple dosing options to individualize patient therapy, including the lowest effective dose that was studied ( . mg e , delivering . mg e /day), to treat vasomotor symptoms associated with menopause. estradiol (e ) gel . % (divigel ® ) is a novel formulation of e consisting of mg e per g transdermal gel for the treatment of vasomotor symptoms associated with menopause. safety and tolerability of e gel . % were evaluated in a large placebo-controlled trial. design: postmenopausal women participated in a -week study comparing placebo to . g/day, . g/day, and . g/day of e gel . %. circulating e and estrone (e ) concentrations were measured. safety analyses included the incidence of adverse events (aes) and clinical laboratory evaluations, including plasma levels of sex hormone binding globulin (shbg). application site tolerability was assessed using the draize scale. results: all doses of e gel . % produced physiologic e :e ratios similar to those seen in premenopausal women. e :e increased from a baseline mean of . to . , . , and . with, respectively, the . , . , and . g/day doses of e gel . %. the most frequently reported aes were breast tenderness and postmenopausal bleeding that appeared to be dose-related and would be expected with increased circulating estrogen concentrations. there were no remarkable changes in hematology, blood chemistry, urinalysis, lipid, coagulation, and carbohydrate values following treatment with e gel . %. shbg levels remained unchanged after weeks of treatment at all doses. the vast majority of patients had no evidence of skin irritation throughout the treatment period. mean draise scale scores after , , and weeks of treatment were . for all treatment groups except for a mean value of . ± . for the . g dose group after weeks of treatment. conclusion: e gel . % is a safe and well-tolerated therapy for the treatment of menopausal symptoms. design: pharmacokinetic parameters, dosing, efficacy, and safety information for divigel ® , elestrin ™ , evamist ™ , estrogel ® , and estrasorb ® were obtained from current prescribing information (obtained from manufacturer websites) and the data were compared. results: together, these new transdermal therapies offer multiple dosing/ delivery options and contain a wide range of e ( . mg to . mg), with the lowest systemic daily delivery of e attained by the divigel ® . g dose. following weeks of treatment, across the dosing options, each treatment significantly reduced both the frequency and severity of moderate to severe vasomotor symptoms (msvms) compared to placebo. change in frequency of msvms ranged from - . (divigel ® . g) to - . (estrasorb ® ); change in severity scores for msvms ranged from - . (divigel ® . g) to - . (divigel ® . g). all treatments are safe and well tolerated. breast tenderness was the only adverse event reported in % of subjects, occurring with all therapies. conclusions: current treatment guidelines recommend using the lowest effective dose of estrogen for the treatment of menopausal symptoms. this side-by-side comparison of the recently available e non-patch transdermal options to the standard transdermal therapies is meant to assist physicians in individualizing treatment for their patients. introduction: cdb- (cdb) is a relatively new progesterone receptor modulator being clinically evaluated for contraception and treatment of fibromas. its use in an intrauterine device/system (ius) has not been reported. in this study we prepared cdb-filled intrauterine devices (cdb-ius) and evaluated their effects on endometrial growth and bleeding patterns in rhesus macaques. methods: short ( . - . cm) lengths of silastic tubing (od . mm), either empty (n= ),or filled with silicone rubber matrix containing % of micronized cdb (n= ), were inserted into the uterine lumens of ovariectomized rhesus macaques. animals were induced to cycle by sequential treatment with systemic estradiol and progesterone (p) as reported (brenner et al, ann ny acad, ) . after . cycles, at the end of the follicular phase, the uterus was removed and processed. results: when systemic p treatment was withdrawn at the end of each cycle, animals with empty ius menstruated normally, while animals with cdb-ius bled little or not at all. over the whole . cycles, animals bearing a blank ius bled for an average of . ± . days while cdb treated animals bled for an average of only ± . days. at the end of treatment, animals exposed to blank ius had mean endometrial wet weights of ± mg while the cdb-treated endometria weighed only ± mg. the proliferation markers ki- and phospho-h were substantially lower in the cdb-ius treated than in the blank treated animals. histologically, the cdb exposed endometria were atrophied with evidence of glandular degeneration while the blank controls were proliferative and normal. summary: in cycling rhesus macaques, a cdb-ius prevented progestational development, blocked menstruation after p withdrawal, and suppressed endometrial proliferation. if these effects are confirmed in women, the cdb-ius could provide estrogen-free and bleed-free contraception, and could help control heavy menstrual bleeding. supported by rr , hd and the population council. introduction: irregular uterine bleeding is a major side effect and cause for discontinuation of ltpoc use. while endometria of ltpoc-exposed women display abnormally enlarged, fragile blood vessels (bv), decreased blood flow and evidence of oxidative stress, the mechanisms by which structural and vasomotor endometrial dysfunction occurs remains unknown, in part by the difficulty of manipulating hormone levels in women. the aims of this study were ) to validate the guinea pig (gp) as a model to study uterine effects of ltpoc and ) to investigate ltpoc-effects on endometrial histology and oxidative stress markers. methods: oophorectomized gps were implanted s.c. with time release pellets containing either placebo (crl,n= ); estradiol (e ,n= ); medroxyprogesterone acetate (mpa,n= ) or e +mpa (n= ). after days, uterine horns were weighed and frozen or paraffin embedded. angiogenesis was assessed by quantitative image analysis of vonwillebrand factor staining and included bv density and size. oxidative stress was detected by isoprostane and -oh-deoxyguanosine ( oxog). apoptosis was investigated by the tunel method. statistical analysis was by -way and -way anova. results: gp uteri were enlarged by both e (p< . ) and mpa (p= . ). effects of mpa on uterine weight differed significantly depending on e levels (p< . ), where mpa opposed the e effect in combined treatments. angiogenesis parameters were similarly impacted upon. thus, mpa alone increased bv density (p= . ) and bv average area (p= . ). the presence of e significantly decreased these parameters (bv density mean± sem: crl: . ± . %, e : . ± . %, mpa: . ± . %, e +mpa: . ± . %, p= . ). these changes were associated with highly elevated -isoprostane content in e +mpa-treated uteri compared to all other groups (p< . ). abnormalities in the e +mpa group were consistent with chromatin redistribution, nuclear pyknosis, karyolysis and increased nuclear oxog staining and a marked increase in tunel labeling. conclusions: ltpoc exposure alters endometrial vascular and tissue morphology consistent with oxidative stress and apoptosis in a complex interplay with endogenous estrogens. the gp is an excellent model for the study of ltpoc effects on the uterus. physiologic and psychological symptoms associated with injectable and oral contraceptive use. abbey b berenson, susan odom, carmen r breitkopf, mahbubur rahman. ob/gyn, utmb, galveston, tx, usa. objective: to compare physiologic and psychological symptoms over mo among users of depomedroxyprogesterone acetate (dmpa), an oral contraceptive with micrograms ethinyl estradiol (oc), and non-hormonal (nh) contraception. methods: a total of women reported the presence of symptoms prior to initiating contraception ( dmpa, oc, nh) and every mo thereafter for mo. longitudinal relationships between symptoms and contraceptives (reference: nh), as well as race/ethnicity (non-hispanic black, non-hispanic white, and hispanic) were assessed by gee-analysis after adjusting for age, visits and baseline status of symptoms. persistence, resolution, and new development of symptoms were noted by method in mo increments for mo and compared with that of nh controls. the gee analyses showed that oc was protective against mastalgia (or= . ), cramping (or= . ), hair loss (or= . ), acne (or= . ), nervousness (or= . ) and mood swings (or= . ). when race was considered, oc was protective for all women against acne, for whites against mastalgia and cramping, and for non-whites against nervousness. for whites, it was a risk factor for bleeding between menses. oc use resulted in resolution of acne within mo in nearly % of those with it at baseline, but no significant resolution after mo. also mastalgia ( % at baseline) was less likely to persist at and mo. bleeding between menses was reported for the first time at mo by % of oc users. dmpa users of all races had an increased risk of missed periods (or= . ), bleeding between menses (or= . ), bleeding > d (or= . ) and loss of libido (or= . ) relative to nh users. it reduced cramping (after mo) and bloating (all mo). racial differences were observed with dmpa protective against mastalgia and mood swings in whites, cramping in whites and hispanics, and bloating in non-whites. it was a risk factor for loss of energy in whites only. neither method affected depressive symptoms. conclusion: side effects of these two methods are mostly related to abnormal bleeding. very low dose pills can be protective against symptoms (mastalgia, cramping, hair loss, acne, nervousness and mood swings) commonly associated with pills while dmpa protects against cramping and bloating. knowledge about racial differences will allow physicians to individualize therapy. counseling should include that some symptoms can develop after mo while resolution often occurs within mo. methods: twenty-four women were enrolled in irb approved prospective, randomized, cross-over trial comparing months of oc (ortho cyclen) vs. tc ortho evra). the daily oc administers micrograms of ee; the weekly tc contains . milligrams of ee. each treatment was followed by months of washout and months of the alternative contraceptive. blood was drawn at baseline and final week of treatment for each arm of the study. ee was quantified by ria, with preceding organic solvent extraction and celite column partition chromatography. data were analyzed by t test with boferroni's correction, p < . . results: after two months of treatment the mean (+/-sem) ee levels for tc = . pg/ml (+/- . ) and oc = . pg/ml (+/- . ). the ee level is not significant different for the two medication (p = . ). conclusions: there is no difference in ee levels with the use of these oral and transdermal contraceptives. this suggests the transdermal contraceptive, despite the lack of the hepatic first pass effect, has similar levels of ethinyl estradiol compared to oc. the continuous elevation in ee seen with the tc route of administration, versus the episodic increases seen with the oc route, raises concerns of constant exposure to ee. this may explain the increased risk of estrogen-induced thrombotic events with this tc route of administration. impact of paracervical block, in combination with general anesthesia, on post-abortion pain. gweneth b lazenby, tod aeby. obstetrics and gynecology, university of hawaii, honolulu, hi, usa. objective to evaluate the impact of paracervical block with a long-acting local anesthetic, in conjunction with general anesthesia, on post-operative pain. methods a power analysis determined patients per arm were needed to demonstrate a significant difference of in mean pain scores. seventy-two patients were allocated to one of two arms using urn randomization. all patients received standardized anesthesia; intravenous sedation for gestational age under weeks and general anesthesia for over weeks. thirty-nine patients were randomized to receive a paracervical block with . % bupivacaine and thirtythree were randomized to no local anesthesia prior to surgical abortion. patients completed visual analog scales for pain and anxiety prior to the procedure, upon awaking, and minutes post-operatively, and prior to discharge. data were analyzed using an anova and students t-test the experimental and control groups were equivelent in age, ethnicity, gravidity, parity, prior abortions, prior vaginal deliveries, prior c-sections, gestational age, number of laminaria, pre-operative and intra-operative dilation, operative time, estimated blood loss, and reported complications. pain and anxiety were not significantly affected by placement of a paracervical block. these data do not support the hypothesized benefit of local anesthesia, prior to surgical abortion under general anesthesia, to reduce post-operative pain. we do not recommend the routine use of a paracervical block to decrease post-operative pain. age, parity, history of abortion and contraceptive choices affect the risk of repeated abortion. oskari heikinheimo, mika gissler, satu suhonen. ob gyn, university of helsinki, helsinki, finland; national research and development centre for welfare and health, helsinki, finland. objective the rate of repeat abortion varies from to % in northern europe. however, risk factors for repeat abortion are poorly understood. we characterized risk factors (demographic, as well as those related to abortion and postabortal contraception) of repeat abortion. design a prospective cohort study of women undergoing medical abortion between august and december . the subjects were followed by means of finnish registry on induced abortions until december ; the follow-up time (mean ± sd) was . ± . months. results altogether ( . %) of the subjects requested repeat abortion within the follow-up time. in univariate analysis previous abortion, parity, young age, smoking and failure to attend the follow-up visit were associated with increased risk of repeat abortion. immediate -in contrast to postponed -initiation of any contraceptive method was linked to lower risk of repeat abortion. in comparison to combined oral contraceptives, use of intrauterine contraception was most efficacious in reducing the risk of another pregnancy termination. in multivariate analysis the effects of young age, parity, previous abortion and type of contraception on the risk of another abortion persisted. conclusions increased focus on young, parous and those with the history of an abortion may be efficacious in decreasing repeat abortion. contraceptive choices made at the time of abortion have an important effect on the rate of reabortion. postabortal use of intrauterine contraception, specifically that of lng-ius, might decrease the rate repeat abortion. objective. to determine the rate of failure and to analyze factors associated with failure for the essure permanent birth control device at the detroit medical center (dmc). methods. a chart review was conducted on patients who underwent essure placement at the dmc from january through june . patient demographics, past medical and surgical history, anesthesia type, procedure time, intraoperative complications, and procedure failures were noted. data were analyzed for statistical significance using spss. results. there were essure procedures attempted at the dmc from january through june . of the attempted procedures, there were failures ( . %). of the failures were attributed to difficulty visualizing the ostia ( %). other causes of failure included expulsion of the device ( ), tubal spasm ( ), uterine perforation ( ), and tubal ostia too large for the device ( ). there were cases of failed placement for undocumented reasons, one case requiring a laparoscopic tubal ligation secondary to postprocedure tubal patency, and post-procedure pregnancies. age, race, body mass index, gavidity, parity, history of sexually transmitted infections, medical history, history of cesarean section, tobacco or illicit drug use, anesthesia type, and physician experience with the procedure were not significantly associated with placement failure or difficulty visualizing the ostia. a longer procedure time was significantly associated with failure ( . vs . min, p = . ), and history of ectopic pregnancy was significantly associated with difficulty visualizing the ostia ( . % vs . %, p = . ). conclusion. the failure rate for placement of the essure permanent birth control device at the dmc is . % with a pregnancy rate of . %. the majority of failures may be attributed to difficulty visualizing the ostia. a history of ectopic gestation was significantly associated with difficulty visualizing the ostia; thus, it may be reasonable to advise these women that success in essure placement may be reduced. introduction. the essure permanent birth control device is a relatively new form of minimally invasive sterilization for women. under hysteroscopic guidance, a dynamically expanding micro-insert is introduced into the proximal portion of the fallopian tube. the micro-insert induces local fibrosis and ultimately occlusion of the tubal lumen. a hysterosalpingogram (hsg) is performed three months after the procedure to confirm bilateral tubal occlusion. objective. to determine the follow-up rate for the post-essure hsg for a clinic population. methods. a retrospective chart review was conducted on university health center (uhc) patients who underwent placement of the essure permanent birth control device at the detroit medical center from january through june . follow-up for the post-essure hsg as well as the result of the hsg were noted for each patient. results. placement of the essure permanent birth control device was attempted in uhc patients of which were successfully completed. of the patients, ten underwent a post-essure hsg ( . %). the hsg was performed three to six months after placement of the essure permanent birth control device. bilateral tubal occlusion was documented in all ten patients. conclusion. despite counseling patients prior to their procedure that a hsg is needed and providing an information sheet, the follow-up rate for the post-essure hsg for this clinic population is only . %. for those in whom a hsg was performed three to six months after essure placement, bilateral tubal occlusion was confirmed in all. steps and or approaches to improve compliance with post-procedure confirmation of tubal occlusion should be employed to increase follow-up in the future. towards fibroids gene therapy: adenovirus mediated delivery of herpes simplex virus thymidine kinase gene/ganciclovir shrinks uterine leiomyoma in the eker rat model. memy hassan, dong zhang, salama salama, cheryl walker, hala el-mazar, ayman al-hendy. ob/gyn, utmb; md anderson; ob/gyn, meharry medical college, nashville, usa. aim: assessment of the efficacy of gene therapy of uterine leiomyoma in the immune-competent eker rat model using adenovirus mediated delivery of herpes simplex- -thymidine kinase gene followed by ganciclivir treatment (ad-tk/gcv). method: female eker rats with mri-confirmed uterine fibroid lesions were randomized to a single treatment with direct intratumor injection of ad-tk/gcv, ad-lacz/gcv, or medium.the tumor volume was evaluated by serial mri scanning and confirmed with caliper measurement at time of euthanasia. sample rats were selected randomly and killed at the following time points; , and days post treatment. samples were collected from tumors, other body organs and blood to assess the safety and efficacy of the treatment. results: ad-tk/gcv treatment produced dramatic shrinkage of the total uterine fibroid volume by % ± , % ± and %± of pretreatment volume at days , and respectively. the tumor size in negative control animals receiving ad-lacz/gcv continued to grow by + %± , + %± , + % ± while receiving media continue to grow by + % ± , + % ± and + % ± at same time points. ad-tk/gcv induced significant increase in caspase activity, bax expression, decrease in bcl and parp proteins expression and increased tunnel apoptosis index. additionally ad-tk/gcv treatment decreased cyclin d and pcna expressions. ad-tk/gcv did not produce any significant change in liver function tests or relative uterine horns weight to total body weight. the adenovirus transfection did not disseminated significantly to other distal organs except to liver and myometrium in limited number of animals. h e staining of non targeted organs did not revealed any sign of tissue damage. ad-transfection increased local cd and cd expressions as well as serum anti-ad antibodies. conclusion: ad-mediated delivery of hsv tk gene by direct intra-tumor inoculation followed by sc treatment of gcv for ten days effectively shrinks uterine leiomyoma lesions in eker rats. this effect is mediated via induction of apoptosis and decreasing the proliferation. the treatment regimen is well tolerated. these studies provide essential preclinical data for the development of gene therapy as an alternative non-surgical treatment option for women with symptomatic uterine fibroids. inhibitors of catechol o-methyl transferase shrinks uterine fibroids in the eker rat model. memy hassan, dong zhang, hala el-mazar, cheryl walker, ayman al-hendy. ob/gyn, utmb; md anderson; ob/gyn, meharry medical college, nashville, usa. background :the sex hormone dependent pattern of uterine leiomyomas and their high content of catechole -o-methy transferase (comt) raise the possibility for the development of novel treatment option using comt inhibitors.aim : to assess the potential therapeutic utility of a synthetic comt inhibitor (ro - ) in the eker rat model of uterine leiomyoma. methods female eker rat were evaluated by mri to confirm the prescence uterine fibroid lesion, then randomized for sc treatment with ro - mg /kg ,twice/ day for days versus vehicle injection.fibroid tumor burden was evaluated by serial mri measurement and confirmed by direct caliper measurement at time of euthanasia. sample animals were euthanized at and weeks. at that time tumor tissue, blood and most of animal organs including long bones were collected and subjected for further evaluations. hours urine samples were collected for evaluation of estrogen (e ) metabolites and bone resorption marker. results:animals treated with ro - exhibited significantly lower uterine fibroid tumor burden ( % and %) of pretreatment volume at and weeks post treatment respectively. conversely, the tumor size in control animals continued to grow and reached %, and % of pretreatment size at the same time points. ro - treatment resulted in an increase in urinary / hydroxy e metabolite ratio. in addition ro - increased bax expression and decreased parp , pcna and cyclind experssions . all ro - treated animals tolerated the treatment protocol with no signs of toxicity. h e staining of different body organs did not reveal any signs of tissue damage. furthermore, there was no significant change in both liver function tests (alt, ast, billirubin) and bone resorption marker , deoxypyridinoline croslinks, between treated and control rats. conclusion ro - , a synthetic selective comt inhibitor, caused immediate arrest of the growth of eker rat uterine leiomyoma. this effect might be in part due to modulation of various estrogen dependent genes regulating leiomyoma apoptosis (parp, bax) and proliferation (pcna, cyclin d ). this anti-estrogenic effect is due to the accumulation of the the antiestrogenic metabolite hydroxy estrogen secondary to comt inhibition.comt inhibitors might present an alternative non-surgical option for the treatment of women with symptomatic uterine fibroids. background development of uterine leiomyomas (fibroids) is the most common pathological feature in the female reproductive tract. they negatively impact patients of virtually every gynecologist. despite such morbidity, leiomyoma development is poorly understood. we have recently demonstrated that leiomyomas have a genomic expression pattern that limits retinoic acid (ra) exposure. our group and others have demonstrated that tgf-beta regulation is altered as well. these two pathways likely play central roles in leiomyoma development. the central feature of uterine leiomyomas, the extracellular matrix (ecm), is regulated by both all-trans retinoic acid and tgf- , focusing on versican as a critical ecm component. human uterine leiomyoma and patient-matched myometrium were obtained from surgical specimens under an irb-approved protocol. these tissues were immortalized and treated with either all-trans retinoic acid, tgf- , or anti-tgf- antibody. rna was isolated for qrt-pcr. human immortalized leiomyoma cells demonstrated the same increased template expression of tgf- ( . ± . fold), retinoic acid metabolizing protein (cyp a ; . ± . ), and versican variant v ( . ± . fold) as was found in the progenitor tissue. when treated with all-trans ra, expression of versican variant v decreased to levels found in myometrial cells ( . ± . fold). conversely, when treated with tgf- , expression of versican variant v increased . ± . fold. to confirm that tgf- was central to the overexpression of v , we treated leiomyoma cells with anti-tgf- antibody, and found that baseline over-expression of v template was decreased to expression levels similar to untreated myometrial cells. finally, we elucidated a link between the ra and tgf-pathways by assessing the impact of ra treatment of tgf- expression, demonstrating that tgf- template decreased to levels comparable to myometrial cell expression ( . ± . fold). the disrupted leiomyoma ecm, of which versican is a central component, defines the leiomyoma phenotype. in this study, the leiomyoma fibrotic phenotype regressed when treated with ra and increased when treated with tgf- , providing the basis for novel therapeutic interventions directed at cell differentiation and ecm formation. objectives: uterine fibroids are the leading cause for hysterectomies in the us. the lack of an appropriate in vitro cell model for the initiation of fibroid growth has hindered advancement in understanding the cellular and molecular basis for the development and progression of uterine fibroids. fibrosis is the underlying mechanism of uterine fibroid formation and myofibroblasts cells are the principal fibrogenic cell type in the uterus. we sought to develop a myofibroblast in vitro cell model for analyzing the initiating molecular events of uterine fibrosis. methods: smooth muscle cells (smcs) were enzymatically isolated from the myometrium of non-pregnant women and cultured in the presence of % serum until % confluent. for the next h cells were cultured in serum-free media followed by replacement with serum containing media. cells were fixed at , , , , m later. cell fine structure and cytoskeletal organization were evaluated by transmission electron microscopy. smooth muscle specific alpha-actin ( -sma) and progesterone receptors (pr) were detected by western blot. results: we observed smc differentiation into myofibroblasts, marked by the presence of notched nuclei ( figure) and the increased expression of -sma m after serum replacement. pr-a and pr-b were detectable at , and m. conclusions: the development of myofibroblasts is important in wound healing and fibrosis. we show for the first time that uterine myofibroblasts can be derived in culture from myometrial smcs. thus, these cells will be utilized as a model for developing "in vitro fibroids". this model will enable the study of myofibroblast activation, cytokine signaling, intracellular regulation of uterine fibrogenesis, production of extracellular matrix proteins and development of antifibrotic drugs. the presence of prs in our model enables us to evaluate pr mediated events in fibroid pathogenesis and treatment. this model will be more useful in determining the molecular biology of fibroid initiation than cell models derived from established fibroids that are already well past their initial stages of development. novel approach to genome-wide expression profiling analysis. liping feng, morgan walls, insuk sohn, millie behera, sin-ho jung, phyllis leppert. obgyn; biostatistics and bioinformatics, duke university medical center, durham, nc, usa. background: microarray studies have examined the differential gene expression between uterine fibroid and normal myometrium. all previous studies considered the fibroid as a whole and analyzed only fold changes. we have developed a novel statistical approach to genome-wide expression analysis comparing two fibroid tissue sites to myometrium. methods: using affymetrix tm u a genechip, we have compared the gene expression between c and e and matched adjacent m. data has been analyzed by considering the specimens per subject and subjects as individuals. we used a block one-way anova method to test if each gene was differentially expressed among the three sites. the p-value is calculated using a permutation method accounting for possible dependency among three lesions. the multiple testing issue was addressed by controlling the false discovery rate. expression values were calculated using the robust multichip average (rma) method. rma estimates are based upon a robust average of background corrected perfect/mismatch (pm) intensities. normalization was done using quantile normalization. expression values were then transformed by taking logarithm base . confirmatory rt-pcr was performed. results: we applied a hierarchical clustering analysis to all raw data sets and then displayed a dendrogram, where the height of each branch point indicates the similarity level at each generated cluster. identical gene expression among sites clusters together. due to a strong site effect, m tissues clustered separately from e and c combined. genes were differentially expressed when we used a . q-value cut off. expression data revealed concordant changes in genes regulating cholesterol biosynthesis, gene transcription, estrogen and extracellular matrix formation when both e and c were examined. cyp was detected and we report for the first time that scc- (a cell cycle-regulated molecule) folliculin and l-selectin are differentially expressed suggesting that they may be involved in the regulation of cell growth and proliferation of uterine fibroids. conclusions: our novel robust analysis of gene expression provides new clues to the relevant pathways of fibroid development. this new statistical approach that can be used in clinical and/or translational studies to identify differentially expressed genes comparing treatment regimens, cells or tissues. objectives: thrombospondin- (tsp- ) is a large matricellular glycoprotein secreted by many cell types. matricellular proteins modulate interactions between cells and their environment, regulate cell adhesion and are expressed during tissue formative processes. they are especially important in fibrosis. tsp- plays an important role in angiogenesis and is an activator of tgf - . in a previous study, we found that differential expression of tsp- in uterine fibroids may contribute to an altered healing process leading to fibrosis. this alteration in tissue response to injury initiates the development of abundant, nonaligned collagen fibrils and changes in other components of the ecm. methods: we measureed the pattern of mrna and protein expression of tsp- by rt-pcr and western blot in an in vitro serum-deprived differentiated myometrial cell (myofibroblast) model. specifically, smooth muscle cells (smcs) were enzymatically isolated from the myometrium of non-pregnant women and grown in primary culture to % confluence. then smc were serum deprived for h and treated back with % serum for , , , and m. cells were collected for rna and protein, and tsp- expression was evaluated. in addition, cells were stained using the combination of anti-cd and anti-smooth muscle -actin or combination of anti-cd d and anti-smooth muscle -actin as well as the appropriate single and double negative controls. stained sections were analyzed using zeiss axio imager widefield fluorescence confocal microscopy. results: tsp- mrna and protein was present in cells in this serumstarvation model after the addition of serum and the expression level remained elevated for m following the addition of serum. fluorescence staining analysis indicated that these cells were positive for human smooth muscle -actin, but negative for leukocyte antigen cd and platelet marker cd d suggesting that the myofibroblasts cells themselves were the source of the tsp- . conclusions: unlike skin wounds, where tsp- is derived from the blood macrophages, monocytes and platelets, differentiated myometrial cells appear to produce tsp- . elucidating the roles of tsp- in myometrium physiology and pathobiology will increase our understanding of the etiology of uterine fibroids and may lead to improved therapies. further studies utilizing this cell model to determine the role of tsp- in the activation of tgf - are indicated. phospholipid s p via gi, rac and rho pathways. yoel smicun, armando wu pimentel, jennifer gilman, david a fishman. obstetrics gynecology, new york university school of medicine, new york, ny, usa. objectives: sphingosine- -phosphate (s p) levels are elevated in serum and ascites of ovarian cancer patients. we have demonstrated that low concentration s p enhances while high dose s p inhibits invasion of epithelial ovarian carcinoma (eoc) cells in a dose and attachment mode dependent manner. we sought to further dissect the pathways by which s p affects invasion, using specific inhibitors. methods: dov eoc cells were pretreated for -hrs with vehicle, . m or m s p and with inhibitors for gi, p -mapk, rac and rock, thereafter cells were detached and tested for invasion towards m lpa chemoattractant in matrigel-coated chambers. conditioned media from pretreated cells and invading cells were quantified for upa activity using colorimetric assays. the significance of results was calculated by student's t-test. results: inhibition of gi mildly increased invasion of both control ( p= . ) and m s p treated cells ( p= . ). inhibition of both p -mapk and rac did not affect m s p treated cells, in contrast invasion of control cells was mildly increased (p= . , . ). inhibition of rock, a protein effector downstream of rho, highly elevated invasion of both control and m s p treated cells ( fold, p= . , fold, p= . ). both upa and gelatinase activities were higher in conditioned media of invading cells than of attached cells. gelatinase activity was enhanced by both concentrations of s p (p= . , . ). ptx fully inhibited gelatinase activity of control and . m s p treated cells, and partially of the m s p treated cells (p< . ). . m s p significantly increased upa activity of attached (p= . ) but not of invading cells. this increase was sensitive to ptx and rac inhibitor. m s p inhibited upa in both attached and invading cells (p= . ), this inhibition was rock dependent. conclusions: these findings suggest a strong inhibition of invasion and upa by the rho pathway, of both control and m s p treated cells. this inhibition is induced partially by upstream gi protein. increased invasion by . m s p is associated with elevation of gelatinase activity through gi, rac and rock pathways. this suggests that attached cells and invading cells affect upa activity through different pathways. objectives: sphingosine- -phosphate (s p) levels are elevated in serum and ascites of epithelial ovarian cancer (eoc) patients. we have demonstrated that invasion of attached eoc cells differentially react to s p as compared to invading cells. we examined the impact of the inhibitors for gi and rac on attached and invading eoc. methods: dov eoc cells were pretreated for -hrs with vehicle, . m or m s p and with inhibitors for gi (pertussis toxin (ptx)), and rac (nsc , (rac-i)), and cells were detached and assayed for invasion towards m lpa in matrigel-coated chambers. to distinguish the response of attached from invading cells, inhibition of cells pretreated with inhibitors was either continued or not in the invasion chambers. conditioned media (cm) of invading cells were quantified for upa and gelatinase activity by fluorometric and colorimetric assays. significance of results was calculated by student's t-test. results: the significant (p= . ) increase of invasion by . m s p was inhibited by both ptx and rac-i, either by pretreatment alone or by continuous treatment (p= . - . ). however, the invasion was higher in cells inhibited continuously than cells inhibited only in dishes. both inhibitors did not affect cells treated with m s p. control cells invasion was increased ( . fold, p= . ) by continuous rac-i treatment. . m s p increased gelatinolysis in cm of invading cells. this and control cells activity was inhibited by ptx pretreatment. continuous treatment with ptx or rac-i elevated and fold gelatinase activity of control and m s p treated cells. in contrast upa activity was inhibited by both . m and m s p. activity of control cells was inhibited by both pretreatment and continuous treatment with both ptx and raci. upa activity of cells treated with . m s p was increased only by pretreatment with ptx and raci. in contrast, in cells treated with m s p upa was inhibited only by continuous inhibition with ptx and raci. conclusions: invasion of s p induced eoc cells correlated with the gelatinase and upa activities in their microenvironment. ptx and rac-i affect attached and invading cells in different manner, inhibition of invasion and gelatinolysis of attached and increased invasion of invading cells. this suggests a dual effect of the gi-rac pathway, inhibiting attached and stimulating invasion via gelatinolysis of invading cells. lysophosphatidic acid (lpa) levels are elevated in the ascites and plasma of early-and late-stage ovarian cancer patients, underscoring the unique role this bioactive lipid plays in the pathobiology of epithelial ovarian cancer (eoc). lpa binding to its cognate g-protein-coupled receptor can transactivate the receptor tyrosine kinase, egfr, which is often overexpressed in ovarian tumors. in the current study, we investigated the role that lpa activation of egfr plays in the processing of the metalloproteinase, mmp- , and eoc dissemination. lpa stimulates tyrosine phosphorylation of egfr in ovarian cancer cells, and egfr kinase activity is required for optimal lpa induction of pro-mmp- activation. lpa-dependent pro-mmp- activation does not require the egfr ligands, amphiregulin, b-cellulin, egf, hb-egf, and tgf-a. we previously reported that when cells are cultured at high density, lpa represses rhoa activity to induce loss of stress fibers, and these changes in actin microfilament organization contribute to pro-mmp- activation. in the current study, inhibition of rho-kinase/rock with y- reversed the repression of lpa-stimulated pro-mmp- processing observed with treatment of the egfr kinase inhibitor, ag . correspondingly, lpa induced the loss of stress fibers, while inhibition of egfr kinase restored stress fiber formation in lpa-treated cells. this suggests that lpa acts through egfr to modulate microfilament organization and pro-mmp- processing. finally, lpa-induced cellular haptotactic migration and invasion are abrogated when egfr kinase activity is blocked. taken together, these results suggest that egfr signaling plays a critical role in lpa regulation of metastatic pathways by mediating changes in the cytoskeleton which impact protease activity. objectives: membrane-derived vesicles are active modulators of tumor dissemination; they promote and contribute to extracellular matrix degradation and tumor cell invasion. we have isolated vesicles from ascites of ovarian cancer patients and from ovarian cancer cells in vitro. here we analyzed the functional consequences of exposure of cancer cells to vesicles derived from a different type of malignancy in order to evaluate their proinvasive properties. methods: ovarian cancer (dov ), breast cancer (mda mb ) and mesothelioma (hp- ) cells were grown in media supplemented with % fbs. after serum deprivation, % vesicle-free fbs was added for hr to induce vesicle release. vesicles were isolated from media by differential centrifugation and quantified with a bradford assay. fusion to cells was followed by fluorescence of the lipophilic tracer dii. each cell line was stimulated with and g/ml of each type of vesicles and tested in a matrigel invasion assay. changes in proliferation were evaluated in an mts assay. gelatin zymography was used to assess matrix metalloproteinase activity of vesicles. results: zymographic analysis showed that vesicles contained mmp- and . fusion experiments showed that all vesicles fused to all cell types. all vesicles induced the invasion of their respective cell types in a dose-dependent manner. when vesicles were used at g/ml they induced significantly high levels of invasion in all cell types tested. at g/ml they significantly inhibited invasion in different cell types. both concentrations of vesicles stimulated cell proliferation in all cell types tested. conclusions: membrane derived vesicles are potent mediators of invasion for mesothelioma, breast and ovarian cancer cells in vitro. this effect occurs in a dose-dependent manner when vesicles induce invasion of the same cell type from which they were isolated. when vesicles are used to induce a different cell type, low concentrations induce invasion while high concentrations inhibit invasion, this effect was independent of cellular proliferation. these results suggest that a novel mechanism may be at play where activation of recognition of self and non-self specific pathways may determine the invasive potential of the tumor cell upon fusion to microvesicles. the identification of signaling pathways responsible for this heterotypic signaling is a current effort. vegfr- as a potential target to inhibit lpa-induced epithelial ovarian cancer (eoc) invasion. sonia dutta, fengqiang wang, elaine barfield, david a fishman. obstetrics and gynecology, new york university school of medicine, new york, ny, usa. objective: vegf and vegf receptors (vegfrs) play important roles in ovarian cancer metastasis. in this study, we examined the expression profiles of vegf and vegfrs (vegfr , vegfr , co-receptor nrp and nrp ) in established eoc cells lines (dov , r , ovca , skov ) and an immortalized normal ovarian epithelium (iose- ). the effect of lysophosphatidic acid (lpa) on vegf and vegfrs expression and the effect of vegfr- silencing by rnai on lpa-induced invasion were also evaluated. methods: vegf and vegfrs expression in ovarian cancer cell lines and normal ovarian epithelium was quantified by real time pcr. cell invasion differentiate into either a pro-tumor or anti-tumor phenotype depending on the specific tumor microenvironment. previously, we described a subgroup of epithelial ovarian cancer (eoc) cells with a functional tlr- -myd -nf-b pathway (type i eoc cells). these cells have constitutive nf-b activity and constitutively secrete il- , il- , mcp- , and gro . we hypothesize that type i eoc cells, but not type ii, can promote macrophage differentiation into a tumor-supporting immune cell. methods: eoc cell conditioned media (cm) was prepared by incubating eoc cells in log-phase growth in optimem for h. migration assay was done using an in vitro cell culture insert with m-size pore and pkh red fluorescentlabeled thp- . cytokine profile of thp- cells co-cultured with eoc cell cm was determined using xmap technology. modulation of response to apoptotic bodies was determined by "pre-educating" thp- cells with eoc cell cm for h prior to exposure to apoptotic bodies ( : ratio with thp- cells). results: monocytes migrate toward eoc cells. however, migration is significantly higher towards type i eoc cells. type i, but not type ii, eoc cells alter monocytes' cytokine profile by inducing the secretion of high levels of progrowth and angiogenic cytokines il- , il- , mcp- , and gro . furthermore, type i eoc cells modify monocytes response to apoptotic bodies by inducing a significant increase in the secretion of il- , il- , mip- , mip- , and gro ( -fold, . -fold, -fold, -fold, and -fold respectively). conclusion: we demonstrate for the first time a differential interaction between two subtypes of eoc cells and monocytes. we showed that the microenvironment created by type i eoc cells is able to modify the function and differentiation of immune cells towards a tumor supporting phenotype. understanding the molecular mechanisms mediating this tumor-immune interaction will help to design appropriate immune therapies that will take into consideration the tumor microenvironment. objectives: the standard of treatment for patients with ovarian cancer (oc) is intravenous combination chemotherapy (ct) after primary cytoreductive surgery. although initial response is above %, most of these patients experience recurrence. the only approach for these patients is salvage ct which may prolong their lives for months. early detection of patients who are not responding to current therapy or are at risk of experiencing an early relapse of disease might improve response rates and survival if alternative therapy is possible. no single predictive marker has yet been proven sufficiently sensitive or specific to find a place in the daily clinic. in the present study we use a newly described biomarker test for the detection of oc (visintin et al clinical cancer research) and evaluated the ability of the test to monitor ct response methods: patients with oc, figo stage i-iv, were included in this study. all patients recieved postsurgery first line combination ct (paclitaxel/carboplatin). samples were evaluated at ) baseline (mean days after surgery), prior to the first cycle of ct, and ) after cycles of ct. μl serum samples were analyzed by multiplex assay (beadlyte® cancer biomarker panel kit) for six markers. changes during ct and differences in markers between patients with short time to progression (ttp) and patients with long ttp were determined. results: positive test for oc was observed in % of the patients evaluated at baseline. all patients had residual disease after surgery. from patients with long ttp, / patients (specificity %) had a negative test after cycles. from the patients with short ttp, / had a positive test (sensitivity %) p= . ( ²). the risk of experiencing a ttp shorter than months when having a positive test after cycles of ct was %. conclusion: we describe for the first time the use of a panel of biomarkers for oc that might have an application for monitoring ct response and risk of relapse. the test detects the presence of residual disease following debulking surgery, and differentiates between long term and short term progression. a longitudinal study is performed to determine how early during ct the test can identify responder versus non responder. ksp inhibitor (arry- ) as an alternative for paclitaxel in myd -positive epithelial ovarian cancer cells. ki h kim, ayesha b alvero, yanhua xie, david trollinger, gil mor. obstetrics, gynecology, and reproductive sciences, yale university school of medicine, new haven, ct, usa; array biopharma inc., boulder, co, usa. background: we previously described that epithelial ovarian cancer (eoc) cells ubiquitously express tlr- , but not the adaptor protein myd . when treated with paclitaxel, a known tlr- ligand, myd -positive eoc cells exhibited nf-b activation, increased secretion of il- , il- , mcp- , and gro , and increased p-erk levels . since majority of eoc patients are given paclitaxel in combination with a platinum drug, it is not only important to distinguish those patients that should not be given paclitaxel; it is also important to identify alternative chemotherapy agents that would benefit this sub-group of patients. the objective of this study is to determine if the ksp inhibitor, arry- , can be an alternative for paclitaxel in myd -positive ovarian cancer patients. methods: myd -positive and myd -negative eoc cell lines isolated from either ascites or tumor tissue were treated with increasing concentrations of arry- ( to nm) or paclitaxel ( . to m) for , , and hours and cell viability was determined using the celltiter aqueous one solution cell proliferation assay. cytokine profiling was performed from supernatants using xmap technology. nf-b activity was determined using a luciferase reporter system. p-erk levels were measured by western blot analysis. results: arry- and paclitaxel exhibited the same cytotoxic effect on myd -negative and positive eoc cells. the ic at h for myd -negative eoc cells was . m and . um for arry- and paclitaxel, respectively. for myd -positive eoc cells, the ic at h was m and m for arry- and paclitaxel, respectively. however, unlike paclitaxel, arry- did not induce nf-b activation or enhance the secretion of il- , il- , mcp- , and gro , and did not induce erk phosphorylation on myd positive cells. conclusions: administration of paclitaxel to patients with a myd -positive tumor could have detrimental effects due to the paclitaxel-induced enhancement of cytokine production which promotes chemoresistance and tumor growth. arry- has similar anti-tumor activity in eoc cells as that of paclitaxel. however, unlike paclitaxel, it does not induce cytokine production in myd positive eoc cells, and therefore, the ksp inhibitor arry- may represent an alternative to paclitaxel in this subgroup of eoc patients. introduction: nf-b activation has been associated with cell proliferation, angiogenesis, metastasis and suppression of apoptosis in ovarian cancer. in addition, nf-b activity induces the production of pro-inflammatory cytokines which may contribute to chemoresistance. inhibition of nf-b may represent a new approach to prevent tumor growth and reverse chemoresistance. in the present study we described the characterization of a novel nf-b inhibitor, eriocalyxin b (erib) that is able to re-establish the apoptotic cascade in chemoresistant ovarian cancer cells by suppressing pro-inflammatory cytokines and antiapoptotic proteins. materials and methods: eoc cell lines were isolated from malignant ovarian cancer ascites. caspase activity was determined by caspase-glotm assay. cytokine production and secretion were determined using multiplex assay. erib effect on cancer cells was evaluated in a time and dose dependent manner using celltiter cell proliferation assay. protein expression was determined by western blot analysis. combination studies were done with paclitaxel and carboplatin in addition to erib treatment. nf-b activity was determined by monitoring the expression of a nf-b luciferase reporter. results: erib decreased cell viability of ovarian cancer cells with an ic of . - μm in hours and was associated to increasing levels of caspases , and activity. intracellular changes induced by erib included: ) inhibition of nf-b activity; ) decrease in cytokine production; ) down regulation of anti-apoptotic proteins xiap and flip, and reversal of resistance to tnf-and fasl-mediated cell death; and ) chemosensitization to carboplatin and paclitaxel. at present, evidence is accumulating regarding the existence of unique populations of specialized tumor-initiating, stem-like cells within various tumor types of distinct origins. these cancer stem cells (csc), with characteristics reminiscent of normal stem cells, are thought to be responsible for driving tumor growth. we propose that ovarian cancers arise from csc, and are using microarray-based technology to identify specific genes/cell surface markers associated with ovarian csc that will permit distinction of these rare cells from the remaining tumor bulk. to identify unique gene signatures associated with an ovarian tumor-initiating cell population, we have utilized an in vivo serial transplantation model. this model selects for primary human ovarian tumor cells with increased tumorigenic capacity, given that time to tumor formation decreases with successive serial transplant despite fewer cells injected. our initial studies used cells derived from a human ovarian clear cell carcinoma, serially transplanted for three passages in nod/scid mice. rna derived from these transplanted tumors was analyzed on human genome microarrays. from these analyses, several differentially expressed genes were identified. the differential expression noted for potential genes of interest is currently being validated by rt-pcr. of particular interest, expression of the transmembrane glycoprotein muc was found to increase both at the rna and protein level with successive transplant of this clear cell carcinoma. further studies are ongoing to determine the functional significance of muc and other identified differentially expressed genes in ovarian clear cell cancer. in addition, we are carrying out parallel microarray analyses in other ovarian tumor subtypes. background recent observations suggest a decreased incidence of neoplastic lesions in hiv infected individuals treated with highly active anti-retroviral therapy comprised of protease inhibitors, such as ritonavir. the polycomb group protein ezh is associated with aggressive human malignancies via transcriptional suppression of dna repair proteins. objective objective of the present study was to assess the antineoplastic impact of ritonavir on epithelial ovarian cancer (eoc) cell lines. methods eoc cell lines (mdah and skov- ) were treated with serial dilutions of ritonavir ( - mm) dissolved in dmso. normal diploid human fibroblasts served as controls. growth curves, apoptosis and cell cycle analysis were performed with cell counting kit- , annexin v and flow cytometry. signal transduction was studied with western blotting. dna double strand breaks (dsb) were induced with . mm etoposide treatment for hours. homologous recombination (hr) repair of dna damage was measured by assessment of rad foci formation in the nucleus of the cells as visualized by fluorescence microscopy according to previously published criteria. untreated eoc cells expressed higher levels of ezh and lower levels of rad and xrcc as compared to controls and in turn, had lower prevalence of rad repair foci formation in response to dsb. serial treatments of eoc cells with ritonavir resulted in a decrease in expression of ezh and an increase in expression of rad and xrcc when compared to untreated eoc cells. after induction of dsb, the rad repair foci formation was significantly more prevalent in eoc cells treated with ritonavir as compared to untreated eoc cells. in addition, ritonavir induced apoptosis in ovarian cancer cell lines by down-regulation of akt pathway and caused g cell cycle arrest mediated by down modulating levels of prb phosphorylation and depleting the cyclindependent kinase and . ritonavir effectively induces apoptosis, cell cycle arrest and improves repair of dna damage by hr in ovarian cancer cell lines. as impaired hr is a key event in causation and progression of neoplastic lesions, ritonavir may have a role in chemoprophylaxis and treatment of human malignancies. a rare case of ovarian cystic lymphangioma. tomer singer, tamer seckin, noa feldman, susan jormark, michael divon. department of obstetrics and gynecology, lenox hill hospital, n.y., ny, usa; department of pathology, lenox hill hospital, n.y., ny, usa. cystic lymphangioma (cl) is a rare, benign malformation of the lymphatic system whose exact etiology remains uncertain. cl may arise in different sites: the spleen, the mediastinum, the axillary region, the retroperitoneum, and the mesentery. retroperitoneal cl is extremely rare and its true incidence is unknown. the majority of cases are symptomatic during childhood. clinical presentation of adult cl is variable and may be misleading. typically, this is a slow-growing tumor and it remains asymptomatic for a long period of time. it is most often found incidentally during abdominal or pelvic imaging studies, surgeries or autopsies. total surgical removal of the lesions with microscopically clear margins is the best approach when it is possible. we report, for the first time, a case of cystic lymphangioma arising from the ovary in a post-menopausal woman and present the feasibility and the advantages of laparoscopic surgery, allowing accurate diagnosis, optimal treatment and minimal risk for the patient. lgsc) is a chemoresistant ovarian neoplasm that has been molecularly linked to low malignant potential tumor (lmp), which often behaves in a non-invasive fashion. micropapillary features within lmp (lmp-mp) are associated with increased invasive behavior. the aim of this study was to determine the differential gene expression of lmp, lmp-mp, and lgsc to identify genes involved in malignant transformation and carcinogenesis. methods: laser capture microdissection was used to isolate epithelial cells from snap-frozen lmp (n= ), lmp-mp (n= ) and lgsc (n= ). rna was extracted, amplified, reverse transcribed to cdna and hybridized to affymetrix u plus arrays. data was analyzed by significance analysis methods: medical records were reviewed for patients who underwent hysterectomy for all indications at wsu hospitals from / / - / / . pathology reports were reviewed to identify the incidence of adenomyosis. data obtained from medial records included: age, race, insurance, bmi, social history, medical history, and presenting symptoms. the correlation between adenomyosis and all the above factors was tested using the appropriate statistical methods. results: patients were included. adenomyosis was confirmed by pathology in patients, an incidence of . %. incidence was not significantly different among races after controlling for parity. . % in african americans, . % in caucasians, and . % in others. incidence was not statistically different between uninsured( . %), privately insured ( . %), and medicaid ( . %) patients. p=. . incidence of adenomyosis was not different between smokers ( . %) and nonsmokers ( . %). p= . . table i shows the correlation between adenomyosis and different risk factors. conclusion: adenomyosis was diagnosed in . % of hysterectomy specimens. race, socioeconomic status or social habits didn't affect its incidence. diabetes and endometrial cancer were negative risk factors for adenomyosis, whereas htn, hypothyroid, breast cancer, fibroids, polyps and endometriosis didn't affect its incidence. menorrhagia, dysmenorrhea, dyspareunia, and chronic pelvic pain but not metrorrhagia had a positive correlation with adenomyosis. there is a protective adipocytokine profile. we hypothesize that this finding may precede some ill-defined threshold of fat mass and/or insulin resistance after which adiponectin decreases. the objective: to correlate reproductive hormone production with menstrual bleeding patterns among women in the menopause transition. methods: a sub-cohort of the swan study consisting of women age - was studied. each woman collected daily first void urine samples for one complete menstrual cycle or days (whichever came first) once a year for years. urine was assayed for excreted levels of fsh, lh, estrogen metabolites and progesterone metabolites which were normalized for creatinine concentration. ovulation was detected by a validated algorithm. menstrual bleeding parameters were derived from daily calendars. correlations between bleeding characteristics, hormone concentrations, and other potential clinical predictors were analyzed using multivariate logistic regression models. results: the cohort was ethnically diverse with a median age of and with % in the early perimenopause at the start of the study. % of all cycles were anovulatory. short cycle intervals (< days) were associated with the early perimenopause (or . , ci . , . ) and with anovulation (or . , ci . , . ). long cycle intervals ( + days) were associated with late perimenopause (or . , ci . , . )and with anovulatory cycles (or . , ci . , . ). both short ( - days) and long ( + days) duration of menstrual bleeding were significantly associated with anovulation (or . and . , respectively). women with anovulatory cycles were less likely to report heavy menstrual bleeding than women with ovulatory cycles. menorrhagia was not associated with steroid hormone concentrations but was associated with obesity (or . , ci . , . ) and with the self-reported presence of fibroids (or . , ci . , . ). conclusions: among women in the menopause transition, abnormalities in timing of menstrual bleeding (cycle intervals or bleeding duration) have a hormonal basis and are frequently associated with anovulation. in contrast, abnormally heavy periods do not have a hormonal basis and are less likely following anovulatory cycles. heavy periods are associated with obesity and fibroids. to undetectable levels. the use is vaginal e is contraindicated, although these women have even higher rates of av. topical testosterone (tt) has successfully treated vulvar atrophy; testosterone receptors are also present in the vaginal epithelium. objective: assess the effect of tt on vaginal maturation index (mi) and relief of av symptoms. methods: postmenopausal women on aromatase inhibitor with symptomatic atrophic vaginitis were enrolled in prospective, irb approved study. estradiol (e) and testosterone (t) levels, av questionnaires (score - ; none to most severe symptoms of dryness, irritation, and dyspareunia), gynecologic exam, and vaginal smears (for ph and vaginal maturation index, mi, by meisels criteria) were performed at baseline and after month of daily treatment with mcg of tt. data was assessed by t test and fischer's exact test; significant p < . . results: e levels were undetectable at baseline and following treatment. t levels increased (mean +/-sem) from baseline ( +/- . ) to treatment ( +/- . ); the difference was not significant; p = . , although one patient had an appreciable rise in serum t level. two av symptoms improved significantly with tt use; comparing baseline to treatment scores: vaginal dryness ( . vs. . ; p = . ) and dyspareunia ( . vs. ; p = . saliva analysis is a convenient, non-invasive and rapid method for assessing estradiol (e ) levels. however, particularly in postmenopausal women, the low salivary e levels are often near or below the sensitivity of available assays, compromising both accuracy and precision. we present results using an extraction step prior to e assay, which concentrates the sample to increase sensitivity and removes potentially interfering substances. morning saliva samples were obtained from premenopausal (mid-luteal phase, n= , ) and postmenopausal women (n= , ) not taking hormones, and from postmenopausal women receiving oral conjugated equine estrogens (cenestin, n= ; premarin, n= ), oral micronized e (estrace, n= ; compounded e , n= ), transdermal e patches (climara, n= ; vivelle, n= ); or topical e cream (compounded e , n= ). e levels were determined by an automated enzyme immunoassay (eia) after solid phase extraction. the functional sensitivity of the assay was determined to be . pg/ml, compared with > pg/ml without extraction. . . . . . oral conjugated equine estrogens; oral e ; e patch; topical cream salivary e levels corresponded with the hormone dosage, suggesting a reliable assessment of unbound e levels with each formulation, dosage and type of estrogen therapy. extraction prior to eia in an automated assay dramatically increased precision and accuracy at low concentrations. omitting the extraction step may have contributed to poor serum versus saliva correlations in other studies. this method may therefore allow reliable monitoring of estrogen therapy without the need for expensive and inconvenient blood tests. the role of fibulin- in pelvic organ prolapse. david d rahn, jesus f acevedo, patrick w keller, lihua marmorstein, r ann word. ob-gyn, ut southwestern, dallas, tx, usa; visual sciences, univ arizona, tuscon, az, usa. objectives: fibulin- (fib- ) is crucial for normal elastic fibers in the protein showed a statistically significant reduction in its expression (p= . ). lox, loxl , and proteins were detected by immunohistochemistry in all three layers of vaginal skin biopsies: ( ) stratified squamous epithelium; ( ) the lamina propria and ( ) the muscularis layer from both patients with pop and controls. significantly, in both groups we detected a numerous macrophages scattered throughout the vaginal thickness which displayed a very strong immunostaining to loxl . patients with severe pop showed reduced expression of proteins regulating collagen and elastin biogenesis. our finding raises the possibility that failure of ecm homeostasis could underlie the etiology of pop in women. fibroblast proliferation is regulated by hoxa : molecular implications for pelvic organ prolapse. kathleen a connell, marsha k guess, richard bercik, hugh s taylor. obstetrics, gynecology reproductive sciences, yale university school of medicine, new haven, ct, usa. objectives: the integrity of the extracellular matrix (ecm) is maintained by a delicate balance between collagen synthesis and degradation. previously, we have demonstrated that hoxa is essential for the development of the uterosacral ligament in mice and regulates the expression of collagen type iii and mmp . we have also shown that hoxa is deficient in the uterosacral ligament of women with pelvic organ prolapse (pop) compared to women with normal support. the exact mechanisms by which hoxa regulates pelvic organ integrity and repair remains to be elucidated. the aim of this study was to determine the effect of hoxa expression on fibroblast proliferation, and its potential role in pop. methods: nih t cells, a murine fibroblast cell line, were seeded onto a six well plate ( x cells/well) and transfected with either a vector carrying a hoxa cdna or empty vector alone as a control. immunohistochemistry using bromodeoxyuridine (brdu) was performed to evaluate cell proliferation. cell division in the uterosacral ligament (usl) was also compared in women with and without pop. usl specimens were obtained at the time of hysterectomy for benign disease. immunohistochemistry was performed on paraffin embedded sections of the usl to evaluate expression of two mitotic markers, cyclin b and phospho-histone . results: constitutive expression of hoxa in murine fibroblasts resulted in significantly higher proliferation. cells transfected with hoxa had a mean brdu incorporation of . + . cells/ cells compared with . + . cells/ cells in controls (p= . ). in the usl obtained from women with and without pop, cell proliferation as determined by cyclin b and phospho-histone expression was not significantly different. cyclin b and phosphor-histone were expressed in comparable numbers of cells in both groups. conclusion: hoxa is necessary for usl development and promotes proliferation of adult fibroblasts. hoxa may have a similar function in vivo in usl, and may regulate fibroblast proliferation during growth and the acute phase response following trauma when fibroblasts are activated to proliferate and remodel the ecm. it is likely that hoxa mediated proliferation of usl fibroblasts contributes to the tensile strength and resilience of these structures and thereby prevents pop. supported by nichd wrhr: k hd - . connective tissue composition, in term of collagen i, iii and proteoglycans content, in support and nonsupport structures of women with uterine prolapse. elisabetta trabucco, gennaro acone, sara d'avino, marco torella, gennaro trezza, annamaria marenna, nicola colacurci, luigi cobellis. department of obstetrics and gynecology, second university of naples, naples, italy; loreto mare hospital, naples, italy. objective. connective tissue consists mainly of collagen and elastic fibers, glycoproteins and proteoglycans (pgs) and is considered an important factor of the support and nonsupport structures of the genitourinary region. it has been already demonstrated altered morphologic features in the pelvic support connective tissue in women with genital prolapse. however, analysis of nonsupport tissue may provide a more accurate reflection of body collagen. the objective of our study was compare the expression of collagen i, iii and four pgs (decorin, fibromodulin, lumican, biglycan), essential for synthesis and regular assembly and diameter of the collagen fibrils, in uterosacral ligaments and in a nonsupport tissue, the uterine cervix, between premenopausal women with uterine prolapse respect to age matched controls. we characterized uterosacral ligaments and uterine cervix of premenopausal women with uterine prolapse and controls. this immunohistochemical study was performed on paraffin-embedded sections. results. uterosacral ligaments of the prolapsed uteri are characterized by a higher expression of collagen iii, decorin, fibromodulin and byglican and a lower quantity of collagen i. no differences in the immunoreactivity of lumican between the two patients groups. the abnormalities of support connective tissue composition are not observed in the uterine cervix of patients with prolapse. conclusions. our results suggest an altered remodeling of connective tissue in the ligaments of premenopausal patients with prolapse, with a significant decrease in collagen i content and an increase in collagen iii and pgs expression . in the prolapse patients this abnormal collagen metabolism and organization, mainly related to the observed change in pgs expression, might affect significantly the tensile strength of the connective tissue and consequently the support that is provided by the suspensory apparatus to the uterus. the alterd remodeling of support tissues, not detectable in nonsupport tissues, may suggest a predominant role of local biomechanical stresses (childbirth, chronic straining) in the pathogenesis of prolapse respect to systemic connective tissue deficiency. objective: to achieve vaginal delivery with minimal maternal injury, vaginal smooth muscle (sm) contractility likely decreases. the objective of this study was to characterize changes in rat vaginal sm contractility in pregnancy that affords circumferential vaginal distension. methods: a tubular segment of the vagina of virgin, late pregnant , immediate and late post vaginal delivery rats were mounted in an organ bath between a force transducer and an adjustable support block. dose response curves to norepinephrine (ne) and potassium (k+) were used to assess contractility. relaxation capacity was determined in ne preconstricted vagina, using cumulative doses of sodium nitroprusside (snp). differences in active vs. passive length-tension curves were used to measure sm contractility relative to the vaginal wall forces. sm -actin levels were measured using quantitative confocal immunofluorescence. results: cumulative doses of ne induced a maximum constriction force of . ± mn/g in virgin vagina while no measurable force was generated in response to ne in late pregnant or postpartum vaginas. virgin vagina relaxed with cumulative doses of snp; no measurable relaxation response was observed from pregnant or postpartum animals. in the presence of the high dose k+ ( mm), virgin vagina generated the greatest contractile force ( . ± mn/g) vs. late pregnant vagina ( . ± . mn/g, p< . ) or immediate post partum vagina ( . ± mn/g, p= . ). four week postpartum k+ induced forces were similar to virgin levels ( . ± mn/g). sm force generation (difference in active vs. passive length tension curves at mm of displacement) was decreased in late pregnancy ( . ± mn/g) compared with virgin ( ± mn/g, p < . ) and week postpartum vagina ( ± mn/g, p < . ). the expression of sm -actin was lowest in late pregnancy ( ± . , p < . ) and immediate postpartum vagina ( ± . , p < . ) relative to virgin ( ± ) with a return to virgin levels week postpartum ( ± ). conclusions: vaginal sm contractility diminishes in pregnancy. the altered contractility is mirrored by a decrease in sm -actin protein expression. near complete recovery to pre-pregnancy levels occurs by weeks post partum. these functional and biochemical changes likely represent maternal adaptations designed to minimize trauma during passage of the fetus(es). background: diagnosis or exclusion of endometriosis usually requires laparoscopy and peritoneal biopsy. we have described a novel and consistent observation of small nerve fibers in the functional layer of eutopic endometrium in women with endometriosis (tokushige et al, ) . these nerve fibers are not present in women without endometriosis. this finding allows the possibility endometriosis in the nude-mouse and inhibited mmp- expression in human endometrial stroma. the present findings may lead to the development of novel treatments of endometriosis involving statins. supported by nih u hd . k-ras actived immunocompetent retrograde menstruation model of endometriosis. ching-wen cheng, , di licence, , cristin g print, , d stephen charnock-jones. , pathology, university of cambridge, cambridge, united kingdom; obstetric gynaecology, university of cambridge, cambridge; department of molecular medicine pathology, university of auckland, auckland, new zealand. objective: to establish a new murine model of endometriosis that mimics the retrograde menstruation theory using immuno-competent mice. method: ovariectomised donor female k-ras v +/-/cre +/+ /rosa r-lacz +/+ transgenic mice were treated sequentially with steroid hormones. to induce decidualization and activation and k-ras transgene, mg/kg b-nf dissolved in maize oil was injected into the uterine lumen. tissue degeneration mimicking menstruation was induced by hormone withdrawal. menstruating endometrial tissue was collected from donor mice hours after last hormone treatment, re-suspended in matrigel, and implanted subcutaneously in female c bl/ recipients. immunohistochemical and morphometric methods were used to characterize the endometriosis-like lesions. microarray analysis (illumina, n= in each group), was used to study the molecular changes in "menstruating" uteri following k-ras activation. result: simple transplantation of decidualised endometrial tissue into immunocompetent animals does not lead to endometriotic lesion development but using tissue with the genetic modification described here overcomes this. viable endometriosis-like lesions are visible days after implantation. these lesions are histologically similar to those seen in man with intact glands, functional blood vessels, leukocyte infiltration and collagen deposition. statistical analysis revealed that transcripts were differentially regulated in the ras activated and control tissue. gene ontology (go) analysis indicated that transcripts associated with epithelial cell function and differentiation were over represented within this gene set (chloride transport and epidermis development) as were those associated with the acute inflammatory response and neutrophil chemotaxis. we developed a new model of endometriosis using immunocompetent mice to mimic retrograde menstruation induced endometriosis. this permits for the first time, the ready use of transgenic and knock-out tools to investigate the cellular and molecular mechanisms underlying endometriosis. since the animals have an intact immune system it also allows the testing of therapeutic agents that modify the inflammatory response. stra is expressed and induced by progesterone in the endometrium and is absent in endometriosis. mary ellen pavone, serdar e bulun, scott s reierstad, joy innes, magdy p milad, you-hong cheng. obstetrics and gynecology, northwestern univeristy, chicago, il, usa. objective: retinoic acid (ra) plays important roles in development, growth and differentiation by regulating the expression of target genes. in the mouse endometrium, ra deficiency leads to hyperkeratinization, while high concentrations of retinoids promote secretory characteristics. this leads many to believe that ra mediates important actions of progesterone in the endometrium and may account for progesterone resistance in endometriosis. the mechanism for regulation of ra production by progesterone is unknown. moreover, the conversion from retinol to retinoic acid is not different between normal endometrium and endometriotic tissue. we hypothesize that retinol intake into cells rather than conversion of retinol to ra is the critical step that determines ra activity in the endometrium and endometriosis. stra , a widely expressed multitransmembrane domain protein and member of a group of "stimulated by retinoic acid" genes, has been identified as the retinol binding protein receptor. it is strongly expressed in adult mice in several tissues including the female genital tract. we hypothesize that ra activity in the endometrium may be regulated by stra . here we investigate the differential expression of stra in the endometrium and endometriosis. design: we studied primary stromal cells isolated from the eutopic endometrium of disease free women and walls of ovarian endometriomas from women with endometriosis with respect to stra expression and regulation by progesterone. materials and methods primary culture of eutopic endometrial and endometriotic stromal cells were treated with - m estradiol (e ), - m r , a progesterone agonist, or vehicle for hours. real-time pcr was employed to quantify stra mrna levels. we treated cells for hours and quantified protein levels by immunoblotting. results basal mrna of stra levels in endometrial cells (n= ), were > , fold higher than those in vehicle-treated endometriotic cells (n= , p<. ). in endometrial stromal cells (n= ), r stimulated stra mrna levels by . - fold. r induced stra protein levels in endometrial stromal cells (n= ). conclusions stra is highly expressed and regulated by progesterone in endometrial stromal cells, whereas it is practically undetectable in endometriotic cells. stra may mediate important actions of progesterone in endometrium. upregulation of aromatase expression in endometrial cells disseminated into the peritoneal cavity (pc) may influence their survival and persistence via local estrogen synthesis. the inducing factors for the upregulation of aromatase in endometriosis are not well defined, but increased expression of sf- has been suggested to play an important role. given that the aromatase substrate androstenedione (a ) is the predominant steroid in peritoneal fluid, we aimed to determine whether a has a role in the regulation of aromatase expression in human endometrium. we found that culture of primary endometrial stromal cells and explants with a ( - nm) for h significantly upregulated expression of aromatase mrna transcripts containing exon iia at their '-ends. moreover, in human endometrial surface epithelial cells (hes), dose-response studies with a ( - nm) revealed that nm a maximally upregulated expression of both aromatase and sf- . when tissue samples were evaluated from women with endometriosis and control endometrium, expression of aromatase mrna mirrored sf- . treatment of hes cells ( h) with tritiated a demonstrated its metabolism to estradiol (e ), testosterone (t), dihydrotestosterone (dht) and androstanediol (a-diol). although equimolar concentrations of a , t and e upregulated aromatase and sf- mrna expression in hes cells, the nonaromatizable androgens, dht and a-diol, had no effect. a positive feedback role of estrogen in aromatase upregulation was suggested by the finding that the estrogen receptor antagonist, ici , , markedly diminished aromatase and sf- mrna expression induced by a . finally, chromatin immunoprecipitation revealed that a significantly enhanced recruitment of sf- to its response element (- bp) upstream of cyp exon iia. collectively, our findings strongly suggest that exposure of endometrial cells within the pc to c -steroids may cause an acute upregulation of cyp gene expression through their aromatization to estrogens. thus, estrogen may play a critical positive feedback role in the pathogenesis of endometriosis. supported by nih r -dk . the hpa axis and depression/anxiety scores in chronic pelvic pain patients. b stegmann, b frank, j gemmill, g chrousos, m ballweg, p stratton. rbmb, nichd/nih, bethesda, md; som, wake forest university, winston-salem, nc; endometriosis association, milwaukee, wi. stress, pain, anxiety and depression adversely affect the hypothalamic-pituitaryadrenal (hpa) axis. we examined the influence of depression and anxiety on the hpa axis response to corticotropin-releasing hormone (crh) testing in women with and without chronic pelvic pain (cpp). methods: healthy women, aged - , with without cpp, with regular menses and off hormonal contraception were studied. none had pelvic infections, untreated depression, manic depression, fibromyalgia, or chronic fatigue syndrome. after ovine crh injection ( mcg/kg), serial blood samples (- , , + , + , + minutes) were obtained for acth and cortisol measurements. depression and anxiety were scored using the duke quality of life questionnaire. hpa response was abnormal if peak acth levels were > pg/ml without a rise in cortisol levels. subject groups were: no pain and normal acth response (npnr), pain and normal acth response (pnr), no pain with an abnormal acth response (npar) and pain with an abnormal acth response (par). student t-test was used for comparisons. results: women ( cpp, controls) had a mean age of . ± . years (range: - ). women responded normally ( pnr, npnr) and women responded abnormally ( par, npar). peak and absolute rise in acth were significantly higher in abnormal (a) vs normal (n) responders; (peak: . a vs . pg/ml n, p< . ; absolute: . a vs . pg/ml n, p= . ). there was a significant difference within groups: peak acth: . par vs . pg/ml pnr, p= . ; . npar vs . pg/ml npnr, p< . ; absolute acth: . par vs . pg/ml pnr, p= . , . npar vs . pg/ml npnr, p< . ). however, total cortisol level was not different between or within the groups ( . a vs . mcg/dl n, p= . ). mean depression score did not differ among cpp patients ( . par vs . pnr, p= . ), but differed among controls ( npar vs . npnr, p= . ). anxiety scores did not differ between or within groups ( . npar vs . npnr p = . ; par vs . pnr, p= . ). conclusions: chronic pelvic pain is associated with an abnormal hpa response, regardless of anxiety or depression symptoms. abnormal hpa responses in control women, however, appear to be influenced by depression. the mechanism and clinical significance of these findings should be explored. support: rbmb/nichd/nih and endometriosis association. objective: the eutopic endometrium in women with endometriosis demonstrates altered endometrial receptivity and altered gene expression. it is unknow if the endometrium is defective giving rise to a predisposition toward endometriosis or alternativly if the endometriosis causes the altered endometrial receptivity. here we created experimental endometriosis in a mouse model through allotransplantation of the uterus to the peritoneal cavity in immunocompetent mice and examined the expression of several markers of endometrial receptivity in the eutopic endometrium. materials and methods: the uterus of -week-old cd female mice was transected at cervicovaginal junction and each horn divided and transplantated into the abdomidnal cavity of eight cd mice. seven controls received sham surgery only. after weeks the uterus was removed, snap-frozen in trizol. total rna was extracted and cdna generated. quantitative real time rt-pcr using sybrgreen was performed and normalized to -actin. fold changes in normalized hoxa , hoxa , bteb , emx , igfbp , integrin - , total progesterone receptor (pr-ab) and progesterone receptor-b (pr-b) were assessed. all experiments were conducted in duplicate, repeated at least three times and compared using mann-whitney rank sum test. results: hoxa , hoxa , bteb , emx , and igfbp mrna expression showed . -fold (p= . ), . -fold (p< . ), . -fold (p= . ), . fold (p= . ), and . -fold (p< . ) decrease in the uterus of mice with experimental endometriosis compared with controls. pr-ab and pr-b mrna showed . -fold (p= . ) and . -fold (p= . ) increase in endometriosis group compared to controls, respectively, however the ratio of pr-b to pr-ab (b/ab) showed no significant change in both groups ( . vs. . , endometriosis vs. control, p> . ). there was no significant change in integrin - mrna expression ( . -fold, p> . ). conclusion: we demonstrate significant changes in multiple markers of endometrial receptivity in eutopic endometrium after induction of endometriosis. these findings suggest that origionally normal endometrium can develop defects with the creation of endometriosis; an abnormal endometrium is not a prerequisite for the development of endometriosis or associated abnormalities. this data also suggest the existance of a signal conduction pathway from endometriosis that alteres endometrial gene expression. endometrial expression patterns of relaxin and relaxin receptor mrna suggest involvement of relaxin in endometriosis. sara s morelli, felice petraglia, gerson weiss, stefano luisi, pasquale florio, jeff gardner, andrea wojtczuk, laura t goldsmith. obstetrics, gynecology and women's health, new jersey medical school, newark, nj, usa; pediatrics, obstetrics and reproductive medicine, university of siena, siena, italy. objective: in normal endometrium, relaxin is a potent inhibitor of matrix metalloproteinases, which have been implicated in the invasive process of endometriosis. we tested the hypothesis that relaxin plays a role in endometriosis by comparing expression of relaxin and its lgr receptor in normal human endometrium to levels in samples from patients with endometriosis. materials and methods: total rnas, extracted from ectopic (n= ) and eutopic (n= ) endometrium of patients with endometriosis and from endometrium of normal controls (n= ), were reverse transcribed into cdnas. real-time pcr was performed using primers previously shown to identify human lgr relaxin receptor mrna, h relaxin mrna, and beta-actin mrna, with sybr-green detection of double stranded dna products. the comparative c t method ( -ct ) determined relative lgr and relaxin mrna expression (normalized to beta-actin mrna expression). relaxin mrna was detectable in normal endometrium from of ( %) control patients. in contrast, relaxin mrna was detectable in a lower proportion of samples [ of ( . %)] from patients with endometriosis, among whom relaxin mrna was detectable in a lower proportion of ectopic samples [ of ( %)] than in eutopic samples [ of ( . %)]. lgr relaxin receptor mrna was detectable in all samples, with lower expression in endometriosis samples than in endometrium from normal controls. relaxin receptor lgr mrna levels vary with cycle phase, with greater expression in the secretory phase (sp) than in proliferative phase (pp): in normal controls . -fold higher levels in the sp than pp; and in endometriosis patients . -fold higher levels in the sp than pp. in both phases, lgr mrna levels were lower in ectopic samples than in either eutopic samples ( . -fold lower in pp and . -fold lower in sp) or endometrium from normal controls ( . -fold lower in pp and . -fold lower in sp). eutopic endometrium had similar lgr mrna expression to controls throughout the cycle. decreased local expression of relaxin and relaxin receptor mrna in ectopic endometrium from patients with endometriosis throughout the menstrual cycle suggests that relaxin may be protective against endometriosis. ovarian reserve after laparoscopic cystectomy of endometriotic ovarian cysts. shinichi hayasaka, takashi murakami. obsterics and gynecology, tohoku university, sendai, japan. objective laparoscopic ovarian cystectomy is generally recommended for endometriotic ovarian cysts because it has been associated with a higher pregnancy rate and a lower recurrent rate. however, residual ovarian function after laparoscopic cystectomy of endometriotic ovarian cysts has been questioned. in this study, we retrospectively evaluated ovarian response to hyperstimulation in women selected for ivf and icsi cycles who previously underwent laparoscopic cystectomy of a monolateral endometriotic ovarian cyst. methods a retrospective review of the patients between january and september was performed. the operated ovary and contralateral intact ovary were compared in terms of number of oocytes retrieved, number of follicles with a mean diameter > mm at the time of hcg administration. the patients who had recurrent endometriotic ovarian cysts were excluded. in total, ten patients were identified. the mean(±sd)number of oocytes retrieved was . ± . in the control ovary and . ± . in the previously operated ovary(p= . ). the mean(±sd)number of follicles with a mean diameter > mm was . ± . in the control ovary and . ± . in the previously operated ovary(p= . ). the age of patients and diameter of the operated ovary had little influence on the difference between the response of the control ovary and one of the previously operated ovary. laparoscopic cystectomy of endometriotic ovarian cysts is associated with a significant reduction in ovarian reserve. background pre-eclampsia (pe) is associated with systemic maternal endothelial dysfunction, which is thought to result from the presence of circulating factors released following placental damage. it is hypothesised that this occurs as a result of reduced oxygen (o ) delivery. some features of placental pathology seen in pe, such as increased apoptosis, can be reproduced by culture of placental explants in % o . metabolomics operates to study 'all' metabolites within a biological system. this strategy has previously identified differences in maternal plasma between normal pregnancies and those complicated by pe. in these experiments we used metabolomics to investigate differences in the metabolic profile of explants cultured in different o tensions. hypothesis the metabolic profile of placental villous explants is altered by culture in different o tensions. methods placental villous explants were cultured with either % serum (n= ) or in serum-free conditions (n= ) for h in %, % and % o . after h, conditioned culture medium and tissue were collected and snap frozen. tissue was homogenised in % ice cold methanol prior to analysis. samples were analysed using gas chromatography-mass spectroscopy (gc-ms). samples cultured at %, % and % o were compared to identify concentration differences in metabolites in conditioned cultured medium and tissue lysate. kruskal-wallis test was used for statistical analysis. due to the large number of metabolites identified, a p value of . was considered significant. results the mean intra-assay variability was . %. there were no differences in the metabolic profile of conditioned culture medium from % and % o . several metabolites differed in culture medium from % compared to % and % o including: deoxyribose, glycerol and threionic acid. these changes were present in both serum and serum-free conditions. using these metabolites alone, culture in % o could be completely discriminated from % and % o . deoxyribose was also elevated in tissue lysate from % o compared to % o . conclusion this metabolomic strategy can identify differences in metabolic profile in placental tissue cultured in % o . these novel compounds may provide further insight into pathophysiology of pe. objective a strategy of metabolic footprinting (the study of extracellular metabolites, which are related to intracellular metabolism) was used to detect a wide array of low molecular weight metabolites. metabolic profiles were employed to differentiate between placental explants cultured at different oxygen tensions. two separate studies were combined to validate initial observations. methods metabolic footprints of placental villous explants, obtained from uncomplicated pregnancies, (n= ) were cultured for h in %, % and % oxygen. conditioned culture medium was then collected and frozen, prior to analysis by uplc/ltq-orbitrap mass spectrometry in both negative and positive ion mode. samples cultured at %, % and % were compared to identify differences in the metabolic profiles. this procedure was repeated for different explants and the results compared across the studies in order to validate initial findings. approximately unique peaks were detected in both sample sets in negative ion mode and peaks in positive ion mode. a number of metabolites were identified as being significantly different using kruskal-wallis (pval< . ) under different oxygen tensions. some differences were seen between the results obtained from both runs due to separate batch preparation of the medium but good reproducibility was obtained for many metabolites between batches. metabolites of biological interest included amongst others -deoxyribose, valine, tyrosine and aspartic acid. the largest differences were those seen between o % and o %. comprehensive metabolic profiles were detected and employed to identify differences in the metabolic footprints of explants cultured under differing oxygen conditions. a number of biologically interesting metabolites were characterized. objective: brain weight and dna content were reduced in leptin deficient (lep ob /lep ob ) mice postnatally. leptin is detected in the sera and its receptors are expressed in the brain of the mouse fetus. we examined the role of leptin in the proliferation and differentiation of neural lineage cells in the mouse fetus. methods: the number of total cells in the cerebral cortex was compared between (lep ob /lep ob ) and wild type fetuses from embryonic day (e) to . the number of brdu positive cells was also compared on e and e . brdu uptake, the ratio of viable colony number to plated cell number, the proportion of multipotent, neuronal or glial progenitor colonies, and the expression levels of hes mrna were compared between leptin-and non-treated neurosphere cells. moreover, we examined stat phosphorylation by leptin stimulation with elisa to investigate whether or not jak/stat pathway transduces the leptin signal in the prenatal period as in the adult. results: lep ob /lep ob fetuses had reduced total cell numbers in the ventricular zone (vz) on e and e , and in the cortical plate on e . the number of brdu-positive cells was reduced in vz of e and e lep ob /lep ob fetuses. brdu uptake and the number of viable colonies were increased by days-leptin treatment in the neurosphere culture. the proportions of glial-restricted and multipotent precursor colonies were increased by leptin, whereas that of oligodendrocyte precursor colonies were decreased. hes mrna expression was enhanced in neurosphere cells by leptin. neither the amount of phosphorylated stat nor that of stat was increased in neurosphere cells by leptin stimulation. conclusion: our study suggests that leptin maintains the neural stem and glial-restricted precursor cells through upregulation of hes expression and increases the number of cerebrocortical cells in the mouse fetus, however, jak/stat pathway does not mediate the leptin signal in undifferentiated neural lineage cells. a vaginal wall, and > % of fib- knockout (ko) mice develop pelvic organ prolapse by weeks of age. in contrast, fib- and - deficiencies do not result in prolapse, and fib- kos die shortly after birth. herein, we evaluated the role of fib- in pelvic organ support. methods: two observers serially measured the degree of vaginal, perineal, and anal prolapse in fib- ko (fib -/-) and in fib -/-mice severity of prolapse was significantly related to age, but not parity, and the average age at diagnosis was wks. fib -/-animals with advanced prolapse had attenuated uterosacral ligaments and descent of the bladder and uterus caudal to the symphysis. pro-mmp ( -fold, p= . ), active mmp ( -fold, p= . ), and prommp ( -fold, p= . ) were increased in vaginal tissues from mice with gross prolapse compared with age-, strain-, and cycle-matched controls. this increase in protease activity, however, was also accompanied by increased expression of fib- and tropoelastin ( . -fold, p< . ). regardless of prolapse maternal morbidtties tf ards-n(%) ( ) acute ren~m f lure-n (%) ( ) car~tovascular ~ysfunrtion-n ("/o) ( ) hepatc fa..lure-n (%) dt sst'dlm atl:d inlfavascul..-coagul tipalhy-n(%) ( ) durallon of !cu suy (days. mean+sd) :t . hospital my (days.. me +sd) . condon, j. c., hardy, d. b., kovaric, k., and mendelson, c. r. ( ) mol endocrinol ( ), - . objective: innervation of the uterine cervix is important for the process of parturition (physiol beh : , ; j histochem cytochem : , jsgi : , ) . the hypogastric nerve is a major pathway that innervates the uterine cervix, yet its contribution to processes associated with cervical ripening and parturition is not known. the objective of this study was to determine the effect of hypogastric nerve transection on processes associated with cervical remodeling and parturition. methods: time-dated pregnant rats were sham-operated or the hypogastric nerve bilaterally transected just anterior to the inferior mesenteric ganglion on day post-breeding. live pups were born spontaneously by all rats at term on days - post-breeding. on the day of birth, the cervix was excised, postfixed overnight, sectioned, and processed to evaluate collagen content and structure (nih image j). sections were also processed by immunohistochemistry to assess cell nuclei density, the census of resident macrophages, and area of tissue that contained nerve fibers. results: hypogastric neurectomy did not affect cell nuclei density, the number of macrophages, or density of nerve fibers in the cervix. the failure of hypogastric nerve transection to affect indices of cervical remodeling is consistent with the finding that duration of pregnancy and timing of birth were not different in sham-operated and hypogastrectomized rats. conclusions: nerve fibers in the hypogastric nerve that innervate the lower uterus and cervix, including sympathetic and neuropeptidergic projections, are thus not essential for birth. these novel findings provide support for the contention that innervation of the uterine cervix other than through the hypogastric nerve contributes to processes associated with cervical ripening and parturition. receptor system in prostaglandin synthesis in pregnancy. eugenie r lumbers, , della m yates, carolyn m mitchell, jonathan j hirst, , tamas zakar. , school of health sciences, university of newcastle, newcastle, nsw, australia; mothers and babies research centre, hunter medical research institute, newcastle, nsw, australia; obstetrics and gynaecology, john hunter hospital, school of medical practice and public health, university of newcastle, newcastle, nsw, australia. the fetal membranes and decidua contain prorenin, which requires proteolytic activation. ngyuen (j clin invest. : ) described a renin/prorenin receptor that conformationally activates prorenin, so that it can form ang i from aogen. in addition, receptor-bound prorenin can stimulate intracellular pathways directly. prostaglandins (pgs) participate in the control of term and preterm labour, and renin can stimulate pg production by amnion and decidua when angiotensin's action is blocked mitchell placenta : ) . the prorenin receptor may mediate these effects. our aim was to find out if the prorenin receptor gene is expressed and colocalised with prorenin and prostaglandin h synthase- (pghs- ) in human placenta, amnion, chorion and decidua. we have also determined if there is any change in the expression of the prorenin and prorenin receptor genes with labor. placentae with attached membranes were collected after term birth either by elective caesarian section or by spontaneous labor, and prorenin, prorenin receptor and pghs- mrna levels were quantitated relative to beta-actin mrna by real-time rt-pcr in amnion, chorion, decidua and placenta. before labor, mean prorenin mrna levels were . times higher in decidua and . times higher in chorion and placenta than in amnion (p< . ). there were no significant changes with labor. proenin receptor mrna was expressed in all tissues. proenin receptor mrna levels in prelabor amnion, chorion and placenta were similar, while levels in decidua were % of those in amnion (p= . ). this low level of prorenin receptor mrna in decidua persisted after labor (p< . ). pghs- mrna expression was highest in amnion; in decidua and placenta it was only and % of amnion. similar differences were present after labor. we conclude that the decidua is the principal source of prorenin within the pregnant uterus, and all gestational tissues are targets for prorenin. decidual prorenin may affect pghs- expression in amnion through the prorenin receptor forming a maternal-fetal paracrine system that stimulates pg production at labor. the immuno-suppressive effects of progesterone on umbilical cord blood mononuclear cells and the effect of culture media estradiol content. nadav schwartz, xiangying xue, christine n metz. obstetrics and gynecology, nyu school of medicine, new york, ny, usa; feinstein institute for medical research, manhasset, ny, usa. objective: progesterone (p ) is known to supress the maternal immune response and similar effects have been seen with fetal cells. we sought to ascertain whether p action was modulated by the phenol red and/or estrogen content of the culture media. methods: umbilical cord blood mononuclear cells were isolated using a density gradient centrifugation. the cells were incubated with p ( - m) hour prior to overnight stimulation with lps. supernatants were assayed for tnf-using elisa. the following culture media were used: a)'red' media: rpmi+ % fbs, b)'yellow' media: phenol red-free rpmi+ % fbs, and c)'clear' media: phenol-free rpmi+ % dextran/charcoal treated fbs. finally, the 'clear' media experiments were repeated with estradiol add-back. results: p suppressed tnf production in all medias. tnf levels were suppressed to . % (p< . ) of controls using 'red' media and to . % (p< . ) in 'yellow' media. the degree of suppression was not as substantial using 'clear' media where tnf levels were . % (p< . ) of control. (fig ) adding estradiol to the 'clear' media had little or no effect on the degree of tnf suppression. (fig ) conclusions: progesterone exerts an immuno-suppressive effect on lpsstimulated fetal mononuclear cells which is more pronounced when using media containing untreated fetal bovine serum. the phenol-red/estradiol content of the culture media did not modulated the p effect. dextran/charcoal treatment of fbs appears to deplete the media of factors other than estradiol that are necessary for p to exert its full effects. culture conditions should be optimized when studying progesterone's effects on immune response. ovine fetal regional blood flow following prenatal dexamethasone (dex) at . gestation (g). antonine d van heesewijk, jan g nijhuis, susan l jenkins, peter w nathanielsz, mark j nijland. ob/gyn, academisch ziekenhuis maastricht, maastricht, netherlands; ob/gyn, cpnr, uthscsa, san antonio, tx, usa. background: pregnant women at risk for premature labor receive antenatal glucocorticoids (gc) to reduce neonatal morbidity and mortality. while fetal blood pressure (bp), heart rate and vascular endothelial and control fetuses (n= ). dispersed adrenal cortical cells ( . x cells/tube; in duplicate) were challenged with - m acth. samples were collected at time (baseline), , , , and min after acth treatment, and tissue and media samples were frozen for determination of cortisol, camp, and star. results: cortisol output (ng/ . x cells) was higher in the lth group compared to the control, (p< . ) at min ( . ± . vs. . ± . ), min ( . ± . vs. . ± . ), and min ( . ± . vs. . ± . ). peak camp levels (fmol/ . x cells) were observed at min but did not differ between control ( . ± . ) and lth ( . ± . ) adrenal cells. western analysis demonstrated that star protein expression was higher in the lth adrenal cortex compared to control (p< . ) at min ( . ± . vs. . ± . ), and at min ( . ± . vs. . ± . ), relative optical density units. conclusions: results from the present study taken together with those of previous in vivo studies suggest that the enhanced cortisol output in lth fetuses is the result of increased adrenal acth sensitivity. this enhanced sensitivity is not due to differences in camp generation. star, which is a key element involved in cholesterol transfer at the level of the mitochondrial membrane appears to play a key role in the enhanced cortisol response to acth in the lth group. (nih grants hd , hd and llu-nih imsd r gm - ). expression. hiroaki itoh, makoto kawamura, shigeo yura, haruta mogami, tsuyoshhi fujii, norimasa sagawa. obstetrics and gynecology, national hospital organization osaka national hospital, osaka, japan; gynecology and obstetrics, kyoto university graduate school of medicine, kyoto, japan; obstetrics and gynecology, mie university school of medicine, tsu, japan. objective: epidemiological evidences suggested that undernutrition in utero develops risk factors for adult cardiovascular disorders, such as blood pressure increase. the present study was designed to prove the hypothesis that maternal iso-caloric high protein diet alleviates blood pressure increase in the adult offspring by affecting placental beta-hydroxysteroid dehydrogenase type ( beta-hsd ) expression. methods: the % calorie restriction was applied to pregnant mice by using either standard protein diet (spd; % casein protein; spd-un) or high protein diet (hpd; % casein protein; spd-un). in some groups, these pregnant mice were sacrificed at . d.p.c.; then maternal and fetal plasma as well as placental tissues were corrected. while in other groups, systolic blood pressure was measured in the offspring at wks. fetal and maternal corticosterone levels were measured by elisa. the placental beta-hsd gene expression was measured by quantitative taqman pcr. results: systolic blood pressure in the spd-un offspring at weeks ( mmhg) was higher than that in spd-nn offspring ( . mmhg). by contrast, systolic blood pressure in the hpd-un offspring ( . mmhg) was similar to that in spd-nn offspring. maternal calorie restriction with spd and hpd caused similar elevation of maternal plasma corticosterone concentrations. by contrast, fetal plasma corticosterone levels in hpd-un offspring ( ng/ml) was lower than those in spd-un offspring ( ng/ml), indicating the amelioration of fetal exposure to high corticosterone by maternal hpd. the placental beta-hsd gene expression in the hpd-un was % higher than that in spd-un, suggesting that maternal hpd augmented placental beta-hsd gene expression in the placenta and probably facilitated the inactivation of maternal cortocsterone in the process of placental transfer to fetal circulation. conclusion: the preset study suggests that maternal high protein ingestion may elevate placental beta-hsd gene expression and ameliorate blood pressure increase in the adult offspring via modification of fetal exposure to maternal corticosterone. elevated cortisol levels at birth exert critical maturational effects through action at glucocorticoid receptors, gr. in contrast, the low cortisol concentrations in the preterm fetus, before the time of increased fetal cortisol secretion, are near to the kd for cortisol at the higher affinity mineralocorticoid receptor, mr. we hypothesized that endogenous cortisol in the fetus exerts physiologic actions at mr in tissues without appreciable cortisol-inactivating enzyme, hsd , including the fetal lung and brain. we therefore tested the effect of infusion of a mr-antagonist, ru (mra, . mg/h over h) into - day fetal sheep. we tested for effects on lung liquid production rate, lung liquid and plasma electrolytes, plasma acth and cortisol, and hematocrit at - h after start of the infusion. although there was no significant difference in the liquid production rate in fetuses infused with ru , the ratio of na + to k + in lung liquid was significantly greater in mra-treated fetuses than in the control fetuses ( . ± . vs . ± . ). plasma acth was significantly greater in the mra-treated fetuses than in control fetuses at h ( ± vs ± pg/ml); in the mra-treated fetuses, acth concentrations at h were greater than in the same fetuses at h ( ± pg/ml), wheras there was no increase in plasma acth in the control fetuses ( h: ± pg/ml). similarly, plasma cortisol concentrations at and h were greater in the mra infused fetuses than in control fetuses ( h: . ± . vs . ± . ng/ml), and cortisol increased significantly over time in the fetuses infused with mra, but not in control fetuses ( h, mra: . ± . , control: . ± . ng/ml). infusion of mra did not alter fetal plasma electrolyte, fetal blood pressure or fetal heart rate. however, fetal packed cell volume was significantly increased at - h of infusion of mra ( h: ± vs ± %) and fetal pco was significantly greater at h of infusion of mra as compared to control fetuses ( . ± . vs . ± . ). these results suggest that endogenous cortisol concentrations in the preterm fetus exert negative feedback control of acth via action at mr in the fetal brain, and play a role in regulation of fetal lung liquid composition and in fetal fluid balance. preparturient increases in fetal acth secretion are cyclooxygenase- (cox- ) dependent. charles e wood. dept. physiology and functional genomics, university of florida college of medicine, gainesville, fl, usa. maturation of the fetal hypothalamus-pituitary-adrenal axis is critical for the timely somatic development of the fetus and readiness for birth. in sheep, increased preparturient activity of the fetal hpa axis appears to be responsible for triggering parturition itself. this study was designed to test the hypothesis that prostaglandin generation, mediated by cox- in the fetal brain, is critically important for stimulation of the preparturient increase in fetal acth secretion. singleton fetal sheep were chronically catheterized with vascular, amniotic, and lateral cerebral ventricular catheters. nimesulide, a selective cox- inhibitor, was infused ( mg/day, n= ), and vehicle ( % dimethylsulfoxide, n= ) was infused. arterial blood samples were drawn from fetuses at hour intervals for measurement of plasma hormone concentrations and arterial blood gases. in the vehicle-treated fetuses, fetal plasma acth, pomc, and cortisol concentrations increased exponentially (p< . by anova) before spontaneous parturition at ± . days. in the nimesulide-treated fetuses, fetal plasma acth and pomc were constant and did not increase prior to parturition (p=ns by anova). in contrast, fetal plasma cortisol increased independent of acth (p< . by anova) and the fetuses were born spontaneously on ± . days gestation. the slight delay in spontaneous parturition in the nimesulide-treated fetuses was statistically significant (p< . by mantel-cox test). intracerebroventricular nimesulide infusion did not decrease fetal plasma concentrations of prostaglandin e , demonstrating that the action of the nimesulide was restricted to the fetal brain. fetal blood gases were normal in all of the fetuses, and there were no differences in blood gases between groups. we conclude that the spontaneous increase in fetal acth and pomc prior to parturition is cox- dependent. however, we also conclude that the increase in fetal plasma cortisol concentration can occur independent of increases in fetal acth, suggesting that the increase in cortisol can result from increases in adrenal sensitivity to acth. the results of this study demonstrate that the timing of parturition in the sheep is not dependent upon increased acth secretion, and the results suggest that parturition is regulated primarily by changes in adrenal sensitivity. expression of extracellular signal-regulated kinase / and p mitogen-antiphosphotyrosine. immunolocalization of inos was performed in the same tissues. hpmec were used to determine effect of sin- ( mm) on inos protein expression ( min, and h). results: inos protein was detected in microvascular endothelium, smooth muscle and syncytiotrophoblast of the placenta. inducible nos levels in placentas from severe preeclampsia were significantly decreased compared to normal (p< . ) but were not different from mild preeclampsia. aligning western blots of anti-phosphotyrosine and inos revealed a phosphorylated band corresponding to the molecular weight of inos. the proportion of tyrosine phosphorylated inos was reduced by % in severe preeclampsia compared with normotensive. preliminary data with ip for phosphotyrosine and crossblotting with inos confirmed this finding. sin- treatment decreased inos protein abundance at and h in hpmec. conclusions: our results demonstrate decreased tyrosine phosphorylation of inos in preeclamptic placenta. phosphorylation of tyrosine in inos has been reported to negatively regulate its activity. we therefore postulate that decreased phosphorylation of inos may be responsible for the increased catalytic activity of the enzyme that we have previously observed. the decreased levels of inos observed on sin- treatment may be due to nitration, an effect analogous to preeclampsia, where the presence of peroxynitrite has been well established. it remains to be seen if protein nitration has an effect on enzyme stability. objective: -isoprostane ( -iso), a prostaglandin-like compound formed in vivo, is a reliable measure of oxidative stress which occurs in response to many different stimuli, including infection. periodontal disease is a chronic oral infection associated with fetal growth restriction and preeclampsia. our objective was to determine if maternal periodontal disease is associated with oxidative stress as measured by -iso. methods: a secondary analysis was conducted using prospective data from the oral conditions and pregnancy study. a cohort of healthy women enrolled < weeks underwent oral health examination and serum sampling. maternal periodontal disease was categorized as healthy, mild, or moderate-severe by clinical criteria. maternal serum was analyzed for -iso by ultra-sensitive elisa. elevated -iso was defined by a value th %tile. maternal factors associated with elevated -iso were determined using chi-square or t-tests as appropriate. a logistic regression model was created to determine adjusted odds ratios ( th %ci) for elevated -iso. results: women had complete data for analysis. the median -iso level (iqr) was , ( - , pg/dl). using bivariate analysis, moderate-severe periodontal disease, non-white race, use of wic/food stamps, unmarried status, obesity, and lack of insurance were associated with elevated -iso. the logistic regression model for elevated -iso is shown below. adjusted or ( th ci)* mild periodontal disease . maternal periodontal disease and utilization of public assistance are associated with oxidative stress in pregnancy. the relationship between periodontal disease, social hardship, oxidative stress, and adverse pregnancy outcome remains to be determined. antioxidant therapy could represent novel therapy for the prevention of adverse pregnancy outcome. severe transient immunological thrombocytopenia in a preeclampsia patient whose bone marrow production of platelets had been restricted. toshihiro yoshimura. obstetrics and gynecology, ntt-west kyushu general hospital, kumamoto-city, kumamoto, japan. introduction: idiopathic myelofibrosis is a chronic myeloproliferative disorder in which clonal haemopoetic stem cell proliferation is accompanied by reactive fibrosis. case report: a -year-old primiparous woman was referred to our hospital for prenatal care after weeks of gestation. her medical history was significant for idiopathic myelofibrosis, and congenital agenesis of the spleen. the laboratory workup showed the patient's white blood cell count to be , / l; hemoglobin, . gm/dl; and platelet count, , / l. her bleeding time was normal ( min). until the th week, the patient's pregnancy was uneventful. during the th week, however, the patient's platelet count declined to , / l, when proteinuria became evident. her bleeding time was prolonged to min. the coagulation system was normal. by the th week, the patient's blood pressure was elevated to / mmhg, and her urinary protein excretion exceeded g/day. our initial diagnosis was thrombocytopenia due to bone marrow suppression. the patient had no history of platelet transfusion, but we expected it would increase the platelet count, particularly since in the absence of destruction by the spleen, the lifespan of the platelets would be prolonged. this was not the case, however. elective induction of labor was carried out after weeks. at the same time, the patient was transfused with units of platelets, but her platelet count remained unchanged ( , / l). it was then that we realized that immunological thrombocytopenia may be involved, and massive doses ( mg/kg) of gamma globulin were given intravenously for one day. thereafter, platelets ( units) were again transfused, this time raising the platelet count to , / l. cesarean section was promptly carried out under general anesthesia. we later found that the patient's serum platelet associated immunoglobulin (paigg) level was positive, though antiplatelet and antinuclear antibodies were negative. the postoperative course was uneventful. the maternal platelet count declined to the pretransfusion level within days after delivery, but gradually increased to the prepregnancy level by weeks. comment: this idiopathic myelofibrosis patient in whom bone marrow production of platelets had been severely restricted demonstrates that immunological destruction may play a major role in the development of thrombocytopenia in preeclampsia. early l-arginine therapy improves notably the fetal growth in preeclamptic women. a randomized controlled trial. keisy lopez-molina, juan c gonzalez-altamirano, julio e valdivia-silva. , oncoinmunología y biología vascular, facultad de medicina, universidad nacional san agustín, arequipa, peru; cardiología y cirugía de tórax, hospital nacional case essalud, arequipa, peru; inmunología, instituto de investigaciones biomédicas, méxico, distrito federal, mexico. objective: to assess the benefit of early administration to l-arginine, the precursor to nitric oxide, on fetal growth. methods: one hundred women with preeclampsia were randomized to receive either l-arginine or placebo until day postpartum. live singleton infants were born after preeclamptic pregnancies and compared those with other control infants. birth size was expressed as the ratio between observed and expected birth weights, and infants smaller than two standard deviations from expected birth weights were classified as small for gestational age (sga). all the women had neither previous precedents of the preeclampsia nor other factors that they cause sga. results: no significant differences existed between the groups with preeclampsia before randomization. preeclampsia was associated with a % ( % confidence interval [ci] %, %) reduction in birth weight. women with hellp syndrome had to leave the study. the risk of sga was three times higher (relative risk [rr] = . ; % ci . , . ) in infants born after preeclampsia without l-arginine therapy than in control pregnancies, and two times higher (relative risk [rr] = . ; % ci . , . ). in infants born after preeclampsia with l-arginine therapy. conclusion: fetal growth improve markedly with l-arginine therapy in women with preeclampsia. introduction: although the pathogenesis of severe preeclampsia (pe) and intrauterine growth restriction (iugr) is poorly defined, inadequate remodeling of uterine spiral arties may promote reperfusion injury leading to focal infarction and reduced nutrient flow between mother and fetus. our goal was to determine whether an intravillous inflammatory cytokine cascade was associated with pe and iugr. methods: immunohistochemistry (ihc) examined il- and il- expression in preterm placentas with severe pe+iugr, and idiopathic preterm controls (ptc) with no evidence of clinical or histological chorioamnionitis. cultures of fibroblasts (fibs) and syncytiotrophoblasts (scts) from term placentas (n= ) were treated with il- and cytokine levels in conditioned media were determined using elisa or multiplex array. results were analyzed by student's t test or anova. results: the gestational age at delivery was not significantly different in pe+iugr and ptc groups ( . ± . vs . ± . wks). ihc of pe+iugr samples revealed intense villus core staining of il- adjacent to infarcts. villi distal to infarcts stained with a lower intensity. in contrast, staining was virtually undetectable in ptc. the pattern of il- staining was nearly identical in pe+iugr and ptc groups, was localized to the syncytium, and did not differ with respect to distance from infarct. to identify cellular targets of il- action, primary cultures of fibs and scts were incubated for h in serum-free medium ± ng/ml il- . by elisa we observed that il- treatment of fibs increased il- levels from ± to ± pg/ g protein (p< . ). similarly, multiplex array revealed that levels of il- , il- , and il- were induced -, -, and -fold, respectively in fibs. the presence of ng/ml il- receptor antagonist reduced the il- -mediated increase in il- levels ± % (p< . ), indicating that this response was mediated through il- receptor. in marked contrast, il- treatment did not significantly affect levels of il- , il- , il- or il- in scts, indicating that this response was cell type-specific. conclusions: these results indicate that increased expression of il- in the placental villus core in pe and iugr may promote an inflammatory cascade in fibs at this site leading to focal infarction and reduced flow of nutrients to the fetus. introduction: in human pregnancy, decreased responsiveness to angiotensin ii (angii) starts in week , promoting an expanded vascular bed. at the same time levels of the vasodilatory hemopexin increases . during pre-eclampsia the vascular bed is contracted and the responsiveness upon angii persists. this may be due to the increased levels of extra cellular atp, a natural inhibitor of hemopexin . in the present study we tested whether extra cellular atp is toxic in the pregnant condition. methods: pregnant rats were infused with either atp ( g/kg bw; n= ) or saline (n= ) on day of pregnancy (permanent jugular vein cannula/ hr infusion). non-pregnant rats (n= ) were infused with atp identically. one day before and , , days after infusion, hr urine and blood samples (wbc count and hemopexin activity) were collected. seven days after the infusion, rats were sacrificed and kidney tissue processed for immunohistology. results: urinary albumin excretion was increased in pregnant atp infused rats only (fig ) . wbc were also increased only in pregnant rats infused with atp (days and vs pre-infusion value). in pregnant atp infused rats intraglomerular influx of monocytes was increased, which correlated with urinary albumin excretion on the same day (r = . ). staining of glomeruli for the angii receptor (at- r) showed decreased at- r expression in control pregnant rats as compared to non-pregnant rats, while at- r expression in atp infused pregnant rats was increased as compared to control pregnant rats. hemopexin activity was increased on days and in control pregnant rats as compared to all other groups. discussion: these data support the notion that atp is toxic exclusively in the pregnant condition. it may be suggested that atp induced an inflammatory response, although the exact mechanism by which atp induced its effects needs further investigation. background: hepatocyte growth factor (hgf) is a multifunctional cytokine that is known to promote division, motility, invasion and morphogenesis of a wide range of cell types and to inhibit apoptosis. the effects of hgf are mediated through its interaction with the tyrosine kinase receptor c-met. in pregnancy hgf/met system is involved in the physiologic growth and development of the feto-placental unit. hgf/met effects are counteracted by transforming growth factor- (tgf- ), that is known to promote progressive fibrosis in human tissues. moreover tgf- plays a major role in trophoblast growth and differentiation. tgf- inhibits hgf expression as well as hgf inhibits tgf- expression. an understanding of the mechanisms regulating placental balance of these growth factors may provide insights into the processes that occur in complications of pregnancy, such as preeclampsia (pe) and fetal growth restriction (fgr). objective: to verify the hypothesis that hgf, c-met and tgf- are differently expressed in tissues of normal and pe placentas. methods: we studied placentas from pregnancies complicated by pe ( with normal fetal growth and with fgr) and placentas from normal pregnancies. from each placenta random samples were excised and rna was extracted; quantitative real-time rt-pcr analysis was used to investigate mrna expression of hgf, c-met and tgf- in pe (with or without fgr) and in normal placentas. results: gestational age, neonatal weight and placental weight were, as expected, lower in pe groups. the mrna expressions of the three molecules are higher in pe than in normal placentas, but the difference is statistically significant only for c-met. the higher values are mainly due to the increase in the pe group without fgr for c-met and tgf- and the increase in the pe-fgr group for hgf. conclusion: increased levels of expression of hgf/met system in pathological placentas could be explained as an attempt to repair placental damages; nevertheless placental regeneration could be inhibited by the increase of tgf- , that promote fibrosis. placental expression of hgf, c-met and tgf- is increased in all pe placentas, but particularly in placentas of pe with normal fetal growth, where placental tissue is probably less compromised. obesity is a risk factor for preeclampsia (pe), but the reason for this risk is unknown. previously, we found that neutrophils infiltrated into the vasculature of pe women released myeloperoxidase (mpo) and matrix metalloproteinase (mmp ), products that can cause oxidative stress and vascular dysfunction. if neutrophils infiltrate the vasculature of obese women and release mpo and/or mmp , this may help explain why they are at risk of developing pe. hypothesis: systemic vascular tissue of obese women will have a significant presence of mpo and mmp as a result of neutrophil infiltration. methods: subcutaneous fat, which is highly vascularized, was obtained at abdominal surgery from normal weight, overweight and obese women. formalin fixed, paraffin embedded μm sections of fat biopsies were stained using immunohistochemistry with specific antibodies for mpo and mmp . data were evaluated for intensity of vessel staining by visual score ( - ), density of staining using image analysis software, and % vessels with neutrophil staining, liberated from serum-free placental villous explant cultures from normal and pe pregnancies on human endothelial cells, and identified candidate mediators of their differential effects on metabolism and behaviour. methods: term placental villous tissue from normal (n= ) or pe (n= ) pregnancies were explanted for days at % oxygen. conditioned h medium (day - ) was applied to human umbilical vein endothelial cells (huvecs). an angiogenesis assay was conducted in which tubule length and number were measured by morphometric imaging following seeding on % matrigel. the effect of explant conditioned medium on endothelial cell metabolism was determined by mtt and bioluminescent atp assay. the release of vasoactive metabolites (nitrite, endothelin- , prostacyclin) from huvecs exposed to this medium was also measured. finally, a luminex bead array was used to screen the explant media for a panel of chemokines/cytokines. results: in the angiogenesis assay, tubule length (p< . , mann-whitney u test) and number (p< . ) were significantly decreased in pe compared to normal pregnancy medium. there was no change in mtt reduction, but endothelial cellular atp levels were also significantly reduced following exposure to pe explant medium (p< . ). both normal and pe-derived medium stimulated huvecs to produce vasoactive metabolites. the following cytokines were detectable in the explant media: interleukin (il)- , il- , gro-, monocyte chemotactic protein- and macrophage inflammatory protein- (mip- ). higher levels of both il- and gro-were present in the normal medium compared to pe (both p< . ). mip- was present in pe conditioned media but undetectable in media generated from normal placental explants. conclusions: these results suggest that pre-eclampsia stimulates the release of soluble factors from the placenta which have adverse effects on endothelial cell angiogenic potential and metabolism. altered levels of several cytokines were detected in the explant medium, and the effects of these on endothelial cell function are currently being addressed. yang gu, davis f lewis, yuping wang. obstetrics and gynecology, lsuhsc-shreveport, shreveport, la, usa. objective: placentas from women with preeclampsia (pe) release more chymotrypsin-like protease (clp). the purpose of this study was to determine if placenta-derived clp was responsible for altering endothelial (ec) barrier function in pe. approaches: endothelial junctional protein complex ( -catenin/ve-cadherin/ p ) expression and junctional protein ve-cadherin distribution were examined in ecs treated with pe placental conditioned medium. methods: ) confluent ecs were treated with pe placental conditioned medium (cm). the association of ec junctional protein complex ve-cadherin/catenin/p was examined by a combined immuno-precipitation (ip) and immuno-blotting (ib) assay, in which total cellular protein was immunoprecipitated with monoclonal antibody against -catenin and then immunoblotted with antibodies against ve-cadherin or p- . ) confluent ecs grown on cover slips were exposed to pe placental cm with or without depletion of chymotrypsin. ec junction protein ve-cadherin distribution was examined by fluorescent microscopy. results: ) ve-cadherin and p are expressed in control ecs but not in ecs exposed to pe cm, which indicate that the junctional protein complex vecadherin/ -catenin/ p is lost in ecs exposed to pe cm. ) ecs exposed to pe placental cm showed a discontinuous distribution and reduced expression for ve-cadherin at cell contact areas. the zipper like structure was lost and cleft was formed at cell-cell contacts. these observations indicate that the homotypic cell-cell adhesion and junction protein intracellular partner complex are disrupted. these disruptive phenomena in cells treated with pe conditioned medium were not present in control cells and in cells treated with cm after depletion of chymotrypsin. conclusion: clp released by the placenta could be a candidate agent that is responsible for disrupting ec integrity and inducing endothelial permeability in pe. (supported by nih grants hl and hd ). introduction: chronic pelvic pain (cpp) syndromes such as endometriosis, irritable bowel syndrome, and interstitial cystitis are associated with visceral hyperalgesia, and often coexist in the same patient. one possible explanation for this phenomenon is viscero-visceral cross-sensitization in which increased nociceptive input from an inflamed pelvic organ sensitizes neurons that receive convergent input from an unaffected organ to the same dorsal root ganglion (drg). nociception induces upregulation of perk and substance p. the purpose of this study was to determine, in a rodent model, whether uterine inflammation increased the number of perk-and sp-positive neurons in sensory ganglia innervating both uterus and colon. methods: colonic and uterine drgs were retrogradely labeled with fluorescent tracer dyes micro-injected into the colon and uterus. ganglia were harvested, cryoprotected and cut for fluorescent microscopy. results:approximately % neurons were colon-specific and % were uterus-specific. among these uterus-or colon-specific neurons, up to % of labeled drg neurons in the l -s levels innervated both visceral organs. uterine inflammation increased the number of perk and sp-immunoreactive neurons in drg neurons innervating colon, uterus, and those innervating both organs. furthermore, this effect was specific as non-retrograde labeled drg neurons did not manifest a significant increase in perk or sp immunoreactive cells. conclusions: localized uterine inflammation leads to increased expression of sp and perk in uterine afferents as well as dichotomizing afferents innervating both uterus and colon, suggesting that viscero-visceral convergence is present at the level of drg primary afferent cell bodies. this visceral sensory integration may underlie the co-morbidity of female pelvic pain disorders and may provide basic information regarding the etiology of cpp syndromes. purpose: success rates of medical and surgical modalities for the treatment of severe fecal incontinence due to obstetric trauma are modest. we tested whether the intrasphincteric injection of autologous myoblasts is clinically safe and feasible for postobstetric fecal incontinence. methods: using ultrasound guidance a suspension of autologous myoblasts was injected in the anal sphincter muscle complex in three women with severe postobstetric fecal incontinence. main outcome measures were safety, feasibility and the wexner grading score. secondary outcome measures were incontinence episodes, rockwood fecal incontinenece quality of life scale, external anal sphincter morphology and anal pressure values. results: all procurement procedures and injections were performed without complications; there were no clinically or laboratory signs of infection or inflammation. three months post myoblast injection, wexner continence grading scores and rockwood incontinence scales were markedly improved and incontinence episodes reduced in all three patients. no change could be observed in sonographic anal sphincter morphology. mean anal squeeze pressure improved. conclusion: autologous myoblast injection for fecal incontinence is clinically feasible and safe; further studies will evaluate the efficacy of this approach. objective: to evaluate whether hysterectomy is associated with a change in postoperative bmi. methods: a retrospective cohort study was conducted of hysterectomies performed for benign indications from june to june . institutional review board approval was obtained. the data were collected by a medical student blinded to the hypothesis. basic demographic data were recorded. body mass index (bmi) was determined at time points: time (immediately postoperatively), time ( weeks to months postoperatively), time ( months to year postoperatively) and time ( to . years postoperatively). one-way anova was performed using ncss statistical software. a bonferroni multiple comparison test (p< . ) was used to compare median changes in bmi from baseline. the starting bmi served as the control group. results: there was a statistically significant increase in bmi at time compared to baseline in all women ( . to . ). when sorted was measured by matrigel invasion assay. small interference rna targeting vegfr- were predesigned by ambion inc. and cells were transfected using ambion siport neofx according to the optimized protocol. results: of the four receptors (vegfr- , vegfr- , nrp- and nrp- ), vegfr- was the predominant receptor that expressed in the more invasive cell lines (dov , skov and r ). lpa, at - m, significantly induced vegfr- expression in dov and skov cells (p< . ), without significantly affecting vegfr- expression. lpa ( - m) also significantly induced the expression of vegf and vegf (p< . ) in dov and skov cells. by small interference rna (sirna) transfection, we demonstrated that inhibition of vegfr- expression could significantly decrease dov cells' invasiveness (p< . ) and moderately decrease skov cells' invasiveness. moreover, silencing of vegfr- by sirna significantly suppressed lpa-induced dov and skov invasion. conclusion: these results suggest that knocking down vegfr- expression by rnai may be an effective strategy to inhibit lpa-induced ovarian cancer metastasis. cancer. fengqiang wang, david fishman. obstetrics and gynecology, new york university school of medicine, new york, ny, usa. objectives: expression of matrix metalloproteinases, such as mmp- and mmp- , has implicated in epithelial ovarian cancer (eoc) invasion and metastasis. osteopontin (opn) was also expressed in various human cancers and associated with tumor progression, invasion and metastasis. in the present study, we examined the correlation of mmp- , mmp- and osteopontin expression with tumor stage in ovarian tumor tissues and normal ovaries using a commercial tissue scan array. methods: complimentary dna (cdna) from normal ovaries (n = ) and ovarian tumors (n = ) of different stages (stage i, n = ; stage ii, n = ; stage iii, n = ; stage iv, n = ) was amplified by real time pcr using gene specific primer pairs for mmp- , mmp- , and osteopontin. gapdh was also amplified as a reference control. the expression level of mmp- , mmp- and osteopontin was calculated as relative expression normalized to that of gapdh in each tissue sample, and the expression in sample that has the lowest target gene expression was arbitrarily set as . the average expression of mmp- in ovarian tumor tissues ( ± ) is fold of that in normal ovaries ( ± , p = . ). using an arbitrarily set standard, of normal ovaries ( . %) has elevated mmp- expression; in ovarian tumor tissues, the percentage is . % ( of ), significantly higher than in normal tissues (p = . ). in early stage tumors (stage i/ii, n = ), of them have elevated mmp- expression ( . %, p = . vs. normal ovaries), whereh the average expression of mmp- is fold of that in normal ovaries (p = . ) and . fold of that in late stage tumors (stage iii/iv, n = ). the mmp- expression in ovarian tumors (n = , ± ) is fold of that in normal ovaries (n = ), however, the difference is not significant (p > . ) due to the extremely high expression of mmp- in two tumor tissues. osteopontin expression in ovarian tumor tissues is . fold of that in normal ovarian tissues ( ± . , n= vs. . ± . , n = , p< . ). in early stage (i/ii) ovarian tumors, osteopontin expression is . fold of that in normal ovaries, while in late stage (iii/iv) ovarian tumors, its expression is . fold of that in normal ovaries (p< . ), suggesting an increase trend associated with disease stages. conclusions: our results suggest that mmp- , mmp- and osteopontin alone or combined may have clinical value for ovarian cancer detection. objectives: -tubulin acetylation has been proposed to control the dynamic nature of microtubule stability. acetylated -tubulin plays a role in regulating microtubule functions in mitosis and cell migration. here, we sought to identify the relationship between post-translational -tubulin acetylation and the expression of epithelial cell adhesion molecules (ep-cam) after exposure to microtubule interacting agents in ovarian cancer cells. methods: epithelial ovarian cancer cell line, hey was treated with microtubule stabilizing agents (taxol, epothilone b and discodermolide), microtubule-destabilizing agent (vinblastine), hdac inhibitor trichostatin a (tsa), anti-metabolite fluorouracil ( fu), or alkalyting agents (cisplatin and carboplatin). cells were separately treated with either ic or -fold ic of each agent, and incubated at °c for h, h and h. acetylation oftubulin and pan--tubulin were evaluated by western blot analysis followed by protein quantification. cell surface ep-cam expression was examined by flow cytometry. results: increased acetylation of -tubulin was seen with taxol, epothilone b, discodermolide, vinblastine and tsa. -tubulin acetylation was time and dose dependent. the highest level of -tubulin acetylation ( . -fold) was observed with vinblastine at -fold ic after h. exposure to microtubule interacting agents and tsa resulted in increased cell surface expression of ep-cam in a time and dose dependent manner. the highest level of cell surface ep-cam expression ( . fold) was observed with -fold ic of vinblastine at h. the increase in acetylated -tubulin and ep-cam expression was clearly detectable after h treatment. this data reveals a similar dose and time dependent increases between the acetylation of -tubulin and the increase of ep-cam expression. conclusions: these data demonstrate the promotion of -tubulin acetylation and cell surface ep-cam expression by a microtubule destabilizing agent and by microtubule stabilizing agents. interestingly, vinblastine induces the highest -tubulin acetylation and ep-cam expression. acetylation of alpha-tubulin may be associated with redistribution of cell surface antigens in ovarian cancer cells. objective: epothilone b (epob) is similar to taxol in its ability to enhance tubulin polymerization and to stabilize microtubules. epob is currently being evaluated as an antitumor agent and is in phase iii clinical trials. the tumor suppressor gene, p plays an important role in the induction of apoptosis by a variety of anticancer drugs. p mitogen-activated protein kinase is activated by a wide array of stress stimuli including chemotherapeutic agents and promotes apoptosis. since both p and p activation induces apoptosis, we hoped to evaluate the relationship between p , p-p and parp cleavage, an early indicator of apoptosis, in ovarian cancer cells after treatment with epob. methods: hey cells were treated with epob ( to nm) for h. parp cleavage product p as well as p-p , p , phospho-p (ser- ), p and survivin were determined by western blot analysis. a wild type ovarian cancer cell line hey, was treated with or without m sb , a specific inhibitor of p-p , followed by treatment with epob ( or nm) for h. lysates were prepared and western blot analysis was performed with polyclonal phospho-p and anti-phospho-p antibodies. results: epob induces p activation and apoptosis demonstrated by increased parp cleavage product. time course studies indicated that phosphorylation of p precedes phosphorylation of p in hey cells. the expression of p targeted gene, p (survivin) were differentially expressed depending on the dose of epob and the duration of drug exposure. pretreatment with specific inhibitor of p markedly inhibited the p phosphorylation at serine . conclusions: nm epob (a concentration that triggered mitotic arrest) causes a decrease in p expression and an increase in survivin expression. epob induces parp cleavage. p inhibitor sb inhibits epo-b -induced p phosphorylation. these results suggest that phosphorylation of p may lead to p activation and these signaling events may be related to epo-b induced cell death in ovarian cells. conclusions: nf-b has been shown to control the expression and function of anti-apoptotic proteins and pro-inflammatory cytokines. in the present study we demonstrated that specific inhibition of nf-b by erib reversed the anti-apoptotic state of chemoresistant ovarian cancer cells, therefore may provide a new potential venue for the treatment of ovarian cancer patients. expression in cervical cancer. madhu chauhan, chandra yallampalli. gynecology, university of texas medical branch, galveston, tx, usa. background: intermedin (imd)/adrenomedullin is a ct/cgrp family peptide. imd is expressed in a variety of tissues such as pituitary, stomach, placenta, uterus, and ovary .we have shown that imd plays a role in a variety of physiological functions, including vasodilatation and fetoplacental growth regulation. further we have data indicating an angiogenic activity of imd in first trimester trophoblast cells. we hypothesize that imd is expressed in cervix and may have a role in the pathology of cervical cancer objective: ) to analyze expression of imd mrna in human cervix, rat cervix from non-pregnant and pregnant rats; ) to assess the differences in the expression of imd in normal human cervix and cervical carcinoma tissues and ) to analyze the effect of imd on the expression of tnf-alpha and il- beta in human epithelial cervical carcinoma cells (hela). methods: groups of sprague-dawley, non-pregnant and pregnant rats on day of gestation were used in this study. cervical tissues were collected from the non-pregnant and pregnant rats, women undergoing hysterectomy and from women diagnosed with cervical carcinoma. total rna was extracted using trizol method. hela cells were grown to % confluency in rpmi medium supplemented with % fbs. the cells were starved in %fbs for hrs followed by treatment with imd ( - m) in presence or absence of imd antagonist, imda ( - m). cells were further incubated for hrs and total rna was extracted using trizol reagent. rna was treated with dnase before performing the reverse transcriptase polymerase chain reaction (rt-pcr). the rt-pcr data was normalized to that of s. results: ) imd mrna is expressed in rat and human cervix and in hela cells. ) expression of imd is elevated in pregnant rat cervix as compared to the non-pregnant. ) imd has no effect on tnf-alpha expression in hela cells treated with imd but caused a decline in the expression of il- beta mrna and, ) expression of imd mrna is significantly elevated (p< . ) in cervical carcinoma as compared to the normal cervix. conclusion: imd is expressed in both rat and human cervix and is elevated in pregnant rat suggesting that it may have a role in cervical function during pregnancy in rat. in addition we demonstrate that imd is involved in the pathology of cervical carcinoma and thus may have a clinical significance in the pathology of cervical cancer. objective: there is minimal information about the impact of treatment delays or dose reductions on chemotherapeutic treatment responses and overall outcomes in ovarian cancer patients. the primary objective of this study was to quantify the impact of treatment delays and dose reductions in an in vivo xenograft ovarian cancer model and to evaluate if the growth factors could improve overall survival. methods: heya- and skov -ip xenograft mouse models to determine the effect of treatment delays or dose reductions on tumor response. results: the results indicated that full dose of paclitaxel ( mg/kg) and carboplatin ( mg/kg) delivered on timeevery daysachieved better tumor response in both aggressive (heya- ) and metastatic (skov -ip ) human ovarian tumors compared to the non-treatment and vehicle groups. in heya- mice, paclitaxel/carboplatin alone and paclitaxel/carboplatin followed by growth factor support agents including pegfilgrastim ( μg/kg) and darbepoetin ( μg/kg) improved overall survival rate. growth factors also improved the tolerability in heya- model. for skov -ip mice, the treatment delays resulted in a significant reduce in overall survival time compared to full dose, on time treatment (p< . ). for the dose reduction group, there was a significant different survival comparing to full dose, on time treatment (p< . ). conclusion: treatment delays had a negative impact on tumor response and overall survival compare with treatment controls. in addition, use of growth factor agents also improved treatment response and tolerability of chemotherapy and ultimately overall survival. - ) . median age at recurrence was ( - ), and median interval to recurrence . months ( - ). ( %) patients died of recurrent disease and ( %) of other causes. site of recurrence and was local in , groin in and distant in . of the local recurrences were managed with wide local excision (wle) radical surgery ( radical excisions with skin flaps, posterior exenterations, anterior exenteration, total exenteration), wle and radiotherapy (rt)/chemort, chemo/chemort/rt, and palliation. groin recurrences were managed surgically ( adjuvant rt), rt alone, chemotherapy alone, and palliation. patients with distant metastases were managed surgically, with rt/chemo/chemort, and palliation. overall median survival after recurrence was months. median survival (months) by site of recurrence was: local , groin , distant , groin or distant . there was a significant difference in survival in local vs groin node recurrence (p< . ), local vs groin and distant (p< . ), and groin node vs distant (p . ). and years after recurrence survival rates were: local % and %, groin % and % distant , . conclusion patients with vscc remain at risk of recurrent disease therefore long term follow up these patients is essential. patients with local recurrence often have a good prognosis. outcomes for groin and distant recurrences are poor, but a proportion of those with groin recurrences can be salvaged. therefore early identification of local and groin recurrences is essential for improving outcomes in recurrent vscc particularly in cases with more conservative management of primary disease. introduction: endocrine disrupting chemicals (edcs) are environmental agents possessing hormone-like activity. exposure to edcs during differentiation can interfere with hormonal signaling, resulting in predisposition to some cancers. it has been suggested that some of these effects may be epigenetic, mediated by changes in dna and histone methylation. we hypothesized that edcs, diethylstilbestrol (des), and bisphenol-a (bpa), may act by altering the expression of dna and histone methyltransferases (dnmts and hmts). methods: ishikawa (endometrial) and mcf (breast) cells were cultured in steroid-free, phenol-free media with bpa ( m), des ( x - m) or vehicle for hours. pregnant cd- mice were treated with intraperitoneal injections of des ( mg/kg) or vehicle on days - of gestation. weeks after birth, offspring were sacrificed and tissue obtained. mrna was extracted, cdna was generated and quantitative real time rt-pcr was performed and normalized to -actin. all experiments were conducted in triplicate, repeated three times and compared using anova. results: dnmts (dnmt , a, and b) and hmts (mll and ezh ) were screened after in vitro exposure to edcs (table ) . ezh mrna expression was significantly increased in ishikawa cells after treatment with des or bpa. a similar response in ezh expression was seen in mcf cells treated with des or bpa. due to the consistent induction of ezh in all cells with either treatment, we examined the effect of des exposure on ezh expression in vivo. in adult mice exposed to des in utero, ezh expression was persistently increased ( . ± . fold, p< . ). conclusions: breast and uterine cell lines show increased expression of ezh in response to bpa or des exposure. this differential expression persists in the adult offspring of mice exposed to des in utero. ezh has been identified as a risk for neoplastic progression in the breast and for increased proliferation in uterine cancers. des induced ezh expression may be a mechanism for the increased incidence of breast and reproductive tract cancers seen in des exposed women. defects in cellular programs that control apoptosis lead to an imbalance of cell proliferation and cell death in ovarian cancer. recent evidence suggests that the use of some anti-inflammatory drugs decreases risk of ovarian cancer. natural curcumin-based anti-inflammatory therapies were shown to be beneficial in preclinical models of ovarian cancer (lin et al., ) . in this study, we examined the effects of plant derived curcumin on cell proliferation, apoptosis, and vegf expression in cultivated ovarian cancer cells. ovarian cancer igrov cells were cultured under standard conditions to study the effects of curcumin on cell kinetics and on vegf expression. cell proliferation was measured by srb and mtt assays. apoptosis was determined by measuring cytoplasmic histone-associated-dna-fragments (cell death detection elisa, roche, germany). vegf gene promoter-reporter activation and real-time quantitative reverse transcription polymerase chain reaction were used. conditioned media concentrations of vegf were measured with a commercially available enzyme-linked immunosorbent assay (quantikine, r d systems, minneapolis, mn). we observed that curcumin dose-dependently suppressed cell growth in igrov cancer cells (ic = um). treated cells showed a - fold increase in dna fragmentation compared to controls. curcumin also resulted in a significant decrease of vegfmrna expression and vegf protein secretion into the conditioned media in a dose-dependent manner. in this study, we have demonstrated that curcumin induces apoptosis, suppresses growth, and inhibits vegf gene and protein expression in an ovarian cancer cell line. experiments are underway to identify specific mechanism of curcumin action. curcumin may act as a chemosensitizing drug by potentiating the antitumor effects of standard treatments including taxols and platins in ovarian cancer. supported by the caldwell family foundation and the vesa w. and william j. hardman, jr. charitable foundation inc. daniel r christie, faheem m shaikh, john a lucas iii, susan l bellis. obsterics and gynecology, university of alabama at birmingham, birmingham, al, usa; physiology and biophysics, university of alabama at birmingham, birmingham, al, usa. introduction: previous work has shown that an upregulation of the enzyme st gal i, which is responsible for the , sialylation of integrins, confers a more tumorigenic/metastatic phenotype to colon carcinoma cell lines. it is not known, however, if this is unique to colon cancer, or if it is more broadly applicable to other forms of cancers. quantitatively increased expression of st gal i has been reported in ovarian cancers versus controls, but no biochemical or functional assays have been described to date. objective: because integrins are involved in cell adhesion and migration, and because metastasis of epithelial ovarian cancers is largely an intraperitoneal dissemination, we hypothesized that upregulation in the expression of st gal i in an ovarian cancer cell line would enhance binding with the extracellular matrix, increase invasiveness, and alter migration. methods: in the present study we forced a stable transduction of st gal i into the ov ovarian tumor cell line, which we found to be lacking the st gal i enzyme, establishing a parental (par), an empty lentiviral vector (ev), and an st gal i expressing (st ) cell line. a collagen i cell adhesion assay was performed and quantified by staining adherent cells and measuring absorbance. a haptotactic collagen i cell migration assay was performed by seeding cells in boyden chambers lined with collagen i concentration gradient, and quantified with cell staining and absorbance measurement. cell invasion through a reconstituted basement membrane (matrigel) was quantified as previously described for the cell migration assay. results: we were able to demonstrate successful creation of the ov -st gal i cell line by western blot analysis. functional assays demonstrated increased adhesion to collagen i (p < . ), increased haptotactic collagen i cell migration (p < . ), and increased invasiveness (p < . ) in the st cell line as compared to par and ev when analyzed by one-way anova. conclusion: this initial study into st gal i in ovarian cancer may have future therapeutic implications, and, in addition, lend greater insight into the intraperitoneal dissemination of disease.of microarrays (sam), arrayassist and binary tree structured vector quantization. differentially expressed genes were integrated with a database of known predicted protein-protein interactions (ophid) and key genes are being validated with real-time pcr and immunohistochemistry. results: unsupervised hierarchical clustering revealed collective clustering of all tumors, irrespective of their pathological classification. sam analysis has highlighted probe sets as differentially expressed between lmp and lmp-mp, probes between lmp and lgsc, and probes between lmp and lmp-mp+lgsc. no differential gene expression was detected between lmp-mp and lgsc. ophid analysis demonstrated gene members of the egfr and mapk / pathways to be differentially regulated between the non-invasive and the invasive tumors. to date, we have successfully validated members of the mapk / pathway-poly adp ribose polymerase (ppol) and traf family associated nf kappa b activator (tank)-using real-time pcr. conclusion: our data demonstrate that although the tumors have related genetic profiles, lmp-mp and lgsc are similar to each other and different from lmp, in keeping with their clinical behavior. members of the mapk / and egfr pathways appear to play a key role in low grade serous cancer. identification of novel genes associated with malignant transformation, may lead to development of more effective targeted therapy for lgsc. background: approximately % of pap smears with the ambiguous diagnosis of atypical squamous cells, cannot exclude high grade (asc-h), are negative for dysplasia in follow up colposcopic examination and biopsy. although hpv testing provides excellent negative predictive value (npv) ( %), the prevalence of high risk hpv infection is high in young women and the positive predictive value (ppv) in asc-h pap smears is no better than cytologic diagnosis alone ( %). recent studies have shown that immunostaining for p ink a , or proex tm c, supports a diagnosis of dysplasia in surgical biopsies. our objective was to determine whether staining for p or proexc provides sufficient predictive value to reliably distinguish high grade dysplasia in asc-h pap smears. design: we retrospectively collected samples from liquid based pap smears diagnosed as asc-h at either ohsu or portland oregon kaiser permanente ( - ). known hsil and negative pap cases were included as immunostaining controls. asc-h cases with followup cervical biopsies (n= ) were included for subsequent immunostaining according to manufacture's instructions. samples were also sequestered for hpv testing (pending). immunostained slides were scored as positive or negative by two independent cytopathologists (as and tm) while blinded to pap diagnoses and surgical biopsy outcomes. results: we observed excellent agreement between pathologists' scores (pairwise kappa statistic . ). the correlation between p and proexc scores was moderate (kappa . ). chi-square analysis comparing staining to biopsy outcome revealed a significant association between proex c positivity and cervical dysplasia (*** p< . jing lin, zhenmin lei, ch v rao. ob/gyn women health, university of louisville health sciences center, louisville, ky, usa. introduction: matrix metalloproteinases (mmps) are a family of highly homologous zinc-dependent endopeptidases, which degrade all kinds of extracellular matrix proteins. the degradation process is required for tissue growth and remodelling. these enzymes are probably involved in uterine growth and devolopment and its differentiated functions. ovarian steroid hormones, estradiol (e ) and progesterone (p ), are known to regulate some of the uterine mmps. since it is now known that lh/hcg can directly regulate uterine growth and devolopment and its functions, we questioned whether these gonadotropins could also regulate mmps in the uterus. methods: sixty day old wild type (wt) and lh receptor knockout (lhrko) mice and -day e and p treated -day old animals were used. in addition, primary cultures of uterine epithelial cells from wt animals were treated for hrs either with no hormone or with a single or a combination of pg/ml of e or p , ng/ml of hcg. then mmp- , - and - mrna levels were quantified by the semiquantitative rt-pcr. results: while the uterine mmp- mrna levels were unaffected, mmp- mrna levels significantly decreased and mmp- mrna levels significantly increased in lhrko animals as compared with wt siblings. e and p treatment reversed mmp- and mmp- changes in lhrko animals. treatment of wt type primary endometrial epithelial cell cultures with hcg had no effect on mmp- mrna levels, but it did increase mmp- mrna levels. this increase was synergistic with both e or p . conclusion: while lh/hcg do not regulate uterine mmp- and mmp- , they seem to co-regulate mmp- with ovarian steroid hormones. cancer. radhika gogoi, christine ardalan, dorota popiolek, leslie gold, john curtin, stephanie v blank, bhavana pothuri. new york university, new york, ny, usa. objective: megesterol, a synthetic progestin with strong androgenic properties, is used in the medical management of patients (pts) with atypical endometrial hyperplasia (aeh) or endometrial carcinoma (emca). our hypothesis was that androgen receptor (ar) expression in the endometrium of aeh and emca pts would correlate with degree of response to treatment. methods: pre-and post-treatment endometrial biopsy specimens were obtained from pts treated with megesterol for aeh or emca. ar expression was investigated by immunohistochemical (ihc) analysis with appropriate positive and negative controls. ihc staining was scored for intensity ( - ) and percentage of positive cells ( - ) in the glandular and stromal compartments by a pathologist, blinded to clinical response data. a composite score utilizing both intensity and percentage of positive cells was calculated. we evaluated pre-and post-treatment ar expression in responders and nonresponders as well as ar expression in emca, aeh and normal endometrium. the mann whitney u and the wilxocon signed rank tests were utilized for statistical analysis. results: eight pts' pre-and post-treatment samples were obtained; with emca and with aeh. three pts had no response, , a partial response and , complete responses. ar expression in emca samples when compared to the normal endometrium was significantly lower in both the glands (mean . vs. ; p< . ) and the stroma (mean . vs. ; p< . ). although there was no statistically significant difference in glandular ar expression in the pretreatment biopsies of responders compared to nonresponders (mean . vs. . ; p= . ), there was a significantly higher level of glandular ar expression in the post treatment biopsies of responders compared to non-responders (mean . vs. ; p< . ). furthermore, we noted a trend towards higher levels of glandular ar expression in the post-treatment versus the pre-treatment biopsies in the responders (mean . vs. . ; p= . ). we noted a significant decrease in ar expression in emca compared to the normal endometrium. increased ar expression after treatment in responders suggests an important role of the ar in pts treated conservatively with progestational therapy, and needs further prospective validation as a means to predict treatment response in these pts. objectives: study the correlation between adenomyosis and some potential non-surgical risk factors. objective: o -methylguanine-dna methyltransferase (mgmt) acts to repair dna damaged by alkylation of guanine residues. mgmt promoter methylation and gene silencing is seen in a variety of cancers and pre-cancerous changes. the loss of mgmt activity is associated with increased sensitivity to alkylating agents and is a favorable prognostic indicator in gliomas. we sought to determine if mgmt promoter methylation plays a role in endometrial cancer. methods: one hundred and twenty primary endometrial cancers were analyzed for mgmt promoter methylation by combined bisulfite restriction analysis (cobra). the cohort included endometrioid endometrial cancers, endometrial tumors of adverse histologic type, and endometrial cancer cell lines. twenty one endometrioid and mixed endometrioid ovarian cancers were also analyzed. a subset of the primary tumors was analyzed for mgmt expression by immunohistochemistry. results: no mgmt promoter methylation was seen in the endometrial cancers evaluated or the endometrial cancer cell lines. one of the ovarian cancers showed methylation. immunohistochemistry for mgmt expression is ongoing. conclusion: mgmt promoter methylation is an infrequent event in endometrial cancer. mgmt expression in tumor cells and repair of alkylguanine residues could explain in part the limited response of endometrial tumors to alkylating chemotherapy. objective: uterine sarcomas ( % of gynecological malignancies) originate from uterine mesenchyma. patients with high-risk disease (i.e. high grade) or at advanced stages have poor -years overall survival (< - %). pelvic radiation and/or chemotherapy have not demonstrated to improve survival. identification of new therapies for this malignancy is a major goal. few in vitro models have been established to test therapeutic agents for uterine sarcoma. here we sought to establish a new human uterine sarcoma cell line and to test the effects of chemotherapeutic drugs: tnf-related apoptosis inducing ligand (trail) used alone or in combination. methods: tissue sample was obtained from a woman with uterine sarcoma undergoing hysterectomy. sarcoma cell lines were established using a published protocol for endometrial cancer. phenotypic characterization was made through the different passages ( to ) by western blot. levels of estrogen (er), progesterone (pr) and trail receptors were also studied (rt-pcr, w-b). cells were incubated with chemotherapy agents (cisplatin, paclitaxel and doxorubicin and trail) and cytotoxicity (mts assays) and apoptosis (flow cytometry, parp cleavage by w-b, and dna laddering) measured. results: human uterine sarcoma cell line was established from a highgrade uterine sarcoma. through the different passages the cell line remains expressing cytokeratin, vimentin, tissue factor, caveolin- and -actin. this cell line expresses low er and pr levels. trail receptors (r and r ) were also detectable (rt-pcr, w-b). cisplatin, paclitaxel and doxorubicin ( um for h) produced low cell cytotoxicity (< %). trail ( ng/ml for h) induced about % cell cytotoxicity. apoptosis was confirmed by parp cleavage. doxorubicin significantly enhances trail mediated cytotoxicity (up to %), this was demonstrated by a significant increase in the sub g /g region in the dna histogram. conclusions: we establish a human uterine sarcoma cell lines using protocols for endometrial cancer. more importantly, we demonstrated that doxorubicin enhances trail effect on this uterine sarcomas cell line. thus, this combination might be considered as a treatment for high-risk uterine sarcomas. (financial support fondecyt ). defining the tumor initiating capacity of cd + human endometrial cancer cells. anne m friel, petra a sergent, christine l cummings, rosemary foster, current data suggest rare subpopulations of cells with tumor initiating capabilities are a common feature of solid tumors. several investigators have recently identified cd , a cell surface marker expressed on primitive cells of neural, hematopoietic, endothelial and epithelial lineages, as a potential tumor initiating cell (tic) marker in solid tumors of the brain, colon and pancreas. in our efforts to investigate such a population in human endometrial cancers (enca), we developed an in vivo model that is based on serial transplants of tics from endometrial tumor explants. serial transplant experiments were initiated in nod/scid mice that were injected with primary human enca cells. a subset of cells derived from the generated tumor was subsequently transplanted into new recipients. tumors formed following serial transplantation retained the original histological phenotype of the primary enca, and the number of cells required to initiate tumor formation was reduced at each successive transplant stage suggesting an increase in the ratio of tics to non-tics. we exploited this model in our initial efforts to investigate the tumor initiating potential of cd -expressing enca cells. to evaluate the tumorigenic potential of cd + cells, the tumor initiation capacity of xenograft derived cd + and cd cells were compared following subcutaneous injection of each population into nod/scid mice. only the cd + fraction was tumorigenic consistent with the hypothesis that tumors are generated and maintained by a subpopulation of cells with phenotypically distinct profiles. we are further investigating the functional significance and characteristics of this fraction in vitro and in vivo. objectives. central issues in tumor biology are the understanding of factors that control tumor cell proliferation and the identification of extracellular matrix cues controlling the signaling transducing repertoire that make cancer cells proliferate and invade the host tissues. among these factors, small leucine-rich proteoglycans (srlps):decorin, fibromodulin, lumican, biglycan, are emerging as powerful modulators of angiogenesis and cell growth, by affecting several key elements including matrix assembly, growth factor binding, and receptor tyrosine kinase activity. recently has been demonstrated that srlps can act as a pan-erbb ligand and, in doing so, down-regulate the activity of one of the most potent oncogenic proteins, erbb , whose overexpression is linked to poor prognosis and increased cancer mortality in breast, ovary, and prostate. since srlps have not been previously investigated in the endometrial cancer biology, we investigated their role in the benign and malignant endometrial neoplasia. method. the sampling has been obtained in women (n= ; mean age ± . ) with endometrial hyperplasia (n= ), polyps(n= ), and cancer(n= ), during therapeutic and diagnostic procedures (hysterectomy, colpohysterectomy, hysteroscopy). physiological endometrial samples (n= ) were obtained from menopausal women (n= ; mean age ± . ), during procedures for others gynaecologic indications. immunohistochemestry was the biology technique used for the detection of srlps.results. in the physiological endometrial samples immunoreactivity for decorin, fibromodulin and biglycan was intense (+++), while it was weak in polyps and hyperplasia (+) and absent (-) in cancer. no significant difference in the staining of lumican between the physiological and pathological samples. conclusions. these results could provide a mechanism by which naturally occurring proteins normally synthesized by fibroblasts and smooth muscle cells, the two key components of the tumor stroma, may play a protective role in the genesis and progression of endometrial neoplasia counteracting the growth of neoplastic cells and suppressing tumor cell-mediated angiogenesis. although further studies are necessary to understand mechanisms whereby srlps might influence endometrial cell growth and survival, these molecules may represent potential target for pharmacological cancer therapy. myostatin is a member of the tgf-beta superfamily of protein and a wellknown inhibitor of skeletal muscle proliferation. the muscular component of the uterus is the myometrium, a tissue that regulates its mass in response to different physiologic conditions under the influence of steroids. we determined the expression of myostatin mrna in immortalized pregnant human myometrial (phm ) cells and we verified its biological activity. functional assays showed myostatin induced phosphorylation of smad- and reduction of proliferation of phm cells in a time and dose-dependent manner. to investigate the physiological relevance of our in vitro findings, the expression of myostatin in rat uterus was examined at various phases of the estrous cycle. for the first time we report that myostatin is expressed in rat uterus and that levels of myostatin mrna change during distinct phases of the estrus cycle. uterine levels of myostatin peaked during late estrous and were the lowest at pro-estrous. to further examine the role of steroids in myostatin regulation, we examined the effects of gonadal steroid administration in ovariectomized (ovx) rats. ovaryectomy increased myostatin expression compared to normal cycling controls. estrogen treatment strong decreased myostatin levels while progesterone produced less robust effects on myostatin expression. these findings taken together suggest an important role for myostatin in the regulation of myometrial functionality. her neu over-expression and pi kinase/akt pathway activation in paget's disease of the vulva. amy stenson, bita behjatnia, jaime shamonki, jianyu rao, amer karam, jonathan berek, oliver dorigo. ob/gyn and pathology, ucla, los angeles, ca, usa; ob/gyn, stanford university, palo alto, ca, usa. background: paget's disease of the vulva is rare with high recurrence rates. treatment of recurrent disease is challenging due to its extent and location. non-surgical approaches have limited clinical efficacy, obviating the need for novel therapies. in contrast to paget's of the breast with well-described overexpression of her neu, molecular features of vulvar paget's are poorly characterized. our objective was to study therapeutic targets in vulvar paget's, including the her neu protein and the phosphorylated oncoprotein akt (pakt). in addition, detailed clinical characteristics were correlated with molecular expression. methods: specimens with vulvar paget's disease were retrieved from ucla's department of pathology. protein expression was evaluated by immunohistochemistry on slides from paraffin embedded tissue using the hercep test (dako) for her neu expression and a standard protocol to assess expression of activated pakt. slides were scored by two independent pathologists based on a nominal scale of (negative) to (strongly positive). clinical data was retrieved via chart review. results: between and , patients with vulvar paget's were identified. median age was yrs ( - yrs). a family history of cancer was found in / ( %), / ( %) were smokers and / ( %) had a history of hormone use. intraepithelial lesions accounted for the majority ( / , %), while / ( %) demonstrated invasion and / ( %) were associated with underlying gi malignancy. / ( %) had at least one recurrence, with median time to recurrence months. so far, specimens were stained for her neu and pakt. overexpression of her neu was found in / ( %.) positive staining for pakt was evident in / ( %.) statistical analysis suggested a correlation between her neu and pakt expression. conclusions: our study demonstrates that overexpression of her neu and activation of the pi kinase/pakt pathway are important features of vulvar paget's disease. to the best of our knowledge, this is the largest series evaluating these molecular pathways in vulvar paget's. our data suggest that her neu and pakt may be important molecular targets for novel therapies using for example the monoclonal antibody trastuzumab directed against her neu, or a pi kinase pathway inhibitor like rapamycin. introduction: uterine leiomyomas are the most frequent tumor of the female reproductive tract and are the primary indication for hysterectomy in women worldwide. these tumors occur in up to % of adult women, and their prevalence is especially high in africa-american women. currently there is no effective and safe medical treatment for uterine fibroids and surgery is the main stay. epigallocatechin gallate (egcg) constitutes the main solid extract of green tea and is believed to contributes most of the antioxidant and antiinflammation capacity of green tea. egcg has been shown to have beneficial effects on prostate cancer and breast cancer by inducing apoptosis and inhibiting proliferation of cancer cells. in this study, we investigated the ability of egcg to inhibit proliferation of human leiomyoma cells (hlm) in vitro. methods: human immortalized leiomyoma cells were cultured at ºc in a humidified atmosphere of % co - % air in smbm medium supplied with %fbs, . % insulin, . % hfgf-b, . % ga- and . % hegf(lonza). the cells were plated at density of × cells/well in -well plates and grown under the same conditions above. the monolayer cultures at approximately % confluence were treated with various concentrations ( , . , . , , , and μm) of egcg (sigma) for days. a fluorometric assay using hoechst dye (sigma) for dna quantitation was conducted at the following designed time points, day , , , and post egcg treatment. the cells were lysed and dna content was determined using hoechst dye solution ( μg/ml). fluorescence was measured after excitation at nm and emission at nm. results: egcg exhibited marked anti-proliferation effect on the growth of hlm cells. compared with untreated control, the inhibitory effect of egcg on hlm cells was observed at μm and peaked at μm concentration. the difference was statistically significant (p = . ). evaluation of the mechanism of action of egcg is cuurently under investigation in the lab.conclusion: egcg significantly inhibited the proliferation of human leiomyoma cells in a dose-depended pattern. egcg may present a potential effective and safe medicinal treatment for uterine fibroids. objective: choriocarcinoma is an aggressive form of germ cell tumor that exhibits rapid growth with early metastases. choriocarcinomas autonomously secrete hcg which acts as an autocrine/paracrine growth factor in these cancers. we hypothesize that a novel hcg antagonist (hcg-ant) can limit tumor expansion by blocking hcg-induced expression of pro-invasive genes. we investigated if hcg-ant could alter rna expression of matrix metalloproteinases (mmp- and mmp- ), which facilitate basement membrane degradation and hence invasion, and metastin (kisspeptin), a strong suppressant of metastasis, in the choriocarcinoma cell line jeg- . design: in vitro experiments using the jeg- cell line. materials and methods: after plating jeg- cells in a well tray overnight in rpmi media containing % fbs, cells were washed twice with pbs and then cultured in ul of serum-free rpmi media containing one of four treatments: ) saline; ) hcg ( iu); ) hcg ( iu) plus hcg-ant ( iu); or ) hcg-ant ( iu). rna was extracted from each well using trizol and reverse transcribed using sensiscript (qiagen). the relative expression of mmp- , mmp- , and metastin mrna was quantified using sybr-green based real time pcr. the expression of the housekeeping gene hprt was used to normalize expression data. results: treatment of jeg- cells with hcg-ant vs. hcg alone reduced expression of mmp- ( . ± . vs. . ± . ) and . hcg-ant reduced mmp- and mmp- expression by % and %, respectively (p< . ). treatment with hcg-ant vs. hcg increased metastin expression ( . ± . vs. . ± . ). metastin expression was increased by % following hcg-ant treatment. conclusion: treatment of the jeg- choriocarcinoma cell line with hcg-ant reversed the increased expression of mmp- and mmp- following treatment with hcg. metastin expression was increased by hcg-ant. this data suggests that hcg antagonism is capable of altering gene expression thereby inhibiting invasion in a choriocarcinoma cell line. the role of hcg-ant as an adjuvant therapy in hcg sensitive tumors offers promise. retinoids, but not progesterone, directly induce differentiation and apoptosis of endometrial cancer cells. you-hong cheng, serdar e bulun. ob/gyn, northwestern university feinberg school of medicine, chicago, il, usa. objectives: the opposing actions of estrogen and progesterone during the menstrual cycle regulate endometrial proliferation, differentiation and secretion. the unopposed action of estrogen contributes to the development of type i endometrial cancer. however, the mechanisms for progesterone protection of estrogen-induced carcinogenesis in endometrium remain unclear. methods and results: in the current study, we demonstrated that retinoids ( -cis retinoic acid (ra) or all-trans ra (atra)) significantly inhibited basal and hormone-stimulated ishikawa cell proliferation by over % using mtt assay. the same experiment indicated that estrogen had no significant effect, whereas progesterone slightly induced, on cell proliferation. cell cycle analysis indicated that atra significantly increased the g /g cell population by % and decreased s phase cells by %, suggesting that ra induces cell cycle arrest at the s phase. knock-down of rar alone or both rar and rxr significantly increased proliferating cell nuclear antigen (pcna) levels in epithelial ishikawa cells, suggesting that ra signaling via rar/rxr activation is critical for normal endometrial growth and differentiation. to determine whether retinoids are naturally secreted by the endometrial stromal cells, we cultured primary stromal cells and analyzed the culture media using hplc. we found that retinol is the predominant retinoid form secreted by stromal cells. the average concentration of retinol in the cultured media of eutopic endometrial stromal cells was approximately to ng/ml/ cells (n= ). although there was less than . ng/ml/ cells of all-trans retinal or atra in the cultured media, we did find a small peak for all-trans retinal and atra in the media using hplc analysis. furthermore, progesterone significantly increased secreted retinol levels from eutopic endometrial stromal cells, but decreased retinol levels secreted from endometriotic stromal cells. retinol is a precursor for ra that is converted to retinal and then to ra. conclusions: we demonstrated that progesterone signaling via pr induces endometrial stromal cells to secrete paracrine retionids that in turn control the phenotype of adjacent epithelial cells. conversely, this interaction is disrupted in diseased endometrial tissues, such as endometrial cancer and endometriosis. the effects of hormonal contraceptives on antimullerian hormone by obesity status. anne z steiner, frank z stanczyk, stan patel, alison edelman. ob/gyn, university of north carolina, chapel hill, nc, usa; ob/gyn, usc keck school of medicine, los angeles, ca, usa; ob/gyn, oregon health and sciences university, portland, or, usa. background: antimullerian hormone (amh) is emerging as a predictor of reproductive potential. serum levels of fsh, a commonly used measure of ovarian reserve, are suppressed with the use of oral contraceptives (ocs) thereby limiting its use. the impact of ocs and on serum amh levels in normal and obese women is unknown. objective: to examine the impact of ocs on serum amh levels by obesity status in reproductive-age women. materials and methods: ovulatory women, ages - years, of normal (< kg/m ; n = ) and obese (> kg/m ; n = ) body mass index (bmi) received a low dose oc ( mcg ethinyl estradiol/ mcg levonorgestrel) for two cycles. serum was obtained at three time points: after days of active pills (cycle , day ), at the end of the -day hormone-free interval (cycle , day ), and during the first week of active pills in cycle (cycle , day , , or ). amh levels were quantified by elisa; fsh and lh levels were determined by chemiluminescent immunoassay. amh at the three time points was compared using repeated measures anova. models were used to assess the relationship between amh and cycle day by obesity status. amh and gonadotropin levels were compared using spearman's correlation. results: amh levels did not differ by oc cycle day (p anova = . ) or by active versus placebo pill use ( . ng/ml ± . vs. . ng/ml ± . , p= . ) among normal bmi women. among obese women, amh levels differed by oc cycle day (p anova = . ), but not by use of active or placebo pill ( . ng/ml ± . vs. . ng/ml ± . , p = . ). across the cycle, cv(standard deviation/mean) averaged . %± . in the obese and . %± . in the normal bmi women ( p= . ). modeling to determine differences in amh throughout the cycles based on obesity status showed a significant interaction (p = . ) and lower amh levels in the obese group (p< . ). among both bmi groups, serum amh and fsh levels did not correlate during active pills or after days of placebo pills. conclusions: in young, normal bmi women serum amh levels do not appear to fluctuate during oc use. among obese women, amh levels are lower and fluctuate significantly. these fluctuations do not appear to mirror changes in gonadotropins and may complicate clinical interpretation of amh. background: menopausal hormone therapy (ht) is a confusing topic for many clinicians and patients. objective: to assess comprehension of basic clinical trial features among prospective participants for the keeps trial, designed to study the effects of ht initiated within years of menopause on chd markers. methods: screening materials were provided giving an overview of study purpose, duration, medications, likelihood of receiving drug vs. placebo, ht related risks and side effects. at a subsequent interview, a -item questionnaire assessed the participant's level of comprehension. a score of % was adequate to complete the informed consent process. those scoring < % were re-counseled and retested. demographic variables determining the likelihood of an initial score > % were evaluated by univariate and multivariable analyses. results: % ( / ) scored > % on initial testing. all women scored > % after re-counseling and retesting. likelihood of an initial response > % correct was unrelated to age or time since menopause. ability to correctly respond was influenced by highest educational level attained. none of women whose furthest educational level was high school scored > % on initial testing, significantly less than those with a college education (p= . ). a higher proportion of college graduates ( / ) scored > % compared to those attaining further education ( / ) (p= . ). comprehension was greatest for study purpose and duration ( / and / correct responses respectively) and least for questions related to results of the whi hormone trial breast cancer and chd. that e alone was not associated with an increased risk of chd ( %) or breast cancer ( %) was poorly understood. conclusion: comprehension of the risks and benefits of ht by potential research subjects is low despite provision of reading materials prior to the informed consent process. (supported by the montefiore medical center/albert einstein college of medicine site for the kronos longevity research institute and k -hd to ns). variation in menopausal symptoms within a sample of hispanic women: swan, the study of women's health across the nation. robin r green, alex j polotsky, aileen p mcginn, carol a derby, rachel p wildman, lhasa ray, kavitha t ram, gerson weiss, nanette f santoro. albert einstein coll of med, bronx, ny; univ of med and dent of new jersey, newark, nj. background: menopausal symptoms are experienced by over % of women. purpose: to describe symptom frequency in a sample of midlife hispanic women from different countries of origin. methods: the study of women's health across the nation (swan) recruited women at baseline who self-identified as hispanic. their baseline responses to validated questionnaires regarding common menopausal symptomatology were examined. symptoms were reported over the previous two weeks and scored on a frequency scale ranging from (not at all) to (every day). for all analyses, symptoms were dichotomized into "absent" vs. "present" variables. responses were correlated with acculturation ( -item scale: marin, hisp j behav sci : , ) and analyzed by sub-ethnicities: central/south american (c/s am), puerto rican (pr), dominican (dr), and cuban (cu). associations between symptoms and sub-ethnicity were tested by chi-square. logistic regression was used to test for associations with acculturation and being us-born. there was significant variation in several menopausal symptoms. while puerto-rican women had the highest likelihood of reporting trouble sleeping (or= . , %ci: . - . ) and headaches (or= . , %ci: . - . ), dominican women were most likely to report vaginal dryness (or= . , %ci: . - . ) acculturation and being us-born did not explain the variation between subethnicities in any of the models tested conclusion: there appear to be significant differences among hispanic women with respect to menopausal symptomatology. these differences were not readily explained by measures of acculturation. (supported by the study of women's health across the nation (swan) has grant support national institutes of health, dhhs, through the national institute on aging, from the national institute of nursing research and the nih office of research on women's health (grants nr ; ag , ag , ag , ag , ag , ag , ag , ag and cd ). purpose: to compare the relative effects of conjugated equine estrogen, raloxifene, and tamoxifen therapies on cognition among women aged years or older. participants and methods: annual modified mini mental state ( ms) examinations were used to assess global cognitive function among the , women enrolled in the two randomized placebo-controlled clinical trials of the women s health initiative memory study (whims) and women enrolled in co-star, the cognitive substudy of the nsabp's study of tamoxifen and raloxifen (star) trial who had baseline ms testing. associations between baseline cognitive risk factors common to both trials and baseline ms scores were assessed and interactions used to examine whether risk factor relationships differed between cohorts. factors for which relationships were similar were used as covariates in analyses comparing ontrial ms scores. factors for which relationships did not appear to be similar were used to stratify analyses. results: compared to placebo, each of the active therapies was associated with a small mean relative deficit in ms scores of . units or less, which was fairly consistent between women with and without prior hysterectomy. overall, relative deficits appeared to be most marked for tamoxifen (unadjusted p= . ) but were also evident for raloxifene (p= . ) and cee (p= . ). conclusions: while unmeasured differences between trials may have confounded our analysis, these findings suggest that both tamoxifen and raloxifene may adversely affect cognitive function in older women. weight gain and increased abdominal fat have been found in women after menopause and is associated with higher levels of leptin, and decreased levels of the cardioprotective adipocytokine adiponectin. at the same time, bmd characteristically decreases. in an effort to determine the evolution and correlates of these changes, we studied postmenopausal women (pm) within years of menopause (age . ± yrs) of normal weight (bmi . ± ) and compared them to weight matched (bmi . ± ) premenopausal (pre) controls (age . ± yrs.) all subjects had bmd and body composition studies by dexa and measurements of leptin, adiponectin, visfatin and retinol-binding protein (rbp .) while total fat was similar in the groups, pm had more trunk and abdominal fat ( ± ; ± g) compared to pre ( ± g p< . ; ± g p< . ) pm also had greater %trunk fat and %central abdominal fat compared to pre, p< . .serum leptin ( . ± . vs ± pg/ml) and visfatin ( . ± vs. . ± . ng/ml) were similar but adiponectin ( ± vs. . ± g/ml) and rbp ( . ± vs. . ± ng/ml) were higher, p< . in pm. while in pre, abdominal fat correlated negatively with adiponectin (p< . ) in pm only leptin correlated with various parameters of fat mass, p< . , and adiponectin did not correlate but correlated positively with age (r . , p< . .) as expected, pm had reduced bmd at the lumbar spine and hip ( . ± . vs. . ± . g/cm ; . ± . vs. . ± . g/cm , p< . ) but there was a correlation between total and trunk fat in pm and lumbar bmd (r . , . , p< . ) but not with hip bmd; or any correlations in pre. there was a correlation between leptin and lumbar bmd in pm (r . , p< . ) but not in pre. in summary, in normal weight early pm, abdominal fat is increased, but only adiponectin and rbp are altered with an increase in the former correlating with age. lumbar bmd correlated with fat mass in pm and is partially explained by increases in leptin. it is suggested that in spite of increasing abdominal fat in normal weight early pm, (which correlates with a higher lumbar bmd) david d rahn, jesus f acevedo, r ann word. ob-gyn, ut southwestern, dallas, tx, usa. objectives: matrix metalloprotease (mmp) activity is increased in the postpartum vagina of wild type (wt) animals, and this activity is accompanied by a burst in elastic fiber synthesis. the mechanisms that precipitate these changes are unclear. the goals of this study were to determine how vaginal distention (such as in parturition) affects elastic fiber homeostasis in the vaginal wall and the potential significance of these changes in the pathogenesis of pelvic organ prolapse. methods: nonpregnant ( ) and pregnant (d ) wt mice underwent either vaginal distention with a μl balloon x min (to simulate parturition) or sham procedure. tissues were obtained at and h for immunoblot analysis, zymography, and histology. distention was also performed in young fbln -/-, fbln +/-, and wts, and prolapse was quantified for weeks. results: distention resulted in marked increases in mmp activity in nonpregnant animals (prommp , . -fold; active mmp , . -fold; prommp , . -fold; active mmp , . -fold; all p < . compared with sham) which was accompanied by fragmented and disrupted elastic fibers in the vaginal wall. abundant amounts of tropoelastin and fibulin- in the nonpregnant vagina were not increased further by distention. in pregnant animals (which normally have suppressed vaginal wall fibulin- and tropoelastin), however, distention resulted in -fold upregulation of both proteins (p< . ). distention also induced increased mmps in pregnant animals (mmp- , . -fold; prommp- , . -fold; active mmp- , . -fold; all p < . ). thirteen young fbln -/-( - wks prior to age of prolapse development), het siblings, and age-matched wts underwent serial exams after ballooning. distention induced rapid increases in perineal bulge and vaginal diameter (within d) in fbln -/mice which never recovered. conclusions: in pregnant mice, vaginal distention results in increased protease activity in the vaginal wall but also increased synthesis of proteins important for elastic fiber assembly. distention may thereby contribute to the burst of elastic fiber synthesis in the postpartum vagina. the finding that distention results in accelerated pelvic organ prolapse in fbln -/animals, but not wt, indicates that distention results in loss of pelvic organ support if elastic fiber synthesis is compromised. further, the data suggest that elastic fiber synthesis is crucial for recovery of the vaginal wall from parturition/distention-induced increases in vaginal protease activity. the molecular etiology of pelvic organ prolapse (pop) is complex and not yet well understood. defects in the connective tissue, such as a decrease in extracellular matrix (ecm) protein content may predispose women to pop. our objective was to study the expression and the enzymatic activity of matrix metalloproteinases (mmps) and their tissue inhibitor (timps) in vaginal tissue of patients with advanced pop and controls. after informed consent, pre-menopausal caucasian patients affected by pop ( grade by pop-q), and control patients (no pop) matched for age and bmi, undergoing vaginal and abdominal hysterectomy respectively were recruited. full thickness anterior vaginal epithelial tissue was obtained from the surgical cuff of patients and controls in the proliferative phase of the menstrual cycle. total protein was extracted using ripa lyses buffer. western immunoblot analysis was performed (patients: n= , controls: n= ) to study the protein expression of mmp- , - , timp- , - , - . gelatin zymography was used to quantify the activity of mmp- and - . immunohistochemical analysis for timp- and - was performed on pfa-fixed vaginal biopsy tissue (n= ) in each group. both patient and control vaginal biopsy samples expressed latent and active forms of mmp- , and mmp- . the protein expression of the kda active form of mmp- was significantly increased in patients with pop compared to controls (p= . ). zymography detected the enzymatic activity of the proform and active form of both mmp- and mmp- . we found a significant increase in gelatinolytic activity of both kda pro-form and kda active forms of mmp- (p= . and p< . respectively) as well as kda active form of mmp- (p< . ) in patients with pop compared to controls. timp- protein expression in vaginal tissue showed a significant reduction in pop patients compared to controls (p= . ). timp- and - immunostaining were mainly localized in the smooth muscle cells at the muscularis layer of the vaginal biopsies. in vaginal tissue of patients with pop, we have shown a decrease in timp protein expression paralleled by an increase in mmp protein expression and activity. these findings reflect an active ecm remodelling that may compromise the structural integrity of the pelvic floor leading to pop. aberrant elastin and collagen synthesis may play a role in the pathogenesis of pelvic organ prolapse (pop). the lysyl oxidase (lox) family of enzymes direct cross linking of collagen and elastin polymers, however to-date no information is available on the expression and localization of these proteins in human vaginal tissue. our objectives were to study the expression and in situ localization of lox, loxl , loxl and loxl proteins in the vaginal tissue of patients with advanced pop and asymptomatic controls. pre-menopausal caucasian patients affected by pop ( grade by pop-q) and control patients (no pop) matched for age and bmi, undergoing vaginal and abdominal hysterectomy respectively were recruited. full thickness anterior vaginal epithelial tissue was obtained from the surgical cuff of patients and controls in the proliferative phase of the menstrual cycle. total protein was extracted using ripa lyses buffer and western immunoblot analysis was performed (patients: n= , controls: n= ). pfa-fixed vaginal biopsy tissues (n= for each group) were used for immunohistochemical study. vaginal biopsy samples from both patient and control groups expressed all four members of lox family proteins: lox ( kda pro-form and kda active form), loxl ( kda pro-form and kda active form), loxl ( kda) and loxl ( kda). the expression of all lox family proteins was reduced in patients with pop compared to controls; however only the pro-form of lox of making the diagnosis of endometriosis from an endometrial biopsy. objective: to evaluate the diagnostic value of examining endometrial biopsy specimens for small nerve fibers in women with pelvic pain or infertility in a double-blinded prospective comparison with laparoscopy. methodology: we undertook to compare the detection of endometrial nerve fibers with laparoscopy and peritoneal biopsy for the diagnosis of endometriosis in women (aged . years; range - years) who presented with chronic pelvic pain or infertility. small nerve fibers were detected in the functional layer of endometrium using immuno-histochemical staining with the highly specific pan-neuronal marker pgp . , using a carefully validated technique and blinded assessment of nerve fiber density. laparoscopic assessment of the presence of endometriosis and peritoneal biopsies was undertaken by three experienced gynecologic endoscopic surgeons. data from these assessments were recorded independently of endometrial findings and maintained under blinded coding until the codes were broken. results: small nerve fibers were detected in all of the women in whom endometriosis was surgically diagnosed and in none of the women in whom endometriosis was excluded at laparoscopy, giving the specificity and sensitivity of %. the density of nerve fibers in the endometriosis cases was . per mm²± . (mean ± sd). conclusions: endometrial biopsy provided a reliability of detection or exclusion of endometriosis which was equal to that of diagnostic laparoscopy carried out by experienced gynecologic laparoscopists. background: erk / are mapk intracellular signaling proteins involved in cell survival and differentiation. endometriosis requires angiogenesis for ectopic implant growth. we hypothesized that the endometriotic peritoneal environment, known to be high in estrogen (e ), vegf, and cytokines such as il- and mcp- , stimulates angiogenesis in human endometrial endothelial cells (heec) through erk signaling. methods: serial sections from normal (n= ) and ectopic (n= ) endometrium were immunostained for total-(t-) and phospho-(p-) erk / and cd (an endothelial progenitor cell marker); results were quantified by computer densitometry and grouped by menstrual phase. cultured normal heec were treated with control, e ( - m), il- , mcp- , and vegf (all ng/ml) for min, and western blotted for p-/t-erk (n= ). heec were treated with peritoneal fluid (pf; diluted : in basal media) from normal (n= ) and endometriotic (n= ) women, with or without pd erk / inhibitor ( m) for h, and cell viability was analyzed by mtt assay. statistical analysis was done with one-way anova. results: tissue staining revealed that ectopic cd + endothelial progenitor cells undergoing angiogenesis (vessel sprouting and/or angiogenic cell cord formation) exhibited the strongest levels of p-erk. heec of ectopic foci showed moderate-high p-erk staining, while surrounding mesothelial capillaries were weak for p-erk. in normal endometrium, p-erk was cycledependent, with low levels in the late secretory phase vs. other phases (p< . ). p-erk of ectopic heec showed no cycle dependence, with moderate-high levels in all phases. t-erk in all tissues was high and invariable. in cultured heec, treatment with pf from endometriotic women significantly increased heec viability after h compared to normal pf (p< . ). this effect was abrogated by erk / inhibitor. among factors known to be high in endometriotic pf, vegf increased p-erk in cultured heec in and min (p< . ), while e , il- , and mcp- had no effect. conclusions: high p-erk found specifically in sprouting endothelial progenitor cells and focal ectopic capillaries suggests that erk / is involved in endometriotic angiogenesis. the peritoneal microenvironment of endometriosis may be persistently stimulating erk-mediated endometrial angiogenesis through vegf. further investigation into erk / inhibitors may offer a novel treatment of endometriosis. the events leading to the development of post operative adhesions are unknown, though recent observations emphasize the crucial role played by the superoxide ion generated by hypoxia. exposure of normal peritoneal fibroblasts to hypoxia caused the development of the adhesion phenotype, which is characterized by increased extracellular matrix molecules and inflammatory cytokines as well as high protein nitration and low nitric oxide. superoxide dismutases (sod) are a family of metalloenzymes that eliminates superoxide by converting it to hydrogen peroxide, protecting mammalian organisms against superoxide. objective: to determine whether sod is differentially expressed between normal peritoneal and adhesion fibroblasts and whether its expression is modulated by hypoxia. methods: fibroblasts from normal peritoneal and adhesion tissues were cultured under normal ( % o ) and hypoxia ( % o ) conditions for and hours. real time rt/pcr was utilized to measure the absolute mrna levels for sod in both cell lines before and after hypoxia exposure. results: normal peritoneal fibroblasts exhibited significantly higher basal mrna levels for sod ( . pg/ grna, p< . ) as compared to adhesion fibroblasts ( . pg/ grna, p< . ). short exposure to hypoxia resulted in a significant increase in sod mrna levels to . and pg/ grna in normal and adhesion fibroblasts respectively, p< . . in contrast, long exposure to hypoxia resulted in a significant decrease in sod mrna levels to . and . pg/ grna in normal peritoneal and adhesion fibroblasts respectively, p< . . conclusion: sod mrna levels are lower in adhesion as compared to normal fibroblasts, both basally and following short and longer exposure to hypoxia. reduced sod expression creates an milieu with greater free radical levels, which leads to the development of the adhesion phenotype. enhancement of sod levels and/or function are likely to be of benefit for reduction of postoperative adhesion development. objectives: development of endometriosis requires ectopic attachment and proliferation of endometrial tissue. the invasive process required to establish endometriosis may involve matrix metalloproteinases (mmps), including mmp- . recently, we have demonstrated that statins inhibit proliferation of endometrial stroma. this study evaluated the effects of simvastatin on a nude mouse model of endometriosis and on modulation of mmp- . methods: proliferative phase human endometrial biopsies were established as organ cultures or utilized for stromal cell isolation. to establish endometriosis in the nude mouse, endometrial tissues were maintained in nm estradiol (e) for hrs and subsequently injected intraperitoneally into ovariectomized nude mice. mice were treated with e ( μg, silastic capsule implants) and simvastatin ( - mg/kg/day) by gavage for days beginning day after tissue injection. control mice received e+vehicle. subsequently, animals were sacrificed and endometrial implants were evaluated. studies of endometrial stroma involved plating the cells in the presence of e ( nm) or e+medroxyprogesterone acetate (mpa; pm) with and without simvastatin ( - μm). some cultures additionally received interleukin- (il- , ng/ml). conditioned media was subjected to western analysis for expression of mmp- . results: in vivo studies demonstrated a dose-dependent inhibitory effect of simvastatin on number and volume of endometrial implants in mice. at the highest dose of simvastatin ( mg/kg/day), the number of endometrial implants was decreased by % and the volume by % in comparison to the control group. isolated stromal cells expressed abundant levels of mmp- following treatment with e, but minimal levels in cultures additionally receiving mpa or simvastatin. although il- induced a dramatic increase in mmp- secretion from cells pretreated with e alone, treatment with either mpa or simvastatin prevented this induction. cultures receiving e+mpa+simvastatin were the most resistant to mmp- induction by il- . objective: to increase awareness of potential presentations of adenomyosis and the differential of a uterine mass. material and method: in a tertiary medical center, a patient was being evaluated for a uterine cystic mass and cyclic dysmenorrhea. the patient is a year old nulliparous woman who has been complaining of cyclic dysmenorrhea for several years. the pain starts with the onset of menses and lasts around weeks. the patient did not improve on contraceptives. the patient had prior laparoscopy and imaging studies which misdiagnosed the mass as either a leiomyoma or an adnexal hemorrhagic cyst. the patient underwent exploratory laparotomy and resection of the mass. results: on ultrasound, the mass appeared as an echodense cyst within the uterus. intraoperatively, a cm thick-walled well circumscribed uterine chocolate cyst was identified. the resection of the cyst was performed in similarly to an ovarian cystectomy. tissue examination confirmed the diagnosis of cystic adenomyosis. following surgical intervention, the patient reported significant improvement of symptoms. conclusion: this case highlights an unusual presentation and treatment of adenomyosis and cyclic dysmenorrhea. cystic adenomyosis should be on the differential of uterine cystic mass, particularly in young women with cyclic dysmenorrhea and menorrhagia. the earlier misdiagnosis probably resulted from the unfamiliarity of physicians with this condition. similar clinical presentations may occur with congenital uterine anomalies (with an obstructed uterine segment) and cystic degeneration of a leiomyoma. the incidence and pathogenesis of cystic adenomyosis are unknown. oral contraceptives may be helpful as a first line therapy. however, resection of the adenomyotic cyst appears to be more effective, particularly in women seeking fertility. objective: it is hypothesized that abnormalities within the eutopic endometrium of females with endometriosis can cause the ectopic growth of the endometrial tissue at extrauterine sites. previous studies have shown that gene expression in eutopic endometrium of women with endometriosis is markedly different from disease-free females. inflammatory cytokines and receptor-dependent kinase pathways are widely recognized as therapeutic targets for immune disorders, which is believed to be the underlying pathogenesis of endometriosis. in this study we asked whether responses of primary eutopic endometrial cell cultures are dysregulated between females with or without endometriosis. methods: a total of biopsies of endometriotic eutopic endometrium (eee) and disease-free endometrium (dfe) were obtained from proliferative phase females. the primary cell cultures established from these biopsies were treated with tnfa to induce expression of inflammatory mediators. parallel cultures were also treated with kinase inhibitors of p , mek, pi k and ikk. after a period of or hours, concentrations of il- , mcp- , and gm-csf in cell culture supernatants were analyzed by elisa. results: in eee, basal concentrations of mcp- and gm-csf were - times higher, while il- was times higher compared to dfe. as expected, tnfa treatment stimulated higher levels of cytokine secretion in dfe mimicking disease state, however, the same treatment had almost no effect on eee. kinase inhibitors were very effective in lowering the cytokine levels of dfe, however, their effect on eee were less dramatic. conclusion: eee expresses higher levels of inflammatory cytokines under basal conditions, which is in concert with the current literature. our results validate that high il- levels in endometrium are diagnostic for females with disease. the increase of gm-csf, il- and mcp- following tnfa treatment was expected in dfe however; tnfa's effect was blunted in eee, which implies that eee are already highly activated. the effect of kinase inhibitors on cytokine production from eee was unaltered relative to dfe, which implies that tnfa stimulated kinase pathways are modified even in eee. endometrial cancer is the fourth leading cause of cancer in women, and hyperplasia and adenocarcinoma are more commonly seen in women with polycystic ovary syndrome (pcos). mig- , a negative inhibitor of egf signaling, is expressed in normal secretory phase endometrium and associated with hyperplasia in mig- knockout mice. objective: we examined and compared endometrium from normal and pcos women for mig- expression and characterized its regulation using in vivo and in vitro models. methods: immunohistochemistry (ihc) and real-time pcr were performed in endometrium from normal (n= ) women and pcos (n= ) women. regulation of mig- was studied in ishikawa and ecc- endometrial cell lines, treated with sex steroids or egf. endometrial xenografts from normal and pcos women were implanted in ovxd rag -(c) immunocompromised mice, treated with e or e + p pellets to mimic a natural cycle. results: mig- protein expression was low in the functionalis of the proliferative phase and high in the secretory phase; this pattern was reversed in the basalis layer. pcos secretory phase endometrium had significantly less mig- protein and mrna than normal endometrium (p < . ). xenografts using pcos samples had paradoxically elevated expression of mig- compared to normal, and appeared unresponsive to steroid treatments. mig- expression in endometrial cell lines was regulated by egf but not by ovarian steroids, e or e + p. conclusions: mig- expression is low in proliferating endometrium and regulated by egf. risk of hyperplasia or cancer in pcos women can be explained by altered expression of mig- . reduced expression mig- provides insight into endometrial function and may lead to better treatment options for disorders of endometrial proliferation including endometriosis, adenomyosis, endometrial hyperplasia and cancer. supported in part by nih-u -hd (sly), u hd (lcg) and u hd (fjd). could androgens influence human luteal cells function? anna tropea, mariateresa orlando, maria francesca gangale, federica romani, isamaria loiudice, federica tiberi, stefania catino, antonio lanzone, rosanna apa. cattedrà di fisiopatologia della riproduzione, università cattolica del s. cuore (ucsc), roma, italy; istituto scientifico internazionale (isi) "paolo vi", ucsc, roma, italy; istituto di ricerca "associazione oasi maria ss onlus", troina (en), italy. in pcos patients reproductive dysfunctions are frequently observed even if ovulation occurs. an impaired luteal function could partially explain this subfertility, since luteal steroidogenesis deficiency and premature luteal degeneration have been described in pcos patients. based on the frequent observation of high plasmatic levels of androgens in pcos, we investigated whether hyperandrogenism could negatively affect luteal function.to this aim, we tested the in vitro effects of androgens on progesterone (p) and on vascular endothelial growth factor (vegf) production by human luteal cells. indeed, vegf is essential for normal luteal development and function being an important regulator of angiogenesis and vascular permeability. since prostaglandins (pgs) play a central role in modulating luteal function, the influence of androgens on pge and pgf a secretion was also investigated. in order to investigate whether testosterone and androstenedione act directly or after local aromatisation, we tested the in vitro effects of estriol, estrone and --estradiol on p, vegf, pge and pgf secretion by human luteal cells. moreover, we tested the effects of testosterone and androstenedione in presence of exemestane -an aromatase inactivator. highly purified human luteal cells were cultured for h with medium alone (control) or in presence of increasing concentrations of testosterone or dihydrotestosterone or androstenedione (from - to - m) or in presence of increasing concentrations of estriol, estrone and --estradiol (from . to ng/ml). moreover, testosterone and androstenedione - m were tested in presence of exemestane (from to nm). in the culture medium vegf, p, pge and pgf secretion was assayed by commercially available elisa kits. in order to evaluate the influence of androgens and estrogens on vegf mrna expression on luteal cells real-time rt-pcr has been performed. our results demonstrated that testosterone, androstenedione and dihydrotestosterone were all able to significantly reduce vegf secretion in human luteal cells, while no effect was seen on vegf mrna expression. androgens were also able to significantly decrease p and pgf secretion, while an increase was observed on pge production. moreover our preliminary results demonstrated that in human isolated luteal cells estriol, estrone and -estradiol at all tested doses are able to mimic androgens effects on p and pge production. on the contrary estrogens were able to increased vegf release. estrogens seemed to have no effect on pgf  released. data regarding exemestane inhibition of testosterone and androstenedione are still in progress. increased levels of androgens were able to decrease luteal vegf, p and pgf release and might be involved in pcos-luteal phase defect. nevertheless, the observed effects could probably be attributed -at least in part -to estrogens locally produced via the aromatase enzyme, rather than be directly mediated by testosterone and androstenedione. the in vitro effect of proghrelin-derived peptides on purified human luteal cells. anna tropea, mariateresa orlando, maria francesca gangale, federica romani, isamaria loiudice, federica tiberi, stefania catino, antonio lanzone, rosanna apa. cattedrà di fisiopatologia della riproduzione, università cattolica del s. cuore (ucsc), roma, italy; istituto scientifico internazionale (isi) "paolo vi", ucsc, roma, italy; istituto di ricerca "associazione oasi maria ss onlus", troina (en), italy. ghrelin, well known mediator of neuroendocrine effects, has been demonstrated to have a role as signal for energy insufficiency. several evidences suggested that ghrelin might also operate as regulator of reproductive function. indeed, we recently demonstrated that both p and vegf released were significantly decreased by ghrelin in isolated human luteal cells. moreover ghrelin was also able to reduce prostaglandin (pg) e and increase pgf luteal cells release. in the present work we investigated whether two ghrelin-related peptides derived by the same ghrelin precursor (unacylated ghrelin and obestatin) might affect human isolated luteal cells function. conventionally regarded as an inert form of ghrelin, unacylated ghrelin has been recently proven as biologically active in specific cellular contexts. obestatin has been suggested to directly control porcine ovarian cell functions. in these assumptions, we investigated whether unacylated ghrelin and obestatin could directly affect luteal progesterone (p) release. moreover, since vascular endothelial growth factor (vegf) is known to play a critical role in luteal development and function, the influence of unacylated ghrelin and obestatin on luteal vegf, pgf and pge production was also analysed. highly purified human luteal cells were incubated for h with medium alone (control) or with hcg ( ng/ml) or with cocl ( μm) or in presence of increasing concentrations of unacylated ghrelin ( - nm) and obestatin ( - nm). in the culture medium vegf, pgf , pge and p secretion was assayed by commercially available elisa kits. moreover real-time rt-pcr has been performed in order to evaluate whether unacylated ghrelin and obestatin could affect vegf mrna in human luteal cells. our preliminary results demonstrated that either unacylated ghrelin or obestatin are able to negatively affect luteal steroidogenesis. moreover, both peptides seemed to increase the release of two luteotropic factors -vegf and pge and to reduce pgf release -a luteolytic prostanoid -from isolated human luteal cells. finally data regarding the expression of vegf mrna are still in progress. our results suggest that unacylated ghrelin and obestatin -two ghrelin-related peptides -could play a role in regulating luteal function affecting both luteal steroidogenesis and luteotropic/luteolytic imbalance. the mechanisms responsible for labor progression have yet to be fully elucidated. in a previous study, over-expression of small conductance calcium-activated k + channel isoform (sk ) in transgenic mice compromised parturition, suggesting a role for sk channels in this process. based on these findings, we hypothesized that sk channel expression is reduced late in pregnancy to enable the uterus to produce the forceful contractions required for parturition. we investigated the effects of sk channel expression on gestation and parturition by comparing transgenic mice over-expressing mice sk (sk t/t ) mice to wild-type (wt). in wt mice, sk transcript and protein are significantly reduced during pregnancy. the force produced by uterine strips from sk t/t mice on the day of delivery was significantly less than wt or sk knockout control (sk dox ), and the contractile force reached wt levels by application of the sk channel inhibitor, apamin. moreover, lipopolysaccharide and ru , which induce pre-term labor in wt mice, failed to result in completion of delivery in sk t/t mice. thus, stimuli that initiate parturition under normal circumstances are insufficient to coordinate the uterine contractions needed for the completion of delivery when sk channel activity is in excess. the mechanism(s) down-regulating this channel in the uterus during pregnancy is unknown. the sk gene promoter region contains two specificity protein (sp) binding sites; ) sp , is a transcription factor known to enhance the transcription of genes in response to estrogen, and ) sp , which competes for the same binding motif as sp , reduces gene expression. sp protein expression in mice uteri dramatically decreases in late pregnancy, while sp protein level remains consistent. our data indicate that sk channels must be downregulated for the gravid uterus to generate labor contractions sufficient for delivery in both term and preterm mice. the changes in sk channel expression may be transcriptionally regulated by sp and sp .